安罗替尼对人胃癌细胞迁移、侵袭的影响及机制

Effects and mechanism of anlotinib on migration and invasion of human gastric cancer cells

目的 探究安罗替尼对人胃癌细胞迁移、侵袭的影响及机制。方法 体外培养人胃癌AGS细胞,取第3代对数生长期AGS细胞,分为对照组(不做干预)、实验组(2μmol/L安罗替尼组、4μmol/L安罗替尼组、8μmol/L安罗替尼组)和抑制剂组[4μmol/L安罗替尼+10μmol/L转化生长因子-β_(1)(TGF-β_(1))/Smads信号通路抑制剂LY2109761],干预24 h。为避免细胞死亡太多干扰实验,抑制剂组选取4μmol/L安罗替尼作为最适浓度加入10μmol/L TGF-β_(1)/Smads通路抑制剂LY2109761进行后续实验。用CCK-8法、倒置显微镜、Transwell小室及Western blotting法对细胞活力、细胞形态、迁移、侵袭能力及上皮间质转化(EMT)、TGF-β_(1)/Smads信号通路相关蛋白表达进行检测。结果 与对照组相比,4、8μmol/L安罗替尼组细胞活力降低(P均<0.05)。实验组人胃癌AGS细胞生长受到抑制,迁移、侵袭能力及TGF-β_(1)、p-Smad3、N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)、纤维黏连蛋白(FN)蛋白表达降低(P均<0.05),Smad7和E-钙黏蛋白(E-cadherin)蛋白表达升高,且呈浓度依赖性(P均<0.05);与4μmol/L安罗替尼组相比,抑制剂组细胞生长受抑,细胞迁移、侵袭能力及TGF-β_(1)、p-Smad3、N-cadherin、Vimentin、FN蛋白表达进一步降低(P均<0.05),Smad7和E-cadherin蛋白表达进一步升高(P均<0.05)。结论 安罗替尼可能通过介导TGF-β_(1)/Smads通路的信号转导抑制人胃癌AGS细胞迁移、侵袭和EMT进程。

Objective To investigate the effects and mechanism of anlotinib on the migration and invasion of human gastric cancer cells.Methods Human gastric cancer AGS cells were cultured in vitro,and the AGS cells of the 3rd gen⁃eration in the logarithmic growth phase were selected.They were divided into the control group(no intervention),experi⁃mental group(2μmol/L anlotinib group,4μmol/L anlotinib group,and 8μmol/L anlotinib group)and inhibitor group[4μmol/L anlotinib+10μmol/L transforming growth factor-β_(1)(TGF-β_(1))/Smads signaling pathway inhibitor LY2109761],which were all treated for 24 h.In order to avoid interference with the experiment of too much cell death,in the inhibitor group,we selected 4μmol/L anlotinib as the optimal concentration and added 10μmol/L TGF-β_(1)/Smads pathway inhibi⁃tor LY2109761 for follow-up study.Cell viability,cell morphology,migration,invasion,and expression levels of epitheli⁃al mesenchymal transformation(EMT)and transforming growth faction-β_(1)(TGF-β_(1))/Smads signaling pathways were ana⁃lyzed by CCK-8,inverted microscopy,Transwell chamber,and Western blotting.Results Compared with the control group,the cell viability of the 4 and 8μmol/L anlotinib groups decreased(both P<0.05).In the experimental group,the growth of AGS cells was inhibited,and the migration and invasion ability and the expression of TGF-β_(1),p-Smad3,N-cad⁃herin,Vimentin and fibronectin(FN)decreased(all P<0.05),and the expression of Smad7 and E-cadherin protein in⁃creased,with a dose-dependent manner(all P<0.05).Compared with the 4μmol/L anlotinib group,the cell growth was inhibited,the cell migration and invasion abilities and the protein expression levels of TGF-β_(1),p-Smad3,N-cadherin,Vimentin and FN further decreased in the inhibitor group(all P<0.05),while the protein expression levels of Smad7 and E-cadherin further increased(both P<0.05).Conclusion Anlotinib can inhibit the migration,invasion and EMT pro⁃cess of AGS cells in human gastric cancer by mediating TGF-β_(1)/Smads pathway signal transduction.