Effects of Diastereoisomers of 5-Formyltetrahydrofolate on Cellular Growth, Sensitivity to 5-Fluoro-2′-deoxyuridine, and Methylenetetrahydrofolate Polyglutamate Levels in HCT-8 Cells

Abstract

We investigated the biological activities of the natural and unnatural diastereoisomers of 5-formyltetrahydrofolate [(6S)- and (6R)-5-HCO-H₄PteGlu, respectively, both 99.99% pure], using a human ileocecal carcinoma cell line (HCT-8). Optimal cell growth could be supported by (6S)-5-HCO-H₄PteGlu at concentrations as low as 1 nm. (6R)-5-HCO-H₄PteGlu did not support growth. Modulation of the in vitro cytotoxicity of 5-fluoro-2′-deoxyuridine (FdUrd) and intracellular (6R)-5,10-methylenetetrahydrofolates [(6R)-CH₂H₄PteGlu_n] pools by (6S)- and (6R)-5-HCO-H₄PteGlu was determined with cells growing in 1 nm (6S)-5-HCO-H₄PteGlu. For the control cells, the concentration of FdUrd inhibiting growth by 50% was 179 nm and the total (6R)-CH₂H₄PteGlu_n was 2.3 pmol/10⁶ cells. When cells were treated with (6S)-5-HCO-H₄PteGlu for 24 h, the 50% inhibition concentration of FdUrd decreased with increasing concentrations of (6S)-5-HCO-H₄PteGlu, and reached a plateau of 36 nm when (6S)-5-HCO-H₄PteGlu was ≥1 µm. The total (6R)-CH₂H₄PteGlu_n pools were augmented by (6S)-5-HCO-H₄PteGlu dose dependently up to 6.8 pmol/10⁶ cells at 1 µm (6S)-5-HCO-H₄PteGlu. (6S)-5-HCO-H₄PteGlu at 10 µm did not further increase the total (6R)-CH₂H₄PteGlu_n, but induced a marked shift in the polyglutamate chain length distribution, with an increase in tri- and tetra-, and a decrease in penta-, hexa-, and heptaglutamate. The down-shift of (6R)-CH₂H₄-PteGlu_n polyglutamate chain length observed after (6S)-5-HCO-H₄PteGlu treatment did not impair the modulation of FdUrd cytotoxicity. Thus shorter chain (6R)-CH₂H₄PteGlu_n (n = 3–4) function as well as longer ones (n = 5–7). (6R)-5-HCO-H₄PteGlu, at 200 µm, had no effect on the cytotoxicity of FdUrd, the total (6R)-CH₂H₄PteGlu_n level, or chain length distribution in the presence or absence of additional (6S)-5-HCO-H₄PteGlu. These results suggest that the high plasma (6R)-5-HCO-H₄PteGlu concentrations (up to 200 µm) achieved in patients following i.v. administration of high doses of (6R,S)-5-HCO-H₄PteGlu probably do not have adverse effects on the modulation of antitumor activity of FdUrd or 5-fluorouracil. Since the optimal dose and schedule of (6S)-5-HCO-H₄PteGlu for modulation of fluoropyrimidines may vary from one cell type to another, introducing high doses of (6R,S)-5-HCO-H₄PteGlu in patients so that the plasma concentration of the natural isomer reaches 10 µm is still recommended.