Abstract: OBJECTIVE： To investigate the role of autophagy in inducing ferroptosis in human colon cancer SW620 cells treated with 5-fluorouracil (5-FU) combined with oxaliplatin (L-OHP). METHODS： The CCK-8 assay was used to assess the effects of different concentrations of 5-FU and L-OHP acting alone on SW620 cell proliferation after 24 h. The IC₅₀ values for both drugs were calculated，and the IC₅₀ concentrations were selected for subsequent combination treatment. SW620 cells were then divided into eight groups： control， 5-FU，L-OHP，5-FU +L-OHP，autophagy inhibitor 3-methyladenine (3-MA)，3-MA+5-FU，3-MA+L-OHP， and the triple combination of 3-MA， 5-FU and L-OHP. Reagent kits were used to detect reactive oxygen species (ROS) and malondialdehyde (MDA) levels in each cell group. Western blot analysis was employed to assess the expression of ferroptosis-related proteins SLC7A11， GPX4， and ACSL4， as well as autophagy-related protein LC3B. Transmission electron microscopy was utilized to observe mitochondrial morphology. RESULTS： Compared with the control group， both 5-FU and L-OHP individually inhibited SW620 cell proliferation (P<0.01). The calculated IC₅₀ values for cell survival under single treatment with 5-FU and L-OHP were 2 971 and 185 μmol/L，respectively. Combined treatment at the IC₅₀ concentrations of both drugs further inhibited SW620 cell proliferation (P<0.01). Both single and combined treatments with 5-FU and L-OHP significantly increased ROS and MDA levels (P<0.01)，while decreasing the ferroptosis-related proteins SLC7A11 and GPX4 (P<0.01)， and increased ACSL4 expression (P<0.05). Following intervention with the autophagy inhibitor 3-MA， compared to the control group， the triple combination group exhibited decreased expression of ferroptosis-related proteins SLC7A11 and GPX4 (P<0.01) and increased ACSL4 expression (P<0.01). In the L-OHP-treated group and the 5-FU and L-OHP group，intracellular autophagy-related protein LC3B-II/I expression was upregulated (P<0.05 or P<0.01). Compared with the 5-FU and/or L-OHP groups，the triple-drug combination group exhibited further reduced cell viability (P<0.01) and significantly elevated MDA and ROS levels (P<0.01). In the 5-FU and/or L-OHP intervention groups supplemented with 3-MA，ACSL4 protein expression increased (P<0.05 or P<0.01)，while SLC7A11 and GPX4 proteins expression decreased (P<0.05 or P<0.01). Transmission electron microscopy revealed that 5-FU and/or L-OHP induced mitochondrial damage in SW620 cells，with more pronounced morphological alterations observed in the combination treatment group.CONCLUSION： 5-FU and L-OHP inhibited proliferation of SW620 colon cancer cells and induced ferroptosis. Co-treatment further enhanced the level of ferroptosis in cancer cells. Inhibition of autophagy potentiated the effect of combined 5-FU and L-OHP treatment.

Key words: colorectal cancer, autophagy, ferroptosis, oxaliplatin, 5-fluorouracil