Yang Hong, Fu Jian-hua, Hu Yi, Huang Wei-zhao, Zheng Bin, Wang Geng
Department of Thoracic Oncology, Cancer Center, Sun Yat-sen University, State Key Laboratory of Oncology in Southern China, Guangzhou 510060, China.
Zhonghua Yi Xue Za Zhi. 2007 Jul 17;87(27):1934-7.
To investigate the relationship between survivin expression and the chemosensitivity in multiple drug resistant human lung cancer cell line
Human lung cancer cells of the parental cell line H460 and multiple drug resistant cell line H460/cDDP were cultured respectively. RT-PCR and Western blotting were used to detect the mRNA and protein expression of survivin. siRNA targeting survivin was transfected into H460/cDDP cells by Liposomes approach. The expression change of survivin protein after transfection was examined by Western blotting. Cisplatin (DDP) and paclitaxel of different concentrations were added into the culture fluid of the H460/cDDP cells respectively. Methyl thiazolyl tetrazolium (MTT) assay was used to calculate the IC(50) value so as to detect the cytotoxicity of cisplatin and paclitaxel to the cells.
The mRNA expression and protein expression of survivin in the H460/cDDP cells were both significantly higher than those in the H460 cells. 24 h, 48 h, and 72 h after transfection of the survivin siRNA the survivin protein expression levels of the H460/cDDP were decreased by 67.0%, 76.2%, and 92.3% respectively. MTT assay showed that the cisplatin IC(50) levels of the H460/cDDP and H460 cells were 6.3 +/- 0.6 mg/L and 0.8 +/- 0.1 mg/L respectively, and the paclitaxel IC(50) levels were 12.7 +/- 1.2 mg/L and 1.5 +/- 0.3 mg/L respectively. 48 h after the transfection of survivin siRNA, the cisplatin and paclitaxel IC(50) levels of the H460/cDDP cells were decreased to 4.0 +/- 0.9 mg/L (P = 0.002) and 8.1 +/- 1.7 mg/L (P = 0.003) respectively.
The survivin expression of the resistant cell line H460/cDDP was up-regulated compared with that of the parental cell line H460. Survivin siRNA sensitized the H460/cDDP cells to both cisplatin and paclitaxel. Survivin participates in the multiple drug resistance mechanism of human lung cancer cells.
探讨生存素表达与多药耐药人肺癌细胞系化疗敏感性之间的关系。
分别培养亲本细胞系H460和多药耐药细胞系H460/cDDP的人肺癌细胞。采用逆转录-聚合酶链反应(RT-PCR)和蛋白质免疫印迹法检测生存素的mRNA和蛋白表达。通过脂质体法将靶向生存素的小干扰RNA(siRNA)转染至H460/cDDP细胞。采用蛋白质免疫印迹法检测转染后生存素蛋白表达的变化。分别向H460/cDDP细胞培养液中加入不同浓度的顺铂(DDP)和紫杉醇。采用甲基噻唑基四氮唑(MTT)法计算半数抑制浓度(IC50)值,以检测顺铂和紫杉醇对细胞的细胞毒性。
H460/cDDP细胞中生存素的mRNA表达和蛋白表达均显著高于H460细胞。转染生存素siRNA后24小时、48小时和72小时,H460/cDDP细胞的生存素蛋白表达水平分别下降了67.0%、76.2%和92.3%。MTT法显示,H460/cDDP细胞和顺铂IC50水平分别为6.3±0.6mg/L和0.8±0.1mg/L,紫杉醇IC50水平分别为12.7±1.2mg/L和1.5±0.3mg/L。转染生存素siRNA后48小时,H460/cDDP细胞的顺铂和紫杉醇IC50水平分别降至4.0±0.9mg/L(P=0.002)和8.1±1.7mg/L(P=0.003)。
与亲本细胞系H460相比,耐药细胞系H460/cDDP的生存素表达上调。生存素siRNA使H460/cDDP细胞对顺铂和紫杉醇均敏感。生存素参与人肺癌细胞的多药耐药机制。