Abstract
Uptake of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates with four or three bridge carbons [compound 1 (C1) and compound 2 (C2), respectively] into solid tumors by the proton-coupled folate transporter (PCFT) represents a novel therapeutic strategy that harnesses the acidic tumor microenvironment. Although these compounds are not substrates for the reduced folate carrier (RFC), the major facilitative folate transporter, RFC expression may alter drug efficacies by affecting cellular tetrahydrofolate (THF) cofactor pools that can compete for polyglutamylation and/or binding to intracellular enzyme targets. Human tumor cells including wild-type (WT) and R5 (RFC-null) HeLa cells express high levels of PCFT protein. C1 and C2 inhibited proliferation of R5 cells 3 to 4 times more potently than WT cells or R5 cells transfected with RFC. Transport of C1 and C2 was virtually identical between WT and R5 cells, establishing that differences in drug sensitivities between sublines were independent of PCFT transport. Steady-state intracellular [3H]THF cofactors derived from [3H]5-formyl-THF were depleted in R5 cells compared with those in WT cells, an effect exacerbated by C1 and C2. Whereas C1 and C2 polyglutamates accumulated to similar levels in WT and R5 cells, there were differences in polyglutamyl distributions in favor of the longest chain length forms. In severe combined immunodeficient mice, the antitumor efficacies of C1 and C2 were greater toward subcutaneous R5 tumors than toward WT tumors, confirming the collateral drug sensitivities observed in vitro. Thus, solid tumor-targeted antifolates with PCFT-selective cellular uptake should have enhanced activities toward tumors lacking RFC function, reflecting contraction of THF cofactor pools.
Footnotes
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The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
This study was supported by the National Institutes of Health National Cancer Institute [Grants CA53535, CA152316, CA125153]; the Barbara Ann Karmanos Cancer Institute; and the Mesothelioma Applied Research Foundation. S.K.D. was supported by a Doctoral Research Award from the Canadian Institute of Health Research.
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS:
- MTX
- methotrexate
- PMX
- pemetrexed
- RFC
- reduced folate carrier
- PCFT
- proton-coupled folate transporter
- FR
- folate receptor
- THF
- tetrahydrofolate
- 5-CHO-THF
- 5-formyl-tetrahydrofolate
- C1
- compound 1
- C2
- compound 2
- hRFC
- human reduced folate carrier
- hPCFT
- human proton-coupled folate transporter
- HA
- hemagglutinin
- WT
- wild-type
- RT
- reverse transcription
- PCR
- polymerase chain reaction
- MES
- 4-morphilinopropane sulfonic acid
- DPBS
- Dulbecco's phosphate-buffered saline
- PIPES
- piperazine-N,N′-bis(2-ethanesulfonic acid)
- HPLC
- high-performance liquid chromatography
- SCID
- severe combined immunodeficient
- LMX
- lometrexol
- RTX
- raltitrexed
- PT523
- Nα-(4-amino-4-deoxypteroyl)-Nδ-hemiphthaloyl-l-ornithine
- PG
- polyglutamate
- FPGS
- folylpolyglutamate synthetase
- ABC
- ATP-binding cassette.
- Received March 29, 2012.
- Accepted June 26, 2012.
- Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
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