咪喹莫特通过Akt/mTOR信号通路诱导人胃癌细胞SGC-7901发生自噬

Imiquimod induces autophagy of human gastric cancer cell SGC-7901 via Akt/mTOR signaling pathway

目的探讨Toll样受体7激动剂咪喹莫特诱导人胃癌细胞SGC-7901发生自噬及其可能的机制。方法体外培养人胃癌细胞株SGC-7901,给与不同浓度咪喹莫特(0,25,50,100μg/ml)处理24 h,RT-PCR及Western blot法检测LC3Ⅱ、Beclin1及p62 mRNA和蛋白表达水平变化。透射电镜下观察100μg/ml咪喹莫特作用12 h自噬小体的形成。不同浓度咪喹莫特处理SGC-7901细胞24 h,Western blot法观察Akt/mTOR通路相关蛋白Akt、mTOR及p70S6K表达水平变化。观察联合应用组(100μg/ml咪喹莫特加100 nmol/L mTOR抑制剂雷帕霉素)与对照组(100μg/ml咪喹莫特)LC3Ⅱ和Beclin1蛋白表达变化,MTT法检测两组细胞增殖活性的改变。结果不同浓度咪喹莫特作用24 h,SGC-7901细胞LC3Ⅱ和Beclin1 mRNA及蛋白表达水平上调,且随浓度增加表达水平相应上升(均P=0.000),p62 mRNA及蛋白表达水平随浓度增加依次下降(均P=0.000)。100μg/ml咪喹莫特作用12 h,透射电镜下可观察到典型的自噬形态学改变。不同浓度咪喹莫特作用24 h,Akt/mTOR信号通路相关蛋白Akt及mTOR磷酸化减少、其下游信号分子p70S6K表达下降。与对照组相比,联合应用雷帕霉素组LC3Ⅱ和Beclin1表达水平上升,而细胞增殖活性显著下降(P<0.05)。结论咪喹莫特能够诱导SGC-7901细胞发生自噬,其可能的机制是通过抑制Akt/mTOR信号通路实现的。

Objective To investigate the autophagy induced by Toll-like receptor 7 agonist imiquimod in human gastric cancer cells SGC-7901 and its possible mechanism.Methods Human gastric cancer cell line SGC-7901 was cultured in vitro and treated with different concentrations of imiquimod(0,25,50,100μg/ml)for 24 h,and then LC3Ⅱ,Beclin1 and p62 were measured by RT-PCR and Western blot.The ultrastructural changes of autophagy were observed under electron microscope after treatment with 100μg/ml imiquimod for 12 h.After treatment with different doses of imiquimod for 24 h,the related proteins of Akt/mTOR signal pathway were measured by Western blot.The protein levels of LC3Ⅱand Beclin1,and the cell viability of SGC-7901 cells were detected after treatment with 100μg/ml imiquimod with or without mTOR inhibitor rapamycin(100 nmol/L)for 24 h.Results The mRNA and protein expression levels of LC3Ⅱand Beclin1 in SGC-7901 cells were upregulated by imiquimod for 24 h,and the expression levels were increased with the increase of imiquimod concentration(all P=0.000),the mRNA and protein expression levels of p62 were decreased with the increase of concentration(all P=0.000).After treatment with 100μg/ml imiquimod for 12 h,many autophagosome-like va-cuoles were found in the cytoplasm of SGC-7901 cells.After treated with different doses of imiquimod for 24 h,the levels of phospho-Akt,phospho-mTOR,and the downstream protein p70S6K were significantly decreased.The levels of LC3Ⅱand Beclin1 in SGC-7901 cells were higher in imiquimod+rapamycin group than inimiquimod,while the proliferation ofSGC-7901 cells was lower(P<0.05).Conclusion Imiquimod can induce the autophagy in SGC-7901 cells through inhibition of Akt/mTOR signaling pathway.