Abstract 4659: Folic acid-metabolizing enzymes regulate 5-fluorouracil anticancer activity on colorectal cancer cell lines

Abstract

Abstract Background: In colorectal cancer chemotherapy, the current standard of care includes 5-fluorouracil (5-FU) and leucovorin (LV) combination therapy. To understand the role of folic acid-metabolizing enzymes in the LV-mediated enhancement of 5-FU's antitumor activity, we suppressed the expression of genes related to folic acid metabolism and evaluated the resultant effect on the proliferation inhibitory effect of 5-fluoro-2′-deoxyuridine (FdUrd). Method: We downregulated the expression of thymidylate synthase (TS), folate receptor 1 (FOLR1), methylenetetrahydrofolate reductase (MTHFR), methylenetetrahydrofolate dehydrogenase (MTHFD), dihydrofolate reductase (DHFR), and phosphoribosylglycinamide formyltransferase (GART) by lipofection of small interfering RNA (siRNA) in DLD-1 and HCT116 cells, and calculated the relative change in IC50 values on 48 hr exposure to FdUrd in comparison with the corresponding IC50 values in control siRNA-treated cells. In cells with TS downregulation, LV-mediated enhancement of sensitivity to FdUrd was also evaluated. Sensitivity to FdUrd was determined using the crystal violet staining method. Results: Treatment with siRNA downregulated the expression of folic acid-metabolizing enzymes. LV enhanced the sensitivity to FdUrd in control siRNA-treated cells in a dose-dependent manner. However, in cells with TS downregulation, LV did not enhance the sensitivity to FdUrd. Downregulation of folic acid-metabolizing enzymes, except TS, decreased the efficacy of FdUrd, although there was little decrease in FdUrd efficacy owing to MTHFR downregulation. Conclusion: The level of TS expression determined the LV requirement, and downregulation of folic acid-metabolizing enzymes, which impaired the folic acid cycle, decreased the efficacy of 5-FU. Comparison of the IC50 values in target siRNA-treated cells and control siRNA-treated cells.DLD-1 cellsHCT116 cellsexpression (%)*IC50 (μM)fold changeexpression (%)*IC50 (μM)fold changeNon-silencing Control siRNA100.000.671.00100.000.251.00TS10.580.010.0114.840.030.12FOLR19.148.6512.9125.122.5910.36MTHFR25.441.432.1348.750.883.52MTHFD9.662.734.0715.061.997.96DHFR8.524.146.1817.231.706.80GART18.905.388.037.102.469.84Values are means (n = 3). *: Expression (%) is the expression at the beginning of FdUrd treatment (at 48 hr after siRNA treatment). Citation Format: Hiroshi Tsukihara, Kenta Tsunekuni, Teiji Takechi. Folic acid-metabolizing enzymes regulate 5-fluorouracil anticancer activity on colorectal cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4659.