Bakuchiol Enhances 5-Fluorouracil Efficacy in Colorectal Cancer Cells via a ROS-Dependent Mechanism Involving Mitochondrial Dysfunction and Apoptosis

Resistance to 5-fluorouracil (5-FU) remains a major limitation in colorectal cancer therapy, prompting the development of combination strategies aimed at improving its efficacy. Bakuchiol (BAK), a natural compound with reported antioxidant and pro-oxidant properties, may modulate redox balance and enhance chemotherapy response. This study compared the effects of 5-FU and BAK, applied as monotherapies and in combination, in DLD-1 and HT-29 colorectal cancer cells. Cytotoxicity assays showed that co-treatment significantly reduced the IC50 of 5-FU, particularly in DLD-1 cells, and revealed an enhanced anticancer effect of the combination treatment compared with either monotherapy. Flow cytometric analyses demonstrated enhanced apoptosis via extrinsic and intrinsic pathways, including increased caspase 8 activity, loss of mitochondrial membrane potential (ΔΨm), activation of caspase 9, and subsequent activation of caspases 3/7. These effects were associated with a pronounced redox imbalance, reflected by increased intracellular reactive oxygen species (ROS) levels, suggesting a central role of oxidative stress in mediating cytotoxicity. Antioxidant pre-treatment attenuated ROS accumulation and reduced apoptosis, confirming a causal relationship. Additionally, autophagy was induced selectively in DLD-1 cells, indicating cell-line-specific differences in redox adaptation. Taken together, BAK enhances 5-FU efficacy through ROS-dependent activation of mitochondrial and caspase-dependent pathways, with stronger effects observed in DLD-1 cells.