Combination of 5‐Fluorouracil and Thymoquinone for Enhanced Cytotoxicity, Genotoxicity and Apoptosis In Colorectal Cancer: In Vitro and In Vivo Studies

Research on the effects of herbal-derived natural active substances on cancer treatment and their combination with conventional treatments has intensified. This study analyzed the cytotoxic, genotoxic, apoptotic, and anticancer effects of combined treatment with 5-Fluorouracil (5-FU) and thymoquinone (TQ) on colorectal cancer. Cytotoxicity was evaluated using the ATP assay, DNA damage was assessed through the comet assay, apoptosis was measured via acridine orange/ethidium bromide staining and annexin V-FITC dye, and the expression of proapoptotic and antiapoptotic proteins was determined by western blot analysis. Transfected LoVo cells were injected subcutaneously into nude mice, and following treatment, oxidative stress and inflammation markers were examined in blood samples, while growth factors and vascularization markers were analyzed in tissue samples. The combination therapy at low concentrations resulted in increased cytotoxicity, DNA damage, apoptosis, and intracellular reactive oxygen species (p < 0.001), while simultaneously decreasing mitochondrial membrane potential and glutathione levels (p < 0.001), in comparison to monotherapy with TQ or 5-FU. Additionally, tissue levels of TGF-β1 and VEGF-α were significantly reduced (p < 0.001). Results demonstrates that while TQ or 5-FU alone have notable anticancer effects, their combination offers greater efficacy in mitigating molecular changes in both In Vitro and In Vivo models. Future studies should focus on optimizing the formulation, understanding the molecular mechanisms, and evaluating the efficacy and safety of the TQ and 5-FU combination across different cancer types.