5-8-dideazaisofolic-acid and Colonic-Neoplasms

The uptake and metabolism of radiolabeled 5,8-dideazaisofolic acid (IAHQ) (N-[p- ([(2-amino-4-hydroxy-6-quinazolinyl)amino]methyl)benzoyl]-L-glutamic acid), a new antifol targeted to thymidylate synthase, has been investigated in the human colon adenocarcinoma cell line HCT-8. [3H]IAHQ uptake was very slow, requiring days to achieve the intracellular level achieved in minutes by [3H]methotrexate. This slow transport of IAHQ was consistent with the long exposures required to achieve cytotoxicity. Intracellular [3H]IAHQ was converted in a concentration-dependent manner to poly-gamma-glutamate derivatives containing between two and four additional glutamate residues. These results are consistent with our hypothesis that IAHQ is a "pro-drug" which must be converted to polyglutamate derivatives before it is a sufficiently potent inhibitor of thymidylate synthase to induce a pyrimidineless state and cell death.

Topics: Adenocarcinoma; Biotransformation; Colonic Neoplasms; Folic Acid Antagonists; Humans; Methotrexate; Polyglutamic Acid; Quinazolines; Thymidylate Synthase; Tumor Cells, Cultured

The clonal cytotoxic effects and mechanism of action of a new series of 2-amino-4-hydroxyquinazoline folate analogues (5,8-dideazafolates) have been assessed using the human colon tumor cell line HCT-8. Of these compounds only 5-methyl-5,8-dideazafolate was potentially more effective than a compound previously identified, 5,8-dideazaisofolate (H-338, NSC 289517). HCT-8 sublines resistant to methotrexate, 5-fluorodeoxyuridine, and H-338 were either minimally or not cross-resistant to the other agents. The cytotoxicity of H-338 was strongly dependent on the time of exposure; at exposure times shorter than 8 h it was essentially nontoxic. Thymidine alone, as well as leucovorin or folic acid, protected against the cytotoxic effects of H-338. This is consistent with thymidylate synthase (TS) as its only locus of action. Studies with dihydrofolate reductase and TS isolated from HCT-8 cells indicated that these quinazolines were weaker inhibitors of dihydrofolate reductase than was methotrexate, but they were not particularly potent TS inhibitors. However, synthetic poly-gamma-glutamate derivatives of quinazolines showed dramatically increased TS, but not dihydrofolate reductase, inhibition. TS inhibition increased as the polyglutamate chain length increased. Using isolated HCT-8 folylpolyglutamate synthetase, all the parent quinazolines containing L-glutamate were found to be substrates. With H-338, the results indicated that tetraglutamate or longer derivatives could be synthesized intracellularly. These results are consistent with our hypothesis that cytotoxicity by such quinazolines necessarily involves "lethal synthesis" from a prodrug; i.e., the nontoxic parent drug must be converted to polyglutamates before TS inhibition and subsequent cytotoxicity can occur.

Topics: Cell Survival; Colonic Neoplasms; Drug Resistance; Drug Screening Assays, Antitumor; Folic Acid Antagonists; Humans; Quinazolines; Structure-Activity Relationship; Tumor Cells, Cultured

None of 13 fresh human tumor samples of various histology cloned in a two-layer agar culture system with 20% undialyzed fetal bovine serum (FBS) showed sensitivity to three antifolates, methotrexate (MTX), trimetrexate and 5,8-dideazaisofolic acid (IAHQ), even after continuous exposure to the highest concentrations (100 microM) for 21 days. In order to investigate this lack of antifolate drug effect, we compared the toxicity of continuous MTX exposure in the human colon carcinoma cell line HCT-8, cloned in a thymidineless medium (RPMI 1640) supplemented with 10% horse serum (HS), 10% fetal bovine serum (FBS), 20% FBS or 20% dialyzed FBS. In the presence of native FBS, when the minimum clone size was set at 30 cells/colony, the survival of HCT-8 cells reached a plateau at approximately 60% of untreated control after exposure to MTX concentrations between 0.1 microM and 100 microM. Only when the minimum clone size was set at 2 X 10(3) cells/colony was the sensitivity of HCT-8 cells to the antimetabolite comparable to that obtained in HS or dialyzed FBS (ED50 values in the range of 0.01 microM). MTX protection experiments indicated that even very small concentrations of thymidine and hypoxanthine together were sufficient to reproduce the pattern of sensitivity to MTX observed under culture conditions with undialyzed FBS. We conclude that for a proper evaluation of MTX cytotoxicity in clonogenic assays, dialyzed FBS and thymidine-less media should be employed; if native FBS is an absolute requirement for growth, only very large colonies (at least 10 cell divisions) should be scored.

Topics: Adenocarcinoma; Animals; Cattle; Cell Adhesion; Colonic Neoplasms; Colony-Forming Units Assay; Culture Media; Dose-Response Relationship, Drug; Humans; Hypoxanthines; Leucovorin; Methotrexate; Neoplastic Stem Cells; Quinazolines; Thymidine; Trimetrexate

In the preceding companion paper, we describe a human colon carcinoma cell line that is resistant in vitro to 5-fluoro-2'-deoxyuridine by virtue of impaired nucleoside transport. Two drug combinations, methotrexate: hypoxanthine: thymidine (dThd) and 5,8-dideazaisofolic acid: dThd, selectively kill these resistant cells with no effect on the sensitive cell population. As little as 0.3 microM dThd was sufficient to completely protect sensitive cells from 5-fluoro-2'-deoxyuridine, and 5,8-dideazaisofolic acid at concentrations that produced over 80% lethality in unprotected cells and the same concentration of dThd in combination with 100 microM hypoxanthine fully protected sensitive cells from greater than 99% methotrexate-induced cell lethality. In contrast, when resistant cells were exposed to these drugs, they were not protected by dThd, or by the combination dThd: hypoxanthine, in concentrations up to 300 times higher than those necessary to prevent sensitive cell kill. Thus, it may be possible to protect normal renewal tissues while obtaining selective tumor cell kill with these two drug combinations in patients whose colon carcinoma cells are resistant to 5-fluoro-2'-deoxyuridine by virtue of defective transport.

Topics: Adenocarcinoma; Antineoplastic Combined Chemotherapy Protocols; Biological Transport; Cell Line; Colonic Neoplasms; Drug Resistance; Floxuridine; Humans; Hypoxanthine; Hypoxanthines; Methotrexate; Nucleosides; Quinazolines; Thymidine

A series of 2-amino-4-hydroxy-quinazolines was synthesized and evaluated as inhibitors of colon adenocarcinoma and the folate-dependent enzymes, thymidylate synthase and dihydrofolate reductase. Of the quinazolines tested, 5,8-dideazaisopteroylglutamate, (IAHQ), when administered at 85 mg/kg on days 2 and 10 after tumor implantation delayed the growth of colon tumor No. 38, and resulted in 6 of 20 tumor-free animals at 90 days. In contrast, methotrexate had no effect on the growth of colon tumor No. 38 at maximally tolerated doses. IAHQ was also active against human colon adenocarcinoma cells (HCT-8) in tissue culture, requiring a concentration of 5 X 10(-7) M to inhibit cell growth 50% after 72 hours continuous exposure. Since IAHQ was an effective substrate for folylpolyglutamate synthetase, we examined the effects of IAHQ and its possible tri-gamma-glutamyl metabolite, 5,8-dideazaisoPteGLu3, on thymidylate synthase and dihydrofolate reductase. Neither IAHQ nor 5,8-dideazaisoPteGlu3 stimulated significant binding of 5-fluorodeoxyuridylate to thymidylate synthase. This was consistent with the observation that IAHQ antagonized the killing of HCT-8 cells by 5-fluorouracil. 5,8 DideazaisoPteGlu3 bound more tightly to thymidylate synthase than dihydrofolate reductase as indicated by Kis of 0.09 and 0.7 microM when deoxyuridylate and dihydropteroylglutamate, respectively, were the variable substrates. Inhibition studies also revealed that binding of IAHQ and 5,8-dideazaisoPteGlu3 to thymidylate synthase is promoted and not antagonized by deoxyuridylate. The data suggests that the biochemical basis for the antitumor effects of IAHQ is the intracellular conversion of IAHQ to poly-gamma-glutamyl metabolites, which inhibit thymidylate synthase via formation of an inhibitor-deoxyuridylate-enzyme complex.

Topics: Adenocarcinoma; Animals; Cell Line; Colonic Neoplasms; DNA Replication; Drug Resistance; Female; Humans; Kinetics; Leukemia; Methotrexate; Methyltransferases; Mice; Mice, Inbred C57BL; Neoplasms, Experimental; Peptide Synthases; Quinazolines; Tetrahydrofolate Dehydrogenase; Thymidylate Synthase

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Cell Line; Colonic Neoplasms; Female; Folic Acid Antagonists; Humans; Leukemia L1210; Methotrexate; Methyltransferases; Mice; Mice, Inbred C57BL; Quinazolines; Thymidylate Synthase