title: Mechanisms of resistance to N-[5-[N-(3,4-dihydro-2-methyl-4- oxoquinazolin-6-ylmethyl)-N-methylamino]-2-thenoyl]-L-glutamic acid (ZD1694), a folate-based thymidylate synthase inhibitor, in the HCT-8 human ileocecal adenocarcinoma cell line.
abstract: N-[5-[N-(3,4-Dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N- methylamino]-2-thenoyl]-L-glutamic acid (ZD1694) is a folate-based thymidylate synthase (TS; EC 2.1.1.45) inhibitor. Metabolism to higher chain length polyglutamates is essential for its optimal cytotoxic effect. A ZD1694-resistant (300-fold) human ileocecal carcinoma cell line (HCT-8/DW2) was developed, and its mechanism of resistance was evaluated. TS activities in situ and TS protein levels in the HCT-8 parental line and HCT-8/DW2 were similar (168 +/- 47 vs 137 +/- 25 pmol/hr/10(6) cells and 2.05 +/- 0.28 vs 2.07 +/- 0.19 pmol/mg protein, respectively). The IC50 values of ZD1694 for TS inhibition in cell-free extracts were similar in both lines, but the IC50 of ZD1694 for TS inhibition in situ in HCT-8/DW2 cells was 27- and 268-fold higher than that in HCT-8 cells at 0 and 24 hr, respectively, after a 2-hr drug exposure. Folylpolyglutamate synthetase (FPGS; EC6.3.2.17) activity was significantly lower in resistant HCT-8/DW2 cells as compared with parental HCT-8 cells (88 +/- 40 vs 1065 +/- 438 pmol/hr/mg protein when ZD1694 was used as substrate). The combined endogenous pool of methylenetetrahydrofolate and tetrahydrofolate in HCT-8/DW2 cells was also decreased. In addition, HCT-8/DW2 cells accumulated lower levels of methotrexate (MTX) in a 2-hr period, although the initial velocity of MTX transport was similar to that in parental HCT-8 cells. The lower level of FPGS activity and the lower level of (anti)folate accumulation in HCT-8/DW2 correlated with drug resistance and with the higher IC50 of ZD1694 for in situ TS inhibition. In addition, drug resistance was also correlated with the rapid recovery of in situ TS activity after drug treatment. In brief, in this highly ZD1694-resistant HCT-8 cell line, resistance is associated with decreased FPGS activity, which, in turn, affects the metabolism of ZD1694 and consequently the extent and duration of in situ TS inhibition by the drug.
authors.affiliation: Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Italy.
authors.forename: K
authors.initials: K
authors.lastname: Lu
authors.forename: M B
authors.initials: MB
authors.lastname: Yin
authors.forename: J J
authors.initials: JJ
authors.lastname: McGuire
authors.forename: E
authors.initials: E
authors.lastname: Bonmassar
authors.forename: Y M
authors.initials: YM
authors.lastname: Rustum
mesh_terms: D002455:Cell Division / Q000187:drug effects; D002474:Cell-Free System; D004305:Dose-Response Relationship, Drug; D004351:Drug Resistance; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D005971:Glutamates / Q000494:pharmacology; D006147:Guanine / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology; D000068437:Pemetrexed; D010453:Peptide Synthases / Q000037:antagonists & inhibitors*; D011624:Pteroylpolyglutamic Acids / Q000378:metabolism; D011799:Quinazolines / Q000494:pharmacology*; D013876:Thiophenes / Q000494:pharmacology*; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors*; D013997:Time Factors; D014407:Tumor Cells, Cultured / Q000187:drug effects
chemical_list: D005493:Folic Acid Antagonists; D005971:Glutamates; D011624:Pteroylpolyglutamic Acids; D011799:Quinazolines; D013876:Thiophenes; D000068437:Pemetrexed; D006147:Guanine; D013940:Thymidylate Synthase; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase; C068874:raltitrexed; D008727:Methotrexate