High Doses Of Ascorbate Research Articles

Ascorbate uptake enables tubular mitophagy to prevent septic AKI by PINK1-PARK2 axis

Ascorbate (Vitamin C) has been proposed as a promising therapeutic agent against sepsis in clinical trials, but there is little experimental evidence on its anti-septic efficacy. We report that Toll-like receptor 4 (TLR4) activation by LPS stimuli augments ascorbate uptake in murine and human tubular cells through upregulation of two ascorbate transporters SVCT-1 and -2 mediated by Fn14/SCFFbxw7α cascade. Ascorbate restriction, or knockout of SVCT-1 and -2, the circumstance reminiscent to blockade of ascorbate uptake, endows tubular cells more vulnerable to the LPS-inducible apoptosis, whereas exogenous administration of ascorbate overrides the ruin execution, for which the PINK1-PARK2, rather than BNIP3-NIX axis is required. Ascorbate increases, while SVCT-1 and -2 knockout or ascorbate restriction dampens tubular mitophagy upon LPS stimuli. Treatment of endotoxemic mice with high-dose ascorbate confers mitophagy and substantial protection against mortality and septic acute kidney injury (AKI). Our work provides a rationale for clinical management of septic AKI with high doses of ascorbate.

Ascorbate kills breast cancer cells by rewiring metabolism via redox imbalance and energy crisis

The idea to use megadoses of ascorbate (vitamin C) for cancer treatment has recently been revived. Despite clear efficacy in animal experimentation, our understanding of the cellular and molecular mechanisms of this treatment is still limited and suggests a combined oxidative and metabolic mechanism behind the selective cytotoxicity of ascorbate towards cancerous cells. To gain more insight into the cellular effects of high doses of ascorbate, we performed a detailed analysis of metabolic changes and cell survival of both luminal and basal-like breast cancer cells treated with ascorbate and revealed a distinctive metabolic shift virtually reversing the Warburg effect and triggering a severe disruption of redox homeostasis. High doses of ascorbate were cytotoxic against MCF7 and MDA-MB231 cells representing luminal and basal-like breast cancer phenotypes. Cell death was dependent on ascorbate-induced oxidative stress and accumulation of ROS, DNA damage, and depletion of essential intracellular co-factors including NAD+/NADH, associated with a multifaceted metabolic rewiring. This included a sharp disruption of glycolysis at the triose phosphate level, a rapid drop in ATP levels, and redirection of metabolites toward lipid droplet accumulation and increased metabolites and enzymatic activity in the pentose phosphate pathway (PPP). High doses of ascorbate also inhibited the TCA cycle and increased oxygen consumption. Together the severe disruptions of the intracellular metabolic homeostasis on multiple levels “redox crisis and energetic catastrophe” consequently trigger a rapid irreversible cell death.

PML/RARa Interferes with NRF2 Transcriptional Activity Increasing the Sensitivity to Ascorbate of Acute Promyelocytic Leukemia Cells.

NRF2 (NF-E2 p45-related factor 2) orchestrates cellular adaptive responses to stress. Its quantity and subcellular location is controlled through a complex network and its activity increases during redox perturbation, inflammation, growth factor stimulation, and energy fluxes. Even before all-trans retinoic acid (ATRA) treatment era it was a common experience that acute promyelocytic leukemia (APL) cells are highly sensitive to first line chemotherapy. Since we demonstrated how high doses of ascorbate (ASC) preferentially kill leukemic blast cells from APL patients, we aimed to define the underlying mechanism and found that promyelocytic leukemia/retinoic acid receptor α (PML/RARa) inhibits NRF2 function, impedes its transfer to the nucleus and enhances its degradation in the cytoplasm. Such loss of NRF2 function alters cell metabolism, demarcating APL tissue from both normal promyelocytes and other acute myeloide leukemia (AML) blast cells. Resistance to ATRA/arsenic trioxide (ATO) treatment is rare but grave and the metabolically-oriented treatment with high doses of ASC, which is highly effective on APL cells and harmless on normal hematopoietic stem cells (HSCs), could be of use in preventing clonal evolution and in rescuing APL-resistant patients.

High Doses of Ascorbate (Vitamin C): A New Frontier in the Treatment of Intraocular Cancer

High Doses of Ascorbate (Vitamin C): A New Frontier in the Treatment of Intraocular Cancer

Modulation of Cytokines in Cancer Patients by Intravenous Ascorbate Therapy.

BackgroundCytokines play an important role in tumor angiogenesis and inflammation. There is evidence in the literature that high doses of ascorbate can reduce inflammatory cytokine levels in cancer patients. The objective of this study was to investigate the effect of treatment by intravenous vitamin C (IVC) on cytokines and tumor markers.Material/MethodsWith the availability of protein array kits allowing assessment of many cytokines in a single sample, we measured 174 cytokines and additional 54 proteins and tumor markers in 12 cancer patients before and after a series of IVC treatments.ResultsPresented results show for our 12 patients the effect of treatment resulted in normalization of many cytokine levels. Cytokines that were most consistently elevated prior to treatments included M-CSF-R, Leptin, EGF, FGF-6, TNF-α, β, TARC, MCP-1,4, MIP, IL-4, 10, IL-4, and TGF-β. Cytokine levels tended to decrease during the course of treatment. These include mitogens (EGF, Fit-3 ligand, HGF, IGF-1, IL-21R) and chemo-attractants (CTAC, Eotaxin, E-selectin, Lymphotactin, MIP-1, MCP-1, TARC, SDF-1), as well as inflammation and angiogenesis factors (FGF-6, IL-1β, TGF-1).ConclusionsWe are able to show that average z-scores for several inflammatory and angiogenesis promoting cytokines are positive, indicating that they are higher than averages for healthy controls, and that their levels decreased over the course of treatment. In addition, serum concentrations of tumor markers decreased during the time period of IVC treatment and there were reductions in cMyc and Ras, 2 proteins implicated in being upregulated in cancer.

Cytotoxic effects of high concentrations of sodium ascorbate on human myeloid cell lines.

The effect of high doses of intravenous (sodium) ascorbate (ASC) in the treatment of cancer has been controversial although there is growing evidence that ASC in high (pharmacologic) concentrations induces dose-dependent pro-apoptotic death of tumor cells, in vitro. Very few data are available on the role of ASC in the treatment of acute myeloid leukemia (AML). Ascorbate behaves as an antioxidant at low (physiologic), and as pro-oxidant at pharmacologic, concentrations, and this may account for the differences reported in different experimental settings, when human myeloid cell lines, such as HL60, were treated with ASC. Considering the myeloid origin of HL60 cells, and previous literature reports showing that some cell lines belonging to the myeloid lineage could be sensitive to the pro-apoptotic effects of high concentrations of ASC, we investigated in more details the effects of high doses (0.5 to 7mM) of ASC in vitro, on a variety of human myeloid cell lines including the following: HL60, U937, NB4, NB4-R4 (retinoic acid [RA]-resistant), NB4/AsR (ATO-resistant) acute promyelocytic leukemia (APL)-derived cell lines, and K562 as well as on normal CD34+ progenitors derived from human cord blood. Our results indicate that all analyzed cell lines including all-trans retinoic acid (ATRA)- and arsenic trioxide (ATO)-resistant ones are highly sensitive to the cytotoxic, pro-oxidant effects of high doses of ASC, with an average 50% lethal concentration (LC50) of 3mM, depending on cell type, ASC concentration, and time of exposure. Conversely, high doses of ASC neither did exert significant cytotoxic effects nor impaired the differentiation potential in cord blood (CB) CD34+ normal cells. Since plasma ASC concentrations within the millimolar (mM) range can be easily and safely reached by intravenous administration, we conclude that phase I/II clinical trials using high doses of ASC should be designed for patients with advanced/refractory AML and APL.

Archaic RDA Methodology for Vitamin C

Frei et al's 2012 review entitled “Authors’ Perspective: What is the Optimum Intake of Vitamin C in Humans” is both flawed and misleading. RCTs are ill suited to determining the RDA, it is debatable that there is sufficient scientific evidence to determine the optimum intake of vitamin C in humans, observations regarding high-doses of ascorbate have been ignored, and there are inaccuracies of fact with respect to the saturation of blood plasma following low dose intake. Until the limitations of current knowledge are recognised it is unwise to set limits on the dose.

Megadoses of Ascorbate as a New Chemotherapeutic Approach in Uveal Melanoma: A Preliminary In Vitro Investigation

Background: Despite the more recent advances, there is still no effective systemic therapy for Uveal Melanoma (UM). However, a better understanding of the role of oxidative stress in cancer, has more recently led to a completely new approach to the systemic therapy of cancer, and modulators of the oxidative balance, such as sodium ascorbate (ASC) or arsenic trioxide (ATO), have already entered advanced phases of preclinical and clinical development. Since high doses of ASC have already demonstrated a strong cytotoxic effect on different human cancer cell lines, we have undertaken the present investigation in order to test the sensitivity of OCM1 and C918 uveal melanoma (UM) cell lines to high doses of ASC, in vitro, as compared to ATO, a pro-oxidant drug which has already undergone extensive in vitro and pre-clinical investigation in UM. Methods: Both OCM1 and C918 UM cell lines have been exposed to increasing doses of either ASC or ATO, to build a dose-response curve around the peak plasma concentrations reached by both chemicals. The assessment of cell count and viability was performed with flow cytometry. Results: Both OCM1 and C918 UM cell lines are highly sensitive to ASC in the range of millimolar (mM) concentrations which can be usually reached by the intravenous injection of high doses of the compound. ATO at the dosages used in this study, never reached the LC50. When the exposure to ASC was reduced to two hours, it still had significant effects on survival of both OCM1 and C918 UM cells. Conclusions: This report shows that ASC is highly cytotoxic for both OCM1 and C918 UM cells, when used in high, pro-oxidant doses. To our knowledge, this is the first report showing that UM cells can be efficiently killed, in vitro, with high doses of ASC.

High Doses of Ascorbate Kill Y79 Retinoblastoma Cells In vitro

Objectives: To tests the sensitivity of Y79 retinoblastoma cell lines to high doses of ascorbate, in vitro, and compare its effects with those of some chemotherapeutic agents routinely employed in the treatment of retinoblastoma.Methods: Y79 retinoblastoma cells have been exposed to increasing doses of either sodium ascorbate (SA) or Melphalan (MEL), to define a dose-response curve around the peak plasma concentrations reached by both chemicals when administered according to the existing therapeutic procedures and protocols. The assessment of cell number and viability was performed, before and after exposure, with both the manual (Trypan Blue Exclusion Test) and automated (flow cytometry) methods. Fluorescence microscopy and direct observation of cells in culture, with inverted microscope, were also performed.Results: Y79 cells are highly sensitive to the cytotoxic effect of SA, with cell viability reduced of over 90% in some experiments. As reported in the literature, this effect is directly cytotoxic and most probably mediated by acute oxidative stress on different cellular components. The same does not apply to Melphalan which, at the doses commonly used for therapeutic purposes, did not show any significant effect on cell viability, in vitro.Conclusion: To our knowledge, this is the first report showing that high doses of SA can actively kill retinoblastoma cells in vitro. While it is not surprising for SA, to show direct cytotoxic effect on tumor cells, the data reported herein represent the first evidence in favor of the possible clinical use of high doses of intravenous SA, to treat children affected by retinoblastoma. Given the many advantages of SA over the chemotherapeutic agents commonly employed to treat cancer (including its almost total absence of toxic or side effects, and its exclusive specificity for cancer cells), it is reasonable to assume, from the data reported herein, that the high doses of intravenous ascorbate, have the potential to represent a real revolution in the treatment of retinoblastoma.

Megadoses of Sodium Ascorbate Efficiently Kill HL60 Cells <i>in Vitro</i>: Comparison with Arsenic Trioxide

Arsenic Trioxide (ATO) is widely acknowledged as the treatment of choice for Acute Promyelocytic Leukemia (APL). It is a “two-sided” drug since it can induce differentiation or kill APL and other tumor cells according to the dosage. Part of the cytotoxic effects of ATO on APL cells is due to its pro-oxidant activity, a characteristic which ATO shares with a number of other compounds, including high doses of ascorbate (ASC). In a comparative investigation on the cytotoxic effects of both ATO and ASC on HL60 (APL) cell lines, in Vitro, we have been able to confirm the known cytotoxic effects of ATO, but, more importantly, we have demonstrated that ASC is significantly more effective than ATO, in killing these cancer cells in Vitro, when the concentrations are maintained within the millimolar (mM) range, i.e. the range of plasma concentrations at which ASC induces oxidative damage to tumor cells. Since these plasma levels can be reached only by the intravenous administration of high doses of ASC, we propose that intravenous high doses of ASC may represent a potentially revolutionary new approach in the management of APL.

Pharmacologic concentrations of ascorbate are achieved by parenteral administration and exhibit antitumoral effects

Recently, it has been proposed that pharmacologic concentrations of ascorbate (vitamin C) can be reached by intravenous injection. Because high doses of ascorbate have been described to possess anticancer effects, the therapeutic potential of these concentrations has been studied, both in vitro and in vivo. By using 2-h exposures, a protocol that mimics a parenteral use, we observed that pharmacologic concentrations of ascorbate killed various cancer cell lines very efficiently (EC 50 ranging from 3 to 7 mM). The mechanism of cytotoxicity is based on the production of extracellular hydrogen peroxide and involves intracellular transition metals. In agreement with what has been previously published, our in vivo results show that both intravenous and intraperitoneal administration of ascorbate induced pharmacologic concentrations (up to 20 mM) in blood. In contrast, the concentrations reached orally remained physiological. According to pharmacokinetic data, parenteral administration of ascorbate decreased the growth rate of a murine hepatoma, whereas oral administration of the same dosage did not. We also report that pharmacologic concentrations of ascorbate did not interfere with but rather reinforced the activity of five important chemotherapeutic drugs. Taken together, these results confirm that oral and parenteral administration of ascorbate are not comparable, the latter resulting in pharmacologic concentrations of ascorbate that exhibit interesting anticancer properties.

Ascorbic acid, cell proliferation, and cell differentiation in culture.

The effects of vitamin C in tissue culture have not been systematically investigated for several reasons: (1) being an essential micronutrient for a limited number of species only (primates, guinea pigs, passeriformis birds, and some reptiles), ascor-bate has not been considered a generally crucial additive of tissue culture media; (2) cultures of primary cells regularly, and those of pertinent cell lines often require supplementation of the media by serum whereby ascorbate is provided at variable but amounts obviously sufficient for proliferation; (3) due to its instability under aerobic conditions and elevated temperatures the actual ascorbate content during the course of a tissue culture remains uncertain if not specifically determined. Also, high doses of ascorbate may be detrimental to cultured cells (see Section 2), particularly in serum-free media. Consequently, supplementation of tissue culture media by vitamin C has been routinely neglected, unless a specific requirement became obvious or a specific scientific problem was to be addressed.

Effect of ascorbate or N-acetylcysteine treatment in a patient with hereditary glutathione synthetase deficiency

A 45-month-old girl with 5-oxoprolinuria (pyroglutamic aciduria), hemolysis, and marked glutathione depletion caused by deficiency of glutathione synthetase was followed before and during treatment with ascorbate or N-acetylcysteine. High doses of ascorbate (0.7 mmol/kg per day) or N-acetylcysteine (6 mmol/kg per day) were given for 1 to 2 weeks without any obvious deleterious side effects. Ascorbate markedly increased lymphocyte (4-fold) and plasma (8-fold) levels of glutathione. N-Acetylcysteine also increased lymphocyte (3.5-fold) and plasma (6-fold) levels of glutathione. After these treatments were discontinued, lymphocyte and plasma glutathione levels decreased rapidly to pretreatment levels. Ascorbate treatment was extended for 1 year, and lymphocyte (4-fold) and plasma (2- to 5-fold) glutathione levels remained elevated above baseline. In parallel, the hematocrit increased from 25.4% to 32.6%, and the reticulocyte count decreased from 11% to 4%. The results demonstrate that ascorbate and N-acetylcysteine can decrease erythrocyte turnover in patients with hereditary glutathione deficiency by increasing glutathione levels. (J P EDIATR 1994;124:229-33)

Ascorbic acid prevents oxidative stress in glutathione-deficient mice: effects on lung type 2 cell lamellar bodies, lung surfactant, and skeletal muscle.

Glutathione deficiency in adult mice leads to lung type 2 cell lamellar body and mitochondrial damage; as reported here, these effects are associated with marked decrease of the levels of phosphatidylcholine (the main component of lung surfactant) in the lung and the bronchoalveolar lining fluid. Severe mitochondrial damage was also found in skeletal muscle. Treatment with ascorbate (1-2 mmol per kg of body weight per day), which led to greatly increased (approximately 2-fold) levels of lung and muscle mitochondrial glutathione, prevented damage to lamellar bodies and mitochondria as well as the decline of phosphatidylcholine levels in lung and alveolar lining fluid. The findings indicate that glutathione deficiency leads to depletion of lung surfactant and that this can be prevented with ascorbate. Administration of ascorbate spares glutathione and prevents cellular damage. Lamellar body degeneration in glutathione deficiency appears to be associated with oxidative damage to the perilamellar membrane, which contains the enzymes required for phosphatidylcholine synthesis. It is notable that although severe glutathione deficiency is lethal to newborn rats, which apparently do not synthesize ascorbate, adult mice are better able to survive such a deficiency because they can synthesize ascorbate. The present studies, which suggest that high doses of ascorbate may be of therapeutic value, emphasize that ascorbate and glutathione have actions in common and that they function together in a physiologically significant antioxidant system.