Atg5 siRNA inhibits autophagy and enhances norcantharidin-induced apoptosis in hepatocellular carcinoma

Abstract

Cantharidin is a terpenoid isolated from Chinese blister beetles, and norcantharidin (NCTD) is a demethylated analog of cantharidin. It has been reported that cantharidin and norcantharidin have anticancer activities. Growing evidence suggests that inhibiting autophagy can induce apoptosis in the human hepatoma cell line HepG2. The objective of the present study was to determine whether inhibition of autophagy enhances NCTD-induced apoptosis in HepG2 cells. HepG2 cells were cultured in DMEM containing NCTD. Autophagy was upregulated in the presence of HBSS media supplemented with Ca2+ and Mg2+ and 10 mM HEPES and downregulated in the presence of 3-methyladenine (3-MA) and Atg5 siRNA. Autophagy, cell viability, and the expression of apoptotic proteins were assessed in HepG2 cells. Our data showed that cell apoptosis generally increased after norcantharidin treatment in HepG2 cells. Expression of LC3-II, an autophagosome marker, increased when cells were treated with HBSS media. It also increased cell viability. However, in the presence of 3-MA and Atg5 siRNA, autophagy was inhibited, LC3-II expression decreased and cell apoptosis increased. There was increased expression of Bax, cytochrome c, cleaved caspase-3, caspase-9 and PARP and the mitochondrial membrane potential was disrupted. Additionally, increased apoptosis was accompanied by increased reactive oxygen species (ROS) production. NCTD has anticancer activity, and Atg5 siRNA-mediated downregulation of autophagy enhanced its anticancer actions due to ROS generation and activation of the mitochondrial apoptosis pathway.