Arenobufagin regulates the p62-Keap1-Nrf2 pathway to induce autophagy-dependent ferroptosis in HepG2 cells.

Hepatocellular carcinoma (HCC) is the most prevalent type of primary liver cancer, accounting for the overwhelming majority of malignant liver tumors. Therefore, how to effectively prevent and cure HCC has become a research hotspot. Many studies have shown that arenobufagin can induce apoptosis, ferroptosis, and autophagy of tumor cells. An increasing number of studies have shown that autophagy is closely linked to ferroptosis. In this study, HepG2 cells and BALB/c nude mice were used as research objects to explore the effect and preliminary mechanism of hepatoma cell autophagy and ferroptosis induced by arenobufagin. We found that arenobufagin can significantly inhibit tumor growth in vivo, and interestingly, we found that arenobufagin inhibited ferroptosis-related proteins Nrf2 and COX-2 in a dose-dependent manner and decreased the levels of reduced glutathione (GSH) and superoxide dismutase (T-SOD) in tissues, while increased the level of reduced malondialdehyde (MDA). In addition, we found that arenobufagin increased the levels of COX-2 and MDA in cells, decreased the levels of Nrf2, GSH, and T-SOD, increased the levels of tissue reactive oxygen species (ROS) and lipid ROS in a dose-dependent manner, and promoted ferroptosis in HepG2 cells. HepG2 cells were preprotected by autophagy inhibitor chloroquine (CQ) and ferroptosis inhibitor deferoxamine (DFO), and then treated with arenobufagin. It was found that CQ partially reversed the changes of COX-2 and Nrf2 expression and lipid peroxidation induced by arenobufagin-induced autophagy and HepG2 cells. Interestingly, CQ partially reversed the inhibition of arenobufagin on cytoplasmic junction protein (Keap1) and heme oxygenase-1 (HO-1) in p62-Keap1-Nrf2 pathway. At the same time, we found that the effect of arenobufagin on oxidative stress of HepG2 cells overexpressed by Nrf2 was significantly less than that of the control group. To sum up, arenobufagin promotes autophagy-dependent ferroptosis of HepG2 cells by inducing autophagy and regulating p62-Keap1-Nrf2 pathway. It is suggested that arenobufagin can be used as a potential intervention therapy.