Decreased expression of the reduced folate carrier and folypolyglutamate synthetase is the basis for acquired resistance to the pemetrexed antifolate (LY231514) in an L1210 murine leukemia cell line.
Pemetrexed (LY231514) is a new-generation antifolate that, in its polyglutamyl forms, is a potent inhibitor of thymidylate synthase and glycinamide ribonucleotide formyltransferase (GAR transformylase). This study explored the mechanisms of resistance to pemetrexed in L1210 murine leukemia cells using chemical mutagenesis with 5-formyltetrahydrofolate (5-formylTHF) as the growth substrate. A cell line, MTA-13, was identified that was 8.5-fold resistant to pemetrexed with comparable cross-resistance to ZD1694 (Tomudex) and lesser cross-resistance (5-fold) to ZD9331 [(2S)-2-(O-fluoro-p-[N-(2,7-dimethyl-4-oxo-3,4-dihydro-quinazolin-6-ylmethyl)-N-(prop-2-ynyl)amino]benzamido)-4-(tetrazol-5-yl)-butyric acid], DDATHF (dideazatetrahydrofolate) (3.5-fold), and methotrexate (MTX) (2.7-fold) but comparable sensitivity to trimetrexate. Influx of pemetrexed, MTX, and 5-formylTHF into MTA-13 cells was decreased by 56, 47, and 38% compared to wild-type cells. Folate receptor expression was negligible in both cell lines. Net drug uptake declined within 15min to a slower, constant rate over the next 45min, reflecting the rate of accumulation of pemetrexed polyglutamate derivatives. This rate in the MTA-13 line was half that of the wild-type cells. Accumulation of 50nM [3H]pemetrexed, 25nM [3H]5-formylTHF, or 50nM [3H]DDATHF after 3 days was decreased to 35, 46, and 56% the level of L1210 cells. The reduced folate carrier (RFC) message and protein were decreased by 50%, and folypolyglutamate synthetase (FPGS) message was decreased by 65% in MTA-13 cells. No mutations were detected in either protein by DNA sequence analysis. There was a slight decrease (approximately 25%) in thymidylate synthase mRNA, without mutations in the protein, and there was no change in GAR transformylase message. The data indicate that resistance to pemetrexed in the MTA-13 cell line was due to changes in both RFC and FPGS expression, two proteins that act in tandem to regulate polyglutamation of folates and antifolates in cells, resulting in cellular depletion of these active pemetrexed congeners.
Wang
Yanhua
Y
Departments of Medicine and Molecular Pharmacology, Albert Einstein College of Medicine Cancer Center, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Zhao
Rongboa
R
Goldman
I David
ID
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics; D015536:Down-Regulation; D019008:Drug Resistance, Neoplasm / Q000502:physiology*; D005492:Folic Acid / Q000378:metabolism; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D020868:Gene Silencing / Q000187:drug effects; D005971:Glutamates / Q000494:pharmacology*; D006147:Guanine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D019877:Hydroxymethyl and Formyl Transferases / Q000096:biosynthesis / Q000235:genetics; D007939:Leukemia L1210 / Q000473:pathology; D026901:Membrane Transport Proteins; D051379:Mice; D000068437:Pemetrexed; D010453:Peptide Synthases / Q000096:biosynthesis* / Q000235:genetics; D051026:Phosphoribosylglycinamide Formyltransferase; D058978:Reduced Folate Carrier Protein; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D005493:Folic Acid Antagonists; D005971:Glutamates; D026901:Membrane Transport Proteins; D058978:Reduced Folate Carrier Protein; C548700:Slc19a1 protein, mouse; D000068437:Pemetrexed; D006147:Guanine; D005492:Folic Acid; D019877:Hydroxymethyl and Formyl Transferases; D051026:Phosphoribosylglycinamide Formyltransferase; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase