Dehydroascorbic acid sensitizes cancer cells to system xc- inhibition-induced ferroptosis by promoting lipid droplet peroxidation.

Since the discovery of ferroptosis, it has been postulated that this type of cell death could be utilized in treatments for cancer. Unfortunately, several highly aggressive tumor models are resistant to the pharmacological induction of ferroptosis. However, with the use of combined therapies, it is possible to recover sensitivity to ferroptosis in certain cellular models. Here, we discovered that co-treatment with the metabolically stable ferroptosis inducer imidazole ketone erastin (IKE) and the oxidized form of vitamin C, dehydroascorbic acid (DHAA), is a powerful therapy that induces ferroptosis in tumor cells previously resistant to IKE-induced ferroptosis. We determined that DHAA and IKE + DHAA delocalize and deplete GPX4 in tumor cells, specifically inducing lipid droplet peroxidation, which leads to ferroptosis. Moreover, in vivo, IKE + DHAA has high efficacy with regard to the eradication of highly aggressive tumors such as glioblastomas. Thus, the use of IKE + DHAA could be an effective and safe therapy for the eradication of difficult-to-treat cancers.

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.

The Safe Soluble Compound Dehydroascorbic Acid Inhibits Various Upstream and Downstream Effectors of PI3K and KRAS Signaling Pathways in Undruggable PIK3CA/KRAS-Mutant Colorectal Cancer Stem-Like Cells.

Efforts to develop effective drugs targeting PI3K and KRAS signaling pathways in PIK3CA/KRAS-mutant colorectal cancer stem cells (CRCSCs) remain challenging. Finding safe compounds that can easily enter CRCSCs with the ability to target metastasis-driver gene CXCR4 and pluripotency network genes as key upstream and downstream effectors of both PI3K and KRAS signaling pathways may provide promising results. PIK3CA/KRAS-mutant CRCSCs display high expression of glucose transporters (GLUTs) on their cell membrane and a glycolytic phenotype providing an opportunity to deliver antiglycolytic compounds into these cells via the GLUTs. CRC patients with low levels of vitamin C in their plasma show a shorter survival suggesting the ability of this vitamin at the physiologic levels for caspase-3 activation and apoptosis in CRCSCs. Vitamin C in an oxidized form (L-dehydroascorbic acid; L-DHA) with antiglycolytic activity can be taken up into CRC cells via the GLUTs. This may provide selective toxicity on CRCSCs and affect CXCR4 and stemness markers genes expression in these cells. To this end, we treated PIK3CA/KRAS-mutant LS174T cells with high glycolytic activity as an attractive model for CRCSCs with L-DHA equal to the pharmacological levels of vitamin C in human plasma, after which cell numbers, metabolic activity, proliferation-rate, CXCR4 and pluripotency network genes expression, caspase-3 activity with apoptosis were evaluated. 48 h post-treatment with 100- to 1000 µM L-DHA, cell numbers were decreased and measured to be 70-47% control. L-DHA with selective toxicity on LS174T cells diminished metabolic activity and cell proliferation-rate to 1.4-0.8 (Control OD = 1.5) and 92-54.5% respectively with no toxicity on PBMCs. L-DHA decreased CXCR4, Bmi-1, Sox-2 and Oct-4 expression to 45%, 85%, 45% and 48% control respectively followed by caspase-3 reactivation by 2.5 to 4.9-fold increases and induction of apoptosis ranging from 0.5% to 58.3% for 100- to 1000 µM L-DHA. According to our data, CRC stem-like cells were highly sensitive to L-DHA in in-vitro. L-DHA selectively targeted LS174T cells and successfully reactivated caspase-3 and apoptosis in these cells. CXCR4, stemness marker genes and metabolic activity appear to be promising targets of L-DHA. Our results may provide a new therapeutic approach to target selectively GLUT-overexpressing PIK3CA/KRAS-mutant CRCSCs using L-DHA with no toxicity on normal cells.

Glutathione and Glutathione-Like Sequences of Opioid and Aminergic Receptors Bind Ascorbic Acid, Adrenergic and Opioid Drugs Mediating Antioxidant Function: Relevance for Anesthesia and Abuse.

Opioids and their antagonists alter vitamin C metabolism. Morphine binds to glutathione (l-γ-glutamyl-l-cysteinyl-glycine), an intracellular ascorbic acid recycling molecule with a wide range of additional activities. The morphine metabolite morphinone reacts with glutathione to form a covalent adduct that is then excreted in urine. Morphine also binds to adrenergic and histaminergic receptors in their extracellular loop regions, enhancing aminergic agonist activity. The first and second extracellular loops of adrenergic and histaminergic receptors are, like glutathione, characterized by the presence of cysteines and/or methionines, and recycle ascorbic acid with similar efficiency. Conversely, adrenergic drugs bind to extracellular loops of opioid receptors, enhancing their activity. These observations suggest functional interactions among opioids and amines, their receptors, and glutathione. We therefore explored the relative binding affinities of ascorbic acid, dehydroascorbic acid, opioid and adrenergic compounds, as well as various control compounds, to glutathione and glutathione-like peptides derived from the extracellular loop regions of the human beta 2-adrenergic, dopamine D1, histamine H1, and mu opioid receptors, as well as controls. Some cysteine-containing peptides derived from these receptors do bind ascorbic acid and/or dehydroascorbic acid and the same peptides generally bind opioid compounds. Glutathione binds not only morphine but also naloxone, methadone, and methionine enkephalin. Some adrenergic drugs also bind to glutathione and glutathione-like receptor regions. These sets of interactions provide a novel basis for understanding some ways that adrenergic, opioid and antioxidant systems interact during anesthesia and drug abuse and may have utility for understanding drug interactions.

Ascorbic acid leads to glycation and interferes with neurite outgrowth.

Ascorbic acid better known as vitamin C, is a reducing carbohydrate needed for a variety of functions in the human body. The most important characteristic of ascorbic acid is the ability to donate two electrons, predestining it as a major player in balancing the physiological redox state and as a necessary cofactor in multiple enzymatic hydroxylation processes. Ascorbic acid can be reversibly oxidized in two steps, leading to semidehydroascorbic acid and dehydroascorbic acid, respectively. Further degradation is irreversible and generates highly reactive carbonyl-intermediates. These intermediates are able to induce glycation of proteins, a non-enzymatic and unspecific reaction of carbonyls with amino groups involved to several age-related diseases. In this study, we investigated the effect of ascorbic acid- and dehydroascorbic acid-induced glycation on PC12 cells, which represent a model for neuronal plasticity. We found that both applications of ascorbic acid or dehydroascorbic acid leads to glycation of cellular proteins, but that ascorbic acid interferes more with viability and neurite outgrowth compared with dehydroascorbic acid.

Role of Zucchini and Its Distinctive Components in the Modulation of Degenerative Processes: Genotoxicity, Anti-Genotoxicity, Cytotoxicity and Apoptotic Effects.

Zucchini (Cucurbita pepo subsp. pepo) is a seasonal vegetable with high nutritional and medical values. Many useful properties of this fruit are attributed to bioactive compounds. Zucchini fruits ("Yellow" and "Light Green" varieties) and four distinctive components (lutein, ß-carotene, zeaxanthin and dehydroascorbic acid) were selected. Firstly, the lutein, ß-carotene, zeaxanthin and dehydroascorbic acid contents were determined in these fruits. Then, in order to evaluate the safety and suitability of their use, different assays were carried out: (i) genotoxicity and anti-genotoxicity tests to determine the safety and DNA-protection against hydrogen peroxide; (ii) cytotoxicity; and (iii) DNA fragmentation and Annexin V/PI (Propidium Iodide) assays to evaluate the pro-apoptotic effect. Results showed that: (i) all the substances were non-genotoxic; (ii) all the substances were anti-genotoxic except the highest concentration of lutein; (iii) "Yellow" zucchini epicarp and mesocarp exhibited the highest cytotoxic activity (IC50 > 0.1 mg/mL and 0.2 mg/mL, respectively); and (iv) "Light Green" zucchini skin induced internucleosomal DNA fragmentation, ß-carotene being the possible molecule responsible for its pro-apoptotic activity. To sum up, zucchini fruit could play a positive role in human health and nutrition due to this fruit and its components were safe, able to inhibit significantly the H2O2-induced damage and exhibit anti-proliferative and pro-apoptotic activities toward HL60 (human promyelocytic leukemia cells) tumor cells. The information generated from this research should be considered when selecting potential accessions for breeding program purposes.

Ascorbic acid, but not dehydroascorbic acid increases intracellular vitamin C content to decrease Hypoxia Inducible Factor -1 alpha activity and reduce malignant potential in human melanoma.

INTRODUCTION: Accumulation of hypoxia inducible factor-1 alpha (HIF-1α) in malignant tissue is known to contribute to oncogenic progression and is inversely associated with patient survival. Ascorbic acid (AA) depletion in malignant tissue may contribute to aberrant normoxic activity of HIF-1α. While AA supplementation has been shown to attenuate HIF-1α function in malignant melanoma, the use of dehydroascorbic acid (DHA) as a therapeutic means to increase intracellular AA and modulate HIF-1α function is yet to be evaluated. Here we compared the ability of AA and DHA to increase intracellular vitamin C content and decrease the malignant potential of human melanoma by reducing the activity of HIF-1α. METHODS: HIF-1α protein accumulation was evaluated by western blot and transcriptional activity was evaluated by reporter gene assay using a HIF-1 HRE-luciferase plasmid. Protein expressions and subcellular localizations of vitamin C transporters were evaluated by western blot and confocal imaging. Intracellular vitamin C content following AA, ascorbate 2-phosphate (A2P), or DHA supplementation was determined using a vitamin C assay. Malignant potential was accessed using a 3D spheroid Matrigel invasion assay. Data was analyzed by One or Two-way ANOVA with Tukey's multiple comparisons test as appropriate with p<0.05 considered significant. RESULTS: Melanoma cells expressed both sodium dependent vitamin C (SVCT) and glucose (GLUT) transporters for AA and DHA transport respectively, however advanced melanomas responded favorably to AA, but not DHA. Physiological glucose conditions significantly impaired intracellular vitamin C accumulation following DHA treatment. Consequently, A2P and AA, but not DHA treated cells demonstrated lower HIF-1α protein expression and activity, and reduced malignant potential. The ability of AA to regulate HIF-1α was dependent on SVCT2 function and SVCT2 was not significantly inhibited at pH representative of the tumor microenvironment. CONCLUSIONS: The use of ascorbic acid as an adjuvant cancer therapy remains under investigated. While AA and A2P were capable of modulating HIF-1α protein accumulation/activity, DHA supplementation resulted in minimal intracellular vitamin C activity with decreased ability to inhibit HIF-1α activity and malignant potential in advanced melanoma. Restoring AA dependent regulation of HIF-1α in malignant cells may prove beneficial in reducing chemotherapy resistance and improving treatment outcomes.

Dehydroascorbic acid-induced endoplasmic reticulum stress and leptin resistance in neuronal cells.

Due to its anti-obesity effects, an adipocyte-derived hormone, leptin, has become important for the treatment of obesity. However, most obese subjects are in a state of leptin resistance, and endoplasmic reticulum (ER) stress is suggested to be involved in the pathophysiology of leptin resistance. Dehydroascorbic acid (DHAA), an oxidized form of vitamin C, was found to be increased in diabetes. In the present study, we investigated the possible effects of DHAA on the activation of ER stress and leptin resistance. A human neuroblastoma cell line, stably transfected with the Ob-Rb leptin receptor (SH-SY5Y-ObRb), was treated with DHAA. We found that DHAA upregulated ER stress-related genes such as GRP78, CHOP, and spliced XBP1. Moreover, leptin-induced STAT3 phosphorylation was hindered by DHAA. These results suggested that increases in the levels of DHAA might be harmful to neurons, contributing to defective leptin-responsive signaling.

Dehydroascorbic Acid Promotes Cell Death in Neurons Under Oxidative Stress: a Protective Role for Astrocytes.

Ascorbic acid (AA), the reduced form of vitamin C, is incorporated into neurons via the sodium ascorbate co-transporter SVCT2. However, this transporter is not expressed in astrocytes, which take up the oxidized form of vitamin C, dehydroascorbic acid (DHA), via the facilitative hexose transporter GLUT1. Therefore, neuron and astrocyte interactions are thought to mediate vitamin C recycling in the nervous system. Although astrocytes are essential for the antioxidant defense of neurons under oxidative stress, a condition in which a large amount of ROS is generated that may favor the extracellular oxidation of AA and the subsequent neuronal uptake of DHA via GLUT3, potentially increasing oxidative stress in neurons. This study analyzed the effects of oxidative stress and DHA uptake on neuronal cell death in vitro. Different analyses revealed the presence of the DHA transporters GLUT1 and GLUT3 in Neuro2a and HN33.11 cells and in cortical neurons. Kinetic analyses confirmed that all cells analyzed in this study possess functional GLUTs that take up 2-deoxyglucose and DHA. Thus, DHA promotes the death of stressed neuronal cells, which is reversed by incubating the cells with cytochalasin B, an inhibitor of DHA uptake by GLUT1 and GLUT3. Additionally, the presence of glial cells (U87 and astrocytes), which promote DHA recycling, reverses the observed cell death of stressed neurons. Taken together, these results indicate that DHA promotes the death of stressed neurons and that astrocytes are essential for the antioxidative defense of neurons. Thus, the astrocyte-neuron interaction may function as an essential mechanism for vitamin C recycling, participating in the antioxidative defense of the brain.

Effect of Glucose on GLUT1-Dependent Intracellular Ascorbate Accumulation and Viability of Thyroid Cancer Cells.

Enhanced glucose requirement of cancer cells is associated with an increased glucose transport across plasma membrane that is mediated by a family of facilitated glucose transporter proteins, named GLUTs. GLUT1 is the main transporter in thyroid cancer cells. Glucose is the principal physiological substrate of GLUT1; however, it is also capable of transporting of oxidized form of vitamin C [i.e., dehydroascorbic acid (DHAA) which inside the cells is reduced to ascorbic acid (AA)]. The objective of this study was to determine the effect of normo-, hypo-, and hyperglycemia conditions on GLUT1-dependent intracellular ascorbate accumulation and viability of thyroid cancer cells. GLUT1 seems to be the main DHAA transporter in thyroid cancer cells because its knockdown by RNAi reduced DHAA accumulation by more than 80%. The results showed that in thyroid cancer cells high glucose inhibits both transport of AA and DHAA. Inhibition of vitamin C transport by glucose had a cytotoxic effect on the cells. However, stabilization of vitamin C in one of 2 forms (i.e., AA or DHAA) abolished this effect. These results suggest that cytotoxic effect is rather associated with extracellular accumulation of vitamin C and changes of its oxidation state than with intracellular level of ascorbate.

Intracellular dehydroascorbic acid inhibits SVCT2-dependent transport of ascorbic acid in mitochondria.

Exposure of U937 cells to low concentrations of L-ascorbic acid (AA) is associated with a prompt cellular uptake and a further mitochondrial accumulation of the vitamin. Under the same conditions, dehydroascorbic acid (DHA) uptake was followed by rapid reduction and accumulation of identical intracellular levels of AA, however, in the absence of significant mitochondrial uptake. This event was instead observed after exposure to remarkably greater concentrations of DHA. Furthermore, experiments performed in isolated mitochondria revealed that DHA transport through hexose transporters and Na(+) -dependent transport of AA were very similar. These results suggest that the different subcellular compartmentalization of the vitamin is mediated by events promoting inhibition of mitochondrial AA transport, possibly triggered by low levels of DHA. We obtained results in line with this notion in intact cells, and more direct evidence in isolated mitochondria. This inhibitory effect was promptly reversible after DHA removal and comparable with that mediated by established inhibitors, as quercetin. The results presented collectively indicate that low intracellular concentrations of DHA, because of its rapid reduction back to AA, are a poor substrate for direct mitochondrial uptake. DHA concentrations, however, appear sufficiently high to mediate inhibition of mitochondrial transport of AA/DHA-derived AA.

U937 cell apoptosis induced by arsenite is prevented by low concentrations of mitochondrial ascorbic acid with hardly any effect mediated by the cytosolic fraction of the vitamin.

Arsenite directly triggers cytochrome c and Smac/DIABLO release in mitochondria isolated from U937 cells. These effects were not observed in mitochondria pre-exposed for 15 min to 10 µM L-ascorbic acid (AA). In other experiments, intact cells treated for 24-72 h with arsenite were found to die by apoptosis through a mechanism involving mitochondrial permeability transition. Pre-exposure (15 min) to low micromolar concentrations of AA and dehydroascorbic acid (DHA), resulting in identical cytosolic levels of the vitamin, had a diverse impact on cell survival, as cytoprotection was only observed after treatment with AA. Also the mitochondrial accumulation of the vitamin was restricted to AA exposure. An additional indication linking cytoprotection to the mitochondrial fraction of the vitamin was obtained in experiments measuring susceptibility to arsenite in parallel with loss of mitochondrial and cytosolic AA at different times after vitamin exposure. Finally, we took advantage of our recent findings that DHA potently inhibits AA transport to demonstrate that DHA abolishes all the protective effects of AA, under the same conditions in which the mitochondrial accumulation of the vitamin is prevented without affecting the overall cellular accumulation of the vitamin.

A novel biological role of dehydroascorbic acid: Inhibition of Na(+)-dependent transport of ascorbic acid.

A U937 cell clone, in which low micromolar concentrations of ascorbic acid (AA) and dehydroascorbic acid (DHA) are taken up at identical rates, was used to investigate possible interactions between transport systems mediating cellular uptake of the two forms of the vitamin. Results obtained with different experimental approaches showed that DHA potently and reversibly inhibits AA uptake through Na(+)-AA cotransporters. Hence, a progressive increase in extracellular DHA concentrations in the presence of a fixed amount of AA caused an initial decrease in the net amount of vitamin C accumulated, and eventually, at higher levels, it caused an accumulation of the vitamin solely based on DHA uptake through hexose transporters. DHA-dependent inhibition of AA uptake was also detected in various other cell types. Taken together, our results provide evidence of a novel biological effect mediated by concentrations of DHA compatible with those produced at inflammatory sites.

The oxidized form of vitamin C, dehydroascorbic acid, regulates neuronal energy metabolism.

Vitamin C is an essential factor for neuronal function and survival, existing in two redox states, ascorbic acid (AA), and its oxidized form, dehydroascorbic acid (DHA). Here, we show uptake of both AA and DHA by primary cultures of rat brain cortical neurons. Moreover, we show that most intracellular AA was rapidly oxidized to DHA. Intracellular DHA induced a rapid and dramatic decrease in reduced glutathione that was immediately followed by a spontaneous recovery. This transient decrease in glutathione oxidation was preceded by an increase in the rate of glucose oxidation through the pentose phosphate pathway (PPP), and a concomitant decrease in glucose oxidation through glycolysis. DHA stimulated the activity of glucose-6-phosphate dehydrogenase, the rate-limiting enzyme of the PPP. Furthermore, we found that DHA stimulated the rate of lactate uptake by neurons in a time- and dose-dependent manner. Thus, DHA is a novel modulator of neuronal energy metabolism by facilitating the utilization of glucose through the PPP for antioxidant purposes.

Vitamin C suppresses cell death in MCF-7 human breast cancer cells induced by tamoxifen.

Vitamin C is generally thought to enhance immunity and is widely taken as a supplement especially during cancer treatment. Tamoxifen (TAM) has both cytostatic and cytotoxic properties for breast cancer. TAM engaged mitochondrial oestrogen receptor beta in MCF-7 cells and induces apoptosis by activation of pro-caspase-8 followed by downstream events, including an increase in reactive oxygen species and the release of pro-apoptotic factors from the mitochondria. In addition to that, TAM binds with high affinity to the microsomal anti-oestrogen-binding site and inhibits cholesterol esterification at therapeutic doses. This study aimed to investigate the role of vitamin C in TAM-mediated apoptosis. Cells were loaded with vitamin C by exposure to dehydroascorbic acid, thereby circumventing in vitro artefacts associated with the poor transport and pro-oxidant effects of ascorbic acid. Pre-treatment with vitamin C caused a dose-dependent attenuation of cytotoxicity, as measured by acridine-orange/propidium iodide (AO/PI) and Annexin V assay after treatment with TAM. Vitamin C dose-dependently protected cancer cells against lipid peroxidation caused by TAM treatment. By real-time PCR analysis, an impressive increase in FasL and tumour necrosis factor-α (TNF-α) mRNA was detected after TAM treatment. In addition, a decrease in mitochondrial transmembrane potential was observed. These results support the hypothesis that vitamin C supplementation during cancer treatment may detrimentally affect therapeutic response.

Mitochondrial ascorbic acid is responsible for enhanced susceptibility of U937 cells to the toxic effects of peroxynitrite.

Otherwise nontoxic levels of peroxynitrite promote toxicity in U937 cells pre-exposed to low micromolar concentrations of l-ascorbic acid (AA). This event was associated with the mitochondrial accumulation of the vitamin and with the early formation of secondary reactive oxygen species and DNA single-strand breaks. The same concentrations of peroxynitrite, however, failed to elicit detectable effects in cells pre-exposed to dehydroascorbic acid (DHA), in which mitochondrial accumulation of vitamin C did not occur despite the identical cytosolic levels. Coherently, oxidation of extracellular AA failed to affect the intracellular concentration of the vitamin, but nevertheless prevented its mitochondrial localization as well as the enhanced response to peroxynitrite. Furthermore, in cells postincubated in vitamin C-free medium, time-dependent loss of mitochondrial AA was paralleled by a progressive decline of susceptibility to peroxynitrite, under the same conditions in which cells retained about half of the initial AA. Using different experimental approaches, we finally showed that the enhancing effects of AA are mediated by events associated with peroxynitrite-dependent superoxide/H2 O2 formation in the mitochondrial respiratory chain. Collectively, these results indicate that mitochondria actively take up vitamin C as AA and respond to otherwise inactive concentrations of peroxynitrite with the mitochondrial formation of secondary species responsible for DNA damage and toxicity. DHA preloading, while leading to the accumulation of identical levels of vitamin C, fails to produce these effects because of the poor mitochondrial accumulation of the vitamin.

Modulation of Dcytb (Cybrd 1) expression and function by iron, dehydroascorbate and Hif-2α in cultured cells.

BACKGROUND: Duodenal cytochrome b (Dcytb) is a mammalian plasma ferric reductase enzyme that catalyses the reduction of ferric to ferrous ion in the process of iron absorption. The current study investigates the relationship between Dcytb, iron, dehydroascorbate (DHA) and Hif-2α in cultured cell lines. METHODS: Dcytb and Hif-2α protein expression was analysed by Western blot technique while gene regulation was determined by quantitative PCR. Functional analyses were carried out by ferric reductase and (59)Fe uptake assays. RESULTS: Iron and dehydroascorbic acid treatment of cells inhibited Dcytb mRNA and protein expression. Desferrioxamine also enhanced Dcytb mRNA level after cells were treated overnight. Dcytb knockdown in HuTu cells resulted in reduced mRNA expression and lowered reductase activity. Preloading cells with DHA (to enhance intracellular ascorbate levels) did not stimulate reductase activity fully in Dcytb-silenced cells, implying a Dcytb-dependence of ascorbate-mediated ferrireduction. Moreover, Hif-2α knockdown in HuTu cells led to a reduction in reductase activity and iron uptake. CONCLUSIONS: Taken together, this study shows the functional regulation of Dcytb reductase activity by DHA and Hif-2α. GENERAL SIGNIFICANCE: Dcytb is a plasma membrane protein that accepts electrons intracellularly from DHA/ascorbic acid for ferrireduction at the apical surface of cultured cells and enterocytes.

Expression and/or activity of the SVCT2 ascorbate transporter may be decreased in many aggressive cancers, suggesting potential utility for sodium bicarbonate and dehydroascorbic acid in cancer therapy.

Hypoxia-inducible factor-1 (HIF-1) is a heterodimer transcription factor whose elevated activity in many cancers helps them to survive under hypoxic conditions and enhances their capacity to grow invasively, establish metastases, and survive chemo- or radiotherapy. Optimal intracellular levels of ascorbate suppress the level and transcriptional activity of HIF-1under normoxic or mildly hypoxic conditions by supporting the activity of proly and asparagyl hydroxylases that target HIF-1alpha. High intracellular ascorbate can also work in various ways to down-regulate activation of NF-kappaB which, like HIF-1 is constitutively active in many cancers and promotes aggressive behavior - in part by promoting transcription of HIF-1alpha. Yet recent evidence suggests that, even in the context of adequate ascorbate nutrition, the intracellular ascorbate content of many aggressive cancers may be supoptimal for effective HIF-1 control. This likely reflects low expression or activity of the SVCT2 ascorbate transporter. The expression of SVCT2 in cancers has so far received little study; but the extracellular acidity characteristic of many tumors would be expected to reduce the activity of this transporter, which has a mildly alkaline pH optimum. Unfortunately, since SVCT2 has a high affinity for ascorbate, and its activity is nearly saturated at normal healthy serum levels of this vitamin, increased oral administration of ascorbate would be unlikely to have much impact on the intracellular ascorbate content of tumors. However, cancers in which HIF-1 is active express high levels of glucose transporters such as GLUT-1, and these transporters can promote influx of dehydroascorbic acid (DHA) via facilitated diffusion; once inside the cell, DHA is rapidly reduced to ascorbate, which effectively is "trapped" within the cell. Hence, episodic intravenous infusions of modest doses of DHA may have potential for optimizing the intracellular ascorbate content of cancers, potentially rendering them less aggressive. Indeed, several published studies have concluded that parenteral DHA--sometimes in quite modest doses--can retard the growth of transplanted tumors in rodents. As an alternative or adjunctive strategy, oral administration of sodium bicarbonate, by normalizing the extracellular pH of tumors, has the potential to boost the activity of SCTV2 in tumor cells, thereby promoting increased ascorbate uptake. Indeed, the utility of oral sodium bicarbonate for suppressing metastasis formation in nude mice xenografted with a human breast cancer has been reported. Hence, oral sodium bicarbonate and intravenous DHA may have the potential to blunt the aggressiveness of certain cancers in which suboptimal intracellular ascorbate levels contribute to elevated HIF-1 activity.

Oxidative stress mediates apoptotic effects of ascorbate and dehydroascorbate in human Myelodysplasia cells in vitro.

The Myelodysplastic Syndromes are stem cell heterogeneous disorders characterized by peripheral cytopenias and hypercellular bone marrow, which can evolute to acute leukaemia. Vitamin C can act as an antioxidant, ascorbic acid (AA) donates two electrons and becomes oxidized to dehydroascorbic acid (DHA). Under physiological conditions, vitamin C predominantly exists in its reduced (AA) form but also exists in trace quantities in the oxidized form (DHA). This study evaluates the therapeutic potential of vitamin C in Myelodysplastic Syndromes (MDSs). F36P cells (MDS cell line) were treated with ascorbate and dehydroascorbate alone and in combination with cytarabine. Cell proliferation and viability were assessed by trypan blue assay and cell death was evaluated by optical microscopy and flow cytometry. The role of reactive oxygen species, mitochondrial membrane potential, BAX, BCL-2 and cytochrome C were also assessed. Vitamin C decreases cell proliferation and viability in a concentration, time and administration dependent-manner inducing cell death by apoptosis, which was shown to be associated to an increased in superoxide production, mitochondrial membrane depolarization. These compounds modulate BCL-2, BAX and cytochrome C release. These results suggest that vitamin C induces cell death trough apoptosis in F36P cells and may be a new therapeutic approach in Myelodysplasia.

Ascorbic acid efficiently enhances neuronal synthesis of norepinephrine from dopamine.

Ascorbic acid enhances synthesis of norepinephrine from dopamine in adrenal chromaffin cells by serving as a co-factor for chromaffin granule dopamine ß-hydroxylase (DßH). However, there is controversy regarding in situ kinetics of the ascorbate effect in chromaffin cells, as well as whether they apply to neuronal cells. In this study we evaluated the stimulation of norepinephrine synthesis from dopamine in cultured SH-SY5Y neuroblastoma cells. These cells contained neither ascorbate nor norepinephrine in culture, but when provided with dopamine, they generated intracellular norepinephrine at rates that were stimulated several-fold by intracellular ascorbate. Ascorbate-induced increases in norepinephrine synthesis in dopamine-treated cells were linear over 60 min, despite saturation of intracellular ascorbate. Norepinephrine accumulation after 60 min of incubation with 100 µM dopamine was half-maximal at intracellular ascorbate concentrations of 0.2-0.5 mM, which fits well with the literature K(m) for ascorbate of DßH using dopamine as a substrate. Moreover, these ascorbate concentrations were generated by initial extracellular ascorbate concentrations of less than 25 µM due to concentrative accumulation by the ascorbate transporter. Treatment with 100 µM dopamine acutely increased cellular superoxide generation, which was prevented by ascorbate loading, but associated with a decrease in intracellular ascorbate when the latter was present at concentrations under 1 mM. These results show that ascorbate promptly enhances norepinephrine synthesis from dopamine by neuronal cells that it does so at physiologic intracellular concentrations in accord with the kinetics of DßH, and that it both protects cells from superoxide and by providing electrons to DßH.