Cinobufagin treatments suppress tumor growth by enhancing the expression of cuproptosis-related genes in liver cancer

Cuproptosis is a recently discovered form of regulated cell death triggered by excess copper (Cu) strongly influenced by the import, export, and intracellular utilization of Cu known as Cu homeostasis. Cinobufagin (CB) is a well-known Chinese medicine for its apoptosis-inducing role; however, its function on cuproptosis is poorly understood. To evaluate the effect of CB on inducing cell death through cuproptosis, we used RNA-seq data of HepG2-treated cells with CB to understand Cuproptosis genes. By using CCK-8 assay, Ross assay, GSH assay, and qRT-PCR, we found that CB could enhance cuproptosis in primary liver cancer cell lines, especially by increasing copper transporters CTR1, CTR2, and LIAS and downregulation of copper efflux transporters ATP7A and ATP7B resulted in increased reactive oxygen species (ROS) production, copper ionophores while reduced intracellular copper chelator glutathione (GSH) synthesis. In conclusion, our findings indicated that CB by increasing cuproptosis-related genes can mediate higher cell cytotoxicity against HepG2 and HUH7 and could provide a new insight into mechanisms of CB as an anti-tumor agent for targeting liver cancer.