WorldWideScience

Sample records for human neutrophil apoptosis

  1. Effect of sevoflurane on human neutrophil apoptosis.

    LENUS (Irish Health Repository)

    Tyther, R

    2012-02-03

    BACKGROUND AND OBJECTIVE: Both chronic occupational exposure to volatile anaesthetic agents and acute in vitro exposure of neutrophils to isoflurane have been shown to inhibit the rate of apoptosis of human neutrophils. It is possible that inhibition of neutrophil apoptosis arises through delaying mitochondrial membrane potential collapse. We assessed mitochondrial depolarization and apoptosis in unexposed neutrophils and neutrophils exposed to sevoflurane in vivo. METHODS: A total of 20 mL venous blood was withdrawn pre- and postinduction of anaesthesia, the neutrophils isolated and maintained in culture. At 1, 12 and 24 h in culture, the percentage of neutrophil apoptosis was assessed by dual staining with annexin V-FITC and propidium iodide. Mitochondrial depolarization was measured using the dual emission styryl dye JC-1. RESULTS: Apoptosis was significantly inhibited in neutrophils exposed to sevoflurane in vivo at 24 (exposed: 38 (12)% versus control: 28 (11)%, P = 0.001), but not at 1 or 12 h, in culture. Mitochondrial depolarization was not delayed in neutrophils exposed to sevoflurane. CONCLUSIONS: The most important findings are that sevoflurane inhibits neutrophil apoptosis in vivo and that inhibition is not mediated primarily by an effect on mitochondrial depolarization.

  2. Mitochondria in neutrophil apoptosis

    NARCIS (Netherlands)

    van Raam, B. J.; Verhoeven, A. J.; Kuijpers, T. W.

    2006-01-01

    Central in the regulation of the short life span of neutrophils are their mitochondria. These organelles hardly contribute to the energy status of neutrophils but play a vital role in the apoptotic process. Not only do the mitochondria contain cytotoxic proteins that are released during apoptosis

  3. Distinct Trypanosoma cruzi isolates induce activation and apoptosis of human neutrophils.

    Directory of Open Access Journals (Sweden)

    Luísa M D Magalhães

    Full Text Available Neutrophils are critical players in the first line of defense against pathogens and in the activation of subsequent cellular responses. We aimed to determine the effects of the interaction of Trypanosoma cruzi with human neutrophils, using isolates of the two major discrete type units (DTUs associated with Chagas' disease in Latin America (clone Col1.7G2 and Y strain, DTU I and II, respectively. Thus, we used CFSE-stained trypomastigotes to measure neutrophil-T. cruzi interaction, neutrophil activation, cytokine expression and death, after infection with Col1.7G2 and Y strain. Our results show that the frequency of CFSE+ neutrophils, indicative of interaction, and CFSE intensity on a cell-per-cell basis were similar when comparing Col1.7G2 and Y strains. Interaction with T. cruzi increased neutrophil activation, as measured by CD282, CD284, TNF and IL-12 expression, although at different levels between the two strains. No change in IL-10 expression was observed after interaction of neutrophils with either strain. We observed that exposure to Y and Col1.7G2 caused marked neutrophil death. This was specific to neutrophils, since interaction of either strain with monocytes did not cause death. Our further analysis showed that neutrophil death was a result of apoptosis, which was associated with an upregulation of TNF-receptor, TNF and FasLigand, but not of Fas. Induction of TNF-associated neutrophil apoptosis by the different T. cruzi isolates may act as an effective common mechanism to decrease the host's immune response and favor parasite survival.

  4. Flow Cytometric Evaluation of Human Neutrophil Apoptosis During Nitric Oxide Generation In Vitro: The Role of Exogenous Antioxidants

    Directory of Open Access Journals (Sweden)

    Zofia Sulowska

    2005-01-01

    in vitro. The effect of exogenous supply of NO donors such as SNP, SIN-1, and GEA-3162 on the course of human neutrophil apoptosis and the role of extracellular antioxidants in this process was investigated. Isolated from peripheral blood, neutrophils were cultured in the presence or absence of NO donor compounds and antioxidants for 8, 12, and 20 hours. Apoptosis of neutrophils was determined in vitro by flow cytometric analysis of cellular DNA content and Annexin V protein binding to the cell surface. Exposure of human neutrophils to GEA-3162 and SIN-1 significantly accelerates and enhances their apoptosis in vitro in a time-dependent fashion. In the presence of SNP, intensification of apoptosis has not been revealed until 12 hours after the culture. The inhibition of GEA-3162- and SIN-1-mediated neutrophil apoptosis by superoxide dismutase (SOD but not by catalase (CAT was observed. Our results show that SOD and CAT can protect neutrophils against NO-donors-induced apoptosis and suggest that the interaction of NO and oxygen metabolites signals may determine the destructive or protective role of NO donor compounds during apoptotic neutrophil death.

  5. Human neutrophil peptide-1 promotes alcohol-induced hepatic fibrosis and hepatocyte apoptosis.

    Directory of Open Access Journals (Sweden)

    Rie Ibusuki

    Full Text Available Neutrophil infiltration of the liver is a typical feature of alcoholic liver injury. Human neutrophil peptide (HNP-1 is an antimicrobial peptide secreted by neutrophils. The aim of this study was to determine if HNP-1 affects ethanol-induced liver injury and to examine the mechanism of liver injury induced by HNP-1.Transgenic (TG mice expressing HNP-1 under the control of a β-actin-based promoter were established. Ethanol was orally administered to HNP-1 TG or wild-type C57BL/6N (WT mice. SK-Hep1 hepatocellular carcinoma cells were used to investigate the effect of HNP-1 on hepatocytes in vitro.After 24 weeks of ethanol intake, hepatic fibrosis and hepatocyte apoptosis were significantly more severe in TG mice than in WT mice. Levels of CD14, TLR4, and IL-6 in liver tissues were higher in TG mice than in WT mice. Apoptosis was accompanied by higher protein levels of caspase-3, caspase-8, and cleaved PARP in liver tissue. In addition, phosphorylated ASK1, ASK1, phosphorylated JNK, JNK1, JNK2, Bax, Bak and Bim were all more abundant in TG mice than in WT mice. In contrast, the level of anti-apoptotic Bcl2 in the liver was significantly lower in TG mice than in WT mice. Analysis of microRNAs in liver tissue showed that miR-34a-5p expression was significantly higher in TG mice than in WT mice. Furthermore, in the presence of ethanol, HNP-1 increased the apoptosis with the decreased level of Bcl2 in a concentration-dependent manner in vitro.HNP-1 secreted by neutrophils may exacerbate alcohol-induced hepatic fibrosis and hepatocyte apoptosis with a decrease in Bcl2 expression and an increase in miR-34a-5p expression.

  6. Effects of ghrelin on the apoptosis of human neutrophils in vitro

    Science.gov (United States)

    Li, Bin; Zeng, Mian; Zheng, Haichong; Huang, Chunrong; He, Wanmei; Lu, Guifang; Li, Xia; Chen, Yanzhu; Xie, Ruijie

    2016-01-01

    Acute respiratory distress syndrome (ARDS) is characterized by lung inflammation and the diffuse infiltration of neutrophils into the alveolar space. Neutrophils are abundant, short-lived leukocytes that play a key role in immune defense against microbial infections. These cells die via apoptosis following the activation and uptake of microbes, and will also enter apoptosis spontaneously at the end of their lifespan if they do not encounter pathogens. Apoptosis is essential for the removal of neutrophils from inflamed tissues and for the timely resolution of neutrophilic inflammation. Ghrelin is an endogenous ligand for the growth hormone (GH) secretagogue receptor, produced and secreted mainly from the stomach. Previous studies have reported that ghrelin exerts anti-inflammatory effects in lung injury through the regulation of the apoptosis of different cell types; however, the ability of ghrelin to regulate alveolar neutrophil apoptosis remains largely undefined. We hypothesized that ghrelin may have the ability to modulate neutrophil apoptosis. In this study, to examine this hypothesis, we investigated the effects of ghrelin on freshly isolated neutrophils in vitro. Our findings demonstrated a decrease in the apoptotic ratio (as shown by flow cytometry), as well as in the percentage of cells with decreased mitochondrial membrane potential (ΔΨm) and in the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labeling-positive rate, accompanied by an increased B-cell lymphoma 2/Bax ratio and the downregulation of cleaved caspase-3 in neutrophils following exposure to lipopolysaccharide (100 ng/ml). However, pre-treatment with ghrelin at a physiological level (100 nM) did not have a notable influence on the neutrophils in all the aforementioned tests. Our findings suggest that ghrelin may not possess the ability to modulate the neutrophil lifespan in vitro. PMID:27431014

  7. Divergent effects of tumor necrosis factor alpha on apoptosis of human neutrophils

    NARCIS (Netherlands)

    van den Berg, J. M.; Weyer, S.; Weening, J. J.; Roos, D.; Kuijpers, T. W.

    2001-01-01

    Apoptosis of neutrophils is a key mechanism to control the intensity of the acute inflammatory response. Previously, the cytokine tumor necrosis factor alpha (TNF-alpha) was reported by some to have pro-apoptotic and by others to have antiapoptotic effects on neutrophils. The aim of this study was

  8. Severe exercise and exercise training exert opposite effects on human neutrophil apoptosis via altering the redox status.

    Directory of Open Access Journals (Sweden)

    Guan-Da Syu

    Full Text Available Neutrophil spontaneous apoptosis, a process crucial for immune regulation, is mainly controlled by alterations in reactive oxygen species (ROS and mitochondria integrity. Exercise has been proposed to be a physiological way to modulate immunity; while acute severe exercise (ASE usually impedes immunity, chronic moderate exercise (CME improves it. This study aimed to investigate whether and how ASE and CME oppositely regulate human neutrophil apoptosis. Thirteen sedentary young males underwent an initial ASE and were subsequently divided into exercise and control groups. The exercise group (n = 8 underwent 2 months of CME followed by 2 months of detraining. Additional ASE paradigms were performed at the end of each month. Neutrophils were isolated from blood specimens drawn at rest and immediately after each ASE for assaying neutrophil spontaneous apoptosis (annexin-V binding on the outer surface along with redox-related parameters and mitochondria-related parameters. Our results showed that i the initial ASE immediately increased the oxidative stress (cytosolic ROS and glutathione oxidation, and sequentially accelerated the reduction of mitochondrial membrane potential, the surface binding of annexin-V, and the generation of mitochondrial ROS; ii CME upregulated glutathione level, retarded spontaneous apoptosis and delayed mitochondria deterioration; iii most effects of CME were unchanged after detraining; and iv CME blocked ASE effects and this capability remained intact even after detraining. Furthermore, the ASE effects on neutrophil spontaneous apoptosis were mimicked by adding exogenous H(2O(2, but not by suppressing mitochondrial membrane potential. In conclusion, while ASE induced an oxidative state and resulted in acceleration of human neutrophil apoptosis, CME delayed neutrophil apoptosis by maintaining a reduced state for long periods of time even after detraining.

  9. Anaplasma phagocytophilum inhibits human neutrophil apoptosis via upregulation of bfl-1, maintenance of mitochondrial membrane potential and prevention of caspase 3 activation.

    Science.gov (United States)

    Ge, Yan; Yoshiie, Kiyotaka; Kuribayashi, Futoshi; Lin, Mingqun; Rikihisa, Yasuko

    2005-01-01

    The inhibition of neutrophil apoptosis plays a central role in human granulocytic anaplasmosis. Intracellular signalling pathways through which the obligatory intracellular bacterium Anaplasma phagocytophilum inhibits the spontaneous apoptosis of human peripheral blood neutrophils were investigated. bfl-1 mRNA levels in uninfected neutrophils after 12 h in culture were reduced to approximately 5-25% of 0 h levels, but remained high in infected neutrophils. The eukaryotic RNA synthesis inhibitor, actinomycin D, prevented the maintenance of bfl-1 mRNA levels by A. phagocytophilum. Differences in mcl-1, bax, bcl-w, bad or bak mRNA levels in infected versus uninfected neutrophils were not remarkable. By using mitochondrial fluorescent dyes, Mitotracker Red and JC-1, it was found that most uninfected neutrophils lost mitochondrial membrane potential after 10-12 h incubation, whereas A. phagocytophilum-infected neutrophils maintained high membrane potential. Caspase 3 activity and the degree of apoptosis were lower in dose-dependent manner in A. phagocytophilum-infected neutrophils at 16 h post infection, as compared to uninfected neutrophils. Anti-active caspase 3 antibody labelling showed less positively stained population in infected neutrophils compared to those in uninfected neutrophils after 12 h incubation. These results suggest that A. phagocytophilum inhibits human neutrophil apoptosis via transcriptional upregulation of bfl-1 and inhibition of mitochondria-mediated activation of caspase 3.

  10. The Natural Stilbenoid Piceatannol Decreases Activity and Accelerates Apoptosis of Human Neutrophils: Involvement of Protein Kinase C

    Directory of Open Access Journals (Sweden)

    Viera Jancinova

    2013-01-01

    Full Text Available Neutrophils are able to release cytotoxic substances and inflammatory mediators, which, along with their delayed apoptosis, have a potential to maintain permanent inflammation. Therefore, treatment of diseases associated with chronic inflammation should be focused on neutrophils; formation of reactive oxygen species and apoptosis of these cells represent two promising targets for pharmacological intervention. Piceatannol, a naturally occurring stilbenoid, has the ability to reduce the toxic action of neutrophils. This substance decreased the amount of oxidants produced by neutrophils both extra- and intracellularly. Radicals formed within neutrophils (fulfilling a regulatory role were reduced to a lesser extent than extracellular oxidants, potentially dangerous for host tissues. Moreover, piceatannol did not affect the phosphorylation of p40phox—a component of NADPH oxidase, responsible for the assembly of functional oxidase in intracellular (granular membranes. The stilbenoid tested elevated the percentage of early apoptotic neutrophils, inhibited the activity of protein kinase C (PKC—the main regulatory enzyme in neutrophils, and reduced phosphorylation of PKC isoforms α, βII, and δ on their catalytic region. The results indicated that piceatannol may be useful as a complementary medicine in states associated with persisting neutrophil activation and with oxidative damage of tissues.

  11. Functional characterization of mitochondria in neutrophils: a role restricted to apoptosis

    NARCIS (Netherlands)

    Maianski, N. A.; Geissler, J.; Srinivasula, S. M.; Alnemri, E. S.; Roos, D.; Kuijpers, T. W.

    2004-01-01

    Mitochondria are known to combine life-supporting functions with participation in apoptosis by controlling caspase activity. Here, we report that in human blood neutrophils the mitochondria are different, because they preserve mainly death-mediating abilities. Neutrophil mitochondria hardly

  12. Bid truncation, Bid/Bax targeting to the mitochondria, and caspase activation associated with neutrophil apoptosis are inhibited by granulocyte colony-stimulating factor

    NARCIS (Netherlands)

    Maianski, Nikolai A.; Roos, Dirk; Kuijpers, Taco W.

    2004-01-01

    Neutrophil apoptosis constitutes a way of managing neutrophil-mediated reactions. It allows coping with infections, but avoiding overt bystander tissue damage. Using digitonin-based subcellular fractionation and Western blotting, we found that spontaneous apoptosis of human neutrophils (after

  13. Bacterial lipoprotein delays apoptosis in human neutrophils through inhibition of caspase-3 activity: regulatory roles for CD14 and TLR-2.

    LENUS (Irish Health Repository)

    Power, Colm P

    2012-02-03

    The human sepsis syndrome resulting from bacterial infection continues to account for a significant proportion of hospital mortality. Neutralizing strategies aimed at individual bacterial wall products (such as LPS) have enjoyed limited success in this arena. Bacterial lipoprotein (BLP) is a major constituent of the wall of diverse bacterial forms and profoundly influences cellular function in vivo and in vitro, and has been implicated in the etiology of human sepsis. Delayed polymorphonuclear cell (PMN) apoptosis is a characteristic feature of human sepsis arising from Gram-negative or Gram-positive bacterial infection. Bacterial wall product ligation and subsequent receptor-mediated events upstream of caspase inhibition in neutrophils remain incompletely understood. BLP has been shown to exert its cellular effects primarily through TLR-2, and it is now widely accepted that lateral associations with the TLRs represent the means by which CD14 communicates intracellular messages. In this study, we demonstrate that BLP inhibits neutrophil mitochondrial membrane depolarization with a subsequent reduction in caspase-3 processing, ultimately leading to a significant delay in PMN apoptosis. Pretreatment of PMNs with an anti-TLR-2 mAb or anti-CD14 mAb prevented BLP from delaying PMN apoptosis to such a marked degree. Combination blockade using both mAbs completely prevented the effects of BLP (in 1 and 10 ng\\/ml concentrations) on PMN apoptosis. At higher concentrations of BLP, the antiapoptotic effects were observed, but were not as pronounced. Our findings therefore provide the first evidence of a crucial role for both CD14 and TLR-2 in delayed PMN apoptosis arising from bacterial infection.

  14. Regulation of apoptosis and priming of neutrophil oxidative burst by diisopropyl fluorophosphate

    Directory of Open Access Journals (Sweden)

    Tsang Jennifer LY

    2010-07-01

    Full Text Available Abstract Background Diisopropyl fluorophosphate (DFP is a serine protease inhibitor that is widely used as an inhibitor of endogenous proteases in in vitro neutrophil studies. Its effects on neutrophil function are unclear. We sought to determine the biological effects of DFP on human neutrophil apoptosis and oxidative burst. Methods We isolated neutrophils from healthy volunteers, incubated them with DFP (2.5 mM, and evaluated neutrophil elastase (NE activity, neutrophil degranulation, apoptosis as reflected in hypodiploid DNA formation and exteriorization of phosphatidylserine (PS, processing and activity of caspases-3 and -8, oxidative burst activity and hydrogen peroxide release. Results Consistent with its activity as a serine protease inhibitor, DFP significantly inhibited NE activity but not the degranulation of azurophilic granules. DFP inhibited constitutive neutrophil apoptosis as reflected in DNA fragmentation, and the processing and activity of caspases-3 and -8. DFP also inhibited priming of neutrophils for oxidative burst activity and hydrogen peroxide release. However, DFP enhanced the exteriorization of PS in a dose-dependent manner. Conclusion We conclude that DFP exerts significant effects on neutrophil inflammatory function that may confound the interpretation of studies that use it for its antiprotease activity. We further conclude that endogenous proteases play a role in the biology of constitutive neutrophil apoptosis.

  15. Human neutrophils in auto-immunity.

    Science.gov (United States)

    Thieblemont, Nathalie; Wright, Helen L; Edwards, Steven W; Witko-Sarsat, Véronique

    2016-04-01

    Human neutrophils have great capacity to cause tissue damage in inflammatory diseases via their inappropriate activation to release reactive oxygen species (ROS), proteases and other tissue-damaging molecules. Furthermore, activated neutrophils can release a wide variety of cytokines and chemokines that can regulate almost every element of the immune system. In addition to these important immuno-regulatory processes, activated neutrophils can also release, expose or generate neoepitopes that have the potential to break immune tolerance and result in the generation of autoantibodies, that characterise a number of human auto-immune diseases. For example, in vasculitis, anti-neutrophil cytoplasmic antibodies (ANCA) that are directed against proteinase 3 or myeloperoxidase are neutrophil-derived autoantigens and activated neutrophils are the main effector cells of vascular damage. In other auto-immune diseases, these neutrophil-derived neoepitopes may arise from a number of processes that include release of granule enzymes and ROS, changes in the properties of components of their plasma membrane as a result of activation or apoptosis, and via the release of Neutrophil Extracellular Traps (NETs). NETs are extracellular structures that contain chromatin that is decorated with granule enzymes (including citrullinated proteins) that can act as neo-epitopes to generate auto-immunity. This review therefore describes the processes that can result in neutrophil-mediated auto-immunity, and the role of neutrophils in the molecular pathologies of auto-immune diseases such as vasculitis, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). We discuss the potential role of NETs in these processes and some of the debate in the literature regarding the role of this phenomenon in microbial killing, cell death and auto-immunity. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Increased lung neutrophil apoptosis and inflammation resolution in nonresponding pneumonia.

    Science.gov (United States)

    Moret, I; Lorenzo, M J; Sarria, B; Cases, E; Morcillo, E; Perpiñá, M; Molina, J M; Menéndez, R

    2011-11-01

    Neutrophil activation state and its relationship with an inflammatory environment in community-acquired pneumonia (CAP) remain insufficiently elucidated. We aimed to evaluate the neutrophil apoptosis and cytokine pattern in CAP patients after 72 h of treatment, and their impact on infection resolution. Apoptosis of blood and bronchoalveolar lavage (BAL) neutrophils was measured in nonresponding CAP (NCAP), in responding CAP (blood only) and in patients without infection (control). Pro-inflammatory (interleukin (IL)-6, IL-8) and anti-inflammatory (IL-10) cytokines were measured. Main outcomes were clinical stability and days of hospitalisation. Basal neutrophil apoptosis was higher in the BAL and blood of NCAP, whereas spontaneous apoptosis (after 24 h culture) was lower. Cytokines in NCAP were higher than in responding CAP and control: IL-6 was increased in BAL and blood, IL-8 in BAL and IL-10 in blood. An increased basal apoptosis (≥20%) in BAL of NCAP was associated with lower systemic IL-10 (p<0.01), earlier clinical stability (p=0.05) and shorter hospital stay (p=0.02). A significant correlation was found for systemic IL-6 and IL-10 with days to reach stability and length of stay. After 72 h of treatment, an increased basal alveolar neutrophil apoptosis might contribute to downregulation of inflammation and to faster clinical stability.

  17. Accelerated apoptosis of neutrophils in familial Mediterranean fever

    DEFF Research Database (Denmark)

    Manukyan, Gayane; Aminov, Rustam; Hakobyan, Gagik

    2015-01-01

    The causative mutations for familial Mediterranean fever (FMF) are located in the MEFV gene, which encodes pyrin. Pyrin modulates the susceptibility to apoptosis via its PYD domain, but how the mutated versions of pyrin affect apoptotic processes are poorly understood. Spontaneous and induced rates...... of systemic neutrophil apoptosis as well as the levels of proteins involved in apoptosis were investigated ex vivo in patients with FMF using flow cytometry and RT-qPCR. The freshly collected neutrophils from the patients in FMF remission displayed a significantly larger number of cells spontaneously entering...... apoptosis compared to control (6.27 ± 2.14 vs. 1.69 ± 0.18%). This elevated ratio was retained after 24 h incubation of neutrophils in the growth medium (32.4 ± 7.41 vs. 7.65 ± 1.32%). Correspondingly, the mRNA level for caspase-3 was also significantly increased under these conditions. In response...

  18. The effect of the anaesthetic agent isoflurane on the rate of neutrophil apoptosis in vitro.

    LENUS (Irish Health Repository)

    Tyther, R

    2012-02-03

    BACKGROUND: Volatile anaesthetic agents influence neutrophil function, and potentially, the inflammatory response to surgery. AIM: The objective of this study was to determine the effect of isoflurane (1-4%) on human polymorphonuclear neutrophil apoptosis in vitro. METHODS: Venous blood from 12 healthy volunteers was exposed to 0, 1, and 4% isoflurane delivered via a 14G Wallace flexihub internal jugular cannula, at a fresh gas flow of 0.51\\/min for 5 minutes. Isolated neutrophils were assessed for apoptosis at 1, 12, and 24 hours in culture using dual staining with annexin V-FITC and propidium iodide (Annexin-V FITC assay). Data were analysed using paired, one-tailed Student\\'s t-tests. p<0.05 was considered significant. RESULTS: At 1 hour apoptosis was inhibited in the 1% (5.1 [6.8]%; p=0.017) and 4% (4.8 [4.5]%; p=0.008) isoflurane groups compared to control (11.3 [6.9]%). At 12 and 24 hours, a dose-dependent inhibition of apoptosis was demonstrated, i.e. 4% > 1% > 0%. CONCLUSION: Human neutrophil apoptosis is inhibited in a concentration-dependent manner in vitro by isoflurane in clinical concentrations.

  19. Arsenic trioxide (AT) is a novel human neutrophil pro-apoptotic agent: effects of catalase on AT-induced apoptosis, degradation of cytoskeletal proteins and de novo protein synthesis.

    Science.gov (United States)

    Binet, François; Cavalli, Hélène; Moisan, Eliane; Girard, Denis

    2006-02-01

    The anti-cancer drug arsenic trioxide (AT) induces apoptosis in a variety of transformed or proliferating cells. However, little is known regarding its ability to induce apoptosis in terminally differentiated cells, such as neutrophils. Because neutropenia has been reported in some cancer patients after AT treatment, we hypothesised that AT could induce neutrophil apoptosis, an issue that has never been investigated. Herein, we found that AT-induced neutrophil apoptosis and gelsolin degradation via caspases. AT did not increase neutrophil superoxide production and did not induce mitochondrial generation of reactive oxygen species. AT-induced apoptosis in PLB-985 and X-linked chronic granulomatous disease (CGD) cells (PLB-985 cells deficient in gp91(phox) mimicking CGD) at the same potency. Addition of catalase, an inhibitor of H2O2, reversed AT-induced apoptosis and degradation of the cytoskeletal proteins gelsolin, alpha-tubulin and lamin B1. Unexpectedly, AT-induced de novo protein synthesis, which was reversed by catalase. Cycloheximide partially reversed AT-induced apoptosis. We conclude that AT induces neutrophil apoptosis by a caspase-dependent mechanism and via de novo protein synthesis. H2O2 is of major importance in AT-induced neutrophil apoptosis but its production does not originate from nicotinamide adenine dinucleotide phosphate dehydrogenase activation and mitochondria. Cytoskeletal structures other than microtubules can now be considered as novel targets of AT.

  20. On the Pharmacology of Oxidative Burst of Human Neutrophils

    Czech Academy of Sciences Publication Activity Database

    Nosáľ, R.; Drábiková, K.; Jančinová, V.; Mačičková, T.; Pečivová, J.; Perečko, T.; Harmatha, Juraj; Šmidrkal, J.

    2015-01-01

    Roč. 64, Suppl 4 (2015), S445-S452 ISSN 0862-8408 Institutional support: RVO:61388963 Keywords : human neutrophils * oxidative burst * chemiluminescence * protein kinase C * apoptosis Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 1.643, year: 2015 http://www.biomed.cas.cz/physiolres/pdf/64/64_S445.pdf

  1. Cytoplasmic lipid bodies of human neutrophilic leukocytes

    International Nuclear Information System (INIS)

    Weller, P.F.; Ackerman, S.J.; Nicholson-Weller, A.; Dvorak, A.M.

    1989-01-01

    The morphology and function of cytoplasmic lipid bodies in human neutrophils were evaluated. By transmission electron microscopy, neutrophil lipid bodies were cytoplasmic inclusions, usually several microns in diameter, that occasionally coalesced to attain a diameter up to 7 microM. Neutrophil lipid bodies were not enveloped by membrane but were often surrounded by a more electron-dense shell at their periphery. Normal peripheral blood neutrophils contained an average of approximately one lipid body per cell. Lipid bodies appeared in greater numbers in neutrophils from inflammatory lesions. Perturbation of neutrophils during conventional methods of cell isolation and purification modestly increased lipid body numbers in neutrophils, whereas incubation of neutrophils with 1 microM oleic acid rapidly induced lipid body formation over 30 to 60 minutes. After granulocytes were incubated for 2 hours with 3H-fatty acids, including arachidonic, oleic, and palmitic acids, electron microscopic autoradiography demonstrated that lipid bodies represented the predominant intracellular sites of localization of each of the three 3H-fatty acids. There was lesser labeling noted in the perinuclear cisterna, but not in cell membranes. Virtually all of each of the three 3H-fatty acids incorporated by the neutrophils were esterified into chromatographically resolved classes of neutral lipids or phospholipids. These findings indicate that cytoplasmic lipid bodies are more prominent in neutrophils in vivo engaged in inflammatory responses and that these organelles in human neutrophils function as sites of deposition of esterified, incorporated fatty acids

  2. Neutrophil and macrophage apoptosis in bronchoalveolar lavage fluid from healthy horses and horses with recurrent airway obstruction (RAO)

    Science.gov (United States)

    2014-01-01

    Background Dysregulation of apoptosis has been implicated in a range of diseases including tumors, neurodegenerative and autoimmine diseases, as well as allergic asthma and chronic obstructive pulmonary disease (COPD) in humans. Although it has a different pathophysiology, delayed apoptosis of various inflammatory cells may play a pivotal role in the development of recurrent airway obstruction (RAO) in horses. Reduction of inflammatory cell apoptosis or a dysregulation of this process could lead to chronic inflammation and tissue injury. Therefore, the aim of this study was to investigate the rate of apoptosis and necrosis of neutrophils and macrophages in bronchoalveolar lavage fluid obtained from seven horses suffering from RAO (study group) and seven control horses. Results We demonstrated that neutrophil/macrophage apoptosis is altered in RAO-affected horses compared with the control group in the BAL fluid. We found a significant difference between the median percentage of early and late apoptosis of neutrophils between the study and control group of horses. Moreover, we found a positive correlation between the rate of apoptosis and the median percentage of macrophages in RAO-affected horses. Conclusion The findings suggest that apoptosis dysregulation may play a significant role in the pathogenesis of RAO. However, further studies are needed to clarify the role of altered apoptosis in the course of equine recurrent airway obstruction. PMID:24460911

  3. Iodinated contrast media induce neutrophil apoptosis through a mitochondrial and caspase mediated pathway.

    LENUS (Irish Health Repository)

    Fanning, N F

    2012-02-03

    Iodinated contrast media (ICM) can induce apoptosis (programmed cell death) in renal, myocardial and endothelial cells. Following intravascular injection, circulating immune cells are exposed to high concentrations of ICM. As neutrophils constitutively undergo apoptosis we hypothesized that ICM may adversely affect neutrophil survival. Our aim was to investigate the effect of ICM on neutrophil apoptosis. Neutrophils were isolated from healthy subjects and cultured in vitro with ionic (diatrizoate and ioxaglate) and non-ionic (iohexol and iotrolan) ICM. The effect of ICM on neutrophil apoptosis in both unstimulated and lipopolysaccharide-stimulated neutrophils was determined by annexin V flow cytometry. The influence of physicochemical properties of the different ICM on apoptosis of neutrophils was also studied. We further investigated the effects of ICM on key intracellular signal pathways, including p38 mitogen-activated protein kinase (MAPK) by Western blotting, and mitochondrial depolarization and caspase activity by flow cytometry. Isoiodine concentrations (20 mg ml(-1)) of ionic (diatrizoate 69.6+\\/-2.9%; ioxaglate 58.9+\\/-2.0%) and non-ionic (iohexol 57.3+\\/-2.9%; iotrolan 57.1+\\/-2.6%) ICM significantly induced neutrophil apoptosis over control levels (47.7+\\/-1.4%). The apoptotic effect of ICM was influenced by their chemical structure, with ionic ICM having a more significant (p<0.01) apoptotic effect than non-ionic ICM (p<0.05). Furthermore, ICM reversed the anti-apoptotic effect of lipopolysaccharide (1000 ng ml(-1)) treated neutrophils to control levels (23.0+\\/-3.5% to 61.2+\\/-5.3%; n=4; p<0.05). These agents induce apoptosis through a p38 MAPK independent pathway that results in mitochondrial depolarization, and is dependent on caspase activation. As neutrophils play a central role in host response to infection and injury, ICM, through induction of neutrophil apoptosis, could have a significant deleterious effect on host immune defence and

  4. Hypertonic Saline Suppresses NADPH Oxidase-Dependent Neutrophil Extracellular Trap Formation and Promotes Apoptosis

    Directory of Open Access Journals (Sweden)

    Ajantha Nadesalingam

    2018-03-01

    Full Text Available Tonicity of saline (NaCl is important in regulating cellular functions and homeostasis. Hypertonic saline is administered to treat many inflammatory diseases, including cystic fibrosis. Excess neutrophil extracellular trap (NET formation, or NETosis, is associated with many pathological conditions including chronic inflammation. Despite the known therapeutic benefits of hypertonic saline, its underlying mechanisms are not clearly understood. Therefore, we aimed to elucidate the effects of hypertonic saline in modulating NETosis. For this purpose, we purified human neutrophils and induced NETosis using agonists such as diacylglycerol mimetic phorbol myristate acetate (PMA, Gram-negative bacterial cell wall component lipopolysaccharide (LPS, calcium ionophores (A23187 and ionomycin from Streptomyces conglobatus, and bacteria (Pseudomonas aeruginosa and Staphylococcus aureus. We then analyzed neutrophils and NETs using Sytox green assay, immunostaining of NET components and apoptosis markers, confocal microscopy, and pH sensing reagents. This study found that hypertonic NaCl suppresses nicotinamide adenine dinucleotide phosphate oxidase (NADPH2 or NOX2-dependent NETosis induced by agonists PMA, Escherichia coli LPS (0111:B4 and O128:B12, and P. aeruginosa. Hypertonic saline also suppresses LPS- and PMA- induced reactive oxygen species production. It was determined that supplementing H2O2 reverses the suppressive effect of hypertonic saline on NOX2-dependent NETosis. Many of the aforementioned suppressive effects were observed in the presence of equimolar concentrations of choline chloride and osmolytes (d-mannitol and d-sorbitol. This suggests that the mechanism by which hypertonic saline suppresses NOX2-dependent NETosis is via neutrophil dehydration. Hypertonic NaCl does not significantly alter the intracellular pH of neutrophils. We found that hypertonic NaCl induces apoptosis while suppressing NOX2-dependent NETosis. In contrast, hypertonic

  5. Human neutrophils facilitate tumor cell transendothelial migration.

    LENUS (Irish Health Repository)

    Wu, Q D

    2012-02-03

    Tumor cell extravasation plays a key role in tumor metastasis. However, the precise mechanisms by which tumor cells migrate through normal vascular endothelium remain unclear. In this study, using an in vitro transendothelial migration model, we show that human polymorphonuclear neutrophils (PMN) assist the human breast tumor cell line MDA-MB-231 to cross the endothelial barrier. We found that tumor-conditioned medium (TCM) downregulated PMN cytocidal function, delayed PMN apoptosis, and concomitantly upregulated PMN adhesion molecule expression. These PMN treated with TCM attached to tumor cells and facilitated tumor cell migration through different endothelial monolayers. In contrast, MDA-MB-231 cells alone did not transmigrate. FACScan analysis revealed that these tumor cells expressed high levels of intercellular adhesion molecule-1 (ICAM-1) but did not express CD11a, CD11b, or CD18. Blockage of CD11b and CD18 on PMN and of ICAM-1 on MDA-MB-231 cells significantly attenuated TCM-treated, PMN-mediated tumor cell migration. These tumor cells still possessed the ability to proliferate after PMN-assisted transmigration. These results indicate that TCM-treated PMN may serve as a carrier to assist tumor cell transendothelial migration and suggest that tumor cells can exploit PMN and alter their function to facilitate their extravasation.

  6. Effects of chronic occupational exposure to anaesthetic gases on the rate of neutrophil apoptosis among anaesthetists.

    LENUS (Irish Health Repository)

    Tyther, R

    2012-02-03

    BACKGROUND AND OBJECTIVE: Volatile anaesthetic agents are known to influence neutrophil function. The aim was to determine the effect of chronic occupational exposure to volatile anaesthetic agents on the rate of neutrophil apoptosis among anaesthetists. To test this hypothesis, we compared the rate of neutrophil apoptosis in anaesthetists who had been chronically exposed to volatile anaesthetic agents with that in unexposed volunteers. METHODS: Venous blood (20 mL) was withdrawn from 24 ASA I-II volunteers, from which neutrophils were isolated, and maintained in culture. At 1, 12 and 24 h in culture, the percentage of neutrophil apoptosis was assessed by dual staining with annexin V-FITC and propidium iodide. RESULTS: At 1 h (but not at 12 and 24 h) in culture, the rate of neutrophil apoptosis was significantly less in the anaesthetists--13.8 (12.9%) versus 34.4 (12.1%) (P = 0.001). CONCLUSIONS: Chronic occupational exposure to volatile anaesthetic agents may inhibit neutrophil apoptosis. This may have implications for anaesthetists and similarly exposed healthcare workers in terms of the adequacy of their inflammatory response.

  7. Comparison of Neutrophil Apoptosis by the Pseudomonas Aeruginosa Exotoxins between Healthy Individuals and Term Infants

    Directory of Open Access Journals (Sweden)

    Soheila Khazaei

    2013-04-01

    Full Text Available Background: Pseudomonas aeruginosa may be colonized in different human tissues and result in some infections potentially. Thus, considering that these bacteria are resistance to most of the current antibiotics, an examination on pathogenesis mechanisms of such bacteria can be effective in controlling the infections developed by it.Materials and Methods: In this project, among 40 blood samples (20 healthy persons, 20 infants, an amount of 5 ml (2 ml in the infants heparinized blood was collected form each and then neutrophils were isolated by a standard method and were counted by neubauer lam. After culturing Pseudomonas bacteria in broth medium, some tubes with densities of 1, 2, 3 and 4 McFarland were prepared and the bacteria were isolated by centrifuge method with 3000rpm for 10 minutes and then its exotoxin were exposed to neutrophils of the groups under study. The effect of time and the bacteria count on the amount of the secreted toxin and in adjacency to neutrophils was measured.Results: There were 11 men and 9 women in the health group and the infants group consisted of 12 boys and 8 girls. Death cell percentage of neutrophils was 100% in the health group and 8.90% in the infants group. Percentage of bacterial growth in the medium 1 and 2 McFarland was zero; in the medium 3 McFarland, it was 12.5% in the healthy group and 1% in the infants group (p<0.10. The average rate of cell death in the minute 15th was different in two groups (68.5% in health group vs. 92.5% in the infants (p<0.0005. Conclusion: This study showed the effect of Pseudomonas bacteria on the development of early cell death in the infants very well. As it was shown, this effect is time-dependent and this cell death (apoptosis is occurred in the infants earlier than health people.

  8. The effect of N-nitrosodimethylamine (NDMA) on Bax and Mcl-1 expression in human neutrophils.

    Science.gov (United States)

    Jablonski, Jakub; Jablonska, Ewa; Leonik, Agnieszka

    2011-12-01

    In the present study we examined a role of pro-apoptotic Bax and anti-apoptotic Mcl-1 proteins, participating in the regulation of intrinsic apoptosis pathway in human neutrophils (PMNs) exposed to N-nitrosodimethylamine (NDMA), the environmental xenobiotic. For the purpose comparison, the same studies were conducted in autologous peripheral blood mononuclear cells (PBMCs). The production of cytochrome c by PMNs was also determined. A deficit of anti-apoptotic Mcl-1 and overexpression of the pro-apoptotic protein Bax suggest that the apoptosis process in human neutrophils exposed to NDMA is dependent on changes in the expression of these proteins. PMNs were more sensitive to NDMA than PBMCs.

  9. Effect of smoking on neutrophil apoptosis in chronic periodontitis: An immunohistochemical study

    Directory of Open Access Journals (Sweden)

    Sachin S Shivanaikar

    2013-01-01

    Full Text Available Context: Periodontal disease is caused by chronic infection inducing an inflammatory reaction leading to breakdown of tooth-supporting tissues. There are various risk factors for the disease, and smoking is one of them. Apoptosis plays a critical role in the regulation of inflammation and host immune response which helps in tissue homeostasis, and a disturbance in this is often associated with disease. The imbalance between the apoptosis and proliferation in the periodontal tissue results in periodontal disease. Neutrophils play an important role in the defense mechanism and are the most abundant immune cells in gingival inflammatory infiltrate in patients suffering from periodontal disease. Neutrophil disorders are associated with rapid destruction of periodontal tissues. Aim: To study the influence of smoking on apoptosis of neutrophils by quantifying them in the gingival connective tissue of smoking and nonsmoking subjects suffering from chronic periodontitis. Materials and Methods: Thirty gingival biopsies were harvested from 15 smoking and 15 nonsmoking subjects who suffered from chronic periodontitis. The apoptosis of neutrophils was assessed and quantified using p53 monoclonal mouse antihuman antibody. Statistical Analysis Used: Chi-square/Fisher′sexact test was used to find the significance of study parameters on a categorical scale between the two groups. Results: Neutrophil apoptosis was significantly more in the group of nonsmokers. There was no statistical difference between plaque and bleeding index, but there was a significant increase in clinical attachment loss among smokers. Conclusions: The study reveals that smoking plays a significant role in the inhibition of neutrophil apoptosis, thereby contributing to the destruction of periodontal tissues in periodontitis.

  10. Superoxide anion production by human neutrophils activated by Trichomonas vaginalis.

    Science.gov (United States)

    Song, Hyun-Ouk; Ryu, Jae-Sook

    2013-08-01

    Neutrophils are the predominant inflammatory cells found in vaginal discharges of patients infected with Trichomonas vaginalis. In this study, we examined superoxide anion (O2 (.-)) production by neutrophils activated by T. vaginalis. Human neutrophils produced superoxide anions when stimulated with either a lysate of T. vaginalis, its membrane component (MC), or excretory-secretory product (ESP). To assess the role of trichomonad protease in production of superoxide anions by neutrophils, T. vaginalis lysate, ESP, and MC were each pretreated with a protease inhibitor cocktail before incubation with neutrophils. Superoxide anion production was significantly decreased by this treatment. Trichomonad growth was inhibited by preincubation with supernatants of neutrophils incubated for 3 hr with T. vaginalis lysate. Furthermore, myeloperoxidase (MPO) production by neutrophils was stimulated by live trichomonads. These results indicate that the production of superoxide anions and MPO by neutrophils stimulated with T. vaginalis may be a part of defense mechanisms of neutrophils in trichomoniasis.

  11. General versus regional anaesthesia for cataract surgery: effects on neutrophil apoptosis and the postoperative pro-inflammatory state.

    LENUS (Irish Health Repository)

    Goto, Y

    2012-02-03

    At clinically relevant concentrations, volatile anaesthetic agents influence neutrophil function. Our hypothesis was that sevoflurane would inhibit neutrophil apoptosis and consequently influence the postoperative pro-inflammatory state. In order to identify selectively the effect of the anaesthetic agent sevoflurane, we studied patients undergoing minimally stimulating (cataract) surgery randomly allocated to receive either sevoflurane (n = 11) or local anaesthesia (n = 12). Venous blood samples were taken immediately prior to anaesthesia and at 1, 8 and 24 h thereafter. The rate of neutrophil apoptosis, plasma concentration of cytokines and differential white cell count were measured. The rates of neutrophil apoptosis and plasma concentrations of IL-1beta, TNF-alpha and IL-8 at each time point were similar in the two groups. IL-6 concentrations increased significantly and to a similar extent compared to preanaesthetic levels at 8 and 24 h. This study demonstrates that sevoflurane does not influence the rate of neutrophil apoptosis, cytokine concentrations and neutrophil count following cataract surgery.

  12. Does chronic occupational exposure to volatile anesthetic agents influence the rate of neutrophil apoptosis?

    LENUS (Irish Health Repository)

    Goto, Y

    2012-02-03

    PURPOSE: The purpose of this preliminary investigation was to determine whether the rate of neutrophil apoptosis in health care workers is influenced by exposure to volatile anesthetic agents. METHODS: Percentage neutrophil apoptosis (Annexin-V FITC assay) was measured in health care workers (n = 20) and unexposed volunteers (n = 10). For the health care workers, time weighted personal exposure monitoring to N2O, sevoflurane and isoflurane was carried out. RESULTS: The sevoflurane and isoflurane concentrations to which health care workers were exposed were less than recommended levels in all 20 cases. Percent apoptosis was less at 24 (but not at one and 12) hr culture in health care workers [50.5 (9.7)%; P = 0.008] than in unexposed volunteers [57.3 (5.1)%]. CONCLUSION: Inhibition of neutrophil apoptosis at 24 hr culture was demonstrated in health care workers chronically exposed to volatile anesthetic agents. Exposure was well below recommended levels in the both scavenged and unscavenged work areas in which the study was carried out. Further study is required to assess the effect of greater degrees of chronic exposure to volatile anesthetic agents on neutrophil apoptosis.

  13. Neutrophils are resistant to Yersinia YopJ/P-induced apoptosis and are protected from ROS-mediated cell death by the type III secretion system.

    Directory of Open Access Journals (Sweden)

    Justin L Spinner

    2010-02-01

    Full Text Available The human innate immune system relies on the coordinated activity of macrophages and polymorphonuclear leukocytes (neutrophils or PMNs for defense against bacterial pathogens. Yersinia spp. subvert the innate immune response to cause disease in humans. In particular, the Yersinia outer protein YopJ (Y. pestis and Y. pseudotuberculosis and YopP (Y. enterocolitica rapidly induce apoptosis in murine macrophages and dendritic cells. However, the effects of Yersinia Yop J/P on neutrophil fate are not clearly defined.In this study, we utilized wild-type and mutant strains of Yersinia to test the contribution of YopJ and YopP on induction of apoptosis in human monocyte-derived macrophages (HMDM and neutrophils. Whereas YopJ and YopP similarly induced apoptosis in HMDMs, interaction of human neutrophils with virulence plasmid-containing Yersinia did not result in PMN caspase activation, release of LDH, or loss of membrane integrity greater than PMN controls. In contrast, interaction of human PMNs with the virulence plasmid-deficient Y. pestis strain KIM6 resulted in increased surface exposure of phosphatidylserine (PS and cell death. PMN reactive oxygen species (ROS production was inhibited in a virulence plasmid-dependent but YopJ/YopP-independent manner. Following phagocytic interaction with Y. pestis strain KIM6, inhibition of PMN ROS production with diphenyleneiodonium chloride resulted in a reduction of PMN cell death similar to that induced by the virulence plasmid-containing strain Y. pestis KIM5.Our findings showed that Yersinia YopJ and/or YopP did not induce pronounced apoptosis in human neutrophils. Furthermore, robust PMN ROS production in response to virulence plasmid-deficient Yersinia was associated with increased PMN cell death, suggesting that Yersinia inhibition of PMN ROS production plays a role in evasion of the human innate immune response in part by limiting PMN apoptosis.

  14. House Dust Mite Allergen Regulates Constitutive Apoptosis of Normal and Asthmatic Neutrophils via Toll-Like Receptor 4.

    Directory of Open Access Journals (Sweden)

    Do Hyung Kim

    Full Text Available House dust mites (HDMs induce allergic diseases such as asthma. Neutrophil apoptosis is an important process of innate immunity, and its dysregulation is associated with asthma. In this study, we examined the effects of HDM on constitutive apoptosis of normal and asthmatic neutrophils. Extract of Dermatophagoides pteronissinus (DP inhibited neutrophil apoptosis, but Dermatophagoides farinae extract had no effect. Anti-apoptotic signaling mediated by DP involves in TLR4, Lyn, PI3K, Akt, ERK, and NF-κB in normal neutrophils. DP delayed cleavage of procaspase 9 and procaspase 3 and the decrease in Mcl-1 expression. Supernatant collected from DP-treated normal neutrophils inhibited the constitutive apoptosis of normal neutrophils, and S100A8 and S100A9 were identified as anti-apoptotic proteins in the supernatant. S100A8 and S100A9 transduced the anti-apoptotic signal via TLR4, Lyn, PI3K, Akt, ERK, and NF-κB. DP also suppressed asthmatic neutrophil apoptosis and induced secretion of S100A8 and S100A9, which delayed the constitutive apoptosis. The anti-apoptotic effects of DP, S100A8 and S100A9 in asthmatic neutrophils are associated with TLR4, Lyn, PI3K, Akt, ERK, and NF-κB. The concentrations of S100A8 and S100A9 were significantly elevated in asthmatic bronchoalveolar lavage fluid (BALF when compared to normal BALF (p<0.01, but not in serum. S100A8 concentration in BALF was positively correlated with the number of BALF neutrophils and negatively correlated with FEV1(%. These findings improve our understanding of the role of HDM in regulation of neutrophil apoptosis in normal individuals and asthmatics and will enable elucidation of asthma pathogenesis.

  15. Characterization of Yersinia pestis Interactions with Human Neutrophils In vitro

    Directory of Open Access Journals (Sweden)

    Sophia C. Dudte

    2017-08-01

    Full Text Available Yersinia pestis is a gram-negative, zoonotic, bacterial pathogen, and the causative agent of plague. The bubonic form of plague occurs subsequent to deposition of bacteria in the skin by the bite of an infected flea. Neutrophils are recruited to the site of infection within the first few hours and interactions between neutrophils and Y. pestis have been demonstrated in vivo. In contrast to macrophages, neutrophils have been considered non-permissive to Y. pestis intracellular survival. Several studies have shown killing of the vast majority of Y. pestis ingested by human neutrophils. However, survival of 10–15% of Y. pestis after phagocytosis by neutrophils is consistently observed. Furthermore, these surviving bacteria eventually replicate within and escape from the neutrophils. We set out to further characterize the interactions between Y. pestis and human neutrophils by (1 determining the effects of known Y. pestis virulence factors on bacterial survival after uptake by neutrophils, (2 examining the mechanisms employed by the neutrophil to kill the majority of intracellular Y. pestis, (3 determining the activation phenotype of Y. pestis-infected neutrophils, and (4 characterizing the Y. pestis-containing phagosome in neutrophils. We infected human neutrophils in vitro with Y. pestis and assayed bacterial survival and uptake. Deletion of the caf1 gene responsible for F1 capsule production resulted in significantly increased uptake of Y. pestis. Surprisingly, while the two-component regulator PhoPQ system is important for survival of Y. pestis within neutrophils, pre-induction of this system prior to infection did not increase bacterial survival. We used an IPTG-inducible mCherry construct to distinguish viable from non-viable intracellular bacteria and determined the association of the Y. pestis-containing phagosome with neutrophil NADPH-oxidase and markers of primary, secondary and tertiary granules. Additionally, we show that inhibition of

  16. Regulation of calcium homeostasis in activated human neutrophils ...

    African Journals Online (AJOL)

    Objectives. The objectives of the current study were to: (i) present an integrated model for the restoration of calcium homeostasis in activated human neutrophils based on current knowledge and recent research; and (ii) identify potential targets for the modulation of calcium fluxes in activated neutrophils based on this model ...

  17. Swell activated chloride channel function in human neutrophils

    Energy Technology Data Exchange (ETDEWEB)

    Salmon, Michael D. [Leukocyte and Ion Channel Research Laboratory, School of Health and Biosciences, University of East London, Stratford Campus, London E15 4LZ (United Kingdom); Ahluwalia, Jatinder, E-mail: j.ahluwalia@uel.ac.uk [Leukocyte and Ion Channel Research Laboratory, School of Health and Biosciences, University of East London, Stratford Campus, London E15 4LZ (United Kingdom)

    2009-04-17

    Non-excitable cells such as neutrophil granulocytes are the archetypal inflammatory immune cell involved in critical functions of the innate immune system. The electron current generated (I{sub e}) by the neutrophil NADPH oxidase is electrogenic and rapidly depolarises the membrane potential. For continuous function of the NADPH oxidase, I{sub e} has to be balanced to preserve electroneutrality, if not; sufficient depolarisation would prevent electrons from leaving the cell and neutrophil function would be abrogated. Subsequently, the depolarisation generated by the neutrophil NADPH oxidase I{sub e} must be counteracted by ion transport. The finding that depolarisation required counter-ions to compensate electron transport was followed by the observation that chloride channels activated by swell can counteract the NADPH oxidase membrane depolarisation. In this mini review, we discuss the research findings that revealed the essential role of swell activated chloride channels in human neutrophil function.

  18. Platelet modulation of human neutrophil functions

    Energy Technology Data Exchange (ETDEWEB)

    McGarrity, S.T.; Hyers, T.M.; Webster, R.O.

    1986-03-01

    The combined presence of platelets (PLTS) and neutrophils (PMN) at inflammatory sites has led to examination of the hypothesis that interaction of these cells modulates their responses to stimuli. Gel-filtered human PLTS (GFP) were found to inhibit N-formyl-met-leu-phe (FMLP) and phorbol myristate acetate (PMA) stimulated PMN O/sub 2//sup -/ generation in a concentration-dependent fashion. The heat-stable inhibitory activity was present in the supernatants of GFP after incubation with FMLP (10/sup -7/M), thrombin (0.5 U/ml) or ADP (20 ..mu..M), suggesting a role for PLT release products. PLT lysates added to PMN produced up to 80% inhibition of O/sub 2//sup -/ generation for PMA and 40% for FMLP. Like GFP, lysates failed to scavenge O/sub 2/..pi.. produced by xanthine oxidase-hypoxanthine. The inhibitory activity could not be ascribed to serotonin or adenosine. PLT lysates failed to compete with /sup 3/H-FMLP for binding to PMN. Sephadex G-200 fractionation of PLT lysates releaved two peaks of inhibitory activity with apparent Mr > 200,000 and < 14,000 Daltons. Pretreatment of PMN with PLT lysates also results in a concentration-dependent inhibition of degranulation provoked by FMLP (2 x 10/sup -7/M) or PMA (2 ng/ml) and PMN chemotaxis to FMLP (10/sup -8/M). These studies indicate that preformed PLT mediator(s) released in response to physiological stimuli may limit tissue damage by PMN at sites of inflammation.

  19. Dopamine induces neutrophil apoptosis through a dopamine D-1 receptor-independent mechanism.

    LENUS (Irish Health Repository)

    Sookhai, S

    2012-02-03

    BACKGROUND: For the normal resolution of an acute inflammatory response, neutrophil (PMN) apoptosis is essential to maintain immune homeostasis and to limit inappropriate host tissue damage. A delay in PMN apoptosis has been implicated in the pathogenesis of the systemic inflammatory response syndrome (SIRS). Dopamine, a biogenic amine with known cardiovascular and neurotransmitter properties, is used in patients with SIRS to maintain hemodynamic stability. We sought to determine whether dopamine may also have immunoregulatory properties capable of influencing PMN apoptosis, function, and activation state in patients with SIRS. METHODS: PMNs were isolated from healthy volunteers and patients with SIRS and treated with varying doses of dopamine and a dopamine D-1 receptor agonist, fenoldopam. PMN apoptosis was assessed every 6 hours with use of propidium iodide DNA staining and PMN function was assessed with use of respiratory burst activity, phagocytosis ability, and CD11a, CD11b, and CD18 receptor expression as functional markers. RESULTS: There was a significant delay in PMN apotosis in patients with SIRS compared with controls. Treatment of isolated PMNs from both healthy controls and patients with SIRS with 10 and 100 mumol\\/L dopamine induced apoptosis. PMN ingestive and cytocidal capacity were both decreased in patients with SIRS compared with controls. Treatment with dopamine significantly increased phagocytic function. Fenoldopam did not induce PMN apoptosis. CONCLUSION: Our data demonstrate for the first time that dopamine induces PMN apoptosis and modulates PMN function both in healthy controls and in patients with SIRS. These results indicate that dopamine may be beneficial during SIRS through a nonhemodynamic PMN-dependent proapoptotic mechanism.

  20. Modulation and Apoptosis of Neutrophil Granulocytes by Extracorporeal Photopheresis in the Treatment of Chronic Graft-Versus-Host Disease.

    Directory of Open Access Journals (Sweden)

    Cindy Franklin

    Full Text Available Chronic graft-versus-host disease (cGVHD is a common side effect of allogeneic stem cell transplantation and a major cause of morbidity and mortality in affected patients. Especially skin, eyes and oral mucosa are affected. This can lead to pain and functional impairment. Extracorporeal photopheresis (ECP is an effective immunomodulatory therapy with minimal side effects but its mode of action is still largely unknown. The objective of the present study was to examine the effects of ECP on neutrophil granulocytes in patients with cGVHD. Analysis of leukocytes from cGVHD patients obtained from the ECP device during treatment showed that neutrophil granulocytes account for the majority of cells treated during ECP. Neutrophils from healthy donors treated in vitro with 8-methoxypsoralen and UVA light as well as neutrophils from buffy coats of patients with cGVHD treated by ECP showed increased apoptosis and decreased half-life. In remaining non-apoptotic cells chemoirradiation resulted in loss of activation markers and reduced effector functions. This was accompanied by an increase in extracellular arginase-1 activity. Additional comparison of neutrophils isolated from blood of cGVHD patients before and 24h after ECP revealed a decreased half-life and reduction of effector functions of post-ECP neutrophils ex vivo. These observations strongly suggest that ECP induces both apoptosis and physiological changes in neutrophils and that these changes also take place in vivo. This study is the first to show that ECP modulates apoptosis and inflammatory activity in neutrophil granulocytes, indicating that neutrophils may significantly contribute to the overall immunomodulatory effects attributed to this treatment.

  1. Effects of Acrolein on Leukotriene Biosynthesis in Human Neutrophils

    OpenAIRE

    Zemski Berry, Karin A.; Henson, Peter M.; Murphy, Robert C.

    2008-01-01

    Acrolein is a toxic, highly reactive α,β-unsaturated aldehyde that is present in high concentrations in cigarette smoke. In the current study, the effect of acrolein on eicosanoid synthesis in stimulated human neutrophils was examined. Eicosanoid synthesis in neutrophils was initiated by priming with granulocyte-macrophage colony-stimulating factor (GM-CSF) and subsequent stimulation with formyl-methionyl-leucyl-phenylalanine (fMLP) and 5-LO products in addition to small amounts of COX produc...

  2. Differential Use of Human Neutrophil Fcγ Receptors for Inducing Neutrophil Extracellular Trap Formation.

    Science.gov (United States)

    Alemán, Omar Rafael; Mora, Nancy; Cortes-Vieyra, Ricarda; Uribe-Querol, Eileen; Rosales, Carlos

    2016-01-01

    Neutrophils (PMN) are the most abundant leukocytes in the blood. PMN migrate from the circulation to sites of infection, where they are responsible for antimicrobial functions. PMN use phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes. NETs are fibers composed of chromatin and neutrophil-granule proteins. Several pathogens, including bacteria, fungi, and parasites, and also some pharmacological stimuli such as phorbol 12-myristate 13-acetate (PMA) are efficient inducers of NETs. Antigen-antibody complexes are also capable of inducing NET formation. However the particular Fcγ receptor involved in triggering this function is a matter of controversy. In order to provide some insight into what Fcγ receptor is responsible for NET formation, each of the two human Fcγ receptors was stimulated individually by specific monoclonal antibodies and NET formation was evaluated. FcγRIIa cross-linking did not promote NET formation. Cross-linking other receptors such as integrins also did not promote NET formation. In contrast FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. NET formation was dependent on NADPH-oxidase, PKC, and ERK activation. These data show that cross-linking FcγRIIIb is responsible for NET formation by the human neutrophil.

  3. Differential Use of Human Neutrophil Fcγ Receptors for Inducing Neutrophil Extracellular Trap Formation

    Directory of Open Access Journals (Sweden)

    Omar Rafael Alemán

    2016-01-01

    Full Text Available Neutrophils (PMN are the most abundant leukocytes in the blood. PMN migrate from the circulation to sites of infection, where they are responsible for antimicrobial functions. PMN use phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs to kill microbes. NETs are fibers composed of chromatin and neutrophil-granule proteins. Several pathogens, including bacteria, fungi, and parasites, and also some pharmacological stimuli such as phorbol 12-myristate 13-acetate (PMA are efficient inducers of NETs. Antigen-antibody complexes are also capable of inducing NET formation. However the particular Fcγ receptor involved in triggering this function is a matter of controversy. In order to provide some insight into what Fcγ receptor is responsible for NET formation, each of the two human Fcγ receptors was stimulated individually by specific monoclonal antibodies and NET formation was evaluated. FcγRIIa cross-linking did not promote NET formation. Cross-linking other receptors such as integrins also did not promote NET formation. In contrast FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. NET formation was dependent on NADPH-oxidase, PKC, and ERK activation. These data show that cross-linking FcγRIIIb is responsible for NET formation by the human neutrophil.

  4. Tumor Associated Neutrophils in Human Lung Cancer

    Science.gov (United States)

    2016-10-01

    tumor innate immune response. anti-tumor adaptive immune response, neutrophil and T cell interaction. ACCOMPLISHMENTS There were no significant...and by producing factors to recruit and acti- vate cells of the innate and adaptive immune system (Mantovani et al., 2011). Given these varying effects...vivo effects on neutro- phil activation (Figure 2, A and B) and cleavage of myeloid and lymphoid cell markers (Supplemental Figure 1, C–G). Once opti

  5. Chemotactic Activity on Human Neutrophils to Streptococcus mutans

    Directory of Open Access Journals (Sweden)

    Tetiana Haniastuti

    2013-07-01

    Full Text Available Objective: The aim of this study was to evaluate chemotactic activity o neutrophil to S. mutans. Chemotaxis assay was performed in blind well chambers. Materials and Methods: Hanks balanced salt solution (HBSS containing 106 S. mutans,  108 S. mutans, 10-8 M fMLP, or HBSS alone were placed in the lower wells of the chamber and covered with polycorbonate membrane filter. Neutrophils suspension (2x105 cells was then placed in the upper compartment. After incubation for 60 mins at 37ºC in a humidified atmosphere with 5% CO2, the filters were removed and stained with Giemsa. Result: ANOVA revealed statistically significant differences among groups (p<0.05, indicating that S. mutans induced neutrophils chemotaxis. The number of neutrophils migration in response to 108 S. mutans and 106 S. mutans were signifiantly greater compared to fMLP (p<0.05. Conclusion: S. mutans may activate human neutrophils, resulting in the chemotaxis of the neutrophils.DOI: 10.14693/jdi.v16i2.99

  6. Peptide secreted by human alveolar macrophages releases neutrophil granule contents

    International Nuclear Information System (INIS)

    MacArthur, C.K.; Miller, E.J.; Cohen, A.B.

    1987-01-01

    A monoclonal antibody was developed against an 8000-kDa enzyme-releasing peptide (ERP) released from human alveolar macrophages. ERP was isolated on an immunoaffinity column containing the antibody bound to staphylococcal protein A-Sepharose, and by autoradiography. Release of ERP from the macrophages is not changed by plastic adherence, phagocytosis, calcium ionophore, or phorbol esters. The peptide was not antigenically similar to interferon-γ, tumor necrosis factor, or interleukin lα or 1β. The release of constituents from azurophilic and specific granules was the main identified biologic function of ERP. ERP was a more effective secretagogue in the untreated neutrophils and f-met-leu-phe was more effective in the cytochalasin B-treated neutrophils. Absorption of ERP from macrophage-conditioned medium removed a small amount of the chemotactic activity; however, the immunopurified peptide was not chemotactic or chemokinetic for neutrophils, and at high concentrations, it suppressed base line chemokinesis. Treatment of washed macrophages with trypsin released active ERP of approximately the same m.w. of spontaneously secreted ERP. These studies showed that human alveolar macrophages release a peptide which is a secretagogue for human neutrophils under conditions which may be encountered in the lungs during certain disease states. Proteolytic enzymes which are free in the lungs may release the peptide and lead to the secretion of neutrophil enzymes

  7. The influence of very low doses of N-nitrosodimethylamine (NDMA) on the apoptosis of rat neutrophils in vivo. The role of reactive oxygen species.

    Science.gov (United States)

    Jablonski, J; Jablonska, E; Chojnowski, M

    2001-08-13

    N-nitrosodimethylamine (NDMA) causes the apoptosis of neutrophils in vitro experiments. This compound also has the ability to stimulate neutrophils for the production of reactive oxygen species. It has been decided to examine more closely whether the apoptosis of neutrophils by NDMA is caused by the influence of the radicals produced by these cells and whether the stimulation to undergo apoptosis of neutrophils is caused by NDMA in either the original form or by its metabolites. The experiment was conducted on rats. The animals were administered a one-time dose of NDMA intragastrically, 1.5 mg/kg. The research was conducted 1,2,4,12 h consecutively following NDMA administration. The concentration of NDMA in blood was evaluated by means of the gas chromatography method. The neutrophils were isolated from blood by means of differential centrifugation. Respiratory burst was assessed in cells, by means of the cytochrome c reduction method. The percentage of cells revealing morphological properties of apoptosis was determined under the fluorescent microscope. It has been observed that the activation of the respiratory burst is caused mainly by non-metabolised NDMA. Probably the non-metabolised molecules of this compound also have a decisive role in the initiation of apoptosis of neutrophils. It can be assumed that the main factor responsible for the apoptosis of neutrophil rats following a one-time NDMA administration is the induction of respiratory burst in neutrophils by this compound.

  8. Effects of acrolein on leukotriene biosynthesis in human neutrophils.

    Science.gov (United States)

    Berry, Karin A Zemski; Henson, Peter M; Murphy, Robert C

    2008-12-01

    Acrolein is a toxic, highly reactive alpha,beta-unsaturated aldehyde that is present in high concentrations in cigarette smoke. In the current study, the effect of acrolein on eicosanoid synthesis in stimulated human neutrophils was examined. Eicosanoid synthesis in neutrophils was initiated by priming with granulocyte-macrophage colony-stimulating factor (GM-CSF) and subsequent stimulation with formyl-methionyl-leucyl-phenylalanine (fMLP) and 5-lipoxygenase (5-LO) products in addition to small amounts of cyclooxygenase (COX) products were detected using LC/MS/MS. A dose-dependent decrease in the formation of 5-LO products was observed in GM-CSF/fMLP-stimulated neutrophils when acrolein (0-50 microM) was present with almost complete inhibition at > or = 25 microM acrolein. The production of COX products was not affected by acrolein in these cells. The effect of acrolein was examined on key parts of the eicosanoid pathway, such as arachidonic acid release, intracellular calcium ion concentration, and adenosine production. In addition, the direct effect of acrolein on 5-LO enzymatic activity was probed using a recombinant enzyme. Some of these factors were affected by acrolein but did not completely explain the almost complete inhibition of 5-LO product formation in GM-CSF/fMLP-treated cells with acrolein. In addition, the effect of acrolein on different stimuli that initiate the 5-LO pathway [platelet-activating factor (PAF)/fMLP, GM-CSF/PAF, opsonized zymosan, and A23187] was examined. Acrolein had no significant effect on the leukotriene production in neutrophils stimulated with PAF/fMLP, GM-CSF/ PAF, or OPZ. Additionally, 50% inhibition of the 5-LO pathway was observed in A23187-stimulated neutrophils. Our results suggest that acrolein has a profound effect on the 5-LO pathway in neutrophils, which may have implications in disease states, such as chronic obstructive pulmonary disease and other pulmonary disease, where both activated neutrophils and acrolein are

  9. Pharmacological intervention with oxidative burst in human neutrophils

    Czech Academy of Sciences Publication Activity Database

    Nosál, R.; Drábiková, K.; Jančinová, V.; Mačičková, T.; Pečivová, J.; Perečko, T.; Harmatha, Juraj

    2017-01-01

    Roč. 10, č. 2 (2017), s. 56-60 ISSN 1337-6853 Institutional support: RVO:61388963 Keywords : human neutrophils * oxidative burst * tharapeutical drugs * natural antioxidants Subject RIV: FR - Pharmacology ; Medidal Chemistry OBOR OECD: Pharmacology and pharmacy https://www.degruyter.com/downloadpdf/j/intox.2017.10.issue-2/intox-2017-0009/intox-2017-0009.pdf

  10. Enhancement by platelets of oxygen radical responses of human neutrophils

    Energy Technology Data Exchange (ETDEWEB)

    McCulloch, K.K.; Powell, J.; Johnson, K.J.; Ward, P.A.

    1986-03-01

    When human blood neutrophils were incubated with immune complexes (consisting of IgG antibody) in the presence of platelets, there was a 2 to 10 fold enhancement in the generation of O-/sub 2/ and H/sub 2/O/sub 2/. This enhancement phenomenon was proportional to the dose of immune complex added and the number of platelets present. The response was not agonist specific since similar enhancement also occurred with the following agonists: phorbol myristate acetate, opsonized zymosan particles and the chemotactic peptide N-formyl-met-leu-phe. The platelet related phenomenon of enhanced O-/sub 2/ generation could not be reproduced by the addition of serotonin, histamine or platelet-derived growth factor and was not affected by prior treatment of platelets with cyclooxygenase inhibitors (indomethacin, piroxicam) or lipoxygenase inhibitors (nafazatrom, BW755C or nordihydroguaiaretic acid). However, activation of platelets by thrombin caused release into the platelet supernatant fluid of a factor that, only in the presence of immune complexes, caused enhanced O-/sub 2/ responses to neutrophils. These data indicate that platelets potentiate oxygen radical responses of human neutrophils and suggest a mechanisms by which platelets may participate in tissue injury which is mediated by oxygen radical products from activated neutrophils.

  11. Enhancement by platelets of oxygen radical responses of human neutrophils

    International Nuclear Information System (INIS)

    McCulloch, K.K.; Powell, J.; Johnson, K.J.; Ward, P.A.

    1986-01-01

    When human blood neutrophils were incubated with immune complexes (consisting of IgG antibody) in the presence of platelets, there was a 2 to 10 fold enhancement in the generation of O- 2 and H 2 O 2 . This enhancement phenomenon was proportional to the dose of immune complex added and the number of platelets present. The response was not agonist specific since similar enhancement also occurred with the following agonists: phorbol myristate acetate, opsonized zymosan particles and the chemotactic peptide N-formyl-met-leu-phe. The platelet related phenomenon of enhanced O- 2 generation could not be reproduced by the addition of serotonin, histamine or platelet-derived growth factor and was not affected by prior treatment of platelets with cyclooxygenase inhibitors (indomethacin, piroxicam) or lipoxygenase inhibitors (nafazatrom, BW755C or nordihydroguaiaretic acid). However, activation of platelets by thrombin caused release into the platelet supernatant fluid of a factor that, only in the presence of immune complexes, caused enhanced O- 2 responses to neutrophils. These data indicate that platelets potentiate oxygen radical responses of human neutrophils and suggest a mechanisms by which platelets may participate in tissue injury which is mediated by oxygen radical products from activated neutrophils

  12. Ascorbic acid transport and accumulation in human neutrophils

    International Nuclear Information System (INIS)

    Washko, P.; Rotrosen, D.; Levine, M.

    1989-01-01

    The transport, accumulation, and distribution of ascorbic acid were investigated in isolated human neutrophils utilizing a new ascorbic acid assay, which combined the techniques of high performance liquid chromatography and coulometric electrochemical detection. Freshly isolated human neutrophils contained 1.0-1.4 mM ascorbic acid, which was localized greater than or equal to 94% to the cytosol, was not protein bound, and was present only as ascorbic acid and not as dehydroascorbic acid. Upon addition of ascorbic acid to the extracellular medium in physiologic amounts, ascorbic acid was accumulated in neutrophils in millimolar concentrations. Accumulation was mediated by a high affinity and a low affinity transporter; both transporters were responsible for maintenance of concentration gradients as large as 50-fold. The high affinity transporter had an apparent Km of 2-5 microns by Lineweaver-Burk and Eadie-Hofstee analyses, and the low affinity transporter had an apparent Km of 6-7 mM by similar analyses. Each transporter was saturable and temperature dependent. In normal human blood the high affinity transporter should be saturated, whereas the low affinity transporter should be in its linear phase of uptake

  13. Autophagy Mediates Interleukin-1β Secretion in Human Neutrophils

    Directory of Open Access Journals (Sweden)

    Leonardo Iula

    2018-02-01

    AEBSF reduced IL-1β secretion. Moreover, IL-1β could be also found colocalizing with elastase, suggesting both some vesicles containing IL-1β intersect azurophil granules content and that serine proteases also regulate IL-1β secretion. Altogether, our findings indicate that an unconventional autophagy-mediated secretory pathway mediates IL-1β secretion in human neutrophils.

  14. Staphylococcus epidermidis strategies to avoid killing by human neutrophils.

    Directory of Open Access Journals (Sweden)

    Gordon Y C Cheung

    2010-10-01

    Full Text Available Staphylococcus epidermidis is a leading nosocomial pathogen. In contrast to its more aggressive relative S. aureus, it causes chronic rather than acute infections. In highly virulent S. aureus, phenol-soluble modulins (PSMs contribute significantly to immune evasion and aggressive virulence by their strong ability to lyse human neutrophils. Members of the PSM family are also produced by S. epidermidis, but their role in immune evasion is not known. Notably, strong cytolytic capacity of S. epidermidis PSMs would be at odds with the notion that S. epidermidis is a less aggressive pathogen than S. aureus, prompting us to examine the biological activities of S. epidermidis PSMs. Surprisingly, we found that S. epidermidis has the capacity to produce PSMδ, a potent leukocyte toxin, representing the first potent cytolysin to be identified in that pathogen. However, production of strongly cytolytic PSMs was low in S. epidermidis, explaining its low cytolytic potency. Interestingly, the different approaches of S. epidermidis and S. aureus to causing human disease are thus reflected by the adaptation of biological activities within one family of virulence determinants, the PSMs. Nevertheless, S. epidermidis has the capacity to evade neutrophil killing, a phenomenon we found is partly mediated by resistance mechanisms to antimicrobial peptides (AMPs, including the protease SepA, which degrades AMPs, and the AMP sensor/resistance regulator, Aps (GraRS. These findings establish a significant function of SepA and Aps in S. epidermidis immune evasion and explain in part why S. epidermidis may evade elimination by innate host defense despite the lack of cytolytic toxin expression. Our study shows that the strategy of S. epidermidis to evade elimination by human neutrophils is characterized by a passive defense approach and provides molecular evidence to support the notion that S. epidermidis is a less aggressive pathogen than S. aureus.

  15. A novel bacterial transport mechanism of Acinetobacter baumannii via activated human neutrophils through interleukin-8.

    Science.gov (United States)

    Kamoshida, Go; Tansho-Nagakawa, Shigeru; Kikuchi-Ueda, Takane; Nakano, Ryuichi; Hikosaka, Kenji; Nishida, Satoshi; Ubagai, Tsuneyuki; Higashi, Shouichi; Ono, Yasuo

    2016-12-01

    Hospital-acquired infections as a result of Acinetobacter baumannii have become problematic because of high rates of drug resistance. Although neutrophils play a critical role in early protection against bacterial infection, their interactions with A. baumannii remain largely unknown. To elucidate the interactions between A. baumannii and human neutrophils, we cocultured these cells and analyzed them by microscopy and flow cytometry. We found that A. baumannii adhered to neutrophils. We next examined neutrophil and A. baumannii infiltration into Matrigel basement membranes by an in vitro transmigration assay. Neutrophils were activated by A. baumannii, and invasion was enhanced. More interestingly, A. baumannii was transported together by infiltrating neutrophils. Furthermore, we observed by live cell imaging that A. baumannii and neutrophils moved together. In addition, A. baumannii-activated neutrophils showed increased IL-8 production. The transport of A. baumannii was suppressed by inhibiting neutrophil infiltration by blocking the effect of IL-8. A. baumannii appears to use neutrophils for transport by activating these cells via IL-8. In this study, we revealed a novel bacterial transport mechanism that A. baumannii exploits human neutrophils by adhering to and inducing IL-8 release for bacterial portage. This mechanism might be a new treatment target. © Society for Leukocyte Biology.

  16. Honey and Apoptosis in Human Gastric Mucosa

    Directory of Open Access Journals (Sweden)

    Alireza Ostadrahimi

    2012-07-01

    Full Text Available Background: Gastric cancer is the fourth most common malignancy in the world. Honey is acomplex mixture of special biological active constituents. Honey possesses antioxidant and antitumorproperties. Nutritional studies have indicated that consumption of honey modulates therisk of developing gastric cancer. On the other hand, apoptosis has been reported to play a decisiverole in precancerous changes. Our chief study was conducted to assess the relationship betweenconsumption of honey and apoptosis in human gastric mucosa.Method: This cross-sectional study was conducted on 98 subjects over 18 years old, referred totwo hospitals in Tabriz, Iran. Subjects were undergone an upper gastrointestinal endoscopy, 62subjects were finally enrolled. Honey consumption was assessed by a Food Frequency Questionnaire(FFQ and apoptosis was detected by TUNEL technique. We tested polynomial curve tofind the best fit between honey consumption and apoptosis.Results: A positive relation between honey consumption and apoptosis was found (P=0.024.Our results indicated that the final and the best fit curve was: apoptosis = 1.714+1.648(honeyamount - 0.533(honey amount2 +1.833×10-5(honey amount7.Conclusion: Honey consumption had positive effects on gastric cancer by inducing apoptosis ingastric mucosa.

  17. Activation of human herpesvirus replication by apoptosis.

    Science.gov (United States)

    Prasad, Alka; Remick, Jill; Zeichner, Steven L

    2013-10-01

    A central feature of herpesvirus biology is the ability of herpesviruses to remain latent within host cells. Classically, exposure to inducing agents, like activating cytokines or phorbol esters that stimulate host cell signal transduction events, and epigenetic agents (e.g., butyrate) was thought to end latency. We recently showed that Kaposi's sarcoma-associated herpesvirus (KSHV, or human herpesvirus-8 [HHV-8]) has another, alternative emergency escape replication pathway that is triggered when KSHV's host cell undergoes apoptosis, characterized by the lack of a requirement for the replication and transcription activator (RTA) protein, accelerated late gene kinetics, and production of virus with decreased infectivity. Caspase-3 is necessary and sufficient to initiate the alternative replication program. HSV-1 was also recently shown to initiate replication in response to host cell apoptosis. These observations suggested that an alternative apoptosis-triggered replication program might be a general feature of herpesvirus biology and that apoptosis-initiated herpesvirus replication may have clinical implications, particularly for herpesviruses that almost universally infect humans. To explore whether an alternative apoptosis-initiated replication program is a common feature of herpesvirus biology, we studied cell lines latently infected with Epstein-Barr virus/HHV-4, HHV-6A, HHV-6B, HHV-7, and KSHV. We found that apoptosis triggers replication for each HHV studied, with caspase-3 being necessary and sufficient for HHV replication. An alternative apoptosis-initiated replication program appears to be a common feature of HHV biology. We also found that commonly used cytotoxic chemotherapeutic agents activate HHV replication, which suggests that treatments that promote apoptosis may lead to activation of latent herpesviruses, with potential clinical significance.

  18. Targeting Neutrophilic Inflammation Using Polymersome-Mediated Cellular Delivery.

    Science.gov (United States)

    Robertson, James D; Ward, Jon R; Avila-Olias, Milagros; Battaglia, Giuseppe; Renshaw, Stephen A

    2017-05-01

    Neutrophils are key effector cells in inflammation and play an important role in neutralizing invading pathogens. During inflammation resolution, neutrophils undergo apoptosis before they are removed by macrophages, but if apoptosis is delayed, neutrophils can cause extensive tissue damage and chronic disease. Promotion of neutrophil apoptosis is a potential therapeutic approach for treating persistent inflammation, yet neutrophils have proven difficult cells to manipulate experimentally. In this study, we deliver therapeutic compounds to neutrophils using biocompatible, nanometer-sized synthetic vesicles, or polymersomes, which are internalized by binding to scavenger receptors and subsequently escape the early endosome through a pH-triggered disassembly mechanism. This allows polymersomes to deliver molecules into the cell cytosol of neutrophils without causing cellular activation. After optimizing polymersome size, we show that polymersomes can deliver the cyclin-dependent kinase inhibitor (R)-roscovitine into human neutrophils to promote apoptosis in vitro. Finally, using a transgenic zebrafish model, we show that encapsulated (R)-roscovitine can speed up inflammation resolution in vivo more efficiently than the free drug. These results show that polymersomes are effective intracellular carriers for drug delivery into neutrophils. This has important consequences for the study of neutrophil biology and the development of neutrophil-targeted therapeutics. Copyright © 2017 The Authors.

  19. Anti-Inflammatory benefits of antibiotic-induced neutrophil apoptosis: tulathromycin induces caspase-3-dependent neutrophil programmed cell death and inhibits NF-kappaB signaling and CXCL8 transcription.

    Science.gov (United States)

    Fischer, Carrie D; Beatty, Jennifer K; Zvaigzne, Cheryl G; Morck, Douglas W; Lucas, Merlyn J; Buret, A G

    2011-01-01

    Clearance of apoptotic neutrophils is a central feature of the resolution of inflammation. Findings indicate that immuno-modulation and induction of neutrophil apoptosis by macrolide antibiotics generate anti-inflammatory benefits via mechanisms that remain obscure. Tulathromycin (TUL), a new antimicrobial agent for bovine respiratory disease, offers superior clinical efficacy for reasons not fully understood. The aim of this study was to identify the immuno-modulating effects of tulathromycin and, in this process, to establish tulathromycin as a new model for characterizing the novel anti-inflammatory properties of antibiotics. Bronchoalveolar lavage specimens were collected from Holstein calves 3 and 24 h postinfection, challenged intratracheally with live Mannheimia haemolytica (2 × 10(7) CFU), and treated with vehicle or tulathromycin (2.5 mg/kg body weight). Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining and enzyme-linked immunosorbent assay (ELISA) revealed that tulathromycin treatment significantly increased leukocyte apoptosis and reduced levels of proinflammatory leukotriene B(4) in M. haemolytica-challenged calves. In vitro, tulathromycin concentration dependently induced apoptosis in freshly isolated bovine neutrophils from healthy steers in a capase-3-dependent manner but failed to induce apoptosis in bovine fibroblasts, epithelial cells, and endothelial cells, as well as freshly isolated bovine blood monocytes and monocyte-derived macrophages. The proapoptotic effects of TUL were also, in part, drug specific; equimolar concentrations of penicillin G, oxytetracycline, and ceftiofur failed to cause apoptosis in bovine neutrophils. In addition, tulathromycin significantly reduced levels of phosphorylated IκBα, nuclear translocation of NF-κB p65, and mRNA levels of proinflammatory interleukin-8 in lipopolysaccharide (LPS)-stimulated bovine neutrophils. The findings illustrate novel mechanisms through which

  20. Computer-assisted image analysis assay of human neutrophil chemotaxis in vitro

    DEFF Research Database (Denmark)

    Jensen, P; Kharazmi, A

    1991-01-01

    We have developed a computer-based image analysis system to measure in-filter migration of human neutrophils in the Boyden chamber. This method is compared with the conventional manual counting techniques. Neutrophils from healthy individuals and from patients with reduced chemotactic activity were....... Another advantage of the assay is that it can be used to show the migration pattern of different populations of neutrophils from both healthy individuals and patients....

  1. Equol Effectively Inhibits Toxic Activity of Human Neutrophils without Influencing Their Viability

    Czech Academy of Sciences Publication Activity Database

    Pažoureková, S.; Lucová, M.; Nosál, R.; Drábiková, K.; Harmatha, Juraj; Šmidrkal, J.; Jančinová, V.

    2016-01-01

    Roč. 97, 3/4 (2016), s. 138-145 ISSN 0031-7012 Institutional support: RVO:61388963 Keywords : neutrophils * equol * chemiluminescence * reactive oxygen species * p40(phox) * apoptosis Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 1.442, year: 2016

  2. p21-Activated kinase (PAK regulates cytoskeletal reorganization and directional migration in human neutrophils.

    Directory of Open Access Journals (Sweden)

    Asako Itakura

    Full Text Available Neutrophils serve as a first line of defense in innate immunity owing in part to their ability to rapidly migrate towards chemotactic factors derived from invading pathogens. As a migratory function, neutrophil chemotaxis is regulated by the Rho family of small GTPases. However, the mechanisms by which Rho GTPases orchestrate cytoskeletal dynamics in migrating neutrophils remain ill-defined. In this study, we characterized the role of p21-activated kinase (PAK downstream of Rho GTPases in cytoskeletal remodeling and chemotactic processes of human neutrophils. We found that PAK activation occurred upon stimulation of neutrophils with f-Met-Leu-Phe (fMLP, and PAK accumulated at the actin-rich leading edge of stimulated neutrophils, suggesting a role for PAK in Rac-dependent actin remodeling. Treatment with the pharmacological PAK inhibitor, PF3758309, abrogated the integrity of RhoA-mediated actomyosin contractility and surface adhesion. Moreover, inhibition of PAK activity impaired neutrophil morphological polarization and directional migration under a gradient of fMLP, and was associated with dysregulated Ca(2+ signaling. These results suggest that PAK serves as an important effector of Rho-family GTPases in neutrophil cytoskeletal reorganization, and plays a key role in driving efficient directional migration of human neutrophils.

  3. Neutrophil-Derived MMP-8 Drives AMPK-Dependent Matrix Destruction in Human Pulmonary Tuberculosis

    Science.gov (United States)

    Ong, Catherine W. M.; Elkington, Paul T.; Brilha, Sara; Ugarte-Gil, Cesar; Tome-Esteban, Maite T.; Tezera, Liku B.; Pabisiak, Przemyslaw J.; Moores, Rachel C.; Sathyamoorthy, Tarangini; Patel, Vimal; Gilman, Robert H.; Porter, Joanna C.; Friedland, Jon S.

    2015-01-01

    Pulmonary cavities, the hallmark of tuberculosis (TB), are characterized by high mycobacterial load and perpetuate the spread of M. tuberculosis. The mechanism of matrix destruction resulting in cavitation is not well defined. Neutrophils are emerging as key mediators of TB immunopathology and their influx are associated with poor outcomes. We investigated neutrophil-dependent mechanisms involved in TB-associated matrix destruction using a cellular model, a cohort of 108 patients, and in separate patient lung biopsies. Neutrophil-derived NF-kB-dependent matrix metalloproteinase-8 (MMP-8) secretion was up-regulated in TB and caused matrix destruction both in vitro and in respiratory samples of TB patients. Collagen destruction induced by TB infection was abolished by doxycycline, a licensed MMP inhibitor. Neutrophil extracellular traps (NETs) contain MMP-8 and are increased in samples from TB patients. Neutrophils lined the circumference of human pulmonary TB cavities and sputum MMP-8 concentrations reflected TB radiological and clinical disease severity. AMPK, a central regulator of catabolism, drove neutrophil MMP-8 secretion and neutrophils from AMPK-deficient patients secrete lower MMP-8 concentrations. AMPK-expressing neutrophils are present in human TB lung biopsies with phospho-AMPK detected in nuclei. These data demonstrate that neutrophil-derived MMP-8 has a key role in the immunopathology of TB and is a potential target for host-directed therapy in this infectious disease. PMID:25996154

  4. Neutrophil-Derived MMP-8 Drives AMPK-Dependent Matrix Destruction in Human Pulmonary Tuberculosis.

    Science.gov (United States)

    Ong, Catherine W M; Elkington, Paul T; Brilha, Sara; Ugarte-Gil, Cesar; Tome-Esteban, Maite T; Tezera, Liku B; Pabisiak, Przemyslaw J; Moores, Rachel C; Sathyamoorthy, Tarangini; Patel, Vimal; Gilman, Robert H; Porter, Joanna C; Friedland, Jon S

    2015-05-01

    Pulmonary cavities, the hallmark of tuberculosis (TB), are characterized by high mycobacterial load and perpetuate the spread of M. tuberculosis. The mechanism of matrix destruction resulting in cavitation is not well defined. Neutrophils are emerging as key mediators of TB immunopathology and their influx are associated with poor outcomes. We investigated neutrophil-dependent mechanisms involved in TB-associated matrix destruction using a cellular model, a cohort of 108 patients, and in separate patient lung biopsies. Neutrophil-derived NF-kB-dependent matrix metalloproteinase-8 (MMP-8) secretion was up-regulated in TB and caused matrix destruction both in vitro and in respiratory samples of TB patients. Collagen destruction induced by TB infection was abolished by doxycycline, a licensed MMP inhibitor. Neutrophil extracellular traps (NETs) contain MMP-8 and are increased in samples from TB patients. Neutrophils lined the circumference of human pulmonary TB cavities and sputum MMP-8 concentrations reflected TB radiological and clinical disease severity. AMPK, a central regulator of catabolism, drove neutrophil MMP-8 secretion and neutrophils from AMPK-deficient patients secrete lower MMP-8 concentrations. AMPK-expressing neutrophils are present in human TB lung biopsies with phospho-AMPK detected in nuclei. These data demonstrate that neutrophil-derived MMP-8 has a key role in the immunopathology of TB and is a potential target for host-directed therapy in this infectious disease.

  5. Induction of hyperresponsiveness in human airway tissue by neutrophils--mechanism of action.

    Science.gov (United States)

    Anticevich, S Z; Hughes, J M; Black, J L; Armour, C L

    1996-05-01

    The two main features of asthma are bronchial hyperresponsiveness and inflammation. The inflammatory response in asthma consists of infiltration and activation of a variety of inflammatory cells including neutrophils. Our previous studies have shown that stimulated neutrophil supernatants cause hyperresponsiveness of human bronchial tissue in vitro. To investigate the effect of the sensitization status of the tissue and the albumin concentration used to prepare supernatants on the response of human bronchial tissue to stimulated neutrophil supernatants. Neutrophil supernatants were prepared from human isolated blood in the presence of varying concentrations of albumin (0%, 0.1% and 4%). Neutrophil supernatants were added to sensitized and non-sensitized human isolated bronchial tissue which was stimulated with electrical field stimulation (EFS) (20 s every 4 min). Receptor antagonists specific for the prostaglandin and thromboxane (10(-7) M GR32191), platelet activating factor (10(-6) M WEB 2086), leukotriene D4 (10(-6) M MK-679) and neurokinin A (10(-7) M SR48968) receptors were used to identify neutrophil products responsible for the effects observed in the bronchial tissue. In non-sensitized human bronchial tissue, stimulated neutrophil supernatants induced a direct contraction in the presence of 0% and 0.1% but not 4% albumin. This contraction was due to leukotriene D4 as MK-679 completely inhibited the contraction. In contrast, stimulated neutrophil supernatants increased responsiveness of sensitized human bronchial tissue to EFS. The increased responsiveness was observed only in the presence of 0.1% albumin, with the site of modulation likely to be prejunctional on the parasympathetic nerve. The increased responsiveness was not inhibited by any of the antagonists tested. Sensitization status of the tissue and albumin concentration effect the responsiveness of human bronchial tissue to stimulated neutrophil supernatant. Our results suggest a possible role for

  6. Synchronisation of glycolytic oscillations in a suspension of human neutrophils

    DEFF Research Database (Denmark)

    Brasen, Jens Christian; Poulsen, Allan K.; Olsen, Lars Folke

    Neutrophils are known to be able to synchronize their production of superoxide. We show that glycolysis is also synchronized in human neutrophils being in suspension and suggest that oscillations in glycolysis are driving the pulsatile production of superoxide. The synchronising agent remains so...... far unknown, however, much evident points to that it might be hydrogen peroxide or an intermediate in glycolysis....

  7. Medium-chain, triglyceride-containing lipid emulsions increase human neutrophil beta2 integrin expression, adhesion, and degranulation

    NARCIS (Netherlands)

    Wanten, G. J.; Geijtenbeek, T. B.; Raymakers, R. A.; van Kooyk, Y.; Roos, D.; Jansen, J. B.; Naber, A. H.

    2000-01-01

    BACKGROUND: To test the hypothesis that lipid emulsions with different triglyceride structures have distinct immunomodulatory properties, we analyzed human neutrophil adhesion and degranulation after lipid incubation. METHODS: Neutrophils, isolated from the blood of 10 healthy volunteers, were

  8. Local anesthetic-induced inhibition of human neutrophil priming: the influence of structure, lipophilicity, and charge

    NARCIS (Netherlands)

    Picardi, Susanne; Cartellieri, Sibylle; Groves, Danja; Hahnenekamp, Klaus; Gerner, Peter; Durieux, Marcel E.; Stevens, Markus F.; Lirk, Philipp; Hollmann, Markus W.

    2013-01-01

    Local anesthetics (LAs) are widely known for inhibition of voltage-gated sodium channels underlying their antiarrhythmic and antinociceptive effects. However, LAs have significant immunomodulatory properties and were shown to affect human neutrophil functions independent of sodium-channel blockade.

  9. Activated human neutrophils release hepatocyte growth factor/scatter factor.

    LENUS (Irish Health Repository)

    McCourt, M

    2012-02-03

    BACKGROUND: Hepatocyte growth factor or scatter factor (HGF\\/SF) is a pleiotropic cytokine that has potent angiogenic properties. We have previously demonstrated that neutrophils (PMN) are directly angiogenic by releasing vascular endothelial growth factor (VEGF). We hypothesized that the acute inflammatory response can stimulate PMN to release HGF. AIMS: To examine the effects of inflammatory mediators on PMN HGF release and the effect of recombinant human HGF (rhHGF) on PMN adhesion receptor expression and PMN VEGF release. METHODS: In the first experiment, PMN were isolated from healthy volunteers and stimulated with tumour necrosis factor-alpha (TNF-alpha), lipopolysaccharide (LPS), interleukin-8 (IL-8), and formyl methionyl-leucyl-phenylalanine (fMLP). Culture supernatants were assayed for HGF using ELISA. In the second experiment, PMN were lysed to measure total HGF release and HGF expression in the PMN was detected by Western immunoblotting. Finally, PMN were stimulated with rhHGF. PMN CD 11a, CD 11b, and CD 18 receptor expression and VEGF release was measured using flow cytometry and ELISA respectively. RESULTS: TNF-alpha, LPS and fMLP stimulation resulted in significantly increased release of PMN HGF (755+\\/-216, 484+\\/-221 and 565+\\/-278 pg\\/ml, respectively) compared to controls (118+\\/-42 pg\\/ml). IL-8 had no effect. Total HGF release following cell lysis and Western blot suggests that HGF is released from intracellular stores. Recombinant human HGF did not alter PMN adhesion receptor expression and had no effect on PMN VEGF release. CONCLUSIONS: This study demonstrates that pro-inflammatory mediators can stimulate HGF release from a PMN intracellular store and that activated PMN in addition to secreting VEGF have further angiogenic potential by releasing HGF.

  10. Neutrophil activation during acetaminophen hepatotoxicity and repair in mice and humans

    Energy Technology Data Exchange (ETDEWEB)

    Williams, C. David; Bajt, Mary Lynn [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Sharpe, Matthew R. [Department of Internal Medicine, University of Kansas Hospital, Kansas City, KS (United States); McGill, Mitchell R. [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States); Farhood, Anwar [Department of Pathology, St. David' s North Austin Medical Center, Austin, TX 78756 (United States); Jaeschke, Hartmut, E-mail: hjaeschke@kumc.edu [Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS (United States)

    2014-03-01

    Following acetaminophen (APAP) overdose there is an inflammatory response triggered by the release of cellular contents from necrotic hepatocytes into the systemic circulation which initiates the recruitment of neutrophils into the liver. It has been demonstrated that neutrophils do not contribute to APAP-induced liver injury, but their role and the role of NADPH oxidase in injury resolution are controversial. C57BL/6 mice were subjected to APAP overdose and neutrophil activation status was determined during liver injury and liver regeneration. Additionally, human APAP overdose patients (ALT: > 800 U/L) had serial blood draws during the injury and recovery phases for the determination of neutrophil activation. Neutrophils in the peripheral blood of mice showed an increasing activation status (CD11b expression and ROS priming) during and after the peak of injury but returned to baseline levels prior to complete injury resolution. Hepatic sequestered neutrophils showed an increased and sustained CD11b expression, but no ROS priming was observed. Confirming that NADPH oxidase is not critical to injury resolution, gp91{sup phox}−/− mice following APAP overdose displayed no alteration in injury resolution. Peripheral blood from APAP overdose patients also showed increased neutrophil activation status after the peak of liver injury and remained elevated until discharge from the hospital. In mice and humans, markers of activation, like ROS priming, were increased and sustained well after active liver injury had subsided. The similar findings between surviving patients and mice indicate that neutrophil activation may be a critical event for host defense or injury resolution following APAP overdose, but not a contributing factor to APAP-induced injury. - Highlights: • Neutrophil (PMN) function increases during liver repair after acetaminophen overdose. • Liver repair after acetaminophen (APAP)-overdose is not dependent on NADPH oxidase. • Human PMNs do not appear

  11. α-1 Antitrypsin regulates human neutrophil chemotaxis induced by soluble immune complexes and IL-8.

    LENUS (Irish Health Repository)

    Bergin, David A

    2010-12-01

    Hereditary deficiency of the protein α-1 antitrypsin (AAT) causes a chronic lung disease in humans that is characterized by excessive mobilization of neutrophils into the lung. However, the reason for the increased neutrophil burden has not been fully elucidated. In this study we have demonstrated using human neutrophils that serum AAT coordinates both CXCR1- and soluble immune complex (sIC) receptor-mediated chemotaxis by divergent pathways. We demonstrated that glycosylated AAT can bind to IL-8 (a ligand for CXCR1) and that AAT-IL-8 complex formation prevented IL-8 interaction with CXCR1. Second, AAT modulated neutrophil chemotaxis in response to sIC by controlling membrane expression of the glycosylphosphatidylinositol-anchored (GPI-anchored) Fc receptor FcγRIIIb. This process was mediated through inhibition of ADAM-17 enzymatic activity. Neutrophils isolated from clinically stable AAT-deficient patients were characterized by low membrane expression of FcγRIIIb and increased chemotaxis in response to IL-8 and sIC. Treatment of AAT-deficient individuals with AAT augmentation therapy resulted in increased AAT binding to IL-8, increased AAT binding to the neutrophil membrane, decreased FcγRIIIb release from the neutrophil membrane, and normalization of chemotaxis. These results provide new insight into the mechanism underlying the effect of AAT augmentation therapy in the pulmonary disease associated with AAT deficiency.

  12. Doxycycline induced photodamage to human neutrophils and tryptophan

    International Nuclear Information System (INIS)

    Sandberg, S.; Glette, J.; Hopen, G.; Solberg, C.O.

    1984-01-01

    Neutrophil function were studied following irradiation (340-380 nm) of the cells in the presence of 22 μM doxycycline. At increasing light fluence the locomotion, chemiluminescence and glucose oxidation (by the hexose monophosphate shunt) of the neutrophils steadily decreased. The photodamage increased with increasing preincubation temperature and time and was enhanced in D 2 O, reduced in azide and abolished in anaerobiosis. Superoxide dismutase, catalase or mannitol did not influence the photodamage. Photooxidation of tryptophan in the presence of doxycycline was increased 9-10-fold in D 2 O and nearly abolished in the presence of 0.25 mM NaN 3 , indicating that singlet oxygen is the most important reactive oxygen species in the doxycycline-induced photodamage. The results may explain some of the features of tetracycline-induced photosensitivity and why other authors have obtained diverging results when studying the influence of tetracyclines on neutrophil functions. (author)

  13. Naloxone inhibits superoxide but not enzyme release by human neutrophils

    Energy Technology Data Exchange (ETDEWEB)

    Simpkins, C.; Alailima, S.; Tate, E.

    1986-03-01

    The release of toxic oxygen metabolites and enzymes by phagocytic cells is thought to play a role in the multisystemic tissue injury of sepsis. Naloxone protects septic animals. We have found that at concentrations administered to animals (10/sup -7/ to 10/sup -4/M), naloxone inhibited (p < .001) the release of superoxide (O/sub 2//sup -/) by human neutrophils (HN), stimulated with N-formyl methionyl leucyl phenylalanine (FMLP). Naloxone had no effect on cell viability. Maximum inhibition was 65% of the total O/sub 2//sup -/ released (13.1 nMoles/8 min/320,000 cells). FMLP-stimulated release of beta-glucoronidase or lysozyme was not altered by naloxone. Naloxone had no effect on the binding of /sup 3/H FMLP to HN. Using /sup 3/H naloxone and various concentrations of unlabeled naloxone higher affinity (K/sub D/ = 12nM) and lower affinity (K/sub D/ = 4.7 x 10/sup -5/) binding sites were detected. The K/sub D/ of the low affinity site corresponded to the ED/sub 50/ for naloxone inhibition of O/sub 2//sup -/ (1 x 10/sup -5/M). Binding to this low affinity site was decreased by (+) naloxone, beta-endorphin and N acetyl beta-endorphin, but not by leu-enkephalin, thyrotropin releasing factor, prostaglandin D/sub 2/ or E/sub 2/. Conclusions: (1) naloxone inhibits FMLP-stimulated O/sub 2/ but not enzyme release, (2) this inhibition is not due to alteration of FMLP receptor binding, (3) naloxone may act via a low affinity binding site which is ligand specific, and (4) a higher affinity receptor is present on HN.

  14. Intracellular accumulation of potent amiloride analogues by human neutrophils

    International Nuclear Information System (INIS)

    Simchowitz, L.; Woltersdorf, O.W. Jr.; Cragoe, E.J. Jr.

    1987-01-01

    The mechanism of uptake of a series of amiloride derivatives by human neutrophils was investigated using [ 14 C]amiloride and the 14 C-labeled 5-(1-hexahydroazepinyl)-6-bromo analogue (BrMM) which is approximately 500-fold more potent than the parent compound at inhibiting Na+/H+ exchange. At an external concentration of 2 microM, the influx of BrMM at 37 degrees C was rapid, reaching a steady state by approximately 20 min. The rate of BrMM uptake (approximately 25 mumol/liter.min) was approximately 90-fold faster than for the same concentration of amiloride, a finding which correlates with differences in lipid partitioning of the two compounds. Uptake was unrelated to specific binding to Na+/H+ exchange transport sites: influx of either drug was nonsaturable whereas amiloride- and BrMM-mediated inhibition of Na+/H+ countertransport obeyed Michaelis-Menten kinetics with apparent Ki values of approximately 75 and approximately 0.2 microM. Entry occurred exclusively via the neutral (uncharged) forms (pK'a 8.40-8.55). Influx was markedly pH-dependent: it was enhanced by extracellular alkalinization and reduced by acidification. Influx was, however, insensitive to large changes in membrane voltage, thereby implying the protonated (charged) species to be impermeant. About 75% of the total intracellular pool of amiloride, but only approximately 25% of BrMM, is contained within the lysosomes, an expected consequence of the partitioning and subsequent trapping of a weak base within this strongly acidic subcellular compartment. With BrMM, there was a relative approximately 60-fold enrichment in the internal/external water concentration ratio of the drug; the value for amiloride was much less, approximately 4. This disparity is consistent with substantial binding of BrMM to internal constituents, presumably to proteins and/or nucleic acids

  15. Naloxone inhibits superoxide but not enzyme release by human neutrophils

    International Nuclear Information System (INIS)

    Simpkins, C.; Alailima, S.; Tate, E.

    1986-01-01

    The release of toxic oxygen metabolites and enzymes by phagocytic cells is thought to play a role in the multisystemic tissue injury of sepsis. Naloxone protects septic animals. We have found that at concentrations administered to animals (10 -7 to 10 -4 M), naloxone inhibited (p 2 - ) by human neutrophils (HN), stimulated with N-formyl methionyl leucyl phenylalanine (FMLP). Naloxone had no effect on cell viability. Maximum inhibition was 65% of the total O 2 - released (13.1 nMoles/8 min/320,000 cells). FMLP-stimulated release of beta-glucoronidase or lysozyme was not altered by naloxone. Naloxone had no effect on the binding of 3 H FMLP to HN. Using 3 H naloxone and various concentrations of unlabeled naloxone higher affinity (K/sub D/ = 12nM) and lower affinity (K/sub D/ = 4.7 x 10 -5 ) binding sites were detected. The K/sub D/ of the low affinity site corresponded to the ED 50 for naloxone inhibition of O 2 - (1 x 10 -5 M). Binding to this low affinity site was decreased by (+) naloxone, beta-endorphin and N acetyl beta-endorphin, but not by leu-enkephalin, thyrotropin releasing factor, prostaglandin D 2 or E 2 . Conclusions: (1) naloxone inhibits FMLP-stimulated O 2 but not enzyme release, (2) this inhibition is not due to alteration of FMLP receptor binding, (3) naloxone may act via a low affinity binding site which is ligand specific, and (4) a higher affinity receptor is present on HN

  16. Regulation of Discrete Functional Responses by Syk and Src Family Tyrosine Kinases in Human Neutrophils

    Directory of Open Access Journals (Sweden)

    Thornin Ear

    2017-01-01

    Full Text Available Neutrophils play a critical role in innate immunity and also influence adaptive immune responses. This occurs in good part through their production of inflammatory and immunomodulatory cytokines, in conjunction with their prolonged survival at inflamed foci. While a picture of the signaling machinery underlying these neutrophil responses is now emerging, much remains to be uncovered. In this study, we report that neutrophils constitutively express various Src family isoforms (STKs, as well as Syk, and that inhibition of these protein tyrosine kinases selectively hinders inflammatory cytokine generation by acting posttranscriptionally. Accordingly, STK or Syk inhibition decreases the phosphorylation of signaling intermediates (e.g., eIF-4E, S6K, and MNK1 involved in translational control. By contrast, delayed apoptosis appears to be independent of either STKs or Syk. Our data therefore significantly extend our understanding of which neutrophil responses are governed by STKs and Syk and pinpoint some signaling intermediates that are likely involved. In view of the foremost role of neutrophils in several chronic inflammatory conditions, our findings identify potential molecular targets that could be exploited for future therapeutic intervention.

  17. Proteome profiling of human neutrophil granule subsets, secretory vesicles, and cell membrane

    DEFF Research Database (Denmark)

    Rørvig, Sara; Østergaard, Ole; Heegaard, Niels Henrik Helweg

    2013-01-01

    granules, SVs, and plasma membrane has been performed before. Here, we performed subcellular fractionation on freshly isolated human neutrophils by nitrogen cavitation and density centrifugation on a four-layer Percoll gradient. Granule subsets were pooled and subjected to SDS-PAGE, and gel pieces were in...... subcellular proteome profiles presented here may be used as a database in combination with the mRNA array database to predict and test the presence and localization of proteins in neutrophil granules and membranes....

  18. Tamoxifen induces apoptotic neutrophil efferocytosis in horses.

    Science.gov (United States)

    Olave, C; Morales, N; Uberti, B; Henriquez, C; Sarmiento, J; Ortloff, A; Folch, H; Moran, G

    2018-03-01

    Macrophages and neutrophils are important cellular components in the process of acute inflammation and its subsequent resolution, and evidence increasingly suggests that they play important functions during the resolution of chronic, adaptive inflammatory processes. Exacerbated neutrophil activity can be harmful to surrounding tissues; this is important in a range of diseases, including allergic asthma and chronic obstructive pulmonary disease in humans, and equine asthma (also known as recurrent airway obstruction (RAO). Tamoxifen (TX) is a non-steroidal estrogen receptor modulator with effects on cell growth and survival. Previous studies showed that TX treatment in horses with induced acute pulmonary inflammation promoted early apoptosis of blood and BALF neutrophils, reduction of BALF neutrophils, and improvement in animals' clinical status. The aim of this study was to describe if TX induces in vitro efferocytosis of neutrophils by alveolar macrophages. Efferocytosis assay, myeloperoxidase (MPO) detection and translocation phosphatidylserine (PS) were performed on neutrophils isolated from peripheral blood samples from five healthy horses. In in vitro samples from heathy horses, TX treatment increases the phenomenon of efferocytosis of peripheral neutrophils by alveolar macrophages. Similar increases in supernatant MPO concentration and PS translocation were observed in TX-treated neutrophils, compared to control cells. In conclusion, these results confirm that tamoxifen has a direct effect on equine peripheral blood neutrophils, through stimulation of the engulfment of apoptotic neutrophils by alveolar macrophages.

  19. Apoptosis modulation in the immune system reveals a role of neutrophils in tissue damage in a murine model of chlamydial genital infection.

    Science.gov (United States)

    Zortel, Tom; Schmitt-Graeff, Annette; Kirschnek, Susanne; Häcker, Georg

    2018-03-07

    Chlamydial infection frequently causes damage to the female genital tract. The precise mechanisms of chlamydial clearance and tissue damage are unknown but studies suggest immunopathology with a particular role of neutrophils. The goal of this study was to understand the contribution of the immune system, in particular neutrophils. Using Chlamydia muridarum, we infected mice with a prolonged immune response due to expression of Bcl-2 in haematopoietic cells (Bcl-2-mice), and mice where mature neutrophils are lacking due to the deletion of Mcl-1 in myeloid cells (LysM-cre-mcl-1-flox-mice; Mcl-1-mice). We monitored bacterial clearance, cellular infiltrate and long-term tissue damage. Both mutant strains showed slightly delayed clearance of the acute infection. Bcl-2-mice had a strongly increased inflammatory infiltrate concerning almost all cell lineages. The infection of Bcl-2-mice caused increased tissue damage. The loss of neutrophils in Mcl-1-mice was associated with substantial quantitative and qualitative alterations of the inflammatory infiltrate. Mcl-1-mice had higher chlamydial burden and reduced tissue damage, including lower incidence of hydrosalpinx and less uterine dilation. Inhibition of apoptosis in the haematopoietic system increases inflammation and tissue damage. Neutrophils have broad functions, including a role in chlamydial clearance and in tissue destruction.

  20. Heterogeneity of Human Neutrophil CD177 Expression Results from CD177P1 Pseudogene Conversion.

    Directory of Open Access Journals (Sweden)

    Zuopeng Wu

    2016-05-01

    Full Text Available Most humans harbor both CD177neg and CD177pos neutrophils but 1-10% of people are CD177null, placing them at risk for formation of anti-neutrophil antibodies that can cause transfusion-related acute lung injury and neonatal alloimmune neutropenia. By deep sequencing the CD177 locus, we catalogued CD177 single nucleotide variants and identified a novel stop codon in CD177null individuals arising from a single base substitution in exon 7. This is not a mutation in CD177 itself, rather the CD177null phenotype arises when exon 7 of CD177 is supplied entirely by the CD177 pseudogene (CD177P1, which appears to have resulted from allelic gene conversion. In CD177 expressing individuals the CD177 locus contains both CD177P1 and CD177 sequences. The proportion of CD177hi neutrophils in the blood is a heritable trait. Abundance of CD177hi neutrophils correlates with homozygosity for CD177 reference allele, while heterozygosity for ectopic CD177P1 gene conversion correlates with increased CD177neg neutrophils, in which both CD177P1 partially incorporated allele and paired intact CD177 allele are transcribed. Human neutrophil heterogeneity for CD177 expression arises by ectopic allelic conversion. Resolution of the genetic basis of CD177null phenotype identifies a method for screening for individuals at risk of CD177 isoimmunisation.

  1. Human Neutrophils Use Different Mechanisms To Kill Aspergillus fumigatus Conidia and Hyphae: Evidence from Phagocyte Defects.

    Science.gov (United States)

    Gazendam, Roel P; van Hamme, John L; Tool, Anton T J; Hoogenboezem, Mark; van den Berg, J Merlijn; Prins, Jan M; Vitkov, Ljubomir; van de Veerdonk, Frank L; van den Berg, Timo K; Roos, Dirk; Kuijpers, Taco W

    2016-02-01

    Neutrophils are known to play a pivotal role in the host defense against Aspergillus infections. This is illustrated by the prevalence of Aspergillus infections in patients with neutropenia or phagocyte functional defects, such as chronic granulomatous disease. However, the mechanisms by which human neutrophils recognize and kill Aspergillus are poorly understood. In this work, we have studied in detail which neutrophil functions, including neutrophil extracellular trap (NET) formation, are involved in the killing of Aspergillus fumigatus conidia and hyphae, using neutrophils from patients with well-defined genetic immunodeficiencies. Recognition of conidia involves integrin CD11b/CD18 (and not dectin-1), which triggers a PI3K-dependent nonoxidative intracellular mechanism of killing. When the conidia escape from early killing and germinate, the extracellular destruction of the Aspergillus hyphae needs opsonization by Abs and involves predominantly recognition via Fcγ receptors, signaling via Syk, PI3K, and protein kinase C to trigger the production of toxic reactive oxygen metabolites by the NADPH oxidase and myeloperoxidase. A. fumigatus induces NET formation; however, NETs did not contribute to A. fumigatus killing. Thus, our findings reveal distinct killing mechanisms of Aspergillus conidia and hyphae by human neutrophils, leading to a comprehensive insight in the innate antifungal response. Copyright © 2016 by The American Association of Immunologists, Inc.

  2. CD177 modulates human neutrophil migration through activation-mediated integrin and chemoreceptor regulation.

    Science.gov (United States)

    Bai, Ming; Grieshaber-Bouyer, Ricardo; Wang, Junxia; Schmider, Angela B; Wilson, Zachary S; Zeng, Liling; Halyabar, Olha; Godin, Matthew D; Nguyen, Hung N; Levescot, Anaïs; Cunin, Pierre; Lefort, Craig T; Soberman, Roy J; Nigrovic, Peter A

    2017-11-09

    CD177 is a glycosylphosphatidylinositol (GPI)-anchored protein expressed by a variable proportion of human neutrophils that mediates surface expression of the antineutrophil cytoplasmic antibody antigen proteinase 3. CD177 associates with β2 integrins and recognizes platelet endothelial cell adhesion molecule 1 (PECAM-1), suggesting a role in neutrophil migration. However, CD177 pos neutrophils exhibit no clear migratory advantage in vivo, despite interruption of in vitro transendothelial migration by CD177 ligation. We sought to understand this paradox. Using a PECAM-1-independent transwell system, we found that CD177 pos and CD177 neg neutrophils migrated comparably. CD177 ligation selectively impaired migration of CD177 pos neutrophils, an effect mediated through immobilization and cellular spreading on the transwell membrane. Correspondingly, CD177 ligation enhanced its interaction with β2 integrins, as revealed by fluorescence lifetime imaging microscopy, leading to integrin-mediated phosphorylation of Src and extracellular signal-regulated kinase (ERK). CD177-driven cell activation enhanced surface β2 integrin expression and affinity, impaired internalization of integrin attachments, and resulted in ERK-mediated attenuation of chemokine signaling. We conclude that CD177 signals in a β2 integrin-dependent manner to orchestrate a set of activation-mediated mechanisms that impair human neutrophil migration. © 2017 by The American Society of Hematology.

  3. Characterization of a receptor for human monocyte-derived neutrophil chemotactic factor/interleukin-8

    International Nuclear Information System (INIS)

    Grob, P.M.; David, E.; Warren, T.C.; DeLeon, R.P.; Farina, P.R.; Homon, C.A.

    1990-01-01

    Monocyte-derived neutrophil chemotactic factor/interleukin-8 (MDNCF/IL-8) is an 8,000-dalton protein produced by monocytes which exhibits activity as a chemoattractant for neutrophils with maximal activity achieved at a concentration of 50 ng/ml. This polypeptide has been iodinated by chloramine-T methodology (350 Ci/mM), and specific receptors for MDNCF/IL-8 have been detected on human neutrophils, U937 cells, THP-1 cells, and dimethyl sulfoxide-differentiated HL-60 cells. The binding of MDNCF/IL-8 to human neutrophils is not inhibited by interleukin-1 alpha, tumor necrosis factor-alpha, insulin, or epidermal growth factor. In addition, chemoattractants such as C5a, fMet-Leu-Phe, leukotriene B4, and platelet-activating factor fail to inhibit binding, suggesting that MDNCF/IL-8 utilizes a unique receptor. The receptor for MDNCF/IL-8 is apparently glycosylated since ligand binding is inhibited by the presence of wheat germ agglutinin, a lectin with a binding specificity for N-acetylglucosamine and neuraminic acid. Steady state binding experiments indicate Kd values of 4 and 0.5 nM and receptor numbers of 75,000 and 7,400 for human neutrophils and differentiated HL-60 cells, respectively. 125I-MDNCF/IL-8 bound to human neutrophils is rapidly internalized and subsequently released from cells as trichloroacetic acid-soluble radioactivity. Affinity labeling experiments suggest that the human neutrophil MDNCF/IL-8 receptor exhibits a mass of approximately 58,000 daltons

  4. Coccidioides Endospores and Spherules Draw Strong Chemotactic, Adhesive, and Phagocytic Responses by Individual Human Neutrophils.

    Directory of Open Access Journals (Sweden)

    Cheng-Yuk Lee

    Full Text Available Coccidioides spp. are dimorphic pathogenic fungi whose parasitic forms cause coccidioidomycosis (Valley fever in mammalian hosts. We use an innovative interdisciplinary approach to analyze one-on-one encounters between human neutrophils and two forms of Coccidioides posadasii. To examine the mechanisms by which the innate immune system coordinates different stages of the host response to fungal pathogens, we dissect the immune-cell response into chemotaxis, adhesion, and phagocytosis. Our single-cell technique reveals a surprisingly strong response by initially quiescent neutrophils to close encounters with C. posadasii, both from a distance (by complement-mediated chemotaxis as well as upon contact (by serum-dependent adhesion and phagocytosis. This response closely resembles neutrophil interactions with Candida albicans and zymosan particles, and is significantly stronger than the neutrophil responses to Cryptococcus neoformans, Aspergillus fumigatus, and Rhizopus oryzae under identical conditions. The vigorous in vitro neutrophil response suggests that C. posadasii evades in vivo recognition by neutrophils through suppression of long-range mobilization and recruitment of the immune cells. This observation elucidates an important paradigm of the recognition of microbes, i.e., that intact immunotaxis comprises an intricate spatiotemporal hierarchy of distinct chemotactic processes. Moreover, in contrast to earlier reports, human neutrophils exhibit vigorous chemotaxis toward, and frustrated phagocytosis of, the large spherules of C. posadasii under physiological-like conditions. Finally, neutrophils from healthy donors and patients with chronic coccidioidomycosis display subtle differences in their responses to antibody-coated beads, even though the patient cells appear to interact normally with C. posadasii endospores.

  5. Solar ultraviolet irradiation induces decorin degradation in human skin likely via neutrophil elastase.

    Science.gov (United States)

    Li, Yong; Xia, Wei; Liu, Ying; Remmer, Henriette A; Voorhees, John; Fisher, Gary J

    2013-01-01

    Exposure of human skin to solar ultraviolet (UV) irradiation induces matrix metalloproteinase-1 (MMP-1) activity, which degrades type I collagen fibrils. Type I collagen is the most abundant protein in skin and constitutes the majority of skin connective tissue (dermis). Degradation of collagen fibrils impairs the structure and function of skin that characterize skin aging. Decorin is the predominant proteoglycan in human dermis. In model systems, decorin binds to and protects type I collagen fibrils from proteolytic degradation by enzymes such as MMP-1. Little is known regarding alterations of decorin in response to UV irradiation. We found that solar-simulated UV irradiation of human skin in vivo stimulated substantial decorin degradation, with kinetics similar to infiltration of polymorphonuclear (PMN) cells. Proteases that were released from isolated PMN cells degraded decorin in vitro. A highly selective inhibitor of neutrophil elastase blocked decorin breakdown by proteases released from PMN cells. Furthermore, purified neutrophil elastase cleaved decorin in vitro and generated fragments with similar molecular weights as those resulting from protease activity released from PMN cells, and as observed in UV-irradiated human skin. Cleavage of decorin by neutrophil elastase significantly augmented fragmentation of type I collagen fibrils by MMP-1. Taken together, these data indicate that PMN cell proteases, especially neutrophil elastase, degrade decorin, and this degradation renders collagen fibrils more susceptible to MMP-1 cleavage. These data identify decorin degradation and neutrophil elastase as potential therapeutic targets for mitigating sun exposure-induced collagen fibril degradation in human skin.

  6. Activation of Triggering Receptor Expressed on Myeloid Cells-1 on Human Neutrophils by Marburg and Ebola Viruses

    National Research Council Canada - National Science Library

    Mohamadzadeh, Mansour; Coberley, Sadie S; Olinger, Gene G; Kalina, Warren V; Ruthel, Gordon; Fullter, Claudette L; Swenson, Dana L; Pratt, William D; Kuhns, Douglas B; Schmaljohn, Alan L

    2006-01-01

    .... Here, we report that MARV and EBOV activate TREM-1 on human neutrophils, resulting in DAP12 phosphorylation, TREM-1 shedding, mobilization of intracellular calcium, secretion of proinflammatory...

  7. Evasion of Human Neutrophil-Mediated Host Defense during Toxoplasma gondii Infection.

    Science.gov (United States)

    Lima, Tatiane S; Gov, Lanny; Lodoen, Melissa B

    2018-02-13

    Neutrophils are a major player in host immunity to infection; however, the mechanisms by which human neutrophils respond to the intracellular protozoan parasite Toxoplasma gondii are still poorly understood. In the current study, we found that, whereas primary human monocytes produced interleukin-1beta (IL-1β) in response to T. gondii infection, human neutrophils from the same blood donors did not. Moreover, T. gondii inhibited lipopolysaccharide (LPS)-induced IL-1β synthesis in human peripheral blood neutrophils. IL-1β suppression required active parasite invasion, since heat-killed or mycalolide B-treated parasites did not inhibit IL-1β release. By investigating the mechanisms involved in this process, we found that T. gondii infection of neutrophils treated with LPS resulted in reduced transcript levels of IL-1β and NLRP3 and reduced protein levels of pro-IL-1β, mature IL-1β, and the inflammasome sensor NLRP3. In T. gondii -infected neutrophils stimulated with LPS, the levels of MyD88, TRAF6, IKKα, IKKβ, and phosphorylated IKKα/β were not affected. However, LPS-induced IκBα degradation and p65 phosphorylation were reduced in T. gondii- infected neutrophils, and degradation of IκBα was reversed by treatment with the proteasome inhibitor MG-132. Finally, we observed that T. gondii inhibited the cleavage and activity of caspase-1 in human neutrophils. These results indicate that T. gondii suppression of IL-1β involves a two-pronged strategy whereby T. gondii inhibits both NF-κB signaling and activation of the NLRP3 inflammasome. These findings represent a novel mechanism of T. gondii evasion of human neutrophil-mediated host defense by targeting the production of IL-1β. IMPORTANCE Toxoplasma gondii is an obligate intracellular parasite that infects approximately one-third of humans worldwide and can invade virtually any nucleated cell in the human body. Although it is well documented that neutrophils infiltrate the site of acute T

  8. Neutrophil-mediated protection of cultured human vascular endothelial cells from damage by growing Candida albicans hyphae

    International Nuclear Information System (INIS)

    Edwards, J.E. Jr.; Rotrosen, D.; Fontaine, J.W.; Haudenschild, C.C.; Diamond, R.D.

    1987-01-01

    Interactions were studied between human neutrophils and cultured human umbilical vein endothelial cells invaded by Candida albicans. In the absence of neutrophils, progressive Candida germination and hyphal growth extensively damaged endothelial cell monolayers over a period of 4 to 6 hours, as determined both by morphological changes and release of 51 Cr from radiolabeled endothelial cells. Monolayers were completely destroyed and replaced by hyphae after 18 hours of incubation. In contrast, when added 2 hours after the monolayers had been infected with Candida, neutrophils selectively migrated toward and attached to hyphae at points of hyphal penetration into individual endothelial cells (observed by time-lapse video-microscopy). Attached neutrophils spread over hyphal surfaces both within and beneath the endothelial cells; neutrophil recruitment to initial sites of leukocyte-Candida-endothelial cell interactions continued throughout the first 60 minutes of observation. Neutrophil spreading and stasis were observed only along Candida hyphae and at sites of Candida-endothelial cell interactions. These events resulted in 58.0% killing of Candida at 2 hours and subsequent clearance of Candida from endothelial cell monolayers, as determined by microcolony counts and morphological observation. On introduction of additional neutrophils to yield higher ratios of neutrophils to endothelial cells (10 neutrophils:1 endothelial cell), neutrophil migration toward hyphal elements continued. Despite retraction or displacement of occasional endothelial cells by invading Candida and neutrophils, most endothelial cells remained intact, viable, and motile as verified both by morphological observations and measurement of 51 Cr release from radiolabeled monolayers

  9. Products of neutrophils and eosinophils increase the responsiveness of human isolated bronchial tissue.

    Science.gov (United States)

    Hallahan, A R; Armour, C L; Black, J L

    1990-05-01

    This study examines the possibility that products of neutrophils and eosinophils could increase the responsiveness of human isolated bronchial tissue. Neutrophils and eosinophils were isolated from the peripheral blood of healthy volunteers. The cells were incubated with 1 microM calcium ionophore A23187 for 10-15 min then centrifuged, the supernatant collected and stored at -70 degrees C. Human bronchial rings (2-3 mm diameter, 3-4 mm long) were prepared from specimens resected at thoracotomy. The tissues were suspended in organ baths under a 1 g load and changes in tension measured isometrically. Stable contractions to bolus doses of histamine (0.1-10 microM) or to electrical field stimulation (40-100 V, 4-16 Hz, 1 ms for 20 s) were established. Supernatant from 106 neutrophils or 105 eosinophils was then added and tissue responsiveness reassessed. Neutrophil supernatant increased tissue responsiveness to histamine and electrical field stimulation by 54 +/- 17% (n = 5, p less than 0.05) and 18 +/- 7% (n = 6, p less than 0.05), respectively. Eosinophil supernatant increased the histamine response by 60 +/- 23% (n = 8, p less than 0.05) while tissue responsiveness to electrical field stimulation was unchanged (n = 3). Thus, as neutrophils and eosinophils can change the responsiveness of human bronchus in vitro it is possible that they do this in vivo and may not simply be temporally related to the development of bronchial hyperresponsiveness.

  10. Neutrophil-induced human bronchial hyperresponsiveness in vitro--pharmacological modulation.

    Science.gov (United States)

    Hughes, J M; McKay, K O; Johnson, P R; Tragoulias, S; Black, J L; Armour, C L

    1993-04-01

    Although it has been postulated that inflammatory cells cause the bronchial hyperresponsiveness which is diagnostic of asthma, until recently there has been little direct evidence of such a link. We have recently shown that calcium ionophore-activated human neutrophils and eosinophils can induce a state of human airway hyperresponsiveness in vitro. In this study we have shown that the anti-inflammatory agent nedocromil sodium, 10(-7) M, inhibited the hyperresponsiveness induced by products released from ionophore activated neutrophils but did not inhibit the release of leukotriene B4 from the same cells. Neutrophil-induced bronchial hyperresponsiveness was also inhibited by pre-treatment of the bronchial tissues with a thromboxane A2 and prostaglandin receptor antagonist, GR32191, 10(-7) M. These findings indicate that cyclooxygenase products are involved in bronchial hyperresponsiveness induced by inflammatory cell products in vitro and that their release can be inhibited by nedocromil sodium.

  11. Localization and Functionality of the Inflammasome in Neutrophils

    DEFF Research Database (Denmark)

    Bakele, Martina; Joos, Melanie; Burdi, Sofia

    2014-01-01

    Neutrophils represent the major fraction of circulating immune cells and are rapidly recruited to sites of infection and inflammation. The inflammasome is a multiprotein complex that regulates the generation of IL-1 family proteins. The precise subcellular localization and functionality...... of the inflammasome in human neutrophils are poorly defined. Here we demonstrate that highly purified human neutrophils express key components of the NOD-like receptor family, pyrin domain containing 3 (NLRP3), and absent in melanoma 2 (AIM2) inflammasomes, particularly apoptosis-associated speck-like protein...... and released as protein, highly purified neutrophils neither expressed nor released IL-1α at baseline or upon stimulation. Upon inflammasome activation, highly purified neutrophils released substantially lower levels of IL-1β protein compared with partially purified neutrophils. Serine proteases and caspases...

  12. Relationship between chemical composition and biological function of Pseudomonas aeruginosa lipopolysaccharide: effect on human neutrophil chemotaxis and oxidative burst

    DEFF Research Database (Denmark)

    Kharazmi, A; Fomsgaard, A; Conrad, R S

    1991-01-01

    There are conflicting data on the effect of bacterial lipopolysaccharides (LPS) on the function of human neutrophils. The present study was designed to examine the relationship between chemical composition and the modulatory effect of LPS on human neutrophil function. LPS was extracted from five...

  13. Brucella abortus Induces the Premature Death of Human Neutrophils through the Action of Its Lipopolysaccharide

    Science.gov (United States)

    Barquero-Calvo, Elías; Mora-Cartín, Ricardo; Arce-Gorvel, Vilma; de Diego, Juana L.; Chacón-Díaz, Carlos; Chaves-Olarte, Esteban; Guzmán-Verri, Caterina; Buret, Andre G.; Gorvel, Jean-Pierre; Moreno, Edgardo

    2015-01-01

    Most bacterial infections induce the activation of polymorphonuclear neutrophils (PMNs), enhance their microbicidal function, and promote the survival of these leukocytes for protracted periods of time. Brucella abortus is a stealthy pathogen that evades innate immunity, barely activates PMNs, and resists the killing mechanisms of these phagocytes. Intriguing clinical signs observed during brucellosis are the low numbers of Brucella infected PMNs in the target organs and neutropenia in a proportion of the patients; features that deserve further attention. Here we demonstrate that B. abortus prematurely kills human PMNs in a dose-dependent and cell-specific manner. Death of PMNs is concomitant with the intracellular Brucella lipopolysaccharide (Br-LPS) release within vacuoles. This molecule and its lipid A reproduce the premature cell death of PMNs, a phenomenon associated to the low production of proinflammatory cytokines. Blocking of CD14 but not TLR4 prevents the Br-LPS-induced cell death. The PMNs cell death departs from necrosis, NETosis and classical apoptosis. The mechanism of PMN cell death is linked to the activation of NADPH-oxidase and a modest but steadily increase of ROS mediators. These effectors generate DNA damage, recruitments of check point kinase 1, caspases 5 and to minor extent of caspase 4, RIP1 and Ca++ release. The production of IL-1β by PMNs was barely stimulated by B. abortus infection or Br-LPS treatment. Likewise, inhibition of caspase 1 did not hamper the Br-LPS induced PMN cell death, suggesting that the inflammasome pathway was not involved. Although activation of caspases 8 and 9 was observed, they did not seem to participate in the initial triggering mechanisms, since inhibition of these caspases scarcely blocked PMN cell death. These findings suggest a mechanism for neutropenia in chronic brucellosis and reveal a novel Brucella-host cross-talk through which B. abortus is able to hinder the innate function of PMN. PMID:25946018

  14. Porphyromonas gingivalis regulates TREM-1 in human polymorphonuclear neutrophils via its gingipains.

    Directory of Open Access Journals (Sweden)

    Nagihan Bostanci

    Full Text Available The Triggering Receptor Expressed on Myeloid cells 1 (TREM-1 is a cell surface receptor of the immunoglobulin superfamily, with the capacity to amplify pro-inflammatory cytokine production and regulate apoptosis. Polymorphonuclear neutrophils (PMNs are the first line of defence against infection, and a major source of TREM-1. Porphyromonas gingivalis is a Gram-negative anaerobe highly implicated in the inflammatory processes governing periodontal disease, which is characterized by the destruction of the tooth-supporting tissues. It expresses a number of virulence factors, including the cysteine proteinases (or gingipains. The aim of this in vitro study was to investigate the effect of P. gingivalis on TREM-1 expression and production by primary human PMNs, and to evaluate the role of its gingipains in this process. After 4 h of challenge, P. gingivalis enhanced TREM-1 expression as identified by quantitative real-time PCR. This was followed by an increase in soluble (sTREM-1 secretion over a period of 18 h, as determined by ELISA. At this time-point, the P. gingivalis-challenged PMNs exhibited diminished TREM-1 cell-membrane staining, as identified by flow cytometry and confocal laser scanning microscopy. Furthermore engagement of TREM-1, by means of anti-TREM-1 antibodies, enhanced the capacity of P. gingivalis to stimulate interleukin (IL-8 production. Conversely, antagonism of TREM-1 using a synthetic peptide resulted in reduction of IL-8 secretion. Using isogenic P. gingivalis mutant strains, we identified the Arg-gingipain to be responsible for shedding of sTREM-1 from the PMN surface, whereas the Lys-gingipain had the capacity to degrade TREM-1. In conclusion, the differential regulation of TREM-1 by the P. gingivalis gingipains may present a novel mechanism by which P. gingivalis manipulates the host innate immune response helping to establish chronic periodontal inflammation.

  15. Cultured rat and purified human Pneumocystis carinii stimulate intra- but not extracellular free radical production in human neutrophils

    DEFF Research Database (Denmark)

    Jensen, T; Aliouat, E M; Lundgren, B

    1998-01-01

    The production of free radicals in human neutrophils was studied in both Pneumocystis carinii derived from cultures of L2 rat lung epithelial-like cells and Pneumocystis carinii purified from human lung. Using the cytochrome C technique, which selectively measured extracellular superoxide...... generation, hardly any free radical production was observed after stimulation with cultured rat-derived P. carinii. A chemiluminescence technique, which separately measured intra- and extracellular free radical production, was subsequently employed to differentiate the free radical generation....... It was established that 1) P. carinii stimulated intra- but not extracellular free radical production in human neutrophils, 2) opsonized cultured rat-derived P. carinii stimulated human neutrophils to a strong intracellular response of superoxide production, and 3) opsonized P. carinii, purified from human lung also...

  16. Aspiration of human neutrophils: effects of shear thinning and cortical dissipation.

    Science.gov (United States)

    Drury, J L; Dembo, M

    2001-12-01

    It is generally accepted that the human neutrophil can be mechanically represented as a droplet of polymeric fluid enclosed by some sort of thin slippery viscoelastic cortex. Many questions remain however about the detailed rheology and chemistry of the interior fluid and the cortex. To address these quantitative issues, we have used a finite element method to simulate the dynamics of neutrophils during micropipet aspiration using various plausible assumptions. The results were then systematically compared with aspiration experiments conducted at eight different combinations of pipet size and pressure. Models in which the cytoplasm was represented by a simple Newtonian fluid (i.e., models without shear thinning) were grossly incapable of accounting for the effects of pressure on the general time scale of neutrophil aspiration. Likewise, models in which the cortex was purely elastic (i.e., models without surface viscosity) were unable to explain the effects of pipet size on the general aspiration rate. Such models also failed to explain the rapid acceleration of the aspiration rate during the final phase of aspiration nor could they account for the geometry of the neutrophil during various phases of aspiration. Thus, our results indicate that a minimal mechanical model of the neutrophil needs to incorporate both shear thinning and surface viscosity to remain valid over a reasonable range of conditions. At low shear rates, the surface dilatation viscosity of the neutrophil was found to be on the order of 100 poise-cm, whereas the viscosity of the interior cytoplasm was on the order of 1000 poise. Both the surface viscosity and the interior viscosity seem to decrease in a similar fashion when the shear rate exceeds approximately 0.05 s(-1). Unfortunately, even models with both surface viscosity and shear thinning studied are still not sufficient to fully explain all the features of neutrophil aspiration. In particular, the very high rate of aspiration during the

  17. Use of CFSE staining of borreliae in studies on the interaction between borreliae and human neutrophils

    Directory of Open Access Journals (Sweden)

    Hytönen Jukka

    2006-10-01

    Full Text Available Abstract Background Species of the tick-transmitted spirochete group Borrelia burgdorferi sensu lato (B. burgdorferi cause Lyme borreliosis. Acute borrelial infection of the skin has unusual characteristics with only a mild local inflammatory response suggesting that the interaction between borreliae and the cells of the first-line defence might differ from that of other bacteria. It has been reported that human neutrophils phagocytose motile borreliae through an unconventional mechanism (tube phagocytosis which is not observed with non-motile borreliae. Therefore, it would be of great interest to visualise the bacteria by a method not affecting motility and viability of borreliae to be able to study their interaction with the cells of the innate immunity. Carboxyfluorescein diacetate, succinimidyl ester (CFSE labelling has been previously used for studying the adhesion of labelled bacteria to host cells and the uptake of labelled substrates by various cells using flow cytometry. Results In this study, CFSE was shown to efficiently stain different genospecies of B. burgdorferi without affecting bacterial viability or motility. Use of CFSE staining allowed subsequent quantification of borreliae associated with human neutrophils with flow cytometry and confocal microscopy. As a result, no difference in association between different borrelial genospecies (Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, or between borreliae and the pyogenic bacterium Streptococcus pyogenes, with neutrophils could be detected. Borrelial virulence, on the other hand, affected association with neutrophils, with significantly higher association of a non-virulent mutant B. burgdorferi sensu stricto strain compared to the parental virulent wild type strain. Conclusion These results suggest that the flow cytometric assay using CFSE labelled borreliae is a valuable tool in the analysis of the interaction between borreliae and human neutrophils. The

  18. Staphylococcus aureus panton-valentine leukocidin is a very potent cytotoxic factor for human neutrophils.

    Directory of Open Access Journals (Sweden)

    Bettina Löffler

    2010-01-01

    Full Text Available The role of the pore-forming Staphylococcus aureus toxin Panton-Valentine leukocidin (PVL in severe necrotizing diseases is debated due to conflicting data from epidemiological studies of community-associated methicillin-resistant S. aureus (CA-MRSA infections and various murine disease-models. In this study, we used neutrophils isolated from different species to evaluate the cytotoxic effect of PVL in comparison to other staphylococcal cytolytic components. Furthermore, to study the impact of PVL we expressed it heterologously in a non-virulent staphylococcal species and examined pvl-positive and pvl-negative clinical isolates as well as the strain USA300 and its pvl-negative mutant. We demonstrate that PVL induces rapid activation and cell death in human and rabbit neutrophils, but not in murine or simian cells. By contrast, the phenol-soluble modulins (PSMs, a newly identified group of cytolytic staphylococcal components, lack species-specificity. In general, after phagocytosis of bacteria different pvl-positive and pvl-negative staphylococcal strains, expressing a variety of other virulence factors (such as surface proteins, induced cell death in neutrophils, which is most likely associated with the physiological clearing function of these cells. However, the release of PVL by staphylococcal strains caused rapid and premature cell death, which is different from the physiological (and programmed cell death of neutrophils following phagocytosis and degradation of virulent bacteria. Taken together, our results question the value of infection-models in mice and non-human primates to elucidate the impact of PVL. Our data clearly demonstrate that PVL acts differentially on neutrophils of various species and suggests that PVL has an important cytotoxic role in human neutrophils, which has major implications for the pathogenesis of CA-MRSA infections.

  19. Alpha-1-antitrypsin is produced by human neutrophil granulocytes and their precursors and liberated during granule exocytosis

    DEFF Research Database (Denmark)

    Clemmensen, Stine N; Jacobsen, Lars C; Rørvig, Sara

    2011-01-01

    Alpha-1-antitrypsin (A1AT) is an important inhibitor of neutrophil proteases including elastase, cathepsin G, and proteinase 3. Transcription profiling data suggest that A1AT is expressed by human neutrophil granulocytes during all developmental stages. A1AT has hitherto only been found associate...... significantly to the antiprotease levels in tissues during inflammation. Impaired binding of neutrophil A1AT to serine proteases in patients with (PI)ZZ mutations may enhance their susceptibility to the development of emphysema....

  20. Monoclonal antibodies to antigens on human neutrophils, activated T lymphocytes, and acute leukemia blast cells

    International Nuclear Information System (INIS)

    Miterev, G.Yu.; Burova, G.F.; Puzhitskaya, M.S.; Danilevich, S.V.; Bulycheva, T.I.

    1987-01-01

    The authors describe the production of two mouse hybridomas secreting monoclonal antibodies to antigenic determinants of the surface membranes of human neutrophils, activated T lymphocytes, and acute leukemic blast cells. The degree of lymphocyte stimulation was estimated from incorporation of 3 H-thymidine with parallel microculture. Monoclonal antibodies of supernatants of hybridoma cultures shown here reacted in both immunofluorescence test and cytotoxicity test with surface membrane antigens on the majority of neutrophils and PHA-activated peripheral blood lymphocytes from healthy subjects, but did not give positive reactions with unactivated lymphocytes, adherent monocytes, erythrocytes, and alloantigen-stimulated lymphocytes

  1. Monoclonal antibodies to antigens on human neutrophils, activated T lymphocytes, and acute leukemia blast cells

    Energy Technology Data Exchange (ETDEWEB)

    Miterev, G.Yu.; Burova, G.F.; Puzhitskaya, M.S.; Danilevich, S.V.; Bulycheva, T.I.

    1987-11-01

    The authors describe the production of two mouse hybridomas secreting monoclonal antibodies to antigenic determinants of the surface membranes of human neutrophils, activated T lymphocytes, and acute leukemic blast cells. The degree of lymphocyte stimulation was estimated from incorporation of /sup 3/H-thymidine with parallel microculture. Monoclonal antibodies of supernatants of hybridoma cultures shown here reacted in both immunofluorescence test and cytotoxicity test with surface membrane antigens on the majority of neutrophils and PHA-activated peripheral blood lymphocytes from healthy subjects, but did not give positive reactions with unactivated lymphocytes, adherent monocytes, erythrocytes, and alloantigen-stimulated lymphocytes.

  2. Legionella phosphatase hydrolyzes phosphatidylinositol 4,5-bisphosphate and inosital triphosphate in human neutrophils

    International Nuclear Information System (INIS)

    Dowling, J.N.; Saha, A.K.; Glew, R.H.

    1987-01-01

    Legionella are facultative intracellular bacterial pathogens which multiply in host phagocytes. L. micdadei cells contain an acid phosphatase (ACP) that blocks superoxide anion production by human neutrophils stimulated with the formylated peptide, fMLP. The possibility that ACP acts by interefering with polyphosphoinositide metabolism and the production of the intracellular second messenger, inositol triphosphate (IP 3 ) was explored. When neutrophil phosphoinositides were labeled with 32 P, incubation of the cells with ACP caused an 85% loss of the labeled phosphatidylinositol-4,5-bisphosphate (PIP 2 ) over 2 h. Treatment of [ 3 H]inositol-labeled neutrophils with ACP for 30 min resulted in a 20% decrease of labeled PIP 2 . Following fMLP stimulation, the fractional reduction in PIP 2 and the fractional increase in IP 3 was the same in ACP-treated and untreated neutrophils, but the total quantity of IP 3 was reduced by ACP pre-treatment. The reduction in IP 3 generated following fMLP stimulation seems to be due primarily to the decreased amount of PIP 2 available for hydrolysis. However, some loss of IP 3 due to direct hydrolysis by ACP cannot be ruled out. The Legionella phosphatase may compromise neutrophil response to the bacteria by hydrolyzing PIP 2 , the prognitor of IP 3 , and by hydrolyzing IP 3 itself

  3. Nucleobindin co-localizes and associates with cyclooxygenase (COX-2 in human neutrophils.

    Directory of Open Access Journals (Sweden)

    Patrick Leclerc

    2008-05-01

    Full Text Available The inducible cyclooxygenase isoform (COX-2 is associated with inflammation, tumorigenesis, as well as with physiological events. Despite efforts deployed in order to understand the biology of this multi-faceted enzyme, much remains to be understood. Nucleobindin (Nuc, a ubiquitous Ca(2+-binding protein, possesses a putative COX-binding domain. In this study, we investigated its expression and subcellular localization in human neutrophils, its affinity for COX-2 as well as its possible impact on PGE(2 biosynthesis. Complementary subcellular localization approaches including nitrogen cavitation coupled to Percoll fractionation, immunofluorescence, confocal and electron microscopy collectively placed Nuc, COX-2, and all of the main enzymes involved in prostanoid synthesis, in the Golgi apparatus and endoplasmic reticulum of human neutrophils. Immunoprecipitation experiments indicated a high affinity between Nuc and COX-2. Addition of human recombinant (hr Nuc to purified hrCOX-2 dose-dependently caused an increase in PGE(2 biosynthesis in response to arachidonic acid. Co-incubation of Nuc with COX-2-expressing neutrophil lysates also increased their capacity to produce PGE(2. Moreover, neutrophil transfection with hrNuc specifically enhanced PGE(2 biosynthesis. Together, these results identify a COX-2-associated protein which may have an impact in prostanoid biosynthesis.

  4. Innate Defense against Influenza A Virus: Activity of Human Neutrophil Defensins and Interactions of Defensins with Surfactant Protein D

    DEFF Research Database (Denmark)

    Hartshorn, Kevan L.; White, Mitchell R.; Tecle, Tesfaldet

    2006-01-01

    Surfactant protein D (SP-D) plays important roles in innate host defense against influenza A virus (IAV) infection, in part by modifying interactions with neutrophils. Human neutrophil defensins (HNPs) inhibit infectivity of enveloped viruses, including IAV. Our goal in this study...

  5. Periodontal bacteria in human carotid atherothrombosis as a potential trigger for neutrophil activation.

    Science.gov (United States)

    Rangé, Hélène; Labreuche, Julien; Louedec, Liliane; Rondeau, Philippe; Planesse, Cynthia; Sebbag, Uriel; Bourdon, Emmanuel; Michel, Jean-Baptiste; Bouchard, Philippe; Meilhac, Olivier

    2014-10-01

    Epidemiological, biological and clinical links between periodontal and cardiovascular diseases are now well established. Several human studies have detected bacterial DNA corresponding to periodontal pathogens in cardiovascular samples. Intraplaque hemorrhage has been associated with a higher risk of atherosclerotic plaque rupture, potentially mediated by neutrophil activation. In this study, we hypothesized that plaque composition may be related to periodontal pathogens. Carotid culprit plaque samples were collected from 157 patients. Macroscopic characterization was performed at the time of collection: presence of blood, lipid core, calcification and fibrosis. Markers of neutrophil activation released by carotid samples were quantified (myeloperoxidase or MPO, cell-free DNA and DNA-MPO complexes). PCR analysis using specific primers for Porphyromonas gingivalis, Aggregatibacter actinomycetemcommitans, Treponema denticola, Prevotella intermedia and Tannerella forsythia was used to detect DNA from periodontal pathogens in carotid tissues. In addition, bacterial lipopolysaccharide (LPS) and Immunoglobulins G against T. forsythia were quantified in atherosclerotic carotid conditioned medium. Intraplaque hemorrhage was present in 73/157 carotid samples and was associated with neutrophil activation, reflected by the release of MPO, cell-free DNA and MPO-DNA complexes. LPS levels were also linked to intraplaque hemorrhage but not with the neutrophil activation markers. Seventy-three percent of the carotid samples were positive for periodontal bacterial DNA. Furthermore, hemoglobin levels were associated with the detection of T. forsythia and neutrophil activation/inflammation markers. This study suggests a potential role of periodontal microorganisms, especially T. forsythia, in neutrophil activation within hemorrhagic atherosclerotic carotid plaques. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  6. Potent inhibition of human neutrophil activations by bractelactone, a novel chalcone from Fissistigma bracteolatum

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Yang-Chang [Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China); Graduate Institute of Integrated Medicine, College of Chinese Medicine, China Medical University, Taichung 404, Taiwan (China); Sureshbabu, Munisamy; Fang, Yao-Ching; Wu, Yi-Hsiu [Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan (China); Lan, Yu-Hsuan [School of Pharmacy, China Medical University, Taichung 404, Taiwan (China); Chang, Fang-Rong [Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China); Chang, Ya-Wen [Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan (China); Hwang, Tsong-Long, E-mail: htl@mail.cgu.edu.tw [Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan (China); Chinese Herbal Medicine Research Team, Healthy Aging Research Center, Chang Gung University, Kweishan, Taoyuan 333, Taiwan (China)

    2013-02-01

    Fissistigma bracteolatum is widely used in traditional medicine to treat inflammatory diseases. However, its active components and mechanisms of action remain unclear. In this study, (3Z)-6,7-dihydroxy-4-methoxy-3-(phenylmethylidene)-5-(3-phenylpropanoyl) -1-benzofuran-2(3H) (bractelactone), a novel chalcone from F. bracteolatum, showed potent inhibitory effects against superoxide anion (O{sub 2}{sup ·−}) production, elastase release, and CD11b expression in formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-induced human neutrophils. However, bractelactone showed only weak inhibition of phorbol myristate acetate-caused O{sub 2}{sup ·−} production. The peak cytosolic calcium concentration ([Ca{sup 2+}]{sub i}) was unaltered by bractelactone in FMLP-induced neutrophils, but the decay time of [Ca{sup 2+}]{sub i} was significantly shortened. In a calcium-free solution, changes in [Ca{sup 2+}]{sub i} caused by the addition of extracellular Ca{sup 2+} were inhibited by bractelactone in FMLP-activated cells. In addition, bractelactone did not alter the phosphorylation of p38 MAPK, ERK, JNK, or AKT or the concentration of cAMP. These results suggest that bractelactone selectively inhibits store-operated calcium entry (SOCE). In agreement with this concept, bractelactone suppressed sustained [Ca{sup 2+}]{sub i} changes in thapsigargin-activated neutrophils. Furthermore, bractelactone did not alter FMLP-induced formation of inositol 1,4,5-triphosphate. Taken together, our results demonstrate that the anti-inflammatory effects of bractelactone, an active ingredient of F. bracteolatum, in human neutrophils are through the selective inhibition of SOCE. Highlights: ► Bractelactone isolated from Fissistigma bracteolatum. ► Bractelactone inhibited FMLP-induced human neutrophil activations. ► Bractelactone had no effect on IP3 formation. ► Bractelactone did not alter MAPKs, AKT, and cAMP pathways. ► Bractelactone inhibited store-operated calcium entry.

  7. Paraquat-induced reactive oxygen species inhibit neutrophil apoptosis via a p38 MAPK/NF-κB-IL-6/TNF-α positive-feedback circuit.

    Directory of Open Access Journals (Sweden)

    Xiaolong Wang

    Full Text Available Paraquat (PQ, a widely used herbicide and potent reactive oxygen species (ROS inducer, can injure multiple tissues and organs, especially the lung. However, the underlying mechanism is still poorly understood. According to previous reports, neutrophil aggregation and excessive ROS production might play pivotal pathogenetic roles. In the present study, we found that PQ could prolong neutrophil lifespan and induce ROS generation in a concentration-independent manner. Activated nuclear factor-κB (NF-κB, p38 mitogen-activated kinase (p38 MAPK, and myeloid cell leukemia sequence 1 (Mcl-1 but not Akt signaling pathways were involved in this process, as well as increasing levels of interleukin-6 (IL-6, tumor necrosis factor-α (TNF-α, and IL-1β. Furthermore, the proinflammatory mediators IL-6 and TNF-α could in turn promote ROS generation, creating a vicious cycle. The existence of such a feedback loop is supported by our finding that neutrophil apoptosis is attenuated by PQ in a concentration-independent manner and could partially explain the clinical dilemma why oxygen therapy will exacerbate PQ induced tissue injury.

  8. Growth factors G-CSF and GM-CSF differentially preserve chemotaxis of neutrophils aging in vitro

    NARCIS (Netherlands)

    Wolach, Baruch; van der Laan, Luc J. W.; Maianski, Nikolai A.; Tool, Anton T. J.; van Bruggen, Robin; Roos, Dirk; Kuijpers, Taco W.

    2007-01-01

    OBJECTIVE: The ability of human neutrophils to migrate was studied during culture in vitro. METHODS: Neutrophils were isolated from human blood and cultured at 37 degrees C. Apoptosis was determined by Annexin-V fluorescein isothiocyanate binding. Receptor expression was measured by fluorescence in

  9. Induction of apoptosis in human breast adenocarcinoma MCF-7 ...

    African Journals Online (AJOL)

    Induction of apoptosis in human breast adenocarcinoma MCF-7 cells by tannic acid and resveratrol. Ahu Soyocak, Didem Turgut Cosan, Ayse Basaran, Hasan Veysi Gunes, Irfan Degirmenci, Fezan Sahin Mutlu ...

  10. Tumor-Associated Neutrophils in Human Lung Cancer

    Science.gov (United States)

    2017-10-01

    markers in humans. The logistical, ethical , and regulatory difficulties in obtaining human tumor tissue for research also act to discourage such...Mouse models of cancer. Annu. Rev. Pathol 6, 95–119 52. Merlo, L.M. et al. (2006) Cancer as an evolutionary and ecological process. Nat. Rev. Cancer...some effect on the phenotype and function of TANs. The logistical, ethical , and regulatory difficulties in obtaining human tumor tissue for research

  11. APOPTOSIS DURING HUMAN FETAL KIDNEY DEVELOPMENT

    Directory of Open Access Journals (Sweden)

    Rade Čukuranović

    2005-01-01

    Full Text Available Kidney morphogenesis is a complex and stepwise process. The formation of mature kidney in mammals is preceded by two primitive embryonic kidneys known as pronephros and mesonephros. Metanephros develops as a result of reciprocal inductive interactions between two primordial mesodermal derivates: ureteric bud, an epithelial outgrowth of the Wolffian duct, and metanephric blastema, a group of mesenchymal cells. The ureteric bud induces the metanephric mesenchyme to differentiate and form nephrons, whilst the metanephric mesenchyme induces the ureteric bud to grow and branch to form collecting ducts. The nephron goes through four developmental stages, which are described as: 1 vesicle, 2 comma-shaped and S-shaped stages, 3 developing capillary loop, and finally 4 maturing glomerulus. Apoptosis (programmed cell death is a predominant form of physiological cell death, by which organism eliminate unwanted or damaged cells. It is the major component of normal development and disease. Apoptosis is the result of series of biochemical processes happening in certain order in a dying cell, among which the most important is activation of enzyme families called caspases which influence different cell components. Apoptosis is characterized by membrane blebbing, shrinkage of the cell, nuclear fragmentation and chromatin condensation. Organelles are preserved almost intact. Cell surface molecules change. A variety of physiological and pathological stimuli can initiate apoptosis. They act via receptor mechanisms, through biochemical agents, or cause DNA and cell membrane damage. Apoptosis is an important component of fetal development. It is thought that apoptosis is the one of the main regulatory events involved in kidney morphogenesis, considering that among great number of developed cells, only a few of them are involved in the developing program by escaping apoptosis. In any period during kidney development about 3 to 5%of cells are apoptotic. Thorough

  12. Attenuated, oncolytic, but not wild-type measles virus infection has pleiotropic effects on human neutrophil function.

    Science.gov (United States)

    Zhang, Yu; Patel, Bella; Dey, Aditi; Ghorani, Ehsan; Rai, Lena; Elham, Mohammed; Castleton, Anna Z; Fielding, Adele K

    2012-02-01

    We previously showed that neutrophils play a role in regression of human tumor xenografts in immunodeficient mice following oncolytic vaccine measles virus (MV-Vac) treatment. In this study, we sought, using normal human neutrophils, to identify potential neutrophil-mediated mechanisms for the attenuated MV-Vac induced effects seen in vivo, by comparison with those consequent on wild-type (WT-MV) infection. Both MV-Vac and WT-MV infected and replicated within neutrophils, despite lack of SLAM expression. In both cases, neutrophils survived longer ex vivo postinfection. Furthermore, MV-Vac (but not WT-MV) infection activated neutrophils and stimulated secretion of several specific antitumor cytokines (IL-8, TNF-α, MCP-1, and IFN-α) via induction of de novo RNA and protein synthesis. In addition, MV-Vac (but not WT-MV) infection caused TRAIL secretion in the absence of de novo synthesis by triggering release of prefabricated TRAIL, via a direct effect upon degranulation. The differences between the outcome of infection by MV-Vac and WT-MV were not entirely explained by differential infection and replication of the viruses within neutrophils. To our knowledge, this is the first demonstration of potential mechanisms of oncolytic activity of an attenuated MV as compared with its WT parent. Furthermore, our study suggests that neutrophils have an important role to play in the antitumor effects of oncolytic MV.

  13. Effects of gadolinium oxide nanoparticles on the oxidative burst from human neutrophil granulocytes

    International Nuclear Information System (INIS)

    Abrikossova, Natalia; Skoglund, Caroline; Ahrén, Maria; Uvdal, Kajsa; Bengtsson, Torbjörn

    2012-01-01

    We have previously shown that gadolinium oxide (Gd 2 O 3 ) nanoparticles are promising candidates to be used as contrast agents in magnetic resonance (MR) imaging applications. In this study, these nanoparticles were investigated in a cellular system, as possible probes for visualization and targeting intended for bioimaging applications. We evaluated the impact of the presence of Gd 2 O 3 nanoparticles on the production of reactive oxygen species (ROS) from human neutrophils, by means of luminol-dependent chemiluminescence. Three sets of Gd 2 O 3 nanoparticles were studied, i.e. as synthesized, dialyzed and both PEG-functionalized and dialyzed Gd 2 O 3 nanoparticles. In addition, neutrophil morphology was evaluated by fluorescent staining of the actin cytoskeleton and fluorescence microscopy. We show that surface modification of these nanoparticles with polyethylene glycol (PEG) is essential in order to increase their biocompatibility. We observed that the as synthesized nanoparticles markedly decreased the ROS production from neutrophils challenged with prey (opsonized yeast particles) compared to controls without nanoparticles. After functionalization and dialysis, more moderate inhibitory effects were observed at a corresponding concentration of gadolinium. At lower gadolinium concentration the response was similar to that of the control cells. We suggest that the diethylene glycol (DEG) present in the as synthesized nanoparticle preparation is responsible for the inhibitory effects on the neutrophil oxidative burst. Indeed, in the present study we also show that even a low concentration of DEG, 0.3%, severely inhibits neutrophil function. In summary, the low cellular response upon PEG-functionalized Gd 2 O 3 nanoparticle exposure indicates that these nanoparticles are promising candidates for MR-imaging purposes. (paper)

  14. Leishmania major surface protease Gp63 interferes with the function of human monocytes and neutrophils in vitro

    DEFF Research Database (Denmark)

    Sørensen, A L; Hey, A S; Kharazmi, A

    1994-01-01

    In the present study the effect of Leishmania major surface protease Gp63 on the chemotaxis and oxidative burst response of human peripheral blood monocytes and neutrophils was investigated. It was shown that prior incubation of cells with Gp63 inhibited chemotaxis of neutrophils but not monocytes...... towards the chemotactic peptide f-met-leu-phe. On the other hand, chemotaxis of both neutrophils and monocytes towards zymosan-activated serum containing C5a was inhibited by Gp63. Monocyte and neutrophil chemiluminescence response to opsonized zymosan was reduced by preincubation of the cells with Gp63...... in a concentration-dependent manner. Notably, monocytes were inhibited to a much greater degree than neutrophils by a given concentration of Gp63, and they were also inhibited at much lower concentrations of the protease. The inhibitory effect of Gp63 on chemotaxis and chemiluminescence was completely abolished...

  15. A role for protein phosphatase-2A in p38 mitogen-activated protein kinase-mediated regulation of the c-Jun NH(2)-terminal kinase pathway in human neutrophils.

    Science.gov (United States)

    Avdi, Natalie J; Malcolm, Kenneth C; Nick, Jerry A; Worthen, G Scott

    2002-10-25

    Human neutrophil accumulation in inflammatory foci is essential for the effective control of microbial infections. Although exposure of neutrophils to cytokines such as tumor necrosis factor-alpha (TNFalpha), generated at sites of inflammation, leads to activation of MAPK pathways, mechanisms responsible for the fine regulation of specific MAPK modules remain unknown. We have previously demonstrated activation of a TNFalpha-mediated JNK pathway module, leading to apoptosis in adherent human neutrophils (Avdi, N. J., Nick, J. A., Whitlock, B. B., Billstrom, M. A., Henson, P. M., Johnson, G. L., and Worthen, G. S. (2001) J. Biol. Chem. 276, 2189-2199). Herein, evidence is presented linking regulation of the JNK pathway to p38 MAPK and the Ser/Thr protein phosphatase-2A (PP2A). Inhibition of p38 MAPK by SB 203580 and M 39 resulted in significant augmentation of TNFalpha-induced JNK and MKK4 (but not MKK7 or MEKK1) activation, whereas prior exposure to a p38-activating agent (platelet-activating factor) diminished the TNFalpha-induced JNK response. TNFalpha-induced apoptosis was also greatly enhanced upon p38 inhibition. Studies with a reconstituted cell-free system indicated the absence of a direct inhibitory effect of p38 MAPK on the JNK module. Neutrophil exposure to the Ser/Thr phosphatase inhibitors okadaic acid and calyculin A induced JNK activation. Increased phosphatase activity following TNFalpha stimulation was shown to be PP2A-associated and p38-dependent. Furthermore, PP2A-induced dephosphorylation of MKK4 resulted in its inactivation. Thus, in neutrophils, p38 MAPK, through a PP2A-mediated mechanism, regulates the JNK pathway, thus determining the extent and nature of subsequent responses such as apoptosis.

  16. Human neutrophil peptides and complement factor Bb in pathogenesis of acquired thrombotic thrombocytopenic purpura.

    Science.gov (United States)

    Cao, Wenjing; Pham, Huy P; Williams, Lance A; McDaniel, Jenny; Siniard, Rance C; Lorenz, Robin G; Marques, Marisa B; Zheng, X Long

    2016-11-01

    Acquired thrombotic thrombocytopenic purpura is primarily caused by the deficiency of plasma ADAMTS13 activity resulting from autoantibodies against ADAMTS13. However, ADAMTS13 deficiency alone is often not sufficient to cause acute thrombotic thrombocytopenic purpura. Infections or systemic inflammation may precede acute bursts of the disease, but the underlying mechanisms are not fully understood. Herein, 52 patients with acquired autoimmune thrombotic thrombocytopenic purpura and 30 blood donor controls were recruited for the study. The plasma levels of human neutrophil peptides 1-3 and complement activation fragments (i.e. Bb, iC3b, C4d, and sC5b-9) were determined by enzyme-linked immunosorbent assays. Univariate analyses were performed to determine the correlation between each biomarker and clinical outcomes. We found that the plasma levels of human neutrophil peptides 1-3 and Bb in patients with acute thrombotic thrombocytopenic purpura were significantly higher than those in the control (Ppurpura patients and the control. We conclude that innate immunity, i.e. neutrophil and complement activation via the alternative pathway, may play a role in the pathogenesis of acute autoimmune thrombotic thrombocytopenic purpura, and a therapy targeted at these pathways may be considered in a subset of these patients. Copyright© Ferrata Storti Foundation.

  17. Indomethacin increases the formation of lipoxygenase products in calcium ionophore stimulated human neutrophils.

    Science.gov (United States)

    Docherty, J C; Wilson, T W

    1987-10-29

    Arachidonic acid metabolism in human neutrophils stimulated in vitro with the calcium ionophore A23187 was studied using combined HPLC and radioimmunoassays. Indomethacin (0.1 and 1.0 microM) caused a 300% increase in LTB4 formation in neutrophils stimulated with A23187. 5-, 12- and 15-HETE levels were also increased. In the presence of exogenous arachidonic acid 1.0 microM Indomethacin caused a 37% increase in LTB4 formation. Acetyl Salicylic Acid and Ibuprofen had no effect on the formation of lipoxygenase metabolites. The effect of indomethacin on LTB4 formation does not appear to be due to a simple redirection of substrate arachidonic acid from the cyclooxygenase to the lipoxygenase pathways.

  18. Amburanins A and B from Amburana cearensis: daphnodorin-type biflavonoids that modulate human neutrophil degranulation

    Energy Technology Data Exchange (ETDEWEB)

    Canuto, Kirley M.; Silveira, Edilberto R., E-mail: edil@ufc.br [Universidade Federal do Ceara (UFCE), Fortaleza, CE (Brazil). Departamento de Quimica Organica e Inorganica; Leal, Luzia K.A.M.; Lopes, Amanda A. [Universidade Federal do Ceara (CEFAC/UFCE), Fortaleza, CE (Brazil). Centro de Estudos Farmaceuticos e Cosmeticos. Departamento de Farmacia; Coleman, Christina M.; Ferreira, Daneel [Department of Pharmacognosy and the Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi, MS (United States)

    2014-04-15

    Two new biflavonoids 3,5,7,4'-tetrahydroxyflavanone-(2→O→4':3→3')-2',4',6',4- tetrahydroxydihydrochalcone (1) and 3,5,7,4'-tetrahydroxyflavanone-(2→O→7:3→8)-3,4',5,7-tetrahydroxyflavone (2), named as amburanin A and amburanin B, respectively, were isolated from the trunk bark of Amburana cearensis, and their structures elucidated on the basis of spectroscopic analysis and by comparison with literature data. The effects of 1 and 2 on the pro-inflammatory response of human neutrophils were investigated (0.1; 1; 25; 50 e 100 μg mL{sup -1}). At concentration higher than 25 μg mL{sup -1}, both compounds suppressed nearly 92% of the neutrophil degranulation and 53% of myeloperoxidase activity, thus indicating that they are potential anti-inflammatory lead compounds. (author)

  19. Amburanins A and B from Amburana cearensis: daphnodorin-type biflavonoids that modulate human neutrophil degranulation

    Energy Technology Data Exchange (ETDEWEB)

    Canuto, Kirley M.; Silveira, Edilberto R., E-mail: edil@ufc.br [Universidade Federal do Ceara (UFCE), Fortaleza, CE (Brazil). Departamento de Quimica Organica e Inorganica; Leal, Luzia K.A.M.; Lopes, Amanda A. [Universidade Federal do Ceara (CEFAC/UFCE), Fortaleza, CE (Brazil). Centro de Estudos Farmaceuticos e Cosmeticos. Departamento de Farmacia; Coleman, Christina M.; Ferreira, Daneel [Department of Pharmacognosy and the Research Institute of Pharmaceutical Sciences, School of Pharmacy, The University of Mississippi, MS (United States)

    2014-04-15

    Two new biflavonoids 3,5,7,4'-tetrahydroxyflavanone-(2→O→4':3→3')-2',4',6',4- tetrahydroxydihydrochalcone (1) and 3,5,7,4'-tetrahydroxyflavanone-(2→O→7:3→8)-3,4',5,7-tetrahydroxyflavone (2), named as amburanin A and amburanin B, respectively, were isolated from the trunk bark of Amburana cearensis, and their structures elucidated on the basis of spectroscopic analysis and by comparison with literature data. The effects of 1 and 2 on the pro-inflammatory response of human neutrophils were investigated (0.1; 1; 25; 50 e 100 μg mL{sup -1}). At concentration higher than 25 μg mL{sup -1}, both compounds suppressed nearly 92% of the neutrophil degranulation and 53% of myeloperoxidase activity, thus indicating that they are potential anti-inflammatory lead compounds. (author)

  20. Amburanins A and B from Amburana cearensis: daphnodorin-type biflavonoids that modulate human neutrophil degranulation

    International Nuclear Information System (INIS)

    Canuto, Kirley M.; Silveira, Edilberto R.

    2014-01-01

    Two new biflavonoids 3,5,7,4'-tetrahydroxyflavanone-(2→O→4':3→3')-2',4',6',4- tetrahydroxydihydrochalcone (1) and 3,5,7,4'-tetrahydroxyflavanone-(2→O→7:3→8)-3,4',5,7-tetrahydroxyflavone (2), named as amburanin A and amburanin B, respectively, were isolated from the trunk bark of Amburana cearensis, and their structures elucidated on the basis of spectroscopic analysis and by comparison with literature data. The effects of 1 and 2 on the pro-inflammatory response of human neutrophils were investigated (0.1; 1; 25; 50 e 100 μg mL -1 ). At concentration higher than 25 μg mL -1 , both compounds suppressed nearly 92% of the neutrophil degranulation and 53% of myeloperoxidase activity, thus indicating that they are potential anti-inflammatory lead compounds. (author)

  1. Minocycline affects human neutrophil respiratory burst and transendothelial migration.

    Science.gov (United States)

    Parenti, Astrid; Indorato, Boris; Paccosi, Sara

    2017-02-01

    This study aimed at investigating the in vitro activity of minocycline and doxycycline on human polymorphonuclear (h-PMN) cell function. h-PMNs were isolated from whole venous blood of healthy subjects; PMN oxidative burst was measured by monitoring ROS-induced oxidation of luminol and transendothelial migration was studied by measuring PMN migration through a monolayer of human umbilical vein endothelial cells. Differences between multiple groups were determined by ANOVA followed by Tukey's multiple comparison test; Student's t test for unpaired data for two groups. Minocycline (1-300 µM) concentration dependently and significantly inhibited oxidative burst of h-PMNs stimulated with 100 nM fMLP. Ten micromolar concentrations, which are superimposable to C max following a standard oral dose of minocycline, promoted a 29.8 ± 4 % inhibition of respiratory burst (P minocycline impaired PMN transendothelial migration, with maximal effect at 100 µM (42.5 ± 7 %, inhibition, n = 5, P minocycline exerted on innate immune h-PMN cell function.

  2. Targeting Neutrophilic Inflammation using Polymersome-Mediated Cellular Delivery

    OpenAIRE

    Robertson, J.D.; Ward, J.R.; Avila-Olias, M.; Battaglia, G.; Renshaw, S.A.

    2017-01-01

    Neutrophils are key effector cells in inflammation and play an important role in neutralizing invading pathogens. During inflammation resolution, neutrophils undergo apoptosis before they are removed by macrophages, but if apoptosis is delayed, neutrophils can cause extensive tissue damage and chronic disease. Promotion of neutrophil apoptosis is a potential therapeutic approach for treating persistent inflammation, yet neutrophils have proven difficult cells to manipulate experimentally. In ...

  3. Mycobacterium tuberculosis Cell Wall Fragments Released upon Bacterial Contact with the Human Lung Mucosa Alter the Neutrophil Response to Infection.

    Science.gov (United States)

    Scordo, Julia M; Arcos, Jesús; Kelley, Holden V; Diangelo, Lauren; Sasindran, Smitha J; Youngmin, Ellie; Wewers, Mark D; Wang, Shu-Hua; Balada-Llasat, Joan-Miquel; Torrelles, Jordi B

    2017-01-01

    In 2016, the World Health Organization reported that one person dies of tuberculosis (TB) every 21 s. A host environment that Mycobacterium tuberculosis ( M.tb ) finds during its route of infection is the lung mucosa bathing the alveolar space located in the deepest regions of the lungs. We published that human lung mucosa, or alveolar lining fluid (ALF), contains an array of hydrolytic enzymes that can significantly alter the M.tb surface during infection by cleaving off parts of its cell wall. This interaction results in two different outcomes: modifications on the M.tb cell wall surface and release of M.tb cell wall fragments into the environment. Typically, one of the first host immune cells at the site of M.tb infection is the neutrophil. Neutrophils can mount an extracellular and intracellular innate immune response to M.tb during infection. We hypothesized that exposure of neutrophils to ALF-induced M.tb released cell wall fragments would prime neutrophils to control M.tb infection better. Our results show that ALF fragments activate neutrophils leading to an increased production of inflammatory cytokines and oxidative radicals. However, neutrophil exposure to these fragments reduces production of chemoattractants (i.e., interleukin-8), and degranulation, with the subsequent reduction of myeloperoxidase release, and does not induce cytotoxicity. Unexpectedly, these ALF fragment-derived modulations in neutrophil activity do not further, either positively or negatively, contribute to the intracellular control of M.tb growth during infection. However, secreted products from neutrophils primed with ALF fragments are capable of regulating the activity of resting macrophages. These results indicate that ALF-induced M.tb fragments could further contribute to the control of M.tb growth and local killing by resident neutrophils by switching on the total oxidative response and limiting migration of neutrophils to the infection site.

  4. Regulation of neutrophil senescence by microRNAs.

    Directory of Open Access Journals (Sweden)

    Jon R Ward

    2011-01-01

    Full Text Available Neutrophils are rapidly recruited to sites of tissue injury or infection, where they protect against invading pathogens. Neutrophil functions are limited by a process of neutrophil senescence, which renders the cells unable to respond to chemoattractants, carry out respiratory burst, or degranulate. In parallel, aged neutrophils also undergo spontaneous apoptosis, which can be delayed by factors such as GMCSF. This is then followed by their subsequent removal by phagocytic cells such as macrophages, thereby preventing unwanted inflammation and tissue damage. Neutrophils translate mRNA to make new proteins that are important in maintaining functional longevity. We therefore hypothesised that neutrophil functions and lifespan might be regulated by microRNAs expressed within human neutrophils. Total RNA from highly purified neutrophils was prepared and subjected to microarray analysis using the Agilent human miRNA microarray V3. We found human neutrophils expressed a selected repertoire of 148 microRNAs and that 6 of these were significantly upregulated after a period of 4 hours in culture, at a time when the contribution of apoptosis is negligible. A list of predicted targets for these 6 microRNAs was generated from http://mirecords.biolead.org and compared to mRNA species downregulated over time, revealing 83 genes targeted by at least 2 out of the 6 regulated microRNAs. Pathway analysis of genes containing binding sites for these microRNAs identified the following pathways: chemokine and cytokine signalling, Ras pathway, and regulation of the actin cytoskeleton. Our data suggest that microRNAs may play a role in the regulation of neutrophil senescence and further suggest that manipulation of microRNAs might represent an area of future therapeutic interest for the treatment of inflammatory disease.

  5. Sulfur mustard primes human neutrophils for increased degranulation and stimulates cytokine release via TRPM2/p38 MAPK signaling

    Energy Technology Data Exchange (ETDEWEB)

    Ham, Hwa-Yong [Department of Pharmacology, Infectious Diseases Medical Research Center, College of Medicine, Hallym University, Chuncheon (Korea, Republic of); Hong, Chang-Won, E-mail: chyj7983@hallym.ac.kr [Department of Chemical and Biological Warfare Research, The Armed Forces Medical Research Institute, Daejeon (Korea, Republic of); Lee, Si-Nae [Department of Pharmacology, Infectious Diseases Medical Research Center, College of Medicine, Hallym University, Chuncheon (Korea, Republic of); Kwon, Min-Soo [Department of Pharmacology, School of Medicine, CHA University, Seongnam (Korea, Republic of); Kim, Yeon-Ja [Department of Pharmacology, Infectious Diseases Medical Research Center, College of Medicine, Hallym University, Chuncheon (Korea, Republic of); Song, Dong-Keun, E-mail: dksong@hallym.ac.kr [Department of Pharmacology, Infectious Diseases Medical Research Center, College of Medicine, Hallym University, Chuncheon (Korea, Republic of)

    2012-01-01

    Sulfur mustard (2,2′-bis-chloroethyl-sulfide; SM) has been a military threat since the World War I. The emerging threat of bioterrorism makes SM a major threat not only to military but also to civilian world. SM injury elicits an inflammatory response characterized by infiltration of neutrophils. Although SM was reported to prime neutrophils, the mechanism has not been identified yet. In the present study, we investigated the mechanism of SM-induced priming in human neutrophils. SM increased [Ca{sup 2+}]{sub i} in human neutrophils in a concentration-dependent fashion. Transient receptor potential melastatin (TRPM) 2 inhibitors (clotrimazole, econazole and flufenamic acid) and silencing of TRPM2 by shRNA attenuated SM-induced [Ca{sup 2+}]{sub i} increase. SM primed degranulation of azurophil and specific granules in response to activation by fMLP as previously reported. SB203580, an inhibitor of p38 MAPK, inhibited SM-induced priming. Neither PD98057, an ERK inhibitor, nor SP600215, a JNK inhibitor, inhibited SM-induced priming. In addition, SM enhanced phosphorylation of NF-kB p65 and release of TNF-α, interleukin (IL)-6 and IL-8. SB203580 inhibited SM-induced NF-kB phosphorylation and cytokine release. These results suggest the involvement of TRPM2/p38 MAPK pathway in SM-induced priming and cytokines release in neutrophils. -- Highlights: ► SM increased [Ca{sup 2+}]{sub i} in human neutrophils through TPRM2-mediated calcium influx. ► SM primed degranulation of azurophil and specific granules. ► SM enhanced p38 MAPK and NF-κB p65 phosphorylation in human neutrophils. ► SM enhanced release of TNF-α, interleukin (IL)-6 and IL-8 from human neutrophils. ► SB203580 inhibited SM-induced priming, NF-κB p65 phosphorylation and cytokine release.

  6. Apoptosis signal-regulating kinase 1 mediates denbinobin-induced apoptosis in human lung adenocarcinoma cells

    Directory of Open Access Journals (Sweden)

    Pan Shiow-Lin

    2009-05-01

    Full Text Available Abstract In the present study, we explore the role of apoptosis signal-regulating kinase 1 (ASK1 in denbinobin-induced apoptosis in human lung adenocarcinoma (A549 cells. Denbinobin-induced cell apoptosis was attenuated by an ASK1 dominant-negative mutant (ASK1DN, two antioxidants (N-acetyl-L-cysteine (NAC and glutathione (GSH, a c-Jun N-terminal kinase (JNK inhibitor (SP600125, and an activator protein-1 (AP-1 inhibitor (curcumin. Treatment of A549 cells with denbinobin caused increases in ASK1 activity and reactive oxygen species (ROS production, and these effects were inhibited by NAC and GSH. Stimulation of A549 cells with denbinobin caused JNK activation; this effect was markedly inhibited by NAC, GSH, and ASK1DN. Denbinobin induced c-Jun phosphorylation, the formation of an AP-1-specific DNA-protein complex, and Bim expression. Bim knockdown using a bim short interfering RNA strategy also reduced denbinobin-induced A549 cell apoptosis. The denbinobin-mediated increases in c-Jun phosphorylation and Bim expression were inhibited by NAC, GSH, SP600125, ASK1DN, JNK1DN, and JNK2DN. These results suggest that denbinobin might activate ASK1 through ROS production to cause JNK/AP-1 activation, which in turn induces Bim expression, and ultimately results in A549 cell apoptosis.

  7. Damage to Aspergillus fumigatus and Rhizopus oryzae Hyphae by Oxidative and Nonoxidative Microbicidal Products of Human Neutrophils In Vitro

    OpenAIRE

    Diamond, Richard D.; Clark, Robert A.

    1982-01-01

    Our previous studies established that human neutrophils could damage and probably kill hyphae of Aspergillus fumigatus and Rhizopus oryzae in vitro, primarily by oxygen-dependent mechanisms active at the cell surface. These studies were extended, again quantitating hyphal damage by reduction in uptake of 14C-labeled uracil or glutamine. Neither A. fumigatus nor R. oryzae hyphae were damaged by neutrophils from patients with chronic granulomatous disease, confirming the importance of oxidative...

  8. Protectin DX, a double lipoxygenase product of DHA, inhibits both ROS production in human neutrophils and cyclooxygenase activities

    Science.gov (United States)

    Liu, Miao; Boussetta, Tarek; Makni-Maalej, Karama; Fay, Michèle; Driss, Fathi; El-Benna, Jamel; Lagarde, Michel; Guichardant, Michel

    2014-01-01

    Neutrophils play a major role in inflammation by releasing large amounts of reactive oxygen species (ROS) produced by NADPH oxidase (NOX) and myeloperoxidase (MPO). This ROS overproduction is mediated by phosphorylation of the NOX subunits with an uncontrolled manner. Therefore, targeting neutrophil subunits would represent a promising strategy to moderate NOX activity, lower ROS, and other inflammatory agents, such as cytokines and leukotrienes, produced by neutrophils. For this purpose, we investigated the effects of protectin DX (PDX) - a docosahexaenoic acid (DHA) di-hydroxylated product which inhibits blood platelet aggregation - on neutrophil activation in vitro. We found that PDX decreases ROS production, inhibits NOX activation and MPO release from neutrophils. We also confirm, that PDX is an anti-aggregatory and anti-inflammatory agent by inhibiting both cyclooxygenase-1 and -2 (COX-1 and COX-2, E.C. 1.14.99.1) as well as COX-2 in lipopolysaccharides (LPS)-treated human neutrophils. However, PDX has no effect on the 5-lipoxygenase pathway that produces the chemotactic agent leukotriene B4 (LTB4). Taken together, our results suggest that PDX could be a protective agent against neutrophil invasion in chronic inflammatory diseases. PMID:24254970

  9. High resolution of heterogeneity among human neutrophil granules: physical, biochemical, and ultrastructural properties of isolated fractions.

    Science.gov (United States)

    Rice, W G; Kinkade, J M; Parmley, R T

    1986-08-01

    Previous studies on the fractionation of human neutrophil granules have identified two major populations: myeloperoxidase (MPO)-containing azurophil, or primary, granules and MPO-deficient specific, or secondary, granules. Peripheral blood neutrophils from individual donors were lysed in sucrose-free media by either hypotonic shock or nitrogen cavitation. Using a novel two-gradient Percoll density centrifugation system, the granule-rich postnuclear supernatant was rapidly (ten minutes) and reproducibly resolved into 13 granule fractions (L1 through L8 and H1 through H5). Granule flotation and recentrifugation experiments on both continuous, self-generated and multiple-step gradients using individual and mixed isolated fractions demonstrated that the banding patterns were isopycnic and nonartifactual. Isolated granules were intact based on the findings that biochemical latency of several granule enzymes was greater than 95%, and thin-sectioned electron micrographs demonstrated intact granule profiles. Biochemical analyses of the granule marker proteins MPO, beta-glucuronidase, lysozyme, and lactoferrin indicated that a number of the fractions were related to the major azurophil and specific granule populations. Lactoferrin was found in ten of 13 fractions (L1 through L8, H1 to H2), whereas MPO was found in every fraction. Consistent with these biochemical data, all fractions exhibited varying degrees of heterogeneity based on ultrastructural morphology and cytochemistry, including diaminobenzidine (DAB) reactivity for peroxidase and periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining for complex glycoconjugates. A variable but significant percentage (23% to 70%) of the granules in fractions L1 through L8 and H1 and H2 showed DAB reactivity, while about 90% of the granules in fractions H3 through H5 were peroxidase positive. These results demonstrated that DAB-reactive granules spanned the entire range of granule size and density. Ultrastructural PA

  10. Augmenter of liver regeneration (ALR) protects human hepatocytes against apoptosis

    International Nuclear Information System (INIS)

    Ilowski, Maren; Kleespies, Axel; Toni, Enrico N. de; Donabauer, Barbara; Jauch, Karl-Walter; Hengstler, Jan G.; Thasler, Wolfgang E.

    2011-01-01

    Research highlights: → ALR decreases cytochrome c release from mitochondria. → ALR protects hepatocytes against apoptosis induction by ethanol, TRAIL, anti-Apo, TGF-β and actinomycin D. → ALR exerts a liver-specific anti-apoptotic effect. → A possible medical usage of ALR regarding protection of liver cells during apoptosis inducing therapies. -- Abstract: Augmenter of liver regeneration (ALR) is known to support liver regeneration and to stimulate proliferation of hepatocytes. However, it is not known if ALR exerts anti-apoptotic effects in human hepatocytes and whether this protective effect is cell type specific. This is relevant, because compounds that protect the liver against apoptosis without undesired effects, such as protection of metastatic tumour cells, would be appreciated in several clinical settings. Primary human hepatocytes (phH) and organotypic cancer cell lines were exposed to different concentrations of apoptosis inducers (ethanol, TRAIL, anti-Apo, TGF-β, actinomycin D) and cultured with or without recombinant human ALR (rhALR). Apoptosis was evaluated by the release of cytochrome c from mitochondria and by FACS with propidium iodide (PI) staining. ALR significantly decreased apoptosis induced by ethanol, TRAIL, anti-Apo, TGF-β and actinomycin D. Further, the anti-apoptotic effect of ALR was observed in primary human hepatocytes and in HepG2 cells but not in bronchial (BC1), colonic (SW480), gastric (GC1) and pancreatic (L3.6PL) cell lines. Therefore, the hepatotrophic growth factor ALR acts in a liver specific manner with regards to both its mitogenic and its anti-apoptotic effect. Unlike the growth factors HGF and EGF, rhALR acts in a liver specific manner. Therefore, ALR is a promising candidate for further evaluation as a possible hepatoprotective factor in clinical settings.

  11. Augmenter of liver regeneration (ALR) protects human hepatocytes against apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Ilowski, Maren [Liver Regeneration Group, Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Kleespies, Axel [Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Toni, Enrico N. de [Department of Medicine II, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Donabauer, Barbara [Liver Regeneration Group, Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Jauch, Karl-Walter [Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Hengstler, Jan G. [Leibniz Research Centre for Working Environment and Human Factors, Technical University, Dortmund (Germany); Thasler, Wolfgang E., E-mail: wolfgang.thasler@med.uni-muenchen.de [Liver Regeneration Group, Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany); Department of Surgery, Grosshadern Hospital, Ludwig Maximilians University, Munich (Germany)

    2011-01-07

    Research highlights: {yields} ALR decreases cytochrome c release from mitochondria. {yields} ALR protects hepatocytes against apoptosis induction by ethanol, TRAIL, anti-Apo, TGF-{beta} and actinomycin D. {yields} ALR exerts a liver-specific anti-apoptotic effect. {yields} A possible medical usage of ALR regarding protection of liver cells during apoptosis inducing therapies. -- Abstract: Augmenter of liver regeneration (ALR) is known to support liver regeneration and to stimulate proliferation of hepatocytes. However, it is not known if ALR exerts anti-apoptotic effects in human hepatocytes and whether this protective effect is cell type specific. This is relevant, because compounds that protect the liver against apoptosis without undesired effects, such as protection of metastatic tumour cells, would be appreciated in several clinical settings. Primary human hepatocytes (phH) and organotypic cancer cell lines were exposed to different concentrations of apoptosis inducers (ethanol, TRAIL, anti-Apo, TGF-{beta}, actinomycin D) and cultured with or without recombinant human ALR (rhALR). Apoptosis was evaluated by the release of cytochrome c from mitochondria and by FACS with propidium iodide (PI) staining. ALR significantly decreased apoptosis induced by ethanol, TRAIL, anti-Apo, TGF-{beta} and actinomycin D. Further, the anti-apoptotic effect of ALR was observed in primary human hepatocytes and in HepG2 cells but not in bronchial (BC1), colonic (SW480), gastric (GC1) and pancreatic (L3.6PL) cell lines. Therefore, the hepatotrophic growth factor ALR acts in a liver specific manner with regards to both its mitogenic and its anti-apoptotic effect. Unlike the growth factors HGF and EGF, rhALR acts in a liver specific manner. Therefore, ALR is a promising candidate for further evaluation as a possible hepatoprotective factor in clinical settings.

  12. Verocytotoxin-induced apoptosis of human microvascular endothelial cells.

    Science.gov (United States)

    Pijpers, A H; van Setten, P A; van den Heuvel, L P; Assmann, K J; Dijkman, H B; Pennings, A H; Monnens, L A; van Hinsbergh, V W

    2001-04-01

    The pathogenesis of the epidemic form of hemolytic uremic syndrome is characterized by endothelial cell damage. In this study, the role of apoptosis in verocytotoxin (VT)-mediated endothelial cell death in human glomerular microvascular endothelial cells (GMVEC), human umbilical vein endothelial cells, and foreskin microvascular endothelial cells (FMVEC) was investigated. VT induced apoptosis in GMVEC and human umbilical vein endothelial cells when the cells were prestimulated with the inflammatory mediator tumor necrosis factor-alpha (TNF-alpha). FMVEC displayed strong binding of VT and high susceptibility to VT under basal conditions, which made them suitable for the study of VT-induced apoptosis without TNF-alpha interference. On the basis of functional (flow cytometry and immunofluorescence microscopy using FITC-conjugated annexin V and propidium iodide), morphologic (transmission electron microscopy), and molecular (agarose gel electrophoresis of cellular DNA fragments) criteria, it was documented that VT induced programmed cell death in microvascular endothelial cells in a dose- and time-dependent manner. Furthermore, whereas partial inhibition of protein synthesis by VT was associated with a considerable number of apoptotic cells, comparable inhibition of protein synthesis by cycloheximide was not. This suggests that additional pathways, independent of protein synthesis inhibition, may be involved in VT-mediated apoptosis in microvascular endothelial cells. Specific inhibition of caspases by Ac-Asp-Glu-Val-Asp-CHO, but not by Ac-Tyr-Val-Ala-Asp-CHO, was accompanied by inhibition of VT-induced apoptosis in FMVEC and TNF-alpha-treated GMVEC. These data indicate that VT can induce apoptosis in human microvascular endothelial cells.

  13. Apoptosis induction of epifriedelinol on human cervical cancer cell line

    African Journals Online (AJOL)

    Background: Present investigation evaluates the antitumor activity of epifriedelinol for the management of cervical cancer by inducing process of apoptosis. Methods: Human Cervical Cancer Cell Line, C33A and HeLa were selected for study and treated with epifriedelinol at a concentration of (50-1000 μg/ml). Cytotoxicity of ...

  14. A stable aspirin-triggered lipoxin A4 analog blocks phosphorylation of leukocyte-specific protein 1 in human neutrophils.

    Science.gov (United States)

    Ohira, Taisuke; Bannenberg, Gerard; Arita, Makoto; Takahashi, Minoru; Ge, Qingyuan; Van Dyke, Thomas E; Stahl, Gregory L; Serhan, Charles N; Badwey, John A

    2004-08-01

    Lipoxins and their aspirin-triggered 15-epimers are endogenous anti-inflammatory agents that block neutrophil chemotaxis in vitro and inhibit neutrophil influx in several models of acute inflammation. In this study, we examined the effects of 15-epi-16-(p-fluoro)-phenoxy-lipoxin A(4) methyl ester, an aspirin-triggered lipoxin A(4)-stable analog (ATLa), on the protein phosphorylation pattern of human neutrophils. Neutrophils stimulated with the chemoattractant fMLP were found to exhibit intense phosphorylation of a 55-kDa protein that was blocked by ATLa (10-50 nM). This 55-kDa protein was identified as leukocyte-specific protein 1, a downstream component of the p38-MAPK cascade in neutrophils, by mass spectrometry, Western blotting, and immunoprecipitation experiments. ATLa (50 nM) also reduced phosphorylation/activation of several components of the p38-MAPK pathway in these cells (MAPK kinase 3/MAPK kinase 6, p38-MAPK, MAPK-activated protein kinase-2). These results indicate that ATLa exerts its anti-inflammatory effects, at least in part, by blocking activation of the p38-MAPK cascade in neutrophils, which is known to promote chemotaxis and other proinflammatory responses by these cells.

  15. Induction of apoptosis by eugenol in human breast cancer cells

    International Nuclear Information System (INIS)

    Vidhya, N.; Niranjali Devaraj, S.

    2011-01-01

    In the present study, potential anticancer effect of eugenol on inhibition of cell proliferation and induction of apoptosis in human MCF-7 breast cancer cells was investigated. Induction of cell death by eugenol was evaluated following MTT assay and monitoring lactate dehydrogenase released into the culture medium for cell viability and cytotoxicity, giemsa staining for morphological alterations, fluorescence microscopy analysis of cells using ethidium bromide and acridine orange and quantitation of DNA fragments for induction of apoptosis. Effect of eugenol on intracellular redox status of the human breast cancer cells was assessed by determining the level of glutathione and lipid peroxidation products (TBARS). Eugenol treatment inhibited the growth and proliferation of human MCF-7 breast cancer cells through induction of cell death, which was dose and time dependent. Microscopic examination of eugenol treated cells showed cell shrinkage, membrane blebbing and apoptotic body formation. Further, eugenol treatment also depleted the level of intracellular glutathione and increased the level of lipid peroxidation. The dose dependent increase in the percentage of apoptotic cells and DNA fragments suggested that apoptosis was involved in eugenol induced cell death and apoptosis might have played a role in the chemopreventive action of eugenol. (author)

  16. Lipopolysaccharide-induced expression of cell surface receptors and cell activation of neutrophils and monocytes in whole human blood

    Directory of Open Access Journals (Sweden)

    N.E. Gomes

    2010-09-01

    Full Text Available Lipopolysaccharide (LPS activates neutrophils and monocytes, inducing a wide array of biological activities. LPS rough (R and smooth (S forms signal through Toll-like receptor 4 (TLR4, but differ in their requirement for CD14. Since the R-form LPS can interact with TLR4 independent of CD14 and the differential expression of CD14 on neutrophils and monocytes, we used the S-form LPS from Salmonella abortus equi and the R-form LPS from Salmonella minnesota mutants to evaluate LPS-induced activation of human neutrophils and monocytes in whole blood from healthy volunteers. Expression of cell surface receptors and reactive oxygen species (ROS and nitric oxide (NO generation were measured by flow cytometry in whole blood monocytes and neutrophils. The oxidative burst was quantified by measuring the oxidation of 2',7'-dichlorofluorescein diacetate and the NO production was quantified by measuring the oxidation of 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. A small increase of TLR4 expression by monocytes was observed after 6 h of LPS stimulation. Monocyte CD14 modulation by LPS was biphasic, with an initial 30% increase followed by a 40% decrease in expression after 6 h of incubation. Expression of CD11b was rapidly up-regulated, doubling after 5 min on monocytes, while down-regulation of CXCR2 was observed on neutrophils, reaching a 50% reduction after 6 h. LPS induced low production of ROS and NO. This study shows a complex LPS-induced cell surface receptor modulation on human monocytes and neutrophils, with up- and down-regulation depending on the receptor. R- and S-form LPS activate human neutrophils similarly, despite the low CD14 expression, if the stimulation occurs in whole blood.

  17. Cytotoxicity towards human endothelial cells, induced by neutrophil myeloperoxidase: protection by ceftazidime

    Directory of Open Access Journals (Sweden)

    M. Mathy-Hartert

    1995-01-01

    Full Text Available We investigated the effects of the antibiotic ceftazidime (CAZ on the cytolytic action of the neutrophil myeloperoxidase–hydrogen peroxide–chloride anion system (MPO/H2O2/Cl−. In this system, myeloperoxidase catalyses the conversion of H2O2 and CI− to the cytotoxic agent HOCl. Stimulated neutrophils can release MPO into the extracellular environment and then may cause tissue injury through direct endothelial cells lysis. We showed that human umbilical vein endothelial cells (HUVEC were capable of taking up active MPO. In presence of H2O2 (10−4 M, this uptake was accompanied by cell lysis. The cytolysis was estimated by the release of 51Cr from HUVEC and expressed as an index of cytotoxicity (IC. Dose dependent protection was obtained for CAZ concentrations ranging from 10−5 to 10−3 M;this can be attributed to inactivation of HOCl by the drug. This protection is comparable to that obtained with methionine and histidine, both of which are known to neutralize HOCl. This protection by CAZ could also be attributed to inactivation of H2O2, but when cytolysis was achieved with H2O2 or O2− generating enzymatic systems, no protection by CAZ was observed. Moreover, the peroxidation activity of MPO (action on H2O2 was not affected by CAZ, while CAZ prevented the chlorination activity of MPO (chlorination of monochlorodimedon. So, we concluded that CAZ acts via HOCl inactivation. These antioxidant properties of CAZ may be clinically useful in pathological situations where excessive activation of neutrophils occurs, such as in sepsis.

  18. Extracellular traps are associated with human and mouse neutrophil and macrophage mediated killing of larval Strongyloides stercoralis.

    Science.gov (United States)

    Bonne-Année, Sandra; Kerepesi, Laura A; Hess, Jessica A; Wesolowski, Jordan; Paumet, Fabienne; Lok, James B; Nolan, Thomas J; Abraham, David

    2014-06-01

    Neutrophils are multifaceted cells that are often the immune system's first line of defense. Human and murine cells release extracellular DNA traps (ETs) in response to several pathogens and diseases. Neutrophil extracellular trap (NET) formation is crucial to trapping and killing extracellular pathogens. Aside from neutrophils, macrophages and eosinophils also release ETs. We hypothesized that ETs serve as a mechanism of ensnaring the large and highly motile helminth parasite Strongyloides stercoralis thereby providing a static target for the immune response. We demonstrated that S. stercoralis larvae trigger the release of ETs by human neutrophils and macrophages. Analysis of NETs revealed that NETs trapped but did not kill larvae. Induction of NETs was essential for larval killing by human but not murine neutrophils and macrophages in vitro. In mice, extracellular traps were induced following infection with S. stercoralis larvae and were present in the microenvironment of worms being killed in vivo. These findings demonstrate that NETs ensnare the parasite facilitating larval killing by cells of the immune system. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  19. Metabolism of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine in the human neutrophil

    International Nuclear Information System (INIS)

    Triggiani, M.; D'Souza, D.M.; Chilton, F.H.

    1991-01-01

    The biosynthesis of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (1-acyl-2-acetyl-GPC) together with that of 1-alkyl-2-acetyl-GPC (platelet-activating factor) has been demonstrated in a variety of inflammatory cells and tissues. It has been hypothesized that the relative proportion of these phospholipids produced upon cell activation may be influenced by their rates of catabolism. We studied the catabolism of 1-acyl-2-acetyl-GPC in resting and activated human neutrophils and compared it to that of 1-alkyl-2-acetyl-GPC. Neutrophils rapidly catabolize both 1-alkyl-2-acetyl-GPC and 1-acyl-2-acetyl-GPC; however, the rate of catabolism of 1-acyl-2-acetyl-GPC is approximately 2-fold higher than that of 1-alkyl-2-acetyl-GPC. In addition, most of 1-acyl-2-acetyl-GPC is catabolized through a pathway different from that of 1-alkyl-2-acetyl-GPC. The main step in the catabolism of 1-acyl-2-acetyl-GPC is the removal of the long chain at the sn-1 position; the long chain residue is subsequently incorporated either into triglycerides or into phosphatidylcholine. The 1-lyso-2-acetyl-GPC formed in this reaction is then further degraded to glycerophosphocholine, choline, or phosphocholine. 1-Acyl-2-acetyl-GPC is also catabolized, to a lesser extent, through deacetylation at the sn-2 position and reacylation with a long chain fatty acid. Stimulation of neutrophils by A23187 results in a higher rate of catabolism of 1-acyl-2-acetyl-GPC by increasing both the removal of the long chain at the sn-1 position and the deacetylation-reacylation at the sn-2 position. In a broken cell preparation, the cytosolic fraction of the neutrophil was shown to contain an enzyme activity which cleaved the sn-1 position of 1-acyl-2-acetyl-GPC and 1-acyl-2-lyso-GPC but not of 1,2-diacyl-GPC

  20. Thrombin Production and Human Neutrophil Elastase Sequestration by Modified Cellulosic Dressings and Their Electrokinetic Analysis

    Directory of Open Access Journals (Sweden)

    Nicolette Prevost

    2011-12-01

    Full Text Available Wound healing is a complex series of biochemical and cellular events. Optimally, functional material design addresses the overlapping acute and inflammatory stages of wound healing based on molecular, cellular, and bio-compatibility issues. In this paper the issues addressed are uncontrolled hemostasis and inflammation which can interfere with the orderly flow of wound healing. In this regard, we review the serine proteases thrombin and elastase relative to dressing functionality that improves wound healing and examine the effects of charge in cotton/cellulosic dressing design on thrombin production and elastase sequestration (uptake by the wound dressing. Thrombin is central to the initiation and propagation of coagulation, and elastase is released from neutrophils that can function detrimentally in a stalled inflammatory phase characteristic of chronic wounds. Electrokinetic fiber surface properties of the biomaterials of this study were determined to correlate material charge and polarity with function relative to thrombin production and elastase sequestration. Human neutrophil elastase sequestration was assessed with an assay representative of chronic wound concentration with cotton gauze cross-linked with three types of polycarboxylic acids and one phosphorylation finish; thrombin production, which was assessed in a plasma-based assay via a fluorogenic peptide substrate, was determined for cotton, cotton-grafted chitosan, chitosan, rayon/polyester, and two kaolin-treated materials including a commercial hemorrhage control dressing (QuickClot Combat Gauze. A correlation in thrombin production to zeta potential was found. Two polycarboxylic acid cross linked and a phosphorylated cotton dressing gave high elastase sequestration.

  1. DioxolaneA3-phosphatidylethanolamines are generated by human platelets and stimulate neutrophil integrin expression

    Directory of Open Access Journals (Sweden)

    Maceler Aldrovandi

    2017-04-01

    Full Text Available Activated platelets generate an eicosanoid proposed to be 8-hydroxy-9,10-dioxolane A3 (DXA3. Herein, we demonstrate that significant amounts of DXA3 are rapidly attached to phosphatidylethanolamine (PE forming four esterified eicosanoids, 16:0p, 18:0p, 18:1p and 18:0a/DXA3-PEs that can activate neutrophil integrin expression. These lipids comprise the majority of DXA3 generated by platelets, are formed in ng amounts (24.3±6.1 ng/2×108 and remain membrane bound. Pharmacological studies revealed DXA3-PE formation involves cyclooxygenase-1 (COX, protease-activated receptors (PAR 1 and 4, cytosolic phospholipase A2 (cPLA2, phospholipase C and intracellular calcium. They are generated primarily via esterification of newly formed DXA3, but can also be formed in vitro via co-oxidation of PE during COX-1 co-oxidation of arachidonate. All four DXA3-PEs were detected in human clots. Purified platelet DXA3-PE activated neutrophil Mac-1 expression, independently of its hydrolysis to the free eicosanoid. This study demonstrates the structures and cellular synthetic pathway for a family of leukocyte-activating platelet phospholipids generated on acute activation, adding to the growing evidence that enzymatic PE oxidation is a physiological event in innate immune cells.

  2. (±)-2-Chloropropionic acid elevates reactive oxygen species formation in human neutrophil granulocytes

    International Nuclear Information System (INIS)

    Aam, B.B.; Fonnum, F.

    2006-01-01

    (±)-2-Chloropropionic acid (2-CPA) is a neurotoxic compound which kills cerebellar granule cells in vivo, and makes cerebellar granule cells in vitro produce reactive oxygen species (ROS). We have studied the effect of 2-CPA on ROS formation in human neutrophil granulocytes in vitro. We found an increased formation of ROS after 2-CPA exposure using three different methods; the fluorescent probe DCFH-DA and the chemiluminescent probes lucigenin and luminol. Four different inhibitors of ROS formation were tested on the cells in combination with 2-CPA to characterize the signalling pathways. The spin-trap s-PBN, the ERK1/2 inhibitor U0126 and the antioxidant Vitamin E inhibited the 2-CPA-induced ROS formation completely, while the mitochondrial transition permeability pore blocker cyclosporine A inhibited the ROS formation partly. We also found that 2-CPA induced an increased nitric oxide production in the cells by using the Griess reagent. The level of reduced glutathione, measured with the DTNB assay, was decreased after exposure to high concentrations of 2-CPA. Western blotting analysis showed that 2-CPA exposure led to an elevated phosphorylation of ERK MAP kinase. This phosphorylation was inhibited by U0126. Based on these experiments it seems like the mechanisms for 2-CPA induced toxicity involves ROS formation and is similar in neutrophil granulocytes as earlier shown in cerebellar granule cells. This also implies that 2-CPA may be immunotoxic

  3. Tumor necrosis factor related apoptosis inducing ligand triggers apoptosis in dividing but not in differentiating human epidermal keratinocytes

    NARCIS (Netherlands)

    Jansen, Bastiaan J. H.; van Ruissen, Fred; Cerneus, Stefanie; Cloin, Wendy; Bergers, Mieke; van Erp, Piet E. J.; Schalkwijk, Joost

    2003-01-01

    Using serial analysis of gene expression we have previously identified the expression of several pro-apoptotic and anti-apoptotic genes in cultured human primary epidermal keratinocytes, including tumor necrosis factor related apoptosis inducing ligand (TRAIL). TRAIL is a potent inducer of apoptosis

  4. Cyclic GMP protects human macrophages against peroxynitrite-induced apoptosis.

    Science.gov (United States)

    Shaw, Catherine A; Webb, David J; Rossi, Adriano G; Megson, Ian L

    2009-05-07

    Nitric oxide (NO) can be both pro- and anti-apoptotic in various cell types, including macrophages. This apparent paradox may result from the actions of NO-related species generated in the microenvironment of the cell, for example the formation of peroxynitrite (ONOO-). In this study we have examined the ability of NO and ONOO- to evoke apoptosis in human monocyte-derived macrophages (MDMvarphi), and investigated whether preconditioning by cyclic guanosine monophosphate (cGMP) is able to limit apoptosis in this cell type. Characterisation of the NO-related species generated by (Z)-1- [2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA/NO) and 1,2,3,4-oxatriazolium, 5-amino-3-(3,4-dichlorophenyl)-, chloride (GEA-3162) was performed by electrochemistry using an isolated NO electrode and electron paramagnetic resonance (EPR) spectrometry. Mononuclear cells were isolated from peripheral blood of healthy volunteers and cultured to allow differentiation into MDMvarphi. Resultant MDMvarphi were treated for 24 h with DETA/NO (100 - 1000 muM) or GEA-3162 (10 - 300 muM) in the presence or absence of BAY 41-2272 (1 muM), isobutylmethylxanthine (IBMX; 1 muM), 1H- [1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ; 20 muM) or 8-bromo-cGMP (1 mM). Apoptosis in MDMvarphi was assessed by flow cytometric analysis of annexin V binding in combination with propidium iodide staining. Electrochemistry and EPR revealed that DETA/NO liberated free NO radical, whilst GEA-3162 concomitantly released NO and O2-, and is therefore a ONOO- generator. NO (DETA/NO) had no effect on cell viability, but ONOO- (GEA-3162) caused a concentration-dependent induction of apoptosis in MDMvarphi. Preconditioning of MDMvarphi with NO in combination with the phosphodiesterase inhibitor, 3-Isobutyl-1-methylxanthine (IBMX), or the NO-independent stimulator of soluble guanylate cyclase, BAY 41-2272, significantly attenuated ONOO--induced apoptosis in a cGMP-dependent manner. These results

  5. Cyclic GMP protects human macrophages against peroxynitrite-induced apoptosis

    Directory of Open Access Journals (Sweden)

    Rossi Adriano G

    2009-05-01

    Full Text Available Abstract Background Nitric oxide (NO can be both pro- and anti-apoptotic in various cell types, including macrophages. This apparent paradox may result from the actions of NO-related species generated in the microenvironment of the cell, for example the formation of peroxynitrite (ONOO-. In this study we have examined the ability of NO and ONOO- to evoke apoptosis in human monocyte-derived macrophages (MDMϕ, and investigated whether preconditioning by cyclic guanosine monophosphate (cGMP is able to limit apoptosis in this cell type. Methods Characterisation of the NO-related species generated by (Z-1- [2-(2-aminoethyl-N-(2-ammonioethylamino]diazen-1-ium-1,2-diolate (DETA/NO and 1,2,3,4-oxatriazolium, 5-amino-3-(3,4-dichlorophenyl-, chloride (GEA-3162 was performed by electrochemistry using an isolated NO electrode and electron paramagnetic resonance (EPR spectrometry. Mononuclear cells were isolated from peripheral blood of healthy volunteers and cultured to allow differentiation into MDMϕ. Resultant MDMϕ were treated for 24 h with DETA/NO (100 – 1000 μM or GEA-3162 (10 – 300 μM in the presence or absence of BAY 41–2272 (1 μM, isobutylmethylxanthine (IBMX; 1 μM, 1H- [1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ; 20 μM or 8-bromo-cGMP (1 mM. Apoptosis in MDMϕ was assessed by flow cytometric analysis of annexin V binding in combination with propidium iodide staining. Results Electrochemistry and EPR revealed that DETA/NO liberated free NO radical, whilst GEA-3162 concomitantly released NO and O2-, and is therefore a ONOO- generator. NO (DETA/NO had no effect on cell viability, but ONOO- (GEA-3162 caused a concentration-dependent induction of apoptosis in MDMϕ. Preconditioning of MDMϕ with NO in combination with the phosphodiesterase inhibitor, 3-Isobutyl-1-methylxanthine (IBMX, or the NO-independent stimulator of soluble guanylate cyclase, BAY 41–2272, significantly attenuated ONOO--induced apoptosis in a cGMP-dependent manner

  6. GMP-140 binds to a glycoprotein receptor on human neutrophils: Evidence for a lectin-like interaction

    International Nuclear Information System (INIS)

    Moore, K.L.; Varki, A.; McEver, R.P.

    1991-01-01

    GMP-140 is a rapidly inducible receptor for neutrophils and monocytes expressed on activated platelets and endothelial cells. It is a member of the selectin family of lectin-like cell surface molecules that mediate leukocyte adhesion. We used a radioligand binding assay to characterize the interaction of purified GMP-140 with human neutrophils. Unstimulated neutrophils rapidly bound [125I]GMP-140 at 4 degrees C, reaching equilibrium in 10-15 min. Binding was Ca2+ dependent, reversible, and saturable at 3-6 nM free GMP-140 with half-maximal binding at approximately 1.5 nM. Receptor density and apparent affinity were not altered when neutrophils were stimulated with 4 beta-phorbol 12-myristate 13-acetate. Treatment of neutrophils with proteases abolished specific binding of [125I]GMP-140. Binding was also diminished when neutrophils were treated with neuraminidase from Vibrio cholerae, which cleaves alpha 2-3-, alpha 2-6-, and alpha 2-8-linked sialic acids, or from Newcastle disease virus, which cleaves only alpha 2-3- and alpha 2-8-linked sialic acids. Binding was not inhibited by an mAb to the abundant myeloid oligosaccharide, Lex (CD15), or by the neoglycoproteins Lex-BSA and sialyl-Lex-BSA. We conclude that neutrophils constitutively express a glycoprotein receptor for GMP-140, which contains sialic acid residues that are essential for function. These findings support the concept that GMP-140 interacts with leukocytes by a lectin-like mechanism

  7. Apoptosis in Pneumovirus Infection

    Directory of Open Access Journals (Sweden)

    Reinout A. Bem

    2013-01-01

    Full Text Available Pneumovirus infections cause a wide spectrum of respiratory disease in humans and animals. The airway epithelium is the major site of pneumovirus replication. Apoptosis or regulated cell death, may contribute to the host anti-viral response by limiting viral replication. However, apoptosis of lung epithelial cells may also exacerbate lung injury, depending on the extent, the timing and specific location in the lungs. Differential apoptotic responses of epithelial cells versus innate immune cells (e.g., neutrophils, macrophages during pneumovirus infection can further contribute to the complex and delicate balance between host defense and disease pathogenesis. The purpose of this manuscript is to give an overview of the role of apoptosis in pneumovirus infection. We will examine clinical and experimental data concerning the various pro-apoptotic stimuli and the roles of apoptotic epithelial and innate immune cells during pneumovirus disease. Finally, we will discuss potential therapeutic interventions targeting apoptosis in the lungs.

  8. Extracellular lipase of Pseudomonas aeruginosa: biochemical characterization and effect on human neutrophil and monocyte function in vitro

    DEFF Research Database (Denmark)

    Jaeger, K E; Kharazmi, A; Høiby, N

    1991-01-01

    concentrations of this lipase preparation were preincubated with human peripheral blood neutrophils and monocytes. The chemotaxis and chemiluminescence of these cells were then determined. It was shown that lipase inhibited the monocyte chemotaxis and chemiluminescence, whereas it had no or very little effect...... on neutrophils. The inhibitory effect was concentration dependent and was abolished by heat treatment of the enzyme at 100 degrees C. Since monocytes are one of the important cells of the host defence system the inhibition of the function of these cells may contribute to the pathogenesis of infections caused...

  9. Gene transfer and expression in human neutrophils. The phox homology domain of p47phox translocates to the plasma membrane but not to the membrane of mature phagosomes

    Directory of Open Access Journals (Sweden)

    Brzezinska Agnieszka A

    2006-12-01

    Full Text Available Abstract Background Neutrophils are non-dividing cells with poor survival after isolation. Consequently, exogenous gene expression in neutrophils is challenging. We report here the transfection of genes and expression of active proteins in human primary peripheral neutrophils using nucleofection. Results Exogenous gene expression in human neutrophils was achieved 2 h post-transfection. We show that neutrophils transfected by nucleofection are functional cells, able to respond to soluble and particulate stimuli. They conserved the ability to undergo physiological processes including phagocytosis. Using this technique, we were able to show that the phox homology (PX domain of p47phox localizes to the plasma membrane in human neutrophils. We also show that RhoB, but not the PX domain of p47phox, is translocated to the membrane of mature phagosomes. Conclusion We demonstrated that cDNA transfer and expression of exogenous protein in human neutrophils is compatible with cell viability and is no longer a limitation for the study of protein function in human neutrophils.

  10. Nanoparticles of barium induce apoptosis in human phagocytes.

    Science.gov (United States)

    Mores, Luana; França, Eduardo Luzia; Silva, Núbia Andrade; Suchara, Eliane Aparecida; Honorio-França, Adenilda Cristina

    2015-01-01

    Nutrients and immunological factors of breast milk are essential for newborn growth and the development of their immune system, but this secretion can contain organic and inorganic toxins such as barium. Colostrum contamination with barium is an important issue to investigate because this naturally occurring element is also associated with human activity and industrial pollution. The study evaluated the administration of barium nanoparticles to colostrum, assessing the viability and functional activity of colostral mononuclear phagocytes. Colostrum was collected from 24 clinically healthy women (aged 18-35 years). Cell viability, superoxide release, intracellular Ca(2+) release, and phagocyte apoptosis were analyzed in the samples. Treatment with barium lowered mononuclear phagocyte viability, increased superoxide release, and reduced intracellular calcium release. In addition, barium increased cell death by apoptosis. These data suggest that nanoparticles of barium in colostrum are toxic to cells, showing the importance of avoiding exposure to this element.

  11. Bystander apoptosis in human cells mediated by irradiated blood plasma

    Energy Technology Data Exchange (ETDEWEB)

    Vinnikov, Volodymyr, E-mail: vlad.vinnikov@mail.ru [Grigoriev Institute for Medical Radiology of the National Academy of Medical Science of Ukraine (Ukraine); Lloyd, David; Finnon, Paul [Centre for Radiation, Chemical and Environmental Hazards of the Health Protection Agency of the United Kingdom (United Kingdom)

    2012-03-01

    Following exposure to high doses of ionizing radiation, due to an accident or during radiotherapy, bystander signalling poses a potential hazard to unirradiated cells and tissues. This process can be mediated by factors circulating in blood plasma. Thus, we assessed the ability of plasma taken from in vitro irradiated human blood to produce a direct cytotoxic effect, by inducing apoptosis in primary human peripheral blood mononuclear cells (PBM), which mainly comprised G{sub 0}-stage lymphocytes. Plasma was collected from healthy donors' blood irradiated in vitro to 0-40 Gy acute {gamma}-rays. Reporter PBM were separated from unirradiated blood with Histopaque and held in medium with the test plasma for 24 h at 37 Degree-Sign C. Additionally, plasma from in vitro irradiated and unirradiated blood was tested against PBM collected from blood given 4 Gy. Apoptosis in reporter PBM was measured by the Annexin V test using flow cytometry. Plasma collected from unirradiated and irradiated blood did not produce any apoptotic response above the control level in unirradiated reporter PBM. Surprisingly, plasma from irradiated blood caused a dose-dependent reduction of apoptosis in irradiated reporter PBM. The yields of radiation-induced cell death in irradiated reporter PBM (after subtracting the respective values in unirradiated reporter PBM) were 22.2 {+-} 1.8% in plasma-free cultures, 21.6 {+-} 1.1% in cultures treated with plasma from unirradiated blood, 20.2 {+-} 1.4% in cultures with plasma from blood given 2-4 Gy and 16.7 {+-} 3.2% in cultures with plasma from blood given 6-10 Gy. These results suggested that irradiated blood plasma did not cause a radiation-induced bystander cell-killing effect. Instead, a reduction of apoptosis in irradiated reporter cells cultured with irradiated blood plasma has implications concerning oncogenic risk from mutated cells surviving after high dose in vivo irradiation (e.g. radiotherapy) and requires further study.

  12. Bystander apoptosis in human cells mediated by irradiated blood plasma

    International Nuclear Information System (INIS)

    Vinnikov, Volodymyr; Lloyd, David; Finnon, Paul

    2012-01-01

    Following exposure to high doses of ionizing radiation, due to an accident or during radiotherapy, bystander signalling poses a potential hazard to unirradiated cells and tissues. This process can be mediated by factors circulating in blood plasma. Thus, we assessed the ability of plasma taken from in vitro irradiated human blood to produce a direct cytotoxic effect, by inducing apoptosis in primary human peripheral blood mononuclear cells (PBM), which mainly comprised G 0 -stage lymphocytes. Plasma was collected from healthy donors’ blood irradiated in vitro to 0–40 Gy acute γ-rays. Reporter PBM were separated from unirradiated blood with Histopaque and held in medium with the test plasma for 24 h at 37 °C. Additionally, plasma from in vitro irradiated and unirradiated blood was tested against PBM collected from blood given 4 Gy. Apoptosis in reporter PBM was measured by the Annexin V test using flow cytometry. Plasma collected from unirradiated and irradiated blood did not produce any apoptotic response above the control level in unirradiated reporter PBM. Surprisingly, plasma from irradiated blood caused a dose-dependent reduction of apoptosis in irradiated reporter PBM. The yields of radiation-induced cell death in irradiated reporter PBM (after subtracting the respective values in unirradiated reporter PBM) were 22.2 ± 1.8% in plasma-free cultures, 21.6 ± 1.1% in cultures treated with plasma from unirradiated blood, 20.2 ± 1.4% in cultures with plasma from blood given 2–4 Gy and 16.7 ± 3.2% in cultures with plasma from blood given 6–10 Gy. These results suggested that irradiated blood plasma did not cause a radiation-induced bystander cell-killing effect. Instead, a reduction of apoptosis in irradiated reporter cells cultured with irradiated blood plasma has implications concerning oncogenic risk from mutated cells surviving after high dose in vivo irradiation (e.g. radiotherapy) and requires further study.

  13. Phagocytosis and killing of Candida albicans by human neutrophils after exposure to structurally different lipid emulsions.

    NARCIS (Netherlands)

    Wanten, G.J.A.; Curfs, J.H.A.J.; Meis, J.F.G.M.; Naber, A.H.J.

    2001-01-01

    BACKGROUND: To test the hypothesis that structurally different lipid emulsions have distinct immune-modulating properties, we analyzed the elimination of Candida albicans by neutrophils after exposure to various emulsions. METHODS: Neutrophils from 8 volunteers were incubated in physiologic 5 mmol/L

  14. Role of ERK1/2 kinase in the expression of iNOS by NDMA in human neutrophils.

    Science.gov (United States)

    Ratajczak-Wrona, Wioletta; Jablonska, Ewa; Garley, Marzena; Jablonski, Jakub; Radziwon, Piotr

    2013-01-01

    Potential role of ERK1/2 kinase in conjunction with p38 in the regulation of inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production, and superoxide anion generation by human neutrophils (PMNs) exposed to N-nitrosodimethylamine (NDMA) was determined. Increased synthesis of NO due to the involvement of iNOS in neutrophils exposed to NDMA was observed. In addition, intensified activation of ERK1/2 and p38 kinases was determined in these cells. Inhibition of kinase regulated by extracellular signals (ERK1/2) pathway, in contrast to p38 pathway, led to an increased production of NO and expression of iNOS in PMNs. Moreover, as a result of inhibition of ERK1/2 pathway, a decreased activation of p38 kinase was observed in neutrophils, while inhibition of p38 kinase did not affect activation of ERK1/2 pathway in these cells. An increased ability to release superoxide anion by the studied PMNs was observed, which decreased after ERK1/2 pathway inhibition. In conclusion, in human neutrophils, ERK1/2 kinase is not directly involved in the regulation of iNOS and NO production induced by NDMA; however, the kinase participates in superoxide anion production in these cells.

  15. Innate Defense against Influenza A Virus: Activity of Human Neutrophil Defensins and Interactions of Defensins with Surfactant Protein D

    DEFF Research Database (Denmark)

    Hartshorn, Kevan L.; White, Mitchell R.; Tecle, Tesfaldet

    2006-01-01

    Surfactant protein D (SP-D) plays important roles in innate host defense against influenza A virus (IAV) infection, in part by modifying interactions with neutrophils. Human neutrophil defensins (HNPs) inhibit infectivity of enveloped viruses, including IAV. Our goal in this study was to characte......Surfactant protein D (SP-D) plays important roles in innate host defense against influenza A virus (IAV) infection, in part by modifying interactions with neutrophils. Human neutrophil defensins (HNPs) inhibit infectivity of enveloped viruses, including IAV. Our goal in this study...... was to characterize antiviral interactions between SP-D and HNPs. Recombinant and/or natural forms of SP-D and related collectins and HNPs were tested for antiviral activity against two different strains of IAV. HNPs 1 and 2 did not inhibit viral hemagglutination activity, but they interfered...... with the hemagglutination-inhibiting activity of SP-D. HNPs had significant viral neutralizing activity against divergent IAV strains. However, the HNPs generally had competitive effects when combined with SP-D in assays using an SP-D-sensitive IAV strain. In contrast, cooperative antiviral effects were noted in some...

  16. Gallic Acid Induces Apoptosis in Human Gastric Adenocarcinoma Cells.

    Science.gov (United States)

    Tsai, Chung-Lin; Chiu, Ying-Ming; Ho, Tin-Yun; Hsieh, Chin-Tung; Shieh, Dong-Chen; Lee, Yi-Ju; Tsay, Gregory J; Wu, Yi-Ying

    2018-04-01

    Gastric cancer is one of the most common malignant cancers with a poor prognosis and high mortality rate worldwide. Current treatment of gastric cancer includes surgery and chemotherapy as the main modalities, but the potentially severe side-effects of chemotherapy present a considerable challenge. Gallic acid is a trihydroxybenzoic acid found to exert an anticancer effect against a variety of cancer cells. The purpose of this study was to determine the anti-cancer activity of Galla chinensis and its main component gallic acid on human gastric adenocarcinoma cells. MTT assay and cell death ELISA were used to determine the apoptotic effect of Gallic Chinensis and gallic acid on human gastric adenocarcinoma cells. To determine the pathway and relevant components by which gallic acid-induced apoptosis is mediated through, cells were transfected with siRNA (Fas, FasL, DR5, p53) using Lipofectamine 2000. Reults: Gallic Chinensis and gallic acid induced apoptosis of human gastric adenocarcinoma cells. Gallic acid induced up-regulation of Fas, FasL, and DR5 expression in AGS cells. Transfection of cells with Fas, FasL, or DR5 siRNA reduced gallic acid-induced cell death. In addition, p53 was shown to be involved in gallic acid-mediated Fas, FasL, and DR5 expression as well as cell apoptosis in AGS cells. These results suggest that gallic acid has a potential role in the treatment of gastric cancer. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  17. Renal cell apoptosis in human lupus nephritis: a histological study

    DEFF Research Database (Denmark)

    Faurschou, M; Penkowa, Milena; Andersen, C B

    2009-01-01

    Nuclear autoantigens from apoptotic cells are believed to drive the immunological response in systemic lupus erythematosus (SLE). Conflicting data exist as to the possible renal origin of apoptotic cells in SLE patients with nephritis. We assessed the level of renal cell apoptosis in kidney...... biopsies from 35 patients with lupus nephritis by means of terminal deoxynucleotidyl-transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-digoxigenin nick end labeling (TUNEL). Five samples of normal kidney tissue served as control specimens. We did not observe apoptotic glomerular cells in any...... cells constitute a quantitatively important source of auto-antibody-inducing nuclear auto-antigens in human lupus nephritis....

  18. Rhein Induces Apoptosis in Human Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Ching-Yao Chang

    2012-01-01

    Full Text Available Human breast cancers cells overexpressing HER2/neu are more aggressive tumors with poor prognosis, and resistance to chemotherapy. This study investigates antiproliferation effects of anthraquinone derivatives of rhubarb root on human breast cancer cells. Of 7 anthraquinone derivatives, only rhein showed antiproliferative and apoptotic effects on both HER2-overexpressing MCF-7 (MCF-7/HER2 and control vector MCF-7 (MCF-7/VEC cells. Rhein induced dose- and time-dependent manners increase in caspase-9-mediated apoptosis correlating with activation of ROS-mediated activation of NF-κB- and p53-signaling pathways in both cell types. Therefore, this study highlighted rhein as processing anti-proliferative activity against HER2 overexpression or HER2-basal expression in breast cancer cells and playing important roles in apoptotic induction of human breast cancer cells.

  19. The effect of cigarette smoking on neutrophil kinetics in human lungs [see comments

    International Nuclear Information System (INIS)

    MacNee, W.; Wiggs, B.; Belzberg, A.S.; Hogg, J.C.

    1989-01-01

    Neutrophils may play a part in the pathogenesis of the centrilobular emphysema associated with cigarette smoking. The capillary bed of the lungs concentrates neutrophils approximately 100-fold with respect to erythrocytes, producing a large pool of marginated cells. We examined the effect of cigarette smoking on the kinetics of this pool of cells, using 99mTc-labeled erythrocytes to measure regional blood velocity and 111In-labeled neutrophils to measure the removal of neutrophils during the first passage through the pulmonary circulation, their subsequent washout from the lungs, and the effect of local blood velocity on the number of neutrophils retained in each lung region. We observed no difference in these measurements between subjects who had never smoked (n = 6) and smokers who did not smoke during the study (n = 12). However, subjects who did smoke during the study (n = 12) had a significantly slower rate of washout of radiolabeled neutrophils from the lung (0.08 +/- 0.04 of the total per minute, as compared with 0.13 +/- 0.06 in smokers who did not smoke during the experiment and 0.14 +/- 0.08 in non-smokers) (P = 0.02). We also observed an increase in the regional retention of labeled neutrophils with respect to blood velocity in 5 of the 12 subjects who smoked during the study, but in none of the other subjects. We conclude that the presence of cigarette smoke in the lungs of some subjects increases the local concentration of neutrophils, and suggest that the lesions that characterize emphysema may be a result of the destruction of lung tissue by neutrophils that remain within pulmonary microvessels

  20. Noradrenaline increases the expression and release of Hsp72 by human neutrophils.

    Science.gov (United States)

    Giraldo, E; Multhoff, G; Ortega, E

    2010-05-01

    The blood concentration of extracellular 72kDa heat shock protein (eHsp72) increases under conditions of stress, including intense exercise. However, the signal(s), source(s), and secretory pathways in its release into the bloodstream have yet to be clarified. The aim of the present study was to evaluate the role of noradrenaline (NA) as a stress signal on the expression and release of Hsp72 by circulating neutrophils (as a source), all within a context of the immunophysiological regulation during exercise-induced stress in sedentary and healthy young (21-26years) women. The expression of Hsp72 on the surface of isolated neutrophils was determined by flow cytometry, and its release by cultured isolated neutrophils was determined by ELISA. Incubation with cmHsp70-FITC showed that neutrophils express Hsp72 on their surface under basal conditions. In addition, cultured isolated neutrophils (37 degrees C and 5% CO(2)) also released Hsp72 under basal conditions, with this release increasing from 10min to 24h in the absence of cell damage. NA at 10(-9)-10(-5)M doubled the percentage of neutrophils expressing Hsp72 after 60min and 24h incubation. NA also stimulated (by about 20%) the release of Hsp72 after 10min of incubation. (1) Hsp72 is expressed on the surface of isolated neutrophils under basal conditions, and this expression is augmented by NA. (2) Isolated neutrophils can also release Hsp72 under cultured basal conditions in the absence of cell death, and NA can increase this release. These results may contribute to confirming the hypothesis that NA can act as a "stress signal" for the increased eHsp72 in the context of exercise stress, with a role for neutrophils as a source for the expression and, to a lesser degree, the release of Hsp72 after activation by NA. Copyright 2010 Elsevier Inc. All rights reserved.

  1. Effect of the dimetilsulfoxido in the response chemiluminescent and the consumption of oxygen of neutrophils activated human

    International Nuclear Information System (INIS)

    Garcia, J.

    2001-01-01

    Dimethylsulfoxide (DMSO), a hydroxyl radical scavenger, exerted a dose dependent inhibition on the luminol and lucigenin-enhanced chemiluminescent responses of human neutrophils activated with soluble and particulate stimulants. DMSO inhibition of the luminol chemiluminescense induced by calcium ionophore A23187 was probably due to OH scavenging, whereas inhibition of the lucigenin chemiluminescence suggested DMSO negatively affects the NADPH-dependent membrane oxidase of neutrophils. In agreement with this, DMSO moderately inhibited O2 consumption in PMN suspensions stimulated with chemotactic peptide and opsonized zymosan-induced luminol chemiluminescense was observed only when added before or in conjunction with stimulants, whereas A23187-induced chemiluminescense was inhibited by DMSO regardless of time of addition. Washing of DMSO-treated PMN resulted in increased luminol enhanced chemiluminescense in response to chemotactic peptide and opsonized zymosan. This is consistent with the idea that DMSO may be interfering with activation of the membrane subunits of the oxidase by translocation and docking of the cytoplasmic, regulatory subunits. These data imply that DMSO inhibits neutrophil chemiluminescense both by OH scavenging and interfering with oxidase activation. Key words:Dimethylsulfoxide, chemiluminescent, luminol, lucigenin,neutrophils [es

  2. Neutrophil Extracellular Traps and Fibrin in Otitis Media: Analysis of Human and Chinchilla Temporal Bones.

    Science.gov (United States)

    Schachern, Patricia A; Kwon, Geeyoun; Briles, David E; Ferrieri, Patricia; Juhn, Steven; Cureoglu, Sebahattin; Paparella, Michael M; Tsuprun, Vladimir

    2017-10-01

    Bacterial resistance in acute otitis can result in bacterial persistence and biofilm formation, triggering chronic and recurrent infections. To investigate the middle ear inflammatory response to bacterial infection in human and chinchilla temporal bones. Six chinchillas underwent intrabullar inoculations with 0.5 mL of 106 colony-forming units (CFUs) of Streptococcus pneumoniae, serotype 2. Two days later, we counted bacteria in middle ear effusions postmortem. One ear from each chinchilla was processed in paraffin and sectioned at 5 µm. The opposite ear was embedded in epoxy resin, sectioned at a thickness of 1 µm, and stained with toluidine blue. In addition, we examined human temporal bones from 2 deceased donors with clinical histories of otitis media (1 with acute onset otitis media, 1 with recurrent infection). Temporal bones had been previously removed at autopsy, processed, embedded in celloidin, and cut at a thickness of 20 µm. Sections of temporal bones from both chinchillas and humans were stained with hematoxylin-eosin and immunolabeled with antifibrin and antihistone H4 antibodies. Histopatological and imminohistochemical changes owing to otitis media. Bacterial counts in chinchilla middle ear effusions 2 days after inoculation were approximately 2 logs above initial inoculum counts. Both human and chinchilla middle ear effusions contained bacteria embedded in a fibrous matrix. Some fibers in the matrix showed positive staining with antifibrin antibody, others with antihistone H4 antibody. In acute and recurrent otitis media, fibrin and neutrophil extracellular traps (NETs) are part of the host inflammatory response to bacterial infection. In the early stages of otitis media the host defense system uses fibrin to entrap bacteria, and NETs function to eliminate bacteria. In chronic otitis media, fibrin and NETs appear to persist.

  3. Selective kallikrein inhibitors alter human neutrophil elastase release during extracorporeal circulation

    NARCIS (Netherlands)

    Wachtfogel, Y.T.; Hack, C.E.; Nuijens, J.H; Kettner, C.; Reilly, T.M.; Knabb, R.M.; Bischoff, Rainer; Tschesche, H.; Wenzel, H.; Kucich, U.

    1995-01-01

    Cardiopulmonary bypass causes hemorrhagic complications and initiates a biochemical and cellular "whole body inflammatory response." This study investigates whether a variety of selective inhibitors of the contact pathway of intrinsic coagulation modulate complement and neutrophil activation during

  4. The Beta-2-Adrenoreceptor Agonists, Formoterol and Indacaterol, but Not Salbutamol, Effectively Suppress the Reactivity of Human Neutrophils In Vitro

    Directory of Open Access Journals (Sweden)

    Ronald Anderson

    2014-01-01

    Full Text Available The clinical relevance of the anti-inflammatory properties of beta-2 agonists remains contentious possibly due to differences in their molecular structures and agonist activities. The current study has compared the effects of 3 different categories of β2-agonists, namely, salbutamol (short-acting, formoterol (long-acting and indacaterol (ultra-long-acting, at concentrations of 1–1000 nM, with human blood neutrophils in vitro. Neutrophils were activated with either N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP, 1 µM or platelet-activating factor (PAF, 200 nM in the absence and presence of the β2-agonists followed by measurement of the generation of reactive oxygen species and leukotriene B4, release of elastase, and expression of the β2-integrin, CR3, using a combination of chemiluminescence, ELISA, colorimetric, and flow cytometric procedures respectively. These were correlated with alterations in the concentrations of intracellular cyclic-AMP and cytosolic Ca2+. At the concentrations tested, formoterol and indacaterol caused equivalent, significant (P<0.05 at 1–10 nM dose-related inhibition of all of the pro-inflammatory activities tested, while salbutamol was much less effective (P<0.05 at 100 nM and higher. Suppression of neutrophil reactivity was accompanied by elevations in intracellular cAMP and accelerated clearance of Ca2+ from the cytosol of activated neutrophils. These findings demonstrate that β2-agonists vary with respect to their suppressive effects on activated neutrophils.

  5. Oxidative burst of human neutrophils is suppressed by N-feruloylserotonin isolated from seeds of Leuzea carthamoides (Wild) DC

    Czech Academy of Sciences Publication Activity Database

    Nosáľ, R.; Perečko, T.; Jančinová, V.; Drábiková, K.; Harmatha, Juraj; Sviteková, K.

    2010-01-01

    Roč. 3, č. 3 (2010), A70-A71 ISSN 1337-6853. [Toxcon 2010, Borderless Toxicology. 15th Interdisciplinary Toxicological Conference & Advanced Toxicological Course. 06.09.-10.09.2010, Stará Lesná - Hotel Academia] R&D Projects: GA ČR(CZ) GA203/07/1227 Institutional research plan: CEZ:AV0Z40550506 Keywords : N-feruloylserotonin * human neutrophils * Leuzea carthamoides Subject RIV: CC - Organic Chemistry

  6. SL-01, an oral derivative of gemcitabine, inhibited human breast cancer growth through induction of apoptosis

    International Nuclear Information System (INIS)

    Li, Yuan-Yuan; Qin, Yi-Zhuo; Wang, Rui-Qi; Li, Wen-Bao; Qu, Xian-Jun

    2013-01-01

    Highlights: •SL-01 is an oral derivative of gemcitabine. •SL-01 possessed activity against human breast cancer growth via apoptotic induction. •SL-01’s activity was more potently than that of gemcitabine. •SL-01 inhibited cancer growth without toxicity to mice. -- Abstract: SL-01 is an oral derivative of gemcitabine that was synthesized by introducing the moiety of 3-(dodecyloxycarbonyl) pyrazine-2-carbonyl at N4-position on cytidine ring of gemcitabine. We aimed to evaluate the efficacy of SL-01 on human breast cancer growth. SL-01 significantly inhibited MCF-7 proliferation as estimated by colorimetric assay. Flow cytometry assay indicated the apoptotic induction and cell cycle arrest in G1 phase. SL-01 modulated the expressions of p-ATM, p53 and p21 and decrease of cyclin D1 in MCF-7 cells. Further experiments were performed in a MCF-7 xenografts mouse model. SL-01 by oral administration strongly inhibited MCF-7 xenografts growth. This effect of SL-01 might arise from its roles in the induction of apoptosis. Immunohistochemistry assay showed the increase of TUNEL staining cells. Western blotting indicated the modulation of apoptotic proteins in SL-01-treated xenografts. During the course of study, there was no evidence of toxicity to mice. In contrast, the decrease of neutrophil cells in peripheral and increase of AST and ALT levels in serum were observed in the gemcitabine-treated mice. Conclusion: SL-01 possessed similar activity against human breast cancer growth with gemcitabine, whereas, with lower toxicity to gemcitabine. SL-01 is a potent oral agent that may supplant the use of gemcitabine

  7. Applying label-free dynamic mass redistribution assay for studying endogenous FPR1 receptor signalling in human neutrophils

    DEFF Research Database (Denmark)

    Christensen, Hanna B; Gloriam, David E; Pedersen, Daniel Sejer

    2017-01-01

    INTRODUCTION: The label-free dynamic mass redistribution-based assay (DMR) is a powerful method for studying signalling pathways of G protein-coupled receptors (GPCRs). Herein we present the label-free DMR assay as a robust readout for pharmacological characterization of formyl peptide receptors...... (FPRs) in human neutrophils. METHODS: Neutrophils were isolated from fresh human blood and their responses to FPR1 and FPR2 agonists, i.e. compound 43, fMLF and WKYMVm were measured in a label-free DMR assay using Epic Benchtop System from Corning®. Obtained DMR traces were used to calculate agonist...... potencies. RESULTS: The potencies (pEC50) of fMLF, WKYMVm and compound 43, determined on human neutrophils using the label-free DMR assay were 8.63, 7.76 and 5.92, respectively. The DMR response to fMLF, but not WKYMVm and compound 43 could be blocked by the FPR1-specific antagonist cyclosporin H...

  8. Proinflammatory effects of bacterial lipoprotein on human neutrophil activation status, function and cytotoxic potential in vitro.

    LENUS (Irish Health Repository)

    Power, C

    2012-02-03

    Bacterial lipoprotein (BLP) is the most abundant protein in gram-negative bacterial cell walls, heavily outweighing lipopolysaccharide (LPS). Herein we present findings demonstrating the potent in vitro effects of BLP on neutrophil (PMN) activation status, function, and capacity to transmigrate an endothelial monolayer. PMNs are the principal effectors of the initial host response to injury or infection and constitute a significant threat to invading bacterial pathogens. The systemic inflammatory response syndrome (SIRS) is characterised by significant host tissue injury mediated, in part, by uncontrolled regulation of PMN cytotoxic activity. We found that BLP-activated human PMN as evidenced by increased CD11b\\/CD18 (Mac-1) expression. Up-regulation of PMN Mac-1 in response to BLP occurred independently of membrane-bound CD14 (mCD14). A similar up-regulation of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells was observed whilst E-Selectin expression was unaffected. PMN transmigration across a human umbilical vein endothelial cell (HUVEC) monolayer was markedly increased after treating either PMN\\'s or HUVEC independently with BLP. This increased transmigration did not occur as a result of any direct effect of BLP on HUVEC monolayer permeability, assessed objectively using the passage of FITC-labeled Dextran-70. BLP primed PMN for enhanced respiratory burst and superoxide anion production in response to PMA, but did not influence phagocytosis of opsonized Escherichia coli. BLP far exceeds LPS as a gram-negative bacterial wall component, these findings therefore implicate BLP as an additional putative mediator of SIRS arising from gram-negative infection.

  9. Aspirin-triggered lipoxin A4 and lipoxin A4 up-regulate transcriptional corepressor NAB1 in human neutrophils.

    Science.gov (United States)

    Qiu, F H; Devchand, P R; Wada, K; Serhan, C N

    2001-12-01

    Aspirin-triggered 15-epi-lipoxin A4 (ATL) is an endogenous lipid mediator that mimics the actions of native lipoxin A4, a putative "stop signal" involved in regulating resolution of inflammation. A metabolically more stable analog of ATL, 15-epi-16-(para-fluoro)-phenoxy-lipoxin A4 analog (ATLa), inhibits neutrophil recruitment in vitro and in vivo and displays potent anti-inflammatory actions. ATLa binds with high affinity to the lipoxin A4 receptor, a G protein-coupled receptor on the surface of leukocytes. In this study, we used freshly isolated human neutrophils to examine ATLa's potential for initiating rapid nuclear responses. Using differential display reverse transcription polymerase chain reaction, we identified a subset of genes that was selectively up-regulated upon short exposure of polymorphonuclear leukocytes to ATLa but not to the chemoattractant leukotriene B4 or vehicle alone. We further investigated ATLa regulation of one of the genes, NAB1, a transcriptional corepressor identified previously as a glucocorticoid-responsive gene in hamster smooth muscle cells. Treatment of human neutrophils with pertussis toxin blocked ATLa up-regulation of NAB1. In addition, ATLa stimulated NAB1 gene expression in murine lung vascular smooth muscle in vivo. These findings provide evidence for rapid transcriptional induction of a cassette of genes via an ATLa-stimulated G protein-coupled receptor pathway that is potentially protective and overlaps with the anti-inflammatory glucocorticoid regulatory circuit.

  10. Data on human neutrophil activation induced by pepducins with amino acid sequences derived from β2AR and CXCR4

    Directory of Open Access Journals (Sweden)

    André Holdfeldt

    2016-09-01

    Full Text Available The data described here is related to the research article titled (Gabl et al., 2016 [1]. Pepducins with peptide sequence derived from one of the intracellular domains of a given G-protein coupled receptor (GPCR can either activate or inhibit cell functions. Here we include data on human neutrophil function induced by pepducins derived from β2AR (ICL3-8 and CXCR4 (ATI-2341, respectively. ICL3-8 exerts neither direct activating effect on the NADPH-oxidase as measured by superoxide release nor inhibitory effect on FPR signaling. ATI-2341 dose-dependently triggers neutrophil activation and these cells were subsequently desensitized in their response to FPR2 specific agonists F2Pal10 and WKYMVM. Moreover, the ATI-2341 response is inhibited by PBP10 and the peptidomimetic Pam-(Lys-betaNSpe6-NH2 (both are FPR2 specific inhibitors, but not to the FPR1 specific inhibitor cyclosporine H.

  11. YKL-40, a mammalian member of the chitinase family, is a matrix protein of specific granules in human neutrophils

    DEFF Research Database (Denmark)

    Volck, B; Price, P A; Johansen, J S

    1998-01-01

    YKL-40, also called human cartilage glycoprotein-39 (HC gp-39), is a member of family 18 glycosyl hydrolases. YKL-40 is secreted by chondrocytes, synovial cells, and macrophages, and recently it has been reported that YKL-40 has a role as an autoantigen in rheumatoid arthritis (RA). The function...... of patients with RA, and the cells are assumed to play a role in joint destruction in that disorder. Therefore, we examined whether neutrophils are a source of YKL-40. YKL-40 was found to colocalize and comobilize with lactoferrin (the most abundant protein of specific granules) but not with gelatinase...... YKL-40 at the myelocyte-metamyelocyte stage, the stage of maturation at which other specific granule proteins are formed. Assuming that YKL-40 has a role as an autoantigen in RA by inducing T cell-mediated autoimmune response, YKL-40 released from neutrophils in the inflamed joint could be essential...

  12. STUDIES IN DYNAMICS OF APOPTOSIS-RELATED SURFACE ANTIGEN (CD95 EXPRESSION ON NEUTROPHILS FROM CERVICAL AND VAGINAL SECRETIONS IN WOMEN WITH CHLAMIDIA INFECTION

    Directory of Open Access Journals (Sweden)

    O. A. Giesinger

    2010-01-01

    Full Text Available CD95 (Fas/APO-1 antigen expression was studied on the surface of neutrophil granulocytes from cervical secretions. Sixty-five female patients with established Chlamydia infection were found to have an increased CD95+ antigen expression following basic therapy. CD95+ receptors on neutrophils in the patients with Chlamydia infection have been shown to return to normal levels following a combined magnetic laser treatment.

  13. Inductive potential of recombinant human granulocyte colony-stimulating factor to mature neutrophils from X-irradiated human peripheral blood hematopoietic progenitor cells

    International Nuclear Information System (INIS)

    Katsumori, Takeo; Yoshino, Hironori; Hayashi, Masako; Takahashi, Kenji; Kashiwakura, Ikuo

    2009-01-01

    Recombinant human granulocyte colony-stimulating factor (rhG-CSF) has been used for treatment of neutropenia. Filgrastim, Nartograstim, and Lenograstim are clinically available in Japan. However, the differences in potential benefit for radiation-induced disorder between these types of rhG-CSFs remain unknown. Therefore, the effects of three different types of rhG-CSFs on granulocyte progenitor cells and expansion of neutrophils from nonirradiated or 2 Gy X-irradiated human CD34 + hematopoietic progenitor cells were examined. For analysis of granulocyte colony-forming units (CFU-G) and a surviving fraction of CFU-G, nonirradiated or X-irradiated CD34 + cells were cultured in methylcellulose containing rhG-CSF. These cells were cultured in serum-free medium supplemented with rhG-CSF, and the expansion and characteristics of neutrophils were analyzed. All three types of rhG-CSFs increased the number of CFU-G in a dose-dependent manner; however, Lenograstim is superior to others because of CFU-G-derived colony formation at relatively low doses. The surviving fraction of CFU-G was independent of the types of rhG-CSFs. Expansion of neutrophils by rhG-CSF was largely attenuated by X-irradiation, though no significant difference in neutrophil number was observed between the three types of rhG-CSFs under both nonirradiation and X-irradiation conditions. In terms of functional characteristics of neutrophils, Lenograstim-induced neutrophils produced high levels of reactive oxygen species compared to Filgrastim, when rhG-CSF was applied to nonirradiated CD34 + cells. In conclusion, different types of rhG-CSFs lead to different effects when rhG-CSF is applied to nonirradiated CD34 + cells, though Filgrastim, Nartograstim, and Lenograstim show equal effects on X-irradiated CD34 + cells. (author)

  14. Anti-Inflammatory Benefits of Antibiotics: Tylvalosin Induces Apoptosis of Porcine Neutrophils and Macrophages, Promotes Efferocytosis, and Inhibits Pro-Inflammatory CXCL-8, IL1α, and LTB4 Production, While Inducing the Release of Pro-Resolving Lipoxin A4 and Resolvin D1

    OpenAIRE

    Ruth Moges; Ruth Moges; Dimitri Desmonts De Lamache; Dimitri Desmonts De Lamache; Saman Sajedy; Bernard S. Renaux; Bernard S. Renaux; Morley D. Hollenberg; Morley D. Hollenberg; Gregory Muench; Elizabeth M. Abbott; Andre G. Buret; Andre G. Buret

    2018-01-01

    Excessive accumulation of neutrophils and their uncontrolled death by necrosis at the site of inflammation exacerbates inflammatory responses and leads to self-amplifying tissue injury and loss of organ function, as exemplified in a variety of respiratory diseases. In homeostasis, neutrophils are inactivated by apoptosis, and non phlogistically removed by neighboring macrophages in a process known as efferocytosis, which promotes the resolution of inflammation. The present study assessed the ...

  15. Oviduct-Specific Expression of Human Neutrophil Defensin 4 in Lentivirally Generated Transgenic Chickens

    Science.gov (United States)

    Liu, Tongxin; Wu, Hanyu; Cao, Dainan; Li, Qingyuan; Zhang, Yaqiong; Li, Ning; Hu, Xiaoxiang

    2015-01-01

    The expression of oviduct-specific recombinant proteins in transgenic chickens is a promising technology for the production of therapeutic biologics in eggs. In this study, we constructed a lentiviral vector encoding an expression cassette for human neutrophil defensin 4 (HNP4), a compound that displays high activity against Escherichia coli, and produced transgenic chickens that expressed the recombinant HNP4 protein in egg whites. After the antimicrobial activity of the recombinant HNP4 protein was tested at the cellular level, a 2.8-kb ovalbumin promoter was used to drive HNP4 expression specifically in oviduct tissues. From 669 injected eggs, 218 chickens were successfully hatched. Ten G0 roosters, with semens identified as positive for the transgene, were mated with wild-type hens to generate G1 chickens. From 1,274 total offspring, fifteen G1 transgenic chickens were positive for the transgene, which was confirmed by PCR and Southern blotting. The results of the Southern blotting and genome walking indicated that a single copy of the HNP4 gene was integrated into chromosomes 1, 2, 3, 4, 6 and 24 of the chickens. As expected, HNP4 expression was restricted to the oviduct tissues, and the levels of both transcriptional and translational HNP4 expression varied greatly in transgenic chickens with different transgene insertion sites. The amount of HNP4 protein expressed in the eggs of G1 and G2 heterozygous transgenic chickens ranged from 1.65 μg/ml to 10.18 μg/ml. These results indicated that the production of transgenic chickens that expressed HNP4 protein in egg whites was successful. PMID:26020529

  16. Human neutrophil antigen profiles in Banjar, Bugis, Champa, Jawa and Kelantan Malays in Peninsular Malaysia.

    Science.gov (United States)

    Manaf, Siti M; NurWaliyuddin, Hanis Z A; Panneerchelvam, Sundararajulu; Zafarina, Zainuddin; Norazmi, Mohd N; Chambers, Geoffrey K; Edinur, Hisham A

    2015-10-01

    Human neutrophil antigens (HNA) are polymorphic and immunogenic proteins involved in the pathogenesis of neonatal alloimmune neutropenia, transfusion-related acute lung injury (TRALI) and transfusion-related alloimmune neutropenia. The characterisation of HNA at a population level is important for predicting the risk of alloimmunisation associated with blood transfusion and gestation and for anthropological studies. Blood samples from 192 healthy, unrelated Malays were collected and genotyped using polymerase chain reaction-sequence specific primers (HNA-1, -3, -4) and polymerase chain reaction-restriction fragment length polymorphisms (HNA-5). The group comprised 30 Banjar, 37 Bugis, 51 Champa, 39 Jawa and 35 Kelantan Malays. The most common HNA alleles in the Malays studied were HNA-1a (0.641-0.765), -3a (0.676-0.867), -4a (0.943-1.000) and -5a (0.529-0.910). According to principal coordinate plots constructed using HNA allele frequencies, the Malay sub-ethnic groups are closely related and grouped together with other Asian populations. The risks of TRALI or neonatal neutropenia were not increased for subjects with HNA-1, -3 and -4 loci even for donor and recipient or pairs from different Malay sub-ethnic groups. Nonetheless, our estimates showed significantly higher risks of HNA alloimmunisation during pregnancy and transfusion between Malays and other genetically differentiated populations such as Africans and Europeans. This study reports HNA allele and genotype frequencies for the five Malay sub-ethnic groups living in Peninsular Malaysia for the first time. These Malay sub-ethnic groups show closer genetic relationships with other Asian populations than with Europeans and Africans. The distributions of HNA alleles in other lineages of people living in Malaysia (e.g. Chinese, Indian and Orang Asli) would be an interesting subject for future study.

  17. Spironolactone induces apoptosis in human mononuclear cells. Association between apoptosis and cytokine suppression

    DEFF Research Database (Denmark)

    Mikkelsen, Martin; Sønder, S U; Nersting, J

    2006-01-01

    preceding apoptosis. An association between the two effects was also seen when testing several SPIR analogues. Contrary to TNF-alpha, the levels of IL-1beta increased in SPIR-treated cultures. However, the amount of IL-1beta in the supernatants depended upon the order of SPIR and LPS addition, as IL-1beta....... In conclusion, suppression of cytokine production by SPIR may be associated with its apoptotic potential, either directly (apoptosis is a consequence of suppressed cytokine production, or vice-versa) or indirectly (suppressed cytokine production and apoptosis are parallel but otherwise unrelated phenomena)....

  18. Human neutrophil clearance of bacterial pathogens triggers anti-microbial γδ T cell responses in early infection.

    Directory of Open Access Journals (Sweden)

    Martin S Davey

    2011-05-01

    Full Text Available Human blood Vγ9/Vδ2 T cells, monocytes and neutrophils share a responsiveness toward inflammatory chemokines and are rapidly recruited to sites of infection. Studying their interaction in vitro and relating these findings to in vivo observations in patients may therefore provide crucial insight into inflammatory events. Our present data demonstrate that Vγ9/Vδ2 T cells provide potent survival signals resulting in neutrophil activation and the release of the neutrophil chemoattractant CXCL8 (IL-8. In turn, Vγ9/Vδ2 T cells readily respond to neutrophils harboring phagocytosed bacteria, as evidenced by expression of CD69, interferon (IFN-γ and tumor necrosis factor (TNF-α. This response is dependent on the ability of these bacteria to produce the microbial metabolite (E-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP, requires cell-cell contact of Vγ9/Vδ2 T cells with accessory monocytes through lymphocyte function-associated antigen-1 (LFA-1, and results in a TNF-α dependent proliferation of Vγ9/Vδ2 T cells. The antibiotic fosmidomycin, which targets the HMB-PP biosynthesis pathway, not only has a direct antibacterial effect on most HMB-PP producing bacteria but also possesses rapid anti-inflammatory properties by inhibiting γδ T cell responses in vitro. Patients with acute peritoneal-dialysis (PD-associated bacterial peritonitis--characterized by an excessive influx of neutrophils and monocytes into the peritoneal cavity--show a selective activation of local Vγ9/Vδ2 T cells by HMB-PP producing but not by HMB-PP deficient bacterial pathogens. The γδ T cell-driven perpetuation of inflammatory responses during acute peritonitis is associated with elevated peritoneal levels of γδ T cells and TNF-α and detrimental clinical outcomes in infections caused by HMB-PP positive microorganisms. Taken together, our findings indicate a direct link between invading pathogens, neutrophils, monocytes and microbe-responsive γδ T cells in

  19. Human Neutrophil Clearance of Bacterial Pathogens Triggers Anti-Microbial γδ T Cell Responses in Early Infection

    Science.gov (United States)

    Roberts, Gareth W.; Heuston, Sinéad; Brown, Amanda C.; Chess, James A.; Toleman, Mark A.; Gahan, Cormac G. M.; Hill, Colin; Parish, Tanya; Williams, John D.; Davies, Simon J.; Johnson, David W.; Topley, Nicholas; Moser, Bernhard; Eberl, Matthias

    2011-01-01

    Human blood Vγ9/Vδ2 T cells, monocytes and neutrophils share a responsiveness toward inflammatory chemokines and are rapidly recruited to sites of infection. Studying their interaction in vitro and relating these findings to in vivo observations in patients may therefore provide crucial insight into inflammatory events. Our present data demonstrate that Vγ9/Vδ2 T cells provide potent survival signals resulting in neutrophil activation and the release of the neutrophil chemoattractant CXCL8 (IL-8). In turn, Vγ9/Vδ2 T cells readily respond to neutrophils harboring phagocytosed bacteria, as evidenced by expression of CD69, interferon (IFN)-γ and tumor necrosis factor (TNF)-α. This response is dependent on the ability of these bacteria to produce the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), requires cell-cell contact of Vγ9/Vδ2 T cells with accessory monocytes through lymphocyte function-associated antigen-1 (LFA-1), and results in a TNF-α dependent proliferation of Vγ9/Vδ2 T cells. The antibiotic fosmidomycin, which targets the HMB-PP biosynthesis pathway, not only has a direct antibacterial effect on most HMB-PP producing bacteria but also possesses rapid anti-inflammatory properties by inhibiting γδ T cell responses in vitro. Patients with acute peritoneal-dialysis (PD)-associated bacterial peritonitis – characterized by an excessive influx of neutrophils and monocytes into the peritoneal cavity – show a selective activation of local Vγ9/Vδ2 T cells by HMB-PP producing but not by HMB-PP deficient bacterial pathogens. The γδ T cell-driven perpetuation of inflammatory responses during acute peritonitis is associated with elevated peritoneal levels of γδ T cells and TNF-α and detrimental clinical outcomes in infections caused by HMB-PP positive microorganisms. Taken together, our findings indicate a direct link between invading pathogens, neutrophils, monocytes and microbe-responsive γδ T cells in early

  20. NR4A orphan nuclear receptor family members, NR4A2 and NR4A3, regulate neutrophil number and survival.

    Science.gov (United States)

    Prince, Lynne R; Prosseda, Svenja D; Higgins, Kathryn; Carlring, Jennifer; Prestwich, Elizabeth C; Ogryzko, Nikolay V; Rahman, Atiqur; Basran, Alexander; Falciani, Francesco; Taylor, Philip; Renshaw, Stephen A; Whyte, Moira K B; Sabroe, Ian

    2017-08-24

    The lifespan of neutrophils is plastic and highly responsive to factors that regulate cellular survival. Defects in neutrophil number and survival are common to both hematologic disorders and chronic inflammatory diseases. At sites of inflammation, neutrophils respond to multiple signals that activate protein kinase A (PKA) signaling, which positively regulates neutrophil survival. The aim of this study was to define transcriptional responses to PKA activation and to delineate the roles of these factors in neutrophil function and survival. In human neutrophil gene array studies, we show that PKA activation upregulates a significant number of apoptosis-related genes, the most highly regulated of these being NR4A2 and NR4A3 Direct PKA activation by the site-selective PKA agonist pair N6/8-AHA (8-AHA-cAMP and N6-MB-cAMP) and treatment with endogenous activators of PKA, including adenosine and prostaglandin E2, results in a profound delay of neutrophil apoptosis and concomitant upregulation of NR4A2/3 in a PKA-dependent manner. NR4A3 expression is also increased at sites of neutrophilic inflammation in a human model of intradermal inflammation. PKA activation also promotes survival of murine neutrophil progenitor cells, and small interfering RNA to NR4A2 decreases neutrophil production in this model. Antisense knockdown of NR4A2 and NR4A3 homologs in zebrafish larvae significantly reduces the absolute neutrophil number without affecting cellular migration. In summary, we show that NR4A2 and NR4A3 are components of a downstream transcriptional response to PKA activation in the neutrophil, and that they positively regulate neutrophil survival and homeostasis. © 2017 by The American Society of Hematology.

  1. Medium-chain, triglyceride-containing lipid emulsions increase human neutrophil beta2 integrin expression, adhesion, and degranulation.

    Science.gov (United States)

    Wanten, G J; Geijtenbeek, T B; Raymakers, R A; van Kooyk, Y; Roos, D; Jansen, J B; Naber, A H

    2000-01-01

    To test the hypothesis that lipid emulsions with different triglyceride structures have distinct immunomodulatory properties, we analyzed human neutrophil adhesion and degranulation after lipid incubation. Neutrophils, isolated from the blood of 10 healthy volunteers, were incubated in medium or physiologic (2.5 mmol/L) emulsions containing long-chain (LCT), medium-chain (MCT), mixed LCT/MCT, or structured (SL) triglycerides. Expression of adhesion molecules and degranulation markers was evaluated by flow cytometry. Also, functional adhesion was investigated by means of a flow cytometric assay using fluorescent beads coated with the integrin ligand intercellular adhesion molecule (ICAM)-1. Although LCT and SL had no effect, LCT/MCT significantly increased expression of the beta2 integrins lymphocyte-function-associated antigen 1 (+18%), macrophage antigen 1 (+387%), p150,95 (+82%), and (alphaDbeta2 (+230%). Degranulation marker expression for azurophilic (CD63, +210%) and specific granules (CD66b, +370%) also significantly increased, whereas L-selectin (CD62L, -70%) decreased. The effects of LCT/MCT were mimicked by the MCT emulsion. ICAM-1 adhesion (% beads bound) was increased by LCT/MCT (34% +/- 4%), whereas LCT (19% +/-3%) and SL (20% +/- 2%) had no effect compared with medium (17% +/- 3%). LCT/MCT and MCT, contrary to LCT and SL emulsions, increased neutrophil beta2 integrin expression, adhesion, and degranulation. Apart from other emulsion constituents, triglyceride chain length might therefore be a key feature in the interaction of lipid emulsions and the phagocyte immune system.

  2. Nimesulide inhibits platelet-activating factor synthesis in activated human neutrophils

    NARCIS (Netherlands)

    Verhoeven, A. J.; Tool, A. T.; Kuijpers, T. W.; Roos, D.

    1993-01-01

    In an inflammatory locus, products of activated neutrophils may be toxic both to the micro-organisms to be eliminated and to the surrounding tissue. In several models of inflammation, nimesulide possesses marked anti-inflammatory properties. The present study was undertaken to further investigate

  3. Antimicrobial Peptide Human Neutrophil Peptide 1 as a Potential Link Between Chronic Inflammation and Ductal Adenocarcinoma of the Pancreas.

    Science.gov (United States)

    Pausch, Thomas; Adolph, Sarah; Felix, Klaus; Bauer, Andrea S; Bergmann, Frank; Werner, Jens; Hartwig, Werner

    Defensins are antimicrobial peptides playing a role in innate immunity, in epithelial cell regeneration, and in carcinogenesis of inflammation-triggered malignancies. We analyzed this role in pancreatic ductal adenocarcinoma (PDAC) in the context of its association with chronic pancreatitis (CP). Human tissue of healthy pancreas, CP, and PDAC was screened for defensins by immunohistochemistry. Defensin α 1 (human neutrophil peptide 1 [HNP-1]) expression was validated using mass spectrometry and microarray analysis. Human neutrophil peptide 1 expression and influences of proinflammatory cytokines (tumor necrosis factor α, interleukin 1β, and interferon γ) were studied in human pancreatic cancer cells (Colo 357, T3M4, PANC-1) and normal human pancreatic duct epithelial cells (HPDE). Accumulation of HNP-1 in malignant pancreatic ductal epithelia was seen. Spectrometry showed increased expression of HNP-1 in CP and even more in PDAC. At RNA level, no significant regulation was found. In cancer cells, HNP-1 expression was significantly higher than in HPDE. Proinflammatory cytokines significantly led to increased HNP-1 levels in culture supernatants and decreased levels in lysates of cancer cells. In HPDE cytokines significantly decreased HNP-1 levels. Inflammatory regulation of HNP-1 in PDAC tissue and cells indicates that HNP-1 may be a link between chronic inflammation and malignant transformation in the pancreas.

  4. fMLP-Induced IL-8 Release Is Dependent on NADPH Oxidase in Human Neutrophils

    Directory of Open Access Journals (Sweden)

    María A. Hidalgo

    2015-01-01

    Full Text Available N-Formyl-methionyl-leucyl-phenylalanine (fMLP and platelet-activating factor (PAF induce similar intracellular signalling profiles; but only fMLP induces interleukin-8 (IL-8 release and nicotinamide adenine dinucleotide phosphate reduced (NADPH oxidase activity in neutrophils. Because the role of ROS on IL-8 release in neutrophils is until now controversial, we assessed if NADPH oxidase is involved in the IL-8 secretions and PI3K/Akt, MAPK, and NF-κB pathways activity induced by fMLP. Neutrophils were obtained from healthy volunteers. IL-8 was measured by ELISA, IL-8 mRNA by qPCR, and ROS production by luminol-amplified chemiluminescence, reduction of ferricytochrome c, and FACS. Intracellular pH changes were detected by spectrofluorescence. ERK1/2, p38 MAPK, and Akt phosphorylation were analysed by immunoblotting and NF-κB was analysed by immunocytochemistry. Hydroxy-3-methoxyaceto-phenone (HMAP, diphenyleneiodonium (DPI, and siRNA Nox2 reduced the ROS and IL-8 release in neutrophils treated with fMLP. HMAP, DPI, and amiloride (a Na+/H+ exchanger inhibitor inhibited the Akt phosphorylation and did not affect the p38 MAPK and ERK1/2 activity. DPI and HMAP reduced NF-κB translocation induced by fMLP. We showed that IL-8 release induced by fMLP is dependent on NADPH oxidase, and ROS could play a redundant role in cell signalling, ultimately activating the PI3K/Akt and NF-κB pathways in neutrophils.

  5. Oxidative burst of circulating neutrophils following traumatic brain injury in human.

    Directory of Open Access Journals (Sweden)

    Yiliu Liao

    Full Text Available Besides secondary injury at the lesional site, Traumatic brain injury (TBI can cause a systemic inflammatory response, which may cause damage to initially unaffected organs and potentially further exacerbate the original injury. Here we investigated plasma levels of important inflammatory mediators, oxidative activity of circulating leukocytes, particularly focusing on neutrophils, from TBI subjects and control subjects with general trauma from 6 hours to 2 weeks following injury, comparing with values from uninjured subjects. We observed increased plasma level of inflammatory cytokines/molecules TNF-α, IL-6 and CRP, dramatically increased circulating leukocyte counts and elevated expression of TNF-α and iNOS in circulating leukocytes from TBI patients, which suggests a systemic inflammatory response following TBI. Our data further showed increased free radical production in leukocyte homogenates and elevated expression of key oxidative enzymes iNOS, COX-2 and NADPH oxidase (gp91(phox in circulating leukocytes, indicating an intense induction of oxidative burst following TBI, which is significantly greater than that in control subjects with general trauma. Furthermore, flow cytometry assay proved neutrophils as the largest population in circulation after TBI and showed significantly up-regulated oxidative activity and suppressed phagocytosis rate for circulating neutrophils following brain trauma. It suggests that the highly activated neutrophils might play an important role in the secondary damage, even outside the injured brain. Taken together, the potent systemic inflammatory response induced by TBI, especially the intensively increase oxidative activity of circulating leukocytes, mainly neutrophils, may lead to a systemic damage, dysfunction/damage of bystander tissues/organs and even further exacerbate secondary local damage. Controlling these pathophysiological processes may be a promising therapeutic strategy and will protect unaffected

  6. Zinc and magnesium ions synergistically inhibit superoxide generation by cultured human neutrophils--a promising candidate formulation for amnioinfusion fluid.

    Science.gov (United States)

    Uchida, Toshiyuki; Itoh, Hiroaki; Nakamura, Yuki; Kobayashi, Yukiko; Hirai, Kyuya; Suzuki, Kazunao; Sugihara, Kazuhiro; Kanayama, Naohiro; Hiramatsu, Mitsuo

    2010-06-01

    Oligohydramnios is often caused by the premature rupturing of membranes and subsequent intrauterine infections, such as chorioamnionitis, in which event oxidative stress is hypothesized to be closely associated with the damage to the fetal organs. The clinical efficiency of amnioinfusion using warmed saline in cases of premature rupture of membranes is still controversial, especially concerning the prognosis for the fetus. In the present study, we found that human amniotic fluid per se suppresses the release of superoxide from cultured human neutrophils, suggesting an acute or chronic shortage of amniotic fluid in cases of premature rupture of membranes can affect the shielding of intrauterine organs from oxidative stress. The aim of this study was to propose a formula of zinc and magnesium ions in saline for amnioinfusion, by assessing antioxidative activities. A combination of 5 microM zinc and 5mM magnesium in saline synergistically inhibited superoxide production by cultured human neutrophils, equivalent to human amniotic fluid. The intraperitoneal administration of this formula significantly improved the survival rate in a rat model of peritonitis compared to the saline control (46.7% vs. 10%). The combination of these metals with saline may thus be a promising formula for an amnioinfusion fluid with the capacity to protect fetal organs from oxidative stress. Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.

  7. The influence of human neutrophils on N-nitrosodimethylamine (NDMA) synthesis.

    Science.gov (United States)

    Jabłoński, Jakub; Jabłońska, Ewa; Iwanowska, Jolanta; Marcińczyk, Magda; Moniuszko-Jakoniuk, Janina

    2006-01-01

    N-nitrozodimethyloamine (NDMA) is a carcinogenic compound that can be formed in vivo. NDMA is synthesized from precursors-amines and nitrosating agents. Nitrosating agents are formed through the reaction of oxide, reactive oxygen species and nitric oxide (NO). Human neutrophils (PMN) are an important source of the most reactive oxygen species as well as of the nitric oxide. The increase in oxygen metabolism of PMN can lead to the increase nitrosating agent and nitroso-forms. Inflammatory process is associated with locally decreased pH that may favor nitrosation reaction. In the present study, we estimated the NDMA synthesis by LPS-stimulated PMN in the presence of the iNOS inhibitor--N-nitro-L-arginine methyl ester (L-NAME). In the nitrosation reaction dimethylamine (DMA) was used as substrat. The viability of the cells was measured by cytometric method. NDMA concentrations the culture media was measured by GCMS method. NO production was estimated by Griess's method. Expression of iNOS was determined by western blotting. Results obtained showed that DMA nitrosation is most effective in pH between 3-4.5. Nonstimulated PMN produced lower concentrations of NO than LPS-stimulated cells (1.27 microg/cm3 and 1.57 microg/cm3, respectively). In the culture of nonstimulated PMN supplemented with DMA, there was NDMA (mean--0.99 ng/cm3). In the culture of LPS-stimulated PMN in the presence of DMA, the concentration of NDMA was higher than in the culture of nonstimulated PMN (median--1.45 ng/cm3). In the supernatants of cells incubated without DMA and with DMA, LPS and L-NAME, no NDMA was detected. These results indicate that PMN can be one of sources of nitrosating agents and can play a role in endogenous NDMA synthesis. Stimulation of PMN can lead to the increase of NDMA concentration following the increase of NO production. Different pathological conditions associated with PMN activation as well as the decreased pH may favor endogenous NDMA synthesis.

  8. Spironolactone induces apoptosis in human mononuclear cells. Association between apoptosis and cytokine suppression

    DEFF Research Database (Denmark)

    Mikkelsen, Martin; Sønder, S U; Nersting, J

    2006-01-01

    Spironolactone (SPIR) has been described to suppress accumulation of pro-inflammatory cytokines. Here, the suppression of TNF-alpha in lipopolysaccharide (LPS)-stimulated mononuclear cell cultures was confirmed. However, SPIR was also found to induce apoptosis, prompting the investigations...... of a possible association between the two effects: The apoptosis-inducing and the cytokine-suppressive effects of SPIR correlated with regard to the effective concentration range. Also, pre-incubation experiments demonstrated a temporal separation of the two effects of ... preceding apoptosis. An association between the two effects was also seen when testing several SPIR analogues. Contrary to TNF-alpha, the levels of IL-1beta increased in SPIR-treated cultures. However, the amount of IL-1beta in the supernatants depended upon the order of SPIR and LPS addition, as IL-1beta...

  9. Angiotensin-(1-7 Promotes Resolution of Neutrophilic Inflammation in a Model of Antigen-Induced Arthritis in Mice

    Directory of Open Access Journals (Sweden)

    Lívia C. Barroso

    2017-11-01

    Full Text Available Defective resolution of inflammation may be crucial for the initiation and development of chronic inflammatory diseases, such as arthritis. Therefore, it has been suggested that therapeutic strategies based on molecules that facilitate inflammation resolution present great potential for the treatment of chronic inflammatory diseases. In this study, we investigated the effects and role of angiotensin-(1-7 [Ang-(1-7] in driving resolution of neutrophilic inflammation in a model of arthritis. For this purpose, male C57BL/6 mice were subjected to antigen-induced arthritis and treated with Ang-(1-7 at the peak of the inflammatory process. Analysis of the number of inflammatory cells, apoptosis, and immunofluorescence for NF-κB was performed in the exudate collected from the knee cavity. Neutrophil accumulation in periarticular tissue was measured by assaying myeloperoxidase activity. Apoptosis of human neutrophil after treatment with Ang-(1-7 was evaluated morphologically and by flow cytometry, and NF-κB phosphorylation by immunofluorescence. Efferocytosis was evaluated in vivo. Therapeutic treatment with Ang-(1-7 at the peak of inflammation promoted resolution, an effect associated with caspase-dependent neutrophils apoptosis and NF-κB inhibition. Importantly, Ang-(1-7 was also able to induce apoptosis of human neutrophils, an effect associated with NF-κB inhibition. The pro-resolving effects of Ang-(1-7 were inhibited by the Mas receptor antagonist A779. Finally, we showed that Ang-(1-7 increased the efferocytic ability of murine macrophages. Our results clearly demonstrate that Ang-(1-7 resolves neutrophilic inflammation in vivo acting in two key step of resolution: apoptosis of neutrophils and their removal by efferocytosis. Ang-(1-7 is a novel mediator of resolution of inflammation.

  10. Selective inhibition of extracellular oxidants liberated from human neutrophils--A new mechanism potentially involved in the anti-inflammatory activity of hydroxychloroquine.

    Science.gov (United States)

    Jančinová, Viera; Pažoureková, Silvia; Lucová, Marianna; Perečko, Tomáš; Mihalová, Danica; Bauerová, Katarína; Nosáľ, Radomír; Drábiková, Katarína

    2015-09-01

    Hydroxychloroquine is used in the therapy of rheumatoid arthritis or lupus erythematosus. Although these diseases are often accompanied by activation of neutrophils, there are still few data relating to the impact of hydroxychloroquine on these cells. We investigated the effect of orally administered hydroxychloroquine on neutrophil oxidative burst in rats with adjuvant arthritis. In human neutrophils, extra- and intracellular formation of oxidants, mobilisation of intracellular calcium and the phosphorylation of proteins regulating NADPH oxidase assembly were analysed. Administration of hydroxychloroquine decreased the concentration of oxidants in blood of arthritic rats. The inhibition was comparable with the reference drug methotrexate, yet it was not accompanied by a reduction in neutrophil count. When both drugs were co-applied, the effect became more pronounced. In isolated human neutrophils, treatment with hydroxychloroquine resulted in reduced mobilisation of intracellular calcium, diminished concentration of external oxidants and in decreased phosphorylation of Ca(2+)-dependent protein kinase C isoforms PKCα and PKCβII, which regulate activation of NADPH oxidase on plasma membrane. On the other hand, no reduction was observed in intracellular oxidants or in the phosphorylation of p40(phox) and PKCδ, two proteins directing the oxidase assembly to intracellular membranes. Hydroxychloroquine reduced neutrophil-derived oxidants potentially involved in tissue damage and protected those capable to suppress inflammation. The observed effects may represent a new mechanism involved in the anti-inflammatory activity of this drug. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Combined activity of post-exercise concentrations of NA and eHsp72 on human neutrophil function: role of cAMP.

    Science.gov (United States)

    Giraldo, Esther; Hinchado, María D; Ortega, Eduardo

    2013-09-01

    Extracellular heat shock proteins of 72 kDa (eHsp72) and noradrenaline (NA) can act as "danger signals" during exercise-induced stress by activating neutrophil function (chemotaxis, phagocytosis, and fungicidal capacity). In addition, post-exercise concentrations of NA increase the expression and release of Hsp72 by human neutrophils, and adrenoreceptors and cAMP are involved in the stimulation of neutrophils by eHsp72. This suggests an interaction between the two molecules in the modulation of neutrophils during exercise-induced stress. Given this context, the aim of the present investigation was to study the combined activity of post-exercise circulating concentrations of NA and eHsp72 on the neutrophil phagocytic process, and to evaluate the role of cAMP as intracellular signal in these effects. Results showed an accumulative stimulation of chemotaxis induced by NA and eHsp72. However, while NA and eHsp72, separately, stimulate the phagocytosis and fungicidal activity of neutrophils, when they act together they do not modify these capacities of neutrophils. Similarly, post-exercise concentrations of NA and eHsp72 separately increased the intracellular level of cAMP, but NA and eHsp72 acting together did not modify the intracellular concentration of cAMP. These results confirm that cAMP can be involved in the autocrine/paracrine physiological regulation of phagocytosis and fungicidal capacity of human neutrophils mediated by NA and eHsp72 in the context of exercise-induced stress. Copyright © 2013 Wiley Periodicals, Inc.

  12. Human retinal pigment epithelial cell-induced apoptosis in activated T cells

    DEFF Research Database (Denmark)

    Jørgensen, A; Wiencke, A K; la Cour, M

    1998-01-01

    human retinal pigment epithelial (RPE) cells can induce apoptosis in activated T cells. METHODS: Fas ligand (FasL) expression was detected by flow cytometry and immunohistochemistry. Cultured RPE cells were cocultured with T-cell lines and peripheral blood lymphocytes for 6 hours to 2 days. Induction...... of apoptosis was detected by 7-amino-actinomycin D and annexin V staining. RESULTS: Retinal pigment epithelial cells expressed FasL and induced apoptosis in activated Fas+ T cells. Blocking of Fas-FasL interaction with antibody strongly inhibited RPE-mediated T-cell apoptosis. Retinal pigment epithelial cells...... induced apoptosis in several activated T-cell populations and T-cell lines, including T-cell antigen receptor (TCR)-CD3-negative T-cell lines. In contrast, RPE cells induced little or no apoptosis in resting peripheral T cells. Major histocompatibility complex (MHC) class II monoclonal antibodies, which...

  13. Isolation and identification of gene mediating radiation-induced apoptosis in human leukemia U937 cells

    International Nuclear Information System (INIS)

    Tong Xin; Luo Ying; Dong Yan; Sun Zhixian

    1998-01-01

    Objective: Increasing evidences suggest that Caspase family proteases play an important role in the effector mechanism of apoptotic cell death. Radiation (IR) can induce apoptosis in tumor cells, so it is very important to isolate and identify the member of the Caspase family proteases involved in IR-induced apoptosis, and this would contribute to the understanding of the mechanism responsible for apoptosis execution. Methods: A PCR approach to isolate genes for IR-induced apoptosis was developed. The approach used degenerated oligonucleotide encoding the highly conserved peptides that were present in all known Caspases. Results: Protease inhibitors special for Caspases could block the apoptotic cell death caused by IR, and Caspase-3 was isolated from irradiated human leukemia U937 cells. Conclusion: Caspases involve in IR-induced apoptosis, and Caspase-3 is the pivotal element of IR-induced apoptosis

  14. Overhauser-enhanced MRI of elastase activity from in vitro human neutrophil degranulation.

    Directory of Open Access Journals (Sweden)

    Elodie Parzy

    Full Text Available Magnetic resonance imaging can reveal exquisite anatomical details. However several diseases would benefit from an imaging technique able to specifically detect biochemical alterations. In this context protease activity imaging is one of the most promising areas of research.We designed an elastase substrate by grafting stable nitroxide free radicals on soluble elastin. This substrate generates a high Overhauser magnetic resonance imaging (OMRI contrast upon digestion by the target proteases through the modulation of its rotational correlation time. The sensitivity is sufficient to generate contrasted images of the degranulation of neutrophils induced by a calcium ionophore from 2×10(4 cells per milliliter, well under the physiological neutrophils concentrations.These ex-vivo experiments give evidence that OMRI is suitable for imaging elastase activity from neutrophil degranulation. Provided that a fast protease-substrate is used these results open the door to better diagnoses of a number of important pathologies (cystic fibrosis, inflammation, pancreatitis by OMRI or Electron Paramagnetic Resonance Imaging in vivo. It also provides a long-expected method to monitor anti-protease treatments efficiency and help pharmaceutical research.

  15. Butyrate down regulates BCL-XL and sensitizes human fibroblasts to radiation and chemotherapy induced apoptosis

    International Nuclear Information System (INIS)

    Chung, Diana H.; Ljungman, Mats; Zhang Fenfen; Chen Feng; McLaughlin, William P.

    1997-01-01

    Purpose/Objective: Butyrate is a short chain fatty acid that has been implicated in the induction of cell cycle arrest, cell differentiation and apoptosis. The purpose of this study was to determine if butyrate treatment sensitizes cells to radiation or chemotherapy induced apoptosis. Materials and Methods: Normal neonatal human diploid fibroblasts were used throughout this study. Apoptosis was scored and quantified using three different methods. First, cell morphology using propidium iodide and fluorescence microscopy was used to qualitatively determine apoptosis and to quantify the percentage of cells undergoing apoptosis. Second, apoptosis induced DNA degradation was scored by quantifying the amount of cells appearing in a sub-G1 peak using fixed and PI-stained cells and flow cytometry. Third, apoptosis-induced DNA degradation was examined by using an assay involving direct lysis of cells in the wells of agarose gels followed by conventional gel electrophoresis. Western blotting was used to quantify the cellular levels of the apoptosis regulators, Bcl-2, Bcl-XL and Bax. Results: Human diploid fibroblasts, which were resistant to radiation induced apoptosis, were found to undergo massive apoptosis when radiation was combined with butyrate treatment. Sensitization was obtained when butyrate was added before or after radiation although the combination of both pre and post-treatment was the most effective. Butyrate was also found to enhance UV light and cisplatin-induced apoptosis. These findings correlated with a reduction of the apoptosis antagonist Bcl-XL. Bcl-XL levels significantly dropped in a time and dose dependent manner. In addition, butyrate effectively blocked UV-induced accumulation of p53. Conclusion: Our results suggest that butyrate may be an attractive agent to use in combination with radiation or chemotherapy to lower the apoptotic threshold of tumor cells, regardless of the p53 status of the tumor cells

  16. INHIBITION OF SPONTANEOUS APOPTOSIS IN HUMAN BREAST CANCER

    Institute of Scientific and Technical Information of China (English)

    邵志敏; 江明; 吴炅; 余黎民; 韩企夏; 张延璆; 沈镇宙

    1996-01-01

    Breast tumorigenesis proceeds through an accumulation of specific genetic alteration. Breast malignant transformation is dependent on not only the rate of cell production but also on apoptcsis,a genetically prograined process of autonomous ceil death. We investigated whether breast tumorigenesis involved an altered susceptibility to apoptosis and proliferation by examining normal breast epithelium and breast cancer sampies. We found there is a great inhibition of spontaneous apoptosis in breast cancer ceils compared with normal breast epithelium. The inhibition of apoptosis in breast cancer may contribute to neoplastic transformation.

  17. Implication of extracellular zinc exclusion by recombinant human calprotectin (MRP8 and MRP14) from target cells in its apoptosis-inducing activity.

    Science.gov (United States)

    Yui, Satoru; Nakatani, Yuichi; Hunter, Michael J; Chazin, Walter J; Yamazaki, Masatoshi

    2002-06-01

    Calprotectin is a calcium-binding and zinc-binding protein complex that is abundant in the cytosol of neutrophils. This factor is composed of 8 and 14 kDa subunits, which have also been termed migration inhibitory factor-related proteins MRP8 and MRP14. We previously reported that rat calprotectin purified from inflammatory neutrophils induces apoptosis of various tumor cells or normal fibroblasts in a zinc-reversible manner. The present study was undertaken to elucidate which subunit is responsible for the apoptosis-inducing activity, and to explore the mechanism of zinc-reversible apoptosis induction. The apoptosis-inducing activity of recombinant human MRP8 (rhMRP8) and recombinant human MRP14 (rhMRP14) was examined against EL-4 lymphoma cells in vitro. To determine whether zinc deprivation by calprotectin was essential for the cytotoxicity, the activity of calprotectin was tested under conditions where physical contact between the factor and the cells was precluded by a low molecular weight cut-off dialysis membrane. The cytotoxicity of rhMRP14 against EL-4 cells was first detected at 10 microM in a standard medium, whereas rhMRP8 caused only marginal cytotoxicity at 40 microM. A mixture of both proteins showed higher specific activity (onset of cytotoxicity at 5 microM). When the cells were cultured in divalent cation-depleted medium, each dose-response curve was shifted to about a four-fold lower concentration range. Calprotectin was found to induce cell death even when the complex and the target cells were separated by dialysis membrane. A membrane-impermeable zinc chelator, diethylenetriamine pentaacetic acid (DTPA), also induced target cell apoptosis in a similar time-course as calprotectin. Moreover, the activities of calprotectin and DTPA were completely inhibited by the presence of zinc ions. These data indicate that calprotectin has higher specific activity to induce apoptosis than the Individual subunits, and that the mechanism is exclusion of zinc

  18. Apoptosis in the human periodontal membrane evaluated in primary and permanent teeth

    DEFF Research Database (Denmark)

    Bille, Marie-Louise Bastholm; Thomsen, Bjarke; Kjær, Inger

    2011-01-01

    that resorption is connected to apoptosis of the epithelial cells of Malassez. The purpose of this study is to localize cells undergoing apoptosis in the periodontal membrane of human primary and permanent teeth. Materials and methods. Human primary and permanent teeth were examined immunohistochemically...... for apoptosis and epithelial cells of Malassez in the periodontal membrane. All teeth examined were extracted in connection with treatment. Results. Apoptosis was seen in close proximity to the root surface and within the epithelial cells of Malassez. This pattern of apoptotis is similar in the periodontal...... membrane in primary and permanent teeth. Conclusions. The inter-relationship between apoptotis and root resorption cannot be concluded from the present study. Apoptosis seen in close proximity to the root surface presumably corresponds to the highly innervated layer of the periodontal membrane...

  19. Assessment of Antioxidant Activity of Spray Dried Extracts of Psidium guajava Leaves by DPPH and Chemiluminescence Inhibition in Human Neutrophils

    Directory of Open Access Journals (Sweden)

    M. R. V. Fernandes

    2014-01-01

    Full Text Available This work evaluated the physicochemical properties and antioxidant activity of spray dried extracts (SDE from Psidium guajava L. leaves. Different drying carriers, namely, maltodextrin, colloidal silicon dioxide, Arabic gum, and β-cyclodextrin at concentrations of 40 and 80% relative to solids content, were added to drying composition. SDE were characterized through determination of the total phenolic, tannins, and flavonoid content. Antioxidant potential of the SDE was assessed by two assays: cellular test that measures the luminol-enhanced chemiluminescence (LumCL produced by neutrophils stimulated with phorbol myristate acetate (PMA and the DPPH radical scavenging (DPPH* method. In both assays the antioxidant activity of the SDE occurred in a concentration-dependent manner and showed no toxicity to the cells. Using the CLlum method, the IC50 ranged from 5.42 to 6.50 µg/mL. The IC50 of the SDE ranged from 7.96 to 8.11 µg/mL using the DPPH• method. Psidium guajava SDE presented significant antioxidant activity; thus they show high potential as an active phytopharmaceutical ingredient. Our findings in human neutrophils are pharmacologically relevant since they indicate that P. guajava SDE is a potential antioxidant and anti-inflammatory agent in human cells.

  20. Assessment of antioxidant activity of spray dried extracts of Psidium guajava leaves by DPPH and chemiluminescence inhibition in human neutrophils.

    Science.gov (United States)

    Fernandes, M R V; Azzolini, A E C S; Martinez, M L L; Souza, C R F; Lucisano-Valim, Y M; Oliveira, W P

    2014-01-01

    This work evaluated the physicochemical properties and antioxidant activity of spray dried extracts (SDE) from Psidium guajava L. leaves. Different drying carriers, namely, maltodextrin, colloidal silicon dioxide, Arabic gum, and β -cyclodextrin at concentrations of 40 and 80% relative to solids content, were added to drying composition. SDE were characterized through determination of the total phenolic, tannins, and flavonoid content. Antioxidant potential of the SDE was assessed by two assays: cellular test that measures the luminol-enhanced chemiluminescence (LumCL) produced by neutrophils stimulated with phorbol myristate acetate (PMA) and the DPPH radical scavenging (DPPH∗ method). In both assays the antioxidant activity of the SDE occurred in a concentration-dependent manner and showed no toxicity to the cells. Using the CLlum method, the IC50 ranged from 5.42 to 6.50 µg/mL. The IC50 of the SDE ranged from 7.96 to 8.11 µg/mL using the DPPH(•) method. Psidium guajava SDE presented significant antioxidant activity; thus they show high potential as an active phytopharmaceutical ingredient. Our findings in human neutrophils are pharmacologically relevant since they indicate that P. guajava SDE is a potential antioxidant and anti-inflammatory agent in human cells.

  1. Apoptosis induced by GanoPoly in human gastric cancer cell line ...

    African Journals Online (AJOL)

    use

    2011-12-12

    Dec 12, 2011 ... ... cancer's active therapy. Key words: Apoptosis, polysaccharide, human gastric cancer cells. ... The active components of polysaccharides are all glucans, which have a ... for the treatment of alleviated fatigue, night sweating,.

  2. Fisetin induces apoptosis through mitochondrial apoptosis pathway in human uveal melanoma cells.

    Science.gov (United States)

    Wang, Kai; Hu, Dan-Ning; Lin, Hui-Wen; Yang, Wei-En; Hsieh, Yi-Hsien; Chien, Hsiang-Wen; Yang, Shun-Fa

    2018-05-01

    Fisetin, a diatery flavonoid, been reported that possess anticancer effects in various cancers. The purpose of the study was to investigate the antitumor effects of fisetin in cultured uveal melanoma cell lines and compared with normal retinal pigment epithelial (RPE) cells. MTT assay was used for evaluating cytotoxic effects of fisetin. Flow cytometry study was used for the determination of apoptosis. JC-1 fluorescent reader was used to determine mitochondrial transmembrane potential changes. The results shown that fisetin dose-dependently decreased the cell viability of uveal melanoma cells but not influenced the cell viability of RPE cells. Apoptosis of uveal melanoma cells was induced by fisetin efficiently. Fisetin inhibited antiapoptotic Bcl-2 family proteins and damaged the mitochondrial transmembrane potential. The levels of proapoptotic Bcl-2 proteins, cytochrome c, and various caspase activities were increased by fisetin. In conclusion, fisetin induces apoptosis of uveal melanoma cells selectively and may be a promising agent to be explored for the treatment of uveal melanoma. © 2018 Wiley Periodicals, Inc.

  3. Transient increase in phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol trisphosphate during activation of human neutrophils

    International Nuclear Information System (INIS)

    Traynor-Kaplan, A.E.; Thompson, B.L.; Harris, A.L.; Taylor, P.; Omann, G.M.; Sklar, L.A.

    1989-01-01

    We recently showed that phosphatidylinositol trisphosphate (PIP3) was present in a unique lipid fraction generated in neutrophils during activation. Here, we demonstrate that the band containing this fraction isolated from thin layer chromatography consists primarily of PIP3 and that only small amounts of radiolabeled PIP3 exist prior to activation. In addition, high performance liquid chromatography of deacylated phospholipids from stimulated cells reveals an increase in a fraction eluting ahead of glycerophosphoinositol 4,5-P2. After removal of the glycerol we found that it coeluted with inositol 1,3,4-P3 when resubjected to high performance liquid chromatography. Thus, we have detected a second, novel form of phosphatidylinositol bisphosphate in activated neutrophils, PI-(3,4)P2. The elevation of PIP3 through the formyl peptide receptor is blocked by pretreatment with pertussis toxin, implicating mediation of the increase in PIP3 by a guanosine triphosphate-binding (G) protein. The rise in PIP3 is not secondary to calcium elevation. Buffering the rise in intracellular calcium did not diminish the increase in PIP3. The elevation of PIP3 appears to occur during activation with physiological agonists, its level varying with the degree of activation. Leukotriene B4, which elicits many of the same responses as stimulation of the formyl peptide receptor but with minimal oxidant production, stimulates a much attenuated rise in PIP3. Isoproterenol, which inhibits oxidant production also reduces the rise in PIP3. Hence formation of PI(3,4)P2 and PIP3 (presumed to be PI(3,4,5)P3) correlates closely with the early events of neutrophil activation

  4. Statins induce apoptosis in rat and human myotube cultures by inhibiting protein geranylgeranylation but not ubiquinone

    International Nuclear Information System (INIS)

    Johnson, Timothy E.; Zhang, Xiaohua; Bleicher, Kimberly B.; Dysart, Gary; Loughlin, Amy F.; Schaefer, William H.; Umbenhauer, Diane R.

    2004-01-01

    Statins are widely used to treat lipid disorders. These drugs are safe and well tolerated; however, in <1% of patients, myopathy and/or rhabdomyolysis can develop. To better understand the mechanism of statin-induced myopathy, we examined the ability of structurally distinct statins to induce apoptosis in an optimized rat myotube model. Compound A (a lactone) and Cerivastatin (an open acid) induced apoptosis, as measured by TUNEL and active caspase 3 staining, in a concentration- and time-dependent manner. In contrast, an epimer of Compound A (Compound B) exhibited a much weaker apoptotic response. Statin-induced apoptosis was completely prevented by mevalonate or geranylgeraniol, but not by farnesol. Zaragozic acid A, a squalene synthase inhibitor, caused no apoptosis on its own and had no effect on Compound-A-induced myotoxicity, suggesting the apoptosis was not a result of cholesterol synthesis inhibition. The geranylgeranyl transferase inhibitors GGTI-2133 and GGTI-2147 caused apoptosis in myotubes; the farnesyl transferase inhibitor FTI-277 exhibited a much weaker effect. In addition, the prenylation of rap1a, a geranylgeranylated protein, was inhibited by Compound A in myotubes at concentrations that induced apoptosis. A similar statin-induced apoptosis profile was seen in human myotube cultures but primary rat hepatocytes were about 200-fold more resistant to statin-induced apoptosis. Although the statin-induced hepatotoxicity could be attenuated with mevalonate, no effect was found with either geranylgeraniol or farnesol. In studies assessing ubiquinone levels after statin treatment in rat and human myotubes, there was no correlation between ubiquinone levels and apoptosis. Taken together, these observations suggest that statins cause apoptosis in myotube cultures in part by inhibiting the geranylgeranylation of proteins, but not by suppressing ubiquinone concentration. Furthermore, the data from primary hepatocytes suggests a cell-type differential

  5. Leptin Regulates Proliferation and Apoptosis in Human Prostate

    Directory of Open Access Journals (Sweden)

    Eduardo Leze

    2012-01-01

    Full Text Available This paper aimed to evaluate the leptin role on the cellular proliferation and the expression of fibroblast growth factor 2, aromatase enzyme, and apoptotic genes in the human prostate tissue. Methods. Fifteen samples of hyperplasic prostate tissue were divided in four symmetric parts maintained in RPMI medium supplemented with 10% fetal bovine serum, 1 ng/mL of gentamicin, and added with 50 ng/mL leptin (L or not (C. After 3 hours of incubation, gene expression was evaluated by real time RT-PCR. Cellular proliferation was evaluated by immunohistochemistry for PCNA. Results. The leptin treatment led to an increase cellular proliferation (C=21.8±0.5; L=64.8±0.9; P<0.0001 and in the expression of Bax (C=0.4±0.1; L=0.9±0.2; P<0.05 while Bcl-2 (C=19.9±5.6; L=5.6±1.8; P<0.05, Bcl-x (C=0.2±0.06; L=0.07±0.02; P<0.05, and aromatase expressions (C=1.9±0.6; L=0.4±0.1; P<0.04 were significantly reduced. Conclusion. Leptin has an important role in maintaining the physiological growth of the prostate since it stimulates both cellular proliferation and apoptosis, with the decrement in the aromatase gene expression.

  6. Inhibition of neutrophil elastase and metalloprotease-9 of human adenocarcinoma gastric cells by chamomile (Matricaria recutita L.) infusion.

    Science.gov (United States)

    Bulgari, Michela; Sangiovanni, Enrico; Colombo, Elisa; Maschi, Omar; Caruso, Donatella; Bosisio, Enrica; Dell'Agli, Mario

    2012-12-01

    This study investigated whether the antiinflammatory effect of chamomile infusion at gastric level could be ascribed to the inhibition of metalloproteinase-9 and elastase. The infusions from capitula and sifted flowers (250-1500 µg/mL) and individual flavonoids (10 µM) were tested on phorbol 12-myristate 13-acetate-stimulated AGS cells and human neutrophil elastase. The results indicate that the antiinflammatory activity associated with chamomile infusions from both the capitula and sifted flowers is most likely due to the inhibition of neutrophil elastase and gastric metalloproteinase-9 activity and secretion; the inhibition occurring in a concentration dependent manner. The promoter activity was inhibited as well and the decrease of metalloproteinase-9 expression was found to be associated with the inhibition of NF-kB driven transcription. The results further indicate that the flavonoid-7-glycosides, major constituents of chamomile flowers, may be responsible for the antiinflammatory action of the chamomile infusion observed here. Copyright © 2012 John Wiley & Sons, Ltd.

  7. Source and role of diacylglycerol formed during phagocytosis of opsonized yeast particles and associated respiratory burst in human neutrophils

    International Nuclear Information System (INIS)

    Della Bianca, V.; Grzeskowiak, M.; Lissandrini, D.; Rossi, F.

    1991-01-01

    The results presented in this paper demonstrate that in human neutrophils phagocytosis of C3b/bi and IgG-opsonized yeast particles is associated with activation of phospholipase D and that this reaction is the main source of diglycerides. The demonstration is based upon the following findings: (1) the challenge of neutrophils with these opsonized particles was followed by a rapid formation of [3H]alkyl-phosphatidic acid [( 3H]alkyl-PA) and [3H]alkyl-diglyceride [( 3H]alkyl-DG) in cells labeled with [3H]alkyl-lyso-phosphatidylcholine; (2) in the presence of ethanol [3H]alkyl-phosphatidylethanol was formed, and accumulation of [3H]alkyl-PA and [3H]alkyl-DG was depressed; (3) propranolol, by inhibiting the dephosphorylation of [3H]alkyl-PA, completely inhibited the accumulation of [3H]alkyl-DG and depressed by about 75% the formation of diglyceride mass. Evidence is also presented that phagocytosis of C3b/bi and IgG-opsonized yeast particles and associated respiratory burst can take place independently of diglyceride formation and of the activity of this second messenger on protein kinase C. In fact: (a) propranolol while completely inhibited the formation of diglyceride mass did not modify either the phagocytosis or respiratory burst; (b) these two processes were insensitive to staurosporine

  8. Anti-proteinase 3 anti-neutrophil cytoplasm autoantibodies recapitulate systemic vasculitis in mice with a humanized immune system.

    Directory of Open Access Journals (Sweden)

    Mark A Little

    Full Text Available Evidence is lacking for direct pathogenicity of human anti-proteinase-3 (PR3 antibodies in development of systemic vasculitis and granulomatosis with polyangiitis (GPA, Wegener's granulomatosis. Progress in study of these antibodies in rodents has been hampered by lack of PR3 expression on murine neutrophils, and by different Fc-receptor affinities for IgG across species. Therefore, we tested whether human anti-PR3 antibodies can induce acute vasculitis in mice with a human immune system. Chimeric mice were generated by injecting human haematopoietic stem cells into irradiated NOD-scid-IL2Rγ⁻/⁻ mice. Matched chimera mice were treated with human IgG from patients with: anti-PR3 positive renal and lung vasculitis; patients with non-vasculitic renal disease; or healthy controls. Six-days later, 39% of anti-PR3 treated mice had haematuria, compared with none of controls. There was punctate bleeding on the surface of lungs of anti-PR3 treated animals, with histological evidence of vasculitis and haemorrhage. Anti-PR3 treated mice had mild pauci-immune proliferative glomerulonephritis, with infiltration of human and mouse leukocytes. In 3 mice (17% more severe glomerular injury was present. There were no glomerular changes in controls. Human IgG from patients with anti-PR3 autoantibodies is therefore pathogenic. This model of anti-PR3 antibody-mediated vasculitis may be useful in dissecting mechanisms of microvascular injury.

  9. Anti-proteinase 3 anti-neutrophil cytoplasm autoantibodies recapitulate systemic vasculitis in mice with a humanized immune system.

    LENUS (Irish Health Repository)

    Little, Mark A

    2012-01-01

    Evidence is lacking for direct pathogenicity of human anti-proteinase-3 (PR3) antibodies in development of systemic vasculitis and granulomatosis with polyangiitis (GPA, Wegener\\'s granulomatosis). Progress in study of these antibodies in rodents has been hampered by lack of PR3 expression on murine neutrophils, and by different Fc-receptor affinities for IgG across species. Therefore, we tested whether human anti-PR3 antibodies can induce acute vasculitis in mice with a human immune system. Chimeric mice were generated by injecting human haematopoietic stem cells into irradiated NOD-scid-IL2Rγ⁻\\/⁻ mice. Matched chimera mice were treated with human IgG from patients with: anti-PR3 positive renal and lung vasculitis; patients with non-vasculitic renal disease; or healthy controls. Six-days later, 39% of anti-PR3 treated mice had haematuria, compared with none of controls. There was punctate bleeding on the surface of lungs of anti-PR3 treated animals, with histological evidence of vasculitis and haemorrhage. Anti-PR3 treated mice had mild pauci-immune proliferative glomerulonephritis, with infiltration of human and mouse leukocytes. In 3 mice (17%) more severe glomerular injury was present. There were no glomerular changes in controls. Human IgG from patients with anti-PR3 autoantibodies is therefore pathogenic. This model of anti-PR3 antibody-mediated vasculitis may be useful in dissecting mechanisms of microvascular injury.

  10. Serum and Glucocorticoid Regulated Kinase 1 (SGK1) Regulates Neutrophil Clearance During Inflammation Resolution

    Science.gov (United States)

    Burgon, Joseph; Robertson, Anne L.; Sadiku, Pranvera; Wang, Xingang; Hooper-Greenhill, Edward; Prince, Lynne R.; Walker, Paul; Hoggett, Emily E.; Ward, Jonathan R.; Farrow, Stuart N.; Zuercher, William J.; Jeffrey, Philip; Savage, Caroline O.; Ingham, Philip W.; Hurlstone, Adam F.; Whyte, Moira K. B.; Renshaw, Stephen A.

    2013-01-01

    The inflammatory response is integral to maintaining health, by functioning to resist microbial infection and repair tissue damage. Large numbers of neutrophils are recruited to inflammatory sites to neutralise invading bacteria through phagocytosis and the release of proteases and reactive oxygen species into the extracellular environment. Removal of the original inflammatory stimulus must be accompanied by resolution of the inflammatory response, including neutrophil clearance, to prevent inadvertent tissue damage. Neutrophil apoptosis and its temporary inhibition by survival signals provides a target for anti-inflammatory therapeutics, making it essential to better understand this process. GM-CSF, a neutrophil survival factor, causes a significant increase in mRNA levels for the known anti-apoptotic protein Serum and Glucocorticoid Regulated Kinase 1 (SGK1). We have characterised the expression patterns and regulation of SGK family members in human neutrophils, and shown that inhibition of SGK activity completely abrogates the anti-apoptotic effect of GM-CSF. Using a transgenic zebrafish model, we have disrupted sgk1 gene function and shown this specifically delays inflammation resolution, without altering neutrophil recruitment to inflammatory sites in vivo. These data suggest SGK1 plays a key role in regulating neutrophil survival signalling, and thus may prove a valuable therapeutic target for the treatment of inflammatory disease. PMID:24431232

  11. Serum and glucocorticoid-regulated kinase 1 regulates neutrophil clearance during inflammation resolution.

    Science.gov (United States)

    Burgon, Joseph; Robertson, Anne L; Sadiku, Pranvera; Wang, Xingang; Hooper-Greenhill, Edward; Prince, Lynne R; Walker, Paul; Hoggett, Emily E; Ward, Jonathan R; Farrow, Stuart N; Zuercher, William J; Jeffrey, Philip; Savage, Caroline O; Ingham, Philip W; Hurlstone, Adam F; Whyte, Moira K B; Renshaw, Stephen A

    2014-02-15

    The inflammatory response is integral to maintaining health by functioning to resist microbial infection and repair tissue damage. Large numbers of neutrophils are recruited to inflammatory sites to neutralize invading bacteria through phagocytosis and the release of proteases and reactive oxygen species into the extracellular environment. Removal of the original inflammatory stimulus must be accompanied by resolution of the inflammatory response, including neutrophil clearance, to prevent inadvertent tissue damage. Neutrophil apoptosis and its temporary inhibition by survival signals provides a target for anti-inflammatory therapeutics, making it essential to better understand this process. GM-CSF, a neutrophil survival factor, causes a significant increase in mRNA levels for the known anti-apoptotic protein serum and glucocorticoid-regulated kinase 1 (SGK1). We have characterized the expression patterns and regulation of SGK family members in human neutrophils and shown that inhibition of SGK activity completely abrogates the antiapoptotic effect of GM-CSF. Using a transgenic zebrafish model, we have disrupted sgk1 gene function and shown this specifically delays inflammation resolution, without altering neutrophil recruitment to inflammatory sites in vivo. These data suggest SGK1 plays a key role in regulating neutrophil survival signaling and thus may prove a valuable therapeutic target for the treatment of inflammatory disease.

  12. Role of oncogene 24p3 neutrophil gelatinase-associated lipocalin (NGAL) in digestive system cancers.

    Science.gov (United States)

    Michalak, Łukasz; Bulska, Magdalena; Kudłacz, Katarzyna; Szcześniak, Piotr

    2016-01-04

    Neutrophil gelatinase-associated lipocalin, known also as 24p3 lipocalin, lipocalin-2 or uterocalin (in mouse), is a small secretory protein binding small molecular weight ligands which takes part in numerous processes including apoptosis induction in leukocytes, iron transport, smell, and prostaglandins and retinol transport [19]. It was discovered in activated neutrophils as a covalent peptide associated with human gelatinase neutrophils [7]. Neutrophil lipocalin is secreted physiologically in the digestive system, respiratory tract, renal tubular cells, liver or immunity system. Systematic (circulated in plasma) neutrophil gelatinase come from multiple sources; it may be synthesized in the liver, secreted from activated neutrophils or macrophages, or derive from atherosclerosis or inflammatory endothelial cells [17]. NGAL is stored secondarily in granulates with lactoferrin, calprotectin or MAC-1, which take part in neutrophils' action and migration [13,19]. NGAL participates in acute and chronic inflammation (production of NGAL is indicated by factors conducive to cancer progression) [13,21]. NGAL levels increase in inflammatory or endothelial damage. NGAL level is measured in blood or urine. It is known as a kidney failure factor [7,20]. NGAL is therefore one of the most promising new generation biomarkers in clinical nephrology [6]. The role of NGAL in digestive system neoplasms has not been explored in detail. However, overexpression of this marker was proved in neoplasms such as esophageal carcinoma, stomach cancer, pancreatic cancer or colon cancer, which may indicate an association between concentration and neoplasm [3].

  13. Estresse oxidativo e aumento da apoptose em neutrófilos de cães com azotemia pré-renal Oxidative stress and increase in apoptosis index of neutrophils from dogs with prerenal azotemia

    Directory of Open Access Journals (Sweden)

    A.C.R.A. Silva

    2013-02-01

    Full Text Available O presente trabalho tem como objetivo testar a hipótese de que, à semelhança do que ocorre na uremia, cães com azotemia pré-renal sofrem estresse oxidativo, o qual está relacionado com alterações do metabolismo oxidativo e apoptose dos neutrófilos. Para tal, foi determinada a peroxidação lipídica pela quantificação do malondialdeído (MDA e o status antioxidante total do plasma de 15 cães normais e 10 com azotemia pré-renal, correlacionando-os com a produção de superóxido e o índice apoptótico dos neutrófilos. As determinações do MDA e do status antioxidante total foram estabelecidas empregando-se um conjunto de reagentes comerciais. Por meio de citometria de fluxo capilar, a produção de superóxido e a apoptose de neutrófilos isolados de sangue periférico foram determinadas utilizando-se a sonda hidroetidina e o sistema anexina V-PE, respectivamente. Cães azotêmicos (26,29±5,32g/L apresentaram menor concentração (p=0,0264 do antioxidante albumina em relação ao grupo-controle (30,36±3,29g/L e também uma menor (p=0,0027 capacidade antioxidante total (2,36±0,32 versus 2,73±0,24mmol/L, enquanto não houve alteração da peroxidação lipídica plasmática e da produção de superóxido neutrofílica. Concluiu-se que, à semelhança do que ocorre na uremia, condições azotêmicas pré-renais no cão causam estresse oxidativo e aceleração da apoptose dos neutrófilos.This study aims to test the hypothesis that, similarly to what occurs in uremia, dogs with prerenal azotemia suffer oxidative stress associated with changes in oxidative metabolism and apoptosis in neutrophils. For this purpose, fifteen normal dogs and ten with prerenal azotemia had lipid peroxidation determined by quantifying the malondialdehyde (MDA and had plasma total antioxidant status evaluated, correlating them with the superoxide production and apoptotic index of neutrophils. MDA and plasma total antioxidant status were determined using

  14. Effect of recombinant human erythropoietin expressions of apoptosis ...

    African Journals Online (AJOL)

    apoptosis genes in rats following traumatic brain injury. Xuesong Yuan1* ... ĸB)-, c-myc-, and Fas/Fasl-positive cells were identified in brain tissues by ... stem cells present in the bone marrow. ... neuronal regeneration [12], lowering toxicity of.

  15. Ebracteolatain A and Ebracteolatain B Induce Apoptosis of Human ...

    African Journals Online (AJOL)

    U937 and HeLa cells, and their anti-cancer mechanisms may be related to apoptosis [7,8]. Ebracteolatain A (EA) and ebracteolatain B (EB) from E. ebracteolata are phloroglucinol derivatives. Phloroglucinol derivatives such as dryofragin and 2,4-bis(2-fluorophenylacetyl) phloroglucinol exhibits anti-cancer effects [9-11].

  16. Renal cell apoptosis in human lupus nephritis: a histological study

    DEFF Research Database (Denmark)

    Faurschou, M; Penkowa, Milena; Andersen, C B

    2009-01-01

    Nuclear autoantigens from apoptotic cells are believed to drive the immunological response in systemic lupus erythematosus (SLE). Conflicting data exist as to the possible renal origin of apoptotic cells in SLE patients with nephritis. We assessed the level of renal cell apoptosis in kidney...

  17. Implication of extracellular zinc exclusion by recombinant human calprotectin (MRP8 and MRP14 from target cells in its apoptosis-inducing activity

    Directory of Open Access Journals (Sweden)

    Satoru Yui

    2002-01-01

    Full Text Available Background: Calprotectin is a calcium-binding and zinc-binding protein complex that is abundant in the cytosol of neutrophils. This factor is composed of 8 and 14 kDa subunits, which have also been termed migration inhibitory factor-related proteins MRP8 and MRP14. We previously reported that rat calprotectin purified from inflammatory neutrophils induces apoptosis of various tumor cells or normal fibroblasts in a zinc-reversible manner.

  18. Suppression of blood monocyte and neutrophil chemotaxis in acute human malaria

    DEFF Research Database (Denmark)

    Nielsen, H; Kharazmi, A; Theander, T G

    1986-01-01

    tested monocyte chemotactic responsiveness in 19 patients with acute primary attack malaria. In addition, the neutrophil chemotaxis was measured in 12 patients. Before the initiation of antimalarial treatment a significant depression of monocyte chemotaxis was observed in approximately half...... of the patients when compared with healthy control subjects. The depression was found in Plasmodium falciparum malaria as well as in P. vivax or P. ovale malaria patients. The defective responsiveness was not receptor specific, since the responses towards casein and zymosan activated serum proved to be equally...... of treatment, and nearly normalized after 7 days (87% of controls). Furthermore, monocyte phagocytic and candidacidal activities were assessed in the same patients on admission and during the follow-up. In contrast to chemotaxis, these functions were normal in all of the patients whenever measured...

  19. The protective effect of resveratrol on human lens epithelial cells against ultraviolet-induced apoptosis

    Directory of Open Access Journals (Sweden)

    Xue - Fang Chen

    2013-06-01

    Full Text Available AIM: To investigate the protective effect of resveratrol on human lens epithelial cells against ultraviolet-induced apoptosis. METHODS:Subcultured human lens epithelial cell line, ultraviolet induced cell apoptosis, 20μmol/L resveratrol pretreated cell, the indicators change was observed: rate of apoptosis was detected by flow cytometry and apoptosis-related factors of caspses-3 and caspase-9 were detected by colorimetric detection, ultrastructure changes were observed under transmission electron microscope. RESULTS: Flow cytometry instrument testing found that resveratrol can suppress the apoptosis induced by ultraviolet irradiation, caspses-3 and caspase-9 content in positive control group were significantly higher than that of the negative control group at the same time period, the difference was statistically significant(P<0.05; caspses-3 and caspase-9 content in experimental group were lower than that in the positive control group at the same time, the difference was statistically significant(P<0.05. In addition, the damage of human lens epithelial cells was alleviated with the incubation time of resveratrol elongated. CONCLUSION:Resveratrol may inhibit ultraviolet-induced apoptosis of human lens epithelial cells, it has preventive function against radioactive cataract, and it can provide reliable evidence for pursuing effective medicine to prevent and treat cataract.

  20. Anti-Inflammatory Benefits of Antibiotics: Tylvalosin Induces Apoptosis of Porcine Neutrophils and Macrophages, Promotes Efferocytosis, and Inhibits Pro-Inflammatory CXCL-8, IL1α, and LTB4 Production, While Inducing the Release of Pro-Resolving Lipoxin A4 and Resolvin D1.

    Science.gov (United States)

    Moges, Ruth; De Lamache, Dimitri Desmonts; Sajedy, Saman; Renaux, Bernard S; Hollenberg, Morley D; Muench, Gregory; Abbott, Elizabeth M; Buret, Andre G

    2018-01-01

    Excessive accumulation of neutrophils and their uncontrolled death by necrosis at the site of inflammation exacerbates inflammatory responses and leads to self-amplifying tissue injury and loss of organ function, as exemplified in a variety of respiratory diseases. In homeostasis, neutrophils are inactivated by apoptosis, and non phlogistically removed by neighboring macrophages in a process known as efferocytosis, which promotes the resolution of inflammation. The present study assessed the potential anti-inflammatory and pro-resolution benefits of tylvalosin, a recently developed broad-spectrum veterinary macrolide derived from tylosin. Recent findings indicate that tylvalosin may modulate inflammation by suppressing NF-κB activation. Neutrophils and monocyte-derived macrophages were isolated from fresh blood samples obtained from 12- to 22-week-old pigs. Leukocytes exposed to vehicle or to tylvalosin (0.1, 1.0, or 10 µg/mL; 0.096-9.6 µM) were assessed at various time points for apoptosis, necrosis, efferocytosis, and changes in the production of cytokines and lipid mediators. The findings indicate that tylvalosin increases porcine neutrophil and macrophage apoptosis in a concentration- and time-dependent manner, without altering levels of necrosis or reactive oxygen species production. Importantly, tylvalosin increased the release of pro-resolving Lipoxin A 4 (LXA 4 ) and Resolvin D1 (RvD 1 ) while inhibiting the production of pro-inflammatory Leukotriene B4 (LTB 4 ) in Ca 2+ ionophore-stimulated porcine neutrophils. Tylvalosin increased neutrophil phospholipase C activity, an enzyme involved in releasing arachidonic acid from membrane stores. Tylvalosin also inhibited pro-inflammatory chemokine (C-X-C motif) ligand 8 (CXCL-8, also known as Interleukin-8) and interleukin-1 alpha (IL-1α) protein secretion in bacterial lipopolysaccharide-stimulated macrophages. Together, these data illustrate that tylvalosin has potent immunomodulatory effects in porcine

  1. Anti-Inflammatory Benefits of Antibiotics: Tylvalosin Induces Apoptosis of Porcine Neutrophils and Macrophages, Promotes Efferocytosis, and Inhibits Pro-Inflammatory CXCL-8, IL1α, and LTB4 Production, While Inducing the Release of Pro-Resolving Lipoxin A4 and Resolvin D1

    Directory of Open Access Journals (Sweden)

    Ruth Moges

    2018-04-01

    Full Text Available Excessive accumulation of neutrophils and their uncontrolled death by necrosis at the site of inflammation exacerbates inflammatory responses and leads to self-amplifying tissue injury and loss of organ function, as exemplified in a variety of respiratory diseases. In homeostasis, neutrophils are inactivated by apoptosis, and non phlogistically removed by neighboring macrophages in a process known as efferocytosis, which promotes the resolution of inflammation. The present study assessed the potential anti-inflammatory and pro-resolution benefits of tylvalosin, a recently developed broad-spectrum veterinary macrolide derived from tylosin. Recent findings indicate that tylvalosin may modulate inflammation by suppressing NF-κB activation. Neutrophils and monocyte-derived macrophages were isolated from fresh blood samples obtained from 12- to 22-week-old pigs. Leukocytes exposed to vehicle or to tylvalosin (0.1, 1.0, or 10 µg/mL; 0.096–9.6 µM were assessed at various time points for apoptosis, necrosis, efferocytosis, and changes in the production of cytokines and lipid mediators. The findings indicate that tylvalosin increases porcine neutrophil and macrophage apoptosis in a concentration- and time-dependent manner, without altering levels of necrosis or reactive oxygen species production. Importantly, tylvalosin increased the release of pro-resolving Lipoxin A4 (LXA4 and Resolvin D1 (RvD1 while inhibiting the production of pro-inflammatory Leukotriene B4 (LTB4 in Ca2+ ionophore-stimulated porcine neutrophils. Tylvalosin increased neutrophil phospholipase C activity, an enzyme involved in releasing arachidonic acid from membrane stores. Tylvalosin also inhibited pro-inflammatory chemokine (C–X–C motif ligand 8 (CXCL-8, also known as Interleukin-8 and interleukin-1 alpha (IL-1α protein secretion in bacterial lipopolysaccharide-stimulated macrophages. Together, these data illustrate that tylvalosin has potent immunomodulatory effects

  2. Modulation of IgE-dependent COX-2 gene expression by reactive oxygen species in human neutrophils.

    Science.gov (United States)

    Vega, Antonio; Chacón, Pedro; Alba, Gonzalo; El Bekay, Rajaa; Martín-Nieto, José; Sobrino, Francisco

    2006-07-01

    Cyclooxygenase (COX) is a key enzyme in prostaglandin (PG) synthesis. Up-regulation of its COX-2 isoform is responsible for the increased PG release, taking place under inflammatory conditions, and also, is thought to be involved in allergic and inflammatory diseases. In the present work, we demonstrate that COX-2 expression becomes highly induced by anti-immunoglobulin E (IgE) antibodies and by antigens in human neutrophils from allergic patients. This induction was detected at mRNA and protein levels and was accompanied by a concomitant PGE(2) and thromboxane A(2) release. We also show evidence that inhibitors of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, such as 4-(2-aminoethyl)benzenesulphonyl fluoride and 4-hydroxy-3-methoxyaceto-phenone, completely cancelled anti-IgE-induced COX-2 protein up-regulation, suggesting that this process is mediated by reactive oxygen species (ROS) derived from NADPH oxidase activity. Moreover, the mitogen-activated protein kinases (MAPKs), p38 and extracellular signal-regulated kinase, and also, the transcription factor, nuclear factor (NF)-kappaB, are involved in the up-regulation of COX-2 expression, as specific chemical inhibitors of these two kinases, such as SB203580 and PD098059, and of the NF-kappaB pathway, such as N(alpha)-benzyloxycarbonyl-l-leucyl-l-leucyl-l-leucinal, abolished IgE-dependent COX-2 induction. Evidence is also presented, using Fe(2)(+)/Cu(2)(+) ions, that hydroxyl radicals generated from hydrogen peroxide through Fenton reactions could constitute candidate modulators able to directly trigger anti-IgE-elicited COX-2 expression through MAPK and NF-kappaB pathways. Present results underscore a new role for ROS as second messengers in the modulation of COX-2 expression by human neutrophils in allergic conditions.

  3. TLR9 and NF-κB are partially involved in activation of human neutrophils by Helicobacter pylori and its purified DNA.

    Directory of Open Access Journals (Sweden)

    Lourdes Alvarez-Arellano

    Full Text Available Helicobacter pylori infection represents one of the most common bacterial infections worldwide. The inflammatory response to this bacterium involves a large influx of neutrophils to the lamina propria of the gastric mucosa. However, little is known about the receptors and molecular mechanisms involved in activation of these neutrophils. In this study, we aimed to determine the role of toll-like receptor 9 (TLR9 in the response of human neutrophils to H. pylori and purified H. pylori DNA (Hp-DNA. Neutrophils were isolated from the blood of adult volunteers and challenged with either H. pylori or Hp-DNA. We found that both, H. pylori and Hp-DNA induced increased expression and release of IL-8. Furthermore, we showed that TLR9 is involved in the induction of IL-8 production by H. pylori and Hp-DNA. IL-8 production induced by H. pylori but not by Hp-DNA was partially mediated by NF-κB. In conclusion, this study showed for first time that both, H. pylori and Hp-DNA activate TLR9 and induce a different inflammatory response that leads to activation of neutrophils.

  4. Apoptosis inhibitor 5 (API-5; AAC-11; FIF) is upregulated in human carcinomas in vivo

    Czech Academy of Sciences Publication Activity Database

    Kočí, Lenka; Chlebová, K.; Hýžďalová, Martina; Hofmanová, Jiřina; Jíra, M.; Kysela, P.; Kozubík, Alois; Kala, Z.; Krejčí, Pavel

    2012-01-01

    Roč. 3, č. 4 (2012), s. 913-916 ISSN 1792-1074 R&D Projects: GA ČR(CZ) GA305/09/1526; GA ČR(CZ) GD303/09/H048; GA ČR(CZ) GAP301/11/1730 Institutional research plan: CEZ:AV0Z50040702 Keywords : apoptosis inhibitor 5 * apoptosis * human carcinoma Subject RIV: BO - Biophysics Impact factor: 0.237, year: 2012

  5. Effect of progesterone receptor status on maspin synthesis via nitric oxide production in neutrophils in human breast cancer.

    Science.gov (United States)

    Ganguly Bhattacharjee, Karabi; Bhattacharyya, Mau; Halder, Umesh Chandra; Jana, Pradipta; Sinha, Asru K

    2014-09-01

    Although progesterone receptor (PR) status, similarly to estrogen receptor status, is of prognostic importance in breast cancer, the involvement of the PR in breast cancer remains obscure. Studies were conducted to determine the function of the PR in neutrophils in the nitric oxide-induced synthesis of maspin, an anti-breast-cancer protein produced in nonmalignant mammary cells and in neutrophils in the circulation. PR status was determined by immunohistochemistry. Maspin synthesis was determined by in-vitro translation of messenger RNA and quantified by enzyme-linked immunosorbent assay. Nitric oxide was determined by the methemoglobin method. It was found that PR status in neutrophils was identical with that in malignant breast tissues. A Scatchard plot for progesterone binding to normal and PR-positive (PR+) neutrophils revealed that whereas normal neutrophils had 11.5 × 10(10) PR sites/cell with K d = 47.619 nM, PR+ neutrophils had 6.6 × 10(10) PR sites/cell with K d = 47.619 nM. The progesterone negative (PR-) neutrophils failed to bind to progesterone. Incubation of normal and PR+ neutrophils with 25 nM progesterone produced 1.317 μM NO and 2.329 nM maspin; the PR+ neutrophils produced 0.72 μM NO and 1.138 nM maspin. The PR- neutrophils failed to produce any NO or maspin in the presence of progesterone. Inhibition of progesterone-induced NO synthesis led to complete inhibition of maspin synthesis in all neutrophils. These results suggest that estrogen and progesterone complement each other in NO-induced maspin synthesis, and do not necessarily antagonize in the synthesis of the anti-breast-cancer protein.

  6. Possible in vivo tolerance of human polymorphonuclear neutrophil to low-grade exercise-induced endotoxaemia

    Directory of Open Access Journals (Sweden)

    G. Camus

    1998-01-01

    Full Text Available To address the question of whether translocation of bacterial lipopolysaccharide (LPS into the blood could be involved in the process of exercise-induced polymorphonuclear neutrophil (PMN activation, 12 healthy male subjects who took part in a sprint triathlon (1.5 km river swim, 40 km bicycle race, 10 km road race were studied. While there was no detectable amount of endotoxin in the blood samples drawn at rest, exercise was followed by the appearance of circulating endotoxin molecules at the end of competition in four subjects, and after one and 24 h recovery in three and seven athletes, respectively. The concentrations of plasma granulocyte myeloperoxidase ([MPO], were significantly higher immediately after exercise and one hour later than baseline values (P<0.001. This variable returned to pre-race levels the day after exercise, despite the presence of detectable amounts of LPS, at that time, in seven athletes. The absence of significant correlation (r=0.26;P=0.383 and temporal association between [MPO]and plasma endotoxin levels led us to conclude that endotoxaemia was not involved in the process of exercise-induced PMN degranulation observed in our subjects.

  7. Apoptosis induced by GanoPoly in human gastric cancer cell line ...

    African Journals Online (AJOL)

    In order to investigate polysaccharide effect on the cultured human gastric cancer cells (SGC7901), DNA ladder, flow cytometry and western blot were used to examine the morpholog, proliferation and apoptosis of human gastric cancer SGC-7901 cells when they were affected by polysaccharide. Results show that ...

  8. Human retinal pigment epithelial cell-induced apoptosis in activated T cells

    DEFF Research Database (Denmark)

    Jørgensen, A; Wiencke, A K; la Cour, M

    1998-01-01

    PURPOSE: The immune privilege of the eye has been thought to be dependent on physical barriers and absence of lymphatic vessels. However, the immune privilege may also involve active immunologic processes, as recent studies have indicated. The purpose of the present study was to investigate whether...... human retinal pigment epithelial (RPE) cells can induce apoptosis in activated T cells. METHODS: Fas ligand (FasL) expression was detected by flow cytometry and immunohistochemistry. Cultured RPE cells were cocultured with T-cell lines and peripheral blood lymphocytes for 6 hours to 2 days. Induction...... of apoptosis was detected by 7-amino-actinomycin D and annexin V staining. RESULTS: Retinal pigment epithelial cells expressed FasL and induced apoptosis in activated Fas+ T cells. Blocking of Fas-FasL interaction with antibody strongly inhibited RPE-mediated T-cell apoptosis. Retinal pigment epithelial cells...

  9. Mitochondrial membrane potential in human neutrophils is maintained by complex III activity in the absence of supercomplex organisation

    NARCIS (Netherlands)

    van Raam, Bram J.; Sluiter, Wim; de Wit, Elly; Roos, Dirk; Verhoeven, Arthur J.; Kuijpers, Taco W.

    2008-01-01

    BACKGROUND: Neutrophils depend mainly on glycolysis for their energy provision. Their mitochondria maintain a membrane potential (Deltapsi(m)), which is usually generated by the respiratory chain complexes. We investigated the source of Deltapsi(m) in neutrophils, as compared to peripheral blood

  10. Mitochondrial membrane potential in human neutrophils is maintained by complex III activity in the absence of supercomplex organisation

    NARCIS (Netherlands)

    B.J. van Raam (Bram); W.J. Sluiter (Wim); F.R.C. de Wit (Frank); D. Roos (Dirk); A.J. Verhoeven (Arthur); T.W. Kuijpers (Taco W.)

    2008-01-01

    textabstractBackground: Neutrophils depend mainly on glycolysis for their enegry provision. Their mitochondria maintain a membrace potential (ΔΨm), which is usually generated by the repiratory chain complexes. We investigated the source of ΔΨm in neutrophils, as compared to peripheral blood

  11. Relationship between human respiratory reactivity and neutrophil metabolism under intermittent hypoxic influences in humans exposed to low-level radiation

    International Nuclear Information System (INIS)

    Serebrovskaya, T.V.; Oberenko, O.A.; Guseva, S.A.

    1996-01-01

    The group of 18 men exposed to radiation during amelioration work in the Chernobyl NPP was examined in the course of adaptation to intermittent hypoxia (rebreathing technique during 10 days of three dayly 5-7 min sessions with 15 min break). The starting level of ventilatory response to hypoxic stimulus (HVR) did not differ from the one in persons living in non-contaminated areas. This hypoxic training (HT) caused the increase of HVR, activity of NADPH-oxidase and cationic protein content in neutrophyls as well as various changes in mieloperoxidase activity. The correlation between respiration reactivity and deviations in neutrophil metabolism under HT was found. 14 refs., 2 figs

  12. Metabolism of isoniazid by neutrophil myeloperoxidase leads to isoniazid-NAD(+) adduct formation: A comparison of the reactivity of isoniazid with its known human metabolites.

    Science.gov (United States)

    Khan, Saifur R; Morgan, Andrew G M; Michail, Karim; Srivastava, Nutan; Whittal, Randy M; Aljuhani, Naif; Siraki, Arno G

    2016-04-15

    The formation of isonicotinyl-nicotinamide adenine dinucleotide (INH-NAD(+)) via the mycobacterial catalase-peroxidase enzyme, KatG, has been described as the major component of the mode of action of isoniazid (INH). However, there are numerous human peroxidases that may catalyze this reaction. The role of neutrophil myeloperoxidase (MPO) in INH-NAD(+) adduct formation has never been explored; this is important, as neutrophils are recruited at the site of tuberculosis infection (granuloma) through infected macrophages' cell death signals. In our studies, we showed that neutrophil MPO is capable of INH metabolism using electron paramagnetic resonance (EPR) spin-trapping and UV-Vis spectroscopy. MPO or activated human neutrophils (by phorbol myristate acetate) catalyzed the oxidation of INH and formed several free radical intermediates; the inclusion of superoxide dismutase revealed a carbon-centered radical which is considered to be the reactive metabolite that binds with NAD(+). Other human metabolites, including N-acetyl-INH, N-acetylhydrazine, and hydrazine did not show formation of carbon-centered radicals, and either produced no detectable free radicals, N-centered free radicals, or superoxide, respectively. A comparison of these free radical products indicated that only the carbon-centered radical from INH is reducing in nature, based on UV-Vis measurement of nitroblue tetrazolium reduction. Furthermore, only INH oxidation by MPO led to a new product (λmax=326nm) in the presence of NAD(+). This adduct was confirmed to be isonicotinyl-NAD(+) using LC-MS analysis where the intact adduct was detected (m/z=769). The findings of this study suggest that neutrophil MPO may also play a role in INH pharmacological activity. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Ghrelin ameliorates the human alveolar epithelial A549 cell apoptosis induced by lipopolysaccharide

    International Nuclear Information System (INIS)

    Huang, Chunrong; Zheng, Haichong; He, Wanmei; Lu, Guifang; Li, Xia; Deng, Yubin; Zeng, Mian

    2016-01-01

    Ghrelin is a gastric acyl-peptide that plays an inhibitory role in cell apoptosis. Herein we investigate the protective effects of ghrelin in LPS-induced apoptosis of human alveolar epithelial A549 cells, along with the possible molecular mechanisms. LPS exposure impaired cell viability and increased apoptosis of A549 cells significantly in concentration- and time-dependent manners embodied in increased Bax and cleaved caspase-3 production, coupled with decreased Bcl-2 levels. Simultaneously, LPS remarkably decreased the expression of phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) and extracellular signal-regulated kinas (ERK) in A549 cells. However, ghrelin'pretreatment ameliorated LPS-caused alterations in the ratio of Bax/Bcl-2 and cleaved caspase-3 expression, whereas activated the PI3K/Akt and ERK signaling. These results demonstrate that ghrelin lightens LPS-induced apoptosis of human alveolar epithelial cells partly through activating the PI3K/Akt and ERK pathway and thereby might benefit alleviating septic ALI. -- Graphical abstract: Ghrelin ameliorates the human alveolar epithelial A549 cells apoptosis induced by lipopolysaccharide partly through activating the PI3K/Akt and ERK pathway. Display Omitted -- Highlights: •It has been observed that LPS insult significantly increased apoptosis in A549 cells. •Both Akt and ERK signaling are critical adapter molecules to mediate the ghrelin-mediated proliferative effect. •Ghrelin may have a therapeutic effect in the prevention of LPS-induced apoptosis.

  14. Ghrelin ameliorates the human alveolar epithelial A549 cell apoptosis induced by lipopolysaccharide

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Chunrong; Zheng, Haichong; He, Wanmei; Lu, Guifang; Li, Xia [Department of Medical Intensive Care Unit, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080 (China); Deng, Yubin, E-mail: dengyub@mail.sysu.edu.cn [Research Center of Translational Medicine, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080 (China); Zeng, Mian, E-mail: zengmian2004@163.com [Department of Medical Intensive Care Unit, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080 (China)

    2016-05-20

    Ghrelin is a gastric acyl-peptide that plays an inhibitory role in cell apoptosis. Herein we investigate the protective effects of ghrelin in LPS-induced apoptosis of human alveolar epithelial A549 cells, along with the possible molecular mechanisms. LPS exposure impaired cell viability and increased apoptosis of A549 cells significantly in concentration- and time-dependent manners embodied in increased Bax and cleaved caspase-3 production, coupled with decreased Bcl-2 levels. Simultaneously, LPS remarkably decreased the expression of phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) and extracellular signal-regulated kinas (ERK) in A549 cells. However, ghrelin'pretreatment ameliorated LPS-caused alterations in the ratio of Bax/Bcl-2 and cleaved caspase-3 expression, whereas activated the PI3K/Akt and ERK signaling. These results demonstrate that ghrelin lightens LPS-induced apoptosis of human alveolar epithelial cells partly through activating the PI3K/Akt and ERK pathway and thereby might benefit alleviating septic ALI. -- Graphical abstract: Ghrelin ameliorates the human alveolar epithelial A549 cells apoptosis induced by lipopolysaccharide partly through activating the PI3K/Akt and ERK pathway. Display Omitted -- Highlights: •It has been observed that LPS insult significantly increased apoptosis in A549 cells. •Both Akt and ERK signaling are critical adapter molecules to mediate the ghrelin-mediated proliferative effect. •Ghrelin may have a therapeutic effect in the prevention of LPS-induced apoptosis.

  15. Ultraviolet B irradiation of human leukaemia HL-60 cells in vitro induces apoptosis

    International Nuclear Information System (INIS)

    Martin, S.J.; Cotter, T.G.

    1991-01-01

    UV radiation is known to be a potent agent for the induction of programmed cell death (apoptosis) in human skin. However, the mechanistic aspects of UV-induced apoptosis remain ill-defined. In this study the effects of varying periods of UV-irradiation on the human leukaemia HL-60 cell line and on five other human cell lines were investigated.HL-60 cells were found to rapidly undergo apoptosis en masse after short periods of UV-irradiation whereas prolonged exposure of these cells to this form of radiation induced a more rapid form of cell death which was suggestive of necrosis, the pathological mode of cell death. UV-induced apoptosis in cell lines was characterized by morphological changes as well as DNA fragmentation into unit multiples of ∼ 200 bp, which was indicative of endogenous endonuclease activation. This DNA fragmentation pattern was not detected in cells immediately after UV-irradiation, and was therefore not the result of direct UV-induced DNA damage. UV-induced apoptosis of the HL-60 cell line was found to require extracellular calcium and to be inhibited in a dose-dependent way by zinc added to the culture medium. (author)

  16. JS-K promotes apoptosis by inducing ROS production in human prostate cancer cells.

    Science.gov (United States)

    Qiu, Mingning; Chen, Lieqian; Tan, Guobin; Ke, Longzhi; Zhang, Sai; Chen, Hege; Liu, Jianjun

    2017-03-01

    Reactive oxygen species (ROS) are chemical species that alter redox status, and are responsible for inducing carcinogenesis. The purpose of the present study was to assess the effects of the glutathione S transferase-activated nitric oxide donor prodrug, JS-K, on ROS accumulation and on proliferation and apoptosis in human prostate cancer cells. Cell proliferation and apoptosis, ROS accumulation and the activation of the mitochondrial signaling pathway were measured. The results demonstrated that JS-K may inhibit prostate cancer cell growth in a dose- and time-dependent manner, and induce ROS accumulation and apoptosis in a dose-dependent manner. With increasing concentrations of JS-K, expression of pro-apoptotic proteins increased, but Bcl-2 expression decreased. Additionally, the antioxidant N-acetylcysteine reversed JS-K-induced cell apoptosis; conversely, the pro-oxidant glutathione disulfide exacerbated JS-K-induced apoptosis. In conclusion, the data suggest that JS-K induces prostate cancer cell apoptosis by increasing ROS levels.

  17. Biological dosimetry: the potential use of radiation-induced apoptosis in human T-lymphocytes

    International Nuclear Information System (INIS)

    Menz, R.; Andres, R.; Larsson, B.; Ozsahin, M.; Crompton, N.E.A.; Trott, K.

    1997-01-01

    An assay for biological dosimetry based on the induction of apoptosis in human T-lymphocytes is described. Radiation-induced apoptosis was assessed by flow cytometric identification of cells displaying apoptosis-associated DNA condensation. CD4 and CD8 T-lymphocytes were analysed. They were recognized on the basis of their cell-surface antigens. Four parameters were measured for both cell types: cell size, granularity, antigen immunofluorescence and DNA content. Apoptosis was quantified as the fraction of CD4-, or CD8-positive cells with a characteristic reduction of cell size and DNA content. At doses below 1 Gy, levels of radiation-induced apoptosis increased for up to 5 days after irradiation. Optimal dose discrimination was observed 4 days after irradiation, at which time the dose-response curves were linear, with a slope of 8% ± 0.5% per 0.1 Gy. In controlled, dose-response experiments the lowest dose level at which the radiation-induced apoptosis frequency was still significantly above control was 0.05 Gy. After 5 days post-irradiation incubation, intra- and interdonor variations were measured and found to be similar; thus, apoptotic levels depend more on the dose than on the donor. The results demonstrate the potential of this assay as a biological dosimeter. (orig.)

  18. LR-90 prevents methylglyoxal-induced oxidative stress and apoptosis in human endothelial cells

    Science.gov (United States)

    Figarola, James L.; Singhal, Jyotsana; Rahbar, Samuel; Awasthi, Sanjay

    2014-01-01

    Methylglyoxal (MGO) is a highly reactive dicarbonyl compound known to induce cellular injury and cytoxicity, including apoptosis in vascular cells. Vascular endothelial cell apoptosis has been implicated in the pathophysiology and progression of atherosclerosis. We investigated whether the advanced glycation end-product inhibitor LR-90 could prevent MGO-induced apoptosis in human umbilical vascular endothelial cells (HUVECs). HUVECs were pre-treated with LR-90 and then stimulated with MGO. Cell morphology, cytotoxicity and apoptosis were evaluated by light microscopy, MTT assay, and Annexin V-FITC and propidium iodide double staining, respectively. Levels of Bax, Bcl-2, cytochrome c, mitogen-activated protein kinases (MAPKs) and caspase activities were assessed by Western blotting. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) were measured with fluorescent probes. LR-90 dose-dependently prevented MGO-associated HUVEC cytotoxicity and apoptotic biochemical changes such as loss of MMP, increased Bax/Bcl-2 protein ratio, mitochondrial cytochrome c release and activation of caspase-3 and 9. Additionally, LR-90 blocked intracellular ROS formation and MAPK (p44/p42, p38, JNK) activation, though the latter seem to be not directly involved in MGO-induced HUVEC apoptosis. LR-90 prevents MGO-induced HUVEC apoptosis by inhibiting ROS and associated mitochondrial-dependent apoptotic signaling cascades, suggesting that LR-90 possess cytoprotective ability which could be beneficial in prevention of diabetic related-atherosclerosis. PMID:24615331

  19. Dose-rate effects for apoptosis and micronucleus formation in gamma-irradiated human lymphocytes

    International Nuclear Information System (INIS)

    Boreham, D.R.; Dolling, J.-A.; Maves, S.R.; Siwarungsun, N.; Mitchel, R.E.J.

    2000-01-01

    We have compared dose-rate effects for γ-radiation-induced apoptosis and micronucleus formation in human lymphocytes. Long-term assessment of individual radiation-induced apoptosis showed little intraindividual variation but significant interindividual variation. The effectiveness of radiation exposure to cause apoptosis or micronucleus formation was reduced by low-dose-rate exposures, but the reduction was apparent at different dose rates for these two end points. Micronucleus formation showed a dose-rate effect when the dose rate was lowered to 0.29 cGy/min, but there was no accompanying cell cycle delay. A further increase in the dose-rate effect was seen at 0.15 cGy/min, but was now accompanied by cell cycle delay. There was no dose-rate effect for the induction of apoptosis until the dose rate was reduced to 0.15 cGy/min, indicating that the mechanisms or signals for processing radiation-induced lesions for these two end points must be different at least in part. There appear to be two mechanisms that contribute to the dose-rate effect for micronucleus formation. One of these does not affect binucleate cell frequency and occurs at dose rates higher than that required to produce a dose-rate effect for apoptosis, and one affects binucleate cell frequency, induced only at the very low dose rate which coincidentally produces a dose-rate effect for apoptosis. Since the dose rate at which cells showed reduced apoptosis as well as a further reduction in micronucleus formation was very low, we conclude that the processing of the radiation-induced lesions that induce apoptosis, and some micronuclei, is very slow in quiescent and PHA-stimulated lymphocytes, respectively. (author)

  20. Dose-rate effects for apoptosis and micronucleus formation in gamma-irradiated human lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Boreham, D.R.; Dolling, J.-A.; Maves, S.R. [Atomic Energy of Canada Limited, Chalk River, Ontario (Canada); Siwarungsun, N. [Chulalongkorn Univ., Bangkok (Thailand); Mitchel, R.E.J. [Atomic Energy of Canada Limited, Chalk River, Ontario (Canada)

    2000-07-01

    We have compared dose-rate effects for {gamma}-radiation-induced apoptosis and micronucleus formation in human lymphocytes. Long-term assessment of individual radiation-induced apoptosis showed little intraindividual variation but significant interindividual variation. The effectiveness of radiation exposure to cause apoptosis or micronucleus formation was reduced by low-dose-rate exposures, but the reduction was apparent at different dose rates for these two end points. Micronucleus formation showed a dose-rate effect when the dose rate was lowered to 0.29 cGy/min, but there was no accompanying cell cycle delay. A further increase in the dose-rate effect was seen at 0.15 cGy/min, but was now accompanied by cell cycle delay. There was no dose-rate effect for the induction of apoptosis until the dose rate was reduced to 0.15 cGy/min, indicating that the mechanisms or signals for processing radiation-induced lesions for these two end points must be different at least in part. There appear to be two mechanisms that contribute to the dose-rate effect for micronucleus formation. One of these does not affect binucleate cell frequency and occurs at dose rates higher than that required to produce a dose-rate effect for apoptosis, and one affects binucleate cell frequency, induced only at the very low dose rate which coincidentally produces a dose-rate effect for apoptosis. Since the dose rate at which cells showed reduced apoptosis as well as a further reduction in micronucleus formation was very low, we conclude that the processing of the radiation-induced lesions that induce apoptosis, and some micronuclei, is very slow in quiescent and PHA-stimulated lymphocytes, respectively. (author)

  1. Demethoxycurcumin Retards Cell Growth and Induces Apoptosis in Human Brain Malignant Glioma GBM 8401 Cells

    Directory of Open Access Journals (Sweden)

    Tzuu-Yuan Huang

    2012-01-01

    Full Text Available Demethoxycurcumin (DMC; a curcumin-related demethoxy compound has been recently shown to display antioxidant and antitumor activities. It has also produced a potent chemopreventive action against cancer. In the present study, the antiproliferation (using the MTT assay, DMC was found to have cytotoxic activities against GBM 8401 cell with IC50 values at 22.71 μM and induced apoptosis effects of DMC have been investigated in human brain malignant glioma GBM 8401 cells. We have studied the mitochondrial membrane potential (MMP, DNA fragmentation, caspase activation, and NF-κB transcriptional factor activity. By these approaches, our results indicated that DMC has produced an inhibition of cell proliferation as well as the activation of apoptosis in GBM 8401 cells. Both effects were observed to increase in proportion with the dosage of DMC treatment, and the apoptosis was induced by DMC in human brain malignant glioma GBM 8401 cells via mitochondria- and caspase-dependent pathways.

  2. A Monoclonal Antibody against Wnt-1 Induces Apoptosis in Human Cancer Cells

    Directory of Open Access Journals (Sweden)

    Biao He

    2004-01-01

    Full Text Available Aberrant activation of the Wingless-type (Wnt/β-catenin signaling pathway is associated with a variety of human cancers. Little is known regarding the role that Wnt ligands play in human carcinogenesis. To test whether a Wnt-1 signal is a survival factor in human cancer cells and thus may serve as a potential cancer therapeutic target, we investigated the effect of inhibition of Wnt-1 signaling in a variety of human cancer cell lines, including non small cell lung cancer, breast cancer, mesothelioma, and sarcoma. Both monoclonal antibody and RNA interference (RNAi were used to inhibit Wnt-1 signaling. We found that incubation of a monoclonal anti-Wnt-1 antibody induced apoptosis and caused downstream protein changes in cancer cells overexpressing Wnt-1. In contrast, apoptosis was not detected in cells lacking or having minimal Wnt-1 expression after the antibody incubation. RNAi targeting of Wnt-1 in cancer cells overexpressing Wnt-1 demonstrated similar downstream protein changes and induction of apoptosis. The antibody also suppressed tumor growth in vivo. Our results indicate that both monoclonal anti-Wnt-1 antibody and Wnt-1 siRNA inhibit Wnt-1 signaling and can induce apoptosis in human cancer cells. These findings hold promise as a novel therapeutic strategy for cancer.

  3. Modulation of neutrophil and monocyte function by recombinant human granulocyte macrophage colony-stimulating factor in patients with lymphoma

    DEFF Research Database (Denmark)

    Kharazmi, A; Nielsen, H; Hovgaard, D

    1991-01-01

    by up to 43-fold. rhGM-CSF treatment did not affect degranulation of the neutrophils as measured by release of vitamin B12 binding protein. Degree of modulation of neutrophil and monocyte function by rhGM-CSF was independent of rhGM-CSF dosages administered. These data suggest that phagocytic defence...... and chemiluminescence responses to f-Met-Leu-Phe, zymosan activated serum (ZAS) and opsonized zymosan (OZ) were determined. It was observed that chemotactic response of neutrophils to f-Met-Leu-Phe and ZAS was reduced, whereas the chemiluminescence response of both cell types to f-Met-Leu-Phe and zymosan was enhanced...

  4. Mechanism research of miR-181 regulating human lens epithelial cell apoptosis

    Directory of Open Access Journals (Sweden)

    Yu Qin

    2015-05-01

    Full Text Available AIM: To investigate the expression of miR-181 in the lens tissue of cataract and the regulating mechanism of miR-181 on apoptosis of human lens epithelial cell.METHODS:Real time q-PCR was used to measure the expression of miR-181 in the anterior lens capsules of age-related cataract and human lens epithelial cell apoptosis model. miR-181 mimic and inhibitor were transfected using Lipofectamine 2 000 to regulate the expression of miR-181, and then Real time q-PCR was used to verify transfection efficiency. Flow cytometry was used to detect the change of cell apoptosis rate. RESULTS: Compared with control group, the expression of miR-181 was significantly higher in both the anterior lens capsules of age-related cataract and human lens epithelial cell apoptosis model; the relative expression of miR-181 in lens epithelial cells transfected with miR-181 mimic was increased, whereas decreased in cells transfected with miR-181 inhibitor; the apoptosis rate of cells transfected with miR-181 mimic was increased, while reduced in miR-181 inhibitor group. Each result was statistically significant(PCONCLUSION: High expression of miR-181 is detected in anterior lens capsule of age-related cataract. miR-181 might play a certain role in the pathogenesis of cataract via promoting human lens epithelial cell apoptosis. miR-181 probably becomes a new approach for the nonoperative treatment of cataract, but the concrete mechanism still needs to be further studied.

  5. Detection of Apoptosis and Necrosis in Normal Human Lung Cells Using 1H NMR Spectroscopy

    Science.gov (United States)

    Shih, Chwen-Ming; Ko, Wun-Chang; Yang, Liang-Yo; Lin, Chien-Ju; Wu, Jui-Sheng; Lo, Tsui-Yun; Wang, Shwu-Huey; Chen, Chien-Tsu

    2005-05-01

    This study aimed to detect apoptosis and necrosis in MRC-5, a normal human lung cell line, by using noninvasive proton nuclear magnetic resonance (1H NMR). Live MRC-5 cells were processed first for 1H NMR spectroscopy; subsequently their types and the percentage of cell death were assessed on a flow cytometer. Cadmium (Cd) and mercury (Hg) induced apoptosis and necrosis in MRC-5 cells, respectively, as revealed by phosphatidylserine externalization on a flow cytometer. The spectral intensity ratio of methylene (CH2) resonance (at 1.3 ppm) to methyl (CH3) resonance (at 0.9 ppm) was directly proportional to the percentage of apoptosis and strongly and positively correlated with PI staining after Cd treatment (r2 = 0.9868, P In contrast, this ratio only increased slightly within 2-h Hg treatment, and longer Hg exposure failed to produce further increase. Following 2-h Hg exposure, the spectral intensity of choline resonance (at 3.2 ppm) was abolished, but this phenomenon was absent in Cd-induced apoptosis. These findings together demonstrate that 1H NMR is a novel tool with a quantitative potential to distinguish apoptosis from necrosis as early as the onset of cell death in normal human lung cells.

  6. Multiple effects of TRAIL in human carcinoma cells: Induction of apoptosis, senescence, proliferation, and cytokine production

    International Nuclear Information System (INIS)

    Levina, Vera; Marrangoni, Adele M.; DeMarco, Richard; Gorelik, Elieser; Lokshin, Anna E.

    2008-01-01

    TRAIL is a death ligand that induces apoptosis in malignant but not normal cells. Recently the ability of TRAIL to induce proliferation in apoptosis-resistant normal and malignant cells was reported. In this study, we analyzed TRAIL effects in apoptosis sensitive MCF7, OVCAR3 and H460 human tumor cell lines. TRAIL at low concentrations preferentially induced cell proliferation. At 100 ng/ml, apoptotic death was readily observed, however surviving cells acquired higher proliferative capacity. TRAIL-stimulated production of several cytokines, IL-8, RANTES, MCP-1 and bFGF, and activation of caspases 1 and 8 was essential for this effect. Antibodies to IL-8, RANTES, and bFGF blocked TRAIL-induced cell proliferation and further stimulated apoptosis. For the first time, we report that high TRAIL concentrations induced cell senescence as determined by the altered morphology and expression of several senescence markers: SA-β-gal, p21 Waf1/Cip1 , p16 INK4a , and HMGA. Caspase 9 inhibition protected TRAIL-treated cells from senescence, whereas inhibition of caspases 1 and 8 increased the yield of SLP cells. In conclusion, in cultured human carcinoma cells, TRAIL therapy results in three functional outcomes, apoptosis, proliferation and senescence. TRAIL-induced proapoptotic and prosurvival responses correlate with the strength of signaling. TRAIL-induced cytokine production is responsible for its proliferative and prosurvival effects

  7. Endogenous acute phase serum amyloid A lacks pro-inflammatory activity, contrasting the two recombinant variants that activate human neutrophils through different receptors

    Directory of Open Access Journals (Sweden)

    Karin eChristenson

    2013-04-01

    Full Text Available Most notable among the acute phase proteins is serum amyloid A (SAA, levels of which can increase 1000-fold during infections, aseptic inflammation, and/or trauma. Chronically elevated SAA levels are associated with a wide variety of pathological conditions, including obesity and rheumatic diseases. Using a recombinant hybrid of the two human SAA isoforms (SAA1 and 2 that does not exist in vivo, numerous in vitro studies have given rise to the notion that acute phase SAA is a pro-inflammatory molecule with cytokine-like properties. It is however unclear whether endogenous acute phase SAA per se mediates pro-inflammatory effects. We tested this in samples from patients with inflammatory arthritis and in a transgenic mouse model that expresses human SAA1. Endogenous human SAA did not drive production of pro-inflammatory IL-8/KC in either of these settings. Human neutrophils derived from arthritis patients displayed no signs of activation, despite being exposed to severely elevated SAA levels in circulation, and SAA-rich sera also failed to activate cells in vitro. In contrast, two recombinant SAA variants (the hybrid SAA and SAA1 both activated human neutrophils, inducing L-selectin shedding, production of reactive oxygen species, and production of IL-8. The hybrid SAA was approximately 100-fold more potent than recombinant SAA1. Recombinant hybrid SAA and SAA1 activated neutrophils through different receptors, with recombinant SAA1 being a ligand for formyl peptide receptor 2 (FPR2. We conclude that even though recombinant SAAs can be valuable tools for studying neutrophil activation, they do not reflect the nature of the endogenous protein.

  8. PARTICIPATION OF TLR4 IN ENGULFMENT OF ESCHERICHIA COLI BY HUMAN BLOOD NEUTROPHILS IN PRESENCE OF LIPOPOLYSACCHARIDES

    Directory of Open Access Journals (Sweden)

    S. V. Zubova

    2012-01-01

    Full Text Available Abstract. TLR4 is a key player in signaling system of host cells. Possible role of TLR4 is actively discussed, e.g. its significance for phagocytosis. A capacity of neutrophils to engulf FITC-labeled E. coli bacteria upon activation with LPS of different origin was studied in presence of anti-TLR4 Mab’s (HTA125 clone. It was shown that, in whole blood, TLR4 does not play any essential role in engulfment of bacteria by the neutrophils. Phagocytic activity of neutrophils in blood increases increased after their priming with E. coli endotoxins. LPS from Rb. сapsulatus did not affect phagocytosis. In presence of endotoxins, the degree of TLR4 involvement in neutrophil phagocytosis depends on LPS structure.

  9. Diosgenin induces apoptosis in IGF-1-stimulated human thyrocytes through two caspase-dependent pathways

    International Nuclear Information System (INIS)

    Mu, Shumin; Tian, Xingsong; Ruan, Yongwei; Liu, Yuantao; Bian, Dezhi; Ma, Chunyan; Yu, Chunxiao; Feng, Mei; Wang, Furong; Gao, Ling; Zhao, Jia-jun

    2012-01-01

    Highlights: ► Diosgenin induces apoptosis in IGF-1-treated thyrocytes through two caspase pathways. ► Diosgenin inhibits FLIP and activates caspase-8 in FAS related-pathway. ► Diosgenin increases ROS, regulates the ratio of Bax/Bcl-2 in mitochondrial pathway. -- Abstract: Insulin-like growth factor-1 (IGF-1) is a growth factor of the thyroid that has been shown in our previous study to possess proliferative and antiapoptotic effects in FRTL-5 cell lines through the upregulation of cyclin D and Fas-associated death domain-like interleukin-1-converting enzyme (FLICE)-inhibitory protein (FLIP). Diosgenin, a natural steroid sapogenin from plants, has been shown to induce apoptosis in many cell lines, with the exception of thyroid cells. In this report, we investigated the apoptotic effect and mechanism of diosgenin in IGF-1-stimulated primary human thyrocytes. Primary human thyrocytes were preincubated with or without IGF-1 for 24 h and subsequently exposed to varying concentrations of diosgenin for different times. We found that diosgenin induced apoptosis in human thyrocytes pretreated with IGF-1 in a dose-dependent manner through the activation of caspase cascades. Moreover, diosgenin inhibited FLIP and activated caspase-8 in the FAS-related apoptotic pathway. Diosgenin increased the production of ROS, regulated the balance of Bax and Bcl-2 and cleaved caspase-9 in the mitochondrial apoptotic pathway. These results indicate that diosgenin induces apoptosis in IGF-1-stimulated primary human thyrocytes through two caspase-dependent pathways.

  10. The role of hypoxia, p53, and apoptosis in human cervical carcinoma pathogenesis

    International Nuclear Information System (INIS)

    Kim, Charlotte Y.; Tsai, Mitchell H.; Osmanian, Cynthia; Calkins, Dennise P.; Graeber, Thomas G.; Greenspan, David L.; Kennedy, Andrew S.; Rinker, Lillian H.; Varia, Mahesh A.; DiPaolo, Joseph A.; Peehl, Donna M.; Raleigh, James A.; Giaccia, Amato J.

    1997-01-01

    Objective: Low oxygen tension in the tumor microenvironment may have an important role during tumor growth, and is of particular prognostic significance in human cervical carcinoma. Because some human papillomavirus (HPV) infections are associated with cervical neoplasia, the relationship between hypoxia and apoptosis in primary cervical epithelial cells containing HPV16 E6 and E7, intact HPV 16 genome, and HPV positive cervical carcinoma cell lines, was examined. In addition, the relationship between hypoxia and apoptosis in spontaneous human cervical carcinomas was determined in situ. Materials and Methods: Primary normal human cervical epithelial cells were infected with retroviral vectors containing HPV16 E6 and E7 or transfected with a plasmid containing the whole HPV 16 genome. Clones were selected in neomycin containing medium. Exponentially growing cells were incubated under aerobic conditions (20% O 2 ), anaerobic conditions (0.02% O 2 ), or irradiated with 6 Gy. Analysis of apoptotic cells was performed by staining with Hoechst dye and propidium iodide and viewing with a fluorescent microscope. To determine the level of expression of the apoptotic modulators p53 and Bax, immunoblots were performed on whole cell extracts from treated cells. A clinical tumor hypoxia study was conducted at the University of North Carolina utilizing pimonidazole, a 2-nitroimidazole compound which binds irreversibly to cellular macromolecules under low oxygen conditions. Nine patients were enrolled with biopsy proven squamous cell carcinoma of the cervix and no prior treatment. Biopsies of the gross tumor were obtained after pimonidazole infusion. Contiguous histological sections were analyzed for hypoxia using a immunohistochemical technique and for apoptosis using TUNEL. Results: In vitro, hypoxia uncoupled p53 from E6 mediated degradation, and stimulated both p53 induction and apoptosis in primary cervical epithelial cells infected with the HPV E6 and E7 genes. In contrast

  11. The Natural Antiangiogenic Compound AD0157 Induces Caspase-Dependent Apoptosis in Human Myeloid Leukemia Cells

    Directory of Open Access Journals (Sweden)

    Melissa García-Caballero

    2017-11-01

    Full Text Available Evasion of apoptosis is a hallmark of cancer especially relevant in the development and the appearance of leukemia drug resistance mechanisms. The development of new drugs that could trigger apoptosis in aggressive hematological malignancies, such as AML and CML, may be considered a promising antileukemic strategy. AD0157, a natural marine pyrrolidinedione, has already been described as a compound that inhibits angiogenesis by induction of apoptosis in endothelial cells. The crucial role played by defects in the apoptosis pathways in the pathogenesis, progression and response to conventional therapies of several forms of leukemia, moved us to analyze the effect of this compound on the growth and death of leukemia cells. In this work, human myeloid leukemia cells (HL60, U937 and KU812F were treated with AD0157 ranging from 1 to 10 μM and an experimental battery was applied to evaluate its apoptogenic potential. We report here that AD0157 was highly effective to inhibit cell growth by promotion of apoptosis in human myeloid leukemia cells, and provide evidence of its mechanisms of action. The apoptogenic activity of AD0157 on leukemia cells was verified by an increased chromatin condensation and DNA fragmentation, and confirmed by an augmentation in the apoptotic subG1 population, translocation of the membrane phosphatidylserine from the inner face of the plasma membrane to the cell surface and by cleavage of the apoptosis substrates PARP and lamin-A. In addition, AD0157 in the low micromolar range significantly enhanced the activities of the initiator caspases-8 and -9, and the effector caspases-3/-7 in a dose-dependent manner. Results presented here throw light on the apoptogenic mechanism of action of AD0157, mediated through caspase-dependent cascades, with an especially relevant role played by mitochondria. Altogether, these results suggest the therapeutic potential of this compound for the treatment of human myeloid leukemia.

  12. The proteolytically stable peptidomimetic Pam-(Lys-βNSpe)6-NH2 selectively inhibits human neutrophil activation via formyl peptide receptor 2.

    Science.gov (United States)

    Skovbakke, Sarah Line; Heegaard, Peter M H; Larsen, Camilla J; Franzyk, Henrik; Forsman, Huamei; Dahlgren, Claes

    2015-01-15

    Immunomodulatory host defense peptides (HDPs) are considered to be lead compounds for novel anti-sepsis and anti-inflammatory agents. However, development of drugs based on HDPs has been hampered by problems with toxicity and low bioavailability due to in vivo proteolysis. Here, a subclass of proteolytically stable HDP mimics consisting of lipidated α-peptide/β-peptoid oligomers was investigated for their effect on neutrophil function. The most promising compound, Pam-(Lys-βNSpe)6-NH2, was shown to inhibit formyl peptide receptor 2 (FPR2) agonist-induced neutrophil granule mobilization and release of reactive oxygen species. The potency of Pam-(Lys-βNSpe)6-NH2 was comparable to that of PBP10, the most potent FPR2-selective inhibitor known. The immunomodulatory effects of structural analogs of Pam-(Lys-βNSpe)6-NH2 emphasized the importance of both the lipid and peptidomimetic parts. By using imaging flow cytometry in primary neutrophils and FPR-transfected cell lines, we found that a fluorescently labeled analog of Pam-(Lys-βNSpe)6-NH2 interacted selectively with FPR2. Furthermore, the interaction between Pam-(Lys-βNSpe)6-NH2 and FPR2 was found to prevent binding of the FPR2-specific activating peptide agonist Cy5-WKYMWM, while the binding of an FPR1-selective agonist was not inhibited. To our knowledge, Pam-(Lys-βNSpe)6-NH2 is the first HDP mimic found to inhibit activation of human neutrophils via direct interaction with FPR2. Hence, we consider Pam-(Lys-βNSpe)6-NH2 to be a convenient tool in the further dissection of the role of FPR2 in inflammation and homeostasis as well as for investigation of the importance of neutrophil stimulation in anti-infective therapy involving HDPs. Copyright © 2014 Elsevier Inc. All rights reserved.

  13. Neutrophils: potential therapeutic targets in tularemia?

    Directory of Open Access Journals (Sweden)

    Lee-Ann H Allen

    2013-12-01

    Full Text Available The central role of neutrophils in innate immunity and host defense has long been recognized, and the ability of these cells to efficiently engulf and kill invading bacteria has been extensively studied, as has the role of neutrophil apoptosis in resolution of the inflammatory response. In the past few years additional immunoregulatory properties of neutrophils were discovered, and it is now clear that these cells play a much greater role in control of the immune response than was previously appreciated. In this regard, it is noteworthy that Francisella tularensis is one of relatively few pathogens that can successfully parasitize neutrophils as well as macrophages, DC and epithelial cells. Herein we will review the mechanisms used by F. tularensis to evade elimination by neutrophils. We will also reprise effects of this pathogen on neutrophil migration and lifespan as compared with other infectious and inflammatory disease states. In addition, we will discuss the evidence which suggests that neutrophils contribute to disease progression rather than effective defense during tularemia, and consider whether manipulation of neutrophil migration or turnover may be suitable adjunctive therapeutic strategies.

  14. Morphological modulation of human fibrosarcoma HT-1080 cells by hydroxybenzoate compounds during apoptosis

    Directory of Open Access Journals (Sweden)

    Jassem G Mahdi

    2015-10-01

    Full Text Available Hydroxybenzoate (HB compounds have shown to modulate the morphology in human fibrosarcoma HT-1080 cells. The changes in HT-1080 cells showed marker signs of apoptosis, which included the condensation of nucleus, cell round, blebbing and the formation of apoptotic bodies. The different stages of apoptosis were assessed microscopically using different staining and immunohistochemical techniques, as well as scanning electron microscopy. In addition, HB compounds increased the expression of caspase-3, which is closely associated with the development of the modulation in HT-1080 cells that are undergoing the programmed cell death. Both acetyl salicylic acid (ASA and HBZn compounds were dose and treatment duration dependent.

  15. Portulaca oleracea extracts protect human keratinocytes and fibroblasts from UV-induced apoptosis.

    Science.gov (United States)

    Lee, Suyeon; Kim, Ki Ho; Park, Changhoon; Lee, Jong-Suk; Kim, Young Heui

    2014-10-01

    Portulaca oleracea extracts, known as Ma Chi Hyun in the traditional Korean medicine, show a variety of biomedical efficacies including those in anti-inflammation and anti-allergy. In this study, we investigate the protective activity of the P. oleracea extracts against UVB-induced damage in human epithelial keratinocytes and fibroblasts by several apoptosis-related tests. The results suggest that P. oleracea extracts have protective effects from UVB-induced apoptosis. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  16. Single-wall carbon nanohorns (SWNHs) inhibited proliferation of human glioma cells and promoted its apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yunjun [The Military General Hospital of Beijing PLA, Affiliated Bayi Brain Hospital (China); Zhang, Jinqian, E-mail: jingwanghou@yahoo.com.cn [Capital Medical University, Institute of Infectious Diseases, Beijing Ditan Hospital (China); Zhao, Ming [Peking University, Department of Chemical Biology, School of Pharmaceutical Sciences (China); Shi, Zujin [Peking University, Beijing National Laboratory for Molecular Sciences, State Key Laboratory of Rare Earth Materials Chemistry and Applications, College of Chemistry and Molecular Engineering (China); Chen, Xin; He, Xihui; Han, Nanyin, E-mail: jingwanghou@sina.com [Peking University, Department of Chemical Biology, School of Pharmaceutical Sciences (China); Xu, Ruxiang, E-mail: everbright999@163.com [The Military General Hospital of Beijing PLA, Affiliated Bayi Brain Hospital (China)

    2013-08-15

    Although single-wall carbon nanohorns (SWNHs) have been demonstrated to accumulate to cytotoxic levels within organs of various animal models and cell types, they have been exploited for cancer therapies. The role of SWNHs in human glioma cell lines was unclear. To address this question, the research about direct role of SWNHs on the growth, proliferation, and apoptosis of human glioma cell lines (U87, U251, and U373) had been performed. Our results indicate that particle size of SWNHs in water is between 342 and 712 nm, the films of SEM show that SWNHs on PS surface are individual particles. SWNHs significantly delayed mitotic entry of human glioma cell lines cells, and inhibited its proliferation in a time- and dose-dependent manner. SWNHs induced a significant increase in G1 phase and inhibition of S phase followed the gradually increasing concentrations. SWNHs in human glioma cell lines cells significantly induced apoptosis followed by their gradually increasing concentrations. The TEM images showed that individual spherical SWNHs particles smaller than 100 nm in diameters were localized inside lysosomes of human glioma cell lines. SWNHs inhibited mitotic entry, growth, and proliferation of human glioma cell lines, and promoted its apoptosis. SWNHs may be a novel opportunity or method for the research on treatment of human glioma.

  17. Single-wall carbon nanohorns (SWNHs) inhibited proliferation of human glioma cells and promoted its apoptosis

    Science.gov (United States)

    Li, Yunjun; Zhang, Jinqian; Zhao, Ming; Shi, Zujin; Chen, Xin; He, Xihui; Han, Nanyin; Xu, Ruxiang

    2013-08-01

    Although single-wall carbon nanohorns (SWNHs) have been demonstrated to accumulate to cytotoxic levels within organs of various animal models and cell types, they have been exploited for cancer therapies. The role of SWNHs in human glioma cell lines was unclear. To address this question, the research about direct role of SWNHs on the growth, proliferation, and apoptosis of human glioma cell lines (U87, U251, and U373) had been performed. Our results indicate that particle size of SWNHs in water is between 342 and 712 nm, the films of SEM show that SWNHs on PS surface are individual particles. SWNHs significantly delayed mitotic entry of human glioma cell lines cells, and inhibited its proliferation in a time- and dose-dependent manner. SWNHs induced a significant increase in G1 phase and inhibition of S phase followed the gradually increasing concentrations. SWNHs in human glioma cell lines cells significantly induced apoptosis followed by their gradually increasing concentrations. The TEM images showed that individual spherical SWNHs particles smaller than 100 nm in diameters were localized inside lysosomes of human glioma cell lines. SWNHs inhibited mitotic entry, growth, and proliferation of human glioma cell lines, and promoted its apoptosis. SWNHs may be a novel opportunity or method for the research on treatment of human glioma.

  18. Apoptosis may determine the release of skeletal alkaline phosphatase activity from human osteoblast-line cells.

    Science.gov (United States)

    Farley, J R; Stilt-Coffing, B

    2001-01-01

    Although quantitative measurement of skeletal alkaline phosphatase (sALP) activity in serum can provide an index of the rate of bone formation, the metabolic process that determines the release of sALP - from the surface of osteoblasts, into circulation-is unknown. The current studies were intended to examine the hypothesis that the release of sALP from human osteoblasts is a consequence of apoptotic cell death. We measured the release of sALP activity from human osteosarcoma (SaOS-2) cells and normal human bone cells, under basal conditions and in response to agents that increased apoptosis (TNF-a, okadiac acid) and agents that inhibit apoptosis (IGF-I, calpain, and caspase inhibitors). Apoptosis was determined by the presence of nucleosomes (histone-associated DNA) in the cytoplasm of the cells by using a commercial kit. The results of these studies showed that TNF-a and okadiac acid caused dose- and time-dependent increases in apoptosis in the SaOS-2 cells (r = 0.78 for doses of TNF-a and r = 0.93 for doses of okadiac acid, P sALP activity (e.g., r = 0.89 for TNF-a and r = 0.75 for okadiac acid, P sALP activity (P sALP activity (P sALP release. The associations between apoptosis and sALP release were not unique to osteosarcoma (i.e., SaOS-2) cells, but also seen with osteoblast-line cells derived from normal human bone. Together, these data demonstrate that the release of sALP activity from human osteoblast-line cells in vitro is associated with, and may be a consequence of, apoptotic cell death. These findings are consistent with the general hypothesis that the appearance of sALP activity in serum may reflect the turnover of osteoblast-line cells.

  19. Noscapine induces mitochondria-mediated apoptosis in human colon cancer cells in vivo and in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Zi-Rong; Liu, Meng; Peng, Xiu-Lan; Lei, Xiao-Fei; Zhang, Ji-Xiang [Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province (China); Dong, Wei-Guo, E-mail: dongwg1966@yahoo.com.cn [Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province (China)

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer Noscapine inhibited cell viability of colon cancer in a time- and dose- dependent manner. Black-Right-Pointing-Pointer G{sub 2}/M phase arrest and chromatin condensation and nuclear fragmentation were induced. Black-Right-Pointing-Pointer Noscapine promoted apoptosis via mitochondrial pathways. Black-Right-Pointing-Pointer Tumorigenicity was inhibited by noscapine. -- Abstract: Noscapine, a phthalide isoquinoline alkaloid derived from opium, has been widely used as a cough suppressant for decades. Noscapine has recently been shown to potentiate the anti-cancer effects of several therapies by inducing apoptosis in various malignant cells without any detectable toxicity in cells or tissues. However, the mechanism by which noscapine induces apoptosis in colon cancer cells remains unclear. The signaling pathways by which noscapine induces apoptosis were investigated in colon cancer cell lines treated with various noscapine concentrations for 72 h, and a dose-dependent inhibition of cell viability was observed. Noscapine effectively inhibited the proliferation of LoVo cells in vitro (IC{sub 50} = 75 {mu}M). This cytotoxicity was reflected by cell cycle arrest at G{sub 2}/M and subsequent apoptosis, as indicated by increased chromatin condensation and fragmentation, the upregulation of Bax and cytochrome c (Cyt-c), the downregulation of survivin and Bcl-2, and the activation of caspase-3 and caspase-9. Moreover, in a xenograft tumor model in mice, noscapine injection clearly inhibited tumor growth via the induction of apoptosis, which was demonstrated using a TUNEL assay. These results suggest that noscapine induces apoptosis in colon cancer cells via mitochondrial pathways. Noscapine may be a safe and effective chemotherapeutic agent for the treatment of human colon cancer.

  20. Noscapine induces mitochondria-mediated apoptosis in human colon cancer cells in vivo and in vitro

    International Nuclear Information System (INIS)

    Yang, Zi-Rong; Liu, Meng; Peng, Xiu-Lan; Lei, Xiao-Fei; Zhang, Ji-Xiang; Dong, Wei-Guo

    2012-01-01

    Highlights: ► Noscapine inhibited cell viability of colon cancer in a time- and dose- dependent manner. ► G 2 /M phase arrest and chromatin condensation and nuclear fragmentation were induced. ► Noscapine promoted apoptosis via mitochondrial pathways. ► Tumorigenicity was inhibited by noscapine. -- Abstract: Noscapine, a phthalide isoquinoline alkaloid derived from opium, has been widely used as a cough suppressant for decades. Noscapine has recently been shown to potentiate the anti-cancer effects of several therapies by inducing apoptosis in various malignant cells without any detectable toxicity in cells or tissues. However, the mechanism by which noscapine induces apoptosis in colon cancer cells remains unclear. The signaling pathways by which noscapine induces apoptosis were investigated in colon cancer cell lines treated with various noscapine concentrations for 72 h, and a dose-dependent inhibition of cell viability was observed. Noscapine effectively inhibited the proliferation of LoVo cells in vitro (IC 50 = 75 μM). This cytotoxicity was reflected by cell cycle arrest at G 2 /M and subsequent apoptosis, as indicated by increased chromatin condensation and fragmentation, the upregulation of Bax and cytochrome c (Cyt-c), the downregulation of survivin and Bcl-2, and the activation of caspase-3 and caspase-9. Moreover, in a xenograft tumor model in mice, noscapine injection clearly inhibited tumor growth via the induction of apoptosis, which was demonstrated using a TUNEL assay. These results suggest that noscapine induces apoptosis in colon cancer cells via mitochondrial pathways. Noscapine may be a safe and effective chemotherapeutic agent for the treatment of human colon cancer.

  1. Apoptosis induction in human lymphocytes after in vitro exposure to cobalt/hard metal compounds

    International Nuclear Information System (INIS)

    Boeck, M. de; Decordier, I.; Lombaert, N.; Cundari, E.; Kirsch-Volders, M.; Lison, D.

    2001-01-01

    Full text: An increased risk of lung cancer is associated with occupational exposure to mixtures of cobalt metal (Co) and tungsten carbide (WC) particles, but apparently not when exposure is to cobalt alone. The mechanism for this increased cancer risk is not fully understood. The evaluation of the in vitro genotoxic effects in lymphocytes exposed to varying cobalt species demonstrated that the WC-Co hard metal mixture is more genotoxic (DNA damage, chromosome/genome mutations) than metallic Co alone. WC alone was not genotoxic. Thus, WC-Co represents a specific (geno)toxic entity. In order to assess the survival of human lymphocytes after in vitro exposure to metallic Co, CoCl 2 , WC and the WC-Co mixture, two apoptosis/necrosis detection methods were applied (annexin V staining and flow cytometry). Annexin-V staining of early apoptotic cells demonstrated a dose- and time dependent induction of apoptosis by metallic Co, CoCl 2 , WC and the WC-Co mixture. The time course of the process varied according to the metal species tested. Metallic Co and CoCl 2 caused a gradually increasing frequency of apoptotic cells with time (up to 24 h). WC-induced apoptosis displayed a typical 6 hour peak, which was not the case for the WC-Co mixture or for Co. Apoptosis induction by the WC-Co mixture was intermediate between that induced by Co and WC separately. Analysis of propidium iodide stained cells by flow cytometry was performed as a later marker for apoptosis induction. Preliminary data indicate similar tendencies of apoptosis induction as those detected by annexin-V. Identification of the apoptotic pathway triggered by the metal compounds was studied by inhibition of the ceramide-apoptosis pathway by fumonisin causing reduction of apoptosis induction for all compounds, but strongest after 6 hour exposure to WC. The use of specific caspase inhibitors will allow to further elucidate the different pathways involved. The current data demonstrating in vitro the apoptosis

  2. Apoptosis induction in human lymphocytes after in vitro exposure to cobalt/hard metal compounds

    Energy Technology Data Exchange (ETDEWEB)

    Boeck, M de; Decordier, I; Lombaert, N; Cundari, E; Kirsch-Volders, M [Vrije Universiteit Brussel, Laboratorium voor Cellulaire Genetica, Brussel (Belgium); Lison, D [Universite catholique de Louvain, Unite de Toxicologie industrielle et Medecine du Travail, Bruxelles (Belgium)

    2001-07-01

    Full text: An increased risk of lung cancer is associated with occupational exposure to mixtures of cobalt metal (Co) and tungsten carbide (WC) particles, but apparently not when exposure is to cobalt alone. The mechanism for this increased cancer risk is not fully understood. The evaluation of the in vitro genotoxic effects in lymphocytes exposed to varying cobalt species demonstrated that the WC-Co hard metal mixture is more genotoxic (DNA damage, chromosome/genome mutations) than metallic Co alone. WC alone was not genotoxic. Thus, WC-Co represents a specific (geno)toxic entity. In order to assess the survival of human lymphocytes after in vitro exposure to metallic Co, CoCl{sub 2}, WC and the WC-Co mixture, two apoptosis/necrosis detection methods were applied (annexin V staining and flow cytometry). Annexin-V staining of early apoptotic cells demonstrated a dose- and time dependent induction of apoptosis by metallic Co, CoCl{sub 2}, WC and the WC-Co mixture. The time course of the process varied according to the metal species tested. Metallic Co and CoCl{sub 2} caused a gradually increasing frequency of apoptotic cells with time (up to 24 h). WC-induced apoptosis displayed a typical 6 hour peak, which was not the case for the WC-Co mixture or for Co. Apoptosis induction by the WC-Co mixture was intermediate between that induced by Co and WC separately. Analysis of propidium iodide stained cells by flow cytometry was performed as a later marker for apoptosis induction. Preliminary data indicate similar tendencies of apoptosis induction as those detected by annexin-V. Identification of the apoptotic pathway triggered by the metal compounds was studied by inhibition of the ceramide-apoptosis pathway by fumonisin causing reduction of apoptosis induction for all compounds, but strongest after 6 hour exposure to WC. The use of specific caspase inhibitors will allow to further elucidate the different pathways involved. The current data demonstrating in vitro the

  3. Staphylococcus aureus resistance to human defensins and evasion of neutrophil killing via the novel virulence factor MprF is based on modification of membrane lipids with L-lysine

    NARCIS (Netherlands)

    Peschel, A.; Jack, R.W.; Otto, M.; Collins, L.V.; Staubitz, P.; Nicholson, G.; Kalbacher, H.; Nieuwenhuizen, W.F.; Jung, G.; Tarkowski, A.; Kessel, K.P.M. van; Strijp, J.A.G. van

    2001-01-01

    Defensins, antimicrobial peptides of the innate immune system, protect human mucosal epithelia and skin against microbial infections and are produced in large amounts by neutrophils. The bacterial pathogen Staphylococcus aureus is insensitive to defensins by virtue of an unknown resistance

  4. Effect of a 2.45-GHz radiofrequency electromagnetic field on neutrophil chemotaxis and phagocytosis in differentiated human HL-60 cells.

    Science.gov (United States)

    Koyama, Shin; Narita, Eijiro; Suzuki, Yoshihisa; Taki, Masao; Shinohara, Naoki; Miyakoshi, Junji

    2015-01-01

    The potential public health risks of radiofrequency (RF) fields have been discussed at length, especially with the use of mobile phones spreading extensively throughout the world. In order to investigate the properties of RF fields, we examined the effect of 2.45-GHz RF fields at the specific absorption rate (SAR) of 2 and 10 W/kg for 4 and 24 h on neutrophil chemotaxis and phagocytosis in differentiated human HL-60 cells. Neutrophil chemotaxis was not affected by RF-field exposure, and subsequent phagocytosis was not affected either compared with that under sham exposure conditions. These studies demonstrated an initial immune response in the human body exposed to 2.45-GHz RF fields at the SAR of 2 W/kg, which is the maximum value recommended by the International Commission for Non-Ionizing Radiation Protection (ICNIRP) guidelines. The results of our experiments for RF-field exposure at an SAR under 10 W/kg showed very little or no effects on either chemotaxis or phagocytosis in neutrophil-like human HL-60 cells. © The Author 2014. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

  5. Induction of factors of apoptosis in human tumor cells by low doses of radon

    International Nuclear Information System (INIS)

    Soto, J.; Martin, A.; Cos, S.; Gonzalez-Lamuno, D.

    1997-01-01

    The possibility of modification of genes related with apoptosis in tumor cells calls for a multidisciplinary experiment which describes the conditions and characteristics of such modification. In this work low radiation doses from radon were used in the irradiation of tumor cells of human mammary glands. After irradiation, the cells incubate for three days, after which they are counted and a total extraction of ARN is effected. Through bimolecular techniques, inverse transcription and polymerase chain reaction, the expression of genes involved in apoptosis is studied. The results found indicate that, in the cell line denominated MCF-7, the genes bcl-2, bcl-xL and bax are expressed. In the irradiated cells, the levels of expression of bcl x increase with respect to the control and induce the expression of the form bcl-xS, the protein of which induces apoptosis

  6. Induction of apoptosis by epigallocatechin-3-gallate in human lymphoblastoid B cells

    International Nuclear Information System (INIS)

    Noda, Chiseko; He, Jinsong; Takano, Tomoko; Tanaka, Chisato; Kondo, Toshinori; Tohyama, Kaoru; Yamamura, Hirohei; Tohyama, Yumi

    2007-01-01

    (-)-Epigallocatechin-3-gallate (EGCG), a major constituent of green tea polyphenols, has been shown to suppress cancer cell proliferation and induce apoptosis. In this study we investigated its efficacy and the mechanism underlying its effect using human B lymphoblastoid cell line Ramos, and effect of co-treatment with EGCG and a chemotherapeutic agent on apoptotic cell death. EGCG induced dose- and time-dependent apoptotic cell death accompanied by loss of mitochondrial transmembrane potential, release of cytochrome c into the cytosol, and cleavage of pro-caspase-9 to its active form. EGCG also enhanced production of intracellular reactive oxygen species (ROS). Pretreatment with diphenylene iodonium chloride, an inhibitor of NAD(P)H oxidase and an antioxidant, partially suppressed both EGCG-induced apoptosis and production of ROS, implying that oxidative stress is involved in the apoptotic response. Furthermore, we showed that combined-treatment with EGCG and a chemotherapeutic agent, etoposide, synergistically induced apoptosis in Ramos cells

  7. Kaempferol Promotes Apoptosis in Human Bladder Cancer Cells by Inducing the Tumor Suppressor, PTEN

    Directory of Open Access Journals (Sweden)

    Liqun Zhou

    2013-10-01

    Full Text Available Kaempferol (Kae, a natural flavonoid, is widely distributed in fruits and vegetables. Previous studies have identified Kae as a possible cancer preventive and therapeutic agent. We found Kae to exhibit potent antiproliferation and anti-migration effects in human bladder cancer EJ cells. Kaempferol robustly induced apoptosis in EJ cells in a dose-dependent manner, as evidenced by increased cleavage of caspase-3. Furthermore, we found Kae-induced apoptosis in EJ cells to be associated with phosphatase and the tensin homolog deleted on the chromosome 10 (PTEN/PI3K/Akt pathway. Kae significantly increased PTEN and decreased Akt phosphorylation. Kae-induced apoptosis was partially attenuated in PTEN-knockdown cells. Our findings indicate that Kae could be an alternative medicine for bladder cancer, based on a PTEN activation mechanism.

  8. Candida albicans escapes from mouse neutrophils

    DEFF Research Database (Denmark)

    Ermert, David; Niemiec, Maria J; Röhm, Marc

    2013-01-01

    is the most widely used model organism. Neutrophils are essential immune cells to prevent opportunistic mycoses. To explore potential differences between the rodent infection model and the human host, we compared the interactions of C. albicans with neutrophil granulocytes from mice and humans. We revealed...

  9. Apoptosis and radiosensitivity induced by N-acety1 phytosphingosine, in human cancer cell line

    International Nuclear Information System (INIS)

    Kim, Y. H.; Kim, K. S.; Han, Y. S.; Jeon, S. J.; Song, J. Y.; Jung, I. S.; Hong, S. H.; Yun, Y. S.; Park, J. S.

    2004-01-01

    Ceramide is a key lipid molecule in signal transduction with a role in various regulatory pathways including differentiation, proliferation and especially apoptosis. Ionizing radiation-induced apoptosis is associated with accumulation of ceramide, and the sphingomyelinase deficiency results in radioresistance. We investigated the exogenous treatment of N-acetyl-phytosphingosine (NAPS), an analogue of N-acetyl-sphingosine (C 2 -Ceramide), and C 2 -ceramide exert apoptotic effect on human T cell lymphoma Jurkat cells and breast cancer cell line MDA-MB-231. NAPS and C 2 -Ceramide has cytotoxic effect in time- and dose-dependent manner, and increased caspase-3, 8 activity. However, NAPS induced apoptosis more effectively, and increased caspase activity induced by NAPS is more higher than C 2 -ceramide. Moreover, NAPS decreased clonogenicity of irradiated cells and increased radiation-induced apoptosis significantly. Increased cell death by irradiation in the presence of NAPS is owing to the increase of caspase activity. These data suggest that NAPS might be used for lead as a new type of radiosensitizing agent increasing radiation-induced apoptosis

  10. Pathway of 3-MCPD-induced apoptosis in human embryonic kidney cells.

    Science.gov (United States)

    Ji, Jian; Zhu, Pei; Sun, Chao; Sun, Jiadi; An, Lu; Zhang, Yinzhi; Sun, Xiulan

    2017-01-01

    3-Chloropropane-1,2-diol (3-MCPD) is a heat-produced contaminant formed during the preparation of soy sauce worldwide. The present investigation was conducted to determine the molecular aspects of 3-MCPD toxicity on human embryonic kidney cells (HEK293). Cell viability and apoptosis were assessed in response to exposure to 3-MCPD using the MTT assay and high-content screening (HCS). DNA damage, intracellular reactive oxygen species (ROS) and apoptosis-related proteins were evaluated. Genes related with apoptosis were detected by qPCR-array for further understanding the 3-MCPD induced cell apoptosis signaling pathway. Our results clearly showed that 3-MCPD treatment inhibits cell proliferation and reactive oxygen species generation. qPCR-array indicated that nine apoptotic genes were up-regulated more than 2-fold and six down-regulated more than 2-fold. Genes associated with the mitochondrial apoptotic pathway, especially BCL2 family genes, changed significantly, indicating that the mitochondrial apoptotic pathway is activated. Death receptor pathway-related genes, TNFRSF11B and TNFRSF1A, changed significantly, indicating that the death receptor pathway is also activated, resulting in the inhibition of cell growth and proliferation as well as induction of apoptosis. To sum up, the experiment results indicated that 3-MCPD induced HEK293 cell toxicity through the death receptor pathway and mitochondrial pathway.

  11. DNA fragmentation and apoptosis induced by safranal in human prostate cancer cell line

    Directory of Open Access Journals (Sweden)

    Saeed Samarghandian

    2013-01-01

    Full Text Available Objectives: Apoptosis, an important mechanism that contributes to cell growth reduction, is reported to be induced by Crocus sativus (Saffron in different cancer types. However, limited effort has been made to correlate these effects to the active ingredients of saffron. The present study was designed to elucidate cytotoxic and apoptosis induction by safranal, the major coloring compound in saffron, in a human prostate cancer cell line (PC-3. Materials and Methods: PC-3 and human fetal lung fibroblast (MRC-5 cells were cultured and exposed to safranal (5, 10, 15, and 20 μg/ml. The 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay was performed to assess cytotoxicity. DNA fragmentation was assessed by gel electrophoresis. Cells were incubated with different concentrations of safranal, and cell morphologic changes and apoptosis were determined by the normal inverted microscope, Annexin V, and propidium iodide, followed by flow cytometric analysis, respectively. Results: MTT assay revealed a remarkable and concentration-dependent cytotoxic effect of safranal on PC-3 cells in comparison with non-malignant cell line. The morphologic alterations of the cells confirmed the MTT results. The IC 50 values against PC-3 cells were found to be 13.0 ΁ 0.07 and 6.4 ΁ 0.09 μg/ml at 48 and 72 h, respectively. Safranal induced an early and late apoptosis in the flow cytometry histogram of treated cells, indicating apoptosis is involved in this toxicity. DNA analysis revealed typical ladders as early as 48 and 72 h after treatment, indicative of apoptosis. Conclusions: Our preclinical study demonstrated a prostate cancer cell line to be highly sensitive to safranal-mediated growth inhibition and apoptotic cell death. Although the molecular mechanisms of safranal action are not clearly understood, it appears to have potential as a therapeutic agent.

  12. BAK overexpression mediates p53-independent apoptosis inducing effects on human gastric cancer cells

    Directory of Open Access Journals (Sweden)

    Liu Jun

    2004-07-01

    Full Text Available Abstract Background BAK (Bcl-2 homologous antagonist/killer is a novel pro-apoptotic gene of the Bcl-2 family. It has been reported that gastric tumors have reduced BAK levels when compared with the normal mucosa. Moreover, mutations of the BAK gene have been identified in human gastrointestinal cancers, suggesting that a perturbation of BAK-mediated apoptosis may contribute to the pathogenesis of gastric cancer. In this study, we explored the therapeutic effects of gene transfer mediated elevations in BAK expression on human gastric cancer cells in vitro. Methods Eukaryotic expression vector for the BAK gene was constructed and transferred into gastric cancer cell lines, MKN-45 (wild-type p53 and MKN-28 (mutant-type p53. RT-PCR and Western Blotting detected cellular BAK gene expression. Cell growth activities were detected by MTT colorimetry and flow cytometry, while apoptosis was assayed by electronic microscopy and TUNEL. Western Blotting and colorimetry investigated cellular caspase-3 activities. Results BAK gene transfer could result in significant BAK overexpression, decreased in vitro growth, cell cycle G0/G1 arrest, and induced apoptosis in gastric cancer cells. In transferred cells, inactive caspase-3 precursor was cleaved into the active subunits p20 and p17, during BAK overexpression-induced apoptosis. In addition, this process occurred equally well in p53 wild-type (MKN-45, or in p53 mutant-type (MKN-28 gastric cancer cells. Conclusions The data presented suggests that overexpression of the BAK gene can lead to apoptosis of gastric cancer cells in vitro, which does not appear to be dependent on p53 status. The action mechanism of BAK mediated apoptosis correlates with activation of caspase-3. This could be served as a potential strategy for further development of gastric cancer therapies.

  13. Autophagy inhibition enhances apigenin-induced apoptosis in human breast cancer cells

    Institute of Scientific and Technical Information of China (English)

    Xuchen Cao; Bowen Liu; Wenfeng Cao; Weiran Zhang; Fei Zhang; Hongmeng Zhao; Ran Meng

    2013-01-01

    Apigenin (4',5,7-trihydroxyflavone) is a member of the flavone subclass of flavonoids present in fruits and vegetables.The involvement of autophagy in the apigenin-induced apoptotic death of human breast cancer cells was investigated.Cell proliferation and viability were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and clonogenic assays.Flow cytometry,fluorescent staining and Western blot analysis were employed to detect apoptosis and autophagy,and the role of autophagy was assessed using autophagy inhibitors.Apigenin dose-and time-dependently repressed the proliferation and clonogenic survival of the human breast cancer T47D and MDA-MB-231 cell lines.The death of T47D and MDA-MB-231 cells was due to apoptosis associated with increased levels of Caspase3,PARP cleavage and Bax/Bcl-2 ratios.The results from flow cytometry and fluorescent staining also verified the occurrence of apoptosis.In addition,the apigenin-treated cells exhibited autophagy,as characterized by the appearance of autophagosomes under fluorescence microscopy and the accumulation of acidic vesicular organelles (AVOs)by flow cytometry.Furthermore,the results of the Western blot analysis revealed that the level of LC3-Ⅱ,the processed form of LC3-Ⅰ,was increased.Treatment with the autophagy inhibitor,3-methyladenine (3-MA),significantly enhanced the apoptosis induced by apigenin,which was accompanied by an increase in the level of PARP cleavage.Similar results were also confirmed by flow cytometry and fluorescence microscopy.These results indicate that apigenin has apoptosis-and autophagy-inducing effects in breast cancer cells.Autophagy plays a cyto-protective role in apigenin-induced apoptosis,and the combination of apigenin and an autophagy inhibitor may be a promising strategy for breast cancer control.

  14. BAK overexpression mediates p53-independent apoptosis inducing effects on human gastric cancer cells

    International Nuclear Information System (INIS)

    Tong, Qiang-Song; Zheng, Li-Duan; Wang, Liang; Liu, Jun; Qian, Wei

    2004-01-01

    BAK (Bcl-2 homologous antagonist/killer) is a novel pro-apoptotic gene of the Bcl-2 family. It has been reported that gastric tumors have reduced BAK levels when compared with the normal mucosa. Moreover, mutations of the BAK gene have been identified in human gastrointestinal cancers, suggesting that a perturbation of BAK-mediated apoptosis may contribute to the pathogenesis of gastric cancer. In this study, we explored the therapeutic effects of gene transfer mediated elevations in BAK expression on human gastric cancer cells in vitro. Eukaryotic expression vector for the BAK gene was constructed and transferred into gastric cancer cell lines, MKN-45 (wild-type p53) and MKN-28 (mutant-type p53). RT-PCR and Western Blotting detected cellular BAK gene expression. Cell growth activities were detected by MTT colorimetry and flow cytometry, while apoptosis was assayed by electronic microscopy and TUNEL. Western Blotting and colorimetry investigated cellular caspase-3 activities. BAK gene transfer could result in significant BAK overexpression, decreased in vitro growth, cell cycle G 0 /G 1 arrest, and induced apoptosis in gastric cancer cells. In transferred cells, inactive caspase-3 precursor was cleaved into the active subunits p20 and p17, during BAK overexpression-induced apoptosis. In addition, this process occurred equally well in p53 wild-type (MKN-45), or in p53 mutant-type (MKN-28) gastric cancer cells. The data presented suggests that overexpression of the BAK gene can lead to apoptosis of gastric cancer cells in vitro, which does not appear to be dependent on p53 status. The action mechanism of BAK mediated apoptosis correlates with activation of caspase-3. This could be served as a potential strategy for further development of gastric cancer therapies

  15. Transmigration of polymorphnuclear neutrophils and monocytes through the human blood-cerebrospinal fluid barrier after bacterial infection in vitro.

    Science.gov (United States)

    Steinmann, Ulrike; Borkowski, Julia; Wolburg, Hartwig; Schröppel, Birgit; Findeisen, Peter; Weiss, Christel; Ishikawa, Hiroshi; Schwerk, Christian; Schroten, Horst; Tenenbaum, Tobias

    2013-02-28

    Bacterial invasion through the blood-cerebrospinal fluid barrier (BCSFB) during bacterial meningitis causes secretion of proinflammatory cytokines/chemokines followed by the recruitment of leukocytes into the CNS. In this study, we analyzed the cellular and molecular mechanisms of polymorphonuclear neutrophil (PMN) and monocyte transepithelial transmigration (TM) across the BCSFB after bacterial infection. Using an inverted transwell filter system of human choroid plexus papilloma cells (HIBCPP), we studied leukocyte TM rates, the migration route by immunofluorescence, transmission electron microscopy and focused ion beam/scanning electron microscopy, the secretion of cytokines/chemokines by cytokine bead array and posttranslational modification of the signal regulatory protein (SIRP) α via western blot. PMNs showed a significantly increased TM across HIBCPP after infection with wild-type Neisseria meningitidis (MC58). In contrast, a significantly decreased monocyte transmigration rate after bacterial infection of HIBCPP could be observed. Interestingly, in co-culture experiments with PMNs and monocytes, TM of monocytes was significantly enhanced. Analysis of paracellular permeability and transepithelial electrical resistance confirmed an intact barrier function during leukocyte TM. With the help of the different imaging techniques we could provide evidence for para- as well as for transcellular migrating leukocytes. Further analysis of secreted cytokines/chemokines showed a distinct pattern after stimulation and transmigration of PMNs and monocytes. Moreover, the transmembrane glycoprotein SIRPα was deglycosylated in monocytes, but not in PMNs, after bacterial infection. Our findings demonstrate that PMNs and monoctyes differentially migrate in a human BCSFB model after bacterial infection. Cytokines and chemokines as well as transmembrane proteins such as SIRPα may be involved in this process.

  16. Postprandial triglyceride-rich lipoproteins promote lipid accumulation and apolipoprotein B-48 receptor transcriptional activity in human circulating and murine bone marrow neutrophils in a fatty acid-dependent manner.

    Science.gov (United States)

    Ortega-Gómez, Almudena; Varela, Lourdes M; López, Sergio; Montserrat de la Paz, Sergio; Sánchez, Rosario; Muriana, Francisco J G; Bermúdez, Beatriz; Abia, Rocío

    2017-09-01

    Postprandial triglyceride-rich lipoproteins (TRLs) promote atherosclerosis. Recent research points the bone marrow (BM) as a primary site in atherosclerosis. We elucidated how the acute administration of monounsaturated fatty acids (MUFAs) MUFAs, omega-3 polyunsaturated fatty acids (PUFAs) PUFAs and saturated fatty acids (SFAs) affects human circulating and murine BM neutrophil lipid accumulation and functionality. Postprandial hypertriglyceridemia was induced in healthy subjects and Apoe -/- mice by the acute administration of dietary fats enriched in MUFAs, PUFAs, or SFAs. Postprandial hypertriglyceridemia increased apolipoprotein-B48 receptor (ApoB48R) transcriptional activity that was linearly correlated with intracellular triglycerides (TGs) TGs accumulation in human circulating and murine BM neutrophils. MUFA and omega-3 PUFAs attenuated ApoB48R gene expression and intracellular TG accumulation compared to SFAs. TRLs induced apoB48R-dependent TG accumulation in human neutrophils ex vivo. Murine BM neutrophils showed a decrease in surface L-selectin and an increase in TNF-α and IL-1β mRNA expressions only after SFAs administration. TRLs enriched in SFAs induced BM neutrophil degranulation ex vivo suggesting cell priming/activation. Postprandial TRLs disrupts the normal biology and function of circulating and BM neutrophils. MUFA- and omega-3 PUFA-rich dietary fats such as virgin olive oil or fish oil has the potential to prevent excessive neutrophil lipid accumulation and activation by targeting the fatty acid composition of TRLs. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. PRAF3 induces apoptosis and inhibits migration and invasion in human esophageal squamous cell carcinoma

    International Nuclear Information System (INIS)

    Shi, Guo-Zhen; Yuan, Yang; Jiang, Guo-Jun; Ge, Zhi-Jun; Zhou, Jian; Gong, De-Jun; Tao, Jing; Tan, Yong-Fei; Huang, Sheng-Dong

    2012-01-01

    Prenylated Rab acceptor 1 domain family member 3 (PRAF3) is involved in the regulation of many cellular processes including apoptosis, migration and invasion. This study was conducted to investigate the effect of PRAF3 on apoptosis, migration and invasion in human esophageal squamous cell carcinoma (ESCC). The expression of PRAF3 mRNA and protein in primary ESCC and the matched normal tissues (57cases) was determined by quantitative RT-PCR and Western blot. Immunohistochemical analysis of PRAF3 expression was carried out in paraffin-embedded sections of ESCC and correlated with clinical features. The role of PRAF3 in apoptosis, migration and invasion was studied in ESCC cell lines of Eca109 and TE-1 through the adenovirus mediated PRAF3 gene transfer. The effect of PRAF3 on apoptosis was analyzed by annexin V-FITC assay. The regulation of PRAF3 on migration was determined by transwell and wounding healing assay, while the cellular invasion was analyzed by matrigel-coated transwell assay. We found that the expression of PRAF3 was significantly down-regulated in ESCC tissue compared with the matched normal tissue and was correlated with the clinical features of pathological grade, tumor stage and lymph node metastasis. Moreover, overexpression of PRAF3 induced cell apoptosis through both caspase-8 and caspase-9 dependent pathways, and inhibited cell migration and invasion by suppressing the activity of both MMP-2 and MMP-9 in human ESCC cell lines. Our data suggest that PRAF3 plays an important role in the regulation of tumor progression and metastasis and serves as a tumor suppressor in human ESCC. We propose that PRAF3 might be used as a potential therapeutic agent for human ESCC

  18. The cystic fibrosis neutrophil: a specialized yet potentially defective cell.

    LENUS (Irish Health Repository)

    Hayes, Elaine

    2012-02-01

    Cystic fibrosis (CF) is one of the commonest genetically inherited diseases in the world. It is characterized by recurrent respiratory tract infections eventually leading to respiratory failure. One of the hallmarks of this disease is a persistent and predominantly neutrophil driven inflammation. Neutrophils provide the first line of defence by killing and digesting phagocytosed bacteria and fungi, yet despite advances in our understanding of the molecular and cellular basis of CF, there remains a paradox of why recruited CF neutrophils fail to eradicate bacterial infections in the lung. This review describes mechanisms involved in neutrophil migration, microbial killing and apoptosis leading to inflammatory resolution. We discuss dysregulated neutrophil activity and consider genetic versus inflammatory neutrophil reprogramming in CF and ultimately pharmacological modulation of the CF neutrophil for therapeutic intervention.

  19. The cystic fibrosis neutrophil: a specialized yet potentially defective cell.

    LENUS (Irish Health Repository)

    Hayes, Elaine

    2011-04-01

    Cystic fibrosis (CF) is one of the commonest genetically inherited diseases in the world. It is characterized by recurrent respiratory tract infections eventually leading to respiratory failure. One of the hallmarks of this disease is a persistent and predominantly neutrophil driven inflammation. Neutrophils provide the first line of defence by killing and digesting phagocytosed bacteria and fungi, yet despite advances in our understanding of the molecular and cellular basis of CF, there remains a paradox of why recruited CF neutrophils fail to eradicate bacterial infections in the lung. This review describes mechanisms involved in neutrophil migration, microbial killing and apoptosis leading to inflammatory resolution. We discuss dysregulated neutrophil activity and consider genetic versus inflammatory neutrophil reprogramming in CF and ultimately pharmacological modulation of the CF neutrophil for therapeutic intervention.

  20. Rel/Nuclear factor-kappa B apoptosis pathways in human cervical cancer cells

    Science.gov (United States)

    Shehata, Marlene F

    2005-01-01

    Cervical cancer is considered a common yet preventable cause of death in women. It has been estimated that about 420 women out of the 1400 women diagnosed with cervical cancer will die during 5 years from diagnosis. This review addresses the pathogenesis of cervical cancer in humans with a special emphasis on the human papilloma virus as a predominant cause of cervical cancer in humans. The current understanding of apoptosis and regulators of apoptosis as well as their implication in carcinogenesis will follow. A special focus will be given to the role of Rel/NF-κB family of genes in the growth and chemotherapeutic treatment of the malignant HeLa cervical cells emphasizing on Xrel3, a cRel homologue. PMID:15857509

  1. Unsaturated long-chain fatty acids induce the respiratory burst of human neutrophils and monocytes in whole blood

    Directory of Open Access Journals (Sweden)

    Osthaus Wilhelm A

    2008-07-01

    Full Text Available Abstract Background It is increasingly recognized that infectious complications in patients treated with total parenteral nutrition (TPN may be caused by altered immune responses. Neutrophils and monocytes are the first line of defence against bacterial and fungal infection through superoxide anion production during the respiratory burst. To characterize the impact of three different types of lipid solutions that are applied as part of TPN formulations, we investigated the unstimulated respiratory burst activation of neutrophils and monocytes in whole blood. Methods Whole blood samples were incubated with LCT (Intralipid®, LCT/MCT (Lipofundin® and LCT-MUFA (ClinOleic® in three concentrations (0.06, 0.3 and 0.6 mg ml-1 for time periods up to one hour. Hydrogen peroxide production during the respiratory burst of neutrophils and monocytes was measured by flow cytometry. Results LCT and LCT-MUFA induced a hydrogen peroxide production in neutrophils and monocytes without presence of a physiological stimulus in contrast to LCT/MCT. Conclusion We concluded that parenteral nutrition containing unsaturated oleic (C18:1 and linoleic (C18:2 acid can induce respiratory burst of neutrophils and monocytes, resulting in an elevated risk of tissue damage by the uncontrolled production of reactive oxygen species. Contradictory observations reported in previous studies may in part be the result of different methods used to determine hydrogen peroxide production.

  2. Conjugated linoleic acid induces apoptosis through estrogen receptor alpha in human breast tissue

    Directory of Open Access Journals (Sweden)

    Liu Suling

    2008-07-01

    Full Text Available Abstract Background Conjugated linoleic acid (CLA, a naturally occurring fatty acid found in ruminant products such as milk and beef, has been shown to possess anti-cancer activities in in vivo animal models and in vitro cell culture systems. In human breast cancer, the overall duration of estrogen exposure is the most important risk factor for developing estrogen-responsive breast cancer. Accordingly, it has been suggested that estrogen exposure reduces apoptosis through the up-regulation of the anti-apoptosis protein, Bcl-2. Bcl-2, an anti-apoptotic protein, regulates apoptosis and plays a crucial role in the development and growth regulation of normal and cancerous cells. Our research interest is to examine the effects of CLA on the induction of apoptosis in human breast tissues. Methods The localization of Bcl-2 in both normal and cancerous human breast tissues was determined by immunohistochemical staining and the Bcl-2 protein expression was tested by western blot analysis. Co-culture of epithelial cells and stromal cells was carried out in the presence or absence of CLA to evaluate apoptosis in the context of a cell-cell interaction. Results The results showed that both normal and cancerous breast tissues were positive for Bcl-2 staining, which was higher overall in mammary ducts but very low in the surrounding stromal compartment. Interestingly, by quantifying the western blot data, basal Bcl-2 protein levels were higher in normal breast epithelial cells than in cancerous epithelial cells. Furthermore, treatment with 17β-estradiol (E2 stimulated growth and up-regulated Bcl-2 expression in estrogen responsive breast epithelial cells; however, these carcinogenic effects were diminished by either CLA or 4-Hydroxytamoxifen (Tam and were suppressed further by the combination of CLA and Tam. In both one cell type cultured and co-culture systems, CLA induced cell apoptosis in ERα transfected MDA-MB-231 cells but not in the wild type MDA

  3. Conjugated linoleic acid induces apoptosis through estrogen receptor alpha in human breast tissue

    International Nuclear Information System (INIS)

    Wang, Li-Shu; Huang, Yi-Wen; Liu, Suling; Yan, Pearlly; Lin, Young C

    2008-01-01

    Conjugated linoleic acid (CLA), a naturally occurring fatty acid found in ruminant products such as milk and beef, has been shown to possess anti-cancer activities in in vivo animal models and in vitro cell culture systems. In human breast cancer, the overall duration of estrogen exposure is the most important risk factor for developing estrogen-responsive breast cancer. Accordingly, it has been suggested that estrogen exposure reduces apoptosis through the up-regulation of the anti-apoptosis protein, Bcl-2. Bcl-2, an anti-apoptotic protein, regulates apoptosis and plays a crucial role in the development and growth regulation of normal and cancerous cells. Our research interest is to examine the effects of CLA on the induction of apoptosis in human breast tissues. The localization of Bcl-2 in both normal and cancerous human breast tissues was determined by immunohistochemical staining and the Bcl-2 protein expression was tested by western blot analysis. Co-culture of epithelial cells and stromal cells was carried out in the presence or absence of CLA to evaluate apoptosis in the context of a cell-cell interaction. The results showed that both normal and cancerous breast tissues were positive for Bcl-2 staining, which was higher overall in mammary ducts but very low in the surrounding stromal compartment. Interestingly, by quantifying the western blot data, basal Bcl-2 protein levels were higher in normal breast epithelial cells than in cancerous epithelial cells. Furthermore, treatment with 17β-estradiol (E 2 ) stimulated growth and up-regulated Bcl-2 expression in estrogen responsive breast epithelial cells; however, these carcinogenic effects were diminished by either CLA or 4-Hydroxytamoxifen (Tam) and were suppressed further by the combination of CLA and Tam. In both one cell type cultured and co-culture systems, CLA induced cell apoptosis in ERα transfected MDA-MB-231 cells but not in the wild type MDA-MB-231 cells. These data, therefore, demonstrate that

  4. Effect of Evodiamine on Inducing Apoptosis of Human Gastric Cancer Cell Line SGC-7901 in Vitro

    Directory of Open Access Journals (Sweden)

    LIU Shao-ping

    2014-09-01

    Full Text Available Objective: To explore the proliferation inhibition and apoptosis-inducing effect of evodiamine in human gastric cancer SGC-7901 cells. Methods: After 48 or 24 h exposure to different concentrations of evodiamine, cell proliferation was analyzed using tetrazolium blue (MTT assay while apoptosis and cell-cycle phase distribution using flow cytometry. Results: In 0.01~30.00 μg/mL range of concentrations, evodiamine inhibited the proliferation of SGC-7901 cells in dose-dependent manner, and the overall mean IC50 was (3.79±0.16 μg/mL; the apoptosis rate was increased from 3.4% to 7.0%, 13.8% and 36.3% at concentrations of 0, 0.5, 1.5 and 30 μg/mL of evodiamine, respectively; the percentage of cells accumulated in G2/M phase was increased from 17.26% to 98.92% in the cells treated with evodiamine for 24 h in 0.01~30.00 μg/mL range of concentrations. Conclusion: Evodiamine can inhibit the proliferation, induce apoptosis in human gastric cancer cell line SGC-7901 in vitro and arrest the cell cycle at the G2/M phase.

  5. In vivo induction of apoptosis in human lymphocytes by therapeutic fractionated total body irradiation

    International Nuclear Information System (INIS)

    Delic, J.; Magdelenat, H.; Barbaroux, C.; Chaillet, M.-P.; Dubray, B.; Fourquet, A.; Cosset, J.-M.; Gluckman, E.; Girinsky, T.

    1995-01-01

    Ionizing radiations have been reported as an in vitro apoptosis initiating stimulus in human lymphocytes. As the cytotoxicity of ionizing radiations and chemotherapeutic agents appears to be dependent on the efficacy of cell death induction, the manipulation of apoptosis initiation might be used as a means to suppress some pathological process. In the present study the in vivo induction of γ-ray mediated programmed cell death in humans is reported. The in vivo induction of apoptosis in peripheral blood lymphocytes (PBL) by ionizing radiations was investigated in 33 patients after each of two sessions (2 Gy and 4 Gy) of fractionated total body irradiation (FTBI) as part of their conditioning regimen before bone marrow transplantation. PBL committed to apoptosis were scored before irradiation (S1), 4 h (S2) and 24 h after 2 Gy (S3, 14-17 h after the second 2 Gy fraction). Nuclear morphology and chromatin-DNA were analysed by fluorescence microscopy immediately after blood sample withdrawal (I) and after 24 h in cell culture medium (II). (author)

  6. Expression of Bcl-2 family proteins and spontaneous apoptosis in normal human testis.

    Science.gov (United States)

    Oldereid, N B; Angelis, P D; Wiger, R; Clausen, O P

    2001-05-01

    We investigated the frequency of spontaneous apoptosis and expression of the Bcl-2 family of proteins during normal spermatogenesis in man. Testicular tissue with both normal morphology and DNA content was obtained from necro-donors and fixed in Bouin's solution. A TdT-mediated dUTP end-labelling method (TUNEL) was used for the detection of apoptotic cells. Expression of apoptosis regulatory Bcl-2 family proteins and of p53 and p21(Waf1) was assessed by immunohistochemistry. Germ cell apoptosis was detected in all testes and was mainly seen in primary spermatocytes and spermatids and in a few spermatogonia. Bcl-2 and Bak were preferentially expressed in the compartments of spermatocytes and differentiating spermatids, while Bcl-x was preferentially expressed in spermatogonia. Bax showed a preferential expression in nuclei of round spermatids, whereas Bad was only seen in the acrosome region of various stages of spermatids. Mcl-1 staining was weak without a particular pattern, whereas expression of Bcl-w, p53 and p21(Waf1) proteins was not detected by immunohistochemistry. The results show that spontaneous apoptosis occurs in all male germ cell compartments in humans. Bcl-2 family proteins are distributed preferentially within distinct germ cell compartments suggesting a specific role for these proteins in the processes of differentiation and maturation during human spermatogenesis.

  7. Acetyl-CoA Carboxylase-α Inhibitor TOFA Induces Human Cancer Cell Apoptosis

    Science.gov (United States)

    Wang, Chun; Xu, Canxin; Sun, Mingwei; Luo, Dixian; Liao, Duan-fang; Cao, Deliang

    2009-01-01

    Acetyl-CoA carboxylase-α (ACCA) is a rate-limiting enzyme in long chain fatty acid synthesis, playing a critical role in cellular energy storage and lipid synthesis. ACCA is upregulated in multiple types of human cancers and small interfering RNA-mediated ACCA silencing in human breast and prostate cancer cells results in oxidative stress and apoptosis. This study reports for the first time that TOFA (5-tetradecyloxy-2-furoic acid), an allosteric inhibitor of ACCA, is cytotoxic to lung cancer cells NCI-H460 and colon carcinoma cells HCT-8 and HCT-15, with an IC50 at approximately 5.0, 5.0, and 4.5 μg/ml, respectively. TOFA at 1.0–20.0 μg/ml effectively blocked fatty acid synthesis and induced cell death in a dose-dependent manner. The cell death was characterized with PARP cleavage, DNA fragmentation, and annexin-V staining, all of which are the features of the apoptosis. Supplementing simultaneously the cells with palmitic acids (100 μM), the end-products of the fatty acid synthesis pathway, prevented the apoptosis induced by TOFA. Taken together, these data suggest that TOFA is a potent cytotoxic agent to lung and colon cancer cells, inducing apoptosis through disturbing their fatty acid synthesis. PMID:19450551

  8. Acetyl-CoA carboxylase-alpha inhibitor TOFA induces human cancer cell apoptosis.

    Science.gov (United States)

    Wang, Chun; Xu, Canxin; Sun, Mingwei; Luo, Dixian; Liao, Duan-Fang; Cao, Deliang

    2009-07-31

    Acetyl-CoA carboxylase-alpha (ACCA) is a rate-limiting enzyme in long chain fatty acid synthesis, playing a critical role in cellular energy storage and lipid synthesis. ACCA is upregulated in multiple types of human cancers and small interfering RNA-mediated ACCA silencing in human breast and prostate cancer cells results in oxidative stress and apoptosis. This study reports for the first time that TOFA (5-tetradecyloxy-2-furoic acid), an allosteric inhibitor of ACCA, is cytotoxic to lung cancer cells NCI-H460 and colon carcinoma cells HCT-8 and HCT-15, with an IC(50) at approximately 5.0, 5.0, and 4.5 microg/ml, respectively. TOFA at 1.0-20.0 microg/ml effectively blocked fatty acid synthesis and induced cell death in a dose-dependent manner. The cell death was characterized with PARP cleavage, DNA fragmentation, and annexin-V staining, all of which are the features of the apoptosis. Supplementing simultaneously the cells with palmitic acids (100 microM), the end-products of the fatty acid synthesis pathway, prevented the apoptosis induced by TOFA. Taken together, these data suggest that TOFA is a potent cytotoxic agent to lung and colon cancer cells, inducing apoptosis through disturbing their fatty acid synthesis.

  9. Characterization of the response chemiluminescence of neutrophils human beings to the hemolysin Escherichia coli alpha

    International Nuclear Information System (INIS)

    Garcia, J.

    2000-01-01

    Escherichia coli alpha hemolysin (AH) evoked a luminol-amplified chemiluminescence (CL) response from human polymorphonuclear leukocytes (PMN). Analysis of kinetic parameters of the PMN CL response to AH established similarities with that of PMN to the calcium ionophore A23187. PMN CL responses to both AH and A23187 were equally decreased by preincubating PMN with A63612, a hidroxamic acid derivative and lipooxigenase inhibitor, showing that the CL response to both hemolysin and ionophore share a common mechanism, probably activation of leukotriene synthesis, due to calcium entry into the cells brought about by AH and A23187. In addition, the CL response of PMN to AH was lowered by the hydroxyl radical scavenger dimethyl sulfoxide, further suggesting arachidonate metabolism is involved in CL response. (Author) [es

  10. Knockdown of HSPA9 induces TP53-dependent apoptosis in human hematopoietic progenitor cells.

    Directory of Open Access Journals (Sweden)

    Tuoen Liu

    Full Text Available Myelodysplastic syndromes (MDS are the most common adult myeloid blood cancers in the US. Patients have increased apoptosis in their bone marrow cells leading to low peripheral blood counts. The full complement of gene mutations that contribute to increased apoptosis in MDS remains unknown. Up to 25% of MDS patients harbor and acquired interstitial deletion on the long arm of chromosome 5 [del(5q], creating haploinsufficiency for a large set of genes including HSPA9. Knockdown of HSPA9 in primary human CD34+ hematopoietic progenitor cells significantly inhibits growth and increases apoptosis. We show here that HSPA9 knockdown is associated with increased TP53 expression and activity, resulting in increased expression of target genes BAX and p21. HSPA9 protein interacts with TP53 in CD34+ cells and knockdown of HSPA9 increases nuclear TP53 levels, providing a possible mechanism for regulation of TP53 by HSPA9 haploinsufficiency in hematopoietic cells. Concurrent knockdown of TP53 and HSPA9 rescued the increased apoptosis observed in CD34+ cells following knockdown of HSPA9. Reduction of HSPA9 below 50% results in severe inhibition of cell growth, suggesting that del(5q cells may be preferentially sensitive to further reductions of HSPA9 below 50%, thus providing a genetic vulnerability to del(5q cells. Treatment of bone marrow cells with MKT-077, an HSPA9 inhibitor, induced apoptosis in a higher percentage of cells from MDS patients with del(5q compared to non-del(5q MDS patients and normal donor cells. Collectively, these findings indicate that reduced levels of HSPA9 may contribute to TP53 activation and increased apoptosis observed in del(5q-associated MDS.

  11. Human-gyrovirus-Apoptin triggers mitochondrial death pathway--Nur77 is required for apoptosis triggering.

    Science.gov (United States)

    Chaabane, Wiem; Cieślar-Pobuda, Artur; El-Gazzah, Mohamed; Jain, Mayur V; Rzeszowska-Wolny, Joanna; Rafat, Mehrdad; Stetefeld, Joerg; Ghavami, Saeid; Los, Marek J

    2014-09-01

    The human gyrovirus derived protein Apoptin (HGV-Apoptin) a homologue of the chicken anemia virus Apoptin (CAV-Apoptin), a protein with high cancer cells selective toxicity, triggers apoptosis selectively in cancer cells. In this paper, we show that HGV-Apoptin acts independently from the death receptor pathway as it induces apoptosis in similar rates in Jurkat cells deficient in either FADD (fas-associated death domain) function or caspase-8 (key players of the extrinsic pathway) and their parental clones. HGV-Apoptin induces apoptosis via the activation of the mitochondrial intrinsic pathway. It induces both mitochondrial inner and outer membrane permebilization, characterized by the loss of the mitochondrial potential and the release into cytoplasm of the pro-apoptotic molecules including apoptosis inducing factor and cytochrome c. HGV-Apoptin acts via the apoptosome, as lack of expression of apoptotic protease-activating factor 1 in murine embryonic fibroblast strongly protected the cells from HGV-Apoptin-induced apoptosis. Moreover, QVD-oph a broad-spectrum caspase inhibitor delayed HGV-Apoptin-induced death. On the other hand, overexpression of the anti-apoptotic BCL-XL confers resistance to HGV-Apoptin-induced cell death. In contrast, cells that lack the expression of the pro-apoptotic BAX and BAK are protected from HGV-Apoptin induced apoptosis. Furthermore, HGV-Apoptin acts independently from p53 signal but triggers the cytoplasmic translocation of Nur77. Taking together these data indicate that HGV-Apoptin acts through the mitochondrial pathway, in a caspase-dependent manner but independently from the death receptor pathway. Copyright © 2014 Neoplasia Press, Inc. Published by Elsevier Inc. All rights reserved.

  12. The effect of tumour necrosis factor-α (TNF-α muteins on human neutrophils in vitro

    Directory of Open Access Journals (Sweden)

    H. Tchorzewski

    1993-01-01

    Full Text Available Tumour necrosis factor-α (TNF-α has been implicated as an important inflammatory mediator. In vitro, TNF-α is reported to activate human polymorphonuclear neutrophils (PMN, inducing responses such as phagocytic activity, degranulation and oxidative metabolism. Biological responses to TNF-α are initiated by its binding to specific cell surface receptors, and various studies have shown that the major TNF receptor species on PMN is the 75 kDa receptor. To verify the suggestion that the receptor binding domain includes the region close to the N-terminus of the TNF-α molecule, four TNF-α derivatives termed muteins were constructed, using a synthetic cDNA fragment substituting the N-terminal 3–7 selected hydrophilic or hydrophobic amino acids in the original TNF-α genomic DNA. Binding of muteins to PMN was assessed using monoclonal antibodies recognizing either the 55 kDa (p55 or the 75 kDa (p75 TNF receptor subtypes. Blocking by muteins of anti-p75 antibody binding to PMN was as expected from their N-terminal amino acid composition and hydrophilic properties. Hydrophilic muteins competed well with anti-TNF receptor antibodies for binding to the p75 receptor. In contrast, hydrophobic muteins were unable to block anti-p75 binding. Similarly, degranulation, chemiluminescence or enhancement of the PMN response to specific stimuli by the muteins correlated with the hydrophilic properties of the muteins. The significance of these observations in relation to the molecular structure of TNF-α is discussed.

  13. Direct interaction between caffeic acid phenethyl ester and human neutrophil elastase inhibits the growth and migration of PANC-1 cells.

    Science.gov (United States)

    Duan, Jianhui; Xiaokaiti, Yilixiati; Fan, Shengjun; Pan, Yan; Li, Xin; Li, Xuejun

    2017-05-01

    Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignant tumors of the digestive system, but the mechanisms of its development and progression are unclear. Inflammation is thought to be fundamental to pancreatic cancer development and caffeic acid phenethyl ester (CAPE) is an active component of honey bee resin or propolis with anti-inflammatory and anticancer activities. We investigated the inhibitory effects of CAPE on cell growth and migration induced by human neutrophil elastase (HNE) and report that HNE induced cancer cell migration at low doses and growth at higher doses. In contrast, lower CAPE doses inhibited migration and higher doses of CAPE inhibited the growth induced by HNE. HNE activity was significantly inhibited by CAPE (7.5-120 µM). Using quantitative real-time PCR and western blotting, we observed that CAPE (18-60 µM) did not affect transcription and translation of α1-antitrypsin (α1-AT), an endogenous HNE inhibitor. However, in an in silico drug target docking model, we found that CAPE directly bound to the binding pocket of HNE (25.66 kcal/mol) according to CDOCKER, and the residue of the catalytic site stabilized the interaction between CAPE and HNE as evidenced by molecular dynamic simulation. Response unit (RU) values of surface plasmon resonance (SPR) significantly increased with incremental CAPE doses (7.5-120 µM), indicating that CAPE could directly bind to HNE in a concentration-dependent manner. Thus, CAPE is an effective inhibitor of HNE via direct interaction whereby it inhibits the migration and growth of PANC-1 cells in a dose-dependent manner.

  14. Dissection of pathways leading to antigen receptor-induced and Fas/CD95-induced apoptosis in human B cells

    NARCIS (Netherlands)

    Lens, S. M.; den Drijver, B. F.; Pötgens, A. J.; Tesselaar, K.; van Oers, M. H.; van Lier, R. A.

    1998-01-01

    To dissect intracellular pathways involved in B cell Ag receptor (BCR)-mediated and Fas-induced human B cell death, we isolated clones of the Burkitt lymphoma cell line Ramos with different apoptosis sensitivities. Selection for sensitivity to Fas-induced apoptosis also selected for clones with

  15. RODENT AND HUMAN NEUROPROGENITOR CELLS FOR HIGH-CONTENT SCREENS OF CHEMICAL EFFECTS ON PROLIFERATION AND APOPTOSIS

    Science.gov (United States)

    The objective of these experiments is to develop high-throughput screens for proliferation and apoptosis in order to compare rodent and human neuroprogenitor cell responses to potential developmental neurotoxicants. Effects of 4 chemicals on proliferation and apoptosis in mouse c...

  16. Thalidomide induces apoptosis in undifferentiated human induced pluripotent stem cells.

    Science.gov (United States)

    Tachikawa, Saoko; Nishimura, Toshinobu; Nakauchi, Hiromitsu; Ohnuma, Kiyoshi

    2017-10-01

    Thalidomide, which was formerly available commercially to control the symptoms of morning sickness, is a strong teratogen that causes fetal abnormalities. However, the mechanism of thalidomide teratogenicity is not fully understood; thalidomide toxicity is not apparent in rodents, and the use of human embryos is ethically and technically untenable. In this study, we designed an experimental system featuring human-induced pluripotent stem cells (hiPSCs) to investigate the effects of thalidomide. These cells exhibit the same characteristics as those of epiblasts originating from implanted fertilized ova, which give rise to the fetus. Therefore, theoretically, thalidomide exposure during hiPSC differentiation is equivalent to that in the human fetus. We examined the effects of thalidomide on undifferentiated hiPSCs and early-differentiated hiPSCs cultured in media containing bone morphogenetic protein-4, which correspond, respectively, to epiblast (future fetus) and trophoblast (future extra-embryonic tissue). We found that only the number of undifferentiated cells was reduced. In undifferentiated cells, application of thalidomide increased the number of apoptotic and dead cells at day 2 but not day 4. Application of thalidomide did not affect the cell cycle. Furthermore, immunostaining and flow cytometric analysis revealed that thalidomide exposure had no effect on the expression of specific markers of undifferentiated and early trophectodermal differentiated cells. These results suggest that the effect of thalidomide was successfully detected in our experimental system and that thalidomide eliminated a subpopulation of undifferentiated hiPSCs. This study may help to elucidate the mechanisms underlying thalidomide teratogenicity and reveal potential strategies for safely prescribing this drug to pregnant women.

  17. Effects of lithium on the functions of human neutrophils and lymphocytes in vitro and in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, R.; Walters, L.; Grabow, G.; Van der Merwe, M.; Van Rensburg, C.E. (Pretoria Univ. (South Africa))

    1982-10-02

    The effects of lithium sulphate (LiSO/sub 4/) at concentrations ranging from 10/sup -7/M to 10/sup -2/M on human polymorphonuclear leucocyte (PMNL) and lymphocyte functions in vitro were investigated. The leucocyte functions assessed were PMNL motility, post-phagocytic hexose-monophosphate shunt activity, myeloperoxidase-mediated iodination of Candida albicans and lymphocyte transformation to mitogens. These same functions as well the results of serological studies were assessed in normal volunteers prior to ingestion of lithium carbonate (LiCO/sub 3/), 2 hours and 24 hours after the ingestion of a single oral dose of 480 mg LiCO/sub 3/ and on the 4th day of ingestion of 2x480 mg LiCO/sub 3/ tablets daily. Incubation of PMNL with LiSO/sub 4/ at concentrations up to 10/sup -3/M had no detectable effects on motility or post-phagocytic metabolic activity. Higher concentrations (10/sup -3/M) inhibited these functions. Likewise, at concentrations up to 1x10/sup -4/M LiSO/sub 4/ had no effects on mitogen-induced transformation of lymphocytes, although higher concentrations did inhibit this activity. These same leucocyte functions were unaffected by ingestion of LiCO/sub 3/. Levels of serum immunoglobulins and complement components, total haemolytic complement activity and salivary lgA values also remained unaltered. In vitro investigations showed that at a concentration of 10/sup -3/M LiSO/sub 4/ had no inhibitory effects on the stimulation of PMNL motility mediated by ascorbate, levamisole and thiamine.

  18. Ultraviolet Radiation Induced Apoptosis in Human Skin In Vivo

    Energy Technology Data Exchange (ETDEWEB)

    Sheehan, J.M.; Young, A.R

    2000-07-01

    Sunburn cells, having many characteristics of apoptotic cells, appear in human skin after exposure to UVB. Time-courses and dose responses for solar simulated radiation (SSR)-induced sunburn cells in human volunteers of skin type II have been determined. For the time-course, two groups of volunteers were exposed to two minimal erythema doses (MED) of SSR. Punch biopsies were obtained from Group 1 immediately, 3, 6, 12, 18 and 24 h after SSR exposure and Group 2 were biopsied immediately, 18, 24, 36, 48 and 72 h after exposure. For the dose-response (Group 3), biopsies were taken 24 h after SSR exposure to 0, 0.25, 0.5, 1, 2 and 3 MED. Sections were stained with H and E and also using TUNEL and analysed by light microscopy. Results show a dose-dependent appearance of SBC after SSR exposure. The time point for maximum SBC counts with both H and E staining and TUNEL staining lie between 24 and 36 h. (author)

  19. Ultraviolet Radiation Induced Apoptosis in Human Skin In Vivo

    International Nuclear Information System (INIS)

    Sheehan, J.M.; Young, A.R.

    2000-01-01

    Sunburn cells, having many characteristics of apoptotic cells, appear in human skin after exposure to UVB. Time-courses and dose responses for solar simulated radiation (SSR)-induced sunburn cells in human volunteers of skin type II have been determined. For the time-course, two groups of volunteers were exposed to two minimal erythema doses (MED) of SSR. Punch biopsies were obtained from Group 1 immediately, 3, 6, 12, 18 and 24 h after SSR exposure and Group 2 were biopsied immediately, 18, 24, 36, 48 and 72 h after exposure. For the dose-response (Group 3), biopsies were taken 24 h after SSR exposure to 0, 0.25, 0.5, 1, 2 and 3 MED. Sections were stained with H and E and also using TUNEL and analysed by light microscopy. Results show a dose-dependent appearance of SBC after SSR exposure. The time point for maximum SBC counts with both H and E staining and TUNEL staining lie between 24 and 36 h. (author)

  20. Early establishment of epithelial apoptosis in the developing human small intestine.

    Science.gov (United States)

    Vachon, P H; Cardin, E; Harnois, C; Reed, J C; Vézina, A

    2000-12-01

    In the adult small intestine, the dynamic renewal of the epithelium is characterized by a sequence of cell production in the crypts, cell maturation and cell migration to the tip of villi, where apoptosis is undertaken. Little is known about enterocytic apoptosis during development. In man, intestinal architectural features and functions are acquired largely by mid-gestation (18-20 wks); the question whether the establishment of enterocytic apoptotic processes parallels or not the acquisition of other intestinal functional features remains open. In the present study, we approached this question by examining enterocytic apoptosis during development of the human jejunum (9-20 wks gestation), using the ISEL (in situ terminal uridine deoxynucleotidyl nick-end labelling) method. Between 9 and 17 wks, apoptotic enterocytes were not evidenced. However, beginning at the 18 wks stage, ISEL-positive enterocytes were regularly observed at the tip of villi. Since the Bcl-2 family of proteins constitutes a critical checkpoint in apoptosis, acting upstream of the apoptotic machinery, we investigated the expression of six Bcl-2 homologs (Bcl-2, Bcl-X(L), Mcl-1, Bax, Bak, Bad) and one non-homologous associated molecule (Bag-1). By immunofluorescence, we found that all homologs analyzed were expressed by enterocytes between 9 and 20 wks. However, Bcl-2 homologs underwent a gradual compartmentalization of epithelial expression along the maturing crypt-villus axis, to establish gradients of expression by 18-20 wks. Western blot analyses indicated that the expression levels of Bcl-2 homologs were modulated during morphogenesis of the crypt-villus axis, in parallel to their gradual compartmentalization of expression. Altogether, these data suggest that regulatory mechanisms of human enterocytic apoptosis become established by mid-gestation (18-20 wks) and coincide with the maturation of the crypt-villus axis of cell proliferation, differentiation and renewal.

  1. Survival and differentiation defects contribute to neutropenia in glucose-6-phosphatase-β (G6PC3) deficiency in a model of mouse neutrophil granulocyte differentiation.

    Science.gov (United States)

    Gautam, S; Kirschnek, S; Gentle, I E; Kopiniok, C; Henneke, P; Häcker, H; Malleret, L; Belaaouaj, A; Häcker, G

    2013-08-01

    Differentiation of neutrophil granulocytes (neutrophils) occurs through several steps in the bone marrow and requires a coordinate regulation of factors determining survival and lineage-specific development. A number of genes are known whose deficiency disrupts neutrophil generation in humans and in mice. One of the proteins encoded by these genes, glucose-6-phosphatase-β (G6PC3), is involved in glucose metabolism. G6PC3 deficiency causes neutropenia in humans and in mice, linked to enhanced apoptosis and ER stress. We used a model of conditional Hoxb8 expression to test molecular and functional differentiation as well as survival defects in neutrophils from G6PC3(-/-) mice. Progenitor lines were established and differentiated into neutrophils when Hoxb8 was turned off. G6PC3(-/-) progenitor cells underwent substantial apoptosis when differentiation was started. Transgenic expression of Bcl-XL rescued survival; however, Bcl-XL-protected differentiated cells showed reduced proliferation, immaturity and functional deficiency such as altered MAP kinase signaling and reduced cytokine secretion. Impaired glucose utilization was found and was associated with ER stress and apoptosis, associated with the upregulation of Bim and Bax; downregulation of Bim protected against apoptosis during differentiation. ER-stress further caused a profound loss of expression and secretion of the main neutrophil product neutrophil elastase during differentiation. Transplantation of wild-type Hoxb8-progenitor cells into irradiated mice allowed differentiation into neutrophils in the bone marrow in vivo. Transplantation of G6PC3(-/-) cells yielded few mature neutrophils in bone marrow and peripheral blood. Transgenic Bcl-XL permitted differentiation of G6PC3(-/-) cells in vivo. However, functional deficiencies and differentiation abnormalities remained. Differentiation of macrophages from Hoxb8-dependent progenitors was only slightly disturbed. A combination of defects in differentiation

  2. β-Glucan induces reactive oxygen species production in human neutrophils to improve the killing of Candida albicans and Candida glabrata isolates from vulvovaginal candidiasis.

    Directory of Open Access Journals (Sweden)

    Patricia de Souza Bonfim-Mendonça

    Full Text Available Vulvovaginal candidiasis (VVC is among the most prevalent vaginal diseases. Candida albicans is still the most prevalent species associated with this pathology, however, the prevalence of other Candida species, such as C. glabrata, is increasing. The pathogenesis of these infections has been intensely studied, nevertheless, no consensus has been reached on the pathogenicity of VVC. In addition, inappropriate treatment or the presence of resistant strains can lead to RVVC (vulvovaginal candidiasis recurrent. Immunomodulation therapy studies have become increasingly promising, including with the β-glucans. Thus, in the present study, we evaluated microbicidal activity, phagocytosis, intracellular oxidant species production, oxygen consumption, myeloperoxidase (MPO activity, and the release of tumor necrosis factor α (TNF-α, interleukin-8 (IL-8, IL-1β, and IL-1Ra in neutrophils previously treated or not with β-glucan. In all of the assays, human neutrophils were challenged with C. albicans and C. glabrata isolated from vulvovaginal candidiasis. β-glucan significantly increased oxidant species production, suggesting that β-glucan may be an efficient immunomodulator that triggers an increase in the microbicidal response of neutrophils for both of the species isolated from vulvovaginal candidiasis. The effects of β-glucan appeared to be mainly related to the activation of reactive oxygen species and modulation of cytokine release.

  3. Holotoxin A1 Induces Apoptosis by Activating Acid Sphingomyelinase and Neutral Sphingomyelinase in K562 and Human Primary Leukemia Cells

    Directory of Open Access Journals (Sweden)

    Seong-Hoon Yun

    2018-04-01

    Full Text Available Marine triterpene glycosides are attractive candidates for the development of anticancer agents. Holotoxin A1 is a triterpene glycoside found in the edible sea cucumber, Apostichopus (Stichopus japonicus. We previously showed that cladoloside C2, the 25(26-dihydro derivative of holotoxin A1, induced apoptosis in human leukemia cells by activating ceramide synthase 6. Thus, we hypothesized that holotoxin A1, which is structurally similar to cladoloside C2, might induce apoptosis in human leukemia cells through the same molecular mechanism. In this paper, we compared holotoxin A1 and cladoloside C2 for killing potency and mechanism of action. We found that holotoxin A1 induced apoptosis more potently than cladoloside C2. Moreover, holotoxin A1 induced apoptosis in K562 cells by activating caspase-8 and caspase-3, but not by activating caspase-9. During holotoxin A1-induced apoptosis, acid sphingomyelinase (SMase and neutral SMase were activated in both K562 cells and human primary leukemia cells. Specifically inhibiting acid SMase and neutral SMаse with chemical inhibitors or siRNAs significantly inhibited holotoxin A1–induced apoptosis. These results indicated that holotoxin A1 might induce apoptosis by activating acid SMase and neutral SMase. In conclusion, holotoxin A1 represents a potential anticancer agent for treating leukemia. Moreover, the aglycone structure of marine triterpene glycosides might affect the mechanism involved in inducing apoptosis.

  4. Cryptolepine, isolated from Sida acuta, sensitizes human gastric adenocarcinoma cells to TRAIL-induced apoptosis.

    Science.gov (United States)

    Ahmed, Firoj; Toume, Kazufumi; Ohtsuki, Takashi; Rahman, Mahmudur; Sadhu, Samir Kumar; Ishibashi, Masami

    2011-01-01

    Bioassay guided separation of Sida acuta whole plants led to the isolation of an alkaloid, cryptolepine (1), along with two kaempferol glycosides (2-3). Compound 1 showed strong activity in overcoming TRAIL-resistance in human gastric adenocarcinoma (AGS) cells at 1.25, 2.5 and 5 μm. Combined treatment of 1 and TRAIL sensitized AGS cells to TRAIL-induced apoptosis at the aforementioned concentrations. Copyright © 2010 John Wiley & Sons, Ltd.

  5. Diosgenin induces apoptosis in IGF-1-stimulated human thyrocytes through two caspase-dependent pathways

    Energy Technology Data Exchange (ETDEWEB)

    Mu, Shumin [Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Hospital Affiliated to Shandong Traditional Chinese Medicine University, Jinan 250011 (China); Institute of Endocrinology, Shandong Academy of Clinical Medicine, Jinan 250021 (China); Tian, Xingsong; Ruan, Yongwei [Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Liu, Yuantao [The Second Hospital of Shandong University, Jinan 250033 (China); Bian, Dezhi [Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Jining Medical College, Jining 272013 (China); Ma, Chunyan [Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Yu, Chunxiao [Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Institute of Endocrinology, Shandong Academy of Clinical Medicine, Jinan 250021 (China); Feng, Mei [Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Wang, Furong [Shandong University of Traditional Chinese Medicine, Jinan 250011 (China); Gao, Ling [Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Institute of Endocrinology, Shandong Academy of Clinical Medicine, Jinan 250021 (China); Zhao, Jia-jun, E-mail: jjzhao@medmail.com.cn [Provincial Hospital Affiliated to Shandong University, Jinan 250021 (China); Institute of Endocrinology, Shandong Academy of Clinical Medicine, Jinan 250021 (China)

    2012-02-10

    Highlights: Black-Right-Pointing-Pointer Diosgenin induces apoptosis in IGF-1-treated thyrocytes through two caspase pathways. Black-Right-Pointing-Pointer Diosgenin inhibits FLIP and activates caspase-8 in FAS related-pathway. Black-Right-Pointing-Pointer Diosgenin increases ROS, regulates the ratio of Bax/Bcl-2 in mitochondrial pathway. -- Abstract: Insulin-like growth factor-1 (IGF-1) is a growth factor of the thyroid that has been shown in our previous study to possess proliferative and antiapoptotic effects in FRTL-5 cell lines through the upregulation of cyclin D and Fas-associated death domain-like interleukin-1-converting enzyme (FLICE)-inhibitory protein (FLIP). Diosgenin, a natural steroid sapogenin from plants, has been shown to induce apoptosis in many cell lines, with the exception of thyroid cells. In this report, we investigated the apoptotic effect and mechanism of diosgenin in IGF-1-stimulated primary human thyrocytes. Primary human thyrocytes were preincubated with or without IGF-1 for 24 h and subsequently exposed to varying concentrations of diosgenin for different times. We found that diosgenin induced apoptosis in human thyrocytes pretreated with IGF-1 in a dose-dependent manner through the activation of caspase cascades. Moreover, diosgenin inhibited FLIP and activated caspase-8 in the FAS-related apoptotic pathway. Diosgenin increased the production of ROS, regulated the balance of Bax and Bcl-2 and cleaved caspase-9 in the mitochondrial apoptotic pathway. These results indicate that diosgenin induces apoptosis in IGF-1-stimulated primary human thyrocytes through two caspase-dependent pathways.

  6. Human herpesvirus 6A induces apoptosis of primary human fetal astrocytes via both caspase-dependent and -independent pathways

    Directory of Open Access Journals (Sweden)

    Gu Bin

    2011-12-01

    Full Text Available Abstract Background Human herpesvirus 6 (HHV-6 is a T-lymphtropic and neurotropic virus that can infect various types of cells. Sequential studies reported that apoptosis of glia and neurons induced by HHV-6 might act a potential trigger for some central nervous system (CNS diseases. HHV-6 is involved in the pathogenesis of encephalitis, multiple sclerosis (MS and fatigue syndrome. However, the mechanisms responsible for the apoptosis of infected CNS cells induced by HHV-6 are poorly understood. In this study, we investigated the cell death processes of primary human fetal astrocytes (PHFAs during productive HHV-6A infection and the underlying mechanisms. Results HHV-6A can cause productive infection in primary human fetal astrocytes. Annexin V-PI staining and electron microscopic analysis indicated that HHV-6A was an inducer of apoptosis. The cell death was associated with activation of caspase-3 and cleavage of poly (ADP-ribose polymerase (PARP, which is known to be an important substrate for activated caspase-3. Caspase-8 and -9 were also significantly activated in HHV-6A-infected cells. Moreover, HHV-6A infection led to Bax up-regulation and Bcl-2 down-regulation. HHV-6A infection increased the release of Smac/Diablo, AIF and cytochrome c from mitochondria to cytosol, which induced apoptosis via the caspase-dependent and -independent pathways. In addition, we also found that anti-apoptotic factors such as IAPs and NF-κB decreased in HHV-6A infected PHFAs. Conclusion This is the first demonstration of caspase-dependent and -independent apoptosis in HHV-6A-infected glial cells. These findings would be helpful in understanding the mechanisms of CNS diseases caused by HHV-6.

  7. Involvement of Endoplasmic Reticulum Stress in Capsaicin-Induced Apoptosis of Human Pancreatic Cancer Cells

    Directory of Open Access Journals (Sweden)

    Shengzhang Lin

    2013-01-01

    Full Text Available Capsaicin, main pungent ingredient of hot chilli peppers, has been shown to have anticarcinogenic effect on various cancer cells through multiple mechanisms. In this study, we investigated the apoptotic effect of capsaicin on human pancreatic cancer cells in both in vitro and in vivo systems, as well as the possible mechanisms involved. In vitro, treatment of both the pancreatic cancer cells (PANC-1 and SW1990 with capsaicin resulted in cells growth inhibition, G0/G1 phase arrest, and apoptosis in a dose-dependent manner. Knockdown of growth arrest- and DNA damage-inducible gene 153 (GADD153, a marker of the endoplasmic-reticulum-stress- (ERS- mediated apoptosis pathway, by specific siRNA attenuated capsaicin-induced apoptosis both in PANC-1 and SW1990 cells. Moreover, in vivo studies capsaicin effectively inhibited the growth and metabolism of pancreatic cancer and prolonged the survival time of pancreatic cancer xenograft tumor-induced mice. Furthermore, capsaicin increased the expression of some key ERS markers, including glucose-regulated protein 78 (GRP78, phosphoprotein kinase-like endoplasmic reticulum kinase (phosphoPERK, and phosphoeukaryotic initiation factor-2α (phospho-eIF2α, activating transcription factor 4 (ATF4 and GADD153 in tumor tissues. In conclusion, we for the first time provide important evidence to support the involvement of ERS in the induction of apoptosis in pancreatic cancer cells by capsaicin.

  8. Nicotine prevents the apoptosis induced by menadione in human lung cancer cells

    International Nuclear Information System (INIS)

    Zhang Tao; Lu Heng; Shang Xuan; Tian Yihao; Zheng Congyi; Wang Shiwen; Cheng Hanhua; Zhou Rongjia

    2006-01-01

    Approximately 50% of long-term cigarette smokers die prematurely from the adverse effects of smoking, including on lung cancer and other illnesses. Nicotine is a main component in tobacco and has been implicated as a potential factor in the pathogenesis of human lung cancer. However, the mechanism of nicotine action in the development of lung cancer remains largely unknown. In the present study, we designed a nicotine-apoptosis system, by pre-treatment of nicotine making lung cancer cell A549 to be in a physiological nicotine environment, and observed that nicotine promoted cell proliferation and prevented the menadione-induced apoptosis, and exerts its role of anti-apoptosis by shift of apoptotic stage induced by menadione from late apoptotic stage to early apoptotic stage, in which NF-κB was up-regulated. Interference analysis of NF-κB in A549 cells showed that knock down of NF-κB resulted in apoptosis promotion and counteracted the protective effect of nicotine. The findings suggest that nicotine has potential effect in lung cancer genesis, especially in patients with undetectable early tumor development and development of specific NF-κB inhibitors would represent a potentially exciting new pharmacotherapy for tobacco-related lung cancer

  9. Resveratrol-Sensitized UVA Induced Apoptosis in Human Keratinocytes through Mitochondrial Oxidative Stress and Pore Opening

    Science.gov (United States)

    Boyer, Jean Z; Jandova, Jana; Janda, Jaroslav; Vleugels, Frank R; Elliott, David; Sligh, James E

    2012-01-01

    Resveratrol (3, 5, 4′-trihydroxy- trans- stilbene), a polyphenol compound, is derived from natural products such as the skin of red grapes, blueberries and cranberries. Resveratrol not only exhibits antioxidant, cardioprotection, and anti-aging properties, but can also inhibit cancer cell growth and induce apoptosis. It has been shown that resveratrol inhibits the activation of Nf-kB and subsequently down regulates the expression of Nf-kB regulated genes such as interleukin-2 and Bcl-2, leading to cell cycle arrest and increased apoptosis in multiple myeloma cells. In the skin, resveratrol has been reported to sensitize keratinocytes to UVA induced apoptosis. However, the effect of resveratrol on opening of the mitochondrial permeability transition pore has not been previously examined. Our data show that UVA (14J/cm2) along with resveratrol causes massive oxidative stress in mitochondria. As a consequence of oxidative stress, the mitochondrial membrane potential decreases which results in opening of the mitochondrial pores ultimately leading to apoptosis in human keratinocytes. These results may have clinical implications for development of future chemotherapeutic treatment for tumors of the skin. PMID:22673012

  10. Laminarin Induces Apoptosis of Human Colon Cancer LOVO Cells through a Mitochondrial Pathway

    Directory of Open Access Journals (Sweden)

    He Zhang

    2012-08-01

    Full Text Available Many scientific studies have shown that laminarin has anti-tumor effects, but the anti-tumor mechanism was unclear. The purpose of this study was to investigate the effect of laminarin on the induction of apoptosis in human colon cancer LOVO cells and the molecular mechanism involved. LOVO cells were treated with different concentrations of laminarin at different times. Morphology observations were performed to determine the effects of laminarin on apoptosis of LOVO cells. Flow cytometry (FCM was used to detect the level of intracellular reactive oxygen species (ROS and pH. Laser scanning confocal microscope (LSCM was used to analyze intracellular calcium ion concentration, mitochondrion permeability transition pore (MPTP and mitochondrial membrane potential (MMP. Western blotd were performed to analyze the expressions of Cyt-C, Caspase-9 and -3. The results showed the apoptosis morphology, which showed cell protuberance, concentrated cytoplasm and apoptotic bodies, was obvious after 72 h treatment. Laminarin treatment for 24 h increased the intracellular level of ROS and Ca2+; decreased pH value; activated intracellular MPTP and decreased MMP in dose-dependent manners. It also induced the release of Cyt-C and the activation of Caspase-9 and -3. In conclusion, laminarin induces LOVO cell apoptosis through a mitochondrial pathway, suggesting that it could be a potent agent for cancer prevention and treatment.

  11. Elevated glucose concentrations promote receptor-independent activation of adherent human neutrophils: an experimental and computational approach

    DEFF Research Database (Denmark)

    Kummer, Ursula; Zobeley, Jürgen; Brasen, Jens Christian

    2007-01-01

    of NO and superoxide formation were observed. However, these changes were not observed for sorbitol, a nonmetabolizable carbohydrate. Glucose transport appears to be important in this process as phloretin interferes with the glucose-specific receptor-independent activation of neutrophils. However, LY83583...

  12. Correlation between the neutrophil-lymphocyte count ratio and bacterial infection in patient with human immunodeficiency virus

    Science.gov (United States)

    Kusnadi, D.; Liwang, M. N. I.; Katu, S.; Mubin, A. H.; Halim, R.

    2018-03-01

    Parameters for starting antibiotic therapy such as CRP andleukocytosis are considered non-specific. Previous studies have shown the Neutrophil-Lymphocyte Count Ratio (NLCR) can serve as the basis of bacterial infection, the level of infection, and the basis of antibiotic therapy. Compared with the Procalcitonin parameter, this NLCR is rapid, an inexpensive and requires no additional sampling. To determine the correlation between The Neutrophil-LymphocyteCount Ratio to bacterial infection in HIV patients. This study was a cross-sectional observational approach to HIV subject at Wahidin Sudirohusodo and Hasanuddin University Hospital. The subjects performed routine blood, microbiology test,and blood Procalcitonin levels tests. Then performed NLCR calculations based on routine blood results. The subjects then grouped the presence or absence of bacterial infection.In 146 study subjects, there were 78 (53.4%) with bacterial infections and 68 (46.6%) without bacterial infection as controls. Subjects with bacterial infections had higher total neutrophils (84.83) compared with non-bacterial infections. Subjects with bacterial infections had total lymphocytes with an average of 8.51 lower than non-bacterial infections. Subjects with bacterial infections had higher NLCR values with an average of 12.80. The Neutrophil-Lymphocyte Count Ratio can become a marker of bacterial infection in HIV patients.

  13. Azurophil granule proteins constitute the major mycobactericidal proteins in human neutrophils and enhance the killing of mycobacteria in macrophages.

    Directory of Open Access Journals (Sweden)

    Prajna Jena

    Full Text Available Pathogenic mycobacteria reside in, and are in turn controlled by, macrophages. However, emerging data suggest that neutrophils also play a critical role in innate immunity to tuberculosis, presumably by their different antibacterial granule proteins. In this study, we purified neutrophil azurophil and specific granules and systematically analyzed the antimycobacterial activity of some purified azurophil and specific granule proteins against M. smegmatis, M. bovis-BCG and M. tuberculosis H37Rv. Using gel overlay and colony forming unit assays we showed that the defensin-depleted azurophil granule proteins (AZP were more active against mycobacteria compared to other granule proteins and cytosolic proteins. The proteins showing antimycobacterial activity were identified by MALDI-TOF mass spectrometry. Electron microscopic studies demonstrate that the AZP disintegrate bacterial cell membrane resulting in killing of mycobacteria. Exogenous addition of AZP to murine macrophage RAW 264.7, THP-1 and peripheral blood monocyte-derived macrophages significantly reduced the intracellular survival of mycobacteria without exhibiting cytotoxic activity on macrophages. Immunofluorescence studies showed that macrophages actively endocytose neutrophil granular proteins. Treatment with AZP resulted in increase in co-localization of BCG containing phagosomes with lysosomes but not in increase of autophagy. These data demonstrate that neutrophil azurophil proteins may play an important role in controlling intracellular survival of mycobacteria in macrophages.

  14. Selection of reliable reference genes for quantitative real-time PCR in human T cells and neutrophils

    Directory of Open Access Journals (Sweden)

    Ledderose Carola

    2011-10-01

    Full Text Available Abstract Background The choice of reliable reference genes is a prerequisite for valid results when analyzing gene expression with real-time quantitative PCR (qPCR. This method is frequently applied to study gene expression patterns in immune cells, yet a thorough validation of potential reference genes is still lacking for most leukocyte subtypes and most models of their in vitro stimulation. In the current study, we evaluated the expression stability of common reference genes in two widely used cell culture models-anti-CD3/CD28 activated T cells and lipopolysaccharide stimulated neutrophils-as well as in unselected untreated leukocytes. Results The mRNA expression of 17 (T cells, 7 (neutrophils or 8 (unselected leukocytes potential reference genes was quantified by reverse transcription qPCR, and a ranking of the preselected candidate genes according to their expression stability was calculated using the programs NormFinder, geNorm and BestKeeper. IPO8, RPL13A, TBP and SDHA were identified as suitable reference genes in T cells. TBP, ACTB and SDHA were stably expressed in neutrophils. TBP and SDHA were also the most stable genes in untreated total blood leukocytes. The critical impact of reference gene selection on the estimated target gene expression is demonstrated for IL-2 and FIH expression in T cells. Conclusions The study provides a shortlist of suitable reference genes for normalization of gene expression data in unstimulated and stimulated T cells, unstimulated and stimulated neutrophils and in unselected leukocytes.

  15. 3-Bromopyruvate induces endoplasmic reticulum stress, overcomes autophagy and causes apoptosis in human HCC cell lines.

    Science.gov (United States)

    Ganapathy-Kanniappan, Shanmugasundaram; Geschwind, Jean-Francois H; Kunjithapatham, Rani; Buijs, Manon; Syed, Labiq H; Rao, Pramod P; Ota, Shinichi; Kwak, Byung Kook; Loffroy, Romaric; Vali, Mustafa

    2010-03-01

    Autophagy, a cellular response to stress, plays a role in resistance to chemotherapy in cancer cells. Resistance renders systemic chemotherapy generally ineffective against human hepatocellular carcinoma (HCC). Recently, we reported that the pyruvate analog 3-bromopyruvate (3-BrPA) promoted tumor cell death by targeting GAPDH. In continuance, we investigated the intracellular response of two human HCC cell lines (Hep3B and SK-Hep1) that differ in their status of key apoptotic regulators, p53 and Fas. 3-BrPA treatment induced endoplasmic reticulum (ER) stress, translation inhibition and apoptosis based on Western blot and qPCR, pulse labeling, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and active caspase-3 in both the cell lines. However, electron microscopy revealed that 3-BrPA treated SK-Hep1 cells underwent classical apoptotic cell death while Hep3B cells initially responded with the protective autophagy that failed to prevent eventual apoptosis. 3-BrPA treatment promotes apoptosis in human HCC cell lines, irrespective of the intracellular response.

  16. Gemcitabine inhibits proliferation and induces apoptosis in human pancreatic cancer PANC-1 cells.

    Science.gov (United States)

    Yong-Xian, Gui; Xiao-Huan, Li; Fan, Zhang; Guo-Fang, Tian

    2016-10-01

    The aim of the study is to investigate the underlying molecular mechanisms by which gemcitabine (gem) inhibits proliferation and induces apoptosis in human pancreatic cancer PANC-1 cells in vitro. After PANC-1 cells had been treated by indicated concentration (0, 5, and 25 mg/L) of gem for 48 h, cell proliferation was evaluated by 3'-(4, 5 dimethyl-thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay; cell morphology was observed by transmission electron microscopy; Expression of c-IAP2 and Bcl-2 proteins was analyzed by Western blot; the activity of caspase-3 and -9 was detected by spectrophotometry. Gem significantly inhibited cell proliferation and could induce apoptosis of human pancreatic cancer PANC-1 cells, with a dose-dependent manner. Western blot analysis showed that gem significantly reduced c-IAP2 and Bcl-2 proteins expression level (P PANC-1 cells. Gem could induce apoptosis of human pancreatic cancer PANC-1 cells, probably through downregulating c-IAP2 and Bcl-2 expression levels, and at the same time activating caspase-3 and -9.

  17. The Efficacy of Dandelion Root Extract in Inducing Apoptosis in Drug-Resistant Human Melanoma Cells

    Directory of Open Access Journals (Sweden)

    S. J. Chatterjee

    2011-01-01

    Full Text Available Notoriously chemoresistant melanoma has become the most prevalent form of cancer for the 25–29 North American age demographic. Standard treatment after early detection involves surgical excision (recurrence is possible, and metastatic melanoma is refractory to immuno-, radio-, and most harmful chemotherapies. Various natural compounds have shown efficacy in killing different cancers, albeit not always specifically. In this study, we show that dandelion root extract (DRE specifically and effectively induces apoptosis in human melanoma cells without inducing toxicity in noncancerous cells. Characteristic apoptotic morphology of nuclear condensation and phosphatidylserine flipping to the outer leaflet of the plasma membrane of A375 human melanoma cells was observed within 48 hours. DRE-induced apoptosis activates caspase-8 in A375 cells early on, demonstrating employment of an extrinsic apoptotic pathway to kill A375 cells. Reactive Oxygen Species (ROS generated from DRE-treated isolated mitochondria indicates that natural compounds in DRE can also directly target mitochondria. Interestingly, the relatively resistant G361 human melanoma cell line responded to DRE when combined with the metabolism interfering antitype II diabetic drug metformin. Therefore, treatment with this common, yet potent extract of natural compounds has proven novel in specifically inducing apoptosis in chemoresistant melanoma, without toxicity to healthy cells.

  18. Genes involved in immunity and apoptosis are associated with human presbycusis based on microarray analysis.

    Science.gov (United States)

    Dong, Yang; Li, Ming; Liu, Puzhao; Song, Haiyan; Zhao, Yuping; Shi, Jianrong

    2014-06-01

    Genes involved in immunity and apoptosis were associated with human presbycusis. CCR3 and GILZ played an important role in the pathogenesis of presbycusis, probably through regulating chemokine receptor, T-cell apoptosis, or T-cell activation pathways. To identify genes associated with human presbycusis and explore the molecular mechanism of presbycusis. Hearing function was tested by pure-tone audiometry. Microarray analysis was performed to identify presbycusis-correlated genes by Illumina Human-6 BeadChip using the peripheral blood samples of subjects. To identify biological process categories and pathways associated with presbycusis-correlated genes, bioinformatics analysis was carried out by Gene Ontology Tree Machine (GOTM) and database for annotation, visualization, and integrated discovery (DAVID). Quantitative RT-PCR (qRT-PCR) was used to validate the microarray data. Microarray analysis identified 469 up-regulated genes and 323 down-regulated genes. Both the dominant biological processes by Gene Ontology (GO) analysis and the enriched pathways by Kyoto encyclopedia of genes and genomes (KEGG) and BIOCARTA showed that genes involved in immunity and apoptosis were associated with presbycusis. In addition, CCR3, GILZ, CXCL10, and CX3CR1 genes showed consistent difference between groups for both the gene chip and qRT-PCR data. The differences of CCR3 and GILZ between presbycusis patients and controls were statistically significant (p < 0.05).

  19. Capsaicin induces cell cycle arrest and apoptosis in human KB cancer cells.

    Science.gov (United States)

    Lin, Chia-Han; Lu, Wei-Cheng; Wang, Che-Wei; Chan, Ya-Chi; Chen, Mu-Kuan

    2013-02-25

    Capsaicin, a pungent phytochemical in a variety of red peppers of the genus Capsicum, has shown an anti-proliferative effect on various human cancer cell lines. In contrast, capsaicin has also been considered to promote the growth of cancer cells. Thus, the effects of capsaicin on various cell types need to be explored. The anti-proliferative effects of capsaicin on human KB cancer cells are still unknown. Therefore, we examined the viability, cell cycle progression, and factors associated with apoptosis in KB cells treated with capsaicin. The cell proliferation/viability and cytotoxicity of KB cells exposed to capsaicin were determined by a sulforhodamine B colorimetric assay and trypan blue exclusion. Apoptosis was detected by Hoechst staining and confirmed by western blot analysis of poly-(ADP-ribose) polymerase cleavage. Cell cycle distribution and changes of the mitochondrial membrane potential were analyzed by flow cytometry. Furthermore, the expression of caspase 3, 8 and 9 was evaluated by immunoblotting. We found that treatment of KB cells with capsaicin significantly reduced cell proliferation/viability and induced cell death in a dose-dependent manner compared with that in the untreated control. Cell cycle analysis indicated that exposure of KB cells to capsaicin resulted in cell cycle arrest at G2/M phase. Capsaicin-induced growth inhibition of KB cells appeared to be associated with induction of apoptosis. Moreover, capsaicin induced disruption of the mitochondrial membrane potential as well as activation of caspase 9, 3 and poly-(ADP-ribose) polymerase in KB cells. Our data demonstrate that capsaicin modulates cell cycle progression and induces apoptosis in human KB cancer cells through mitochondrial membrane permeabilization and caspase activation. These observations suggest an anti-cancer activity of capsaicin.

  20. Induction of apoptosis by pinostrobin in human cervical cancer cells: Possible mechanism of action.

    Directory of Open Access Journals (Sweden)

    Alka Jaudan

    Full Text Available Pinostrobin (PN is a naturally occurring dietary bioflavonoid, found in various medicinal herbs/plants. Though anti-cancer potential of many such similar constituents has been demonstrated, critical biochemical targets and exact mechanism for their apoptosis-inducing actions have not been fully elucidated. The present study was aimed to investigate if PN induced apoptosis in cervical cancer cells (HeLa of human origin. It is demonstrated that PN at increasing dose effectivity reduced the cell viability as well as GSH and NO2- levels. Condensed nuclei with fragmented chromatin and changes in mitochondrial matrix morphology clearly indicated the role of mitochondria in PN induced apoptosis. A marked reduction in mitochondrial membrane potential and increased ROS production after PN treatment showed involvement of free radicals, which in turn further augment ROS levels. PN treatment resulted in DNA damage, which could have been triggered by an increase in ROS levels. Decrease in apoptotic cells in the presence of caspase 3 inhibitor in PN-treated cells suggested that PN induced apoptosis via caspase dependent pathways. Additionally, a significant increase in the expression of proteins of extrinsic (TRAIL R1/DR4, TRAIL R2/DR5, TNF RI/TNFRSF1A, FADD, Fas/TNFRSF6 and intrinsic pathway (Bad, Bax, HTRA2/Omi, SMAC/Diablo, cytochrome C, Pro-Caspase-3, Cleaved Caspase-3 was observed in the cells exposed to PN. Taken together, these observations suggest that PN efficiently induces apoptosis through ROS mediated extrinsic and intrinsic dependent signaling pathways, as well as ROS mediated mitochondrial damage in HeLa cells.

  1. Rhein induces apoptosis of human gastric cancer SGC-7901 cells via an intrinsic mitochondrial pathway

    Directory of Open Access Journals (Sweden)

    Yiwen Li

    2012-11-01

    Full Text Available Rhein is a primary anthraquinone found in the roots of a traditional Chinese herb, rhubarb, and has been shown to have some anticancer effects. The aim of the present study was to investigate the effect of rhein on the apoptosis of the human gastric cancer line SGC-7901 and to identify the mechanism involved. SGC-7901 cells were cultured and treated with rhein (0, 50, 100, 150, and 200 µM for 24, 48, or 72 h. Relative cell viability assessed by the MTT assay after treatment was 100, 99, 85, 79, 63% for 24 h; 100, 98, 80, 51, 37% for 48 h, and 100, 97, 60, 36, 15% for 72 h, respectively. Cell apoptosis was detected with TUNEL staining and quantified with flow cytometry using annexin FITC-PI staining at 48 h after 100, 200 and 300 µm rhein. The percentage of apoptotic cells was 7.3, 21.9, 43.5%, respectively. We also measured the mRNA levels of caspase-3 and -9 using real-time PCR. Treatment with 100 µM rhein for 48 h significantly increased mRNA expression of caspase-3 and -9. The levels of apoptosis-related proteins including Bcl-2, Bax, Bcl-xL, and pro-caspase-3 were evaluated in rhein-treated cells. Rhein increased the Bax:Bcl-2 ratio but decreased the protein levels of Bcl-xL and pro-caspase-3. Moreover, rhein significantly increased the expression of cytochrome c and apoptotic protease activating factor 1, two critical components involved in mitochondrial pathway-mediated apoptosis. We conclude that rhein inhibits SGC-7901 proliferation by inducing apoptosis and this antitumor effect of rhein is mediated in part by an intrinsic mitochondrial pathway.

  2. Rhein induces apoptosis of human gastric cancer SGC-7901 cells via an intrinsic mitochondrial pathway

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yiwen; Xu, Yuqing [Department of Oncology,Second Affiliated Hospital, Harbin Medical University, Nangang District, Harbin, Heilongjiang (China); Lei, Bo [Department of Breast Surgery, Third Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang (China); Wang, Wenxiu [Department of Oncology,Second Affiliated Hospital, Harbin Medical University, Nangang District, Harbin, Heilongjiang (China); Ge, Xin; Li, Jingrui [Department of General Surgery, Heilongjiang Province Hospital, Harbin, Heilongjiang (China)

    2012-08-03

    Rhein is a primary anthraquinone found in the roots of a traditional Chinese herb, rhubarb, and has been shown to have some anticancer effects. The aim of the present study was to investigate the effect of rhein on the apoptosis of the human gastric cancer line SGC-7901 and to identify the mechanism involved. SGC-7901 cells were cultured and treated with rhein (0, 50, 100, 150, and 200 µM) for 24, 48, or 72 h. Relative cell viability assessed by the MTT assay after treatment was 100, 99, 85, 79, 63% for 24 h; 100, 98, 80, 51, 37% for 48 h, and 100, 97, 60, 36, 15% for 72 h, respectively. Cell apoptosis was detected with TUNEL staining and quantified with flow cytometry using annexin FITC-PI staining at 48 h after 100, 200 and 300 µm rhein. The percentage of apoptotic cells was 7.3, 21.9, 43.5%, respectively. We also measured the mRNA levels of caspase-3 and -9 using real-time PCR. Treatment with 100 µM rhein for 48 h significantly increased mRNA expression of caspase-3 and -9. The levels of apoptosis-related proteins including Bcl-2, Bax, Bcl-xL, and pro-caspase-3 were evaluated in rhein-treated cells. Rhein increased the Bax:Bcl-2 ratio but decreased the protein levels of Bcl-xL and pro-caspase-3. Moreover, rhein significantly increased the expression of cytochrome c and apoptotic protease activating factor 1, two critical components involved in mitochondrial pathway-mediated apoptosis. We conclude that rhein inhibits SGC-7901 proliferation by inducing apoptosis and this antitumor effect of rhein is mediated in part by an intrinsic mitochondrial pathway.

  3. Nickel nanowires induced and reactive oxygen species mediated apoptosis in human pancreatic adenocarcinoma cells

    Directory of Open Access Journals (Sweden)

    Kleve MG

    2011-07-01

    Full Text Available Md. Zakir Hossain1, Maurice G Kleve21Applied Biosciences (Bionanotechnology Research, Department of Applied Science, 2Molecular Biotechnology and Microscopy Laboratory, Department of Biology, University of Arkansas at Little Rock, Little Rock, Arkansas, USABackground: The ability to evade apoptosis is one of the key properties of cancer. The apoptogenic effect of nickel nanowires (Ni NWs on cancer cell lines has never been adequately addressed. Due to the unique physicochemical characteristics of Ni NWs, we envision the development of a novel anticancer therapeutics specifically for pancreatic cancer. Thus, we investigated whether Ni NWs induce ROS-mediated apoptosis in human pancreatic adenocarcinoma (Panc-1 cells. Methods: In this study Ni NWs were fabricated using the electrodeposition method. Synthesized Ni NWs were physically characterized by energy dispersive X-ray analysis, UV-Vis spectroscopy of NanoDrop 2000 (UV-Vis, magnetization study, scanning electron microscopy, and transmission electron microscopy. Assessment of morphological apoptotic characteristics by phase contrast microscopy (PCM, Ni-NWs-induced apoptosis staining with ethidium bromide (EB and acridine orange (AO followed by fluorescence microscopy (FM was performed. For molecular biological and biochemical characterization, Panc-1 cell culture and cytotoxic effect of Ni NWs were determined by using 3-(4, 5-dimethylthiazol-2-yl-2, 5-diphenyl tetrazolium bromide (MTT assay. Quantitative apoptosis was analyzed by flow cytometry staining with propidium iodide through cell cycle arrest and generation of ROS using 2', 7'-dichlorofluorescein diacetate fluorescence intensity. In all experiments, Panc-1 cancer cells without any treatment were used as the negative controls.Results: The intracellular uptake of Ni NWs through endocytosis by Panc-1 cells was observed by PCM. EB and AO staining of FM and MTT assay qualitatively and quantitatively confirmed the extent of apoptosis. Flow

  4. Vitamin C: A Novel Regulator of Neutrophil Extracellular Trap Formation

    Directory of Open Access Journals (Sweden)

    Ramesh Natarajan

    2013-08-01

    Full Text Available Introduction: Neutrophil extracellular trap (NET formation was recently identified as a novel mechanism to kill pathogens. However, excessive NET formation in sepsis can injure host tissues. We have recently shown that parenteral vitamin C (VitC is protective in sepsis. Whether VitC alters NETosis is unknown. Methods: We used Gulo−/− mice as they lack the ability to synthesize VitC. Sepsis was induced by intraperitoneal infusion of a fecal stem solution (abdominal peritonitis, FIP. Some VitC deficient Gulo−/− mice received an infusion of ascorbic acid (AscA, 200 mg/kg 30 min after induction of FIP. NETosis was assessed histologically and by quantification for circulating free DNA (cf-DNA in serum. Autophagy, histone citrullination, endoplasmic reticulum (ER stress, NFκB activation and apoptosis were investigated in peritoneal PMNs. Results: Sepsis produced significant NETs in the lungs of VitC deficient Gulo−/− mice and increased circulating cf-DNA. This was attenuated in the VitC sufficient Gulo−/− mice and in VitC deficient Gulo−/− mice infused with AscA. Polymorphonuclear neutrophils (PMNs from VitC deficient Gulo−/− mice demonstrated increased activation of ER stress, autophagy, histone citrullination, and NFκB activation, while apoptosis was inhibited. VitC also significantly attenuated PMA induced NETosis in PMNs from healthy human volunteers.

  5. Chlorella vulgaris Induces Apoptosis of Human Non-Small Cell Lung Carcinoma (NSCLC) Cells.

    Science.gov (United States)

    Zhang, Zhi-Dong; Liang, Kai; Li, Kun; Wang, Guo-Quan; Zhang, Ke-Wei; Cai, Lei; Zhai, Shui-Ting; Chou, Kuo-Chen

    2017-01-01

    Chlorella vulgaris (C. vulgaris), a unicellular green microalga, has been widely used as a food supplement and reported to have antioxidant and anticancer properties. The current study was designed to assess the cytotoxic, apoptotic, and DNA-damaging effects of C. vulgaris growth factor (CGF), hot water C. vulgaris extracts, inlung tumor A549 and NCI-H460 cell lines. A549 cells, NCI-H460 cells, and normal human fibroblasts were treated with CGF at various concentrations (0-300 μg/ml) for 24 hr. The comet assay and γH2AX assay showed DNA damage in A549 and NCI-H460 cells upon CGF exposure. Evaluation of apoptosis by the TUNEL assay and DNA fragmentation analysis by agarose gel electrophoresis showed that CGF induced apoptosis in A549 and NCI-H460 cells. Chlorella vulgaris hot water extract induced apoptosis and DNA damage in human lung carcinoma cells. CGF can thus be considered a potential cytotoxic or genotoxic drug for treatment of lung carcinoma. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  6. [Effects of metformin on human oral cancer KB cell proliferation and apoptosis in vitro].

    Science.gov (United States)

    Wang, Fang; Xu, Jincheng; Xia, Fei; Liu, Zhe; Zhao, Surong; Liu, Hao; Jiang, Zhiwen

    2014-02-01

    To investigate the effects of metformin on the proliferation and apoptosis of human oral cancer cell line KB in vitro. Human oral cancer cell line KB was exposed to different doses of metformin (0, 1.25, 2.5, 5, 10, and 20 mmol/L), and the changes in cell viability were detected using MTT assay. Colony formation of the cells was observed following an 8-day metformin exposure. The changes in mitochondrial membrane potential were measured by JC-1 assay, and PI staining was used to observe the cell apoptosis. Western blotting was employed to detect the changes in the protein expressions of GRP78 and activated caspase-3. Metformin exposure caused time- and dose-dependent suppression of KB cell proliferation, and exposure to 5 mmol/L metformin for 24, 48 and 72 h resulted in cell survival rates of 68.0%, 36.9%, and 14.5%, respectively. Metformin significantly inhibited KB cell colony formation. Exposure of the cells to increased concentrations of metformin gradually increased the apoptotic rate and decreased mitochondrial membrane potential. Metformin caused an initial up-regulation followed by a down-regulation of GRP78 expression in KB cells and increased the expression of activated caspase-3. Metformin can inhibit the proliferation and induce apoptosis of KB cells, the mechanism of which may involve the activation of the mitochondrial apoptotic pathway and endoplasmic reticulum stress.

  7. Human Adipose Derived Stem Cells Induced Cell Apoptosis and S Phase Arrest in Bladder Tumor

    Directory of Open Access Journals (Sweden)

    Xi Yu

    2015-01-01

    Full Text Available The aim of this study was to determine the effect of human adipose derived stem cells (ADSCs on the viability and apoptosis of human bladder cancer cells. EJ and T24 cells were cocultured with ADSCs or cultured with conditioned medium of ADSCs (ADSC-CM, respectively. The cell counting and colony formation assay showed ADSCs inhibited the proliferation of EJ and T24 cells. Cell viability assessment revealed that the secretions of ADSCs, in the form of conditioned medium, were able to decrease cancer cell viability. Wound-healing assay suggested ADSC-CM suppressed migration of T24 and EJ cells. Moreover, the results of the flow cytometry indicated that ADSC-CM was capable of inducing apoptosis of T24 cells and inducing S phase cell cycle arrest. Western blot revealed ADSC-CM increased the expression of cleaved caspase-3 and cleaved PARP, indicating that ADSC-CM induced apoptosis in a caspase-dependent way. PTEN/PI3K/Akt pathway and Bcl-2 family proteins were involved in the mechanism of this reaction. Our study indicated that ADSCs may provide a promising and practicable manner for bladder tumor therapy.

  8. Growth and apoptosis of human natural killer cell neoplasms: role of interleukin-2/15 signaling.

    Science.gov (United States)

    Yamasaki, Satoshi; Maeda, Motoi; Ohshima, Koichi; Kikuchi, Masahiro; Otsuka, Teruhisa; Harada, Mine

    2004-10-01

    Interleukin (IL)-15 plays an important role in the survival of human natural killer (NK) cells. We investigated IL-2/15 signaling in NK cell neoplasms from five patients and in five cell lines (NK-92, KHYG-1, SNK-6, HANK1 and MOTN-1) compared to mature peripheral NK cells from 10 healthy subjects. Apoptosis of NK cell lines was prevented by addition of IL-15 in vitro. Blocking IL-2/15Rbeta on IL-2-stimulated NK-92 cells resulted in reduced expression of Bcl-X(L) and phosphorylated Stat5, which paralleled early apoptosis without altering Bcl-2 expression. These data add IL-2/15Rbeta to the list of factors important for the survival of NK cell neoplasms.

  9. Geoditin A Induces Oxidative Stress and Apoptosis on Human Colon HT29 Cells

    Directory of Open Access Journals (Sweden)

    Wing-Keung Liu

    2010-01-01

    Full Text Available Geoditin A, an isomalabaricane triterpene isolated from the marine sponge Geodia japonica, has been demonstrated to dissipate mitochondrial membrane potential, activate caspase 3, decrease cytoplasmic proliferating cell nuclear antigen (PCNA, and induce apoptosis of leukemia cells, but the underlying mechanism remains unclear [1]. In this study, we found fragmentation of Golgi structure, suppression of transferrin receptor expression, production of oxidants, and DNA fragmentation in human colon cancer HT29 cells after treatment with geoditin A for 24 h. This apoptosis was not abrogated by chelation of intracellular iron with salicylaldehyde isonicotinoyl hydrazone (SIH, but suppressed by N-acetylcysteine (NAC, a thiol antioxidant and GSH precursor, indicating that the cytotoxic effect of geoditin A is likely mediated by a NAC-inhibitable oxidative stress. Our results provide a better understanding of the apoptotic properties and chemotherapeutical potential of this marine triterpene.

  10. A protocol to assess cell cycle and apoptosis in human and mouse pluripotent cells

    Directory of Open Access Journals (Sweden)

    Edel Michael J

    2011-04-01

    Full Text Available Abstract Embryonic stem cells (ESC and induced pluripotent stem cells (iPSCs present a great opportunity to treat and model human disease as a cell replacement therapy. There is a growing pressure to understand better the signal transduction pathways regulating pluripotency and self-renewal of these special cells in order to deliver a safe and reliable cell based therapy in the near future. Many signal transduction pathways converge on two major cell functions associated with self-renewal and pluripotency: control of the cell cycle and apoptosis, although a standard method is lacking across the field. Here we present a detailed protocol to assess the cell cycle and apoptosis of ESC and iPSCs as a single reference point offering an easy to use standard approach across the field.

  11. The small molecule calactin induces DNA damage and apoptosis in human leukemia cells.

    Science.gov (United States)

    Lee, Chien-Chih; Lin, Yi-Hsiung; Chang, Wen-Hsin; Wu, Yang-Chang; Chang, Jan-Gowth

    2012-09-01

    We purified calactin from the roots of the Chinese herb Asclepias curassavica L. and analyzed its biologic effects in human leukemia cells. Our results showed that calactin treatment caused DNA damage and resulted in apoptosis. Increased phosphorylation levels of Chk2 and H2AX were observed and were reversed by the DNA damage inhibitor caffeine in calactin-treated cells. In addition, calactin treatment showed that a decrease in the expression of cell cycle regulatory proteins Cyclin B1, Cdk1, and Cdc25C was consistent with a G2/M phase arrest. Furthermore, calactin induced extracellular signal-regulated kinase (ERK) phosphorylation, activation of caspase-3, caspase-8, and caspase-9, and PARP cleavage. Pretreatment with the ERK inhibitor PD98059 significantly blocked the loss of viability in calactin-treated cells. It is indicated that calactin-induced apoptosis may occur through an ERK signaling pathway. Our data suggest that calactin is a potential anticancer compound.

  12. Advanced Role of Neutrophils in Common Respiratory Diseases

    Directory of Open Access Journals (Sweden)

    Jinping Liu

    2017-01-01

    Full Text Available Respiratory diseases, always being a threat towards the health of people all over the world, are most tightly associated with immune system. Neutrophils serve as an important component of immune defense barrier linking innate and adaptive immunity. They participate in the clearance of exogenous pathogens and endogenous cell debris and play an essential role in the pathogenesis of many respiratory diseases. However, the pathological mechanism of neutrophils remains complex and obscure. The traditional roles of neutrophils in severe asthma, chronic obstructive pulmonary diseases (COPD, pneumonia, lung cancer, pulmonary fibrosis, bronchitis, and bronchiolitis had already been reviewed. With the development of scientific research, the involvement of neutrophils in respiratory diseases is being brought to light with emerging data on neutrophil subsets, trafficking, and cell death mechanism (e.g., NETosis, apoptosis in diseases. We reviewed all these recent studies here to provide you with the latest advances about the role of neutrophils in respiratory diseases.

  13. Echinophora platyloba DC (Apiaceae crude extract induces apoptosis in human prostate adenocarcinoma cells (PC 3

    Directory of Open Access Journals (Sweden)

    Fatemeh Zare Shahneh

    2014-10-01

    Full Text Available Background: Prostate cancer is the second leading malignancy worldwide and the second prominent cause of cancer-related deaths among men. Therefore, there is a serious necessity for finding advanced alternative therapeutic measures against this lethal malignancy. In this article, we report the cytotoxicity and the mechanism of cell death of the methanolic extract prepared from Echinophora platyloba DC plant against human prostate adenocarcinoma PC 3 cell line and Human Umbilical Vein Endothelial Cells HUVEC cell line. Methods: Cytotoxicity and viability of the methanolic extract were assessed by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay and dye exclusion assay. Cell death enzyme-linked immunosorbent assay (ELISA was employed to quantify the nucleosome production resulting from nuclear DNA fragmentation during apoptosis and determine whether the mechanism involves induction of apoptosis or necrosis. The cell death was identified as apoptosis using terminal deoxynucleotidyl transferase (TdT-mediated dUTP nick end labeling (TUNEL assay and DNA fragmentation gel electrophoresis. Results: E. platyloba could decrease cell viability in malignant cells in a dose- and time-dependent manner. The IC50 values against PC 3 were determined as 236.136 ± 12.4, 143.400 ± 7.2, and 69.383 ± 1.29 μg/ml after 24, 36, and 48 h, respectively, but there was no significant activity in HUVEC normal cell (IC50 > 800 μg/ml. Morphological characterizations and DNA laddering assay showed that the methanolic extract treated cells displayed marked apoptotic characteristics such as nuclear fragmentation, appearance of apoptotic bodies, and DNA laddering fragment. Increase in an early apoptotic population was observed in a dose-dependent manner. PC 3 cell death elicited by the extract was found to be apoptotic in nature based a clear indication of TUNEL assay and gel electrophoresis DNA fragmentation, which is a hallmark of apoptosis

  14. Induction of apoptosis by Armillaria mellea constituent armillarikin in human hepatocellular carcinoma

    Directory of Open Access Journals (Sweden)

    Chen YJ

    2016-08-01

    Full Text Available Yu-Jen Chen,1–4 Chien-Chih Chen,5 Huey-Lan Huang6 1Department of Medical Research, 2Department of Radiation Oncology, Mackay Memorial Hospital, 3Institute of Traditional Medicine, School of Medicine, National Yang-Ming University, 4Institute of Pharmacology, Taipei Medical University, Taipei, 5Department of Biotechnology, HungKuang University, Taichung, 6Department of Bioscience Technology, College of Health Science, Chang Jung Christian University, Tainan, Taiwan Abstract: Armillaria mellea is a honey mushroom often used in the traditional Chinese medicine “Tianma”. Currently, this medicinal mushroom is also used as a dietary supplement in numerous Western and Eastern countries. Armillarikin was isolated from A. mellea, and we previously discovered that it induced cytotoxicity in human leukemia cells. In this study, we further investigated the cytotoxicity of armillarikin against liver and intrahepatic bile duct cancer cells. Armillarikin was cytotoxic against human hepatocellular carcinoma Huh7, HA22T, and HepG2 cells based on the 3-(4,5-dimethylthiazol-2-yl-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl-2H-tetrazolium and alamarBlue® assays. Armillarikin treatment also induced the collapse of the mitochondrial transmembrane potential of these cells. Furthermore, armillarikin-induced apoptotic cell death was demonstrated by sub-G1 chromosomal DNA formation by using flow cytometry. In addition, the apoptosis was inhibited by the pan-caspase inhibitor, Z-VAD-fmk. Immunoblotting also revealed the armillarikin-induced activation of procaspase-3, -8, and -9 and upregulation of the apoptosis- and cell cycle arrest-related phospho-histones 2 and 3, respectively. Moreover, reactive oxygen species scavengers also inhibited the armillarikin-induced apoptosis in human hepatocellular carcinoma, suggesting that reactive oxygen species formation played an important role in the armillarikin-induced apoptosis of human hepatocellular carcinoma. In

  15. Azurophil granule proteins constitute the major mycobactericidal proteins in human neutrophils and enhance the killing of mycobacteria in macrophages

    DEFF Research Database (Denmark)

    Jena, Prajna; Mohanty, Soumitra; Mohanty, Tirthankar

    2012-01-01

    Pathogenic mycobacteria reside in, and are in turn controlled by, macrophages. However, emerging data suggest that neutrophils also play a critical role in innate immunity to tuberculosis, presumably by their different antibacterial granule proteins. In this study, we purified neutrophil azurophil...... and specific granules and systematically analyzed the antimycobacterial activity of some purified azurophil and specific granule proteins against M. smegmatis, M. bovis-BCG and M. tuberculosis H37Rv. Using gel overlay and colony forming unit assays we showed that the defensin-depleted azurophil granule...... proteins (AZP) were more active against mycobacteria compared to other granule proteins and cytosolic proteins. The proteins showing antimycobacterial activity were identified by MALDI-TOF mass spectrometry. Electron microscopic studies demonstrate that the AZP disintegrate bacterial cell membrane...

  16. Immune modulation by neutrophil subsets

    NARCIS (Netherlands)

    Kamp, V.M.

    2013-01-01

    We show that human neutrophils can suppress T-cell proliferation in acute systemic inflammation and thus have anti-inflammatory functions, next to their well-known pro-inflammatory functions. The suppression is mediated by ROS production and integrin MAC-1, which are also important for the

  17. Suppression of oxidative burst in human neutrophils with the naturally occurring serotonin derivative isomer from Leuzea carthamoides

    Czech Academy of Sciences Publication Activity Database

    Nosáľ, R.; Perečko, T.; Jančinová, V.; Drábiková, K.; Harmatha, Juraj; Sviteková, K.

    2010-01-01

    Roč. 31, Suppl.2 (2010), s. 69-72 ISSN 0172-780X R&D Projects: GA ČR(CZ) GA203/07/1227 Institutional research plan: CEZ:AV0Z40550506 Keywords : N-feruloylserotonin * neutrophil chemiluminescence * protein kinase C Subject RIV: CC - Organic Chemistry Impact factor: 1.621, year: 2010 http://node.nel.edu

  18. Neutrophils at work

    DEFF Research Database (Denmark)

    Nauseef, William M; Borregaard, Niels

    2014-01-01

    In this Review we discuss data demonstrating recently recognized aspects of neutrophil homeostasis in the steady state, granulopoiesis in 'emergency' conditions and interactions of neutrophils with the adaptive immune system. We explore in vivo observations of the recruitment of neutrophils from ...

  19. Zinc oxide nanoparticles induce apoptosis and autophagy in human ovarian cancer cells

    Directory of Open Access Journals (Sweden)

    Bai D

    2017-09-01

    Full Text Available Ding-Ping Bai,1,* Xi-Feng Zhang,2,* Guo-Liang Zhang,3,4 Yi-Fan Huang,1 Sangiliyandi Gurunathan5 1Fujian Key Laboratory of Traditional Chinese Veterinary Medicine and Animal Health, Fujian Agriculture and Forestry University, Fuzhou, China; 2College of Biological and Pharmaceutical Engineering, Wuhan Polytechnic University, Wuhan, China; 3Dong-E-E-Jiao Co., Ltd., Shandong, China; 4National Engineering Research Center for Gelatin-based Traditional Chinese Medicine, Shandong, China; 5Department of Stem Cell and Regenerative Biotechnology, Konkuk University, Seoul, Republic of Korea *These authors contributed equally to this work Background: Zinc oxide nanoparticles (ZnO NPs are frequently used in industrial products such as paint, surface coating, and cosmetics, and recently, they have been explored in biologic and biomedical applications. Therefore, this study was undertaken to investigate the effect of ZnO NPs on cytotoxicity, apoptosis, and autophagy in human ovarian cancer cells (SKOV3. Methods: ZnO NPs with a crystalline size of 20 nm were characterized with various analytical techniques, including ultraviolet-visible spectroscopy, X-ray diffraction, transmission electron microscopy, Fourier transform infrared spectroscopy, and atomic force microscopy. The cytotoxicity, apoptosis, and autophagy were examined using a series of cellular assays. Results: Exposure of cells to ZnO NPs resulted in a dose-dependent loss of cell viability, and the characteristic apoptotic features such as rounding and loss of adherence, enhanced reactive oxygen species generation, and loss of mitochondrial membrane potential were observed in the ZnO NP-treated cells. Furthermore, the cells treated with ZnO NPs showed significant double-strand DNA breaks, which are gained evidences from significant number of γ-H2AX and Rad51 expressed cells. ZnO NP-treated cells showed upregulation of p53 and LC3, indicating that ZnO NPs are able to upregulate apoptosis and autophagy

  20. The cardiac glycoside oleandrin induces apoptosis in human colon cancer cells via the mitochondrial pathway.

    Science.gov (United States)

    Pan, Li; Zhang, Yuming; Zhao, Wanlu; Zhou, Xia; Wang, Chunxia; Deng, Fan

    2017-07-01

    Evidence indicates that the cardiac glycoside oleandrin exhibits cytotoxic activity against several different types of cancer. However, the specific mechanisms underlying oleandrin-induced anti-tumor effects remain largely unknown. The present study examined the anti-cancer effect and underlying mechanism of oleandrin on human colon cancer cells. The cytotoxicity and IC50 of five small molecule compounds (oleandrin, neriifolin, strophanthidin, gitoxigenin, and convallatoxin) in human colon cancer cell line SW480 cells and normal human colon cell line NCM460 cells were determined by cell counting and MTT assays, respectively. Apoptosis was determined by staining cells with annexin V-FITC and propidium iodide, followed by flow cytometry. Intracellular Ca 2+ was determined using Fluo-3 AM,glutathione (GSH) levels were measured using a GSH detection kit,and the activity of caspase-3, -9 was measured using a peptide substrate. BAX, pro-caspase-3, -9, cytochrome C and BCL-2 expression were determined by Western blotting. Oleandrin significantly decreased cell viabilities in SW480, HCT116 and RKO cells. The IC50 for SW480 cells was 0.02 µM, whereas for NCM460 cells 0.56 µM. More interestingly, the results of flow cytometry showed that oleandrin potently induced apoptosis in SW480 and RKO cells. Oleandrin downregulated protein expression of pro-caspase-3, -9, but enhanced caspase-3, -9 activities. These effects were accompanied by upregulation of protein expression of cytochrome C and BAX, and downregulation of BCL-2 protein expression in a concentration-dependent manner. Furthermore, oleandrin increased intracellular Ca 2+ concentration, but decreased GSH concentration in the cells. The present results suggest that oleandrin induces apoptosis in human colorectal cancer cells via the mitochondrial pathway. Our findings provide new insight into the mechanism of anti-cancer property of oleandrin.

  1. Trichodermin induces cell apoptosis through mitochondrial dysfunction and endoplasmic reticulum stress in human chondrosarcoma cells

    International Nuclear Information System (INIS)

    Su, Chen-Ming; Wang, Shih-Wei; Lee, Tzong-Huei; Tzeng, Wen-Pei; Hsiao, Che-Jen; Liu, Shih-Chia; Tang, Chih-Hsin

    2013-01-01

    Chondrosarcoma is the second most common primary bone tumor, and it responds poorly to both chemotherapy and radiation treatment. Nalanthamala psidii was described originally as Myxosporium in 1926. This is the first study to investigate the anti-tumor activity of trichodermin (trichothec-9-en-4-ol, 12,13-epoxy-, acetate), an endophytic fungal metabolite from N. psidii against human chondrosarcoma cells. We demonstrated that trichodermin induced cell apoptosis in human chondrosarcoma cell lines (JJ012 and SW1353 cells) instead of primary chondrocytes. In addition, trichodermin triggered endoplasmic reticulum (ER) stress protein levels of IRE1, p-PERK, GRP78, and GRP94, which were characterized by changes in cytosolic calcium levels. Furthermore, trichodermin induced the upregulation of Bax and Bid, the downregulation of Bcl-2, and the dysfunction of mitochondria, which released cytochrome c and activated caspase-3 in human chondrosarcoma. In addition, animal experiments illustrated reduced tumor volume, which led to an increased number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells and an increased level of cleaved PARP protein following trichodermin treatment. Together, this study demonstrates that trichodermin is a novel anti-tumor agent against human chondrosarcoma cells both in vitro and in vivo via mitochondrial dysfunction and ER stress. - Highlights: • Trichodermin induces chondrosarcoma apoptosis. • ER stress is involved in trichodermin-induced cell death. • Trichodermin induces chondrosarcoma death in vivo.

  2. Trichodermin induces cell apoptosis through mitochondrial dysfunction and endoplasmic reticulum stress in human chondrosarcoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Su, Chen-Ming [Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan (China); Wang, Shih-Wei [Department of Medicine, Mackay Medical College, New Taipei City, Taiwan (China); Lee, Tzong-Huei [Graduate Institute of Pharmacognosy, Taipei Medical University, Taipei, Taiwan (China); Tzeng, Wen-Pei [Graduate Institute of Sports and Health, National Changhua University of Education, Changhua, Taiwan (China); Hsiao, Che-Jen [School of Respiratory Therapy, College of Medicine, Taipei Medical University, Taipei, Taiwan (China); Liu, Shih-Chia [Department of Orthopaedics, Mackay Memorial Hospital, Taipei, Taiwan (China); Tang, Chih-Hsin, E-mail: chtang@mail.cmu.edu.tw [Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan (China); Department of Pharmacology, School of Medicine, China Medical University, Taichung, Taiwan (China); Department of Biotechnology, College of Health Science, Asia University, Taichung, Taiwan (China)

    2013-10-15

    Chondrosarcoma is the second most common primary bone tumor, and it responds poorly to both chemotherapy and radiation treatment. Nalanthamala psidii was described originally as Myxosporium in 1926. This is the first study to investigate the anti-tumor activity of trichodermin (trichothec-9-en-4-ol, 12,13-epoxy-, acetate), an endophytic fungal metabolite from N. psidii against human chondrosarcoma cells. We demonstrated that trichodermin induced cell apoptosis in human chondrosarcoma cell lines (JJ012 and SW1353 cells) instead of primary chondrocytes. In addition, trichodermin triggered endoplasmic reticulum (ER) stress protein levels of IRE1, p-PERK, GRP78, and GRP94, which were characterized by changes in cytosolic calcium levels. Furthermore, trichodermin induced the upregulation of Bax and Bid, the downregulation of Bcl-2, and the dysfunction of mitochondria, which released cytochrome c and activated caspase-3 in human chondrosarcoma. In addition, animal experiments illustrated reduced tumor volume, which led to an increased number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells and an increased level of cleaved PARP protein following trichodermin treatment. Together, this study demonstrates that trichodermin is a novel anti-tumor agent against human chondrosarcoma cells both in vitro and in vivo via mitochondrial dysfunction and ER stress. - Highlights: • Trichodermin induces chondrosarcoma apoptosis. • ER stress is involved in trichodermin-induced cell death. • Trichodermin induces chondrosarcoma death in vivo.

  3. Induction of apoptosis by hydrolyzable tannins from Eugenia jambos L. on human leukemia cells.

    Science.gov (United States)

    Yang, L L; Lee, C Y; Yen, K Y

    2000-08-31

    Eugenia jambos L. (Myrtaceae) is an antipyretic and anti-inflammatory herb of Asian folk medicine. A 70% acetone extract exerted the strongest cytotoxic effects on human leukemia cells (HL-60) from a preliminary screening of 15 plants. The cytotoxic principles were separated by bio-assay-guided fractionation to HL-60 cells; two hydrolyzable tannins (1-O-galloyl castalagin and casuarinin) were isolated from the 70% acetone extract. All significantly inhibited human promyelocytic leukemia cell line HL-60 and showed less cytotoxicity to human adenocarcinoma cell line SK-HEP-1 and normal cell lines of human lymphocytes and Chang liver cells. Thus, these compounds were exhibited the dose-dependent manner in HL-60 cells and the IC(50) were 10.8 and 12.5 microM, respectively. Flow cytometric analysis demonstrated the presence of apoptotic cells with low DNA content, a decrease of cell population at G(2)/M phase, and a concomitant increase of cell population at G(1) phase. The apoptosis induced by these two compounds was also demonstrated by DNA fragmentation assay and microscopic observation. These results suggest that the cytotoxic mechanism of both antitumor principle constituents might be the induction of apoptosis in HL-60 cells.

  4. 3'-Azido-3'-deoxythymidine (AZT) induces apoptosis and alters metabolic enzyme activity in human placenta

    International Nuclear Information System (INIS)

    Collier, Abby C.; Helliwell, Rachel J.A.; Keelan, Jeffrey A.; Paxton, James W.; Mitchell, Murray D.; Tingle, Malcolm D.

    2003-01-01

    The anti-HIV drug 3'-azido-3'-deoxythymidine (AZT) is the drug of choice for preventing maternal-fetal HIV transmission during pregnancy. Our aim was to assess the cytotoxic effects of AZT on human placenta in vitro. The mechanisms of AZT-induced effects were investigated using JEG-3 choriocarcinoma cells and primary explant cultures from term and first-trimester human placentas. Cytotoxicity measures included trypan blue exclusion, MTT, and reactive oxygen species (ROS) assays. Apoptosis was measured with an antibody specific to cleaved caspase-3 and by rescue of cells by the general caspase inhibitor Boc-D-FMK. The effect of AZT on the activities of glutathione-S-transferase, β-glucuronidase, UDP-glucuronosyl transferase, cytochrome P450 (CYP) 1A, and CYP reductase (CYPR) in the placenta was assessed using biochemical assays and immunoblotting. AZT increased ROS levels, decreased cellular proliferation rates, was toxic to mitochondria, and initiated cell death by a caspase-dependent mechanism in the human placenta in vitro. In the absence of serum, the effects of AZT were amplified in all the models used. AZT also increased the amounts of activity of GST, β-glucuronidase, and CYP1A, whereas UGT and CYPR were decreased. We conclude that AZT causes apoptosis in the placenta and alters metabolizing enzymes in human placental cells. These findings have implications for the safe administration of AZT in pregnancy with respect to the maintenance of integrity of the maternal-fetal barrier

  5. Apoptotic-like tumor cells and apoptotic neutrophils in mitochondrion-rich gastric adenocarcinomas: a comparative study with light and electronmicroscopy between these two forms of cell death

    Directory of Open Access Journals (Sweden)

    Antonio Venuti

    2013-04-01

    Full Text Available Mitochondrion-rich adenocarcinomas represent a rare variant of gastric adenocarcinomas composed predominantly of columnar adenocarcinoma cells with eosinophilic cytoplasm, a strong supranuclear immunoreactivity for antimitochondrial antibody, and a marked neutrophil infiltration associated to tumor cell death. The purpose of this work is to investigate, using correlated light and electron microscopy, mitochondrion-rich gastric adenocarcinomas focusing on the nature of the death in neoplastic cells and in infiltrating neutrophils. Adenocarcinoma cells, single or in small clusters, showed convoluted nuclei, irregularly condensed chromatin, loss of microvilli, and nuclear envelope dilatation. No nuclear fragmentation was observed in these dying cells and the plasma membrane did not show signs of disruption. These ultrastructural findings represent intermediate aspects between apoptosis and necrosis and are compatible with apoptosis-like programmed cell death. By contrast, some infiltrating neutrophils showed ultrastructural signs of classic apoptosis such as chromatin condensation into compact geometric (globular, crescent-shaped figures, tightly packed cytoplasmic granules and intact cell membrane. Our study provides ultrastructural evidence of apoptosis-like tumour cell death in mitochondrion-rich gastric carcinomas and confirms that stereotyped outcome either as apoptosis or necrosis of tumor cells cannot always be expected in human neoplasms.

  6. Fluorine-induced apoptosis and lipid peroxidation in human hair follicles in vitro.

    Science.gov (United States)

    Wang, Zheng-hui; Li, Xiao-li; Yang, Zhuang-qun; Xu, Min

    2010-12-01

    Fluoride is an essential trace element for human body; however, exposure to high amounts of fluoride has been documented to be correlated with an increasing risk of hair loss. To date, little is known about the mechanism(s) of how fluoride affects hair follicles. Here, we demonstrated that middle (1.0 mmol/L) and high (10.0 mmol/L) concentrations of sodium fluoride (NaF) significantly inhibited hair follicle elongation in vitro, but low NaF (0.1 mmol/L) showed little influence. Moreover, treatment with high levels of NaF resulted in a marked increase in terminal dUTP nick end labeling-positive cells in the outer layer of the outer root sheath, the dermal sheath, and the lower bulb matrix surrounding dermal papilla. Furthermore, the enhanced apoptosis was coupled with an increased oxidative stress manifested as higher malondialdehyde content. Additionally, the presence of selenium considerably antagonized the effects of middle NaF on hair follicles, with regard to either the suppression of hair growth or the induction of oxidative stress and apoptosis. In conclusion, exposure to high levels of fluoride compromises hair follicle growth and accelerate cell apoptosis in vitro. The toxicity of fluoride can be reduced by selenium, at least partially via the suppression of intracellular oxidative stress.

  7. Regorafenib Induces Apoptosis and Inhibits Metastatic Potential of Human Bladder Carcinoma Cells.

    Science.gov (United States)

    Hsu, Fei-Ting; Sun, Cho-Chin; Wu, Chia-Hsing; Lee, Yen-Ju; Chiang, Chih-Hung; Wang, Wei-Shu

    2017-09-01

    The aim of the present study was to verify the effects of regorafenib on apoptosis and metastatic potential in TSGH 8301 human bladder carcinoma cells in vitro. Cells were treated with different concentration of regorafenib for different periods of time. Effects of regorafenib on cell viability, apoptosis pathways, metastatic potential, and expression of metastatic and anti-apoptotic proteins were evaluated with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay, flow cytometry, cell migration and invasion assay, and western blotting. We found regorafenib significantly reduced cell viability, cell migration and invasion, and expression of metastatic and anti-apoptotic proteins. In addition, regorafenib significantly induced accumulation of sub-G 1 phase cells, loss of mitochondrial membrane potential, and expression of active caspase-3 and caspase-8. These results show that regorafenib not only induces apoptosis, but also inhibits metastatic potential in bladder cancer TSGH 8301 cells in vitro. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  8. Effects of Raloxifene on the Proliferation and Apoptosis of Human Aortic Valve Interstitial Cells

    Directory of Open Access Journals (Sweden)

    Zhimin Fu

    2016-01-01

    Full Text Available We aimed to explore the effects of raloxifene (RAL on the proliferation and apoptosis of human aortic valve interstitial cells (AVICs. Different concentrations of RAL were used to act on AVICs. MTS kit is used to test the effects of different concentrations of RAL on the proliferation of AVICs. Cell cycle and apoptosis test used flow cytometry after seven-day treatment. The relative expression levels of caspase-3 and caspase-8 are tested with RT-qPCR and Western blot. The results of MTS testing revealed that the absorbance value (OD value of the cells in the concentration groups of 10 and 100 nmol/L RAL at a wavelength of 490 nm at five, seven, and nine days significantly decreased compared with that in the control group. Meanwhile, the results of flow cytometry of the cells collected after seven days showed that the ratio of the S stage and the cell apoptosis rate of AVICs can be significantly reduced by RAL in the concentration groups of 10 and 100 nmol/L. The mRNA and protein expressions of caspase-3 and caspase-8 were significantly decreased compared with those in the control group. This study laid the foundation for further treatment of aortic valve disease by using RAL.

  9. [Apoptosis of human lung carcinoma cell line GLC-82 induced by high power electromagnetic pulse].

    Science.gov (United States)

    Cao, Xiao-zhe; Zhao, Mei-lan; Wang, De-wen; Dong, Bo

    2002-09-01

    Electromagnetic pulse (EMP) could be used for sterilization of food and the efficiency is higher than 2450 MHz continuous microwave done. This study was designed to evaluate the effect of electromagnetic pulse (EMP) on apoptosis of human lung carcinoma cell line GLC-82, so that to explore and develop therapeutic means for cancer. The injury changes in GLC-82 cells after irradiated with EMP (electric field intensity was 60 kV/m, 5 pulses/2 min) were analyzed by cytometry, MTT chronometry, and flow cytometry. The immunohistochemical SP staining was used to determine the expressions of bcl-2 protein and p53 protein. The stained positive cells were analyzed by CMIAS-II image analysis system at a magnification 400. All data were analyzed by SPSS8.0 software. EMP could obviously inhibited proliferation and activity of lung carcinoma cell line GLC-82. The absorbance value (A570) of MTT decreased immediately, at 0 h, 1 h, and 6 h after the GLC-82 cells irradiated by EMP as compared with control group. The highest apoptosis rate was found to reach 13.38% by flow cytometry at 6 h after EMP irradiation. Down-regulation of bcl-2 expression and up-regulation of p53 expression were induced by EMP. EMP promotes apoptosis of GLC-82 cells. At same time, EMP can down-regulate bcl-2 expression and up-regulate p53 expression in GLC-82 cells. The bcl-2 and the p53 protein may involve the apoptotic process.

  10. Viability, Apoptosis, Proliferation, Activation, and Cytokine Secretion of Human Keratoconus Keratocytes after Cross-Linking

    Directory of Open Access Journals (Sweden)

    Xuefei Song

    2015-01-01

    Full Text Available Purpose. The purpose of this study was to determine the impact of cross-linking (CXL on viability, apoptosis, proliferation, activation, and cytokine secretion of human keratoconus (KC keratocytes, in vitro. Methods. Primary KC keratocytes were cultured in DMEM/Ham’s F12 medium supplemented with 10% FCS and underwent UVA illumination (370 nm, 2 J/cm2 during exposure to 0.1% riboflavin and 20% Dextran in PBS. Twenty-four hours after CXL, viability was assessed using Alamar blue assay; apoptosis using APO-DIRECT Kit; proliferation using ELISA-BrdU kit; and CD34 and alpha-smooth muscle actin (α-SMA expression using flow cytometry. Five and 24 hours after CXL, FGFb, HGF, TGFβ1, VEGF, KGF, IL-1β, IL-6, and IL-8 secretion was measured using enzyme-linked-immunoabsorbent assay (ELISA. Results. Following CXL, cell viability and proliferation decreased (P0.06. Five hours after CXL, FGFb secretion increased significantly (P=0.037; however no other cytokine secretion differed significantly from controls after 5 or 24 hours (P>0.12. Conclusions. Cross-linking decreases viability, triggers apoptosis, and inhibits proliferation, without an impact on multipotent hematopoietic stem cell transformation and myofibroblastic transformation of KC keratocytes. CXL triggers FGFb secretion of KC keratocytes transiently (5 hours, normalizing after 24 hours.

  11. Bee venom induces apoptosis and suppresses matrix metaloprotease-2 expression in human glioblastoma cells

    Directory of Open Access Journals (Sweden)

    Mohsen Sisakht

    Full Text Available Abstract Glioblastoma is the most common malignant brain tumor representing with poor prognosis, therapy resistance and high metastasis rate. Increased expression and activity of matrix metalloproteinase-2, a member of matrix metalloproteinase family proteins, has been reported in many cancers including glioblastoma. Inhibition of matrix metalloproteinase-2 expression has resulted in reduced aggression of glioblastoma tumors in several reports. In the present study, we evaluated effect of bee venom on expression and activity of matrix metalloproteinase-2 as well as potential toxicity and apoptogenic properties of bee venom on glioblastoma cells. Human A172 glioblastoma cells were treated with increasing concentrations of bee venom. Then, cell viability, apoptosis, matrix metalloproteinase-2 expression, and matrix metalloproteinase-2 activity were measured using MMT assay, propidium iodide staining, real time-PCR, and zymography, respectively. The IC50 value of bee venom was 28.5 µg/ml in which it leads to decrease of cell viability and induction of apoptosis. Incubation with bee venom also decreased the expression of matrix metalloproteinase-2 in this cell line (p < 0.05. In zymography, there was a reverse correlation between bee venom concentration and total matrix metalloproteinase-2 activity. Induction of apoptosis as well as inhibition of matrix metalloproteinase-2 activity and expression can be suggested as molecular mechanisms involved in cytotoxic and antimetastatic effects of bee venom against glioblastoma cells.

  12. RITA inhibits growth of human hepatocellular carcinoma through induction of apoptosis.

    Science.gov (United States)

    Wang, Haihe; Chen, Guofu; Wang, Hongzhi; Liu, Chunbo

    2013-01-01

    RBP-J-interacting and tubulin-associated (RITA) is a novel RBP-J-interacting protein that downregulates Notch-mediated transcription. The current study focuses on the antitumor effect of RITA in human hepatocellular carcinoma (HCC) and aims to explore its molecular mechanism. Thirty paired HCC and adjacent non-tumoral liver samples were analyzed by real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). RITA overexpression was induced by transfection of a pcDNA3.1-Flag-RITA plasmid into HepG2 cells. RITA knockdown was achieved by siRNA transfection. mRNA and protein expression of target genes were quantified by qRT-PCR and Western blotting, respectively. Cell proliferation and apoptosis were measured using MTT assay and flow cytometry. Our results demonstrate that adjacent nontumoral liver samples exhibited increased RITA expression compared to HCC tissues (p RITA levels were associated with tumor differentiation status. Overexpression of RITA suppressed cell proliferation and promoted early apoptosis, while its silencing promoted cell growth dramatically (p RITA overexpression upregulated p53 and reduced cyclin E levels, whereas silencing of RITA had the opposite effect on p53 and cyclin E expression. Our in vitro results represent the first evidence that RITA might suppress tumor growth and induce apoptosis in HCCs, and may be a potent antitumoral agent for HCC treatment that deserves further exploration.

  13. Viral single-strand DNA induces p53-dependent apoptosis in human embryonic stem cells.

    Science.gov (United States)

    Hirsch, Matthew L; Fagan, B Matthew; Dumitru, Raluca; Bower, Jacquelyn J; Yadav, Swati; Porteus, Matthew H; Pevny, Larysa H; Samulski, R Jude

    2011-01-01

    Human embryonic stem cells (hESCs) are primed for rapid apoptosis following mild forms of genotoxic stress. A natural form of such cellular stress occurs in response to recombinant adeno-associated virus (rAAV) single-strand DNA genomes, which exploit the host DNA damage response for replication and genome persistence. Herein, we discovered a unique DNA damage response induced by rAAV transduction specific to pluripotent hESCs. Within hours following rAAV transduction, host DNA damage signaling was elicited as measured by increased gamma-H2AX, ser15-p53 phosphorylation, and subsequent p53-dependent transcriptional activation. Nucleotide incorporation assays demonstrated that rAAV transduced cells accumulated in early S-phase followed by the induction of apoptosis. This lethal signaling sequalae required p53 in a manner independent of transcriptional induction of Puma, Bax and Bcl-2 and was not evident in cells differentiated towards a neural lineage. Consistent with a lethal DNA damage response induced upon rAAV transduction of hESCs, empty AAV protein capsids demonstrated no toxicity. In contrast, DNA microinjections demonstrated that the minimal AAV origin of replication and, in particular, a 40 nucleotide G-rich tetrad repeat sequence, was sufficient for hESC apoptosis. Our data support a model in which rAAV transduction of hESCs induces a p53-dependent lethal response that is elicited by a telomeric sequence within the AAV origin of replication.

  14. Viral single-strand DNA induces p53-dependent apoptosis in human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Matthew L Hirsch

    Full Text Available Human embryonic stem cells (hESCs are primed for rapid apoptosis following mild forms of genotoxic stress. A natural form of such cellular stress occurs in response to recombinant adeno-associated virus (rAAV single-strand DNA genomes, which exploit the host DNA damage response for replication and genome persistence. Herein, we discovered a unique DNA damage response induced by rAAV transduction specific to pluripotent hESCs. Within hours following rAAV transduction, host DNA damage signaling was elicited as measured by increased gamma-H2AX, ser15-p53 phosphorylation, and subsequent p53-dependent transcriptional activation. Nucleotide incorporation assays demonstrated that rAAV transduced cells accumulated in early S-phase followed by the induction of apoptosis. This lethal signaling sequalae required p53 in a manner independent of transcriptional induction of Puma, Bax and Bcl-2 and was not evident in cells differentiated towards a neural lineage. Consistent with a lethal DNA damage response induced upon rAAV transduction of hESCs, empty AAV protein capsids demonstrated no toxicity. In contrast, DNA microinjections demonstrated that the minimal AAV origin of replication and, in particular, a 40 nucleotide G-rich tetrad repeat sequence, was sufficient for hESC apoptosis. Our data support a model in which rAAV transduction of hESCs induces a p53-dependent lethal response that is elicited by a telomeric sequence within the AAV origin of replication.

  15. The nonstructural protein NP1 of human bocavirus 1 induces cell cycle arrest and apoptosis in Hela cells

    International Nuclear Information System (INIS)

    Sun, Bin; Cai, Yingyue; Li, Yongshu; Li, Jingjing; Liu, Kaiyu; Li, Yi; Yang, Yongbo

    2013-01-01

    Human bocavirus type 1 (HBoV1) is a newly identified pathogen associated with human respiratory tract illnesses. Previous studies demonstrated that proteins of HBoV1 failed to cause cell death, which is considered as a possible common feature of bocaviruses. However, our work showed that the NP1 of HBoV1 induced apoptotic cell death in Hela cells in the absence of viral genome replication and expression of other viral proteins. Mitochondria apoptotic pathway was involved in the NP1-induced apoptosis that was confirmed by apoptotic characteristics including morphological changes, DNA fragmentation and caspase activation. We also demonstrated that the cell cycle of NP1-transfected Hela cells was transiently arrested at G2/M phase followed by rapid appearance of apoptosis and that the N terminal domain of NP1 was critical to its nuclear localization and function in apoptosis induction in Hela cells. These findings might provide alternative information for further study of mechanism of HBoV1 pathogenesis. - Highlights: ► NP1 protein of HBoV1 induced apoptosis in Hela cells was first reported. ► NP1 induced-apoptosis followed the cell cycle arrest at G2/M phase. ► The NP1 induced-apoptosis was mediated by mitochondrion apoptotic pathway. ► N terminal of NP1 was critical for apoptosis induction and nuclear localization

  16. The nonstructural protein NP1 of human bocavirus 1 induces cell cycle arrest and apoptosis in Hela cells

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Bin; Cai, Yingyue; Li, Yongshu [College of Life Science, Central China Normal University, Wuhan 430079, Hubei (China); Li, Jingjing [College of Life Science, Hubei Normal University, Huangshi 435002, Hubei (China); Liu, Kaiyu [College of Life Science, Central China Normal University, Wuhan 430079, Hubei (China); Li, Yi, E-mail: johnli2668@hotmail.com [College of Life Science, Central China Normal University, Wuhan 430079, Hubei (China); Bioengineering Department, Wuhan Bioengineering Institute, Wuhan 430415, Hubei (China); Yang, Yongbo, E-mail: yongboyang@mail.ccnu.edu.cn [College of Life Science, Central China Normal University, Wuhan 430079, Hubei (China)

    2013-05-25

    Human bocavirus type 1 (HBoV1) is a newly identified pathogen associated with human respiratory tract illnesses. Previous studies demonstrated that proteins of HBoV1 failed to cause cell death, which is considered as a possible common feature of bocaviruses. However, our work showed that the NP1 of HBoV1 induced apoptotic cell death in Hela cells in the absence of viral genome replication and expression of other viral proteins. Mitochondria apoptotic pathway was involved in the NP1-induced apoptosis that was confirmed by apoptotic characteristics including morphological changes, DNA fragmentation and caspase activation. We also demonstrated that the cell cycle of NP1-transfected Hela cells was transiently arrested at G2/M phase followed by rapid appearance of apoptosis and that the N terminal domain of NP1 was critical to its nuclear localization and function in apoptosis induction in Hela cells. These findings might provide alternative information for further study of mechanism of HBoV1 pathogenesis. - Highlights: ► NP1 protein of HBoV1 induced apoptosis in Hela cells was first reported. ► NP1 induced-apoptosis followed the cell cycle arrest at G2/M phase. ► The NP1 induced-apoptosis was mediated by mitochondrion apoptotic pathway. ► N terminal of NP1 was critical for apoptosis induction and nuclear localization.

  17. 3-Bromopyruvate and sodium citrate induce apoptosis in human gastric cancer cell line MGC-803 by inhibiting glycolysis and promoting mitochondria-regulated apoptosis pathway.

    Science.gov (United States)

    Guo, Xingyu; Zhang, Xiaodong; Wang, Tingan; Xian, Shulin; Lu, Yunfei

    2016-06-17

    Cancer cells are mainly dependent on glycolysis to generate adenosine triphosphate (ATP) and intermediates required for cell growth and proliferation. Thus, inhibition of glycolysis might be of therapeutic value in antitumor treatment. Our previously studies had found that both 3-bromopyruvate (BP) and sodium citrate (SCT) can inhibit tumor growth and proliferation in vitro and in vivo. However, the mechanism involved in the BP and SCT mediated antitumor activity is not entirely clear. In this work, it is demonstrated that BP inhibits the enzyme hexokinase (HK) activity and SCT suppresses the phosphofructokinase (PFK) activity respectively, both the two agents decrease viability, ATP generation and lactate content in the human gastric cancer cell line MGC-803. These effects are directly correlated with blockage of glycolysis. Furthermore, BP and SCT can induce the characteristic manifestations of mitochondria-regulated apoptosis, such as down-regulation of anti-apoptosis proteins Bcl-2 and Survivin, up-regulation of pro-apoptosis protein Bax, activation of caspase-3, as well as leakage of cytochrome c (Cyt-c). In summary, our results provided evidences that BP and SCT inhibit the MGC-803 cells growth and proliferation might be correlated with inhibiting glycolysis and promoting mitochondria-regulated apoptosis. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  18. Induction of apoptosis in cultured human proximal tubule cells by fumonisins and fumonisin metabolites

    Energy Technology Data Exchange (ETDEWEB)

    Seefelder, W; Humpf, H -U; Schwerdt, G; Freudinger, R; Gekle, M

    2003-10-15

    Fumonisin B{sub 1} (FB{sub 1}) causes apoptosis in a variety of cell types and tissues but the apoptotic potential of other fumonisins and fumonisin metabolites has not been determined and the underlying mechanisms are not completely understood. In our studies we exposed human proximal tubule-derived cells (IHKE cells) to FB{sub 1}, fumonisin B{sub 2} (FB{sub 2}), fumonisin B{sub 3} (FB{sub 3}), hydrolyzed fumonisin B{sub 1} (HFB{sub 1}) and N-palmitoyl-hydrolyzed fumonisin B{sub 1} (N-Pal-HFB{sub 1}) and investigated caspase-3 activation, chromatin condensation and DNA fragmentation. Exposure to 10 {mu}mol/L FB{sub 1} for 24 h led to a significant increase in caspase-3 activity, chromatin condensation and to DNA fragmentation. All other tested compounds did not show any significant activation of caspase-3 activity nor chromatin condensation and DNA-fragmentation. Furthermore, we examined if a sphinganine accumulation is correlated with an induction of apoptosis in IHKE cells. Therefore we used a liquid chromatography/electrospray ionization-mass spectrometry(LC/ESI-MS)-method using phytosphingosine as an internal standard to determine sphinganine and sphingosine concentrations in IHKE cells. Whereas a significant increase of sphinganine (up to 7000% compared to control cells) was observed with all fumonisin-derivates, sphingosine levels nearly remained unchanged indicating that all substrates inhibited ceramide synthase effectively. These results demonstrate that all compounds let to increased sphinganine levels in IHKE cells but only FB{sub 1} was able to induce apoptosis. We conclude that the inhibition of the ceramide synthase is not per se a predictor whether or not fumonisins induce apoptosis.

  19. Induction of apoptosis in cultured human proximal tubule cells by fumonisins and fumonisin metabolites

    International Nuclear Information System (INIS)

    Seefelder, W.; Humpf, H.-U.; Schwerdt, G.; Freudinger, R.; Gekle, M.

    2003-01-01

    Fumonisin B 1 (FB 1 ) causes apoptosis in a variety of cell types and tissues but the apoptotic potential of other fumonisins and fumonisin metabolites has not been determined and the underlying mechanisms are not completely understood. In our studies we exposed human proximal tubule-derived cells (IHKE cells) to FB 1 , fumonisin B 2 (FB 2 ), fumonisin B 3 (FB 3 ), hydrolyzed fumonisin B 1 (HFB 1 ) and N-palmitoyl-hydrolyzed fumonisin B 1 (N-Pal-HFB 1 ) and investigated caspase-3 activation, chromatin condensation and DNA fragmentation. Exposure to 10 μmol/L FB 1 for 24 h led to a significant increase in caspase-3 activity, chromatin condensation and to DNA fragmentation. All other tested compounds did not show any significant activation of caspase-3 activity nor chromatin condensation and DNA-fragmentation. Furthermore, we examined if a sphinganine accumulation is correlated with an induction of apoptosis in IHKE cells. Therefore we used a liquid chromatography/electrospray ionization-mass spectrometry(LC/ESI-MS)-method using phytosphingosine as an internal standard to determine sphinganine and sphingosine concentrations in IHKE cells. Whereas a significant increase of sphinganine (up to 7000% compared to control cells) was observed with all fumonisin-derivates, sphingosine levels nearly remained unchanged indicating that all substrates inhibited ceramide synthase effectively. These results demonstrate that all compounds let to increased sphinganine levels in IHKE cells but only FB 1 was able to induce apoptosis. We conclude that the inhibition of the ceramide synthase is not per se a predictor whether or not fumonisins induce apoptosis

  20. Terpinen-4-ol Induces Apoptosis in Human Nonsmall Cell Lung Cancer In Vitro and In Vivo

    Directory of Open Access Journals (Sweden)

    Chieh-Shan Wu

    2012-01-01

    Full Text Available Terpinen-4-ol, a monoterpene component of the essential oils of several aromatic plants, exhibits antitumor effects. In this study, the antitumor effects of terpinen-4-ol and the cellular and molecular mechanisms responsible for it were evaluated and studied, respectively on human nonsmall cell lung cancer (NSCLC cells. Our results indicated that terpinen-4-ol elicited a dose-dependent cytotoxic effect, as determined by MTT assay. Increased sub-G1 population and annexin-V binding, activation of caspases 9 and 3, cleavage of poly(ADPribose polymerase (PARP, and a decrease of mitochondrial membrane potential (MMP indicated involvement of the mitochondrial apoptotic pathway in terpinen-4-ol-treated A549 and CL1-0 cells. Elevation of the Bax/Bcl-2 ratio and a decrease in IAP family proteins XIAP and survivin were also observed following terpinen-4-ol treatment. Notably, terpinen-4-ol was able to increase p53 levels in A549 and CL1-0 cells. Diminution of p53 by RNA interference induced necrosis instead of apoptosis in A549 cells following terpinen-4-ol treatment, indicating that terpinen-4-ol-elicited apoptosis is p53-dependent. Moreover, intratumoral administration of terpinen-4-ol significantly suppressed the growth of s.c. A549 xenografts by inducing apoptosis, as confirmed by TUNEL assay. Collectively, these data provide insight into the molecular mechanisms underlying terpinen-4-ol-induced apoptosis in NSCLC cells, rendering this compound a potential anticancer drug for NSCLC.

  1. Cadmium telluride quantum dots induce apoptosis in human breast cancer cell lines.

    Science.gov (United States)

    Naderi, Saeed; Zare, Hakimeh; Taghavinia, Nima; Irajizad, Azam; Aghaei, Mahmoud; Panjehpour, Mojtaba

    2018-05-01

    Semiconductor quantum dots (QDs), especially those containing cadmium, have undergone marked improvements and are now widely used nanomaterials in applicable biological fields. However, great concerns exist regarding their toxicity in biomedical applications. Because of the lack of sufficient data regarding the toxicity mechanism of QDs, this study aimed to evaluate the cytotoxicity of three types of QDs: CdTe QDs, high yield CdTe QDs, and CdTe/CdS core/shell QDs on two human breast cancer cell lines MDA-MB468 and MCF-7. The breast cancer cells were treated with different concentrations of QDs, and cell viability was evaluated via MTT assay. Hoechst staining was applied for observation of morphological changes due to apoptosis. Apoptotic DNA fragmentation was visualized by the agarose gel electrophoresis assay. Flow cytometric annexin V/propidium iodide (PI) measurement was used for apoptosis detection. A significant decrease in cell viability was observed after QDs treatment ( p < 0.05). Apoptotic bodies and chromatin condensation was observed by Hoechst staining. DNA fragmentation assay demonstrated a DNA ladder profile in the exposed cells and also annexin V/PI flow cytometry confirmed apoptosis in a dose-dependent manner. Our results revealed that CdTe, high yield CdTe, and CdTe/CdS core/shell QDs induce apoptosis in breast cancer cell lines in a dose-dependent manner. This study would help realizing the underlying cytotoxicity mechanism, at least partly, of CdTe QDs and may provide information for the development of nanotoxicology and safe use of biological applications of QDs.

  2. Chronic neutrophilic leukemia.

    Science.gov (United States)

    Bredeweg, Arthur; Burch, Micah; Krause, John R

    2018-01-01

    Chronic neutrophilic leukemia is a rare myeloproliferative disorder characterized by a sustained peripheral blood neutrophilia, absence of the BCR/ABL oncoprotein, bone marrow hypercellularity with less than 5% myeloblasts and normal neutrophil maturation, and no dysplasia. This leukemia has been associated with mutations in the colony-stimulating factor 3 receptor (CSF3R) that may activate this receptor, leading to the proliferation of neutrophils that are the hallmark of chronic neutrophilic leukemia. We present a case of chronic neutrophilic leukemia and discuss the criteria for diagnosis and the significance of mutations found in this leukemia.

  3. Untangling the Roles of Anti-Apoptosis in Regulating Programmed Cell Death using Humanized Yeast Cells

    International Nuclear Information System (INIS)

    Clapp, Caitlin; Portt, Liam; Khoury, Chamel; Sheibani, Sara; Eid, Rawan; Greenwood, Matthew; Vali, Hojatollah; Mandato, Craig A.; Greenwood, Michael T.

    2012-01-01

    Genetically programmed cell death (PCD) mechanisms, including apoptosis, are important for the survival of metazoans since it allows, among things, the removal of damaged cells that interfere with normal function. Cell death due to PCD is observed in normal processes such as aging and in a number of pathophysiologies including hypoxia (common causes of heart attacks and strokes) and subsequent tissue reperfusion. Conversely, the loss of normal apoptotic responses is associated with the development of tumors. So far, limited success in preventing unwanted PCD has been reported with current therapeutic approaches despite the fact that inhibitors of key apoptotic inducers such as caspases have been developed. Alternative approaches have focused on mimicking anti-apoptotic processes observed in cells displaying increased resistance to apoptotic stimuli. Hormesis and pre-conditioning are commonly observed cellular strategies where sub-lethal levels of pro-apoptotic stimuli lead to increased resistance to higher or lethal levels of stress. Increased expression of anti-apoptotic sequences is a common mechanism mediating these protective effects. The relevance of the latter observation is exemplified by the observation that transgenic mice overexpressing anti-apoptotic genes show significant reductions in tissue damage following ischemia. Thus strategies aimed at increasing the levels of anti-apoptotic proteins, using gene therapy or cell penetrating recombinant proteins are being evaluated as novel therapeutics to decrease cell death following acute periods of cell death inducing stress. In spite of its functional and therapeutic importance, more is known regarding the processes involved in apoptosis than anti-apoptosis. The genetically tractable yeast Saccharomyces cerevisiae has emerged as an exceptional model to study multiple aspects of PCD including the mitochondrial mediated apoptosis observed in metazoans. To increase our knowledge of the process of anti-apoptosis

  4. Effects of hydroxyapatite nanoparticles on proliferation and apoptosis of human breast cancer cells (MCF-7)

    International Nuclear Information System (INIS)

    Meena, Ramovatar; Kesari, Kavindra Kumar; Rani, Madhu; Paulraj, R.

    2012-01-01

    The study aimed to correlate cell proliferation inhibition with oxidative stress and p53 protein expression in cancerous cells. Hydroxyapatite (HAP) (Ca 10 (PO 4 ) 6 (OH) 2 ) is the essential component of inorganic composition in human bone. It has been found to have obvious inhibitory function on growth of many kinds of tumor cells and its nanoparticle has stronger anti-cancerous effect than macromolecule microparticles. Human breast cancer cells (MCF-7) were cultured and treated with HAP nanoparticles at various concentrations. Cells viability was detected with MTT colorimetric assay. The morphology of the cancerous cells was performed by transmission electron microscopy and the expression of a cell apoptosis related gene (p53) was determined by ELISA assay and flow cytometry (FCM). The intracellular reactive oxygen species (ROS) level in HAP exposed cells was measured by H 2 DCFDA staining. DNA damage was measured by single-cell gel electrophoresis assay. The statistical analysis was done by one way ANOVA. The cellular proliferation inhibition rate was significantly (p < 0.05) increasing in a dose-dependent manner of HAP nanoparticles. Cell apoptotic characters were observed after MCF-7 cells were treated by HAP nanoparticles for 48 h. Moreover, ELISA assay and FCM shows a dose-dependent activation of p53 in MCF-7 cells treated with nanoHAP. These causative factors of the above results may be justified by an overproduction of ROS. In this study, a significant (p < 0.05) increase in the level of intracellular ROS in HAP-treated cells was observed. This study shows that HAP inhibits the growth of human breast cancer MCF-7 cells as well as induces cell apoptosis. This study shows that HAP NPs Induce the production of intracellular reactive oxygen species and activate p53, which may be responsible for DNA damage and cell apoptosis.

  5. The effect of calcium phosphate nanoparticles on hormone production and apoptosis in human granulosa cells

    Directory of Open Access Journals (Sweden)

    Gao Li

    2010-04-01

    Full Text Available Abstract Objectives Although many nanomaterials are being used in academia, industry and daily life, there is little understanding about the effects of nanoparticles on the reproductive health of vertebral animals, including human beings. An experimental study was therefore performed here to explore the effect of calcium phosphate nanoparticles on both steroid hormone production and apoptosis in human ovarian granulosa cells. Methods Calcium phosphate nanoparticles uptaking was evaluated by transmission electron microscopy (TEM. The cell cycle was assessed with propidium iodide-stained cells (distribution of cells in G0/G1, S, and G2/M phases by flow cytometry. The pattern of cell death (necrosis and apoptosis was analyzed by flow cytometry with annexin V-FITC/PI staining. The expression of mRNAs encoding P450scc, P450arom and StAR were determined by RT-PCR. Progesterone and estradiol levels were measured by radioimmunoassay. Results TEM results confirmed that calcium phosphate nanoparticles could enter into granulosa cells, and distributed in the membranate compartments, including lysosome and mitochondria and intracellular vesicles. The increased percentage of cells in S phase when cultured with nanoparticles indicated that there was an arrest at the checkpoint from phase S-to-G2/M (from 6.28 +/- 1.55% to 11.18 +/- 1.73%, p Conclusion Calcium phosphate nanoparticles interfered with cell cycle of cultured human ovarian granulosa cells thus increasing cell apoptosis. This pilot study suggested that effects of nanoparticles on ovarian function should be extensively investigated.

  6. Proteomic investigation into betulinic acid-induced apoptosis of human cervical cancer HeLa cells.

    Science.gov (United States)

    Xu, Tao; Pang, Qiuying; Zhou, Dong; Zhang, Aiqin; Luo, Shaman; Wang, Yang; Yan, Xiufeng

    2014-01-01

    Betulinic acid is a pentacyclic triterpenoid that exhibits anticancer functions in human cancer cells. This study provides evidence that betulinic acid is highly effective against the human cervical cancer cell line HeLa by inducing dose- and time-dependent apoptosis. The apoptotic process was further investigated using a proteomics approach to reveal protein expression changes in HeLa cells following betulinic acid treatment. Proteomic analysis revealed that there were six up- and thirty down-regulated proteins in betulinic acid-induced HeLa cells, and these proteins were then subjected to functional pathway analysis using multiple analysis software. UDP-glucose 6-dehydrogenase, 6-phosphogluconate dehydrogenase decarboxylating, chain A Horf6-a novel human peroxidase enzyme that involved in redox process, was found to be down-regulated during the apoptosis process of the oxidative stress response pathway. Consistent with our results at the protein level, an increase in intracellular reactive oxygen species was observed in betulinic acid-treated cells. The proteins glucose-regulated protein and cargo-selection protein TIP47, which are involved in the endoplasmic reticulum pathway, were up-regulated by betulinic acid treatment. Meanwhile, 14-3-3 family proteins, including 14-3-3β and 14-3-3ε, were down-regulated in response to betulinic acid treatment, which is consistent with the decrease in expression of the target genes 14-3-3β and 14-3-3ε. Furthermore, it was found that the antiapoptotic bcl-2 gene was down-regulated while the proapoptotic bax gene was up-regulated after betulinic acid treatment in HeLa cells. These results suggest that betulinic acid induces apoptosis of HeLa cells by triggering both the endoplasmic reticulum pathway and the ROS-mediated mitochondrial pathway.

  7. Simultaneous human papilloma virus type 16 E7 and cdk inhibitor p21 expression induces apoptosis and cathepsin B activation

    DEFF Research Database (Denmark)

    Kaznelson, Dorte Wissing; Bruun, Silas; Monrad, Astrid

    2004-01-01

    Human papillomavirus type 16 (HPV-16) is the major risk factor for development of cervical cancer. The major oncoprotein E7 enhances cell growth control. However, E7 has in some reports been shown to induce apoptosis suggesting that there is a delicate balance between cell proliferation and induc......Human papillomavirus type 16 (HPV-16) is the major risk factor for development of cervical cancer. The major oncoprotein E7 enhances cell growth control. However, E7 has in some reports been shown to induce apoptosis suggesting that there is a delicate balance between cell proliferation......, possibly because of conflicting growth control. Interestingly, E7/p21-induced cell death is associated with the activation of a newly identified mediator of apoptosis, namely cathepsin B. Activation of the cellular caspases is undetectable in cells undergoing E7/p21-induced apoptosis. To our knowledge...

  8. BMP signaling in the human fetal ovary is developmentally regulated and promotes primordial germ cell apoptosis.

    Science.gov (United States)

    Childs, Andrew J; Kinnell, Hazel L; Collins, Craig S; Hogg, Kirsten; Bayne, Rosemary A L; Green, Samira J; McNeilly, Alan S; Anderson, Richard A

    2010-08-01

    Primordial germ cells (PGCs) are the embryonic precursors of gametes in the adult organism, and their development, differentiation, and survival are regulated by a combination of growth factors collectively known as the germ cell niche. Although many candidate niche components have been identified through studies on mouse PGCs, the growth factor composition of the human PGC niche has not been studied extensively. Here we report a detailed analysis of the expression of components of the bone morphogenetic protein (BMP) signaling apparatus in the human fetal ovary, from postmigratory PGC proliferation to the onset of primordial follicle formation. We find developmentally regulated and reciprocal patterns of expression of BMP2 and BMP4 and identify germ cells to be the exclusive targets of ovarian BMP signaling. By establishing long-term cultures of human fetal ovaries in which PGCs are retained within their physiological niche, we find that BMP4 negatively regulates postmigratory PGC numbers in the human fetal ovary by promoting PGC apoptosis. Finally, we report expression of both muscle segment homeobox (MSX)1 and MSX2 in the human fetal ovary and reveal a selective upregulation of MSX2 expression in human fetal ovary in response to BMP4, suggesting this gene may act as a downstream effector of BMP-induced apoptosis in the ovary, as in other systems. These data reveal for the first time growth factor regulation of human PGC development in a physiologically relevant context and have significant implications for the development of cultures systems for the in vitro maturation of germ cells, and their derivation from pluripotent stem cells.

  9. Resistance to asbestos-induced apoptosis with continuous exposure to crocidolite on a human T cell

    Energy Technology Data Exchange (ETDEWEB)

    Maeda, Megumi [Department of Biofunctional Chemistry, Graduate School of Natural Science and Technology, Okayama University, 1-1-1 Tsushima-Naka, Okayama 700-8530 (Japan); Department of Hygiene, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-0192 (Japan); Yamamoto, Shoko [Department of Hygiene, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-0192 (Japan); Chen, Ying [Division of Pneumoconiosis, School of Public Health, China Medical University, 92 North 2nd, Heping District, Shenyang 110001 (China); Kumagai-Takei, Naoko [Department of Hygiene, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-0192 (Japan); Hayashi, Hiroaki [Department of Hygiene, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-0192 (Japan); Department of Dermatology, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-0192 (Japan); Matsuzaki, Hidenori; Lee, Suni; Hatayama, Tamayo; Miyahara, Naomi; Katoh, Minako [Department of Hygiene, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-0192 (Japan); Hiratsuka, Juni-ichi [Department of Radiation Oncology, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-0192 (Japan); Nishimura, Yasumitsu [Department of Hygiene, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-0192 (Japan); Otsuki, Takemi, E-mail: takemi@med.kawasaki-m.ac.jp [Department of Hygiene, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-0192 (Japan)

    2012-07-01

    We have been investigating the immunological effects of asbestos. The establishment of a low-dose and continuously exposed human T cell line, HTLV-1 immortalized MT-2, to chrysotile (CB) revealed reduction of CXCR3 chemokine receptor and production of IFN-{gamma} that caused a decline of tumor immunity. These effects were coupled with upregulation of IL-10, TGF-{beta}, and BCL-2 in asbestos-exposed patients. To observe the immunological effects of crocidolite (CR) on human T cells, a trial to establish a low-dose and continuously exposed model was conducted and compared with a previously reported CB-exposed model (MT-2CB). Transient exposure of MT-2 original cells to CB or CR induced a similar level of apoptosis and growth inhibition. The establishment of a continuously exposed subline to CR (MT-2CR) revealed resistance against CR-induced apoptosis and upregulation of the BCL-2/BAX ratio similar to that recorded for MT-2CB. Both sublines showed reduced production of IFN-{gamma}, TNF-{alpha}, and IL-6 with increased IL-10. cDNA microarray with network/pathway analyses focusing on transcription factors revealed that many similar factors related to cell proliferation were involved following continuous exposure to asbestos in both MT-2CB and MT-2CR. These results indicate that both CB and CR fibers affect human T cells with similar degrees even though the carcinogenic activity of these substances differs due to their chemical and physical forms. Trials to identify early detection markers for asbestos exposure or the occurrence of asbestos-inducing malignancies using these findings may lead to the development of clinical tools for asbestos-related diseases and chemoprevention that modifies the reduced tumor immunity. - Highlights: Black-Right-Pointing-Pointer Comparison of effects of chrysotile and crocidolite on human T cell was done. Black-Right-Pointing-Pointer Both fibers caused apoptosis of T cells by transient exposure. Black-Right-Pointing-Pointer T cells

  10. SurR9C84A protects and recovers human cardiomyocytes from hypoxia induced apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Ashok, Ajay [Nanomedicine-Laboratory of Immunology and Molecular Biomedical Research (NLIMBR), School of Medicine (SoM), Faculty of Health, Centre for Molecular and Medical Research - C-MMR, Deakin University, Waurn Ponds, Victoria 3216 (Australia); Department of Pathology, Case Western Reserve University, 2103 Cornell Rd. WRB 5128, Cleveland, OH 44106-7288 (United States); Kanwar, Jagat Rakesh [Nanomedicine-Laboratory of Immunology and Molecular Biomedical Research (NLIMBR), School of Medicine (SoM), Faculty of Health, Centre for Molecular and Medical Research - C-MMR, Deakin University, Waurn Ponds, Victoria 3216 (Australia); Krishnan, Uma Maheswari [Centre for Nanotechnology & Advanced Biomaterials (CeNTAB), School of Chemical & Biotechnology (SCBT), SASTRA University, Thanjavur 613401 (India); Kanwar, Rupinder Kaur, E-mail: rupinder.kanwar@deakin.edu.au [Nanomedicine-Laboratory of Immunology and Molecular Biomedical Research (NLIMBR), School of Medicine (SoM), Faculty of Health, Centre for Molecular and Medical Research - C-MMR, Deakin University, Waurn Ponds, Victoria 3216 (Australia)

    2017-01-01

    Survivin, as an anti-apoptotic protein and a cell cycle regulator, is recently gaining importance for its regenerative potential in salvaging injured hypoxic cells of vital organs such as heart. Different strategies are being employed to upregulate survivin expression in dying hypoxic cardiomyocytes. We investigated the cardioprotective potential of a cell permeable survivin mutant protein SurR9C84A, for the management of hypoxia mediated cardiomyocyte apoptosis, in a novel and clinically relevant model employing primary human cardiomyocytes (HCM). The aim of this research work was to study the efficacy and mechanism of SurR9C84A facilitated cardioprotection and regeneration in hypoxic HCM. To mimic hypoxic microenvironment in vitro, well characterized HCM were treated with 100 µm (48 h) cobalt chloride to induce hypoxia. Hypoxia induced (HI) HCM were further treated with SurR9C84A (1 µg/mL) in order to analyse its cardioprotective efficacy. Confocal microscopy showed rapid internalization of SurR9C84A and scanning electron microscopy revealed the reinstatement of cytoskeleton projections in HI HCM. SurR9C84A treatment increased cell viability, reduced cell death via, apoptosis (Annexin-V assay), and downregulated free cardiac troponin T and MMP-9 expression. SurR9C84A also upregulated the expression of proliferation markers (PCNA and Ki-67) and downregulated mitochondrial depolarization and ROS levels thereby, impeding cell death. Human Apoptosis Array further revealed that SurR9C84A downregulated expression of pro-apoptotic markers and augmented expression of HSPs and HTRA2/Omi. SurR9C84A treatment led to enhanced levels of survivin, VEGF, PI3K and pAkt. SurR9C84A proved non-toxic to normoxic HCM, as validated through unaltered cell proliferation and other marker levels. Its pre-treatment exhibited lesser susceptibility to hypoxia/damage. SurR9C84A holds a promising clinical potential for human cardiomyocyte survival and proliferation following hypoxic injury

  11. Molecular mechanism implicated in Pemetrexed-induced apoptosis in human melanoma cells

    Directory of Open Access Journals (Sweden)

    Buqué Aitziber

    2012-04-01

    Full Text Available Abstract Background Metastatic melanoma is a lethal skin cancer and its incidence is rising every year. It represents a challenge for oncologist, as the current treatment options are non-curative in the majority of cases; therefore, the effort to find and/or develop novel compounds is mandatory. Pemetrexed (Alimta®, MTA is a multitarget antifolate that inhibits folate-dependent enzymes: thymidylate synthase, dihydrofolate reductase and glycinamide ribonucleotide formyltransferase, required for de novo synthesis of nucleotides for DNA replication. It is currently used in the treatment of mesothelioma and non-small cell lung cancer (NSCLC, and has shown clinical activity in other tumors such as breast, colorectal, bladder, cervical, gastric and pancreatic cancer. However, its effect in human melanoma has not been studied yet. Results In the current work we studied the effect of MTA on four human melanoma cell lines A375, Hs294T, HT144 and MeWo and in two NSCLC cell lines H1299 and Calu-3. We have found that MTA induces DNA damage, S-phase cell cycle arrest, and caspase- dependent and –independent apoptosis. We show that an increment of the intracellular reactive oxygen species (ROS and p53 is required for MTA-induced cytotoxicity by utilizing N-Acetyl-L-Cysteine (NAC to blockage of ROS and p53-defective H1299 NSCLC cell line. Pretreatment of melanoma cells with NAC significantly decreased the DNA damage, p53 up-regulation and cytotoxic effect of MTA. MTA was able to induce p53 expression leading to up-regulation of p53-dependent genes Mcl-1 and PIDD, followed by a postranscriptional regulation of Mcl-1 improving apoptosis. Conclusions We found that MTA induced DNA damage and mitochondrial-mediated apoptosis in human melanoma cells in vitro and that the associated apoptosis was both caspase-dependent and –independent and p53-mediated. Our data suggest that MTA may be of therapeutic relevance for the future treatment of human malignant melanoma.

  12. Neutrophils in critical illness.

    Science.gov (United States)

    McDonald, Braedon

    2018-03-01

    During critical illness, dramatic alterations in neutrophil biology are observed including abnormalities of granulopoeisis and lifespan, cell trafficking and antimicrobial effector functions. As a result, neutrophils transition from powerful antimicrobial protectors into dangerous mediators of tissue injury and organ dysfunction. In this article, the role of neutrophils in the pathogenesis of critical illness (sepsis, trauma, burns and others) will be explored, including pathological changes to neutrophil function during critical illness and the utility of monitoring aspects of the neutrophil phenotype as biomarkers for diagnosis and prognostication. Lastly, we review findings from clinical trials of therapies that target the harmful effects of neutrophils, providing a bench-to-bedside perspective on neutrophils in critical illness.

  13. β-Elemene-induced autophagy protects human gastric cancer cells from undergoing apoptosis

    International Nuclear Information System (INIS)

    Liu, Jing; Zhang, Ye; Qu, Jinglei; Xu, Ling; Hou, Kezuo; Zhang, Jingdong; Qu, Xiujuan; Liu, Yunpeng

    2011-01-01

    β-Elemene, a compound found in an herb used in traditional Chinese medicine, has shown promising anti-cancer effects against a broad spectrum of tumors. The mechanism by which β-elemene kills cells remains unclear. The aim of the present study is to investigate the anti-tumor effect of β-elemene on human gastric cancer cells and the molecular mechanism involved. β-Elemene inhibited the viability of human gastric cancer MGC803 and SGC7901 cells in a dose-dependent manner. The suppression of cell viability was due to the induction of apoptosis. A robust autophagy was observed in the cells treated with β-elemene; it was characterized by the increase of punctate LC3 dots, the cellular morphology, and the increased levels of LC3-II protein. Further study showed that β-elemene treatment up-regulated Atg5-Atg12 conjugated protein but had little effect on other autophagy-related proteins. PI3K/Akt/mTOR/p70S6K1 activity was inhibited by β-elemene. Knockdown of Beclin 1 with small interfering RNA, or co-treatment with the autophagy inhibitor, 3-methyladenine or chlorochine enhanced significantly the antitumor effects of β-elemene. Our data provides the first evidence that β-elemene induces protective autophagy and prevents human gastric cancer cells from undergoing apoptosis. A combination of β-elemene with autophagy inhibitor might thus be a useful therapeutic option for advanced gastric cancer

  14. Immunosenescence of Polymorphonuclear Neutrophils

    Directory of Open Access Journals (Sweden)

    Inga Wessels

    2010-01-01

    Full Text Available All immune cells are affected by aging, contributing to the high susceptibility to infections and increased mortality observed in the elderly. The effect of aging on cells of the adaptive immune system is well documented. In contrast, knowledge concerning age-related defects of polymorphonuclear neutrophils (PMN is limited. During the past decade, it has become evident that in addition to their traditional role as phagocytes, neutrophils are able to secrete a wide array of immunomodulating molecules. Their importance is underlined by the finding that genetic defects that lead to neutropenia increase susceptibility to infections. Whereas there is consistence about the constant circulating number of PMN throughout aging, the abilities of tissue infiltration, phagocytosis, and oxidative burst of PMN from aged donors are discussed controversially. Furthermore, there are numerous discrepancies between in vivo and in vitro results, as well as between results for murine and human PMN. Most of the reported functional changes can be explained by defective signaling pathways, but further research is required to get a detailed insight into the underlying molecular mechanisms. This could form the basis for drug development in order to prevent or treat age-related diseases, and thus to unburden the public health systems.

  15. Inhibition of PAF-induced expression of CD11b and shedding of L-selectin on human neutrophils and eosinophils by the type IV selective PDE inhibitor, rolipram

    NARCIS (Netherlands)

    Dijkhuizen, B; deMonchy, JGR; Dubois, AEJ; Gerritsen, J; Kauffman, HF

    We quantitatively determined whether the selective phosphodiesterase (PDE) inhibitor, rolipram, inhibits changes in the adhesion molecules CD11b and L-selectin on platelet-activating factor (PAF)-stimulated human neutrophils and eosinophils in vitro. Incubations were performed in human whole blood

  16. Fisetin Induces Apoptosis Through p53-Mediated Up-Regulation of DR5 Expression in Human Renal Carcinoma Caki Cells

    OpenAIRE

    Kyoung-jin Min; Ju-Ock Nam; Taeg Kyu Kwon

    2017-01-01

    Fisetin is a natural compound found in fruits and vegetables such as strawberries, apples, cucumbers, and onions. Since fisetin can elicit anti-cancer effects, including anti-proliferation and anti-migration, we investigated whether fisetin induced apoptosis in human renal carcinoma (Caki) cells. Fisetin markedly induced sub-G1 population and cleavage of poly (ADP-ribose) polymerase (PARP), which is a marker of apoptosis, and increased caspase activation. We found that pan-caspase inhibitor (...

  17. Fem1b, a proapoptotic protein, mediates proteasome inhibitor-induced apoptosis of human colon cancer cells.

    Science.gov (United States)

    Subauste, M Cecilia; Sansom, Owen J; Porecha, Nehal; Raich, Natacha; Du, Liqin; Maher, Joseph F

    2010-02-01

    In the treatment of colon cancer, the development of resistance to apoptosis is a major factor in resistance to therapy. New molecular approaches to overcome apoptosis resistance, such as selectively upregulating proapoptotic proteins, are needed in colon cancer therapy. In a mouse model with inactivation of the adenomatous polyposis coli (Apc) tumor suppressor gene, reflecting the pathogenesis of most human colon cancers, the gene encoding feminization-1 homolog b (Fem1b) is upregulated in intestinal epithelium following Apc inactivation. Fem1b is a proapoptotic protein that interacts with apoptosis-inducing proteins Fas, tumor necrosis factor receptor-1 (TNFR1), and apoptotic protease activating factor-1 (Apaf-1). Increasing Fem1b expression induces apoptosis of cancer cells, but effects on colon cancer cells have not been reported. Fem1b is a homolog of feminization-1 (FEM-1), a protein in Caenorhabditis elegans that is regulated by proteasomal degradation, but whether Fem1b is likewise regulated by proteasomal degradation is unknown. Herein, we found that Fem1b protein is expressed in primary human colon cancer specimens, and in malignant SW620, HCT-116, and DLD-1 colon cancer cells. Increasing Fem1b expression, by transfection of a Fem1b expression construct, induced apoptosis of these cells. We found that proteasome inhibitor treatment of SW620, HCT-116, and DLD-1 cells caused upregulation of Fem1b protein levels, associated with induction of apoptosis. Blockade of Fem1b upregulation with morpholino antisense oligonucleotide suppressed the proteasome inhibitor-induced apoptosis of these cells. In conclusion, the proapoptotic protein Fem1b is downregulated by the proteasome in malignant colon cancer cells and mediates proteasome inhibitor-induced apoptosis of these cells. Therefore, Fem1b could represent a novel molecular target to overcome apoptosis resistance in therapy of colon cancer.

  18. Involvement of ERK, Bcl-2 family and caspase 3 in recombinant human activin A-induced apoptosis in A549

    International Nuclear Information System (INIS)

    Wang Baiding; Feng Yuling; Song Xingbo; Liu Qingqing; Ning Yunye; Ou Xuemei; Yang Jie; Zhang Xiaohong; Wen, Fuqiang

    2009-01-01

    Background: Activins are members of the transforming growth factor-β (TGF-β) superfamily. Previous studies have shown that activin A may have a central role in regulating both apoptosis and proliferation. However, direct studies of recombination human activin A on human NSCLC A549 cells have not yet been reported. The purpose of this study was to investigate whether activin A could induce apoptosis in A549 cells and the possible mechanisms via which it worked. Methods: Cellular apoptosis induced by activin A was detected by TUNEL assay and the levels of protein expression were detected by western blot. Results: Recombination human activin A induced apoptosis in human NSCLC A549 cells in a concentrate-dependent manner. Activin A-induced A549 apoptosis was accompanied by the up-regulation of Bax, Bad and Bcl-Xs and down-regulation of Bcl-2. Moreover, activin A treatment increased the expression of its typeII receptors, activated ERK and caspase 3 in A549. These results clearly demonstrate that the induction of apoptosis by activin-A involves multiple cellular/molecular pathways and strongly suggest that pro- and anti-apoptotic Bcl-2 family proteins and caspase 3 participate in activin A-induced apoptotic process in A549 cells. On the other hand, activin A treatment had little effect on primary human small airway epithelial cells (SAECs). Conclusion: Recombination human activin A induced apoptosis in A549 cells, at least partially, through ERK and mitochondrial pathway. The result that activin A did not affect the normal SAEC revealed activin A might be considered as a potential anticancer agent and worthy of further studies

  19. Effect of Isolation Techniques on Viability of Bovine Blood Neutrophils

    Directory of Open Access Journals (Sweden)

    P. Sláma

    2006-01-01

    Full Text Available The effect of selected isolation methods on the viability of neutrophil granulocytes (neutrophils from the blood of healthy Holstein x Bohemian Red Pied crossbred heifers was evaluated. Two methods of neutrophil isolation were used: a neutrophil isolation on the basis of hypotonic erythrocyte lysis (in two variants: after the erythrocyte lysis proper, the cells were centrifuged at either 200 g or 1000 g, and b neutrophil isolation with FACS Lysing Solution as the lysing agent. The viability of the isolated neutrophils was evaluated on the basis of apoptosis and necrosis. The results obtained with flow cytometry (FCM suggest that, from the isolation techniques used, the method based on FACS Lysing Solution impaired the neutrophil viability least. After the application of this method, 5.36 ± 2.15% of neutrophils were apoptotic and 0.51 ± 0.12% were necrotic. In contrast, when the hypotonic erythrocyte lysis was used, the proportion of apoptotic neutrophils amounted to 42.14 ± 7.12% and 49.00 ± 14.70%, respectively, and 41.12 ± 5.55% and 36.91 ± 24.38% respectively of necrotic neutrophils (P < 0.01. This was also confirmed by the light microscopy. After the isolation with FASC Lysing Solution, 1.92 ± 1.74% of neutrophils were apoptotic and 1.05 ± 0.76% were necrotic, as distinct from after the hypotonic erythrocyte lysis where 9.43 ± 3.69% of neutrophils were apoptotic and 12.67 ± 4.74% of necrotic after centrifugation at 200 g, while 12.60 ± 4.35 were apoptotic and 14.96 ± 12.64% were necrotic after centrifugation at 1000 g. It follows from the above-mentioned data that hypotonic lysis is not a suitable method for the isolation of neutrophils, as the method itself markedly affects cell viability.

  20. Androgen-Sensitized Apoptosis of HPr-1AR Human Prostate Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Congcong Chen

    Full Text Available Androgen receptor (AR signaling is crucial to the development and homeostasis of the prostate gland, and its dysregulation mediates common prostate pathologies. The mechanisms whereby AR regulates growth suppression and differentiation of luminal epithelial cells in the prostate gland and proliferation of malignant versions of these cells have been investigated in human and rodent adult prostate. However, the cellular stress response of human prostate epithelial cells is not well understood, though it is central to prostate health and pathology. Here, we report that androgen sensitizes HPr-1AR and RWPE-AR human prostate epithelial cells to cell stress agents and apoptotic cell death. Although 5α-dihydrotestosterone (DHT treatment alone did not induce cell death, co-treatment of HPr-1AR cells with DHT and an apoptosis inducer, such as staurosporine (STS, TNFt, or hydrogen peroxide, synergistically increased cell death in comparison to treatment with each apoptosis inducer by itself. We found that the synergy between DHT and apoptosis inducer led to activation of the intrinsic/mitochondrial apoptotic pathway, which is supported by robust cleavage activation of caspase-9 and caspase-3. Further, the dramatic depolarization of the mitochondrial membrane potential that we observed upon co-treatment with DHT and STS is consistent with increased mitochondrial outer membrane permeabilization (MOMP in the pro-apoptotic mechanism. Interestingly, the synergy between DHT and apoptosis inducer was abolished by AR antagonists and inhibitors of transcription and protein synthesis, suggesting that AR mediates pro-apoptotic synergy through transcriptional regulation of MOMP genes. Expression analysis revealed that pro-apoptotic genes (BCL2L11/BIM and AIFM2 were DHT-induced, whereas pro-survival genes (BCL2L1/BCL-XL and MCL1 were DHT-repressed. Hence, we propose that the net effect of these AR-mediated expression changes shifts the balance of BCL2-family proteins

  1. Fatty acid synthase inhibition triggers apoptosis during S phase in human cancer cells.

    Science.gov (United States)

    Zhou, Weibo; Simpson, P Jeanette; McFadden, Jill M; Townsend, Craig A; Medghalchi, Susan M; Vadlamudi, Aravinda; Pinn, Michael L; Ronnett, Gabriele V; Kuhajda, Francis P

    2003-11-01

    C75, an inhibitor of fatty acid synthase (FAS), induces apoptosis in cultured human cancer cells. Its proposed mechanism of action linked high levels of malonyl-CoA after FAS inhibition to potential downstream effects including inhibition of carnitine palmitoyltransferase-1 (CPT-1) with resultant inhibition of fatty acid oxidation. Recent data has shown that C75 directly stimulates CPT-1 increasing fatty acid oxidation in MCF-7 human breast cancer cells despite inhibitory concentrations of malonyl-CoA. In light of these findings, we have studied fatty acid metabolism in MCF7 human breast cancer cells to elucidate the mechanism of action of C75. We now report that: (a) in the setting of increased fatty acid oxidation, C75 inhibits fatty acid synthesis; (b) C273, a reduced form of C75, is unable to inhibit fatty acid synthesis and is nontoxic to MCF7 cells; (c) C75 and 5-(tetradecyloxy)-2-furoic acid (TOFA), an inhibitor of acetyl-CoA carboxylase, both cause a significant reduction of fatty acid incorporation into phosphatidylcholine, the major membrane phospholipid, within 2 h; (d) pulse chase studies with [(14)C]acetate labeling of membrane lipids show that both C75 and TOFA accelerate the decay of (14)C-labeled lipid from membranes within 2 h; (e) C75 also promotes a 2-3-fold increase in oxidation of membrane lipids within 2 h; and (f) because interference with phospholipid synthesis during S phase is known to trigger apoptosis in cycling cells, we performed double-labeled terminal deoxynucleotidyltransferase-mediated nick end labeling and BrdUrd analysis with both TOFA and C75. C75 triggered apoptosis during S phase, whereas TOFA did not. Moreover, application of TOFA 2 h before C75 blocked the C75 induced apoptosis, whereas etomoxir did not. Taken together these data indicate that FAS inhibition and its downstream inhibition of phospholipid production is a necessary part of the mechanism of action of C75. CPT-1 stimulation does not likely play a role in the

  2. Lipoxin A4 and lipoxin B4 stimulate the release but not the oxygenation of arachidonic acid in human neutrophils: Dissociation between lipid remodeling and adhesion

    Energy Technology Data Exchange (ETDEWEB)

    Nigam, S.; Fiore, S.; Luscinskas, F.W.; Serhan, C.N. (Brigham and Women' s Hospital, Boston, MA (USA))

    1990-06-01

    The profiles of actions of lipoxin A4 (LXA4) and lipoxin B4 (LXB4), two lipoxygenase-derived eicosanoids, were examined with human neutrophils. At nanomolar concentrations, LXA4 and LXB4 each stimulated the release of (1-14C)arachidonic acid from esterified sources in neutrophils. Lipoxin-induced release of (1-14C)arachidonic acid was both dose- and time-dependent and was comparable to that induced by the chemotactic peptide f-met-leu-phe. Time-course studies revealed that lipoxin A4 and lipoxin B4 each induced a biphasic release of (1-14C)arachidonic acid, which was evident within seconds (5-15 sec) in its initial phase and minutes (greater than 30 sec) in the second phase. In contrast, the all-trans isomers of LXA4 and LXB4 did not provoke (1-14C)AA release. Lipoxin-induced release of arachidonic acid was inhibited by prior treatment of the cells with pertussis toxin but not by its beta-oligomers, suggesting the involvement of guaninine nucleotide-binding regulatory proteins in this event. Dual radiolabeling of neutrophil phospholipid classes with (1-14C)arachidonic acid and (3H)palmitic acid showed that phosphatidylcholine was a major source of lipoxin-induced release of (1-14C)arachidonic acid. They also demonstrated that lipoxins rapidly stimulate both formation of phosphatidic acid as well as phospholipid remodeling. Although both LXA4 and LXB4 (10(-8)-10(-6) M) stimulated the release of (1-14C)arachidonic acid, neither compound evoked its oxygenation by either the 5- or 15-lipoxygenase pathways (including the formation of LTB4, 20-COOH-LTB4, 5-HETE, or 15-HETE). LXA4 and LXB4 (10(-7) M) each stimulated the elevation of cytosolic Ca2+ as monitored with Fura 2-loaded cells, albeit to a lesser extent than equimolar concentrations of FMLP. Neither lipoxin altered the binding of (3H)LTB4 to its receptor on neutrophils.

  3. NF-kappa B modulation is involved in celastrol induced human multiple myeloma cell apoptosis.

    Directory of Open Access Journals (Sweden)

    Haiwen Ni

    Full Text Available Celastrol is an active compound extracted from the root bark of the traditional Chinese medicine Tripterygium wilfordii Hook F. To investigate the effect of celastrol on human multiple myeloma cell cycle arrest and apoptosis and explore its molecular mechanism of action. The activity of celastrol on LP-1 cell proliferation was detected by WST-8 assay. The celastrol-induced cell cycle arrest was analyzed by flow cytometry after propidium iodide staining. Nuclear translocation of the nuclear factor kappa B (NF-κB was observed by fluorescence microscope. Celastrol inhibited cell proliferation of LP-1 myeloma cell in a dose-dependent manner with IC50 values of 0.8817 µM, which was mediated through G1 cell cycle arrest and p27 induction. Celastrol induced apoptosis in LP-1 and RPMI 8226 myeloma cells in a time and dose dependent manner, and it involved Caspase-3 activation and NF-κB pathway. Celastrol down-modulated antiapoptotic proteins including Bcl-2 and survivin expression. The expression of NF-κB and IKKa were decreased after celastrol treatment. Celastrol effectively blocked the nuclear translocation of the p65 subunit and induced human multiple myeloma cell cycle arrest and apoptosis by p27 upregulation and NF-kB modulation. It has been demonstrated that the effect of celastrol on NF-kB was HO-1-independent by using zinc protoporphyrin-9 (ZnPPIX, a selective heme oxygenase inhibitor. From the results, it could be inferred that celastrol may be used as a NF-kB inhibitor to inhibit myeloma cell proliferation.

  4. miR-16 promotes the apoptosis of human cancer cells by targeting FEAT

    International Nuclear Information System (INIS)

    Liang, Hongwei; Fu, Zheng; Jiang, Xueyuan; Wang, Nan; Wang, Feng; Wang, Xueliang; Zhang, Suyang; Wang, Yanbo; Yan, Xin; Guan, Wen-xian; Zhang, Chen-Yu; Zen, Ke; Zhang, Yujing; Chen, Xi; Zhou, Guangxin

    2015-01-01

    Although human cancers have heterogeneous combinations of altered oncogenes, some crucial genes are universally dysregulated in most cancers. One such gene, FEAT (faint expression in normal tissues, aberrant overexpression in tumors), is uniformly overexpressed in a variety of human cancers and plays an important role in tumorigenesis by suppressing apoptosis. However, the precise molecular mechanism through which FEAT is upregulated during tumorigenesis remains largely unknown. In this study, we used bioinformatic analyses to search for miRNAs that potentially target FEAT. We examined the expression of FEAT protein level by western blotting and miR-16 level by qRT-PCR assay. Cancer cell lines (A549, MCF-7 and Huh-7) with miR-16 upregulation and FEAT silencing were established and the effects on apoptosis of cancer cells in vitro were assessed. Luciferase reporter assay was also performed to investigate the interaction between miR-16 and FEAT. We identified a specific target site for miR-16 in the 3′-untranslated region (3′-UTR) of FEAT. Consistent with the bioinformatic analyses, we identified an inverse correlation between the miR-16 and FEAT protein levels in lung cancer, breast cancer, and hepatocellular cancer tissues. We then experimentally validated miR-16 as a direct regulator of FEAT using cell transfection and luciferase assays. Finally, we demonstrated that the repression of FEAT by miR-16 promoted the apoptosis of cancer cells. Our findings provide the first clues regarding the role of miR-16 as a tumor suppressor in cancer cells through the inhibition of FEAT translation. The online version of this article (doi:10.1186/s12885-015-1458-8) contains supplementary material, which is available to authorized users

  5. RBBP6: a potential biomarker of apoptosis induction in human cervical cancer cell lines

    Directory of Open Access Journals (Sweden)

    Moela P

    2016-07-01

    Full Text Available Pontsho Moela, Lesetja Raymond Motadi Department of Biochemistry, North-West University, Potchefstroom, South Africa Abstract: Overexpression of RBBP6 in cancers of the colon, lung, and esophagus makes it a potential target in anticancer therapy. This is especially important because RBBP6 associates with the tumor suppressor gene p53, the inactivation of which has been linked to over 50% of all cancer types. However, the expression of RBBP6 in cancer and its interaction with p53 are yet to be understood in order to determine whether or not RBBP6 is cancer promoting and therefore a potential biomarker. In this study, we manipulated RBBP6 expression levels followed by treatment with either camptothecin or γ-aminobutyric acid in cervical cancer cells to induce apoptosis or cell cycle arrest. We began by staining human cervical cancer tissue sections with anti-RBBP6 monoclonal antibody to evaluate the extent of expression of RBBP6 in patients’ specimens. We followed on with silencing the overexpression of RBBP6 and treatment with anticancer agents to evaluate how the specimens respond to combinational therapy. Apoptosis induction was evaluated through confocal microscope, and flow cytometry using annexin V staining, and also by checking the mitochondrial and caspase-3/7 activity. Cell cycle arrest was evaluated using flow cytometry through staining with propidium iodide. RBBP6 was highly expressed in cervical cancer tissue sections that were in stage II or III of development. Silencing RBBP6 followed by treatment with γ-aminobutyric acid and camptothecin seems to sensitize cells to apoptosis induction rather than cell cycle arrest. Overexpression of RBBP6 seems to promote S-phase in cell cycle and cell proliferation. These results predict a proliferative role of RBBP6 in cancer progression rather than as a cancer-causing gene. Furthermore, sensitization of cells to camptothecin-induced apoptosis by RBBP6 targeting suggests a promising tool for

  6. Hypoxia causes IL-8 secretion, Charcot Leyden crystal formation, and suppression of corticosteroid-induced apoptosis in human eosinophils.

    Science.gov (United States)

    Porter, L M; Cowburn, A S; Farahi, N; Deighton, J; Farrow, S N; Fiddler, C A; Juss, J K; Condliffe, A M; Chilvers, E R

    2017-06-01

    Inflamed environments are typically hypercellular, rich in pro-inflammatory cytokines, and profoundly hypoxic. While the effects of hypoxia on neutrophil longevity and function have been widely studied, little is known about the consequences of this stimulus on eosinophils. We sought to investigate the effects of hypoxia on several key aspects of eosinophil biology, namely secretion, survival, and their sensitivity to glucocorticosteroids (GCS), agents that normally induce eosinophil apoptosis. Eosinophils derived from patients with asthma/atopy or healthy controls were incubated under normoxia and hypoxia, with or without glucocorticoids. Activation was measured by flow cytometry, ELISA of cultured supernatants, and F-actin staining; apoptosis and efferocytosis by morphology and flow cytometry; and GCS efficacy by apoptosis assays and qPCR. Hypoxic incubation (3 kPa) caused (i) stabilization of HIF-2α and up-regulation of hypoxia-regulated genes including BNIP3 (BCL2/adenovirus E1B 19-kDa protein-interacting protein 3) and GLUT1 (glucose transporter 1); (ii) secretion of pre-formed IL-8, and Charcot Leyden crystal (CLC) formation, which was most evident in eosinophils derived from atopic and asthmatic donors; (iii) enhanced F-actin formation; (iv) marked prolongation of eosinophil lifespan (via a NF-κB and Class I PI3-kinase-dependent mechanism); and (v) complete abrogation of the normal pro-apoptotic effect of dexamethasone and fluticasone furoate. This latter effect was evident despite preservation of GCS-mediated gene transactivation under hypoxia. These data indicate that hypoxia promotes an eosinophil pro-inflammatory phenotype by enhancing eosinophil secretory function, delaying constitutive apoptosis, and importantly, antagonizing the normal pro-apoptotic effect of GCS. As eosinophils typically accumulate at sites that are relatively hypoxic, particularly during periods of inflammation, these findings may have important implications to understanding the

  7. Establishment and characterization of GSA-1, a human cell line highly susceptible to apoptosis after free-fall

    International Nuclear Information System (INIS)

    Nomura, Jun; Himeda, Jyuni; Chen, Zheng; Sugaya, Shigeru; Takahashi, Shunji; Kita, Kazuko; Ichinose, Masaharu; Suzuki, Nobuo

    2002-01-01

    The induction of apoptosis by microgravity and/or gravity-changing stress is considered to be one of the important causes of cell death, although the molecular mechanisms of the apoptotic event remain unclarified. In this study, we established a cell line,GSA-1, from ethyl methanesulfonate-treated human RSa cells. GSA-1 cells were highly susceptible to apoptosis after a free-fall; 24.4% of these cells underwent apoptosis after free-fall, compared with only 6% of the RSa cells. The apoptosis of GSA-1 cells was augmented by ultraviolet (UV, principally 254-nm wavelength) irradiation before free-fall to a greater extents than those in RSa cells. The molecular mechanisms of apoptosis included p53 and Bax proteins; the expression of nuclear p53 and cytoplasmic Bax in GSA-1 cells increased at 4 h after free-fall irrespective of irradiation. In addition, the rate of removal of cyclobutane pyrimidine dimer (CPD) in UV-irradiated GSA-1 cells was higher in cells exposed to free-fall than in those under the l-G condition. Our results suggested that in GSA-1 cells, free-fall accelerates apoptosis, and that this process is associated with the accumulation of p53 and Bax, as well as CPD removal. Thus, GSA-1 cells should be useful for investigating the mechanism of cellular response, including the induction of apoptosis under gravity-changing stress. (author)

  8. Apoptosis and autophagy induced by pyropheophorbide-α methyl ester-mediated photodynamic therapy in human osteosarcoma MG-63 cells.

    Science.gov (United States)

    Huang, Qiu; Ou, Yun-Sheng; Tao, Yong; Yin, Hang; Tu, Ping-Hua

    2016-06-01

    Pyropheophorbide-α methyl ester (MPPa) was a second-generation photosensitizer with many potential applications. Here, we explored the impact of MPPa-mediated photodynamic therapy (MPPa-PDT) on the apoptosis and autophagy of human osteosarcoma (MG-63) cells as well as the relationships between apoptosis and autophagy of the cells, and investigated the related molecular mechanisms. We found that MPPa-PDT demonstrated the ability to inhibit MG-63 cell viability in an MPPa concentration- and light dose-dependent manner, and to induce apoptosis via the mitochondrial apoptosis pathway. Additionally, MPPa-PDT could also induce autophagy of MG-63 cell. Meanwhile, the ROS scavenger N-acetyl-L-cysteine (NAC) and the Jnk inhibitor SP600125 were found to inhibit the MPPa-PDT-induced autophagy, and NAC could also inhibit Jnk phosphorylation. Furthermore, pretreatment with the autophagy inhibitor 3-methyladenine or chloroquine showed the potential in reducing the apoptosis rate induced by MPPa-PDT in MG-63 cells. Our results indicated that the mitochondrial pathway was involved in MPPa-PDT-induced apoptosis of MG-63 cells. Meanwhile the ROS-Jnk signaling pathway was involved in MPPa-PDT-induced autophagy, which further promoted the apoptosis in MG-63 cells.

  9. Lundep, a sand fly salivary endonuclease increases Leishmania parasite survival in neutrophils and inhibits XIIa contact activation in human plasma.

    Directory of Open Access Journals (Sweden)

    Andrezza C Chagas

    2014-02-01

    Full Text Available Neutrophils are the host's first line of defense against infections, and their extracellular traps (NET were recently shown to kill Leishmania parasites. Here we report a NET-destroying molecule (Lundep from the salivary glands of Lutzomyia longipalpis. Previous analysis of the sialotranscriptome of Lu. longipalpis showed the potential presence of an endonuclease. Indeed, not only was the cloned cDNA (Lundep shown to encode a highly active ss- and dsDNAse, but also the same activity was demonstrated to be secreted by salivary glands of female Lu. longipalpis. Lundep hydrolyzes both ss- and dsDNA with little sequence specificity with a calculated DNase activity of 300000 Kunitz units per mg of protein. Disruption of PMA (phorbol 12 myristate 13 acetate- or parasite-induced NETs by treatment with recombinant Lundep or salivary gland homogenates increases parasite survival in neutrophils. Furthermore, co-injection of recombinant Lundep with metacyclic promastigotes significantly exacerbates Leishmania infection in mice when compared with PBS alone or inactive (mutagenized Lundep. We hypothesize that Lundep helps the parasite to establish an infection by allowing it to escape from the leishmanicidal activity of NETs early after inoculation. Lundep may also assist blood meal intake by lowering the local viscosity caused by the release of host DNA and as an anticoagulant by inhibiting the intrinsic pathway of coagulation.

  10. Comparison of the effect of timegadine, levamisole, and D-penicillamine on human neutrophil metabolism of endogenous arachidonic acid and chemotaxis

    Energy Technology Data Exchange (ETDEWEB)

    Nielsen, O.H.; Ahnfelt-Roenne, I. Department of Pharmacology, Leo Pharmaceutical Products, Ballerup; Elmgreen, J.

    1988-01-01

    The effect of timegadine, a novel experimental antirheumatic drug, on human neutrophil (PMN) 5-lipoxygenase activity and leukotriene B/sub 4/ (LTB/sub 4/) chemotaxis was compared with that of two second-line antiinflammatory drugs, D-penicillamine and levamisole. 1-/sup 14/C-Arachidonic acid (AA) was incorporated into the purified cells until steady state conditions were obtained. After preincubation with serial dilutions of the three drugs, AA release and metabolism was stimulated with calcium ionophore A23187. The radioactive eicosanoids released were extracted and separated by thinlayer chromatography, followed by autoradiography and quantitative laser densitometry. Chemotaxi of PMNs towards LTB/sub 4/ was measured in a modified Boyden chamber. Timegardine showed dose-dependent inhibition of both the 5-lipoxygenase pathway (IC50 3.4 x 10/sup -5/ M), and of chemotaxis (IC50 3 x 10/sup -4/ M). Inhibition of the release of AA from phospholipids, however, occurred only at therapeutically irrelevant doses (millimolar concentrations). Levamisole and D-penicillamine did not inhibit any of the cell functions investigated. Inhibition of both neutrophil motility and cellular synthesis of pro-inflammatory eicosanoids, may thus contribute to the clinical effects of timegadine in rheumatoid arthritis.

  11. Inhibiting ROS-TFEB-Dependent Autophagy Enhances Salidroside-Induced Apoptosis in Human Chondrosarcoma Cells.

    Science.gov (United States)

    Zeng, Wei; Xiao, Tao; Cai, Anlie; Cai, Weiliang; Liu, Huanhuan; Liu, Jingling; Li, Jie; Tan, Miduo; Xie, Li; Liu, Ying; Yang, Xiangcheng; Long, Yi

    2017-01-01

    Autophagy modulation has been considered a potential therapeutic strategy for human chondrosarcoma, and a previous study indicated that salidroside exhibits significant anti-carcinogenic activity. However, the ability of salidroside to induce autophagy and its role in human chondrosarcoma cell death remains unclear. We exposed SW1353 cells to different concentrations of salidroside (0.5, 1 and 2 mM) for 24 h. RT-PCR, Western-blotting, Immunocytofluorescence, and Luciferase Reporter Assays were used to evaluate whether salidroside activated the TFEB-dependent autophagy. We show that salidroside induced significant apoptosis in the human chondrosarcoma cell line SW1353. In addition, we demonstrate that salidroside-induced an autophagic response in SW1353 cells, as evidenced by the upregulation of LC3-II and downregulation of P62. Moreover, pharmacological or genetic blocking of autophagy enhanced salidroside -induced apoptosis, indicating the cytoprotective role of autophagy in salidroside-treated SW1353 cells. Salidroside also induced TFEB (Ser142) dephosphorylation, subsequently to activated TFEB nuclear translocation and increase of TFEB reporter activity, which contributed to lysosomal biogenesis and the expression of autophagy-related genes. Importantly, we found that salidroside triggered the generation of ROS in SW1353 cells. Furthermore, NAC, a ROS scavenger, abrogated the effects of salidroside on TFEB-dependent autophagy. These data demonstrate that salidroside increased TFEB-dependent autophagy by activating ROS signaling pathways in human chondrosarcoma cells. These data also suggest that blocking ROS-TFEB-dependent autophagy to enhance the activity of salidroside warrants further attention in treatment of human chondrosarcoma cells. © 2017 The Author(s). Published by S. Karger AG, Basel.

  12. Milk fermented by Propionibacterium freudenreichii induces apoptosis of HGT-1 human gastric cancer cells.

    Directory of Open Access Journals (Sweden)

    Fabien J Cousin

    Full Text Available Gastric cancer is one of the most common cancers in the world. The "economically developed countries" life style, including diet, constitutes a risk factor favoring this cancer. Diet modulation may lower digestive cancer incidence. Among promising food components, dairy propionibacteria were shown to trigger apoptosis of human colon cancer cells, via the release of short-chain fatty acids acetate and propionate.A fermented milk, exclusively fermented by P. freudenreichii, was recently designed. In this work, the pro-apoptotic potential of this new fermented milk was demonstrated on HGT-1 human gastric cancer cells. Fermented milk supernatant induced typical features of apoptosis including chromatin condensation, formation of apoptotic bodies, DNA laddering, cell cycle arrest and emergence of a subG1 population, phosphatidylserine exposure at the plasma membrane outer leaflet, reactive oxygen species accumulation, mitochondrial transmembrane potential disruption, caspase activation and cytochrome c release. Remarkably, this new fermented milk containing P. freudenreichii enhanced the cytotoxicity of camptothecin, a drug used in gastric cancer chemotherapy.Such new probiotic fermented milk may thus be useful as part of a preventive diet designed to prevent gastric cancer and/or as a food supplement to potentiate cancer therapeutic treatments.

  13. Evodiamine Induces Apoptosis and Inhibits Migration of HCT-116 Human Colorectal Cancer Cells

    Directory of Open Access Journals (Sweden)

    Lv-Cui Zhao

    2015-11-01

    Full Text Available Evodiamine (EVO exhibits strong anti-cancer effects. However, the effect of EVO on the human colorectal cancer cell line HCT-116 has not been explored in detail, and its underlying molecular mechanisms remain unknown. In the present study, cell viability was assessed by Cell Counting Kit-8 (CCK-8. Cell cycle and apoptosis were measured by flow cytometry, and morphological changes in the nucleus were examined by fluorescence microscopy and Hoechst staining. Cell motility was detected by Transwell assay. ELISA was used to assess the protein levels of autocrine motility factor (AMF in the cell supernatant, and protein expression was determined by Western blotting. Our results showed that EVO inhibited the proliferation of HCT-116 cells, caused accumulation of cells in S and G2/M phases, and reduced the levels of the secreted form of AMF. The protein levels of tumor suppressor protein (p53, Bcl-2 Associated X protein (Bax, B cell CLL/lymphoma-2 (Bcl-2, phosphoglucose isomerase (PGI, phosphorylated signal transducers and activators of transcription 3 (p-STAT3 and matrix metalloproteinase 3 (MMP3 were altered in cells treated with EVO. Taken together, our results suggest that EVO modulates the activity of the p53 signaling pathway to induce apoptosis and downregulate MMP3 expression by inactivating the JAK2/STAT3 pathway through the downregulation of PGI to inhibit migration of HCT-116 human colorectal cancer cells.

  14. Sildenafil Prevents Apoptosis of Human First-Trimester Trophoblast Cells Exposed to Oxidative Stress

    Science.gov (United States)

    Bolnick, Jay M.; Kilburn, Brian A.; Bolnick, Alan D.; Diamond, Michael P.; Singh, Manvinder; Hertz, Michael; Dai, Jing

    2015-01-01

    Human first-trimester trophoblast cells proliferate at low O2, but survival is compromised by oxidative stress, leading to uteroplacental insufficiency. The vasoactive drug, sildenafil citrate (Viagra, Sigma, St Louis, Missouri), has proven useful in reducing adverse pregnancy outcomes. An important biological function of this pharmaceutical is its action as an inhibitor of cyclic guanosine monophosphate (cGMP) phosphodiesterase type 5 activity, which suggests that it could have beneficial effects on trophoblast survival. To investigate whether sildenafil can prevent trophoblast cell death, human first-trimester villous explants and the HTR-8/SVneo cytotrophoblast cell line were exposed to hypoxia and reoxygenation (H/R) to generate oxidative stress, which induces apoptosis. Apoptosis was optimally inhibited during H/R by 350 ng/mL sildenafil. Sildenafil-mediated survival was reversed by l-NG-nitro-l-arginine methyl ester hydrochloride or cGMP antagonist, indicating a dependence on both nitric oxide (NO) and cGMP. Indeed, either a cGMP agonist or an NO generator was cytoprotective independent of sildenafil. These findings suggest a novel intervention route for patients with recurrent pregnancy loss or obstetrical placental disorders. PMID:25431453

  15. Milk Fermented by Propionibacterium freudenreichii Induces Apoptosis of HGT-1 Human Gastric Cancer Cells

    Science.gov (United States)

    Cousin, Fabien J.; Jouan-Lanhouet, Sandrine; Dimanche-Boitrel, Marie-Thérèse; Corcos, Laurent; Jan, Gwénaël

    2012-01-01

    Background Gastric cancer is one of the most common cancers in the world. The “economically developed countries” life style, including diet, constitutes a risk factor favoring this cancer. Diet modulation may lower digestive cancer incidence. Among promising food components, dairy propionibacteria were shown to trigger apoptosis of human colon cancer cells, via the release of short-chain fatty acids acetate and propionate. Methodology/Principal Findings A fermented milk, exclusively fermented by P. freudenreichii, was recently designed. In this work, the pro-apoptotic potential of this new fermented milk was demonstrated on HGT-1 human gastric cancer cells. Fermented milk supernatant induced typical features of apoptosis including chromatin condensation, formation of apoptotic bodies, DNA laddering, cell cycle arrest and emergence of a subG1 population, phosphatidylserine exposure at the plasma membrane outer leaflet, reactive oxygen species accumulation, mitochondrial transmembrane potential disruption, caspase activation and cytochrome c release. Remarkably, this new fermented milk containing P. freudenreichii enhanced the cytotoxicity of camptothecin, a drug used in gastric cancer chemotherapy. Conclusions/Significance Such new probiotic fermented milk may thus be useful as part of a preventive diet designed to prevent gastric cancer and/or as a food supplement to potentiate cancer therapeutic treatments. PMID:22442660

  16. Mycobacterium tuberculosis infection causes different levels of apoptosis and necrosis in human macrophages and alveolar epithelial cells.

    Science.gov (United States)

    Danelishvili, Lia; McGarvey, Jeffery; Li, Yong-Jun; Bermudez, Luiz E

    2003-09-01

    Mycobacterium tuberculosis interacts with macrophages and epithelial cells in the alveolar space of the lung, where it is able to invade and replicate in both cell types. M. tuberculosis-associated cytotoxicity to these cells has been well documented, but the mechanisms of host cell death are not well understood. We examined the induction of apoptosis and necrosis of human macrophages (U937) and type II alveolar epithelial cells (A549) by virulent (H37Rv) and attenuated (H37Ra) M. tuberculosis strains. Apoptosis was determined by both enzyme-linked immunosorbent assay (ELISA) and TdT-mediated dUTP nick end labelling (TUNEL) assay, whereas necrosis was evaluated by the release of lactate dehydrogenase (LDH). Both virulent and attenuated M. tuberculosis induced apoptosis in macrophages; however, the attenuated strain resulted in significantly more apoptosis than the virulent strain after 5 days of infection. In contrast, cytotoxicity of alveolar cells was the result of necrosis, but not apoptosis. Although infection with M. tuberculosis strains resulted in apoptosis of 14% of the cells on the monolayer, cell death associated with necrosis was observed in 59% of alveolar epithelial cells after 5 days of infection. Infection with M. tuberculosis suppressed apoptosis of alveolar epithelial cells induced by the kinase inhibitor, staurosporine. Because our findings suggest that M. tuberculosis can modulate the apoptotic response of macrophages and epithelial cells, we carried out an apoptosis pathway-specific cDNA microarray analysis of human macrophages and alveolar epithelial cells. Whereas the inhibitors of apoptosis, bcl-2 and Rb, were upregulated over 2.5-fold in infected (48 h) alveolar epithelial cells, the proapoptotic genes, bad and bax, were downregulated. The opposite was observed when U937 macrophages were infected with M. tuberculosis. Upon infection of alveolar epithelial cells with M. tuberculosis, the generation of apoptosis, as determined by the

  17. The lipidated peptidomimetic Lau-[(S)-Aoc]-(Lys-βNphe)6-NH2 is a novel formyl peptide receptor 2 agonist that activates both human and mouse neutrophil NADPH-oxidase

    DEFF Research Database (Denmark)

    Holdfeldt, Andre; Skovbakke, Sarah Line; Winther, Malene

    2016-01-01

    Neutrophils expressing formyl peptide receptor 2 (FPR2) play key roles in host defense, immune regulation, and resolution of inflammation. Consequently, the search for FPR2-specific modulators has attracted much attention due to its therapeutic potential. Earlier described agonists......2 (F2M2), showing comparable potency in activating human and mouse neutrophils by inducing a rise in intracellular Ca2+ concentration and assembly of the superoxide-generating NADPH oxidase. This FPR2/Fpr2 agonist contains a headgroup consisting of a 2-aminooctanoic acid (Aoc) residue acylated......2 signaling as well as for development of prophylactic immunomodulatory therapy. This novel class of cross-species FPR2/Fpr2 agonists should enable translation of results obtained with mouse neutrophils (and disease models) into enhanced understanding of human inflammatory and immune diseases....

  18. Apoptosis and inflammation

    Directory of Open Access Journals (Sweden)

    C. Haanen

    1995-01-01

    Full Text Available During the last few decades it has been recognized that cell death is not the consequence of accidental injury, but is the expression of a cell suicide programme. Kerr et al. (1972 introduced the term apoptosis. This form of cell death is under the influence of hormones, growth factors and cytokines, which depending upon the receptors present on the target cells, may activate a genetically controlled cell elimination process. During apoptosis the cell membrane remains intact and the cell breaks into apoptotic bodies, which are phagocytosed. Apoptosis, in contrast to necrosis, is not harmful to the host and does not induce any inflammatory reaction. The principal event that leads to inflammatory disease is cell damage, induced by chemical/physical injury, anoxia or starvation. Cell damage means leakage of cell contents into the adjacent tissues, resulting in the capillary transmigration of granulocytes to the injured tissue. The accumulation of neutrophils and release of enzymes and oxygen radicals enhances the inflammatory reaction. Until now there has been little research into the factors controlling the accumulation and the tissue load of granulocytes and their histotoxic products in inflammatory processes. Neutrophil apoptosis may represent an important event in the control of intlamtnation. It has been assumed that granulocytes disintegrate to apoptotic bodies before their fragments are removed by local macrophages. Removal of neutrophils from the inflammatory site without release of granule contents is of paramount importance for cessation of inflammation. In conclusion, apoptotic cell death plays an important role in inflammatory processes and in the resolution of inflammatory reactions. The facts known at present should stimulate further research into the role of neutrophil, eosinophil and macrophage apoptosis in inflammatory diseases.

  19. Transient p53 suppression increases reprogramming of human fibroblasts without affecting apoptosis and DNA damage

    DEFF Research Database (Denmark)

    Rasmussen, Mikkel Aabech; Holst, Bjørn; Tümer, Zeynep

    2014-01-01

    The discovery of human-induced pluripotent stem cells (iPSCs) has sparked great interest in the potential treatment of patients with their own in vitro differentiated cells. Recently, knockout of the Tumor Protein 53 (p53) gene was reported to facilitate reprogramming but unfortunately also led...... to genomic instability. Here, we report that transient suppression of p53 during nonintegrative reprogramming of human fibroblasts leads to a significant increase in expression of pluripotency markers and overall number of iPSC colonies, due to downstream suppression of p21, without affecting apoptosis...... and DNA damage. Stable iPSC lines generated with or without p53 suppression showed comparable expression of pluripotency markers and methylation patterns, displayed normal karyotypes, contained between 0 and 5 genomic copy number variations and produced functional neurons in vitro. In conclusion...

  20. Dynamic interactions of neutrophils and biofilms

    Directory of Open Access Journals (Sweden)

    Josefine Hirschfeld

    2014-12-01

    Full Text Available Background: The majority of microbial infections in humans are biofilm-associated and difficult to treat, as biofilms are highly resistant to antimicrobial agents and protect themselves from external threats in various ways. Biofilms are tenaciously attached to surfaces and impede the ability of host defense molecules and cells to penetrate them. On the other hand, some biofilms are beneficial for the host and contain protective microorganisms. Microbes in biofilms express pathogen-associated molecular patterns and epitopes that can be recognized by innate immune cells and opsonins, leading to activation of neutrophils and other leukocytes. Neutrophils are part of the first line of defense and have multiple antimicrobial strategies allowing them to attack pathogenic biofilms. Objective/design: In this paper, interaction modes of neutrophils with biofilms are reviewed. Antimicrobial strategies of neutrophils and the counteractions of the biofilm communities, with special attention to oral biofilms, are presented. Moreover, possible adverse effects of neutrophil activity and their biofilm-promoting side effects are discussed. Results/conclusion: Biofilms are partially, but not entirely, protected against neutrophil assault, which include the processes of phagocytosis, degranulation, and formation of neutrophil extracellular traps. However, virulence factors of microorganisms, microbial composition, and properties of the extracellular matrix determine whether a biofilm and subsequent microbial spread can be controlled by neutrophils and other host defense factors. Besides, neutrophils may inadvertently contribute to the physical and ecological stability of biofilms by promoting selection of more resistant strains. Moreover, neutrophil enzymes can degrade collagen and other proteins and, as a result, cause harm to the host tissues. These parameters could be crucial factors in the onset of periodontal inflammation and the subsequent tissue breakdown.

  1. Fisetin inhibits cellular proliferation and induces mitochondria-dependent apoptosis in human gastric cancer cells.

    Science.gov (United States)

    Sabarwal, Akash; Agarwal, Rajesh; Singh, Rana P

    2017-02-01

    The anticancer effects of fisetin, a dietary agent, are largely unknown against human gastric cancer. Herein, we investigated the mechanisms of fisetin-induced inhibition of growth and survival of human gastric carcinoma AGS and SNU-1 cells. Fisetin (25-100 μM) caused significant decrease in the levels of G1 phase cyclins and CDKs, and increased the levels of p53 and its S15 phosphorylation in gastric cancer cells. We also observed that growth suppression and death of non-neoplastic human intestinal FHs74int cells were minimally affected by fisetin. Fisetin strongly increased apoptotic cells and showed mitochondrial membrane depolarization in gastric cancer cells. DNA damage was observed as early as 3 h after fisetin treatment which was accompanied with gamma-H2A.X(S139) phosphorylation and cleavage of PARP. Fisetin-induced apoptosis was observed to be independent of p53. DCFDA and MitoSOX analyses showed an increase in mitochondrial ROS generation in time- and dose-dependent fashion. It also increased cellular nitrite and superoxide generation. Pre-treatment with N-acetyl cysteine (NAC) inhibited ROS generation and also caused protection from fisetin-induced DNA damage. The formation of comets were observed in only fisetin treated cells which was blocked by NAC pre-treatment. Further investigation of the source of ROS, using mitochondrial respiratory chain (MRC) complex inhibitors, suggested that fisetin caused ROS generation specifically through complex I. Collectively, these results for the first time demonstrated that fisetin possesses anticancer potential through ROS production most likely via MRC complex I leading to apoptosis in human gastric carcinoma cells. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  2. Toona Sinensis Extracts Induced Cell Cycle Arrest and Apoptosis in the Human Lung Large Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Cheng-Yuan Wang

    2010-02-01

    Full Text Available Toona sinensis extracts have been shown to exhibit anti-cancer effects in human ovarian cancer cell lines, human promyelocytic leukemia cells and human lung adenocarcinoma. Its safety has also been confirmed in animal studies. However, its anti-cancer properties in human lung large cell carcinoma have not been studied. Here, we used a powder obtained by freeze-drying the super-natant of centrifuged crude extract from Toona sinensis leaves (TSL-1 to treat the human lung carcinoma cell line H661. Cell viability was evaluated by the 3-(4-,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide assay. Flow cytometry analysis revealed that TSL-1 blocked H661 cell cycle progression. Western blot analysis showed decreased expression of cell cycle proteins that promote cell cycle progression, including cyclin-dependent kinase 4 and cyclin D1, and increased the expression of proteins that inhibit cell cycle progression, including p27. Furthermore, flow cytometry analysis showed that TSL-1 induced H661 cell apoptosis. Western blot analysis showed that TSL-1 reduced the expression of the anti-apoptotic protein B-cell lymphoma 2, and degraded the DNA repair protein, poly(ADP-ribose polymerase. TSL-1 shows potential as a novel therapeutic agent or for use as an adjuvant for treating human lung large cell carcinoma.

  3. Raddeanin A induces human gastric cancer cells apoptosis and inhibits their invasion in vitro

    International Nuclear Information System (INIS)

    Xue, Gang; Zou, Xi; Zhou, Jin-Yong; Sun, Wei; Wu, Jian; Xu, Jia-Li; Wang, Rui-Ping

    2013-01-01

    Highlights: •Raddeanin A is a triterpenoid saponin in herb medicine Anemone raddeana Regel. •Raddeanin A can inhibit 3 kinds of gastric cancer cells’ proliferation and invasion. •Caspase-cascades’ activation indicates apoptosis induced by Raddeanin A. •MMPs, RECK, Rhoc and E-cad are involved in Raddeanin A-induced invasion inhibition. -- Abstract: Raddeanin A is one of the triterpenoid saponins in herbal medicine Anemone raddeana Regel which was reported to suppress the growth of liver and lung cancer cells. However, little was known about its effect on gastric cancer (GC) cells. This study aimed to investigate its inhibitory effect on three kinds of different differentiation stage GC cells (BGC-823, SGC-7901 and MKN-28) in vitro and the possible mechanisms. Proliferation assay and flow cytometry demonstrated Raddeanin A’s dose-dependent inhibitory effect and determined its induction of cells apoptosis, respectively. Transwell assay, wounding heal assay and cell matrix adhesion assay showed that Raddeanin A significantly inhibited the abilities of the invasion, migration and adhesion of the BGC-823 cells. Moreover, quantitative real time PCR and Western blot analysis found that Raddeanin A increased Bax expression while reduced Bcl-2, Bcl-xL and Survivin expressions and significantly activated caspase-3, caspase-8, caspase-9 and poly-ADP ribose polymerase (PARP). Besides, Raddeanin A could also up-regulate the expression of reversion inducing cysteine rich protein with Kazal motifs (RECK), E-cadherin (E-cad) and down-regulate the expression of matrix metalloproteinases-2 (MMP-2), MMP-9, MMP-14 and Rhoc. In conclusion, Raddeanin A inhibits proliferation of human GC cells, induces their apoptosis and inhibits the abilities of invasion, migration and adhesion, exhibiting potential to become antitumor drug

  4. Raddeanin A induces human gastric cancer cells apoptosis and inhibits their invasion in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Xue, Gang [Department of Oncology, Nanjing University of Chinese Medicine, Nanjing (China); Zou, Xi [Department of Oncology, Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Traditional Chinese Medicine, Nanjing (China); Zhou, Jin-Yong [Laboratory Center, Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Traditional Chinese Medicine, Nanjing (China); Sun, Wei [Department of Oncology, Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Traditional Chinese Medicine, Nanjing (China); Wu, Jian [Laboratory Center, Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Traditional Chinese Medicine, Nanjing (China); Xu, Jia-Li [Department of Oncology, Nanjing University of Chinese Medicine, Nanjing (China); Wang, Rui-Ping, E-mail: ruipingwang61@hotmail.com [Department of Oncology, Affiliated Hospital of Nanjing University of Chinese Medicine, Jiangsu Province Hospital of Traditional Chinese Medicine, Nanjing (China)

    2013-09-20

    Highlights: •Raddeanin A is a triterpenoid saponin in herb medicine Anemone raddeana Regel. •Raddeanin A can inhibit 3 kinds of gastric cancer cells’ proliferation and invasion. •Caspase-cascades’ activation indicates apoptosis induced by Raddeanin A. •MMPs, RECK, Rhoc and E-cad are involved in Raddeanin A-induced invasion inhibition. -- Abstract: Raddeanin A is one of the triterpenoid saponins in herbal medicine Anemone raddeana Regel which was reported to suppress the growth of liver and lung cancer cells. However, little was known about its effect on gastric cancer (GC) cells. This study aimed to investigate its inhibitory effect on three kinds of different differentiation stage GC cells (BGC-823, SGC-7901 and MKN-28) in vitro and the possible mechanisms. Proliferation assay and flow cytometry demonstrated Raddeanin A’s dose-dependent inhibitory effect and determined its induction of cells apoptosis, respectively. Transwell assay, wounding heal assay and cell matrix adhesion assay showed that Raddeanin A significantly inhibited the abilities of the invasion, migration and adhesion of the BGC-823 cells. Moreover, quantitative real time PCR and Western blot analysis found that Raddeanin A increased Bax expression while reduced Bcl-2, Bcl-xL and Survivin expressions and significantly activated caspase-3, caspase-8, caspase-9 and poly-ADP ribose polymerase (PARP). Besides, Raddeanin A could also up-regulate the expression of reversion inducing cysteine rich protein with Kazal motifs (RECK), E-cadherin (E-cad) and down-regulate the expression of matrix metalloproteinases-2 (MMP-2), MMP-9, MMP-14 and Rhoc. In conclusion, Raddeanin A inhibits proliferation of human GC cells, induces their apoptosis and inhibits the abilities of invasion, migration and adhesion, exhibiting potential to become antitumor drug.

  5. Experimental research on recombinant human endostatin-induced cardiomyocyte apoptosis in rats

    Directory of Open Access Journals (Sweden)

    Jing QIN

    2014-03-01

    Full Text Available Objective To explore the recombinant human endostatin (rh-ES-induced cardiotoxicity in rats and its mechanism. Methods Twenty four female Wistar rats were randomly divided into four groups (6 each. Rats in low, moderate and high dose group received rh-ES with a dosage of 3, 6 and 12mg/(kg·d, respectively, by intraperitoneal injection, and rats in control group received the same amount of normal saline alone. Half of rats in each group were sacrificed by spinal dislocation after 4 weeks and 8 weeks of the treatment. Pathomorphologic and ultrastructural changes in rat's myocardial tissue were evaluated by light microscopy and transmission electron microscopy. Cardiomyocyte apoptosis was detected with TdT-mediated dUTP nick end labeling (TUNEL assay. Microvessel density (MVD in myocardial tissue was measured by immunohistochemically marking endothelial cell with CD34. Results No pathomorphologic and ultrastrucural changes were found under light microscope and transmission electron microscope in the low dose and moderate dose groups, but cardiomyocyte damage were found in the high dose group. TUNEL assay revealed more apoptotic cells in high and moderate (only 8 weeks dose groups than in control group (P=0.033, P=0.000, and the apoptosis index was highest in the high dose group at 8 weeks. In addition, compared with the control group, MVD significantly increased in high dose groups at 4 weeks and 8 weeks (P<0.05. Conclusions rh-ES induces the cardiotoxicity in rats, and cardiomyocyte apoptosis is involved in the pathological course of cardiac toxicity. DOI: 10.11855/j.issn.0577-7402.2014.01.02

  6. Je-Chun-Jun induced apoptosis of human cervical carcinoma HeLa cells

    Institute of Scientific and Technical Information of China (English)

    Han-jung CHAE; Kyung-mi PARK; Geun-youn LEE; Gi-seup JEONG; Hyung-rae PARK; Hyung-min KIM; Soo-wan CHAE; Shim-keun YOO; Hyung-ryong KIM

    2004-01-01

    AIM: To study the mechanism of Je-Chun-Jun (JCJ)-inducing the apoptosis of the human cervical carcinoma,HeLa cells. METHODS: The cell viability was assessed using MTT assay. The optical density was measured at 570 nm. The caspase activity was measured using 50 mmol/L of fluorogenic substrate, AC-DEVD-AMC (caspase3), AC-VEID-AMC (caspase-8) or AC-LEHD-AFC (caspase-9). To confirm the expression of proteins, Western blotting was performed. To detect the characteristic of apoptosis chromatin condensation, HeLa cells were stained with Hoechst 33258 in the presence of JCJ. For the cell cycle analysis, HeLa cells were incubated with Propidium iodide (PI) solution. Fluorescence intensity of cell cycle was measured using flow cytometry system. RESULTS:The loss of viability occurred following the exposure of 10 g/L JCJ. Cells treated with 10 g/L JCJ for 3 d exhibited the apoptotic morphology (brightly blue-fluorescent condensed nuclei by Hoechst 33258-staining) and the reduction of cell volume. Cells incubated with JCJ for 48 h were arrested at the G1 phase of cell cycle and their G1 checkpoint related gene products such as cyclin D1 were transiently decreased. We showed that JCJ induced the p38 MAPK activation in HeLa cells. The p38 MAPK inhibitor, SB203580 protected Hela cells from the JCJ-induced death as well as intervened the JCJ-induced accumulation of cells at the G1 phase. In contrast, MEK1 (-ERK upstream) inhibitor, PD98059 had no effect on HeLa cells. CONCLUSION: JCJ induced cell cycle arrest and apoptosis of HeLa cells through p38 MAPK pathway.

  7. Apoptosis-like death in bacteria induced by HAMLET, a human milk lipid-protein complex.

    Directory of Open Access Journals (Sweden)

    Anders P Hakansson

    Full Text Available BACKGROUND: Apoptosis is the primary means for eliminating unwanted cells in multicellular organisms in order to preserve tissue homeostasis and function. It is characterized by distinct changes in the morphology of the dying cell that are orchestrated by a series of discrete biochemical events. Although there is evidence of primitive forms of programmed cell death also in prokaryotes, no information is available to suggest that prokaryotic death displays mechanistic similarities to the highly regulated programmed death of eukaryotic cells. In this study we compared the characteristics of tumor and bacterial cell death induced by HAMLET, a human milk complex of alpha-lactalbumin and oleic acid. METHODOLOGY/PRINCIPAL FINDINGS: We show that HAMLET-treated bacteria undergo cell death with mechanistic and morphologic similarities to apoptotic death of tumor cells. In Jurkat cells and Streptococcus pneumoniae death was accompanied by apoptosis-like morphology such as cell shrinkage, DNA condensation, and DNA degradation into high molecular weight fragments of similar sizes, detected by field inverse gel electrophoresis. HAMLET was internalized into tumor cells and associated with mitochondria, causing a rapid depolarization of the mitochondrial membrane and bound to and induced depolarization of the pneumococcal membrane with similar kinetic and magnitude as in mitochondria. Membrane depolarization in both systems required calcium transport, and both tumor cells and bacteria were found to require serine protease activity (but not caspase activity to execute cell death. CONCLUSIONS/SIGNIFICANCE: Our results suggest that many of the morphological changes and biochemical responses associated with apoptosis are present in prokaryotes. Identifying the mechanisms of bacterial cell death has the potential to reveal novel targets for future antimicrobial therapy and to further our understanding of core activation mechanisms of cell death in eukaryote cells.

  8. Apoptosis-like death in bacteria induced by HAMLET, a human milk lipid-protein complex.

    Science.gov (United States)

    Hakansson, Anders P; Roche-Hakansson, Hazeline; Mossberg, Ann-Kristin; Svanborg, Catharina

    2011-03-10

    Apoptosis is the primary means for eliminating unwanted cells in multicellular organisms in order to preserve tissue homeostasis and function. It is characterized by distinct changes in the morphology of the dying cell that are orchestrated by a series of discrete biochemical events. Although there is evidence of primitive forms of programmed cell death also in prokaryotes, no information is available to suggest that prokaryotic death displays mechanistic similarities to the highly regulated programmed death of eukaryotic cells. In this study we compared the characteristics of tumor and bacterial cell death induced by HAMLET, a human milk complex of alpha-lactalbumin and oleic acid. We show that HAMLET-treated bacteria undergo cell death with mechanistic and morphologic similarities to apoptotic death of tumor cells. In Jurkat cells and Streptococcus pneumoniae death was accompanied by apoptosis-like morphology such as cell shrinkage, DNA condensation, and DNA degradation into high molecular weight fragments of similar sizes, detected by field inverse gel electrophoresis. HAMLET was internalized into tumor cells and associated with mitochondria, causing a rapid depolarization of the mitochondrial membrane and bound to and induced depolarization of the pneumococcal membrane with similar kinetic and magnitude as in mitochondria. Membrane depolarization in both systems required calcium transport, and both tumor cells and bacteria were found to require serine protease activity (but not caspase activity) to execute cell death. Our results suggest that many of the morphological changes and biochemical responses associated with apoptosis are present in prokaryotes. Identifying the mechanisms of bacterial cell death has the potential to reveal novel targets for future antimicrobial therapy and to further our understanding of core activation mechanisms of cell death in eukaryote cells.

  9. Effect of Flavopiridol on Radiation-induced Apoptosis of Human Laryngeal and Lung Cancer Cells

    International Nuclear Information System (INIS)

    Kim, Suzy; Kwon, Eun Kyung; Lee, B. S.; Lee, Seung Hee; Park, B. S.; Wu, Hong Gyun

    2007-01-01

    Purpose: To investigate the flavopiridol effect on radiation-induced apoptosis and expression of apoptosisrelated genes of human laryngeal and lung cancer cells. Materials and Methods: A human laryngeal cancer cell line, AMC-HN3 and a human lung cancer cell line, NCI-H460, were used in the study. The cells were divided into four groups according to the type of treatment: 1) control groups; 2) cells that were only irradiated; 3) cells treated only with flavopiridol; 4) cells treated with flavopiridol and radiation simultaneously. The cells were irradiated with 10 Gy of X-rays using a 4 MV linear accelerator. Flavopiridol was administered to the media at a concentration of 100 nM for 24 hours. We compared the fraction of apoptotic cells of each group 24 hours after the initiation of treatment. The fraction of apoptotic cells was detected by measurement of the sub-G1 fractions from a flow cytometric analysis. The expression of apoptosis-regulating genes, including cleaved caspase-3, cleaved PARP (poly (ADP-ribose) polymerase), p53, p21, cyclin D1, and phosphorylated Akt (protein kinase B) were analyzed by Western blotting. Results: The sub-G1 fraction of cells was significantly increased in the combination treatment group, as compared to cells exposed to radiation alone or flavopiridol alone. Western blotting also showed an increased expression of cleaved caspase-3 and cleaved PARP expression in cells of the combination treatment group, as compared with cells exposed to radiation alone or flavopiridol alone. Treatment with flavopiridol down regulated cyclin D1 expression of both cell lines but its effect on p53 and p21 expression was different according to each individual cell line. Flavopiridol did not affect the expression of phophorylated Akt in both cell lines. Conclusion: Treatment with flavopiridol increased radiation-induced apoptosis of both the human laryngeal and lung cancer cell lines. Flavopiridol effects on p53 and p21 expression were different according

  10. Matrix Metalloproteinase-9/Neutrophil Gelatinase-Associated Lipocalin Complex Activity in Human Glioma Samples Predicts Tumor Presence and Clinical Prognosis

    Directory of Open Access Journals (Sweden)

    Ming-Fa Liu

    2015-01-01

    Full Text Available Matrix metalloproteinase-9/neutrophil gelatinase-associated lipocalin (MMP-9/NGAL complex activity is elevated in brain tumors and may serve as a molecular marker for brain tumors. However, the relationship between MMP-9/NGAL activity in brain tumors and patient prognosis and treatment response remains unclear. Here, we compared the clinical characteristics of glioma patients with the MMP-9/NGAL activity measured in their respective tumor and urine samples. Using gelatin zymography assays, we found that MMP-9/NGAL activity was significantly increased in tumor tissues (TT and preoperative urine samples (Preop-1d urine. Activity was reduced by seven days after surgery (Postop-1w urine and elevated again in cases of tumor recurrence. The MMP-9/NGAL status correlated well with MRI-based tumor assessments. These findings suggest that MMP-9/NGAL activity could be a novel marker to detect gliomas and predict the clinical outcome of patients.

  11. 3-Bromopyruvate and sodium citrate induce apoptosis in human gastric cancer cell line MGC-803 by inhibiting glycolysis and promoting mitochondria-regulated apoptosis pathway

    International Nuclear Information System (INIS)

    Guo, Xingyu; Zhang, Xiaodong; Wang, Tingan; Xian, Shulin; Lu, Yunfei

    2016-01-01

    Cancer cells are mainly dependent on glycolysis to generate adenosine triphosphate (ATP) and intermediates required for cell growth and proliferation. Thus, inhibition of glycolysis might be of therapeutic value in antitumor treatment. Our previously studies had found that both 3-bromopyruvate (BP) and sodium citrate (SCT) can inhibit tumor growth and proliferation in vitro and in vivo. However, the mechanism involved in the BP and SCT mediated antitumor activity is not entirely clear. In this work, it is demonstrated that BP inhibits the enzyme hexokinase (HK) activity and SCT suppresses the phosphofructokinase (PFK) activity respectively, both the two agents decrease viability, ATP generation and lactate content in the human gastric cancer cell line MGC-803. These effects are directly correlated with blockage of glycolysis. Furthermore, BP and SCT can induce the characteristic manifestations of mitochondria-regulated apoptosis, such as down-regulation of anti-apoptosis proteins Bcl-2 and Survivin, up-regulation of pro-apoptosis protein Bax, activation of caspase-3, as well as leakage of cytochrome c (Cyt-c). In summary, our results provided evidences that BP and SCT inhibit the MGC-803 cells growth and proliferation might be correlated with inhibiting glycolysis and promoting mitochondria-regulated apoptosis. -- Highlights: •Blockage of glycolysis might be a novel way to anticancer. •Both 3-bromopyruvate and sodium citrate could inhibit glycolysis and regulate mitochondrial pathway in cancer cells. •Both 3-bromopyruvate and sodium citrate would be the novel agents on treatment of gastric cancer.

  12. 3-Bromopyruvate and sodium citrate induce apoptosis in human gastric cancer cell line MGC-803 by inhibiting glycolysis and promoting mitochondria-regulated apoptosis pathway

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Xingyu; Zhang, Xiaodong; Wang, Tingan, E-mail: moonsonlife@yahoo.com; Xian, Shulin; Lu, Yunfei, E-mail: doctorlife@126.com

    2016-06-17

    Cancer cells are mainly dependent on glycolysis to generate adenosine triphosphate (ATP) and intermediates required for cell growth and proliferation. Thus, inhibition of glycolysis might be of therapeutic value in antitumor treatment. Our previously studies had found that both 3-bromopyruvate (BP) and sodium citrate (SCT) can inhibit tumor growth and proliferation in vitro and in vivo. However, the mechanism involved in the BP and SCT mediated antitumor activity is not entirely clear. In this work, it is demonstrated that BP inhibits the enzyme hexokinase (HK) activity and SCT suppresses the phosphofructokinase (PFK) activity respectively, both the two agents decrease viability, ATP generation and lactate content in the human gastric cancer cell line MGC-803. These effects are directly correlated with blockage of glycolysis. Furthermore, BP and SCT can induce the characteristic manifestations of mitochondria-regulated apoptosis, such as down-regulation of anti-apoptosis proteins Bcl-2 and Survivin, up-regulation of pro-apoptosis protein Bax, activation of caspase-3, as well as leakage of cytochrome c (Cyt-c). In summary, our results provided evidences that BP and SCT inhibit the MGC-803 cells growth and proliferation might be correlated with inhibiting glycolysis and promoting mitochondria-regulated apoptosis. -- Highlights: •Blockage of glycolysis might be a novel way to anticancer. •Both 3-bromopyruvate and sodium citrate could inhibit glycolysis and regulate mitochondrial pathway in cancer cells. •Both 3-bromopyruvate and sodium citrate would be the novel agents on treatment of gastric cancer.

  13. Impact of Salinomycin on human cholangiocarcinoma: induction of apoptosis and impairment of tumor cell proliferation in vitro

    Directory of Open Access Journals (Sweden)

    Lieke Thorsten

    2012-10-01

    Full Text Available Abstract Background Cholangiocarcinoma (CC is a primary liver cancer with increasing incidence worldwide. Despite all efforts made in past years, prognosis remains to be poor. At least in part, this might be explained by a pronounced resistance of CC cells to undergo apoptosis. Thus, new therapeutic strategies are imperatively required. In this study we investigated the effect of Salinomycin, a polyether ionophore antibiotic, on CC cells as an appropriate agent to treat CC. Salinomycin was quite recently identified to induce apoptosis in cancer stem cells and to overcome apoptosis-resistance in several leukemia-cells and other cancer cell lines of different origin. Methods To delineate the effects of Salinomycin on CC, we established an in vitro cell culture model using three different human CC cell lines. After treatment apoptosis as well as migration and proliferation behavior was assessed and additional cell cycle analyses were performed by flowcytometry. Results By demonstrating Annexin V and TUNEL positivity of human CC cells, we provide evidence that Salinomycin reveals the capacity to break apoptosis-resistance in CC cells. Furthermore, we are able to demonstrate that the non-apoptotic cell fraction is characterized by sustainable impaired migration and proliferation. Cell cycle analyses revealed G2-phase accumulation of human CC cells after treatment with Salinomycin. Even though apoptosis is induced in two of three cell lines of CC cells, one cell line remained unaffected in regard of apoptosis but revealed as the other CC cells decreased proliferation and migration. Conclusion In this study, we are able to demonstrate that Salinomycin is an effective agent against previously resistant CC cells and might be a potential candidate for the treatment of CC in the future.

  14. Transcutaneous application of carbon dioxide (CO2 induces mitochondrial apoptosis in human malignant fibrous histiocytoma in vivo.

    Directory of Open Access Journals (Sweden)

    Yasuo Onishi

    Full Text Available Mitochondria play an essential role in cellular energy metabolism and apoptosis. Previous studies have demonstrated that decreased mitochondrial biogenesis is associated with cancer progression. In mitochondrial biogenesis, peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α regulates the activities of multiple nuclear receptors and transcription factors involved in mitochondrial proliferation. Previously, we showed that overexpression of PGC-1α leads to mitochondrial proliferation and induces apoptosis in human malignant fibrous histiocytoma (MFH cells in vitro. We also demonstrated that transcutaneous application of carbon dioxide (CO(2 to rat skeletal muscle induces PGC-1α expression and causes an increase in mitochondrial proliferation. In this study, we utilized a murine model of human MFH to determine the effect of transcutaneous CO(2 exposure on PGC-1α expression, mitochondrial proliferation and cellular apoptosis. PGC-1α expression was evaluated by quantitative real-time PCR, while mitochondrial proliferation was assessed by immunofluorescence staining and the relative copy number of mitochondrial DNA (mtDNA was assessed by real-time PCR. Immunofluorescence staining and DNA fragmentation assays were used to examine mitochondrial apoptosis. We also evaluated the expression of mitochondrial apoptosis related proteins, such as caspases, cytochorome c and Bax, by immunoblot analysis. We show that transcutaneous application of CO(2 induces PGC-1α expression, and increases mitochondrial proliferation and apoptosis of tumor cells, significantly reducing tumor volume. Proteins involved in the mitochondrial apoptotic cascade, including caspase 3 and caspase 9, were elevated in CO(2 treated tumors compared to control. We also observed an enrichment of cytochrome c in the cytoplasmic fraction and Bax protein in the mitochondrial fraction of CO(2 treated tumors, highlighting the involvement of mitochondria in apoptosis

  15. NiO nanoparticles induce apoptosis through repressing SIRT1 in human bronchial epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Duan, Wei-Xia; He, Min-Di; Mao, Lin [Department of Occupational Health, Third Military Medical University, Chongqing 400038 (China); Qian, Feng-Hua [Department of Hematology, Southwest Hospital, Third Military Medical University, Chongqing 400038 (China); Li, Yu-Ming [Institute of Hepatobiliary Surgery, XinQiao Hospital, Third Military Medical University, Chongqing 400038 (China); Pi, Hui-Feng; Liu, Chuan; Chen, Chun-Hai; Lu, Yong-Hui; Cao, Zheng-Wang; Zhang, Lei; Yu, Zheng-Ping [Department of Occupational Health, Third Military Medical University, Chongqing 400038 (China); Zhou, Zhou, E-mail: lunazhou00@163.com [Department of Occupational Health, Third Military Medical University, Chongqing 400038 (China)

    2015-07-15

    With application of nano-sized nickel-containing particles (Nano-Ni) expanding, the health concerns about their adverse effects on the pulmonary system are increasing. However, the mechanisms for the pulmonary toxicity of these materials remain unclear. In the present study, we focused on the impacts of NiO nanoparticles (NiONPs) on sirtuin1 (SIRT1), a NAD-dependent deacetylase, and investigated whether SIRT1 was involved in NiONPs-induced apoptosis. Although the NiONPs tended to agglomerate in fluid medium, they still entered into the human bronchial epithelial cells (BEAS-2B) and released Ni{sup 2+} inside the cells. NiONPs at doses of 5, 10, and 20 μg/cm{sup 2} inhibited the cell viability. NiONPs' produced cytotoxicity was demonstrated through an apoptotic process, indicated by increased numbers of Annexin V positive cells and caspase-3 activation. The expression of SIRT1 was markedly down-regulated by the NiONPs, accompanied by the hyperacetylation of p53 (tumor protein 53) and overexpression of Bax (Bcl-2-associated X protein). However, overexpression of SIRT1 through resveratrol treatment or transfection clearly attenuated the NiONPs-induced apoptosis and activation of p53 and Bax. Our results suggest that the repression of SIRT1 may underlie the NiONPs-induced apoptosis via p53 hyperacetylation and subsequent Bax activation. Because SIRT1 participates in multiple biologic processes by deacetylation of dozens of substrates, this knowledge of the impact of NiONPs on SIRT1 may lead to an improved understanding of the toxic mechanisms of Nano-Ni and provide a molecular target to antagonize Nano-Ni toxicity. - Highlights: • NiONPs were taken up by BEAS-2B cells and released Ni{sup 2+}. • NiONPs produced cytotoxicity was demonstrated through an apoptotic process. • NiONPs repressed SIRT1 expression and activated p53 and Bax. • Overexpression of SIRT1 attenuated NiONPs-induced apoptosis via deacetylation p53.

  16. Role of EGFR transactivation in preventing apoptosis in Pseudomonas aeruginosa-infected human corneal epithelial cells.

    Science.gov (United States)

    Zhang, Jing; Li, Hui; Wang, Jinzhao; Dong, Zheng; Mian, Shahzad; Yu, Fu-Shin X

    2004-08-01

    To determine the role of epidermal growth factor (EGF) receptor (EGFR)-mediated signaling pathways in preventing infection-induced apoptosis in human corneal epithelial cells (HCECs). Epithelial monolayers of a telomerase-immortalized HCEC line, HUCL, and primary culture of HCECs were infected with Pseudomonas aeruginosa in the presence of the EGFR inhibitor tyrphostin AG1478, the extracellular signal-regulated kinase (ERK) inhibitor U0126, the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, the heparin-binding EGF-like growth factor (HB-EGF) antagonist CRM197, the HB-EGF neutralizing antibody, or the matrix metalloproteinase inhibitor GM6001. The activation of EGFR was analyzed by immunoprecipitation using EGFR antibodies, followed by Western blot analysis with phosphotyrosine antibody. Phosphorylation of ERK and Akt, a major substrate of PI3K, and generation of cleaved caspase-3 and poly (ADP-ribose) polymerase (PARP) were determined by Western blot analysis. Apoptotic cells were characterized by positive staining of active caspase-3, loss of mitochondrial cytochrome c, and condensation of chromosomes. Apoptosis was also confirmed by measuring caspase-3 activity and assessing the generation of cleaved caspase-3 and PARP. P. aeruginosa infection of HUCL cells resulted in EGFR activation and EGFR-dependent ERK1/2 and PI3K phosphorylation. Inhibition of EGFR, ERK1/2, and PI3K activities with kinase-specific inhibitors (AG1478, U0126, and LY294002, respectively) resulted in an increase in the number of apoptotic cells, in elevated cellular caspase-3 activity, and/or in increased cleaved PARP in P. aeruginosa-infected HUCL cells or primary culture of HCECs. Blocking HB-EGF ectodomain shedding by inhibition of matrix metalloproteinase-mediated proteolysis, downregulation of HB-EGF, or neutralization of its activity retarded infection-induced EGFR transactivation and, as a consequence, increased infection-induced HUCL apoptosis. Bacterial infection of HCECs induces

  17. Role of EGFR Transactivation in Preventing Apoptosis in Pseudomonas aeruginosa–Infected Human Corneal Epithelial Cells

    Science.gov (United States)

    Zhang, Jing; Li, Hui; Wang, Jinzhao; Dong, Zheng; Mian, Shahzad; Yu, Fu-Shin X.

    2009-01-01

    PURPOSE To determine the role of epidermal growth factor (EGF) receptor (EGFR)–mediated signaling pathways in preventing infection-induced apoptosis in human corneal epithelial cells (HCECs). METHODS Epithelial monolayers of a telomerase-immortalized HCEC line, HUCL, and primary culture of HCECs were infected with Pseudomonas aeruginosa in the presence of the EGFR inhibitor tyrphostin AG1478, the extracellular signal-regulated kinase (ERK) inhibitor U0126, the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, the heparin-binding EGF-like growth factor (HB-EGF) antagonist CRM197, the HB-EGF neutralizing antibody, or the matrix metalloproteinase inhibitor GM6001. The activation of EGFR was analyzed by immunoprecipitation using EGFR antibodies, followed by Western blot analysis with phosphotyrosine antibody. Phosphorylation of ERK and Akt, a major substrate of PI3K, and generation of cleaved caspase-3 and poly (ADP-ribose) polymerase (PARP) were determined by Western blot analysis. Apoptotic cells were characterized by positive staining of active caspase-3, loss of mitochondrial cytochrome c, and condensation of chromosomes. Apoptosis was also confirmed by measuring caspase-3 activity and assessing the generation of cleaved caspase-3 and PARP. RESULTS P. aeruginosa infection of HUCL cells resulted in EGFR activation and EGFR-dependent ERK1/2 and PI3K phosphorylation. Inhibition of EGFR, ERK1/2, and PI3K activities with kinase-specific inhibitors (AG1478, U0126, and LY294002, respectively) resulted in an increase in the number of apoptotic cells, in elevated cellular caspase-3 activity, and/or in increased cleaved PARP in P. aeruginosa–infected HUCL cells or primary culture of HCECs. Blocking HB-EGF ectodomain shedding by inhibition of matrix metalloproteinase–mediated proteolysis, downregulation of HB-EGF, or neutralization of its activity retarded infection-induced EGFR transactivation and, as a consequence, increased infection-induced HUCL apoptosis

  18. Jaceosidin Induces Apoptosis in Human Ovary Cancer Cells through Mitochondrial Pathway

    Directory of Open Access Journals (Sweden)

    Wen Lv

    2008-01-01

    Full Text Available We examined the antiproliferation effect of Jaceosidin (4′, 5, 7-trihydroxy-3′, 6-dimethoxyflavone isolated from the herb of Artemisia vestita Wall on several human cancer cell lines. Jaceosidin significantly reduced the proliferation of CAOV-3, SKOV-3, HeLa, and PC3 cells in a concentration-dependent manner. A time-dependent inhibition was also observed in CAOV-3 cells by Jaceosidin. By flow cytometric analysis, we found that Jaceosidin treatment resulted in an increased apoptosis in CAOV-3 cells. The cells treated with Jaceosidin exhibited a decreased mitochondrial membrane potential. Jaceosidin also increased the level of cleaved caspase-9 and induced the cleavage of caspase-3 and poly (ADP-ribose polymerase (PARP, while caspase-3 inhibitor Z-DEVD-FMK significantly reversed the proapoptotic effect of Jaceosidin in CAOV-3 cells. Moreover, Jaceosidin elevated the level of cytochrome c in cytosol. These findings suggest that the anticancer effect of Jaceosidin may be contributed by an induction of apoptosis involving cytochrome c release from mitochondria to cytosol.

  19. Orlistat Reduces Proliferation and Enhances Apoptosis in Human Pancreatic Cancer Cells (PANC-1).

    Science.gov (United States)

    Sokolowska, Ewa; Presler, Malgorzata; Goyke, Elzbieta; Milczarek, Ryszard; Swierczynski, Julian; Sledzinski, Tomasz

    2017-11-01

    Pancreatic cancer is a disease with very poor prognosis, and none of currently available pharmacotherapies have proven to be efficient in this indication. The aim of this study was to analyze the expression of fatty acid synthase (FASN) gene as a potential therapeutic target in proliferating human pancreatic cancer cells (PANC-1), and verify if orlistat, originally developed as an anti-obesity drug, inhibits PANC-1 proliferation. The effects of orlistat on gene expression, lipogenesis, proliferation and apoptosis was studied in PANC-1 cell culture. Expression of FASN increased during proliferation of PANC-1. Inhibition of FASN by orlistat resulted in a significant reduction of PANC-1 proliferation and enhanced apoptosis of these cells. This study showed, to our knowledge for the first time, that orlistat exhibits significant antitumor activity against PANC-1 cells. This implies that orlistat analogs with good oral bioavailability may find application in pharmacotherapy of pancreatic cancer. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  20. Effects of disulfiram on apoptosis in PANC-1 human pancreatic cancer cell line.

    Science.gov (United States)

    Dastjerdi, M Nikbakht; Babazadeh, Z; Rabbani, M; Gharagozloo, M; Esmaeili, A; Narimani, M

    2014-01-01

    Pancreatic carcinoma is currently considered as a rapidly progressive and fatal disease, and is typically diagnosed late in its natural course. It is characterized by a poor diagnosis and lack of response to conventional therapy. Recent studies have suggested that disulfiram (DSF), a member of the dithiocarbamate family, may have antitumor activity. This study aimed to evaluate the in vitro effect of DSF on apoptosis in human pancreatic cancerous cell line (PANC-1). PANC-1 cells were cultured and treated with DSF at doses of 5, 10, 13 μM for 24 h and apoptosis was measured. Methylation specific PCR (MS-PCR) and real-time quantitative PCR were carried out to detect the methylation pattern and to estimate the mRNA expression levels of RASSF1A, p21 and Bax. MS-PCR analysis demonstrated that no unmethylated band was apeared in PANC-1 cell line after DSF treatments. The real-time quantitative PCR results showed no significant mRNA expression for RASSF1A (p>0.05); whereas p21 and Bax expression were significantly (pPANC-1 through p21 and Bax pathway but not through RASSF1A.

  1. Cytotoxicity and apoptosis induction by e-cigarette fluids in human gingival fibroblasts.

    Science.gov (United States)

    Sancilio, Silvia; Gallorini, Marialucia; Cataldi, Amelia; di Giacomo, Viviana

    2016-04-01

    Electronic cigarettes (e-cigarettes) are generally acknowledged as a safer alternative to the use of combusted tobacco products. Nevertheless, there are increasing conflicting claims concerning the effect of these novel industrial products on the health of e-cigarettes users. The aim of this work was to investigate the effects of the liquids of e-cigarettes on human gingival fibroblasts (HGFs) and to compare the effects of nicotine-containing fluid to the fluid itself. HGFs were treated with different concentrations (0-5 mg/mL) of fluids of e-cigarettes for different times (0-72 h) and cytotoxicity was analyzed by MTT assay. Fluids were administered also after being vaped (e.g., warmed into the cartomizer). Apoptosis occurrence and Bax expression were evaluated by flow cytometry; ROS production was analyzed by fluorescence optical microscopy. Both nicotine-containing and nicotine-free fluids induced an increased ROS production after 24 h, along with an increased Bax expression, followed by apoptosis occurrence after 48 h of exposure. The cytotoxicity exerted on HGFs by e-cigarettes fluids is not entirely ascribable to nicotine. Since the e-cigarettes are advertised as a safer alternative to traditional ones, especially for the possibility of "smoking" nicotine-free fluids, further studies are necessary to clarify the mechanism involved in the occurrence of cytotoxicity exerted by such compounds. Our results suggest a role for e-cigarette fluids in the pathogenesis of oral diseases, such as periodontitis.

  2. Edaravone protects against hyperosmolarity-induced oxidative stress and apoptosis in primary human corneal epithelial cells.

    Directory of Open Access Journals (Sweden)

    Yanwei Li

    Full Text Available An increase in the osmolarity of tears induced by excessive evaporation of the aqueous tear phase is a major pathological mechanism behind dry eye. Exposure of epithelial cells on the surface of the human eye to hyperosmolarity leads to oxidative stress, mitochondrial dysfunction, and apoptosis. Edaravone, a hydroxyl radical scavenging agent, is clinically used to reduce neuronal damage following ischemic stroke. In this study, we found that treatment with hyperosmotic media at 400 and 450 mOsM increased the levels of ROS and mitochondrial oxidative damage, which were ameliorated by edaravone treatment in a dose-dependent manner. We also found that edaravone could improve mitochondrial function in HCEpiCs by increasing the levels of ATP and mitochondrial membrane potential. MTT and LDH assays indicated that edaravone could attenuate hyperosmolarity-induced cell death. It was found that edaravone prevented apoptosis by decreasing the level of cleaved caspase-3, and attenuating the release of cytochrome C. Mechanistically, we found that edaravone augmented the expression of Nrf2 and its target genes, such as HO-1, GPx-1, and GCLC.

  3. Edaravone protects against hyperosmolarity-induced oxidative stress and apoptosis in primary human corneal epithelial cells.

    Science.gov (United States)

    Li, Yanwei; Liu, Haifeng; Zeng, Wei; Wei, Jing

    2017-01-01

    An increase in the osmolarity of tears induced by excessive evaporation of the aqueous tear phase is a major pathological mechanism behind dry eye. Exposure of epithelial cells on the surface of the human eye to hyperosmolarity leads to oxidative stress, mitochondrial dysfunction, and apoptosis. Edaravone, a hydroxyl radical scavenging agent, is clinically used to reduce neuronal damage following ischemic stroke. In this study, we found that treatment with hyperosmotic media at 400 and 450 mOsM increased the levels of ROS and mitochondrial oxidative damage, which were ameliorated by edaravone treatment in a dose-dependent manner. We also found that edaravone could improve mitochondrial function in HCEpiCs by increasing the levels of ATP and mitochondrial membrane potential. MTT and LDH assays indicated that edaravone could attenuate hyperosmolarity-induced cell death. It was found that edaravone prevented apoptosis by decreasing the level of cleaved caspase-3, and attenuating the release of cytochrome C. Mechanistically, we found that edaravone augmented the expression of Nrf2 and its target genes, such as HO-1, GPx-1, and GCLC.

  4. Edaravone protects human peripheral blood lymphocytes from γ-irradiation-induced apoptosis and DNA damage.

    Science.gov (United States)

    Chen, Liming; Liu, Yinghui; Dong, Liangliang; Chu, Xiaoxia

    2015-03-01

    Radiation-induced cellular injury is attributed primarily to the harmful effects of free radicals, which play a key role in irradiation-induced apoptosis. In this study, we investigated the radioprotective efficacy of edaravone, a licensed clinical drug and a powerful free radical scavenger that has been tested against γ-irradiation-induced cellular damage in cultured human peripheral blood lymphocytes in studies of various diseases. Edaravone was pre-incubated with lymphocytes for 2 h prior to γ-irradiation. It was found that pretreatment with edaravone increased cell viability and inhibited generation of γ-radiation-induced reactive oxygen species (ROS) in lymphocytes exposed to 3 Gy γ-radiation. In addition, γ-radiation decreased antioxidant enzymatic activity, such as superoxide dismutase and glutathione peroxidase, as well as the level of reduced glutathione. Conversely, treatment with 100 μM edaravone prior to irradiation improved antioxidant enzyme activity and increased reduced glutathione levels in irradiated lymphocytes. Importantly, we also report that edaravone reduced γ-irradiation-induced apoptosis through downregulation of Bax, upregulation of Bcl-2, and consequent reduction of the Bax:Bcl-2 ratio. The current study shows edaravone to be an effective radioprotector against γ-irradiation-induced cellular damage in lymphocytes in vitro. Finally, edaravone pretreatment significantly reduced DNA damage in γ-irradiated lymphocytes, as measured by comet assay (% tail DNA, tail length, tail moment, and olive tail moment) (p edaravone offers protection from radiation-induced cytogenetic alterations.

  5. Enhancement of esculetin on Taxol-induced apoptosis in human hepatoma HepG2 cells

    International Nuclear Information System (INIS)

    Kuo, H.-C.; Lee, H.-J.; Hu, C.-C.; Shun, H.-I; Tseng, T.-H.

    2006-01-01

    The potential use of low dose chemotherapy has been appealing since lower dosages are more attainable during cancer therapy and cause less toxicity in patients. Combination therapy of Taxol, a promising frontline chemotherapy agent, with natural anti-tumor agents that are considerably less toxic with a capability of activating additional apoptotic signals or inhibiting survival signals may provide a rational molecular basis for novel chemotherapeutic strategies. Esculetin, a well-known lipoxygenase inhibitor, showed an inhibitory effect on the cell cycle progression of HL-60 cells in our previous study. In this report, the effects of a concomitant administration of esculetin and Taxol were investigated in human hepatoma HepG2 cells. Firstly, esculetin alone could exert an antiproliferation effect together with an inhibitory effect on the activation of ERKs and p38 MAPK. As compared to the treatment with Taxol only, a co-administration with esculetin and Taxol could result in a further enhancement of apoptosis as revealed by DNA fragmentation assay and Annexin-V-based assay. Meanwhile, immunoblotting analysis also showed that the co-administration of esculetin and Taxol could increase the expression of Bax and the cytosolic release of cytochrome C and enhance the expression of Fas and Fas ligand while the activation of caspase-8 and caspase-3 was also increased. Finally, the ERK cascade was proven to be involved in the enhancement of esculetin on the Taxol-induced apoptosis

  6. Acacia catechu ethanolic bark extract induces apoptosis in human oral squamous carcinoma cells

    Directory of Open Access Journals (Sweden)

    Thangavelu Lakshmi

    2017-01-01

    Full Text Available Oral cancer is in approximately 30% of all cancers in India. This study was conducted to evaluate the cytotoxic activity of ethanolic extract of Acacia catechu bark (ACB against human squamous cell carcinoma cell line-25 (SCC-25. Cytotoxic effect of ACB extract was determined by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium Bromide assay. A. catechu extract was treated SCC-25 cells with 25 and 50 μg/mL for 24 h. Apoptosis markers such as caspases-8 and 9, bcl-2, bax, and cytochrome c (Cyt-c were done by RT-PCR. Morphological changes of ACB treated cells were evaluated using acridine orange/ethidium bromide (AO/EB dual staining. Nuclear morphology and DNA fragmentation were evaluated using propidium iodide (PI staining. Further, cell cycle analysis was performed using flow cytometry. A. catechu treatment caused cytotoxicity in SCC-25 cells with an IC50 of 52.09 μg/mL. Apoptotic marker gene expressions were significantly increased on ACB treatment. Staining with AO/EB and PI shows membrane blebbing and nuclear membrane distortion, respectively, and it confirms the apoptosis induction in SCC-25 cells. These results suggest that ACB extract can be used as a modulating agent in oral squamous cell carcinoma.

  7. Echinacoside Induces Apoptosis in Human SW480 Colorectal Cancer Cells by Induction of Oxidative DNA Damages

    Directory of Open Access Journals (Sweden)

    Liwei Dong

    2015-06-01

    Full Text Available Echinacoside is a natural compound with potent reactive oxygen species (ROS-scavenging and anti-oxidative bioactivities, which protect cells from oxidative damages. As cancer cells are often under intense oxidative stress, we therefore tested if Echinacoside treatment would promote cancer development. Surprisingly, we found that Echinacoside significantly inhibited the growth and proliferation of a panel of cancer cell lines. Treatment of the human SW480 cancer cells with Echinacoside resulted in marked apoptosis and cell cycle arrest, together with a significant increase in active caspase 3 and cleaved PARP, and upregulation of the G1/S-CDK blocker CDKN1B (p21. Interestingly, immunocytochemistry examination of drug-treated cancer cells revealed that Echinacoside caused a significant increase of intracellular oxidized guanine, 8-oxoG, and dramatic upregulation of the double-strand DNA break (DSB-binding protein 53BP1, suggesting that Echinacoside induced cell cycle arrest and apoptosis in SW480 cancer cells via induction of oxidative DNA damages. These results establish Echinacoside as a novel chemical scaffold for development of anticancer drugs.

  8. Differentiation of Human Malignant Melanoma Cells that Escape Apoptosis After Treatment with 9-Nitrocamptothecin In Vitro

    Directory of Open Access Journals (Sweden)

    Panayotis Pantazis

    1999-08-01

    Full Text Available After in-vitro exposure to 0.05 μmol/L 9-nitrocamptothecin (9NC for periods of time longer than 5 days, 65% to 80% of the human malignant melanoma SB1 B cells die by apoptosis, whereas the remaining cells are arrested at the G2-phase of the cell cycle. Upon discontinuation of exposure to 9NC the G2-arrested cells resume cell cycling or remain arrested depending on the duration of 9NC exposure. In contrast to cycling malignant cells, the cells irreversibly arrested at G2 exhibit features of normal-like cells, the melanocytes, as assessed by the appearance of dendrite-like structures; loss of proliferative activity; synthesis of the characteristic pigment, melanin; and, particularly, loss of tumorigenic ability after xenografting in immunodeficient mice. Further, the expression of the cyclin-dependent kinase inhibitor p16 is upregulated in the 9NC-treated, G1-arrested, but downregulated in density G1-arrested cells, whereas the reverse is observed in the expression of another cyclin-dependent kinase inhibitor, p21. These results suggest that malignant melanoma SB1B cells that escape 9NC-induced death by apoptosis undergo differentiation toward nonmalignant, normal-like cells.

  9. DETECTION OF OXIDATIVE STRESS, APOPTOSIS AND MOLECULAR LESIONS IN HUMAN OVARIAN CANCER CELLS

    Directory of Open Access Journals (Sweden)

    H. I. Falfushynska

    2016-05-01

    Full Text Available Background. Ovarian cancer has the highest mortality rate of gynaecological cancers. This is partly due to the lack of effective screening markers. Indices of oxidative stress are well-recognized prognostic criteria for tumorous transformation of tissue, but their value depends on the type of tumor and the stage of its development. Objective. The aim of this study is to clarify the relationship between antioxidant/pro-oxidant ratio and the signs of molecular lesions and apoptosis rate in blood of ovarian cancer patients and non-cancer ones. Results. The ovarian cancer group is marked by antioxidant/prooxidant balance shifting to oxidative damage in blood as the consequence of overexpression of oxyradicals (by 300%. Higher level of glutathione (by 366%, lower level of metallothioneins (by 65% as well as higher level of lipid peroxidation (by 174% and protein carbonyls (by 186% in blood of ovarian cancer patients compared to the normal ovarian group have been observed. The signs of cytotoxicity are determined in blood of ovarian cancer patients: an increased (compared to control level of DNA fragmentation (by 160%, choline esterase (up to twice, higher rate of both caspase dependent and caspase independent lysosomal mediated apoptosis. Conclusions. Cathepsin D activity both total and free, choline esterase activity, TBA-reactive substance and protein carbonyls level in blood could be used as the predictive markers of worse prognosis and the signs of human ovarian cancer.

  10. Epothilone B induces extrinsic pathway of apoptosis in human SKOV-3 ovarian cancer cells.

    Science.gov (United States)

    Rogalska, Aneta; Gajek, Arkadiusz; Marczak, Agnieszka

    2014-06-01

    The molecular mechanisms underlying epothilone B (EpoB) induced apoptosis were investigated in SKOV-3 human ovarian cancer cells. The aim of this research was to compare EpoB's, which belongs to the new class of anticancer drugs, with paclitaxel's (PTX) ability to induce apoptosis. The mode of cell death was assessed colorimetrically, fluorimetrically and by immunoblot analyses through measuring DNA fragmentation, the level of intracellular calcium, the level of cytochrome c, TRAIL, the cleavage of poly(ADP-ribose) polymerase (PARP) and the activation of caspase-9, -8 and -3. EpoB leads to an increase of the cytosolic level of cytochrome c after 4 h of cell treatment. After 24 and 48 h of cell treatment the level of intracellular calcium also increased by about 21% and 24% respectively. Moreover, EpoB, similarly to PTX, promoted the expression of TRAIL in lymphocytes, although high TRAIL expression on tumor cells was detected only after adding EpoB to SKOV-3 cells. EpoB mediates caspases-8 and -3 activation, which is independent of the reduction in the amount of caspase-9. Epitope-specific monoclonal and polyclonal antibodies revealed characteristic apoptotic changes that included cleavage of the 116 kDa PARP polypeptide to 25 kDa fragments. The results of our study show that EpoB induces mainly the extrinsic pathway. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Compound A398, a novel podophyllotoxin analogue: cytotoxicity and induction of apoptosis in human leukemia cells.

    Directory of Open Access Journals (Sweden)

    Alethéia L Silveira

    Full Text Available Despite advances in oncology research, cancer is one of the leading causes of death worldwide. Thus, there is a demand for the development of more selective and effective antitumor agents. This study showed that A398, a novel podophyllotoxin analogue, was cytotoxic to the HT-29, MCF-7, MOLT-4 and HL-60 tumor cell lines, being less active in human peripheral blood mononuclear cells and normal cell lines FGH and IEC-6. Tests using the HepG2 lineage indicated that its metabolites do not contribute to its cytotoxicity. In the HL-60 cells, A398 induced apoptosis in a time and concentration-dependent manner, promoting mitochondrial depolarization, inhibition of Bcl-2, phosphatidylserine exposure, activation of caspases -8, -9 and -3, and DNA fragmentation. The production of reactive oxygen species does not seem to be a crucial event for the apoptotic process. Pretreatment with specific inhibitors of kinases ERK1/2, JNK and p38 resulted in an increased percentage of death induced by A398. These results indicate that the compound induced apoptosis through activation of intrinsic and extrinsic death pathways with the mechanism involving the inhibition of the MAPKs and Bcl-2. Taken together, our findings suggest that A398 has an anticancer potential, proving itself to be a candidate for preclinical studies.

  12. Blockage of JNK pathway enhances arsenic trioxide-induced apoptosis in human keratinocytes

    International Nuclear Information System (INIS)

    Huang, H.-S.; Liu, Z.-M.; Hong, D.-Y.

    2010-01-01

    Arsenic is well known as a carcinogen predisposing humans to some severe diseases and also as an effective medicine for treating acute promyelocytic leukemia, syphilis, and psoriasis. Multiple active mechanisms, including cell cycle arrest and apoptosis, have been proposed in therapy; however, the opposing effects of arsenic remain controversial. Our previous study found that arsenic trioxide (ATO)-induced activation of p21 WAF1/CIP1 (p21) led to A431 cell death through the antagonistic effects of the signaling of ERK1/2 and JNK1. In the current study, the inhibitory effects of JNK1 on ATO-induced p21 expression were explored. Over-expression of JNK1 in A431 cells could inhibit p21 expression, which was associated with HDAC1 and TGIF. Using the GST pull-down assay and fluorescence resonance energy transfer analysis, N-terminal domain (amino acids 1-108) of TGIF, critical to its binding with c-Jun, was found. Using reporter assays, requirement of the C-terminal domain (amino acids 138-272) of TGIF to suppress ATO-induced p21 expression was observed. Thus, the domains of TGIF that carried out its inhibitory effects on p21 were identified. Finally, treatment with JNK inhibitor SP600125 could enhance ATO-induced apoptosis of HaCaT keratinocytes by using flow cytometry.

  13. Silencing glypican-3 expression induces apoptosis in human hepatocellular carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Shiyuan [The Second Hospital of Lanzhou University, Lanzhou 730030, Gansu (China); Key Laboratory of Digestive System Tumors of Gansu Province, Lanzhou 730030, Gansu (China); Li, Yumin, E-mail: liym@lzu.edu.cn [The Second Hospital of Lanzhou University, Lanzhou 730030, Gansu (China); Key Laboratory of Digestive System Tumors of Gansu Province, Lanzhou 730030, Gansu (China); Chen, Wei [Department of Biochemistry and Molecular Biology, School of Basic Medical Science, Lanzhou University, Lanzhou 730000, Gansu (China); Zheng, Pengfei [The Second Hospital of Lanzhou University, Lanzhou 730030, Gansu (China); Key Laboratory of Digestive System Tumors of Gansu Province, Lanzhou 730030, Gansu (China); Liu, Tao; He, Wenting; Zhang, Junqiang; Zeng, Xiangting [Key Laboratory of Digestive System Tumors of Gansu Province, Lanzhou 730030, Gansu (China)

    2012-03-23

    Highlights: Black-Right-Pointing-Pointer RNA interference GPC3 induces apoptosis in human hepatocellular carcinoma cell. Black-Right-Pointing-Pointer Silencing GPC3 resulted in the release of cytochrome c and activation of caspase-3. Black-Right-Pointing-Pointer GPC3 modulates the Bax/Bcl-2/cytochrome c/caspase-3 apoptotic signaling pathway. Black-Right-Pointing-Pointer Knockdown of GPC3 is a novel approach to HCC treatment. -- Abstract: Hepatocellular carcinoma (HCC) is one of the most common internal malignant tumors. Glypican-3 (GPC3) is involved in the biological and molecular events in the tumorigenesis of HCC. We used RNA interference to evaluate the molecular effects of GPC3 suppression at the translational level and demonstrated for the first time that GPC3 silencing results in a significant elevation of the Bax/Bcl-2 ratio, the release of cytochrome c from mitochondria and the activation of caspase-3. The results suggest that GPC3 regulates cell proliferation by enhancing the resistance to apoptosis through the dysfunction of the Bax/Bcl-2/cytochrome c/caspase-3 signaling pathway and therefore plays a critical role in the tumorigenesis of HCC. Thus, the knockdown of GPC3 should be further investigated as an attractive novel approach for the targeted gene therapy of HCC.

  14. A newly isolated probiotic Enterococcus faecalis strain from vagina microbiota enhances apoptosis of human cancer cells.

    Science.gov (United States)

    Nami, Y; Abdullah, N; Haghshenas, B; Radiah, D; Rosli, R; Yari Khosroushahi, A

    2014-08-01

    This study aimed to describe probiotic properties and bio-therapeutic effects of newly isolated Enterococcus faecalis from the human vaginal tract. The Enterococcus faecalis strain was originally isolated from the vaginal microbiota of Iranian women and was molecularly identified using 16SrDNA gene sequencing. Some biochemical methodologies were preliminarily used to characterize the probiotic potential of Ent. faecalis, including antibiotic susceptibility, antimicrobial activity, as well as acid and bile resistance. The bio-therapeutic effects of this strain's secreted metabolites on four human cancer cell lines (AGS, HeLa, MCF-7 and HT-29) and one normal cell line (HUVEC) were evaluated by cytotoxicity assay and apoptosis scrutiny. The characterization results demonstrated into the isolated bacteria strain revealed probiotic properties, such as antibiotic susceptibility, antimicrobial activity and resistance under conditions similar to those in the gastrointestinal tract. Results of bio-therapeutic efficacy assessments illustrated acceptable apoptotic effects on four human cancer cell lines and negligible side effects on assayed normal cell line. Our findings revealed that the apoptotic effect of secreted metabolites mainly depended on proteins secreted by Ent. faecalis on different cancer cells. These proteins can induce the apoptosis of cancer cells. The metabolites produced by this vaginal Ent. faecalis strain can be used as alternative pharmaceutical compounds with promising therapeutic indices because they are not cytotoxic to normal mammalian cells. Accordingly, the physicochemical, structural and functional properties of the secreted anticancer substances should be further investigated before using them as anticancer therapeutics. This study aim to screen total bacterial secreted metabolites as a wealthy source to find the new active compounds to introduce as anticancer therapeutics in the future. © 2014 The Society for Applied Microbiology.

  15. Cadmium Chloride Induces DNA Damage and Apoptosis of Human Liver Carcinoma Cells via Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Anthony Skipper

    2016-01-01

    Full Text Available Cadmium is a heavy metal that has been shown to cause its toxicity in humans and animals. Many documented studies have shown that cadmium produces various genotoxic effects such as DNA damage and chromosomal aberrations. Ailments such as bone disease, renal damage, and several forms of cancer are attributed to overexposure to cadmium.  Although there have been numerous studies examining the effects of cadmium in animal models and a few case studies involving communities where cadmium contamination has occurred, its molecular mechanisms of action are not fully elucidated. In this research, we hypothesized that oxidative stress plays a key role in cadmium chloride-induced toxicity, DNA damage, and apoptosis of human liver carcinoma (HepG2 cells. To test our hypothesis, cell viability was determined by MTT assay. Lipid hydroperoxide content stress was estimated by lipid peroxidation assay. Genotoxic damage was tested by the means of alkaline single cell gel electrophoresis (Comet assay. Cell apoptosis was measured by flow cytometry assessment (Annexin-V/PI assay. The result of MTT assay indicated that cadmium chloride induces toxicity to HepG2 cells in a concentration-dependent manner, showing a 48 hr-LD50 of 3.6 µg/mL. Data generated from lipid peroxidation assay resulted in a significant (p < 0.05 increase of hydroperoxide production, specifically at the highest concentration tested. Data obtained from the Comet assay indicated that cadmium chloride causes DNA damage in HepG2 cells in a concentration-dependent manner. A strong concentration-response relationship (p < 0.05 was recorded between annexin V positive cells and cadmium chloride exposure. In summary, these in vitro studies provide clear evidence that cadmium chloride induces oxidative stress, DNA damage, and programmed cell death in human liver carcinoma (HepG2 cells.

  16. Characterization of Total Phenolic Constituents from the Stems of Spatholobus suberectus Using LC-DAD-MSn and Their Inhibitory Effect on Human Neutrophil Elastase Activity

    Directory of Open Access Journals (Sweden)

    Yiming Li

    2013-06-01

    Full Text Available Spatholobus suberectus Dunn, belonging to the legume family (Fabaceae, has been used as a Traditional Chinese Medicine for the treatment of anemia, menoxenia and rheumatism. A limited number of studies report that various types of flavonoids are the main characteristic constituents of this herb. We have now found that S. suberectus contains about 2% phenolic components and characterized the major phenolic components as homogeneous B-type procyanidin conjugates using a liquid chromatography with diode-array detection-ESI mass spectrometry (LC-DAD/ESI-MS method. This is the first report on occurrence of most B-type procyanidins in this herb. Moreover, the total phenolics extract was assayed for inhibitory activity on human neutrophil elastase and its IC50 was found to be 1.33 μg/mL.

  17. Synthesis and evaluation of the potential deleterious effects of ZnO nanomaterials (nanoneedles and nanoflowers) on blood components, including albumin, erythrocytes and human isolated primary neutrophils

    Energy Technology Data Exchange (ETDEWEB)

    Pastrello, Bruna [São Paulo State University (UNESP), Department of Chemistry, Faculty of Sciences (Brazil); Paracatu, Luana Chiquetto [São Paulo State University (UNESP), Department of Clinical Analysis, School of Pharmaceutical Sciences (Brazil); Carvalho Bertozo, Luiza de [São Paulo State University (UNESP), Department of Chemistry, Faculty of Sciences (Brazil); Paino, Iêda Maria Martinez [University of São Paulo (USP), Nanomedicine and Nanotoxicology Group, Physics Institute of São Carlos (IFSC) (Brazil); Lisboa-Filho, Paulo Noronha [São Paulo State University (UNESP), Department of Physics, Faculty of Sciences (Brazil); Ximenes, Valdecir Farias, E-mail: vfximenes@fc.unesp.br [São Paulo State University (UNESP), Department of Chemistry, Faculty of Sciences (Brazil)

    2016-07-15

    The application of zinc oxide (ZnO) nanoparticles in biomaterials has increased significantly in the recent years. Here, we aimed to study the potential deleterious effects of ZnO on blood components, including human serum albumin (HSA), erythrocytes and human isolated primary neutrophils. To test the influence of the morphology of the nanomaterials, ZnO nanoneedles (ZnO-nn) and nanoflowers (ZnO-nf) were synthesized. The zeta potential and mean size of ZnO-nf and ZnO-nn suspensions in phosphate-buffered saline were −10.73 mV and 3.81 nm and −5.27 mV and 18.26 nm, respectively. The incubation of ZnO with HSA did not cause its denaturation as verified by the absence of significant alterations in the intrinsic and extrinsic fluorescence and in the circular dichroism spectrum of the protein. The capacity of HSA as a drug carrier was not affected as verified by employing site I and II fluorescent markers. Neither type of ZnO was able to provoke the activation of neutrophils, as verified by lucigenin- and luminol-dependent chemiluminescence and by the extracellular release of hydrogen peroxide. ZnO-nf, but not ZnO-nn, induced the haemolysis of erythrocytes. In conclusion, our results reinforce the concept that ZnO nanomaterials are relatively safe for usage in biomaterials. A potential exception is the capacity of ZnO-nf to promote the lysis of erythrocytes, a discovery that shows the importance of the morphology in the toxicity of nanoparticles.

  18. Selective induction of apoptosis in human gastric cancer cells by Lactobacillus kefiri (PFT), a novel kefir product.

    Science.gov (United States)

    Ghoneum, Mamdooh; Felo, Nouran

    2015-10-01

    The present study was undertaken to evaluate the effect of Lactobacillus kefiri (PFT), a novel kefir product, on apoptosis of gastric cancer cells (AGS), breast cancer cells (4T1), and human peripheral blood mononuclear cells (PBMCs). Cells were cultured with PFT and apoptosis was determined by flow cytometry using 7-AAD dye and cytospin preparation. Mitochondrial dysfunction and expression of Bcl2 were monitored by flow cytometry. Results showed that PFT induced apoptosis in AGS gastric cancer cells in a dose-dependent manner. Apoptosis was detected at a concentration of 0.3 mg/ml (20.8%), increased to 25.8% at 0.6 mg/ml, 37% at 1.2 mg/ml, 53.1% at 2.5 mg/ml, and peaked at 66.3% at 5.0 mg/ml. Apoptosis is associated with the decreased polarization of mitochondrial membrane potential (MMP) and decreased Bcl2 expression. PFT-treated AGS cells manifested membrane blebbing, nuclear condensation, and fragmentation as identified in cytospin cytocentrifuge Giemsa stained preparations. On the other hand, flow cytometry analysis showed that PFT did not induce apoptosis in 4T1 breast cancer cells nor in PBMCs. These results suggest that PFT is safe for white blood cells and selectively induces apoptotic effects in gastric cancer cells. Hence, it may have potential as a therapeutic agent for the treatment of gastric cancers.

  19. Bee venom induces apoptosis through intracellular Ca2+ -modulated intrinsic death pathway in human bladder cancer cells.

    Science.gov (United States)

    Ip, Siu-Wan; Chu, Yung-Lin; Yu, Chun-Shu; Chen, Po-Yuan; Ho, Heng-Chien; Yang, Jai-Sing; Huang, Hui-Ying; Chueh, Fu-Shin; Lai, Tung-Yuan; Chung, Jing-Gung

    2012-01-01

    To focus on bee venom-induced apoptosis in human bladder cancer TSGH-8301 cells and to investigate its signaling pathway to ascertain whether intracellular calcium iron (Ca(2+)) is involved in this effect. Bee venom-induced cytotoxic effects, productions of reactive oxygen species and Ca(2+) and the level of mitochondrial membrane potential (ΔΨm) were analyzed by flow cytometry. Apoptosis-associated proteins were examined by Western blot analysis and confocal laser microscopy. Bee venom-induced cell morphological changes and decreased cell viability through the induction of apoptosis in TSGH-8301 cell were found. Bee venom promoted the protein levels of Bax, caspase-9, caspase-3 and endonuclease G. The enhancements of endoplasmic reticulum stress-related protein levels were shown in bee venom-provoked apoptosis of TSGH-8301 cells. Bee venom promoted the activities of caspase-3, caspase-8, and caspase-9, increased Ca(2+) release and decreased the level of ΔΨm. Co-localization of immunofluorescence analysis showed the releases of endonuclease G and apoptosis-inducing factor trafficking to nuclei for bee venom-mediated apoptosis. The images revealed evidence of nuclear condensation and formation of apoptotic bodies by 4',6-diamidino-2-phenylindole staining and DNA gel electrophoresis showed the DNA fragmentation in TSGH-8301 cells. Bee venom treatment induces both caspase-dependent and caspase-independent apoptotic death through intracellular Ca(2+) -modulated intrinsic death pathway in TSGH-8301 cells. © 2011 The Japanese Urological Association.

  20. Fisetin Induces Apoptosis Through p53-Mediated Up-Regulation of DR5 Expression in Human Renal Carcinoma Caki Cells

    Directory of Open Access Journals (Sweden)

    Kyoung-jin Min

    2017-08-01

    Full Text Available Fisetin is a natural compound found in fruits and vegetables such as strawberries, apples, cucumbers, and onions. Since fisetin can elicit anti-cancer effects, including anti-proliferation and anti-migration, we investigated whether fisetin induced apoptosis in human renal carcinoma (Caki cells. Fisetin markedly induced sub-G1 population and cleavage of poly (ADP-ribose polymerase (PARP, which is a marker of apoptosis, and increased caspase activation. We found that pan-caspase inhibitor (z-VAD-fmk inhibited fisetin-induced apoptosis. In addition, fisetin induced death receptor 5 (DR5 expression at the transcriptional level, and down-regulation of DR5 by siRNA blocked fisetin-induced apoptosis. Furthermore, fisetin induced p53 protein expression through up-regulation of protein stability, whereas down-regulation of p53 by siRNA markedly inhibited fisetin-induced DR5 expression. In contrast, fisetin induced up-regulation of CHOP expression and reactive oxygen species production, which had no effect on fisetin-induced apoptosis. Taken together, our study demonstrates that fisetin induced apoptosis through p53 mediated up-regulation of DR5 expression at the transcriptional level.

  1. Fisetin Induces Apoptosis Through p53-Mediated Up-Regulation of DR5 Expression in Human Renal Carcinoma Caki Cells.

    Science.gov (United States)

    Min, Kyoung-Jin; Nam, Ju-Ock; Kwon, Taeg Kyu

    2017-08-02

    Fisetin is a natural compound found in fruits and vegetables such as strawberries, apples, cucumbers, and onions. Since fisetin can elicit anti-cancer effects, including anti-proliferation and anti-migration, we investigated whether fisetin induced apoptosis in human renal carcinoma (Caki) cells. Fisetin markedly induced sub-G1 population and cleavage of poly (ADP-ribose) polymerase (PARP), which is a marker of apoptosis, and increased caspase activation. We found that pan-caspase inhibitor (z-VAD-fmk) inhibited fisetin-induced apoptosis. In addition, fisetin induced death receptor 5 (DR5) expression at the transcriptional level, and down-regulation of DR5 by siRNA blocked fisetin-induced apoptosis. Furthermore, fisetin induced p53 protein expression through up-regulation of protein stability, whereas down-regulation of p53 by siRNA markedly inhibited fisetin-induced DR5 expression. In contrast, fisetin induced up-regulation of CHOP expression and reactive oxygen species production, which had no effect on fisetin-induced apoptosis. Taken together, our study demonstrates that fisetin induced apoptosis through p53 mediated up-regulation of DR5 expression at the transcriptional level.