Effect And Mechanism Of PDB-1 On Proliferation Apoptosis And Autophagy In A549 Human Lung Cancer Cells

PDB-1 which was found by the research group for the first time is a new C-27-carboxylated-lupane-triterpenoid derivative isolated from Potentilla discolor Bunge and is a type of compound that is rarely found in natural sources.Previous studies have found that this compound has a significant selective inhibitory effect on tumor cells.Therefore,this study focused on clarifying the molecular mechanism of the effects of PDB-1 on the inhibition of proliferation and the induction of apoptosis,autophagy in A549 cells.The cytotoxic activity of PDB-1 and the positive control drug 10-hydroxycamptothecin against five cancer cell lines(HepG2,Hela,MCF-7,A549 and 231 cells)and human normal bronchial epithelial cells HBE were analyzed.It was found that PDB-1 is less toxic to human normal bronchial epithelial cells HBE.Compared with other tumor cells,PDB-1 has the strongest cytotoxicity to A549 cells(IC50 was 7.848±1.93 ?M),and the effect is better than 10-Hydroxycamptothecin.Therefore,A549 cells were selected as subjects and the subsequent studies were performed.In this paper,the effect of PDB-1 on the proliferation,apoptosis and autophagy of A549 human lung cancer cells was systematically studied.Generally,human lung adenocarcinoma A549 cells are treated with PDB-1 for different times and the cell survival rate is measured by the CCK-8 assay.The intracellular reactive oxygen species,mitochondrial membrane potential assay,cell cycle assay,Annexin V-FITC /PI assay,MDC staining were performed in A549 cell by induced PDB-1.The mRNA and protein expression of cell cycle,apoptosis and autophagy related-factors were detected by RT-qPCR and Western-blot.The results showed that PDB-1 inhibited A549 cells proliferation and colony formation in a dose-and time-dependent manner.PDB-1 inhibits the expression of cycle-related proteins cyclin B1 and CDK1,and promote the expression of cyclin-dependent kinase inhibitor p21,indicating that the decrease in the viability of A549 cells is caused by G2 / M cell cycle arrest.With the increase of PDB-1 treatment concentrations,the intracellular active oxygen content showed a dose-dependent increase,the mitochondrial membrane potential decreased.PDB-1 dose-dependently increased the apoptosis of A549 cells,accompanied by an increase in the Bax / Bcl-2 ratio and an increase in the expression level of caspase-3 /caspase-9.Similarly,PDB-1 induces cell autophagy by increasing the expression of LC3 II and Beclin-1.PI3 K / Akt / mTOR signaling pathway is closely related to the process of cell growth and survival,our research shows that PDB-1 significantly inhibits the PI3 K / Akt cell survival signaling pathway,while a specific PI3 K inhibitor(LY294002)enhances the expression of apoptosis-and autophagy-related proteins induced by PDB-1.These results suggest that PDB-1 as a new triterpenoid can inhibit the proliferation,promote apoptosis and autophagy of A549 human lung cancer cells,and has less cytotoxic activity on human normal bronchial epithelial cells HBE.Therefore,PDB-1 has the potential to be developed as a drug for the treatment of lung cancer,and can be used as a candidate drug for further in-depth research.