Abstract
BACKGROUND: Methyl-cantharidimide (MCA), a cantharidin (CTD) analog, has been approved in China for the clinical treatment of liver cancer. Previous studies have shown that MCA can induce both apoptosis and cell cycle arrest in cancer cells. However, the precise molecular mechanisms underlying its anticancer activity remain largely unclear. METHODS: The effects of MCA were evaluated in liver cancer cell lines using colony formation assays. Bioinformatic analysis was performed to explore molecular mechanisms, with validation via immunoblotting and immunofluorescence. Reactive oxygen species (ROS) production was measured to assess oxidative stress induction. A 3D multicellular tumor spheroid (MCTS) model was used to mimic tumor-like in vitro conditions, and a HepG2 xenograft mouse model was employed to investigate in vivo efficacy. RESULTS: MCA significantly inhibited colony formation in liver cancer cells. Bioinformatic profiling suggested that downregulation of cyclin-dependent kinase 1 (CDK1) may mediate MCA-induced cell cycle arrest, which was confirmed by suppressed CDK1 expression in both immunoblotting and immunofluorescence assays. MCA also increased intracellular ROS levels. Further analyses revealed that MCA-induced apoptosis occurred through a ROS-dependent oxeiptosis pathway, rather than the conventional caspase-dependent route. In the MCTS model, MCA maintained its cytotoxicity, demonstrating efficacy under more physiologically relevant conditions. In the in vivo xenograft mouse model, MCA treatment led to a significant reduction in tumor size. CONCLUSIONS: MCA exerts anticancer effects in liver cancer cells by inducing cell cycle arrest via CDK1 downregulation and promoting apoptosis through a ROS-dependent oxeiptosis pathway. These findings provide mechanistic insight into MCA’s antitumor activity and support its potential as a therapeutic agent for liver cancer. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13046-026-03669-8.