β-Thujaplicin induces autophagic cell death, apoptosis, and cell cycle arrest through ROS-mediated Akt and p38/ERK MAPK signaling in human hepatocellular carcinoma

Hepatocellular carcinoma (HCC), a common liver malignancy worldwide, has high morbidity and mortality. beta-Thujaplicin, a tropolone derivative, has been used in some health-care products and clinical adjuvant drugs, but its use for HCC is unknown. In this study, we found that beta-Thujaplicin inhibits the growth of HCC cells, but not normal liver cells, with nanomolar potency. Mechanistically, we found that beta-Thujaplicin could induce autophagy, as judged by western blot, confocal microscopy, and transmission electron microscopy. Further using beta-Thujaplicin combined with an autophagy blocker or agonist treatment HepG2 cells, we found that beta-Thujaplicin induced autophagic cell death (ACD) mediated by ROS caused inhibition of the Akt-mTOR signaling pathway. Moreover, beta-Thujaplicin triggered HepG2 apoptosis and increased cleaved PARP1, cleaved caspase-3, and Bax/Bcl-2 ratio, which indicated that beta-Thujaplicin induced apoptosis mediated by the mitochondrial-dependent pathway. We also found that increased expression of p21 and decreased expression of CDK7, Cyclin D1, and Cyclin A2 participating in beta-Thujaplicin caused the S-phase arrest. It seems that beta-Thujaplicin exerts these functions by ROS-mediated p38/ERK MAPK but not by JNK signaling pathway activation. Consistent with in vitro findings, our in vivo study verified that beta-Thujaplicin treatment significantly reduced HepG2 tumor xenograft growth. Taken together these findings suggest that beta-Thujaplicin have an ability of anti-HCC cells and may conducively promote the development of novel anti-cancer agents.