Overexpression Of Superoxide Dismutase Research Articles

Overexpression of CuZnSOD in median preoptic nucleus improves cardiac function after myocardial infarction in the rat

It has been shown that central overexpression of the superoxide scavenging enzyme, Cu/Zn superoxide dismutase (SOD), in the subfornical organ improves cardiac function in a mouse model of heart failure (HF). A downstream hypothalamic site, the median preoptic nucleus (MnPO), may act as a relay center for reactive oxygen species to act as mediators in the pathophysiology of HF. To test the hypothesis that elevated superoxide in the MnPO is related to the pathophysiology of HF and decreased cardiac function, we injected adenovirus encoding CuZnSOD (AdSOD, n=2) or control empty vector (AdE, n=3) into the MnPO of normal rats. Subsequently, rats were subjected to coronary artery ligation to create a myocardial infarct (MI) of the left ventricle. Cardiac function was monitored at 2 week intervals via echocardiography. Upon completion, rat brains were examined for SOD expression in MnPO via immunofluorescence and histopathological analyses of cardiac infarct size were conducted. Baseline (EF) ejection fractions (%) of AdSOD and AdE rats were 62±4 and 67±2, respectively. Two weeks after MI, EF was significantly decreased in both groups of rats (AdSOD: 38±3, AdE: 48±4). In contrast by 6 weeks post MI, EF had improved to 52±1 in AdSOD rats yet was only 41±6 in AdE rats. In conclusion, despite decreases in EF early after MI, overexpression of SOD in the MnPO was related to an improvement in left ventricular function 6 weeks post MI.

Overexpression of Mn superoxide dismutase attenuates age related up‐regulation of TGF‐ß and remodeling in the aging heart

Aging results in accumulation of extracellular matrix proteins and impairment of cardiac function. Oxidative stress is a potential mechanism in age‐associated cardiac fibrosis. We have previously show exercise training reduces fibrosis and expression of transforming growth factor‐beta (TGF‐ß1) in the aging heart, concomitant with a reduction in oxidative stress and upregulation of the manganese‐isoform of superoxide dismutase (MnSOD). However, the causal relationship between MnSOD and TGF‐ß1 in the aging heart is unknown. Thus, we tested the hypothesis that overexpression of Mn‐SOD would reduce TGF‐ß1 and markers of fibrosis in the aging heart. Old (27 mo) wild type (OWT) animals had larger cardiac myocyte cross‐sectional areas (CSA) that young (4 mo) wild type mice (YWT), an effect reduced in Old Mn‐SOD transgenic mice animals (Otg). TGF‐β1 and collagen I abundance was higher in OWT vs. YWT, while overexpression of MnSOD attenuated TGF‐β1 and collagen I in hearts of old mice. Matrix metalloproteinase‐9 (MMP‐9) was lower in OWT, with age dependent suppression of MMP‐9 prevented by Mn‐SOD. Results indicate that overexpression of Mn‐SOD overexpression protects against age‐related upregulation of TGF‐ß1 and fibrosis, suggestive that elevation of MnSOD with exercise training may indeed be cardioprotective against fibrosis and remodeling. Supported by AHA 0855158F; NIH AR054084

Does Scavenging of Mitochondrial Superoxide Attenuate Cancer Prosurvival Signaling Pathways?

It has been previously suggested that overexpression of mitochondrial superoxide dismutase (SOD) attenuates cancer development; however, the exact mechanism remains unclear. In this work, we have studied the direct effect of the mitochondria-targeted superoxide scavenger, (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (mitoTEMPO), on B16-F0 mouse melanoma cells and tumor growth in a nude mouse model of human melanoma. We show that scavenging of mitochondrial superoxide inhibited cell growth, reduced viability, and induced apoptosis in melanoma cells, but did not affect nonmalignant skin fibroblasts. Diminished mitochondrial superoxide inhibited redox-dependent Akt, restored activity of mitochondrial pyruvate dehydrogenase, and reduced HIF1-α and lactate dehydrogenase expression in cancer cells. Suppression of glycolysis in mitoTEMPO-treated melanoma cells resulted in a significant drop of cellular adenosine-5'-triphosphate and induced cell death. In vivo mitoTEMPO treatment effectively suppressed growth of established tumor in the mouse model of human melanoma. Therefore, our data lead to the hypothesis that scavenging of mitochondrial superoxide selectively inhibits redox-sensitive survival and metabolic pathways, resulting in cancer cell death. In contrast to existing anticancer therapies, inhibition of mitochondrial superoxide may represent a novel specific anticancer treatment with reduced cytotoxic side effects.

Losartan Reduces Oxidative Stress Within the Rostral Ventrolateral Medulla of Rats With Renovascular Hypertension

Previous studies showed that the microinjection of antioxidants or the overexpression of superoxide dismutase within the rostral ventrolateral medulla (RVLM) reduces hypertension and sympathoexcitation in the 2-kidney, 1-clip (2K-1C) model. In this study, we hypothesized that angiotensin II (ANG II) type 1 receptor (AT1R) is involved in the oxidative stress within the RVLM and contributes to cardiovascular dysfunction in renovascular hypertension. Losartan (30mg/kg/day, oral gavage) was administered for 7 consecutive days by week 5 after implantation of the clip (gap width = 0.2mm). Mean arterial pressure, baroreflex, and renal sympathetic nerve activity (rSNA) were evaluated. Superoxide production was evaluated by dihydroethidium (DHE) staining within the RVLM and within a control area. Systemic oxidative stress was characterized by measurement of thiobarbituric acid reactive substances (TBARS) and total glutathione (tGSH) in the blood. AT1R blockade significantly (P < 0.05) reduced hypertension by approximately 20% (n = 11) and sympathoexcitation to the kidneys by approximately 41% (n = 6) in the 2K-1C rats. Losartan treatment increased the baroreflex sensitivity of rSNA to pressor (67%) and depressor (140%) stimuli in the 2K-1C rats. AT1R blockade caused a significant (66%) reduction in DHE staining within the RVLM but not within the control area, reduced plasma TBARS (from 1.6±0.1 to 1.0±0.1 nmol/ml), and increased tGSH (from 3.4±0.4 to 5.2±0.3 μmol/g Hb) in the 2K-1C group only. Our findings suggest that the beneficial effects of ANG II blockade in renovascular hypertension are partly due to preferential reduction of oxidative stress in the RVLM.

Induction of reactive oxygen species generation inhibits epithelial–mesenchymal transition and promotes growth arrest in prostate cancer cells

Oxidative stress is one causative factor of the pathogenesis and aggressiveness of most of the cancer types, including prostate cancer (CaP). A moderate increase in reactive oxygen species (ROS) induces cell proliferation whereas excessive amounts of ROS promote apoptosis. In this study, we explored the pro-oxidant property of 3,9-dihydroxy-2-prenylcoumestan (psoralidin [pso]), a dietary agent, on CaP (PC-3 and C4-2B) cells. Pso greatly induced ROS generation (more than 20-fold) that resulted in the growth inhibition of CaP cells. Overexpression of anti-oxidant enzymes superoxide dismutase 1 (SOD1), SOD2, and catalase, or pretreatment with the pharmacological inhibitor N-acetylcysteine (NAC) significantly attenuated both pso-mediated ROS generation and pso-mediated growth inhibition in CaP cells. Furthermore, pso administration significantly inhibited the migratory and invasive property of CaP cells by decreasing the transcription of β-catenin, and slug, which promote epithelial-mesenchymal transition (EMT), and by concurrently inducing E-cadherin expression in CaP cells. Pso-induced ROS generation in CaP cells resulted in loss of mitochondrial membrane potential, cytochrome-c release, and activation of caspase-3 and -9 and poly (ADP-ribose) polymerase (PARP), which led to apoptosis. On the other hand, overexpression of anti-oxidants rescued pso-mediated effects on CaP cells. These findings suggest that increasing the threshold of intracellular ROS could prevent or treat CaP growth and metastasis.

IFNγ-mediated inhibition of cell proliferation through increased PKCδ-induced overexpression of EC-SOD

Extracellular superoxide dismutase (EC-SOD) overexpression modulates cellular responses such as tumor cell suppression and is induced by IFNγ. Therefore, we examined the role of EC-SOD in IFNγ-mediated tumor cell suppression. We observed that the dominant-negative protein kinase C delta (PKCδ) suppresses IFNγ-induced EC-SOD expression in both keratinocytes and melanoma cells. Our results also showed that PKCδ-induced ECSOD expression was reduced by pretreatment with a PKCspecific inhibitor or a siRNA against PKCδ. PKCδ-induced ECSOD expression suppressed cell proliferations by the up-regulation of p21 and Rb, and the downregulation of cyclin A and D. Finally, we demonstrated that increased expression of EC-SOD drastically suppressed lung melanoma proliferation in an EC-SOD transgenic mouse via p21 expression. In summary, our findings suggest that IFNγ-induced EC-SOD expression occurs via activation of PKCδ. Therefore, the upregulation of EC-SOD may be effective for prevention of various cancers, including melanoma, via cell cycle arrest. [BMB Reports 2012; 45(11): 659-664]

SOD1 gene transfer into paraventricular nucleus attenuates hypertension and sympathetic activity in spontaneously hypertensive rats

Excessive sympathetic activity contributes to the initiation and progression of hypertension. Reactive oxygen species in the paraventricular nucleus (PVN) is involved in sympathetic overdrive and hypertension. The present study was designed to investigate whether superoxide dismutase 1 (SOD1) overexpression in the PVN attenuated sympathetic activation and hypertension. Adenoviral vectors containing human SOD1 or null adenoviral vectors were microinjected into the PVN of Wistar rats and spontaneously hypertensive rats (SHR). Significant depressor effects were observed from weeks 1 to 4 after SOD1 gene transfer in SHR. Acute experiments were carried out at the end of the 3rd week. In the PVN, superoxide anion and angiotensin II levels were increased while SOD1 activity and protein expression were decreased in SHR, which were attenuated by SOD1 overexpression in the PVN. However, SOD1 overexpression had no significant effect on the SOD2 activity in the PVN. The blood pressure response to ganglionic blockade, cardiac sympathetic nerve activity, and cardiac sympathetic afferent reflex (CSAR) were enhanced, and the plasma norepinephrine level was increased in SHR, which were prevented by SOD1 gene transfer in the PVN. Furthermore, SOD1 overexpression decreased the ratio of left ventricular weight to body weight, cross-sectional areas of myocardial cells, media thickness, and the media/lumen ratio of small arteries in the heart in SHR. These results indicate that SOD1 overexpression in the PVN reduces arterial blood pressure, attenuates excessive sympathetic activity and CSAR, and improves myocardial and vascular remodeling in SHR.

Cardiomyocyte-restricted overexpression of extracellular superoxide dismutase increases nitric oxide bioavailability and reduces infarct size after ischemia/reperfusion

Increased levels of extracellular superoxide dismutase (ecSOD) induced by preconditioning or gene therapy protect the heart from ischemia/reperfusion injury. To elucidate the mechanism responsible for this action, we studied the effects of increased superoxide scavenging on nitric oxide (NO) bioavailability in a cardiac myocyte-specific ecSOD transgenic (Tg) mouse. Results indicated that ecSOD overexpression increased cardiac myocyte-specific ecSOD activity 27.5-fold. Transgenic ecSOD was localized to the sarcolemma and, notably, the cytoplasm of cardiac myocytes. Ischemia/reperfusion injury was attenuated in ecSOD Tg hearts, in which infarct size was decreased and LV functional recovery was improved. Using the ROS spin trap, DMPO, electron paramagnetic resonance (EPR) spectroscopy demonstrated a significant decrease in ROS in Tg hearts during the first 20 min of reperfusion. This decrease in ROS was accompanied by an increase in NO production determined by EPR using the NO spin trap, Fe-MGD. Attenuated ROS in ecSOD Tg myocytes was also supported by decreased production of peroxynitrite (ONOO−). Increased NO bioavailability was confirmed by attenuated guanylate cyclase-dependent (p-VASP) signaling. In conclusion, attenuation of ROS levels by cardiac-specific ecSOD overexpression increases NO bioavailability in response to ischemia/reperfusion and protects against reperfusion injury. These findings are the first to demonstrate increased NO bioavailability with attenuation of ROS by direct measurement of these reactive species (EPR, reactive fluorescent dyes) with cardiac-specific ecSOD expression. This is also the first indication that the predominantly extracellular SOD isoform is capable of cytosolic localization that affects myocardial intracellular signal transduction and function.Electronic supplementary materialThe online version of this article (doi:10.1007/s00395-012-0305-1) contains supplementary material, which is available to authorized users.

Protective effects of astaxanthin on capillary regression in atrophied soleus muscle of rats

The capillary regression in skeletal muscles associated with a chronic decrease in activity is related to a dysfunction of endocapillary cells induced by over-expression of oxidative stress. We hypothesized that treatment with astaxanthin, an antioxidant, would attenuate the oxidative stress induced by decreased skeletal muscle use, and that this attenuation would prevent the associated capillary regression. The purpose of the present study was to investigate the antioxidant and preventive effects of astaxanthin on capillary regression in the soleus muscle during hindlimb unloading. Twenty-four adult male Wistar rats were assigned randomly either to a control, control plus astaxanthin treatment, hindlimb unloaded or hindlimb unloaded plus astaxanthin treatment group for 7 days. Hindlimb unloading resulted in a decrease in mean soleus absolute weight, capillary number, volume and luminal diameter. The accumulation of reactive oxygen species and the over-expression of superoxide dismutase (SOD-1), a decrease in the levels of vascular endothelial growth factor (VEGF) and its receptors, an inhibition of the angiopoietin pathway and an increase of thrombospondin-1 (TSP-1), as an anti-angiogenic factor were showed. Administration of astaxanthin attenuated the changes in SOD-1 and VEGF, up-regulated the angiogenic factors and reduced the capillary regression in the soleus of hindlimb unloaded rats. In addition, the VEGF-to-TSP1 ratio was higher in the astaxanthin treated groups than in the control and HU groups. These results suggest that astaxanthin may be an effective treatment to counter the detrimental effects of a chronic decrease in skeletal muscle use on the capillary network and associated angiogenic pathways.

Nox4- and Nox2-dependent oxidant production is required for VEGF-induced SERCA cysteine-674 S-glutathiolation and endothelial cell migration

Endothelial cell (EC) migration in response to vascular endothelial growth factor (VEGF) is a critical step in both physiological and pathological angiogenesis. Although VEGF signaling has been extensively studied, the mechanisms by which VEGF-dependent reactive oxygen species (ROS) production affects EC signaling are not well understood. The aim of this study was to elucidate the involvement of Nox2- and Nox4-dependent ROS in VEGF-mediated EC Ca2+ regulation and migration. VEGF induced migration of human aortic ECs into a scratch wound over 6 h, which was inhibited by overexpression of either catalase or superoxide dismutase (SOD). EC stimulation by micromolar concentrations of H2O2 was inhibited by catalase, but also unexpectedly by SOD. Both VEGF and H2O2 increased S-glutathiolation of SERCA2b and increased Ca2+ influx into EC, and these events could be blocked by overexpression of catalase or overexpression of SERCA2b in which the reactive cysteine-674 was mutated to a serine. In determining the source of VEGF-mediated ROS production, our studies show that specific knockdown of either Nox2 or Nox4 inhibited VEGF-induced S-glutathiolation of SERCA, Ca2+ influx, and EC migration. Treatment with H2O2 induced S-glutathiolation of SERCA and EC Ca2+ influx, overcoming the knockdown of Nox4, but not Nox2, and Amplex red measurements indicated that Nox4 is the source of H2O2. These results demonstrate that VEGF stimulates EC migration through increased S-glutathiolation of SERCA and Ca2+ influx in a Nox4- and H2O2-dependent manner, requiring Nox2 downstream.

Over-expression of PaSOD in transgenic potato enhances photosynthetic performance under drought

Drought stress enhances the production of superoxide radical (O2 ._) and superoxide dismutase catalyses dismutation of it to H2O2 and O2, and hence provides a first line of defense against oxidative stress. Over-expression of a cytosolic copper-zinc superoxide dismutase, cloned from Potentilla atrosanguinea (PaSOD), in potato (Solanum tuberosum ssp. tuberosum L. cv. Kufri Sutlej) resulted in enhanced net photosynthetic rates (PN) and stomatal conductance (gs) compared to that in the wild type (WT) plants under control (irrigated) as well as drought stress conditions. Drought stress declined leaf water potential, PN, gs, photosystem II activity, and chlorophyll content, but increased proline and O2 ._ content more in WT than transgenic potato plants (SS5). The significantly higher SOD activity in SS5 coincided well with lower O2 ._ content suggesting its role in maintaining higher gs and PN in transgenic potato plants.

Cloning, overexpression, purification, and characterization of a new iron superoxide dismutase fromJatropha curcas

A novel iron superoxide dismutase (Fe-SOD) gene from Jatropha curcas was cloned and expressed in Escherichia coli BL21 (DE3). This recombinant enzyme was isolated by a combined procedure involving immobilized metal-ion affinity chromatography and ion-exchange chromatography. The apparent molecular mass of this purified enzyme (designated as JcFe-SOD) was 35 kDa on SDS-PAGE. The full-length complementary DNA sequence of JcFe-SOD contained a 918-bp open reading frame encoding a 305-amino-acid precursor of 34.589 kDa. The result of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry showed that the purified enzyme may own two forms: a dimer and a monomer. The enzyme was relatively stable and showed 54% activity when incubated in 70°C for 60 Min. JcFe-SOD was found to have good pH stability in the pH range of 5.5-9.5 at 25°C over 1 H incubation. The activity of this enzyme was gradually inhibited by increasing concentration of H₂O₂, 2-mercaptoethanol, and ethylenediaminetetraacetic acid. An assay of the atomic absorption spectrum showed the presence of 0.41 atom of Fe in each SOD subunit.

Overexpression of manganese superoxide dismutase ameliorates high-fat diet-induced insulin resistance in rat skeletal muscle

Elevated mitochondrial reactive oxygen species have been suggested to play a causative role in some forms of muscle insulin resistance. However, the extent of their involvement in the development of diet-induced insulin resistance remains unclear. To investigate, manganese superoxide dismutase (MnSOD), a key mitochondrial-specific enzyme with antioxidant modality, was overexpressed, and the effect on in vivo muscle insulin resistance induced by a high-fat (HF) diet in rats was evaluated. Male Wistar rats were maintained on chow or HF diet. After 3 wk, in vivo electroporation (IVE) of MnSOD expression and empty vectors was undertaken in right and left tibialis cranialis (TC) muscles, respectively. After one more week, insulin action was evaluated using hyperinsulinemic euglycemic clamp, and tissues were subsequently analyzed for antioxidant enzyme capacity and markers of oxidative stress. MnSOD mRNA was overexpressed 4.5-fold, and protein levels were increased by 70%, with protein detected primarily in the mitochondrial fraction of muscle fibers. This was associated with elevated MnSOD and glutathione peroxidase activity, indicating that the overexpressed MnSOD was functionally active. The HF diet significantly reduced whole body and TC muscle insulin action, whereas overexpression of MnSOD in HF diet animals ameliorated this reduction in TC muscle glucose uptake by 50% (P < 0.05). Decreased protein carbonylation was seen in MnSOD overexpressing TC muscle in HF-treated animals (20% vs. contralateral control leg, P < 0.05), suggesting that this effect was mediated through an altered redox state. Thus interventions causing elevation of mitochondrial antioxidant activity may offer protection against diet-induced insulin resistance in skeletal muscle.

Synergistic protection against hyperoxia-induced lung injury by neutrophils blockade and EC-SOD overexpression

BackgroundOxygen may damage the lung directly via generation of reactive oxygen species (ROS) or indirectly via the recruitment of inflammatory cells, especially neutrophils. Overexpression of extracellular superoxide dismutase (EC-SOD) has been shown to protect the lung against hyperoxia in the newborn mouse model. The CXC-chemokine receptor antagonist (Antileukinate) successfully inhibits neutrophil influx into the lung following a variety of pulmonary insults. In this study, we tested the hypothesis that the combined strategy of overexpression of EC-SOD and inhibiting neutrophil influx would reduce the inflammatory response and oxidative stress in the lung after acute hyperoxic exposure more efficiently than either single intervention.MethodsNeonate transgenic (Tg) (with an extra copy of hEC-SOD) and wild type (WT) were exposed to acute hyperoxia (95% FiO2 for 7 days) and compared to matched room air groups. Inflammatory markers (myeloperoxidase, albumin, number of inflammatory cells), oxidative markers (8-isoprostane, ratio of reduced/oxidized glutathione), and histopathology were examined in groups exposed to room air or hyperoxia. During the exposure, some mice received a daily intraperitoneal injection of Antileukinate.ResultsAntileukinate-treated Tg mice had significantly decreased pulmonary inflammation and oxidative stress compared to Antileukinate-treated WT mice (p < 0.05) or Antileukinate-non-treated Tg mice (p < 0.05).ConclusionCombined strategy of EC-SOD and neutrophil influx blockade may have a therapeutic benefit in protecting the lung against acute hyperoxic injury.

Longevity by design

Sagi and Kim used genome engineering to explore how lifespan could be enhanced in Caenorhabditis elegans. As expected, the individual overexpression of known C. elegans longevity-related genes increased lifespan. Importantly, this was also achieved by zebrafish transgenes that provide biochemical functions not found in C. elegans, such as an antibacterial lysozyme and a mitochondrial uncoupling protein. Combining up to four manipulations resulted in lifespan increases of up to 130%. The authors also characterized physiological features of long-lived strains, such as overexpression of superoxide dismutase 3 (SOD-3), as a possible rapid readout for longevity.

Activation of AMP-activated protein kinase alleviates High-glucose-induced dysfunction of brain microvascular endothelial cell tight-junction dynamics

The blood–brain barrier, formed by specialized brain endothelial cells that are interconnected by tight junctions, strictly regulates paracellular permeability to maintain an optimal extracellular environment for brain homeostasis. Diabetes is known to compromise the blood–brain barrier, although the underlying mechanism remains unknown. The aim of this study was to elucidate the molecular mechanisms underlying disruption of the blood–brain barrier in diabetes and to determine whether activation of AMP-activated protein kinase prevents diabetes-induced blood–brain barrier dysfunction. Exposure of human brain microvascular endothelial cells to high glucose (25mmol/L d-glucose), but not to high osmotic conditions (20mmol/L l-glucose plus 5mmol/L d-glucose), for 2h to 1 week significantly increased the permeability of the blood–brain barrier in parallel with lowered expression levels of zonula occludens-1, occludin, and claudin-5, three proteins that are essential to maintaining endothelial cell tight junctions. In addition, high glucose significantly increased the generation of superoxide anions. Adenoviral overexpression of superoxide dismutase or catalase significantly attenuated the high-glucose-induced reduction of endothelial cell tight-junction proteins. Furthermore, administration of apocynin reversed the effects of high glucose on endothelial cell tight-junction proteins. Finally, activation of AMP-activated protein kinase with 5-amino-4-imidazole carboxamide riboside or adenoviral overexpression of constitutively active AMP-activated protein kinase mutants abolished both the induction of NAD(P)H oxidase-derived superoxide anions and the tight-junction protein degradation induced by high glucose. We conclude that high glucose increases blood–brain barrier dysfunction in diabetes through induction of superoxide anions and that the activation of AMP-activated protein kinase protects the integrity of the blood–brain barrier by suppressing the induction of NAD(P)H oxidase-derived superoxide anions.

Nutrient‐dependent requirement for SOD1 in lifespan extension by protein restriction in Drosophila melanogaster

Reactive oxygen species (ROS) modulate aging and aging-related diseases. Dietary composition is critical in modulating lifespan. However, how ROS modulate dietary effects on lifespan remains poorly understood. Superoxide dismutase 1 (SOD1) is a major cytosolic enzyme responsible for scavenging superoxides. Here we investigated the role of SOD1 in lifespan modulation by diet in Drosophila. We found that a high sugar-low protein (HS-LP) diet or low-calorie diet with low-sugar content, representing protein restriction, increased lifespan but not resistance to acute oxidative stress in wild-type flies, relative to a standard base diet. A low sugar-high protein diet had an opposite effect. Our genetic analysis indicated that SOD1 overexpression or dfoxo deletion did not alter lifespan patterns of flies responding to diets. However, sod1 reduction blunted lifespan extension by the HS-LP diet but not the low-calorie diet. HS-LP and low-calorie diets both reduced target of rapamycin (TOR) signaling and only the HS-LP diet increased oxidative damage. sod1 knockdown did not affect phosphorylation of S6 kinase, suggesting that SOD1 acts in parallel with or downstream of TOR signaling. Surprisingly, rapamycin decreased lifespan in sod1 mutant but not wild-type males fed the standard, HS-LP, and low-calorie diets, whereas antioxidant N-acetylcysteine only increased lifespan in sod1 mutant males fed the HS-LP diet, when compared to diet-matched controls. Our findings suggest that SOD1 is required for lifespan extension by protein restriction only when dietary sugar is high and support the context-dependent role of ROS in aging and caution the use of rapamycin and antioxidants in aging interventions.

XIAP Inhibition and Generation of Reactive Oxygen Species Enhances TRAIL Sensitivity in Inflammatory Breast Cancer Cells

We recently identified superoxide dismutase (SOD) overexpression and decreased induction of reactive oxygen species (ROS)-mediated apoptosis in models of inflammatory breast cancer (IBC) cells with acquired therapeutic resistance. This population of cells has high expression of X-linked inhibitor of apoptosis protein (XIAP), which inhibits both extrinsic and intrinsic apoptosis pathways. We therefore wanted to evaluate the effect of classical apoptosis-inducing agent TRAIL, a proapoptotic receptor agonist that selectively triggers death receptor (DR)-mediated apoptosis in cancer cells, in the IBC acquired resistance model. XIAP levels and subsequent inhibition of caspase activity inversely correlated with TRAIL sensitivity in our models of IBC. These include SUM149, a basal-type cell line isolated from primary IBC tumors and isogenic SUM149-derived lines rSUM149 and SUM149 wtXIAP, models of acquired therapeutic resistance with endogenous and exogenous XIAP overexpression, respectively. Inhibition of XIAP function using embelin, a plant-derived cell permeable small molecule, in combination with TRAIL caused a synergistic decrease in cell viability. Embelin treatment resulted in activation of extracellular signal-regulated kinase (ERK)1/2 and ROS accumulation, which correlated with downregulation of antioxidant protein SOD1 and consumption of redox modulator reduced glutathione in the XIAP-overexpressing cells. Simultaneous treatment with an SOD mimic, which protects against ROS accumulation, reversed the decrease in cell viability caused by embelin + TRAIL treatment. Embelin primes IBC cells for TRAIL-mediated apoptosis by its direct action on the anti-caspase activity of XIAP and by shifting the cellular redox balance toward oxidative stress-mediated apoptosis. Thus, ROS modulators represent a novel approach to enhance efficacy of TRAIL-based treatment protocols in IBC.

Dantrolene is neuroprotective in vitro, but does not affect survival in SOD1G93A mice

Amyotrophic Lateral Sclerosis (ALS) is a devastating progressive neurodegenerative disease. One of the proposed disease mechanisms is excitotoxicity, in which excessive cytosolic calcium causes neuronal death. Although most calcium may originate from the extracellular space through activation of calcium-permeable AMPA receptors, we investigated in this study the contribution of endoplasmic reticulum calcium release by blocking the ryanodine receptor (RyR) using dantrolene. In vitro, dantrolene provides a significant protection to motor neurons exposed to a brief excitotoxic insult. However, daily administration of dantrolene to mice overexpressing superoxide dismutase 1 glycine to alanine at position 93 (SOD1G93A) does affect neither survival nor the number of motor neurons and ubiquitin aggregates indicating that calcium release through RyRs does not contribute to the selective motor neuron death in this animal model for ALS.

Isocitrate dehydrogenase 1 is downregulated during early skin tumorigenesis which can be inhibited by overexpression of manganese superoxide dismutase

Isocitrate dehydrogenase 1 (IDH1), a cytosolic enzyme that converts isocitrate to alpha-ketoglutarate, has been shown to be dysregulated during tumorigenesis. However, at what stage of cancer development IDH1 is dysregulated and how IDH1 may affect cell transformation and tumor promotion during early stages of cancer development are unclear. We used a skin cell transformation model and mouse skin epidermal tissues to study the role of IDH1 in early skin tumorigenesis. Our studies demonstrate that both the tumor promoter TPA and UVC irradiation decreased expression and activity levels of IDH1, not IDH2, in the tumor promotable JB6 P+ cell model. Skin epidermal tissues treated with dimethylbenz[α]anthracene/TPA also showed decreases in IDH1 expression and activity. In non-promotable JB6 P-cells, IDH1 was upregulated upon TPA treatment, whereas IDH2 was maintained at similar levels with TPA treatment. Interestingly, IDH1 knockdown enhanced, whereas IDH1 overexpression suppressed, TPA-induced cell transformation. Finally, manganese superoxide dismutase overexpression suppressed tumor promoter induced decreases in IDH1 expression and mitochondrial respiration, while intracellular alpha-ketoglutarate levels were unchanged. These results suggest that decreased IDH1 expression in early stage skin tumorigenesis is highly correlated with tumor promotion. In addition, oxidative stress might contribute to IDH1 inactivation, because manganese superoxide dismutase, a mitochondrial antioxidant enzyme, blocked decreases in IDH1 expression and activity.