Abstract:

Objective  To investigate the effects of autophagy on adenanthin-induced death of human hepatocellular carcinoma (HCC) Bel-7402 cells. Methods  Bel-7402 cells were treated by adenanthin of 9 μmol/L for 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 h. Expressions of autophagy related proteins Beclin-1 and LC3 were detected by Western blotting. The distribution of expression of endogenous LC3 protein in Bel-7402 cells treated by adenanthin of 9 μmol/L for 0, 2, and 4 h was detected by immunofluorescence.The cell line that the expression of Beclin-1 gene was specifically intervened was constructed. The effects of low expression of Beclin-1 on changes of autophagy activity, cell growth, and cell death induced by adenanthin were analyzed and observed by western blotting and light microscopy, respectively.  CCK-8 was adopted to detect the effects of wild type and ATG5 knockout (ATG5^-/-) mouse embryonic fibroblasts (MEFs) on the inhibition of cell growth induced by adenanthin of different concentrations. The difference of sensitivity to adenanthin between wild type and ATG5 knock out MEFs was analyzed by comparing the IC₅₀. Results  Western blotting and immunofluorescence showed that adenanthin significantly increased the autophagy activity of Bel-7402 cells. Knockout of Beclin-1 did not remarkably affect adenanthin-induced autophagy activity and cell death. Compared with wild type MEFs, ATG5^-/- MEFs were more resistant to the inhibition of cell growth induced by adenanthin. Conclusion Adenanthin can significantly increase the autophagy activity of Bel-7402 cells and promote adenanthin-induced cell death. But the increase of autophagy activity is independent of the expression of Beclin-1.

Key words: adenanthin, autophagy, hepatocellular carcinoma, cell death