Noncanonical function of folate through folate receptor 1 during neural tube formation.

Folate supplementation reduces the occurrence of neural tube defects (NTDs), birth defects consisting in the failure of the neural tube to form and close. The mechanisms underlying NTDs and their prevention by folate remain unclear. Here we show that folate receptor 1 (FOLR1) is necessary for the formation of neural tube-like structures in human-cell derived neural organoids. FOLR1 knockdown in neural organoids and in Xenopus laevis embryos leads to NTDs that are rescued by pteroate, a folate precursor that is unable to participate in metabolism. We demonstrate that FOLR1 interacts with and opposes the function of CD2-associated protein, molecule essential for apical endocytosis and turnover of C-cadherin in neural plate cells. In addition, folates increase Ca2+ transient frequency, suggesting that folate and FOLR1 signal intracellularly to regulate neural plate folding. This study identifies a mechanism of action of folate distinct from its vitamin function during neural tube formation.

Folate regulation of planar cell polarity pathway and F-actin through folate receptor alpha.

Folate deficiency contribute to neural tube defects (NTDs) which could be rescued by folate supplementation. However, the underlying mechanisms are still not fully understood. Besides, there is considerable controversy concerning the forms of folate used for supplementation. To address this controversy, we prepared culture medium with different forms of folate, folic acid (FA), and 5-methyltetrahydrofolate (5mTHF), at concentrations of 5 µM, 500 nM, 50 nM, and folate free, respectively. Mouse embryonic fibroblasts (MEFs) were treated with different folates continuously for three passages, and cell proliferation and F-actin were monitored. We determined that compared to 5mTHF, FA showed stronger effects on promoting cell proliferation and F-actin formation. We also found that FOLR1 protein level was positively regulated by folate concentration and the non-canonical Wnt/planar cell polarity (PCP) pathway signaling was significantly enriched among different folate conditions in RNA-sequencing analyses. We demonstrated for the first time that FOLR1 could promote the transcription of Vangl2, one of PCP core genes. The transcription of Vangl2 was down-regulated under folate-deficient condition, which resulted in a decrease in PCP activity and F-actin formation. In summary, we identified a distinct advantage of FA in cell proliferation and F-actin formation over 5mTHF, as well as demonstrating that FOLR1 could promote transcription of Vangl2 and provide a new mechanism by which folate deficiency can contribute to the etiology of NTDs.

A Paternal Methylation Error in the Congenital Hydrocephalic Texas (H-Tx) Rat Is Partially Rescued with Natural Folate Supplements.

Folate deficiencies, folate imbalance and associated abnormal methylation are associated with birth defects, developmental delays, neurological conditions and diseases. In the hydrocephalic Texas (H-Tx) rat, 10-formyl tetrahydrofolate dehydrogenase (FDH) is reduced or absent from the CSF and the nuclei of cells in the brain and liver and this is correlated with decreased DNA methylation. In the present study, we tested whether impaired folate metabolism or methylation exists in sexually mature, unaffected H-Tx rats, which may explain the propagation of hydrocephalus in their offspring. We compared normal Sprague Dawley (SD, n = 6) rats with untreated H-Tx (uH-Tx, n = 6 and folate-treated H-Tx (TrH-Tx, n = 4). Structural abnormalities were observed in the testis of uH-Tx rats, with decreased methylation, increased demethylation, and cell death, particularly of sperm. FDH and FRα protein expression was increased in uH-Tx males but not in folate-treated males but tissue folate levels were unchanged. 5-Methylcytosine was significantly reduced in untreated and partially restored in treated individuals, while 5-hydroxymethylcytosine was not significantly changed. Similarly, a decrease in DNA-methyltransferase-1 expression in uH-Tx rats was partially reversed with treatment. The data expose a significant germline methylation error in unaffected adult male H-Tx rats from which hydrocephalic offspring are obtained. Reduced methylation in the testis and sperm was partially recovered by treatment with folate supplements leading us to conclude that this neurological disorder may not be completely eradicated by maternal supplementation alone.

Multimodal image-guided folic acid targeted Ag-based quantum dots for the combination of selective methotrexate delivery and photothermal therapy.

Multifunctional quantum dots (QDs) with photothermal therapy (PTT) potential loaded with an anticancer drug and labelled with a targeting agent can be highly effective nano-agents for tumour specific, image-guided PTT/chemo combination therapy of cancer. Ag-chalcogenides are promising QDs with good biocompatibility. Ag2S QDs are popular theranostic agents for imaging in near-infrared with PTT potential. However, theranostic applications of AgInS2 QDs emitting in the visible region and its PTT potential need to be explored. Here, we first present a simple synthesis of small, glutathione (GSH) coated AgInS2 QDs with peak emission at 634 nm, 21% quantum yield, and excellent long-term stability without an inorganic shell. Ag2S-GSH QDs emitting in the near-infrared region (peak emission = 822 nm) were also produced. Both QDs were tagged with folic acid (FA) and conjugated with methotrexate (MTX). About 3-fold higher internalization of FA-tagged QDs by folate-receptor (FR) overexpressing HeLa cells than HT29 and A549 cells was observed. Delivery of MTX by QD-FA-MTX reduced the IC50 of the drug from 10 µg/mL to 2.5-5 µg/mL. MTX release was triggered at acidic pH, which was further enhanced with local temperature increase created by laser irradiation. Irradiation of AgInS2-GSH QDs at 640 nm (300 mW) for 10 min, caused about 10 °C temperature increase but did not cause any thermal ablation of cells. On the other hand, Ag2S-GSH-FA based PTT effectively and selectively killed HeLa cells with 10 min 808 nm laser irradiation via mostly necrosis with an IC50 of 5 µg Ag/mL. Under the same conditions, IC50 of MTX was reduced to 0.21 µg/mL if Ag2S-GSH-FA.

Nanotoxicological study of downconversion Y2 O3 :Eu3+ luminescent nanoparticles functionalized with folic acid for cancer cells bioimaging.

Luminescent lanthanide downconversion nanoparticles (DCNPs) provide a combination of high luminescence intensity, sharp emission peaks with narrow bandwidth and a large Stokes' shift, leading to high-performance biomedical applications mainly for imaging. The purpose of this study is to present a nanotoxicological study of DCNPs Y2 O3 codoped with Eu3+ and functionalized with folic acid (FA). These assessments include cytotoxicity, genotoxicity, hemocompatibility, and in vitro inflammatory studies. We demonstrated by flow cytometry and confocal microscope the internalization of FA-DCNPs in breast cancer and melanoma cells. They were synthesized by sol-gel method and coated with a thin silica shell to make them biocompatible; also they were functionalized with amino groups and FA ligands that bind to the folate receptors (FR) located on the surface of the cancer cells studied. This functionalization enables the DCNPs to be internalized into the cancer cells via endocytosis by the conjugation FA-FR. The DCNPs were characterized with transmission electron microscope, Fourier transform infrared spectroscopy and photoluminescence. The nanotoxicological assessments demonstrated that both nanoparticles (bare and functionalized) are no cytotoxic and no genotoxic at the tested concentrations (0.01-20 µg/mL) in three cell lines (breast, skin cancer, and osteoblasts). Also they are hemocompatible and do not exert nitric oxide production in vitro by macrophages. The FA-DCNPs were clearly localized into the cell cytoplasm with bright red luminescence. Thus, herein we present a complete nanotoxicological study of FA-DCNPs Y2 O3 codoped with Eu3+ and we conclude that these nanoparticles are biocompatible and can be further used for cancer cells bioimaging.

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.

Hybrid micelles containing methotrexate-conjugated polymer and co-loaded with microRNA-124 for rheumatoid arthritis therapy.

Purpose: Methotrexate (MTX) is a first-line drug for rheumatoid arthritis (RA)therapy. However, MTX monotherapy often results in irreversible joint damage due to its slow onset of action and long duration. microRNA-124 (miR-124) has shown direct bone protection activity against RA. A co-delivery system for MTX and microRNA combination may provide therapeutic synergy. Methods: Methotrexate-conjugated polymer hybrid micelles (M-PHMs) were prepared by self-assembly of two functional amphiphilic polymers (MTX-PEI-LA and mPEG-LA) at an optimized weight ratio. Incorporation of microRNA was achieved through electrostatic interactions between microRNA and cationic polymer MTX-PEI-LA. Cellular uptake, endosome escape, biodistribution, and therapeutic efficacy of M-PHMs/miR-124 complexes were investigated and evaluated in RAW264.7 cells and a rat adjuvant-induced arthritis (AIA) model. Results: M-PHMs/miR-124 complexes exhibited folate receptor-mediated uptake in activated RAW264.7 cells. miR-124 was able to escape from the endosome and down-regulate nuclear factor of activated T cells cytoplasmic1 (NFATc1). M-PHMs/miR-124 complexes accumulated in inflamed joints of AIA rats and showed superior therapeutic efficacy through both anti-inflammatory effect and direct bone protective effect. Combination of miR-124 and MTX in these micelles induced disease remission. Conclusions: M-PHMs/miR-124 was highly effective against RA through therapeutic synergy. Additional studies are warranted to further investigate its therapeutic potential and delineate its mechanisms of action.

Augmented cytotoxic effects of paclitaxel by curcumin induced overexpression of folate receptor-α for enhanced targeted drug delivery in HeLa cells.

BACKGROUND: New targeted therapies are intended to minimize the toxic effects and maximize destruction of tumor cells. Folate is a membrane-bound receptor that plays a vital role in the uptake of anti-folate molecules aimed for efficient drug delivery of anti-folate drugs. PURPOSE: The present study is aimed at the modulation of the expression of folate receptor by curcumin that enhances the intake, cytotoxicity and anticancer effects of paclitaxel in HeLa cells. MATERIALS AND METHODS: HeLa cells were pretreated with curcumin and treated with paclitaxel. We measured the cell viability, uptake of radiolabelled folic acid and paclitaxel, Folate receptor -alpha (FR-α) protein expression by immunocytochemistry and western blot and FR-α mRNA expression by qualitative and quantitative analysis. RESULTS: This study shows that curcumin (10 - 50 µM) causes significantly increased cytotoxicity in a dose and time dependent manner. It also enhances the intake of radiolabeled folic acid and paclitaxel 3-4 folds in HeLa cells. The pretreatment of HeLa cells with curcumin shows statistically significant of cell death by paclitaxel. The quantitative RT-PCR demonstrates the expression of FR- α mRNA upon curcumin treatment. Furthermore, immunochemistry and western blotting analysis proved that curcumin enhances expression the FR- α in HeLa cells. CONCLUSION: Our study proved that the molecular mechanism of curcumin enhances the upregulation of FR - α mRNA and protein expression in HeLa cells. Therefore, a combination of curcumin and paclitaxel at less concentration may be a targeting strategy for FR-targeted drug delivery providing a better therapeutic intervention of cancer.

Evaluation of Prophylactic Corticosteroid Eye Drop Use in the Management of Corneal Abnormalities Induced by the Antibody-Drug Conjugate Mirvetuximab Soravtansine.

PURPOSE: Reversible, low-grade ocular adverse events (AE) are associated with administration of mirvetuximab soravtansine, a folate receptor alpha (FRα)-targeted antibody-drug conjugate undergoing phase III clinical evaluation in platinum-resistant ovarian cancer. This study investigated the underlying mechanisms of ocular toxicity and evaluated primary prophylactic use of corticosteroid eye drops in patients receiving mirvetuximab soravtansine. PATIENTS AND METHODS: Target expression in the human eye was determined by IHC. The ocular toxicity profile of mirvetuximab soravtansine was assessed preclinically using Dutch-Belted rabbits. In a phase I clinical study, patients with ovarian cancer were treated with 6 mg/kg mirvetuximab soravtansine intravenously once every 3 weeks, including one expansion cohort with corticosteroid eye drops administered daily for the first 10 days of each treatment cycle. RESULTS: FRα expression was absent from human corneal tissues. Ocular abnormalities in the rabbit eye appeared phenotypically consistent with off-target effects on the cornea. Forty patients were enrolled in the expansion cohort. Reversible grade 1 or 2 blurred vision and keratopathy occurred in 16 (40%) and 12 (30%) patients, respectively; no grade 3/4 ocular events were observed. Compared with those patients who did not receive primary prophylaxis, corticosteroid eye drop use resulted in fewer dose reductions (5% vs. 15%) and none discontinued due to ocular AEs. CONCLUSIONS: Preclinical modeling was predictive of the corneal-related symptoms seen in some patients dosed with mirvetuximab soravtansine. Primary prophylactic use of topical corticosteroid eye drops resulted in a trend toward symptomatic improvement and a reduction in ocular AE-related dose modifications in patients treated with mirvetuximab soravtansine.

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.

Folate homeostasis in epileptic rats.

Folate is involved in metabolic processes and it has been implicated in both aggravation and amelioration of seizures. The aim of the current work was to study the effect of chronic temporal lobe epilepsy (TLE) on the plasma and brain concentrations of folate and on its uptake carriers in the brain - the reduced folate carrier (RFC), folate receptor α (FRα) and proton coupled folate transporter (PCFT). We utilized the rat lithium pilocarpine model for TLE. Approximately two months following status epilepticus, rats with spontaneous recurrent seizures (SRS) were sacrificed for brain and plasma folate concentration analyses and folate uptake carrier expression studies. RT-PCR and western blot analyses were utilized for quantification of folate carriers' mRNAs and proteins, respectively. The distribution of folate carriers in the brain was studied using immunohistochemistry. In the SRS rats we found lower plasma concentrations (10 ±â€¯0.9 in control vs. 6.6 ±â€¯1.6 ng/ml in SRS, P < 0.05), but preserved cortical and increased hippocampal levels of folate (0.5 ±â€¯0.1 in control vs. 0.9 ±â€¯0.2 ng/mg in SRS, P = 0.055). Hippocampus - to - plasma ratio of folate concentration was 3-fold higher in the SRS group, compared with the controls (0.13 ±â€¯0.03 vs. 0.04 ±â€¯0.02, respectively; P < 0.01). mRNA and protein levels of the folate uptake carriers did not differ between SRS rats and controls. However, immunofluorescent staining quantification revealed that the emission intensity of both RFC and FRα was elevated 8-fold and 4-fold, respectively, in hippocampal CA1 neurons of SRS rats, compared to controls (P < 0.01). PCFT was unquantifiable. If corroborated by complementary research in humans, the findings of this study may be utilized clinically for supplemental therapy planning, in imaging the epileptic focus, and for drug delivery into the epileptic brain. Further studies are required for better elucidating the clinical and mechanistic significance of altered folate balances in the epileptic brain.

Folinic acid improves verbal communication in children with autism and language impairment: a randomized double-blind placebo-controlled trial.

We sought to determine whether high-dose folinic acid improves verbal communication in children with non-syndromic autism spectrum disorder (ASD) and language impairment in a double-blind placebo control setting. Forty-eight children (mean age 7 years 4 months; 82% male) with ASD and language impairment were randomized to receive 12 weeks of high-dose folinic acid (2 mg kg-1 per day, maximum 50 mg per day; n=23) or placebo (n=25). Children were subtyped by glutathione and folate receptor-α autoantibody (FRAA) status. Improvement in verbal communication, as measured by a ability-appropriate standardized instrument, was significantly greater in participants receiving folinic acid as compared with those receiving placebo, resulting in an effect of 5.7 (1.0,10.4) standardized points with a medium-to-large effect size (Cohen's d=0.70). FRAA status was predictive of response to treatment. For FRAA-positive participants, improvement in verbal communication was significantly greater in those receiving folinic acid as compared with those receiving placebo, resulting in an effect of 7.3 (1.4,13.2) standardized points with a large effect size (Cohen's d=0.91), indicating that folinic acid treatment may be more efficacious in children with ASD who are FRAA positive. Improvements in subscales of the Vineland Adaptive Behavior Scale, the Aberrant Behavior Checklist, the Autism Symptom Questionnaire and the Behavioral Assessment System for Children were significantly greater in the folinic acid group as compared with the placebo group. There was no significant difference in adverse effects between treatment groups. Thus, in this small trial of children with non-syndromic ASD and language impairment, treatment with high-dose folinic acid for 12 weeks resulted in improvement in verbal communication as compared with placebo, particularly in those participants who were positive for FRAAs.

Folate receptors and neural tube closure.

Neural tube defects (NTD) are among the most common human congenital malformations, affecting 0.5-8.0/1000 of live births. Human clinical trials have shown that periconceptional folate supplementation significantly decreases the occurrence of NTD in offspring. However, the mechanism by which folate acts on NTD remains largely unknown. Folate receptor (Folr) is one of the three membrane proteins that mediate cellular uptake of folates. Recent studies suggest that mouse Folr1 (formerly referred to as Fbp1) is essential for neural tube closure. Therefore, we examined spatial and temporal expression patterns of Folr1 in developing mouse embryos, showing a close association between Folr1 and anterior neural tube closure. Transient transgenic analysis was performed using lacZ as a reporter; we identified a 1.1-kb enhancer that directs lacZ expression in the neural tube and optic vesicle in a manner that is similar to endogenous Folr1. The 1.1-kb enhancer sequences were highly conserved between humans and mice, suggesting that human FOLR1 is associated with anterior neural tube closure in humans. Several experimental studies in mice and human epidemiological and genetics studies have suggested that folate receptor abnormalities are involved in a portion of human NTDs, although the solo defect of FOLR1 did not cause NTD.

Folate receptor 1 is necessary for neural plate cell apical constriction during Xenopus neural tube formation.

Folate supplementation prevents up to 70% of neural tube defects (NTDs), which result from a failure of neural tube closure during embryogenesis. The elucidation of the mechanisms underlying folate action has been challenging. This study introduces Xenopus laevis as a model to determine the cellular and molecular mechanisms involved in folate action during neural tube formation. We show that knockdown of folate receptor 1 (Folr1; also known as FRα) impairs neural tube formation and leads to NTDs. Folr1 knockdown in neural plate cells only is necessary and sufficient to induce NTDs. Folr1-deficient neural plate cells fail to constrict, resulting in widening of the neural plate midline and defective neural tube closure. Pharmacological inhibition of folate action by methotrexate during neurulation induces NTDs by inhibiting folate interaction with its uptake systems. Our findings support a model in which the folate receptor interacts with cell adhesion molecules, thus regulating the apical cell membrane remodeling and cytoskeletal dynamics necessary for neural plate folding. Further studies in this organism could unveil novel cellular and molecular events mediated by folate and lead to new ways of preventing NTDs.

Targeting of herbal bioactives through folate receptors: a novel concept to enhance intracellular drug delivery in cancer therapy.

Targeted drug delivery through folate receptor (FR) has emerged as a most biocompatible, target oriented, and non-immunogenic cargoes for the delivery of anticancer drugs. FRs are highly overexpressed in many tumor cells (like ovarian, lung, breast, kidney, brain, endometrial, and colon cancer), and targeting them through conjugates bearing specific ligand with encapsulated nanodrug moiety is undoubtedly, a promising approach toward tumor targeting. Folate, being an endogenous ligand, can be exploited well to affect various cellular events occurring during the progress of tumor, in a more natural and definite way. Thus, the aim of the review lies in summarizing the advancements taken place in the drug delivery system of different therapeutics through FRs and to refine its role as an endogenous ligand, in targeting of synthetic as well as natural bioactives. The review also provides an update on the patents received on the folate-based drug delivery system.

Prevention of behavioral deficits in rats exposed to folate receptor antibodies: implication in autism.

Folate receptor alpha (FRα) autoantibodies have been associated with fetal abnormalities and cerebral folate deficiency-related developmental disorders. Over 70% of the children with autism spectrum disorders (ASD) are positive for these autoantibodies and high-dose folinic acid is beneficial in treating these children. Here we show that antibodies (Abs) to the rat FRα administered during gestation produce communication, learning and cognitive deficits in a rat model that can be prevented by folinic acid and dexamethasone. FRα Ab can trigger inflammation as well as block folate transport to the fetus and to the developing brain to produce the functional deficits. In humans, exposure to FRα autoantibodies during fetal development and infancy could contribute to brain dysfunction such as that seen in ASD and other developmental disorders. Identifying women positive for the autoantibody and treating them with high-dose folinic acid along with other interventions to lower the autoantibody titer are effective strategies that may be considered to reduce the risk of having a child with developmental deficits.

Solubility enhancement and targeted delivery of a potent anticancer flavonoid analogue to cancer cells using ligand decorated dendrimer nano-architectures.

Conventional chemotherapy using small molecule drugs is marred by several challenges such as short half-life, low therapeutic index and adverse systemic side effects. In this regard, targeted therapies using ligand directed polyamidoamine (PAMAM) dendrimers could be a promising strategy to specifically deliver anticancer drugs to cancer cells overexpressing complementary receptor binding domains. The aim of this study was to utilize folate decorated PAMAM to enhance the aqueous solubility of a highly hydrophobic but very potent anticancer flavonoid analogue, 3,4-difluorobenzylidene diferuloylmethane (CDF) and to deliver it specifically to folate receptor overexpressing cervical cancer cells (HeLa) and ovarian cancer cells (SKOV3). As compared to the non-targeted formulation, the targeted formulation exhibited significant anticancer activity with higher accumulation in folate receptor overexpressing cells, larger population of apoptotic cancer cells, elevated expression of tumor suppressor phosphatase and tensin homolog (PTEN), and inhibition of nuclear factor kappa B (NFκB) which further confirmed the targeting ability and the promising anticancer activity of the folate based nanoformulation.

Folate Receptor Alpha Upregulates Oct4, Sox2 and Klf4 and Downregulates miR-138 and miR-let-7 in Cranial Neural Crest Cells.

Prenatal folic acid (FA) supplementation prevents neural tube defects. Folate receptor alpha (FRα) is critical for embryonic development, including neural crest (NC) development. Previously we showed that FRα translocates to the nucleus in response to FA, where it acts as a transcription factor. In this study, we examined if FA through interaction with FRα regulates stem cell characteristics of cranial neural crest cells (CNCCs)-critical for normal development. We hypothesized that FRα upregulates coding genes and simultaneously downregulates non-coding miRNA which targets coding genes in CNCCs. Quantitative RT-PCR and chromatin immunoprecipitation showed that FRα upregulates Oct4, Sox2, and Klf4 by binding to their cis-regulator elements-5' enhancer/promoters defined by H3K27Ac and p300 occupancy. FA via FRα downregulates miRNAs, miR-138 and miR-let-7, which target Oct4 and Trim71 (an Oct4 downstream effector), respectively. Co-immunoprecipitation data suggests that FRα interacts with the Drosha-DGCR8 complex to affect pre-miRNA processing. Transfecting anti-miR-138 or anti-miR-let-7 into non-proliferating neural crest cells (NCCs) derived from Splotch (Sp-/- ), restored their proliferation potential. In summary, these results suggest a novel pleiotropic role of FRα: (a) direct activation of Oct4, Sox2, and Klf4 genes; and (b) repression of biogenesis of miRNAs that target these genes or their effector molecules. Stem Cells 2016;34:2721-2732.

The metabolic basis for developmental disorders due to defective folate transport.

Folates are essential in the intermediary metabolism of amino acids, synthesis of nucleotides and for maintaining methylation reactions. They are also linked to the production of neurotransmitters through GTP needed for the synthesis of tetrahydrobiopterin. During pregnancy, folate is needed for fetal development. Folate deficiency during this period has been linked to increased risk of neural tube defects. Disturbances of folate metabolism due to genetic abnormalities or the presence of autoantibodies to folate receptor alpha (FRα) can impair physiologic processes dependent on folate, resulting in a variety of developmental disorders including cerebral folate deficiency syndrome and autism spectrum disorders. Overall, adequate folate status has proven to be important during pregnancy as well as neurological development and functioning in neonates and children. Treatment with pharmacologic doses of folinic acid has led to reversal of some symptoms in many children diagnosed with cerebral folate deficiency syndrome and autism, especially in those positive for autoantibodies to FRα. Thus, as the brain continues to develop throughout fetal and infant life, it can be affected and become dysfunctional due to a defective folate transport contributing to folate deficiency. Treatment and prevention of these disorders can be achieved by identification of those at risk and supplementation with folinic acid.

Assessment of the specificity of a new folate-targeted photosensitizer for peritoneal metastasis of epithelial ovarian cancer to enable intraperitoneal photodynamic therapy. A preclinical study.

BACKGROUND: Ovarian cancer's prognosis remains dire after primary therapy. Recurrence rate is disappointingly high as 60% of women with epithelial ovarian cancer considered in remission will develop recurrent disease within 5 years. Special attention to undetected peritoneal metastasis during surgery is necessary as they are the main predictive factors of recurrences. Folate Receptor α (FRα) shows promising prospects in targeting ovarian cancerous cells and intraperitoneal photodynamic therapy (PDT) could be a solution in addition to macroscopic cytoreductive surgery to treat peritoneal micrometastasis. The aim of this preclinical study is to assess the specificity of a folate-targeted photosensitizer for ovarian peritoneal micrometastasis. METHODS: We used the NuTu-19 epithelial ovarian cancer cell line to induce peritoneal carcinomatosis in female Fischer 344 rats. Three groups of 6 rats were studied (Control (no photosensitizer)/Non-conjugated photosensitizer (Porph)/Folate-conjugated photosensitizer (Porph-s-FA)). Four hours after the administration of the photosensitizer, animals were sacrificed and intraperitoneal organs tissues were sampled. FRα tissue expression was evaluated by immunohistochemistry. Tissue incorporation of photosensitizers was assessed by confocal microscopy and tissue quantification. RESULTS: FRα is overexpressed in tumor, ovary, and liver whereas, peritoneum, colon, small intestine, and kidney do not express it. Cytoplasmic red endocytosis vesicles observed by confocal microscopy are well correlated to FRα tissue expression. Photosensitizer tissue quantification shows a mean tumor-to-normal tissue ratio of 9.6. CONCLUSION: We demonstrated that this new generation folate-targeted photosensitizer is specific of epithelial ovarian peritoneal metastasis and may allow the development of efficient and safe intraperitoneal PDT procedure.