Abstract
The proton-coupled folate transporter (PCFT) is a proton-folate symporter with an acidic pH optimum. By real-time reverse transcription-polymerase chain reaction, PCFT was expressed in the majority of 53 human tumor cell lines, with the highest levels in Caco-2 (colorectal adenocarcinoma), SKOV3 (ovarian), and HepG2 (hepatoma) cells. A novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolate (compound 1) was used to establish whether PCFT can deliver cytotoxic drug under pH conditions that mimic the tumor microenvironment. Both 1 and pemetrexed (Pmx) inhibited proliferation of R1-11-PCFT4 HeLa cells engineered to express PCFT without the reduced folate carrier (RFC) and of HepG2 cells expressing both PCFT and RFC. Unlike Pmx, 1 did not inhibit proliferation of R1-11-RFC6 HeLa cells, which express RFC without PCFT. Treatment of R1-11-PCFT4 cells at pH 6.8 with 1 or Pmx inhibited colony formation with dose and time dependence. Transport of [3H]compound 1 into R1-11-PCFT4 and HepG2 cells was optimal at pH 5.5 but appreciable at pH 6.8. At pH 6.8, [3H]compound 1 was metabolized to 3H-labeled polyglutamates. Glycinamide ribonucleotide formyltransferase (GARFTase) in R1-11-PCFT4 cells was inhibited by 1 at pH 6.8, as measured by an in situ GARFTase assay, and was accompanied by substantially reduced ATP levels. Compound 1 caused S-phase accumulation and a modest level of apoptosis. An in vivo efficacy trial with severe combined immunodeficient mice implanted with subcutaneous HepG2 tumors showed that compound 1 was active. Our findings suggest exciting new therapeutic possibilities to selectively deliver novel antifolate drugs via transport by PCFT over RFC by exploiting the acidic tumor microenvironment.
Footnotes
↵ 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; the Mesothelioma Applied Research Foundation; and a Doctoral Research Award from the Canadian Institutes of Health Research (to S.K.D.).
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.111.073833.
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ABBREVIATIONS:
- Mtx
- methotrexate
- Pmx
- pemetrexed
- GAR
- β-glycinamide ribonucleotide
- GARFTase
- β-glycinamide ribonucleotide formyltransferase
- RFC
- reduced folate carrier
- FR
- folate receptor
- PCFT
- proton-coupled folate transporter
- h
- human
- LCV
- leucovorin
- RT-PCR
- reverse transcription-polymerase chain reaction
- GAPDH
- glyceraldehyde-3-phosphate dehydrogenase
- dFBS
- dialyzed fetal bovine serum
- DMSO
- dimethyl sulfoxide
- PIPES
- 25 mM piperazine-N,N′-bis(2-ethanesulfonic acid
- DPBS
- Dulbecco's phosphate-buffered saline
- MES
- 2-(N-morpholino)ethanesulfonic acid
- HPLC
- high-performance liquid chromatography
- FITC
- fluorescein isothiocyanate
- PI
- propidium iodide
- SCID
- severe combined immunodeficiency
- ALL
- acute lymphoblastic leukemia
- CHO
- Chinese hamster ovary
- MTAP
- methylthioadenosine phosphorylase.
- Received May 27, 2011.
- Accepted September 22, 2011.
- Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics
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