β-Ionone Synergizes with 5-Fluorouracil to Inhibit Gastric Cancer Progression through PAX6-Mediated Cell Cycle Arrest

Aims Overcoming 5-fluorouracil (5-FU) resistance remains a challenge in gastric cancer (GC) therapy. Combining natural compounds like β-ionone (BI) with 5-FU is a promising strategy. While BI exhibits anti-tumor activity, its synergy with 5-FU and the underlying mechanism are unclear. This study investigated the synergistic anti-tumor effects of BI and 5-FU in GC, focusing on the role of PAX6-mediated cell cycle arrest. Methods The effects of BI and 5-FU, alone or combined, on human GC cells (MKN45, AGS) were assessed in vitro (MTT/MB assays, EdU flow cytometry, cell cycle analysis, spheroid formation, Western blot, immunofluorescence, co-immunoprecipitation, siRNA, GSK-3β inhibitor CHIR-99021) and in vivo (MKN45 xenografts in BALB/c nude mice; tumor growth, HE/TUNEL staining, IHC, Western blot). Results BI synergized with 5-FU to suppress GC cell viability, DNA synthesis, tumor spheroid formation, and stemness. The combination induced cell cycle arrest and significantly downregulated proliferating cell nuclear antigen (PCNA), cell cycle proteins, transcription factor PAX6, and GSK-3β. Interaction between PAX6 and GSK-3β was confirmed. The GSK-3β inhibitor CHIR-99021 altered cell cycle distribution, supporting the pathway's role. In vivo, the BI/5-FU combination potently inhibited xenograft tumor growth, increased apoptosis, and decreased PCNA, PAX6, and GSK-3β expression. Conclusions BI significantly enhances the efficacy of 5-FU against gastric cancer. This synergy is mechanistically linked to the inhibition of the transcription factor PAX6 and subsequent modulation of GSK-3β, leading to cell cycle arrest and inhibition of tumor progression.