Reactive Oxygen Species Is Essential for Cycloheximide to Sensitize Lexatumumab-Induced Apoptosis in Hepatocellular Carc...
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Title: Reactive Oxygen Species Is Essential for Cycloheximide to Sensitize Lexatumumab-Induced Apoptosis in Hepatocellular Carcinoma Cells
Physical Description: Journal Article
Creator: Zhao, Xiangxuan
Cao, Mengde
Liu, Joy J.
Zhu, Haizhen
Nelson, David R.
Liu, Chen
Publisher: plos one
Place of Publication: plos one
Publication Date: February 10, 2011
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Abstract: This study aims to investigate apoptosis induced by lexatumumab (Lexa) in hepatocellular carcinoma (HCC) cells. We assessed the sensitivity of HCC cell lines and normal human hepatocytes to Lexa and explored the sensitization of HCC cells to Lexa-induced apoptosis by cycloheximide (CHX). Our data indicated that CHX sensitized HCC cell lines to Lexa-induced apoptosis, whereas treatment using solely CHX or Lexa was ineffective. The sequential treatment of CHX followed by Lexa dramatically induced caspase-dependent apoptosis in HCC cells and had synergistically increased intracellular rates of reactive oxygen species (ROS). Additionally, when ROS production was blocked by N-acetyl-L-cysteine (NAC), HCC cells were protected against Lexa and CHX combination treatment-induced apoptosis. ROS generation induced by combination treatment of Lexa and CHX triggered pro-apoptotic protein Bax oligomerization, conformation change, and translocation to mitochondria, which resulted in the release of cytochrome c and subsequent cell death. Furthermore, HSP90 was involved in mediating Lexa and CHX combination treatment-induced ROS increase and apoptotic death. More importantly, we observed that combination treatment of Lexa and CHX did not cause apoptotic toxicity in normal human primary hepatocytes. These results suggest that Lexa and CHX combination treatment merits investigation for the development of therapies for patients with HCC.
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 research grants from the National Institutes of Health: R01CA133086 to CL. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding was received for this study. Publication of this article was funded in part by the University of Florida Open-Access Publishing Fund.
Citation/Reference: Zhao X, Cao M, Liu JJ, Zhu H, Nelson DR, et al. (2011) Reactive Oxygen Species Is Essential for Cycloheximide to Sensitize Lexatumumab-Induced Apoptosis in Hepatocellular Carcinoma Cells. PLoS ONE 6(2): e16966. doi:10.1371/journal.pone.0016966
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Source Institution: University of Florida Institutional Repository
Holding Location: University of Florida
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System ID: IR00000573:00001

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ReactiveOxygenSpeciesIsEssentialforCycloheximide toSensitizeLexatumumab-InducedApoptosisin HepatocellularCarcinomaCellsXiangxuanZhao1,MengdeCao2,JoyJ.Liu1,HaizhenZhu1¤,DavidR.Nelson2,ChenLiu1*1 DepartmentsofPathology,Immunology,andLaboratoryMedicine,UniversityofFloridaCollegeofMedicine,Gainesville,Florida,UnitedStatesof America, 2 DepartmentofMedicine,UniversityofFlorida,Gainesville,Florida,UnitedStatesofAmericaAbstractThisstudyaimstoinvestigateapoptosisinducedbylexatumumab(Lexa)inhepatocellularcarcinoma(HCC)cells.Weassessed thesensitivityofHCCcelllinesandnormalhumanhepatocytestoLexaandexploredthesensitizationofHCCcellstoLexainducedapoptosisbycycloheximide(CHX).OurdataindicatedthatCHXsensitizedHCCcelllinestoLexa-inducedapoptosis, whereastreatmentusingsolelyCHXorLexawasineffective.ThesequentialtreatmentofCHXfollowedbyLexadramatically inducedcaspase-dependentapoptosisinHCCcellsandhadsynergisticallyincreasedintracellularratesofreactiveoxygen species(ROS).Additionally,whenROSproductionwasblockedbyN-acetyl-L-cysteine(NAC),HCCcellswereprotectedagainst LexaandCHXcombinationtreatment-inducedapoptosis.ROSgenerationinducedbycombinationtreatmentofLexaandCHX triggeredpro-apoptoticproteinBaxoligomerization,conformationchange,andtranslocationtomitochondria,whichresulted inthereleaseofcytochromecandsubsequentcelldeath.Furthermore,HSP90wasinvolvedinmediatingLexaandCHX combinationtreatment-inducedROSincreaseandapoptoticdeath.Moreimportantly,weobservedthatcombination treatmentofLexaandCHXdidnotcauseapoptotictoxicityinnormalhumanprimaryhepatocytes.Theseresultssuggestthat LexaandCHXcombinationtreatmentmeritsinvestigationforthedevelopmentoftherapiesforpatientswithHCC.Citation: ZhaoX,CaoM,LiuJJ,ZhuH,NelsonDR,etal.(2011)ReactiveOxygenSpeciesIsEssentialforCycloheximidetoSensitizeLexatumumab-Induced ApoptosisinHepatocellularCarcinomaCells.PLoSONE6(2):e16966.doi:10.1371/journal.pone.0016966 Editor: WaelEl-Rifai,VanderbiltUniversityMedicalCenter,UnitedStatesofAmerica Received September22,2010; Accepted January18,2011; Published February10,2011 Copyright: 2011Zhaoetal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermits unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited. Funding: ThisworkwassupportedbyresearchgrantsfromtheNationalInstitutesofHealth:R01CA133086toCL.Thefundershadnoroleinstudydesign,data collectionandanalysis,decisiontopublish,orpreparationofthemanuscript.Noadditionalexternalfundingwasreceivedforthisstudy.Publica tionofthisarticle wasfundedinpartbytheUniversityofFloridaOpen-AccessPublishingFund. CompetingInterests: Theauthorshavedeclaredthatnocompetinginterestsexist. *E-mail:liu@pathology.ufl.edu ¤Currentaddress:StateKeyLaboratoryofChemo/BiosensingandChemometrics,SchoolofBiology,HunanUniversity,Changsha,ChinaIntroductionHepatocellularcancerisoneofthefivemostcommoncancers worldwideandisfatalinmorethan90%ofpatients[1].Currently, therearenoeffectivetherapiesforlivercancerotherthansurgical resectionorlivertransplantationintheearlystagesoftumor development.Suchtreatmentsonlyapplytoasmallpercentageof patients,whilethemajoritydiewithin6monthsofdiagnosis[2]. Therefore,newtherapeuticstrategiesareurgentlyneeded. Targetingdeathreceptoractivation-mediatedcelldeathis quicklybecomingoneofthemostpromisingstrategiesforanticancertherapy[3,4].Anoverwhelmingnumberofstudieshave demonstratedthattheadministrationofdeathreceptoragonists canselectivelyinduceapoptosisintumorcellsandsignificantly inhibitxenografthumantumorgrowth[5–8].Amongthedeath receptoragonists,lexatumumab(Lexa)wasdevelopedasa potentialhumanizedanti-deathreceptor5(DR5)monoclonal antibody.IthasbeenshownthatLexaspecificallybindstodeath receptor5andinducesapoptosisinanumberoftumorcelllines, includingrenalcellcarcinoma(RCC)[9],humanmyelomacell lines(HMCL)[10],andmalignantpleuralmesothelioma(MPM) [11].Differentresearchershavealsoreportedthatcombination treatmentwithagonisticdeathreceptor5mAbsandchemotherapeuticdrugsexertasynergisticapoptoticeffectinsometumorcell lines,suchaslymphoma[12–14],breastcancer[15],colorectal cancer[16],andmalignantmesothelioma[11].Nevertheless,it remainsunknownwhetherLexacaninduceapoptosisin hepatocellularcarcinoma(HCC)cellsorwhetherithasapoptotic toxicitytonormalhepatocytes. Inthepresentstudy,wearethefirsttoshowdataindicatingthat LexacansignificantlyinduceapoptosisinresistantHCCcelllines inthepresenceofcycloheximide(CHX).Weprovideevidenceto demonstratethattreatmentcombiningLexaandCHXinduces caspase-dependentapoptosisinHCCcells.Intracellularreactive oxygenspecies(ROS)generation,Bax/Bakactivation,andheat shockprotein90(HSP90)inactivationareinvolvedinkillingthe HCCcells.Moreimportantly,wefoundthatLexaandCHX combinationtreatmenthasnoapparentapoptotictoxicityto normalhumanhepatocytes.MaterialsandMethods CellcultureandreagentsHumanhepatocellularcarcinomacelllines,Huh7andLH86 weregrowninDulbecco’sModifiedEagle’sMedium(DMEM) with10%fetalbovineserum(Sigma,St.Louis,MO)and antibiotics(100U/mlpenicillinand100mg/mlstreptomycin)at 37 u Cin5%CO2.Normalprimaryhumanhepatocyteswere PLoSONE|www.plosone.org1February2011|Volume6|Issue2|e16966

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obtainedfromCellzDirectInc(Austin,TX).Thecellswere culturedinDMEM/F12(1:1)culturemedium.Thehuman normalhepatocytesusedwereatleast90%viablebefore treatment.Anti-caspase8,anti-caspase10,anti-caspase3,anticytochromec,anti-HSP90,anti-Bcl-xL,anti-IKKb ,anti-I k Ba anti-p-I k Ba ,anti-Mcl-1,anti-Bak,anti-DR4,anti-DR5,andantiBidprimaryantibodieswereobtainedfromCellSignaling Technology(Beverly,MA);Dihydroethidium(DHED),N-acetylL-cysteine(NAC),Bis(maleimido)hexane(BMH)/DSS,DMAG17,MitoTracker(Red)CMXRos,IKKinhibitor,NEMO-binding domainpeptide(NBD):MAPKinhibitor:PD98059,P38inhibitor: SB203580,andJNKinhibitor:SP600125wereobtainedfrom Invitrogen(Carlsbad,CA);antib -actin,anti-Bax6A7monoclonal antibodies,Hoechst33258,and4 9 ,6 9 -Diamidino-2-phenylindole (DAPI)wereobtainedfromSigma(St.Louis,MO);z-IETD-FMK andz-VAD-FMKwereobtainedfromCalbiochem(SanDiego, CA).Anti-Bax(N-20)primarypolyclonalantibody,goatantirabbithorseradishperoxidase(HRP)conjugatedsecondary antibody,Goatanti-rabbitsecondaryantibodyconjugatedwith FITC,andproteinGplus-agarosewerepurchasedfromSanta CruzBiotechnology(SantaCruz,CA).TheAnnexin-Vapoptosis detectionkitwasobtainedfromBDBioscience(SanDiego,CA). LexatumumabwaskindlyprovidedbyHumanGenomeScience Inc.[12,17].HoechststainingassayApoptosiswasdeterminedthroughnuclearmorphologychange. Aftertreatmentwithdifferentstimuli,cellswerestainedwith Hoechst33258at37 u Cfor10min.CellularDNAfragmentation/ nucleuscondensationwasdetectedusinganOlympusfluorescent microscope.Apoptoticcelldeathratiowasassessedthrough calculatingthenumberofapoptoticcellswithcondensednucleiin sixtoeightrandomlyselectedareas.DNAladderassayADNAladderassaywasperformedaspreviouslydescribed[18] withmodification.Briefly,cellstreatedunderdifferentconditions wereharvestedandwashedwith1 6 phosphatebufferedsaline (PBS).Cellpelletswereresuspendedwithlysisbuffer(1%NP-40in 20mMEDTA,50mMTris-HCl,pH7.5).Thesampleswere centrifugedat1,600 6 g for5minandsupernatantswere transferredtonewtubes.SDS(finalconcentration1%)and RNaseA(finalconcentration5mg/ml)wereaddedtothe supernatants,andthemixturewasincubatedat56 u Cfor2h. AfteradditionofproteinaseK(finalconcentration2.5mg/ml),the sampleswereincubatedat37 u Cfor2h.DNAwasprecipitated withtheadditionof10Mammoniumacetateandethanol, washedoncewith70%ethanolandthendissolvedinwaterand separatedbyelectrophoresisin1%agarosegel.Fluorescenceassistedcellsorting(FACS)assayApoptosiswasmeasuredusingtheAnnexin-Vdetectionkit accordingtothemanufacturer’sinstructions.Flowcytometric analysiswasperformedtomonitorthegreenfluorescenceofthe FITC-conjugatedAnnexin-VandtheredfluorescenceofDNAboundpropidiumiodide(PI).AlldatawereanalyzedwithaCell Questsoftware(BD).WesternblottinganalysisCellswereharvestedandwashedtwicewith1 6 PBS.Thecell pelletswereresuspendedinlysisbuffercontainingNonidetP-40 (10mMHEPES,pH7.4,2mMEGTA,0.5%NonidetP-40, 1mMNaF,1mMNaVO4,1mMphenylmethylsulfonylfluoride, 1mMdithiothreitol,50mg/mltrypsininhibitor,10mg/ml aprotinin,andleupeptin)andincubatedonicefor30min.After centrifugationat12,000 6 g at4 u Cfor15min,thesupernatantwas transferredtoanewtubeandtheproteinconcentrationwas determined.Equivalentsamples(20mgofprotein)weresubjected toSDS-PAGEon12%gels.Theproteinsweretransferredto nitrocellulosemembranesandprobedwiththeindicatedprimary antibodies,followedbytheappropriatesecondaryantibodies conjugatedwithhorseradishperoxidase.Immunoreactivebands weredetectedusingenhancedchemiluminescence(ECL)(Pierce, Rockford,IL).Themolecularsizesoftheproteinsdetectedwere determinedbycomparisonwithprestainedproteinmarkers(BioRad,Hercules,CA).Immuno-fluoresencestainingImmunostainingwasperformedasdescribedpreviously[19] withmodification.Briefly,cellsseededoncover-slipsinculture dishesweretreatedwithLexainthepresenceofCHXfor6h. Mito-Tracker(Red)wasdirectlyaddedtoculturesandincubated for15min.Cellswerewashedwith1 6 PBS,fixedwith3.7% paraformaldehyde(PFA)at37 u Cfor15min,andthenquenched with50mMNH4Cl.AfterwashingwithPBStwice,cellswere permeabilizedwith0.2%TritonX-100inPBS,thenblockedwith PBScontaining5%goatserumfor30min.Cellswerefurther incubatedwithprimaryantibodycytochromecovernightat4 u C, followedbystainingwithFITCconjugatedgoatanti-rabbitIgG (1:200)for2hatroomtemperature.Thecover-slipswerethen extensivelywashedandmountedinMowiolwithDAPIdye. FluorescentimageswereobtainedusinganOlympusfluorescent microscope.Baxcross-linkingInvitroproteincross-linkingwascarriedoutaspreviously described[20]withminormodification.Cellstreatedwith differentconditionswereharvested.Celllysateswereprepared afterlysiswithHEPES-CHAPSlysisbuffer(20mMHEPES pH7.2,150mMNaCl,2mMEDTA,10mMglucose,2% CHAPS).Bis(maleimido)hexane(BMH)substrateordimethyl sulfoxide(DMSO)wasaddedatafinalconcentrationof2mM. Thesampleswereincubatedatroomtemperaturefor30minand quenchedbytheadditionof1MTris-HCl(pH7.5).Aninvivo proteincrosslinkingassaywasperformedaspreviouslydescribed. FollowingtreatmentswithLexaorLexacombinedwithCHX, cellswerewashedwithconjugatingbuffercontaining150mM NaCl,20mMHEPES(pH7.2),1.5mMMgCl2,and10mM glucose.DSSinDMSOwasaddedforafinalconcentrationof 2mM.Afterreactionatroomtemperaturefor30min,thecrosslinkerwasquenchedbytheadditionof1MTris-HCl(pH7.5)for afinalconcentrationof20mM.Sampleswerethensolubilizedin 1%NP-40andcentrifugedat12,000 6 g for10min.A20-ml aliquotfromthe400mlcross-linkedlysatewasanalyzedby WesternblottingwithBax(N-20)polyclonalantibody.ROSdetectionTheintracellularproductionofROSwasmeasuredby fluorogenicprobedihydroethidium-(DHED)basedfluorescent staining.Briefly,aftervarioustreatments,cellswereincubated withDHED(5mM)for30minat37 u C,thenrapidlyrinsedtwice with1 6 PBSandobservedunderfluorescentmicroscope.siRNAknockdownsiRNAknockdownwasperformedaspreviouslydescribed[21]. Smart-poolpre-designedsiRNAduplexestargetedagainsthumanCHXandLexaCombination-InducedApoptosisinHCC PLoSONE|www.plosone.org2February2011|Volume6|Issue2|e16966

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Figure1.LexaandCHXcombinationtreatmentinducesapoptosisinHCCcells.A, Huh7cellsweretreatedwithTNFa (10ng/ml)orLexa (1mg/ml)forindicatedtimes.CelllysateswerepreparedandsubjectedtoWesternblottingtodetecttheexpressionofI k Ba andphospho-I k Ba b actinproteinlevelswereusedasanequalproteinloadingcontrol(Lexa,lexatumumab). B ,Huh7cellsweretreatedwithDMSO(Con),CHX(10mg/ml), Lexa(1mg/ml),oracombinationofLexa(1mg/ml)andCHX(10mg/ml).ApoptosiswasmeasuredbynucleardyeHoechst33258stainingtolabelDNA CHXandLexaCombination-InducedApoptosisinHCC PLoSONE|www.plosone.org3February2011|Volume6|Issue2|e16966

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HSP90mRNAwerefromCellSignalingTechnology(Beverly, MA).HCCcellswereplatedatadensityof1 6 106cells/wellin6wellplates(BDBioscience,SanDiego,CA).Nextday,cellswere transfectedwith100nMsiRNAduplexmixturesfor24hinthe presenceoflipo-fectamineRNAiMax(Invitrogen,Carlsbad,CA) accordingtothemanufacturer’sinstructions.Anon-specific randomsiRNA(CellSignalingBiotechnology,Beverly,MA)was alsotransfectedatthesameconcentrationascontrol.SubcellularfractionationThisassaywasperformedasdescribedpreviously[22].Cells wereharvestedandresuspendedinthreevolumesofhypotonic buffer(210mMsucrose,70mMmannitol,10mMHEPES (pH7.4),1mMEDTA)containing1mMphenylmethylsulfonyl fluoride(PMSF),50mg/mltrypsininhibitor,10mg/mlleupeptin, 5mg/mlaprotinin,and10mg/mlpepstatin.AftergentlehomogenizationwithaDouncehomogenizer,celllysateswerecentrifugedat1000 6 g for5mintoremoveunbrokencellsandnuclei. Thepostnuclearsupernatantwascentrifugedat10,000 6 g topellet themitochondria-enrichedheavymembranefraction.The supernatantwasfurthercentrifugedat100,000 6 g toobtainthe cytosolicfraction.A30-mlaliquotfromthe500mlcytosolic fractionwasanalyzedbyWesternblottingwithanti-cytochromec monoclonalantibody.DetectionofBaxconformationchangeThisassayswasperformedasdescribedpreviously[23].Cells werelysedwithChapslysisbuffer(10mMHEPES(pH7.4), 150mMNaCl,and1%Chaps)containing1mMphenylmethylsulfonylfluoride(PMSF),50mg/mltrypsininhibitor,10mg/ml leupeptin,1mg/mlaprotinin,and5mg/mlpepstatin.Thecell lysateswerenormalizedforproteincontentand500mgoftotal proteinwasincubatedwith2mgofanti-Bax6A7monoclonal antibodyin500mlofChapslysisbufferat4 u Cfor4h.Then, 40mlofproteinGplus-agarosewereaddedintothereactionsand incubatedat4 u Cforanadditional2h.Followingthreewashings inChapslysisbuffer,beadswereboiledinloadingbuffer,andthe conformationallychangedBaxproteinintheimmunoprecipitates wassubjectedtoSDS–PAGE(15%gel)andimmunoblotanalysis withanti-Baxpolyclonalantibodyasdescribedabove.Results LexainducesapoptosisinHCCcellsinthepresenceof CHXIncreasingevidenceindicatedthatmanycancertypesincluding breastcancercells,lungcancercells,andhepatomacells,could developresistancetoTRAIL-inducedapoptosisthroughDR5mediatedactivationofNFk Bsignalpathway[24–26].Here,to investigatewhetherLexatriggersNFk BactivationinHCCcell lines,Huh7cellsweretreatedwithTNFa orLexaforthe indicatedtimes.WesternblottingresultsindicatedthatTNFa inducedI k -Ba decreaseaftertreatmentfor15minandLexadid thatwithtreatmentfor60min.However,comparedwithTNFa LexacouldnotpromoteI k -Ba phosphorylationat60min (Fig.1A);thusLexa,aDR5specificmonoclonalantibodyunlike multifunctionalTNFa ,didnotactivatetheanti-apoptoticNFk B signalpathwaythroughbindingtoDR5.Then,wetestedwhether HCCcellsaresensitivetoLexa-inducedcelldeath.Huh7and LH86cellsweretreatedwithLexa(1mg/ml)forupto6h. ApoptosisassaysindicatedthatbothHCCcelllineswereresistant toLexa(seeLexa-treatedgroupsinFig.1B,1C,and1DforHuh7 cells,andFig.1Eand1FforLH86cells). IthasbeenshownthatchemotherapeuticreagentCHX,a proteinsynthesisinhibitor,canenhanceTRAIL-inducedapoptosis intumorcellssuchasrenalcancer[27],coloncancer[28],and lungcarcinoma[29].AscellsdidnotundergoapoptosiswithLexa singletreatment,weexaminedwhetherCHXcanalterthe insensitivityofHCCcellstoLexa.Apoptosisassaysindicatedthat pre-treatmentwithCHX(10mg/mlfor30min)followedbyLexa (1mg/mlfor6h)couldsignificantlyenhanceapoptosisinduction inbothHuh7andLH86cells(LexaandCHXcombination treatedgroupsinFig.1B,1C,and1DforHuh7cells,andFig.1E and1FforLH86cells).TofurtherconfirmLexa-induced apoptosiscouldbeenhancedbyCHXpre-treatment,weevaluated caspaseactivationandthecleavageofthepro-apoptoticprotein BidthroughWesternblottinganalysis.OurdataindicatedthatcotreatmentwithLexaandCHXfor6hsignificantlyinduced proteolyticprocessingofcaspase8,10,and3,aswellasBid (Fig.1G,rightpanel),butsingletreatmentwitheitherLexaor CHXcouldnotresultincleavedbandsoractivesubunitsfrom procaspase8,procaspase10,orprocaspase3.Additionally,Bid cleavagewasnotprogressivelyprocessedafter3h(Fig.1G,left panel),supportingtheassumptionthattheselivercancercellsare resistanttoLexa-inducedapoptosis.Toinvestigatethepossible signalingpathwaysunderlyingtheresistanceofHCCcellstoLexa, weusedthespecificinhibitorsNBD,PD98059,SB203580,and SP600125torespectivelyinhibittheIKK,MAPK,P38,andJNK signalingpathways.AsshowninFig.1H,incomparisonwith positivecontrolthatCHXandLexacombinationtreatmentinducedcaspase3cleavages,theotherfourinhibitorsdidnot reversetheinsensitivityofHCCcellstoLexa-inducedapoptosis. TheseresultssuggestthatLexaandCHXcombinationtreatment synergisticallyinducesapoptosisinHCCcells.LexaandCHXcombinationtreatmentinducescaspase dependentapoptosisDeathreceptor-mediatedapoptosiscanbeclassifiedaseither caspasedependentorcaspaseindependent[30,31].Toexamine whetherLexaandCHXcombination-inducedapoptosisisfragmentation(nuclearmorphologychanges).(Apoptoticcellsweremarkedwitharrows). C ,Huh7cellsweretreatedwithDMSO(Con),CHX(10mg/ ml),Lexa(1mg/ml),orcombinationtreatmentofLexa(1mg/ml)andCHX(10mg/ml)andapoptosiswasevaluatedthroughAnnexinVandPIdouble stainingbasedFACSanalysisusingtheAnnexin-Vassaykit(see’materialsandmethods’). D ,Thepercentageofapoptoticcellswerecharacterizedas thosethatstainedwithAnnexin-V.Datarepresentthemeanvaluesofthreeindependentexperiments(*p 0.05). E ,LH86cellsweretreatedwith DMSO(Con),CHX(10mg/ml),Lexa(1mg/ml),oracombinationofLexa(1mg/ml)andCHX(10mg/ml).ApoptosiswasmeasuredasinFig.1B (Apoptoticcellsweremarkedwitharrows). F ,LH86cellsweretreatedwithvariousstimuliasinFig.1E.ApoptosiswasmeasuredthroughFACS analysisasinFig.1Cand1DandstatisticalanalysiswasperformedtoshowpercentageofAnnexin-Vpositivecellsforapoptosisratio.Datarepresen t themeanvaluesofthreeindependentexperiments(**p 0.05). G ,Huh7cellsweretreatedwithLexa(1mg/ml)orcombinationofLexa(1mg/ml)and CHX(10mg/ml)forupto6handharvested.CelllysateswerepreparedforWesternblottingtodetectthecleavagesofcaspase8,caspase10,caspase 3,andBid. b -actinproteinlevelsweresetupasloadingcontrolforequaltotalproteinamounts. H ,Huh7cellstreatedwithindicatedconditionsfor 6hwereharvestedandcelllysateswerepreparedandsubjectedtoWesternblottinganalysis.Caspase3activationwasevaluatedthroughdetecting cleavedbandswithspecificantibody. b -actinproteinlevelsweresetupasloadingcontrolforequaltotalproteinamounts.Eachexperimentwas conductedintriplicateorduplicateandrepeatedtwiceindependently. doi:10.1371/journal.pone.0016966.g001 CHXandLexaCombination-InducedApoptosisinHCC PLoSONE|www.plosone.org4February2011|Volume6|Issue2|e16966

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controlledbycaspaseactivation,Huh7cellswerepre-incubated withpan-caspaseinhibitorz-VAD-FMK(50mM)for1h,followed byvariousstimuliasindicated.DNAladderassays(Fig.2A),DNA fragmentationanalysis(Fig.2Band2C)andFACSanalysis (Fig.2Dand2E)demonstratedthatapoptosisinducedby combinationtreatmentwithLexaandCHXweretotallyblocked bypan-caspaseinhibitorz-VAD-FMK,suggestingthatCHXsensitizedandLexa-inducedapoptosisiscaspasedependent. Meanwhile,wemeasuredtheeffectsofcaspase8specificinhibitor z-IETD-FMKonapoptosisinducedbyLexaandCHXcombinationtreatment.AsshowninFig.2A,2B,2C,2D,and2E, blockadeofcaspase8activationcompletelyabolishedLexaand CHXcombination-inducedapoptosisinHuh7cells.Taken together,theseresultssuggestthatcaspaseactivationplaysakey roleinLexa-mediatedapoptosisinductioninHCCcells.LexaandCHXcombinationtreatment-inducedapoptosis isregulatedbyROSToassesswhetherROSplaysamajorroleinregulatingLexa andCHXcombination-inducedapoptosis,HCCcellsweretreated withLexaandCHXfor6h.IntracellularROSlevelswere determinedbyaDHED-basedfluorescenceassay.Asshownin Fig.3A,LexaandCHXcombinationtreatmentinducedROS dramaticincreaseinapoptoticcells,butnotinnon-apoptoticcells, suggestingROSmaycontributetoCHX-andLexa-induced apoptosis.TofurtherdeterminewhethergenerationofROScan regulateapoptosis,N-acetyl-L-cysteine(NAC),aprecursorof reducedglutathione(GSH)widelyusedasathiol-containing antioxidanttoscavengeintracellularROS,wasaddedbefore administrationofLexaandCHX.AsshowninFig.3B,theROS increaseinducedbycombinedtreatmentofLexaandCHXwas completelynegated.Moreimportantly,apoptosisassaysindicated thattheblockadeofROSbyNACcanalsoprotectcellsagainst Lexa-andCHX-inducedapoptosis(Fig.3C,3D,3E,and3F). TheseresultssuggestthatLexaandCHXcombinationtreatmentinducedROSincreasecantriggerapoptosisinHCCcells.EffectsofcombinationtreatmentwithLexaandCHXon proteinexpressioninHCCcellsSinceCHXisaproteinsynthesisinhibitor,itmaysensitizeLexa toinduceapoptosisthroughdown-regulatinganti-apoptotic proteins.Weanalyzedtheapoptosisassociatedproteinexpression changesinHCCcellstreatedwithLexaaloneorcombinationtreatedwithLexaandCHXforupto6h.Westernblottingresults showedthattherewerenodecreasesinanti-apoptoticmolecules Bcl-xLorsurvivinexpression;ofthepro-apoptoticproteins,both BadandBimweredown-regulated,whileBakwasdown-regulated withLexasingletreatmentandup-regulatedwithcombination treatment.Additionally,proteinlevelsofpro-apoptoticBaxwere notaffected,andtheexpressionlevelsofdeathreceptorsDR4and Figure2.LexaandCHXcombinationtreatmentinducescaspase-dependentapoptosis.A ,Huh7cellsweretreatedwithdifferent conditionsasindicated.ApoptosiswastestedbyDNAladderassaysasdescribedin‘MaterialsandMethods’. B ,Huh7cellsweretreatedwithvarious stimuliasindicated(Lexa,(1mg/ml),CHX,(10mg/ml),orcaspase8inhibitorIETD(50mM)).ApoptoticcellswereexaminedasdescribedinFig.1B (representativeapoptoticcellsweremarkedwitharrows). C ,Huh7cellsweretreatedwithconditionsasindicatedandapoptoticratiowasdetermined bycountingcellswithapoptoticnucleiasdescribedin‘MaterialsandMethods’.Datarepresentthemeanvaluesofthreeindependentexperiments (*p 0.05). D ,Huh7cellsweretreatedwithvariousconditionsasindicated(Lexa,(1mg/ml),CHX,(10mg/ml),IETD(50mM),z-VAD(50mM)).Apoptosis wasevaluatedasinFig.1C. E ,Huh7cellsweretreatedwithdifferentconditionsasindicatedandapoptosispercentagewasdeterminedasinFig.1D. Datarepresentthemeanvaluesofthreeindependentexperiments(**p 0.05). doi:10.1371/journal.pone.0016966.g002 CHXandLexaCombination-InducedApoptosisinHCC PLoSONE|www.plosone.org5February2011|Volume6|Issue2|e16966

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Figure3.LexaandCHXcombinationtreatmentinducesapoptosisviaROS.A ,Huh7cellsweretreatedwiththecombinationofLexa(1mg/ml)and CHX(10mg/ml).NucleiwerestainedbyHoechst33258(right panel-blue,thinarrowmarksthenucleusinnon-ap optoticcells;thickarrowmarksapoptotic condensednuclei).IntracellularROSlevelwasassessedbyDHED-basedfluo rescentstaining(middlepanel-red,thinarrowmarksROSinnon-apoptot iccells, thickarrowmarksROSinapoptoticcells),andmergedfigure(left)toshowth atROSlevelsinnon-apoptoticcellsarelowerthanthatinapoptoticcell s(white boxesmarkedareaswereenlarged(Zoomin)). B ,Huh7cellsweretreatedwithDMSO(Con),Lexa(1mg/ml),CHX(10mg/ml),NAC(10mM),acombinationof Lexa(1mg/ml)andCHX(10mg/ml)intheabsenceorpresenceofNAC(10mM).Intracellul arROSlevelwasmeasuredwithDHEDdye(redfluorescence). C Huh7cellsweretreatedwithvari ousstimuliasindicatedandapoptosiswasassessedbyDNAladder. D ,Huh7cellstreatedwithDMSO(Con),Lexa(1mg/ml), CHX(10mg/ml),NAC(10mM),thecombinationofLexa(1mg/ml)andCHX(10mg/ml)intheabsenceorthepresenceofNAC(10mM)for6h.Thencells werestainedwithHoechst33258totestapoptoticcelldeath(r epresentativeapoptoticce llsaremarkedwitharrows). E and F ,Huh7cellsweretreatedwith conditionsasindicated.Apoptosi swasevaluatedasinFig.1Cand1D.Datarepresentth emeanvaluesofthreeindependentexperiments(**p 0.05). doi:10.1371/journal.pone.0016966.g003 CHXandLexaCombination-InducedApoptosisinHCC PLoSONE|www.plosone.org6February2011|Volume6|Issue2|e16966

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DR5showednosignificantchanges(Fig.4).Theseresultssuggest thattheinhibitorCHXcouldnotcompletelyblockprotein synthesisstimulatedbyLexainHCCcellsandthatapoptosis inducedbycombinationtreatmentofLexaandCHXmaybe mediatedbyBax/Bakactivation.CombinationtreatmentofLexaandCHXtriggersthe activationofBaxItiswellknownthatundervariouscellularstresses,proapoptoticBax/Bakcanbeactivatedthrougholigomerizationand conformationchange,resultinginmitochondria-dependentapoptosis[32–35].Therefore,weexaminedwhetherLexaandCHX co-treatmentcanactivateBaxthrougholigomerizationinHCC cells.OurresultsrevealedthatinthepresenceofCHX(10mg/ml), Baxdimerizationwasup-regulatedbyLexainHCCcells(Fig.5A, 5B,and5C).TofurthertestwhetherROSwasinvolvedinBax dimerization,cellswerepre-incubatedwithNACbeforethe additionofLexaandCHX.AsshowninFig.5C,pre-treatment withNACresultedinsignificantinhibitionofBaxdimerization inducedbycombinationtreatmentofLexaandCHX. NextweattemptedtotestthepossibilitythatintracellularROS couldinduceBaxtoundergoconformationchangeinHCCcells combination-treatedwithLexaandCHX.Tothisend,aBax monoclonalantibody6A7thatspecificallyrecognizesconformationallychangedBaxwasusedtodoimmunoprecipitation.As showninFig.5D,combinationtreatmentofLexaandCHXcould induceBaxconformationchangeinbothLH86andHuh7HCC cells.Asexpected,withtheadditionofantioxidantsNACtoblock ROS,conformationallychangedBaxwassignificantdecreased. Afteroligomerizationandconformationalchange,theactivated Baxcantranslocatetomitochondria,whichresultsincytochrome crelease.Inourstudy,weobservedtransientlytransfectedGFPBaxtranslocationtomitochondriaincellstreatedwithLexaand CHXfor6h(Fig.6A).Next,westudiedcytochromecrelease frommitochondriaincellswithcombinationaltreatmentofLexa andCHX.Immunofluorescencestainingresultsindicatedthatin cellstreatedwithLexaandCHX,cytochromecwasreleasedand diffusedintothecytosol(Fig.6B,center),butthisprocesswas blockedbyNAC(Fig.6B,rightpanel).Theseobservationswere consistentwiththeresultspresentedabove:LexaandCHXcotreatment-inducedapoptosiscanbenegatedbyNAC(Fig.3C,3D, 3E,and3F).Meanwhile,subcellularfractionationassayalso showedLexaandCHXcombinationtreatmentinducedcytochromecreleaseinHCCcells,whereas,NACaddition significantlycouldblockthisprocess(Fig.6C).Takentogether, theseresultssuggestthatLexaandCHXcombinationtreatmentinducedROSgenerationresultsinapoptosisviatheactivationof Bax.HSP90isinvolvedinLexaandCHXcombination treatment-inducedROSandapoptosisHeatshockprotein90(HSP90)mayconferasurvivaladvantage totumorcellsthroughassociationwithseveralregulatorsof apoptosis[36–38].HSP90bindingtoitsclientproteinmayprotect againstphysiologicaloxidativestress[39,40].Here,ourdata demonstratedthatCHXspecificallydecreasedHSP90protein expression(Fig.7A).Then,weaskedwhetherHSP90playsakey roleinregulatingLexaandCHXcombination-inducedapoptosis. UsingDMAG-17,aspecificHSP90inhibitortoblockitsactivity, weobservedthatHSP90inhibitionsignificantlyenhancedLexa treatment-inducedapoptoticcelldeath(Fig.7Band7D)andROS production(Fig.7Cand7D)inHCCcells,suggestingHSP90may beinvolvedinLexa-inducedROSgeneration.Ithasalsobeen reportedthatDMAG-17caninduceapoptosisintumorcells throughdown-regulatingIKKs,Mcl-1,andsurvivinexpression [41].However,inthisstudy,apoptosisassaysindicatedthatHCC cellsareinsensitivetoDMAG-17singletreatment(Fig.7Band 7D).FurtherWesternblottingresultsshowedthatproteinlevelsof IKKb weredecreased;I k Ba andtheNFk Btargetedproteins survivinwereincreased;Mcl-1,anotheranti-apoptoticprotein, wasnotaffectedbyDMAG-17treatment(Fig.7E).Finally,to furtherconfirmthatHSP90inactivationiscriticalforLexainducedapoptosis,HCCcellsweretransientlytransfectedwith HSP90specificsiRNAtoknockdownHSP90expressionandwere treatedwithLexa.ApoptosisassaysindicatedthatHSP90 knockdown(Fig.7F)significantlysensitizedLexa-inducedapoptosisinHCCcells(Fig.7G,and7H).Theseresultssuggestthat HSP90canbetargetedbyCHXtoregulatecellsensitivityto Lexa-inducedapoptosis.LexaandCHXcombinationtreatmenthasnoapoptotic effectsonnormalhumanhepatocytesAmajorbarrierfortheclinicaluseofTNFa orTRAIListheir potentialtoxicitytonormalhumancells[42–45].Inthisstudy,we examinedwhetherLexaandCHXcombinationtreatmentcan selectivelyinduceapoptosisinHCCcells.AsshowninFig.8A, combinationtreatmentwithLexa(1mg/ml)andCHX(10mg/ml) inducedcaspase8activationinHCCcells,butnotinnormal humanhepatocytes.Tofurtherevaluatewhynormalhepatocytes areinsensitivetoCHXandLexaco-treatment,weexaminedthe expressionofanti-apoptoticandpro-apoptoticproteinsinHCC cellsandnormalhumanhepatocytes.IncomparisonwithHCC cells,levelsofpro-apoptoticBaxandBakwereundetectablein normalhumanhepatocytes,anti-apoptoticBcl-xLlevelswere Figure4.ProteinexpressioninHCCcellstreatedwithLexaor thecombinationofLexaandCHX. LH86cellsweretreatedwith Lexa(1mg/ml)orpre-treatedwithCHX(10mg/ml)followedbyLexa (1mg/ml)forupto6h.Cellswereharvestedandcelllysateswere preparedforWesternblotting.Bcl-xL,survivin,Bim,Bad,Bak,Bax,DR4, andDR5weredetectedwithspecificantibodiesrespectively. b -actin wasdetectedwithantib -actinmouseantibodyforanequalprotein loadingcontrol. doi:10.1371/journal.pone.0016966.g004 CHXandLexaCombination-InducedApoptosisinHCC PLoSONE|www.plosone.org7February2011|Volume6|Issue2|e16966

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similarinallthecelllinestested,andBidexpressionlevelswere muchhigherinHCCcellsthanthoseinnormalhuman hepatocytes.However,DR5proteininnormalhumanhepatocytes showedmuchgreaterexpressioncomparedwiththatinHCCcells (Fig.8B).Theseobservationssuggestthattheinsensitivityof humannormallivercellstoLexaandCHXcombination treatmentisnotcontrolledbydeathreceptor5(DR5)expression levelonthecellularmembrane,butratherdeterminedbythe expressionlevelsofintracellularpro-apoptoticproteins,including Bax,Bak,andBid.Takentogether,theseresultssuggestthatCHX canselectivelyenhanceLexatoinduceapoptosisinHCCcells.DiscussionInthepresentstudy,weinvestigatedapoptosisinducedby combinationtreatmentwithLexaandCHXinresistantHCC cells.Ourresultsdemonstratedthatpre-incubationwithCHX couldrenderresistantHCCcellssensitivetomonoclonalantibody Lexa-inducedapoptosis.LexaandCHXcombinationtreatmentinducedapoptosiswastriggeredbyintracellularROSproduction. ROSaccumulationincellsresultedinpro-apoptoticBax/Bak activationthrougholigomerizationandconformationchange. HSP90mayactasaswitchcontrolledbyCHXtoregulate intracellularROSlevelsuponcelldeathstimulation.More interestingly,thecombinationtreatmentdidnotinduceapoptosis innormalhumanhepatocytescomparingwiththeHCCcells underidenticalconditions.Thesefindingsarepromisingforthe clinicaluseofCHXandLexaasanovelchemotherapeuticoption inthetreatmentofpatientswithHCC. Lexa,ahumanizedmonoclonalantibody,specificallybindsto DR5toactivatedeathsignalsandhasalongerhalflife invivo renderingitasapotentialkilleroftumorcells.Here,weused Figure5.LexaandCHXco-treatment-inducedROSincreasetriggersBaxactivation.A ,LH86cellsweretreatedwithLexa(1mg/ml)orpretreatedwithCHX(10mg/ml)followedbyLexa(1mg/ml)forupto6h.Cellswerethentreatedwiththecross-linkingagentDSS.Baxproteinswere resolvedbySDS-PAGEanddetectedbyWesternblottingusingaBaxpolyclonalantibody.A23kDabandrepresentsBaxmonomers,andbandsat 46kDa,detectedonlyinthepresenceofDSS,representBaxhomodimers.RelativeintensityofBaxdimerbandswasanalyzedbyImageJsoftware. B Huh7cellsweretreatedwithacombinationofLexa(1mg/ml)andCHX(10mg/ml)forupto6h.Celllysateswerepreparedandincubatedwithcross linkerBMHasdescribedinMaterialsandMethods.Celllysatesandcrosslinkermixturewasboiledwith1 6 SDSloadingbufferandBaxoligomerization wasanalyzedbyWesternblotting.TheBaxdimerwasrecognizedbyBaxpolyclonalantibody.RelativeintensityofBaxdimerbandswasquantifiedby ImageJsoftware. C ,Huh7cellstreatedwithconditionsasindicatedandwereharvestedandextractswerepreparedforWesternblottinganalysisto analyzeBaxdimerformation.RelativeintensityofBaxdimerbandswasqualifiedbyImageJsoftware.D,LH86(rightpanel)orHuh7(leftpanel)cells treatedwithvariousstimuliasindicatedwereharvestedandlysedinChapslysisbufferandsubjectedtoimmunoprecipitationwithanti-Bax6A7 antibodyfordetectionofconformationallychangedBaxprotein.CelllysateobtainedbyNP-40lysiswasusedaspositivecontrol.Inaddition,theto tal lysateswereapplieddirectlytoSDS-PAGE/Westernblottinganalysiswithspecificanti-Baxpolyclonalantibodyandseveredasloadingcontrol. doi:10.1371/journal.pone.0016966.g005 CHXandLexaCombination-InducedApoptosisinHCC PLoSONE|www.plosone.org8February2011|Volume6|Issue2|e16966

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HCCcellsasamodeltoexaminetheapoptoticeffectsofLexa. Ourresultsshowedthatfirst,comparedwithpositivecontrol TNFa ,I k Ba phosphorylationcouldnotbedetectedinHCC cellsaftertreatmentwithLexa,suggestingthatspecificbindingto DR5avoidsactivationoftheNFk BsignalingpathwayinHCC cells.Thisdiffersfromtheothertwocytokines(TNFa and TRAIL)thatcaninduceactivationofNFk Btoproduceantiapoptoticsignalsthroughbindingtomembrane[46–48].Nevertheless,wedidobserveLexacouldreduceI k Ba expressionin HCCcells,ofwhichthesignificanceremainstobeclarifiedinthe future.Second,singletreatmentofLexashowednolethaltoxicity toHCCcells,whichisconsistentwithpreviousstudiesreporting thatHCCcellsareresistanttoTRAIL[49–51].Third,we observedthatpre-incubationwithCHXcouldsensitizeHCCcells toLexa-inducedapoptosis.Itisthereforelikelythatbesides inhibitionofNFk B,therearesomeothermechanismsthrough whichCHXcansensitizeLexatoinduceapoptosisinHCCcells. SeveralgroupshavereportedthatactivationofDR5could signalnon-apoptoticpathwaysthroughIKK,MAPK,P38,and JNKsignalingpathwaystoneutralizeTRAIL-inducedapoptosis [52–54].Ourdatademonstratedthatspecificinhibitors,including NBD,PD98059,SB203580,andSP600125,thatrespectively blocktheabovementionedanti-apoptoticsignalingpathways, failedtoreversetheresistanceofHCCcellstoapoptosisinduced byLexa,suggestingthatCHXsensitizingcellstoLexalethal toxicitydoesnotrequireblockinganti-apoptoticeffectsfromthe activationofthesesignalingpathways. Asapan-proteinsynthesisinhibitor,CHXmayinhibitthe activityofsomeapoptosisassociatedproteinsbydecreasingtheir expressions.However,ourresultsdemonstratedthatCHXcould notcompletelyblockproteinsynthesisbecauseunderCHXand Lexaco-treatment,Bakproteinwassignificantlyincreased, suggestingthatBax/Bakmaybeactivatedtoresultinapoptotic celldeath.Asexpected,ourstudiesshowedthatLexaandCHX co-treatmentinducedBaxoligomerization,conformational change,andtranslocation,whichresultedincytochromecrelease andapoptosis.IthasbeenreportedthatBax/Bakcanbeactivated byintracellularROStoinducecelldeathinhumancolorectal cancercells[55,56].Similarlyinthisstudy,whenHCCcellswere co-treatedwithCHXandLexa,weobservedROSincreasein apoptoticcells,butnotinnon-apoptoticones.Moreover,the blockadeofROSwithNACnotonlypreventedBaxoligomerizationandconformationalchanges,butalsodecreasedapoptosis significantly.TheseresultssuggestthatgenerationofROSplaysa majorroleinCHXandLexaco-treatment-inducedapoptosis. Ourfindingsareinagreementwithastudythatreportedthatthe generationofROSinducedmitochondria-dependentapoptosisin coloncancercellsthroughBaxactivation[57]. Incomparisontotreatmentwitheitheragentalone,LexainducedROSaccumulationoccurredonlyinthepresenceof CHX,whichsuggeststhatCHXmaymodifysomeintracellular signalingpathway(s)ormolecule(s)thatactasaswitchtocontrol LexatoinduceROSgenerationOurresultsbasedonthis hypothesisindicatedthatpretreatmentofcellswithCHX decreasedHSP90expressionsignificantly,andHSP90inactivation byspecificinhibitorDMAG-17notonlyresultedinaLexainducedROSincrease,butalsosensitizedLexa-inducedapoptosis. Moreover,inhibitionofHSP90activitythroughgenesilencingby specificsiRNAsignificantlysensitizedLexa-inducedapoptosis. Thesefindingsarealsoinagreementwithareportshowingthat blockadeofHSP90sensitizedprostatecancercellstoTRAIL[58]. Meanwhile,wehaveobservedthatDMAG-17singletreatment couldnotinduceHCCcellstoundergoapoptosis.Furtheranalysis indicatedthatalthoughIKKb wasdecreasedbyDMAG-17,the NFk BtargetedproteinsincludingsurvivinandMcl-1werenot down-regulated.Thisisincontrasttoapreviousstudywhich reportedthatDMAG-17inducedapoptosisinchroniclymphocyticleukemia(CLL)cellsbydecreasingNFk Btargetedgene transcription[59]. Inordertobringthedeathreceptoragonistsclosertoclinical therapy,weevaluatedthesafetyofCHXandLexacombination treatmentinnormalhumanhepatocytes.Ourdataindicatedthat normalhumanhepatocytesareresistanttothiscombination treatment.ComparisonofDR5expressionlevelsandLexa Figure6.LexaandCHXco-treatmentinducesBaxtranslocation andcytochromecreleaseinHCCcells.A ,Huh7cellswere transfectedwithGFP-N1-Baxplasmid(0.25mg/ml).After10hposttransfection,cellsweretreatedwithLexa(1mg/ml)inthepresenceof CHX(10mg/ml)ornot.GFP-N1-Baxtranslocation(green)wasobserved underfluorescentmicroscope.MitochondriawerestainedwithMito Tracker(red)CMXRos(con,control;mito,mitochondria).Nuclearwas stainedwithDAPI(blue). B ,Huh7cellsculturedoncoverslipswere treatedwithLexa(1mg/ml)andCHX(10mg/ml)inthepresence(right panel)orabsence(middlepanel)ofNAC(10mM)for6h.Fluorescence immunostainingwasperformedtolabelcytochromecwithanticytochromecprimaryantibodyandFITCconjugatedgoatanti-mouse secondaryantibody(green). C ,Huh7cellswereexposedtodifferent conditionsasindicatedandsubjectedtosubcellularfractionation.Then, thecytosolicfractionswereanalyzedbyWesternblottingwithantibody specificforcytochromec. b -actinwasdetectedwithantib -actinmouse antibodyforequalproteinloadingcontrol. doi:10.1371/journal.pone.0016966.g006 CHXandLexaCombination-InducedApoptosisinHCC PLoSONE|www.plosone.org9February2011|Volume6|Issue2|e16966

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sensitivityofthenormalhepatocytesandHCCcellsusedinthis studydidnotrevealaconsistentrelationship,suggestingthatLexa sensitivityisnotdependentondeathreceptorexpressionlevelsand thatotherintracellularmechanismsmaycontroldeathsignal transductioninresistantcells.Asdeathreceptor-mediatedcell deathcanberegulatedbyBcl-2familyBH3onlyproteins,suchas Bax,Bak,andBid.Here,wefoundthatexpressionlevelsofthe BH3onlyproteinsBaxandBakwereundetectableinnormal humanhepatocytes,whiletheHCCcellsshowedclearhigh expressionoftheseproteins.Thisobviousdifferencemaybeakey elementinprotectingnormalcellsagainstthetoxicityofLexaand CHXcombinationtreatment.Inaddition,toextendtheuseof LexatotreatHCC,wetestedtheeffectsofcombinationofthe deathreceptoragonistswithotherchemotherapeuticreagentssuch ascisplatinordoxorubicin.Ourdatashowedthatonly doxorubicincouldsensitizeLexa-inducedapoptosisinHCCcells (seeFigureS1andFigureS2).Theunderlyingmechanismremains tobeinvestigated. Insummary,wehavefoundthatHCCcellsaregenerally resistanttoLexa,andCHXcansensitizeHCCcellstoLexaFigure7.HSP90isinvolvedinLexaandCHXco-treatment-inducedROSincreaseandapoptosis.A ,Huh7cellsweretreatedwithCHX (10mg/ml)ornot.CelllysateswerepreparedforWesternblotting.Anti-HSP90,anti-Bax,anti-Bcl-xLpolyclonalantibodieswereusedtodetectHSP90, BaxandBcl-xLproteinlevelsrespectively. b -actinwasusedasanequalproteinloadingcontrol.RelativeintensitiesofbandsinFig.7Awereanalyzed byImageJsoftware. B ,Huh7cellsweretreatedwithDMSO(Con),Lexa(1mg/ml),CHX(10mg/ml),thecombinationofLexa(1mg/ml)andCHX (10mg/ml),DMAG(0.5mg/ml),orthecombinationofLexa(1mg/ml)andDMAG(0.5mg/ml)for6h(CellsweretreatedwithcombinationofLexaand CHXaspositivecontrol).Apoptosiswasassessedbynuclearmorphologicalchanges. C ,Huh7cellsweretreatedasinFig.7BandintracellularROS levelwasdetectedbyfluorescentstainingwithDFHD(CellsweretreatedwithcombinationofLexaandCHXaspositivecontrol). D ,Huh7cellswere treatedwithvariousconditionsasindicated(Ctrl,control;L,Lexa(1mg/ml);C,CHX(10mg/ml),D,DMAG-17(0.5mg/ml);L + D,thecombinationofLexa (1mg/ml)andDMAG-17(0.5mg/ml);L + C,thecombinationofLexa(1mg/ml)andCHX(10mg/ml).CellswithbothROSpositiveandDNA fragmentationwerecounted.Datarepresentthemeanvaluesofthreeindependentexperiments(*p 0.05;**p 0.05). E ,Huh7cellsweretreated withdifferentdosageofDMAG-17for24h.CellswereharvestedandcelllysateswerepreparedforWesternblotting.IKKb ,I k Ba ,survivinandMcl-1 proteinlevelsweredetectedusingspecificantibodiesrespectively. b -actinwasdetectedbyantib -actinmouseantibodyforanequalproteinloading control. F ,Huh7cellsweretransfectedwithsynthesizedrandomsiRNA(ascontrol)andHSP90specificsiRNA,andafter48hpost-transfectioncells weresubjectedtoWesternblottinganalysiswithHSP90polyclonalantibody. b -actinwasusedasanequalproteinloadingcontrol.Theintensityof immunoblotbandsFig.7FwasanalyzedbyImageJsoftware. G ,Huh7cellsweretransfectedwithsynthesizedrandomcontrolsiRNAandHSP90 siRNA,and48hpost-transfection,cellsweretreatedwithLexaornotandthensubjectedtoHoechststainingforapoptosisanalysis(RC,random siRNAcontrol;RS + L,randomsiRNAcellstreatedwithLexa(1mg/ml);HSC,HSP90siRNAcontrol;HS + L,HSP90siRNAcellstreatedwithLexa(1mg/ml)). H ,Huh7cellstreatedasinFig.7GwereharvestedandsubjectedtoWesternblottinganalysiswithcaspase3(forcleavedbandsonly)polyclonal antibody. b -actinwasdetectedwithantib -actinmouseantibodyforanequalproteinloadingcontrol. doi:10.1371/journal.pone.0016966.g007 CHXandLexaCombination-InducedApoptosisinHCC PLoSONE|www.plosone.org10February2011|Volume6|Issue2|e16966

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inducedapoptosis.Theunderlyingmechanismofactionfor combinationtreatmentisrelatedtoROSgenerationandBax activation.Ourstudyalsodemonstratesthatnormalhepatocytes areresistanttothecombinationtherapy.Wesuggestthat administrationofCHXfollowedbyLexashouldbeconsidered forfurtherinvestigationforthetreatmentofpatientswithHCC.SupportingInformationFigureS1Cisplatin(Cis)couldnotsensitizeLexatumumab(Lexa)-inducedapoptosisinHCCcells. Huh7cells weretreatedwithDMSO(control),Lexa(1mg/ml),differentdoses ofcisplatin(1-20 m g/ml),oracombinationofLexaandcisplatinas indicated.ApoptosiswasmeasuredbynucleardyeHoechst33258 stainingtolabelDNAfragmentation(nuclearmorphological changes).NotethatLexa,cisplatin,orthecombinationtreatment ofLexaandcisplatinhadnoapoptotictoxicitytoHCCcells. (TIF)FigureS2Doxorubicin(Dox)sensitizesLexatumumab (Lexa)-inducedapoptosisinHCCcells. Huh7cellswere treatedwithDMSO(control),Lexa(1mg/ml),differentdosesof doxorubicin(0.25-2 m M),oracombinationofLexaand doxorubicinasindicated.Apoptosiswasmeasuredbynuclear dyeHoechst33258stainingtolabelDNAfragmentation(nuclear morphologicalchanges).NotethateitherLexaordoxorubicin couldnotinduceapoptosisinHCCcells.However,the combinationtreatmentofLexaanddoxorubicininduced significantapoptosisinHCCcells. (TIF)AcknowledgmentsWegivethankstoDr.RobinHumphreysandDr.ManiSubramanianof HumanGenomeScienceforprovidingLexaandTonWangforeditingthe paper.AuthorContributionsConceivedanddesignedtheexperiments:XXZCL.Performedthe experiments:XXZ.Analyzedthedata:XXZCL.Contributedreagents/ materials/analysistools:XXZCL.Wrotethepaper:XXZCL.References1.JemalA,SiegelR,WardE,HaoY,XuJ,etal.(2009)Cancerstatistics,2009. CACancerJClin59:225–249. 2.DeufficS,PoynardT,BuffatL,ValleronAJ(1998)Trendsinprimaryliver cancer.Lancet351:214–215. 3.RussoM,MupoA,SpagnuoloC,RussoGL(2010)Exploringdeathreceptor pathwaysasselectivetargetsincancertherapy.BiochemPharmacol80:674–682. 4.WiezorekJ,HollandP,GravesJ(2010)Deathreceptoragonistsasatargeted therapyforcancer.ClinCancerRes16:1701–1708. 5.CamidgeDR,HerbstRS,GordonMS,EckhardtSG,KurzrockR,etal.(2010)A phaseIsafetyandpharmacokineticstudyofthedeathreceptor5agonisticantibody PRO95780inpatientswithadvancedmalignancies.ClinCancerRes16:1256–1263. 6.Forero-TorresA,ShahJ,WoodT,PoseyJ,CarlisleR,etal.(2010)PhaseItrial ofweeklytigatuzumab,anagonistichumanizedmonoclonalantibodytargeting deathreceptor5(DR5).CancerBiotherRadiopharm25:13–19. 7.LeeSH,ParkDW,SungES,ParkHR,KimJK,etal.(2010)Humanizationof anagonisticanti-deathreceptor4singlechainvariablefragmentantibodyand avidity-mediatedenhancementofitscelldeath-inducingactivity.MolImmunol 47:816–824. 8.SungES,ParkKJ,LeeSH,JangYS,ParkSK,etal.(2009)Anovelagonistic antibodytohumandeathreceptor4inducesapoptoticcelldeathinvarious tumorcellswithoutcytotoxicityinhepatocytes.MolCancerTher8:2276– 2285. 9.ZhangL,ZhangX,BarrisfordGW,OlumiAF(2007)Lexatumumab(TRAILreceptor2mAb)inducesexpressionofDR5andpromotesapoptosisinprimary andmetastaticrenalcellcarcinomainamouseorthotopicmodel.CancerLett 251:146–157. 10.MenoretE,Gomez-BougieP,Geffroy-LuseauA,DanielsS,MoreauP,etal. (2006)Mcl-1LcleavageisinvolvedinTRAIL-R1-andTRAIL-R2-mediated Figure8.LexaandCHXco-treatmenthasnolethaleffectsonnormalhumanhepatocytes.A ,Freshlypreparednormalhumanprimary hepatocyteswereseededonsix-wellplatesandtreatedaftertwodayswithLexa(1mg/ml)andCHX(10mg/ml)forupto6h.Huh7cellstreated underthesameconditionswereusedasapositivecontrol.Caspase8activationwasmeasuredthroughdetectingthecleavedbandsbyWestern blotting. B ,CelllysatesfromnormalhumanhepatocytesandHCCcellsLH86andHuh7werepreparedandWesternblottingwasperformedtodetect Bcl-xL,Bax,Bak,Bim,Bid,andDR5withspecificantibodies. doi:10.1371/journal.pone.0016966.g008 CHXandLexaCombination-InducedApoptosisinHCC PLoSONE|www.plosone.org11February2011|Volume6|Issue2|e16966

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apoptosisinducedbyHGS-ETR1andHGS-ETR2humanmAbsinmyeloma cells.Blood108:1346–1352. 11.BelyanskayaLL,MartiTM,Hopkins-DonaldsonS,KurtzS,Felley-BoscoE, etal.(2007)HumanagonisticTRAILreceptorantibodiesMapatumumaband Lexatumumabinduceapoptosisinmalignantmesotheliomaandactsynergisticallywithcisplatin.MolCancer6:66. 12.LusterTA,CarrellJA,McCormickK,SunD,HumphreysR(2009) Mapatumumabandlexatumumabin duceapoptosisinTRAIL-R1and TRAIL-R2antibody-resistantNSCLCcelllineswhentreatedincombination withbortezomib.MolCancerTher8:292–302.1535-7163. 13.MaddipatlaS,Hernandez-IlizaliturriFJ,KnightJ,CzuczmanMS(2007) AugmentedantitumoractivityagainstB-celllymphomabyacombinationof monoclonalantibodiestargetingTRAIL-R1andCD20.ClinCancerRes13: 4556–4564. 14.SmithMR,JinF,JoshiI(2007)Bortezomibsensitizesnon-Hodgkin’slymphoma cellstoapoptosisinducedbyantibodiestotumornecrosisfactorrelated apoptosis-inducingligand(TRAIL)receptorsTRAIL-R1andTRAIL-R2.Clin CancerRes13:5528s–5534s. 15.LuM,StroheckerA,ChenF,KwanT,BosmanJ,etal.(2008)Aspirinsensitizes cancercellstoTRAIL-inducedapoptosisbyreducingsurvivinlevels.Clin CancerRes14:3168–3176. 16.GongJ,YangD,KohanimS,HumphreysR,BroemelingL,etal.(2006)Novel invivoimagingshowsup-regulationofdeathreceptorsbypaclitaxeland correlateswithenhancedantitumoreffectsofreceptoragonistantibodies.Mol CancerTher5:2991–3000.1535-7163. 17.NawrockiST,CarewJS,DouglasL,ClevelandJL,HumphreysR,etal.(2007) Histonedeacetylaseinhibitorsenhancelexatumumab-inducedapoptosisviaa p21Cip1-dependentdecreaseinsurvivinlevels.CancerRes67:6987–6994. 18.HerrmannM,LorenzHM,VollR,GrunkeM,WoithW,etal.(1994)Arapid andsimplemethodfortheisolationofapoptoticDNAfragments.NucleicAcids Res22:5506–5507. 19.MoraR,AbschuetzA,KeesT,DokicI,JoschkoN,etal.(2009)TNF-alpha-and TRAIL-resistantgliomacellsundergoautophagy-dependentcelldeathinduced byactivatedmicroglia.Glia57:561–581. 20.SundararajanR,WhiteE(2001)E1B19KblocksBaxoligomerizationand tumornecrosisfactoralpha-mediatedapoptosis.JVirol75:7506–7516. 21.TianC,MurrinLC,ZhengJC(2009)Mitochondrialfragmentationisinvolved inmethamphetamine-inducedcelldeathinrathippocampalneuralprogenitor cells.PLoSOne4:e5546.10.1371/journal.pone.0005546[doi]. 22.ChenQ,GongB,AlmasanA(2000)Distinctstagesofcytochromecreleasefrom mitochondria:evidenceforafeedbackamplificationlooplinkingcaspase activationtomitochondrialdysfunctioningenotoxicstressinducedapoptosis. CellDeathDiffer7:227–233. 23.YamaguchiH,ParanawithanaSR,LeeMW,HuangZ,BhallaKN,etal.(2002) EpothiloneBanalogue(BMS-247550)-mediatedcytotoxicitythroughinduction ofBaxconformationalchangeinhumanbreastcancercells.CancerRes62: 466–471. 24.ChenJJ,ChouCW,ChangYF,ChenCC(2008)Proteasomeinhibitorsenhance TRAIL-inducedapoptosisthroughtheintronicregulationofDR5:involvement ofNF-kappaBandreactiveoxygenspecies-mediatedp53activation.JImmunol 180:8030–8039. 25.LinT,DingZ,LiN,XuJ,LuoG,etal.(2010)2-tellurium-bridged{beta}cyclodextrin,athioredoxinreductaseinhibitor,sensitizeshumanbreastcancer cellstoTRAIL-inducedapoptosisthroughDR5inductionandNF-{kappa}B suppression.Carcinogenesis.bgq234[pii];10.1093/carcin/bgq234[doi]. 26.ShigenoM,NakaoK,IchikawaT,SuzukiK,KawakamiA,etal.(2003) Interferon-alphasensitizeshumanhepatomacellstoTRAIL-inducedapoptosis throughDR5upregulationandNF-kappaBinactivation.Oncogene22: 1653–1662. 27.BrooksAD,SayersTJ(2005)ReductionoftheantiapoptoticproteincFLIP enhancesthesusceptibilityofhumanrenalcancercellstoTRAILapoptosis. CancerImmunolImmunother54:499–505. 28.HernandezA,WangQD,SchwartzSA,EversBM(2001)Sensitizationof humancoloncancercellstoTRAIL-mediatedapoptosis.JGastrointestSurg5: 56–65. 29.TakenagaK(1984)Enhancementoflung-colonizingabilityofclonedlowmetastaticLewislungcarcinomacellsbytreatmentwithhighlypolar compounds.IntJCancer34:83–89. 30.GrotzerMA,EggertA,ZuzakTJ,JanssAJ,MarwahaS,etal.(2000)Resistance toTRAIL-inducedapoptosisinprimitiveneuroectodermalbraintumorcells correlateswithalossofcaspase-8expression.Oncogene19:4604–4610. 31.SeolDW,LiJ,SeolMH,ParkSY,TalanianRV,etal.(2001)Signalingevents triggeredbytumornecrosisfactor-relatedapoptosis-inducingligand(TRAIL): caspase-8isrequiredforTRAIL-inducedapoptosis.CancerRes61:1138–1143. 32.ChaudhariAA,SeolJW,KimSJ,LeeYJ,KangHS,etal.(2007)Reactive oxygenspeciesregulateBaxtranslocationandmitochondrialtransmembrane potential,apossiblemechanismforenhancedTRAIL-inducedapoptosisby CCCP.OncolRep18:71–76. 33.delMM-S,Corbalan-GarciaS,Gomez-FernandezJC(2001)Conformationof theC-terminaldomainofthepro-apoptoticproteinBaxandmutantsandits interactionwithmembranes.Biochemistry40:9983–9992. 34.KimS,LeeTJ,LeemJ,ChoiKS,ParkJW,etal.(2008)Sanguinarine-induced apoptosis:generationofROS,down-regulationofBcl-2,c-FLIP,andsynergy withTRAIL.JCellBiochem104:895–907. 35.NechushtanA,SmithCL,HsuYT,YouleRJ(1999)ConformationoftheBax C-terminusregulatessubcellularlocationandcelldeath.EMBOJ18: 2330–2341. 36.GaoF,HuXY,XieXJ,XuQY,WangYP,etal.(2010)Heatshockprotein90 protectsratmesenchymalstemcellsagainsthypoxiaandserumdeprivationinducedapoptosisviathePI3K/AktandERK1/2pathways.JZhejiangUniv SciB11:608–617. 37.PerottiC,LiuR,ParuselCT,BocherN,SchultzJ,etal.(2008)Heatshock protein-90-alpha,aprolactin-STAT5targetgeneidentifiedinbreastcancer cells,isinvolvedinapoptosisregulation.BreastCancerRes10:R94. 38.ZhaoC,WangE(2004)Heatshockprotein90suppressestumornecrosisfactor alphainducedapoptosisbypreventingthecleavageofBidinNIH3T3 fibroblasts.CellSignal16:313–321. 39.LiuDH,YuanHY,CaoCY,GaoZP,ZhuBY,etal.(2007)Heatshockprotein 90actsasamolecularchaperoneinlate-phaseactivationofextracellularsignalregulatedkinase1/2stimulatedbyoxidativestressinvascularsmoothmuscle cells.ActaPharmacolSin28:1907–1913. 40.Rodriguez-SinovasA,BoenglerK,CabestreroA,GresP,MorenteM,etal. (2006)Translocationofconnexin43totheinnermitochondrialmembraneof cardiomyocytesthroughtheheatshockprotein90-dependentTOMpathway anditsimportanceforcardioprotection.CircRes99:93–101. 41.RoblesAI,WrightMH,GandhiB,FeisSS,HaniganCL,etal.(2006)Scheduledependentsynergybetweentheheatshockprotein90inhibitor17-(dimethylaminoethylamino)-17-demethoxygeldanamycinanddoxorubicinrestoresapoptosistop53-mutantlymphomacelllines.ClinCancerRes12:6547–6556.42.ChopraDP,MenardRE,JanuszewskiJ,MattinglyRR(2004)TNF-alphamediatedapoptosisinnormalhumanprostateepithelialcellsandtumorcell lines.CancerLett203:145–154. 43.KoornstraJJ,KleibeukerJH,vanGeelenCM,RijckenFE,HollemaH,etal. (2003)ExpressionofTRAIL(TNF-relatedapoptosis-inducingligand)andits receptorsinnormalcolonicmucosa,adenomas,andcarcinomas.JPathol200: 327–335. 44.MoriE,ThomasM,MotokiK,NakazawaK,TaharaT,etal.(2004)Human normalhepatocytesaresusceptibletoapoptosissignalmediatedbybothTRAILR1andTRAIL-R2.CellDeathDiffer11:203–207. 45.NesterovA,IvashchenkoY,KraftAS(2002)Tumornecrosisfactor-related apoptosis-inducingligand(TRAIL)triggersapoptosisinnormalprostate epithelialcells.Oncogene21:1135–1140. 46.HsuH,XiongJ,GoeddelDV(1995)TheTNFreceptor1-associatedprotein TRADDsignalscelldeathandNF-kappaBactivation.Cell81:495–504. 47.LirdprapamongkolK,SakuraiH,SuzukiS,KoizumiK,PrangsaengtongO, etal.(2010)VanillinEnhancesTRAIL-InducedApoptosisinCancerCells throughInhibitionofNF-{kappa}BActivation.InVivo24:501–506. 48.SchutzeS,WiegmannK,MachleidtT,KronkeM(1995)TNF-induced activationofNF-kappaB.Immunobiology193:193–203. 49.HeS,ChenY,ChenX,ZhaoY,WangH,etal.(2005)Antitumoreffectsof solubleTRAILinhumanhepatocellularcarcinoma.JHuazhongUnivSci TechnologMedSci25:51–54. 50.HeSQ,ChenY,ChenXP,ZhangWG,WangHP,etal.(2003)[Antitumor effectsofsolubleTRAILinhumanhepatocellularcarcinoma].ZhonghuaZhong LiuZaZhi25:116–119. 51.YamanakaT,ShirakiK,SugimotoK,ItoT,FujikawaK,etal.(2000) ChemotherapeuticagentsaugmentTRAIL-inducedapoptosisinhuman hepatocellularcarcinomacelllines.Hepatology32:482–490. 52.AbdollahiT,RobertsonNM,AbdollahiA,LitwackG(2005)Inhibitionof TRAIL-inducedapoptosisbyIL-8ismediatedbythep38-MAPKpathwayin OVCAR3cells.Apoptosis10:1383–1393. 53.DieterleA,OrthR,DaubrawaM,GrotemeierA,AlersS,etal.(2009)TheAkt inhibitortriciribinesensitizesprostatecarcinomacellstoTRAIL-induced apoptosis.IntJCancer125:932–941. 54.MahalingamD,KeaneM,PirianovG,MehmetH,SamaliA,etal.(2009) DifferentialactivationofJNK1isoformsbyTRAILreceptorsmodulate apoptosisofcoloncancercelllines.BrJCancer100:1415–1424. 55.HuangF,NieC,YangY,YueW,RenY,etal.(2009)SeleniteinducesredoxdependentBaxactivationandapoptosisincolorectalcancercells.FreeRadic BiolMed46:1186–1196. 56.PatsosHA,GreenhoughA,HicksDJ,AlKM,CollardTJ,etal.(2010)The endogenouscannabinoid,anandamide,inducesCOX-2-dependentcelldeathin apoptosis-resistantcoloncancercells.IntJOncol37:187–193. 57.SuCC,LinJG,LiTM,ChungJG,YangJS,etal.(2006)Curcumin-induced apoptosisofhumancoloncancercolo205cellsthroughtheproductionofROS, Ca2 + andtheactivationofcaspase-3.AnticancerRes26:4379–4389. 58.MaY,LakshmikanthanV,LewisRW,KumarMV(2006)Sensitizationof TRAIL-resistantcellsbyinhibitionofheatshockprotein90withlow-dose geldanamycin.MolCancerTher5:170–178. 59.HertleinE,WagnerAJ,JonesJ,LinTS,MaddocksKJ,etal.(2010)17-DMAG targetsthenuclearfactor-kappaBfamilyofproteinstoinduceapoptosisin chroniclymphocyticleukemia:clinicalimplicationsofHSP90inhibition.Blood 116:45–53.CHXandLexaCombination-InducedApoptosisinHCC PLoSONE|www.plosone.org12February2011|Volume6|Issue2|e16966


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