卵巢癌顺铂敏感与耐药细胞ClC-3蛋白表达及通道功能的差异

Difference of ClC-3 protein expression and channel function between cisplatin-sensitive and -resistant ovarian cancer cells

目的:从分子和细胞水平研究卵巢癌顺铂敏感(a2780)及耐药(a2780cp)细胞中ClC-3氯通道蛋白表达及通道功能的差异。方法:MTT法检测人卵巢癌a2780和a2780cp细胞对顺铂敏感性的差异;real-time PCR法检测ClC氯通道家族在a2780和a2780cp细胞中的mRNA表达;Western blot检测ClC-3蛋白的表达;免疫荧光法观察ClC-3蛋白在a2780和a2780cp细胞的分布;全细胞膜片钳技术记录细胞氯电流。结果:(1) a2780和a2780cp细胞对顺铂的敏感性存在差异,其IC_(50)值分别为5μmol/L和20μmol/L(P<0.01);(2)对ClC氯通道家族mRNA表达的检测结果显示,a2780和a2780cp细胞主要表达ClC-3,且在a2780cp细胞中ClC-3 mRNA表达显著减少(P<0.01);(3)在蛋白水平上,与a2780细胞相比,a2780cp细胞的ClC-3蛋白表达量显著降低(P<0.01);(4)免疫荧光显示ClC-3在a2780细胞中主要分布在胞膜上,而在a2780cp细胞中则主要表达在胞质内;(5)顺铂能激活a2780细胞的氯通道,产生ClC-3介导的氯电流,但在a2780cp细胞中则不能;(6)ClC-3 siRNA处理a2780细胞后,顺铂诱导的氯电流则不再出现。结论:ClC-3氯通道在顺铂敏感和耐药的卵巢癌细胞蛋白分布、表达和功能上存在差异,可能是引起顺铂耐药的潜在机制之一。

AIM:To study the difference of ClC-3 chloride channel protein expression and channel function between cisplatin-sensitive(a2780)and-resistant(a2780cp)ovarian cancer cells.METHODS:The inhibition of a2780 and a2780cp cell proliferation induced by cisplatin were detected by MTT assay.The mRNA and protein expressions of ClC chloride channel families in a2780 cells and a2780cp cells were detected by real-time PCR and Western blot,respectively.The distribution of ClC-3 protein in a2780 cells and a2780cp cells were analyzed by immunofluorescence staining.The whole cell patch-clamp technique was used to record the chloride current in the cells.RESULTS:The sensitivities of a2780 cells and a2780cp cells to cisplatin were different.The IC 50 values of a2780 cells and a2780cp cells to cisplatin were 5μmol/L and 20μmol/L,respectively(P<0.01).The a2780 cells and a2780cp cells mainly expressed ClC-3 in ClC families.However,the mRNA expression of ClC-3 was much lower in a2780cp cells than that in a2780 cells(P<0.01).Compared with a2780 cells,the protein expression of ClC-3 in a2780cp cells was also significantly decreased(P<0.01).ClC-3 protein was mainly distributed on the membrane in a2780 cells,while was in cytoplasma in a2780cp cells.Cisplatin activated the chloride channel and induced the chloride current in the a2780 cells,but not in the a2780cp cells.Cisplatin did not induced the chloride current in a2780 cells treated with ClC-3 siRNA.CONCLUSION:The differences in protein distribution,expression and function of ClC-3 chloride channel were observed in cisplatin-sensitive and-resistant ovarian cancer cells,which may be one of the underlying mechanisms of cisplatin resistance.