胡桃醌通过Akt/mTOR/p70S6K通路调控肺癌细胞A549自噬的研究

Regulation of Autophagy of Lung Cancer Cell A549 by Juglone via Akt/mTOR/p70S6K Pathway

目的:研究胡桃醌通过Akt/mTOR/p70S6K通路调控肺癌细胞A549自噬,发挥其抗肺癌作用的机制。方法:MTT法检测胡桃醌对A549细胞存活率的影响。透射电镜和细胞自噬染色检测试剂盒(MDC)观察胡桃醌诱导A549细胞自噬。蛋白免疫印迹法检测自噬标志蛋白Beclin-1、LC3Ⅱ和LC3I蛋白和自噬经典通路Akt/mTOR/p70S6K相关蛋白的表达水平。RT-PCR法检测A549细胞中Beclin-1和LC3ⅡmRNA表达水平。结果:MTT结果表明胡桃醌抑制A549细胞增殖,IC 50=10μmol/L;透射电镜和MDC实验发现胡桃醌可以诱导A549细胞产生自噬;与对照组相比,胡桃醌可以显著增加Beclin-1蛋白表达水平、LC3Ⅱ/LC3I比值和Beclin-1、LC3ⅡmRNA表达水平(P<0.05,P<0.01);胡桃醌干预后,与对照组相比,Akt、mTOR和p70S6K蛋白的表达水平基本不变,但Akt、mTOR和p70S6K磷酸化蛋白的表达水平显著降低(P<0.05,P<0.01)。结论:胡桃醌可能通过Akt/mTOR/p70S6K通路调控肺癌细胞A549自噬。

Objective:To study the mechanism of juglone regulating autophagy of lung cancer cells A549 through Akt/mTOR/p70S6K pathway and its anti-lung cancer effect.Methods:MTT assay was used to detect the effect of juglone on the survival rate of A549 cells.Transmission electron microscopy(TEM)and autophagy staining kit(MDC)were used to observe juglone induced autophagy in A549 cells.The expression levels of autophagy marker Beclin-1,LC3Ⅱand LC3I proteins,and classical autophagy pathway Akt/mTOR/p70S6K related proteins were detected by Western blotting.Beclin-1 and LC3ⅡmRNA expression levels of A549 cells were detected by RT-PCR.Results:MTT results showed that juglone inhibited the proliferation of A549 cells and IC 50=10 mol/L;the results of TEM and MDC experiments showed that juglone induced autophagy in A549 cells.Compared with the control group,juglone significantly increased Beclin-1 protein expression,LC3Ⅱ/LC3I ratio and the mRNA expression levels of Beclin-1 and LC3ⅡmRNA(P<0.05).Compared with the control group,the protein expression levels of Akt,mTOR and p70S6K were basically unchanged after juglone intervention,but the expression levels of phosphorylated proteins of Akt,mTOR and p70S6K were significantly decreased(P<0.05).Conclusion:Juglone regulates autophagy in lung cancer cell A549 possibly via Akt/mTOR/p70S6K pathway.