Survivin Inhibition Is Critical for Bcl-2 Inhibitor-Induced Apoptosis in Hepatocellular Carcinoma Cells
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Title: Survivin Inhibition Is Critical for Bcl-2 Inhibitor-Induced Apoptosis in Hepatocellular Carcinoma Cells
Physical Description: Journal Article
Creator: Zhao, Xiangxuan
Ogunwobi, Olorunseun O.
Liu, Chen
Publisher: plos one
Place of Publication: plos one
Publication Date: August 1, 2011
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Abstract: Our study aims to study the therapeutic effects of a novel Bcl-2 inhibitor, ABT-263, on hepatocellular carcinoma (HCC) and to provide primary preclinical data for future clinical trial with ABT-263. In this study we showed that Bcl-xL and survivin were up-regulated in HCC cell lines and human liver cancer tissues. Clinic used ABT-263 single treatment had no apoptotic effects on HCC cells whereas higher doses of ABT-263 did. Interestingly, the combination treatment of ABT-263 with survivin inhibitor YM-155 could result in significant apoptosis in HCC cells. Survivin inhibition through gene silencing significantly enhanced ABT-263 to induce apoptosis in HCC cells. We found that low dose of ABT-263 single treatment resulted in ERK activation and survivin up-regulation, which might be involved in the resistance of HCC cells to ABT-263 since blockade of ERK activation sensitized ABT-263-induced apoptosis. Importantly, ABT-263 and YM-155 combination treatment had no apoptotic effects on normal human hepatocytes. Taken together, these data suggest the combination treatment of Bcl-2 inhibitor and survivin inhibition may have a great potential for liver cancer therapy.
Acquisition: Collected for University of Florida's Institutional Repository by the UFIR Self-Submittal tool. Submitted by Xiangxuan Zhao.
Publication Status: Published
Funding: This work was supported by the National Institutes of Health grant R01CA133086 to CL. Publication of this article was funded in part by the University of Florida Open-Access Publishing Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Citation/Reference: Zhao X, Ogunwobi OO, Liu C (2011) Survivin Inhibition Is Critical for Bcl-2 Inhibitor-Induced Apoptosis in Hepatocellular Carcinoma Cells. PLoS ONE 6(8): e21980. doi:10.1371/journal.pone.0021980
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Source Institution: University of Florida Institutional Repository
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SurvivinInhibitionIsCriticalforBcl-2Inhibitor-Induced ApoptosisinHepatocellularCarcinomaCellsXiangxuanZhao,OlorunseunO.Ogunwobi,ChenLiu *DepartmentofPathology,ImmunologyandLaboratoryMedicine,UniversityofFlorida,Gainesville,Florida,UnitedStatesofAmericaAbstractOurstudyaimstostudythetherapeuticeffectsofanovelBcl-2inhibitor,ABT-263,onhepatocellularcarcinoma(HCC)and toprovideprimarypreclinicaldataforfutureclinicaltrialwithABT-263.InthisstudyweshowedthatBcl-xLandsurvivin wereup-regulatedinHCCcelllinesandhumanlivercancertissues.ClinicusedABT-263singletreatmenthadnoapoptotic effectsonHCCcellswhereashigherdosesofABT-263did.Interestingly,thecombinationtreatmentofABT-263withsurvivin inhibitorYM-155couldresultinsignificantapoptosisinHCCcells.Survivininhibitionthroughgenesilencingsignificantly enhancedABT-263toinduceapoptosisinHCCcells.WefoundthatlowdoseofABT-263singletreatmentresultedinERK activationandsurvivinup-regulation,whichmightbeinvolvedintheresistanceofHCCcellstoABT-263sinceblockadeof ERKactivationsensitizedABT-263-inducedapoptosis.Importantly,ABT-263andYM-155combinationtreatmenthadno apoptoticeffectsonnormalhumanhepatocytes.Takentogether,thesedatasuggestthecombinationtreatmentofBcl-2 inhibitorandsurvivininhibitionmayhaveagreatpotentialforlivercancertherapy.Citation: ZhaoX,OgunwobiOO,LiuC(2011)SurvivinInhibitionIsCriticalforBcl-2Inhibitor-InducedApoptosisinHepatocellularCarcinomaCells.PLoSONE 6(8): e21980.doi:10.1371/journal.pone.0021980 Editor: CarloGaetano,IstitutoDermopaticodell’Immacolata,Italy Received February7,2011; Accepted June14,2011; Published August1,2011 Copyright: 2011Zhaoetal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermits unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited. Funding: ThisworkwassupportedbytheNationalInstitutesofHealthgrantR01CA133086toCL.PublicationofthisarticlewasfundedinpartbytheUniversity ofFloridaOpen-AccessPublishingFund.Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationof the manuscript. CompetingInterests: Theauthorshavedeclaredthatnocompetinginterestsexist. *E-mail:liu@pathology.ufl.eduIntroductionCurrently,therearenoeffectivetherapiesforlivercancerother thansurgicalresectionorlivertransplantationintheearlystagesof tumordevelopment.However,suchtreatmentsonlyapplytoa smallpercentageofpatientswhilethemajoritiesdiewithin12 monthsofdiagnosis.Therefore,newtherapeuticstrategiesare urgentlyneeded. Overwhelmingevidenceareshowingthatup-regulationofthe anti-apoptoticproteinsoftheBcl-2familyisoneofthe mechanismsemployedbycancercellstoevadeapoptosis[1–3]. Smallmoleculeinhibitorshavenowbeendesignedtocombatthis abilityofcancercells.Ofall,ABT-263isanovelsecond generationorallybio-availableBcl-2homologydomain3(BH3)mimeticthatinhibitstheanti-apoptoticBcl-2familyproteins(Bcl2,Bcl-xL,andBclv )[4].Ithasbeenshowntobeeffectivein inducingapoptosisandtumorregressioninsmallcelllungcancer, acutelymphoblasticleukemia,multiplemyelomaandlymphoid malignancieseitherasasingleagentorincombinationwithother agents[5–8].However,thetherapeuticeffectsofABT-263onliver cancersarenotknown. Meanwhile,survivin,anotheranti-apoptoticproteinisknownto beover-expressedinmostcancersincludinglivercancers[9,10]. Consequently,ithasbeenbecomingaveryattractivetargetin cancertherapy.YM-155,anovelsmallimidazolium-basedagent wasshowntohavespecificactivityagainstsurvivinwithno associatedsystemictoxicity[11,12].YM-155hasdemonstrateda safeprofileandanti-tumoractivityinaphaseItrialthatincluded patientswithnon-smallcelllungcancer,esophagealcancer, prostatecancercellsandnon-Hodgkinlymphoma[13–16]. SimilartoABT-263,theanti-tumoreffectsofYM-155onHCC remainunknown. Inthepresentstudy,weexaminedtheapoptoticeffectsofABT263treatmentonHCCcells.Weprovidedevidenceforthefirst timetoshowthatlowdosesofABT-263treatmentcouldnot induceapoptosisinHCCcells.However,pre-incubationwith survivininhibitorYM-155orsurvivingenesilencingbysiRNA sensitizedHCCcellstoABT-263-inducedapoptosis.ERK activationandsurvivinup-regulationmaycontributetothe insensitivityofHCCcellstoABT-263.ABT-263andYM-155 combinationtreatmenthadnoapoptotictoxicitytonormal humanhepatocytes.Collectively,thesefindingsprovideanovel frameworkforunderstandingthemechanismofactionofABT263andpossiblytherationalintegrationoftwoagentsintoantiHCCregimens.MaterialsandMethods CellcultureandreagentsEthicsstatement:withtheapprovalbytheUniversityofFlorida GainesvilleHealthScienceCenterInstitutionalReviewBoard (IRB-01)andwithinformedwrittenconsent,livercancertissues andnon-tumorlivertissuesfromsamepatientsrespectivelywere collectedandanalyzed.TheLH86humanHCCcelllinewas establishedinourlaboratoryaspreviouslydescribed[17].Huh7 cellswereobtainedfromtheAmericanTypeCultureCollection (ATCC)(Manassas,VA).HumanhepatocellularcarcinomaHuh7 andLH86cellsweregrowninDulbecco’sModifiedEagle’s Medium(DMEM)with10%fetalbovineserum(Sigma,St.Louis, MO)andantibiotics(100U/mlpenicillinand100mg/ml PLoSONE|www.plosone.org1August2011|Volume6|Issue8|e21980

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streptomycin)at37 u Cin5%CO2.Normalprimaryhuman hepatocyteswereobtainedfromCellzDirectInc(Austin,TX).The cellswereculturedinDMEM/F12(1:1)culturemedium.The humannormalhepatocytesusedwereatleast90%viablebefore treatment.CellsincultureweretreatedwithABT-263aloneor YM-155alone(bothdissolvedinDMSO)oracombinationof both.Untreatedcellswerealwaysusedascontrolsandexposedto equalvolumeofDMSOasfortreatedcells.ABT-263andYM155werepurchasedfromSelleckChemical(Houston,TX); Hoechst33258,antib -actinantibody,andcrystalvioletwere obtainedfromSigma(StLoius,MO);PD98059werefrom Calbiochem(Gibbstown,NJ);anti-caspase9,anti-caspase3,antiBcl-xL,anti-Mcl-1,anti-survivin,anti-Bad,anti-Bax,anti-Bak, anti-ERK,andanti-p-ERKpolyclonalprimaryantibodieswere obtainedfromCellSignalingTechnology(Beverly,MA).WesternblottingCellsweregrowninamonolayerin6-wellplatesandtreated withdrugsasmentionedaboveuntil60%-70%confluent.Cells wereharvested,washedtwicewith1 6 PBSandresuspendedin lysisbuffercontainingNonidetP-40(10mMHEPES,pH7.4, 2mMEGTA,0.5%NonidetP-40,1mMNaF,1mMNaVO4, 1mMphenylmethylsulfonylfluoride,1mMdithiothreitol,50mg/ mltrypsininhibitor,10mg/mlaprotinin,andleupeptin)and incubatedonicefor30min.Aftercentrifugationat12,000 6 g at 4 u Cfor15min,thesupernatantwastransferredtoanewtubeand theproteinconcentrationwasdetermined.Equivalentsamples (20mgofprotein)weresubjectedtoSDS-PAGEon12%gels.The proteinsweretransferredtonitrocellulosemembranesandprobed withthespecificprimaryantibodies.TheImmunoreactive proteinswerevisualizedbyincubatinginHRP-conjugated secondaryantibodies.Chemiluminescencewasdetectedby incubatinginanequal-partsmixtureoftheSuperSignalWest Picostableperoxidesolutionandluminol/enhancersolution (Pierce,Rockford,IL)andsubsequentlyusinganimageprocessing machine.Themolecularsizesoftheproteinsdetectedwere determinedbycomparisonwithprestainedproteinmarkers,BioRad(Hercules,CA).HoechststainingCellswereseededontocoverslipsin6-wellplatesin10%FBScontainingmediumandthentreatedwithABT-263orYM-155as mentionedabove.Afterremovalofculturemediumcellswere exposedtostainingsolutioncontainingHoechst33258(1mg/ml)at 37 u Cfor10–30min.Cellswithchromatincondensationwere visualizedandphotographedusingadigitalfluorescencemicroscope(Olympus)30minafteradditionofthestainingsolution. Chromatincondensationisthemostcharacteristicfeatureof apoptosis.Apoptoticcelldeathratiowasassessedbycountingthe numberofapoptoticcellswithcondensednucleiinsixtoeight randomlyselectedareas.SmallinterferingRNA(siRNA)transfectionCellsweregrowninamonolayerin6-wellplatesuntil60%– 70%confluent.SiRNAtransfectionwasperformedaccordingto themanufacturer’sinstructions.Briefly,cellswereplatedata densityof0.5 6 106cells/wellin6-wellplates.Cellswere transfectedwith100nMsiRNAduplexmixture(Cellsignaling Biotechnology,Beverly,MA)for24hinthepresenceof lipofectamineRNAiMax(InvitrogenInc.,Carlsbad,CA).AnonspecificcontrolsiRNA(CellsignalingBiotechnology,Beverly,MA) wasalsotransfectedatthesameconcentrationasthenegative control.ColonyformationassayColonyformationassaywasperformedasdescribedpreviously [18,19]withmodification.Cellswereseededin6-wellplatesata densityof1000cells.CellswereuntreatedortreatedwithABT263,YM-155,orcombinationofABT-263andYM-155for48h. Afterbeingrinsedwithfreshmedium,cellswereallowedtogrow for14daystoformcolonies,whichwerethenstainedwithcrystal violet(0.4g/L;Sigma).Clonogenicassaywasusedtoelucidatethe possibledifferencesinlong-termeffectsthecombinationon humanHCCcells.StatisticalanalysisStatisticalanalysiswasperformedusingStudent’s t testanalysis, with P values 0.05consideredsignificant.Results Bcl-xLisup-regulatedinhumanHCCcelllinesandliver cancertissuesTounderstandtherolesofBcl-2familyofproteinsinHCC,liver cancertissuesfrommorethan10differentpatientsandnon-tumor livertissuesfromsamepatientsrespectivelywerecollectedand analyzedforproteinexpressionthroughWesternblotting.Asshown inFigure1A,comparedwithnormallivertissues,therewas consistentup-regulationofBcl-xLandsurvivinproteinexpressionin threerepresentativelivertumortissues,whereasMcl-1wasdownregulated.Meanwhile,Bcl-xL,survivinandMcl-1proteinexpressionwasexaminedinnormalhumanhepatocytesandtwoHCCcell lines:LH86andHuh7.Consistentwithdatafromthehumantissue study,thetwohumanlivercancercelllineshadincreased expressionofBcl-xLandsurvivinincomparisontothenormal primaryhepatocytes.WhileMcl-1wasdown-regulatedinbothliver tumortissuesandHCCcelllines(Figure1B).Theseresultssuggest thatBcl-2familyproteinmemberBcl-xLandsurvivinmaybevery importantforthemetastasisordevelopmentofHCC.HCCcellsareresistanttolowdosesofABT-263GiventheelevatedexpressionofBcl-xLinHCCcells,we evaluatedthetherapeuticeffectsofABT-263,apotentBcl-2family inhibitorthathasbeenusedfortreatmentofothercancerssuchas small-celllungcancerandlymphoidmalignanciesclinically.A previousreporthasshownthatABT-263peakandefficacyplasma concentrationswere7.7mM[4].Thus,inourstudy,cellswere untreatedortreatedwithdifferentdosesofABT-263(0–20mM) for24h.ApoptosisassayswereperformedbyHoechst33258 stainingfornuclearmorphologyandWesternblottingforcaspases activation.AsshowninFigure2Aand2B,and2C,thehigherdose ofABT-263(10–20mM)treatmentfor24hinducedsignificant DNAfragmentationandcaspase9or3cleavageactivationin HCCcells,whereaslowerconcentrationsofABT-263(0–2.5mM) hadnoapoptotictoxicitytoHCCcells.Theseresultssuggestthat HCCcellsarerelativelyresistanttolowdosesofABT-263.SurvivinYM-155sensitizesABT-263-inducedapoptosisin HCCcellsSurvivinhasbeenreportedtobehighlyexpressedinlivercancer cells,whichcontributestothemulti-drugresistanceandtumor recurrences[20–23].Inthisstudy,wealsoobservedthatsurvivin over-expressedinHCC.Tofindoutthemechanismunderliningthe relativeresistanceofHCCcellstolowdoseABT-263,weexamined whethersurvivinisinvolvedintheresistanceofHCCcellstoBcl-2 inhibitor.BothLH86andHuh7cellsweretreatedwithABT-263 (1mM),YM-155(1mM),orpre-treatedwithYM-155(1mM)forSurvivinInhibitioninABT-263-InducedApoptosis PLoSONE|www.plosone.org2August2011|Volume6|Issue8|e21980

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1hfollowedbyABT-263(1mM).AsshowninFigure3A,3B,3C, 3D,3Eand3F,neitherABT-263(1mM)norYM-155(1mM)single treatmentwasabletoinduceapoptoticeventsinHCCcells. Surprisingly,acombinationtreatmentofABT-263(1mM)with YM-155(1mM)induceddramaticapoptosiswithin6h.Additional experimentsweredonetodeterminetheanti-tumoractivitiesof combinationofABT-263andYM-155inhibitionwhenanalyzedby invitrocolonyformationassays.AsshowninFigure3Gand3F, mediandoseofABT-263andYM-155combinationtreatment significantlyreducedHCCcellproliferation.Theseresultssuggest thatsurvivininhibitorYM-155cansensitizeABT-263toinduce apoptosisinHCCcells.Survivindown-regulationsensitizesABT-263toinduce apoptosisinHCCcellsYM-155hasbeenreportedbeabletoinhibitsurvivinexpression attranscriptionlevel[24].Toconfirmwhethertheapoptosis enhancingeffectsobservedinFigure3aremediatedbysurvivin Figure1.Bcl-xLisup-regulatedinlivertumortissuesandHCCcells.A .Lysatesfromnormallivertissuesandlivertumortissuesofdifferent patientswerepreparedandsubjectedtoWesternblotting.Bcl-xL,Mcl-1,andsurvivinexpressionsweredetectedwithspecificantibodies. b -actin proteinlevelswereassessedasanequalproteinloadingcontrol(P1,P2,andP3:patientnumber). B .Celllysatesfromhumannormalprimary hepatocytesandHCCcellsLH86andHuh7werepreparedandWesternblottingwasperformedtodetectBcl-xL,Mcl-1,andsurvivinexpressionwith specificantibodies. b -actinproteinlevelswereassessedasanequalproteinloadingcontrol. doi:10.1371/journal.pone.0021980.g001 Figure2.HCCcellsareresistanttolowdosesofABT-263.A. LH86and B. Huh7cellsweretreatedwithABT-263(0–20mM)forupto24h. ApoptosiswasmeasuredthroughHoechststainingtoshowapoptoticcellswithcondensednucleiasdescribedin‘materialsandmethods’. (representativeapoptoticcellsweremarkedwithwhitearrowsinABT-263treatmentpanel). C. HCCcellsweretreatedwithincreasingdosesofABT263asindicatedforupto24h.ThencellswereharvestedandcelllysateswerepreparedandsubjectedtoWesternblotting.Caspaseactivationwas assessedthroughdetectingthecleavedbandsofcaspase9andcaspase3. b -actinproteinlevelswereusedasanequalproteinloadingcontrol. doi:10.1371/journal.pone.0021980.g002 SurvivinInhibitioninABT-263-InducedApoptosis PLoSONE|www.plosone.org3August2011|Volume6|Issue8|e21980

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inhibitiononproteinlevel,weexaminedtheproteinexpressionsin HCCcellstreatedwithABT-263(1mM),YM-155(1mM),orthe combinationofYM-155(1mM)andABT-263(1mM).Asshown inFigure4A,thecombinationtreatmentofHCCcellswithABT263(1mM)andYM-155(1mM)forupto6hhasnoeffectsonthe expressionsofeitheranti-apoptoticproteinBcl-xLorproapoptoticproteinsincludingBad,Bak,andBax.However,as expected,thepresenceofYM-155significantlydecreasedsurvivin proteinexpression(Figure4B,leftthirdlane).Co-treatmentofcells withABT-263(1mM)andYM-155(1mM)inducedaneven greaterdecreaseinsurvivinproteinexpression(Figure4B,right twolanes)thanthatofYM-155itselfdid.However,weindeed observedthatABT-263singletreatmentfor3hresultedin survivinincrease(Figure4B).Tofurtherdeterminesurvivin inhibitionplaysacriticalroleinsensitizingABT-263toinduce apoptosisinHCCcells,wedown-regulatedsurvivinexpressionin HCCcellsbysiRNAduplexestargetedagainsthumansurvivin mRNA,andthenexaminedtheexpressionofsurvivinbyWestern blotting(Figure4C)andapoptoticeventsafterABT-263 treatments.TheresultsdemonstratedthatABT-263induced significantapoptosisinthesurvivinsiRNA-transfectedcells,but notinsiRNARandom-transfected(control)cells(Figure4D,4E, and4F).Inaddition,Mcl-1wasalsodown-regulatedincells treatedwithYM-155orthecombinationofABT-263andYM155(Figure4A).ToexplorewhetherMcl-1decreasecontributed toregulateYM-155andABT-263co-treatment-inducedapoptosis,weover-expressedMcl-1inHCCcellsandfounditcouldnot neutralizethecombinationtriggeredcytotoxicity(datanotshown). Figure3.YM-155sensitizesABT-263-inducedapoptosisinHCCcells.A. LH86and B. Huh7cellswereuntreatedortreatedwithABT263(1mM),YM-155(1mM)orcombinationofABT-263(1mM)andYM-155(1mM)forupto6h.ThenapoptoticcellswereassessedasinFigure2Aand 2B(representativeapoptoticcellsweremarkedwithwhitearrows). C. LH86and D. Huh7cellswereuntreatedortreatedwithABT-263(1mM),YM155(1mM)orcombinationofABT-263(1mM)andYM-155(1mM)for6h.Cellswithapoptoticnucleiwerecountedtodeterminecelldeathratio (*p 0.05,**p 0.05). E. LH86cellsand F. Huh7cellsweretreatedasindicatedandcelllysateswerepreparedandsubjectedtoWesternblotting. Apoptosiswasevaluatedthroughcaspase3activation. b -actinwasusedasanequalproteinloadingcontrol. G. LH86cellsgrowninsix-wellplate wereuntreated(control)ortreatedwithdifferentconditionsasindicatedfor48h.Afterrinsedwithfreshculturemediumfor3times,cellswere culturedforanothertwoweeks.Cellcolonyformationassayswereperformedwithcrystalvioletstaining. H. colonynumberwerecountedtoshow combinationtreatmentwithABT-263andYM-155resultedinreductionofclonogenesis( # p 0.05). doi:10.1371/journal.pone.0021980.g003 SurvivinInhibitioninABT-263-InducedApoptosis PLoSONE|www.plosone.org4August2011|Volume6|Issue8|e21980

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Takentogether,theseresultssuggestthatsurvivindown-regulation playsacriticalroleforanti-cancerdrugBcl-2inhibitortoinduce apoptosisinHCCcells.Bcl-xLknockingdownenhancessurvivininhibitor YM-155-inducedapoptotictoxicityinHCCcellsTofurtherdeterminesurvivininhibitioncanreversethe insensitivityofHCCcellsinthepresenceofBcl-xLactivity blockade.Wedown-regulatedBcl-xLproteinexpressioninHCC cellsbysiRNAduplexestargetedagainsthumanBcl-xLmRNA andthentreatedcellswithYM-155.KnockdowneffectsofBcl-xL wereexaminedthroughWesternblottingandapoptosiswas assessedthroughnuclearstainingandcaspaseactivation.Asshown inFigure5A,comparedwithRandomsiRNA(negativecontrol), Bcl-xLproteinexpressionwasdramaticallyreduced.Moreover, apoptoticdatademonstratedthatYM-155inducedsignificant DNAfragmentationandcaspase3cleavagesintheBcl-xLsiRNAtransfectedcells,butnotinsiRNAcontrol-transfectedcells (Figure5B,5C,and5D).Theseresultssuggestthatsurvivin inhibitionandBcl-xLdown-regulationissufficienttoinduce apoptosisinHCCcells.ABT-263inducesERKactivationandsurvivin up-regulationinHCCcellsDys-regulationandactivationofRaf-MEK-ERK-survivinhas beenshownassociatedwithanti-apoptoticcapabilityofcancer cells[25–28].Consequently,inordertounderstandwhylower doseABT-263couldnotinduceapoptosisofHCCcells,we examinedtheeffectsofABT-263onERK-survivinsignalpathway activationinHCCcells.Ononehand,ourdataindicatedthat Figure4.Survivindown-regulationsensitizesABT-263-inducedapoptosisinHCCcells.A. LH86cellsweretreatedasindicatedandcell lysateswerepreparedforWesternblotting.Pro-apoptoticproteins:Bax,Bad,andBakandanti-apoptoticproteinsBcl-xLandMcl-1wereassessed withspecificantibodiesrespectively. b -actinwasdetectedandservedasanequalproteinloadingcontrol. B. LH86cellswereuntreatedortreated withABT-263(1mM),YM-155(1mM)orcombinationofABT-263(1mM)andYM155(1mM)forupto6hasindicated.Thencellswereharvestedand celllysateswerepreparedforWesternblotting.Anti-survivinandanti-Bcl-xLpolyclonalantibodieswereusedtoassessproteinlevelsforsurviv inand Bcl-xLrespectively. b -actinwasusedasanequalproteinloadingcontrol.Thebandintensitiesofsurvivin,Bcl-xL,and b -actinwasqualifiedwithImage Jsoftware. C. LH86cellsweretransientlytransfectedwithsynthesizedrandomsiRNA(control)orsurvivinspecificsiRNAduplexes,and48hposttransfection,cellsweresubjectedtoWesternblottinganalysiswithanti-survivinpolyclonalantibody. b -actinwasusedasanequalproteinloading control. D. LH86cellsweretransfectedwithsynthesizedrandomcontrolsiRNAorsurvivinspecificsiRNA,and48hpost-transfection,cellswere untreatedortreatedwithABT-263(1mM)for24handthensubjectedtoHoechststainingtoshowapoptoticcellswithcondensednuclei (representativeapoptoticcellsweremarkedwithwhitearrows). E. LH86cellsweretreatedasinFigure4Dandapoptosiswasmeasuredasin Figure2A.Statisticalanalysiswasperformedforapoptosisratiobycountingthenumberofcellswithapoptoticnuclei(*p 0.05). F. LH86cellstreated asinFigure4DwereharvestedandcelllysateswerepreparedandsubjectedtoWesternblotting.Apoptosiswasdeterminedthroughcaspase3 activation. b -actinwasusedasanequalproteinloadingcontrol. doi:10.1371/journal.pone.0021980.g004 SurvivinInhibitioninABT-263-InducedApoptosis PLoSONE|www.plosone.org5August2011|Volume6|Issue8|e21980

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treatmentofcellswithABT-263(1–2.5mM)for1hcouldresult intheincreaseofphosphorylatedERK(p-ERK)butnotERK (Figure6A).Ontheotherhand,asshowninFigure6Bor Figure4B,ABT-263(1mM)administrationcouldresultin survivinexpressionincrease.Thus,inanattempttoknowif ERK-survivinactivationcouldprotectcellsagainstABT-263 toxicity,cellswereuntreatedortreatedwithABT-263(1mM), PD98059(50mM),orpre-treatedwithPD98059(50mM) followedbyABT-263(1mM).AsshowninFigure6Cand6D, blockingERKactivationwithspecificinhibitorPD98059 enhancedABT-263-indcuedapoptosisinHCCcells.Tofurther determineERKanti-apoptoticeffectsonABT-263treatedHCC cells,weknockeddownERKexpressionthroughsiRNA mediatedgenesilencing(Figure6E)andthenadministrated ABT-263.SimilarresultsrevealedthatERKdepletionsensitized ABT-263-inducedapoptosis(Figure6F).Theseresultssuggestthe activationofERK-survivinmayrendercellstoberesistanttolow doseABT-263-inducedapoptosis.ABT-263hasnoapoptotictoxicitytonormalhuman hepatocytesFinally,weaskedthequestionwhetherABT-263,YM-155,or thecombinationtreatmentofABT-263andYM-155istoxicto normalhumanhepatocytes invitro .AsshowninFigure7,ABT263,YM-155singletreatment,orthecombinationtreatmentof ABT-263andYM-155hadnoapoptotictoxicityinnormalhuman hepatocytes,whereas,inpositivecontrol,thecombination treatmentinducedsignificantapoptosisinlivercancercells.These Figure5.Bcl-xLdown-regulationenhancesYM-155-inducedapoptotictoxicityinHCCcells.A. Huh7cellsweretransientlytransfected withsynthesizedrandomsiRNA(control)orBcl-xLspecificsiRNAduplexes,and48hpost-transfection,cellsweresubjectedtoWesternblotting analysiswithanti-Bcl-xLpolyclonalantibody. b -actinwasusedasanequalproteinloadingcontrol. B. Huh7cellsweretransfectedwithsynthesized randomcontrolsiRNAorBcl-xLspecificsiRNA,and48hpost-transfection,cellswereuntreatedortreatedwithYM-155(1mM)for24handthen subjectedtoHoechststainingtoshowapoptoticcellswithcondensednuclei(representativeapoptoticcellsweremarkedwithwhitearrows). C. Huh7cellsweretreatedasinwithYM-155asindicatedandapoptosiswasmeasuredasinFigure2A.Statisticalanalysiswasperformedforapoptosis ratiobycountingthenumberofcellswithapoptoticnuclei(*p 0.05). D. Huh7cellstreatedwereharvestedandcelllyateswerepreparedand subjectedtoWesternblotting.Apoptosiswasdeterminedthroughcaspase3activation. b -actinwasusedasanequalproteinloadingcontrol. doi:10.1371/journal.pone.0021980.g005 SurvivinInhibitioninABT-263-InducedApoptosis PLoSONE|www.plosone.org6August2011|Volume6|Issue8|e21980

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resultssuggestthatcombinationtreatmentofBcl-2andsurvivin inhibitorsissafetonormalhumanlivercells.DiscussionInthepresentstudy,wefirstprovideevidencetoshowthat relativelowdosesofABT-263couldnotpromoteapoptosisin humanHCCcelllines.Significantapoptoticeffectswereobserved whenABT-263wasusedalongwithYM-155.SurvivindownregulationappearstobecriticaltosensitizeABT-263-induced apoptosisinHCCcells.ERKactivationandsurvivinupregulationinducedbyLowdoseofABT-263mayprotectHCC cellsagainstapoptosis.ThiscombinationtreatmentofABT-263 andYM-155hasnoapoptotictoxicitytonormalhuman hepatocytes. ItiswellknownthatBcl-2familymembersareanti-apoptotic proteinsandplaycriticalrolesintumorgenesis.Hence,itis possibletoinduceapoptosisbytargetingthemwithinhibitorssuch asABT-263.Thisstrategyhasbeenshowntobepromisingin treatmentofanumberofcancerssuchassmallcelllungcancer, acutelymphoblasticleukemia,multiplemyeloma,andlymphoid malignancies[29–32].However,itwasunknownwhethertheuse Figure6.ABT-263inducesactivationofERKandsurvivinup-regulationinHCCcells.A. LH86cellsweretreatedwithdifferentdosesof ABT-263asindicatedfor1h.CellswereharvestedandcelllysateswerepreparedforWesternblotting.Phospho-ERKandERKproteinlevelswere examinedwithspecificantibodies. b -actinwasassessedandservedasanequalproteinloadingcontrol. B ,LH86cellswereuntreatedortreatedwith ABT-263(1mM)for1h.CellswereharvestedandcelllysateswerepreparedforWesternblotting.Survivinexpressionlevelwasexaminedwithspecific antibodies. b -actinwasassessedandservedasanequalproteinloadingcontrol. C. LH86cellswerenottreated(control)ortreatedwithABT263(1mM),ERKspecificinhibitorPD98059(50mM),orpre-treatedwithPD98059(50mM)for1hfollowedbyABT-263(1mM)for24h.Apoptosiswas determinedbyHoechststainingtoshowcellswithapoptoticnuclei(representativeapoptoticcellswerelabeledwithwhitearrows;PD:PD98059). D. LH86cellsweretreatedasinFigure6C,apoptosiswasassessedbynuclearstainingandcellswithapoptoticnucleiwerecountedasdescribedin ‘materialsandmethods’(*p 0.05). E. LH86cellswereuntransfectedortransientlytransfectedwithsynthesizedrandomsiRNA(control)orERK specificsiRNAduplexes,and48hpost-transfection,cellsweresubjectedtoWesternblottinganalysiswithanti-ERKpolyclonalantibody. b -actinwas usedasanequalproteinloadingcontrol. F. LH86cellsweretransfectedwithsynthesizedrandomcontrolsiRNAorERKspecificsiRNA,and48hposttransfection,cellswereuntreatedortreatedwithABT-263(1mM)for24handthensubjectedtoHoechststainingtoshowapoptoticcellswith condensednuclei(representativeapoptoticcellsweremarkedwithwhitearrows). doi:10.1371/journal.pone.0021980.g006 SurvivinInhibitioninABT-263-InducedApoptosis PLoSONE|www.plosone.org7August2011|Volume6|Issue8|e21980

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ofABT-263maybeaneffectiveapproachinHCCtreatment. Inourstudy,wefirstlyshowedthatBcl-2familyproteinBcl-xL isdown-regulatedinnormaldi fferentiatedtissuesornormal humanhepatocyteswhereasisover-expressedinthemajorityof malignancies.ThisisconsistentwithpreviousreportsthatBcl-2 familyproteinswereelevatedinlivertumorcells[33–37].In addition,unlikeBcl-xL,anotherBcl-2familyanti-apoptotic protein,Mcl-1,isdecreasedintumortissuesandcelllinesin comparisonwiththatinnormallivertissuesandcells,ofwhich underliningreasonandfunctionremainsfurtherstudy;Bcl-2 proteinexpressionisundetectableineitherlivertumoror normalsamples,whichisinagreementwithreportsthatBcl-2 israrelyexpressedinlivertumo rcells[38,39].Secondly,wedid observethatveryhighdosesofABT-263couldinduce apoptosisinHCCcells.However,thereareconcernsabout thesideeffectsofmanychemotherapeuticagentsincluding thoseinclinicaluse.Thus,lowerdosesofABT-263-induced apoptosiswereexaminedinHCCcells.Unfortunately,we foundthatHCCcellswereresistanttolowconcentrationof ABT-263.Ourresultsarenotinagreementwiththestudies, whichsuggestthatlowdosesofABT-263weresufficientto induceapoptosisincancercells includingsmall-celllungcancer andlymphomacelllines[5,8].Thisimpliesapossibilitythatin sometypesofcancercells,monotherapyviainhibitionofantiapoptoticBcl-2familyproteinact ivityisnotsufficienttotrigger apoptosis.Unfortunately,inso far,thereisnoreporttoshowthe reasonwhyanumberofcancercellscannotbekilledbyBcl-2 inhibitor.Inourstudy,withanattempttounderstandthe insensitivityofHCCcellsto lowdoseofABT-263-induced apoptosis,wefoundthatanotheranti-apoptoticprotein survivinisup-regulatedinlive rtumortissuesorcelllines,but rarelyexpressedinmostnorma ltissuesandundetectablein normallivercells,suggestingtha titmayfacilitatecellresistant tolowdosesofABT-263treatment.Ourdatashowedthatin combinationwiththesurvivininhibitorYM-155,verylowdose ofABT-263inducedsignificantapoptosisinHCCcellsonlyin 6h.Moreover,wefoundthatknockdownofBcl-xL significantlyenhancedYM-155-inducedapoptotictoxicity, whichfurthersupportedBcl-xLi nhibition-inducedapoptosis needssurvivininhibition.Our findingsareinagreementwith studiesthatreportedthatYM-155couldenhanceradiation therapeuticagents-induceda poptosisintumorcells[40]. Meanwhile,wefoundthatthelowdoseofneitherofthese agentswasabletoinduceapoptoticcelldeathofHCCcells. Third,wefoundthatthesub-lethaldoseofABT-263andYM155thatcombinedtoeffectivelyi nduceapoptoticcelldeathin theHCCcellswasnotcytotoxictohumannormalprimary hepatocytes.Collectively,thesedataareveryinterestingand encouragingbecausetheyshowthatlowdosesofbothABT-263 andYM-155maybeusedtoeffectivelytreatHCCwithno associatedhepatotoxicity. Weareinterestedindeterminingthemechanismsofaction underlyingtheobservedeffectsnotedabove.Ononehand,our experimentsshowedthatABT-263,YM-155,orABT-263and YM-155combinationtreatmenthadnoeffectsonexpressionof pro-apoptoticproteinssuchasBad,Bax,andBakorantiapoptoticBcl-xL.However,YM-155treatmentcouldsignificantlyinducesurvivinproteindecrease,butnotthatofantiapoptoticproteinBcl-xL,suggestingthatYM-155maysensitize ABT-263-inducedapoptosisviasurvivindown-regulation.On theotherhand,survivinknock-downthroughsiRNAsignificantlyenhancedlowdoseofABT-263toinduceapoptosisin HCCcells,whichfurtherconfirmedthatsurvivininactivation indeedplayscriticalrolestodeterminecellsensitivitytoBcl-2 inhibitortoxicity.Meanwhile,weobservedthatMcl-1wasalso decreasedbyeitherYM-155singleorcombinationtreatmentof ABT-263andYM-155,suggestingitmaycontributetosensitize ABT-263-inducedapoptosisinHCCcells.However,further experimentsshowedthatover-expressionofMcl-1couldnot protectHCCcellsagainstABT-263andYM-155co-treatmentinducedapoptosis.Thus,Mcl-1appearstoplaynorolein regulatingapoptosis-inducedbyABT-263andYM-155combination. IthasbeenreportedthattheactivationofRaf-MEK-ERKsurvivinsignalingpathwayco uldprotectendothelialcells againstgamma-radiation-inducedapoptosis[41].Itispossible thatincubationoflowdoseA BT-263couldactivateERKsurvivinanti-apoptoticsignalpathwaytoneutralizeitsBcl-2 inhibitioneffects.Inourstudy,weobservedthatlowdosesof ABT-263singletreatmentcouldr esultinincreasedphosphorylationofERKintheLH86cellsandsurvivinup-regulation, suggestinglowdoseABT-263cana ctivateERK-survivinsignal pathway.Thesedatamayfirstprovideanimportantanddirect explanationastowhylowdoseABT-263isunabletoinduce apoptosis.Moreimportantl y,eitherusingspecificERK inhibitorPD98059topre-treatcellsorusingsiRNAtoknock downERKproteinexpressionsign ificantlysensitizedsub-lethal ABT-263-inducedapoptosisin HCCcells,suggestingthatERK anti-apoptoticsignalpathwaya ctivationcouldrendercells resistanttothisBcl-2inhibitortoxicity.Thesefindingsarein agreementwithpreviousreportsthatERK-survivinsignal pathwaycanregulatechemotherapeuticreagents-inducedapoptosis[25,26].Finally,ourresultsdemonstratednormalhuman hepatocytesareresistanttot hecombinationofABT-263and YM-155,ofwhichthemechanismunderlyingremainstobe investigated. Overall,thedatafromthisstudyhaveledtothefollowing conclusions:lowdosesofABT-263alonecouldnotinduce apoptosisinHCCcells.Survivininhibitionsensitizedthisbcl-2 inhibitortoinduceapoptosisinHCCcells,butnotinnormal humanprimaryhepatocytes.ERKsignalpathwayactivationmay beinvolvedintheprotectionofcellsagainstapoptosisexertedby Bcl-2inhibitor.SinceABT-263andYM-155havealreadyshown promisingresultsinclinicaltrialsinothertypesofcancers,the currentdatamayprovideabasicclueforclinicaltrialoftheircombinationinHCCtreatment. Figure7.ABT-263hasnoapoptotictoxicitytonormalhuman hepatocytes.A. Freshlypreparednormalhumanhepatocyteswere grownin6-wellplates.Aftertwodayslater,cellsweretreatedwith variousconditionsasindicated.Apoptosiswereassessedthrough Westernblottingtodetectactivationofcaspase3withspecific antibody.Celllysatesfromhepatoblastomacells(HB01)treatedwitha combinationofABT-263(1mM)andYM-155(1mM)forupto6hwere setupasapositivecontrol. b -actinwasdetectedandservedasanequal proteinloadingcontrol. doi:10.1371/journal.pone.0021980.g007 SurvivinInhibitioninABT-263-InducedApoptosis PLoSONE|www.plosone.org8August2011|Volume6|Issue8|e21980

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