Investigating the Radiosensitizing Effects of CF10 for the Treatment of Rectal Cancer

Abstract

Rectal cancer remains a major clinical challenge due to high recurrence rates, limited effectiveness of existing radiosensitizers, and significant treatment-related toxicities. The fluoropyrimidine 5-fluorouracil (5FU) is the current standard for chemoradiation but is constrained by incomplete efficacy and dose-limiting adverse effects. CF10 (AraC-FdUMP), an investigational polymeric fluoropyrimidine, exhibits dual inhibition of thymidylate synthase (TS) and topoisomerase I (TOP1), offering a novel mechanism for radiosensitization in colorectal cancer. This thesis systematically compares the radiosensitizing properties of CF10 and 5FU in colorectal cancer models. The specific objectives are to: Assess the effects of both agents, alone and in combination with ionizing radiation, on clonogenic survival; Evaluate their impact on cell cycle dynamics; Quantify DNA double-strand break (DSB) induction under various treatment regimens.Experiments were conducted in HCT116 and LS174T colorectal cancer cell lines. Clonogenic survival was evaluated via colony formation assays after graded doses of drugs and X-ray irradiation; cell cycle changes were measured by propidium iodide flow cytometry; and DSBs were assessed using γH2AX/PI staining and flow cytometry. Two-way ANOVA and post hoc multiple comparisons were used to analyze the data. Results demonstrate that ionizing radiation was the primary determinant of colony survival across both models; however, at higher concentrations, CF10 consistently exhibited greater cytotoxicity than 5FU. Evidence of radiosensitization was observed with high-dose CF10 in moderate and high seeding densities of HCT116 and at high density in LS174T. Mechanistically, CF10 induced rapid and sustained disruption of cell cycle organization, characterized by S-phase accumulation and elevated apoptosis with loss of G1-phase dominance, while 5FU rendered minimal effect on cell cycle dynamics. Importantly, CF10 combined with radiation led to significantly greater and longer-lasting DNA damage—confirmed by persistent γ-H2AX signal—than 5FU in both cell models, with the strongest effect in LS174T. Collectively, these findings establish CF10 as a more potent cytotoxic and radiosensitizing agent than 5FU in colorectal cancer cell lines. The enhanced DNA damage and cell cycle disruption mediated by CF10 suggest a synergistic mechanism with radiation, supporting further development of CF10 as a promising therapeutic partner in rectal cancer chemoradiation regimens.