摘要
目的:研究异甘草素(isoliquiritigenin,ISL)对人肾透明细胞癌786-O细胞的抗癌作用,并探讨其可能存在的分子机制。方法:通过噻唑蓝(thiazolyl blue tetrazolium bromide,MTT)比色法检测ISL(0,10,25,50,75,100μmol·L~(-1))对786-O细胞增殖的作用;细胞划痕和Transwell实验检测ISL对786-O细胞迁移、侵袭能力的影响;吖啶橙染色,Ad-GFP-LC3转染实验观察细胞自噬状态;蛋白免疫印迹法(Western blot)检测自噬相关蛋白的表达,并分析磷脂酰肌醇-3-激酶(phosphatidylinositol-3-kinase,PI3K)/蛋白激酶B(protein kinase B,Akt)/哺乳动物雷帕霉素蛋白(mammalian target of rapamycin,m TOR)信号通路变化,探讨其可能的作用机制。结果:MTT比色法结果显示,与空白组比较,ISL能够明显抑制786-O细胞的增殖,且呈时间-剂量依赖性(P<0.05);划痕和Transwell实验结果表明,与空白组比较,ISL能够抑制785-O细胞的迁移和侵袭(P<0.05);吖啶橙染色和Ad-GFP-LC3转染实验表明ISL能够诱导786-O细胞发生自噬。此外,与空白组比较,ISL能够诱导细胞内自噬标志物-Ⅱ(LC3-Ⅱ),自噬相关蛋白Beclin1,Atg5蛋白表达(P<0.05),并显著下调p62蛋白表达(P<0.05),当加入自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)可逆转上述现象(P<0.05);同时ISL能够抑制Akt,m TOR的磷酸化水平(P<0.05),加入抑制剂LY294002和雷帕霉素(rapamycin,Rap),LC3-Ⅱ,Beclin1,Atg5蛋白表达明显上调(P<0.05),p62蛋白表达明显下调(P<0.05)。结论:ISL能够抑制肾透明细胞癌786-O细胞的增殖、迁移和侵袭,并通过抑制PI3K/Akt/m TOR信号通路诱导细胞发生自噬。
Objective:To investigate the anticancer effect of isoliquiritigenin(ISL)on human clear cell renal cell carcinoma 786-O cells,and explore its possible molecular mechanism.Method:Thiazolyl blue tetrazolium bromide(MTT)assay was used to detect effect of ISL(0,10,25,50,75,100μmol·L~(-1))on proliferation of 786-O cells.The effect of ISL on migration and invasion of 786-O cells was detected by cell scratch test and Transwell assay.The autophagy was observed under the fluorescence microscope through acridine orange staining and Ad-GFP-LC3 transfection experiment.Western blot was used to detect the expression of autophagy related protein and analyze the changes of phosphatidylinositol-3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(m TOR)signaling pathway to explore the possible mechanism.Result:MTT results showed that ISL could significantly inhibit the proliferation of 786-O cells in a time-dose dependent manner(P<0.05).The results of scratch and Transwell experiments indicated that ISL could inhibit the migration and invasion of 786-O cells(P<0.05).Acridine orange staining and ad-GFP-LC3 transfection showed that ISL can induce autophagy in 786-O cells.Besides,ISL can induce LC3-Ⅱ,Beclin1,Atg5 protein expressions(P<0.05),and significantly reduce p62 protein expression(P<0.05);autophagy inhibitor 3-methyl adenine(3-MA)can be added to reverse the above phenomena(P<0.05).Meanwhile,ISL can inhibit phosphorylation of Akt,m TOR levels(P<0.05),inhibitor LY294002 and rapamycin(Rap)can be added to significantly reregulate LC3-Ⅱ,Beclin1,Atg5 protein expressions(P<0.05)and significantly down-regulate p62 protein expression(P<0.05).Conclusion:ISL can inhibit the proliferation,migration and invasion of clear cell renal carcinoma 786-O cells,and induce autophagy by inhibiting the PI3K/Akt/m TOR signaling pathway.
引文
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