Reduced Folate Carrier Expression Research Articles

Protective effects of pyrroloquinoline quinone in brain folate deficiency

BackgroundFolates (Vitamin B9) are critical for normal neurodevelopment and function, with transport mediated by three major pathways: folate receptor alpha (FRα), proton-coupled folate transporter (PCFT), and reduced folate carrier (RFC). Cerebral folate uptake primarily occurs at the blood-cerebrospinal fluid barrier (BCSFB) through concerted actions of FRα and PCFT, with impaired folate transport resulting in the neurological disorder cerebral folate deficiency (CFD). Increasing evidence suggests that disorders associated with CFD also present with neuroinflammation, oxidative stress, and mitochondrial dysfunction, however the role of brain folate deficiency in inducing these abnormalities is not well-understood. Our laboratory has identified the upregulation of RFC by nuclear respiratory factor 1 (NRF-1) at the blood–brain barrier (BBB) once indirectly activated by the natural compound pyrroloquinoline quinone (PQQ). PQQ is also of interest due to its anti-inflammatory, antioxidant, and mitochondrial biogenesis effects. In this study, we examined the effects of folate deficiency and PQQ treatment on inflammatory and oxidative stress responses, and changes in mitochondrial function.MethodsPrimary cultures of mouse mixed glial cells exposed to folate-deficient (FD) conditions and treated with PQQ were analyzed for changes in gene expression of the folate transporters, inflammatory markers, oxidative stress markers, and mitochondrial DNA (mtDNA) content through qPCR analysis. Changes in cellular reactive oxygen species (ROS) levels were analyzed in vitro through a DCFDA assay. Wildtype (C57BL6/N) mice exposed to FD (0 mg/kg folate), or control (2 mg/kg folate) diets underwent a 10-day (20 mg/kg/day) PQQ treatment regimen and brain tissues were collected and analyzed.ResultsFolate deficiency resulted in increased expression of inflammatory and oxidative stress markers in vitro and in vivo, with increased cellular ROS levels observed in mixed glial cells as well as a reduction of mitochondrial DNA (mtDNA) content observed in FD mixed glial cells. PQQ treatment was able to reverse these changes, while increasing RFC expression through activation of the PGC-1α/NRF-1 signaling pathway.ConclusionThese results demonstrate the effects of brain folate deficiency, which may contribute to the neurological deficits commonly seen in disorders of CFD. PQQ may represent a novel treatment strategy for disorders associated with CFD, as it can increase folate uptake, while in parallel reversing many abnormalities that arise with brain folate deficiency.

The evolution of folate supplementation - from one size for all to personalized, precision, poly-paths.

Folate is a crucial nutrient that supports physiological functions. Low folate levels is a risk factor for several diseases, including cardiovascular diseases and neural tube defects. The most used folate supplement is folic acid, a synthetic oxidative form, and folic acid grain fortification is a success story of public health. However, the metabolic conversion of folic acid to bioactive tetrahydrofolate requires several enzymes and cofactors. Therefore, these factors influence its bioavailability and efficacy. In contrast, 5-methyltetrahydrofolate is used directly and participates in one-carbon metabolism, and the use of 5-methyltetrahydrofolate as an alternative folate supplement has increased. The metabolism of 5-methyltetrahydrofolate is primarily dependent on the transmembrane transporter, reduced folate carrier (RFC), and the RFC gene SLC19A1 variant is a functional polymorphism that affects folate status indexes. Recent studies demonstrated that the expression of RFC and cystathionine β-synthase, another enzyme required for homocysteine clearance, increases significantly by supplementation with calcitriol (vitamin D3), suggesting that calcitriol intake promotes the bioavailability of folate and has synergistic effects in homocysteine clearance. The advancements in biomedical and cohort studies and clinical trials have enhanced our understanding of the critical roles of folate and the regulation of one-carbon metabolism. We anticipate that the field of folate supplementation is poised to evolve from one size for all to personalized, precision, poly-paths (3Ps), which is a critical measure to meet individual needs, maximize health benefits, and minimize side effects.

Abstract 4903: Levels of folate transporters impact the compartmentalization of one-carbon metabolism in the mitochondria vs cytosol providing a unique vulnerability to SHMT inhibition

Abstract These studies characterize the critical role of the Reduced Folate Carrier (RFC) and the Proton Coupled Folate Transporter (PCFT) as determinants of cancer cell reliance on mitochondrial vs cytosolic one carbon (C1) metabolism, to identify a unique susceptibility towards SHMT1 and SHMT2 inhibition in cancer cells. C1 metabolism is frequently reprogrammed in cancer cells to provide nucleotides, amino acids, and glutathione, and to maintain redox homeostasis for proliferation. The mitochondrial C1 converting enzymes serine hydroxymethyltransferase (SHMT) 2 and methylene tetrahydrofolate (THF) dehydrogenase 2 (MTHFD2) are among the top overexpressed metabolic enzymes in human cancers, suggesting these are important cancer-specific targets. We previously discovered novel 5-substituted pyrrolo[3,2-d]pyrimidine antifolates (with AGF347 being the lead) that potently inhibit mitochondrial SHMT2, as well as SHMT1. Folates are essential cofactors for C1 converting reactions; folates and antifolates primarily rely on the major facilitative folate transporters RFC and PCFT for internalization by tumor cells. In these studies, we explore the effects of folate transporter expression on the compartmentalization of C1 metabolism in the mitochondria and cytosol and the potential impact on SHMT1 and SHMT2 inhibition. We used a tetracycline inducible system for RFC expression in RFC/PCFT-null HeLa cells, with and without constitutive PCFT, and characterized the impact on transport and accumulation of folates and AGF347 in response to folate transporter expression. Increased accumulation of folates was observed with increasing RFC in the absence of PCFT. In the presence of PCFT, baseline folate accumulation was substantial and independent of RFC expression, indicating a major role of PCFT as a facilitative folate transporter. SHMT2 catalyzes the conversion of serine and THF to glycine and 5, 10-methylene THF in the mitochondria, while SHMT1 catalyzes the reverse reaction in the cytosol, producing THF and serine. To determine how folate transporter expression affects the C1 flux through SHMT1 vs SHMT2, [2,3,3-2H]serine tracer experiments were performed; increased mitochondrial C1 flux through SHMT2 was observed with increasing RFC in the absence of PCFT, whereas the mitochondrial flux through SHMT2 predominated in the presence of PCFT. By cell proliferation assays it was discovered that cells with lower RFC expression exhibit a hypersensitive phenotype towards AGF347 in the absence of PCFT, indicating a unique therapeutic opportunity for targeting SHMT forms in cancer cells with low folate transport activity. Here we identify RFC and PCFT as key determinants for the compartmentalization of C1 flux in the mitochondria vs the cytosol, in turn affecting the sensitivity of cancer cells towards SHMT inhibition. Citation Format: Mathew Joseph Schneider, Carrie O'connor, Xun Bao, Md. Junayed Nayeen, Zhanjun Hou, Jing Li, Aleem Gangjee, Larry H. Matherly. Levels of folate transporters impact the compartmentalization of one-carbon metabolism in the mitochondria vs cytosol providing a unique vulnerability to SHMT inhibition. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4903.

Interaction between dolutegravir and folate transporters and receptor in human and rodent placenta.

SummaryBackgroundDue to the critical role of folates in neurodevelopment, it is important to understand potential interactions between anti-HIV drugs used during pregnancy, and folate delivery pathways in the placenta. This study investigates the effect of dolutegravir (DTG) exposure on the functional expression of the reduced folate carrier (RFC), proton-coupled folate transporter (PCFT), and folate receptor-α (FRα) in the placenta.MethodsHuman placental cell lines, human placental explants, and a pregnant mouse model treated with clinically relevant concentrations of DTG were used. Gene and protein expression were assessed by qPCR, immunoblot and immunohistochemical assays. Folate transport function was measured by applying radioisotope-based transport assays.FindingsIn placental cells, clinically relevant DTG exposure for 3h or 6h was associated with a modest but significant reduction in the expression of RFC and PCFT both at the mRNA and protein levels, as well as decreased uptake of RFC and PCFT substrates [3H]-methotrexate and [3H]-folic acid, respectively. In pregnant mice, DTG administration was associated with an increase in both placental RFC and PCFT mRNA expression, accompanied by a decrease in placental FRα mRNA under folate-deficient dietary conditions.InterpretationThese findings demonstrate a potential interaction between DTG and folate transport pathways in the placenta, particularly in vivo, under folate deficient conditions, potentially impacting folate delivery to the foetus in the context of DTG-based ART during pregnancy.FundingFunded by Ontario HIV Treatment Network, grant #506657; and Eunice Kennedy Shriver National Institute of Child Health & Human Development of the National Institutes of Health, award #R01HD104553.

Folate Transporters Are Found Throughout the Colon of Humans and Their mRNA Expression Appears to Be Unaffected by Low Dose Folic Acid Supplementation

ObjectivesA large depot of folate resides in the colon and can exceed dietary intake. Little is known about the capacity of the colon to absorb folate. We aimed to determine the expression of key folate transporters, reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT), throughout the colon of healthy adults and the impact of low dose folic acid (FA) supplementation. MethodsIn this 16 wk open-labelled randomized control trial, healthy adults (n = 25) from a colonoscopy waiting list were randomized to receive daily a multivitamin plus a 400 μg FA (400FA, n = 12) or no FA supplement (0FA, n = 13). Subjects were provided with low FA bread and pasta and instructions how to follow a low FA containing diet. Six 24-hr diet recalls were administered throughout the study and blood samples were collected at baseline, 8 and 16 wk. At colonoscopy (16 wk), 4 tissue biopsies were collected from the terminal ileum, cecum, ascending and descending colon. Blood folates were determined by microbial assay; mRNA levels of folate transporters were assessed using qPCR. ResultsOne subject was removed from analysis due to missing data (0FA). No group differences in age, sex, BMI, dietary intake, vitamin B12, red blood cell (RBC) and plasma folate levels at baseline were observed. Mean ± SD FA supplement adherence was 92 ± 12% and 96 ± 9% at 8 and 16 wk, respectively. Subjects in the 400FA group had higher total folate intake (P < 0.05) and higher RBC and plasma folate levels (P < 0.01) at 8 and 16 wk compared to 0FA subjects. RBC values at 16 wk were 1764 ± 636 and 865 ± 190 nmol/L in the 400FA and 0FA group, respectively. RFC and PCFT were detected in terminal ileum and colon biopsies (n = 96) and their mRNA levels in each section did not differ between groups. However, expression of RFC was markedly higher than PCFT across all biopsy sections (P < 0.05) and highest in the terminal ileum, compared to the cecum, ascending and descending colon in both groups (P < 0.05). ConclusionsWe demonstrated expression of folate transporters, RFC and PCFT, throughout the human colon suggesting their potential contribution to overall folate absorption. mRNA expressions were not affected by a low dose FA supplement. A deeper understanding of how FA and folate status affect colonic transporter regulation may inform future revisions of folate intake recommendations. Funding SourcesNatural Sciences and Engineering Research Council.

Tumor Reliance on Cytosolic versus Mitochondrial One-Carbon Flux Depends on Folate Availability

Folate metabolism supplies one-carbon (1C) units for biosynthesis and methylation and has long been a target for cancer chemotherapy. Mitochondrial serine catabolism is considered the sole contributor of folate-mediated 1C units in proliferating cancer cells. Here, we show that under physiological folate levels in the cell environment, cytosolic serine-hydroxymethyltransferase (SHMT1) is the predominant source of 1C units in a variety of cancers, while mitochondrial 1C flux is overly repressed. Tumor-specific reliance on cytosolic 1C flux is associated with poor capacity to retain intracellular folates, which is determined by the expression of SLC19A1, which encodes the reduced folate carrier (RFC). We show that silencing SHMT1 in cells with low RFC expression impairs pyrimidine biosynthesis and tumor growth invivo. Overall, our findings reveal major diversity in cancer cell utilization of the cytosolic versus mitochondrial folate cycle across tumors and SLC19A1 expression as a marker for increased reliance on SHMT1.

Nuclear respiratory factor 1 (NRF-1) upregulates the expression and function of reduced folate carrier (RFC) at the blood-brain barrier.

Folates are important for neurodevelopment and cognitive function. Folate transport across biological membranes is mediated by three major pathways: folate receptor alpha (FRα), proton-coupled folate transporter (PCFT), and reduced folate carrier (RFC). Brain folate transport primarily occurs at the choroid plexus through FRα and PCFT; inactivation of these transport systems results in suboptimal folate levels in the cerebrospinal fluid (CSF) causing childhood neurological disorders. Our group has reported that upregulation of RFC at the blood-brain barrier (BBB) through interactions with specific transcription factors, that is, vitamin D receptor (VDR) could increase brain folate delivery. This study investigates the role of nuclear respiratory factor 1 (NRF-1) in the regulation of RFC at the BBB. Activation of NRF-1/PGC-1α signaling through treatment with its specific ligand, pyrroloquinoline quinone (PQQ), significantly induced RFC expression and transport activity in hCMEC/D3 cells. In contrast, transfection with NRF-1 or PGC-1α targeting siRNA downregulated RFC functional expression in the same cell system. Applying chromatin immunoprecipitation (ChIP) assay, we further demonstrated that PQQ treatment increased NRF-1 binding to putative NRF-1 binding sites within the SLC19A1 promoter, which encodes for RFC. Additionally, in vivo treatment of wild type mice with PQQ-induced RFC expression in isolated mouse brain capillaries. Together, these findings demonstrate that NRF-1/PGC-1α activation by PQQ upregulates RFC functional expression at the BBB and could potentially enhance brain folate uptake.

The anti-tumor activity of pralatrexate (PDX) correlates with the expression of RFC and DHFR mRNA in preclinical models of multiple myeloma.

Multiple myeloma (MM) is the second most common hematologic malignancy. While major advances have been made in the disease, it is still incurable. Although antifolate-based drugs are not commonly used to treat myeloma, new generation analogs with distinct patterns of preclinical and clinical activity may offer an opportunity to identify new classes of potentially active drugs. Pralatrexate (PDX), which was approved for the treatment of relapsed or refractory peripheral T-cell lymphoma in 2009, may be one such drug. Pralatrexate exhibits a potency and pattern of activity distinct from its predecessors like methotrexate (MTX). We sought to understand the activity and mechanisms of resistance of multiple myeloma to these drugs, which could also offer potential strategies for selective use of the drug. We demonstrate that PDX and MTX both induce a significant decrease in cell viability in the low nanomolar range, with PDX exhibiting a more potent effect. We identified a series of myeloma cell lines exhibiting markedly different patterns of sensitivity to the drugs, with some lines frankly resistant, and others exquisitely sensitive. These differences were largely attributed to the basal RFC (Reduced Folate Carrier) mRNA expression levels. RFC mRNA expression correlated directly with rates of drug uptake, with the most sensitive lines exhibiting the most significant intracellular accumulation of pralatrexate. This mechanism explains the widely varying patterns of sensitivity and resistance to pralatrexate in multiple myeloma cell lines. These findings could have implications for this class of drugs and their role in the treatment of multiple myeloma.

Decreased placental folate transporter expression and activity in first and second trimester in obese mothers

Obese women have an approximately twofold higher risk to deliver an infant with neural tube defects (NTDs) despite folate supplementation. Placental transfer of folate is mediated by folate receptor alpha (FR-α), proton coupled folate transporter (PCFT), and reduced folate carrier (RFC). Decreased placental transport may contribute to NTDs in obese women. Serum folate levels were measured and placental tissue was collected from 13 women with normal BMI (21.9±1.9) and 11 obese women (BMI 33.1±2.8) undergoing elective termination at 8–22 weeks of gestation. The syncytiotrophoblast microvillous plasma membranes (MVM) were isolated using homogenization, magnesium precipitation, and differential centrifugation. MVM expression of FR-α, PCFT and RFC was determined by western blot. Folate transport capacity was assessed using radiolabeled methyl-tetrahydrofolate and rapid filtration techniques. Differences in expression and transport capacity were adjusted for gestational age and maternal age in multivariable regression models. P<.05 was considered statistically significant. Serum folate levels were not significantly different between groups. Placental MVM folate transporter expression did not change with gestational age. MVM RFC (−19%) and FR-α (−17%) expression was significantly reduced in placentas from obese women (P<.05). MVM folate transporter activity was reduced by−52% (P<.05) in obese women. These differences remained after adjustment for gestational age. There was no difference in mTOR signaling between groups. In conclusion, RFC and FR alpha expression and transporter activity in the placental MVM are significantly reduced in obese women in early pregnancy. These results may explain the higher incidence of NTDs in infants of obese women with adequate serum folate.

Microbial Metabolic Capacity for Intestinal Folate Production and Modulation of Host Folate Receptors.

Microbial metabolites, including B complex vitamins contribute to diverse aspects of human health. Folate, or vitamin B9, refers to a broad category of biomolecules that include pterin, para-aminobenzoic acid (pABA), and glutamate subunits. Folates are required for DNA synthesis and epigenetic regulation. In addition to dietary nutrients, the gut microbiota has been recognized as a source of B complex vitamins, including folate. This study evaluated the predicted folate synthesis capabilities in the genomes of human commensal microbes identified in the Human Microbiome Project and folate production by representative strains of six human intestinal bacterial phyla. Bacterial folate synthesis genes were ubiquitous across 512 gastrointestinal reference genomes with 13% of the genomes containing all genes required for complete de novo folate synthesis. An additional 39% of the genomes had the genetic capacity to synthesize folates in the presence of pABA, an upstream intermediate that can be obtained through diet or from other intestinal microbes. Bacterial folate synthesis was assessed during exponential and stationary phase growth through the evaluation of expression of select folate synthesis genes, quantification of total folate production, and analysis of folate polyglutamylation. Increased expression of key folate synthesis genes was apparent in exponential phase, and increased folate polyglutamylation occurred during late stationary phase. Of the folate producers, we focused on the commensal Lactobacillus reuteri to examine host–microbe interactions in relation to folate and examined folate receptors in the physiologically relevant human enteroid model. RNAseq data revealed segment-specific folate receptor distribution. Treatment of human colonoid monolayers with conditioned media (CM) from wild-type L. reuteri did not influence the expression of key folate transporters proton-coupled folate transporter (PCFT) or reduced folate carrier (RFC). However, CM from L. reuteri containing a site-specific inactivation of the folC gene, which prevents the bacteria from synthesizing a polyglutamate tail on folate, significantly upregulated RFC expression. No effects were observed using L. reuteri with a site inactivation of folC2, which results in no folate production. This work sheds light on the contributions of microbial folate to overall folate status and mammalian host metabolism.

Upregulation of reduced folate carrier by vitamin D enhances brain folate uptake in mice lacking folate receptor alpha

Folates are critical for central nervous system function. Folate transport is mediated by 3 major pathways, reduced folate carrier (RFC), proton-coupled folate transporter (PCFT), and folate receptor alpha (FRα/Folr1), known to be regulated by ligand-activated nuclear receptors. Cerebral folate delivery primarily occurs at the choroid plexus through FRα and PCFT; inactivation of these transport systems can result in very low folate levels in the cerebrospinal fluid causing childhood neurodegenerative disorders. These disorders have devastating effects in young children, and current therapeutic approaches are not sufficiently effective. Our group has previously reported in vitro that functional expression of RFC at the blood-brain barrier (BBB) and its upregulation by the vitamin D nuclear receptor (VDR) could provide an alternative route for brain folate uptake. In this study, we further demonstrated in vivo, using Folr1 knockout (KO) mice, that loss of FRα led to a substantial decrease of folate delivery to the brain and that pretreatment of Folr1 KO mice with the VDR activating ligand, calcitriol (1,25-dihydroxyvitamin D3), resulted in over a 6-fold increase in [13C5]-5-formyltetrahydrofolate ([13C5]-5-formylTHF) concentration in brain tissues, with levels comparable to wild-type animals. Brain-to-plasma concentration ratio of [13C5]-5-formylTHF was also significantly higher in calcitriol-treated Folr1 KO mice (15-fold), indicating a remarkable enhancement in brain folate delivery. These findings demonstrate that augmenting RFC functional expression at the BBB could effectively compensate for the loss of Folr1-mediated folate uptake at the choroid plexus, providing a therapeutic approach for neurometabolic disorders caused by defective brain folate transport.

Expression of Folate Transport Proteins in a Mouse Model of 4NQO‐Induced Oral Cancer

IntroductionHead and neck cancer is the sixth most common cancer worldwide. Risk factors include tobacco exposure, alcohol use, male sex, and a poor diet. Administration of 4‐nitroquinoline‐1‐oxide (4NQO) in drinking water causes oral cancer in mice, and mimics the effects of tobacco use. Folate is essential for nucleotide biosynthesis as well as epigenetic regulation. Folate is imported by folate receptors (FR), proton‐coupled folate transporters (PCFT), and the reduced folate carrier (RFC). This study aimed to investigate the effects of a high saturated fat (HF) or low fat (LF) diet, sex, and 4NQO treatment on expression of folate transport proteins in mice tongues. We hypothesized that there would be a decreased expression of these proteins in the HNSCC tongue samples compared to the control tongue samples.Materials and MethodsFive week old mice (C57BI/6, 36 per sex) were provided either a low saturated fat diet (10 kcal% fat; LF) or a high fat diet (60 kcal% fat, HF). After one week, mice were then randomly assigned to 17 weeks on one of three water treatment groups: water alone (control); propylene glycol in water (1.25%, PG‐water); or 4NQO in PG‐water (50 mg/ml; 4NQO). After 17 weeks, the mice were provided water alone for six more weeks but maintained on their diet. Tongues from euthanized animals were fixed in formalin and processed for histological examination. Immunohistochemistry was performed to detect the expression of FR, PCFT, and RFC in these samples. Slides were visually graded, and the total score for the expression of each protein was acquired by adding the intensity grade (0–3) and distribution grade (0–3). Statistical analysis was performed using Graphpad Prism 7.0. All animal work was in compliance with the Institutional Animal Care and Use Committee at Midwestern University, Downers Grove, IL.ResultsCompared to the control tongues, there was decreased expression of all three folate transport proteins in tumors (p&lt;0.001). The total score (intensity + distribution) in oral cancer versus control tissue for FR, RFC, and PCFT, respectively were: 3.21 ± 0.10 vs 4.48 + 0.14; 4.38 ± 0.11 vs 5.13 ± 0.10; and 4.22 ± 0.11 vs 5.65 ± 0.69. All slides displayed a 100% distribution rate of FR, PCFT, and RFC in the epithelium. There were no significant differences in folate transport protein expression in males versus females. In the 4NQO tongues, a statistically significant decrease in FR expression was observed between the HF diet group and the LF group (3.97 ± 0.20 vs 3.05 ± 0.10, p &lt;0.0002). No differences were observed in the expression of RFC and PCFT proteins with regards to diet or sex.ConclusionWe measured decreased expression in all three folate transport proteins in oral cancers when compared to normal tongue epithelium. We speculate that mutations leading to decreased folate import can promote carcinogenesis through epigenetic mechanisms.Support or Funding InformationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Decreased expression of folate transport proteins in oral cancer

The purpose of this study was to assess the expression of the 3 major folate transporters-folate receptors (FRs), reduced folate carrier (RFC), and proton-coupled folate transporter (PCFT)-in oral squamous cell carcinoma (OSCC). We hypothesized that patterns of expression of folate transporters would be different in OSCC compared with normal oral epithelium. We used immunohistochemistry to examine the expression of FR, RFC, and PCFT in 15 primary specimens collected from patients with OSCC, 2 human cadaveric samples of OSCC, and 12 normal human cadaveric oral tissues from a medical gross anatomy laboratory. Possible correlations between the expression of each folate transporter and patients' clinical data were determined. All 3 folate transporters were highly expressed in normal oral epithelium. In contrast, OSCC samples generally demonstrated low expression of FR, RFC, and PCFT, with wide distribution in the invading cancer cells. There were no differences in folate transporter expression between OSCC samples collected from patients and from human cadavers. The lowest expression of FR and PCFT characterized less-differentiated tumors, and the lowest expression of RFC correlated with higher lymph node involvement. Human oral cancer samples expressed decreased amounts of all 3 major folate transport proteins compared with controls from normal cadaveric oral tissues.

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.

Regulation of Reduced Folate Carrier (RFC) by Vitamin D Receptor at the Blood-Brain Barrier.

Folates are essential for brain development and function. Folate transport in mammalian tissues is mediated by three major folate transport systems, i.e., reduced folate carrier (RFC), proton-coupled folate transporter (PCFT), and folate receptor alpha (FRα), known to be regulated by ligand-activated nuclear receptors, such as vitamin D receptor (VDR). Folate uptake at the choroid plexus, which requires the actions of both FRα and PCFT, is critical to cerebral folate delivery. Inactivating FRα or PCFT mutations cause severe cerebral folate deficiency resulting in early childhood neurodegeneration. The objective of this study was to investigate the role of RFC in folate uptake at the level of the blood-brain barrier (BBB) and its potential regulation by VDR. We detected robust expression of RFC in different in vitro BBB model systems, particularly in immortalized cultures of human cerebral microvascular endothelial cells (hCMEC/D3) and isolated mouse brain capillaries. [3H]-methotrexate uptake by hCMEC/D3 cells at pH 7.4 was inhibited by PT523 and pemetrexed, antifolates with high affinity for RFC. We also showed that activation of VDR through calcitriol (1,25-dihydroxyvitamin D3) exposure up-regulates RFC mRNA and protein expression as well as function in hCMEC/D3 cells and isolated mouse brain capillaries. We further demonstrated that RFC expression could be down-regulated by VDR-targeting siRNA, further confirming the role of VDR in the direct regulation of this folate transporter. Together, these data suggest that augmenting RFC functional expression could constitute a novel strategy for enhancing brain folate delivery for the treatment of neurometabolic disorders caused by loss of FRα or PCFT function.

Methotrexate influx via folate transporters into alveolar epithelial cell line A549

Methotrexate (MTX), a drug used for the treatment of certain cancers as well as rheumatoid arthritis, sometimes induces serious interstitial lung injury. Although lung toxicity of MTX is related to its accumulation, the information concerning MTX transport in the lungs is lacking. In this study, we investigated the mechanisms underlying MTX influx into human alveolar epithelial cell line A549. MTX influx into A549 cells was time-, pH-, and temperature-dependent and showed saturation kinetics. The influx was inhibited by folic acid with IC50 values of 256.1 μM at pH 7.4 and 1.6 μM at pH 5.5, indicating that the mechanisms underlying MTX influx would be different at these pHs. We then examined the role of two folate transporters in MTX influx, reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT). The expression of RFC and PCFT mRNAs in A549 cells was confirmed by reverse transcription polymerase chain reaction. In addition, MTX influx was inhibited by thiamine monophosphate, an RFC inhibitor, at pH 7.4, and by sulfasalazine, a PCFT inhibitor, at pH 5.5. These results indicated that RFC and PCFT are predominantly involved in MTX influx into A549 cells at pH 7.4 and pH 5.5, respectively.

Significance of thymidylate synthase expression for resistance to pemetrexed in pulmonary adenocarcinoma

Pemetrexed (PEM), a multi-targeted antifolate, has promising clinical activity in non-squamous non-small cell lung cancer. However, the majority of patients eventually acquire resistance to PEM. To evaluate the resistant mechanisms, the A549 lung adenocarcinoma cell line was exposed to stepwise increasing PEM concentrations of 1.6, 6.4 and 16 μM to establish three PEM-resistant lung cancer cell lines, A549/PEM-1.6, -6.4 and -16. Growth inhibition was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Expression of the genes encoding thymidylate synthase (TS), reduced folate carrier (RFC) and folypoly-γ-glutamate synthetase (FPGS) were analyzed by quantitative real-time reverse transcription polymerase chain reaction. The three A549 cell lines showed more resistance to PEM (3.7-, 17.3− and 38.0-fold, respectively) compared with that of the parental cell line, which also showed cross-resistance to cisplatin, but not to docetaxel, vinorelbine and 5-Fluorouracil (5-FU). TS gene expression was significantly increased in three PEM-resistant cells, relative to that of the parental cells, in a PEM dose-dependent manner. Knockdown of TS expression with siRNA enhanced the cytotoxicity of PEM in A549/PEM-16 cells. By contrast, the levels of RFC and FPGS gene expression in A549/PEM-1.6 and -6.4 cells were significantly decreased, whereas the levels of the two genes were restored in A549/PEM-16 cells. In summary, PEM-resistant A549 cells remained sensitive to docetaxel, vinorelbine and 5-FU. TS expression appeared to be associated with resistance to PEM, which may be a predictive marker for PEM sensitivity in lung adenocarcinoma.

Pre-Clinical Analysis Of The Novel Antifolate Pralatrexate In Multiple Myeloma Reveals Functional Biomarkers Correlated With Response

Multiple myeloma (MM) is a neoplasia of plasma cells and is the second most common hematologic malignancy. Current combination therapies for MM typically include proteasome inhibitors (PIs) and/or immunomodulatory drugs (ImiDs). Although these regimens are effective, relapse and resistance to these agents are inevitable. There is a need to develop new classes of drugs with activity against MM and to identify biomarkers associated with response to these agents, in order to tailor salvage therapy for individuals most likely to respond and minimize exposure to therapies that are unlikely to be of benefit. We investigated these principles with the novel antifolate pralatrexate (PDX) in laboratory models of MM. Antifolates such as methotrexate (MTX) are not commonly used in the treatment of MM; however, combination chemotherapy regimens containing MTX have demonstrated clinical efficacy. In addition, case reports of the application of intrathecal MTX have shown activity against MM in the central nervous system. PDX was designed to be a preferred substrate for the reduced folate carrier (RFC) and folylpolyglutamate synthetase (FPGS) as compared to MTX, and it is approved for the treatment of peripheral T cell lymphoma. PDX induced intrinsic apoptosis with greater potency than MTX in a subset of human myeloma cell lines (HMCLs). Interestingly, other HMCLs were resistance to antifolate-induced apoptosis. This dichotomy among HMCLs provided a platform to identify functional biomarkers associated with sensitivity or resistance to PDX-induced apoptosis. We examined expression of the genes that are determinants of antifolate actions: RFC, FPGS, gamma-glutamyl hydrolase (GGH), and dihydrofolate reductase (DHFR) in sensitive and resistant HMCLs. Higher pre-treatment levels of RFC mRNA by qRT-PCR correlated with increased sensitivity to PDX and higher rates of RFC-mediated transport. We then quantified mRNA for folate metabolism genes by qRT-PCR in total RNA from CD138-selected MM cells from patients and detected a broad range of expression for RFC, indicating that further studies with primary MM cells are warranted to assess RFC expression as a biomarker of antifolate sensitivity. Interestingly, PDX-resistant HMCLs also demonstrated dose-dependent increases in DHFR protein, one of the major antifolate targets, after treatment whereas PDX-sensitive HMCLs did not. Messenger RNA expression was unchanged under these conditions, suggesting a translational or post-translational mechanism for this activity. Sensitivity to PDX-induced apoptosis was not suppressed by co-incubation with recombinant human interleukin-6 or by co-culture with HS-5 bone marrow stromal cells. Finally, we used a previously validated bioluminescent xenograft mouse model to examine the effects of PDX in vivo. PDX-sensitive MM.1s cells stably-transduced with a dual cassette construct expressing both green fluorescent protein (GFP) and luciferase were introduced by tail-vein injection into NOD/Shi-scid/IL-2Rγnull (NOG) mice. These cells engrafted in the marrow of the femur, pelvis, vertebral bodies, and sternum, recapitulating human disease, and tumor burden was quantified by in vivo imaging after luciferin injection. PDX treated mice demonstrated reductions in tumor burden compared to vehicle treated controls. These results demonstrate that the novel antifolate PDX has potent activity against a subset of HMCLs in vitro and in vivo. Baseline mRNA expression of RFC and post-treatment protein levels of DHFR correlated with response in HMCLs. These findings support further clinical studies to assess the potential role of PDX in the treatment of MM coupled with correlative studies to validate the clinical utility of the biomarkers identified in HMCLs. PDX may be an effective addition to individualized therapy for relapsed multiple myeloma. Disclosures: O'Connor: Spectrum Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees; Allos Pharmaceuticals: Consultancy.

Reduced folate carrier (RFC) as a predictive marker for response to pemetrexed in advanced non-small cell lung cancer (NSCLC)

RFC is the major transport system in mammalian cells for folate cofactors and antifolate therapeutics. The aim of this study was to assess the predictive value of RFC expression in patients receiving pemetrexed for advanced NSCLC. The study was carried out in a population of 48 patients with advanced NSCLC which have received pemetrexed monotherapy in second and third line. RFC expression was assessed using a two-step model of immunohistochemical staining in paraffin-embedded tissue samples. RFC expression was detected in 16 (33 %) patients. In the global population, the median progression free survival (PFS) and the median overall survival (OS) were 3.3 and 6.5 months respectively. The subgroup of patients with expression of RFC had a tendency to better median PFS (4.5 vs 2.8 months; p = 0.926) and median OS (11.7 vs 4.8; p = 0.150). In patients with adenocarcinoma histology and RFC expression median OS after treatment with pemetrexed was 14.4 months versus 5.0 in those with adenocarcinoma but without RFC expression (p = 0.039). These results suggest the possible relation between RFC expression and response to treatment with antifolates (pemetrexed) independently of the tumor histology. Further studies are required to confirm these results.