Reactive Oxygen Species Depletion Research Articles

Sichuan pepper attenuates H2O2-induced apoptosis via antioxidant activity and up-regulating heme oxygenase-1 gene expression in primary rat hepatocytes

Sichuan pepper, a well-known traditional Chinese spice, is popularly used in Chinese cuisine. In this study, the antioxidant activities of Sichuan pepper water extract (SP) were evaluated both chemically [in 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH) assay] and biologically [in 2,2′-azobis-(2-amidinopropane) (AAPH)-induced haemolysis assay]. The protective effect of SP against H2O2-induced cytotoxicity in primary Sprague Dawley rat hepatocytes was investigated by using cell viability assay, nuclear staining, intracellular reactive oxygen species (ROS) measurement and flow cytometry. It was found to possess high total phenolic content as assessed by the Folin–Ciocalteu method. The antiapoptotic effect of SP may be mediated, in part, by significantly inhibiting intracellular ROS generation and depletion of the mitochondrial membrane potential. SP was also found to enhance the cellular antioxidant defense system through up-regulating heme oxygenase-1 expression. SP is an effective antioxidant that can be consumed regularly as a functional food for the prevention of oxidative stress-related liver diseases. Practical applications The present study demonstrated that Sichuan pepper water extract (SP) could enhance the cellular antioxidant ability in hepatocytes. SP is an effective antioxidant that can be consumed regularly as a functional food for the prevention of oxidative stress-related liver diseases.

Antiapoptotic effects of caspase inhibitors on H2O2-treated lung cancer cells concerning oxidative stress and GSH.

Exogenous hydrogen peroxide (H2O2) induces oxidative stress and apoptosis in cancer cells. This study evaluated the antiapoptotic effects of pan-caspase and caspase-3, -8, or -9 inhibitors on H2O2-treated Calu-6 and A549 lung cancer cells in relation to reactive oxygen species (ROS) and glutathione (GSH). Treatment with 50-500 μM H2O2 inhibited the growth of Calu-6 and A549 cells at 24h and induced apoptosis in these cells. All the tested caspase inhibitors significantly prevented cell death in H2O2-treated lung cancer cells. H2O2 increased intracellular ROS levels, including that of O 2·- , at 1 and 24h. It also increased the activity of catalase but decreased the activity of SOD. In addition, H2O2 triggered GSH deletion in Calu-6 and A549 cells at 24h. It reduced GSH levels in Calu-6 cells at 1h but increased them at 24h. Caspase inhibitors decreased O 2·- levels in H2O2-treated Calu-6 cells at 1h and these inhibitors decreased ROS levels, including that of O 2·- , in H2O2-treated A549 cells at 24h. Caspase inhibitors partially attenuated GSH depletion in H2O2-treated A549 cells and increased GSH levels in these cells at 24h. However, the inhibitors did not affect GSH deletion and levels in Calu-6 cells at 24h. In conclusion, H2O2 induced caspase-dependent apoptosis in Calu-6 and A549 cells, which was accompanied by increases in ROS and GSH depletion. The antiapoptotic effects of caspase inhibitors were somewhat related to the suppression of H2O2-induced oxidative stress and GSH depletion.

A novel benzimidazole derivative, MBIC inhibits tumor growth and promotes apoptosis via activation of ROS-dependent JNK signaling pathway in hepatocellular carcinoma.

A prior screening programme carried out using MTT assay by our group identified a series of novel benzimidazole derivatives, among which Methyl 2-(5-fluoro-2-hydroxyphenyl)-1H- benzo[d]imidazole-5-carboxylate (MBIC) showed highest anticancer efficacy compared to that of chemotherapeutic agent, cisplatin. In the present study, we found that MBIC inhibited cell viability in different hepatocellular carcinoma (HCC) cell lines without exerting significant cytotoxic effects on normal liver cells. Annexin V-FITC/PI flow cytometry analysis and Western blotting results indicated that MBIC can induce apoptosis in HCC cells, which was found to be mediated through mitochondria associated proteins ultimately leading to the activation of caspase-3. The exposure to MBIC also resulted in remarkable impairment of HCC cell migration and invasion. In addition, treatment with MBIC led to a rapid generation of reactive oxygen species (ROS) and substantial activation of c-Jun-N-terminal kinase (JNK). The depletion of ROS by N-Acetyl cysteine (NAC) partially blocked MBIC-induced apoptosis and JNK activation in HCC cells. Finally, MBIC significantly inhibited tumor growth at a dose of 25 mg/kg in an orthotopic HCC mouse model. Taken together, these results demonstrate that MBIC may inhibit cell proliferation via ROS-mediated activation of the JNK signaling cascade in HCC cells.

Sibutramine provokes apoptosis of aortic endothelial cells through altered production of reactive oxygen and nitrogen species

Overdose administration of sibutramine, a serotonin-noradrenalin reuptake inhibitor, is considered to elicit severe side effects including hypertension, whose pathogenic mechanism remains unclear. Here, we found that 48-h incubation with >10μM sibutramine provokes apoptosis of human aortic endothelial (HAE) cells. Treatment with the lethal concentration of sibutramine facilitated production of reactive oxygen species (ROS), altered expression of endoplasmic reticulum stress response genes (heat shock protein 70 and C/EBP homologous protein), and inactivated 26S proteasome-based proteolysis. The treatment also decreased cellular level of nitric oxide (NO) through lowering of expression and activity of endothelial NO synthase. These results suggest that ROS production and depletion of NO are crucial events in the apoptotic mechanism and may be linked to the pathogenesis of vasoconstriction elicited by the drug. Compared to sibutramine, its metabolites (N-desmethylsibutramine and N-didesmethylsibutramine) were much less cytotoxic to HAE cells, which hardly metabolized sibutramine. In contrast, both the drug and metabolites showed low cytotoxicity to hepatic HepG2 cells with high metabolic potency and expression of cytochrome P450 (CYP) 3A4. The cytotoxicity of sibutramine to HepG2 and Chang Liver cells was remarkably augmented by inhibition and knockdown of CYP3A4. This study also suggests an inverse relationship between sibutramine cytotoxicity and CYP3A4-mediated metabolism into the N-desmethyl metabolites.

Shift in aggregation, ROS generation, antioxidative defense, lysozyme and acetylcholinesterase activities in the cells of an Indian freshwater sponge exposed to washing soda (sodium carbonate)

Washing soda, chemically identified as anhydrous sodium carbonate, is a popular cleaning agent among the rural and urban populations of India which often contaminates the freshwater ponds and lakes, the natural habitat of sponge Eunapius carteri. Present investigation deals with estimation of cellular aggregation, generation of ROS and activities of antioxidant enzymes, lysozyme and acetylcholinesterase in the cells of E. carteri under the environmentally realistic concentrations of washing soda. Prolonged treatment of washing soda inhibited the degree of cellular aggregation. Experimental exposure of 8 and 16mg/l of sodium carbonate for 48h elevated the physiological level of reactive oxygen species (ROS) generation in the agranulocytes, semigranulocytes and granulocytes of E. carteri, whereas, treatment of 192h inhibited the ROS generation in three cellular morphotypes. Activities of superoxide dismutase, catalase and glutathione-S-transferase were recorded to be inhibited under prolonged exposure of washing soda. Washing soda mediated inhibition of ROS generation and depletion in the activities of antioxidant enzymes were indicative to an undesirable shift in cytotoxic status and antioxidative defense in E. carteri. Inhibition in the activity of lysozyme under the treatment of sodium carbonate was suggestive to a severe impairment of the innate immunological efficiency of E. carteri distributed in the washing soda contaminated habitat. Washing soda mediated inhibition in the activity of acetylcholinesterase indicated its neurotoxicity in E. carteri. Washing soda, a reported environmental contaminant, affected adversely the immunophysiological status of E. carteri with reference to cellular aggregation, oxidative stress, antioxidative defense, lysozyme and acetylcholinesterase activity.

Effects of combined exposure to formaldehyde and benzene on immune cells in the blood and spleen in Balb/c mice.

Formaldehyde and benzene are the two major indoor air pollutants due to their prevalence and toxicity. This study aimed to explore the toxic effect on the spleen and relevant immune responses of Balb/c mice caused by exposure to a combination of formaldehyde and benzene. Balb/c mice were divided randomly into five groups (n=9/group): blank control group (Ctrl); solvent ([corn] Oil) control; formaldehyde only (FA, 3mg/m(3)); benzene only (BZ, 150mg/kg BW); and, formaldehyde+benzene group (FA+BZ). Exposures were performed for 8h/day, 5 day/week, for 2 weeks. Tail blood was collected after the final exposure; 24-h later, the mice were euthanized to permit assessment of a variety of immune endpoints. The endpoints' three areas were: (1) in living mice, body weight and delayed-type hypersensitivity (DTH) responses; (2) in blood, immune cell counts and serum antibody levels (serum hemagglutination); and, (3) in spleen samples, reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), caspase-3 (cell apoptosis) levels and lymphocyte proliferation. In this study we fund (1) BZ and FA+BZ exposure can lead to the reduction in the number of some immune cells in peripheral blood; (2) Formaldehyde has certain synergistic effects on benzene-induced cytotoxicity in peripheral blood, (3) FA, BZ and FA+BZ exposure can lead to ROS and GSH depletion in spleen cells, and spleen cell apoptosis (caspase-3 increased) may be one of the downstream events, decreased splenic lymphocyte proliferation; and (4) the FA+BZ combined exposure can lead to the decreased body weight, serum antibody level (by serum hemagglutination assay).

Optimal ROS Signaling Is Critical for Nuclear Reprogramming.

Efficient nuclear reprogramming of somatic cells to pluripotency requires activation of innate immunity. Because innate immune activation triggers reactive oxygen species (ROS) signaling, we sought to determine whether there was a role of ROS signaling in nuclear reprogramming. We examined ROS production during the reprogramming of doxycycline (dox)-inducible mouse embryonic fibroblasts (MEFs) carrying the Yamanaka factors (Oct4, Sox2, Klf4, and c-Myc [OSKM]) into induced pluripotent stem cells (iPSCs). ROS generation was substantially increased with the onset of reprogramming. Depletion of ROSvia antioxidants or Nox inhibitors substantiallydecreased reprogramming efficiency. Similarly, both knockdown and knockout of p22(phox)-a critical subunit of the Nox (1-4) complex-decreased reprogramming efficiency. However, excessive ROS generation using genetic and pharmacological approaches also impaired reprogramming. Overall, our data indicate that ROS signaling is activated earlywith nuclear reprogramming, and optimal levels of ROS signaling are essential to induce pluripotency.

Mitochondrial ROS regulate thermogenic energy expenditure and sulfenylation of UCP1.

Brown adipose tissue (BAT) can dissipate chemical energy as heat through thermogenic respiration, which requires uncoupling protein 1 (UCP1)1,2. Thermogenesis from BAT and beige adipose can combat obesity and diabetes3, encouraging investigation of factors that control UCP1-dependent respiration in vivo. Herein we show that acutely activated BAT thermogenesis is defined by a substantial increase in mitochondrial reactive oxygen species (ROS) levels. Remarkably, this process supports in vivo BAT thermogenesis, as pharmacological depletion of mitochondrial ROS results in hypothermia upon cold exposure, and inhibits UCP1-dependent increases in whole body energy expenditure. We further establish that thermogenic ROS alter BAT cysteine thiol redox status to drive increased respiration, and Cys253 of UCP1 is a key target. UCP1 Cys253 is sulfenylated during thermogenesis, while mutation of this site desensitizes the purine nucleotide inhibited state of the carrier to adrenergic activation and uncoupling. These studies identify BAT mitochondrial ROS induction as a mechanism that drives UCP1-dependent thermogenesis and whole body energy expenditure, which opens the way to develop improved therapeutic strategies for combating metabolic disorders.

Uric acid enhances PKC-dependent eNOS phosphorylation and mediates cellular ER stress: A mechanism for uric acid-induced endothelial dysfunction.

The mechanism by which hyperuricemia induced-endothelial dysfunction contributes to cardiovascular diseases (CVDs) is not yet fully understood. In the present study, we used uric acid (UA) to trigger endothelial dysfunction in cultured endothelial cells, and investigated the effects of induced reactive oxygen species (ROS) generation, endoplasmic reticulum (ER) stress induction, and the protein kinase C (PKC)-dependent endothelial nitric oxide synthase (eNOS) signaling pathway. Human umbilical vein endothelial cells (HUVECs) were incubated with 6, 9 or 12 mg/dl UA, ROS scavenger polyethylene glycol-superoxide dismutase (PEG-SOD), ER stress inhibitor 4-phenylbutyric acid (4-PBA), and PKC inhibitor polymyxin B for 6–48 h. Nitric oxide (NO) production, eNOS activity, intracellular ROS, ER stress levels, and the interaction between eNOS and calmodulin (CaM) and cytosolic calcium levels were assessed using fluorescence microscopy and western blot analysis. Apoptosis was assessed by annexin V staining. UA increased HUVEC apoptosis and reduced eNOS activity and NO production in a dose- and time-dependent manner. Intracellular ROS was elevated after 3 h, while ER stress level increased after 6 h. UA did not alter intracellular Ca2+, CaM, or eNOS concentration, or eNOS Ser1177 phosphorylation. However, PKC-dependent eNOS phosphorylation at Thr495 was greatly enhanced, and consequently interaction between eNOS and CaM was reduced. Cellular ROS depletion, ER stress inhibition and PKC activity reduction inhibited the effect of UA on eNOS activity, NO release and apoptosis in HUVECs. Thus, we concluded that UA induced HUVEC apoptosis and endothelial dysfunction by triggering oxidative and ER stress through PKC/eNOS-mediated eNOS activity and NO production.

Early bioelectric activities mediate redox-modulated regeneration.

Reactive oxygen species (ROS) and electric currents modulate regeneration; however, the interplay between biochemical and biophysical signals during regeneration remains poorly understood. We investigate the interactions between redox and bioelectric activities during tail regeneration in Xenopus laevis tadpoles. We show that inhibition of NADPH oxidase-mediated production of ROS, or scavenging or blocking their diffusion into cells, impairs regeneration and consistently regulates the dynamics of membrane potential, transepithelial potential (TEP) and electric current densities (JI) during regeneration. Depletion of ROS mimics the altered TEP and JI observed in the non-regenerative refractory period. Short-term application of hydrogen peroxide (H2O2) rescues (from depleted ROS) and induces (from the refractory period) regeneration, TEP increase and JI reversal. H2O2 is therefore necessary for and sufficient to induce regeneration and to regulate TEP and JI Epistasis assays show that voltage-gated Na+ channels act downstream of H2O2 to modulate regeneration. Altogether, these results suggest a novel mechanism for regeneration via redox-bioelectric orchestration.

Abstract 677: Possible role of nimbolide in prevention and therapy of prostate cancer

Abstract Prostate cancer is one of the most commonly diagnosed cancers worldwide. Current therapies for metastatic prostate cancer are only marginally effective, and hence novel treatment modalities are urgently needed. Considerable evidence suggests that chronic inflammation plays a pivotal role in the development and progression of prostate cancer. Thus agents that can suppress these inflammatory mediators could form the basis of novel therapy for prostate cancer patients. In our study, we focused on analyzing the potential anticancer effects of nimbolide, a terpenoid lactone derived from Azadirachta indica (Neem tree) against prostate cancer. Data from the in vitro studies indicated that nimbolide could inhibit cell proliferation, induce apoptosis and suppress cellular invasion and migration. Interestingly, nimbolide also abrogated the activation of pro-inflammatory STAT3 transcription factor, and this effect was found to be mediated via an increased production of reactive oxygen species (ROS), whereas depletion of ROS attenuated p-STAT3 inhibitory effects of the drug. The in vivo efficacy of nimbolide was also analyzed in transgenic adenocarcinoma of mouse prostate (TRAMP) model, in which this triterpenoid significantly suppressed the tumor progression and growth without exhibiting any significant adverse effects. Overall our findings indicate that nimbolide exhibits significant anticancer effects in prostate cancer, and these effects may be mediated at least in part through the modulation of STAT3 signaling pathway. Citation Format: Jingwen Zhang, Muthu K. Shanmugam, Amudha Deivasigamani, Alan P. Kumar, Kam M. Hui, Gautam Sethi. Possible role of nimbolide in prevention and therapy of prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 677. doi:10.1158/1538-7445.AM2015-677

The variable chemotherapeutic response of Malabaricone-A in leukemic and solid tumor cell lines depends on the degree of redox imbalance

The variable chemotherapeutic response of Malabaricone-A in leukemic and solid tumor cell lines depends on the degree of redox imbalance

Abstract 424: Hyper-Inflammatory Macrophages in Coronary Artery Disease and Rheumatoid Arthritis; A Signature of CCL18, Krüppel-like Factor 2 and 4 and Oxidative Stress Response Genes

Background: Autoimmune disease is an independent risk factor for accelerated coronary artery disease (CAD). Patients with rheumatoid arthritis (RA) have a 2-fold increased risk for CAD, comparable to the impact of diabetes mellitus. Abnormalities in the immune system contribute to both RA and CAD pathogenesis and defining these might elucidate disease mechanisms. We studied inflammatory macrophages in RA and CAD to identify shared and disease-specific molecular pathways. Methods: Patients with CAD and a history of myocardial infarction, patients with RA who satisfied the 2010 RA Classification Criteria and age-matched controls were enrolled. Monocytes isolated from peripheral blood were differentiated into macrophages and further polarized into M1 or M2 with IFNγ and lipopolysaccharide or IL-4 and IL-13, respectively. The expression of 55 genes was measured by quantitative PCR. Intracellular reactive oxygen species (ROS) were quantified using CellROX and the oxidant superoxide was scavenged using the SOD mimetic Tempol. Results: Both CAD and RA macrophages exhibited a gene expression signature of excess inflammatory activity when compared to controls. Besides the upregulation of cytokine genes, the hyper-inflammatory signature included the loss of the inflammatory suppressors Krüppel-like factor (KLF)-2 and KLF-4, and the gain of the chemokine CCL18 and the oxidative stress response gene, NAD(P)H:quinone oxidoreductase 1 (NQO1). Quantification of intracellular ROS confirmed increased oxidative stress in the patient-derived macrophages. To examine whether superoxide affects production of CCL18, cells were treated with Tempol. ROS depletion normalized the excess production of CCL18. Conclusion: Pro-inflammatory macrophages in RA and CAD share molecular abnormalities characterized by excessive cytokines and chemokine production, the loss of negative regulators and the gain of oxidative stress responses. Unbalanced ROS production is upstream of abnormal chemokine production and correcting redox balance in macrophages may be helpful in suppressing the hyper-inflammatory phenotype of these cells in CAD and in the CAD-prone autoimmune disease RA.

ROS signaling by NADPH oxidase 5 modulates the proliferation and survival of prostate carcinoma cells.

Prostate cancer (PCa) is the most commonly diagnosed cancer and second leading cause of male cancer death in Western nations. Thus, new treatment modalities are urgently needed. Elevated production of reactive oxygen species (ROS) by NADPH oxidase (Nox) enzymes is implicated in tumorigenesis of the prostate and other tissues. However, the identity of the Nox enzyme(s) involved in prostate carcinogenesis remains largely unknown. Analysis of radical prostatectomy tissue samples and benign and malignant prostate epithelial cell lines identified Nox5 as an abundantly expressed Nox isoform. Consistently, immunohistochemical staining of a human PCa tissue microarray revealed distinct Nox5 expression in epithelial cells of benign and malignant prostatic glands. shRNA‐mediated knockdown of Nox5 impaired proliferation of Nox5‐expressing (PC‐3, LNCaP) but not Nox5‐negative (DU145) PCa cell lines. Similar effects were observed upon ROS ablation via the antioxidant N‐acetylcysteine confirming ROS as the mediators. In addition, Nox5 silencing increased apoptosis of PC‐3 cells. Concomitantly, protein kinase C zeta (PKCζ) protein levels and c‐Jun N‐terminal kinase (JNK) phosphorylation were reduced. Moreover, the effect of Nox5 knockdown on PC‐3 cell proliferation could be mimicked by pharmacological inhibition of JNK. Collectively, these data indicate that Nox5 is expressed at functionally relevant levels in the human prostate and clinical PCa. Moreover, findings herein suggest that Nox5‐derived ROS and subsequent depletion of PKCζ and JNK inactivation play a critical role in modulating intracellular signaling cascades involved in the proliferation and survival of PCa cells. © 2014 The Authors. Molecular Carcinogenesis published by Wiley Periodicals, Inc.

Differentially Expressed Genes and Pathways in Endothelial Cells Exposed to Hemin or Plasma from Patients with Sickle Cell Disease: A Meta-Analysis of Gene Expression Studies

Introduction: Vaso-occlusion and chronic hemolysis are recognized as the most important pathogenic mechanisms of sickle cell disease (SCD), feeding a vicious circle that leads to acute and chronic complications. Although phenotypic aspects of this pro-inflammatory state have been described in detail, much less is known about the upstream pathways that activate and perpetuate inflammation in SCD. It has been known for more than 50 years that patients with SCD present higher plasma concentrations of heme. More recently, the role of heme as a mediator of inflammation in SCD has been confirmed in relevant models, suggesting that free heme can be a trigger for both microvascular occlusion and acute chest syndrome (ACS). In the past, microarray-based gene expression experiments have been used to study the effects of heme on endothelial cells (EC), as well as gene expression signatures of SCD. These studies can be analyzed in combination, since original raw data are now collected in public archives. In fact, it has been shown that by analyzing data from multiple experiments by meta-analysis, biases and artifacts between datasets can be cancelled out, potentially allowing true relationships to stand out. In order to gain insights into the cellular and molecular pathways activated by heme in endothelial cells (EC), as well as about their potential relevance in SCD, we performed a meta-analysis of microarray-based gene expression studies involving heme, EC and SCD.Material and methods: Microarray data were identified by searching two public databases (GEO and Array-Express) using the following search criteria: (“sickle cell disease” and “homo sapiens”). Eleven studies were identified, of which two were selected for our meta-analysis (GSE1849; GSE25014). One study evaluated the effect of heme 5µM in human primary pulmonary artery (PAECs) and microvascular EC (PMVECs) (12 samples), while the other study evaluated the effect of plasma from SCD patients (9 patients in steady state and 12 patients during ACS) in PAECs. To perform the meta-analysis we used INMEX, an integrative web-based tool for meta-analysis of expression data. For the meta-analysis, we applied a combining rank orders method based in the RankProd package. Genes with expression fold-changes (FC) in the same direction (either up or down) of 1.4 in at least one study were selected as candidates for differentially expressed (cDE) genes. Selected genes were ranked based on p value, and a p value ≤0.05 was considered statistically significant. To further understand functions of the subset of genes that were cDE in both studies, we performed gene ontology enrichment analysis. The functional analysis was undertaken using INMEX, and confirmed in other three gene set analysis tools (Pathway Commons, WikiPathways and KEGG). Only pathways that were identified in more than one tool were considered in the analysis.Results: Two different meta-analysis were performed. Gene expression data from heme-stimulated EC was compared to: (i) data from EC stimulated by plasma from SCD patients at steady-state; or (ii) data from EC stimulated by plasma obtained during ACS. In the first (heme x steady-state) and second (heme x ACS) analysis, 799 and 786 genes were consistently up- or down-regulated in both studies. The up- and down-regulated genes with the lowest p values were C2CD4A (C2 calcium-dependent domain containing 4A), and KLHL23 (kelch-like family member 23), respectively. In addition, genes associated with depletion of reactive oxygen species and coagulation activation were also identified. The most significant pathways identified in the gene set analysis were “IL5-mediated signaling events” (heme x steady-state; p= 0.0012) and “MAPK signaling pathway” (heme x ACS; p= 0.0073512) respectively.Results and conclusion: Genes and pathways that are DE both in EC stimulated by heme or by plasma from SCD patients could be relevant elements in the pathogenesis of inflammation in SCD. Heme has been shown to increase the generation of ROS and to induce the expression of inflammatory and pro-coagulant proteins by EC. The results of our meta-analysis are consistent with these effects. Therefore, the comprehensive list of genes and pathways identified in our study could help the generation new hypothesis about the mechanisms involved in heme-induced activation and perpetuation of inflammation in SCD. DisclosuresNo relevant conflicts of interest to declare.

Hyperglycemia and angiotensin II cooperate to enhance collagen I deposition by cardiac fibroblasts through a ROS-STAT3-dependent mechanism

Cardiac fibroblasts significantly contribute to diabetes-induced structural and functional changes in the myocardium. The objective of the present study was to determine the effects of high glucose (alone or supplemented with angiotensin II) in the activation of the JAK2/STAT3 pathway and its involvement in collagen I production by cardiac fibroblasts. We observed that the diabetic environment 1) enhanced tyrosine phosphorylation of JAK2 and STAT3; 2) induced nuclear localization of tyrosine phosphorylated STAT3 through a reactive oxygen species-mediated mechanism, with angiotensin II stimulation further enhancing STAT3 nuclear accumulation; and 3) stimulated collagen I production. The effects were inhibited by depletion of reactive oxygen species or silencing of STAT3 in high glucose alone or supplemented with exogenous angiotensin II. Combined, our data demonstrate that increased collagen I deposition in the setting of high glucose occurred through a reactive oxygen species- and STAT3-dependent mechanism. Our results reveal a novel role for STAT3 as a key signaling molecule of collagen I production in cardiac fibroblasts exposed to a diabetic environment.

Suberoylanilide hydroxamic acid-induced HeLa cell death is closely correlated with oxidative stress and thioredoxin 1 levels

Suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase (HDAC) inhibitor which has anticancer effects. We evaluated the growth inhibitory effects of SAHA on HeLa cervical cancer cells in relation to reactive oxygen species (ROS) levels. SAHA inhibited the growth of HeLa cells with an IC(50) of approximately 10 µM at 24 h, and induced apoptosis which was accompanied by the cleavage of PARP, caspase-3 activation and loss of mitochondrial membrane potential (MMP; ∆ψ(m)). All the tested caspase inhibitors prevented HeLa cell death induced by SAHA whereas TNF-α intensified apoptotic cell death in SAHA-treated HeLa cells. With respect to ROS and glutathione (GSH) levels, SAHA increased ROS levels, especially mitochondrial O(2)•- in HeLa cells and also induced GSH depletion. Caspase inhibitors reduced the levels of ROS and GSH depletion in SAHA-treated HeLa cells whereas TNF-α enhanced the levels in these cells. The well-known antioxidant N-acetyl cysteine (NAC) attenuated cell death and an increase in ROS levels was caused by SAHA. Moreover, SAHA decreased the levels of thioredoxin 1 (Trx1) in HeLa cells. While the downregulation of Trx1 enhanced cell death and ROS levels in SAHA-treated HeLa cells, the overexpression of Trx1 attenuated the levels in these cells. In conclusion, SAHA inhibited the growth of HeLa cell via caspase-dependent apoptosis, which was influenced by the mitochondrial O(2)•- and Trx1 levels.

Anticancer effect of salidroside on A549 lung cancer cells through inhibition of oxidative stress and phospho-p38 expression.

Oxidative stress is important in carcinogenesis and metastasis. Salidroside, a phenylpropanoid glycoside isolated from Rhodiola rosea L., shows potent antioxidant properties. The aim of the present study was to investigate the roles of salidroside in cell proliferation, the cell cycle, apoptosis, invasion and epithelial-mesenchymal transition (EMT) in A549 cells. The human alveolar adenocarcinoma cell line, A549, was incubated with various concentrations of salidroside (0, 1, 5, 10 and 20 μg/ml) and cell proliferation was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Propidium iodide (PI) staining was used to determine the cell cycle by flow cytometry. Cell apoptosis was detected by Annexin V-fluorescein isothiocyanate and PI double-staining, and tumor invasion was detected by Boyden chamber invasion assay. Western blot analysis was performed to detect the expression of EMT markers, Snail and phospho-p38. The results showed that salidroside significantly reduced the proliferation of A549 cells, inhibited cell cycle arrest in the G0/G1 phase and induced apoptosis. Salidroside inhibited transforming growth factor-β-induced tumor invasion and suppressed the protein expression of Snail. As an antioxidant, salidroside inhibited the intracellular reactive oxygen species (ROS) formation in a dose-dependent manner in A549 cells, and depletion of intracellular ROS by vitamin C suppressed apoptosis by salidroside treatment. Salidroside was also found to inhibit the expression of phospho-p38 in A549 cells. In conclusion, salidroside inhibits cell proliferation, the cell cycle and metastasis and induces apoptosis, which may be due to its interference in the intracellular ROS generation, thereby, downregulating the ROS-phospho-p38 signaling pathway.

The role of oxidative stress in pro-inflammatory activation of human endothelial cells on Ti6Al4V alloy

Inflammation is an important step in the early phase of tissue regeneration around an implanted metallic orthopaedic device. However, prolonged inflammation, which can be induced by metallic corrosion products, can lead to aseptic loosening and implant failure. Cells in peri-implant tissue as well as metal corrosion can induce reactive oxygen species (ROS) formation, thus contributing to an oxidative microenvironment around an implant. Understanding cellular reactions to implant-induced oxidative stress and inflammatory activation is important to help prevent an adverse response to metallic materials. In an earlier study we have shown that endothelial cells grown on Ti6Al4V alloy are subjected to oxidative stress. Since endothelial cells play a critical role in inflammation, in this study we examined the role of oxidative stress in their response to pro-inflammatory activation. Therefore, we stimulated endothelial cells in contact with Ti6Al4V with tumour necrosis factor-α (TNF-α) and monitored the expression of inflammation-associated molecules, such as E-selectin, intercellular adhesion molecule-1 (ICAM-1) and interleukin-8 (IL-8). The induction of these proteins was lower in endothelial cells on Ti6Al4V compared to control tissue culture conditions. There was, however, a discrepancy in pro-inflammatory activation at protein compared to mRNA level in the cells on Ti6Al4V. To examine the role of oxidative stress in this response we utilized different ROS scavengers and showed that ROS depletion improved cellular response to TNF-α on Ti6Al4V. These results could contribute to developing strategies to improve tissue response to metal implants.

ROS Are Required for Mouse Spermatogonial Stem Cell Self-Renewal

Reactive oxygen species (ROS) generation is implicated in stem cell self-renewal in several tissues but is thought to be detrimental for spermatogenesis as well as spermatogonial stem cells (SSCs). Using cultured SSCs, we show that ROS are generated via the AKT and MEK signaling pathways under conditions where the growth factors glial cell line-derived neurotrophic factor and fibroblast growth factor 2 drive SSC self-renewal and, instead, stimulate self-renewal at physiological levels. SSCs depleted of ROS stopped proliferating, but they showed enhanced self-renewal when ROS levels were increased by the addition of hydrogen peroxide, which induced the phosphorylation of stress kinases p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal kinase (JNK). Moreover, ROS depletion invivo decreased SSC number in the testis, and NADPH oxidase 1 (Nox1)-deficient SSCs exhibited reduced self-renewal division upon serial transplantation. These results suggest that ROS generated by Nox1 play critical roles in SSC self-renewal via the activation of the p38 MAPK and JNK pathways.