Tumor necrosis factor alpha-mediated nitric oxide production enhances manganese superoxide dismutase nitration and mitochondrial dysfunction in primary neurons: an insight into the role of glial cells.

Tumor necrosis factor-alpha (TNF-alpha), a ubiquitous pro-inflammatory cytokine, is an important mediator in the immune-neuroendocrine system that affects the CNS. The present study demonstrates that treatment with TNF-alpha activates microglia to increase TNF-alpha production in primary cultures of glial cells isolated from wild-type (WT) mice and mice deficient in the inducible form of nitric oxide synthase (iNOSKO). However, mitochondrial dysfunction in WT neurons occurs at lower concentrations of TNF-alpha when neurons are directly treated with TNF-alpha or co-cultured with TNF-alpha-treated microglia than iNOSKO neurons similarly treated. Immunofluorescent staining of primary neurons co-cultured with TNF-alpha-treated microglia reveals that the antioxidant enzyme in mitochondria, manganese superoxide dismutase (MnSOD), is co-localized with nitrotyrosine in WT but not in iNOSKO primary neuronal cells. Importantly, the percentage of surviving neurons is significantly reduced in WT neurons compared with iNOSKO neurons under identical treatment conditions. Together, the results suggest that TNF-alpha activates microglia to produce high levels of TNF-alpha and that production of nitric oxide (NO) in neurons is an important factor affecting MnSOD nitration and subsequent mitochondrial dysfunction.

A neuronal model of Alzheimer's disease: an insight into the mechanisms of oxidative stress-mediated mitochondrial injury.

Alzheimer's disease (AD) is associated with beta-amyloid accumulation, oxidative stress and mitochondrial dysfunction. However, the effects of genetic mutation of AD on oxidative status and mitochondrial manganese superoxide dismutase (MnSOD) production during neuronal development are unclear. To investigate the consequences of genetic mutation of AD on oxidative damages and production of MnSOD during neuronal development, we used primary neurons from new born wild-type (WT/WT) and amyloid precursor protein (APP) (NLh/NLh) and presenilin 1 (PS1) (P264L) knock-in mice (APP/PS1) which incorporated humanized mutations in the genome. Increasing levels of oxidative damages, including protein carbonyl, 4-hydroxynonenal (4-HNE) and 3-nitrotyrosine (3-NT), were accompanied by a reduction in mitochondrial membrane potential in both developing and mature APP/PS1 neurons compared with WT/WT neurons suggesting mitochondrial dysfunction under oxidative stress. Interestingly, developing APP/PS1 neurons were significantly more resistant to beta-amyloid 1-42 treatment, whereas mature APP/PS1 neurons were more vulnerable than WT/WT neurons of the same age. Consistent with the protective function of MnSOD, developing APP/PS1 neurons have increased MnSOD protein and activity, indicating an adaptive response to oxidative stress in developing neurons. In contrast, mature APP/PS1 neurons exhibited lower MnSOD levels compared with mature WT/WT neurons indicating that mature APP/PS1 neurons lost the adaptive response. Moreover, mature APP/PS1 neurons had more co-localization of MnSOD with nitrotyrosine indicating a greater inhibition of MnSOD by nitrotyrosine. Overexpression of MnSOD or addition of MnTE-2-PyP(5+) (SOD mimetic) protected against beta-amyloid-induced neuronal death and improved mitochondrial respiratory function. Together, the results demonstrate that compensatory induction of MnSOD in response to an early increase in oxidative stress protects developing neurons against beta-amyloid toxicity. However, continuing development of neurons under oxidative damage conditions may suppress the expression of MnSOD and enhance cell death in mature neurons.

RelB regulates manganese superoxide dismutase gene and resistance to ionizing radiation of prostate cancer cells.

The relationship between NF-kappaB and resistance to radiation treatment in many tumor cell types has been generally well recognized. However, which members of the NF-kappaB family contribute to radiation resistance is unclear. In the present study, we demonstrate that RelB plays an important radioprotective role in aggressive prostate cancer cells, in part by the induction of antioxidant and antiapoptotic manganese superoxide dismutase (MnSOD) gene. RelB is both constitutively present and is inducible by radiation in aggressive prostate cancer cells. Using ectopically expressed dominant negative inhibitor, p100 mutant, and the siRNA approach, we demonstrate that selective inhibition of RelB significantly decreases the levels of MnSOD resulting in a significant increase in the sensitivity of prostate cancer cells to radiation treatment. These results demonstrate that RelB plays an important role in redox regulation of the cell and protects aggressive prostate cancer cells against radiation-induced cell death. Thus, inhibition of RelB could be a novel mechanism to radiosensitize prostate cancer.

The protective role of manganese superoxide dismutase against adriamycin-induced acute cardiac toxicity in transgenic mice.

Adriamycin (ADR) is a potent anticancer drug known to cause severe cardiac toxicity. Although ADR generates free radicals, the role of free radicals in the development of cardiac toxicity and the intracellular target for ADR-induced cardiac toxicity are still not well understood. We produced three transgenic mice lines expressing increased levels of human manganese superoxide dismutase (MnSOD), a mitochondrial enzyme, as an animal model to investigate the role of ADR-mediated free radical generation in mitochondria. The human MnSOD was expressed, functionally active, and properly transported into mitochondria in the heart of transgenic mice. The levels of copper-zinc SOD, catalase, and glutathione peroxidase did not change in the transgenic mice. Electron microscopy revealed dose-dependent ultrastructural alterations with marked mitochondrial damage in nontransgenic mice treated with ADR, but not in the transgenic littermates. Biochemical analysis indicated that the levels of serum creatine kinase and lactate dehydrogenase in ADR-treated mice were significantly greater in nontransgenic than their transgenic littermates expressing a high level of human MnSOD after ADR treatment. These results support a major role for free radical generation in ADR toxicity as well as suggesting mitochondria as the critical site of cardiac injury.

A novel redox regulator, MnTnBuOE-2-PyP5+, enhances normal hematopoietic stem/progenitor cell function.

The signaling of reactive oxygen species (ROS) is essential for the maintenance of normal cellular function. However, whether and how ROS regulate stem cells are unclear. Here, we demonstrate that, in transgenic mice expressing the human manganese superoxide dismutase (MnSOD) gene, a scavenger of ROS in mitochondria, the number and function of mouse hematopoietic stem/progenitor cells (HSPC) under physiological conditions are enhanced. Importantly, giving MnTnBuOE-2-PyP5+(MnP), a redox- active MnSOD mimetic, to mouse primary bone marrow cells or to C57B/L6 mice significantly enhances the number of HSPCs. Mechanistically, MnP reduces superoxide to hydrogen peroxide, which activates intracellular Nrf2 signaling leading to the induction of antioxidant enzymes, including MnSOD and catalase, and mitochondrial uncoupling protein 3. The results reveal a novel role of ROS signaling in regulating stem cell function, and suggest a possible beneficial effect of MnP in treating pathological bone marrow cell loss and in increasing stem cell population for bone marrow transplantation.

Manganese superoxide dismutase deficiency triggers mitochondrial uncoupling and the Warburg effect.

This corrects the article DOI: 10.1038/onc.2014.355.

Complementary DNA encoding human colon cancer manganese superoxide dismutase and the expression of its gene in human cells.

Manganese superoxide dismutase (MnSOD) is a member of a family of metalloenzymes that catalyze the dismutation of the superoxide anion to H2O2. It has been shown that MnSOD activity in tumor cells is lower than that in their normal counterparts. To investigate the molecular basis for the reduced level of MnSOD activity in human tumor cells, the primary structure of human MnSOD has been determined from complementary DNA (cDNA) isolated from a human colon carcinoma (HT-29) cDNA library. The sequence of the mature protein is composed of 198 amino acids preceded by a 24-amino acid leader peptide. DNA sequence analysis revealed that the translated region of the human tumor MnSOD is virtually identical to the MnSOD sequence isolated from normal human sources but exhibits differences in both the 5'- and 3'-untranslated regions. DNA blot analysis of genomic DNA isolated from HT-29, simian virus-transformed human lung fibroblast (SV-40/WI-38), and parental human lung fibroblast (WI-38) cells showed an identical pattern of hybridization to that of MnSOD cDNA. RNA blot analysis revealed that tumor cells have lower levels of MnSOD mRNA. However, the half-life of the mRNA was the same (approximately 10 h) in tumor and normal cells. Immunological measurement of the level of MnSOD in both normal and tumor cells also showed a reduced level of MnSOD protein in the tumor cells. These results suggest that the reduced level of MnSOD activity observed in human tumor cells is not due to a defect in the primary structure of the MnSOD protein, a change in the dosage of the MnSOD gene, or a decrease in the stability of MnSOD mRNA in tumor cells but rather is due to a defect or defects in the expression of the gene.

Effect of the Bowman-Birk protease inhibitor on the expression of oncogenes in the irradiated rat colon.

In this study, we tested the influence of i.p. Bowman-Birk protease inhibitor (BBI) administration on oncogene expression in unirradiated and irradiated rat colonic mucosa. Total cellular RNA was collected from the colonic mucosa, and the levels of c-myc, c-fos, c-Ha-ras, c-EGFR, and c-actin mRNA were examined by standard dot and Northern blot analyses. The data demonstrate that BBI is capable of preventing radiation-induced overexpression of c-myc and c-fos without interfering with the constitutive expression of these 2 genes. It was also determined that BBI did not interfere with either radiation-induced overexpression of c-Ha-ras and c-EGFR or the constitutive expression of c-Ha-ras, c-EGFR, or c-actin. The data demonstrate that the anticarcinogenic BBI selectively inhibits the overexpression of c-myc and c-fos while not affecting crypt cell proliferation. These results suggest that a protease is involved in the pathway for enhanced c-myc and c-fos expression and that protease inhibitors such as BBI can interrupt this pathway.

Overexpression of manganese superoxide dismutase selectively modulates the activity of Jun-associated transcription factors in fibrosarcoma cells.

Manganese superoxide dismutase (MnSOD) is reduced in a variety of tumor cells and has been proposed to be a new type of tumor suppressor gene. The mechanism(s) by which MnSOD suppresses cancer development is currently unknown. However, expression of this antioxidant might play a significant role in maintaining cellular redox status. The relationship between MnSOD expression and modulation of DNA-binding activity and transcriptional activation of redox-sensitive oncoproteins and tumor suppressor proteins was studied in a murine fibrosarcoma cell line (FSa-II). Electrophoretic mobility shift assay and transcriptional activation studies revealed an inverse correlation between MnSOD expression and activity of c-jun-associated transcription factors, activator protein 1 and cyclic AMP-responsive element binding protein. Furthermore, expression of an activator protein 1 target gene, bcl-xL, was decreased in MnSOD-transfected cell lines. The results suggest that overexpression of MnSOD may exert its tumor suppressor activity, in part, by modulation of specific oncogenes.

Suppression of fibrosarcoma metastasis by elevated expression of manganese superoxide dismutase.

A mouse fibrosarcoma cell line (FSa-II), which exhibits low endogenous levels of manganese superoxide dismutase, was transfected with a human manganese superoxide dismutase complementary DNA. Fifty clones were screened for manganese superoxide dismutase activity by the superoxide dismutase activity gel assay. Activity of the positive clones was measured by the nitro blue tetrazolium-reduction assay in the presence of cyanide. Three cell lines exhibiting a range of activity were chosen to be transplanted into syngeneic mice. The results indicated that the metastasis rate for all transfected cells was significantly less than that of control cells.

Manganese-containing superoxide dismutase overexpression causes phenotypic reversion in SV40-transformed human lung fibroblasts.

Manganese superoxide dismutase (MnSOD) has been found to be low in a wide range of tumor cells as well as in vitro-transformed cell lines and has been implicated as a new type of tumor suppressor gene. The relationship between MnSOD activity and the malignant phenotype was studied by transfection of MnSOD cDNA into the SV40-transformed human fibroblast cell line WI-38 VA13 subline 2RA. The integration and expression of the exogenous MnSOD cDNA was confirmed in three selected clones with a 2-3.5-fold increase in MnSOD activity. The effect of elevated expression of MnSOD on the cell phenotype was determined by observing growth characteristics. Compared with the parental and neo control cells, the MnSOD-overexpressing clones had a slower growth rate, lower plating efficiency, increased anchorage dependence, and morphological differences. These changes were correlated strongly with the level of MnSOD activity. The results suggest that an increase of MnSOD activity can reverse part of the malignant phenotype in SV40-transformed human fibroblast cells. A possible mechanism is that overexpression of MnSOD might alter the intracellular redox state by modulation of the balance of reactive oxygen species.

Expression of manganese superoxide dismutase reduces tumor control radiation dose: gene-radiotherapy.

This study investigated the in vitro and in vivo radiation response of tumor cells transfected with human manganese superoxide dismutase (MnSOD) cDNA. A major objective was to test the potential tumor suppressive effect of MnSOD in vivo. Tumor cells studied were an in vitro line derived from a murine spontaneous fibrosarcoma, FSa-II, which expressed an undetectable MnSOD activity. These cells were transfected with pSV2-NEO plasmid (NEO line) or cotransfected with MnSOD plasmid plus pSV2-NEO plasmid (SOD lines) as described previously. The cell lines used were SOD-L and SOD-H, which expressed, respectively, low and high MnSOD activities after transfection, and NEO and parental FSa-II controls. Both SOD-L and SOD-H cell lines were slightly more resistant to ionizing radiation than were the two control cell lines when irradiated in vitro in the presence of oxygen. The dose-modifying factors calculated at the survival level of 0.01 were 1.13 and 1.15 for the SOD-L and SOD-H cells, respectively. To investigate potential tumor suppressive effects, animal tumors of 4 mm diameter were irradiated in vivo under hypoxic conditions, and the radiation dose to control one-half of the irradiated tumors (TCD50) was determined for each tumor. The TCD50S obtained on the basis of the tumor control rate in 120 days after irradiation were substantially lower for the SOD-H and SOD-L tumors compared to the NEO tumors. They were 22.9, 28.6, and 47.5 Gy for SOD-H, SOD-L and NEO tumors, respectively. To analyze these data, survival curves were obtained for hypoxic cells by irradiating NEO and SOD-H tumors under hypoxic conditions in vivo and assaying in vitro. Analysis of these curves suggests that the decrease in the TCD50S of SOD tumors is attributable to the reduced tumorigenicity in these tumors. The hypoxic cell survival curves also showed that SOD did not protect cells from radiation in the absence of oxygen. Electron microscopy showed no morphological differences between these cells. These results suggest that the fraction of tumorigenic cells could be reduced by expression of MnSOD, resulting in a substantial decrease in the TCD50.

Suppression of the malignant phenotype of human glioma cells by overexpression of manganese superoxide dismutase.

Manganese superoxide dismutase (MnSOD) has been previously shown to suppress the malignant phenotype of human melanoma and breast cancer cells. To test the possible role of MnSOD in glioma malignancy, MnSOD was overexpressed in wild type human glioma U118 cells and subcloned U118-9 cells by transfection of human MnSOD cDNA. The MnSOD-transfected cell lines demonstrated expression of exogenous (plasmid) MnSOD mRNA, increase in MnSOD immunoreactive protein, and a three- to eightfold increase in MnSOD enzymatic activity. The MnSOD overexpressing cell lines became less malignant as demonstrated by requiring a higher serum concentration to grow in vitro and much slower tumor growth in nude mice than the parental and neo control cell lines. These findings further support the hypothesis that MnSOD may be a tumor suppressor gene in a wide variety of human tumors.

Phenotypic changes induced in human breast cancer cells by overexpression of manganese-containing superoxide dismutase.

Human manganese containing superoxide dismutase (MnSOD) cDNA was transfected into a human breast cancer cell line (MCF-7) in order to examine the effect of increased functional MnSOD on the cellular phenotype. A MnSOD-overexpressing clone was compared to control vector-transfected cells and to wild type MCF-7 cells. Southern blotting indicated incorporation of MnSOD cDNA into genomic DNA in the MnSOD overexpressing cell line. The MnSOD overexpressing cell line showed a 5.7-fold increase in MnSOD activity compared to wild type MCF-7 cells. Similar increases in MnSOD immunoreactive protein and mRNA levels were observed by Western and Northern blotting as well as using RT-PCR. The plating efficiency of cells grown in different concentrations of serum (1 to 20%) was decreased in the MnSOD overexpressing cell line. The clonogenic fraction in soft agar culture was also decreased after MnSOD cDNA transfection. When inoculated in nude mice, tumor growth was markedly inhibited in MnSOD overexpressing cells compared to wild type MCF-7 cells or plasmid control cells. These results support the hypothesis that increased MnSOD expression suppresses the malignant phenotype of human breast cancer cells and suggests that the MnSOD gene is a tumor suppressor gene in human breast cancer.

Mutations in the promoter reveal a cause for the reduced expression of the human manganese superoxide dismutase gene in cancer cells.

Manganese superoxide dismutase (MnSOD) has been shown to play an important role in preventing the development of cancer. MnSOD activity is reduced in many transformed cells and tumor tissues. We previously showed that the reduced level of MnSOD activity in cancer cells was not due to a defect in the primary structure of MnSOD protein, but rather was due to defects in gene expression. To elucidate the cause for the reduced expression of human MnSOD in cancer, we investigated the nucleotide sequence in the regulatory region of the MnSOD gene in a normal human cell line and various human tumor cell lines. A DNA fragment spanning 3.4 kb 5' flanking region of the MnSOD gene isolated from a normal human genomic DNA library was used to determine the DNA sequence of MnSOD promoter. PCR primers were used for amplification of the 3.4 kb 5' flanking region of the human MnSOD gene in cancer cells. Sequence analysis identified three heterozygous mutations in the proximal region of the promoter in five human tumor cell lines. These mutations, clustered around the GC-rich region of the human MnSOD promoter, change the binding pattern of AP-2 and lead to a reduction in transcription activity using a luciferase reporter assay system. These results suggest that the reduced level of MnSOD expression in some tumor cells is, at least in part, due to a defect in the DNA sequence of the promoter region.

Manganese superoxide dismutase deficiency triggers mitochondrial uncoupling and the Warburg effect.

Manganese superoxide dismutase (MnSOD) is a mitochondrially localized primary antioxidant enzyme, known to be essential for the survival of aerobic life and to have important roles in tumorigenesis. Here, we show that MnSOD deficiency in skin tissues of MnSOD-heterozygous knockout (Sod2(+/-)) mice leads to increased expresson of uncoupling proteins (UCPs). When MnSOD is deficient, superoxide radical and its resulting reactive oxygen species (ROS) activate ligand binding to peroxisome proliferator-activated receptor alpha (PPARα), suggesting that the activation of PPARα signaling is a major mechanism underlying MnSOD-dependent UCPs expression that consequently triggers the PI3K/Akt/mTOR pathway, leading to increased aerobic glycolysis. Knockdown of UCPs and mTOR suppresses lactate production and increases ATP levels, suggesting that UCPs contribute to increased glycolysis. These results highlight the existence of a free radical-mediated mechanism that activates mitochondria uncoupling to reduce ROS production, which precedes the glycolytic adaptation described as the Warburg Effect.

Mitochondrial SOD2 regulates epithelial-mesenchymal transition and cell populations defined by differential CD44 expression.

Epithelial-mesenchymal transition (EMT) promotes cancer cell invasion, metastasis and treatment failure. EMT may be activated in cancer cells by reactive oxygen species (ROS). EMT may promote conversion of a subset of cancer cells from a CD44(low)-CD24(high) (CD44L) epithelial phenotype to a CD44(high)-CD24(-/low) (CD44H) mesenchymal phenotype, the latter associated with increased malignant properties of cancer cells. ROS are required for cells undergoing EMT, although excessive ROS may induce cell death or senescence; however, little is known as to how cellular antioxidant capabilities may be regulated during EMT. Mitochondrial superoxide dismutase 2 (SOD2) is frequently overexpressed in oral and esophageal cancers. Here, we investigate mechanisms of SOD2 transcriptional regulation in EMT, as well as the functional role of this antioxidant in EMT. Using well-characterized genetically engineered oral and esophageal human epithelial cell lines coupled with RNA interference and flow cytometric approaches, we find that transforming growth factor (TGF)-ß stimulates EMT, resulting in conversion of CD44L to CD44H cells, the latter of which display SOD2 upregulation. SOD2 induction in transformed keratinocytes was concurrent with suppression of TGF-ß-mediated induction of both ROS and senescence. SOD2 gene expression appeared to be transcriptionally regulated by NF-κB and ZEB2, but not ZEB1. Moreover, SOD2-mediated antioxidant activity may restrict conversion of CD44L cells to CD44H cells at the early stages of EMT. These data provide novel mechanistic insights into the dynamic expression of SOD2 during EMT. In addition, we delineate a functional role for SOD2 in EMT via the influence of this antioxidant upon distinct CD44L and CD44H subsets of cancer cells that have been implicated in oral and esophageal tumor biology.

Overproduction of human Mn-superoxide dismutase modulates paraquat-mediated toxicity in mammalian cells.

Manganese superoxide dismutase (MnSOD) is a nuclear encoded mitochondrial matrix enzyme that functions to scavenge superoxide radicals. The human MnSOD cDNA under the transcriptional control of a human beta-actin promoter was introduced into mouse C3H10T1/2 cells by cotransfection with a recombinant plasmid containing the NeoR selectable marker. C3H10T1/2 transformants (C3H-SOD) were obtained that expressed high levels of authentic enzymatically active human MnSOD. Overexpression of the MnSOD gene did not affect the protein levels of CuZnSOD, catalase (CAT), or glutathione peroxidase (GPX) in the transformants. Treatment of cells with paraquat was less toxic to the C3H-SOD cells than to the control cells. These results are consistent with the possibility that superoxide radicals are mediators of paraquat cytotoxicity.

Antioxidant and oxidative status in tissues of manganese superoxide dismutase transgenic mice.

Manganese superoxide dismutase (Mn-SOD) plays an important role in attenuating free radical-induced oxidative damage. The purpose of this research was to determine if increased expression of Mn-SOD gene alters intracellular redox status. Twelve week old male B6C3 mice, engineered to express human Mn-SOD in multiple organs, and their nontransgenic littermates were assessed for oxidative stress and antioxidant status in heart, brain, lung, skeletal muscle, liver, and kidney. Relative to their nontransgenic littermates, transgenic mice had significantly (p <.01) higher activity of Mn-SOD in heart, skeletal muscle, lung, and brain. Copper, zinc (Cu,Zn)-SOD activity was significantly higher in kidney, whereas catalase activity was lower in brain and liver. The activities of selenium (Se)-GSH peroxidase and non-Se-GSH peroxidase, and levels of vitamin E, ascorbic acid and GSH were not significantly different in any tissues measured between Mn-SOD transgenic mice and their nontransgenic controls. The levels of malondialdehyde were significantly lower in the muscle and heart of Mn-SOD mice, and conjugated dienes and protein carbonyls were not altered in any tissues measured. The results obtained showed that expression of human SOD gene did not systematical alter antioxidant systems or adversely affect the redox state of the transgenic mice. The results also suggest that expression of human SOD gene confers protection against peroxidative damage to membrane lipids.

Expression of manganese superoxide dismutase promotes cellular differentiation.

Manganese superoxide dismutase (MnSOD) is a nuclear encoded mitochondrial matrix enzyme that scavenges toxic superoxide radicals. It has been shown that increased generation of reactive oxygen species is associated with the differentiation of microorganisms. To test the hypothesis that the ability of mitochondrial superoxide dismutase to neutralize a cellular hyperoxidant state is important for differentiation of mammalian cells, we examined the effect of transfection of MnSOD into mouse embryo fibroblasts on cellular differentiation. C3H10T1/2 cells served as a model for differentiation because these cells can be triggered to differentiate into myoblasts, adipocytes, and chondrocytes by treatment with 5-azacytidine. In this report, myoblast differentiation was defined by the presence of multinucleated cells, appearance of Z-bands, and expression of actin and desmin in the differentiated cells. Transfection of MnSOD gene was found to greatly enhance differentiation of C3H10T1/2 cells into myoblasts by 5-azacytidine. This result identifies MnSOD as an important factor for cell differentiation and supports a role for reactive oxygen species in the process of cellular differentiation.