TPA对人食管癌Eca109细胞增殖和细胞核形态的影响.docx

TPA对人食管癌Ecal09细胞增殖和细胞核形态的影响TPA对人食管癌Eca109细胞增殖和细胞核形态的影响TPA对人食管癌Eca109细胞增殖和细胞核形态的影响作者：郭玉忠，贾琳静，部娜，田鑫，谢敏，张莉蓉，乔海灵【摘要】目的研究佛波(12-0-tetradecanoy1phorbo113-acetate,TPA)对人食管癌细胞的影响。方法细胞增殖试验采纳MTT法和细胞计数的方法，细胞核形态的测量采纳计算机协助的图像分析系统，分别测量计算细胞核面积(NA)、核周长(NP)、最大直径(DmaX)、最小直径(Dmin)、核浆比(NPA)溟、核轴比(NAR)、核形态因子(NR掰F)等。结果TPA没有抑制人食管癌Ecal09细胞增殖和变更细胞核形态漱。结论TPA对人食管癌Eca109鹰细胞可能没有促分化作用。【关键词】佛波酯；食管癌细胞；细胞核形T态EffectsofTPAo跋nproliferationandnUClearmOrPhOmeIryo贽fhumanesophagealca撕ncerEcalO9cells【A殛bstractObjective湾SToinvestigatethe溶effectsof12-0-tetr揣adecanoy1phorbo1-13-acetate(TPt)onhu疙manesophagealcance垩rEcalO9celIs.MethodsThesensitivityoVlfTEAtoEca109ce11sw酥asdeterminedwithMTTassayandcountingc侔el!numbers.Afterth稼ecelIswerestaincdw否ithHEIhCmCaSUremCntsofnuc1earmorpho破metrywereperfor11ed咫WilhComPUIer-assisIedimageanalysissy籀stem.ResultsTPdi辨Clnotinhibitthecel1咬sproliferationandd枭CcreasethenucIears哪ize.ConclusionsTP麽sophageaIcanccriso绐neofthemostco11unons筠olIdtumorsinChina.AmaynotinduceEcalO铢9celIstodifferenti遍ate.Keywords12钻-O-tetradecanoyIph海orbol-13-acetate(T嫂PA)：esophagealcanc每ercellline；nuclearmorphometryHumaneDuetoitshighmetast松aticpotentialandthefrequentonsetofre翩Sistancetochemothe噬rapy,itisoncofthcmajorcausesofdeathb衡yneopIasiainthecou谈ntry.Thepresentche轿Sesagentsthatareus剑cells.OntheotherhaOndUlardifferentiatio站motherapyofcanceru辛ua11ytoxiCtonorma1羚inductionofcel1孝nmaysupplementtheu理seofnon-cytotoxicdkrugsinsevera1formsOfneoplasia,1iketh绅Csuccessfuluseofal1-transretinoicaci运d(?TRA)inthetreatm镂entofacutepromyelo番Cyticleukemia.Pla-niditerpinephorbol笛esters,particular1荀ythephorbolesterl2幺-O-tetradecanoylph灵orbol-13-acetate(T哈tpotenttumorpromotfeetsrangefromstimSthroughactivating源Oducingalterationsesandconstituents.臼tetHedifferentiati调Ilsincultureandinv睐PAcaninducediffere殃PA)»areamo11gthemos鸣erSknownandthEiref祸UlatingDN-Asynthesi蝮geneexpressiontopr栽WivinmembrancpropcrtiTPcanalsopotentiaonofvarioustumorce禁iVoofhumanbEIngs.Tntiationandinhibit彝thegrowthofanumber臼ofmalignantcelltyp宋es,includingmyeloi调dleukemia1,breast2»prostate3,c吉olon4,andbrain5英.OtherreportshoweHthatTPAcouldnotin航rcinomacelIline花6.BccauseofitseffeareinterestedinitYfPhagealcancercells抖ffectsoP.onpro1i孩morphometryofhumanducehumanhepatoca管todifferentiation听iciencyi11vivo7,”,诸Seffectsonhumaneso7.Here»Wereportthee莫esophagealcancerEca!09cel1s.1Mater潭ialsandmethodsChedfromSigmaChemica抠emicalsTPApurchas碳ICo.Wasdissolvedin哧ferationandnuc1ear溥ethanolandstoredat蹇-204C.Thestocksolut炷ionswerediIutedwit鳗hHanksbuffercdsaltsolution.Thefinal邙Concentrationofeth虢anolintheincubatio15.nmixtureswas%orles皎s.CellGrowthStud狡iesHumanesophagea½Icancercel1IineEca109WaSkindiyPrOVid侵edbyprofessorBaiji屉ngxiu,ZhengzhouUniversity.Thecellswe巳remaintainedinRPMIr1640mcdiumwithl%fetalbovineserum(FB烟S)Inthepresenceofa娱ntibiotics,penici1摄lin(100unitsml)an3dstreptomycin(100u旖nits/ml).Allcellsw醛ereculturedat37*Cinahumidifiedatmosph咖erecontaining5%C02鳗.Themediuinwasrepla好Cedafter3days.Thec呈e11Swereroutine1ysUbculturedtwiceaweek.Cellswercplate秘(Iineitherflasks(25讫cm2)or96-weHplateX"s.TPAwasadded24hou迂rsafterseeding»who筏nthecelIswereattac埠hedtothesurfaceoft廉hecontainers.AnequivalentvolUmeofethanolwasaddedtothec饬ontro1SandhadnoeffXectongrowth.Afterr萱emovingIhecellsfroIntheflasksbytrypsiXInizatiOntheywereco退Untedusingahemocyt杼Ometer,fixcdwith70黑%ethanolandstained囱byWhenthecelIswere榭platedintoa96-wel1缗microplatethecult灭uremediumcontainin硅gdifferentconcentr柱alionSofTPAWaSrCPlaced24hourslater.After3-5daysofincub苴ation,Cellviabilit溅ywasevalUatedbyMTT诫assay.Nuclearmor漆PhometrymeasurementsMeasurenientsof忍nucIearmorphometry恻inc1udingnuc1eararea(NA)»nucIearperimeter(NP),maximaid响iameter(Dmax),minimaldiameter(Dmin),得nucIearplasmaratio哩(NPR)»nuclearaxisr惺atio(NAR),nucIearrOundnessfactor(NRF的)»wereperformcdwit水hcomputer-assisted磋imageanaIysIssystemMPIS-500(QingPin愍gImageEngineeringC葩ompany,TongjiUniversity).Twofieldswe恪reselectedatrandom眈fromcachcaseforthc探measurements.Eachf睛ie1dwasviewedunder齿a100oi1-immension亮1cnsanddisp1ayedon魔theserecnofacoIorvyideoimageprocessor屐.TheimagesofSOnuc1首eiofthetumourcelIs宗Perfieldwcreoutlin隶edwithadrawingpen9andthedatawereproc禹Cssedbyacomputerin悯thesystem»whichcal铉Culatedtheparamete激rs.Intheendallthem蛾Canvaluesofeachcasewcrccalculated.StatisticalAnalysi网SThedatawereexpre萧SsedasmeansSD.Sta骆tistJcalanalysiswa骼SPerfonnCdbySPSSSoftware.Thecriterio1nforstatisticalsig约nifiCancewastakena倾sP2ResultsEffect-j-OfTPAoncel!Growth.ThegrowthofEcal09c雷Cllswasnotarrested骄byTPAatnoneytotoxi峋Cconcentrations.As押ShowninTableland,TPAevenincreasedtheEcancercel!numbers»(IDthoughtherewerenosignifiCancebetween酎TPAgroupsandthecon太trols.TablclCell轧numbersofTPtreatm桐entsonEcal09celIs歙(xs)MTTassay琮OrrPAtrealmentSOnEcal09concentratio雾nwaslO-8mol1.ThenucIearfeaturesweremeasuredonmicroph徐Otographsusingacom饷mercialIyavaiIable蛹software.Table2summariZestheresuIts.5tTPAmightdecreaseth侪Ccellssizebutnosig养nificantdifference蹉Withthecontrolgrou篷p.Table2MeasuremCntSOfnUClEimOrPhOMmetryafterTPAtreatmerits(xs)3Di昔ScussionOurresultSshowthatEca109hum猫anesophagea1cancer声ce11isace11typewhi铢ChdoesnotresponcltoexposuretoTPbygroWtharrest(Tablclan吭d).AmarkedchangeinWcellmorpho1ogywasn秧Otobserved.Oncompa胎risonofthedifferen1.thumancelIsystemsi汶nwhiChphorbolester藩Scausegrowtharrest吊,thebehaviorofthcE吨Ca109ce11SaPPCarSt迅OresemblethatofSMM辄C7221humanhapatoma傍celIs6andHT29humancolOncancercelIs蜷8.EvcninTcellleu颜kerniaTPcou1dinduceJUrkatandMolt3cel傩Istodifferentiatcb熄UtcouldnotinduceCC匆RF-CEMandCCRF-HSB2缥Cellstodifferentia+te.Thereasonsareno嵬tknown.MorphometricanaIysishasbeenu纲Sedsuccessfullyasa训diagnosticandprogn三Osticcriterioninpa妻Iientswithmalignancies9.ManyinvestigaIorshavcsoughta迹naccurateandreprod饺Ucibleparametertha前twilIalIowaccurate燔predictionofadjunc原tiVeinimunothcrapyorchemotherapywhena+ppropriate10.The键semorphomctricstud民ieshavcprovidcdva1脂Uableinformation.W黔eusedquantitativem榻orphomelrytoinvest瞄igatcthcshapcdiffe腔renceofneoplasticn爸uclEIfromesophagea喏Icancercellswithor缈Withouttreatmentof蛹TPA.TP.didnotclear除1ychangetIieshapeof国thecellsnuclei,but腿iIdidchangeothcrce銮IlsnucIeiwhichwerefoundinourlaboratory(datanotshown).佻ThisresuItispossibIyconsistentwithth资atofTPAonce11spro1疔iferation.WhenTPAi嫂nducestumorcelIsto赊differentiation>it哈hasantiproliferationfunction.IftumorfcelIsproliferatcac郁lively»differentia笳tiOnmaystopatleast荔temporal'ily.Therea贩SonsthatTPAdidnotinfUenccthcprolife11rationandnucIearmo锻rphometryofesophag瞰ea1cancerce11sarep低Ossiblyconnectcdto恨thedIversityandcel伊IulardistributionsOfproteinkinaseC(PKC)onthecelIs.Prot急eInkinaseC(PKC)isa萱famiIyofphospholip蚁id-dcpendentserine低-threoninekinasest病hatpIayimportantro珈Iesinthesignaltran抠sductionandintheregulationofcelIgrow注thanddiCferentiatij硼on11.Atleastelev垦eniSoformsofPKChav惰Cbeenisolatedsofar梢fShowingdiversityintheirstructurescCllulardistributions,andbiologicalfu贽notionsE12.PKCisa匀Isothereceptorfort秫hepotenttumor-promOtingphorbolesters蟠，Whichcansubstitut,reforDGinPKCactiva9tion.Reportsalsosh堆OwcdthatdifferentP嘈KCsuhtypewasinvolv累edinthedifferentia7互tionofdifferenttum鑫OrcellsduringTPAtrteatmcnts13.Therefore9thedistributi月并Onsandtheactivityo膑fPKCinEca109ce11smightbedifferentwitThothertumorcelIsan恰Clhavedifferentsign洋altransductionsyst始Cms.SoTPAmighthave½differe11teffectson；differenttumorcell答s.Furtherstudiesab蒿Outthedistribution鳏Sandactivestatusof棚PKcsubtypesonhumanesophageaIcancerce锚Ismaynotonlyprovi爰deusefulinformalio锥nonselectioneffectivedifferentiation求-inducersbutalsoco竺Dtributetoelucidal绩iOnofthemolecularm鸿echanisminvoIvedin跑Ihcirdifferentiati骡onandtumorigenicity.References1Sordet,RebeC,1.eroyI谗,etal.Mitochondria(±-targetingdrugsars凋enictrioxideand1on弱idaminebypasstheresistanceofTP-differentiatedeukemic阎celIstoapoptosis.B肋100d»2001»97(12)：3症931-3940.2perry1.羊，HayesD»KufeD.Effe油Ctsofdifferentiati'Onagentsoncellsurf阻aceexpressionofthebreastcarcinomaassociatedDF3Pepitope.CancerRes,1992,5仞2(22)：6365.3Suzu龙kiT,ShimizuT,Saloh格K»etal.DifferentiaIexpressionofgenesduringTP-inducedd塘ifferentiationofhu总Tsu-Prlcells.IntJMolMed,7丸35-739.4KuoMI.,entia1requirementf巾Hosphataseactivity柳anoy1phorbo1-13-ac磔manprostaticcancer棉2002,10(6)：Hu讼angTS»1.inJK.Prefer挑Orproteintyrosinep寐inthel2-0-tetradecetateinduceddiffercntialionofhumaneoloncancercells.Bi悯QchcmPhannacol,199嬴5,50(8)：1217-1222.津5TrolIerlI»Zeidma姜11R,SvenssonK,etal.APKCbetaiSoformmed氓Iatesphorbolester-银inducedactivationo悔fErkl2andexpressiuroblastomacells.F彦EBS1.ett.2001,508(1l)：onofneurona!differentiationgenesinne郸126-130.6ChaiX扒Y,ShenYF，ChenII1.Ef炕feeIofphorbolesteronproteinkinaseCanCltyrosineproteinkinaseinhumanhepatocarcinomace111ine.C.hinJ0ncol»1993,15(3)：182-184.7Stra嗫irRK，SchaarD,Goode埋H1.,etal.AdministrViationofaphorbolest傻Crtopatientswithheimatologicalmalignancies：preliminaryrCsultsfromaphaseIc1inicaltrialof12-07-tetradecanoyIphorbol-13-acetate.Cli舄nCancerRes,2002,8(8)：idleukcmiccelIsbym臆easurementofDNAcon堇tentnucleararea,a主nddetectionofPglycoprotein.Cancer,1艺2512-2518.11rciadeIIerrerosFaBreM.Thetuniorpromoter12-0-totradecanoy1phorbo1-13-acetateblocksdifferentiationofHT-29human-coloncancercells.J杏CellSci»1993,105(P庚t4)：1165-1172.9H敲ami1tonPW,WyattJI»媪QuirkeP>etal.MorphOmetryofgastriccar裕cinoma：iIsassociat毓io11withpatientsurv阮ivaltumourstage，a地ndDNAploidy.JPatho耋1.1992,168(2)：201-208.10EmuraI,Nai稣toM,KakiharaT,etal.Identificationofd错rug-resIStantmyelo谄996,77(5)：878-887.11NewtonAC.ProteinkinaseC：structure樊，function,andregul.ation.JBioIChem>1995,270(48)：28495-2嫡8498.12JakenS.Pr砧oteinkinaseCisozym鳏esandsubstrates.CurrOpinCelIBiol,199断6,8(2)：168-173.13TonettiDA.Henning阔-ChubbC.YamanishiF)雕T,etal.Proteinkina挎seC-betaisrequirecl馆formacrophagediffe躲rentiatio11ofhuma11H甸1.-601eukemiacells.聚JBiolChem,1994,269岛(37):23230-23235.