Hannah J. Zhang

Activation of Matrix Metalloproteinase-2 by Overexpression of Manganese Superoxide Dismutase in Human Breast Cancer MCF-7 Cells Involves Reactive Oxygen Species

Matrix metalloproteinases (MMPs) participate in cell migration and remodeling processes by affecting the extracellular matrix. MMP-2 is thought to be involved in cancer cell invasiveness. It has been proposed that the activity of MMP-2 can be modulated by intracellular reactive oxygen species (ROS)/reactive nitrogen species. We hypothesized that manganese superoxide dismutase (MnSOD) could mediate MMP-2 activity by changing the intracellular ROS level and that nitric oxide (⋅NO) may be involved in this process. Human breast cancer MCF-7 cells were stably transfected with plasmids containing MnSOD cDNA. A 2–30-fold increase of MnSOD protein and activity was observed in four clones. Our data demonstrated that overexpression of MnSOD stimulated the activation of MMP-2 with a corresponding elevation of ROS. A decrease in ROS by ebselen, a glutathione peroxidase mimetic, or by transduction of adenovirus containing human catalase or glutathione peroxidase cDNA abolished the effect of MnSOD on MMP-2 activation. Treatment of MCF-7 cells with antimycin A or rotenone increased intracellular ROS production and MMP-2 activation simultaneously. Our data also showed a suppression of endothelial nitric-oxide synthase expression that was accompanied by decreased ⋅NO production in MnSOD-overexpressing cells. However, the changes in endothelial nitric-oxide synthase and⋅NO did not correlate with the MnSOD activity. Corresponding changes of MMP-2 activity after the addition of a NOS inhibitor (N G-amino-l-arginine) or a⋅NO donor ((Z)-1-[(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate) to the cells suggested the possibility that ⋅NO may be involved in the MnSOD-mediated MMP-2 activation pathway. These results indicate that MnSOD induces MMP-2 activity by regulation of intracellular ROS and imply that signaling pathways involving ⋅NO may also be involved in the MnSOD mediation of MMP-2 activity.

Heat-induced liver injury in old rats is associated with exaggerated oxidative stress and altered transcription factor activation

A decline in stress tolerance is a hallmark of aging. For instance, older organisms showed extensive hepatic damage, along with increased morbidity and mortality, after environmental heating. We hypothesized that hyperthermic challenge would produce exaggerated oxidative stress in old animals, leading to increased hepatic injury. After a heat‐stress protocol, time‐course changes in reactive oxygen species (ROS) levels, oxidative damage markers, glutathione (GSH)/glutathione disulfide (GSSG) ratios, and activation of stress‐response transcription factors (AP‐1 and NF‐κB) were measured in young and old rats. A small, transient increase in hepatic oxidative damage, with minimal injury, was observed in young rats. However, old rats showed widespread hepatic injury that was manifested over a 24 h period after heating. This pathology was preceded by elevated steady‐state levels of ROS, along with large increases in lipid peroxidation products, prolonged hepatic DNA oxidation damage, aberrant GSH/GSSG profiles, and altered activation patterns for AP‐1. These data indicate that young animals have an effective oxidation‐reduction buffering system in the liver that provides protection from oxidative damage to intracellular macromolecules under stress conditions. In sharp contrast, an environmental challenge in older animals produces exaggerated oxidative stress and alterations in signal transduction pathways, which can contribute to cellular dysfunction and age‐related reductions in stress tolerance.

Manganese superoxide dismutase suppresses hypoxic induction of hypoxia-inducible factor-1|[alpha]| and vascular endothelial growth factor

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that governs cellular responses to reduced O2 availability by mediating crucial homeostatic processes. HIF-1 is composed of an HIF-1α subunit and an HIF-1β subunit. HIF-1α is degraded following enzyme-dependent hydroxylation of prolines of HIF-1α in the presence of molecular oxygen, Fe2+, α-ketoglutarate, and ascorbate. These cofactors contribute to the redox environment of cells. The antioxidant enzyme manganese superoxide dismutase (MnSOD) also modulates the cellular redox environment. Here we show that MnSOD suppressed hypoxic accumulation of HIF-1α protein in human breast carcinoma MCF-7 cells. This suppression was biphasic depending on MnSOD activity. At low levels of MnSOD activity, HIF-1α protein accumulated under hypoxic conditions. At moderate levels of MnSOD activity (two- to six-fold increase compared to parent cells), these accumulations were blocked. However, at higher levels of MnSOD activity (>6-fold increase), accumulation of HIF-1α protein was again observed. This biphasic modulation was observed under both 1 and 4% O2. Coexpression of mitochondrial hydrogen peroxide-removing proteins prevented the accumulation of HIF-1α protein in cells with high levels of MnSOD; this effect demonstrates that the restabilization of HIF-1α observed in high MnSOD overexpressors is probably due to hydrogen peroxide, most likely produced from MnSOD. Hypoxic induction of vascular endothelial growth factor (VEGF) protein was also suppressed by elevated MnSOD activity and its levels reflected HIF-1α protein levels. These observations demonstrated that HIF-1α accumulation and VEGF expression could be modulated by the antioxidant enzyme MnSOD.

Redox modulation of the liver with chronic antioxidant enzyme mimetic treatment prevents age-related oxidative damage associated with environmental stress

A reduction in stress tolerance is a hallmark of the aging process, and the lowered functional capacity observed in aged organisms is associated with an increased rate of oxidative stress and a greater susceptibility of aged tissues to oxidative injury. In this report, we show that chronic systemic administration of a superoxide dismutase (SOD)/catalase mimetic (EUK‐189), delivered over a 1 month period via osmotic pump, prevents heat stress‐induced liver injury by dramatically decreasing oxidative damage in aged animals. Widespread liver injury was present in old but not young vehicle‐treated rats in response to a 2 day heating protocol. However, SOD/catalase mimetic treatment markedly decreased the hyperthermia‐induced liver injury associated in old animals. The reversal of damage with EUK‐189 was associated with an improvement in intracellular redox status and a striking reduction in hepatocellular lipid peroxidation. EUK‐189 treatment also blocked the activation of activator protein‐1 (AP‐1), which is a redox‐sensitive early response transcription factor involved in the regulation of cellular stress responses. These results demonstrate that oxidative stress plays a unique role in age‐related hyperthermic injury and suggest that therapeutic strategies aimed at improving redox potential, such as chronic SOD/catalase mimetic treatment, can prevent the oxidative‐mediated damage associated with environmental stress.

Aging Results in Increased Autophagy of Mitochondria and Protein Nitration in Rat Hepatocytes Following Heat Stress

The natural breakdown of cells, tissues, and organ systems is a significant consequence of aging and is at least partially caused by a decreased ability to tolerate environmental stressors. Based on quantitative ultrastructural analysis using transmission electron microscopy and computer imaging, we show significant differences in hepatocyte morphology between young and old rats during a 48-hr recovery period following a 2-day heat stress protocol. Mitochondrial injury was greater overall in old compared with young rats. Autophagy was observed in both young and old rats, with autophagy greater overall in old compared with young hepatocytes. Lipid peroxidation and protein nitration were evaluated by localization and quantification of 4-hydroxy-2-nonenal (4-HNE)–modified protein adducts and 3-nitrotyrosine (3-NT) levels, respectively. Levels of 3-NT but not 4-HNE-protein adducts were significantly elevated in hepatocytes of old rats in comparison with young at 90 min after heat stress, suggesting a major role for reactive nitrogen species in the pathology observed at this time point. These results show a differential response of hepatocyte mitochondria to heat stress with aging, as well as greater levels of both autophagic and nitrative damage in old vs young hepatocytes. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

MnSOD up-regulates maspin tumor suppressor gene expression in human breast and prostate cancer cells.

Manganese superoxide dismutase (MnSOD) is an antioxidant enzyme with tumor suppressor activity; however, the molecular mechanisms of MnSOD antitumor effects remain unclear. We hypothesized that MnSOD activity in cancer cells might cause downstream changes in the expression of other tumor suppressor genes. To determine whether maspin, a tumor suppressor gene that inhibits breast cancer cell invasion and metastasis, might be a target of MnSOD, we forced MnSOD expression in several human breast and prostate cancer cell lines by adenovirus-mediated gene transfer and measured maspin mRNA expression. Forced expression of MnSOD caused maspin mRNA to accumulate in a dose-dependent manner in both human breast and prostate cancer cells. Normal p53 was not necessary to mediate the effect of MnSOD because MnSOD up-regulated maspin in cells that harbor wild-type p53 and in cells that harbor mutant p53. Moreover, the effects of MnSOD on maspin were not due to demethylation of the maspin promoter. Analyses of maspin promoter activity, transcriptional run-on, and mRNA stability showed that maspin mRNA stability was the major mechanism for maspin up-regulation by MnSOD. Our findings identify a mechanism underlying MnSOD antitumor effects and provide evidence to support MnSOD as a genetic therapy in the treatment of human breast and prostate cancers.

USE OF COMMERCIAL ANTIBODIES FOR DETECTION OF THE PRIMARY ANTIOXIDANT ENZYMES

Fourteen commercial antibodies against human antioxidant enzymes were tested on whole cell lysates by Western analysis for specificity and species crossreactivity. All antibodies, except one, recognized pure protein antigen. All four catalase antibodies were of high quality, and they could also recognize the catalases from rat, mouse, dog, and hamster cells. Two CuZnSOD antibodies were very specific for CuZnSOD protein. They could also crossreact with CuZnSOD from rat, mouse, and hamster cells, but not from dog cells. All five MnSOD antibodies detected only very high levels of MnSOD. We believe that they could not properly be used in immunohistochemistry. Three GPX antibodies could not detect the specific GPX band from cell lysates. We believe that it is difficult to use these GPX antibodies in both Western blotting and immunohistochemistry.

Redox Regulation of Adenovirus-Induced AP-1 Activation by Overexpression of Manganese-Containing Superoxide Dismutase

ABSTRACT Adenovirus gene therapy is a promising tool in the clinical treatment of many genetic and acquired diseases. However, it has also caused pathogenic effects in organs such as the liver. The redox-sensitive transcription factors AP-1 and NF-κB have been implicated in these effects. To study the mechanisms of adenovirus-mediated AP-1 and NF-κB activation and the possible involvement of oxidative stress in adenovirus transduction, rats were injected with either replication-defective recombinant adenovirus with DNA containing the cytomegalovirus promoter region only (AdCMV), adenovirus containing human manganese-containing superoxide dismutase (MnSOD) cDNA (AdMnSOD), or vehicle. Compared to vehicle and AdCMV transduction, MnSOD gene transfer yielded a fivefold increase in liver MnSOD activity 7 days postinjection. Gel shift assay showed that AdCMV transduction induced DNA binding activity for AP-1 but not NF-κB. MnSOD overexpression abolished this activation. Western blotting analysis of c-Fos and c-Jun suggested that up-regulation of c-fos and c-jun gene expression does not directly contribute to the induction of AP-1 activation. Glutathione/glutathione disulfide ratios were decreased by adenovirus transduction and restored by MnSOD overexpression. The AP-1 binding activity that was induced by AdCMV was decreased by immunoprecipitation of Ref-1 protein. Ref-1 involvement was confirmed by restoration of AP-1 binding activity after the immunoprecipitated Ref-1 protein had been added back. AP-1 DNA binding activity was also elevated in control and AdMnSOD-injected rats after addition of the immunoprecipitated Ref-1 protein. These data indicate that cellular transduction by recombinant adenovirus stimulates AP-1 DNA binding activity. Furthermore, our results suggest that MnSOD overexpression decreases AP-1 DNA binding activity by regulating intracellular redox status, with the possible involvement of Ref-1 in this redox-sensitive pathway.

v-Ha-Ras Overexpression Induces Superoxide Production and Alters Levels of Primary Antioxidant Enzymes

Reactive oxygen species have been shown to play important roles in v-Ha-Ras mitogenic signaling. We hypothesized that v-Ha-Ras overexpression would induce superoxide production, and therefore modify expression of the primary antioxidant enzyme system. We have demonstrated that immortal rat kidney epithelial cells stably transduced with constitutively active v-Ha-ras produced significantly larger amounts of superoxide radical than wild-type or vector-transfected control cells. The levels of the primary antioxidant enzymes copper- and zinc-containing superoxide dismutase, manganese-containing superoxide dismutase, catalase, and glutathione peroxidase were increased in the superoxide-overproducing cells. DNA-binding activities of the transcription factors activator protein-1, activator protein-2, and nuclear factor-kappaB were all enhanced in the superoxide-overproducing cells. These v-Ha-ras transduced cells also had a shortened cell doubling time and higher plating efficiency, and displayed greater constitutive levels of phosphorylated mitogen-activated protein kinases. These data demonstrate that v-Ha-Ras overexpression increases superoxide production and this apparently affects a wide variety of cell signaling and redox systems.

Differential Regulation of Hepatic Heme Oxygenase-1 Protein With Aging and Heat Stress

Increased expression of heme oxygenase-1 (HO-1) in response to physiological stress is considered to be a protective response, which may be altered with aging. In this study, HO-1 expression was assessed following heat stress by immunoblotting of liver homogenates and isolated hepatocytes from young (6 months) and old (24 months) Fischer 344 rats and by immunohistochemistry. Livers of old rats showed higher baseline levels of HO-1, which was predominately localized to Kupffer cells. After heat stress, young animals showed a greater relative increase in hepatic HO-1, part of which was caused by increased numbers of nonparenchymal cells that were immunoreactive to HO-1. Consistent with these data, HO-1 was significantly upregulated after hyperthermia in vitro only in hepatocytes from young rats. Hence, aging alters stress-induced expression of HO-1 in a cell-specific manner, which may contribute to the diminished stress tolerance observed in older organisms.