Synergistic effect of 5-fluorouracil and the small molecule Wnt/β-catenin inhibitor iCRT3 on Caco-2 colorectal cancer cells in vitro

<h2>Abstract</h2> Although 5-fluorouracil (5-FU) is a cornerstone of colorectal cancer (CRC) treatment, its efficacy is often limited by resistance. Wnt/β-catenin signalling plays a crucial role in CRC carcinogenesis and resistance, as Wnt expression is upregulated in 5-FU-resistant cells, protecting them from cell cycle arrest and apoptosis, thereby contributing to drug resistance. The small molecule inhibitor β-catenin responsive transcription inhibitor 3 (iCRT3) disrupts Wnt/β-catenin signalling and may enhance CRC sensitivity to 5-FU, overcoming resistance. In this study, the cytotoxic effects of 5-FU and iCRT3 were investigated using the Caco-2 colon adenocarcinoma cell line, marking the first investigation of their combined effects. To this end, the half-maximal inhibitory concentration (IC₅₀) values were determined using the MTT assay. Subsequently, the drugs were combined in different ways, and drug combination index (DCI) calculations were performed to evaluate their interaction. iCRT3 was found to be 2.45-fold more potent than 5-FU (p = 0.1982). Drug combination significantly increased the IC₅₀ compared to 5-FU, with a 40.95-fold increase (p = 0.0022) when 5-FU was fixed (2.56 μM) and a 43.5-fold increase (p = 0.0023) when iCRT3 was fixed (2.41 μM). Two-way ANOVA showed significant impacts from both drug concentration (50.93 %) and treatment condition (25.31 %) on cell viability (p < 0.0001). DCI analysis confirmed strong synergism with fixed 5-FU (DCI = 0.154) and synergism with fixed iCRT3 (DCI = 0.618), indicating that combining 5-FU and iCRT3 could be a promising strategy for CRC treatment, warranting further investigation.