DNA修复及细胞凋亡与人卵巢癌细胞对顺铂耐药关系的研究
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摘要
目的(1)观察顺铂导致人卵巢癌SKOV3细胞药物耐受后细胞凋亡情况。探讨DNA错配修复基因hMLH1、hMSH2的在人卵巢癌细胞SKOV3及耐顺铂人卵巢癌细胞SKOV3/DDP中的转录与翻译表达变化。
(2)探讨耐顺铂人卵巢癌细胞SKOV3/DDP中凋亡基因bax、bcl-2、caspase9的转录与翻译表达变化,为进一步研究凋亡途径异常提供实验室依据。
方法(1)在第一部分细胞培养实验中,采用MTT法、Hoechst免疫荧光染色观察顺铂对卵巢癌SKOV3细胞和卵巢癌顺铂耐药SKOV3/DDP细胞凋亡情况,检测SKOV3/DDP细胞耐药性。使用RT-PCR技术及Western blotting技术分别检测顺铂敏感性人卵巢癌SKOV3细胞及耐顺铂人卵巢癌SKOV3/DDP细胞中错配修复基因hMLH1和hMSH2的转录与翻译表达变化情况。
(2)在第二部分实验中,使用RT-PCR技术及Western-blotting技术分别检测顺铂敏感性人卵巢癌SKOV3细胞及耐顺铂人卵巢癌SKOV3/DDP细胞中凋亡相关基因bcl-2、bax、caspase9的转录与翻译表达变化情况。
结果(1)同一顺铂浓度作用下,耐顺铂人卵巢癌细胞的凋亡率明显低于顺铂敏感性人卵巢癌细胞。顺铂敏感性人卵巢癌细胞的错配修复基因hMLH1和hMSH2的mRNA及蛋白表达水平均显著高于耐顺铂人卵巢癌细胞。
(2)顺铂敏感性人卵巢癌细胞的凋亡相关基因bax、caspase9的mRNA及蛋白表达水平均显著高于耐顺铂人卵巢癌细胞,而bcl-2的mRNA及蛋白表达水平反而在耐顺铂人卵巢癌细胞有明显增高。
结论错配修复基因hMLH1和hMSH2在耐顺铂人卵巢癌细胞中低表达可能是导致顺铂耐药发生的重要原因。凋亡基因bcl-2、bax、caspase9在耐顺铂人卵巢癌细胞中异常表达可能与顺铂耐药的发生有直接相关性。
Objective (1)Observation of cisplatin led to SKOV3 human ovarian cancer cell apoptosis after drug resistance。To investigate the roles of hMLH1 and hMSH2 gene in cisplatin resistance in human ovarian cancer. (2)To investigate the roles of bax,bcl-2 and caspase9 gene in cisplatin resistance in human ovarian cancer. To provide the apoptosis way for laboratory further study.
Methods (1)The chemosensitivity to cisplatin was measured by MTT assay. The apoptosis induced by DDP was tested by Hoechst fluorescence analysis. The gene expression of hMLH1 and hMLH2 at transcription and translation levels in SKOV3 and SKOV3/DDP were measured by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, respectively. (2)The gene expression of bax,bcl-2 and caspase9 at transcription and translation levels in SKOV3 and SKOV3/DDP were measured by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, respectively.
Results (1)The apopototic rate of SKOV3/DDP was markedly reduced compared to that of SKOV3 after cisplatin treatment. Both mRNA and protein levles of hMSH2 in SKOV3 were significantly higher than those observed in SKOV3/DDP. (2)Both mRNA and protein levles of bax and caspase9 in SKOV3 were significantly higher than those observed in SKOV3/DDP. mRNA and protein levles of bcl-2 in SKOV3 were significantly lower than those observed in SKOV3/DDP
Conclusion The inhibition of hMLH1 and hMSH2 gene expression may contribute to the occurrence of cisplatin resistance in human ovarian cancer.Also The inhibition of bax,bcl-2 and caspase9 gene expression may contribute to the occurrence of cisplatin resistance in human ovarian cancer
(2)探讨耐顺铂人卵巢癌细胞SKOV3/DDP中凋亡基因bax、bcl-2、caspase9的转录与翻译表达变化,为进一步研究凋亡途径异常提供实验室依据。
方法(1)在第一部分细胞培养实验中,采用MTT法、Hoechst免疫荧光染色观察顺铂对卵巢癌SKOV3细胞和卵巢癌顺铂耐药SKOV3/DDP细胞凋亡情况,检测SKOV3/DDP细胞耐药性。使用RT-PCR技术及Western blotting技术分别检测顺铂敏感性人卵巢癌SKOV3细胞及耐顺铂人卵巢癌SKOV3/DDP细胞中错配修复基因hMLH1和hMSH2的转录与翻译表达变化情况。
(2)在第二部分实验中,使用RT-PCR技术及Western-blotting技术分别检测顺铂敏感性人卵巢癌SKOV3细胞及耐顺铂人卵巢癌SKOV3/DDP细胞中凋亡相关基因bcl-2、bax、caspase9的转录与翻译表达变化情况。
结果(1)同一顺铂浓度作用下,耐顺铂人卵巢癌细胞的凋亡率明显低于顺铂敏感性人卵巢癌细胞。顺铂敏感性人卵巢癌细胞的错配修复基因hMLH1和hMSH2的mRNA及蛋白表达水平均显著高于耐顺铂人卵巢癌细胞。
(2)顺铂敏感性人卵巢癌细胞的凋亡相关基因bax、caspase9的mRNA及蛋白表达水平均显著高于耐顺铂人卵巢癌细胞,而bcl-2的mRNA及蛋白表达水平反而在耐顺铂人卵巢癌细胞有明显增高。
结论错配修复基因hMLH1和hMSH2在耐顺铂人卵巢癌细胞中低表达可能是导致顺铂耐药发生的重要原因。凋亡基因bcl-2、bax、caspase9在耐顺铂人卵巢癌细胞中异常表达可能与顺铂耐药的发生有直接相关性。
Objective (1)Observation of cisplatin led to SKOV3 human ovarian cancer cell apoptosis after drug resistance。To investigate the roles of hMLH1 and hMSH2 gene in cisplatin resistance in human ovarian cancer. (2)To investigate the roles of bax,bcl-2 and caspase9 gene in cisplatin resistance in human ovarian cancer. To provide the apoptosis way for laboratory further study.
Methods (1)The chemosensitivity to cisplatin was measured by MTT assay. The apoptosis induced by DDP was tested by Hoechst fluorescence analysis. The gene expression of hMLH1 and hMLH2 at transcription and translation levels in SKOV3 and SKOV3/DDP were measured by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, respectively. (2)The gene expression of bax,bcl-2 and caspase9 at transcription and translation levels in SKOV3 and SKOV3/DDP were measured by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, respectively.
Results (1)The apopototic rate of SKOV3/DDP was markedly reduced compared to that of SKOV3 after cisplatin treatment. Both mRNA and protein levles of hMSH2 in SKOV3 were significantly higher than those observed in SKOV3/DDP. (2)Both mRNA and protein levles of bax and caspase9 in SKOV3 were significantly higher than those observed in SKOV3/DDP. mRNA and protein levles of bcl-2 in SKOV3 were significantly lower than those observed in SKOV3/DDP
Conclusion The inhibition of hMLH1 and hMSH2 gene expression may contribute to the occurrence of cisplatin resistance in human ovarian cancer.Also The inhibition of bax,bcl-2 and caspase9 gene expression may contribute to the occurrence of cisplatin resistance in human ovarian cancer
引文
[1] Ahmedin Jemal,Rebecca Siegel,Elizabeth Ward,et a1.Cancer Statistics[J]. CA Cancer J Clin.2008,58:71-96
[2] Qi Wang, Zhijuan He ,Jianhua Gao , et a1. S100P sensitizes ovarian cancer cells to carboplatin and paclitaxel in vitro[J]. Cancer Letters.2008,272:277–284
[3] Wang D,Lippard S. Cellular processing of platinum anticancer drugs[J]. Nat.Rev. DrugDiscov. 2005,4:307–320
[4]刘国艳.DNA修复相关基因与卵巢癌耐药研究进展[J].国际妇产科学杂志.2008,35(4):248-251
[5] IshikawaT,Zhang SS,Qinx,et al、DNA repair and cancer:lessens from mutant mouse models[J].Cancer Sci.2004,95(2):112-117
[6] Bernstein C,Bernstein H,Payne CM,et a1.DNA repair/proapoptotic dual role proteins in five major DNA repair pathways : fail-safe protection against carcinogenesis[J].Murat Res.2OO2,511(2)145—178
[7] Peters D,Freund J,Ochs RL.Genome-wide transcriptional analysis of carboplatin response in chemosensitive and chemoresistant ovarian cancer cells[J].Mol Cancer Ther.2005,4(10):1605-1616
[8] Ming-Hong Tsai,Woei-Horng Fang,Shu-Wha Lin,et a1.Mitochondrial Genomic Instability in Colorectal Cancer: No Correlation to Nuclear Microsatellite Instability and Allelic Deletion of hMSH2, hMLH1, and p53 Genes, but Prediction of Better Survival for Dukes’Stage C Disease [J]. Annals of Surgical Oncology.2009,16:2918-2925
[9] Peltomaki P. Deficient DNA mismatch repair: a common etiologic factor for colon cancer [J]. Human Molecular Genetics.2001,10:735-740
[10]杨晓奎,郑芳,陈建华等.凋亡相关蛋白的表达及Caspase-3活性改变与人卵巢癌细胞顺铂耐药的关系.癌症. [J].2002,21(12):1288-1291
[11] Cheng NL,Janumyan YM,Didion L,et a1.Bcl-2 inhi-bition of T-cell proliferation is related to prolonged T-cell survival[J].Oncogene.2004,23:3770—3780)
[1] MA Jianmei ,FAN Kai ,HU Shaowei.The Efect of Helicobacter Pylori Infection on Expression of hMSH2 , hMLH1 and p53 in Gastric Carcin0genesis.The Chinese-German Journal of Clinical Oncology.2005,4(4):209-212
[2] Hui ZHANG,1 Shiqian ZHANG,1 Jing CUI, Expression and promoter methylation status of mismatch repair gene hMLH1 and hMSH2 in epithelial ovarian cancer. Australian and New Zealand Journal of Obstetrics and Gynaecology. 2008,48: 505–509
[3] Kuraguchi M , Yang K, Wong E , et al . The distinct spectra of tumor-associated Apc mutations in mismatch repair-deficient Apc1638N mice define the roles of MSH3 and MSH6 in DNA repair and intestinal tumorigenesis. Cancer Res , 2001 , 61 (21) :7934～7942
[4]刘卓吴建新. DNA错配修复系统组成和功能的研究进展.[J].现代生物医学进展,2008,1160-1165
[5] Picard SF, Franco N, Sergent C, et a1. Analysis of microsatellite instability in acquired drug resistanxe human tunor cell lines [J]. Oncol Rep,2002, 9 (5):97l-976
[6]李梅,吕申.错配修复功能缺失对肿瘤细胞耐药性的影响[J].现代肿瘤医学,2007,15(9):1348-1340
[7] Fishel R. The selection for mismatch repair defects in hereditary nonpolyposis colorectal cancer: revising the mutator hypothesis [J]. Cancer Res,2001,15 (20):7369-7374
[8]王晓莉,彭芝兰,王和等.野生型p53基因转染对卵巢癌细胞生长及药物敏感性影响.四川肿瘤防治.2001,14(3):134-137
[9]倪虹,糜若然,王德华.封闭野生型p53表达是卵巢癌细胞药物敏感性增加的机制.天津医药.2004,32(9):537-539
[10]杨晓葵,郑芳,刑辉等.细胞色素c介导的半胱天冬氨酸蛋白酶-3活性改变与人卵巢癌顺铂耐药的关系.2002,24(6):544-547
[11] Chancy SG, Campbell SL, Temple B, et al. Protein interactions with platium-DNA adducts: from structure to function[J]. Inorg Biochem. 2004,98 (10):l55l-l559
[12]马丁.卵巢癌多耐药机制的探讨.中华妇幼临床医学杂志(电子版)2007, 3(4):184-188
[1] Bronner CE,Baker SM ,Morrison PT,et a1.Mutations in the DNA mismatch repair gene homologue hMl Hl is associated with HNPCC[J].Nature,1994,368(6468):258—261.
[2] Liu B,Parsons RE,Hamilton SR,et a1.hMSH2 mutations in hereditary nonpolyposis coloreetal cancer kindreds[J] . CancerRes , 1994 , 54(17) :4590—4594.
[3] Back MJ,Kang H,Kim SE,et al.Expression of hMLH1 is inactivated in the gastric adenomas with enhanced microsatellite instability[J].Br J Cancer,2001,85(8):1147-I152.
[4]吴俚蓉. DNA错配修复基因hMLH1与肿瘤及化疗耐药相关性的研究进展.临床肿瘤学杂志2009年3月第l卷第3期:274-278.
[5] Bronner CE,Baker SM,Morrison PT,et a1.Mutations in the DNA mismatch epair gene homologue hMLHI is associated with HNPCC.Nature,1994;368:258—261.
[6] Eshleman JR, Markowitz SD. Mismatch repair defects in human carcinogenesis. Hum Mol Genet, 1996, 5:1489-1494.
[7]刘晓蓉.人类错配修复基因突变在肿瘤发生中的作用研究新进展[J].国外医学遗传学分册,2001,24(5);301.
[8] Lrpez de Saro FJ,Marinus MG,Modrich P,et a1.The beta sliding clamp binds to multiple sites within MutL and MutS[J].J Biol Chem,2006,281(20):14340—14349.
[9]张爱凤,张师前,位玲霞. 5-氮-2’-脱氧胞苷对人卵巢癌细胞SKOV3凋亡及HMLH1和HMSH2表达的影响.基础医学与临床. 2007,27(4):412-416.
[10] Xinarianos G,Scott FM,Liloglou T,et a1.hMLHI and hMSH2 expression correlates with allelic imbalance on Chromosome 3p in non—small cell lung carcinomas[J].Cancer Res,2000 Aug 1,60(15):4216—4221.
[11]庹新兰,臼明,刘丽江.错配修复基因hMLH1、hMSH2及p53在肺癌组织中的表达及意义.中国热带医学,2007, 7(4):521-523
[12] Wang Yi-Ching,Lu Yung-Pin,Tseng Ruo-Chia,et a1.Inactivation of hMLH1 and hMSH2 by promoter methylation in non-smal cell lung tumors and matched sputum samples [J].J Clin Ivest,2003,111:887—895.
[2] Qi Wang, Zhijuan He ,Jianhua Gao , et a1. S100P sensitizes ovarian cancer cells to carboplatin and paclitaxel in vitro[J]. Cancer Letters.2008,272:277–284
[3] Wang D,Lippard S. Cellular processing of platinum anticancer drugs[J]. Nat.Rev. DrugDiscov. 2005,4:307–320
[4]刘国艳.DNA修复相关基因与卵巢癌耐药研究进展[J].国际妇产科学杂志.2008,35(4):248-251
[5] IshikawaT,Zhang SS,Qinx,et al、DNA repair and cancer:lessens from mutant mouse models[J].Cancer Sci.2004,95(2):112-117
[6] Bernstein C,Bernstein H,Payne CM,et a1.DNA repair/proapoptotic dual role proteins in five major DNA repair pathways : fail-safe protection against carcinogenesis[J].Murat Res.2OO2,511(2)145—178
[7] Peters D,Freund J,Ochs RL.Genome-wide transcriptional analysis of carboplatin response in chemosensitive and chemoresistant ovarian cancer cells[J].Mol Cancer Ther.2005,4(10):1605-1616
[8] Ming-Hong Tsai,Woei-Horng Fang,Shu-Wha Lin,et a1.Mitochondrial Genomic Instability in Colorectal Cancer: No Correlation to Nuclear Microsatellite Instability and Allelic Deletion of hMSH2, hMLH1, and p53 Genes, but Prediction of Better Survival for Dukes’Stage C Disease [J]. Annals of Surgical Oncology.2009,16:2918-2925
[9] Peltomaki P. Deficient DNA mismatch repair: a common etiologic factor for colon cancer [J]. Human Molecular Genetics.2001,10:735-740
[10]杨晓奎,郑芳,陈建华等.凋亡相关蛋白的表达及Caspase-3活性改变与人卵巢癌细胞顺铂耐药的关系.癌症. [J].2002,21(12):1288-1291
[11] Cheng NL,Janumyan YM,Didion L,et a1.Bcl-2 inhi-bition of T-cell proliferation is related to prolonged T-cell survival[J].Oncogene.2004,23:3770—3780)
[1] MA Jianmei ,FAN Kai ,HU Shaowei.The Efect of Helicobacter Pylori Infection on Expression of hMSH2 , hMLH1 and p53 in Gastric Carcin0genesis.The Chinese-German Journal of Clinical Oncology.2005,4(4):209-212
[2] Hui ZHANG,1 Shiqian ZHANG,1 Jing CUI, Expression and promoter methylation status of mismatch repair gene hMLH1 and hMSH2 in epithelial ovarian cancer. Australian and New Zealand Journal of Obstetrics and Gynaecology. 2008,48: 505–509
[3] Kuraguchi M , Yang K, Wong E , et al . The distinct spectra of tumor-associated Apc mutations in mismatch repair-deficient Apc1638N mice define the roles of MSH3 and MSH6 in DNA repair and intestinal tumorigenesis. Cancer Res , 2001 , 61 (21) :7934～7942
[4]刘卓吴建新. DNA错配修复系统组成和功能的研究进展.[J].现代生物医学进展,2008,1160-1165
[5] Picard SF, Franco N, Sergent C, et a1. Analysis of microsatellite instability in acquired drug resistanxe human tunor cell lines [J]. Oncol Rep,2002, 9 (5):97l-976
[6]李梅,吕申.错配修复功能缺失对肿瘤细胞耐药性的影响[J].现代肿瘤医学,2007,15(9):1348-1340
[7] Fishel R. The selection for mismatch repair defects in hereditary nonpolyposis colorectal cancer: revising the mutator hypothesis [J]. Cancer Res,2001,15 (20):7369-7374
[8]王晓莉,彭芝兰,王和等.野生型p53基因转染对卵巢癌细胞生长及药物敏感性影响.四川肿瘤防治.2001,14(3):134-137
[9]倪虹,糜若然,王德华.封闭野生型p53表达是卵巢癌细胞药物敏感性增加的机制.天津医药.2004,32(9):537-539
[10]杨晓葵,郑芳,刑辉等.细胞色素c介导的半胱天冬氨酸蛋白酶-3活性改变与人卵巢癌顺铂耐药的关系.2002,24(6):544-547
[11] Chancy SG, Campbell SL, Temple B, et al. Protein interactions with platium-DNA adducts: from structure to function[J]. Inorg Biochem. 2004,98 (10):l55l-l559
[12]马丁.卵巢癌多耐药机制的探讨.中华妇幼临床医学杂志(电子版)2007, 3(4):184-188
[1] Bronner CE,Baker SM ,Morrison PT,et a1.Mutations in the DNA mismatch repair gene homologue hMl Hl is associated with HNPCC[J].Nature,1994,368(6468):258—261.
[2] Liu B,Parsons RE,Hamilton SR,et a1.hMSH2 mutations in hereditary nonpolyposis coloreetal cancer kindreds[J] . CancerRes , 1994 , 54(17) :4590—4594.
[3] Back MJ,Kang H,Kim SE,et al.Expression of hMLH1 is inactivated in the gastric adenomas with enhanced microsatellite instability[J].Br J Cancer,2001,85(8):1147-I152.
[4]吴俚蓉. DNA错配修复基因hMLH1与肿瘤及化疗耐药相关性的研究进展.临床肿瘤学杂志2009年3月第l卷第3期:274-278.
[5] Bronner CE,Baker SM,Morrison PT,et a1.Mutations in the DNA mismatch epair gene homologue hMLHI is associated with HNPCC.Nature,1994;368:258—261.
[6] Eshleman JR, Markowitz SD. Mismatch repair defects in human carcinogenesis. Hum Mol Genet, 1996, 5:1489-1494.
[7]刘晓蓉.人类错配修复基因突变在肿瘤发生中的作用研究新进展[J].国外医学遗传学分册,2001,24(5);301.
[8] Lrpez de Saro FJ,Marinus MG,Modrich P,et a1.The beta sliding clamp binds to multiple sites within MutL and MutS[J].J Biol Chem,2006,281(20):14340—14349.
[9]张爱凤,张师前,位玲霞. 5-氮-2’-脱氧胞苷对人卵巢癌细胞SKOV3凋亡及HMLH1和HMSH2表达的影响.基础医学与临床. 2007,27(4):412-416.
[10] Xinarianos G,Scott FM,Liloglou T,et a1.hMLHI and hMSH2 expression correlates with allelic imbalance on Chromosome 3p in non—small cell lung carcinomas[J].Cancer Res,2000 Aug 1,60(15):4216—4221.
[11]庹新兰,臼明,刘丽江.错配修复基因hMLH1、hMSH2及p53在肺癌组织中的表达及意义.中国热带医学,2007, 7(4):521-523
[12] Wang Yi-Ching,Lu Yung-Pin,Tseng Ruo-Chia,et a1.Inactivation of hMLH1 and hMSH2 by promoter methylation in non-smal cell lung tumors and matched sputum samples [J].J Clin Ivest,2003,111:887—895.