Paris Saponin Ⅶ,a Direct Activator Of AMPK,Induces Autophagy And Exhibits Therapeutic Potential In Non-Small-Cell Lung Cancer

Background: Lung cancer is the leading cause of cancer-related death worldwide.Non-small cell lung cancer(NSCLC)accounts for more than 85% of all lung cancer types.The treatments for NSCLC mainly include surgery,chemotherapy,radiotherapy and targeted therapy However,current treatment methods still cannot effectively control tumor progression.Finding new treatment strategies and targeted drugs is critical to NSCLC.Autophagy is a highly conserved and lysosome-dependent degradation process in eukaryotes.Numerous studies have shown that excessive autophagy could induce death in tumor cells.Targeting autophagy becomes a novel strategy for tumor therapy.AMPactivated kinase(AMPK)is a critical upstream signal protein of autophagy.AMPK activates autophagy through inhibiting Mammalian target of rapamycin complex1(mTORC1).Paris Saponin Ⅶ(PSⅦ)is a biologically active natural compound extracted from the rhizome of Trillium tschonoskii Maxim,which has anti-inflammatory and anti-tumor effects.In this study,we demonstrated that PSⅦ induced autophagy in NSCLC cells by directly activating AMPK and played a therapeutic effect.Objective: To explore the mechanism of PSⅦ inhibiting NSCLC cells and to provide a preclinical basis for the discovery of new NSCLC therapeutic drugs.Methods: MTT,trypan blue assay and clone formation assay were used to detect the cytotoxic effect of PSⅦ on NSCLC cells in vitro.Flow cytometry,DAPI staining and Western blot were used to detect the effect of PSⅦ on apoptosis of NSCLC cells.Autophagosome formation assay,Western blot and autophagy inhibitors were used to investigate the effect of PSⅦ on f autophagy in NSCLC cells.Western blot,AMPK kinase activity assay and AMPK inhibitor were used to detect the effect of PSⅦ on the upstream AMPK/mTOR signaling pathway of autophagy.The molecular docking,microscale thermophoresis and drug affinity responsive target stability assays were used to detect the direct affinity between PSⅦ and AMPK.Results: PSⅦ inhibited the growth and proliferation of NSCLC cells in a dosedependent and time-dependent manner.PSⅦ inhibited the clonal formation of NSCLC cells.PSⅦ induced autophagy and blocked autophagy flux in NSCLC cells.PSⅦ increased the phosphorylation of AMPK and decreased the phosphorylation of mTORC1complex(mTOR,P70S6 K and 4EBP1)in NSCLC cells.PSⅦ activated AMPK kinase activity.The specific inhibitor of AMPK reversed the effect of PSⅦ on cell proliferation,autophagy,and apoptosis.PSⅦ activated AMPK by directly binding to the drug allosteric and metabolic sites of AMPK.Conclusion: Here,we clarified the therapeutic mechanism of PSⅦ in NSCLC,that is,as a direct activator of AMPK to induce autophagy,thereby inhibiting cell growth and inducing apoptosis.