Dual Oxidase-Induced Sustained Generation of Hydrogen Peroxide Contributes to Pharmacologic Ascorbate-Induced Cytotoxicity.

Pharmacologic ascorbate treatment (P-AscH-, high-dose, intravenous vitamin C) results in a transient short-term increase in the flux of hydrogen peroxide that is preferentially cytotoxic to cancer cells versus normal cells. This study examines whether an increase in hydrogen peroxide is sustained posttreatment and potential mechanisms involved in this process. Cellular bioenergetic profiling following treatment with P-AscH- was examined in tumorigenic and nontumorigenic cells. P-AscH- resulted in sustained increases in the rate of cellular oxygen consumption (OCR) and reactive oxygen species (ROS) in tumor cells, with no changes in nontumorigenic cells. Sources for this increase in ROS and OCR were DUOX 1 and 2, which are silenced in pancreatic ductal adenocarcinoma, but upregulated with P-AscH- treatment. An inducible catalase system, to test causality for the role of hydrogen peroxide, reversed the P-AscH--induced increases in DUOX, whereas DUOX inhibition partially rescued P-AscH--induced toxicity. In addition, DUOX was significantly downregulated in pancreatic cancer specimens compared with normal pancreas tissues. Together, these results suggest that P-AscH--induced toxicity may be enhanced by late metabolic shifts in tumor cells, resulting in a feed-forward mechanism for generation of hydrogen peroxide and induction of metabolic stress through enhanced DUOX expression and rate of oxygen consumption. SIGNIFICANCE: A high dose of vitamin C, in addition to delivering an acute exposure of H2O2 to tumor cells, activates DUOX in pancreatic cancer cells, which provide sustained production of H2O2.

Pharmacologic Ascorbate Primes Pancreatic Cancer Cells for Death by Rewiring Cellular Energetics and Inducing DNA Damage.

The clinical potential of pharmacologic ascorbate (P-AscH-; intravenous delivery achieving mmol/L concentrations in blood) as an adjuvant in cancer therapy is being reevaluated. At mmol/L concentrations, P-AscH- is thought to exhibit anticancer activity via generation of a flux of H2O2 in tumors, which leads to oxidative distress. Here, we use cell culture models of pancreatic cancer to examine the effects of P-AscH- on DNA damage, and downstream consequences, including changes in bioenergetics. We have found that the high flux of H2O2 produced by P-AscH- induces DNA damage. In response to this DNA damage, we observed that PARP1 is hyperactivated. Using our unique absolute quantitation, we found that P-AscH- mediated the overactivation of PARP1, which results in consumption of NAD+, and subsequently depletion of ATP leading to mitotic cell death. We have also found that Chk1 plays a major role in the maintenance of genomic integrity following treatment with P-AscH-. Hyperactivation of PARP1 and DNA repair are ATP-consuming processes. Using a Seahorse XF96 analyzer, we demonstrated that the severe decrease in ATP after challenging with P-AscH- is because of increased demand, not changes in the rate of production. Genetic deletion and pharmacologic inhibition of PARP1 preserved both NAD+ and ATP; however, the toxicity of P-AscH- remained. These data indicate that disruption of bioenergetics is a secondary factor in the toxicity of P-AscH-; damage to DNA appears to be the primary factor. IMPLICATIONS: Efforts to leverage P-AscH- in cancer therapy should first focus on DNA damage.

Enhanced Pharmacological Ascorbate Oxidation Radiosensitizes Pancreatic Cancer.

Pharmacologic ascorbate (P-AscH-) is emerging as a promising adjuvant for advanced pancreatic cancer. P-AscH- generates hydrogen peroxide (H2O2), leading to selective cancer cell cytotoxicity. Catalytic manganoporphyrins, such as MnT4MPyP, can increase the rate of oxidation of P-AscH-, thereby increasing the flux of H2O2, resulting in increased cytotoxicity. We hypothesized that a multimodal treatment approach, utilizing a combination of P-AscH-, ionizing radiation and MnT4MPyP, would result in significant flux of H2O2 and pancreatic cancer cytotoxicity. P-AscH- with MnT4MPyP increased the rate of oxidation of P-AscH- and produced radiosensitization in all pancreatic cancer cell lines tested. Three-dimensional (3D) cell cultures demonstrated resistance to P-AscH-, radiation or MnT4MPyP treatments alone; however, combined treatment with P-AscH- and MnT4MPyP resulted in the inhibition of tumor growth, particularly when also combined with radiation. In vivo experiments using a murine model demonstrated an increased rate of ascorbate oxidation when combinations of P-AscH- with MnT4MPyP were given, thus acting as a radiosensitizer. The translational potential was demonstrated by measuring increased ascorbate oxidation ex vivo, whereby MnT4MPyP was added exogenously to plasma samples from patients treated with P-AscH- and radiation. Combination treatment utilizing P-AscH-, manganoporphyrin and radiation results in significant cytotoxicity secondary to enhanced ascorbate oxidation and an increased flux of H2O2. This multimodal approach has the potential to be an effective treatment for pancreatic ductal adenocarcinoma.