Thymidine and hypoxanthine protection patterns of the folic acid-enhanced synergies for combinations of trimetrexate plus a polyglutamylatable inhibitor of purine or thymidylate synthesis against human ileocecal HCT-8 cells

Abstract

In order to examine the intracellular locus of the folic acid (PteGlu)-enhanced synergies of trimetrexate (TMQ) plus the thymidylate synthase (TS) inhibitor, raltitrexed (RTX), and TMQ plus the glycinamide ribonucleotide formyltransferase (GARFT) inhibitor, AG2034, comprehensive protection studies with thymidine (dThd) and hypoxanthine (HX) were conducted in a 96-well plate cell growth inhibition (sulforhodamine B) assay. Current modeling techniques were extended to characterize these protection patterns involving multiple-agent interaction. Wild-type human ileocecal HCT-8 cells and DW2, a subline deficient in folylpoly-gamma-glutamate synthetase (FPGS) were individually treated for 96 h with TMQ, AG2034 and a 1:1 mixture of TMQ:AG2034 or with TMQ, RTX, and a 1:1 mixture of TMQ:RTX in the presence of PteGlu (2.3 or 40 micro M) and the protection agents (10 micro M dThd and/or 100 micro M HX). Drug treatments were randomly assigned to wells. Both isobols and 3-dimensional concentration-effect surfaces were used to assess the nature and the intensity of drug interactions. The structural Hill model was fitted to data with weighted non-linear regression for most cases. A so-called 'double Hill' model was sometimes more appropriate when a plateau in the middle of the concentration-effect curve was found. In HCT-8 and DW2 cells at 2.3 and 40 micro M PteGlu, inhibition of DHFR by TMQ induced antithymidylate and antipurine effects; AG2034 and RTX selectively inhibited de novo purine or thymidine synthesis, respectively. dThd protection increased the PteGlu-enhancement of the TMQ + AG2034 synergy, whereas HX protection increased the PteGlu-enhancement of the TMQ + RTX synergy. The PteGlu-enhanced synergies of TMQ + AG2034 and TMQ + RTX occur primarily through inhibition of purine synthesis and inhibition of thymidylate synthesis, respectively. These results further substantiate the hypothesis that the nonpolyglutamylatable DHFR inhibitor, TMQ, acts as a modulator by decreasing the protection by PteGlu of cells against the polyglutamylatable AG2034 and RTX.