Network-level reprogramming of cell death pathways in colorectal cancer cells by combined thymoquinone and 5-fluorouracil treatment.

Introduction: Colorectal cancer remains a leading cause of cancer-related mortality, with resistance to 5-fluorouracil (5-FU) posing a major therapeutic challenge. Thymoquinone (TQ), a bioactive compound derived from Methods: RKO colorectal cancer cells were treated with TQ, 5-FU, or their combination for 24 h, followed by genome-wide transcriptomic profiling using oligonucleotide microarrays. Drug interaction effects were assessed using a deviation-from-additivity model. Selected genes were validated by RT-qPCR and Western blotting, and functional relevance was evaluated using bioinformatics analyses. Results: Combined treatment induced extensive network-level reprogramming of pathways associated with apoptosis, cellular stress response, and proliferation. Although no classical transcriptional synergy was observed, the interaction between TQ and 5-FU resulted in coordinated modulation of overlapping signaling networks. Key regulatory genes, including Conclusion: TQ acts as a context-dependent modulator of chemotherapy response, reshaping cell death and stress-related signaling networks rather than directly enhancing cytotoxicity. These findings highlight the potential of TQ to influence therapeutic responses in fluoropyrimidine-based treatment of colorectal cancer and support further functional and