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Our data show that ascorbic acid selectively killed cancer but not normal cells, using concentrations that could only be achieved by i.v. administration

Ascorbate-mediated cell death was due to protein-dependent extracellular H2O2 generation, via ascorbate radical formation from ascorbate as the electron donor. Like glucose, when ascorbate is infused i.v., the resulting pharmacologic concentrations should distribute rapidly in the extracellular water space (42). We showed that such pharmacologic ascorbate concentrations in media, as a surrogate for extracellular fluid, generated ascorbate radical and H2O2. In contrast, the same pharmacologic ascorbate concentrations in whole blood generated little detectable ascorbate radical and no detectable H2O2. These findings can be accounted for by efficient and redundant H2O2 catabolic pathways in whole blood (e.g., catalase and glutathione peroxidase) relative to those in media or extracellular fluid

ascorbic acid administered i.v. in pharmacologic concentrations may serve as a pro-drug for H2O2 delivery to the extracellular milieu

H2O2 generated in blood is normally removed by catalase and glutathione peroxidase within red blood cells, with internal glutathione providing reducing equivalents

The electron source for glutathione is NADPH from the pentose shunt, via glucose-6-phosphate dehydrogenase. If activity of this enzyme is diminished, the predicted outcome is impaired H2O2 removal causing intravascular hemolysis, the observed clinical finding.

Only recently has it been understood that the discordant clinical findings can be explained by previously unrecognized fundamental pharmacokinetics properties of ascorbate

Intracellular transport of ascorbate is tightly controlled in relation to extracellular concentration

Intravenous ascorbate infusion is expected to drastically change extracellular but not intracellular concentrations

For i.v. ascorbate to be clinically useful in killing cancer cells, pharmacologic but not physiologic extracellular concentrations should be effective, independent of intracellular ascorbate concentrations.

It is unknown why ascorbate, via H2O2, killed some cancer cells but not normal cells.

There was no correlation with ascorbate-induced cell death and glutathione, catalase activity, or glutathione peroxidase activity.

H2O2, as the product of pharmacologic ascorbate concentrations, has potential therapeutic uses in addition to cancer treatment, especially in infections

Neutrophils generate H2O2 from superoxide,

i.v. ascorbate is effective in some viral infections

H2O2 is toxic to hepatitis C

Use of ascorbate as an H2O2-delivery system against sensitive pathogens, viral or bacterial, has substantial clinical implications that deserve rapid exploration.

Recent pharmacokinetics studies in men and women show that 10 g of ascorbate given i.v. is expected to produce plasma concentrations of nearly 6 mM, which are >25-fold higher than those concentrations from the same oral dose

As much as a 70-fold difference in plasma concentrations is expected between oral and i.v. administration,

Complementary and alternative medicine practitioners worldwide currently use ascorbate i.v. in some patients, in part because there is no apparent harm

Human Burkitt's lymphoma cells

We first investigated whether ascorbate in pharmacologic concentrations selectively affected the survival of cancer cells by studying nine cancer cell lines

Clinical pharmacokinetics analyses show that pharmacologic concentrations of plasma ascorbate, from 0.3 to 15 mM, are achievable only from i.v. administration

plasma ascorbate concentrations from maximum possible oral doses cannot exceed 0.22 mM because of limited intestinal absorption

For five of the nine cancer cell lines, ascorbate concentrations causing a 50% decrease in cell survival (EC50 values) were less than 5 mM, a concentration easily achievable from i.v. infusion

All tested normal cells were insensitive to 20 mM ascorbate.

Lymphoma cells were selected because of their sensitivity to ascorbate

As ascorbate concentration increased, the pattern of death changed from apoptosis to pyknosis/necrosis, a pattern suggestive of H2O2-mediated cell death

Apoptosis occurred by 6 h after exposure, and cell death by pyknosis was ≈90% at 14 h after exposure

In contrast to lymphoma cells, there was little or no killing of normal lymphocytes and monocytes by ascorbate

Ascorbate is transported into cells as such by sodium-dependent transporters, whereas dehydroascorbic acid is transported into cells by glucose transporters and then immediately reduced internally to ascorbate

Whether or not intracellular ascorbate was preloaded, extracellular ascorbate induced the same amount and type of death.

extracellular ascorbate in pharmacologic concentrations mediates death of lymphoma cells by apoptosis and pyknosis/necrosis, independently of intracellular ascorbate.

H2O2 as the effector species mediating pharmacologic ascorbate-induced cell death

Superoxide dismutase was not protective

Because these data implicated H2O2 in cell killing, we added H2O2 to lymphoma cells and studied death patterns using nuclear staining (19, 28). The death patterns found with exogenous H2O2 exposure were similar to those found with ascorbate

For both ascorbate and H2O2, death changed from apoptosis to pyknosis/necrosis as concentrations increased

Sensitivity to direct exposure to H2O2 was greater in lymphoma cells compared with normal lymphocytes and normal monocytes

There was no association between the EC50 for ascorbate-mediated cell death and intracellular glutathione concentrations, catalase activity, or glutathione peroxidase activity

H2O2 generation was dependent on time, ascorbate concentration, and the presence of trace amounts of serum in media

ascorbate radical is a surrogate marker for H2O2 formation.

whatever H2O2 is generated should be removed by glutathione peroxidase and catalase within red blood cells, because H2O2 is membrane permeable

The data are consistent with the hypothesis that ascorbate in pharmacologic concentrations is a pro-drug for H2O2 generation in the extracellular milieu but not in blood.

The occurrence of one predicted complication, oxalate kidney stones, is controversial

In patients with glucose-6-phosphate dehydrogenase deficiency, i.v. ascorbate is contraindicated because it causes intravascular hemolysis

ascorbate at pharmacologic concentrations in blood is a pro-drug for H2O2 delivery to tissues.

ascorbate, an electron-donor in such reactions, ironically initiates pro-oxidant chemistry and H2O2 formation

data here showed that ascorbate initiated H2O2 formation extracellularly, but H2O2 targets could be either intracellular or extracellular, because H2O2 is membrane permeant

More than 100 patients have been described, presumably without glucose-6-phosphate dehydrogenase deficiency, who received 10 g or more of i.v. ascorbate with no reported adverse effects other than tumor lysis

The GKI can be useful in determining the success of dietary therapies that shift glucose- and lactate-based metabolism to ketone-based metabolism

Alzheimer’s disease, Parkinson’s disease, traumatic brain injury, chronic inflammatory disease, and epilepsy

The zone of metabolic management is likely entered with GKI values between 1 and 2 for humans

Optimal management is predicted for values approaching 1.0, and blood glucose and ketone values should be measured 2–3 hours postprandial, twice a day if possible

Preclinical studies have demonstrated a clear linkage between GKI and therapeutic efficacy

the Warburg effect (aerobic fermentation of glucose) is a common metabolic malady expressed in nearly all neoplastic cells of these and other malignant tumors

Aerobic fermentation (Warburg effect) is necessary to compensate for the insufficiency of mitochondrial oxidative phosphorylation in the cells of most tumors

Normal brain cells gradually transition from the metabolism of glucose to the metabolism of ketone bodies (primarily β-hydroxybutyrate and acetoacetate) for energy when circulating glucose levels become limiting

Ketone bodies bypass the glycolytic pathway in the cytoplasm and are metabolized directly to acetyl CoA in the mitochondria

Tumor cells are less capable than normal cells in metabolizing ketone bodies for energy due to their mitochondrial defects

daily activities and emotional stress can cause blood glucose levels to vary making it difficult for some people to enter the predicted zone of metabolic management

a clear association of the GKI to the therapeutic action of calorie restriction against distal invasion, proliferation, and angiogenesis in the VM-M3 model of glioblastoma

The results suggest that GKI levels that approach 1.0 are therapeutic for managing brain tumor growth

Therapeutic efficacy of the KD or calorie restriction is greater with lower GKI values than with higher values

molecule), cancer cells upregulate glucose receptors and significantly increase glucose uptake in an attempt to ‘catch up

early carcinogenesis often occurs in a hypoxic microenvironment, the transformed cells have to rely on anaerobic GLY for energy production.

Hypoxia-inducible factor (HIF) is activated in hypoxic conditions

evidence suggests that transformation to a glycolytic phenotype offers resistance to apoptosis

non-small cell lung cancer, breast cancer and glioblastoma

Dichloroacetate activated the pyruvate dehydrogenase, which resulted in increased delivery of pyruvate into the mitochondria

DCA increased GO and depolarised the mitochondria, returning the membrane potential towards the levels of the non-cancer cells, without affecting the mitochondria of non-cancerous cells

induction of apoptosis by DCA in non-small cell lung cancer, breast cancer and glioblastoma cell lines

DCA was shown to induce apoptosis in endometrial (Wong et al, 2008) and prostate (Cao et al, 2008) cancer cells

The health benefits following exercise training are elicited by gene expression changes in skeletal muscle, which are fundamental to the remodeling process

there is increasing evidence that more short-term environmental factors can influence DNA methylation

dietary factors have the potency to alter the degree of DNA methylation in different tissues, 9,10 including skeletal muscle

In one study, a single bout of endurance-type exercise was shown to affect methylation at a few promoter CpG sites

In the context of diabetes, exercise training has been shown to affect genome-wide methylation pattern in skeletal muscle,13 as well as in adipose tissue.

physiological stressors can indeed affect DNA methylation

training intervention reshapes the epigenome and induces significant changes in DNA methylation

the findings from this tightly controlled human study strongly suggest that the regulation and maintenance of exercise training adaptation is to a large degree associated to epigenetic changes, especially in regulatory enhancer regions

Endurance training [after training (T2) vs. before training (T1)] induced significant (false discovery rate, FDR< 0.05) methylation changes at 4919 sites across the genome in the trained leg

identified 4076 differentially expressed genes

a complementary approach revealed that over 600 CpG sites correlated to the increase in citrate synthase activity, an objective measure of training response (Figure S4 and Dataset S14). This might imply that some of these sites could influence the degree of training response.

As expected by a physiological environmental trigger on adult tissue, the observed effect size on DNA methylation was small in comparison to disease states such as cancer

a preferential localization outside of CpG Islands/Shelves/Shores

endurance training especially influences enhancers

negative correlation was more prominent for probes in promoter/5′UTR/1st exon regions, while gene bodies had a stronger peak of positive correlation

The significant changes in DNA methylation, that primarily occurred in enhancer regions, were to a large extent associated with relevant changes in gene expression

The main findings of this study were that 3 months of endurance training in healthy human volunteers induced significant methylation changes at almost 5000 sites across the genome and significant differential expression of approximately 4000 genes

DMPs that increased in methylation were mainly associated to structural remodeling of the muscle and glucose metabolism, while the DMPs with decreased methylation were associated to inflammatory/immunological processes and transcriptional regulation

This suggests that the changes in methylation seen with training were not a random effect across the genome but rather a controlled process that likely contributes to skeletal muscle adaptation to endurance training

Correlation of the changes in DNA methylation to the changes in gene expression showed that the majority of significant methylation/expression pairs were found in the groups representing either increases in expression with a concomitant decrease in methylation or vice versa

The fraction of genes showing both significant decrease in methylation and upregulation was 7.5% of the DEGs or 2.3% of all genes detected in muscle tissue with at least one measured DNA methylation position. Correspondingly, 7.0% of the DEGs or 2.1% of all genes showed both significant increase in methylation and downregulation

we show that DNA methylation changes are associated to gene expression changes in roughly 20% of unique genes that significantly changed with training

Examples of structural genes include COL4A1, COL4A2 and LAMA4. These genes have also been identified as important for differences in responsiveness to endurance training

methylation status could be part of the mechanism behind variable training response

Among the metabolic genes, MDH1 catalyzes the reversible oxidation of malate to oxaloacetate, utilizing the NAD/NADH cofactor system in the citric acid cycle and NDUFA8 plays an important role in transferring electrons from NADH to the respiratory chain

PPP1R12A,

In the present study, methylation predominantly changed in enhancer regions with enrichment for binding motifs for different transcription factors suggesting that enhancer methylation may be highly relevant also in exercise biology

Of special interest in the biology of endurance training may be that MRFs, through binding to the PGC-1α core promoter, can regulate this well-studied co-factor for mitochondrial biogenesis

That endurance training led to an increased methylation in enhancer regions containing motifs for the MRFs and MEFs is somewhat counterintuitive since it should lead to the repression of the action of the above discussed transcription factors

decrease with training in this study, including CDCH15, MYH3, TNNT2, RYR1 and SH3GLB1

expression of MEF2A itself decreased with training

this study demonstrates that the transcriptional alterations in skeletal muscle in response to a long-term endurance exercise intervention are coupled to DNA methylation changes

We suggest that the training-induced coordinated epigenetic reprogramming mainly targets enhancer regions, thus contributing to differences in individual response to lifestyle interventions

a physiological health-enhancing stimulus can induce highly consistent modifications in DNA methylation that are associated to gene expression changes concordant with observed phenotypic adaptations

exercise changed both DNA methylation and expression of a number of genes, including ADIPOR1, ADIPOR2, and BDKRB2, encoding receptors for adiponectin and bradykinin, respectively, which both regulate metabolism in muscle

we cannot draw a conclusion as to whether differential expression is a consequence rather than a cause of changes in methylation

ageing is associated with increased DNA methylation and decreased expression of genes involved in oxidative phosphorylation in human muscle

exercise can induce genome-wide epigenetic changes in human muscle and that the response may differ in people with different genetic predispositions to metabolic disease