MCL-1 siRNA通过线粒体凋亡途径部分逆转耐顺铂结肠癌细胞系SW480的耐药性

目的 探讨顺铂耐药结肠癌细胞的MCL-1表达水平与顺铂耐药性的关系。方法 用MTT法检测顺铂耐药结肠癌细胞系SW480(SW480-R)对顺铂的敏感性。通过检测siRNA下调SW480-R 细胞MCL-1的表达作用观察其对细胞耐药性的影响。Western blot试验检测SW480-R 细胞Bcl-2家族蛋白及线粒体来源促凋亡因子的表达水平。Annexin V/PI染色检测SW480-R 细胞的凋亡。结果 SW480-R细胞相比于常规SW480细胞对顺铂的敏感性显著下降，western blot结果表明SW480-R细胞的MCL-1水平显著上调而其他Bcl-2蛋白家族成员(Bcl-2，Bcl-xl，BIM，BAK，BAX)表达水平变化不明显。体外转染MCL-1 siRNA能逆转SW480-R细胞的耐药性，提高顺铂对SW480-R的杀伤活性。在SW480-R细胞中，MCL-1 siRNA能促进线粒体来源的促凋亡因子(细胞色素C、凋亡诱导因子、Smac/DIABLO)在顺铂治疗下从线粒体中释放到细胞质中，进而诱导耐药肿瘤细胞发生凋亡。结论 MCL-1的高表达可能是结肠癌细胞产生顺铂耐药性的重要机制，MCL-1基因沉默能通过线粒体凋亡途径逆转耐顺铂结肠癌细胞系SW480的耐药性。

MCL-1 siRNA Reverses the Chemoresistance of Cisplatin-resistant SW480 Cells via the Pathway of Mitochondrial Apoptosis

OBJECTIVE To investigate the relationship between the expression of MCL-1 and the cisplatin-resistance in cisplatin-resistant colon cancer cells. METHODS MTT assay was performed to evaluate the sensitivity of cisplatin-resistant SW480(SW480-R) cells to cisplatin. RNA interference was used to down-regulate the expression of MCL-1, detecting the role of MCL-1 siRNA in chemoresistance in SW480-R. Western blot analysis was performed to measure the expression of BCL-2 family proteins and mitochondria-derived pro-apoptotic proteins. The apoptosis of SW480-R cells was determined by Annexin V/PI staining. RESULTS The sensitivity of SW480-R to cisplatin was significantly reduced compared with the routine SW480 cells. The results of western blot assay indicated the level of MCL-1 was significantly up-regulated in SW480-R, whereas the other Bcl-2 family proteins (Bcl-2, Bcl-xl, BIM, BAK, and BAX) were not influenced obviously. Transfection of MCL-1 siNRA in vitro could reverse the drug-resistance in SW480-R, enhancing the anti-tumor effect of cisplatin. Furthermore, the MCL-1 siNRA promoted the release of mitochondrial-derived pro-apoptotic factors (cytochrome C, Smac/DIABLO, apoptosis inducing factor) into cytoplasm in SW480-R cells treated with cisplatin, leading to the induction of apoptosis. CONCLUSION Overexpression of MCL-1 is one of the most important mechanisms for the induction of chemoresistance in colon cancer. Knockdown of MCL-1 can reverse the chemoresistance of cisplatin-resistant SW480 cells via the pathway of mitochondrial apoptosis.