Expression Of Proton-coupled Folate Transporter Research Articles

Functional Characterization of Reduced Folate Carrier and Protein-Coupled Folate Transporter for Antifolates Accumulation in Non-Small Cell Lung Cancer Cells.

Antifolates are important for chemotherapy in non-small cell lung cancer (NSCLC). They mainly rely on reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT) to enter cells. PCFT is supposed to be the dominant transporter of the two in tumors, as it operates optimally at acidic pH and has limited transport activity at physiological pH, whereas RFC operates optimally at neutral pH. In this study, we found RFC showed a slightly pH-dependent uptake of antifolates, with similar affinity values at pH 7.4 and 6.5. PCFT showed a highly pH-dependent uptake of antifolates, with an optimum pH of 6.0 for pemetrexed and 5.5 for methotrexate. The Michaelis-Menten constant (Km ) value of PCFT for pemetrexed at pH 7.4 was more than 10 times higher than that at pH 6.5. Interestingly, we found that antifolate accumulations mediated by PCFT at acidic pH were significantly affected by the efflux transporter, breast cancer resistance protein (BCRP). The highest pemetrexed concentration was observed at pH 7.0-7.4 after a 60-minute accumulation in PCFT-expressing cells, which was further evidenced by the cytotoxicity of pemetrexed, with the IC50 value of pemetrexed at pH 7.4 being one-third of that at pH 6.5. In addition, the in vivo study indicated that increasing PCFT and RFC expression significantly enhanced the antitumor efficacy of pemetrexed despite the high expression of BCRP. These results suggest that both RFC and PCFT are important for antifolates accumulation in NSCLC, although there is an acidic microenvironment and high BCRP expression in tumors. SIGNIFICANCE STATEMENT: Evaluating the role of reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT) on antifolates accumulation in non-small cell lung cancer (NSCLC) is necessary for new drug designs. By using cell models, we found both RFC and PCFT were important for antifolates accumulation in NSCLC. Breast cancer resistance protein (BCRP) significantly affected PCFT-mediated antifolates accumulation at acidic pH but not RFC-mediated pemetrexed accumulation at physiological pH. High expression of PCFT or RFC enhanced the cytotoxicity and antitumor effect of pemetrexed.

Cell-Free Expression of Proton-Coupled Folate Transporter in the Presence of Nanodiscs.

Proton coupled folate transporter (PCFT) is an integral membrane protein with 12 transmembrane segments localized to the plasma membrane. PCFT is the main route by which folate, vitamin B9, from dietary sources enters mammalian cells in the small intestine. Loss-of-function mutations in this membrane transport protein cause hereditary folate malabsorption, and upregulation of PCFT has been reported in cancer cells. Currently, a complete translocation mechanism of folate via PCFT is still missing. To reveal this mechanism via studies of structural architecture and structure-function relationships, soluble and stable PCFT in a phospholipid bilayer environment is needed. We therefore develop an approach to screen lipid environments in which PCFT is most soluble. Traditional in vitro expression and reconstitution into lipid bilayers of integral membrane proteins requires separate steps, which are costly and time-consuming. In this chapter, we describe a protocol for in vitro translation of PCFT into preformed lipid nanodiscs using a cell-free expression system, which helps to accelerate and reduce the cost of the sample preparation.

Intraluminal Behavior of Various Transporter Substrates in the Rat Gastrointestinal Tract.

The aim of this study was to evaluate the intraluminal behavior of various transporter substrates in different regions of the gastrointestinal (GI) tract. Drug solutions containing non-absorbable FITC-dextran 4000 (FD-4), were orally administered to rats. Residual water was sampled from the GI regions to measure the luminal drug concentration. Cephalexin (CEX), a substrate of the proton-coupled oligopeptide transporter, was absorbed rapidly, and no drug was detected in the lower small intestine. Saquinavir (SQV) was primarily absorbed in the upper region. However, unlike CEX, SQV was detected even in the lower segment probably due to the efflux of SQV via P-glycoprotein (P-gp). The concentration of methotrexate (MTX) showed a similar pattern to that of non-absorbable FD-4. The low absorption of MTX was probably due to efflux via several efflux transporters, and the limited expression of proton-coupled folate transporter, an absorptive transporter for MTX, in the upper region. This study revealed that the luminal concentration pattern of each drug differed considerably depending on the site because of the different absorption properties and luminal volumes. Although further investigation using a specific transporter inhibitor or transporter-knockout animals are necessary to clarify the actual contribution of each transporter to the drug absorption, this information will be valuable in evaluating transporter-mediated drug absorption in in vitro transport studies for ensuring optimal drug concentrations.

Impact of nanodisc lipid composition on cell-free expression of proton-coupled folate transporter.

The Proton-Coupled Folate Transporter (PCFT) is a transmembrane transport protein that controls the absorption of dietary folates in the small intestine. PCFT also mediates uptake of chemotherapeutically used antifolates into tumor cells. PCFT has been identified within lipid rafts observed in phospholipid bilayers of plasma membranes, a micro environment that is altered in tumor cells. The present study aimed at investigating the impact of different lipids within Lipid-protein nanodiscs (LPNs), discoidal lipid structures stabilized by membrane scaffold proteins, to yield soluble PCFT expression in an E. coli lysate-based cell-free transcription/translation system. In the absence of detergents or lipids, we observed PCFT quantitatively as precipitate in this system. We then explored the ability of LPNs to support solubilized PCFT expression when present during in-vitro translation. LPNs consisted of either dimyristoyl phosphatidylcholine (DMPC), palmitoyl-oleoyl phosphatidylcholine (POPC), or dimyristoyl phosphatidylglycerol (DMPG). While POPC did not lead to soluble PCFT expression, both DMPG and DMPC supported PCFT translation directly into LPNs, the latter in a concentration dependent manner. The results obtained through this study provide insights into the lipid preferences of PCFT. Membrane-embedded or solubilized PCFT will enable further studies with diverse biophysical approaches to enhance the understanding of the structure and molecular mechanism of folate transport through PCFT.

Impact of hypoxia on chemoresistance of mesothelioma mediated by the proton-coupled folate transporter, and preclinical activity of new anti-LDH-A compounds

BackgroundExpression of proton-coupled folate transporter (PCFT) is associated with survival of mesothelioma patients treated with pemetrexed, and is reduced by hypoxia, prompting studies to elucidate their correlation.MethodsModulation of glycolytic gene expression was evaluated by PCR arrays in tumour cells and primary cultures growing under hypoxia, in spheroids and after PCFT silencing. Inhibitors of lactate dehydrogenase (LDH-A) were tested in vitro and in vivo. LDH-A expression was determined in tissue microarrays of radically resected malignant pleural mesothelioma (MPM, N = 33) and diffuse peritoneal mesothelioma (DMPM, N = 56) patients.ResultsOverexpression of hypoxia marker CAIX was associated with low PCFT expression and decreased MPM cell growth inhibition by pemetrexed. Through integration of PCR arrays in hypoxic cells and spheroids and following PCFT silencing, we identified the upregulation of LDH-A, which correlated with shorter survival of MPM and DMPM patients. Novel LDH-A inhibitors enhanced spheroid disintegration and displayed synergistic effects with pemetrexed in MPM and gemcitabine in DMPM cells. Studies with bioluminescent hypoxic orthotopic and subcutaneous DMPM athymic-mice models revealed the marked antitumour activity of the LDH-A inhibitor NHI-Glc-2, alone or combined with gemcitabine.ConclusionsThis study provides novel insights into hypoxia/PCFT-dependent chemoresistance, unravelling the potential prognostic value of LDH-A, and demonstrating the preclinical activity of LDH-A inhibitors.

RX-3117 (Fluorocyclopentenyl-Cytosine)-Mediated Down-Regulation of DNA Methyltransferase 1 Leads to Protein Expression of Tumor-Suppressor Genes and Increased Functionality of the Proton-Coupled Folate Carrier.

(1) Background: RX-3117 (fluorocyclopentenyl-cytosine) is a cytidine analog that inhibits DNA methyltransferase 1 (DNMT1). We investigated the mechanism and potential of RX-3117 as a demethylating agent in several in vitro models. (2) Methods: we used western blotting to measure expression of several proteins known to be down-regulated by DNA methylation: O6-methylguanine-DNA methyltransferase (MGMT) and the tumor-suppressor genes, p16 and E-cadherin. Transport of methotrexate (MTX) mediated by the proton-coupled folate transporter (PCFT) was used as a functional assay. (3) Results: RX-3117 treatment decreased total DNA-cytosine-methylation in A549 non-small cell lung cancer (NSCLC) cells, and induced protein expression of MGMT, p16 and E-cadherin in A549 and SW1573 NSCLC cells. Leukemic CCRF-CEM cells and the MTX-resistant variant (CEM/MTX, with a deficient reduced folate carrier) have a very low expression of PCFT due to promoter hypermethylation. In CEM/MTX cells, pre-treatment with RX-3117 increased PCFT-mediated MTX uptake 8-fold, and in CEM cells 4-fold. With the reference hypomethylating agent 5-aza-2′-deoxycytidine similar values were obtained. RX-3117 also increased PCFT gene expression and PCFT protein. (4) Conclusion: RX-3117 down-regulates DNMT1, leading to hypomethylation of DNA. From the increased protein expression of tumor-suppressor genes and functional activation of PCFT, we concluded that RX-3117 might have induced hypomethylation of the promotor.

Targeted Delivery of Nanoparticulate Cytochrome C into Glioma Cells Through the Proton-Coupled Folate Transporter.

In this study, we identified the proton-coupled folate transporter (PCFT) as a route for targeted delivery of drugs to some gliomas. Using the techniques of confocal imaging, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and small interfering (siRNA) knockdown against the PCFT, we demonstrated that Gl261 and A172 glioma cells, but not U87 and primary cultured astrocytes, express the PCFT, which provides selective internalization of folic acid (FA)-conjugated cytochrome c-containing nanoparticles (FA-Cyt c NPs), followed by cell death. The FA-Cyt c NPs (100 µg/mL), had no cytotoxic effects in astrocytes but caused death in glioma cells, according to their level of expression of PCFT. Whole-cell patch clamp recording revealed FA-induced membrane currents in FA-Cyt c NPs-sensitive gliomas, that were reduced by siRNA PCFT knockdown in a similar manner as by application of FA-Cyt c NPs, indicating that the PCFT is a route for internalization of FA-conjugated NPs in these glioma cells. Analysis of human glioblastoma specimens revealed that at least 25% of glioblastomas express elevated level of either PCFT or folate receptor (FOLR1). We conclude that the PCFT provides a mechanism for targeted delivery of drugs to some gliomas as a starting point for the development of efficient methods for treating gliomas with high expression of PCFT and/or FOLR1.

High-fat diet consumption reduces hepatic folate transporter expression via nuclear respiratory factor-1.

Folate is an essential micronutrient for biological function. The liver, a primary organ for folate metabolism and storage, plays an important role in folate homeostasis. Proton-coupled folate transporter (PCFT) and reduced folate carrier (RFC) are the major folate transporters responsible for folate uptake at basolateral membrane of hepatocytes. Low serum folate levels are frequently associated with obesity. We investigated the mechanism that regulated folate status in a mouse model with diet-induced obesity. Mice (C57BL/6J) were fed a high-fat diet (60% kcal fat) for 8weeks. Mice displayed increased hepatic lipid accumulation and decreased folate levels in the liver and serum compared to mice fed a normal chow diet (10% kcal fat). High-fat diet-fed mice had low expression of PCFT and RFC and decreased nuclear respiratory factor-1 (NRF-1)/DNA-binding activity. Treatment with NRF-1 siRNA or palmitic acid reduced folate transporter expression in hepatocytes. Inhibition of NRF-1 mediated folate transporter expression significantly reduced intracellular folate levels. These results suggest that chronic consumption of high-fat diets impairs folate transporter expression via NRF-1-dependent mechanism, leading to reduced hepatic folate storage. Understanding the regulation of folate homeostasis in obesity may have an important implication in current guideline of folate intake. KEY MESSAGES: Serum and liver folate levels are decreased in diet-induced obese mice. Chronic high-fat diet consumption impairs expression of hepatic PCFT and RFC. NRF-1 regulates hepatic folate transporters expression and folate levels.

Folate transporter expression in placenta from pregnancies complicated with birth defects.

Folate plays a fundamental role for fetal development, participating in cell division, embryogenesis, and fetal growth. The fetus depends on maternal supply of folate across the placenta. The objective of this study was to compare the expression of Folate Receptor-α (FR-α), Reduced Folate Carrier (RFC), and Proton Coupled Folate Transporter (PCFT) in placentas from pregnancies complicated with birth defects (BD) and controls. Case-control study, including placentas of BD-complicated pregnancies (n = 25) and a control group (n = 25). We determined the placental expression of FR-α, RFC, and PCFT by immunohistochemistry. Optical density was measured to obtain a relative quantification of the expression. The expression of PCFT was greater in placentas from pregnancies complicated with BD than in those from the control group (p < .01). The expression of FR-α and RFC was not different between groups. The expression of PCFT in placentas from BD-complicated pregnancies is increased, possibly as an adaptive response to increase the folate flux at the maternal-fetal interface.

Evaluation of the effects of antiepileptic drugs on folic acid uptake by human placental choriocarcinoma cells

Folate status during pregnancy is important for fetal development and health. The placenta plays an important role in supplying the fetus with folate. Most women with epilepsy continue their medication during pregnancy. In the present study, we aimed to evaluate the effects of 16 antiepileptic drugs, clinically used for treatment of epilepsy, on folic acid uptake in two in vitro placental models, BeWo and JEG-3 cells. Short-term exposure to antiepileptic drugs had no effects on [3H]-folic acid uptake by BeWo cells. However, long-term exposure (24h) to valproic acid (VPA) increased [3H]-folic acid uptake by BeWo and JEG-3 cells. VPA treatment for 24h increased folate receptor-α (FRα) and proton-coupled folate transporter (PCFT) mRNA expression; however, it did not affect reduced folate carrier expression. These results suggested that the increase in folic acid uptake after exposure to VPA can be attributed to the induction of FRα and PCFT expression. Furthermore, the present study showed that exposure to clinical concentrations of oxcarbazepine and stiripentol reduced the viability of BeWo cells. Therefore, the findings of the present study may contribute to better understanding of the mechanisms of toxicity of antiepileptic drugs, and estimation of their potential risk to fetus.

Expression in Sf9 insect cells, purification and functional reconstitution of the human proton-coupled folate transporter (PCFT, SLC46A1).

The proton-coupled folate transporter (PCFT) provides an essential uptake route for the vitamin folic acid (B9) in mammals. In addition, it is currently of high interest for targeting chemotherapeutic agents to tumors due to the increased folic acid requirement of rapidly dividing tumor cells as well as the upregulated PCFT expression in several tumors. To understand its function, determination of its atomic structure and molecular mechanism of transport are essential goals that require large amounts of functional PCFT. Here, we present a high-level heterologous expression system for human PCFT using a recombinant baculovirus and Spodoptera frugiperda (Sf9) insect cells. We demonstrate folate transport functionality along the PCFT expression, isolation, and purification process. Importantly, purified PCFT transports folic acid after reconstitution. We thus succeeded in overcoming heterologous expression as a major bottleneck of PCFT research. The availability of an overexpression system for human PCFT provides the basis for future biochemical, biophysical and structural studies.

Expression, Purification and Functional Characterization of Human Proton-Coupled Folate Transporter (SLC46A1)

Folate cofactors play important roles in hundreds of metabolic reactions in a cell such as synthesis of protein and DNA precursors. The human proton-coupled folate transporter (PCFT) is the only means for absorption of dietary folates. PCFT also transports anti-cancer agents such as Pemetrexed and Methotrexate. Inadequate supply of folates and impaired PCFT function is associated with many disorders such as cancer, hereditary folate malabsorption, hypercysteinemia, heart diseases, obesity, anemia, neural tube defects and Alzheimer's disease. It is therefore important to study structural and functional characteristics of PCFT, to understand folate-homeostasis mechanisms and to develop better folate-based therapies. The primary requirement in structural and functional characterization of mammalian membrane proteins such as PCFT is the availability of good expression systems and methods to obtain large quantities of purified protein. To address this primary concern, we are establishing an efficient system to over-express PCFT and developing methods to obtain purified PCFT in good yields. We have expressed human PCFT in insect, yeast, and bacterial cells. We have successfully purified PCFT to homogeneity. Importantly, we also demonstrate that the heterologously-expressed PCFT is functional. Our findings are significant because 12-transmembrane helical, mammalian membrane proteins such as PCFT are notoriously difficult to express in versatile prokaryotic and single-celled eukaryotic systems. Furthermore, due to their predominant hydrophobic character and requirement of detergent and/or lipid environment for their stability, such membrane proteins are difficult to purify. Each of the three PCFT expression systems that we have developed offers unique advantages over another depending on the intended use of PCFT. Our findings will be significant for the advancement of knowledge concerning both the structure and function of PCFT through basic science research as well as for industrial applications including design and screening of PCFT-targeted agents.

Abstract 4335: Role of proton-coupled folate transporter expression in resistance of mesothelioma patients treated with pemetrexed

Abstract Background: Approximately 40% of malignant pleural mesothelioma (MPM) patients respond to the combination treatment with the antifolate pemetrexed (PMX) and platinum compounds. Several studies highlighted the predictive role of thymidylate synthase (TS), but additional resistance mechanisms are poorly understood. This study aimed to evaluate the role of the reduced folate carrier (RFC/SLC19A1) and proton-coupled folate transporter (PCFT/SLC46A1) as potential biological markers of intrinsic PMX-resistance in MPM. Materials and methods: PCFT promoter methylation was evaluated by combined bisulfite restriction analysis as well as MethyLight PCR in 14 cell lines and 10 MPM tissues, while PCFT and RFC mRNA levels were studied by Real-Time-PCR in MPM specimens from a test and a validation cohort of 73 and 51 PMX-treated patients, respectively. PCFT protein expression was evaluated by immunohistochemistry in a tissue microarray with representative MPM core biopsies from 35 patients. Data were analyzed by t-test, Fisher/log-rank test and Cox proportional hazards models. Results: PCFT expression inversely correlated with promoter methylation and treatment with 5-Aza-2′-deoxycytidine (5-Aza-dC) induced its marked restoration in MPM cells. Patients with low PCFT mRNA levels had significantly shorter overall survival (OS), in both the test (median OS, 11.3 vs. 17.6 months, P = 0.01) and in the validation cohort (OS, 12.6 vs. 30.3 months, P = 0.02). Multivariate analysis, including age, sex, EORTC prognostic score and histology, confirmed PCFT prognostic relevance (HR, 2.1 and 2.4 in the test and validation cohorts, respectively). Low PCFT protein expression was also associated with significantly shorter OS (11.8 vs. 30.3 months, P = 0.02). Remarkably, the subgroup of patients with both low PCFT and high TS mRNA levels (30% of the patients in the test cohort) had the worse prognosis (median OS, 5.5 months, P&amp;lt;0.01), while no correlation was found in a series of 40 MPM patients not PMX-treated. No significant associations were found between RFC and clinical outcome. Conclusions: These results define PCFT as a novel prognostic factor for MPM patients treated with PMX. Prospective trials for the validation of the prognostic/predictive role of PCFT in MPM patients treated with PMX-based regimens are warranted. Furthermore preclinical data suggest that exposure to 5-Aza-dC might restore PCFT expression and result in efficient eradication of antifolate-resistant cells. Citation Format: Elisa Giovannetti, Paolo A. Zucali, Yehuda G. Assaraf, Niccola Funel, Maria Gemelli, Michal Stark, Leticia G. Leon, Zhanjun Hou, Matteo Perrino, Larry H. Matherly, Godefridus J. Peters. Role of proton-coupled folate transporter expression in resistance of mesothelioma patients treated with pemetrexed. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4335. doi:10.1158/1538-7445.AM2015-4335

The Human Proton-Coupled Folate Transporter: Determination of Conformation and Identification of the Folate-Binding Pocket

Folate cofactors play crucial roles in hundreds of reactions in cells including DNA and protein synthesis. The human proton-coupled folate transporter (PCFT) is the only means of absorption of dietary folates in humans. PCFT expression and function is associated with many disorders including hereditary folate malabsorption, neural tube defects, Down syndrome, cancer, heart diseases, Alzheimer's and Parkinson's disease. Upregulation of PCFT expression in tumor cells is of significant consideration for development of PCFT-targeted chemotherapeutic agents. However, not much is known about the structure and function of PCFT, which contributes to the low clinical success rate of folate-based agents. To address this gap in the knowledge we performed extensive Cys-mutagenesis studies. We analyzed 40 residues towards the extracellular face of PCFT, 35 positions towards the cytoplasmic face of PCFT and 28 positions along the proposed folate-binding pocket of PCFT. Based on the accessibility studies of the extracellular face of PCFT we determined loop-helix boundaries of this face and identified the glycerol-3-phosphate transporter (PDB#1PW4) and tripeptide-proton symporter (PDB#4APS) as the best templates for modeling PCFT. Based on the accessibility studies of the cytoplasmic face of PCFT, we identified loop-helix boundaries of this face. Here, we also show that our accessibility studies support the hypothesis that PCFT is present predominantly in an inward-open conformation in the absence of substrate (pH 7.5 and no folic acid). We also show that the folate-binding pocket of PCFT is formed by residues present in PCFT transmembrane helices I, IV, V, X and XI. Our results are of high significance in understanding the details of folate-homeostasis mechanisms and in design of PCFT-targeted therapeutic and diagnostic agents.

Experimentally Defined Structural Model of the Human Proton-Coupled Folate Transporter

Folate cofactors, Vitamin B9, play crucial roles in more than a hundred one-carbon metabolism reactions in mammalian cells. Humans cannot synthesize folates de novo and absorption through the diet is the only source of this vitamin. The proton-coupled folate transporter (PCFT) mediates this uptake in the upper small intestine by a pH-dependent process. PCFT also transports folates into the central nervous system. Point mutations in the PCFT gene cause Hereditary Folate Malabsorption (HFM), with associated hematological and neurological defects due to impaired folate transport. Certain solid tumor cell lines express high levels of PCFT mRNA. Importantly, the overall expression of PCFT is limited to only certain tissues in humans. Therefore, PCFT is a molecular target for specific delivery of anti-folate chemotherapeutic agents to tumor cells. Unfortunately, the success rate of anti-folate agents to reach clinical use is very low due to their side-effects. A structural model of PCFT can aid the development of specific anti-folate agents for their PCFT-targeted delivery and thus minimize their side-effects. To optimize and verify an initial structural model of PCFT, we applied a wide array of experimental approaches: solvent accessibility profiling through substituted cysteine accessibility scanning, studying the helix packing, and assaying the functionality of PCFT mutants. We complemented the experimental data with theoretical approaches for structure prediction such as homology modeling/threading, ligand docking and an extensive review of other secondary transporters and folate transport proteins. With these combined approaches, we have developed a structural model of PCFT that accurately reflects our experimental observations. This model forms a basis to understand the impacts of HFM mutations, and to develop folate analogues for folate deficiency intervention. Additionally, it will aid the design of PCFT structure-based anti-folate agents for the treatment of cancer.

Thymidylate synthase inhibitors for thoracic tumors

Abstract Thymidylate synthase (TS) is a valid target for treatment of non-small cell lung cancer (NSCLC) and mesothelioma (MPM). The TS inhibitor pemetrexed (PMX) is now commonly used in combination with cisplatin (CDDP) or carboplatin, in the first-line setting for both of these lethal diseases. A combination of another TS inhibitor, Tomudex with CDDP, demonstrated similar activity in MPM patients. However, the efficacy of TS inhibitors is limited by uptake, metabolism and target affinity. While uptake of most antifolates is mediated by the reduced folate carrier, PMX is also a good substrate for the proton-coupled folate transporter (PCFT), which displays optimal activity at the acidic pH of the tumor microenvironment. NSCLC and MPM have a variable expression of PCFT, which might be caused by the differential methylation status of the PCFT promoter, resulting in decreased anticancer activity. PMX and TDX activity also depends on polyglutamylation, catalyzed by folylpolyglutamate synthetase (FPGS), which results in intracellular retention and thus enhanced cytotoxicity. We demonstrated that resistance to the classical antifolate methotrexate in human leukemia cells with a marked loss of FPGS activity was associated with impaired splicing of FPGS pre-mRNA. Moreover, we recently showed that the sensitivity of MPM patients to PMX-carboplatin is related to tumor TS expression. Patients with low TS mRNA levels had a significantly longer overall survival (20 vs. 7 months) compared with patients with high expression. Intriguingly, recent trials demonstrated that histology plays an important role in the sensitivity of NSCLC patients, and PMX-CDDP is now approved only for adenocarcinoma patients. These results might be partly explained by the higher TS expression detected in lung tumors of squamous histology. In summary, the clinical success of TS inhibitors may depend on biomarker-driven patient selection, and further studies should evaluate the genetic/epigenetic factors involved in the modulation of these biomarkers in the preclinical and clinical setting.

Inhibition of the Proton-Coupled Folate Transporter (PCFT-SLC46A1) by Bicarbonate and Other Anions

The proton-coupled folate transporter (PCFT) plays a key role in intestinal folate absorption, and loss-of-function mutations in the gene encoding this transporter are the molecular basis for hereditary folate malabsorption. Using a stable transfectant with high expression of PCFT, physiologic levels of bicarbonate produced potent and rapidly reversible inhibition of PCFT-mediated transport at neutral pH. Bisulfite and nitrite also inhibited PCFT function at neutral pH, whereas sulfate, nitrate, and phosphate had no impact at all. At weakly acidic pH (6.5), bisulfite and nitrite exhibited much stronger inhibition of PCFT-mediated transport, whereas sulfate and nitrate remained noninhibitory. Inhibition by bisulfite and nitrite at pH 6.5 was associated with a marked decrease in the influx Vmax and collapse of the transmembrane proton gradient attributed to the diffusion of the protonated forms into these cells. Monocarboxylates such as pyruvate and acetate also collapsed the pH gradient and were also inhibitory, whereas citrate and glycine neither altered the proton gradient nor inhibited PCFT-mediated transport. These observations add another dimension to the unfavorable pH environment for PCFT function in systemic tissues: the presence of high concentrations of bicarbonate.

Adaptive transport of folic acid across renal epithelia in folate-deficient rats

Folate (vitamin B(9)) is an essential vitamin for a wide spectrum of biochemical reactions; however, unlike bacteria and plants, mammals are devoid of folate biosynthesis and thus must obtain this cofactor from exogenous sources. The activities of folate transporters on the kidneys play an important role in conserving folate excretion and reabsorption across the apical membrane of the renal proximal tubules. The different transport system activities may become identifiable in response to external stimuli, such as folate availability and exposure to chemotherapeutic agents. We have explored the effect of folate deficiency on the activity and expression of folate transporters in rat kidneys. Wistar rats were fed a folate-containing diet (2 mg folic acid kg(-1) diet) or a folic acid-free diet over a 3-month period, and mechanisms of folate transport were studied in renal brush border membrane vesicles and basolateral membrane vesicles. The renal folate uptake process is saturable and pH dependent, and it involves the folate receptor and reduced folate carrier (RFC) systems and possibly the proton coupled folate transporter (PCFT) system. We found that folate deficiency increased the renal brush border membrane and basolateral folate uptake by increasing the number of transporter molecules. The observed up-regulation of mRNA expression was also associated with a significant increase in RFC and PCFT expression at the protein level.

Neonatal and maternal iron status, but not serum folate, is related to placental expression of the proton coupled folate transporter (PCFT)

The proton coupled folate transporter (PCFT) has been identified as a major tissue folate transporter but this protein also exhibits a low‐affinity for heme iron (Fe). This study aimed to characterize placental PCFT expression in relation to maternal and neonatal folate and Fe status in 104 pregnant adolescents (ages 13–18y) enrolled in a prospective study examining effects of maternal nutrition on neonatal health. Fe status indicators (hemoglobin, serum ferritin (SF), soluble transferrin receptor (sTfR), total body Fe) were assessed at mid‐gestation (wks 23.4±3.4), delivery (wks 39.9±1.3) and in cord blood. Expression of PCFT, heme oxygenase (HO)‐1 and HO‐2 were assessed by Western blot in placental tissue collected at delivery. Placental PCFT was associated with neonatal sTfR (p=0.01, n=91, R2=0.07) and hepcidin (p=0.04, n=83, R2=0.05). Significant relationships were found between expression of PCFT with HO‐1 (p=0.009, n=55, R2=0.12) and HO‐ 2 (p=0.006, n=50, R2=0.15). Adolescents with depleted Fe stores (SF&lt;12μg/L) at delivery had elevated PCFT expression compared to Fe sufficient adolescents (p=0.03, n=91). Placental PCFT was not related to folate levels in maternal mid‐gestation sera or in cord sera. A multivariate model that included neonatal sTfR and placental HO‐1 explained 20% variation in placental PCFT (n=48, p=0.0027). Placental PCFT expression was related to neonatal and maternal Fe status and placental HO‐1 expression, suggesting an involvement of PCFT in placental Fe utilization and transport. The lack of relationship of PCFT with maternal and cord serum folate suggests a folate‐independent regulation of PCFT expression in the human placenta. Funded by USDA: 2005‐35200 &amp; 2008‐0857Grant Funding Source : USDA

Regulation of proton-coupled folate transporter in retinal Müller cells by the antipsoriatic drug monomethylfumarate

Fumaric acid esters are used to treat psoriasis, an inflammatory skin disease characterized by keratinocyte proliferation. Inflammation and proliferation are hallmarks of retinal disease; hence, fumaric acid esters may have therapeutic value in retinal pathology. In diseased retinas, Müller glial cells (MCs) undergo reactive gliosis, a hyperproliferative state. MCs take up folate, a vitamin necessary for cell proliferation, via the proton-coupled folate transporter (PCFT). Here we examined the effect of monomethylfumarate (MMF), the active metabolite of fumaric acid esters, on expression and function of PCFT in MCs. Primary MCs, isolated from neonatal mouse retinas, were treated with MMF, and PCFT function was monitored by measuring uptake of radiolabeled methyltetrahydrofolate (MTF) at pH 5.5. Dose-response and time-course analyses were performed to identify optimal conditions for maximal effect. The influence of MMF treatment on kinetic parameters of PCFT was studied, and PCFT expression was analyzed at the mRNA and protein level. MTF uptake in MCs decreased by ˜50% following 18 h treatment with 1 mM MMF. This effect was specific to fumaric acid esters. MMF treatment decreased the maximal velocity of the transporter without altering substrate affinity. The decrease in PCFT function following MMF treatment was accompanied by attenuated PCFT expression. This is the first report that an antipsoriatic compound can regulate folate transport in MCs and may have potential for the treatment of reactive gliosis in retinal disease.