Cantharidic acid causes mitochondrial dysfunction via the Nrf2/HO-1/GPX4 pathway to inhibit colorectal cancer progression.

BACKGROUND: Cantharidic acid (CA) is a cantharidin analog and has antitumor effects. This study aimed to investigate the antitumor activity of CA against colorectal cancer (CRC) and the underlying mechanisms of this activity. METHODS: The impact of CA on the viability of the normal FHC cell line was evaluated by the CCK-8 assay. The malignant behavior of CRC cells was determined using the CCK-8 assay, colony formation assay, Transwell assay, and an LDH commercial kit. The surviving and apoptotic cell numbers were examined via flow cytometry and calcein-AM/PI staining. The ultrastructure of the mitochondria was observed, and the concentration of mtDNA was detected via RT-qPCR. A subcutaneous xenograft tumor model in nude mice was established, and pathological staining was used to assess apoptosis and changes in protein expression. Western blotting was used to evaluate the levels of nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1)/glutathione peroxidase 4 (GPX4) pathway-related proteins and mitochondrial damage-related proteins. RESULTS: CA did not significantly affect FHC viability but was able to reduce CRC cell viability, decrease colony-forming ability, inhibit migration and invasion, and induce apoptosis. CA treatment disrupted mitochondrial morphology and structure and caused a decrease in mitochondrial membrane potential, ATP production, and mtDNA concentration. Treatment with Mito-TEMPO (a mitochondrion-targeted antioxidant) reversed the effects of CA treatment on the above metrics. Furthermore, CA blocked the Nrf2/HO-1/GPX4 pathway, whereas the Nrf2 agonist TBHQ alleviated CA-induced mitochondrial dysfunction. CA treatment decreased the volume and mass of tumor tissue, inhibited cell proliferation, and promoted apoptosis. Notably, CA also led to mitochondrial dysfunction in vivo, which was effectively mitigated by TBHQ. CONCLUSION: CA blocks the Nrf2/HO-1/GPX4 pathway, causing mitochondrial dysfunction and apoptosis, and thus inhibits the malignant progression of CRC. CA has potential as a therapeutic agent for CRC.