Therapeutic Targeting of a Novel 6-Substituted Pyrrolo [2,3-d]pyrimidine Thienoyl Antifolate to Human Solid Tumors Based on Selective Uptake by the Proton-Coupled Folate Transporter

Graduate Program in Cancer Biology, Wayne State University School of Medicine, Detroit, Michigan, USA.
Molecular pharmacology (Impact Factor: 4.13). 09/2011; 80(6):1096-107. DOI: 10.1124/mol.111.073833
Source: PubMed


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 [(3)H]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, [(3)H]compound 1 was metabolized to (3)H-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.

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Available from: Aleem Gangjee, May 08, 2015
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    • "RFC mediates folate transport into systemic tissues whereas PCFT plays a key role in intestinal folate absorption and folate transport from blood across the choroid plexus into the cerebrospinal fluid (Zhao et al., 2011). RFC and PCFT are also ubiquitously expressed in human cancers (Zhao et al., 2004b; Kugel Desmoulin et al., 2011). FR-a is expressed in epithelia (proximal renal tubule, choroid plexus, retinal pigment epithelium) (Elnakat and Ratnam, 2004; Kamen and Smith, 2004) and is widely expressed in epithelial cancers (Weitman et al., 1992; Parker et al., 2005); FR-b is expressed in hematopoietic malignancies (Ross et al., 1999). "
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    ABSTRACT: The reduced folate carrier (RFC), the proton-coupled folate transporter (PCFT) and folate receptors are folate-specific transporters. Antifolates currently in the clinic such as pemetrexed, methotrexate and pralatrexate are transported into tumor cells primarily via RFC. Folic acid conjugated to cytotoxics, a new class of antineoplastics, are transported into cells via folate receptor (FR) mediated endocytosis. To better define the role of PCFT in antifolate resistance, a methotrexate-resistant cell line, M160-8, was selected from a HeLa subline in which the RFC gene was deleted and PCFT was highly overexpressed. These cells were cross-resistant to pemetrexed. PCFT function, as well as the PCFT mRNA level, in M160-8 cells was barely detectable while FR-α function and mRNA level were increased as compared to the parent cells. While pemetrexed rapidly associated with folate receptor and was internalized within endosomes in M160-8 cells, consistent with folate receptor-mediated transport, subsequent pemetrexed and (6S)5-formyltetrahydrofolate export into the cytosol was markedly impaired. In contrast, M160-8 cells were collaterally sensitive to EC0905, a folic acid-desacetylvinblastine monohydrazide conjugate also transported by FR- mediated endocytosis. However, in this case a sulfhydryl bond is cleaved to release the lipophilic cytotoxic moiety into the endosome, which passively diffuses out of the endosome into the cytosol. Hence, resistance to pemetrexed in M160-8 cells was due to entrapment of drug within the endosome due to the absence of PCFT under conditions in which folate receptor cycling function was intact.
    Preview · Article · Nov 2013 · Molecular pharmacology
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    • "We found previously that the novel 6-substituted pyrrolo[2,3d]pyrimidine thienoyl antifolates C1 and C2 (Fig. 2A) were potent (nanomolar) inhibitors of proliferation in cells engineered to express hPCFT in the absence of hRFC or FRs (Wang et al., 2010Wang et al., , 2011Kugel Desmoulin et al., 2011), suggesting that C1 and C2 are substrates for hPCFT-mediated cellular uptake. In engineered cell lines, C1 and C2 seemed to be poorly transported by hRFC (Wang et al., 2010Wang et al., , 2011Kugel Desmoulin et al., 2011). Both analogs induced current at 90 mV and pH 5.5 in Xenopus laevis oocytes microinjected with hPCFT cRNAs, and both were competitive inhibitors of[ 3 H]MTX transport in hPCFT transfectants from pH 5.5 to pH 6.8 (Kugel Desmoulin et al., 2011;). "
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    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 [(3)H]THF cofactors derived from [(3)H]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.
    Preview · Article · Jun 2012 · Molecular pharmacology
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    ABSTRACT: The proton-coupled folate transporter (PCFT; SLC46A1) is a proton-folate symporter that is abundantly expressed in solid tumors and normal tissues, such as duodenum. The acidic pH optimum for PCFT is relevant to intestinal absorption of folates and could afford a means of selectively targeting tumors with novel cytotoxic antifolates. PCFT is a member of the major facilitator superfamily of transporters. Because major facilitator superfamily members exist as homo-oligomers, we tested this for PCFT because such structures could be significant to PCFT mechanism and regulation. By transiently expressing PCFT in reduced folate carrier- and PCFT-null HeLa (R1-11) cells and chemical cross-linking with 1,1-methanediyl bismethanethiosulfonate and Western blotting, PCFT species with molecular masses approximating those of the PCFT dimer and higher order oligomers were detected. Blue native polyacrylamide gel electrophoresis identified PCFT dimer, trimer, and tetramer forms. PCFT monomers with hemagglutinin and His(10) epitope tags were co-expressed in R1-11 cells, solubilized, and bound to nickel affinity columns, establishing their physical associations. Co-expressing YPet and ECFP*-tagged PCFT monomers enabled transport and fluorescence resonance energy transfer in plasma membranes of R1-11 cells. Combined wild-type (WT) and inactive mutant P425R PCFTs were targeted to the cell surface by surface biotinylation/Western blots and confocal microscopy and functionally exhibited a "dominant-positive" phenotype, implying positive cooperativity between PCFT monomers and functional rescue of mutant by WT PCFT. Our results demonstrate the existence of PCFT homo-oligomers and imply their functional and regulatory impact. Better understanding of these higher order PCFT structures may lead to therapeutic applications related to folate uptake in hereditary folate malabsorption, and delivery of PCFT-targeted chemotherapy drugs for cancer.
    No preview · Article · Dec 2011 · Journal of Biological Chemistry
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