BAM15 Enhances Regorafenib Cytotoxicity in a Canine Osteosarcoma Cell Model

Osteosarcoma is an aggressive primary bone malignancy affecting both humans and dogs, and canine osteosarcoma represents a valuable comparative model for investigating novel therapeutic strategies. Targeting tumour metabolism may enhance the efficacy of existing anticancer treatments. This study investigated whether the mitochondrial uncoupler BAM15 modifies the response of canine D17 osteosarcoma cells to regorafenib. Cells were treated with BAM15 (20 µM) or regorafenib (REG; IC₂₅, 8 µM; IC₅₀, 16 µM) alone or pretreated with BAM15 before regorafenib exposure. Treatment effects were assessed using viability assays, Annexin V/propidium iodide staining, cell-cycle analysis, Seahorse XF bioenergetic profiling, quantitative PCR, and Western blotting. BAM15 alone did not significantly affect cell metabolic activity. Combined treatment with BAM15 and regorafenib produced a significantly greater reduction in cell metabolic activity than regorafenib alone at IC₅₀, whereas no significant difference between treatments was observed at IC₂₅. Combined treatment at IC₅₀ increased late apoptotic and necrotic populations and disrupted cell-cycle distribution. BAM15 reduced oxidative phosphorylation-derived ATP production without detectable glycolytic compensation but did not further suppress mitochondrial ATP production beyond regorafenib effect alone. Combined treatment also induced transcriptional changes in stress-adaptation-related genes, while protein analysis revealed reduced BAX and BAX/BCL-2 ratio. These exploratory findings provide preliminary evidence that mitochondrial uncoupling by BAM15 may modify the response to regorafenib and support further mechanistic investigation and validation in additional tumour and non-malignant canine models.