Cells contain a large number of antioxidants to prevent or repair the damage caused by ROS, as well as to regulate redox-sensitive signaling pathways General protocols are described to measure the antioxidant enzyme activity of superoxide dismutase (SOD), catalase, and glutathione peroxidase. The SODs convert superoxide radical into hydrogen peroxide and molecular oxygen, while the catalase and peroxidases convert hydrogen peroxide into water. In this way, two toxic species, superoxide radical and hydrogen peroxide, are converted to the harmless product water. Western blots, activity gels and activity assays are various methods used to determine protein and activity in both cells and tissue depending on the amount of protein needed for each assay. Other techniques including immunohistochemistry and immunogold can further evaluate the levels of the various antioxidant enzymes in tissue and cells. In general, these assays require 24 to 48 hours to complete.
Proline oxidase (POX), localized on inner mitochondrial membranes, is encoded by a p53-induced gene and metabolically participates in p53-induced apoptosis. Previously, we showed that POX catalyzed the generation of reactive oxygen species (ROS). We and others have demonstrated that overexpression of POX, independent of p53, causes apoptotic cell death in a variety of cancer cells. But a necessary role for ROS remains uncertain. Therefore, we asked whether superoxide dismutases (SOD) and catalase (CAT), important antioxidant enzymes, might interfere with the POX-dependent induction of apoptosis. In this study, we used DLD-1 colorectal cancer cells stably transfected with the POX gene under the control of a tetracycline-inducible promoter. When doxycycline was removed from the culture medium and the expression of POX was induced, apoptotic cell death was initiated. To examine the importance of the ROS-dependent component of the pathway, we infected DLD-1 POX cells with recombinant adenoviruses containing MnSOD, CuZnSOD, CAT or varying combinations of these adenoviruses followed by induced expression of POX. The expression of MnSOD inhibited POX-induced apoptosis, but others did not. Mechanistically, mitochondria-localized MnSOD dramatically reduced the release of cytochrome c to cytosol by POX. Compared with control cells, MnSOD-expressing DLD-1 POX cells generated a higher concentration of H2O2 owing to dismutation of superoxide radicals, which was elevated by POX. Thus, these data further suggest that the generation of superoxide radicals plays a crucial role in POX-induced apoptosis and the process is partially blocked by MnSOD.
The interaction between epithelial cells and the extracellular matrix is crucial for tissue architecture and function and is compromised during cancer progression. Dystroglycan is a membrane receptor that mediates interactions between cells and basement membranes in various epithelia. In many epithelium-derived cancers, -dystroglycan is expressed, but ␣-dystroglycan is not detected. Here we report that ␣-dystroglycan is correctly expressed and trafficked to the cell membrane but lacks laminin binding as a result of the silencing of the like-acetylglucosaminyltransferase (LARGE) gene in a cohort of highly metastatic epithelial cell lines derived from breast, cervical, and lung cancers. Exogenous expression of LARGE in these cancer cells restores the normal glycosylation and laminin binding of ␣-dystroglycan, leading to enhanced cell adhesion and reduced cell migration in vitro. Our findings demonstrate that LARGE repression is responsible for the defects in dystroglycan-mediated cell adhesion that are observed in epithelium-derived cancer cells and point to a defect of dystroglycan glycosylation as a factor in cancer progression.Normal epithelial cells are tightly associated with one another and with the underlying basement membrane to maintain tissue architecture and function. During cancer progression, primitive cancer cells escape from this control by modifying the binding affinities of their cell membrane receptors. Several receptors have been described as important for this process. Of these, the integrins are the best studied (1). The receptor dystroglycan has been reported to be required for the development and maintenance of epithelial tissues (2, 3). A direct requirement for dystroglycan in epithelia is further demonstrated by the profound effect that loss of dystroglycan expression has on cell polarity and laminin binding in cultured mammary epithelial cells (4, 5). However, dystroglycan is not only important in the establishment and maintenance of epithelial structure. Associations have also been made between the loss of ␣-dystroglycan immunoreactivity and cancer progression in tumors of epithelial origin, including breast, colon, cervix, and prostate cancers (4, 6 -9). The dystroglycan loss of function could thus serve as an effective means by which cancerous cells modify their adhesion to the extracellular matrix (ECM). 2Dystroglycan is a ubiquitously expressed cell membrane protein that plays a key function in cellular integrity, linking the intracellular cytoskeleton to the extracellular matrix. The dystroglycan gene encodes a preprotein that is cleaved into two peptides (10). The C-terminal component, known as -dystroglycan, is embedded within the cell membrane, whereas the N-terminal component, ␣-dystroglycan, is present within the extracellular periphery but remains associated with -dystroglycan through non-covalent bonds. -Dystroglycan binds to actin (11), dystrophin (11), utrophin (11), and Grb2 (12) through its C-terminal intracellular domain. ␣-Dystroglycan, on the other hand, binds to E...
Objective-We tested the hypothesis that deficiency of cellular glutathione peroxidase (GPx-1) enhances susceptibility to endothelial dysfunction in mice with moderate hyperhomocysteinemia. Methods and Results-Mice that were wild type (Gpx1 ϩ/ϩ ), heterozygous (Gpx1 ϩ/Ϫ ), or homozygous (Gpx1 Ϫ/Ϫ ) for the mutated Gpx1 allele were fed a control diet or a high-methionine diet for 17 weeks. Plasma total homocysteine was elevated in mice on the high-methionine diet compared with mice on the control diet (23Ϯ3 versus 6Ϯ0.3 mol/L, respectively; PϽ0.001) and was not influenced by Gpx1 genotype. In mice fed the control diet, maximal relaxation of the aorta in response to the endothelium-dependent dilator acetylcholine (10 Ϫ5 mol/L) was similar in Gpx1 ϩ/ϩ , Gpx1 ϩ/Ϫ , and Gpx1 Ϫ/Ϫ mice, but relaxation to lower concentrations of acetylcholine was selectively impaired in Gpx1 Ϫ/Ϫ mice (PϽ0.05 versus Gpx1 ϩ/ϩ mice). In mice fed the high-methionine diet, relaxation to low and high concentrations of acetylcholine was impaired in Gpx1 PϽ0.05). No differences in vasorelaxation to nitroprusside or papaverine were observed between Gpx1 ϩ/ϩ and Gpx1 Ϫ/Ϫ mice fed either diet. Dihydroethidium fluorescence, a marker of superoxide, was elevated in Gpx1 Ϫ/Ϫ mice fed the high-methionine diet (PϽ0.05 versus Gpx1 ϩ/ϩ mice fed the control diet). Conclusions-These findings demonstrate that deficiency of GPx-1 exacerbates endothelial dysfunction in hyperhomocysteinemic mice and provide support for the hypothesis that hyperhomocysteinemia contributes to endothelial dysfunction through a peroxide-dependent oxidative mechanism. Key Words: endothelium Ⅲ homocysteine Ⅲ nitric oxide Ⅲ peroxide H yperhomocysteinemia is an emerging risk factor for cardiovascular events and venous thrombosis, 1,2 but the mechanisms responsible for the vascular pathology of hyperhomocysteinemia are still incompletely understood. Like many other cardiovascular risk factors, hyperhomocysteinemia produces endothelial dysfunction, which is possibly due to oxidative inactivation of endothelium-derived NO. 3,4 The oxidative stress hypothesis 4 is supported by the observation that auto-oxidation of homocysteine in vitro generates reactive oxygen species (ROS), including hydrogen peroxide and superoxide, and promotes oxidation of LDL. 5-7 Treatment of cultured endothelial cells with homocysteine decreases bioavailable NO 8,9 and produces cytotoxicity mediated by hydrogen peroxide. 10 Homocysteine also indirectly contributes to oxidative stress by inhibiting the expression of antioxidant enzymes, such as cellular glutathione peroxidase (GPx-1), an effect that may sensitize one to the toxic effects of homocysteine-derived hydrogen peroxides and lipid peroxides. 8,11 However, the role of oxidative stress in the vascular dysfunction of hyperhomocysteinemia in vivo is less clear, and its clinical importance has been questioned. 12 Attempts to demonstrate elevated levels of oxidation products in humans with hyperhomocysteinemia have produced conflicting results. [13][14][15][16] R...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.