Sodium Butyrate Enhances 5-Fluorouracil Sensitivity Through Thymidylate Synthase Regulation and Cell-Cycle Modulation in Gastric Cancer.

5-Fluorouracil (5-FU) is a first-line chemotherapy for gastric cancer (GC), but its efficacy is often limited by acquired resistance mediated by upregulation of thymidylate synthase (TS). Sodium butyrate (NaB), a microbiota-derived short-chain fatty acid (SCFA), has shown potential in colorectal cancer, but its role in overcoming 5-FU resistance in GC remains undefined. Therefore, we investigated the molecular mechanisms of NaB in GC cell lines (AGS and KATO-III) by evaluating cell viability, migration, apoptosis, mitochondrial membrane potential, cell cycle, and gene expressions related to DNA synthesis, cell cycle, and cellular stress. NaB decreased cell viability, impaired cellular aggregation, and induced apoptosis and mitochondrial dysfunction. Furthermore, NaB synergized with 5-FU, enhancing growth inhibition and apoptotic effects. Notably, NaB significantly downregulated TS expression at both mRNA and protein levels and also reduced gene expression such as TK1 and FOXM1. Cell-cycle analysis indicated distinct responses between AGS (p53-wild-type) and KATO-III (p53-null) cells. In conclusion, as a fermentation product of dietary fiber, NaB represents an accessible nutritional adjuvant. Dietary or probiotic strategies to boost systemic butyrate levels could be combined with standard chemotherapy to improve response rates, mitigate resistance, and support patient outcomes in GC.