Overexpression Of Manganese Superoxide Dismutase Research Articles

Contribution of reactive oxygen species to the anticancer activity of aminoalkanol derivatives of xanthone

SummaryReactive oxygen species (ROS) are critically involved in the action of anticancer agents. In this study, we investigated the role of ROS in the anticancer mechanism of new aminoalkanol derivatives of xanthone. Most xanthones used in the study displayed significant pro-oxidant effects similar to those of gambogic acid, one of the most active anticancer xanthones. The pro-oxidant activity of our xanthones was shown both directly (by determination of ROS induction, effects on the levels of intracellular antioxidants, and expression of antioxidant enzymes) and indirectly by demonstrating that the overexpression of manganese superoxide dismutase decreases ROS-mediated cell senescence. We also observed that mitochondrial dysfunction and cellular apoptosis enhancement correlated with xanthone-induced oxidative stress. Finally, we showed that the use of the antioxidant N-acetyl-L-cysteine partly reversed these effects of aminoalkanol xanthones. Our results demonstrated that novel aminoalkanol xanthones mediated their anticancer activity primarily through ROS elevation and enhanced oxidative stress, which led to mitochondrial cell death stimulation; this mechanism was similar to the activity of gambogic acid.

Nitric Oxide Signaling in Neurodegeneration and Cell Death.

In this tribute to Solomon H. Snyder (Sol) we discuss the mechanisms by which nitric oxide (NO) kills neurons. We provide a historical perspective regarding the discovery that glutamate excitotoxicity is mediated by NO. It also contains a discussion of the discovery that neuronal nitric oxide synthase (nNOS) catalytic activity accounts for NADPH diaphorase activity and its localization in the central nervous system. NADPH diaphorase/nNOS neurons are unique in that they are resistant to toxic effects of excess glutamate and that they are resistant to neurodegeneration in a variety of neurodegenerative diseases. NADPH diaphorase/nNOS neurons are resistant to neurotoxicity and neurodegeneration through the overexpression of manganese superoxide dismutase. The review also delves into the mechanisms by which NO kills neurons including NO's activation of the glyceraldehyde-3-phosphate dehydrogenase-dependent cell pathway. In addition, there is a review of parthanatos in which NO combines with the superoxide anion ( [Formula: see text] ) to form peroxynitrite (ONOO-) that damages DNA and activates poly (ADP-ribose) (PAR) polymerase (PARP). This ultimately leads to activation of the PARP-dependent apoptosis-inducing factor-associated nuclease, the final executioner in NO-dependent cell death. Finally, there is a discussion of potential targets that are under development that target the mechanisms by which NO kills neurons.

Inhibition of NFE2L2-Antioxidant Response Element Pathway by Mitochondrial Reactive Oxygen Species Contributes to Development of Cardiomyopathy and Left Ventricular Dysfunction in Chagas Disease.

We investigated the effects of mitochondrial reactive oxygen species (mtROS) on nuclear factor (erythroid 2)-like 2 (NFE2L2) transcription factor activity during Trypanosoma cruzi (Tc) infection and determined whether enhancing the mtROS scavenging capacity preserved the heart function in Chagas disease. C57BL/6 wild type (WT, female) mice infected with Tc exhibited myocardial loss of mitochondrial membrane potential, complex II (CII)-driven coupled respiration, and ninefold increase in mtROS production. In vitro and in vivo studies showed that Tc infection resulted in an ROS-dependent decline in the expression, nuclear translocation, antioxidant response element (ARE) binding, and activity of NFE2L2, and 35-99% decline in antioxidants' (gamma-glutamyl cysteine synthase [γGCS], heme oxygenase-1 [HO1], glutamate-cysteine ligase modifier subunit [GCLM], thioredoxin (Trx), glutathione S transferase [GST], and NAD(P)H dehydrogenase, quinone 1 [NQO1]) expression. An increase in myocardial and mitochondrial oxidative adducts, myocardial interventricular septum thickness, and left ventricle (LV) mass, a decline in LV posterior wall thickness, and disproportionate synthesis of collagens (COLI/COLIII), αSMA, and SM22α were noted in WT.Tc mice. Overexpression of manganese superoxide dismutase (MnSOD) in cultured cells (HeLa or cardiomyocytes) and MnSODtg mice preserved the NFE2L2 transcriptional activity and antioxidant/oxidant balance, and cardiac oxidative and fibrotic pathology were significantly decreased in MnSODtg.Tc mice. Importantly, echocardiography finding of a decline in LV systolic (stroke volume, cardiac output, ejection fraction) and diastolic (early/late peak filling ratio, myocardial performance index) function in WT.Tc mice was abolished in MnSODtg.Tc mice. Innovation and Conclusion: The mtROS inhibition of NFE2L2/ARE pathway constitutes a key mechanism in signaling the fibrotic gene expression and evolution of chronic cardiomyopathy. Preserving the NFE2L2 activity arrested the mitochondrial and cardiac oxidative stress, cardiac fibrosis, and heart failure in Chagas disease. Antioxid. Redox Signal. 27, 550-566.

Low glucose induces mitochondrial reactive oxygen species via fatty acid oxidation in bovine aortic endothelial cells.

Aims/IntroductionOverproduction of reactive oxygen species (ROS) in endothelial cells (ECs) plays a pivotal role in endothelial dysfunction. Mitochondrial ROS (mtROS) is one of the key players in the pathogenesis of diabetic vascular complications. Hypoglycemia is linked to increased ROS production and vascular events; however, the underlying mechanisms remain unclear. In the present study, we aimed to determine whether and how low glucose (LG) mediates mtROS generation in ECs, and to examine the impact of LG‐induced mtROS on endothelial dysfunction.Materials and MethodsMetabolomic profiling, cellular oxygen consumption rate, mtROS, endothelial nitric oxide synthase phosphorylation, and the expression of vascular cell adhesion molecule‐1 or intercellular adhesion molecule‐1 were evaluated in bovine aortic ECs.ResultsWe found that LG increased mtROS generation in ECs; which was suppressed by overexpression of manganese superoxide dismutase. Comprehensive metabolic analysis using capillary electrophoresis‐mass spectrometry and oxygen consumption rate assessment showed that the pathway from fatty acid to acetyl‐CoA through fatty acid oxidation was upregulated in ECs under LG conditions. In addition, etomoxir, a specific inhibitor of the free fatty acid transporter, decreased LG‐induced mtROS production. These results suggested that LG increased mtROS generation through activation of fatty acid oxidation. We further revealed that LG inhibited endothelial nitric oxide synthase phosphorylation, and increased the expression of vascular cell adhesion molecule‐1 and intercellular adhesion molecule‐1. These effects were suppressed either by overexpression of manganese superoxide dismutase or by treatment with etomoxir.ConclusionsThe activation of fatty acid oxidation followed by mtROS production could be one of the causes for endothelial dysfunction during hypoglycemia.

Cytosolic calcium mediates RIP1/RIP3 complex-dependent necroptosis through JNK activation and mitochondrial ROS production in human colon cancer cells

Necroptosis is a form of programmed necrosis mediated by signaling complexes with receptor-interacting protein 1 (RIP1) and RIP3 kinases as the main mediators. However, the underlying execution pathways of this phenomenon have yet to be elucidated in detail. In this study, a RIP1/RIP3 complex was formed in 2-methoxy-6-acetyl-7-methyljuglone (MAM)-treated HCT116 and HT29 colon cancer cells. With this formation, mitochondrial reactive oxygen species (ROS) levels increased, mitochondrial depolarization occurred, and ATP concentrations decreased. This process was identified as necroptosis. This finding was confirmed by experiments showing that MAM-induced cell death was attenuated by the pharmacological or genetic blockage of necroptosis signaling, including RIP1 inhibitor necrostatin-1s (Nec-1s) and siRNA-mediated gene silencing of RIP1 and RIP3, but was unaffected by caspase inhibitor z-vad-fmk or necrosis inhibitor 2-(1H-Indol-3-yl)-3-pentylamino-maleimide (IM54). Transmission electron microscopy (TEM) analysis further revealed the ultrastructural features of MAM-induced necroptosis. MAM-induced RIP1/RIP3 complex triggered necroptosis through cytosolic calcium (Ca2+) accumulation and sustained c-Jun N-terminal kinase (JNK) activation. Both calcium chelator BAPTA-AM and JNK inhibitor SP600125 could attenuate necroptotic features, including mitochondrial ROS elevation, mitochondrial depolarization, and ATP depletion. 2-thenoyltrifluoroacetone (TTFA), which is a mitochondrial complex II inhibitor, was found to effectively reverse both MAM induced mitochondrial ROS generation and cell death, indicating the complex II was the ROS-producing site. The essential role of mitochondrial ROS was confirmed by the protective effect of overexpression of manganese superoxide dismutase (MnSOD). MAM-induced necroptosis was independent of TNFα, p53, MLKL, and lysosomal membrane permeabilization. In summary, our study demonstrated that RIP1/RIP3 complex-triggered cytosolic calcium accumulation is a critical mediator in MAM-induced necroptosis through sustained JNK activation and mitochondrial ROS production. Our study also provided new insights into the molecular regulation of necroptosis in human colon cancer cells.

MnSOD mediated by HSV vectors in the periaqueductal gray suppresses morphine withdrawal in rats.

Morphine appears to be the most active metabolite of heroin; therefore, the effects of morphine are important in understanding the ramifications of heroin abuse. Opioid physical dependence (withdrawal response) may have very long-lasting effects on the motivation for reward, including the incubation of cue-induced drug-seeking behavior. However, the exact mechanisms of morphine withdrawal (MW) are not clear yet, and its treatment remains elusive. Periaqueductal gray (PAG) is one of the important sites in the pathogenesis of MW. Here, we used recombinant herpes simplex virus (HSV) vectors that encode the sod2 gene expressing manganese superoxide dismutase (MnSOD) to evaluate its therapeutic potential in MW. Microinjection of HSV vectors expressing MnSOD into the PAG reduced the MW syndrome. MnSOD vectors suppressed the upregulated mitochondrial superoxide, and endoplasmic reticulum stress markers (glucose-related protein 78 (GRP78) and activating transcription factor 6 alpha (ATF6α)) in the PAG induced by MW. Immunostaining showed that mitochondrial superoxide, GRP78 and ATF6α were colocalized with neuronal nuclei (a neuronal-specific marker), suggesting that they are located in the neurons in the PAG. These results suggest that overexpression of MnSOD by HSV vectors may relieve opioid dependence. This study may provide a novel therapeutic approach to morphine physical withdrawal response.

Overexpression of manganese superoxide dismutase in mouse liver leads to defects in oxidative phosphorylation

Non‐alcoholic fatty liver disease (NAFLD) is a chronic disease that is characterized by inflammation in the liver. This inflammation is due primarily to an increase in reactive oxygen species, and therefore antioxidant therapy can reduce the severity of the disease. However, these antioxidants have also been met with negative side effects, which warrants further investigation. Manganese superoxide dismutase, or MnSOD, is a mitochondrial antioxidant enzyme that converts then reduces superoxide levels by converting them into hydrogen peroxide. Hydrogen peroxide is then detoxified by a number of mitochondrial antioxidant enzymes. Although genetic overexpression of SOD2 could be beneficial to the treatment of NAFLD, preliminary data has shown that mice with SOD2 overexpression actually demonstrate defects in oxidative phosphorylation. These defects include reductions in levels of complex II and V measured by blue native PAGE, and reductions in levels of complex II, III and IV measured by SDS‐PAGE. Western blot results for reduction in levels of complex I, II, and III are under investigation. These data indicate that antioxidant therapy may have unexpected complications that should be considered before administration of these drugs.

Estrogen Protects the Female Heart from Ischemia/Reperfusion Injury through Manganese Superoxide Dismutase Phosphorylation by Mitochondrial p38β at Threonine 79 and Serine 106

A collective body of evidence indicates that estrogen protects the heart from myocardial ischemia/reperfusion (I/R) injury, but the underlying mechanism remains incompletely understood. We have previously delineated a novel mechanism of how 17β-estradiol (E2) protects cultured neonatal rat cardiomyocytes from hypoxia/reoxygenation (H/R) by identifying a functionally active mitochondrial pool of p38β and E2-driven upregulation of manganese superoxide dismutase (MnSOD) activity via p38β, leading to the suppression of reactive oxygen species (ROS) and apoptosis. Here we investigate these cytoprotective actions of E2 in vivo. Left coronary artery ligation and reperfusion was used to produce I/R injury in ovariectomized (OVX) female mice and in estrogen receptor (ER) null female mice. E2 treatment in OVX mice reduced the left ventricular infarct size accompanied by increased activity of mitochondrial p38β and MnSOD. I/R-induced infarct size in ERα knockout (ERKO), ERβ knockout (BERKO) and ERα and β double knockout (DERKO) female mice was larger than that in wild type (WT) mice, with little difference among ERKO, BERKO, and DERKO. Loss of both ERα and ERβ led to reduced activity of mitochondrial p38β and MnSOD at baseline and after I/R. The physical interaction between mitochondrial p38β and MnSOD in the heart was detected by co-immunoprecipitation (co-IP). Threonine 79 (T79) and serine 106 (S106) of MnSOD were identified to be phosphorylated by p38β in kinase assays. Overexpression of WT MnSOD in cardiomyocytes reduced ROS generation during H/R, while point mutation of T79 and S106 of MnSOD to alanine abolished its antioxidative function. We conclude that the protective effects of E2 and ER against cardiac I/R injury involve the regulation of MnSOD via posttranslational modification of the dismutase by p38β.

Promotion of Survival and Engraftment of Transplanted Adipose Tissue-Derived Stromal and Vascular Cells by Overexpression of Manganese Superoxide Dismutase.

Promotion of Survival and Engraftment of Transplanted Adipose Tissue-Derived Stromal and Vascular Cells by Overexpression of Manganese Superoxide Dismutase.

Promotion of Survival and Engraftment of Transplanted Adipose Tissue-Derived Stromal and Vascular Cells by Overexpression of Manganese Superoxide Dismutase.

Short-term persistence of transplanted cells during early post-implant period limits clinical efficacy of cell therapy. Poor cell survival is mainly due to the harsh hypoxic microenvironment transplanted cells face at the site of implantation and to anoikis, driven by cell adhesion loss. We evaluated the hypothesis that viral-mediated expression of a gene conferring hypoxia resistance to cells before transplant could enhance survival of grafted cells in early stages after implant. We used adipose tissue as cell source because it consistently provides high yields of adipose-tissue-derived stromal and vascular cells (ASCs), suitable for regenerative purposes. Luciferase positive cells were transduced with lentiviral vectors expressing either green fluorescent protein as control or human manganese superoxide dismutase (SOD2). Cells were then exposed in vitro to hypoxic conditions, mimicking cell transplantation into an ischemic site. Cells overexpressing SOD2 displayed survival rates significantly greater compared to mock transduced cells. Similar results were also obtained in vivo after implantation into syngeneic mice and assessment of cell engraftment by in vivo bioluminescent imaging. Taken together, these findings suggest that ex vivo gene transfer of SOD2 into ASCs before implantation confers a cytoprotective effect leading to improved survival and engraftment rates, therefore enhancing cell therapy regenerative potential.

Hyperglycemia Induces Cellular Hypoxia through Production of Mitochondrial ROS Followed by Suppression of Aquaporin-1.

We previously proposed that hyperglycemia-induced mitochondrial reactive oxygen species (mtROS) generation is a key event in the development of diabetic complications. Interestingly, some common aspects exist between hyperglycemia and hypoxia-induced phenomena. Thus, hyperglycemia may induce cellular hypoxia, and this phenomenon may also be involved in the pathogenesis of diabetic complications. In endothelial cells (ECs), cellular hypoxia increased after incubation with high glucose (HG). A similar phenomenon was observed in glomeruli of diabetic mice. HG-induced cellular hypoxia was suppressed by mitochondria blockades or manganese superoxide dismutase (MnSOD) overexpression, which is a specific SOD for mtROS. Overexpression of MnSOD also increased the expression of aquaporin-1 (AQP1), a water and oxygen channel. AQP1 overexpression in ECs suppressed hyperglycemia-induced cellular hypoxia, endothelin-1 and fibronectin overproduction, and apoptosis. Therefore, hyperglycemia-induced cellular hypoxia and mtROS generation may promote hyperglycemic damage in a coordinated manner.

Manganese superoxide dismutase mediates anoikis resistance and tumor metastasis in nasopharyngeal carcinoma.

Metastatic cancer cells are able to survive the loss of attachment to the extracellular matrix (ECM) by developing resistance to anoikis, a specialized form of apoptosis. Here we investigated resistance to anoikis in nasopharyngeal carcinoma cells (NPC). When detached in culture, the highly metastatic S18 NPC cell line exhibited strong resistance to anoikis, as compared to the poorly metastatic S26 NPC cell line. With loss of attachment, S18 cells had lower levels of reactive oxygen species (ROS) and higher levels of manganese superoxide dismutase (MnSOD), an essential mitochondrial antioxidant enzyme. MnSOD knockdown increased the levels of ROS and diminished resistance to anoikis in S18 cells. Conversely, removal of reactive oxygen species (ROS) using NAC or overexpression of MnSOD in S26 cells induced resistance to anoikis. Blocking β-catenin through RNA interference down-regulated MnSOD expression and enhanced anoikis in S18 cells, while β-catenin overexpression enhanced MnSOD expression and suppressed anoikis in S26 cells. In addition, knockdown of MnSOD in S18 cells reduced colony formation in vitro and ameliorated lung metastasis in vivo. In patients with NPC, MnSOD expression was positively correlated with pathologic tumor stages and negatively correlated with overall survival. These results establish MnSOD as a key mediator of anoikis resistance and tumor metastasis and suggest that β-catenin/MnSOD could be a therapeutic target in NPC.

Manganese-superoxide dismutase (Mn-SOD) overexpression is a common event in colorectal cancers with mitochondrial microsatellite instability.

Mitochondrial displacement loop (D-loop) is a hot spot for mitochondrial DNA (mtDNA) alterations that effects cellular reactive oxygen species (ROS) generation. Manganese-superoxide dismutase (Mn-SOD) is a major antioxidant enzyme that protects cells from ROS-mediated damage. In the present study, we investigated the relationship between sequence alterations of mitochondrial D-loop and Mn-SOD expression in colorectal cancer (CRC). Genotyping of entire mitochondrial D-loop (1124bp) was carried out on mtDNA of analogous tumor and normal tissues from 35 CRC patients of south Indian origin by PCR-sequencing analysis. Tumor-specific large-scale mtDNA deletions and Mn-SOD expression was analyzed by PCR and Western blot analysis, respectively. We identified 87 polymorphisms in the D-loop region of tumor and/or control tissues. Polymorphisms were predominantly located in hypervariable region I (67.9%) than in II (32.1%) of D-loop. Significantly increased mtDNA microsatellite instability (mtMSI) [310'C' insertion (P = 0.00001) and T16189C (P = 0.0007)] and elevated Mn-SOD expression was observed in tumor tissues compared with controls. Interestingly, mtMSI was significantly high in tumors with Mn-SOD overexpression. Tumor-specific large-scale mtDNA deletions were not observed in CRC tissues. In conclusion, mtMSI and Mn-SOD overexpression are a common event in CRC. The analysis of mtMSI and/or Mn-SOD expression might help to identify patients at high risk for disease outcome, thereby helping to refine therapeutic decisions in CRC.

Molecular cloning and characterization of two manganese superoxide dismutases from Miscanthus × giganteus

Six MnSOD genes were isolated from five Miscanthus species. MgMnSOD1 functions in mitochondria and MgMnSOD1 seems to be the main MnSOD gene involved in stress response of M. × giganteus. Miscanthus × giganteus is a promising biomass energy crop with advantages of vigorous growth, high yield, low fertilizer and pesticide inputs. However, poor overwinter ability limits its widespread cultivation. Moreover, narrow genetic base may increase the risk of susceptibility to diseases and pests. Manganese superoxide dismutase (MnSOD), an important antioxidant enzyme involved in stress tolerance is able to protect plant cells from accumulated reactive oxygen species by converting superoxide to peroxide and oxygen. In many plants, overexpression of MnSOD has shown the ability to enhance the resistance to various stresses. This article describes the studies performed in an attempt to elucidate the molecular and enzymatic properties of MnSODs in M. × giganteus. MnSOD genes from M. × giganteus (MgMnSOD1, MgMnSOD2), M. lutarioriparia (MlMnSOD), M. sacchariflora (MsaMnSOD), M. sinensis (MsiMnSOD), and M. floridulus (MfMnSOD) were cloned and sequenced. The sequence analysis and expression patterns of MgMnSOD1 and MgMnSOD2 suggest that they were orthologous genes which were inherited from the two parents, M. sacchariflora and M. sinensis, respectively. In addition, MgMnSOD1 is predicted to be the main MnSOD gene involved in stress response of M. × giganteus. The activity of purified recombinant MgMnSOD1 was 1854.79 ± 39.98 U mg(-1) (mean ± SD). Further enzymatic assays revealed that the protein exhibited an outstanding thermal stability. MgMnSOD1 is predicted to be targeted to mitochondria and involved in removing the superoxide radical generated by respiration. The presence and sequences of other SOD isozymes transcripts were also investigated in this study.

Abstract 5103: Loss of p27 upregulates MnSOD in a STAT3-dependent manner, disrupts intracellular redox activity and enhances cell migration

Abstract Cell migration is a dynamic process that is central to a variety of physiological functions as well as disease pathogenesis. The modulation of cell migration by p27 (officially known as CDKN1B) has been reported, but the exact mechanism(s) whereby p27 interacts with downstream effectors that control cell migration have not been elucidated. By systematically comparing p27+/+ mouse embryonic fibroblasts (MEFs) with genetically ablated p272/2 MEFs using wound-healing, transwell and time-lapse microscopic analyses, we provide direct evidence that p27 inhibits both directional and random cell migration. Identical results were obtained with normal and cancer epithelial cells using complementary knockdown and overexpression approaches. Additional studies revealed that overexpression of manganese superoxide dismutase (MnSOD, officially known as SOD2) and reduced intracellular oxidation played a key role in increased cell migration in p27-deficient cells. Furthermore, we identified signal transducer and activator of transcription 3 (STAT3) as the transcription factor responsible for p27-regulated MnSOD expression, which was further mediated by ERK- and ATF1-dependent transactivation of the cAMP response element (CRE) within the Stat3 promoter. Collectively, our data strongly indicate that p27 plays a crucial negative role in cell migration by inhibiting MnSOD expression in a STAT3-dependent manner. Citation Format: Jing An, Dongyun Zhang, Chuanshu Huang. Loss of p27 upregulates MnSOD in a STAT3-dependent manner, disrupts intracellular redox activity and enhances cell migration. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5103. doi:10.1158/1538-7445.AM2015-5103

Abstract 582: Overexpression of Mnsod Reduces Aortic Valve Calcification Through Repression of Pro-osteogenic Signaling

Increased BMP signaling and markers of osteogenic differentiation are strongly associated with regions of valvular calcification in aortic valves from humans with end-stage calcific aortic valve stenosis. While oxidative stress is also markedly elevated in these regions, it is unknown whether increasing antioxidant capacity can reduce pro-osteogenic signaling and valvular calcification. Therefore, we hypothesized that overexpression of manganese superoxide dismutase (MnSOD) can reduce pro-osteogenic signaling in the aortic valves from hypercholesterolemic mice, ultimately resulting in reduced valvular calcification and improved valvular function. We used Ldlr -/- /ApoB 100/100 (LA) mice that were wild-type for MnSOD (LA-MnSOD 0/0 ) or overexpressing MnSOD (LA-MnSOD Tg/0 ) and fed a western diet (TD88137) for six months. We first measured mRNA levels of the BMP ligands BMP2 and BMP4 (qRT-PCR), both of which were unchanged between LA-MnSOD 0/0 and LA-MNSOD Tg/0 mice. While there was a small but significant increase in Runx2 mRNA levels in LA-MnSOD Tg/0 mice compared to wild-type littermates (1.22±0.09, 1.0±0.09, qRT-PCR, p < 0.05.) gene expression levels of late stage osteoblast marker Sp7 were reduced by approximately 50% in LA-MnSOD Tg/0 compared to wild-type littermates (0.53±0.15, 1.0±0.24, qRT-PCR, p < 0.05). Sp7 protein levels (immunohistochemistry) were also significantly reduced in LA-MnSOD Tg/0 compared to wild-type littermates (164.5±12.3 RFU vs 259.6±10.4 RFU, p < 0.05). Critically, aortic valve calcification (Alizarin Red) was also significantly reduced in LA-MnSOD Tg/0 mice compared to their LA-MnSOD 0/0 littermates. Functionally, aortic valve cusp separation distance (high resolution echocardiography) was slightly improved in LA-MnSOD Tg/0 mice compared to LA-MnSOD 0/0 mice (0.98±0.04 mm vs 0.91±0.03 mm, p = n.s.). Collectively, these data suggest that increasing mitochondrial antioxidant capacity can reduce pro-osteogenic signaling and attenuate aortic valve calcification, and may be a potential therapeutic target to slow the progression of valvular calcification and dysfunction in patients with calcific aortic valve stenosis.

Novel role of mitochondrial manganese superoxide dismutase in STAT3 dependent pluripotency of mouse embryonic stem cells.

Leukemia Inhibitory Factor (LIF)/Signal transducer and activator of transcription 3 (STAT3) signaling pathway maintains the stemness and pluripotency of mouse embryonic stem cells (mESCs). Detailed knowledge on key intermediates in this pathway as well as any parallel pathways is largely missing. We initiated our study by investigating the effect of small molecule Curcumin on various signalling pathways essential for self-renewal. Curcumin sustained the LIF independent self-renewal of mESCs and induced pluripotent stem cells (miPSCs) in a STAT3 activity dependent manner. Gene expression analysis showed LIF/STAT3 and redox signaling components to be majorly modulated. Amongst ROS genes, expression of Manganese Superoxide Dismutase (MnSOD) specifically relied on STAT3 signaling as evidenced by STAT3 inhibition and reporter assay. The silencing of MnSOD, but not Cu-ZnSOD expression, resulted in the loss of mESC pluripotency in presence of LIF, and the overexpression of MnSOD is sufficient for maintaining the expression of pluripotent genes in the absence of STAT3 signaling. Finally, we demonstrate MnSOD to stabilize the turnover of pluripotent proteins at the post-translational level by modulating proteasomal activity. In conclusion, our findings unravel a novel role of STAT3 mediated MnSOD in the self-renewal of mESCs.

Cellular redox status determines sensitivity to BNIP3-mediated cell death in cardiac myocytes.

The atypical BH3-only protein Bcl-2/adenovirus E1B 19-kDa interacting protein 3 (BNIP3) is an important regulator of hypoxia-mediated cell death. Interestingly, the susceptibility to BNIP3-mediated cell death differs between cells. In this study we examined whether there are mechanistic differences in BNIP3-mediated cell death between neonatal and adult cardiac myocytes. We discovered that BNIP3 is a potent inducer of cell death in neonatal myocytes, whereas adult myocytes are remarkably resistant to BNIP3. When exploring the potential underlying basis for the resistance, we discovered that adult myocytes express significantly higher levels of the mitochondrial antioxidant manganese superoxide dismutase (MnSOD) than neonatal myocytes. Overexpression of MnSOD confers resistance to BNIP3-mediated cell death in neonatal myocytes. In contrast, the presence of a pharmacological MnSOD inhibitor, 2-methoxyestradiol, results in increased sensitivity to BNIP3-mediated cell death in adult myocytes. Cotreatment with the mitochondria-targeted antioxidant MitoTEMPO or the MnSOD mimetic manganese (III) tetrakis (4-benzoic acid) porphyrin chloride abrogates the increased cell death by 2-methoxyestradiol. Moreover, increased oxidative stress also restores the ability of BNIP3 to induce cell death in adult myocytes. Taken together, these data indicate that redox status determines cell susceptibility to BNIP3-mediated cell death. These findings are clinically relevant, given that pediatric hearts are known to be more vulnerable than the adult heart to ischemic injury. Our studies provide important insight into why pediatric hearts are more sensitive to ischemic injury and may help in the clinical management of childhood heart disease.

Manganese superoxide dismutase promotes interaction of actin, S100A4 and Talin, and enhances rat gastric tumor cell invasion.

It has been demonstrated that cancer cells are under high levels of oxidative stress and express high levels of Manganese superoxide dismutase (MnSOD) to protect themselves and support the anabolic metabolism needed for growth and cell motility. The aim of this study was to identify proteins that may have a correlation with invasion and redox regulation by mitochondrial reactive oxygen species (ROS). MnSOD scavenges superoxide anions generated from mitochondria and is an important regulator of cellular redox status. Oxidative posttranslational modification of cysteine residues is a key mechanism that regulates protein structure and function. We hypothesized that MnSOD regulates intracellular reduced thiol status and promotes cancer invasion. A proteomic thiol-labeling approach with 5-iodoacetamidofluorescein was used to identify changes in intracellular reduced thiol-containing proteins. Our results demonstrate that overexpression of MnSOD maintained the major structural protein, actin, in a reduced state, and enhanced the invasion ability in gastric mucosal cancer cells, RGK1. We also found that the expression of Talin and S100A4 were increased in MnSOD-overexpressed RGK1 cells. Moreover, Talin bound not only with actin but also with S100A4, suggesting that the interaction of these proteins may, in part, contribute to the invasive ability of rat gastric cancer.

MnSOD overexpression confers cisplatin resistance in lung adenocarcinoma via the NF-κB/Snail/Bcl-2 pathway

Manganese superoxide dismutase (MnSOD) has been shown to be associated with doxorubicin resistance in gastric cancer cells, but the underlying mechanism of MnSOD in drug resistance remains unclear. A recent study indicated that NF-κB activation by MnSOD promoted tumor malignancy in lung adenocarcinoma. Therefore, we hypothesized that MnSOD-mediated NF-κB activation might confer cisplatin resistance in lung adenocarcinoma via the NF-κB/Bcl-2/Snail pathway. Here, the inhibition concentration of cisplatin with 50% cell viability (IC50) was positively correlated with MnSOD expression and its activity in a panel of lung adenocarcinoma cells. The IC50 value was markedly increased and decreased by MnSOD overexpression and knockdown, respectively, in lung cancer cells. Mechanistically, an increase in Bcl-2 by MnSOD-mediated NF-κB activation confers greater cisplatin resistance than cIAP2, Bcl-xL, Mcl-1, and Snail. MnSOD-mediated cisplatin resistance can be overcome by a Bcl-2 antagonist (ABT-199) or IKKβ inhibitor (curcumin) in cells and xenograft tumors. MnSOD expression was positively correlated with nuclear p65 protein and Bcl-2 mRNA expression in tumors from patients with lung adenocarcinomas. A retrospective study indicated that it was more common for MnSOD-positive, nuclear p65-positive, or high Bcl-2 mRNA tumors to have an unfavorable response to cisplatin-based chemotherapy than their counterparts. Therefore, we suggest that ABT-199 or curcumin may be potentially useful to improve tumor regression and chemotherapeutic response in patients with MnSOD/Bcl-2-positive tumors.