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.

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.

Aberrant redox biology and epigenetic reprogramming: Co-conspirators across multiple human diseases.

An epigenetic landscape encompasses a series of dynamic interconnected mechanisms working together to fashion a diverse set of phenotypes from a singular genotype. The epigenetic plasticity observed in disease and development is facilitated by enzymes that create and remove covalent modifications to DNA and histones. Several important discoveries within the past decade have revealed that epigenetic control mechanisms are subject to redox regulation and mitochondrial-to-nuclear retrograde signaling. This has led to our current understanding that the writers and erasers of the epigenome are influenced by several levels of redox and metabolic control including the bioavailability of oxygen, nutrients, and metabolite co-factors necessary for optimal enzyme activity. Thus, these enzymes perceive a cell's redox state, metabolic status, and environmental signals to influence chromatin structure and accessibility to the transcriptional apparatus. Not only are the activities of epigenetic enzymes affected by cellular redox conditions, but also, in feedback loop fashion, genes encoding antioxidant enzymes as well as prooxidant enzymes can be altered in their expression patterns by epigenetic silencing mechanisms. The altered expression of the anti- and prooxidant genes can then contribute to the onset or progression of disease. Epigenetic regulation of gene expression by the confluence of redox biology and gene-environment interactions is an active area of research and our understanding of these links continues to evolve. Given the emergent importance of crosstalk between redox biology and epigenetic regulatory mechanisms, it is timely that this issue should explore the current state of knowledge on this topic and how changes in metabolism and redox flux can result in tectonic shifts of the epigenetic landscape.

The epigenetic and morphogenetic effects of molecular oxygen and its derived reactive species in development.

The development of multicellular organisms involves the unpacking of a complex genetic program. Extensive characterization of discrete developmental steps has revealed the genetic program is controlled by an epigenetic state. Shifting the epigenome is a group of epigenetic enzymes that modify DNA and proteins to regulate cell type specific gene expression. While the role of these modifications in development has been established, the input(s) responsible for electing changes in the epigenetic state remains unknown. Development is also associated with dynamic changes in cellular metabolism, redox, free radical production, and oxygen availability. It has previously been postulated that these changes are causal in development by affecting gene expression. This suggests that oxygen is a morphogenic compound that impacts the removal of epigenetic marks. Likewise, metabolism and reactive oxygen species influence redox signaling through iron and glutathione to limit the availability of key epigenetic cofactors such as α-ketoglutarate, ascorbate, NAD+ and S-adenosylmethionine. Given the close relationship between these cofactors and epigenetic marks it seems likely that the two are linked. Here we describe how changing these inputs might affect the epigenetic state during development to drive gene expression. Combined, these cofactors and reactive oxygen species constitute the epigenetic landscape guiding cells along differing developmental paths.

Succinate Accumulation Links Mitochondrial MnSOD Depletion to Aberrant Nuclear DNA Methylation and Altered Cell Fate.

Previous studies showed that human cell line HEK293 lacking mitochondrial superoxide dismutase (MnSOD) exhibited decreased succinate dehydrogenase (SDH) activity, and mice lacking MnSOD displayed significant reductions in SDH and aconitase activities. Since MnSOD has significant effects on SDH activity, and succinate is a key regulator of TET enzymes needed for proper differentiation, we hypothesized that SOD2 loss would lead to succinate accumulation, inhibition of TET activity, and impaired erythroid precursor differentiation. To test this hypothesis, we genetically disrupted the SOD2 gene using the CRISPR/Cas9 genetic strategy in a human erythroleukemia cell line (HEL 92.1.7) capable of induced differentiation toward an erythroid phenotype. Cells obtained in this manner displayed significant inhibition of SDH activity and ~10-fold increases in cellular succinate levels compared to their parent cell controls. Furthermore, SOD2 -/- cells exhibited significantly reduced TET enzyme activity concomitant with decreases in genomic 5-hmC and corresponding increases in 5-mC. Finally, when stimulated with δ-aminolevulonic acid (δ-ALA), SOD2 -/- HEL cells failed to properly differentiate toward an erythroid phenotype, likely due to failure to complete the necessary global DNA demethylation program required for erythroid maturation. Together, our findings support the model of an SDH/succinate/TET axis and a role for succinate as a retrograde signaling molecule of mitochondrial origin that significantly perturbs nuclear epigenetic reprogramming and introduce MnSOD as a governor of the SDH/succinate/TET axis.

Extracellular superoxide dismutase (SOD3) regulates oxidative stress at the vitreoretinal interface.

Oxidative stress is a pathogenic feature in vitreoretinal disease. However, the ability of the inner retina to manage metabolic waste and oxidative stress is unknown. Proteomic analysis of antioxidants in the human vitreous, the extracellular matrix opposing the inner retina, identified superoxide dismutase-3 (SOD3) that localized to a unique matrix structure in the vitreous base and cortex. To determine the role of SOD3, Sod3-/- mice underwent histological and clinical phenotyping. Although the eyes were structurally normal, at the vitreoretinal interface Sod3-/- mice demonstrated higher levels of 3-nitrotyrosine, a key marker of oxidative stress. Pattern electroretinography also showed physiological signaling abnormalities within the inner retina. Vitreous biopsies and epiretinal membranes collected from patients with diabetic vitreoretinopathy (DVR) and a mouse model of DVR showed significantly higher levels of nitrates and/or 3-nitrotyrosine oxidative stress biomarkers suggestive of SOD3 dysfunction. This study analyzes the molecular pathways that regulate oxidative stress in human vitreous substructures. The absence or dysregulation of the SOD3 antioxidant at the vitreous base and cortex results in increased oxidative stress and tissue damage to the inner retina, which may underlie DVR pathogenesis and other vitreoretinal diseases.

Limitations of oxygen delivery to cells in culture: An underappreciated problem in basic and translational research.

Molecular oxygen is one of the most important variables in modern cell culture systems. Fluctuations in its concentration can affect cell growth, differentiation, signaling, and free radical production. In order to maintain culture viability, experimental validity, and reproducibility, it is imperative that oxygen levels be consistently maintained within physiological "normoxic" limits. Use of the term normoxia, however, is not consistent among scientists who experiment in cell culture. It is typically used to describe the atmospheric conditions of a standard incubator, not the true microenvironment to which the cells are exposed. This error may lead to the situation where cells grown in a standard "normoxic" oxygen concentration may actually be experiencing a wide range of conditions ranging from hyperoxia to near-anoxic conditions at the cellular level. This apparent paradox is created by oxygen's sluggish rate of diffusion through aqueous medium, and the generally underappreciated effects that cell density, media volume, and barometric pressure can have on pericellular oxygen concentration in a cell culture system. This review aims to provide an overview of this phenomenon we have termed "consumptive oxygen depletion" (COD), and includes a basic review of the physics, potential consequences, and alternative culture methods currently available to help circumvent this largely unrecognized problem.

Mitochondrial Superoxide Increases Age-Associated Susceptibility of Human Dermal Fibroblasts to Radiation and Chemotherapy.

Elderly cancer patients treated with ionizing radiation (IR) or chemotherapy experience more frequent and greater normal tissue toxicity relative to younger patients. The current study demonstrates that exponentially growing fibroblasts from elderly (old) male donor subjects (70, 72, and 78 years) are significantly more sensitive to clonogenic killing mediated by platinum-based chemotherapy and IR (∼70%-80% killing) relative to young fibroblasts (5 months and 1 year; ∼10%-20% killing) and adult fibroblasts (20 years old; ∼10%-30% killing). Old fibroblasts also displayed significantly increased (2-4-fold) steady-state levels of O2•-, O2 consumption, and mitochondrial membrane potential as well as significantly decreased (40%-50%) electron transport chain (ETC) complex I, II, IV, V, and aconitase (70%) activities, decreased ATP levels, and significantly altered mitochondrial structure. Following adenoviral-mediated overexpression of SOD2 activity (5-7-fold), mitochondrial ETC activity and aconitase activity were restored, demonstrating a role for mitochondrial O2•- in these effects. Old fibroblasts also demonstrated elevated levels of endogenous DNA damage that were increased following treatment with IR and chemotherapy. Most importantly, treatment with the small-molecule, superoxide dismutase mimetic (GC4419; 0.25 µmol/L) significantly mitigated the increased sensitivity of old fibroblasts to IR and chemotherapy and partially restored mitochondrial function without affecting IR or chemotherapy-induced cancer cell killing. These results support the hypothesis that age-associated increased O2•- and resulting DNA damage mediate the increased susceptibility of old fibroblasts to IR and chemotherapy that can be mitigated by GC4419. Cancer Res; 77(18); 5054-67. ©2017 AACR.

Extracellular superoxide dismutase and its role in cancer.

Reactive oxygen species (ROS) are increasingly recognized as critical determinants of cellular signaling and a strict balance of ROS levels must be maintained to ensure proper cellular function and survival. Notably, ROS is increased in cancer cells. The superoxide dismutase family plays an essential physiological role in mitigating deleterious effects of ROS. Due to the compartmentalization of ROS signaling, EcSOD, the only superoxide dismutase in the extracellular space, has unique characteristics and functions in cellular signal transduction. In comparison to the other two intracellular SODs, EcSOD is a relatively new comer in terms of its tumor suppressive role in cancer and the mechanisms involved are less well understood. Nevertheless, the degree of differential expression of this extracellular antioxidant in cancer versus normal cells/tissues is more pronounced and prevalent than the other SODs. A significant association of low EcSOD expression with reduced cancer patient survival further suggests that loss of extracellular redox regulation promotes a conducive microenvironment that favors cancer progression. The vast array of mechanisms reported in mediating deregulation of EcSOD expression, function, and cellular distribution also supports that loss of this extracellular antioxidant provides a selective advantage to cancer cells. Moreover, overexpression of EcSOD inhibits tumor growth and metastasis, indicating a role as a tumor suppressor. This review focuses on the current understanding of the mechanisms of deregulation and tumor suppressive function of EcSOD in cancer.

Allele-specific interaction between glutathione peroxidase 1 and manganese superoxide dismutase affects the levels of Bcl-2, Sirt3 and E-cadherin.

Manganese superoxide dismutase (MnSOD) is a mitochondrial-resident enzyme that reduces superoxide to hydrogen peroxide (H2O2), which can be further reduced to water by glutathione peroxidase (GPX1). Data from human studies have indicated that common polymorphisms in both of these proteins are associated with the risk of several cancers, including breast cancer. Moreover, polymorphisms in MnSOD and GPX1 were shown to interact to increase the risk of breast cancer. To gain an understanding of the molecular mechanisms behind these observations, we engineered human MCF-7 breast cancer cells to exclusively express GPX1 and/or MnSOD alleles and investigated the consequences on the expression of several proteins associated with cancer aetiology. Little or no effect was observed on the ectopic expression of these genes on the phosphorylation of Akt, although allele-specific effects and interactions were observed for the impact on the levels of Bcl-2, E-cadherin and Sirt3. The patterns observed were not consistent with the steady-state levels of H2O2 determined in the transfected cells. These results indicate plausible contributing factors to the effects of allelic variations on cancer risk observed in human epidemiological studies.

O2⋠- and H2O2-Mediated Disruption of Fe Metabolism Causes the Differential Susceptibility of NSCLC and GBM Cancer Cells to 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.

Extracellular superoxide dismutase inhibits hepatocyte growth factor-mediated breast cancer-fibroblast interactions.

We have previously shown tumor suppressive effects of extracellular superoxide dismutase, EcSOD in breast cancer cells. In this study, an RTK signaling array revealed an inhibitory effect of EcSOD on c-Met phosphorylation and its downstream kinase c-Abl in MDA-MB231 cells. Moreover, an extracellular protein array showed that thrombospondin 1 (TSP-1), a scavenger of the c-Met ligand, hepatocyte growth factor (HGF) is significantly up-regulated in EcSOD overexpressing cells (Ec.20). We further determined the effects of EcSOD on HGF/c-Met-mediated cancer-fibroblast interactions by co-culturing normal fibroblasts (RMF) or RMF which overexpresses HGF (RMF-HGF) with MDA-MB231 cells. We observed that while RMF-HGF significantly promoted Matrigel growth of MDA-MB231, overexpression of EcSOD inhibited the HGF-stimulated growth. Similarly, a SOD mimetic, MnTE-2-PyP, inhibited HGF-induced growth and invasion of MDA-MB231. In addition, a long-term heterotypic co-culture study not only showed that Ec.20 cells are resistant to RMF-HGF-induced invasive stimulation but RMF-HGF that were co-cultured with Ec.20 cells showed an attenuated phenotype, suggesting an oxidative-mediated reciprocal interaction between the two cell types. In addition, we demonstrated that RMF-HGF showed an up-regulation of an ROS-generating enzyme, NADPH oxidase 4 (Nox4). Targeting this pro-oxidant significantly suppressed the activated phenotype of RMF-HGF in a collagen contraction assay, suggesting that RMF-HGF contributes to the oxidative tumor microenvironment. We have further shown that scavenging ROS with EcSOD significantly inhibited RMF-HGF-stimulated orthotopic tumor growth of MDA-MB231. This study suggests the loss of EcSOD in breast cancer plays a pivotal role in promoting the HGF/c-Met-mediated cancer-fibroblast interactions.

Histone deacetylation contributes to low extracellular superoxide dismutase expression in human idiopathic pulmonary arterial hypertension.

Epigenetic mechanisms, including DNA methylation and histone acetylation, regulate gene expression in idiopathic pulmonary arterial hypertension (IPAH). These mechanisms can modulate expression of extracellular superoxide dismutase (SOD3 or EC-SOD), a key vascular antioxidant enzyme, and loss of vascular SOD3 worsens outcomes in animal models of pulmonary arterial hypertension. We hypothesized that SOD3 gene expression is decreased in patients with IPAH due to aberrant DNA methylation and/or histone deacetylation. We used lung tissue and pulmonary artery smooth muscle cells (PASMC) from subjects with IPAH at transplantation and from failed donors (FD). Lung SOD3 mRNA expression and activity was decreased in IPAH vs. FD. In contrast, mitochondrial SOD (Mn-SOD or SOD2) protein expression was unchanged and intracellular SOD activity was unchanged. Using bisulfite sequencing in genomic lung or PASMC DNA, we found the methylation status of the SOD3 promoter was similar between FD and IPAH. Furthermore, treatment with 5-aza-2'-deoxycytidine did not increase PASMC SOD3 mRNA, suggesting DNA methylation was not responsible for PASMC SOD3 expression. Though total histone deacetylase (HDAC) activity, histone acetyltransferase (HAT) activity, acetylated histones, and acetylated SP1 were similar between IPAH and FD, treatment with two selective class I HDAC inhibitors increased SOD3 only in IPAH PASMC. Class I HDAC3 siRNA also increased SOD3 expression. Trichostatin A, a pan-HDAC inhibitor, decreased proliferation in IPAH, but not in FD PASMC. These data indicate that histone deacetylation, specifically via class I HDAC3, decreases SOD3 expression in PASMC and HDAC inhibitors may protect IPAH in part by increasing PASMC SOD3 expression.

SSRI use and clinical outcomes in epithelial ovarian cancer.

Selective serotonin reuptake inhibitor (SSRI) use is common among ovarian cancer patients. We examined the effect of SSRIs on survival and progression in ovarian cancer patients and effects of 5-HT on ovarian cancer cell (OCC) proliferation. Ovarian cancer patients from a 6-site study between 1994 and 2010 were included. Cox proportional hazards models were used for multivariate analysis. SSRI use was associated with decreased time to disease recurrence (HR 1.3, CI 1.0-1.6, p=0.03), but not overall survival (HR 1.1, CI 0.9-1.3, p=0.56). Compared to normal ovarian cells, most OCCs had elevated 5-HT2A receptor mRNA expression (up to 1600 fold greater expression). Clonogenic survival increased in cells treated with 10 uM (1.6 fold, p<0.001) and 20uM (1.9 fold, p=0.018) 5-HT. Mice receiving 5-HT injections had increases in tumor weight (p=0.07) and nodules (p=0.08) with increased Ki67 expression. Injections with sertraline doubled mean tumor weight in mice (p=0.16). 5-HT and sertraline both increased Ki67 expression in mouse tumors (p < 0.001).Patients using SSRIs had significantly decreased time to disease progression. It is possible that SSRIs alter serotonin levels in the tumor microenvironment, resulting in activation of proliferation pathways. Further characterization of serotonergic pathways in ovarian cancer is recommended to demonstrate safety of these medications.

Succinate dehydrogenase activity regulates PCB3-quinone-induced metabolic oxidative stress and toxicity in HaCaT human keratinocytes.

Polychlorinated biphenyls (PCBs) and their metabolites are environmental pollutants that are known to have adverse health effects. 1-(4-Chlorophenyl)-benzo-2,5-quinone (4-ClBQ), a quinone metabolite of 4-monochlorobiphenyl (PCB3, present in the environment and human blood) is toxic to human skin keratinocytes, and breast and prostate epithelial cells. This study investigates the hypothesis that 4-ClBQ-induced metabolic oxidative stress regulates toxicity in human keratinocytes. Results from Seahorse XF96 Analyzer showed that the 4-ClBQ treatment increased extracellular acidification rate, proton production rate, oxygen consumption rate and ATP content, indicative of metabolic oxidative stress. Results from a q-RT-PCR assay showed significant increases in the mRNA levels of hexokinase 2 (hk2), pyruvate kinase M2 (pkm2) and glucose-6-phosphate dehydrogenase (g6pd), and decreases in the mRNA levels of succinate dehydrogenase (complex II) subunit C and D (sdhc and sdhd). Pharmacological inhibition of G6PD-activity enhanced the toxicity of 4-ClBQ, suggesting that the protective function of the pentose phosphate pathway is functional in 4-ClBQ-treated cells. The decrease in sdhc and sdhd expression was associated with a significant decrease in complex II activity and increase in mitochondrial levels of ROS. Overexpression of sdhc and sdhd suppressed 4-ClBQ-induced inhibition of complex II activity, increase in mitochondrial levels of ROS, and toxicity. These results suggest that the 4-ClBQ treatment induces metabolic oxidative stress in HaCaT cells, and while the protective function of the pentose phosphate pathway is active, inhibition of complex II activity sensitizes HaCaT cells to 4-ClBQ-induced toxicity.

SOD2 targeted gene editing by CRISPR/Cas9 yields Human cells devoid of MnSOD.

To date no models exist to study MnSOD deficiency in human cells. To address this deficiency, we created a SOD2-null human cell line that is completely devoid of detectable MnSOD protein expression and enzyme activity. We utilized the CRISPR/Cas9 system to generate biallelic SOD2 disruption in HEK293T cells. These SOD2-null cells exhibit impaired clonogenic activity, which was rescued by either treatment with GC4419, a pharmacological small-molecule mimic of SOD, or growth in hypoxia. The phenotype of these cells is primarily characterized by impaired mitochondrial bioenergetics. The SOD2-null cells displayed perturbations in their mitochondrial ultrastructure and preferred glycolysis as opposed to oxidative phosphorylation to generate ATP. The activities of mitochondrial complex I and II were both significantly impaired by the absence of MnSOD activity, presumably from disruption of the Fe/S centers in NADH dehydrogenase and succinate dehydrogenase subunit B by the aberrant redox state in the mitochondrial matrix of SOD2-null cells. By creating this model we provide a novel tool with which to study the consequences of lack of MnSOD activity in human cells.

Disruption of thioredoxin metabolism enhances the toxicity of transforming growth factor ß-activated kinase 1 (TAK1) inhibition in KRAS-mutated colon cancer cells.

Transforming growth factor ß-activated kinase 1 (TAK1) is critical for survival of many KRAS mutated colorectal cancer cells, and TAK1 inhibition with 5Z-7-oxozeaenol has been associated with oxidative stress leading to tumor cell killing. When SW 620 and HCT 116 human colon cancer cells were treated with 5µM 5Z-7-oxozeaenol, cell viability, growth, and clonogenic survival were significantly decreased. Consistent with TAK1 inhibition being causally related to thiol-mediated oxidative stress, 10mM N-acetylcysteine (NAC) partially reversed the growth inhibitory effects of 5Z-7-oxozeaenol. In addition, 5Z-7-oxozeaenol also increased steady-state levels of H2DCFDA oxidation as well as increased levels of total glutathione (GSH) and glutathione disulfide (GSSG). Interestingly, depletion of GSH using buthionine sulfoximine did not significantly potentiate 5Z-7-oxozeaenol toxicity in either cell line. In contrast, pre-treatment of cells with auranofin (Au) to inhibit thioredoxin reductase activity significantly increased levels of oxidized thioredoxin as well as sensitized cells to 5Z-7-oxozeaenol-induced growth inhibition and clonogenic cell killing. These results were confirmed in SW 620 murine xenografts, where treatment with 5Z-7-oxozeaenol or with Au plus 5Z-7-oxozeaenol significantly inhibited growth, with Au plus 5Z-7-oxozeaenol trending toward greater growth inhibition compared to 5Z-7-oxozeaenol alone. These results support the hypothesis that thiol-mediated oxidative stress is causally related to TAK1-induced colon cancer cell killing. In addition, these results support the hypothesis that thioredoxin metabolism is a critical target for enhancing colon cancer cell killing via TAK1 inhibition and could represent an effective therapeutic strategy in patients with these highly resistant tumors.