Rafoxanide disrupts mitochondrial homeostasis through VDAC1 modulation in colorectal cancer cells.

Functional mitochondria are essential for cancer cells, as they sustain oxidative phosphorylation, metabolic flexibility and survival. Targeting mitochondrial homeostasis has therefore emerged as a promising strategy to sensitize cancer cells to cell death. Rafoxanide is a halogenated salicylanilide originally developed as a veterinary anthelmintic and described to exert mitochondrial uncoupling activity in parasitic organisms. Although rafoxanide has been shown to exert potent antitumor activity against colorectal cancer (CRC), the mechanisms underlying this effect remain incompletely understood. Here, we investigated the impact of rafoxanide on mitochondrial function and stress responses in CRC cells. Rafoxanide rapidly impaired mitochondrial respiration, reducing basal and maximal oxygen consumption and ATP-related respiration, and induced a progressive but reversible dissipation of mitochondrial membrane potential. Integrated transcriptomic, proteomic, and metabolomic analyses revealed that prolonged rafoxanide exposure resulted in sustained mitochondrial dysfunction, failure of metabolic adaptation, and release of cytochrome c from the mitochondria into the cytosol. Mechanistically, rafoxanide inhibited mitochondrial respiratory chain complexes I and III, leading to a rapid increase in total cellular reactive oxygen species. This redox imbalance promoted voltage-dependent anion channel (VDAC1) oligomerization and mitochondrial outer membrane permeabilization. Notably, mitochondrial superoxide production was reduced at later time points, consistent with the loss of mitochondrial membrane potential rather than the absence of a cellular oxidative stress response. Finally, proteomic analysis of colonic lesions from a murine model of sporadic CRC, as well as human CRC explants and intestinal organoids, confirmed that rafoxanide consistently alters mitochondrial protein expression and function across in vitro, in vivo, and ex vivo systems. In conclusion, our results identify rafoxanide as a modulator of mitochondrial homeostasis that induces redox-dependent VDAC1 activation and progressive mitochondrial dysfunction in CRC cells, providing mechanistic insight into its antitumor activity and supporting further exploration of mitochondrial stress modulation as a therapeutic strategy in CRC.