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Synergistic Growth Inhibition of Rat Hepatoma Cells Exposed in Vitro to N¹⁰-Propargyl-5,8-dideazafolate with Methotrexate or the Lipophilic Antifolates Trimetrexate or Metoprine¹

Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York 12201

The growth inhibitory effects of combinations of antifolates on hepatoma cells in culture have been examined. In these studies methotrexate or the lipophilic inhibitors of dihydrofolate reductase were used with the thymidylate synthase inhibitor N¹⁰-propargyl-5,8-dideazafolate (PDDF). Under certain conditions partial growth inhibition by methotrexate and trimetrexate is reduced by noninhibitory to slightly inhibitory concentrations (< 1 µM) of PDDF. At somewhat higher concentrations (1.6–4 µM) of PDDF, synergy is observed with methotrexate, trimetrexate, or metoprine. Trimetrexate exerted greater synergistic effects than methotrexate. A noninhibitory concentration of trimetrexate (2 nM) in combination with a partially inhibitory concentration of PDDF reduced growth by 93%. Metoprine was capable of replacing trimetrexate and exhibits slightly greater inhibitory activity in combination than trimetrexate. Both metoprine and trimetrexate in combination with PDDF caused synergistic inhibition of the de novo synthesis of thymidylate in intact cells as measured by tritium release from [5-³H]deoxyuridine. Clonal assays were used to demonstrate synergy between trimetrexate or metoprine and PDDF, attesting to the cytotoxic properties of this combination. Thymidine alone can protect against both the synergistic combination of trimetrexate or metoprine and PDDF and PDDF alone, but has only a weak protective effect on toxic concentrations of trimetrexate and metoprine. These observations suggest that growth inhibition is mediated by the activity of N¹⁰-propargyl-5,8-dideazafolate on thymidylate synthase. These results are discussed with regard to the mechanism of inhibition of thymidylate synthase by the 5,8-dideazafolates and the possibility of enhancing the inhibitory activity of this class of compounds by using them with inhibitors of dihydrofolate reductase.

¹ This work was supported by NIH Grant CA25933 by the National Cancer Institute, USPHS-Department of Health and Human Services.

² To whom requests for reprints should be addressed.

Received 3/30/87. Revised 6/29/87. Accepted 7/14/87.