Cell Compartmentalization in Redox Signaling

From a growing body of evidence on the role of Reactive Oxygen Species as intracellular signaling molecules, the concept starts to emerge that cell responses to redox changes are function of the intracellular site where oxidants are produced and/or meet their molecular targets. In particular,a major distinction between oxidative events in the cytosolic versus the mitochondrial compartment appears to exist in terms of physiological stimuli, signaling mechanisms and functional consequences. Experimental data supporting this view are reviewed here, and the potential implications of this new perspective in redox signaling are discussed

[8] Determination of intracellular reactive oxygen species as function of cell density

Reactive oxygen species (ROS) such as superoxide (O2–) hydrogen peroxide (H2O2), and nitric oxide (NO), generated by activated phagocytes, are crucial for the immune response against pathogens. It has become evident that ROS, while responsible for indiscriminate oxidative cell damage when released in excess amounts, can also, at low concentrations and in very limited time frames, behave as fine modulators of important intracellular events, such as protein phosphorylation and gene transcription, through which the cell response to environmental stimuli is triggered. In view of these considerations, a protocol in which the intracellular production of oxygen species grown at different plating densities is evaluated in single cells by flow cytometry after cell loading with the peroxide-sensitive probe dichlorofluorescein diacetate (DCF-DA) and following rapid detachment from the substrate. A flow chart summarizing the basic protocol for determination of intracellular ROS at different plating densities is also presented. A method to evaluate the intracellular content of oxidants as a function of cell density—that is, in cells plated at different degree of confluence is developed. The method is based on the cytofluorimetric analysis of the oxidative modification of a widely used probe, dichlorofluorescein diacetate that gains a bright green fluorescence on interaction with hydrogen peroxide and organic peroxides

Mitochondrial superoxide dismutase: a promising target for new anticancer therapies

Compelling experimental and epidemiological evidence involves oxygen radicals in carcinogenesis, acting reactive oxygen species both as endogenous genotoxins during cell initiation and as messenger molecules in mitogenesis and in tumor promotion. Moreover, oxidants stimulate neoangiogenesis, which is a prerequisite for tumor growth. However, while several natural as well as synthetic antioxidant compounds appear to be chemopreventive in mutagenicity assays, antioxidant-based treatments for the prevention or cure of cancer have led to non-conclusive if not disappointing results. This is likely due to the fact that oxygen radicals have also a major role in the natural defences against the propagation of cancer cells, i.e. tumor cell apoptosis and immune surveillance, and mediate the beneficial cytotoxic effect of both the chemo-and radio-therapy. In recent years, the mitochondrial antioxidant enzyme, Manganous Superoxide Dismutase (MnSOD), has received a growing attention as a negative modulator of cellular apoptosis and as a survival factor for cancer cells. In fact, while overexpression of this enzyme in cancer cells decreases proliferation and tumor incidence in transgenic models, it is clear that even small amounts of this enzyme are crucial for cell resistance to inflammatory stimuli and anticancer drugs, and prevent oncogene-induced apoptosis triggered by the tumor suppressor protein p53. A previously unexpected oncogenic potential of MnSOD is also suggested by the elevated levels of this enzyme in several classes of human neoplasms, in a fashion which often correlates with the degree of their malignancy. This review focuses on the debated issue of the pro- and/or anti-tumoral effect of MnSOD, with special emphasis on recent observations suggesting that pharmacological inhibition of MnSOD may represent an effective strategy to selectively kill cancer cells and to circumvent their resistance to the commonly used anticancer treatments

Reactive oxygen species as downstream mediators of angiogenic signaling by vascular endothelial growth factor receptor-2/KDR

Recent evidence shows the involvement of reactive oxygen species (ROS) in the mitogenic cascade initiated by the tyrosine kinase receptors of several growth factor peptides. We have asked whether also the vascular endothelial growth factor (VEGF) utilizes ROS as messenger intermediates downstream of the VEGF receptor-2 (VEGFR-2)/KDR receptor given that the proliferation of endothelial cells during neoangiogenesis is physiologically regulated by oxygen and likely by its derivative species. In porcine aortic endothelial cells stably expressing human KDR, receptor activation by VEGF is followed by a rapid increase in the intracellular generation of hydrogen peroxide as revealed by the peroxide-sensitive probe dichlorofluorescein diacetate. Genetic and pharmacological studies suggest that such oxidant burst requires as upstream events the activation of phosphatidylinositol 3-kinase and the small GTPase Rac-1 and is likely initiated by lipoxygenases. Interestingly, ROS generation in response to VEGF is not blocked but rather potentiated by endothelial nitric-oxide synthase inhibitors diphenyleneiodonium and N(G)methyl-l-arginine, ruling out the possibility of nitric oxide being the oxidant species here detected in VEGF-stimulated cells. Inhibition of KDR-dependent generation of ROS attenuates early signaling events including receptor autophosphorylation and binding to a phospholipase C-gamma-glutathione S-transferase fusion protein. Moreover, catalase, the lipoxygenase inhibitor nordihydroguaiaretic acid, the synthetic ROS scavenger EUK-134, and phosphatidylinositol 3-kinase inhibitor wortmannin all reduce ERK phosphorylation in response to VEGF, and antioxidants prevent VEGF-dependent mitogenesis. Finally, cell culture and stimulation in a nearly anoxic environment mimic the effect of ROS scavenger on receptor and ERK phosphorylation, reinforcing the idea that ROS are necessary components of the mitogenic signaling cascade initiated by KDR. These data identify ROS as a new class of intracellular angiogenic mediators and may represent a potential premise for new antioxidant-based antiangiogenic therapies

Increased growth capacity of cervical‐carcinoma cells over‐expressing manganous superoxide dismutase

Increases in the expression of manganese‐dependent superoxide dismutase (MnSOD) have been detected in several classes of human and experimental tumors and appear to correlate with poorer prognosis in human neuro‐epithelial, ovarian and cervical tumors. To delineate the relevance of MnSOD expression to tumor‐cell growth and survival, a human MnSOD cDNA was over‐expressed in the HeLa cervical‐carcinoma cell line. MnSOD over‐expression had marginal effects on the growth of HeLa cells in standard medium, but markedly protected the cells from growth suppression and cell death in conditions of serum deprivation. Serum starvation did not affect expression of endogenous MnSOD in wild‐type HeLa cells, but was associated with increases in cell death and in the generation of intracellular oxygen radicals. By contrast, in HT29 colon‐carcinoma cells, which are relatively resistant to growth‐factor withdrawal, serum deprivation was associated with increases in MnSOD expression and activity. Together these observations suggest that MnSOD provides a mechanism for counteracting the intracellular oxidative processes that impair cell growth and viability in the context of growth‐factor withdrawal and, in this context, may promote tumor‐cell survival in vivo in conditions normally unfavorable to cell growth. Int. J. Cancer 82:145–150, 1999. © 1999 Wiley‐Liss, Inc

Redox Regulation of cAMP-responsive Element-binding Protein and Induction of Manganous Superoxide Dismutase in Nerve Growth Factor-dependent Cell Survival

Reactive oxygen species (ROS) act as both signaling molecules and mediators of cell damage in the nervous system and are implicated in the pathogenesis of neurodegenerative diseases. Neurotrophic factors such as the nerve-derived growth factor (NGF) support neuronal survival during development and promote regeneration after neuronal injury through the activation of intracellular signals whose molecular effectors and downstream targets are still largely unknown. Here we present evidence that early oxidative signals initiated by NGF in PC12 cells, an NGF-responsive cell line, play a critical role in preventing apoptosis induced by serum deprivation. This redox-signaling cascade involves phosphatidylinositol 3-kinase, the small GTPase Rac-1, and the transcription factor cAMP-responsive element-binding protein (CREB), a molecule essential to promote NGF-dependent survival. We found that ROS are necessary for NGF-dependent phosphorylation of CREB, an event directly correlated with CREB activity, whereas hydrogen peroxide induces a robust CREB phosphorylation. Cells exposed to NGF show a late decrease in the intracellular content of ROS when compared with untreated cells and increased expression of the mitochondrial antioxidant enzyme manganese superoxide dismutase, a general inhibitor of cell death. Accordingly, serum deprivation-induced apoptosis was selectively inhibited by low concentrations of the mitochondrially targeted antioxidant Mito Q (mitoquinol/mitoquinone). Taken together, these data demonstrate that the oxidant-dependent activation of CREB is a component of NGF survival signaling in PC12 cells and outline an intriguing circuitry by which a cytosolic redox cascade promotes cell survival at least in part by increasing mitochondrial resistance to oxidative stress

Abrogation of hepatocyte apoptosis and early appearance of liver dysplasia in ethanol-fed p53-deficient mice

Ethanol consumption represents a major risk factor for cancer development, and a significant fraction of hepatocarcinomas arises in alcoholic liver cirrhosis. Increasing evidence indicates that ethanol acts as a tumor promoter on genetically initiated cells, by increasing the intracellular concentration of reactive oxygen species and promoting tissue necrosis/regeneration and cell proliferation. The tumor suppressor p53 restrains the expansion of carcinogen-initiated cells by inducing cell cycle arrest and apoptosis; accordingly, p53-deficient mice develop spontaneous and chemically induced neoplasms at a much higher frequency than normal mice. In normal mice exposed to a subacute (3 weeks) ethanol intoxication, a significant increase in the number of apoptotic hepatocytes was observed in concomitance with the up-regulation of the mitochondrial superoxide scavenger MnSOD, a reliable indicator of oxidative stress. Cell death occurred in the absence of liver inflammation and necrosis. Ethanol-induced hepatocyte apoptosis was completely abrogated in the p53 null background, suggesting that the tumor suppressor is necessary for hepatocyte death by ethanol. Accordingly, p53 -/- MEF were, unlike wild type cells, completely insensitive up to 0.5 M ethanol in the culture medium. Strikingly, marked and widespread signs of dysplasia, with nuclear pleomorphisms and initial loss of normal architecture, heralding malignant transformation, were scored in all the mutant mice exposed to ethanol, but not in the control-fed littermates nor in ethanol-fed normal mice. These observations suggest that p53-dependent apoptosis restrains the tumorigenic effect of ethanol on liver cells, in agreement with the frequent loss of p53 function in HCC, and reveal an unexpected carcinogenic potential of alcohol which appears to be independent from the induction of cirrhosis and hepatocyte regeneration.Fil: Pani, Giovambattista. Università Cattolica del Sacro Cuore; ItaliaFil: Fusco, Salvatore. Università Cattolica del Sacro Cuore; ItaliaFil: Colavitti, Renata. Università Cattolica del Sacro Cuore; ItaliaFil: Borrello, Silvia. Università Cattolica del Sacro Cuore; ItaliaFil: Maggiano, Nicola. Università Cattolica del Sacro Cuore; ItaliaFil: Cravero, Amerys A.M.. Universidad de Buenos Aires; ArgentinaFil: Farré, Stella Maris. Universidad de Buenos Aires; ArgentinaFil: Galeotti, Tommaso. Università Cattolica del Sacro Cuore; ItaliaFil: Koch, Osvaldo Raul. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentin