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

H2 O2

160 Myeloid Leukemia

 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,

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

The pharmacokinetic studies of Levine and Padayatty [28, 29], on Vitamin C, indicate that after oral administration of 200 mg of the nutrient, the maximum plasma concentrations obtained, are not superior to 70–80 μM. This is due to a "tight control," operated by several different mechanisms, including, among others: bioavailability, intestinal absorption, tissue accumulation, renal reabsorption and excretion, and utilization rate as a function of homeostasis. On the contrary, when Vitamin C is administered intravenously, "tight control" is bypassed, until

Therefore, according to these data, the intravenous administration of Vitamin C is the only way to achieve plasma concentrations in the order of millimoles, necessary to kill cancer cells.

**a.** The results obtained by intravenous administration of Vitamin C, do not show the same large effects reported by Robinson, feeding squamous cell carcinoma implanted mice,

**b.** Abram Hoffer [53] used oral high doses of Vitamin C in cancer patients and obtained essentially the same significant results as Cameron and Pauling, Cameron and Campbell

**c.** Although it is presently believed that only injected Vitamin C delivers the concentrations needed to produce an anti-tumor effect, neither the legendary scientist Linus Pauling nor the consultant surgeon, Ewan Cameron, seemed to know the difference between oral and intravenous administration. In fact, in their clinical trial, the protocol started with a few days of 10 grams of intravenous Vitamin C, followed by 10 grams of oral Vitamin C for the whole life. Interestingly, Cameron and Campbell, who had already reported on the successful treatment of cancer with *oral* Vitamin C, had already observed that "*… with increasing experience, we tend now to believe that the intravenous regime is probably unnecessary as a routine measure, and need only to be employed in situations where vomiting, anorexia, or other* 

**d.** Plasma concentrations above the 400 μM have been reported, after the administration of a

renal excretion restores equilibrium, depending on the dose administered [51].

However, this view is in disagreement with the following evidences:

*complications of malignancy, preclude oral administration*" [58];

single dose of oral liposomal Vitamin C [60];

as in the case of the use of Vitamin C as an anticancer compound [48].

kill cancer cells, is still far from being fully elucidated.

**6. Oral vs. intravenous Vitamin C**

with large doses of the nutrient [52];

[54–58], and Murata [59];

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

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 necessary to kill cancer cells in APL.

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

**Figure 1.** The microphotographs refer to the cytomorphologic modifications of HL60 cell lines (human acute promyelocytic leukemia—APL) exposed for 2 h to increasing concentrations of Vitamin C. It is evident that by increasing the concentration of Vitamin C in the medium (from 1 to 5 mM), APL cells show an increasing degree of morphologic alterations indicating progressive cell death (apoptosis, autophagy, autoschizis). With the Hoechst/PI fluorescent staining, vital cells are colored in blue, while dead/apoptotic cells are stained in red. M.G.G. = May Grunwald Giemsa cell staining; Hoechst33342/Propidium Iodide (PI) = Vital Staining; C = control (untreated) sample; 1 mM, 3 mM, 5 mM = Vitamin C at 1, 3, and 5 mM in the culture medium; original magnification: 400×.

**Figure 2.** Viability profile of (Human) acute promyelocytic leukemia (APL) cell line (HL60) exposed for 2 h to increasing concentrations of Viability profile of (Human) acute promyelocytic leukemia (APL) cell line (HL60) exposed for 2 h to increasing concentrations of Vitamin C. (Flow Cytometry analysis: see text). The percentage of dead cells in the plates is proportional to the concentration of Vitamin C in the medium. C = control (untreated) sample; 1 mM, 3 mM, 5 mM = Vitamin C at 1, 3, and 5 mM in the culture medium.

its "toxic" effects last for hours (days), even when the nutrient has been removed from the culture medium. This is in agreement with the notion that both normal and leukemic white blood cells tend to concentrate Vitamin C [70–73] to levels that are 10–100 fold higher than plasma [74, 75], and it is in contrast with the view that hydrogen peroxide forms outside the tumor cells [31, 32].

Neutrophils, in particular, accumulate Vitamin C via the sodium-dependent Vitamin C cotransporter 2 (SVCT2) [76], and have intracellular levels of 1–2 mM [77]. Therefore, while there is agreement on the fact that in solid tumors, Vitamin C, initially oxidized to dehydroascorbic acid (DHAA), is internalized by the cell, via GLUT 1 and 4, and finally reduced

**Figure 4.** The figure illustrates **Table 1**. Highlighted with colored circles, the LC50 for each human myeloid leukemia

CONTR. VC 0.5mM VC 1mM VC 3mM VC 5mM VC7mM

HL60 NB4 K562 U937 NB4-R1 NB4/AS

CONTR. VC 1mM VC 3mM VC 5mM VC7mM

**Cytotoxic eect of Vitamin C on human myeloid leukemia cell lines**

High Doses of Vitamin C and Leukemia: In Vitro Update http://dx.doi.org/10.5772/intechopen.71484 163

HL60 K562 U937 NB4-R1 NB4/AS

**Figure 3.** The figure illustrates **Table 1**. The diagram shows the almost uniform decrease in the number of vital leukemic

**Cytotoxic eect of Vitamin C on human myeloid leukemia cell lines**

> **O O O O OO**

NB4 VC 0.5mM

cells in the culture medium, after exposing them to increasing concentrations of Vitamin C, for 2 h.

0

0

cell line tested.

500

1,000

1,500

2,000

2,500

3,000

3,500

500

1,000

1,500

2,000

2,500

3,000

3,500

**Cytotoxic eect of Vitamin C on human myeloid**

**Figure 3.** The figure illustrates **Table 1**. The diagram shows the almost uniform decrease in the number of vital leukemic cells in the culture medium, after exposing them to increasing concentrations of Vitamin C, for 2 h.

**Figure 4.** The figure illustrates **Table 1**. Highlighted with colored circles, the LC50 for each human myeloid leukemia cell line tested.

its "toxic" effects last for hours (days), even when the nutrient has been removed from the culture medium. This is in agreement with the notion that both normal and leukemic white blood cells tend to concentrate Vitamin C [70–73] to levels that are 10–100 fold higher than plasma [74, 75], and it is in contrast with the view that hydrogen peroxide forms outside the

C 1mM 3mM 5mM

C 1mM 3mM

**Figure 1.** The microphotographs refer to the cytomorphologic modifications of HL60 cell lines (human acute promyelocytic leukemia—APL) exposed for 2 h to increasing concentrations of Vitamin C. It is evident that by increasing the concentration of Vitamin C in the medium (from 1 to 5 mM), APL cells show an increasing degree of morphologic alterations indicating progressive cell death (apoptosis, autophagy, autoschizis). With the Hoechst/PI fluorescent staining, vital cells are colored in blue, while dead/apoptotic cells are stained in red. M.G.G. = May Grunwald Giemsa cell staining; Hoechst33342/Propidium Iodide (PI) = Vital Staining; C = control (untreated) sample; 1 mM, 3 mM,

5 mM = Vitamin C at 1, 3, and 5 mM in the culture medium; original magnification: 400×.

5mM

**Figure 2.** Viability profile of (Human) acute promyelocytic leukemia (APL) cell line (HL60) exposed for 2 h to increasing concentrations of Viability profile of (Human) acute promyelocytic leukemia (APL) cell line (HL60) exposed for 2 h to increasing concentrations of Vitamin C. (Flow Cytometry analysis: see text). The percentage of dead cells in the plates is proportional to the concentration of Vitamin C in the medium. C = control (untreated) sample; 1 mM, 3 mM, 5 mM =

tumor cells [31, 32].

Hoechst/PI

M.G.G.

162 Myeloid Leukemia

Vitamin C at 1, 3, and 5 mM in the culture medium.

Neutrophils, in particular, accumulate Vitamin C via the sodium-dependent Vitamin C cotransporter 2 (SVCT2) [76], and have intracellular levels of 1–2 mM [77]. Therefore, while there is agreement on the fact that in solid tumors, Vitamin C, initially oxidized to dehydroascorbic acid (DHAA), is internalized by the cell, via GLUT 1 and 4, and finally reduced


HIF-1 is a heterodimeric transcription factor discovered in 1991 [80], and is composed of two subunits, α and β. The HIF-1α subunit is oxygen sensitive and it is induced by hypoxic conditions, which are very common in cancer. Direct transcriptional targets of HIF-1 include genes regulating, among others, growth and apoptosis, cell migration, energy metabolism, angiogenesis, vasomotor regulation, matrix and barrier functions, and transport of metal ions

High Doses of Vitamin C and Leukemia: In Vitro Update http://dx.doi.org/10.5772/intechopen.71484 165

In normoxic conditions, the HIF-1α unit is downregulated by Vitamin C dependent hydroxylases, while in hypoxic conditions (such as those existing in many different types of cancer), HIF-1α hydroxylation is repressed with consequent increase in HIF-dependent gene tran-

More importantly, since Vitamin C stimulates HIF-1α prolyl hydroxylases, low levels of Vitamin C promote tumor growth and progression, by reducing HIF-1α hydroxylation [83], thereby stabilizing HIF1-α. On the contrary, high levels of HIF render cancer cells more sensitive to Vitamin C-induced toxicity. To confirm this view, Kuiper and Coll. [84] have recently found an inverse relationship between intra-tumor levels of Vitamin C and HIF activity in

In 1925, Otto Warburg observed that cancer cells manifest increased rates of lactate production under aerobic conditions ("Warburg Effect") or, in other words, they preferentially utilize glycolysis, instead of oxidative phosphorylation, for metabolism even in the presence of

"Hypoxia" (low oxygen concentration) is a hallmark of solid tumors, usually occurring at the center of the tumor mass, where blood vessels are abnormal or insufficient to supply adequate

In response to the reduced oxygen tension, the HIF is activated to mediate the primary tran-

As previously mentioned, HIFs regulate angiogenesis, cell survival, proliferation, apoptosis, adhesion, and metabolism by transcriptionally activating downstream targets such as vascular endothelial growth factor and erythropoietin. Therefore, HIF (HIF1, in particular) plays a major role in tumor growth, and clinical data suggest that the upregulation of HIF, as determined by the low oxygen tension, is usually associated with increased mortality in a number of different cancers [92–94], and may represent a relevant target for new therapeutic

The role of HIF-1α in leukemia, and in particular in acute myeloid leukemia (AML), has only recently emerged and it is still somewhat controversial. One possible explanation for this delayed interest in the role of hypoxia in leukemia could be the fact that leukemia is not considered a "solid" tumor, and therefore, the role of oxygen, in its pathogenesis, has been

scription, neo-angiogenesis, and tumor growth and progression [82].

both endometrial cancer [85] and colorectal carcinoma (CRC) [86].

scriptional adaption to hypoxic stress in cancer cells [90, 91].

and glucose [81].

oxygen [87, 88].

amounts of oxygen [89].

approaches to the disease [95–97].

**9. The HIF pathway in leukemia**

The cell lines used in this experiment are variants of human myeloid leukemia cells, and include: HL60, NB4, K562, U937, NB4-R1, NB4/As. It is evident that the total number of cells in culture decreases by increasing the concentration of Vitamin C in the culture medium. C = control (untreated) sample; VC = Vitamin C; VC 0.5 mM, VC 1 mM, VC 3 mM, VC 5 mM = Vitamin C at 0.5, 1, 3, and 5 mM in the culture medium.

**Table 1.** The number of vital cells after 2 h of exposure to increasing concentrations of Vitamin C in the culture medium.

again to ascorbic acid, with consumption of GSH; this may not be the case in acute myeloid leukemia.

More importantly, the parallel exposure of normal hematopoietic precursors (CD34+), isolated from cord blood, to Vitamin C, at the concentrations that are cytotoxic for leukemic cells did not affect their survival, or impair their capacity to proliferate and differentiate in response to myeloid growth factors. These data confirm that Vitamin C is harmless for normal hematopoietic precursors and therefore highly selective in its anticancer/antileukemic effect.
