The inverse relationship between reduced folate carrier function and pemetrexed activity in a human colon cancer cell line.
Pemetrexed, a new generation antifolate recently approved for the treatment of mesothelioma and non-small cell lung cancer, is an excellent substrate for the reduced folate carrier (RFC). To explore the carrier's effect on pemetrexed activity, RFC was inactivated in HCT-15 colon cancer cells by mutagenesis and PT632 selective pressure. A clone (PT1) was obtained with a glycine to arginine substitution at amino acid 401, resulting in the loss of RFC function. PT1 cells were resistant to PT632 (178-fold), methotrexate (4-fold), and ZD1694 (Tomudex, raltitrexed; 20-fold), but were 3-fold collaterally sensitive to pemetrexed when grown in 25 nmol/L of 5-formyltetrahydrofolate. PT1 cells transfected with wild-type RFC had antifolate sensitivities comparable to that of wild-type HCT-15 cells, indicating that the RFC mutation was the sole basis for resistance. Folate pools were contracted in PT1 cells by 32% or 60%, as measured by radiolabeling intracellular folates or by an enzyme binding assay, respectively. This was reflected in marked (6.5-fold) collateral sensitivity to trimetrexate. The initial uptake of pemetrexed in PT1 cells was markedly reduced ( approximately 85%) but intracellular pemetrexed levels increased to approximately 60% and approximately 70% to that of wild-type cells after 2 hours and 6 days, respectively. There was increased pemetrexed inhibition of glycinamide ribonucleotide transformylase and, to a lesser extent, thymidylate synthase in PT1 cells growing in 5-formyltetrahydrofolate based on nucleoside protection analyses. Hence, loss of RFC function leads to collateral sensitivity to pemetrexed in HCT-15 cells, likely due to cellular folate pool contraction resulting in partial preservation of pemetrexed polyglutamylation and increased target enzyme inhibition. micro
Chattopadhyay
Shrikanta
S
Department of Medicine, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Zhao
Rongbao
R
Krupenko
Sergey A
SA
Krupenko
Natalia
N
Goldman
I David
ID
D019943:Amino Acid Substitution; D000970:Antineoplastic Agents / Q000378:metabolism*; D001120:Arginine / Q000737:chemistry / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000235:genetics / Q000378:metabolism*; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D058973:Folate Receptors, GPI-Anchored; D005493:Folic Acid Antagonists / Q000494:pharmacology; D005971:Glutamates / Q000378:metabolism*; D005998:Glycine / Q000737:chemistry / Q000235:genetics; D006147:Guanine / Q000819:agonists / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D002955:Leucovorin / Q000378:metabolism; D008727:Methotrexate / Q000378:metabolism; D009154:Mutation; D000068437:Pemetrexed; D051026:Phosphoribosylglycinamide Formyltransferase / Q000037:antagonists & inhibitors; D011687:Purines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D011799:Quinazolines / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D013876:Thiophenes / Q000378:metabolism; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors
D000970:Antineoplastic Agents; D002352:Carrier Proteins; D058973:Folate Receptors, GPI-Anchored; D005493:Folic Acid Antagonists; D005971:Glutamates; D011687:Purines; D011743:Pyrimidines; D011799:Quinazolines; D011956:Receptors, Cell Surface; D013876:Thiophenes; D000068437:Pemetrexed; D006147:Guanine; D001120:Arginine; D013940:Thymidylate Synthase; D051026:Phosphoribosylglycinamide Formyltransferase; C068874:raltitrexed; D002955:Leucovorin; D005998:Glycine; D008727:Methotrexate