Research Article Identification of Target Genes Involved

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 502568 8 pageshttpdxdoiorg1011552013502568

Research ArticleIdentification of Target Genes Involved inthe Antiproliferative Effect of Enzyme-Modified GinsengExtract in HepG2 Hepatocarcinoma Cell

Sung-Il Jang1 Yeon-Weol Lee1 Chong-Kwan Cho1 Hwa-Seung Yoo1 and Jun-Hyeog Jang2

1 East-West Cancer Center Dunsan Oriental Hospital of Daejeon University Daejeon 302-122 Republic of Korea2Department of Biochemistry Inha University School of Medicine Incheon 400-712 Republic of Korea

Correspondence should be addressed to Hwa-Seung Yoo altyhsdjukr and Jun-Hyeog Jang juhjanginhaackr

Received 1 June 2013 Accepted 20 August 2013

Academic Editor Qing He

Copyright copy 2013 Sung-Il Jang 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

Ginsenosides are ginseng saponins which are the major biologically active components of Panax ginseng often metabolizedby intestinal bacteria into more effective forms In this study we found that the antiproliferative activity of ginseng increasedafter enzymatic processing of ginseng saponin (50 inhibitory concentration [IC

50] gt30 120583gmL) which may be the result of the

accumulation of minor saponins such as Rh1 Rg3 compound K and PPT constituents in ginseng saponin Using the AgilentPrimeViewHumanGene Expression Array we found that the expression of several genes involved in apoptosis (caspase-4 AnnexinA2 HSPA9 AIFM1 UQCRC2 and caspase-7) were increased in HepG2 human hepatocarcinoma cells after their treatment withenzyme-modified ginseng extract (EMGE) Furthermore several genes implicated in cell cycle progression (CDCA3 CDCA8CABLES2 CDC25B CNNM3 and CCNK) showed decreased expression in HepG2 cells treated with EMGE Finally from flowcytometric analysis we found that EMGE-treated HepG2 cells showed increased apoptotic sub-G1 population (24) comparedwith that observed in DMSO-treated control cells (16) Taken together our results suggest that EMGE induces anticancer activitythrough the induction of apoptosis-related genes and cell cycle arrest via decreased expression of cell cycle regulatory genes

1 Introduction

Panax ginseng (Korean ginseng) has been used as a traditionalmedicine for the treatment of cancer and has been shownto inhibit tumor cell proliferation and tumor growth toinduce differentiation and apoptosis and to inhibit cancer cellinvasion [1ndash4] The major biologically active components ofPanax ginseng are a series of saponin glycosides collectivelyknown as the ginsenosides a group of steroidal saponinsOver 50 ginsenosides have been isolated and identified fromginseng saponins to date [5]

Ginsenosides are characterized by a steroid-like skeletonconsisting of 4 trans-rings with modifications that dependon the type (eg glucose maltose and fructose) and numberof sugar moieties as well as the sites of attachment of thehydroxyl group (eg C-3 C-6 or C-20) Based on chemicalstructural characteristics ginseng saponins can be dividedinto protopanaxadiol and protopanaxatriol groups except for

ginsenoside Ro which is derived from an oleanolic group Inthe protopanaxadiol group sugars are attached to the 120573-OHat C-3 and another ndashOHat C-20 as found in Rb1 Rb2 Rc RdRg3 and Rh2 In the protopanaxatriol group sugar residuesare attached to the 120572-OH at C-6 with another ndashOH at C-20examples being Re Rg1 Rg2 Rh1 and Rf

Recent studies have indicated that intestinal bacteriacleave the oligosaccharide connected to the aglycon stepwisefrom the terminal sugar to form the ginsenoside metabo-lite 20S-protopanaxadiol 20-O-120573-d-glucopyranoside (com-pound K) after oral administration of ginseng extract inboth humans and rats [6] Wakabayashi et al have revealedthat the antitumor activities of ginsenoside following oraladministration are attributable to the metabolites formed bycolonic bacteria-mediated deglycosylation [7] In additioncompound K is known to exhibit cytotoxicity by the induc-tion of apoptosis and cell cycle arrest at the G

1phase by a

caspase-dependent pathway via mitochondrial disruption in

2 Evidence-Based Complementary and Alternative Medicine

tumor cells and by reversing multidrug resistance in tumorcells [8ndash10] The combined treatment of compound K andradiation enhances human lung cancer cell death [8 11]

Various transformation methods including mild acidhydrolysis enzymatic conversion and microbial conversionhave been tested for their ability to transform ginsenosidesto enhance their physiological effectiveness In this studywe investigated the antiproliferative effects and possiblemechanism of action of EMGE that were transformed byenzyme treatment by using the human hepatocarcinoma cellline HepG2

2 Materials and Methods

21 Cell Culture HepG2 cells were maintained in DulbeccorsquosModified Eaglersquos Medium (DMEM) supplemented with 10heat-inactivated fetal calf serum (Invitrogen Carlsbad CA)100 unitsmL penicillin G sodium 100120583gmL streptomycinsulfate and 025120583gmL amphotericin B (Invitrogen Carls-bad CA) in a 5 CO

2atmosphere at 37∘C Confluent cells

were detached with 025 trypsin-EDTA for 5min andaliquots were subcultured Human hepatoma HepG2 cellswere purchased from the Korean Cell Line Bank (KCLBSeoul Republic of Korea)

22 Preparation of Enzyme-Modified Ginseng Extract(EMGE) The root of regular ginseng (4 years old) waspurchased from GampV Korea A total of 20 g of pulverizedginseng root powder were suspended in 380mL of distilledwater and then sterilized by heating at 121∘C for 15min Toreduce the complexity of components in ginseng root theextract was fractionated via extraction with water methanoland butanol Among the fractions ginseng butanol extractcontained the compounds with specific anticancer activityIn addition the suspension was treated with aliquotsof filter-sterilized commercial enzymes (laminarinase100mg pectinase 100mg) with an equimolar ratio (1 1specific activity unit) For these treatments the mixturewas incubated at 40∘C for 2 days and then evaporated todryness at 60∘C These enzyme-modified ginseng powderswere then suspended in 400mL of 80 (vv) methanol andsubjected to ultrasonication for 5min followed by filtrationthrough Whatman No 2 Filter Paper The wet powder onthe filter paper was then collected suspended treated in anultrasound bath and filtered in the same manner once againafter which the two filtrates were combined and evaporatedto dryness at 50∘CThemethanol extract dissolved in 200mLof distilled water was washed with 200mL of ethyl acetatein a separation funnel and then extracted with 200mL ofbutanol Next the butanol extract was evaporated to drynessat 50∘C and then dissolved to a concentration of 10 (wv)in 70 ethanol

23 HPLC Analysis of Ginsenosides Standard ginsenosidesincluding Rg1 Re Rf Rh1 Rb1 Rc Rb2 f1 Rd Rg3(S)Rg3(R) PPT Com K Rh2 and PPD were obtained fromChromaDex Inc (Irvine CA) and analyzed using an AcquityUPLC system (Waters Milford MA) with an Acquity BEH

C18HPLC columnThemobile phase consisted of solution A

(CH3CN HPLC-grade JT Baker NJ) and solution B (Milli-

Q H2O Millipore MA) The flow rate was set at 06mLmin

and the injection of volume was fixed at 2 1 The separationof ginsenosides was performed using an isocratic gradientof 100 solution A for 27min The column temperaturewas maintained at room temperature during the separationand the UV diode array detection was set at 203 nm Toconfirm a reliable retention time of sample ginsenosidethe individual ginsenosides were identified and quantifiedbased on comparison with the retention times of standardginsenosides

24 Cell Viability Assay The effect of EMGE on cell viabilitywas determined using the 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazoliumbromide (MTT a yellow tetrazole) assaywhich quantifies the number of viable cells Cells wereseeded (2 times 104 cellswell) in 24-well flat-bottomed plates(Nunc EU) After incubation at 37∘C for 24 h the mediumwas replaced with EMGE at the appropriate concentrationsControl cells were treatedwithDMSOat a final concentrationof less than 02 After further 24 h incubation cells werewashed 3 times with Dulbeccorsquos phosphate-buffered saline(DPBS) and 500 120583L MTT solution (5mgmL in PBS) wasadded to each well After incubating for 4 h the mediumwas removed and 200 120583L of formazan (crystals dissolved inDMSO) was added to the cells The absorbance of each wellwas read at 540 nm using a microplate reader

25 RNA Extraction and cDNA Synthesis Total RNA wasextracted using the Easy-spin RNA Extraction kit (iNtRONSeoul Republic of Korea) according to the manufacturerrsquosinstructions The purity of RNA was assessed by measuringthe absorption at 260 and 280 nm (values of the ratio of119860260119860280

of 19ndash21 were considered acceptable) and byethidium bromide staining of 18 S and 28 S RNA by gelelectrophoresis RNA concentrations were determined fromthe 119860

260 Two micrograms of total RNA were reverse-

transcribed in a 20 120583L reaction mixture containing 50 unitsof SuperScript II reverse transcriptase (Invitrogen Carls-bad CA) 5 120583M DTT 40 units of RNaseOUT recombinantribonuclease inhibitor (Invitrogen Carlsbad CA) 05120583Mof random hexanucleotide primers and 500120583M of dNTPmixture The reverse transcription reaction was carried outat 50∘C for 60min The reaction was terminated by heatingthe reaction mixture at 70∘C for 15min and the cDNA wasstored at minus20∘C

26 Quantitative Real-Time Reverse Transcriptase PolymeraseChain Reaction (RT-PCR) Overexpression of the targetgenes was confirmed by quantitative real-time RT-PCR Allreal-time PCR analyses were performed on an ABI Step OneReal-time PCR system (Applied Biosystems) Each reactioncontained 01 120583M of each primer 10 120583L of 2x SYBR GreenPCRmastermix (includingAmpliTaqGoldDNApolymerasewith buffer dNTPs mix SYBR Green I dye ROX dyeand 10mm MgCl

2Applied Biosystems) and 1 120583L of the

template cDNA in a 20120583L total reaction volume The typical

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Primer sequences for real-time RT-PCR experiments

Genes Sense (51015840-31015840) Antisense (51015840-31015840)Caspase-4 GAAGGACAAACCCAAGGTCA AAGAGGCCACTTCCAAGGATCyp450 TGATAAGCACGTTGCAGGAG CAAAGCAAATGGCACAGATGCaspase-7 AGACCTCATCCGGTCAAGTG TTATCAGACAGCGCAACAGCCyclin M GAAGCGGAAGGAGGAGTTCT CGGGCTGAATACATCCACTTCyclin K GACATCTGCCACCAAATCCT GCACTTGTGGTGTAGGCTGAGAPDH TGGAAGGACTCATGACCACA TTCAGCTCAGGGATGACCTT

Table 2 HPLC analysis of EMGE

(mgg)Rg1 Re Rf Rh1 Rb1 Rc Rb2 F1 Rd Rg3 ComK Rh2

Control (ginseng) 92 72 25 mdash 77 47 23 20 06 mdash mdash mdashEMGE 06 10 mdash 27 27 mdash mdash mdash mdash 95 47 27

amplification program included activation of the enzyme at94∘C for 10min followed by 40 cycles of denaturation at94∘C for 15 s then annealing and extension at 60∘C for 1minThe 119862

119879(cycle threshold) value for each gene was determined

using the automated threshold analysis function of the ABIinstrument and normalizing to 119862

119879(G3PDH) to obtain d119862119879(=

119862119879(G3PDH) minus 119862119879(test)) The difference of 119899 between the 2119862

119879

or d119862119879values indicates a 2119899-fold difference in the amount

of the target sequence between the 2 cDNA samples beingcompared The primers used in quantitative PCR are shownin Table 1

27 Microarray Assay Gene expression was analyzed withAgilent PrimeView Human Gene Expression Array contain-ing 36000 genes transcript and variants via Unigene anno-tation (Affymetrix Santa Clara CA) which are comprisedof over 45000 probe sets representing approximately 38500well-characterized human genes For each gene 11 pairs ofoligonucleotide probes were synthesized in situ on the arraysBiotinylated cRNA was prepared according to the standardAffymetrix protocol from 100 ng of total RNA (ExpressionAnalysis Technical Manual 2001 Affymetrix) Followingfragmentation 12 120583g of aRNAwas hybridized for 16 h at 45∘Con a GeneChip Human Genome Array GeneChips werewashed and stained in the Affymetrix Fluidics Station 450GeneChips were scanned using the Affymetrix GeneChipScanner 3000 7G The data were analyzed with MicroarraySuite version 50 using theAffymetrix default analysis settingsand global scaling as the normalizationmethodThe trimmedmean target intensity of each array was arbitrarily set to 100The normalized and log transformed intensity values werethen analyzed usingGeneSpringGX 1151 (Agilent Technolo-gies CA) Fold-change filters included the requirement thatthe genes be present in at least 200 of controls for geneswith upregulated expression and less than 50 of controlsfor genes with downregulated expression Hierarchical clus-ter analysis using GeneSpring GX 1151 (Agilent technolo-gies CA) identified clustered groups as those that behavedsimilarly across experiments The clustering algorithm wasEuclidean distance with average linkage

28 Cell Count and Flow Cytometry Analysis Cell countswere performed using a hemocytometer Approximately 1 times106 HepG2 cells were suspended in 100 120583L of PBS and 200120583Lof 95 ethanol was added while vortexing The cells wereincubated at 4∘C for 1 h washed with PBS and resuspendedin 250120583L of 112 sodium citrate buffer (pH 84) togetherwith 125120583g of RNase Incubation was continued at 37∘Cfor 30min The cellular DNA was then stained by applying250120583L of propidium iodide (50 120583gmL) for 30min at roomtemperatureThe stained cells were analyzed by fluorescence-activated cell sorting (FACS) flow cytometry (FACScannerBecton Dickinson) for relative DNA content based on redfluorescence

3 Results

31 Chromatographic Analysis of Ginseng Saponin during theEnzyme Treatment Ginseng saponins (ginsenosides) havebeen regarded as the principal components responsible forthe pharmacological activities of ginseng and a large numberof ginsenoside derivatives have been identified in Panaxginseng [5] Recently cytotoxic effects of minor saponinssuch as Rh1 and Rh2 on the growth of various cancercells and also inhibitory effects of human intestinal bacterialsaponin metabolites such as compound K and protopanax-atriol (PPT) on the growth invasion and migration oftumor cells have been reported [12ndash16] High-performanceliquid chromatography (HPLC) analysis was performed todetermine the changes in chemical constituent in ginsengsaponin following enzyme treatment HPLC results showedthe appearance of minor ginsenosides (Rh1 Rg3 and com-pound K) indicative of EMGE (Table 2)

32 Antiproliferative Effects of EMGE on HepG2 Hepatocarci-noma Cells Ginsenosides have been shown to inhibit tumorcell proliferation and tumor growth and to induce differenti-ation and apoptosis [17] To determine the antiproliferativeeffects of EMGE on HepG2 human hepatocarcinoma cellswe performed the MTT assay EMGE inhibited HepG2 cellgrowth in a dose-dependentmanner at 148 222 333 50 and


Table 3 Genes with highly upregulated expression due to EMGE in HepG2

Symbol Gene description Fold change GenBank IDCYP1A1 Cytochrome P450 family 1 subfamily A 200 NM000499HGD Homogentisate 12-dioxygenase 153 NM000187AQP3 Aquaporin 3 145 NM004925IL8 Interleukin 8 143 NM000584ANXA2 Annexin A2 111 NM001002857HSPA9 Heat shock 70 kDa protein 9 105 NM004134CASP4 Caspase-4 104 NM001225SMAD2 SMAD family member 2 98 NM001003652MAPK6 Mitogen-activated protein kinase 6 93 NM002748KLF6 Kruppel-like factor 6 90 NM001160124CTNNB1 Catenin (cadherin-associated protein) beta 1 88 kda 83 NM001098209AIFM1 Apoptosis-inducing factor mitochondrion-associated 1 78 NM001130846PPHLN1 Periphilin 1 75 NM001143787UQCRC2 Ubiquinol-cytochrome c reductase core protein II 74 NM003366IL6ST Interleukin 6 signal transducer 64 NM001190981IFITM1 Interferon-induced transmembrane protein 1 51 NM003641CASP7 Caspase-7 50 NM001227

0

20

40

60

80

100

120

0 438 658 987 148 222 333 50 100

Cel

l pro

lifer

atio

n ac

tivity

()

Concentration (120583gmL)

ControlMGX

Figure 1 Antiproliferative effects of EMGE on human HepG2hepatocarcinoma cells Cell viability at the indicated concentrationsof EMGE at 24 hwas assessed by theMTT testThe treated cells wereused for total RNA isolation followed by microarray analysis

100 120583gmL at 24 h The concentration at which 50 growthinhibition (IC

50) occurredwas 30 120583gmL (Figure 1)However

exposure of HepG2 cells to 30120583gmL nontreated ginsengsaponin extract showed less growth inhibition (approxi-mately 20) Thus these results suggest that enzymaticprocessing of ginseng saponin increased its antiproliferativeeffects on HepG2 cells

33 Identification of Differentially Expressed Genes inHepG2 Human Hepatocarcinoma Cells Treated with EMGEAlthough ginseng has been used as a traditional medicinefor the treatment of cancer the underlying mechanisms forthe varying activities observed following exposure to gin-seng are largely unclear To identify relevant alterations ofgene expression associated with treatment of EMGE we

analyzed the gene expression profiles of HepG2 cells byusing Affymetrix Gene Chip arrays containing more than38500 genes From the results of Affymetrix Gene Chip arrayanalysis we detected 31 differentially expressed genes exhibit-ing at least a 5-fold change following treatment with EMGEOf these 17 had significantly upregulated expression and 14had significantly downregulated expression when comparingtreatment with the EMGE versus the control treatment(Tables 3 and 4)

Several genes involved in apoptosis (caspase-4 AnnexinA2HSPA9AIFM1UQCRC2 and caspase-7) were expressedat higher levels following treatment with EMGE (Table 3)The expression of genes involved in the regulation of cellsignaling (MAPK6 SMAD2 IL6ST KLF6 and IFITM1) alsoincreased Furthermore several genes implicated in cell adhe-sion (CTNNB1) transport (AQP3 IFITM1) and the immuneresponse (IL8) showed increased expression in HepG2 hu-man hepatocarcinoma cells that were treated with EMGE

In contrast 14 genes showed reduced expression inHepG2 human hepatocarcinoma cells treated with EMGEThere was an overrepresentation of members of genesinvolved in cell cycle progression (CDCA3 CDCA8CABLES2 CDC25B CNNM3 and CCNK) and cellularmetabolism (CS ALDH3B1 and NPRL3) Table 4 lists thenames of genes exhibiting a reduction in expression wherethe difference was gt5-fold This group of genes that wereexpressed very differently may serve as an important targetin EMGE-mediated anticancer activity in human hepatocar-cinoma cells

34 Real-Time RT-PCR Validation of Microarray Data Toconfirm the results obtained using microarrays we choose todetermine if the expression of 5 target genes was regulatedby EMGE in HepG2 cells by quantitative real-time RT-PCR analysis These genes included 3 genes with highlyupregulated expression (cyp450 caspase-4 and caspase-7)


Table 4 Genes with highly downregulated expression due to EMGE in HepG2

Symbol Gene description Fold change GenBank IDCDCA3 Cell division cycle associated 3 minus84 NM031299CS Citrate synthase minus80 NM004077CDCA8 Cell division cycle associated 8 minus77 NM018101CABLES2 Cdk5 and Abl enzyme substrate 2 minus74 NM031215CDC25B Cell division cycle 25 homolog B minus66 NM004358CNNM3 Cyclin M3 minus62 NM017623ZNF362 Zinc finger protein 362 minus58 NM152493CCNK Cyclin K minus58 NM001099402RAD51AP1 RAD51 associated protein 1 minus58 NM001130862LEAP2 Liver expressed antimicrobial peptide 2 minus53 NM052971HEPACAM Hepatocyte cell adhesion molecule minus51 NM152722ALDH3B1 Aldehyde dehydrogenase 3 family member B1 minus50 NM000694NPRL3 Nitrogen permease regulator-like 3 minus50 NM001039476DOT1L DOT1-like histone H3 methyltransferase minus50 NM032482

and 2 with highly downregulated expression (Cyclin K andCyclin M) To accurately quantify the expression of these 5genes GAPDH expression was amplified and the mean datain each case was used to normalize the result

As shown in Figures 2 and 3 the expression level of the3 selected upregulated genes (cyp450 (73-fold) caspase-4 (6-fold) and caspase-7 (3-fold)) significantly increased upontreatment with EMGE Conversely the expression of the 2downregulated genes (Cyclin M (4-fold) Cyclin K (12-fold))decreased These results indicate that the real-time RT-PCRresults are highly consistent with the microarray data

35 Effect of EMGE on Cell Cycle Distribution and Apoptosisin HepG2 Hepatocarcinoma Cells To determine whetherEMGE has an effect on cell cycle arrest apoptosis orboth in human HepG2 hepatocarcinoma cells cells treatedwith DMSO or EMGE for 12 or 24 h were subjected toflow cytometric analysis after DNA staining Histograms offlow cytometric data are shown in Figure 4 Control cells(DMSO-treated) progressed through the cell cycle normallyIn contrast EMGE-treated HepG2 cells showed increasedapoptotic sub-G1 populations (24) compared with thatobserved in DMSO-treated control cells (16)These resultssuggest that EMGE induces antiproliferative effects inHepG2cells mainly through apoptotic mechanisms

4 Discussion

We found that the enzymatic processing of ginseng saponincould increase the content of active constituents and enhanceits anticancer activity presumably because of the productionof minor saponins such as Rh1 Rg3 compound K andPPT constituents in ginseng saponin In this study we usedcDNA microarray analysis to detect target genes involved inthe anticancer activity of EMGE Microarray (DNA chip) isa 2D array of microscopic DNA spots for high-throughputexpression screeningWe found that several genes involved inapoptosis (caspase-4 Annexin A2HSPA9 AIFM1 UQCRC2and caspase-7) as well as several genes involved in cell

signaling (MAPK6 SMAD2 IL6ST KLF6 and IFITM1) wereexpressed at higher levels after treatment with EMGE Fur-thermore several genes implicated in cell cycle progression(CDCA3CDCA8CABLES2CDC25BCNNM3 andCCNK)and metabolism (CS ALDH3B1 and NPRL3) exhibiteddecreased expression in HepG2 human hepatocarcinomacells treated with EMGE Thus the apoptosis-related geneswith upregulated expression and cell cycle-related genes withdownregulated expression could be important target genesinvolved in the anticancer activity of EMGE

In this study caspase-4 and caspase-7 which are com-ponents of the cell-intrinsic apoptosis machinery were sig-nificantly induced by EMGE Caspase-4 has a functionalrole in endoplasmic-reticulum-stress- (ERS-) induced apop-tosis in humans [18] Upregulation of caspase-4 has beendemonstrated to mediate apoptosis in human bronchialepithelial cells [19] human neuroblastoma SKN-SH cells andhuman esophageal squamous carcinoma EC109 [20] Thusthe induction of apoptosis-related genes such as caspase-4and caspase-7 in cells treated with EMGE may contribute tothe antiproliferative activity of EMGE

The cytochrome P450 1A1 (CYP1A1) is a member of thecytochrome P450 monooxygenase superfamily which playsan important role in xenobiotic metabolism as well as incarcinogenesis [21] In this study CYP1A1 level was signif-icantly increased after treatment with EMGE The CYP1A1enzymes mediate the bioactivation of carcinogens foundboth in the human diet and in the environment includingcompounds such as polycyclic aromatic hydrocarbons suchas 3-methylcholanthrene benzo(a)pyrene polyhalogenatedaromatic hydrocarbons and certain congeners of polyhalo-genated biphenyls [22] Our finding that the EMGE increasedthe expression of CYP1A1 is consistent with the previousresults showing the induction of CYP1A1 expression byginsenosides Rg1 and Rb1 [23]

Cell cycle progression is a tightly regulated processrequiring regulatory factors such as CDCs and cyclins Thuslack of cell cycle machinery molecules often causes cell cyclearrest leading to cell growth inhibition apoptosis or both


3

2

1

Control EMGE

Caspase-7Re

lativ

e exp

ress

ion

0

(a)

3

2

1

Control EMGE

Caspase-4

Relat

ive e

xpre

ssio

n

4

5

0

(b)

40

20

Control EMGE

CytP450

Relat

ive e

xpre

ssio

n

60

80

0

(c)

Figure 2 Quantitative real-time RT-PCR analysis of 3 genes with upregulated expression cyp450 caspase-4 and caspase-7

15

10

05

Control EMGE

Cyclin K

Relat

ive e

xpre

ssio

n

0

(a)

Control EMGE

Cyclin M15

10

05Relat

ive e

xpre

ssio

n

0

(b)

Figure 3 Quantitative real-time RT-PCR analysis of 2 downregulated genes Cyclin K and Cyclin M

Our results showing the decreased expression of severalgenes implicated in cell cycle regulation (CDCA3 CDCA8CABLES2 CDC25B CNNM3 and CCNK) in HepG2 cellstreated with EMGE suggest that the induction of cell cycle

arrest by EMGE may contribute to the antiproliferative acti-vity of EMGE

In summary we found that the anticancer activity ofginseng was increased by enzymatic processing of ginseng


Control

PISub-G1 16G1 618S 93G2M 271

200

160

120

80

40

00 200 400 600 800 1000

Cou

nts

(a)

EMGE

PISub-G1 24G1 516S 91G2M 153

200

160

120

80

40

00 200 400 600 800 1000

Cou

nts

(b)

Figure 4 FACS analysis of PI-stained nuclei of HepG2 cells treated with EMGE Cells were treated with DMSO (005) or EMGE (50120583gmL)for 24 h and cell cycle analysis was conducted as described in Section 2 Data are representative of 3 independent experiments

saponin whichmay be the result of the relative accumulationof minor saponins such as Rh1 Rg3 compound K andPPT constituents in ginseng saponin Moreover the resultsof microarray analysis suggest that EMGE induces anticanceractivity through the induction of apoptosis-related genesand the decreased expression of cell cycle regulatory genesHowever these results do not exclude the role of other genesthat may be involved in anticancer activity

Acknowledgments

This work was supported by the National Research Founda-tion Korea (NRF) Grant funded by the Korean Government(MOE) (2010-0022628 2013R1A1A2011375) and Inha Univer-sity Grant

References

[1] I S An S An K J Kwon Y J Kim and S Bae ldquoGinsenosideRh2 mediates changes in the microRNA expression profileof human non-small cell lung cancer A549 cellsrdquo OncologyReports vol 29 no 2 pp 523ndash528 2013

[2] A D Kim K A Kang R Zhang et al ldquoGinseng saponinmetabolite induces apoptosis in MCF-7 breast cancer cellsthrough the modulation of AMP-activated protein kinaserdquoEnvironmental Toxicology and Pharmacology vol 30 no 2 pp134ndash140 2010

[3] H Yuan H Quan Y Zhang S H Kim and S Chung ldquo20(S)-ginsenoside Rg3-induced apoptosis in HT-29 colon cancercells is associated with AMPK signaling pathwayrdquo MolecularMedicine Reports vol 3 no 5 pp 825ndash831 2010

[4] X Pan H Guo J Han et al ldquoGinsenoside Rg3 attenuates cellmigration via inhibition of aquaporin 1 expression in PC-3Mprostate cancer cellsrdquo European Journal of Pharmacology vol683 no 1ndash3 pp 27ndash34 2012

[5] O Tanaka and R Kasai ldquoSaponins of ginseng and relatedplantsrdquo Fortschritte der Chemie Organischer Naturstoffe vol 46pp 1ndash76 1984

[6] H Hasegawa J Sung S Matsumiya and M Uchiyama ldquoMainginseng saponin metabolites formed by intestinal bacteriardquoPlanta Medica vol 62 no 5 pp 453ndash457 1996

[7] C Wakabayashi H Hasegawa J Murata and I Saiki ldquoIn vivoantimetastatic action of ginseng protopanaxadiol saponins isbased on their intestinal bacterial metabolites after oral admin-istrationrdquo Oncology Research vol 9 no 8 pp 411ndash417 1997

[8] K A Kang Y W Kim S U Kim et al ldquoG1 phase arrest of thecell cycle by a ginsengmetabolite compoundK inU937 humanmonocytic leukamia cellsrdquo Archives of Pharmacal Research vol28 no 6 pp 685ndash690 2005

[9] H Hasegawa S Matsumiya M Uchiyama et al ldquoInhibitoryeffect of some triterpenoid saponins on glucose transport intumor cells and its application to in vitro cytotoxic and antiviralactivitiesrdquo Planta Medica vol 60 no 3 pp 240ndash243 1994

[10] BH Lee S J Lee J HHur et al ldquoIn vitro antigenotoxic activityof novel ginseng saponin metabolites formed by intestinalbacteriardquo Planta Medica vol 64 no 6 pp 500ndash503 1998

[11] S Chae K A Kang W Y Chang et al ldquoEffect of compound Kametabolite of ginseng saponin combined with 120574-ray radiationin human lung cancer cells in vitro and in vivordquo Journal ofAgricultural and Food Chemistry vol 57 no 13 pp 5777ndash57822009

[12] E BaeM J HanM Choo S Park andD Kim ldquoMetabolism of20(S)- and 20(R)-ginsenoside Rg3 by human intestinal bacteriaand its relation to in vitro biological activitiesrdquo Biological ampPharmaceutical Bulletin vol 25 no 1 pp 58ndash63 2002

[13] S Lee J Sung S Lee C Moon and B Lee ldquoAntitumor activityof a novel ginseng saponin metabolite in human pulmonaryadenocarcinoma cells resistant to cisplatinrdquo Cancer Letters vol144 no 1 pp 39ndash43 1999

[14] S Odashima T Ohta and H Kohno ldquoControl of phenotypicexpression of cultured B16 melanoma cells by plant glycosidesrdquoCancer Research vol 45 no 6 pp 2781ndash2784 1985

[15] J A Park K Y Lee Y J Oh K Kim and S K Lee ldquoActivationof caspase-3 protease via a Bcl-2-insensitive pathway during theprocess of ginsenoside Rh2-induced apoptosisrdquo Cancer Lettersvol 121 no 1 pp 73ndash81 1997


[16] C Wakabayashi K Murakami H Hasegawa J Murata andI Saiki ldquoAn intestinal bacterial metabolite of ginseng pro-topanaxadiol saponins has the ability to induce apoptosis intumor cellsrdquo Biochemical and Biophysical Research Communi-cations vol 246 no 3 pp 725ndash730 1998

[17] T K Yun Y S Lee Y H Lee S I Kim and H Y Yun ldquoAnti-carcinogenic effect of Panax ginseng CA Meyer and identifica-tion of active compoundsrdquo Journal of Korean Medical Sciencevol 16 pp S6ndashS18 2001

[18] J Hitomi T Katayama Y Eguchi et al ldquoInvolvement ofcaspase-4 in endoplasmic reticulum stress-induced apoptosisand A120573-induced cell deathrdquo Journal of Cell Biology vol 165 no3 pp 347ndash356 2004

[19] Y Tagawa N Hiramatsu A Kasai et al ldquoInduction of apoptosisby cigarette smoke via ROS-dependent endoplasmic reticulumstress andCCAATenhancer-binding protein-homologous pro-tein (CHOP)rdquo Free Radical Biology and Medicine vol 45 no 1pp 50ndash59 2008

[20] B Liang X Song G Liu et al ldquoInvolvement of TR3Nur77translocation to the endoplasmic reticulum in ER stress-induced apoptosisrdquo Experimental Cell Research vol 313 no 13pp 2833ndash2844 2007

[21] F J Gonzalez and H V Gelboin ldquoRole of human cytochromesP450 in the metabolic activation of chemical carcinogens andtoxinsrdquo Drug Metabolism Reviews vol 26 no 1-2 pp 165ndash1831994

[22] M S Denison and S R Nagy ldquoActivation of the aryl hydrocar-bon receptor by structurally diverse exogenous and endogenouschemicalsrdquoAnnual Review of Pharmacology and Toxicology vol43 pp 309ndash334 2003

[23] YWang X Ye Z Ma et al ldquoInduction of cytochrome P450 1A1expression by ginsenoside Rg1 and Rb1 in HepG2 cellsrdquo Euro-pean Journal of Pharmacology vol 601 no 1ndash3 pp 73ndash78 2008

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


tumor cells and by reversing multidrug resistance in tumorcells [8ndash10] The combined treatment of compound K andradiation enhances human lung cancer cell death [8 11]

Various transformation methods including mild acidhydrolysis enzymatic conversion and microbial conversionhave been tested for their ability to transform ginsenosidesto enhance their physiological effectiveness In this studywe investigated the antiproliferative effects and possiblemechanism of action of EMGE that were transformed byenzyme treatment by using the human hepatocarcinoma cellline HepG2

2 Materials and Methods

21 Cell Culture HepG2 cells were maintained in DulbeccorsquosModified Eaglersquos Medium (DMEM) supplemented with 10heat-inactivated fetal calf serum (Invitrogen Carlsbad CA)100 unitsmL penicillin G sodium 100120583gmL streptomycinsulfate and 025120583gmL amphotericin B (Invitrogen Carls-bad CA) in a 5 CO

2atmosphere at 37∘C Confluent cells

were detached with 025 trypsin-EDTA for 5min andaliquots were subcultured Human hepatoma HepG2 cellswere purchased from the Korean Cell Line Bank (KCLBSeoul Republic of Korea)

22 Preparation of Enzyme-Modified Ginseng Extract(EMGE) The root of regular ginseng (4 years old) waspurchased from GampV Korea A total of 20 g of pulverizedginseng root powder were suspended in 380mL of distilledwater and then sterilized by heating at 121∘C for 15min Toreduce the complexity of components in ginseng root theextract was fractionated via extraction with water methanoland butanol Among the fractions ginseng butanol extractcontained the compounds with specific anticancer activityIn addition the suspension was treated with aliquotsof filter-sterilized commercial enzymes (laminarinase100mg pectinase 100mg) with an equimolar ratio (1 1specific activity unit) For these treatments the mixturewas incubated at 40∘C for 2 days and then evaporated todryness at 60∘C These enzyme-modified ginseng powderswere then suspended in 400mL of 80 (vv) methanol andsubjected to ultrasonication for 5min followed by filtrationthrough Whatman No 2 Filter Paper The wet powder onthe filter paper was then collected suspended treated in anultrasound bath and filtered in the same manner once againafter which the two filtrates were combined and evaporatedto dryness at 50∘CThemethanol extract dissolved in 200mLof distilled water was washed with 200mL of ethyl acetatein a separation funnel and then extracted with 200mL ofbutanol Next the butanol extract was evaporated to drynessat 50∘C and then dissolved to a concentration of 10 (wv)in 70 ethanol

23 HPLC Analysis of Ginsenosides Standard ginsenosidesincluding Rg1 Re Rf Rh1 Rb1 Rc Rb2 f1 Rd Rg3(S)Rg3(R) PPT Com K Rh2 and PPD were obtained fromChromaDex Inc (Irvine CA) and analyzed using an AcquityUPLC system (Waters Milford MA) with an Acquity BEH

C18HPLC columnThemobile phase consisted of solution A

(CH3CN HPLC-grade JT Baker NJ) and solution B (Milli-

Q H2O Millipore MA) The flow rate was set at 06mLmin

and the injection of volume was fixed at 2 1 The separationof ginsenosides was performed using an isocratic gradientof 100 solution A for 27min The column temperaturewas maintained at room temperature during the separationand the UV diode array detection was set at 203 nm Toconfirm a reliable retention time of sample ginsenosidethe individual ginsenosides were identified and quantifiedbased on comparison with the retention times of standardginsenosides

24 Cell Viability Assay The effect of EMGE on cell viabilitywas determined using the 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazoliumbromide (MTT a yellow tetrazole) assaywhich quantifies the number of viable cells Cells wereseeded (2 times 104 cellswell) in 24-well flat-bottomed plates(Nunc EU) After incubation at 37∘C for 24 h the mediumwas replaced with EMGE at the appropriate concentrationsControl cells were treatedwithDMSOat a final concentrationof less than 02 After further 24 h incubation cells werewashed 3 times with Dulbeccorsquos phosphate-buffered saline(DPBS) and 500 120583L MTT solution (5mgmL in PBS) wasadded to each well After incubating for 4 h the mediumwas removed and 200 120583L of formazan (crystals dissolved inDMSO) was added to the cells The absorbance of each wellwas read at 540 nm using a microplate reader

25 RNA Extraction and cDNA Synthesis Total RNA wasextracted using the Easy-spin RNA Extraction kit (iNtRONSeoul Republic of Korea) according to the manufacturerrsquosinstructions The purity of RNA was assessed by measuringthe absorption at 260 and 280 nm (values of the ratio of119860260119860280

of 19ndash21 were considered acceptable) and byethidium bromide staining of 18 S and 28 S RNA by gelelectrophoresis RNA concentrations were determined fromthe 119860

260 Two micrograms of total RNA were reverse-

transcribed in a 20 120583L reaction mixture containing 50 unitsof SuperScript II reverse transcriptase (Invitrogen Carls-bad CA) 5 120583M DTT 40 units of RNaseOUT recombinantribonuclease inhibitor (Invitrogen Carlsbad CA) 05120583Mof random hexanucleotide primers and 500120583M of dNTPmixture The reverse transcription reaction was carried outat 50∘C for 60min The reaction was terminated by heatingthe reaction mixture at 70∘C for 15min and the cDNA wasstored at minus20∘C

26 Quantitative Real-Time Reverse Transcriptase PolymeraseChain Reaction (RT-PCR) Overexpression of the targetgenes was confirmed by quantitative real-time RT-PCR Allreal-time PCR analyses were performed on an ABI Step OneReal-time PCR system (Applied Biosystems) Each reactioncontained 01 120583M of each primer 10 120583L of 2x SYBR GreenPCRmastermix (includingAmpliTaqGoldDNApolymerasewith buffer dNTPs mix SYBR Green I dye ROX dyeand 10mm MgCl

2Applied Biosystems) and 1 120583L of the

template cDNA in a 20120583L total reaction volume The typical










119879(G3PDH) to obtain d119862119879(=


119879





3 Results






0

20

40

60

80

100

120

0 438 658 987 148 222 333 50 100

Cel

l pro

lifer

atio

n ac

tivity

()


ControlMGX
















4 Discussion







3

2

1

Control EMGE

Caspase-7Re

lativ

e exp

ress

ion

0

(a)

3

2

1

Control EMGE

Caspase-4

Relat

ive e

xpre

ssio

n

4

5

0

(b)

40

20

Control EMGE

CytP450

Relat

ive e

xpre

ssio

n

60

80

0

(c)


15

10

05

Control EMGE

Cyclin K

Relat

ive e

xpre

ssio

n

0

(a)

Control EMGE

Cyclin M15

10

05Relat

ive e

xpre

ssio

n

0

(b)






Control

PISub-G1 16G1 618S 93G2M 271

200

160

120

80

40

00 200 400 600 800 1000

Cou

nts

(a)

EMGE

PISub-G1 24G1 516S 91G2M 153

200

160

120

80

40

00 200 400 600 800 1000

Cou

nts

(b)



Acknowledgments


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























119879(G3PDH) to obtain d119862119879(=


119879





3 Results






0

20

40

60

80

100

120

0 438 658 987 148 222 333 50 100

Cel

l pro

lifer

atio

n ac

tivity

()


ControlMGX
















4 Discussion







3

2

1

Control EMGE

Caspase-7Re

lativ

e exp

ress

ion

0

(a)

3

2

1

Control EMGE

Caspase-4

Relat

ive e

xpre

ssio

n

4

5

0

(b)

40

20

Control EMGE

CytP450

Relat

ive e

xpre

ssio

n

60

80

0

(c)


15

10

05

Control EMGE

Cyclin K

Relat

ive e

xpre

ssio

n

0

(a)

Control EMGE

Cyclin M15

10

05Relat

ive e

xpre

ssio

n

0

(b)






Control

PISub-G1 16G1 618S 93G2M 271

200

160

120

80

40

00 200 400 600 800 1000

Cou

nts

(a)

EMGE

PISub-G1 24G1 516S 91G2M 153

200

160

120

80

40

00 200 400 600 800 1000

Cou

nts

(b)



Acknowledgments


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















0

20

40

60

80

100

120

0 438 658 987 148 222 333 50 100

Cel

l pro

lifer

atio

n ac

tivity

()


ControlMGX
















4 Discussion







3

2

1

Control EMGE

Caspase-7Re

lativ

e exp

ress

ion

0

(a)

3

2

1

Control EMGE

Caspase-4

Relat

ive e

xpre

ssio

n

4

5

0

(b)

40

20

Control EMGE

CytP450

Relat

ive e

xpre

ssio

n

60

80

0

(c)


15

10

05

Control EMGE

Cyclin K

Relat

ive e

xpre

ssio

n

0

(a)

Control EMGE

Cyclin M15

10

05Relat

ive e

xpre

ssio

n

0

(b)






Control

PISub-G1 16G1 618S 93G2M 271

200

160

120

80

40

00 200 400 600 800 1000

Cou

nts

(a)

EMGE

PISub-G1 24G1 516S 91G2M 153

200

160

120

80

40

00 200 400 600 800 1000

Cou

nts

(b)



Acknowledgments


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















4 Discussion







3

2

1

Control EMGE

Caspase-7Re

lativ

e exp

ress

ion

0

(a)

3

2

1

Control EMGE

Caspase-4

Relat

ive e

xpre

ssio

n

4

5

0

(b)

40

20

Control EMGE

CytP450

Relat

ive e

xpre

ssio

n

60

80

0

(c)


15

10

05

Control EMGE

Cyclin K

Relat

ive e

xpre

ssio

n

0

(a)

Control EMGE

Cyclin M15

10

05Relat

ive e

xpre

ssio

n

0

(b)






Control

PISub-G1 16G1 618S 93G2M 271

200

160

120

80

40

00 200 400 600 800 1000

Cou

nts

(a)

EMGE

PISub-G1 24G1 516S 91G2M 153

200

160

120

80

40

00 200 400 600 800 1000

Cou

nts

(b)



Acknowledgments


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















3

2

1

Control EMGE

Caspase-7Re

lativ

e exp

ress

ion

0

(a)

3

2

1

Control EMGE

Caspase-4

Relat

ive e

xpre

ssio

n

4

5

0

(b)

40

20

Control EMGE

CytP450

Relat

ive e

xpre

ssio

n

60

80

0

(c)


15

10

05

Control EMGE

Cyclin K

Relat

ive e

xpre

ssio

n

0

(a)

Control EMGE

Cyclin M15

10

05Relat

ive e

xpre

ssio

n

0

(b)






Control

PISub-G1 16G1 618S 93G2M 271

200

160

120

80

40

00 200 400 600 800 1000

Cou

nts

(a)

EMGE

PISub-G1 24G1 516S 91G2M 153

200

160

120

80

40

00 200 400 600 800 1000

Cou

nts

(b)



Acknowledgments


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Control

PISub-G1 16G1 618S 93G2M 271

200

160

120

80

40

00 200 400 600 800 1000

Cou

nts

(a)

EMGE

PISub-G1 24G1 516S 91G2M 153

200

160

120

80

40

00 200 400 600 800 1000

Cou

nts

(b)



Acknowledgments


References






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article Identification of Target Genes Involved