**3. How much Vitamin C to treat leukemia? The concept of "mega-doses"**

In 1949, Frederik Klenner first reported the successful treatment of bulbar poliomyelitis, with high doses of Vitamin C administered by intramuscular, intravenous, and oral route [12]. Klenner had also established clinical protocols using massive doses of Vitamin C to treat a number of different viral conditions, but only more than two decades later, Stone formally defined the concept and rationale for the use of "*mega-doses*" of Vitamin C. In particular, Stone observed that man and only a few other species do not produce their own Vitamin C, while the great majority of mammals do, according to their physiologic requirements [3]. This observation led the author to hypothesize that due to either insufficient intake or increased consumption of the nutrient, or both, man could easily undergo a condition that he defined "*hypoascorbemia*." Hypoascorbemia is a reduced amount of circulating Vitamin C (also called "ascorbic acid"), due to the lack of the enzyme L-gulonolactone oxidase (GLO), as a consequence of an "inborn error of carbohydrate metabolism" [13–15]. This defect, now very well acknowledged and characterized [16], led Stone to hypothesize that to be in good health, man needs mega-doses of Vitamin C (several grams a day) [17, 18], rather than doses in the order of milligrams, as stated by the Recommended Daily Allowances (RDAs) [19].

The rationale behind the use of mega-doses of Vitamin C was further refined by the chemist and twofold Nobel Prize, Linus Pauling. Pauling soon became an enthusiastic supporter of the use of this nutrient, in high doses, not only to prevent disease [20–23], but also to treat a number of pathologic conditions, ranging from common cold [24, 25] to cancer [26] and AIDS [27].

## **4. Intravenous Vitamin C and cancer**

**5.** enhancing the absorption of non-heme iron;

er among the natural antioxidants);

156 Myeloid Leukemia

**9.** reducing the risk of premature death;

blood cells, as compared to plasma.

and Zipf, in 1955 [8].

rate of 25 g or more per day.

**11.** preventing the development of nitrosamines.

the assumption and use of this fundamental nutrient.

**2. Vitamin C and leukemia: historical background**

**6.** promoting the synthesis of collagen (its most widely known physiological function);

**8.** protecting DNA from damage due to free radicals and mutagens;

**10.** fighting off widespread environmental pollutants; and

**7.** neutralizing free radicals (it is a reducing agent, "scavenger" of free radicals, and a found-

Though ubiquitous, ascorbate is not produced by humans, guinea pigs, some primates, a particular type of fruit eating bat, the majority of fishes and birds [2], who depend on diet for

The first mention of the therapeutic potentialities of Vitamin C in leukemia, can be found in the book "*The healing Factor: Vitamin C against disease*," written by the biochemist Irwin Stone, in 1974 [3]. In his book, Stone refers to a study, performed in 1936 by Stephen and Hawley [4], demonstrating, for the first time, that when the blood is separated into plasma, red blood cells, and white blood cells, there is a 20- to 30-fold concentration of Vitamin C in the white

Following this report, Barkhan and Howard, by studying a few cases of chronic myelogenous and lymphatic leukemia, added the evidence that leukemic patients have substantially lower than normal plasma levels of Vitamin C [5]. As noted by Stone, although this knowledge could suggest the use of Vitamin C as a therapeutic agent, in leukemia, the first clinical trials

Later on, Vogt, in a literature review [6], confirmed that there are high deficits of Vitamin C in leukemic patients, as also confirmed by the reports of Kyhos and Coll. [7] in 1945, and Waldo

According to Stone [3], leukemia reduces the body stores of Vitamin C to very low levels, and any residual Vitamin C circulating in the blood is scavenged and locked in the excessive numbers of leukocytes characterizing this disorder. As a direct consequence, the plasma levels of Vitamin C are reduced to zero or close thereto, and tissues are no longer being supplied with

Stone [3] defined "*biochemical scurvy*" as the condition of insufficient Vitamin C supply to body tissues, and proposed that its correction required the administration of Vitamin C at a

showed contrasting results, due to the inappropriately *low doses* administered.

this most important metabolite, since it is accumulated in leukocytes.

Studies on dose-concentration relationship in humans, performed by Levine and co-workers [28], revealed that at oral doses exceeding 250 mg/day, the plasma levels of Vitamin C reach a plateau, and any further increase in the amount administered by mouth, does not determine significant increase in plasma concentration. This is due to multiple "control" mechanisms, including, among others, intestinal absorption, tissue accumulation, renal reabsorption and excretion, and utilization. On the contrary, the intravenous administration of high doses of Vitamin C, bypassing the above control mechanisms, allows plasma concentrations that are 100-fold or higher than maximally tolerated oral doses, and the peak could last for hours within the millimolar (mM) range [29].

More precisely, beyond being inactivated directly by ROS (including H<sup>2</sup>

death [43].

**5. High doses of Vitamin C and H2**

as a pro-oxidant, leading to the formation of H<sup>2</sup>

hydrogen peroxide [45, 46].

O2

tion of H<sup>2</sup>

"*thus decreasing or destroying the ability of the cancer cells to detoxify H2*

significant [33–35], and other mechanisms should be hypothesized.

motes both production and decomposition of H<sup>2</sup>

reaction, which prevents the accumulation of H<sup>2</sup>

chelating agents, which remove iron from the medium [47].

lytic iron may not be strictly necessary for the production of H<sup>2</sup>

is also hindered by the depletion of nicotinamide adenine dinucleotide (NAD+), caused by the activation of the DNA repairing enzyme, poly(ADP-ribose) polymerase (PARP), induced by damaged DNA. In fact, the increased production of ROS, in cancer cells, due to the high doses of Vitamin C, produces increased DNA damage and consequent activation of PARP. PARP, in turn, consumes NAD+ with consequent NAD+ depletion, ATP depletion, and cancer cell

**O2**

The view that Vitamin C in high concentrations, administered by intravenous infusion, acts

cells, is not new. In 1969, Benade and co-workers had already demonstrated that Vitamin C could selectively kill cancer cells, without harming normal cells. The authors suggested that the cytotoxic effect of ascorbate could be due ("*in major part*") to the intracellular generation of toxic hydrogen peroxide produced upon oxidation of Vitamin C, by the cells. This view was corroborated by the fact that the toxicity of Vitamin C was greatly enhanced by the concomitant administration of 3-amino-1, 2, 4-triazole (ATA) that inhibits the enzyme catalase,

scientific reports confirmed that human cancer cells have low levels of antioxidant enzymes (including, among others, catalase and glutathione peroxidase), and therefore cannot detoxify

According to the pro-oxidant theory, Vitamin C in high concentrations induces the produc-

strates by iron and hydrogen peroxide, in which trivalent iron (Fe3+) plays a fundamental role. However, since Fenton-like reactions are usually controlled, in vivo, because of iron sequestration by metal binding proteins, the pro-oxidant effect of Vitamin C, in vivo, may be scarcely

Other authors, using two prostate cancer cell lines (LNCaP and PC-3) have shown that iron at physiological concentrations in cell culture medium and human plasma abrogates the anticancer/cytotoxic effects of Vitamin C. In particular, at physiological concentrations, iron pro-

Vitamin C. Therefore, for an optimal anticancer effect, Vitamin C should be administered with

On the other hand, Vitamin C readily undergoes pH-dependent autoxidation producing hydrogen peroxide, and catalytic metals only accelerate the oxidation process. Therefore, cata-

(oxidation in the absence of catalytic metals) occurs via the ascorbate di-anion (Asc2−). In particular, at pH 7.0, 99.9% of ascorbate (Vitamin C) is in the form of mono-anion (AscH−). Asc2− increases by a factor ten, with one unit increase in the pH. Therefore, while the production of

O2

O2

through a Fenton-like reaction. This reaction is the oxidation of organic sub-

O2

O2

High Doses of Vitamin C and Leukemia: In Vitro Update http://dx.doi.org/10.5772/intechopen.71484

, thus inducing oxidative damage to cancer

*O2*

, the latter being mediated by a Fenton

, thus abolishing the cytotoxic effect of

. This auto oxidation process

O2

 *effectively*" [44]. Further

), GAPDH function

159

More importantly, at plasma concentrations easily achievable by intravenous administration (5–10 mM for 1–2 h), Vitamin C induced death in 75% of 48 cancer cell lines tested in vitro [30], but had no toxic effect on human peripheral white blood cells, fibroblasts, or epithelial cells. This selective cytotoxic effect would be achieved since at high doses, parenteral ascorbate is a *peroxide delivery system* for the generation of sustainable ascorbate radical and H<sup>2</sup> O2 . H<sup>2</sup> O2 would be produced in the extracellular space, with consequent oxidative damage to cancer cells [31, 32]. Therefore, Vitamin C in high doses would be cytotoxic to cancer cells because of its *pro-oxidant*, rather than anti-oxidant effect, even though some authors remark that the pro-oxidant activity of Vitamin C, may not be relevant, in vivo [33–35].

More recently, Yun and co-workers [36], by investigating the effects of high doses of Vitamin C on KRAS and BRAF mutants cells derived from colorectal cancer (CRC), have further refined the mechanistic explanation of the anticancer properties of Vitamin C. In particular, according to the authors, the death of KRAS and BRAF cell mutants of CRC is not caused by the Vitamin C itself, but rather, by its oxidized form, dehydroascorbic acid (DHAA). While Vitamin C enter cells though specific receptors, called sodium-Vitamin C co-transporters (SVCTs) [37], DHAA competes with glucose, for intracellular uptake by glucose transporters (GLUT), mainly 1 and 4 subtype receptors [38, 39].

Interestingly, both KRAS and BRAF activating mutations are responsible for the upregulation of GLUT1 expression in different types of cancer, including CRC [40, 41].

However, as reported by Yun and Coll. [36], the upregulation of GLUT-1 expression is not always associated with increased sensitivity of tumor cell lines to the cytotoxic effects of DHAA.

Further investigation into the metabolic makeup of KRAS and BRAF mutations CRC-derived cell lines, showed an accumulation of glycolytic intermediates upstream glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and a contemporary depletion of the metabolites downstream GAPDH. This finding indicates an inhibition or severe reduction of GAPDH activity, which appears to be the key of the cytotoxic effect of DHA.

In summary, the data reported by Yun and Coll. on the effect of DHAA on CRC cell lines, indicate that in glycolysis-addicted KRAS and BRAF mutated cell lines, high amounts of DHAA are transported into the cancer cells, through the overexpressed GLUT-1 receptors. The exceeding amounts of intracellular DHAA are then reduced again to Vitamin C with consequent consumption of glutathione (GSH), redox imbalance, and oxidative stress. Oxidative stress, in turn, causes GAPDH inactivation, with inhibition of glycolysis, and energetic crisis, ultimately leading to cell death [42].

More precisely, beyond being inactivated directly by ROS (including H<sup>2</sup> O2 ), GAPDH function is also hindered by the depletion of nicotinamide adenine dinucleotide (NAD+), caused by the activation of the DNA repairing enzyme, poly(ADP-ribose) polymerase (PARP), induced by damaged DNA. In fact, the increased production of ROS, in cancer cells, due to the high doses of Vitamin C, produces increased DNA damage and consequent activation of PARP. PARP, in turn, consumes NAD+ with consequent NAD+ depletion, ATP depletion, and cancer cell death [43].

#### **5. High doses of Vitamin C and H2 O2**

plateau, and any further increase in the amount administered by mouth, does not determine significant increase in plasma concentration. This is due to multiple "control" mechanisms, including, among others, intestinal absorption, tissue accumulation, renal reabsorption and excretion, and utilization. On the contrary, the intravenous administration of high doses of Vitamin C, bypassing the above control mechanisms, allows plasma concentrations that are 100-fold or higher than maximally tolerated oral doses, and the peak could last for hours

More importantly, at plasma concentrations easily achievable by intravenous administration (5–10 mM for 1–2 h), Vitamin C induced death in 75% of 48 cancer cell lines tested in vitro [30], but had no toxic effect on human peripheral white blood cells, fibroblasts, or epithelial cells. This selective cytotoxic effect would be achieved since at high doses, parenteral ascorbate is

would be produced in the extracellular space, with consequent oxidative damage to cancer cells [31, 32]. Therefore, Vitamin C in high doses would be cytotoxic to cancer cells because of its *pro-oxidant*, rather than anti-oxidant effect, even though some authors remark that the

More recently, Yun and co-workers [36], by investigating the effects of high doses of Vitamin C on KRAS and BRAF mutants cells derived from colorectal cancer (CRC), have further refined the mechanistic explanation of the anticancer properties of Vitamin C. In particular, according to the authors, the death of KRAS and BRAF cell mutants of CRC is not caused by the Vitamin C itself, but rather, by its oxidized form, dehydroascorbic acid (DHAA). While Vitamin C enter cells though specific receptors, called sodium-Vitamin C co-transporters (SVCTs) [37], DHAA competes with glucose, for intracellular uptake by glucose transporters

Interestingly, both KRAS and BRAF activating mutations are responsible for the upregulation

However, as reported by Yun and Coll. [36], the upregulation of GLUT-1 expression is not always associated with increased sensitivity of tumor cell lines to the cytotoxic effects of

Further investigation into the metabolic makeup of KRAS and BRAF mutations CRC-derived cell lines, showed an accumulation of glycolytic intermediates upstream glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and a contemporary depletion of the metabolites downstream GAPDH. This finding indicates an inhibition or severe reduction of GAPDH

In summary, the data reported by Yun and Coll. on the effect of DHAA on CRC cell lines, indicate that in glycolysis-addicted KRAS and BRAF mutated cell lines, high amounts of DHAA are transported into the cancer cells, through the overexpressed GLUT-1 receptors. The exceeding amounts of intracellular DHAA are then reduced again to Vitamin C with consequent consumption of glutathione (GSH), redox imbalance, and oxidative stress. Oxidative stress, in turn, causes GAPDH inactivation, with inhibition of glycolysis, and energetic crisis,

O2 . H<sup>2</sup> O2

a *peroxide delivery system* for the generation of sustainable ascorbate radical and H<sup>2</sup>

pro-oxidant activity of Vitamin C, may not be relevant, in vivo [33–35].

of GLUT1 expression in different types of cancer, including CRC [40, 41].

activity, which appears to be the key of the cytotoxic effect of DHA.

(GLUT), mainly 1 and 4 subtype receptors [38, 39].

ultimately leading to cell death [42].

DHAA.

within the millimolar (mM) range [29].

158 Myeloid Leukemia

The view that Vitamin C in high concentrations, administered by intravenous infusion, acts as a pro-oxidant, leading to the formation of H<sup>2</sup> O2 , thus inducing oxidative damage to cancer cells, is not new. In 1969, Benade and co-workers had already demonstrated that Vitamin C could selectively kill cancer cells, without harming normal cells. The authors suggested that the cytotoxic effect of ascorbate could be due ("*in major part*") to the intracellular generation of toxic hydrogen peroxide produced upon oxidation of Vitamin C, by the cells. This view was corroborated by the fact that the toxicity of Vitamin C was greatly enhanced by the concomitant administration of 3-amino-1, 2, 4-triazole (ATA) that inhibits the enzyme catalase, "*thus decreasing or destroying the ability of the cancer cells to detoxify H2 O2 effectively*" [44]. Further scientific reports confirmed that human cancer cells have low levels of antioxidant enzymes (including, among others, catalase and glutathione peroxidase), and therefore cannot detoxify hydrogen peroxide [45, 46].

According to the pro-oxidant theory, Vitamin C in high concentrations induces the production of H<sup>2</sup> O2 through a Fenton-like reaction. This reaction is the oxidation of organic substrates by iron and hydrogen peroxide, in which trivalent iron (Fe3+) plays a fundamental role. However, since Fenton-like reactions are usually controlled, in vivo, because of iron sequestration by metal binding proteins, the pro-oxidant effect of Vitamin C, in vivo, may be scarcely significant [33–35], and other mechanisms should be hypothesized.

Other authors, using two prostate cancer cell lines (LNCaP and PC-3) have shown that iron at physiological concentrations in cell culture medium and human plasma abrogates the anticancer/cytotoxic effects of Vitamin C. In particular, at physiological concentrations, iron promotes both production and decomposition of H<sup>2</sup> O2 , the latter being mediated by a Fenton reaction, which prevents the accumulation of H<sup>2</sup> O2 , thus abolishing the cytotoxic effect of Vitamin C. Therefore, for an optimal anticancer effect, Vitamin C should be administered with chelating agents, which remove iron from the medium [47].

On the other hand, Vitamin C readily undergoes pH-dependent autoxidation producing hydrogen peroxide, and catalytic metals only accelerate the oxidation process. Therefore, catalytic iron may not be strictly necessary for the production of H<sup>2</sup> O2 . This auto oxidation process (oxidation in the absence of catalytic metals) occurs via the ascorbate di-anion (Asc2−). In particular, at pH 7.0, 99.9% of ascorbate (Vitamin C) is in the form of mono-anion (AscH−). Asc2− increases by a factor ten, with one unit increase in the pH. Therefore, while the production of H2 O2 may be scarcely relevant in the absence of catalytic iron (as in the "Fenton chemistry"), it may become considerable when the concentration of ascorbate is in the order of the millimoles, as in the case of the use of Vitamin C as an anticancer compound [48].

**e.** At times of stress or illnesses (including cancer), the body may absorb extra Vitamin C, as demonstrated by the principle of "bowel tolerance" to the nutrient administered by mouth. According to this principle, when the body is saturated with Vitamin C, slight diarrhea may appear, due to intestinal elimination of the nutrient. However, during stress or disease, the amount of oral Vitamin C a patient can tolerate, before the appearance of

High Doses of Vitamin C and Leukemia: In Vitro Update http://dx.doi.org/10.5772/intechopen.71484 161

**f.** This means that the "tight control" hypothesized by Levine and Padayatty, over the plasma concentration of Vitamin C, is either inexistent or relative to disease conditions or stress. To achieve the maximum plasma levels, a typical person may need 20 g of oral Vitamin C spread throughout the day (3–4 g every 4 h); but cancer patients may require far more [62]. Such massive intake may result in plasma concentrations that the tumor may

**g.** More recently, the paradigm according to which antioxidants inhibit tumorigenesis predominantly by decreasing ROS-mediated DNA damage and mutations [63, 64] has been challenged by experimental data. Antioxidants such as N-acetylcysteine (NAC) and Vitamin C exerts their anti-tumorigenic activity by downregulating HIF-1α [65]. Interestingly, these data were obtained not by injecting, but by simply feeding mice with large amounts of NAC or Vitamin C. These findings validate the role of oral administration of Vitamin C

As we have previously demonstrated, high ("pharmacologic") concentrations of Vitamin C (in the form of the sodium salt of ascorbic acid) are capable of eliciting a clear-cut pro-apoptotic/cytotoxic effect on human promyelocytic leukemia-derived cell lines (HL60), in vitro [66] (**Figures 1** and **2**). This effect is already evident at concentrations of Vitamin C of 1 mM in the

Since clinical investigations using high doses of Vitamin C to treat cancer, have reported plasma levels of more than 30 [67], and up to 49 mM [68], it seems reasonable to conclude that using high amounts of Vitamin C, administered by intravenous injection, is not strictly neces-

Further investigations in leukemia, performed by our research group, have shown that a plasma concentration of 3 mM of Vitamin C in the culture medium, is sufficient to kill more than a half of the cells in culture (LC50) in a number of different human myeloid leukemia cell lines [69] (**Figures 3** and **4**) (**Table 1**). It is of interest to consider that according to our protocol, the leukemic cells are exposed to Vitamin C for no more than 2 h, then accurately "washed," re-suspended in fresh culture medium, without Vitamin C, and further incubated for additional 18–24 h, before the evaluation of cell survival and apoptosis. Given the results obtained, it is reasonable to conclude that the Vitamin C added to the culture medium (in the form of sodium ascorbate) is rapidly internalized by the leukemic cells, and

diarrhea, increases in proportion with the severity of the condition [61];

absorb, generating hydrogen peroxide that kills cancer cells;

(and other antioxidants) in fighting cancer.

sary to kill cancer cells in APL.

**7. Vitamin C and leukemia: an in vitro update**

culture medium, and it is proportional to the amount of Vitamin C.

Finally, accumulating evidence suggests that cancer cells produce high amounts of hydrogen peroxide [49], and hydrogen peroxide itself is a powerful carcinogen, associated with mutagenic potential [50]. Therefore, the role of Vitamin C as a pro-drug of hydrogen peroxide, to kill cancer cells, is still far from being fully elucidated.
