The Role of Oxidative Stress in Ceftazidime-Induced Colorectal Cancer Cell Death

Abstract

Background: Colorectal cancer is the third most common cancer globally in terms of incidence and the second leading cause of cancer-related death. Despite advances in the current therapies, resistance, toxicities, and costs remain significant challenges. Drug repurposing has become a practical approach for identifying novel therapeutic applications for available drugs with verified and accepted safety profiles. Antibiotics have attracted attention for their diverse biological properties beyond antimicrobial activity. An in vivo and in vitro study conducted in Dr. El Najjar's lab found that CFT is an excellent candidate for repurposing. Among nine antibiotics tested against DLD-1 colon cancer cells, only CFT induced apoptosis in vitro and in vivo without being taken up by cells. Preliminary docking analysis also showed that CFT binds to Organic Anion Transporter 2 (OAT2) and Organic Cation Transporter 2 (OCTN2), which are, respectively, overexpressed by 30% and 60%. This data might imply that CFT binding to OAT2 and OCTN2 may induce a downstream signaling pathway leading to apoptosis. Moreover, conflicting data exists on the oxidant or antioxidant effect of CFT. Nevertheless, whether CFT induces apoptosis via ROS production, binding to OAT2/OCTN2 transporters, or both remains unclear. Given that many chemotherapies inhibit cell proliferation by inducing oxidative stress and considering the potential oxidative role of CFT[2, 3], the hypothesis is that CFT induces its inhibitory effects by inducing oxidative stress, binding to OAT2/OCTN2, or both.

Aim: This thesis explores oxidative stress induction and the roles of OCTN2 and OAT2 transporters in CFT-induced cell death in DLD-1, a colorectal cancer cell line.

Materials and methods: To determine the role of ROS in the CFT-inhibitory effect in CRC cells, cells were treated with 5-FU and CFT and analyzed using the DCFH-DA assay, MTT assay, and propidium iodide (PI) staining in the presence and absence of N-acetyl cysteine, a universal radical scavenger. Additionally, MTT assay and PI staining were used to assess the involvement of transporters in CFT’s effect by using indomethacin and meldonium inhibitors of OAT2 and OCTN2, respectively. Statistical analysis was performed using Microsoft Excel (Microsoft 365) and GraphPad Prism 8 (version 10.5.0; GraphPad Software Inc., La Jolla, CA, United States). 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: This study revealed that 5-FU and CFT induced strong oxidative stress, but CFT's effect was abolished by NAC pretreatment, whereas 5-FU’s persisted. This was confirmed by DCFH-DA, MTT, and PI, which revealed that the inhibitory effect of CFT on DLD-1 cells was ROS-dependent, whereas that of 5-FU was not. Additionally, 5-FU induced an S-phase arrest, followed by reductions in G0/G1 and G2/M phases, and an elevated pre-G0 phase, resulting in a significant distortion of the cell cycle. CFT, on the other hand, caused slight distortion of the cell cycle, with an S-phase arrest, mild reductions in G0/G1, and an elevated preG0. MTT assay and PI analysis in the presence of OCTN2 inhibitor, meldonium, and OAT2 inhibitor, indomethacin, revealed a complete reversal of CFT’s effect both at 48 and 72 hours, while 5-FU’s effect was not affected. This indicated that CFT’s effect was transporter-dependent, whereas 5-FU was not.

Conclusion: The findings from this study revealed that CFT and 5-FU exert their cytotoxic effects through different mechanisms. Most importantly, in DLD-1 cells, a cell line resistant to 5-FU, CFT inhibits cell growth via ROS generation and the involvement of OAT2/OCTN2. The contribution of OAT2 and OCTN2 to CFT’s inhibitory provides a therapeutic relevance because non-cancerous cells have lower expression of these transporters and are less sensitive to oxidative stress; therefore, this can reduce off-target toxicity. Finally, the data further confirms the potential of repurposing CFT as a new anticancer drug against CRC.