Therapeutic Effect of Quinacrine, an Antiprotozoan Drug ... · visualized using DAPI. After washing with PBS, cover slips were mounted with mounting solution (Vector Laboratories)

Cell Death and Survival

Therapeutic Effect of Quinacrine, anAntiprotozoan Drug, by Selective Suppressionof p-CHK1/2 in p53-Negative Malignant CancersSoyoung Park1, Ah-Young Oh1, Jung-Hyun Cho1, Min-Ho Yoon1, Tae-Gyun Woo1,So-mi Kang1, Ho-Young Lee2, Youn-Jin Jung3, and Bum-Joon Park1

Abstract

Quinacrine (QNC), antiprotozoan drug commonly usedagainst Malaria and Giardiasis, has been recently tried for rheu-matics and prion diseases via drug repositioning. In addition,several reports suggest antitumor effects of QNC through sup-pression of NF-kB and activation of p53. This study demonstratesthe anticancer effect of QNC via a novel pathway through theelimination of checkpoint kinase 1/2 (Chk1/2) under p53-inac-tivated conditions. Inhibition of p53 by PFT-aor siRNApromotesQNC-induced apoptosis in normal fibroblast and p53-intactcancer cells. Considering that Chk1/2 kinases exert an essentialrole in the control of cell cycle, inhibition of Chk1/2 byQNCmayinduce cell death via uncontrolled cell cycle progression. Indeed,

QNC reduces Chk1/2 expression under p53-impaired cancer cellsand induces cell death in the G2–M phase. QNC increases thebinding between p-Chk1/2 and b-TrCP and promotes protea-some-dependent degradation. Moreover, QNC treatment dis-played antitumor effects in a Villin-Cre;p53þ/LSL-R172H intestinalcancermousemodel systemaswell asHCT116p53�/� xenografts.

Implications: QNC has been used for the past over 70 yearswithout obvious side effects, as such it is a plausible drug candi-date for relapsed cancers, small-cell lung cancer, breast cancer aswell as various p53-inactivated human malignancies. Mol CancerRes; 16(6); 935–46. �2018 AACR.

IntroductionDespite intensive investigation of anticancer drug develop-

ment, cancer incidence and mortality are continuouslyincreased by extension of life span. In addition, cancer relapseratio and resistance against typical anticancer drugs areincreased. Relapsed cancers after radiation and chemotherapy,in general, show defects in DNA damage-induced cell deathpathways, particularly, p53-dependent pathways via geneticmutations or epigenetic alternations (1, 2). Hyper-activatedATM/ATR-related DNA repair systems also provide resistanceagainst DNA-damaging agents (3). Thus, inhibition of DNArepair systems is a plausible therapeutic strategy for relapsedcancers and p53-inactivated cancers.

Quinacrine (QNC) is widely used as an antiprotozoa drug;however, new applications of QNC are being proposed. Previousliterature has reported favorable effects of QNC on autoimmunediseases (4) and prion diseases (5). Moreover, an antitumoral

effect of QNC has been suggested (6). Concerning anticancereffects, activation of p53 and inhibition of NF-kB are suggested(7), becauseQNChas an acridine orange-like ring structure that issupposed to intercalate DNA (8, 9). However, unlike other DNAintercalating agents, such as DAPI and EtBr, it is not considered astrong DNA-intercalating agent or carcinogen. Indeed, despitelong time usage of QNC since World War II, there is littleepidemiological evidence to support an increase in cancer inci-dence inQNC-treatedpatients (10).However, we cannot alsofindepidemiological study about low cancer incidence in QNC-trea-ted patients. These facts are very curious because the cancerprevention effect of rapamycin, a widely used antibacterial drug,has been revealed by epidemiological follow-up (11). Indeed,RAD001 has been developed as an anticancer drug based on thebiological effect of rapamycin (12). These facts suggest twopossibilities: (i) QNC is not related with human cancer incidenceand prevention and (ii) QNC could be associated with cancerunder special condition. However, first possibility could bepotentially excluded because QNC-mediated antitumor effectshave been reported (13).

Cancer cells are often depicted as trying to escape from acontrolling environment and transform to a single cell-like state(14). Thus, we hypothesized that the antiprotozoa drugs such asantimalaria drugs might show meaningful anticancer effects. Totest this hypothesis, the effect of well-known antiprotozoa che-micals on human cancer cell lines was evaluated. Discovery of alead candidate could potentially be applied to human patients,with minimal delay, as these chemicals have already beenapproved by the FDA.

In summary, this study revealed thatQNChas antitumor effectson p53-deficient malignant cancers in vitro and in vivo. Mecha-nistically, QNC promoted p-Chk1/2 degradation and eliminated

1Department of Molecular Biology, College of Natural Science, Pusan NationalUniversity, Busan, Republic of Korea. 2Department of Nuclear Medicine, SeoulNational University BundangHospital, Seongnam, Republic of Korea. 3College ofPharmacy, Pusan National University, Busan, Republic of Korea.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

S. Park and A.-Y. Oh contributed equally to this article.

Corresponding Author: Bum-Joon Park, Pusan National University, 30Jangjeon-dong, Geumjeong-gu, Busan 609-735, Republic of Korea. Phone:82-51-510-2220; Fax: 82-51-513-9258; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-17-0511

�2018 American Association for Cancer Research.

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minimal cell-cycle checkpoint. Thus, p53-deficient cells wereeasily affected by QNC-induced cytotoxicity. These results strong-ly suggest that QNC would be a plausible candidate drug againstrelapsed human malignancies.

Materials and MethodsMice

All experimental procedures using laboratory animals wereapproved by the animal care committee of Pusan NationalUniversity. Athymic nude mice were maintained undertemperature- and light-controlled conditions (20�C–23�C,12 hours/12 hours light/dark cycle) and provided autoclavedfood and water ad libitum.

In vivo treatment studiesFor xenografts, HCT 116 p53�/� (1� 107) cells were seeded in

the right and left flanks of nude mice. After a week, randomlypicked tumor-bearing mice were treated with PBS (n ¼ 3) as acontrol, QNC (10 mg/kg, n ¼ 4) and (20 mg/kg, n ¼ 4) byintraperitoneal (i.p.) injection three times a week. Tumor size andbody weight were measured twice a week. To generate Villin-Cre;p53þ/LSL-R172Hmice, Villin-Cremice were crossed with Villin-Cre;p53þ/LSL-R172H mice. Villin-Cre;p53þ/LSL-R172H mice (4-month-old, n ¼ 5) were treated with carrier (n ¼ 2) as a control andQNC (20mg/kg, n¼ 3) by i.p. injection three times a week. Bodyweights were measured twice a week.

Cell culture and reagentsA549, H1299, HEK 293, MCF7, MDA-MB231, and MDA-

MB468 were purchased and authenticated from the ATCC andmaintained in RPMI-1640 or Dulbecco's Modified Eaglemedium(DMEM) supplemented with 10% FBS and antibiotics. ACHNwere obtained and authenticated from Korea cell line bank(KCLB) and maintained in DMEMmedium containing 10% FBSand antibiotics. HCT 116 p53þ/þ, p53þ/� and p53�/� cells werekindly provided by Dr. B. Vogelstein (Johns Hopkins University,Baltimore, MD) andmaintained inMcCoy's 5A (modified)medi-um containing 10% FBS and antibiotics. MEF cells were isolatedfrom 14.5-day embryos using a standard protocol and cultured inMEM/EBSS medium containing 15% FBS and 1% antibiotics.Mycoplasma tests and DNA finger printing were performed every6months (Mycoplasma tests) and 2 years (last test was performed2016). Cells were passaged no longer than 2 months. QNC,SB202190, 5-Fluorouracil (5-Fu), 4,7-Dichloroquinoline, and4-Hydroxytamoxifen (4-OHT) were purchased from Sigma-Aldrich. Adriamycin, Ptfithrin-a, LY294002, U0126, and MG132were obtained from Calbiochem and rapamycin, gefitinib, anderlotinib were provided by LC Laboratory. PD98059 was pur-chased from StressGen and ABT-737, SB216763, and imatinibwere obtained from Selleckchem.

Immunoblot analysisProtein was extracted using RIPA buffer (50 mmol/L Tris-Cl,

150 mmol/L NaCl, 1% NP-40, 0.1% SDS, and 10% sodiumdeoxycholate) and heated with sample buffer for inactivation(heated at 95�C for 7 minutes). Protein samples were appliedto sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and immunoblot analysis was carried out fol-lowing a general protocol. In brief, the samples were transferredonto a PVDF membrane and blocked with 3% skimmed milk

before incubation with primary antibodies. The membraneswere washed three times and incubated with HRP conjugatesecondary antibodies for 1 hour at room temperature. Proteinbands were detected by chemiluminescence with ECL kit(Advansta). Antibodies against Actin (sc-1616), GFP (sc-9996),Chk2 (sc-9064), His (sc-8036), GST (sc-138), NF2 (sc-331),b-catenin (sc-7963), HA (sc-7392), b-TrCP (sc-390629), Chk1(sc-8408), a-tubulin (sc-8035), and p53DO1 (sc-126) were pur-chased from Santa Cruz Biotechnology. p-Chk1 (2341), p-Chk2(2661), p-Erk (9101), and t-p53(9282) were obtained from Cellsignaling Technology. Anti-Mre11 (gtx70212) was provided byGeneTex and anti-FLAG (F3165) and anti-Myc (M5546) wasobtained from Sigma-Aldrich.

ImmunofluorescenceCells were cultured on cover slips and transfected with the

indicated vectors and treated with the indicated chemicals. Cellswere fixed with MeOH for 30 minutes and the nucleus wasvisualized using DAPI. After washing with PBS, cover slips weremounted with mounting solution (Vector Laboratories). Imageswere captured through fluorescence microscopy.

Transfection and siRNAspeGFP-Chk1, pcDNA3 NF2-FLAG (Isoform 1 of merlin),

pcDNA3 NF2-NTERM-FLAG 1-332 AA (15), b-TrCP-Myc, b-TrCPWD2-7-Myc (b-TrCP -DN), CDC4-Myc, CDC4 R465C-Myc(CDC4-DN), and Skp2-Myc were purchased from Addgene.HA-p53 vectors kindly provided by Dr. S. Kim (Seoul NationalUniversity, Seoul, Republic of Korea). VHL-FLAGandSiah1-FLAGvectors were obtained from Dr. Y.J. Jung (Pusan National Uni-versity, Busan, Republic of Korea) and from Dr. Lashuel (QatarFoundation), respectively. GFP-fused Chk2 expression vector wasobtained by Dr. N.C. Ha (Seoul National University, Seoul,Republic of Korea). Transfections were performed using the jetPEItransfection agent (Polyplus Transfection) following the manu-facturer's protocol. In brief, vectors (1.5 mg) were mixed with1.5 mL of jetPEI reagent in 150 mmol/L NaCl solution. Afterincubation for 20 minutes at room temperature, the mixture wasadded to the cell. After 3 hours, the serum-free medium wasreplaced with 10% FBS-containing medium. For in vitro geneknockdown, siRNAs against indicated targets were generated bya custom service (Cosmo Genetech). The siRNA target sequencefor b-TrCP is described below: 50-GCGACATAGTTTACAGAGATTCAAGAGA TCTCTGTAAACTATGTCGC-30. For siRNA transfec-tions, the INTERFERin transfection reagent (Polyplus) was used.In brief, 1.5 pmol/L (21 ng) of siRNA duplexes weremixed with 4mL of INTERFERin in 200 mLmediumwithout serum. Themixturewas homogenized by vortexing and incubated for 10 minutes atroom temperature. After the incubation, themixturewas added tothe cell and incubated at least 48 hours.

Immunoprecipitation and pulldownTo determine the interaction between proteins, pulldown

and immunoprecipitation (IP) assays were performed. Forpulldown assay, GST-Chk1 or Ni-b-TrCP were incubated withcell lysate in RIPA buffer for 30 minutes at 4�C. For IP, cellswere lysed in the RIPA buffer. Whole cell lysates were incubatedwith indicated primary antibodies for 1 hour at 4�C and thenwith protein A/G-conjugated agarose beads (Invitrogen) for1 hour. After centrifugation, beads with bound proteins were

Park et al.

Mol Cancer Res; 16(6) June 2018 Molecular Cancer Research936




washed with RIPA buffer and precipitated proteins weredetected by immunoblot.

Cell viabilityBriefly, cells were treated with varying concentrations of

drug for the indicated duration. The exposed cells were thenincubated with 0.5 mg/mL of MTT [3-(4, 5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] solution (Calbio-chem) for 4 hours at 37�C. After removing the supernatant,the precipitate was dissolved in dimethyl sulfoxide (DMSO)and quantified by measuring the absorbance on a spectropho-tometer at 540 nm.

Luciferase assayTo examine NF-kB promoter activity, NF-kB-luc vectors were

transfected into cells for 24 hours, and cells were treated with theindicated chemicals. After washing with wash buffer (Promega),the cells were lysed by lysis buffer (Promega). The luciferaseactivity was determined by a luminometer (MicroDigital).

Apoptosis assayCells were seeded on 6-well plates and incubated 12 hours with

DMSO (as control) or 5 mmol/L QNC. After treatment, cells werestained using the Annexin V-FITC apoptosis detection kit(B32117, Biotool). A minimum of 10,000 cells were analyzedby FACS (Beckman Coulter).

Histology and immunostaining.Tissue specimens were fixed in 4% paraformaldehyde and

embedded in paraffin. The paraffin blocks were sectioned at5 mm by Leica microtome and transferred onto adhesive-coatedslides (Marienfeld Laboratory Glassware). After deparaffini-zation and rehydration, tissue sections were stained withhematoxylin and eosin (H&E). Immunohistochemistry wasperformed on tissue sections using polyclonal rabbit anti-Ki67or mouse monoclonal p53 (Pab1801) antibodies. In brief,sections were gradually deparaffinized and rehydrated usingxylene and alcohols and rinsed in PBS. Antigen retrieval wasperformed with 10 mmol/L sodium citrate (pH 6.0) 2 times at95�C for 10 minutes each. Endogenous peroxidase activity wasblocked with 3% hydrogen peroxide for 10 minutes. Primaryantibodies (1:200) were incubated overnight at 4�C and HRP-conjugate secondary antibodies were incubated for 4 hours. Thevisualization was performed using DAB Peroxidase (HRP)Substrate Kit (Vector Labs).

Image acquisition of [18F] FDG PET/CTTo acquire [18F] -FDGPET/CT images, eachmousewas fasted at

least 6 hours. [18F] FDG (500 � 23 uCi) was intravenouslyadministered through the tail vein. After administration of [18F]FDG, mice were placed in a cage with dim light for 60 minutes.Each mouse was maintained under anesthesia with isoflurane(2.5% flow rate) for the duration of the scan. Animals werepositioned prone in the standard mouse bed. Limbs were posi-tioned lateral to the body to acquire uniform CT images. Wholebrain CT images were acquired with a Micro-PET/CT scanner(nanoPET/CT, Bioscan Inc.). For CT image acquisition, the X-raysource was set to 200 mA and 45 kVp with 0.5mm. The CT imageswere reconstructed using cone-beam reconstruction with a Sheppfilter with the cutoff at the Nyquist frequency and a binning factor

of 4, resulting in an image matrix of 480� 480� 632 and a voxelsize of 125 mm.

ResultsChemical screening

To test our hypothesis that antiprotozoa drugs (SupplementaryFig. S1A) have anticancer effects, MTT assays were performedusing human breast cancer cell lines, MDA-MB468, and MCF7(Fig. 1A and B). This initial screen revealed that QNC, comparedwith previously well-known anticancer drugs or chemical inhibi-tors, suppressed the cell viability of MDA-MB468 cells (Fig. 1B),but not MCF7 (Fig. 1A). As MDA-MB468 cells are considered amore aggressive type of breast cancer thanMCF7 cells (15), it wasspeculated that QNC might be effective against more aggressivetypes of cancer. Indeed, QNC at two different concentrationssuppressed the viability of MDA-MB468 cells with dramatic effect(Fig. 1C), compared with MCF7 cells (Fig. 1D). Similar resultswere obtained by counting viable cells using Tryphan blue dyeexclusion (Supplementary Fig. S1B). To address how QNC sup-presses cell viability, cells were stained with FITC-Annexin V, awell confirmed early apoptosismarker (17), and staining intensitywas measured by FACS. QNC increased the annexin V-positivestaining in MDA-MB468 cells (more than 50%; Fig. 1E), com-pared with MCF7 cells (Fig. 1E). To collect more evidence aboutthe anticancer effects ofQNConhumanmalignancies, additionalcell lines were tested. Reduced cell viability in response to QNCwas observed in small-cell lung cancer cell lines (SupplementaryFig. S1C). To further extend this, more cell lines (detailed infor-mation in Supplementary Fig. S1D) were tested: two mesotheli-oma cell lines (H28 andH2452), a renal cancer cell line (ACHN),and another aggressive human breast cancer cell line (MDA-MB231) all of which consistently displayed reduced viability withQNC treatment (Fig. 1F). However, other anticancer drugs such asHer2/neu inhibitors (Iressa and Tarceva) and an mTOR inhibitor(rapamycin) did not suppress the viability of these cell lines(Fig. 1F). In addition, QNCdid not show synergistic or additionaleffects with other conventional anticancer drugs such as EGFRkinase inhibitors (Supplementary Fig. S2A and B). Althoughautophagy has been suggested as the molecular mechanismof QNC-induced cell death, drugs that affect autophagy suchas RAD001, rapamycin, and 3-MA (inhibitor of autophagy;refs. 18–20) did not alter the QNC-induced cell death (Supple-mentary Fig. S2C).

The relevance of anticancer effect of QNC and p53 statusTodetermine if p53 contributes to theQNC-induced anticancer

effects, we compared the response with QNC in HCT116 and itsisogenic HCT116 p53�/� cell line. In contrast to previous reportsthat QNC induces p53 (6, 7), p53-deficient HCT116 cells weremore sensitive to QNC than parental HCT116 cells with intactp53 (Fig. 2A). Indeed, suppression of p53 expression via siRNA(Supplementary Fig. S2D) promoted QNC-induced cell death inp53-intact A549 andMCF7 cells (Fig. 2B). PFT-a, a p53 transcrip-tion inhibitor, also sensitized A549 and MCF7 cells to QNC-induced cell death (Supplementary Fig. S2E). QNC-induced celldeathwas also observed in normal humanfibroblast by treatmentwith PFT-a (Supplementary Fig. S3A). These results indicated thatQNC can selectively induce cell death under p53-inactivatedcondition. To confirm the effect of p53 on QNC-induced celldeath, cell viability was measured in MEF cells, obtained from

Antitumor Effect of QNC in p53-Negative Cancers

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Figure 1.

Anticancer effect of chloroquine relative chemicals. A and B, Chemical screening of various anticancer drugs and antiprotozoa drugs by MTT assay. Among variouschemicals, QNC suppressed viability of MDA-MB468 cells. In contrast, it did not show in less aggressive type of cancer, MCF7 cells. Cells were incubated withindicated chemicals for 24 hours and viability was determined by MTT assay. ��, P < 0.005. C and D, QNC suppressed cell viability of MDA-MB468 but notMCF7. Treating with another antiprotozoa drug, primaquine could not inhibit both cells' viability. After incubation with chemicals for 24 hours, the cell viabilitywas monitored by MTT assay. �� , P < 0.005, N.S., not significance. E, Apoptotic signals from QNC-treated MDA-MB468 and MCF7 cells were detected usingAnnexin V-FITC staining. Comparing with MCF7 cells, MDA-MB468 cells showed increased Annexin V-FITC stained cells by QNC treatment. Cells were treatedwith QNC of 5 mmol/L for 12 hours and assessed apoptosis by Annexin V-FITC staining (early apoptosis) and PI (late apoptosis). Numbers in each panelindicate percentage in total cells. F, All aggressive cancer cells including mesothelioma cell lines (H28 and H2452), RCC cell line (ACHN), and anotheraggressive human breast cancer cell line (MDA-MB231) were very sensitive to QNC. Cells were incubated with chemicals for 36 hours. Then, cell viability wasmeasured by MTT assay. � , 0.005 < P < 0.05; �� , P < 0.005.

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Figure 2.

Anticancer effect of Quinacrine under p53-deficient condition. A, Cell viability was gradually suppressed depending on QNC concentration especially inp53-deficient HCT116 cells compared with p53-intact HCT116 cells. HCT116 cells were treated with QNC for 24 hours and cell viability was determined withMTT assay. ��, P < 0.005. B, Inhibition of p53 using siRNA can promote QNC-induced cell death in p53-WT A549 and MCF7 cells. A549 and MCF7 cellswere transfected with si-scramble (si-con) or si-p53 for 24 hours. Subsequently, cells were treated with QNC for 24 hours and cell viability wasmeasured by MTT assay. � , 0.005 < P < 0.05 and �� , P < 0.005. N.S., not significance. C and D, p53 mutant MEF cells' viability was more inhibited byQNC compared with p53 intact condition. MEF cells obtained form UBC-ER-Cre; p53þ/LSL-R172H knock in mouse were pretreated with or without 4-OHT.Subsequently, cells were treated with QNC of indicated dose for 6 or 12 hours. Then immunoblot and MTT assay were performed. � , 0.005 < P < 0.05. E, QNCreduced tumor growth of HCT116 p53�/� cells engrafted to nude mouse. HCT116 p53�/� cells were inoculated subcutaneously into athymic nude miceand two concentrations of QNC or PBS were injected three times a week intraperitoneally (i.p.) for 8 weeks. Tumor volume was monitored twice a week.F, ACHN, MDA-MB231, and MDA-MB468 cells showed a stair-like dropped-down phase in cell death by QNC treatment. These dropped-down pointsinduced by QNC associated with cell-cycle progression (about 12–24 hours). In contrast, it did not show in MCF7 cells. After treatment with 5mmol/L QNC forindicated time, cell viability was measured by MTT assay. � , 0.005 < P < 0.05; �� , P < 0.005.






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conditional Ubc-Cre-ER; p53þ/LSL-R172H knock-in mice (21).LSL-p53R172H mouse cells express wild-type p53 before activa-tion of Cre recombinase because of a stop codon between theLoxP site and p53 mutation which when expressed by activationof Ubc-Cre-ER acts as dominant negative (22). Thus, 4-OHTtreatment induced mutant p53 expression (Fig. 2C) and consis-tently reduced expression of p21 a well-known p53 target(Fig. 2C). Under p53 mutant expressed condition, QNC inducedcell death (Fig. 2D). To test the in vivo antitumor effects ofQNC, HCT116 p53�/� cells were inoculated in athymic nudemice and injected with QNC (10 or 20 mg/kg) three times perweeks via i.p. for 8 weeks. As shown in Fig. 2E, HCT116 p53�/�

tumor growth was suppressed by QNC injection without bodyweight loss (Supplementary Fig. S3B). These results stronglysuggest that QNC suppresses tumor growth under p53-impairedconditions. Although suppression of NF-kB has been suggested asan important mechanism for the antitumor effects of QNC (6, 7),reduced NF-kB transcription was not observed by luciferase assayat the concentrations (i.e., 1 mmol/L and 5 mmol/L) used in thestudy. However, reduced NF-kB-luc activity at high concentra-tions (>10 mmol/L) was observed (Supplementary Fig. S3C).Thus, considering that QNC could induce cell death at concen-trations of 1 to 5 mmol/L (i.e, less than needed to suppress NF-kBactivity), it appears that NF-kB activity is not critical for theantitumoral effects of QNC. Interestingly, the renal cell lineACHN responded to QNC, despite expressing wild-type p53. Ithas been reported that in ACHN cells p53 is inactivated by NF2deletion (23). Consistent with our previous report (24), trans-fection of NF2 could induce p53 expression (SupplementaryFig. S3D). In addition, QNC-induced cell death was also sup-pressed by NF2 transfection (Supplementary Fig. S3E). This resultsuggests that QNC might be effective in NF2-deficient cancerssuch as RCCormesothelioma. To better understand the dynamicsofQNC-induced cell death, cell viability wasmonitored in a time-dependent manner. Interestingly, QNC induced cell death from12 to 24 hours in MBA-MB468 cells (Fig. 2F). Considering thatcell-cycle progression is generally about 12 to 24 hours, cell deathby QNC appears to coincide with cell-cycle progression, in par-ticular, S-to-M phase. In other cell lines (e.g., ACHN and MDA-MB231), cell death was detected from 32 to 36 hours and wouldpotentially couple with cell cycle (2 cycle or more; Fig. 2F).However, MCF7 did not show a stair-like dropped-down phase,but rather a gradual decrease of cell viability was detected. Toconfirm a potential cell-cycle–dependent cell death, cell viability

was measured after cell-cycle inhibition at M-phase using Taxol.Blocking cell-cycle progression in M-phase diminished the QNC-induced cell death (Supplementary Fig. S3F). These results sug-gested that QNC-induced cell death is potentially regulated in acell-cycle phase-dependent manner. Indeed, previous literatureshowed that QNC can promote S-G2–M cell-cycle progression inp53 intact lung cancer cell lines with alteration of cell cycle andDNA repair-related gene expression via microarray analysis (13).

Reduction of checkpoint kinase 1/2 activity by QNCThe potential for QNC-induced cell death to be dependent on

p53 and the cell cycle prompted further investigation using theTCGA database (through www.cbioportal.org; ref. 25). In severalkinds of human cancers, upregulation of p-Chk1/2 or total-Chk2inp53-mutated cancerswas found (Supplementary Fig. S4A). Thissuggested that Chk1/2 activitiesmay be required for proper cancercell growth under p53-inactivated condition. Indeed, Chk1/2 arekey regulators of S-G2–M phase and are also well conserved insingle cell organisms to mammalian systems (26–28). In addi-tion, it has been proposed that inhibition of cell-cycle checkpointkinases can enhance the sensitivity to anticancer drugs under p53-deficient condition (29–31). To explore the possibility, cell via-bility was determined in p53-deficient and -proficient cells afterelimination of Chk1/2. Although single knockdown of Chk1 orChk2 did not induce cell death, cotreatment of siRNAs againstChk1 andChk2 (Supplementary Fig. S4B) did induce cell death inp53-null HCT116 (Supplementary Fig. S4C). Thus, the effect ofQNC on Chk1/2 activity was determined. As shown in Fig. 3A,both p-Chk1 and 2 were suppressed by QNC in UV-treatedconditions. Similar results were obtained in Adriamycin-treatedHCT116 cells (Supplementary Fig. S5A). Next, a dose-dependentreduction of activated p-Chk1/2 was detected upon QNC treat-ment (Fig. 3B).Of note, a rapid and selective reduction of p-Chk1/2 without an obvious reduction of total Chk1/2 was seen(Fig. 3C). Reduction of p-Chk1/2 in the human lung epithelialcell line, WI-26, transformed by Large T was also observed(Supplementary Fig. S5B). These results indicated that QNCcould reduce p-Chk1/2. Next, the engagement of p53 on QNC-induced Chk1/2 reduction was assessed. Compared withHCT116 cells, p-Chk1 and 2 were rapidly reduced in HCT116p53�/� cells (Fig. 3D). To confirm this, p-Chk2 was measured inMCF7 cells. Although 50 mmol/L of QNC could suppress p-Chk2,reduction of p-Chk2 was detected from 10 mmol/L of QNC onlyin PFT-a–treated cells (Supplementary Fig. S5C). Similarly, the

Figure 3.Anticancer effect of Quinacrine via Chk1/2. A, Among various antiprotozoa drugs, QNC notably suppressed endogenous p-Chk1/2 protein levels althoughChk1/2 was activated by UV treatment. HCT116 p53þ/� and isogenic HCT116 p53�/� cells were pretreated with or without UV. Subsequently, cells weretreated with indicated chemicals of 10 mmol/L for 4 hours and immunoblot was performed. Actin was used for loading control. B, QNC induceddose-dependent reduction effect and can selectively eliminate activated p-Chk1/2 by Adriamycin treatment. Chk2-GFP was transfected to HCT116 cells.Subsequently cells were pretreated with or without Adriamycin and were treated with QNC of indicated dosage for 6 hours. Then immunoblot analysiswas performed. C, QNC induced time-dependent reduction effect and can selectively eliminate activated p-Chk1/2 by Adriamycin treatment. HCT116p53þ/�/ cell was pretreated with Adriamycin and measured the expression of p-Chk1/2 and total Chk1 following 5 mmol/L QNC-incubated time. D, In responseto QNC, p-Chk2 expression was more rapidly reduced in HCT116 p53�/� than HCT116. Cells were treated with QNC of indicated dosage for 4 hours.Then immunoblot analysis was performed. E, Unlike p53 WT, abnormal status of p53 could not block the QNC-induced cell death. Wild-type p53, p53 R175H,and Chk2-GFP were transfected to HCT116 P53�/� cells and subsequently QNC were treated of indicated dosage for 4 hours. Then immunoblot analysiswas performed. F, QNC can reduce exogenous Chk2 expression but not Mre11 and p18, upstream regulator of Chk2 signaling. Indicated vectors weretransfected to HEK293 cells and QNC (10 mmol/L) was treated for 4 hours. Only Chk2 expression was decreased by QNC. G, Chk2 reduction bytreatment of QNC was detected by immunofluorescence. After transfected with Chk2-GFP (green), Cells were stained with DAPI (blue) to visualize thenucleus. H, Chk2 reduction by treatment of QNC was inhibited by pretreatment with the proteasome inhibitor, MG132. After transfected with Chk2-GFP,cells were pretreated with or without MG132 and were treated with QNC of 5 mmol/L for indicated time. Subsequently immunoblot was performed.I, Treatment with QNC promoted ubiquitination of Chk1/2. After transfected with ubiquitin and Chk2-GFP or Chk1-GFP, cells were pretreated with orwithout MG132 and were treated with QNC of 5 mmol/L for 2 hours. Subsequently, immunoprecipitation with a GFP antibody was performed.





www.cbioportal.org


rapid reduction of p-Chk2 in PFT-a–treated MCF7 cells couldobserved (Supplementary Fig. S5C). Finally, transfection ofwild-type p53, but not mutant p53, could block the reductionof Chk2 (Fig. 3E) and QNC-induced cell death (SupplementaryFig. S5D). These results implied that QNC can preferably elim-inate activated Chk1/2.

QNC promoted Chk1/2 destabilizationTo address how QNC suppresses p-Chk1/2, the expression of

Chk2 was measured. QNC could reduce Chk2 expression in p53-inactivated HEK293 cells (Fig. 3F). However, MRE11, anupstream regulator of Chk2 (32, 33), or p18, an activator of ATMkinase (34), were not reduced by QNC (Fig. 3F). Reduction ofChk2 could also be observed via IF (Fig. 3G). However, anotherantiprotozoan drug, primaquine (PQ) did not suppress Chk2expression (Supplementary Fig. S5E), indicating that reduction ofChk2 may be specific to QNC. To understand how Chk2 wasreduced by QNC, protein stability was evaluated. QNC-inducedChk2 turnover was inhibited by the proteasome inhibitor,MG132 (Fig. 3H). However, GFP (control protein), RAD50, andMRE11 expression was not reduced by QNC (Fig. 3H) suggestingthat QNC might selectively reduce the protein half-life of Chk2.Indeed, expression of MRE11 or RAD50 were not altered by QNCin the presence of PFT-a (Supplementary Fig. S5F) and anMRE11inhibitor did not abolish QNC-reduced cell viability (Supple-mentary Fig. S5G). Chk1 expression was also reduced in a QNCdose-dependent (Supplementary Fig. S6A) and time-dependent(Supplementary Fig. S6B) manner. To confirm the reduction ofChk1/2 via proteasome-mediated degradation, ubiquitinationassays were performed. Treatment of QNC could promote ubi-quitination of Chk1/2 (Fig. 3I), without an overall increase intotal ubiquitination (Supplementary Fig. S6C). These resultssuggest that QNC selectively promotes Chk1/2 degradation viathe proteasome. Under p53-deficient conditions, Chk1/2 wouldbe rapidly phosphorylated because of rapid cell-cycle progression.Thus, QNC might induce reduction of total Chk1/2, althoughQNC only suppressed p-Chk1/2.

QNC promote Chk1/2 degradation using b-TrCPTo further investigate the mechanism, Chk2 expression was

measured in several E3 ligase transfected cells. Among the testedE3 ligases, b-TrCP, CDC4, and Siah1 reduced Chk2 expression(Fig. 4A). Indeed, transfection of b-TrCP and CDC4 promotedChk2 degradation in response toQNC (Supplementary Fig. S7A).In contrast, Siah1 eliminated Chk2 expression, regardless of QNCtreatment (Supplementary Fig. S7A). Next, the interactionbetween E3 ligases and Chk1 was evaluated via GST-pulldown.QNC as well as PQ enhanced the binding between Chk1 andb-TrCP or CDC4; while Siah1 did not associate with Chk1 (Fig.4B). This result indicated that Siah1 is not responsible for theobserved QNC-induced Chk1 reduction. To determine which E3ligase is essential for QNC-induced Chk1/2 reduction, we firstblocked the CDC4 activity using a dominant negative CDC4(CDC4-DN). However, CDC4-DN did not completely block theQNC-induced Chk2 reduction (Supplementary Fig. S7B),although it could increase basal GFP-Chk2 expression (Supple-mentary Fig. S7A and S7B). Next, we knockdowned b-TrCP andchecked the Chk1/2 expression. Elimination of b-TrCP blockedthe QNC-induced reduction of exogenous Chk2 (Fig. 4C) as wellas endogenous Chk1/2 (Supplementary Fig. S7C). DN-b-TrCPalso blocked the QNC-induced Chk2 reduction (Supplementary

Fig. S7D), implying that b-TrCP could be responsible for QNC-induced Chk1/2 reduction. To confirm this, the pulldown assayand immunoprecipitation assay were performed again usingrecombinant b-TrCP and b-TrCP antibody. QNC increased thebinding between Chk1/2 and b-TrCP (Fig. 4D; SupplementaryFig. S7E) in a dose-dependent manner (Supplementary Fig. S7F).However, inhibition of GSK3b, upstream kinase for b-TrCP sub-strate such as b-catenin, did not block the QNC-induced Chk1/2reduction (Supplementary Fig. S7G and H). These results implythat Chk1 and 2 are not canonical targets of b-TrCP. In ourprevious results, it was demonstrated that PQ also promoted thebinding of Chk1 and b-TrCP (Fig. 4B)without cell death (Fig. 1A–D). Thus, PQ could be competitive inhibitor against QNC, if itbinds to same site. Importantly, QNC and PQ have a very similarchemical structure (Supplementary Fig. S1A). Consistent with thishypothesis, pretreatment of PQ blocked the QNC-induced p-Chk2 reduction (Fig. 4E). However, high concentrations of QNCcould overcome this PQ effect (Fig. 4F). These results suggest thatQNC is very selectively anchored to Chk1/2 and promote protea-some-dependent degradation.

Antitumor effect of QNC in intestinal tumor modelTo explore the antitumor effects of QNC in vivo, an intestinal

cancer mouse model (VP mice) was created Villin-Cre;p53þ/LSL-

R172H (Supplementary Fig. S8A). After 4 months, QNC wasinjected (20 mg/kg, i.p.) three times per week for 22 weeks intothese VP mice (Supplementary Fig. S8A). Gross morphology andbody weight did not show detectable differences between controlandQNC-injectedmice (Supplementary Fig. S8B). From the PET-CT analysis, tumors were detected in control mice (VP13 andVP17; Fig. 5A) but disappeared in QNC-treated mice (Fig. 5A;right). Indeed, tumor numbers detected by PET-CT were reducedby QNC-treatment (Fig. 5B). Intestinal histology was analyzedand tumors were observable from 3 to 5 sites in the control mice(Fig. 5C; Supplementary Fig. S9). Indeed, invasive or diffusedcancer cells were detected in control VP mouse tissues (Fig. 5C;Supplementary Fig. S9A–S9D). In contrast, QNC-treated miceonly showed overgrowing intestinal epithelial cells (Fig. 5C;Supplementary Fig. S10A–S10D). QNC treatment also reducedthe cell proliferation of intestinal villi (Fig. 5D). These resultsstrongly support the notion that QNC would be a plausibletreatment strategy for p53-negative cancers.

DiscussionIn this study, it was found that QNC, a drug previously used to

fight malaria and protozoa (10), possesses a very interestinganticancer property though inhibition of Chk1/2 under p53-deficient or inactivated conditions. As mentioned above, a newstrategy for drug development is the repurposing of old chemicalsthrough mining of new biological effects in other diseases (35).Our purpose is tofindnewanticancer drugs fromold drugs,whichis a very economic and smart approach for drugs already approvedby FDA with reported toxicities. Importantly, preclinical investi-gation is not generally required for application of clinical trials.With regard to QNC, it has been widely used to combat malariasince World War II and is now used for anti-Giardiasis (10). Inaddition, QNC has been repositioned for prion-related neurodi-seases (5), anti-inflammation (e.g., SLE; ref. 4), and cancer (6).

Concerning the anticancer effects of QNC, previous literaturesuggested that activation of p53 and inhibition of NF-kB aremain

Park et al.





Figure 4.

Quinacrine enhances b-TrCP activity. A, Chk2 expression test in several E3 ligase transfected cells. Among them, only b-TrCP, CDC4, and Siah1 reduced theChk2 expression. Vectors were transfected to HEK293 cells for 24 hours. Then immunoblot analysis was performed. B, Chk1 can bind to b-TrCP andCDC4, but not Siah1. And both QNC and Primaquine (PQ) could enhance binding affinity of Chk1-bead with b-TrCP and CDC4. Cell lysates transfectedwith indicated vectors were incubated with GST-Chk1 bead and indicated chemicals. Bound proteins were eluted and immunoblot analysis was performed.C, Elimination of b-TrCP by RNA interference blocked the QNC-induced reduction of Chk2. Vectors and siRNA were transfected to HEK293 cells andQNC (5 mmol/L) was treated for indicated time. Then immunoblot analysis was performed. D, Chk1/2 binding to Ni-b-TrCP bead was enhanced by QNC. Celllysates transfected with indicated vectors were incubated with Ni-b-TrCP bead and QNC. Bound proteins were eluted and immunoblot analysis wasperformed. E and F, Pretreatment of PQ blocked the QNC-induced p-Chk2 reduction. PQ was used as binding competitor of QNC. E, Cells werepretreated with Adriamycin and then were treated with PQ. Subsequently QNC were treated of 10 mmol/L for indicated time. Then immunoblot analysiswas performed. F, Chk2-GFP was transfected to HCT116 cells. Subsequently, cells were pretreated with or without PQ and were treated with QNC ofindicated dosage for 6 hours. Then immunoblot analysis was performed.






Figure 5.

Anticancer effect of Quinacrine in p53 mutant cancer model. A, Through the PET/CT analysis, QNC suppressed the tumor formation in the Villin-Cre;p53þ/LSL-R172H mouse model. Compared with control mice (VP13 and VP17), QNC-injected mice (VP15, VP20, and VP21) did not show tumor formation. Thearrow indicates the localization of the tumor. B, the number of tumors, based on PET-CT image. C, H&E staining of Villin-Cre;p53þ/LSL-R172H mouseintestines from control and QNC-injected mice. Compared with control mice (VP13 and VP17), QNC-injected mice (VP15, VP20, and VP21) did not showtumor regions. Cancer regions of control mice and hyperplastic regions of treated mice were presented as enlarged images. D, Immunohistochemistry(IHC) staining for Ki-67 and hematoxylin counterstain in Villin-Cre;p53þ/LSL-R172H mouse intestines of control and QNC-injected mice. The intestines of controlgroup mice had more Ki-67 staining than QNC-injected mice.

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pathways (6, 7). Considering the chemical structure and itsintercalating property into DNA, p53 activation by QNC isvery plausible hypothesis. However, at effective concentration(5–10 mmol/L), p53 was not induced (Fig. 3D). Rather, in ourhands, QNC promoted cell death under p53-deficient conditions(Fig. 2). In addition, QNC did not suppress NF-kB activity.Although, while we and others have already observed the anti-cancer activity of QNC, the underlying mechanism is not clearlydemonstrated.

The current results revealed that QNC induced cell deathunder p53-deficient conditions via rapid degradation ofChk1/2 (Fig. 6). Chk1/2 is a well-conserved cell-cycle regu-lator from single-cell organisms, such as yeast, to humans andis speculated to perform the basic cell-cycle regulation. Incontrast, the physiological role of p53 in multicellular sys-tems appears to be important for maintaining cellularhomeostasis and determination of which cell will die orsurvive (36). Thus, under p53-intact conditions, Chk1/2activity seems to be not essential. However, in cancer cellsthat try to escape the systematic control and move to poten-tially behave more like a single-cell system, Chk1/2-mediatedminimum cell-cycle regulation would be critical for cellsurvival. Our results suggest that elimination of Chk1/2 willlead the cancer cells to a more chaotic cell-cycle progressionand end in cell death. Which is why we believe that QNCcan work on protozoa and cancer. Indeed, we did not findtriple-mutant cancers (i.e., p53-;Chk1-;Chk2-) in the humancancer cell lines tested.

As p53-mediated cell regulatory systems may be collapsed atthe end stage of cancer or in relapsed cancers, which ATM/ATR-mediated DNA repair systems and Chk1/2 activity are elevated,QNC-induced Chk1/2 degradation would be a useful approachfor therapeutic effect.

This study reveals that QNC induces cell death under p53-inactivated conditions via rapid degradation of Chk1/2. As QNChas been used clinically after determination of proper treatmentprotocol, it could be used as a cancer drug, in particular, p53-deficient end-stage or relapsed human cancers including GBM,SCLC, and NF2-deficient cancers.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: Y. Jung, B.-J. ParkDevelopment of methodology: M.-H. Yoon, T.-G. Woo, H.-Y. Lee, B.-J. ParkAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Park, H.-Y. LeeAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Park, J.-H. Cho, H.-Y. Lee, B.-J. ParkWriting, review, and/or revision of the manuscript: S. Park, A.-Y. Oh,B.-J. ParkAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. Park, J.-H. Cho, S. Kang, Y. JungStudy supervision: B.-J. Park

AcknowledgmentsThis research was supported by Basic Science Research Program through

the National Research Foundation of Korea (NRF) funded by the Ministryof Science and ICT (NRF-2017R1A2B2007355; B.-J. Park) and by Ministryof Education (NRF-2017R1A6A3A11035837; A.-Y. Oh).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received September 14, 2017; revisedDecember 22, 2017; acceptedMarch 8,2018; published first March 15, 2018.

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Figure 6.

The schematic diagram illustrates proposed mode of action of QNC. QNC could reduce p-Chk1/2 protein level by enhancing b-TrCP activity. Thus, especially underp53-deficient conditions, QNC can induce cancer cell death.






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2018;16:935-946. Published OnlineFirst March 15, 2018.Mol Cancer Res Soyoung Park, Ah-Young Oh, Jung-Hyun Cho, et al. CancersSelective Suppression of p-CHK1/2 in p53-Negative Malignant Therapeutic Effect of Quinacrine, an Antiprotozoan Drug, by

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