The inverse relationship between reduced folate carrier function and pemetrexed activity in a human colon cancer cell line

Shrikanta Chattopadhyay, Rongbao Zhao, Sergery A. Krupenko, Natalia Krupenko, I. David Goldman

Research output: Contribution to journalArticle

39 Citations (Scopus)

Abstract

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 (∼85%) but intracellular pemetrexed levels increased to ∼60% and ∼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.

Original languageEnglish (US)
Pages (from-to)438-449
Number of pages12
JournalMolecular Cancer Therapeutics
Volume5
Issue number2
DOIs
StatePublished - Feb 2006

Fingerprint

Pemetrexed
Reduced Folate Carrier Protein
Colonic Neoplasms
Cell Line
Folic Acid
Folic Acid Antagonists
Leucovorin
Phosphoribosylglycinamide Formyltransferase
Trimetrexate
Thymidylate Synthase
Mesothelioma
Enzyme Assays
Amino Acid Substitution
Nucleosides
Methotrexate
Non-Small Cell Lung Carcinoma
Mutagenesis
Glycine
Arginine

ASJC Scopus subject areas

  • Oncology
  • Drug Discovery
  • Pharmacology

Cite this

The inverse relationship between reduced folate carrier function and pemetrexed activity in a human colon cancer cell line. / Chattopadhyay, Shrikanta; Zhao, Rongbao; Krupenko, Sergery A.; Krupenko, Natalia; Goldman, I. David.

In: Molecular Cancer Therapeutics, Vol. 5, No. 2, 02.2006, p. 438-449.

Research output: Contribution to journalArticle

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