KatarzynaLubecka-Pietruszewskaa,⇑,1,AgnieszkaKaufman-Szymczyka,1,BarbaraStefanskab,KrystynaFabianowska-MajewskaaabDepartmentofBiomedicalChemistry,MedicalUniversityofLodz,6/8MazowieckaStreet,92-215Lodz,PolandDepartmentofPharmacologyandTherapeutics,McGillUniversity,3655SirWilliamOslerPromenade,Montreal,QC,CanadaH3G1Y6articleinfoabstract
Folate,oneofthemoststudieddietarycompounds,hasrecentlybecomethemaintopicofdebatesonfoodfortification.Althoughlowfolatelevelsmaybeassociatedwithincreasedriskofcancerdevelop-ment,simultaneouslyseveralreportsindicateadetrimentaleffectsmediatedbyhighfolateconcentra-tions.Usingthemethylationsensitiverestrictionanalysis(MSRA)andreal-timeRT-PCRwetestedtheeffectoffolicacidonDNApromotermethylationandexpressionofPTEN,APCandRARbeta2tumoursup-pressorgenesinMCF-7andMDA-MB-231breastcancercelllineswithdifferentinvasivecapacity.Thetestedgenesencodeproteinsinvolvedinregulationofoncogenicintracellularsignalingpathways.Theresultsshowthattheincreasingconcentrationsoffolicacidleadtoadose-dependentdown-regulationoftumoursuppressorgeneswhichmaybelinkedtotheincreasedDNAmethylationdetectedwithintheirpromoterregions.Theeffectsweremoreremarkableinnon-invasiveMCF-7cellswherewealsoobserved30%up-regulationofDNMT1expressionatthehighestfolateconcentrationused.Ourfindingsshowthatcautionneedtobeusedwhenintroducingfolicacidsupplementationsinceitmayleadtocancerprogression.Ó2012ElsevierInc.Allrightsreserved.Articlehistory:Received12November2012Availableonline3December2012Keywords:FolicacidEpigeneticregulationofgenetranscriptionDNAmethylationBreastcancer1.IntroductionFolicacidisawater-solublevitaminB9presentinavarietyoffoodsincludinglentils,okra,beans,asparagus,spinach,broccoli,andavocado.Sinceadequatefolateintakewasshowntoreducetheriskofcancer[1],cardiovasculardisease[2]andprotectfromneuraltubedefects[3],supplementationforwomenintendingtobecomepregnantanddietaryfortificationhavebeenintroducedinmultiplecountries.Folateconstitutesoneofthecoenzymesofone-carbonmetabolism[4].Afterdietaryintake,itisconvertedtotetrahydrofolatethatisinvolvedinremethylationofhomocyste-inetomethioninewhichisaprecursorofS-adenosylmethionine(SAM),primarymethylgroupdonorformostmethylationreac-tions,includingDNA[5,6].DNAmethylationinnormalcellsisimplicatedinoncogenerepression,thecontrolofexpressionofgenescrucialforcellproliferation,differentiation,andnormaldevelopmentaswellasinparentalimprinting,Xchromosomeinactivation,andchromosomalintegrity[7,8].Animalstudiesandclinicalobservationsfromthelastdecadesuggestthatfolateplaysadualroleincarcinogenesisdepending⇑Correspondingauthor.Fax:+48426784277.E-mailaddress:katarzyna.lubecka-pietruszewska@umed.lodz.pl(K.Lubecka-Pietruszewska).1Theseauthorscontributedequallytothiswork.0006-291X/$-seefrontmatterÓ2012ElsevierInc.Allrightsreserved.http://dx.doi.org/10.1016/j.bbrc.2012.11.103onthetiming,doseandindividualconditions,forexampletheage[9–11].Itiswellknownthatfolatedeficiencyleadstoincreaseincancerriskbydisturbinghomeostasisofone-carbonmetabo-lism,therebyleadingtoperturbationofSAMsynthesisandsubse-quentalterationsinDNAmethylation.Numerousstudiesshowedacausalroleoffolatedeficiencyinthedevelopmentofdifferenttypesofcancer,suchascolonandrectum,esophagus,gastric,pan-creaticandbreastcancer[1,11–15].Interventiontrialsconductedinordertoassesstheeffectoffolicacidsupplementationontheriskofcancerdevelopmentdeliveredinconsistentresultswithcon-cludeddecreasedriskofcolorectalcancer[16,17].Ontheotherhand,theliteraturedatafromthelastdecadedemonstratethathighdosesoffolatemayincreasecancerriskandpromotecancerprogression[17–19].DNAmethylationisanepigeneticDNAmodificationwhichpar-ticipatesinregulationofgeneexpressionwithoutchangesinunderlyingDNAsequence.AlterationsinDNAmethylationpat-ternshavebeenreportedinmanymalignanciesandhavebeenshowntobeimplicatedincancerinitiationandprogression.Thehallmarksofcancercellsarehypermethylationandsilencingoftu-moursuppressorgenes[20–25],hypomethylationandactivationofoncogenesandpro-metastaticgenesaswellasglobalDNAhypomethylation[26].InourpreviousstudieswithMCF-7cells,weobserveddiffer-encesinDNApromotermethylationstatesdependingonfolicacid624K.Lubecka-Pietruszewskaetal./BiochemicalandBiophysicalResearchCommunications430(2013)623–628concentrationincellculturemedia.Thepresentstudiesfocusonevaluationwhetherfolicacidsupplementationcanaffectpromotermethylationandtranscriptionalactivitiesofthreetumoursup-pressorgenes,PTEN,APCandRARbeta2,inhumanbreastcancercelllines,andwhetherfolicacidmaybeusedinepigenetictherapyofbreastcancer.Inordertotestourhypothesis,weestimatetheef-fectsoffolicacidonDNApromotermethylationandexpressionoftheabovetumoursuppressorgenesaswellasDNMT1expres-sioninMCF-7andMDA-MB-231breastcancercelllineswithdis-tinctinvasiveandmetastaticpotentials.Thetestedgenesencodeproteinsthatparticipateindown-regulationofintracellularonco-genicsignalingpathways.PTENisinvolvedinregulationofPI3K/AktandRas/MAPK/AP-1pathways,whereasAPCcontrolsWnt-1/betacatenincascade.TheactionofRARbetaismostlymediatedbyitsreceptors.Theligand/receptorcomplexactsasatranscrip-tionalfactorbindingtoresponsiveelementswithingenesregulat-ingcellcycle,differentiationandapoptosis.Thethreeselectedtumoursuppressorgenesareoftenepigeneticallysilencedincan-certissuesandcelllines[20–25].Forinstance,PTENandAPCpro-moterhypermethylationwithconcomitantreductionofexpressiononmRNAlevelwasdetectedinbreasttumoursandbreastcancercelllines[22,23,25].Similarly,promoterhypermethylationofRARbeta2wasassociatedwithpartialorcompletesuppressionofthegenetranscriptionalactivityinbreastcancer[24].Theresultsofthepresentstudies,whichfocusontheevaluationoffolicacideffectsonmethylationandexpressionoftheselectedtumoursuppressorgenes,revealthatrisingfolateconcentrationsmaypromotebreastcancerprogression.OurfindingsshouldalsobetakenintoconsiderationwhenonewishestoinvestigatetheroleofDNAmethylationinvitroasthepresenceoffolicacidincul-turemediummayaffectfinaloutcome.2.Materialsandmethods2.1.Reagents,cellculture,RNAandDNAisolationandpurificationReagentsforRNAandDNApurificationandfolicacidcalciumsaltwerepurchasedfromSigma–AldrichCo(Poland),endonucle-aseHpaIIandEco72IfromFermentas(Lituania).Folicacidwasdis-solvedinwaterattheconcentration1mg/ml(1.96mM).Humanbreastadenocarcinomacelllines,MCF-7andMDA-MB-231,fromAmericanTypeCultureCollection,ATCC(LGCStandards)andEuropeanCollectionofCellCultures,ECACC(Salisbury,UK)wereculturedfor96hinEMEMmedium(MEMEaglewithEarle’sBSS,withoutL-glutamine,Lonza)andL15medium(Leibovitz’sL15mediumwithoutL-glutamine,Lonza),respectively.Thesemediaweresupplementedwith:2mML-glutamine;0.01mg/mlbovineinsulin(onlyforMCF-7cells)(Sigma–Aldrich,St.Louis,MO,USA);10%(andforMDA-MB-231cells–15%)fetalbovineserum(FBS);1U/mlpenicillin,and1lg/mlstreptomycin(Gibco,Scot-land,UK).Cellsweregrownfor96hat37°Cinahumidifiedatmo-sphereof5%CO2,exceptforMDA-MB-231cellswhichwereincubatedwithoutCO2.Mediausedforbothcelllinescontained1mg/loffolicacid(controlsamples,CFA,controlfolicacidconcen-tration).Fortheexperiments,theconcentrationoffolicacidwasincreasedto4mg/l(LFA,lowerfolicacidconcentration)and8mg/l(HFA,higherfolicacidconcentration).Cellviabilitywasestimatedwithtrypanblue(Sigma–Aldrich)exclusiontest.Additionally,theviabilityofcellswereconfirmedandcompletedbyapplyingflowcytometryanalysis(FACSCaliburflowcytometer,BectonDickinson),usingannexinV/propidiumio-dideassays,accordingtothemanufacturer’sprotocol.CellularDNAfromthebreastcancercelllineswasisolatedafter20hofincubationwithproteinaseK,followedbyextractionusingphenol:chloroform:isoamylalcohol(25:24:1)mixture(Sigma–Al-drich)accordingtothemanufacturer’sprotocol.PureDNAwasdi-lutedinTEbufferandstoredatÀ20°C.TotalRNAfromthetestedcellswasisolatedusingTRIZOL(Invitrogen,LifeTechnologies,Carlsbad,CA,USA)accordingtothemanufacturer’sprotocol.IsolatedRNAwasdissolvedinwatercontaining1%DEPC(ribonucleaseinhibitor)andstoredatÀ70°C.2.2.MethylationgeneanalysisThemethylationstatusofPTEN,RARbeta2andAPCpromoterswasestimatedusingmethylation-sensitiverestrictionanalysis(MSRA)accordingtoIwase’smethod[27].TheMSRAanalysisin-cludedfoursteps:(i)digestionofcellularDNAwithendonucleasethatrecognizesonlynon-methylatedsequence,(ii)PCRamplifica-tionofdigestedDNA,(iii)electrophoreticanalysisofamplifiedpro-moterfragments,and(iv)densitometricquantitativeanalysisofthebandintensity.GenomicDNA(0.5lg)wasincubatedwith20UofHpaIIorEco72Irestrictionenzymesat37°Covernight.HpaIIrecognizesnon-methylatedC;CGGsequencelocatedwithinPTENandRARbeta2promoterfragments,whereasEco72Icutsnon-methylatedCAC;GTGsequencewithinAPCpromoterfrag-ment.Twocontrolsofdigestionreaction,asamplewithoutanen-zymeandMspI-digestedsample,wereincubatedinthesameconditions.Afterincubation,controlanddigestedDNAwereampli-fiedinPCRusingthefollowingprimersfortheselectedpromoterfragments:PTEN(GenBankaccessionno.AF143312;chr:10q23.3;ampliconlength214bp[22,23]:(forward)50-cagccgttcggaggatt-attc-30and(reverse)50-gggcttcttctgcaggatgg-30;RARbeta2(Gen-Bankaccessionno.X56849;chr:3p24;ampliconlength295bp[24]:(forward)50-ctcgctgcctgcctctctgg-30and(reverse)50-gcgttctcggcatcccagtc-30;APC(GenBankaccessionno.U02509;chr:5q21-q22;ampliconlength317bp[25]:(forward)50-ctagg-caggctgtgcggttg-30and(reverse)50-cggtttaagacagtgcgagg-30.ThereactionmixtureforPCRwaspreparedasdescribedprevi-ously[28],andwascarriedoutinTpersonalThermalCycler(Biom-etra,Goettingen,Germany)at95°Cfor5min,cycledfor1minat94°C,1minatannealingtemperature(61.1°C,58.4°Cand61.1°C,forPTEN,RARbeta2andAPCpromoterfragments,respec-tively)and1minat70°C(30cycles),followedbya10minexten-sionat72°C.TheamplifiedPCRproductswerefractionedona6%polyacrylamidegel,stainedwithethidiumbromideandvisualizedunderUVillumination.Fordensitometricanalysisofbandintensi-tiestheQuantityOnesoftware(Bio-RadLaboratoriesLtd.,UK)wasused.Methylationlevelineachsamplewascalculatedbasedondensitometricanalysisandexpressedasapercentageofundi-gestedDNAafterthecomparisonofbandintensitiesfordigestedandundigestedDNA.Thepercentageofmethylationinhibitionwasevaluatedbycomparisonofmethylationlevelincontrolcellsthatgrewinthepresenceof1mg/lfolicacidandincellstreatedwithfolicacidatconcentration4or8mg/l.2.3.cDNAsynthesisandreal-timePCR(QPCR)TotalRNAwasisolatedusingTRIZOLÒ(Invitrogen,Lifetechnol-ogies,Carlsbad,USA)andcDNAwassynthesizedusing:2lgofto-talRNA;6llofrandomhexamers,5llofoligo(dT)15(Promega,Madison,USA)andImProm-IIreversetranscriptase(Promega)accordingtomanufacturer’sprotocols.AllQPCRreactionswerecarriedoutinaRotor-GeneTG-3000machine(CorbettResearch,Australia).Thereactionmixturepre-paredaccordingtomanufacturer’sprotocolcomprisedthefollow-ingprimers:PTEN(forward)50-cgaactggtgtaatgatatgt-30and(reverse)50-catgaacttgtcttcccgt-30;RARbeta(forward)50-ttcaag-caagcctcacatgtttcca-30and(reverse)50-aggtaattacacgctctgcacctt-tag-30;APC(forward)50-tgcgagaagttggaagtgtgaaagcattg-30and(reverse)50-tgacaaattccataaggcactcaatacgc-30;DNMT1(forward)K.Lubecka-Pietruszewskaetal./BiochemicalandBiophysicalResearchCommunications430(2013)623–62862550-accgcccctggccaaagccattg-30and(reverse)50-agcagcttcctcctcctttattttagctgag-30.Afteraninitial2mindenatur-ationstepat94°C,amplificationconsistedof50cycleswereper-formedunderthefollowingconditions:30sat94°C,15satannealingtemperature(50°Cand56°CforPTENandRARbeta,respectively,60°CforAPCandDNMT1),and30sofelongationat72°C.Therelativeexpressionofeachtestedgenewasnormalizedtothegeometricmeanoffourhousekeepinggenes,RPS17(40SribosomalproteinS17),RPLP0(60SacidicribosomalproteinP0),H3F3A(H3histonefamily3A)andBMG(beta2-microglobulin),accordingtoPfaffl’smethod[29].2.4.StatisticalanalysisDatawereassessedbyone-wayanalysisofvariance(ANOVA)followedbyTukey’sposthoctest.Eachvaluerepresentsthemean±SDofthreeindependentexperiments.TheresultswereconsideredasstatisticallysignificantwhenP<0.05.3.Results3.1.FolicacideffectsonviabilityofMCF-7andMDA-MB-231cellsAsmeasuredbythetrypanblueexclusiontest,itwasobservedthatfolicaciddidnotaffectgrowthandviabilityofthecellsinbothcelllines(datanotshown).Thecellviabilityprofilesaccordingtoflowcytometryarepre-sentedinFig.1.Folicacidat4and8mg/lconcentrationsincreasedthenumberofapoptoticcellsby12–14%inMCF-7cellline.Over32%ofallapoptoticcellsshowedactivecaspase-3whichindicatesthesignificantinvolvementofcaspase-dependentapoptoticpath-way.TheincubationwiththetestedcompounddidnotinduceapoptosisininvasiveMDA-MB-231cells.3.2.PromotermethylationandexpressionlevelsofthetestedgenesinthecontrolcellsDNAmethylationlevelofthetestedfragmentwithinPTENpromoterincontrolMCF-7andMDA-MB-231cellsthatgrewinthepresenceof1mg/lfolicacidwasestimatedtobeapproxi-mately34%and70%,respectively.APCpromoterfragmentwasmethylatedat62%and41%,whereasRARbeta2at42%and57%inMCF-7andMDA-MB-231cells,respectively(Fig.2AandB).Real-timePCRrevealedthatinvasiveMDA-MB-231cellsshowasignificantlylowerexpressionofalltestedgenesby33%,74%and73%,respectively,forPTEN,RARbeta2andAPCincomparisonwithMCF-7cells(Fig.3).Although,DNMT1expressionwassimi-larinbothcelllines(Fig.3),lowerPTENandRARbeta2expressionwasassociatedwithhigherDNAmethylationwithinpromotersofthesegenesinMDA-MB-231cellsascomparedwithMCF-7cells(Figs.2and3).3.3.DNAmethylationandexpressionofPTEN,APCandRARbeta2aftertreatmentwith4and8mg/lfolicacid3.3.1.MCF-7cellsInnon-invasiveandER-positiveMCF-7breastcancercells,theincreasingconcentrationsoffolicacidledtoelevationofDNAmethylationofalltestedpromoterfragmentswiththehighestchangeinPTENpromoter(over142%at4mg/land144%at8mg/lfolicacid,Fig.2A).WealsoobservedaconcomitantdecreaseinexpressionofthetestedgenesonmRNAlevel.Themostrobustdown-regulationby58%wasdetectedforRARbetaafterchallengewith8mg/lfolicacid(Fig.2A).Furthermore,thealterationsinmethylationandexpressionlevelsafterthetreatmentswereasso-ciatedwithupto30%increaseinDNMT1expressionasmeasuredbyQPCR(Fig.4A).Fig.1.Flowcytometrydataanalysisfornon-invasiveMCF-7(A)andinvasiveMDA-MB-231(B)cellsculturedwithfolicacid(viablecells–whitebars;necroticcells–blackbars;cellsinlateapoptosis–mediumgraybars;cellsinearlyapoptosis–lightgraybars).CFAt0,controlcellsusedforexperiments(time0h)[1mg/l];CFA,controlfolicacidconcentration[1mg/l];LFA,lowerfolicacidconcentration[4mg/l];HFA,higherfolicacidconcentration[8mg/l].Datarepresentthemean±S.D.ofthreeindependentexperiments.StatisticalanalyseswereperformedbyANOVAfollowedbyTukey’sposthoctest.Meanvalueaftertreatmentwassignificantlydifferentfromthecontrol:⁄P<0.05,⁄⁄P<0.01.626K.Lubecka-Pietruszewskaetal./BiochemicalandBiophysicalResearchCommunications430(2013)623–628Fig.2.FolicacideffectsonpromotermethylationstatusandgeneexpressiononmRNAlevelinMCF-7(A)andMDA-MB-231(B)cells.PromotermethylationlevelandrelativeexpressionofthetestedgenesinMCF-7andMDA-MB-231cellsculturedat1mg/l(control,lightgray),4mg/l(mediumgray)and8mg/l(darkgray)folicacidwereestimatedasdescribedinSection2.Datarepresentthemean±S.D.ofthreeindependentexperiments.StatisticalanalyseswereperformedbyANOVAfollowedbyTukey’sposthoctest.Meanvalueaftertreatmentwassignificantlydifferentfromthecontrol:⁄P<0.05,⁄⁄P<0.01.withinAPCpromoterhoweveritwasnotaccompaniedbyasignif-icantchangeingeneexpression.Interestingly,therewasaremark-abledifferenceinthelevelofPTENpromoterhypermethylation(Fig.2)andDNMT1down-regulationbetweenMDA-MB-231andMCF-7cells(Fig.4).4.DiscussionHumanclinicalandepidemiologicaldataalongwithanimalstudieshavesuggestedthatfolatemayplayaprotectiveroleincarcinogenesis,particularlyincolorectalcancer[16].However,re-centepidemiologicalstudieshaveindicatedthathighfolateintake,mainlyinitssyntheticformpresentinsupplementsandfortifiedfoods,mayincreasetheriskofbreastcancer[30,31].ThelargeobservationstudyofcancerscreeningtrialcohortperformedbyStolzenberg-Solomonshowedastatisticallysignificant32%in-creaseinbreastcancerriskinpostmenopausalwomenconsuminghigherfolatelevel[19].Becauseofcontradictoryresultsofseveralepidemiologicalinvestigationsoftheeffectsoffolateintakeonbreastcancerrisk,aswellasthepremisethatfolicacidmaymodifycancerriskthroughitsinvolvementinregulationofDNAmethylation,wehaveundertakenthepresentstudy.OurgoalwastoelucidatetheinfluenceoffolicacidonmethylationandexpressionofPTEN,APCandRARbeta2tumoursuppressorgenesinbreastcancer.WeestablishedrelationsbetweenDNAmethylationandexpressionofthetestedgenesandDNMT1expressioninnon-invasiveandinvasivecellswithdifferentERstatus.Fig.3.RelativeexpressionofPTEN,RARbeta,APCandDNMT1inMDA-MB-231cellsincomparisonwithMCF-7cells.ExpressionlevelofeachgeneinMDA-MB-231cellswascomparedwithitsexpressioninMCF-7cells(control)andshowedasafoldchange.Datarepresentthemean±S.D.ofthreeindependentexperiments.Statis-ticalanalyseswereperformedbyANOVAfollowedbyTukey’sposthoctest.Meanvalueaftertreatmentwassignificantlydifferentfromthecontrol:⁄P<0.05,⁄⁄P<0.01.3.3.2.MDA-MB-231cellsIntheinvasiveandER-negativeMDA-MB-231breastcancercellline,thesupplementationofculturemediawith4or8mg/lfolicacidledtoanincreaseinDNAmethylationofthetestedpromoterfragmentsalthoughtoalesserextentascomparedtoMCF-7cells(Fig.2B).Themostrelevant30%hypermethylationwasrevealedK.Lubecka-Pietruszewskaetal./BiochemicalandBiophysicalResearchCommunications430(2013)623–628627Fig.4.TheeffectoffolicacidonDNAmethyltransferase1(DNMT1)expressioninMCF-7(A)andMDA-MB-231(B)cells.RelativeexpressionofDNMT1inMCF-7andMDA-MB-231cellsculturedat1mg/l(control,CFA),4mg/l(LFA)and8mg/l(HFA)folicacidconcentrationswereestimatedasdescribedinSection2.Datarepresentthemean±S.D.ofthreeindependentexperiments.StatisticalanalyseswereperformedbyANOVAfollowedbyTukey’sposthoctest.Meanvalueaftertreatmentwassignificantlydifferentfromthecontrol:⁄P<0.05,⁄⁄P<0.01.Ourfindingsindicatethatinbothbreastcancercelllines,non-invasiveER-positiveMCF-7cellsandinvasiveER-negativeMDA-MB-231cells,folicacidat4and8mg/lconcentrationscausedanincreaseinmethylationofPTENpromoterwithinthetestedfrag-mentandsilencingofgenetranscription(Fig.2AandB)ascom-paredtocontrolcellsgrowinginthepresenceof1mg/lfolate.TheextentofhypermethylationwasmuchhigherandstatisticallyrelevantinMCF-7cellsalthoughthefinalPTENmethylationlevelinbothcelllineswasthesame,approximately80%,aftertreatmentwitheither4mg/lor8mg/lfolicacid.ItneedstobeemphasizedthatbeforetreatmentsPTENpromoterwasmethylatedat70%and30%inMDA-MB-231andMCF-7cells,respectively,whichmightexplainmuchweakereffectsobservedintheinvasivecells.ThealterationsinPTENpromotermethylationwereassociatedwithdiminutionofgeneexpressiononmRNAlevelinbothbreastcancercelllineshoweverthechangewasmuchlowerinMDA-MB-231cells.SimilarchangesinpromotermethylationandgeneexpressionafterexposuretofolicacidweredetectedforAPCandRARbeta2tumoursuppressorgenes.Inbothinvasiveandnon-inva-sivecells,hypermethylationofAPCandRARbeta2promoterswasconcomitantwiththesegenestranscriptionaldown-regulationthatwasstatisticallysignificantinMCF-7non-invasivecells.FolicacidexertedadosedependenteffectonAPCmethylation,particu-larlyinnon-invasivecells.TheincreaseinRARbeta2promotermethylationinMDA-MB-231cellswasnotassociatedwithchangesingeneexpression.ItsuggeststhattranscriptionalactivityofRARbeta2isregulatedbyanotherthanDNAmethylationmech-anismattheadvancedinvasivestageofbreastcancer.Interest-ingly,folicacidatthehighestconcentrationusedinthestudyledto30%increaseinDNMT1expressioninMCF-7cellswhilecausingonlyaslightelevationinMDA-MB-231cells(Fig.4AandB).Toourbestknowledge,thisisthefirststudyfocusingontheef-fectsofhighfolicacidconcentrationsonmethylationandexpres-sionofPTEN,RARbeta2andAPCtumoursuppressorgenesinbreastcancer.MostofotherstudiesthataddressedfolateandDNAmethylationwereundertakentoexaminegenomicDNAmethylationlevelthatislessinformativeandmaybemisleading.Genome-wideDNAmethylationisdynamicandinvolvesbothdemethylationofoncogenesandhypermethylationoftumoursup-pressorgenesduringcarcinogenesis.Hence,evaluatingglobalDNAmethylationlevelaveragesthesealterationsinDNAmethylationpatterns.Moreover,accordingtoKim’sdatathefinaleffectsoffo-lateonDNAmethylationstatusarehighlycomplexanddependentonacelltype,targetorgan,stateoftransformation,age,sexandlifestyleoftheindividuals[32].Ourresultsindicatethatrisingcon-centrationsoffolicaciddeepenpromoterhypermethylationoftu-moursuppressorgeneswhichwasalsoobservedinotherstudiesofourgrouponK562humanerythroleukemiccellline.InK562cells,transcriptionalsilencingofthetestedgeneswasmuchstrongerthaninthebreastcancercellsthatmightbepartiallyexplainedbyahighproliferationrateofthesecells(unpublisheddata).OurobservationthathighconcentrationoffolicacidledtoincreaseinmethylationoftumoursuppressorgenesisconsistentwithBer-ner’sresultsforESR1,p16andp15genesinCaco-2colonadenocar-cinomacells[33].Ourfindingshaveprovidedevidencethatfolicacidmayinduceapoptoticcelldeathinnon-invasiveMCF-7breastcancercellline,whereasinvasiveMDA-MB-231cellsarenotresponsivetofolicacidasapro-apoptoticagent.FolicacidwasreportedbeforeasaninducerofapoptosisinhumangastriccancercelllinesMKN-45andMKN-28[34].Inconclusion,inthepresentstudywedemonstratethatfolicacidatincreasingconcentrationsimpairstranscriptionalactivitiesofthetestedtumoursuppressorgenesthatisconcomitantwithin-creasedDNAmethylationwithintheirpromoters.Thehighestfo-lateconcentrationusedinourexperimentscausedinductionofDNMT1expression.Probably,theseobservationsmayberelatedtotheeffectoffolicacidactiononSAMpool.Ourfindingsconfirmotherauthors’datashowingthatfolicacidsupplementationmayleadtodown-regulationofthetestedtumoursuppressorgenes,whatmaypromoteprogressionofbreastneoplasia[9].Itshouldbetakenintoaccountinanticancertherapywheredietenrichedwithsyntheticvitaminsisoftenrecommended.AcknowledgmentsTheresearchwassupportedbytheMedicalUniversityofLodz–grantsnos.503/6-099-01/503-01and502-03/6-099-01/502-64-057.TheauthorsaregratefultoProfessorPiotrSmolewskiandDrBarbaraCebula(DepartmentofExperimentalHaematology,Medi-calUniversityofLodz,Poland)forthepossibilitytoperformtheflowcytometryanalysis.References[1]S.C.Larsson,E.Giovannucci,A.Wolk,Folateintake,MTHFRpolymorphisms,andriskofesophageal,gastric,andpancreaticcancer:ameta-analysis,Gastroenterology131(2006)1271–1283.[2]L.A.Bazzano,K.Reynolds,K.N.Holder,etal.,Effectoffolicacidsupplementationonriskofcardiovasculardiseases:ameta-analysisofrandomizedcontrolledtrials,JAMA296(22)(2006)2720–2726.[3]J.L.Mills,C.Signore,Neuraltubedefectratesbeforeandafterfoodfortificationwithfolicacid,BirthDefectsRes.A.Clin.Mol.Teratol.70(11)(2004)844–845.[4]L.B.Bailey3rd,J.F.Gregory,Folatemetabolismandrequirements,J.Nutr.10(1999)779–782.[5]J.Selhub,Folate,vitaminB12andvitaminB6andonecarbonmetabolism,J.Nutr.HealthAging6(2002)39–42.[6]B.Stefanska,H.Karlic,F.Varga,etal.,Epigeneticmechanismsinanti-canceractionsofbioactivefoodcomponents–theimplicationsincancerprevention,Br.J.Pharmacol.167(2)(2012)279–297.[7]A.Hermann,H.Gowher,A.Jeltsch,BiochemistryandbiologyofmammalianDNAmethyltransferases,CellMol.LifeSci.61(19–20)(2004)2571–2587.628K.Lubecka-Pietruszewskaetal./BiochemicalandBiophysicalResearchCommunications430(2013)623–628[8]M.Szyf,P.Pakneshan,S.A.Rabbani,DNAmethylationandbreastcancer,Biochem.Pharmacol.68(6)(2004)1187–1197.[9]Y.I.Kim,Doesahighfolateintakeincreasetheriskofbreastcancer?,NutrRev.64(2006)468–475.[10]S.W.Choi,S.Friso,M.K.Keyes,etal.,FolatesupplementationincreasesgenomicDNAmethylationintheliverofolderrats,Br.J.Nutr.93(2005)31–35.[11]S.W.Choi,S.Friso,Interactionsbetweenfolateandagingforcarcinogenesis,Clin.Chem.Lab.Met.43(2005)1151–1157.[12]Y.I.Kim,Roleoffolateincoloncancerdevelopmentandprogression,J.Nutr.133(2003)3731S–3739S.[13]S.C.Larsson,E.Giovannucci,A.Wolk,Folateandriskofbreastcancer:ameta-analysis,J.Natl.CancerInst.99(2007)64–76.[14]K.Wu,E.A.Platz,W.C.Willett,etal.,Arandomizedtrialonfolicacidsupplementationandriskofrecurrentcolorectaladenoma,Am.J.Clin.Nutr.90(2009)1623–1631.[15]T.E.Rohan,M.G.Jain,G.R.Howe,etal.,Dietaryfolateconsumptionandbreastcancerrisk,J.Natl.CancerInst.92(2000)266–269.[16]R.Jaszewski,S.Misra,M.Tobi,etal.,Folicsupplementationinhibitsrecurrenceofcolorectaladenomas:arandomizedchemopreventiontrial,WorldJ.Gastroenterol.14(2008)4492–4498.[17]B.F.Cole,J.A.Baron,R.S.Sandler,etal.,Folicacidforthepreventionofcolorectaladenomas:arandomizedclinicaltrial,JAMA297(2007)2351–2359.[18]J.C.Figueiredo,M.V.Grau,R.W.Haile,etal.,Folicacidandriskofprostatecancer:resultsfromarandomizedclinicaltrial,J.Natl.CancerInst.101(2009)432–435.[19]R.Z.Stolzenberg-Solomon,S.C.Chang,M.F.Leitzmann,etal.,Folateintake,alcoholuse,andpostmenopausalbreastcancerriskintheprostate,lung,colorectal,andovariancancerscreeningtrial,Am.J.Clin.Nutr.83(2006)895–904.[20]B.Stefanska,P.Salame,A.Bednarek,etal.,Comparativeeffectsofretinoicacid,vitaminDandresveratrolaloneandincombinationwithadenosineanaloguesonmethylationandexpressionofPTENtumoursuppressorgeneinbreastcancercells,Br.J.Nutr.107(2012)781–790.[21]B.Stefanska,K.Rudnicka,A.Bednarek,etal.,Hypomethylationandinductionofretinoicacidbeta2(RARbeta2)genebyconcurrentactionofadenosineanaloguesandnaturalcompoundsinbreastcancercells,Eur.J.Pharmacol.638(2010)47–53.[22]J.M.Garcia,J.Silva,C.Pena,etal.,PromotermethylationofthePTENgeneisacommonmolecularchangeinbreastcancer,GenesChromosomesCancer41(2004)117–124.[23]S.Khan,T.Kumagai,J.Vora,etal.,PTENpromoterismethylatedinproportionofinvasivebreastcancers,Int.J.Cancer.112(2004)407–410.[24]M.Widschwendter,J.Berger,M.Hermann,etal.,Methylationandsilencingoftheretinoicacidreceptor-beta2geneinbreastcancer,J.Natl.CancerInst.92(2000)826–832.[25]Z.Jin,G.Tamura,T.Tsuchiya,etal.,Adenomatouspolyposiscoli(APC)genepromoterhypermethylationinprimarybreastcancer,Br.J.Cancer.85(2001)69–73.[26]B.Stefanska,J.Huang,B.Bhattacharyya,etal.,Definitionoftheland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