Immunomodulatory effect of betulin and its derivatives on IL-6 expression in colorectal cancer cell lines and molecular docking insights.

Purpose: This study investigated the immunomodulatory and therapeutic potential of betulin and its derivatives (EB5 and ECH147) in colorectal cancer (CRC), focusing on their effects on IL-6 expression at the molecular level and their possible application as diagnostic and therapeutic tools. Methods: Human CRC cell lines (HT-29, RKO, SW1116) and normal colonocytes (CCD-841CoN) were treated with betulin, EB5, ECH147, cisplatin, and 5-fluorouracil (10 μg/mL) for 2, 8, and 24 h. IL-6 mRNA levels were measured by RT-qPCR in real time, and IL-6 protein was quantified using a proximity ligation immunoassay. Molecular docking was performed using IL-6 structure (PDB ID: 1ALU). Statistical significance was evaluated using Kruskal-Wallis and post hoc rank tests. Results: IL-6 expression was undetectable in HT-29 and RKO cells, both harboring the BRAF V600E mutation. ECH147 and EB5 derivatives significantly decreased IL-6 mRNA and protein levels in SW1116 and CCD-841CoN cells at 8 and 24 h. Molecular docking analysis revealed that ECH147 formed a stable hydrogen bond, suggesting direct binding. Conclusion: Structural modification of betulin enhances its molecular therapeutic activity, with phosphonate derivative ECH147 showing the strongest decrease in IL-6 levels. These findings suggest that IL-6 downregulation can serve as a molecular biomarker for drug efficacy, while ECH147 represents a promising candidate for targeted molecular therapy in CRC. This dual diagnostic-therapeutic approach highlights the potential of betulin derivatives in advancing precision medicine for IL-6-mediated pathways.