槲皮素通过Bim-Bak/Bax途径提高顺铂耐药膀胱癌细胞对顺铂敏感性的研究

Quercetin Increased the Sensitivity of Cisplatin-resistant Bladder Cancer Cells to Cisplatin Through the Bim-Bak/Bax Pathway

关键词:  槲皮素  顺铂  膀胱癌  Bim  凋亡

Key words:  quercetin  cisplatin  bladder cancer  Bim  apoptosis

摘要:

目的 探讨槲皮素能否逆转膀胱癌细胞对顺铂的耐药性并研究其机制。方法 MTT法检测顺铂耐药T24膀胱癌细胞在顺铂和槲皮素处理下的细胞活力。流式细胞实验检测顺铂耐药T24膀胱癌细胞在顺铂和槲皮素处理下的细胞凋亡。Western blot试验检测顺铂耐药T24膀胱癌细胞在顺铂和槲皮素处理下Bim、活化caspase-9、活化caspase-3的表达水平以及细胞色素c、Smac/DIABLO的释放。免疫共沉淀实验检测顺铂耐药T24膀胱癌细胞在顺铂和槲皮素处理下Bim蛋白与Bak、Bax蛋白的相互作用。结果 相比于常规T24细胞，顺铂耐药T24细胞对顺铂的敏感性显著下降。MTT和流式细胞实验结果表明槲皮素能显著促进顺铂对耐药T24细胞的杀伤活性和凋亡诱导活性。免疫共沉淀和Western blot试验结果表明槲皮素能显著上调顺铂耐药T24细胞中Bim蛋白的表达，从而通过与Bak、Bax蛋白的相互作用促进顺铂对肿瘤细胞线粒体膜孔道的开放，诱导细胞色素c和Smac/DIABLO从线粒体中释放到细胞质中，最终引起caspase-9和caspase-3的活化。结论 槲皮素通过Bim-Bak/Bax途径提高顺铂耐药膀胱癌细胞对顺铂的敏感性。

Abstract:

OBJECTIVE To investigate the role and mechanisms of quercetin in reversing the cisplatin-resistance in the bladder cancer cells. METHODS MTT assay was performed to measure the viability of cisplatin-resistant T24 bladder cancer cells treated with quercetin and cisplatin. Flow cytometry analysis was performed to measure the apoptosis of cisplatin-resistant T24 bladder cancer cells treated with quercetin and cisplatin. Western blot analysis was performed to evaluate the expression of Bim, cleaved caspase-9, cleaved caspase-3, and release of cytochrome C and Smac/DIABLO in the cisplatin-resistant T24 bladder cancer cells treated with quercetin and cisplatin. Co-immunoprecipitation analysis was performed to evaluate the interaction of Bim with Bak and Bax in the cisplatin-resistant T24 bladder cancer cells treated with quercetin and cisplatin. RESULTS The sensitivity of cisplatin-resistant T24 cells to cisplatin was significantly lower than the routine T24 cells. The results of MTT assay and flow cytometry analysis showed that combination with quercetin could significantly promote the cisplatin-induced cell death and apoptosis in the cisplatin-resistant T24 cells. The results of co-immunoprecipitation and western blot analysis showed that quercetin significantly up-regulated the expression of Bim, which could interact with Bak and Bax, leading to the opening of mitochondrial membrane pore and the subsequent release of cytochrome C and Smac/DIABLO to the cytoplasm, and finally, the caspase-9 and caspase-3 was triggered. CONCLUSION Quercetin rises the sensitivity of cisplatin-resistant bladder cancer cells to cisplatin through the Bim-Bak/Bax pathway.