Superoxide Levels In Cells Research Articles

P 023 - Differential antioxidant protection against oxidants by esculetin or quercetin in human leukemia NB4 cells

Esculetin (6,7-dihydrocoumarin) and the flavonoid quercetin (3,5,7,3',4' pentahydroxyflavone) show different pharmacological properties probably related to effects on redox homeostasis. Human leukemia NB4 cells were preincubated for 30 min or 2 h with either 100 μM esculetin or 25 μM quercetin. The cells were then treated with 1 mM H2O2 or 250 μM tert-butyl hydroperoxide (t-BHP) for 1 h. Pretreatments with quercetin prevented loss of cell viability (impermeability to PI) induced by H2O2 or t-BHP. Treatment with 1 mM H2O2 for 1 h produced lower apoptotic cells (26%) than 250 μM t-BHP (38%). Pretreatments with esculetin increased the apoptosis induced by H2O2 but reduced significantly the apoptosis produced by t-BHP. Quercetin reduced apoptosis produced by H2O2 and wholly protects against apoptosis induced by t-BHP. Superoxide was increased by treatment with H2O2 or t-BHP. Esculetin or quercetin increased the levels of superoxide in cells treated with H2O2 but reduced them in cells treated with t-BHP. Esculetin but not quercetin almost prevented peroxide production by H2O2. Our results show different effects of antioxidant treatment on leukemia cells and possible differential applications for antitumor therapy with either of those antioxidant compounds used.

Inhibitors of Catalase-Amyloid Interactions Protect Cells from β-Amyloid-Induced Oxidative Stress and Toxicity

Compelling evidence shows a strong correlation between accumulation of neurotoxic β-amyloid (Aβ) peptides and oxidative stress in the brains of patients afflicted with Alzheimer disease (AD). One hypothesis for this correlation involves the direct and harmful interaction of aggregated Aβ peptides with enzymes responsible for maintaining normal, cellular levels of reactive oxygen species (ROS). Identification of specific, destructive interactions of Aβ peptides with cellular anti-oxidant enzymes would represent an important step toward understanding the pathogenicity of Aβ peptides in AD. This report demonstrates that exposure of human neuroblastoma cells to cytotoxic preparations of aggregated Aβ peptides results in significant intracellular co-localization of Aβ with catalase, an anti-oxidant enzyme responsible for catalyzing the degradation of the ROS intermediate hydrogen peroxide (H(2)O(2)). These catalase-Aβ interactions deactivate catalase, resulting in increased cellular levels of H(2)O(2). Furthermore, small molecule inhibitors of catalase-amyloid interactions protect the hydrogen peroxide-degrading activity of catalase in Aβ-rich environments, leading to reduction of the co-localization of catalase and Aβ in cells, inhibition of Aβ-induced increases in cellular levels of H(2)O(2), and reduction of the toxicity of Aβ peptides. These studies, thus, provide evidence for the important role of intracellular catalase-amyloid interactions in Aβ-induced oxidative stress and propose a novel molecular strategy to inhibit such harmful interactions in AD.

Cu, Zn Superoxide Dismutase and NADP(H) Homeostasis Are Required for Tolerance of Endoplasmic Reticulum Stress inSaccharomyces cerevisiae

Genome-wide screening for sensitivity to chronic endoplasmic reticulum (ER) stress induced by dithiothreitol and tunicamycin (TM) identified mutants deleted for Cu, Zn superoxide dismutase (SOD) function (SOD1, CCS1) or affected in NADPH generation via the pentose phosphate pathway (TKL1, RPE1). TM-induced ER stress led to an increase in cellular superoxide accumulation and an increase in SOD1 expression and Sod1p activity. Prior adaptation of the hac1 mutant deficient in the unfolded protein response (UPR) to the superoxide-generating agent paraquat reduced cell death under ER stress. Overexpression of the ER oxidoreductase Ero1p known to generate hydrogen peroxide in vitro, did not lead to increased superoxide levels in cells subjected to ER stress. The mutants lacking SOD1, TKL1, or RPE1 exhibited decreased UPR induction under ER stress. Sensitivity of the sod1 mutant to ER stress and decreased UPR induction was partially rescued by overexpression of TKL1 encoding transketolase. These data indicate an important role for SOD and cellular NADP(H) in cell survival during ER stress, and it is proposed that accumulation of superoxide affects NADP(H) homeostasis, leading to reduced UPR induction during ER stress.

Mitochondrial Production of Reactive Oxygen Species Mediate Dicumarol-induced Cytotoxicity in Cancer Cells

Dicumarol is a naturally occurring anticoagulant derived from coumarin that induces cytotoxicity and oxidative stress in human pancreatic cancer cells (Cullen, J. J., Hinkhouse, M. M., Grady, M., Gaut, A. W., Liu, J., Zhang, Y., Weydert, C. J. D., Domann, F. E., and Oberley, L. W. (2003) Cancer Res. 63, 5513-5520). Although dicumarol has been used as an inhibitor of the two-electron reductase NAD(P)H:quinone oxidoreductase (NQO1), dicumarol is also thought to affect quinone-mediated electron transfer reactions in the mitochondria, leading to the production of superoxide (O2*-) and hydrogen peroxide (H(2)O(2)). We hypothesized that mitochondrial production of reactive oxygen species mediates the increased susceptibility of pancreatic cancer cells to dicumarol-induced metabolic oxidative stress. Dicumarol decreased clonogenic survival equally in both MDA-MB-468 NQO1(-) and MDA-MB-468 NQO1+ breast cancer cells. Dicumarol decreased clonogenic survival in the transformed fibroblast cell line IMRSV-90 compared with the IMR-90 cell line. Dicumarol, with the addition of mitochondrial electron transport chain blockers, decreased clonogenic cell survival in human pancreatic cancer cells and increased superoxide levels. Dicumarol with the mitochondrial electron transport chain blocker antimycin A decreased clonogenic survival and increased superoxide levels in cells with functional mitochondria but had little effect on cancer cells without functional mitochondria. Overexpression of manganese superoxide dismutase and mitochondrial-targeted catalase with adenoviral vectors reversed the dicumarol-induced cytotoxicity and reversed fluorescence of the oxidation-sensitive probe. We conclude mitochondrial production of reactive oxygen species mediates the increased susceptibility of cancer cells to dicumarol-induced cytotoxicity.