Down-regulation of Rab 5C-dependent Endocytosis and Glycolysis in Cisplatin-resistant Ovarian Cancer Cell Lines

Drug resistance poses a major challenge to ovarian cancer treatment. Understanding mechanisms of drug resistance is important for finding new therapeutic targets. In the present work, a cisplatin-resistant ovarian cancer cell line A2780-DR was established with a resistance index of 6.64. The cellular accumulation of cisplatin was significantly reduced in A2780-DR cells as compared to A2780 cells consistent with the general character of drug resistance. Quantitative proteomic analysis identified 340 differentially expressed proteins between A2780 and A2780-DR cells, which involve in diverse cellular processes, including metabolic process, cellular component biogenesis, cellular processes and stress responses. Expression levels of Ras-related proteins Rab 5C and Rab 11B in A2780-DR cells were lower than those in A2780 cells as confirmed by real-time quantitative PCR and western blotting. The short hairpin (sh)RNA-mediated knockdown of Rab 5C in A2780 cells resulted in markedly increased resistance to cisplatin whereas overexpression of Rab 5C in A2780-DR cells increases sensitivity to cisplatin, demonstrating that Rab 5C-dependent endocytosis plays an important role in cisplatin resistance. Our results also showed that expressions of glycolytic enzymes PKM, GPI, Aldolase, LDH, and PGK were down-regulated in drug resistant cells, indicating drug resistance in ovarian cancer is directly associated with a decrease in glycolysis. Furthermore, it was found that glutathione reductase were up-regulated in A2780-DR, while vimentin, HSP90, and Annexin A1 and A2 were down-regulated. Taken together, our results suggest that drug resistance in ovarian cancer cell line A2780 is caused by multifactorial traits, including the down-regulation of Rab 5C-dependent endocytosis of cisplatin, glycolytic enzymes and vimentin, and up-regulation of antioxidant proteins, suggesting Rab 5C is a potential target for treatment of drug-resistant ovarian cancer. This constitutes a further step towards a comprehensive understanding of drug resistance in ovarian cancer.

耐药是卵巢癌治疗面临的重大挑战。阐明耐药机制对发掘新治疗靶点具有重要意义。本研究成功构建顺铂耐药卵巢癌细胞系A2780-DR，其耐药指数为6.64。与亲本细胞A2780相比，A2780-DR细胞内顺铂蓄积量显著降低，这符合耐药细胞的一般特性。定量蛋白质组学分析显示，A2780与A2780-DR细胞间存在340个差异表达蛋白，这些蛋白参与多种细胞生物学过程，包括代谢过程、细胞组分生物发生、细胞进程及应激反应。经实时定量聚合酶链反应（real-time quantitative PCR）与蛋白质印迹（western blotting）验证，A2780-DR细胞中Ras相关蛋白Rab 5C与Rab 11B的表达水平低于亲本A2780细胞。在A2780细胞中通过短发夹RNA（shRNA）介导敲低Rab 5C的表达，可显著提升其对顺铂的耐药性；而在A2780-DR细胞中过表达Rab 5C则可增强其对顺铂的敏感性，这表明依赖Rab 5C的内吞作用在顺铂耐药过程中发挥重要作用。本研究结果还显示，耐药细胞中糖酵解酶PKM、GPI、醛缩酶（Aldolase）、LDH及PGK的表达均下调，提示卵巢癌耐药与糖酵解水平降低直接相关。此外，本研究发现A2780-DR细胞中谷胱甘肽还原酶表达上调，而波形蛋白（vimentin）、热休克蛋白90（HSP90）及膜联蛋白A1、A2（Annexin A1/A2）的表达则下调。综上，本研究结果表明，卵巢癌细胞系A2780的耐药性由多因素共同介导，包括依赖Rab 5C的顺铂内吞作用下调、糖酵解酶及波形蛋白表达下调，以及抗氧化蛋白表达上调，提示Rab 5C可作为耐药性卵巢癌治疗的潜在靶点。本研究为全面解析卵巢癌耐药机制迈出了重要一步。