重楼皂苷Ⅰ通过激活AMPK信号通路诱导非小细胞肺癌自噬的作用机制

The Mechanism of Polyphyllin Ⅰ Inducing Autophagy in Non-small Cell Lung Cancer by Activating AMPK Signaling Pathway

目的:本文旨在探讨重楼皂苷Ⅰ (Polyphyllin I,PPI)抑制非小细胞肺癌(non-small-cell lung cancer,NSCLC)生长的作用及机制。方法:通过MTT测定法及克隆形成实验检测PPI对2种不同肺癌细胞系(PC9和H460)体外增殖能力的影响;通过Western Blot和免疫荧光实验进行自噬检测;通过Western Blot检测PPI对AMPK/mTOR及其下游信号通路的影响;通过Western Blot检测AMPK抑制剂Compound C (CC)对PPI诱导的自噬激活的影响;通过分子对接分析PPI与AMPK的结合作用;通过微量热泳动实验和基于靶点稳定性的药物结合实验检测PSVII与AMPK的亲和力。结果:PPI在低微摩尔浓度剂量下能够显著抑制NSCLC细胞的增殖;PPI能够诱导NSCLC细胞发生自噬;PPI能够在NSCLC细胞中激活AMPK/mTOR信号通路;CC能够逆转PPI诱导的自噬激活;PPI与AMPK具有直接结合作用。结论:PPI通过直接靶向活化AMPK诱导NSCLC细胞发生自噬进而抑制NSCLC细胞生长和增殖。

Objective The aim of this study was to investigate the inhibitory effect and mechanism of polyphyllin I(PPI)on the growth of non-small cell lung cancer(NSCLC).Methods MTT and soft agar colony formation assay were used to detect the effect of PPI on the in vitro proliferation of two different NSCLC cell lines(PC9 and H460).Western Blot and immunofluorescence were used to detect the autophagy.Western Blot was used to detect the influence of PPI on AMPK/mTOR and its downstream signal pathway and Western Blot was used to determine the effect of AMPK inhibitor Compound C(CC)on PPI induced autophagy.Molecular docking was used to analyze the binding effect of PPI and AMPK.The affinity between PPI and MAPK was determined by microscale thermophoresis assay and drug binding assay based on target stability.Results PPI could significantly inhibit the proliferation of NSCLC cells at low micromolar doses.PPI induced autophagy in NSCLC cells.PPI activated the AMPK/mTOR signaling pathway in NSCLC cells.CC reversed PPI induced autophagy activation.PPI had a direct binding effect with AMPK.Conclusion PPI directly targets and activates AMPK to induce autophagy in NSCLC cells,thereby inhibiting the growth and proliferation of NSCLC cells.