Abstract

Tumor cells are more sensitive to methionine restriction than normal tissues, a phenomenon known as methionine auxotrophy. Previous studies showed that 5-fluorouracil and methionine restriction act synergistically against a variety of tumors. The purpose of the current studies was to determine the molecular mechanism(s) underlying this synergy. 5-Fluorouracil is known to inhibit thymidylate synthase (TS), a key enzyme that transfers a methyl group from 5,10-methylene-tetrahydrofolate to dUMP during nucleotide biosynthesis. We found that methionine restriction reduced 5,10-methylene-tetrahydrofolate levels by 75% and selectively inhibited TS activity in PC-3 human prostate cancer cells within 24hr, whereas it did not in normal prostate epithelial cells. The observed fall in TS activity was accompanied by a commensurate reduction in TS protein levels as determined by western blot analysis. In contrast, 5-fluorouracil inhibited TS activity by >90% but increased TS protein levels. This increase was abrogated by methionine restriction. Surprisingly, methionine restriction increased 3H-leucine incorporation in PC-3 cells over the first 24hr, suggesting that reduction of TS levels was not simply due to global protein synthesis inhibition. Methionine restriction also significantly reduced the ratio of dUMP to dTTP in PC-3 cells, creating an imbalanced nucleotide pool. These results suggest that synergy between methionine restriction and 5-fluorouracil is attributable to multiple factors, including depletion of reduced folates, selective inhibition of TS, and creation of an imbalanced nucleotide pool. Dietary and/or enzymatic methionine restriction combined with 5-fluoruracil has great promise as a novel treatment for advanced cancer.