姜黄素抑制耐紫杉醇食管癌细胞的上皮间质转化作用及机制探讨

目的 建立耐紫杉醇食管癌(EC)细胞系EC9706/PTX,观察姜黄素对EC9706/PTX细胞上皮间质转化(EMT)的抑制作用并探讨其机制,为耐药EC的治疗提供理论依据.方法 用中等浓度间歇作用法建立耐紫杉醇EC细胞EC9706/PTX,四甲基偶氮唑蓝(MTT)法测定细胞耐药指数及交叉耐药性,检测不同浓度姜黄素对EC9706/PTX细胞增殖的抑制.使用细胞骨架染色、划痕实验、Transwell侵袭实验检测姜黄素对EC9706/PTX细胞形态变化、迁移和侵袭能力的影响.荧光定量PCR及蛋白免疫印迹(Western blot)检测姜黄素对EC9706/PTX细胞中EMT相关分子标志物E-钙黏蛋白、N-钙黏蛋白、波形蛋白、纤维连接蛋白在mRNA和蛋白水平的表达影响.Western blot检测姜黄素对EC9706/PTX细胞中转录因子NF-κB p65及Snail在蛋白水平的表达影响.结果 EC9706/PTX对紫杉醇的耐药指数为28.4,对顺铂、阿霉素产生交叉耐药性,姜黄素能够抑制EC9706/PTX细胞的增殖.在20 μmol/L浓度的姜黄素作用下,EC9706/PTX细胞的迁移和侵袭能力明显降低.荧光定量PCR及West-ern blot检测显示,细胞耐药后E-钙黏蛋白的表达明显下调,而N-钙黏蛋白表达则明显上调,姜黄素逆转了这一现象.Westernblot检测提示,EC细胞发生耐药及EMT后,NF-κB p65及Snail蛋白的表达增强,姜黄素阻断了这一作用.结论 姜黄素能够抑制紫杉醇耐药EC细胞的增殖并且能够逆转紫杉醇耐药EC细胞的EMT现象,其机制可能是通过抑制NF-κB-Snail信号通路实现的.

The inhibition effects of curcumin on EMT of PTX-resistant esophageal cancer cell line and its mechanism

Objective To establish the effect of curcumin on PTX-resistant esophageal cancer cell line EC9706/PTX and to investigate the mechanism of curcumin on the epithelial stromalization (EMT) of EC9706/PTX cells.Methods EC9706/PTX cells were established by medium concentration intermittent method.The drug resistance index and cross resistance were measured by MTT assay.The inhibitory effects of different concentrations of curcumin on EC9706/PTX cell proliferation were detected.The effects of curcumin on the morphological changes,migration and invasion of EC9706/PTX cells were examined by cytostatic staining,scratching and transwell invasion assay.The effects of curcumin on the expression of E-cadherin,N-cadherin,vimentin and fibronectin in EC9706/PTX cells at mRNA and protein levels were detected by fluorescence quantitative PCR and Western blot.The effect of curcumin on the expressions of NF-κB p65 and Snail in EC9706/PTX cells were detected by Western blot.Results The drug resistance index of EC9706/PTX was 28.4,which was cross-resistant to cisplatin and doxorubicin.Curcumin could inhibit the proliferation of EC9706/PTX cells.The migration and invasion of EC9706/PTX cells were significantly decreased under the action of curcumin at 20 μmol/L concentration.Fluorescence quantitative PCR and Western blot analysis showed that the expression of Ecadherin was down-regulated and the expression of N-cadherin was up-regulated,and curcumin reversed this phenomenon.Western blot analysis showed that the expression of NF-κB p65 and Snail protein was enhanced after PTX-resistant generated in EC cell,but curcumin reversed this phenomenon.Conclusion Curcumin can inhibit the proliferation,migration and invasion of EC9706/PTX cells.The mechanism maybe that curcumin inhibits the NF-κB-Snail pathway.