Pharmacologic ascorbic acid concentrations selectively kill cancer cells: action as a pro-drug to deliver hydrogen peroxide to tissues

Abstract

Human pharmacokinetics data indicate that i.v. ascorbic acid (ascorbate) in pharmacologic concentrations could have an unanticipated role in cancer treatment. Our goals here were to test whether ascorbate killed cancer cells selectively, and if so, to determine mechanisms, using clinically relevant conditions. Cell death in 10 cancer and 4 normal cell types was measured by using 1-h exposures. Normal cells were unaffected by 20 mM ascorbate, whereas 5 cancer lines had EC(50) values of <4 mM, a concentration easily achievable i.v. Human lymphoma cells were studied in detail because of their sensitivity to ascorbate (EC(50) of 0.5 mM) and suitability for addressing mechanisms. Extracellular but not intracellular ascorbate mediated cell death, which occurred by apoptosis and pyknosis/necrosis. Cell death was independent of metal chelators and absolutely dependent on H(2)O(2) formation. Cell death from H(2)O(2) added to cells was identical to that found when H(2)O(2) was generated by ascorbate treatment. H(2)O(2) generation was dependent on ascorbate concentration, incubation time, and the presence of 0.5-10% serum, and displayed a linear relationship with ascorbate radical formation. Although ascorbate addition to medium generated H(2)O(2), ascorbate addition to blood generated no detectable H(2)O(2) and only trace detectable ascorbate radical. Taken together, these data indicate that ascorbate at concentrations achieved only by i.v. administration may be a pro-drug for formation of H(2)O(2), and that blood can be a delivery system of the pro-drug to tissues. These findings give plausibility to i.v. ascorbic acid in cancer treatment, and have unexpected implications for treatment of infections where H(2)O(2) may be beneficial.

Fig. 1.

Effects of pharmacologic ascorbic acid concentrations on cancer and normal cells. Concentrations in this and all figures indicate final concentrations. (A) EC₅₀ values of ascorbate in human and mouse cancer cells and normal human cells. All cells were treated with ascorbate for 1 h, washed, and recultured without ascorbate. EC₅₀ values were determined 18-22 h later by using Hoechst/PI for human Burkitt's lymphoma cells (JLP119), MTT and Hoechst/PI for normal lymphocytes and monocytes, and MTT for all other cells (see Materials and Methods). (B) Colony formation of cancer cells in soft agar after a 1-h treatment with 5 mM ascorbate. Surviving fraction, expressed in log scale, indicates the number of treated colonies compared with matched untreated control cells.

Fig. 2.

Effects of ascorbic acid on human Burkitt's lymphoma cells. Cells were treated for 1 h, washed, and recultured without ascorbate. Amounts and types of cell death were determined 18-22 h later by nuclear staining with Hoechst/PI. Types of cell death: necrosis (black), pyknosis/necrosis (gray), early apoptosis (blue), and late apoptosis (red). (A) Amount and type of cell death as a function of external ascorbate concentration. (B) Time course and type of cell death after 1 h external ascorbate (2 mM). (C) Cell death as a function of external ascorbate concentration in human Burkitt's lymphoma cells (♦), normal lymphocytes (▪), and normal monocytes (▴). (D) Cell death as a function of external ascorbate (♦) or dehydroascorbic acid (□) concentrations (1-h incubation). (E) Type and amount of cell death with 2 mM ascorbate treatment, in cells previously loaded to contain 3 mM ascorbate (right), compared with unloaded cells (left).

Fig. 3.

Extracellular ascorbate kills human Burkitt's lymphoma cells by generating H₂O₂. Cell death determined and symbolized as in Fig. 2; H₂O₂ measured by oxygen electrode (see Materials and Methods). (A) Effects of reactive oxygen species quenchers/scavengers, reducing agent, and metal chelators on ascorbate-mediated cell death. The following (final concentrations) were preincubated with cells for 30 min before exposure to ascorbate (2 mM): catalase (100 μg/ml); tetrakis (4-benzoic acid) meso-substituted manganoporphyrin (MnTBAP) (100 μM); Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) (500 μM); diethylene-triamine-pentaacetic acid (DTPA) (1 mM); and N,N′-bis(2-hydroxybenzyl)ethyl-enediamine-N,N′-diacetic acid (HBED) (50 μM). (B) Type and amount of cell death as function of added H₂O₂ (final concentrations). (C) Cell death as a function of added H₂O₂ for 1 h (♦) or mean H₂O₂ concentration generated by 0.2-2 mM ascorbate during a 1-h incubation (▵). (D) Cell death in human Burkitt's lymphoma cells (♦), normal lymphocytes (▪), and normal monocytes (▴) as function of added H₂O₂ (final concentrations).

Fig. 4.

Enhancing factors for ascorbate-mediated H₂O₂ generation in cell culture medium. H₂O₂ was measured by oxygen electrode, and ascorbate radical was measured by electron paramagnetic resonance. (A) H₂O₂ formation as function of time and ascorbate concentration: 0.2 mM (×), 0.5 mM (▴), 1 mM (□), and 2 mM (♦). (B) H₂O₂ formation as a function of the percentage of FBS for 1 h (2 mM ascorbate). (C) H₂O₂ formation as a function of ascorbate radical formation (0.2-2 mM ascorbate, 1-h incubation).

Fig. 5.

Human blood inhibits H₂O₂ and ascorbate radical generation from ascorbate. Ascorbate radical was measured by electron paramagnetic resonance, H₂O₂ was measured by oxygen electrode, and cell death was measured and displayed as in Fig. 2. (A) Ascorbate radical formation as function of ascorbate concentrations added to blood (♦) or medium (▴). (B) H₂O₂ generated by ascorbate concentrations added to blood (♦) or medium (▴) (1-h incubation), and H₂O₂ measured in blood immediately after the addition of indicated concentrations (▵). (C) Human Burkitt's lymphoma cell death in the presence or absence of red blood cells (RBC) at 25% or 50% hematocrit (HCT) (2 mM ascorbate, 1-h treatment).