Pharmacologic doses of ascorbate act as a prooxidant and decrease growth of aggressive tumor xenografts in mice

Abstract

Ascorbic acid is an essential nutrient commonly regarded as an antioxidant. In this study, we showed that ascorbate at pharmacologic concentrations was a prooxidant, generating hydrogen-peroxide-dependent cytotoxicity toward a variety of cancer cells in vitro without adversely affecting normal cells. To test this action in vivo, normal oral tight control was bypassed by parenteral ascorbate administration. Real-time microdialysis sampling in mice bearing glioblastoma xenografts showed that a single pharmacologic dose of ascorbate produced sustained ascorbate radical and hydrogen peroxide formation selectively within interstitial fluids of tumors but not in blood. Moreover, a regimen of daily pharmacologic ascorbate treatment significantly decreased growth rates of ovarian (P < 0.005), pancreatic (P < 0.05), and glioblastoma (P < 0.001) tumors established in mice. Similar pharmacologic concentrations were readily achieved in humans given ascorbate intravenously. These data suggest that ascorbate as a prodrug may have benefits in cancers with poor prognosis and limited therapeutic options.

Fig. 1.

Relative cytotoxicity of ascorbate on cancer and normal cells. (A) Cells (1 × 10⁴) in logarithmic growth phase were cultured in recommended growth media containing 10% FCS and exposed to serial dilutions of ascorbate (0–20 mM, pH 7) for 2 h and washed and cultured for an additional 24–48 h in growth medium in the absence of ascorbate. EC₅₀ values indicate the concentration of ascorbate that reduced survival by 50% determined by viability assays as previously described (4, 9). EC₅₀ values for 13 of 43 cells were previously shown (4). (B) Ovarian carcinoma-Ovcar5 (▴), pancreatic carcinoma-Pan02 (●), glioblastoma-9L (■), and normal human fibroblast-CCD34SK (□) were exposed to ascorbate (pH 7) for 1 h as described in A. Addition of catalase (600 units/ml) before ascorbate (10 mM) ameliorated cytoxicity equivalently for all cells tested (○). Data in A and B represent mean values of six determinations ± SD.

Fig. 2.

Impact of pharmacological ascorbate on tumor growth. Tumors were grown in the flanks of athymic mice to a volume of ≈50 ± 10 mm³, and treatment commenced with either ascorbate (4 g per kilogram of body weight) or osmotically equivalent saline by i.p. injection as indicated. Data (± SEM) show growth curves and final tumor weight with either saline (□) or ascorbate (■) treatments in mice bearing Ovcar5 (A and B), Pan02 (C and D) and 9L (E and F) tumors. P values were calculated by unpaired t-test: *, P < 0.01; **, P < 0.005; ***, P < 0.001.

Fig. 3.

Real-time quantification of ascorbate prodrug metabolism in vivo. Mice were anesthetized and maintained for microdialysis as previously described in Materials and Methods. Separate probes implanted into either tumor tissue or s.c. spaces were perfused (1 μl/min) with sterile saline solution. A single dose of ascorbate (4 g per kilogram of body weight, pH 7) was given by i.p. injection at 0 min, and probe eluates were collected simultaneously from each site in 30-min intervals. Blood was collected from the tail vein into heparinized hematocrit tubes, and analytes were determined as single point measures every 30 min. (A and B) Ascorbate and ascorbate radical concentrations in blood (●), s.c. (□), and tumor (■) extracellular fluids. (C) Ascorbate radical in blood (●), s.c. (□), or tumor (■) extracellular fluid as a function of ascorbate concentrations for all time points (± SEM) are shown. (Inset) Previous data (dashed box) were added from studies of rat-administered ascorbate (either 0.25 or 0.5 g per kilogram of body weight) [Reproduced with permission from Chen Q, et al. (5) (Copyright 2007)]. (D) Formation of H₂O₂ in s.c. (□) or tumor (■) extracellular fluid. (E) Peak plasma concentrations of ascorbate in human subjects who received escalating doses of i.v. ascorbate.

Fig. 4.

Proposed mechanism for tumorcidal actions of pharmacological ascorbate. Ascorbate (AA) distributes from the blood to the tumor extracellular fluid compartments after i.v. administration. In the tumor interstitium, ascorbate is oxidized to ascorbate radical (AA^•) by a metalloprotein catalyst (M), which donates an electron (e⁻) to oxygen forming superoxide radical (O₂⁻) and ultimately the tumorcidal effector H₂O_2. In blood, these reactions are minimized (4, 5).