Study

?218?cJIM2004;10(3):218—220

    ;StudyOllTa)【oIinInhibitingHumanLeukemiaCellProliferationand

    ;ZHAOXiao—ying(赵小英),ZHANGXiao—hong(张晓红),

    ;XULei(徐磊),andZHANGXing(张行)

    ;ABS.I_F{ACobjective:ToexploretheeffectsofTaxolininhibitinghumanleukemiak562cellproliferation

    ;andinducingapoptosisinvitro.Methods:HumanleukemiaK562cellsweretreatedwithTaxolofdifferent

    ;concentrationsfor12.72hrs.CellproliferationwasevaluatedbyMTTassayandmorphologicalchangesof

    ;apoptosiswereexaminedbymicroscopy.Cellapoptosiswasdeterminedbyflowcytometry(FCM)andDNA

    ;gelelectrophoresis.Results:GrowthofK562cellswasinhibitedbyTaxolwithanIC5.valueof0.84pg/mI. ;Typicalnuclearcondensationandapoptosisbodieswereobservedasearlyas24hrsaftera0.5ug/mlTaxol ;treatment:ApoptoticrateoftheTaxo1.treatedK562cellsincreasedfrOm3.7%t024.O%in24hrs.NoDNA

    ;ladderwasobservedbyDNAgelelectrODhOresis.Conclusion:TaxolcouldinhibitK562cellgrowthandin—

    ;duceapoptosisinvitro.

    ;KEYWORD8Taxol,leukemia,K562cellIiRe,cellapoptosis

    ;Paclitaxel(Taxo1)isanaturalproduct

    ;foundinthebarkofyewtreessuchasTaxus

    ;baccataandTaxusbrevifolia.Becauseofits

    tumoractivity,Taxolhasbeen ;potentanti—

    ;extensivelytestedasachemotherapeutica—

    ;gentagainstmalignanttumorsandhasbeen

    ;provenclinicallyeffectiveintreatingbreast

    ;andovariancancer.Despiteitspotentialan—

    ;ti-tumoractivity,Taxolhasnotbeensys—

    ;tematicallyinvestigatedregardingitsappli—

    ;cationintreatingIeukemiabothinIaborato—

    ;ryandclinicaItrials.Inthepresentstudy,

    ;theauthorsexaminedtheanti—proliferation

    ;andpromotingapoptosiseffectofTaxolon

    ;culturedhumanIeukemiaK562cells.

    ;METHoDS

    ;eellCulture

    ;K562cellswereculturedinRPMI1640

    ;mediumsupplementedwith10%fetalcalf

;serum.at37.Cinanincubatorwithhumidi—

    ;fledatmospherecontaining5%CO2. ;Reagents

    ;TaxolwasgivenfromSichuanTaiji ;PharmaceuticalCo.Itwasdissolvedin ;O.9sodiumchloridetotheinitialconcen—

    ;trationof6Oyg/mlanddilutedtodesired ;concentrationswiththecellculturemedium. ;TheK562humanacuteerythroleukemiacell ;linewasprovidedbyCancerInstitute,Zhe—

    ;iiangUniversity.RPMI1640andfeta1calf ;serumwerepurchasedfromHycloneCo. ;andHangzhouEverygreenBiotech,Ltd.re—

    ;spectively.The3-4,5一dimethylthiazol一2,5一

    ;diphenyltetrazolium(MTT)andpropidium ;iodide(PI)werepurchasedfromHuamei ;BioandSigmaCo.respectively.

    ;ProliferationAssay

    ;MTTwasusedtomeasurecellprolifer—

    ;ation(1).K562cellsuspension(2×10cells/ ;m1)wasinoculatedintoeachwellofa96一

    ;wellmicrotiterplate.Eachwellwaspre—

    ;loadedwith100”loftheculturemedium

    ;containingTaxolofvariousconcentrations ;(O.125,O.25,O.5,1.O,2.O,4.0/zg/m1). ;Foreveryconcentrationthreewellswere ;used.Cellswerencubatedfor72hrsat37. ;CinaCO,incubator.Cellgrowthandvia—

    ;bilitywasassayedbyadding2OtAMTT ;with5mg/m1.After72hrsofincubation, ;coloredformazanconvertedfromMTTby ;viablecellswasdirectlyquantifiedusingan ;enzyme—linkedassayreaderat570nm. ;CelI

    ;cells/m1)

    ;0.5,1.0

    ;Assays

    ;morphology:K562cells(1X10

    ;weregrowninmediumwith0.25,

    ;and2.0yg/mlTaxolfor12to24

    ;DepartmentofHematology,TheSecondHospital,College

    ;ofMedicine,ZhejiangUniversity,Hangzhou(310009),

    ;China

    ;Correspondenceto:ZHAOXiao-ying,Tel:0571—87783802,

;E-mail:zhaoxy0731@yahoo.corn

    ;

    ;hrs.Right—Giemsastainwasusedtodetect ;thecharacteristicmorphologicalchangesof ;apoptosis.

    ;DNAIadderinganalysis:K562cel】s

    ;weregrowninmediumcontaining0.25, ;0.5.1.0and2.0g/mlTaxolforl2,24. ;48,or72hrs.CelIswereharvestedbya ;5-mincentrifu窟ati0nat2000r/minand

    ;washedtwiceinPBS.Thecellpellets(2× ;10cells/’m1)wereresuspendedii1600?lof

    ;laddering1ysisbuffer(10mmo1LTs—

    ;HCl,10mmol/’LEDTA.75retool/LNaC1.

    ;5g/LSDS)andRNase5ml(10mg/’m1)

    ;andincubatedat56.Cfor2hrs.Theprotein ;wasremovedfromthecelI】ysatebyadding

    ;l5lproteinaseK(10mg/’m1)andineuba—

    ;tionat37.Cfor24hrs.TotalcellularDNA ;wasisolatedusingI1vdroxvbenzeneand ;chloroform.DNAfragmentationpattern ;“w-asexaminedby2agarosegelelectropho—

    ;resisfor4hrsat60V.

    ;Flowcytometry:Apoptosisw-astested ;aspreviouslydescribed.K562cel1s(1× ;10cells/m1)weregrowninmediumcontai—

    ;ning0.25,0.5,1.0,and2.0>g/mlTaxoI ;for24hrs.AtthesametimethecontroIthat ;didnotcontainTaxo1wasfound.Cellswere ;fixedbycoldalcoholandstoredat4.C.Be ;foreanalysisbyFCM.theceIlsweretreated ;withRNase(10mg/m1)for10minat37.C. ;Afterstainingwith50gmlPIfor30min, ;thecellsweremeasuredbvFCMandthere—

    ;suItswereanalyzedusingsoftwareCellquest ;3.1andMuhieycle3.0.

    ;Slatistlcalhlalysis

    ;StatisficaIevaluation,includingtlest ;andwelghtedlinearregresstonwereper ;formedwithSPSSforWindows(10.0). ;Differenceswereconsideredsignificantif ;valuesofP<O.05.

    ;RESULTS

    ;T缸oIInhibit$CellProliferationinCultured

;K562Cells

    ;ProliferationofK562cellswassup—

    ;pressedobviouslyaftera72hrsTaxoltreat一

    ;2l9

    ;ment.TheinhibitoryeffectofTaxo1on ;K562cellproliferationwasdose-dependent ;atarangefrom0.125to1.0tzg.

    ;,”m1(Table

    ;1)andtheIC.valuewas0.84t~g./’m1.The

    ;K562cellstreatedwith0.5t~g/mlTaxolde—

    ;creasedincellgrowthandviabilitywassta ;tisticallysignificant(P一0.001)whencorn—

    ;paredwiththeuntreatedcontro1.When ;1.0gmlTaXO1wasused,53.52ofcell ;pronferationwasinhibited.

    ;Table1.Inhibitor)’EffectofTaxolon

    ;K562CelIProljferation

    ;Notes:’P<0.05,.’P<0.01一corllparedwiththe ;COIllrO】

    ;TaXOllnduoesApoptoslsInK562Cells ;Typicalapoptoticbodieswerepresent ;intheK562ceilstreatedwith0.5#g/”ml

    ;Taxolfor24hrs.Nuclearcondensation, ;chromosomefragmentationandapoptosis ;bodieswereobservedbyinverted

    ;mIcroscopy.

    ;AB鬣Fig.1.MorphologicalChangesofK562Cells ;Note:A:Control}B:05gmlTaxol

    ;AsmeasuredbyFCM,theapoptotic

    ;rateincreasedto24following0.5#g. ;/ml

    ;Taxo1treatmentfor24hrs.andincompad—

    ;sonitshowedanapoptoticrateof3.7inthe ;control,i.e.theuntreatedK562cells.Charac—

    ;teristicDNA[addering,however,itwasnot ;observedintheTaxoltreatedK562cells. ;

    ;?

    ;22O?

    ;DlSCUSSION

    ;Taxolhasbeenprovedtobeeffectivea- ;gainstavarietyofsolidtumorsandhasbeen ;successfullyusedtotreatmalignantdiseases ;suchasbreastcancer,ovariancancer,and

;lungcancer.Althoughtheunderlyingmo—

    ;lecularandcellularmechanismsstillremain ;unclear.theanti—tumorefficacyofTaxolis

    ;evidentlyrelatedtoitsabilitytoinducecell ;apoptosis,topromotetumornecrosis,and ;toenhancehostimmunity’.Asaninitial

    ;steptowardsdeterminingwhetherTaxolcan ;alsobeusedtotreatleukemia,aformof ;malignanttumor,weinvestigatedtheanti—

    ;proliferativeeffectofTaxolonculturedhu—

    ;manleukemiacells.

    ;OurresultsshowedthatTaxolwasable ;toinhibittheproliferationofK562cellswith ;anIC5ovalueof0.84/~g/m1.Apoptosiswas ;triggered,asmorphologicalchangescharac—

    ;teristicofapoptosissuchasnuclearconden——

    ;sationandchromosomefragmentationwere ;evidentinTaxol—treatedK562cells.Anap—

    ;optoticrateof24wasreachedwithin24 ;hrsfollowingtreatmentwith0.5/,g/ml ;Taxo1.ThesedatasuggestedthatTaxo1cy—

    ;totoxiceffectonK562cellsmightbelinked ;toapoptosis.Interestingly,noDNAladder ;wasobservedbyDNAgelelectroDhoresis ;whenK562cellswereexposedto0.5/,g/ml ;Taxolfor24hrs.Internucleosomaldouble—

    ;strandDNAcleavage,whichischaracterized ;byformationofDNAladdersinagarose ;gels,hasbeenconsideredoneofthehall—

    ;marksofapoptosis.However,severalstud—

    ;iessuggestedthatdetectionofDNAladders ;isnotnecessarilyanaccurateindicatorofap—

    ;optosis,sinceapoptosiscanalsooccurwith ;onlysingle—strandbreaks(,?.Thelackof ;DNA1adderbutthepresenceofextensive ;single—strandbreakswasreportedwhen ;K562cellsunderwentapoptosis.Itispos—

    ;siblethatthisphenomenoniscausedbyei—

    ;thertheinhibitionortheabsenceofaspecif—

    ;icendonucleaseactivityinK562thatlack ;double—strandDNAcleavage’”.

    ;cJIM2004:10(3):218—220

    ;0urstudyalsoshowedthatTaxolwas ;abletoexertitsanti—proliferationeffecton

    ;K562cellsataverylowconcentration(e.g. ;0.125/~g/m1).When1.0/~g/mlTaxolwas

    ;used,theproliferationof53.52cellswas

    ;inhibited.Alamleta1.reportedpoorlevels ;ofinvivoinductionofapoptosisfroma

    ;phaseIclinicalstudyofpaclitaxeltherapy ;in26leukemicpatients.Theirdatasugges—

    ;tedthattheresponsesofCD34negativeand

    ;CD34positiveacutemyeloid1eukemia

    ;(AML)cellstopaclitaxelarequitediffer—

    ;ent.CD34negativecells,suchasHL60and

    ;U937,tendstobemoreresponsivetothe

    ;inhibitoryeffectofTaxol’.Inordertoac—

    ;curatelyevaluatethetherapeuticpotentialof ;Taxolonleukemia,weproposethat,inthe

    ;futureleukemiapatientsshouldbepre—

    ;screenedandgroupedintodifferentcell

    ;typesandimmunetypesforclinicaltreat—

    ;mentwithTaxo】.

    ;REFERENCES

    ;1.ZhangYS,ZhouH,HanFL,eta1.Studyoninvitro ;killingactivityofDAAO/D-AlosystemtoK562cel1.Clin ;JHematol2002;23(1):12—15.

    ;2.NicolettiI,MiglioratiG,PagliacciMC,eta1.Arapid ;andmethodformeasuringthymocyteapoptosisbypropid—

    ;iumiodidestainingandflowcytometry.JImmunolMeth—

    ;ods1991;139:271—279.

    ;3.WangHY,LiYM,LiuGQ.Advanceinstudyonmecha- ;nismofantitumoractionbyTaxo1.AdvanceonPharmacy ;1999:23(4):209—214.

    ;4.CohenGM,SunXM,SnowdenRT,eta1.Keymorpho—

    ;logicalfeaturesofapoptosismayoccurintheabsenceof ;internuc|eosoma1DNAfragmentation.BiochemJ1992: ;286(Pt2):331—334.

    ;5.GoochJL,YeeD.Strain-specificdifferencesinformation ;ofapoptoticDNAladdersinMCF-7breastcancercells. ;CancerLett1999:144(1):31—37.

    ;6.McGahonA,BissonnetteR,SchmittM,eta1.BCR—ABL

    ;maintainsresistanceofchronicmyelogenousleukemia ;cellstoapoptoticcelldeath.Blood1994:83(5):1179—

    ;1187.

    ;7.WalkerPR,SikorskaM.Newaspectsofthemechanism ;ofDNAfragmentationinapoptosis.BiochemCellBiol ;1997;75(4):287—299.

    ;8.ALAlamiO,SammonsJ,MartinJH,eta1.Divergent ;effectofTaxolonproliferation.apoptosisandnitricoxide ;productioninMHH225CD34positiveandU937CDa4neg—

    ;ativehumanleukemiacells.LeukRe81998;22(1O): ;934—945.

    ;(ReceivedMay8,2004)

    ;

    ;