Targeting NAD(P)H:Quinone Oxidoreductase (NQO1) in Pancreatic Cancer.

NAD(P)H: Quinone oxidoreductase (NQO1) functions as an important part of cellular antioxidant defense by detoxifying quinones, thus preventing the formation of reactive oxygen species. The aims of our study were to determine if NQO1 is elevated in pancreatic cancer specimens and pancreatic cancer cell lines and if so, would compounds previously demonstrated to redox cycle with NQO1 be effective in killing pancreatic cancer cells. Immunohistochemistry of resected pancreatic specimens demonstrated an increased immunoreactivity for NQO1 in pancreatic cancer and pancreatic intraepithelial neoplasia (PanIN) specimens versus normal human pancreas. Immunocytochemistry and Western immunoblots demonstrated inceased immunoreactivity in pancreatic cancer cells when compared to a near normal immortalized human pancreatic ductal epithelial cell line and a colonic epithelial cell line. Streptonigrin, a compound known to cause redox cycling in the presence of NQO1, decreased clonogenic survival and decreased anchorage-independent growth in soft agar. Streptonigrin had little effect on cell lines with absent or reduced levels of NQO1. The effects of streptonigrin were reversed in pancreatic cancer cells pretreated with dicumarol, a known inhibitor of NQO1. NQO1 may be a therapeutic target in pancreatic cancer where survival is measured in months. © 2006 Wiley-Liss, Inc.

Antioxidant proteins and reactive oxygen species are decreased in a murine epidermal side population with stem cell-like characteristics.

Reactive oxygen species (ROS) and antioxidants are essential to maintain a redox balance within tissues and cells. Intracellular ROS regulate key cellular functions such as proliferation, differentiation and apoptosis through cellular signaling, and response to injury. The redox environment is particularly important for stem/progenitor cells, as their self-renewal and differentiation has been shown to be redox sensitive. However, not much is known about ROS and antioxidant protein function in freshly isolated keratinocytes, notably the different keratinocyte subpopulations. Immunostaining of neonatal cutaneous sections revealed that antioxidant enzymes [catalase, SOD2, gluthatione peroxidase-1 (GPx)] and ROS are localized predominantly to the epidermis. We isolated keratinocyte subpopulations and found lower levels of SOD2, catalase and GPx, as well as decreased SOD and catalase activity in an epidermal side population with stem cell-like characteristics (EpSPs) compared to more differentiated (Non-SP) keratinocytes. EpSPs also exhibited less mitochondrial area, fewer peroxisomes and produced lower levels of ROS than Non-SPs. Finally, EpSPs were more resistant to UV radiation than their progeny. Together, our data indicate ROS and antioxidant levels are decreased in stem-like EpSPs.

Increased levels of superoxide and H2O2 mediate the differential susceptibility of cancer cells versus normal cells to glucose deprivation.

Cancer cells, relative to normal cells, demonstrate increased sensitivity to glucose-deprivation-induced cytotoxicity. To determine whether oxidative stress mediated by O(2)(*-) and hydroperoxides contributed to the differential susceptibility of human epithelial cancer cells to glucose deprivation, the oxidation of DHE (dihydroethidine; for O(2)(*-)) and CDCFH(2) [5- (and 6-)carboxy-2',7'-dichlorodihydrofluorescein diacetate; for hydroperoxides] was measured in human colon and breast cancer cells (HT29, HCT116, SW480 and MB231) and compared with that in normal human cells [FHC cells, 33Co cells and HMECs (human mammary epithelial cells)]. Cancer cells showed significant increases in DHE (2-20-fold) and CDCFH(2) (1.8-10-fold) oxidation, relative to normal cells, that were more pronounced in the presence of the mitochondrial electron-transport-chain blocker, antimycin A. Furthermore, HCT116 and MB231 cells were more susceptible to glucose-deprivation-induced cytotoxicity and oxidative stress, relative to 33Co cells and HMECs. HT29 cells were also more susceptible to 2DG (2-deoxyglucose)-induced cytotoxicity, relative to FHC cells. Overexpression of manganese SOD (superoxide dismutase) and mitochondrially targeted catalase significantly protected HCT116 and MB231 cells from glucose-deprivation-induced cytotoxicity and oxidative stress and also protected HT29 cells from 2DG-induced cytotoxicity. These results show that cancer cells (relative to normal cells) demonstrate increased steady-state levels of ROS (reactive oxygen species; i.e. O(2)(*-) and H(2)O(2)) that contribute to differential susceptibility to glucose-deprivation-induced cytotoxicity and oxidative stress. These studies support the hypotheses that cancer cells increase glucose metabolism to compensate for excess metabolic production of ROS and that inhibition of glucose and hydroperoxide metabolism may provide a biochemical target for selectively enhancing cytotoxicity and oxidative stress in human cancer cells.

Superoxide signaling mediates N-acetyl-L-cysteine-induced G1 arrest: regulatory role of cyclin D1 and manganese superoxide dismutase.

Thiol antioxidants, including N-acetyl-L-cysteine (NAC), are widely used as modulators of the intracellular redox state. We investigated the hypothesis that NAC-induced reactive oxygen species (ROS) signaling perturbs cellular proliferation by regulating the cell cycle regulatory protein cyclin D1 and the ROS scavenging enzyme Mn-superoxide dismutase (MnSOD). When cultured in media containing NAC, mouse fibroblasts showed G(1) arrest with decreased cyclin D1 protein levels. The absence of a NAC-induced G(1) arrest in fibroblasts overexpressing cyclin D1 (or a nondegradable mutant of cyclin D1-T286A) indicates that cyclin D1 regulates this G(1) arrest. A delayed response to NAC exposure was an increase in both MnSOD protein and activity. NAC-induced G(1) arrest is exacerbated in MnSOD heterozygous fibroblasts. Results from electron spin resonance spectroscopy and flow cytometry measurements of dihydroethidine fluorescence showed an approximately 2-fold to 3-fold increase in the steady-state levels of superoxide (O(2)(*-)) in NAC-treated cells compared with control. Scavenging of O(2)(*-) with Tiron reversed the NAC-induced G(1) arrest. These results show that an O(2)(*-) signaling pathway regulates NAC-induced G(1) arrest by decreasing cyclin D1 protein levels and increasing MnSOD activity.

Epigenetic silencing of SOD2 by histone modifications in human breast cancer cells.

Many breast cancer cells typically exhibit lower expression of manganese superoxide dismutase (MnSOD) compared to the normal cells from which they arise. This decrease can often be attributed to a defect in the transcription of SOD2, the gene encoding MnSOD; however, the mechanism responsible for this change remains unclear. Here, we describe how altered histone modifications and a repressive chromatin structure constitute an epigenetic process to down regulate SOD2 in human breast carcinoma cell lines. Utilizing chromatin immunoprecipitation (ChIP) we observed decreased levels of dimethyl H3K4 and acetylated H3K9 at key regulatory elements of the SOD2 gene. Consistent with these results, we show that loss of these histone modifications creates a repressive chromatin structure at SOD2. Transcription factor ChIP experiments revealed that this repressive chromatin structure influences the binding of SP-1, AP-1, and NFkappaB to SOD2 regulatory cis-elements in vivo. Lastly, we show that treatment with the histone deacetylase inhibitors trichostatin A and sodium butyrate can reactivate SOD2 expression in breast cancer cell lines. Taken together, these results indicate that epigenetic silencing of SOD2 could be facilitated by changes in histone modifications and represent one mechanism leading to the altered expression of MnSOD observed in many breast cancers.

2-Deoxyglucose combined with wild-type p53 overexpression enhances cytotoxicity in human prostate cancer cells via oxidative stress.

Overexpression of the tumor suppressor gene, wild-type p53 (wtp53), using adenoviral vectors (Adp53) has been suggested to kill cancer cells by hydroperoxide-mediated oxidative stress [1,2] and nutrient distress induced by the glucose analog, 2-deoxyglucose (2DG), has been suggested to enhance tumor cell killing by agents that induce oxidative stress via disrupting hydroperoxide metabolism [3,4]. In the current study clonogenic cell killing of PC-3 and DU-145 human prostate cancer cells (lacking functional p53) mediated by 4 h exposure to 50 plaque forming units (pfus)/cell of Adp53 (that caused the enforced overexpression of wtp53) was significantly enhanced by treatment with 2DG. Accumulation of glutathione disulfide was found to be significantly greater in both cell lines treated with 2DG+Adp53 and both cell lines treated with 2DG+Adp53 showed a approximately 2-fold increases in dihydroethidine (DHE) and 5-(and-6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (CDCFH(2)) oxidation, indicative of increased steady-state levels of O(2)(.-) and hydroperoxides, respectively. Finally, overexpression of catalase or glutathione peroxidase using adenoviral vectors partially, but significantly, protected DU-145 cells from the toxicity induced by 2DG+Adp53 treatment. These results show that treatment of human prostate cancer cells with the combination of 2DG (a nutrient stress) and overexpression of the tumor suppressor gene, wtp53, enhances clonogenic cell killing by a mechanism that involves oxidative stress as well as allowing for the speculation that inhibitors of glucose and hydroperoxide metabolism can be used in combination with Adp53 gene therapy to enhance therapeutic responses.

Increased oxidative stress created by adenoviral MnSOD or CuZnSOD plus BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) inhibits breast cancer cell growth.

Superoxide dismutases (SODs) have been found to decrease tumor formation and angiogenesis. SOD gene therapy, as with many other gene transfer strategies, may not completely inhibit tumor growth on its own. Thus, concomitant therapies are necessary to completely control the spread of this disease. We hypothesized that intratumoral injection of AdSOD in combination with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) chemotherapy would synergistically inhibit breast cancer growth. Our data indicate that BCNU when combined with SOD overexpression increased oxidative stress as suggested by elevated glutathione disulfide (GSSG) production in one of three breast cancer cell lines tested, at least in part due to glutathione reductase (GR) inactivation. The increased oxidative stress caused by BCNU combined with adenovirally expressed SODs, manganese or copper zinc SOD, decreased growth and survival in the three cell lines tested in vitro, but had the largest effect in the MDA-MB231 cell line, which showed the largest amount of oxidative stress. Delivery of MnSOD and BCNU intratumorally completely inhibited MDA-MB231 xenograft growth and increased nude mouse survival in vivo. Intravenous (iv) BCNU, recapitulating clinical usage, and intratumoral AdMnSOD delivery, to provide tumor specificity, provided similar decreased growth and survival in our nude mouse model. This cancer therapy produced impressive results, suggesting the potential use of oxidative stress-induced growth inhibitory treatments for breast cancer patients.

Modulation of reactive oxygen species in pancreatic cancer.

PURPOSE: The aim of the present study was to compare the effects of the three different forms of the antioxidant enzyme superoxide dismutase [i.e., manganese superoxide dismutase (MnSOD), copper zinc superoxide dismutase (CuZnSOD), and extracellular superoxide dismutase (EcSOD)] on the malignant phenotype of human pancreatic cancer. EXPERIMENTAL DESIGN: Human pancreatic cancer cell lines were infected with adenoviral vectors containing the cDNAs for three different forms of the antioxidant enzyme SOD. Intratumoral injections of the adenoviral vectors were used in nude mice with human tumor xenografts. RESULTS: Increases in immunoreactive protein and enzymatic activity were seen after infections with the AdMnSOD, AdCuZnSOD, or AdEcSOD constructs. Increased SOD activity decreased superoxide levels and increased hydrogen peroxide levels. Increasing SOD levels correlated with increased doubling time. Cell growth and plating efficiency decreased with increasing amounts of the adenoviral constructs, with the AdCuZnSOD vector having the greatest effect in decreasing in vitro tumor growth. In contrast, inhibiting endogenous SOD with small interfering RNA increased superoxide levels and promoted tumor growth. Of the three SODs, tumors grew the slowest and survival was increased the greatest in nude mice injected with the AdEcSOD construct. CONCLUSIONS: Scavenging plasma membrane-generated superoxide may prove beneficial for suppression of pancreatic cancer growth.

Manganese superoxide dismutase-mediated gene expression in radiation-induced adaptive responses.

Antioxidant enzymes are critical in oxidative stress responses. Radioresistant variants isolated from MCF-7 human carcinoma cells following fractionated ionizing radiation (MCF+FIR cells) or overexpression of manganese superoxide dismutase (MCF+SOD cells) demonstrated dose-modifying factors at 10% isosurvival of 1.8 and 2.3, respectively. MCF+FIR and MCF-7 cells (exposed to single-dose radiation) demonstrated 5- to 10-fold increases in MnSOD activity, mRNA, and immunoreactive protein. Radioresistance in MCF+FIR and MCF+SOD cells was reduced following expression of antisense MnSOD. DNA microarray analysis and immunoblotting identified p21, Myc, 14-3-3 zeta, cyclin A, cyclin B1, and GADD153 as genes constitutively overexpressed (2- to 10-fold) in both MCF+FIR and MCF+SOD cells. Radiation-induced expression of these six genes was suppressed in fibroblasts from Sod2 knockout mice (-/-) as well as in MCF+FIR and MCF+SOD cells expressing antisense MnSOD. Inhibiting NF-kappa B transcriptional activity in MCF+FIR cells, by using mutant I kappa B alpha, inhibited radioresistance as well as reducing steady-state levels of MnSOD, 14-3-3 zeta, GADD153, cyclin A, and cyclin B1 mRNA. In contrast, mutant I kappa B alpha was unable to inhibit radioresistance or reduce 14-3-3 zeta, GADD153, cyclin A, and cyclin B1 mRNAs in MCF+SOD cells, where MnSOD overexpression was independent of NF-kappa B. These results support the hypothesis that NF-kappa B is capable of regulating the expression of MnSOD, which in turn is capable of increasing the expression of genes that participate in radiation-induced adaptive responses.

Manganese superoxide dismutase overexpression inhibits the growth of androgen-independent prostate cancer cells.

This study investigates the role of the antioxidant enzyme manganese superoxide dismutase (MnSOD) in androgen-independent human prostate cancer (PC-3) cells' growth rate in vitro and in vivo. MnSOD levels were found to be lower in parental PC-3 cells compared to nonmalignant, immortalized human prostate epithelial cells (P69SV40T). To unravel the role of MnSOD in the prostate cancer phenotype, PC-3 cells were stably transfected with MnSOD cDNA plasmid. The MnSOD protein and activity levels in clones overexpressing MnSOD were increased seven- to eightfold. These cell lines showed elongated cell doubling time, reduced anchorage-independent growth in soft agar compared to parental PC-3 (Wt) cells, and reduced growth rate of PC-3 tumor xenografts in athymic nude mice. Flow cytometric studies showed an increase in membrane potential in the MnSOD-overexpressing clone (Mn32) compared to Wt and Neo cells. Also, production of extracellular H(2)O(2) was increased in the MnSOD-overexpressing clones. As determined by DNA cell cycle analysis, the proportion of cells in G(1) phase was enhanced by MnSOD overexpression. Therefore, MnSOD not only regulates cell survival but also affects PC-3 cell proliferation by retarding G(1) to S transition. Our results are consistent with MnSOD being a tumor suppressor gene in human prostate cancer.

Manganese superoxide dismutase suppresses hypoxic induction of hypoxia-inducible factor-1alpha 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-1alpha subunit and an HIF-1beta subunit. HIF-1alpha is degraded following enzyme-dependent hydroxylation of prolines of HIF-1alpha in the presence of molecular oxygen, Fe2+, alpha-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-1alpha protein in human breast carcinoma MCF-7 cells. This suppression was biphasic depending on MnSOD activity. At low levels of MnSOD activity, HIF-1alpha 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-1alpha 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-1alpha protein in cells with high levels of MnSOD; this effect demonstrates that the restabilization of HIF-1alpha 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-1alpha protein levels. These observations demonstrated that HIF-1alpha accumulation and VEGF expression could be modulated by the antioxidant enzyme MnSOD.

Suppression of the malignant phenotype in pancreatic cancer by overexpression of phospholipid hydroperoxide glutathione peroxidase.

Phospholipid glutathione peroxidase (PhGPx) reduces lipid hydroperoxides generated in biomembranes and also uses a wide range of reducing cofactors in addition to glutathione. PhGPx is synthesized as a mitochondrial PhGPx form (L-form) and as a nonmitochondrial PhGPx form (S-form). Our aims were to determine whether overexpression of PhGPx altered pancreatic tumor cell behavior. Pancreatic cancer cell lines were found by Western blotting to have diminished levels of PhGPx-immunoreactive protein compared with normal human pancreas. To normalize the levels of this protein, PhGPx was overexpressed in MIA PaCa-2 and AsPC-1 human pancreatic cancer cells by infection with an adenovirus-PhGPx L-form construct (AdPhGPx- L-form) (0-200 MOI) or with an adenovirus-PhGPx S-form construct (AdPhGPx-S-form) (0-200 MOI), and cell growth, plating efficiency, and growth in soft agar were determined. Pancreatic cancer cells were also injected subcutaneously into nude mice and tumor volume was calculated. Single direct injections of the adenoviral- PhGPx constructs were made into preestablished tumors. In vitro, AdPhGPx-S-form demonstrated 80% tumor growth inhibition, whereas AdPhGPx-L-form demonstrated 95% tumor growth inhibition. Ad- PhGPx-L-form or AdPhGPx-S-form also decreased plating efficiency and growth in soft agar. AdPhGPx-Lform decreased in vivo tumor growth to a greater extent than did AdPhGPx-S-form. Because of the growthinhibitory effects of PhGPx, lipid hydroperoxides may play an important role in the growth of pancreatic cancer.

Delayed radioprotection by nuclear transcription factor kappaB -mediated induction of manganese superoxide dismutase in human microvascular endothelial cells after exposure to the free radical scavenger WR1065.

The free radical scavenger WR1065 (SH) is the active thiol form of the clinically approved cytoprotector amifostine. At doses of 40 microM and 4 mM it can activate the redox-sensitive nuclear transcription factor kappaB (NFkappaB) and elevate the expression of the antioxidant gene manganese superoxide dismutase (MnSOD) in human microvascular endothelial cells (HMEC). MnSOD contains binding motifs for a number of transcription factors other than NFkappaB and codes for a potent antioxidant enzyme localized in the mitochondria that is known to confer enhanced radiation resistance to cells. The purpose of this study was to determine the effect of WR1065 exposure on the various transcription factors known to affect MnSOD expression along with the subsequent kinetics of intracellular elevation of MnSOD protein levels and associated change in sensitivity to ionizing radiation in HMEC. Cells were grown to confluence and exposed to WR1065 for 30 min. Affects on the transcription factors AP1, AP2, CREB, NFkappaB, and Sp1 were monitored as a function of time ranging from 30 min to 4 h after drug exposure using a gel-shift assay. Only NFkappaB exhibited a marked activation and that occurred 30 min following the cessation of drug exposure. MnSOD protein levels, as determined by Western blot analysis, increased up to 16-fold over background control levels by 16 h following drug treatment, and remained at 10-fold or higher levels for an additional 32 h. MnSOD activity was evaluated using a gel-based assay and was found to be active throughout this time period. HMEC were irradiated with X-rays either in the presence of 40 microM or 4 mM WR1065 or 24 h after its removal when MnSOD levels were most elevated. No protection was observed for cells irradiated in the presence of 40 microM WR1065. In contrast, a 4 mM dose of WR1065 afforded an increase in cell survival by a factor of 2. A "delayed radioprotective" effect was, however, observed when cells were irradiated 24 h later, regardless of the concentration of WR1065 used. This effect is characterized as an increase in survival at the 2 Gy dose point, i.e., a 40% increase in survival, and an increase in the initial slope of the survival curve by a factor of 2. The anti-inflammatory sesquiterpene lactone, Helenalin, is an effective inhibitor of NFkappaB activation. HMEC were exposed to Helenalin for 2 h at a nontoxic concentration of 5 microM prior to exposure to WR1065. This treatment not only inhibited activation of NFkappaB by WR1065, but also inhibited the subsequent elevation of MnSOD and the delayed radioprotective effect. A persistent marked elevation of MnSOD in cells following their exposure to a thiol-containing reducing agent such as WR1065 can result in an elevated resistance to the cytotoxic effects of ionizing radiation and represents a novel radioprotection paradigm.

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.

Enzymatic activity is necessary for the tumor-suppressive effects of MnSOD.

The antioxidant protein manganese-containing superoxide dismutase (MnSOD) has been found to be a new type of tumor-suppressor protein. Overexpression of the cDNA for this gene in various types of cancer via plasmid transfection or adenovirus transduction leads to growth suppression both in vitro and in vivo. The growth-suppressive effect of MnSOD overexpression has been presumed to be due to the enzymatic activity of the MnSOD protein, but could be due to a number of other mechanisms, including a regulatory effect of the RNA or protein produced. To examine this question, we used site-directed mutagenesis to produce a mutant form of human MnSOD that has a leucine at amino acid 26 in the active site rather than the usual histidine. We demonstrate that plasmid transfection or adenoviral transduction of this mutant MnSOD cDNA leads to a large increase in immunoreactive MnSOD protein, but little or no increase in enzymatic activity. In contrast, overexpression of wild-type MnSOD leads to cells with both increased MnSOD protein and activity. Overexpression of wild-type, but not mutant, MnSOD leads to decreased plating efficiency and growth. These results clearly demonstrate that the tumor-suppressive effect of MnSOD protein is largely due to its enzymatic activity.

Inactivation of primary antioxidant enzymes in mouse keratinocytes by photodynamically generated singlet oxygen.

Cellular antioxidant enzymes protect against damage caused by exposure to endogenous or exogenous prooxidants. Singlet oxygen ((1)O(2)) is a reactive form of oxygen that can be produced in vivo either in normal and pathophysiologic conditions or by photosensitizing chemicals, as during photodynamic treatment. We hypothesized that photodynamically generated (1)O(2) would decrease the enzymatic activities of cellular antioxidants. To test this hypothesis, we treated cultured mouse epidermal keratinocytes with the photosensitizer Photofrin plus visible light to produce (1)O(2), and then measured CuZnSOD, MnSOD, and catalase activities with both ingel and spectrophotometric enzyme activity assays. Our results demonstrated that the enzymatic activities of cellular CuZnSOD, MnSOD, and catalase were significantly decreased after keratinocytes were treated with Photofrin plus visible light. By contrast, the enzymatic activities of cellular CuZnSOD, MnSOD, and catalase were unaffected in control cells treated with Photofrin only or visible light only. Despite the decreased levels of enzymatic activities, the protein levels of all three primary antioxidant enzymes remained constant after photodynamic treatment, as determined by Western blotting. L-Histidine, a (1)O(2) quencher, protected against the inactivation of cellular CuZnSOD, MnSOD, and catalase enzymes induced by photodynamically generated (1)O(2). The conclusion from these experiments is that the primary cellular antioxidant enzymes CuZnSOD, MnSOD, and catalase can be inactivated by photodynamically generated (1)O(2) in nucleated mammalian cells. These findings may be useful in the future development of antineoplastic adjuvant therapies that use photodynamic generation of (1)O(2) to inactivate antioxidant defenses with a goal of sensitizing tumor cells to prooxidant-generating drugs.

Chronic antioxidant enzyme mimetic treatment differentially modulates hyperthermia-induced liver HSP70 expression with aging.

One postulated mechanism for the reduction in stress tolerance with aging is a decline in the regulation of stress-responsive genes, such as inducible heat shock protein 72 (HSP70). Increased levels of oxidative stress are also associated with aging, but it is unclear what impact a prooxidant environment might have on HSP70 gene expression. This study utilized a superoxide dismutase/catalase mimetic (Eukarion-189) to evaluate the impact of a change in redox environment on age-related HSP70 responses to a physiologically relevant heat challenge. Results demonstrate that liver HSP70 mRNA and protein levels are reduced in old compared with young rats at selected time points over a 48-h recovery period following a heat-stress protocol. While chronic systemic administration of Eukarion-189 suppressed hyperthermia-induced liver HSP70 mRNA expression in both age groups, HSP70 protein accumulation was blunted in old rats but not in their young counterparts. These data suggest that a decline in HSP70 mRNA levels may be responsible for the reduction in HSP70 protein observed in old animals after heat stress. Furthermore, improvements in redox status were associated with reduced HSP70 mRNA levels in both young and old rats, but differential effects were manifested on protein expression, suggesting that HSP70 induction is differentially regulated with aging. These findings highlight the integrated mechanisms of stress protein regulation in eukaryotic organisms responding to environmental stress, which likely involve interactions between a wide range of cellular signals.

Vitamin E inhibits abdominal aortic aneurysm formation in angiotensin II-infused apolipoprotein E-deficient mice.

BACKGROUND: Abdominal aortic aneurysms (AAAs) in humans are associated with locally increased oxidative stress and activity of NADPH oxidase. We investigated the hypothesis that vitamin E, an antioxidant with documented efficacy in mice, can attenuate AAA formation during angiotensin II (Ang II) infusion in apolipoprotein E-deficient mice. METHODS AND RESULTS: Six-month-old male apolipoprotein E-deficient mice were infused with Ang II at 1000 ng/kg per minute for 4 weeks via osmotic minipumps while consuming either a regular diet or a diet enriched with vitamin E (2 IU/g of diet). After 4 weeks, abdominal aortic weight and maximal diameter were determined, and aortic tissues were sectioned and examined using biochemical and histological techniques. Vitamin E attenuated formation of AAA, decreasing maximal aortic diameter by 24% and abdominal aortic weight by 34% (P<0.05, respectively). Importantly, animals treated with vitamin E showed a 44% reduction in the combined end point of fatal+nonfatal aortic rupture (P<0.05). Vitamin E also decreased aortic 8-isoprostane content (a marker of oxidative stress) and reduced both aortic macrophage infiltration and osteopontin expression (P<0.05, respectively). Vitamin E treatment had no significant effect on the extent of aortic root atherosclerosis, activation of matrix metalloproteinases 2 or 9, serum lipid profile, or systolic blood pressure. CONCLUSIONS: Vitamin E ameliorates AAAs and reduces the combined end point of fatal+nonfatal aortic rupture in this animal model. These findings are consistent with the concept that oxidative stress plays a pivotal role in Ang II-driven AAA formation in hyperlipidemic mice.

Dual-expressing adenoviral vectors encoding the sodium iodide symporter for use in noninvasive radiological imaging of therapeutic gene transfer.

INTRODUCTION: Noninvasive analysis of therapeutic transgene expression is important for the development of clinical translational gene therapy strategies against cancer. To image p53 and MnSOD gene transfer noninvasively, we used radiologically detectable dual-expressing adenoviral vectors with the human sodium iodide symporter (hNIS) as the reporter gene. METHODS: Dual-expressing adenoviral vectors were constructed with hNIS cloned into E3 region and therapeutic genes, either MnSOD or p53, recombined into the E1 region. Steady-state mRNA levels of hNIS were evaluated by real-time polymerase chain reaction. hNIS function was determined by iodide uptake assay and MnSOD, and p53 protein levels were assessed by Western blots. RESULTS: 125I- accumulation resulting from hNIS expression in both Ad-p53-hNIS- and Ad-MnSOD-hNIS-infected MDA-MB-435 cells could be visualized clearly on phosphorimaging autoradiograph. Iodide accumulation increased with increasing adenovirus titer, and there was a linear correlation between iodide uptake and dose. p53 and MnSOD protein levels increased as a function of adenovirus titer, and there was a direct positive correlation between p53 and MnSOD expression and hNIS function. P53 and MnSOD overexpression inhibited cell growth in the dual-expressing adenoviral vector-infected cells. CONCLUSIONS: Radiological detection of hNIS derived from dual-expressing adenoviral vectors is a highly effective method to monitor therapeutic gene transfer and expression in a noninvasive manner.

Overexpression of copper zinc superoxide dismutase suppresses human glioma cell growth.

Copper zinc superoxide dismutase (CuZnSOD) is an essential primary antioxidant enzyme that converts superoxide radical to hydrogen peroxide and molecular oxygen in the cytoplasm. Cytosolic glutathione peroxidase (GPx) converts hydrogen peroxide into water. The overall goal of the present study was to explore the possible role of the antioxidant enzyme CuZnSOD in expression of the malignant phenotype. We hypothesized that overexpression of CuZnSOD would lead to the suppression of at least part of the human malignant phenotype. To test this hypothesis, human CuZnSOD cDNA was transfected into U118-9 human malignant glioma cells. CuZnSOD activity levels increased 1.5-, 2.0-, 2.6-, and 3.5-fold, respectively, in four table transfected cell lines compared with wild type and vector controls. Overexpression of CuZnSOD altered cellular antioxidant enzyme profiles, including those of manganese superoxide dismutase, catalase, and GPx. The transfected clone with the highest CuZnSOD:GPx ratio (3.5) showed a 42% inhibition of tumor cell growth in vitro. The decreased rate of tumor cell growth in vitro was strongly correlated with the enzyme activity ratio of CuZnSOD:GPx. Glioma cells that stably overexpressed CuZnSOD demonstrated additional suppressive effects on the malignant phenotype when compared with the parental cells and vector controls. These cells showed decreased plating efficiency, elongated cell population doubling time, lower clonogenic fraction in soft agar, and, more significantly, inhibition of tumor formation in nude mice. This work suggested that CuZnSOD is a new tumor suppressor gene. Increased intracellular ROS levels were found in cells with high activity ratios of CuZnSOD:GPx. Change in the cellular redox status, especially change attributable to the accumulation of hydrogen peroxide or other hydroperoxides, is a possible reason to explain the suppression of tumor growth observed in CuZnSOD-overexpressing cells.