Investigating the Role of Oxidative Stress in the Synergistic Interaction Between Linezolid and 5-Fluorouracil in Colorectal Cancer Cell Lines

Abstract

Background: Colorectal cancer (CRC) remains one of the leading causes of cancer-related mortality worldwide, with 5-fluorouracil (5-FU)-based chemotherapy representing the cornerstone of systemic treatment. However, resistance to 5-FU and dose-limiting toxicity necessitate the identification of novel combination strategies capable of enhancing efficacy while reducing chemotherapeutic dose. Drug repurposing has emerged as a cost-effective approach, with several antibiotics demonstrating the capacity to potentiate chemotherapy response. Linezolid (LIN), an oxazolidinone antibiotic, selectively inhibits mitochondrial protein synthesis through binding to the mitochondrial ribosome, impairing electron transport chain function and reducing expression of oxidative phosphorylation subunits. Preliminary data from Dr. Nahed El Najjar's lab, based on combination index analysis, demonstrated that LIN synergizes 5-FU's effect in HCT-116 and DLD-1 colorectal cancer cell lines at clinically relevant concentrations. Given LIN’s established capacity to impair mitochondrial function, a ROS-mediated mechanism is hypothesized to underlie the observed synergy. The present study was designed to directly investigate this hypothesis

Aims: This study aimed to investigate whether the enhanced anti-proliferative interaction between LIN and 5-FU in HCT-116 and DLD-1 CRC cells is mediated through ROS.

Materials and methods: HCT-116 and DLD-1 colorectal cancer cell lines were employed. Six treatment conditions were applied: untreated control (CTRL); 5-FU alone (8 µM for HCT-116; 16 µM for DLD-1); LIN at 2 µg/mL (LIN2); LIN at 10 µg/mL (LIN10); and the combinations 5-FU + LIN2 (K2) and 5-FU + LIN10 (K10). Intracellular ROS generation was quantified using the DCFH-DA flow cytometry assay. Metabolic activity was assessed using MTT assays at 48 and 72 hours with each condition tested with and without the ROS scavenger N-acetylcysteine (NAC; 4 mM). Combination effects were evaluated against the Bliss independence reference model, with the expected remaining metabolic activity calculated for each independent experiment and compared with the observed value. Statistical analysis was performed using Microsoft Excel (Microsoft 365) and IBM SPSS Statistics. Data was presented as mean ± standard error of the mean. A one-way or two-way ANOVA test was used to test for statistically relevant differences.

Results: DCFH-DA flow cytometry revealed that the combinations of LIN and 5-FU generated higher intracellular ROS levels than 5-FU alone in both cell lines, reaching 322.83% ± 81.48 (K2) and 448.44% ± 23.86 (K10) of untreated control in HCT-116 cells, and 306.21% ± 34.38 (K2) and 301.20% ± 20.79 (K10) in DLD-1 cells. In DLD-1 cells the combinations also generated significantly more ROS than LIN alone (K2 vs LIN2, p < 0.001; K10 vs LIN10, p < 0.05), whereas in HCT-116 cells this difference did not reach significance, reflecting the already high ROS-inducing capacity of LIN in this line (LIN10 alone, 411.06% ± 41.14). To determine whether ROS was contributing to the enhanced effect of the combinations, MTT assays were performed with and without NAC. At 48 hours, both combinations produced significantly greater inhibition of metabolic activity than 5-FU monotherapy in HCT-116 cells (K2, 52.82% vs 42.96%, p < 0.05; K10, 55.74% vs 42.96%, p = 0.001), and K2 did so in DLD-1 cells (68.67% vs 52.87%, p < 0.01). Co-treatment with NAC selectively attenuated the anti-metabolic effects of the combination groups but not of the individual drugs, reducing inhibition in HCT-116 cells from 52.82% to 37.24% for K2 and from 55.74% to 42.38% for K10 (both p < 0.0001), and in DLD-1 cells from 68.67% to 55.47% for K2 (p = 0.001), confirming that this combination-specific component is specifically ROS-dependent rather than reflecting a generalized cytotoxic effect. Formal evaluation against the Bliss independence model indicated that the combined effects were additive.

Conclusion: DCFH-DA flow cytometry confirmed additive intracellular ROS generation in cells treated with the LIN and 5-FU combinations. MTT assays demonstrated that pharmacological scavenging of this ROS with NAC selectively abolished the synergistic effect without affecting single-agent responses. Collectively, these findings establish that the synergistic interaction between LIN and 5-FU in CRC cells is mechanistically dependent on ROS generation. The comparable effect of the combination to double the 5-FU concentration supports the potential of LIN as a repurposed adjuvant to 5-FU and highlights a ROS-mediated mitochondrial mechanism as a promising target for combination strategies in CRC treatment.