Dehydroepiandrosterone-α-2-Deoxyglucoside Exhibits Enhanced Anticancer Effects in MCF-7 Breast Cancer Cells and Inhibits Glucose-6-Phosphate Dehydrogenase Activity.

In the pentose phosphate pathway, dehydroepiandrosterone (DHEA) uncompetitively inhibits glucose-6-phosphate dehydrogenase (G6PD), reducing NADPH production and increasing oxidative stress, which can influence the onset and/or progression of several diseases, including cancer. 2-Deoxy-D-glucose (2-DG), a glucose mimetic, competes with glucose for cellular uptake, inhibiting glycolysis and competing with glucose-6-phosphate (G-6-P) for G6PD activity. In this study, we report that DHEA-α-2-DG (5), an α-covalent conjugate of DHEA and 2-DG, exhibits better anticancer activity than DHEA, 2-DG, DHEA +2-DG, and polydatin in MCF-7 cells, and reduces NADPH/NADP+ ratio in cellular assays. In vitro enzyme kinetics and molecular docking studies showed that 5 uncompetitively inhibits human G6PD activity and binds to the structural NADP+ site but not to the catalytic NADP+ site. Further combining 5 with the FDA-approved drug tamoxifen enhanced its cytotoxicity against MCF-7 cells, suggesting that it could serve as a candidate for combination of drug strategies.

3-Nitropropionic Acid Enhances Ferroptotic Cell Death via NOX2-Mediated ROS Generation in STHdhQ111 Striatal Cells Carrying Mutant Huntingtin.

Huntington's disease (HD) is a hereditary neurodegenerative disease that involves an expansion of the CAG repeats of the Huntingtin (HTT) gene, but the disease onset and progression do not necessarily correspond to the extent of CAG repeats. Decreased mitochondrial complex II activity has also been reported to be closely associated with disease pathogenesis. Here, we examined the mechanism of cell death induced by 3-nitropropionic acid (3-NP), a mitochondrial complex II inhibitor, using striatal cells (STHdhQ111 cells) derived from HD model mice with mutant HTT carrying the CAG repeat extended. Treatment with 3-NP (5 mM) enhanced cell death in STHdhQ111 compared to STHdhQ7 cells with normal HTT. Ferrostatin-1, an inhibitor of ferroptosis, and deferoxamine, an iron chelator, markedly inhibited 3-NP-induced cell death in both the STHdh cell lines. On the other hands, cell death was not abrogated by a broad-spectrum caspase inhibitor, Z-VAD-FMK, indicating that this cell death was caspase-independent. Cell death caused by 3-NP is suggested to be due to ferroptosis. Furthermore, 3-NP-induced cell death was markedly inhibited by GSK2795039, a reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) inhibitor, suggesting that cell death is mainly mediated by intracellular superoxide anion (O2-) production through NOX2. Furthermore, a mitochondria-targeted superoxide dismutase mimetic (Mito-TEMPO), partially inhibited 3-NP-induced cell death, suggesting that O2- production in the mitochondria is partially responsible for cell death. These results indicate that 3-NP-induced cell death in the STHdhQ111 cells is caspase-independent, non-apoptotic, and that ferroptotic cell death is mainly induced via NOX2 activation.

Microbial redox coenzyme engineering and applications in biosynthesis.

Many biosynthetic processes, either in vivo or in vitro, involve redox reactions catalyzed by oxidoreductases - which depend on coenzymes as electron carriers. Redox balance is regulated mainly by coenzymes NAD(P)+ and NAD(P)H and is essential for biosynthesis. New techniques for the regulation and regeneration of coenzymes have recently advanced our understanding of, and demonstrated promising applications in, synthetic biology.

Polyoxypregnanes as safe, potent, and specific ABCB1-inhibitory pro-drugs to overcome multidrug resistance in cancer chemotherapy in vitro and in vivo.

Multidrug resistance (MDR) mediated by ATP binding cassette subfamily B member 1 (ABCB1) is significantly hindering effective cancer chemotherapy. However, currently, no ABCB1-inhibitory drugs have been approved to treat MDR cancer clinically, mainly due to the inhibitor specificity, toxicity, and drug interactions. Here, we reported that three polyoxypregnanes (POPs) as the most abundant constituents of Marsdenia tenacissima (M. tenacissima) were novel ABCB1-modulatory pro-drugs, which underwent intestinal microbiota-mediated biotransformation in vivo to generate active metabolites. The metabolites at non-toxic concentrations restored chemosensitivity in ABCB1-overexpressing cancer cells via inhibiting ABCB1 efflux activity without changing ABCB1 protein expression, which were further identified as specific non-competitive inhibitors of ABCB1 showing multiple binding sites within ABCB1 drug cavity. These POPs did not exhibit ABCB1/drug metabolizing enzymes interplay, and their repeated administration generated predictable pharmacokinetic interaction with paclitaxel without obvious toxicity in vivo. We further showed that these POPs enhanced the accumulation of paclitaxel in tumors and overcame ABCB1-mediated chemoresistance. The results suggested that these POPs had the potential to be developed as safe, potent, and specific pro-drugs to reverse ABCB1-mediated MDR. Our work also provided scientific evidence for the use of M. tenacissima in combinational chemotherapy.

Astragaloside IV Ameliorates Cognitive Impairment and Neuroinflammation in an Oligomeric Aß Induced Alzheimer's Disease Mouse Model via Inhibition of Microglial Activation and NADPH Oxidase Expression.

Microglial activation and neuroinflammation induced by amyloid ß (Aß) play pivotal roles in Alzheimer's disease (AD) pathogenesis. Astragaloside IV (AS-IV) is one of the major active compounds of the traditional Chinese medicine Astmgali Radix. It has been reported that AS-IV could protect against Aß-induced neuroinflammation and cognitive impairment, but the underlying mechanisms need to be further clarified. In this study, the therapeutic effects of AS-IV were investigated in an oligomeric Aß (oAß) induced AD mice model. The effects of AS-IV on microglial activation, neuronal damage and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase expression were further studied. Different doses of AS-IV were administered intragastrically once a day after intracerebroventricularly oAß injection. Results of behavioral experiments including novel object recognition (NOR) test and Morris water maze (MWM) test revealed that AS-IV administration could significantly ameliorate oAß-induced cognitive impairment in a dose dependent manner. Enzyme linked immunosorbent assay (ELISA) results showed that increased levels of reactive oxygen species (ROS), tumor necrosis factor α (TNF-α), interleukin-1ß (IL-1ß) and IL-6 in hippocampal tissues induced by oAß injection were remarkably inhibited after AS-IV treatment. OAß induced microglial activation and neuronal damage was significantly suppressed in AS-IV-treated mice brain, observed in immunohistochemistry results. Furthermore, oAß upregulated protein expression of NADPH oxidase subunits gp91phox, p47phox, p22phox and p67phox were remarkably reduced by AS-IV in Western blotting assay. These results revealed that AS-IV could ameliorate oAß-induced cognitive impairment, neuroinflammation and neuronal damage, which were possibly mediated by inhibition of microglial activation and down-regulation of NADPH oxidase protein expression. Our findings provide new insights of AS-IV for the treatment of neuroinflammation related diseases such as AD.

Protein Kinases Signaling in Pancreatic Beta-cells Death and Type 2 Diabetes.

Type 2 diabetes (T2D) is a worldwide serious public health problem. Insulin resistance and ß-cell failure are the two major components of T2D pathology. In addition to defective endoplasmic reticulum (ER) stress signaling due to glucolipotoxicity, ß-cell dysfunction or ß-cell death initiates the deleterious vicious cycle observed in T2D. Although the primary cause is still unknown, overnutrition that contributes to the induction of the state of low-grade inflammation, and the activation of various protein kinases-related metabolic pathways are main factors leading to T2D. In this chapter following subjects, which have critical checkpoints regarding ß-cell fate and protein kinases pathways are discussed; hyperglycemia-induced ß-cell failure, chronic accumulation of unfolded protein in ß-cells, the effect of intracellular reactive oxygen species (ROS) signaling to insulin secretion, excessive saturated free fatty acid-induced ß-cell apoptosis, mitophagy dysfunction, proinflammatory responses and insulin resistance, and the reprogramming of ß-cell for differentiation or dedifferentiation in T2D. There is much debate about selecting proposed therapeutic strategies to maintain or enhance optimal ß-cell viability for adequate insulin secretion in T2D. However, in order to achieve an effective solution in the treatment of T2D, more intensive clinical trials are required on newer therapeutic options based on protein kinases signaling pathways.

N-Methyl-D-Aspartate Receptor Signaling-Protein Kinases Crosstalk in Cerebral Ischemia.

Although stroke is very often the cause of death worldwide, the burden of ischemic and hemorrhagic stroke varies between regions and over time regarding differences in prognosis, prevalence of risk factors, and treatment strategies. Excitotoxicity, oxidative stress, dysfunction of the blood-brain barrier, neuroinflammation, and lysosomal membrane permeabilization, sequentially lead to the progressive death of neurons. In this process, protein kinases-related checkpoints tightly regulate N-methyl-D-aspartate (NMDA) receptor signaling pathways. One of the major hallmarks of cerebral ischemia is excitotoxicity, characterized by overactivation of glutamate receptors leading to intracellular Ca2+ overload and ultimately neuronal death. Thus, reduced expression of postsynaptic density-95 protein and increased protein S-nitrosylation in neurons is responsible for neuronal vulnerability in cerebral ischemia. In this chapter death-associated protein kinases, cyclin-dependent kinase 5, endoplasmic reticulum stress-induced protein kinases, hyperhomocysteinemia-related NMDA receptor overactivation, ephrin-B-dependent amplification of NMDA-evoked neuronal excitotoxicity and lysosomocentric hypothesis have been discussed.Consequently, ample evidences have demonstrated that enhancing extrasynaptic NMDA receptor activity triggers cell death after stroke. In this context, considering the dual roles of NMDA receptors in both promoting neuronal survival and mediating neuronal damage, selective augmentation of NR2A-containing NMDA receptor activation in the presence of NR2B antagonist may constitute a promising therapy for stroke.

Alcoholic liver disease-from steatosis to cirrhosis - a biochemistry approach.

Nowadays, chronic alcoholism and its health implications represent a global concern. Over three million deaths are linked to chronic alcohol intake every year. This article aims to spread awareness about the negative impact ethanol can have on almost every organ in the body, especially the liver. Understanding ethanol metabolism and the cellular pathways through which alcohol increases liver oxidative stress may prevent a broad spectrum of hepatic lesions such as steatosis, steatohepatitis, and, ultimately, cirrhosis. After a short review of ethanol metabolism and liver oxidative stress, each hepatic lesion will be individually discussed regarding the mechanism of apparition, treatment, and future targeted therapies.

Regulation of Antioxidant Stress-Responsive Transcription Factor Nrf2 Target Gene in the Reduction of Radiation Damage by the Thrombocytopenia Drug Romiplostim.

Ionizing radiation induces severe oxidative stress, resulting in individual death by acute radiation syndrome. The nuclear factor-erythroid-2-related factor 2 (Nrf2) plays an important role in the antioxidant response pathway. Recently, romiplostim (RP), an idiopathic thrombocytopenic purpura therapeutic drug, was reported to completely rescue mice exposed to lethal total-body irradiation (TBI). However, the details underlying the mechanism for reducing radiation damage remain largely unknown. To elucidate the involvement of the master redox regulator Nrf2 in the radio-mitigative efficacy of RP on TBI-induced oxidative stress, expression of Nrf2 target genes in hematopoietic tissues such as bone marrow, spleen, and lung from mice treated with RP for three consecutive days after 7 Gy of X-ray TBI was analyzed. RP promoted the recovery of bone marrow cells from day 10 and the significant up-regulation of reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) dehydrogenase quinone 1 (Nqo1), glutamate-cysteine ligase catalytic subunit (Gclc) and glutamate-cysteine ligase modifier subunit (Gclm) was observed compared to the TBI mice. RP also promoted the recovery of splenic cells on day 18, and the significant up-regulation of Nqo1, Gclc and Gclm in spleen both on day 10 and 18 and Nqo1 and Gclm in lung on day 10 was observed compared to the TBI mice. The present study suggests that the radio-mitigative effects of RP indicates on the activation of Nrf2 target genes involved in redox regulation and the antioxidative function, especially Nqo1, Gclc and Gclm. It is indicating the importance of these genes in the maintenance of biological homeostasis in response to radiation-induced oxidative stress.

Active fractions of mannoproteins derived from yeast cell wall stimulate innate and acquired immunity of adult and elderly dogs.

Nutritional intervention in older dogs aims to increase lifespan and improve life quality as well as delay the development of diseases related to ageing. It is believed that active fractions of mannoproteins (AFMs) obtained through extraction and fractionation of yeast cell walls (Saccharomyces cerevisiae) may beneficially modulate the immune system. However, studies that have evaluated this component and the effects of ageing on the immune system of dogs are scarce. This study aimed to evaluate the immunological effects of AFMs in adult and elderly dogs. Three extruded iso-nutrient experimental diets were formulated: without addition of AFM (T0); with AFM at 400 mg/kg (T400); and with AFM at 800 mg/kg (T800). Thirty-six beagle dogs were used, and six experimental treatments, resulting in combinations of age (adult and elderly) and diet (T0, T400, and T800), were evaluated. On days zero, 14, and 28, blood samples were obtained for leucocyte phenotyping and phagocytosis assays. On days zero and 28, a lymphoproliferation test, quantification of reactive oxygen (H2O2) and nitrogen (NO) intermediate production, evaluation of faecal immunoglobulin A (IgA) content, and a delayed cutaneous hypersensitivity test (DCHT) were performed. Statistical analyses were performed with SAS software. Repeated measure variance analyses were performed, and means were compared by the Tukey test. Values of P ≤ 0.05 were considered significant, and values of P ≤ 0.10 were considered tendencies. Dogs fed T400 tended to have higher neutrophilic phagocytic activity than dogs fed T800 (P = 0.073). Regarding reactive oxygen intermediates, bacterial lipopolysaccharide (LPS)-stimulated neutrophils from animals that were fed T400 had a tendency to produce more H2O2 than those from animals fed the control diet (P = 0.093). Elderly dogs, when compared to adult dogs, had lower absolute T and B lymphocyte counts, lower auxiliary T lymphocyte counts, and higher cytotoxic T lymphocyte counts (P < 0.05). A significant effect of diet, age, and time with saline inoculation was noted for the DCHT. There was no effect of diet or age on faecal IgA content in dogs. This study suggests beneficial effects of mannoproteins on the specific and nonspecific immune responses in adult and elderly dogs.

Celastrol Alleviates Aortic Valve Calcification Via Inhibition of NADPH Oxidase 2 in Valvular Interstitial Cells.

This study sought to investigate whether reactive oxygen species (ROS)-generating reduced nicotinamide adenine dinucleotide phosphate oxidase 2 (Nox2) contributes to calcific aortic valve disease (CAVD) or whether celastrol, a natural Nox2 inhibitor, may provide potential therapeutic target for CAVD. CAVD is an active and cellular-driven fibrocalcific process characterized by differentiation of aortic valvular interstitial cells (AVICs) toward an osteogenic-like phenotype. ROS levels increase in calcified aortic valves, while the sources of ROS and their roles in the pathogenesis of CAVD are elusive. The roles of Nox2 and the effects of celastrol were studied using cultured porcine AVICs in vitro and a rabbit CAVD model in vivo. Nox2 proteins were significantly upregulated in human aortic valves with CAVD. In vitro, Nox2 was markedly induced upon stimulation of AVICs with osteogenic medium, along with the increases in ROS production and calcium nodule formation. Celastrol significantly decreased calcium deposition of AVICs by 35%, with a reduction of ROS generation. Knockdown of endogenous Nox2 substantially suppressed AVIC calcification by 39%, the inhibitory effect being similar to celastrol treatment. Mechanistically, either celastrol treatment or knockdown of Nox2 significantly inhibited glycogen synthase kinase 3 beta/ß-catenin signaling, leading to attenuation of fibrogenic and osteogenic responses of AVICs. In a rabbit CAVD model, administration of celastrol significantly reduced aortic valve ROS production, fibrosis, calcification, and severity of aortic stenosis, with less left ventricular dilatation and better preserved contractile function. Upregulation of Nox2 is critically involved in CAVD. Celastrol is effective to alleviate CAVD, likely through the inhibition of Nox2-mediated glycogen synthase kinase 3 beta/ß-catenin pathway in AVICs.

Transfer of glucose hydrogens via acetyl-CoA, malonyl-CoA, and NADPH to fatty acids during de novo lipogenesis.

Deuterated water (2H2O) is widely used for measuring de novo lipogenesis (DNL). 2H is incorporated into fatty acids via exchange between body water and the hydrogens of acetyl-CoA, malonyl-CoA, and NADPH. Previous studies concluded that these exchanges are incomplete; therefore, fatty acid 2H enrichment requires correcting. In mice, we measured the 2H enrichment of fatty acid positions 2 and 3 and methyl hydrogens from [U-2H7]glucose to determine 2H transfer from glucose to fatty acid via malonyl-CoA, NADPH, and acetyl-CoA, respectively. Positional fatty acid 2H enrichments were compared with 13C enrichment of the same sites from an equivalent amount of [U-13C6]glucose provided alongside the [U-2H7]glucose tracer. Transfer of glucose 2H to fatty acid position 2 and methyl sites was low (2H enrichment of 0.06 ± 0.01 and 0.14 ± 0.01 relative to 13C) indicating extensive exchange at both malonyl- and acetyl-CoA, respectively. Transfer of glucose 2H into fatty acid position 3 was more extensive (0.46 ± 0.04 relative to 13C, P < 10-5 vs. position 2), indicating a more limited exchange of those glucose hydrogens that were transferred via NADPH. However, mice provided with [U-13C6]glucose and 2H2O had equivalent 2H enrichments of fatty acid positions 2 and 3, suggesting that in this setting, NADPH and body water 2H had exchanged extensively. This is explained by contributions of substrates other than exogenous glucose to DNL coupled with their extensive 2H enrichment from 2H2O prior to DNL. Under such conditions, 2H enrichment of fatty acids from 2H2O does not need correction.

NOX1 Negatively Modulates Fibulin-5 in Vascular Smooth Muscle Cells to Affect Aortic Dissection.

Aortic dissection (AD) diseases are characterized by degeneration of the aortic media. Oxidative stress plays a crucial role in the development of AD. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (NOX1) deficiency reduces the incidence of aortic dissection induced by angiotensin II, but its mechanism remains to be further elucidated. The expression of Fibulin-5 is decreased in patients with AD, but its upstream mechanism is still unclear. This study was to clarify the relationship between NOX1 and Fibulin-5 in the AD. Results showed that the expressions of NOX1 and Fibulin-5 were increased and decreased in the AD, respectively. Next, by employing gain- and loss-of-function approaches in vitro, NOX1 negatively regulated Fibulin-5 in the vascular smooth muscle cells. Moreover, the blunted activity of NOX1 with VAS2870 could upregulate the expression of Fibulin-5. These findings indicate NOX1 is a negative modulator of Fibulin-5 in the AD.

The protective effect of puerarin on angiotensin II-induced aortic aneurysm formation by the inhibition of NADPH oxidase activation and oxidative stress-triggered AP-1 signaling pathways.

Puerarin, an active ingredient of Pueraria lobata, has a range of pharmacological effects and excellent pharmacodynamic properties. In the present study, the effect of puerarin on angiotensin II-induced aortic aneurysm formation and the potential underlying molecular mechanisms were examined. The results revealed that puerarin significantly suppressed the viability, and induced the apoptosis, of aneurysm-inducing cells in a time- and dose-dependent manner. Furthermore, treatment with puerarin significantly suppressed the production of reactive oxygen species (ROS) and the expression of matrix metalloproteinase-2 (MMP-2) protein in aneurysm cells. Puerarin treatment significantly increased caspase-9 and -3 activity, induced the protein expression of phosphorylated (p)-Jun and inhibited the protein expression of activator protein 1 (AP-1) in aneurysm cells. It was also demonstrated that Puerarin significantly suppressed the reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase activity in aneurysm cells. Therefore, it was demonstrated that puerarin on suppressed the cell growth of angiotensin II-induced aortic aneurysm formation by affecting the rate of apoptosis, the generation of ROS, MMP-2, AP-1 and p-Jun protein expression and NADPH oxidase.

Chlorophytum comosum-bacteria interactions for airborne benzene remediation: Effect of native endophytic Enterobacter sp. EN2 inoculation and blue-red LED light.

This study was performed to determine the effect of plant-endophytic Enterobacter sp. EN2 interactions and blue-red LED light conditions on gaseous benzene removal by plants. It was found that under consecutive benzene fumigation for three cycles (18 days), inoculation of the strain EN2 into sterilized and non-sterilized native C. comosum resulted in significantly increased gaseous benzene removal compared to that in non-inoculated groups under the same light conditions (P < 0.05). Remarkably, EN2 colonization in inoculated plants under LED conditions was higher than under fluorescence conditions as the EN2 could grow better under LED conditions. Strain EN2 possesses NADPH that is used to facilitate benzene degradation and modulate plant growth under benzene stress by bacterial IAA production and ACC deaminase activity; higher IAA and lower ethylene levels were found in inoculated plants compared to non-inoculated ones. These contributed to better benzene removal efficiency. Interestingly, under fumigation for 16 cycles (67 days), there was no difference in gaseous benzene removal between inoculated plants and non-inoculated plants under the same light conditions at initial benzene concentrations of 5 ppm. This is probably due to EN2 reaching maximum growth under all treatments. However, C. comosum exhibited better benzene removal under LED conditions than under fluorescence conditions during 16 cycles, possibly due to better photosynthetic performance and plant growth, leading to more NADPH, and eventually enhanced benzene removal efficiency. Hence, the most efficient acceleration of benzene removal was provided by inoculation of strain EN2 onto C. comosum under blue-red LED light conditions.

Organic Solute Transporter α-ß Protects Ileal Enterocytes From Bile Acid-Induced Injury.

BACKGROUND & AIMS: Ileal bile acid absorption is mediated by uptake via the apical sodium-dependent bile acid transporter (ASBT), and export via the basolateral heteromeric organic solute transporter α-ß (OSTα-OSTß). In this study, we investigated the cytotoxic effects of enterocyte bile acid stasis in Ostα-/- mice, including the temporal relationship between intestinal injury and initiation of the enterohepatic circulation of bile acids. METHODS: Ileal tissue morphometry, histology, markers of cell proliferation, gene, and protein expression were analyzed in male and female wild-type and Ostα-/- mice at postnatal days 5, 10, 15, 20, and 30. Ostα-/-Asbt-/- mice were generated and analyzed. Bile acid activation of intestinal Nrf2-activated pathways was investigated in Drosophila. RESULTS: As early as day 5, Ostα-/- mice showed significantly increased ileal weight per length, decreased villus height, and increased epithelial cell proliferation. This correlated with premature expression of the Asbt and induction of bile acid-activated farnesoid X receptor target genes in neonatal Ostα-/- mice. Expression of reduced nicotinamide adenine dinucleotide phosphate oxidase-1 and Nrf2-anti-oxidant responsive genes were increased significantly in neonatal Ostα-/- mice at these postnatal time points. Bile acids also activated Nrf2 in Drosophila enterocytes and enterocyte-specific knockdown of Nrf2 increased sensitivity of flies to bile acid-induced toxicity. Inactivation of the Asbt prevented the changes in ileal morphology and induction of anti-oxidant response genes in Ostα-/- mice. CONCLUSIONS: Early in postnatal development, loss of Ostα leads to bile acid accumulation, oxidative stress, and a restitution response in ileum. In addition to its essential role in maintaining bile acid homeostasis, Ostα-Ostß functions to protect the ileal epithelium against bile acid-induced injury. NCBI Gene Expression Omnibus: GSE99579.

Identification and Characterization of a Novel NADPH Oxidase 1 (Nox1) Inhibitor That Suppresses Proliferation of Colon and Stomach Cancer Cells.

Reactive oxygen species (ROS) generated by reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox)1 mediate cellular signalings involved in normal physiological processes, and aberrant control of Nox1 has been implicated in the pathogenesis of various diseases. Therefore, Nox1 could have great potential as a therapeutic target. Here, we identified a novel Nox1 inhibitor, NOS31 secreted from Stretomyces sp. and analyzed its chemical structure. Furthermore, NOS31 was found to selectively inhibit Nox1-mediated ROS generation, with only a marginal effect on other Nox isoforms (Nox2-5) and no ROS scavenging activity. This compound blocked both Nox organizer 1 (NOXO1)/Nox activator 1 (NOXA1)-dependent and phorbol 12-myristate 13-acetate-stimulated Nox1-mediated ROS production in colon cancer cells. NOS31 inhibited the proliferation of several colon carcinoma and gastric cancer cell lines that upregulate the Nox1 system, whereas it had no appreciable effect on normal cells with low levels of Nox1. The finding suggests that NOS31 is a unique, potent Nox1 inhibitor of microbial origin and raises its possibility as a therapeutic agent for inhibiting gastrointestinal cancer cell growth.

Sialic acid-binding immunoglobulin-like lectin 8 (Siglec-8) is an activating receptor mediating ß2-integrin-dependent function in human eosinophils.

BACKGROUND: Siglec-8 is a CD33 subfamily cell-surface receptor selectively expressed on human eosinophils. After cytokine priming, Siglec-8 mAb or glycan ligand binding causes eosinophil apoptosis associated with reactive oxygen species (ROS) production. Most CD33-related Siglecs function as inhibitory receptors, but the ability of Siglec-8 to stimulate eosinophil ROS production and apoptosis suggests that Siglec-8 might instead function as an activating receptor. OBJECTIVE: We sought to determine the role of IL-5 priming and identify the signaling molecules involved in Siglec-8 function for human eosinophils. METHODS: We used an mAb and/or a multimeric synthetic sulfated sialoglycan ligand recognizing Siglec-8 in combination with integrin blocking antibodies, pharmacologic inhibitors, phosphoproteomics, and Western blot analysis to define the necessity of various proteins involved in Siglec-8 function for human eosinophils. RESULTS: Cytokine priming was required to elicit the unanticipated finding that Siglec-8 engagement promotes rapid ß2-integrin-dependent eosinophil adhesion. Also novel was the finding that this adhesion was necessary for subsequent ROS production and apoptosis. Siglec-8-mediated ROS was generated through reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation because pretreatment of eosinophils with catalase (an extracellular superoxide scavenger) or NSC 23766 (a Rac GTPase inhibitor) completely inhibited Siglec-8-mediated eosinophil apoptosis. Finally, engagement of Siglec-8 on IL-5-primed eosinophils resulted in increased phosphorylation of Akt, p38, and c-Jun N-terminal kinase 1 that was also ß2-integrin dependent; pharmacologic inhibition of these kinases completely prevented Siglec-8-mediated eosinophil apoptosis. CONCLUSIONS: These data demonstrate that Siglec-8 functions uniquely as an activating receptor on IL-5-primed eosinophils through a novel pathway involving regulation of ß2-integrin-dependent adhesion, NADPH oxidase, and a subset of protein kinases.

Neutrophils as Components of Mucosal Homeostasis.

Inflammatory responses in the intestinal mucosa inevitably result in the recruitment of neutrophils (polymorphonuclear leukocytes [PMNs]). Epithelial cells that line the mucosa play an integral role in the recruitment, maintenance, and clearance of PMNs at sites of inflammation. The consequences of such PMN-epithelial interactions often determine tissue responses and, ultimately, organ function. For this reason, there is significant interest in understanding how PMNs function in the mucosa during inflammation. Recent studies have shown that PMNs play a more significant role in molding of the immune response than previously thought. Here, we review the recent literature regarding the contribution of PMNs to the development and resolution of inflammation, with an emphasis on the role of the tissue microenvironment and pathways for promoting epithelial restitution. These studies highlight the complex nature of inflammatory pathways and provide important insight into the difficulties of treating mucosal inflammation.