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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

Under steady-state conditions, intracellular GSH is maintained at millimolar concentrations, which keeps cells in a reduced environment and serves as the principal intracellular redox buffer when cells are subjected to an oxidative stressor including H2O2 (26). Glutathione peroxidase (GPx) activity catalyzes the reduction of H2O2 to water with the conversion of GSH to glutathione disulfide (GSSG). Under steady-state conditions, GSSG is recycled back to GSH by glutathione disulfide reductase using reducing equivalents from NADPH. However, under conditions of increased H2O2 flux, this recycling mechanism may become overwhelmed leading to a depletion of intracellular GSH (27, 28).

ascorbate radiosensitization can create an overwhelming oxidative stress to pancreatic cancer cells resulting in oxidation/depletion of the GSH intracellular redox buffer, resulting in cell death.

Treatment with the combination of ascorbate + IR significantly delayed tumor growth compared to controls or ascorbate alone

Ascorbate + IR also significantly increased overall survival compared to controls, IR alone or ascorbate alone

54% of mice treated with the combination of IR + ascorbate had no measurable tumors

Glutathione is a measurable marker indicative of the oxidation state of the thiol redox buffer in cells. In severe systemic oxidative stress, the GSSG/2GSH couple may become oxidized, i.e. the concentration of GSH decreases and GSSG may increase because the capacity to recycle GSSG to GSH becomes rate-limiting

This suggests that the very high levels of pharmacological ascorbate in these experiments may have a pro-oxidant toward red blood cells as seen by a decrease in the capacity of the intracellular redox buffer

These data support the hypothesis that ascorbate radiosensitization does not cause an increase in oxidative damage from lipid-derived aldehydes to other organs.

Our current study demonstrates the potential for pharmacological ascorbate as a radiosensitizer in the treatment of pancreatic cancer.

pharmacological ascorbate enhances IR-induced cell killing and DNA fragmentation leading to induction of apoptosis in HL60 leukemia cells

pharmacological ascorbate significantly decreases clonogenic survival and inhibits the growth of all pancreatic cancer cell lines as a single agent, as well as sensitizes cancer cells to IR

Hurst et al. demonstrated that pharmacological ascorbate combined with IR leads to increased numbers of double-strand DNA breaks and cell cycle arrest when compared to either treatment alone

pharmacological ascorbate could serve as a “pro-drug” for the delivery of H2O2 to tumors

the double-strand breaks induced by H2O2 were more slowly repaired

The combination of ascorbate and IR provide two distinct mechanisms of action: ascorbate-induced toxicity due to extracellular production of H2O2 that then diffuses into cells and causes damage to DNA, protein, and lipids; and radiation-induced toxicity as a result of ROS-induced damage to DNA. In addition, redox metal metals like Fe2+ may play an important role in ascorbate-induced cytotoxicity. By catalyzing the oxidation of ascorbate, labile iron can enhance the rate of formation of H2O2; labile iron can also react with H2O2. Recently our group has demonstrated that pharmacological ascorbate and IR increase the labile iron in tumor homogenates from this murine model of pancreatic cancer

we demonstrated that ascorbate or IR alone decreased tumor growth, but the combination treatment further inhibited tumor growth, indicating that pharmacological ascorbate is an effective radiosensitizer in vivo

data suggest that pharmacological ascorbate may protect the gut locally by decreasing IR-induced damage to the crypt cells, and systemically, by ameliorating increases in TNF-α

pharmacokinetic data indicate that intravenous administration of ascorbate can bypass this tight control resulting in highly elevated plasma levels

ascorbate readily oxidizes to produce H2O2, pharmacological ascorbate has been proposed as a prodrug for the delivery of H2O2 to tumors

Ascorbate is an excellent reducing agent and readily undergoes two consecutive, one-electron oxidations to form ascorbate radical (Asc•−) and dehydroascorbic acid (DHA)

Ascorbate oxidizes readily. The rate of oxidation is dependent on pH and is accelerated by catalytic metals

In near-neutral buffers with contaminating metals, the oxidation and subsequent loss of ascorbate can be very rapid

Ascorbate is required for maintaining iron in the ferrous state

In the presence of catalytic metal ions, ascorbate can also exert pro-oxidant effects

Ascorbate is an excellent one-electron reducing agent that can reduce ferric (Fe3+) to ferrous (Fe2+) iron, while being oxidized to ascorbate radical

In a classic Fenton reaction, Fe2+ reacts with H2O2 to generate Fe3+ and the very oxidizing hydroxyl radical

e presence of ascorbate can allow the recycling of Fe3+ back to Fe2+, which in turn will catalyze the formation of highly reactive oxidants from H2O2

Depending on concentrations, the effects of ascorbate on models of lipid peroxidation can be pro- or antioxidant

pharmacologic ascorbate as a prooxidant drug for therapeutic use.

Recently we reported that pharmacologic ascorbic acid concentrations produced H2O2 concentrations of ≥25 μM, causing cancer cell death in vitro

We found that H2O2 concentrations generated in vivo were those that caused cancer cell death in vitro

When ascorbate was given parenterally, Asc•−, the product of a loss of one electron from ascorbate, was detected preferentially in extracellular fluid compared with blood

Asc•− generation in extracellular fluid depended on the ascorbate dose and the resulting concentrations

With i.v. administration of ascorbate, Asc•− concentrations were as much as 12-fold greater in extracellular fluid compared to blood and approached 250 nM

In blood, such Asc•− concentrations were never produced and were always <50 nM

These data are all consistent with the hypothesis that pharmacologic ascorbate concentrations in vivo serve as a prodrug for selective delivery of H2O2 to the extracellular space

After oral ingestion, control of intracellular and extracellular ascorbate concentrations is mediated by three mechanisms: intestinal absorption, tissue transport, and renal reabsorption

intestinal absorption, or bioavailability, declines at doses >200 mg

corresponding to plasma concentrations of ≈60 μM

at approximately this concentration, the ascorbate tissue transporter SVCT2 approaches Vmax, and tissues appear to be saturated

also at ≈60 μM, renal reabsorption approaches saturation, and excess ascorbate is excreted in urine

Parenteral administration bypasses tight control

When tight control is bypassed, H2O2 forms in the extracellular space

in vivo validation of ascorbate as a prodrug for selective H2O2 formation

Temporarily bypassing tight control with parenteral administration of ascorbate allows H2O2 to form in discrete time periods only, decreasing likelihood of harm, and provides a pharmacologic basis for therapeutic use of i.v. ascorbate

H2O2 formation results in selective cytotoxicity

Tumor cells are killed with exposure to H2O2 for ≤30 min

In vitro, killing is mediated by H2O2 rather than Asc•−

In addition to cancer treatment, another potential therapeutic use is for treatment of infections. H2O2 concentrations of 25–50 μM are bacteriostatic

virally infected cells may also be candidates

subnormal plasma ascorbic acid levels are a predictable feature in patients with severe sepsis

Ascorbic acid depletion in sepsis results from ascorbic acid consumption by the reduction of plasma free iron, ascorbic acid consumption by the scavenging of aqueous free radicals (peroxyl radicals), and by the destruction of the oxidized form of ascorbic acid dehydroascorbic acid

Sepsis further inhibits intracellular reduction of dehydroascorbic acid, producing acute intracellular ascorbic acid depletion

Ascorbic acid treated patients in this study exhibited rapid and sustained increases in plasma ascorbic acid levels using an intermittent every six hours administration protocol

Septic ascorbic acid-deficient neutrophils fail to undergo normal apoptosis. Rather, they undergo necrosis thereby releasing hydrolytic enzymes in tissue beds, thus contributing to organ injury

We speculate that intravenous ascorbic acid acts to restore neutrophil ascorbic acid levels

Repletion of ascorbic acid in this way allows for normal apoptosis, thus, preventing the release of organ damaging hydrolytic enzymes.

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.

A treatment dose of 4 g ascorbate/kg body weight either once or twice daily did not produce any discernible adverse effects

Xenograft experiments showed that parenteral ascorbate as the only treatment significantly decreased both tumor growth and weight by 41–53%

Peak plasma concentrations of ascorbate approached 30 mM

Pharmacologic concentrations of ascorbate decreased tumor volumes 41–53% in diverse cancer types known for both their aggressive growth and limited treatment options.

Our findings showed that pharmacologic ascorbic acid concentrations were cytotoxic to many types of cancer cells in vitro (Fig. 1A) and significantly impeded tumor progression in vivo without toxicity to normal tissues

The amelioration of ascorbate cytotoxicity in vitro by the addition of catalase was consistent among sensitive cancer cells (Fig. 1B) and points unambiguously to H2O2 generation in the extracellular medium

the current in vivo data support that pharmacologic ascorbate concentrations, which can readily be achieved in humans (Fig. 3E), diminished growth of several aggressive cancer types in mice (Fig. 2) without causing apparent adverse effects.

These intratumoral H2O2 concentrations of >125 μM persisted for >3 h after ascorbate administration

These data suggest that PAA may show a therapeutic advantage to blood cancers vs solid tumors because of the communication between tumor cells and blood plasma

These results strongly suggest that the mechanism of PAA killing of MM cells is indeed iron-dependent

These results suggest that PAA administration in SMM may be able to prevent progression to symtomatic MM

A recent study by Yun and colleagues demonstrated that vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting GAPDH, but spares normal cells

RAS family genes show the most frequent mutations in MM. KRAS, NRAS and BRAF are mutated in 22%, 20% and 7% of MM samples

the disease stage rather than the mutation of RAS and/or BRAF is the major predictive factor for PAA sensitivity in MM treatment

Other molecular mechanisms including ATP depletion and ATM-AMPK signaling have been reported to explain PAA-induced cell death

our pilot study also suggested that PAA could overcome drug resistance to bortezomib in MM cells

Our findings complement reported studies and further address the mechanism of action using clinical samples in which we observed that PAA killed tumor cells with high iron content, suggesting that iron might be the initiator of PAA cytotoxicity

combination of PAA with standard therapeutic drugs, such as melphalan, may significantly reduce the dose of melphalan needed

Combined treatment of reduced dose melphalan with PAA achieved a significantly longer progression-free survival than the same dose of melphalan alone.

These data also suggest that the bone marrow suppression induced by high-dose melphalan can be ameliorated by the combination of PAA with lower dose of melphalan because of the lack of toxicity of PAA on normal cells with low iron content.

if creatinine clearance is <30 mL/min, high dose ascorbic acid should be not administrated.

In MM preclinical and clinical studies, ascorbate was used as an adjunct drug and showed controversial results (Harvey et al., 2009, Perrone et al., 2009, Held et al., 2013, Sharma et al., 2012, Nakano et al., 2011, Takahashi, 2010, Sharma et al., 2009, Qazilbash et al., 2008). However, none of these tests used pharmacological doses of ascorbate and intravenous administration

Multiple myeloma (MM) is a plasma cell neoplasm.

Cameron and Pauling reported that high doses of vitamin C increased survival of patients with cancer

pharmacologically dosed ascorbic acid (PAA) 50–100 g (Chen et al., 2008, Padayatty et al., 2004, Hoffer et al., 2008, Padayatty et al., 2006, Welsh et al., 2013), administered intravenously, has potent anti-cancer activity and its role as anti-cancer therapy is being studied at the University of Iowa and in other centers

In the presence of catalytic metal ions like iron, PAA administered intravenously exerts pro-oxidant effects leading to the formation of highly reactive oxygen species (ROS), resulting in cell death

the labile iron pool (LIP) is significantly elevated in MM cells

The survival of CD138+ cells in vitro was significantly decreased following PAA treatment in all 9 MM

In contrast, no significant change of cell viability was observed in CD138− BM cells from the same patients

The same effect of PAA was also observed in the SMM patients

no response to PAA was detected in CD138+ cells from the 2 MGUS patients

the combination of melphalan plus PAA showed greater tumor burden reduction than each drug alone, suggesting a synergistic activity between these two drugs

Both catalase and NAC protect cells from oxidative damage

cells pretreated with NAC and catalase became resistant to PAA even at high doses

adding deferoxamine (DFO), an iron chelator, to OCI-MY5 cells before PAA treatment was also sufficient to prevent PAA-induced cellular death

iron is essential for PAA to achieve its anti-cancer activity

PAA induced early necrosis (Fig. 3Fig. 3A, 60 min) followed by late apoptosis

results further indicated that PAA induced mitochondria-mediated apoptosis

PAA by reacting with LIP and generating ROS induces mitochondria-mediated apoptosis in which AIF1 cleavage is important for cell death.

ROS and H2O2 are well known factors mediating PAA-induced cancer cell death

PAA was sensitive to all 9 MMs and 2 SMMs

related to intracellular hydrogen peroxide generation

only be obtained by intravenous administration of AA

Preferentially kills neoplastic cells

Is virtually non-toxic at any dosage

Does not suppress the immune system, unlike most chemotherapy agents

Increases animal and human resistance to infectious agents by enhancing lymphocyte blastogenesis, enhancing cellular immunity, strengthening the extracellular matrix, and enhancing bactericidal activity of neutrophils and modulation of complement protein

Strengthens the structural integrity of the extracellular matrix which is responsible for stromal resistance to malignant invasiveness

1969, researchers at the NCI reported AA was highly toxic to Ehrlich ascites cells in vitro

In 1977, Bram et al reported preferential AA toxicity for several malignant melanoma cell lines, including four human-derived lines

Noto et al reported that AA plus vitamin K3 had growth inhibiting action against three human tumor cell lines at non-toxic levels

Metabolites of AA have also shown antitumor activity in vitro

The AA begins to reduce cell proliferation in the tumor cell line at the lowest concentration, 1.76 mg/dl, and is completely cytotoxic to the cells at 7.04 mg/dl

the normal cells grew at an enhanced rate at the low dosages (1.76 and 3.52 mg/dl)

preferential toxicity of AA for tumor cells. >95% toxicity to human endometrial adenocarcinoma and pancreatic tumor cells (ATCC AN3-CA and MIA PaCa-2) occurred at 20 and 30 mg/dl, respectively.

No toxicity or inhibition was demonstrated in the normal, human skin fibroblasts (ATCC CCD 25SK) even at the highest concentration of 50 mg/dl.

the use of very high-dose intravenous AA for the treatment of cancer was proposed as early as 1971

Cameron and Pauling have published extensive suggestive evidence for prolonged life in terminal cancer patients orally supplemented (with and without initial intravenous AA therapy) with 10 g/day of AA

AA, plasma levels during infusion were not monitored,

the long-term, oral dosage used in those experiments (10 g/day), while substantial and capable of producing immunostimulatory and extracellular matrix modulation effects, was not high enough to achieve plasma concentrations that are generally cytotoxic to tumor cells in culture

This low cytotoxic level of AA is exceedingly rare

5 — 40 mg/dl of AA is required in vitro to kill 100% of tumor cells within 3 days. The 100% kill levels of 30 mg/dl for the endometrial carcinoma cells and 40 mg/dl for the pancreatic carcinoma cells in Figure 2 are typical

normal range (95% range) of 0.39-1.13 mg/dl

1 h after beginning his first 8-h infusion of 115 g AA (Merit Pharmaceuticals, Los Angeles, CA), the plasma AA was 3.7 mg/dl and at 5 h was 19 mg/dl. During his fourth 8-h infusion, 8 days later, the 1 h plasma level was 158 mg/dl and 5 h was 185 mg/dl

plasma levels of over 100 mg/dl have been maintained in 3 patients for more than 5 h using continuous intravenous infusion

In rare instances of patients with widely disseminated and rapidly proliferating tumors, intravenous AA administration (10 — 45 g/day) precipitated widespread tumor hemorrhage and necrosis, resulting in death

Although the outcomes were disastrous in these cases, they are similar to the description of tumor-necrosis-factor-induced hemorrhage and necrosis in mice (52) and seem to demonstrate the ability of AA to kill tumor cells in vivo.

toxic effects of AA on one normal cell line were observed at 58.36 mg/dl and the lack of side effects in patients maintaining >100 mg/dl plasma levels

Although it is very rare, tumor necrosis, hemorrhage, and subsequent death should be the highest priority concern for the safety of intravenous AA for cancer patients.

Klenner, who reported no ill effects of dosages as high as 150 g intravenously over a 24-h period

Cathcart (55) who describes no ill effects with doses of up to 200 g/d in patients with various pathological conditions

following circumstances: renal insufficiency, chronic hemodialysis patients, unusual forms of iron overload, and oxalate stone formers

Screening for red cell glucose-6-phosphate dehydrogenase deficiency, which can give rise to hemolysis of red blood cells under oxidative stress (57), should also be performed

any cancer therapy should be started at a low dosage to ensure that tumor hemorrhage does not occur.

patient is orally supplementing between infusions

a scorbutic rebound effect can be avoided with oral supplementation. Because of the possibility of a rebound effect, measurement of plasma levels during the periods between infusions should be performed to ensure that no such effect takes place

Every effort should be made to monitor plasma AA levels when a patient discontinues intravenous AA therapy.

The H2O2 produced this way (Reactions 1–3) seems to be key to ascorbate's antitumor effect because H2O2 causes cancer cells to undergo apoptosis, pyknosis, and necrosis

In contrast, normal cells are considerably less vulnerable to H2O2

The reason for the increased sensitivity of tumor cells to H2O2 is not clear but may be due to lower antioxidant defenses

In fact, a lower capacity to destroy H2O2—e.g., by catalase, peroxiredoxins, and GSH peroxidases—may cause tumor cells to grow and proliferate more rapidly than normal cells in response to low concentrations of H2O2

These observations, combined with the inhibitory effect on xenograft growth, provide the proof of concept that millimolar concentrations of extracellular ascorbate, achievable by i.p. injection or i.v. infusion in experimental animals and humans, respectively, exert pro-oxidant, antitumor effects in vivo.

They also show that the concentration of the ascorbyl radical correlates with the concentration of H2O2 in interstitial fluid, whereas no H2O2 can be detected in blood or plasma

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

Vitamin C may serve as a pro-drug for hydrogen peroxide delivery to extravascular tissues, but without the presence of hydrogen peroxide in blood

not all cancer cells were killed by ascorbic acid in vitro

Intravascular hemolysis was reported after massive vitamin C administration in people with glucose-6-phosphate dehydrogenase deficiency

Administration of high-dose vitamin C to patients with systemic iron overload may increase iron absorption and represents a contraindication

Ascorbic acid is metabolized to oxalate, and 2 cases of acute oxalate nephropathy were reported in patients with pre-existing renal insufficiency given massive intravenous doses of vitamin C

Rare cases of acute tumour hemorrhage and necrosis were reported in patients with advanced cancer within a few days of starting high-dose intravenous vitamin C therapy, although this was not independently verified by pathologic review