Extracellular H2O2 Generation Research Articles

The Protective Effects of α-Mangostin Attenuate Sodium Iodate-Induced Cytotoxicity and Oxidative Injury via Mediating SIRT-3 Inactivation via the PI3K/AKT/PGC-1α Pathway.

It is well known that age-related macular degeneration (AMD) is an irreversible neurodegenerative disease that can cause blindness in the elderly. Oxidative stress-induced retinal pigment epithelial (RPE) cell damage is a part of the pathogenesis of AMD. In this study, we evaluated the protective effect and mechanisms of alpha-mangostin (α-mangostin, α-MG) against NaIO3-induced reactive oxygen species (ROS)-dependent toxicity, which activates apoptosis in vivo and in vitro. MTT assay and flow cytometry demonstrated that the pretreatment of ARPE-19 cells with α-MG (0, 3.75, 7.5, and 15 μM) significantly increased cell viability and reduced apoptosis from NaIO3-induced oxidative stress in a concentration-dependent manner, which was achieved by the inhibition of Bax, cleaved PARP-1, cleaved caspase-3 protein expression, and enhancement of Bcl-2 protein. Furthermore, pre-incubation of ARPE-19 cells with α-MG markedly inhibited the intracellular ROS and extracellular H2O2 generation via blocking of the abnormal enzyme activities of superoxide dismutase (SOD), the downregulated levels of catalase (CAT), and the endogenous antioxidant, glutathione (GSH), which were regulated by decreasing PI3K-AKT-PGC-1α-STRT-3 signaling in ARPE-19 cells. In addition, our in vivo results indicated that α-MG improved retinal deformation and increased the thickness of both the outer nuclear layer and inner nuclear layer by inhibiting the expression of cleaved caspase-3 protein. Taken together, our results suggest that α-MG effectively protects human ARPE-19 cells from NaIO3-induced oxidative damage via antiapoptotic and antioxidant effects.

NKX2.5 is expressed in papillary thyroid carcinomas and regulates differentiation in thyroid cells

BackgroundNKX2.5 is a transcription factor transiently expressed during thyroid organogenesis. Recently, several works have pointed out the oncogenic role of NKX2.5 in a variety of tumors. We therefore hypothesized that NKX2.5 could also play a role in thyroid cancer.MethodsThe validation of NKX2.5 expression was assessed by immunohistochemistry analysis in a Brazilian case series of 10 papillary thyroid carcinoma (PTC) patients. Then, the long-term prognostic value of NKX2.5 and its correlation with clinicopathologic features of 51 PTC patients was evaluated in a cohort with 10-years follow-up (1990–1999). Besides, the effect of NKX2.5 overexpression on thyroid differentiation markers and function was also investigated in a non-tumor thyroid cell line (PCCL3).ResultsNKX2.5 was shown to be expressed in most PTC samples (8/10, case series; 27/51, cohort). Patients who had tumors expressing NKX2.5 showed lower rates of persistence/recurrence (p = 0.013). Overexpression of NKX2.5 in PCCL3 cells led to: 1) downregulation of thyroid differentiation markers (thyrotropin receptor, thyroperoxidase and sodium-iodide symporter); 2) reduced iodide uptake; 3) increased extracellular H2O2 generation, dual oxidase 1 mRNA levels and activity of DuOx1 promoter.ConclusionsIn summary, NKX2.5 is expressed in most PTC samples analyzed and its presence correlates to better prognosis of PTC. In vitro, NKX2.5 overexpression reduces the expression of thyroid differentiation markers and increases ROS production. Thus, our data suggests that NKX2.5 could play a role in thyroid carcinogenesis.

Influence of Stem Cell Therapy on Thyroid Function and Reactive Oxygen Species Production in Diabetic Rats.

Cell therapy with mesenchymal stem cells (MSC) has been proposed for the treatment of diabetes mellitus (DM). It is known that the prevalence of thyroid disease is higher among diabetic patients than in general population. Therefore, our aim was to investigate the effect of the treatment with MSC on thyroid function and ROS generation in an experimental model of type 1 DM. Adult male Wistar rats were divided into the following groups: control, DM (80 mg/kg BW streptozotocin, iv.) and DM+MSC. MSC treatment occurred 4 weeks after DM induction and the animals were euthanized 4 weeks after MSC administration. We also evaluated the effect of co-culture with MSC or extracellular vesicles (EV) obtained from these cells on the rat thyroid cell line PCCL3 exposed to high glucose. Thyroid H2O2 generation was increased in DM, which was reversed by MSC treatment. These changes paralled a significant DuOx1 mRNA increase. The incubation of PCCL3 with high glucose increased extracellular H2O2 generation, which was reversed by both the co-culture with MSC and EV. Even though MSC treatment normalized thyroid ROS generation, serum thyroid hormone (TH) concentration remained low, along with increased serum TSH concentrations. Thyroperoxidase (TPO) activity, was reduced in DM, and MSC treatment did not normalize TPO. Therefore, we conclude that the treatment with MSC was able to reverse the increased thyroid H2O2 generation in diabetic animals and in PCCL3 cells exposed to high glucose, an effect probably mediated by EV produced by these cells, acting in a paracrine fashion.

Abstract 2698: IL-17A alone or in combination with IL-4/IFN-γ induces Duox2/DuoxA2 expression and reactive oxygen production in human colon and pancreatic cancer cells

Abstract NADPH oxidase (NOX)-derived reactive oxygen species (ROS) contribute significantly to inflammation-associated carcinogenesis. Expression of dual oxidase 2 (Duox2), one of seven members of the NOX gene family, is up-regulated in inflammatory bowel disease, chronic pancreatitis, and in many human malignancies including carcinomas of the colon, pancreas, prostate, lung, and breast. Previously, we demonstrated that Stat1 and/or NF-kB play a critical role in modulating the enhanced expression of Duox2, and its cognate maturation factor DuoxA2, by the pro-inflammatory mediators IFN-γ and lipopolysaccharide in human pancreatic cancer cells. This cytokine-mediated increase in Duox2 expression was responsible for a significant increase in tumor cell H2O2 production, DNA damage, and HIF-1α and VEGFA upregulation in pancreatic cancer cell lines. Using the T84 human colon cancer line, and BxPC-3, AsPC-1, and HTB134 pancreatic cancer cell lines, we now report that the pro-inflammatory cytokine IL-17A, pivotal in the adaptive immune response against various pathogens, and in the promotion of colon and pancreatic cancer progression, strongly induces Duox2/DuoxA2 mRNA and protein expression (alone or in combination with IL-4 or IFN-γ). In BxPC-3 cells, IL-17A enhances the expression of Duox2/DuoxA2 in a time- and concentration-dependent manner, which leads to a significant increase in the generation of extracellular H2O2. When IL-17A is combined with IL-4 in either T-84 or AsPc-1 lines, Duox2-mediated ROS formation is sufficient to produce a DNA damage response, indicated by the strong induction of γH2AX expression; moreover, in AsPc-1 cells, Duox2-specific siRNA significantly attenuated cytokine-induced ROS production and γH2AX phosphorylation. IL-17A in combination with IL-4 also significantly increases Stat3 phosphorylation and promotes proliferation in AsPC-1 cells. Ongoing cellular experiments are focused on whether the cytokine-mediated upregulation of Duox2/DuoxA2 is responsible for Stat3 activation and cell growth promotion in the K-ras mutated AsPC-1 cells. Bio-informatic analysis with the TCGA data set, as well as RT-PCR using surgically-resected human colon cancer samples compared to adjacent, uninvolved colonic epithelium revealed that Duox2 was significantly upregulated in human colon cancers. These studies, in concert with our previous data demonstrating high-level Duox protein expression in pancreatitis, pancreatic intra-epithelial neoplasia (PanIN), and frank pancreatic cancer, strongly suggest that ROS, produced as a consequence of cytokine-related Duox2 up-regulation, may be associated with the development of inflammation-related gastrointestinal malignancies. Citation Format: James H. Doroshow, Yongzhong Wu, Jiamo Lu, Smitha Antony, Jennifer Meitzler, Agnes Juhasz, Guojian Jiang, Iris Dahan, Mariam Konate, Krishnendu Roy. IL-17A alone or in combination with IL-4/IFN-γ induces Duox2/DuoxA2 expression and reactive oxygen production in human colon and pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2698. doi:10.1158/1538-7445.AM2017-2698

Antioxidative response in variegated Pelargonium zonale leaves and generation of extracellular H2O2 in (peri)vascular tissue induced by sunlight and paraquat

In this study we exposed variegated leaves of Pelargonium zonale to strong sunlight (>1100μmolm−2s−1 of photosynthetically active radiation) with and without paraquat (Pq), with the aim to elucidate the mechanisms of H2O2 regulation in green and white tissues with respect to the photosynthetically-dependent generation of reactive oxygen species (ROS). Sunlight induced marked accumulation of H2O2 in the apoplast of vascular and (peri)vascular tissues only in green sectors. This effect was enhanced by the addition of Pq. In the presence of diphenyl iodide, an NADPH oxidase inhibitor, H2O2 accumulation was abolished. Distinct light-induced responses were observed: in photosynthetic cells, sunlight rapidly provoked ascorbate (Asc) biosynthesis and an increase of glutathione reductase (GR) and catalase activities, while in non-photosynthetic cells, early up-regulation of soluble ascorbate peroxidase, dehydroascorbate reductase (DHAR) and GR activities was observed. Paraquat addition stimulated DHAR and GR activities in green sectors, while in white sectors activities of monodehydroascorbate reductase, DHAR and class III peroxidases, as well as Asc content rapidly increased. Differential antioxidative responses in the two tissues in the frame of their contrasting metabolisms, and the possible role of (peri)vascular H2O2 in signaling were discussed.

Disulfiram anti-cancer efficacy without copper overload is enhanced by extracellular H2O2 generation: antagonism by tetrathiomolybdate

Cu/Zn superoxide dismutases (SODs) like the extracellular SOD3 and cytoplasmic SOD1 regulate cell proliferation by generating hydrogen peroxide (H2O2). This pro-oxidant inactivates essential cysteine residues in protein tyrosine phosphatases (PTP) helping receptor tyrosine kinase activation by growth factor signaling, and further promoting downstream MEK/ERK linked cell proliferation. Disulfiram (DSF), currently in clinical cancer trials is activated by copper chelation, being potentially capable of diminishing the copper dependent activation of MEK1/2 and SOD1/SOD3 and promoting reactive oxygen species (ROS) toxicity. However, copper (Cu) overload may occur when co-administered with DSF, resulting in toxicity and mutagenicity against normal tissue, through generation of the hydroxyl radical (•OH) by the Fenton reaction. To investigate: a) whether sub-toxic DSF efficacy can be increased without Cu overload against human melanoma cells with unequal BRAF(V600E) mutant status and Her2-overexpressing SKBR3 breast cancer cells, by increasing H2O2 from exogenous SOD; b) to compare the anti-tumor efficacy of DSF with that of another clinically used copper chelator, tetrathiomolybdate (TTM). a) without copper supplementation, exogenous SOD potentiated sub-toxic DSF toxicity antagonized by sub-toxic TTM or by the anti-oxidant N-acetylcysteine; b) exogenous glucose oxidase, another H2O2 generator resembled exogenous SOD in potentiating sub-toxic DSF. potentiation of sub-lethal DSF toxicity by extracellular H2O2 against the human tumor cell lines investigated, only requires basal Cu and increased ROS production, being unrelated to non-specific or TTM copper chelator sequestration. These findings emphasize the relevance of extracellular H2O2 as a novel mechanism to improve disulfiram anticancer effects minimizing copper toxicity.

Astrocytic phospholipase A2 contributes to neuronal glutamate toxicity

The role of astrocytes in glutamate toxicity has been controversial. Here, we show that astrocytes in neuron–astrocyte co-cultures increased neuronal sensitivity to chronic glutamate exposure but not to acute exposure. Enhanced neuronal toxicity by chronic exposure was dependent on astrocyte cell numbers. A reduced generation of extracellular H2O2 induced by glutamate was observed in co-cultures. Further, neuronal glutamate toxicity was not suppressed by NADPH oxidase (Nox) inhibitors, catalase or Nox4 knockdown in co-cultures, whereas these compounds effectively reduced the toxicity in pure neuron cultures. Instead, the intracellular scavenger of reactive oxygen species, N-acetylcysteine (NAC), reduced neuronal cytotoxicity in co-cultures, whereas catalase worked in pure neuron cultures. Lipoxygenase (LOX) inhibitors attenuated neuronal glutamate toxicity in co-cultures but not in pure neuron cultures. Neuronal 5-LOX activity was increased only in co-cultures, whereas 12-LOX activity was increased in both types of cultures. The cyclooxygenase (COX) inhibitors, indomethacin and NS-398, and the phospholipase A2 (PLA2) inhibitors, LY311727 and MAFP, more effectively reduced neuronal glutamate toxicity in co-cultures than in pure neuron cultures. However, in co-cultures, pre-treating neurons and astrocytes with the same inhibitors generated opposite results. COX inhibitors suppressed neuronal glutamate toxicity in pre-treated neurons rather than astrocytes, whereas PLA2 inhibitors reduced the toxicity in pre-treated astrocytes rather than neurons. Gene-specific knockdown of PLA2 confirmed these results. Knockdown of cPLA2α and/or sPLA2-V in astrocytes rather than in neurons more effectively reduced glutamate toxicity in co-cultures. These findings suggest that astrocytic PLA2 activity increases neuronal sensitivity to chronic glutamate exposure in neuron–astrocyte co-cultures.

Abstract 5358: IL-4/IL-13 induce Duox2/DuoxA2 expression and reactive oxygen production in human pancreatic and colon cancer cells

Abstract NADPH oxidase (NOX)-derived reactive oxygen species (ROS) contribute significantly to inflammation-associated carcinogenesis. Expression of dual oxidase 2 (Duox2), one of seven members of the NOX gene family, is up-regulated in inflammatory bowel disease, chronic pancreatitis, and in many human malignancies including carcinomas of the prostate, lung, and breast. Previously, we demonstrated that Stat1 and/or NF-κB play a critical role in modulating the enhanced expression of Duox2, and its cognate maturation factor DuoxA2, by IFN-γ and lipopolysaccharide in human pancreatic cancer cells. This cytokine-mediated increase in Duox2 expression is responsible for a consequent significant increase in tumor cell H2O2 production and DNA damage. Using Caco2 and T84 human colon cancer lines, and BxPC-3 pancreatic cancer cells, we now report that two other pro-inflammatory cytokines, IL-4 and IL-13, also strongly induce Duox2/DuoxA2 RNA and protein expression (>5-10-fold after 24h treatment with 50 ng/ml cytokine). In the BxPC-3 line, IL-4 exposure activates the Stat6 signaling pathway, enhances expression of Duox2/DuoxA2 in a time- and concentration-dependent manner, and leads to a significant increase in the generation of extracellular H2O2. The transcription and translation inhibitors actinomycin D and cyclohexamide both individually suppress IL-4-induced Duox2 expression, suggesting that IL-4-mediated Duox2 up-regulation is transcriptionally regulated, but that new protein synthesis is also necessary. We found that Stat6 is essential for IL-4-induced Duox2 expression because silencing Stat6 with Stat6-specific siRNA significantly attenuates IL-4-induced Duox2 RNA and protein levels in BxPC-3 cells. To evaluate the potential role of Duox2 in inflammation-associated human cancers further, immunohistochemical studies were performed to semi-quantitatively examine Duox expression using human pancreatic and colon cancer tissue arrays. We found that a majority of samples of pancreatic intra-epithelial neoplasia and frank pancreatic cancer demonstrated high-level expression of Duox , often in association with inflammatory cell infiltrates; high-level Duox expression was also found in most colorectal cancer samples. These studies, in concert with our previous experiments, strongly suggest that ROS, produced as a consequence of cytokine-induced Duox up-regulation, may be associated with the development of inflammation-related gastrointestinal malignancies. Note: This abstract was not presented at the meeting. Citation Format: Yongzhong Wu, James H. Doroshow. IL-4/IL-13 induce Duox2/DuoxA2 expression and reactive oxygen production in human pancreatic and colon cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5358. doi:10.1158/1538-7445.AM2014-5358

Salinity induced the changes of root growth and antioxidative responses in two wheat cultivars

This study aimed to investigate the inhibitory mechanism of root growth and to compare antioxidative responses in two wheat cultivars, drought-tolerant Ningchun and drought-sensitive Xihan, exposed to different NaCl concentrations. Ningchun exhibited lower germination rate, seedling growth, and lipid peroxidation than Xihan when exposed to salinity. The loss of cell viability was correlated with the inhibition of root growth induced by NaCl stress. Moreover, treatments with H2O2 scavenger dimethylthiourea and catalase (CAT) partly blocked salinity-induced negative effects on root growth and cell viability. Besides, the enhancement of superoxide radical and H2O2 levels, and the stimulation of CAT and diamine oxidase (DAO) as well as the inhibition of glutathione reductase (GR) were observed in two wheat roots treated with salinity. However, hydroxyl radical content increased only in Xihan roots under NaCl treatment, and the changes of soluble peroxidase (POD), ascorbate peroxidase (APX), superoxide dismutase (SOD), and cell-wall-bound POD activities were different in drought-tolerant Ningchun and drought-sensitive Xihan exposed to different NaCl concentrations. In conclusion, salinity might induce the loss of cell viability via a pathway associated with extracellular H2O2 generation, which was the primary reason leading to the inhibition of root growth in two wheat cultivars. Here, it was also suggested that increased H2O2 accumulation in the roots of drought-tolerant Ningchun might be due to decreased POD and GR activities as well as enhanced cell-wall-bound POD and DAO ones, while the inhibition of APX and GR as well as the stimulation of SOD and DAO was responsible for the elevation of H2O2 level in drought-sensitive Xihan roots.

PI3Kγ contributes to MEK1/2 activation in oxidative glutamate toxicity via PDK1

The role of phosphoinositide 3-kinase (PI3K) in oxidative glutamate toxicity is not clear. Here, we investigate its role in HT22 mouse hippocampal cells and primary cortical neuronal cultures, showing that inhibitors of PI3K, LY294002, and wortmannin suppress extracellular hydrogen peroxide (H₂O₂) generation and increase cell survival during glutamate toxicity in HT22 cells. The mitogen-activated protein kinase kinase (MEK) inhibitor U0126 also reduced glutamate-induced H₂O₂ generation and inhibited phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. LY294002 was seen to abolish phosphorylation of both ERK1/2 and Akt. A small interfering RNA (siRNA) study showed that PI3Kβ and PI3Kγ, rather than PI3Kα and PI3Kδ, contribute to glutamate-induced H₂O₂ generation and cell death. PI3Kγ knockdown also inhibited glutamate-induced ERK1/2 phosphorylation, whereas transfection with the constitutively active form of human PI3Kγ (PI3Kγ-CAAX) triggered MEK1/2 and ERK1/2 phosphorylation and H₂O₂ generation without glutamate exposure. This H₂O₂ generation was reduced by inhibition of MEK. Transfection with kinase-dead 3-phosphoinositide-dependent protein kinase 1 (PDK1-KD) reduced glutamate-induced ERK1/2 phosphorylation and H₂O₂ generation. Accordingly, cotransfection of cells with PDK1-KD and PI3Kγ-CAAX suppressed PI3Kγ-CAAX-triggered ERK1/2 phosphorylation and H₂O₂ generation. These results suggest that activation of PI3Kγ induces ERK1/2 phosphorylation, leading to extracellular H₂O₂ generation via PDK1 in oxidative glutamate toxicity.

Do ascorbic acid and viscum album improve PFS in NSCLC?

3102 Background: Intravenous vitamin C achieves 70 times higher plasma concentrations of ascorbate as a similar oral dose. Such pharmacologic concentrations(0.3-20mM) kill cancer cells but not normal cells, through extracellular H2O2 generation. Ascorbate treatment depleted ATP and induced autophagy in prostate cancer cells; gemcitabine with ascorbate resulted in 50% inhibition of growth in pancreatic tumor xenografts in mice models. A phase I/II study for pancreatic cancer patients is underway. Viscum album (mistletoe) is widely used in Europe as complementary therapy. Its cytotoxic and immunomodulatory effects are poorly understood. Additional treatment with subcutaneous mistletoe injections improved quality of life in gastric cancer patients in a study. Methods: We report the case of a 54 year old male smoker with adenocarcinoma of the lung (pT4N2M1a), EGFR/RAS/ALK negative. After 5 cycles of chemotherapy (carboplatin/taxol x 3, then carboplatin/pemetrexed x 2) scans showed continued progression. The patient made an informed decision to stop further chemotherapy and initiated complementary medical means, receiving high dose IV vitamin C (75 g, 2 x week) and subcutaneous mistletoe injections every other day, for the last seven months. Results: Six months into the non-standard therapy, a PET/CT shows a PR by RECIST and decreased FDG activity of the biopsy proven primary and metastatic site. The patient feels well, a year now after being diagnosed with advanced NSCLC. Conclusions: Whether the PR and the surprisingly long PFS is due to IV ascorbic acid and/or mistletoe is unknown. A review of the literature reveals reports of similar astonishing clinical results after treatment with either agent. A registry and biorepository of cases treated with alternative modalities may discover markers and biological mechanisms to explain these surprising reports.

Secreted protein acidic and rich in cysteine (SPARC) is upregulated by transforming growth factor (TGF)-β and is required for TGF-β-induced hydrogen peroxide production in fibroblasts

BackgroundIdiopathic pulmonary fibrosis (IPF) is a poorly understood progressive disease characterized by the recurrent damage of alveolar epithelial cells as well as inappropriate expansion and activation of fibroblasts resulting in pronounced extracellular matrix (ECM) deposition. Although recent studies have indicated the involvement of secreted protein acidic and rich in cysteine (SPARC), a matricellular protein regulating ECM deposition, in the pathogenesis of fibrosis, factors regulating SPARC expression or roles of SPARC in fibrosis have not been fully elucidated.ResultsAmong the profibrotic factors examined in cultured fibroblasts, we showed that SPARC expression was upregulated mainly by transforming growth factor (TGF)-β. We also showed that expression of SPARC in the lung was upregulated in the murine bleomycin-induced pulmonary fibrosis model, which was inhibited by TGF-β receptor I inhibitor. Knockdown of SPARC in fibroblasts using siRNA or treatment with the antioxidant N-acetylcysteine attenuated epithelial cell injury induced by TGF-β-activated fibroblasts in a coculture system. We also demonstrated that SPARC was required for hydrogen peroxide (H2O2) production in fibroblasts treated with TGF-β. Furthermore, TGF-β activated integrin-linked kinase (ILK), which was inhibited by SPARC siRNA. Knockdown of ILK attenuated extracellular H2O2 generation in TGF-β-stimulated fibroblasts. Our results indicated that SPARC is upregulated by TGF-β and is required for TGF-β-induced H2O2 production via activation of ILK, and this H2O2 production from fibroblasts is capable of causing epithelial cell injury.ConclusionsThe results presented in this study suggest that SPARC plays a role in epithelial damage in the IPF lung via enhanced H2O2 production from fibroblasts activated by TGF-β. Therefore, SPARC inhibition may prevent epithelial injury in IPF lung and represent a potential therapeutic approach for IPF.

Thyroid hydrogen peroxide production is enhanced by the Th2 cytokines, IL-4 and IL-13, through increased expression of the dual oxidase 2and its maturation factorDUOXA2

The dual oxidases (DUOX) 1and 2constitute the major components of the thyroid H2O2-generating system required for thyroid hormone synthesis. With their maturation factor, DUOXA1 or DUOXA2, they share the same bidirectional promoter allowing coexpression of DUOX/DUOXA in the same tissue. However, the molecular mechanisms regulating their transcription in the human thyroid gland are not well characterized yet. Inflammatory molecules associated with autoimmune thyroid diseases have been shown to repress the thyroid function by down-regulating the expression of the major thyroid differentiation markers. These findings led us to investigate the effects of the main cytokines involved in Hashimoto thyroiditis (IFN-γ) and Graves' diseases (IL-4/IL-13) on the transcriptional regulation of DUOX and their corresponding DUOXA genes in thyroid cells. Human thyrocytes exposed to the Th2 cytokines IL-4 and IL-13 showed up-regulation of DUOX2 and DUOXA2 genes but not DUOX1/DUOXA1. The DUOX2/DUOXA2 induction was rapid and associated with a significant increase of calcium-stimulated extracellular H2O2 generation. IFN-γ treatment inhibited DUOX gene expression and repressed the Th2 cytokine-dependent DUOX2/DUOXA2 expression. In another DUOX-expressing model, the human intestinal Caco-2 cell line, expression of DUOX2 and DUOXA2 mRNA was also positively modulated by IL-4 and IL-13. Analysis of the IL-4 signaling pathway revealed that the JAK1-STAT6 cascade activated by the IL-4 type 2receptor is required for DUOX2/DUOXA2 induction. The present data open new perspectives for a better understanding of the pathophysiology of thyroid autoimmune diseases considering DUOX2-mediated oxidative damages.

PI3K–ERK1/2 activation contributes to extracellular H2O2 generation in amyloid β toxicity

Amyloid β peptide (Aβ) induces hydrogen peroxide (H2O2) and superoxide generation, leading to neuronal death. Many studies have shown the involvement of NADPH oxidase, but the isotype-specific role was not assessed. Moreover, the activation status of phosphoinositide 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK) 1/2 is unclear in extracellular H2O2 generation. In this paper, we showed that Aβ1–42 induced extracellular H2O2 generation and the resulting cytotoxicity in a concentration-dependent manner. Nox2- and Nox4-specific siRNAs suppressed H2O2 and superoxide generation. LY294002 and U0126, inhibitors of PI3K and ERK1/2, respectively, reduced H2O2 generation in concentration-dependent manners. Furthermore, PI3K activation is responsible for ERK1/2 phosphorylation. An additional increase in H2O2 generation and corresponding cytotoxicity was observed after treatment with Aβ1–42 and glutamate. These results suggest that Aβ1–42 enhances the neuronal vulnerability to oxidative injury in Alzheimer's disease (AD) by increasing H2O2 generation.

Hyperglycemia compromises mitochondrial function

Mitochondrial dysfunction has a significant role in the development and complications of diabetic cardiomyopathy. Hyperglycemia‐induced mitochondrial oxidative stress has been suggested as one contributing factor. The impact of hyperglycemia was studied using H9c2 myotubes (a cardiac‐derived cell line) and Cyt C‐ cells (an embryonic cell line having reduced levels of cytochrome C). Cells were maintained in the presence of 5.5, 16.5, or 35.0 mM glucose for up to 13 days. Hyperglycemia significantly increased protein levels in the Cyt C‐ cells but not the H9c2 cells. Functional tests of mitochondria (succinate dehydrogenase and ATP generation) suggested that although the H9c2 myotubes were sensitive to hyperglycemia, the Cyt C‐ cells were not. Hyperglycemia did not significantly alter generation of extracellular H2O2 in either cell line. However, intramitochondrial superoxide (mtSO) levels (detected using MitoSox) was increased in the H9c2 cells following 13 days of hyperglycemia. Conversely, up to13 days of hyperglycemia did not significantly altered mtSO levels in the Cyt C‐ cells. Hyperglycemia increased H9c2 caspase 3/7 activity that appeared to track mtSO levels. These results suggest that mitochondrial function may be compromised by hyperglycemia as a consequence of altered intramitochondrial oxidative stress. Supported in part by the New York Medical College Research Endowment Fund.

Novel Action of Paclitaxel against Cancer Cells: Bystander Effect Mediated by Reactive Oxygen Species

Generation of reactive oxygen species (ROS) has been observed in cancer cells treated with paclitaxel, but the underlying mechanisms and therapeutic implications remain unclear. In the present study, we showed that paclitaxel promoted ROS generation through enhancing the activity of NADPH oxidase (NOX) associated with plasma membranes. Treatment of breast cancer cells caused an increased translocation of Rac1, a positive regulatory protein of NOX, to the membrane fraction. The paclitaxel-induced ROS generation occurred rapidly within several hours of drug exposure, with O(2)(-) and H(2)O(2) accumulation mainly outside the cells while the intracellular ROS remained unchanged. Importantly, the increase in extracellular ROS caused lethal damage to the bystander cancer cells not exposed to paclitaxel, as shown by two different methods using coculture systems where the bystander cells were differentiated from the paclitaxel-treated cells by fluorescent or radioactive labeling. This cytotoxic bystander effect was also observed with other microtubule-targeted agents vincristine and taxotere but not with 5-fluorouracil or doxorubicin. This toxic bystander effect was enhanced by CuZnSOD that converts O(2)(-) to H(2)O(2) and was abolished by a catalase that eliminates H(2)O(2). Furthermore, paclitaxel was able to induce an almost complete inhibition of proliferation of the bystander cells in the coculture system. Our study revealed a novel mechanism by which paclitaxel induces toxic bystander effect through generation of extracellular H(2)O(2) from the membrane-associated NOX. This may contribute to the potent anticancer activity of paclitaxel and provide a novel basis to improve the clinical use of this important drug.

H2O2 plays different roles in determining penetration failure in three diverse plant-fungal interactions.

Fungal plant pathogens that attempt to penetrate and feed on living cells frequently trigger a localized plant defence response that results in fungal penetration failure. In the current study we demonstrate that breakdown products of the cell wall released by the localized application of hemicellulase elicit localized responses including, sequentially, extracellular H2O2 generation; accumulation of phenolic compounds; and cross-linking of proteins in the cell wall. In a detailed time-course study of three plant-fungus interactions that result in a high frequency of penetration failure, only one plant-fungus combination displayed a similar profile of responses to that induced by localized cell-wall degradation. The additional generation of extracellular O2- in one interaction, and the absence of phenolic compounds in the cell wall in another, demonstrate that plant responses to the penetration process may be influenced by activities of the penetrating fungus. Significantly, H2O2 generation was the only response detected in all three plant-fungal combinations at the correct time and place to account for penetration failure, and in all three combinations the enzymatic removal of H2O2 resulted in increased penetration success. Pharmacological studies suggest that in two of the three interactions, H2O2 generation required cytoskeletal involvement but was independent of transcription or translation, although inhibition of the latter processes increased fungal penetration. In at least one of these two interactions, the data suggest that H2O2 generation and new gene expression act within the same penetration-inhibiting pathway, possibly through the involvement of phenolic materials. However, enzymatic removal of H2O2 from the third interaction almost completely eliminated penetration failure, while interference with cytoplasmic processes had no effect, suggesting that H2O2 generation in this system did not require protoplast involvement and, alone, was necessary and sufficient to account for fungal penetration failure.

Intracellular generation of reactive oxygen species in endothelial cells exposed to anoxia-reoxygenation.

Reactive oxygen species (ROS) play an important role in the pathogenesis of ischemia-reperfusion injury. Extracellular H2O2 generation from bovine pulmonary artery endothelial cells (EC) is known to increase in response to anoxia-reoxygenation (A-R). To determine potential sources of intracellular ROS formation in EC in response to A-R, a fluorometric assay based on the oxidation of 2',7'-dichlorofluorescin was used. Intracellular ROS production declined 40% during 6 h of anoxia (P < 0.05). After A-R, the rates of intracellular ROS formation increased to 148 +/- 9% (P < 0.001) that of normoxic EC (100 +/- 3%). In EC exposed to A-R, allopurinol and NG-methyl-L-arginine (L-NMMA), inhibitors of xanthine oxidase (XO) and nitric oxide synthase (NOS), respectively, reduced intracellular ROS formation by 25 +/- 1% (P < 0.001) and 36 +/- 4% (P < 0.01). Furthermore, at low doses (i.e., 20 microM), deferoxamine and diethylenetriaminepentaacetic acid (DTPA) significantly inhibited intracellular ROS formation. However, at 100 microM, only deferoxamine caused further reduction in DCF fluorescence. In summary, EC respond to A-R by generating increased amounts of XO- and NOS-derived intracellular ROS. The inhibition, to a similar extent, caused by allopurinol and L-NMMA, as well as the effect of deferoxamine and DTPA suggest that the ROS detected is peroxynitrite. Based on these findings and previous work, we conclude that EC generate ROS in response to A-R from at least two different sources: a plasma membrane-bound NADPH oxidase-like enzyme that releases H2O2 extracellularly and XO, which generates intracellular O2-, which in turn may react with nitric oxide to form peroxynitrite.

Regulation of hydrogen peroxide generation in cultured endothelial cells.

Endogenous hydrogen peroxide (H2O2) release from aortic endothelial cells was studied in the presence of antioxidant enzyme inhibitors, mitochondrial inhibitors, a microsomal cytochrome P-450 inhibitor, and after oxidative stress induced with H2O2 or menadione. Extracellular H2O2 generation was determined spectrofluorometrically using 3-methoxy-4-hydroxy phenylacetic acid, and intracellular H2O2 production (in or near peroxisomes) was measured indirectly using aminotriazole, which inactivates catalase in the presence of H2O2. Extracellular H2O2 release was 0.079 +/- 0.005 nmol/min/mg protein in Hanks' balanced salt solution, was constant during a 120-min incubation period, and was not affected by the cell passage number. The half-life for catalase inactivation with aminotriazole was 23 min. Inhibition of catalase, glutathione reductase, or gamma-glutamylcysteine synthetase did not change the rate of extracellular release of H2O2. Furthermore, inhibition of the mitochondrial respiratory chain (rotenone, antimycin A) or microsomal cytochrome P-450 (8-methoxypsoralen) did not change extracellular H2O2 release or intracellular H2O2 production (at peroxisomes) by endothelial cells or cells in which glutathione reductase was inactivated. When the cells were exposed to exogenous H2O2 (30 microM), extracellular H2O2 was scavenged primarily by the glutathione redox pathway. Exogenously added H2O2 (100 microM) changed intracellular H2O2 production (in or near peroxisomes) only when the glutathione redox cycle was inactivated. Menadione (20 microM), which undergoes intracellular redox cycling, increased extracellular H2O2 release almost 4-fold to 0.3 nmol/min/mg protein. Furthermore, menadione increased peroxisomal H2O2 levels and decreased the half-life for catalase inactivation in the presence of aminotriazole to 13 min. Catalase inhibition increased extracellular H2O2 release during menadione treatment, indicating that H2O2 can diffuse across the plasma membrane during oxidant stress.(ABSTRACT TRUNCATED AT 250 WORDS)