Overcoming Acquired 5-FU Resistance in Gastric Cancer: Oleuropein Targets Proliferation, Migration and Survival Pathways

5-Fluorouracil (5-FU) remains a cornerstone in gastric cancer (GC) treatment; however, chemoresistance limits its efficacy. Oleuropein (OL), a phenolic compound in olive leaf extract, has emerged as a low-toxicity candidate with antitumor-potential. A 5-FU-resistant AGS subline (AGS-FUR) was established via stepwise drug exposure. The effects of OL-only or in combination with 5-FU, on proliferation (xCELLigence), apoptosis (Annexin V/PI), ROS, colony formation, migration, and EMT/CSC gene expression and proteomic analyses were assessed in AGS and AGS-FUR cells. OL exhibited dose- and time-dependent anti-proliferative effects, with IC50 values of 73.86µM in AGS cells and 94.58µM in AGS-FUR cells (p<0.0001). Combination therapy showed additive efficacy, significantly increasing apoptosis (p<0.0001) and partially reversing resistance-associated survival. Notably, OL reduced ROS even in AGS-FUR cells but retained proapoptotic effects, suggesting a redox-independent mechanism. In resistant cells unresponsive to 5-FU, combined treatment suppressed colony formation 22.2-fold (p<0.0001). OL also downregulated the CSC markers CD133 and NANOG (p<0.05) and inhibited migration/EMT by reducing wound closure and repressing the effects of 5-FU alone on the expression of CDH2 (22.2-fold; p<0.0001) and ZEB1 (3.83-fold; p<0.05). Proteomic profiling identified 3.392 proteins, of which 232 were highly confidently differentially expressed. Resistance is associated with alterations in chromosome organization, mitochondrial function, and RNA processing. Strikingly, OL suppressed protein levels in AGS cells but upregulated them in AGS-FUR cells, indicating opposing cell line-specific regulation. Overall, OL enhances 5-FU efficacy by impairing proliferation, stemness, and invasion while modulating EMT and apoptosis, underscoring its potential as an adjuvant for overcoming GC chemoresistance.