Correlation between augmentative effects of leucovorin on 5-fluorouracil and thymidylate synthase inhibition

Background We studied the relationship between the augmentative effects of leucovorin (LV) on 5-fluorouracil (5-FU) and the inhibition rate of thymidylate synthase (TS) activity in Colon 26 murine colon carcinoma cells and L1210 murine leukemia cells. Methods In cytotoxic and TS inhibition studies, cells were exposed for 24 hours to varied concentrations of 5-FU alone or in combination with 1 or 10 μmol/L LV. Cytotoxicity was determined by colony forming efficiency or trypan blue dye exclusion, and TS inhibition rate was determined by [6-³H]-5-FdUMP binding assay. A growth inhibition curve was constructed for longer exposures. Results For tumor cell growth inhibition and cytotoxicity, the augmentative effect was observed when the lower 5-FU concentrations (0.1 μg/mL) were used. There was no difference in the effects between the low (1 μmol/L) and high (10 μmol/L) LV doses. Normally, TS enzyme levels rise acutely when cells are exposed to 5-FU. Both cell lines displayed an increase in TS after exposure to 5-FU. This exposure to 5-FU resulted in the maintenance of free enzyme. LV was able to increase the ternary complex and TS inhibition rate with little induction of TS, however, the inhibition rate was not dependent on doses of LV. Conclusions It was concluded that the augmentative effect of LV at a concentration of 0.1 μg/mL 5-FU occurred at the clinically achievable levels of 1 μmol/L of LV, and there was no difference in the effect between the low and high doses. TS was inhibited effectively by 5-FU and the addition of LV, without a marked induction of TS.