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

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

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.

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.

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.

Redox proteomic identification of HNE-bound mitochondrial proteins in cardiac tissues reveals a systemic effect on energy metabolism after doxorubicin treatment.

Doxorubicin (DOX), one of the most effective anticancer drugs, is known to generate progressive cardiac damage, which is due, in part, to DOX-induced reactive oxygen species (ROS). The elevated ROS often induce oxidative protein modifications that result in alteration of protein functions. This study demonstrates that the level of proteins adducted by 4-hydroxy-2-nonenal (HNE), a lipid peroxidation product, is significantly increased in mouse heart mitochondria after DOX treatment. A redox proteomics method involving two-dimensional electrophoresis followed by mass spectrometry and investigation of protein databases identified several HNE-modified mitochondrial proteins, which were verified by HNE-specific immunoprecipitation in cardiac mitochondria from the DOX-treated mice. The majority of the identified proteins are related to mitochondrial energy metabolism. These include proteins in the citric acid cycle and electron transport chain. The enzymatic activities of the HNE-adducted proteins were significantly reduced in DOX-treated mice. Consistent with the decline in the function of the HNE-adducted proteins, the respiratory function of cardiac mitochondria as determined by oxygen consumption rate was also significantly reduced after DOX treatment. Treatment with Mn(III) meso-tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin, an SOD mimic, averted the doxorubicin-induced mitochondrial dysfunctions as well as the HNE-protein adductions. Together, the results demonstrate that free radical-mediated alteration of energy metabolism is an important mechanism mediating DOX-induced cardiac injury, suggesting that metabolic intervention may represent a novel approach to preventing cardiac injury after chemotherapy.

Manganese superoxide dismutase is a mitochondrial fidelity protein that protects Polγ against UV-induced inactivation.

Manganese superoxide dismutase is a nuclear encoded primary antioxidant enzyme localized exclusively in the mitochondrial matrix. Genotoxic agents, such as ultraviolet (UV) radiation, generates oxidative stress and cause mitochondrial DNA (mtDNA) damage. The mtDNA polymerase (Polγ), a major constituent of nucleoids, is responsible for the replication and repair of the mitochondrial genome. Recent studies suggest that the mitochondria contain fidelity proteins and MnSOD constitutes an integral part of the nucleoid complex. However, it is not known whether or how MnSOD participates in the mitochondrial repair processes. Using skin tissue from C57BL/6 mice exposed to UVB radiation, we demonstrate that MnSOD has a critical role in preventing mtDNA damage by protecting the function of Polγ. Quantitative-PCR analysis shows an increase in mtDNA damage after UVB exposure. Immunofluorescence and immunoblotting studies demonstrate p53 translocation to the mitochondria and interaction with Polγ after UVB exposure. The mtDNA immunoprecipitation assay with Polγ and p53 antibodies in p53(+/+) and p53(-/-) mice demonstrates an interaction between MnSOD, p53 and Polγ. The results suggest that these proteins form a complex for the repair of UVB-associated mtDNA damage. The data also demonstrate that UVB exposure injures the mtDNA D-loop in a p53-dependent manner. Using MnSOD-deficient mice we demonstrate that UVB-induced mtDNA damage is MnSOD dependent. Exposure to UVB results in nitration and inactivation of Polγ, which is prevented by addition of the MnSOD mimetic Mn(III)TE-2-PyP(5+). These results demonstrate for the first time that MnSOD is a fidelity protein that maintains the activity of Polγ by preventing UVB-induced nitration and inactivation of Polγ. The data also demonstrate that MnSOD has a role along with p53 to prevent mtDNA damage.

Doxorubicin-induced central nervous system toxicity and protection by xanthone derivative of Garcinia mangostana.

Doxorubicin (Dox) is a potent, broad-spectrum chemotherapeutic drug used around the world. Despite its effectiveness, it has a wide range of toxic side effects, many of which most likely result from its inherent pro-oxidant activity. It has been reported that Dox has toxic effects on normal tissues, including brain tissue. The present study tested the protective effect of a xanthone derivative of Garcinia Mangostana against Dox-induced neuronal toxicity. Xanthone can prevent Dox from causing mononuclear cells to increase the level of tumor necrosis factor-alpha (TNFα). We show that xanthone given to mice before Dox administration suppresses protein carbonyl, nitrotyrosine and 4-hydroxy-2'-nonenal (4HNE)-adducted proteins in brain tissue. The levels of the pro-apoptotic proteins p53 and Bax and the anti-apoptotic protein Bcl-xL were significantly increased in Dox-treated mice compared with the control group. Consistent with the increase of apoptotic markers, the levels of caspase-3 activity and TUNEL-positive cells were also increased in Dox-treated mice. Pretreatment with xanthone suppressed Dox-induced increases in all indicators of injury tested. Together, the results suggest that xanthone prevents Dox-induced central nervous system toxicity, at least in part, by suppression of Dox-mediated increases in circulating TNFα. Thus, xanthone is a good candidate for prevention of systemic effects resulting from reactive oxygen generating anticancer therapeutics.

Alterations in brain antioxidant enzymes and redox proteomic identification of oxidized brain proteins induced by the anti-cancer drug adriamycin: implications for oxidative stress-mediated chemobrain.

Adriamycin (ADR) is a chemotherapeutic for the treatment of solid tumors. This quinone-containing anthracycline is well known to produce large amounts of reactive oxygen species (ROS) in vivo. A common complaint of patients undergoing long-term treatment with ADR is somnolence, often referred to as "chemobrain." While ADR itself does not cross the blood brain barrier (BBB), we recently showed that ADR administration causes a peripheral increase in tumor necrosis factor alpha (TNF-alpha), which migrates across the BBB and leads to inflammation and oxidative stress in brain, most likely contributing to the observed decline in cognition. In the current study, we measured levels of the antioxidant glutathione (GSH) in brains of mice injected intraparitoneally (i.p.) with ADR, as well as the levels and activities of several enzymes involved in brain GSH metabolism. We observed significantly decreased GSH levels, as well as altered GSH/GSSG ratio in brains of ADR treated mice relative to saline-treated controls. Also observed in brains of ADR treated mice were increased levels of glutathione peroxidase (GPx), glutathione-S-transferase (GST), and glutathione reductase (GR). We also observed increased activity of GPx, but a significant reduction in GST and GR activity in mice brain, 72 h post i.p. injection of ADR (20 mg/kg body weight). Furthermore, we used redox proteomics to identify specific proteins that are oxidized and/or have differential levels in mice brains as a result of a single i.p. injection of ADR. Visinin like protein 1 (VLP1), peptidyl prolyl isomerase 1 (Pin1), and syntaxin 1 (SYNT1) showed differential levels in ADR treated mice relative to saline-treated controls. Triose phosphate isomerase (TPI), enolase, and peroxiredoxin 1 (PRX-1) showed significantly increased specific carbonylation in ADR treated mice brain. These results further support the notion ADR induces oxidative stress in brain despite not crossing the BBB, and that antioxidant intervention may prevent ADR-induced cognitive 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.

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.

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.

TPA-activated transcription of the human MnSOD gene: role of transcription factors Sp-1 and Egr-1.

Induction of manganese superoxide dismutase (MnSOD) in response to oxidative stress has been well established in animals, tissues, and cell culture. However, the role of the human MnSOD (hMnSOD) promoter in stimulus-dependent activation of transcription is unknown. The hMnSOD promoter lacks both a TATA and a CAAT box but possesses several GC motifs. In a previous study, we showed that the basal promoter contains multiple Sp1 and AP-2 binding sites and that Sp1 is essential for the constitutive expression of the hMnSOD gene. In this study, we identified an Egr-1 binding site in the basal promoter of hMnSOD. We also found that the basal promoter is responsive to 12-O-tetradecanoylphorbol-13-acetate (TPA)-activated hMnSOD transcription in the human hepatocarcinoma cell line HepG2. The contributions of these binding sites and the roles of the transcription factors Egr-1, AP-2, and Sp1 in the activation of hMnSOD transcription by TPA were investigated by site-directed mutation analysis, Western blotting, and overexpression of transcription factors. The results showed that Sp1 plays a positive role for both basal and TPA-activated hMnSOD transcription, whereas overexpression of Egr-1 has a negative role in the basal promoter activity without any effect on TPA-mediated activation of hMnSOD transcription.

Overexpression of MnSOD protects murine fibrosarcoma cells (FSa-II) from apoptosis and promotes a differentiation program upon treatment with 5-azacytidine: involvement of MAPK and NFkappaB pathways.

Stable transfection of neomycin and human manganese superoxide dismutase (MnSOD2) expression plasmids into a murine fibrosarcoma cell line (FSa-II) was previously done in our laboratory. Treatment with 10 microM 5-azacytidine induced apoptosis in the control cell line (NEO), whereas the MnSOD-overexpressing cell line (SOD-H) demonstrated differentiated-appearing morphology. The levels of the myogenic transcription factor, MyoD, and the muscle-specific marker, alpha-actin, were increased over time with 5-azacytidine treatment in the SOD-H cell line. Nuclear transcription factor NFkappaB was activated in the SOD-H cell line, whereas inhibition of NFkappaB activation reduced the levels of MyoD and alpha-actin. Members of mitogen-activated protein kinase pathway and the Raf1/MEK/ERK cascade were shown to play a positive role in this event. Overexpression of MnSOD not only can protect cells from the toxic effects of 5-azacytidine, but can also promote the fibrosarcoma cells to enter a differentiation program.

Overexpression of manganese superoxide dismutase gene suppresses spontaneous apoptosis without a resultant alteration in in vivo growth of the mouse fibrosarcoma, FSa-II.

The relationship between spontaneous apoptosis and overexpression of manganese superoxide dismutase (MnSOD) gene was examined in vivo. The mouse fibrosarcoma cells expressing high MnSOD activities due to transfection with the human MnSOD cDNA (SOD-H), or the fibrosarcoma cells transfected with the selectable marker alone (NEO), were transplanted into immune-deficient Fox Chase SCID C.B-17/Icr-scid Jcl mice. Apoptosis in tumors was visually quantified by the in situ end-labeling method. The number of apoptotic cells in the SOD-H tumors was significantly less than that in the NEO tumors. The tumor growth time of the SOD-H tumors to grow from 34 to 500 mm3 in one-half of the mice was slightly longer than that of the NEO tumors, but the difference was not statistically significant. These results suggest that overexpression of MnSOD gene is involved in the suppression of spontaneous apoptosis, without a resultant alteration in the tumor growth.

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.

An intronic NF-kappaB element is essential for induction of the human manganese superoxide dismutase gene by tumor necrosis factor-alpha and interleukin-1beta.

Tumor necrosis factor-alpha (TNF) and interleukin-1beta (IL-1) are cytokines that induce expression of various genes through activation of the redox-sensitive transcription factor nuclear factor-kappaB (NF-kappaB). We have previously cloned the entire human MnSOD (SOD2) gene and found several NF-kappaB-binding sites in the 5' and 3' flanking and intronic regions. To test whether these putative NF-kappaB-binding sites are able to respond to TNF and IL-1, we performed induction analysis using various deletion constructs ligated to a luciferase reporter gene. We found that the 5' and 3' flanking regions containing several NF-kappaB-binding sites do not mediate MnSOD induction by TNF or IL-1. When a 342-bp intron 2 fragment containing NF-kappaB, C/EBP, and NF-1 binding sites was linked to the basal promoter of the SOD2 gene, transcriptional activities were significantly increased in response to TNF and IL-1 in an orientation- and position-independent manner. To accurately identify the element that is most critical for the enhancer activity, deletions and specific mutations of each individual site were studied. The results indicated that the NF-kappaB binding site is essential but not sufficient for TNF- or IL-1-mediated induction. Furthermore, NF-kappaB elements in the 5' and 3' flanking regions could be made to function in TNF or IL-1 induction when they were transposed to the intronic fragment. Taken together, these results suggest that an NF-kappaB element and its location in the SOD2 gene is critical for TNF/IL-1-mediated induction. However, a complex interaction between NF-kappaB and other transcription elements is needed for a high-level induction.

Overexpression of manganese superoxide dismutase protects against mitochondrial-initiated poly(ADP-ribose) polymerase-mediated cell death.

Mitochondria have recently been shown to serve a central role in programmed cell death. In addition, reactive oxygen species (ROS) have been implicated in cell death pathways upon treatment with a variety of agents; however, the specific cellular source of the ROS generation is unknown. We hypothesize that mitochondria-derived free radicals play a critical role in apoptotic cell death. To directly test this hypothesis, we treated murine fibrosarcoma cell lines, which expressed a range of mitochondrial manganese superoxide dismutase (MnSOD) activities, with respiratory chain inhibitors. Apoptosis was confirmed by DNA fragmentation analysis and electron microscopy. MnSOD overexpression specifically protected against cell death upon treatment with rotenone or antimycin. We examined bcl-x(L), p53 and poly(ADP-ribose) polymerase (PARP) to identify specific cellular pathways that might contribute to the mitochondrial-initiated ROS-mediated cell death. Cells overexpressing MnSOD contained less bcl-x(L) within the mitochondria compared to control (NEO) cells, therefore excluding the role of bcl-x(L). p53 was undetectable by Western analysis and examination of the proapoptotic protein bax, a p53 target gene, did not increase with treatment. Activation of caspase-3 (CPP-32) occurred in the NEO cells independent of cytochrome c release from the mitochondria. PARP, a target protein of CPP-32 activity, was cleaved to a 64 kDa fragment in the NEO cells prior to generation of nucleosomal fragments. Taken together, these findings suggest that mitochondrial-mediated ROS generation is a key event by which inhibition of respiration causes cell death, and identifies CPP-32 and the PARP-linked pathway as targets of mitochondrial-derived ROS-induced cell death.

Exacerbation of damage and altered NF-kappaB activation in mice lacking tumor necrosis factor receptors after traumatic brain injury.

Tumor necrosis factor alpha (TNFalpha) is widely expressed in both neurons and glia and has been shown to be upregulated after traumatic brain injury (TBI). TNFalpha receptor activation results in activation of the transcription factor nuclear factor kappaB (NF-kappaB), which may serve an antiapoptotic role via the induction of target genes manganese superoxide dismutase (MnSOD) and/or calbindin. In the present study, we used a controlled cortical impact model of TBI with pertinent lines of transgenic mice to combine both morphological characterization and molecular analysis to elucidate the role of TNFalpha after TBI. Measurements of both the lesion volume and the blood-brain barrier breach indicated exacerbations in mice rendered genetically deficient in both the p55 and p75 TNFalpha receptors (TNFR-KO) compared with wild-type animals. Additionally, animals genetically altered to overexpress MnSOD showed a significant decrease in lesion volume compared with that of control littermates, whereas no alterations were observed in mice lacking the calcium-binding protein calbindin D28k. Analysis of NF-kappaB activation and relative levels of MnSOD revealed delayed responses in the injured cortex of TNFR-KO animals compared with wild-type animals, implying that endogenous TNFalpha may be neuroprotective after TBI.

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 protects mitochondrial complex I against adriamycin-induced cardiomyopathy in transgenic mice.

Adriamycin (ADR) is a potent anticancer drug that causes severe cardiomyopathy. We have previously demonstrated that ADR-induced ultrastructural mitochondrial injury in the heart was attenuated in manganese superoxide dismutase (MnSOD) transgenic mice. To further investigate the biochemical mechanisms by which MnSOD protected mitochondria against ADR-induced damage, cardiac mitochondrial function and activities were evaluated. The results showed that ADR caused significant decrease in state 3 respiration and respiratory control ratio using both complex I and II substrates in nontransgenic mice. In transgenic mice, state 3 respiration for complex I substrates remained unaffected by ADR, but was reduced for complex II substrate. Complex I activity was significantly decreased in nontransgenic, but not in transgenic mice after ADR treatment, suggesting that mitochondrial complex I is sensitive to inactivation by superoxide radicals. The activities of complex II and mitochondrial creatine kinase were decreased by ADR in both nontransgenic and transgenic mice. These results support our previous observations on the protective role of MnSOD on the ultrastructural damage of the heart after ADR treatment and extend the understanding of its mechanisms in mitochondria.