Effect of methotrexate and 5-fluorouracil on de novo thymidylate synthesis in human colon carcinoma cell line, Caco-2

Although colon cancers respond poorly to chemotherapeutic agents, 5-fluorouracil (5-FU) is considered the most effective single agent in the treatment of advanced colon cancer, whereas methotrexate has been reported as an ineffective agent. Bur both 5-FU and methotrexate inhibit de novo thymidylate synthesis. IN the present study we assessed the sensitivity of folate-dependent thymidylate synthesis to methotrexate and 5-FU in Caco-2, a human colon carcinoma cell line. Sensitivity was assessed indirectly, by the deoxyuridine suppression test and directly, by the degree of inhibition of [H-3]deoxyuridine incorporation into deoxyribonucleic acid (DNA). Methotrexate or 5-fluorodexyuridine resulted in a significant decrease in suppression of [H-3]thymidine incorporation by exogenous deoxyuridine. [H-3]deoxyuridine incorporation was also inhibited by the two agents. Inhibition was dose dependent and 50% inhibition occurred at about 2.5 mu mol/L methotrexate and 25 mu mol/L 5-FU. In a second study, the effect of methotrexate and 5-FU on [H-3]deoxyuridine incorporation into DNA was assessed under conditions in which a Caco-2 cell monolayer was exposed to the agents either at the apical or at the basolateral membrane side. Under these conditions, inhibition was also dose dependent and cells were more sensitive to basolateral exposure to both methotrexate and 5-FU (P < 0.05). The data suggest that both methotrexate and 5-FU are effective inhibitors of thymidylate synthesis in Caco-2 cells. Determining the degree of inhibition of deoxyuridine incorporation into DNA is an effective method for evaluating these agents' effect on de novo thymidylate synthesis. Further studies are required to determine if these inhibitory effects also hold for colon cancer tissue biopsies and whether the reported differences in therapeutic efficacy between the two agents can be manifested in vitro.