RESUMEN
Folic acid is a nutrient essential for embryonic development. Folate deficiency can cause embryonic lethality or neural tube defects and orofacial anomalies. Folate receptor 1 (Folr1) is a folate binding protein that facilitates the cellular uptake of dietary folate. To better understand the biological processes affected by folate deficiency, gene expression profiles of gestational day 9.5 (gd9.5) Folr1-/- embryos were compared to those of gd9.5 Folr1+/+ embryos. The expression of 837 genes/ESTs was found to be differentially altered in Folr1-/- embryos, relative to those observed in wild-type embryos. The 837 differentially expressed genes were subjected to Ingenuity Pathway Analysis. Among the major biological functions affected in Folr1-/- mice were those related to 'digestive system development/function', 'cardiovascular system development/function', 'tissue development', 'cellular development', and 'cell growth and differentiation', while the major canonical pathways affected were those associated with blood coagulation, embryonic stem cell transcription and cardiomyocyte differentiation (via BMP receptors). Cellular proliferation, apoptosis and migration were all significantly affected in the Folr1-/- embryos. Cranial neural crest cells (NCCs) and neural tube explants, grown under folate-deficient conditions, exhibited marked reduction in directed migration that can be attributed, in part, to an altered cytoskeleton caused by perturbations in F-actin formation and/or assembly. The present study revealed that several developmentally relevant biological processes were compromised in Folr1-/- embryos.
Asunto(s)
Diferenciación Celular , Embrión de Mamíferos/metabolismo , Receptor 1 de Folato/fisiología , Ácido Fólico/metabolismo , Regulación del Desarrollo de la Expresión Génica , Cresta Neural/metabolismo , Defectos del Tubo Neural/patología , Animales , Embrión de Mamíferos/citología , Femenino , Perfilación de la Expresión Génica , Edad Gestacional , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Cresta Neural/patología , Defectos del Tubo Neural/genética , Defectos del Tubo Neural/metabolismoRESUMEN
Elevated plasma homocysteine (Hcy) levels are associated with endothelial dysfunction. Previous studies have indicated the role of endoplasmic reticulum (ER) stress in Hcyinduced endothelial injury. However, the mechanism of action remains unclear. In the current study, the role of human folate receptor α (hFRα) in Hcyinduced ER stress was investigated, along with endothelial injury in human umbilical vein endothelial cells (HUVECs). The results showed that a moderate dose of Hcy induced morphological injury and reduced viability of HUVECs. Furthermore, moderatedose Hcy reduced hFRα expression in HUVECs. Notably, hFRα inhibition by RNA interference resulted in activation of the protein kinase RNAlike endoplasmic reticulum kinase pathway and increased apoptosis of HUVECs. In conclusion, this study demonstrated the presence of hFRα in HUVECs, and indicated its role in the regulation of ER stress and cell injury by Hcy.
Asunto(s)
Estrés del Retículo Endoplásmico , Retículo Endoplásmico/metabolismo , Receptor 1 de Folato/fisiología , Homocisteína/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Apoptosis , Biomarcadores/metabolismo , Células Cultivadas , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Interferencia de ARNRESUMEN
Increased levels of plasma cysteine predispose to obesity and metabolic disturbances. Our recent genetic analyses in spontaneously hypertensive rats (SHR) revealed mutated Folr1 (folate receptor 1) on chromosome 1 as a quantitative trait gene associated with reduced folate levels, hypercysteinemia and metabolic disturbances. The Folr1 gene is closely linked to the Folh1 (folate hydrolase 1) gene which codes for an enzyme involved in the hydrolysis of dietary polyglutamyl folates in the intestine. In the current study, we obtained evidence that Folh1 mRNA of the BN (Brown Norway) origin is weakly but significantly expressed in the small intestine. Next we analyzed the effects of the Folh1 alleles on folate and sulfur amino acid levels and consecutively on glucose and lipid metabolism using SHR-1 congenic sublines harboring either Folr1 BN and Folh1 SHR alleles or Folr1 SHR and Folh1 BN alleles. Both congenic sublines when compared to SHR controls, exhibited significantly reduced folate clearance and lower plasma cysteine and homocysteine levels which was associated with significantly decreased serum glucose and insulin concentrations and reduced adiposity. These results strongly suggest that, in addition to Folr1, the Folh1 gene also plays an important role in folate and sulfur amino acid levels and affects glucose and lipid metabolism in the rat.
Asunto(s)
Receptor 1 de Folato/fisiología , Glutamato Carboxipeptidasa II/fisiología , Síndrome Metabólico/genética , Síndrome Metabólico/metabolismo , Animales , Animales Congénicos , Masculino , Estrés Oxidativo/fisiología , Ratas , Ratas Endogámicas BN , Ratas Endogámicas SHRRESUMEN
Structure-activity relationships for cellular uptake and inhibition of cell proliferation were studied for 2-amino-4-oxo-6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates in which the terminal l-glutamate of the parent structure (7) was replaced by natural or unnatural amino acids. Compounds 7 and 10-13 were selectively inhibitory toward folate receptor (FR) α-expressing Chinese hamster ovary (CHO) cells. Antiproliferative effects of compounds 7 and 9-13 toward FRα- and FRß-expressing CHO cells were only partly reflected in binding affinities to FRα and FRß or in the docking scores with molecular models of FRα and FRß. Compounds 7 and 11 were potent inhibitors of glycinamide ribonucleotide formyltransferase in de novo purine biosynthesis in KB human tumor cells. These studies establish for the first time the importance of the α- and γ-carboxylic acid groups, the length of the amino acid, and the conformation of the side chain for transporter binding and biological activity of 6-substituted pyrrolo[2,3-d]pyrimidine thienoyl antifolates.
Asunto(s)
Receptor 1 de Folato/fisiología , Receptor 2 de Folato/fisiología , Antagonistas del Ácido Fólico/síntesis química , Transportadores de Ácido Fólico/fisiología , Pirimidinas/síntesis química , Animales , Células CHO , Cricetinae , Cricetulus , Antagonistas del Ácido Fólico/farmacología , Humanos , Células KB , Modelos Moleculares , Pirimidinas/farmacología , Relación Estructura-ActividadRESUMEN
Folate receptor alpha (FRalpha) is overexpressed on ovarian cancer cells and is a promising molecular target for ovarian cancer gene therapy, but there was still no related report. In this study, folate modified cationic liposomes (F-PEG-CLPs) for ovarian cancer gene delivery were developed for the first time. Folate-poly(ethylene glycol)-succinate-cholesterol (F-PEG-suc-Chol) was firstly synthesized and then used to prepare folate-targeted cationic liposomes/plasmid DNA complexes (F-targeted lipoplexes). F-targeted lipoplexes were prepared by post-insertion method, and displayed membrane structure by transmission electron microscopy observation with the diameter of 193 nm-200 nm and the zeta potential of 35 mV-38 mV. DNase degradation experiments showed that plasmid DNA could be effectively shielded by F-targeted lipoplexes in vitro. F-targeted lipoplexes could transfer gene into human ovarian carcinoma cell line SKOV-3, and 0.1% F-PEG-CLPs composed by DOTAP/Chol/mPEG-Chol/F-PEG-suc-Chol (50:45:5:0.1, molar ratio) had the highest transfection efficiency. The transfection activity of F-targeted lipoplexes could be competitively inhibited by free folic acid, demonstrating that folate-FRalpha interaction caused high transfection efficiency of F-targeted lipoplexes. The uptake mechanism of F-targeted lipoplexes was further validated on human oral carcinoma cell line KB and human liver carcinoma cell line HepG2. The concentration-dependent and time-dependent cytotoxicity of targeted material F-PEG-suc-Chol was observed by MTT assay on SKOV-3 cell and its application would not increase the cytotoxicity of F-targeted lipoplexes in SKOV-3 cells. All the data indicated that F-PEG-CLPs would be a promising gene vector targeting for ovarian cancer therapy.