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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

of the approximately 108 cannabinoids produced by C. sativa, Δ9-tetrahydrocannabinol (thc) is the most relevant because of its high potency and abundance in plant preparations

Tetrahydrocannabinol exerts a wide variety of biologic effects by mimicking endogenous substances—the endocannabinoids anandamide3 and 2-arachidonoylglycerol4,5—that engage specific cell-surface cannabinoid receptors

the cb2 receptor was initially described to be present in the immune system6, but was more recently shown to also be expressed in cells from other origins

transient receptor potential cation channel subfamily V, member 1

orphan G protein–coupled receptor 55

Most of the effects produced by cannabinoids in the nervous system and in non-neural tissues rely on cb1 receptor activation

two major cannabinoid-specific receptors—cb1 and cb2

cardiovascular tone, energy metabolism, immunity, and reproduction

cannabinoids are well known to exert palliative effects in cancer patients

best-established use is the inhibition of chemotherapy-induced nausea and vomiting

thc and other cannabinoids exhibit antitumour effects in a wide array of animal models of cancer

cannabinoid receptors and their endogenous ligands are both generally upregulated in tumour tissue compared with non-tumour tissue

cb2 promotes her2 (human epidermal growth factor receptor 2) pro-oncogenic signalling in breast cancer

pharmacologic activation of cannabinoid receptors decreases tumour growth

endocannabinoid signalling can also have a tumour-suppressive role

pharmacologic stimulation of cb receptors is, in most cases, antitumourigenic. Nonetheless, a few reports have proposed a tumour-promoting effect of cannabinoids

most prevalent effect is the induction of cancer cell death by apoptosis and the inhibition of cancer cell proliferation

impair tumour angiogenesis and block invasion and metastasis

thc and other cannabinoids induce the apoptotic death of glioma cells by cb1- and cb2-dependent stimulation

Autophagy is primarily a cytoprotective mechanism, although its activation can also lead to cell death

autophagy is important for cannabinoid antineoplastic activity

autophagy is upstream of apoptosis in the mechanism of cannabinoid-induced cell death

the effect of cannabinoids in hormone- dependent tumours might rely, at least in part, on the ability to interfere with the activation of growth factor receptors

glioma cells), pharmacologic blockade of either cb1 or cb2 prevents cannabinoid-induced cell death with similar efficacy

other types of cancer cells (pancreatic48, breast24, or hepatic43 carcinoma cells, for example), antagonists of cb2 but not of cb1 inhibit cannabinoid antitumour actions

thc promotes cancer cell death in a cb1- or cb2-dependent manner (or both) at lower concentrations

cannabidiol (cbd), a phytocannabinoid with a low affinity for cannabinoid receptors15, and other marijuana-derived cannabinoids57 have also been proposed to promote the apoptotic death of cancer cells acting independently of the cb1 and cb2 receptors

In cancer cells, cannabinoids block the activation of the vascular endothelial growth factor (vegf) pathway, an inducer of angiogenesi

In vascular endothelial cells, cannabinoid receptor activation inhibits proliferation and migration, and induces apoptosis

cb1 or cb2 receptor agonists (or both) reduce the formation of distant tumour masses in animal models of both induced and spontaneous metastasis, and inhibit adhesion, migration, and invasiveness of glioma64, breast65,66, lung67,68, and cervical68 cancer cells in culture

the ceramide/p8–regulated pathway plays a general role in the antitumour activity of cannabinoids targeting cb1 and cb2

cbd, by acting independently of the cb1 and cb2 receptors, produces a remarkable anti-tumour effect—including reduction of invasiveness and metastasis

cannabinoids can also enhance immune system–mediated tumour surveillance in some contexts

ability of thc to reduce inflammation75,76, an effect that might prevent certain types of cancer

recent observations suggest that the combined administration of cannabinoids with other anticancer drugs acts synergistically to reduce tumour growth

combined administration of gemcitabine (the benchmark agent for the treatment of pancreatic cancer) and various cannabinoid agonists synergistically reduced the viability of pancreatic cancer cells

Other reports indicated that anandamide and HU-210 might also enhance the anticancer activity of paclitaxel89 and 5-fluorouracil90 respectively

Combined administration of thc and cbd enhances the anticancer activity of thc and reduces the dose of thc needed to induce its tumour growth-inhibiting activity

Preclinical animal models have yielded data indicating that systemic (oral or intraperitoneal) administration of cannabinoids effectively decreases tumour growth

Combinations of cannabinoids with classical chemotherapeutic drugs such as the alkylating agent temozolomide (the benchmark agent for the management of glioblastoma80,84) have been shown to produce a strong anticancer action in animal models

pharmacologic inhibition of egfr, erk83, or akt enhances the cell-death-promoting action of thc in glioma cultures (unpublished observations by the authors), which suggests that targeting egfr and the akt and erk pathways could enhance the antitumour effect of cannabinoids

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

Eighty-five percent of lung cancers are non-small-cell lung cancer (NSCLC)

Laboratory studies have demonstrated anticancer effects of vitamin D metabolites on three critical phases in the development of breast tumors: differentiation, apoptosis, and angiogenesis