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

Abstract

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.

FHL1 regulates myoblast differentiation and autophagy through its interaction with LC3

“…Consistent with this finding, the levels of cleaved caspase‐3 and PARP were higher following FHL1 silencing, further indicating that autophagy was inhibited and contributes to myofiber degeneration and weakness. Similar results were reported by Xiong et al () in which Atg5 silencing increased the expression of cleaved caspase‐3, caspase‐9, and PARP, leading to the inhibition of autophagy and enhanced apoptosis in hepatocellular carcinoma cells. Autophagy is a complex regulatory process.…”

Regulation of autophagy by Ca2+

“…The inhibition of autophagy in HepG2 cells has pro-apoptotic effects, partly through enhanced ROS generation and activation of the mitochondrial apoptotic pathway, although the exact function of autophagy in apoptosis remains to be studied further [87]. Recent reports revealed that oxidative stress-triggered autophagic cell death is independent of apoptosis [87], suggesting that the processes of apoptosis and pro-survival autophagy are interrelated in a complicated manner or occur independently of each other. Therefore, the interplay between autophagy and apoptosis is controversial, and the scenario of autophagy in deciding cell destiny is elusive.…”

Cantharidin suppresses cell growth and migration, and activates autophagy in human non-small cell lung cancer cells

“…It has been reported that Cantharidin (CTD), an active chemical compound isolated from the blister beetle (Coleoptera: Meloidae), serves a notable role in promoting autophagy and suppressing hepatocellular carcinoma (13). Consequently, one aim of the present study was to investigate the association between CTD and autophagy.…”