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Study (2)

By Jeremy Parker,2014-07-19 00:34
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Study (2)study,Study

    Study

?218?cJIM2004;10(3):218220

    ;StudyOllTa)oIinInhibitingHumanLeukemiaCellProliferationand

    ;ZHAOXiaoying(赵小英),ZHANGXiaohong(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,

    ;theauthorsexaminedtheantiproliferation

    ;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.The3-4,5dimethylthiazol2,5

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

    ;ProliferationAssay

    ;MTTwasusedtomeasurecellprolifer

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

    ;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 ;enzymelinkedassayreaderat570nm. ;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:057187783802,

;E-mail:zhaoxy0731@yahoo.corn

    ;

    ;hrs.RightGiemsastainwasusedtodetect ;thecharacteristicmorphologicalchangesof ;apoptosis.

    ;DNAIadderinganalysis:K562cels

    ;weregrowninmediumcontaining0.25, ;0.5.1.0and2.0g/mlTaxolforl2,24. ;48,or72hrs.CelIswereharvestedbya ;5-mincentrifuati0nat2000r/minand

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

    ;laddering1ysisbuffer(10mmo1LTs

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

    ;5g/LSDS)andRNase5ml(10mg/’m1)

    ;andincubatedat56.Cfor2hrs.Theprotein ;wasremovedfromthecelIysatebyadding

    ;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

    ;ToIInhibit$CellProliferationinCultured

;K562Cells

    ;ProliferationofK562cellswassup

    ;pressedobviouslyaftera72hrsTaxoltreat

    ;2l9

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

    ;,”m1(Table

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

    ;K562cellstreatedwith0.5t~g/mlTaxolde

    ;creasedincellgrowthandviabilitywassta ;tisticallysignificant(P0.001)whencorn

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

    ;Table1.Inhibitor)’EffectofTaxolon

    ;K562CelIProljferation

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

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

    ;Taxolfor24hrs.Nuclearcondensation, ;chromosomefragmentationandapoptosis ;bodieswereobservedbyinverted

    ;mIcroscopy.

    ;ABFig.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.theantitumorefficacyofTaxolis

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

    ;steptowardsdeterminingwhetherTaxolcan ;alsobeusedtotreatleukemia,aformof ;malignanttumor,weinvestigatedtheanti

    ;proliferativeeffectofTaxolonculturedhu

    ;manleukemiacells.

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

    ;teristicofapoptosissuchasnuclearconden——

    ;sationandchromosomefragmentationwere ;evidentinTaxoltreatedK562cells.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 ;onlysinglestrandbreaks(,?.Thelackof ;DNA1adderbutthepresenceofextensive ;singlestrandbreakswasreportedwhen ;K562cellsunderwentapoptosis.Itispos

    ;siblethatthisphenomenoniscausedbyei

    ;thertheinhibitionortheabsenceofaspecif

    ;icendonucleaseactivityinK562thatlack ;double—strandDNAcleavage’”.

    ;cJIM2004:10(3):218220

    ;0urstudyalsoshowedthatTaxolwas ;abletoexertitsantiproliferationeffecton

    ;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

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

    ;iumiodidestainingandflowcytometry.JImmunolMeth

    ;ods1991;139:271279.

    ;3.WangHY,LiYM,LiuGQ.Advanceinstudyonmecha- ;nismofantitumoractionbyTaxo1.AdvanceonPharmacy ;1999:23(4):209214.

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

    ;logicalfeaturesofapoptosismayoccurintheabsenceof ;internuc|eosoma1DNAfragmentation.BiochemJ1992: ;286(Pt2):331334.

    ;5.GoochJL,YeeD.Strain-specificdifferencesinformation ;ofapoptoticDNAladdersinMCF-7breastcancercells. ;CancerLett1999:144(1):3137.

    ;6.McGahonA,BissonnetteR,SchmittM,eta1.BCRABL

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    ;7.WalkerPR,SikorskaM.Newaspectsofthemechanism ;ofDNAfragmentationinapoptosis.BiochemCellBiol ;1997;75(4):287299.

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

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    ;(ReceivedMay8,2004)

    ;

    ;

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