端粒酶线粒体转位与肝癌细胞多药耐药的相关性研究

Translocation of telomerase into mitochondria and multidrug resistance in human hepatocellular carcinoma cells

目的:本研究旨在观察人端粒酶(human telomerase)线粒体转位对肿瘤多药耐药的影响。方法:分别采用顺铂(cisplatin,CDDP)大剂量冲击和间歇诱导的方法处理人肝癌SK-Hep1细胞株,建立具有不同程度耐药指数(resistance index,RI)的人肝癌耐药细胞株;采用CCK-8(cell counting kit-8)法检测各组细胞的药物敏感性,计算半数细胞抑制浓度(half inhibitory concentration,IC50)和RI;FCM法检测细胞周期及凋亡率;免疫荧光染色检测人端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)在细胞核和线粒体中的分布;蛋白质印迹法检测hTERT蛋白在全细胞和线粒体中的表达情况;实时定量基因扩增荧光检测系统(real-time quantitative PCR detecting system,Q-PCR)检测SK-Hep1亲本细胞株和各耐药细胞株的端粒相对长度;实时荧光定量-PCR(real-time fluorogenic quantitative-PCR,RFQ-PCR)检测线粒体DNA(mitochondrial DNA,mtDNA)的氧化损伤;JC-1染色法检测线粒体的膜电位。结果:获得3株对CDDP具有不同程度RI的SK-Hep1细胞,分别命名为SK-Hep1/CDDP1、SK-Hep1/CDDP2和SK-Hep1/CDDP3细胞,CCK-8检测结果显示,它们对CDDP的RI值分别为7.51、9.31和13.76,并对多柔比星和5-氟尿嘧啶有交叉耐药作用。FCM法、免疫荧光染色法、Q-PCR系统、RFQ-PCR法及JC-1染色法的检测结果显示,随着RI值的增加,各耐药细胞株的凋亡率明显降低,hTERT逐渐从细胞核转位到了线粒体中,细胞中的端粒明显缩短,损伤的mtDNA逐渐减少,线粒体膜电位降低。结论:多药耐药肿瘤细胞中端粒酶线粒体转位增加,线粒体端粒酶可发挥保护线粒体的作用。肿瘤细胞通过端粒酶的线粒体保护功能抵抗凋亡。因此,端粒酶线粒体转位可能是肿瘤细胞多药耐药的另一重要的作用机制。

Objective： To investigate the effect of translocation of human telomerase into mitochondria on multidrug resistance （MDR） of human hepatocellular carcinoma SK-Hepl cells in vitro. Methods： SK- Hepl cells were treated with high-dose cisplatin （CDDP） pulse therapy and intermittent selection to establish CDDP-resistant cell sublines with different resistance index （RI）. The drug sensitivities of chemoresistance SK-Hepl cells and their parental cells were examined by cell counting kit-8 （CCK-8） assay, and the half inhibitory concentration （ICs0） and resistance index （RI） were calculated. The cell cycle distribution and apoptosis rate were detected by flow cytometry （FCM）. The distributions of human telomerase reverse transcriptase （hTERT） in nuclei and mitochondria were detected by immunofluorescence staining, and the expressions of hTERT protein in whole cell and mitochondria were detected by Western blotting. The oxidative damage of mitochondria DNA （mtDNA） was examined by real-time fluorogenic quantitative-PCR （RFQ-PCR）. The mJtocbondrial membrane potential was detected by JC-1 staining. The relative length of telomere was measured by real-time quantitative PCR detection system. Results： Three CDDP-resistant sublines SK-Hepl/CDDP1, SK-Hepl/CDDP2 and SK-Hepl/CDDP3 were established in vitro. The RIs of SK-Hepl/CDDP1, SK-Hepl/CDDP2 and SK-Hepl/CDDP3 cells were 7.51, 9.31 and 13.76, respectively, and the CCK-8 assay showed that the three cell sublines exhibited cross-resistance to adramycin and 5-fuorouracil. The results of FCM, immunofluorescence staining, real- time quantitative PCR detection system, RFQ-PCR andJC-1 staining showed that the apoptosis rate of chemoresistant cells was significantly decreased as the RI value was increased. The hTERT was gradually translocated from nucleus into mitochondria, the telomere length of chemoresistant cells was shortened, the mtDNA damage was reduced, and the mitochondrial membrane potential was also decreased. Conclusion： The translocation of telomerase into mitochondria is more frequent in multiple drug-resistant cells, and it can protect mitochondrial function. The apoptosis of tumor cells is inhibited through mitochondrial protection by telomerase. Therefore, translocation of telomerase into mitochondria might be an additional important mechanism in multiple drug-resistance of tumor cells.