青蒿琥酯通过Akt/Snail信号通路逆转结直肠癌细胞上皮间充质转化

Mechanism of Artesunate in Reversing Epithelial-mesenchymal Transition and Akt/Snail Signaling Pathway in Colorectal Carcinoma

目的:探讨青蒿琥酯(ART)对结直肠癌HCT-8细胞上皮-间充质转化(EMT)的作用,并探讨ART对结直肠癌细胞迁移,侵袭,EMT能力和蛋白激酶B(Akt)/Snail信号通路的影响。方法:采用噻唑蓝(MTT)比色法检测不同药物浓度的ART对HCT-8细胞增殖的影响,采用伤口愈合实验和transwell实验,检测ART对结直肠癌HCT-8细胞迁移侵袭能力的影响。免疫荧光双重染色法检测不同ART浓度对肿瘤细胞HCT-8细胞中EMT相关蛋白波形蛋白(vimentin),E-钙黏蛋白(E-cadherin)分布情况的影响。采用蛋白免疫印迹法(Western blot)检测蛋白N-钙黏蛋白(N-cadherin),vimentin,E-cadherin的蛋白表达情况的影响以及对Akt/Snail信号途径中相关蛋白Akt1,磷酸化Akt1(p-Akt1)和Snail1表达的影响。结果:不同浓度的ART作用于HCT-8后,计算细胞增殖抑制率,并绘制出量效曲线,得出ART对HCT-8细胞的半数抑制浓度(IC_(50))为(16.67±1.95)μmol·L^(-1),且与药物质量浓度呈剂量依赖关系,由此实验处理组分为4组,空白组(0μmol·L^(-1)),ART低剂量组(2μmol·L^(-1)),ART中剂量组(10μmol·L^(-1)),ART高剂量组(50μmol·L^(-1));与空白组比较,ART组可显著抑制HCT-8细胞的迁移和侵袭能力(P<0.05);与空白组比较,ART组中相关蛋白E-cadherin表达明显上调,vimentin和N-cadherin表达明显下调,且p-Akt1和Snail1的表达水平明显降低从而抑制EMT(P<0.05)。结论:ART可以抑制EMT引起的迁移和侵袭,其机制可能与抑制Akt/Snail通路活化从而逆转EMT有关。

Objective:To investigate the effects of artesunate(ART)on epithelial-mesenchymal transformation(EMT)of colorectal cancer HCT-8 cells,and explore the effects of ART on cell migration,invasion,EMT ability,and protein kinase B(Akt)/Snail signaling pathway of colorectal cancer.Method:3-(4-5-Dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide(MTT)assay was used to detect the effects of ART at different concentrations on the proliferation of HCT-8 cells.Wound healing assay and Transwell assay were used respectively to detect the effects of ART on migration and invasion of colorectal cancer cells.The effects of different concentrations of ART on the distribution of EMT-related proteins vimentin and E-cadherin in HCT-8 cells were detected by double-immunofluorescent staining.The effects of ART on protein expression levels of EMT markers E-cadherin,vimentin and N-cadherin in HCT-8 cells and the expression of Akt1,p-Akt1,and Snail1 in the Akt/Snail signaling pathway were determined by Western blot.Result:The dose-dependent inhibitory effects of ART on the proliferation of HCT-8 cells were determined and the inhibition rate was calculated.A dose-response curve was plotted accordingly.The half-maximal inhibitory concentration(IC_(50))of ART on HCT-8 cells was(16.67±1.95)μmol·L^(-1).The following four groups were set up:a control group(0μmol·L^(-1)),and low-,medium-,and high-dose ART groups(2,10,50μmol·L^(-1)).Compared with the results in the control group,ART inhibited the migration and invasion of HCT-8 cells(P<0.05).Specifically,the expression of E-cadherin in HCT-8 cells was significantly up-regulated,and that of vimentin and N-cadherin was significantly down-regulated(P<0.05).The expression levels of p-Akt1 and Snail1 were significantly decreased after ART treatment,thus inhibiting EMT(P<0.05).Conclusion:The findings of this study suggested that ART inhibited the EMT-triggered migration and invasion of HCT-8 cells presumedly by inhibiting the activation of the Akt/Snail pathway to reverse EMT.