三羟基异黄酮通过AMPK和mTOR信号通路对宫颈癌细胞抑制作用的研究

Inhibitory effect of genistein on cervical carcinoma cell through AMPK and mTOR signaling pathway

目的:探讨三羟基异黄酮对人宫颈癌Caski细胞株生长凋亡的影响及机制。方法:磺酰罗丹明B(SRB)比色法检测不同浓度三羟基异黄酮(GEN)干预对Hela,SiHa,Caski,HeLa-S3,HeLa229及C-33A等宫颈癌细胞株生长增殖的影响。流式细胞术检测细胞周期和线粒体膜电位变化情况,逆转录-聚合酶链反应(RT-PCR)和Western blot检测细胞内一磷酸腺苷(AMP)激活的蛋白激酶(AMPK)和哺乳动物雷帕霉素靶蛋白(mTOR)信号通路相关基因的信使核糖核酸和蛋白水平的表达。免疫沉淀法检测三羟基异黄酮对Caski细胞结节性硬化症基因1/结节性硬化症基因2(TSC2/TSC1)复合物组装的影响。结果:三羟基异黄酮的抗癌效能次序是Caski>Hela>C-33A>SiHa>HeLa-S3>HeLa229;三羟基异黄酮彻底阻滞了Caski细胞周期进程,并诱导其凋亡。三羟基异黄酮没有改变G1调节蛋白的mRNA表达水平,表明蛋白翻译受到抑制,但转录水平没受到影响。三羟基异黄酮诱导组装TSC1/TSC2,并通过抑制包括mTOR(Ser2448)、p70S6K(Thr421/Ser424和Thr389)和4E-BP1(Thr37/Thr46和Thr70)等蛋白质磷酸化导致细胞蛋白质合成受阻。三羟基异黄酮干预还引起宫颈癌细胞AMPK活性升高。AMPK抑制剂复合物C显著逆转三羟基异黄酮干预效果的结果表明AMPK通路是三羟基异黄酮抑制宫颈癌细胞增殖过程中的关键途径。此外,线粒体膜电势和线粒体含量下降表明三羟基异黄酮引起线粒体氧化应激。结论:三羟基异黄酮干预Caski细胞后,引起线粒体应激反应,激活AMPK信号通路,使线粒体膜电位下降、TSC1/TSC2复合物形成增加及mTOR介导的蛋白质翻译通路受阻,最终导致宫颈癌Caski细胞周期G1期阻滞,并诱导其凋亡。

AIM： To observe the effect of genistein on growth and apoptosis of cervical carcinoma Caski cell lines , and to explore its potential mechanisms. METHODS： Cervical car- cinoma cell lines Hela, SiHa, Caski, HeLa-S3, HeLa229 and C-33A were cultured and treated with genistein at different concentration and at different time points. The effect of genistein on cervical carcinoma cell lines proliferation was studied by means of Sulforhodamine B （SRB）, the cell cycle and mitochondrial membrane po- tential were detected with flow eytometry; RT- PCR and western blot were used to detect the expressions of AMPK and mTOR signaling pathway related gene at mRNA and protein lev- els. Determination of TSC1/TSC2 association by immunoprecipitation assay. RESULTS： The rank order of genistein potency against cervical carci- noma is Caski〉Hela〉 C-33A 〉 SiHa〉HeLa- S3 〉 HeLa229. Genistein completely abolished cell-cycle progression released from double-thy- midine-block synchronization and caused a sub- sequent apoptosis. The data were supported by down-regulation and reduced nuclear transloca- tion of Gl-regulator proteins, including cyclin D1, cyclin E, Cdk4 and Cdk2. Further analysis showed that the mRNA expressions of the G1- regulator proteins were not modified by Methyl jasmonate, indicating an inhibition of transla-tional but not transcriptional levels. Genistein induced the assembly of tuberous sclerosis com- plex （TSC1/TSC2）, leading to the blockade of cellular protein synthesis through inhibition of protein phosphorylation including roTOR （Ser2448）, pTOS6K （ Thr421/Ser424 and Thr389 ） and 4E-BP1 （ Thr37/Thr46 and ThrT0）. Furthermore, the AMPK activity was elevated by genistein. Compound C, a selective AMPK inhibitor, significantly reversed genistein-mediated effects suggesting the crucial role of AMPK. Besides, the loss of mitochondri- al membrane potential and depletion of mito- chondrial content indicated the mitochondrial stress caused by genistein. CONCLUSION. Genistein induces anticancer signaling cascades in a sequential manner. The exposure of cells to genistein induces mitochondrial stress and acti- vation of AMPK that further induces the loss of mitochondrial membrane potential and activates TSC1/TSC2 association. Consequently, the mTOR mediated translational pathways are blocked, leading to G1 arrest of the cell-cycle and subsequent cell death.