Redox-regulation of intrinsic prion expression in multicellular prostate tumor spheroids

The cellular function of the intrinsic prion protein (PrPc) remains largely unknown. In the present study PrPc expression was investigated in multicellular prostate tumor spheroids and was correlated to the intracellular redox state as evaluated using the fluorescent dye 2'7'-dichlorodihydrofluorescein diacetate (H2DCFDA). In small tumor spheroids (diameter 100 +/- 20 microm) reactive oxygen species (ROS) levels were increased as compared with large (diameter 250 +/- 50 microm) spheroids. ROS generation was mediated by the mitochondrial respiratory chain and a NADPH oxidaselike enzyme, because carbonylcyanide-m-chlorophenylhydrazone (CCCP), rotenone, and diphenylene iodonium chloride (DPI) significantly reduced ROS levels. The elevated ROS were correlated to an increased expression of PrPc, Cu/Zn superoxide dismutase (SOD-1), and catalase in small as compared with large spheroids. In large tumor spheroids, PrPc was predominantly expressed in the peripheral cell layers and colocalized with SOD-1 and catalase. Raising intracellular ROS in large tumor spheroids by hydrogen peroxide, menadione, buthionine sulfoximine (BSO), and incubation in glutamine-reduced medium increased PrPc expression. In small spheroids PrPc was downregulated after incubation with the radical scavengers dehydroascorbate (DHA) and vitamin E. Our data indicate that PrPc expression in tumor spheroids is related to the intracellular redox state and may participate in antioxidative defense.     

Caveolin-1 is a negative regulator of NADPH oxidase-derived reactive oxygen species

Changes in the expression and function of caveolin-1 (Cav-1) have been proposed as a pathogenic mechanism underlying many cardiovascular diseases. Cav-1 binds to and regulates the activity of numerous signaling proteins via interactions with its scaffolding domain. In endothelial cells, Cav-1 has been shown to reduce reactive oxygen species (ROS) production, but whether Cav-1 regulates the activity of NADPH oxidases (Noxes), a major source of cellular ROS, has not yet been shown. Herein, we show that Cav-1 is primarily expressed in the endothelium and adventitia of pulmonary arteries (PAs) and that Cav-1 expression is reduced in isolated PAs from multiple models of pulmonary artery hypertension (PH). Reduced Cav-1 expression correlates with increased ROS production in the adventitia of hypertensive PA. In vitro experiments revealed a significant ability of Cav-1 and its scaffolding domain to inhibit Nox1–5 activity and it was also found that Cav-1 binds to Nox5 and Nox2 but not Nox4. In addition to posttranslational actions, in primary cells, Cav-1 represses the mRNA and protein expression of Nox2 and Nox4 through inhibition of the NF-κB pathway. Last, in a mouse hypoxia model, the genetic ablation of Cav-1 increased the expression of Nox2 and Nox4 and exacerbated PH. Together, these results suggest that Cav-1 is a negative regulator of Nox function via two distinct mechanisms, acutely through direct binding and chronically through alteration of expression levels. Accordingly, the loss of Cav-1 expression in cardiovascular diseases such as PH may account for the increased Nox activity and greater production of ROS.     

Induction of reactive oxygen species from isolated rat glomeruli by protein kinase C activation and TNF-alpha stimulation, and effects of a phosphodiesterase inhibitor

Diabetic nephropathy is a major complication of diabetes leading to end-stage renal disease, which requires hemodialysis. Although the mechanism by which it progresses is largely unknown, the role of hyperglycemia-derived oxidative stress has recently been the focus of attention as the cause of diabetic complications. Constituent cells of the renal glomeruli have the capacity to release reactive oxygen species (ROS) upon stimulation of NADPH oxidase activated by protein kinase C (PKC). Hyperglycemia and insulin resistance in the diabetic state are often associated with activation of PKC and tumor necrosis factor (TNF)-alpha, respectively. The aim of this study is to clarify the signaling pathway leading to ROS production by PKC and TNF-alpha in rat glomeruli. Isolated rat glomeruli were stimulated with phorbol 12-myristate 13-acetate (PMA) and TNF-alpha, and the amount of ROS was measured using a chemiluminescence method. Stimulation with PMA (10 ng/ml) generated ROS with a peak value of 136+/-1.2 cpm/mg protein (mean+/-SEM). The PKC inhibitor H-7, the NADPH oxidase inhibitor diphenylene iodonium and the phosphatidylinositol-3 (PI-3) kinase inhibitor wortmannin inhibited PMA-induced ROS production by 100%, 100% and 80%, respectively. In addition, TNF-alpha stimulated ROS production (283+/-5.8/mg protein/20 min). The phosphodiesterase inhibitor cilostazol activates protein kinase A and is reported to improve albuminuria in diabetic rats. Cilostazol (100 microg/ml) inhibited PMA, and TNF-alpha-induced ROS production by 78+/-1.8, and 19+/-2.7%, respectively. The effects of cilostazol were not additive with wortmannin. Cilostazol arrests oxidative stress induced by PKC activation by inhibiting the PI-3 kinase-dependent pathway, and may thus prevent the development of diabetic nephropathy.     

Molecular interaction of NADPH oxidase 1 with betaPix and Nox Organizer 1

It is well established that growth-factor-induced reactive oxygen species (ROS) act as second messengers in cell signaling. We have previously reported that betaPix, a guanine nucleotide exchange factor for Rac, interacts with NADPH oxidase 1 (Nox1) leading to EGF-induced ROS generation. Here, we report the identification of the domains of Nox1 and betaPix responsible for the interaction between the two proteins. GST pull-down assays show that the PH domain of betaPix binds to the FAD-binding region of Nox1. We also show that overexpression of the PH domain of betaPix results in inhibition of superoxide anion generation in response to EGF. Additionally, NADPH oxidase Organizer 1 (NoxO1) is shown to interact with the NADPH-binding region of Nox1. These results suggest that the formation of the complex consisting of Nox1, betaPix, and NoxO1 is likely to be a critical step in EGF-induced ROS generation.     

Fatty acids as modulators of the cellular production of reactive oxygen species

Long-chain nonesterified ("free") fatty acids (FFA) and some of their derivatives and metabolites can modify intracellular production of reactive oxygen species (ROS), in particular O(2)(-) and H(2)O(2). In mitochondria, FFA exert a dual effect on ROS production. Because of slowing down the rate of electron flow through Complexes I and III of the respiratory chain due to interaction within the complex subunit structure, and between Complexes III and IV due to release of cytochrome c from the inner membrane, FFA increase the rate of ROS generation in the forward mode of electron transport. On the other hand, due to their protonophoric action on the inner mitochondrial membrane ("mild uncoupling effect"), FFA strongly decrease ROS generation in the reverse mode of electron transport. In the plasma membrane of phagocytic neutrophils and a number of other types of cells, polyunsaturated FFA stimulate O(2)(-) generation by NADPH oxidase. These effects of FFA can modulate signaling functions of ROS and be, at least partly, responsible for their proapoptotic effects in several types of cells.     

Effect of atrial natriuretic peptide on reactive oxygen species-induced by hydrogen peroxide in THP-1 monocytes: Role in cell growth,migration and cytokine release

Atrial natriuretic peptide (ANP), a cardiovascular hormone, elicits different biological actions in the immune system. The aim of the present study was to investigate in THP-1 monocytes the ANP effect on hydrogen peroxide (H₂O₂)-induced Reactive Oxygen Species (ROS), cell proliferation and migration. A significant increase of H₂O₂-dependent ROS production was induced by physiological concentration of ANP (10⁻¹⁰ M). The ANP action was partially affected by cell pretreatment with PD98059, an inhibitor of mitogen activated-protein kinases (MAPK) as well as by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K) and totally suppressed by diphenylene iodonium (DPI), an inhibitor of the enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. The hormone effect was mimicked by cANF and an ANP/NPR-C signaling pathway was studied using pertussis toxin (PTX). A significant increase of H₂O₂-induced cell migration was observed after ANP (10⁻¹⁰ M) treatment, conversely a decrease of THP-1 proliferation, due to cell death, was found. Both ANP actions were partially prevented by DPI. Moreover, H₂O₂-induced release of IL-9, TNF-α, MIP-1α and MIP-1β was not counteracted by DPI, whereas no effect was observed in any experimental condition for both IL-6 and IL-1β. Our results support the view that ANP can play a key role during the inflammatory process.     

Phosphorylation of serine282 in NADPH oxidase activator 1 by Erk desensitizes EGF-induced ROS generation

Accumulating evidence indicates that protein phosphorylation regulates Nox activity. In this report, we show that serine282 residue of Nox activator 1 (NoxA1) is phosphorylated by Erk in response to EGF resulting in desensitization of Nox1 activity. Specifically, murine NoxA1 is detected as two independent protein bands in SDS PAGE, and the form of protein with higher mobility shifted to and merged with the one with lower mobility in response to EGF treatment. Pretreatment with PD98059 resulted in inhibition of NoxA1 migration in response to EGF indicating that Erk was involved in the process. Site-directed mutagenesis showed that S282A mutant but not S239A mutant failed to respond to EGF, demonstrating that serine282 is the target amino acid of Erk. Expression of S282A mutant of NoxA1 in these cells led to increased superoxide anion production in response to EGF compared to expression of the wild type, whereas the expression of S282E, a phosphomimetic mutant, resulted in significantly decreased superoxide anion generation. We also tested whether the phosphorylation of serine282 of NoxA1 affects Rac activation. Expression of S282A mutant NoxA1 up-regulated the Rac activity, whereas expression of S282E mutant led to the abrogation of Rac activation. Taken together, these results demonstrate that phosphorylation of NoxA1 is a part of the feedback mechanism that functions through activation of Rac with a net outcome of negative modulation of Nox1 activity.     

Characterization of N-glycosylation sites on the extracellular domain of NOX1/NADPH oxidase

Extensive evidence demonstrates the pathophysiological importance of NOX1, the catalytic subunit of superoxide-generating enzyme NADPH oxidase, as a source of reactive oxygen species in nonphagocytic cells. However, the biochemical properties of NOX1 have not been extensively characterized due to a lack of specific immunological tools. We used a newly raised NOX1 polyclonal antibody to investigate posttranslational modifications of NOX1 overexpressed in cultured cells and in the colon, where endogenous NOX1 is highly expressed. Immunoblots of lysates from cells expressing NOX1 revealed a doublet of 56 and 60 kDa accompanied by a broad band of 60–90 kDa. Based on differential sensitivity to glycosidases, the doublet was identified as two high-mannose-type glycoforms of NOX1, whereas the broad band represented NOX1 with complex-type N-linked oligosaccharides. Deglycosylated NOX1 migrated at ~53 kDa and N-glycosylation was demonstrated in NOX1 derived from both rat and human. Site-directed mutagenesis identified N-glycosylation sites at Asn¹⁶¹ and Asn²⁴¹ on the extracellular loop of mouse NOX1. Elimination of N-glycosylation on NOX1 did not affect its electron transferase activity, protein stability, targeting to the cell surface, or localization in F-actin-positive membrane protrusions. Taken together, these data identify the two specific sites of N-linked glycosylation of murine NOX1 and demonstrate that they are not required for normal enzyme activity, protein stability, and membrane trafficking. As is true for NOX2, the contribution of glycosylation in NOX1 to its biologic function(s) merits further study.     

Ionizing irradiation induces apoptotic damage of salivary gland acinar cells via NADPH oxidase 1-dependent superoxide generation

Reactive oxygen species (ROS) have important roles in various physiological processes. Recently, several novel homologues of the phagocytic NADPH oxidase have been discovered and this protein family is now designated as the Nox family. We investigated the involvement of Nox family proteins in ionizing irradiation-induced ROS generation and impairment in immortalized salivary gland acinar cells (NS-SV-AC), which are radiosensitive, and immortalized ductal cells (NS-SV-DC), which are radioresistant. Nox1-mRNA was upregulated by γ-ray irradiation in NS-SV-AC, and the ROS level in NS-SV-AC was increased to approximately threefold of the control level after 10 Gy irradiation. The increase of ROS level in NS-SV-AC was suppressed by Nox1-siRNA-transfection. In parallel with the suppression of ROS generation and Nox1-mRNA expression by Nox1-siRNA, ionizing irradiation-induced apoptosis was strongly decreased in Nox1-siRNA-transfected NS-SV-AC. There were no large differences in total SOD or catalase activities between NS-SV-AC and NS-SV-DC although the post-irradiation ROS level in NS-SV-AC was higher than that in NS-SV-DC. In conclusion, these results indicate that Nox1 plays a crucial role in irradiation-induced ROS generation and ROS-associated impairment of salivary gland cells and that Nox1 gene may be targeted for preservation of the salivary gland function from radiation-induced impairment.     

Reactive oxygen species are involved in insulin-dependent regulation of autophagy in primary rat podocytes

Autophagy is an intracellular defense mechanism responsible for the turnover of damaged or non-functional cellular constituents. This process provides cells with energy and essential compounds under unfavorable environmental conditions—such as oxidative stress and hyperglycemia, which are both observed in diabetes. The most common diabetes complication is diabetic nephropathy (DN), which can lead to renal failure. This condition often includes impaired podocyte function. Here we investigated autophagic activity in rat podocytes cultured with a high insulin concentration (300 nM). Autophagy was activated after 60 min of insulin stimulation. Moreover, this effect was abolished following pharmacological (apocynin) or genetic (siRNA) inhibition of NAD(P)H oxidase activity, indicating that insulin-dependent autophagy stimulation involved reactive oxygen species (ROS). We also observed a continuous and time-dependent increase of podocyte albumin permeability in response to insulin, and this process was slightly improved by autophagy inhibition following short-term insulin exposure. Our results suggest that insulin may be a factor affecting the development of diabetic nephropathy.     

Involvement of Mst1 in tumor necrosis factor-alpha-induced apoptosis of endothelial cells

Mammalian sterile 20-kinase 1 (Mst1), a member of the sterile-20 family protein kinase, plays an important role in the induction of apoptosis. However, little is know about the physiological activator of Mst1 and the role of Mst1 in endothelial cells (ECs). We examined whether Mst1 is involved in the tumor necrosis factor (TNF)-α-induced apoptosis of ECs. Western blot analysis revealed that TNF-α induced activation of caspase 3 and Mst1 in a time- and dose-dependent manner. TNF-α-induced Mst1 activation is almost completely prevented by pretreatment with Z-DEVD-FMK, a caspase 3 inhibitor. Nuclear staining with Hoechst 33258 and fluorescence-activated cell sorting of propidium iodide-stained cells showed that TNF-α induced apoptosis of EC. Diphenyleneiodonium, an inhibitor of NADPH oxidase, and N-acetylcysteine, a potent antioxidant, also inhibited TNF-α-induced activation of Mst1 and caspase 3, as well as apoptosis. Knockdown of Mst1 expression by short interfering RNA attenuated TNF-α-induced apoptosis but not cleavage of caspase 3. These results suggest that Mst1 plays an important role in the induction of TNF-α-induced apoptosis of EC. However, positive feedback mechanism between Mst1 and caspase 3, which was shown in the previous studies, was not observed. Inhibition of Mst1 function may be beneficial for maintaining the endothelial integrity and inhibition of atherogenesis.     

Cisplatin induces production of reactive oxygen species via NADPH oxidase activation in human prostate cancer cells

Abstract

This study aimed to examine the roles of reactive oxygen species (ROS) in cisplatin treatment of human prostate cancer cells; hormone-sensitive LNCaP and hormone-refractory PC3 and DU145 cells. Intracellular levels of ROS and H₂O₂ were measured and visualized using specific fluorescent probes. NADPH oxidase (NOX) activity was detected by lucigenin chemiluminescence assay. Expression levels of NOX isoforms were determined by semi-quantitative RT-PCR. Cisplatin treatment increased the intracellular levels of ROS and H₂O₂ in three prostate cancer cell lines. The increase was transient and robust in hormone-sensitive LNCaP cells compared with hormone-refractory PC3 and DU145 cells. Consistent with these findings, the NOX activity induced by cisplatin was higher in LNCaP cells than in PC3 and DU145 cells. Expression pattern of NOX isoforms varied among three cell lines and the NOX activity was independent of NOX expression. Taken together, we have shown that cisplatin induces production of ROS and H₂O₂ via NOX activation in human prostate cancer cell lines, which is most prominent in hormone-sensitive LNCaP cells.     

Analysis of Dichlorodihydrofluorescein and Dihydrocalcein as Probes for the Detection of Intracellular Reactive Oxygen Species

Dihydrocalcein (H₂-calcein) is recommended as a superior probe for intracellular radical (ROS) detection as different to dichlorodihydrofluorescein (H₂-DCF), its oxidation product calcein is thought not to leak out of cells. We determined whether H₂-calcein is a useful tool to measure ROS in vascular smooth muscle cells. In vitro, both compounds were oxidized by peroxynitrite, hydroxyl radicals and peroxidase, but not hydrogen peroxide or nitric oxide. The intracellular half-life of calcein was several hours whereas that of DCF was approximately 5?min. Intracellular ROS, as generated by the angiotensin II (Ang II)-activated NADPH oxidase, did not increase the oxidation of H₂-calcein but increased the oxidation of H₂-DCF by approximately 50%. Similar changes were detected using electron spin resonance spectroscopy. Inhibition of the NADPH oxidase using gp91ds-tat prevented the Ang II-induced increase in DCF fluorescence, without affecting cells loaded with H₂-calcein. Diphenylene iodonium (DPI), which inhibits all flavin-dependent enzymes, including those in the respiratory chain, had little effect on the basal but prevented the Ang II-induced oxidation of H₂-DCF. In contrast, DPI inhibited H₂-calcein oxidation in non-stimulated cells by almost 50%. Blockade of respiratory chain complex I inhibited H₂-calcein oxidation, whereas inhibitors of complex III were without effect. Calcein accumulated in the mitochondria, whereas DCF was localized in the cytoplasm. In submitochondrial particles, H₂-calcein, but not H₂-DCF inhibited complex I activity.

These observations indicate that H₂-DCF is an indicator for intracellular ROS, whereas the oxidation of H₂-calcein most likely occurs as a consequence of direct electron transfer to mitochondrial complex I.     

Reactive oxygen species damaged human serum albumin in patients with type 1 diabetes mellitus: biochemical and immunological studies

The role of hydroxyl radical (.OH) damaged human serum albumin (HSA) in type 1 diabetes has been investigated in the present study. Hydroxyl radical induced modification on HSA has been studied by UV absorption spectroscopy, ANS fluorescence and carbonyl estimation. Hydroxyl radical modified HSA was found to be highly immunogenic in rabbits as compared to native HSA. The binding characteristics of circulating autoantibodies in type 1 diabetes patients against native and modified HSA were assessed. Diabetes patients (n=31) were examined by direct binding ELISA and the results were compared with healthy age-matched controls (n=22). High degree of specific binding by 54.8% of patients sera towards .OH modified HSA, in comparison to its native analogue (p<0.05) was observed. Sera from those type 1 diabetes patients having smoking history, high aging with high degree of disease showed substantially stronger binding to .OH modified HSA over native HSA in particular. Normal human sera showed negligible binding with either antigen. Competitive inhibition ELISA reiterates the direct binding results. Gel retardation assay further substantiated the enhanced recognition of modified HSA by circulating autoantibodies in diabetes patients. The increase in total serum protein carbonyl levels in the diabetes patients was largely due to an increase in oxidized albumin. HSA of diabetes mellitus patients (DM-HSA) and normal subjects (normal-HSA) were purified on a Sephacryl S-200 HR column. Spectroscopic analysis confirmed that the DM-HSA samples contained higher levels of carbonyls than normal-HSA (p<0.001). DM-HSA was conformationally altered, with more exposure of its hydrophobic regions. Collectively, the oxidation of plasma proteins, especially HSA, might enhance oxidative stress in type 1 diabetes mellitus patients.     

植物乙烯生物合成过程中活性氧的作用

大量的研究结果表明,活性氧参与植物乙烯生物合成过程具有明显的普遍性,超氧阴离子自由基是参与乙烯生物合成过程的主要活性氧。近年来研究的焦点主要从乙烯生物合成的关键调控酶ACC合酶及ACC氧化酶的酶活性、酶动力学特性、酶蛋白空间结构、酶基因表达水平等方面来阐明活性氧调控植物乙烯生物合成的机制。最新的研究表明：植物在各种正常或应激的生长条件下首先诱导了活性氧产生水平的变化,活性氧在基因或蛋白质水平上影响ACC合酶和ACC氧化酶的活性水平,从而调节乙烯的生物合成。本文首次综述了活性氧影响植物乙烯生物合成过程的最新研究进展,并对活性氧在植物乙烯生物合成中具有诱导与抑制并存的“双重性”作用进行了探讨。     

Involvement of NADPH oxidase 1 in UVB-induced cell signaling and cytotoxicity in human keratinocytes

Members of NADPH oxidase (Nox) enzyme family are important sources of reactive oxygen species (ROS) and are known to be involved in several physiological functions in response to various stimuli including UV irradiation. UVB-induced ROS have been associated with inflammation, cytotoxicity, cell death, or DNA damage in human keratinocytes. However, the source and the role of UVB-induced ROS remain undefined.Here, we show that Nox1 is involved in UVB-induced p38/MAPK activation and cytotoxicity via ROS generation in keratinocytes. Nox1 knockdown or inhibitor decreased UVB-induced ROS production in human keratinocytes. Nox1 knockdown impaired UVB-induced p38 activation, accompanied by reduced IL-6 levels and attenuated cell toxicity. Treatment of cells with N-acetyl-L-cysteine (NAC), a potent ROS scavenger, suppressed p38 activation as well as consequent IL-6 production and cytotoxicity in response to UVB exposure. p38 inhibitor also suppressed UVB-induced IL-6 production and cytotoxicity. Furthermore, the blockade of IL-6 production by IL-6 neutralizing antibody reduced UVB-induced cell toxicity.In vivo assay using wild-type mice, the intradermal injection of lysates from UVB-irradiated control cells, but not from UVB-irradiated Nox1 knockdown cells, induced inflammatory swelling and IL-6 production in the skin of ears. Moreover, administration of Nox1 inhibitor suppressed UVB-induced increase in IL-6 mRNA expression in mice skin.Collectively, these data suggest that Nox1-mediated ROS production is required for UVB-induced cytotoxicity and inflammation through p38 activation and inflammatory cytokine production, such as IL-6. Thus, our findings suggest Nox1 as a therapeutic target for cytotoxicity and inflammation in response to UVB exposure.     

Identification of the Arabidopsis dry2/sqe1‐5 mutant reveals a central role for sterols in drought tolerance and regulation of reactive oxygen species

Squalene epoxidase enzymes catalyse the conversion of squalene into 2,3‐oxidosqualene, the precursor of cyclic triterpenoids. Here we report that the Arabidopsis drought hypersensitive/squalene epoxidase 1‐5 (dry2/sqe1‐5) mutant, identified by its extreme hypersensitivity to drought stress, has altered stomatal responses and root defects because of a point mutation in the SQUALENE EPOXIDASE 1 (SQE1) gene. GC‐MS analysis indicated that the dry2/sqe1‐5 mutant has altered sterol composition in roots but wild‐type sterol composition in shoots, indicating an essential role for SQE1 in root sterol biosynthesis. Importantly, the stomatal and root defects of the dry2/sqe1‐5 mutant are associated with altered production of reactive oxygen species. As RHD2 NADPH oxidase is de‐localized in dry2/sqe1‐5 root hairs, we propose that sterols play an essential role in the localization of NADPH oxidases required for regulation of reactive oxygen species, stomatal responses and drought tolerance.