Antcin B and its ester derivative from Antrodia camphorata induce apoptosis in hepatocellular carcinoma cells involves enhancing oxidative stress coincident with activation of intrinsic and extrinsic apoptotic pathway

The triterpenoids methylantcinate B (MAB) and antcin B (AB), isolated from the medicinal mushroom Antrodia camphorata, have been identified as strong cytotoxic agents against various type of cancer cells; however, the mechanisms of MAB- and AB-induced cytotoxicity have not been adequately explored. This study investigated the roles of caspase cascades, reactive oxygen species (ROS), DNA damage, mitochondrial disruption, and Bax and Bcl-2 proteins in MAB- and AB-induced apoptosis of hepatocellular carcinoma (HCC) HepG2 cells. Here, we showed that MAB and AB induced apoptosis in HepG2 cells, as characterized by increased DNA fragmentation, cleavage of PARP, sub-G1 population, chromatin condensation, loss of mitochondrial membrane potential, and release of cytochrome c. Increasing the levels of caspase-2, -3, -8, and -9 activities was involved in MAB- and AB-induced apoptosis, and they could be attenuated by inhibitors of specific caspases, indicating that MAB and AB triggered the caspase-dependent apoptotic pathway. Additionally, the enhanced apoptotic effect correlates with high expression of Fas, Fas ligand, as well as Bax and decreased protein levels of Bcl- _XL and Bcl-2, suggesting that both the extrinsic and intrinsic apoptosis pathways were involved in the apoptotic processes. Incubation of HepG2 cells with antioxidant enzymes superoxide dismutase and catalase and antioxidants N-acetylcysteine and ascorbic acid attenuated the ROS generation and apoptosis induced by MAB and AB, which indicate that ROS plays a pivotal role in cell death. NADPH oxidase activation was observed in MAB- and AB-stimulated HepG2 cells; however, inhibition of such activation by diphenylamine significantly blocked MAB- and AB-induced ROS production and increased cell viability. Taken together, our results provide the first evidence that triterpenoids MAB and AB induced a NADPH oxidase-provoked oxidative stress and extrinsic and intrinsic apoptosis as a critical mechanism of cause cell death in HCC cells.