论著基因芯片技术分析斑蝥素对肝癌细胞细胞毒作用的分子机.pdf

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1、论著基因芯片技术分析斑蝥素对肝癌细胞细胞毒作用的分子机制胡和平13,张俊平2,应康3,肖振宇2,吴红梅3,毛裕民3,吴孟超1(1.第二军医大学东方肝胆外科医院消化内科,上海200438;2.第二军医大学药学院药理学教研室,上海200433;3.复旦大学生命科学院遗传工程国家重点实验室,上海200433)摘要目的:研究抗癌药斑蝥素对肝癌细胞细胞毒作用的分子机制。方法:应用基因芯片技术检测12.5mol?L斑蝥素作用于肝癌Q GY7703细胞24 h后基因表达谱的变化。结果:斑蝥素抑制细胞表达参与细胞周期进程基因(如p27、ref21、DN Apolym erase delta、X RCC9等)、

2、能量代谢基因(如m alate dehyd rogenase、A D P?A T P translocase等)、致瘤活性基因(如c2myc、tre等)以及肿瘤特异表达基因(如bladder cancer related p rotein等)。相反,斑蝥素促进了多种细胞生长抑制基因(如B CRA2、B TG2、dual2specif icity p rotein phosphatase等)以及凋亡相关基因(如A TL2derived PM A2responsive pep tide等)的表达。结论:斑蝥素改变调控细胞周期进程、细胞增殖、能量代谢以及凋亡级联反应的基因表达可能是其细胞毒作用的机制

3、。关键词斑蝥素;基因表达谱;基因芯片;肝癌;Q GY7703细胞中图分类号R 735.7文献标识码A文章编号02582879X(2003)0620645205Gene m icroarrays in detecting molecular mechan ism s of cantharidin-mediated cytotoxicity on humanhepatic cancer cellsHU He2Ping13,ZHAN G Jun2Ping2,YI N G Kang3,X I AO Zhen2Yu2,WUHong2M ei3,MAO Yu2M in3,WU M eng2Chao1(1.

4、Department of Gastroenterology,Eastern Hepatobiliary Surgery Hospital,Second M ilitary M edical U niversity,Shanghai200438,China;2.Department of Pharmacology,School of Pharmacy,Second M ilitaryM edical U niversity,Shanghai 200433;3.State Key L aboratory of Genetic Engineering,School of L ife Science

5、,Fudan U niversity,Shanghai 200433)ABSTRACTObjective:To study the molecular mechanism s of antitumor agent cantharidin on human cancer cells.M eth2ods:Gene expression profile was identified by cDNA m icroarrays in hepatic cancer Q GY27703 cells exposed to cantharidin(12.5mol?L)for 24 h.Results:Canth

6、aridin2treated cells had decreased expression of genes coding for proteins involved incell cycle progress(p27,DN A polym erase delta,X RCC,ref21),energy metabolism(m alate dehyd rogenase,A D P?A T P translo2case),oncogenic activation(c2myc,tre)and tumor2specific expression(bladder cancer related p r

7、otein).In contrast,these treatedcells overexpressed several genes encoding intracellular grow th2inhibitory proteins(B CRA2,B TG2,dual2specif icity p roteinphosphatase)and proapoptotic genes(A TL2derived PM A2responsive pep tide).Conclusion:These findings suggest that alter2ations in specific genes

8、involved in modulating the cell cycle progress,cell proliferation,energy metabolism,and apoptosis cas2cadesmay be responsible for cantharidin2mediated cytotoxicity.KEY WORDScantharidin;hepatic cancer;cells;gene expression profiling;cDNA m icroarray;Q GY7703 cellsA cad J SecM ilM ed U niv,2003,24(6):

9、6452649 斑蝥素(cantharidin,CTD)为传统中药斑蝥的活性成分,临床上对多种肿瘤有疗效,特别是肝癌和白血病,具有升高白细胞而无骨髓抑制的特点1,2。斑蝥素的抗癌作用机制目前尚不清楚,有研究发现可能与其抑制蛋白磷酸酶有关3,但是其分子作用机制还不确定。基因芯片技术的发展为大规模分析基因表达提供了有效的工具4,5。本研究应用基因芯片技术分析斑蝥素作用于人肝癌细胞系Q GY7703后的基因表达谱变化,探讨其抗癌的分子作用机制。1材料和方法1.1细胞培养和药物处理Q GY7703肝癌细胞系购于中国科学院上海细胞研究所,用含10%胎牛血清(GibcoBRL)的RPM I 1640培养液

10、(Sigma公司)传代培养。细胞用胰蛋白酶消化后,接种于75m l培养瓶中,生长达90%融合后,弃上清,更换含10%胎牛血清的新鲜培养液。对照组只加药物溶剂(0.1%M e2SO),药 物 组 加 入CTD(Sigma公 司,12.5mol?L),细胞置于37,5%CO2孵箱培养24 h。1.2RNA制备和芯片杂交用异硫氰酸胍和酚氯仿试剂一步法抽提对照组和药物组细胞总RNA,用O ligotex mRNA kit(Q iagen)分离mRNA,再用R iboGreenRNAquantitationkit(M olecular546第 二 军 医 大 学 学 报Acad J Sec M il M

11、 ed U niv2003 Jun;24(6)基金项目上海市临床医学中心发展基金(ZX01B05).作者简介胡和平(19612),男(汉族),硕士,副教授,硕士生导师.3Corresponding author.E2mail: 1995-2005 Tsinghua Tongfang Optical Disc Co.,Ltd.All rights reserved.Probes,Eugene,OR)测定mRNA含量。药物及溶剂对照组mRNA分别用cDNA一链合 成 掺 入 荧 光 标 记dU TP(Cy52dU TP?Cy32dU TP)的方法制备cDNA探针,混合后在密封仓内与12800型人c

12、DNA基因芯片(U nited Gene Hold2ings,L td.,PRC)42杂交16 h。结束后按BioDoor洗片技术进行洗涤和晾干。再用ScanA rray 3000(General Scanning公司)扫描芯片,用I maGene3.0软件(BioD iscovery,Inc.)分析Cy3、Cy5两种荧光信号的强度和比值7。每次芯片杂交重复2次。选择两次杂交比值均-2或者-2作为判定药物作用下肿瘤细胞系基因表达差异标准。2结果2.1斑蝥素对细胞周期进程相关基因表达的抑制作用基因芯片结果表明,斑蝥素能显著抑制肝癌细胞系Q GY7703表达细胞周期相关基因,这些基因功能包括参与细

13、胞有丝分裂、核酸合成、DNA复制和修复以及RNA加工和蛋白质合成(表1)。表1斑蝥素对细胞周期相关基因表达的影响Tab 1Effects of cantharidin on expression of genes involved in cell cycle progressGeneAccession numberFunctionFold changeM itosisChrom atin2specif ic transcrip tion elongation f actorNM2007192Transcription factor-3.9P27AB001740Kinetochore-3.3Ge

14、neral transcrip tion f actor3A(GT F3A)D32257Transcription factor-2.5Chrom atin assem bly f actor2?P150U 20979Chromatin assembly-2.4K inesin superf am ily m otor K IF4AF071592Kinetochore-2.2Centrom ere autoantigen CM 95724Kinetochore-2.0DNA replicationD inb1AB027564DNA polymerase-5.5DN A ligase?M 360

15、67DNA ligase-3.7DN A polym erase deltaU 21090DNA polymerase and exonuclease-3.5D ihyd rop terid ine reductaseM 16447Nucleotide synthesis-3.4DN A polym erase epsilonl09561DNA polymerase-3.2P rim ase,polypep tide1(49 000)NM2000946DNA pri mase-3.0R ibonucleotide reductaseM1X59543Nucleotide synthesis-2.

16、9M ethy lenetetrahyd rof olate dehyd rogenaseJ04031Folate metabolism regulator-2.5Putative gene containing theM CM3AL 034343DNA replication licensing factor-2.5R ep lication p rotein A(70 000)M 63488Single2stranded DNA binding protein-2.4CT P synthetaseX52142Nucleotide synthesis-2.3R ep lication p r

17、otein A(32 000)J05249Single2stranded DNA binding protein-2.1R ep lication f actor C(36 000)L 07540PCNA clamp formation-2.0DNA repairR ef21(A PEX nuclease)S43127Apurinic endonuclease-7.5X rcc9NM2004629DNA postreplication repair or cell-6.0cycle checkpoint controlE rcc2X52221Correcting the sensitivity

18、 to UV radiation and-5.5defective nucleotide excision repairR eca2like p rotein(H rec2)U 92074DNA recombination and repair-3.7DN A2dependent p rotein kinaseU 47077Double2strand break repair-3.5cataly tic subunit(DN A2Pkcs)and V(D)J recombinationM uts,E.coli,hom olog of,6(H m sh6)U 73737M ismatch2bin

19、ding factor-3.4RN A p rocessing and p rotein synthesis nucleolinM 60858Rrna transcriptional control,ribosome-6.9maturation and assembly,and ribosomalcomponents transportation40S ribosom al p rotein S3X55715Ribosomal protein and DNA repair-5.0H istidy l2trna synthetase hom olog(H O3)U 18937Protein sy

20、nthesis-4.2Cdna(w eakly sim ilar to putative p re2m rnaAK001751RNA splicing-3.8sp licing f actor RN A helicase)P re2m rna sp licing f actor S F2p32M 69039RNA splicing-3.5Putative trna synthetase2like p roteinU 07424Protein synthesis-3.4646第二军医大学学报2003年6月,第24卷 1995-2005 Tsinghua Tongfang Optical Disc

21、 Co.,Ltd.All rights reserved.(续前表)GeneAccession numberFunctionFold changeSm all nuclear ribonucleop rotein polypep tide A(SN R PA1)NM2003090Protein synthesis-3.2RN A polym eraseU 37689RNA transcription-2.9V aly l2T rna synthetase2(VA RS2)NM2006295Protein synthesis-2.8R ibosom al p rotein S14M 13934P

22、rotein synthesis-2.7N ucleolar p rotein hnop56Y12065Ribosome biogenesis-2.5R ibosom al p rotein L3X06323Protein synthesis-2.3Eukaryotic translation initiation f actor(E if3)U 78525Protein synthesis-2.3Sm all nuclear ribonucleop rotein D1polypep tide(SN R PD1)U 78525Protein synthesis-2.3Sm all nuclea

23、r ribonucleop rotein polypep tide F(SN R PF)NM2003095Protein synthesis-2.2Isoleucy l2T rna synthetaseU 04953Protein synthesis-2.1T ranslation initiation f actor E if3P40U 54559Protein synthesis-2.02.2斑蝥素对细胞增殖相关基因表达的抑制作用斑蝥素能抑制多种癌基因、原癌基因的表达,也能抑制具有促细胞增殖作用的基因(如肝癌衍生生长因子)或肿瘤特异表达基因的表达(表2)。表2斑蝥素对细胞增值相关基因表达的

24、影响Tab 2Effects of cantharindin on expression of proliferation-related genesGeneAccession numberFunctionFold change(D aud i)translocated t(8;14)c2myc oncogeneK02276Oncogene-6.1T re oncogene(ubiquitin2specif ic p rotease6;US P6)X63547Oncogene-5.1PCA F2associated f actor400(PA F400)AF110377Cofactor for

25、 both the c2myc and E1A?E2F-5.0oncogenic transcription factor pathwaysP rostatic carcinom a oncogene PT I21NM2001403Oncogene that could affect protein translation-4.3in human prostate and other tissuesB ladder cancer related p rotein(10 000)(B C10)NM2006698Overexpressed in carcinomas-3.8H epatom a2d

26、erived g row th f actorD16431Heparin2binding protein w ith m itogenic activity-3.7U biquitin p rotease(U nph)p roto2oncogeneU 20657Proto2oncogene-3.52.3斑蝥素对能量代谢基因表达的抑制作用斑蝥素抑制参与糖酵解、脂肪酸 氧化和三羧酸循环的代谢酶基因,呼吸链电子传递系统部分成分的基因以及ADP?A TP转运子基因的表达(表3)。表3斑蝥素对能量代谢基因表达的影响Tab 3Effects of cantharindin on expression of

27、 genes involved in energymetabolismGeneAccession numberFunctionFold changeM alate dehyd rogenaseD55654Tricarboxylic acid cycle-5.3A cy l2coa synthetaseD88308Fatty acidoxidation-3.3AD P?A T P translocaseJ03592ADP?ATP transportation-3.2T ransketolase(tk)L 12711Glycolysis-3.0M itochond rial A T P synth

28、ase subunit9U 09813Respiratory electron transport chain component-2.8N ADH dehyd rogenase Fe2S p rotein2(N DU FS2)NM2004550Respiratory electron transport chain component-2.7Phosphog lycerate m utaseJ04173Glycolysis-2.3M itochond rial A T P synthase c subunitX69908Respiratory electron transport chain

29、 component-2.2U biquinol2cy tochrom e c reductaseNM2006004Respiratory electron transport chain component-2.2hinge p rotein(UQCRH)M itochond rial enoy l2coa hyd ratase?32D16480Tricarboxylic acid cycle-2.2hyd roxyacy l2coa dehyd rogenese alphaA cy l2coenzym e A dehyd rogenase,NM2000016Fatty acidoxidat

30、ion-2.2C24to C212straight chain12phosphof ructokinase(PFKL)X15573Glycolysis-2.1N ADH dehyd rogenase1alphaNM2002488Respiratory electron transport chain component-2.1subcomp lex,2(N DU FA2)Isocitrate dehyd rogenaseX69433Tricarboxylic acid cycle-2.0746第6期.胡和平,等.基因芯片技术分析斑蝥素对肝癌细胞细胞毒作用的分子机制 1995-2005 Tsin

31、ghua Tongfang Optical Disc Co.,Ltd.All rights reserved.2.4斑蝥素对凋亡相关基因和细胞生长抑制基因表达的诱导作用斑蝥素能诱导肝癌细胞系Q GY7703细胞表达促凋亡基因和细胞生长抑制基因(表4)。此外,斑蝥素还能抑制肝癌细胞系Q GY7703细胞表达电压依赖阴离子通道(VDAC)、线粒体外膜蛋白转运受体(TOM)基因。表4斑蝥素对凋亡相关基因和细胞生长抑制基因表达的影响Tab 4Effects of cantharindin on expression of proapoptotic genes and growth-inhibitory

32、 genesGeneAccession numberFunctionFold changeApoptosis related genesA TL2derived PM A2responsive pep tide(N oxa)D90070Apoptosis2related gene23.7D eath2associated p rotein kinase1(DA PK1)NM2004938Positive mediators of the programmed cell3.8death induced by gamma2interferonP rog ramm ed cell death10(P

33、DCD10)NM2007217Apoptosis2related gene3.0B cl210p roteinAJ006288Apoptosis2related gene2.6V oltage2dependent anion channel isof orm1(VDA C)L 06132Pathway for movement of adenine nucleotides-7.7through the m itochondrial outer membranePutative m itochond rial outer m em braneAF026031U nknown-5.1p rotei

34、n import recep tor(htom)Growth inhibitorsD ual2specif icity p rotein phosphataseU 15932Deactivation of m itogen2or stress242.0activated protein kinasesB RCA2reg ion,m rna sequence CG003U 50534Tumor suppressor4.2B2cell translocation gene2(B TG2)U 72649BTG12related grow th inhibitor3.9A ntagonizer of

35、myc transcrip tional activity(M ad)L 06895Tumor suppressor3.4T um or necrosis f actor recep torM 32315M ediator of apoptosis or death3.2B RCA2reg ionU 50529Tumor suppressor2.6B2cell translocation gene1(B TG1)X61123Tumor suppressor2.23讨论基因芯片检测结果显示,斑蝥素作用于肝癌细胞后可显著抑制细胞周期进程相关基因。这些基因的功能涉及细胞有丝分裂、DNA复制、DNA修

36、复、RNA转录和加工以及蛋白质的合成。例如GT F3A基因编码的蛋白为锌指蛋白,参与RNA聚合酶转录5S RNA的过程,还与DNA和RNA结合,参与活性染色质的装配7;DN A polym erase delta基因编码的DNA聚合酶 不仅参与DNA的复制,还参与DNA核苷酸的剪切修复,同时对维持DNA复制的精确性有重要作用8;nucleolin基因编码的蛋白参与调控RNA聚合酶?转录核糖体RNA,还参与核糖体成熟和装配以及核糖体成分在核质的转运9。众所周知,DNA复制、修复、RNA转录和蛋白质合成是细胞周期进程所必需,基因组的忠实复制是细胞存活的基础。因此,斑蝥素抑制调控这些特异的细胞周期进

37、程相关基因可能是其引起细胞生长停止,甚至最终导致细胞死亡的原因。诱导表达生长抑制基因或抑制表达生长促进基因均可调控细胞的增殖。斑蝥素不仅能抑制具有促进细胞增殖作用的基因表达(如肝癌衍生生长因子),而且能抑制多种癌基因、原癌基因或肿瘤特异表达基因的表达(表2)。例如癌基因c2myc编码的蛋白具有强大的促增殖作用和致瘤性,过表达c2myc可减少细胞分化和促进细胞周期进程。前列腺癌基因PT I21可影响蛋白质翻译,促进前列腺和其他组织癌的发展10。因此抑制这些生长促进基因的表达可能与斑蝥素抑制肿瘤细胞增殖的作用有关。此外,斑蝥素处理的肝癌细胞系Q GY7703细胞过表达了多种肿瘤抑制基因如B TG1

38、、B TG2、B CRA2和dual2specif icity p rotein phosphatase。B CRA2不同于癌基因B CRA1,是一个抑癌基因11,12。dual2specif icity p rotein phosphatase编码的蛋白起负调节丝裂原激活的蛋白激酶家族(MA PK?ERK,SA PK?JN K,P38)作用,抑制细胞增殖和分化。因此,斑蝥素可能部分通过诱导产生这些生长抑制基因表达达到抑制肿瘤细胞生长的作用。斑蝥素还可显著诱导促进细胞凋亡基因的表达。例如,促凋亡基因A TL2derived PM A2respon2sive pep tide(N ox a)是本

39、研究结果中差异表达上调较高的基因之一。有报道,N ox a编码的蛋白与Bcl22846第二军医大学学报2003年6月,第24卷 1995-2005 Tsinghua Tongfang Optical Disc Co.,Ltd.All rights reserved.抗凋亡家族成员相互作用,引起caspase29的激活而导致细胞凋亡13。此外,研究发现,voltage2depen2dent anion channel(VDA C)和putative m itochond ri2al outer m em brane p rotein import recep tor(hTOM)基因在斑蝥素处理的

40、Q GY7703细胞中表达下调。VDA C编码的蛋白具有转运阴离子、膜孔形成作用以及在细胞凋亡时释放细胞色素C的作用,神经NMB细胞凋亡与VDA C表达减少有关14。hTOM编码的蛋白为线粒体易位酶,能转运核编码的线粒体蛋白。斑蝥素抑制其表达的最终功能尚不清楚,推测可能与其影响细胞死亡有关。本研究发现,斑蝥素能显著抑制糖酵解、脂肪酸氧化以及三羧酸循环中的一些关键酶基因,还能抑制电子传递呼吸链中复合体成分以及A TP合成酶基因表达。此外还能抑制在细胞内能量代谢中起重要作用的ADP?A TP易位酶的表达,这些结果提示,斑蝥素抑制这些能量代谢基因的表达可能最终导致细胞的死亡。总之,斑蝥素作用于肝癌细

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48、the Bcl22 fam ily and candidate mediator of p532inducedapoptosisJ.S cience,2000,288(5468):105321058.14 Premkumar A,Si mantov R.M itochondrial voltage2dependentanion channel is involved in dopam ine2induced apoptosisJ.JN eurochem,2002,82(2):3452352.收稿日期2003202213修回日期2003204225本文编辑余党会(上接第628页)可结合胞嘧啶25

49、 2单核苷酸酶细胞化学反应再作分类;没有反应的可能是急性淋巴细胞或巨核细胞白血病,可进一步观察血小板过氧化物酶反应,呈阳性反应的必定是巨核细胞白血病,无反应的可能是急性淋巴细胞白血病。另外,白血病细胞还可用CD系列抗体进行胶金标记作进一步鉴别和分类。长期以来,我室依靠上述多项电镜技术对疑难白血病、肾脏病、肌病、肝脏某些代谢性疾病及某些皮肤病的诊断作了大量工作,积累了丰富的经验。运用综合电镜技术对下列肿瘤的诊断和鉴别诊断也有重要作用:(1)黑素瘤和肉瘤、腺癌和间质瘤;(2)恶性淋巴瘤和小细胞癌;(3)恶性纤维组织细胞瘤、平滑肌瘤、神经母细胞瘤、梭形细胞癌等。收稿日期2002210215修回日期2003201218本文编辑尹茶,邓晓群946第6期.胡和平,等.基因芯片技术分析斑蝥素对肝癌细胞细胞毒作用的分子机制 1995-2005 Tsinghua Tongfang Optical Disc Co.,Ltd.All rights reserved.