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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

Patients with higher inflammation or tumor burdens, as measured by CRP levels or tumor antigen levels, tend to show lower peak plasma ascorbate levels after IVC.

Patients with metastatic tumors tend to achieve lower post infusion plasma ascorbate levels than those with localized tumors.

Meta-analyses of clinical studies involving cancer and vitamins also conclude that antioxidant supplementation does not interfere with the efficacy of chemotherapeutic regiments

Most of the prostate cancer patients studied, 75±19% (95% confidence), showed reductions in PSA levels during the course of their IVC therapy

Laboratory studies suggest that, at high concentrations, ascorbate does not interfere with chemotherapy or irradiation and may enhance efficacy in some situations

Cameron and Pauling observed fourfold survival times in terminal cancer patients treated with intravenous ascorbate infusions followed by oral supplementation

The inflammatory microenvironment of cancer cells leads to increasing oxidative stress, which apparently depletes vitamin C, resulting in lower plasma ascorbate concentrations in blood samples post IVC infusion. Another explanation for this finding may be that cancers are themselves more metabolically active in their uptake of vitamin C, causing subjects to absorb more of the vitamin, and as a results show lower plasma ascorbate concentrations in blood post IVC infusion.

patients with severely elevated CRP levels attain plasma ascorbate concentrations after IVC infusions that are only 65% of those attained for subjects with normal CRP levels

The finding of decreased plasma ascorbate levels in cancer patients may relate to the molecular structure of ascorbic acid; in particular, the similarity of its oxidized form, dihydroascorbic acid, to glucose

Since tumor have increased requirement for glucose [67], transport of dehydroascorbate into the cancer cells via glucose transport molecules and ascorbate through sodium-dependent transporter may be elevated

Increased accumulation of ascorbic acid in the tumor site was supported by measurements of the level of ascorbic acid in tumors in animal experiments

patients with advanced malignancies may have lower level of ascorbic acid in tissue, creating a higher demand for the vitamin C

IVC therapy appears to reduce CRP levels in cancer patients.

CRP concentrations directly correlate with disease activity in many cases and can contribute to disease progression through a range of pro-inflammatory properties.

Being an exquisitely sensitive marker of systemic inflammation and tissue damage, CRP is very useful in screening for organic disease and monitoring treatment responses

ncreases in CRP concentrations have been associated with poorer prognosis of survival in cancer patients, particularly with advance disease independent of tumor stage

Regarding inflammation, 73±13% of subjects (95% confidence) showed a reduction in CRP levels during therapy. This was an even more dramatic 86±13% (95% confidence) in subjects who started therapy with CRP levels above 10 mg/L

patients treated by IVC with follow-up several year showed that suppression of inflammation in cancer patients by high-dose IVC is feasible and potentially beneficial

Inflammation is a marker of high cancer risk, and poor treatment outcome

The subjects with highly elevated CRP concentrations have a three-fold elevation “all-cause” mortality risk and a twenty-eight fold increase in cancer mortality risk

cancer patients may need higher doses to achieve a given plasma concentration.

patients with lower vitamin C levels may see more distribution of intravenously administered ascorbate into tissues and thus attain less in plasma.

When treating patients with IVC, the first treatment likely serves to replenish depleted tissue stores, if those subjects were vitamin C deficient at the beginning of the treatment. Then, in subsequent treatments, with increasing doses, higher plasma concentrations can be attained. On-going treatments serve to progressively reduce oxidative stress in cancer patients.

large doses given intravenously may result in maximum plasma concentrations of roughly 30 mM, a level that has been shown to be sufficient for preferential cytotoxicity against cancer cells

oral intake of vitamin C exceeded 200 mg administered once daily, it was difficult to increase plasma and tissue concentrations above roughly 200 μM.

whereas the expression of SVCT-2 is ubiquitous

differential sensitivity to VC may result from variations in VC flow into cells, which is dependent on SVCT-2 expression.

high-dose VC significantly impaired both the tumorspheres initiation (Fig. 4d, e) and the growth of established tumorspheres derived from HCC cells (Fig. 4f, g) in a time-dependent and dose-dependent manner.

Hepatocellular carcinoma (HCC)

The antioxidant, N-acetyl-L-cysteine (NAC), preventing VC-induced ROS production (a ROS scavenger), completely restored the viability and colony formation among VC-treated cells

DNA double-strand damage was found following VC treatment

DNA damage was prevented by NAC

Interestingly, the combination of VC and cisplatin was even more effective in reducing tumor growth and weight

Consistent with the in vitro results, stemness-related genes expressions in tumor xenograft were remarkably reduced after VC or VC+cisplatin treatment, whereas conventional cisplatin therapy alone led to the increase of CSCs

VC is one of the numerous common hepatoprotectants.

Interestingly, at extracellular concentrations greater than 1 mM, VC induces strong cytotoxicity to cancer cells including liver cancer cells

we hypothesized that intravenous VC might reduce the risk of recurrence in HCC patients after curative liver resection.

Intriguingly, the 5-year disease-free survival (DFS) for patients who received intravenous VC was 24%, as opposed to 15% for no intravenous VC-treated patients

Median DFS time for VC users was 25.2 vs. 18 months for VC non-users

intravenous VC use is linked to improved DFS in HCC patients.

In this study, based on the elevated expression of SVCT-2, which is responsible for VC uptake, in liver CSCs, we revealed that clinically achievable concentrations of VC preferentially eradicated liver CSCs in vitro and in vivo

Additionally, we found that intravenous VC reduced the risk of post-surgical HCC progression in a retrospective cohort study.

Three hundred thirty-nine participants (55.3%) received 2 g intravenous VC for 4 or more days after initial hepatectomy

As the key protein responsible for VC uptake in the liver, SVCT-2 played crucial roles in regulating the sensitivity to ascorbate-induced cytotoxicity

we also observed that SVCT-2 was highly expressed in human HCC samples and preferentially elevated in liver CSCs

SVCT-2 might serve as a potential CSC marker and therapeutic target in HCC

CSCs play critical roles in regulating tumor initiation, relapse, and chemoresistance

we revealed that VC treatment dramatically reduced the self-renewal ability, expression levels of CSC-associated genes, and percentages of CSCs in HCC, indicating that CSCs were more susceptible to VC-induced cell death

as a drug for eradicating CSCs, VC may represent a promising strategy for treatment of HCC, alone or particularly in combination with chemotherapeutic drugs

In HCC, we found that VC-generated ROS caused genotoxic stress (DNA damage) and metabolic stress (ATP depletion), which further activated the cyclin-dependent kinase inhibitor p21, leading to G2/M phase cell cycle arrest and caspase-dependent apoptosis in HCC cells

we demonstrated a synergistic effect of VC and chemotherapeutic drug cisplatin on killing HCC both in vitro and in vivo

Intravenous VC has also been reported to reduce chemotherapy-associated toxicity of carboplatin and paclitaxel in patients,38 but the specific mechanism needs further investigation

Our retrospective cohort study also showed that intravenous VC use (2 g) was related to the improved DFS in HCC patients after initial hepatectomy

several clinical trials of high-dose intravenous VC have been conducted in patients with advanced cancer and have revealed improved quality of life and prolonged OS

high-dose VC was not toxic to immune cells and major immune cell subpopulations in vivo

high recurrence rate and heterogeneity

tumor progression, metastasis, and chemotherapy-resistance

SVCT-2 was highly expressed in HCC samples in comparison to peri-tumor tissues

high expression (grade 2+/3+) of SVCT-2 was in agreement with poorer overall survival (OS) of HCC patients (Fig. 1c) and more aggressive tumor behavior

SVCT-2 is enriched in liver CSCs

these data suggest that SVCT-2 is preferentially expressed in liver CSCs and is required for the maintenance of liver CSCs.

pharmacologic concentrations of plasma VC higher than 0.3 mM are achievable only from i.v. administration

The viabilities of HCC cells were dramatically decreased after exposure to VC in dose-dependent manner

VC and cisplatin combination further caused cell apoptosis in tumor xenograft

These results verify that VC inhibits tumor growth in HCC PDX models and SVCT-2 expression level is associated with VC response

qPCR and IHC analysis demonstrated that expression levels of CSC-associated genes and percentages of CSCs in PDXs dramatically declined after VC treatment, confirming the inhibitory role of VC in liver CSCs