5-Fluorouracil (5-FU) is a cornerstone chemotherapeutic agent for gastric cancer (GC), but its clinical efficacy is often limited by suboptimal antitumor activity and dose-related toxic side effects in patients. Combining natural compounds such as β-ionone (BI) with 5-FU has emerged as a promising strategy to enhance the therapeutic efficacy of GC chemotherapy while reducing the required dosage of 5-FU and mitigating its systemic toxicity. While BI exhibits anti-tumor activities, its synergy with 5-FU and the underlying mechanism are still unclear. This study was investigated the synergistic anti-tumor effects of BI and 5-FU in GC, focusing on the role of PAX6-mediated cell cycle arrest.

The effects of BI and 5-FU, alone or combined, on human GC cells (MKN45 cells and AGS cells) were assessed in vitro (MTT/MB assays, EdU flow cytometry, cell cycle analysis, spheroid formation, Western blot, immunofluorescence, co-immunoprecipitation, siRNA, and GSK-3β inhibitor - CHIR-99021) and in vivo (MKN45 cell xenografts in a BALB/c nude mouse model, tumor growth, HE/TUNEL staining, IHC, Western blot).

BI significantly synergized with 5-FU to suppress the GC cell viability, DNA synthesis, tumor spheroid formation, and stemness. Their combination significantly induced the cell cycle arrest and significantly downregulated the expression of proliferating cell nuclear antigen (PCNA), cell cycle proteins, transcription factor PAX6, and GSK-3β proteins in MKN45 cells. Interaction between PAX6 and GSK-3β was confirmed by Co-IP. A GSK-3β inhibitor - CHIR-99,021 also altered the cell cycle distribution, recovrying effects of BI with 5-FU in MKN45 cells. In addition, BI combined with 5-FU potently inhibited MKN45 cell xenograft growth, increased apoptosis, and decreased the expression of PCNA, PAX6, and GSK-3β in MKN45 cell xenografts.

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.