Inhibition of cell growth by overexpression of manganese superoxide dismutase (MnSOD) in human pancreatic carcinoma

Manganese superoxide dismutase (MnSOD) levels have been found to be low in human pancreatic cancer [Pancreas26, (2003), 23] and human pancreatic cancer cell lines [Cancer Res.63, (2003), 1297] when compared to normal human pancreas. We hypothesized that stable overexpression of pancreatic cancer cells with MnSOD cDNA would alter the malignant phenotype. MIA PaCa-2 cells were stably transfected with a pcDNA3 plasmid containing sense human MnSOD cDNA or containing no MnSOD insert by using the lipofectAMINE method. G418-resistant colonies were isolated, grown and maintained. Overexpression of MnSOD was confirmed in two selected clones with a 2-4-fold increase in MnSOD immunoreactive protein. Compared with the parental and neo control cells, the MnSOD-overexpressing clones had decreased growth rates, growth in soft agar and plating efficiency in vitro, while in vivo, the MnSOD-overexpressing clones had slower growth in nude mice. These results suggest that MnSOD may be a tumor suppressor gene in human pancreatic cancer.     

Induction of apoptosis by adenovirus-mediated manganese superoxide dismutase overexpression in SV-40-transformed human fibroblasts

Although mitochondrial reactive oxygen species (ROS) have been implicated both as an initiator and as an effector of apoptosis, the exact role of mitochondrial ROS has been difficult to establish due to the lack of an appropriate experimental system where ROS could be specifically generated from mitochondria and subsequent effects on cells analyzed. In this study, a manganese superoxide dismutase (MnSOD) activity-mediated apoptosis model was established and characterized. It was shown that despite early increases in the steady-state levels of ROS upon MnSOD overexpression, cellular oxidative damage was decreased significantly at later time points. Alterations in levels of peroxiredoxin (Prxn1) protein preceded the onset of apoptosis after MnSOD overexpression. A time course study demonstrated that increases in MnSOD activity prior to the onset of apoptosis correlated with alterations in the levels of nitration of tyrosine residue(s) of MnSOD protein. A direct correlation between MnSOD activity and the degree of apoptosis was demonstrated using a mutant MnSOD with decreased activity. The current study supports a causative role of mitochondrial ROS leading to the onset of apoptosis. The MnSOD activity-mediated apoptosis model described here could be further utilized to study mitochondrial apoptotic pathways.     

Modulation of antioxidant enzymes,reactive oxygen species,and glutathione levels in manganese superoxide dismutase-overexpressing NIH/3T3 fibroblasts during the cell cycle

NIH/3T3 mouse embryo fibroblasts were transfected with the cDNA for manganese superoxide dismutase (MnSOD). Previous studies showed characteristic unique AE profiles in nonsynchronous populations of parental, control plasmid-transfected, and MnSOD-overexpressing NIH/3T3 cell lines. However, the present study showed that during S and M phases of the cell cycle, antioxidant enzyme (AE) levels were altered in MnSOD-overexpressing cell lines towards levels in S and M phases of parental and control plasmid-transfected cells. Because of the demonstration that MnSOD overexpression inhibits cell growth in both nonmalignant and malignant cells, the present study was designed to measure AEs, reactive oxygen species (ROS), and glutathione levels in various phases of the cell cycle in both parental NIH/3T3 cells and NIH/3T3 cells overexpressing MnSOD, to try to determine whether AEs, ROS, and glutathione levels could have a possible regulatory role in cell cycle progression. In all cell lines studied, ROS levels were lower in M than S phase of the cell cycle. Total glutathione and glutathione disulfide levels were greatly increased during the M phase of the cell cycle compared with quiescence and S phase in all cell lines studied. This suggests that oxidative stress exists in M phase of the cell cycle with total glutathione levels increased to decrease oxidative stress. Analysis of MnSOD-overexpressing cell clones showed a correlation of decreased cell growth with an increase in ROS in S phase of the cell cycle and a decrease in glutathione in mitosis. The data strongly suggest that specific levels of cell redox state are necessary for cells to successfully progress through the various phases of the cell cycle. J. Cell. Physiol. 177:148–160, 1998. © 1998 Wiley-Liss, Inc.     

Amplification and overexpression of the met gene in spontaneously transformed NIH3T3 mouse fibroblasts.

We have identified a class of transformed NIH3T3 mouse fibroblasts that arise at low frequencies in transfection experiments with DNA from both neoplastic and non-neoplastic cells and that may result from a low level of spontaneous transformation of NIH3T3 cells. DNA from the transformed cells was unable to transform NIH3T3 cells in a second cycle of transfection and, where examined, the cells showed no evidence for the uptake of the transfected DNA sequences. The results of Southern analyses demonstrate that a mouse homologue of the human met oncogene is amplified 4- to 8-fold in 7 of 10 lines of these transformed NIH3T3 mouse fibroblasts. The cells containing the amplified gene also exhibit at least a 20-fold overexpression of an 8.5-kb mRNA that is homologous to met. To test the hypothesis that met encodes a growth factor receptor, we examined the binding of platelet-derived growth factor, epidermal growth factor, insulin-like growth factor I and gastrin-releasing peptide to transformed and non-transformed NIH3T3 cells. The results show that there is no significant elevation of the binding of these growth factors to cells containing amplification and overexpression of met.     

Regulation by cytokines of extracellular superoxide dismutase and other superoxide dismutase isoenzymes in fibroblasts.

The influence of cytokines on extracellular superoxide dismutase (EC-SOD) expression by human dermal fibroblasts was investigated. The expression was markedly stimulated by interferon-gamma (IFN-gamma), was varying between fibroblast lines stimulated or depressed by interleukin-1 alpha (IL-1 alpha), was intermediately depressed by tumor necrosis factor-alpha (TNF-alpha), and markedly depressed by transforming growth factor-beta (TGF-beta). TNF-alpha, however, enhanced the stimulation by a high dose of IFN-gamma, whereas TGF-beta markedly depressed the stimulations given by IFN-gamma and IL-1 alpha. The ratio between the maximal stimulation and depression observed was around 30-fold. The responses were generally slow and developed over periods of several days. There were no effects of IFN-alpha, IL-2, IL-3, IL-4, IL-6, IL-8, granulocyte-macrophage colony-stimulating factor, human growth hormone, Escherichia coli lipopolysaccharide, leukotriene B4, prostaglandin E2, formylmethionylleucylphenylalanine, platelet-activating factor, and indomethacin. The cytokines influencing the EC-SOD expression are also known to influence superoxide production by leukocytes and other cell types, and the EC-SOD response pattern is roughly compatible with the notion that its function is to protect cells against extracellular superoxide radicals. The results show that EC-SOD is a participant in the complex inflammatory response orchestrated by cytokines. The CuZn-SOD activity of the fibroblasts was not influenced by any of the cytokines, whereas the Mn-SOD activity was depressed by TGF-beta. TNF-alpha, IL-1 alpha, and IFN-gamma stimulated the Mn-SOD activity, as previously known, and these responses were reduced by TGF-beta. The different responses of the three SOD isoenzymes illustrate their different physiological roles.     

Invited review: manganese superoxide dismutase in disease

Manganese superoxide dismutase (MnSOD) is essential for life as dramatically illustrated by the neonatal lethality of mice that are deficient in MnSOD. In addition, mice expressing only 50% of the normal compliment of MnSOD demonstrate increased susceptibility to oxidative stress and severe mitochondrial dysfunction resulting from elevation of reactive oxygen species. Thus, it is important to know the status of both MnSOD protein levels and activity in order to assess its role as an important regulator of cell biology.

Numerous studies have shown that MnSOD can be induced to protect against pro-oxidant insults resulting from cytokine treatment, ultraviolet light, irradiation, certain tumors, amyotrophic lateral sclerosis, and ischemia/reperfusion. In addition, overexpression of MnSOD has been shown to protect against pro-apoptotic stimuli as well as ischemic damage. Conversely, several studies have reported declines in MnSOD activity during diseases including cancer, aging, progeria, asthma, and transplant rejection. The precise biochemical/molecular mechanisms involved with this loss in activity are not well understood. Certainly, MnSOD gene expression or other defects could play a role in such inactivation. However, based on recent findings regarding the susceptibility of MnSOD to oxidative inactivation, it is equally likely that post-translational modification of MnSOD may account for the loss of activity. Our laboratory has recently demonstrated that MnSOD is tyrosine nitrated and inactivated during human kidney allograft rejection and human pancreatic ductal adenocarcinoma. We have determined that peroxynitrite (ONOO^-) is the only known biological oxidant competent to inactivate enzymatic activity, to nitrate critical tyrosine residues, and to induce dityrosine formation in MnSOD. Tyrosine nitration and inactivation of MnSOD would lead to increased levels of superoxide and concomitant increases in ONOO^- within the mitochondria which, could lead to tyrosine nitration/oxidation of key mitochondrial proteins and ultimately mitochondrial dysfunction and cell death. This article assesses the important role of MnSOD activity in various pathological states in light of this potentially lethal positive feedback cycle involving oxidative inactivation.     

Inhibition of fibroblast chemotaxis by superoxide dismutase

Superoxide radicals are known to be important mediators in chronic inflammatory and fibrotic processes, in which accumulation of fibroblasts is thought to play a major role in the pathogenetic events. The enzyme superoxide dismutase removes these radicals by a catalytic reaction. Chemotactic response of human fibroblasts and fibrosarcoma-derived cells (HT-1080) to fibroblast conditioned medium, fibronectin and platelet-derived growth factor was inhibited in a dose-dependent manner in the presence of superoxide dismutase, while random migration, cell proliferation, cell viability and synthesis of collagen and non-collagenous proteins was not altered. In contrast, phorbol myristate acetate, an inducer of superoxide generation, stimulated the chemotactic movement of fibroblasts to the attractants. Evidence for the formation of superoxide is provided by the reduction of tetrazolium salt by activated fibroblasts which could be inhibited by superoxide dismutase. Thus, it is concluded that superoxide in small amounts is involved in the mechanism of fibroblast chemotaxis. Superoxide dismutase may, therefore, reduce fibroblast migration into sites of injury or inflammation.     

HSP25 overexpression attenuates oxidative stress-induced apoptosis: roles of ERK1/2 signaling and manganese superoxide dismutase

HSP25 has been shown to induce resistance to radiation and oxidative stress; however, its exact mechanisms remain unclear. In the present study, a high concentration of H2O2 was found to induce DNA fragmentation in L929 mouse fibroblast cells, and HSP25 overexpression attenuated this phenomenon. To elucidate the mechanisms of H2O2-mediated cell death, ERK1/2, p38 MAPK, and JNK1/2 phosphorylation in the cells after treatment with H2O2 were examined. ERK1/2 and JNK1/2 were activated by H2O2; ERK1/2 activation was inhibited in HSP25-overexpressed cells, while JNK1/2 was indifferent. Inhibition of ERK1/2 activation by treatment of the cells with PD98059 or dominant-negative ERK2 transfection blocked H2O2-induced cell death; similarly treated HSP25-overexpressed cells were not at all affected. Moreover, inhibition of JNK1/2 by dominant-negative JNK1 or JNK2 transfection did not affect H2O2-mediated cell death in control cells. Dominant-negative Ras or Raf transfection inhibited H2O2-mediated ERK1/2 activation and cell death in control cells. On the contrary, HSP25-overexpressed cells did not show any differences. Upstream pathways of H2O2-mediated ERK1/2 activation and cell death involved both tyrosine kinase (PDGFbeta receptor and Src) and PKCdelta, while in HSP25-overexpressed cells these kinases did not respond to H2O2 treatment. Since HSP25 overexpression reduced reactive oxygen species (ROS), increased manganese superoxide dismutase (MnSOD) gene expression, and increased enzyme activity, involvement of MnSOD in HSP25-mediated attenuation of H2O2-mediated ERK1/2 activation and cell death was examined. Blockage of MnSOD with antisense oligonucleotides prevented DNA fragmentation and returned the ERK1/2 activation to the control level. Indeed, when MnSOD was overexpressed in L929 cells, similar to in HSP25-overexpressed cells, DNA fragmentation and ERK1/2 activation were reduced. From the above results, we suggest for the first time that reduced oxidative damage by HSP25 was due to MnSOD-mediated downregulation of ERK1/2.     

Hydrogen bonding in human manganese superoxide dismutase containing 3-fluorotyrosine

Incorporation of 3-fluorotyrosine and site-specific mutagenesis has been utilized with Fourier transform infrared (FTIR) spectroscopy and x-ray crystallography to elucidate active-site structure and the role of an active-site residue Tyr34 in human manganese superoxide dismutase (MnSOD). Calculated harmonic frequencies at the B3LYP/6-31G** level of theory for L-tyrosine and its 3-fluorine substituted analog are compared to experimental frequencies for vibrational mode assignments. Each of the nine tyrosine residues in each of the four subunits of the homotetramer of human MnSOD was replaced with 3-fluorotyrosine. The crystal structures of the unfluorinated and fluorinated wild-type MnSOD are nearly superimposable with the root mean-square deviation for 198 alpha-carbon atoms at 0.3 A. The FTIR data show distinct vibrational modes arising from 3-fluorotyrosine in MnSOD. Comparison of spectra for wild-type and Y34F MnSOD showed that the phenolic hydroxyl of Tyr34 is hydrogen bonded, acting as a proton donor in the active site. Comparison with crystal structures demonstrates that the hydroxyl of Tyr34 is a hydrogen bond donor to an adjacent water molecule; this confirms the participation of Tyr34 in a network of residues and water molecules that extends from the active site to the adjacent subunit.     

Ca influx evoked by inositol-3,4,5,6-tetrakisphosphate in ras-transformed NIH/3T3 fibroblasts

Infusion of inositol-3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P₄) from the patch pipette into the cytoplasm, produced a biphasic intracellular free Ca²⁺ concentration ([Ca²⁺]_i) increase in ras-transformed NIH/3T3 (DT) cells. The Ins(3,4,5,6)P₄-induced increase in DT cells depended upon extracellular Ca²⁺ and was enhanced by membrane hyperpolarization. Identical [Ca²⁺]_i increases were observed with intracellular application of inositol-1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P₄) and inositol-1,3,4,6-tetrakisphosphate but not with inositol-1,2,4,5-tetrakisphosphate, inositol-1,4,5-trisphosphate or inositol-1,3,4,5,6-pentakisphosphate. Stimulation of DT cells with bradykinin increased the levels of Ins(3,4,5,6)P₄ and Ins(1,3,4,5)P₄. These results suggest that Ins(3,4,5,6)P₄ may serve as a second messenger for continuous Ca²⁺ influx along with other tetrakisphosphates downstream from bradykinin receptors in DT cells.     

Radioprotection by short-term oxidative preconditioning: Role of manganese superoxide dismutase

Manganese superoxide dismutase (MnSOD) is vital to the protection of mitochondria and cells against oxidative stress. Earlier, we demonstrated that catalytically active homo-tetramer of MnSOD can be stabilized by oxidative cross-linking. Here we report that this effect may be translated into increased radioresistance of mouse embryonic cells (MECs) by pre-exposure to oxidative stress. Pre-treatment of MECs with antimycin A, rotenone or H₂O₂ increased their survival after irradiation. Using MnSOD siRNA, we show that MECs with decreased MnSOD levels displayed a lowered ability to preconditioning. Thus oxidative preconditioning may be used for targeted regulation of MnSOD.

Structured summary

MINT-7288408: MnSOD (uniprotkb:P04179) and MnSOD (uniprotkb:P04179) physically interact (MI:0915) by zymography (MI:0512)     

Lowered level of translatable messenger RNAs for manganese superoxide dismutase in human fibroblasts transformed by SV 40

The activity of manganese superoxide dismutase (MnSOD) revealed by specific staining after gel electrophoresis of cell extracts, is decreased in human fibroblasts transformed by SV40. The decrease in enzyme activity is attributable to decreased amount of enzyme protein as determined by radial immunodiffusion. Total fibroblast RNAs were translated in the presence of (35S) methionine in a cell-free translation system and the neo synthesized proteins submitted to immunoprecipitation with an anti MnSOD antiserum. Gel electrophoresis of the immunoprecipitated material followed by fluorography shows that MnSOD is translated as a peptide which is 2000 daltons larger than the mature enzyme subunit. This precursor (pre-MnSOD) is processed in vitro to mature MnSOD by the action of an isolated mitochondrial preparation. Levels of translatable MnSOD mRNA in normal and SV 40 transformed cells were compared in terms of the radioactivities incorporated into pre MnSOD bands. The results indicate that the decreased amount of MnSOD in SV 40 transformed fibroblasts is due to a decreased level of translatable mRNA for MnSOD.     

Overexpression of manganese or copper-zinc superoxide dismutase inhibits breast cancer growth

We have studied the effects of overexpression of superoxide dismutase (SOD), a tumor suppressor protein that dismutes superoxide radical to H2O2, on breast cancer cell growth in vitro and xenograft growth in vivo. No previous work has directly compared the growth-suppressive effects of manganese SOD (MnSOD) and copper-zinc SOD (CuZnSOD). We hypothesized that either adenoviral MnSOD (AdMnSOD) or adenoviral CuZnSOD (AdCuZnSOD) gene therapy would suppress the growth of human breast cancer cells. After determining the antioxidant profiles of three human breast cell lines, MCF 10A, MDA-MB231, and MCF-7, we measured the effects of MnSOD or CuZnSOD overexpression on cell growth and survival in vitro and in vivo. Results demonstrated that infection with AdMnSOD or AdCuZnSOD increased the activity of the respective enzyme in all three cell lines. In vitro, overexpression of MnSOD or CuZnSOD decreased not only cell growth but also clonogenic survival in a dose- and transgene-dependent manner. In vivo, treatment of tumors with AdMnSOD or AdCuZnSOD decreased xenograft growth compared to controls. The first direct comparison of MnSOD to CuZnSOD overexpression indicated that CuZnSOD and MnSOD were similarly effective at suppressing cancer cell growth.     

Induction of mitochondrial manganese superoxide dismutase by interleukin 1

Interleukin 1 (IL 1) inhibits the growth of human melanoma A375 cells. To identify the subcellular events preceding inhibition of growth by IL 1, we have examined the effect of IL 1 on protein synthesis caused by A375 cells. IL 1 selectively and predominantly induced a 25-kDa polypeptide (p25) in A375 cells after 12 h. On subcellular fractionation, p25 was exclusively located in the 10,000 x g-pelleted (mitochondria-enriched) fraction. To identify the p25 moiety, it was purified to homogeneity by sequential chromatography on DEAE-Sephacel and reverse-phase, high-pressure liquid chromatography and its amino-terminal amino acid sequence was determined. The sequence of the 35 amino-terminal amino acids of the p25 moiety was identical to that of human manganese superoxide dismutase (Mn SOD). The enzymatic activities of SOD were induced only in the mitochondria-enriched fraction of IL 1-treated A375 cells. However, IL 1 also induced Mn SOD in normal human skin fibroblasts and peripheral blood mononuclear cells, whose growth was stimulated by IL 1. The results show that induction of Mn SOD by IL 1 is a common biochemical event in IL 1-responsive cells.     

Inhibition of cytotoxicity by intracellular superoxide dismutase supplementation

The role of intracellular oxyradicals in H2O2 and neutrophil-induced cytotoxicity is suggested by previous studies showing protection by inhibitors such as deferroxamine, dimethylthiourea, and dimethyl sulfoxide. In the current studies, the role of intracellular O2- is specifically examined by evaluating the effects of intracellular superoxide dismutase (SOD) supplementation on cytotoxicity of rat pulmonary artery endothelial cells induced by H2O2 and activated neutrophils. To minimize in vitro manipulation, supplementation was accomplished by incubating endothelial cells in the presence of SOD (1-20 mg/mL). Increases up to greater than 17-fold the baseline SOD activity were achievable using this approach, with uptake being maximal after 6 h of incubation. This increase was resistant to trypsin digestion, suggesting the intracellular location of SOD. Compared to controls, SOD-supplemented cells showed significantly increased resistance to killing by H2O2 and activated neutrophils. Inactive SOD failed to provide protection. The degree of protection was dependent on the dose of cytotoxic agent and the extent of SOD supplementation. The results provide new evidence that intracellular O2- participates in the killing process induced by these two stimuli. The intracellular source of O2- remains to be determined, although previous studies suggest xanthine oxidase as a likely candidate.