摘要
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20μmol/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Western 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。Western blot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。
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 E- cadherin 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.
出处
《重庆医学》
CAS
北大核心
2017年第34期4753-4756,4762,共5页
Chongqing medicine
基金
河南省科技厅基础与前沿项目(112300410044)
关键词
紫杉醇
食管肿瘤
姜黄素
上皮间质化
paclitaxcel
esophageal neoplasms
curcumin
epithelial-mesenchymal transition
