Dihydrofolate reductase binding and cellular uptake of nonpolyglutamatable antifolates: correlates of cytotoxicity toward methotrexate-sensitive and -resistant human head and neck squamous carcinoma cells.
Several mechanisms have been demonstrated to be independently involved in methotrexate (MTX) resistance, including increased dihydrofolate reductase (DHFR) activity, decreased membrane transport, and decreased conversion to noneffluxing polyglutamates by folylpolyglutamate synthetase. We conducted the present study to test the hypothesis that nonpolyglutamatable antifolates with an N delta-hemiphthaloyl-L-ornithine side chain could be more potent than MTX against MTX-sensitive and -resistant human carcinoma cells via tighter DHFR binding, more efficient cellular uptake, the ability to bypass defective polyglutamation, or a combination. Two nonpolyglutamatable antifolates, N alpha-(4-amino-4-deoxypteroyl)-N delta-hemiphthaloyl-L-ornithine (PT523) and the new B-ring analogue N alpha-[4-[N-(2,4-diamino-5-chloroquinazolin-6-yl)methyl]amino] benzoyl-N delta-hemiphthaloyl-L-ornithine (PT619), were tested as inhibitors of purified recombinant human DHFR and were found to bind somewhat better to the enzyme than MTX as determined by competitive radioligand binding assay. PT523 and PT619 were 9- and 14-fold, respectively, more active than MTX as inhibitors of parental SCC25 human and neck squamous carcinoma cell growth in 72-hr cultures. Moreover, there was an even greater increase in relative potency against two previously described MTX-resistant cell lines with an increased DHFR content and a decreased ability to convert MTX to polyglutamates: SCC25/R1 (selected with MTX) and SCC25/CP (selected with cisplatin but collaterally resistant to MTX). Both PT523 and PT619 very efficiently inhibited [3H]MTX uptake by SCC25 cells in a 1-hr assay, with PT523 being 11-fold more potent and PT619 being 17-fold more potent than MTX. Greater inhibition of [3H]MTX uptake with PT523 and PT619 than with MTX was also observed in SCC25/R1 and SCC/CP cells. However, the increase in activity of PT523 and PT619 relative to MTX in uptake experiments was less than that in growth-inhibition assays, especially for SCC25/CP cells. This suggested that additional cytotoxicity determinants may exist in these resistant cells.
D000964:Antimetabolites, Antineoplastic / Q000493:pharmacokinetics* / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000378:metabolism / Q000493:pharmacokinetics* / Q000494:pharmacology; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D019008:Drug Resistance, Neoplasm; D004354:Drug Screening Assays, Antitumor; D005493:Folic Acid Antagonists / Q000378:metabolism / Q000493:pharmacokinetics* / Q000494:pharmacology; D006258:Head and Neck Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008727:Methotrexate / Q000493:pharmacokinetics / Q000494:pharmacology*; D009952:Ornithine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology; D011622:Pterins / Q000378:metabolism / Q000493:pharmacokinetics* / Q000494:pharmacology; D011624:Pteroylpolyglutamic Acids / Q000378:metabolism; D011799:Quinazolines / Q000494:pharmacology*; D013762:Tetrahydrofolate Dehydrogenase / Q000187:drug effects / Q000378:metabolism*; D014316:Tritium; D014407:Tumor Cells, Cultured
D000964:Antimetabolites, Antineoplastic; D000970:Antineoplastic Agents; D005493:Folic Acid Antagonists; C096334:PT 619; D011622:Pterins; D011624:Pteroylpolyglutamic Acids; D011799:Quinazolines; D014316:Tritium; C085590:N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine; D009952:Ornithine; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate