HT抗增殖和抗凋亡

Cytotechnology(2009)59:45–53DOI10.1007/s10616-009-9191-2 SPECIALISSUEJAACT Anti-proliferativeandapoptoticeffectsofoleuropeinandhydroxytyrosolonhumanbreastcancerMCF-7cells JunkyuHanÆTerenceP.N.TaloreteÆParidaYamadaÆHirokoIsoda Received:22September2008/Accepted:6March2009/Publishedonline:8April2009ÓSpringerScience+BusinessMediaB.V.2009 AbstractOliveoilintakehasbeenshowntoinducesignificantlevelsofapoptosisinvariouscancercells.Theseanti-cancerpropertiesarethoughttobemed-iatedbyphenoliccompoundspresentinolive.Thesebeneficialhealtheffectsofolivehavebeenattributed,atleastinpart,tothepresenceofoleuropeinandhydroxytyrosol.Inthisstudy,oleuropeinandhydroxytyrosol,majorphenoliccompoundofoliveoil,wasstudiedforitseffectsongrowthinMCF-7humanbreastcancercellsusingassaysforproliferation(MTTassay),cellviability(GuavaViaCountassay),cellapoptosis,cellcycle(flowcytometry).Oleuropeinorhydroxytyrosoldecreasedcellviability,inhibitedcellproliferation,andinducedcellapoptosisinMCF-7cells.ResultofMTTassayshowedthat200lg/mLofoleuropeinor50lg/mLofhydroxytyrosolremark-ablyreducedcellviabilityofMCF-7cells.OleuropeinorhydroxytyrosoldecreaseofthenumberofMCF-7cellsbyinhibitingtherateofcellproliferationandinducingcellapoptosis.AlsohydroxytyrosolandoleuropeinexhibitedstatisticallysignificantblockofG1toSphasetransitionmanifestedbytheincreaseofcellnumberinG0/G1phase. KeywordsMCF-7cellsÁOleuropeinÁHydroxytyrosolÁApoptosis Introduction TheincidenceofcancerinMediterraneancountriesislowerthanintherestofEuropeancountriesandtheUnitedStates(Keysetal.1981).Thisismostlydescribedbythelowerrateofthelargebowel,breast,endometrial,andprostatecancersbyanumberofepidemiologicalstudies,andthemajorreasonforthis,apartfrompossiblegeneticfactors,isattributedtothedietarypractices.ThetraditionalMediterraneandietischaracterizedbyhighconsumptionoffoodsofplantorigin,relativelylowconsumptionofredmeat,andhighconsumptionofoliveoilanditsproducts.Thereareanumberofstudiesonhealthbeneficialeffectsofoliveoil.Severalstudieshavebeenreportedthatoliveoilismorefavorableagainstcancerthanotherformsofaddedlipidsduetoitshighcontentofmonounsaturatedfattyacids(Owenetal.2000a,b;VisioliandGalli2001).Inrecentstudies,dietcontaining15%oliveoilcouldsignificantlyreducepre-cancerouslesionsinratbreastandcolon(Martin-Morenoetal.1994;Coronaetal.2007;Paulaetal.2007).However,similaramountofsoyoildidnothavesuchaprotectiveeffectaswasexpected(LaVecchiaetal.1998).Furthermore,theincidenceofbreastcancerwas70%lessintheratsgroupfedoliveoilthanintheratsgroupfedsaffloweroil(Owen J.HanÁT.P.N.TaloreteÁP.YamadaÁH.Isoda(&)GraduateSchoolofLifeandEnvironmentalSciences,UniversityofTsukuba,Tennodai1-1-1,Tsukuba,Ibaraki305-8572,Japan e-mail:isoda@sakura.cc.tsukuba.ac.jpJ.Han e-mail:jhan@sakura.cc.tsukuba.ac.jp 123 46etal.2000a,b).Thesedatasuggestthatcancerpreventiveeffectofoliveoilisnotonlyattributedtothe‘‘good’’fatcontent.Recently,growingevidenceshowthatminorcompoundsinoliveoilmaytakeapartincancerprotectionaswell,andmoreattentionispaidtoitsphenoliccompounds. Thephenoliccompoundsofoliveoilandleafareacomplexmixtureofcompoundsthatinclude3,4-dihydroxyphenylethanol(hydroxytyrosol),4-hydroxy-phenylethanol(tyrosol),4-hydroxyphenylaceticacid,protocatechuicacid,caffeicacidandp-coumaricacid,amongothers(Litridouetal.1997;Caponioetal.1999).Theconcentrationofthephenolicfractionisseveraltimeshigherinoliveleafthaninoilandvariesdependingonthecultivarandclimate(Tables1,2;Servilietal.1999;Ryanetal.2002;Abazaetal2005).Invivoandinvitrostudiessuggestthatthesebioactivecompoundsexhibitpowerfulantioxidantactivity(Visiolietal.2000;LeTutourandGuedon1992).Oneofvariousphenoliccom-poundshydroxytyrosolseemstobeamongthemostimportantones.Itispresentinfreeformandasaconstituentofcomplexmoleculessuchasoleuropeininleavesandfruits.Bothhydroxytyrosolandoleu-ropeinhavebeenshowntopossessanti-inflamma-tory,bactericidalandbacteriostaticactivities(Yangetal.2007).Someinvivostudiesonoliveleafhaveshownthatitsextractcandecreasebloodpressureanddilatethecoronaryarteriessurroundingtheheart(TuckandHayball2002).Moreover,hydroxytyrosolhasbeenshowntohaveanti-cancereffectonhumancolonadenocarcinomaHT-29cellsandhumanpro-myelocyticleukemiaHL-60cells(Fabianietal.2002,2006),haveanti-melanogenesisactivity,whereasoleuropeininhibitedcellgrowthofLN-18,poorlydifferentiatedglioblastoma;TF-1a,erythro-leukemia;786-O,renalcelladenocarcinoma;T-47D,infiltratingductalcarcinomaofthebreastpleuraleffusion;RPMI-7951,malignantmelanomaoftheskin-lymphnodemetastasis;andLoVo,colorectaladenocarcinomacells(HamdiandHamdi2005).Eventhoughanticancerpropertiesofoleuropeinandhydroxytyrosolwereconfirmedinvitrowithdifferentcelllines,studiesoftheirprotectiveeffectfrombreastcancerhavenotbeendemonstrated.Sincemanyepidemiologicalstudiessuggestthepossiblecorrela-tionbetweenoliveproductsconsumptionandinci-denceofbreastcancerherewetriedtoelucidatethepossibleeffectofthemainphenoliccompounds 123 Cytotechnology(2009)59:45–53 Table1Theconcentrationsofmajorphenoliccompoundsinextra-virginoliveoil(Servilietal.1999;Ryanetal.2002;Abazaetal2005)PhenoliccompoundConcentration(mg/kg)Hydroxytyrosol14.42±3.01Oleuropein2.04±0.78Oleuropeinagycone14.42±3.01Tyrosol27.45±4.05Apigenin 15.80±4.51 Dataexpressedinmg/kgSEM Table2Theconcentrationsofmajorphenoliccompoundsinoliveleaf(Servilietal.1999;Ryanetal.2002;Abazaetal2005) PhenoliccompoundConcentration(mg/kg)Hydroxytyrosol219±3Oleuropein2,231±52Demethyloleuropein984±47Verbascoside27.45±4.05Rutin 15.80±4.51Luteolin7-o-glucoside15.80±4.51 Dataexpressedinmg/kgSEM hydroxytyrosolandoleuropeinofoliveleafonbreastcancerusinghumanbreastcancercelllineMCF-7. MaterialsandmethodsChemicals Dulbeco’sModifiedEagle’sMedium(DMEM),fetalbovineserum(FBS)andtrypsinwerepurchasedfromSigmaChemicalCo.(St.Louis,MO,USA).3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazoliumbro-mide(MTT)waspurchasedfromWako(Osaka,Japan).OleuropeinandhydroxytyrosolwerepurchasedfromFunakoshi(Tokyo,Japan).ViaCountAssayreagent,propidiumiodide,sulforhodamine-valyl-alanyl-aspar-tyl-fluoromethyl-ketoneand7-amino-actinomycinDwerepurchasedfromGEHealthcare(WI,USA).Oliveleafextractpreparation FreshgreenoliveleaveswerecollectedfromtheolivetreegrowninTunisia.Dryoliveleaves(10g) Cytotechnology(2009)59:45–53wereextractedwith70%ethanol(100mL)for2weeks.Extractedsolution[10%(w/v)]wasfiltratedwithafilterunit(Millipore,poresize;0.22lm).Cellculture MCF-7cellswereobtainedfromAmericanTypeCultureCollection(ATCC),andmaintainedinDMEM,supplementedwith10%FBS(Sigma),1%5,000units/mLpenicillin,5,000lg/mLstreptomycin(Sigma).Thecellswereincubatedinanatmosphereof5%CO2at37°C.Allcellsusedinthisstudywerebetweenpassages5and15.Growthinhibitionassay MCF-7cellswereseededin96-wellplatesat29103cells/wellin100lLofDMEMwith10%FBS.After24hthecellsweretreatedwitholiveleafextract[0.001,0.01,0.02,0.1%(w/v)]for12h.AlsoMCF-7cellswereseededin96-wellplatesat29103,29104,29105cells/wellin100lLofDMEMwith10%FBS.After24hthecellsweretreatedwitholeuropein(25,50,100,200lg/mL)orhydroxytyrosol(6.25,12.5,25,50lg/mL)for12h.ThetreatedcellswerewashedwithPBSand10lLofMTTsolution(5mg/mLMTTinPBS)wasaddedtoeachwell.Cellswereincubatedfor12hat37°Cattheendofwhich100lL10%SDSwasaddedandincubatedfurtherfor6hat37°C.Opticaldensitiesat570nmweremeasuredusingaplatereader(Power-scanHT;DainipponPharmaceutical,Osaka,Japan).Therelativecellviabilityinpercentagewascalcu-latedas(A570oftreatedsamples/A570ofuntreatedsamples)9100.Cellproliferationassay Cellswereroutinelyseededintosix-wellplatesat29104cells/wellandincubatedat37°Cfor24h.Cellsweretreatedinfreshmediumandcellswereincubatedwitholeuropein(200lg/mL)orhydroxy-tyrosol(50lg/mL)for3,6,and12h.Attheendofeachtimeperiod,thecellsweretrypsinizedtoproduceasinglecellsuspension,andtheviablecellnumberineachwellwascountedusingtheViaCountAssay(GuavaTechnologies,Hayward,CA). 47 Multicaspaseassaybyflowcytometry Apoptosiswasassessedusingaflowcytometrybasedmulticaspaseassaykit(GuavaTechnologies,Burlin-game,CA).Themulticaspaseassayisbasedonmeasurementofcaspaseenzymesactivatedduringapoptosis.MCF-7cells(29105cells/plate)wereplatedontissueculturedishes(100mm)in10%FBSmedium.Cellswereincubatedwitholeuropein(200lg/mL)orhydroxytyrosol(50lg/mL)for3,6and12h.Cellsweretrypsinized,washedinPBSandstainedwithsulforhodamine-valyl-alanyl-aspartyl-fluoromethyl-ketone(SR-VAD-FMK),and7-amino-actinomycinD(7-AAD)accordingtothemanufac-turesinstructions.CellpopulationswerequantifiedusingGuavapersonalcytometer(GuavaTechnolo-gies,Hayward,CA).Cellcycleanalysis MCF-7cells(29105cells/plate)wereexposedtoconcentrationsofoleuropein(100lg/mL)orhydroxytyrosol(25lg/mL)for24handthetreatedcellswerecollectedbytrypsinisation.Thecellswerefixedwith70%ethanolandstoredat-20°C.Onthedayofanalysis,thecellswerewashedwithPBSandsuspendedin250lLofPBS.OnemLofphosphate–citratebufferwasaddedtothecellsuspensionandthesuspensionwasincubatedatroomtemperaturefor5mintofacilitatetheextractionoflowmolecularweightDNA.Followingcentrifugationthecellswereresuspendedin500lLDNAstainingsolution(20lg/mLpropidiumiodide,200lg/mLDNasefreeRNaseand0.1%TritonX-100)andincubatedinthedarkatroomtemperaturefor30min.Cellcycledistributionwasdeterminedbyfluorescence-acti-vatedcellsortinganalysisofpropidiumiodide-stainedethanol-fixedcellsusingaGuavaEasyCyte(GuavaTechnologies,Hayward,CA). Results Oliveleafextracthascytotoxiceffect ForthescreeningofanticancereffectofoliveleafweusedoliveleafextracttotreatbreastcancerMCF-7cells.MTTassayresultshaveshownthatafter48htreatmentwiththeextractatthedosageof0.1% 123 48cancercellproliferationwasinhibitedto60%com-paredtothevehicletreatedcontrolcells(Fig.1).Theothertreatmentconcentrationsdidnotshowcytotoxiceffect.Sincethemajorphenoliccompoundsofoliveleafareoleuropeinandhydroxytyrosol,insubsequentanticancerassaysweassumedthatthiscytotoxiceffectcouldbemainlyduetopresenceofthesecompoundsandtriedtoelucidatetheirtumorsup-pressiveeffects. Hydroxytyrosolandoleuropeininhibitcellproliferation Thetime-dependentchangeinMCF-7cellviabilitywasdeterminedusingtheMTTassay.AsshowninFig.2,therewasareductionincellviabilityinatime-andconcentration-dependantmannerafteranexposuretohydroxytyrosolandoleuropein.Initialcellnumberhadagreatinfluenceoncytotoxiceffectsofthesecompounds.200lg/mLofoleuropeinand50lg/mLofhydroxytyrosoltreatmenthadthegreatestcytotoxiceffectson29103cells/wellincludingcellgrowtharrestinalmost90%oftreatedcellsafter12hofincubationincomparisontothecontrols.Whereasindoubledandtripledcellnumbertreatmenttheeffecthasdiminishedbutthecom-poundsstillcouldinducecellgrowtharrest.Theresultsofflowcytometryanalysisshowedthesame Fig.1Cytotoxiceffectofoliveleafextract.InfluenceofinitialcellnumberofhumanbreastcancerMCF-7cellsincubatedwitholiveleafextract[0.1,0.02,0.01,0.001%(w/v)].CellviabilitywasdeterminedwiththeMTTassay.Results(±SD)representtheaverageofthreeindependent experiments 123 Cytotechnology(2009)59:45–53 Fig.2InhibitionofMCF-7cellproliferation.Influenceofinitialcellnumberontheconcentration-dependentdecreaseinviabilityofhumanbreastcancerMCF-7cellsincubatedwitholeuropeinaandhydroxytyrosolb.CellviabilitywasdeterminedwiththeMTTassay.Results(±SD)representtheaverageofthreeindependentexperiments trendaswithMTTassay.AsshowninFig.3,thepercentageofdeadcellsincreasedsignificantlyafter12hoftreatment,andtheinhibitionrateofcellproliferationwassimilarwithMTTassayresultsforbothcompounds.However,thefollowingconcentra-tionsofoleuropeinandhydroxytyrosolhadbeenshowntobenon-toxicforprimarycells,suchasnormalhumangingivalGN61fibroblastsandnormalhumanneutrophils.Oleuropeindidnotshowcyto-toxicityata100mMconcentration.Also,hydroxy-tyrosoldidnotshowcytotoxicityata200mMconcentration(BabichandVisioli2003).MorphologychangeofMCF-7cells Duringourtrialswithcellcytotoxicityassaysweobservedsignificantmorphologicalchangesinthecells(29103cells/well)incubatedwith200lg/mLoleuropein.Cellshrinkage,chromatincondensationandformationofapoptoticbodieswereclearly defined. Cytotechnology(2009)59:45–53Fig.3EffectofoleuropeinandhydroxytyrosolontheviabilityofMCF-7cells.ViabilityofMCF-7cells(29104cells/well)incubatedwitholeuropein(200lg/mL)andhydroxytyrosol(50lg/mL)for3,6,and12hasdeterminedbytheGuavaViaCountAssay.Resultsrepresenttheaverageoftwoindependent determinations Thesamepatternwasalsoobservedincellsincubatedwith50lg/mLhydroxytyrosolfor12h,suggestingapoptoticcelldeathofMCF-7cells(Fig.4).Sincemorphologicalfeaturesofapoptosiswerewellnoticedduringabovementionedconcentrationrangeswetriedtousetheserangesthroughoutthestudy,unlessstated.Hydroxytyrosolandoleuropeininducecellapoptosis Sincecaspaseactivationistheclassicfeatureofapoptosisweperformedmulticaspaseassayusingfluorochromeconjugatedcaspaseinhibitorsulforho-damine-valyl-alanyl-aspartyl-fluoromethylketone(SR-VAD-FMK).Thisinhibitoriscellpermeableandnon-cytotoxicandbindstomultiplecaspases 49 Fig.4EffectofoleuropeinandhydroxytyrosolonMCF-7cellsmorphology.MCF-7cells(29103cells/well)incubatedwith200lg/mLoleuropeinaorwith50lg/mLhydroxytyro-solbfor3,6,12h activatedduringapoptosis.Alsothecellimpermeabledye7-aminoactinomycinD(7-ADD)wasincludedintheassaytodistinguishdeadcellsfromapoptoticones(Georgetal.2007).Fromthemulticaspaseassaywecouldobservethatcaspaseswereactivatedsoonaftertreatmentwithbothcompounds,besides,induc-tionoftheenzymes’activationwasgreaterinoleuropeintreatedcells(29.11%)comparetothehydroxytyrosoltreatedcells(11.64%)atthebegin-ningofthetreatment.Nevertheless,both compounds 123 50inducedsignificanttimedependantcaspasesactiva-tiontocomparedtothecontrolcells.After12hthemajorityofthecellswereapoptoticforbothcompoundtreatedcells(Fig.5). HydroxytyrosolandoleuropeininduceG1cellcyclearrestinMCF-7cells BecauseithasbeenreportedthatthecellsundergoingapoptosisarearrestedinG1/G0phaseofcelldivisionphasewecouldverifytheinductionofapoptosisofbreastcancercellsbyhydroxytyrosolandoleuropeinusingcellcycleanalysis.InthisassaywetreatedMCF-7cellswith100lg/mLofoleuropeinand25lg/mLofhydroxytyrosol.Whencellsweretreatedfor24hwithchangedconcentrationranges,thecellcyclearrestatG1phasewasevident(41.1and45.7%inoleuropeinandhydroxytyrosoltreatedcells,respectively),accompanyingadecreaseinG2/Mphasewhencomparedwiththeuntreatedcontrolcells(27.1%inuntreatedcells;Fig.6).FromthisresultwesuggestthatapoptoticcelldeathofMCF-7cellsafterhydroxytyrosolandoleuropeintreatmentwasaccom-paniedwiththeblockofcellcycleintheG1phase. Fig.5Effectofhydroxytyrosolandoleuropeinoninductionofcellapoptosis.MulticaspaseassayofhumanbreastcancerMCF-7cellsincubatedfor3,6and12hwith200lg/mLoleuropeinor50lg/mLhydroxytyrosol.Resultsrepresenttheaverageoftwoindependentdeterminations 123 Cytotechnology(2009)59:45–53 Discussion ManyvegetablefoodsinMediterraneandietcontainsubstancespossessinganticancerproperties(Visiolietal.1998).Amongthemoliveproductssuchasoilareobviouslythesubstantiallydifferentcharacteris-ticsfromwesterndiet.Unlikerefinedoils,oliveoilhasasignificantcontentofpolyphenoliccompounds.Eventhoughrecentstudieshavedemonstratedthatphenoliccompoundsofoliveoilmayplayaroleincancerprotectionthemainsourceofthesecom-poundsisoliveleafratherthanoliveoil.Anditisnotsurprisingthatoliveleafhasalonghistoryforitsmedicinalremedies.Hydroxytyrosolandtheseco-iridoidoleuropeinarestudiedwellfortheirbiologicalactivitiesamongolivephenolics.Itshouldbenotedthatwhiletocopherols,phenolicacids,phenolicalcoholsandflavonoidsarepresentinmanyfruitsandvegetablesbelongingtoseveralbotanicalfami-lies,secoiridoidsarepresentexclusivelyinplantsofthefamilyofOlearacea.Anticancerpropertieshydroxytyrosolandoleuropeinwereconfirmedinvitroondifferentcelllinesstudies.But,breastcancercelllineshavenotbeendemonstrated. Inthisstudywehavedemonstratedthathydroxy-tyrosolandoleuropeinofoliveleaveinhibitthecellproliferationofbreastcancercellsandinduceapop-toticcelldeath.TheresultsofMTTassayhaveshownthatbothhydroxytyrosolandoleuropeinhavecyto-toxiceffectonMCF-7cellsinadosedependantmanner.Alsotherewasnotmuchdifferenceincellgrowthinhibitioneffectbetweenbothcompounds.SinceMTTassayisbasedonthemetabolicactivityofthecellswetriedtoconfirmthecytotoxiceffectbyflowcytometryanalysis.ResultsofflowcytometricanalysisalsoconfirmedthatthephenoliccompoundsreducedcellviabilitywiththesametrendasMTTassayresults.Interestingly,thecellgrowtharrestwasmoreeffectivewhencellswereseededatalowcellnumber(Fig.2).Itisprobablycausedbythecontactsurfacewithhydroxytyrosoloroleuropein.Thecontactsurfacewithhydroxytyrosoloroleuropeinatsmallnumberofinitialcellseedingiswiderthanthelargenumberofinitialcellseeding.Thesedatamaysuggestthatphenoliccompoundsofoliveleafhavehealthprotectiveeffectsratherthanhealingeffects.Constantconsumptionofoliveproductssuchasleafteaandoilmayenhancetheseprotectiveeffectsthus,suggestingtheirdailyconsumptionismore effective Cytotechnology(2009)59:45–53Fig.6Effectofhydroxytyrosoland oleuropeinonMCF-7cellcycle.CellcyclekineticsofhumanbreastcancerMCF-7cellsincubatedfor12,24and48hwith100lg/mLoleuropeinor25lg/mLhydroxytyrosol.Resultsrepresenttheaverage(%)oftwoindependentdeterminations 51 thantheinfrequentintake.Alsoinvivostudieshaveshownthatsoonafteroraladministrationthecom-poundsappearedintheplasmasuggestingtheirgoodabsorptionfromtheintestines,buttheentirequantityadministeredwasnotfoundintheurinewhichmaysuggestaccumulationinorganssuchasbreastorerythrocytes(Elisaetal.2005). Duringourtrialsusingcellcytotoxicityassaysweobservedsignificantmorphologicalchangesinthecells(Fig.4).Cellshrinkage,chromatincondensationandformationofapoptoticbodieswereclearlyobservedsuggestingthecellgrowtharrestcouldbeduetoapoptosis.Apoptosishasbeenwellcharacter-izedandisthoughttoproceedthroughaseriesofconservedandwellstudiedsteps. Thecaspases,cysteineproteases,areofcentralimportanceintheapoptoticsignalingnetworkwhichisactivatedinmostcasesofapoptoticcelldeath.Actually,strictlydefined,celldeathonlycanbeclassifiedtofollowaclassicalapoptoticmodeifexecutionofcelldeathisdependentoncaspaseactivity.Ourmulticaspaseactivityassayrevealedthatwithcompounds’treatmenttheproportionofcaspaseenzymesactivatedcellsincreasedtime-dependently,indicatingtheactivationoftheseenzymesinthecells(Fig.5). Tofurtherelucidateourhypothesisofapoptoticcelldeathofbreastcancercellsbyhydroxytyrosolandoleuropeinwetriedtouseothermethodsforitsdetection.SincetheG1/G0cellcyclearresthasbeenshowntocorrelateswellwithapoptosisdeterminedbyothermeansweexaminedsub-G1DNAcontentbyflowcytometryanalysisfollowingtreatmentwithtwophenoliccompounds.OurdataconfirmedthatthecellstreatedwithhydroxytyrosolandoleuropeinexhibitedstatisticallysignificantblockofG1toSphasetransitionmanifestedbytheincreaseofcellnumberinG0/G1phase.Althoughtheoleuropeinnotablyinducedlesscellcyclearrestthanhydroxy-tyrosolitwasstillcapabletosignificantlypromotebreastcancerapoptoticcelldeath. Themolecularmechanismofhydroxytyrosolandoleuropeininducedapoptoticcelldeathisnotclear.Yet,thesecompoundshavehighantioxidantactivi-ties.Inarecentstudy,theantioxidantactivityofa - 123 52tocopherolandphenolicextractsfromolivesandoliveoilwascomparedovertime.Itwasdemon-stratedthatafterinitiationofradicalformationthescavengeractivityofa-tocopherolwashigherbutsoonterminatedwhereastheextractfromolivesandoilcontainedcompoundscontinuedtoreducetheconcentrationoftheseradicalsandwithtimetheextractsoftheolivesweremuchmoreactivethana-tocopherol(KeceliandGordon2001).Ontheotherhand,someinvestigatorsobservedthatphenoliccompoundswithhighreducingabilitycanbenotonlyantioxidantsbutalsopro-oxidants,thusgener-atingreactiveoxygenspecies(ROS)(WlodekandSteven2001).Itisinterestingtonotethatmanyhumancancercelltypesexistinahighlyoxidativestateduetodecreasedantioxidantprotectiveenzymelevelscomparedtotheirnormaltissuecounterparts.Thus,cancercellsmaybemoresensitivetoanygeneratedROSwithinthecells.GrowingevidenceexistthatROSmayevenactivateapoptoticdeathpathways.Zhaoetal.(1997)observedthatvitaminEinducedDNAsynthesisarrestandstimulatedapop-tosisinMCF-7cells(WlodekandSteven2001).Alsomostchemotherapeuticagentssuchasradiation,alkylatingagents,anthracyclinesinduceapoptoticdeathincancercellsthroughgeneratingROS.Thiscouldbethepossiblemechanismofactionofantioxidantsfortumorsuppressiveeffect. Inconclusion,ourresultshaveshownthatmainphenoliccompoundsofoliveleafhydroxytyrosolandoleuropeininducedapoptoticcelldeathofhumanbreastcancerMCF-7cells.OurfindingsmaysupportthebreastcancerprotectiveresultsofepidemiologicalstudiesofMediterraneandietinrelationtooliveproducts’consumption.Eventhoughthecancerpro-tectiveeffectofthemainoliveoilcomponentoleicacidingeneregulationwasdemonstratedfurtherstudiesareneededtoexploremolecularmechanismsoftumorsuppressiveeffectofotherolivetreeproductssuchasoliveleafbecausetheyalsocouldbepotentinducersofsucheffectifconsumedfrequently. 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