Expression Of Folate Transporters Research Articles

Antiretroviral drug dolutegravir induces inflammation at the mouse brain barriers.

Integrase strand transfer inhibitors (INSTIs) based antiretroviral therapy (ART) is currently used as first-line regimen to treat HIV infection. Despite its high efficacy and barrier to resistance, ART-associated neuropsychiatric adverse effects remain a major concern. Recent studies have identified a potential interaction between the INSTI, dolutegravir (DTG), and folate transport pathways at the placental barrier. We hypothesized that such interactions could also occur at the two major blood-brain interfaces: blood-cerebrospinal fluid barrier (BCSFB) and blood-brain barrier (BBB). To address this question, we evaluated the effect of two INSTIs, DTG and bictegravir (BTG), on folate transporters and receptor expression at the mouse BCSFB and the BBB invitro, exvivo and invivo. We demonstrated that DTG but not BTG significantly downregulated the mRNA and/or protein expression of folate transporters (RFC/SLC19A1, PCFT/SLC46A1) in human and mouse BBB models invitro, and mouse brain capillaries exvivo. Our invivo study further revealed a significant downregulation in Slc19a1 and Slc46a1 mRNA expression at the BCSFB and the BBB following a 14-day DTG oral treatment in C57BL/6 mice. However, despite the observed downregulatory effect of DTG in folate transporters/receptor at both brain barriers, a 14-day oral treatment of DTG-based ART did not significantly alter the brain folate level in animals. Interestingly, DTG treatment robustly elevated the mRNA and/or protein expression of pro-inflammatory cytokines and chemokines (Cxcl1, Cxcl2, Cxcl3, Il6, Il23, Il12) in primary cultures of mouse brain microvascular endothelial cells (BBB). DTG oral treatment also significantly upregulated proinflammatory cytokines and chemokine (Il6, Il1β, Tnfα, Ccl2) at the BCSFB in mice. We additionally observed a downregulated mRNA expression of drug efflux transporters (Abcc1, Abcc4, and Abcb1a) and tight junction protein (Cldn3) at the CP isolated from mice treated with DTG. Despite the structural similarities, BTG only elicited minor effects on the markers of interest at both the BBB and BCSFB. In summary, our current data demonstrates that DTG but not BTG strongly induced inflammatory responses in a rodent BBB and BCSFB model. Together, these data provide valuable insights into the mechanism of DTG-induced brain toxicity, which may contribute to the pathogenesis of DTG-associated neuropsychiatric adverse effect.

Antiretroviral drug dolutegravir induces inflammation at the mouse brain barriers.

Integrase strand transfer inhibitors (INSTIs) based antiretroviral therapy (ART) is currently used as first-line regimen to treat HIV infection. Despite its high efficacy and barrier to resistance, ART-associated neuropsychiatric adverse effects remain a major concern. Recent studies have identified a potential interaction between the INSTI, dolutegravir (DTG), and folate transport pathways at the placental barrier. We hypothesized that such interactions could also occur at the two major blood-brain interfaces: blood-cerebrospinal fluid barrier (BCSFB) and blood-brain barrier (BBB). To address this question, we evaluated the effect of two INSTIs, DTG and bictegravir (BTG), on folate transporters and receptor expression at the mouse BCSFB and the BBB invitro, exvivo and invivo. We demonstrated that DTG but not BTG significantly downregulated the mRNA and/or protein expression of folate transporters (RFC/SLC19A1, PCFT/SLC46A1) in human and mouse BBB models invitro, and mouse brain capillaries exvivo. Our invivo study further revealed a significant downregulation in Slc19a1 and Slc46a1 mRNA expression at the BCSFB and the BBB following a 14-day DTG oral treatment in C57BL/6 mice. However, despite the observed downregulatory effect of DTG in folate transporters/receptor at both brain barriers, a 14-day oral treatment of DTG-based ART did not significantly alter the brain folate level in animals. Interestingly, DTG treatment robustly elevated the mRNA and/or protein expression of pro-inflammatory cytokines and chemokines (Cxcl1, Cxcl2, Cxcl3, Il6, Il23, Il12) in primary cultures of mouse brain microvascular endothelial cells (BBB). DTG oral treatment also significantly upregulated proinflammatory cytokines and chemokine (Il6, Il1β, Tnfα, Ccl2) at the BCSFB in mice. We additionally observed a downregulated mRNA expression of drug efflux transporters (Abcc1, Abcc4, and Abcb1a) and tight junction protein (Cldn3) at the CP isolated from mice treated with DTG. Despite the structural similarities, BTG only elicited minor effects on the markers of interest at both the BBB and BCSFB. In summary, our current data demonstrates that DTG but not BTG strongly induced inflammatory responses in a rodent BBB and BCSFB model. Together, these data provide valuable insights into the mechanism of DTG-induced brain toxicity, which may contribute to the pathogenesis of DTG-associated neuropsychiatric adverse effect.

Abstract 4730: Pharmacodynamic determinants of anti tumor activity of SHMT2 inhibitors

Abstract These studies investigate factors that impact the antitumor activity of SHMT2 targeting including drug accumulation, metabolism, expression of one carbon (C1) related enzymes, and tumor hypoxia. Mitochondrial C1 metabolism is upregulated in a variety of cancer types including leukemias and solid tumors. The mitochondrial C1 enzymes serine hydroxymethyltransferase (SHMT) 2 and 5,10-methylene tetrahydrofolate (THF) dehydrogenase 2 (MTHFD2) are among the top 5 overexpressed metabolic enzymes in tumors vs normal tissues in a wide range of tumor types. Thus, SHMT2 and MTHFD2 are potential therapeutic targets for cancer, sparing normal tissues that are less reliant on mitochondrial C1 metabolism. SHMT2 metabolizes serine and THF to produce glycine and 5,10-methylene THF; MTHFD2 metabolizes 5,10-methylene THF to NADH and 10-formyl THF which is converted to formate by MTHFD1L. Thus, mitochondrial C1 metabolism is the principal source of C1 units for cellular biosynthesis and provides the majority of glycine for protein, purine and glutathione synthesis. We previously discovered novel 5-substituted pyrrolo[3,2-d]pyrimidine antifolate compounds which exhibit primary inhibition at mitochondrial SHMT2 along with cytosolic SHMT1 and de novo purine biosynthesis at glycinamide ribonucleotide and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferases. The lead compound AGF347 is taken up into cells by the facilitative transporters reduced folate carrier (SLC19A1) and the proton coupled folate transporter (SLC46A1) and like folate cofactors is metabolized to polyglutamate (PG) forms by folylpolyglutamate synthase (FPGS) which promotes retention in cytosolic and mitochondrial compartments and target enzyme engagement. We investigated the impact of folate transporter expression on metabolism to PGs for SHMT2 targeted antifolates. Folate transporter expression inversely correlated with anti-tumor efficacy of SHMT2 targeted antifolates and overexpression of FPGS resulted in dramatically increased sensitivities. However, for pyrazolopyran inhibitors of SHMT2 (e.g. SHIN1) which enter cells by diffusion and are not metabolized to polyglutamates, increased transporter and FPGS levels dramatically decreased anti-tumor activity. As SHMT2 is a target of HIF1α, we studied the impact of multitargeting SHMT2 and purine biosynthesis by pyrrolo[3,2-d]pyrimidine antifolates under hypoxic conditions. Our results establish that multi-targeting SHMT2 and purine biosynthesis preserves anti-tumor activity under hypoxia. Collectively, our results demonstrate that SHMT2 targeted pyrrolo[3,2-d]pyrimidine antifolates exhibit increased antitumor efficacies in cells expressing low folate transporter expression and is further enhanced by increased FPGS activity. Additionally, we find multitargeting of SHMT2 and de novo purine biosynthesis manifests as antitumor activity even under hypoxic conditions. Citation Format: Mathew Joseph Schneider, Carrie O'Connor, Xun Bao, Md. Junayed Nayeen, Zhanjun Hou, Jing Li, Aleem Gangjee, Larry Matherly. Pharmacodynamic determinants of anti tumor activity of SHMT2 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4730.

Vitamin D promotes the folate transport and metabolism in zebrafish (Danio rerio).

Vitamin D (VD) is a fat-soluble sterol that possesses a wide range of physiological functions. The present study aimed to evaluate the effects of VD on folate metabolism in zebrafish and further investigated the underlying mechanism. Wild-type (WT) zebrafish were fed with a diet containing 0 IU/kg VD3 or 800 IU/kg VD3 for 3 wk. Meanwhile, cyp2r1 mutant zebrafish with impaired VD metabolism was used as another model of VD deficiency. Our results showed that VD deficiency in zebrafish suppressed the gene expression of folate transporters, including reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT) in the intestine. Moreover, VD influenced the gene expression of several enzymes related to cellular folate metabolism in the intestine and liver of zebrafish. Importantly, VD-deficient zebrafish contained a remarkably lower level of folate content in the liver. Notably, VD was incapable of altering folate metabolism in zebrafish when gut microbiota was depleted by antibiotic treatment. Further studies proved that gut commensals from VD-deficient fish displayed a lower capacity to produce folate than those from WT fish. Our study revealed the potential correlation between VD and folate metabolism in zebrafish, and gut microbiota played a key role in VD-regulated folate metabolism in zebrafish.NEW & NOTEWORTHY Our study has identified that VD influences intestinal uptake and transport of folate in zebrafish while also altering hepatic folate metabolism and storage. Interestingly, the regulatory effects of VD on folate transport and metabolism diminished after the gut flora was interrupted by antibiotic treatment, suggesting that the regulatory effects of VD on folate metabolism in zebrafish are most likely dependent on the intestinal flora.

Effects of folate deficiency and sex on carcinogenesis in a mouse model of oral cancer.

To investigate the effects of dietary folate and sex on histopathology of oral squamous cell carcinoma in mice. Mice (C57Bl/6, 30/sex) were fed either a deficient folate or sufficient folate diet. Vehicle or 4-nitroquinoline1-oxide (50 μg/mL) in vehicle were administered in drinking water for 20 weeks, followed by 6 weeks of regular drinking water. Oral lesions were observed weekly. Tongues were studied for histopathologic changes. Immunohistochemical techniques were used to measure cell proliferation (Ki67+), and to quantify expression of folate receptor, reduced folate carrier, and proton-coupled folate transporter. T cells, macrophages, and neutrophils were counted and normalized to area. All 4NQO-treated mice developed oral tumors. Dietary folate level did not affect tumor burden. More tumors were observed on the ventral aspect of the tongue than in other locations within the oral cavity. 4-nitroquinoline-1-oxide-treated mice displayed 27%-46% significantly lower expression of all three folate transport proteins; diet and sex had no effect on folate transporter expression. T-cell and neutrophil infiltration in tongues were 9.1-fold and 18.1-fold increased in the 4-nitroquinoline-1-oxide-treated mouse tongues than in controls. Treatment with 4NQO was the primary factor in determining cancer development, decreased folate transport expression, and lymphoid cell infiltration.

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.

Stage specific gene expression of folate mediated one-carbon metabolism enzymes and transporters in buffalo oocytes and pre-implantation embryos

DNA synthesis and methylations are crucial during pre-implantation embryonic development, and are mediated by one-carbon metabolism of folates. Folates, transported into the cells via folate receptors (FOLR1 and FOLR2) and carriers (SLC19A1), are metabolized by various enzymes involved in folate-methionine cycle. However, the variations in temporal expression of folate transporters and folate-methionine cycle enzymes during pre-implantation embryo development is obscure. Thus, the present study aimed to investigate the differential expression of the genes for folate transporters and folate-methionine cycle enzymes. We also examined the expression of folate transport proteins in different pre-implantation development stages. Immature buffalo oocytes were matured in maturation medium followed by in vitro fertilization and culture at standard culture conditions. The temporal pattern of gene expression in buffalo, when compared to previous studies, indicated an inter-specific variation. The transcripts of some enzymes and folate transporters were significantly upregulated after zygotic genome activation. The transcripts as well as proteins for FOLR1, FOLR2 and SLC19A1 were present in oocytes and all the pre-implantation embryo stages. FOLR1 was present in the nuclei of different stages of developing embryos but not in the metaphase (MII) oocytes. As a result, the present study advocates the existence of active folate transport in buffalo oocytes and pre-implantation embryos. The data provided by the analysis of differential gene expression of folate transporters and metabolic enzymes would likely contribute to a better understanding of the role of folates in embryo development as well as advancements in assisted reproductive technologies.

Folate supplementation during oocyte maturation positively impacts the folate-methionine metabolism in pre-implantation embryos

Folic acid is vital for DNA synthesis and methylations through one-carbon (C1) metabolism. Thus, it is essential for cell division during embryonic development. Although the oocytes contain endogenous pool of folates for development, the present study investigated the effect of external folic acid supplementation on oocyte maturation, blastocyst development and the expression of folate transporters as well as folate metabolism enzymes in oocytes and pre-implantation embryos of goat. Immature goat oocytes, matured in maturation medium comprising different folic acid concentrations (0, 10, 50, 100 and 150 μM), were in vitro fertilized and cultured. Cumulus expansion markers (PTX3 and PTGS2) in cumulus cells were highly upregulated after 50 μM folic acid supplementation indicating higher degree of maturation. Supplementation of 50 μM folic acid during oocyte maturation resulted in significantly higher blastocyst production rate, reduction in intracellular ROS levels as well as upregulation of the transcripts for folate transporters and key folate-methionine cycle enzymes in comparison to control. The present study demonstrates the existence of active folate-methionine cycle in oocytes and pre-implantation goat embryos. Supplementation of 50 μM folic acid in maturation medium improves oocyte maturation, the blastocyst production rate, reduces ROS production as well as upregulate the expression of FOLR1 and folate metabolism enzyme, MTR.

Ischemia-Reperfusion Injury Reduces Kidney Folate Transporter Expression and Plasma Folate Levels.

Acute or chronic kidney disease can cause micronutrient deficiency. Patients with end-stage renal disease, kidney transplantation or on dialysis have reduced circulating levels of folate, an essential B vitamin. However, the molecular mechanism is not well understood. Reabsorption of folate in renal proximal tubules through folate transporters is an important process to prevent urinary loss of folate. The present study investigated the impact of acute kidney injury (AKI) on folate transporter expression and the underlying mechanism. AKI was induced in Sprague-Dawley rats that were subjected to kidney ischemia (45 min)-reperfusion (24 h). Both male and female rats displayed kidney injury and low plasma folate levels compared with sham-operated rats. The plasma folate levels were inversely correlated to plasma creatinine levels. There was a significant increase in neutrophil gelatinase-associated lipocalin (NGAL) and IL-6 mRNA expression in the kidneys of rats with ischemia-reperfusion, indicating kidney injury and increased inflammatory cytokine expression. Ischemia-reperfusion decreased mRNA and protein expression of folate transporters including folate receptor 1 (FOLR1) and reduced folate carrier (RFC); and inhibited transcription factor Sp1/DNA binding activity in the kidneys. Simulated ischemia-reperfusion through hypoxia-reoxygenation or Sp1 siRNA transfection in human proximal tubular cells inhibited folate transporter expression and reduced intracellular folate levels. These results suggest that ischemia-reperfusion injury downregulates renal folate transporter expression and decreases folate uptake by tubular cells, which may contribute to low folate status in AKI. In conclusion, ischemia-reperfusion injury can downregulate Sp1 mediated-folate transporter expression in tubular cells, which may reduce folate reabsorption and lead to low folate status.

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.

Effect of imbalance in folate and vitamin B12 in maternal/parental diet on global methylation and regulatory miRNAs

DNA methylation, a central component of the epigenetic network is altered in response to nutritional influences. In one-carbon cycle, folate acts as a one-carbon carrier and vitamin B12 acts as co-factor for the enzyme methionine synthase. Both folate and vitamin B12 are the important regulators of DNA methylation which play an important role in development in early life. Previous studies carried out in this regard have shown the individual effects of these vitamins but recently the focus has been to study the combined effects of both the vitamins during pregnancy. Therefore, this study was planned to elucidate the effect of the altered dietary ratio of folate and B12 on the expression of transporters, related miRNAs and DNA methylation in C57BL/6 mice. Female mice were fed diets with 9 combinations of folate and B12 for 4 weeks. They were mated and off-springs born (F1) were continued on the same diet for 6 weeks post-weaning. Maternal and fetal (F2) tissues were collected at day 20 of gestation. Deficient state of folate led to an increase in the expression of folate transporters in both F1 and F2 generations, however, B12 deficiency (BDFN) also led to an increase in the expression in both the generations. B12 transporters/proteins were found to be increased with B12 deficiency in F1 and F2 generations except for TC-II in the kidney which was found to be decreased in the F1 generation. miR-483 was found to be increased with all conditions of folate and B12 in both F1 and F2 generations, however, deficient conditions of B12 led to an increase in the expression of miR-221 in both F1 and F2 generations. The level of miR-133 was found to be increased in BDFN group in F1 generation however; in F2 generation the change in expression was tissue and sex-specific. Global DNA methylation was decreased with deficiency of both folate and B12 in maternal tissues (F1) but increased with folate deficiency in placenta (F1) and under all conditions in fetal tissues (F2). DNA methyltransferases were overall found to be increased with deficiency of folate and B12 in both F1 and F2 generations. Results suggest that the dietary ratio of folate and B12 resulted in altered expression of transporters, miRNAs, and genomic DNA methylation in association with DNMTs.

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.

Abstract 4791: Impact of folate transport redundancies on the therapy with tumor-targeted and untargeted antifolates

Abstract Folates participate in one-carbon (C1) transfer reactions in normal and cancer cells. Antifolate therapeutics disrupt cytosolic C1 metabolism, and have long been a mainstay for the therapy of a number of cancers. There are three major folate transporter systems in human tissues and tumors, including the reduced folate carrier (RFC), folate receptors (FRs) and proton-coupled folate transporter (PCFT). RFC is broadly expressed in tissues and tumors and is characterized by a neutral pH optimum. PCFT has more limited tissue distribution but is widely expressed in human solid tumors and exhibits an acidic pH optimum. FRα is expressed in a subset of solid tumors including epithelial ovarian cancer (EOC) but shows limited expression in most normal tissues. We previously discovered novel 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl compounds typified by AGF94 which show selective cellular uptake by FRα and/or PCFT over RFC, in contrast to the classical antifolates methotrexate (MTX) and pemetrexed (PMX) which are transported by both PCFT and RFC, and to a lesser extent by FRα. We showed that AGF94 had enhanced antitumor activities by targeting cytosolic C1 metabolism to PCFT-expressing tumors but lacking RFC, reflecting contraction of cellular tetrahydrofolate pools. In EOC cells, AGF94 showed substantial in vitro inhibition of cell proliferation independent of FRα expression, as long as PCFT was present. We systematically explored the impact of folate transporter redundancies among PCFT, FRα and RFC in determining anti-tumor efficacy of novel tumor-targeted and classical antifolates. Towards this goal, we engineered cell line models from PCFT-, FR-, and RFC-null HeLa cells to express doxycycline (DOX)-inducible FRα or RFC. We used these models to constitutively express PCFT together with DOX-inducible FRα or RFC. These were characterized for transporter expression and function, as well as intracellular folate levels, with/without DOX induction. The relative contributions of RFC, PCFT and FRα to transport function were evaluated from pH 5.5 to 7.4, with radiolabeled MTX and AGF347 which targets both cytosolic and mitochondrial C1 metabolism. We assessed antiproliferative activities of classical antifolates (PMX, MTX, PT523) versus tumor-targeted compounds (e.g., AGF94, AGF102, AGF347) in these models with/without DOX. Our results establish that co-expression of the major folate transporters can have variable and surprisingly disparate and substantial impacts on anti-tumor efficacies of both classical and tumor-targeted antifolates. Our study identified critical determinants of anti-tumor activity with classical and tumor-targeted antifolates, including relative levels of folate transporter expression and transporter specificity, and the impact of intracellular folate levels and extracellular pH. Citation Format: Zhanjun Hou, Carrie O’Connor, Changwen Ning, Adrianne Wallace-Povirk, Josephine Frühauf, Md. Junayed Nayeen, Aleem Gangjee, Larry H. Matherly. Impact of folate transport redundancies on the therapy with tumor-targeted and untargeted antifolates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4791.

Temporal expression of genes involved in folate metabolism and transport during placental development, preeclampsia and neural tube defects.

Folate is an essential micronutrient during pregnancy. The differential expression of genes related to folate transport and metabolism during the advancing gestation and pregnancy complications is not well established. Hence, we studied the gene expression of folate metabolism and transport proteins in the placenta with advancing gestation, preeclampsia and neural tube defects (NTD). The expression of folate transporters and enzymes involved in folate metabolism in the placenta with advancing gestation and pregnancy-related disorders were studied by 2-step RT-PCR. Folate levels were estimated by microbiological assay using Lactobacillus casei. Significant changes in levels of placental folate metabolizing enzymes were found in both physiological and pathological pregnancies during advancing gestation. Expression of methyltetrahydrofolate reductase (MTHFR) (p < 0.001) and cystathionine-β-synthase (CBS) (p < 0.001) was decreased while that of methionine synthase (MS) (p < 0.001) was increased with advancing gestation. A much-reduced expression of MTHFR (p < 0.01) and an abnormally high expression of methionine synthase reductase (p < 0.001) were observed in the NTD group. In NTDs, there was an adaptive up-regulation of folate transporters mainly reduced folate carrier (p < 0.001) and folate receptor alpha (p < 0.001). MTHFR expression showed a strong positive correlation (r = 0.96, p < 0.01) with folate levels in placenta. Pregnant women with preeclampsia had low expression of MS (p < 0.01) in association with low folate levels. Placental folate metabolizing enzymes exhibited a differential pattern during advancing gestation. Deficient folate status in association with alteration in expression of enzymes involved in folate metabolism might be associated with pregnancy complications such as preeclampsia and NTDs.

The Impact of Maternal Pre-Pregnancy Body Weight and Gestational Diabetes on Markers of Folate Metabolism in the Placenta.

Dietary methyl donors, including folate, may modify the placenta and size at birth but the influence of maternal body weight has not been widely investigated. We therefore examined whether maternal or fetal folate status, together with indices of placental folate transport, were modulated by either maternal pre-pregnancy body mass index (BMI i.e., overweight: 25 ≤ BMI < 30 or obesity: BMI ≥ 30 kg/m2) and/or gestational diabetes mellitus (GD). We utilised a sub-sample of 135 pregnant women participating in the Spanish PREOBE survey for our analysis (i.e., 59 healthy normal weight, 29 overweight, 22 obese and 25 GD). They were blood sampled at 34 weeks gestation, and, at delivery, when a placental sample was taken together with maternal and cord blood. Placental gene expression of folate transporters and DNA methyltransferases (DNMT) were all measured. Folate plasma concentrations were determined with an electro-chemiluminescence immunoassay. Food diaries indicated that folate intake was unaffected by BMI or GD and, although all women maintained normal folate concentrations (i.e., 5–16 ng/mL), higher BMIs were associated with reduced maternal folate concentrations at delivery. Umbilical cord folate was not different, reflecting an increased concentration gradient between the mother and her fetus. Placental mRNA abundance for the folate receptor alpha (FOLR1) was reduced with obesity, whilst DNMT1 was increased with raised BMI, responses that were unaffected by GD. Multi-regression analysis to determine the best predictors for placental FOLR1 indicated that pre-gestational BMI had the greatest influence. In conclusion, the placenta’s capacity to maintain fetal folate supply was not compromised by either obesity or GD.

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.

Down-regulation of placental folate transporters in intrauterine growth restriction

Folate deficiency in pregnancy is associated with neural tube defects, restricted fetal growth and fetal programming of diseases later in life. Fetal folate availability is dependent on maternal folate levels and placental folate transport capacity, mediated by two key transporters, Folate Receptor-α and Reduced Folate Carrier (RFC). We tested the hypothesis that intrauterine growth restriction (IUGR) is associated with decreased folate transporter expression and activity in isolated syncytiotrophoblast microvillous plasma membranes (MVM). Women with pregnancies complicated by IUGR (birth weight <3rd percentile, mean birth weight 1804±110 g, gestational age 35.7±0.61 weeks, n=25) and women delivering an appropriately-for gestational age infant (control group, birth weight 25th–75th centile, mean birth weight 2493±216 g, gestational age 33.9±0.95 weeks, n=19) were recruited and placentas were collected at delivery. MVM was isolated and folate transporter protein expression was measured using Western blot and transporter activity was determined using radiolabelled methyltetrahydrofolic acid and rapid filtration. Whereas the expression of FR-α was unaffected, MVM RFC protein expression was significantly decreased in the IUGR group (−34%, P<.05). IUGR MVM had a significantly lower folate uptake compared to the control group (−38%, P<.05). In conclusion, placental folate transport capacity is decreased in IUGR, which may contribute to the restricted fetal growth and intrauterine programming of childhood and adult disease. These findings suggest that continuation of folate supplementation in the second and third trimester is of particular importance in pregnancies complicated by IUGR.

Decreased protein expression of folate transporters in early pregnancy placentas of obese women

Decreased protein expression of folate transporters in early pregnancy placentas of obese women