线粒体融合在卵巢癌顺铂耐药中的作用及其机制研究

Ovarian cancer is one of the most common cancers with high mortality in female reproductive system. Although cisplatin has dominated the chemotherapy of ovarian cancer, the majority of patients will experience disease recurrence following cisplatin resistance. Our recent studies show mitochondrial dynamics may regulate the antineoplastic activity of cisplatin (Int J Oncol. 2015; 46(2):691-700). However, its mechanism still remains to be elucidated. In the present study, we will investigate the role of mitochondrial fusion in the resistance of ovarian cancer to cisplatin and its underlying mechanism. In preliminary experiments, we found that the expression of Mfn1, 2 and OPA1 were increased in SKOV3/DDP cells, on the contrary, the expression of mitochondrial fission protein Drp1 was decreased. Obvious mitochondrial fusion was also observed in SKOV3/DDP cells. In experiments of cisplatin stress, mitochondrial fusion proteins Mfn1, 2 and OPA1were all increased in SKOV3 cells at day 4 after treatment with 3.3μM cisplatin, but fission protein Drp1 was decreased. Moreover, the enforced expression of GFP-Mfn1 in SKOv3 cell or knockdown of Mfn1 in SKOv3/DDP cells not only affected mitochondrial fusion, but also influenced cisplatin-induced decrease in mitochondrial membrane potential, ROS production and cell death. These results suggest that mitochondrial fusion might contribute to cisplatin resistance in ovarian cancer via regulating intrinsic apoptotic pathway. To demonstrate the hypothesis, we will further examine the expression of mitochondrial fusion and fission proteins in other cisplatin resistant ovarian cancer cell lines, and investigate the role of mitochondrial fusion in cisplatin resistance in ovarian cancer cells and ovarian cancer bearing mice. Therefore, this research may provide possible new therapeutic target to prevent cisplatin resistance in ovarian cancer.

卵巢癌是常见女性生殖系统肿瘤，铂类药是其重要化疗药物，但易耐药失效。我们研究发现线粒体变化参与顺铂抗瘤活性（Int J Oncol.2015;46(2):691-700），但机制不清楚。本课题将探讨线粒体融合在卵巢癌顺铂耐药中的作用机制。预实验发现：SKOv3/DDP中线粒体融合蛋白Mfn1、2和OPA1表达比SKOV3显著升高，分裂蛋白表达下降，线粒体融合增强；顺铂压力实验时，线粒体融合蛋白在3.3μM顺铂处理SKOV3第4天均表达增加，而分裂蛋白下降；敲低或过表达Mfn1可通过线粒体融合改变顺铂诱导的线粒体膜电位下降、ROS产生和细胞死。这提示线粒体融合可能通过调节细胞凋亡途径参与卵巢癌顺铂耐药机制。为证明上述假说，将继续检测其它顺铂耐药卵巢癌细胞中线粒体形态调节蛋白水平；并通过细胞和动物实验深入研究线粒体融合在卵巢癌顺铂耐药中的作用机制。故本课题将为解决卵巢癌顺铂耐药问题提供新思路。

卵巢癌是常见女性生殖系统肿瘤，铂类药是其重要化疗药物之一，但容易产生化疗耐药而失效。我们课题组以前的研究发现线粒体动态变化参与了顺铂抗瘤活性，但在卵巢癌中的作用及其机制不清楚。本课题主要是研究线粒体融合是否通过稳定线粒体膜电位、降低胞内ROS产生，并抑制内源性凋亡途径，从而参与卵巢癌的顺铂耐药机制。实验结果发现，与非耐药卵巢癌细胞SKOv3相比，顺铂耐药SKOv3/DDP细胞中线粒体分裂蛋白Drp1表达水平显著降低，而线粒体融合蛋白Mfn2显著上调；顺铂压力实验时，3.3μM顺铂处理非耐药SKOV3可使Drp1在第4天开始显著下调，线粒体融合蛋白Mfn1和OPA-1在第4天均表达上调，Mfn2在第2天即表达增加；线粒体形态分析也发现SKOv3/DDP细胞内线粒体融合程度明显强于SKOv3细胞；然后我们利用siRNA分别敲低SKOv3细胞中Drp1和SKOv3/DDP中Mfn2，敲低Drp1后SKOv3细胞中线粒体长度显著增长，而敲低SKOv3/DDP内Mfn2显著降低了细胞内线粒体长度；敲低Drp1能降低顺铂诱导的SKOv3细胞ROS水平、caspase剪切、Bax表达，而敲低Mfn2能显著增加顺铂诱导的SKOv3/DDP细胞线粒体膜电位降低，ROS产生、Caspase剪切和Bax表达；有趣的是，SKOv3/DDP的ATP水平显著低于SKOv3细胞，敲低Mfn2能有效增加SKOv3/DDP细胞的ATP水平；划痕和Transwell实验均显示SKOv3细胞的迁移能力强于SKOv3/DDP细胞，敲低Drp1或Mfn2分别能减弱SKOv3细胞迁移能力和增强SKOv3/DDP迁移能力。上述实验结果说明了线粒体融合在卵巢癌顺铂化疗耐药中的重要作用及其机制，这为进一步提高卵巢癌顺铂化疗敏感性提供了以线粒体为靶点的新思路。