Being ranked as the seventh most common cancer and the third lethal cause in cancer all over the world according to GLOBOCAN 2020, there were approximately 905,677 new liver cancer cases and 830,180 deaths annually (Sung et al., 2021). The main type of primary liver cancer was HCC, which comprises more than 80% of the cases (Yang et al., 2019). Chronic Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), alcohol abuse, diabetes and exposure to aflatoxin B1 are risk factors for HCC (Kulik and El-Serag, 2019). Besides, Surgical resection, liver transplantation, radio-embolization, radiofrequency ablation, chemotherapy, targeted therapy and immunotherapy are commonly used for HCC patients (Lee et al., 2020). However, the 5-year survival rate of HCC is 18% globally and even as low as 12% in China (Craig et al., 2020).

Endoplasmic reticulum (ER) is responsible for the synthesis, folding, processing, transportation of protein, the storage of Ca²⁺ (Oakes, 2020), and participate in lipid metabolism. The disorder of Ca²⁺ and accumulation of unfolded proteins in ER lumen, which is called ER stress (ERS), can be caused by reactive oxygen species (ROS), hypoxia and nutrient deprivation, as they could result in the disorder of cellular redox regulation (Kim and Kim, 2018). Unfolded protein response (UPR), mediated by inosito-requiring enzyme 1 (IRE1), protein kinase RNA-like ER kinase (PERK) and activating transcription factor 6 (ATF6), can alleviate ERS and maintain intracellular homeostasis (Marciniak, 2019). In addition, excessive and persistent ERS will lead to the binding of glucose-regulated protein 78 (GRP78) or binding protein (BIP) to the unfolded protein and promote the activation of IRE1, PERK and ATF6 pathways, thereby activating the pro-apoptotic pathway to induce cancer cell death (Chen and Cubillos-Ruiz, 2021; Eugene et al., 2020; Zachariah et al., 2021). Recently, a large number of studies have confirmed the role of ERS in nonalcoholic steatohepatitis, chronic liver disease and HCC (Hernández-Alvarez et al., 2019; Lebeaupin et al., 2018; Liu et al., 2019; Wu et al., 2021; Xia et al., 2020). Therefore, it is necessary to find effective ERS inducers for the treatment of HCC.

In recent years, an increasing number of researchers have realized that some natural compounds can kill cancer cells, and exploring the molecular mechanisms would also be interesting by which we can find out how these compounds work against tumors. Cantharis vesicatoria (blister beetles) has been used as a traditional Chinese folk medicine for more than 2000 years (Puerto Galvis et al., 2013). Cantharidin (CTD) is an anticancer active ingredient isolated from Cantharis vesicatoria. Current studies have reported that the anticancer mechanisms of cantharidin and its derivatives include inhibition of invasion and metastasis of tumor cells (Hsia et al., 2016), induction of apoptosis (Wang et al., 2018), regulation of cell cycle (Ye et al., 2017), induction of autophagy (Sun et al., 2017). Magnesium Demethylcantharidate (MDC) is a derivative of cantharidin with anti-HCC activity (Liu et al., 2021). Because there are two methyl groups less than cantharidin, the toxicity of MDC is greatly reduced, and its LD₅₀ is much higher than cantharidin (Li and Casida, 1992; Liu et al., 2021). But the mechanism of MDC remains to be further studied. In particular, whether MDC can cause ER stress of HCC were still unknown. In this study, we elucidated the anticancer mechanism of MDC in two HCC cell lines through ERS signaling pathway. Our study suggests that MDC is a potential drug for the treatment of HCC.

HCC is a serious threat to human health due to its high incidence and mortality (He et al., 2021; Piñero et al., 2020). In recent decades, many researchers found that certain natural products can kill HCC cells, and it would also be interesting to explore the anti-tumor molecular mechanisms. Cantharidin is one of natural products that can significantly inhibit the proliferation of HCC cells both in vitro and in vivo (Wang et al., 2000; Zhu et al., 2020). MDC is a novel agent derived from cantharidin, which can inhibit the invasion and metastasis of HCC cells by activating transcription factor FOXO1 (Liu et al., 2021). However, the mechanism of MDC needs further investigation. In particular, whether MDC can cause ER stress of HCC remains unknown.

It is known that apoptosis includes intrinsic and extrinsic pathways. The intrinsic pathway can be triggered by ERS, excess reactive oxygen species (Kesavardhana et al., 2020). ERS is the adaptive response of cells to external adverse stimuli. In the early stage, ERS can inhibit cell apoptosis, which is beneficial to cell growth. However, when ERS is too intense and persistent, it can induce apoptosis and therefore promote cell death (Fernández et al., 2015). In this study, we observed the elevation of Bax/Bcl-2 levels, and the caspase-9 and caspase-3 were cleaved after HCC cells were treated with MDC (Fig. 2B), which indicated that intrinsic apoptosis occurred in HCC cells. The increased expression of BIP was observed through western blot, leading to the phosphorylation of IRE1 and further proved that MDC induced apoptosis is generated through ERS signaling pathway (Fig. 3). Phosphorylated IRE1 can promote the synthesis of spliced X-box binding protein1 (XBP1s), and activate ASK1/JNK signal to activate C/EBP homologous protein (CHOP), Caspase12 and Bax (Liu et al., 2020). When CHOP and Caspase12 elevated, the expression of Bcl-2 is inhibited, then, cleaved Caspase-9 was released, and cleaved Caspase-3 was further released, inducing apoptosis (Meng et al., 2019).

Overall, we demonstrated that MDC could induce apoptosis of HCC cells through the ERS pathway. This finding suggest that MDC may be a potential anti-liver cancer drug.