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

ABSTRACT: 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.

SUBMITTER: Buranasudja V

PROVIDER: S-EPMC6774825 | biostudies-literature | 2019 Oct

REPOSITORIES: biostudies-literature

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

Buranasudja Visarut V Doskey Claire M CM Gibson Adrienne R AR Wagner Brett A BA Du Juan J Gordon David J DJ Koppenhafer Stacia L SL Cullen Joseph J JJ Buettner Garry R GR

Molecular cancer research : MCR 20190723 10

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 downs ...[more]

PMID: 31337671

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Project description:: 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 (H2O2)-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, P = 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, P < 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.

Project description:Rationale: Ascorbate is an essential micronutrient known for redox functions at normal physiologic concentrations. In recent decades, pharmacological ascorbate has been found to selectively kill tumour cells. However, the dosing frequency of pharmacologic ascorbate in humans has not yet been defined. Methods: We determined that among five hepatic cell lines, Huh-7 cells were the most sensitive to ascorbate. The effects of high-dose ascorbate on hepatoma were therefore assessed using Huh-7 cells and xenograft tumour mouse model. Results: In Huh-7 cells, ascorbate induced a significant increase in the percentage of cells in the G0/G1 phase, apoptosis and intracellular levels of ROS. High doses of ascorbate (4.0 pmol cell-1), but not low doses of ascorbate (1.0 pmol cell-1), also served as a pro-drug that killed hepatoma cells by altering mitochondrial respiration. Furthermore, in a Huh-7 cell xenograft tumour mouse model, intraperitoneal injection of ascorbate (4.0 g/kg/3 days) but not a lower dose of ascorbate (2.0 g/kg/3 days) significantly inhibited tumour growth. Gene array analysis of HCC tumour tissue from xenograft mice given IP ascorbate (4.0 g/kg/3 days) identified changes in the transcript levels of 192 genes/ncRNAs involved in insulin receptor signalling, metabolism and mitochondrial respiration. Consistent with the array data, gene expression levels of AGER, DGKK, ASB2, TCP10L2, Lnc-ALCAM-3, and Lnc-TGFBR2-1 were increased 2.05-11.35 fold in HCC tumour tissue samples from mice treated with high-dose ascorbate, and IHC staining analysis also verified that AGER/RAGE and DGKK proteins were up-regulated, which implied that AGER/RAGE and DGKK activation might be related to oxidative stress, leading to hepatoma cell death. Conclusions: Our studies identified multiple mechanisms are responsible for the anti-tumour activity of ascorbate and suggest high doses of ascorbate with less frequency will act as a novel therapeutic agent for liver cancer in vivo.

Dataset Information

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

Publications

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

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