Role of vitamin C and SVCT2 in neurogenesis.

Different studies have established the fundamental role of vitamin C in proliferation, differentiation, and neurogenesis in embryonic and adult brains, as well as in in vitro cell models. To fulfill these functions, the cells of the nervous system regulate the expression and sorting of sodium-dependent vitamin C transporter 2 (SVCT2), as well as the recycling of vitamin C between ascorbic acid (AA) and dehydroascorbic acid (DHA) via a bystander effect. SVCT2 is a transporter preferentially expressed in neurons and in neural precursor cells. In developmental stages, it is concentrated in the apical region of the radial glia, and in adult life, it is expressed preferentially in motor neurons of the cerebral cortex, starting on postnatal day 1. In neurogenic niches, SVCT2 is preferentially expressed in precursors with intermediate proliferation, where a scorbutic condition reduces neuronal differentiation. Vitamin C is a potent epigenetic regulator in stem cells; thus, it can induce the demethylation of DNA and histone H3K27m3 in the promoter region of genes involved in neurogenesis and differentiation, an effect mediated by Tet1 and Jmjd3 demethylases, respectively. In parallel, it has been shown that vitamin C induces the expression of stem cell-specific microRNA, including the Dlk1-Dio3 imprinting region and miR-143, which promotes stem cell self-renewal and suppresses de novo expression of the methyltransferase gene Dnmt3a. The epigenetic action of vitamin C has also been evaluated during gene reprogramming of human fibroblasts to induced pluripotent cells, where it has been shown that vitamin C substantially improves the efficiency and quality of reprogrammed cells. Thus, for a proper effect of vitamin C on neurogenesis and differentiation, its function as an enzymatic cofactor, modulator of gene expression and antioxidant is essential, as is proper recycling from DHA to AA by various supporting cells in the CNS.

Hybrid nanomaterial composed of chitosan, curcumin, ZnO and TiO2 for antibacterial therapies.

Metal nanoparticles have been tremendously utilised, such as; antibacterial and anticancer agents. Although metal nanoparticles exhibits antibacterial and anticancer activity, but the drawback of toxicity on normal cells limits their clinical applications. Therefore, improving the bioactivity of hybrid nanomaterial (HNM) and minimizing toxicity is of paramount importance for biomedical applications. Herein, a facile and simple double precipitation method was used to develop biocompatible and multifunctional HNM from antimicrobial chitosan, curcumin, ZnO and TiO2. In HNM, biomolecules chitosan and curcumin were used to control the toxicity of ZnO and TiO2 and improve their biocidal properties. The cytotxicological properties of the HNM was studied against human breast cancer (MDA-MB-231) and fibroblast (L929) cell lines. The antimicrobial activity of the HNM was examined against Escherichia coli and Staphylococcus aureus bacteria, via the well-diffusion method. In addition, the antioxidant property was evaluated by the radical scavenging method. These findings actively, support the ZTCC HNM potential, as an innovative biocidal agent for applications in the clinical and healthcare sectors.

LOX-1 Activation by oxLDL Induces AR and AR-V7 Expression via NF-κB and STAT3 Signaling Pathways Reducing Enzalutamide Cytotoxic Effects.

The oxidized low-density lipoprotein receptor 1 (LOX-1) is one of the most important receptors for modified LDLs, such as oxidated (oxLDL) and acetylated (acLDL) low-density lipoprotein. LOX-1 and oxLDL are fundamental in atherosclerosis, where oxLDL/LOX1 promotes ROS generation and NF-κB activation inducing the expression of IL-6, a STAT3 activator. Furthermore, LOX-1/oxLDL function has been associated with other diseases, such as obesity, hypertension, and cancer. In prostate cancer (CaP), LOX-1 overexpression is associated with advanced stages, and its activation by oxLDL induces an epithelial-mesenchymal transition, increasing angiogenesis and proliferation. Interestingly, enzalutamide-resistant CaP cells increase the uptake of acLDL. Enzalutamide is an androgen receptor (AR) antagonist for castration-resistant prostate cancer (CRPC) treatment, and a high percentage of patients develop a resistance to this drug. The decreased cytotoxicity is promoted in part by STAT3 and NF-κB activation that induces the secretion of the pro-inflammatory program and the expression of AR and its splicing variant AR-V7. Here, we demonstrate for the first time that oxLDL/LOX-1 increases ROS levels and activates NF-κB, inducing IL-6 secretion and the activation of STAT3 in CRPC cells. Furthermore, oxLDL/LOX1 increases AR and AR-V7 expression and decreases enzalutamide cytotoxicity in CRPC. Thus, our investigation suggests that new factors associated with cardiovascular pathologies, such as LOX-1/oxLDL, may also promote important signaling axes for the progression of CRPC and its resistance to drugs used for its treatment.

Vitamin C Deficiency Reduces Neurogenesis and Proliferation in the SVZ and Lateral Ventricle Extensions of the Young Guinea Pig Brain.

Although scurvy, the severe form of vitamin C deficiency, has been almost eradicated, the prevalence of subclinical vitamin C deficiency is much higher than previously estimated and its impact on human health might not be fully understood. Vitamin C is an essential molecule, especially in the central nervous system where it performs numerous, varied and critical functions, including modulation of neurogenesis and neuronal differentiation. Although it was originally considered to occur only in the embryonic brain, it is now widely accepted that neurogenesis also takes place in the adult brain. The subventricular zone (SVZ) is the neurogenic niche where the largest number of new neurons are born; however, the effect of vitamin C deficiency on neurogenesis in this key region of the adult brain is unknown. Therefore, through BrdU labeling, immunohistochemistry, confocal microscopy and transmission electron microscopy, we analyzed the proliferation and cellular composition of the SVZ and the lateral ventricle (LVE) of adult guinea pigs exposed to a vitamin-C-deficient diet for 14 and 21 days. We found that neuroblasts in the SVZ and LVE were progressively and significantly decreased as the days under vitamin C deficiency elapsed. The neuroblasts in the SVZ and LVE decreased by about 50% in animals with 21 days of deficiency; this was correlated with a reduction in BrdU positive cells in the SVZ and LVE. In addition, the reduction in neuroblasts was not restricted to a particular rostro-caudal area, but was observed throughout the LVE. We also found that vitamin C deficiency altered cellular morphology at the ultrastructural level, especially the cellular and nuclear morphology of ependymal cells of the LVE. Therefore, vitamin C is essential for the maintenance of the SVZ cell populations required for normal activity of the SVZ neurogenic niche in the adult guinea pig brain. Based on our results from the guinea pig brain, we postulate that vitamin C deficiency could also affect neurogenesis in the human brain.

Is IIIG9 a New Protein with Exclusive Ciliary Function? Analysis of Its Potential Role in Cancer and Other Pathologies.

The identification of new proteins that regulate the function of one of the main cellular phosphatases, protein phosphatase 1 (PP1), is essential to find possible pharmacological targets to alter phosphatase function in various cellular processes, including the initiation and development of multiple diseases. IIIG9 is a regulatory subunit of PP1 initially identified in highly polarized ciliated cells. In addition to its ciliary location in ependymal cells, we recently showed that IIIG9 has extraciliary functions that regulate the integrity of adherens junctions. In this review, we perform a detailed analysis of the expression, localization, and function of IIIG9 in adult and developing normal brains. In addition, we provide a 3D model of IIIG9 protein structure for the first time, verifying that the classic structural and conformational characteristics of the PP1 regulatory subunits are maintained. Our review is especially focused on finding evidence linking IIIG9 dysfunction with the course of some pathologies, such as ciliopathies, drug dependence, diseases based on neurological development, and the development of specific high-malignancy and -frequency brain tumors in the pediatric population. Finally, we propose that IIIG9 is a relevant regulator of PP1 function in physiological and pathological processes in the CNS.

Glioblastoma Invasiveness and Collagen Secretion Are Enhanced by Vitamin C.

Aims: Glioblastoma (GB) is one of the most aggressive brain tumors. These tumors modify their metabolism, increasing the expression of glucose transporters, GLUTs, which incorporate glucose and the oxidized form of vitamin C, dehydroascorbic acid (DHA). We hypothesized that GB cells preferentially take up DHA, which is intracellularly reduced and compartmentalized into the endoplasmic reticulum (ER), promoting collagen biosynthesis and an aggressive phenotype. Results: Our results showed that GB cells take up DHA using GLUT1, while GLUT3 and sodium-dependent vitamin C transporter 2 (SVCT2) are preferably intracellular. Using a baculoviral system and reticulum-enriched extracts, we determined that SVCT2 is mainly located in the ER and corresponds to a short isoform. Ascorbic acid (AA) was compartmentalized, stimulating collagen IV secretion and increasing in vitro and in situ cell migration. Finally, orthotopic xenografts induced in immunocompetent guinea pigs showed that vitamin C deficiency retained collagen, reduced blood vessel invasion, and affected glomeruloid vasculature formation, all pathological conditions associated with malignancy. Innovation and Conclusion: We propose a functional role for vitamin C in GB development and progression. Vitamin C is incorporated into the ER of GB cells, where it favors the synthesis of collagen, thus impacting tumor development. Collagen secreted by tumor cells favors the formation of the glomeruloid vasculature and enhances perivascular invasion. Antioxid. Redox Signal. 37, 538-559.

Vitamin C deficient reduces proliferation in a human periventricular tumor stem cell-derived glioblastoma model.

Glioblastoma multiforme (GBM) is the most common and aggressive brain tumor with a median survival of 14.6 months. GBM is highly resistant to radio- and chemotherapy, and remains without a cure; hence, new treatment strategies are constantly sought. Vitamin C, an essential micronutrient and antioxidant, was initially described as an antitumor molecule; however, several studies have shown that it can promote tumor progression and angiogenesis. Thus, considering the high concentrations of vitamin C present in the brain, our aim was to study the effect of vitamin C deficiency on the progression of GBM using a GBM model generated by the stereotactic injection of human GBM cells (U87-MG or HSVT-C3 cells) in the subventricular zone of guinea pig brain. Initial characterization of U87-MG and HSVT-C3 cells showed that HSVT-C3 are highly proliferative, overexpress p53, and are resistant to ferroptosis. To induce intraperiventricular tumors, animals received control or a vitamin C-deficient diet for 3 weeks, after which histopathological and confocal microscopy analyses were performed. We demonstrated that the vitamin C-deficient condition reduced the glomeruloid vasculature and microglia/macrophage infiltration in U87-MG tumors. Furthermore, tumor size, proliferation, glomeruloid vasculature, microglia/macrophage infiltration, and invasion were reduced in C3 tumors carried by vitamin C-deficient guinea pigs. In conclusion, the effect of the vitamin C deficiency was dependent on the tumor cell used for GBM induction. HSVT-C3 cells, a cell line with stem cell features isolated from a human subventricular GBM, showed higher sensitivity to the deficient condition; however, vitamin C deficiency displayed an antitumor effect in both GBM models analyzed.

Vitamin C Recycling Regulates Neurite Growth in Neurospheres Differentiated In Vitro.

The reduced form of vitamin C, ascorbic acid (AA), has been related with gene expression and cell differentiation in the cerebral cortex. In neurons, AA is mainly oxidized to dehydroascorbic acid (DHA); however, DHA cannot accumulate intracellularly because it induces metabolic changes and cell death. In this context, it has been proposed that vitamin C recycling via neuron-astrocyte coupling maintains AA levels and prevents DHA parenchymal accumulation. To date, the role of this mechanism during the outgrowth of neurites is unknown. To stimulate neuronal differentiation, adhered neurospheres treated with AA and retinoic acid (RA) were used. Neuritic growth was analyzed by confocal microscopy, and the effect of vitamin C recycling (bystander effect) in vitro was studied using different cells. AA stimulates neuritic growth more efficiently than RA. However, AA is oxidized to DHA in long incubation periods, generating a loss in the formation of neurites. Surprisingly, neurite growth is maintained over time following co-incubation of neurospheres with cells that efficiently capture DHA. In this sense, astrocytes have high capacity to recycle DHA and stimulate the maintenance of neurites. We demonstrated that vitamin C recycling in vitro regulates the morphology of immature neurons during the differentiation and maturation processes.

Maytenus disticha Extract and an Isolated ß-Dihydroagarofuran Induce Mitochondrial Depolarization and Apoptosis in Human Cancer Cells by Increasing Mitochondrial Reactive Oxygen Species.

Maytenus disticha (Hook F.), belonging to the Celastraceae family, is an evergreen shrub, native of the central southern mountains of Chile. Previous studies demonstrated that the total extract of M. disticha (MD) has an acetylcholinesterase inhibitory activity along with growth regulatory and insecticidal activities. ß-Dihydroagarofurans sesquiterpenes are the most active components in the plant. However, its activity in cancer has not been analyzed yet. Here, we demonstrate that MD has a cytotoxic activity on breast (MCF-7), lung (PC9), and prostate (C4-2B) human cancer cells with an IC50 (µg/mL) of 40, 4.7, and 5 µg/mL, respectively, an increasing Bax/Bcl2 ratio, and inducing a mitochondrial membrane depolarization. The ß-dihydroagarofuran-type sesquiterpene (MD-6), dihydromyricetin (MD-9), and dihydromyricetin-3-O-ß-glucoside (MD-10) were isolated as the major compounds from MD extracts. From these compounds, only MD-6 showed cytotoxic activity on MCF-7, PC9, and C4-2B with an IC50 of 31.02, 17.58, and 42.19 µM, respectively. Furthermore, the MD-6 increases cell ROS generation, and MD and MD-6 induce a mitochondrial superoxide generation and apoptosis on MCF-7, PC9, and C4-2B, which suggests that the cytotoxic effect of MD is mediated in part by the ß-dihydroagarofuran-type that induces apoptosis by a mitochondrial dysfunction.

Cytoarchitecture, Proliferative Activity and Neuroblast Migration in the Subventricular Zone and Lateral Ventricle Extension of the Adult Guinea Pig Brain.

In the mouse brain, neuroblasts generated in the subventricular zone (SVZ) migrate to the olfactory bulb (OB) through the rostral migratory stream (RMS). Although the RMS is not present in the human brain, a migratory pathway that is organized around a ventricular cavity that reaches the OB has been reported. A similar cavity, the lateral ventricle extension (LVE), is found in the adult guinea pig brain. Therefore, we analyzed cytoarchitecture, proliferative activity and precursor cell migration in the SVZ and LVE of 1-, 6- and 12-month-old guinea pigs. In young animals, we used confocal spectral and transmission electron microscopy to identify neuroblasts, astrocytes, and progenitor cells in the SVZ and LVE. Analysis of peroxidase diffusion demonstrated that the LVE was a continuous cavity lined by ependymal cells and surrounded by neuroblasts. Precursor cells were mostly located in the SVZ and migrated from the SVZ to the OB through the LVE. Finally, analysis of 6- and 12-month-old guinea pigs revealed that the LVE was preserved in older animals; however, the number of neurogenic cells was significantly reduced. Consequently, we propose that the guinea pig brain may be used as a new neurogenic model with increased similarity to humans, given that the LVE connects the LV with the OB, as has been described in humans, and that the LVE works a migratory pathway. Stem Cells 2016;34:2574-2586.

SVCT2 Overexpression in Neuroblastoma Cells Induces Cellular Branching that is Associated with ERK Signaling.

Expression of the sodium and ascorbic acid (AA) cotransporter SVCT2 is induced during the period of cellular arborization and synaptic maturation of early postnatal (P1-P5) rat cerebral neurons. The physiological importance of the transporter for neurons is evidenced by the lethality and delayed neuronal differentiation detected in mice with ablation of SVCT2. The mechanism(s) involved in these defects and the role of SVCT2 in neuronal branching have not been determined yet. To address this, we used lentiviral expression vectors to increase the levels of SVCT2 in N2a cells and analyzed the effects on neurite formation. Expression of a fusion protein containing the human SVCT2wt and EYFP induced an increase in the number of MAP2+ neurites and filopodia in N2a cells. Overexpression of SVCT2 and treatment with AA promoted ERK1/2 phosphorylation. Our data suggest that enhanced expression of the high affinity AA transporter SVCT2, which tightly regulates intracellular AA concentrations, induces neuronal branching that then activates key signaling pathways that are involved in the differentiation and maturation of cortical neurons during postnatal development.

Vitamin C Transporters, Recycling and the Bystander Effect in the Nervous System: SVCT2 versus Gluts.

Vitamin C is an essential micronutrient in the human diet; its deficiency leads to a number of symptoms and ultimately death. After entry into cells within the central nervous system (CNS) through sodium vitamin C transporters (SVCTs) and facilitative glucose transporters (GLUTs), vitamin C functions as a neuromodulator, enzymatic cofactor, and reactive oxygen species (ROS) scavenger; it also stimulates differentiation. In this review, we will compare the molecular and structural aspects of vitamin C and glucose transporters and their expression in endothelial or choroid plexus cells, which form part of the blood-brain barrier and blood-cerebrospinal fluid (CSF) barrier, respectively. Additionally, we will describe SVCT and GLUT expression in different cells of the brain as well as SVCT2 distribution in tanycytes and astrocytes of the hypothalamic region. Finally, we will describe vitamin C recycling in the brain, which is mediated by a metabolic interaction between astrocytes and neurons, and the role of the "bystander effect" in the recycling mechanism of vitamin C in both normal and pathological conditions.

SVCT2 vitamin C transporter expression in progenitor cells of the postnatal neurogenic niche.

Known as a critical antioxidant, recent studies suggest that vitamin C plays an important role in stem cell generation, proliferation and differentiation. Vitamin C also enhances neural differentiation during cerebral development, a function that has not been studied in brain precursor cells. We observed that the rat neurogenic niche is structurally organized at day 15 of postnatal development, and proliferation and neural differentiation increase at day 21. In the human brain, a similar subventricular niche was observed at 1-month of postnatal development. Using immunohistochemistry, sodium-vitamin C cotransporter 2 (SVCT2) expression was detected in the subventricular zone (SVZ) and rostral migratory stream (RMS). Low co-distribution of SVCT2 and ßIII-tubulin in neuroblasts or type-A cells was detected, and minimal co-localization of SVCT2 and GFAP in type-B or precursor cells was observed. Similar results were obtained in the human neurogenic niche. However, BrdU-positive cells also expressed SVCT2, suggesting a role of vitamin C in neural progenitor proliferation. Primary neurospheres prepared from rat brain and the P19 teratocarcinoma cell line, which forms neurospheres in vitro, were used to analyze the effect of vitamin C in neural stem cells. Both cell types expressed functional SVCT2 in vitro, and ascorbic acid (AA) induced their neural differentiation, increased ßIII-tubulin and SVCT2 expression, and amplified vitamin C uptake.

Superoxide-dependent uptake of vitamin C in human glioma cells.

Glioblastomas are lethal brain tumors that resist current cytostatic therapies. Vitamin C may antagonize the effects of reactive oxygen species (ROS) generating therapies; however, it is often used to reduce therapy-related side effects despite its effects on therapy or tumor growth. Because the mechanisms of vitamin C uptake in gliomas are currently unknown, we evaluated the expression of the sodium-vitamin C cotransporter (SVCT) and facilitative hexose transporter (GLUT) families in human glioma cells. In addition, as microglial cells can greatly infiltrate high-grade gliomas (constituting up to 45% of cells in glioblastomas), the effect of TC620 glioma cell interactions with microglial-like HL60 cells on vitamin C uptake (Bystander effect) was determined. Although glioma cells expressed high levels of the SVCT isoform-2 (SVCT2), low functional activity, intracellular localization and the expression of the dominant-negative isoform (dnSVCT2) were observed. The increased glucose metabolic activity of glioma cells was evident by the high 2-Deoxy-d-glucose and dehydroascorbic acid (DHA) uptake rates through the GLUT isoform-1 (GLUT1), the main DHA transporter in glioblastoma. Co-culture of glioma cells and activated microglial-like HL60 cells resulted in extracellular ascorbic acid oxidation and high DHA uptake by glioma cells. This Bystander effect may explain the high antioxidative potential observed in high-grade gliomas. This study strongly suggests that the Bystander effect, that is, glioma cell interaction with oxidant-producing microglia, could be an important mechanism for glioma vitamin C loading in the absence of functional sodium-vitamin C cotransporter 2 (SVCT2) expression. The high cellular vitamin C load in glioma cells results from a high uptake of extracellular dehydroascorbic acid (DHA) generated by neighboring microglia. This Bystander effect may explain the high antioxidative potential observed in high-grade gliomas, considering that high-grade gliomas may be the only neoplasm where oxidant-producing microglia can almost equal the number of tumor cells.

Typical and atypical stem cells in the brain, vitamin C effect and neuropathology.

Stem cells are considered a valuable cellular resource for tissue replacement therapies in most brain disorders. Stem cells have the ability to self-replicate and differentiate into numerous cell types, including neurons, oligodendrocytes and astrocytes. As a result, stem cells have been considered the "holy grail" of modern medical neuroscience. Despite their tremendous therapeutic potential, little is known about the mechanisms that regulate their differentiation. In this review, we analyze stem cells in embryonic and adult brains, and illustrate the differentiation pathways that give origin to most brain cells. We also evaluate the emergent role of the well known anti-oxidant, vitamin C, in stem cell differentiation. We believe that a complete understanding of all molecular players, including vitamin C, in stem cell differentiation will positively impact on the use of stem cell transplantation for neurodegenerative diseases.

Typical and atypical stem cells in the brain, vitamin C effect and neuropathology

Stem cells are considered a valuable cellular resource for tissue replacement therapies in most brain disorders. Stem cells have the ability to self-replicate and differentiate into numerous cell types, including neurons, oligodendrocytes and astrocytes. As a result, stem cells have been considered the "holy grail" of modern medical neuroscience. Despite their tremendous therapeutic potential, little is known about the mechanisms that regulate their differentiation. In this review, we analyze stem cells in embryonic and adult brains, and illustrate the differentiation pathways that give origin to most brain cells. We also evaluate the emergent role of the well known anti-oxidant, vitamin C, in stem cell differentiation. We believe that a complete understanding of all molecular players, including vitamin C, in stem cell differentiation will positively impact on the use of stem cell transplantation for neurodegenerative diseases.

Insulin regulates GLUT1-mediated glucose transport in MG-63 human osteosarcoma cells.

Osteosarcoma is the most common type of malignant bone cancer, accounting for 35% of primary bone malignancies. Because cancer cells utilize glucose as their primary energy substrate, the expression and regulation of glucose transporters (GLUT) may be important in tumor development and progression. GLUT expression has not been studied previously in human osteosarcoma cell lines. Furthermore, although insulin and insulin-like growth factor (IGF-I) play an important role in cell proliferation and tumor progression, the role of these hormones on GLUT expression and glucose uptake, and their possible relation to osteosarcoma, have also not been studied. We determined the effect of insulin and IGF-I on GLUT expression and glucose transport in three well-characterized human osteosarcoma cell lines (MG-63, SaOs-2, and U2-Os) using immunocytochemical, RT-PCR and functional kinetic analyses. Furthermore we also studied GLUT isoform expression in osteosarcoma primary tumors and metastases by in situ hybridization and immunohistochemical analyses. RT-PCR and immunostaining show that GLUT1 is the main isoform expressed in the cell lines and tissues studied, respectively. Immunocytochemical analysis shows that although insulin does not affect levels of GLUT1 expression it does induce a translocation of the transporter to the plasma membrane. This translocation is associated with increased transport of glucose into the cell. GLUT1 is the main glucose transporter expressed in osteosarcoma, furthermore, this transporter is regulated by insulin in human MG-63 cells. One possible mechanism through which insulin is involved in cancer progression is by increasing the amount of glucose available to the cancer cell.