Studies over the last funding period were directed to elucidating the molecular basis for transport-associated acquired resistance to antifolates in murine leukemia cells and gaining insight into the structure-function of the murine RFC (RFC). The current proposal represents an evolution of this work, based on recent studies from this laboratory, that have characterized ubiquitously expressed, prominent, folate transport activities with low-pH optima in human solid tumor cell lines that contribute to the activity of antifolates and play a role in intestinal absorption of folates. The data indicate that there are at least two types of low-pH folate transport activities typified by (i) rat intestinal epithelial cells, (IEC-6) in which the major portion, but not all, of the low pH activity is linked in some way to RFC expression and (ii) Hela cells in which the low-pH activity is entirely RFC- independent. The objectives of the proposed studies are to explore mechanisms underlying transport-related antifolate resistance in human solid tumor cell lines and to better define the basis for, and pharmacological importance of, low-pH folate transport routes and their relationship to RFC. The new generation antifolate, pemetrexed, is emphasized in these studies because of increasing evidence of its clinical utility, its apparent high affinity for low pH transporter(s), and its other novel properties. The studies proposed: (i) assess the prevalence, and characterize the properties of, folate transport activities with low-pH optima; (ii) develop RFC-deficient human solid tumor cell lines in order to distinguish between RFC-associated, and RFC-independent, low pH transport activities, to provide further insight into the structure-function of human RFC, and how this impacts on antifolate activities; (iii) select human tumor cell lines for primary resistance to pemetrexed in order to assess the roles of RFC and/or low-pH transport activities, assess cross-resistance patterns, and determine the role of putative pemetrexed enzyme targets in drug activity; (iv) clarify mechanisms by which RFC function is related to low-pH transport activity in cells of small intestinal origin, and possibly tumors that may be identified; (v) implement methodologies for cloning the low pH. RFC-independent, transporter in Hela cells. The proposed studies are supported by preliminary data and variety of cell lines that have been developed with impaired RFC function, altered low pH-transport activity, or other unique properties that will facilitate this work.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Drug Discovery and Molecular Pharmacology Study Section (DMP)
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Forry, Suzanne L
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Albert Einstein College of Medicine
Internal Medicine/Medicine
Schools of Medicine
United States
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Zhao, Rongbao; Najmi, Mitra; Aluri, Srinivas et al. (2018) Concentrative Transport of Antifolates Mediated by the Proton-Coupled Folate Transporter (SLC46A1); Augmentation by a HEPES Buffer. Mol Pharmacol 93:208-215
Zhao, Rongbao; Najmi, Mitra; Aluri, Srinivas et al. (2017) Impact of posttranslational modifications of engineered cysteines on the substituted cysteine accessibility method: evidence for glutathionylation. Am J Physiol Cell Physiol 312:C517-C526
Zhao, Rongbao; Aluri, Srinivas; Goldman, I David (2017) The proton-coupled folate transporter (PCFT-SLC46A1) and the syndrome of systemic and cerebral folate deficiency of infancy: Hereditary folate malabsorption. Mol Aspects Med 53:57-72
Aluri, Srinivas; Zhao, Rongbao; Fiser, Andras et al. (2017) Residues in the eighth transmembrane domain of the proton-coupled folate transporter (SLC46A1) play an important role in defining the aqueous translocation pathway and in folate substrate binding. Biochim Biophys Acta Biomembr 1859:2193-2202
Najmi, Mitra; Zhao, Rongbao; Fiser, Andras et al. (2016) Role of the tryptophan residues in proton-coupled folate transporter (PCFT-SLC46A1) function. Am J Physiol Cell Physiol 311:C150-7
Zhao, Rongbao; Najmi, Mitra; Fiser, Andras et al. (2016) Identification of an Extracellular Gate for the Proton-coupled Folate Transporter (PCFT-SLC46A1) by Cysteine Cross-linking. J Biol Chem 291:8162-72
Visentin, Michele; Unal, Ersin Selcuk; Najmi, Mitra et al. (2015) Identification of Tyr residues that enhance folate substrate binding and constrain oscillation of the proton-coupled folate transporter (PCFT-SLC46A1). Am J Physiol Cell Physiol 308:C631-41
Zhao, Rongbao; Visentin, Michele; Goldman, I David (2015) Determinants of the activities of antifolates delivered into cells by folate-receptor-mediated endocytosis. Cancer Chemother Pharmacol 75:1163-73
Zhao, Rongbao; Diop-Bove, Ndeye; Goldman, I David (2014) Enhanced receptor-mediated endocytosis and cytotoxicity of a folic acid-desacetylvinblastine monohydrazide conjugate in a pemetrexed-resistant cell line lacking folate-specific facilitative carriers but with increased folate receptor expression. Mol Pharmacol 85:310-21
Visentin, Michele; Unal, Ersin Selcuk; Goldman, I David (2014) The impact of 5-formyltetrahydrofolate on the anti-tumor activity of pralatrexate, as compared to methotrexate, in HeLa cells in vitro. Cancer Chemother Pharmacol 73:1055-62

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