重楼皂苷A直接靶向活化AMPK诱导结直肠癌细胞自噬

Polyphyllin A directly activates AMPK to induce autophagy in colorectal cancer cells

目的:探讨重楼皂苷A(Polyphyllin A,PPA)对结直肠癌细胞自噬的影响及其潜在机制。方法:选用结直肠癌RKO和HRT18细胞株作为细胞模型。用不同浓度PPA处理RKO细胞和HRT18细胞,MTT法检测细胞活力,筛选最佳实验浓度;集落形成实验检测细胞克隆能力;免疫印迹法检测微管相关蛋白LC3-Ⅱ表达、腺苷酸活化蛋白激酶(AMP-activated protein kinase,AMPK)和雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)表达及其磷酸化;免疫荧光检测自噬体形成;分子对接分析PPA与AMPK相互间作用。结果:与0μmol/L组相比,PPA各浓度组细胞活力明显降低(P<0.05)。与0μmol/L组相比,PPA组两株细胞克隆形成能力明显降低(P<0.05);随着PPA浓度逐渐增加,两株细胞中LC3-Ⅱ表达明显上调(P<0.05),自噬体发生聚集,总AMPK、mTOR蛋白无明显变化(P>0.05),而磷酸化AMPK升高、磷酸化mTOR蛋白降低(P<0.05);PPA与AMPK存在直接结合作用。结论:PPA通过直接靶向活化AMPK诱导结直肠癌细胞发生自噬。

Objective:To investigate the effect of Polyphyllin A(PPA)on autophage of colorectal cancer(CRC)cells and its potential mechanism.Methods:Colorectal cancer RKO and HRT18 cells were selected as cell model.RKO and HRT18 cells were treated with different concentrations of PPA,MTT assay was used to detect the cell viability,and to screen the optimal experimental concentration.Colony formation assay was used to detect cell cloning ability.Western blotting was used to detect the expression of microtubule-associated proteinⅡlight chain-3(LC3-Ⅱ),expression of AMP-activated protein kinase(AMPK)and mammalian target of rapamycin(mTOR)and its phosphorylation.In addition,immunofluorescence was used to detect autophagosome formation.Molecular docking was used to analyze the interaction of PPA and AMPK.Results:Compared with the 0μmol/L group,the cell viability in each concentration group of PPA was significantly reduced(P<0.05).Compared with the 0μmol/L group,the cloning ability of the two cell lines was significantly reduced(P<0.05).With the gradual increase of PPA concentration,the expression of LC3-Ⅱin the two cell lines gradually increased,and autophagosomes aggregated.The expression of total AMPK and mTOR protein did not change significantly(P>0.05),while the phosphorylation of AMPK was increased and the phosphorylation of mTOR was decreased(P<0.05).The molecular docking showed that PPA directly combined with AMPK.Conclusion:PPA induced autophagy in CRC cells by directly targeting and activating AMPK.