Regulation of SIRT1/AMPK axis is critically involved in gallotannin-induced senescence and impaired autophagy leading to cell death in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is one of the most fatal malignancies with high mortality worldwide. Here the underlying antitumor mechanism of gallotannin was elucidated in HCC cells. Gallotannin suppressed viability and colony formation, increased subG1 portion and also induced senescence via upregulation of p21, G0/G1 arrest and higher SA-β-gal activity in HepG2 and SK-Hep1 cells. However, pan-caspase inhibitor Z-VAD-FMK reversed the ability of gallotannin to activate caspase 3 at 48 h after treatment in two HCC cells. Of note, gallotannin also induced autophagic features by increasing LC3 punctae, LC3B-II conversion, autophagic vacuoles and decreasing the expression of Beclin1 in two HCC cells. Furthermore, autophagy flux assay using GFP-mRFP-LC3 plasmid revealed increased yellowish color and late autophagy inhibitor CQ or NH4Cl enhanced cytotoxicity, LC3B-II conversion, and LC3 punctae in gallotannin-treated HepG2 and SK-Hep1 cells compared to early autophagy inhibitor 3-MA or wortmannin. Interestingly, gallotannin attenuated the expression of SIRT1 and mTOR and activated phosphorylation of AMPK in two HCC cells. Furthermore, AMPK activator AICAR significantly enhanced SA-β-gal activity and antiproliferation induced by gallotannin, while AMPK inhibitor compound C did not in two HCC cells. Consistently, LC3B-II conversion by gallotannin was not shown in AMPKα1 -/- MEF cells compared to WT AMPK +/+ MEF cells. Consistently, gallotannin reduced in vivo growth of HepG2 cells implanted in NCr nude mice along with decreased expression of PCNA and SIRT1 and increased AMPKα1 and TUNEL. Overall, these findings highlight evidence that regulation of SIRT1/AMPK is critically involved in gallotannin-induced senescence and impaired autophagy leading to cell death in HCC cells.