Pharmacologic Ascorbate Radiosensitizes Pancreatic Cancer but Radioprotects Normal Tissue: The Role of Oxidative Stress-Induced Lipid Peroxidation.

The toxicity of ionizing radiation limits its effectiveness in the treatment of pancreatic ductal adenocarcinoma. Pharmacologic ascorbate (P-AscH-) has been shown to radiosensitize pancreatic cancer cells while simultaneously radioprotecting normal cells. We hypothesize that P-AscH- protects the small intestine while radiosensitizing pancreatic cancer cells partially through an oxidative stress mechanism. Duodenal samples from pancreaticoduodenectomy specimens of patients who underwent radio-chemotherapy ± P-AscH- and mouse tumor and jejunal samples treated with radiation ± P-AscH- were evaluated. Pancreatic cancer and non-tumorigenic cells were treated with radiation ± P-AscH- to assess lipid peroxidation. To determine the mechanism, pancreatic cancer cells were treated with selenomethionine or RSL3, an inhibitor of glutathione peroxidase 4 (GPx4). Radiation-induced decreases in villi length and increases in 4-HNE immunofluorescence were reversed with P-AscH- in human duodenum. In vivo, radiation-induced decreases in villi length and increased collagen deposition were reversed in P-AscH--treated jejunal samples. P-AscH- and radiation increased BODIPY oxidation in pancreatic cancer cells but not in non-tumorigenic cells. Selenomethionine increased GPx4 protein and activity in pancreatic cancer and reversed P-AscH--induced toxicity and lipid peroxidation. RSL3 treatment inhibited GPx4 activity and increased lipid peroxidation. Differences in oxidative stress may play a role in radioprotecting normal cells while radiosensitizing pancreatic cancer cells when treated with P-AscH-.

Magnetic Resonance Imaging of Iron Metabolism with T2* Mapping Predicts an Enhanced Clinical Response to Pharmacologic Ascorbate in Patients with GBM.

PURPOSE: Pharmacologic ascorbate (P-AscH-) is hypothesized to be an iron (Fe)-dependent tumor-specific adjuvant to chemoradiation in treating glioblastoma (GBM). This study determined the efficacy of combining P-AscH- with radiation and temozolomide in a phase II clinical trial while simultaneously investigating a mechanism-based, noninvasive biomarker in T2* mapping to predict GBM response to P-AscH- in humans. PATIENTS AND METHODS: The single-arm phase II clinical trial (NCT02344355) enrolled 55 subjects, with analysis performed 12 months following the completion of treatment. Overall survival (OS) and progression-free survival (PFS) were estimated with the Kaplan-Meier method and compared across patient subgroups with log-rank tests. Forty-nine of 55 subjects were evaluated using T2*-based MRI to assess its utility as an Fe-dependent biomarker. RESULTS: Median OS was estimated to be 19.6 months [90% confidence interval (CI), 15.7-26.5 months], a statistically significant increase compared with historic control patients (14.6 months). Subjects with initial T2* relaxation < 50 ms were associated with a significant increase in PFS compared with T2*-high subjects (11.2 months vs. 5.7 months, P < 0.05) and a trend toward increased OS (26.5 months vs. 17.5 months). These results were validated in preclinical in vitro and in vivo model systems. CONCLUSIONS: P-AscH- combined with temozolomide and radiotherapy has the potential to significantly enhance GBM survival. T2*-based MRI assessment of tumor iron content is a prognostic biomarker for GBM clinical outcomes. See related commentary by Nabavizadeh and Bagley, p. 255.

Hydrogen Peroxide Mediates Pharmacological Ascorbate Induced Radio-Sensitization of Pancreatic Cancer Cells by Enhancing G2-accumulation and Reducing Cyclin B1 Protein Levels.

Pharmacological ascorbate (P-AscH-, high dose, intravenous vitamin C) preferentially sensitizes human pancreas ductal adenocarcinoma (PDAC) cells to radiation-induced toxicity compared to non-tumorigenic epithelial cells. Radiation-induced G2-checkpoint activation contributes to the resistance of cancer cells to DNA damage induced toxicity. We hypothesized that P-AscH- induced radio-sensitization of PDAC cells is mediated by perturbations in the radiation induced activation of the G2-checkpoint pathway. Both non-tumorigenic pancreatic ductal epithelial and PDAC cells display decreased clonogenic survival and increased doubling times after radiation treatment. In contrast, the addition of P-AscH- to radiation increases clonogenic survival and decreases the doubling time of non-tumorigenic epithelial cells but decreasing clonogenic survival and increasing the doubling time of PDAC cells. Results from the mitotic index and propidium iodide assays showed that while the P-AscH- treatments did not affect radiation-induced G2-checkpoint activation, it enhanced G2-accumulation. The addition of catalase reverses the increases in G2-accumulation, indicating a peroxide-mediated mechanism. In addition, P-AscH- treatment of PDAC cells suppresses radiation-induced accumulation of cyclin B1 protein levels. Both translational and post-translational pathways appear to regulate cyclin B1 protein levels after the combination treatment of PDAC cells with P-AscH- and radiation. The protein changes seen are reversed by the addition of catalase suggesting that hydrogen peroxide mediates P-AscH- induced radiation sensitization of PDAC cells by enhancing G2-accumulation and reducing cyclin B1 protein levels.

Pharmacologic Ascorbate and DNMT Inhibitors Increase DUOX Expression and Peroxide-Mediated Toxicity in Pancreatic Cancer.

Recent studies have demonstrated an important role for vitamin C in the epigenetic regulation of cancer-related genes via DNA demethylation by the ten-eleven translocation (TET) methylcytosine dioxygenase enzymes. DNA methyltransferase (DNMT) reverses this, increasing DNA methylation and decreasing gene expression. Dual oxidase (DUOX) enzymes produce hydrogen peroxide (H2O2) in normal pancreatic tissue but are silenced in pancreatic cancer (PDAC). Treatment of PDAC with pharmacologic ascorbate (P-AscH-, intravenous, high dose vitamin C) increases DUOX expression. We hypothesized that inhibiting DNMT may act synergistically with P-AscH- to further increase DUOX expression and cytotoxicity of PDAC. PDAC cells demonstrated dose-dependent increases in DUOX mRNA and protein expression when treated with DNMT inhibitors. PDAC cells treated with P-AscH- + DNMT inhibitors demonstrated increased DUOX expression, increased intracellular oxidation, and increased cytotoxicity in vitro and in vivo compared to either treatment alone. These findings suggest a potential therapeutic, epigenetic mechanism to treat PDAC.

Pharmacological ascorbate induces sustained mitochondrial dysfunction.

Pharmacological ascorbate (P-AscH-; high dose given intravenously) generates H2O2 that is selectively cytotoxic to cancer compared to normal cells. The RAS-RAF-ERK1/2 is a major signaling pathway in cancers carrying RAS mutations and is known to be activated by H2O2. Activated ERK1/2 also phosphorylates the GTPase dynamin-related protein (Drp1), which then stimulates mitochondrial fission. Although early generation of H2O2 leads to cytotoxicity of cancer cells, we hypothesized that sustained increases in H2O2 activate ERK-Drp1 signaling, leading to an adaptive response; inhibition of this pathway would enhance the toxicity of P-AscH-. Increases in phosphorylated ERK and Drp1 induced by P-AscH- were reversed with genetic and pharmacological inhibitors of ERK and Drp1, as well as in cells lacking functional mitochondria. P-AscH- increased Drp1 colocalization to mitochondria, decreased mitochondrial volume, increased disconnected components, and decreased mitochondrial length, suggesting an increase in mitochondrial fission 48 h after treatment with P-AscH-. P-AscH- decreased clonogenic survival; this was enhanced by genetic and pharmacological inhibition of both ERK and Drp1. In murine tumor xenografts, the combination of P-AscH- and pharmacological inhibition of Drp1 increased overall survival. These results suggest that P-AscH- induces sustained changes in mitochondria, through activation of the ERK/Drp1 signaling pathway, an adaptive response. Inhibition of this pathway enhanced the toxicity P-AscH- to cancer cells.

The role of mitochondria in pharmacological ascorbate-induced toxicity.

At pharmacological levels, ascorbate (P-AscH-) acts as a pro-oxidant by generating H2O2, depleting ATP in sensitive cells leading to cell death. The aim of this study was to determine the role of ATP production by oxidative phosphorylation or glycolysis in mechanisms of resistance to P-AscH-induced cell death. Pancreatic cancer cells were used to generate ρ0 cells by mitochondrial overexpression of the Y147A mutant uracil-N-glycosylase or Herpes Simplex Virus protein. The ρ0 phenotype was confirmed by probing for mitochondrial DNA, mitochondrial DNA-encoded cytochrome c oxidase subunit 2, and monitoring the rate of oxygen consumption. In ρ0 cells, glycolysis accounted for 100% of ATP production as there was no mitochondrial oxygen consumption. Even though the activities of H2O2-removing antioxidant enzymes were similar in both the parental and ρ0 clones, P-AscH- -induced clonogenic cell death in ρ0 cells showed more resistance than the parental cell line. In addition, P-AscH- induced more DNA damage and more consumption of NAD+ and greater decreases in the production of ATP in the parental cell line compared to the ρ0 cells. Thus, cancer cells that largely use oxidative phosphorylation to generate ATP may be more sensitive to P-AscH- compared with cells that are glycolysis-dependent.

Pharmacological Ascorbate Enhances Chemotherapies in Pancreatic Ductal Adenocarcinoma.

OBJECTIVES: Pharmacological ascorbate (P-AscH - , high-dose, intravenous vitamin C) has shown promise as an adjuvant therapy for pancreatic ductal adenocarcinoma (PDAC) treatment. The objective of this study was to determine the effects of P-AscH - when combined with PDAC chemotherapies. METHODS: Clonogenic survival, combination indices, and DNA damage were determined in human PDAC cell lines treated with P-AscH - in combination with 5-fluorouracil, paclitaxel, or FOLFIRINOX (combination of leucovorin, 5-fluorouracil, irinotecan, oxaliplatin). Tumor volume changes, overall survival, blood analysis, and plasma ascorbate concentration were determined in vivo in mice treated with P-AscH - with or without FOLFIRINOX. RESULTS: P-AscH - combined with 5-fluorouracil, paclitaxel, or FOLFIRINOX significantly reduced clonogenic survival in vitro. The DNA damage, measured by γH2AX protein expression, was increased after treatment with P-AscH - , FOLFIRINOX, and their combination. In vivo, tumor growth rate was significantly reduced by P-AscH - , FOLFIRINOX, and their combination. Overall survival was significantly increased by the combination of P-AscH - and FOLFIRINOX. Treatment with P-AscH - increased red blood cell and hemoglobin values but had no effect on white blood cell counts. Plasma ascorbate concentrations were significantly elevated in mice treated with P-AscH - with or without FOLFIRINOX. CONCLUSIONS: The addition of P-AscH - to standard of care chemotherapy has the potential to be an effective adjuvant for PDAC treatment.

Pharmacological ascorbate as a novel therapeutic strategy to enhance cancer immunotherapy.

Pharmacological ascorbate (i.e., intravenous infusions of vitamin C reaching ~ 20 mM in plasma) is under active investigation as an adjuvant to standard of care anti-cancer treatments due to its dual redox roles as an antioxidant in normal tissues and as a prooxidant in malignant tissues. Immune checkpoint inhibitors (ICIs) are highly promising therapies for many cancer patients but face several challenges including low response rates, primary or acquired resistance, and toxicity. Ascorbate modulates both innate and adaptive immune functions and plays a key role in maintaining the balance between pro and anti-inflammatory states. Furthermore, the success of pharmacological ascorbate as a radiosensitizer and a chemosensitizer in pre-clinical studies and early phase clinical trials suggests that it may also enhance the efficacy and expand the benefits of ICIs.

Auranofin and Pharmacologic Ascorbate as Radiomodulators in the Treatment of Pancreatic Cancer.

Pancreatic cancer accounts for nearly one fourth of all new cancers worldwide. Little progress in the development of novel or adjuvant therapies has been made over the past few decades and new approaches to the treatment of pancreatic cancer are desperately needed. Pharmacologic ascorbate (P-AscH-, high-dose, intravenous vitamin C) is being investigated in clinical trials as an adjunct to standard-of-care chemoradiation treatments. In vitro, P-AscH- has been shown to sensitize cancer cells to ionizing radiation in a manner that is dependent on the generation of H2O2 while simultaneously protecting normal tissue from radiation damage. There is renewed interest in Auranofin (Au), an FDA-approved medication utilized in the treatment of rheumatoid arthritis, as an anti-cancer agent. Au inhibits the thioredoxin antioxidant system, thus increasing the overall peroxide burden on cancer cells. In support of current literature demonstrating Au's effectiveness in breast, colon, lung, and ovarian cancer, we offer additional data that demonstrate the effectiveness of Au alone and in combination with P-AscH- and ionizing radiation in pancreatic cancer treatment. Combining P-AscH- and Au in the treatment of pancreatic cancer may confer multiple mechanisms to increase H2O2-dependent toxicity amongst cancer cells and provide a promising translatable avenue by which to enhance radiation effectiveness and improve patient outcomes.

Pharmacological ascorbate improves the response to platinum-based chemotherapy in advanced stage non-small cell lung cancer.

PURPOSE: Platinum-based chemotherapy with or without immunotherapy is the mainstay of treatment for advanced stage non-small cell lung cancer (NSCLC) lacking a molecular driver alteration. Pre-clinical studies have reported that pharmacological ascorbate (P-AscH-) enhances NSCLC response to platinum-based therapy. We conducted a phase II clinical trial combining P-AscH- with carboplatin-paclitaxel chemotherapy. EXPERIMENTAL DESIGN: Chemotherapy naïve advanced stage NSCLC patients received 75 g ascorbate twice per week intravenously with carboplatin and paclitaxel every three weeks for four cycles. The primary endpoint was to improve tumor response per Response Evaluation Criteria in Solid Tumors (RECIST) v1.1 compared to the historical control of 20%. The trial was conducted as an optimal Simon's two-stage design. Blood samples were collected for exploratory analyses. RESULTS: The study enrolled 38 patients and met its primary endpoint with an objective response rate of 34.2% (p = 0.03). All were confirmed partial responses (cPR). The disease control rate was 84.2% (stable disease + cPR). Median progression-free and overall survival were 5.7 months and 12.8 months, respectively. Treatment-related adverse events (TRAE) included one grade 5 (neutropenic fever) and five grade 4 events (cytopenias). Cytokine and chemokine data suggest that the combination elicits an immune response. Immunophenotyping of peripheral blood mononuclear cells demonstrated an increase in effector CD8 T-cells in patients with a progression-free survival (PFS) ≥ 6 months. CONCLUSIONS: The addition of P-AscH- to platinum-based chemotherapy improved tumor response in advanced stage NSCLC. P-AscH- appears to alter the host immune response and needs further investigation as a potential adjuvant to immunotherapy.

Enhancing the Biopsychosocial Approach to Perioperative Care: A Pilot Randomized Trial of the Perioperative Pain Self-management (PePS) Intervention.

OBJECTIVE: The current study aimed to pilot the PePS intervention, based on principles of cognitive behavioral therapy (CBT), to determine feasibility and preliminary efficacy for preventing chronic pain and long-term opioid use. SUMMARY BACKGROUND DATA: Surgery can precipitate the development of both chronic pain and long-term opioid use. CBT can reduce distress and improve functioning among patients with chronic pain. Adapting CBT to target acute pain management in the postoperative period may impact longer-term postoperative outcomes. METHODS: This was a mixed-methods randomized controlled trial in a mixed surgical sample with assignment to standard care or PePS, with primary outcomes at 3-months postsurgery. The sample consisted of rural-dwelling United States Military Veterans. RESULTS: Logistic regression analyses found a significant effect of PePS on odds of moderate-severe pain (on average over the last week) at 3-months postsurgery, controlling for preoperative moderate-severe pain: Adjusted odds ratio = 0.25 (95% CI: 0.07-0.95, P < 0.05). At 3-months postsurgery, 15% (6/39) of standard care participants and 2% (1/45) of PePS participants used opioids in the prior seven days: Adjusted Odds ratio = 0.10 (95% CI: 0.01-1.29, P = .08). Changes in depression, anxiety, and pain catastrophizing were not significantly different between arms. CONCLUSIONS: The findings from this study support the feasibility and preliminary efficacy of the PePS intervention.

Utilization of Pharmacological Ascorbate to Enhance Hydrogen Peroxide-Mediated Radiosensitivity in Cancer Therapy.

Interest in the use of pharmacological ascorbate as a treatment for cancer has increased considerably since it was introduced by Cameron and Pauling in the 1970s. Recently, pharmacological ascorbate has been used in preclinical and early-phase clinical trials as a selective radiation sensitizer in cancer. The results of these studies are promising. This review summarizes data on pharmacological ascorbate (1) as a safe and efficacious adjuvant to cancer therapy; (2) as a selective radiosensitizer of cancer via a mechanism involving hydrogen peroxide; and (3) as a radioprotector in normal tissues. Additionally, we present new data demonstrating the ability of pharmacological ascorbate to enhance radiation-induced DNA damage in glioblastoma cells, facilitating cancer cell death. We propose that pharmacological ascorbate may be a general radiosensitizer in cancer therapy and simultaneously a radioprotector of normal tissue.

Impact of EcSOD Perturbations in Cancer Progression.

Reactive oxygen species (ROS) are a normal byproduct of cellular metabolism and are required components in cell signaling and immune responses. However, an imbalance of ROS can lead to oxidative stress in various pathological states. Increases in oxidative stress are one of the hallmarks in cancer cells, which display an altered metabolism when compared to corresponding normal cells. Extracellular superoxide dismutase (EcSOD) is an antioxidant enzyme that catalyzes the dismutation of superoxide anion (O2-) in the extracellular environment. By doing so, this enzyme provides the cell with a defense against oxidative damage by contributing to redox balance. Interestingly, EcSOD expression has been found to be decreased in a variety of cancers, and this loss of expression may contribute to the development and progression of malignancies. In addition, recent compounds can increase EcSOD activity and expression, which has the potential for altering this redox signaling and cellular proliferation. This review will explore the role that EcSOD expression plays in cancer in order to better understand its potential as a tool for the detection, predicted outcomes and potential treatment of malignancies.

Catalase Modulates the Radio-Sensitization of Pancreatic Cancer Cells by Pharmacological Ascorbate.

Pancreatic cancer cells (PDACs) are more susceptible to an oxidative insult than normal cells, resulting in greater cytotoxicity upon exposure to agents that increase pro-oxidant levels. Pharmacological ascorbate (P-AscH-), i.e., large amounts given intravenously (IV), generates significant fluxes of hydrogen peroxide (H2O2), resulting in the killing of PDACs but not normal cells. Recent studies have demonstrated that P-AscH- radio-sensitizes PDAC but is a radioprotector to normal cells and tissues. Several mechanisms have been hypothesized to explain the dual roles of P-AscH- in radiation-induced toxicity including the activation of nuclear factor-erythroid 2-related factor 2 (Nrf2), RelB, as well as changes in bioenergetic profiles. We have found that P-AscH- in conjunction with radiation increases Nrf2 in both cancer cells and normal cells. Although P-AscH- with radiation decreases RelB in cancer cells vs. normal cells, the knockout of RelB does not radio-sensitize PDACs. Cellular bioenergetic profiles demonstrate that P-AscH- with radiation increases the ATP demand/production rate (glycolytic and oxidative phosphorylation) in both PDACs and normal cells. Knocking out catalase results in P-AscH- radio-sensitization in PDACs. In a phase I trial where P-AscH- was included as an adjuvant to the standard of care, short-term survivors had higher catalase levels in tumor tissue, compared to long-term survivors. These data suggest that P-AscH- radio-sensitizes PDACs through increased peroxide flux. Catalase levels could be a possible indicator for how tumors will respond to P-AscH-.

Magnetic resonance imaging (MRI) of pharmacological ascorbate-induced iron redox state as a biomarker in subjects undergoing radio-chemotherapy.

Pharmacological ascorbate (P-AscH-) combined with standard of care (SOC) radiation and temozolomide is being evaluated in a phase 2 clinical trial (NCT02344355) in the treatment of glioblastoma (GBM). Previously published data demonstrated that paramagnetic iron (Fe3+) catalyzes ascorbate's oxidation to form diamagnetic iron (Fe2+). Because paramagnetic Fe3+ may influence relaxation times observed in MR imaging, quantitative MR imaging of P-AscH--induced changes in redox-active Fe was assessed as a biomarker for therapy response. Gel phantoms containing either Fe3+ or Fe2+ were imaged with T2* and quantitative susceptibility mapping (QSM). Fifteen subjects receiving P-AscH- plus SOC underwent T2* and QSM imaging four weeks into treatment. Subjects were scanned: pre-P-AscH- infusion, post-P-AscH- infusion, and post-radiation (3-4 h between scans). Changes in T2* and QSM relaxation times in tumor and normal tissue were calculated and compared to changes in Fe3+ and Fe2+ gel phantoms. A GBM mouse model was used to study the relationship between the imaging findings and the labile iron pool. Phantoms containing Fe3+ demonstrated detectable changes in T2* and QSM relaxation times relative to Fe2+ phantoms. Compared to pre-P-AscH-, GBM T2* and QSM imaging were significantly changed post-P-AscH- infusion consistent with conversion of Fe3+ to Fe2+. No significant changes in T2* or QSM were observed in normal brain tissue. There was moderate concordance between T2* and QSM changes in both progression free survival and overall survival. The GBM mouse model showed similar results with P-AscH- inducing greater changes in tumor labile iron pools compared to the normal tissue. CONCLUSIONS: T2* and QSM MR-imaging responses are consistent with P-AscH- reducing Fe3+ to Fe2+, selectively in GBM tumor volumes and represent a potential biomarker of response. This study is the first application using MR imaging in humans to measure P-AscH--induced changes in redox-active iron.

Pharmacological ascorbate inhibits pancreatic cancer metastases via a peroxide-mediated mechanism.

Pharmacological ascorbate (P-AscH-, high-dose, intravenous vitamin C) is cytotoxic to tumor cells in doses achievable in humans. Phase I studies in pancreatic cancer (PDAC) utilizing P-AscH- have demonstrated increases in progression free survival, suggesting a reduction in metastatic disease burden. The purpose of this study was to determine the effects of P-AscH- on metastatic PDAC. Several in vitro and in vivo mechanisms involved in PDAC metastases were investigated following treatment with P-AscH-. Serum from PDAC patients in clinical trials with P-AscH- were tested for the presence and quantity of circulating tumor cell-derived nucleases. P-AscH- inhibited invasion, basement membrane degradation, decreased matrix metalloproteinase expression, as well as clonogenic survival and viability during exposure to fluid shear stress. In vivo, P-AscH- significantly decreased formation of ascites, tumor burden over time, circulating tumor cells, and hepatic metastases. Both in vitro and in vivo findings were reversed with the addition of catalase suggesting that the effect of P-AscH- on metastatic disease is mediated by hydrogen peroxide. Finally, P-AscH- decreased CTC-derived nucleases in subjects with stage IV PDAC in a phase I clinical trial. We conclude that P-AscH- attenuates the metastatic potential of PDAC and may prove to be effective for treating advanced disease.

Arachidonate 12-lipoxygenase and 12-hydroxyeicosatetraenoic acid contribute to stromal aging-induced progression of pancreatic cancer.

The incidence of pancreatic cancer increases with age, suggesting that chronological aging is a significant risk factor for this disease. Fibroblasts are the major nonmalignant cell type in the stroma of human pancreatic ductal adenocarcinoma (PDAC). In this study, we investigated whether the chronological aging of normal human fibroblasts (NHFs), a previously underappreciated area in pancreatic cancer research, influences the progression and therapeutic outcomes of PDAC. Results from experiments with murine xenografts and 2D and 3D co-cultures of NHFs and PDAC cells revealed that older NHFs stimulate proliferation of and confer resistance to radiation therapy of PDAC. MS-based metabolite analysis indicated that older NHFs have significantly increased arachidonic acid 12-lipoxygenase (ALOX12) expression and elevated levels of its mitogenic metabolite, 12-(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12-(S)-HETE) compared with their younger counterparts. In co-cultures with older rather than with younger NHFs, PDAC cells exhibited increases in mitogen-activated protein kinase signaling and cellular metabolism, as well as a lower oxidation state that correlated with their enhanced proliferation and resistance to radiation therapy. Expression of ALOX12 was found to be significantly lower in PDAC cell lines and tumor biopsies, suggesting that PDAC cells rely on a stromal supply of mitogens for their proliferative needs. Pharmacological (hydroxytyrosol) and molecular (siRNA) interventions of ALOX12 in older NHFs suppressed their ability to stimulate proliferation of PDAC cells. We conclude that chronological aging of NHFs contributes to PDAC progression and that ALOX12 and 12-(S)-HETE may be potential stromal targets for interventions that seek to halt progression and improve therapy outcomes.

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.