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 Ca 2+ and Mg 2+ 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. IntroductionLiver cancer is the sixth most frequent cancer and the second leading cause of cancer-related deaths worldwide. Hepatocellular carcinoma (HCC) represents 90% of all primary liver cancers (1-3). The 5-year survival rate for HCC is <30% and the recurrence rate is ~70% (4,5). Current HCC treatments, including surgical resection, liver transplantation, chemotherapy, or immuno-biological cancer therapies, are typically not very effective.Cantharidin is a terpenoid isolated from Chinese blister beetles, and norcantharidin (NCTD) is a demethylated analog of cantharidin that has anticancer activity in breast cancer, lung cancer, leukemia, colon and liver cancer. One possible explanation for the anticancer actions of NCTD is its inhibition of protein phosphatases, through which G0/G1 or G2/M arrest is triggered. Thus, apoptosis is subsequently induced via ROS generation and the mitochondrial pathway (6,7).Autophagy, an important pathophysiological process, is crucial for cell development, differentiation, survival and homeostasis. Additionally, autophagy has an important role in liver cancer. Previous studies have shown that autophagy is induced in liver cancer.

Cinobufagin is a cardiotoxic bufanolide steroid secreted by the Asiatic toad Bufo gargarizans. Cinobufagin is one of the active ingredients in the anticancer Chinese medicine called Chan Su, which was demonstrated to be an effective treatment for gastric cancer. Increasing evidence shows that inhibition of autophagy has a pro-apoptotic effect on human gastric cancer cells. The aim of the present study was to investigate the relationship between cinobufagin, autophagy and apoptosis in gastric cancer. Autophagy was induced or inhibited in the human gastric cancer cell line SGC-7901 by incubation in HBSS media or by treatment with 3-methyladenine or ATG5 siRNA, respectively. Following treatment, the levels of apoptosis, apoptotic proteins, reactive oxygen species (ROS), and mitochondrial membrane potential were compared between the conditions. As anticipated, we found that cinobufagin increased apoptosis in SGC-7901 cells. Notably, inhibition of autophagy, monitored by the absence of the autophagosome marker LC3-II, also enhanced cell apoptosis. This effect was reversed when autophagy was induced by incubation in HBSS media. Enhanced expression of pro-apoptotic indicators, including BAX, cytosolic cytochrome c, cleaved PARP, caspase-3 and caspase-9, was detected when autophagy was suppressed. Increased pro-apoptotic protein expression was accompanied by disrupted mitochondrial membrane potential and elevated ROS production. Altogether, these data suggest that inhibition of autophagy enhances the anticancer action of cinobufagin through increased apoptosis of gastric cancer cells. Moreover, these effects may be partly mediated by ROS generation and the activation of the mitochondrial programmed cell death pathway.

Abstract. There is growing evidence indicating that autophagy plays a protective role in liver ischemia/reperfusion (IR) injury. Heme oxygenase-1 (HO-1) can also prevent liver IR injury by limiting inflammation and inducing an anti-apoptotic response. Autophagy also plays a crucial role in liver IR injury. The aim of the present study was to investigate the role of HO-1 in liver IR injury and the association between HO-1, autophagy and apoptotic pathways. IR simulation was performed using buffalo rat liver (BRL) cells, and HO-1 activity was either induced by hemin (HIR group) or inhibited by zinc protoporphyrin (ZnPP) (ZIR group). In the HIR and ZIR group, the expression of HO-1 and autophagy-related genes [light chain 3-Ⅱ (LC3-Ⅱ)] was assessed by RT-qPCR and the protein expression of caspases, autophagy-related genes and genes associated with apoptotic pathways (Bax) was detected by western blot anlaysis. The results of RT-PCR revealed the genetically decreased expression of HO-1 and autophagy-related genes in the ZIR group. Similar results were obtained by western blot analysis and immunofluorescence. An ultrastructural analysis revealed a lower number of autophagosomes in the ZIR group; in the HIR group, the number of autophagosomes was increased. The expression of Bax and cytosolic cytochrome c was increased, while that of Bcl-2 was decreased following treatment of the cells with ZnPP prior to IR simulation; the oppostie occurred in the HIR group. Cleaved caspase-3, caspase-9 and poly(ADPribose) polymerase (PARP) protein were activated in the IR and ZIR groups. The disruption of mitochondrial membrane potential was also observed in the ZIR group. In general, the downregulation of HO-1 reduced autophagy and activated the mitochondrial apoptotic pathway.