Rapid Production Of Reactive Oxygen Species Research Articles

Chemiluminescence-Based Assay to Monitor Early Oxidative Bursts in Soybean (Glycine max) Lateral Roots.

The reactive oxygen species (ROS) burst assay is a valuable tool for studying pattern-triggered immunity (PTI) in plants. During PTI, the interaction between pathogen recognition receptors (PRRs) and pathogen-associated molecular patterns (PAMPs) leads to the rapid production of ROS in the apoplastic space. The resultant ROS can be measured using a chemiluminescent approach that involves the usage of horseradish peroxidase and luminol. Although several methods and protocols are available to detect early ROS bursts in leaf tissues, no dedicated method is available for root tissues. Here, we have established a reliable method to measure the PAMP-triggered ROS burst response in soybean lateral roots. In plants, lateral roots are the potential entry and colonization sites for pathogens in the rhizosphere. We have used important PAMPs such as chitohexaose, flagellin 22 peptide fragment, and laminarin to validate our method. In addition, we provide a detailed methodology for the isolation and application of fungal cell wall components to monitor the oxidative burst in soybean lateral roots. Furthermore, we provide methodology for performing ROS burst assays in soybean leaf discs with laminarin and fungal cell walls. This approach could also be applied to leaf and root tissues of other plant species to study the PTI response upon elicitor treatment. © 2023 Wiley Periodicals LLC. Basic Protocol: Reactive oxygen species (ROS) burst assay in soybean lateral root tissues Alternate Protocol: ROS burst assay in soybean leaf discs Support Protocol: Isolating fungal cell wall fractions.

The regulation of generation rate facilitates the win–win of ROS yield and utilization efficiency in heterogeneous persulfate catalytic oxidation system

Heterogeneous catalytic oxidation is one of the most effective methods to degrade various and complex organic pollutants in aqueous solution. Increasing reactive oxygen species (ROS) yield has always been the main objective to improve the oxidation efficiency. However, the mass production of ROS in a short time is inevitably faced with self-quenching and ineffective consumption. Herein, Cu-based composite oxides (CuOMOx) (M = Ca, Mg, Zn, Mn) were synthesized and used for catalytic activation of permonosulfate (PMS). Although the introduction of unreducible metal oxides significantly improved the activation efficiency of PMS, the abnormal phenomenon of lower substrate degradation efficiency and PMS stoichiometry appeared. The underlying mechanism is that the rapid production of ROS results in a mismatch of the production/effective consumption ratio, which causes massive ROS disappeared naturally before attacking the substrate. In this scenario, reasonable regulation of ROS generation rate by phosphate addition improved the utilization efficiency of ROS without affecting its yield. Our finding is beneficial to achieve a win–win situation of ROS yield and utilization efficiency in heterogeneous catalytic oxidation systems.

Quantitatively Assessing the Respiratory Burst in Innate Immune Cells.

The respiratory burst is a rapid cellular consumption of oxygen resulting in abundant production of reactive oxygen species (ROS), most often associated with primary mediators of innate immunity, neutrophils and macrophages. These myeloid cells convert ROS into potent antimicrobial oxidants that efficiently kill pathogens. The respiratory burst also can have destructive consequences, as ROS are well known to support chronic inflammation and aberrant autoimmune responses. Interestingly, ROS perform conflicting roles in the tumor microenvironment; ROS and derived cytotoxic products can destroy cancer cells but also suppress important tumor-fighting functions of T cells or natural killer cells, or yield mutagenized proteins that can promote tumorigenesis or support tumor cell growth. Moreover, high numbers of neutrophils or macrophages in tumors are associated with poor prognosis. Therefore, accurate and quantitative assays to assess the respiratory burst are an important tool for measuring ROS production by neutrophils or cells of the monocyte/macrophage system, each recently identified in the tumor microenvironment. Described are methods to derive mouse or human models of neutrophils or macrophages, which are then used in a detailed assay to quantitatively measure ROS produced by either cell type using luminescence-enhanced reagents and a multi-well platform along with different stimulants that cause rapid ROS production.

Omics analyses indicate sdhC/D act as hubs of early response of E. coli to antibiotics.

In recent years, the phenomenon of microbial resistance has become increasingly serious. The generation of reactive oxygen species (ROS) during the bactericidal process of antibiotics has attracted great interest, but little research has been done on the generation of ROS in the early stage of antibiotic action. We confirmed the rapid production of ROS by flow cytometry and transmission electron microscopy (TEM). GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis indicated that the oxidative phosphorylation pathway is the key pathway of ROS production. Protein-protein interaction (PPI) network results indicate that sdhC/D are key genes in the oxidative phosphorylation pathway. The overexpression of sdhC/D resulted in a lower survival rate than the control strain after antibiotic treatments, which might be due to excess ROS induced by sdhC/D overexpression. The production of superoxide anion in the overexpress strain was significantly higher than that in the control strain, which further verified the importance of sdhC/D in the ROS release of bacteria. Current results showed that bacteria produce large amounts of ROS in the early stage of gentamicin and ampicillin action, and the regulation patterns of genes in the key pathway were consistent. sdhC/D are key genes in the early ROS release process of bacteria. Our study provides a basis for the search of ROS-related enhancers of antimicrobial action.

Flexible, mesoporous, and monodispersed metallic cobalt-embedded inorganic nanofibrous membranes enable ultra-fast and high-efficiency killing of bacteria

Severe emerging infectious diseases, deriving from the contamination of pathogenic bacteria, are threatening human’s survival and development. Metal-embedded inorganic porous material with large mesopores and high flexibility, has proven to be one of the most promising activators of peroxymonosulfate (PMS) to generate reactive oxygen species (ROS) for the high-efficiency pathogenic bacteria elimination. However, there still exists huge challenges to develop such materials. Herein, a robust strategy was reported to create flexible metallic cobalt (Co)-embedded inorganic nanofibrous membranes (CINMs) with interconnected mesoporous structures and monodispersed Co nanoparticles, for the first time, by double-template electrospinning approach and in situ carbonization reduction method. The monodispersed Co particles throughout fibers could activate PMS to effectively produce ROS, enabling the complete bacterial inactivation with a 7 log reduction within only 3 min. Benefitting from the integrated features of high porosity, robust mechanical property, and rapid ROS production, the obtained CINMs exhibit an ultra-high bactericidal efficiency of 99.99999% and a high permeate flux of 3.4 × 104 L m−2 h−1 merely driven by gravity (≈1.2 kPa). Moreover, a novel CINMs/PMS-based spray type sterilizer was successfully created for the convenient and high-efficiency sterilization of solid surfaces, which could completely inactivate bacteria on the surfaces after being sprayed for 10 s. The successful synthesis of such fascinating materials may shed light on designing new types of inorganic nanofiber-based antimicrobials for various public health and environment protection.

Increasing Pyruvate Concentration Enhances Conidial Thermotolerance in the Entomopathogenic Fungus Metarhizium robertsii.

The fungal entomopathogens Metarhizium spp. have been developed as environmentally friendly mycoinsecticides. However, heat stress severely reduces the viability of Metarhizium conidia in the field, which is an important obstacle to the successful use of these mycoinsecticides. Heat treatment induces rapid accumulation of pyruvate, which timely scavenges heat-induced ROS (reactive oxygen species) in hyphal cells of M. robertsii. However, in heat-treated conidia, pyruvate accumulation occurs later than the rapid production of ROSs, which could harm the conidial cells. In the present study, a transgenic M. robertsii strain was constructed with the pyruvate kinases gene overexpressed during conidiation. Two independent transformants of the transgenic strain produced conidia under optimal conditions with elevated pyruvate concentration. This inhibits the rapid heat-induced ROS production and prevents the collapse of mitochondrial membrane potential, thereby increasing conidial tolerance to heat stress. In conclusion, the tolerance of M. robertsii conidia to heat stress was improved by increasing the conidial pyruvate concentration, which could be translated into a more effective pest control.

Calcium-mediated oxidative stress: a common mechanism in tight junction disruption by different types of cellular stress.

The role of reactive oxygen species (ROS) in osmotic stress, dextran sulfate sodium (DSS) and cyclic stretch-induced tight junction (TJ) disruption was investigated in Caco-2 cell monolayers in vitro and restraint stress-induced barrier dysfunction in mouse colon in vivo Live cell imaging showed that osmotic stress, cyclic stretch and DSS triggered rapid production of ROS in Caco-2 cell monolayers, which was blocked by depletion of intracellular Ca2+ by 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Knockdown of CaV1.3 or TRPV6 channels blocked osmotic stress and DSS-induced ROS production and attenuated TJ disruption and barrier dysfunction. N-Acetyl l-cysteine (NAC) and l-NG-Nitroarginine methyl ester (l-NAME) blocked stress-induced TJ disruption and barrier dysfunction. NAC and l-NAME also blocked stress-induced activation of c-Jun N-terminal kinase (JNK) and c-Src. ROS was colocalized with the mitochondrial marker in stressed cells. Cyclosporin A blocked osmotic stress and DSS-induced ROS production, barrier dysfunction, TJ disruption and JNK activation. Mitochondria-targeted Mito-TEMPO blocked osmotic stress and DSS-induced barrier dysfunction and TJ disruption. Chronic restraint stress in mice resulted in the elevation of intracellular Ca2+, activation of JNK and c-Src, and disruption of TJ in the colonic epithelium. Furthermore, corticosterone administration induced JNK and c-Src activation, TJ disruption and protein thiol oxidation in colonic mucosa. The present study demonstrates that oxidative stress is a common signal in the mechanism of TJ disruption in the intestinal epithelium by different types of cellular stress in vitro and bio behavioral stress in vivo.

Reactive Oxygen Species Mediated Prostaglandin E2 Contributes to Acute Response of Epithelial Injury.

Reactive oxygen species (ROS) generated after tissue injury play a crucial role during wound healing through initiating acute inflammation, clarifying infection and dead tissue, and mediating various intracellular signal transduction. Prostaglandin E2 (PGE2) has been identified as one of the major factors responsible for inflammation and tissue repair. In this study, we tested our hypothesis that ROS produced by damaged human keratinocytes induces the synthesis of PGE2. In vitro epithelial wounding model was used to observe the production of ROS and secretion of PGE2 as well as the involved signal pathway. The mechanical injury caused the rapid production of ROS in in vitro cultured keratinocytes, which was significantly blocked by an inhibitor of nicotinamide adenine dinucleotide phosphate oxidase. The increased intracellular ROS caused by mechanical injury stimulates PGE2 production in a time-dependent manner via the activation of cyclooxygenase-2 (COX-2), which was stimulated by phosphorylation of extracellular signal-regulated protein kinase (ERK). These results indicate ROS-induced ERK activation leading to the activation of COX-2 and the synthesis of PGE2 in human keratinocytes responding to mechanical injury in the acute phase.

Similarities and Differences in Global Gene Expression Profiles between Herbicide and Pathogen-Induced PSII Inhibition

Plant pathogens, and photosynthesis inhibiting herbicides, can both damage photosystem II (PSII), causing it to be highly sensitive to damage by light energy, and to release high levels of reactive oxygen species (ROS). This photoinhibition of PSII could possibly be the source of the strong oxidative burst associated with the pathogeninduced, hypersensitive defense response (HR). To examine a possible mechanism of how the HR-associated ROS burst could originate from PSII inhibition, we compared the transcriptome responses in soybean undergoing photoinhibition induced by HR, to soybean undergoing photoinhibition induced by the herbicide bentazon, which specially stops PSII electron flow by preventing QB from binding to D1. Most genes shared similar expression patterns between HR and bentazon treatments; however, interesting differences were also observed. The most striking differences were seen with genes related to photosynthesis, where these genes were uniformly down regulated in HR, but were mostly up in response to bentazon. Another interesting difference was seen in genes of the phenylpropanoid pathway. These defense-related genes were mostly down or non-responsive to bentazon, but were generally induced in response to pathogen-induced HR, showing that soybeans activate the phenylpropanoidbased phytoalexins independent of PSII inhibition. We conclude that the PSII inhibition occurring during the HR is not being triggered simply by the inhibition of electron flow through the photosystem centers. Instead, it is more likely that the initial triggers of the HR halt the repair of damaged PSII which leads to enhancing photoinhibition and contributing the rapid production of ROS, sealing the fate of cells undergoing HR; and the triggers independently induce specific aspects of defenses such as the phenylpropanoid pathway.

Porphyromonas gingivalis-induced reactive oxygen species activate JAK2 and regulate production of inflammatory cytokines through c-Jun.

Pathogen-induced reactive oxygen species (ROS) play a crucial role in host innate immune responses through regulating the quality and quantity of inflammatory mediators. However, the underlying molecular mechanisms of this effect have yet to be clarified. In this study, we examined the mechanism of action of ROS stimulated by Porphyromonas gingivalis in gingival epithelial cells. P. gingivalis induced the rapid production of ROS, which lead to the phosphorylation of JAK2 and increased levels of secreted proinflammatory cytokines interleukin-6 (IL-6) and IL-1β. Neutralization of ROS by N-acetyl-l-cysteine (NAC) abrogated the phosphorylation of JAK2 and suppressed the production of IL-6 and IL-1β. ROS-mediated phosphorylation of JAK2 induced the phosphoactivation of c-Jun amino-terminal protein kinase (JNK) and the downstream transcriptional regulator c-Jun. Inhibition of JAK2, either pharmacologically or by small interfering RNA (siRNA), reduced both the phosphorylation of these molecules and the production of proinflammatory cytokines in response to P. gingivalis. Furthermore, pharmacological inhibition or siRNA-mediated gene silencing of JNK or c-Jun mimicked the effect of JAK2 inhibition to suppress P. gingivalis-induced IL-6 and IL-1β levels. The results show that ROS-mediated activation of JAK2 is required for P. gingivalis-induced inflammatory cytokine production and that the JNK/c-Jun signaling axis is involved in the ROS-dependent regulation of IL-1β and IL-6 production.

Prolonged Production of Reactive Oxygen Species in Response to B Cell Receptor Stimulation Promotes B Cell Activation and Proliferation

We have investigated the intracellular sources and physiological function of reactive oxygen species (ROS) produced in primary B cells in response to BCR stimulation. BCR stimulation of primary resting murine B cells induced the rapid production of ROS that occurred within minutes and was maintained for at least 24 h after receptor stimulation. While the early production of ROS (0-2 h) was dependent on the Nox2 isoform of NADPH oxidase, at later stages of B cell activation (6-24 h) ROS were generated by a second pathway, which appeared to be dependent on mitochondrial respiration. B cells from mice deficient in the Nox2 NADPH oxidase complex lacked detectable early production of extracellular and intracellular ROS after BCR stimulation but had normal proximal BCR signaling and BCR-induced activation and proliferation in vitro and mounted normal or somewhat elevated Ab responses in vivo. In contrast, neutralizing both pathways of BCR-derived ROS with the scavenger N-acetylcysteine resulted in impaired in vitro BCR-induced activation and proliferation and attenuated BCR signaling through the PI3K pathway at later times. These results indicate that the production of ROS downstream of the BCR is derived from at least two distinct cellular sources and plays a critical role at the later stages of B cell activation by promoting sustained BCR signaling via the PI3K pathway, which is needed for effective B cell responses to Ag.

Correlation of Different NADPH Oxidase Homologues with Late Endothelial Progenitor Cell Senescence Induced by Angiotensin II: Effect of Telmisartan

Involvement of different NADPH oxidase (NOX) homologues in late endothelial progenitor cell (EPC) senescence induced by angiotensin II (Ang II) remains rarely studied systemically. The goal of our study was to determine NOX homologues which are correlated with late EPCs senescence induced by Ang II. The inhibitory effect of telmisartan was also studied. Late EPCs were obtained from mononuclear cells isolated from peripheral venous blood. Stimulated by Ang II with telmisartan (Tel) or VAS2870 pretreatment or siRNA prior silencing, NOX was detected by RT-PCR and Western blot. Cell senescence was measured by the acidic β-galactosidase activity assay and cell cycle analysis. Intracellular reactive oxygen species (ROS) were analyzed by flow cytometer based on DCFH-DA. A bi-phasic change existed in NOX level after Ang II stimulation. Translocated NOX5 was correlated with early and rapid ROS production, but it contributed little to EPCs senescence. NOX2 and NOX4 were correlated with the late and slow phase and contributed greatly to EPCs senescence. There were no significant changes in NOX1 or NOX3. Telmisartan effectively depressed NOX change and delayed late EPCs senescence. Ang II accelerates late EPCs senescence mainly via increased ROS originating from NOX2 and NOX4 up-regulation or translocated NOX5. Telmisartan effectively inhibited that cascade reaction and delayed EPCs senescence.

Perception of the Arabidopsis Danger Signal Peptide 1 Involves the Pattern Recognition Receptor AtPEPR1 and Its Close Homologue AtPEPR2

Plasma membrane-borne pattern recognition receptors, which recognize microbe-associated molecular patterns and endogenous damage-associated molecular patterns, provide the first line of defense in innate immunity. In plants, leucine-rich repeat receptor kinases fulfill this role, as exemplified by FLS2 and EFR, the receptors for the microbe-associated molecular patterns flagellin and elongation factor Tu. Here we examined the perception of the damage-associated molecular pattern peptide 1 (AtPep1), an endogenous peptide of Arabidopsis identified earlier and shown to be perceived by the leucine-rich repeat protein kinase PEPR1. Using seedling growth inhibition, elicitation of an oxidative burst and induction of ethylene biosynthesis, we show that wild type plants and the pepr1 and pepr2 mutants, affected in PEPR1 and in its homologue PEPR2, are sensitive to AtPep1, but that the double mutant pepr1/pepr2 is completely insensitive. As a central body of our study, we provide electrophysiological evidence that at the level of the plasma membrane, AtPep1 triggers a receptor-dependent transient depolarization through activation of plasma membrane anion channels, and that this effect is absent in the double mutant pepr1/pepr2. The double mutant also fails to respond to AtPep2 and AtPep3, two distant homologues of AtPep1 on the basis of homology screening, implying that the PEPR1 and PEPR2 are responsible for their perception too. Our findings provide a basic framework to study the biological role of AtPep1-related danger signals and their cognate receptors.

Role of reactive oxygen species (ROS) in Mycobacterium bovis bacillus Calmette Guérin-mediated up-regulation of the human cathelicidin LL-37 in A549 cells

The human cathelicidin LL-37 is one of the major antimicrobial peptides of the non-specific innate immune system in Mycobacterium tuberculosis infection. Its expression has been reported in epithelial cells infected with mycobacteria. However, the underlying molecular mechanisms by which Mycobacterium bovis bacillus Calmette-Guérin (BCG) triggers gene transcription of cathelicidin have not been elucidated. The objective of this study was to investigate the role of reactive oxygen species (ROS) in the M. bovis BCG-mediated up-regulation of the antimicrobial peptide cathelicidin LL-37 in human epithelial cells. Infection of A549 cells with M. bovis BCG led to a rapid ROS production. Importantly, blockade of ROS by preincubation of cells with the general ROS scavenger N-acetyl-l-cysteine (NAC) or the NADPH oxidase inhibitor DPI significantly reduced M. bovis BCG-induced up-regulation of cathelicidin LL-37 mRNA expression as determined by semi-quantitative RT-PCR or real-time PCR. In contrast, the xanthine oxidase inhibitor allopurinol did not affect M. bovis BCG-mediated up-regulation of cathelicidin LL-37 mRNA. Moreover, M. bovis BCG-mediated cathelicidin LL-37 mRNA expression was significantly blocked by the effect of the mitochondrial electron transfer chain subunit I inhibitor rotenone and H 2O 2 scavenging enzyme catalase. In addition, M. bovis BCG-induced cathelicidin LL-37 protein secretion was inhibited by the addition of NAC, DPI, and the selective inhibitor of NADPH oxidase apocynin. Our results collectively indicate that M. bovis BCG-mediated up-regulation of cathelicidin is influenced by NADPH/ROS signaling pathways. In conclusion, these findings demonstrate a novel regulatory mechanism for the expression of cathelicidin LL-37 in human epithelial cells stimulated with M. bovis BCG.

Innate immune responses toMycobacterium ulceransvia toll-like receptors and dectin-1 in human keratinocytes

Mycobacterium ulcerans (MU), an environmental pathogen, causes Buruli ulcer, a severe skin disease. We hypothesized that epidermal keratinocytes might not be a simple barrier, but play a role during MU infection through pattern-recognition receptors expressed in keratinocytes. We found that keratinocyte Toll-like receptors (TLRs) 2 and 4 and Dectin-1 actively participate in the innate immune response to MU, which includes the internalization of bacteria, the production of reactive oxygen species (ROS), and the expression of chemokines and LL-37. Human keratinocytes constitutively expressed TLRs 2 and 4 and induced Dectin-1 in response to MU. Exposing keratinocytes to MU resulted in rapid ROS production, which in turn contributed to the mRNA and protein expression of LL-37. In addition, TLR2, Dectin-1 and, to an extent, TLR4 are essential for the MU-mediated expression of CXCL8, CCL2 and LL-37 in keratinocytes. Furthermore, confocal analysis showed that the Dectin-1 is necessary for keratinocytes to internalize bacilli. Importantly, blockade of ROS and LL-37 significantly increased the intracellular MU growth in keratinocytes, suggesting an important role of these mediators for cutaneous innate immune responses. Our results demonstrate that TLR2, TLR4 and Dectin-1 actively sense, internalize and respond in an innate way to MU in human epidermal keratinocytes.

The Nuclear Receptor Interacting Factor-3 Transcriptional Coregulator Mediates Rapid Apoptosis in Breast Cancer Cells through Direct and Bystander-Mediated Events

We previously reported that amino acids 20 to 50 of nuclear receptor interacting factor-3 mediates rapid apoptosis in breast cancer cell lines but not in cells derived from other tissues. We refer to this short region as death domain-1 (DD1). Small interfering RNA studies indicated that DD1-mediated apoptosis is caspase-2 dependent. In this study, we examined DD1-mediated apoptosis in more detail and generated stable caspase-2 knockdown breast cancer cells. These cells are resistant to DD1-mediated apoptosis. Time-lapse movies suggested that DD1-mediated apoptosis also leads to a "bystander effect." We found that within 5 h of DD1 expression, breast cancer cells release a factor(s) into the medium that leads to apoptosis of naive breast cancer cells or DD1-resistant cells (e.g., HeLa). The DD1-expressing caspase-2 knockdown cells also release a factor(s) that kills other cells, indicating that this effect is not dependent on the apoptogenic process. The bystander effect seems dependent on the production of reactive oxygen species (ROS). These and other studies indicate that DD1 expression in breast cancer cells leads to at least two death signals: one involving the rapid production of ROS and/or other soluble factors that directly or indirectly leads to a bystander effect and a second caspase-2-dependent process that leads to apoptosis in cells in which DD1 is expressed.

D-501036, a novel selenophene-based triheterocycle derivative, exhibits potentin vitroandin vivoantitumoral activity which involves DNA damage and ataxia telangiectasia–mutated nuclear protein kinase activation

D-501036 [2,5-bis(5-hydroxymethyl-2-selenienyl)-3-hydroxymethyl-N-methylpyrrole] is herein identified as a novel antineoplastic agent with a broad spectrum of antitumoral activity against several human cancer cells and an IC(50) value in the nanomolar range. The IC(50) values for D-501036 in the renal proximal tubule, normal bronchial epithelial, and fibroblast cells were >10 mumol/L. D-501036 exhibited no cross-resistance with vincristine- and paclitaxel-resistant cell lines, whereas a low level of resistance toward the etoposide-resistant KB variant was observed. Cell cycle analysis established that D-501036 treatment resulted in a dose-dependent accumulation in S phase with concomitant loss of both the G(0)-G(1) and G(2)-M phase in both Hep 3B and A-498 cells. Pulsed-field gel electrophoresis showed D-501036-induced, concentration-dependent DNA breaks in both Hep 3B and A-498 cells. These breaks did not involve interference with either topoisomerase-I and topoisomerase-II function or DNA binding. Rapid reactive oxygen species production and formation of Se-DNA adducts were evident following exposure of cells to D-501036, indicating that D-501036-mediated DNA breaks were attributable to the induction of reactive oxygen species and DNA adduct formation. Moreover, D-501036-induced DNA damage activated ataxia telangiectasia-mutated nuclear protein kinase, leading to hyperphosphorylation of Chk1, Chk2, and p53, decreased expression of CDC25A, and up-regulation of p21(WAF1) in both p53-proficient and p53-deficient cells. Collectively, the results indicate that D-501036-induced cell death was associated with DNA damage-mediated induction of ataxia telangiectasia-mutated activation, and p53-dependent and -independent apoptosis pathways. Notably, D-501036 shows potent activity against the growth of xenograft tumors of human renal carcinoma A-498 cells. Thus, D-501036 is a promising anticancer compound that has strong potential for the management of human cancers.

Rapid reactive oxygen species production by mitochondria in endothelial cells exposed to tumor necrosis factor-alpha is mediated by ceramide.

Tumor necrosis factor (TNF)-alpha increases mitochondrial reactive oxygen species (ROS) production in tumor cells and hepatocytes. However, whether TNF-alpha stimulates mitochondrial ROS production in endothelial cells (EC) has not yet been reported. We studied the effect of TNF-alpha on mitochondrial ROS generation in EC and the signaling pathways involved. Cultured human umbilical vein EC (HUVEC) were studied by fluorescence microscopy, using dichlorodihydrofluorescein diacetate (DCFH-DA) as a marker of ROS production and propidium iodide uptake for cell viability. TNF-alpha increased DCFH oxidation in HUVEC dose-dependently. To determine the source of ROS, the mitochondrial respiratory chain inhibitors rotenone + thenoyltrifluoroacetone (TTFA), which inhibit electron entry to ubiquinone, and antimycin A (AA), a blocker of ubisemiquinone, were used. Rotenone and TTFA inhibited (n = 7, P < 0.05), whereas AA increased (118% in 3 min; n = 4, P < 0.01) ROS generation in HUVEC. In contrast, ROS production was not abolished by the nicotinamide adenine dinucleotide phosphate-dependent oxidase inhibitor diphenylene iodonium, by the xanthine oxidase inhibitor allopurinol, nor by the nitric oxide and cyclooxygenase pathway inhibitors N(omega)-nitro-L-arginine and mefenamic acid. In addition, TNF-alpha-induced ROS production was inhibited by the acidic sphingomyelinase inhibitor desipramine (5 microM; -80%, n = 4, P < 0.01) and totally blocked by the ceramide-activated protein kinase (CAPK) inhibitor dimethylaminopurine (1 mM; n = 6, P < 0.05). Thus, TNF-alpha induces mitochondrial ROS production in HUVEC that primarily occurs at the ubisemiquinone site and is mediated by ceramide-dependent signaling pathways involving CAPK.