Folylpolyglutamate synthase and γ-glutamyl hydrolase regulate leucovorin-enhanced 5-fluorouracil anticancer activity

2815 5-Fluorouracil (5-FU) plus leucovorin (LV) is a standard chemotherapy regimen for colorectal cancer. 5-FU exerts its anticancer activity mainly by inhibiting thymidylate synthase (TS) via the formation of a ternary complex of TS, 5,10-methylenetetrahydrofolate (CH 2 THF) and 5-fluoro-29-deoxyuridine 59-monophosphate (FdUMP), which is the active form of 5-FU. Although LV enhances the antitumor activity of 5-FU by supplying CH 2 THF and stabilizing the ternary complex, the factors that determine the LV-mediated enhancement of the effect of 5-FU have remained unknown. We investigated the roles of folylpolyglutamate synthase (FPGS) and γ-glutamyl hydrolase (GGH), which are the main enzymes involved in folate metabolism, in the effect of LV in human colon cancer cell lines. 5-FU-based drug S-1 (6.9 mg/kg) and LV (20 mg/kg) were administered orally for 14 consecutive days to nude mice bearing human colon tumor xenografts COL-1 and KM20C. LV significantly enhanced the inhibitory effect of S-1 on COL-1 tumor growth ( P 2 THF and its direct precursor tetrahydrofolate (THF)) in both tumors ( P P P 3 H] folate level after the addition of [ 3 H] LV in cells treated with FPGS siRNA was reduced by 76% compared with cells treated with control siRNA. On the other hand, treatment with GGH siRNA tended to increase the [ 3 H] folate level compared with cells treated with control siRNA. The downregulation of FPGS by siRNA reduced the enhancement of FdUrd -induced cytotoxicity elicited by LV, whereas the downregulation of GGH by siRNA increased cellular sensitivity to FdUrd combined with LV. These results indicate that the expression of FPGS and GGH within tumor cells regulated the effectiveness of LV in enhancing the antitumor activity of 5-FU, by regulating folate levels. These results also suggest that the levels of FPGS and GGH expression in tumors could predict the effectiveness of LV in combination with 5-FU chemotherapy for colorectal cancer.