薯蓣皂苷通过调控PI3K/AKT信号通路抑制人肺腺癌细胞H1299上皮间质转化

Dioscin Inhibits Epithelial-mesenchymal Transition in Human Lung Adenocarcinoma H1299 Cells by Regulating PI3K/AKT Signaling Pathway

目的研究薯蓣皂苷(Dioscin,Dio)对人肺腺癌细胞H1299上皮间质转化的影响,并初步探讨其作用机制。方法采用不同浓度Dio(1、2、4、8μmol·L^(-1))处理H1299细胞24、48 h,CCK8法检测细胞的增殖能力。选择低于IC_(50)浓度的Dio(1、2μmol·L^(-1))作用于H1299细胞,采用细胞划痕实验和Transwell穿膜实验观察不同浓度Dio对H1299细胞迁移能力的影响;采用铺有Matrigel胶的Transwell实验检测Dio对H1299细胞侵袭能力的影响;Western Blot法检测Dio对H1299细胞中E-cadherin、Zonula occluden 1(ZO-1)、ZEB1、Cludin-1、N-cadherin、Vimentin、β-catenin、PI3K、AKT、P-AKT蛋白表达的影响;免疫荧光法检测间质标志物Vimentin在细胞中的定位及表达情况。结果 Dio干预24、48 h后的IC_(50)值分别为3.50、3.21μmol·L^(-1),且呈现浓度依赖性。Dio干预后与空白对照组比较,Dio 1、2μmol·L^(-1)浓度组的细胞愈合率显著降低(P<0.05,P<0.001);Dio 1、2μmol·L^(-1)浓度组均能显著抑制H1299细胞的迁移能力(P <0.001),并显著抑制H1299细胞体外侵袭能力(P <0.001);Dio 1、2μmol·L^(-1)浓度组能明显上调E-cadherin、ZO-1和Cludin-1蛋白的表达(P <0.05,P <0.01,P <0.001),下调N-cadherin、Vimentin、ZEB1、β-catenin蛋白及PI3K/AKT信号通路蛋白PI3K、AKT、P-AKT的表达(P <0.05,P <0.01,P <0.001);Dio 1、2μmol·L^(-1)浓度组免疫荧光定位表达结果显示Vimentin表达明显降低。结论 Dio能有效抑制人肺腺癌H1299细胞增殖,低于IC_(50)浓度的Dio(1、2μmol·L^(-1))能明显抑制H1299细胞的迁移与侵袭能力,可能与通过调控PI3K/AKT信号通路有关。

Objective To investigate roles of dioscin(Dio) on epithelial-mesenchymal transition of lungadenocarcinoma H1299 cells,and the mechanism of its role on migration and invasion. Methods H1299 cells weretreated with Dio at a range of 1 to 8 μmol·L^-1for 24 h and 48 h respectively. Cell proliferation was assessed byCCK8 assay. Cell migration at different concentrations of Dio(1,2 μmol·L^-1) on H1299 cells was assayed by thescratch wound healing and Transwell chamber assay. And Matrigel cell invasion assay was used to detect the effect ofDio on the invasion of H1299 cells. Western Blot was used to detect the protein levels of E-cadherin, Zonulaoccluden 1(ZO-1),ZEB1,Cludin-1,N-cadherin,Vimentin,β-catenin,PI3 K,AKT,P-AKT in the cells.Immunofluorescence was used to detect the localization and expression of interstitial marker Vimentin in cells.Results IC50 values were 3.50 μmol·L^-1and 3.21 μmol·L^-1after cells were treated with Dio for 24 h and 48 h,which showed a dose dependent manner. Compared with the control,the scratch wound healing of the cells were significantly decreased after treating with Dio(1,2 μmol·L^-1)(P<0.05,P<0.001);and we observed that Dio(1,2 μmol·L^-1) significantly suppressed the migration(P < 0.001) and invasion(P < 0.001) activities of H1299 cells. What’s more,the levels of E-cadherin,ZO-1 and Cludin-1 proteins’ expression in treatment groups weresignificantly up-regulated compared with the control(P<0.05, P<0.01, P<0.001), and the levels of N-cadherin, Vimentin, ZEB1, β-catenin, PI3 K, AKT, P-AKT protein expression in treatment groups weresignificantly up-regulated compared with the control(P<0.05, P<0.01, P<0.001). Conclusion Our datademonstrated that Dio can effectively inhibit the proliferation of human lung adenocarcinoma H1299 cells. Dio doses(1,2 μmol·L^-1) lower than IC50 can significantly inhibit the migration and invasion of H1299 cells,which may berelated to the regulation of PI3K/AKT signaling pathway.