Since the discovery of vitamin C, the number of its known biological functions is continually expanding. Both the names ascorbic acid and vitamin C reflect its antiscorbutic properties due to its ...role in the synthesis of collagen in connective tissues. Ascorbate acts as an electron-donor keeping iron in the ferrous state thereby maintaining the full activity of collagen hydroxylases; parallel reactions with a variety of dioxygenases affect the expression of a wide array of genes, for example via the HIF system, as well as via the epigenetic landscape of cells and tissues. In fact, all known physiological and biochemical functions of ascorbate are due to its action as an electron donor. The ability to donate one or two electrons makes AscH− an excellent reducing agent and antioxidant. Ascorbate readily undergoes pH-dependent autoxidation producing hydrogen peroxide (H2O2). In the presence of catalytic metals this oxidation is accelerated. In this review, we show that the chemical and biochemical nature of ascorbate contribute to its antioxidant as well as its prooxidant properties. Recent pharmacokinetic data indicate that intravenous (i.v.) administration of ascorbate bypasses the tight control of the gut producing highly elevated plasma levels; ascorbate at very high levels can act as prodrug to deliver a significant flux of H2O2 to tumors. This new knowledge has rekindled interest and spurred new research into the clinical potential of pharmacological ascorbate. Knowledge and understanding of the mechanisms of action of pharmacological ascorbate bring a rationale to its use to treat disease especially the use of i.v. delivery of pharmacological ascorbate as an adjuvant in the treatment of cancer.
Cells contain a large number of antioxidants to prevent or repair the damage caused by reactive oxygen species, as well as to regulate redox-sensitive signaling pathways. General protocols are ...described to measure the antioxidant enzyme activity of superoxide dismutase (SOD), catalase and glutathione peroxidase. The SODs convert superoxide radical into hydrogen peroxide and molecular oxygen, whereas the catalase and peroxidases convert hydrogen peroxide into water. In this way, two toxic species, superoxide radical and hydrogen peroxide, are converted to the harmless product water. Western blots, activity gels and activity assays are various methods used to determine protein and activity in both cells and tissue depending on the amount of protein required for each assay. Other techniques including immunohistochemistry and immunogold can further evaluate the levels of the various antioxidant enzymes in tissues and cells. In general, these assays require 24-48 h to complete.
Ascorbate (AscH
−
) functions as a versatile reducing agent. At pharmacological doses (P-AscH
−
; plasma AscH
−
≥≈20 mM), achievable through intravenous delivery, oxidation of P-AscH
−
can produce a ...high flux of H
2
O
2
in tumors. Catalase is the major enzyme for detoxifying high concentrations of H
2
O
2
. We hypothesize that sensitivity of tumor cells to P-AscH
−
compared to normal cells is due to their lower capacity to metabolize H
2
O
2
. Rate constants for removal of H
2
O
2
(
k
cell
) and catalase activities were determined for 15 tumor and 10 normal cell lines of various tissue types. A differential in the capacity of cells to remove H
2
O
2
was revealed, with the average
k
cell
for normal cells being twice that of tumor cells. The ED
50
(50% clonogenic survival) of P-AscH
−
correlated directly with
k
cell
and catalase activity. Catalase activity could present a promising indicator of which tumors may respond to P-AscH
−
.
•
Ascorbate oxidizes in cell culture medium to generate a flux of H
2
O
2
.
•
The rate constants for removal of extracellular H
2
O
2
are on average 2-fold higher in normal cells than in cancer cells.
•
The ED
50
of high-dose ascorbate correlated with the ability of tumor cells to remove extracellular H
2
O
2
.
•
The response to pharmacological ascorbate in murine-models of pancreatic cancer paralleled the
in vitro
results.
fx1
: Chemoradiation therapy is the mainstay for treatment of locally advanced, borderline resectable pancreatic cancer. Pharmacologic ascorbate (P-AscH
, i.e., intravenous infusions of ascorbic acid, ...vitamin C), but not oral ascorbate, produces high plasma concentrations capable of selective cytotoxicity to tumor cells. In doses achievable in humans, P-AscH
decreases the viability and proliferative capacity of pancreatic cancer via a hydrogen peroxide (H
O
)-mediated mechanism. In this study, we demonstrate that P-AscH
radiosensitizes pancreatic cancer cells but inhibits radiation-induced damage to normal cells. Specifically, radiation-induced decreases in clonogenic survival and double-stranded DNA breaks in tumor cells, but not in normal cells, were enhanced by P-AscH
, while radiation-induced intestinal damage, collagen deposition, and oxidative stress were also reduced with P-AscH
in normal tissue. We also report on our first-in-human phase I trial that infused P-AscH
during the radiotherapy "beam on." Specifically, treatment with P-AscH
increased median overall survival compared with our institutional average (21.7 vs. 12.7 months,
= 0.08) and the E4201 trial (21.7 vs. 11.1 months). Progression-free survival in P-AscH
-treated subjects was also greater than our institutional average (13.7 vs. 4.6 months,
< 0.05) and the E4201 trial (6.0 months). Results indicated that P-AscH
in combination with gemcitabine and radiotherapy for locally advanced pancreatic adenocarcinoma is safe and well tolerated with suggestions of efficacy. Because of the potential effect size and minimal toxicity, our findings suggest that investigation of P-AscH
efficacy is warranted in a phase II clinical trial. SIGNIFICANCE: These findings demonstrate that pharmacologic ascorbate enhances pancreatic tumor cell radiation cytotoxicity in addition to offering potential protection from radiation damage in normal surrounding tissue, making it an optimal agent for improving treatment of locally advanced pancreatic adenocarcinoma.
Pharmacological ascorbate has been shown to induce toxicity in a wide range of cancer cell lines. Pharmacological ascorbate in animal models has shown promise for use in cancer treatment. At ...pharmacological concentrations the oxidation of ascorbate produces a high flux of H2O2 via the formation of ascorbate radical (Asc•-). The rate of oxidation of ascorbate is principally a function of the level of catalytically active metals. Iron in cell culture media contributes significantly to the rate of H2O2 generation. We hypothesized that increasing intracellular iron would enhance ascorbate-induced cytotoxicity and that iron chelators could modulate the catalytic efficiency with respect to ascorbate oxidation. Treatment of cells with the iron-chelators deferoxamine (DFO) or dipyridyl (DPD) in the presence of 2mM ascorbate decreased the flux of H2O2 generated by pharmacological ascorbate and reversed ascorbate-induced toxicity. Conversely, increasing the level of intracellular iron by preincubating cells with Fe-hydroxyquinoline (HQ) increased ascorbate toxicity and decreased clonogenic survival. These findings indicate that redox metal metals, e.g., Fe3+/Fe2+, have an important role in ascorbate-induced cytotoxicity. Approaches that increase catalytic iron could potentially enhance the cytotoxicity of pharmacological ascorbate in vivo.
Display omitted
•Catalytic metals are central in determining the rate of oxidation of ascorbate and production of H2O2.•Extracellular catalytic iron is vital to the cellular toxicity of pharmacological ascorbate.•Increasing intracellular labile iron enhances the toxicity of pharmacological ascorbate.
Pharmacological ascorbate has been proposed as a potential anti-cancer agent when combined with radiation and chemotherapy. The anti-cancer effects of ascorbate are hypothesized to involve the ...autoxidation of ascorbate leading to increased steady-state levels of H2O2; however, the mechanism(s) for cancer cell-selective toxicity remain unknown. The current study shows that alterations in cancer cell mitochondrial oxidative metabolism resulting in increased levels of O2⋅− and H2O2 are capable of disrupting intracellular iron metabolism, thereby selectively sensitizing non-small-cell lung cancer (NSCLC) and glioblastoma (GBM) cells to ascorbate through pro-oxidant chemistry involving redox-active labile iron and H2O2. In addition, preclinical studies and clinical trials demonstrate the feasibility, selective toxicity, tolerability, and potential efficacy of pharmacological ascorbate in GBM and NSCLC therapy.
Display omitted
•High-dose ascorbate sensitizes NSCLC and GBM cells to radio-chemotherapy•O2⋅− and H2O2 increase labile iron causing cancer cell-selective ascorbate toxicity•Therapeutic levels of ascorbate are achievable and well tolerated in GBM and NSCLC•Cancer cell oxidative metabolism can be targeted with ascorbate for cancer therapy
Schoenfeld et al. show that cancer cells are selectively sensitive to ascorbate due to their altered redox-active iron metabolism. They present preclinical and clinical data demonstrating the feasibility, tolerability, and potential efficacy of pharmacological ascorbate for treating glioblastoma and non-small cell lung cancer.
Pharmacologic concentrations of ascorbate may be effective in cancer therapeutics. We hypothesized that ascorbate concentrations achievable with i.v. dosing would be cytotoxic in pancreatic cancer ...for which the 5-year survival is <3%.
Pancreatic cancer cell lines were treated with ascorbate (0, 5, or 10 mmol/L) for 1 hour, then viability and clonogenic survival were determined. Pancreatic tumor cells were delivered s.c. into the flank region of nude mice and allowed to grow at which time they were randomized to receive either ascorbate (4 g/kg) or osmotically equivalent saline (1 mol/L) i.p. for 2 weeks.
There was a time- and dose-dependent increase in measured H(2)O(2) production with increased concentrations of ascorbate. Ascorbate decreased viability in all pancreatic cancer cell lines but had no effect on an immortalized pancreatic ductal epithelial cell line. Ascorbate decreased clonogenic survival of the pancreatic cancer cell lines, which was reversed by treatment of cells with scavengers of H(2)O(2). Treatment with ascorbate induced a caspase-independent cell death that was associated with autophagy. In vivo, treatment with ascorbate inhibited tumor growth and prolonged survival.
These results show that pharmacologic doses of ascorbate, easily achievable in humans, may have potential for therapy in pancreatic cancer.
The prognosis for patients diagnosed with pancreatic cancer remains dismal, with less than 3% survival at 5 years. Recent studies have demonstrated that high-dose, intravenous pharmacological ...ascorbate (ascorbic acid, vitamin C) induces cytotoxicity and oxidative stress selectively in pancreatic cancer cells vs. normal cells, suggesting a promising new role of ascorbate as a therapeutic agent. At physiologic concentrations, ascorbate functions as a reducing agent and antioxidant. However, when pharmacological ascorbate is given intravenously, it is possible to achieve millimolar plasma concentration. At these pharmacological levels, and in the presence of catalytic metal ions, ascorbate can induce oxidative stress through the generation of hydrogen peroxide (H2O2). Recent in vitro and in vivo studies have demonstrated ascorbate oxidation occurs extracellularly, generating H2O2 flux into cells resulting in oxidative stress. Pharmacologic ascorbate also inhibits the growth of pancreatic tumor xenografts and displays synergistic cytotoxic effects when combined with gemcitabine in pancreatic cancer. Phase I trials of pharmacological ascorbate in pancreatic cancer patients have demonstrated safety and potential efficacy. In this chapter, we will review the mechanism of ascorbate-induced cytotoxicity, examine the use of pharmacological ascorbate in treatment and assess the current data supporting its potential as an adjuvant in pancreatic cancer.
PDF
