Exploratory Analysis of the Effects of Thymoquinone Alone and in Combination with 5-Fluorouracil on Inflammatory Mediators in Colorectal Cancer Cells: An In Vitro and In Silico Study

Colorectal cancer (CRC) remains one of the leading causes of cancer-related morbidity and mortality worldwide. Chronic inflammation plays a pivotal role in CRC development and progression, with cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), and interleukin-8 (IL-8) representing key mediators of tumor-promoting inflammation. This study aimed to evaluate the effects of thymoquinone (TQ), 5-fluorouracil (5-FU), and their combination on the expression of COX-2, IL-6, and IL-8 at both the mRNA and protein levels in colorectal cancer cell lines (SW1116 and RKO) and normal colon epithelial cells (CCD-841CoN). Although numerical changes in inflammation-associated mediator expression were observed following TQ monotherapy, none of the TQ-monotherapy comparisons versus the untreated control reached statistical significance after correction for multiple comparisons. Notably, combined TQ/5-FU treatment significantly reduced COX-2 protein levels in SW1116 cells compared with the untreated control. Differences between transcript and protein responses were observed across the investigated cell lines and treatment conditions. However, since both endpoints were assessed at a single time point, the mechanisms underlying these differences cannot be conclusively determined from the present data. Exploratory molecular docking analysis was additionally performed to examine potential direct interactions of TQ with COX-2, IL-6, and IL-8. The predicted docking scores ranged from −5.1 to −6.6 kcal/mol, with the most favorable predicted interaction observed for COX-2. These computational findings are hypothesis-generating and should be interpreted independently of the experimentally observed changes in target expression. Collectively, the present findings demonstrate treatment- and cell-line-dependent response patterns in inflammation-associated mediators, while providing no statistically significant evidence of an effect of TQ monotherapy versus the untreated control under the present experimental conditions.