Mitochondrial Manganese Superoxide Research Articles

Abstract 446: Estrogen and Estrogen Receptor Protect the Female Heart From Ischemia/reperfusion Injury by Increasing the Activity of Mitochondrial p38β and MnSOD

Background: We have previously demonstrated a novel in-vitro mechanism of how 17β-estradiol (E2) protects cultured neonatal cardiomyocytes from H/R by identifying a functionally active mitochondrial pool of p38β and E2-driven upregulation of manganese superoxide (MnSOD) activity via p38β. This protective mechanism leads to E2-mediated suppression of reactive oxygen species and attenuates apoptosis. However, little is known about these cytoprotective actions of E2 in the heart after myocardial I/R in vivo . The aim of this study is to determine whether the cardioprotective effects of E2 involve the interaction of mitochondrial p38β and MnSOD in the heart in vivo . Methods and Results: Left coronary ligation for 45 min and reperfusion for 24 hr was used to produce myocardial I/R injury in ovariectomized female mice with or without E2 pellet (0.1 mg/pellet). TTC staining showed that the infarct size was approximately 30% of the left ventricle at risk in untreated mice after I/R. The infarct size was reduced by more than half in the E2-treated group. Western blotting showed that the phosphorylation of p38β (p-p38β) in the heart homogenate was upregulated by E2, compared to the untreated mice, while the total p38β protein level was not altered. Mitochondrial p-p38β isolated from the hearts decreased after I/R, while E2 administration attenuated this effect. The total protein level of MnSOD decreased after I/R, regardless of E2 treatment. However, the MnSOD activity was normalized by E2 treatment, suggesting that E2 specifically increased the enzymatically active subset of MnSOD. Physical interaction between mitochondrial p38β and MnSOD was found by using co-immunoprecipitation from the hearts of the studied animals. Estrogen receptor (ER) α, β and double knockout female mice exhibited larger infarct size after I/R injury than that of WT mice while no significant difference was observed between the knockout mice. Loss of both ERs led to reduced activity of mitochondrial p38β and MnSOD at the baseline and after myocardial I/R. Conclusion: This work offers in-vivo evidence to our previously detailed mechanistic data that the protective effects of estrogen and ER against cardiac I/R injury involve the regulation of MnSOD activity via mitochondrial p38β.

Bidirectional Regulation of Manganese Superoxide Dismutase (MnSOD) on the Radiosensitivity of Esophageal Cancer Cells

The mitochondrial antioxidant protein manganese superoxide dismutase (MnSOD) may represent a new type of tumor suppressor protein. Overexpression of the cDNA of this gene by plasmid or recombinant lentiviral transfection in various types of cancer leads to growth suppression both in vitro and in vivo. We previously determined that changes in MnSOD expression had bidirectional effects on adriamycin (ADR) when combined with nitric oxide (NO). Radiation induces free radicals in a manner similar to ADR, so we speculated that MnSOD combined with NO would also have a bidirectional effect on cellular radiosensitivity. To examine this hypothesis, TE-1 human esophageal squamous carcinoma cells were stably transfected using lipofectamine with a pLenti6-DEST plasmid containing human MnSOD cDNA at moderate to high overexpression levels or with no MnSOD insert. Blastidicin-resistant colonies were isolated, grown, and maintained in culture. We found that moderate overexpression of MnSOD decreased growth rates, plating efficiency, and increased apoptosis. However, high overexpression increased growth rates, plating efficiency, and decreased apoptosis. When combined with NO, moderate overexpression of MnSOD increased the radiosensitivity of esophageal cancer cells, whereas high MnSOD overexpression had the opposite effect. This finding suggests a potential new method to kill certain radioresistant tumors and to provide radioresistance to normal cells.

Abstract LB-67: COX-2/PGE2: molecular ambassadors of Kaposi's sarcoma associated herpes virus (KSHV) oncoprotein-v-FLIP

Abstract Kaposi's sarcoma herpesvirus latent oncoprotein viral FLICE-like inhibitory protein (v-FLIP) or K13, a potent activator of NF-κB has well established roles in KSHV latency and oncogenesis. KSHV induced COX-2 represents a novel strategy employed by KSHV to promote latency and inflammation/angiogenesis/invasion. Here, we demonstrate that v-FLIP/K13 promotes tumorigenic effects via induction of host protein COX-2 and its inflammatory metabolite PGE2 in an NF-κB dependent manner. In addition to our previous studies demonstrating COX-2/PGE2's role in transcriptional regulation of KSHV latency promoter and latent gene expression, current study adds to the complexity that though LANA-1 (latency associated nuclear antigen) is utilizing COX-2/PGE2 as critical factors for its transcriptional regulation, it is the v-FLIP/K13 gene in the KSHV latency cluster that maintains continuous COX-2/PGE2 levels in infected cells. We demonstrate that COX-2 inhibition, via its chemical inhibitors (NS-398 or celecoxib), reduced v-FLIP/K13 mediated NF-κB induction, and extracellular matrix (ECM) interaction mediated FAK/Src/AKT, Rac-1 activation, mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD) levels, and subsequently down-regulated detachment-induced apoptosis (anoikis) resistance. COX-2 inhibition in v-FLIP/K13-Human microvascular endothelial cells (HMVECs) reduced inflammation and invasion/metastasis related genes along with reduced anchorage independent colony formation via modulating ‘extrinsic’ as well as ‘intrinsic’ cell death pathways. COX-2 blockade in v-FLIP/K13-HMVECs drastically augmented cell death induced by removal of essential growth/survival factors secreted in the microenvironment. Transformed cells obtained from anchorage independent colonies of COX-2 inhibitor treated v-FLIP/K13-HMVEC cells expressed lowered levels of endothelial mesenchymal transition (EndMT) genes such as slug, snail and twist, and higher expression of the tumor suppressor gene E-cadherin. Taken together, our study provides strong evidences that FDA approved COX-2 inhibitors have great implications in blocking tumorigenic events linked to KSHV's oncogenic protein v-FLIP/K13. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-67. doi:1538-7445.AM2012-LB-67

Abstract P237: Exercise Training Prevents Hypercholesterolemia-Induced Cardiac Mitochondrial Dysfunction

Hypercholesterolemia has been suggested to have direct negative effects on myocardial function due to increased reactive oxygen species (ROS) generation and increased myocyte death. Mitochondrial permeability transition (MPT) is a significant mediator of cell death, which is enhanced by ROS generation and attenuated by exercise training. The purpose of this study was to investigate the effect of hypercholesterolemia on the MPT response of cardiac mitochondria. We hypothesized that familial hypercholesterolemic (FH) pigs would have an enhanced MPT response, and that exercise training could reverse this phenotype. FH pigs were obtained from the University of Wisconsin. Control, normolipidemic farm pigs were maintained on standard pig chow. After 4 months on a high-fat diet, the FH pigs were switched to the standard pig chow, and randomized to sedentary or exercise groups. The exercise group underwent a progressive treadmill-based training program for 4 months. At the end of the training protocol the animals were sacrificed and the heart removed. MPT was assessed by mitochondrial swelling in response to Ca2+. Protein nitrotyrosylation, GSH levels, and antioxidant enzyme expression were also examined. FH pigs did show an increased MPT response despite no change in the expression of putative MPT pore components adenine nucleotide translocase (ANT), mitochondrial phosphate carrier (PiC), and cyclophilin-D (CypD). FH also caused increased oxidative stress, depicted by increased protein nitrotyrosylation and decreased GSH levels. This was associated with concomitant decreases in the expression of mitochondrial antioxidant enzymes manganese superoxide dismutase (MnSOD) and thioredoxin-2 (Trx2). However, chronic exercise training was able to normalize the MPT response in FH pigs, reduce oxidative stress, and increase MnSOD expression. We conclude that hypercholesterolemia causes increased oxidative stress and enhances the MPT response in the porcine myocardium, and that exercise training can correct for both the increased oxidative stress and MPT alterations observed with hypercholesterolemia.

Hepatocyte-specific deficiency of SOD-2 causes liver failure and promotes tumorigenesis

In the mitochondrial matrix manganese superoxide dismutase (SOD-2) is the essential antioxidant enzyme that degrades reactive oxygen species. The liver has the highest specific MnSOD activity, which implies its important role for maintaining proper organ function and to prevent pathological situations such as cancer. In line with this, we generated hepatocyte-specific SOD-2 knockout mice and found that that ROS levels were enhanced in livers of SOD-2 lacking mice which were reduced in size and displayed signs of liver failure such as intracellular protein droplets, increased apoptotic bodies and Bax levels as well as multinuclear hepatocytes. Further, the zonation of glutamine synthetase, glucokinase and phosphoenolpyruvate carboxykinase was no longer preserved. Serum levels of enzymes indicating liver damage such as glutamate oxalacetate aminotransferase, glutamate pyruvate aminotransferase, cholinesterase, gamma glutamyltransferase, alkaline phosphatase were elevated compared to the control animals. In addition, bilirubin and albumin levels were also enhanced. Further, by using diethylnitrosamine as tumor-inducing agent we could show that tumor development was accelarated in mice with hepatocyte-specific lack of SOD-2.

In situ reduction of oxidative damage, increased cell turnover, and delay of mitochondrial injury by overexpression of manganese superoxide dismutase in a multistage skin carcinogenesis model.

To study early subcellular pathologic changes of tumorigenesis in mouse skin and possible modulation by overexpression of the mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD), skin keratinocytes from nontransgenic (Ntg) and transgenic (TgH) mice overexpressing MnSOD topically treated with one dose of 7,12-dimethylbenz(a)anthracene (DMBA) and a subsequent dose of 12-O-tetradecanoylphorbol 13-acetate (TPA) were analyzed in situ for levels of MnSOD and the oxidative damage product 4-hydroxy-2-nonenal (4HNE)-modified proteins using specific antibodies and immunogold electron microscopy. At all selected time points analyzed after TPA treatment, there was more MnSOD immunoreactive protein in mitochondria of keratinocytes of TgH mice than Ntg mice. Compared with untreated groups, there was a large increase in 4HNE-modified proteins at 6-24 h after TPA treatment, and this increase was larger in Ntg than TgH mice. Indices of mitosis and apoptosis of keratinocytes were greater in DMBA/TPA-treated TgH than Ntg mouse skin. Mitochondrial injury detected by transmission electron microscopy was delayed in keratinocytes of TgH compared with Ntg mice. The present study demonstrated that overexpression of MnSOD not only protected cells from oxidative damage, but also affected cell turnover kinetics. Thus, previously identified reduction in papilloma formation observed in TgH mice is correlated with mitochondrial events.

Adrenocortical Expression of MnSOD

The mitochondrial matrix antioxidant manganese superoxide dismutase (MnSOD) is part of a cascade of components that protects the cell from cytotoxic reactive oxygen species (ROS) and previous investigations have predominately concentrated on this role. However, recent studies on the roles of redox state and ROS suggest that they may have a role in cellular regulation. Using a cDNA clone we generated, northern blot analysis localised the expression of MnSOD predominantly to the zona fasciculata and reticularis (zfr) in rat adrenals. To test whether this correlated with MnSOD activity, assays were conducted on adrenal tissue from rats maintained on a low sodium diet or betamethasone treated and compared with that of untreated controls. MnSOD activity was enhanced in zfr adrenal mitochondria from rats that were ACTH treated or on a low sodium diet. Betamethasone had no effect, and cytosolic/membrane SOD activities were unchanged by all of the treatments. The identification of MnSOD as a hormone inducible component in the adrenal cortex is consistent with the concept that the redox state and/or ROS are involved in the specific regulation of zfr function.

Processing of a chimeric protein in chloroplasts is different in transgenic maize and tobacco plants.

Transgenic maize (Zea mays L.) and tobacco (Nicotiana tabacum Petit Havana SR1) plants have been generated, which overproduce a mitochondrial Nicotiana plumbaginifolia manganese superoxide dismutase (MnSOD) in chloroplasts. For this, the mature MnSOD-coding sequence was fused to a chloroplast transit peptide from a Pisum sativum ribulose-1,5-bisphosphate carboxylase (Rubisco) gene and expression of the chimeric gene was driven by the cauliflower mosaic virus (CaMV) 35S promoter. The transgenic MnSOD gene product was correctly targeted to the chloroplasts both in maize and tobacco. However, despite the use of the CaMV 35S promoter, the MnSOD was predominantly localized in the chloroplasts of the bundle sheath cells of maize. Furthermore, the transit peptide was cleaved off at a different position in maize and tobacco.