ABSTRACT
BACKGROUND: Folates are a family of B9 vitamins essential for normal growth and development in the central nervous system (CNS). Transport of folates is mediated by three major transport proteins: folate receptor alpha (FRα), proton-coupled folate transporter (PCFT), and reduced folate carrier (RFC). Brain folate uptake occurs at the choroid plexus (CP) epithelium through coordinated actions of FRα and PCFT, or directly into brain parenchyma at the vascular blood-brain barrier (BBB), mediated by RFC. Impaired folate transport can occur due to loss of function mutations in FRα or PCFT, resulting in suboptimal CSF folate levels. Our previous reports have demonstrated RFC upregulation by nuclear respiratory factor-1 (NRF-1) once activated by the natural compound pyrroloquinoline quinone (PQQ). More recently, we have identified folate transporter localization at the arachnoid barrier (AB). The purpose of the present study was to further characterize folate transporters localization and function in AB cells, as well as their regulation by NRF-1/PGC-1α signaling and folate deficiency. METHODS: In immortalized mouse AB cells, polarized localization of RFC and PCFT was assessed by immunocytochemical analysis, with RFC and PCFT functionality examined with transport assays. The effects of PQQ treatment on changes in RFC functional expression were also investigated. Mouse AB cells grown in folate-deficient conditions were assessed for changes in gene expression of the folate transporters, and other key transporters and tight junction proteins. RESULTS: Immunocytochemical analysis revealed apical localization of RFC at the mouse AB epithelium, with PCFT localized on the basolateral side and within intracellular compartments. PQQ led to significant increases in RFC functional expression, mediated by activation of the NRF-1/PGC-1α signalling cascade. Folate deficiency led to significant increases in expression of RFC, MRP3, P-gp, GLUT1 and the tight junction protein claudin-5. CONCLUSION: These results uncover the polarized expression of RFC and PCFT at the AB, with induction of RFC functional expression by activation of the NRF-1/PGC-1α signalling pathway and folate deficiency. These results suggest that the AB may contribute to the flow of folates into the CSF, representing an additional pathway when folate transport at the CP is impaired.
Subject(s)
Folic Acid , Proton-Coupled Folate Transporter , Animals , Folic Acid/metabolism , Mice , Proton-Coupled Folate Transporter/metabolism , Proton-Coupled Folate Transporter/genetics , Folate Receptor 1/metabolism , Folate Receptor 1/genetics , Biological Transport/physiology , Reduced Folate Carrier Protein/metabolism , Reduced Folate Carrier Protein/genetics , Folic Acid Deficiency/metabolism , Blood-Brain Barrier/metabolism , Blood-Brain Barrier/drug effects , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/metabolism , Choroid Plexus/metabolismABSTRACT
Folates are essential to the maintenance of normal life activities in almost all organisms. Proton-coupled folate transporter (PCFT), belonging to the major facilitator superfamily, is one of the three major folate transporter types widely studied in mammals. However, information about plant PCFTs is limited. Here, a genome-wide identification of maize PCFTs was performed, and two PCFTs, ZmMFS_1-62 and ZmMFS_1-73, were functionally investigated. Both proteins contained the typical 12 transmembrane helixes with N- and C-termini located in the cytoplasm, and were localized in the plasma membrane. Molecular docking analysis indicated their binding activity with folates via hydrogen bonding. Interference with ZmMFS_1-62 and ZmMFS_1-73 in maize seedlings through virus-induced gene silencing disrupted folate homeostasis, mainly in the roots, and reduced tolerance to drought and salt stresses. Moreover, a molecular chaperone protein, ZmHSP20, was found to interact with ZmMFS_1-62 and ZmMFS_1-73, and interference with ZmHSP20 in maize seedlings also led to folate disruption and increased sensitivity to drought and salt stresses. Overall, this is the first report of functional identification of maize PCFTs, which play essential roles in salt and drought stress tolerance, thereby linking folate metabolism with abiotic stress responses in maize.
Subject(s)
Drought Resistance , Plant Proteins , Proton-Coupled Folate Transporter , Zea mays , Drought Resistance/genetics , Folic Acid/metabolism , Gene Expression Regulation, Plant , Plant Proteins/metabolism , Plant Proteins/genetics , Proton-Coupled Folate Transporter/metabolism , Proton-Coupled Folate Transporter/genetics , Salt Tolerance/genetics , Zea mays/metabolism , Zea mays/geneticsABSTRACT
Myelomeningocele (MMC) is a congenital disease. For a long time, molecular mechanism of MMC, the role of folate receptor and transporter proteins remain unclear. Folate from maternal lumen to developing embryo is carried out with the help of folate transporters (SLC46A1, SLC19A1, FOLH1 and SLC25A32) and folate receptor (FOLR1, FOLR2 and FOLR3). Due to the loss of function of these important genes, complications can facilitate the risk of MMC. This study focused on the mutational analysis of FOLR1 and FOLR2 genes in children suffering from MMC. Myelomeningocele is a rare disorder so twenty blood samples from the children were collected. Primers of selected exons for FOLR1 and FOLR2 genes were designed with the help of PrimerFox software. Extracted DNA was amplified, and PCR based mutational analysis was done to check any type of mutation/SNPs in these genes. Sanger sequencing method was performed to confirm mutation in FOLR1 and FOLR2 genes. The results showed that certain environmental factors (smoking, low socio-economic status of mother bearing MMC fetus) were found to be significantly (P<0.05) associated with MMC but no mutation in the selected exons of FOLR1 and FOLR2 genes was detected. Thus, genetic variations in the folate transporter gene may have no role in the progression of MMC in the studied population.
Subject(s)
Folate Receptor 2 , Meningomyelocele , Child , Humans , Meningomyelocele/genetics , Carrier Proteins/genetics , Exons/genetics , Folic Acid/metabolism , Folate Receptor 1/genetics , Proton-Coupled Folate Transporter/genetics , Folate Receptor 2/geneticsABSTRACT
Insufficient dietary folate intake, hereditary malabsorption, or defects in folate transport may lead to combined immunodeficiency (CID). Although loss of function mutations in the major intestinal folate transporter PCFT/SLC46A1 was shown to be associated with CID, the evidence for pathogenic variants of RFC/SLC19A1 resulting in immunodeficiency was lacking. We report two cousins carrying a homozygous pathogenic variant c.1042 G > A, resulting in p.G348R substitution who showed symptoms of immunodeficiency associated with defects of folate transport. SLC19A1 expression by peripheral blood mononuclear cells (PBMC) was quantified by real-time qPCR and immunostaining. T cell proliferation, methotrexate resistance, NK cell cytotoxicity, Treg cells and cytokine production by T cells were examined by flow cytometric assays. Patients were treated with and benefited from folinic acid. Studies revealed normal NK cell cytotoxicity, Treg cell counts, and naive-memory T cell percentages. Although SLC19A1 mRNA and protein expression were unaltered, remarkably, mitogen induced-T cell proliferation was significantly reduced at suboptimal folic acid and supraoptimal folinic acid concentrations. In addition, patients' PBMCs were resistant to methotrexate-induced apoptosis supporting a functionally defective SLC19A1. This study presents the second pathogenic SLC19A1 variant in the literature, providing the first experimental evidence that functionally defective variants of SLC19A1 may present with symptoms of immunodeficiency.
Subject(s)
Immunologic Deficiency Syndromes , Leucovorin , Reduced Folate Carrier Protein , Humans , Folic Acid/genetics , Folic Acid/metabolism , Leucovorin/therapeutic use , Leucovorin/metabolism , Leukocytes, Mononuclear/metabolism , Methotrexate/pharmacology , Methotrexate/therapeutic use , Proton-Coupled Folate Transporter/genetics , Proton-Coupled Folate Transporter/metabolism , Reduced Folate Carrier Protein/genetics , Immunologic Deficiency Syndromes/drug therapy , Immunologic Deficiency Syndromes/genetics , Immunologic Deficiency Syndromes/metabolismABSTRACT
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.
Subject(s)
Folic Acid Deficiency , Proton-Coupled Folate Transporter , Animals , Folic Acid/metabolism , Folic Acid Deficiency/metabolism , Lipids , Mammals/metabolism , Mutation , Proton-Coupled Folate Transporter/chemistry , Proton-Coupled Folate Transporter/genetics , Proton-Coupled Folate Transporter/metabolismABSTRACT
BACKGROUND: Hereditary folate malabsorption-a rare disorder caused by impairment of the folate transporter-can develop into severe folate deficiency manifesting as megaloblastic anemia and occasionally thrombocytopenia. Reportedly, megaloblastic anemia can manifest with hemorrhagic episodes, possibly due to ineffective platelet production and platelet dysfunction. However, life-threatening hemorrhage events in hereditary folate malabsorption have not been well investigated. CASE PRESENTATION: A 3-month-old Japanese boy was transferred to our hospital due to thrombocytopenia and severe megaloblastic anemia. During a thorough examination of hematopoietic abnormalities, the patient suddenly went into cardiac arrest due to pulmonary hemorrhage. Although intravenous folate supplementation was started soon after the identification of folate deficiency, the patient died of circulatory defect and multiple organ failure. The cause of pulmonary hemorrhage, such as respiratory infection, could not be confirmed. Genetic investigation revealed a mutation in the SLC46A1 gene to be the cause of the hereditary folate malabsorption. CONCLUSION: We report an infantile case of hereditary folate malabsorption that progressed to lethal pulmonary hemorrhage before folate deficiency was identified. Clinicians should consider that megaloblastic anemia could lead to severe bleeding without warning, and that nutrient supplementation should be initiated as soon as possible.
Subject(s)
Anemia, Megaloblastic , Thrombocytopenia , Anemia, Megaloblastic/etiology , Folic Acid/therapeutic use , Folic Acid Deficiency , Hemorrhage/etiology , Humans , Infant , Malabsorption Syndromes , Male , Proton-Coupled Folate Transporter/genetics , Thrombocytopenia/complicationsABSTRACT
Background: Drug metabolism genes are involved in the in vivo metabolic processing of drugs. In previous research, we found that a high-fat diet affected the transcript levels of mouse hepatic genes responsible for drug metabolism. Aims: Our research intends to discover the drug metabolism genes that are dysregulated at the transcriptome level in nonalcoholic fatty liver disease (NAFLD). Methods: We analyzed the transcriptome for drug metabolism genes of 35 human liver tissues obtained during laparoscopic cholecystectomy. Additionally, we imported transcriptome data from mice fed a high-fat diet in previous research and two open-access Gene Expression Omnibus (GEO) datasets (GSE63067 and GSE89632). Then, using quantitative real-time polymerase chain reaction (qRT-PCR), we cross-linked the differentially expressed genes (DEGs) in clinical and animal samples and validated the common genes. Results: In this study, we identified 35 DEGs, of which 33 were up-regulated and two were down-regulated. Moreover, we found 71 DEGs (39 up- and 32 down-regulated), 276 DEGs (157 up- and 119 down-regulated), and 158 DEGs (117 up- and 41 down-regulated) in the GSE63067, GSE89632, and high-fat diet mice, respectively. Of the 35 DEGs, nine co-regulated DEGs were found in the Venn diagram (CYP20A1, CYP2U1, SLC9A6, SLC26A6, SLC31A1, SLC46A1, SLC46A3, SULT1B1, and UGT2A3). Conclusion: Nine significant drug metabolism genes were identified in NAFLD. Future research should investigate the impacts of these genes on drug dose adjustment in patients with NAFLD. Clinical Trial Registration: http://www.chictr.org.cn, identifier ChiCTR2100041714.
Subject(s)
Non-alcoholic Fatty Liver Disease , Humans , Mice , Animals , Non-alcoholic Fatty Liver Disease/genetics , Non-alcoholic Fatty Liver Disease/metabolism , Transcriptome , Gene Expression Profiling , Diet, High-Fat/adverse effects , Cytochrome P450 Family 2/genetics , Cytochrome P450 Family 2/metabolism , Proton-Coupled Folate Transporter/genetics , Proton-Coupled Folate Transporter/metabolism , Sulfate Transporters/genetics , Sulfate Transporters/metabolism , Antiporters/genetics , Antiporters/metabolismABSTRACT
This study revealed the importance of serine 318 (S318) residue for proton-coupled folate transporter (PCFT, SLC46A1) functioning. Substitution of S318 with arginine or lysine impaired transport of methotrexate (MTX), but substitution with alanine (has a simple side chain structure), or cysteine (structurally similar to serine), had no significant effect on MTX transport. The initial uptake rate of MTX by S318A and S318C mutant at pH 5.0, followed by Michaelis-Menten kinetics with a Km value of approximately 2.3 µM (for S318A) and 2.9 µM (for S318C), was similar to that of the wild-type. The normalized Vmax value of the S318A mutant, calculated by dividing the Vmax value by the Western blot protein band's relative intensity, was approximately 2-fold greater than that of the wild-type. The normalized Vmax value of the S318C mutant was approximately 0.8-fold smaller than the wild-type. Results obtained showed that the substitution of S318 with basic amino acid residues results in the loss of transport activity, even though PCFT mutants are expressed at the cell membrane. Alternatively, the substitution of S318 with neutral amino acids did not significantly affect the transport function of PCFT.
Subject(s)
Methotrexate , Proton-Coupled Folate Transporter , Folic Acid , HeLa Cells , Humans , Proton-Coupled Folate Transporter/genetics , Serine/geneticsABSTRACT
Valproate (VPA), an antiepileptic drug, is known to inhibit histone deacetylases (HDACs). Exposure to VPA during pregnancy increases several fetal risks. The maintenance of folate level during pregnancy is essential for adequate fetal development, and the placenta plays a critical role in supplying nutrients to the fetus. The aim of this study was to elucidate the effects of VPA on the gene expression of folate carriers and metabolizing enzymes in the rat placenta at both mid and late gestation periods. Pregnant rats were orally administered VPA on a single day or 4 days (repeated administration). Gene expression of folate carriers (Folr1, Slc19a1, Slc46a1) and metabolizing enzymes (Cth, Mtr, Mtrr, Mthfr, Dhfr) was assessed in the placenta on gestational day (GD) 13 or GD20. In the control rats, the expression of Folr1, Slc46a1, Cth, and Mthfr tended to be upregulated, whereas that of Mtrr and Dhfr was downregulated during gestation; the expression of Slc19a1 and Mtr did not change. Repeated VPA administration reduced the placental expression of Folr1and Mtr on GD20 and increased the expression of Dhfr on GD13 compared with the control. These findings indicate that administration of VPA alters the placental gene expression of folate carriers and metabolism-related enzymes.
Subject(s)
Placenta , Valproic Acid , Animals , Anticonvulsants/therapeutic use , Female , Folic Acid , Histone Deacetylase Inhibitors/pharmacology , Membrane Transport Proteins , Minor Histocompatibility Antigens , Pregnancy , Proton-Coupled Folate Transporter/genetics , Rats , Reduced Folate Carrier Protein/geneticsSubject(s)
Folic Acid Deficiency/genetics , Malabsorption Syndromes/genetics , Consanguinity , Female , Folic Acid/cerebrospinal fluid , Folic Acid/metabolism , Folic Acid/therapeutic use , Folic Acid Deficiency/diagnosis , Humans , Infant , Magnetic Resonance Imaging , Malabsorption Syndromes/diagnosis , Microcephaly/genetics , Neurologic Examination , Proton-Coupled Folate Transporter/genetics , Seizures/etiology , Tomography, X-Ray Computed , Treatment OutcomeABSTRACT
There are three major folate uptake systems in human tissues and tumors, including the reduced folate carrier (RFC), folate receptors (FRs) and proton-coupled folate transporter (PCFT). We studied the functional interrelationships among these systems for the novel tumor-targeted antifolates AGF94 (transported by PCFT and FRs but not RFC) and AGF102 (selective for FRs) versus the classic antifolates pemetrexed, methotrexate and PT523 (variously transported by FRs, PCFT and RFC). We engineered HeLa cell models to express FRα or RFC under control of a tetracycline-inducible promoter with or without constitutive PCFT. We showed that cellular accumulations of extracellular folates were determined by the type and levels of the major folate transporters, with PCFT and RFC prevailing over FRα, depending on expression levels and pH. Based on patterns of cell proliferation in the presence of the inhibitors, we established transport redundancy for RFC and PCFT in pemetrexed uptake, and for PCFT and FRα in AGF94 uptake; uptake by PCFT predominated for pemetrexed and FRα for AGF94. For methotrexate and PT523, uptake by RFC predominated even in the presence of PCFT or FRα. For both classic (methotrexate, PT523) and FRα-targeted (AGF102) antifolates, anti-proliferative activities were antagonized by PCFT, likely due to its robust activity in mediating folate accumulation. Collectively, our findings describe a previously unrecognized interplay among the major folate transport systems that depends on transporter levels and extracellular pH, and that determines their contributions to the uptake and anti-tumor efficacies of targeted and untargeted antifolates.
Subject(s)
Folate Receptor 1/genetics , Folic Acid/metabolism , Neoplasms/drug therapy , Proton-Coupled Folate Transporter/genetics , Reduced Folate Carrier Protein/genetics , Biological Transport/genetics , Cell Proliferation/drug effects , Folate Receptor 1/metabolism , Folic Acid/genetics , Folic Acid Antagonists/pharmacology , HeLa Cells , Humans , Methotrexate/pharmacology , Neoplasms/genetics , Neoplasms/metabolism , Ornithine/analogs & derivatives , Ornithine/pharmacology , Pemetrexed/pharmacology , Proton-Coupled Folate Transporter/metabolism , Pterins/pharmacology , Reduced Folate Carrier Protein/metabolismABSTRACT
The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes hepatic toxicity associated with prominent lipid accumulation in humans. Here, the authors report that the lysosomal copper transporter SLC46A3 is induced by TCDD and underlies the hepatic lipid accumulation in mice, potentially via effects on mitochondrial function. SLC46A3 was localized to the lysosome where it modulated intracellular copper levels. Forced expression of hepatic SLC46A3 resulted in decreased mitochondrial membrane potential and abnormal mitochondria morphology consistent with lower copper levels. SLC46A3 expression increased hepatic lipid accumulation similar to the known effects of TCDD exposure in mice and humans. The TCDD-induced hepatic triglyceride accumulation was significantly decreased in Slc46a3-/- mice and was more pronounced when these mice were fed a high-fat diet, as compared to wild-type mice. These data are consistent with a model where lysosomal SLC46A3 induction by TCDD leads to cytosolic copper deficiency resulting in mitochondrial dysfunction leading to lower lipid catabolism, thus linking copper status to mitochondrial function, lipid metabolism and TCDD-induced liver toxicity.
Subject(s)
Copper Transport Proteins/metabolism , Copper/metabolism , Cytosol/metabolism , Homeostasis , Lysosomes/metabolism , Proton-Coupled Folate Transporter/metabolism , Animals , Copper Transport Proteins/genetics , Cytosol/drug effects , Green Fluorescent Proteins/metabolism , Hepatocytes/drug effects , Hepatocytes/metabolism , Hepatocytes/ultrastructure , Homeostasis/drug effects , Ions , Liver/metabolism , Lysosomes/drug effects , Male , Membrane Potential, Mitochondrial/drug effects , Mice, Inbred C57BL , Mice, Knockout , Models, Biological , Polychlorinated Dibenzodioxins/toxicity , Proton-Coupled Folate Transporter/genetics , Receptors, Aryl Hydrocarbon/metabolism , Substrate Specificity/drug effects , Superoxide Dismutase/metabolism , Triglycerides/metabolismABSTRACT
BACKGROUND AND OBJECTIVE: Membrane solute carrier transporters play an important role in the transport of a wide spectrum of substrates including anticancer drugs and cancer-related physiological substrates. This study aimed to assess the prognostic relevance of gene expression and genetic variability of selected solute carrier transporters in breast cancer. METHODS: Gene expression was determined by quantitative real-time polymerase chain reaction. All SLC46A1 and SLCO1A2 exons and surrounding non-coding sequences in DNA extracted from the blood of patients with breast cancer (exploratory phase) were analyzed by next-generation sequencing technology. Common variants (minor allele frequency ≥ 5%) with in silico-predicted functional relevance were further analyzed in a large cohort of patients with breast cancer (n = 815) and their prognostic and predictive potential was estimated (validation phase). RESULTS: A gene expression and bioinformatics analysis suggested SLC46A1 and SLCO1A2 to play a putative role in the prognosis of patients with breast cancer. In total, 135 genetic variants (20 novel) were identified in both genes in the exploratory phase. Of these variants, 130 were non-coding, three missense, and two synonymous. One common variant in SLCO1A2 and four variants in SLC46A1 were predicted to be pathogenic by in silico programs and subsequently validated. A SLC46A1 haplotype block composed of rs2239911-rs2239910-rs8079943 was significantly associated with ERBB2/HER2 status and disease-free survival of hormonally treated patients. CONCLUSIONS: This study revealed the prognostic value of a SLC46A1 haplotype block for breast cancer that should be further studied.
Subject(s)
Breast Neoplasms/genetics , Genetic Variation , Organic Anion Transporters/genetics , Proton-Coupled Folate Transporter/genetics , Biomarkers, Tumor/genetics , Female , Gene Expression Regulation, Neoplastic , Haplotypes , High-Throughput Nucleotide Sequencing , Humans , Prognosis , Real-Time Polymerase Chain Reaction , Sequence Analysis, DNA , Survival AnalysisABSTRACT
Potential risks to the fetus or infant should be considered prior to medication during pregnancy and lactation. It is essential to evaluate the exposure levels of drugs and their related factors in addition to toxicological effects. Epilepsy is one of the most common neurological complications in pregnancy; some women continue to use antiepileptic drugs (AEDs) to control seizures. Benzodiazepines (BZDs) are widely prescribed for several women who experience symptoms such as anxiety and insomnia during the postpartum period. In this review, we describe the 1) transport mechanisms of AEDs across the placenta and the effects of these drugs on placental transporters, and 2) the transfer of BZDs into breast milk. Our findings indicated that carrier systems were involved in the uptake of gabapentin (GBP) and lamotrigine (LTG) in placental trophoblast cell lines. SLC7A5 was the main contributor to GBP transport in placental cells. LTG was transported by a carrier that was sensitive to chloroquine, imipramine, quinidine, and verapamil. Short-term exposure to 16 AEDs had no effect on folic acid uptake in placental cells. However, long-term exposure to valproic acid (VPA) affected the expression of folate carriers (FOLR1, SLC46A1). Furthermore, VPA administration changed the expression levels of various transporters in rat placenta, suggesting that sensitivity to VPA differed across gestational stages. Lastly, we developed a method for quantifying eight BZDs in human breast milk and plasma using LC/MS/MS, and successfully applied it to quantify alprazolam in breast milk and plasma donated by a lactating woman.
Subject(s)
Anticonvulsants/metabolism , Benzodiazepines/metabolism , Biological Transport/genetics , Breast Feeding , Gabapentin/metabolism , Lactation/metabolism , Lamotrigine/metabolism , Large Neutral Amino Acid-Transporter 1/physiology , Maternal-Fetal Exchange , Milk, Human/metabolism , Placenta/metabolism , Valproic Acid/metabolism , Anticonvulsants/adverse effects , Benzodiazepines/adverse effects , Benzodiazepines/therapeutic use , Cell Line , Epilepsy/drug therapy , Female , Folate Receptor 1/genetics , Folate Receptor 1/metabolism , Gabapentin/adverse effects , Gene Expression/drug effects , Humans , Lamotrigine/adverse effects , Pregnancy , Pregnancy Complications/drug therapy , Proton-Coupled Folate Transporter/genetics , Proton-Coupled Folate Transporter/metabolism , Valproic Acid/adverse effectsABSTRACT
The proton-coupled folate transporter (PCFT, SLC46A1) is required for folate intestinal absorption and transport across the choroid plexus. Recent work has identified a F392V mutation causing hereditary folate malabsorption. However, the residue properties responsible for this loss of function remains unknown. Using site-directed mutagenesis, we observed complete loss of function with charged (Lys, Asp, and Glu) and polar (Thr, Ser, and Gln) Phe-392 substitutions and minimal function with some neutral substitutions; however, F392M retained full function. Using the substituted-cysteine accessibility method (with N-biotinyl aminoethyl methanethiosulfonate labeling), Phe-392 mutations causing loss of function, although preserving membrane expression and trafficking, also resulted in loss of accessibility of the substituted cysteine in P314C-PCFT located within the aqueous translocation pathway. F392V function and accessibility of the P314C cysteine were restored by insertion of a G305L (suppressor) mutation. A S196L mutation localized in proximity to Gly-305 by homology modeling was inactive. However, when inserted into the inactive F392V scaffold, function was restored (mutually compensatory mutations), as was accessibility of the P314C cysteine residue. Reduced function, documented with F392H PCFT, was due to a 15-fold decrease in methotrexate influx Vmax, accompanied by a decreased influx Kt (4.5-fold) and Ki (3-fold). The data indicate that Phe-392 is required for rapid oscillation of the carrier among its conformational states and suggest that this is achieved by dampening affinity of the protein for its folate substrates. F392V and other inactivating Phe-392 PCFT mutations lock the protein in its inward-open conformation. Reach (length) and hydrophobicity of Phe-392 appear to be features required for full activity.
Subject(s)
Proton-Coupled Folate Transporter/metabolism , Amino Acid Sequence , Animals , Biological Transport , Cysteine/chemistry , Cysteine/metabolism , Folic Acid Deficiency/pathology , HeLa Cells , Humans , Kinetics , Malabsorption Syndromes/pathology , Methotrexate/metabolism , Mutagenesis, Site-Directed , Protein Structure, Tertiary , Proton-Coupled Folate Transporter/chemistry , Proton-Coupled Folate Transporter/geneticsABSTRACT
BACKGROUND: Expression 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. METHODS: Modulation 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. RESULTS: Overexpression 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. CONCLUSIONS: This 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.
Subject(s)
Antigens, Neoplasm/genetics , Carbonic Anhydrase IX/genetics , Enzyme Inhibitors/administration & dosage , L-Lactate Dehydrogenase/genetics , Mesothelioma, Malignant/drug therapy , Peritoneal Neoplasms/drug therapy , Pleural Neoplasms/drug therapy , Proton-Coupled Folate Transporter/genetics , Animals , Antigens, Neoplasm/metabolism , Carbonic Anhydrase IX/metabolism , Cell Culture Techniques , Cell Hypoxia , Cell Line, Tumor , Deoxycytidine/administration & dosage , Deoxycytidine/analogs & derivatives , Deoxycytidine/pharmacology , Drug Resistance, Neoplasm/drug effects , Drug Synergism , Enzyme Inhibitors/pharmacology , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Mesothelioma, Malignant/genetics , Mesothelioma, Malignant/metabolism , Mice , Pemetrexed/administration & dosage , Pemetrexed/pharmacology , Peritoneal Neoplasms/genetics , Peritoneal Neoplasms/metabolism , Pleural Neoplasms/genetics , Pleural Neoplasms/metabolism , Proton-Coupled Folate Transporter/metabolism , Xenograft Model Antitumor Assays , GemcitabineABSTRACT
(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.
Subject(s)
Cytidine/analogs & derivatives , DNA (Cytosine-5-)-Methyltransferase 1/metabolism , Proton-Coupled Folate Transporter/metabolism , Tumor Suppressor Proteins/metabolism , Cell Line, Tumor , Cytidine/pharmacology , DNA (Cytosine-5-)-Methyltransferase 1/genetics , DNA Methylation/drug effects , Dose-Response Relationship, Drug , Gene Expression Regulation/drug effects , Humans , Methotrexate/pharmacology , Proton-Coupled Folate Transporter/genetics , Tumor Suppressor Proteins/geneticsABSTRACT
Folate receptor alpha (FRα) is overexpressed in >80% of epithelial ovarian cancer (EOC). Accordingly, folate is attracting attention as a targeting ligand for EOC. For EOC patients, paclitaxel (PTX) is generally used as a first-line chemotherapeutic agent in combination with platinum-based drugs. Cyclodextrin (CyD) is a potential new formulation vehicle for PTX that could replace Cremophor-EL, a traditional formulation vehicle that causes significant side effects, including neutropenia. Several years ago, folate-appended ß-CyD (Fol-c1 -ß-CyD) was developed as an FRα-targeting drug carrier, but its efficacy as a treatment for EOC remains to be determined. In this study, we assessed the antitumor activity of PTX in Fol-c1 -ß-CyD (PTX/Fol-c1 -ß-CyD) in EOC-derived cell lines. We found that PTX/Fol-c1 -ß-CyD killed not only FRα-expressing cells but also FRα-negative cells. In the FRα-negative A2780 cells, knockdown of proton-coupled folate transporter (PCFT) significantly decreased the cytotoxicity of PTX/Fol-c1 -ß-CyD, whereas knockdown of FRα did not. By contrast, knockdown of either FRα or proton-coupled folate transporter (PCFT) decreased the cytotoxicity of PTX/Fol-c1 -ß-CyD in FRα-expressing SK-OV-3 cells. Furthermore, the cytotoxicity of PTX/Fol-c1 -ß-CyD in A2780 cells was increased at acidic pH, and this increase was suppressed by PCFT inhibitor. In mice intraperitoneally inoculated with FRα-expressing or PCFT-expressing EOC cells, intraperitoneal administration of PTX/Fol-c1 -ß-CyD significantly suppressed the growth of both types of EOC cells relative to PTX alone, without inducing a significant change in the neutrophil/white blood cell ratio. Our data suggest that Fol-c1 -ß-CyD targets not only FRα but also PCFT, and can efficiently deliver anticancer drugs to EOC cells in the peritoneal cavity.
Subject(s)
Carcinoma, Ovarian Epithelial/metabolism , Drug Carriers/chemistry , Drug Delivery Systems/methods , Folic Acid/chemistry , Ovarian Neoplasms/metabolism , Proton-Coupled Folate Transporter/metabolism , beta-Cyclodextrins/chemistry , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Carcinoma, Ovarian Epithelial/drug therapy , Carcinoma, Ovarian Epithelial/pathology , Cell Line, Tumor , Cell Survival/drug effects , Drug Carriers/administration & dosage , Female , Folate Receptor 1/genetics , Folate Receptor 1/metabolism , Folic Acid/administration & dosage , Gene Expression , Humans , Mice , Molecular Structure , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/pathology , Paclitaxel/administration & dosage , Paclitaxel/chemistry , Paclitaxel/pharmacology , Proton-Coupled Folate Transporter/genetics , Xenograft Model Antitumor Assays , beta-Cyclodextrins/administration & dosageABSTRACT
Human proton-coupled folate transporter (hPCFT/SLC46A1) has recently been found to be inhibited by myricetin by a sustained mechanism, raising a concern that the inhibition might lead to malabsorption of folates in the intestine, where hPCFT works for their epithelial uptake. However, rat PCFT (rPCFT) has more recently been found not to be inhibited by myricetin. Prompted by this finding, we attempted to determine the amino acid residue involved in that by analyses comparing between hPCFT and rPCFT. In the initial analysis, chimeric constructs prepared from hPCFT and rPCFT were examined for myricetin sensitivity to determine the hPCFT segment involved in the sensitivity. Focusing on the thereby determined segment from 83rd to 186th amino acid residue, hPCFT mutants having a designated amino acid residue replaced with its counterpart in rPCFT were prepared for the subsequent analysis. Among them, only G158N-substituted hPCFT was found to be transformed to be insensitive to myricetin and, accordingly, oppositely N158G-substituted rPCFT was transformed to be sensitive to myricetin. These results indicate the critical role of Gly158 in the myricetin sensitivity of hPCFT. This finding would help advance the elucidation of the mechanism of the myricetin-induced inhibition of hPCFT and manage the potential risk arising from that.
Subject(s)
Flavonoids/pharmacology , Glycine/genetics , Proton-Coupled Folate Transporter/genetics , Amino Acid Substitution , Folic Acid/pharmacokinetics , HEK293 Cells , Humans , Intestinal Absorption/drug effects , Intestinal Absorption/physiology , Mutagenesis, Site-Directed , Proton-Coupled Folate Transporter/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
Offspring of dams exposed to excess folic acid during the perigestational period have been shown by us to be predisposed to metabolic dysfunction revealed by hyperglycemia, glucose intolerance, increased insulin and decreased adiponectin in late adulthood. This work aims to characterize adipocyte phenotype and expression profile of genes in the regulation of lipid and glucose metabolism in visceral adipose tissue and in skeletal muscle. From mating until weaning, a recommended dose of folic acid for pregnancy (C, 2 mg of folic acid per kg of diet) or a high folic acid dose (HFA, 40 mg of folic acid per kg of diet) was administered to Sprague-Dawley females. At 10 months of age progeny were divided into groups fed the standard chow (C/STD and HFA/STD) and groups fed the standard chow plus drinking water with 10% fructose (C/FRU and HFA/FRU), as an additional metabolic challenge. Adipocyte morphology and quantification of key genes involved in lipid and glucose metabolism were studied in visceral adipose tissue and skeletal muscle of 13 months old offspring. HFA exposure led to an enlargement of visceral adipose cells most likely mediated by an upregulation of lipoprotein lipase, and it tended to downregulate Glut4 in visceral adipose tissue and skeletal muscle. Fructose exposure in a background of perigestational excess folic acid, but not in controls, induced an upregulation of lipogenesis pathway genes and it decreased jejunal expression of the proton-coupled folate transporter (Pcft1). In addition, fructose exposure led to a downregulation of jejunal Sglt1 in control animals. Our data suggest that high folic acid exposure during the perigestational period caused morphologic and genic alterations related to insulin resistant states indicating that this intervention may act as an effective programmer of long-term metabolic dysfunction.