Review ArticleRedox Modulating NRF2 A Potential Mediator of Cancer StemCell Resistance

In-geun Ryoo Sang-hwan Lee and Mi-Kyoung Kwak

College of Pharmacy The Catholic University of Korea Bucheon Gyeonggi-do 420-743 Republic of Korea

Correspondence should be addressed to Mi-Kyoung Kwak mkwakcatholicackr

Received 5 June 2015 Accepted 27 July 2015

Academic Editor Amit Tyagi

Copyright copy 2016 In-geun Ryoo et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Tumors contain a distinct small subpopulation of cells that possess stem cell-like characteristicsThese cells have been called cancerstem cells (CSCs) and are thought to be responsible for anticancer drug resistance and tumor relapse after therapy Emergingevidence indicates that CSCs share many properties such as self-renewal and quiescence with normal stem cells In particularCSCs and normal stem cells retain low levels of reactive oxygen species (ROS) which can contribute to stem cell maintenance andresistance to stressful tumor environments Current literatures demonstrate that the activation of ataxia telangiectasia mutated(ATM) and forkhead box O3 (FoxO3) is associated with the maintenance of low ROS levels in normal stem cells such ashematopoietic stem cells However the importance of ROS signaling in CSC biology remains poorly understood Recent studiesdemonstrate that nuclear factor-erythroid 2-related factor 2 (NRF2) amaster regulator of the cellular antioxidant defense system isinvolved in the maintenance of quiescence survival and stress resistance of CSCs Here we review the recent findings on the rolesof NRF2 in maintenance of the redox state and multidrug resistance in CSCs focusing on how NRF2-mediated ROS modulationinfluences the growth and resistance of CSCs

1 Introduction

Reactive oxygen species (ROS) are highly proactivemoleculesderived from molecular oxygen and include free radicalssuch as hydrogen peroxide (H

2O2) superoxide anion (O2

minus)and hydroxyl radical (OH∙) Under normal physiologicalconditions low-to-moderate levels of ROS play a criticalrole in cellular development and signaling However excessROS levels which can be caused by metabolic dysfunctionor environmental stress conditions can lead to peroxida-tion of cellular macromolecules such as lipids proteinsand nucleic acids [1 2] These ROS-induced byproductseventually trigger cellular senescence carcinogenesis or celldeath Interestingly mammalian cells have developed tightlyregulated antioxidant systems for protection against ROS-induced oxidative damage For example the superoxideanion a product of mitochondrial dysfunction is convertedto H2O2by superoxide dismutases (SODs) H

2O2is then

decomposed to oxygen and water by catalase or glutathioneperoxidases (GPXs) [3 4]

Multiple lines of evidence suggest that cancer cells possesshigher levels of intracellular ROS than normal cells [5 6]Elevated ROS levels in cancer can be utilized to promote cellproliferation invasiveness and metastasis [6ndash9] There areseveral underlying mechanisms involved in ROS elevation incancer cells First activated oncogenes can trigger ROS pro-duction through upregulation of ROS-generating enzymessuch as NADPH oxidases (NOXs) [10 11] The RAS onco-gene increases NOX1 expression via the extracellular signal-regulated kinases (ERK) [10] or mitogen-activated proteinkinase (MAPK) signaling pathways [11] in human cancersOverexpression of the c-MYC oncogene in normal humanfibroblasts induces DNA damage by increasing ROS levels[12] Mutation of mitochondrial DNA (mtDNA) is a majorcause of ROS elevation in cancer cells Polyak et al found thatseven out of ten colorectal cancer cell lines retained somaticmutations in mtDNA most of these mutations were detectedin mitochondrial genes such as those encoding cytochrome coxidases 1ndash3 which has potential implications with respect toincrease in mitochondrial ROS [13] Cancer cells have their

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 2428153 14 pageshttpdxdoiorg10115520162428153

2 Oxidative Medicine and Cellular Longevity

own adaptation mechanisms against increased ROS such asupregulation of ROS scavenging systems As a result of thesesystems malignant transformed cells can utilize ROS as asignal for tumor progression and metastasis [5 14]

Recent studies are expanding our knowledge about thebiological implications of ROS in cancer stem cells (CSCs)which are small subpopulation of cancer cells responsible fortumorigenesis and tumor progression and relapse Based onincreasing evidence for the role of ROS in stem cell biologylower levels of cellular ROS are considered beneficial forthe maintenance of quiescence and chemoradioresistanceof CSCs [15] In this review we show current findingsillustrating the relationship between ROS and CSC biologyand present emerging evidence that nuclear factor-erythroid2- (NF-E2-) related factor 2 (NRF2) may play a role in CSCgrowth and resistance

2 CSCs and Resistance to EnvironmentalStress and Chemotherapy

Tumors contain a small population of cells with stem cellproperties namely CSCs or tumor-initiating cells (TICs)[16 17] These cells are known to play a crucial role in tumormaintenance and relapse In the 1990s the first experimentalevidence of CSCs was introduced by Bonnet and Dick [18]In acute myeloid leukemia (AML) it appeared that 01 to1 of the total cell population had tumor-initiating activityThis subpopulation exhibited a CD34+CD38minus phenotypeand was capable of tumor reconstitution after transplantationinto nonobese diabeticsevere combined immune-deficient(NODSCID)mice [18] Since thenmultiple lines of evidencehave revealed that theCSCpopulation exists in different typesof solid tumors including brain breast and colon cancers[19ndash21]

CSCs are characterized by their self-renewal and differen-tiation capacity similar to normal stem cells [16] Markers ofembryonic stem cells (ESCs) such as octamer-binding tran-scription factor 4 (OCT4) Nanog homeobox (NANOG) andSRY (sex determining region Y)-box 2 (SOX2) are expressedin CSCs and the Wnt120573-catenin Hedgehog and Notchpathways are implicated in the self-renewal of CSCs [22ndash26]Several CSC-specific surfacemarkers have been identified forthe detection and isolation of CSCs from the tumor massCD44+CD24minus phenotypic cells were isolated from breastcancer tissues and breast carcinoma cell lines andwere shownto exhibit self-renewal and high tumorigenic capacity [27]The CD133+ subpopulation from brain tumors demonstratedstem cell properties and showed tumor-initiating capabilityin NODSCID mouse brains [20]

CSCs are considered to be one of themain causes of tumorrecurrence after therapy CSC resistance to conventionalanticancer drug therapies and radiotherapy is attributed toincreased expression of ROS scavenging molecules drugtransporters and enhanced DNA repair capacity [28ndash30] Ithas been reported that CSCs contain low levels of endogenousROS compared to those seen in non-CSCs [31 32] Inprimary AMLs a subpopulation of low ROS-producing cellsdemonstrated characteristics of CSCs including quiescence

and a CD34+CD38minus phenotype [31] Moreover Chang et alobserved that this population of low ROS-producing cellsexhibited increased expression of stem cell markers (OCT4and NANOG) and higher chemoresistance and tumorigenic-ity than the population of high ROS-producing cells in headand neck cancer [32]

The ATP-binding cassette transporter (ABC transporter)family is known to induce multidrug resistance by activelytransporting intracellular drugs to outside of the cell [33 34]P-glycoprotein (P-gp) multidrug resistance-associated pro-teins (MRPs) and breast cancer associated protein (BCRP)belong to the ABC transporter family Since many types ofanticancer agents are substrates of these ABC transportersenhanced expression of P-gp MRPs and BCRP is stronglyassociatedwith the chemoresistant phenotype of CSCs Basedon this ABC transporters are often used as a CSC surfacemarker [28 35] The side population (SP) which is a fractionof cells that expresses a high level of BCRP can be isolatedfrom cancer cells using fluorogenic dye Hoechst 33342As Hoechst 33342 dye is a substrate of BCRP the BCRPoverexpressing cells exclude this fluorescent dye and therebya fraction of cells with low fluorescence can be isolated fromnon-SP cellsThis method is now widely used to isolate CSCsfrom cancer cell lines and specimens using a flow cytometry[36]

3 Role of ROS in Stem Cells

Stem cells can be broadly classified into two categoriesadult stem cells (eg haematopoietic stem cells (HSCs) andneural stem cells) and ESCs Under homeostatic conditionsthese stem cells particularly adult stem cells are generallymaintained in a quiescent state However stem cells are ableto escape quiescence and enter the cell cycle for proliferationwhen they are exposed to metabolic changes [37ndash40]

ROS are considered as important signaling molecules instem cell biology They play a key role in stem cell mainte-nance by preserving quiescence and protecting againstenvironmental stress [37 38] Recent stem cell studieshave demonstrated that stem cells contain low levels ofintracellular ROS and this redox status was found to becritical for regulation of stem cell quiescence and self-renewal Murine ESCs exhibited low levels of intracellularROS compared to differentiated murine cells due to theincreased level of GSH and thioredoxin (TXN) system [41]Furthermore HSCs containing low levels of ROS (ie cellswith low fluorescent activity following the incubation with2101584071015840-dichlorodihydrofluorescein diacetate (DCFDA) a cell-permeable ROS sensing fluorogenic dye) were highly qui-escent and expressed relatively high levels of NOTCH1 andBCRP compared to high DCFDA fluorescent cells Highlevels of ROS are cytotoxic since ROS accumulation in HSCscan lead to cellular prematurity and senescence [42 43]

ROS have been reported to be involved in stem cell differ-entiation Bone marrow mesenchymal stem cells (MSCs) arefound in the bone marrow together with HSCs and have thepotential to differentiate to adipocytes osteocytes and chon-drocytes It was shown that humanMSCs are highly resistant

Oxidative Medicine and Cellular Longevity 3

to ROSThis phenomenon was linked to low levels of cellularROS and high levels of SODs catalase GPX1 and GSH inMSCs [38 40] Elevated antioxidant molecules appear to playa crucial role in the protection of stem cells against oxidativestress However under certain circumstances NOX-derivedROS are associated with MSC differentiation The treatmentof MSCs with antioxidants or interfering RNA of NOX4 pre-vented adipocyte differentiation of MSCs via cAMP responseelement-binding protein (CREB) inhibition Similarly ESCdifferentiation to the cardiac lineage was dependent onNOX4-derived ROS [44] These findings indicate that ROSare important for stem cell fate determination for quiescenceor differentiation

4 Redox Signaling Molecules in Stem Cells

It has been reported that multiple signaling molecules areinvolved in ROS-mediated regulation of stem cells (Figure 1)First ataxia telangiectasia mutated (ATM) plays a criticalrole in controlling ROS levels in stem cells ATM a ser-inethreonine protein kinase is a known regulator of theDNA damage response and contributes to the regulation ofcellular ROS ATM is known to regulate ROS via modulationof AMPK-mTOR pathway or NADPH production [45 46]Ito et al showed that atmminusminus mice developed bone marrowfailure after 24 weeks of age due to a depletion of HSCsIn this study HSCs in atm knockout mice showed higherlevels of ROS than wild type mice which presumably causeda reduction in the self-renewal activity of HSCs Howeverthe treatment of mice with antioxidant N-acetylcysteine(NAC) restored HSC reconstitution in atm knockout mice byreducing ROS in HSCs confirming the critical role of ROSin HSCs maintenance [47] Similarly in another study NACtreatment prevented hypersensitivity of atmminusminusmice to X-rayirradiation and senescence of atmminusminus embryonic fibroblasts[48] Cosentino et al have presented amolecularmechanisticrole for ATM demonstrating that ATM activation promotesthe binding of heat shock protein 27 (HSP27) to glucose-6-phosphate dehydrogenase (G6PDH) which can result inG6PDH activation and subsequent NADPH increase [46]

The forkhead box O (FoxO) transcription factor familyis also implicated in redox regulation of stem cells TheFoxO family including FoxO1 FoxO3 FoxO4 and FoxO6is a key regulator of cell survival proliferation DNA repairand apoptosis FoxO1 and FoxO3 are reported to upregulatethe expression of GSH biosynthetic enzymes and SODsand therefore are associated with cellular protection againstoxidative stress [49 50] Particularly FoxO3 has been knownto play crucial roles in cytoprotection of stem cells includingHSCs [51ndash53] Loss of FoxO3a which regulates the expressionof antioxidant enzymes such as catalases and SOD2 led toROS accumulation and thus a higher rate of cell cyclingand a loss of quiescence in HSCs [51] In foxo1foxo3afoxo4triple-knockout mice the number of HSCs was substantiallydecreased and apoptotic HSCs were increased through ROSelevation [52] Notably Yalcin et al provided a link betweenATMand the FoxOprotein in ROS regulation of stem cells Infoxo3minusminusHSCs ATMexpressionwas diminished compared to

Quiescenceself-renewal

Senescenceapoptosis

cell cycle progressionLow ROS

High ROSATM uarr FoxO uarr

PI3KAKT uarr

FoxO darr

Figure 1 Involvement of ROS in normal stem cell quiescence andself-renewal In normal stem cells modulation of ROS levels candetermine quiescence and cell fate progression At low ROS levelswhich are maintained by ATM and FoxO signaling stem cellsremain quiescent and self-renewal activity is enhancedOn the otherhand increased ROS levels result in cell cycle progression cellularsenescence and apoptosis The PI3K-AKT pathway is known toelevate ROS levels by negative regulation of FoxO

normalHSCs suggesting that FoxO3 repressed ROS viaATMregulation [53] Similar to HSCs foxo-deficient neural stemcells demonstrated a decline in self-renewal capacity due toincreased cellular ROS levels [54 55]

The phosphoinositide 3-kinase (PI3K)AKT pathway isanother ROS regulator in normal stem cells In particularPI3KAKT signaling associates with FoxO transcription fac-tors to mediate ROS regulation Activated AKT promotesFoxO phosphorylation resulting in the nuclear export andcytoplasmic degradation of FoxO through the proteasome[56 57] Therefore ak11990512 double knockout HSCs displayedincreased quiescence and low cellular ROS levels [58] Con-sistently persistent activation of the PI3KAKT pathway inphosphatase and tensin homolog (PTEN) deleted HSCs ledto defective quiescence resulting in cellular senescence [59]Based on the above observations the PI3KAKTpathway andFoxOATMpathway exhibit opposite roles in ROS regulationof stem cells

Hypoxia-inducible factors (HIFs) are transcription fac-tors that respond to hypoxic conditions [60] They are alsocritical factors for the maintenance of stem cells HSCscultured in hypoxic conditions displayed a higher colonyformation capacity and high HIF levels positively regulatedthe pluripotency of human ESCs by activating stemnesstranscription factors such as OCT4 SOX2 and NANOG[61 62] Moreover in neuronal stem cells in a hypoxicenvironment accumulated HIF1120572 promoted Wnt120573-cateninpathway activation [63] The involvement of HIFs in stemcell biology is mediated by ROS Takubo et al observed thathif1120572minusminus HSCs contain high levels of ROS which could beassociated with a loss of HSC quiescence and an induction ofcellular senescence [64] In agreement with this finding thesuppression of HIF1120572 and HIF2120572 in HSCs led to increasedROS generation via mitochondrial metabolic shift and con-sequently induced cellular senescence and apoptosis Scav-enging ROS byNAC treatment could restore HSC quiescenceand function in stem cells withHIF1120572 andHIF2120572 suppression[65]

5 Involvement of ROS in CSC Biology

Very few studies have investigated the involvement of redoxchange in CSC biology compared to that in cancer cellsor normal stem cells However it has been reported that

4 Oxidative Medicine and Cellular Longevity

CSCs appear to share several ROS-associated propertieswith normal stem cells CSCs are known to contain lowerlevels of ROS compared to non-CSCs CD44+CD24minuslowbreast cancer CSCs isolated from MDA-MB-231 and MCF7mammospheres were relatively more resistant to radiationand this was associated with lower levels of ROS afterradiation [66] Diehn et al observed that ROS levels inCD44+CD24minus breast cancer CSCs were lower than in non-CSCs and the expression levels of the modulatory subunit ofglutamate cysteine ligase (GCLM the rate-limiting enzymeof GSH synthesis) and FoxO1 were high Treatment of theCD44+CD24minus subpopulationwith buthionine sulphoximine(BSO) an inhibitor of GSH synthesis resulted in reducedcolony forming capacity and increased sensitivity to radiationtherapy through an increase in ROS [67] In a study usinghuman AML specimens leukemic stem cells with low levelsof ROS majorly contributed to stem cell quiescence bymaintaining a low rate of oxidative phosphorylation andmetabolism [31] A leukemia with a high amount of leukemicstem cells showed low levels of ROS and increased expressionof GPX3 compared to tumors with a low frequency ofleukemic stem cells This study demonstrated that GPX3levels positively correlated with poor prognostic outcome inAML patients [68] Glioma stem cells within the tumor masshave low levels of cellular ROS although they are located in ahypoxic environment A proposed molecular mechanism ofthis phenomenon was the significantly upregulated expres-sion of peroxiredoxin 4 (PRDX4) in glioma stem cells [69]

Evidence is indicating that low ROS levels in CSCsresult from the intrinsic characteristics of CSCs Cell surfacemarkers of CSCs including CD44 and CD13 are found tobe involved in ROS regulation Ishimoto et al demonstratedthat a variant isoform of CD44 (CD44v) can bind to thecystineglutamate exchange transporter xCT and activatescysteine uptake to enhance GSH synthesis in gastrointestinalCSCs [70] The expression of antioxidant genes such asGPX12 was significantly increased in CD44+ gastric tumorcells In addition knockdown of CD44 in mice led toROS increase p38MAPK activation and cellular senescencethat are related to p21 expression In a subsequent studythe same group demonstrated that the number of CD44+cells increased with neoadjuvant chemotherapy in head andneck squamous cell carcinoma (HNSCC) patients TheseCD44+ undifferentiated cancer cells displayed high xCTexpression GSH upregulation and low cellular ROS levelsAblation of xCT by siRNA or sulfasalazine treatment (xCT-mediated cystine transport inhibitor) induced differentiationof HNSCCCSCs both in vitro and in vivo [71] CD13 has beenidentified as a surface marker for liver CSCs In liver cancercell lines including Huh7 and PLCPRF5 CD13 positive cellspredominated the SP fraction and were mainly in the G0G1phase of the cell cycle Additionally resistance to anticancerdrugs or radiation in the CD13 positive cell fraction wasmuch higher than that observed in the CD13 negative cellfraction Direct comparison of ROS levels between the twocell fractions revealed that the CD13 positive cell fractioncontains lower levels of ROS and expresses higher levelsof GCLM [72] In another study by the same group CD13expression reduced transforming growth factor-120573 (TGF-120573)

induced ROS production and promoted survival of liverCSCs [73]

It has been demonstrated that signaling pathwaysinvolved in ROS regulation of normal stem cells also playa role in CSC biology The nuclear expression levels ofFoxO3a was high in chronic myeloid leukemia-initiatingcells and the transplantation of leukemic stem cells derivedfrom foxo3a knockout mice significantly reduced their abilityto cause myeloid leukemia in an animal model [74] Thisstudy also revealed that TGF-120573 is a crucial regulator ofFoxO3a activity In the SP of MCF-7 breast cancer cellsactivation of the PI3Kmammalian target rapamycin (mTOR)signaling pathway was important for tumorigenecity of theseCSCs and knockdown of PI3K or mTOR led to ablatedtumorigenecity [75] When CD133+CD44+ prostate cancercells were grown in sphere-forming conditions activatedPI3KAKT signaling was found to be critical for maintainingCSCs [76] CD44+CD24minusor low cells isolated from breastcancer cell lines and breast cancer patient specimens wereradioresistant and this resistant phenotype was associatedwith ATM signaling activation [77]

6 NRF2 as a Key Molecule forRedox Homeostasis

In 1990 Rushmore and Pickett discovered the enhancersequence in the rat gsta2 gene promoter as a response elementto 120573-naphthoflavone and t-butylhydroquinone (t-BHQ) andnamed it antioxidant responsive element (ARE) [78] Subse-quent studies revealed that ARE is commonly involved in thetranscription of multiple antioxidant and detoxifying genesincluding glutamate-cysteine ligase (GCL) glutathione S-transferase (GST) and NAD(P)H quinone oxidoreductase-1 (NQO-1) [79 80] Based on sequence homology betweenARE and MAF-recognition element (MRE) further studieshypothesized that small MAF and bZIP caprsquonrsquocollar (CNC)transcription factors may interact with ARE [81 82] AmongbZIP CNC transcription factors NRF2 was found to play acrucial role in ARE regulation in which inducible expressionof NQO1 and GST was ablated in t-butylhydroxy anisole-treated nrf2 null mice in contrast to the observation in wildtype mice [83] After this report numerous studies haveelucidated a wide spectrum of protective effects of NRF2signaling against various stressors For example sensitivityto benzo[a]pyrene-induced carcinogenesis was significantlygreater in nrf2-knockout mice than in wild type mice [84]

To account for the protective effects of Nrf2 comparativeanalyses of gene expression patterns were carried out innrf2-deficient and wild type mice following treatment withNrf2 activators In global gene analysis of dithiolethione-administered mouse livers Nrf2 was found to governthe expression of xenobiotic-detoxifying enzymes GSH-generating systems antioxidant proteins and the molec-ular chaperone-26S proteasome [85] Similarly Hu et aldemonstrated that detoxifying enzymes antioxidants drugtransporters stress response proteins and some signalingmolecules serve as Nrf2-dependent and isothiocyanate-inducible genes in mouse liver [86] It has now been firmly

Oxidative Medicine and Cellular Longevity 5

established thatNRF2 regulates divergent genes to coordinatexenobiotic detoxification and redox homeostasis [85 87ndash89]In its function as a regulator of cellular redox homeostasisNRF2 elevates the expression of GCL and the cysteinetransporter xCT to increase cellular GSH levels NRF2 alsoenhances regeneration of reduced GSH by upregulatingGPX and GSH reductase (GSR) Expression of thioredoxin1 (TXN1) thioredoxin reductase 1 (TXNRD1) and perox-iredoxin 16 which can reduce oxidized protein thiols isalso under the control of NRF2 In addition the levels ofNADPH a cofactor of many antioxidant enzymes such asGSR and TXNRD can be increased by NRF2 (reviewedin Hayes and Dinkova-Kostova [90]) The expression ofmultiple NADPH generating enzymes such as G6PDH and6-phosphogluconate dehydrogenase is upregulated by NRF2Additionally the role of NRF2 in ABC transporter expressionfor xenobiotic detoxification is notable The basal expressionlevel of Mrp1 was relatively lower in nrf2-deficient fibroblaststhan that in wild type fibroblasts and the treatment ofmice with Nrf2 activating diethyl maleate increased Mrp1expression in the liver [91] Levels of MDR1 MRP23 andBCRP were elevated following oltipraz treatment in pri-mary human hepatocytes [92] Sulforaphane (SFN) treatmentenhanced the levels ofMDR1 BCRP andMRP2 in the blood-brain barrier of rats [93] Our recent study showed thatgenetic activation of NRF2 via KEAP1 silencing increases theexpression ofMDR1MRP23 and BCRP in human proximaltubular epithelial cells [94] As direct molecular evidencefunctional AREs have been identified in human MRP3 [95]and BCRP genes [96]

Kelch-like ECH-associated protein 1 (KEAP1) a cysteine-rich actin-binding protein is the main negative regulatorof NRF2 activity [83 97] Under quiescent conditionsNRF2 remains inactive by forming a complex with KEAP1in the cytoplasm NRF2 is subject to ubiquitination andKEAP1-induced proteasomal degradation through the Cullin3 (CUL3) based E3 ligase KEAP1 has three major domainsas follows (i) The BTB domain is associated with KEAP1homodimerization (ii) the IVR domain plays a role inregulation ofKEAP1 activity and (iii) theKelchDGRdomainmediates binding with NRF2 [83 90 98ndash101] The bindingof NRF2 with KEAP1 has been described as the ldquohinge andlatchrdquo model where one molecule of NRF2 interacts with theKelchDGR domains of the KEAP1 dimer through conservedmotifs called ETGE (DN-X-E-TS-G-E) and DLG (L-X-X-Q-D-X-D-L-G) [102ndash104] In these reports it was shown thatthe binding affinity of the ETGE motif to KEAP1 is muchhigher than that of the DLG motif It has therefore beenshown that ldquolatchrdquo binding of the NRF2 DLG motif is easilybroken by modifications of KEAP1 cysteine residues by ROSor electrophiles In turn disrupted DLG binding of NRF2 toKEAP1 leads to the blockade of ubiquitination and furtherdegradation of NRF2 resulting in nuclear translocation ofNRF2 It has been demonstrated that the sulfhydryl groups ofmultiple cysteine residues of KEAP1 can be directly modifiedby oxidationreduction or alkylation In particular Cys151Cys273 and Cys288 were found to be essential for theregulation of NRF2 activity [99 104ndash106] Mutation of theCys273 or Cys288 residue of KEAP1 ablated its ability to

suppress NRF2 activity leading to accumulation of the NRF2protein [99 104]

In addition to KEAP1-mediated stability regulationNRF2 activity can be modulated at multiple steps First itis noticeable that NRF2 activity is regulated at the transcrip-tional step Functional AREs were identified in the murinenrf2 gene promoter and were involved in the autoregulationof NRF2 through transcriptional activation [107] Moreovera single nucleotide polymorphism in the ARE-like sequencesof the human NRF2 promoter was associated with increasedlung cancer susceptibility [108] Second it was shown thatNRF2 activity is regulated by posttranslationalmodificationsStudies indicate that NRF2 activation involves phosphoryla-tion signaling mediated by multiple kinase pathways such asMAPK protein kinase C (PKC) PI3K and protein kinaseRNA-like endoplasmic reticulum kinase (PERK) [109ndash111]Meanwhile glycogen synthase kinase-3 (GSK-3) a consti-tutively active serinethreonine kinase was found to inhibitNRF2 activity [112] Last NRF2 activity is increased byseveral intrinsic proteins such as p21 and p62 [113 114] Forexample p62 a linker protein of ubiquitinated proteins toautophagy degradation binds to the KEAP1 protein andinterferes with the binding of NRF2 to KEAP1 resulting inNRF2 stabilization

7 Emerging Role of NRF2 in Cancer Biology

Continuous or fatal stimuli such as toxic chemicals and excessROS disrupt cellular homeostasis causing macromoleculardamage and alterations in cell cycle and growth signalingwhich can eventually result in carcinogenesis The NRF2pathway deserved significant attention in the area of cancerbiology because numerous studies have demonstrated thatactivation of the NRF2 pathway decreases the sensitivity ofcells to carcinogens [115ndash117] For instance the burden ofgastric neoplasia caused by benzo[a]pyrene was effectivelyattenuated by the Nrf2 activator oltipraz in wild type micewhereas nrf2 knockout mice did not show any protectiveeffect of oltipraz [84] Similarly the incidence of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) induced urinary blad-der carcinomawas greater in nrf2 knockoutmice than in wildtype mice and oltipraz treatment reduced tumor incidenceonly in wild type mice [118]

Although it has been firmly confirmed that NRF2 activa-tion can protect cells against a wide range of toxicants andstressors aberrant activation of NRF2 has been associatedwith several types of cancers NRF2 levels were constitutivelyelevated in cancer cell lines and tumor samples of the lungbreast esophagus endometrial cancers and prostate cancers[119ndash125] Molecular mechanisms involved in constitutiveNRF2 activation include the following (i) somatic mutationsof KEAP1 or NRF2 (ii) epigenetic silencing of the KEAP1gene (iii) aberrant accumulation of proteins that competewith NRF2 for KEAP1 binding and (iv) oncogene-mediatedoverexpression of NRF2 [101 126] First somatic mutationsof KEAP1-NRF2 have been reported in an initial study byPadmanabhan et al [127] This study identified mutations

6 Oxidative Medicine and Cellular Longevity

in the KelchDGR domain of KEAP1 in lung cancer celllines as well as lung cancer tissue samples and demonstratedthat these mutant KEAP1 proteins lost their NRF2 repressivefunction which resulted in NRF2 accumulation Singh et alalso demonstrated that the KelchDGR and IVR domainsof the KEAP1 gene contain multiple somatic mutations andthese mutations were identified in 19 of tumor specimensfrom non-small cell lung cancer patients [123] In gallbladdercancer 4 of 13 patients harbored KEAP1 mutations [122]Shibata et al reported that NRF2 somatic mutations werefound in 107 of primary lung cancer patients and 272of primary head and neck cancer patients [122] Notablythesemutationswere primarily located in theDLG andETGEmotifs and eventually led to the loss of a proper interactionbetween the NRF2 protein and KEAP1 Second CpG islandhypermethylation in the KEAP1 promoter resulted in lowKEAP1 expression in lung cancer cell lines and tumor samples[124]Third in humanhepatocellular carcinoma p62 positivecellular aggregates were found with a frequency of 25 andmost of these tumors retained higher levels of NRF2 andits target gene expression [128] Fourth oncogenes have alsobeen shown to play a role in NRF2 signaling Oncogenicactivation of KRAS (KRASG12D) c-MYC (c-MYCERT12) andBRAF (BRAFV619E) elevates the transcript levels of NRF2 andits target gene expression [129]

It is now widely accepted that aberrant activation ofNRF2 can enhance cancer cell survival and growth in oxi-dizing tumor environments and further promote chemoradioresistance Indeed the prognosis of cancer patientsnegatively correlated with NRF2 levels in the tumor [122130] The favorable effect of NRF2 overexpression on tumorsurvival and growth can be attributed to the increase inNRF2 target antioxidant proteins and their counteractiveeffect on oxidative stress For instance GSH which is adirect target molecule of NRF2 has been shown to be criticalfor cell proliferation [131 132] In addition to its antioxi-dant contribution Mitsuishi et al provided direct evidencedemonstrating that NRF2 alters the cellular metabolism inrelation to anabolic pathways to accelerate cell proliferation[133] Multiple metabolic genes such as those involvedin the pentose phosphate pathway were upregulated byNRF2 through ARE and these changes promoted purinesynthesis glutamine metabolism and NADPH productionfor enhanced cell proliferation

Constitutively high levels of NRF2 have been associatedwith chemoresistance as well as radioresistance Cancer cellswith high NRF2 activity were less sensitive to cytotoxicchemotherapeutics such as cisplatin doxorubicin and 5-fluorouracil through facilitated detoxification of anticanceragents and enhanced antioxidant capacity [101] It is there-fore hypothesized that NRF2 inhibition can enhance thechemosensitivity of cancers NRF2 siRNA could suppresscancer resistance to cisplatin topoisomerase inhibitors and5-fluorouracil [101 122 134 135] Cancer cells with constitu-tively high NRF2 were protected against 120574-radiation inducedtoxicity Moreover siRNA-mediated inhibition of NRF2 innon-small cell lung cancer cell lines substantially enhancedradiosensitivity [136] Additionally NRF2 expression was

increased during the acquisition of chemoresistance In ourprevious study doxorubicin-selected ovarian cancer cellsdemonstrated increased expression of NRF2 and its targetgenes forGSH synthesis andNRF2 inhibition in this resistantcell line could restore doxorubicin sensitivity [137]

Our understanding of the role of NRF2 in cancer cell sig-naling has expanded In particular the relationship betweenoncogenic signaling and NRF2 is noteworthy As men-tioned earlier activation of oncogenes such as KRAS and c-MYC increased the expression of NRF2 presumably throughoncogene-mediated ROS increase and this phenomenonappears to contribute to the maintenance of reduced redoxhomeostasis in cancer cells [129] In ERBB2 (Her2Neu) over-expressing ovarian cancer cells the stable silencing of NRF2repressed ERBB2 expression and its downstream signalingand retarded tumor growth Therefore the inhibition ofNRF2 could sensitize these cells to taxol therapy by repressingERBB2 expression [138] Moreover NRF2 was shown to beassociated with HIF signaling which is a critical factor fortumor angiogenesis When NRF2 was stably knocked downin colon carcinoma cell lines hypoxia-inducible HIF-1120572accumulation was abrogated and consequently angiogenesisand tumor growth were significantly suppressed in NRF2knockdown tumors compared to the control group [139] Intype 2 papillary renal cell cancer which is characterized byloss of the fumarate hydratase gene and consequentmetabolicalteration accumulated fumarate was associated with tumorprogression via NRF2 signaling Fumarate was shown tomodify KEAP1 cysteine residues and elevate NRF2 levelswhich contributed to the growth and progression of type 2papillary renal cell cancer [140] These accumulating linesof evidence suggest that once cells are transformed to theneoplastic stage cancer cells utilize NRF2 signaling to adaptto the stressful tumor environment and to promote survivaland further cancer progression (Figure 2)

8 Involvement of NRF2 Signaling inStem Cell Quiescence and Differentiation

There is considerable evidence to suggest that NRF2 playsa role in normal stem cell biology [141ndash145] For exampleNRF2 activation in HSCs plays a critical role in not only themaintenance of quiescence but also in the determination ofdifferentiation fate [141 145] In Drosophila intestinal stemcells constitutive Nrf2 activation sustained quiescence byreducing the levels of ROS via upregulation of antioxidantgenes such as gclc However in the case of KEAP1-mediatedNrf2 repression high levels of intracellular ROS facilitatedan ablation of the quiescent state in intestinal stem cellsand age-related degeneration in the intestinal epithelium[146] Similarly low intracellular ROS levels are requiredfor the maintenance of quiescence in human airway basalstem cells (ABSCs) When exposed to exogenous ROSquiescent ABSCs enter the proliferation stage Changes inROS levels activate the NRF2-Notch pathway which resultsin self-renewal and protection of ABSCs from ROS-inducedhyperproliferation and senescence Moreover the quiescent

Oxidative Medicine and Cellular Longevity 7

NRF2 sMaf

GCLCGCLMNQO1SODs

Antioxidant enzymes Drug transportersMetabolic enzymes

ARE

Tumor growthprogression

MDRsMRPsBCRP

G6PDPGDME1

Cell survival Drug effluxProliferation

Target genes

Figure 2 Implications of NRF2 signaling in cancer NRF2 coordi-nates the expression of genes associated with cellular redox regu-lation metabolism and xenobiotic efflux and thereby its aberrantactivation promotes cancer cell survival proliferation and anti-cancer drug resistance GCLM glutamate cysteine ligase modifiersubunit GCLC glutamate cysteine ligase catalytic subunit NQO1NAD(P)Hquinone oxidoreductase 1 SODs superoxide dismutasesG6PDH glucose-6-phosphate dehydrogenase PGD phosphoglu-conate dehydrogenase ME1 malic enzyme 1 MDRs multidrugresistance proteinsMRPsmultidrug resistance-associated proteinsBCRP breast cancer resistance protein

state of ABSCs was maintained by NRF2 activation [147]In osteoclast progenitor cells hydrogen sulfide (H

2S) inhib-

ited human osteoclast differentiation by NRF2-dependentinduction of peroxiredoxin 1 and NQO1 These resultswere further confirmed using NRF2 activators includingsulforaphane and t-BHQ [148] It is also notable that NRF2participates in the regulation of cell fate determination ofHSCsMurakami et al demonstrated that HSCs derived fromKEAP1-deficientmice exhibited preferred differentiation intothe granulocyte-monocyte lineage rather than differentiatinginto the erythroid-lymphoid lineage [145]

Up to now numerous studies have demonstrated thatNRF2 plays a protective role against various stressors instem cells In neural stem cells overexpression of NRF2or pharmacological NRF2 activation prevented necroticcell death [149] In an animal study nrf2-deficient miceshowed defective stem cell function HSCs from nrf2minusminus miceexpressed lower levels of prosurvival cytokines and exhib-ited spontaneous apoptosis [150] Ionizing radiation-inducedmyelosuppression and mortality were mitigated throughNRF2-mediated Notch signaling activation in HSCs [142]Similarly resveratrol-inducedNRF2 expression improved thesurvival of cardiac stem cells and consequently regeneratedinfarcted myocardium [151 152] The heme oxygenase 1(HO-1) inducer cobalt protoporphyrin (CoPP) elicited anantiapoptotic effect on cardiac stem cells via activation of the

ERK-NRF2 pathway [153] In neural stem cells NRF2 activa-tion by melatonin or t-BHQ ameliorated lipopolysaccharide(LPS) orH

2O2induced cell death [149 154] In addition amy-

loid 120573-mediated neural stem cell death could be alleviated byexogenous NRF2 transduction which was accompanied byincreased expression of GCLC NQO-1 andHO-1This studyalso demonstrated that neuronal differentiation of neuralstem cells is enhanced by NRF2 activation [155] Similar toneural stem cells NRF2 has a protective role against hypoxicand oxidative stress conditions in undifferentiated MSCsTreatment of the murine mesenchymal stem cell line withadrenaline increased the mRNA expression of nrf2 gclc andxCT leading to an increase in GSH levels and the preventionof ROS-induced cytotoxicity [156 157]

9 Potential Implication of NRF2 inCSC Maintenance and Resistance

The role of NRF2 in CSC biology is now beginning tobe unveiled Similar to the case of normal stem cells itwas shown that NRF2 contributes to CSC stemness bymaintaining their self-renewal capacity and protecting themfrom chemoradiotherapy Achuthan et al established stablechemotherapy-resistant breast cancer cells and observed thatthese cells expressed higher levels of CD133 and OCT-4indicating that these cells exhibit CSC phenotype [34] Ofnote it was shown that ROS levels were relatively low inthese drug-selected cells presumably due to higher levelsof antioxidant enzymes such as SOD1 and GPX12 NRF2protein stabilizationwas associatedwith high levels of antiox-idant enzymes As an underlying molecular mechanismdiminished proteasome activity and increased p21 levelsappear to stabilize the NRF2 protein in these stem-like cellsEvidently p21 knockdown repressed the mammosphere-forming potential of these stem-like breast cancer cellsSimilarly a study by Zhu et al showed the involvement ofNRF2 in glioblastoma stem cells that were isolated fromhuman surgical glioblastoma specimens NRF2 knockdownin glioblastoma stem cells inhibited cell proliferation andneurosphere formation and further suppressed SOX2 expres-sion Moreover NRF2 knockdown changed the cell cycledistribution to the G2 phase and significantly attenuated thetumorigenecity of glioblastoma stem cells [158 159] Theseresults are providing evidence that NRF2 is necessary formaintenance of the self-renewal capacity of glioblastomastem cells

On the other hand activation of NRF2 signaling has beendemonstrated in different types of CSC models includinglung esophageal breast ovarian and colon CSCs and thisis closely correlated with themaintenance of low intracellularROS levels and chemoresistance of CSCs [160ndash165] In lungand esophageal cancer cells cigarette smoke condensateincreased the SP as well as BCRP expression which arehallmarks of CSCs [165] Promoter analysis revealed thatBCRP expression was associated with elevated levels ofNRF2 aryl hydrocarbon receptor (AhR) and specificityprotein 1 (SP1) Additionally this study demonstrated thatmithramycin diminished BCRP expression via repression of

8 Oxidative Medicine and Cellular Longevity

Low ROS

Increased drug efflux

SurvivalChemoresistance

CSC

TumorRecurrenceProgression

Radioresistance

PERKuarr

p21uarr

p62uarr

Proteasome darr

NRF2 uarr

Figure 3 Potential roles of NRF2 signaling in CSCs InCSC-like cellmodels NRF2 is activated through multiple molecular mechanismsin a context-dependent manner The upregulation of competingproteins such as p62 and p21 activation of PERK or repressedproteasome function were shown to enhance NRF2 activity inthese models Elevated NRF2 levels in CSCs can contribute tothe maintenance of low ROS by upregulating multiple antioxidantgenes In addition NRF2-mediated expression of ABC transporterselicits efflux of anticancer drugs from cancer cells Overall activatedNRF2 signaling facilitates CSCs survival and stress resistance andconsequently it can be suggested that CSCs with high NRF2 activityplay a crucial role in tumor recurrence and further progression

NRF2 AhR and SP1 and thereby inhibiting the expressionof genes associated with CSC-related pathways resulting inreduced proliferation and tumorigenecity Similarly it wasreported that lung cancer SP cells exhibited high levels ofNRF2 and BCRP expression These SP cells were highlytumorigenic and possessed self-renewal capacity comparedto non-SP cells [164] Emmink et al performed a proteomeanalysis on collected secretome from highly tumorigenicCSCs and their corresponding nontumorigenic differentiatedcells both of which were established from human colorectalspecimens [160] Subsequent bioinformatic analysis revealedthat the CSC secretome contained a large amount of proteinsassociated with cell survival and protein quality controlcompared to differentiated tumor cells Notably the CSCsecretome contained an NRF2 antioxidant and detoxifyingprotein signature in that it included elevated levels ofGCLC GPX23 and TXNRD1 This study provided novelevidence that CSCs secrete NRF2 target antioxidant proteinsto counteract extracellular stressors and chemotherapeuticsIn patients with ovarian clear cell carcinoma the expressionof the CSC maker aldehyde dehydrogenase-1 (ALDH1) wasstrongly correlated with an advanced clinical stage andreduced progression free survival [161] Ovarian clear cellcarcinoma cells with high ALDH1 expressionmaintained lowlevels of ROS compared to ALDH-low cells and these cellswere shown to express higher levels of NRF2 and its targetgenes

Two recent studies have demonstratedNRF2 activation insphere cultures of breast cancer cells that is one of models ofCSCs Wu et al showed that mammospheres derived fromMCF7 and MDA-MB231 breast cancer cell lines exhibitedlower ROS levels compared to their monolayer counterpartsThey also showed that levels of NRF2 and target genes such

as NQO1 and GCLM were elevated in mammospheres [163]Similarly our group has shown substantially elevated NRF2protein levels along with increased expression of antioxidantgenes (eg HO-1 and GPX2) and drug efflux transporters(eg MRP2 and BCRP) in sphere cultures of breast cancercells NRF2 accumulation was also observed in sphere-cultured ovarian and colon cancer cells However shRNA-mediated downregulation ofNRF2 led to decreased chemore-sistance of mammospheres presumably due to reduced levelsof antioxidant genes and drug transporters High ROS levelsin NRF2 knockdown mammospheres caused sphere growthretardation and apoptosis Coherently ablation of ABCtransporter induction in NRF2 knockdown mammospheressensitized to anticancer agents [162] Additionally this studyprovided evidence that increased NRF2 protein expression inmammospheres can be linked to 26S proteasome reductionand p62 accumulation In particular knockdown of p62 inMCF7 mammospheres significantly attenuated NRF2 eleva-tion

Surviving dedifferentiated breast cancer cells after chem-otherapy treatment retained high levels of NRF2 activationsimilar to other CSCs However NRF2 activation was medi-ated by a noncanonical pathway Levels of PERKwere high indedifferentiated cancer cells and this in turn phosphorylatedand activated NRF2 signaling to maintain low cellular ROSlevels and to express ABC transporters In agreement withthese findings clinical observations revealed that the PERKpathway gene signature is related to chemoresistance andreduced patient survival [166]

10 Concluding Remarks

Recent studies have started to uncover the role of ROS signal-ing in the biology of CSCs which is related to tumorigenecitytumor progression and relapse Expression of the transcrip-tion factor NRF2 a master regulator of antioxidant genesexpression is increased in different models of CSCs and thiselevation is likely to promote CSC maintenance and survivalin an oxidizing tumormicroenvironment In additionNRF2-mediated overexpression of ABC transporters particularlythe CSC marker BCRP may play a critical role in themultidrug resistance of CSCsThese findings combined withthe increasing evidence showing the alteration of KEAP1-NRF2 signaling in cancer cells suggest a novel role of NRF2in CSC maintenance and survival (Figure 3)

One important question that arises from the current stud-ies is whether it is possible to design CSC-targeted therapiesthrough regulation of theNRF2 pathway and its related redoxhomeostasis inCSCs Recent studies provide several potentialclues for addressing this question the naturally occurringalkaloid brusatol could reduce the growth and chemoresis-tance of breast CSCs [163] Treatment ofmammospheres withbrusatol elevated ROS levels and promoted taxol-inducedgrowth retardation and cell death The NRF2 repressivemechanism of brusatol has not been clearly elucidated but itappears to be independent of KEAP1-mediated degradation[167] In addition to brusatol natural compounds such as

Oxidative Medicine and Cellular Longevity 9

chrysin apigenin luteolin and trigonelline are known toinhibit NRF2 signaling in several types of cancer cells [102168ndash170] and therefore the development of NRF2 inhibitorswith characterized modes of action will enable efficient tar-geting of the redox homeostasis system as well as multidrugresistance systems in CSCs

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This studywas financially supported by theNational ResearchFoundation (NRF) funded by the Ministry of Science ICT ampFuture Planning (NRF-2013R1A2A2A01015497)

References

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[2] P Storz ldquoForkhead homeobox type O transcription factorsin the responses to oxidative stressrdquo Antioxidants amp RedoxSignaling vol 14 no 4 pp 593ndash605 2011

[3] R S Balaban S Nemoto and T Finkel ldquoMitochondria oxi-dants and agingrdquo Cell vol 120 no 4 pp 483ndash495 2005

[4] L A Sena and N S Chandel ldquoPhysiological roles of mitochon-drial reactive oxygen speciesrdquoMolecular Cell vol 48 no 2 pp158ndash167 2012

[5] P T Schumacker ldquoReactive oxygen species in cancer cells liveby the sword die by the swordrdquo Cancer Cell vol 10 no 3 pp175ndash176 2006

[6] S Toyokuni K Okamoto J Yodoi and H Hiai ldquoPersistentoxidative stress in cancerrdquo FEBS Letters vol 358 no 1 pp 1ndash3 1995

[7] L Behrend G Henderson and R Zwacka ldquoReactive oxy-gen species in oncogenic transformationrdquo Biochemical SocietyTransactions vol 31 no 6 pp 1441ndash1444 2003

[8] E O Hileman J Liu M Albitar M J Keating and P HuangldquoIntrinsic oxidative stress in cancer cells a biochemical basis fortherapeutic selectivityrdquo Cancer Chemotherapy and Pharmacol-ogy vol 53 no 3 pp 209ndash219 2004

[9] H PelicanoD Carney and PHuang ldquoROS stress in cancer cellsand therapeutic implicationsrdquo Drug Resistance Updates vol 7no 2 pp 97ndash110 2004

[10] Y Adachi Y Shibai J Mitsushita W H Shang K Hirose andT Kamata ldquoOncogenic Ras upregulates NADPH oxidase 1 geneexpression through MEK-ERK-dependent phosphorylation ofGATA-6rdquo Oncogene vol 27 no 36 pp 4921ndash4932 2008

[11] J Mitsushita J D Lambeth and T Kamata ldquoThe superoxide-generating oxidase Nox1 is functionally required for Ras onco-gene transformationrdquoCancer Research vol 64 no 10 pp 3580ndash3585 2004

[12] O Vafa M Wade S Kern et al ldquoc-Myc can induce DNAdamage increase reactive oxygen species and mitigate p53function a mechanism for oncogene-induced genetic instabil-ityrdquoMolecular Cell vol 9 no 5 pp 1031ndash1044 2002

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[17] B S Zhou H Zhang M Damelin K G Geles J C Grindleyand P B Dirks ldquoTumour-initiating cells challenges and oppor-tunities for anticancer drug discoveryrdquo Nature Reviews DrugDiscovery vol 8 no 10 pp 806ndash823 2009

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10 Oxidative Medicine and Cellular Longevity

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[31] E Lagadinou A Sach K Callahan et al ldquoBCL-2 inhibitiontargets oxidative phosphorylation and selectively eradicatesquiescent human leukemia stem cellsrdquo Cell Stem Cell vol 12no 3 pp 329ndash341 2013

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[35] L Zinzi M Contino M Cantore E Capparelli M Leopoldoand N A Colabufo ldquoABC transporters in CSCs membranesas a novel target for treating tumor relapserdquo Frontiers inPharmacology vol 5 article 163 2014

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[39] V A Rafalski E Mancini and A Brunet ldquoEnergy metabolismand energy-sensing pathways in mammalian embryonic andadult stem cell faterdquo Journal of Cell Science vol 125 no 23 pp5597ndash5608 2012

[40] K Wang T Zhang Q Dong E C Nice C Huang and Y WeildquoRedox homeostasis the linchpin in stem cell self-renewal anddifferentiationrdquo Cell Death and Disease vol 4 no 3 p e5372013

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[43] S Ghaffari ldquoOxidative stress in the regulation of normal andneoplastic hematopoiesisrdquo Antioxidants amp Redox Signaling vol10 no 11 pp 1923ndash1940 2008

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[45] A Alexander S-L Cai J Kim et al ldquoATM signals to TSC2in the cytoplasm to regulate mTORC1 in response to ROSrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 107 no 9 pp 4153ndash4158 2010

[46] C Cosentino D Grieco and V Costanzo ldquoATM activates thepentose phosphate pathway promoting anti-oxidant defenceand DNA repairrdquo The EMBO Journal vol 30 no 3 pp 546ndash555 2011

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[49] Y Wang Y Zhou and D T Graves ldquoFOXO transcription fac-tors their clinical significance and regulationrdquoBioMed ResearchInternational vol 2014 Article ID 925350 13 pages 2014

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[51] K Miyamoto K Y Araki K Naka et al ldquoFoxo3a is essentialfor maintenance of the hematopoietic stem cell poolrdquo Cell StemCell vol 1 no 1 pp 101ndash112 2007

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[55] V M Renault V A Rafalski A A Morgan et al ldquoFoxO3regulates neural stem cell homeostasisrdquo Cell Stem Cell vol 5no 5 pp 527ndash539 2009

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[58] M M Juntilla V D Patil M Calamito R P Joshi M JBirnbaum and G A Koretzky ldquoAKT1 and AKT2 maintainhematopoietic stem cell function by regulating reactive oxygenspeciesrdquo Blood vol 115 no 20 pp 4030ndash4038 2010

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stem cells through Wnt120573-catenin signallingrdquo Nature Cell Biol-ogy vol 12 no 10 pp 1007ndash1013 2010

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[64] K Takubo N Goda W Yamada et al ldquoRegulation of the HIF-1alpha level is essential for hematopoietic stem cellsrdquo Cell StemCell vol 7 no 3 pp 391ndash402 2010

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[67] M Diehn R W Cho N A Lobo et al ldquoAssociation of reactiveoxygen species levels and radioresistance in cancer stem cellsrdquoNature vol 458 no 7239 pp 780ndash783 2009

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[69] S Kim C Kwon and I Nakano ldquoDetoxification of oxidativestress in glioma stem cells mechanism clinical relevance andtherapeutic developmentrdquo Journal of Neuroscience Research vol92 no 11 pp 1419ndash1424 2014

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[72] N Haraguchi H Ishii K Mimori et al ldquoCD13 is a therapeutictarget in human liver cancer stem cellsrdquo Journal of ClinicalInvestigation vol 120 no 9 pp 3326ndash3339 2010

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[74] K Naka T Hoshii T Muraguchi et al ldquoTGF-120573ndashFOXO sig-nalling maintains leukaemia-initiating cells in chronic myeloidleukaemiardquo Nature vol 463 no 7281 pp 676ndash680 2010

[75] J Zhou J Wulfkuhle H Zhang et al ldquoActivation of thePTENmTORSTAT3 pathway in breast cancer stem-like cellsis required for viability and maintenancerdquo Proceedings of theNational Academy of Sciences vol 104 no 41 pp 16158ndash161632007

[76] A Dubrovska S Kim R J Salamone et al ldquoThe role ofPTENAktPI3K signaling in the maintenance and viability ofprostate cancer stem-like cell populationsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 106 no 1 pp 268ndash273 2009

[77] H Yin and J Glass ldquoThe phenotypic radiation resistance ofCS44+CD24minusor low breast cancer cells is mediated through theenhanced activation of ATM signalingrdquo PLoS ONE vol 6 no9 Article ID e24080 2011

[78] T H Rushmore and C Pickett ldquoTranscriptional regulation ofthe rat glutathione S-transferase Ya subunit gene Characteri-zation of a xenobiotic-responsive element controlling inducibleexpression by phenolic antioxidantsrdquo The Journal of BiologicalChemistry vol 265 no 24 pp 14648ndash14653 1990

[79] Y Li and A Jaiswal ldquoRegulation of humanNAD (P)H quinoneoxidoreductase gene Role of AP1 binding site contained withinhuman antioxidant response elementrdquoThe Journal of BiologicalChemistry vol 267 no 21 pp 15097ndash15104 1992

[80] T Prestera P Talalay J Alam et al ldquoParallel induction of hemeoxygenase-1 and chemoprotective phase 2 enzymes by elec-trophiles and antioxidants regulation by upstream antioxidant-responsive elements (ARE)rdquoMolecular Medicine vol 1 no 7 p827 1995

[81] K Itoh K IgarashiNHayashi et al ldquoCloning and characteriza-tion of a novel erythroid cell-derived CNC family transcriptionfactor heterodimerizing with the small Maf family proteinsrdquoMolecular and Cellular Biology vol 15 no 8 pp 4184ndash41931995

[82] R Venugopal and A K Jaiswal ldquoNrf1 and Nrf2 positivelyand c-Fos and Fra1 negatively regulate the human antioxidantresponse element-mediated expression of NAD(P)Hquinoneoxidoreductase1 generdquo Proceedings of the National Academy ofSciences vol 93 no 25 pp 14960ndash14965 1996

[83] K Itoh T Chiba S Takahashi et al ldquoAn Nrf2small Mafheterodimer mediates the induction of phase II detoxifyingenzyme genes through antioxidant response elementsrdquo Bio-chemical and Biophysical Research Communications vol 236no 2 pp 313ndash322 1997

[84] M Ramos-Gomez M-K Kwak P M Dolan et al ldquoSensitivityto carcinogenesis is increased and chemoprotective efficacy ofenzyme inducers is lost in nrf2 transcription factor-deficientmicerdquo Proceedings of the National Academy of Sciences vol 98no 6 pp 3410ndash3415 2001

[85] M-K Kwak N Wakabayashi K Itoh H Motohashi MYamamoto and T W Kensler ldquoModulation of gene expressionby cancer chemopreventive dithiolethiones through the Keap1-Nrf2 pathway Identification of novel gene clusters for cellsurvivalrdquo The Journal of Biological Chemistry vol 278 no 10pp 8135ndash8145 2003

[86] R Hu C Xu G Shen et al ldquoGene expression profiles inducedby cancer chemopreventive isothiocyanate sulforaphane in theliver of C57BL6J mice and C57BL6JNrf2 (minusminus) micerdquo CancerLetters vol 243 no 2 pp 170ndash192 2006

[87] S Chanas Q Jiang and M McMahon ldquoLoss of the Nrf2transcription factor causes a marked reduction in constitutiveand inducible expression of the glutathione S-transferase Gsta1Gsta2 Gstm1 Gstm2 Gstm3 and Gstm4 genes in the livers ofmale and female micerdquo Biochemical Journal vol 365 no 2 pp405ndash416 2002

[88] H-Y Cho S P Reddy A DeBiase M Yamamoto and SR Kleeberger ldquoGene expression profiling of NRF2-mediatedprotection against oxidative injuryrdquo Free Radical Biology andMedicine vol 38 no 3 pp 325ndash343 2005

[89] G Shen C Xu R Hu et al ldquoModulation of nuclear factorE2-related factor 2-mediated gene expression in mice liver andsmall intestine by cancer chemopreventive agent curcuminrdquoMolecular Cancer Therapeutics vol 5 no 1 pp 39ndash51 2006

[90] J D Hayes and A T Dinkova-Kostova ldquoThe Nrf2 regulatorynetwork provides an interface between redox and intermediarymetabolismrdquo Trends in Biochemical Sciences vol 39 no 4 pp199ndash218 2014

[91] A Hayashi H Suzuki K Itoh M Yamamoto and Y SugiyamaldquoTranscription factor Nrf2 is required for the constitutive andinducible expression of multidrug resistance-associated protein1 in mouse embryo fibroblastsrdquo Biochemical and BiophysicalResearch Communications vol 310 no 3 pp 824ndash829 2003

[92] E Jigorel M Le Vee C Boursier-Neyret Y Parmentier andO Fardel ldquoDifferential regulation of sinusoidal and canalicularhepatic drug transporter expression by xenobiotics activating

12 Oxidative Medicine and Cellular Longevity

drug-sensing receptors in primary human hepatocytesrdquo DrugMetabolism and Disposition vol 34 no 10 pp 1756ndash1763 2006

[93] X Wang C R Campos J C Peart et al ldquoNrf2 upregulatesATP binding cassette transporter expression and activity atthe blood-brain and blood-spinal cord barriersrdquo Journal ofNeuroscience vol 34 no 25 pp 8585ndash8593 2014

[94] H Jeong I Ryoo andMKwak ldquoRegulation of the expression ofrenal drug transporters in KEAP1-knockdown human tubularcellsrdquo Toxicology in Vitro vol 29 no 5 pp 884ndash892 2015

[95] M J Canet M D Merrell B G Harder et al ldquoIdentificationof a functional antioxidant response element within the eighthintron of the human ABCC

3generdquo Drug Metabolism and

Disposition vol 43 no 1 pp 93ndash99 2014[96] A Singh H Wu P Zhang C Happel J Ma and S Biswal

ldquoExpression of ABCG2 (BCRP) is regulated by Nrf2 in cancercells that confers side population and chemoresistance pheno-typerdquo Molecular Cancer Therapeutics vol 9 no 8 pp 2365ndash2376 2010

[97] N Wakabayashi K Itoh J Wakabayashi et al ldquoKeap1-nullmutation leads to postnatal lethality due to constitutive Nrf2activationrdquo Nature Genetics vol 35 no 3 pp 238ndash245 2003

[98] S B Cullinan J D Gordan J Jin J W Harper and J A DiehlldquoThe Keap1-BTB protein is an adaptor that bridges Nrf2 to aCul3-based E3 ligase oxidative stress sensing by a Cul3-Keap1ligaserdquoMolecular and Cellular Biology vol 24 no 19 pp 8477ndash8486 2004

[99] D D Zhang and M Hannink ldquoDistinct cysteine residues inkeap1 are required for keap1-dependent ubiquitination of nrf2and for stabilization of nrf2 by chemopreventive agents andoxidative stressrdquoMolecular and Cellular Biology vol 23 no 22pp 8137ndash8151 2003

[100] L M Zipper and R T Mulcahy ldquoThe Keap1 BTBPOZ dimer-ization function is required to sequester Nrf2 in cytoplasmrdquoThe Journal of Biological Chemistry vol 277 no 39 pp 36544ndash36552 2002

[101] M C Jaramillo and D D Zhang ldquoThe emerging role of theNrf2-Keap1 signaling pathway in cancerrdquoGenesampDevelopmentvol 27 no 20 pp 2179ndash2191 2013

[102] K I Tong Y Katoh H Kusunoki K Itoh T Tanaka and MYamamoto ldquoKeap1 recruits Neh2 through binding to ETGEand DLG motifs characterization of the two-site molecularrecognition modelrdquoMolecular and Cellular Biology vol 26 no8 pp 2887ndash2900 2006

[103] K I Tong A Kobayashi F Katsuoka andM Yamamoto ldquoTwo-site substrate recognition model for the Keap1-Nrf2 system ahinge and latch mechanismrdquo Biological Chemistry vol 387 no10-11 pp 1311ndash1320 2006

[104] K I Tong B Padmanabhan A Kobayashi et al ldquoDifferentelectrostatic potentials define ETGE and DLG motifs as hingeand latch in oxidative stress responserdquo Molecular and CellularBiology vol 27 no 21 pp 7511ndash7521 2007

[105] M Kobayashi L Li N Iwamoto et al ldquoThe antioxidant defensesystemKeap1-Nrf2 comprises amultiple sensingmechanism forresponding to a wide range of chemical compoundsrdquoMolecularand Cellular Biology vol 29 no 2 pp 493ndash502 2009

[106] T Yamamoto T Suzuki A Kobayashi et al ldquoPhysiological sig-nificance of reactive cysteine residues of Keap1 in determiningNrf2 activityrdquoMolecular and Cellular Biology vol 28 no 8 pp2758ndash2770 2008

[107] M K Kwak K Itoh M Yamamoto and T W KenslerldquoEnhanced expression of the transcription factorNrf2 by cancer

chemopreventive agents role of antioxidant response element-like sequences in the nrf2 promoterrdquo Molecular and CellularBiology vol 22 no 9 pp 2883ndash2892 2002

[108] T Suzuki T Shibata K Takaya et al ldquoRegulatory nexus ofsynthesis and degradation deciphers cellular Nrf2 expressionlevelsrdquoMolecular and Cellular Biology vol 33 no 12 pp 2402ndash2412 2013

[109] S B Cullinan D Zhang M Hannink E Arvisais R JKaufman and J A Diehl ldquoNrf2 is a direct PERK substrateand effector of PERK-dependent cell survivalrdquo Molecular andCellular Biology vol 23 no 20 pp 7198ndash7209 2003

[110] W Li and A Kong ldquoMolecular mechanisms of Nrf2-mediatedantioxidant responserdquo Molecular Carcinogenesis vol 48 no 2pp 91ndash104 2009

[111] Y J Surh ldquoCancer chemoprevention with dietary phytochemi-calsrdquo Nature Reviews Cancer vol 3 no 10 pp 768ndash780 2003

[112] M Salazar A I Rojo D Velasco R M de Sagarra and ACuadrado ldquoGlycogen synthase kinase-3beta inhibits the xeno-biotic and antioxidant cell response by direct phosphorylationand nuclear exclusion of the transcription factor Nrf2rdquo Journalof Biological Chemistry vol 281 no 21 pp 14841ndash14851 2006

[113] W Chen Z Sun X J Wang et al ldquoDirect interactionbetween Nrf2 and p21(Cip1WAF1) upregulates the Nrf2-mediated antioxidant responserdquo Molecular Cell vol 34 no 6pp 663ndash673 2009

[114] M Komatsu H Kurokawa S Waguri et al ldquoThe selectiveautophagy substrate p62 activates the stress responsive tran-scription factor Nrf2 through inactivation of Keap1rdquoNature CellBiology vol 12 no 3 pp 213ndash223 2010

[115] J D Hayes M McMahon S Chowdhry and A T Dinkova-Kostova ldquoCancer chemoprevention mechanisms mediatedthrough the Keap1-Nrf2 pathwayrdquoAntioxidants amp Redox Signal-ing vol 13 no 11 pp 1713ndash1748 2010

[116] R Hu C L Saw R Yu and A T Kong ldquoRegulation ofNF-E2-related factor 2 signaling for cancer chemopreventionantioxidant coupled with antiinflammatoryrdquo Antioxidants ampRedox Signaling vol 13 no 11 pp 1679ndash1698 2010

[117] M Kwak and T W Kensler ldquoTargeting NRF2 signaling for can-cer chemopreventionrdquo Toxicology and Applied Pharmacologyvol 244 no 1 pp 66ndash76 2010

[118] K Iida K Itoh Y Kumagai et al ldquoNrf2 is essential for thechemopreventive efficacy of oltipraz against urinary bladdercarcinogenesisrdquo Cancer Research vol 64 no 18 pp 6424ndash64312004

[119] T Jiang N Chen F Zhao et al ldquoHigh levels of Nrf2 deter-mine chemoresistance in type II endometrial cancerrdquo CancerResearch vol 70 no 13 pp 5486ndash5496 2010

[120] Y R Kim J EOhM S Kim et al ldquoOncogenicNRF2mutationsin squamous cell carcinomas of oesophagus and skinrdquo TheJournal of Pathology vol 220 no 4 pp 446ndash451 2010

[121] T Shibata A Kokubu M Gotoh et al ldquoGenetic alterationof Keap1 confers constitutive Nrf2 activation and resistance tochemotherapy in gallbladder cancerrdquoGastroenterology vol 135no 4 pp 1358e4ndash1368e4 2008

[122] T Shibata T Ohta K I Tong et al ldquoCancer related mutationsin NRF2 impair its recognition by Keap1-Cul3 E3 ligase andpromote malignancyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 36 pp13568ndash13573 2008

[123] A Singh V Misra R K Thimmulappa et al ldquoDysfunctionalKEAP1-NRF2 interaction in non-small-cell lung cancerrdquo PLoSMedicine vol 3 no 10 article e420 2006

Oxidative Medicine and Cellular Longevity 13

[124] R Wang J An F Ji H Jiao H Sun and D Zhou ldquoHyper-methylation of the Keap1 gene in human lung cancer cell linesand lung cancer tissuesrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 1 pp 151ndash154 2008

[125] P Zhang A Singh S Yegnasubramanian et al ldquoLoss ofKelch-like ECH-associated protein 1 function in prostate cancercells causes chemoresistance and radioresistance and promotestumor growthrdquoMolecular Cancer Therapeutics vol 9 no 2 pp336ndash346 2010

[126] J D Hayes and M McMahon ldquoNRF2 and KEAP1 mutationspermanent activation of an adaptive response in cancerrdquo Trendsin Biochemical Sciences vol 34 no 4 pp 176ndash188 2009

[127] B Padmanabhan K I Tong T Ohta et al ldquoStructural basis fordefects of Keap1 activity provoked by its pointmutations in lungcancerrdquoMolecular Cell vol 21 no 5 pp 689ndash700 2006

[128] Y Inami S Waguri A Sakamoto et al ldquoPersistent activation ofNrf2 throughp62 in hepatocellular carcinoma cellsrdquoTheJournalof Cell Biology vol 193 no 2 pp 275ndash284 2011

[129] G M DeNicola F A Karreth T J Humpton et al ldquoOncogene-induced Nrf2 transcription promotes ROS detoxification andtumorigenesisrdquo Nature vol 475 no 7354 pp 106ndash109 2011

[130] L M Solis C Behrens W Dong et al ldquoNrf2 and Keap1abnormalities in non-small cell lung carcinoma and associationwith clinicopathologic featuresrdquo Clinical Cancer Research vol16 no 14 pp 3743ndash3753 2010

[131] N M Reddy S R Kleeberger H Y Cho et al ldquoDeficiency inNrf2-GSH signaling impairs type II cell growth and enhancessensitivity to oxidantsrdquoAmerican Journal of Respiratory Cell andMolecular Biology vol 37 no 1 pp 3ndash8 2007

[132] N M Reddy S R Kleeberger M Yamamoto et al ldquoGeneticdissection of the Nrf2-dependent redox signaling-regulatedtranscriptional programs of cell proliferation and cytoprotec-tionrdquo Physiological Genomics vol 32 no 1 pp 74ndash81 2007

[133] Y Mitsuishi K Taguchi Y Kawatani et al ldquoNrf2 redirectsglucose and glutamine into anabolic pathways in metabolicreprogrammingrdquo Cancer Cell vol 22 no 1 pp 66ndash79 2012

[134] J Cho SManandharH LeeH Park andMKwak ldquoRole of theNrf2-antioxidant system in cytotoxicity mediated by anticancercisplatin implication to cancer cell resistancerdquo Cancer Lettersvol 260 no 1-2 pp 96ndash108 2008

[135] T Ohta K Iijima M Miyamoto et al ldquoLoss of Keap1 functionactivates Nrf2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008

[136] A Singh M Bodas N Wakabayashi F Bunz and S BiswalldquoGain of Nrf2 function in non-small-cell lung cancer cellsconfers radioresistancerdquoAntioxidantsampRedox Signaling vol 13no 11 pp 1627ndash1637 2010

[137] G-S Shim S Manandhar D-H Shin T Kim and M KwakldquoAcquisition of doxorubicin resistance in ovarian carcinomacells accompanies activation of the NRF2 pathwayrdquo Free RadicalBiology and Medicine vol 47 no 11 pp 1619ndash1631 2009

[138] S Manandhar B Choi K Jung et al ldquoNRF2 inhibitionrepresses ErbB2 signaling in ovarian carcinoma cells implica-tions for tumor growth retardation and docetaxel sensitivityrdquoFree Radical Biology and Medicine vol 52 no 9 pp 1773ndash17852012

[139] T Kim E-g Hur S J Kang et al ldquoNRF2 blockade suppressescolon tumor angiogenesis by inhibiting hypoxia-induced acti-vation ofHIF-1120572rdquoCancer Research vol 71 no 6 pp 2260ndash22752011

[140] L Kinch N V Grishin and J Brugarolas ldquoSuccination of Keap1and activation of Nrf2-dependent antioxidant pathways in FH-deficient papillary renal cell carcinoma type 2rdquo Cancer Cell vol20 no 4 pp 418ndash420 2011

[141] J J Tsai J A Dudakov K Takahashi et al ldquoNrf2 regulateshaematopoietic stem cell functionrdquo Nature Cell Biology vol 15no 3 pp 309ndash316 2013

[142] J H Kim R KThimmulappa V Kumar et al ldquoNRF2-mediatedNotch pathway activation enhances hematopoietic reconsti-tution following myelosuppressive radiationrdquo The Journal ofClinical Investigation vol 124 no 2 pp 730ndash741 2014

[143] J P Chute ldquoNRF2 mitigates radiation-induced hematopoieticdeathrdquo The Journal of Clinical Investigation vol 124 no 3 pp960ndash961 2014

[144] J Jang Y Wang H Kim M A Lalli and K S KosikldquoNrf2 a regulator of the proteasome controls self-renewal andpluripotency in human embryonic stem cellsrdquo Stem Cells vol32 no 10 pp 2616ndash2625 2014

[145] S Murakami R Shimizu P Romeo M Yamamoto and HMotohashi ldquoKeap1-Nrf2 system regulates cell fate determina-tion of hematopoietic stem cellsrdquo Genes to Cells vol 19 no 3pp 239ndash253 2014

[146] C E Hochmuth B Biteau D Bohmann and H Jasper ldquoRedoxregulation by Keap1 and Nrf2 controls intestinal stem cellproliferation inDrosophilardquoCell Stem Cell vol 8 no 2 pp 188ndash199 2011

[147] M K Paul B Bisht D O Darmawan et al ldquoDynamic changesin intracellular ROS levels regulate airway basal stem cellhomeostasis through Nrf2-dependent notch signalingrdquo CellStem Cell vol 15 no 2 pp 199ndash214 2014

[148] L Gambari G Lisignoli L Cattini C Manferdini A Facchiniand F Grassi ldquoSodium hydrosulfide inhibits the differentiationof osteoclast progenitor cells via NRF2-dependent mechanismrdquoPharmacological Research vol 87 pp 99ndash112 2014

[149] J Li D Johnson M Calkins L Wright C Svendsen andJ Johnson ldquoStabilization of Nrf2 by tBHQ confers protectionagainst oxidative stress-induced cell death in human neuralstem cellsrdquo Toxicological Sciences vol 83 no 2 pp 313ndash3282005

[150] A AMerchant A SinghWMatsui and S Biswal ldquoThe redox-sensitive transcription factor Nrf2 regulatesmurine hematopoi-etic stem cell survival independently of ROS levelsrdquo Blood vol118 no 25 pp 6572ndash6579 2011

[151] N Gurusamy D Ray I Lekli and D K Das ldquoRed wineantioxidant resveratrol-modified cardiac stem cells regener-ate infarcted myocardiumrdquo Journal of Cellular and MolecularMedicine vol 14 no 9 pp 2235ndash2239 2010

[152] N Gorbunov G Petrovski N Gurusamy D Ray D H Kimand D K Das ldquoRegeneration of infarcted myocardium withresveratrol-modified cardiac stem cellsrdquo Journal of Cellular andMolecular Medicine vol 16 no 1 pp 174ndash184 2012

[153] C Cai L Teng D Vu et al ldquoThe heme oxygenase 1 inducer(CoPP) protects human cardiac stem cells against apoptosisthrough activation of the extracellular signal-regulated kinase(ERK)NRF2 signaling pathway and cytokine releaserdquo TheJournal of Biological Chemistry vol 287 no 40 pp 33720ndash33732 2012

[154] J Song S M Kang K M Lee and J E Lee ldquoThe protectiveeffect of melatonin on neural stem cell against LPS-inducedinflammationrdquo BioMed Research International vol 2015 ArticleID 854359 13 pages 2015

14 Oxidative Medicine and Cellular Longevity

[155] V Karkkainen Y Pomeshchik E Savchenko et al ldquoNrf2regulates neurogenesis and protects neural progenitor cellsagainst A120573 toxicityrdquo STEM CELLS vol 32 no 7 pp 1904ndash19162014

[156] M Mohammadzadeh R Halabian A Gharehbaghian et alldquoNrf-2 overexpression in mesenchymal stem cells reducesoxidative stress-induced apoptosis and cytotoxicityrdquo Cell Stressand Chaperones vol 17 no 5 pp 553ndash565 2012

[157] Y Takahata T Takarada M Iemata et al ldquoFunctional expres-sion of 1205732 adrenergic receptors responsible for protectionagainst oxidative stress through promotion of glutathione syn-thesis after Nrf2 upregulation in undifferentiated mesenchymalC3H10T12 stem cellsrdquo Journal of Cellular Physiology vol 218no 2 pp 268ndash275 2009

[158] J Zhu H Wang Y Fan et al ldquoKnockdown of nuclear factorerythroid 2-related factor 2 by lentivirus induces differentiationof glioma stem-like cellsrdquo Oncology Reports vol 32 no 3 pp1170ndash1178 2014

[159] J Zhu H Wang Q Sun et al ldquoNrf2 is required to maintain theself-renewal of glioma stem cellsrdquo BMC Cancer vol 13 no 1article 380 2013

[160] B L Emmink A Verheem W J Van Houdt et al ldquoThe secre-tome of colon cancer stem cells contains drug-metabolizingenzymesrdquo Journal of Proteomics vol 91 pp 84ndash96 2013

[161] T Mizuno N Suzuki H Makino et al ldquoCancer stem-like cellsof ovarian clear cell carcinoma are enriched in the ALDH-highpopulation associated with an accelerated scavenging system inreactive oxygen speciesrdquo Gynecologic Oncology vol 137 no 2pp 299ndash305 2015

[162] I-G Ryoo B-H Choi and M-K Kwak ldquoActivation of NRF2by p62 and proteasome reduction in sphere-forming breastcarcinoma cellsrdquo Oncotarget vol 6 no 10 pp 8167ndash8184 2015

[163] T Wu B G Harder P K Wong J E Lang and D D ZhangldquoOxidative stress mammospheres and Nrf2-new implicationfor breast cancer therapyrdquoMolecular Carcinogenesis 2014

[164] B Yang Y Ma and Y Liu ldquoElevated expression of Nrf-2 andABCG2 involved in multi-drug resistance of lung cancer SPcellsrdquo Drug Research 2015

[165] M Zhang A Mathur Y Zhang et al ldquoMithramycin repressesbasal and cigarette smoke-induced expression of abcg2 andinhibits stem cell signaling in lung and esophageal cancer cellsrdquoCancer Research vol 72 no 16 pp 4178ndash4192 2012

[166] C A Del Vecchio Y Feng E S Sokol et al ldquoDe-differentiationconfers multidrug resistance via noncanonical PERK-Nrf2signalingrdquo PLoS Biology vol 12 no 9 Article ID e1001945 2014

[167] D Ren N F Villeneuve T Jiang et al ldquoBrusatol enhancesthe efficacy of chemotherapy by inhibiting the Nrf2-mediateddefense mechanismrdquo Proceedings of the National Academy ofSciences of the United States of America vol 108 no 4 pp 1433ndash1438 2011

[168] A Arlt S Sebens S Krebs et al ldquoInhibition of the Nrf2 tran-scription factor by the alkaloid trigonelline renders pancreaticcancer cells more susceptible to apoptosis through decreasedproteasomal gene expression and proteasome activityrdquo Onco-gene vol 32 no 40 pp 4825ndash4835 2013

[169] A-M Gao Z-P Ke F Shi G Sun and H Chen ldquoChrysinenhances sensitivity of BEL-7402ADM cells to doxorubicin bysuppressing PI3KAktNrf2 and ERKNrf2 pathwayrdquo Chemico-Biological Interactions vol 206 no 1 pp 100ndash108 2013

[170] A M Gao Z P Ke J N Wang J Yang S Chen and HChen ldquoApigenin sensitizes doxorubicin-resistant hepatocellular

carcinoma BEL-7402ADM cells to doxorubicin via inhibitingPI3KAktNrf2 pathwayrdquo Carcinogenesis vol 34 no 8 pp1806ndash1814 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Oxidative Medicine and Cellular Longevity

own adaptation mechanisms against increased ROS such asupregulation of ROS scavenging systems As a result of thesesystems malignant transformed cells can utilize ROS as asignal for tumor progression and metastasis [5 14]

Recent studies are expanding our knowledge about thebiological implications of ROS in cancer stem cells (CSCs)which are small subpopulation of cancer cells responsible fortumorigenesis and tumor progression and relapse Based onincreasing evidence for the role of ROS in stem cell biologylower levels of cellular ROS are considered beneficial forthe maintenance of quiescence and chemoradioresistanceof CSCs [15] In this review we show current findingsillustrating the relationship between ROS and CSC biologyand present emerging evidence that nuclear factor-erythroid2- (NF-E2-) related factor 2 (NRF2) may play a role in CSCgrowth and resistance

2 CSCs and Resistance to EnvironmentalStress and Chemotherapy

Tumors contain a small population of cells with stem cellproperties namely CSCs or tumor-initiating cells (TICs)[16 17] These cells are known to play a crucial role in tumormaintenance and relapse In the 1990s the first experimentalevidence of CSCs was introduced by Bonnet and Dick [18]In acute myeloid leukemia (AML) it appeared that 01 to1 of the total cell population had tumor-initiating activityThis subpopulation exhibited a CD34+CD38minus phenotypeand was capable of tumor reconstitution after transplantationinto nonobese diabeticsevere combined immune-deficient(NODSCID)mice [18] Since thenmultiple lines of evidencehave revealed that theCSCpopulation exists in different typesof solid tumors including brain breast and colon cancers[19ndash21]

CSCs are characterized by their self-renewal and differen-tiation capacity similar to normal stem cells [16] Markers ofembryonic stem cells (ESCs) such as octamer-binding tran-scription factor 4 (OCT4) Nanog homeobox (NANOG) andSRY (sex determining region Y)-box 2 (SOX2) are expressedin CSCs and the Wnt120573-catenin Hedgehog and Notchpathways are implicated in the self-renewal of CSCs [22ndash26]Several CSC-specific surfacemarkers have been identified forthe detection and isolation of CSCs from the tumor massCD44+CD24minus phenotypic cells were isolated from breastcancer tissues and breast carcinoma cell lines andwere shownto exhibit self-renewal and high tumorigenic capacity [27]The CD133+ subpopulation from brain tumors demonstratedstem cell properties and showed tumor-initiating capabilityin NODSCID mouse brains [20]

CSCs are considered to be one of themain causes of tumorrecurrence after therapy CSC resistance to conventionalanticancer drug therapies and radiotherapy is attributed toincreased expression of ROS scavenging molecules drugtransporters and enhanced DNA repair capacity [28ndash30] Ithas been reported that CSCs contain low levels of endogenousROS compared to those seen in non-CSCs [31 32] Inprimary AMLs a subpopulation of low ROS-producing cellsdemonstrated characteristics of CSCs including quiescence

and a CD34+CD38minus phenotype [31] Moreover Chang et alobserved that this population of low ROS-producing cellsexhibited increased expression of stem cell markers (OCT4and NANOG) and higher chemoresistance and tumorigenic-ity than the population of high ROS-producing cells in headand neck cancer [32]

The ATP-binding cassette transporter (ABC transporter)family is known to induce multidrug resistance by activelytransporting intracellular drugs to outside of the cell [33 34]P-glycoprotein (P-gp) multidrug resistance-associated pro-teins (MRPs) and breast cancer associated protein (BCRP)belong to the ABC transporter family Since many types ofanticancer agents are substrates of these ABC transportersenhanced expression of P-gp MRPs and BCRP is stronglyassociatedwith the chemoresistant phenotype of CSCs Basedon this ABC transporters are often used as a CSC surfacemarker [28 35] The side population (SP) which is a fractionof cells that expresses a high level of BCRP can be isolatedfrom cancer cells using fluorogenic dye Hoechst 33342As Hoechst 33342 dye is a substrate of BCRP the BCRPoverexpressing cells exclude this fluorescent dye and therebya fraction of cells with low fluorescence can be isolated fromnon-SP cellsThis method is now widely used to isolate CSCsfrom cancer cell lines and specimens using a flow cytometry[36]

3 Role of ROS in Stem Cells

Stem cells can be broadly classified into two categoriesadult stem cells (eg haematopoietic stem cells (HSCs) andneural stem cells) and ESCs Under homeostatic conditionsthese stem cells particularly adult stem cells are generallymaintained in a quiescent state However stem cells are ableto escape quiescence and enter the cell cycle for proliferationwhen they are exposed to metabolic changes [37ndash40]

ROS are considered as important signaling molecules instem cell biology They play a key role in stem cell mainte-nance by preserving quiescence and protecting againstenvironmental stress [37 38] Recent stem cell studieshave demonstrated that stem cells contain low levels ofintracellular ROS and this redox status was found to becritical for regulation of stem cell quiescence and self-renewal Murine ESCs exhibited low levels of intracellularROS compared to differentiated murine cells due to theincreased level of GSH and thioredoxin (TXN) system [41]Furthermore HSCs containing low levels of ROS (ie cellswith low fluorescent activity following the incubation with2101584071015840-dichlorodihydrofluorescein diacetate (DCFDA) a cell-permeable ROS sensing fluorogenic dye) were highly qui-escent and expressed relatively high levels of NOTCH1 andBCRP compared to high DCFDA fluorescent cells Highlevels of ROS are cytotoxic since ROS accumulation in HSCscan lead to cellular prematurity and senescence [42 43]

ROS have been reported to be involved in stem cell differ-entiation Bone marrow mesenchymal stem cells (MSCs) arefound in the bone marrow together with HSCs and have thepotential to differentiate to adipocytes osteocytes and chon-drocytes It was shown that humanMSCs are highly resistant

Oxidative Medicine and Cellular Longevity 3

to ROSThis phenomenon was linked to low levels of cellularROS and high levels of SODs catalase GPX1 and GSH inMSCs [38 40] Elevated antioxidant molecules appear to playa crucial role in the protection of stem cells against oxidativestress However under certain circumstances NOX-derivedROS are associated with MSC differentiation The treatmentof MSCs with antioxidants or interfering RNA of NOX4 pre-vented adipocyte differentiation of MSCs via cAMP responseelement-binding protein (CREB) inhibition Similarly ESCdifferentiation to the cardiac lineage was dependent onNOX4-derived ROS [44] These findings indicate that ROSare important for stem cell fate determination for quiescenceor differentiation

4 Redox Signaling Molecules in Stem Cells

It has been reported that multiple signaling molecules areinvolved in ROS-mediated regulation of stem cells (Figure 1)First ataxia telangiectasia mutated (ATM) plays a criticalrole in controlling ROS levels in stem cells ATM a ser-inethreonine protein kinase is a known regulator of theDNA damage response and contributes to the regulation ofcellular ROS ATM is known to regulate ROS via modulationof AMPK-mTOR pathway or NADPH production [45 46]Ito et al showed that atmminusminus mice developed bone marrowfailure after 24 weeks of age due to a depletion of HSCsIn this study HSCs in atm knockout mice showed higherlevels of ROS than wild type mice which presumably causeda reduction in the self-renewal activity of HSCs Howeverthe treatment of mice with antioxidant N-acetylcysteine(NAC) restored HSC reconstitution in atm knockout mice byreducing ROS in HSCs confirming the critical role of ROSin HSCs maintenance [47] Similarly in another study NACtreatment prevented hypersensitivity of atmminusminusmice to X-rayirradiation and senescence of atmminusminus embryonic fibroblasts[48] Cosentino et al have presented amolecularmechanisticrole for ATM demonstrating that ATM activation promotesthe binding of heat shock protein 27 (HSP27) to glucose-6-phosphate dehydrogenase (G6PDH) which can result inG6PDH activation and subsequent NADPH increase [46]

The forkhead box O (FoxO) transcription factor familyis also implicated in redox regulation of stem cells TheFoxO family including FoxO1 FoxO3 FoxO4 and FoxO6is a key regulator of cell survival proliferation DNA repairand apoptosis FoxO1 and FoxO3 are reported to upregulatethe expression of GSH biosynthetic enzymes and SODsand therefore are associated with cellular protection againstoxidative stress [49 50] Particularly FoxO3 has been knownto play crucial roles in cytoprotection of stem cells includingHSCs [51ndash53] Loss of FoxO3a which regulates the expressionof antioxidant enzymes such as catalases and SOD2 led toROS accumulation and thus a higher rate of cell cyclingand a loss of quiescence in HSCs [51] In foxo1foxo3afoxo4triple-knockout mice the number of HSCs was substantiallydecreased and apoptotic HSCs were increased through ROSelevation [52] Notably Yalcin et al provided a link betweenATMand the FoxOprotein in ROS regulation of stem cells Infoxo3minusminusHSCs ATMexpressionwas diminished compared to

Quiescenceself-renewal

Senescenceapoptosis

cell cycle progressionLow ROS

High ROSATM uarr FoxO uarr

PI3KAKT uarr

FoxO darr

Figure 1 Involvement of ROS in normal stem cell quiescence andself-renewal In normal stem cells modulation of ROS levels candetermine quiescence and cell fate progression At low ROS levelswhich are maintained by ATM and FoxO signaling stem cellsremain quiescent and self-renewal activity is enhancedOn the otherhand increased ROS levels result in cell cycle progression cellularsenescence and apoptosis The PI3K-AKT pathway is known toelevate ROS levels by negative regulation of FoxO

normalHSCs suggesting that FoxO3 repressed ROS viaATMregulation [53] Similar to HSCs foxo-deficient neural stemcells demonstrated a decline in self-renewal capacity due toincreased cellular ROS levels [54 55]

The phosphoinositide 3-kinase (PI3K)AKT pathway isanother ROS regulator in normal stem cells In particularPI3KAKT signaling associates with FoxO transcription fac-tors to mediate ROS regulation Activated AKT promotesFoxO phosphorylation resulting in the nuclear export andcytoplasmic degradation of FoxO through the proteasome[56 57] Therefore ak11990512 double knockout HSCs displayedincreased quiescence and low cellular ROS levels [58] Con-sistently persistent activation of the PI3KAKT pathway inphosphatase and tensin homolog (PTEN) deleted HSCs ledto defective quiescence resulting in cellular senescence [59]Based on the above observations the PI3KAKTpathway andFoxOATMpathway exhibit opposite roles in ROS regulationof stem cells

Hypoxia-inducible factors (HIFs) are transcription fac-tors that respond to hypoxic conditions [60] They are alsocritical factors for the maintenance of stem cells HSCscultured in hypoxic conditions displayed a higher colonyformation capacity and high HIF levels positively regulatedthe pluripotency of human ESCs by activating stemnesstranscription factors such as OCT4 SOX2 and NANOG[61 62] Moreover in neuronal stem cells in a hypoxicenvironment accumulated HIF1120572 promoted Wnt120573-cateninpathway activation [63] The involvement of HIFs in stemcell biology is mediated by ROS Takubo et al observed thathif1120572minusminus HSCs contain high levels of ROS which could beassociated with a loss of HSC quiescence and an induction ofcellular senescence [64] In agreement with this finding thesuppression of HIF1120572 and HIF2120572 in HSCs led to increasedROS generation via mitochondrial metabolic shift and con-sequently induced cellular senescence and apoptosis Scav-enging ROS byNAC treatment could restore HSC quiescenceand function in stem cells withHIF1120572 andHIF2120572 suppression[65]

5 Involvement of ROS in CSC Biology

Very few studies have investigated the involvement of redoxchange in CSC biology compared to that in cancer cellsor normal stem cells However it has been reported that

4 Oxidative Medicine and Cellular Longevity

CSCs appear to share several ROS-associated propertieswith normal stem cells CSCs are known to contain lowerlevels of ROS compared to non-CSCs CD44+CD24minuslowbreast cancer CSCs isolated from MDA-MB-231 and MCF7mammospheres were relatively more resistant to radiationand this was associated with lower levels of ROS afterradiation [66] Diehn et al observed that ROS levels inCD44+CD24minus breast cancer CSCs were lower than in non-CSCs and the expression levels of the modulatory subunit ofglutamate cysteine ligase (GCLM the rate-limiting enzymeof GSH synthesis) and FoxO1 were high Treatment of theCD44+CD24minus subpopulationwith buthionine sulphoximine(BSO) an inhibitor of GSH synthesis resulted in reducedcolony forming capacity and increased sensitivity to radiationtherapy through an increase in ROS [67] In a study usinghuman AML specimens leukemic stem cells with low levelsof ROS majorly contributed to stem cell quiescence bymaintaining a low rate of oxidative phosphorylation andmetabolism [31] A leukemia with a high amount of leukemicstem cells showed low levels of ROS and increased expressionof GPX3 compared to tumors with a low frequency ofleukemic stem cells This study demonstrated that GPX3levels positively correlated with poor prognostic outcome inAML patients [68] Glioma stem cells within the tumor masshave low levels of cellular ROS although they are located in ahypoxic environment A proposed molecular mechanism ofthis phenomenon was the significantly upregulated expres-sion of peroxiredoxin 4 (PRDX4) in glioma stem cells [69]

Evidence is indicating that low ROS levels in CSCsresult from the intrinsic characteristics of CSCs Cell surfacemarkers of CSCs including CD44 and CD13 are found tobe involved in ROS regulation Ishimoto et al demonstratedthat a variant isoform of CD44 (CD44v) can bind to thecystineglutamate exchange transporter xCT and activatescysteine uptake to enhance GSH synthesis in gastrointestinalCSCs [70] The expression of antioxidant genes such asGPX12 was significantly increased in CD44+ gastric tumorcells In addition knockdown of CD44 in mice led toROS increase p38MAPK activation and cellular senescencethat are related to p21 expression In a subsequent studythe same group demonstrated that the number of CD44+cells increased with neoadjuvant chemotherapy in head andneck squamous cell carcinoma (HNSCC) patients TheseCD44+ undifferentiated cancer cells displayed high xCTexpression GSH upregulation and low cellular ROS levelsAblation of xCT by siRNA or sulfasalazine treatment (xCT-mediated cystine transport inhibitor) induced differentiationof HNSCCCSCs both in vitro and in vivo [71] CD13 has beenidentified as a surface marker for liver CSCs In liver cancercell lines including Huh7 and PLCPRF5 CD13 positive cellspredominated the SP fraction and were mainly in the G0G1phase of the cell cycle Additionally resistance to anticancerdrugs or radiation in the CD13 positive cell fraction wasmuch higher than that observed in the CD13 negative cellfraction Direct comparison of ROS levels between the twocell fractions revealed that the CD13 positive cell fractioncontains lower levels of ROS and expresses higher levelsof GCLM [72] In another study by the same group CD13expression reduced transforming growth factor-120573 (TGF-120573)

induced ROS production and promoted survival of liverCSCs [73]

It has been demonstrated that signaling pathwaysinvolved in ROS regulation of normal stem cells also playa role in CSC biology The nuclear expression levels ofFoxO3a was high in chronic myeloid leukemia-initiatingcells and the transplantation of leukemic stem cells derivedfrom foxo3a knockout mice significantly reduced their abilityto cause myeloid leukemia in an animal model [74] Thisstudy also revealed that TGF-120573 is a crucial regulator ofFoxO3a activity In the SP of MCF-7 breast cancer cellsactivation of the PI3Kmammalian target rapamycin (mTOR)signaling pathway was important for tumorigenecity of theseCSCs and knockdown of PI3K or mTOR led to ablatedtumorigenecity [75] When CD133+CD44+ prostate cancercells were grown in sphere-forming conditions activatedPI3KAKT signaling was found to be critical for maintainingCSCs [76] CD44+CD24minusor low cells isolated from breastcancer cell lines and breast cancer patient specimens wereradioresistant and this resistant phenotype was associatedwith ATM signaling activation [77]

6 NRF2 as a Key Molecule forRedox Homeostasis

In 1990 Rushmore and Pickett discovered the enhancersequence in the rat gsta2 gene promoter as a response elementto 120573-naphthoflavone and t-butylhydroquinone (t-BHQ) andnamed it antioxidant responsive element (ARE) [78] Subse-quent studies revealed that ARE is commonly involved in thetranscription of multiple antioxidant and detoxifying genesincluding glutamate-cysteine ligase (GCL) glutathione S-transferase (GST) and NAD(P)H quinone oxidoreductase-1 (NQO-1) [79 80] Based on sequence homology betweenARE and MAF-recognition element (MRE) further studieshypothesized that small MAF and bZIP caprsquonrsquocollar (CNC)transcription factors may interact with ARE [81 82] AmongbZIP CNC transcription factors NRF2 was found to play acrucial role in ARE regulation in which inducible expressionof NQO1 and GST was ablated in t-butylhydroxy anisole-treated nrf2 null mice in contrast to the observation in wildtype mice [83] After this report numerous studies haveelucidated a wide spectrum of protective effects of NRF2signaling against various stressors For example sensitivityto benzo[a]pyrene-induced carcinogenesis was significantlygreater in nrf2-knockout mice than in wild type mice [84]

To account for the protective effects of Nrf2 comparativeanalyses of gene expression patterns were carried out innrf2-deficient and wild type mice following treatment withNrf2 activators In global gene analysis of dithiolethione-administered mouse livers Nrf2 was found to governthe expression of xenobiotic-detoxifying enzymes GSH-generating systems antioxidant proteins and the molec-ular chaperone-26S proteasome [85] Similarly Hu et aldemonstrated that detoxifying enzymes antioxidants drugtransporters stress response proteins and some signalingmolecules serve as Nrf2-dependent and isothiocyanate-inducible genes in mouse liver [86] It has now been firmly

Oxidative Medicine and Cellular Longevity 5

established thatNRF2 regulates divergent genes to coordinatexenobiotic detoxification and redox homeostasis [85 87ndash89]In its function as a regulator of cellular redox homeostasisNRF2 elevates the expression of GCL and the cysteinetransporter xCT to increase cellular GSH levels NRF2 alsoenhances regeneration of reduced GSH by upregulatingGPX and GSH reductase (GSR) Expression of thioredoxin1 (TXN1) thioredoxin reductase 1 (TXNRD1) and perox-iredoxin 16 which can reduce oxidized protein thiols isalso under the control of NRF2 In addition the levels ofNADPH a cofactor of many antioxidant enzymes such asGSR and TXNRD can be increased by NRF2 (reviewedin Hayes and Dinkova-Kostova [90]) The expression ofmultiple NADPH generating enzymes such as G6PDH and6-phosphogluconate dehydrogenase is upregulated by NRF2Additionally the role of NRF2 in ABC transporter expressionfor xenobiotic detoxification is notable The basal expressionlevel of Mrp1 was relatively lower in nrf2-deficient fibroblaststhan that in wild type fibroblasts and the treatment ofmice with Nrf2 activating diethyl maleate increased Mrp1expression in the liver [91] Levels of MDR1 MRP23 andBCRP were elevated following oltipraz treatment in pri-mary human hepatocytes [92] Sulforaphane (SFN) treatmentenhanced the levels ofMDR1 BCRP andMRP2 in the blood-brain barrier of rats [93] Our recent study showed thatgenetic activation of NRF2 via KEAP1 silencing increases theexpression ofMDR1MRP23 and BCRP in human proximaltubular epithelial cells [94] As direct molecular evidencefunctional AREs have been identified in human MRP3 [95]and BCRP genes [96]

Kelch-like ECH-associated protein 1 (KEAP1) a cysteine-rich actin-binding protein is the main negative regulatorof NRF2 activity [83 97] Under quiescent conditionsNRF2 remains inactive by forming a complex with KEAP1in the cytoplasm NRF2 is subject to ubiquitination andKEAP1-induced proteasomal degradation through the Cullin3 (CUL3) based E3 ligase KEAP1 has three major domainsas follows (i) The BTB domain is associated with KEAP1homodimerization (ii) the IVR domain plays a role inregulation ofKEAP1 activity and (iii) theKelchDGRdomainmediates binding with NRF2 [83 90 98ndash101] The bindingof NRF2 with KEAP1 has been described as the ldquohinge andlatchrdquo model where one molecule of NRF2 interacts with theKelchDGR domains of the KEAP1 dimer through conservedmotifs called ETGE (DN-X-E-TS-G-E) and DLG (L-X-X-Q-D-X-D-L-G) [102ndash104] In these reports it was shown thatthe binding affinity of the ETGE motif to KEAP1 is muchhigher than that of the DLG motif It has therefore beenshown that ldquolatchrdquo binding of the NRF2 DLG motif is easilybroken by modifications of KEAP1 cysteine residues by ROSor electrophiles In turn disrupted DLG binding of NRF2 toKEAP1 leads to the blockade of ubiquitination and furtherdegradation of NRF2 resulting in nuclear translocation ofNRF2 It has been demonstrated that the sulfhydryl groups ofmultiple cysteine residues of KEAP1 can be directly modifiedby oxidationreduction or alkylation In particular Cys151Cys273 and Cys288 were found to be essential for theregulation of NRF2 activity [99 104ndash106] Mutation of theCys273 or Cys288 residue of KEAP1 ablated its ability to

suppress NRF2 activity leading to accumulation of the NRF2protein [99 104]

In addition to KEAP1-mediated stability regulationNRF2 activity can be modulated at multiple steps First itis noticeable that NRF2 activity is regulated at the transcrip-tional step Functional AREs were identified in the murinenrf2 gene promoter and were involved in the autoregulationof NRF2 through transcriptional activation [107] Moreovera single nucleotide polymorphism in the ARE-like sequencesof the human NRF2 promoter was associated with increasedlung cancer susceptibility [108] Second it was shown thatNRF2 activity is regulated by posttranslationalmodificationsStudies indicate that NRF2 activation involves phosphoryla-tion signaling mediated by multiple kinase pathways such asMAPK protein kinase C (PKC) PI3K and protein kinaseRNA-like endoplasmic reticulum kinase (PERK) [109ndash111]Meanwhile glycogen synthase kinase-3 (GSK-3) a consti-tutively active serinethreonine kinase was found to inhibitNRF2 activity [112] Last NRF2 activity is increased byseveral intrinsic proteins such as p21 and p62 [113 114] Forexample p62 a linker protein of ubiquitinated proteins toautophagy degradation binds to the KEAP1 protein andinterferes with the binding of NRF2 to KEAP1 resulting inNRF2 stabilization

7 Emerging Role of NRF2 in Cancer Biology

Continuous or fatal stimuli such as toxic chemicals and excessROS disrupt cellular homeostasis causing macromoleculardamage and alterations in cell cycle and growth signalingwhich can eventually result in carcinogenesis The NRF2pathway deserved significant attention in the area of cancerbiology because numerous studies have demonstrated thatactivation of the NRF2 pathway decreases the sensitivity ofcells to carcinogens [115ndash117] For instance the burden ofgastric neoplasia caused by benzo[a]pyrene was effectivelyattenuated by the Nrf2 activator oltipraz in wild type micewhereas nrf2 knockout mice did not show any protectiveeffect of oltipraz [84] Similarly the incidence of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) induced urinary blad-der carcinomawas greater in nrf2 knockoutmice than in wildtype mice and oltipraz treatment reduced tumor incidenceonly in wild type mice [118]

Although it has been firmly confirmed that NRF2 activa-tion can protect cells against a wide range of toxicants andstressors aberrant activation of NRF2 has been associatedwith several types of cancers NRF2 levels were constitutivelyelevated in cancer cell lines and tumor samples of the lungbreast esophagus endometrial cancers and prostate cancers[119ndash125] Molecular mechanisms involved in constitutiveNRF2 activation include the following (i) somatic mutationsof KEAP1 or NRF2 (ii) epigenetic silencing of the KEAP1gene (iii) aberrant accumulation of proteins that competewith NRF2 for KEAP1 binding and (iv) oncogene-mediatedoverexpression of NRF2 [101 126] First somatic mutationsof KEAP1-NRF2 have been reported in an initial study byPadmanabhan et al [127] This study identified mutations

6 Oxidative Medicine and Cellular Longevity

in the KelchDGR domain of KEAP1 in lung cancer celllines as well as lung cancer tissue samples and demonstratedthat these mutant KEAP1 proteins lost their NRF2 repressivefunction which resulted in NRF2 accumulation Singh et alalso demonstrated that the KelchDGR and IVR domainsof the KEAP1 gene contain multiple somatic mutations andthese mutations were identified in 19 of tumor specimensfrom non-small cell lung cancer patients [123] In gallbladdercancer 4 of 13 patients harbored KEAP1 mutations [122]Shibata et al reported that NRF2 somatic mutations werefound in 107 of primary lung cancer patients and 272of primary head and neck cancer patients [122] Notablythesemutationswere primarily located in theDLG andETGEmotifs and eventually led to the loss of a proper interactionbetween the NRF2 protein and KEAP1 Second CpG islandhypermethylation in the KEAP1 promoter resulted in lowKEAP1 expression in lung cancer cell lines and tumor samples[124]Third in humanhepatocellular carcinoma p62 positivecellular aggregates were found with a frequency of 25 andmost of these tumors retained higher levels of NRF2 andits target gene expression [128] Fourth oncogenes have alsobeen shown to play a role in NRF2 signaling Oncogenicactivation of KRAS (KRASG12D) c-MYC (c-MYCERT12) andBRAF (BRAFV619E) elevates the transcript levels of NRF2 andits target gene expression [129]

It is now widely accepted that aberrant activation ofNRF2 can enhance cancer cell survival and growth in oxi-dizing tumor environments and further promote chemoradioresistance Indeed the prognosis of cancer patientsnegatively correlated with NRF2 levels in the tumor [122130] The favorable effect of NRF2 overexpression on tumorsurvival and growth can be attributed to the increase inNRF2 target antioxidant proteins and their counteractiveeffect on oxidative stress For instance GSH which is adirect target molecule of NRF2 has been shown to be criticalfor cell proliferation [131 132] In addition to its antioxi-dant contribution Mitsuishi et al provided direct evidencedemonstrating that NRF2 alters the cellular metabolism inrelation to anabolic pathways to accelerate cell proliferation[133] Multiple metabolic genes such as those involvedin the pentose phosphate pathway were upregulated byNRF2 through ARE and these changes promoted purinesynthesis glutamine metabolism and NADPH productionfor enhanced cell proliferation

Constitutively high levels of NRF2 have been associatedwith chemoresistance as well as radioresistance Cancer cellswith high NRF2 activity were less sensitive to cytotoxicchemotherapeutics such as cisplatin doxorubicin and 5-fluorouracil through facilitated detoxification of anticanceragents and enhanced antioxidant capacity [101] It is there-fore hypothesized that NRF2 inhibition can enhance thechemosensitivity of cancers NRF2 siRNA could suppresscancer resistance to cisplatin topoisomerase inhibitors and5-fluorouracil [101 122 134 135] Cancer cells with constitu-tively high NRF2 were protected against 120574-radiation inducedtoxicity Moreover siRNA-mediated inhibition of NRF2 innon-small cell lung cancer cell lines substantially enhancedradiosensitivity [136] Additionally NRF2 expression was

increased during the acquisition of chemoresistance In ourprevious study doxorubicin-selected ovarian cancer cellsdemonstrated increased expression of NRF2 and its targetgenes forGSH synthesis andNRF2 inhibition in this resistantcell line could restore doxorubicin sensitivity [137]

Our understanding of the role of NRF2 in cancer cell sig-naling has expanded In particular the relationship betweenoncogenic signaling and NRF2 is noteworthy As men-tioned earlier activation of oncogenes such as KRAS and c-MYC increased the expression of NRF2 presumably throughoncogene-mediated ROS increase and this phenomenonappears to contribute to the maintenance of reduced redoxhomeostasis in cancer cells [129] In ERBB2 (Her2Neu) over-expressing ovarian cancer cells the stable silencing of NRF2repressed ERBB2 expression and its downstream signalingand retarded tumor growth Therefore the inhibition ofNRF2 could sensitize these cells to taxol therapy by repressingERBB2 expression [138] Moreover NRF2 was shown to beassociated with HIF signaling which is a critical factor fortumor angiogenesis When NRF2 was stably knocked downin colon carcinoma cell lines hypoxia-inducible HIF-1120572accumulation was abrogated and consequently angiogenesisand tumor growth were significantly suppressed in NRF2knockdown tumors compared to the control group [139] Intype 2 papillary renal cell cancer which is characterized byloss of the fumarate hydratase gene and consequentmetabolicalteration accumulated fumarate was associated with tumorprogression via NRF2 signaling Fumarate was shown tomodify KEAP1 cysteine residues and elevate NRF2 levelswhich contributed to the growth and progression of type 2papillary renal cell cancer [140] These accumulating linesof evidence suggest that once cells are transformed to theneoplastic stage cancer cells utilize NRF2 signaling to adaptto the stressful tumor environment and to promote survivaland further cancer progression (Figure 2)

8 Involvement of NRF2 Signaling inStem Cell Quiescence and Differentiation

There is considerable evidence to suggest that NRF2 playsa role in normal stem cell biology [141ndash145] For exampleNRF2 activation in HSCs plays a critical role in not only themaintenance of quiescence but also in the determination ofdifferentiation fate [141 145] In Drosophila intestinal stemcells constitutive Nrf2 activation sustained quiescence byreducing the levels of ROS via upregulation of antioxidantgenes such as gclc However in the case of KEAP1-mediatedNrf2 repression high levels of intracellular ROS facilitatedan ablation of the quiescent state in intestinal stem cellsand age-related degeneration in the intestinal epithelium[146] Similarly low intracellular ROS levels are requiredfor the maintenance of quiescence in human airway basalstem cells (ABSCs) When exposed to exogenous ROSquiescent ABSCs enter the proliferation stage Changes inROS levels activate the NRF2-Notch pathway which resultsin self-renewal and protection of ABSCs from ROS-inducedhyperproliferation and senescence Moreover the quiescent

Oxidative Medicine and Cellular Longevity 7

NRF2 sMaf

GCLCGCLMNQO1SODs

Antioxidant enzymes Drug transportersMetabolic enzymes

ARE

Tumor growthprogression

MDRsMRPsBCRP

G6PDPGDME1

Cell survival Drug effluxProliferation

Target genes

Figure 2 Implications of NRF2 signaling in cancer NRF2 coordi-nates the expression of genes associated with cellular redox regu-lation metabolism and xenobiotic efflux and thereby its aberrantactivation promotes cancer cell survival proliferation and anti-cancer drug resistance GCLM glutamate cysteine ligase modifiersubunit GCLC glutamate cysteine ligase catalytic subunit NQO1NAD(P)Hquinone oxidoreductase 1 SODs superoxide dismutasesG6PDH glucose-6-phosphate dehydrogenase PGD phosphoglu-conate dehydrogenase ME1 malic enzyme 1 MDRs multidrugresistance proteinsMRPsmultidrug resistance-associated proteinsBCRP breast cancer resistance protein

state of ABSCs was maintained by NRF2 activation [147]In osteoclast progenitor cells hydrogen sulfide (H

2S) inhib-

ited human osteoclast differentiation by NRF2-dependentinduction of peroxiredoxin 1 and NQO1 These resultswere further confirmed using NRF2 activators includingsulforaphane and t-BHQ [148] It is also notable that NRF2participates in the regulation of cell fate determination ofHSCsMurakami et al demonstrated that HSCs derived fromKEAP1-deficientmice exhibited preferred differentiation intothe granulocyte-monocyte lineage rather than differentiatinginto the erythroid-lymphoid lineage [145]

Up to now numerous studies have demonstrated thatNRF2 plays a protective role against various stressors instem cells In neural stem cells overexpression of NRF2or pharmacological NRF2 activation prevented necroticcell death [149] In an animal study nrf2-deficient miceshowed defective stem cell function HSCs from nrf2minusminus miceexpressed lower levels of prosurvival cytokines and exhib-ited spontaneous apoptosis [150] Ionizing radiation-inducedmyelosuppression and mortality were mitigated throughNRF2-mediated Notch signaling activation in HSCs [142]Similarly resveratrol-inducedNRF2 expression improved thesurvival of cardiac stem cells and consequently regeneratedinfarcted myocardium [151 152] The heme oxygenase 1(HO-1) inducer cobalt protoporphyrin (CoPP) elicited anantiapoptotic effect on cardiac stem cells via activation of the

ERK-NRF2 pathway [153] In neural stem cells NRF2 activa-tion by melatonin or t-BHQ ameliorated lipopolysaccharide(LPS) orH

2O2induced cell death [149 154] In addition amy-

loid 120573-mediated neural stem cell death could be alleviated byexogenous NRF2 transduction which was accompanied byincreased expression of GCLC NQO-1 andHO-1This studyalso demonstrated that neuronal differentiation of neuralstem cells is enhanced by NRF2 activation [155] Similar toneural stem cells NRF2 has a protective role against hypoxicand oxidative stress conditions in undifferentiated MSCsTreatment of the murine mesenchymal stem cell line withadrenaline increased the mRNA expression of nrf2 gclc andxCT leading to an increase in GSH levels and the preventionof ROS-induced cytotoxicity [156 157]

9 Potential Implication of NRF2 inCSC Maintenance and Resistance

The role of NRF2 in CSC biology is now beginning tobe unveiled Similar to the case of normal stem cells itwas shown that NRF2 contributes to CSC stemness bymaintaining their self-renewal capacity and protecting themfrom chemoradiotherapy Achuthan et al established stablechemotherapy-resistant breast cancer cells and observed thatthese cells expressed higher levels of CD133 and OCT-4indicating that these cells exhibit CSC phenotype [34] Ofnote it was shown that ROS levels were relatively low inthese drug-selected cells presumably due to higher levelsof antioxidant enzymes such as SOD1 and GPX12 NRF2protein stabilizationwas associatedwith high levels of antiox-idant enzymes As an underlying molecular mechanismdiminished proteasome activity and increased p21 levelsappear to stabilize the NRF2 protein in these stem-like cellsEvidently p21 knockdown repressed the mammosphere-forming potential of these stem-like breast cancer cellsSimilarly a study by Zhu et al showed the involvement ofNRF2 in glioblastoma stem cells that were isolated fromhuman surgical glioblastoma specimens NRF2 knockdownin glioblastoma stem cells inhibited cell proliferation andneurosphere formation and further suppressed SOX2 expres-sion Moreover NRF2 knockdown changed the cell cycledistribution to the G2 phase and significantly attenuated thetumorigenecity of glioblastoma stem cells [158 159] Theseresults are providing evidence that NRF2 is necessary formaintenance of the self-renewal capacity of glioblastomastem cells

On the other hand activation of NRF2 signaling has beendemonstrated in different types of CSC models includinglung esophageal breast ovarian and colon CSCs and thisis closely correlated with themaintenance of low intracellularROS levels and chemoresistance of CSCs [160ndash165] In lungand esophageal cancer cells cigarette smoke condensateincreased the SP as well as BCRP expression which arehallmarks of CSCs [165] Promoter analysis revealed thatBCRP expression was associated with elevated levels ofNRF2 aryl hydrocarbon receptor (AhR) and specificityprotein 1 (SP1) Additionally this study demonstrated thatmithramycin diminished BCRP expression via repression of

8 Oxidative Medicine and Cellular Longevity

Low ROS

Increased drug efflux

SurvivalChemoresistance

CSC

TumorRecurrenceProgression

Radioresistance

PERKuarr

p21uarr

p62uarr

Proteasome darr

NRF2 uarr

Figure 3 Potential roles of NRF2 signaling in CSCs InCSC-like cellmodels NRF2 is activated through multiple molecular mechanismsin a context-dependent manner The upregulation of competingproteins such as p62 and p21 activation of PERK or repressedproteasome function were shown to enhance NRF2 activity inthese models Elevated NRF2 levels in CSCs can contribute tothe maintenance of low ROS by upregulating multiple antioxidantgenes In addition NRF2-mediated expression of ABC transporterselicits efflux of anticancer drugs from cancer cells Overall activatedNRF2 signaling facilitates CSCs survival and stress resistance andconsequently it can be suggested that CSCs with high NRF2 activityplay a crucial role in tumor recurrence and further progression

NRF2 AhR and SP1 and thereby inhibiting the expressionof genes associated with CSC-related pathways resulting inreduced proliferation and tumorigenecity Similarly it wasreported that lung cancer SP cells exhibited high levels ofNRF2 and BCRP expression These SP cells were highlytumorigenic and possessed self-renewal capacity comparedto non-SP cells [164] Emmink et al performed a proteomeanalysis on collected secretome from highly tumorigenicCSCs and their corresponding nontumorigenic differentiatedcells both of which were established from human colorectalspecimens [160] Subsequent bioinformatic analysis revealedthat the CSC secretome contained a large amount of proteinsassociated with cell survival and protein quality controlcompared to differentiated tumor cells Notably the CSCsecretome contained an NRF2 antioxidant and detoxifyingprotein signature in that it included elevated levels ofGCLC GPX23 and TXNRD1 This study provided novelevidence that CSCs secrete NRF2 target antioxidant proteinsto counteract extracellular stressors and chemotherapeuticsIn patients with ovarian clear cell carcinoma the expressionof the CSC maker aldehyde dehydrogenase-1 (ALDH1) wasstrongly correlated with an advanced clinical stage andreduced progression free survival [161] Ovarian clear cellcarcinoma cells with high ALDH1 expressionmaintained lowlevels of ROS compared to ALDH-low cells and these cellswere shown to express higher levels of NRF2 and its targetgenes

Two recent studies have demonstratedNRF2 activation insphere cultures of breast cancer cells that is one of models ofCSCs Wu et al showed that mammospheres derived fromMCF7 and MDA-MB231 breast cancer cell lines exhibitedlower ROS levels compared to their monolayer counterpartsThey also showed that levels of NRF2 and target genes such

as NQO1 and GCLM were elevated in mammospheres [163]Similarly our group has shown substantially elevated NRF2protein levels along with increased expression of antioxidantgenes (eg HO-1 and GPX2) and drug efflux transporters(eg MRP2 and BCRP) in sphere cultures of breast cancercells NRF2 accumulation was also observed in sphere-cultured ovarian and colon cancer cells However shRNA-mediated downregulation ofNRF2 led to decreased chemore-sistance of mammospheres presumably due to reduced levelsof antioxidant genes and drug transporters High ROS levelsin NRF2 knockdown mammospheres caused sphere growthretardation and apoptosis Coherently ablation of ABCtransporter induction in NRF2 knockdown mammospheressensitized to anticancer agents [162] Additionally this studyprovided evidence that increased NRF2 protein expression inmammospheres can be linked to 26S proteasome reductionand p62 accumulation In particular knockdown of p62 inMCF7 mammospheres significantly attenuated NRF2 eleva-tion

Surviving dedifferentiated breast cancer cells after chem-otherapy treatment retained high levels of NRF2 activationsimilar to other CSCs However NRF2 activation was medi-ated by a noncanonical pathway Levels of PERKwere high indedifferentiated cancer cells and this in turn phosphorylatedand activated NRF2 signaling to maintain low cellular ROSlevels and to express ABC transporters In agreement withthese findings clinical observations revealed that the PERKpathway gene signature is related to chemoresistance andreduced patient survival [166]

10 Concluding Remarks

Recent studies have started to uncover the role of ROS signal-ing in the biology of CSCs which is related to tumorigenecitytumor progression and relapse Expression of the transcrip-tion factor NRF2 a master regulator of antioxidant genesexpression is increased in different models of CSCs and thiselevation is likely to promote CSC maintenance and survivalin an oxidizing tumormicroenvironment In additionNRF2-mediated overexpression of ABC transporters particularlythe CSC marker BCRP may play a critical role in themultidrug resistance of CSCsThese findings combined withthe increasing evidence showing the alteration of KEAP1-NRF2 signaling in cancer cells suggest a novel role of NRF2in CSC maintenance and survival (Figure 3)

One important question that arises from the current stud-ies is whether it is possible to design CSC-targeted therapiesthrough regulation of theNRF2 pathway and its related redoxhomeostasis inCSCs Recent studies provide several potentialclues for addressing this question the naturally occurringalkaloid brusatol could reduce the growth and chemoresis-tance of breast CSCs [163] Treatment ofmammospheres withbrusatol elevated ROS levels and promoted taxol-inducedgrowth retardation and cell death The NRF2 repressivemechanism of brusatol has not been clearly elucidated but itappears to be independent of KEAP1-mediated degradation[167] In addition to brusatol natural compounds such as

Oxidative Medicine and Cellular Longevity 9

chrysin apigenin luteolin and trigonelline are known toinhibit NRF2 signaling in several types of cancer cells [102168ndash170] and therefore the development of NRF2 inhibitorswith characterized modes of action will enable efficient tar-geting of the redox homeostasis system as well as multidrugresistance systems in CSCs

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This studywas financially supported by theNational ResearchFoundation (NRF) funded by the Ministry of Science ICT ampFuture Planning (NRF-2013R1A2A2A01015497)

References

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[2] P Storz ldquoForkhead homeobox type O transcription factorsin the responses to oxidative stressrdquo Antioxidants amp RedoxSignaling vol 14 no 4 pp 593ndash605 2011

[3] R S Balaban S Nemoto and T Finkel ldquoMitochondria oxi-dants and agingrdquo Cell vol 120 no 4 pp 483ndash495 2005

[4] L A Sena and N S Chandel ldquoPhysiological roles of mitochon-drial reactive oxygen speciesrdquoMolecular Cell vol 48 no 2 pp158ndash167 2012

[5] P T Schumacker ldquoReactive oxygen species in cancer cells liveby the sword die by the swordrdquo Cancer Cell vol 10 no 3 pp175ndash176 2006

[6] S Toyokuni K Okamoto J Yodoi and H Hiai ldquoPersistentoxidative stress in cancerrdquo FEBS Letters vol 358 no 1 pp 1ndash3 1995

[7] L Behrend G Henderson and R Zwacka ldquoReactive oxy-gen species in oncogenic transformationrdquo Biochemical SocietyTransactions vol 31 no 6 pp 1441ndash1444 2003

[8] E O Hileman J Liu M Albitar M J Keating and P HuangldquoIntrinsic oxidative stress in cancer cells a biochemical basis fortherapeutic selectivityrdquo Cancer Chemotherapy and Pharmacol-ogy vol 53 no 3 pp 209ndash219 2004

[9] H PelicanoD Carney and PHuang ldquoROS stress in cancer cellsand therapeutic implicationsrdquo Drug Resistance Updates vol 7no 2 pp 97ndash110 2004

[10] Y Adachi Y Shibai J Mitsushita W H Shang K Hirose andT Kamata ldquoOncogenic Ras upregulates NADPH oxidase 1 geneexpression through MEK-ERK-dependent phosphorylation ofGATA-6rdquo Oncogene vol 27 no 36 pp 4921ndash4932 2008

[11] J Mitsushita J D Lambeth and T Kamata ldquoThe superoxide-generating oxidase Nox1 is functionally required for Ras onco-gene transformationrdquoCancer Research vol 64 no 10 pp 3580ndash3585 2004

[12] O Vafa M Wade S Kern et al ldquoc-Myc can induce DNAdamage increase reactive oxygen species and mitigate p53function a mechanism for oncogene-induced genetic instabil-ityrdquoMolecular Cell vol 9 no 5 pp 1031ndash1044 2002

[13] K Polyak Y Li H Zhu et al ldquoSomatic mutations of themitochondrial genome in human colorectal tumoursrdquo NatureGenetics vol 20 no 3 pp 291ndash293 1998

[14] D Trachootham J Alexandre and P Huang ldquoTargeting can-cer cells by ROS-mediated mechanisms a radical therapeuticapproachrdquo Nature Reviews Drug Discovery vol 8 no 7 pp579ndash591 2009

[15] X Shi Y Zhang J Zheng and J Pan ldquoReactive oxygen speciesin cancer stem cellsrdquoAntioxidantsampRedox Signaling vol 16 no11 pp 1215ndash1228 2012

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[17] B S Zhou H Zhang M Damelin K G Geles J C Grindleyand P B Dirks ldquoTumour-initiating cells challenges and oppor-tunities for anticancer drug discoveryrdquo Nature Reviews DrugDiscovery vol 8 no 10 pp 806ndash823 2009

[18] D Bonnet and J E Dick ldquoHuman acute myeloid leukemiais organized as a hierarchy that originates from a primitivehematopoietic cellrdquo Nature Medicine vol 3 no 7 pp 730ndash7371997

[19] C A OrsquoBrien A Pollett S Gallinger and J E Dick ldquoAhuman colon cancer cell capable of initiating tumour growth inimmunodeficient micerdquo Nature vol 445 no 7123 pp 106ndash1102006

[20] S K Singh C Hawkins I D Clarke et al ldquoIdentification ofhuman brain tumour initiating cellsrdquo Nature vol 432 no 7015pp 396ndash401 2004

[21] M Al-Hajj M S Wicha A Benito-Hernandez S J Morrisonand M F Clarke ldquoProspective identification of tumorigenicbreast cancer cellsrdquo Proceedings of the National Academy ofSciences vol 100 no 7 pp 3983ndash3988 2003

[22] S-H Chiou M-L Wang Y-T Chou et al ldquoCoexpression ofOct4 andNanog enhancesmalignancy in lung adenocarcinomaby inducing cancer stem cell-like properties and epithelial-mesenchymal transdifferentiationrdquoCancer Research vol 70 no24 pp 10433ndash10444 2010

[23] F Yu J Li H Chen et al ldquoKruppel-like factor 4 (KLF4) isrequired for maintenance of breast cancer stem cells and for cellmigration and invasionrdquoOncogene vol 30 no 18 pp 2161ndash21722011

[24] L Vermeulen E M Felipe De Sousa M van der Heijden et alldquoWnt activity defines colon cancer stem cells and is regulatedby the microenvironmentrdquo Nature Cell Biology vol 12 no 5pp 468ndash476 2010

[25] C Zhao A Chen C H Jamieson et al ldquoHedgehog signallingis essential for maintenance of cancer stem cells in myeloidleukaemiardquo Nature vol 458 no 7239 pp 776ndash779 2009

[26] G Farnie and R B Clarke ldquoMammary stem cells and breastcancermdashrole of Notch signallingrdquo Stem Cell Reviews vol 3 no2 pp 169ndash175 2007

[27] D Ponti ldquoIsolation and in vitro propagation of tumorigenicbreast cancer cells with stemprogenitor cell propertiesrdquoCancerResearch vol 65 no 13 pp 5506ndash5511 2005

[28] M Dean T Fojo and S Bates ldquoTumour stem cells and drugresistancerdquo Nature Reviews Cancer vol 5 no 4 pp 275ndash2842005

[29] C Blanpain M Mohrin P A Sotiropoulou and E PassegueldquoDNA-damage response in tissue-specific and cancer stemcellsrdquo Cell Stem Cell vol 8 no 1 pp 16ndash29 2011

10 Oxidative Medicine and Cellular Longevity

[30] A A Dayem H Choi J Kim and S Cho ldquoRole of oxidativestress in stem cancer and cancer stem cellsrdquo Cancers vol 2 no2 pp 859ndash884 2010

[31] E Lagadinou A Sach K Callahan et al ldquoBCL-2 inhibitiontargets oxidative phosphorylation and selectively eradicatesquiescent human leukemia stem cellsrdquo Cell Stem Cell vol 12no 3 pp 329ndash341 2013

[32] C Chang Y Chen S Chou et al ldquoDistinct subpopulations ofhead and neck cancer cells with different levels of intracellularreactive oxygen species exhibit diverse stemness proliferationand chemosensitivityrdquoCancer Research vol 74 no 21 pp 6291ndash6305 2014

[33] MMGottesman T Fojo and S E Bates ldquoMultidrug resistancein cancer role of ATPndashdependent transportersrdquoNature ReviewsCancer vol 2 no 1 pp 48ndash58 2002

[34] S Achuthan T R Santhoshkumar J Prabhakar S A Nair andM R Pillai ldquoDrug-induced senescence generates chemoresis-tant stemlike cells with low reactive oxygen speciesrdquoThe Journalof Biological Chemistry vol 286 no 43 pp 37813ndash37829 2011

[35] L Zinzi M Contino M Cantore E Capparelli M Leopoldoand N A Colabufo ldquoABC transporters in CSCs membranesas a novel target for treating tumor relapserdquo Frontiers inPharmacology vol 5 article 163 2014

[36] A Hadnagy L Gaboury R Beaulieu and D Balicki ldquoSPanalysis may be used to identify cancer stem cell populationsrdquoExperimental Cell Research vol 312 no 19 pp 3701ndash3710 2006

[37] C L Bigarella R Liang and S Ghaffari ldquoStem cells and theimpact of ROS signalingrdquo Development vol 141 no 22 pp4206ndash4218 2014

[38] P Chaudhari Z Ye and Y Y Jang ldquoRoles of reactive oxygenspecies in the fate of stem cellsrdquoAntioxidants ampRedox Signalingvol 20 no 12 pp 1881ndash1890 2014

[39] V A Rafalski E Mancini and A Brunet ldquoEnergy metabolismand energy-sensing pathways in mammalian embryonic andadult stem cell faterdquo Journal of Cell Science vol 125 no 23 pp5597ndash5608 2012

[40] K Wang T Zhang Q Dong E C Nice C Huang and Y WeildquoRedox homeostasis the linchpin in stem cell self-renewal anddifferentiationrdquo Cell Death and Disease vol 4 no 3 p e5372013

[41] G Saretzki L Armstrong A Leake and et al ldquoStress defensein murine embryonic stem cells is superior to that of variousdifferentiated murine cellsrdquo Stem Cells vol 22 no 6 pp 962ndash971 2004

[42] Y Y Jang and S J Sharkis ldquoA low level of reactive oxygen speciesselects for primitive hematopoietic stem cells that may reside inthe low-oxygenic nicherdquo Blood vol 110 no 8 pp 3056ndash30632007

[43] S Ghaffari ldquoOxidative stress in the regulation of normal andneoplastic hematopoiesisrdquo Antioxidants amp Redox Signaling vol10 no 11 pp 1923ndash1940 2008

[44] M Schmelter B Ateghang S Helmig M Wartenberg and HSauer ldquoEmbryonic stem cells utilize reactive oxygen speciesas transducers of mechanical strain-induced cardiovasculardifferentiationrdquoThe FASEB Journal vol 20 no 8 pp 1182ndash11842006

[45] A Alexander S-L Cai J Kim et al ldquoATM signals to TSC2in the cytoplasm to regulate mTORC1 in response to ROSrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 107 no 9 pp 4153ndash4158 2010

[46] C Cosentino D Grieco and V Costanzo ldquoATM activates thepentose phosphate pathway promoting anti-oxidant defenceand DNA repairrdquo The EMBO Journal vol 30 no 3 pp 546ndash555 2011

[47] K Ito A Hirao F Arai et al ldquoRegulation of oxidative stress byATM is required for self-renewal of haematopoietic stem cellsrdquoNature vol 431 no 7011 pp 997ndash1002 2004

[48] K Ito K Takubo F Arai et al ldquoRegulation of reactive oxygenspecies by Atm is essential for proper response to DNA double-strand breaks in lymphocytesrdquoThe Journal of Immunology vol178 no 1 pp 103ndash110 2007

[49] Y Wang Y Zhou and D T Graves ldquoFOXO transcription fac-tors their clinical significance and regulationrdquoBioMed ResearchInternational vol 2014 Article ID 925350 13 pages 2014

[50] E Lam R Francis and M Petkovic ldquoFOXO transcriptionfactors key regulators of cell faterdquo Biochemical Society Trans-actions vol 34 no 5 pp 722ndash726 2006

[51] K Miyamoto K Y Araki K Naka et al ldquoFoxo3a is essentialfor maintenance of the hematopoietic stem cell poolrdquo Cell StemCell vol 1 no 1 pp 101ndash112 2007

[52] Z Tothova R Kollipara B J Huntly et al ldquoFoxOs are criticalmediators of hematopoietic stem cell resistance to physiologicoxidative stressrdquo Cell vol 128 no 2 pp 325ndash339 2007

[53] S Yalcin X Zhang J P Luciano et al ldquoFoxo3 is essential for theregulation of ataxia telangiectasia mutated and oxidative stress-mediated homeostasis of hematopoietic stem cellsrdquoThe Journalof Biological Chemistry vol 283 no 37 pp 25692ndash25705 2008

[54] J Paik Z Ding R Narurkar et al ldquoFoxOs cooperatively regu-late diverse pathways governing neural stem cell homeostasisrdquoCell Stem Cell vol 5 no 5 pp 540ndash553 2009

[55] V M Renault V A Rafalski A A Morgan et al ldquoFoxO3regulates neural stem cell homeostasisrdquo Cell Stem Cell vol 5no 5 pp 527ndash539 2009

[56] A Brunet A Bonni M J Zigmond et al ldquoAkt promotescell survival by phosphorylating and inhibiting a Forkheadtranscription factorrdquo Cell vol 96 no 6 pp 857ndash868 1999

[57] A Brunet F Kanai J Stehn and et al ldquo14-3-3 transits tothe nucleus and participates in dynamic nucleocytoplasmictransportrdquo Journal of Cell Biology vol 156 no 5 pp 817ndash8282002

[58] M M Juntilla V D Patil M Calamito R P Joshi M JBirnbaum and G A Koretzky ldquoAKT1 and AKT2 maintainhematopoietic stem cell function by regulating reactive oxygenspeciesrdquo Blood vol 115 no 20 pp 4030ndash4038 2010

[59] O H Yilmaz R Valdez B K Theisen et al ldquoPten depen-dence distinguishes haematopoietic stem cells from leukaemia-initiating cellsrdquo Nature vol 441 no 7092 pp 475ndash482 2006

[60] S N Greer J L Metcalf Y Wang and M Ohh ldquoThe updatedbiology of hypoxia-inducible factorrdquoTheEMBO Journal vol 31no 11 pp 2448ndash2460 2012

[61] C E Forristal K L Wright N A Hanley R O Oreffo and FD Houghton ldquoHypoxia inducible factors regulate pluripotencyand proliferation in human embryonic stem cells cultured atreduced oxygen tensionsrdquo Reproduction vol 139 no 1 pp 85ndash97 2009

[62] K L Covello J Kehler H Yu et al ldquoHIF-2120572 regulates Oct-4effects of hypoxia on stem cell function embryonic develop-ment and tumor growthrdquo Genes amp Development vol 20 no5 pp 557ndash570 2006

[63] J Mazumdar W T OrsquoBrien R S Johnson et al ldquoO2regulates

stem cells through Wnt120573-catenin signallingrdquo Nature Cell Biol-ogy vol 12 no 10 pp 1007ndash1013 2010

Oxidative Medicine and Cellular Longevity 11

[64] K Takubo N Goda W Yamada et al ldquoRegulation of the HIF-1alpha level is essential for hematopoietic stem cellsrdquo Cell StemCell vol 7 no 3 pp 391ndash402 2010

[65] FKocabas J Zheng SThet et al ldquoMeis1 regulates themetabolicphenotype and oxidant defense of hematopoietic stem cellsrdquoBlood vol 120 no 25 pp 4963ndash4972 2012

[66] T M Phillips W H McBride and F Pajonk ldquoThe responseof CD24minus119897119900119908CD44+ breast cancer-initiating cells to radiationrdquoJNCI Journal of the National Cancer Institute vol 98 no 24 pp1777ndash1785 2006

[67] M Diehn R W Cho N A Lobo et al ldquoAssociation of reactiveoxygen species levels and radioresistance in cancer stem cellsrdquoNature vol 458 no 7239 pp 780ndash783 2009

[68] O Herault K J Hope E Deneault et al ldquoA role for GPx3in activity of normal and leukemia stem cellsrdquo The Journal ofExperimental Medicine vol 209 no 5 pp 895ndash901 2012

[69] S Kim C Kwon and I Nakano ldquoDetoxification of oxidativestress in glioma stem cells mechanism clinical relevance andtherapeutic developmentrdquo Journal of Neuroscience Research vol92 no 11 pp 1419ndash1424 2014

[70] T Ishimoto O Nagano T Yae et al ldquoCD44 variant regulatesredox status in cancer cells by stabilizing the xCT subunit ofsystem xc(-) and thereby promotes tumor growthrdquo Cancer Cellvol 19 no 3 pp 387ndash400 2011

[71] M Yoshikawa K Tsuchihashi T Ishimoto et al ldquoxCT inhibi-tion depletes CD44v-expressing tumor cells that are resistantto EGFR-targeted therapy in head and neck squamous cellcarcinomardquo Cancer Research vol 73 no 6 pp 1855ndash1866 2013

[72] N Haraguchi H Ishii K Mimori et al ldquoCD13 is a therapeutictarget in human liver cancer stem cellsrdquo Journal of ClinicalInvestigation vol 120 no 9 pp 3326ndash3339 2010

[73] H M Kim N Haraguchi H Ishii et al ldquoIncreased CD13expression reduces reactive oxygen species promoting sur-vival of liver cancer stem cells via an epithelial-mesenchymaltransition-like phenomenonrdquo Annals of Surgical Oncology vol19 supplement 3 pp S539ndashS548 2012

[74] K Naka T Hoshii T Muraguchi et al ldquoTGF-120573ndashFOXO sig-nalling maintains leukaemia-initiating cells in chronic myeloidleukaemiardquo Nature vol 463 no 7281 pp 676ndash680 2010

[75] J Zhou J Wulfkuhle H Zhang et al ldquoActivation of thePTENmTORSTAT3 pathway in breast cancer stem-like cellsis required for viability and maintenancerdquo Proceedings of theNational Academy of Sciences vol 104 no 41 pp 16158ndash161632007

[76] A Dubrovska S Kim R J Salamone et al ldquoThe role ofPTENAktPI3K signaling in the maintenance and viability ofprostate cancer stem-like cell populationsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 106 no 1 pp 268ndash273 2009

[77] H Yin and J Glass ldquoThe phenotypic radiation resistance ofCS44+CD24minusor low breast cancer cells is mediated through theenhanced activation of ATM signalingrdquo PLoS ONE vol 6 no9 Article ID e24080 2011

[78] T H Rushmore and C Pickett ldquoTranscriptional regulation ofthe rat glutathione S-transferase Ya subunit gene Characteri-zation of a xenobiotic-responsive element controlling inducibleexpression by phenolic antioxidantsrdquo The Journal of BiologicalChemistry vol 265 no 24 pp 14648ndash14653 1990

[79] Y Li and A Jaiswal ldquoRegulation of humanNAD (P)H quinoneoxidoreductase gene Role of AP1 binding site contained withinhuman antioxidant response elementrdquoThe Journal of BiologicalChemistry vol 267 no 21 pp 15097ndash15104 1992

[80] T Prestera P Talalay J Alam et al ldquoParallel induction of hemeoxygenase-1 and chemoprotective phase 2 enzymes by elec-trophiles and antioxidants regulation by upstream antioxidant-responsive elements (ARE)rdquoMolecular Medicine vol 1 no 7 p827 1995

[81] K Itoh K IgarashiNHayashi et al ldquoCloning and characteriza-tion of a novel erythroid cell-derived CNC family transcriptionfactor heterodimerizing with the small Maf family proteinsrdquoMolecular and Cellular Biology vol 15 no 8 pp 4184ndash41931995

[82] R Venugopal and A K Jaiswal ldquoNrf1 and Nrf2 positivelyand c-Fos and Fra1 negatively regulate the human antioxidantresponse element-mediated expression of NAD(P)Hquinoneoxidoreductase1 generdquo Proceedings of the National Academy ofSciences vol 93 no 25 pp 14960ndash14965 1996

[83] K Itoh T Chiba S Takahashi et al ldquoAn Nrf2small Mafheterodimer mediates the induction of phase II detoxifyingenzyme genes through antioxidant response elementsrdquo Bio-chemical and Biophysical Research Communications vol 236no 2 pp 313ndash322 1997

[84] M Ramos-Gomez M-K Kwak P M Dolan et al ldquoSensitivityto carcinogenesis is increased and chemoprotective efficacy ofenzyme inducers is lost in nrf2 transcription factor-deficientmicerdquo Proceedings of the National Academy of Sciences vol 98no 6 pp 3410ndash3415 2001

[85] M-K Kwak N Wakabayashi K Itoh H Motohashi MYamamoto and T W Kensler ldquoModulation of gene expressionby cancer chemopreventive dithiolethiones through the Keap1-Nrf2 pathway Identification of novel gene clusters for cellsurvivalrdquo The Journal of Biological Chemistry vol 278 no 10pp 8135ndash8145 2003

[86] R Hu C Xu G Shen et al ldquoGene expression profiles inducedby cancer chemopreventive isothiocyanate sulforaphane in theliver of C57BL6J mice and C57BL6JNrf2 (minusminus) micerdquo CancerLetters vol 243 no 2 pp 170ndash192 2006

[87] S Chanas Q Jiang and M McMahon ldquoLoss of the Nrf2transcription factor causes a marked reduction in constitutiveand inducible expression of the glutathione S-transferase Gsta1Gsta2 Gstm1 Gstm2 Gstm3 and Gstm4 genes in the livers ofmale and female micerdquo Biochemical Journal vol 365 no 2 pp405ndash416 2002

[88] H-Y Cho S P Reddy A DeBiase M Yamamoto and SR Kleeberger ldquoGene expression profiling of NRF2-mediatedprotection against oxidative injuryrdquo Free Radical Biology andMedicine vol 38 no 3 pp 325ndash343 2005

[89] G Shen C Xu R Hu et al ldquoModulation of nuclear factorE2-related factor 2-mediated gene expression in mice liver andsmall intestine by cancer chemopreventive agent curcuminrdquoMolecular Cancer Therapeutics vol 5 no 1 pp 39ndash51 2006

[90] J D Hayes and A T Dinkova-Kostova ldquoThe Nrf2 regulatorynetwork provides an interface between redox and intermediarymetabolismrdquo Trends in Biochemical Sciences vol 39 no 4 pp199ndash218 2014

[91] A Hayashi H Suzuki K Itoh M Yamamoto and Y SugiyamaldquoTranscription factor Nrf2 is required for the constitutive andinducible expression of multidrug resistance-associated protein1 in mouse embryo fibroblastsrdquo Biochemical and BiophysicalResearch Communications vol 310 no 3 pp 824ndash829 2003

[92] E Jigorel M Le Vee C Boursier-Neyret Y Parmentier andO Fardel ldquoDifferential regulation of sinusoidal and canalicularhepatic drug transporter expression by xenobiotics activating

12 Oxidative Medicine and Cellular Longevity

drug-sensing receptors in primary human hepatocytesrdquo DrugMetabolism and Disposition vol 34 no 10 pp 1756ndash1763 2006

[93] X Wang C R Campos J C Peart et al ldquoNrf2 upregulatesATP binding cassette transporter expression and activity atthe blood-brain and blood-spinal cord barriersrdquo Journal ofNeuroscience vol 34 no 25 pp 8585ndash8593 2014

[94] H Jeong I Ryoo andMKwak ldquoRegulation of the expression ofrenal drug transporters in KEAP1-knockdown human tubularcellsrdquo Toxicology in Vitro vol 29 no 5 pp 884ndash892 2015

[95] M J Canet M D Merrell B G Harder et al ldquoIdentificationof a functional antioxidant response element within the eighthintron of the human ABCC

3generdquo Drug Metabolism and

Disposition vol 43 no 1 pp 93ndash99 2014[96] A Singh H Wu P Zhang C Happel J Ma and S Biswal

ldquoExpression of ABCG2 (BCRP) is regulated by Nrf2 in cancercells that confers side population and chemoresistance pheno-typerdquo Molecular Cancer Therapeutics vol 9 no 8 pp 2365ndash2376 2010

[97] N Wakabayashi K Itoh J Wakabayashi et al ldquoKeap1-nullmutation leads to postnatal lethality due to constitutive Nrf2activationrdquo Nature Genetics vol 35 no 3 pp 238ndash245 2003

[98] S B Cullinan J D Gordan J Jin J W Harper and J A DiehlldquoThe Keap1-BTB protein is an adaptor that bridges Nrf2 to aCul3-based E3 ligase oxidative stress sensing by a Cul3-Keap1ligaserdquoMolecular and Cellular Biology vol 24 no 19 pp 8477ndash8486 2004

[99] D D Zhang and M Hannink ldquoDistinct cysteine residues inkeap1 are required for keap1-dependent ubiquitination of nrf2and for stabilization of nrf2 by chemopreventive agents andoxidative stressrdquoMolecular and Cellular Biology vol 23 no 22pp 8137ndash8151 2003

[100] L M Zipper and R T Mulcahy ldquoThe Keap1 BTBPOZ dimer-ization function is required to sequester Nrf2 in cytoplasmrdquoThe Journal of Biological Chemistry vol 277 no 39 pp 36544ndash36552 2002

[101] M C Jaramillo and D D Zhang ldquoThe emerging role of theNrf2-Keap1 signaling pathway in cancerrdquoGenesampDevelopmentvol 27 no 20 pp 2179ndash2191 2013

[102] K I Tong Y Katoh H Kusunoki K Itoh T Tanaka and MYamamoto ldquoKeap1 recruits Neh2 through binding to ETGEand DLG motifs characterization of the two-site molecularrecognition modelrdquoMolecular and Cellular Biology vol 26 no8 pp 2887ndash2900 2006

[103] K I Tong A Kobayashi F Katsuoka andM Yamamoto ldquoTwo-site substrate recognition model for the Keap1-Nrf2 system ahinge and latch mechanismrdquo Biological Chemistry vol 387 no10-11 pp 1311ndash1320 2006

[104] K I Tong B Padmanabhan A Kobayashi et al ldquoDifferentelectrostatic potentials define ETGE and DLG motifs as hingeand latch in oxidative stress responserdquo Molecular and CellularBiology vol 27 no 21 pp 7511ndash7521 2007

[105] M Kobayashi L Li N Iwamoto et al ldquoThe antioxidant defensesystemKeap1-Nrf2 comprises amultiple sensingmechanism forresponding to a wide range of chemical compoundsrdquoMolecularand Cellular Biology vol 29 no 2 pp 493ndash502 2009

[106] T Yamamoto T Suzuki A Kobayashi et al ldquoPhysiological sig-nificance of reactive cysteine residues of Keap1 in determiningNrf2 activityrdquoMolecular and Cellular Biology vol 28 no 8 pp2758ndash2770 2008

[107] M K Kwak K Itoh M Yamamoto and T W KenslerldquoEnhanced expression of the transcription factorNrf2 by cancer

chemopreventive agents role of antioxidant response element-like sequences in the nrf2 promoterrdquo Molecular and CellularBiology vol 22 no 9 pp 2883ndash2892 2002

[108] T Suzuki T Shibata K Takaya et al ldquoRegulatory nexus ofsynthesis and degradation deciphers cellular Nrf2 expressionlevelsrdquoMolecular and Cellular Biology vol 33 no 12 pp 2402ndash2412 2013

[109] S B Cullinan D Zhang M Hannink E Arvisais R JKaufman and J A Diehl ldquoNrf2 is a direct PERK substrateand effector of PERK-dependent cell survivalrdquo Molecular andCellular Biology vol 23 no 20 pp 7198ndash7209 2003

[110] W Li and A Kong ldquoMolecular mechanisms of Nrf2-mediatedantioxidant responserdquo Molecular Carcinogenesis vol 48 no 2pp 91ndash104 2009

[111] Y J Surh ldquoCancer chemoprevention with dietary phytochemi-calsrdquo Nature Reviews Cancer vol 3 no 10 pp 768ndash780 2003

[112] M Salazar A I Rojo D Velasco R M de Sagarra and ACuadrado ldquoGlycogen synthase kinase-3beta inhibits the xeno-biotic and antioxidant cell response by direct phosphorylationand nuclear exclusion of the transcription factor Nrf2rdquo Journalof Biological Chemistry vol 281 no 21 pp 14841ndash14851 2006

[113] W Chen Z Sun X J Wang et al ldquoDirect interactionbetween Nrf2 and p21(Cip1WAF1) upregulates the Nrf2-mediated antioxidant responserdquo Molecular Cell vol 34 no 6pp 663ndash673 2009

[114] M Komatsu H Kurokawa S Waguri et al ldquoThe selectiveautophagy substrate p62 activates the stress responsive tran-scription factor Nrf2 through inactivation of Keap1rdquoNature CellBiology vol 12 no 3 pp 213ndash223 2010

[115] J D Hayes M McMahon S Chowdhry and A T Dinkova-Kostova ldquoCancer chemoprevention mechanisms mediatedthrough the Keap1-Nrf2 pathwayrdquoAntioxidants amp Redox Signal-ing vol 13 no 11 pp 1713ndash1748 2010

[116] R Hu C L Saw R Yu and A T Kong ldquoRegulation ofNF-E2-related factor 2 signaling for cancer chemopreventionantioxidant coupled with antiinflammatoryrdquo Antioxidants ampRedox Signaling vol 13 no 11 pp 1679ndash1698 2010

[117] M Kwak and T W Kensler ldquoTargeting NRF2 signaling for can-cer chemopreventionrdquo Toxicology and Applied Pharmacologyvol 244 no 1 pp 66ndash76 2010

[118] K Iida K Itoh Y Kumagai et al ldquoNrf2 is essential for thechemopreventive efficacy of oltipraz against urinary bladdercarcinogenesisrdquo Cancer Research vol 64 no 18 pp 6424ndash64312004

[119] T Jiang N Chen F Zhao et al ldquoHigh levels of Nrf2 deter-mine chemoresistance in type II endometrial cancerrdquo CancerResearch vol 70 no 13 pp 5486ndash5496 2010

[120] Y R Kim J EOhM S Kim et al ldquoOncogenicNRF2mutationsin squamous cell carcinomas of oesophagus and skinrdquo TheJournal of Pathology vol 220 no 4 pp 446ndash451 2010

[121] T Shibata A Kokubu M Gotoh et al ldquoGenetic alterationof Keap1 confers constitutive Nrf2 activation and resistance tochemotherapy in gallbladder cancerrdquoGastroenterology vol 135no 4 pp 1358e4ndash1368e4 2008

[122] T Shibata T Ohta K I Tong et al ldquoCancer related mutationsin NRF2 impair its recognition by Keap1-Cul3 E3 ligase andpromote malignancyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 36 pp13568ndash13573 2008

[123] A Singh V Misra R K Thimmulappa et al ldquoDysfunctionalKEAP1-NRF2 interaction in non-small-cell lung cancerrdquo PLoSMedicine vol 3 no 10 article e420 2006

Oxidative Medicine and Cellular Longevity 13

[124] R Wang J An F Ji H Jiao H Sun and D Zhou ldquoHyper-methylation of the Keap1 gene in human lung cancer cell linesand lung cancer tissuesrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 1 pp 151ndash154 2008

[125] P Zhang A Singh S Yegnasubramanian et al ldquoLoss ofKelch-like ECH-associated protein 1 function in prostate cancercells causes chemoresistance and radioresistance and promotestumor growthrdquoMolecular Cancer Therapeutics vol 9 no 2 pp336ndash346 2010

[126] J D Hayes and M McMahon ldquoNRF2 and KEAP1 mutationspermanent activation of an adaptive response in cancerrdquo Trendsin Biochemical Sciences vol 34 no 4 pp 176ndash188 2009

[127] B Padmanabhan K I Tong T Ohta et al ldquoStructural basis fordefects of Keap1 activity provoked by its pointmutations in lungcancerrdquoMolecular Cell vol 21 no 5 pp 689ndash700 2006

[128] Y Inami S Waguri A Sakamoto et al ldquoPersistent activation ofNrf2 throughp62 in hepatocellular carcinoma cellsrdquoTheJournalof Cell Biology vol 193 no 2 pp 275ndash284 2011

[129] G M DeNicola F A Karreth T J Humpton et al ldquoOncogene-induced Nrf2 transcription promotes ROS detoxification andtumorigenesisrdquo Nature vol 475 no 7354 pp 106ndash109 2011

[130] L M Solis C Behrens W Dong et al ldquoNrf2 and Keap1abnormalities in non-small cell lung carcinoma and associationwith clinicopathologic featuresrdquo Clinical Cancer Research vol16 no 14 pp 3743ndash3753 2010

[131] N M Reddy S R Kleeberger H Y Cho et al ldquoDeficiency inNrf2-GSH signaling impairs type II cell growth and enhancessensitivity to oxidantsrdquoAmerican Journal of Respiratory Cell andMolecular Biology vol 37 no 1 pp 3ndash8 2007

[132] N M Reddy S R Kleeberger M Yamamoto et al ldquoGeneticdissection of the Nrf2-dependent redox signaling-regulatedtranscriptional programs of cell proliferation and cytoprotec-tionrdquo Physiological Genomics vol 32 no 1 pp 74ndash81 2007

[133] Y Mitsuishi K Taguchi Y Kawatani et al ldquoNrf2 redirectsglucose and glutamine into anabolic pathways in metabolicreprogrammingrdquo Cancer Cell vol 22 no 1 pp 66ndash79 2012

[134] J Cho SManandharH LeeH Park andMKwak ldquoRole of theNrf2-antioxidant system in cytotoxicity mediated by anticancercisplatin implication to cancer cell resistancerdquo Cancer Lettersvol 260 no 1-2 pp 96ndash108 2008

[135] T Ohta K Iijima M Miyamoto et al ldquoLoss of Keap1 functionactivates Nrf2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008

[136] A Singh M Bodas N Wakabayashi F Bunz and S BiswalldquoGain of Nrf2 function in non-small-cell lung cancer cellsconfers radioresistancerdquoAntioxidantsampRedox Signaling vol 13no 11 pp 1627ndash1637 2010

[137] G-S Shim S Manandhar D-H Shin T Kim and M KwakldquoAcquisition of doxorubicin resistance in ovarian carcinomacells accompanies activation of the NRF2 pathwayrdquo Free RadicalBiology and Medicine vol 47 no 11 pp 1619ndash1631 2009

[138] S Manandhar B Choi K Jung et al ldquoNRF2 inhibitionrepresses ErbB2 signaling in ovarian carcinoma cells implica-tions for tumor growth retardation and docetaxel sensitivityrdquoFree Radical Biology and Medicine vol 52 no 9 pp 1773ndash17852012

[139] T Kim E-g Hur S J Kang et al ldquoNRF2 blockade suppressescolon tumor angiogenesis by inhibiting hypoxia-induced acti-vation ofHIF-1120572rdquoCancer Research vol 71 no 6 pp 2260ndash22752011

[140] L Kinch N V Grishin and J Brugarolas ldquoSuccination of Keap1and activation of Nrf2-dependent antioxidant pathways in FH-deficient papillary renal cell carcinoma type 2rdquo Cancer Cell vol20 no 4 pp 418ndash420 2011

[141] J J Tsai J A Dudakov K Takahashi et al ldquoNrf2 regulateshaematopoietic stem cell functionrdquo Nature Cell Biology vol 15no 3 pp 309ndash316 2013

[142] J H Kim R KThimmulappa V Kumar et al ldquoNRF2-mediatedNotch pathway activation enhances hematopoietic reconsti-tution following myelosuppressive radiationrdquo The Journal ofClinical Investigation vol 124 no 2 pp 730ndash741 2014

[143] J P Chute ldquoNRF2 mitigates radiation-induced hematopoieticdeathrdquo The Journal of Clinical Investigation vol 124 no 3 pp960ndash961 2014

[144] J Jang Y Wang H Kim M A Lalli and K S KosikldquoNrf2 a regulator of the proteasome controls self-renewal andpluripotency in human embryonic stem cellsrdquo Stem Cells vol32 no 10 pp 2616ndash2625 2014

[145] S Murakami R Shimizu P Romeo M Yamamoto and HMotohashi ldquoKeap1-Nrf2 system regulates cell fate determina-tion of hematopoietic stem cellsrdquo Genes to Cells vol 19 no 3pp 239ndash253 2014

[146] C E Hochmuth B Biteau D Bohmann and H Jasper ldquoRedoxregulation by Keap1 and Nrf2 controls intestinal stem cellproliferation inDrosophilardquoCell Stem Cell vol 8 no 2 pp 188ndash199 2011

[147] M K Paul B Bisht D O Darmawan et al ldquoDynamic changesin intracellular ROS levels regulate airway basal stem cellhomeostasis through Nrf2-dependent notch signalingrdquo CellStem Cell vol 15 no 2 pp 199ndash214 2014

[148] L Gambari G Lisignoli L Cattini C Manferdini A Facchiniand F Grassi ldquoSodium hydrosulfide inhibits the differentiationof osteoclast progenitor cells via NRF2-dependent mechanismrdquoPharmacological Research vol 87 pp 99ndash112 2014

[149] J Li D Johnson M Calkins L Wright C Svendsen andJ Johnson ldquoStabilization of Nrf2 by tBHQ confers protectionagainst oxidative stress-induced cell death in human neuralstem cellsrdquo Toxicological Sciences vol 83 no 2 pp 313ndash3282005

[150] A AMerchant A SinghWMatsui and S Biswal ldquoThe redox-sensitive transcription factor Nrf2 regulatesmurine hematopoi-etic stem cell survival independently of ROS levelsrdquo Blood vol118 no 25 pp 6572ndash6579 2011

[151] N Gurusamy D Ray I Lekli and D K Das ldquoRed wineantioxidant resveratrol-modified cardiac stem cells regener-ate infarcted myocardiumrdquo Journal of Cellular and MolecularMedicine vol 14 no 9 pp 2235ndash2239 2010

[152] N Gorbunov G Petrovski N Gurusamy D Ray D H Kimand D K Das ldquoRegeneration of infarcted myocardium withresveratrol-modified cardiac stem cellsrdquo Journal of Cellular andMolecular Medicine vol 16 no 1 pp 174ndash184 2012

[153] C Cai L Teng D Vu et al ldquoThe heme oxygenase 1 inducer(CoPP) protects human cardiac stem cells against apoptosisthrough activation of the extracellular signal-regulated kinase(ERK)NRF2 signaling pathway and cytokine releaserdquo TheJournal of Biological Chemistry vol 287 no 40 pp 33720ndash33732 2012

[154] J Song S M Kang K M Lee and J E Lee ldquoThe protectiveeffect of melatonin on neural stem cell against LPS-inducedinflammationrdquo BioMed Research International vol 2015 ArticleID 854359 13 pages 2015

14 Oxidative Medicine and Cellular Longevity

[155] V Karkkainen Y Pomeshchik E Savchenko et al ldquoNrf2regulates neurogenesis and protects neural progenitor cellsagainst A120573 toxicityrdquo STEM CELLS vol 32 no 7 pp 1904ndash19162014

[156] M Mohammadzadeh R Halabian A Gharehbaghian et alldquoNrf-2 overexpression in mesenchymal stem cells reducesoxidative stress-induced apoptosis and cytotoxicityrdquo Cell Stressand Chaperones vol 17 no 5 pp 553ndash565 2012

[157] Y Takahata T Takarada M Iemata et al ldquoFunctional expres-sion of 1205732 adrenergic receptors responsible for protectionagainst oxidative stress through promotion of glutathione syn-thesis after Nrf2 upregulation in undifferentiated mesenchymalC3H10T12 stem cellsrdquo Journal of Cellular Physiology vol 218no 2 pp 268ndash275 2009

[158] J Zhu H Wang Y Fan et al ldquoKnockdown of nuclear factorerythroid 2-related factor 2 by lentivirus induces differentiationof glioma stem-like cellsrdquo Oncology Reports vol 32 no 3 pp1170ndash1178 2014

[159] J Zhu H Wang Q Sun et al ldquoNrf2 is required to maintain theself-renewal of glioma stem cellsrdquo BMC Cancer vol 13 no 1article 380 2013

[160] B L Emmink A Verheem W J Van Houdt et al ldquoThe secre-tome of colon cancer stem cells contains drug-metabolizingenzymesrdquo Journal of Proteomics vol 91 pp 84ndash96 2013

[161] T Mizuno N Suzuki H Makino et al ldquoCancer stem-like cellsof ovarian clear cell carcinoma are enriched in the ALDH-highpopulation associated with an accelerated scavenging system inreactive oxygen speciesrdquo Gynecologic Oncology vol 137 no 2pp 299ndash305 2015

[162] I-G Ryoo B-H Choi and M-K Kwak ldquoActivation of NRF2by p62 and proteasome reduction in sphere-forming breastcarcinoma cellsrdquo Oncotarget vol 6 no 10 pp 8167ndash8184 2015

[163] T Wu B G Harder P K Wong J E Lang and D D ZhangldquoOxidative stress mammospheres and Nrf2-new implicationfor breast cancer therapyrdquoMolecular Carcinogenesis 2014

[164] B Yang Y Ma and Y Liu ldquoElevated expression of Nrf-2 andABCG2 involved in multi-drug resistance of lung cancer SPcellsrdquo Drug Research 2015

[165] M Zhang A Mathur Y Zhang et al ldquoMithramycin repressesbasal and cigarette smoke-induced expression of abcg2 andinhibits stem cell signaling in lung and esophageal cancer cellsrdquoCancer Research vol 72 no 16 pp 4178ndash4192 2012

[166] C A Del Vecchio Y Feng E S Sokol et al ldquoDe-differentiationconfers multidrug resistance via noncanonical PERK-Nrf2signalingrdquo PLoS Biology vol 12 no 9 Article ID e1001945 2014

[167] D Ren N F Villeneuve T Jiang et al ldquoBrusatol enhancesthe efficacy of chemotherapy by inhibiting the Nrf2-mediateddefense mechanismrdquo Proceedings of the National Academy ofSciences of the United States of America vol 108 no 4 pp 1433ndash1438 2011

[168] A Arlt S Sebens S Krebs et al ldquoInhibition of the Nrf2 tran-scription factor by the alkaloid trigonelline renders pancreaticcancer cells more susceptible to apoptosis through decreasedproteasomal gene expression and proteasome activityrdquo Onco-gene vol 32 no 40 pp 4825ndash4835 2013

[169] A-M Gao Z-P Ke F Shi G Sun and H Chen ldquoChrysinenhances sensitivity of BEL-7402ADM cells to doxorubicin bysuppressing PI3KAktNrf2 and ERKNrf2 pathwayrdquo Chemico-Biological Interactions vol 206 no 1 pp 100ndash108 2013

[170] A M Gao Z P Ke J N Wang J Yang S Chen and HChen ldquoApigenin sensitizes doxorubicin-resistant hepatocellular

carcinoma BEL-7402ADM cells to doxorubicin via inhibitingPI3KAktNrf2 pathwayrdquo Carcinogenesis vol 34 no 8 pp1806ndash1814 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Oxidative Medicine and Cellular Longevity

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Oxidative Medicine and Cellular Longevity 3

to ROSThis phenomenon was linked to low levels of cellularROS and high levels of SODs catalase GPX1 and GSH inMSCs [38 40] Elevated antioxidant molecules appear to playa crucial role in the protection of stem cells against oxidativestress However under certain circumstances NOX-derivedROS are associated with MSC differentiation The treatmentof MSCs with antioxidants or interfering RNA of NOX4 pre-vented adipocyte differentiation of MSCs via cAMP responseelement-binding protein (CREB) inhibition Similarly ESCdifferentiation to the cardiac lineage was dependent onNOX4-derived ROS [44] These findings indicate that ROSare important for stem cell fate determination for quiescenceor differentiation

4 Redox Signaling Molecules in Stem Cells

It has been reported that multiple signaling molecules areinvolved in ROS-mediated regulation of stem cells (Figure 1)First ataxia telangiectasia mutated (ATM) plays a criticalrole in controlling ROS levels in stem cells ATM a ser-inethreonine protein kinase is a known regulator of theDNA damage response and contributes to the regulation ofcellular ROS ATM is known to regulate ROS via modulationof AMPK-mTOR pathway or NADPH production [45 46]Ito et al showed that atmminusminus mice developed bone marrowfailure after 24 weeks of age due to a depletion of HSCsIn this study HSCs in atm knockout mice showed higherlevels of ROS than wild type mice which presumably causeda reduction in the self-renewal activity of HSCs Howeverthe treatment of mice with antioxidant N-acetylcysteine(NAC) restored HSC reconstitution in atm knockout mice byreducing ROS in HSCs confirming the critical role of ROSin HSCs maintenance [47] Similarly in another study NACtreatment prevented hypersensitivity of atmminusminusmice to X-rayirradiation and senescence of atmminusminus embryonic fibroblasts[48] Cosentino et al have presented amolecularmechanisticrole for ATM demonstrating that ATM activation promotesthe binding of heat shock protein 27 (HSP27) to glucose-6-phosphate dehydrogenase (G6PDH) which can result inG6PDH activation and subsequent NADPH increase [46]

The forkhead box O (FoxO) transcription factor familyis also implicated in redox regulation of stem cells TheFoxO family including FoxO1 FoxO3 FoxO4 and FoxO6is a key regulator of cell survival proliferation DNA repairand apoptosis FoxO1 and FoxO3 are reported to upregulatethe expression of GSH biosynthetic enzymes and SODsand therefore are associated with cellular protection againstoxidative stress [49 50] Particularly FoxO3 has been knownto play crucial roles in cytoprotection of stem cells includingHSCs [51ndash53] Loss of FoxO3a which regulates the expressionof antioxidant enzymes such as catalases and SOD2 led toROS accumulation and thus a higher rate of cell cyclingand a loss of quiescence in HSCs [51] In foxo1foxo3afoxo4triple-knockout mice the number of HSCs was substantiallydecreased and apoptotic HSCs were increased through ROSelevation [52] Notably Yalcin et al provided a link betweenATMand the FoxOprotein in ROS regulation of stem cells Infoxo3minusminusHSCs ATMexpressionwas diminished compared to

Quiescenceself-renewal

Senescenceapoptosis

cell cycle progressionLow ROS

High ROSATM uarr FoxO uarr

PI3KAKT uarr

FoxO darr

Figure 1 Involvement of ROS in normal stem cell quiescence andself-renewal In normal stem cells modulation of ROS levels candetermine quiescence and cell fate progression At low ROS levelswhich are maintained by ATM and FoxO signaling stem cellsremain quiescent and self-renewal activity is enhancedOn the otherhand increased ROS levels result in cell cycle progression cellularsenescence and apoptosis The PI3K-AKT pathway is known toelevate ROS levels by negative regulation of FoxO

normalHSCs suggesting that FoxO3 repressed ROS viaATMregulation [53] Similar to HSCs foxo-deficient neural stemcells demonstrated a decline in self-renewal capacity due toincreased cellular ROS levels [54 55]

The phosphoinositide 3-kinase (PI3K)AKT pathway isanother ROS regulator in normal stem cells In particularPI3KAKT signaling associates with FoxO transcription fac-tors to mediate ROS regulation Activated AKT promotesFoxO phosphorylation resulting in the nuclear export andcytoplasmic degradation of FoxO through the proteasome[56 57] Therefore ak11990512 double knockout HSCs displayedincreased quiescence and low cellular ROS levels [58] Con-sistently persistent activation of the PI3KAKT pathway inphosphatase and tensin homolog (PTEN) deleted HSCs ledto defective quiescence resulting in cellular senescence [59]Based on the above observations the PI3KAKTpathway andFoxOATMpathway exhibit opposite roles in ROS regulationof stem cells

Hypoxia-inducible factors (HIFs) are transcription fac-tors that respond to hypoxic conditions [60] They are alsocritical factors for the maintenance of stem cells HSCscultured in hypoxic conditions displayed a higher colonyformation capacity and high HIF levels positively regulatedthe pluripotency of human ESCs by activating stemnesstranscription factors such as OCT4 SOX2 and NANOG[61 62] Moreover in neuronal stem cells in a hypoxicenvironment accumulated HIF1120572 promoted Wnt120573-cateninpathway activation [63] The involvement of HIFs in stemcell biology is mediated by ROS Takubo et al observed thathif1120572minusminus HSCs contain high levels of ROS which could beassociated with a loss of HSC quiescence and an induction ofcellular senescence [64] In agreement with this finding thesuppression of HIF1120572 and HIF2120572 in HSCs led to increasedROS generation via mitochondrial metabolic shift and con-sequently induced cellular senescence and apoptosis Scav-enging ROS byNAC treatment could restore HSC quiescenceand function in stem cells withHIF1120572 andHIF2120572 suppression[65]

5 Involvement of ROS in CSC Biology

Very few studies have investigated the involvement of redoxchange in CSC biology compared to that in cancer cellsor normal stem cells However it has been reported that

4 Oxidative Medicine and Cellular Longevity

CSCs appear to share several ROS-associated propertieswith normal stem cells CSCs are known to contain lowerlevels of ROS compared to non-CSCs CD44+CD24minuslowbreast cancer CSCs isolated from MDA-MB-231 and MCF7mammospheres were relatively more resistant to radiationand this was associated with lower levels of ROS afterradiation [66] Diehn et al observed that ROS levels inCD44+CD24minus breast cancer CSCs were lower than in non-CSCs and the expression levels of the modulatory subunit ofglutamate cysteine ligase (GCLM the rate-limiting enzymeof GSH synthesis) and FoxO1 were high Treatment of theCD44+CD24minus subpopulationwith buthionine sulphoximine(BSO) an inhibitor of GSH synthesis resulted in reducedcolony forming capacity and increased sensitivity to radiationtherapy through an increase in ROS [67] In a study usinghuman AML specimens leukemic stem cells with low levelsof ROS majorly contributed to stem cell quiescence bymaintaining a low rate of oxidative phosphorylation andmetabolism [31] A leukemia with a high amount of leukemicstem cells showed low levels of ROS and increased expressionof GPX3 compared to tumors with a low frequency ofleukemic stem cells This study demonstrated that GPX3levels positively correlated with poor prognostic outcome inAML patients [68] Glioma stem cells within the tumor masshave low levels of cellular ROS although they are located in ahypoxic environment A proposed molecular mechanism ofthis phenomenon was the significantly upregulated expres-sion of peroxiredoxin 4 (PRDX4) in glioma stem cells [69]

Evidence is indicating that low ROS levels in CSCsresult from the intrinsic characteristics of CSCs Cell surfacemarkers of CSCs including CD44 and CD13 are found tobe involved in ROS regulation Ishimoto et al demonstratedthat a variant isoform of CD44 (CD44v) can bind to thecystineglutamate exchange transporter xCT and activatescysteine uptake to enhance GSH synthesis in gastrointestinalCSCs [70] The expression of antioxidant genes such asGPX12 was significantly increased in CD44+ gastric tumorcells In addition knockdown of CD44 in mice led toROS increase p38MAPK activation and cellular senescencethat are related to p21 expression In a subsequent studythe same group demonstrated that the number of CD44+cells increased with neoadjuvant chemotherapy in head andneck squamous cell carcinoma (HNSCC) patients TheseCD44+ undifferentiated cancer cells displayed high xCTexpression GSH upregulation and low cellular ROS levelsAblation of xCT by siRNA or sulfasalazine treatment (xCT-mediated cystine transport inhibitor) induced differentiationof HNSCCCSCs both in vitro and in vivo [71] CD13 has beenidentified as a surface marker for liver CSCs In liver cancercell lines including Huh7 and PLCPRF5 CD13 positive cellspredominated the SP fraction and were mainly in the G0G1phase of the cell cycle Additionally resistance to anticancerdrugs or radiation in the CD13 positive cell fraction wasmuch higher than that observed in the CD13 negative cellfraction Direct comparison of ROS levels between the twocell fractions revealed that the CD13 positive cell fractioncontains lower levels of ROS and expresses higher levelsof GCLM [72] In another study by the same group CD13expression reduced transforming growth factor-120573 (TGF-120573)

induced ROS production and promoted survival of liverCSCs [73]

It has been demonstrated that signaling pathwaysinvolved in ROS regulation of normal stem cells also playa role in CSC biology The nuclear expression levels ofFoxO3a was high in chronic myeloid leukemia-initiatingcells and the transplantation of leukemic stem cells derivedfrom foxo3a knockout mice significantly reduced their abilityto cause myeloid leukemia in an animal model [74] Thisstudy also revealed that TGF-120573 is a crucial regulator ofFoxO3a activity In the SP of MCF-7 breast cancer cellsactivation of the PI3Kmammalian target rapamycin (mTOR)signaling pathway was important for tumorigenecity of theseCSCs and knockdown of PI3K or mTOR led to ablatedtumorigenecity [75] When CD133+CD44+ prostate cancercells were grown in sphere-forming conditions activatedPI3KAKT signaling was found to be critical for maintainingCSCs [76] CD44+CD24minusor low cells isolated from breastcancer cell lines and breast cancer patient specimens wereradioresistant and this resistant phenotype was associatedwith ATM signaling activation [77]

6 NRF2 as a Key Molecule forRedox Homeostasis

In 1990 Rushmore and Pickett discovered the enhancersequence in the rat gsta2 gene promoter as a response elementto 120573-naphthoflavone and t-butylhydroquinone (t-BHQ) andnamed it antioxidant responsive element (ARE) [78] Subse-quent studies revealed that ARE is commonly involved in thetranscription of multiple antioxidant and detoxifying genesincluding glutamate-cysteine ligase (GCL) glutathione S-transferase (GST) and NAD(P)H quinone oxidoreductase-1 (NQO-1) [79 80] Based on sequence homology betweenARE and MAF-recognition element (MRE) further studieshypothesized that small MAF and bZIP caprsquonrsquocollar (CNC)transcription factors may interact with ARE [81 82] AmongbZIP CNC transcription factors NRF2 was found to play acrucial role in ARE regulation in which inducible expressionof NQO1 and GST was ablated in t-butylhydroxy anisole-treated nrf2 null mice in contrast to the observation in wildtype mice [83] After this report numerous studies haveelucidated a wide spectrum of protective effects of NRF2signaling against various stressors For example sensitivityto benzo[a]pyrene-induced carcinogenesis was significantlygreater in nrf2-knockout mice than in wild type mice [84]

To account for the protective effects of Nrf2 comparativeanalyses of gene expression patterns were carried out innrf2-deficient and wild type mice following treatment withNrf2 activators In global gene analysis of dithiolethione-administered mouse livers Nrf2 was found to governthe expression of xenobiotic-detoxifying enzymes GSH-generating systems antioxidant proteins and the molec-ular chaperone-26S proteasome [85] Similarly Hu et aldemonstrated that detoxifying enzymes antioxidants drugtransporters stress response proteins and some signalingmolecules serve as Nrf2-dependent and isothiocyanate-inducible genes in mouse liver [86] It has now been firmly

Oxidative Medicine and Cellular Longevity 5

established thatNRF2 regulates divergent genes to coordinatexenobiotic detoxification and redox homeostasis [85 87ndash89]In its function as a regulator of cellular redox homeostasisNRF2 elevates the expression of GCL and the cysteinetransporter xCT to increase cellular GSH levels NRF2 alsoenhances regeneration of reduced GSH by upregulatingGPX and GSH reductase (GSR) Expression of thioredoxin1 (TXN1) thioredoxin reductase 1 (TXNRD1) and perox-iredoxin 16 which can reduce oxidized protein thiols isalso under the control of NRF2 In addition the levels ofNADPH a cofactor of many antioxidant enzymes such asGSR and TXNRD can be increased by NRF2 (reviewedin Hayes and Dinkova-Kostova [90]) The expression ofmultiple NADPH generating enzymes such as G6PDH and6-phosphogluconate dehydrogenase is upregulated by NRF2Additionally the role of NRF2 in ABC transporter expressionfor xenobiotic detoxification is notable The basal expressionlevel of Mrp1 was relatively lower in nrf2-deficient fibroblaststhan that in wild type fibroblasts and the treatment ofmice with Nrf2 activating diethyl maleate increased Mrp1expression in the liver [91] Levels of MDR1 MRP23 andBCRP were elevated following oltipraz treatment in pri-mary human hepatocytes [92] Sulforaphane (SFN) treatmentenhanced the levels ofMDR1 BCRP andMRP2 in the blood-brain barrier of rats [93] Our recent study showed thatgenetic activation of NRF2 via KEAP1 silencing increases theexpression ofMDR1MRP23 and BCRP in human proximaltubular epithelial cells [94] As direct molecular evidencefunctional AREs have been identified in human MRP3 [95]and BCRP genes [96]

Kelch-like ECH-associated protein 1 (KEAP1) a cysteine-rich actin-binding protein is the main negative regulatorof NRF2 activity [83 97] Under quiescent conditionsNRF2 remains inactive by forming a complex with KEAP1in the cytoplasm NRF2 is subject to ubiquitination andKEAP1-induced proteasomal degradation through the Cullin3 (CUL3) based E3 ligase KEAP1 has three major domainsas follows (i) The BTB domain is associated with KEAP1homodimerization (ii) the IVR domain plays a role inregulation ofKEAP1 activity and (iii) theKelchDGRdomainmediates binding with NRF2 [83 90 98ndash101] The bindingof NRF2 with KEAP1 has been described as the ldquohinge andlatchrdquo model where one molecule of NRF2 interacts with theKelchDGR domains of the KEAP1 dimer through conservedmotifs called ETGE (DN-X-E-TS-G-E) and DLG (L-X-X-Q-D-X-D-L-G) [102ndash104] In these reports it was shown thatthe binding affinity of the ETGE motif to KEAP1 is muchhigher than that of the DLG motif It has therefore beenshown that ldquolatchrdquo binding of the NRF2 DLG motif is easilybroken by modifications of KEAP1 cysteine residues by ROSor electrophiles In turn disrupted DLG binding of NRF2 toKEAP1 leads to the blockade of ubiquitination and furtherdegradation of NRF2 resulting in nuclear translocation ofNRF2 It has been demonstrated that the sulfhydryl groups ofmultiple cysteine residues of KEAP1 can be directly modifiedby oxidationreduction or alkylation In particular Cys151Cys273 and Cys288 were found to be essential for theregulation of NRF2 activity [99 104ndash106] Mutation of theCys273 or Cys288 residue of KEAP1 ablated its ability to

suppress NRF2 activity leading to accumulation of the NRF2protein [99 104]

In addition to KEAP1-mediated stability regulationNRF2 activity can be modulated at multiple steps First itis noticeable that NRF2 activity is regulated at the transcrip-tional step Functional AREs were identified in the murinenrf2 gene promoter and were involved in the autoregulationof NRF2 through transcriptional activation [107] Moreovera single nucleotide polymorphism in the ARE-like sequencesof the human NRF2 promoter was associated with increasedlung cancer susceptibility [108] Second it was shown thatNRF2 activity is regulated by posttranslationalmodificationsStudies indicate that NRF2 activation involves phosphoryla-tion signaling mediated by multiple kinase pathways such asMAPK protein kinase C (PKC) PI3K and protein kinaseRNA-like endoplasmic reticulum kinase (PERK) [109ndash111]Meanwhile glycogen synthase kinase-3 (GSK-3) a consti-tutively active serinethreonine kinase was found to inhibitNRF2 activity [112] Last NRF2 activity is increased byseveral intrinsic proteins such as p21 and p62 [113 114] Forexample p62 a linker protein of ubiquitinated proteins toautophagy degradation binds to the KEAP1 protein andinterferes with the binding of NRF2 to KEAP1 resulting inNRF2 stabilization

7 Emerging Role of NRF2 in Cancer Biology

Continuous or fatal stimuli such as toxic chemicals and excessROS disrupt cellular homeostasis causing macromoleculardamage and alterations in cell cycle and growth signalingwhich can eventually result in carcinogenesis The NRF2pathway deserved significant attention in the area of cancerbiology because numerous studies have demonstrated thatactivation of the NRF2 pathway decreases the sensitivity ofcells to carcinogens [115ndash117] For instance the burden ofgastric neoplasia caused by benzo[a]pyrene was effectivelyattenuated by the Nrf2 activator oltipraz in wild type micewhereas nrf2 knockout mice did not show any protectiveeffect of oltipraz [84] Similarly the incidence of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) induced urinary blad-der carcinomawas greater in nrf2 knockoutmice than in wildtype mice and oltipraz treatment reduced tumor incidenceonly in wild type mice [118]

Although it has been firmly confirmed that NRF2 activa-tion can protect cells against a wide range of toxicants andstressors aberrant activation of NRF2 has been associatedwith several types of cancers NRF2 levels were constitutivelyelevated in cancer cell lines and tumor samples of the lungbreast esophagus endometrial cancers and prostate cancers[119ndash125] Molecular mechanisms involved in constitutiveNRF2 activation include the following (i) somatic mutationsof KEAP1 or NRF2 (ii) epigenetic silencing of the KEAP1gene (iii) aberrant accumulation of proteins that competewith NRF2 for KEAP1 binding and (iv) oncogene-mediatedoverexpression of NRF2 [101 126] First somatic mutationsof KEAP1-NRF2 have been reported in an initial study byPadmanabhan et al [127] This study identified mutations

6 Oxidative Medicine and Cellular Longevity

in the KelchDGR domain of KEAP1 in lung cancer celllines as well as lung cancer tissue samples and demonstratedthat these mutant KEAP1 proteins lost their NRF2 repressivefunction which resulted in NRF2 accumulation Singh et alalso demonstrated that the KelchDGR and IVR domainsof the KEAP1 gene contain multiple somatic mutations andthese mutations were identified in 19 of tumor specimensfrom non-small cell lung cancer patients [123] In gallbladdercancer 4 of 13 patients harbored KEAP1 mutations [122]Shibata et al reported that NRF2 somatic mutations werefound in 107 of primary lung cancer patients and 272of primary head and neck cancer patients [122] Notablythesemutationswere primarily located in theDLG andETGEmotifs and eventually led to the loss of a proper interactionbetween the NRF2 protein and KEAP1 Second CpG islandhypermethylation in the KEAP1 promoter resulted in lowKEAP1 expression in lung cancer cell lines and tumor samples[124]Third in humanhepatocellular carcinoma p62 positivecellular aggregates were found with a frequency of 25 andmost of these tumors retained higher levels of NRF2 andits target gene expression [128] Fourth oncogenes have alsobeen shown to play a role in NRF2 signaling Oncogenicactivation of KRAS (KRASG12D) c-MYC (c-MYCERT12) andBRAF (BRAFV619E) elevates the transcript levels of NRF2 andits target gene expression [129]

It is now widely accepted that aberrant activation ofNRF2 can enhance cancer cell survival and growth in oxi-dizing tumor environments and further promote chemoradioresistance Indeed the prognosis of cancer patientsnegatively correlated with NRF2 levels in the tumor [122130] The favorable effect of NRF2 overexpression on tumorsurvival and growth can be attributed to the increase inNRF2 target antioxidant proteins and their counteractiveeffect on oxidative stress For instance GSH which is adirect target molecule of NRF2 has been shown to be criticalfor cell proliferation [131 132] In addition to its antioxi-dant contribution Mitsuishi et al provided direct evidencedemonstrating that NRF2 alters the cellular metabolism inrelation to anabolic pathways to accelerate cell proliferation[133] Multiple metabolic genes such as those involvedin the pentose phosphate pathway were upregulated byNRF2 through ARE and these changes promoted purinesynthesis glutamine metabolism and NADPH productionfor enhanced cell proliferation

Constitutively high levels of NRF2 have been associatedwith chemoresistance as well as radioresistance Cancer cellswith high NRF2 activity were less sensitive to cytotoxicchemotherapeutics such as cisplatin doxorubicin and 5-fluorouracil through facilitated detoxification of anticanceragents and enhanced antioxidant capacity [101] It is there-fore hypothesized that NRF2 inhibition can enhance thechemosensitivity of cancers NRF2 siRNA could suppresscancer resistance to cisplatin topoisomerase inhibitors and5-fluorouracil [101 122 134 135] Cancer cells with constitu-tively high NRF2 were protected against 120574-radiation inducedtoxicity Moreover siRNA-mediated inhibition of NRF2 innon-small cell lung cancer cell lines substantially enhancedradiosensitivity [136] Additionally NRF2 expression was

increased during the acquisition of chemoresistance In ourprevious study doxorubicin-selected ovarian cancer cellsdemonstrated increased expression of NRF2 and its targetgenes forGSH synthesis andNRF2 inhibition in this resistantcell line could restore doxorubicin sensitivity [137]

Our understanding of the role of NRF2 in cancer cell sig-naling has expanded In particular the relationship betweenoncogenic signaling and NRF2 is noteworthy As men-tioned earlier activation of oncogenes such as KRAS and c-MYC increased the expression of NRF2 presumably throughoncogene-mediated ROS increase and this phenomenonappears to contribute to the maintenance of reduced redoxhomeostasis in cancer cells [129] In ERBB2 (Her2Neu) over-expressing ovarian cancer cells the stable silencing of NRF2repressed ERBB2 expression and its downstream signalingand retarded tumor growth Therefore the inhibition ofNRF2 could sensitize these cells to taxol therapy by repressingERBB2 expression [138] Moreover NRF2 was shown to beassociated with HIF signaling which is a critical factor fortumor angiogenesis When NRF2 was stably knocked downin colon carcinoma cell lines hypoxia-inducible HIF-1120572accumulation was abrogated and consequently angiogenesisand tumor growth were significantly suppressed in NRF2knockdown tumors compared to the control group [139] Intype 2 papillary renal cell cancer which is characterized byloss of the fumarate hydratase gene and consequentmetabolicalteration accumulated fumarate was associated with tumorprogression via NRF2 signaling Fumarate was shown tomodify KEAP1 cysteine residues and elevate NRF2 levelswhich contributed to the growth and progression of type 2papillary renal cell cancer [140] These accumulating linesof evidence suggest that once cells are transformed to theneoplastic stage cancer cells utilize NRF2 signaling to adaptto the stressful tumor environment and to promote survivaland further cancer progression (Figure 2)

8 Involvement of NRF2 Signaling inStem Cell Quiescence and Differentiation

There is considerable evidence to suggest that NRF2 playsa role in normal stem cell biology [141ndash145] For exampleNRF2 activation in HSCs plays a critical role in not only themaintenance of quiescence but also in the determination ofdifferentiation fate [141 145] In Drosophila intestinal stemcells constitutive Nrf2 activation sustained quiescence byreducing the levels of ROS via upregulation of antioxidantgenes such as gclc However in the case of KEAP1-mediatedNrf2 repression high levels of intracellular ROS facilitatedan ablation of the quiescent state in intestinal stem cellsand age-related degeneration in the intestinal epithelium[146] Similarly low intracellular ROS levels are requiredfor the maintenance of quiescence in human airway basalstem cells (ABSCs) When exposed to exogenous ROSquiescent ABSCs enter the proliferation stage Changes inROS levels activate the NRF2-Notch pathway which resultsin self-renewal and protection of ABSCs from ROS-inducedhyperproliferation and senescence Moreover the quiescent

Oxidative Medicine and Cellular Longevity 7

NRF2 sMaf

GCLCGCLMNQO1SODs

Antioxidant enzymes Drug transportersMetabolic enzymes

ARE

Tumor growthprogression

MDRsMRPsBCRP

G6PDPGDME1

Cell survival Drug effluxProliferation

Target genes

Figure 2 Implications of NRF2 signaling in cancer NRF2 coordi-nates the expression of genes associated with cellular redox regu-lation metabolism and xenobiotic efflux and thereby its aberrantactivation promotes cancer cell survival proliferation and anti-cancer drug resistance GCLM glutamate cysteine ligase modifiersubunit GCLC glutamate cysteine ligase catalytic subunit NQO1NAD(P)Hquinone oxidoreductase 1 SODs superoxide dismutasesG6PDH glucose-6-phosphate dehydrogenase PGD phosphoglu-conate dehydrogenase ME1 malic enzyme 1 MDRs multidrugresistance proteinsMRPsmultidrug resistance-associated proteinsBCRP breast cancer resistance protein

state of ABSCs was maintained by NRF2 activation [147]In osteoclast progenitor cells hydrogen sulfide (H

2S) inhib-

ited human osteoclast differentiation by NRF2-dependentinduction of peroxiredoxin 1 and NQO1 These resultswere further confirmed using NRF2 activators includingsulforaphane and t-BHQ [148] It is also notable that NRF2participates in the regulation of cell fate determination ofHSCsMurakami et al demonstrated that HSCs derived fromKEAP1-deficientmice exhibited preferred differentiation intothe granulocyte-monocyte lineage rather than differentiatinginto the erythroid-lymphoid lineage [145]

Up to now numerous studies have demonstrated thatNRF2 plays a protective role against various stressors instem cells In neural stem cells overexpression of NRF2or pharmacological NRF2 activation prevented necroticcell death [149] In an animal study nrf2-deficient miceshowed defective stem cell function HSCs from nrf2minusminus miceexpressed lower levels of prosurvival cytokines and exhib-ited spontaneous apoptosis [150] Ionizing radiation-inducedmyelosuppression and mortality were mitigated throughNRF2-mediated Notch signaling activation in HSCs [142]Similarly resveratrol-inducedNRF2 expression improved thesurvival of cardiac stem cells and consequently regeneratedinfarcted myocardium [151 152] The heme oxygenase 1(HO-1) inducer cobalt protoporphyrin (CoPP) elicited anantiapoptotic effect on cardiac stem cells via activation of the

ERK-NRF2 pathway [153] In neural stem cells NRF2 activa-tion by melatonin or t-BHQ ameliorated lipopolysaccharide(LPS) orH

2O2induced cell death [149 154] In addition amy-

loid 120573-mediated neural stem cell death could be alleviated byexogenous NRF2 transduction which was accompanied byincreased expression of GCLC NQO-1 andHO-1This studyalso demonstrated that neuronal differentiation of neuralstem cells is enhanced by NRF2 activation [155] Similar toneural stem cells NRF2 has a protective role against hypoxicand oxidative stress conditions in undifferentiated MSCsTreatment of the murine mesenchymal stem cell line withadrenaline increased the mRNA expression of nrf2 gclc andxCT leading to an increase in GSH levels and the preventionof ROS-induced cytotoxicity [156 157]

9 Potential Implication of NRF2 inCSC Maintenance and Resistance

The role of NRF2 in CSC biology is now beginning tobe unveiled Similar to the case of normal stem cells itwas shown that NRF2 contributes to CSC stemness bymaintaining their self-renewal capacity and protecting themfrom chemoradiotherapy Achuthan et al established stablechemotherapy-resistant breast cancer cells and observed thatthese cells expressed higher levels of CD133 and OCT-4indicating that these cells exhibit CSC phenotype [34] Ofnote it was shown that ROS levels were relatively low inthese drug-selected cells presumably due to higher levelsof antioxidant enzymes such as SOD1 and GPX12 NRF2protein stabilizationwas associatedwith high levels of antiox-idant enzymes As an underlying molecular mechanismdiminished proteasome activity and increased p21 levelsappear to stabilize the NRF2 protein in these stem-like cellsEvidently p21 knockdown repressed the mammosphere-forming potential of these stem-like breast cancer cellsSimilarly a study by Zhu et al showed the involvement ofNRF2 in glioblastoma stem cells that were isolated fromhuman surgical glioblastoma specimens NRF2 knockdownin glioblastoma stem cells inhibited cell proliferation andneurosphere formation and further suppressed SOX2 expres-sion Moreover NRF2 knockdown changed the cell cycledistribution to the G2 phase and significantly attenuated thetumorigenecity of glioblastoma stem cells [158 159] Theseresults are providing evidence that NRF2 is necessary formaintenance of the self-renewal capacity of glioblastomastem cells

On the other hand activation of NRF2 signaling has beendemonstrated in different types of CSC models includinglung esophageal breast ovarian and colon CSCs and thisis closely correlated with themaintenance of low intracellularROS levels and chemoresistance of CSCs [160ndash165] In lungand esophageal cancer cells cigarette smoke condensateincreased the SP as well as BCRP expression which arehallmarks of CSCs [165] Promoter analysis revealed thatBCRP expression was associated with elevated levels ofNRF2 aryl hydrocarbon receptor (AhR) and specificityprotein 1 (SP1) Additionally this study demonstrated thatmithramycin diminished BCRP expression via repression of

8 Oxidative Medicine and Cellular Longevity

Low ROS

Increased drug efflux

SurvivalChemoresistance

CSC

TumorRecurrenceProgression

Radioresistance

PERKuarr

p21uarr

p62uarr

Proteasome darr

NRF2 uarr

Figure 3 Potential roles of NRF2 signaling in CSCs InCSC-like cellmodels NRF2 is activated through multiple molecular mechanismsin a context-dependent manner The upregulation of competingproteins such as p62 and p21 activation of PERK or repressedproteasome function were shown to enhance NRF2 activity inthese models Elevated NRF2 levels in CSCs can contribute tothe maintenance of low ROS by upregulating multiple antioxidantgenes In addition NRF2-mediated expression of ABC transporterselicits efflux of anticancer drugs from cancer cells Overall activatedNRF2 signaling facilitates CSCs survival and stress resistance andconsequently it can be suggested that CSCs with high NRF2 activityplay a crucial role in tumor recurrence and further progression

NRF2 AhR and SP1 and thereby inhibiting the expressionof genes associated with CSC-related pathways resulting inreduced proliferation and tumorigenecity Similarly it wasreported that lung cancer SP cells exhibited high levels ofNRF2 and BCRP expression These SP cells were highlytumorigenic and possessed self-renewal capacity comparedto non-SP cells [164] Emmink et al performed a proteomeanalysis on collected secretome from highly tumorigenicCSCs and their corresponding nontumorigenic differentiatedcells both of which were established from human colorectalspecimens [160] Subsequent bioinformatic analysis revealedthat the CSC secretome contained a large amount of proteinsassociated with cell survival and protein quality controlcompared to differentiated tumor cells Notably the CSCsecretome contained an NRF2 antioxidant and detoxifyingprotein signature in that it included elevated levels ofGCLC GPX23 and TXNRD1 This study provided novelevidence that CSCs secrete NRF2 target antioxidant proteinsto counteract extracellular stressors and chemotherapeuticsIn patients with ovarian clear cell carcinoma the expressionof the CSC maker aldehyde dehydrogenase-1 (ALDH1) wasstrongly correlated with an advanced clinical stage andreduced progression free survival [161] Ovarian clear cellcarcinoma cells with high ALDH1 expressionmaintained lowlevels of ROS compared to ALDH-low cells and these cellswere shown to express higher levels of NRF2 and its targetgenes

Two recent studies have demonstratedNRF2 activation insphere cultures of breast cancer cells that is one of models ofCSCs Wu et al showed that mammospheres derived fromMCF7 and MDA-MB231 breast cancer cell lines exhibitedlower ROS levels compared to their monolayer counterpartsThey also showed that levels of NRF2 and target genes such

as NQO1 and GCLM were elevated in mammospheres [163]Similarly our group has shown substantially elevated NRF2protein levels along with increased expression of antioxidantgenes (eg HO-1 and GPX2) and drug efflux transporters(eg MRP2 and BCRP) in sphere cultures of breast cancercells NRF2 accumulation was also observed in sphere-cultured ovarian and colon cancer cells However shRNA-mediated downregulation ofNRF2 led to decreased chemore-sistance of mammospheres presumably due to reduced levelsof antioxidant genes and drug transporters High ROS levelsin NRF2 knockdown mammospheres caused sphere growthretardation and apoptosis Coherently ablation of ABCtransporter induction in NRF2 knockdown mammospheressensitized to anticancer agents [162] Additionally this studyprovided evidence that increased NRF2 protein expression inmammospheres can be linked to 26S proteasome reductionand p62 accumulation In particular knockdown of p62 inMCF7 mammospheres significantly attenuated NRF2 eleva-tion

Surviving dedifferentiated breast cancer cells after chem-otherapy treatment retained high levels of NRF2 activationsimilar to other CSCs However NRF2 activation was medi-ated by a noncanonical pathway Levels of PERKwere high indedifferentiated cancer cells and this in turn phosphorylatedand activated NRF2 signaling to maintain low cellular ROSlevels and to express ABC transporters In agreement withthese findings clinical observations revealed that the PERKpathway gene signature is related to chemoresistance andreduced patient survival [166]

10 Concluding Remarks

Recent studies have started to uncover the role of ROS signal-ing in the biology of CSCs which is related to tumorigenecitytumor progression and relapse Expression of the transcrip-tion factor NRF2 a master regulator of antioxidant genesexpression is increased in different models of CSCs and thiselevation is likely to promote CSC maintenance and survivalin an oxidizing tumormicroenvironment In additionNRF2-mediated overexpression of ABC transporters particularlythe CSC marker BCRP may play a critical role in themultidrug resistance of CSCsThese findings combined withthe increasing evidence showing the alteration of KEAP1-NRF2 signaling in cancer cells suggest a novel role of NRF2in CSC maintenance and survival (Figure 3)

One important question that arises from the current stud-ies is whether it is possible to design CSC-targeted therapiesthrough regulation of theNRF2 pathway and its related redoxhomeostasis inCSCs Recent studies provide several potentialclues for addressing this question the naturally occurringalkaloid brusatol could reduce the growth and chemoresis-tance of breast CSCs [163] Treatment ofmammospheres withbrusatol elevated ROS levels and promoted taxol-inducedgrowth retardation and cell death The NRF2 repressivemechanism of brusatol has not been clearly elucidated but itappears to be independent of KEAP1-mediated degradation[167] In addition to brusatol natural compounds such as

Oxidative Medicine and Cellular Longevity 9

chrysin apigenin luteolin and trigonelline are known toinhibit NRF2 signaling in several types of cancer cells [102168ndash170] and therefore the development of NRF2 inhibitorswith characterized modes of action will enable efficient tar-geting of the redox homeostasis system as well as multidrugresistance systems in CSCs

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This studywas financially supported by theNational ResearchFoundation (NRF) funded by the Ministry of Science ICT ampFuture Planning (NRF-2013R1A2A2A01015497)

References

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[2] P Storz ldquoForkhead homeobox type O transcription factorsin the responses to oxidative stressrdquo Antioxidants amp RedoxSignaling vol 14 no 4 pp 593ndash605 2011

[3] R S Balaban S Nemoto and T Finkel ldquoMitochondria oxi-dants and agingrdquo Cell vol 120 no 4 pp 483ndash495 2005

[4] L A Sena and N S Chandel ldquoPhysiological roles of mitochon-drial reactive oxygen speciesrdquoMolecular Cell vol 48 no 2 pp158ndash167 2012

[5] P T Schumacker ldquoReactive oxygen species in cancer cells liveby the sword die by the swordrdquo Cancer Cell vol 10 no 3 pp175ndash176 2006

[6] S Toyokuni K Okamoto J Yodoi and H Hiai ldquoPersistentoxidative stress in cancerrdquo FEBS Letters vol 358 no 1 pp 1ndash3 1995

[7] L Behrend G Henderson and R Zwacka ldquoReactive oxy-gen species in oncogenic transformationrdquo Biochemical SocietyTransactions vol 31 no 6 pp 1441ndash1444 2003

[8] E O Hileman J Liu M Albitar M J Keating and P HuangldquoIntrinsic oxidative stress in cancer cells a biochemical basis fortherapeutic selectivityrdquo Cancer Chemotherapy and Pharmacol-ogy vol 53 no 3 pp 209ndash219 2004

[9] H PelicanoD Carney and PHuang ldquoROS stress in cancer cellsand therapeutic implicationsrdquo Drug Resistance Updates vol 7no 2 pp 97ndash110 2004

[10] Y Adachi Y Shibai J Mitsushita W H Shang K Hirose andT Kamata ldquoOncogenic Ras upregulates NADPH oxidase 1 geneexpression through MEK-ERK-dependent phosphorylation ofGATA-6rdquo Oncogene vol 27 no 36 pp 4921ndash4932 2008

[11] J Mitsushita J D Lambeth and T Kamata ldquoThe superoxide-generating oxidase Nox1 is functionally required for Ras onco-gene transformationrdquoCancer Research vol 64 no 10 pp 3580ndash3585 2004

[12] O Vafa M Wade S Kern et al ldquoc-Myc can induce DNAdamage increase reactive oxygen species and mitigate p53function a mechanism for oncogene-induced genetic instabil-ityrdquoMolecular Cell vol 9 no 5 pp 1031ndash1044 2002

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10 Oxidative Medicine and Cellular Longevity

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[40] K Wang T Zhang Q Dong E C Nice C Huang and Y WeildquoRedox homeostasis the linchpin in stem cell self-renewal anddifferentiationrdquo Cell Death and Disease vol 4 no 3 p e5372013

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[43] S Ghaffari ldquoOxidative stress in the regulation of normal andneoplastic hematopoiesisrdquo Antioxidants amp Redox Signaling vol10 no 11 pp 1923ndash1940 2008

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[46] C Cosentino D Grieco and V Costanzo ldquoATM activates thepentose phosphate pathway promoting anti-oxidant defenceand DNA repairrdquo The EMBO Journal vol 30 no 3 pp 546ndash555 2011

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[49] Y Wang Y Zhou and D T Graves ldquoFOXO transcription fac-tors their clinical significance and regulationrdquoBioMed ResearchInternational vol 2014 Article ID 925350 13 pages 2014

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[51] K Miyamoto K Y Araki K Naka et al ldquoFoxo3a is essentialfor maintenance of the hematopoietic stem cell poolrdquo Cell StemCell vol 1 no 1 pp 101ndash112 2007

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[54] J Paik Z Ding R Narurkar et al ldquoFoxOs cooperatively regu-late diverse pathways governing neural stem cell homeostasisrdquoCell Stem Cell vol 5 no 5 pp 540ndash553 2009

[55] V M Renault V A Rafalski A A Morgan et al ldquoFoxO3regulates neural stem cell homeostasisrdquo Cell Stem Cell vol 5no 5 pp 527ndash539 2009

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[57] A Brunet F Kanai J Stehn and et al ldquo14-3-3 transits tothe nucleus and participates in dynamic nucleocytoplasmictransportrdquo Journal of Cell Biology vol 156 no 5 pp 817ndash8282002

[58] M M Juntilla V D Patil M Calamito R P Joshi M JBirnbaum and G A Koretzky ldquoAKT1 and AKT2 maintainhematopoietic stem cell function by regulating reactive oxygenspeciesrdquo Blood vol 115 no 20 pp 4030ndash4038 2010

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stem cells through Wnt120573-catenin signallingrdquo Nature Cell Biol-ogy vol 12 no 10 pp 1007ndash1013 2010

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[64] K Takubo N Goda W Yamada et al ldquoRegulation of the HIF-1alpha level is essential for hematopoietic stem cellsrdquo Cell StemCell vol 7 no 3 pp 391ndash402 2010

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[69] S Kim C Kwon and I Nakano ldquoDetoxification of oxidativestress in glioma stem cells mechanism clinical relevance andtherapeutic developmentrdquo Journal of Neuroscience Research vol92 no 11 pp 1419ndash1424 2014

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[72] N Haraguchi H Ishii K Mimori et al ldquoCD13 is a therapeutictarget in human liver cancer stem cellsrdquo Journal of ClinicalInvestigation vol 120 no 9 pp 3326ndash3339 2010

[73] H M Kim N Haraguchi H Ishii et al ldquoIncreased CD13expression reduces reactive oxygen species promoting sur-vival of liver cancer stem cells via an epithelial-mesenchymaltransition-like phenomenonrdquo Annals of Surgical Oncology vol19 supplement 3 pp S539ndashS548 2012

[74] K Naka T Hoshii T Muraguchi et al ldquoTGF-120573ndashFOXO sig-nalling maintains leukaemia-initiating cells in chronic myeloidleukaemiardquo Nature vol 463 no 7281 pp 676ndash680 2010

[75] J Zhou J Wulfkuhle H Zhang et al ldquoActivation of thePTENmTORSTAT3 pathway in breast cancer stem-like cellsis required for viability and maintenancerdquo Proceedings of theNational Academy of Sciences vol 104 no 41 pp 16158ndash161632007

[76] A Dubrovska S Kim R J Salamone et al ldquoThe role ofPTENAktPI3K signaling in the maintenance and viability ofprostate cancer stem-like cell populationsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 106 no 1 pp 268ndash273 2009

[77] H Yin and J Glass ldquoThe phenotypic radiation resistance ofCS44+CD24minusor low breast cancer cells is mediated through theenhanced activation of ATM signalingrdquo PLoS ONE vol 6 no9 Article ID e24080 2011

[78] T H Rushmore and C Pickett ldquoTranscriptional regulation ofthe rat glutathione S-transferase Ya subunit gene Characteri-zation of a xenobiotic-responsive element controlling inducibleexpression by phenolic antioxidantsrdquo The Journal of BiologicalChemistry vol 265 no 24 pp 14648ndash14653 1990

[79] Y Li and A Jaiswal ldquoRegulation of humanNAD (P)H quinoneoxidoreductase gene Role of AP1 binding site contained withinhuman antioxidant response elementrdquoThe Journal of BiologicalChemistry vol 267 no 21 pp 15097ndash15104 1992

[80] T Prestera P Talalay J Alam et al ldquoParallel induction of hemeoxygenase-1 and chemoprotective phase 2 enzymes by elec-trophiles and antioxidants regulation by upstream antioxidant-responsive elements (ARE)rdquoMolecular Medicine vol 1 no 7 p827 1995

[81] K Itoh K IgarashiNHayashi et al ldquoCloning and characteriza-tion of a novel erythroid cell-derived CNC family transcriptionfactor heterodimerizing with the small Maf family proteinsrdquoMolecular and Cellular Biology vol 15 no 8 pp 4184ndash41931995

[82] R Venugopal and A K Jaiswal ldquoNrf1 and Nrf2 positivelyand c-Fos and Fra1 negatively regulate the human antioxidantresponse element-mediated expression of NAD(P)Hquinoneoxidoreductase1 generdquo Proceedings of the National Academy ofSciences vol 93 no 25 pp 14960ndash14965 1996

[83] K Itoh T Chiba S Takahashi et al ldquoAn Nrf2small Mafheterodimer mediates the induction of phase II detoxifyingenzyme genes through antioxidant response elementsrdquo Bio-chemical and Biophysical Research Communications vol 236no 2 pp 313ndash322 1997

[84] M Ramos-Gomez M-K Kwak P M Dolan et al ldquoSensitivityto carcinogenesis is increased and chemoprotective efficacy ofenzyme inducers is lost in nrf2 transcription factor-deficientmicerdquo Proceedings of the National Academy of Sciences vol 98no 6 pp 3410ndash3415 2001

[85] M-K Kwak N Wakabayashi K Itoh H Motohashi MYamamoto and T W Kensler ldquoModulation of gene expressionby cancer chemopreventive dithiolethiones through the Keap1-Nrf2 pathway Identification of novel gene clusters for cellsurvivalrdquo The Journal of Biological Chemistry vol 278 no 10pp 8135ndash8145 2003

[86] R Hu C Xu G Shen et al ldquoGene expression profiles inducedby cancer chemopreventive isothiocyanate sulforaphane in theliver of C57BL6J mice and C57BL6JNrf2 (minusminus) micerdquo CancerLetters vol 243 no 2 pp 170ndash192 2006

[87] S Chanas Q Jiang and M McMahon ldquoLoss of the Nrf2transcription factor causes a marked reduction in constitutiveand inducible expression of the glutathione S-transferase Gsta1Gsta2 Gstm1 Gstm2 Gstm3 and Gstm4 genes in the livers ofmale and female micerdquo Biochemical Journal vol 365 no 2 pp405ndash416 2002

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[89] G Shen C Xu R Hu et al ldquoModulation of nuclear factorE2-related factor 2-mediated gene expression in mice liver andsmall intestine by cancer chemopreventive agent curcuminrdquoMolecular Cancer Therapeutics vol 5 no 1 pp 39ndash51 2006

[90] J D Hayes and A T Dinkova-Kostova ldquoThe Nrf2 regulatorynetwork provides an interface between redox and intermediarymetabolismrdquo Trends in Biochemical Sciences vol 39 no 4 pp199ndash218 2014

[91] A Hayashi H Suzuki K Itoh M Yamamoto and Y SugiyamaldquoTranscription factor Nrf2 is required for the constitutive andinducible expression of multidrug resistance-associated protein1 in mouse embryo fibroblastsrdquo Biochemical and BiophysicalResearch Communications vol 310 no 3 pp 824ndash829 2003

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12 Oxidative Medicine and Cellular Longevity

drug-sensing receptors in primary human hepatocytesrdquo DrugMetabolism and Disposition vol 34 no 10 pp 1756ndash1763 2006

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[94] H Jeong I Ryoo andMKwak ldquoRegulation of the expression ofrenal drug transporters in KEAP1-knockdown human tubularcellsrdquo Toxicology in Vitro vol 29 no 5 pp 884ndash892 2015

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3generdquo Drug Metabolism and

Disposition vol 43 no 1 pp 93ndash99 2014[96] A Singh H Wu P Zhang C Happel J Ma and S Biswal

ldquoExpression of ABCG2 (BCRP) is regulated by Nrf2 in cancercells that confers side population and chemoresistance pheno-typerdquo Molecular Cancer Therapeutics vol 9 no 8 pp 2365ndash2376 2010

[97] N Wakabayashi K Itoh J Wakabayashi et al ldquoKeap1-nullmutation leads to postnatal lethality due to constitutive Nrf2activationrdquo Nature Genetics vol 35 no 3 pp 238ndash245 2003

[98] S B Cullinan J D Gordan J Jin J W Harper and J A DiehlldquoThe Keap1-BTB protein is an adaptor that bridges Nrf2 to aCul3-based E3 ligase oxidative stress sensing by a Cul3-Keap1ligaserdquoMolecular and Cellular Biology vol 24 no 19 pp 8477ndash8486 2004

[99] D D Zhang and M Hannink ldquoDistinct cysteine residues inkeap1 are required for keap1-dependent ubiquitination of nrf2and for stabilization of nrf2 by chemopreventive agents andoxidative stressrdquoMolecular and Cellular Biology vol 23 no 22pp 8137ndash8151 2003

[100] L M Zipper and R T Mulcahy ldquoThe Keap1 BTBPOZ dimer-ization function is required to sequester Nrf2 in cytoplasmrdquoThe Journal of Biological Chemistry vol 277 no 39 pp 36544ndash36552 2002

[101] M C Jaramillo and D D Zhang ldquoThe emerging role of theNrf2-Keap1 signaling pathway in cancerrdquoGenesampDevelopmentvol 27 no 20 pp 2179ndash2191 2013

[102] K I Tong Y Katoh H Kusunoki K Itoh T Tanaka and MYamamoto ldquoKeap1 recruits Neh2 through binding to ETGEand DLG motifs characterization of the two-site molecularrecognition modelrdquoMolecular and Cellular Biology vol 26 no8 pp 2887ndash2900 2006

[103] K I Tong A Kobayashi F Katsuoka andM Yamamoto ldquoTwo-site substrate recognition model for the Keap1-Nrf2 system ahinge and latch mechanismrdquo Biological Chemistry vol 387 no10-11 pp 1311ndash1320 2006

[104] K I Tong B Padmanabhan A Kobayashi et al ldquoDifferentelectrostatic potentials define ETGE and DLG motifs as hingeand latch in oxidative stress responserdquo Molecular and CellularBiology vol 27 no 21 pp 7511ndash7521 2007

[105] M Kobayashi L Li N Iwamoto et al ldquoThe antioxidant defensesystemKeap1-Nrf2 comprises amultiple sensingmechanism forresponding to a wide range of chemical compoundsrdquoMolecularand Cellular Biology vol 29 no 2 pp 493ndash502 2009

[106] T Yamamoto T Suzuki A Kobayashi et al ldquoPhysiological sig-nificance of reactive cysteine residues of Keap1 in determiningNrf2 activityrdquoMolecular and Cellular Biology vol 28 no 8 pp2758ndash2770 2008

[107] M K Kwak K Itoh M Yamamoto and T W KenslerldquoEnhanced expression of the transcription factorNrf2 by cancer

chemopreventive agents role of antioxidant response element-like sequences in the nrf2 promoterrdquo Molecular and CellularBiology vol 22 no 9 pp 2883ndash2892 2002

[108] T Suzuki T Shibata K Takaya et al ldquoRegulatory nexus ofsynthesis and degradation deciphers cellular Nrf2 expressionlevelsrdquoMolecular and Cellular Biology vol 33 no 12 pp 2402ndash2412 2013

[109] S B Cullinan D Zhang M Hannink E Arvisais R JKaufman and J A Diehl ldquoNrf2 is a direct PERK substrateand effector of PERK-dependent cell survivalrdquo Molecular andCellular Biology vol 23 no 20 pp 7198ndash7209 2003

[110] W Li and A Kong ldquoMolecular mechanisms of Nrf2-mediatedantioxidant responserdquo Molecular Carcinogenesis vol 48 no 2pp 91ndash104 2009

[111] Y J Surh ldquoCancer chemoprevention with dietary phytochemi-calsrdquo Nature Reviews Cancer vol 3 no 10 pp 768ndash780 2003

[112] M Salazar A I Rojo D Velasco R M de Sagarra and ACuadrado ldquoGlycogen synthase kinase-3beta inhibits the xeno-biotic and antioxidant cell response by direct phosphorylationand nuclear exclusion of the transcription factor Nrf2rdquo Journalof Biological Chemistry vol 281 no 21 pp 14841ndash14851 2006

[113] W Chen Z Sun X J Wang et al ldquoDirect interactionbetween Nrf2 and p21(Cip1WAF1) upregulates the Nrf2-mediated antioxidant responserdquo Molecular Cell vol 34 no 6pp 663ndash673 2009

[114] M Komatsu H Kurokawa S Waguri et al ldquoThe selectiveautophagy substrate p62 activates the stress responsive tran-scription factor Nrf2 through inactivation of Keap1rdquoNature CellBiology vol 12 no 3 pp 213ndash223 2010

[115] J D Hayes M McMahon S Chowdhry and A T Dinkova-Kostova ldquoCancer chemoprevention mechanisms mediatedthrough the Keap1-Nrf2 pathwayrdquoAntioxidants amp Redox Signal-ing vol 13 no 11 pp 1713ndash1748 2010

[116] R Hu C L Saw R Yu and A T Kong ldquoRegulation ofNF-E2-related factor 2 signaling for cancer chemopreventionantioxidant coupled with antiinflammatoryrdquo Antioxidants ampRedox Signaling vol 13 no 11 pp 1679ndash1698 2010

[117] M Kwak and T W Kensler ldquoTargeting NRF2 signaling for can-cer chemopreventionrdquo Toxicology and Applied Pharmacologyvol 244 no 1 pp 66ndash76 2010

[118] K Iida K Itoh Y Kumagai et al ldquoNrf2 is essential for thechemopreventive efficacy of oltipraz against urinary bladdercarcinogenesisrdquo Cancer Research vol 64 no 18 pp 6424ndash64312004

[119] T Jiang N Chen F Zhao et al ldquoHigh levels of Nrf2 deter-mine chemoresistance in type II endometrial cancerrdquo CancerResearch vol 70 no 13 pp 5486ndash5496 2010

[120] Y R Kim J EOhM S Kim et al ldquoOncogenicNRF2mutationsin squamous cell carcinomas of oesophagus and skinrdquo TheJournal of Pathology vol 220 no 4 pp 446ndash451 2010

[121] T Shibata A Kokubu M Gotoh et al ldquoGenetic alterationof Keap1 confers constitutive Nrf2 activation and resistance tochemotherapy in gallbladder cancerrdquoGastroenterology vol 135no 4 pp 1358e4ndash1368e4 2008

[122] T Shibata T Ohta K I Tong et al ldquoCancer related mutationsin NRF2 impair its recognition by Keap1-Cul3 E3 ligase andpromote malignancyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 36 pp13568ndash13573 2008

[123] A Singh V Misra R K Thimmulappa et al ldquoDysfunctionalKEAP1-NRF2 interaction in non-small-cell lung cancerrdquo PLoSMedicine vol 3 no 10 article e420 2006

Oxidative Medicine and Cellular Longevity 13

[124] R Wang J An F Ji H Jiao H Sun and D Zhou ldquoHyper-methylation of the Keap1 gene in human lung cancer cell linesand lung cancer tissuesrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 1 pp 151ndash154 2008

[125] P Zhang A Singh S Yegnasubramanian et al ldquoLoss ofKelch-like ECH-associated protein 1 function in prostate cancercells causes chemoresistance and radioresistance and promotestumor growthrdquoMolecular Cancer Therapeutics vol 9 no 2 pp336ndash346 2010

[126] J D Hayes and M McMahon ldquoNRF2 and KEAP1 mutationspermanent activation of an adaptive response in cancerrdquo Trendsin Biochemical Sciences vol 34 no 4 pp 176ndash188 2009

[127] B Padmanabhan K I Tong T Ohta et al ldquoStructural basis fordefects of Keap1 activity provoked by its pointmutations in lungcancerrdquoMolecular Cell vol 21 no 5 pp 689ndash700 2006

[128] Y Inami S Waguri A Sakamoto et al ldquoPersistent activation ofNrf2 throughp62 in hepatocellular carcinoma cellsrdquoTheJournalof Cell Biology vol 193 no 2 pp 275ndash284 2011

[129] G M DeNicola F A Karreth T J Humpton et al ldquoOncogene-induced Nrf2 transcription promotes ROS detoxification andtumorigenesisrdquo Nature vol 475 no 7354 pp 106ndash109 2011

[130] L M Solis C Behrens W Dong et al ldquoNrf2 and Keap1abnormalities in non-small cell lung carcinoma and associationwith clinicopathologic featuresrdquo Clinical Cancer Research vol16 no 14 pp 3743ndash3753 2010

[131] N M Reddy S R Kleeberger H Y Cho et al ldquoDeficiency inNrf2-GSH signaling impairs type II cell growth and enhancessensitivity to oxidantsrdquoAmerican Journal of Respiratory Cell andMolecular Biology vol 37 no 1 pp 3ndash8 2007

[132] N M Reddy S R Kleeberger M Yamamoto et al ldquoGeneticdissection of the Nrf2-dependent redox signaling-regulatedtranscriptional programs of cell proliferation and cytoprotec-tionrdquo Physiological Genomics vol 32 no 1 pp 74ndash81 2007

[133] Y Mitsuishi K Taguchi Y Kawatani et al ldquoNrf2 redirectsglucose and glutamine into anabolic pathways in metabolicreprogrammingrdquo Cancer Cell vol 22 no 1 pp 66ndash79 2012

[134] J Cho SManandharH LeeH Park andMKwak ldquoRole of theNrf2-antioxidant system in cytotoxicity mediated by anticancercisplatin implication to cancer cell resistancerdquo Cancer Lettersvol 260 no 1-2 pp 96ndash108 2008

[135] T Ohta K Iijima M Miyamoto et al ldquoLoss of Keap1 functionactivates Nrf2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008

[136] A Singh M Bodas N Wakabayashi F Bunz and S BiswalldquoGain of Nrf2 function in non-small-cell lung cancer cellsconfers radioresistancerdquoAntioxidantsampRedox Signaling vol 13no 11 pp 1627ndash1637 2010

[137] G-S Shim S Manandhar D-H Shin T Kim and M KwakldquoAcquisition of doxorubicin resistance in ovarian carcinomacells accompanies activation of the NRF2 pathwayrdquo Free RadicalBiology and Medicine vol 47 no 11 pp 1619ndash1631 2009

[138] S Manandhar B Choi K Jung et al ldquoNRF2 inhibitionrepresses ErbB2 signaling in ovarian carcinoma cells implica-tions for tumor growth retardation and docetaxel sensitivityrdquoFree Radical Biology and Medicine vol 52 no 9 pp 1773ndash17852012

[139] T Kim E-g Hur S J Kang et al ldquoNRF2 blockade suppressescolon tumor angiogenesis by inhibiting hypoxia-induced acti-vation ofHIF-1120572rdquoCancer Research vol 71 no 6 pp 2260ndash22752011

[140] L Kinch N V Grishin and J Brugarolas ldquoSuccination of Keap1and activation of Nrf2-dependent antioxidant pathways in FH-deficient papillary renal cell carcinoma type 2rdquo Cancer Cell vol20 no 4 pp 418ndash420 2011

[141] J J Tsai J A Dudakov K Takahashi et al ldquoNrf2 regulateshaematopoietic stem cell functionrdquo Nature Cell Biology vol 15no 3 pp 309ndash316 2013

[142] J H Kim R KThimmulappa V Kumar et al ldquoNRF2-mediatedNotch pathway activation enhances hematopoietic reconsti-tution following myelosuppressive radiationrdquo The Journal ofClinical Investigation vol 124 no 2 pp 730ndash741 2014

[143] J P Chute ldquoNRF2 mitigates radiation-induced hematopoieticdeathrdquo The Journal of Clinical Investigation vol 124 no 3 pp960ndash961 2014

[144] J Jang Y Wang H Kim M A Lalli and K S KosikldquoNrf2 a regulator of the proteasome controls self-renewal andpluripotency in human embryonic stem cellsrdquo Stem Cells vol32 no 10 pp 2616ndash2625 2014

[145] S Murakami R Shimizu P Romeo M Yamamoto and HMotohashi ldquoKeap1-Nrf2 system regulates cell fate determina-tion of hematopoietic stem cellsrdquo Genes to Cells vol 19 no 3pp 239ndash253 2014

[146] C E Hochmuth B Biteau D Bohmann and H Jasper ldquoRedoxregulation by Keap1 and Nrf2 controls intestinal stem cellproliferation inDrosophilardquoCell Stem Cell vol 8 no 2 pp 188ndash199 2011

[147] M K Paul B Bisht D O Darmawan et al ldquoDynamic changesin intracellular ROS levels regulate airway basal stem cellhomeostasis through Nrf2-dependent notch signalingrdquo CellStem Cell vol 15 no 2 pp 199ndash214 2014

[148] L Gambari G Lisignoli L Cattini C Manferdini A Facchiniand F Grassi ldquoSodium hydrosulfide inhibits the differentiationof osteoclast progenitor cells via NRF2-dependent mechanismrdquoPharmacological Research vol 87 pp 99ndash112 2014

[149] J Li D Johnson M Calkins L Wright C Svendsen andJ Johnson ldquoStabilization of Nrf2 by tBHQ confers protectionagainst oxidative stress-induced cell death in human neuralstem cellsrdquo Toxicological Sciences vol 83 no 2 pp 313ndash3282005

[150] A AMerchant A SinghWMatsui and S Biswal ldquoThe redox-sensitive transcription factor Nrf2 regulatesmurine hematopoi-etic stem cell survival independently of ROS levelsrdquo Blood vol118 no 25 pp 6572ndash6579 2011

[151] N Gurusamy D Ray I Lekli and D K Das ldquoRed wineantioxidant resveratrol-modified cardiac stem cells regener-ate infarcted myocardiumrdquo Journal of Cellular and MolecularMedicine vol 14 no 9 pp 2235ndash2239 2010

[152] N Gorbunov G Petrovski N Gurusamy D Ray D H Kimand D K Das ldquoRegeneration of infarcted myocardium withresveratrol-modified cardiac stem cellsrdquo Journal of Cellular andMolecular Medicine vol 16 no 1 pp 174ndash184 2012

[153] C Cai L Teng D Vu et al ldquoThe heme oxygenase 1 inducer(CoPP) protects human cardiac stem cells against apoptosisthrough activation of the extracellular signal-regulated kinase(ERK)NRF2 signaling pathway and cytokine releaserdquo TheJournal of Biological Chemistry vol 287 no 40 pp 33720ndash33732 2012

[154] J Song S M Kang K M Lee and J E Lee ldquoThe protectiveeffect of melatonin on neural stem cell against LPS-inducedinflammationrdquo BioMed Research International vol 2015 ArticleID 854359 13 pages 2015

14 Oxidative Medicine and Cellular Longevity

[155] V Karkkainen Y Pomeshchik E Savchenko et al ldquoNrf2regulates neurogenesis and protects neural progenitor cellsagainst A120573 toxicityrdquo STEM CELLS vol 32 no 7 pp 1904ndash19162014

[156] M Mohammadzadeh R Halabian A Gharehbaghian et alldquoNrf-2 overexpression in mesenchymal stem cells reducesoxidative stress-induced apoptosis and cytotoxicityrdquo Cell Stressand Chaperones vol 17 no 5 pp 553ndash565 2012

[157] Y Takahata T Takarada M Iemata et al ldquoFunctional expres-sion of 1205732 adrenergic receptors responsible for protectionagainst oxidative stress through promotion of glutathione syn-thesis after Nrf2 upregulation in undifferentiated mesenchymalC3H10T12 stem cellsrdquo Journal of Cellular Physiology vol 218no 2 pp 268ndash275 2009

[158] J Zhu H Wang Y Fan et al ldquoKnockdown of nuclear factorerythroid 2-related factor 2 by lentivirus induces differentiationof glioma stem-like cellsrdquo Oncology Reports vol 32 no 3 pp1170ndash1178 2014

[159] J Zhu H Wang Q Sun et al ldquoNrf2 is required to maintain theself-renewal of glioma stem cellsrdquo BMC Cancer vol 13 no 1article 380 2013

[160] B L Emmink A Verheem W J Van Houdt et al ldquoThe secre-tome of colon cancer stem cells contains drug-metabolizingenzymesrdquo Journal of Proteomics vol 91 pp 84ndash96 2013

[161] T Mizuno N Suzuki H Makino et al ldquoCancer stem-like cellsof ovarian clear cell carcinoma are enriched in the ALDH-highpopulation associated with an accelerated scavenging system inreactive oxygen speciesrdquo Gynecologic Oncology vol 137 no 2pp 299ndash305 2015

[162] I-G Ryoo B-H Choi and M-K Kwak ldquoActivation of NRF2by p62 and proteasome reduction in sphere-forming breastcarcinoma cellsrdquo Oncotarget vol 6 no 10 pp 8167ndash8184 2015

[163] T Wu B G Harder P K Wong J E Lang and D D ZhangldquoOxidative stress mammospheres and Nrf2-new implicationfor breast cancer therapyrdquoMolecular Carcinogenesis 2014

[164] B Yang Y Ma and Y Liu ldquoElevated expression of Nrf-2 andABCG2 involved in multi-drug resistance of lung cancer SPcellsrdquo Drug Research 2015

[165] M Zhang A Mathur Y Zhang et al ldquoMithramycin repressesbasal and cigarette smoke-induced expression of abcg2 andinhibits stem cell signaling in lung and esophageal cancer cellsrdquoCancer Research vol 72 no 16 pp 4178ndash4192 2012

[166] C A Del Vecchio Y Feng E S Sokol et al ldquoDe-differentiationconfers multidrug resistance via noncanonical PERK-Nrf2signalingrdquo PLoS Biology vol 12 no 9 Article ID e1001945 2014

[167] D Ren N F Villeneuve T Jiang et al ldquoBrusatol enhancesthe efficacy of chemotherapy by inhibiting the Nrf2-mediateddefense mechanismrdquo Proceedings of the National Academy ofSciences of the United States of America vol 108 no 4 pp 1433ndash1438 2011

[168] A Arlt S Sebens S Krebs et al ldquoInhibition of the Nrf2 tran-scription factor by the alkaloid trigonelline renders pancreaticcancer cells more susceptible to apoptosis through decreasedproteasomal gene expression and proteasome activityrdquo Onco-gene vol 32 no 40 pp 4825ndash4835 2013

[169] A-M Gao Z-P Ke F Shi G Sun and H Chen ldquoChrysinenhances sensitivity of BEL-7402ADM cells to doxorubicin bysuppressing PI3KAktNrf2 and ERKNrf2 pathwayrdquo Chemico-Biological Interactions vol 206 no 1 pp 100ndash108 2013

[170] A M Gao Z P Ke J N Wang J Yang S Chen and HChen ldquoApigenin sensitizes doxorubicin-resistant hepatocellular

carcinoma BEL-7402ADM cells to doxorubicin via inhibitingPI3KAktNrf2 pathwayrdquo Carcinogenesis vol 34 no 8 pp1806ndash1814 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Oxidative Medicine and Cellular Longevity

CSCs appear to share several ROS-associated propertieswith normal stem cells CSCs are known to contain lowerlevels of ROS compared to non-CSCs CD44+CD24minuslowbreast cancer CSCs isolated from MDA-MB-231 and MCF7mammospheres were relatively more resistant to radiationand this was associated with lower levels of ROS afterradiation [66] Diehn et al observed that ROS levels inCD44+CD24minus breast cancer CSCs were lower than in non-CSCs and the expression levels of the modulatory subunit ofglutamate cysteine ligase (GCLM the rate-limiting enzymeof GSH synthesis) and FoxO1 were high Treatment of theCD44+CD24minus subpopulationwith buthionine sulphoximine(BSO) an inhibitor of GSH synthesis resulted in reducedcolony forming capacity and increased sensitivity to radiationtherapy through an increase in ROS [67] In a study usinghuman AML specimens leukemic stem cells with low levelsof ROS majorly contributed to stem cell quiescence bymaintaining a low rate of oxidative phosphorylation andmetabolism [31] A leukemia with a high amount of leukemicstem cells showed low levels of ROS and increased expressionof GPX3 compared to tumors with a low frequency ofleukemic stem cells This study demonstrated that GPX3levels positively correlated with poor prognostic outcome inAML patients [68] Glioma stem cells within the tumor masshave low levels of cellular ROS although they are located in ahypoxic environment A proposed molecular mechanism ofthis phenomenon was the significantly upregulated expres-sion of peroxiredoxin 4 (PRDX4) in glioma stem cells [69]

Evidence is indicating that low ROS levels in CSCsresult from the intrinsic characteristics of CSCs Cell surfacemarkers of CSCs including CD44 and CD13 are found tobe involved in ROS regulation Ishimoto et al demonstratedthat a variant isoform of CD44 (CD44v) can bind to thecystineglutamate exchange transporter xCT and activatescysteine uptake to enhance GSH synthesis in gastrointestinalCSCs [70] The expression of antioxidant genes such asGPX12 was significantly increased in CD44+ gastric tumorcells In addition knockdown of CD44 in mice led toROS increase p38MAPK activation and cellular senescencethat are related to p21 expression In a subsequent studythe same group demonstrated that the number of CD44+cells increased with neoadjuvant chemotherapy in head andneck squamous cell carcinoma (HNSCC) patients TheseCD44+ undifferentiated cancer cells displayed high xCTexpression GSH upregulation and low cellular ROS levelsAblation of xCT by siRNA or sulfasalazine treatment (xCT-mediated cystine transport inhibitor) induced differentiationof HNSCCCSCs both in vitro and in vivo [71] CD13 has beenidentified as a surface marker for liver CSCs In liver cancercell lines including Huh7 and PLCPRF5 CD13 positive cellspredominated the SP fraction and were mainly in the G0G1phase of the cell cycle Additionally resistance to anticancerdrugs or radiation in the CD13 positive cell fraction wasmuch higher than that observed in the CD13 negative cellfraction Direct comparison of ROS levels between the twocell fractions revealed that the CD13 positive cell fractioncontains lower levels of ROS and expresses higher levelsof GCLM [72] In another study by the same group CD13expression reduced transforming growth factor-120573 (TGF-120573)

induced ROS production and promoted survival of liverCSCs [73]

It has been demonstrated that signaling pathwaysinvolved in ROS regulation of normal stem cells also playa role in CSC biology The nuclear expression levels ofFoxO3a was high in chronic myeloid leukemia-initiatingcells and the transplantation of leukemic stem cells derivedfrom foxo3a knockout mice significantly reduced their abilityto cause myeloid leukemia in an animal model [74] Thisstudy also revealed that TGF-120573 is a crucial regulator ofFoxO3a activity In the SP of MCF-7 breast cancer cellsactivation of the PI3Kmammalian target rapamycin (mTOR)signaling pathway was important for tumorigenecity of theseCSCs and knockdown of PI3K or mTOR led to ablatedtumorigenecity [75] When CD133+CD44+ prostate cancercells were grown in sphere-forming conditions activatedPI3KAKT signaling was found to be critical for maintainingCSCs [76] CD44+CD24minusor low cells isolated from breastcancer cell lines and breast cancer patient specimens wereradioresistant and this resistant phenotype was associatedwith ATM signaling activation [77]

6 NRF2 as a Key Molecule forRedox Homeostasis

In 1990 Rushmore and Pickett discovered the enhancersequence in the rat gsta2 gene promoter as a response elementto 120573-naphthoflavone and t-butylhydroquinone (t-BHQ) andnamed it antioxidant responsive element (ARE) [78] Subse-quent studies revealed that ARE is commonly involved in thetranscription of multiple antioxidant and detoxifying genesincluding glutamate-cysteine ligase (GCL) glutathione S-transferase (GST) and NAD(P)H quinone oxidoreductase-1 (NQO-1) [79 80] Based on sequence homology betweenARE and MAF-recognition element (MRE) further studieshypothesized that small MAF and bZIP caprsquonrsquocollar (CNC)transcription factors may interact with ARE [81 82] AmongbZIP CNC transcription factors NRF2 was found to play acrucial role in ARE regulation in which inducible expressionof NQO1 and GST was ablated in t-butylhydroxy anisole-treated nrf2 null mice in contrast to the observation in wildtype mice [83] After this report numerous studies haveelucidated a wide spectrum of protective effects of NRF2signaling against various stressors For example sensitivityto benzo[a]pyrene-induced carcinogenesis was significantlygreater in nrf2-knockout mice than in wild type mice [84]

To account for the protective effects of Nrf2 comparativeanalyses of gene expression patterns were carried out innrf2-deficient and wild type mice following treatment withNrf2 activators In global gene analysis of dithiolethione-administered mouse livers Nrf2 was found to governthe expression of xenobiotic-detoxifying enzymes GSH-generating systems antioxidant proteins and the molec-ular chaperone-26S proteasome [85] Similarly Hu et aldemonstrated that detoxifying enzymes antioxidants drugtransporters stress response proteins and some signalingmolecules serve as Nrf2-dependent and isothiocyanate-inducible genes in mouse liver [86] It has now been firmly

Oxidative Medicine and Cellular Longevity 5

established thatNRF2 regulates divergent genes to coordinatexenobiotic detoxification and redox homeostasis [85 87ndash89]In its function as a regulator of cellular redox homeostasisNRF2 elevates the expression of GCL and the cysteinetransporter xCT to increase cellular GSH levels NRF2 alsoenhances regeneration of reduced GSH by upregulatingGPX and GSH reductase (GSR) Expression of thioredoxin1 (TXN1) thioredoxin reductase 1 (TXNRD1) and perox-iredoxin 16 which can reduce oxidized protein thiols isalso under the control of NRF2 In addition the levels ofNADPH a cofactor of many antioxidant enzymes such asGSR and TXNRD can be increased by NRF2 (reviewedin Hayes and Dinkova-Kostova [90]) The expression ofmultiple NADPH generating enzymes such as G6PDH and6-phosphogluconate dehydrogenase is upregulated by NRF2Additionally the role of NRF2 in ABC transporter expressionfor xenobiotic detoxification is notable The basal expressionlevel of Mrp1 was relatively lower in nrf2-deficient fibroblaststhan that in wild type fibroblasts and the treatment ofmice with Nrf2 activating diethyl maleate increased Mrp1expression in the liver [91] Levels of MDR1 MRP23 andBCRP were elevated following oltipraz treatment in pri-mary human hepatocytes [92] Sulforaphane (SFN) treatmentenhanced the levels ofMDR1 BCRP andMRP2 in the blood-brain barrier of rats [93] Our recent study showed thatgenetic activation of NRF2 via KEAP1 silencing increases theexpression ofMDR1MRP23 and BCRP in human proximaltubular epithelial cells [94] As direct molecular evidencefunctional AREs have been identified in human MRP3 [95]and BCRP genes [96]

Kelch-like ECH-associated protein 1 (KEAP1) a cysteine-rich actin-binding protein is the main negative regulatorof NRF2 activity [83 97] Under quiescent conditionsNRF2 remains inactive by forming a complex with KEAP1in the cytoplasm NRF2 is subject to ubiquitination andKEAP1-induced proteasomal degradation through the Cullin3 (CUL3) based E3 ligase KEAP1 has three major domainsas follows (i) The BTB domain is associated with KEAP1homodimerization (ii) the IVR domain plays a role inregulation ofKEAP1 activity and (iii) theKelchDGRdomainmediates binding with NRF2 [83 90 98ndash101] The bindingof NRF2 with KEAP1 has been described as the ldquohinge andlatchrdquo model where one molecule of NRF2 interacts with theKelchDGR domains of the KEAP1 dimer through conservedmotifs called ETGE (DN-X-E-TS-G-E) and DLG (L-X-X-Q-D-X-D-L-G) [102ndash104] In these reports it was shown thatthe binding affinity of the ETGE motif to KEAP1 is muchhigher than that of the DLG motif It has therefore beenshown that ldquolatchrdquo binding of the NRF2 DLG motif is easilybroken by modifications of KEAP1 cysteine residues by ROSor electrophiles In turn disrupted DLG binding of NRF2 toKEAP1 leads to the blockade of ubiquitination and furtherdegradation of NRF2 resulting in nuclear translocation ofNRF2 It has been demonstrated that the sulfhydryl groups ofmultiple cysteine residues of KEAP1 can be directly modifiedby oxidationreduction or alkylation In particular Cys151Cys273 and Cys288 were found to be essential for theregulation of NRF2 activity [99 104ndash106] Mutation of theCys273 or Cys288 residue of KEAP1 ablated its ability to

suppress NRF2 activity leading to accumulation of the NRF2protein [99 104]

In addition to KEAP1-mediated stability regulationNRF2 activity can be modulated at multiple steps First itis noticeable that NRF2 activity is regulated at the transcrip-tional step Functional AREs were identified in the murinenrf2 gene promoter and were involved in the autoregulationof NRF2 through transcriptional activation [107] Moreovera single nucleotide polymorphism in the ARE-like sequencesof the human NRF2 promoter was associated with increasedlung cancer susceptibility [108] Second it was shown thatNRF2 activity is regulated by posttranslationalmodificationsStudies indicate that NRF2 activation involves phosphoryla-tion signaling mediated by multiple kinase pathways such asMAPK protein kinase C (PKC) PI3K and protein kinaseRNA-like endoplasmic reticulum kinase (PERK) [109ndash111]Meanwhile glycogen synthase kinase-3 (GSK-3) a consti-tutively active serinethreonine kinase was found to inhibitNRF2 activity [112] Last NRF2 activity is increased byseveral intrinsic proteins such as p21 and p62 [113 114] Forexample p62 a linker protein of ubiquitinated proteins toautophagy degradation binds to the KEAP1 protein andinterferes with the binding of NRF2 to KEAP1 resulting inNRF2 stabilization

7 Emerging Role of NRF2 in Cancer Biology

Continuous or fatal stimuli such as toxic chemicals and excessROS disrupt cellular homeostasis causing macromoleculardamage and alterations in cell cycle and growth signalingwhich can eventually result in carcinogenesis The NRF2pathway deserved significant attention in the area of cancerbiology because numerous studies have demonstrated thatactivation of the NRF2 pathway decreases the sensitivity ofcells to carcinogens [115ndash117] For instance the burden ofgastric neoplasia caused by benzo[a]pyrene was effectivelyattenuated by the Nrf2 activator oltipraz in wild type micewhereas nrf2 knockout mice did not show any protectiveeffect of oltipraz [84] Similarly the incidence of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) induced urinary blad-der carcinomawas greater in nrf2 knockoutmice than in wildtype mice and oltipraz treatment reduced tumor incidenceonly in wild type mice [118]

Although it has been firmly confirmed that NRF2 activa-tion can protect cells against a wide range of toxicants andstressors aberrant activation of NRF2 has been associatedwith several types of cancers NRF2 levels were constitutivelyelevated in cancer cell lines and tumor samples of the lungbreast esophagus endometrial cancers and prostate cancers[119ndash125] Molecular mechanisms involved in constitutiveNRF2 activation include the following (i) somatic mutationsof KEAP1 or NRF2 (ii) epigenetic silencing of the KEAP1gene (iii) aberrant accumulation of proteins that competewith NRF2 for KEAP1 binding and (iv) oncogene-mediatedoverexpression of NRF2 [101 126] First somatic mutationsof KEAP1-NRF2 have been reported in an initial study byPadmanabhan et al [127] This study identified mutations

6 Oxidative Medicine and Cellular Longevity

in the KelchDGR domain of KEAP1 in lung cancer celllines as well as lung cancer tissue samples and demonstratedthat these mutant KEAP1 proteins lost their NRF2 repressivefunction which resulted in NRF2 accumulation Singh et alalso demonstrated that the KelchDGR and IVR domainsof the KEAP1 gene contain multiple somatic mutations andthese mutations were identified in 19 of tumor specimensfrom non-small cell lung cancer patients [123] In gallbladdercancer 4 of 13 patients harbored KEAP1 mutations [122]Shibata et al reported that NRF2 somatic mutations werefound in 107 of primary lung cancer patients and 272of primary head and neck cancer patients [122] Notablythesemutationswere primarily located in theDLG andETGEmotifs and eventually led to the loss of a proper interactionbetween the NRF2 protein and KEAP1 Second CpG islandhypermethylation in the KEAP1 promoter resulted in lowKEAP1 expression in lung cancer cell lines and tumor samples[124]Third in humanhepatocellular carcinoma p62 positivecellular aggregates were found with a frequency of 25 andmost of these tumors retained higher levels of NRF2 andits target gene expression [128] Fourth oncogenes have alsobeen shown to play a role in NRF2 signaling Oncogenicactivation of KRAS (KRASG12D) c-MYC (c-MYCERT12) andBRAF (BRAFV619E) elevates the transcript levels of NRF2 andits target gene expression [129]

It is now widely accepted that aberrant activation ofNRF2 can enhance cancer cell survival and growth in oxi-dizing tumor environments and further promote chemoradioresistance Indeed the prognosis of cancer patientsnegatively correlated with NRF2 levels in the tumor [122130] The favorable effect of NRF2 overexpression on tumorsurvival and growth can be attributed to the increase inNRF2 target antioxidant proteins and their counteractiveeffect on oxidative stress For instance GSH which is adirect target molecule of NRF2 has been shown to be criticalfor cell proliferation [131 132] In addition to its antioxi-dant contribution Mitsuishi et al provided direct evidencedemonstrating that NRF2 alters the cellular metabolism inrelation to anabolic pathways to accelerate cell proliferation[133] Multiple metabolic genes such as those involvedin the pentose phosphate pathway were upregulated byNRF2 through ARE and these changes promoted purinesynthesis glutamine metabolism and NADPH productionfor enhanced cell proliferation

Constitutively high levels of NRF2 have been associatedwith chemoresistance as well as radioresistance Cancer cellswith high NRF2 activity were less sensitive to cytotoxicchemotherapeutics such as cisplatin doxorubicin and 5-fluorouracil through facilitated detoxification of anticanceragents and enhanced antioxidant capacity [101] It is there-fore hypothesized that NRF2 inhibition can enhance thechemosensitivity of cancers NRF2 siRNA could suppresscancer resistance to cisplatin topoisomerase inhibitors and5-fluorouracil [101 122 134 135] Cancer cells with constitu-tively high NRF2 were protected against 120574-radiation inducedtoxicity Moreover siRNA-mediated inhibition of NRF2 innon-small cell lung cancer cell lines substantially enhancedradiosensitivity [136] Additionally NRF2 expression was

increased during the acquisition of chemoresistance In ourprevious study doxorubicin-selected ovarian cancer cellsdemonstrated increased expression of NRF2 and its targetgenes forGSH synthesis andNRF2 inhibition in this resistantcell line could restore doxorubicin sensitivity [137]

Our understanding of the role of NRF2 in cancer cell sig-naling has expanded In particular the relationship betweenoncogenic signaling and NRF2 is noteworthy As men-tioned earlier activation of oncogenes such as KRAS and c-MYC increased the expression of NRF2 presumably throughoncogene-mediated ROS increase and this phenomenonappears to contribute to the maintenance of reduced redoxhomeostasis in cancer cells [129] In ERBB2 (Her2Neu) over-expressing ovarian cancer cells the stable silencing of NRF2repressed ERBB2 expression and its downstream signalingand retarded tumor growth Therefore the inhibition ofNRF2 could sensitize these cells to taxol therapy by repressingERBB2 expression [138] Moreover NRF2 was shown to beassociated with HIF signaling which is a critical factor fortumor angiogenesis When NRF2 was stably knocked downin colon carcinoma cell lines hypoxia-inducible HIF-1120572accumulation was abrogated and consequently angiogenesisand tumor growth were significantly suppressed in NRF2knockdown tumors compared to the control group [139] Intype 2 papillary renal cell cancer which is characterized byloss of the fumarate hydratase gene and consequentmetabolicalteration accumulated fumarate was associated with tumorprogression via NRF2 signaling Fumarate was shown tomodify KEAP1 cysteine residues and elevate NRF2 levelswhich contributed to the growth and progression of type 2papillary renal cell cancer [140] These accumulating linesof evidence suggest that once cells are transformed to theneoplastic stage cancer cells utilize NRF2 signaling to adaptto the stressful tumor environment and to promote survivaland further cancer progression (Figure 2)

8 Involvement of NRF2 Signaling inStem Cell Quiescence and Differentiation

There is considerable evidence to suggest that NRF2 playsa role in normal stem cell biology [141ndash145] For exampleNRF2 activation in HSCs plays a critical role in not only themaintenance of quiescence but also in the determination ofdifferentiation fate [141 145] In Drosophila intestinal stemcells constitutive Nrf2 activation sustained quiescence byreducing the levels of ROS via upregulation of antioxidantgenes such as gclc However in the case of KEAP1-mediatedNrf2 repression high levels of intracellular ROS facilitatedan ablation of the quiescent state in intestinal stem cellsand age-related degeneration in the intestinal epithelium[146] Similarly low intracellular ROS levels are requiredfor the maintenance of quiescence in human airway basalstem cells (ABSCs) When exposed to exogenous ROSquiescent ABSCs enter the proliferation stage Changes inROS levels activate the NRF2-Notch pathway which resultsin self-renewal and protection of ABSCs from ROS-inducedhyperproliferation and senescence Moreover the quiescent

Oxidative Medicine and Cellular Longevity 7

NRF2 sMaf

GCLCGCLMNQO1SODs

Antioxidant enzymes Drug transportersMetabolic enzymes

ARE

Tumor growthprogression

MDRsMRPsBCRP

G6PDPGDME1

Cell survival Drug effluxProliferation

Target genes

Figure 2 Implications of NRF2 signaling in cancer NRF2 coordi-nates the expression of genes associated with cellular redox regu-lation metabolism and xenobiotic efflux and thereby its aberrantactivation promotes cancer cell survival proliferation and anti-cancer drug resistance GCLM glutamate cysteine ligase modifiersubunit GCLC glutamate cysteine ligase catalytic subunit NQO1NAD(P)Hquinone oxidoreductase 1 SODs superoxide dismutasesG6PDH glucose-6-phosphate dehydrogenase PGD phosphoglu-conate dehydrogenase ME1 malic enzyme 1 MDRs multidrugresistance proteinsMRPsmultidrug resistance-associated proteinsBCRP breast cancer resistance protein

state of ABSCs was maintained by NRF2 activation [147]In osteoclast progenitor cells hydrogen sulfide (H

2S) inhib-

ited human osteoclast differentiation by NRF2-dependentinduction of peroxiredoxin 1 and NQO1 These resultswere further confirmed using NRF2 activators includingsulforaphane and t-BHQ [148] It is also notable that NRF2participates in the regulation of cell fate determination ofHSCsMurakami et al demonstrated that HSCs derived fromKEAP1-deficientmice exhibited preferred differentiation intothe granulocyte-monocyte lineage rather than differentiatinginto the erythroid-lymphoid lineage [145]

Up to now numerous studies have demonstrated thatNRF2 plays a protective role against various stressors instem cells In neural stem cells overexpression of NRF2or pharmacological NRF2 activation prevented necroticcell death [149] In an animal study nrf2-deficient miceshowed defective stem cell function HSCs from nrf2minusminus miceexpressed lower levels of prosurvival cytokines and exhib-ited spontaneous apoptosis [150] Ionizing radiation-inducedmyelosuppression and mortality were mitigated throughNRF2-mediated Notch signaling activation in HSCs [142]Similarly resveratrol-inducedNRF2 expression improved thesurvival of cardiac stem cells and consequently regeneratedinfarcted myocardium [151 152] The heme oxygenase 1(HO-1) inducer cobalt protoporphyrin (CoPP) elicited anantiapoptotic effect on cardiac stem cells via activation of the

ERK-NRF2 pathway [153] In neural stem cells NRF2 activa-tion by melatonin or t-BHQ ameliorated lipopolysaccharide(LPS) orH

2O2induced cell death [149 154] In addition amy-

loid 120573-mediated neural stem cell death could be alleviated byexogenous NRF2 transduction which was accompanied byincreased expression of GCLC NQO-1 andHO-1This studyalso demonstrated that neuronal differentiation of neuralstem cells is enhanced by NRF2 activation [155] Similar toneural stem cells NRF2 has a protective role against hypoxicand oxidative stress conditions in undifferentiated MSCsTreatment of the murine mesenchymal stem cell line withadrenaline increased the mRNA expression of nrf2 gclc andxCT leading to an increase in GSH levels and the preventionof ROS-induced cytotoxicity [156 157]

9 Potential Implication of NRF2 inCSC Maintenance and Resistance

The role of NRF2 in CSC biology is now beginning tobe unveiled Similar to the case of normal stem cells itwas shown that NRF2 contributes to CSC stemness bymaintaining their self-renewal capacity and protecting themfrom chemoradiotherapy Achuthan et al established stablechemotherapy-resistant breast cancer cells and observed thatthese cells expressed higher levels of CD133 and OCT-4indicating that these cells exhibit CSC phenotype [34] Ofnote it was shown that ROS levels were relatively low inthese drug-selected cells presumably due to higher levelsof antioxidant enzymes such as SOD1 and GPX12 NRF2protein stabilizationwas associatedwith high levels of antiox-idant enzymes As an underlying molecular mechanismdiminished proteasome activity and increased p21 levelsappear to stabilize the NRF2 protein in these stem-like cellsEvidently p21 knockdown repressed the mammosphere-forming potential of these stem-like breast cancer cellsSimilarly a study by Zhu et al showed the involvement ofNRF2 in glioblastoma stem cells that were isolated fromhuman surgical glioblastoma specimens NRF2 knockdownin glioblastoma stem cells inhibited cell proliferation andneurosphere formation and further suppressed SOX2 expres-sion Moreover NRF2 knockdown changed the cell cycledistribution to the G2 phase and significantly attenuated thetumorigenecity of glioblastoma stem cells [158 159] Theseresults are providing evidence that NRF2 is necessary formaintenance of the self-renewal capacity of glioblastomastem cells

On the other hand activation of NRF2 signaling has beendemonstrated in different types of CSC models includinglung esophageal breast ovarian and colon CSCs and thisis closely correlated with themaintenance of low intracellularROS levels and chemoresistance of CSCs [160ndash165] In lungand esophageal cancer cells cigarette smoke condensateincreased the SP as well as BCRP expression which arehallmarks of CSCs [165] Promoter analysis revealed thatBCRP expression was associated with elevated levels ofNRF2 aryl hydrocarbon receptor (AhR) and specificityprotein 1 (SP1) Additionally this study demonstrated thatmithramycin diminished BCRP expression via repression of

8 Oxidative Medicine and Cellular Longevity

Low ROS

Increased drug efflux

SurvivalChemoresistance

CSC

TumorRecurrenceProgression

Radioresistance

PERKuarr

p21uarr

p62uarr

Proteasome darr

NRF2 uarr

Figure 3 Potential roles of NRF2 signaling in CSCs InCSC-like cellmodels NRF2 is activated through multiple molecular mechanismsin a context-dependent manner The upregulation of competingproteins such as p62 and p21 activation of PERK or repressedproteasome function were shown to enhance NRF2 activity inthese models Elevated NRF2 levels in CSCs can contribute tothe maintenance of low ROS by upregulating multiple antioxidantgenes In addition NRF2-mediated expression of ABC transporterselicits efflux of anticancer drugs from cancer cells Overall activatedNRF2 signaling facilitates CSCs survival and stress resistance andconsequently it can be suggested that CSCs with high NRF2 activityplay a crucial role in tumor recurrence and further progression

NRF2 AhR and SP1 and thereby inhibiting the expressionof genes associated with CSC-related pathways resulting inreduced proliferation and tumorigenecity Similarly it wasreported that lung cancer SP cells exhibited high levels ofNRF2 and BCRP expression These SP cells were highlytumorigenic and possessed self-renewal capacity comparedto non-SP cells [164] Emmink et al performed a proteomeanalysis on collected secretome from highly tumorigenicCSCs and their corresponding nontumorigenic differentiatedcells both of which were established from human colorectalspecimens [160] Subsequent bioinformatic analysis revealedthat the CSC secretome contained a large amount of proteinsassociated with cell survival and protein quality controlcompared to differentiated tumor cells Notably the CSCsecretome contained an NRF2 antioxidant and detoxifyingprotein signature in that it included elevated levels ofGCLC GPX23 and TXNRD1 This study provided novelevidence that CSCs secrete NRF2 target antioxidant proteinsto counteract extracellular stressors and chemotherapeuticsIn patients with ovarian clear cell carcinoma the expressionof the CSC maker aldehyde dehydrogenase-1 (ALDH1) wasstrongly correlated with an advanced clinical stage andreduced progression free survival [161] Ovarian clear cellcarcinoma cells with high ALDH1 expressionmaintained lowlevels of ROS compared to ALDH-low cells and these cellswere shown to express higher levels of NRF2 and its targetgenes

Two recent studies have demonstratedNRF2 activation insphere cultures of breast cancer cells that is one of models ofCSCs Wu et al showed that mammospheres derived fromMCF7 and MDA-MB231 breast cancer cell lines exhibitedlower ROS levels compared to their monolayer counterpartsThey also showed that levels of NRF2 and target genes such

as NQO1 and GCLM were elevated in mammospheres [163]Similarly our group has shown substantially elevated NRF2protein levels along with increased expression of antioxidantgenes (eg HO-1 and GPX2) and drug efflux transporters(eg MRP2 and BCRP) in sphere cultures of breast cancercells NRF2 accumulation was also observed in sphere-cultured ovarian and colon cancer cells However shRNA-mediated downregulation ofNRF2 led to decreased chemore-sistance of mammospheres presumably due to reduced levelsof antioxidant genes and drug transporters High ROS levelsin NRF2 knockdown mammospheres caused sphere growthretardation and apoptosis Coherently ablation of ABCtransporter induction in NRF2 knockdown mammospheressensitized to anticancer agents [162] Additionally this studyprovided evidence that increased NRF2 protein expression inmammospheres can be linked to 26S proteasome reductionand p62 accumulation In particular knockdown of p62 inMCF7 mammospheres significantly attenuated NRF2 eleva-tion

Surviving dedifferentiated breast cancer cells after chem-otherapy treatment retained high levels of NRF2 activationsimilar to other CSCs However NRF2 activation was medi-ated by a noncanonical pathway Levels of PERKwere high indedifferentiated cancer cells and this in turn phosphorylatedand activated NRF2 signaling to maintain low cellular ROSlevels and to express ABC transporters In agreement withthese findings clinical observations revealed that the PERKpathway gene signature is related to chemoresistance andreduced patient survival [166]

10 Concluding Remarks

Recent studies have started to uncover the role of ROS signal-ing in the biology of CSCs which is related to tumorigenecitytumor progression and relapse Expression of the transcrip-tion factor NRF2 a master regulator of antioxidant genesexpression is increased in different models of CSCs and thiselevation is likely to promote CSC maintenance and survivalin an oxidizing tumormicroenvironment In additionNRF2-mediated overexpression of ABC transporters particularlythe CSC marker BCRP may play a critical role in themultidrug resistance of CSCsThese findings combined withthe increasing evidence showing the alteration of KEAP1-NRF2 signaling in cancer cells suggest a novel role of NRF2in CSC maintenance and survival (Figure 3)

One important question that arises from the current stud-ies is whether it is possible to design CSC-targeted therapiesthrough regulation of theNRF2 pathway and its related redoxhomeostasis inCSCs Recent studies provide several potentialclues for addressing this question the naturally occurringalkaloid brusatol could reduce the growth and chemoresis-tance of breast CSCs [163] Treatment ofmammospheres withbrusatol elevated ROS levels and promoted taxol-inducedgrowth retardation and cell death The NRF2 repressivemechanism of brusatol has not been clearly elucidated but itappears to be independent of KEAP1-mediated degradation[167] In addition to brusatol natural compounds such as

Oxidative Medicine and Cellular Longevity 9

chrysin apigenin luteolin and trigonelline are known toinhibit NRF2 signaling in several types of cancer cells [102168ndash170] and therefore the development of NRF2 inhibitorswith characterized modes of action will enable efficient tar-geting of the redox homeostasis system as well as multidrugresistance systems in CSCs

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This studywas financially supported by theNational ResearchFoundation (NRF) funded by the Ministry of Science ICT ampFuture Planning (NRF-2013R1A2A2A01015497)

References

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[2] P Storz ldquoForkhead homeobox type O transcription factorsin the responses to oxidative stressrdquo Antioxidants amp RedoxSignaling vol 14 no 4 pp 593ndash605 2011

[3] R S Balaban S Nemoto and T Finkel ldquoMitochondria oxi-dants and agingrdquo Cell vol 120 no 4 pp 483ndash495 2005

[4] L A Sena and N S Chandel ldquoPhysiological roles of mitochon-drial reactive oxygen speciesrdquoMolecular Cell vol 48 no 2 pp158ndash167 2012

[5] P T Schumacker ldquoReactive oxygen species in cancer cells liveby the sword die by the swordrdquo Cancer Cell vol 10 no 3 pp175ndash176 2006

[6] S Toyokuni K Okamoto J Yodoi and H Hiai ldquoPersistentoxidative stress in cancerrdquo FEBS Letters vol 358 no 1 pp 1ndash3 1995

[7] L Behrend G Henderson and R Zwacka ldquoReactive oxy-gen species in oncogenic transformationrdquo Biochemical SocietyTransactions vol 31 no 6 pp 1441ndash1444 2003

[8] E O Hileman J Liu M Albitar M J Keating and P HuangldquoIntrinsic oxidative stress in cancer cells a biochemical basis fortherapeutic selectivityrdquo Cancer Chemotherapy and Pharmacol-ogy vol 53 no 3 pp 209ndash219 2004

[9] H PelicanoD Carney and PHuang ldquoROS stress in cancer cellsand therapeutic implicationsrdquo Drug Resistance Updates vol 7no 2 pp 97ndash110 2004

[10] Y Adachi Y Shibai J Mitsushita W H Shang K Hirose andT Kamata ldquoOncogenic Ras upregulates NADPH oxidase 1 geneexpression through MEK-ERK-dependent phosphorylation ofGATA-6rdquo Oncogene vol 27 no 36 pp 4921ndash4932 2008

[11] J Mitsushita J D Lambeth and T Kamata ldquoThe superoxide-generating oxidase Nox1 is functionally required for Ras onco-gene transformationrdquoCancer Research vol 64 no 10 pp 3580ndash3585 2004

[12] O Vafa M Wade S Kern et al ldquoc-Myc can induce DNAdamage increase reactive oxygen species and mitigate p53function a mechanism for oncogene-induced genetic instabil-ityrdquoMolecular Cell vol 9 no 5 pp 1031ndash1044 2002

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10 Oxidative Medicine and Cellular Longevity

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[31] E Lagadinou A Sach K Callahan et al ldquoBCL-2 inhibitiontargets oxidative phosphorylation and selectively eradicatesquiescent human leukemia stem cellsrdquo Cell Stem Cell vol 12no 3 pp 329ndash341 2013

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[40] K Wang T Zhang Q Dong E C Nice C Huang and Y WeildquoRedox homeostasis the linchpin in stem cell self-renewal anddifferentiationrdquo Cell Death and Disease vol 4 no 3 p e5372013

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[42] Y Y Jang and S J Sharkis ldquoA low level of reactive oxygen speciesselects for primitive hematopoietic stem cells that may reside inthe low-oxygenic nicherdquo Blood vol 110 no 8 pp 3056ndash30632007

[43] S Ghaffari ldquoOxidative stress in the regulation of normal andneoplastic hematopoiesisrdquo Antioxidants amp Redox Signaling vol10 no 11 pp 1923ndash1940 2008

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[46] C Cosentino D Grieco and V Costanzo ldquoATM activates thepentose phosphate pathway promoting anti-oxidant defenceand DNA repairrdquo The EMBO Journal vol 30 no 3 pp 546ndash555 2011

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[49] Y Wang Y Zhou and D T Graves ldquoFOXO transcription fac-tors their clinical significance and regulationrdquoBioMed ResearchInternational vol 2014 Article ID 925350 13 pages 2014

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[51] K Miyamoto K Y Araki K Naka et al ldquoFoxo3a is essentialfor maintenance of the hematopoietic stem cell poolrdquo Cell StemCell vol 1 no 1 pp 101ndash112 2007

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[53] S Yalcin X Zhang J P Luciano et al ldquoFoxo3 is essential for theregulation of ataxia telangiectasia mutated and oxidative stress-mediated homeostasis of hematopoietic stem cellsrdquoThe Journalof Biological Chemistry vol 283 no 37 pp 25692ndash25705 2008

[54] J Paik Z Ding R Narurkar et al ldquoFoxOs cooperatively regu-late diverse pathways governing neural stem cell homeostasisrdquoCell Stem Cell vol 5 no 5 pp 540ndash553 2009

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[58] M M Juntilla V D Patil M Calamito R P Joshi M JBirnbaum and G A Koretzky ldquoAKT1 and AKT2 maintainhematopoietic stem cell function by regulating reactive oxygenspeciesrdquo Blood vol 115 no 20 pp 4030ndash4038 2010

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stem cells through Wnt120573-catenin signallingrdquo Nature Cell Biol-ogy vol 12 no 10 pp 1007ndash1013 2010

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[64] K Takubo N Goda W Yamada et al ldquoRegulation of the HIF-1alpha level is essential for hematopoietic stem cellsrdquo Cell StemCell vol 7 no 3 pp 391ndash402 2010

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[72] N Haraguchi H Ishii K Mimori et al ldquoCD13 is a therapeutictarget in human liver cancer stem cellsrdquo Journal of ClinicalInvestigation vol 120 no 9 pp 3326ndash3339 2010

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[74] K Naka T Hoshii T Muraguchi et al ldquoTGF-120573ndashFOXO sig-nalling maintains leukaemia-initiating cells in chronic myeloidleukaemiardquo Nature vol 463 no 7281 pp 676ndash680 2010

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[76] A Dubrovska S Kim R J Salamone et al ldquoThe role ofPTENAktPI3K signaling in the maintenance and viability ofprostate cancer stem-like cell populationsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 106 no 1 pp 268ndash273 2009

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[79] Y Li and A Jaiswal ldquoRegulation of humanNAD (P)H quinoneoxidoreductase gene Role of AP1 binding site contained withinhuman antioxidant response elementrdquoThe Journal of BiologicalChemistry vol 267 no 21 pp 15097ndash15104 1992

[80] T Prestera P Talalay J Alam et al ldquoParallel induction of hemeoxygenase-1 and chemoprotective phase 2 enzymes by elec-trophiles and antioxidants regulation by upstream antioxidant-responsive elements (ARE)rdquoMolecular Medicine vol 1 no 7 p827 1995

[81] K Itoh K IgarashiNHayashi et al ldquoCloning and characteriza-tion of a novel erythroid cell-derived CNC family transcriptionfactor heterodimerizing with the small Maf family proteinsrdquoMolecular and Cellular Biology vol 15 no 8 pp 4184ndash41931995

[82] R Venugopal and A K Jaiswal ldquoNrf1 and Nrf2 positivelyand c-Fos and Fra1 negatively regulate the human antioxidantresponse element-mediated expression of NAD(P)Hquinoneoxidoreductase1 generdquo Proceedings of the National Academy ofSciences vol 93 no 25 pp 14960ndash14965 1996

[83] K Itoh T Chiba S Takahashi et al ldquoAn Nrf2small Mafheterodimer mediates the induction of phase II detoxifyingenzyme genes through antioxidant response elementsrdquo Bio-chemical and Biophysical Research Communications vol 236no 2 pp 313ndash322 1997

[84] M Ramos-Gomez M-K Kwak P M Dolan et al ldquoSensitivityto carcinogenesis is increased and chemoprotective efficacy ofenzyme inducers is lost in nrf2 transcription factor-deficientmicerdquo Proceedings of the National Academy of Sciences vol 98no 6 pp 3410ndash3415 2001

[85] M-K Kwak N Wakabayashi K Itoh H Motohashi MYamamoto and T W Kensler ldquoModulation of gene expressionby cancer chemopreventive dithiolethiones through the Keap1-Nrf2 pathway Identification of novel gene clusters for cellsurvivalrdquo The Journal of Biological Chemistry vol 278 no 10pp 8135ndash8145 2003

[86] R Hu C Xu G Shen et al ldquoGene expression profiles inducedby cancer chemopreventive isothiocyanate sulforaphane in theliver of C57BL6J mice and C57BL6JNrf2 (minusminus) micerdquo CancerLetters vol 243 no 2 pp 170ndash192 2006

[87] S Chanas Q Jiang and M McMahon ldquoLoss of the Nrf2transcription factor causes a marked reduction in constitutiveand inducible expression of the glutathione S-transferase Gsta1Gsta2 Gstm1 Gstm2 Gstm3 and Gstm4 genes in the livers ofmale and female micerdquo Biochemical Journal vol 365 no 2 pp405ndash416 2002

[88] H-Y Cho S P Reddy A DeBiase M Yamamoto and SR Kleeberger ldquoGene expression profiling of NRF2-mediatedprotection against oxidative injuryrdquo Free Radical Biology andMedicine vol 38 no 3 pp 325ndash343 2005

[89] G Shen C Xu R Hu et al ldquoModulation of nuclear factorE2-related factor 2-mediated gene expression in mice liver andsmall intestine by cancer chemopreventive agent curcuminrdquoMolecular Cancer Therapeutics vol 5 no 1 pp 39ndash51 2006

[90] J D Hayes and A T Dinkova-Kostova ldquoThe Nrf2 regulatorynetwork provides an interface between redox and intermediarymetabolismrdquo Trends in Biochemical Sciences vol 39 no 4 pp199ndash218 2014

[91] A Hayashi H Suzuki K Itoh M Yamamoto and Y SugiyamaldquoTranscription factor Nrf2 is required for the constitutive andinducible expression of multidrug resistance-associated protein1 in mouse embryo fibroblastsrdquo Biochemical and BiophysicalResearch Communications vol 310 no 3 pp 824ndash829 2003

[92] E Jigorel M Le Vee C Boursier-Neyret Y Parmentier andO Fardel ldquoDifferential regulation of sinusoidal and canalicularhepatic drug transporter expression by xenobiotics activating

12 Oxidative Medicine and Cellular Longevity

drug-sensing receptors in primary human hepatocytesrdquo DrugMetabolism and Disposition vol 34 no 10 pp 1756ndash1763 2006

[93] X Wang C R Campos J C Peart et al ldquoNrf2 upregulatesATP binding cassette transporter expression and activity atthe blood-brain and blood-spinal cord barriersrdquo Journal ofNeuroscience vol 34 no 25 pp 8585ndash8593 2014

[94] H Jeong I Ryoo andMKwak ldquoRegulation of the expression ofrenal drug transporters in KEAP1-knockdown human tubularcellsrdquo Toxicology in Vitro vol 29 no 5 pp 884ndash892 2015

[95] M J Canet M D Merrell B G Harder et al ldquoIdentificationof a functional antioxidant response element within the eighthintron of the human ABCC

3generdquo Drug Metabolism and

Disposition vol 43 no 1 pp 93ndash99 2014[96] A Singh H Wu P Zhang C Happel J Ma and S Biswal

ldquoExpression of ABCG2 (BCRP) is regulated by Nrf2 in cancercells that confers side population and chemoresistance pheno-typerdquo Molecular Cancer Therapeutics vol 9 no 8 pp 2365ndash2376 2010

[97] N Wakabayashi K Itoh J Wakabayashi et al ldquoKeap1-nullmutation leads to postnatal lethality due to constitutive Nrf2activationrdquo Nature Genetics vol 35 no 3 pp 238ndash245 2003

[98] S B Cullinan J D Gordan J Jin J W Harper and J A DiehlldquoThe Keap1-BTB protein is an adaptor that bridges Nrf2 to aCul3-based E3 ligase oxidative stress sensing by a Cul3-Keap1ligaserdquoMolecular and Cellular Biology vol 24 no 19 pp 8477ndash8486 2004

[99] D D Zhang and M Hannink ldquoDistinct cysteine residues inkeap1 are required for keap1-dependent ubiquitination of nrf2and for stabilization of nrf2 by chemopreventive agents andoxidative stressrdquoMolecular and Cellular Biology vol 23 no 22pp 8137ndash8151 2003

[100] L M Zipper and R T Mulcahy ldquoThe Keap1 BTBPOZ dimer-ization function is required to sequester Nrf2 in cytoplasmrdquoThe Journal of Biological Chemistry vol 277 no 39 pp 36544ndash36552 2002

[101] M C Jaramillo and D D Zhang ldquoThe emerging role of theNrf2-Keap1 signaling pathway in cancerrdquoGenesampDevelopmentvol 27 no 20 pp 2179ndash2191 2013

[102] K I Tong Y Katoh H Kusunoki K Itoh T Tanaka and MYamamoto ldquoKeap1 recruits Neh2 through binding to ETGEand DLG motifs characterization of the two-site molecularrecognition modelrdquoMolecular and Cellular Biology vol 26 no8 pp 2887ndash2900 2006

[103] K I Tong A Kobayashi F Katsuoka andM Yamamoto ldquoTwo-site substrate recognition model for the Keap1-Nrf2 system ahinge and latch mechanismrdquo Biological Chemistry vol 387 no10-11 pp 1311ndash1320 2006

[104] K I Tong B Padmanabhan A Kobayashi et al ldquoDifferentelectrostatic potentials define ETGE and DLG motifs as hingeand latch in oxidative stress responserdquo Molecular and CellularBiology vol 27 no 21 pp 7511ndash7521 2007

[105] M Kobayashi L Li N Iwamoto et al ldquoThe antioxidant defensesystemKeap1-Nrf2 comprises amultiple sensingmechanism forresponding to a wide range of chemical compoundsrdquoMolecularand Cellular Biology vol 29 no 2 pp 493ndash502 2009

[106] T Yamamoto T Suzuki A Kobayashi et al ldquoPhysiological sig-nificance of reactive cysteine residues of Keap1 in determiningNrf2 activityrdquoMolecular and Cellular Biology vol 28 no 8 pp2758ndash2770 2008

[107] M K Kwak K Itoh M Yamamoto and T W KenslerldquoEnhanced expression of the transcription factorNrf2 by cancer

chemopreventive agents role of antioxidant response element-like sequences in the nrf2 promoterrdquo Molecular and CellularBiology vol 22 no 9 pp 2883ndash2892 2002

[108] T Suzuki T Shibata K Takaya et al ldquoRegulatory nexus ofsynthesis and degradation deciphers cellular Nrf2 expressionlevelsrdquoMolecular and Cellular Biology vol 33 no 12 pp 2402ndash2412 2013

[109] S B Cullinan D Zhang M Hannink E Arvisais R JKaufman and J A Diehl ldquoNrf2 is a direct PERK substrateand effector of PERK-dependent cell survivalrdquo Molecular andCellular Biology vol 23 no 20 pp 7198ndash7209 2003

[110] W Li and A Kong ldquoMolecular mechanisms of Nrf2-mediatedantioxidant responserdquo Molecular Carcinogenesis vol 48 no 2pp 91ndash104 2009

[111] Y J Surh ldquoCancer chemoprevention with dietary phytochemi-calsrdquo Nature Reviews Cancer vol 3 no 10 pp 768ndash780 2003

[112] M Salazar A I Rojo D Velasco R M de Sagarra and ACuadrado ldquoGlycogen synthase kinase-3beta inhibits the xeno-biotic and antioxidant cell response by direct phosphorylationand nuclear exclusion of the transcription factor Nrf2rdquo Journalof Biological Chemistry vol 281 no 21 pp 14841ndash14851 2006

[113] W Chen Z Sun X J Wang et al ldquoDirect interactionbetween Nrf2 and p21(Cip1WAF1) upregulates the Nrf2-mediated antioxidant responserdquo Molecular Cell vol 34 no 6pp 663ndash673 2009

[114] M Komatsu H Kurokawa S Waguri et al ldquoThe selectiveautophagy substrate p62 activates the stress responsive tran-scription factor Nrf2 through inactivation of Keap1rdquoNature CellBiology vol 12 no 3 pp 213ndash223 2010

[115] J D Hayes M McMahon S Chowdhry and A T Dinkova-Kostova ldquoCancer chemoprevention mechanisms mediatedthrough the Keap1-Nrf2 pathwayrdquoAntioxidants amp Redox Signal-ing vol 13 no 11 pp 1713ndash1748 2010

[116] R Hu C L Saw R Yu and A T Kong ldquoRegulation ofNF-E2-related factor 2 signaling for cancer chemopreventionantioxidant coupled with antiinflammatoryrdquo Antioxidants ampRedox Signaling vol 13 no 11 pp 1679ndash1698 2010

[117] M Kwak and T W Kensler ldquoTargeting NRF2 signaling for can-cer chemopreventionrdquo Toxicology and Applied Pharmacologyvol 244 no 1 pp 66ndash76 2010

[118] K Iida K Itoh Y Kumagai et al ldquoNrf2 is essential for thechemopreventive efficacy of oltipraz against urinary bladdercarcinogenesisrdquo Cancer Research vol 64 no 18 pp 6424ndash64312004

[119] T Jiang N Chen F Zhao et al ldquoHigh levels of Nrf2 deter-mine chemoresistance in type II endometrial cancerrdquo CancerResearch vol 70 no 13 pp 5486ndash5496 2010

[120] Y R Kim J EOhM S Kim et al ldquoOncogenicNRF2mutationsin squamous cell carcinomas of oesophagus and skinrdquo TheJournal of Pathology vol 220 no 4 pp 446ndash451 2010

[121] T Shibata A Kokubu M Gotoh et al ldquoGenetic alterationof Keap1 confers constitutive Nrf2 activation and resistance tochemotherapy in gallbladder cancerrdquoGastroenterology vol 135no 4 pp 1358e4ndash1368e4 2008

[122] T Shibata T Ohta K I Tong et al ldquoCancer related mutationsin NRF2 impair its recognition by Keap1-Cul3 E3 ligase andpromote malignancyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 36 pp13568ndash13573 2008

[123] A Singh V Misra R K Thimmulappa et al ldquoDysfunctionalKEAP1-NRF2 interaction in non-small-cell lung cancerrdquo PLoSMedicine vol 3 no 10 article e420 2006

Oxidative Medicine and Cellular Longevity 13

[124] R Wang J An F Ji H Jiao H Sun and D Zhou ldquoHyper-methylation of the Keap1 gene in human lung cancer cell linesand lung cancer tissuesrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 1 pp 151ndash154 2008

[125] P Zhang A Singh S Yegnasubramanian et al ldquoLoss ofKelch-like ECH-associated protein 1 function in prostate cancercells causes chemoresistance and radioresistance and promotestumor growthrdquoMolecular Cancer Therapeutics vol 9 no 2 pp336ndash346 2010

[126] J D Hayes and M McMahon ldquoNRF2 and KEAP1 mutationspermanent activation of an adaptive response in cancerrdquo Trendsin Biochemical Sciences vol 34 no 4 pp 176ndash188 2009

[127] B Padmanabhan K I Tong T Ohta et al ldquoStructural basis fordefects of Keap1 activity provoked by its pointmutations in lungcancerrdquoMolecular Cell vol 21 no 5 pp 689ndash700 2006

[128] Y Inami S Waguri A Sakamoto et al ldquoPersistent activation ofNrf2 throughp62 in hepatocellular carcinoma cellsrdquoTheJournalof Cell Biology vol 193 no 2 pp 275ndash284 2011

[129] G M DeNicola F A Karreth T J Humpton et al ldquoOncogene-induced Nrf2 transcription promotes ROS detoxification andtumorigenesisrdquo Nature vol 475 no 7354 pp 106ndash109 2011

[130] L M Solis C Behrens W Dong et al ldquoNrf2 and Keap1abnormalities in non-small cell lung carcinoma and associationwith clinicopathologic featuresrdquo Clinical Cancer Research vol16 no 14 pp 3743ndash3753 2010

[131] N M Reddy S R Kleeberger H Y Cho et al ldquoDeficiency inNrf2-GSH signaling impairs type II cell growth and enhancessensitivity to oxidantsrdquoAmerican Journal of Respiratory Cell andMolecular Biology vol 37 no 1 pp 3ndash8 2007

[132] N M Reddy S R Kleeberger M Yamamoto et al ldquoGeneticdissection of the Nrf2-dependent redox signaling-regulatedtranscriptional programs of cell proliferation and cytoprotec-tionrdquo Physiological Genomics vol 32 no 1 pp 74ndash81 2007

[133] Y Mitsuishi K Taguchi Y Kawatani et al ldquoNrf2 redirectsglucose and glutamine into anabolic pathways in metabolicreprogrammingrdquo Cancer Cell vol 22 no 1 pp 66ndash79 2012

[134] J Cho SManandharH LeeH Park andMKwak ldquoRole of theNrf2-antioxidant system in cytotoxicity mediated by anticancercisplatin implication to cancer cell resistancerdquo Cancer Lettersvol 260 no 1-2 pp 96ndash108 2008

[135] T Ohta K Iijima M Miyamoto et al ldquoLoss of Keap1 functionactivates Nrf2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008

[136] A Singh M Bodas N Wakabayashi F Bunz and S BiswalldquoGain of Nrf2 function in non-small-cell lung cancer cellsconfers radioresistancerdquoAntioxidantsampRedox Signaling vol 13no 11 pp 1627ndash1637 2010

[137] G-S Shim S Manandhar D-H Shin T Kim and M KwakldquoAcquisition of doxorubicin resistance in ovarian carcinomacells accompanies activation of the NRF2 pathwayrdquo Free RadicalBiology and Medicine vol 47 no 11 pp 1619ndash1631 2009

[138] S Manandhar B Choi K Jung et al ldquoNRF2 inhibitionrepresses ErbB2 signaling in ovarian carcinoma cells implica-tions for tumor growth retardation and docetaxel sensitivityrdquoFree Radical Biology and Medicine vol 52 no 9 pp 1773ndash17852012

[139] T Kim E-g Hur S J Kang et al ldquoNRF2 blockade suppressescolon tumor angiogenesis by inhibiting hypoxia-induced acti-vation ofHIF-1120572rdquoCancer Research vol 71 no 6 pp 2260ndash22752011

[140] L Kinch N V Grishin and J Brugarolas ldquoSuccination of Keap1and activation of Nrf2-dependent antioxidant pathways in FH-deficient papillary renal cell carcinoma type 2rdquo Cancer Cell vol20 no 4 pp 418ndash420 2011

[141] J J Tsai J A Dudakov K Takahashi et al ldquoNrf2 regulateshaematopoietic stem cell functionrdquo Nature Cell Biology vol 15no 3 pp 309ndash316 2013

[142] J H Kim R KThimmulappa V Kumar et al ldquoNRF2-mediatedNotch pathway activation enhances hematopoietic reconsti-tution following myelosuppressive radiationrdquo The Journal ofClinical Investigation vol 124 no 2 pp 730ndash741 2014

[143] J P Chute ldquoNRF2 mitigates radiation-induced hematopoieticdeathrdquo The Journal of Clinical Investigation vol 124 no 3 pp960ndash961 2014

[144] J Jang Y Wang H Kim M A Lalli and K S KosikldquoNrf2 a regulator of the proteasome controls self-renewal andpluripotency in human embryonic stem cellsrdquo Stem Cells vol32 no 10 pp 2616ndash2625 2014

[145] S Murakami R Shimizu P Romeo M Yamamoto and HMotohashi ldquoKeap1-Nrf2 system regulates cell fate determina-tion of hematopoietic stem cellsrdquo Genes to Cells vol 19 no 3pp 239ndash253 2014

[146] C E Hochmuth B Biteau D Bohmann and H Jasper ldquoRedoxregulation by Keap1 and Nrf2 controls intestinal stem cellproliferation inDrosophilardquoCell Stem Cell vol 8 no 2 pp 188ndash199 2011

[147] M K Paul B Bisht D O Darmawan et al ldquoDynamic changesin intracellular ROS levels regulate airway basal stem cellhomeostasis through Nrf2-dependent notch signalingrdquo CellStem Cell vol 15 no 2 pp 199ndash214 2014

[148] L Gambari G Lisignoli L Cattini C Manferdini A Facchiniand F Grassi ldquoSodium hydrosulfide inhibits the differentiationof osteoclast progenitor cells via NRF2-dependent mechanismrdquoPharmacological Research vol 87 pp 99ndash112 2014

[149] J Li D Johnson M Calkins L Wright C Svendsen andJ Johnson ldquoStabilization of Nrf2 by tBHQ confers protectionagainst oxidative stress-induced cell death in human neuralstem cellsrdquo Toxicological Sciences vol 83 no 2 pp 313ndash3282005

[150] A AMerchant A SinghWMatsui and S Biswal ldquoThe redox-sensitive transcription factor Nrf2 regulatesmurine hematopoi-etic stem cell survival independently of ROS levelsrdquo Blood vol118 no 25 pp 6572ndash6579 2011

[151] N Gurusamy D Ray I Lekli and D K Das ldquoRed wineantioxidant resveratrol-modified cardiac stem cells regener-ate infarcted myocardiumrdquo Journal of Cellular and MolecularMedicine vol 14 no 9 pp 2235ndash2239 2010

[152] N Gorbunov G Petrovski N Gurusamy D Ray D H Kimand D K Das ldquoRegeneration of infarcted myocardium withresveratrol-modified cardiac stem cellsrdquo Journal of Cellular andMolecular Medicine vol 16 no 1 pp 174ndash184 2012

[153] C Cai L Teng D Vu et al ldquoThe heme oxygenase 1 inducer(CoPP) protects human cardiac stem cells against apoptosisthrough activation of the extracellular signal-regulated kinase(ERK)NRF2 signaling pathway and cytokine releaserdquo TheJournal of Biological Chemistry vol 287 no 40 pp 33720ndash33732 2012

[154] J Song S M Kang K M Lee and J E Lee ldquoThe protectiveeffect of melatonin on neural stem cell against LPS-inducedinflammationrdquo BioMed Research International vol 2015 ArticleID 854359 13 pages 2015

14 Oxidative Medicine and Cellular Longevity

[155] V Karkkainen Y Pomeshchik E Savchenko et al ldquoNrf2regulates neurogenesis and protects neural progenitor cellsagainst A120573 toxicityrdquo STEM CELLS vol 32 no 7 pp 1904ndash19162014

[156] M Mohammadzadeh R Halabian A Gharehbaghian et alldquoNrf-2 overexpression in mesenchymal stem cells reducesoxidative stress-induced apoptosis and cytotoxicityrdquo Cell Stressand Chaperones vol 17 no 5 pp 553ndash565 2012

[157] Y Takahata T Takarada M Iemata et al ldquoFunctional expres-sion of 1205732 adrenergic receptors responsible for protectionagainst oxidative stress through promotion of glutathione syn-thesis after Nrf2 upregulation in undifferentiated mesenchymalC3H10T12 stem cellsrdquo Journal of Cellular Physiology vol 218no 2 pp 268ndash275 2009

[158] J Zhu H Wang Y Fan et al ldquoKnockdown of nuclear factorerythroid 2-related factor 2 by lentivirus induces differentiationof glioma stem-like cellsrdquo Oncology Reports vol 32 no 3 pp1170ndash1178 2014

[159] J Zhu H Wang Q Sun et al ldquoNrf2 is required to maintain theself-renewal of glioma stem cellsrdquo BMC Cancer vol 13 no 1article 380 2013

[160] B L Emmink A Verheem W J Van Houdt et al ldquoThe secre-tome of colon cancer stem cells contains drug-metabolizingenzymesrdquo Journal of Proteomics vol 91 pp 84ndash96 2013

[161] T Mizuno N Suzuki H Makino et al ldquoCancer stem-like cellsof ovarian clear cell carcinoma are enriched in the ALDH-highpopulation associated with an accelerated scavenging system inreactive oxygen speciesrdquo Gynecologic Oncology vol 137 no 2pp 299ndash305 2015

[162] I-G Ryoo B-H Choi and M-K Kwak ldquoActivation of NRF2by p62 and proteasome reduction in sphere-forming breastcarcinoma cellsrdquo Oncotarget vol 6 no 10 pp 8167ndash8184 2015

[163] T Wu B G Harder P K Wong J E Lang and D D ZhangldquoOxidative stress mammospheres and Nrf2-new implicationfor breast cancer therapyrdquoMolecular Carcinogenesis 2014

[164] B Yang Y Ma and Y Liu ldquoElevated expression of Nrf-2 andABCG2 involved in multi-drug resistance of lung cancer SPcellsrdquo Drug Research 2015

[165] M Zhang A Mathur Y Zhang et al ldquoMithramycin repressesbasal and cigarette smoke-induced expression of abcg2 andinhibits stem cell signaling in lung and esophageal cancer cellsrdquoCancer Research vol 72 no 16 pp 4178ndash4192 2012

[166] C A Del Vecchio Y Feng E S Sokol et al ldquoDe-differentiationconfers multidrug resistance via noncanonical PERK-Nrf2signalingrdquo PLoS Biology vol 12 no 9 Article ID e1001945 2014

[167] D Ren N F Villeneuve T Jiang et al ldquoBrusatol enhancesthe efficacy of chemotherapy by inhibiting the Nrf2-mediateddefense mechanismrdquo Proceedings of the National Academy ofSciences of the United States of America vol 108 no 4 pp 1433ndash1438 2011

[168] A Arlt S Sebens S Krebs et al ldquoInhibition of the Nrf2 tran-scription factor by the alkaloid trigonelline renders pancreaticcancer cells more susceptible to apoptosis through decreasedproteasomal gene expression and proteasome activityrdquo Onco-gene vol 32 no 40 pp 4825ndash4835 2013

[169] A-M Gao Z-P Ke F Shi G Sun and H Chen ldquoChrysinenhances sensitivity of BEL-7402ADM cells to doxorubicin bysuppressing PI3KAktNrf2 and ERKNrf2 pathwayrdquo Chemico-Biological Interactions vol 206 no 1 pp 100ndash108 2013

[170] A M Gao Z P Ke J N Wang J Yang S Chen and HChen ldquoApigenin sensitizes doxorubicin-resistant hepatocellular

carcinoma BEL-7402ADM cells to doxorubicin via inhibitingPI3KAktNrf2 pathwayrdquo Carcinogenesis vol 34 no 8 pp1806ndash1814 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

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Disease Markers

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BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ObesityJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

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Research and TreatmentAIDS

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Oxidative Medicine and Cellular Longevity 5

established thatNRF2 regulates divergent genes to coordinatexenobiotic detoxification and redox homeostasis [85 87ndash89]In its function as a regulator of cellular redox homeostasisNRF2 elevates the expression of GCL and the cysteinetransporter xCT to increase cellular GSH levels NRF2 alsoenhances regeneration of reduced GSH by upregulatingGPX and GSH reductase (GSR) Expression of thioredoxin1 (TXN1) thioredoxin reductase 1 (TXNRD1) and perox-iredoxin 16 which can reduce oxidized protein thiols isalso under the control of NRF2 In addition the levels ofNADPH a cofactor of many antioxidant enzymes such asGSR and TXNRD can be increased by NRF2 (reviewedin Hayes and Dinkova-Kostova [90]) The expression ofmultiple NADPH generating enzymes such as G6PDH and6-phosphogluconate dehydrogenase is upregulated by NRF2Additionally the role of NRF2 in ABC transporter expressionfor xenobiotic detoxification is notable The basal expressionlevel of Mrp1 was relatively lower in nrf2-deficient fibroblaststhan that in wild type fibroblasts and the treatment ofmice with Nrf2 activating diethyl maleate increased Mrp1expression in the liver [91] Levels of MDR1 MRP23 andBCRP were elevated following oltipraz treatment in pri-mary human hepatocytes [92] Sulforaphane (SFN) treatmentenhanced the levels ofMDR1 BCRP andMRP2 in the blood-brain barrier of rats [93] Our recent study showed thatgenetic activation of NRF2 via KEAP1 silencing increases theexpression ofMDR1MRP23 and BCRP in human proximaltubular epithelial cells [94] As direct molecular evidencefunctional AREs have been identified in human MRP3 [95]and BCRP genes [96]

Kelch-like ECH-associated protein 1 (KEAP1) a cysteine-rich actin-binding protein is the main negative regulatorof NRF2 activity [83 97] Under quiescent conditionsNRF2 remains inactive by forming a complex with KEAP1in the cytoplasm NRF2 is subject to ubiquitination andKEAP1-induced proteasomal degradation through the Cullin3 (CUL3) based E3 ligase KEAP1 has three major domainsas follows (i) The BTB domain is associated with KEAP1homodimerization (ii) the IVR domain plays a role inregulation ofKEAP1 activity and (iii) theKelchDGRdomainmediates binding with NRF2 [83 90 98ndash101] The bindingof NRF2 with KEAP1 has been described as the ldquohinge andlatchrdquo model where one molecule of NRF2 interacts with theKelchDGR domains of the KEAP1 dimer through conservedmotifs called ETGE (DN-X-E-TS-G-E) and DLG (L-X-X-Q-D-X-D-L-G) [102ndash104] In these reports it was shown thatthe binding affinity of the ETGE motif to KEAP1 is muchhigher than that of the DLG motif It has therefore beenshown that ldquolatchrdquo binding of the NRF2 DLG motif is easilybroken by modifications of KEAP1 cysteine residues by ROSor electrophiles In turn disrupted DLG binding of NRF2 toKEAP1 leads to the blockade of ubiquitination and furtherdegradation of NRF2 resulting in nuclear translocation ofNRF2 It has been demonstrated that the sulfhydryl groups ofmultiple cysteine residues of KEAP1 can be directly modifiedby oxidationreduction or alkylation In particular Cys151Cys273 and Cys288 were found to be essential for theregulation of NRF2 activity [99 104ndash106] Mutation of theCys273 or Cys288 residue of KEAP1 ablated its ability to

suppress NRF2 activity leading to accumulation of the NRF2protein [99 104]

In addition to KEAP1-mediated stability regulationNRF2 activity can be modulated at multiple steps First itis noticeable that NRF2 activity is regulated at the transcrip-tional step Functional AREs were identified in the murinenrf2 gene promoter and were involved in the autoregulationof NRF2 through transcriptional activation [107] Moreovera single nucleotide polymorphism in the ARE-like sequencesof the human NRF2 promoter was associated with increasedlung cancer susceptibility [108] Second it was shown thatNRF2 activity is regulated by posttranslationalmodificationsStudies indicate that NRF2 activation involves phosphoryla-tion signaling mediated by multiple kinase pathways such asMAPK protein kinase C (PKC) PI3K and protein kinaseRNA-like endoplasmic reticulum kinase (PERK) [109ndash111]Meanwhile glycogen synthase kinase-3 (GSK-3) a consti-tutively active serinethreonine kinase was found to inhibitNRF2 activity [112] Last NRF2 activity is increased byseveral intrinsic proteins such as p21 and p62 [113 114] Forexample p62 a linker protein of ubiquitinated proteins toautophagy degradation binds to the KEAP1 protein andinterferes with the binding of NRF2 to KEAP1 resulting inNRF2 stabilization

7 Emerging Role of NRF2 in Cancer Biology

Continuous or fatal stimuli such as toxic chemicals and excessROS disrupt cellular homeostasis causing macromoleculardamage and alterations in cell cycle and growth signalingwhich can eventually result in carcinogenesis The NRF2pathway deserved significant attention in the area of cancerbiology because numerous studies have demonstrated thatactivation of the NRF2 pathway decreases the sensitivity ofcells to carcinogens [115ndash117] For instance the burden ofgastric neoplasia caused by benzo[a]pyrene was effectivelyattenuated by the Nrf2 activator oltipraz in wild type micewhereas nrf2 knockout mice did not show any protectiveeffect of oltipraz [84] Similarly the incidence of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) induced urinary blad-der carcinomawas greater in nrf2 knockoutmice than in wildtype mice and oltipraz treatment reduced tumor incidenceonly in wild type mice [118]

Although it has been firmly confirmed that NRF2 activa-tion can protect cells against a wide range of toxicants andstressors aberrant activation of NRF2 has been associatedwith several types of cancers NRF2 levels were constitutivelyelevated in cancer cell lines and tumor samples of the lungbreast esophagus endometrial cancers and prostate cancers[119ndash125] Molecular mechanisms involved in constitutiveNRF2 activation include the following (i) somatic mutationsof KEAP1 or NRF2 (ii) epigenetic silencing of the KEAP1gene (iii) aberrant accumulation of proteins that competewith NRF2 for KEAP1 binding and (iv) oncogene-mediatedoverexpression of NRF2 [101 126] First somatic mutationsof KEAP1-NRF2 have been reported in an initial study byPadmanabhan et al [127] This study identified mutations

6 Oxidative Medicine and Cellular Longevity

in the KelchDGR domain of KEAP1 in lung cancer celllines as well as lung cancer tissue samples and demonstratedthat these mutant KEAP1 proteins lost their NRF2 repressivefunction which resulted in NRF2 accumulation Singh et alalso demonstrated that the KelchDGR and IVR domainsof the KEAP1 gene contain multiple somatic mutations andthese mutations were identified in 19 of tumor specimensfrom non-small cell lung cancer patients [123] In gallbladdercancer 4 of 13 patients harbored KEAP1 mutations [122]Shibata et al reported that NRF2 somatic mutations werefound in 107 of primary lung cancer patients and 272of primary head and neck cancer patients [122] Notablythesemutationswere primarily located in theDLG andETGEmotifs and eventually led to the loss of a proper interactionbetween the NRF2 protein and KEAP1 Second CpG islandhypermethylation in the KEAP1 promoter resulted in lowKEAP1 expression in lung cancer cell lines and tumor samples[124]Third in humanhepatocellular carcinoma p62 positivecellular aggregates were found with a frequency of 25 andmost of these tumors retained higher levels of NRF2 andits target gene expression [128] Fourth oncogenes have alsobeen shown to play a role in NRF2 signaling Oncogenicactivation of KRAS (KRASG12D) c-MYC (c-MYCERT12) andBRAF (BRAFV619E) elevates the transcript levels of NRF2 andits target gene expression [129]

It is now widely accepted that aberrant activation ofNRF2 can enhance cancer cell survival and growth in oxi-dizing tumor environments and further promote chemoradioresistance Indeed the prognosis of cancer patientsnegatively correlated with NRF2 levels in the tumor [122130] The favorable effect of NRF2 overexpression on tumorsurvival and growth can be attributed to the increase inNRF2 target antioxidant proteins and their counteractiveeffect on oxidative stress For instance GSH which is adirect target molecule of NRF2 has been shown to be criticalfor cell proliferation [131 132] In addition to its antioxi-dant contribution Mitsuishi et al provided direct evidencedemonstrating that NRF2 alters the cellular metabolism inrelation to anabolic pathways to accelerate cell proliferation[133] Multiple metabolic genes such as those involvedin the pentose phosphate pathway were upregulated byNRF2 through ARE and these changes promoted purinesynthesis glutamine metabolism and NADPH productionfor enhanced cell proliferation

Constitutively high levels of NRF2 have been associatedwith chemoresistance as well as radioresistance Cancer cellswith high NRF2 activity were less sensitive to cytotoxicchemotherapeutics such as cisplatin doxorubicin and 5-fluorouracil through facilitated detoxification of anticanceragents and enhanced antioxidant capacity [101] It is there-fore hypothesized that NRF2 inhibition can enhance thechemosensitivity of cancers NRF2 siRNA could suppresscancer resistance to cisplatin topoisomerase inhibitors and5-fluorouracil [101 122 134 135] Cancer cells with constitu-tively high NRF2 were protected against 120574-radiation inducedtoxicity Moreover siRNA-mediated inhibition of NRF2 innon-small cell lung cancer cell lines substantially enhancedradiosensitivity [136] Additionally NRF2 expression was

increased during the acquisition of chemoresistance In ourprevious study doxorubicin-selected ovarian cancer cellsdemonstrated increased expression of NRF2 and its targetgenes forGSH synthesis andNRF2 inhibition in this resistantcell line could restore doxorubicin sensitivity [137]

Our understanding of the role of NRF2 in cancer cell sig-naling has expanded In particular the relationship betweenoncogenic signaling and NRF2 is noteworthy As men-tioned earlier activation of oncogenes such as KRAS and c-MYC increased the expression of NRF2 presumably throughoncogene-mediated ROS increase and this phenomenonappears to contribute to the maintenance of reduced redoxhomeostasis in cancer cells [129] In ERBB2 (Her2Neu) over-expressing ovarian cancer cells the stable silencing of NRF2repressed ERBB2 expression and its downstream signalingand retarded tumor growth Therefore the inhibition ofNRF2 could sensitize these cells to taxol therapy by repressingERBB2 expression [138] Moreover NRF2 was shown to beassociated with HIF signaling which is a critical factor fortumor angiogenesis When NRF2 was stably knocked downin colon carcinoma cell lines hypoxia-inducible HIF-1120572accumulation was abrogated and consequently angiogenesisand tumor growth were significantly suppressed in NRF2knockdown tumors compared to the control group [139] Intype 2 papillary renal cell cancer which is characterized byloss of the fumarate hydratase gene and consequentmetabolicalteration accumulated fumarate was associated with tumorprogression via NRF2 signaling Fumarate was shown tomodify KEAP1 cysteine residues and elevate NRF2 levelswhich contributed to the growth and progression of type 2papillary renal cell cancer [140] These accumulating linesof evidence suggest that once cells are transformed to theneoplastic stage cancer cells utilize NRF2 signaling to adaptto the stressful tumor environment and to promote survivaland further cancer progression (Figure 2)

8 Involvement of NRF2 Signaling inStem Cell Quiescence and Differentiation

There is considerable evidence to suggest that NRF2 playsa role in normal stem cell biology [141ndash145] For exampleNRF2 activation in HSCs plays a critical role in not only themaintenance of quiescence but also in the determination ofdifferentiation fate [141 145] In Drosophila intestinal stemcells constitutive Nrf2 activation sustained quiescence byreducing the levels of ROS via upregulation of antioxidantgenes such as gclc However in the case of KEAP1-mediatedNrf2 repression high levels of intracellular ROS facilitatedan ablation of the quiescent state in intestinal stem cellsand age-related degeneration in the intestinal epithelium[146] Similarly low intracellular ROS levels are requiredfor the maintenance of quiescence in human airway basalstem cells (ABSCs) When exposed to exogenous ROSquiescent ABSCs enter the proliferation stage Changes inROS levels activate the NRF2-Notch pathway which resultsin self-renewal and protection of ABSCs from ROS-inducedhyperproliferation and senescence Moreover the quiescent

Oxidative Medicine and Cellular Longevity 7

NRF2 sMaf

GCLCGCLMNQO1SODs

Antioxidant enzymes Drug transportersMetabolic enzymes

ARE

Tumor growthprogression

MDRsMRPsBCRP

G6PDPGDME1

Cell survival Drug effluxProliferation

Target genes

Figure 2 Implications of NRF2 signaling in cancer NRF2 coordi-nates the expression of genes associated with cellular redox regu-lation metabolism and xenobiotic efflux and thereby its aberrantactivation promotes cancer cell survival proliferation and anti-cancer drug resistance GCLM glutamate cysteine ligase modifiersubunit GCLC glutamate cysteine ligase catalytic subunit NQO1NAD(P)Hquinone oxidoreductase 1 SODs superoxide dismutasesG6PDH glucose-6-phosphate dehydrogenase PGD phosphoglu-conate dehydrogenase ME1 malic enzyme 1 MDRs multidrugresistance proteinsMRPsmultidrug resistance-associated proteinsBCRP breast cancer resistance protein

state of ABSCs was maintained by NRF2 activation [147]In osteoclast progenitor cells hydrogen sulfide (H

2S) inhib-

ited human osteoclast differentiation by NRF2-dependentinduction of peroxiredoxin 1 and NQO1 These resultswere further confirmed using NRF2 activators includingsulforaphane and t-BHQ [148] It is also notable that NRF2participates in the regulation of cell fate determination ofHSCsMurakami et al demonstrated that HSCs derived fromKEAP1-deficientmice exhibited preferred differentiation intothe granulocyte-monocyte lineage rather than differentiatinginto the erythroid-lymphoid lineage [145]

Up to now numerous studies have demonstrated thatNRF2 plays a protective role against various stressors instem cells In neural stem cells overexpression of NRF2or pharmacological NRF2 activation prevented necroticcell death [149] In an animal study nrf2-deficient miceshowed defective stem cell function HSCs from nrf2minusminus miceexpressed lower levels of prosurvival cytokines and exhib-ited spontaneous apoptosis [150] Ionizing radiation-inducedmyelosuppression and mortality were mitigated throughNRF2-mediated Notch signaling activation in HSCs [142]Similarly resveratrol-inducedNRF2 expression improved thesurvival of cardiac stem cells and consequently regeneratedinfarcted myocardium [151 152] The heme oxygenase 1(HO-1) inducer cobalt protoporphyrin (CoPP) elicited anantiapoptotic effect on cardiac stem cells via activation of the

ERK-NRF2 pathway [153] In neural stem cells NRF2 activa-tion by melatonin or t-BHQ ameliorated lipopolysaccharide(LPS) orH

2O2induced cell death [149 154] In addition amy-

loid 120573-mediated neural stem cell death could be alleviated byexogenous NRF2 transduction which was accompanied byincreased expression of GCLC NQO-1 andHO-1This studyalso demonstrated that neuronal differentiation of neuralstem cells is enhanced by NRF2 activation [155] Similar toneural stem cells NRF2 has a protective role against hypoxicand oxidative stress conditions in undifferentiated MSCsTreatment of the murine mesenchymal stem cell line withadrenaline increased the mRNA expression of nrf2 gclc andxCT leading to an increase in GSH levels and the preventionof ROS-induced cytotoxicity [156 157]

9 Potential Implication of NRF2 inCSC Maintenance and Resistance

The role of NRF2 in CSC biology is now beginning tobe unveiled Similar to the case of normal stem cells itwas shown that NRF2 contributes to CSC stemness bymaintaining their self-renewal capacity and protecting themfrom chemoradiotherapy Achuthan et al established stablechemotherapy-resistant breast cancer cells and observed thatthese cells expressed higher levels of CD133 and OCT-4indicating that these cells exhibit CSC phenotype [34] Ofnote it was shown that ROS levels were relatively low inthese drug-selected cells presumably due to higher levelsof antioxidant enzymes such as SOD1 and GPX12 NRF2protein stabilizationwas associatedwith high levels of antiox-idant enzymes As an underlying molecular mechanismdiminished proteasome activity and increased p21 levelsappear to stabilize the NRF2 protein in these stem-like cellsEvidently p21 knockdown repressed the mammosphere-forming potential of these stem-like breast cancer cellsSimilarly a study by Zhu et al showed the involvement ofNRF2 in glioblastoma stem cells that were isolated fromhuman surgical glioblastoma specimens NRF2 knockdownin glioblastoma stem cells inhibited cell proliferation andneurosphere formation and further suppressed SOX2 expres-sion Moreover NRF2 knockdown changed the cell cycledistribution to the G2 phase and significantly attenuated thetumorigenecity of glioblastoma stem cells [158 159] Theseresults are providing evidence that NRF2 is necessary formaintenance of the self-renewal capacity of glioblastomastem cells

On the other hand activation of NRF2 signaling has beendemonstrated in different types of CSC models includinglung esophageal breast ovarian and colon CSCs and thisis closely correlated with themaintenance of low intracellularROS levels and chemoresistance of CSCs [160ndash165] In lungand esophageal cancer cells cigarette smoke condensateincreased the SP as well as BCRP expression which arehallmarks of CSCs [165] Promoter analysis revealed thatBCRP expression was associated with elevated levels ofNRF2 aryl hydrocarbon receptor (AhR) and specificityprotein 1 (SP1) Additionally this study demonstrated thatmithramycin diminished BCRP expression via repression of

8 Oxidative Medicine and Cellular Longevity

Low ROS

Increased drug efflux

SurvivalChemoresistance

CSC

TumorRecurrenceProgression

Radioresistance

PERKuarr

p21uarr

p62uarr

Proteasome darr

NRF2 uarr

Figure 3 Potential roles of NRF2 signaling in CSCs InCSC-like cellmodels NRF2 is activated through multiple molecular mechanismsin a context-dependent manner The upregulation of competingproteins such as p62 and p21 activation of PERK or repressedproteasome function were shown to enhance NRF2 activity inthese models Elevated NRF2 levels in CSCs can contribute tothe maintenance of low ROS by upregulating multiple antioxidantgenes In addition NRF2-mediated expression of ABC transporterselicits efflux of anticancer drugs from cancer cells Overall activatedNRF2 signaling facilitates CSCs survival and stress resistance andconsequently it can be suggested that CSCs with high NRF2 activityplay a crucial role in tumor recurrence and further progression

NRF2 AhR and SP1 and thereby inhibiting the expressionof genes associated with CSC-related pathways resulting inreduced proliferation and tumorigenecity Similarly it wasreported that lung cancer SP cells exhibited high levels ofNRF2 and BCRP expression These SP cells were highlytumorigenic and possessed self-renewal capacity comparedto non-SP cells [164] Emmink et al performed a proteomeanalysis on collected secretome from highly tumorigenicCSCs and their corresponding nontumorigenic differentiatedcells both of which were established from human colorectalspecimens [160] Subsequent bioinformatic analysis revealedthat the CSC secretome contained a large amount of proteinsassociated with cell survival and protein quality controlcompared to differentiated tumor cells Notably the CSCsecretome contained an NRF2 antioxidant and detoxifyingprotein signature in that it included elevated levels ofGCLC GPX23 and TXNRD1 This study provided novelevidence that CSCs secrete NRF2 target antioxidant proteinsto counteract extracellular stressors and chemotherapeuticsIn patients with ovarian clear cell carcinoma the expressionof the CSC maker aldehyde dehydrogenase-1 (ALDH1) wasstrongly correlated with an advanced clinical stage andreduced progression free survival [161] Ovarian clear cellcarcinoma cells with high ALDH1 expressionmaintained lowlevels of ROS compared to ALDH-low cells and these cellswere shown to express higher levels of NRF2 and its targetgenes

Two recent studies have demonstratedNRF2 activation insphere cultures of breast cancer cells that is one of models ofCSCs Wu et al showed that mammospheres derived fromMCF7 and MDA-MB231 breast cancer cell lines exhibitedlower ROS levels compared to their monolayer counterpartsThey also showed that levels of NRF2 and target genes such

as NQO1 and GCLM were elevated in mammospheres [163]Similarly our group has shown substantially elevated NRF2protein levels along with increased expression of antioxidantgenes (eg HO-1 and GPX2) and drug efflux transporters(eg MRP2 and BCRP) in sphere cultures of breast cancercells NRF2 accumulation was also observed in sphere-cultured ovarian and colon cancer cells However shRNA-mediated downregulation ofNRF2 led to decreased chemore-sistance of mammospheres presumably due to reduced levelsof antioxidant genes and drug transporters High ROS levelsin NRF2 knockdown mammospheres caused sphere growthretardation and apoptosis Coherently ablation of ABCtransporter induction in NRF2 knockdown mammospheressensitized to anticancer agents [162] Additionally this studyprovided evidence that increased NRF2 protein expression inmammospheres can be linked to 26S proteasome reductionand p62 accumulation In particular knockdown of p62 inMCF7 mammospheres significantly attenuated NRF2 eleva-tion

Surviving dedifferentiated breast cancer cells after chem-otherapy treatment retained high levels of NRF2 activationsimilar to other CSCs However NRF2 activation was medi-ated by a noncanonical pathway Levels of PERKwere high indedifferentiated cancer cells and this in turn phosphorylatedand activated NRF2 signaling to maintain low cellular ROSlevels and to express ABC transporters In agreement withthese findings clinical observations revealed that the PERKpathway gene signature is related to chemoresistance andreduced patient survival [166]

10 Concluding Remarks

Recent studies have started to uncover the role of ROS signal-ing in the biology of CSCs which is related to tumorigenecitytumor progression and relapse Expression of the transcrip-tion factor NRF2 a master regulator of antioxidant genesexpression is increased in different models of CSCs and thiselevation is likely to promote CSC maintenance and survivalin an oxidizing tumormicroenvironment In additionNRF2-mediated overexpression of ABC transporters particularlythe CSC marker BCRP may play a critical role in themultidrug resistance of CSCsThese findings combined withthe increasing evidence showing the alteration of KEAP1-NRF2 signaling in cancer cells suggest a novel role of NRF2in CSC maintenance and survival (Figure 3)

One important question that arises from the current stud-ies is whether it is possible to design CSC-targeted therapiesthrough regulation of theNRF2 pathway and its related redoxhomeostasis inCSCs Recent studies provide several potentialclues for addressing this question the naturally occurringalkaloid brusatol could reduce the growth and chemoresis-tance of breast CSCs [163] Treatment ofmammospheres withbrusatol elevated ROS levels and promoted taxol-inducedgrowth retardation and cell death The NRF2 repressivemechanism of brusatol has not been clearly elucidated but itappears to be independent of KEAP1-mediated degradation[167] In addition to brusatol natural compounds such as

Oxidative Medicine and Cellular Longevity 9

chrysin apigenin luteolin and trigonelline are known toinhibit NRF2 signaling in several types of cancer cells [102168ndash170] and therefore the development of NRF2 inhibitorswith characterized modes of action will enable efficient tar-geting of the redox homeostasis system as well as multidrugresistance systems in CSCs

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This studywas financially supported by theNational ResearchFoundation (NRF) funded by the Ministry of Science ICT ampFuture Planning (NRF-2013R1A2A2A01015497)

References

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[2] P Storz ldquoForkhead homeobox type O transcription factorsin the responses to oxidative stressrdquo Antioxidants amp RedoxSignaling vol 14 no 4 pp 593ndash605 2011

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[4] L A Sena and N S Chandel ldquoPhysiological roles of mitochon-drial reactive oxygen speciesrdquoMolecular Cell vol 48 no 2 pp158ndash167 2012

[5] P T Schumacker ldquoReactive oxygen species in cancer cells liveby the sword die by the swordrdquo Cancer Cell vol 10 no 3 pp175ndash176 2006

[6] S Toyokuni K Okamoto J Yodoi and H Hiai ldquoPersistentoxidative stress in cancerrdquo FEBS Letters vol 358 no 1 pp 1ndash3 1995

[7] L Behrend G Henderson and R Zwacka ldquoReactive oxy-gen species in oncogenic transformationrdquo Biochemical SocietyTransactions vol 31 no 6 pp 1441ndash1444 2003

[8] E O Hileman J Liu M Albitar M J Keating and P HuangldquoIntrinsic oxidative stress in cancer cells a biochemical basis fortherapeutic selectivityrdquo Cancer Chemotherapy and Pharmacol-ogy vol 53 no 3 pp 209ndash219 2004

[9] H PelicanoD Carney and PHuang ldquoROS stress in cancer cellsand therapeutic implicationsrdquo Drug Resistance Updates vol 7no 2 pp 97ndash110 2004

[10] Y Adachi Y Shibai J Mitsushita W H Shang K Hirose andT Kamata ldquoOncogenic Ras upregulates NADPH oxidase 1 geneexpression through MEK-ERK-dependent phosphorylation ofGATA-6rdquo Oncogene vol 27 no 36 pp 4921ndash4932 2008

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10 Oxidative Medicine and Cellular Longevity

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[43] S Ghaffari ldquoOxidative stress in the regulation of normal andneoplastic hematopoiesisrdquo Antioxidants amp Redox Signaling vol10 no 11 pp 1923ndash1940 2008

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[46] C Cosentino D Grieco and V Costanzo ldquoATM activates thepentose phosphate pathway promoting anti-oxidant defenceand DNA repairrdquo The EMBO Journal vol 30 no 3 pp 546ndash555 2011

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[53] S Yalcin X Zhang J P Luciano et al ldquoFoxo3 is essential for theregulation of ataxia telangiectasia mutated and oxidative stress-mediated homeostasis of hematopoietic stem cellsrdquoThe Journalof Biological Chemistry vol 283 no 37 pp 25692ndash25705 2008

[54] J Paik Z Ding R Narurkar et al ldquoFoxOs cooperatively regu-late diverse pathways governing neural stem cell homeostasisrdquoCell Stem Cell vol 5 no 5 pp 540ndash553 2009

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stem cells through Wnt120573-catenin signallingrdquo Nature Cell Biol-ogy vol 12 no 10 pp 1007ndash1013 2010

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[79] Y Li and A Jaiswal ldquoRegulation of humanNAD (P)H quinoneoxidoreductase gene Role of AP1 binding site contained withinhuman antioxidant response elementrdquoThe Journal of BiologicalChemistry vol 267 no 21 pp 15097ndash15104 1992

[80] T Prestera P Talalay J Alam et al ldquoParallel induction of hemeoxygenase-1 and chemoprotective phase 2 enzymes by elec-trophiles and antioxidants regulation by upstream antioxidant-responsive elements (ARE)rdquoMolecular Medicine vol 1 no 7 p827 1995

[81] K Itoh K IgarashiNHayashi et al ldquoCloning and characteriza-tion of a novel erythroid cell-derived CNC family transcriptionfactor heterodimerizing with the small Maf family proteinsrdquoMolecular and Cellular Biology vol 15 no 8 pp 4184ndash41931995

[82] R Venugopal and A K Jaiswal ldquoNrf1 and Nrf2 positivelyand c-Fos and Fra1 negatively regulate the human antioxidantresponse element-mediated expression of NAD(P)Hquinoneoxidoreductase1 generdquo Proceedings of the National Academy ofSciences vol 93 no 25 pp 14960ndash14965 1996

[83] K Itoh T Chiba S Takahashi et al ldquoAn Nrf2small Mafheterodimer mediates the induction of phase II detoxifyingenzyme genes through antioxidant response elementsrdquo Bio-chemical and Biophysical Research Communications vol 236no 2 pp 313ndash322 1997

[84] M Ramos-Gomez M-K Kwak P M Dolan et al ldquoSensitivityto carcinogenesis is increased and chemoprotective efficacy ofenzyme inducers is lost in nrf2 transcription factor-deficientmicerdquo Proceedings of the National Academy of Sciences vol 98no 6 pp 3410ndash3415 2001

[85] M-K Kwak N Wakabayashi K Itoh H Motohashi MYamamoto and T W Kensler ldquoModulation of gene expressionby cancer chemopreventive dithiolethiones through the Keap1-Nrf2 pathway Identification of novel gene clusters for cellsurvivalrdquo The Journal of Biological Chemistry vol 278 no 10pp 8135ndash8145 2003

[86] R Hu C Xu G Shen et al ldquoGene expression profiles inducedby cancer chemopreventive isothiocyanate sulforaphane in theliver of C57BL6J mice and C57BL6JNrf2 (minusminus) micerdquo CancerLetters vol 243 no 2 pp 170ndash192 2006

[87] S Chanas Q Jiang and M McMahon ldquoLoss of the Nrf2transcription factor causes a marked reduction in constitutiveand inducible expression of the glutathione S-transferase Gsta1Gsta2 Gstm1 Gstm2 Gstm3 and Gstm4 genes in the livers ofmale and female micerdquo Biochemical Journal vol 365 no 2 pp405ndash416 2002

[88] H-Y Cho S P Reddy A DeBiase M Yamamoto and SR Kleeberger ldquoGene expression profiling of NRF2-mediatedprotection against oxidative injuryrdquo Free Radical Biology andMedicine vol 38 no 3 pp 325ndash343 2005

[89] G Shen C Xu R Hu et al ldquoModulation of nuclear factorE2-related factor 2-mediated gene expression in mice liver andsmall intestine by cancer chemopreventive agent curcuminrdquoMolecular Cancer Therapeutics vol 5 no 1 pp 39ndash51 2006

[90] J D Hayes and A T Dinkova-Kostova ldquoThe Nrf2 regulatorynetwork provides an interface between redox and intermediarymetabolismrdquo Trends in Biochemical Sciences vol 39 no 4 pp199ndash218 2014

[91] A Hayashi H Suzuki K Itoh M Yamamoto and Y SugiyamaldquoTranscription factor Nrf2 is required for the constitutive andinducible expression of multidrug resistance-associated protein1 in mouse embryo fibroblastsrdquo Biochemical and BiophysicalResearch Communications vol 310 no 3 pp 824ndash829 2003

[92] E Jigorel M Le Vee C Boursier-Neyret Y Parmentier andO Fardel ldquoDifferential regulation of sinusoidal and canalicularhepatic drug transporter expression by xenobiotics activating

12 Oxidative Medicine and Cellular Longevity

drug-sensing receptors in primary human hepatocytesrdquo DrugMetabolism and Disposition vol 34 no 10 pp 1756ndash1763 2006

[93] X Wang C R Campos J C Peart et al ldquoNrf2 upregulatesATP binding cassette transporter expression and activity atthe blood-brain and blood-spinal cord barriersrdquo Journal ofNeuroscience vol 34 no 25 pp 8585ndash8593 2014

[94] H Jeong I Ryoo andMKwak ldquoRegulation of the expression ofrenal drug transporters in KEAP1-knockdown human tubularcellsrdquo Toxicology in Vitro vol 29 no 5 pp 884ndash892 2015

[95] M J Canet M D Merrell B G Harder et al ldquoIdentificationof a functional antioxidant response element within the eighthintron of the human ABCC

3generdquo Drug Metabolism and

Disposition vol 43 no 1 pp 93ndash99 2014[96] A Singh H Wu P Zhang C Happel J Ma and S Biswal

ldquoExpression of ABCG2 (BCRP) is regulated by Nrf2 in cancercells that confers side population and chemoresistance pheno-typerdquo Molecular Cancer Therapeutics vol 9 no 8 pp 2365ndash2376 2010

[97] N Wakabayashi K Itoh J Wakabayashi et al ldquoKeap1-nullmutation leads to postnatal lethality due to constitutive Nrf2activationrdquo Nature Genetics vol 35 no 3 pp 238ndash245 2003

[98] S B Cullinan J D Gordan J Jin J W Harper and J A DiehlldquoThe Keap1-BTB protein is an adaptor that bridges Nrf2 to aCul3-based E3 ligase oxidative stress sensing by a Cul3-Keap1ligaserdquoMolecular and Cellular Biology vol 24 no 19 pp 8477ndash8486 2004

[99] D D Zhang and M Hannink ldquoDistinct cysteine residues inkeap1 are required for keap1-dependent ubiquitination of nrf2and for stabilization of nrf2 by chemopreventive agents andoxidative stressrdquoMolecular and Cellular Biology vol 23 no 22pp 8137ndash8151 2003

[100] L M Zipper and R T Mulcahy ldquoThe Keap1 BTBPOZ dimer-ization function is required to sequester Nrf2 in cytoplasmrdquoThe Journal of Biological Chemistry vol 277 no 39 pp 36544ndash36552 2002

[101] M C Jaramillo and D D Zhang ldquoThe emerging role of theNrf2-Keap1 signaling pathway in cancerrdquoGenesampDevelopmentvol 27 no 20 pp 2179ndash2191 2013

[102] K I Tong Y Katoh H Kusunoki K Itoh T Tanaka and MYamamoto ldquoKeap1 recruits Neh2 through binding to ETGEand DLG motifs characterization of the two-site molecularrecognition modelrdquoMolecular and Cellular Biology vol 26 no8 pp 2887ndash2900 2006

[103] K I Tong A Kobayashi F Katsuoka andM Yamamoto ldquoTwo-site substrate recognition model for the Keap1-Nrf2 system ahinge and latch mechanismrdquo Biological Chemistry vol 387 no10-11 pp 1311ndash1320 2006

[104] K I Tong B Padmanabhan A Kobayashi et al ldquoDifferentelectrostatic potentials define ETGE and DLG motifs as hingeand latch in oxidative stress responserdquo Molecular and CellularBiology vol 27 no 21 pp 7511ndash7521 2007

[105] M Kobayashi L Li N Iwamoto et al ldquoThe antioxidant defensesystemKeap1-Nrf2 comprises amultiple sensingmechanism forresponding to a wide range of chemical compoundsrdquoMolecularand Cellular Biology vol 29 no 2 pp 493ndash502 2009

[106] T Yamamoto T Suzuki A Kobayashi et al ldquoPhysiological sig-nificance of reactive cysteine residues of Keap1 in determiningNrf2 activityrdquoMolecular and Cellular Biology vol 28 no 8 pp2758ndash2770 2008

[107] M K Kwak K Itoh M Yamamoto and T W KenslerldquoEnhanced expression of the transcription factorNrf2 by cancer

chemopreventive agents role of antioxidant response element-like sequences in the nrf2 promoterrdquo Molecular and CellularBiology vol 22 no 9 pp 2883ndash2892 2002

[108] T Suzuki T Shibata K Takaya et al ldquoRegulatory nexus ofsynthesis and degradation deciphers cellular Nrf2 expressionlevelsrdquoMolecular and Cellular Biology vol 33 no 12 pp 2402ndash2412 2013

[109] S B Cullinan D Zhang M Hannink E Arvisais R JKaufman and J A Diehl ldquoNrf2 is a direct PERK substrateand effector of PERK-dependent cell survivalrdquo Molecular andCellular Biology vol 23 no 20 pp 7198ndash7209 2003

[110] W Li and A Kong ldquoMolecular mechanisms of Nrf2-mediatedantioxidant responserdquo Molecular Carcinogenesis vol 48 no 2pp 91ndash104 2009

[111] Y J Surh ldquoCancer chemoprevention with dietary phytochemi-calsrdquo Nature Reviews Cancer vol 3 no 10 pp 768ndash780 2003

[112] M Salazar A I Rojo D Velasco R M de Sagarra and ACuadrado ldquoGlycogen synthase kinase-3beta inhibits the xeno-biotic and antioxidant cell response by direct phosphorylationand nuclear exclusion of the transcription factor Nrf2rdquo Journalof Biological Chemistry vol 281 no 21 pp 14841ndash14851 2006

[113] W Chen Z Sun X J Wang et al ldquoDirect interactionbetween Nrf2 and p21(Cip1WAF1) upregulates the Nrf2-mediated antioxidant responserdquo Molecular Cell vol 34 no 6pp 663ndash673 2009

[114] M Komatsu H Kurokawa S Waguri et al ldquoThe selectiveautophagy substrate p62 activates the stress responsive tran-scription factor Nrf2 through inactivation of Keap1rdquoNature CellBiology vol 12 no 3 pp 213ndash223 2010

[115] J D Hayes M McMahon S Chowdhry and A T Dinkova-Kostova ldquoCancer chemoprevention mechanisms mediatedthrough the Keap1-Nrf2 pathwayrdquoAntioxidants amp Redox Signal-ing vol 13 no 11 pp 1713ndash1748 2010

[116] R Hu C L Saw R Yu and A T Kong ldquoRegulation ofNF-E2-related factor 2 signaling for cancer chemopreventionantioxidant coupled with antiinflammatoryrdquo Antioxidants ampRedox Signaling vol 13 no 11 pp 1679ndash1698 2010

[117] M Kwak and T W Kensler ldquoTargeting NRF2 signaling for can-cer chemopreventionrdquo Toxicology and Applied Pharmacologyvol 244 no 1 pp 66ndash76 2010

[118] K Iida K Itoh Y Kumagai et al ldquoNrf2 is essential for thechemopreventive efficacy of oltipraz against urinary bladdercarcinogenesisrdquo Cancer Research vol 64 no 18 pp 6424ndash64312004

[119] T Jiang N Chen F Zhao et al ldquoHigh levels of Nrf2 deter-mine chemoresistance in type II endometrial cancerrdquo CancerResearch vol 70 no 13 pp 5486ndash5496 2010

[120] Y R Kim J EOhM S Kim et al ldquoOncogenicNRF2mutationsin squamous cell carcinomas of oesophagus and skinrdquo TheJournal of Pathology vol 220 no 4 pp 446ndash451 2010

[121] T Shibata A Kokubu M Gotoh et al ldquoGenetic alterationof Keap1 confers constitutive Nrf2 activation and resistance tochemotherapy in gallbladder cancerrdquoGastroenterology vol 135no 4 pp 1358e4ndash1368e4 2008

[122] T Shibata T Ohta K I Tong et al ldquoCancer related mutationsin NRF2 impair its recognition by Keap1-Cul3 E3 ligase andpromote malignancyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 36 pp13568ndash13573 2008

[123] A Singh V Misra R K Thimmulappa et al ldquoDysfunctionalKEAP1-NRF2 interaction in non-small-cell lung cancerrdquo PLoSMedicine vol 3 no 10 article e420 2006

Oxidative Medicine and Cellular Longevity 13

[124] R Wang J An F Ji H Jiao H Sun and D Zhou ldquoHyper-methylation of the Keap1 gene in human lung cancer cell linesand lung cancer tissuesrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 1 pp 151ndash154 2008

[125] P Zhang A Singh S Yegnasubramanian et al ldquoLoss ofKelch-like ECH-associated protein 1 function in prostate cancercells causes chemoresistance and radioresistance and promotestumor growthrdquoMolecular Cancer Therapeutics vol 9 no 2 pp336ndash346 2010

[126] J D Hayes and M McMahon ldquoNRF2 and KEAP1 mutationspermanent activation of an adaptive response in cancerrdquo Trendsin Biochemical Sciences vol 34 no 4 pp 176ndash188 2009

[127] B Padmanabhan K I Tong T Ohta et al ldquoStructural basis fordefects of Keap1 activity provoked by its pointmutations in lungcancerrdquoMolecular Cell vol 21 no 5 pp 689ndash700 2006

[128] Y Inami S Waguri A Sakamoto et al ldquoPersistent activation ofNrf2 throughp62 in hepatocellular carcinoma cellsrdquoTheJournalof Cell Biology vol 193 no 2 pp 275ndash284 2011

[129] G M DeNicola F A Karreth T J Humpton et al ldquoOncogene-induced Nrf2 transcription promotes ROS detoxification andtumorigenesisrdquo Nature vol 475 no 7354 pp 106ndash109 2011

[130] L M Solis C Behrens W Dong et al ldquoNrf2 and Keap1abnormalities in non-small cell lung carcinoma and associationwith clinicopathologic featuresrdquo Clinical Cancer Research vol16 no 14 pp 3743ndash3753 2010

[131] N M Reddy S R Kleeberger H Y Cho et al ldquoDeficiency inNrf2-GSH signaling impairs type II cell growth and enhancessensitivity to oxidantsrdquoAmerican Journal of Respiratory Cell andMolecular Biology vol 37 no 1 pp 3ndash8 2007

[132] N M Reddy S R Kleeberger M Yamamoto et al ldquoGeneticdissection of the Nrf2-dependent redox signaling-regulatedtranscriptional programs of cell proliferation and cytoprotec-tionrdquo Physiological Genomics vol 32 no 1 pp 74ndash81 2007

[133] Y Mitsuishi K Taguchi Y Kawatani et al ldquoNrf2 redirectsglucose and glutamine into anabolic pathways in metabolicreprogrammingrdquo Cancer Cell vol 22 no 1 pp 66ndash79 2012

[134] J Cho SManandharH LeeH Park andMKwak ldquoRole of theNrf2-antioxidant system in cytotoxicity mediated by anticancercisplatin implication to cancer cell resistancerdquo Cancer Lettersvol 260 no 1-2 pp 96ndash108 2008

[135] T Ohta K Iijima M Miyamoto et al ldquoLoss of Keap1 functionactivates Nrf2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008

[136] A Singh M Bodas N Wakabayashi F Bunz and S BiswalldquoGain of Nrf2 function in non-small-cell lung cancer cellsconfers radioresistancerdquoAntioxidantsampRedox Signaling vol 13no 11 pp 1627ndash1637 2010

[137] G-S Shim S Manandhar D-H Shin T Kim and M KwakldquoAcquisition of doxorubicin resistance in ovarian carcinomacells accompanies activation of the NRF2 pathwayrdquo Free RadicalBiology and Medicine vol 47 no 11 pp 1619ndash1631 2009

[138] S Manandhar B Choi K Jung et al ldquoNRF2 inhibitionrepresses ErbB2 signaling in ovarian carcinoma cells implica-tions for tumor growth retardation and docetaxel sensitivityrdquoFree Radical Biology and Medicine vol 52 no 9 pp 1773ndash17852012

[139] T Kim E-g Hur S J Kang et al ldquoNRF2 blockade suppressescolon tumor angiogenesis by inhibiting hypoxia-induced acti-vation ofHIF-1120572rdquoCancer Research vol 71 no 6 pp 2260ndash22752011

[140] L Kinch N V Grishin and J Brugarolas ldquoSuccination of Keap1and activation of Nrf2-dependent antioxidant pathways in FH-deficient papillary renal cell carcinoma type 2rdquo Cancer Cell vol20 no 4 pp 418ndash420 2011

[141] J J Tsai J A Dudakov K Takahashi et al ldquoNrf2 regulateshaematopoietic stem cell functionrdquo Nature Cell Biology vol 15no 3 pp 309ndash316 2013

[142] J H Kim R KThimmulappa V Kumar et al ldquoNRF2-mediatedNotch pathway activation enhances hematopoietic reconsti-tution following myelosuppressive radiationrdquo The Journal ofClinical Investigation vol 124 no 2 pp 730ndash741 2014

[143] J P Chute ldquoNRF2 mitigates radiation-induced hematopoieticdeathrdquo The Journal of Clinical Investigation vol 124 no 3 pp960ndash961 2014

[144] J Jang Y Wang H Kim M A Lalli and K S KosikldquoNrf2 a regulator of the proteasome controls self-renewal andpluripotency in human embryonic stem cellsrdquo Stem Cells vol32 no 10 pp 2616ndash2625 2014

[145] S Murakami R Shimizu P Romeo M Yamamoto and HMotohashi ldquoKeap1-Nrf2 system regulates cell fate determina-tion of hematopoietic stem cellsrdquo Genes to Cells vol 19 no 3pp 239ndash253 2014

[146] C E Hochmuth B Biteau D Bohmann and H Jasper ldquoRedoxregulation by Keap1 and Nrf2 controls intestinal stem cellproliferation inDrosophilardquoCell Stem Cell vol 8 no 2 pp 188ndash199 2011

[147] M K Paul B Bisht D O Darmawan et al ldquoDynamic changesin intracellular ROS levels regulate airway basal stem cellhomeostasis through Nrf2-dependent notch signalingrdquo CellStem Cell vol 15 no 2 pp 199ndash214 2014

[148] L Gambari G Lisignoli L Cattini C Manferdini A Facchiniand F Grassi ldquoSodium hydrosulfide inhibits the differentiationof osteoclast progenitor cells via NRF2-dependent mechanismrdquoPharmacological Research vol 87 pp 99ndash112 2014

[149] J Li D Johnson M Calkins L Wright C Svendsen andJ Johnson ldquoStabilization of Nrf2 by tBHQ confers protectionagainst oxidative stress-induced cell death in human neuralstem cellsrdquo Toxicological Sciences vol 83 no 2 pp 313ndash3282005

[150] A AMerchant A SinghWMatsui and S Biswal ldquoThe redox-sensitive transcription factor Nrf2 regulatesmurine hematopoi-etic stem cell survival independently of ROS levelsrdquo Blood vol118 no 25 pp 6572ndash6579 2011

[151] N Gurusamy D Ray I Lekli and D K Das ldquoRed wineantioxidant resveratrol-modified cardiac stem cells regener-ate infarcted myocardiumrdquo Journal of Cellular and MolecularMedicine vol 14 no 9 pp 2235ndash2239 2010

[152] N Gorbunov G Petrovski N Gurusamy D Ray D H Kimand D K Das ldquoRegeneration of infarcted myocardium withresveratrol-modified cardiac stem cellsrdquo Journal of Cellular andMolecular Medicine vol 16 no 1 pp 174ndash184 2012

[153] C Cai L Teng D Vu et al ldquoThe heme oxygenase 1 inducer(CoPP) protects human cardiac stem cells against apoptosisthrough activation of the extracellular signal-regulated kinase(ERK)NRF2 signaling pathway and cytokine releaserdquo TheJournal of Biological Chemistry vol 287 no 40 pp 33720ndash33732 2012

[154] J Song S M Kang K M Lee and J E Lee ldquoThe protectiveeffect of melatonin on neural stem cell against LPS-inducedinflammationrdquo BioMed Research International vol 2015 ArticleID 854359 13 pages 2015

14 Oxidative Medicine and Cellular Longevity

[155] V Karkkainen Y Pomeshchik E Savchenko et al ldquoNrf2regulates neurogenesis and protects neural progenitor cellsagainst A120573 toxicityrdquo STEM CELLS vol 32 no 7 pp 1904ndash19162014

[156] M Mohammadzadeh R Halabian A Gharehbaghian et alldquoNrf-2 overexpression in mesenchymal stem cells reducesoxidative stress-induced apoptosis and cytotoxicityrdquo Cell Stressand Chaperones vol 17 no 5 pp 553ndash565 2012

[157] Y Takahata T Takarada M Iemata et al ldquoFunctional expres-sion of 1205732 adrenergic receptors responsible for protectionagainst oxidative stress through promotion of glutathione syn-thesis after Nrf2 upregulation in undifferentiated mesenchymalC3H10T12 stem cellsrdquo Journal of Cellular Physiology vol 218no 2 pp 268ndash275 2009

[158] J Zhu H Wang Y Fan et al ldquoKnockdown of nuclear factorerythroid 2-related factor 2 by lentivirus induces differentiationof glioma stem-like cellsrdquo Oncology Reports vol 32 no 3 pp1170ndash1178 2014

[159] J Zhu H Wang Q Sun et al ldquoNrf2 is required to maintain theself-renewal of glioma stem cellsrdquo BMC Cancer vol 13 no 1article 380 2013

[160] B L Emmink A Verheem W J Van Houdt et al ldquoThe secre-tome of colon cancer stem cells contains drug-metabolizingenzymesrdquo Journal of Proteomics vol 91 pp 84ndash96 2013

[161] T Mizuno N Suzuki H Makino et al ldquoCancer stem-like cellsof ovarian clear cell carcinoma are enriched in the ALDH-highpopulation associated with an accelerated scavenging system inreactive oxygen speciesrdquo Gynecologic Oncology vol 137 no 2pp 299ndash305 2015

[162] I-G Ryoo B-H Choi and M-K Kwak ldquoActivation of NRF2by p62 and proteasome reduction in sphere-forming breastcarcinoma cellsrdquo Oncotarget vol 6 no 10 pp 8167ndash8184 2015

[163] T Wu B G Harder P K Wong J E Lang and D D ZhangldquoOxidative stress mammospheres and Nrf2-new implicationfor breast cancer therapyrdquoMolecular Carcinogenesis 2014

[164] B Yang Y Ma and Y Liu ldquoElevated expression of Nrf-2 andABCG2 involved in multi-drug resistance of lung cancer SPcellsrdquo Drug Research 2015

[165] M Zhang A Mathur Y Zhang et al ldquoMithramycin repressesbasal and cigarette smoke-induced expression of abcg2 andinhibits stem cell signaling in lung and esophageal cancer cellsrdquoCancer Research vol 72 no 16 pp 4178ndash4192 2012

[166] C A Del Vecchio Y Feng E S Sokol et al ldquoDe-differentiationconfers multidrug resistance via noncanonical PERK-Nrf2signalingrdquo PLoS Biology vol 12 no 9 Article ID e1001945 2014

[167] D Ren N F Villeneuve T Jiang et al ldquoBrusatol enhancesthe efficacy of chemotherapy by inhibiting the Nrf2-mediateddefense mechanismrdquo Proceedings of the National Academy ofSciences of the United States of America vol 108 no 4 pp 1433ndash1438 2011

[168] A Arlt S Sebens S Krebs et al ldquoInhibition of the Nrf2 tran-scription factor by the alkaloid trigonelline renders pancreaticcancer cells more susceptible to apoptosis through decreasedproteasomal gene expression and proteasome activityrdquo Onco-gene vol 32 no 40 pp 4825ndash4835 2013

[169] A-M Gao Z-P Ke F Shi G Sun and H Chen ldquoChrysinenhances sensitivity of BEL-7402ADM cells to doxorubicin bysuppressing PI3KAktNrf2 and ERKNrf2 pathwayrdquo Chemico-Biological Interactions vol 206 no 1 pp 100ndash108 2013

[170] A M Gao Z P Ke J N Wang J Yang S Chen and HChen ldquoApigenin sensitizes doxorubicin-resistant hepatocellular

carcinoma BEL-7402ADM cells to doxorubicin via inhibitingPI3KAktNrf2 pathwayrdquo Carcinogenesis vol 34 no 8 pp1806ndash1814 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Oxidative Medicine and Cellular Longevity

in the KelchDGR domain of KEAP1 in lung cancer celllines as well as lung cancer tissue samples and demonstratedthat these mutant KEAP1 proteins lost their NRF2 repressivefunction which resulted in NRF2 accumulation Singh et alalso demonstrated that the KelchDGR and IVR domainsof the KEAP1 gene contain multiple somatic mutations andthese mutations were identified in 19 of tumor specimensfrom non-small cell lung cancer patients [123] In gallbladdercancer 4 of 13 patients harbored KEAP1 mutations [122]Shibata et al reported that NRF2 somatic mutations werefound in 107 of primary lung cancer patients and 272of primary head and neck cancer patients [122] Notablythesemutationswere primarily located in theDLG andETGEmotifs and eventually led to the loss of a proper interactionbetween the NRF2 protein and KEAP1 Second CpG islandhypermethylation in the KEAP1 promoter resulted in lowKEAP1 expression in lung cancer cell lines and tumor samples[124]Third in humanhepatocellular carcinoma p62 positivecellular aggregates were found with a frequency of 25 andmost of these tumors retained higher levels of NRF2 andits target gene expression [128] Fourth oncogenes have alsobeen shown to play a role in NRF2 signaling Oncogenicactivation of KRAS (KRASG12D) c-MYC (c-MYCERT12) andBRAF (BRAFV619E) elevates the transcript levels of NRF2 andits target gene expression [129]

It is now widely accepted that aberrant activation ofNRF2 can enhance cancer cell survival and growth in oxi-dizing tumor environments and further promote chemoradioresistance Indeed the prognosis of cancer patientsnegatively correlated with NRF2 levels in the tumor [122130] The favorable effect of NRF2 overexpression on tumorsurvival and growth can be attributed to the increase inNRF2 target antioxidant proteins and their counteractiveeffect on oxidative stress For instance GSH which is adirect target molecule of NRF2 has been shown to be criticalfor cell proliferation [131 132] In addition to its antioxi-dant contribution Mitsuishi et al provided direct evidencedemonstrating that NRF2 alters the cellular metabolism inrelation to anabolic pathways to accelerate cell proliferation[133] Multiple metabolic genes such as those involvedin the pentose phosphate pathway were upregulated byNRF2 through ARE and these changes promoted purinesynthesis glutamine metabolism and NADPH productionfor enhanced cell proliferation

Constitutively high levels of NRF2 have been associatedwith chemoresistance as well as radioresistance Cancer cellswith high NRF2 activity were less sensitive to cytotoxicchemotherapeutics such as cisplatin doxorubicin and 5-fluorouracil through facilitated detoxification of anticanceragents and enhanced antioxidant capacity [101] It is there-fore hypothesized that NRF2 inhibition can enhance thechemosensitivity of cancers NRF2 siRNA could suppresscancer resistance to cisplatin topoisomerase inhibitors and5-fluorouracil [101 122 134 135] Cancer cells with constitu-tively high NRF2 were protected against 120574-radiation inducedtoxicity Moreover siRNA-mediated inhibition of NRF2 innon-small cell lung cancer cell lines substantially enhancedradiosensitivity [136] Additionally NRF2 expression was

increased during the acquisition of chemoresistance In ourprevious study doxorubicin-selected ovarian cancer cellsdemonstrated increased expression of NRF2 and its targetgenes forGSH synthesis andNRF2 inhibition in this resistantcell line could restore doxorubicin sensitivity [137]

Our understanding of the role of NRF2 in cancer cell sig-naling has expanded In particular the relationship betweenoncogenic signaling and NRF2 is noteworthy As men-tioned earlier activation of oncogenes such as KRAS and c-MYC increased the expression of NRF2 presumably throughoncogene-mediated ROS increase and this phenomenonappears to contribute to the maintenance of reduced redoxhomeostasis in cancer cells [129] In ERBB2 (Her2Neu) over-expressing ovarian cancer cells the stable silencing of NRF2repressed ERBB2 expression and its downstream signalingand retarded tumor growth Therefore the inhibition ofNRF2 could sensitize these cells to taxol therapy by repressingERBB2 expression [138] Moreover NRF2 was shown to beassociated with HIF signaling which is a critical factor fortumor angiogenesis When NRF2 was stably knocked downin colon carcinoma cell lines hypoxia-inducible HIF-1120572accumulation was abrogated and consequently angiogenesisand tumor growth were significantly suppressed in NRF2knockdown tumors compared to the control group [139] Intype 2 papillary renal cell cancer which is characterized byloss of the fumarate hydratase gene and consequentmetabolicalteration accumulated fumarate was associated with tumorprogression via NRF2 signaling Fumarate was shown tomodify KEAP1 cysteine residues and elevate NRF2 levelswhich contributed to the growth and progression of type 2papillary renal cell cancer [140] These accumulating linesof evidence suggest that once cells are transformed to theneoplastic stage cancer cells utilize NRF2 signaling to adaptto the stressful tumor environment and to promote survivaland further cancer progression (Figure 2)

8 Involvement of NRF2 Signaling inStem Cell Quiescence and Differentiation

There is considerable evidence to suggest that NRF2 playsa role in normal stem cell biology [141ndash145] For exampleNRF2 activation in HSCs plays a critical role in not only themaintenance of quiescence but also in the determination ofdifferentiation fate [141 145] In Drosophila intestinal stemcells constitutive Nrf2 activation sustained quiescence byreducing the levels of ROS via upregulation of antioxidantgenes such as gclc However in the case of KEAP1-mediatedNrf2 repression high levels of intracellular ROS facilitatedan ablation of the quiescent state in intestinal stem cellsand age-related degeneration in the intestinal epithelium[146] Similarly low intracellular ROS levels are requiredfor the maintenance of quiescence in human airway basalstem cells (ABSCs) When exposed to exogenous ROSquiescent ABSCs enter the proliferation stage Changes inROS levels activate the NRF2-Notch pathway which resultsin self-renewal and protection of ABSCs from ROS-inducedhyperproliferation and senescence Moreover the quiescent

Oxidative Medicine and Cellular Longevity 7

NRF2 sMaf

GCLCGCLMNQO1SODs

Antioxidant enzymes Drug transportersMetabolic enzymes

ARE

Tumor growthprogression

MDRsMRPsBCRP

G6PDPGDME1

Cell survival Drug effluxProliferation

Target genes

Figure 2 Implications of NRF2 signaling in cancer NRF2 coordi-nates the expression of genes associated with cellular redox regu-lation metabolism and xenobiotic efflux and thereby its aberrantactivation promotes cancer cell survival proliferation and anti-cancer drug resistance GCLM glutamate cysteine ligase modifiersubunit GCLC glutamate cysteine ligase catalytic subunit NQO1NAD(P)Hquinone oxidoreductase 1 SODs superoxide dismutasesG6PDH glucose-6-phosphate dehydrogenase PGD phosphoglu-conate dehydrogenase ME1 malic enzyme 1 MDRs multidrugresistance proteinsMRPsmultidrug resistance-associated proteinsBCRP breast cancer resistance protein

state of ABSCs was maintained by NRF2 activation [147]In osteoclast progenitor cells hydrogen sulfide (H

2S) inhib-

ited human osteoclast differentiation by NRF2-dependentinduction of peroxiredoxin 1 and NQO1 These resultswere further confirmed using NRF2 activators includingsulforaphane and t-BHQ [148] It is also notable that NRF2participates in the regulation of cell fate determination ofHSCsMurakami et al demonstrated that HSCs derived fromKEAP1-deficientmice exhibited preferred differentiation intothe granulocyte-monocyte lineage rather than differentiatinginto the erythroid-lymphoid lineage [145]

Up to now numerous studies have demonstrated thatNRF2 plays a protective role against various stressors instem cells In neural stem cells overexpression of NRF2or pharmacological NRF2 activation prevented necroticcell death [149] In an animal study nrf2-deficient miceshowed defective stem cell function HSCs from nrf2minusminus miceexpressed lower levels of prosurvival cytokines and exhib-ited spontaneous apoptosis [150] Ionizing radiation-inducedmyelosuppression and mortality were mitigated throughNRF2-mediated Notch signaling activation in HSCs [142]Similarly resveratrol-inducedNRF2 expression improved thesurvival of cardiac stem cells and consequently regeneratedinfarcted myocardium [151 152] The heme oxygenase 1(HO-1) inducer cobalt protoporphyrin (CoPP) elicited anantiapoptotic effect on cardiac stem cells via activation of the

ERK-NRF2 pathway [153] In neural stem cells NRF2 activa-tion by melatonin or t-BHQ ameliorated lipopolysaccharide(LPS) orH

2O2induced cell death [149 154] In addition amy-

loid 120573-mediated neural stem cell death could be alleviated byexogenous NRF2 transduction which was accompanied byincreased expression of GCLC NQO-1 andHO-1This studyalso demonstrated that neuronal differentiation of neuralstem cells is enhanced by NRF2 activation [155] Similar toneural stem cells NRF2 has a protective role against hypoxicand oxidative stress conditions in undifferentiated MSCsTreatment of the murine mesenchymal stem cell line withadrenaline increased the mRNA expression of nrf2 gclc andxCT leading to an increase in GSH levels and the preventionof ROS-induced cytotoxicity [156 157]

9 Potential Implication of NRF2 inCSC Maintenance and Resistance

The role of NRF2 in CSC biology is now beginning tobe unveiled Similar to the case of normal stem cells itwas shown that NRF2 contributes to CSC stemness bymaintaining their self-renewal capacity and protecting themfrom chemoradiotherapy Achuthan et al established stablechemotherapy-resistant breast cancer cells and observed thatthese cells expressed higher levels of CD133 and OCT-4indicating that these cells exhibit CSC phenotype [34] Ofnote it was shown that ROS levels were relatively low inthese drug-selected cells presumably due to higher levelsof antioxidant enzymes such as SOD1 and GPX12 NRF2protein stabilizationwas associatedwith high levels of antiox-idant enzymes As an underlying molecular mechanismdiminished proteasome activity and increased p21 levelsappear to stabilize the NRF2 protein in these stem-like cellsEvidently p21 knockdown repressed the mammosphere-forming potential of these stem-like breast cancer cellsSimilarly a study by Zhu et al showed the involvement ofNRF2 in glioblastoma stem cells that were isolated fromhuman surgical glioblastoma specimens NRF2 knockdownin glioblastoma stem cells inhibited cell proliferation andneurosphere formation and further suppressed SOX2 expres-sion Moreover NRF2 knockdown changed the cell cycledistribution to the G2 phase and significantly attenuated thetumorigenecity of glioblastoma stem cells [158 159] Theseresults are providing evidence that NRF2 is necessary formaintenance of the self-renewal capacity of glioblastomastem cells

On the other hand activation of NRF2 signaling has beendemonstrated in different types of CSC models includinglung esophageal breast ovarian and colon CSCs and thisis closely correlated with themaintenance of low intracellularROS levels and chemoresistance of CSCs [160ndash165] In lungand esophageal cancer cells cigarette smoke condensateincreased the SP as well as BCRP expression which arehallmarks of CSCs [165] Promoter analysis revealed thatBCRP expression was associated with elevated levels ofNRF2 aryl hydrocarbon receptor (AhR) and specificityprotein 1 (SP1) Additionally this study demonstrated thatmithramycin diminished BCRP expression via repression of

8 Oxidative Medicine and Cellular Longevity

Low ROS

Increased drug efflux

SurvivalChemoresistance

CSC

TumorRecurrenceProgression

Radioresistance

PERKuarr

p21uarr

p62uarr

Proteasome darr

NRF2 uarr

Figure 3 Potential roles of NRF2 signaling in CSCs InCSC-like cellmodels NRF2 is activated through multiple molecular mechanismsin a context-dependent manner The upregulation of competingproteins such as p62 and p21 activation of PERK or repressedproteasome function were shown to enhance NRF2 activity inthese models Elevated NRF2 levels in CSCs can contribute tothe maintenance of low ROS by upregulating multiple antioxidantgenes In addition NRF2-mediated expression of ABC transporterselicits efflux of anticancer drugs from cancer cells Overall activatedNRF2 signaling facilitates CSCs survival and stress resistance andconsequently it can be suggested that CSCs with high NRF2 activityplay a crucial role in tumor recurrence and further progression

NRF2 AhR and SP1 and thereby inhibiting the expressionof genes associated with CSC-related pathways resulting inreduced proliferation and tumorigenecity Similarly it wasreported that lung cancer SP cells exhibited high levels ofNRF2 and BCRP expression These SP cells were highlytumorigenic and possessed self-renewal capacity comparedto non-SP cells [164] Emmink et al performed a proteomeanalysis on collected secretome from highly tumorigenicCSCs and their corresponding nontumorigenic differentiatedcells both of which were established from human colorectalspecimens [160] Subsequent bioinformatic analysis revealedthat the CSC secretome contained a large amount of proteinsassociated with cell survival and protein quality controlcompared to differentiated tumor cells Notably the CSCsecretome contained an NRF2 antioxidant and detoxifyingprotein signature in that it included elevated levels ofGCLC GPX23 and TXNRD1 This study provided novelevidence that CSCs secrete NRF2 target antioxidant proteinsto counteract extracellular stressors and chemotherapeuticsIn patients with ovarian clear cell carcinoma the expressionof the CSC maker aldehyde dehydrogenase-1 (ALDH1) wasstrongly correlated with an advanced clinical stage andreduced progression free survival [161] Ovarian clear cellcarcinoma cells with high ALDH1 expressionmaintained lowlevels of ROS compared to ALDH-low cells and these cellswere shown to express higher levels of NRF2 and its targetgenes

Two recent studies have demonstratedNRF2 activation insphere cultures of breast cancer cells that is one of models ofCSCs Wu et al showed that mammospheres derived fromMCF7 and MDA-MB231 breast cancer cell lines exhibitedlower ROS levels compared to their monolayer counterpartsThey also showed that levels of NRF2 and target genes such

as NQO1 and GCLM were elevated in mammospheres [163]Similarly our group has shown substantially elevated NRF2protein levels along with increased expression of antioxidantgenes (eg HO-1 and GPX2) and drug efflux transporters(eg MRP2 and BCRP) in sphere cultures of breast cancercells NRF2 accumulation was also observed in sphere-cultured ovarian and colon cancer cells However shRNA-mediated downregulation ofNRF2 led to decreased chemore-sistance of mammospheres presumably due to reduced levelsof antioxidant genes and drug transporters High ROS levelsin NRF2 knockdown mammospheres caused sphere growthretardation and apoptosis Coherently ablation of ABCtransporter induction in NRF2 knockdown mammospheressensitized to anticancer agents [162] Additionally this studyprovided evidence that increased NRF2 protein expression inmammospheres can be linked to 26S proteasome reductionand p62 accumulation In particular knockdown of p62 inMCF7 mammospheres significantly attenuated NRF2 eleva-tion

Surviving dedifferentiated breast cancer cells after chem-otherapy treatment retained high levels of NRF2 activationsimilar to other CSCs However NRF2 activation was medi-ated by a noncanonical pathway Levels of PERKwere high indedifferentiated cancer cells and this in turn phosphorylatedand activated NRF2 signaling to maintain low cellular ROSlevels and to express ABC transporters In agreement withthese findings clinical observations revealed that the PERKpathway gene signature is related to chemoresistance andreduced patient survival [166]

10 Concluding Remarks

Recent studies have started to uncover the role of ROS signal-ing in the biology of CSCs which is related to tumorigenecitytumor progression and relapse Expression of the transcrip-tion factor NRF2 a master regulator of antioxidant genesexpression is increased in different models of CSCs and thiselevation is likely to promote CSC maintenance and survivalin an oxidizing tumormicroenvironment In additionNRF2-mediated overexpression of ABC transporters particularlythe CSC marker BCRP may play a critical role in themultidrug resistance of CSCsThese findings combined withthe increasing evidence showing the alteration of KEAP1-NRF2 signaling in cancer cells suggest a novel role of NRF2in CSC maintenance and survival (Figure 3)

One important question that arises from the current stud-ies is whether it is possible to design CSC-targeted therapiesthrough regulation of theNRF2 pathway and its related redoxhomeostasis inCSCs Recent studies provide several potentialclues for addressing this question the naturally occurringalkaloid brusatol could reduce the growth and chemoresis-tance of breast CSCs [163] Treatment ofmammospheres withbrusatol elevated ROS levels and promoted taxol-inducedgrowth retardation and cell death The NRF2 repressivemechanism of brusatol has not been clearly elucidated but itappears to be independent of KEAP1-mediated degradation[167] In addition to brusatol natural compounds such as

Oxidative Medicine and Cellular Longevity 9

chrysin apigenin luteolin and trigonelline are known toinhibit NRF2 signaling in several types of cancer cells [102168ndash170] and therefore the development of NRF2 inhibitorswith characterized modes of action will enable efficient tar-geting of the redox homeostasis system as well as multidrugresistance systems in CSCs

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This studywas financially supported by theNational ResearchFoundation (NRF) funded by the Ministry of Science ICT ampFuture Planning (NRF-2013R1A2A2A01015497)

References

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[2] P Storz ldquoForkhead homeobox type O transcription factorsin the responses to oxidative stressrdquo Antioxidants amp RedoxSignaling vol 14 no 4 pp 593ndash605 2011

[3] R S Balaban S Nemoto and T Finkel ldquoMitochondria oxi-dants and agingrdquo Cell vol 120 no 4 pp 483ndash495 2005

[4] L A Sena and N S Chandel ldquoPhysiological roles of mitochon-drial reactive oxygen speciesrdquoMolecular Cell vol 48 no 2 pp158ndash167 2012

[5] P T Schumacker ldquoReactive oxygen species in cancer cells liveby the sword die by the swordrdquo Cancer Cell vol 10 no 3 pp175ndash176 2006

[6] S Toyokuni K Okamoto J Yodoi and H Hiai ldquoPersistentoxidative stress in cancerrdquo FEBS Letters vol 358 no 1 pp 1ndash3 1995

[7] L Behrend G Henderson and R Zwacka ldquoReactive oxy-gen species in oncogenic transformationrdquo Biochemical SocietyTransactions vol 31 no 6 pp 1441ndash1444 2003

[8] E O Hileman J Liu M Albitar M J Keating and P HuangldquoIntrinsic oxidative stress in cancer cells a biochemical basis fortherapeutic selectivityrdquo Cancer Chemotherapy and Pharmacol-ogy vol 53 no 3 pp 209ndash219 2004

[9] H PelicanoD Carney and PHuang ldquoROS stress in cancer cellsand therapeutic implicationsrdquo Drug Resistance Updates vol 7no 2 pp 97ndash110 2004

[10] Y Adachi Y Shibai J Mitsushita W H Shang K Hirose andT Kamata ldquoOncogenic Ras upregulates NADPH oxidase 1 geneexpression through MEK-ERK-dependent phosphorylation ofGATA-6rdquo Oncogene vol 27 no 36 pp 4921ndash4932 2008

[11] J Mitsushita J D Lambeth and T Kamata ldquoThe superoxide-generating oxidase Nox1 is functionally required for Ras onco-gene transformationrdquoCancer Research vol 64 no 10 pp 3580ndash3585 2004

[12] O Vafa M Wade S Kern et al ldquoc-Myc can induce DNAdamage increase reactive oxygen species and mitigate p53function a mechanism for oncogene-induced genetic instabil-ityrdquoMolecular Cell vol 9 no 5 pp 1031ndash1044 2002

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10 Oxidative Medicine and Cellular Longevity

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[40] K Wang T Zhang Q Dong E C Nice C Huang and Y WeildquoRedox homeostasis the linchpin in stem cell self-renewal anddifferentiationrdquo Cell Death and Disease vol 4 no 3 p e5372013

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[43] S Ghaffari ldquoOxidative stress in the regulation of normal andneoplastic hematopoiesisrdquo Antioxidants amp Redox Signaling vol10 no 11 pp 1923ndash1940 2008

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[46] C Cosentino D Grieco and V Costanzo ldquoATM activates thepentose phosphate pathway promoting anti-oxidant defenceand DNA repairrdquo The EMBO Journal vol 30 no 3 pp 546ndash555 2011

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[49] Y Wang Y Zhou and D T Graves ldquoFOXO transcription fac-tors their clinical significance and regulationrdquoBioMed ResearchInternational vol 2014 Article ID 925350 13 pages 2014

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[54] J Paik Z Ding R Narurkar et al ldquoFoxOs cooperatively regu-late diverse pathways governing neural stem cell homeostasisrdquoCell Stem Cell vol 5 no 5 pp 540ndash553 2009

[55] V M Renault V A Rafalski A A Morgan et al ldquoFoxO3regulates neural stem cell homeostasisrdquo Cell Stem Cell vol 5no 5 pp 527ndash539 2009

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[58] M M Juntilla V D Patil M Calamito R P Joshi M JBirnbaum and G A Koretzky ldquoAKT1 and AKT2 maintainhematopoietic stem cell function by regulating reactive oxygenspeciesrdquo Blood vol 115 no 20 pp 4030ndash4038 2010

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stem cells through Wnt120573-catenin signallingrdquo Nature Cell Biol-ogy vol 12 no 10 pp 1007ndash1013 2010

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[72] N Haraguchi H Ishii K Mimori et al ldquoCD13 is a therapeutictarget in human liver cancer stem cellsrdquo Journal of ClinicalInvestigation vol 120 no 9 pp 3326ndash3339 2010

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[74] K Naka T Hoshii T Muraguchi et al ldquoTGF-120573ndashFOXO sig-nalling maintains leukaemia-initiating cells in chronic myeloidleukaemiardquo Nature vol 463 no 7281 pp 676ndash680 2010

[75] J Zhou J Wulfkuhle H Zhang et al ldquoActivation of thePTENmTORSTAT3 pathway in breast cancer stem-like cellsis required for viability and maintenancerdquo Proceedings of theNational Academy of Sciences vol 104 no 41 pp 16158ndash161632007

[76] A Dubrovska S Kim R J Salamone et al ldquoThe role ofPTENAktPI3K signaling in the maintenance and viability ofprostate cancer stem-like cell populationsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 106 no 1 pp 268ndash273 2009

[77] H Yin and J Glass ldquoThe phenotypic radiation resistance ofCS44+CD24minusor low breast cancer cells is mediated through theenhanced activation of ATM signalingrdquo PLoS ONE vol 6 no9 Article ID e24080 2011

[78] T H Rushmore and C Pickett ldquoTranscriptional regulation ofthe rat glutathione S-transferase Ya subunit gene Characteri-zation of a xenobiotic-responsive element controlling inducibleexpression by phenolic antioxidantsrdquo The Journal of BiologicalChemistry vol 265 no 24 pp 14648ndash14653 1990

[79] Y Li and A Jaiswal ldquoRegulation of humanNAD (P)H quinoneoxidoreductase gene Role of AP1 binding site contained withinhuman antioxidant response elementrdquoThe Journal of BiologicalChemistry vol 267 no 21 pp 15097ndash15104 1992

[80] T Prestera P Talalay J Alam et al ldquoParallel induction of hemeoxygenase-1 and chemoprotective phase 2 enzymes by elec-trophiles and antioxidants regulation by upstream antioxidant-responsive elements (ARE)rdquoMolecular Medicine vol 1 no 7 p827 1995

[81] K Itoh K IgarashiNHayashi et al ldquoCloning and characteriza-tion of a novel erythroid cell-derived CNC family transcriptionfactor heterodimerizing with the small Maf family proteinsrdquoMolecular and Cellular Biology vol 15 no 8 pp 4184ndash41931995

[82] R Venugopal and A K Jaiswal ldquoNrf1 and Nrf2 positivelyand c-Fos and Fra1 negatively regulate the human antioxidantresponse element-mediated expression of NAD(P)Hquinoneoxidoreductase1 generdquo Proceedings of the National Academy ofSciences vol 93 no 25 pp 14960ndash14965 1996

[83] K Itoh T Chiba S Takahashi et al ldquoAn Nrf2small Mafheterodimer mediates the induction of phase II detoxifyingenzyme genes through antioxidant response elementsrdquo Bio-chemical and Biophysical Research Communications vol 236no 2 pp 313ndash322 1997

[84] M Ramos-Gomez M-K Kwak P M Dolan et al ldquoSensitivityto carcinogenesis is increased and chemoprotective efficacy ofenzyme inducers is lost in nrf2 transcription factor-deficientmicerdquo Proceedings of the National Academy of Sciences vol 98no 6 pp 3410ndash3415 2001

[85] M-K Kwak N Wakabayashi K Itoh H Motohashi MYamamoto and T W Kensler ldquoModulation of gene expressionby cancer chemopreventive dithiolethiones through the Keap1-Nrf2 pathway Identification of novel gene clusters for cellsurvivalrdquo The Journal of Biological Chemistry vol 278 no 10pp 8135ndash8145 2003

[86] R Hu C Xu G Shen et al ldquoGene expression profiles inducedby cancer chemopreventive isothiocyanate sulforaphane in theliver of C57BL6J mice and C57BL6JNrf2 (minusminus) micerdquo CancerLetters vol 243 no 2 pp 170ndash192 2006

[87] S Chanas Q Jiang and M McMahon ldquoLoss of the Nrf2transcription factor causes a marked reduction in constitutiveand inducible expression of the glutathione S-transferase Gsta1Gsta2 Gstm1 Gstm2 Gstm3 and Gstm4 genes in the livers ofmale and female micerdquo Biochemical Journal vol 365 no 2 pp405ndash416 2002

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[89] G Shen C Xu R Hu et al ldquoModulation of nuclear factorE2-related factor 2-mediated gene expression in mice liver andsmall intestine by cancer chemopreventive agent curcuminrdquoMolecular Cancer Therapeutics vol 5 no 1 pp 39ndash51 2006

[90] J D Hayes and A T Dinkova-Kostova ldquoThe Nrf2 regulatorynetwork provides an interface between redox and intermediarymetabolismrdquo Trends in Biochemical Sciences vol 39 no 4 pp199ndash218 2014

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12 Oxidative Medicine and Cellular Longevity

drug-sensing receptors in primary human hepatocytesrdquo DrugMetabolism and Disposition vol 34 no 10 pp 1756ndash1763 2006

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[94] H Jeong I Ryoo andMKwak ldquoRegulation of the expression ofrenal drug transporters in KEAP1-knockdown human tubularcellsrdquo Toxicology in Vitro vol 29 no 5 pp 884ndash892 2015

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3generdquo Drug Metabolism and

Disposition vol 43 no 1 pp 93ndash99 2014[96] A Singh H Wu P Zhang C Happel J Ma and S Biswal

ldquoExpression of ABCG2 (BCRP) is regulated by Nrf2 in cancercells that confers side population and chemoresistance pheno-typerdquo Molecular Cancer Therapeutics vol 9 no 8 pp 2365ndash2376 2010

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[98] S B Cullinan J D Gordan J Jin J W Harper and J A DiehlldquoThe Keap1-BTB protein is an adaptor that bridges Nrf2 to aCul3-based E3 ligase oxidative stress sensing by a Cul3-Keap1ligaserdquoMolecular and Cellular Biology vol 24 no 19 pp 8477ndash8486 2004

[99] D D Zhang and M Hannink ldquoDistinct cysteine residues inkeap1 are required for keap1-dependent ubiquitination of nrf2and for stabilization of nrf2 by chemopreventive agents andoxidative stressrdquoMolecular and Cellular Biology vol 23 no 22pp 8137ndash8151 2003

[100] L M Zipper and R T Mulcahy ldquoThe Keap1 BTBPOZ dimer-ization function is required to sequester Nrf2 in cytoplasmrdquoThe Journal of Biological Chemistry vol 277 no 39 pp 36544ndash36552 2002

[101] M C Jaramillo and D D Zhang ldquoThe emerging role of theNrf2-Keap1 signaling pathway in cancerrdquoGenesampDevelopmentvol 27 no 20 pp 2179ndash2191 2013

[102] K I Tong Y Katoh H Kusunoki K Itoh T Tanaka and MYamamoto ldquoKeap1 recruits Neh2 through binding to ETGEand DLG motifs characterization of the two-site molecularrecognition modelrdquoMolecular and Cellular Biology vol 26 no8 pp 2887ndash2900 2006

[103] K I Tong A Kobayashi F Katsuoka andM Yamamoto ldquoTwo-site substrate recognition model for the Keap1-Nrf2 system ahinge and latch mechanismrdquo Biological Chemistry vol 387 no10-11 pp 1311ndash1320 2006

[104] K I Tong B Padmanabhan A Kobayashi et al ldquoDifferentelectrostatic potentials define ETGE and DLG motifs as hingeand latch in oxidative stress responserdquo Molecular and CellularBiology vol 27 no 21 pp 7511ndash7521 2007

[105] M Kobayashi L Li N Iwamoto et al ldquoThe antioxidant defensesystemKeap1-Nrf2 comprises amultiple sensingmechanism forresponding to a wide range of chemical compoundsrdquoMolecularand Cellular Biology vol 29 no 2 pp 493ndash502 2009

[106] T Yamamoto T Suzuki A Kobayashi et al ldquoPhysiological sig-nificance of reactive cysteine residues of Keap1 in determiningNrf2 activityrdquoMolecular and Cellular Biology vol 28 no 8 pp2758ndash2770 2008

[107] M K Kwak K Itoh M Yamamoto and T W KenslerldquoEnhanced expression of the transcription factorNrf2 by cancer

chemopreventive agents role of antioxidant response element-like sequences in the nrf2 promoterrdquo Molecular and CellularBiology vol 22 no 9 pp 2883ndash2892 2002

[108] T Suzuki T Shibata K Takaya et al ldquoRegulatory nexus ofsynthesis and degradation deciphers cellular Nrf2 expressionlevelsrdquoMolecular and Cellular Biology vol 33 no 12 pp 2402ndash2412 2013

[109] S B Cullinan D Zhang M Hannink E Arvisais R JKaufman and J A Diehl ldquoNrf2 is a direct PERK substrateand effector of PERK-dependent cell survivalrdquo Molecular andCellular Biology vol 23 no 20 pp 7198ndash7209 2003

[110] W Li and A Kong ldquoMolecular mechanisms of Nrf2-mediatedantioxidant responserdquo Molecular Carcinogenesis vol 48 no 2pp 91ndash104 2009

[111] Y J Surh ldquoCancer chemoprevention with dietary phytochemi-calsrdquo Nature Reviews Cancer vol 3 no 10 pp 768ndash780 2003

[112] M Salazar A I Rojo D Velasco R M de Sagarra and ACuadrado ldquoGlycogen synthase kinase-3beta inhibits the xeno-biotic and antioxidant cell response by direct phosphorylationand nuclear exclusion of the transcription factor Nrf2rdquo Journalof Biological Chemistry vol 281 no 21 pp 14841ndash14851 2006

[113] W Chen Z Sun X J Wang et al ldquoDirect interactionbetween Nrf2 and p21(Cip1WAF1) upregulates the Nrf2-mediated antioxidant responserdquo Molecular Cell vol 34 no 6pp 663ndash673 2009

[114] M Komatsu H Kurokawa S Waguri et al ldquoThe selectiveautophagy substrate p62 activates the stress responsive tran-scription factor Nrf2 through inactivation of Keap1rdquoNature CellBiology vol 12 no 3 pp 213ndash223 2010

[115] J D Hayes M McMahon S Chowdhry and A T Dinkova-Kostova ldquoCancer chemoprevention mechanisms mediatedthrough the Keap1-Nrf2 pathwayrdquoAntioxidants amp Redox Signal-ing vol 13 no 11 pp 1713ndash1748 2010

[116] R Hu C L Saw R Yu and A T Kong ldquoRegulation ofNF-E2-related factor 2 signaling for cancer chemopreventionantioxidant coupled with antiinflammatoryrdquo Antioxidants ampRedox Signaling vol 13 no 11 pp 1679ndash1698 2010

[117] M Kwak and T W Kensler ldquoTargeting NRF2 signaling for can-cer chemopreventionrdquo Toxicology and Applied Pharmacologyvol 244 no 1 pp 66ndash76 2010

[118] K Iida K Itoh Y Kumagai et al ldquoNrf2 is essential for thechemopreventive efficacy of oltipraz against urinary bladdercarcinogenesisrdquo Cancer Research vol 64 no 18 pp 6424ndash64312004

[119] T Jiang N Chen F Zhao et al ldquoHigh levels of Nrf2 deter-mine chemoresistance in type II endometrial cancerrdquo CancerResearch vol 70 no 13 pp 5486ndash5496 2010

[120] Y R Kim J EOhM S Kim et al ldquoOncogenicNRF2mutationsin squamous cell carcinomas of oesophagus and skinrdquo TheJournal of Pathology vol 220 no 4 pp 446ndash451 2010

[121] T Shibata A Kokubu M Gotoh et al ldquoGenetic alterationof Keap1 confers constitutive Nrf2 activation and resistance tochemotherapy in gallbladder cancerrdquoGastroenterology vol 135no 4 pp 1358e4ndash1368e4 2008

[122] T Shibata T Ohta K I Tong et al ldquoCancer related mutationsin NRF2 impair its recognition by Keap1-Cul3 E3 ligase andpromote malignancyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 36 pp13568ndash13573 2008

[123] A Singh V Misra R K Thimmulappa et al ldquoDysfunctionalKEAP1-NRF2 interaction in non-small-cell lung cancerrdquo PLoSMedicine vol 3 no 10 article e420 2006

Oxidative Medicine and Cellular Longevity 13

[124] R Wang J An F Ji H Jiao H Sun and D Zhou ldquoHyper-methylation of the Keap1 gene in human lung cancer cell linesand lung cancer tissuesrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 1 pp 151ndash154 2008

[125] P Zhang A Singh S Yegnasubramanian et al ldquoLoss ofKelch-like ECH-associated protein 1 function in prostate cancercells causes chemoresistance and radioresistance and promotestumor growthrdquoMolecular Cancer Therapeutics vol 9 no 2 pp336ndash346 2010

[126] J D Hayes and M McMahon ldquoNRF2 and KEAP1 mutationspermanent activation of an adaptive response in cancerrdquo Trendsin Biochemical Sciences vol 34 no 4 pp 176ndash188 2009

[127] B Padmanabhan K I Tong T Ohta et al ldquoStructural basis fordefects of Keap1 activity provoked by its pointmutations in lungcancerrdquoMolecular Cell vol 21 no 5 pp 689ndash700 2006

[128] Y Inami S Waguri A Sakamoto et al ldquoPersistent activation ofNrf2 throughp62 in hepatocellular carcinoma cellsrdquoTheJournalof Cell Biology vol 193 no 2 pp 275ndash284 2011

[129] G M DeNicola F A Karreth T J Humpton et al ldquoOncogene-induced Nrf2 transcription promotes ROS detoxification andtumorigenesisrdquo Nature vol 475 no 7354 pp 106ndash109 2011

[130] L M Solis C Behrens W Dong et al ldquoNrf2 and Keap1abnormalities in non-small cell lung carcinoma and associationwith clinicopathologic featuresrdquo Clinical Cancer Research vol16 no 14 pp 3743ndash3753 2010

[131] N M Reddy S R Kleeberger H Y Cho et al ldquoDeficiency inNrf2-GSH signaling impairs type II cell growth and enhancessensitivity to oxidantsrdquoAmerican Journal of Respiratory Cell andMolecular Biology vol 37 no 1 pp 3ndash8 2007

[132] N M Reddy S R Kleeberger M Yamamoto et al ldquoGeneticdissection of the Nrf2-dependent redox signaling-regulatedtranscriptional programs of cell proliferation and cytoprotec-tionrdquo Physiological Genomics vol 32 no 1 pp 74ndash81 2007

[133] Y Mitsuishi K Taguchi Y Kawatani et al ldquoNrf2 redirectsglucose and glutamine into anabolic pathways in metabolicreprogrammingrdquo Cancer Cell vol 22 no 1 pp 66ndash79 2012

[134] J Cho SManandharH LeeH Park andMKwak ldquoRole of theNrf2-antioxidant system in cytotoxicity mediated by anticancercisplatin implication to cancer cell resistancerdquo Cancer Lettersvol 260 no 1-2 pp 96ndash108 2008

[135] T Ohta K Iijima M Miyamoto et al ldquoLoss of Keap1 functionactivates Nrf2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008

[136] A Singh M Bodas N Wakabayashi F Bunz and S BiswalldquoGain of Nrf2 function in non-small-cell lung cancer cellsconfers radioresistancerdquoAntioxidantsampRedox Signaling vol 13no 11 pp 1627ndash1637 2010

[137] G-S Shim S Manandhar D-H Shin T Kim and M KwakldquoAcquisition of doxorubicin resistance in ovarian carcinomacells accompanies activation of the NRF2 pathwayrdquo Free RadicalBiology and Medicine vol 47 no 11 pp 1619ndash1631 2009

[138] S Manandhar B Choi K Jung et al ldquoNRF2 inhibitionrepresses ErbB2 signaling in ovarian carcinoma cells implica-tions for tumor growth retardation and docetaxel sensitivityrdquoFree Radical Biology and Medicine vol 52 no 9 pp 1773ndash17852012

[139] T Kim E-g Hur S J Kang et al ldquoNRF2 blockade suppressescolon tumor angiogenesis by inhibiting hypoxia-induced acti-vation ofHIF-1120572rdquoCancer Research vol 71 no 6 pp 2260ndash22752011

[140] L Kinch N V Grishin and J Brugarolas ldquoSuccination of Keap1and activation of Nrf2-dependent antioxidant pathways in FH-deficient papillary renal cell carcinoma type 2rdquo Cancer Cell vol20 no 4 pp 418ndash420 2011

[141] J J Tsai J A Dudakov K Takahashi et al ldquoNrf2 regulateshaematopoietic stem cell functionrdquo Nature Cell Biology vol 15no 3 pp 309ndash316 2013

[142] J H Kim R KThimmulappa V Kumar et al ldquoNRF2-mediatedNotch pathway activation enhances hematopoietic reconsti-tution following myelosuppressive radiationrdquo The Journal ofClinical Investigation vol 124 no 2 pp 730ndash741 2014

[143] J P Chute ldquoNRF2 mitigates radiation-induced hematopoieticdeathrdquo The Journal of Clinical Investigation vol 124 no 3 pp960ndash961 2014

[144] J Jang Y Wang H Kim M A Lalli and K S KosikldquoNrf2 a regulator of the proteasome controls self-renewal andpluripotency in human embryonic stem cellsrdquo Stem Cells vol32 no 10 pp 2616ndash2625 2014

[145] S Murakami R Shimizu P Romeo M Yamamoto and HMotohashi ldquoKeap1-Nrf2 system regulates cell fate determina-tion of hematopoietic stem cellsrdquo Genes to Cells vol 19 no 3pp 239ndash253 2014

[146] C E Hochmuth B Biteau D Bohmann and H Jasper ldquoRedoxregulation by Keap1 and Nrf2 controls intestinal stem cellproliferation inDrosophilardquoCell Stem Cell vol 8 no 2 pp 188ndash199 2011

[147] M K Paul B Bisht D O Darmawan et al ldquoDynamic changesin intracellular ROS levels regulate airway basal stem cellhomeostasis through Nrf2-dependent notch signalingrdquo CellStem Cell vol 15 no 2 pp 199ndash214 2014

[148] L Gambari G Lisignoli L Cattini C Manferdini A Facchiniand F Grassi ldquoSodium hydrosulfide inhibits the differentiationof osteoclast progenitor cells via NRF2-dependent mechanismrdquoPharmacological Research vol 87 pp 99ndash112 2014

[149] J Li D Johnson M Calkins L Wright C Svendsen andJ Johnson ldquoStabilization of Nrf2 by tBHQ confers protectionagainst oxidative stress-induced cell death in human neuralstem cellsrdquo Toxicological Sciences vol 83 no 2 pp 313ndash3282005

[150] A AMerchant A SinghWMatsui and S Biswal ldquoThe redox-sensitive transcription factor Nrf2 regulatesmurine hematopoi-etic stem cell survival independently of ROS levelsrdquo Blood vol118 no 25 pp 6572ndash6579 2011

[151] N Gurusamy D Ray I Lekli and D K Das ldquoRed wineantioxidant resveratrol-modified cardiac stem cells regener-ate infarcted myocardiumrdquo Journal of Cellular and MolecularMedicine vol 14 no 9 pp 2235ndash2239 2010

[152] N Gorbunov G Petrovski N Gurusamy D Ray D H Kimand D K Das ldquoRegeneration of infarcted myocardium withresveratrol-modified cardiac stem cellsrdquo Journal of Cellular andMolecular Medicine vol 16 no 1 pp 174ndash184 2012

[153] C Cai L Teng D Vu et al ldquoThe heme oxygenase 1 inducer(CoPP) protects human cardiac stem cells against apoptosisthrough activation of the extracellular signal-regulated kinase(ERK)NRF2 signaling pathway and cytokine releaserdquo TheJournal of Biological Chemistry vol 287 no 40 pp 33720ndash33732 2012

[154] J Song S M Kang K M Lee and J E Lee ldquoThe protectiveeffect of melatonin on neural stem cell against LPS-inducedinflammationrdquo BioMed Research International vol 2015 ArticleID 854359 13 pages 2015

14 Oxidative Medicine and Cellular Longevity

[155] V Karkkainen Y Pomeshchik E Savchenko et al ldquoNrf2regulates neurogenesis and protects neural progenitor cellsagainst A120573 toxicityrdquo STEM CELLS vol 32 no 7 pp 1904ndash19162014

[156] M Mohammadzadeh R Halabian A Gharehbaghian et alldquoNrf-2 overexpression in mesenchymal stem cells reducesoxidative stress-induced apoptosis and cytotoxicityrdquo Cell Stressand Chaperones vol 17 no 5 pp 553ndash565 2012

[157] Y Takahata T Takarada M Iemata et al ldquoFunctional expres-sion of 1205732 adrenergic receptors responsible for protectionagainst oxidative stress through promotion of glutathione syn-thesis after Nrf2 upregulation in undifferentiated mesenchymalC3H10T12 stem cellsrdquo Journal of Cellular Physiology vol 218no 2 pp 268ndash275 2009

[158] J Zhu H Wang Y Fan et al ldquoKnockdown of nuclear factorerythroid 2-related factor 2 by lentivirus induces differentiationof glioma stem-like cellsrdquo Oncology Reports vol 32 no 3 pp1170ndash1178 2014

[159] J Zhu H Wang Q Sun et al ldquoNrf2 is required to maintain theself-renewal of glioma stem cellsrdquo BMC Cancer vol 13 no 1article 380 2013

[160] B L Emmink A Verheem W J Van Houdt et al ldquoThe secre-tome of colon cancer stem cells contains drug-metabolizingenzymesrdquo Journal of Proteomics vol 91 pp 84ndash96 2013

[161] T Mizuno N Suzuki H Makino et al ldquoCancer stem-like cellsof ovarian clear cell carcinoma are enriched in the ALDH-highpopulation associated with an accelerated scavenging system inreactive oxygen speciesrdquo Gynecologic Oncology vol 137 no 2pp 299ndash305 2015

[162] I-G Ryoo B-H Choi and M-K Kwak ldquoActivation of NRF2by p62 and proteasome reduction in sphere-forming breastcarcinoma cellsrdquo Oncotarget vol 6 no 10 pp 8167ndash8184 2015

[163] T Wu B G Harder P K Wong J E Lang and D D ZhangldquoOxidative stress mammospheres and Nrf2-new implicationfor breast cancer therapyrdquoMolecular Carcinogenesis 2014

[164] B Yang Y Ma and Y Liu ldquoElevated expression of Nrf-2 andABCG2 involved in multi-drug resistance of lung cancer SPcellsrdquo Drug Research 2015

[165] M Zhang A Mathur Y Zhang et al ldquoMithramycin repressesbasal and cigarette smoke-induced expression of abcg2 andinhibits stem cell signaling in lung and esophageal cancer cellsrdquoCancer Research vol 72 no 16 pp 4178ndash4192 2012

[166] C A Del Vecchio Y Feng E S Sokol et al ldquoDe-differentiationconfers multidrug resistance via noncanonical PERK-Nrf2signalingrdquo PLoS Biology vol 12 no 9 Article ID e1001945 2014

[167] D Ren N F Villeneuve T Jiang et al ldquoBrusatol enhancesthe efficacy of chemotherapy by inhibiting the Nrf2-mediateddefense mechanismrdquo Proceedings of the National Academy ofSciences of the United States of America vol 108 no 4 pp 1433ndash1438 2011

[168] A Arlt S Sebens S Krebs et al ldquoInhibition of the Nrf2 tran-scription factor by the alkaloid trigonelline renders pancreaticcancer cells more susceptible to apoptosis through decreasedproteasomal gene expression and proteasome activityrdquo Onco-gene vol 32 no 40 pp 4825ndash4835 2013

[169] A-M Gao Z-P Ke F Shi G Sun and H Chen ldquoChrysinenhances sensitivity of BEL-7402ADM cells to doxorubicin bysuppressing PI3KAktNrf2 and ERKNrf2 pathwayrdquo Chemico-Biological Interactions vol 206 no 1 pp 100ndash108 2013

[170] A M Gao Z P Ke J N Wang J Yang S Chen and HChen ldquoApigenin sensitizes doxorubicin-resistant hepatocellular

carcinoma BEL-7402ADM cells to doxorubicin via inhibitingPI3KAktNrf2 pathwayrdquo Carcinogenesis vol 34 no 8 pp1806ndash1814 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Oxidative Medicine and Cellular Longevity 7

NRF2 sMaf

GCLCGCLMNQO1SODs

Antioxidant enzymes Drug transportersMetabolic enzymes

ARE

Tumor growthprogression

MDRsMRPsBCRP

G6PDPGDME1

Cell survival Drug effluxProliferation

Target genes

Figure 2 Implications of NRF2 signaling in cancer NRF2 coordi-nates the expression of genes associated with cellular redox regu-lation metabolism and xenobiotic efflux and thereby its aberrantactivation promotes cancer cell survival proliferation and anti-cancer drug resistance GCLM glutamate cysteine ligase modifiersubunit GCLC glutamate cysteine ligase catalytic subunit NQO1NAD(P)Hquinone oxidoreductase 1 SODs superoxide dismutasesG6PDH glucose-6-phosphate dehydrogenase PGD phosphoglu-conate dehydrogenase ME1 malic enzyme 1 MDRs multidrugresistance proteinsMRPsmultidrug resistance-associated proteinsBCRP breast cancer resistance protein

state of ABSCs was maintained by NRF2 activation [147]In osteoclast progenitor cells hydrogen sulfide (H

2S) inhib-

ited human osteoclast differentiation by NRF2-dependentinduction of peroxiredoxin 1 and NQO1 These resultswere further confirmed using NRF2 activators includingsulforaphane and t-BHQ [148] It is also notable that NRF2participates in the regulation of cell fate determination ofHSCsMurakami et al demonstrated that HSCs derived fromKEAP1-deficientmice exhibited preferred differentiation intothe granulocyte-monocyte lineage rather than differentiatinginto the erythroid-lymphoid lineage [145]

Up to now numerous studies have demonstrated thatNRF2 plays a protective role against various stressors instem cells In neural stem cells overexpression of NRF2or pharmacological NRF2 activation prevented necroticcell death [149] In an animal study nrf2-deficient miceshowed defective stem cell function HSCs from nrf2minusminus miceexpressed lower levels of prosurvival cytokines and exhib-ited spontaneous apoptosis [150] Ionizing radiation-inducedmyelosuppression and mortality were mitigated throughNRF2-mediated Notch signaling activation in HSCs [142]Similarly resveratrol-inducedNRF2 expression improved thesurvival of cardiac stem cells and consequently regeneratedinfarcted myocardium [151 152] The heme oxygenase 1(HO-1) inducer cobalt protoporphyrin (CoPP) elicited anantiapoptotic effect on cardiac stem cells via activation of the

ERK-NRF2 pathway [153] In neural stem cells NRF2 activa-tion by melatonin or t-BHQ ameliorated lipopolysaccharide(LPS) orH

2O2induced cell death [149 154] In addition amy-

loid 120573-mediated neural stem cell death could be alleviated byexogenous NRF2 transduction which was accompanied byincreased expression of GCLC NQO-1 andHO-1This studyalso demonstrated that neuronal differentiation of neuralstem cells is enhanced by NRF2 activation [155] Similar toneural stem cells NRF2 has a protective role against hypoxicand oxidative stress conditions in undifferentiated MSCsTreatment of the murine mesenchymal stem cell line withadrenaline increased the mRNA expression of nrf2 gclc andxCT leading to an increase in GSH levels and the preventionof ROS-induced cytotoxicity [156 157]

9 Potential Implication of NRF2 inCSC Maintenance and Resistance

The role of NRF2 in CSC biology is now beginning tobe unveiled Similar to the case of normal stem cells itwas shown that NRF2 contributes to CSC stemness bymaintaining their self-renewal capacity and protecting themfrom chemoradiotherapy Achuthan et al established stablechemotherapy-resistant breast cancer cells and observed thatthese cells expressed higher levels of CD133 and OCT-4indicating that these cells exhibit CSC phenotype [34] Ofnote it was shown that ROS levels were relatively low inthese drug-selected cells presumably due to higher levelsof antioxidant enzymes such as SOD1 and GPX12 NRF2protein stabilizationwas associatedwith high levels of antiox-idant enzymes As an underlying molecular mechanismdiminished proteasome activity and increased p21 levelsappear to stabilize the NRF2 protein in these stem-like cellsEvidently p21 knockdown repressed the mammosphere-forming potential of these stem-like breast cancer cellsSimilarly a study by Zhu et al showed the involvement ofNRF2 in glioblastoma stem cells that were isolated fromhuman surgical glioblastoma specimens NRF2 knockdownin glioblastoma stem cells inhibited cell proliferation andneurosphere formation and further suppressed SOX2 expres-sion Moreover NRF2 knockdown changed the cell cycledistribution to the G2 phase and significantly attenuated thetumorigenecity of glioblastoma stem cells [158 159] Theseresults are providing evidence that NRF2 is necessary formaintenance of the self-renewal capacity of glioblastomastem cells

On the other hand activation of NRF2 signaling has beendemonstrated in different types of CSC models includinglung esophageal breast ovarian and colon CSCs and thisis closely correlated with themaintenance of low intracellularROS levels and chemoresistance of CSCs [160ndash165] In lungand esophageal cancer cells cigarette smoke condensateincreased the SP as well as BCRP expression which arehallmarks of CSCs [165] Promoter analysis revealed thatBCRP expression was associated with elevated levels ofNRF2 aryl hydrocarbon receptor (AhR) and specificityprotein 1 (SP1) Additionally this study demonstrated thatmithramycin diminished BCRP expression via repression of

8 Oxidative Medicine and Cellular Longevity

Low ROS

Increased drug efflux

SurvivalChemoresistance

CSC

TumorRecurrenceProgression

Radioresistance

PERKuarr

p21uarr

p62uarr

Proteasome darr

NRF2 uarr

Figure 3 Potential roles of NRF2 signaling in CSCs InCSC-like cellmodels NRF2 is activated through multiple molecular mechanismsin a context-dependent manner The upregulation of competingproteins such as p62 and p21 activation of PERK or repressedproteasome function were shown to enhance NRF2 activity inthese models Elevated NRF2 levels in CSCs can contribute tothe maintenance of low ROS by upregulating multiple antioxidantgenes In addition NRF2-mediated expression of ABC transporterselicits efflux of anticancer drugs from cancer cells Overall activatedNRF2 signaling facilitates CSCs survival and stress resistance andconsequently it can be suggested that CSCs with high NRF2 activityplay a crucial role in tumor recurrence and further progression

NRF2 AhR and SP1 and thereby inhibiting the expressionof genes associated with CSC-related pathways resulting inreduced proliferation and tumorigenecity Similarly it wasreported that lung cancer SP cells exhibited high levels ofNRF2 and BCRP expression These SP cells were highlytumorigenic and possessed self-renewal capacity comparedto non-SP cells [164] Emmink et al performed a proteomeanalysis on collected secretome from highly tumorigenicCSCs and their corresponding nontumorigenic differentiatedcells both of which were established from human colorectalspecimens [160] Subsequent bioinformatic analysis revealedthat the CSC secretome contained a large amount of proteinsassociated with cell survival and protein quality controlcompared to differentiated tumor cells Notably the CSCsecretome contained an NRF2 antioxidant and detoxifyingprotein signature in that it included elevated levels ofGCLC GPX23 and TXNRD1 This study provided novelevidence that CSCs secrete NRF2 target antioxidant proteinsto counteract extracellular stressors and chemotherapeuticsIn patients with ovarian clear cell carcinoma the expressionof the CSC maker aldehyde dehydrogenase-1 (ALDH1) wasstrongly correlated with an advanced clinical stage andreduced progression free survival [161] Ovarian clear cellcarcinoma cells with high ALDH1 expressionmaintained lowlevels of ROS compared to ALDH-low cells and these cellswere shown to express higher levels of NRF2 and its targetgenes

Two recent studies have demonstratedNRF2 activation insphere cultures of breast cancer cells that is one of models ofCSCs Wu et al showed that mammospheres derived fromMCF7 and MDA-MB231 breast cancer cell lines exhibitedlower ROS levels compared to their monolayer counterpartsThey also showed that levels of NRF2 and target genes such

as NQO1 and GCLM were elevated in mammospheres [163]Similarly our group has shown substantially elevated NRF2protein levels along with increased expression of antioxidantgenes (eg HO-1 and GPX2) and drug efflux transporters(eg MRP2 and BCRP) in sphere cultures of breast cancercells NRF2 accumulation was also observed in sphere-cultured ovarian and colon cancer cells However shRNA-mediated downregulation ofNRF2 led to decreased chemore-sistance of mammospheres presumably due to reduced levelsof antioxidant genes and drug transporters High ROS levelsin NRF2 knockdown mammospheres caused sphere growthretardation and apoptosis Coherently ablation of ABCtransporter induction in NRF2 knockdown mammospheressensitized to anticancer agents [162] Additionally this studyprovided evidence that increased NRF2 protein expression inmammospheres can be linked to 26S proteasome reductionand p62 accumulation In particular knockdown of p62 inMCF7 mammospheres significantly attenuated NRF2 eleva-tion

Surviving dedifferentiated breast cancer cells after chem-otherapy treatment retained high levels of NRF2 activationsimilar to other CSCs However NRF2 activation was medi-ated by a noncanonical pathway Levels of PERKwere high indedifferentiated cancer cells and this in turn phosphorylatedand activated NRF2 signaling to maintain low cellular ROSlevels and to express ABC transporters In agreement withthese findings clinical observations revealed that the PERKpathway gene signature is related to chemoresistance andreduced patient survival [166]

10 Concluding Remarks

Recent studies have started to uncover the role of ROS signal-ing in the biology of CSCs which is related to tumorigenecitytumor progression and relapse Expression of the transcrip-tion factor NRF2 a master regulator of antioxidant genesexpression is increased in different models of CSCs and thiselevation is likely to promote CSC maintenance and survivalin an oxidizing tumormicroenvironment In additionNRF2-mediated overexpression of ABC transporters particularlythe CSC marker BCRP may play a critical role in themultidrug resistance of CSCsThese findings combined withthe increasing evidence showing the alteration of KEAP1-NRF2 signaling in cancer cells suggest a novel role of NRF2in CSC maintenance and survival (Figure 3)

One important question that arises from the current stud-ies is whether it is possible to design CSC-targeted therapiesthrough regulation of theNRF2 pathway and its related redoxhomeostasis inCSCs Recent studies provide several potentialclues for addressing this question the naturally occurringalkaloid brusatol could reduce the growth and chemoresis-tance of breast CSCs [163] Treatment ofmammospheres withbrusatol elevated ROS levels and promoted taxol-inducedgrowth retardation and cell death The NRF2 repressivemechanism of brusatol has not been clearly elucidated but itappears to be independent of KEAP1-mediated degradation[167] In addition to brusatol natural compounds such as

Oxidative Medicine and Cellular Longevity 9

chrysin apigenin luteolin and trigonelline are known toinhibit NRF2 signaling in several types of cancer cells [102168ndash170] and therefore the development of NRF2 inhibitorswith characterized modes of action will enable efficient tar-geting of the redox homeostasis system as well as multidrugresistance systems in CSCs

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This studywas financially supported by theNational ResearchFoundation (NRF) funded by the Ministry of Science ICT ampFuture Planning (NRF-2013R1A2A2A01015497)

References

[1] C R Reczek and N S Chandel ldquoROS-dependent signaltransductionrdquo Current Opinion in Cell Biology vol 33 pp 8ndash132015

[2] P Storz ldquoForkhead homeobox type O transcription factorsin the responses to oxidative stressrdquo Antioxidants amp RedoxSignaling vol 14 no 4 pp 593ndash605 2011

[3] R S Balaban S Nemoto and T Finkel ldquoMitochondria oxi-dants and agingrdquo Cell vol 120 no 4 pp 483ndash495 2005

[4] L A Sena and N S Chandel ldquoPhysiological roles of mitochon-drial reactive oxygen speciesrdquoMolecular Cell vol 48 no 2 pp158ndash167 2012

[5] P T Schumacker ldquoReactive oxygen species in cancer cells liveby the sword die by the swordrdquo Cancer Cell vol 10 no 3 pp175ndash176 2006

[6] S Toyokuni K Okamoto J Yodoi and H Hiai ldquoPersistentoxidative stress in cancerrdquo FEBS Letters vol 358 no 1 pp 1ndash3 1995

[7] L Behrend G Henderson and R Zwacka ldquoReactive oxy-gen species in oncogenic transformationrdquo Biochemical SocietyTransactions vol 31 no 6 pp 1441ndash1444 2003

[8] E O Hileman J Liu M Albitar M J Keating and P HuangldquoIntrinsic oxidative stress in cancer cells a biochemical basis fortherapeutic selectivityrdquo Cancer Chemotherapy and Pharmacol-ogy vol 53 no 3 pp 209ndash219 2004

[9] H PelicanoD Carney and PHuang ldquoROS stress in cancer cellsand therapeutic implicationsrdquo Drug Resistance Updates vol 7no 2 pp 97ndash110 2004

[10] Y Adachi Y Shibai J Mitsushita W H Shang K Hirose andT Kamata ldquoOncogenic Ras upregulates NADPH oxidase 1 geneexpression through MEK-ERK-dependent phosphorylation ofGATA-6rdquo Oncogene vol 27 no 36 pp 4921ndash4932 2008

[11] J Mitsushita J D Lambeth and T Kamata ldquoThe superoxide-generating oxidase Nox1 is functionally required for Ras onco-gene transformationrdquoCancer Research vol 64 no 10 pp 3580ndash3585 2004

[12] O Vafa M Wade S Kern et al ldquoc-Myc can induce DNAdamage increase reactive oxygen species and mitigate p53function a mechanism for oncogene-induced genetic instabil-ityrdquoMolecular Cell vol 9 no 5 pp 1031ndash1044 2002

[13] K Polyak Y Li H Zhu et al ldquoSomatic mutations of themitochondrial genome in human colorectal tumoursrdquo NatureGenetics vol 20 no 3 pp 291ndash293 1998

[14] D Trachootham J Alexandre and P Huang ldquoTargeting can-cer cells by ROS-mediated mechanisms a radical therapeuticapproachrdquo Nature Reviews Drug Discovery vol 8 no 7 pp579ndash591 2009

[15] X Shi Y Zhang J Zheng and J Pan ldquoReactive oxygen speciesin cancer stem cellsrdquoAntioxidantsampRedox Signaling vol 16 no11 pp 1215ndash1228 2012

[16] T Reya S J Morrison M F Clarke et al ldquoStem cells cancerand cancer stem cellsrdquo Nature vol 414 no 6859 pp 105ndash1112001

[17] B S Zhou H Zhang M Damelin K G Geles J C Grindleyand P B Dirks ldquoTumour-initiating cells challenges and oppor-tunities for anticancer drug discoveryrdquo Nature Reviews DrugDiscovery vol 8 no 10 pp 806ndash823 2009

[18] D Bonnet and J E Dick ldquoHuman acute myeloid leukemiais organized as a hierarchy that originates from a primitivehematopoietic cellrdquo Nature Medicine vol 3 no 7 pp 730ndash7371997

[19] C A OrsquoBrien A Pollett S Gallinger and J E Dick ldquoAhuman colon cancer cell capable of initiating tumour growth inimmunodeficient micerdquo Nature vol 445 no 7123 pp 106ndash1102006

[20] S K Singh C Hawkins I D Clarke et al ldquoIdentification ofhuman brain tumour initiating cellsrdquo Nature vol 432 no 7015pp 396ndash401 2004

[21] M Al-Hajj M S Wicha A Benito-Hernandez S J Morrisonand M F Clarke ldquoProspective identification of tumorigenicbreast cancer cellsrdquo Proceedings of the National Academy ofSciences vol 100 no 7 pp 3983ndash3988 2003

[22] S-H Chiou M-L Wang Y-T Chou et al ldquoCoexpression ofOct4 andNanog enhancesmalignancy in lung adenocarcinomaby inducing cancer stem cell-like properties and epithelial-mesenchymal transdifferentiationrdquoCancer Research vol 70 no24 pp 10433ndash10444 2010

[23] F Yu J Li H Chen et al ldquoKruppel-like factor 4 (KLF4) isrequired for maintenance of breast cancer stem cells and for cellmigration and invasionrdquoOncogene vol 30 no 18 pp 2161ndash21722011

[24] L Vermeulen E M Felipe De Sousa M van der Heijden et alldquoWnt activity defines colon cancer stem cells and is regulatedby the microenvironmentrdquo Nature Cell Biology vol 12 no 5pp 468ndash476 2010

[25] C Zhao A Chen C H Jamieson et al ldquoHedgehog signallingis essential for maintenance of cancer stem cells in myeloidleukaemiardquo Nature vol 458 no 7239 pp 776ndash779 2009

[26] G Farnie and R B Clarke ldquoMammary stem cells and breastcancermdashrole of Notch signallingrdquo Stem Cell Reviews vol 3 no2 pp 169ndash175 2007

[27] D Ponti ldquoIsolation and in vitro propagation of tumorigenicbreast cancer cells with stemprogenitor cell propertiesrdquoCancerResearch vol 65 no 13 pp 5506ndash5511 2005

[28] M Dean T Fojo and S Bates ldquoTumour stem cells and drugresistancerdquo Nature Reviews Cancer vol 5 no 4 pp 275ndash2842005

[29] C Blanpain M Mohrin P A Sotiropoulou and E PassegueldquoDNA-damage response in tissue-specific and cancer stemcellsrdquo Cell Stem Cell vol 8 no 1 pp 16ndash29 2011

10 Oxidative Medicine and Cellular Longevity

[30] A A Dayem H Choi J Kim and S Cho ldquoRole of oxidativestress in stem cancer and cancer stem cellsrdquo Cancers vol 2 no2 pp 859ndash884 2010

[31] E Lagadinou A Sach K Callahan et al ldquoBCL-2 inhibitiontargets oxidative phosphorylation and selectively eradicatesquiescent human leukemia stem cellsrdquo Cell Stem Cell vol 12no 3 pp 329ndash341 2013

[32] C Chang Y Chen S Chou et al ldquoDistinct subpopulations ofhead and neck cancer cells with different levels of intracellularreactive oxygen species exhibit diverse stemness proliferationand chemosensitivityrdquoCancer Research vol 74 no 21 pp 6291ndash6305 2014

[33] MMGottesman T Fojo and S E Bates ldquoMultidrug resistancein cancer role of ATPndashdependent transportersrdquoNature ReviewsCancer vol 2 no 1 pp 48ndash58 2002

[34] S Achuthan T R Santhoshkumar J Prabhakar S A Nair andM R Pillai ldquoDrug-induced senescence generates chemoresis-tant stemlike cells with low reactive oxygen speciesrdquoThe Journalof Biological Chemistry vol 286 no 43 pp 37813ndash37829 2011

[35] L Zinzi M Contino M Cantore E Capparelli M Leopoldoand N A Colabufo ldquoABC transporters in CSCs membranesas a novel target for treating tumor relapserdquo Frontiers inPharmacology vol 5 article 163 2014

[36] A Hadnagy L Gaboury R Beaulieu and D Balicki ldquoSPanalysis may be used to identify cancer stem cell populationsrdquoExperimental Cell Research vol 312 no 19 pp 3701ndash3710 2006

[37] C L Bigarella R Liang and S Ghaffari ldquoStem cells and theimpact of ROS signalingrdquo Development vol 141 no 22 pp4206ndash4218 2014

[38] P Chaudhari Z Ye and Y Y Jang ldquoRoles of reactive oxygenspecies in the fate of stem cellsrdquoAntioxidants ampRedox Signalingvol 20 no 12 pp 1881ndash1890 2014

[39] V A Rafalski E Mancini and A Brunet ldquoEnergy metabolismand energy-sensing pathways in mammalian embryonic andadult stem cell faterdquo Journal of Cell Science vol 125 no 23 pp5597ndash5608 2012

[40] K Wang T Zhang Q Dong E C Nice C Huang and Y WeildquoRedox homeostasis the linchpin in stem cell self-renewal anddifferentiationrdquo Cell Death and Disease vol 4 no 3 p e5372013

[41] G Saretzki L Armstrong A Leake and et al ldquoStress defensein murine embryonic stem cells is superior to that of variousdifferentiated murine cellsrdquo Stem Cells vol 22 no 6 pp 962ndash971 2004

[42] Y Y Jang and S J Sharkis ldquoA low level of reactive oxygen speciesselects for primitive hematopoietic stem cells that may reside inthe low-oxygenic nicherdquo Blood vol 110 no 8 pp 3056ndash30632007

[43] S Ghaffari ldquoOxidative stress in the regulation of normal andneoplastic hematopoiesisrdquo Antioxidants amp Redox Signaling vol10 no 11 pp 1923ndash1940 2008

[44] M Schmelter B Ateghang S Helmig M Wartenberg and HSauer ldquoEmbryonic stem cells utilize reactive oxygen speciesas transducers of mechanical strain-induced cardiovasculardifferentiationrdquoThe FASEB Journal vol 20 no 8 pp 1182ndash11842006

[45] A Alexander S-L Cai J Kim et al ldquoATM signals to TSC2in the cytoplasm to regulate mTORC1 in response to ROSrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 107 no 9 pp 4153ndash4158 2010

[46] C Cosentino D Grieco and V Costanzo ldquoATM activates thepentose phosphate pathway promoting anti-oxidant defenceand DNA repairrdquo The EMBO Journal vol 30 no 3 pp 546ndash555 2011

[47] K Ito A Hirao F Arai et al ldquoRegulation of oxidative stress byATM is required for self-renewal of haematopoietic stem cellsrdquoNature vol 431 no 7011 pp 997ndash1002 2004

[48] K Ito K Takubo F Arai et al ldquoRegulation of reactive oxygenspecies by Atm is essential for proper response to DNA double-strand breaks in lymphocytesrdquoThe Journal of Immunology vol178 no 1 pp 103ndash110 2007

[49] Y Wang Y Zhou and D T Graves ldquoFOXO transcription fac-tors their clinical significance and regulationrdquoBioMed ResearchInternational vol 2014 Article ID 925350 13 pages 2014

[50] E Lam R Francis and M Petkovic ldquoFOXO transcriptionfactors key regulators of cell faterdquo Biochemical Society Trans-actions vol 34 no 5 pp 722ndash726 2006

[51] K Miyamoto K Y Araki K Naka et al ldquoFoxo3a is essentialfor maintenance of the hematopoietic stem cell poolrdquo Cell StemCell vol 1 no 1 pp 101ndash112 2007

[52] Z Tothova R Kollipara B J Huntly et al ldquoFoxOs are criticalmediators of hematopoietic stem cell resistance to physiologicoxidative stressrdquo Cell vol 128 no 2 pp 325ndash339 2007

[53] S Yalcin X Zhang J P Luciano et al ldquoFoxo3 is essential for theregulation of ataxia telangiectasia mutated and oxidative stress-mediated homeostasis of hematopoietic stem cellsrdquoThe Journalof Biological Chemistry vol 283 no 37 pp 25692ndash25705 2008

[54] J Paik Z Ding R Narurkar et al ldquoFoxOs cooperatively regu-late diverse pathways governing neural stem cell homeostasisrdquoCell Stem Cell vol 5 no 5 pp 540ndash553 2009

[55] V M Renault V A Rafalski A A Morgan et al ldquoFoxO3regulates neural stem cell homeostasisrdquo Cell Stem Cell vol 5no 5 pp 527ndash539 2009

[56] A Brunet A Bonni M J Zigmond et al ldquoAkt promotescell survival by phosphorylating and inhibiting a Forkheadtranscription factorrdquo Cell vol 96 no 6 pp 857ndash868 1999

[57] A Brunet F Kanai J Stehn and et al ldquo14-3-3 transits tothe nucleus and participates in dynamic nucleocytoplasmictransportrdquo Journal of Cell Biology vol 156 no 5 pp 817ndash8282002

[58] M M Juntilla V D Patil M Calamito R P Joshi M JBirnbaum and G A Koretzky ldquoAKT1 and AKT2 maintainhematopoietic stem cell function by regulating reactive oxygenspeciesrdquo Blood vol 115 no 20 pp 4030ndash4038 2010

[59] O H Yilmaz R Valdez B K Theisen et al ldquoPten depen-dence distinguishes haematopoietic stem cells from leukaemia-initiating cellsrdquo Nature vol 441 no 7092 pp 475ndash482 2006

[60] S N Greer J L Metcalf Y Wang and M Ohh ldquoThe updatedbiology of hypoxia-inducible factorrdquoTheEMBO Journal vol 31no 11 pp 2448ndash2460 2012

[61] C E Forristal K L Wright N A Hanley R O Oreffo and FD Houghton ldquoHypoxia inducible factors regulate pluripotencyand proliferation in human embryonic stem cells cultured atreduced oxygen tensionsrdquo Reproduction vol 139 no 1 pp 85ndash97 2009

[62] K L Covello J Kehler H Yu et al ldquoHIF-2120572 regulates Oct-4effects of hypoxia on stem cell function embryonic develop-ment and tumor growthrdquo Genes amp Development vol 20 no5 pp 557ndash570 2006

[63] J Mazumdar W T OrsquoBrien R S Johnson et al ldquoO2regulates

stem cells through Wnt120573-catenin signallingrdquo Nature Cell Biol-ogy vol 12 no 10 pp 1007ndash1013 2010

Oxidative Medicine and Cellular Longevity 11

[64] K Takubo N Goda W Yamada et al ldquoRegulation of the HIF-1alpha level is essential for hematopoietic stem cellsrdquo Cell StemCell vol 7 no 3 pp 391ndash402 2010

[65] FKocabas J Zheng SThet et al ldquoMeis1 regulates themetabolicphenotype and oxidant defense of hematopoietic stem cellsrdquoBlood vol 120 no 25 pp 4963ndash4972 2012

[66] T M Phillips W H McBride and F Pajonk ldquoThe responseof CD24minus119897119900119908CD44+ breast cancer-initiating cells to radiationrdquoJNCI Journal of the National Cancer Institute vol 98 no 24 pp1777ndash1785 2006

[67] M Diehn R W Cho N A Lobo et al ldquoAssociation of reactiveoxygen species levels and radioresistance in cancer stem cellsrdquoNature vol 458 no 7239 pp 780ndash783 2009

[68] O Herault K J Hope E Deneault et al ldquoA role for GPx3in activity of normal and leukemia stem cellsrdquo The Journal ofExperimental Medicine vol 209 no 5 pp 895ndash901 2012

[69] S Kim C Kwon and I Nakano ldquoDetoxification of oxidativestress in glioma stem cells mechanism clinical relevance andtherapeutic developmentrdquo Journal of Neuroscience Research vol92 no 11 pp 1419ndash1424 2014

[70] T Ishimoto O Nagano T Yae et al ldquoCD44 variant regulatesredox status in cancer cells by stabilizing the xCT subunit ofsystem xc(-) and thereby promotes tumor growthrdquo Cancer Cellvol 19 no 3 pp 387ndash400 2011

[71] M Yoshikawa K Tsuchihashi T Ishimoto et al ldquoxCT inhibi-tion depletes CD44v-expressing tumor cells that are resistantto EGFR-targeted therapy in head and neck squamous cellcarcinomardquo Cancer Research vol 73 no 6 pp 1855ndash1866 2013

[72] N Haraguchi H Ishii K Mimori et al ldquoCD13 is a therapeutictarget in human liver cancer stem cellsrdquo Journal of ClinicalInvestigation vol 120 no 9 pp 3326ndash3339 2010

[73] H M Kim N Haraguchi H Ishii et al ldquoIncreased CD13expression reduces reactive oxygen species promoting sur-vival of liver cancer stem cells via an epithelial-mesenchymaltransition-like phenomenonrdquo Annals of Surgical Oncology vol19 supplement 3 pp S539ndashS548 2012

[74] K Naka T Hoshii T Muraguchi et al ldquoTGF-120573ndashFOXO sig-nalling maintains leukaemia-initiating cells in chronic myeloidleukaemiardquo Nature vol 463 no 7281 pp 676ndash680 2010

[75] J Zhou J Wulfkuhle H Zhang et al ldquoActivation of thePTENmTORSTAT3 pathway in breast cancer stem-like cellsis required for viability and maintenancerdquo Proceedings of theNational Academy of Sciences vol 104 no 41 pp 16158ndash161632007

[76] A Dubrovska S Kim R J Salamone et al ldquoThe role ofPTENAktPI3K signaling in the maintenance and viability ofprostate cancer stem-like cell populationsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 106 no 1 pp 268ndash273 2009

[77] H Yin and J Glass ldquoThe phenotypic radiation resistance ofCS44+CD24minusor low breast cancer cells is mediated through theenhanced activation of ATM signalingrdquo PLoS ONE vol 6 no9 Article ID e24080 2011

[78] T H Rushmore and C Pickett ldquoTranscriptional regulation ofthe rat glutathione S-transferase Ya subunit gene Characteri-zation of a xenobiotic-responsive element controlling inducibleexpression by phenolic antioxidantsrdquo The Journal of BiologicalChemistry vol 265 no 24 pp 14648ndash14653 1990

[79] Y Li and A Jaiswal ldquoRegulation of humanNAD (P)H quinoneoxidoreductase gene Role of AP1 binding site contained withinhuman antioxidant response elementrdquoThe Journal of BiologicalChemistry vol 267 no 21 pp 15097ndash15104 1992

[80] T Prestera P Talalay J Alam et al ldquoParallel induction of hemeoxygenase-1 and chemoprotective phase 2 enzymes by elec-trophiles and antioxidants regulation by upstream antioxidant-responsive elements (ARE)rdquoMolecular Medicine vol 1 no 7 p827 1995

[81] K Itoh K IgarashiNHayashi et al ldquoCloning and characteriza-tion of a novel erythroid cell-derived CNC family transcriptionfactor heterodimerizing with the small Maf family proteinsrdquoMolecular and Cellular Biology vol 15 no 8 pp 4184ndash41931995

[82] R Venugopal and A K Jaiswal ldquoNrf1 and Nrf2 positivelyand c-Fos and Fra1 negatively regulate the human antioxidantresponse element-mediated expression of NAD(P)Hquinoneoxidoreductase1 generdquo Proceedings of the National Academy ofSciences vol 93 no 25 pp 14960ndash14965 1996

[83] K Itoh T Chiba S Takahashi et al ldquoAn Nrf2small Mafheterodimer mediates the induction of phase II detoxifyingenzyme genes through antioxidant response elementsrdquo Bio-chemical and Biophysical Research Communications vol 236no 2 pp 313ndash322 1997

[84] M Ramos-Gomez M-K Kwak P M Dolan et al ldquoSensitivityto carcinogenesis is increased and chemoprotective efficacy ofenzyme inducers is lost in nrf2 transcription factor-deficientmicerdquo Proceedings of the National Academy of Sciences vol 98no 6 pp 3410ndash3415 2001

[85] M-K Kwak N Wakabayashi K Itoh H Motohashi MYamamoto and T W Kensler ldquoModulation of gene expressionby cancer chemopreventive dithiolethiones through the Keap1-Nrf2 pathway Identification of novel gene clusters for cellsurvivalrdquo The Journal of Biological Chemistry vol 278 no 10pp 8135ndash8145 2003

[86] R Hu C Xu G Shen et al ldquoGene expression profiles inducedby cancer chemopreventive isothiocyanate sulforaphane in theliver of C57BL6J mice and C57BL6JNrf2 (minusminus) micerdquo CancerLetters vol 243 no 2 pp 170ndash192 2006

[87] S Chanas Q Jiang and M McMahon ldquoLoss of the Nrf2transcription factor causes a marked reduction in constitutiveand inducible expression of the glutathione S-transferase Gsta1Gsta2 Gstm1 Gstm2 Gstm3 and Gstm4 genes in the livers ofmale and female micerdquo Biochemical Journal vol 365 no 2 pp405ndash416 2002

[88] H-Y Cho S P Reddy A DeBiase M Yamamoto and SR Kleeberger ldquoGene expression profiling of NRF2-mediatedprotection against oxidative injuryrdquo Free Radical Biology andMedicine vol 38 no 3 pp 325ndash343 2005

[89] G Shen C Xu R Hu et al ldquoModulation of nuclear factorE2-related factor 2-mediated gene expression in mice liver andsmall intestine by cancer chemopreventive agent curcuminrdquoMolecular Cancer Therapeutics vol 5 no 1 pp 39ndash51 2006

[90] J D Hayes and A T Dinkova-Kostova ldquoThe Nrf2 regulatorynetwork provides an interface between redox and intermediarymetabolismrdquo Trends in Biochemical Sciences vol 39 no 4 pp199ndash218 2014

[91] A Hayashi H Suzuki K Itoh M Yamamoto and Y SugiyamaldquoTranscription factor Nrf2 is required for the constitutive andinducible expression of multidrug resistance-associated protein1 in mouse embryo fibroblastsrdquo Biochemical and BiophysicalResearch Communications vol 310 no 3 pp 824ndash829 2003

[92] E Jigorel M Le Vee C Boursier-Neyret Y Parmentier andO Fardel ldquoDifferential regulation of sinusoidal and canalicularhepatic drug transporter expression by xenobiotics activating

12 Oxidative Medicine and Cellular Longevity

drug-sensing receptors in primary human hepatocytesrdquo DrugMetabolism and Disposition vol 34 no 10 pp 1756ndash1763 2006

[93] X Wang C R Campos J C Peart et al ldquoNrf2 upregulatesATP binding cassette transporter expression and activity atthe blood-brain and blood-spinal cord barriersrdquo Journal ofNeuroscience vol 34 no 25 pp 8585ndash8593 2014

[94] H Jeong I Ryoo andMKwak ldquoRegulation of the expression ofrenal drug transporters in KEAP1-knockdown human tubularcellsrdquo Toxicology in Vitro vol 29 no 5 pp 884ndash892 2015

[95] M J Canet M D Merrell B G Harder et al ldquoIdentificationof a functional antioxidant response element within the eighthintron of the human ABCC

3generdquo Drug Metabolism and

Disposition vol 43 no 1 pp 93ndash99 2014[96] A Singh H Wu P Zhang C Happel J Ma and S Biswal

ldquoExpression of ABCG2 (BCRP) is regulated by Nrf2 in cancercells that confers side population and chemoresistance pheno-typerdquo Molecular Cancer Therapeutics vol 9 no 8 pp 2365ndash2376 2010

[97] N Wakabayashi K Itoh J Wakabayashi et al ldquoKeap1-nullmutation leads to postnatal lethality due to constitutive Nrf2activationrdquo Nature Genetics vol 35 no 3 pp 238ndash245 2003

[98] S B Cullinan J D Gordan J Jin J W Harper and J A DiehlldquoThe Keap1-BTB protein is an adaptor that bridges Nrf2 to aCul3-based E3 ligase oxidative stress sensing by a Cul3-Keap1ligaserdquoMolecular and Cellular Biology vol 24 no 19 pp 8477ndash8486 2004

[99] D D Zhang and M Hannink ldquoDistinct cysteine residues inkeap1 are required for keap1-dependent ubiquitination of nrf2and for stabilization of nrf2 by chemopreventive agents andoxidative stressrdquoMolecular and Cellular Biology vol 23 no 22pp 8137ndash8151 2003

[100] L M Zipper and R T Mulcahy ldquoThe Keap1 BTBPOZ dimer-ization function is required to sequester Nrf2 in cytoplasmrdquoThe Journal of Biological Chemistry vol 277 no 39 pp 36544ndash36552 2002

[101] M C Jaramillo and D D Zhang ldquoThe emerging role of theNrf2-Keap1 signaling pathway in cancerrdquoGenesampDevelopmentvol 27 no 20 pp 2179ndash2191 2013

[102] K I Tong Y Katoh H Kusunoki K Itoh T Tanaka and MYamamoto ldquoKeap1 recruits Neh2 through binding to ETGEand DLG motifs characterization of the two-site molecularrecognition modelrdquoMolecular and Cellular Biology vol 26 no8 pp 2887ndash2900 2006

[103] K I Tong A Kobayashi F Katsuoka andM Yamamoto ldquoTwo-site substrate recognition model for the Keap1-Nrf2 system ahinge and latch mechanismrdquo Biological Chemistry vol 387 no10-11 pp 1311ndash1320 2006

[104] K I Tong B Padmanabhan A Kobayashi et al ldquoDifferentelectrostatic potentials define ETGE and DLG motifs as hingeand latch in oxidative stress responserdquo Molecular and CellularBiology vol 27 no 21 pp 7511ndash7521 2007

[105] M Kobayashi L Li N Iwamoto et al ldquoThe antioxidant defensesystemKeap1-Nrf2 comprises amultiple sensingmechanism forresponding to a wide range of chemical compoundsrdquoMolecularand Cellular Biology vol 29 no 2 pp 493ndash502 2009

[106] T Yamamoto T Suzuki A Kobayashi et al ldquoPhysiological sig-nificance of reactive cysteine residues of Keap1 in determiningNrf2 activityrdquoMolecular and Cellular Biology vol 28 no 8 pp2758ndash2770 2008

[107] M K Kwak K Itoh M Yamamoto and T W KenslerldquoEnhanced expression of the transcription factorNrf2 by cancer

chemopreventive agents role of antioxidant response element-like sequences in the nrf2 promoterrdquo Molecular and CellularBiology vol 22 no 9 pp 2883ndash2892 2002

[108] T Suzuki T Shibata K Takaya et al ldquoRegulatory nexus ofsynthesis and degradation deciphers cellular Nrf2 expressionlevelsrdquoMolecular and Cellular Biology vol 33 no 12 pp 2402ndash2412 2013

[109] S B Cullinan D Zhang M Hannink E Arvisais R JKaufman and J A Diehl ldquoNrf2 is a direct PERK substrateand effector of PERK-dependent cell survivalrdquo Molecular andCellular Biology vol 23 no 20 pp 7198ndash7209 2003

[110] W Li and A Kong ldquoMolecular mechanisms of Nrf2-mediatedantioxidant responserdquo Molecular Carcinogenesis vol 48 no 2pp 91ndash104 2009

[111] Y J Surh ldquoCancer chemoprevention with dietary phytochemi-calsrdquo Nature Reviews Cancer vol 3 no 10 pp 768ndash780 2003

[112] M Salazar A I Rojo D Velasco R M de Sagarra and ACuadrado ldquoGlycogen synthase kinase-3beta inhibits the xeno-biotic and antioxidant cell response by direct phosphorylationand nuclear exclusion of the transcription factor Nrf2rdquo Journalof Biological Chemistry vol 281 no 21 pp 14841ndash14851 2006

[113] W Chen Z Sun X J Wang et al ldquoDirect interactionbetween Nrf2 and p21(Cip1WAF1) upregulates the Nrf2-mediated antioxidant responserdquo Molecular Cell vol 34 no 6pp 663ndash673 2009

[114] M Komatsu H Kurokawa S Waguri et al ldquoThe selectiveautophagy substrate p62 activates the stress responsive tran-scription factor Nrf2 through inactivation of Keap1rdquoNature CellBiology vol 12 no 3 pp 213ndash223 2010

[115] J D Hayes M McMahon S Chowdhry and A T Dinkova-Kostova ldquoCancer chemoprevention mechanisms mediatedthrough the Keap1-Nrf2 pathwayrdquoAntioxidants amp Redox Signal-ing vol 13 no 11 pp 1713ndash1748 2010

[116] R Hu C L Saw R Yu and A T Kong ldquoRegulation ofNF-E2-related factor 2 signaling for cancer chemopreventionantioxidant coupled with antiinflammatoryrdquo Antioxidants ampRedox Signaling vol 13 no 11 pp 1679ndash1698 2010

[117] M Kwak and T W Kensler ldquoTargeting NRF2 signaling for can-cer chemopreventionrdquo Toxicology and Applied Pharmacologyvol 244 no 1 pp 66ndash76 2010

[118] K Iida K Itoh Y Kumagai et al ldquoNrf2 is essential for thechemopreventive efficacy of oltipraz against urinary bladdercarcinogenesisrdquo Cancer Research vol 64 no 18 pp 6424ndash64312004

[119] T Jiang N Chen F Zhao et al ldquoHigh levels of Nrf2 deter-mine chemoresistance in type II endometrial cancerrdquo CancerResearch vol 70 no 13 pp 5486ndash5496 2010

[120] Y R Kim J EOhM S Kim et al ldquoOncogenicNRF2mutationsin squamous cell carcinomas of oesophagus and skinrdquo TheJournal of Pathology vol 220 no 4 pp 446ndash451 2010

[121] T Shibata A Kokubu M Gotoh et al ldquoGenetic alterationof Keap1 confers constitutive Nrf2 activation and resistance tochemotherapy in gallbladder cancerrdquoGastroenterology vol 135no 4 pp 1358e4ndash1368e4 2008

[122] T Shibata T Ohta K I Tong et al ldquoCancer related mutationsin NRF2 impair its recognition by Keap1-Cul3 E3 ligase andpromote malignancyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 36 pp13568ndash13573 2008

[123] A Singh V Misra R K Thimmulappa et al ldquoDysfunctionalKEAP1-NRF2 interaction in non-small-cell lung cancerrdquo PLoSMedicine vol 3 no 10 article e420 2006

Oxidative Medicine and Cellular Longevity 13

[124] R Wang J An F Ji H Jiao H Sun and D Zhou ldquoHyper-methylation of the Keap1 gene in human lung cancer cell linesand lung cancer tissuesrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 1 pp 151ndash154 2008

[125] P Zhang A Singh S Yegnasubramanian et al ldquoLoss ofKelch-like ECH-associated protein 1 function in prostate cancercells causes chemoresistance and radioresistance and promotestumor growthrdquoMolecular Cancer Therapeutics vol 9 no 2 pp336ndash346 2010

[126] J D Hayes and M McMahon ldquoNRF2 and KEAP1 mutationspermanent activation of an adaptive response in cancerrdquo Trendsin Biochemical Sciences vol 34 no 4 pp 176ndash188 2009

[127] B Padmanabhan K I Tong T Ohta et al ldquoStructural basis fordefects of Keap1 activity provoked by its pointmutations in lungcancerrdquoMolecular Cell vol 21 no 5 pp 689ndash700 2006

[128] Y Inami S Waguri A Sakamoto et al ldquoPersistent activation ofNrf2 throughp62 in hepatocellular carcinoma cellsrdquoTheJournalof Cell Biology vol 193 no 2 pp 275ndash284 2011

[129] G M DeNicola F A Karreth T J Humpton et al ldquoOncogene-induced Nrf2 transcription promotes ROS detoxification andtumorigenesisrdquo Nature vol 475 no 7354 pp 106ndash109 2011

[130] L M Solis C Behrens W Dong et al ldquoNrf2 and Keap1abnormalities in non-small cell lung carcinoma and associationwith clinicopathologic featuresrdquo Clinical Cancer Research vol16 no 14 pp 3743ndash3753 2010

[131] N M Reddy S R Kleeberger H Y Cho et al ldquoDeficiency inNrf2-GSH signaling impairs type II cell growth and enhancessensitivity to oxidantsrdquoAmerican Journal of Respiratory Cell andMolecular Biology vol 37 no 1 pp 3ndash8 2007

[132] N M Reddy S R Kleeberger M Yamamoto et al ldquoGeneticdissection of the Nrf2-dependent redox signaling-regulatedtranscriptional programs of cell proliferation and cytoprotec-tionrdquo Physiological Genomics vol 32 no 1 pp 74ndash81 2007

[133] Y Mitsuishi K Taguchi Y Kawatani et al ldquoNrf2 redirectsglucose and glutamine into anabolic pathways in metabolicreprogrammingrdquo Cancer Cell vol 22 no 1 pp 66ndash79 2012

[134] J Cho SManandharH LeeH Park andMKwak ldquoRole of theNrf2-antioxidant system in cytotoxicity mediated by anticancercisplatin implication to cancer cell resistancerdquo Cancer Lettersvol 260 no 1-2 pp 96ndash108 2008

[135] T Ohta K Iijima M Miyamoto et al ldquoLoss of Keap1 functionactivates Nrf2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008

[136] A Singh M Bodas N Wakabayashi F Bunz and S BiswalldquoGain of Nrf2 function in non-small-cell lung cancer cellsconfers radioresistancerdquoAntioxidantsampRedox Signaling vol 13no 11 pp 1627ndash1637 2010

[137] G-S Shim S Manandhar D-H Shin T Kim and M KwakldquoAcquisition of doxorubicin resistance in ovarian carcinomacells accompanies activation of the NRF2 pathwayrdquo Free RadicalBiology and Medicine vol 47 no 11 pp 1619ndash1631 2009

[138] S Manandhar B Choi K Jung et al ldquoNRF2 inhibitionrepresses ErbB2 signaling in ovarian carcinoma cells implica-tions for tumor growth retardation and docetaxel sensitivityrdquoFree Radical Biology and Medicine vol 52 no 9 pp 1773ndash17852012

[139] T Kim E-g Hur S J Kang et al ldquoNRF2 blockade suppressescolon tumor angiogenesis by inhibiting hypoxia-induced acti-vation ofHIF-1120572rdquoCancer Research vol 71 no 6 pp 2260ndash22752011

[140] L Kinch N V Grishin and J Brugarolas ldquoSuccination of Keap1and activation of Nrf2-dependent antioxidant pathways in FH-deficient papillary renal cell carcinoma type 2rdquo Cancer Cell vol20 no 4 pp 418ndash420 2011

[141] J J Tsai J A Dudakov K Takahashi et al ldquoNrf2 regulateshaematopoietic stem cell functionrdquo Nature Cell Biology vol 15no 3 pp 309ndash316 2013

[142] J H Kim R KThimmulappa V Kumar et al ldquoNRF2-mediatedNotch pathway activation enhances hematopoietic reconsti-tution following myelosuppressive radiationrdquo The Journal ofClinical Investigation vol 124 no 2 pp 730ndash741 2014

[143] J P Chute ldquoNRF2 mitigates radiation-induced hematopoieticdeathrdquo The Journal of Clinical Investigation vol 124 no 3 pp960ndash961 2014

[144] J Jang Y Wang H Kim M A Lalli and K S KosikldquoNrf2 a regulator of the proteasome controls self-renewal andpluripotency in human embryonic stem cellsrdquo Stem Cells vol32 no 10 pp 2616ndash2625 2014

[145] S Murakami R Shimizu P Romeo M Yamamoto and HMotohashi ldquoKeap1-Nrf2 system regulates cell fate determina-tion of hematopoietic stem cellsrdquo Genes to Cells vol 19 no 3pp 239ndash253 2014

[146] C E Hochmuth B Biteau D Bohmann and H Jasper ldquoRedoxregulation by Keap1 and Nrf2 controls intestinal stem cellproliferation inDrosophilardquoCell Stem Cell vol 8 no 2 pp 188ndash199 2011

[147] M K Paul B Bisht D O Darmawan et al ldquoDynamic changesin intracellular ROS levels regulate airway basal stem cellhomeostasis through Nrf2-dependent notch signalingrdquo CellStem Cell vol 15 no 2 pp 199ndash214 2014

[148] L Gambari G Lisignoli L Cattini C Manferdini A Facchiniand F Grassi ldquoSodium hydrosulfide inhibits the differentiationof osteoclast progenitor cells via NRF2-dependent mechanismrdquoPharmacological Research vol 87 pp 99ndash112 2014

[149] J Li D Johnson M Calkins L Wright C Svendsen andJ Johnson ldquoStabilization of Nrf2 by tBHQ confers protectionagainst oxidative stress-induced cell death in human neuralstem cellsrdquo Toxicological Sciences vol 83 no 2 pp 313ndash3282005

[150] A AMerchant A SinghWMatsui and S Biswal ldquoThe redox-sensitive transcription factor Nrf2 regulatesmurine hematopoi-etic stem cell survival independently of ROS levelsrdquo Blood vol118 no 25 pp 6572ndash6579 2011

[151] N Gurusamy D Ray I Lekli and D K Das ldquoRed wineantioxidant resveratrol-modified cardiac stem cells regener-ate infarcted myocardiumrdquo Journal of Cellular and MolecularMedicine vol 14 no 9 pp 2235ndash2239 2010

[152] N Gorbunov G Petrovski N Gurusamy D Ray D H Kimand D K Das ldquoRegeneration of infarcted myocardium withresveratrol-modified cardiac stem cellsrdquo Journal of Cellular andMolecular Medicine vol 16 no 1 pp 174ndash184 2012

[153] C Cai L Teng D Vu et al ldquoThe heme oxygenase 1 inducer(CoPP) protects human cardiac stem cells against apoptosisthrough activation of the extracellular signal-regulated kinase(ERK)NRF2 signaling pathway and cytokine releaserdquo TheJournal of Biological Chemistry vol 287 no 40 pp 33720ndash33732 2012

[154] J Song S M Kang K M Lee and J E Lee ldquoThe protectiveeffect of melatonin on neural stem cell against LPS-inducedinflammationrdquo BioMed Research International vol 2015 ArticleID 854359 13 pages 2015

14 Oxidative Medicine and Cellular Longevity

[155] V Karkkainen Y Pomeshchik E Savchenko et al ldquoNrf2regulates neurogenesis and protects neural progenitor cellsagainst A120573 toxicityrdquo STEM CELLS vol 32 no 7 pp 1904ndash19162014

[156] M Mohammadzadeh R Halabian A Gharehbaghian et alldquoNrf-2 overexpression in mesenchymal stem cells reducesoxidative stress-induced apoptosis and cytotoxicityrdquo Cell Stressand Chaperones vol 17 no 5 pp 553ndash565 2012

[157] Y Takahata T Takarada M Iemata et al ldquoFunctional expres-sion of 1205732 adrenergic receptors responsible for protectionagainst oxidative stress through promotion of glutathione syn-thesis after Nrf2 upregulation in undifferentiated mesenchymalC3H10T12 stem cellsrdquo Journal of Cellular Physiology vol 218no 2 pp 268ndash275 2009

[158] J Zhu H Wang Y Fan et al ldquoKnockdown of nuclear factorerythroid 2-related factor 2 by lentivirus induces differentiationof glioma stem-like cellsrdquo Oncology Reports vol 32 no 3 pp1170ndash1178 2014

[159] J Zhu H Wang Q Sun et al ldquoNrf2 is required to maintain theself-renewal of glioma stem cellsrdquo BMC Cancer vol 13 no 1article 380 2013

[160] B L Emmink A Verheem W J Van Houdt et al ldquoThe secre-tome of colon cancer stem cells contains drug-metabolizingenzymesrdquo Journal of Proteomics vol 91 pp 84ndash96 2013

[161] T Mizuno N Suzuki H Makino et al ldquoCancer stem-like cellsof ovarian clear cell carcinoma are enriched in the ALDH-highpopulation associated with an accelerated scavenging system inreactive oxygen speciesrdquo Gynecologic Oncology vol 137 no 2pp 299ndash305 2015

[162] I-G Ryoo B-H Choi and M-K Kwak ldquoActivation of NRF2by p62 and proteasome reduction in sphere-forming breastcarcinoma cellsrdquo Oncotarget vol 6 no 10 pp 8167ndash8184 2015

[163] T Wu B G Harder P K Wong J E Lang and D D ZhangldquoOxidative stress mammospheres and Nrf2-new implicationfor breast cancer therapyrdquoMolecular Carcinogenesis 2014

[164] B Yang Y Ma and Y Liu ldquoElevated expression of Nrf-2 andABCG2 involved in multi-drug resistance of lung cancer SPcellsrdquo Drug Research 2015

[165] M Zhang A Mathur Y Zhang et al ldquoMithramycin repressesbasal and cigarette smoke-induced expression of abcg2 andinhibits stem cell signaling in lung and esophageal cancer cellsrdquoCancer Research vol 72 no 16 pp 4178ndash4192 2012

[166] C A Del Vecchio Y Feng E S Sokol et al ldquoDe-differentiationconfers multidrug resistance via noncanonical PERK-Nrf2signalingrdquo PLoS Biology vol 12 no 9 Article ID e1001945 2014

[167] D Ren N F Villeneuve T Jiang et al ldquoBrusatol enhancesthe efficacy of chemotherapy by inhibiting the Nrf2-mediateddefense mechanismrdquo Proceedings of the National Academy ofSciences of the United States of America vol 108 no 4 pp 1433ndash1438 2011

[168] A Arlt S Sebens S Krebs et al ldquoInhibition of the Nrf2 tran-scription factor by the alkaloid trigonelline renders pancreaticcancer cells more susceptible to apoptosis through decreasedproteasomal gene expression and proteasome activityrdquo Onco-gene vol 32 no 40 pp 4825ndash4835 2013

[169] A-M Gao Z-P Ke F Shi G Sun and H Chen ldquoChrysinenhances sensitivity of BEL-7402ADM cells to doxorubicin bysuppressing PI3KAktNrf2 and ERKNrf2 pathwayrdquo Chemico-Biological Interactions vol 206 no 1 pp 100ndash108 2013

[170] A M Gao Z P Ke J N Wang J Yang S Chen and HChen ldquoApigenin sensitizes doxorubicin-resistant hepatocellular

carcinoma BEL-7402ADM cells to doxorubicin via inhibitingPI3KAktNrf2 pathwayrdquo Carcinogenesis vol 34 no 8 pp1806ndash1814 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

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Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

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Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Research and TreatmentAIDS

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Gastroenterology Research and Practice

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

8 Oxidative Medicine and Cellular Longevity

Low ROS

Increased drug efflux

SurvivalChemoresistance

CSC

TumorRecurrenceProgression

Radioresistance

PERKuarr

p21uarr

p62uarr

Proteasome darr

NRF2 uarr

Figure 3 Potential roles of NRF2 signaling in CSCs InCSC-like cellmodels NRF2 is activated through multiple molecular mechanismsin a context-dependent manner The upregulation of competingproteins such as p62 and p21 activation of PERK or repressedproteasome function were shown to enhance NRF2 activity inthese models Elevated NRF2 levels in CSCs can contribute tothe maintenance of low ROS by upregulating multiple antioxidantgenes In addition NRF2-mediated expression of ABC transporterselicits efflux of anticancer drugs from cancer cells Overall activatedNRF2 signaling facilitates CSCs survival and stress resistance andconsequently it can be suggested that CSCs with high NRF2 activityplay a crucial role in tumor recurrence and further progression

NRF2 AhR and SP1 and thereby inhibiting the expressionof genes associated with CSC-related pathways resulting inreduced proliferation and tumorigenecity Similarly it wasreported that lung cancer SP cells exhibited high levels ofNRF2 and BCRP expression These SP cells were highlytumorigenic and possessed self-renewal capacity comparedto non-SP cells [164] Emmink et al performed a proteomeanalysis on collected secretome from highly tumorigenicCSCs and their corresponding nontumorigenic differentiatedcells both of which were established from human colorectalspecimens [160] Subsequent bioinformatic analysis revealedthat the CSC secretome contained a large amount of proteinsassociated with cell survival and protein quality controlcompared to differentiated tumor cells Notably the CSCsecretome contained an NRF2 antioxidant and detoxifyingprotein signature in that it included elevated levels ofGCLC GPX23 and TXNRD1 This study provided novelevidence that CSCs secrete NRF2 target antioxidant proteinsto counteract extracellular stressors and chemotherapeuticsIn patients with ovarian clear cell carcinoma the expressionof the CSC maker aldehyde dehydrogenase-1 (ALDH1) wasstrongly correlated with an advanced clinical stage andreduced progression free survival [161] Ovarian clear cellcarcinoma cells with high ALDH1 expressionmaintained lowlevels of ROS compared to ALDH-low cells and these cellswere shown to express higher levels of NRF2 and its targetgenes

Two recent studies have demonstratedNRF2 activation insphere cultures of breast cancer cells that is one of models ofCSCs Wu et al showed that mammospheres derived fromMCF7 and MDA-MB231 breast cancer cell lines exhibitedlower ROS levels compared to their monolayer counterpartsThey also showed that levels of NRF2 and target genes such

as NQO1 and GCLM were elevated in mammospheres [163]Similarly our group has shown substantially elevated NRF2protein levels along with increased expression of antioxidantgenes (eg HO-1 and GPX2) and drug efflux transporters(eg MRP2 and BCRP) in sphere cultures of breast cancercells NRF2 accumulation was also observed in sphere-cultured ovarian and colon cancer cells However shRNA-mediated downregulation ofNRF2 led to decreased chemore-sistance of mammospheres presumably due to reduced levelsof antioxidant genes and drug transporters High ROS levelsin NRF2 knockdown mammospheres caused sphere growthretardation and apoptosis Coherently ablation of ABCtransporter induction in NRF2 knockdown mammospheressensitized to anticancer agents [162] Additionally this studyprovided evidence that increased NRF2 protein expression inmammospheres can be linked to 26S proteasome reductionand p62 accumulation In particular knockdown of p62 inMCF7 mammospheres significantly attenuated NRF2 eleva-tion

Surviving dedifferentiated breast cancer cells after chem-otherapy treatment retained high levels of NRF2 activationsimilar to other CSCs However NRF2 activation was medi-ated by a noncanonical pathway Levels of PERKwere high indedifferentiated cancer cells and this in turn phosphorylatedand activated NRF2 signaling to maintain low cellular ROSlevels and to express ABC transporters In agreement withthese findings clinical observations revealed that the PERKpathway gene signature is related to chemoresistance andreduced patient survival [166]

10 Concluding Remarks

Recent studies have started to uncover the role of ROS signal-ing in the biology of CSCs which is related to tumorigenecitytumor progression and relapse Expression of the transcrip-tion factor NRF2 a master regulator of antioxidant genesexpression is increased in different models of CSCs and thiselevation is likely to promote CSC maintenance and survivalin an oxidizing tumormicroenvironment In additionNRF2-mediated overexpression of ABC transporters particularlythe CSC marker BCRP may play a critical role in themultidrug resistance of CSCsThese findings combined withthe increasing evidence showing the alteration of KEAP1-NRF2 signaling in cancer cells suggest a novel role of NRF2in CSC maintenance and survival (Figure 3)

One important question that arises from the current stud-ies is whether it is possible to design CSC-targeted therapiesthrough regulation of theNRF2 pathway and its related redoxhomeostasis inCSCs Recent studies provide several potentialclues for addressing this question the naturally occurringalkaloid brusatol could reduce the growth and chemoresis-tance of breast CSCs [163] Treatment ofmammospheres withbrusatol elevated ROS levels and promoted taxol-inducedgrowth retardation and cell death The NRF2 repressivemechanism of brusatol has not been clearly elucidated but itappears to be independent of KEAP1-mediated degradation[167] In addition to brusatol natural compounds such as

Oxidative Medicine and Cellular Longevity 9

chrysin apigenin luteolin and trigonelline are known toinhibit NRF2 signaling in several types of cancer cells [102168ndash170] and therefore the development of NRF2 inhibitorswith characterized modes of action will enable efficient tar-geting of the redox homeostasis system as well as multidrugresistance systems in CSCs

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This studywas financially supported by theNational ResearchFoundation (NRF) funded by the Ministry of Science ICT ampFuture Planning (NRF-2013R1A2A2A01015497)

References

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[2] P Storz ldquoForkhead homeobox type O transcription factorsin the responses to oxidative stressrdquo Antioxidants amp RedoxSignaling vol 14 no 4 pp 593ndash605 2011

[3] R S Balaban S Nemoto and T Finkel ldquoMitochondria oxi-dants and agingrdquo Cell vol 120 no 4 pp 483ndash495 2005

[4] L A Sena and N S Chandel ldquoPhysiological roles of mitochon-drial reactive oxygen speciesrdquoMolecular Cell vol 48 no 2 pp158ndash167 2012

[5] P T Schumacker ldquoReactive oxygen species in cancer cells liveby the sword die by the swordrdquo Cancer Cell vol 10 no 3 pp175ndash176 2006

[6] S Toyokuni K Okamoto J Yodoi and H Hiai ldquoPersistentoxidative stress in cancerrdquo FEBS Letters vol 358 no 1 pp 1ndash3 1995

[7] L Behrend G Henderson and R Zwacka ldquoReactive oxy-gen species in oncogenic transformationrdquo Biochemical SocietyTransactions vol 31 no 6 pp 1441ndash1444 2003

[8] E O Hileman J Liu M Albitar M J Keating and P HuangldquoIntrinsic oxidative stress in cancer cells a biochemical basis fortherapeutic selectivityrdquo Cancer Chemotherapy and Pharmacol-ogy vol 53 no 3 pp 209ndash219 2004

[9] H PelicanoD Carney and PHuang ldquoROS stress in cancer cellsand therapeutic implicationsrdquo Drug Resistance Updates vol 7no 2 pp 97ndash110 2004

[10] Y Adachi Y Shibai J Mitsushita W H Shang K Hirose andT Kamata ldquoOncogenic Ras upregulates NADPH oxidase 1 geneexpression through MEK-ERK-dependent phosphorylation ofGATA-6rdquo Oncogene vol 27 no 36 pp 4921ndash4932 2008

[11] J Mitsushita J D Lambeth and T Kamata ldquoThe superoxide-generating oxidase Nox1 is functionally required for Ras onco-gene transformationrdquoCancer Research vol 64 no 10 pp 3580ndash3585 2004

[12] O Vafa M Wade S Kern et al ldquoc-Myc can induce DNAdamage increase reactive oxygen species and mitigate p53function a mechanism for oncogene-induced genetic instabil-ityrdquoMolecular Cell vol 9 no 5 pp 1031ndash1044 2002

[13] K Polyak Y Li H Zhu et al ldquoSomatic mutations of themitochondrial genome in human colorectal tumoursrdquo NatureGenetics vol 20 no 3 pp 291ndash293 1998

[14] D Trachootham J Alexandre and P Huang ldquoTargeting can-cer cells by ROS-mediated mechanisms a radical therapeuticapproachrdquo Nature Reviews Drug Discovery vol 8 no 7 pp579ndash591 2009

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[29] C Blanpain M Mohrin P A Sotiropoulou and E PassegueldquoDNA-damage response in tissue-specific and cancer stemcellsrdquo Cell Stem Cell vol 8 no 1 pp 16ndash29 2011

10 Oxidative Medicine and Cellular Longevity

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[31] E Lagadinou A Sach K Callahan et al ldquoBCL-2 inhibitiontargets oxidative phosphorylation and selectively eradicatesquiescent human leukemia stem cellsrdquo Cell Stem Cell vol 12no 3 pp 329ndash341 2013

[32] C Chang Y Chen S Chou et al ldquoDistinct subpopulations ofhead and neck cancer cells with different levels of intracellularreactive oxygen species exhibit diverse stemness proliferationand chemosensitivityrdquoCancer Research vol 74 no 21 pp 6291ndash6305 2014

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[34] S Achuthan T R Santhoshkumar J Prabhakar S A Nair andM R Pillai ldquoDrug-induced senescence generates chemoresis-tant stemlike cells with low reactive oxygen speciesrdquoThe Journalof Biological Chemistry vol 286 no 43 pp 37813ndash37829 2011

[35] L Zinzi M Contino M Cantore E Capparelli M Leopoldoand N A Colabufo ldquoABC transporters in CSCs membranesas a novel target for treating tumor relapserdquo Frontiers inPharmacology vol 5 article 163 2014

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[37] C L Bigarella R Liang and S Ghaffari ldquoStem cells and theimpact of ROS signalingrdquo Development vol 141 no 22 pp4206ndash4218 2014

[38] P Chaudhari Z Ye and Y Y Jang ldquoRoles of reactive oxygenspecies in the fate of stem cellsrdquoAntioxidants ampRedox Signalingvol 20 no 12 pp 1881ndash1890 2014

[39] V A Rafalski E Mancini and A Brunet ldquoEnergy metabolismand energy-sensing pathways in mammalian embryonic andadult stem cell faterdquo Journal of Cell Science vol 125 no 23 pp5597ndash5608 2012

[40] K Wang T Zhang Q Dong E C Nice C Huang and Y WeildquoRedox homeostasis the linchpin in stem cell self-renewal anddifferentiationrdquo Cell Death and Disease vol 4 no 3 p e5372013

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[42] Y Y Jang and S J Sharkis ldquoA low level of reactive oxygen speciesselects for primitive hematopoietic stem cells that may reside inthe low-oxygenic nicherdquo Blood vol 110 no 8 pp 3056ndash30632007

[43] S Ghaffari ldquoOxidative stress in the regulation of normal andneoplastic hematopoiesisrdquo Antioxidants amp Redox Signaling vol10 no 11 pp 1923ndash1940 2008

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[45] A Alexander S-L Cai J Kim et al ldquoATM signals to TSC2in the cytoplasm to regulate mTORC1 in response to ROSrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 107 no 9 pp 4153ndash4158 2010

[46] C Cosentino D Grieco and V Costanzo ldquoATM activates thepentose phosphate pathway promoting anti-oxidant defenceand DNA repairrdquo The EMBO Journal vol 30 no 3 pp 546ndash555 2011

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[48] K Ito K Takubo F Arai et al ldquoRegulation of reactive oxygenspecies by Atm is essential for proper response to DNA double-strand breaks in lymphocytesrdquoThe Journal of Immunology vol178 no 1 pp 103ndash110 2007

[49] Y Wang Y Zhou and D T Graves ldquoFOXO transcription fac-tors their clinical significance and regulationrdquoBioMed ResearchInternational vol 2014 Article ID 925350 13 pages 2014

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[52] Z Tothova R Kollipara B J Huntly et al ldquoFoxOs are criticalmediators of hematopoietic stem cell resistance to physiologicoxidative stressrdquo Cell vol 128 no 2 pp 325ndash339 2007

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[54] J Paik Z Ding R Narurkar et al ldquoFoxOs cooperatively regu-late diverse pathways governing neural stem cell homeostasisrdquoCell Stem Cell vol 5 no 5 pp 540ndash553 2009

[55] V M Renault V A Rafalski A A Morgan et al ldquoFoxO3regulates neural stem cell homeostasisrdquo Cell Stem Cell vol 5no 5 pp 527ndash539 2009

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[58] M M Juntilla V D Patil M Calamito R P Joshi M JBirnbaum and G A Koretzky ldquoAKT1 and AKT2 maintainhematopoietic stem cell function by regulating reactive oxygenspeciesrdquo Blood vol 115 no 20 pp 4030ndash4038 2010

[59] O H Yilmaz R Valdez B K Theisen et al ldquoPten depen-dence distinguishes haematopoietic stem cells from leukaemia-initiating cellsrdquo Nature vol 441 no 7092 pp 475ndash482 2006

[60] S N Greer J L Metcalf Y Wang and M Ohh ldquoThe updatedbiology of hypoxia-inducible factorrdquoTheEMBO Journal vol 31no 11 pp 2448ndash2460 2012

[61] C E Forristal K L Wright N A Hanley R O Oreffo and FD Houghton ldquoHypoxia inducible factors regulate pluripotencyand proliferation in human embryonic stem cells cultured atreduced oxygen tensionsrdquo Reproduction vol 139 no 1 pp 85ndash97 2009

[62] K L Covello J Kehler H Yu et al ldquoHIF-2120572 regulates Oct-4effects of hypoxia on stem cell function embryonic develop-ment and tumor growthrdquo Genes amp Development vol 20 no5 pp 557ndash570 2006

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stem cells through Wnt120573-catenin signallingrdquo Nature Cell Biol-ogy vol 12 no 10 pp 1007ndash1013 2010

Oxidative Medicine and Cellular Longevity 11

[64] K Takubo N Goda W Yamada et al ldquoRegulation of the HIF-1alpha level is essential for hematopoietic stem cellsrdquo Cell StemCell vol 7 no 3 pp 391ndash402 2010

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[66] T M Phillips W H McBride and F Pajonk ldquoThe responseof CD24minus119897119900119908CD44+ breast cancer-initiating cells to radiationrdquoJNCI Journal of the National Cancer Institute vol 98 no 24 pp1777ndash1785 2006

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[69] S Kim C Kwon and I Nakano ldquoDetoxification of oxidativestress in glioma stem cells mechanism clinical relevance andtherapeutic developmentrdquo Journal of Neuroscience Research vol92 no 11 pp 1419ndash1424 2014

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[72] N Haraguchi H Ishii K Mimori et al ldquoCD13 is a therapeutictarget in human liver cancer stem cellsrdquo Journal of ClinicalInvestigation vol 120 no 9 pp 3326ndash3339 2010

[73] H M Kim N Haraguchi H Ishii et al ldquoIncreased CD13expression reduces reactive oxygen species promoting sur-vival of liver cancer stem cells via an epithelial-mesenchymaltransition-like phenomenonrdquo Annals of Surgical Oncology vol19 supplement 3 pp S539ndashS548 2012

[74] K Naka T Hoshii T Muraguchi et al ldquoTGF-120573ndashFOXO sig-nalling maintains leukaemia-initiating cells in chronic myeloidleukaemiardquo Nature vol 463 no 7281 pp 676ndash680 2010

[75] J Zhou J Wulfkuhle H Zhang et al ldquoActivation of thePTENmTORSTAT3 pathway in breast cancer stem-like cellsis required for viability and maintenancerdquo Proceedings of theNational Academy of Sciences vol 104 no 41 pp 16158ndash161632007

[76] A Dubrovska S Kim R J Salamone et al ldquoThe role ofPTENAktPI3K signaling in the maintenance and viability ofprostate cancer stem-like cell populationsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 106 no 1 pp 268ndash273 2009

[77] H Yin and J Glass ldquoThe phenotypic radiation resistance ofCS44+CD24minusor low breast cancer cells is mediated through theenhanced activation of ATM signalingrdquo PLoS ONE vol 6 no9 Article ID e24080 2011

[78] T H Rushmore and C Pickett ldquoTranscriptional regulation ofthe rat glutathione S-transferase Ya subunit gene Characteri-zation of a xenobiotic-responsive element controlling inducibleexpression by phenolic antioxidantsrdquo The Journal of BiologicalChemistry vol 265 no 24 pp 14648ndash14653 1990

[79] Y Li and A Jaiswal ldquoRegulation of humanNAD (P)H quinoneoxidoreductase gene Role of AP1 binding site contained withinhuman antioxidant response elementrdquoThe Journal of BiologicalChemistry vol 267 no 21 pp 15097ndash15104 1992

[80] T Prestera P Talalay J Alam et al ldquoParallel induction of hemeoxygenase-1 and chemoprotective phase 2 enzymes by elec-trophiles and antioxidants regulation by upstream antioxidant-responsive elements (ARE)rdquoMolecular Medicine vol 1 no 7 p827 1995

[81] K Itoh K IgarashiNHayashi et al ldquoCloning and characteriza-tion of a novel erythroid cell-derived CNC family transcriptionfactor heterodimerizing with the small Maf family proteinsrdquoMolecular and Cellular Biology vol 15 no 8 pp 4184ndash41931995

[82] R Venugopal and A K Jaiswal ldquoNrf1 and Nrf2 positivelyand c-Fos and Fra1 negatively regulate the human antioxidantresponse element-mediated expression of NAD(P)Hquinoneoxidoreductase1 generdquo Proceedings of the National Academy ofSciences vol 93 no 25 pp 14960ndash14965 1996

[83] K Itoh T Chiba S Takahashi et al ldquoAn Nrf2small Mafheterodimer mediates the induction of phase II detoxifyingenzyme genes through antioxidant response elementsrdquo Bio-chemical and Biophysical Research Communications vol 236no 2 pp 313ndash322 1997

[84] M Ramos-Gomez M-K Kwak P M Dolan et al ldquoSensitivityto carcinogenesis is increased and chemoprotective efficacy ofenzyme inducers is lost in nrf2 transcription factor-deficientmicerdquo Proceedings of the National Academy of Sciences vol 98no 6 pp 3410ndash3415 2001

[85] M-K Kwak N Wakabayashi K Itoh H Motohashi MYamamoto and T W Kensler ldquoModulation of gene expressionby cancer chemopreventive dithiolethiones through the Keap1-Nrf2 pathway Identification of novel gene clusters for cellsurvivalrdquo The Journal of Biological Chemistry vol 278 no 10pp 8135ndash8145 2003

[86] R Hu C Xu G Shen et al ldquoGene expression profiles inducedby cancer chemopreventive isothiocyanate sulforaphane in theliver of C57BL6J mice and C57BL6JNrf2 (minusminus) micerdquo CancerLetters vol 243 no 2 pp 170ndash192 2006

[87] S Chanas Q Jiang and M McMahon ldquoLoss of the Nrf2transcription factor causes a marked reduction in constitutiveand inducible expression of the glutathione S-transferase Gsta1Gsta2 Gstm1 Gstm2 Gstm3 and Gstm4 genes in the livers ofmale and female micerdquo Biochemical Journal vol 365 no 2 pp405ndash416 2002

[88] H-Y Cho S P Reddy A DeBiase M Yamamoto and SR Kleeberger ldquoGene expression profiling of NRF2-mediatedprotection against oxidative injuryrdquo Free Radical Biology andMedicine vol 38 no 3 pp 325ndash343 2005

[89] G Shen C Xu R Hu et al ldquoModulation of nuclear factorE2-related factor 2-mediated gene expression in mice liver andsmall intestine by cancer chemopreventive agent curcuminrdquoMolecular Cancer Therapeutics vol 5 no 1 pp 39ndash51 2006

[90] J D Hayes and A T Dinkova-Kostova ldquoThe Nrf2 regulatorynetwork provides an interface between redox and intermediarymetabolismrdquo Trends in Biochemical Sciences vol 39 no 4 pp199ndash218 2014

[91] A Hayashi H Suzuki K Itoh M Yamamoto and Y SugiyamaldquoTranscription factor Nrf2 is required for the constitutive andinducible expression of multidrug resistance-associated protein1 in mouse embryo fibroblastsrdquo Biochemical and BiophysicalResearch Communications vol 310 no 3 pp 824ndash829 2003

[92] E Jigorel M Le Vee C Boursier-Neyret Y Parmentier andO Fardel ldquoDifferential regulation of sinusoidal and canalicularhepatic drug transporter expression by xenobiotics activating

12 Oxidative Medicine and Cellular Longevity

drug-sensing receptors in primary human hepatocytesrdquo DrugMetabolism and Disposition vol 34 no 10 pp 1756ndash1763 2006

[93] X Wang C R Campos J C Peart et al ldquoNrf2 upregulatesATP binding cassette transporter expression and activity atthe blood-brain and blood-spinal cord barriersrdquo Journal ofNeuroscience vol 34 no 25 pp 8585ndash8593 2014

[94] H Jeong I Ryoo andMKwak ldquoRegulation of the expression ofrenal drug transporters in KEAP1-knockdown human tubularcellsrdquo Toxicology in Vitro vol 29 no 5 pp 884ndash892 2015

[95] M J Canet M D Merrell B G Harder et al ldquoIdentificationof a functional antioxidant response element within the eighthintron of the human ABCC

3generdquo Drug Metabolism and

Disposition vol 43 no 1 pp 93ndash99 2014[96] A Singh H Wu P Zhang C Happel J Ma and S Biswal

ldquoExpression of ABCG2 (BCRP) is regulated by Nrf2 in cancercells that confers side population and chemoresistance pheno-typerdquo Molecular Cancer Therapeutics vol 9 no 8 pp 2365ndash2376 2010

[97] N Wakabayashi K Itoh J Wakabayashi et al ldquoKeap1-nullmutation leads to postnatal lethality due to constitutive Nrf2activationrdquo Nature Genetics vol 35 no 3 pp 238ndash245 2003

[98] S B Cullinan J D Gordan J Jin J W Harper and J A DiehlldquoThe Keap1-BTB protein is an adaptor that bridges Nrf2 to aCul3-based E3 ligase oxidative stress sensing by a Cul3-Keap1ligaserdquoMolecular and Cellular Biology vol 24 no 19 pp 8477ndash8486 2004

[99] D D Zhang and M Hannink ldquoDistinct cysteine residues inkeap1 are required for keap1-dependent ubiquitination of nrf2and for stabilization of nrf2 by chemopreventive agents andoxidative stressrdquoMolecular and Cellular Biology vol 23 no 22pp 8137ndash8151 2003

[100] L M Zipper and R T Mulcahy ldquoThe Keap1 BTBPOZ dimer-ization function is required to sequester Nrf2 in cytoplasmrdquoThe Journal of Biological Chemistry vol 277 no 39 pp 36544ndash36552 2002

[101] M C Jaramillo and D D Zhang ldquoThe emerging role of theNrf2-Keap1 signaling pathway in cancerrdquoGenesampDevelopmentvol 27 no 20 pp 2179ndash2191 2013

[102] K I Tong Y Katoh H Kusunoki K Itoh T Tanaka and MYamamoto ldquoKeap1 recruits Neh2 through binding to ETGEand DLG motifs characterization of the two-site molecularrecognition modelrdquoMolecular and Cellular Biology vol 26 no8 pp 2887ndash2900 2006

[103] K I Tong A Kobayashi F Katsuoka andM Yamamoto ldquoTwo-site substrate recognition model for the Keap1-Nrf2 system ahinge and latch mechanismrdquo Biological Chemistry vol 387 no10-11 pp 1311ndash1320 2006

[104] K I Tong B Padmanabhan A Kobayashi et al ldquoDifferentelectrostatic potentials define ETGE and DLG motifs as hingeand latch in oxidative stress responserdquo Molecular and CellularBiology vol 27 no 21 pp 7511ndash7521 2007

[105] M Kobayashi L Li N Iwamoto et al ldquoThe antioxidant defensesystemKeap1-Nrf2 comprises amultiple sensingmechanism forresponding to a wide range of chemical compoundsrdquoMolecularand Cellular Biology vol 29 no 2 pp 493ndash502 2009

[106] T Yamamoto T Suzuki A Kobayashi et al ldquoPhysiological sig-nificance of reactive cysteine residues of Keap1 in determiningNrf2 activityrdquoMolecular and Cellular Biology vol 28 no 8 pp2758ndash2770 2008

[107] M K Kwak K Itoh M Yamamoto and T W KenslerldquoEnhanced expression of the transcription factorNrf2 by cancer

chemopreventive agents role of antioxidant response element-like sequences in the nrf2 promoterrdquo Molecular and CellularBiology vol 22 no 9 pp 2883ndash2892 2002

[108] T Suzuki T Shibata K Takaya et al ldquoRegulatory nexus ofsynthesis and degradation deciphers cellular Nrf2 expressionlevelsrdquoMolecular and Cellular Biology vol 33 no 12 pp 2402ndash2412 2013

[109] S B Cullinan D Zhang M Hannink E Arvisais R JKaufman and J A Diehl ldquoNrf2 is a direct PERK substrateand effector of PERK-dependent cell survivalrdquo Molecular andCellular Biology vol 23 no 20 pp 7198ndash7209 2003

[110] W Li and A Kong ldquoMolecular mechanisms of Nrf2-mediatedantioxidant responserdquo Molecular Carcinogenesis vol 48 no 2pp 91ndash104 2009

[111] Y J Surh ldquoCancer chemoprevention with dietary phytochemi-calsrdquo Nature Reviews Cancer vol 3 no 10 pp 768ndash780 2003

[112] M Salazar A I Rojo D Velasco R M de Sagarra and ACuadrado ldquoGlycogen synthase kinase-3beta inhibits the xeno-biotic and antioxidant cell response by direct phosphorylationand nuclear exclusion of the transcription factor Nrf2rdquo Journalof Biological Chemistry vol 281 no 21 pp 14841ndash14851 2006

[113] W Chen Z Sun X J Wang et al ldquoDirect interactionbetween Nrf2 and p21(Cip1WAF1) upregulates the Nrf2-mediated antioxidant responserdquo Molecular Cell vol 34 no 6pp 663ndash673 2009

[114] M Komatsu H Kurokawa S Waguri et al ldquoThe selectiveautophagy substrate p62 activates the stress responsive tran-scription factor Nrf2 through inactivation of Keap1rdquoNature CellBiology vol 12 no 3 pp 213ndash223 2010

[115] J D Hayes M McMahon S Chowdhry and A T Dinkova-Kostova ldquoCancer chemoprevention mechanisms mediatedthrough the Keap1-Nrf2 pathwayrdquoAntioxidants amp Redox Signal-ing vol 13 no 11 pp 1713ndash1748 2010

[116] R Hu C L Saw R Yu and A T Kong ldquoRegulation ofNF-E2-related factor 2 signaling for cancer chemopreventionantioxidant coupled with antiinflammatoryrdquo Antioxidants ampRedox Signaling vol 13 no 11 pp 1679ndash1698 2010

[117] M Kwak and T W Kensler ldquoTargeting NRF2 signaling for can-cer chemopreventionrdquo Toxicology and Applied Pharmacologyvol 244 no 1 pp 66ndash76 2010

[118] K Iida K Itoh Y Kumagai et al ldquoNrf2 is essential for thechemopreventive efficacy of oltipraz against urinary bladdercarcinogenesisrdquo Cancer Research vol 64 no 18 pp 6424ndash64312004

[119] T Jiang N Chen F Zhao et al ldquoHigh levels of Nrf2 deter-mine chemoresistance in type II endometrial cancerrdquo CancerResearch vol 70 no 13 pp 5486ndash5496 2010

[120] Y R Kim J EOhM S Kim et al ldquoOncogenicNRF2mutationsin squamous cell carcinomas of oesophagus and skinrdquo TheJournal of Pathology vol 220 no 4 pp 446ndash451 2010

[121] T Shibata A Kokubu M Gotoh et al ldquoGenetic alterationof Keap1 confers constitutive Nrf2 activation and resistance tochemotherapy in gallbladder cancerrdquoGastroenterology vol 135no 4 pp 1358e4ndash1368e4 2008

[122] T Shibata T Ohta K I Tong et al ldquoCancer related mutationsin NRF2 impair its recognition by Keap1-Cul3 E3 ligase andpromote malignancyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 36 pp13568ndash13573 2008

[123] A Singh V Misra R K Thimmulappa et al ldquoDysfunctionalKEAP1-NRF2 interaction in non-small-cell lung cancerrdquo PLoSMedicine vol 3 no 10 article e420 2006

Oxidative Medicine and Cellular Longevity 13

[124] R Wang J An F Ji H Jiao H Sun and D Zhou ldquoHyper-methylation of the Keap1 gene in human lung cancer cell linesand lung cancer tissuesrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 1 pp 151ndash154 2008

[125] P Zhang A Singh S Yegnasubramanian et al ldquoLoss ofKelch-like ECH-associated protein 1 function in prostate cancercells causes chemoresistance and radioresistance and promotestumor growthrdquoMolecular Cancer Therapeutics vol 9 no 2 pp336ndash346 2010

[126] J D Hayes and M McMahon ldquoNRF2 and KEAP1 mutationspermanent activation of an adaptive response in cancerrdquo Trendsin Biochemical Sciences vol 34 no 4 pp 176ndash188 2009

[127] B Padmanabhan K I Tong T Ohta et al ldquoStructural basis fordefects of Keap1 activity provoked by its pointmutations in lungcancerrdquoMolecular Cell vol 21 no 5 pp 689ndash700 2006

[128] Y Inami S Waguri A Sakamoto et al ldquoPersistent activation ofNrf2 throughp62 in hepatocellular carcinoma cellsrdquoTheJournalof Cell Biology vol 193 no 2 pp 275ndash284 2011

[129] G M DeNicola F A Karreth T J Humpton et al ldquoOncogene-induced Nrf2 transcription promotes ROS detoxification andtumorigenesisrdquo Nature vol 475 no 7354 pp 106ndash109 2011

[130] L M Solis C Behrens W Dong et al ldquoNrf2 and Keap1abnormalities in non-small cell lung carcinoma and associationwith clinicopathologic featuresrdquo Clinical Cancer Research vol16 no 14 pp 3743ndash3753 2010

[131] N M Reddy S R Kleeberger H Y Cho et al ldquoDeficiency inNrf2-GSH signaling impairs type II cell growth and enhancessensitivity to oxidantsrdquoAmerican Journal of Respiratory Cell andMolecular Biology vol 37 no 1 pp 3ndash8 2007

[132] N M Reddy S R Kleeberger M Yamamoto et al ldquoGeneticdissection of the Nrf2-dependent redox signaling-regulatedtranscriptional programs of cell proliferation and cytoprotec-tionrdquo Physiological Genomics vol 32 no 1 pp 74ndash81 2007

[133] Y Mitsuishi K Taguchi Y Kawatani et al ldquoNrf2 redirectsglucose and glutamine into anabolic pathways in metabolicreprogrammingrdquo Cancer Cell vol 22 no 1 pp 66ndash79 2012

[134] J Cho SManandharH LeeH Park andMKwak ldquoRole of theNrf2-antioxidant system in cytotoxicity mediated by anticancercisplatin implication to cancer cell resistancerdquo Cancer Lettersvol 260 no 1-2 pp 96ndash108 2008

[135] T Ohta K Iijima M Miyamoto et al ldquoLoss of Keap1 functionactivates Nrf2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008

[136] A Singh M Bodas N Wakabayashi F Bunz and S BiswalldquoGain of Nrf2 function in non-small-cell lung cancer cellsconfers radioresistancerdquoAntioxidantsampRedox Signaling vol 13no 11 pp 1627ndash1637 2010

[137] G-S Shim S Manandhar D-H Shin T Kim and M KwakldquoAcquisition of doxorubicin resistance in ovarian carcinomacells accompanies activation of the NRF2 pathwayrdquo Free RadicalBiology and Medicine vol 47 no 11 pp 1619ndash1631 2009

[138] S Manandhar B Choi K Jung et al ldquoNRF2 inhibitionrepresses ErbB2 signaling in ovarian carcinoma cells implica-tions for tumor growth retardation and docetaxel sensitivityrdquoFree Radical Biology and Medicine vol 52 no 9 pp 1773ndash17852012

[139] T Kim E-g Hur S J Kang et al ldquoNRF2 blockade suppressescolon tumor angiogenesis by inhibiting hypoxia-induced acti-vation ofHIF-1120572rdquoCancer Research vol 71 no 6 pp 2260ndash22752011

[140] L Kinch N V Grishin and J Brugarolas ldquoSuccination of Keap1and activation of Nrf2-dependent antioxidant pathways in FH-deficient papillary renal cell carcinoma type 2rdquo Cancer Cell vol20 no 4 pp 418ndash420 2011

[141] J J Tsai J A Dudakov K Takahashi et al ldquoNrf2 regulateshaematopoietic stem cell functionrdquo Nature Cell Biology vol 15no 3 pp 309ndash316 2013

[142] J H Kim R KThimmulappa V Kumar et al ldquoNRF2-mediatedNotch pathway activation enhances hematopoietic reconsti-tution following myelosuppressive radiationrdquo The Journal ofClinical Investigation vol 124 no 2 pp 730ndash741 2014

[143] J P Chute ldquoNRF2 mitigates radiation-induced hematopoieticdeathrdquo The Journal of Clinical Investigation vol 124 no 3 pp960ndash961 2014

[144] J Jang Y Wang H Kim M A Lalli and K S KosikldquoNrf2 a regulator of the proteasome controls self-renewal andpluripotency in human embryonic stem cellsrdquo Stem Cells vol32 no 10 pp 2616ndash2625 2014

[145] S Murakami R Shimizu P Romeo M Yamamoto and HMotohashi ldquoKeap1-Nrf2 system regulates cell fate determina-tion of hematopoietic stem cellsrdquo Genes to Cells vol 19 no 3pp 239ndash253 2014

[146] C E Hochmuth B Biteau D Bohmann and H Jasper ldquoRedoxregulation by Keap1 and Nrf2 controls intestinal stem cellproliferation inDrosophilardquoCell Stem Cell vol 8 no 2 pp 188ndash199 2011

[147] M K Paul B Bisht D O Darmawan et al ldquoDynamic changesin intracellular ROS levels regulate airway basal stem cellhomeostasis through Nrf2-dependent notch signalingrdquo CellStem Cell vol 15 no 2 pp 199ndash214 2014

[148] L Gambari G Lisignoli L Cattini C Manferdini A Facchiniand F Grassi ldquoSodium hydrosulfide inhibits the differentiationof osteoclast progenitor cells via NRF2-dependent mechanismrdquoPharmacological Research vol 87 pp 99ndash112 2014

[149] J Li D Johnson M Calkins L Wright C Svendsen andJ Johnson ldquoStabilization of Nrf2 by tBHQ confers protectionagainst oxidative stress-induced cell death in human neuralstem cellsrdquo Toxicological Sciences vol 83 no 2 pp 313ndash3282005

[150] A AMerchant A SinghWMatsui and S Biswal ldquoThe redox-sensitive transcription factor Nrf2 regulatesmurine hematopoi-etic stem cell survival independently of ROS levelsrdquo Blood vol118 no 25 pp 6572ndash6579 2011

[151] N Gurusamy D Ray I Lekli and D K Das ldquoRed wineantioxidant resveratrol-modified cardiac stem cells regener-ate infarcted myocardiumrdquo Journal of Cellular and MolecularMedicine vol 14 no 9 pp 2235ndash2239 2010

[152] N Gorbunov G Petrovski N Gurusamy D Ray D H Kimand D K Das ldquoRegeneration of infarcted myocardium withresveratrol-modified cardiac stem cellsrdquo Journal of Cellular andMolecular Medicine vol 16 no 1 pp 174ndash184 2012

[153] C Cai L Teng D Vu et al ldquoThe heme oxygenase 1 inducer(CoPP) protects human cardiac stem cells against apoptosisthrough activation of the extracellular signal-regulated kinase(ERK)NRF2 signaling pathway and cytokine releaserdquo TheJournal of Biological Chemistry vol 287 no 40 pp 33720ndash33732 2012

[154] J Song S M Kang K M Lee and J E Lee ldquoThe protectiveeffect of melatonin on neural stem cell against LPS-inducedinflammationrdquo BioMed Research International vol 2015 ArticleID 854359 13 pages 2015

14 Oxidative Medicine and Cellular Longevity

[155] V Karkkainen Y Pomeshchik E Savchenko et al ldquoNrf2regulates neurogenesis and protects neural progenitor cellsagainst A120573 toxicityrdquo STEM CELLS vol 32 no 7 pp 1904ndash19162014

[156] M Mohammadzadeh R Halabian A Gharehbaghian et alldquoNrf-2 overexpression in mesenchymal stem cells reducesoxidative stress-induced apoptosis and cytotoxicityrdquo Cell Stressand Chaperones vol 17 no 5 pp 553ndash565 2012

[157] Y Takahata T Takarada M Iemata et al ldquoFunctional expres-sion of 1205732 adrenergic receptors responsible for protectionagainst oxidative stress through promotion of glutathione syn-thesis after Nrf2 upregulation in undifferentiated mesenchymalC3H10T12 stem cellsrdquo Journal of Cellular Physiology vol 218no 2 pp 268ndash275 2009

[158] J Zhu H Wang Y Fan et al ldquoKnockdown of nuclear factorerythroid 2-related factor 2 by lentivirus induces differentiationof glioma stem-like cellsrdquo Oncology Reports vol 32 no 3 pp1170ndash1178 2014

[159] J Zhu H Wang Q Sun et al ldquoNrf2 is required to maintain theself-renewal of glioma stem cellsrdquo BMC Cancer vol 13 no 1article 380 2013

[160] B L Emmink A Verheem W J Van Houdt et al ldquoThe secre-tome of colon cancer stem cells contains drug-metabolizingenzymesrdquo Journal of Proteomics vol 91 pp 84ndash96 2013

[161] T Mizuno N Suzuki H Makino et al ldquoCancer stem-like cellsof ovarian clear cell carcinoma are enriched in the ALDH-highpopulation associated with an accelerated scavenging system inreactive oxygen speciesrdquo Gynecologic Oncology vol 137 no 2pp 299ndash305 2015

[162] I-G Ryoo B-H Choi and M-K Kwak ldquoActivation of NRF2by p62 and proteasome reduction in sphere-forming breastcarcinoma cellsrdquo Oncotarget vol 6 no 10 pp 8167ndash8184 2015

[163] T Wu B G Harder P K Wong J E Lang and D D ZhangldquoOxidative stress mammospheres and Nrf2-new implicationfor breast cancer therapyrdquoMolecular Carcinogenesis 2014

[164] B Yang Y Ma and Y Liu ldquoElevated expression of Nrf-2 andABCG2 involved in multi-drug resistance of lung cancer SPcellsrdquo Drug Research 2015

[165] M Zhang A Mathur Y Zhang et al ldquoMithramycin repressesbasal and cigarette smoke-induced expression of abcg2 andinhibits stem cell signaling in lung and esophageal cancer cellsrdquoCancer Research vol 72 no 16 pp 4178ndash4192 2012

[166] C A Del Vecchio Y Feng E S Sokol et al ldquoDe-differentiationconfers multidrug resistance via noncanonical PERK-Nrf2signalingrdquo PLoS Biology vol 12 no 9 Article ID e1001945 2014

[167] D Ren N F Villeneuve T Jiang et al ldquoBrusatol enhancesthe efficacy of chemotherapy by inhibiting the Nrf2-mediateddefense mechanismrdquo Proceedings of the National Academy ofSciences of the United States of America vol 108 no 4 pp 1433ndash1438 2011

[168] A Arlt S Sebens S Krebs et al ldquoInhibition of the Nrf2 tran-scription factor by the alkaloid trigonelline renders pancreaticcancer cells more susceptible to apoptosis through decreasedproteasomal gene expression and proteasome activityrdquo Onco-gene vol 32 no 40 pp 4825ndash4835 2013

[169] A-M Gao Z-P Ke F Shi G Sun and H Chen ldquoChrysinenhances sensitivity of BEL-7402ADM cells to doxorubicin bysuppressing PI3KAktNrf2 and ERKNrf2 pathwayrdquo Chemico-Biological Interactions vol 206 no 1 pp 100ndash108 2013

[170] A M Gao Z P Ke J N Wang J Yang S Chen and HChen ldquoApigenin sensitizes doxorubicin-resistant hepatocellular

carcinoma BEL-7402ADM cells to doxorubicin via inhibitingPI3KAktNrf2 pathwayrdquo Carcinogenesis vol 34 no 8 pp1806ndash1814 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Oxidative Medicine and Cellular Longevity 9

chrysin apigenin luteolin and trigonelline are known toinhibit NRF2 signaling in several types of cancer cells [102168ndash170] and therefore the development of NRF2 inhibitorswith characterized modes of action will enable efficient tar-geting of the redox homeostasis system as well as multidrugresistance systems in CSCs

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This studywas financially supported by theNational ResearchFoundation (NRF) funded by the Ministry of Science ICT ampFuture Planning (NRF-2013R1A2A2A01015497)

References

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[2] P Storz ldquoForkhead homeobox type O transcription factorsin the responses to oxidative stressrdquo Antioxidants amp RedoxSignaling vol 14 no 4 pp 593ndash605 2011

[3] R S Balaban S Nemoto and T Finkel ldquoMitochondria oxi-dants and agingrdquo Cell vol 120 no 4 pp 483ndash495 2005

[4] L A Sena and N S Chandel ldquoPhysiological roles of mitochon-drial reactive oxygen speciesrdquoMolecular Cell vol 48 no 2 pp158ndash167 2012

[5] P T Schumacker ldquoReactive oxygen species in cancer cells liveby the sword die by the swordrdquo Cancer Cell vol 10 no 3 pp175ndash176 2006

[6] S Toyokuni K Okamoto J Yodoi and H Hiai ldquoPersistentoxidative stress in cancerrdquo FEBS Letters vol 358 no 1 pp 1ndash3 1995

[7] L Behrend G Henderson and R Zwacka ldquoReactive oxy-gen species in oncogenic transformationrdquo Biochemical SocietyTransactions vol 31 no 6 pp 1441ndash1444 2003

[8] E O Hileman J Liu M Albitar M J Keating and P HuangldquoIntrinsic oxidative stress in cancer cells a biochemical basis fortherapeutic selectivityrdquo Cancer Chemotherapy and Pharmacol-ogy vol 53 no 3 pp 209ndash219 2004

[9] H PelicanoD Carney and PHuang ldquoROS stress in cancer cellsand therapeutic implicationsrdquo Drug Resistance Updates vol 7no 2 pp 97ndash110 2004

[10] Y Adachi Y Shibai J Mitsushita W H Shang K Hirose andT Kamata ldquoOncogenic Ras upregulates NADPH oxidase 1 geneexpression through MEK-ERK-dependent phosphorylation ofGATA-6rdquo Oncogene vol 27 no 36 pp 4921ndash4932 2008

[11] J Mitsushita J D Lambeth and T Kamata ldquoThe superoxide-generating oxidase Nox1 is functionally required for Ras onco-gene transformationrdquoCancer Research vol 64 no 10 pp 3580ndash3585 2004

[12] O Vafa M Wade S Kern et al ldquoc-Myc can induce DNAdamage increase reactive oxygen species and mitigate p53function a mechanism for oncogene-induced genetic instabil-ityrdquoMolecular Cell vol 9 no 5 pp 1031ndash1044 2002

[13] K Polyak Y Li H Zhu et al ldquoSomatic mutations of themitochondrial genome in human colorectal tumoursrdquo NatureGenetics vol 20 no 3 pp 291ndash293 1998

[14] D Trachootham J Alexandre and P Huang ldquoTargeting can-cer cells by ROS-mediated mechanisms a radical therapeuticapproachrdquo Nature Reviews Drug Discovery vol 8 no 7 pp579ndash591 2009

[15] X Shi Y Zhang J Zheng and J Pan ldquoReactive oxygen speciesin cancer stem cellsrdquoAntioxidantsampRedox Signaling vol 16 no11 pp 1215ndash1228 2012

[16] T Reya S J Morrison M F Clarke et al ldquoStem cells cancerand cancer stem cellsrdquo Nature vol 414 no 6859 pp 105ndash1112001

[17] B S Zhou H Zhang M Damelin K G Geles J C Grindleyand P B Dirks ldquoTumour-initiating cells challenges and oppor-tunities for anticancer drug discoveryrdquo Nature Reviews DrugDiscovery vol 8 no 10 pp 806ndash823 2009

[18] D Bonnet and J E Dick ldquoHuman acute myeloid leukemiais organized as a hierarchy that originates from a primitivehematopoietic cellrdquo Nature Medicine vol 3 no 7 pp 730ndash7371997

[19] C A OrsquoBrien A Pollett S Gallinger and J E Dick ldquoAhuman colon cancer cell capable of initiating tumour growth inimmunodeficient micerdquo Nature vol 445 no 7123 pp 106ndash1102006

[20] S K Singh C Hawkins I D Clarke et al ldquoIdentification ofhuman brain tumour initiating cellsrdquo Nature vol 432 no 7015pp 396ndash401 2004

[21] M Al-Hajj M S Wicha A Benito-Hernandez S J Morrisonand M F Clarke ldquoProspective identification of tumorigenicbreast cancer cellsrdquo Proceedings of the National Academy ofSciences vol 100 no 7 pp 3983ndash3988 2003

[22] S-H Chiou M-L Wang Y-T Chou et al ldquoCoexpression ofOct4 andNanog enhancesmalignancy in lung adenocarcinomaby inducing cancer stem cell-like properties and epithelial-mesenchymal transdifferentiationrdquoCancer Research vol 70 no24 pp 10433ndash10444 2010

[23] F Yu J Li H Chen et al ldquoKruppel-like factor 4 (KLF4) isrequired for maintenance of breast cancer stem cells and for cellmigration and invasionrdquoOncogene vol 30 no 18 pp 2161ndash21722011

[24] L Vermeulen E M Felipe De Sousa M van der Heijden et alldquoWnt activity defines colon cancer stem cells and is regulatedby the microenvironmentrdquo Nature Cell Biology vol 12 no 5pp 468ndash476 2010

[25] C Zhao A Chen C H Jamieson et al ldquoHedgehog signallingis essential for maintenance of cancer stem cells in myeloidleukaemiardquo Nature vol 458 no 7239 pp 776ndash779 2009

[26] G Farnie and R B Clarke ldquoMammary stem cells and breastcancermdashrole of Notch signallingrdquo Stem Cell Reviews vol 3 no2 pp 169ndash175 2007

[27] D Ponti ldquoIsolation and in vitro propagation of tumorigenicbreast cancer cells with stemprogenitor cell propertiesrdquoCancerResearch vol 65 no 13 pp 5506ndash5511 2005

[28] M Dean T Fojo and S Bates ldquoTumour stem cells and drugresistancerdquo Nature Reviews Cancer vol 5 no 4 pp 275ndash2842005

[29] C Blanpain M Mohrin P A Sotiropoulou and E PassegueldquoDNA-damage response in tissue-specific and cancer stemcellsrdquo Cell Stem Cell vol 8 no 1 pp 16ndash29 2011

10 Oxidative Medicine and Cellular Longevity

[30] A A Dayem H Choi J Kim and S Cho ldquoRole of oxidativestress in stem cancer and cancer stem cellsrdquo Cancers vol 2 no2 pp 859ndash884 2010

[31] E Lagadinou A Sach K Callahan et al ldquoBCL-2 inhibitiontargets oxidative phosphorylation and selectively eradicatesquiescent human leukemia stem cellsrdquo Cell Stem Cell vol 12no 3 pp 329ndash341 2013

[32] C Chang Y Chen S Chou et al ldquoDistinct subpopulations ofhead and neck cancer cells with different levels of intracellularreactive oxygen species exhibit diverse stemness proliferationand chemosensitivityrdquoCancer Research vol 74 no 21 pp 6291ndash6305 2014

[33] MMGottesman T Fojo and S E Bates ldquoMultidrug resistancein cancer role of ATPndashdependent transportersrdquoNature ReviewsCancer vol 2 no 1 pp 48ndash58 2002

[34] S Achuthan T R Santhoshkumar J Prabhakar S A Nair andM R Pillai ldquoDrug-induced senescence generates chemoresis-tant stemlike cells with low reactive oxygen speciesrdquoThe Journalof Biological Chemistry vol 286 no 43 pp 37813ndash37829 2011

[35] L Zinzi M Contino M Cantore E Capparelli M Leopoldoand N A Colabufo ldquoABC transporters in CSCs membranesas a novel target for treating tumor relapserdquo Frontiers inPharmacology vol 5 article 163 2014

[36] A Hadnagy L Gaboury R Beaulieu and D Balicki ldquoSPanalysis may be used to identify cancer stem cell populationsrdquoExperimental Cell Research vol 312 no 19 pp 3701ndash3710 2006

[37] C L Bigarella R Liang and S Ghaffari ldquoStem cells and theimpact of ROS signalingrdquo Development vol 141 no 22 pp4206ndash4218 2014

[38] P Chaudhari Z Ye and Y Y Jang ldquoRoles of reactive oxygenspecies in the fate of stem cellsrdquoAntioxidants ampRedox Signalingvol 20 no 12 pp 1881ndash1890 2014

[39] V A Rafalski E Mancini and A Brunet ldquoEnergy metabolismand energy-sensing pathways in mammalian embryonic andadult stem cell faterdquo Journal of Cell Science vol 125 no 23 pp5597ndash5608 2012

[40] K Wang T Zhang Q Dong E C Nice C Huang and Y WeildquoRedox homeostasis the linchpin in stem cell self-renewal anddifferentiationrdquo Cell Death and Disease vol 4 no 3 p e5372013

[41] G Saretzki L Armstrong A Leake and et al ldquoStress defensein murine embryonic stem cells is superior to that of variousdifferentiated murine cellsrdquo Stem Cells vol 22 no 6 pp 962ndash971 2004

[42] Y Y Jang and S J Sharkis ldquoA low level of reactive oxygen speciesselects for primitive hematopoietic stem cells that may reside inthe low-oxygenic nicherdquo Blood vol 110 no 8 pp 3056ndash30632007

[43] S Ghaffari ldquoOxidative stress in the regulation of normal andneoplastic hematopoiesisrdquo Antioxidants amp Redox Signaling vol10 no 11 pp 1923ndash1940 2008

[44] M Schmelter B Ateghang S Helmig M Wartenberg and HSauer ldquoEmbryonic stem cells utilize reactive oxygen speciesas transducers of mechanical strain-induced cardiovasculardifferentiationrdquoThe FASEB Journal vol 20 no 8 pp 1182ndash11842006

[45] A Alexander S-L Cai J Kim et al ldquoATM signals to TSC2in the cytoplasm to regulate mTORC1 in response to ROSrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 107 no 9 pp 4153ndash4158 2010

[46] C Cosentino D Grieco and V Costanzo ldquoATM activates thepentose phosphate pathway promoting anti-oxidant defenceand DNA repairrdquo The EMBO Journal vol 30 no 3 pp 546ndash555 2011

[47] K Ito A Hirao F Arai et al ldquoRegulation of oxidative stress byATM is required for self-renewal of haematopoietic stem cellsrdquoNature vol 431 no 7011 pp 997ndash1002 2004

[48] K Ito K Takubo F Arai et al ldquoRegulation of reactive oxygenspecies by Atm is essential for proper response to DNA double-strand breaks in lymphocytesrdquoThe Journal of Immunology vol178 no 1 pp 103ndash110 2007

[49] Y Wang Y Zhou and D T Graves ldquoFOXO transcription fac-tors their clinical significance and regulationrdquoBioMed ResearchInternational vol 2014 Article ID 925350 13 pages 2014

[50] E Lam R Francis and M Petkovic ldquoFOXO transcriptionfactors key regulators of cell faterdquo Biochemical Society Trans-actions vol 34 no 5 pp 722ndash726 2006

[51] K Miyamoto K Y Araki K Naka et al ldquoFoxo3a is essentialfor maintenance of the hematopoietic stem cell poolrdquo Cell StemCell vol 1 no 1 pp 101ndash112 2007

[52] Z Tothova R Kollipara B J Huntly et al ldquoFoxOs are criticalmediators of hematopoietic stem cell resistance to physiologicoxidative stressrdquo Cell vol 128 no 2 pp 325ndash339 2007

[53] S Yalcin X Zhang J P Luciano et al ldquoFoxo3 is essential for theregulation of ataxia telangiectasia mutated and oxidative stress-mediated homeostasis of hematopoietic stem cellsrdquoThe Journalof Biological Chemistry vol 283 no 37 pp 25692ndash25705 2008

[54] J Paik Z Ding R Narurkar et al ldquoFoxOs cooperatively regu-late diverse pathways governing neural stem cell homeostasisrdquoCell Stem Cell vol 5 no 5 pp 540ndash553 2009

[55] V M Renault V A Rafalski A A Morgan et al ldquoFoxO3regulates neural stem cell homeostasisrdquo Cell Stem Cell vol 5no 5 pp 527ndash539 2009

[56] A Brunet A Bonni M J Zigmond et al ldquoAkt promotescell survival by phosphorylating and inhibiting a Forkheadtranscription factorrdquo Cell vol 96 no 6 pp 857ndash868 1999

[57] A Brunet F Kanai J Stehn and et al ldquo14-3-3 transits tothe nucleus and participates in dynamic nucleocytoplasmictransportrdquo Journal of Cell Biology vol 156 no 5 pp 817ndash8282002

[58] M M Juntilla V D Patil M Calamito R P Joshi M JBirnbaum and G A Koretzky ldquoAKT1 and AKT2 maintainhematopoietic stem cell function by regulating reactive oxygenspeciesrdquo Blood vol 115 no 20 pp 4030ndash4038 2010

[59] O H Yilmaz R Valdez B K Theisen et al ldquoPten depen-dence distinguishes haematopoietic stem cells from leukaemia-initiating cellsrdquo Nature vol 441 no 7092 pp 475ndash482 2006

[60] S N Greer J L Metcalf Y Wang and M Ohh ldquoThe updatedbiology of hypoxia-inducible factorrdquoTheEMBO Journal vol 31no 11 pp 2448ndash2460 2012

[61] C E Forristal K L Wright N A Hanley R O Oreffo and FD Houghton ldquoHypoxia inducible factors regulate pluripotencyand proliferation in human embryonic stem cells cultured atreduced oxygen tensionsrdquo Reproduction vol 139 no 1 pp 85ndash97 2009

[62] K L Covello J Kehler H Yu et al ldquoHIF-2120572 regulates Oct-4effects of hypoxia on stem cell function embryonic develop-ment and tumor growthrdquo Genes amp Development vol 20 no5 pp 557ndash570 2006

[63] J Mazumdar W T OrsquoBrien R S Johnson et al ldquoO2regulates

stem cells through Wnt120573-catenin signallingrdquo Nature Cell Biol-ogy vol 12 no 10 pp 1007ndash1013 2010

Oxidative Medicine and Cellular Longevity 11

[64] K Takubo N Goda W Yamada et al ldquoRegulation of the HIF-1alpha level is essential for hematopoietic stem cellsrdquo Cell StemCell vol 7 no 3 pp 391ndash402 2010

[65] FKocabas J Zheng SThet et al ldquoMeis1 regulates themetabolicphenotype and oxidant defense of hematopoietic stem cellsrdquoBlood vol 120 no 25 pp 4963ndash4972 2012

[66] T M Phillips W H McBride and F Pajonk ldquoThe responseof CD24minus119897119900119908CD44+ breast cancer-initiating cells to radiationrdquoJNCI Journal of the National Cancer Institute vol 98 no 24 pp1777ndash1785 2006

[67] M Diehn R W Cho N A Lobo et al ldquoAssociation of reactiveoxygen species levels and radioresistance in cancer stem cellsrdquoNature vol 458 no 7239 pp 780ndash783 2009

[68] O Herault K J Hope E Deneault et al ldquoA role for GPx3in activity of normal and leukemia stem cellsrdquo The Journal ofExperimental Medicine vol 209 no 5 pp 895ndash901 2012

[69] S Kim C Kwon and I Nakano ldquoDetoxification of oxidativestress in glioma stem cells mechanism clinical relevance andtherapeutic developmentrdquo Journal of Neuroscience Research vol92 no 11 pp 1419ndash1424 2014

[70] T Ishimoto O Nagano T Yae et al ldquoCD44 variant regulatesredox status in cancer cells by stabilizing the xCT subunit ofsystem xc(-) and thereby promotes tumor growthrdquo Cancer Cellvol 19 no 3 pp 387ndash400 2011

[71] M Yoshikawa K Tsuchihashi T Ishimoto et al ldquoxCT inhibi-tion depletes CD44v-expressing tumor cells that are resistantto EGFR-targeted therapy in head and neck squamous cellcarcinomardquo Cancer Research vol 73 no 6 pp 1855ndash1866 2013

[72] N Haraguchi H Ishii K Mimori et al ldquoCD13 is a therapeutictarget in human liver cancer stem cellsrdquo Journal of ClinicalInvestigation vol 120 no 9 pp 3326ndash3339 2010

[73] H M Kim N Haraguchi H Ishii et al ldquoIncreased CD13expression reduces reactive oxygen species promoting sur-vival of liver cancer stem cells via an epithelial-mesenchymaltransition-like phenomenonrdquo Annals of Surgical Oncology vol19 supplement 3 pp S539ndashS548 2012

[74] K Naka T Hoshii T Muraguchi et al ldquoTGF-120573ndashFOXO sig-nalling maintains leukaemia-initiating cells in chronic myeloidleukaemiardquo Nature vol 463 no 7281 pp 676ndash680 2010

[75] J Zhou J Wulfkuhle H Zhang et al ldquoActivation of thePTENmTORSTAT3 pathway in breast cancer stem-like cellsis required for viability and maintenancerdquo Proceedings of theNational Academy of Sciences vol 104 no 41 pp 16158ndash161632007

[76] A Dubrovska S Kim R J Salamone et al ldquoThe role ofPTENAktPI3K signaling in the maintenance and viability ofprostate cancer stem-like cell populationsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 106 no 1 pp 268ndash273 2009

[77] H Yin and J Glass ldquoThe phenotypic radiation resistance ofCS44+CD24minusor low breast cancer cells is mediated through theenhanced activation of ATM signalingrdquo PLoS ONE vol 6 no9 Article ID e24080 2011

[78] T H Rushmore and C Pickett ldquoTranscriptional regulation ofthe rat glutathione S-transferase Ya subunit gene Characteri-zation of a xenobiotic-responsive element controlling inducibleexpression by phenolic antioxidantsrdquo The Journal of BiologicalChemistry vol 265 no 24 pp 14648ndash14653 1990

[79] Y Li and A Jaiswal ldquoRegulation of humanNAD (P)H quinoneoxidoreductase gene Role of AP1 binding site contained withinhuman antioxidant response elementrdquoThe Journal of BiologicalChemistry vol 267 no 21 pp 15097ndash15104 1992

[80] T Prestera P Talalay J Alam et al ldquoParallel induction of hemeoxygenase-1 and chemoprotective phase 2 enzymes by elec-trophiles and antioxidants regulation by upstream antioxidant-responsive elements (ARE)rdquoMolecular Medicine vol 1 no 7 p827 1995

[81] K Itoh K IgarashiNHayashi et al ldquoCloning and characteriza-tion of a novel erythroid cell-derived CNC family transcriptionfactor heterodimerizing with the small Maf family proteinsrdquoMolecular and Cellular Biology vol 15 no 8 pp 4184ndash41931995

[82] R Venugopal and A K Jaiswal ldquoNrf1 and Nrf2 positivelyand c-Fos and Fra1 negatively regulate the human antioxidantresponse element-mediated expression of NAD(P)Hquinoneoxidoreductase1 generdquo Proceedings of the National Academy ofSciences vol 93 no 25 pp 14960ndash14965 1996

[83] K Itoh T Chiba S Takahashi et al ldquoAn Nrf2small Mafheterodimer mediates the induction of phase II detoxifyingenzyme genes through antioxidant response elementsrdquo Bio-chemical and Biophysical Research Communications vol 236no 2 pp 313ndash322 1997

[84] M Ramos-Gomez M-K Kwak P M Dolan et al ldquoSensitivityto carcinogenesis is increased and chemoprotective efficacy ofenzyme inducers is lost in nrf2 transcription factor-deficientmicerdquo Proceedings of the National Academy of Sciences vol 98no 6 pp 3410ndash3415 2001

[85] M-K Kwak N Wakabayashi K Itoh H Motohashi MYamamoto and T W Kensler ldquoModulation of gene expressionby cancer chemopreventive dithiolethiones through the Keap1-Nrf2 pathway Identification of novel gene clusters for cellsurvivalrdquo The Journal of Biological Chemistry vol 278 no 10pp 8135ndash8145 2003

[86] R Hu C Xu G Shen et al ldquoGene expression profiles inducedby cancer chemopreventive isothiocyanate sulforaphane in theliver of C57BL6J mice and C57BL6JNrf2 (minusminus) micerdquo CancerLetters vol 243 no 2 pp 170ndash192 2006

[87] S Chanas Q Jiang and M McMahon ldquoLoss of the Nrf2transcription factor causes a marked reduction in constitutiveand inducible expression of the glutathione S-transferase Gsta1Gsta2 Gstm1 Gstm2 Gstm3 and Gstm4 genes in the livers ofmale and female micerdquo Biochemical Journal vol 365 no 2 pp405ndash416 2002

[88] H-Y Cho S P Reddy A DeBiase M Yamamoto and SR Kleeberger ldquoGene expression profiling of NRF2-mediatedprotection against oxidative injuryrdquo Free Radical Biology andMedicine vol 38 no 3 pp 325ndash343 2005

[89] G Shen C Xu R Hu et al ldquoModulation of nuclear factorE2-related factor 2-mediated gene expression in mice liver andsmall intestine by cancer chemopreventive agent curcuminrdquoMolecular Cancer Therapeutics vol 5 no 1 pp 39ndash51 2006

[90] J D Hayes and A T Dinkova-Kostova ldquoThe Nrf2 regulatorynetwork provides an interface between redox and intermediarymetabolismrdquo Trends in Biochemical Sciences vol 39 no 4 pp199ndash218 2014

[91] A Hayashi H Suzuki K Itoh M Yamamoto and Y SugiyamaldquoTranscription factor Nrf2 is required for the constitutive andinducible expression of multidrug resistance-associated protein1 in mouse embryo fibroblastsrdquo Biochemical and BiophysicalResearch Communications vol 310 no 3 pp 824ndash829 2003

[92] E Jigorel M Le Vee C Boursier-Neyret Y Parmentier andO Fardel ldquoDifferential regulation of sinusoidal and canalicularhepatic drug transporter expression by xenobiotics activating

12 Oxidative Medicine and Cellular Longevity

drug-sensing receptors in primary human hepatocytesrdquo DrugMetabolism and Disposition vol 34 no 10 pp 1756ndash1763 2006

[93] X Wang C R Campos J C Peart et al ldquoNrf2 upregulatesATP binding cassette transporter expression and activity atthe blood-brain and blood-spinal cord barriersrdquo Journal ofNeuroscience vol 34 no 25 pp 8585ndash8593 2014

[94] H Jeong I Ryoo andMKwak ldquoRegulation of the expression ofrenal drug transporters in KEAP1-knockdown human tubularcellsrdquo Toxicology in Vitro vol 29 no 5 pp 884ndash892 2015

[95] M J Canet M D Merrell B G Harder et al ldquoIdentificationof a functional antioxidant response element within the eighthintron of the human ABCC

3generdquo Drug Metabolism and

Disposition vol 43 no 1 pp 93ndash99 2014[96] A Singh H Wu P Zhang C Happel J Ma and S Biswal

ldquoExpression of ABCG2 (BCRP) is regulated by Nrf2 in cancercells that confers side population and chemoresistance pheno-typerdquo Molecular Cancer Therapeutics vol 9 no 8 pp 2365ndash2376 2010

[97] N Wakabayashi K Itoh J Wakabayashi et al ldquoKeap1-nullmutation leads to postnatal lethality due to constitutive Nrf2activationrdquo Nature Genetics vol 35 no 3 pp 238ndash245 2003

[98] S B Cullinan J D Gordan J Jin J W Harper and J A DiehlldquoThe Keap1-BTB protein is an adaptor that bridges Nrf2 to aCul3-based E3 ligase oxidative stress sensing by a Cul3-Keap1ligaserdquoMolecular and Cellular Biology vol 24 no 19 pp 8477ndash8486 2004

[99] D D Zhang and M Hannink ldquoDistinct cysteine residues inkeap1 are required for keap1-dependent ubiquitination of nrf2and for stabilization of nrf2 by chemopreventive agents andoxidative stressrdquoMolecular and Cellular Biology vol 23 no 22pp 8137ndash8151 2003

[100] L M Zipper and R T Mulcahy ldquoThe Keap1 BTBPOZ dimer-ization function is required to sequester Nrf2 in cytoplasmrdquoThe Journal of Biological Chemistry vol 277 no 39 pp 36544ndash36552 2002

[101] M C Jaramillo and D D Zhang ldquoThe emerging role of theNrf2-Keap1 signaling pathway in cancerrdquoGenesampDevelopmentvol 27 no 20 pp 2179ndash2191 2013

[102] K I Tong Y Katoh H Kusunoki K Itoh T Tanaka and MYamamoto ldquoKeap1 recruits Neh2 through binding to ETGEand DLG motifs characterization of the two-site molecularrecognition modelrdquoMolecular and Cellular Biology vol 26 no8 pp 2887ndash2900 2006

[103] K I Tong A Kobayashi F Katsuoka andM Yamamoto ldquoTwo-site substrate recognition model for the Keap1-Nrf2 system ahinge and latch mechanismrdquo Biological Chemistry vol 387 no10-11 pp 1311ndash1320 2006

[104] K I Tong B Padmanabhan A Kobayashi et al ldquoDifferentelectrostatic potentials define ETGE and DLG motifs as hingeand latch in oxidative stress responserdquo Molecular and CellularBiology vol 27 no 21 pp 7511ndash7521 2007

[105] M Kobayashi L Li N Iwamoto et al ldquoThe antioxidant defensesystemKeap1-Nrf2 comprises amultiple sensingmechanism forresponding to a wide range of chemical compoundsrdquoMolecularand Cellular Biology vol 29 no 2 pp 493ndash502 2009

[106] T Yamamoto T Suzuki A Kobayashi et al ldquoPhysiological sig-nificance of reactive cysteine residues of Keap1 in determiningNrf2 activityrdquoMolecular and Cellular Biology vol 28 no 8 pp2758ndash2770 2008

[107] M K Kwak K Itoh M Yamamoto and T W KenslerldquoEnhanced expression of the transcription factorNrf2 by cancer

chemopreventive agents role of antioxidant response element-like sequences in the nrf2 promoterrdquo Molecular and CellularBiology vol 22 no 9 pp 2883ndash2892 2002

[108] T Suzuki T Shibata K Takaya et al ldquoRegulatory nexus ofsynthesis and degradation deciphers cellular Nrf2 expressionlevelsrdquoMolecular and Cellular Biology vol 33 no 12 pp 2402ndash2412 2013

[109] S B Cullinan D Zhang M Hannink E Arvisais R JKaufman and J A Diehl ldquoNrf2 is a direct PERK substrateand effector of PERK-dependent cell survivalrdquo Molecular andCellular Biology vol 23 no 20 pp 7198ndash7209 2003

[110] W Li and A Kong ldquoMolecular mechanisms of Nrf2-mediatedantioxidant responserdquo Molecular Carcinogenesis vol 48 no 2pp 91ndash104 2009

[111] Y J Surh ldquoCancer chemoprevention with dietary phytochemi-calsrdquo Nature Reviews Cancer vol 3 no 10 pp 768ndash780 2003

[112] M Salazar A I Rojo D Velasco R M de Sagarra and ACuadrado ldquoGlycogen synthase kinase-3beta inhibits the xeno-biotic and antioxidant cell response by direct phosphorylationand nuclear exclusion of the transcription factor Nrf2rdquo Journalof Biological Chemistry vol 281 no 21 pp 14841ndash14851 2006

[113] W Chen Z Sun X J Wang et al ldquoDirect interactionbetween Nrf2 and p21(Cip1WAF1) upregulates the Nrf2-mediated antioxidant responserdquo Molecular Cell vol 34 no 6pp 663ndash673 2009

[114] M Komatsu H Kurokawa S Waguri et al ldquoThe selectiveautophagy substrate p62 activates the stress responsive tran-scription factor Nrf2 through inactivation of Keap1rdquoNature CellBiology vol 12 no 3 pp 213ndash223 2010

[115] J D Hayes M McMahon S Chowdhry and A T Dinkova-Kostova ldquoCancer chemoprevention mechanisms mediatedthrough the Keap1-Nrf2 pathwayrdquoAntioxidants amp Redox Signal-ing vol 13 no 11 pp 1713ndash1748 2010

[116] R Hu C L Saw R Yu and A T Kong ldquoRegulation ofNF-E2-related factor 2 signaling for cancer chemopreventionantioxidant coupled with antiinflammatoryrdquo Antioxidants ampRedox Signaling vol 13 no 11 pp 1679ndash1698 2010

[117] M Kwak and T W Kensler ldquoTargeting NRF2 signaling for can-cer chemopreventionrdquo Toxicology and Applied Pharmacologyvol 244 no 1 pp 66ndash76 2010

[118] K Iida K Itoh Y Kumagai et al ldquoNrf2 is essential for thechemopreventive efficacy of oltipraz against urinary bladdercarcinogenesisrdquo Cancer Research vol 64 no 18 pp 6424ndash64312004

[119] T Jiang N Chen F Zhao et al ldquoHigh levels of Nrf2 deter-mine chemoresistance in type II endometrial cancerrdquo CancerResearch vol 70 no 13 pp 5486ndash5496 2010

[120] Y R Kim J EOhM S Kim et al ldquoOncogenicNRF2mutationsin squamous cell carcinomas of oesophagus and skinrdquo TheJournal of Pathology vol 220 no 4 pp 446ndash451 2010

[121] T Shibata A Kokubu M Gotoh et al ldquoGenetic alterationof Keap1 confers constitutive Nrf2 activation and resistance tochemotherapy in gallbladder cancerrdquoGastroenterology vol 135no 4 pp 1358e4ndash1368e4 2008

[122] T Shibata T Ohta K I Tong et al ldquoCancer related mutationsin NRF2 impair its recognition by Keap1-Cul3 E3 ligase andpromote malignancyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 36 pp13568ndash13573 2008

[123] A Singh V Misra R K Thimmulappa et al ldquoDysfunctionalKEAP1-NRF2 interaction in non-small-cell lung cancerrdquo PLoSMedicine vol 3 no 10 article e420 2006

Oxidative Medicine and Cellular Longevity 13

[124] R Wang J An F Ji H Jiao H Sun and D Zhou ldquoHyper-methylation of the Keap1 gene in human lung cancer cell linesand lung cancer tissuesrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 1 pp 151ndash154 2008

[125] P Zhang A Singh S Yegnasubramanian et al ldquoLoss ofKelch-like ECH-associated protein 1 function in prostate cancercells causes chemoresistance and radioresistance and promotestumor growthrdquoMolecular Cancer Therapeutics vol 9 no 2 pp336ndash346 2010

[126] J D Hayes and M McMahon ldquoNRF2 and KEAP1 mutationspermanent activation of an adaptive response in cancerrdquo Trendsin Biochemical Sciences vol 34 no 4 pp 176ndash188 2009

[127] B Padmanabhan K I Tong T Ohta et al ldquoStructural basis fordefects of Keap1 activity provoked by its pointmutations in lungcancerrdquoMolecular Cell vol 21 no 5 pp 689ndash700 2006

[128] Y Inami S Waguri A Sakamoto et al ldquoPersistent activation ofNrf2 throughp62 in hepatocellular carcinoma cellsrdquoTheJournalof Cell Biology vol 193 no 2 pp 275ndash284 2011

[129] G M DeNicola F A Karreth T J Humpton et al ldquoOncogene-induced Nrf2 transcription promotes ROS detoxification andtumorigenesisrdquo Nature vol 475 no 7354 pp 106ndash109 2011

[130] L M Solis C Behrens W Dong et al ldquoNrf2 and Keap1abnormalities in non-small cell lung carcinoma and associationwith clinicopathologic featuresrdquo Clinical Cancer Research vol16 no 14 pp 3743ndash3753 2010

[131] N M Reddy S R Kleeberger H Y Cho et al ldquoDeficiency inNrf2-GSH signaling impairs type II cell growth and enhancessensitivity to oxidantsrdquoAmerican Journal of Respiratory Cell andMolecular Biology vol 37 no 1 pp 3ndash8 2007

[132] N M Reddy S R Kleeberger M Yamamoto et al ldquoGeneticdissection of the Nrf2-dependent redox signaling-regulatedtranscriptional programs of cell proliferation and cytoprotec-tionrdquo Physiological Genomics vol 32 no 1 pp 74ndash81 2007

[133] Y Mitsuishi K Taguchi Y Kawatani et al ldquoNrf2 redirectsglucose and glutamine into anabolic pathways in metabolicreprogrammingrdquo Cancer Cell vol 22 no 1 pp 66ndash79 2012

[134] J Cho SManandharH LeeH Park andMKwak ldquoRole of theNrf2-antioxidant system in cytotoxicity mediated by anticancercisplatin implication to cancer cell resistancerdquo Cancer Lettersvol 260 no 1-2 pp 96ndash108 2008

[135] T Ohta K Iijima M Miyamoto et al ldquoLoss of Keap1 functionactivates Nrf2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008

[136] A Singh M Bodas N Wakabayashi F Bunz and S BiswalldquoGain of Nrf2 function in non-small-cell lung cancer cellsconfers radioresistancerdquoAntioxidantsampRedox Signaling vol 13no 11 pp 1627ndash1637 2010

[137] G-S Shim S Manandhar D-H Shin T Kim and M KwakldquoAcquisition of doxorubicin resistance in ovarian carcinomacells accompanies activation of the NRF2 pathwayrdquo Free RadicalBiology and Medicine vol 47 no 11 pp 1619ndash1631 2009

[138] S Manandhar B Choi K Jung et al ldquoNRF2 inhibitionrepresses ErbB2 signaling in ovarian carcinoma cells implica-tions for tumor growth retardation and docetaxel sensitivityrdquoFree Radical Biology and Medicine vol 52 no 9 pp 1773ndash17852012

[139] T Kim E-g Hur S J Kang et al ldquoNRF2 blockade suppressescolon tumor angiogenesis by inhibiting hypoxia-induced acti-vation ofHIF-1120572rdquoCancer Research vol 71 no 6 pp 2260ndash22752011

[140] L Kinch N V Grishin and J Brugarolas ldquoSuccination of Keap1and activation of Nrf2-dependent antioxidant pathways in FH-deficient papillary renal cell carcinoma type 2rdquo Cancer Cell vol20 no 4 pp 418ndash420 2011

[141] J J Tsai J A Dudakov K Takahashi et al ldquoNrf2 regulateshaematopoietic stem cell functionrdquo Nature Cell Biology vol 15no 3 pp 309ndash316 2013

[142] J H Kim R KThimmulappa V Kumar et al ldquoNRF2-mediatedNotch pathway activation enhances hematopoietic reconsti-tution following myelosuppressive radiationrdquo The Journal ofClinical Investigation vol 124 no 2 pp 730ndash741 2014

[143] J P Chute ldquoNRF2 mitigates radiation-induced hematopoieticdeathrdquo The Journal of Clinical Investigation vol 124 no 3 pp960ndash961 2014

[144] J Jang Y Wang H Kim M A Lalli and K S KosikldquoNrf2 a regulator of the proteasome controls self-renewal andpluripotency in human embryonic stem cellsrdquo Stem Cells vol32 no 10 pp 2616ndash2625 2014

[145] S Murakami R Shimizu P Romeo M Yamamoto and HMotohashi ldquoKeap1-Nrf2 system regulates cell fate determina-tion of hematopoietic stem cellsrdquo Genes to Cells vol 19 no 3pp 239ndash253 2014

[146] C E Hochmuth B Biteau D Bohmann and H Jasper ldquoRedoxregulation by Keap1 and Nrf2 controls intestinal stem cellproliferation inDrosophilardquoCell Stem Cell vol 8 no 2 pp 188ndash199 2011

[147] M K Paul B Bisht D O Darmawan et al ldquoDynamic changesin intracellular ROS levels regulate airway basal stem cellhomeostasis through Nrf2-dependent notch signalingrdquo CellStem Cell vol 15 no 2 pp 199ndash214 2014

[148] L Gambari G Lisignoli L Cattini C Manferdini A Facchiniand F Grassi ldquoSodium hydrosulfide inhibits the differentiationof osteoclast progenitor cells via NRF2-dependent mechanismrdquoPharmacological Research vol 87 pp 99ndash112 2014

[149] J Li D Johnson M Calkins L Wright C Svendsen andJ Johnson ldquoStabilization of Nrf2 by tBHQ confers protectionagainst oxidative stress-induced cell death in human neuralstem cellsrdquo Toxicological Sciences vol 83 no 2 pp 313ndash3282005

[150] A AMerchant A SinghWMatsui and S Biswal ldquoThe redox-sensitive transcription factor Nrf2 regulatesmurine hematopoi-etic stem cell survival independently of ROS levelsrdquo Blood vol118 no 25 pp 6572ndash6579 2011

[151] N Gurusamy D Ray I Lekli and D K Das ldquoRed wineantioxidant resveratrol-modified cardiac stem cells regener-ate infarcted myocardiumrdquo Journal of Cellular and MolecularMedicine vol 14 no 9 pp 2235ndash2239 2010

[152] N Gorbunov G Petrovski N Gurusamy D Ray D H Kimand D K Das ldquoRegeneration of infarcted myocardium withresveratrol-modified cardiac stem cellsrdquo Journal of Cellular andMolecular Medicine vol 16 no 1 pp 174ndash184 2012

[153] C Cai L Teng D Vu et al ldquoThe heme oxygenase 1 inducer(CoPP) protects human cardiac stem cells against apoptosisthrough activation of the extracellular signal-regulated kinase(ERK)NRF2 signaling pathway and cytokine releaserdquo TheJournal of Biological Chemistry vol 287 no 40 pp 33720ndash33732 2012

[154] J Song S M Kang K M Lee and J E Lee ldquoThe protectiveeffect of melatonin on neural stem cell against LPS-inducedinflammationrdquo BioMed Research International vol 2015 ArticleID 854359 13 pages 2015

14 Oxidative Medicine and Cellular Longevity

[155] V Karkkainen Y Pomeshchik E Savchenko et al ldquoNrf2regulates neurogenesis and protects neural progenitor cellsagainst A120573 toxicityrdquo STEM CELLS vol 32 no 7 pp 1904ndash19162014

[156] M Mohammadzadeh R Halabian A Gharehbaghian et alldquoNrf-2 overexpression in mesenchymal stem cells reducesoxidative stress-induced apoptosis and cytotoxicityrdquo Cell Stressand Chaperones vol 17 no 5 pp 553ndash565 2012

[157] Y Takahata T Takarada M Iemata et al ldquoFunctional expres-sion of 1205732 adrenergic receptors responsible for protectionagainst oxidative stress through promotion of glutathione syn-thesis after Nrf2 upregulation in undifferentiated mesenchymalC3H10T12 stem cellsrdquo Journal of Cellular Physiology vol 218no 2 pp 268ndash275 2009

[158] J Zhu H Wang Y Fan et al ldquoKnockdown of nuclear factorerythroid 2-related factor 2 by lentivirus induces differentiationof glioma stem-like cellsrdquo Oncology Reports vol 32 no 3 pp1170ndash1178 2014

[159] J Zhu H Wang Q Sun et al ldquoNrf2 is required to maintain theself-renewal of glioma stem cellsrdquo BMC Cancer vol 13 no 1article 380 2013

[160] B L Emmink A Verheem W J Van Houdt et al ldquoThe secre-tome of colon cancer stem cells contains drug-metabolizingenzymesrdquo Journal of Proteomics vol 91 pp 84ndash96 2013

[161] T Mizuno N Suzuki H Makino et al ldquoCancer stem-like cellsof ovarian clear cell carcinoma are enriched in the ALDH-highpopulation associated with an accelerated scavenging system inreactive oxygen speciesrdquo Gynecologic Oncology vol 137 no 2pp 299ndash305 2015

[162] I-G Ryoo B-H Choi and M-K Kwak ldquoActivation of NRF2by p62 and proteasome reduction in sphere-forming breastcarcinoma cellsrdquo Oncotarget vol 6 no 10 pp 8167ndash8184 2015

[163] T Wu B G Harder P K Wong J E Lang and D D ZhangldquoOxidative stress mammospheres and Nrf2-new implicationfor breast cancer therapyrdquoMolecular Carcinogenesis 2014

[164] B Yang Y Ma and Y Liu ldquoElevated expression of Nrf-2 andABCG2 involved in multi-drug resistance of lung cancer SPcellsrdquo Drug Research 2015

[165] M Zhang A Mathur Y Zhang et al ldquoMithramycin repressesbasal and cigarette smoke-induced expression of abcg2 andinhibits stem cell signaling in lung and esophageal cancer cellsrdquoCancer Research vol 72 no 16 pp 4178ndash4192 2012

[166] C A Del Vecchio Y Feng E S Sokol et al ldquoDe-differentiationconfers multidrug resistance via noncanonical PERK-Nrf2signalingrdquo PLoS Biology vol 12 no 9 Article ID e1001945 2014

[167] D Ren N F Villeneuve T Jiang et al ldquoBrusatol enhancesthe efficacy of chemotherapy by inhibiting the Nrf2-mediateddefense mechanismrdquo Proceedings of the National Academy ofSciences of the United States of America vol 108 no 4 pp 1433ndash1438 2011

[168] A Arlt S Sebens S Krebs et al ldquoInhibition of the Nrf2 tran-scription factor by the alkaloid trigonelline renders pancreaticcancer cells more susceptible to apoptosis through decreasedproteasomal gene expression and proteasome activityrdquo Onco-gene vol 32 no 40 pp 4825ndash4835 2013

[169] A-M Gao Z-P Ke F Shi G Sun and H Chen ldquoChrysinenhances sensitivity of BEL-7402ADM cells to doxorubicin bysuppressing PI3KAktNrf2 and ERKNrf2 pathwayrdquo Chemico-Biological Interactions vol 206 no 1 pp 100ndash108 2013

[170] A M Gao Z P Ke J N Wang J Yang S Chen and HChen ldquoApigenin sensitizes doxorubicin-resistant hepatocellular

carcinoma BEL-7402ADM cells to doxorubicin via inhibitingPI3KAktNrf2 pathwayrdquo Carcinogenesis vol 34 no 8 pp1806ndash1814 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Behavioural Neurology

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Disease Markers

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BioMed Research International

OncologyJournal of

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Oxidative Medicine and Cellular Longevity

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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ObesityJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Research and TreatmentAIDS

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

10 Oxidative Medicine and Cellular Longevity

[30] A A Dayem H Choi J Kim and S Cho ldquoRole of oxidativestress in stem cancer and cancer stem cellsrdquo Cancers vol 2 no2 pp 859ndash884 2010

[31] E Lagadinou A Sach K Callahan et al ldquoBCL-2 inhibitiontargets oxidative phosphorylation and selectively eradicatesquiescent human leukemia stem cellsrdquo Cell Stem Cell vol 12no 3 pp 329ndash341 2013

[32] C Chang Y Chen S Chou et al ldquoDistinct subpopulations ofhead and neck cancer cells with different levels of intracellularreactive oxygen species exhibit diverse stemness proliferationand chemosensitivityrdquoCancer Research vol 74 no 21 pp 6291ndash6305 2014

[33] MMGottesman T Fojo and S E Bates ldquoMultidrug resistancein cancer role of ATPndashdependent transportersrdquoNature ReviewsCancer vol 2 no 1 pp 48ndash58 2002

[34] S Achuthan T R Santhoshkumar J Prabhakar S A Nair andM R Pillai ldquoDrug-induced senescence generates chemoresis-tant stemlike cells with low reactive oxygen speciesrdquoThe Journalof Biological Chemistry vol 286 no 43 pp 37813ndash37829 2011

[35] L Zinzi M Contino M Cantore E Capparelli M Leopoldoand N A Colabufo ldquoABC transporters in CSCs membranesas a novel target for treating tumor relapserdquo Frontiers inPharmacology vol 5 article 163 2014

[36] A Hadnagy L Gaboury R Beaulieu and D Balicki ldquoSPanalysis may be used to identify cancer stem cell populationsrdquoExperimental Cell Research vol 312 no 19 pp 3701ndash3710 2006

[37] C L Bigarella R Liang and S Ghaffari ldquoStem cells and theimpact of ROS signalingrdquo Development vol 141 no 22 pp4206ndash4218 2014

[38] P Chaudhari Z Ye and Y Y Jang ldquoRoles of reactive oxygenspecies in the fate of stem cellsrdquoAntioxidants ampRedox Signalingvol 20 no 12 pp 1881ndash1890 2014

[39] V A Rafalski E Mancini and A Brunet ldquoEnergy metabolismand energy-sensing pathways in mammalian embryonic andadult stem cell faterdquo Journal of Cell Science vol 125 no 23 pp5597ndash5608 2012

[40] K Wang T Zhang Q Dong E C Nice C Huang and Y WeildquoRedox homeostasis the linchpin in stem cell self-renewal anddifferentiationrdquo Cell Death and Disease vol 4 no 3 p e5372013

[41] G Saretzki L Armstrong A Leake and et al ldquoStress defensein murine embryonic stem cells is superior to that of variousdifferentiated murine cellsrdquo Stem Cells vol 22 no 6 pp 962ndash971 2004

[42] Y Y Jang and S J Sharkis ldquoA low level of reactive oxygen speciesselects for primitive hematopoietic stem cells that may reside inthe low-oxygenic nicherdquo Blood vol 110 no 8 pp 3056ndash30632007

[43] S Ghaffari ldquoOxidative stress in the regulation of normal andneoplastic hematopoiesisrdquo Antioxidants amp Redox Signaling vol10 no 11 pp 1923ndash1940 2008

[44] M Schmelter B Ateghang S Helmig M Wartenberg and HSauer ldquoEmbryonic stem cells utilize reactive oxygen speciesas transducers of mechanical strain-induced cardiovasculardifferentiationrdquoThe FASEB Journal vol 20 no 8 pp 1182ndash11842006

[45] A Alexander S-L Cai J Kim et al ldquoATM signals to TSC2in the cytoplasm to regulate mTORC1 in response to ROSrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 107 no 9 pp 4153ndash4158 2010

[46] C Cosentino D Grieco and V Costanzo ldquoATM activates thepentose phosphate pathway promoting anti-oxidant defenceand DNA repairrdquo The EMBO Journal vol 30 no 3 pp 546ndash555 2011

[47] K Ito A Hirao F Arai et al ldquoRegulation of oxidative stress byATM is required for self-renewal of haematopoietic stem cellsrdquoNature vol 431 no 7011 pp 997ndash1002 2004

[48] K Ito K Takubo F Arai et al ldquoRegulation of reactive oxygenspecies by Atm is essential for proper response to DNA double-strand breaks in lymphocytesrdquoThe Journal of Immunology vol178 no 1 pp 103ndash110 2007

[49] Y Wang Y Zhou and D T Graves ldquoFOXO transcription fac-tors their clinical significance and regulationrdquoBioMed ResearchInternational vol 2014 Article ID 925350 13 pages 2014

[50] E Lam R Francis and M Petkovic ldquoFOXO transcriptionfactors key regulators of cell faterdquo Biochemical Society Trans-actions vol 34 no 5 pp 722ndash726 2006

[51] K Miyamoto K Y Araki K Naka et al ldquoFoxo3a is essentialfor maintenance of the hematopoietic stem cell poolrdquo Cell StemCell vol 1 no 1 pp 101ndash112 2007

[52] Z Tothova R Kollipara B J Huntly et al ldquoFoxOs are criticalmediators of hematopoietic stem cell resistance to physiologicoxidative stressrdquo Cell vol 128 no 2 pp 325ndash339 2007

[53] S Yalcin X Zhang J P Luciano et al ldquoFoxo3 is essential for theregulation of ataxia telangiectasia mutated and oxidative stress-mediated homeostasis of hematopoietic stem cellsrdquoThe Journalof Biological Chemistry vol 283 no 37 pp 25692ndash25705 2008

[54] J Paik Z Ding R Narurkar et al ldquoFoxOs cooperatively regu-late diverse pathways governing neural stem cell homeostasisrdquoCell Stem Cell vol 5 no 5 pp 540ndash553 2009

[55] V M Renault V A Rafalski A A Morgan et al ldquoFoxO3regulates neural stem cell homeostasisrdquo Cell Stem Cell vol 5no 5 pp 527ndash539 2009

[56] A Brunet A Bonni M J Zigmond et al ldquoAkt promotescell survival by phosphorylating and inhibiting a Forkheadtranscription factorrdquo Cell vol 96 no 6 pp 857ndash868 1999

[57] A Brunet F Kanai J Stehn and et al ldquo14-3-3 transits tothe nucleus and participates in dynamic nucleocytoplasmictransportrdquo Journal of Cell Biology vol 156 no 5 pp 817ndash8282002

[58] M M Juntilla V D Patil M Calamito R P Joshi M JBirnbaum and G A Koretzky ldquoAKT1 and AKT2 maintainhematopoietic stem cell function by regulating reactive oxygenspeciesrdquo Blood vol 115 no 20 pp 4030ndash4038 2010

[59] O H Yilmaz R Valdez B K Theisen et al ldquoPten depen-dence distinguishes haematopoietic stem cells from leukaemia-initiating cellsrdquo Nature vol 441 no 7092 pp 475ndash482 2006

[60] S N Greer J L Metcalf Y Wang and M Ohh ldquoThe updatedbiology of hypoxia-inducible factorrdquoTheEMBO Journal vol 31no 11 pp 2448ndash2460 2012

[61] C E Forristal K L Wright N A Hanley R O Oreffo and FD Houghton ldquoHypoxia inducible factors regulate pluripotencyand proliferation in human embryonic stem cells cultured atreduced oxygen tensionsrdquo Reproduction vol 139 no 1 pp 85ndash97 2009

[62] K L Covello J Kehler H Yu et al ldquoHIF-2120572 regulates Oct-4effects of hypoxia on stem cell function embryonic develop-ment and tumor growthrdquo Genes amp Development vol 20 no5 pp 557ndash570 2006

[63] J Mazumdar W T OrsquoBrien R S Johnson et al ldquoO2regulates

stem cells through Wnt120573-catenin signallingrdquo Nature Cell Biol-ogy vol 12 no 10 pp 1007ndash1013 2010

Oxidative Medicine and Cellular Longevity 11

[64] K Takubo N Goda W Yamada et al ldquoRegulation of the HIF-1alpha level is essential for hematopoietic stem cellsrdquo Cell StemCell vol 7 no 3 pp 391ndash402 2010

[65] FKocabas J Zheng SThet et al ldquoMeis1 regulates themetabolicphenotype and oxidant defense of hematopoietic stem cellsrdquoBlood vol 120 no 25 pp 4963ndash4972 2012

[66] T M Phillips W H McBride and F Pajonk ldquoThe responseof CD24minus119897119900119908CD44+ breast cancer-initiating cells to radiationrdquoJNCI Journal of the National Cancer Institute vol 98 no 24 pp1777ndash1785 2006

[67] M Diehn R W Cho N A Lobo et al ldquoAssociation of reactiveoxygen species levels and radioresistance in cancer stem cellsrdquoNature vol 458 no 7239 pp 780ndash783 2009

[68] O Herault K J Hope E Deneault et al ldquoA role for GPx3in activity of normal and leukemia stem cellsrdquo The Journal ofExperimental Medicine vol 209 no 5 pp 895ndash901 2012

[69] S Kim C Kwon and I Nakano ldquoDetoxification of oxidativestress in glioma stem cells mechanism clinical relevance andtherapeutic developmentrdquo Journal of Neuroscience Research vol92 no 11 pp 1419ndash1424 2014

[70] T Ishimoto O Nagano T Yae et al ldquoCD44 variant regulatesredox status in cancer cells by stabilizing the xCT subunit ofsystem xc(-) and thereby promotes tumor growthrdquo Cancer Cellvol 19 no 3 pp 387ndash400 2011

[71] M Yoshikawa K Tsuchihashi T Ishimoto et al ldquoxCT inhibi-tion depletes CD44v-expressing tumor cells that are resistantto EGFR-targeted therapy in head and neck squamous cellcarcinomardquo Cancer Research vol 73 no 6 pp 1855ndash1866 2013

[72] N Haraguchi H Ishii K Mimori et al ldquoCD13 is a therapeutictarget in human liver cancer stem cellsrdquo Journal of ClinicalInvestigation vol 120 no 9 pp 3326ndash3339 2010

[73] H M Kim N Haraguchi H Ishii et al ldquoIncreased CD13expression reduces reactive oxygen species promoting sur-vival of liver cancer stem cells via an epithelial-mesenchymaltransition-like phenomenonrdquo Annals of Surgical Oncology vol19 supplement 3 pp S539ndashS548 2012

[74] K Naka T Hoshii T Muraguchi et al ldquoTGF-120573ndashFOXO sig-nalling maintains leukaemia-initiating cells in chronic myeloidleukaemiardquo Nature vol 463 no 7281 pp 676ndash680 2010

[75] J Zhou J Wulfkuhle H Zhang et al ldquoActivation of thePTENmTORSTAT3 pathway in breast cancer stem-like cellsis required for viability and maintenancerdquo Proceedings of theNational Academy of Sciences vol 104 no 41 pp 16158ndash161632007

[76] A Dubrovska S Kim R J Salamone et al ldquoThe role ofPTENAktPI3K signaling in the maintenance and viability ofprostate cancer stem-like cell populationsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 106 no 1 pp 268ndash273 2009

[77] H Yin and J Glass ldquoThe phenotypic radiation resistance ofCS44+CD24minusor low breast cancer cells is mediated through theenhanced activation of ATM signalingrdquo PLoS ONE vol 6 no9 Article ID e24080 2011

[78] T H Rushmore and C Pickett ldquoTranscriptional regulation ofthe rat glutathione S-transferase Ya subunit gene Characteri-zation of a xenobiotic-responsive element controlling inducibleexpression by phenolic antioxidantsrdquo The Journal of BiologicalChemistry vol 265 no 24 pp 14648ndash14653 1990

[79] Y Li and A Jaiswal ldquoRegulation of humanNAD (P)H quinoneoxidoreductase gene Role of AP1 binding site contained withinhuman antioxidant response elementrdquoThe Journal of BiologicalChemistry vol 267 no 21 pp 15097ndash15104 1992

[80] T Prestera P Talalay J Alam et al ldquoParallel induction of hemeoxygenase-1 and chemoprotective phase 2 enzymes by elec-trophiles and antioxidants regulation by upstream antioxidant-responsive elements (ARE)rdquoMolecular Medicine vol 1 no 7 p827 1995

[81] K Itoh K IgarashiNHayashi et al ldquoCloning and characteriza-tion of a novel erythroid cell-derived CNC family transcriptionfactor heterodimerizing with the small Maf family proteinsrdquoMolecular and Cellular Biology vol 15 no 8 pp 4184ndash41931995

[82] R Venugopal and A K Jaiswal ldquoNrf1 and Nrf2 positivelyand c-Fos and Fra1 negatively regulate the human antioxidantresponse element-mediated expression of NAD(P)Hquinoneoxidoreductase1 generdquo Proceedings of the National Academy ofSciences vol 93 no 25 pp 14960ndash14965 1996

[83] K Itoh T Chiba S Takahashi et al ldquoAn Nrf2small Mafheterodimer mediates the induction of phase II detoxifyingenzyme genes through antioxidant response elementsrdquo Bio-chemical and Biophysical Research Communications vol 236no 2 pp 313ndash322 1997

[84] M Ramos-Gomez M-K Kwak P M Dolan et al ldquoSensitivityto carcinogenesis is increased and chemoprotective efficacy ofenzyme inducers is lost in nrf2 transcription factor-deficientmicerdquo Proceedings of the National Academy of Sciences vol 98no 6 pp 3410ndash3415 2001

[85] M-K Kwak N Wakabayashi K Itoh H Motohashi MYamamoto and T W Kensler ldquoModulation of gene expressionby cancer chemopreventive dithiolethiones through the Keap1-Nrf2 pathway Identification of novel gene clusters for cellsurvivalrdquo The Journal of Biological Chemistry vol 278 no 10pp 8135ndash8145 2003

[86] R Hu C Xu G Shen et al ldquoGene expression profiles inducedby cancer chemopreventive isothiocyanate sulforaphane in theliver of C57BL6J mice and C57BL6JNrf2 (minusminus) micerdquo CancerLetters vol 243 no 2 pp 170ndash192 2006

[87] S Chanas Q Jiang and M McMahon ldquoLoss of the Nrf2transcription factor causes a marked reduction in constitutiveand inducible expression of the glutathione S-transferase Gsta1Gsta2 Gstm1 Gstm2 Gstm3 and Gstm4 genes in the livers ofmale and female micerdquo Biochemical Journal vol 365 no 2 pp405ndash416 2002

[88] H-Y Cho S P Reddy A DeBiase M Yamamoto and SR Kleeberger ldquoGene expression profiling of NRF2-mediatedprotection against oxidative injuryrdquo Free Radical Biology andMedicine vol 38 no 3 pp 325ndash343 2005

[89] G Shen C Xu R Hu et al ldquoModulation of nuclear factorE2-related factor 2-mediated gene expression in mice liver andsmall intestine by cancer chemopreventive agent curcuminrdquoMolecular Cancer Therapeutics vol 5 no 1 pp 39ndash51 2006

[90] J D Hayes and A T Dinkova-Kostova ldquoThe Nrf2 regulatorynetwork provides an interface between redox and intermediarymetabolismrdquo Trends in Biochemical Sciences vol 39 no 4 pp199ndash218 2014

[91] A Hayashi H Suzuki K Itoh M Yamamoto and Y SugiyamaldquoTranscription factor Nrf2 is required for the constitutive andinducible expression of multidrug resistance-associated protein1 in mouse embryo fibroblastsrdquo Biochemical and BiophysicalResearch Communications vol 310 no 3 pp 824ndash829 2003

[92] E Jigorel M Le Vee C Boursier-Neyret Y Parmentier andO Fardel ldquoDifferential regulation of sinusoidal and canalicularhepatic drug transporter expression by xenobiotics activating

12 Oxidative Medicine and Cellular Longevity

drug-sensing receptors in primary human hepatocytesrdquo DrugMetabolism and Disposition vol 34 no 10 pp 1756ndash1763 2006

[93] X Wang C R Campos J C Peart et al ldquoNrf2 upregulatesATP binding cassette transporter expression and activity atthe blood-brain and blood-spinal cord barriersrdquo Journal ofNeuroscience vol 34 no 25 pp 8585ndash8593 2014

[94] H Jeong I Ryoo andMKwak ldquoRegulation of the expression ofrenal drug transporters in KEAP1-knockdown human tubularcellsrdquo Toxicology in Vitro vol 29 no 5 pp 884ndash892 2015

[95] M J Canet M D Merrell B G Harder et al ldquoIdentificationof a functional antioxidant response element within the eighthintron of the human ABCC

3generdquo Drug Metabolism and

Disposition vol 43 no 1 pp 93ndash99 2014[96] A Singh H Wu P Zhang C Happel J Ma and S Biswal

ldquoExpression of ABCG2 (BCRP) is regulated by Nrf2 in cancercells that confers side population and chemoresistance pheno-typerdquo Molecular Cancer Therapeutics vol 9 no 8 pp 2365ndash2376 2010

[97] N Wakabayashi K Itoh J Wakabayashi et al ldquoKeap1-nullmutation leads to postnatal lethality due to constitutive Nrf2activationrdquo Nature Genetics vol 35 no 3 pp 238ndash245 2003

[98] S B Cullinan J D Gordan J Jin J W Harper and J A DiehlldquoThe Keap1-BTB protein is an adaptor that bridges Nrf2 to aCul3-based E3 ligase oxidative stress sensing by a Cul3-Keap1ligaserdquoMolecular and Cellular Biology vol 24 no 19 pp 8477ndash8486 2004

[99] D D Zhang and M Hannink ldquoDistinct cysteine residues inkeap1 are required for keap1-dependent ubiquitination of nrf2and for stabilization of nrf2 by chemopreventive agents andoxidative stressrdquoMolecular and Cellular Biology vol 23 no 22pp 8137ndash8151 2003

[100] L M Zipper and R T Mulcahy ldquoThe Keap1 BTBPOZ dimer-ization function is required to sequester Nrf2 in cytoplasmrdquoThe Journal of Biological Chemistry vol 277 no 39 pp 36544ndash36552 2002

[101] M C Jaramillo and D D Zhang ldquoThe emerging role of theNrf2-Keap1 signaling pathway in cancerrdquoGenesampDevelopmentvol 27 no 20 pp 2179ndash2191 2013

[102] K I Tong Y Katoh H Kusunoki K Itoh T Tanaka and MYamamoto ldquoKeap1 recruits Neh2 through binding to ETGEand DLG motifs characterization of the two-site molecularrecognition modelrdquoMolecular and Cellular Biology vol 26 no8 pp 2887ndash2900 2006

[103] K I Tong A Kobayashi F Katsuoka andM Yamamoto ldquoTwo-site substrate recognition model for the Keap1-Nrf2 system ahinge and latch mechanismrdquo Biological Chemistry vol 387 no10-11 pp 1311ndash1320 2006

[104] K I Tong B Padmanabhan A Kobayashi et al ldquoDifferentelectrostatic potentials define ETGE and DLG motifs as hingeand latch in oxidative stress responserdquo Molecular and CellularBiology vol 27 no 21 pp 7511ndash7521 2007

[105] M Kobayashi L Li N Iwamoto et al ldquoThe antioxidant defensesystemKeap1-Nrf2 comprises amultiple sensingmechanism forresponding to a wide range of chemical compoundsrdquoMolecularand Cellular Biology vol 29 no 2 pp 493ndash502 2009

[106] T Yamamoto T Suzuki A Kobayashi et al ldquoPhysiological sig-nificance of reactive cysteine residues of Keap1 in determiningNrf2 activityrdquoMolecular and Cellular Biology vol 28 no 8 pp2758ndash2770 2008

[107] M K Kwak K Itoh M Yamamoto and T W KenslerldquoEnhanced expression of the transcription factorNrf2 by cancer

chemopreventive agents role of antioxidant response element-like sequences in the nrf2 promoterrdquo Molecular and CellularBiology vol 22 no 9 pp 2883ndash2892 2002

[108] T Suzuki T Shibata K Takaya et al ldquoRegulatory nexus ofsynthesis and degradation deciphers cellular Nrf2 expressionlevelsrdquoMolecular and Cellular Biology vol 33 no 12 pp 2402ndash2412 2013

[109] S B Cullinan D Zhang M Hannink E Arvisais R JKaufman and J A Diehl ldquoNrf2 is a direct PERK substrateand effector of PERK-dependent cell survivalrdquo Molecular andCellular Biology vol 23 no 20 pp 7198ndash7209 2003

[110] W Li and A Kong ldquoMolecular mechanisms of Nrf2-mediatedantioxidant responserdquo Molecular Carcinogenesis vol 48 no 2pp 91ndash104 2009

[111] Y J Surh ldquoCancer chemoprevention with dietary phytochemi-calsrdquo Nature Reviews Cancer vol 3 no 10 pp 768ndash780 2003

[112] M Salazar A I Rojo D Velasco R M de Sagarra and ACuadrado ldquoGlycogen synthase kinase-3beta inhibits the xeno-biotic and antioxidant cell response by direct phosphorylationand nuclear exclusion of the transcription factor Nrf2rdquo Journalof Biological Chemistry vol 281 no 21 pp 14841ndash14851 2006

[113] W Chen Z Sun X J Wang et al ldquoDirect interactionbetween Nrf2 and p21(Cip1WAF1) upregulates the Nrf2-mediated antioxidant responserdquo Molecular Cell vol 34 no 6pp 663ndash673 2009

[114] M Komatsu H Kurokawa S Waguri et al ldquoThe selectiveautophagy substrate p62 activates the stress responsive tran-scription factor Nrf2 through inactivation of Keap1rdquoNature CellBiology vol 12 no 3 pp 213ndash223 2010

[115] J D Hayes M McMahon S Chowdhry and A T Dinkova-Kostova ldquoCancer chemoprevention mechanisms mediatedthrough the Keap1-Nrf2 pathwayrdquoAntioxidants amp Redox Signal-ing vol 13 no 11 pp 1713ndash1748 2010

[116] R Hu C L Saw R Yu and A T Kong ldquoRegulation ofNF-E2-related factor 2 signaling for cancer chemopreventionantioxidant coupled with antiinflammatoryrdquo Antioxidants ampRedox Signaling vol 13 no 11 pp 1679ndash1698 2010

[117] M Kwak and T W Kensler ldquoTargeting NRF2 signaling for can-cer chemopreventionrdquo Toxicology and Applied Pharmacologyvol 244 no 1 pp 66ndash76 2010

[118] K Iida K Itoh Y Kumagai et al ldquoNrf2 is essential for thechemopreventive efficacy of oltipraz against urinary bladdercarcinogenesisrdquo Cancer Research vol 64 no 18 pp 6424ndash64312004

[119] T Jiang N Chen F Zhao et al ldquoHigh levels of Nrf2 deter-mine chemoresistance in type II endometrial cancerrdquo CancerResearch vol 70 no 13 pp 5486ndash5496 2010

[120] Y R Kim J EOhM S Kim et al ldquoOncogenicNRF2mutationsin squamous cell carcinomas of oesophagus and skinrdquo TheJournal of Pathology vol 220 no 4 pp 446ndash451 2010

[121] T Shibata A Kokubu M Gotoh et al ldquoGenetic alterationof Keap1 confers constitutive Nrf2 activation and resistance tochemotherapy in gallbladder cancerrdquoGastroenterology vol 135no 4 pp 1358e4ndash1368e4 2008

[122] T Shibata T Ohta K I Tong et al ldquoCancer related mutationsin NRF2 impair its recognition by Keap1-Cul3 E3 ligase andpromote malignancyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 36 pp13568ndash13573 2008

[123] A Singh V Misra R K Thimmulappa et al ldquoDysfunctionalKEAP1-NRF2 interaction in non-small-cell lung cancerrdquo PLoSMedicine vol 3 no 10 article e420 2006

Oxidative Medicine and Cellular Longevity 13

[124] R Wang J An F Ji H Jiao H Sun and D Zhou ldquoHyper-methylation of the Keap1 gene in human lung cancer cell linesand lung cancer tissuesrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 1 pp 151ndash154 2008

[125] P Zhang A Singh S Yegnasubramanian et al ldquoLoss ofKelch-like ECH-associated protein 1 function in prostate cancercells causes chemoresistance and radioresistance and promotestumor growthrdquoMolecular Cancer Therapeutics vol 9 no 2 pp336ndash346 2010

[126] J D Hayes and M McMahon ldquoNRF2 and KEAP1 mutationspermanent activation of an adaptive response in cancerrdquo Trendsin Biochemical Sciences vol 34 no 4 pp 176ndash188 2009

[127] B Padmanabhan K I Tong T Ohta et al ldquoStructural basis fordefects of Keap1 activity provoked by its pointmutations in lungcancerrdquoMolecular Cell vol 21 no 5 pp 689ndash700 2006

[128] Y Inami S Waguri A Sakamoto et al ldquoPersistent activation ofNrf2 throughp62 in hepatocellular carcinoma cellsrdquoTheJournalof Cell Biology vol 193 no 2 pp 275ndash284 2011

[129] G M DeNicola F A Karreth T J Humpton et al ldquoOncogene-induced Nrf2 transcription promotes ROS detoxification andtumorigenesisrdquo Nature vol 475 no 7354 pp 106ndash109 2011

[130] L M Solis C Behrens W Dong et al ldquoNrf2 and Keap1abnormalities in non-small cell lung carcinoma and associationwith clinicopathologic featuresrdquo Clinical Cancer Research vol16 no 14 pp 3743ndash3753 2010

[131] N M Reddy S R Kleeberger H Y Cho et al ldquoDeficiency inNrf2-GSH signaling impairs type II cell growth and enhancessensitivity to oxidantsrdquoAmerican Journal of Respiratory Cell andMolecular Biology vol 37 no 1 pp 3ndash8 2007

[132] N M Reddy S R Kleeberger M Yamamoto et al ldquoGeneticdissection of the Nrf2-dependent redox signaling-regulatedtranscriptional programs of cell proliferation and cytoprotec-tionrdquo Physiological Genomics vol 32 no 1 pp 74ndash81 2007

[133] Y Mitsuishi K Taguchi Y Kawatani et al ldquoNrf2 redirectsglucose and glutamine into anabolic pathways in metabolicreprogrammingrdquo Cancer Cell vol 22 no 1 pp 66ndash79 2012

[134] J Cho SManandharH LeeH Park andMKwak ldquoRole of theNrf2-antioxidant system in cytotoxicity mediated by anticancercisplatin implication to cancer cell resistancerdquo Cancer Lettersvol 260 no 1-2 pp 96ndash108 2008

[135] T Ohta K Iijima M Miyamoto et al ldquoLoss of Keap1 functionactivates Nrf2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008

[136] A Singh M Bodas N Wakabayashi F Bunz and S BiswalldquoGain of Nrf2 function in non-small-cell lung cancer cellsconfers radioresistancerdquoAntioxidantsampRedox Signaling vol 13no 11 pp 1627ndash1637 2010

[137] G-S Shim S Manandhar D-H Shin T Kim and M KwakldquoAcquisition of doxorubicin resistance in ovarian carcinomacells accompanies activation of the NRF2 pathwayrdquo Free RadicalBiology and Medicine vol 47 no 11 pp 1619ndash1631 2009

[138] S Manandhar B Choi K Jung et al ldquoNRF2 inhibitionrepresses ErbB2 signaling in ovarian carcinoma cells implica-tions for tumor growth retardation and docetaxel sensitivityrdquoFree Radical Biology and Medicine vol 52 no 9 pp 1773ndash17852012

[139] T Kim E-g Hur S J Kang et al ldquoNRF2 blockade suppressescolon tumor angiogenesis by inhibiting hypoxia-induced acti-vation ofHIF-1120572rdquoCancer Research vol 71 no 6 pp 2260ndash22752011

[140] L Kinch N V Grishin and J Brugarolas ldquoSuccination of Keap1and activation of Nrf2-dependent antioxidant pathways in FH-deficient papillary renal cell carcinoma type 2rdquo Cancer Cell vol20 no 4 pp 418ndash420 2011

[141] J J Tsai J A Dudakov K Takahashi et al ldquoNrf2 regulateshaematopoietic stem cell functionrdquo Nature Cell Biology vol 15no 3 pp 309ndash316 2013

[142] J H Kim R KThimmulappa V Kumar et al ldquoNRF2-mediatedNotch pathway activation enhances hematopoietic reconsti-tution following myelosuppressive radiationrdquo The Journal ofClinical Investigation vol 124 no 2 pp 730ndash741 2014

[143] J P Chute ldquoNRF2 mitigates radiation-induced hematopoieticdeathrdquo The Journal of Clinical Investigation vol 124 no 3 pp960ndash961 2014

[144] J Jang Y Wang H Kim M A Lalli and K S KosikldquoNrf2 a regulator of the proteasome controls self-renewal andpluripotency in human embryonic stem cellsrdquo Stem Cells vol32 no 10 pp 2616ndash2625 2014

[145] S Murakami R Shimizu P Romeo M Yamamoto and HMotohashi ldquoKeap1-Nrf2 system regulates cell fate determina-tion of hematopoietic stem cellsrdquo Genes to Cells vol 19 no 3pp 239ndash253 2014

[146] C E Hochmuth B Biteau D Bohmann and H Jasper ldquoRedoxregulation by Keap1 and Nrf2 controls intestinal stem cellproliferation inDrosophilardquoCell Stem Cell vol 8 no 2 pp 188ndash199 2011

[147] M K Paul B Bisht D O Darmawan et al ldquoDynamic changesin intracellular ROS levels regulate airway basal stem cellhomeostasis through Nrf2-dependent notch signalingrdquo CellStem Cell vol 15 no 2 pp 199ndash214 2014

[148] L Gambari G Lisignoli L Cattini C Manferdini A Facchiniand F Grassi ldquoSodium hydrosulfide inhibits the differentiationof osteoclast progenitor cells via NRF2-dependent mechanismrdquoPharmacological Research vol 87 pp 99ndash112 2014

[149] J Li D Johnson M Calkins L Wright C Svendsen andJ Johnson ldquoStabilization of Nrf2 by tBHQ confers protectionagainst oxidative stress-induced cell death in human neuralstem cellsrdquo Toxicological Sciences vol 83 no 2 pp 313ndash3282005

[150] A AMerchant A SinghWMatsui and S Biswal ldquoThe redox-sensitive transcription factor Nrf2 regulatesmurine hematopoi-etic stem cell survival independently of ROS levelsrdquo Blood vol118 no 25 pp 6572ndash6579 2011

[151] N Gurusamy D Ray I Lekli and D K Das ldquoRed wineantioxidant resveratrol-modified cardiac stem cells regener-ate infarcted myocardiumrdquo Journal of Cellular and MolecularMedicine vol 14 no 9 pp 2235ndash2239 2010

[152] N Gorbunov G Petrovski N Gurusamy D Ray D H Kimand D K Das ldquoRegeneration of infarcted myocardium withresveratrol-modified cardiac stem cellsrdquo Journal of Cellular andMolecular Medicine vol 16 no 1 pp 174ndash184 2012

[153] C Cai L Teng D Vu et al ldquoThe heme oxygenase 1 inducer(CoPP) protects human cardiac stem cells against apoptosisthrough activation of the extracellular signal-regulated kinase(ERK)NRF2 signaling pathway and cytokine releaserdquo TheJournal of Biological Chemistry vol 287 no 40 pp 33720ndash33732 2012

[154] J Song S M Kang K M Lee and J E Lee ldquoThe protectiveeffect of melatonin on neural stem cell against LPS-inducedinflammationrdquo BioMed Research International vol 2015 ArticleID 854359 13 pages 2015

14 Oxidative Medicine and Cellular Longevity

[155] V Karkkainen Y Pomeshchik E Savchenko et al ldquoNrf2regulates neurogenesis and protects neural progenitor cellsagainst A120573 toxicityrdquo STEM CELLS vol 32 no 7 pp 1904ndash19162014

[156] M Mohammadzadeh R Halabian A Gharehbaghian et alldquoNrf-2 overexpression in mesenchymal stem cells reducesoxidative stress-induced apoptosis and cytotoxicityrdquo Cell Stressand Chaperones vol 17 no 5 pp 553ndash565 2012

[157] Y Takahata T Takarada M Iemata et al ldquoFunctional expres-sion of 1205732 adrenergic receptors responsible for protectionagainst oxidative stress through promotion of glutathione syn-thesis after Nrf2 upregulation in undifferentiated mesenchymalC3H10T12 stem cellsrdquo Journal of Cellular Physiology vol 218no 2 pp 268ndash275 2009

[158] J Zhu H Wang Y Fan et al ldquoKnockdown of nuclear factorerythroid 2-related factor 2 by lentivirus induces differentiationof glioma stem-like cellsrdquo Oncology Reports vol 32 no 3 pp1170ndash1178 2014

[159] J Zhu H Wang Q Sun et al ldquoNrf2 is required to maintain theself-renewal of glioma stem cellsrdquo BMC Cancer vol 13 no 1article 380 2013

[160] B L Emmink A Verheem W J Van Houdt et al ldquoThe secre-tome of colon cancer stem cells contains drug-metabolizingenzymesrdquo Journal of Proteomics vol 91 pp 84ndash96 2013

[161] T Mizuno N Suzuki H Makino et al ldquoCancer stem-like cellsof ovarian clear cell carcinoma are enriched in the ALDH-highpopulation associated with an accelerated scavenging system inreactive oxygen speciesrdquo Gynecologic Oncology vol 137 no 2pp 299ndash305 2015

[162] I-G Ryoo B-H Choi and M-K Kwak ldquoActivation of NRF2by p62 and proteasome reduction in sphere-forming breastcarcinoma cellsrdquo Oncotarget vol 6 no 10 pp 8167ndash8184 2015

[163] T Wu B G Harder P K Wong J E Lang and D D ZhangldquoOxidative stress mammospheres and Nrf2-new implicationfor breast cancer therapyrdquoMolecular Carcinogenesis 2014

[164] B Yang Y Ma and Y Liu ldquoElevated expression of Nrf-2 andABCG2 involved in multi-drug resistance of lung cancer SPcellsrdquo Drug Research 2015

[165] M Zhang A Mathur Y Zhang et al ldquoMithramycin repressesbasal and cigarette smoke-induced expression of abcg2 andinhibits stem cell signaling in lung and esophageal cancer cellsrdquoCancer Research vol 72 no 16 pp 4178ndash4192 2012

[166] C A Del Vecchio Y Feng E S Sokol et al ldquoDe-differentiationconfers multidrug resistance via noncanonical PERK-Nrf2signalingrdquo PLoS Biology vol 12 no 9 Article ID e1001945 2014

[167] D Ren N F Villeneuve T Jiang et al ldquoBrusatol enhancesthe efficacy of chemotherapy by inhibiting the Nrf2-mediateddefense mechanismrdquo Proceedings of the National Academy ofSciences of the United States of America vol 108 no 4 pp 1433ndash1438 2011

[168] A Arlt S Sebens S Krebs et al ldquoInhibition of the Nrf2 tran-scription factor by the alkaloid trigonelline renders pancreaticcancer cells more susceptible to apoptosis through decreasedproteasomal gene expression and proteasome activityrdquo Onco-gene vol 32 no 40 pp 4825ndash4835 2013

[169] A-M Gao Z-P Ke F Shi G Sun and H Chen ldquoChrysinenhances sensitivity of BEL-7402ADM cells to doxorubicin bysuppressing PI3KAktNrf2 and ERKNrf2 pathwayrdquo Chemico-Biological Interactions vol 206 no 1 pp 100ndash108 2013

[170] A M Gao Z P Ke J N Wang J Yang S Chen and HChen ldquoApigenin sensitizes doxorubicin-resistant hepatocellular

carcinoma BEL-7402ADM cells to doxorubicin via inhibitingPI3KAktNrf2 pathwayrdquo Carcinogenesis vol 34 no 8 pp1806ndash1814 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Oxidative Medicine and Cellular Longevity 11

[64] K Takubo N Goda W Yamada et al ldquoRegulation of the HIF-1alpha level is essential for hematopoietic stem cellsrdquo Cell StemCell vol 7 no 3 pp 391ndash402 2010

[65] FKocabas J Zheng SThet et al ldquoMeis1 regulates themetabolicphenotype and oxidant defense of hematopoietic stem cellsrdquoBlood vol 120 no 25 pp 4963ndash4972 2012

[66] T M Phillips W H McBride and F Pajonk ldquoThe responseof CD24minus119897119900119908CD44+ breast cancer-initiating cells to radiationrdquoJNCI Journal of the National Cancer Institute vol 98 no 24 pp1777ndash1785 2006

[67] M Diehn R W Cho N A Lobo et al ldquoAssociation of reactiveoxygen species levels and radioresistance in cancer stem cellsrdquoNature vol 458 no 7239 pp 780ndash783 2009

[68] O Herault K J Hope E Deneault et al ldquoA role for GPx3in activity of normal and leukemia stem cellsrdquo The Journal ofExperimental Medicine vol 209 no 5 pp 895ndash901 2012

[69] S Kim C Kwon and I Nakano ldquoDetoxification of oxidativestress in glioma stem cells mechanism clinical relevance andtherapeutic developmentrdquo Journal of Neuroscience Research vol92 no 11 pp 1419ndash1424 2014

[70] T Ishimoto O Nagano T Yae et al ldquoCD44 variant regulatesredox status in cancer cells by stabilizing the xCT subunit ofsystem xc(-) and thereby promotes tumor growthrdquo Cancer Cellvol 19 no 3 pp 387ndash400 2011

[71] M Yoshikawa K Tsuchihashi T Ishimoto et al ldquoxCT inhibi-tion depletes CD44v-expressing tumor cells that are resistantto EGFR-targeted therapy in head and neck squamous cellcarcinomardquo Cancer Research vol 73 no 6 pp 1855ndash1866 2013

[72] N Haraguchi H Ishii K Mimori et al ldquoCD13 is a therapeutictarget in human liver cancer stem cellsrdquo Journal of ClinicalInvestigation vol 120 no 9 pp 3326ndash3339 2010

[73] H M Kim N Haraguchi H Ishii et al ldquoIncreased CD13expression reduces reactive oxygen species promoting sur-vival of liver cancer stem cells via an epithelial-mesenchymaltransition-like phenomenonrdquo Annals of Surgical Oncology vol19 supplement 3 pp S539ndashS548 2012

[74] K Naka T Hoshii T Muraguchi et al ldquoTGF-120573ndashFOXO sig-nalling maintains leukaemia-initiating cells in chronic myeloidleukaemiardquo Nature vol 463 no 7281 pp 676ndash680 2010

[75] J Zhou J Wulfkuhle H Zhang et al ldquoActivation of thePTENmTORSTAT3 pathway in breast cancer stem-like cellsis required for viability and maintenancerdquo Proceedings of theNational Academy of Sciences vol 104 no 41 pp 16158ndash161632007

[76] A Dubrovska S Kim R J Salamone et al ldquoThe role ofPTENAktPI3K signaling in the maintenance and viability ofprostate cancer stem-like cell populationsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 106 no 1 pp 268ndash273 2009

[77] H Yin and J Glass ldquoThe phenotypic radiation resistance ofCS44+CD24minusor low breast cancer cells is mediated through theenhanced activation of ATM signalingrdquo PLoS ONE vol 6 no9 Article ID e24080 2011

[78] T H Rushmore and C Pickett ldquoTranscriptional regulation ofthe rat glutathione S-transferase Ya subunit gene Characteri-zation of a xenobiotic-responsive element controlling inducibleexpression by phenolic antioxidantsrdquo The Journal of BiologicalChemistry vol 265 no 24 pp 14648ndash14653 1990

[79] Y Li and A Jaiswal ldquoRegulation of humanNAD (P)H quinoneoxidoreductase gene Role of AP1 binding site contained withinhuman antioxidant response elementrdquoThe Journal of BiologicalChemistry vol 267 no 21 pp 15097ndash15104 1992

[80] T Prestera P Talalay J Alam et al ldquoParallel induction of hemeoxygenase-1 and chemoprotective phase 2 enzymes by elec-trophiles and antioxidants regulation by upstream antioxidant-responsive elements (ARE)rdquoMolecular Medicine vol 1 no 7 p827 1995

[81] K Itoh K IgarashiNHayashi et al ldquoCloning and characteriza-tion of a novel erythroid cell-derived CNC family transcriptionfactor heterodimerizing with the small Maf family proteinsrdquoMolecular and Cellular Biology vol 15 no 8 pp 4184ndash41931995

[82] R Venugopal and A K Jaiswal ldquoNrf1 and Nrf2 positivelyand c-Fos and Fra1 negatively regulate the human antioxidantresponse element-mediated expression of NAD(P)Hquinoneoxidoreductase1 generdquo Proceedings of the National Academy ofSciences vol 93 no 25 pp 14960ndash14965 1996

[83] K Itoh T Chiba S Takahashi et al ldquoAn Nrf2small Mafheterodimer mediates the induction of phase II detoxifyingenzyme genes through antioxidant response elementsrdquo Bio-chemical and Biophysical Research Communications vol 236no 2 pp 313ndash322 1997

[84] M Ramos-Gomez M-K Kwak P M Dolan et al ldquoSensitivityto carcinogenesis is increased and chemoprotective efficacy ofenzyme inducers is lost in nrf2 transcription factor-deficientmicerdquo Proceedings of the National Academy of Sciences vol 98no 6 pp 3410ndash3415 2001

[85] M-K Kwak N Wakabayashi K Itoh H Motohashi MYamamoto and T W Kensler ldquoModulation of gene expressionby cancer chemopreventive dithiolethiones through the Keap1-Nrf2 pathway Identification of novel gene clusters for cellsurvivalrdquo The Journal of Biological Chemistry vol 278 no 10pp 8135ndash8145 2003

[86] R Hu C Xu G Shen et al ldquoGene expression profiles inducedby cancer chemopreventive isothiocyanate sulforaphane in theliver of C57BL6J mice and C57BL6JNrf2 (minusminus) micerdquo CancerLetters vol 243 no 2 pp 170ndash192 2006

[87] S Chanas Q Jiang and M McMahon ldquoLoss of the Nrf2transcription factor causes a marked reduction in constitutiveand inducible expression of the glutathione S-transferase Gsta1Gsta2 Gstm1 Gstm2 Gstm3 and Gstm4 genes in the livers ofmale and female micerdquo Biochemical Journal vol 365 no 2 pp405ndash416 2002

[88] H-Y Cho S P Reddy A DeBiase M Yamamoto and SR Kleeberger ldquoGene expression profiling of NRF2-mediatedprotection against oxidative injuryrdquo Free Radical Biology andMedicine vol 38 no 3 pp 325ndash343 2005

[89] G Shen C Xu R Hu et al ldquoModulation of nuclear factorE2-related factor 2-mediated gene expression in mice liver andsmall intestine by cancer chemopreventive agent curcuminrdquoMolecular Cancer Therapeutics vol 5 no 1 pp 39ndash51 2006

[90] J D Hayes and A T Dinkova-Kostova ldquoThe Nrf2 regulatorynetwork provides an interface between redox and intermediarymetabolismrdquo Trends in Biochemical Sciences vol 39 no 4 pp199ndash218 2014

[91] A Hayashi H Suzuki K Itoh M Yamamoto and Y SugiyamaldquoTranscription factor Nrf2 is required for the constitutive andinducible expression of multidrug resistance-associated protein1 in mouse embryo fibroblastsrdquo Biochemical and BiophysicalResearch Communications vol 310 no 3 pp 824ndash829 2003

[92] E Jigorel M Le Vee C Boursier-Neyret Y Parmentier andO Fardel ldquoDifferential regulation of sinusoidal and canalicularhepatic drug transporter expression by xenobiotics activating

12 Oxidative Medicine and Cellular Longevity

drug-sensing receptors in primary human hepatocytesrdquo DrugMetabolism and Disposition vol 34 no 10 pp 1756ndash1763 2006

[93] X Wang C R Campos J C Peart et al ldquoNrf2 upregulatesATP binding cassette transporter expression and activity atthe blood-brain and blood-spinal cord barriersrdquo Journal ofNeuroscience vol 34 no 25 pp 8585ndash8593 2014

[94] H Jeong I Ryoo andMKwak ldquoRegulation of the expression ofrenal drug transporters in KEAP1-knockdown human tubularcellsrdquo Toxicology in Vitro vol 29 no 5 pp 884ndash892 2015

[95] M J Canet M D Merrell B G Harder et al ldquoIdentificationof a functional antioxidant response element within the eighthintron of the human ABCC

3generdquo Drug Metabolism and

Disposition vol 43 no 1 pp 93ndash99 2014[96] A Singh H Wu P Zhang C Happel J Ma and S Biswal

ldquoExpression of ABCG2 (BCRP) is regulated by Nrf2 in cancercells that confers side population and chemoresistance pheno-typerdquo Molecular Cancer Therapeutics vol 9 no 8 pp 2365ndash2376 2010

[97] N Wakabayashi K Itoh J Wakabayashi et al ldquoKeap1-nullmutation leads to postnatal lethality due to constitutive Nrf2activationrdquo Nature Genetics vol 35 no 3 pp 238ndash245 2003

[98] S B Cullinan J D Gordan J Jin J W Harper and J A DiehlldquoThe Keap1-BTB protein is an adaptor that bridges Nrf2 to aCul3-based E3 ligase oxidative stress sensing by a Cul3-Keap1ligaserdquoMolecular and Cellular Biology vol 24 no 19 pp 8477ndash8486 2004

[99] D D Zhang and M Hannink ldquoDistinct cysteine residues inkeap1 are required for keap1-dependent ubiquitination of nrf2and for stabilization of nrf2 by chemopreventive agents andoxidative stressrdquoMolecular and Cellular Biology vol 23 no 22pp 8137ndash8151 2003

[100] L M Zipper and R T Mulcahy ldquoThe Keap1 BTBPOZ dimer-ization function is required to sequester Nrf2 in cytoplasmrdquoThe Journal of Biological Chemistry vol 277 no 39 pp 36544ndash36552 2002

[101] M C Jaramillo and D D Zhang ldquoThe emerging role of theNrf2-Keap1 signaling pathway in cancerrdquoGenesampDevelopmentvol 27 no 20 pp 2179ndash2191 2013

[102] K I Tong Y Katoh H Kusunoki K Itoh T Tanaka and MYamamoto ldquoKeap1 recruits Neh2 through binding to ETGEand DLG motifs characterization of the two-site molecularrecognition modelrdquoMolecular and Cellular Biology vol 26 no8 pp 2887ndash2900 2006

[103] K I Tong A Kobayashi F Katsuoka andM Yamamoto ldquoTwo-site substrate recognition model for the Keap1-Nrf2 system ahinge and latch mechanismrdquo Biological Chemistry vol 387 no10-11 pp 1311ndash1320 2006

[104] K I Tong B Padmanabhan A Kobayashi et al ldquoDifferentelectrostatic potentials define ETGE and DLG motifs as hingeand latch in oxidative stress responserdquo Molecular and CellularBiology vol 27 no 21 pp 7511ndash7521 2007

[105] M Kobayashi L Li N Iwamoto et al ldquoThe antioxidant defensesystemKeap1-Nrf2 comprises amultiple sensingmechanism forresponding to a wide range of chemical compoundsrdquoMolecularand Cellular Biology vol 29 no 2 pp 493ndash502 2009

[106] T Yamamoto T Suzuki A Kobayashi et al ldquoPhysiological sig-nificance of reactive cysteine residues of Keap1 in determiningNrf2 activityrdquoMolecular and Cellular Biology vol 28 no 8 pp2758ndash2770 2008

[107] M K Kwak K Itoh M Yamamoto and T W KenslerldquoEnhanced expression of the transcription factorNrf2 by cancer

chemopreventive agents role of antioxidant response element-like sequences in the nrf2 promoterrdquo Molecular and CellularBiology vol 22 no 9 pp 2883ndash2892 2002

[108] T Suzuki T Shibata K Takaya et al ldquoRegulatory nexus ofsynthesis and degradation deciphers cellular Nrf2 expressionlevelsrdquoMolecular and Cellular Biology vol 33 no 12 pp 2402ndash2412 2013

[109] S B Cullinan D Zhang M Hannink E Arvisais R JKaufman and J A Diehl ldquoNrf2 is a direct PERK substrateand effector of PERK-dependent cell survivalrdquo Molecular andCellular Biology vol 23 no 20 pp 7198ndash7209 2003

[110] W Li and A Kong ldquoMolecular mechanisms of Nrf2-mediatedantioxidant responserdquo Molecular Carcinogenesis vol 48 no 2pp 91ndash104 2009

[111] Y J Surh ldquoCancer chemoprevention with dietary phytochemi-calsrdquo Nature Reviews Cancer vol 3 no 10 pp 768ndash780 2003

[112] M Salazar A I Rojo D Velasco R M de Sagarra and ACuadrado ldquoGlycogen synthase kinase-3beta inhibits the xeno-biotic and antioxidant cell response by direct phosphorylationand nuclear exclusion of the transcription factor Nrf2rdquo Journalof Biological Chemistry vol 281 no 21 pp 14841ndash14851 2006

[113] W Chen Z Sun X J Wang et al ldquoDirect interactionbetween Nrf2 and p21(Cip1WAF1) upregulates the Nrf2-mediated antioxidant responserdquo Molecular Cell vol 34 no 6pp 663ndash673 2009

[114] M Komatsu H Kurokawa S Waguri et al ldquoThe selectiveautophagy substrate p62 activates the stress responsive tran-scription factor Nrf2 through inactivation of Keap1rdquoNature CellBiology vol 12 no 3 pp 213ndash223 2010

[115] J D Hayes M McMahon S Chowdhry and A T Dinkova-Kostova ldquoCancer chemoprevention mechanisms mediatedthrough the Keap1-Nrf2 pathwayrdquoAntioxidants amp Redox Signal-ing vol 13 no 11 pp 1713ndash1748 2010

[116] R Hu C L Saw R Yu and A T Kong ldquoRegulation ofNF-E2-related factor 2 signaling for cancer chemopreventionantioxidant coupled with antiinflammatoryrdquo Antioxidants ampRedox Signaling vol 13 no 11 pp 1679ndash1698 2010

[117] M Kwak and T W Kensler ldquoTargeting NRF2 signaling for can-cer chemopreventionrdquo Toxicology and Applied Pharmacologyvol 244 no 1 pp 66ndash76 2010

[118] K Iida K Itoh Y Kumagai et al ldquoNrf2 is essential for thechemopreventive efficacy of oltipraz against urinary bladdercarcinogenesisrdquo Cancer Research vol 64 no 18 pp 6424ndash64312004

[119] T Jiang N Chen F Zhao et al ldquoHigh levels of Nrf2 deter-mine chemoresistance in type II endometrial cancerrdquo CancerResearch vol 70 no 13 pp 5486ndash5496 2010

[120] Y R Kim J EOhM S Kim et al ldquoOncogenicNRF2mutationsin squamous cell carcinomas of oesophagus and skinrdquo TheJournal of Pathology vol 220 no 4 pp 446ndash451 2010

[121] T Shibata A Kokubu M Gotoh et al ldquoGenetic alterationof Keap1 confers constitutive Nrf2 activation and resistance tochemotherapy in gallbladder cancerrdquoGastroenterology vol 135no 4 pp 1358e4ndash1368e4 2008

[122] T Shibata T Ohta K I Tong et al ldquoCancer related mutationsin NRF2 impair its recognition by Keap1-Cul3 E3 ligase andpromote malignancyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 36 pp13568ndash13573 2008

[123] A Singh V Misra R K Thimmulappa et al ldquoDysfunctionalKEAP1-NRF2 interaction in non-small-cell lung cancerrdquo PLoSMedicine vol 3 no 10 article e420 2006

Oxidative Medicine and Cellular Longevity 13

[124] R Wang J An F Ji H Jiao H Sun and D Zhou ldquoHyper-methylation of the Keap1 gene in human lung cancer cell linesand lung cancer tissuesrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 1 pp 151ndash154 2008

[125] P Zhang A Singh S Yegnasubramanian et al ldquoLoss ofKelch-like ECH-associated protein 1 function in prostate cancercells causes chemoresistance and radioresistance and promotestumor growthrdquoMolecular Cancer Therapeutics vol 9 no 2 pp336ndash346 2010

[126] J D Hayes and M McMahon ldquoNRF2 and KEAP1 mutationspermanent activation of an adaptive response in cancerrdquo Trendsin Biochemical Sciences vol 34 no 4 pp 176ndash188 2009

[127] B Padmanabhan K I Tong T Ohta et al ldquoStructural basis fordefects of Keap1 activity provoked by its pointmutations in lungcancerrdquoMolecular Cell vol 21 no 5 pp 689ndash700 2006

[128] Y Inami S Waguri A Sakamoto et al ldquoPersistent activation ofNrf2 throughp62 in hepatocellular carcinoma cellsrdquoTheJournalof Cell Biology vol 193 no 2 pp 275ndash284 2011

[129] G M DeNicola F A Karreth T J Humpton et al ldquoOncogene-induced Nrf2 transcription promotes ROS detoxification andtumorigenesisrdquo Nature vol 475 no 7354 pp 106ndash109 2011

[130] L M Solis C Behrens W Dong et al ldquoNrf2 and Keap1abnormalities in non-small cell lung carcinoma and associationwith clinicopathologic featuresrdquo Clinical Cancer Research vol16 no 14 pp 3743ndash3753 2010

[131] N M Reddy S R Kleeberger H Y Cho et al ldquoDeficiency inNrf2-GSH signaling impairs type II cell growth and enhancessensitivity to oxidantsrdquoAmerican Journal of Respiratory Cell andMolecular Biology vol 37 no 1 pp 3ndash8 2007

[132] N M Reddy S R Kleeberger M Yamamoto et al ldquoGeneticdissection of the Nrf2-dependent redox signaling-regulatedtranscriptional programs of cell proliferation and cytoprotec-tionrdquo Physiological Genomics vol 32 no 1 pp 74ndash81 2007

[133] Y Mitsuishi K Taguchi Y Kawatani et al ldquoNrf2 redirectsglucose and glutamine into anabolic pathways in metabolicreprogrammingrdquo Cancer Cell vol 22 no 1 pp 66ndash79 2012

[134] J Cho SManandharH LeeH Park andMKwak ldquoRole of theNrf2-antioxidant system in cytotoxicity mediated by anticancercisplatin implication to cancer cell resistancerdquo Cancer Lettersvol 260 no 1-2 pp 96ndash108 2008

[135] T Ohta K Iijima M Miyamoto et al ldquoLoss of Keap1 functionactivates Nrf2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008

[136] A Singh M Bodas N Wakabayashi F Bunz and S BiswalldquoGain of Nrf2 function in non-small-cell lung cancer cellsconfers radioresistancerdquoAntioxidantsampRedox Signaling vol 13no 11 pp 1627ndash1637 2010

[137] G-S Shim S Manandhar D-H Shin T Kim and M KwakldquoAcquisition of doxorubicin resistance in ovarian carcinomacells accompanies activation of the NRF2 pathwayrdquo Free RadicalBiology and Medicine vol 47 no 11 pp 1619ndash1631 2009

[138] S Manandhar B Choi K Jung et al ldquoNRF2 inhibitionrepresses ErbB2 signaling in ovarian carcinoma cells implica-tions for tumor growth retardation and docetaxel sensitivityrdquoFree Radical Biology and Medicine vol 52 no 9 pp 1773ndash17852012

[139] T Kim E-g Hur S J Kang et al ldquoNRF2 blockade suppressescolon tumor angiogenesis by inhibiting hypoxia-induced acti-vation ofHIF-1120572rdquoCancer Research vol 71 no 6 pp 2260ndash22752011

[140] L Kinch N V Grishin and J Brugarolas ldquoSuccination of Keap1and activation of Nrf2-dependent antioxidant pathways in FH-deficient papillary renal cell carcinoma type 2rdquo Cancer Cell vol20 no 4 pp 418ndash420 2011

[141] J J Tsai J A Dudakov K Takahashi et al ldquoNrf2 regulateshaematopoietic stem cell functionrdquo Nature Cell Biology vol 15no 3 pp 309ndash316 2013

[142] J H Kim R KThimmulappa V Kumar et al ldquoNRF2-mediatedNotch pathway activation enhances hematopoietic reconsti-tution following myelosuppressive radiationrdquo The Journal ofClinical Investigation vol 124 no 2 pp 730ndash741 2014

[143] J P Chute ldquoNRF2 mitigates radiation-induced hematopoieticdeathrdquo The Journal of Clinical Investigation vol 124 no 3 pp960ndash961 2014

[144] J Jang Y Wang H Kim M A Lalli and K S KosikldquoNrf2 a regulator of the proteasome controls self-renewal andpluripotency in human embryonic stem cellsrdquo Stem Cells vol32 no 10 pp 2616ndash2625 2014

[145] S Murakami R Shimizu P Romeo M Yamamoto and HMotohashi ldquoKeap1-Nrf2 system regulates cell fate determina-tion of hematopoietic stem cellsrdquo Genes to Cells vol 19 no 3pp 239ndash253 2014

[146] C E Hochmuth B Biteau D Bohmann and H Jasper ldquoRedoxregulation by Keap1 and Nrf2 controls intestinal stem cellproliferation inDrosophilardquoCell Stem Cell vol 8 no 2 pp 188ndash199 2011

[147] M K Paul B Bisht D O Darmawan et al ldquoDynamic changesin intracellular ROS levels regulate airway basal stem cellhomeostasis through Nrf2-dependent notch signalingrdquo CellStem Cell vol 15 no 2 pp 199ndash214 2014

[148] L Gambari G Lisignoli L Cattini C Manferdini A Facchiniand F Grassi ldquoSodium hydrosulfide inhibits the differentiationof osteoclast progenitor cells via NRF2-dependent mechanismrdquoPharmacological Research vol 87 pp 99ndash112 2014

[149] J Li D Johnson M Calkins L Wright C Svendsen andJ Johnson ldquoStabilization of Nrf2 by tBHQ confers protectionagainst oxidative stress-induced cell death in human neuralstem cellsrdquo Toxicological Sciences vol 83 no 2 pp 313ndash3282005

[150] A AMerchant A SinghWMatsui and S Biswal ldquoThe redox-sensitive transcription factor Nrf2 regulatesmurine hematopoi-etic stem cell survival independently of ROS levelsrdquo Blood vol118 no 25 pp 6572ndash6579 2011

[151] N Gurusamy D Ray I Lekli and D K Das ldquoRed wineantioxidant resveratrol-modified cardiac stem cells regener-ate infarcted myocardiumrdquo Journal of Cellular and MolecularMedicine vol 14 no 9 pp 2235ndash2239 2010

[152] N Gorbunov G Petrovski N Gurusamy D Ray D H Kimand D K Das ldquoRegeneration of infarcted myocardium withresveratrol-modified cardiac stem cellsrdquo Journal of Cellular andMolecular Medicine vol 16 no 1 pp 174ndash184 2012

[153] C Cai L Teng D Vu et al ldquoThe heme oxygenase 1 inducer(CoPP) protects human cardiac stem cells against apoptosisthrough activation of the extracellular signal-regulated kinase(ERK)NRF2 signaling pathway and cytokine releaserdquo TheJournal of Biological Chemistry vol 287 no 40 pp 33720ndash33732 2012

[154] J Song S M Kang K M Lee and J E Lee ldquoThe protectiveeffect of melatonin on neural stem cell against LPS-inducedinflammationrdquo BioMed Research International vol 2015 ArticleID 854359 13 pages 2015

14 Oxidative Medicine and Cellular Longevity

[155] V Karkkainen Y Pomeshchik E Savchenko et al ldquoNrf2regulates neurogenesis and protects neural progenitor cellsagainst A120573 toxicityrdquo STEM CELLS vol 32 no 7 pp 1904ndash19162014

[156] M Mohammadzadeh R Halabian A Gharehbaghian et alldquoNrf-2 overexpression in mesenchymal stem cells reducesoxidative stress-induced apoptosis and cytotoxicityrdquo Cell Stressand Chaperones vol 17 no 5 pp 553ndash565 2012

[157] Y Takahata T Takarada M Iemata et al ldquoFunctional expres-sion of 1205732 adrenergic receptors responsible for protectionagainst oxidative stress through promotion of glutathione syn-thesis after Nrf2 upregulation in undifferentiated mesenchymalC3H10T12 stem cellsrdquo Journal of Cellular Physiology vol 218no 2 pp 268ndash275 2009

[158] J Zhu H Wang Y Fan et al ldquoKnockdown of nuclear factorerythroid 2-related factor 2 by lentivirus induces differentiationof glioma stem-like cellsrdquo Oncology Reports vol 32 no 3 pp1170ndash1178 2014

[159] J Zhu H Wang Q Sun et al ldquoNrf2 is required to maintain theself-renewal of glioma stem cellsrdquo BMC Cancer vol 13 no 1article 380 2013

[160] B L Emmink A Verheem W J Van Houdt et al ldquoThe secre-tome of colon cancer stem cells contains drug-metabolizingenzymesrdquo Journal of Proteomics vol 91 pp 84ndash96 2013

[161] T Mizuno N Suzuki H Makino et al ldquoCancer stem-like cellsof ovarian clear cell carcinoma are enriched in the ALDH-highpopulation associated with an accelerated scavenging system inreactive oxygen speciesrdquo Gynecologic Oncology vol 137 no 2pp 299ndash305 2015

[162] I-G Ryoo B-H Choi and M-K Kwak ldquoActivation of NRF2by p62 and proteasome reduction in sphere-forming breastcarcinoma cellsrdquo Oncotarget vol 6 no 10 pp 8167ndash8184 2015

[163] T Wu B G Harder P K Wong J E Lang and D D ZhangldquoOxidative stress mammospheres and Nrf2-new implicationfor breast cancer therapyrdquoMolecular Carcinogenesis 2014

[164] B Yang Y Ma and Y Liu ldquoElevated expression of Nrf-2 andABCG2 involved in multi-drug resistance of lung cancer SPcellsrdquo Drug Research 2015

[165] M Zhang A Mathur Y Zhang et al ldquoMithramycin repressesbasal and cigarette smoke-induced expression of abcg2 andinhibits stem cell signaling in lung and esophageal cancer cellsrdquoCancer Research vol 72 no 16 pp 4178ndash4192 2012

[166] C A Del Vecchio Y Feng E S Sokol et al ldquoDe-differentiationconfers multidrug resistance via noncanonical PERK-Nrf2signalingrdquo PLoS Biology vol 12 no 9 Article ID e1001945 2014

[167] D Ren N F Villeneuve T Jiang et al ldquoBrusatol enhancesthe efficacy of chemotherapy by inhibiting the Nrf2-mediateddefense mechanismrdquo Proceedings of the National Academy ofSciences of the United States of America vol 108 no 4 pp 1433ndash1438 2011

[168] A Arlt S Sebens S Krebs et al ldquoInhibition of the Nrf2 tran-scription factor by the alkaloid trigonelline renders pancreaticcancer cells more susceptible to apoptosis through decreasedproteasomal gene expression and proteasome activityrdquo Onco-gene vol 32 no 40 pp 4825ndash4835 2013

[169] A-M Gao Z-P Ke F Shi G Sun and H Chen ldquoChrysinenhances sensitivity of BEL-7402ADM cells to doxorubicin bysuppressing PI3KAktNrf2 and ERKNrf2 pathwayrdquo Chemico-Biological Interactions vol 206 no 1 pp 100ndash108 2013

[170] A M Gao Z P Ke J N Wang J Yang S Chen and HChen ldquoApigenin sensitizes doxorubicin-resistant hepatocellular

carcinoma BEL-7402ADM cells to doxorubicin via inhibitingPI3KAktNrf2 pathwayrdquo Carcinogenesis vol 34 no 8 pp1806ndash1814 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

12 Oxidative Medicine and Cellular Longevity

drug-sensing receptors in primary human hepatocytesrdquo DrugMetabolism and Disposition vol 34 no 10 pp 1756ndash1763 2006

[93] X Wang C R Campos J C Peart et al ldquoNrf2 upregulatesATP binding cassette transporter expression and activity atthe blood-brain and blood-spinal cord barriersrdquo Journal ofNeuroscience vol 34 no 25 pp 8585ndash8593 2014

[94] H Jeong I Ryoo andMKwak ldquoRegulation of the expression ofrenal drug transporters in KEAP1-knockdown human tubularcellsrdquo Toxicology in Vitro vol 29 no 5 pp 884ndash892 2015

[95] M J Canet M D Merrell B G Harder et al ldquoIdentificationof a functional antioxidant response element within the eighthintron of the human ABCC

3generdquo Drug Metabolism and

Disposition vol 43 no 1 pp 93ndash99 2014[96] A Singh H Wu P Zhang C Happel J Ma and S Biswal

ldquoExpression of ABCG2 (BCRP) is regulated by Nrf2 in cancercells that confers side population and chemoresistance pheno-typerdquo Molecular Cancer Therapeutics vol 9 no 8 pp 2365ndash2376 2010

[97] N Wakabayashi K Itoh J Wakabayashi et al ldquoKeap1-nullmutation leads to postnatal lethality due to constitutive Nrf2activationrdquo Nature Genetics vol 35 no 3 pp 238ndash245 2003

[98] S B Cullinan J D Gordan J Jin J W Harper and J A DiehlldquoThe Keap1-BTB protein is an adaptor that bridges Nrf2 to aCul3-based E3 ligase oxidative stress sensing by a Cul3-Keap1ligaserdquoMolecular and Cellular Biology vol 24 no 19 pp 8477ndash8486 2004

[99] D D Zhang and M Hannink ldquoDistinct cysteine residues inkeap1 are required for keap1-dependent ubiquitination of nrf2and for stabilization of nrf2 by chemopreventive agents andoxidative stressrdquoMolecular and Cellular Biology vol 23 no 22pp 8137ndash8151 2003

[100] L M Zipper and R T Mulcahy ldquoThe Keap1 BTBPOZ dimer-ization function is required to sequester Nrf2 in cytoplasmrdquoThe Journal of Biological Chemistry vol 277 no 39 pp 36544ndash36552 2002

[101] M C Jaramillo and D D Zhang ldquoThe emerging role of theNrf2-Keap1 signaling pathway in cancerrdquoGenesampDevelopmentvol 27 no 20 pp 2179ndash2191 2013

[102] K I Tong Y Katoh H Kusunoki K Itoh T Tanaka and MYamamoto ldquoKeap1 recruits Neh2 through binding to ETGEand DLG motifs characterization of the two-site molecularrecognition modelrdquoMolecular and Cellular Biology vol 26 no8 pp 2887ndash2900 2006

[103] K I Tong A Kobayashi F Katsuoka andM Yamamoto ldquoTwo-site substrate recognition model for the Keap1-Nrf2 system ahinge and latch mechanismrdquo Biological Chemistry vol 387 no10-11 pp 1311ndash1320 2006

[104] K I Tong B Padmanabhan A Kobayashi et al ldquoDifferentelectrostatic potentials define ETGE and DLG motifs as hingeand latch in oxidative stress responserdquo Molecular and CellularBiology vol 27 no 21 pp 7511ndash7521 2007

[105] M Kobayashi L Li N Iwamoto et al ldquoThe antioxidant defensesystemKeap1-Nrf2 comprises amultiple sensingmechanism forresponding to a wide range of chemical compoundsrdquoMolecularand Cellular Biology vol 29 no 2 pp 493ndash502 2009

[106] T Yamamoto T Suzuki A Kobayashi et al ldquoPhysiological sig-nificance of reactive cysteine residues of Keap1 in determiningNrf2 activityrdquoMolecular and Cellular Biology vol 28 no 8 pp2758ndash2770 2008

[107] M K Kwak K Itoh M Yamamoto and T W KenslerldquoEnhanced expression of the transcription factorNrf2 by cancer

chemopreventive agents role of antioxidant response element-like sequences in the nrf2 promoterrdquo Molecular and CellularBiology vol 22 no 9 pp 2883ndash2892 2002

[108] T Suzuki T Shibata K Takaya et al ldquoRegulatory nexus ofsynthesis and degradation deciphers cellular Nrf2 expressionlevelsrdquoMolecular and Cellular Biology vol 33 no 12 pp 2402ndash2412 2013

[109] S B Cullinan D Zhang M Hannink E Arvisais R JKaufman and J A Diehl ldquoNrf2 is a direct PERK substrateand effector of PERK-dependent cell survivalrdquo Molecular andCellular Biology vol 23 no 20 pp 7198ndash7209 2003

[110] W Li and A Kong ldquoMolecular mechanisms of Nrf2-mediatedantioxidant responserdquo Molecular Carcinogenesis vol 48 no 2pp 91ndash104 2009

[111] Y J Surh ldquoCancer chemoprevention with dietary phytochemi-calsrdquo Nature Reviews Cancer vol 3 no 10 pp 768ndash780 2003

[112] M Salazar A I Rojo D Velasco R M de Sagarra and ACuadrado ldquoGlycogen synthase kinase-3beta inhibits the xeno-biotic and antioxidant cell response by direct phosphorylationand nuclear exclusion of the transcription factor Nrf2rdquo Journalof Biological Chemistry vol 281 no 21 pp 14841ndash14851 2006

[113] W Chen Z Sun X J Wang et al ldquoDirect interactionbetween Nrf2 and p21(Cip1WAF1) upregulates the Nrf2-mediated antioxidant responserdquo Molecular Cell vol 34 no 6pp 663ndash673 2009

[114] M Komatsu H Kurokawa S Waguri et al ldquoThe selectiveautophagy substrate p62 activates the stress responsive tran-scription factor Nrf2 through inactivation of Keap1rdquoNature CellBiology vol 12 no 3 pp 213ndash223 2010

[115] J D Hayes M McMahon S Chowdhry and A T Dinkova-Kostova ldquoCancer chemoprevention mechanisms mediatedthrough the Keap1-Nrf2 pathwayrdquoAntioxidants amp Redox Signal-ing vol 13 no 11 pp 1713ndash1748 2010

[116] R Hu C L Saw R Yu and A T Kong ldquoRegulation ofNF-E2-related factor 2 signaling for cancer chemopreventionantioxidant coupled with antiinflammatoryrdquo Antioxidants ampRedox Signaling vol 13 no 11 pp 1679ndash1698 2010

[117] M Kwak and T W Kensler ldquoTargeting NRF2 signaling for can-cer chemopreventionrdquo Toxicology and Applied Pharmacologyvol 244 no 1 pp 66ndash76 2010

[118] K Iida K Itoh Y Kumagai et al ldquoNrf2 is essential for thechemopreventive efficacy of oltipraz against urinary bladdercarcinogenesisrdquo Cancer Research vol 64 no 18 pp 6424ndash64312004

[119] T Jiang N Chen F Zhao et al ldquoHigh levels of Nrf2 deter-mine chemoresistance in type II endometrial cancerrdquo CancerResearch vol 70 no 13 pp 5486ndash5496 2010

[120] Y R Kim J EOhM S Kim et al ldquoOncogenicNRF2mutationsin squamous cell carcinomas of oesophagus and skinrdquo TheJournal of Pathology vol 220 no 4 pp 446ndash451 2010

[121] T Shibata A Kokubu M Gotoh et al ldquoGenetic alterationof Keap1 confers constitutive Nrf2 activation and resistance tochemotherapy in gallbladder cancerrdquoGastroenterology vol 135no 4 pp 1358e4ndash1368e4 2008

[122] T Shibata T Ohta K I Tong et al ldquoCancer related mutationsin NRF2 impair its recognition by Keap1-Cul3 E3 ligase andpromote malignancyrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 36 pp13568ndash13573 2008

[123] A Singh V Misra R K Thimmulappa et al ldquoDysfunctionalKEAP1-NRF2 interaction in non-small-cell lung cancerrdquo PLoSMedicine vol 3 no 10 article e420 2006

Oxidative Medicine and Cellular Longevity 13

[124] R Wang J An F Ji H Jiao H Sun and D Zhou ldquoHyper-methylation of the Keap1 gene in human lung cancer cell linesand lung cancer tissuesrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 1 pp 151ndash154 2008

[125] P Zhang A Singh S Yegnasubramanian et al ldquoLoss ofKelch-like ECH-associated protein 1 function in prostate cancercells causes chemoresistance and radioresistance and promotestumor growthrdquoMolecular Cancer Therapeutics vol 9 no 2 pp336ndash346 2010

[126] J D Hayes and M McMahon ldquoNRF2 and KEAP1 mutationspermanent activation of an adaptive response in cancerrdquo Trendsin Biochemical Sciences vol 34 no 4 pp 176ndash188 2009

[127] B Padmanabhan K I Tong T Ohta et al ldquoStructural basis fordefects of Keap1 activity provoked by its pointmutations in lungcancerrdquoMolecular Cell vol 21 no 5 pp 689ndash700 2006

[128] Y Inami S Waguri A Sakamoto et al ldquoPersistent activation ofNrf2 throughp62 in hepatocellular carcinoma cellsrdquoTheJournalof Cell Biology vol 193 no 2 pp 275ndash284 2011

[129] G M DeNicola F A Karreth T J Humpton et al ldquoOncogene-induced Nrf2 transcription promotes ROS detoxification andtumorigenesisrdquo Nature vol 475 no 7354 pp 106ndash109 2011

[130] L M Solis C Behrens W Dong et al ldquoNrf2 and Keap1abnormalities in non-small cell lung carcinoma and associationwith clinicopathologic featuresrdquo Clinical Cancer Research vol16 no 14 pp 3743ndash3753 2010

[131] N M Reddy S R Kleeberger H Y Cho et al ldquoDeficiency inNrf2-GSH signaling impairs type II cell growth and enhancessensitivity to oxidantsrdquoAmerican Journal of Respiratory Cell andMolecular Biology vol 37 no 1 pp 3ndash8 2007

[132] N M Reddy S R Kleeberger M Yamamoto et al ldquoGeneticdissection of the Nrf2-dependent redox signaling-regulatedtranscriptional programs of cell proliferation and cytoprotec-tionrdquo Physiological Genomics vol 32 no 1 pp 74ndash81 2007

[133] Y Mitsuishi K Taguchi Y Kawatani et al ldquoNrf2 redirectsglucose and glutamine into anabolic pathways in metabolicreprogrammingrdquo Cancer Cell vol 22 no 1 pp 66ndash79 2012

[134] J Cho SManandharH LeeH Park andMKwak ldquoRole of theNrf2-antioxidant system in cytotoxicity mediated by anticancercisplatin implication to cancer cell resistancerdquo Cancer Lettersvol 260 no 1-2 pp 96ndash108 2008

[135] T Ohta K Iijima M Miyamoto et al ldquoLoss of Keap1 functionactivates Nrf2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008

[136] A Singh M Bodas N Wakabayashi F Bunz and S BiswalldquoGain of Nrf2 function in non-small-cell lung cancer cellsconfers radioresistancerdquoAntioxidantsampRedox Signaling vol 13no 11 pp 1627ndash1637 2010

[137] G-S Shim S Manandhar D-H Shin T Kim and M KwakldquoAcquisition of doxorubicin resistance in ovarian carcinomacells accompanies activation of the NRF2 pathwayrdquo Free RadicalBiology and Medicine vol 47 no 11 pp 1619ndash1631 2009

[138] S Manandhar B Choi K Jung et al ldquoNRF2 inhibitionrepresses ErbB2 signaling in ovarian carcinoma cells implica-tions for tumor growth retardation and docetaxel sensitivityrdquoFree Radical Biology and Medicine vol 52 no 9 pp 1773ndash17852012

[139] T Kim E-g Hur S J Kang et al ldquoNRF2 blockade suppressescolon tumor angiogenesis by inhibiting hypoxia-induced acti-vation ofHIF-1120572rdquoCancer Research vol 71 no 6 pp 2260ndash22752011

[140] L Kinch N V Grishin and J Brugarolas ldquoSuccination of Keap1and activation of Nrf2-dependent antioxidant pathways in FH-deficient papillary renal cell carcinoma type 2rdquo Cancer Cell vol20 no 4 pp 418ndash420 2011

[141] J J Tsai J A Dudakov K Takahashi et al ldquoNrf2 regulateshaematopoietic stem cell functionrdquo Nature Cell Biology vol 15no 3 pp 309ndash316 2013

[142] J H Kim R KThimmulappa V Kumar et al ldquoNRF2-mediatedNotch pathway activation enhances hematopoietic reconsti-tution following myelosuppressive radiationrdquo The Journal ofClinical Investigation vol 124 no 2 pp 730ndash741 2014

[143] J P Chute ldquoNRF2 mitigates radiation-induced hematopoieticdeathrdquo The Journal of Clinical Investigation vol 124 no 3 pp960ndash961 2014

[144] J Jang Y Wang H Kim M A Lalli and K S KosikldquoNrf2 a regulator of the proteasome controls self-renewal andpluripotency in human embryonic stem cellsrdquo Stem Cells vol32 no 10 pp 2616ndash2625 2014

[145] S Murakami R Shimizu P Romeo M Yamamoto and HMotohashi ldquoKeap1-Nrf2 system regulates cell fate determina-tion of hematopoietic stem cellsrdquo Genes to Cells vol 19 no 3pp 239ndash253 2014

[146] C E Hochmuth B Biteau D Bohmann and H Jasper ldquoRedoxregulation by Keap1 and Nrf2 controls intestinal stem cellproliferation inDrosophilardquoCell Stem Cell vol 8 no 2 pp 188ndash199 2011

[147] M K Paul B Bisht D O Darmawan et al ldquoDynamic changesin intracellular ROS levels regulate airway basal stem cellhomeostasis through Nrf2-dependent notch signalingrdquo CellStem Cell vol 15 no 2 pp 199ndash214 2014

[148] L Gambari G Lisignoli L Cattini C Manferdini A Facchiniand F Grassi ldquoSodium hydrosulfide inhibits the differentiationof osteoclast progenitor cells via NRF2-dependent mechanismrdquoPharmacological Research vol 87 pp 99ndash112 2014

[149] J Li D Johnson M Calkins L Wright C Svendsen andJ Johnson ldquoStabilization of Nrf2 by tBHQ confers protectionagainst oxidative stress-induced cell death in human neuralstem cellsrdquo Toxicological Sciences vol 83 no 2 pp 313ndash3282005

[150] A AMerchant A SinghWMatsui and S Biswal ldquoThe redox-sensitive transcription factor Nrf2 regulatesmurine hematopoi-etic stem cell survival independently of ROS levelsrdquo Blood vol118 no 25 pp 6572ndash6579 2011

[151] N Gurusamy D Ray I Lekli and D K Das ldquoRed wineantioxidant resveratrol-modified cardiac stem cells regener-ate infarcted myocardiumrdquo Journal of Cellular and MolecularMedicine vol 14 no 9 pp 2235ndash2239 2010

[152] N Gorbunov G Petrovski N Gurusamy D Ray D H Kimand D K Das ldquoRegeneration of infarcted myocardium withresveratrol-modified cardiac stem cellsrdquo Journal of Cellular andMolecular Medicine vol 16 no 1 pp 174ndash184 2012

[153] C Cai L Teng D Vu et al ldquoThe heme oxygenase 1 inducer(CoPP) protects human cardiac stem cells against apoptosisthrough activation of the extracellular signal-regulated kinase(ERK)NRF2 signaling pathway and cytokine releaserdquo TheJournal of Biological Chemistry vol 287 no 40 pp 33720ndash33732 2012

[154] J Song S M Kang K M Lee and J E Lee ldquoThe protectiveeffect of melatonin on neural stem cell against LPS-inducedinflammationrdquo BioMed Research International vol 2015 ArticleID 854359 13 pages 2015

14 Oxidative Medicine and Cellular Longevity

[155] V Karkkainen Y Pomeshchik E Savchenko et al ldquoNrf2regulates neurogenesis and protects neural progenitor cellsagainst A120573 toxicityrdquo STEM CELLS vol 32 no 7 pp 1904ndash19162014

[156] M Mohammadzadeh R Halabian A Gharehbaghian et alldquoNrf-2 overexpression in mesenchymal stem cells reducesoxidative stress-induced apoptosis and cytotoxicityrdquo Cell Stressand Chaperones vol 17 no 5 pp 553ndash565 2012

[157] Y Takahata T Takarada M Iemata et al ldquoFunctional expres-sion of 1205732 adrenergic receptors responsible for protectionagainst oxidative stress through promotion of glutathione syn-thesis after Nrf2 upregulation in undifferentiated mesenchymalC3H10T12 stem cellsrdquo Journal of Cellular Physiology vol 218no 2 pp 268ndash275 2009

[158] J Zhu H Wang Y Fan et al ldquoKnockdown of nuclear factorerythroid 2-related factor 2 by lentivirus induces differentiationof glioma stem-like cellsrdquo Oncology Reports vol 32 no 3 pp1170ndash1178 2014

[159] J Zhu H Wang Q Sun et al ldquoNrf2 is required to maintain theself-renewal of glioma stem cellsrdquo BMC Cancer vol 13 no 1article 380 2013

[160] B L Emmink A Verheem W J Van Houdt et al ldquoThe secre-tome of colon cancer stem cells contains drug-metabolizingenzymesrdquo Journal of Proteomics vol 91 pp 84ndash96 2013

[161] T Mizuno N Suzuki H Makino et al ldquoCancer stem-like cellsof ovarian clear cell carcinoma are enriched in the ALDH-highpopulation associated with an accelerated scavenging system inreactive oxygen speciesrdquo Gynecologic Oncology vol 137 no 2pp 299ndash305 2015

[162] I-G Ryoo B-H Choi and M-K Kwak ldquoActivation of NRF2by p62 and proteasome reduction in sphere-forming breastcarcinoma cellsrdquo Oncotarget vol 6 no 10 pp 8167ndash8184 2015

[163] T Wu B G Harder P K Wong J E Lang and D D ZhangldquoOxidative stress mammospheres and Nrf2-new implicationfor breast cancer therapyrdquoMolecular Carcinogenesis 2014

[164] B Yang Y Ma and Y Liu ldquoElevated expression of Nrf-2 andABCG2 involved in multi-drug resistance of lung cancer SPcellsrdquo Drug Research 2015

[165] M Zhang A Mathur Y Zhang et al ldquoMithramycin repressesbasal and cigarette smoke-induced expression of abcg2 andinhibits stem cell signaling in lung and esophageal cancer cellsrdquoCancer Research vol 72 no 16 pp 4178ndash4192 2012

[166] C A Del Vecchio Y Feng E S Sokol et al ldquoDe-differentiationconfers multidrug resistance via noncanonical PERK-Nrf2signalingrdquo PLoS Biology vol 12 no 9 Article ID e1001945 2014

[167] D Ren N F Villeneuve T Jiang et al ldquoBrusatol enhancesthe efficacy of chemotherapy by inhibiting the Nrf2-mediateddefense mechanismrdquo Proceedings of the National Academy ofSciences of the United States of America vol 108 no 4 pp 1433ndash1438 2011

[168] A Arlt S Sebens S Krebs et al ldquoInhibition of the Nrf2 tran-scription factor by the alkaloid trigonelline renders pancreaticcancer cells more susceptible to apoptosis through decreasedproteasomal gene expression and proteasome activityrdquo Onco-gene vol 32 no 40 pp 4825ndash4835 2013

[169] A-M Gao Z-P Ke F Shi G Sun and H Chen ldquoChrysinenhances sensitivity of BEL-7402ADM cells to doxorubicin bysuppressing PI3KAktNrf2 and ERKNrf2 pathwayrdquo Chemico-Biological Interactions vol 206 no 1 pp 100ndash108 2013

[170] A M Gao Z P Ke J N Wang J Yang S Chen and HChen ldquoApigenin sensitizes doxorubicin-resistant hepatocellular

carcinoma BEL-7402ADM cells to doxorubicin via inhibitingPI3KAktNrf2 pathwayrdquo Carcinogenesis vol 34 no 8 pp1806ndash1814 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Oxidative Medicine and Cellular Longevity 13

[124] R Wang J An F Ji H Jiao H Sun and D Zhou ldquoHyper-methylation of the Keap1 gene in human lung cancer cell linesand lung cancer tissuesrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 1 pp 151ndash154 2008

[125] P Zhang A Singh S Yegnasubramanian et al ldquoLoss ofKelch-like ECH-associated protein 1 function in prostate cancercells causes chemoresistance and radioresistance and promotestumor growthrdquoMolecular Cancer Therapeutics vol 9 no 2 pp336ndash346 2010

[126] J D Hayes and M McMahon ldquoNRF2 and KEAP1 mutationspermanent activation of an adaptive response in cancerrdquo Trendsin Biochemical Sciences vol 34 no 4 pp 176ndash188 2009

[127] B Padmanabhan K I Tong T Ohta et al ldquoStructural basis fordefects of Keap1 activity provoked by its pointmutations in lungcancerrdquoMolecular Cell vol 21 no 5 pp 689ndash700 2006

[128] Y Inami S Waguri A Sakamoto et al ldquoPersistent activation ofNrf2 throughp62 in hepatocellular carcinoma cellsrdquoTheJournalof Cell Biology vol 193 no 2 pp 275ndash284 2011

[129] G M DeNicola F A Karreth T J Humpton et al ldquoOncogene-induced Nrf2 transcription promotes ROS detoxification andtumorigenesisrdquo Nature vol 475 no 7354 pp 106ndash109 2011

[130] L M Solis C Behrens W Dong et al ldquoNrf2 and Keap1abnormalities in non-small cell lung carcinoma and associationwith clinicopathologic featuresrdquo Clinical Cancer Research vol16 no 14 pp 3743ndash3753 2010

[131] N M Reddy S R Kleeberger H Y Cho et al ldquoDeficiency inNrf2-GSH signaling impairs type II cell growth and enhancessensitivity to oxidantsrdquoAmerican Journal of Respiratory Cell andMolecular Biology vol 37 no 1 pp 3ndash8 2007

[132] N M Reddy S R Kleeberger M Yamamoto et al ldquoGeneticdissection of the Nrf2-dependent redox signaling-regulatedtranscriptional programs of cell proliferation and cytoprotec-tionrdquo Physiological Genomics vol 32 no 1 pp 74ndash81 2007

[133] Y Mitsuishi K Taguchi Y Kawatani et al ldquoNrf2 redirectsglucose and glutamine into anabolic pathways in metabolicreprogrammingrdquo Cancer Cell vol 22 no 1 pp 66ndash79 2012

[134] J Cho SManandharH LeeH Park andMKwak ldquoRole of theNrf2-antioxidant system in cytotoxicity mediated by anticancercisplatin implication to cancer cell resistancerdquo Cancer Lettersvol 260 no 1-2 pp 96ndash108 2008

[135] T Ohta K Iijima M Miyamoto et al ldquoLoss of Keap1 functionactivates Nrf2 and provides advantages for lung cancer cellgrowthrdquo Cancer Research vol 68 no 5 pp 1303ndash1309 2008

[136] A Singh M Bodas N Wakabayashi F Bunz and S BiswalldquoGain of Nrf2 function in non-small-cell lung cancer cellsconfers radioresistancerdquoAntioxidantsampRedox Signaling vol 13no 11 pp 1627ndash1637 2010

[137] G-S Shim S Manandhar D-H Shin T Kim and M KwakldquoAcquisition of doxorubicin resistance in ovarian carcinomacells accompanies activation of the NRF2 pathwayrdquo Free RadicalBiology and Medicine vol 47 no 11 pp 1619ndash1631 2009

[138] S Manandhar B Choi K Jung et al ldquoNRF2 inhibitionrepresses ErbB2 signaling in ovarian carcinoma cells implica-tions for tumor growth retardation and docetaxel sensitivityrdquoFree Radical Biology and Medicine vol 52 no 9 pp 1773ndash17852012

[139] T Kim E-g Hur S J Kang et al ldquoNRF2 blockade suppressescolon tumor angiogenesis by inhibiting hypoxia-induced acti-vation ofHIF-1120572rdquoCancer Research vol 71 no 6 pp 2260ndash22752011

[140] L Kinch N V Grishin and J Brugarolas ldquoSuccination of Keap1and activation of Nrf2-dependent antioxidant pathways in FH-deficient papillary renal cell carcinoma type 2rdquo Cancer Cell vol20 no 4 pp 418ndash420 2011

[141] J J Tsai J A Dudakov K Takahashi et al ldquoNrf2 regulateshaematopoietic stem cell functionrdquo Nature Cell Biology vol 15no 3 pp 309ndash316 2013

[142] J H Kim R KThimmulappa V Kumar et al ldquoNRF2-mediatedNotch pathway activation enhances hematopoietic reconsti-tution following myelosuppressive radiationrdquo The Journal ofClinical Investigation vol 124 no 2 pp 730ndash741 2014

[143] J P Chute ldquoNRF2 mitigates radiation-induced hematopoieticdeathrdquo The Journal of Clinical Investigation vol 124 no 3 pp960ndash961 2014

[144] J Jang Y Wang H Kim M A Lalli and K S KosikldquoNrf2 a regulator of the proteasome controls self-renewal andpluripotency in human embryonic stem cellsrdquo Stem Cells vol32 no 10 pp 2616ndash2625 2014

[145] S Murakami R Shimizu P Romeo M Yamamoto and HMotohashi ldquoKeap1-Nrf2 system regulates cell fate determina-tion of hematopoietic stem cellsrdquo Genes to Cells vol 19 no 3pp 239ndash253 2014

[146] C E Hochmuth B Biteau D Bohmann and H Jasper ldquoRedoxregulation by Keap1 and Nrf2 controls intestinal stem cellproliferation inDrosophilardquoCell Stem Cell vol 8 no 2 pp 188ndash199 2011

[147] M K Paul B Bisht D O Darmawan et al ldquoDynamic changesin intracellular ROS levels regulate airway basal stem cellhomeostasis through Nrf2-dependent notch signalingrdquo CellStem Cell vol 15 no 2 pp 199ndash214 2014

[148] L Gambari G Lisignoli L Cattini C Manferdini A Facchiniand F Grassi ldquoSodium hydrosulfide inhibits the differentiationof osteoclast progenitor cells via NRF2-dependent mechanismrdquoPharmacological Research vol 87 pp 99ndash112 2014

[149] J Li D Johnson M Calkins L Wright C Svendsen andJ Johnson ldquoStabilization of Nrf2 by tBHQ confers protectionagainst oxidative stress-induced cell death in human neuralstem cellsrdquo Toxicological Sciences vol 83 no 2 pp 313ndash3282005

[150] A AMerchant A SinghWMatsui and S Biswal ldquoThe redox-sensitive transcription factor Nrf2 regulatesmurine hematopoi-etic stem cell survival independently of ROS levelsrdquo Blood vol118 no 25 pp 6572ndash6579 2011

[151] N Gurusamy D Ray I Lekli and D K Das ldquoRed wineantioxidant resveratrol-modified cardiac stem cells regener-ate infarcted myocardiumrdquo Journal of Cellular and MolecularMedicine vol 14 no 9 pp 2235ndash2239 2010

[152] N Gorbunov G Petrovski N Gurusamy D Ray D H Kimand D K Das ldquoRegeneration of infarcted myocardium withresveratrol-modified cardiac stem cellsrdquo Journal of Cellular andMolecular Medicine vol 16 no 1 pp 174ndash184 2012

[153] C Cai L Teng D Vu et al ldquoThe heme oxygenase 1 inducer(CoPP) protects human cardiac stem cells against apoptosisthrough activation of the extracellular signal-regulated kinase(ERK)NRF2 signaling pathway and cytokine releaserdquo TheJournal of Biological Chemistry vol 287 no 40 pp 33720ndash33732 2012

[154] J Song S M Kang K M Lee and J E Lee ldquoThe protectiveeffect of melatonin on neural stem cell against LPS-inducedinflammationrdquo BioMed Research International vol 2015 ArticleID 854359 13 pages 2015

14 Oxidative Medicine and Cellular Longevity

[155] V Karkkainen Y Pomeshchik E Savchenko et al ldquoNrf2regulates neurogenesis and protects neural progenitor cellsagainst A120573 toxicityrdquo STEM CELLS vol 32 no 7 pp 1904ndash19162014

[156] M Mohammadzadeh R Halabian A Gharehbaghian et alldquoNrf-2 overexpression in mesenchymal stem cells reducesoxidative stress-induced apoptosis and cytotoxicityrdquo Cell Stressand Chaperones vol 17 no 5 pp 553ndash565 2012

[157] Y Takahata T Takarada M Iemata et al ldquoFunctional expres-sion of 1205732 adrenergic receptors responsible for protectionagainst oxidative stress through promotion of glutathione syn-thesis after Nrf2 upregulation in undifferentiated mesenchymalC3H10T12 stem cellsrdquo Journal of Cellular Physiology vol 218no 2 pp 268ndash275 2009

[158] J Zhu H Wang Y Fan et al ldquoKnockdown of nuclear factorerythroid 2-related factor 2 by lentivirus induces differentiationof glioma stem-like cellsrdquo Oncology Reports vol 32 no 3 pp1170ndash1178 2014

[159] J Zhu H Wang Q Sun et al ldquoNrf2 is required to maintain theself-renewal of glioma stem cellsrdquo BMC Cancer vol 13 no 1article 380 2013

[160] B L Emmink A Verheem W J Van Houdt et al ldquoThe secre-tome of colon cancer stem cells contains drug-metabolizingenzymesrdquo Journal of Proteomics vol 91 pp 84ndash96 2013

[161] T Mizuno N Suzuki H Makino et al ldquoCancer stem-like cellsof ovarian clear cell carcinoma are enriched in the ALDH-highpopulation associated with an accelerated scavenging system inreactive oxygen speciesrdquo Gynecologic Oncology vol 137 no 2pp 299ndash305 2015

[162] I-G Ryoo B-H Choi and M-K Kwak ldquoActivation of NRF2by p62 and proteasome reduction in sphere-forming breastcarcinoma cellsrdquo Oncotarget vol 6 no 10 pp 8167ndash8184 2015

[163] T Wu B G Harder P K Wong J E Lang and D D ZhangldquoOxidative stress mammospheres and Nrf2-new implicationfor breast cancer therapyrdquoMolecular Carcinogenesis 2014

[164] B Yang Y Ma and Y Liu ldquoElevated expression of Nrf-2 andABCG2 involved in multi-drug resistance of lung cancer SPcellsrdquo Drug Research 2015

[165] M Zhang A Mathur Y Zhang et al ldquoMithramycin repressesbasal and cigarette smoke-induced expression of abcg2 andinhibits stem cell signaling in lung and esophageal cancer cellsrdquoCancer Research vol 72 no 16 pp 4178ndash4192 2012

[166] C A Del Vecchio Y Feng E S Sokol et al ldquoDe-differentiationconfers multidrug resistance via noncanonical PERK-Nrf2signalingrdquo PLoS Biology vol 12 no 9 Article ID e1001945 2014

[167] D Ren N F Villeneuve T Jiang et al ldquoBrusatol enhancesthe efficacy of chemotherapy by inhibiting the Nrf2-mediateddefense mechanismrdquo Proceedings of the National Academy ofSciences of the United States of America vol 108 no 4 pp 1433ndash1438 2011

[168] A Arlt S Sebens S Krebs et al ldquoInhibition of the Nrf2 tran-scription factor by the alkaloid trigonelline renders pancreaticcancer cells more susceptible to apoptosis through decreasedproteasomal gene expression and proteasome activityrdquo Onco-gene vol 32 no 40 pp 4825ndash4835 2013

[169] A-M Gao Z-P Ke F Shi G Sun and H Chen ldquoChrysinenhances sensitivity of BEL-7402ADM cells to doxorubicin bysuppressing PI3KAktNrf2 and ERKNrf2 pathwayrdquo Chemico-Biological Interactions vol 206 no 1 pp 100ndash108 2013

[170] A M Gao Z P Ke J N Wang J Yang S Chen and HChen ldquoApigenin sensitizes doxorubicin-resistant hepatocellular

carcinoma BEL-7402ADM cells to doxorubicin via inhibitingPI3KAktNrf2 pathwayrdquo Carcinogenesis vol 34 no 8 pp1806ndash1814 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

14 Oxidative Medicine and Cellular Longevity

[155] V Karkkainen Y Pomeshchik E Savchenko et al ldquoNrf2regulates neurogenesis and protects neural progenitor cellsagainst A120573 toxicityrdquo STEM CELLS vol 32 no 7 pp 1904ndash19162014

[156] M Mohammadzadeh R Halabian A Gharehbaghian et alldquoNrf-2 overexpression in mesenchymal stem cells reducesoxidative stress-induced apoptosis and cytotoxicityrdquo Cell Stressand Chaperones vol 17 no 5 pp 553ndash565 2012

[157] Y Takahata T Takarada M Iemata et al ldquoFunctional expres-sion of 1205732 adrenergic receptors responsible for protectionagainst oxidative stress through promotion of glutathione syn-thesis after Nrf2 upregulation in undifferentiated mesenchymalC3H10T12 stem cellsrdquo Journal of Cellular Physiology vol 218no 2 pp 268ndash275 2009

[158] J Zhu H Wang Y Fan et al ldquoKnockdown of nuclear factorerythroid 2-related factor 2 by lentivirus induces differentiationof glioma stem-like cellsrdquo Oncology Reports vol 32 no 3 pp1170ndash1178 2014

[159] J Zhu H Wang Q Sun et al ldquoNrf2 is required to maintain theself-renewal of glioma stem cellsrdquo BMC Cancer vol 13 no 1article 380 2013

[160] B L Emmink A Verheem W J Van Houdt et al ldquoThe secre-tome of colon cancer stem cells contains drug-metabolizingenzymesrdquo Journal of Proteomics vol 91 pp 84ndash96 2013

[161] T Mizuno N Suzuki H Makino et al ldquoCancer stem-like cellsof ovarian clear cell carcinoma are enriched in the ALDH-highpopulation associated with an accelerated scavenging system inreactive oxygen speciesrdquo Gynecologic Oncology vol 137 no 2pp 299ndash305 2015

[162] I-G Ryoo B-H Choi and M-K Kwak ldquoActivation of NRF2by p62 and proteasome reduction in sphere-forming breastcarcinoma cellsrdquo Oncotarget vol 6 no 10 pp 8167ndash8184 2015

[163] T Wu B G Harder P K Wong J E Lang and D D ZhangldquoOxidative stress mammospheres and Nrf2-new implicationfor breast cancer therapyrdquoMolecular Carcinogenesis 2014

[164] B Yang Y Ma and Y Liu ldquoElevated expression of Nrf-2 andABCG2 involved in multi-drug resistance of lung cancer SPcellsrdquo Drug Research 2015

[165] M Zhang A Mathur Y Zhang et al ldquoMithramycin repressesbasal and cigarette smoke-induced expression of abcg2 andinhibits stem cell signaling in lung and esophageal cancer cellsrdquoCancer Research vol 72 no 16 pp 4178ndash4192 2012

[166] C A Del Vecchio Y Feng E S Sokol et al ldquoDe-differentiationconfers multidrug resistance via noncanonical PERK-Nrf2signalingrdquo PLoS Biology vol 12 no 9 Article ID e1001945 2014

[167] D Ren N F Villeneuve T Jiang et al ldquoBrusatol enhancesthe efficacy of chemotherapy by inhibiting the Nrf2-mediateddefense mechanismrdquo Proceedings of the National Academy ofSciences of the United States of America vol 108 no 4 pp 1433ndash1438 2011

[168] A Arlt S Sebens S Krebs et al ldquoInhibition of the Nrf2 tran-scription factor by the alkaloid trigonelline renders pancreaticcancer cells more susceptible to apoptosis through decreasedproteasomal gene expression and proteasome activityrdquo Onco-gene vol 32 no 40 pp 4825ndash4835 2013

[169] A-M Gao Z-P Ke F Shi G Sun and H Chen ldquoChrysinenhances sensitivity of BEL-7402ADM cells to doxorubicin bysuppressing PI3KAktNrf2 and ERKNrf2 pathwayrdquo Chemico-Biological Interactions vol 206 no 1 pp 100ndash108 2013

[170] A M Gao Z P Ke J N Wang J Yang S Chen and HChen ldquoApigenin sensitizes doxorubicin-resistant hepatocellular

carcinoma BEL-7402ADM cells to doxorubicin via inhibitingPI3KAktNrf2 pathwayrdquo Carcinogenesis vol 34 no 8 pp1806ndash1814 2013

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom