Study On Plumbagin Induced Apoptosis And Autophagy In Hepatocellular Carcinoma And Its Signal Regulation

Objective: In this study,human hepatocellular carcinoma SMMC-7721 cells and BEL-7404 cells were used as research objects to explore: The effects of Plumbagin(PL)on the apoptosis and autophagy of hepatocellular carcinoma cells and its mechanism;The effects of PL on autophagy of hepatocellular carcinoma cells and their related signalling pathways;To elucidate the molecular mechanism of PL induced hepatocellular carcinoma apoptosis and autophagy,and provide more scientific basis for the development of PL against hepatocellular carcinoma.Methods: The vitality of hepatocellular carcinoma cells in each group was measured by MTT method.Inhibitors Z-VAD-FMK and IM-54 were treated with PL after incubation and compared with the control group;AO/EB staining was used to observe the acid autophagosomes(AVOs)and apoptosis of human hepatocellular carcinoma cells.Transmission electron microscopy was used to observe cell apoptosis and autophagy;Immunofluorescence detection of the expression of autophagy protein LC3 in hepatocellular carcinoma cells;Real-time quantitative PCR detection of m RNA expression levels of autophagy-related genes Beclin1,ATG5,and ATG7;Western blot was used to detect the expression of autophagy-related proteins(LC3-?,Beclin1,ATG5 proteins)and autophagy-related signalling pathway proteins(PI3K,Akt,m TOR,and p38 proteins)from the PI3 K / Akt / m TOR pathway and the p38 MAPK pathway,respectively.To analyze the effect of PL on autophagy and autophagy-related signalling pathways in hepatocellular carcinoma.Results: With the increase of PL concentration and prolonged treatment time,the relative viability of human hepatocellular carcinoma SMMC-7721 cells and BEL-7404 cells gradually decreased,showing a time-dose dependence;Under the transmission electron microscope,the apoptotic morphology of hepatocellular carcinoma cells after PL treatment was distinct,and autophagosomes appeared;After AO/EB staining,PL group showed a bright red fluorescence intensity.This indicates that PL induces the formation of autophagolysosomes in SMMC-7721 cells and BEL-7404 cells and promotes apoptosis;Immunofluorescence showed that the red fluorescence of LC3 protein was relatively concentrated in the PL group.The increase in the expression level of LC3 protein indicated that PL could induce autophagy in SMMC-7721 cells;Western blot results showed that the expression of autophagy-related proteins Beclin1,ATG5 and ATG7 protein increased significantly with increasing PL dose.RT-PCR showed that the m RNA expression of autophagy-related protein Beclin-1,ATG5 and ATG7 protein also increased significantly with increasing PL dose;Apoptosis and autophagy-related signalling pathway proteins p-PI3 K,p-Akt,and p-m TOR protein gradually decreased with the increase of PL concentration and had statistical significance.It is suggested that in human hepatocellular carcinoma SMMC-7721 cells and BEL-7404 cells,PL may activate autophagy and induce apoptosis by inhibiting the PI3 K / Akt / m TOR signaling pathway;p38 MAPK signaling pathway is closely related to autophagy.This study showed that when the expression of p-p38 decreased,the expression of LC3-II protein increased.This indicates that in human hepatocellular carcinoma SMMC-7721 cells and BEL-7404 cells,PL may inhibit p38 MAPK signaling pathway to activate autophagy.Conclusion: PL can significantly inhibit human hepatocellular carcinoma SMMC-7721 cells and BEL-7404 cells proliferation and induce apoptosis,and it has a time-dose-dependent effect;meanwhile,PL may activate human hepatocellular carcinoma cells by inhibiting the activity of the PI3 K / Akt / m TOR signalling pathway.Also,PL may inhibit p38 MAPK signaling pathway to activate autophagy.This is of great significance to improve the clinical efficacy of PL in the treatment of hepatocellular carcinoma and improve the quality of life of patients.