Flavoprotein Inhibitor Research Articles

Involvement of a flavoprotein, acetohydroxyacid synthase, in growth and riboflavin production in riboflavin-overproducing Ashbya gossypii mutant

BackgroundPreviously, we isolated a riboflavin-overproducing Ashbya gossypii mutant (MT strain) and discovered some mutations in genes encoding flavoproteins. Here, we analyzed the riboflavin production in the MT strain, in view of flavoproteins, which are localized in the mitochondria.ResultsIn the MT strain, mitochondrial membrane potential was decreased compared with that in the wild type (WT) strain, resulting in increased reactive oxygen species. Additionally, diphenyleneiodonium (DPI), a universal flavoprotein inhibitor, inhibited riboflavin production in the WT and MT strains at 50 µM, indicating that some flavoproteins may be involved in riboflavin production. The specific activities of NADH and succinate dehydrogenases were significantly reduced in the MT strain, but those of glutathione reductase and acetohydroxyacid synthase were increased by 4.9- and 25-fold, respectively. By contrast, the expression of AgGLR1 gene encoding glutathione reductase was increased by 32-fold in the MT strain. However, that of AgILV2 gene encoding the catalytic subunit of acetohydroxyacid synthase was increased by only 2.1-fold. These results suggest that in the MT strain, acetohydroxyacid synthase, which catalyzes the first reaction of branched-chain amino acid biosynthesis, is vital for riboflavin production. The addition of valine, which is a feedback inhibitor of acetohydroxyacid synthase, to a minimal medium inhibited the growth of the MT strain and its riboflavin production. In addition, the addition of branched-chain amino acids enhanced the growth and riboflavin production in the MT strain.ConclusionThe significance of branched-chain amino acids for riboflavin production in A. gossypii is reported and this study opens a novel approach for the effective production of riboflavin in A. gossypii.

NADPH oxidase 4 is dispensable for skin myofibroblast differentiation and wound healing

Differentiation of fibroblasts to myofibroblasts is governed by the transforming growth factor beta (TGF-β) through a mechanism involving redox signaling and generation of reactive oxygen species (ROS). Myofibroblasts synthesize proteins of the extracellular matrix (ECM) and display a contractile phenotype. Myofibroblasts are predominant contributors of wound healing and several pathological states, including fibrotic diseases and cancer. Inhibition of the ROS-generating enzyme NADPH oxidase 4 (NOX4) has been proposed to mitigate fibroblast to myofibroblast differentiation and to offer a therapeutic option for the treatment of fibrotic diseases. In this study, we addressed the role of NOX4 in physiological wound healing and in TGF-β-induced myofibroblast differentiation. We explored the phenotypic changes induced by TGF-β in primary skin fibroblasts isolated from Nox4-deficient mice by immunofluorescence, Western blotting and RNA sequencing. Mice deficient for Cyba, the gene coding for p22phox, a key subunit of NOX4 were used for confirmatory experiments as well as human primary skin fibroblasts. In vivo, the wound healing was similar in wild-type and Nox4-deficient mice. In vitro, despite a strong upregulation following TGF-β treatment, Nox4 did not influence skin myofibroblast differentiation although a putative NOX4 inhibitor GKT137831 and a flavoprotein inhibitor diphenylene iodonium mitigated this mechanism. Transcriptomic analysis revealed upregulation of the mitochondrial protein Ucp2 and the stress-response protein Hddc3 in Nox4-deficient fibroblasts, which had however no impact on fibroblast bioenergetics. Altogether, we provide extensive evidence that NOX4 is dispensable for wound healing and skin fibroblast to myofibroblast differentiation, and suggest that another H2O2-generating flavoprotein drives this mechanism.

Diphenyleneiodonium Triggers Cell Death of Acute Myeloid Leukemia Cells by Blocking the Mitochondrial Respiratory Chain, and Synergizes with Cytarabine.

Simple SummaryAcute myeloid leukemia (AML) is an aggressive heterogeneous cancer of the blood, of which 70% of cases develop relapse. Relapse is mainly due to chemoresistant leukemic cells (LCs) that are characterized by high mitochondrial oxidative phosphorylation (OxPhos) status, i.e., cells that are dependent on the mitochondrial respiratory chain (MRC) function. The aim of our study was to determine whether diphenyleneiodonium (DPI)—known as a potent inhibitor of flavoproteins—could be used to target AML cells. Herein, we demonstrate that DPI disrupts the mitochondrial function of AML cell lines. Interestingly, we found that cells with high OxPhos are more sensitive to the apoptotic effects of DPI. Moreover, we showed that DPI sensitizes AML cell lines to cytarabine (Ara-C) treatment, suggesting that MRC inhibitors could be employed to target LCs that are resistant to this chemotherapeutic agent.Acute myeloid leukemia (AML) is characterized by the accumulation of undifferentiated blast cells in the bone marrow and blood. In most cases of AML, relapse frequently occurs due to resistance to chemotherapy. Compelling research results indicate that drug resistance in cancer cells is highly dependent on the intracellular levels of reactive oxygen species (ROS). Modulating ROS levels is therefore a valuable strategy to overcome the chemotherapy resistance of leukemic cells. In this study, we evaluated the efficiency of diphenyleneiodonium (DPI)—a well-known inhibitor of ROS production—in targeting AML cells. Results showed that although inhibiting cytoplasmic ROS production, DPI also triggered an increase in the mitochondrial ROS levels, caused by the disruption of the mitochondrial respiratory chain. We also demonstrated that DPI blocks mitochondrial oxidative phosphorylation (OxPhos) in a dose-dependent manner, and that AML cells with high OxPhos status are highly sensitive to treatment with DPI, which synergizes with the chemotherapeutic agent cytarabine (Ara-C). Thus, our results suggest that targeting mitochondrial function with DPI might be exploited to target AML cells with high OxPhos status.

218 Knockout p47phox reduces angiotensin ii-induced cardiac oxidative stress and hypertrophy

Oxidative stress due to the activation of a Nox2-containing NADPH oxidase is involved in Angiotensin II (AngII)-induced cardiovascular dysfunction. p47phox is a key regulatory subunit of Nox2. However, the role...

The Pyruvate and α-Ketoglutarate Dehydrogenase Complexes of Pseudomonas aeruginosa Catalyze Pyocyanin and Phenazine-1-carboxylic Acid Reduction via the Subunit Dihydrolipoamide Dehydrogenase

Phenazines are a class of redox-active molecules produced by diverse bacteria and archaea. Many of the biological functions of phenazines, such as mediating signaling, iron acquisition, and redox homeostasis, derive from their redox activity. Although prior studies have focused on extracellular phenazine oxidation by oxygen and iron, here we report a search for reductants and catalysts of intracellular phenazine reduction in Pseudomonas aeruginosa Enzymatic assays in cell-free lysate, together with crude fractionation and chemical inhibition, indicate that P. aeruginosa contains multiple enzymes that catalyze the reduction of the endogenous phenazines pyocyanin and phenazine-1-carboxylic acid in both cytosolic and membrane fractions. We used chemical inhibitors to target general enzyme classes and found that an inhibitor of flavoproteins and heme-containing proteins, diphenyleneiodonium, effectively inhibited phenazine reduction in vitro, suggesting that most phenazine reduction derives from these enzymes. Using natively purified proteins, we demonstrate that the pyruvate and α-ketoglutarate dehydrogenase complexes directly catalyze phenazine reduction with pyruvate or α-ketoglutarate as electron donors. Both complexes transfer electrons to phenazines through the common subunit dihydrolipoamide dehydrogenase, a flavoprotein encoded by the gene lpdG Although we were unable to co-crystallize LpdG with an endogenous phenazine, we report its X-ray crystal structure in the apo-form (refined to 1.35 Å), bound to NAD+ (1.45 Å), and bound to NADH (1.79 Å). In contrast to the notion that phenazines support intracellular redox homeostasis by oxidizing NADH, our work suggests that phenazines may substitute for NAD+ in LpdG and other enzymes, achieving the same end by a different mechanism.

Activation of Signaling Cascades by Weak Extremely Low Frequency Electromagnetic Fields

Background/Aims: Results from recent studies suggest that extremely low frequency magnetic fields (ELF-MF) interfere with intracellular signaling pathways related to proliferative control. The mitogen-activated protein kinases (MAPKs), central signaling components that regulate essentially all stimulated cellular processes, include the extracellular signal-regulated kinases 1/2 (ERK1/2) that are extremely sensitive to extracellular cues. Anti-phospho-ERK antibodies serve as a readout for ERK1/2 activation and are able to detect minute changes in ERK stimulation. The objective of this study was to explore whether activation of ERK1/2 and other signaling cascades can be used as a readout for responses of a variety of cell types, both transformed and non-transformed, to ELF-MF. Methods: We applied ELF-MF at various field strengths and time periods to eight different cell types with an exposure system housed in a tissue culture incubator and followed the phosphorylation of MAPKs and Akt by western blotting. Results: We found that the phosphorylation of ERK1/2 is increased in response to ELF-MF. However, the phosphorylation of ERK1/2 is likely too low to induce ELF-MF-dependent proliferation or oncogenic transformation. The p38 MAPK was very slightly phosphorylated, but JNK or Akt were not. The effect on ERK1/2 was detected for exposures to ELF-MF strengths as low as 0.15 µT and was maximal at ∼10 µT. We also show that ERK1/2 phosphorylation is blocked by the flavoprotein inhibitor diphenyleneiodonium, indicating that the response to ELF-MF may be exerted via NADP oxidase similar to the phosphorylation of ERK1/2 in response to microwave radiation. Conclusions: Our results further indicate that cells are responsive to ELF-MF at field strengths much lower than previously suspected and that the effect may be mediated by NADP oxidase. However, the small increase in ERK1/2 phosphorylation is probably insufficient to affect proliferation and oncogenic transformation. Therefore, the results cannot be regarded as proof of the involvement of ELF-MF in cancer in general or childhood leukemia in particular.

Cloning, Characteristics, and Functional Analysis of Rabbit NADPH Oxidase 5.

Background: Nox5 was the last member of the Nox enzyme family to be identified. Functionally distinct from the other Nox isoforms, our understanding of its physiological significance has been hampered by the absence of Nox5 in mouse and rat genomes. Nox5 is present in the genomes of other species such as the rabbit that have broad utility as models of cardiovascular disease. However, the mRNA sequence, characteristics, and functional analysis of rabbit Nox5 has not been fully defined and were the goals of the current study.Methods: Rabbit Nox5 was amplified from rabbit tissue, cloned, and sequenced. COS-7 cells were employed for expression and functional analysis via Western blotting and measurements of superoxide. We designed and synthesized miRNAs selectively targeting rabbit Nox5. The nucleotide and amino acid sequences of rabbit Nox5 were aligned with those of putative rabbit isoforms (X1, X2, X3, and X4). A phylogenetic tree was generated based on the mRNA sequence for Nox5 from rabbit and other species.Results: Sequence alignment revealed that the identified rabbit Nox5 was highly conserved with the predicted sequence of rabbit Nox5. Cell based experiments reveal that rabbit Nox5 was robustly expressed and produced superoxide at rest and in a calcium and PMA-dependent manner that was susceptible to superoxide dismutase and the flavoprotein inhibitor, DPI. miRNA-1 was shown to be most effective in down-regulating the expression of rabbit Nox5. Phylogenetic analysis revealed a close relationship between rabbit and armadillo Nox5. Rabbit Nox5 was relatively closely related to human Nox5, but lies in a distinct cluster.Conclusion: Our study establishes the suitability of the rabbit as a model organism to further our understanding of the role of Nox5 in cardiovascular and other diseases and provides new information on the genetic relationship of Nox5 genes in different species.

The Characteristics and Regulatory Mechanisms of Superoxide Generation from eNOS Reductase Domain.

In addition to superoxide (O2 .-) generation from nitric oxide synthase (NOS) oxygenase domain, a new O2 .- generation site has been identified in the reductase domain of inducible NOS (iNOS) and neuronal NOS (nNOS). Cysteine S-glutathionylation in eNOS reductase domain also induces O2 .- generation from eNOS reductase domain. However, the characteristics and regulatory mechanism of the O2 .- generation from NOS reductase domain remain unclear. We cloned and purified the wild type bovine eNOS (WT eNOS), a mutant of Serine 1179 replaced with aspartic acid eNOS (S1179D eNOS), which mimics the negative charge caused by phosphorylationand truncated eNOS reductase domain (eNOS RD). Both WT eNOS and S1179D eNOS generated significant amount of O2 .- in the absence of BH4 and L-arginine. The capacity of O2 .- generation from S1179D eNOS was significantly higher than that of WT eNOS (1.74:1). O2 .- generation from both WT eNOS and S1179D eNOS were not completely inhibited by 100nM tetrahydrobiopterin(BH4). This BH4 un-inhibited O2 .- generation from eNOS was blocked by 10mM flavoprotein inhibitor, diphenyleneiodonium (DPI). Purified eNOS reductase domain protein confirmed that this BH4 un-inhibited O2 .- generation originates at the FMN or FAD/NADPH binding site of eNOS reductase domain. DEPMPO-OOH adduct EPR signals and NADPH consumptions analyses showed that O2 .- generation from eNOS reductase domain was regulated by Serine 1179 phosphorylation and DPI, but not by L-arginine, BH4 or calmodulin (CaM). In addition to the heme center of eNOS oxygenase domain, we confirmed another O2 .- generation site in the eNOS reductase domain and characterized its regulatory properties.

Characterization of activity-dependent changes in flavoprotein fluorescence in cerebellar slices from juvenile rats

Flavoprotein autofluorescence signals attributed to neuronal metabolism have been used to assess synaptic function. Here, we characterized flavoprotein autofluorescence responses in the molecular layer of rat cerebellar slices. High frequency stimulation elicited a transient fluorescence increase (peak phase) that was followed by a longer-lasting fluorescence decrease (valley phase). The peak phase was restricted to the molecular layer, whereas the valley phase extended into the Purkinje cell layer and a portion of the granule cell layer. Responses were abolished by either the Na+ channel antagonist, tetrodotoxin, or a combination of the AMPA receptor antagonists, NBQX and GIKI-53655, and were also reduced by a flavoprotein inhibitor (diphenyleneiodonium). These findings are consistent with responses being mediated by an increase in mitochondrial activity triggered by increased energy demands evoked by AMPA receptor-mediated synaptic transmission. The GABAA receptor antagonist picrotoxin did not significantly influence evoked responses. Likewise, exogenous application of ethanol, at concentrations known to increase GABAA receptor-mediated synaptic transmission at Purkinje cells, did not modify peak responses. These observations indicate that flavoprotein autofluorescence imaging could be useful to assess the coupling between glutamatergic synaptic transmission and neuronal metabolism in cerebellar slices.

NO-donating oximes relax corpora cavernosa through mechanisms other than those involved in arterial relaxation.

Erectile dysfunction (ED), as well as many cardiovascular diseases, is associated with impaired nitric oxide (NO) bioavailability. Recently, oxime derivatives have emerged as vasodilators due to their NO-donating capacities. However, whether these oximes offer therapeutic perspectives as an alternative NO delivery strategy for the treatment of ED is unexplored. This study aims to analyze the influence of formaldoxime (FAL), formamidoxime (FAM), and cinnamaldoxime (CAOx) on corporal tension and to elucidate the underlying molecular mechanisms. Organ bath studies were carried out measuring isometric tension on isolated mice corpora cavernosa (CC), thoracic aorta, and femoral artery. After contraction with norepinephrine (NOR), cumulative concentration-response curves of FAL, FAM, and CAOx (100 nmol/L-1 mmol/L) were performed. FAL-/FAM-induced relaxations were evaluated in the absence/presence of various inhibitors of different molecular pathways. FAL, FAM, and CAOx relax isolated CC as well as aorta and femoral artery from mice. ODQ (soluble guanylyl cyclase-inhibitor), diphenyliodonium chloride (nonselective flavoprotein inhibitor), and 7-ethoxyresorufin (inhibitor of CYP450 1A1 and NADPH-dependent reductases) substantially blocked the FAL-/FAM-induced relaxation in the arteries but not in CC. Only a small inhibition of the FAM response in CC was observed with ODQ. This study shows for the first time that NO-donating oximes relax mice CC. Therefore, oximes are a new group of molecules with potential for the treatment of ED. However, the underlying mechanism(s) of the FAL-/FAM-induced corporal relaxation clearly differ(s) from the one(s) involved in arterial vasorelaxation.

Role of Nicotinamide Adenine Dinucleotide Phosphate Oxidase in Mediating Vesicant-Induced Interleukin-6 Secretion in Human Airway Epithelial Cells

Aerosolized exposure to the chemical warfare vesicant sulfur mustard and its analog nitrogen mustard (HN2) is known to induce airway lesions associated with secretion of proinflammatory cytokines such as IL-6. We have shown recently that HN2 challenge induced IL-6 secretion in human airway epithelial cells, a process mediated via epidermal growth factor receptor (EGFR) signaling. In this study, we evaluated the role of redox signaling in regulating HN2-induced, EGFR-mediated IL-6 secretions in primary cultured normal human bronchial epithelial cells (NHBECs) in the air-liquid interface. HN2-induced EGFR phosphorylation and IL-6 secretion in NHBECs were inhibited by the antioxidant N-acetyl-L-cysteine (NAC) and by the flavoprotein inhibitor diphenyleneiodonium chloride (DPI). These observations suggested that the inflammatory response in NHBECs after HN2 challenge was mediated via oxidative stress. HN2 exposure induced increased reactive oxygen species (ROS) formation and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in NHBECs, findings that were inhibited by NAC and DPI treatment. Among NADPH oxidase isoforms, mRNA expression of dual oxidase (DUOX)1 and DUOX2 were up-regulated by HN2. Furthermore, knockdown of DUOX1 or DUOX2 by short hairpin RNA resulted in inhibition of ROS generation, EGFR pathway activation, and IL-6 secretion in NHBECs. These results provide evidence that redox signaling plays a pivotal role in the HN2-induced airway inflammation and underscore the importance of DUOX1 and DUOX2 in vesicant-induced IL-6 secretion in human airway epithelial cells.

NADPH Oxidase 4 Deficiency Reduces Aquaporin-2 mRNA Expression in Cultured Renal Collecting Duct Principal Cells via Increased PDE3 and PDE4 Activity

The final control of renal water reabsorption occurs in the collecting duct (CD) and relies on regulated expression of aquaporin-2 (AQP2) in principal CD cells. AQP2 transcription is primarily induced by type 2 vasopressin receptor (V2R)-cAMP-protein kinase A (PKA) signaling but also by other factors, including TonEBP and NF-κB. NAPDH oxidase 4 (NOX4) represents a major source of reactive oxygen species (ROS) in the kidney. Because NOX-derived ROS may alter PKA, TonEBP and NF-κB activity, we examined the effects of NOX4 depletion on AQP2 expression. Depleted NOX4 expression by siRNA (siNOX4) in mpkCCDcl4 cells attenuated increased AQP2 mRNA expression by arginine vasopressin (AVP) but not by hypertonicity, which induces both TonEBP and NF-κB activity. AVP-induced AQP2 expression was similarly decreased by the flavoprotein inhibitor diphenyleneiodonium. siNOX4 altered neither TonEBP nor NF-κB activity but attenuated AVP-inducible cellular cAMP concentration, PKA activity and CREB phosphorylation as well as AQP2 mRNA expression induced by forskolin, a potent activator of adenylate cyclase. The repressive effect of siNOX4 on AVP-induced AQP2 mRNA expression was abolished by the non-selective phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) and was significantly decreased by selective PDE antagonists cilostamide and rolipram, but not vinpocetine, which respectively target PDE3, PDE4 and PDE1. Thus, by inhibiting PDE3 and PDE4 activity NOX4-derived ROS may contribute to V2R-cAMP-PKA signaling and enhance AQP2 transcription.

Src tyrosine kinase mediates platelet-derived growth factor BB-induced and redox-dependent migration in metanephric mesenchymal cells

The adult kidney is derived from the interaction between the metanephric blastema and the ureteric bud. Platelet-derived growth factor (PDGF) receptor β is essential for the development of the mature glomerular tuft, as mice deficient for this receptor lack mesangial cells. This study investigated the role of Src tyrosine kinase in PDGF-mediated reactive oxygen species (ROS) generation and migration of metanephric mesenchymal cells (MMCs). Cultured embryonic MMCs from wild-type and PDGF receptor-deficient embryos were established. Migration was determined via wound-healing assay. Unlike PDGF AA, PDGF BB-induced greater migration in MMCs with respect to control. This was abrogated by neutralizing an antibody to PDGF BB. Phosphatidylinositol 3-kinase (PI3K) inhibitors suppressed PDGF BB-induced migration. Conversely, mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) inhibitors had no effect. Src inhibitors inhibited PDGF-induced cell migration, PI3K activity, and Akt phosphorylation. Adenoviral dominant negative Src (AD DN Src) abrogated PDGF BB-induced Akt phosphorylation. Hydrogen peroxide stimulated cell migration. PDGF BB-induced wound closure was inhibited by the antioxidants N-acetyl-l-cysteine, tiron, and the flavoprotein inhibitor diphenyleneiodonium. These cells express the NADPH oxidase homolog Nox4. Inhibiting Nox4 with antisense oligonucleotides or small interfering RNA (siRNA) suppressed PDGF-induced wound closure. Inhibition of Src with siRNA reduced PDGF BB-induced ROS generation as assessed by 2',7'-dichlorodihydrofluorescein diacetate fluorescence. Furthermore, PDGF BB-stimulated ROS generation and migration were similarly suppressed by Ad DN Src. In MMCs, PDGF BB-induced migration is mediated by PI3K and Src in a redox-dependent manner involving Nox4. Src may be upstream to PI3K and Nox4.

Potent inhibition of nitroglycerin bioactivation by diphenyleneiodonium (DIP)

Background Aldehyde dehydrogenase-2 (ALDH2) catalyzes vascular bioactivation of the antianginal drug nitroglycerin (GTN) to yield nitric oxide (NO) or a related species that activates soluble guanylate cyclase (sGC), resulting in cGMPmediated vasodilation [1]. Accordingly, established ALDH2 inhibitors attenuate GTN-induced vasorelaxation in vitro and in vivo. However, the ALDH2 hypothesis has not been reconciled with early studies demonstrating potent inhibition of the GTN response by diphenyleneiodonium (DPI) [2], a widely used inhibitor of flavoproteins, in particular NADPH oxidases. We addressed this issue and investigated the effects of DPI on GTN-induced relaxation of rat aortic rings and the function of purified ALDH2.

Potent Inhibition of Aldehyde Dehydrogenase-2 by Diphenyleneiodonium: Focus on Nitroglycerin Bioactivation

Aldehyde dehydrogenase-2 (ALDH2) catalyzes vascular bioactivation of the antianginal drug nitroglycerin (GTN) to yield nitric oxide (NO) or a related species that activates soluble guanylate cyclase (sGC), resulting in cGMP-mediated vasodilation. Accordingly, established ALDH2 inhibitors attenuate GTN-induced vasorelaxation in vitro and in vivo. However, the ALDH2 hypothesis has not been reconciled with early studies demonstrating potent inhibition of the GTN response by diphenyleneiodonium (DPI), a widely used inhibitor of flavoproteins, in particular NADPH oxidases. We addressed this issue and investigated the effects of DPI on GTN-induced relaxation of rat aortic rings and the function of purified ALDH2. DPI (0.3 µM) inhibited the high affinity component of aortic relaxation to GTN without affecting the response to NO, indicating that the drug interfered with GTN bioactivation. Denitration and bioactivation of 1-2 µM GTN, assayed as 1,2-glycerol dinitrate formation and activation of purified sGC, respectively, were inhibited by DPI with a half-maximally active concentration of about 0.2 µM in a GTN-competitive manner. Molecular modeling indicated that DPI binds to the catalytic site of ALDH2, and this was confirmed by experiments showing substrate-competitive inhibition of the dehydrogenase and esterase activities of the enzyme. Our data identify ALDH2 as highly sensitive target of DPI and explain inhibition of GTN-induced relaxation by this drug observed previously. In addition, the data provide new evidence for the essential role of ALDH2 in GTN bioactivation and may have implications to other fields of ALDH2 research, such as hepatic ethanol metabolism and cardiac ischemia/reperfusion injury.

Effects of iodonium-class flavin dehydrogenase inhibitors on growth, reactive oxygen production, cell cycle progression, NADPH oxidase 1 levels, and gene expression in human colon cancer cells and xenografts

Iodonium-class flavoprotein dehydrogenase inhibitors have been demonstrated to possess antiproliferative potential and to inhibit reactive oxygen production in human tumor cells, although the mechanism(s) that explains the relationship between altered cell growth and the generation of reactive oxygen species (ROS) remains an area of active investigation. Because of the ability of these compounds to inhibit the activity of flavoprotein-containing epithelial NADPH oxidases, we chose to examine the effects of several iodonium-class flavoprotein inhibitors on human colon cancer cell lines that express high, functional levels of a single such oxidase (NADPH oxidase 1, or Nox1). We found that diphenyleneiodonium (DPI), di-2-thienyliodonium (DTI), and iodonium diphenyl inhibited the growth of Caco2, HT-29, and LS-174T colon cancer cells at concentrations (10–250nM for DPI, 0.5–2.5μM for DTI, and 155nM to 10μM for iodonium diphenyl) substantially lower than needed for DU145 human prostate cancer cells, which do not possess functional NADPH oxidase activity. Drug treatment was associated with decreased H2O2 production and diminished intracellular ROS levels, lasting up to 24h, after short-term (1-h) exposure to the iodonium analogs. Decreased tumor cell proliferation was caused, in part, by a profound block in cell cycle progression at the G1/S interface in both LS-174T and HT-29 cells exposed to either DPI or DTI; and the G1 block was produced, for LS-174T cells, by upregulation of p27 and a drug concentration-related decrease in the expression of cyclins D1, A, and E that was partially prevented by exogenous H2O2. Not only did DPI and DTI decrease intracellular ROS, they both also significantly decreased the mRNA expression levels of Nox1, potentially contributing to the prolonged reduction in tumor cell reactive oxygen levels. We also found that DPI and DTI significantly decreased the growth of both HT-29 and LS-174T human tumor xenografts, at dose levels that produced peak plasma concentrations similar to those utilized for our in vitro experiments. These findings suggest that iodonium analogs have therapeutic potential for NADPH oxidase-containing human colon cancers in vivo and that at least part of their antineoplastic mechanism of action may be related to targeting Nox1.

Endothelial Nitric Oxide Synthase-Independent Release of Nitric Oxide in the Aorta of the Spontaneously Hypertensive Rat

In the aorta of male spontaneously hypertensive rats (SHR), but not in that of normotensive Wistar-Kyoto rats (WKY), contractions to phenylephrine obtained in the presence of L-NAME [inhibitor of nitric oxide synthase (NOS)] and indomethacin (inhibitor of cyclooxygenase) are inhibited by an unknown endothelium-derived factor. The present study aimed to identify the mechanism underlying this endothelium-dependent inhibition in the SHR aorta. Aortic rings of male SHR and WKY, with and without endothelium, were suspended in organ chambers in the presence of indomethacin and L-NAME for the measurement of isometric tension. Contractions to phenylephrine were smaller in SHR aortae with endothelium than in those without, but were similar in the two types of preparations of WKY aortae. The endothelium-dependent, NOS-independent inhibition of phenylephrine-induced contraction was abolished by oxyhemoglobin [extracellular NO scavenger], carboxy-PTIO (NO scavenger) and ODQ (inhibitor of soluble guanylyl cyclase). It was unmasked not only by indomethacin but also by apocynin (antioxidant), but inhibited by diphenyleneiodonium (inhibitor of flavoproteins including cytochrome P450 reductase). The cytochrome P450 reductase protein expression was similar in SHR and WKY aortae. However, the level of nitrate and nitrite, substrates of cytochrome P450 reductase, were higher in SHR than WKY plasma and aortae. Therefore, in SHR but not WKY aortae, eNOS-independent NO is formed by cytochrome P450 reductase.

H2O2 Production Downstream of FLT3 Is Mediated by p22phox in the Endoplasmic Reticulum and Is Required for STAT5 Signalling

The internal tandem duplication (ITD) of the juxtamembrane region of the FLT3 receptor has been associated with increased reactive oxygen species (ROS) generation in acute myeloid leukemia (AML). How this elevated level of ROS contributes to the leukemic phenotype, however, remains poorly understood. In this work we show that ROS in the FLT3-ITD expressing AML cell line MV4-11 is reduced by treatment with PKC412, an inhibitor of FLT3, DPI, a flavoprotein inhibitor, and VAS2870, a Nox specific inhibitor, suggesting that ROS production is both FLT3 and NADPH oxidase dependent. The majority of these ROS co-localize to the endoplasmic reticulum (ER), as determined with the H2O2-specific aryl-boronate dye Peroxyorange 1, which also corresponds to co-localization of p22phox. Moreover, knocking down p22phox dramatically reduces H2O2 after 24 hours in the ER, without affecting mitochondrial ROS. Significantly, the FLT3 inhibitor PKC412 reduces H2O2 in FLT3-ITD expressing cell lines (MV4-11, MOLM-13) through reduction of p22phox over 24 hours. Reduced p22phox is achieved by proteasomal degradation and is prevented upon GSK3-β inhibition. Knockdown of p22phox resulted in reduced STAT5 signalling and reduced Pim-1 levels in the cells after 24 hours. Thus, we have shown that FLT3 driven H2O2 production in AML cells is mediated by p22phox and is critical for STAT5 signalling.

Abstract 2060: NADPH oxidase NOX5 regulates the expression of the cell cycle inhibitor p27kip1 in the human melanoma UACC-257 cancer cells

Abstract NADPH oxidase (NOX) family enzymes (NOX1-5 and DUOX1-2) generate reactive oxygen species (ROS) and modulate cellular pathways that regulate proliferation. While high doses of ROS are clearly toxic, the effects of low doses of NOX-mediated ROS as signaling molecules are less clear. Recent work has proposed that NOX5 plays a role in cell proliferation in Hairy cell leukemia, Barrett's esophageal adenocarcinoma and in prostate cancer cells, yet relatively little is known of the signaling pathways mediated by NOX5. In this study we explored the functional role of NOX5 in cancer cells obtained from the NCI-60 cell line panel. Initial screening of the NCI-60 cancer cell line panel for expression of the Nox family members by real time PCR revealed several melanoma cell lines that express Nox5. Of the NOX5 expressing NCI-60 human cancer cell lines, subsequent studies were carried out in human UACC-257 melanoma cancer cells. Two molecular approaches were undertaken to determine the functional role of NOX5 in UACC-257 cells: 1) generating NOX5 over-expressing stable clones and 2) transiently silencing endogenous NOX5 and the stably over-expressed NOX5. Redox-sensitive assays for both intra- and extracellular ROS production confirmed that NOX5 is functional and generates superoxide in both UACC-257 cells and stable Nox5-over-expressing clones. NOX5 can be activated both by the protein kinase C activating phorbol ester PMA and by the Ca2+ ionophore ionomycin. Furthermore, this enzymatic activity is calcium- and flavin dehydrogenase-dependent; both the calcium chelator BAPTA-AM and the flavoprotein inhibitor DPI (200nM) significantly decreased NOX5-mediated ROS production. Silencing NOX5 over-expression significantly inhibited both endogenous and PMA-stimulated ROS production, suggesting that the ROS production is NOX5-mediated. Furthermore, knockdown of both endogenous and over-expressed NOX5 in UACC-257 cells resulted in increased p27Kip1 expression. Conversely, over-expression of NOX5 in UACC-257 cells resulted in decreased p27Kip1 expression both at the mRNA and protein levels. As the AKT signaling pathways are known to modulate p27Kip1 levels, we observed that knockdown of both endogenous and over-expressed NOX5 in UACC-257 cells resulted in decreased Akt and GSK-3β phosphorylation. Studies to characterize additional mechanisms of translational and transcriptional regulation of p27Kip1 by NOX5 are ongoing. In summary, our findings suggest that NOX5 expression could contribute to cell proliferation in human cancers in part due to the generation of high local concentrations of extracellular ROS that regulate the Akt/GSK-3β/p27Kip1 signaling network. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2060. doi:1538-7445.AM2012-2060

Nrf3-Pla2g7 Interaction Plays an Essential Role in Smooth Muscle Differentiation From Stem Cells

Phospholipase A2, group 7 (Pla2g7) is an important mediator in cardiovascular development and diseases because of its divergent physiological and pathological functions in inflammation and oxidative stress. However, little is known about the functional role of Pla2g7 in smooth muscle cell (SMC) differentiation from stem cells. In the present study, embryonic stem cells were cultivated on collagen IV-coated plates to allow SMC differentiation. Pla2g7 gene expression and activity were upregulated significantly following 4 to 14 days of cell differentiation and colocalized with SMC differentiation markers in the differentiated SMCs. Knockdown of Pla2g7 resulted in downregulation of smooth muscle-specific markers in vitro and impairment of SMC differentiation in vivo, whereas enforced expression of Pla2g7 enhanced SMC differentiation and increased reactive oxygen species generation. Importantly, enforced expression of Pla2g7 significantly increased the binding of serum response factor to SMC differentiation gene promoters, resulting in SMC differentiation, which was abolished by free radical scavenger and flavoprotein inhibitor of NADPH oxidase but not hydrogen peroxide inhibitor. Moreover, we demonstrated that nuclear factor erythroid 2-related factor 3 (Nrf3) regulates Pla2g7 gene expression through direct binding to the promoter regions of Pla2g7 gene. Our findings demonstrated that Pla2g7 plays a crucial physiological role in SMC differentiation from stem cells, and the fine interactions between Nrf3 and Pla2g7 are essential for SMC differentiation.