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Sample records for interleukin-27 inhibits human

  1. Elevated interleukin-27 levels in human neonatal macrophages regulate indoleamine dioxygenase in a STAT-1 and STAT-3-dependent manner.

    PubMed

    Jung, Joo-Yong; Gleave Parson, Madeline; Kraft, Jennifer D; Lyda, Logan; Kobe, Brianna; Davis, Celestia; Robinson, Jembber; Peña, Maria Marjorette O; Robinson, Cory M

    2016-09-01

    Microbial infections are a major cause of infant mortality as a result of limitations in immune defences. Interleukin-27 (IL-27) is a heterodimeric cytokine produced primarily by leucocytes and is immunosuppressive toward lymphocytes and leucocytes. Our laboratory demonstrated that human neonatal macrophages express IL-27 more abundantly than adult macrophages. Similarly in mice, IL-27 expression is elevated early in life and maintained through infancy. To determine IL-27-regulated mechanisms that may limit immunity, we evaluated the expression of a number of genes in response to this cytokine in primary human neonatal macrophages. Indoleamine 2,3-dioxygenase (IDO) gene expression was increased dose-responsively by IL-27. We have previously demonstrated inhibition of T-cell proliferation and cytokine production by neonatal macrophage-generated IL-27, and IDO is often implicated in this negative regulation. An increase in IDO protein was demonstrated by immunofluorescence microscopy and was consistent with increased enzyme activity following treatment with IL-27. Inclusion of a soluble receptor to neutralize endogenous IL-27, decreased IDO expression and activity compared with untreated macrophages. In response to IL-27, neonatal macrophages phosphorylate signal transdcuer and activator of transcription 1 (STAT-1) and STAT-3. Both transcription factors are recruited to the IDO regulatory region. STAT-3 dominates during steady-state regulation by lower levels of endogenous IL-27 production. A shift to enhanced STAT-1 recruitment occurs during increased levels of exogenously supplied IL-27. These data suggest an interesting interplay of STAT-1 and STAT-3 to regulate IDO activity and immunosuppression in response to different levels of IL-27 in the microenvironment of the immune response that may further our understanding of this interesting cytokine. PMID:27238498

  2. Interleukin-27 inhibits vaccine-enhanced pulmonary disease following respiratory syncytial virus infection by regulating cellular memory responses.

    PubMed

    Zeng, Ruihong; Zhang, Huixian; Hai, Yan; Cui, Yuxiu; Wei, Lin; Li, Na; Liu, Jianxun; Li, Caixia; Liu, Ying

    2012-04-01

    Respiratory syncytial virus (RSV) is the most important cause of lower respiratory tract disease in young children. In the 1960s, infants vaccinated with formalin-inactivated RSV developed a more severe disease characterized by excessive inflammatory immunopathology in lungs upon natural RSV infection. The fear of causing the vaccine-enhanced disease (VED) is an important obstacle for development of safe and effective RSV vaccines. The recombinant vaccine candidate G1F/M2 immunization also led to VED. It has been proved that cellular memory induced by RSV vaccines contributed to VED. Interleukin-27 (IL-27) and IL-23 regulate Th1, Th17, and/or Th2 cellular immune responses. In this study, mice coimmunized with pcDNA3-IL-27 and G1F/M2 were fully protected and, importantly, did not develop vaccine-enhanced inflammatory responses and immunopathology in lungs after RSV challenge, which was correlated with moderate Th1-, suppressed Th2-, and Th17-like memory responses activated by RSV. In contrast, G1F/M2- or pcDNA3-IL-23+G1F/M2-immunized mice, in which robust Th2- and Th17-like memory responses were induced, developed enhanced pulmonary inflammation and severe immunopathology. Mice coimmunized with G1F/M2 and the two cytokine plasmids exhibited mild inflammatory responses as well as remarkable Th1-, suppressed Th2-, and Th17-like memory responses. These results suggested that Th1-, Th2-, and Th17-like memory responses and, in particular, excessive Th2- and Th17-like memory responses were closely associated with VED; IL-27 may inhibit VED following respiratory syncytial virus infection by regulating cellular memory responses. PMID:22301139

  3. Potential clinical application of interleukin-27 as an antitumor agent

    PubMed Central

    Yoshimoto, Takayuki; Chiba, Yukino; Furusawa, Jun-Ichi; Xu, Mingli; Tsunoda, Ren; Higuchi, Kaname; Mizoguchi, Izuru

    2015-01-01

    Cancer immunotherapies such as sipuleucel-T and ipilimumab are promising new treatments that harness the power of the immune system to fight cancer and achieve long-lasting remission. Interleukin (IL)-27, a member of the IL-12 heterodimeric cytokine family, has pleiotropic functions in the regulation of immune responses with both pro-inflammatory and anti-inflammatory properties. Evidence obtained using a variety of preclinical mouse models indicates that IL-27 possesses potent antitumor activity against various types of tumors through multiple mechanisms without apparent adverse effects. These mechanisms include those mediated not only by CD8+ T cells, natural killer cells and macrophages, but also by antibody-dependent cell-mediated cytotoxicity, antiangiogenesis, direct antiproliferative effects, inhibition of expression of cyclooxygenase-2 and prostaglandin E2, and suppression of epithelial–mesenchymal transition, depending on the characteristics of individual tumors. However, the endogenous role of IL-27 subunits and one of its receptor subunits, WSX-1, in the susceptibility to tumor development after transplantation of tumor cell lines or endogenously arising tumors seems to be more complicated. IL-27 functions as a double-edged sword: IL-27 increases IL-10 production and the expression of programmed death ligand 1 and T-cell immunoglobulin and mucin domain-3, and promotes the generation of regulatory T cells, and IL-27 receptor α singling enhances transformation; IL-27 may augment protumor effects as well. Here, we review both facets of IL-27, antitumor effects and protumor effects, and discuss the potential clinical application of IL-27 as an antitumor agent. PMID:26132605

  4. Interleukin-27 (IL-27) Mediates Susceptibility to Visceral Leishmaniasis by Suppressing the IL-17-Neutrophil Response.

    PubMed

    Quirino, Gustavo F S; Nascimento, Manuela S L; Davoli-Ferreira, Marcela; Sacramento, Lais A; Lima, Mikhael H F; Almeida, Roque P; Carregaro, Vanessa; Silva, João Santana

    2016-08-01

    The relationship established between Leishmania infantum and the vertebrate host can lead to a self-healing infection or to the manifestation of visceral leishmaniasis, a chronic systemic infection associated with high rates of mortality. We hypothesized that regulatory cytokines, such as interleukin-27 (IL-27), play a role in susceptibility to L. infantum infection. IL-27 is a heterodimeric cytokine composed of IL-27p28 and EBi3 subunits which, when combined, bind to IL-27R, leading to STAT-1 and -3 activation, playing a role in the regulation of the immune response. We observed in this work that IL-27 regulates the Th1/Th17 profiles in a mouse model of visceral leishmaniasis (VL) caused by L. infantum We showed here that the pathogen recognition by endosomal Toll-like receptors triggers a type I interferon (IFN) response, which acts through the type I IFN receptor and interferon regulatory factor 1 to induce IL-27 production by macrophages. Furthermore, IL-27 plays a major regulatory role in vivo, because Ebi3(-/-) mice can efficiently control parasite replication despite reduced levels of IFN-γ compared to wild-type mice. On the other hand, the absence of Ebi3 leads to exacerbated IL-17A production in the infected organs as well as in a coculture system, suggesting a direct regulatory action of IL-27 during L. infantum infection. As a consequence of exacerbated IL-17A in Ebi3(-/-) mice, a greater neutrophil influx was observed in the target organs, playing a role in parasite control. Thus, this work unveiled the molecular steps of IL-27 production after L. infantum infection and demonstrated its regulatory role in the IL-17A-neutrophil axis. PMID:27245409

  5. Protective role of Interleukin 27 (IL-27) gene polymorphisms in patients with ulcerative colitis.

    PubMed

    Yamamoto-Furusho, Jesús K; Posadas-Sánchez, Rosalinda; Alvarez-León, Edith; Vargas-Alarcón, Gilberto

    2016-04-01

    Ulcerative colitis (UC) is a chronic condition of unknown etiology and a polygenic disease. The interleukin 27 (IL-27) have been implicated in the pathogenesis of several autoimmune diseases including inflammatory bowel disease. Several polymorphisms of IL-27 have been associated with several types of cancer and immune disorders. The aim of the present study was to evaluate the association between IL-27 gene polymorphisms and the development of UC. Four polymorphisms of IL-27p28 gene (rs181206, rs26528, rs17855750, and rs40837) and three of the Epstein-Barr virus-induced gene 3 (EBI3) (rs428253, rs4740, and rs4905) were genotyped by 5' exonuclease TaqMan genotyping assays on an ABI Prism 7900HT Fast Real-Time PCR System in 375 Mexican patients with UC and 1599 Mexican Mestizo healthy unrelated individuals. IL-27 levels were determined in 458 healthy controls. Under recessive model adjusted by age and gender, the IL-27p28 rs17855750 polymorphism was associated with decreased risk of developing UC (OR=0.27, 95% CI: 0.06-1.13, P=0.031). On the other hand, under recessive models adjusted by age and gender, the EBI3 rs428253 (OR=0.54, 95% CI: 0.29-0.99, P=0.035), rs4740 (OR=0.60, 95% CI: 0.36-1.01, P=0.046) and rs4905 (OR=0.59, 95% CI: 0.35-1.01, P=0.043) were associated with decreased risk of developing UC. Similar levels of IL-27 were observed among the genotypes of the studied polymorphisms. IL-27 polymorphisms might play a protective role for the development of UC in the Mexican population. PMID:26905929

  6. Human Cytomegalovirus Inhibits Erythropoietin Production

    PubMed Central

    Dzabic, Mensur; Bakker, Frank; Davoudi, Belghis; Jeffery, Hannah; Religa, Piotr; Bojakowski, Krzysztof; Yaiw, Koon-Chu; Rahbar, Afsar; Söderberg-Naucler, Cecilia

    2014-01-01

    Anemia is a feature of CKD and a complication of renal transplantation, often caused by impaired production of erythropoietin. The kidney is a target organ for human cytomegalovirus (hCMV) in such patients, but it is not known whether hCMV effects erythropoietin production. We found that kidneys from patients with CKD were positive for hCMV protein and that blood levels of hCMV IgG inversely correlated with red blood cell count. In mice, systemic murine cytomegalovirus infection decreased serum erythropoietin levels. In human erythropoietin-producing cells, hCMV inhibited hypoxia-induced expression of erythropoietin mRNA and protein. hCMV early gene expression was responsible, as ultraviolet-inactivated virus had no effect and valganciclovir treatment showed that late gene expression was nonessential. Hypoxia-induced gene transcription is controlled by the transcription factors hypoxia-inducible transcription factor (HIF)-1α and HIF2α, which are constitutively produced but stable only under low oxygen conditions. We found that hCMV inhibited constitutive production of HIF2α mRNA. HIF2α is thought to be the master regulator of erythropoietin transcription. Single-cell analysis revealed that nuclear accumulation of HIF2α was inhibited in hCMV-infected cells, and the extent of inhibition correlated with hCMV protein expression. Our findings suggest that renal hCMV infection could induce or exacerbate anemia in patients. PMID:24722450

  7. Administration of DNA Encoding the Interleukin-27 Gene Augments Antitumour Responses through Non-adaptive Immunity.

    PubMed

    Li, Q; Sato, A; Shimozato, O; Shingyoji, M; Tada, Y; Tatsumi, K; Shimada, H; Hiroshima, K; Tagawa, M

    2015-10-01

    DNA-mediated immunization of a tumour antigen is a possible immunotherapy for cancer, and interleukin (IL)-27 has diverse functions in adaptive immunity. In this study, we examined whether IL-27 DNA administration enhanced antitumour effects in mice vaccinated with DNA encoding a putative tumour antigen, β-galactosidase (β-gal). An intramuscular injection of cardiotoxin before DNA administration facilitated the exogenous gene expression. In mice received β-gal and IL-27 DNA, growth of β-gal-positive P815 tumours was retarded and survival of the mice was prolonged. Development of β-gal-positive Colon 26 tumours was suppressed by vaccination of β-gal DNA and further inhibited by additional IL-27 DNA administration or IL-12 family cytokines. Nevertheless, a population of β-gal-specific CD8(+) T cells did not increase, and production of anti-β-gal antibody was not enhanced by IL-27 DNA administration. Spleen cells from mice bearing IL-27-expressing Colon 26 tumours showed greater YAC-1-targeted cytotoxicity although CD3(-)/DX5(+) natural killer (NK) cell numbers remained unchanged. Recombinant IL-27 enhanced YAC-1-targeted cytotoxicity of IL-2-primed splenic NK cells and augmented a phosphorylation of signal transducer and activator of transcription 3 and an expression of perforin. These data collectively indicate that IL-27 DNA administration activates NK cells and augments vaccination effects of DNA encoding a tumour antigen through non-adaptive immune responses. PMID:26095954

  8. Allosteric antibody inhibition of human hepsin protease.

    PubMed

    Koschubs, Tobias; Dengl, Stefan; Dürr, Harald; Kaluza, Klaus; Georges, Guy; Hartl, Christiane; Jennewein, Stefan; Lanzendörfer, Martin; Auer, Johannes; Stern, Alvin; Huang, Kuo-Sen; Packman, Kathryn; Gubler, Ueli; Kostrewa, Dirk; Ries, Stefan; Hansen, Silke; Kohnert, Ulrich; Cramer, Patrick; Mundigl, Olaf

    2012-03-15

    Hepsin is a type II transmembrane serine protease that is expressed in several human tissues. Overexpression of hepsin has been found to correlate with tumour progression and metastasis, which is so far best studied for prostate cancer, where more than 90% of such tumours show this characteristic. To enable improved future patient treatment, we have developed a monoclonal humanized antibody that selectively inhibits human hepsin and does not inhibit other related proteases. We found that our antibody, hH35, potently inhibits hepsin enzymatic activity at nanomolar concentrations. Kinetic characterization revealed non-linear, slow, tight-binding inhibition. This correlates with the crystal structure we obtained for the human hepsin-hH35 antibody Fab fragment complex, which showed that the antibody binds hepsin around α3-helix, located far from the active centre. The unique allosteric mode of inhibition of hH35 is distinct from the recently described HGFA (hepatocyte growth factor activator) allosteric antibody inhibition. We further explain how a small change in the antibody design induces dramatic structural rearrangements in the hepsin antigen upon binding, leading to complete enzyme inactivation. PMID:22132769

  9. Inhibition in the Human Auditory Cortex

    PubMed Central

    Inui, Koji; Nakagawa, Kei; Nishihara, Makoto; Motomura, Eishi; Kakigi, Ryusuke

    2016-01-01

    Despite their indispensable roles in sensory processing, little is known about inhibitory interneurons in humans. Inhibitory postsynaptic potentials cannot be recorded non-invasively, at least in a pure form, in humans. We herein sought to clarify whether prepulse inhibition (PPI) in the auditory cortex reflected inhibition via interneurons using magnetoencephalography. An abrupt increase in sound pressure by 10 dB in a continuous sound was used to evoke the test response, and PPI was observed by inserting a weak (5 dB increase for 1 ms) prepulse. The time course of the inhibition evaluated by prepulses presented at 10–800 ms before the test stimulus showed at least two temporally distinct inhibitions peaking at approximately 20–60 and 600 ms that presumably reflected IPSPs by fast spiking, parvalbumin-positive cells and somatostatin-positive, Martinotti cells, respectively. In another experiment, we confirmed that the degree of the inhibition depended on the strength of the prepulse, but not on the amplitude of the prepulse-evoked cortical response, indicating that the prepulse-evoked excitatory response and prepulse-evoked inhibition reflected activation in two different pathways. Although many diseases such as schizophrenia may involve deficits in the inhibitory system, we do not have appropriate methods to evaluate them; therefore, the easy and non-invasive method described herein may be clinically useful. PMID:27219470

  10. BROMODICHLOROMETHANE INHIBITS HUMAN PLACENTAL TROPHOBLAST DIFFERENTIATION

    EPA Science Inventory

    BROMODICHLOROMETHANE INHIBITS HUMAN PLACENTAL
    TROPHOBLAST DIFFERENTIATION
    Jiangang Chen, Twanda L. Thirkill, Peter N. Lohstroh, Susan R. Bielmeier, Michael
    G. Narotsky, Deborah S. Best, Randy A. Harrison, Kala Natarajan, Rex A. Pegram,
    Bill L. Lasley, and Gordon C. Do...

  11. Peptide inhibition of human cytomegalovirus infection

    PubMed Central

    2011-01-01

    Background Human cytomegalovirus (HCMV) is the most prevalent congenital viral infection in the United States and Europe causing significant morbidity and mortality to both mother and child. HCMV is also an opportunistic pathogen in immunocompromised individuals, including human immunodeficiency virus (HIV)- infected patients with AIDS, and solid organ and allogeneic stem cell transplantation recipients. Current treatments for HCMV-associated diseases are insufficient due to the emergence of drug-induced resistance and cytotoxicity, necessitating novel approaches to limit HCMV infection. The aim of this study was to develop therapeutic peptides targeting glycoprotein B (gB), a major glycoprotein of HCMV that is highly conserved across the Herpesviridae family, that specifically inhibit fusion of the viral envelope with the host cell membrane preventing HCMV entry and infection. Results Using the Wimley-White Interfacial Hydrophobicity Scale (WWIHS), several regions within gB were identified that display a high potential to interact with lipid bilayers of cell membranes and hydrophobic surfaces within proteins. The ability of synthetic peptides analogous to WWIHS-positive sequences of HCMV gB to inhibit viral infectivity was evaluated. Human foreskin fibroblasts (HFF) were infected with the Towne-GFP strain of HCMV (0.5 MOI), preincubated with peptides at a range of concentrations (78 nm to 100 μM), and GFP-positive cells were visualized 48 hours post-infection by fluorescence microscopy and analyzed quantitatively by flow cytometry. Peptides that inhibited HCMV infection demonstrated different inhibitory concentration curves indicating that each peptide possesses distinct biophysical properties. Peptide 174-200 showed 80% inhibition of viral infection at a concentration of 100 μM, and 51% and 62% inhibition at concentrations of 5 μM and 2.5 μM, respectively. Peptide 233-263 inhibited infection by 97% and 92% at concentrations of 100 μM and 50 μM, respectively

  12. Bromodichloromethane inhibits human placental trophoblast differentiation.

    PubMed

    Chen, Jiangang; Thirkill, Twanda L; Lohstroh, Peter N; Bielmeier, Susan R; Narotsky, Michael G; Best, Deborah S; Harrison, Randy A; Natarajan, Kala; Pegram, Rex A; Overstreet, James W; Lasley, Bill L; Douglas, Gordon C

    2004-03-01

    Epidemiological data suggest an association between exposures to bromodichloromethane (BDCM), a trihalomethane found in drinking water as a result of drinking water disinfection, and an increased risk of spontaneous abortion. We previously hypothesized that BDCM targets the placenta and showed that the secretion of chorionic gonadotrophin (CG) was reduced in primary cultures of human term syncytiotrophoblasts exposed to BDCM. In the present study we extend this observation by evaluating the effects of BDCM on the morphological differentiation of mononucleated cytotrophoblast cells to multinucleated syncytiotrophoblast-like colonies. Addition of BDCM to cytotrophoblast cultures inhibited the subsequent formation of multinucleated colonies in a dose-dependent manner, as determined by immunocytochemical staining for desmosomes and nuclei. The effect was seen at BDCM concentrations between 0.02 and 2 mM and was confirmed by quantitative image analysis. Secretion of bioactive and immunoreactive chorionic gonadotropin was also significantly inhibited in a dose-dependent manner under these culture conditions, and cellular levels of CG were also reduced. Trophoblast viability was not compromised by exposure to BDCM. We conclude that BDCM disrupts syncytiotrophoblast formation and inhibits CG secretion in vitro. Although other tissue targets are not ruled out, these data substantiate the idea that BDCM targets the placenta and could have implications for understanding the adverse pregnancy outcomes associated with BDCM exposure in humans. PMID:14691210

  13. PCB126 Inhibits Adipogenesis of Human Preadipocytes

    PubMed Central

    Gadupudi, Gopi; Gourronc, Francoise A.; Ludewig, Gabriele; Robertson, Larry W.; Klingelhutz, Aloysius J.

    2014-01-01

    Emerging evidence indicates that persistent organic pollutants (POPs), including polychlorinated biphenyls (PCBs), are involved in the development of diabetes. Dysfunctional adipocytes play a significant role in initiating insulin resistance. Preadipocytes make up a large portion of adipose tissue and are necessary for the generation of functional mature adipocytes through adipogenesis. PCB126 is a dioxin-like PCB and a potent aryl hydrocarbon receptor (AhR) agonist. We hypothesized that PCB126 may be involved in the development of diabetes through disruption of adipogenesis. Using a newly developed human preadipocyte cell line called NPAD (Normal PreADipocytes), we found that exposure of preadipocytes to PCB126 resulted in significant reduction in their subsequent ability to fully differentiate into adipocytes, more so than when the cells were exposed to PCB126 during differentiation. Reduction in differentiation by PCB126 was associated with downregulation of transcript levels of a key adipocyte transcription factor, PPARγ, and late adipocyte differentiation genes. An AhR antagonist, CH223191, blocked this effect. These studies indicate that preadipocytes are particularly sensitive to the effects of PCB126 and suggest that AhR activation inhibits PPARγ transcription and subsequent adipogenesis. Our results validate the NPAD cell line as a useful model for studying the effects of POPs on adipogenesis. PMID:25304490

  14. Pain inhibits pain; human brainstem mechanisms.

    PubMed

    Youssef, A M; Macefield, V G; Henderson, L A

    2016-01-01

    Conditioned pain modulation is a powerful analgesic mechanism, occurring when a painful stimulus is inhibited by a second painful stimulus delivered at a different body location. Reduced conditioned pain modulation capacity is associated with the development of some chronic pain conditions and the effectiveness of some analgesic medications. Human lesion studies show that the circuitry responsible for conditioned pain modulation lies within the caudal brainstem, although the precise nuclei in humans remain unknown. We employed brain imaging to determine brainstem sites responsible for conditioned pain modulation in 54 healthy individuals. In all subjects, 8 noxious heat stimuli (test stimuli) were applied to the right side of the mouth and brain activity measured using functional magnetic resonance imaging. This paradigm was then repeated. However, following the fourth noxious stimulus, a separate noxious stimulus, consisting of an intramuscular injection of hypertonic saline into the leg, was delivered (conditioning stimulus). During this test and conditioning stimulus period, 23 subjects displayed conditioned pain modulation analgesia whereas 31 subjects did not. An individual's analgesic ability was not influenced by gender, pain intensity levels of the test or conditioning stimuli or by psychological variables such as pain catastrophizing or fear of pain. Brain images were processed using SPM8 and the brainstem isolated using the SUIT toolbox. Significant increases in signal intensity were determined during each test stimulus and compared between subjects that did and did not display CPM analgesia (p<0.05, small volume correction). The expression of analgesia was associated with reduction in signal intensity increases during each test stimulus in the presence of the conditioning stimulus in three brainstem regions: the caudalis subdivision of the spinal trigeminal nucleus, i.e., the primary synapse, the region of the subnucleus reticularis dorsalis and in the

  15. Copper, aluminum, iron and calcium inhibit human acetylcholinesterase in vitro.

    PubMed

    Pohanka, Miroslav

    2014-01-01

    Acetylcholinesterase (AChE) is an important part of cholinergic nerves where it participates in termination of neurotransmission. AChE can be inhibited by e.g. some Alzheimer disease drugs, nerve agents, and secondary metabolites. In this work, metal salts aluminum chloride, calcium chloride, cupric chloride, ferric chloride, potassium chloride, magnesium chloride and sodium chloride were tested for their ability to inhibit AChE. Standard Ellman assay based on human recombinant AChE was done and inhibition was measured using Dixon plot. No inhibition was proved for sodium, potassium and magnesium ions. However, aluminum, cupric, ferric and calcium ions were able to inhibit AChE via noncompetitive mechanism of inhibition. Though the inhibition is much weaker when compared to e.g. drugs with noncompetitive mechanism of action, biological relevance of the findings can be anticipated. PMID:24473150

  16. Oxathiazolones Selectively Inhibit the Human Immunoproteasome over the Constitutive Proteasome

    PubMed Central

    2014-01-01

    Selective inhibitors for the human immunoproteasome LMP7 (β5i) subunit over the constitutive proteasome hold promise for the treatment of autoimmune and inflammatory diseases and hematologic malignancies. Here we report that oxathiazolones inhibit the immunoproteasome β5i with up to 4700-fold selectivity over the constitutive proteasome, are cell permeable, and inhibit proteasomes inside cells. PMID:24900849

  17. Allosteric Inhibition of Human Immunodeficiency Virus Integrase

    PubMed Central

    Gupta, Kushol; Brady, Troy; Dyer, Benjamin M.; Malani, Nirav; Hwang, Young; Male, Frances; Nolte, Robert T.; Wang, Liping; Velthuisen, Emile; Jeffrey, Jerry; Van Duyne, Gregory D.; Bushman, Frederic D.

    2014-01-01

    HIV-1 replication in the presence of antiviral agents results in evolution of drug-resistant variants, motivating the search for additional drug classes. Here we report studies of GSK1264, which was identified as a compound that disrupts the interaction between HIV-1 integrase (IN) and the cellular factor lens epithelium-derived growth factor (LEDGF)/p75. GSK1264 displayed potent antiviral activity and was found to bind at the site occupied by LEDGF/p75 on IN by x-ray crystallography. Assays of HIV replication in the presence of GSK1264 showed only modest inhibition of the early infection steps and little effect on integration targeting, which is guided by the LEDGF/p75·IN interaction. In contrast, inhibition of late replication steps was more potent. Particle production was normal, but particles showed reduced infectivity. GSK1264 promoted aggregation of IN and preformed LEDGF/p75·IN complexes, suggesting a mechanism of inhibition. LEDGF/p75 was not displaced from IN during aggregation, indicating trapping of LEDGF/p75 in aggregates. Aggregation assays with truncated IN variants revealed that a construct with catalytic and C-terminal domains of IN only formed an open polymer associated with efficient drug-induced aggregation. These data suggest that the allosteric inhibitors of IN are promising antiviral agents and provide new information on their mechanism of action. PMID:24904063

  18. Interleukin 27R regulates CD4+ T cell phenotype and impacts protective immunity during Mycobacterium tuberculosis infection

    PubMed Central

    Torrado, Egidio; Fountain, Jeffrey J.; Liao, Mingfeng; Tighe, Michael; Reiley, William W.; Lai, Rachel P.; Meintjes, Graeme; Pearl, John E.; Chen, Xinchun; Zak, Daniel E.; Thompson, Ethan G.; Aderem, Alan; Ghilardi, Nico; Solache, Alejandra; McKinstry, K. Kai; Strutt, Tara M.; Wilkinson, Robert J.; Swain, Susan L.

    2015-01-01

    CD4+ T cells mediate protection against Mycobacterium tuberculosis (Mtb); however, the phenotype of protective T cells is undefined, thereby confounding vaccination efforts. IL-27 is highly expressed during human tuberculosis (TB), and absence of IL-27R (Il27ra) specifically on T cells results in increased protection. IL-27R deficiency during chronic Mtb infection does not impact antigen-specific CD4+ T cell number but maintains programmed death-1 (PD-1), CD69, and CD127 expression while reducing T-bet and killer cell lectin-like receptor G1 (KLRG1) expression. Furthermore, T-bet haploinsufficiency results in failure to generate KLRG1+, antigen-specific CD4+ T cells, and in improved protection. T cells in Il27ra−/− mice accumulate preferentially in the lung parenchyma within close proximity to Mtb, and antigen-specific CD4+ T cells lacking IL-27R are intrinsically more fit than intact T cells and maintain IL-2 production. Improved fitness of IL-27R–deficient T cells is not associated with increased proliferation but with decreased expression of cell death–associated markers. Therefore, during Mtb infection, IL-27R acts intrinsically on T cells to limit protection and reduce fitness, whereas the IL-27R–deficient environment alters the phenotype and location of T cells. The significant expression of IL-27 in TB and the negative influence of IL-27R on T cell function demonstrate the pathway by which this cytokine/receptor pair is detrimental in TB. PMID:26282876

  19. Interleukin 27R regulates CD4+ T cell phenotype and impacts protective immunity during Mycobacterium tuberculosis infection.

    PubMed

    Torrado, Egidio; Fountain, Jeffrey J; Liao, Mingfeng; Tighe, Michael; Reiley, William W; Lai, Rachel P; Meintjes, Graeme; Pearl, John E; Chen, Xinchun; Zak, Daniel E; Thompson, Ethan G; Aderem, Alan; Ghilardi, Nico; Solache, Alejandra; McKinstry, K Kai; Strutt, Tara M; Wilkinson, Robert J; Swain, Susan L; Cooper, Andrea M

    2015-08-24

    CD4+ T cells mediate protection against Mycobacterium tuberculosis (Mtb); however, the phenotype of protective T cells is undefined, thereby confounding vaccination efforts. IL-27 is highly expressed during human tuberculosis (TB), and absence of IL-27R (Il27ra) specifically on T cells results in increased protection. IL-27R deficiency during chronic Mtb infection does not impact antigen-specific CD4+ T cell number but maintains programmed death-1 (PD-1), CD69, and CD127 expression while reducing T-bet and killer cell lectin-like receptor G1 (KLRG1) expression. Furthermore, T-bet haploinsufficiency results in failure to generate KLRG1+, antigen-specific CD4+ T cells, and in improved protection. T cells in Il27ra(-/-) mice accumulate preferentially in the lung parenchyma within close proximity to Mtb, and antigen-specific CD4+ T cells lacking IL-27R are intrinsically more fit than intact T cells and maintain IL-2 production. Improved fitness of IL-27R-deficient T cells is not associated with increased proliferation but with decreased expression of cell death-associated markers. Therefore, during Mtb infection, IL-27R acts intrinsically on T cells to limit protection and reduce fitness, whereas the IL-27R-deficient environment alters the phenotype and location of T cells. The significant expression of IL-27 in TB and the negative influence of IL-27R on T cell function demonstrate the pathway by which this cytokine/receptor pair is detrimental in TB. PMID:26282876

  20. Bee venom inhibits growth of human cervical tumors in mice.

    PubMed

    Lee, Hye Lim; Park, Sang Ho; Kim, Tae Myoung; Jung, Yu Yeon; Park, Mi Hee; Oh, Sang Hyun; Yun, Hye Seok; Jun, Hyung Ok; Yoo, Hwan Soo; Han, Sang-Bae; Lee, Ung Soo; Yoon, Joo Hee; Song, Min Jong; Hong, Jin Tae

    2015-03-30

    We studied whether bee venom (BV) inhibits cervical tumor growth through enhancement of death receptor (DR) expressions and inactivation of nuclear factor kappa B (NF-κB) in mice. In vivo study showed that BV (1 mg/kg) inhibited tumor growth. Similar inhibitory effects of BV on cancer growth in primary human cervical cancer cells were also found. BV (1-5 μg/ml) also inhibited the growth of cancer cells, Ca Ski and C33Aby the induction of apoptotic cell death in a dose dependent manner. Agreed with cancer cell growth inhibition, expression of death receptors; FAS, DR3 and DR6, and DR downstream pro-apoptotic proteins including caspase-3 and Bax was concomitantly increased, but the NF-κB activity and the expression of Bcl-2 were inhibited by treatment with BV in tumor mice, human cancer cell and human tumor samples as well as cultured cancer cells. In addition, deletion of FAS, DR3 and DR6 by small interfering RNA significantly reversed BV-induced cell growth inhibitory effects as well as NF-κB inactivation. These results suggest that BV inhibits cervical tumor growth through enhancement of FAS, DR3 and DR6 expression via inhibition of NF-κB pathway. PMID:25730901

  1. Inhibition of human papillomavirus expression using DNAzymes.

    PubMed

    Benítez-Hess, María Luisa; Reyes-Gutiérrez, Pablo; Alvarez-Salas, Luis Marat

    2011-01-01

    Deoxyribozymes (DXZs) are catalytic oligodeoxynucleotides capable of performing diverse functions including the specific cleavage of a target RNA. These molecules represent a new type of therapeutic oligonucleotides combining the efficiency of ribozymes and the intracellular endurance and simplicity of modified antisense oligonucleotides. Commonly used DXZs include the 8-17 and 10-23 motifs, which have been engineered to destroy disease-associated genes with remarkable efficiency. Targeting DXZs to disease-associated transcripts requires extensive biochemical testing to establish target RNA accessibility, catalytic efficiency, and nuclease sensibility. The usage of modified nucleotides to render nuclease-resistance DXZs must be counterweighted against deleterious consequences on catalytic activity. Further intracellular testing is required to establish the effect of microenvironmental conditions on DXZ activity and off-target issues. Application of modified DXZs to cervical cancer results in specific growth inhibition, cell death, and apoptosis. Thus, DXZs represent a highly effective antisense moiety with minimal secondary effects. PMID:21748650

  2. Genistein suppresses FLT4 and inhibits human colorectal cancer metastasis.

    PubMed

    Xiao, Xiao; Liu, Zhiguo; Wang, Rui; Wang, Jiayin; Zhang, Song; Cai, Xiqiang; Wu, Kaichun; Bergan, Raymond C; Xu, Li; Fan, Daiming

    2015-02-20

    Dietary consumption of genistein, found in soy, has been associated with a potentially protective role in colorectal cancer (CRC) development and progression. Herein we demonstrate that genistein will inhibit human CRC cell invasion and migration, that it does so at non-cytotoxic concentrations and we demonstrate this in multiple human CRC cell lines. After orthotopic implantation of human CRC tumors into mice, oral genistein did not inhibit tumor growth, but did inhibit distant metastasis formation, and was non-toxic to mice. Using a qPCR array, we screened for genistein-induced changes in gene expression, followed by Western blot confirmation, demonstrating that genistein downregulated matrix metalloproteinase 2 and Fms-Related Tyrosine Kinase 4 (FLT4; vascular endothelial growth factor receptor 3). After demonstrating that genistein suppressed neo-angiogenesis in mouse tumors, we examined FLT4 expression in primary CRC and adjacent normal colonic tissue from 60 human subjects, demonstrating that increased FLT4 significantly correlates with increased stage and decreased survival. In summary, we demonstrate for the first time that genistein inhibits human CRC metastasis at dietary, non-toxic, doses. FLT4 is identified as a marker of metastatic disease, and as a response marker for small molecule therapeutics that inhibit CRC metastasis. PMID:25605009

  3. Mechanisms of inhibition of Cryptococcus neoformans by human lymphocytes.

    PubMed Central

    Levitz, S M; North, E A; Dupont, M P; Harrison, T S

    1995-01-01

    Recently, our laboratory and others have demonstrated that human peripheral blood T and NK lymphocytes directly inhibit the growth of Cryptococcus neoformans. In this study, we further define the conditions under which lymphocyte-mediated fungistasis against C. neoformans occurs and examine whether mechanisms implicated in lymphocyte-mediated activities against other target cells are also involved in anticryptococcal activity. The addition of whole or broken heat-killed C. neoformans modestly inhibited lymphocyte-mediated fungistasis, whereas other particulates had no effect. The hydroxyl radical scavenger catechin, but not diethyl urea or propyl gallate, profoundly inhibited fungistasis. Salicylic acid inhibited fungistasis in a dose-dependent fashion. However, two other cyclooxygenase inhibitors, piroxicam and indomethacin, had no effect, suggesting that the mechanism of inhibition by salicylic acid was cyclooxygenase independent. Reagent prostaglandin E2, at concentrations shown by others to inhibit NK cell-mediated bactericidal and tumorlytic activities, had no effect on lymphocyte-mediated fungistasis. The addition of selected monoclonal antibodies or ligands reactive with receptors on human lymphocytes had no significant effect on lymphocyte-mediated fungistasis. Acapsular, small-capsuled, and large-capsuled C. neoformans organisms were inhibited by lymphocytes to an approximately equal extent. These data demonstrate that lymphocyte-mediated activity against C. neoformans proceeds regardless of the presence of capsule and by mechanisms at least in part dissimilar from those seen with other target cells. PMID:7642290

  4. Ozone selectively inhibits growth of human cancer cells

    SciTech Connect

    Sweet, F.; Kao, M.S.; Lee, S.C.; Hagar, W.L.; Sweet, W.E.

    1980-08-01

    The growth of human cancer cells from lung, breast, and uterine tumors was selectively inhibited in a dose-dependent manner by ozone at 0.3 to 0.8 part per million of ozone in ambient air during 8 days of culture. Human lung diploid fibroblasts served as noncancerous control cells. The presence of ozone at 0.3 to 0.5 part per million inhibited cancer cell growth 40 and 60 percent, respectively. The noncancerous lung cells were unaffected at these levels. Exposure to ozone at 0.8 part per million inhibited cancer cell growth more than 90 percent and control cell growth less than 50 percent. Evidently, the mechanisms for defense against ozone damage are impaired in human cancer cells.

  5. Human central nervous system myelin inhibits neurite outgrowth.

    PubMed

    Ng, W P; Cartel, N; Roder, J; Roach, A; Lozano, A

    1996-05-13

    In vitro and animal studies have identified molecules in mammalian CNS myelin which inhibit neuritic extension and which may be responsible, at least in part, for the lack of axonal regeneration after injury in the injured brain, optic nerve and spinal cord. To determine whether such inhibitory activity may be present in human CNS myelin, we used a bioassay to characterize neurite outgrowth on this substrate. Human CNS myelin strongly inhibited neuritic outgrowth from newborn rat dorsal root ganglion neurons and NG-108-15 cells, a neuroblastoma-glioma hybrid cell line. Similar but less potent inhibitory activity was identified in human gray matter. The CNS myelin inhibition of neuritic outgrowth appeared to be dependent on direct contact between the myelin substrate and neurites. The inhibitory activity in human CNS myelin closely resembled that described in adult rodents. Inhibition of neurite growth by human CNS myelin in this in vitro bioassay mirrors the lack of regeneration in vivo and can be used as a model to develop strategies designed to enhance axonal regeneration and neural recovery. PMID:8782892

  6. Inhibition of Human Neutrophil Elastase by Pentacyclic Triterpenes

    PubMed Central

    Feng, Li; Liu, Xiaoyu; Zhu, Weiliang; Guo, Fujiang; YingchunWu; Wang, Rui; Chen, Kaixian; Huang, Cheng; Li, Yiming

    2013-01-01

    Scope Inhibiting human neutrophil elastase (HNE) is a promising strategy for treating inflammatory lung diseases, such as H1N1 and SARS virus infections. The use of sivelestat, the only clinically registered synthesized HNE inhibitor, is largely limited by its risk of organ toxicity because it irreversibly inhibits HNE. Therefore, potent reversible HNE inhibitors are promising alternatives to sivelestat. Methods and Results An in vitro HNE inhibition assay was employed to screen a series of triterpenes. Six pentacyclic triterpenes, but not tetracyclic triterpenes, significantly inhibited HNE. Of these pentacyclic triterpenes, ursolic acid exhibited the highest inhibitory potency (IC50 = 5.51 µM). The HNE inhibitory activity of ursolic acid was further verified using a mouse model of acute smoke-induced lung inflammation. The results of nuclear magnetic resonance and HNE inhibition kinetic analysis showed that the pentacyclic triterpenes competitively and reversibly inhibited HNE. Molecular docking experiments indicated that the molecular scaffold, 28-COOH, and a double bond at an appropriate location in the pentacyclic triterpenes are important for their inhibitory activity. Conclusion Our results provide insights into the effects of pentacyclic triterpenes on lung inflammatory actions through reversible inhibition of HNE activity. PMID:24376583

  7. Ketamine inhibits human sperm function by Ca(2+)-related mechanism.

    PubMed

    He, Yuanqiao; Zou, Qianxing; Li, Bingda; Chen, Houyang; Du, Xiaohong; Weng, Shiqi; Luo, Tao; Zeng, Xuhui

    2016-09-01

    Ketamine, a dissociative anesthetic, which was widely used in human and animal medicine, has become a popular recreational drug, as it can induce hallucinatory effects. Ketamine abuse can cause serious damage to many aspects of the organism, mainly reflected in the nervous system and urinary system. It has also been reported that ketamine can impair the male genital system. However, the detailed effect of ketamine on human spermatozoa remains unclear. Thus, we investigated the in vitro effects of ketamine on human sperm functions, to elucidate the underlying mechanism. Human sperm were treated in vitro with different concentrations of ketamine (0, 0.125, 0.25, 0.5, 1 g/L). The results showed that 0.25-1 g/L ketamine inhibited sperm total motility, progressive motility and linear velocity, in a dose-dependent manner. In addition, the sperm's ability to penetrate viscous medium and the progesterone-induced acrosome reaction were significantly inhibited by ketamine. Ketamine did not affect sperm viability, capacitation and spontaneous acrosome reaction. The intracellular calcium concentration ([Ca(2+)]i), which is a central factor in the regulation of human sperm function, was decreased by ketamine (0.125-1 g/L) in a dose-dependent manner. Furthermore, the currents of the sperm-specific Ca(2+) channel, CatSper, which modulates Ca(2+) influx in sperm, were inhibited by ketamine (0.125-1 g/L) in a dose-dependent manner. Our findings suggest that ketamine induces its toxic effects on human sperm functions by reducing sperm [Ca(2+)]i through inhibition of CatSper channel. PMID:27143628

  8. Sesquiterpenoid Lactones in Tanacetum huronense Inhibit Human Glioblastoma Cell Proliferation.

    PubMed

    Dissanayake, Amila A; Bejcek, Bruce E; Zhang, Chuan-Rui; Nair, Muraleedharan G

    2016-05-01

    Tanacetum huronense (Lake Huron tansy), which is native to the upper Midwest region of USA and Canada, was examined for the presence of anticancer compounds using an in vitro human tumor cell proliferation inhibition assay, with glioblastoma derived cell line U-87 MG. Bioassay-directed purification of the ethyl acetate extract of the aerial portion of this plant identified six active sesquiterpenoid lactones (1-6). Among these, compounds 5 and 6 are new structural analogs. One of the most abundant isolates, tanacin (4), exhibited the greatest inhibition with an IC50 value of 4.5 μg/mL. PMID:27319121

  9. Kinetics of inhibition of platelet calpain II by human kininogens.

    PubMed Central

    Bradford, H N; Schmaier, A H; Colman, R W

    1990-01-01

    The plasma kininogens, high-molecular-mass and low-molecular-mass kininogens, are the most potent plasma inhibitors of platelet calpain. We explored the kinetic mechanisms for kininogen inhibition of calpain by comparing calpain inactivation by human high-molecular-mass kininogen (HK) and human low-molecular-mass kininogen (LK). With a [14C]methylated alpha-casein substrate, the inhibition of calpain by HK did not follow classic Michaelis-Menten kinetics. With the use of a fluorogenic assay with the dipeptide substrate for calpain, 3-carboxypropionyl-leucyltyrosine 7-(4-methyl)coumarylamide, the inhibition by HK and LK fitted a kinetic model of tight-binding inhibition. LK was found to be a non-competitive inhibitor of platelet calpain with a Ki of 2.7 nM. HK showed mixed non-competitive inhibition of calpain with a Ki of 2.3 nM in the absence of substrate and Ki of 0.71 nM in the presence of saturating substrate, almost 4-fold tighter than LK. Proteolysis of HK by plasma and tissue kallikreins did not influence its ability to inhibit calpain. Digestion of the HK light chain by Factor XIa also did not alter its calpain-inhibitory function. These studies indicate that the kininogens are tight-binding non-competitive inhibitors of platelet calpain, the inhibitory domain in each case being mainly on the heavy chain. The light chain of HK appears to influence its kinetic behaviour. Images Fig. 1. Fig. 2. PMID:2396995

  10. IL-17 Inhibits Chondrogenic Differentiation of Human Mesenchymal Stem Cells

    PubMed Central

    Kondo, Masahiro; Yamaoka, Kunihiro; Sonomoto, Koshiro; Fukuyo, Shunsuke; Oshita, Koichi; Okada, Yosuke; Tanaka, Yoshiya

    2013-01-01

    Objective Mesenchymal stem cells (MSCs) can differentiate into cells of mesenchymal lineages, such as osteoblasts and chondrocytes. Here we investigated the effects of IL-17, a key cytokine in chronic inflammation, on chondrogenic differentiation of human MSCs. Methods Human bone marrow MSCs were pellet cultured in chondrogenic induction medium containing TGF-β3. Chondrogenic differentiation was detected by cartilage matrix accumulation and chondrogenic marker gene expression. Results Over-expression of cartilage matrix and chondrogenic marker genes was noted in chondrogenic cultures, but was inhibited by IL-17 in a dose-dependent manner. Expression and phosphorylation of SOX9, the master transcription factor for chondrogenesis, were induced within 2 days and phosphorylated SOX9 was stably maintained until day 21. IL-17 did not alter total SOX9 expression, but significantly suppressed SOX9 phosphorylation in a dose-dependent manner. At day 7, IL-17 also suppressed the activity of cAMP-dependent protein kinase A (PKA), which is known to phosphorylate SOX9. H89, a selective PKA inhibitor, also suppressed SOX9 phosphorylation, expression of chondrogenic markers and cartilage matrix, and also decreased chondrogenesis. Conclusions IL-17 inhibited chondrogenesis of human MSCs through the suppression of PKA activity and SOX9 phosphorylation. These results suggest that chondrogenic differentiation of MSCs can be inhibited by a mechanism triggered by IL-17 under chronic inflammation. PMID:24260226

  11. Connexin43 Inhibition Prevents Human Vein Grafts Intimal Hyperplasia

    PubMed Central

    Longchamp, Alban; Allagnat, Florent; Alonso, Florian; Kuppler, Christopher; Dubuis, Céline; Ozaki, Charles-Keith; Mitchell, James R.; Berceli, Scott; Corpataux, Jean-Marc

    2015-01-01

    Venous bypass grafts often fail following arterial implantation due to excessive smooth muscle cells (VSMC) proliferation and consequent intimal hyperplasia (IH). Intercellular communication mediated by Connexins (Cx) regulates differentiation, growth and proliferation in various cell types. Microarray analysis of vein grafts in a model of bilateral rabbit jugular vein graft revealed Cx43 as an early upregulated gene. Additional experiments conducted using an ex-vivo human saphenous veins perfusion system (EVPS) confirmed that Cx43 was rapidly increased in human veins subjected ex-vivo to arterial hemodynamics. Cx43 knock-down by RNA interference, or adenoviral-mediated overexpression, respectively inhibited or stimulated the proliferation of primary human VSMC in vitro. Furthermore, Cx blockade with carbenoxolone or the specific Cx43 inhibitory peptide 43gap26 prevented the burst in myointimal proliferation and IH formation in human saphenous veins. Our data demonstrated that Cx43 controls proliferation and the formation of IH after arterial engraftment. PMID:26398895

  12. Human erythrocytes inhibit complement-mediated solubilization of immune complexes by human serum

    SciTech Connect

    Dorval, B.L.

    1987-01-01

    The aim of this study was to develop an autologus human system to evaluate the effects of human erythrocytes on solubilization of immune complex precipitates (IC) by human serum. Incubation of IC with fresh human serum or guinea pig serum resulted in solubilization of IC. When packed erythrocytes were added to human serum or guinea pig serum binding of IC to the erythrocyte occurred and IC solubilization was inhibited significantly (p <.025). Sheep erythrocytes did not bind IC or inhibit IC solubilization. To evaluate the role of human erythrocyte complement receptor (CR1) on these findings, human erythrocytes were treated with trypsin or anti-CR1 antibodies. Both treatments abrogated IC binding to human erythrocytes but did not affect the ability of the human erythrocyte to inhibit IC solubilization. Radioimmunoassay was used to measure C3, C4 and C5 activation in human serum after incubation with IC, human erythrocytes, human erythrocytes plus IC, whole blood or in whole blood plus IC.

  13. Inhibition of human immunodeficiency virus type 1 activity by purified human breast milk mucin (MUC1) in an inhibition assay.

    PubMed

    Habte, Habtom H; de Beer, Corena; Lotz, Zoë E; Tyler, Marilyn G; Kahn, Delawir; Mall, Anwar S

    2008-01-01

    It has been reported that breast-feeding is responsible for approximately 40% of the HIV transmissions from HIV-positive mothers to children. Human breast milk, however, is known to contain numerous biologically active components which protect breast-fed infants against bacteria, viruses, and toxins. The purpose of this study was to purify and characterize breast milk mucin and to determine its anti-HIV-1 activity in an HIV inhibition assay. Sepharose CL-4B column chromatography and caesium chloride isopycnic density gradient purification were used to isolate and purify the mucin. Following Western blotting and amino acid analysis, an HIV-1 inhibition assay was carried out to determine the anti-HIV-1 activity of crude breast milk and purified milk mucin (MUC1) by incubating them with HIV-1 prior to infection of the human T lymphoblastoid cell line (CEM SS cells). SDS-PAGE analysis of the mucin, together with its amino acid composition and Western blotting, suggested that this purified mucin from human breast milk was MUC1. The HIV inhibition assay revealed that while the purified milk mucin (MUC1) inhibited the HIV-1 activity by approximately 97%, there was no inhibition of the HIV-1 activity by crude breast milk. Although the reason for this is not clear, it is likely that because the MUC1 in crude milk is enclosed by fat globules, there may not be any physical contact between the mucin and the virus in the crude breast milk. Thus, there is a need to free the mucin from the fat globules for it to be effective against the virus. PMID:17878743

  14. Inhibition of human immunodeficiency virus type-1 by cdk inhibitors.

    PubMed

    Guendel, Irene; Agbottah, Emmanuel T; Kehn-Hall, Kylene; Kashanchi, Fatah

    2010-01-01

    Current therapy for human immunodeficiency virus (HIV-1) infection relies primarily on the administration of anti-retroviral nucleoside analogues, either alone or in combination with HIV-protease inhibitors. Although these drugs have a clinical benefit, continuous therapy with the drugs leads to drug-resistant strains of the virus. Recently, significant progress has been made towards the development of natural and synthetic agents that can directly inhibit HIV-1 replication or its essential enzymes. We previously reported on the pharmacological cyclin-dependent kinase inhibitor (PCI) r-roscovitine as a potential inhibitor of HIV-1 replication. PCIs are among the most promising novel antiviral agents to emerge over the past few years. Potent activity on viral replication combined with proliferation inhibition without the emergence of resistant viruses, which are normally observed in HAART patients; make PCIs ideal candidates for HIV-1 inhibition. To this end we evaluated twenty four cdk inhibitors for their effect on HIV-1 replication in vitro. Screening of these compounds identified alsterpaullone as the most potent inhibitor of HIV-1 with activity at 150 nM. We found that alsterpaullone effectively inhibits cdk2 activity in HIV-1 infected cells with a low IC50 compared to control uninfected cells. The effects of alsterpaullone were associated with suppression of cdk2 and cyclin expression. Combining both alsterpaullone and r-roscovitine (cyc202) in treatment exhibited even stronger inhibitory activities in HIV-1 infected PBMCs. PMID:20334651

  15. Inhibition of Human Immunodeficiency Virus Replication by Antisense Oligodeoxynucleotides

    NASA Astrophysics Data System (ADS)

    Goodchild, John; Agrawal, Sudhir; Civeira, Maria P.; Sarin, Prem S.; Sun, Daisy; Zamecnik, Paul C.

    1988-08-01

    Twenty different target sites within human immunodeficiency virus (HIV) RNA were selected for studies of inhibition of HIV replication by antisense oligonucleotides. Target sites were selected based on their potential capacity to block recognition functions during viral replication. Antisense oligomers complementary to sites within or near the sequence repeated at the ends of retrovirus RNA (R region) and to certain splice sites were most effective. The effect of antisense oligomer length on inhibiting virus replication was also investigated, and preliminary toxicity studies in mice show that these compounds are toxic only at high levels. The results indicate potential usefulness for these oligomers in the treatment of patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex either alone or in combination with other drugs.

  16. GUDC inhibits cytochrome c release from human cholangiocyte mitochondria.

    PubMed

    Que, F G; Phan, V A; Phan, V H; LaRusso, N F; Gores, G J

    1999-05-15

    Although ursodeoxycholic acid (UDC) is considered effective treatment for primary biliary cirrhosis (PBC), its mechanism of action is unclear. We tested the hypothesis that UDC is taken up by cholangiocytes and inhibits caspase 3-dependent apoptosis. We used the human cholangiocyte cell line (H69) and assessed it for expression and function of an apical sodium-dependent bile acid transporter (ASBT) by RT-PCR and uptake of tritiated taurocholic acid. We experimentally induced apoptosis in H69 cells using beauvericin (BV) and determined caspase 3 activation using a fluorogenic substrate and mitochondrial cytochrome c release (CC) into the cytosol by immunoblot analysis. We found that a functional ASBT is expressed by H69 cells as demonstrated by RT-PCR and bile acid uptake studies. Exposure of H69 cells to BV induced apoptosis in 39.4 +/- 1.3% of cells at 2 h (0.23 +/- 0.2% in controls). In contrast, when H69 cells were preincubated with GUDC (50 mM) for 24 h and then exposed to BV, apoptosis was inhibited by 23% (P < 0.03). In cholangiocytes pretreated with GUDC for 24 h and those treated with BV for 2 h, caspase 3-like activity was reduced by 79% and mitochondrial CC release was inhibited. In summary, the human cholangiocyte cell line H69 possesses a functional bile acid transporter, and GUDC decreases BV-induced apoptosis and inhibits activity of caspase 3 protease by blocking CC release from mitochondria. These preliminary results are consistent with our hypothesis that the beneficial effect of UDC on PBC may involve decreased apoptosis after GUDC uptake by cholangiocytes. PMID:10329102

  17. Medial olivocochlear efferent reflex inhibition of human cochlear nerve responses.

    PubMed

    Lichtenhan, J T; Wilson, U S; Hancock, K E; Guinan, J J

    2016-03-01

    Inhibition of cochlear amplifier gain by the medial olivocochlear (MOC) efferent system has several putative roles: aiding listening in noise, protection against damage from acoustic overexposure, and slowing age-induced hearing loss. The human MOC reflex has been studied almost exclusively by measuring changes in otoacoustic emissions. However, to help understand how the MOC system influences what we hear, it is important to have measurements of the MOC effect on the total output of the organ of Corti, i.e., on cochlear nerve responses that couple sounds to the brain. In this work we measured the inhibition produced by the MOC reflex on the amplitude of cochlear nerve compound action potentials (CAPs) in response to moderate level (52-60 dB peSPL) clicks from five, young, normal hearing, awake, alert, human adults. MOC activity was elicited by 65 dB SPL, contralateral broadband noise (CAS). Using tympanic membrane electrodes, approximately 10 h of data collection were needed from each subject to yield reliable measurements of the MOC reflex inhibition on CAP amplitudes from one click level. The CAS produced a 16% reduction of CAP amplitude, equivalent to a 1.98 dB effective attenuation (averaged over five subjects). Based on previous reports of efferent effects as functions of level and frequency, it is possible that much larger effective attenuations would be observed at lower sound levels or with clicks of higher frequency content. For a preliminary comparison, we also measured MOC reflex inhibition of DPOAEs evoked from the same ears with f2's near 4 kHz. The resulting effective attenuations on DPOAEs were, on average, less than half the effective attenuations on CAPs. PMID:26364824

  18. Prolonged cyclic strain inhibits human endothelial cell growth.

    PubMed

    Peyton, Kelly J; Liu, Xiao-ming; Durante, William

    2016-01-01

    The vascular endothelium is continuously exposed to cyclic mechanical strain due to the periodic change in vessel diameter as a result of pulsatile blood flow. Since emerging evidence indicates the cyclic strain plays an integral role in regulating endothelial cell function, the present study determined whether application of a physiologic regimen of cyclic strain (6% at 1 hertz) influences the proliferation of human arterial endothelial cells. Prolonged exposure of human dermal microvascular or human aortic endothelial cells to cyclic strain for up to 7 days resulted in a marked decrease in cell growth. The strain-mediated anti-proliferative effect was associated with the arrest of endothelial cells in the G2/M phase of the cell cycle, did not involve cell detachment or cytotoxicity, and was due to the induction of p21. Interestingly, the inhibition in endothelial cell growth was independent of the strain regimen since prolonged application of constant or intermittent 6% strain was also able to block endothelial cell proliferation. The ability of chronic physiologic cyclic strain to inhibit endothelial cell growth represents a previously unrecognized mechanism by which hemodynamic forces maintain these cells in a quiescent, non-proliferative state. PMID:26709656

  19. Human milk mucin inhibits rotavirus replication and prevents experimental gastroenteritis.

    PubMed Central

    Yolken, R H; Peterson, J A; Vonderfecht, S L; Fouts, E T; Midthun, K; Newburg, D S

    1992-01-01

    Acute gastrointestinal infections due to rotaviruses and other enteric pathogens are major causes of morbidity and mortality in infants and young children throughout the world. Breast-feeding can reduce the rate of serious gastroenteritis in infants; however, the degrees of protection offered against rotavirus infection vary in different populations. The mechanisms associated with milk-mediated protection against viral gastroenteritis have not been fully elucidated. We have isolated a macromolecular component of human milk that inhibits the replication of rotaviruses in tissue culture and prevents the development of gastroenteritis in an animal model system. Purification of the component indicates that the antiviral activity is associated with an acidic fraction (pI = 4.0-4.6), which is free of detectable immunoglobulins. Furthermore, high levels of antiviral activity are associated with an affinity-purified complex of human milk mucin. Deglycosylation of the mucin complex results in the loss of antiviral activity. Further purification indicated that rotavirus specifically binds to the milk mucin complex as well as to the 46-kD glycoprotein component of the complex. Binding to the 46-kD component was substantially reduced after chemical hydrolysis of sialic acid. We have documented that human milk mucin can bind to rotavirus and inhibit viral replication in vitro and in vivo. Variations in milk mucin glycoproteins may be associated with different levels of protection against infection with gastrointestinal pathogens. Images PMID:1331178

  20. Imeglimin prevents human endothelial cell death by inhibiting mitochondrial permeability transition without inhibiting mitochondrial respiration.

    PubMed

    Detaille, D; Vial, G; Borel, A-L; Cottet-Rouselle, C; Hallakou-Bozec, S; Bolze, S; Fouqueray, P; Fontaine, E

    2016-01-01

    Imeglimin is the first in a new class of oral glucose-lowering agents, having recently completed its phase 2b trial. As Imeglimin did show a full prevention of β-cell apoptosis, and since angiopathy represents a major complication of diabetes, we studied Imeglimin protective effects on hyperglycemia-induced death of human endothelial cells (HMEC-1). These cells were incubated in several oxidative stress environments (exposure to high glucose and oxidizing agent tert-butylhydroperoxide) which led to mitochondrial permeability transition pore (PTP) opening, cytochrome c release and cell death. These events were fully prevented by Imeglimin treatment. This protective effect on cell death occurred without any effect on oxygen consumption rate, on lactate production and on cytosolic redox or phosphate potentials. Imeglimin also dramatically decreased reactive oxygen species production, inhibiting specifically reverse electron transfer through complex I. We conclude that Imeglimin prevents hyperglycemia-induced cell death in HMEC-1 through inhibition of PTP opening without inhibiting mitochondrial respiration nor affecting cellular energy status. Considering the high prevalence of macrovascular and microvascular complications in type 2 diabetic subjects, these results together suggest a potential benefit of Imeglimin in diabetic angiopathy. PMID:27551496

  1. Imeglimin prevents human endothelial cell death by inhibiting mitochondrial permeability transition without inhibiting mitochondrial respiration

    PubMed Central

    Detaille, D; Vial, G; Borel, A-L; Cottet-Rouselle, C; Hallakou-Bozec, S; Bolze, S; Fouqueray, P; Fontaine, E

    2016-01-01

    Imeglimin is the first in a new class of oral glucose-lowering agents, having recently completed its phase 2b trial. As Imeglimin did show a full prevention of β-cell apoptosis, and since angiopathy represents a major complication of diabetes, we studied Imeglimin protective effects on hyperglycemia-induced death of human endothelial cells (HMEC-1). These cells were incubated in several oxidative stress environments (exposure to high glucose and oxidizing agent tert-butylhydroperoxide) which led to mitochondrial permeability transition pore (PTP) opening, cytochrome c release and cell death. These events were fully prevented by Imeglimin treatment. This protective effect on cell death occurred without any effect on oxygen consumption rate, on lactate production and on cytosolic redox or phosphate potentials. Imeglimin also dramatically decreased reactive oxygen species production, inhibiting specifically reverse electron transfer through complex I. We conclude that Imeglimin prevents hyperglycemia-induced cell death in HMEC-1 through inhibition of PTP opening without inhibiting mitochondrial respiration nor affecting cellular energy status. Considering the high prevalence of macrovascular and microvascular complications in type 2 diabetic subjects, these results together suggest a potential benefit of Imeglimin in diabetic angiopathy. PMID:27551496

  2. Inhibition of recombinant human maltase glucoamylase by salacinol and derivatives.

    PubMed

    Rossi, Elena J; Sim, Lyann; Kuntz, Douglas A; Hahn, Dagmar; Johnston, Blair D; Ghavami, Ahmad; Szczepina, Monica G; Kumar, Nag S; Sterchi, Erwin E; Nichols, Buford L; Pinto, B M; Rose, David R

    2006-06-01

    Inhibitors targeting pancreatic alpha-amylase and intestinal alpha-glucosidases delay glucose production following digestion and are currently used in the treatment of Type II diabetes. Maltase-glucoamylase (MGA), a family 31 glycoside hydrolase, is an alpha-glucosidase anchored in the membrane of small intestinal epithelial cells responsible for the final step of mammalian starch digestion leading to the release of glucose. This paper reports the production and purification of active human recombinant MGA amino terminal catalytic domain (MGAnt) from two different eukaryotic cell culture systems. MGAnt overexpressed in Drosophila cells was of quality and quantity suitable for kinetic and inhibition studies as well as future structural studies. Inhibition of MGAnt was tested with a group of prospective alpha-glucosidase inhibitors modeled after salacinol, a naturally occurring alpha-glucosidase inhibitor, and acarbose, a currently prescribed antidiabetic agent. Four synthetic inhibitors that bind and inhibit MGAnt activity better than acarbose, and at comparable levels to salacinol, were found. The inhibitors are derivatives of salacinol that contain either a selenium atom in place of sulfur in the five-membered ring, or a longer polyhydroxylated, sulfated chain than salacinol. Six-membered ring derivatives of salacinol and compounds modeled after miglitol were much less effective as MGAnt inhibitors. These results provide information on the inhibitory profile of MGAnt that will guide the development of new compounds having antidiabetic activity. PMID:16817895

  3. Monomethylarsonous acid inhibited endogenous cholesterol biosynthesis in human skin fibroblasts

    SciTech Connect

    Guo, Lei; Xiao, Yongsheng; Wang, Yinsheng

    2014-05-15

    Human exposure to arsenic in drinking water is a widespread public health concern, and such exposure is known to be associated with many human diseases. The detailed molecular mechanisms about how arsenic species contribute to the adverse human health effects, however, remain incompletely understood. Monomethylarsonous acid [MMA(III)] is a highly toxic and stable metabolite of inorganic arsenic. To exploit the mechanisms through which MMA(III) exerts its cytotoxic effect, we adopted a quantitative proteomic approach, by coupling stable isotope labeling by amino acids in cell culture (SILAC) with LC-MS/MS analysis, to examine the variation in the entire proteome of GM00637 human skin fibroblasts following acute MMA(III) exposure. Among the ∼ 6500 unique proteins quantified, ∼ 300 displayed significant changes in expression after exposure with 2 μM MMA(III) for 24 h. Subsequent analysis revealed the perturbation of de novo cholesterol biosynthesis, selenoprotein synthesis and Nrf2 pathways evoked by MMA(III) exposure. Particularly, MMA(III) treatment resulted in considerable down-regulation of several enzymes involved in cholesterol biosynthesis. In addition, real-time PCR analysis showed reduced mRNA levels of select genes in this pathway. Furthermore, MMA(III) exposure contributed to a distinct decline in cellular cholesterol content and significant growth inhibition of multiple cell lines, both of which could be restored by supplementation of cholesterol to the culture media. Collectively, the present study demonstrated that the cytotoxicity of MMA(III) may arise, at least in part, from the down-regulation of cholesterol biosynthesis enzymes and the resultant decrease of cellular cholesterol content. - Highlights: • MMA(III)-induced perturbation of the entire proteome of GM00637 cells is studied. • Quantitative proteomic approach revealed alterations of multiple cellular pathways. • MMA(III) inhibits de novo cholesterol biosynthesis. • MMA

  4. Organophosphate inhibition of human heart muscle cholinesterase isoenzymes.

    PubMed

    Chemnitius, J M; Sadowski, R; Winkel, H; Zech, R

    1999-05-14

    The rate of acetylcholine hydrolysis of mammalian heart muscle influences cardiac responses to vagal innervation. We characterized cholinesterases of human left ventricular heart muscle with respect to both substrate specificity and irreversible inhibition kinetics with the organophosphorus inhibitor N,N'-di-isopropylphosphorodiamidic fluoride (mipafox). Specimens were obtained postmortem from three men and four women (61 +/- 5 years) with no history of cardiovascular disease. Myocardial choline ester hydrolyzing activity was determined with acetylthiocholine (ASCh; 1.25 mM), acetyl-beta-methylthiocholine (AbetaMSCh; 2.0 mM), and butyrylthiocholine (BSCh; 30 mM). After irreversible and covalent inhibition (60 min; 25 degrees C) with a wide range of mipafox concentrations (50 nM-5 mM), residual choline ester hydrolyzing activities were fitted to a sum of up to five exponentials using weighted least-squares non-linear curve fitting. In each ease, quality of curve fitting reached its optimum on the basis of a four component model. Final classification of heart muscle cholinesterases was achieved according to substrate hydrolysis patterns (nmol/min per g wet weight) and to second-order organophosphate inhibition rate constants k2 (1/mol per min); one choline ester hydrolyzing enzyme was identified as acetylcholinesterase (AChE; k2/mipafox = 6.1 (+/- 0.8) x 10(2)), and one as butyrylcholinesterase (BChE; k2/mipafox = 5.3 (+/- 1.1) x 10(3)). An enzyme exhibiting both ChE-like substrate specificity and relative resistance to mipafox inhibition (k2/mipafox = 5.2 (+/- 1.0) x 10(-1)) was classified as atypical cholinesterase. PMID:10421452

  5. GSK-3 inhibition overcomes chemoresistance in human breast cancer.

    PubMed

    Ugolkov, Andrey; Gaisina, Irina; Zhang, Jin-San; Billadeau, Daniel D; White, Kevin; Kozikowski, Alan; Jain, Sarika; Cristofanilli, Massimo; Giles, Francis; O'Halloran, Thomas; Cryns, Vincent L; Mazar, Andrew P

    2016-10-01

    Glycogen Synthase Kinase-3β (GSK-3β), a serine/threonine protein kinase, is an emerging therapeutic target in the treatment of human breast cancer. In this study, we demonstrate that the pharmacological inhibition of GSK-3 by two novel small molecule GSK-3 inhibitors, 9-ING-41 and 9-ING-87, reduced the viability of breast cancer cells but had little effect on non-tumorigenic cell growth. Moreover, treatment with 9-ING-41 enhanced the antitumor effect of irinotecan (CPT-11) against breast cancer cells in vitro. We next established two patient-derived xenograft tumor models (BC-1 and BC-2) from metastatic pleural effusions obtained from patients with progressive, chemorefractory breast cancer and demonstrated that 9-ING-41 also potentiated the effect of the chemotherapeutic drug CPT-11 in vivo, leading to regression of established BC-1 and BC-2 tumors in mice. Our results suggest that the inhibition of GSK-3 is a promising therapeutic approach to overcome chemoresistance in human breast cancer, and identify the GSK-3 inhibitor 9-ING-41 as a candidate targeted agent for metastatic breast cancer therapy. PMID:27424289

  6. Inhibition of BET bromodomains alleviates inflammation in human RPE cells.

    PubMed

    Hytti, M; Tokarz, P; Määttä, E; Piippo, N; Korhonen, E; Suuronen, T; Honkakoski, P; Kaarniranta, K; Lahtela-Kakkonen, M; Kauppinen, A

    2016-06-15

    Bromodomain-containing proteins are vital for controlling the expression of many pro-inflammatory genes. Consequently, compounds capable of inhibiting specific bromodomain-facilitated protein-protein interactions would be predicted to alleviate inflammation, making them valuable agents in the treatment of diseases caused by dysregulated inflammation, such as age-related macular degeneration. Here, we assessed the ability of known inhibitors JQ-1, PFI-1, and IBET-151 to protect from the inflammation and cell death caused by etoposide exposure in the human retinal pigment epithelial cell line, ARPE-19. The potential anti-inflammatory effects of the bromodomain inhibitors were assessed by ELISA (enzyme-linked immunosorbent assay) profiling. The involvement of NF-κB and SIRT1 in inflammatory signaling was monitored by ELISA and western blotting. Furthermore, SIRT1 was knocked down using a specific siRNA or inhibited by EX-527 to elucidate its role in the inflammatory reaction. The bromodomain inhibitors effectively decreased etoposide-induced release of IL-6 and IL-8. This anti-inflammatory effect was not related to SIRT1 activity, although all bromodomain inhibitors decreased the extent of acetylation of p53 at the SIRT1 deacetylation site. Overall, since bromodomain inhibitors display anti-inflammatory properties in human retinal pigment epithelial cells, these compounds may represent a new way of alleviating the inflammation underlying the onset of age-related macular degeneration. PMID:27106081

  7. Sickle erythrocytes inhibit human endothelial cell DNA synthesis

    SciTech Connect

    Weinstein, R.; Zhou, M.A.; Bartlett-Pandite, A.; Wenc, K. )

    1990-11-15

    Patients with sickle cell anemia experience severe vascular occlusive phenomena including acute pain crisis and cerebral infarction. Obstruction occurs at both the microvascular and the arterial level, and the clinical presentation of vascular events is heterogeneous, suggesting a complex etiology. Interaction between sickle erythrocytes and the endothelium may contribute to vascular occlusion due to alteration of endothelial function. To investigate this hypothesis, human vascular endothelial cells were overlaid with sickle or normal erythrocytes and stimulated to synthesize DNA. The erythrocytes were sedimented onto replicate monolayers by centrifugation for 10 minutes at 17 g to insure contact with the endothelial cells. Incorporation of 3H-thymidine into endothelial cell DNA was markedly inhibited during contact with sickle erythrocytes. This inhibitory effect was enhanced more than twofold when autologous sickle plasma was present during endothelial cell labeling. Normal erythrocytes, with or without autologous plasma, had a modest effect on endothelial cell DNA synthesis. When sickle erythrocytes in autologous sickle plasma were applied to endothelial monolayers for 1 minute, 10 minutes, or 1 hour and then removed, subsequent DNA synthesis by the endothelial cells was inhibited by 30% to 40%. Although adherence of sickle erythrocytes to the endothelial monolayers was observed under these experimental conditions, the effect of sickle erythrocytes on endothelial DNA synthesis occurred in the absence of significant adherence. Hence, human endothelial cell DNA synthesis is partially inhibited by contact with sickle erythrocytes. The inhibitory effect of sickle erythrocytes occurs during a brief (1 minute) contact with the endothelial monolayers, and persists for at least 6 hours of 3H-thymidine labeling.

  8. Is Peroxiredoxin II's peroxidase activity strongly inhibited in human erythrocytes?

    PubMed

    Benfeitas, Rui; Selvaggio, Gianluca; Antunes, Fernando; Coelho, Pedro; Salvador, Armindo

    2014-10-01

    H2O2 elimination in human erythrocytes is mainly carried out by catalase (Cat), glutathione peroxidase (GPx1) and the more recently discovered peroxiredoxin 2 (Prx2). However, the contribution of Prx2 to H2O2 consumption is still unclear. Prx2's high reactivity with H2O2 (kPrx2=10×10(7) M(-1)s(-1), kCat =7×10(7) M(-1)s(-1), kGPx1 =4×10(7) M(-1)s(-1)) and high abundance ([Prx2]= 570µM, [Cat]= 32µM, [GPx1]= 1µM) suggest that under low H2O2 supply rates it should consume >99% of the H2O2. However, extensive evidence indicates that in intact erythrocytes Prx2 contributes no more than Cat to H2O2 consumption. In order for this to be attained, Prx2's effective rate constant with H2O2would have to be just ~10(5) M(-1)s(-1), much lower than that determined in multiple experiments with the purified proteins. Nevertheless, nearly all Prx2 is oxidized within 1min of exposing erythrocytes to a H2O2 bolus, which is inconsistent with an irreversible inhibition. A mathematical model of the H2O2 metabolism in human erythrocytes [Benfeitas et al. (2014) Free Radic. Biol. Med.] where Prx2 either has a low kPrx2 or is subject to a strong (>99%) but readily reversible inhibition achieves quantitative agreement with detailed experimental observations of the responses of the redox status of Prx2 in human erythrocytes and suggests functional advantages of this design (see companion abstract). By contrast, a variant where Prx2 is fully active with kPrx2=10(8) M(-1)s(-1) shows important qualitative discrepancies. Altogether, these results suggest that Prx2's peroxidase activity is strongly inhibited in human erythrocytes. We acknowledge fellowship SFRH/BD/51199/2010, grants PEst-C/SAU/LA0001/2013-2014, PEst-OE/QUI/UI0612/2013, PEst-OE/QUI/UI0313/2014, and FCOMP-01-0124-FEDER-020978 (PTDC/QUI-BIQ/119657/2010) co-financed by FEDER through the COMPETE program and by FCT. PMID:26461310

  9. Effect of human plasma on the reactivation of sarin-inhibited human erythrocyte acetylcholinesterase.

    PubMed

    Worek, F; Eyer, P; Kiderlen, D; Thiermann, H; Szinicz, L

    2000-03-01

    The reactivation of organophosphate-inhibited acetylcholinesterase (AChE) by oximes inevitably results in the formation of highly reactive phosphoryloximes (POX), which are able to re-inhibit the enzyme. In this study, the dependence of POX formation on AChE concentration was investigated with sarin-inhibited human erythrocyte AChE (EryAChE). A marked dependence was found with obidoxime but not with the experimental oxime HI 6, suggesting great differences in the decomposition rates of the respective POXs. At a physiological erythrocyte content the reactivation of EryAChE was markedly affected by POX with obidoxime and pralidoxime (2-PAM) but not with the newer oximes HI 6 and HLö 7. Addition of extensively dialysed, sarin-treated human plasma reduced the reactivation by obidoxime and 2-PAM even more. Obidoxime and 2-PAM were superior to HI 6 and HLö 7 in reactivating butyrylcholinesterase (BChE). This effect was pronounced in diluted plasma, but was obscured in concentrated plasma, probably because of re-inhibition by the generated POX. Addition of native erythrocytes to sarin-treated plasma resulted in marked inhibition of EryAChE in the presence of obidoxime, suggesting a higher affinity of the POX for EryAChE. The results indicate that obidoxime and 2-PAM may reactivate sarin-inhibited AChE insufficiently due to re-inhibition by the POX formed. In addition, the re-inhibition of Ery-AChE may be aggravated by the POX that is produced during BChE reactivation. These reactions must be regarded as therapeutically detrimental and disqualify those oximes which are capable of forming stable POX by reactivation of BChE. PMID:10817663

  10. Oxidative stress inhibits distant metastasis by human melanoma cells

    PubMed Central

    Piskounova, Elena; Agathocleous, Michalis; Murphy, Malea M.; Hu, Zeping; Huddlestun, Sara E.; Zhao, Zhiyu; Leitch, A. Marilyn; Johnson, Timothy M.; DeBerardinis, Ralph J.; Morrison, Sean J.

    2015-01-01

    Solid cancer cells commonly enter the blood and disseminate systemically but are highly inefficient at forming distant metastases for poorly understood reasons. We studied human melanomas that differed in their metastasis histories in patients and in their capacity to metastasize in NSG mice. All melanomas had high frequencies of cells that formed subcutaneous tumours, but much lower percentages of cells that formed tumours after intravenous or intrasplenic transplantation, particularly among inefficient metastasizers. Melanoma cells in the blood and visceral organs experienced oxidative stress not observed in established subcutaneous tumours. Successfully metastasizing melanomas underwent reversible metabolic changes during metastasis that increased their capacity to withstand oxidative stress, including increased dependence upon NADPH-generating enzymes in the folate pathway. Anti-oxidants promoted distant metastasis in NSG mice. Folate pathway inhibition using low-dose methotrexate, ALDH1L2 knockdown, or MTHFD1 knockdown inhibited distant metastasis without significantly affecting the growth of subcutaneous tumors in the same mice. Oxidative stress thus limits distant metastasis by melanoma cells in vivo. PMID:26466563

  11. Inhibition of human natural killer (NK) cytotoxicity by Candida albicans

    SciTech Connect

    Zunino, S.; Hudig, D.

    1986-03-01

    Experiments were initiated to determine whether human NK cells are cytotoxic to C. albicans with similar activity observed for mouse NK cells against the yeast Paracoccidiodes brasiliensis. In 48 hour assays using limiting dilutions of C. albicans, strain 3153A, mononuclear leukocytes with NK activity had only marginal effects on yeast outgrowth, whereas granulocytes killed most of the yeast. However, these yeast were able to block NK activity in 4 hr /sup 51/Cr release assays with K562 cells, at yeast to K562 ratios of 10:1 and 100:1. Yeast pretreated with the serum of the majority of donors blocked the NK activity more than untreated yeast. Two of the 7 donors did not enhance NK inhibition after pretreatment of the yeast with their serum. Serum antibody to C. albicans and complement consumption by the yeast correlated with the relative efficiency of NK inhibition for most donors. This report suggests that there may be in vivo interactions between NK cells of the immune system and opportunistic fungal pathogens, which may compromise NK cell function.

  12. N-acetylcysteine inhibits muscle fatigue in humans.

    PubMed Central

    Reid, M B; Stokić, D S; Koch, S M; Khawli, F A; Leis, A A

    1994-01-01

    N-acetylcysteine (NAC) is a nonspecific antioxidant that selectively inhibits acute fatigue of rodent skeletal muscle stimulated at low (but not high) tetanic frequencies and that decreases contractile function of unfatigued muscle in a dose-dependent manner. The present experiments test the hypothesis that NAC pretreatment can inhibit acute muscular fatigue in humans. Healthy volunteers were studied on two occasions each. Subjects were pretreated with NAC 150 mg/kg or 5% dextrose in water by intravenous infusion. The subject then sat in a chair with surface electrodes positioned over the motor point of tibialis anterior, an ankle dorsiflexor of mixed-fiber composition. The muscle was stimulated to contract electrically (40-55 mA, 0.2-ms pulses) and force production was measured. Function of the unfatigued muscle was assessed by measuring the forces produced during maximal voluntary contractions (MVC) of ankle dorsiflexor muscle groups and during electrical stimulation of tibialis anterior at 1, 10, 20, 40, 80, and 120 Hz (protocol 1). Fatigue was produced using repetitive tetanic stimulations at 10 Hz (protocol 1) or 40 Hz (protocol 2); intermittent stimulations subsequently were used to monitor recovery from fatigue. The contralateral leg then was studied using the same protocol. Pretreatment with NAC did not alter the function of unfatigued muscle; MVC performance and the force-frequency relationship of tibialis anterior were unchanged. During fatiguing contractions stimulated at 10 Hz, NAC increased force output by approximately 15% (P < 0.0001), an effect that was evident after 3 min of repetitive contraction (P < 0.0125) and persisted throughout the 30-min protocol. NAC had no effect on fatigue induced using 40 Hz stimuli or on recovery from fatigue. N-acetylcysteine pretreatment can improve performance of human limb muscle during fatiguing exercise, suggesting that oxidative stress plays a causal role in the fatigue process and identifying antioxidant

  13. Inhibition of Human Colon Cancer Growth by Antibody-Directed Human LAK Cells in SCID Mice

    NASA Astrophysics Data System (ADS)

    Takahashi, Hiroshi; Nakada, Tetsuya; Puisieux, Isabelle

    1993-03-01

    Advanced human colon cancer does not respond to lymphokine-activated killer (LAK) cells. In order to direct cytotoxic cells to the tumor, human LAK cells linked with antibodies to a tumor cell surface antigen were tested with established hepatic metastases in severe combined immunodeficient (SCID) mice. These cells had increased uptake into the tumor and suppression of tumor growth as compared with LAK cells alone, thereby improving the survival of tumor-bearing mice. Thus, tumor growth can be inhibited by targeted LAK cells, and SCID mice can be used to test the antitumor properties of human effector cells.

  14. Monomethylarsonous acid inhibited endogenous cholesterol biosynthesis in human skin fibroblasts.

    PubMed

    Guo, Lei; Xiao, Yongsheng; Wang, Yinsheng

    2014-05-15

    Human exposure to arsenic in drinking water is a widespread public health concern, and such exposure is known to be associated with many human diseases. The detailed molecular mechanisms about how arsenic species contribute to the adverse human health effects, however, remain incompletely understood. Monomethylarsonous acid [MMA(III)] is a highly toxic and stable metabolite of inorganic arsenic. To exploit the mechanisms through which MMA(III) exerts its cytotoxic effect, we adopted a quantitative proteomic approach, by coupling stable isotope labeling by amino acids in cell culture (SILAC) with LC-MS/MS analysis, to examine the variation in the entire proteome of GM00637 human skin fibroblasts following acute MMA(III) exposure. Among the ~6500 unique proteins quantified, ~300 displayed significant changes in expression after exposure with 2 μM MMA(III) for 24 h. Subsequent analysis revealed the perturbation of de novo cholesterol biosynthesis, selenoprotein synthesis and Nrf2 pathways evoked by MMA(III) exposure. Particularly, MMA(III) treatment resulted in considerable down-regulation of several enzymes involved in cholesterol biosynthesis. In addition, real-time PCR analysis showed reduced mRNA levels of select genes in this pathway. Furthermore, MMA(III) exposure contributed to a distinct decline in cellular cholesterol content and significant growth inhibition of multiple cell lines, both of which could be restored by supplementation of cholesterol to the culture media. Collectively, the present study demonstrated that the cytotoxicity of MMA(III) may arise, at least in part, from the down-regulation of cholesterol biosynthesis enzymes and the resultant decrease of cellular cholesterol content. PMID:24625837

  15. DMSO inhibits human platelet activation through cyclooxygenase-1 inhibition. A novel agent for drug eluting stents?

    SciTech Connect

    Asmis, Lars; Tanner, Felix C.; Sudano, Isabella; Luescher, Thomas F.; Camici, Giovanni G.

    2010-01-22

    Background: DMSO is routinely infused together with hematopoietic cells in patients undergoing myeloablative therapy and was recently found to inhibit smooth muscle cells proliferation and arterial thrombus formation in the mouse by preventing tissue factor (TF), a key activator of the coagulation cascade. This study was designed to investigate whether DMSO prevents platelet activation and thus, whether it may represent an interesting agent to be used on drug eluting stents. Methods and results: Human venous blood from healthy volunteers was collected in citrated tubes and platelet activation was studied by cone and platelet analyzer (CPA) and rapid-platelet-function-assay (RPFA). CPA analysis showed that DMSO-treated platelets exhibit a lower adherence in response to shear stress (-15.54 {+-} 0.9427%, n = 5, P < 0.0001 versus control). Additionally, aggregometry studies revealed that DMSO-treated, arachidonate-stimulated platelets had an increased lag phase (18.0% {+-} 4.031, n = 9, P = 0.0004 versus control) as well as a decreased maximal aggregation (-6.388 {+-} 2.212%, n = 6, P = 0.0162 versus control). Inhibitory action of DMSO could be rescued by exogenous thromboxane A2 and was mediated, at least in part, by COX-1 inhibition. Conclusions: Clinically relevant concentrations of DMSO impair platelet activation by a thromboxane A2-dependent, COX-1-mediated effect. This finding may be crucial for the previously reported anti-thrombotic property displayed by DMSO. Our findings support a role for DMSO as a novel drug to prevent not only proliferation, but also thrombotic complications of drug eluting stents.

  16. Isocyanides inhibit human heme oxygenases at the verdoheme stage.

    PubMed

    Evans, John P; Kandel, Sylvie; Ortiz de Montellano, Paul R

    2009-09-22

    Heme oxygenases (HO) catalyze the oxidative cleavage of heme to generate biliverdin, CO, and free iron. In humans, heme oxygenase-1 (hHO-1) is overexpressed in tumor tissues, where it helps to protect cancer cells from anticancer agents, while HOs in fungal pathogens, such as Candida albicans, function as the primary means of iron acquisition. Thus, HO can be considered a potential therapeutic target for certain diseases. In this study, we have examined the equilibrium binding of three isocyanides, isopropyl, n-butyl, and benzyl, to the two major human HO isoforms (hHO-1 and hHO-2), Candida albicans HO (CaHmx1), and human cytochrome P450 CYP3A4 using electronic absorption spectroscopy. Isocyanides coordinate to both ferric and ferrous HO-bound heme, with tighter binding by the more hydrophobic isocyanides and 200-300-fold tighter binding to the ferrous form. Benzyl isocyanide was the strongest ligand to ferrous heme in all the enzymes. Because the dissociation constants (KD) of the ligands for ferrous heme-hHO-1 were below the limit of accuracy for equilibrium titrations, stopped-flow kinetic experiments were used to measure the binding parameters of the isocyanides to ferrous hHO-1. Steady-state activity assays showed that benzyl isocyanide was the most potent uncompetitive inhibitor with respect to heme with a KI = 0.15 microM for hHO-1. Importantly, single turnover assays revealed that the reaction was completely stopped by coordination of the isocyanide to the verdoheme intermediate rather than to the ferric heme complex. Much tighter binding of the inhibitor to the verdoheme intermediate differentiates it from inhibition of, for example, CYP3A4 and offers a possible route to more selective inhibitor design. PMID:19694439

  17. Acrylamide inhibits cellular differentiation of human neuroblastoma and glioblastoma cells.

    PubMed

    Chen, Jong-Hang; Chou, Chin-Cheng

    2015-08-01

    This study explores human neuroblastoma (SH-SY5Y) and human glioblastoma (U-1240 MG) cellular differentiation changes under exposure to acrylamide (ACR). Differentiation of SH-SY5Y and U-1240 MG cells were induced by retinoic acid (RA) and butyric acid (BA), respectively. Morphological observations and MTT assay showed that the induced cellular differentiation and cell proliferation were inhibited by ACR in a time- and dose-dependent manner. ACR co-treatment with RA attenuated SH-SY5Y expressions of neurofilament protein-L (NF-L), microtubule-associated protein 1b (MAP1b; 1.2 to 0.7, p < 0.001), MAP2c (2.2 to 0.8, p < 0.05), and Janus kinase1 (JAK1; 1.9 to 0.6, p < 0.001), while ACR co-treatment with BA attenuated U-1240 MG expressions of glial fibrillary acidic protein (GFAP), MAP1b (1.2 to 0.6, p < 0.001), MAP2c (1.5 to 0.7, p < 0.01), and JAK1 (2.1 to 0.5, p < 0.001), respectively. ACR also decreased the phosphorylation of extracellular-signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK) in U-1240 MG cells, while caffeine reversed this suppression of ERK and JNK phosphorylation caused by ACR treatment. These results showed that RA-induced neurogenesis of SH-SY5Y and BA-induced astrogliogenesis of U-1240 MG cells were attenuated by ACR and were associated with down-regulation of MAPs expression and JAK-STAT signaling. PMID:25959841

  18. Dicinnamoylquinides in roasted coffee inhibit the human adenosine transporter.

    PubMed

    de Paulis, Tomas; Schmidt, Dennis E; Bruchey, Aleksandra K; Kirby, Michael T; McDonald, Michael P; Commers, Patricia; Lovinger, David M; Martin, Peter R

    2002-05-10

    Preliminary screening of a minor, non-xanthine constituent of roasted coffee, 3,4-diferuloyl-1,5-quinolactone (DIFEQ), showed inhibition of the adenosine transporter at low micromolar concentration. DIFEQ is a neutral derivative of the chlorogenic acids, i.e. isomeric mono- and di-substituted coumaroyl-, caffeoyl-, and feruloyl-esters of quinic acid, formed in the roasting process of coffee. Displacement of the adenosine transporter antagonist [(3)H](S)-(nitrobenzyl)-6-thioinosine binding by DIFEQ in cultured U-937 cell preparations, expressing the human adenosine transporter protein (hENT1), showed a K(i) of 0.96+/-0.13 microM. Extracts of regular and decaffeinated coffee showed binding activities equivalent to 30-40 mg DIFEQ per three cups of coffee. Acute administration of a high dose of DIFEQ (100 mg/kg i.p.) reduced open field locomotion in mice for 20 min in correlation with brain levels of DIFEQ. Both 3,4-dicaffeoyl-1,5-quinide and 3,4-dicoumaroyl-1,5-quinide, two close structural analogs of DIFEQ also present in roasted coffee, showed similar affinities for the adenosine transporter, while the corresponding 3- and 4-mono caffeoyl- and feruloyl-quinides were one to two orders of magnitudes less active. This suggests that 3,4-dicinnamoyl-1,5-quinides in coffee could have the potential to raise extra-cellular adenosine levels, thereby counteracting the stimulant effect of caffeine. PMID:12065074

  19. Selective Inhibition of Collagen Prolyl 4-Hydroxylase in Human Cells

    PubMed Central

    Vasta, James D.; Andersen, Kristen A.; Deck, Kathryn M.; Nizzi, Christopher P.; Eisenstein, Richard S.; Raines, Ronald T.

    2016-01-01

    Collagen is the most abundant protein in animals. Its overproduction is associated with fibrosis and cancer metastasis. The stability of collagen relies on post-translational modifications, the most prevalent being the hydroxylation of collagen strands by collagen prolyl 4-hydroxylases (CP4Hs). Catalysis by CP4Hs enlists an iron cofactor to convert proline residues to 4 hydroxyproline residues, which are essential for the conformational stability of mature collagen. Ethyl 3,4-dihydroxybenzoate (EDHB) is commonly used as a “P4H” inhibitor in cells, but suffers from low potency, poor selectivity, and off-target effects that cause iron deficiency. Dicarboxylates of 2,2′-bipyridine are among the most potent known CP4H inhibitors but suffer from a high affinity for free iron. A screen of biheteroaryl compounds revealed that replacing one pyridyl group with a thiazole moiety retains potency and enhances selectivity. A diester of 2 (5-carboxythiazol-2-yl)pyridine-5-carboxylic acid is bioavailable to human cells and inhibits collagen biosynthesis at concentrations that neither cause general toxicity nor disrupt iron homeostasis. These data anoint a potent and selective probe for CP4H and a potential lead for the development of a new class of antifibrotic and antimetastatic agents. PMID:26535807

  20. Downregulation of CCR1 inhibits human hepatocellular carcinoma cell invasion

    SciTech Connect

    Wu Xiaofeng; Fan Jia; E-mail: jiafan99@yahoo.com; Wang Xiaoying; Zhou Jian; Qiu Shuangjian; Yu Yao; Liu Yinkun; Tang Zhaoyou

    2007-04-20

    CC chemokine receptor 1 (CCR1) has an important role in the recruitment of leukocytes to the site of inflammation. The migration and metastasis of tumor cells shares many similarities with leukocyte trafficking, which is mainly regulated by chemokine receptor-ligand interactions. CCR1 is highly expressed in hepatocellular carcinoma (HCC) cells and tissues with unknown functions. In this study, we silenced CCR1 expression in the human HCC cell line HCCLM3 using artificial microRNA (miRNA)-mediated RNA interference (RNAi) and examined the invasiveness and proliferation of CCR1-silenced HCCLM3 cells and the matrix metalloproteinase (MMP) activity. The miRNA-mediated knockdown expression of CCR1 significantly inhibited the invasive ability of HCCLM3 cells, but had only a minor effect on the cellular proliferation rate. Moreover, CCR1 knockdown significantly reduced the secretion of MMP-2. Together, these findings indicate that CCR1 has an important role in HCCLM3 invasion and that CCR1 might be a new target of HCC treatment.

  1. Enterobacter cloacae inhibits human norovirus infectivity in gnotobiotic pigs

    PubMed Central

    Lei, Shaohua; Samuel, Helen; Twitchell, Erica; Bui, Tammy; Ramesh, Ashwin; Wen, Ke; Weiss, Mariah; Li, Guohua; Yang, Xingdong; Jiang, Xi; Yuan, Lijuan

    2016-01-01

    Human noroviruses (HuNoVs) are the leading cause of epidemic gastroenteritis worldwide. Study of HuNoV biology has been hampered by the lack of an efficient cell culture system. Recently, enteric commensal bacteria Enterobacter cloacae has been recognized as a helper in HuNoV infection of B cells in vitro. To test the influences of E. cloacae on HuNoV infectivity and to determine whether HuNoV infects B cells in vivo, we colonized gnotobiotic pigs with E. cloacae and inoculated pigs with 2.74 × 104 genome copies of HuNoV. Compared to control pigs, reduced HuNoV shedding was observed in E. cloacae colonized pigs, characterized by significantly shorter duration of shedding in post-inoculation day 10 subgroup and lower cumulative shedding and peak shedding in individual pigs. Colonization of E. cloacae also reduced HuNoV titers in intestinal tissues and in blood. In both control and E. cloacae colonized pigs, HuNoV infection of enterocytes was confirmed, however infection of B cells was not observed in ileum, and the entire lamina propria in sections of duodenum, jejunum, and ileum were HuNoV-negative. In summary, E. cloacae inhibited HuNoV infectivity, and B cells were not a target cell type for HuNoV in gnotobiotic pigs, with or without E. cloacae colonization. PMID:27113278

  2. Protease activity, localization and inhibition in the human hair follicle

    PubMed Central

    Bhogal, R K; Mouser, P E; Higgins, C A; Turner, G A

    2014-01-01

    Synopsis Objective In humans, the process of hair shedding, referred to as exogen, is believed to occur independently of the other hair cycle phases. Although the actual mechanisms involved in hair shedding are not fully known, it has been hypothesized that the processes leading to the final step of hair shedding may be driven by proteases and/or protease inhibitor activity. In this study, we investigated the presence of proteases and protease activity in naturally shed human hairs and assessed enzyme inhibition activity of test materials. Methods We measured enzyme activity using a fluorescence-based assay and protein localization by indirect immunohistochemistry (IHC). We also developed an ex vivo skin model for measuring the force required to pull hair fibres from skin. Results Our data demonstrate the presence of protease activity in the tissue material surrounding club roots. We also demonstrated the localization of specific serine protease protein expression in human hair follicle by IHC. These data provide evidence demonstrating the presence of proteases around the hair club roots, which may play a role during exogen. We further tested the hypothesis that a novel protease inhibitor system (combination of Trichogen® and climbazole) could inhibit protease activity in hair fibre club root extracts collected from a range of ethnic groups (UK, Brazil, China, first-generation Mexicans in the USA, Thailand and Turkey) in both males and females. Furthermore, we demonstrated that this combination is capable of increasing the force required to remove hair in an ex vivo skin model system. Conclusion These studies indicate the presence of proteolytic activity in the tissue surrounding the human hair club root and show that it is possible to inhibit this activity with a combination of Trichogen® and climbazole. This technology may have potential to reduce excessive hair shedding. Résumé Objectif Chez l'homme, le processus de perte de cheveux, désigné comme exog

  3. 4-Methoxycinnamaldehyde inhibited human respiratory syncytial virus in a human larynx carcinoma cell line.

    PubMed

    Wang, Kuo Chih; Chang, Jung San; Chiang, Lien Chai; Lin, Chun Ching

    2009-09-01

    4-Methoxycinnamaldehyde, an active constituent of Agastache rugosa, was examined for its cytoprotective activity against RSV by XTT method in human larynx carcinoma cell line. 4-Methoxycinnamaldehyde could effectively inhibit cytopathic effect of RSV (p<0.0001) with an estimated IC(50) of 0.055microg/ml and a selectivity index (SI) of 898.2. 4-Methoxycinnamaldehyde (0.03microg/ml) could inhibit viral entrance by interfering viral attachment (IC(50) of 0.06microg/ml; p<0.0001) and internalization (IC(50) of 0.01microg/ml; p<0.0001). 4-Methoxycinnamaldehyde significantly increased the basal production of IFN (p=0.0015), but not the virus-induced IFN production. Therefore, its cytoprotective activity against RSV was not mediated by interferon. In conclusion, 4-methoxycinnamaldehyde might be helpful to manage the disease induced by RSV infection. PMID:19303275

  4. Bovine lactoferrin digested with human gastrointestinal enzymes inhibits replication of human echovirus 5 in cell culture.

    PubMed

    Furlund, Camilla B; Kristoffersen, Anja B; Devold, Tove G; Vegarud, Gerd E; Jonassen, Christine M

    2012-07-01

    Many infant formulas are enriched with lactoferrin (Lf) because of its claimed beneficial effects on health. Native bovine Lf (bLf) is known to inhibit in vitro replication of human enteroviruses, a group of pathogenic viruses that replicate in the gut as their primary infection site. On the basis of a model digestion and human gastrointestinal enzymes, we hypothesized that bLf could retain its antiviral properties against enterovirus in the gastrointestinal tract, either as an intact protein or through bioactive peptide fragments released by digestive enzymes. To test our hypothesis, bLf was digested with human gastric juice and duodenal juice in a 2-step in vitro digestion model. Two gastric pH levels and reduction conditions were used to simulate physiological conditions in adults and infants. The antiviral activity of native bLf and of the digested fractions was studied on echovirus 5 in vitro, using various assay conditions, addressing several mechanisms for replication inhibition. Both native and digested bLf fractions revealed a significant inhibitory effect, when added before or simultaneously with the virus onto the cells. Furthermore, a significant stronger sustained antiviral effect was observed when bLf was fully digested in the gastric phase with fast pH reduction to 2.5, compared with native bLf, suggesting the release of antiviral peptides from bLf during the human digestion process. In conclusion, this study demonstrates that bLf may have a role in the prevention of human gastrointestinal virus infection under physiological conditions and that food containing bLf may protect against infection in vivo. PMID:22901558

  5. Fluoride inhibits the antimicrobial peroxidase systems in human whole saliva.

    PubMed

    Hannuksela, S; Tenovuo, J; Roger, V; Lenander-Lumikari, M; Ekstrand, J

    1994-01-01

    Fluoride (F-) ions at concentrations present in vivo at the plaque/enamel interface (0.05-10 mM) inhibited the activities of lactoperoxidase (LP), myeloperoxidase (MP) and total salivary peroxidase (TSP) in a pH- and dose-dependent way. The inhibition was observed only at pH < or = 6.5 and with F- concentrations > or = 0.1 mM. At pH 5.5 LP activity was inhibited by 85% and MP by 34% with 10 mM F-. TSP activity was also inhibited only at low pH (5.5) by approximately 25%. Furthermore, the generation of the actual antimicrobial agent in vivo, hypothiocyanite (HOSCN/OSCN-), of the oral peroxidase systems was inhibited by F-, again at low pH (5.0-5.5) both in buffer (by 45%) and in saliva (by 15%). This inhibition was observed only with the highest F- concentrations studied (5-10 mM). Fluoridated toothpaste (with 0.10 or 0.14% F) mixed with saliva did not inhibit TSP or HOSCN/OSCN- generation. This may have been due to the 'buffering' effect of toothpaste which did not allow salivary pH to drop below 5.9. We conclude that the F- ions in acidic fluoride products, e.g. in gels or varnishes (but not in toothpastes), may have the potential to locally inhibit the generation of a nonimmune host defense factor, HOSCN/OSCN/SCN-, produced by oral peroxidase systems. The possible clinical significance of this finding remains to be shown. PMID:7850846

  6. Lipoxin A4 inhibits phosphoinositide hydrolysis in human neutrophils.

    PubMed

    Grandordy, B M; Lacroix, H; Mavoungou, E; Krilis, S; Crea, A E; Spur, B W; Lee, T H

    1990-03-30

    Lipoxins (LX) are trihydroxytetraene metabolites derived from arachidonic acid via an interaction between the 5- and 15-lipoxygenases. Preincubation of [3H] myo-inositol labeled PMN with 10-7M and 10-5M LXA4 for 1 minute at 37 degrees C resulted in a concentration dependent inhibition of the generation of [3H] IP3 and [3H] IP in cells subsequently stimulated by increasing doses of LTB4 or FMLP for 1 minute at 37 degrees C. Preincubation of PMN with LXA4 did not inhibit specific binding of [3H] LTB4 to PMN. These results indicate that LXA4 inhibits chemotactic factor-induced phosphoinositide hydrolysis at a post-receptor level. PMID:2157419

  7. The inhibition of the Human Immunodeficiency Virus type 1 activity by crude and purified human pregnancy plug mucus and mucins in an inhibition assay

    PubMed Central

    Habte, Habtom H; de Beer, Corena; Lotz, Zoë E; Tyler, Marilyn G; Schoeman, Leann; Kahn, Delawir; Mall, Anwar S

    2008-01-01

    Background The female reproductive tract is amongst the main routes for Human Immunodeficiency Virus (HIV) transmission. Cervical mucus however is known to protect the female reproductive tract from bacterial invasion and fluid loss and regulates and facilitates sperm transport to the upper reproductive tract. The purpose of this study was to purify and characterize pregnancy plug mucins and determine their anti-HIV-1 activity in an HIV inhibition assay. Methods Pregnancy plug mucins were purified by caesium chloride density-gradient ultra-centrifugation and characterized by Western blotting analysis. The anti-HIV-1 activities of the crude pregnancy plug mucus and purified pregnancy plug mucins was determined by incubating them with HIV-1 prior to infection of the human T lymphoblastoid cell line (CEM SS cells). Results The pregnancy plug mucus had MUC1, MUC2, MUC5AC and MUC5B. The HIV inhibition assay revealed that while the purified pregnancy plug mucins inhibit HIV-1 activity by approximately 97.5%, the crude pregnancy plug mucus failed to inhibit HIV-1 activity. Conclusion Although it is not clear why the crude sample did not inhibit HIV-1 activity, it may be that the amount of mucins in the crude pregnancy plug mucus (which contains water, mucins, lipids, nucleic acids, lactoferrin, lysozyme, immunoglobulins and ions), is insufficient to cause viral inhibition or aggregation. PMID:18489743

  8. Sampangine inhibits heme biosynthesis in both yeast and human

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The azaoxoaporphine alkaloid sampangine exhibits strong antiproliferation activity in various organisms. Previous studies suggested that it somehow affects heme metabolism and stimulates production of reactive oxygen species (ROS). In this study, we show that inhibition of heme biosynthesis is the p...

  9. Molecular sequelae of proteasome inhibition in human multiple myeloma cells

    PubMed Central

    Mitsiades, Nicholas; Mitsiades, Constantine S.; Poulaki, Vassiliki; Chauhan, Dharminder; Fanourakis, Galinos; Gu, Xuesong; Bailey, Charles; Joseph, Marie; Libermann, Towia A.; Treon, Steven P.; Munshi, Nikhil C.; Richardson, Paul G.; Hideshima, Teru; Anderson, Kenneth C.

    2002-01-01

    The proteasome inhibitor PS-341 inhibits IκB degradation, prevents NF-κB activation, and induces apoptosis in several types of cancer cells, including chemoresistant multiple myeloma (MM) cells. PS-341 has marked clinical activity even in the setting of relapsed refractory MM. However, PS-341-induced apoptotic cascade(s) are not yet fully defined. By using gene expression profiling, we characterized the molecular sequelae of PS-341 treatment in MM cells and further focused on molecular pathways responsible for the anticancer actions of this promising agent. The transcriptional profile of PS-341-treated cells involved down-regulation of growth/survival signaling pathways, and up-regulation of molecules implicated in proapoptotic cascades (which are both consistent with the proapoptotic effect of proteasome inhibition), as well as up-regulation of heat-shock proteins and ubiquitin/proteasome pathway members (which can correspond to stress responses against proteasome inhibition). Further studies on these pathways showed that PS-341 decreases the levels of several antiapoptotic proteins and triggers a dual apoptotic pathway of mitochondrial cytochrome c release and caspase-9 activation, as well as activation of Jun kinase and a Fas/caspase-8-dependent apoptotic pathway [which is inhibited by a dominant negative (decoy) Fas construct]. Stimulation with IGF-1, as well as overexpression of Bcl-2 or constitutively active Akt in MM cells also modestly attenuates PS-341-induced cell death, whereas inhibitors of the BH3 domain of Bcl-2 family members or the heat-shock protein 90 enhance tumor cell sensitivity to proteasome inhibition. These data provide both insight into the molecular mechanisms of antitumor activity of PS-341 and the rationale for future clinical trials of PS-341, in combination with conventional and novel therapies, to improve patient outcome in MM. PMID:12391322

  10. Delineation of Interfaces on Human Alpha-Defensins Critical for Human Adenovirus and Human Papillomavirus Inhibition

    PubMed Central

    Wiens, Mayim E.; Lu, Wuyuan; Smith, Jason G.

    2014-01-01

    Human α-defensins are potent anti-microbial peptides with the ability to neutralize bacterial and viral targets. Single alanine mutagenesis has been used to identify determinants of anti-bacterial activity and binding to bacterial proteins such as anthrax lethal factor. Similar analyses of α-defensin interactions with non-enveloped viruses are limited. We used a comprehensive set of human α-defensin 5 (HD5) and human neutrophil peptide 1 (HNP1) alanine scan mutants in a combination of binding and neutralization assays with human adenovirus (AdV) and human papillomavirus (HPV). We have identified a core of critical hydrophobic residues that are common determinants for all of the virus-defensin interactions that were analyzed, while specificity in viral recognition is conferred by specific surface-exposed charged residues. The hydrophobic residues serve multiple roles in maintaining the tertiary and quaternary structure of the defensins as well as forming an interface for virus binding. Many of the important solvent-exposed residues of HD5 group together to form a critical surface. However, a single discrete binding face was not identified for HNP1. In lieu of whole AdV, we used a recombinant capsid subunit comprised of penton base and fiber in quantitative binding studies and determined that the anti-viral potency of HD5 was a function of stoichiometry rather than affinity. Our studies support a mechanism in which α-defensins depend on hydrophobic and charge-charge interactions to bind at high copy number to these non-enveloped viruses to neutralize infection and provide insight into properties that guide α-defensin anti-viral activity. PMID:25188351

  11. Beta 1,4-oligoglucosides inhibit the binding of Aspergillus fumigatus conidia to human monocytes.

    PubMed

    Kan, V L; Bennett, J E

    1991-05-01

    The binding of Aspergillus fumigatus conidia to human monocytes is mediated by a barley beta-glucan-inhibitable receptor. The simplest linkages in this glucan are present in the disaccharides laminaribiose (beta 1,3) and cellobiose (beta 1,4). Although laminaribiose gave strong inhibition of conidial binding to monocytes, cellobiose and oligosaccharides with beta 1,4-linked glucose residues were more potent as specific inhibitors of this binding over similar concentrations. Increasing the number of beta 1,4-linked glucose residues led to greater inhibition of conidial binding by human monocytes. PMID:2019764

  12. Selectivity for inhibition of nilotinib on the catalytic activity of human UDP-glucuronosyltransferases.

    PubMed

    Ai, Limei; Zhu, Liangliang; Yang, Lu; Ge, Guangbo; Cao, Yunfeng; Liu, Yong; Fang, Zhongze; Zhang, Yanyan

    2014-04-01

    1. Nilotinib, a tyrosine kinase inhibitor, could potently inhibit SN-38 glucuronidation mainly catalyzed by UDP-glucuronosyltransferase (UGT) 1A1. This study was designed to investigate whether nilotinib can be used as a selective inhibitor of UGT1A1 in human liver. 2. Assays with recombinant UGTs indicated that nilotinib could strongly inhibit the activity of UGT1A1 and decreased the activity of extra-hepatic UGT1A7 to a much lesser extent. The inhibition on 4-methylumbelliferone (4Mu) glucuronidation by recombinant UGT1A1 obeyed competitive inhibition mechanism, with a kinetic constants (Ki) value of 0.17 μM. Assays with human liver microsomes (HLM) demonstrated that nilotinib could selectively inhibit estradiol-3-O-glucuronidation (E2-3-O-glucuronidation), a probe reaction of UGT1A1. Kinetic studies displayed that the inhibition on E2-3-O-glucuronidation followed non-competitive inhibition model, different from the inhibition on 4Mu glucuronidation. The Ki values were calculated to be 0.14 and 0.53 μM, depending on the enzyme sources of recombinant UGT1A1 or HLM, respectively. 3. Given that UGT1A7 is an extra-hepatic enzyme, this study indicates that nilotinib can be used as a selective inhibitor of UGT1A1 in human liver. PMID:24066723

  13. Gossypol enantiomers potently inhibit human placental 3β-hydroxysteroid dehydrogenase 1 and aromatase activities.

    PubMed

    Dong, Yaoyao; Mao, Baiping; Li, Linxi; Guan, Hongguo; Su, Ying; Li, Xiaoheng; Lian, Qingquan; Huang, Ping; Ge, Ren-Shan

    2016-03-01

    Gossypol is a chemical isolated from cotton seeds. It exists as (+) or (-) enantiomer and has been tested for anticancer, abortion-inducing, and male contraception. Progesterone formed from pregnenolone by 3β-hydroxysteroid dehydrogenase 1 (HSD3B1) and estradiol from androgen by aromatase (CYP19A1) are critical for the maintenance of pregnancy or associated with some cancers. In this study we compared the potencies of (+)- and (-)-gossypol enantiomers in the inhibition of HSD3B1 and aromatase activities as well as progesterone and estradiol production in human placental JEG-3 cells. (+) Gossypol showed potent inhibition on human placental HSD3B1 with IC50 value of 2.3 μM, while (-) gossypol weakly inhibited it with IC50 over 100 μM. In contrast, (-) gossypol moderately inhibited CYP19A1 activity with IC50 of 23 μM, while (+) gossypol had no inhibition when the highest concentration (100 μM) was tested. (+) Gossypol enantiomer competitively inhibited HSD3B1 against substrate pregnenolone and showed mixed mode against NAD(+). (-) Gossypol competitively inhibited CYP19A1 against substrate testosterone. Gossypol enantiomers showed different potency related to their inhibition on human HSD3B1 and CYP19A1. Whether gossypol enantiomer is used alone or in combination relies on its application and beneficial effects. PMID:26709042

  14. Inhibition of mitogen stimulated growth of human colon cancer cells by interferon.

    PubMed Central

    Hamburger, A. W.; Condon, M. E.; O'Donnell, K.

    1988-01-01

    Recombinant human interferon alpha inhibits growth of a human colon cancer cell line, Colo 205. To explore the mechanisms of IFN induced growth inhibition, quiescent Colo 205 cells were stimulated to proliferate in serum-free media by defined growth factors. Addition of insulin, transferrin and selenium (ITS) stimulated DNA synthesis, as measured by 3H-thymidine incorporation, in a dose-dependent manner. IFN-alpha (at concentrations greater than 100 U ml-1) inhibited ITS stimulated DNA synthesis by 63%. Inhibition of cell cycle traverse was confirmed by flow cytometric analysis. Although IFN inhibited growth of ITS-treated cells, steady state levels of c-myc mRNA remained above levels observed in unstimulated cells. IFN inhibited DNA synthesis only when added prior to mitogen stimulation. IFN, added 6 h after exposure of quiescent cells to ITS, failed to inhibit cell growth. Addition of increasing concentrations of ITS failed to overcome the IFN-induced growth inhibition. These results suggest IFN may inhibit cell growth in part by antagonizing the action of growth factors. Images Figure 4 PMID:3166905

  15. Targeting Notch1 inhibits invasion and angiogenesis of human breast cancer cells via inhibition Nuclear Factor-κB signaling

    PubMed Central

    Liu, Yuan; Su, Chuanfu; Shan, Yuqing; Yang, Shouxiang; Ma, Guifeng

    2016-01-01

    Notch-1, a type-1 transmembrane protein, plays critical roles in the pathogenesis and progression of human malignancies, including breast cancer; however, the precise mechanism by which Notch-1 causes tumor cell invasion and angiogenesis remain unclear. Nuclear factor-κB (NF-κB), interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMP) are critically involved in the processes of tumor cell invasion and metastasis, we investigated whether targeting Notch-1 could be mechanistically associated with the down-regulation of NF-κB, IL-8, VEGF, and MMP-9, resulting in the inhibition of invasion and angiogenesis of breast cancer cells. Our data showed that down-regulation of Notch-1 leads to the inactivation of NF-κB activity and inhibits the expression of its target genes, such as IL-8, VEGF and MMP-9. We also found that down-regulation of Notch-1 decreased cell invasion, and vice versa Consistent with these results, we also found that the down-regulation of Notch-1 not only decreased MMP-9 mRNA and its protein expression but also inhibited MMP-9 active form. Moreover, conditioned medium from Notch-1 siRNA-transfected breast cancer cells showed reduced levels of IL-8 and VEGF and, in turn, inhibited the tube formation of HUVECs, suggesting that down-regulation of Notch-1 leads to the inhibition of angiogenesis. Furthermore, conditioned medium from Notch-1 cDNA-transfected breast cancer cells showed increased levels of IL-8 and VEGF and, in turn, promoted the tube formation of HUVECs, suggesting that Notch-1 overexpression leads to the promotion of angiogenesis.We therefore concluded that down-regulation of Notch-1 leads to the inactivation NF-κB and its target genes (IL-8, MMP-9 and VEGF), resulting in the inhibition of invasion and angiogenesis. PMID:27398151

  16. Selective serotonin reuptake inhibitors and theophylline metabolism in human liver microsomes: potent inhibition by fluvoxamine.

    PubMed Central

    Rasmussen, B B; Maënpää, J; Pelkonen, O; Loft, S; Poulsen, H E; Lykkesfeldt, J; Brøsen, K

    1995-01-01

    1. Fluvoxamine and seven other selective serotonin reuptake inhibitors (SRRI) were tested for their ability to inhibit a number of human cytochrome P450 isoforms (CYPs). 2. None of the drugs showed potent inhibition of CYP2A6 (coumarin 7-hydroxylase) or CYP2E1 (chlorzoxazone 6-hydroxylase), while norfluoxetine was the only potent inhibitor of CYP3A having IC50 values of 11 microM and 19 microM for testosterone 6 beta-hydroxylase and cortisol 6 beta-hydroxylase, respectively. 3. Norfluoxetine, sertraline and fluvoxamine inhibited CYP1A1 (7-ethoxyresorufin O-deethylase) in microsomes from human placenta (IC50 values 29 microM, 35 microM and 80 microM, respectively). Fluvoxamine was a potent inhibitor of CYP1A2-mediated 7-ethoxyresorufin O-deethylase activity (IC50 = 0.3 microM) in human liver. 4. In microsomes from three human livers fluvoxamine potently inhibited all pathways of theophylline biotransformation, the apparent inhibitor constant, Ki, was 0.07-0.13 microM, 0.05-0.10 microM and 0.16-0.29 microM for inhibition of 1-methylxanthine, 3-methylxanthine and 1,3-dimethyluric acid formation, respectively. Seven other SSRIs showed either weak or no inhibition of theophylline metabolism. 5. Ethanol inhibited the formation of 1,3-dimethyluric acid with K(i) value of 300 microM, a value which is consistent with inhibition of CYP2E1. Ethanol and fluvoxamine both inhibited 8-hydroxylation by about 45% and, in combination, the compounds decreased the formation of 1,3-dimethyluric acid by 90%, indicating that CYP1A2 and CYP2E1 are equally important isoforms for the 8-hydroxylation of theophylline. 6. It is concluded that pharmacokinetic interaction between fluvoxamine and theophylline is due to potent inhibition of CYP1A2. PMID:7742153

  17. Actein Inhibits Cell Proliferation and Migration in Human Osteosarcoma

    PubMed Central

    Chen, Zhi; Wu, Jingdong; Guo, Qinghao

    2016-01-01

    Background Osteosarcoma is one of the most common malignant bone cancers worldwide. Although the traditional chemotherapies have made some progression in the past decades, the mortality of osteosarcoma in children and adolescent is very high. Herein, the role of actein in osteosarcoma was explored. Material/Methods Cell viability assay was performed in osteosarcoma cell lines 143B and U2OS. Colony formation analysis was included when cells were treated with different doses of actin. Cell cycle assay was conducted to further examine the role of actein. Cell apoptotic rate and the relative activities of caspase-3, caspase-8, and caspase-9 were detected in 143B and U2OS osteosarcoma cells. Moreover, transwell assays were used to explore the effects of actein on cell metastasis. Results Actein significantly inhibited osteosarcoma cell viability in a time- and dose-dependent manner. Actein also dramatically suppressed the colony formation ability in osteosarcoma143B and U2OS cells. It was revealed that osteosarcoma cells were arrested in G0/G1 phase in the cell cycle progression and induced to apoptosis by administration of actein. The activities of pro-apoptotic factors such as caspase-3 and caspase-9 were significantly increased by actein. Furthermore, administration of actein decreased cell migrated and invasive abilities in both 143B and U2OS cell lines. Conclusions Actein inhibits tumor growth by inducing cell apoptosis in osteosarcoma. The inhibitive roles of actein in cell proliferation, migration and invasion suggest that actein may serve as a potential therapeutic agent in the treatment of osteosarcoma. PMID:27173526

  18. Bovine Lactoferrampin, Human Lactoferricin, and Lactoferrin 1-11 Inhibit Nuclear Translocation of HIV Integrase.

    PubMed

    Wang, Winston Yan; Wong, Jack Ho; Ip, Denis Tsz Ming; Wan, David Chi Cheong; Cheung, Randy Chifai; Ng, Tzi Bun

    2016-08-01

    This study aimed to investigate fragments derived from human and bovine lactoferrins for ability to inhibit nuclear translocation of HIV-1 integrase. It was shown that human lactoferricin, human lactoferrin 1-11, and bovine lactoferrampin reduced nuclear distribution of HIV-1 integrase. Bovine lactoferrampin could inhibit both the activity and nuclear translocation of HIV-1 integrase. Human lactoferrampin, bovine lactoferricin, and bovine lactoferrin 1-11 had no effect on HIV-1 integrase nuclear translocation. Human lactoferrampin which inhibited the activity of integrase did not prevent its nuclear translocation. Human lactoferricin and lactoferrin 1-11 did not inhibit HIV-1 integrase nuclear translocation despite their ability to attenuate the enzyme activity. The discrepancy between the findings on reduction of HIV-1 activity and inhibition of nuclear translocation of HIV-1 integrase was due to the different mechanisms involved. A similar reasoning can also be applied to the different inhibitory potencies of the milk peptides on different HIV enzymes, i.e., nuclear translocation. PMID:27022750

  19. DIETARY ISOTHIOCYANATE IBERIN INHIBITS GROWTH AND INDUCES APOPTOSIS IN HUMAN GLIOBLASTOMA CELLS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this study, we evaluated the antiproliferative and proapoptotic effects of the isothiocyanate iberin, a bioactive agent in Brassicaceae species, in human glioblastoma cells. The human glioblastoma cell cultures were treated with different concentrations of iberin and tested for growth inhibition...

  20. Inhibition of phospholipase A/sub 2/ from human plasma by sodium bisulfite

    SciTech Connect

    Wiggins, C.W.; Franson, R.C.

    1987-05-01

    The anti-oxidant sodium bisulfite has been shown to inhibit acid active(lysosomal), non-Ca/sup + +/-dependent phospholipase A/sub 2/ (PLA/sub 2/), and to interact reversibly with unsaturated fatty acids, altering their chromatographic mobility. The authors examined the effect of bisulfite on neutral active, Ca/sup + +/-dependent PLA/sub 2/ from human plasma. Using (1-/sup 14/C)oleate-labelled autoclaved E. coli as substrate, PLA/sub 2/ activity was inhibited in a dose-dependent manner by bisulfite. Maximal inhibition occurred at 100..mu..M bisulfite. Preincubation of plasma for 0-30 minutes with bisulfite resulted in a time-dependent increase in PLA/sub 2/ inhibition. Preincubation of substrate with bisulfite had no such effect. When the plasma PLA/sub 2/ was purified 25-fold by SP-Sephadex chromatography it was no longer inhibited by bisulfite. The SP-Sephadex wash through fraction, which contained greater than 95% of the applied protein but not PLA/sub 2/ activity, did not inhibit the purified enzyme. When incubated with bisulfite however, the SP-wash through fraction produced dose-dependent inhibition of the purified enzyme. These results indicate that sodium bisulfite inhibits human plasma PLA/sub 2/, in vitro, indirectly by interaction with a factor(s) present in plasma and suggests that anti-oxidants may similarly influence expression of extracellular PLA/sub 2/ in vivo.

  1. Curcumin Inhibits Rift Valley Fever Virus Replication in Human Cells*

    PubMed Central

    Narayanan, Aarthi; Kehn-Hall, Kylene; Senina, Svetlana; Lundberg, Lindsay; Van Duyne, Rachel; Guendel, Irene; Das, Ravi; Baer, Alan; Bethel, Laura; Turell, Michael; Hartman, Amy Lynn; Das, Bhaskar; Bailey, Charles; Kashanchi, Fatah

    2012-01-01

    Rift Valley fever virus (RVFV) is an arbovirus that is classified as a select agent, an emerging infectious virus, and an agricultural pathogen. Understanding RVFV-host interactions is imperative to the design of novel therapeutics. Here, we report that an infection by the MP-12 strain of RVFV induces phosphorylation of the p65 component of the NFκB cascade. We demonstrate that phosphorylation of p65 (serine 536) involves phosphorylation of IκBα and occurs through the classical NFκB cascade. A unique, low molecular weight complex of the IKK-β subunit can be observed in MP-12-infected cells, which we have labeled IKK-β2. The IKK-β2 complex retains kinase activity and phosphorylates an IκBα substrate. Inhibition of the IKK complex using inhibitors impairs viral replication, thus alluding to the requirement of an active IKK complex to the viral life cycle. Curcumin strongly down-regulates levels of extracellular infectious virus. Our data demonstrated that curcumin binds to and inhibits kinase activity of the IKK-β2 complex in infected cells. Curcumin partially exerts its inhibitory influence on RVFV replication by interfering with IKK-β2-mediated phosphorylation of the viral protein NSs and by altering the cell cycle of treated cells. Curcumin also demonstrated efficacy against ZH501, the fully virulent version of RVFV. Curcumin treatment down-regulated viral replication in the liver of infected animals. Our data point to the possibility that RVFV infection may result in the generation of novel versions of host components (such as IKK-β2) that, by virtue of altered protein interaction and function, qualify as unique therapeutic targets. PMID:22847000

  2. Antisense inhibition of hyaluronan synthase-2 in human osteosarcoma cells inhibits hyaluronan retention and tumorigenicity

    SciTech Connect

    Nishida, Yoshihiro . E-mail: ynishida@med.nagoya-u.ac.jp; Knudson, Warren; Knudson, Cheryl B.; Ishiguro, Naoki

    2005-07-01

    Osteosarcoma is a common malignant bone tumor associated with childhood and adolescence. The results of numerous studies have suggested that hyaluronan plays an important role in regulating the aggressive behavior of various types of cancer cells. However, no studies have addressed hyaluronan with respect to osteosarcomas. In this investigation, the mRNA expression copy number of three mammalian hyaluronan synthases (HAS) was determined using competitive RT-PCR in the osteoblastic osteosarcoma cell line, MG-63. MG-63 are highly malignant osteosarcoma cells with an abundant hyaluronan-rich matrix. The results demonstrated that HAS-2 is the predominant HAS in MG-63. Accumulation of intracellular hyaluronan increased in association with the proliferative phase of these cells. The selective inhibition of HAS-2 mRNA in MG-63 cells by antisense phosphorothioate oligonucleotides resulted in reduced hyaluronan accumulation by these cells. As expected, the reduction in hyaluronan disrupted the assembly of cell-associated matrices. However, of most interest, coincident with the reduction in hyaluronan, there was a substantial decrease in cell proliferation, a decrease in cell motility and a decrease in cell invasiveness. These data suggest that hyaluronan synthesized by HAS-2 in MG-63 plays a crucial role in osteosarcoma cell proliferation, motility, and invasion.

  3. Crystal structure of the thioesterase domain of human fatty acid synthase inhibited by orlistat

    SciTech Connect

    Pemble,C.; Johnson, L.; Kridel, S.; Lowther, W.

    2007-01-01

    Human fatty acid synthase (FAS) is uniquely expressed at high levels in many tumor types. Pharmacological inhibition of FAS therefore represents an important therapeutic opportunity. The drug Orlistat, which has been approved by the US Food and Drug Administration, inhibits FAS, induces tumor cell-specific apoptosis and inhibits the growth of prostate tumor xenografts. We determined the 2.3-{angstrom}-resolution crystal structure of the thioesterase domain of FAS inhibited by Orlistat. Orlistat was captured in the active sites of two thioesterase molecules as a stable acyl-enzyme intermediate and as the hydrolyzed product. The details of these interactions reveal the molecular basis for inhibition and suggest a mechanism for acyl-chain length discrimination during the FAS catalytic cycle. Our findings provide a foundation for the development of new cancer drugs that target FAS.

  4. Coordinate inhibition of expression of several genes for protein subunits of human nuclear RNase P

    PubMed Central

    Kovrigina, Elizaveta; Wesolowski, Donna; Altman, Sidney

    2003-01-01

    The deliberate inhibition of expression of one of the protein subunits (Rpp38) of human nuclear RNase P is achievable by using external guide sequence (EGS) technology. Both the protein product and the mRNA are greatly reduced 24 h after transient transfection with a gene coding for an appropriate EGS. Control experiments indicated that four other protein subunits of RNase P and their RNAs are also inhibited with no external manipulation. The remaining RNase P proteins, their mRNAs, and the RNA subunit of RNase P all are unchanged. Several short nucleotide sequences adjacent to the ORFs for the inhibited genes are similar and could be targets for transcriptional repression. The explanation of coordinate inhibition of the expression of the product of one particular gene by the transfection of an EGS (or RNA interference) requires some care in terms of interpreting phenotypic effects because, in our case, several gene products that are not targeted are also inhibited. PMID:12552092

  5. Vasoactive Intestinal Peptide Inhibits Human Small-Cell Lung Cancer Proliferation in vitro and in vivo

    NASA Astrophysics Data System (ADS)

    Maruno, Kaname; Absood, Afaf; Said, Sami I.

    1998-11-01

    Small-cell lung carcinoma (SCLC) is an aggressive, rapidly growing and metastasizing, and highly fatal neoplasm. We report that vasoactive intestinal peptide inhibits the proliferation of SCLC cells in culture and dramatically suppresses the growth of SCLC tumor-cell implants in athymic nude mice. In both cases, the inhibition was mediated apparently by a cAMP-dependent mechanism, because the inhibition was enhanced by the adenylate cyclase activator forskolin and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine in proportion to increases in intracellular cAMP levels, and the inhibition was abolished by selective inhibition of cAMP-dependent protein kinase. If confirmed in clinical trials, this antiproliferative action of vasoactive intestinal peptide may offer a new and promising means of suppressing SCLC in human subjects, without the toxic side effects of chemotherapeutic agents.

  6. Inhibition of human pathogenic fungi by ethnobotanically selected plant extracts.

    PubMed

    Ficker, Christine E; Arnason, J T; Vindas, P S; Alvarez, L P; Akpagana, K; Gbéassor, M; De Souza, C; Smith, M L

    2003-02-01

    In this study, 36 extracts derived from 29 plant species selected using an ethnobotanical approach were tested for antifungal activity against a taxonomically diverse group of 13 human pathogenic fungi. We compared the inhibitory characteristics of these plant extracts with those of the commonly used antifungals, amphotericin B and ketoconazole, and the plant-derived antifungal, berberine. Several plant extracts, notably those from Zingiber officinale (ginger) and Juglans cinerea (butternut), had pronounced antifungal activity against a wide variety of fungi, including strains that were highly resistant to amphotericin B and ketoconazole. Further exploration of Z. officinale as an antifungal is warranted as this species is generally regarded as safe for human consumption. PMID:12588480

  7. The E5 oncoprotein of human papillomavirus type 16 inhibits the acidification of endosomes in human keratinocytes.

    PubMed Central

    Straight, S W; Herman, B; McCance, D J

    1995-01-01

    The human papillomavirus type 16 E5 oncoprotein possesses mitogenic activity that acts synergistically with epidermal growth factor (EGF) in human keratinocytes and inhibits the degradation of the EGF receptor in endosomal compartments after ligand-stimulated endocytosis. One potential explanation for these observations is that E5 inhibits the acidification of endosomes. This may be mediated through the 16-kDa component of the vacuolar proton-ATPase, since animal and human papillomavirus E5 proteins bind this subunit protein. Using a ratio-imaging technique to determine endosomal pH, we found that the acidification of endosomes in E5-expressing keratinocytes was delayed at least fourfold compared with normal human keratinocytes and endosomes in some cells never completely acidified. Furthermore, E5 expression increased the resistance of keratinocytes to protein synthesis inhibition by diphtheria toxin, a process dependent on efficient endosomal acidification. Finally, artificially inhibiting endosomal acidification with chloroquine during the endocytosis of EGF receptors in keratinocytes demonstrated many of the same effects as the expression of human papillomavirus type 16 E5, including prolonged retention of undegraded EGF receptors in intracellular vesicles. PMID:7707548

  8. Inhibition of Prevotella and Capnocytophaga immunoglobulin A1 proteases by human serum.

    PubMed

    Frandsen, E V; Kjeldsen, M; Kilian, M

    1997-07-01

    Oral Prevotella and Capnocytophaga species, regularly isolated from periodontal pockets and associated with extraoral infections, secret specific immunoglobulin A1 (IgA1) proteases cleaving human IgA1 in the hinge region into intact Fab and Fc fragments. To investigate whether these enzymes are subject to inhibition in vivo in humans, we tested 34 sera from periodontally diseased and healthy individuals in an enzyme-linked immunosorbent assay for the presence and titers of inhibition of seven Prevotella and Capnocytophaga proteases. All or nearly all of the sera inhibited the IgA1 protease activity of Prevotella buccae, Prevotella oris, and Prevotella loescheii. A minor proportion of the sera inhibited Prevotella buccalis, Prevotella denticola, and Prevotella melaninogenica IgA1 proteases, while no sera inhibited Capnocytophaga ochracea IgA1 protease. All inhibition titers were low, ranging from 5 to 55, with titer being defined as the reciprocal of the dilution of serum causing 50% inhibition of one defined unit of protease activity. No correlation between periodontal disease status and the presence, absence, or titer of inhibition was observed. The nature of the low titers of inhibition in all sera of the IgA1 proteases of P. buccae, P. oris, and P. loescheii was further examined. In size exclusion chromatography, inhibitory activity corresponded to the peak volume of IgA. Additional inhibition of the P. oris IgA1 protease was found in fractions containing both IgA and IgG. Purification of the IgG fractions of five sera by passage of the sera on a protein G column resulted in recovery of inhibitory IgG antibodies against all three IgA1 proteases, with the highest titer being for the P. oris enzyme. These finding indicate that inhibitory activity is associated with enzyme-neutralizing antibodies. PMID:9220164

  9. Peptides in common bean fractions inhibit human colorectal cancer cells.

    PubMed

    Luna Vital, Diego A; González de Mejía, Elvira; Dia, Vermont P; Loarca-Piña, Guadalupe

    2014-08-15

    The aim of this study was to characterize peptides present in common bean non-digestible fractions (NDF) produced after enzymatic digestion and determine their antiproliferative action on human colorectal cancer cells. Five NDF peptides represented 70% of total protein (GLTSK, LSGNK, GEGSGA, MPACGSS and MTEEY) with antiproliferative activity on human colon cancer cells. Based on the antiproliferative effect, HCT116 cell line was most sensitive to bean Azufrado Higuera (IC50=0.53 mg/ml) and RKO to Bayo Madero (IC50=0.51 mg/ml) peptide extracts. Both cultivars increased significantly (p<0.05) the expression of p53 in HCT116 by 76% and 68%, respectively. Azufrado Higuera modified the expression of cell cycle regulation proteins p21 and cyclin B1. Bayo Madero modified the expression of mitochondrial activated apoptotic proteins BAD, cytC, c-casp3, Survivin, BIRC7. Results suggest that peptides present in common bean NDF contributed to the antiproliferative effect on human colorectal cancer cells by modifying molecules involved in either cell cycle arrest or apoptosis. PMID:24679790

  10. Structural Basis of Human CYP51 Inhibition by Antifungal Azoles

    SciTech Connect

    Strushkevich, Natallia; Usanov, Sergey A.; Park, Hee-Won

    2010-09-22

    The obligatory step in sterol biosynthesis in eukaryotes is demethylation of sterol precursors at the C14-position, which is catalyzed by CYP51 (sterol 14-alpha demethylase) in three sequential reactions. In mammals, the final product of the pathway is cholesterol, while important intermediates, meiosis-activating sterols, are produced by CYP51. Three crystal structures of human CYP51, ligand-free and complexed with antifungal drugs ketoconazole and econazole, were determined, allowing analysis of the molecular basis for functional conservation within the CYP51 family. Azole binding occurs mostly through hydrophobic interactions with conservative residues of the active site. The substantial conformational changes in the B{prime} helix and F-G loop regions are induced upon ligand binding, consistent with the membrane nature of the protein and its substrate. The access channel is typical for mammalian sterol-metabolizing P450 enzymes, but is different from that observed in Mycobacterium tuberculosis CYP51. Comparison of the azole-bound structures provides insight into the relative binding affinities of human and bacterial P450 enzymes to ketoconazole and fluconazole, which can be useful for the rational design of antifungal compounds and specific modulators of human CYP51.

  11. Mechanism of selective inhibition of human immunodeficiency virus by ingenol triacetate.

    PubMed Central

    Fujiwara, M; Ijichi, K; Tokuhisa, K; Katsuura, K; Shigeta, S; Konno, K; Wang, G Y; Uemura, D; Yokota, T; Baba, M

    1996-01-01

    Ingenol 3,5,20-triacetate (ITA), one of the ingenol derivatives, is a selective inhibitor of human immunodeficiency virus (HIV) replication in vitro. ITA inhibited the replication of HIV strains in MT-4 cells at concentrations of 0.051 to 0.65 microM. This concentration was approximately 10(3)-fold lower than its cytotoxic threshold. The mechanism of action of ITA is primarily attributed to the inhibition of viral adsorption to the host cells, but it is distinct from the mechanism of inhibition by other adsorption inhibitors. PMID:8787923

  12. Potent inhibition by star fruit of human cytochrome P450 3A (CYP3A) activity.

    PubMed

    Hidaka, Muneaki; Fujita, Ken-ichi; Ogikubo, Tetsuya; Yamasaki, Keishi; Iwakiri, Tomomi; Okumura, Manabu; Kodama, Hirofumi; Arimori, Kazuhiko

    2004-06-01

    There has been very limited information on the capacities of tropical fruits to inhibit human cytochrome P450 3A (CYP3A) activity. Thus, the inhibitory effects of tropical fruits on midazolam 1'-hydroxylase activity of CYP3A in human liver microsomes were evaluated. Eight tropical fruits such as common papaw, dragon fruit, kiwi fruit, mango, passion fruit, pomegranate, rambutan, and star fruit were tested. We also examined the inhibition of CYP3A activity by grapefruit (white) and Valencia orange as controls. The juice of star fruit showed the most potent inhibition of CYP3A. The addition of a star fruit juice (5.0%, v/v) resulted in the almost complete inhibition of midazolam 1'-hydroxylase activity (residual activity of 0.1%). In the case of grape-fruit, the residual activity was 14.7%. The inhibition depended on the amount of fruit juice added to the incubation mixture (0.2-6.0%, v/v). The elongation of the preincubation period of a juice from star fruit (1.25 or 2.5%, v/v) with the microsomal fraction did not alter the CYP3A inhibition, suggesting that the star fruit did not contain a mechanism-based inhibitor. Thus, we discovered filtered extracts of star fruit juice to be inhibitors of human CYP3A activity in vitro. PMID:15155547

  13. Inhibition by sildenafil of contractility of isolated non-pregnant human myometrium

    PubMed Central

    Winston, Aruldhas Blessed; Vazhudhi, Kaysina; Sen, Sumalya; Thomas, Elsy; Benjamin, Santhosh; Peedicayil, Jacob

    2015-01-01

    Objective: To investigate the ability of sildenafil to inhibit the contractility of isolated non pregnant human myometrium. Materials and Methods: The inhibitory effect of three concentrations (3, 10, and 30 µM) of sildenafil on 55 mM KCl-induced contractility of isolated non-pregnant human myometrium was studied. The ability of the guanylyl cyclase inhibitor ODQ (10 µM), the adenylyl cyclase inhibitor MDL-12,330A (10 µM), the non-specific potassium channel blocker TEA (2 mM), and the calcium-sensitive potassium (BKCa) channel blocker iberiotoxin (100 nM) to reverse the inhibition of 10 µM sildenafil on KCl-induced myometrial contractility was also studied. Results: Sildenafil produced a concentration-dependent inhibition of KCl-induced myometrial contractility that was statistically significant at all three concentrations of sildenafil used. The inhibition by 10 µM sildenafil of KCl-induced myometrial contractility was not reversed by the concurrent administration of ODQ or MDL-12,330A. The inhibition of 10 µM sildenafil of myometrial contractility was partially reversed by concurrent administration of TEA and totally and significantly reversed by the concurrent administration of iberiotoxin. Conclusions: These results suggest that sildenafil inhibits the contractility of isolated non-pregnant human myometrium. The results suggest that sildenafil does so by opening BKCa channels. PMID:26311996

  14. Human Medial Frontal Cortex Mediates Unconscious Inhibition of Voluntary Action

    PubMed Central

    Sumner, Petroc; Nachev, Parashkev; Morris, Peter; Peters, Andrew M.; Jackson, Stephen R.; Kennard, Christopher; Husain, Masud

    2007-01-01

    Summary Within the medial frontal cortex, the supplementary eye field (SEF), supplementary motor area (SMA), and pre-SMA have been implicated in the control of voluntary action, especially during motor sequences or tasks involving rapid choices between competing response plans. However, the precise roles of these areas remain controversial. Here, we study two extremely rare patients with microlesions of the SEF and SMA to demonstrate that these areas are critically involved in unconscious and involuntary motor control. We employed masked-prime stimuli that evoked automatic inhibition in healthy people and control patients with lateral premotor or pre-SMA damage. In contrast, our SEF/SMA patients showed a complete reversal of the normal inhibitory effect—ocular or manual—corresponding to the functional subregion lesioned. These findings imply that the SEF and SMA mediate automatic effector-specific suppression of motor plans. This automatic mechanism may contribute to the participation of these areas in the voluntary control of action. PMID:17553420

  15. Chlorogenic Acid Inhibits Human Platelet Activation and Thrombus Formation

    PubMed Central

    Fuentes, Eduardo; Caballero, Julio; Alarcón, Marcelo; Rojas, Armando; Palomo, Iván

    2014-01-01

    Background Chlorogenic acid is a potent phenolic antioxidant. However, its effect on platelet aggregation, a critical factor in arterial thrombosis, remains unclear. Consequently, chlorogenic acid-action mechanisms in preventing platelet activation and thrombus formation were examined. Methods and Results Chlorogenic acid in a dose-dependent manner (0.1 to 1 mmol/L) inhibited platelet secretion and aggregation induced by ADP, collagen, arachidonic acid and TRAP-6, and diminished platelet firm adhesion/aggregation and platelet-leukocyte interactions under flow conditions. At these concentrations chlorogenic acid significantly decreased platelet inflammatory mediators (sP-selectin, sCD40L, CCL5 and IL-1β) and increased intraplatelet cAMP levels/PKA activation. Interestingly, SQ22536 (an adenylate cyclase inhibitor) and ZM241385 (a potent A2A receptor antagonist) attenuated the antiplatelet effect of chlorogenic acid. Chlorogenic acid is compatible to the active site of the adenosine A2A receptor as revealed through molecular modeling. In addition, chlorogenic acid had a significantly lower effect on mouse bleeding time when compared to the same dose of aspirin. Conclusions Antiplatelet and antithrombotic effects of chlorogenic acid are associated with the A2A receptor/adenylate cyclase/cAMP/PKA signaling pathway. PMID:24598787

  16. SKI knockdown inhibits human melanoma tumor growth in vivo.

    PubMed

    Chen, Dahu; Lin, Qiushi; Box, Neil; Roop, Dennis; Ishii, Shunsuke; Matsuzaki, Koichi; Fan, Tao; Hornyak, Thomas J; Reed, Jon A; Stavnezer, Ed; Timchenko, Nikolai A; Medrano, Estela E

    2009-12-01

    The SKI protein represses the TGF-beta tumor suppressor pathway by associating with the Smad transcription factors. SKI is upregulated in human malignant melanoma tumors in a disease-progression manner and its overexpression promotes proliferation and migration of melanoma cells in vitro. The mechanisms by which SKI antagonizes TGF-beta signaling in vivo have not been fully elucidated. Here we show that human melanoma cells in which endogenous SKI expression was knocked down by RNAi produced minimal orthotopic tumor xenograft nodules that displayed low mitotic rate and prominent apoptosis. These minute tumors exhibited critical signatures of active TGF-beta signaling including high levels of nuclear Smad3 and p21(Waf-1), which are not found in the parental melanomas. To understand how SKI promotes tumor growth we used gain- and loss-of-function approaches and found that simultaneously to blocking the TGF-beta-growth inhibitory pathway, SKI promotes the switch of Smad3 from tumor suppression to oncogenesis by favoring phosphorylations of the Smad3 linker region in melanoma cells but not in normal human melanocytes. In this context, SKI is required for preventing TGF-beta-mediated downregulation of the oncogenic protein c-MYC, and for inducing the plasminogen activator inhibitor-1, a mediator of tumor growth and angiogenesis. Together, the results indicate that SKI exploits multiple regulatory levels of the TGF-beta pathway and its deficiency restores TGF-beta tumor suppressor and apoptotic activities in spite of the likely presence of oncogenic mutations in melanoma tumors. PMID:19845874

  17. Leptin inhibits steroid biosynthesis by human granulosa-lutein cells.

    PubMed

    Ghizzoni, L; Barreca, A; Mastorakos, G; Furlini, M; Vottero, A; Ferrari, B; Chrousos, G P; Bernasconi, S

    2001-06-01

    Absence of leptin secretion compromises reproductive function and fertility in the ob/ob mouse which, when given leptin, shows a rise in serum LH levels and becomes fertile. Recently, the long and active isoform of the leptin receptor was detected in the ovary, indicating that leptin may also show direct gonad-related activity. To examine this, we studied the effect of graded doses of human leptin on estradiol (E2) and progesterone (P4) concentrations in the culture media of human granulosa-lutein cells obtained from follicular fluid of women undergoing in vitro fertilization. We also evaluated the mRNA expression of steroidogenic acute regulatory protein (StAR), aromatase, and cytochrome P450 17alpha (CYP17) in these cells at baseline and after exposure to leptin. Estradiol levels were significantly decreased in the media 24 hours after incubation of the cells with increasing hLeptin concentrations (10(-11) - 10(-7) mol/l). The maximal 30% decrease in E2 production was caused by the 10(-9) mol/l hLeptin concentration; however, P4 levels in the media were not influenced by leptin. Exposure of granulosa-lutein cells to 10(-9) mol/l hLeptin did not produce any measurable changes on StAR, aromatase, or CYP17 mRNA expression. When hLeptin (10(-9) mol/l) was co-incubated with increasing concentrations of hCG (1.25 - 10 mlU/ml), IGF-II (15-60 ng/ml) or 1-6 desaminated IGF-II (deslGF-II; 15-60 ng/ml), it did not modify the elevation of E2 concentrations caused by each of the different stimuli. We conclude that leptin suppresses E2 secretion by human granulosa-lutein cells but does not impair the stimulatory effects of hCG and IGFs on these cells. Leptin may play a minor, but direct regulatory role on unstimulated human ovarian steroidogenesis by interfering with either the translational or post-translational steps of the baseline CYP17 and/or aromatase synthesis and/or the activation of the enzymes. PMID:11456279

  18. Group A streptococcal peptidoglycan-polysaccharide inhibits phagocytic activity of human polymorphonuclear leukocytes.

    PubMed Central

    Leong, P A; Cohen, M S

    1984-01-01

    Injection of sterile aqueous preparations of the peptidoglycan-polysaccharide of group A streptococci (PG-APS) produces chronic inflammation in several animal models. Chronic bacterial infection may be involved in some aspects of the pathogenesis of inflammation associated with the accumulation of PG-APS. Accordingly, the effect of PG-APS on human neutrophil (polymorphonuclear leukocyte [PMN]) bactericidal activity was studied with the supposition that this interaction may contribute to the inflammation observed. Concentrations of PG-APS greater than 10 micrograms/ml inhibited the ability of PMNs to kill Staphylococcus aureus. This inhibition was not due to a cytotoxic effect of PG-APS on PMNs, nor did PG-APS inhibit PMN metabolism required for the formation of microbicidal oxygen reduction products. PG-APS concentrations of 10 micrograms/ml or greater in the presence of 10% normal serum inhibited the attachment of bacteria to PMNs by 49% as compared with control cell populations. The concentrations of PG-APS required to inhibit uptake of Staphylococcus aureus were identical to those required for inhibition of PMN bactericidal activity. This inhibition did not occur in the presence of serum-free medium or medium with sera that had been heated to inactivate complement. These results show that PG-APS interacts with serum to inhibit PMN-mediated killing of S. aureus, most probably by interfering with bacterial uptake. PMID:6378796

  19. Suppression of autophagy augments the radiosensitizing effects of STAT3 inhibition on human glioma cells

    SciTech Connect

    Yuan, Xiaopeng; Du, Jie; Hua, Song; Zhang, Haowen; Gu, Cheng; Wang, Jie; Yang, Lei; Huang, Jianfeng; Yu, Jiahua Liu, Fenju

    2015-01-15

    Radiotherapy is an essential component of the standard therapy for newly diagnosed glioblastoma. To increase the radiosensitivity of glioma cells is a feasible solution to improve the therapeutic effects. It has been suggested that inhibition of signal transducer and activator of transcription 3 (STAT3) can radiosensitize glioma cells, probably via the activation of mitochondrial apoptotic pathway. In this study, human malignant glioma cells, U251 and A172, were treated with an STAT3 inhibitor, WP1066, or a short hairpin RNA plasmid targeting STAT3 to suppress the activation of STAT3 signaling. The radiosensitizing effects of STAT3 inhibition were confirmed in glioma cells. Intriguingly, combination of ionizing radiation exposure and STAT3 inhibition triggered a pronounced increase of autophagy flux. To explore the role of autophagy, glioma cells were treated with 3-methyladenine or siRNA for autophagy-related gene 5, and it was demonstrated that inhibition of autophagy further strengthened the radiosensitizing effects of STAT3 inhibition. Accordingly, more apoptotic cells were induced by the dual inhibition of autophagy and STAT3 signaling. In conclusion, our data revealed a protective role of autophagy in the radiosensitizing effects of STAT3 inhibition, and inhibition of both autophagy and STAT3 might be a potential therapeutic strategy to increase the radiosensitivity of glioma cells. - Highlights: • Inactivation of STAT3 signaling radiosensitizes malignant glioma cells. • STAT3 inhibition triggers a significant increase of autophagy flux induced by ionizing radiation in glioma cells. • Suppression of autophagy further strengthens the radiosensitizing effects of STAT3 inhibition in glioma cells. • Dual inhibition of autophagy and STAT3 induce massive apoptotic cells upon exposure to ionizing radiation.

  20. Inhibition of human arginase I by substrate and product analogues

    SciTech Connect

    Di Costanzo, Luigi; Ilies, Monica; Thorn, Katherine J.; Christianson, David W.

    2010-06-21

    Human arginase I is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of l-arginine to generate l-ornithine and urea. We demonstrate that N-hydroxy-l-arginine (NOHA) binds to this enzyme with K{sub d} = 3.6 {micro}M, and nor-N-hydroxy-l-arginine (nor-NOHA) binds with K{sub d} = 517 nM (surface plasmon resonance) or K{sub d} {approx} 50 nM (isothermal titration calorimetry). Crystals of human arginase I complexed with NOHA and nor-NOHA afford 2.04 and 1.55 {angstrom} resolution structures, respectively, which are significantly improved in comparison with previously-determined structures of the corresponding complexes with rat arginase I. Higher resolution structures clarify the binding interactions of the inhibitors. Finally, the crystal structure of the complex with l-lysine (K{sub d} = 13 {micro}M) is reported at 1.90 {angstrom} resolution. This structure confirms the importance of hydrogen bond interactions with inhibitor {alpha}-carboxylate and {alpha}-amino groups as key specificity determinants of amino acid recognition in the arginase active site.

  1. Humanized-VHH Transbodies that Inhibit HCV Protease and Replication

    PubMed Central

    Jittavisutthikul, Surasak; Thanongsaksrikul, Jeeraphong; Thueng-in, Kanyarat; Chulanetra, Monrat; Srimanote, Potjanee; Seesuay, Watee; Malik, Aijaz Ahmad; Chaicumpa, Wanpen

    2015-01-01

    There is a need for safe and broadly effective anti-HCV agents that can cope with genetic multiplicity and mutations of the virus. In this study, humanized-camel VHHs to genotype 3a HCV serine protease were produced and were linked molecularly to a cell penetrating peptide, penetratin (PEN). Human hepatic (Huh7) cells transfected with the JFH-1 RNA of HCV genotype 2a and treated with the cell penetrable nanobodies (transbodies) had a marked reduction of the HCV RNA intracellularly and in their culture fluids, less HCV foci inside the cells and less amounts of HCV core antigen in culture supernatants compared with the infected cells cultured in the medium alone. The PEN-VHH-treated-transfected cells also had up-regulation of the genes coding for the host innate immune response (TRIF, TRAF3, IRF3, IL-28B and IFN-β), indicating that the cell penetrable nanobodies rescued the host innate immune response from the HCV mediated-suppression. Computerized intermolecular docking revealed that the VHHs bound to residues of the protease catalytic triad, oxyanion loop and/or the NS3 N-terminal portion important for non-covalent binding of the NS4A protease cofactor protein. The so-produced transbodies have high potential for testing further as a candidate for safe, broadly effective and virus mutation tolerable anti-HCV agents. PMID:25903832

  2. The structure and inhibition of human diamine oxidase†,‡

    PubMed Central

    McGrath, Aaron P; Hilmer, Kimberly M; Collyer, Charles A; Shepard, Eric M; Elmore, Bradley O.; Brown, Doreen E; Dooley, David M; Guss, J Mitchell

    2009-01-01

    Humans have three functioning genes that code for copper-containing amine oxidases. The product of the AOC1 gene is a so-called diamine oxidase (hDAO), named for its substrate preference for diamines, particularly histamine. hDAO has been cloned and expressed in insect cells and the structure of the native enzyme determined by X-ray crystallography to a resolution of 1.8 Å. The homodimeric structure has the archetypal amine oxidase fold. Two active sites, one in each subunit, are characterized by the presence of a copper ion and a topaquinone residue formed by the post-translational modification of a tyrosine. Although hDAO shares 37.9 % sequence identity with another human copper amine oxidase, semicarbazide sensitive amine oxidase or vascular adhesion protein-1, its substrate binding pocket and entry channel are distinctly different in accord with the different substrate specificities. The structures of two inhibitor complexes of hDAO, berenil and pentamidine, have been refined to resolutions of 2.1 Å and 2.2 Å, respectively. They bind non-covalently in the active site channel. The inhibitor binding suggests that an aspartic acid residue, conserved in all diamine oxidases but absent from other amine oxidases, is responsible for the diamine specificity by interacting with the second amino group of preferred diamine substrates. PMID:19764817

  3. Humanized-VHH transbodies that inhibit HCV protease and replication.

    PubMed

    Jittavisutthikul, Surasak; Thanongsaksrikul, Jeeraphong; Thueng-In, Kanyarat; Chulanetra, Monrat; Srimanote, Potjanee; Seesuay, Watee; Malik, Aijaz Ahmad; Chaicumpa, Wanpen

    2015-04-01

    There is a need for safe and broadly effective anti-HCV agents that can cope with genetic multiplicity and mutations of the virus. In this study, humanized-camel VHHs to genotype 3a HCV serine protease were produced and were linked molecularly to a cell penetrating peptide, penetratin (PEN). Human hepatic (Huh7) cells transfected with the JFH-1 RNA of HCV genotype 2a and treated with the cell penetrable nanobodies (transbodies) had a marked reduction of the HCV RNA intracellularly and in their culture fluids, less HCV foci inside the cells and less amounts of HCV core antigen in culture supernatants compared with the infected cells cultured in the medium alone. The PEN-VHH-treated-transfected cells also had up-regulation of the genes coding for the host innate immune response (TRIF, TRAF3, IRF3, IL-28B and IFN-β), indicating that the cell penetrable nanobodies rescued the host innate immune response from the HCV mediated-suppression. Computerized intermolecular docking revealed that the VHHs bound to residues of the protease catalytic triad, oxyanion loop and/or the NS3 N-terminal portion important for non-covalent binding of the NS4A protease cofactor protein. The so-produced transbodies have high potential for testing further as a candidate for safe, broadly effective and virus mutation tolerable anti-HCV agents. PMID:25903832

  4. Inhibition of Human Arginase I by Substrate adn Product Analogues

    SciTech Connect

    L Di Costanzo; M Ilies; K Thorn; D Christianson

    2011-12-31

    Human arginase I is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of L-arginine to generate L-ornithine and urea. We demonstrate that N-hydroxy-L-arginine (NOHA) binds to this enzyme with K(d)=3.6 microM, and nor-N-hydroxy-L-arginine (nor-NOHA) binds with K(d)=517 nM (surface plasmon resonance) or K(d) approximately 50 nM (isothermal titration calorimetry). Crystals of human arginase I complexed with NOHA and nor-NOHA afford 2.04 and 1.55 A resolution structures, respectively, which are significantly improved in comparison with previously-determined structures of the corresponding complexes with rat arginase I. Higher resolution structures clarify the binding interactions of the inhibitors. Finally, the crystal structure of the complex with L-lysine (K(d)=13 microM) is reported at 1.90 A resolution. This structure confirms the importance of hydrogen bond interactions with inhibitor alpha-carboxylate and alpha-amino groups as key specificity determinants of amino acid recognition in the arginase active site.

  5. In vitro growth inhibition of human cancer cells by novel honokiol analogs.

    PubMed

    Lin, Jyh Ming; Prakasha Gowda, A S; Sharma, Arun K; Amin, Shantu

    2012-05-15

    Honokiol possesses many pharmacological activities including anti-cancer properties. Here in, we designed and synthesized honokiol analogs that block major honokiol metabolic pathway which may enhance their effectiveness. We studied their cytotoxicity in human cancer cells and evaluated possible mechanism of cell cycle arrest. Two analogs, namely 2 and 4, showed much higher growth inhibitory activity in A549 human lung cancer cells and significant increase of cell population in the G0-G1 phase. Further elucidation of the inhibition mechanism on cell cycle showed that analogs 2 and 4 inhibit both CDK1 and cyclin B1 protien levels in A549 cells. PMID:22533983

  6. Individual strains of Lactobacillus paracasei differentially inhibit human basophil and mouse mast cell activation

    PubMed Central

    Cassard, Lydie; Lalanne, Ana Inés; Garault, Peggy; Cotillard, Aurélie; Chervaux, Christian; Wels, Michiel; Smokvina, Tamara

    2016-01-01

    Abstract Introduction The microbiota controls a variety of biological functions, including immunity, and alterations of the microbiota in early life are associated with a higher risk of developing allergies later in life. Several probiotic bacteria, and particularly lactic acid bacteria, were described to reduce both the induction of allergic responses and allergic manifestations. Although specific probiotic strains were used in these studies, their protective effects on allergic responses also might be common for all lactobacilli. Methods To determine whether allergic effector cells inhibition is a common feature of lactobacilli or whether it varies among lactobacilli strains, we compared the ability of 40 strains of the same Lactobacillus paracasei species to inhibit IgE‐dependent mouse mast cell and human basophil activation. Results We uncovered a marked heterogeneity in the inhibitory properties of the 40 Lactobacillus strains tested. These segregated into three to four clusters depending on the intensity of inhibition. Some strains inhibited both mouse mast cell and human basophil activation, others strains inhibited only one cell type and another group induced no inhibition of activation for either cell type. Conclusions Individual Lactobacillus strains of the same species differentially inhibit IgE‐dependent activation of mouse mast cells and human basophils, two cell types that are critical in the onset of allergic manifestations. Although we failed to identify specific bacterial genes associated with inhibition by gene‐trait matching analysis, our findings demonstrate the complexity of the interactions between the microbiota and the host. These results suggest that some L. paracasei strains might be more beneficial in allergies than others strains and provide the bases for a rational screening of lactic acid bacteria strains as next‐generation probiotics in the field of allergy. PMID:27621812

  7. Use of Human Plasma Samples to Identify Circulating Drug Metabolites that Inhibit Cytochrome P450 Enzymes.

    PubMed

    Eng, Heather; Obach, R Scott

    2016-08-01

    Drug interactions elicited through inhibition of cytochrome P450 (P450) enzymes are important in pharmacotherapy. Recently, greater attention has been focused on not only parent drugs inhibiting P450 enzymes but also on possible inhibition of these enzymes by circulating metabolites. In this report, an ex vivo method whereby the potential for circulating metabolites to be inhibitors of P450 enzymes is described. To test this method, seven drugs and their known plasma metabolites were added to control human plasma at concentrations previously reported to occur in humans after administration of the parent drug. A volume of plasma for each drug based on the known inhibitory potency and time-averaged concentration of the parent drug was extracted and fractionated by high-pressure liquid chromatography-mass spectrometry, and the fractions were tested for inhibition of six human P450 enzyme activities (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4). Observation of inhibition in fractions that correspond to the retention times of metabolites indicates that the metabolite has the potential to contribute to P450 inhibition in vivo. Using this approach, norfluoxetine, hydroxyitraconazole, desmethyldiltiazem, desacetyldiltiazem, desethylamiodarone, hydroxybupropion, erythro-dihydrobupropion, and threo-dihydrobupropion were identified as circulating metabolites that inhibit P450 activities at a similar or greater extent as the parent drug. A decision tree is presented outlining how this method can be used to determine when a deeper investigation of the P450 inhibition properties of a drug metabolite is warranted. PMID:27271369

  8. Inhibition behavior of fructus psoraleae's ingredients towards human carboxylesterase 1 (hCES1).

    PubMed

    Sun, Dong-Xue; Ge, Guang-Bo; Dong, Pei-Pei; Cao, Yun-Feng; Fu, Zhi-Wei; Ran, Rui-Xue; Wu, Xue; Zhang, Yan-Yan; Hua, Hui-Ming; Zhao, Zhenying; Fang, Zhong-Ze

    2016-06-01

    1. Fructus psoraleae (FP) is the dried ripe seeds of Psoralea corylifolia L. (Fabaceae) widely used in Asia, and has been reported to exert important biochemical and pharmacological activities. The adverse effects of FP remain unclear. The present study aims to determine the inhibition of human carboxylesterase 1 (CES1) by FP's major ingredients, including neobavaisoflavone, corylifolinin, coryfolin, psoralidin, corylin and bavachinin. 2. The probe substrate of CES1 2-(2-benzoyl-3-methoxyphenyl) benzothiazole (BMBT) was derived from 2-(2-hydroxy-3-methoxyphenyl) benzothiazole (HMBT), and human liver microsomes (HLMs)-catalyzed BMBT metabolism was used to phenotype the activity of CES1. In silico docking method was employed to explain the inhibition mechanism. 3. All the tested compounds exerted strong inhibition towards the activity of CES1 in a concentration-dependent behavior. Furthermore, the inhibition kinetics was determined for the inhibition of neobavaisoflavone, corylifolinin, coryfolin, corylin and bavachinin towards CES1. Both Dixon and Lineweaver-Burk plots showed that neobavaisoflavone, corylifolinin, coryfolin and corylin noncompetitively inhibited the activity of CES1, and bavachinin competitively inhibited the activity of CES1. The inhibition kinetic parameters (Ki) were calculated to be 5.3, 9.4, 1.9, 0.7 and 0.5 μM for neobavaisoflavone, corylifolinin, coryfolin, corylin and bavachinin, respectively. In conclusion, the inhibition behavior of CES1 by the FP's constituents was given in this article, indicating the possible adverse effects of FP through the disrupting CES1-catalyzed metabolism of endogenous substances and xenobiotics. PMID:26560012

  9. Intracellular Adenosine Inhibits IgE-Dependent Degranulation of Human Skin Mast Cells

    PubMed Central

    Gomez, Gregorio; Nardone, Vincent; Lotfi-Emran, Sahar; Zhao, Wei; Schwartz, Lawrence B.

    2015-01-01

    Purpose Adenosine (ADO) can enhance and inhibit mast cell degranulation. Potentiation of degranulation occurs at relatively low concentrations of ADO (10−6–10−5 M) through triggering of A3AR, whereas, inhibition occurs at higher concentrations of ADO reportedly through triggering of A2aAR. However, the discrepancy in the concentration of ADO that inhibits degranulation and that required to trigger ADORs suggests a different mechanism. The purpose of this study is to determine the mechanism by which ADO inhibits human mast cell degranulation. Methods We compare the effectiveness of A2aAR specific antagonist ZM241385 and equilibrative nucleoside transporter inhibitors Dipyridamole and NBMPR in preventing ADO-mediated inhibition of FcεRI-induced degranulation of human skin mast cells (hSMCs). Western blotting is done to analyze the effect of ADO on FcεRI-induced Syk phosphorylation. Results Dipyridamole and NBMPR completely and dose-dependently prevented ADO from inhibiting FcεRI-induced degranulation in all hSMC preparations. In contrast, ZM241385 at 10−5 M was effective in only 3 of 10 hSMC preparations. Moreover, NBMPR was effective even in those hSMC preparations not responsive to ZM241385. ADO inhibited degranulation induced by FcεRI crosslinking, but not that induced by complement component 5a (C5a), Substance P or calcium ionophore. Accordingly, ADO significantly attenuated FcεRI-induced phosphorylation of Syk at the critical activating tyrosine (Y525). Conclusion Blocking the influx of ADO, but not A2aAR signals, is necessary and sufficient to prevent ADO from inhibiting FcεRI-induced mast cell degranulation. Thus, ADO specifically inhibits FcεRI-induced degranulation of hSMCs primarily by an intracellular mechanism that requires its influx via equilibrative nucleoside transporter 1 (ENT1). PMID:24122028

  10. Effect of local acetylcholinesterase inhibition on sweat rate in humans

    NASA Technical Reports Server (NTRS)

    Shibasaki, M.; Crandall, C. G.

    2001-01-01

    ACh is the neurotransmitter responsible for increasing sweat rate (SR) in humans. Because ACh is rapidly hydrolyzed by acetylcholinesterase (AChE), it is possible that AChE contributes to the modulation of SR. Thus the primary purpose of this project was to identify whether AChE around human sweat glands is capable of modulating SR during local application of various concentrations of ACh in vivo, as well as during a heat stress. In seven subjects, two microdialysis probes were placed in the intradermal space of the forearm. One probe was perfused with the AChE inhibitor neostigmine (10 microM); the adjacent membrane was perfused with the vehicle (Ringer solution). SR over both membranes was monitored via capacitance hygrometry during microdialysis administration of various concentrations of ACh (1 x 10(-7)-2 M) and during whole body heating. SR was significantly greater at the neostigmine-treated site than at the control site during administration of lower concentrations of ACh (1 x 10(-7)-1 x 10(-3) M, P < 0.05), but not during administration of higher concentrations of ACh (1 x 10(-2)-2 M, P > 0.05). Moreover, the core temperature threshold for the onset of sweating at the neostigmine-treated site was significantly reduced relative to that at the control site. However, no differences in SR were observed between sites after 35 min of whole body heating. These results suggest that AChE is capable of modulating SR when ACh concentrations are low to moderate (i.e., when sudomotor activity is low) but is less effective in governing SR after SR has increased substantially.

  11. Inhibition of human cytochrome P450 enzymes by the natural hepatotoxin safrole.

    PubMed

    Ueng, Yune-Fang; Hsieh, Chih-Hang; Don, Ming-Jaw

    2005-05-01

    The hepatotoxin, safrole is a methylenedioxy phenyl compound, found in sassafras oil and certain other essential oils. Recombinant cytochrome P450 (CYP, P450) and human liver microsomes were studied to investigate the selective inhibitory effects of safrole on human P450 enzymes and the mechanisms of action. Using Escherichia coli-expressed human P450, our results demonstrated that safrole was a non-selective inhibitor of CYP1A2, CYP2A6, CYP2D6, CYP2E1, and CYP3A4 in the IC(50) order CYP2E1 < CYP1A2 < CYP2A6 < CYP3A4 < CYP2D6. Safrole strongly inhibited CYP1A2, CYP2A6, and CYP2E1 activities with IC(50) values less than 20 microM. Safrole caused competitive, non-competitive, and non-competitive inhibition of CYP1A2, CYP2A6 and CYP2E1 activities, respectively. The inhibitor constants were in the order CYP1A2 < CYP2E1 < CYP2A6. In human liver microsomes, 50 microM safrole strongly inhibited 7-ethoxyresorufin O-deethylation, coumarin hydroxylation, and chlorzoxazone hydroxylation activities. These results revealed that safrole was a potent inhibitor of human CYP1A2, CYP2A6, and CYP2E1. With relatively less potency, CYP2D6 and CYP3A4 were also inhibited. PMID:15778010

  12. Replication of Many Human Viruses Is Refractory to Inhibition by Endogenous Cellular MicroRNAs

    PubMed Central

    Bogerd, Hal P.; Skalsky, Rebecca L.; Kennedy, Edward M.; Furuse, Yuki; Whisnant, Adam W.; Flores, Omar; Schultz, Kimberly L. W.; Putnam, Nicole; Barrows, Nicholas J.; Sherry, Barbara; Scholle, Frank; Garcia-Blanco, Mariano A.; Griffin, Diane E.

    2014-01-01

    ABSTRACT The issue of whether viruses are subject to restriction by endogenous microRNAs (miRNAs) and/or by virus-induced small interfering RNAs (siRNAs) in infected human somatic cells has been controversial. Here, we address this question in two ways. First, using deep sequencing, we demonstrate that infection of human cells by the RNA virus dengue virus (DENV) or West Nile virus (WNV) does not result in the production of any virus-derived siRNAs or viral miRNAs. Second, to more globally assess the potential of small regulatory RNAs to inhibit virus replication, we used gene editing to derive human cell lines that lack a functional Dicer enzyme and that therefore are unable to produce miRNAs or siRNAs. Infection of these cells with a wide range of viruses, including DENV, WNV, yellow fever virus, Sindbis virus, Venezuelan equine encephalitis virus, measles virus, influenza A virus, reovirus, vesicular stomatitis virus, human immunodeficiency virus type 1, or herpes simplex virus 1 (HSV-1), failed to reveal any enhancement in the replication of any of these viruses, although HSV-1, which encodes at least eight Dicer-dependent viral miRNAs, did replicate somewhat more slowly in the absence of Dicer. We conclude that most, and perhaps all, human viruses have evolved to be resistant to inhibition by endogenous human miRNAs during productive replication and that dependence on a cellular miRNA, as seen with hepatitis C virus, is rare. How viruses have evolved to avoid inhibition by endogenous cellular miRNAs, which are generally highly conserved during metazoan evolution, remains to be determined. IMPORTANCE Eukaryotic cells express a wide range of small regulatory RNAs, including miRNAs, that have the potential to inhibit the expression of mRNAs that show sequence complementarity. Indeed, previous work has suggested that endogenous miRNAs have the potential to inhibit viral gene expression and replication. Here, we demonstrate that the replication of a wide range of

  13. Inhibition of human ornithine decarboxylase activity by enantiomers of difluoromethylornithine.

    PubMed Central

    Qu, Ning; Ignatenko, Natalia A; Yamauchi, Phillip; Stringer, David E; Levenson, Corey; Shannon, Patrick; Perrin, Scott; Gerner, Eugene W

    2003-01-01

    Racemic difluoromethylornithine (D/L-DFMO) is an inhibitor of ODC (ornithine decarboxylase), the first enzyme in eukaryotic polyamine biosynthesis. D/L-DFMO is an effective anti-parasitic agent and inhibitor of mammalian cell growth and development. Purified human ODC-catalysed ornithine decarboxylation is highly stereospecific. However, both DFMO enantiomers suppressed ODC activity in a time- and concentration-dependent manner. ODC activity failed to recover after treatment with either L- or D-DFMO and dialysis to remove free inhibitor. The inhibitor dissociation constant (K(D)) values for the formation of enzyme-inhibitor complexes were 28.3+/-3.4, 1.3+/-0.3 and 2.2+/-0.4 microM respectively for D-, L- and D/L-DFMO. The differences in these K(D) values were statistically significant ( P <0.05). The inhibitor inactivation constants (K(inact)) for the irreversible step were 0.25+/-0.03, 0.15+/-0.03 and 0.15+/-0.03 min(-1) respectively for D-, L- and D/L-DFMO. These latter values were not statistically significantly different ( P >0.1). D-DFMO was a more potent inhibitor (IC50 approximately 7.5 microM) when compared with D-ornithine (IC50 approximately 1.5 mM) of ODC-catalysed L-ornithine decarboxylation. Treatment of human colon tumour-derived HCT116 cells with either L- or D-DFMO decreased the cellular polyamine contents in a concentration-dependent manner. These results show that both enantiomers of DFMO irreversibly inactivate ODC and suggest that this inactivation occurs by a common mechanism. Both enantiomers form enzyme-inhibitor complexes with ODC, but the probability of formation of these complexes is 20 times greater for L-DFMO when compared with D-DFMO. The rate of the irreversible reaction in ODC inactivation is similar for the L- and D-enantiomer. This unexpected similarity between DFMO enantiomers, in contrast with the high degree of stereospecificity of the substrate ornithine, appears to be due to the alpha-substituent of the inhibitor. The D

  14. Interactions between glutamate, dopamine, and the neuronal signature of response inhibition in the human striatum.

    PubMed

    Lorenz, Robert C; Gleich, Tobias; Buchert, Ralph; Schlagenhauf, Florian; Kühn, Simone; Gallinat, Jürgen

    2015-10-01

    Response inhibition is a basic mechanism in cognitive control and dysfunctional in major psychiatric disorders. The neuronal mechanisms are in part driven by dopamine in the striatum. Animal data suggest a regulatory role of glutamate on the level of the striatum. We used a trimodal imaging procedure of the human striatum including F18-DOPA positron emission tomography, proton magnetic resonance spectroscopy, and functional magnetic resonance imaging of a stop signal task. We investigated dopamine synthesis capacity and glutamate concentration in vivo and their relation to functional properties of response inhibition. A mediation analysis revealed a significant positive association between dopamine synthesis capacity and inhibition-related neural activity in the caudate nucleus. This relationship was significantly mediated by striatal glutamate concentration. Furthermore, stop signal reaction time was inversely related to striatal activity during inhibition. The data show, for the first time in humans, an interaction between dopamine, glutamate, and the neural signature of response inhibition in the striatum. This finding stresses the importance of the dopamine-glutamate interaction for behavior and may facilitate the understanding of psychiatric disorders characterized by impaired response inhibition. PMID:26177932

  15. Shikonin Inhibits Inflammatory Cytokine Production in Human Periodontal Ligament Cells.

    PubMed

    Shindo, Satoru; Hosokawa, Yoshitaka; Hosokawa, Ikuko; Ozaki, Kazumi; Matsuo, Takashi

    2016-06-01

    Shikonin, which is derived from Lithospermum erythrorhizon, a herb used in traditional medicine, has long been considered to be a useful treatment for various diseases in traditional oriental medicine. Shikonin has recently been reported to have several pharmacological properties, e.g., it has anti-microbial, anti-tumor, and anti-inflammatory effects. The aim of this study was to examine whether shikonin is able to influence the production of interleukin (IL)-6, IL-8, and/or chemokine C-C motif ligand (CCL)20, which contribute to the pathogenesis of periodontal disease, in human periodontal ligament cells (HPDLC). The production levels of IL-6, IL-8, and CCL20 in HPDLC were determined using an ELISA. Western blot analysis was used to detect nuclear factor kappa B (NF-κB) pathway activation in HPDLC. Shikonin prevented IL-1β- or tumor necrosis factor (TNF)-α-mediated IL-6, IL-8, and CCL20 production in HPDLC. Moreover, we found that shikonin suppressed the phosphorylation and degradation of inhibitor of kappa B-alpha (IκB-α) in IL-1β- or TNF-α-stimulated HPDLC. These findings suggest that shikonin could have direct beneficial effects against periodontal disease by reducing IL-6, IL-8, and CCL20 production in periodontal lesions. PMID:27072015

  16. Inhibition of plasma vasopressin after drinking in dehydrated humans

    NASA Technical Reports Server (NTRS)

    Geelen, G.; Keil, L. C.; Kravik, S. E.; Wade, C. E.; Thrasher, T. N.; Barnes, P. R.; Pyka, G.; Nesvig, C.; Greenleaf, J. E.

    1984-01-01

    The effects of nonosmotic and nonvolumetric factors on vasopressin secretion in dehydrated humans has been investigated experimentally, before and after drinking. The subjects of the experiment were five adult men and three adult women weighing 69-77 kg. In order to determine the influence of nonosmotic and nonvolumetric factors on vasopressin secretion, measurements were obtained of the following blood hematological indices: serum Na(+) content; serum K(+) content; osmolality; and hemoglobin. Measurements of hematocrit, plasma arginine vasopressin (AVP), aldosterone, and renin activity were also obtained. It is found that dehydration increased mean serum Na(+) content, osmolality,and AVP. No significant changes were observed in renin activity, hemoglobin, hematocrit, or plasma volume, while plasma aldosterone increased from 11.1 ng/dl after dehydration to 15.6 ng/dl between 30 and 60 min after drinking. A rapid fall of AVP content following rehydration occurred in the absence of changes in the primary regulators of AVP osmolality and plasma volume, with no change in blood pressure. On the basis of the experimental results, it is suggested that oropharyngeal factors may be the mechanism, for the observed decrease in AVP following rehydration.

  17. Trichinella spiralis Paramyosin Binds Human Complement C1q and Inhibits Classical Complement Activation

    PubMed Central

    Sun, Ran; Zhao, Xi; Wang, Zixia; Yang, Jing; Zhao, Limei; Zhan, Bin; Zhu, Xinping

    2015-01-01

    Background Trichinella spiralis expresses paramyosin (Ts-Pmy) as a defense mechanism. Ts-Pmy is a functional protein with binding activity to human complement C8 and C9 and thus plays a role in evading the attack of the host’s immune system. In the present study, the binding activity of Ts-Pmy to human complement C1q and its ability to inhibit classical complement activation were investigated. Methods and Findings The binding of recombinant and natural Ts-Pmy to human C1q were determined by ELISA, Far Western blotting and immunoprecipitation, respectively. Binding of recombinant Ts-Pmy (rTs-Pmy) to C1q inhibited C1q binding to IgM and consequently inhibited C3 deposition. The lysis of antibody-sensitized erythrocytes (EAs) elicited by the classical complement pathway was also inhibited in the presence of rTs-Pmy. In addition to inhibiting classical complement activation, rTs-Pmy also suppressed C1q binding to THP-1-derived macrophages, thereby reducing C1q-induced macrophages migration. Conclusion Our results suggest that T. spiralis paramyosin plays an important role in immune evasion by interfering with complement activation through binding to C1q in addition to C8 and C9. PMID:26720603

  18. Sequence specific inhibition of human type II phospholipase A2 enzyme activity by phosphorothioate oligonucleotides.

    PubMed Central

    Bennett, C F; Chiang, M Y; Wilson-Lingardo, L; Wyatt, J R

    1994-01-01

    Phosphorothioate oligonucleotides were identified which directly inhibited human type II phospholipase A2 (PLA2) enzyme activity in a sequence specific manner. The minimum pharmacophore common to all oligonucleotides which inhibited PLA2 enzyme activity consisted of two sets of three or more consecutive guanosine residues in a row. These oligonucleotides appear to form G quartets resulting in the formation of oligonucleotide aggregates. Additionally, a phosphorothioate backbone was required to be effective inhibitors of type II PLA2. The activity of one oligodeoxynucleotide, IP 3196 (5'-GGGTGGGTATAGAAGGGCTCC-3') has been characterized in more detail. IP 3196 inhibited PLA2 enzyme activity when the substrate was presented in the form of a phospholipid bilayer but not when presented in the form of a mixed micelle with anionic detergents. Human type II PLA2 was 50-fold more sensitive to inhibition by IP 3196 than venom and pancreatic type I enzymes. These data demonstrate that phosphorothioate oligonucleotides can specifically inhibit human type II PLA2 enzyme activity in a sequence specific manner. PMID:8065936

  19. Highly chlorinated PCBs inhibit the human xenobiotic response mediated by the steroid and xenobiotic receptor (SXR).

    PubMed Central

    Tabb, Michelle M; Kholodovych, Vladyslav; Grün, Felix; Zhou, Changcheng; Welsh, William J; Blumberg, Bruce

    2004-01-01

    Polychlorinated biphenyls (PCBs) are a family of persistent organic contaminants suspected to cause adverse effects in wildlife and humans. In rodents, PCBs bind to the aryl hydrocarbon (AhR) and pregnane X receptors (PXR) inducing the expression of catabolic cytochrome p450 enzymes of the CYP1A and 3A families. We found that certain highly chlorinated PCBs are potent activators of rodent PXR but antagonize its human ortholog, the steroid and xenobiotic receptor (SXR), inhibiting target gene induction. Thus, exposure to PCBs may blunt the human xenobiotic response, inhibiting the detoxification of steroids, bioactive dietary compounds, and xenobiotics normally mediated by SXR. The antagonistic PCBs are among the most stable and abundant in human tissues. These findings have important implications for understanding the biologic effects of PCB exposure and the use of animal models to predict the attendant risk. PMID:14754570

  20. Comparative sensitivity of human and rat neural cultures to chemical-induced inhibition of neurite outgrowth

    SciTech Connect

    Harrill, Joshua A.; Freudenrich, Theresa M.; Robinette, Brian L.; Mundy, William R.

    2011-11-15

    There is a need for rapid, efficient and cost-effective alternatives to traditional in vivo developmental neurotoxicity testing. In vitro cell culture models can recapitulate many of the key cellular processes of nervous system development, including neurite outgrowth, and may be used as screening tools to identify potential developmental neurotoxicants. The present study compared primary rat cortical cultures and human embryonic stem cell-derived neural cultures in terms of: 1) reproducibility of high content image analysis based neurite outgrowth measurements, 2) dynamic range of neurite outgrowth measurements and 3) sensitivity to chemicals which have been shown to inhibit neurite outgrowth. There was a large increase in neurite outgrowth between 2 and 24 h in both rat and human cultures. Image analysis data collected across multiple cultures demonstrated that neurite outgrowth measurements in rat cortical cultures were more reproducible and had higher dynamic range as compared to human neural cultures. Human neural cultures were more sensitive than rat cortical cultures to chemicals previously shown to inhibit neurite outgrowth. Parallel analysis of morphological (neurite count, neurite length) and cytotoxicity (neurons per field) measurements were used to detect selective effects on neurite outgrowth. All chemicals which inhibited neurite outgrowth in rat cortical cultures did so at concentrations which did not concurrently affect the number of neurons per field, indicating selective effects on neurite outgrowth. In contrast, more than half the chemicals which inhibited neurite outgrowth in human neural cultures did so at concentrations which concurrently decreased the number of neurons per field, indicating that effects on neurite outgrowth were secondary to cytotoxicity. Overall, these data demonstrate that the culture models performed differently in terms of reproducibility, dynamic range and sensitivity to neurite outgrowth inhibitors. While human neural

  1. Simvastatin Inhibits Glucose Metabolism and Legumain Activity in Human Myotubes

    PubMed Central

    Smith, Robert; Solberg, Rigmor; Jacobsen, Linn Løkken; Voreland, Anette Larsen; Rustan, Arild Christian; Thoresen, G. Hege; Johansen, Harald Thidemann

    2014-01-01

    Simvastatin, a HMG-CoA reductase inhibitor, is prescribed worldwide to patients with hypercholesterolemia. Although simvastatin is well tolerated, side effects like myotoxicity are reported. The mechanism for statin-induced myotoxicity is still poorly understood. Reports have suggested impaired mitochondrial dysfunction as a contributor to the observed myotoxicity. In this regard, we wanted to study the effects of simvastatin on glucose metabolism and the activity of legumain, a cysteine protease. Legumain, being the only known asparaginyl endopeptidase, has caspase-like properties and is described to be involved in apoptosis. Recent evidences indicate a regulatory role of both glucose and statins on cysteine proteases in monocytes. Satellite cells were isolated from the Musculus obliquus internus abdominis of healthy human donors, proliferated and differentiated into polynuclear myotubes. Simvastatin with or without mevalonolactone, farnesyl pyrophosphate or geranylgeranyl pyrophosphate were introduced on day 5 of differentiation. After 48 h, cells were either harvested for immunoblotting, ELISA, cell viability assay, confocal imaging or enzyme activity analysis, or placed in a fuel handling system with [14C]glucose or [3H]deoxyglucose for uptake and oxidation studies. A dose-dependent decrease in both glucose uptake and oxidation were observed in mature myotubes after exposure to simvastatin in concentrations not influencing cell viability. In addition, simvastatin caused a decrease in maturation and activity of legumain. Dysregulation of glucose metabolism and decreased legumain activity by simvastatin points out new knowledge about the effects of statins on skeletal muscle, and may contribute to the understanding of the myotoxicity observed by statins. PMID:24416446

  2. Inhibition of RNA Polymerase II Transcription in Human Cells by Synthetic DNA-Binding Ligands

    NASA Astrophysics Data System (ADS)

    Dickinson, Liliane A.; Gulizia, Richard J.; Trauger, John W.; Baird, Eldon E.; Mosier, Donald E.; Gottesfeld, Joel M.; Dervan, Peter B.

    1998-10-01

    Sequence-specific DNA-binding small molecules that can permeate human cells potentially could regulate transcription of specific genes. Multiple cellular DNA-binding transcription factors are required by HIV type 1 for RNA synthesis. Two pyrrole--imidazole polyamides were designed to bind DNA sequences immediately adjacent to binding sites for the transcription factors Ets-1, lymphoid-enhancer binding factor 1, and TATA-box binding protein. These synthetic ligands specifically inhibit DNA-binding of each transcription factor and HIV type 1 transcription in cell-free assays. When used in combination, the polyamides inhibit virus replication by >99% in isolated human peripheral blood lymphocytes, with no detectable cell toxicity. The ability of small molecules to target predetermined DNA sequences located with RNA polymerase II promoters suggests a general approach for regulation of gene expression, as well as a mechanism for the inhibition of viral replication.

  3. Inhibition of Sendai virus fusion with phospholipid vesicles and human erythrocyte membranes by hydrophobic peptides

    SciTech Connect

    Kelsey, D.R.; Flanagan, T.D.; Young, J.E.; Yeagle, P.L. )

    1991-06-01

    Hydrophobic di- and tripeptides which are capable of inhibiting enveloped virus infection of cells are also capable of inhibiting at least three different types of membrane fusion events. Large unilamellar vesicles (LUV) of N-methyl dioleoylphosphatidylethanolamine (N-methyl DOPE), containing encapsulated 1-aminonaphthalene-3,6,8-trisulfonic acid (ANTS) and/or p-xylene bis(pyridinium bromide) (DPX), were formed by extrusion. Vesicle fusion and leakage were then monitored with the ANTS/DPX fluorescence assay. Sendai virus fusion with lipid vesicles and Sendai virus fusion with human erythrocyte membranes were measured by following the relief of fluorescence quenching of virus labeled with octadecylrhodamine B chloride (R18). This study found that the effectiveness of the peptides carbobenzoxy-L-Phe-L-Phe (Z-L-Phe-L-Phe), Z-L-Phe, Z-D-Phe, and Z-Gly-L-Phe-L-Phe in inhibiting N-methyl DOPE LUV fusion or fusion of virus with N-methyl DOPE LUV also paralleled their reported ability to block viral infectivity. Furthermore, Z-D-Phe-L-PheGly and Z-Gly-L-Phe inhibited Sendai virus fusion with human erythrocyte membranes with the same relative potency with which they inhibited vesicle-vesicle and virus-vesicle fusion. The evidence suggests a mechanism by which these peptides exert their inhibition of plaque formation by enveloped viruses. This class of inhibitors apparently acts by inhibiting fusion of the viral envelope with the target cell membrane, thereby preventing viral infection. The physical pathway by which these peptides inhibit membrane fusion was investigated. {sup 31}P nuclear magnetic resonance (NMR) of proposed intermediates in the pathway for membrane fusion in LUV revealed that the potent fusion inhibitor Z-D-Phe-L-PheGly selectively altered the structure (or dynamics) of the hypothesized fusion intermediates and that the poor inhibitor Z-Gly-L-Phe did not.

  4. Inhibition of human platelet function in vitro and ex vivo by acetaminophen.

    PubMed

    Lages, B; Weiss, H J

    1989-03-15

    The effects of acetaminophen (APAP) in vitro, or ex vivo following APAP ingestion, on human platelet aggregation, 14C-5HT secretion, and thromboxane B2 (TxB2) formation were assessed. APAP added in vitro to citrated platelet-rich plasma (PRP) inhibited aggregation, secretion, and TxB2 formation induced by collagen, epinephrine, arachidonate, and the ionophore A23187, but had no effect on the responses induced by the endoperoxide analog U44069. Arachidonate-induced responses were inhibited by lower concentrations of APAP than were the responses to the other agonists. In PRP obtained 1 hour after ingestion of 650 mg or 1000 mg APAP, arachidonate-induced TxB2 formation was inhibited by 40-99% in five subjects tested, whereas inhibition of collagen- or epinephrine-induced TxB2 formation was less consistent. Aggregation and secretion responses were not altered by APAP ingestion in 4 of the 5 subjects, but were inhibited in the remaining subject, who had the highest plasma APAP levels. In contrast to aspirin and indomethacin, APAP-induced inhibition of collagen-stimulated TxB2 formation could be partially overcome with increasing collagen concentrations. No such partial correction occurred with epinephrine, however. In washed platelet suspensions labeled with 3H-arachidonate, both APAP and aspirin inhibited the formation of labeled PGD2 and PGE2, as well as TxB2. These results suggest that APAP acts in human platelets as a reversible inhibitor of cyclo-oxygenase, as found previously in other tissues, and that recent APAP ingestion can, on occasion, produce inhibition of platelet functional responses measured in vitro. PMID:2499947

  5. Blockade of MUC1 expression by glycerol guaiacolate inhibits proliferation of human breast cancer cells.

    PubMed

    Smith, J S; Colon, J; Madero-Visbal, R; Isley, B; Konduri, S D; Baker, C H

    2010-10-01

    We sought to determine whether administration of glycerol guaiacolate at an optimal biological dose inhibits human breast cancer cell growth. Human breast cancer MCF-7 and ZR-75-1 cells were treated with glycerol guaiacolate and the therapeutic efficacy and biological activity of this drug was investigated on breast cancer cell growth. MCF-7 cells were injected into the mammary fat pad of overectamized female athymic nude mice. Ten days later, animals were treated with daily intraperitoneal injections of glycerol guaiacolate for six weeks. Tumor size and volume was monitored and immunohistochemistry analysis on MUC1, p21 and ki-67 was performed. Glycerol guaiacolate decreased breast cancer cell growth in a dose-dependent manner, decreased cell migration, and caused G1 cell cycle arrest. Our results demonstrate that glycerol guaiacolate inhibits MUC1 protein and mRNA expression levels and significantly increased p21 expression in human breast cancer cells as well as induced PARP cleavage. Similarly, glycerol guaiacolate inhibited breast tumor growth in vivo as well as enhanced p21 expression and decreased breast tumor cell proliferation (ki-67 expression). Collectively, our results demonstrate that glycerol guaiacolate decreased MUC1 expression and enhanced cell growth inhibition by inducing p21 expression in breast cancer cells. These findings suggest that glycerol guaiacolate may provide a novel and effective approach for the treatment of human breast cancer. PMID:21184665

  6. Inhibition of Nuclear Factor-Kappa B Activation Decreases Survival of Mycobacterium tuberculosis in Human Macrophages

    PubMed Central

    Chmura, Kathryn; Ovrutsky, Alida R.; Su, Wen-Lin; Griffin, Laura; Pyeon, Dohun; McGibney, Mischa T.; Strand, Matthew J.; Numata, Mari; Murakami, Seiji; Gaido, Loretta; Honda, Jennifer R.; Kinney, William H.; Oberley-Deegan, Rebecca E.; Voelker, Dennis R.; Ordway, Diane J.; Chan, Edward D.

    2013-01-01

    Nuclear factor-kappa B (NFκB) is a ubiquitous transcription factor that mediates pro-inflammatory responses required for host control of many microbial pathogens; on the other hand, NFκB has been implicated in the pathogenesis of other inflammatory and infectious diseases. Mice with genetic disruption of the p50 subunit of NFκB are more likely to succumb to Mycobacterium tuberculosis (MTB). However, the role of NFκB in host defense in humans is not fully understood. We sought to examine the role of NFκB activation in the immune response of human macrophages to MTB. Targeted pharmacologic inhibition of NFκB activation using BAY 11-7082 (BAY, an inhibitor of IκBα kinase) or an adenovirus construct with a dominant-negative IκBα significantly decreased the number of viable intracellular mycobacteria recovered from THP-1 macrophages four and eight days after infection. The results with BAY were confirmed in primary human monocyte-derived macrophages and alveolar macrophages. NFκB inhibition was associated with increased macrophage apoptosis and autophagy, which are well-established killing mechanisms of intracellular MTB. Inhibition of the executioner protease caspase-3 or of the autophagic pathway significantly abrogated the effects of BAY. We conclude that NFκB inhibition decreases viability of intracellular MTB in human macrophages via induction of apoptosis and autophagy. PMID:23634218

  7. Inhibition of nuclear factor-kappa B activation decreases survival of Mycobacterium tuberculosis in human macrophages.

    PubMed

    Bai, Xiyuan; Feldman, Nicole E; Chmura, Kathryn; Ovrutsky, Alida R; Su, Wen-Lin; Griffin, Laura; Pyeon, Dohun; McGibney, Mischa T; Strand, Matthew J; Numata, Mari; Murakami, Seiji; Gaido, Loretta; Honda, Jennifer R; Kinney, William H; Oberley-Deegan, Rebecca E; Voelker, Dennis R; Ordway, Diane J; Chan, Edward D

    2013-01-01

    Nuclear factor-kappa B (NFκB) is a ubiquitous transcription factor that mediates pro-inflammatory responses required for host control of many microbial pathogens; on the other hand, NFκB has been implicated in the pathogenesis of other inflammatory and infectious diseases. Mice with genetic disruption of the p50 subunit of NFκB are more likely to succumb to Mycobacterium tuberculosis (MTB). However, the role of NFκB in host defense in humans is not fully understood. We sought to examine the role of NFκB activation in the immune response of human macrophages to MTB. Targeted pharmacologic inhibition of NFκB activation using BAY 11-7082 (BAY, an inhibitor of IκBα kinase) or an adenovirus construct with a dominant-negative IκBα significantly decreased the number of viable intracellular mycobacteria recovered from THP-1 macrophages four and eight days after infection. The results with BAY were confirmed in primary human monocyte-derived macrophages and alveolar macrophages. NFκB inhibition was associated with increased macrophage apoptosis and autophagy, which are well-established killing mechanisms of intracellular MTB. Inhibition of the executioner protease caspase-3 or of the autophagic pathway significantly abrogated the effects of BAY. We conclude that NFκB inhibition decreases viability of intracellular MTB in human macrophages via induction of apoptosis and autophagy. PMID:23634218

  8. Antiretroviral Agents Inhibit Infection of Human Cells by Porcine Endogenous Retroviruses

    PubMed Central

    Powell, S. K.; Gates, M. E.; Langford, G.; Gu, M.-L.; Lockey, C.; Long, Z.; Otto, E.

    2000-01-01

    The efficacy of antiretroviral drugs against porcine endogenous retroviruses (PERV) that may be harbored in pig organs intended for transplantation was examined in human cells in vitro. The nucleoside analogs zidovudine and dideoxyinosine were found to effectively inhibit PERV replication. PMID:11083652

  9. Müllerian inhibiting substance preferentially inhibits stem/progenitors in human ovarian cancer cell lines compared with chemotherapeutics

    PubMed Central

    Wei, Xiaolong; Dombkowski, David; Meirelles, Katia; Pieretti-Vanmarcke, Rafael; Szotek, Paul P.; Chang, Henry L.; Preffer, Frederic I.; Mueller, Peter R.; Teixeira, Jose; MacLaughlin, David T.; Donahoe, Patricia K.

    2010-01-01

    Cancer stem cells are proposed to be tumor-initiating cells capable of tumorigenesis, recurrence, metastasis, and drug resistance, and, like somatic stem cells, are thought to be capable of unlimited self-renewal and, when stimulated, proliferation and differentiation. Here we select cells by expression of a panel of markers to enrich for a population with stem cell-like characteristics. A panel of eight was initially selected from 95 human cell surface antigens as each was shared among human ovarian primary cancers, ovarian cancer cell lines, and normal fimbria. A total of 150 combinations of markers were reduced to a panel of three—CD44, CD24, and Epcam—which selected, in three ovarian cancer cell lines, those cells which best formed colonies. Cells expressing CD44, CD24, and Epcam exhibited stem cell characteristics of shorter tumor-free intervals in vivo after limiting dilution, and enhanced migration in invasion assays in vitro. Also, doxorubicin, cisplatin, and paclitaxel increased this enriched population which, conversely, was significantly inhibited by Müllerian inhibiting substance (MIS) or the MIS mimetic SP600125. These findings demonstrate that flow cytometry can be used to detect a population which shows differential drug sensitivity, and imply that treatment of patients can be individualized to target both stem/progenitor cell enriched and nonenriched subpopulations. The findings also suggest that this population, amenable to isolation by flow cytometry, can be used to screen for novel treatment paradigms, including biologic agents such as MIS, which will improve outcomes for patients with ovarian cancer. PMID:20952655

  10. PARP-1 inhibition influences the oxidative stress response of the human lens.

    PubMed

    Smith, Andrew J O; Ball, Simon S R; Bowater, Richard P; Wormstone, I Michael

    2016-08-01

    Poly(ADP-ribose) polymerase-1 (PARP-1) is best characterised for its involvement in DNA repair. PARP-1 activity is also linked to cell fate, confounding its roles in maintaining genome integrity. The current study assessed the functional roles of PARP-1 within human lens cells in response to oxidative stress. The human lens epithelial cell line FHL124 and whole human lens cultures were used as experimental systems. Hydrogen peroxide (H2O2) was employed to induce oxidative stress and cell death was assessed by LDH release. The functional influence of PARP-1 was assessed using targeted siRNA and chemical inhibition (by AG14361). Immunocytochemistry and western blotting were used to assess PARP-1 expression and the alkaline comet assay determined the levels of DNA strand breaks. PARP-1 was generally observed in the cell nucleus in both the FHL124 cell line and whole human lenses. PARP-1 inhibition rendered FHL124 cells more susceptible to H2O2-induced DNA strand breaks. Interestingly, reduction of PARP-1 activity significantly inhibited H2O2-induced cell death relative to control cells. Inhibition of PARP-1 in whole human lenses resulted in a reduced level of lens opacity and cell death following exposure to H2O2 relative to matched pair controls. Thus, we show that PARP-1 could play a role in the fate of human lens cells, and these first observations in human lenses suggest that it could impact on lens opacity. Further studies are required to elucidate the regulatory processes that give rise to these effects. PMID:26990173

  11. PARP-1 inhibition influences the oxidative stress response of the human lens

    PubMed Central

    Smith, Andrew J.O.; Ball, Simon S.R.; Bowater, Richard P.; Wormstone, I. Michael

    2016-01-01

    Poly(ADP-ribose) polymerase-1 (PARP-1) is best characterised for its involvement in DNA repair. PARP-1 activity is also linked to cell fate, confounding its roles in maintaining genome integrity. The current study assessed the functional roles of PARP-1 within human lens cells in response to oxidative stress. The human lens epithelial cell line FHL124 and whole human lens cultures were used as experimental systems. Hydrogen peroxide (H2O2) was employed to induce oxidative stress and cell death was assessed by LDH release. The functional influence of PARP-1 was assessed using targeted siRNA and chemical inhibition (by AG14361). Immunocytochemistry and western blotting were used to assess PARP-1 expression and the alkaline comet assay determined the levels of DNA strand breaks. PARP-1 was generally observed in the cell nucleus in both the FHL124 cell line and whole human lenses. PARP-1 inhibition rendered FHL124 cells more susceptible to H2O2-induced DNA strand breaks. Interestingly, reduction of PARP-1 activity significantly inhibited H2O2-induced cell death relative to control cells. Inhibition of PARP-1 in whole human lenses resulted in a reduced level of lens opacity and cell death following exposure to H2O2 relative to matched pair controls. Thus, we show that PARP-1 could play a role in the fate of human lens cells, and these first observations in human lenses suggest that it could impact on lens opacity. Further studies are required to elucidate the regulatory processes that give rise to these effects. PMID:26990173

  12. CO{sub 2} impairs peroxynitrite-mediated inhibition of human caspase-3

    SciTech Connect

    Ascenzi, Paolo . E-mail: ascenzi@uniroma3.it; Marino, Maria; Menegatti, Enea

    2006-10-13

    Peroxynitrite (ONOO{sup -}) is a transient powerful oxidant produced in vivo as the reaction of nitrogen monoxide ({sup ?}NO) with superoxide (O2?-). The peroxynitrite reactivity is modulated by carbon dioxide (CO{sub 2}) which enhances the peroxynitrite-mediated nitration of aromatics and partially impairs the oxidation of thiols. Here, the effect of CO{sub 2} on the peroxynitrite-mediated inhibition of human caspase-3, the execution enzyme of the apoptotic cascade, is reported. Peroxynitrite inhibits the catalytic activity of human caspase-3 by oxidizing the S{gamma} atom of the Cys catalytic residue. In the absence of CO{sub 2}, 1.0 equivalent of peroxynitrite inactivates 1.0 equivalent of human caspase-3. In the presence of the physiological concentration of CO{sub 2} (=1.3x10{sup -3}M), 1.0 equivalent of peroxynitrite inactivates only 0.38 equivalents of human caspase-3. Peroxynitrite affects the k{sub cat} value of the human caspase-3 catalyzed hydrolysis of N-acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin, without altering K{sub m}. Both in the absence and presence of CO{sub 2}, the reducing agent dithiothreitol does not prevent human caspase-3 inhibition by peroxynitrite and does not reverse the peroxynitrite-induced inactivation of human caspase-3. These results represent First evidence for modulation of peroxynitrite-mediated inhibition of cysteine proteinase action by CO{sub 2}, supporting the role of CO{sub 2} in fine tuning of cell processes (e.g., apoptosis)

  13. 5-azacytidine and 5-azadeoxycytidine inhibit human immunodeficiency virus type 1 replication in vitro.

    PubMed Central

    Bouchard, J; Walker, M C; Leclerc, J M; Lapointe, N; Beaulieu, R; Thibodeau, L

    1990-01-01

    Chemotherapeutic agents which affect the integration, stability, or inducibility of the human immunodeficiency virus (HIV) provirus would have considerable value in treating acquired immunodeficiency syndrome. Two nucleoside analogs of cytosine, 5-azacytidine and 5-azadeoxycytidine, which seem to have such value because of their capabilities to affect both the stability and the methylation patterns of the nucleic acids into which they are incorporated, were tested for their ability to inhibit the replication of HIV type 1 (HIV-1) in human CEM T cells in vitro. 5-Azadeoxycytidine (1 microM) completely inhibited HIV replication in CEM cells, by the criteria of reduced viral antigen expression and decreased supernatant reverse transcriptase activity, with little toxicity for the treated cells. 5-azacytidine (1 microM) also inhibited HIV replication, but less effectively. When added 2 or more h after CEM cells were infected with HIV-1, both 5-azacytosine derivatives were less effective than they were when added at the time of infection. Even 2 h of exposure to 5-azadeoxycytidine was sufficient for inhibition of HIV replication. Although long exposure to either analog at concentrations of 1 microM would result in pronounced cellular cytotoxicity, the the fact that short exposures to the same dose of drug inhibit HIV replication but are not toxic for the cells implies that cellular toxicity itself is not an important mechanism of the antiviral action of the analogs. PMID:1691617

  14. The Aryl Hydrocarbon Receptor Ligand ITE Inhibits TGFβ1-Induced Human Myofibroblast Differentiation

    PubMed Central

    Lehmann, Geniece M.; Xi, Xia; Kulkarni, Ajit A.; Olsen, Keith C.; Pollock, Stephen J.; Baglole, Carolyn J.; Gupta, Shikha; Casey, Ann E.; Huxlin, Krystel R.; Sime, Patricia J.; Feldon, Steven E.; Phipps, Richard P.

    2011-01-01

    Fibrosis can occur in any human tissue when the normal wound healing response is amplified. Such amplification results in fibroblast proliferation, myofibroblast differentiation, and excessive extracellular matrix deposition. Occurrence of these sequelae in organs such as the eye or lung can result in severe consequences to health. Unfortunately, medical treatment of fibrosis is limited by a lack of safe and effective therapies. These therapies may be developed by identifying agents that inhibit critical steps in fibrotic progression; one such step is myofibroblast differentiation triggered by transforming growth factor-β1 (TGFβ1). In this study, we demonstrate that TGFβ1-induced myofibroblast differentiation is blocked in human fibroblasts by a candidate endogenous aryl hydrocarbon receptor (AhR) ligand 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). Our data show that ITE disrupts TGFβ1 signaling by inhibiting the nuclear translocation of Smad2/3/4. Although ITE functions as an AhR agonist, and biologically persistent AhR agonists, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, cause severe toxic effects, ITE exhibits no toxicity. Interestingly, ITE effectively inhibits TGFβ1-driven myofibroblast differentiation in AhR−/− fibroblasts: Its ability to inhibit TGFβ1 signaling is AhR independent. As supported by the results of this study, the small molecule ITE inhibits myofibroblast differentiation and may be useful clinically as an antiscarring agent. PMID:21406171

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

    SciTech Connect

    Wu, Yang-Chang; Sureshbabu, Munisamy; Fang, Yao-Ching; Wu, Yi-Hsiu; Lan, Yu-Hsuan; Chang, Fang-Rong; Chang, Ya-Wen; Hwang, Tsong-Long

    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.

  16. BET bromodomain inhibition rescues erythropoietin differentiation of human erythroleukemia cell line UT7

    SciTech Connect

    Goupille, Olivier; Penglong, Tipparat; Lefevre, Carine; Granger, Marine; Kadri, Zahra; Fucharoen, Suthat; Maouche-Chretien, Leila; Leboulch, Philippe; Chretien, Stany

    2012-12-07

    Highlights: Black-Right-Pointing-Pointer UT7 erythroleukemia cells are known to be refractory to differentiate. Black-Right-Pointing-Pointer Brief JQ1 treatment initiates the first steps of erythroid differentiation program. Black-Right-Pointing-Pointer Engaged UT7 cells then maturate in the presence of erythropoietin. Black-Right-Pointing-Pointer Sustained JQ1 treatment inhibits both proliferation and erythroid differentiation. -- Abstract: Malignant transformation is a multistep process requiring oncogenic activation, promoting cellular proliferation, frequently coupled to inhibition of terminal differentiation. Consequently, forcing the reengagement of terminal differentiation of transformed cells coupled or not with an inhibition of their proliferation is a putative therapeutic approach to counteracting tumorigenicity. UT7 is a human leukemic cell line able to grow in the presence of IL3, GM-CSF and Epo. This cell line has been widely used to study Epo-R/Epo signaling pathways but is a poor model for erythroid differentiation. We used the BET bromodomain inhibition drug JQ1 to target gene expression, including that of c-Myc. We have shown that only 2 days of JQ1 treatment was required to transitory inhibit Epo-induced UT7 proliferation and to restore terminal erythroid differentiation. This study highlights the importance of a cellular erythroid cycle break mediated by c-Myc inhibition before initiation of the erythropoiesis program and describes a new model for BET bromodomain inhibitor drug application.

  17. Pirfenidone inhibits p38-mediated generation of procoagulant microparticles by human alveolar epithelial cells.

    PubMed

    Neri, Tommaso; Lombardi, Stefania; Faìta, Francesca; Petrini, Silvia; Balìa, Cristina; Scalise, Valentina; Pedrinelli, Roberto; Paggiaro, Pierluigi; Celi, Alessandro

    2016-08-01

    Pirfenidone is a drug recently approved for idiopathic pulmonary fibrosis but its mechanisms of action are partially unknown. We have previously demonstrated that the airways of patients with idiopathic pulmonary fibrosis contain procoagulant microparticles that activate coagulation factor X to its active form, Xa, a proteinase that signals fibroblast growth and differentiation, thus potentially contributing to the pathogenesis of the disease. We also reported that in vitro exposure of human alveolar cells to H2O2 causes microparticle generation. Since p38 activation is involved in microparticle generation in some cell models and p38 inhibition is one of the mechanisms of action of pirfenidone, we investigated the hypothesis that H2O2-induced generation of microparticles by alveolar cells is dependent on p38 phosphorylation and is inhibited by pirfenidone. H2O2 stimulation of alveolar cells caused p38 phosphorylation that was inhibited by pirfenidone. The drug also inhibited H2O2 induced microparticle generation as assessed by two independent methods (solid phase thrombin generation and flow cytometry). The shedding of microparticle-bound tissue factor activity was also inhibited by pirfenidone. Inhibition of p38-mediated generation of procoagulant microparticle is a previously unrecognized mechanism of action of the antifibrotic drug, pirfenidone. PMID:27237042

  18. Sensory gating, inhibition control and gamma oscillations in the human somatosensory cortex

    PubMed Central

    Cheng, Chia-Hsiung; Chan, Pei-Ying S.; Niddam, David M.; Tsai, Shang-Yueh; Hsu, Shih-Chieh; Liu, Chia-Yih

    2016-01-01

    Inhibiting the responses to irrelevant stimuli is an essential component of human cognitive function. Pre-attentive auditory sensory gating (SG), an attenuated neural activation to the second identical stimulus, has been found to be related to the performance of higher-hierarchical brain function. However, it remains unclear whether other cortical regions, such as somatosensory cortex, also possess similar characteristics, or if such a relationship is modality-specific. This study used magnetoencephalography to record neuromagnetic responses to paired-pulse electrical stimulation to median nerve in 22 healthy participants. Somatosensory SG ratio and cortical brain oscillations were obtained and compared with the behavioral performance of inhibition control, as evaluated by somatosensory and auditory Go-Nogo tasks. The results showed that somatosensory P35m SG ratio correlated with behavioral performance of inhibition control. Such relationship was also established in relation to the auditory Go-Nogo task. Finally, a higher frequency value of evoked gamma oscillations was found to relate to a better somatosensory SG ability. In conclusion, our data provided an empirical link between automatic cortical inhibition and behavioral performance of attentive inhibition control. This study invites further research on the relationships among gamma oscillations, neurophysiological indices, and behavioral performance in clinical populations in terms of SG or cortical inhibition. PMID:26843358

  19. Inhibition of human topoisomerase II in vitro by bioactive benzene metabolites

    SciTech Connect

    Frantz, C.E.; Chen, Hongwei; Eastmond, D.A.

    1996-12-01

    Benzene is a clastogenic and carcinogenic agent that induces acute myelogenous leukemia in humans and multiple types of tumors in animals. Previous research has indicated that benzene must first be metabolized to one or more bioactive species to exert its myelotoxic and genotoxic effects. To better understand the possible role of individual benzene metabolites in the leukemogenic process, as well as to further investigate inhibition of topoisomerase 11 by benzene metabolites, a series of known and putative benzene metabolites, phenol, 4{prime}4-biphenol, 2,2{prime}-biphenol, hydroquinone, catechol, 1,2,4-benzenetriol, 1,4-benzoquinone, and trans-trans-muconaldehyde were tested for inhibitory effects in vitro on the human topoisomerase 11 enzyme. With minor modifications of the standard assay conditions, 1,4-benzoquinone and trans-trans-muconaldehyde were shown to be directly inhibitory, whereas all of the phenolic metabolites were shown to inhibit enzymatic activity following bioactivation using a peroxidase activation system. The majority of compounds tested inhibited topoisomerase 11 at concentrations at or below 10 pM. These results confirm and expand upon previous findings from our laboratory and indicate that many of the metabolites of benzene could potentially interfere with topoisomerase 11. Since other inhibitors of topoisomerase 11 have been shown to induce leukemia in humans, inhibition of this enzyme by benzene metabolites may also play a role in the carcinogenic effects of benzene. 48 refs., 4 tabs.

  20. Selective Inhibition of Bakuchicin Isolated from Psoralea corylifolia on CYP1A in Human Liver Microsomes

    PubMed Central

    Kim, Sun Joo; Oh, Heung Chan; Kim, Youn-Chul; Jeong, Gil-Saeng; Lee, Sangkyu

    2016-01-01

    Bakuchicin is a furanocoumarin isolated from Psoralea corylifolia and shows several biological activities. Although there have been studies on the biological effects of bakuchicin, its modulation potency of CYP activities has not been previously investigated. Here, we investigated the inhibitory effects of bakuchicin on the activities of CYP isoforms by using a cocktail of probe substrates in pooled human liver microsomes (HLMs) and human recombinant cDNA-expressed CYP. Bakuchicin strongly inhibited CYP1A-mediated phenacetin O-deethylation with an IC50 value of 0.43 μM in HLMs. It was confirmed by human recombinant cDNA-expressed CYP1A1 and CYP1A2 with a Ki value of 0.11 μM and 0.32 μM, respectively. A Lineweaver-Burk plot indicated that the inhibition mechanism of bakuchicin was competitive inhibition. Overall, this is the first study to investigate the potential CYP1A1 and CYP1A2 inhibition associated with bakuchicin and to report its competitive inhibitory effects on HLMs. PMID:26977174

  1. Dimethyl sulfoxide at high concentrations inhibits non-selective cation channels in human erythrocytes.

    PubMed

    Nardid, Oleg A; Schetinskey, Miroslav I; Kucherenko, Yuliya V

    2013-03-01

    Dimethyl sulfoxide (DMSO), a by-product of the pulping industry, is widely used in biological research, cryobiology and medicine. On cellular level DMSO was shown to suppress NMDA-AMPA channels activation, blocks Na+ channel activation and attenuates Ca2+ influx (Lu and Mattson 2001). In the present study we explored the whole-cell patch-clamp to examine the acute effect of high concentrations of DMSO (0.1-2 mol/l) on cation channels activity in human erythrocytes. Acute application of DMSO (0.1-2 mol/l) dissolved in Cl--containing saline buffer solution significantly inhibited cation conductance in human erythrocytes. Inhibition was concentration-dependent and had an exponential decay profile. DMSO (2 mol/l) induced cation inhibition in Cl-- containing saline solutions of: 40.3 ± 3.9% for K+, 35.4 ± 3.1% for Ca2+ and 47.4 ± 1.9% for NMDG+. Substitution of Cl- with gluconate- increased the inhibitory effect of DMSO on the Na+ current. Inhibitory effect of DMSO was neither due to high permeability of erythrocytes to DMSO nor to an increased tonicity of the bath media since no effect was observed in 2 mol/l glycerol solution. In conclusion, we have shown that high concentrations of DMSO inhibit the non-selective cation channels in human erythrocytes and thus protect the cells against Na+ and Ca2+ overload. Possible mechanisms of DMSO effect on cation conductance are discussed. PMID:23531832

  2. Inhibition of human anthracycline reductases by emodin - A possible remedy for anthracycline resistance.

    PubMed

    Hintzpeter, Jan; Seliger, Jan Moritz; Hofman, Jakub; Martin, Hans-Joerg; Wsol, Vladimir; Maser, Edmund

    2016-02-15

    The clinical application of anthracyclines, like daunorubicin and doxorubicin, is limited by two factors: dose-related cardiotoxicity and drug resistance. Both have been linked to reductive metabolism of the parent drug to their metabolites daunorubicinol and doxorubicinol, respectively. These metabolites show significantly less anti-neoplastic properties as their parent drugs and accumulate in cardiac tissue leading to chronic cardiotoxicity. Therefore, we aimed to identify novel and potent natural inhibitors for anthracycline reductases, which enhance the anticancer effect of anthracyclines by preventing the development of anthracycline resistance. Human enzymes responsible for the reductive metabolism of daunorubicin were tested for their sensitivity towards anthrachinones, in particular emodin and anthraflavic acid. Intense inhibition kinetic data for the most effective daunorubicin reductases, including IC50- and Ki-values, the mode of inhibition, as well as molecular docking, were compiled. Subsequently, a cytotoxicity profile and the ability of emodin to reverse daunorubicin resistance were determined using multiresistant A549 lung cancer and HepG2 liver cancer cells. Emodin potently inhibited the four main human daunorubicin reductases in vitro. Further, we could demonstrate that emodin is able to synergistically sensitize human cancer cells towards daunorubicin at clinically relevant concentrations. Therefore, emodin may yield the potential to enhance the therapeutic effectiveness of anthracyclines by preventing anthracycline resistance via inhibition of the anthracycline reductases. In symphony with its known pharmacological properties, emodin might be a compound of particular interest in the management of anthracycline chemotherapy efficacy and their adverse effects. PMID:26773812

  3. Selective inhibition of leukotriene C/sub 4/ synthesis in human neutrophils by ethacrynic acid

    SciTech Connect

    Leung, K.H.

    1986-05-29

    Addition of glutathione S-transferase inhibitors, ethyacrynic acid (ET), caffeic acid (CA), and ferulic acid (FA) to human neutrophils led to inhibition of leukotriene C/sub 4/ (LTC/sub 4/) synthesis induced by calcium ionophore A23187. ET is the most specific of these inhibitors for it had little effect on LTB/sub 4/, PGE/sub 2/, and 5-HETE synthesis. The inhibition of LTC/sub 4/ was irreversible and time dependent. ET also had little effect on /sup 3/H-AA release from A23187-stimulated neutrophils.

  4. Eicosapentaenoic acid inhibits UV-induced MMP-1 expression in human dermal fibroblasts.

    PubMed

    Kim, Hyeon Ho; Shin, Chung Min; Park, Chi-Hyun; Kim, Kyu Han; Cho, Kwang Hyun; Eun, Hee Chul; Chung, Jin Ho

    2005-08-01

    Ultraviolet (UV) irradiation regulates UV-responsive genes, including matrix metalloproteinases (MMPs). Moreover, UV-induced MMPs cause connective tissue damage and the skin to become wrinkled and aged. Here, we investigated the effect of eicosapentaenoic acid (EPA), a dietary omega-3 fatty acid, on UV-induced MMP-1 expression in human dermal fibroblasts (HDFs). We found that UV radiation increases MMP-1 expression and that this is mediated by p44 and p42 MAP kinase (ERK) and Jun-N-terminal kinase (JNK) activation but not by p38 activation. Pretreatment of HDFs with EPA inhibited UV-induced MMP-1 expression in a dose-dependent manner and also inhibited the UV-induced activation of ERK and JNK by inhibiting ERK kinase (MEK1) and SAPK/ERK kinase 1 (SEK1) activation, respectively. Moreover, inhibition of ERK and JNK by EPA resulted in the decrease of c-Fos expression and c-Jun phosphorylation/expression induced by UV, respectively, which led to the inhibition of UV-induced activator protein-1 DNA binding activity. This inhibitory effect of EPA on MMP-1 was not mediated by an antioxidant effect. We also found that EPA inhibited 12-O-tetradecanoylphorbol-13-acetate- or tumor necrosis factor-alpha-induced MMP-1 expression in HDFs and UV-induced MMP-1 expression in HaCaT cells. In conclusion, our results demonstrate that EPA can inhibit UV-induced MMP-1 expression by inhibiting the MEK1/ERK/c-Fos and SEK1/JNK/c-Jun pathways. Therefore, EPA is a potential agent for the prevention and treatment of skin aging. PMID:15930517

  5. Inhibition of human and rat CYP1A1 enzyme by grapefruit juice compounds.

    PubMed

    Santes-Palacios, Rebeca; Romo-Mancillas, Antonio; Camacho-Carranza, Rafael; Espinosa-Aguirre, Jesús Javier

    2016-09-01

    Cytochrome P4501A1 is involved in the metabolism of carcinogenic polycyclic aromatic hydrocarbons; therefore, its inhibition interferes with the carcinogenesis process induced by these compounds in rats. The human and rat CYP1A1 differ by 21% in amino acid sequence, including the active site of the enzyme; this difference may be an important factor when results obtained using animal models are interpolated to humans. Based on its previously reported CYP inhibitory properties, we studied the effects of two molecules contained within grapefruit juice, naringenin and 6',7'-dihydroxybergamottin, on human and rat CYP1A1 activity. For this purpose, the kinetics of inhibition as well as computational simulations were used. Naringenin and 6',7'-dihydroxybergamottin were found to be competitive inhibitors of human and rat CYP1A1. Additionally, naringenin exerted a mixed type inhibition effect on rat CYP1A1. Computational docking showed that inhibitors might block the oxidation of 7-ethoxyresorufin by binding to the CYP1A1 active site. Our results demonstrate the differences in CYP inhibitory mechanisms for the same molecule when CYP from different species are considered. PMID:27444380

  6. Human Cerberus Prevents Nodal-Receptor Binding, Inhibits Nodal Signaling, and Suppresses Nodal-Mediated Phenotypes

    PubMed Central

    Aykul, Senem; Ni, Wendi; Mutatu, Washington; Martinez-Hackert, Erik

    2015-01-01

    The Transforming Growth Factor-ß (TGFß) family ligand Nodal is an essential embryonic morphogen that is associated with progression of breast and other cancers. It has therefore been suggested that Nodal inhibitors could be used to treat breast cancers where Nodal plays a defined role. As secreted antagonists, such as Cerberus, tightly regulate Nodal signaling during embryonic development, we undertook to produce human Cerberus, characterize its biochemical activities, and determine its effect on human breast cancer cells. Using quantitative methods, we investigated the mechanism of Nodal signaling, we evaluated binding of human Cerberus to Nodal and other TGFß family ligands, and we characterized the mechanism of Nodal inhibition by Cerberus. Using cancer cell assays, we examined the ability of Cerberus to suppress aggressive breast cancer cell phenotypes. We found that human Cerberus binds Nodal with high affinity and specificity, blocks binding of Nodal to its signaling partners, and inhibits Nodal signaling. Moreover, we showed that Cerberus profoundly suppresses migration, invasion, and colony forming ability of Nodal expressing and Nodal supplemented breast cancer cells. Taken together, our studies provide mechanistic insights into Nodal signaling and Nodal inhibition with Cerberus and highlight the potential value of Cerberus as anti-Nodal therapeutic. PMID:25603319

  7. Inhibition of ZEB1 expression induces redifferentiation of adult human β cells expanded in vitro

    PubMed Central

    Sintov, Elad; Nathan, Gili; Knoller, Sarah; Pasmanik-Chor, Metsada; Russ, Holger A.; Efrat, Shimon

    2015-01-01

    In-vitro expansion of functional adult human β-cells is an attractive approach for generating insulin-producing cells for transplantation. However, human islet cell expansion in culture results in loss of β-cell phenotype and epithelial-mesenchymal transition (EMT). This process activates expression of ZEB1 and ZEB2, two members of the zinc-finger homeobox family of E-cadherin repressors, which play key roles in EMT. Downregulation of ZEB1 using shRNA in expanded β-cell-derived (BCD) cells induced mesenchymal-epithelial transition (MET), β-cell gene expression, and proliferation attenuation. In addition, inhibition of ZEB1 expression potentiated redifferentiation induced by a combination of soluble factors, as judged by an improved response to glucose stimulation and a 3-fold increase in the fraction of C-peptide-positive cells to 60% of BCD cells. Furthermore, ZEB1 shRNA led to increased insulin secretion in cells transplanted in vivo. Our findings suggest that the effects of ZEB1 inhibition are mediated by attenuation of the miR-200c target genes SOX6 and SOX2. These findings, which were reproducible in cells derived from multiple human donors, emphasize the key role of ZEB1 in EMT in cultured BCD cells and support the value of ZEB1 inhibition for BCD cell redifferentiation and generation of functional human β-like cells for cell therapy of diabetes. PMID:26264186

  8. HIV protease inhibitor nelfinavir inhibits growth of human melanoma cells by induction of cell cycle arrest.

    PubMed

    Jiang, Wei; Mikochik, Peter J; Ra, Jin H; Lei, Hanqin; Flaherty, Keith T; Winkler, Jeffrey D; Spitz, Francis R

    2007-02-01

    HIV protease inhibitors (HIV PI) are a class of antiretroviral drugs that are designed to target the viral protease. Unexpectedly, this class of drugs is also reported to have antitumor activity. In this study, we have evaluated the in vitro activity of nelfinavir, a HIV PI, against human melanoma cells. Nelfinavir inhibits the growth of melanoma cell lines at low micromolar concentrations that are clinically attainable. Nelfinavir promotes apoptosis and arrests cell cycle at G(1) phase. Cell cycle arrest is attributed to inhibition of cyclin-dependent kinase 2 (CDK2) and concomitant dephosphorylation of retinoblastoma tumor suppressor. We further show that nelfinavir inhibits CDK2 through proteasome-dependent degradation of Cdc25A phosphatase. Our results suggest that nelfinavir is a promising candidate chemotherapeutic agent for advanced melanoma, for which novel and effective therapies are urgently needed. PMID:17283158

  9. Inhibition of human copper trafficking by a small molecule significantly attenuates cancer cell proliferation

    PubMed Central

    Wang, Jing; Luo, Cheng; Shan, Changliang; You, Qiancheng; Lu, Junyan; Elf, Shannon; Zhou, Yu; Wen, Yi; Vinkenborg, Jan L.; Fan, Jun; Kang, Heebum; Lin, Ruiting; Han, Dali; Xie, Yuxin; Karpus, Jason; Chen, Shijie; Ouyang, Shisheng; Luan, Chihao; Zhang, Naixia; Ding, Hong; Merkx, Maarten; Liu, Hong; Chen, Jing; Jiang, Hualiang; He, Chuan

    2016-01-01

    Copper is a transition metal that plays critical roles in many life processes. Controlling the cellular concentration and trafficking of copper offers a route to disrupt these processes. Here we report small molecules that inhibit the human copper-trafficking proteins Atox1 and CCS, and so provide a selective approach to disrupt cellular copper transport. The knockdown of Atox1 and CCS or their inhibition leads to a significantly reduced proliferation of cancer cells, but not of normal cells, as well as to attenuated tumour growth in mouse models. We show that blocking copper trafficking induces cellular oxidative stress and reduces levels of cellular ATP. The reduced level of ATP results in activation of the AMP-activated protein kinase that leads to reduced lipogenesis. Both effects contribute to the inhibition of cancer cell proliferation. Our results establish copper chaperones as new targets for future developments in anticancer therapies. PMID:26587712

  10. Inhibition of excision-repair of ultraviolet damage in human cells by exposure to methyl methanesulfonate.

    PubMed

    Park, S D; Choi, K H; Hong, S W; Cleaver, J E

    1981-07-01

    Unscheduled DNA synthesis and excision of pyrimidine dimers in human cells exposed to ultraviolet let were inhibited by exposure to methyl methanesulfonate (MMS, 1-2 mM), but repair of MMS damage was not inhibited by UV light. Because the pathways for excision of pyrimidine dimers and alkylation damage have previously been shown to be different, this observation implies a direct effect of alkylation on repair enzymes. We estimate that if inhibition is due to protein alkylation, the UV repair system must present an extremely large target to alkylation and may involve a complex of protein subunits in the order of 1 million daltons such that 1 or more alkylations occur per complex at the concentrations used. These results also indicate that the method of exposing cells to 2 DNA-damaging agents to determine whether they are repaired by common or different pathways can be quite unreliable because of other effects on the repair systems themselves. PMID:7196494

  11. Inhibition of human copper trafficking by a small molecule significantly attenuates cancer cell proliferation.

    PubMed

    Wang, Jing; Luo, Cheng; Shan, Changliang; You, Qiancheng; Lu, Junyan; Elf, Shannon; Zhou, Yu; Wen, Yi; Vinkenborg, Jan L; Fan, Jun; Kang, Heebum; Lin, Ruiting; Han, Dali; Xie, Yuxin; Karpus, Jason; Chen, Shijie; Ouyang, Shisheng; Luan, Chihao; Zhang, Naixia; Ding, Hong; Merkx, Maarten; Liu, Hong; Chen, Jing; Jiang, Hualiang; He, Chuan

    2015-12-01

    Copper is a transition metal that plays critical roles in many life processes. Controlling the cellular concentration and trafficking of copper offers a route to disrupt these processes. Here we report small molecules that inhibit the human copper-trafficking proteins Atox1 and CCS, and so provide a selective approach to disrupt cellular copper transport. The knockdown of Atox1 and CCS or their inhibition leads to a significantly reduced proliferation of cancer cells, but not of normal cells, as well as to attenuated tumour growth in mouse models. We show that blocking copper trafficking induces cellular oxidative stress and reduces levels of cellular ATP. The reduced level of ATP results in activation of the AMP-activated protein kinase that leads to reduced lipogenesis. Both effects contribute to the inhibition of cancer cell proliferation. Our results establish copper chaperones as new targets for future developments in anticancer therapies. PMID:26587712

  12. A Hairpin Ribozyme Inhibits Expression of Diverse Strains of Human Immunodeficiency Virus Type 1

    NASA Astrophysics Data System (ADS)

    Yu, Mang; Ojwang, Joshua; Yamada, Osamu; Hampel, Arnold; Rapapport, Jay; Looney, David; Wong-Staal, Flossie

    1993-07-01

    Ribozymes have enormous potential as antiviral agents. We have previously reported that a hairpin ribozyme expressed under the control of the β-actin promoter that cleaves human immunodeficiency virus type 1 (HIV-1) RNA in the leader sequence can inhibit HIV-1 (pHXB2gpt) expression. For such a ribozyme in a retroviral vector delivery system to be useful in gene therapy for the treatment of HIV-1 infection, it must be able to inhibit the expression of multiple HIV-1 strains. We have now cloned this ribozyme into various regular expression vectors (including retroviral vectors) by using various gene expression control strategies. Here we show by transient transfection that inhibition of expression of diverse strains of HIV-1 can be achieved by this ribozyme expressed in the proper vectors. These data further support the potential of this hairpin ribozyme as a therapeutic agent for HIV-1.

  13. Inhibition of human copper trafficking by a small molecule significantly attenuates cancer cell proliferation

    NASA Astrophysics Data System (ADS)

    Wang, Jing; Luo, Cheng; Shan, Changliang; You, Qiancheng; Lu, Junyan; Elf, Shannon; Zhou, Yu; Wen, Yi; Vinkenborg, Jan L.; Fan, Jun; Kang, Heebum; Lin, Ruiting; Han, Dali; Xie, Yuxin; Karpus, Jason; Chen, Shijie; Ouyang, Shisheng; Luan, Chihao; Zhang, Naixia; Ding, Hong; Merkx, Maarten; Liu, Hong; Chen, Jing; Jiang, Hualiang; He, Chuan

    2015-12-01

    Copper is a transition metal that plays critical roles in many life processes. Controlling the cellular concentration and trafficking of copper offers a route to disrupt these processes. Here we report small molecules that inhibit the human copper-trafficking proteins Atox1 and CCS, and so provide a selective approach to disrupt cellular copper transport. The knockdown of Atox1 and CCS or their inhibition leads to a significantly reduced proliferation of cancer cells, but not of normal cells, as well as to attenuated tumour growth in mouse models. We show that blocking copper trafficking induces cellular oxidative stress and reduces levels of cellular ATP. The reduced level of ATP results in activation of the AMP-activated protein kinase that leads to reduced lipogenesis. Both effects contribute to the inhibition of cancer cell proliferation. Our results establish copper chaperones as new targets for future developments in anticancer therapies.

  14. Sampangine Inhibits Heme Biosynthesis in both Yeast and Human ▿ †

    PubMed Central

    Huang, Zhiwei; Chen, Kaifu; Xu, Tao; Zhang, Jianhuai; Li, Yongxiang; Li, Wei; Agarwal, Ameeta K.; Clark, Alice M.; Phillips, John D.; Pan, Xuewen

    2011-01-01

    The azaoxoaporphine alkaloid sampangine exhibits strong antiproliferation activity in various organisms. Previous studies suggested that it somehow affects heme metabolism and stimulates production of reactive oxygen species (ROS). In this study, we show that inhibition of heme biosynthesis is the primary mechanism of action by sampangine and that increases in the levels of reactive oxygen species are secondary to heme deficiency. We directly demonstrate that sampangine inhibits heme synthesis in the yeast Saccharomyces cerevisiae. It also causes accumulation of uroporphyrinogen and its decarboxylated derivatives, intermediate products of the heme biosynthesis pathway. Our results also suggest that sampangine likely works through an unusual mechanism—by hyperactivating uroporhyrinogen III synthase—to inhibit heme biosynthesis. We also show that the inhibitory effect of sampangine on heme synthesis is conserved in human cells. This study also reveals a surprising essential role for the interaction between the mitochondrial ATP synthase and the electron transport chain. PMID:21908598

  15. Malathion-induced inhibition of human plasma cholinesterase studied by the fluorescence spectroscopy method

    NASA Astrophysics Data System (ADS)

    Pavelkić, V. M.; Krinulović, K. S.; Savić, J. Z.; Ilić, M. A.

    2008-05-01

    The in vitro effect of technical grade malathion was assessed via the kinetic parameters of human plasma butyrylcholinesterase (BChE) using N-methylindoxyl acetate as a substrate for BChE. An inhibitor kinetics study demonstrated the existence of a biphasic inhibition curve, indicating high-and low-affinity binding sites of malathion. The IC 50 values as calculated from the experimental inhibition curves were 1.33 × 10-9 and 1.48 × 10-5 M for the high-and low-affinity binding sites, respectively; Hill’s analysis gave 1.29 × 10-9 and 1.38 × 10-6 M. The Cornish-Bowden plots and their secondary plots indicated that the nature of inhibition was of mixed type with the predominant competitive character of both affinity binding sites.

  16. Antisense oligodeoxynucleotide against human telomerase reverse transcriptase inhibits the proliferation of Eca-109 esophageal carcinoma cells

    PubMed Central

    FAN, XIANG-KUI; YAN, RUI-HUA; LI, BAO-JIANG; CHEN, XIANG-MING; WEI, LIN; WANG, ZHOU

    2014-01-01

    Previous studies have demonstrated that the growth of tumor cells may be inhibited by antisense oligonucleotides (ASODNs) targeted against human telomerase (hTR) or human telomerase reverse transcriptase (hTERT), resulting in antitumor activity in a wide variety of tumors. However, few studies have investigated the effect of hTERT gene-targeted ASODNs on telomerase activity and cell proliferation in human esophageal cancer. In the present study, an MTT assay was used to determine the growth inhibition rate of Eca-109 cells treated with a hTERT-targeted phosphorothioate-ASODN (PS-ASODN). An inverted microscope was used to observe the morphologic changes of the cells following treatment with 5 μM PS-ASODN for 10 days. Telomerase activity was detected using the silver staining semi-quantitative telomeric repeat amplification protocol (TRAP) assay. Following treatment with the PS-ASODN (1–5 μmol/l), the proliferation of the Eca-109 cells was inhibited. The differences in inhibition rate between the PS-ASODN and blank control groups were statistically significant (P<0.05) when the concentration of the PS-ASODN was ≥2 μmol/l, whereas no statistically significant difference was identified between the non-specific-ASODN and blank control groups. The inhibition rate increased gradually as the concentration of the PS-ASODN increased and with time, suggesting that the PS-ASODN inhibited the growth of Eca-109 cells in a concentration-dependent, time-dependent and sequence-specific manner. The growth rate of the cells incubated with the PS-ASODN was reduced compared with that of the control cells. Cells treated with the PS-ASODN became round, suspended and reduced in size. The PS-ASODN was also found to inhibit telomerase activity. The ability of the PS-ASODN to inhibit the telomerase activity and cell proliferation of the Eca-109 cell line suggests that ASODNs have the potential to be novel therapeutic agents for the treatment of esophageal cancer. PMID:25187833

  17. NADPH- and iron-dependent lipid peroxidation inhibit aromatase activity in human placental microsomes.

    PubMed

    Milczarek, Ryszard; Sokołowska, Ewa; Hallmann, Anna; Kaletha, Krystian; Klimek, Jerzy

    2008-06-01

    During pregnancy placenta is the most significant source of lipid hydroperoxides and other reactive oxygen species (ROS). The increased production of lipid peroxides and other ROS is often linked to pre-eclampsia. It is already proved that placental endoplasmic reticulum may be an important place of lipid peroxides and superoxide radical production. In the present study we revealed that NADPH- and iron-dependent lipid peroxidation in human placental microsomes (HPM) inhibit placental aromatase--a key enzyme of estrogen biosynthesis in human placenta. We showed that significant inhibition of this enzyme is caused by small lipid peroxidation (TBARS (thiobarbituric acid-reactive substances)<4nmol/mg microsomal protein (m.p.)). More intensive lipid peroxidation (TBARS>9nmol/mg microsomal protein) diminishes aromatase activity to value being less than 5% of initial value. NADPH- and iron-dependent lipid peroxidation also causes disappearance of cytochrome P450 parallel to observed aromatase activity inhibition. EDTA, alpha-tocopherol, MgCl(2) and superoxide dismutase (SOD) prevent aromatase activity inhibition and cytochrome P450(AROM) degradation. Mannitol and catalase have not effect on TBARS synthesis, aromatase activity and cytochrome P450 degradation. In view of the above we postulate that the inhibition of aromatase activity observed is mainly a consequence of cytochrome P450(AROM) degradation induced by lipid radicals. The role of hydroxyl radical in cytochrome P450 degradation is negligible in our experimental conditions. The results presented here also suggest that the inhibition of aromatase activity can also take place in placenta at in vivo conditions. PMID:18499441

  18. Prolyl oligopeptidase inhibition-induced growth arrest of human gastric cancer cells

    SciTech Connect

    Suzuki, Kanayo; Sakaguchi, Minoru; Tanaka, Satoshi; Yoshimoto, Tadashi; Takaoka, Masanori

    2014-01-03

    Highlights: •We examined the effects of prolyl oligopeptidase (POP) inhibition on p53 null gastric cancer cell growth. •POP inhibition-induced cell growth suppression was associated with an increase in a quiescent G{sub 0} state. •POP might regulate the exit from and/or reentry into the cell cycle. -- Abstract: Prolyl oligopeptidase (POP) is a serine endopeptidase that hydrolyzes post-proline peptide bonds in peptides that are <30 amino acids in length. We recently reported that POP inhibition suppressed the growth of human neuroblastoma cells. The growth suppression was associated with pronounced G{sub 0}/G{sub 1} cell cycle arrest and increased levels of the CDK inhibitor p27{sup kip1} and the tumor suppressor p53. In this study, we investigated the mechanism of POP inhibition-induced cell growth arrest using a human gastric cancer cell line, KATO III cells, which had a p53 gene deletion. POP specific inhibitors, 3-((4-[2-(E)-styrylphenoxy]butanoyl)-L-4-hydroxyprolyl)-thiazolidine (SUAM-14746) and benzyloxycarbonyl-thioprolyl-thioprolinal, or RNAi-mediated POP knockdown inhibited the growth of KATO III cells irrespective of their p53 status. SUAM-14746-induced growth inhibition was associated with G{sub 0}/G{sub 1} cell cycle phase arrest and increased levels of p27{sup kip1} in the nuclei and the pRb2/p130 protein expression. Moreover, SUAM-14746-mediated cell cycle arrest of KATO III cells was associated with an increase in the quiescent G{sub 0} state, defined by low level staining for the proliferation marker, Ki-67. These results indicate that POP may be a positive regulator of cell cycle progression by regulating the exit from and/or reentry into the cell cycle by KATO III cells.

  19. Characterization of the PGE receptor subtype mediating inhibition of superoxide production in human neutrophils.

    PubMed Central

    Talpain, E; Armstrong, R A; Coleman, R A; Vardey, C J

    1995-01-01

    1. The aims of this study were to characterize the EP receptor subtype mediating the inhibition of superoxide anion generation by formyl methionyl leucine phenylalanine (FMLP)-stimulated human neutrophils, and to test the hypothesis that adenosine 3':5'-cyclic monophosphate (cyclic AMP) is the second messenger mediating the inhibition of the neutrophil by prostaglandin (PG)E2. 2. PGE2 (0.001-10 microM) inhibited FMLP (100 nM)-induced O2-generation from human peripheral blood neutrophils in a concentration-dependent manner, with an EC50 of 0.15 +/- 0.03 microM, and a maximum effect ranging from 36-84% (mean inhibition of 68.7 +/- 2.5%, n = 32). 3. The EP2-receptor agonists, misoprostol, 11-deoxy PGE1, AH13205 and butaprost, all at 10 microM, inhibited O2- generation, causing 95.5 +/- 2.9%, 56.8 +/- 5.2%, 37.1 +/- 6.6% and 18.9 +/- 4.4% inhibition respectively, the latter two being much less effective than PGE2. Similarly, the EP1-receptor agonist, 17-phenyl PGE2 (10 microM), and the EP3/EP1-receptor agonist, sulprostone (10 microM), also inhibited O2- generation, causing 32.2 +/- 7.0% and 15.3 +/- 3.4% inhibition respectively. 4. The non-selective phosphodiesterase inhibitor, isobutyl methylxanthine (IBMX, 0.25 mM) inhibited the FMLP response by 54.5 +/- 5.0%. In addition, IBMX shifted concentration-effect curves for PGE2, misoprostol, 11-deoxy PGE1, butaprost, and AH 13205 to the left, to give EC50s of 0.04 +/- 0.03 (n = 13), 0.07 +/- 0.03 (n = 4), 0.08 +/- 0.03 (n = 4), 0.33 +/- 0.13 (n = 4) and 0.41 +/- 0.2 microM (n = 3) respectively, allowing equieffective concentration-ratios (EECs, PGE2 = 1) of 11.5, 5.3, 50.7 and 12.7 to be calculated.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7606349

  20. Induction and inhibition of NAD(P)H: quinone reductase in murine and human skin.

    PubMed

    Merk, H; Jugert, F; Bonnekoh, B; Mahrle, G

    1991-01-01

    The purpose of this study was to characterize the human cutaneous NAD(P)H: quinone reductase (NQR) activity by known inhibitors of different reductases and to compare it with the murine skin and liver NQR activity. This enzyme plays a major role in the defence of cells against oxygen stress because it inhibits the 1-electron reduction of quinones to semiquinones and their subsequent oxidation to quinones termed as quinone redox cycle. It belongs to the aromatic hydrocarbon-responsive (Ah) battery. This gene battery includes Cyp1a1 (cytochrome P-450 IA1), Cyp1a2 (cytochrome P-450 IA2) and Nmo-1 [NAD(P)H: quinone reductase]. In the skin cytochrome P-450 IA1-dependent activity is about 1-5% compared to the corresponding activity in the liver, whereas NQR has the same activity in skin and liver. NQR was determined in the cytoplasm of murine skin, liver, and human keratinocytes using 2,6-dichlorophenolindophenol as the substrate. The Ah-receptor binding compounds, such as coal tar constituents, or 3-methylcholanthrene induce cytochrome P-450-dependent activities such as aryl hydrocarbon hydroxylase or 7-ethoxyresorufin-O-de-ethylase and NQR, whereas butyl hydroxytoluol, which does not bind to the Ah receptor, induces only NQR. For inhibition studies several known inhibitors of dihydrodiol dehydrogenase, aldo-keto and carbonyl reductase activities were used. There was a similar pattern of inhibition of the basal and induced activity in all tissues investigated. Pyrazole, progesterone and phenobarbital did not inhibit, whereas dicoumarol, rutin and indomethacin inhibited NQR activity in murine skin and liver as well as in human keratinocytes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1768430

  1. Selective Time- and NADPH-Dependent Inhibition of Human CYP2E1 by Clomethiazole.

    PubMed

    Stresser, David M; Perloff, Elke S; Mason, Andrew K; Blanchard, Andrew P; Dehal, Shangara S; Creegan, Timothy P; Singh, Ritu; Gangl, Eric T

    2016-08-01

    The sedative clomethiazole (CMZ) has been used in Europe since the mid-1960s to treat insomnia and alcoholism. It has been previously demonstrated in clinical studies to reversibly inhibit human CYP2E1 in vitro and decrease CYP2E1-mediated elimination of chlorzoxazone. We have investigated the selectivity of CMZ inhibition of CYP2E1 in pooled human liver microsomes (HLMs). In a reversible inhibition assay of the major drug-metabolizing cytochrome P450 (P450) isoforms, CYP2A6 and CYP2E1 exhibited IC50 values of 24 µM and 42 µM, respectively with all other isoforms exhibiting values >300 µM. When CMZ was preincubated with NADPH and liver microsomal protein for 30 minutes before being combined with probe substrates, however, more potent inhibition was observed for CYP2E1 and CYP2B6 but not CYP2A6 or other P450 isoforms. The substantial increase in potency of CYP2E1 inhibition upon preincubation enables the use of CMZ to investigate the role of human CYP2E1 in xenobiotic metabolism and provides advantages over other chemical inhibitors of CYP2E1. The KI and kinact values obtained with HLM-catalyzed 6-hydroxylation of chlorzoxazone were 40 µM and 0.35 minute(-1), respectively, and similar to values obtained with recombinant CYP2E1 (41 µM, 0.32 minute(-1)). The KI and kinact values, along with other parameters, were used in a mechanistic static model to explain earlier observations of a profound decrease in the rate of chlorzoxazone elimination in volunteers despite the absence of detectable CMZ in blood. PMID:27149898

  2. Inhibition of eicosanoid formation in human polymorphonuclear leukocytes by high concentrations of magnesium ions.

    PubMed

    Ludwig, P; Petrich, K; Schewe, T; Diezel, W

    1995-12-01

    The cutaneous antiinflammatory action of Dead-Sea brine is thought to be due to magnesium ions. To elucidate their mode of action, we studied the influence of isotonic solutions containing high concentrations of Mg2+ (up to 115mM) on the formation of 5-lipoxygenase-derived eicosanoids in human polymorphonuclear leukocytes. The cells were stimulated by either ionophore A23187 or the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine. We observed a pronounced inhibition of the formation of leukotriene B4 and 5-hydroxyeicosatetraenoic acid from either added [1-14C] or endogenously liberated arachidonic acid. In the latter case, the sum of arachidonic acid and its oxygenation products was also markedly diminished. The inhibitory effects of Mg2+ depended in a reciprocal manner on the concentration of Ca2+ in the incubation medium. An unspecific damage to cells as reason for the inhibitory effects was excluded. Human recombinant 5-lipoxygenase was also inhibited by Mg2+ in the same concentration range (IC50 16 mM). These data suggest that high concentrations of Mg2+ inhibit the eicosanoid metabolism both at the level of the liberation of arachidonic acid and by direct inhibition of the 5-lipoxygenase enzyme. PMID:9072050

  3. Resistance to telomerase inhibition by human squamous cell carcinoma cell lines.

    PubMed

    Bojovic, Bojana; Crowe, David L

    2011-04-01

    Telomeres are nucleoprotein structures at the ends of chromosomes that are composed of a repetitive G rich sequence and telomeric binding proteins. Telomeres prevent the degradation of chromosomal ends and protect against inappropriate recombination. Telomere attrition involves a tumor suppressor pathway that limits the replication of premalignant cells. The loss of telomeric DNA with each round of replication leads to growth arrest accompanied by senescence or apoptosis. Many tumor cells activate the telomerase gene to bypass senescence. Telomerase is a multisubunit ribonucleoprotein that uses an RNA template to catalyze the addition of telomeric DNA to chromosomal ends. Overexpression of the TERT subunit leads to telomere lengthening and extension of the replicative lifespan. Dominant-negative telomerase has been shown to inhibit telomerase activity in many tumor cell lines, and this is associated with telomere shortening and apoptosis. Additionally, pharmacological telomerase inhibitors have been developed which lead to progressive telomere shortening and programmed cell death. In this study, we report a series of human squamous cell carcinoma cell lines that have high telomerase activity and short telomeres. Dominant-negative telomerase expression and pharmacological telomerase inhibition failed to completely inhibit enzymatic activity which was accompanied by the lack of telomere shortening. These cells continued to proliferate and demonstrated fewer responsive genes when treated with a pharmacological telomerase inhibitor. We concluded that some human squamous cell carcinoma cell lines are resistant to telomerase inhibition. PMID:21305252

  4. Inhibition of Wnt Signaling by Silymarin in Human Colorectal Cancer Cells.

    PubMed

    Eo, Hyun Ji; Park, Gwang Hun; Jeong, Jin Boo

    2016-07-01

    Silymarin from milk thistle (Silybum marianum) has been reported to show an anti-cancer activity. In previous study, we reported that silymarin induces cyclin D1 proteasomal degradation through NF-κB-mediated threonine-286 phosphorylation. However, mechanism for the inhibition of Wnt signaling by silymarin still remains unanswered. Thus, we investigated whether silymarin affects Wnt signaling in human colorectal cancer cells to elucidate the additional anti-cancer mechanism of silymarin. Transient transfection with a TOP and FOP FLASH luciferase construct indicated that silymarin suppressed the transcriptional activity of β-catenin/TCF. Silymarin treatment resulted in a decrease of intracellular β-catenin protein but not mRNA. The inhibition of proteasome by MG132 and GSK3β inhibition by SB216763 blocked silymarin-mediated downregulation of β-catenin. In addition, silymarin increased phosphorylation of β-catenin and a point mutation of S33Y attenuated silymarin-mediated β-catenin downregulation. In addition, silymarin decreased TCF4 and increased Axin expression in both protein and mRNA level. From these results, we suggest that silymarin-mediated downregulation of β-catenin and TCF4 may result in the inhibition of Wnt signaling in human colorectal cancer cells. PMID:27068260

  5. Inhibition of Wnt Signaling by Silymarin in Human Colorectal Cancer Cells

    PubMed Central

    Eo, Hyun Ji; Park, Gwang Hun; Jeong, Jin Boo

    2016-01-01

    Silymarin from milk thistle (Silybum marianum) has been reported to show an anti-cancer activity. In previous study, we reported that silymarin induces cyclin D1 proteasomal degradation through NF-κB-mediated threonine-286 phosphorylation. However, mechanism for the inhibition of Wnt signaling by silymarin still remains unanswered. Thus, we investigated whether silymarin affects Wnt signaling in human colorectal cancer cells to elucidate the additional anti-cancer mechanism of silymarin. Transient transfection with a TOP and FOP FLASH luciferase construct indicated that silymarin suppressed the transcriptional activity of β-catenin/TCF. Silymarin treatment resulted in a decrease of intracellular β-catenin protein but not mRNA. The inhibition of proteasome by MG132 and GSK3β inhibition by SB216763 blocked silymarin-mediated downregulation of β-catenin. In addition, silymarin increased phosphorylation of β-catenin and a point mutation of S33Y attenuated silymarin-mediated β-catenin downregulation. In addition, silymarin decreased TCF4 and increased Axin expression in both protein and mRNA level. From these results, we suggest that silymarin-mediated downregulation of β-catenin and TCF4 may result in the inhibition of Wnt signaling in human colorectal cancer cells. PMID:27068260

  6. Human Cytomegalovirus Inhibition by Cardiac Glycosides: Evidence for Involvement of the hERG Gene

    PubMed Central

    Kapoor, Arun; Cai, Hongyi; Forman, Michael; He, Ran; Shamay, Meir

    2012-01-01

    Infection with human cytomegalovirus (HCMV) continues to be a major threat for pregnant women and the immunocompromised population. Although several anti-HCMV therapies are available, the development of new anti-HCMV agents is highly desired. There is growing interest in identifying compounds that might inhibit HCMV by modulating the cellular milieu. Interest in cardiac glycosides (CG), used in patients with congestive heart failure, has increased because of their established anticancer and their suggested antiviral activities. We report that the several CG—digoxin, digitoxin, and ouabain—are potent inhibitors of HCMV at nM concentrations. HCMV inhibition occurred prior to DNA replication, but following binding to its cellular receptors. The levels of immediate early, early, and late viral proteins and cellular NF-κB were significantly reduced in CG-treated cells. The activity of CG in infected cells correlated with the expression of the potassium channel gene, hERG. CMV infection upregulated hERG, whereas CG significantly downregulated its expression. Infection with mouse CMV upregulated mouse ERG (mERG), but treatment with CG did not inhibit virus replication or mERG transcription. These findings suggest that CG may inhibit HCMV by modulating human cellular targets associated with hERG and that these compounds should be studied for their antiviral activities. PMID:22777050

  7. ERP44 inhibits human lung cancer cell migration mainly via IP3R2

    PubMed Central

    Zhai, Kui; Chang, Yan; Yuan, Qi; Yao, Kai-Tai; Ji, Guangju

    2016-01-01

    Cancer cell migration is involved in tumour metastasis. However, the relationship between calcium signalling and cancer migration is not well elucidated. In this study, we used the human lung adenocarcinoma A549 cell line to examine the role of endoplasmic reticulum protein 44 (ERP44), which has been reported to regulate calcium release inside of the endoplasmic reticulum (ER), in cell migration. We found that the inositol 1,4,5-trisphosphate receptors (IP3Rs/ITPRs) inhibitor 2-APB significantly inhibited A549 cell migration by inhibiting cell polarization and pseudopodium protrusion, which suggests that Ca2+ is necessary for A549 cell migration. Similarly, the overexpression of ERP44 reduced intracellular Ca2+ release via IP3Rs, altered cell morphology and significantly inhibited the migration of A549 cells. These phenomena were primarily dependent on IP3R2 because wound healing in A549 cells with IP3R2 rather than IP3R1 or IP3R3 siRNA was markedly inhibited. Moreover, the overexpression of ERP44 did not affect the migration of the human neuroblastoma cell line SH-SY5Y, which mainly expresses IP3R1. Based on the above observations, we conclude that ERP44 regulates A549 cell migration mainly via an IP3R2-dependent pathway. PMID:27347718

  8. 3-Bromopyruvate inhibits cell proliferation and induces apoptosis in CD133+ population in human glioma.

    PubMed

    Xu, Dong-Qiang; Tan, Xiao-Yu; Zhang, Bao-Wei; Wu, Tao; Liu, Ping; Sun, Shao-Jun; Cao, Yin-Guang

    2016-03-01

    The study was aimed to investigate the role of 3-bromopyruvate in inhibition of CD133+ U87 human glioma cell population growth. The results demonstrated that 3-bromopyruvate inhibited the viability of both CD133+ and parental cells derived from U87 human glioma cell line. However, the 3-bromopyruvate-induced inhibition in viability was more prominent in CD133+ cells at 10 μM concentration after 48 h. Treatment of CD133+ cells with 3-bromopyruvate caused reduction in cell population and cell size, membrane bubbling, and degradation of cell membranes. Hoechst 33258 staining showed condensation of chromatin material and fragmentation of DNA in treated CD133+ cells after 48 h. 3-Bromopyruvate inhibited the migration rate of CD133+ cells significantly compared to the parental cells. Flow cytometry revealed that exposure of CD133+ cells to 3-bromopyruvate increased the cell population in S phase from 24.5 to 37.9 % with increase in time from 12 to 48 h. In addition, 3-bromopyruvate significantly enhanced the expression of Bax and cleaved caspase 3 in CD133+ cells compared to the parental cells. Therefore, 3-bromopyruvate is a potent chemotherapeutic agent for the treatment of glioma by targeting stem cells selectively. PMID:26453119

  9. Recombinant human diamine oxidase activity is not inhibited by ethanol, acetaldehyde, disulfiram, diethyldithiocarbamate or cyanamide.

    PubMed

    Bartko, Johann; Gludovacz, Elisabeth; Petroczi, Karin; Borth, Nicole; Jilma, Bernd; Boehm, Thomas

    2016-08-01

    Human diamine oxidase (hDAO, EC 1.4.3.22) is the key enzyme in the degradation of extracellular histamine. Consumption of alcohol is a known trigger of mast cell degranulation in patients with mast cell activation syndrome. Ethanol may also interfere with enzymatic histamine degradation, but reports on the effects on DAO activity are controversial. There are also conflicting reports whether disulfiram, an FDA-approved agent in the treatment of alcohol dependence, inhibits DAO. We therefore investigated the inhibitory potential of ethanol and disulfiram and their metabolites on recombinant human DAO (rhDAO) in three different assay systems. Relevant concentrations of ethanol, acetaldehyde, and acetate did not inhibit rhDAO activity in an in vitro assay system using horseradish peroxidase (HRP) -mediated luminol oxidation. The aldehyde dehydrogenase (ALDH; EC 1.2.1.3) inhibitors cyanamide and its dimer dicyanamide also had no effect on DAO activity. In one assay system, the irreversible ALDH inhibitor disulfiram and its main metabolite diethyldithiocarbamate seemed to inhibit DAO activity. However, the decreased product formation was not due to a direct block of DAO activity but resulted from inhibition of peroxidase employed in the coupled system. Our in vitro data do not support a direct blocking effect of ethanol, disulfiram, and their metabolites on DAO activity in vivo. PMID:27401969

  10. Inhibition of human GLUT1 and GLUT5 by plant carbohydrate products; insights into transport specificity.

    PubMed

    George Thompson, Alayna M; Iancu, Cristina V; Nguyen, Thi Thanh Hanh; Kim, Doman; Choe, Jun-yong

    2015-01-01

    Glucose transporters GLUT1 (transports glucose) and GLUT5 (transports fructose), in addition to their functions in normal metabolism, have been implicated in several diseases including cancer and diabetes. While GLUT1 has several inhibitors, none have been described for GLUT5. By transport activity assays we found two plant products, rubusoside (from Rubus suavissimus) and astragalin-6-glucoside (a glycosylated derivative of astragalin, from Phytolacca americana) that inhibited human GLUT5. These plants are utilized in traditional medicine: R. suavissimus for weight loss and P. americana for cancer treatment, but the molecular interactions of these products are unknown. Rubusoside also inhibited human GLUT1, but astragalin-6-glucoside did not. In silico analysis of rubusoside:protein interactions pinpointed a major difference in substrate cavity between these transporters, a residue that is a tryptophan in GLUT1 but an alanine in GLUT5. Investigation of mutant proteins supported the importance of this position in ligand specificity. GLUT1W388A became susceptible to inhibition by astragalin-6-glucoside and resistant to rubusoside. GLUT5A396W transported fructose and also glucose, and maintained inhibition by rubusoside and astragalin-6-glucoside. Astragalin-6-glucoside can serve as a starting point in the design of specific inhibitors for GLUT5. The application of these studies to understanding glucose transporters and their interaction with substrates and ligands is discussed. PMID:26306809

  11. Inhibition of human GLUT1 and GLUT5 by plant carbohydrate products; insights into transport specificity

    PubMed Central

    George Thompson, Alayna M.; Iancu, Cristina V.; Nguyen, Thi Thanh Hanh; Kim, Doman; Choe, Jun-yong

    2015-01-01

    Glucose transporters GLUT1 (transports glucose) and GLUT5 (transports fructose), in addition to their functions in normal metabolism, have been implicated in several diseases including cancer and diabetes. While GLUT1 has several inhibitors, none have been described for GLUT5. By transport activity assays we found two plant products, rubusoside (from Rubus suavissimus) and astragalin-6-glucoside (a glycosylated derivative of astragalin, from Phytolacca americana) that inhibited human GLUT5. These plants are utilized in traditional medicine: R. suavissimus for weight loss and P. americana for cancer treatment, but the molecular interactions of these products are unknown. Rubusoside also inhibited human GLUT1, but astragalin-6-glucoside did not. In silico analysis of rubusoside:protein interactions pinpointed a major difference in substrate cavity between these transporters, a residue that is a tryptophan in GLUT1 but an alanine in GLUT5. Investigation of mutant proteins supported the importance of this position in ligand specificity. GLUT1W388A became susceptible to inhibition by astragalin-6-glucoside and resistant to rubusoside. GLUT5A396W transported fructose and also glucose, and maintained inhibition by rubusoside and astragalin-6-glucoside. Astragalin-6-glucoside can serve as a starting point in the design of specific inhibitors for GLUT5. The application of these studies to understanding glucose transporters and their interaction with substrates and ligands is discussed. PMID:26306809

  12. Inhibition of the Differentiation of Monocyte-Derived Dendritic Cells by Human Gingival Fibroblasts

    PubMed Central

    Séguier, Sylvie; Tartour, Eric; Guérin, Coralie; Couty, Ludovic; Lemitre, Mathilde; Lallement, Laetitia; Folliguet, Marysette; Naderi, Samah El; Terme, Magali; Badoual, Cécile; Lafont, Antoine; Coulomb, Bernard

    2013-01-01

    We investigated whether gingival fibroblasts (GFs) can modulate the differentiation and/or maturation of monocyte-derived dendritic cells (DCs) and analyzed soluble factors that may be involved in this immune modulation. Experiments were performed using human monocytes in co-culture with human GFs in Transwell® chambers or using monocyte cultures treated with conditioned media (CM) from GFs of four donors. The four CM and supernatants from cell culture were assayed by ELISA for cytokines involved in the differentiation of dendritic cells, such as IL-6, VEGF, TGFβ1, IL-13 and IL-10. The maturation of monocyte-derived DCs induced by LPS in presence of CM was also studied. Cell surface phenotype markers were analyzed by flow cytometry. In co-cultures, GFs inhibited the differentiation of monocyte-derived DCs and the strength of this blockade correlated with the GF/monocyte ratio. Conditioned media from GFs showed similar effects, suggesting the involvement of soluble factors produced by GFs. This inhibition was associated with a lower stimulatory activity in MLR of DCs generated with GFs or its CM. Neutralizing antibodies against IL-6 and VEGF significantly (P<0.05) inhibited the inhibitory effect of CM on the differentiation of monocytes-derived DCs and in a dose dependent manner. Our data suggest that IL-6 is the main factor responsible for the inhibition of DCs differentiation mediated by GFs but that VEGF is also involved and constitutes an additional mechanism. PMID:23936476

  13. Buthionine sulfoximine inhibition of cystine uptake and glutathione biosynthesis in human lung carcinoma cells

    SciTech Connect

    Brodie, A.E.; Reed, D.J.

    1985-03-15

    Intracellular glutathione (GSH) content of human lung carcinoma cells, A549, in log phase was 25 +/- 5 nmol/10(6) cells, which is considerably higher than that reported in other tumor cells. After partial depletion of GSH with diethyl maleate (DEM), addition of cystine to the medium allowed full resynthesis of GSH in 4 hr, cysteine in the same time period led to less resynthesis, and methionine provided minimal resynthesis. Using cystine as the sole sulfur source and with buthionine sulfoximine (BSO, 5 mM) included in the medium after cells were depleted with DEM, inhibition of both cystine uptake and resynthesis of GSH occurred. BSO inhibited (/sup 35/S)cystine uptake (as early as 10 min) in a concentration-dependent process, ranging from a 28% decrease for 1 microM BSO to an 85% decrease for 100 microM BSO compared to the control cells after 240 min of incubation. In addition, GSH resynthesis from (/sup 35/S)cystine for 240 min was inhibited in a parallel dose-dependent manner, in that 1 microM BSO caused a 27% decrease and 100 microM BSO provided a 75% decrease from control values. BSO did not inhibit the uptake of (/sup 35/S)methionine, but inhibited the low amount of resynthesis of GSH when methionine was the sole sulfur source. BSO did not inhibit the uptake of arginine, phenylalanine, and leucine. DL-, L-, and methyl ester-BSO each inhibited (/sup 35/S)cystine uptake and incorporation into GSH to a similar extent. The half-life of GSH was 3.5 +/- 0.4 hr in A549 cells that were grown in complete medium with GSH synthesis occurring.

  14. Heparin fragments inhibit human vascular smooth muscle cell proliferation in vitro

    SciTech Connect

    Selden, S.C.; Johnson, W.V.; Maciag, T.

    1986-03-01

    The authors have examined the effect of heparin on human abdominal aortic smooth muscle cell growth. Cell proliferation was inhibited by more than 90% at a concentration of 20 ..mu..g/ml in a 12 day growth assay using heparin from Sigma, Upjohn or Calbiochem. Additionally, 200 ..mu..g/ml Upjohn heparin inhibits /sup 3/H-thymidine incorporation by 50% in short term assays using serum or purified platelet-derived growth factor (25-100ng/ml) to initiate the cell cycle. Homogeneous size classes of heparin fragments were prepared by nitrous acid cleavage and BioGel P-10 filtration chromatography. Deca-, octa-, hexa-, tetra-, and di-saccharides inhibited proliferation by 90% at concentrations of 280, 320, 260, 180 and 100 ..mu..g/ml, respectively, in a 12 day growth assay. These data confirm the work of Castellot et.al. and extend the range of inhibitory fragments down to the tetra- and di-saccharide size. These data suggest, therefore, that di-saccharide subunit of heparin is sufficient to inhibit vascular smooth muscle cell proliferation. The authors are now examining the role of the anhydromannose moiety on the reducing end of the nitrous acid generated fragments as a possible mediator of heparin-induced inhibition of vascular smooth muscle cell proliferation.

  15. RNase P Ribozymes Inhibit the Replication of Human Cytomegalovirus by Targeting Essential Viral Capsid Proteins

    PubMed Central

    Yang, Zhu; Reeves, Michael; Ye, Jun; Trang, Phong; Zhu, Li; Sheng, Jingxue; Wang, Yu; Zen, Ke; Wu, Jianguo; Liu, Fenyong

    2015-01-01

    An engineered RNase P-based ribozyme variant, which was generated using the in vitro selection procedure, was used to target the overlapping mRNA region of two proteins essential for human cytomegalovirus (HCMV) replication: capsid assembly protein (AP) and protease (PR). In vitro studies showed that the generated variant, V718-A, cleaved the target AP mRNA sequence efficiently and its activity was about 60-fold higher than that of wild type ribozyme M1-A. Furthermore, we observed a reduction of 98%–99% in AP/PR expression and an inhibition of 50,000 fold in viral growth in cells with V718-A, while a 75% reduction in AP/PR expression and a 500-fold inhibition in viral growth was found in cells with M1-A. Examination of the antiviral effects of the generated ribozyme on the HCMV replication cycle suggested that viral DNA encapsidation was inhibited and as a consequence, viral capsid assembly was blocked when the expression of AP and PR was inhibited by the ribozyme. Thus, our study indicates that the generated ribozyme variant is highly effective in inhibiting HCMV gene expression and blocking viral replication, and suggests that engineered RNase P ribozyme can be potentially developed as a promising gene-targeting agent for anti-HCMV therapy. PMID:26114473

  16. Nordihydroguaiaretic Acid Inhibits Insulin-Like Growth Factor Signaling, Growth, and Survival in Human Neuroblastoma Cells

    PubMed Central

    Meyer, Gary E.; Chesler, Louis; Liu, Dandan; Gable, Karissa; Maddux, Betty A.; Goldenberg, David D.; Youngren, Jack F.; Goldfine, Ira D.; Weiss, William A.; Matthay, Katherine K.; Rosenthal, Stephen M.

    2010-01-01

    Neuroblastoma is a common pediatric malignancy that metastasizes to the liver, bone, and other organs. Children with metastatic disease have a less than 50% chance of survival with current treatments. Insulin-like growth factors (IGFs) stimulate neuroblastoma growth, survival, and motility, and are expressed by neuroblastoma cells and the tissues they invade. Thus, therapies that disrupt the effects of IGFs on neuroblastoma tumorigenesis may slow disease progression. We show that NVP-AEW541, a specific inhibitor of the IGF-I receptor (IGF-IR), potently inhibits neuroblastoma growth in vitro. Nordihydroguaiaretic acid (NDGA), a phenolic compound isolated from the creosote bush (Larrea divaricata), has anti-tumor properties against a number of malignancies, has been shown to inhibit the phosphorylation and activation of the IGF-IR in breast cancer cells, and is currently in Phase I trials for prostate cancer. In the present study in neuroblastoma, NDGA inhibits IGF-I-mediated activation of the IGF-IR and disrupts activation of ERK and Akt signaling pathways induced by IGF-I. NDGA inhibits growth of neuroblastoma cells and induces apoptosis at higher doses, causing IGF-I-resistant activation of caspase-3 and a large increase in the fraction of sub-G0 cells. In addition, NDGA inhibits the growth of xenografted human neuroblastoma tumors in nude mice. These results indicate that NDGA may be useful in the treatment of neuroblastoma and may function in part via disruption of IGF-IR signaling. PMID:17486636

  17. Modulation of recurrent inhibition from knee extensors to ankle motoneurones during human walking

    PubMed Central

    Lamy, Jean-Charles; Iglesias, Caroline; Lackmy, Alexandra; Nielsen, Jens Bo; Katz, Rose; Marchand-Pauvert, Véronique

    2008-01-01

    The neural control for muscle coordination during human locomotion involves spinal and supraspinal networks, but little is known about the exact mechanisms implicated. The present study focused on modulation of heteronymous recurrent inhibition from knee extensors to ankle motoneurones at different times in the gait cycle, when quadriceps (Quad) muscle activity overlaps that in tibialis anterior (TA) and soleus (Sol). The effects of femoral nerve stimulation on ankle motoneurones were investigated during treadmill walking and during tonic co-contraction of Quad and TA/Sol while standing. Recurrent inhibition of TA motoneurones depended on the level of background EMG, and was similar during walking and standing for matched background EMG levels. On the other hand, recurrent inhibition in Sol was reduced in early stance, with respect to standing, and enhanced in late stance. Reduced inhibition in Sol was also observed when Quad was coactivated with TA around the time of heel contact, compared to standing at matched background EMG levels in the two muscles. The modulation of recurrent inhibition of Sol during walking might reflect central and/or peripheral control of the Renshaw cells. These modulations could be implicated in the transition phases, from swing to stance to assist Sol activation during the stance phase, and from stance to swing, for its deactivation. PMID:18936080

  18. Telomerase inhibition effectively targets mouse and human AML stem cells and delays relapse following chemotherapy.

    PubMed

    Bruedigam, Claudia; Bagger, Frederik O; Heidel, Florian H; Paine Kuhn, Catherine; Guignes, Solene; Song, Axia; Austin, Rebecca; Vu, Therese; Lee, Erwin; Riyat, Sarbjit; Moore, Andrew S; Lock, Richard B; Bullinger, Lars; Hill, Geoffrey R; Armstrong, Scott A; Williams, David A; Lane, Steven W

    2014-12-01

    Acute myeloid leukemia (AML) is an aggressive and lethal blood cancer maintained by rare populations of leukemia stem cells (LSCs). Selective targeting of LSCs is a promising approach for treating AML and preventing relapse following chemotherapy, and developing such therapeutic modalities is a key priority. Here, we show that targeting telomerase activity eradicates AML LSCs. Genetic deletion of the telomerase subunit Terc in a retroviral mouse AML model induces cell-cycle arrest and apoptosis of LSCs, and depletion of telomerase-deficient LSCs is partially rescued by p53 knockdown. Murine Terc(-/-) LSCs express a specific gene expression signature that can be identified in human AML patient cohorts and is positively correlated with patient survival following chemotherapy. In xenografts of primary human AML, genetic or pharmacological inhibition of telomerase targets LSCs, impairs leukemia progression, and delays relapse following chemotherapy. Altogether, these results establish telomerase inhibition as an effective strategy for eliminating AML LSCs. PMID:25479751

  19. Theophylline prevents NAD{sup +} depletion via PARP-1 inhibition in human pulmonary epithelial cells

    SciTech Connect

    Moonen, Harald J.J. . E-mail: h.moonen@grat.unimaas.nl; Geraets, Liesbeth; Vaarhorst, Anika; Bast, Aalt; Wouters, Emiel F.M.; Hageman, Geja J.

    2005-12-30

    Oxidative DNA damage, as occurs during exacerbations in chronic obstructive pulmonary disease (COPD), highly activates the nuclear enzyme poly(ADP-ribose)polymerase-1 (PARP-1). This can lead to cellular depletion of its substrate NAD{sup +}, resulting in an energy crisis and ultimately in cell death. Inhibition of PARP-1 results in preservation of the intracellular NAD{sup +} pool, and of NAD{sup +}-dependent cellular processes. In this study, PARP-1 activation by hydrogen peroxide decreased intracellular NAD{sup +} levels in human pulmonary epithelial cells, which was found to be prevented in a dose-dependent manner by theophylline, a widely used compound in the treatment of COPD. This enzyme inhibition by theophylline was confirmed in an ELISA using purified human PARP-1 and was found to be competitive by nature. These findings provide new mechanistic insights into the therapeutic effect of theophylline in oxidative stress-induced lung pathologies.

  20. Telomerase Inhibition Effectively Targets Mouse and Human AML Stem Cells and Delays Relapse Following Chemotherapy

    PubMed Central

    Bruedigam, Claudia; Bagger, Frederik O.; Heidel, Florian H.; Kuhn, Catherine Paine; Guignes, Solene; Song, Axia; Austin, Rebecca; Vu, Therese; Lee, Erwin; Riyat, Sarbjit; Moore, Andrew S.; Lock, Richard B.; Bullinger, Lars; Hill, Geoffrey R.; Armstrong, Scott A.; Williams, David A.; Lane, Steven W.

    2014-01-01

    SUMMARY Acute myeloid leukemia (AML) is an aggressive and lethal blood cancer maintained by rare populations of leukemia stem cells (LSCs). Selective targeting of LSCs is a promising approach for treating AML and preventing relapse following chemotherapy, and developing such therapeutic modalities is a key priority. Here, we show that targeting telomerase activity eradicates AML LSCs. Genetic deletion of the telomerase subunit Terc in a retroviral mouse AML model induces cell cycle arrest and apoptosis of LSCs, and depletion of telomerase-deficient LSCs is partially rescued by p53 knockdown. Murine Terc−/− LSCs express a specific gene expression signature that can be identified in human AML patient cohorts and is positively correlated with patient survival following chemotherapy. In xenografts of primary human AML, genetic or pharmacological inhibition of telomerase targets LSCs, impairs leukemia progression, and delays relapse following chemotherapy. Together, these results establish telomerase inhibition as an effective strategy for eliminating AML LSCs. PMID:25479751

  1. A large blood pressure-raising effect of nitric oxide synthase inhibition in humans

    NASA Technical Reports Server (NTRS)

    Sander, M.; Chavoshan, B.; Victor, R. G.; Blomqvist, C. G. (Principal Investigator)

    1999-01-01

    In experimental animals, systemic administration of nitric oxide synthase (NOS) inhibitors causes large increases in blood pressure that are in part sympathetically mediated. The aim of this study was to determine the extent to which these conclusions can be extrapolated to humans. In healthy normotensive humans, we measured blood pressure in response to two NOS inhibitors, NG-monomethyl-L-arginine (L-NMMA) and NG-nitro-L-arginine methyl ester (L-NAME), the latter of which recently became available for use in humans. The major new findings are 3-fold. First, L-NAME produced robust increases in blood pressure that were more than 2 times larger than those previously reported in humans with L-NMMA and approximated those seen in experimental animals. L-NAME (4 mg/kg) raised mean arterial pressure by 24+/-2 mm Hg (n=27, P<0.001), whereas in subjects who received both inhibitors, a 12-fold higher dose of L-NMMA (50 mg/kg) raised mean arterial pressure by 15+/-2 mm Hg (n=4, P<0.05 vs L-NAME). Second, the L-NAME-induced increases in blood pressure were caused specifically by NOS inhibition because they were reversed by L-arginine (200 mg/kg, n=12) but not D-arginine (200 mg/kg, n=6) and because NG-nitro-D-arginine methyl ester (4 mg/kg, n=5) had no effect on blood pressure. Third, in humans, there is an important sympathetic component to the blood pressure-raising effect of NOS inhibition. alpha-Adrenergic blockade with phentolamine (0.2 mg/kg, n=9) attenuated the L-NAME-induced increase in blood pressure by 40% (P<0.05). From these data, we conclude that pharmacological inhibition of NOS causes large increases in blood pressure that are in part sympathetically mediated in humans as well as experimental animals.

  2. TASK-1 current is inhibited by phosphorylation during human and canine chronic atrial fibrillation.

    PubMed

    Harleton, Erin; Besana, Alessandra; Chandra, Parag; Danilo, Peter; Rosen, Tove S; Rosen, Michael R; Argenziano, Michael; Robinson, Richard B; Feinmark, Steven J

    2015-01-15

    Atrial fibrillation (AF) is a common arrhythmia with significant morbidities and only partially adequate therapeutic options. AF is associated with atrial remodeling processes, including changes in the expression and function of ion channels and signaling pathways. TWIK protein-related acid-sensitive K+ channel (TASK)-1, a two-pore domain K+ channel, has been shown to contribute to action potential repolarization as well as to the maintenance of resting membrane potential in isolated myocytes, and TASK-1 inhibition has been associated with the induction of perioperative AF. However, the role of TASK-1 in chronic AF is unknown. The present study investigated the function, expression, and phosphorylation of TASK-1 in chronic AF in atrial tissue from chronically paced canines and in human subjects. TASK-1 current was present in atrial myocytes isolated from human and canine hearts in normal sinus rhythm but was absent in myocytes from humans with AF and in canines after the induction of AF by chronic tachypacing. The addition of phosphatase to the patch pipette rescued TASK-1 current from myocytes isolated from AF hearts, indicating that the change in current is phosphorylation dependent. Western blot analysis showed that total TASK-1 protein levels either did not change or increased slightly in AF, despite the absence of current. In studies of perioperative AF, we have shown that phosphorylation of TASK-1 at Thr383 inhibits the channel. However, phosphorylation at this site was unchanged in atrial tissue from humans with AF or in canines with chronic pacing-induced AF. We conclude that phosphorylation-dependent inhibition of TASK-1 is associated with AF, but the phosphorylation site responsible for this inhibition remains to be identified. PMID:25437921

  3. Anthraquinone emodin inhibits human cancer cell invasiveness by antagonizing P2X7 receptors.

    PubMed

    Jelassi, Bilel; Anchelin, Monique; Chamouton, Julie; Cayuela, María Luisa; Clarysse, Lucie; Li, Junying; Goré, Jacques; Jiang, Lin-Hua; Roger, Sébastien

    2013-07-01

    The adenosine 5'-triphosphate (ATP)-gated Ca(2+)-permeable channel P2X7 receptor (P2X7R) is strongly upregulated in many tumors and cancer cells, and has an important role in cancer cell invasion associated with metastases. Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is an anthraquinone derivative originally isolated from Rheum officinale Baill known for decades to possess anticancer properties. In this study, we examined the effects of emodin on P2X7R-dependent Ca(2+) signaling, extracellular matrix degradation, and in vitro and in vivo cancer cell invasiveness using highly aggressive human cancer cells. Inclusion of emodin at doses ≤10 µM in cell culture had no or very mild effect on the cell viability. ATP elicited increases in intracellular Ca(2+) concentration were reduced by 35 and 60% by 1 and 10 µM emodin, respectively. Emodin specifically inhibited P2X7R-mediated currents with an IC50 of 3 µM and did not inhibit the currents mediated by the other human P2X receptors heterologously expressed in human embryonic kidney (HEK293T) cells. ATP-induced increase in gelatinolytic activity, in cancer cell invasiveness in vitro and in cell morphology changes were prevented by 1 µM emodin. Furthermore, such ATP-evoked effects and inhibition by emodin were almost completely ablated in cancer cells transfected with P2X7R-specific small interfering RNA (siRNA) but not with scrambled siRNA. Finally, the in vivo invasiveness of the P2X7R-positive MDA-MB-435s breast cancer cells, assessed using a zebrafish model of micrometastases, was suppressed by 40 and 50% by 1 and 10 µM emodin. Taken together, these results provide consistent evidence to indicate that emodin inhibits human cancer cell invasiveness by specifically antagonizing the P2X7R. PMID:23524196

  4. Diosgenin inhibits IL-1β-induced expression of inflammatory mediators in human osteoarthritis chondrocytes.

    PubMed

    Wang, Leisheng; Ma, Tian; Zheng, Yanpin; Lv, Shiqiao; Li, Yu; Liu, Shaoxian

    2015-01-01

    It is well known that the inflammatory cytokines play important roles in osteoarthritis (OA). Diosgenin is a steroidal saponin found in several plants including Solanum and Dioscorea species and possesses diverse biological activities including anti-inflammatory properties. However, the role of diosgenin in inflammatory responses in OA chondrocytes is still unclear. Therefore, in this study, we investigated the anti-inflammatory properties of diosgenin in human OA chondrocytes. We found that diosgenin inhibited the production of nitric oxide (NO) and prostaglandin E2 (PGE2) induced by interleukin-1-beta (IL-1β). Diosgenin significantly inhibited the IL-1β-stimulated expression of metalloproteinase-3 (MMP-3), MMP-13, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in human OA chondrocytes. In addition, diosgenin suppressed the degradation of IκB-α in IL-1β-induced human OA chondrocytes. Taken together, this study showed that diosgenin can effectively inhibit the IL-1β-induced expression of inflammatory mediators, suggesting that diosgenin may be a potential agent in the treatment of OA. PMID:26191174

  5. [Inhibition of adherence of Corynebacterium diphtheriae to human buccal epithelium by glycoside hydrolases from marine hydrobiontes].

    PubMed

    Zaporozhets, T S; Makarenkova, I D; Bakunina, I Iu; Burtseva, Iu V; Kusaĭkin, M I; Balabanova, L A; Zviagintseva, T N; Besednova, N N; Rasskazov, V A

    2010-01-01

    A possibility of adhesion inhibition of Corynebacterium diphtheriae to human buccal epithelium by glycoside hydrolases of marine hydrobiontes was investigated using alpha-galactosidase from marine bacterium Pseudoalteromonas sp. KMM 701, total enzyme preparation and beta-1,3-glucanase from marine fungi Chaetomium, total enzyme preparation and beta-1,3-glucanase from marine mollusk Littorina kurila, and total enzyme preparation from crystalline style of marine mollusk Spisula sachalinensis were used. The enzymes were added to test-tubes containing buccal epithelial cells and/or the toxigenic bacterial strain C. diphtheriae No 1129, v. gravis. All the investigated enzymes were able to abort C. diphtheriae adherence, to human buccal epithelocytes. Inhibition of adhesion was more pronounced in the case of treatment of epithelocytes with highly purified enzymes of marine hydrobiontes in comparison with total enzyme preparations. The significant inhibition of C. diphtheriae adhesion was observed when the enzymes were added to the epithelocytes with the attached microorganisms. The results obtained show that glycoside hydrolases of marine hydrobiontes degrade any carbohydrates expressed on cell surface of bacterium or human buccal epithelocytes, impair unique lectin-carbohydrate interaction and prevent the adhesion. PMID:20695214

  6. Diosgenin inhibits IL-1β-induced expression of inflammatory mediators in human osteoarthritis chondrocytes

    PubMed Central

    Wang, Leisheng; Ma, Tian; Zheng, Yanpin; Lv, Shiqiao; Li, Yu; Liu, Shaoxian

    2015-01-01

    It is well known that the inflammatory cytokines play important roles in osteoarthritis (OA). Diosgenin is a steroidal saponin found in several plants including Solanum and Dioscorea species and possesses diverse biological activities including anti-inflammatory properties. However, the role of diosgenin in inflammatory responses in OA chondrocytes is still unclear. Therefore, in this study, we investigated the anti-inflammatory properties of diosgenin in human OA chondrocytes. We found that diosgenin inhibited the production of nitric oxide (NO) and prostaglandin E2 (PGE2) induced by interleukin-1-beta (IL-1β). Diosgenin significantly inhibited the IL-1β-stimulated expression of metalloproteinase-3 (MMP-3), MMP-13, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in human OA chondrocytes. In addition, diosgenin suppressed the degradation of IκB-α in IL-1β-induced human OA chondrocytes. Taken together, this study showed that diosgenin can effectively inhibit the IL-1β-induced expression of inflammatory mediators, suggesting that diosgenin may be a potential agent in the treatment of OA. PMID:26191174

  7. Tormentic Acid Inhibits IL-1β-Induced Inflammatory Response in Human Osteoarthritic Chondrocytes.

    PubMed

    Yang, Yang; Wang, Yawei; Wang, Yumin; Zhao, Meng; Jia, Haobo; Li, Bing; Xing, Dan

    2016-06-01

    The pro-inflammatory cytokine interleukin-1beta (IL-1β) plays critical roles in pathogenesis of osteoarthritis (OA). Tormentic acid (TA), a triterpene isolated from Rosa rugosa, has anti-inflammatory activity. However, the anti-inflammatory effect of TA on OA is still unclear. So, in the present study, we examined the effect of TA on IL-1β-induced inflammatory response in primary human OA chondrocytes. Our results demonstrated that TA significantly decreased the IL-1β-stimulated expression of matrix metalloproteinase-3 (MMP-3) and MMP-13. It also inhibited the IL-1β-induced expression of inducible nitric oxide (NO) synthase (iNOS) and cyclooxygenase-2 (COX-2), as well as the production of NO and prostaglandin E2 (PGE2) in human OA chondrocytes. Furthermore, TA greatly inhibited the IL-1β-induced NF-κB activation. In conclusion, this study is the first to demonstrate the anti-inflammatory activity of TA in human OA chondrocytes. TA significantly inhibits the IL-1β-induced inflammatory response by suppressing the NF-κB signaling pathway. Thus, TA may be a potential agent in the treatment of OA. PMID:27102898

  8. Inhibition of human dendritic cell activation by hydroethanolic but not lipophilic extracts of turmeric (Curcuma longa).

    PubMed

    Krasovsky, Joseph; Chang, David H; Deng, Gary; Yeung, Simon; Lee, Mavis; Leung, Ping Chung; Cunningham-Rundles, Susanna; Cassileth, Barrie; Dhodapkar, Madhav V

    2009-03-01

    Turmeric has been extensively utilized in Indian and Chinese medicine for its immune-modulatory properties. Dendritic cells (DCs) are antigen-presenting cells specialized to initiate and regulate immunity. The ability of DCs to initiate immunity is linked to their activation status. The effects of turmeric on human DCs have not been studied. Here we show that hydroethanolic (HEE) but not lipophilic "supercritical" extraction (SCE) of turmeric inhibits the activation of human DCs in response to inflammatory cytokines. Treatment of DCs with HEE also inhibits the ability of DCs to stimulate the mixed lymphocyte reaction (MLR). Importantly, the lipophilic fraction does not synergize with the hydroethanolic fraction for the ability of inhibiting DC maturation. Rather, culturing of DCs with the combination of HEE and SCE leads to partial abrogation of the effects of HEE on the MLR initiated by DCs. These data provide a mechanism for the anti-inflammatory properties of turmeric. However, they suggest that these extracts are not synergistic and may contain components with mutually antagonistic effects on human DCs. Harnessing the immune effects of turmeric may benefit from specifically targeting the active fractions. PMID:19034830

  9. Inhibition of adipocytogenesis by canonical WNT signaling in human mesenchymal stem cells

    SciTech Connect

    Shen, Longxiang; Glowacki, Julie; Zhou, Shuanhu

    2011-08-01

    The WNT signaling pathway plays important roles in the self-renewal and differentiation of mesenchymal stem cells (MSCs). Little is known about WNT signaling in adipocyte differentiation of human MSCs. In this study, we tested the hypothesis that canonical and non-canonical WNTs differentially regulate in vitro adipocytogenesis in human MSCs. The expression of adipocyte gene PPAR{gamma}2, lipoprotein lipase, and adipsin increased during adipocytogenesis of hMSCs. Simultaneously, the expression of canonical WNT2, 10B, 13, and 14 decreased, whereas non-canonical WNT4 and 11 increased, and WNT5A was unchanged. A small molecule WNT mimetic, SB-216763, increased accumulation of {beta}-catenin protein, inhibited induction of WNT4 and 11 and inhibited adipocytogenesis. In contrast, knockdown of {beta}-catenin with siRNA resulted in spontaneous adipocytogenesis. These findings support the view that canonical WNT signaling inhibits and non-canonical WNT signaling promotes adipocytogenesis in adult human marrow-derived mesenchymal stem cells.

  10. Inhibition of apolipoprotein B and triglyceride secretion in human hepatoma cells (HepG2).

    PubMed

    Haghpassand, M; Wilder, D; Moberly, J B

    1996-07-01

    Apolipoprotein B (apoB), the major protein component of triglyceride-rich lipoproteins, is assembled into a lipoprotein particle via a complex, multistep process. Recent studies indicate that triglyceride-rich lipoprotein assembly requires the activity of the heterodimeric protein, microsomal triglyceride transfer protein (MTP). We identified a novel inhibitor of apolipoprotein B secretion using the human hepatoma cell line, HepG2. CP-10447, a derivative of the hypnotic drug methaqualone (Quaalude), inhibited apoB secretion from HepG2 cells with an IC50 of approximately 5 microM. CP-10447 also inhibited apoB secretion from Caco-2 cells, a model of intestinal lipoprotein production. In experiments using [3H]glycerol as a precursor for triglyceride synthesis, CP-10447 (20 microM) inhibited radiolabeled triglyceride secretion by approximately 83% (P < 0.0001) in HepG2 cells and 76% (P < 0.05) in Caco-2 cells with no effect on radiolabel incorporation into cellular triglyceride, indicating that CP-10447 inhibited triglyceride secretion without affecting triglyceride synthesis. RNA solution hybridization assay indicated that CP-10447 did not affect apoB or apoA-I mRNA levels. Pulse-chase experiments in HepG2 cells confirmed that CP-10447 inhibited the secretion of apoB (not its synthesis) without affecting secretion of total proteins or albumin and suggested that CP-10447 stimulates the early intracellular degradation of apoB in the endoplasmic reticulum (ER). Further studies demonstrated that CP-10447 is a potent inhibitor of human liver microsomal triglyceride transfer activity (IC50 approximately 1.7 microM) in an in vitro assay containing artificial liposomes and partially purified human MTP. These data suggest that CP-10447 may inhibit apoB and triglyceride secretion by inhibiting MTP activity and stimulating the early ER degradation of apoB. CP-10447 should provide a useful tool for further study of the mechanisms of apoB secretion and triglyceride

  11. Metal inhibition of human alkylpurine-DNA-N-glycosylase activityin base excision repair

    SciTech Connect

    Wang, Ping; Guliaev, Anton B.; Hang, Bo

    2006-02-28

    Cadmium (Cd{sup 2+}), nickel (Ni{sup 2+}) and cobalt (Co{sup 2+}) are human and/or animal carcinogens. Zinc (Zn{sup 2+}) is not categorized as a carcinogen, and rather an essential element to humans. Metals were recently shown to inhibit DNA repair proteins that use metals for their function and/or structure. Here we report that the divalent ions Cd{sup 2+}, Ni{sup 2+}, and Zn{sup 2+} can inhibit the activity of a recombinant human N-methylpurine-DNA glycosylase (MPG) toward a deoxyoligonucleotide with ethenoadenine (var epsilonA). MPG removes a variety of toxic/mutagenic alkylated bases and does not require metal for its catalytic activity or structural integrity. At concentrations starting from 50 to 1000 {micro}M, both Cd{sup 2+} and Zn{sup 2+} showed metal-dependent inhibition of the MPG catalytic activity. Ni{sup 2+} also inhibited MPG, but to a lesser extent. Such an effect can be reversed with EDTA addition. In contrast, Co{sup 2+} and Mg{sup 2+} did not inhibit the MPG activity in the same dose range. Experiments using HeLa cell-free extracts demonstrated similar patterns of inactivation of the var epsilonA excision activity by the same metals. Binding of MPG to the substrate was not significantly affected by Cd{sup 2+}, Zn{sup 2+}, and Ni{sup 2+} at concentrations that show strong inhibition of the catalytic function, suggesting that the reduced catalytic activity is not due to altered MPG binding affinity to the substrate. Molecular dynamics (MD) simulations with Zn{sup 2+} showed that the MPG active site has a potential binding site for Zn{sup 2+}, formed by several catalytically important and conserved residues. Metal binding to such a site is expected to interfere with the catalytic mechanism of this protein. These data suggest that inhibition of MPG activity may contribute to metal genotoxicity and depressed repair of alkylation damage by metals in vivo.

  12. Sildenafil inhibits the growth of human colorectal cancer in vitro and in vivo

    PubMed Central

    Mei, Xiao-Long; Yang, Yang; Zhang, Yao-Jun; Li, Yong; Zhao, Jin-Ming; Qiu, Jian-Ge; Zhang, Wen-Ji; Jiang, Qi-Wei; Xue, You-Qiu; Zheng, Di-Wei; Chen, Yao; Qin, Wu-Ming; Wei, Meng-Ning; Shi, Zhi

    2015-01-01

    Colorectal cancer is the third most common human cancer with frequent overexpression of the cGMP-specific phosphodiesterase 5 (PDE5). In the present study, we investigated that the anticancer effect of sildenafil on human colorectal cancer in vitro and in vivo, which is a potent and selective inhibitor of PDE5 for the treatment of erectile dysfunction and pulmonary arterial hypertension in the clinic. Sildenafil significantly induced cell growth inhibition, cell cycle arrest and apoptosis of human colorectal cancer with increased intracellular reactive oxidative specie (ROS) levels, which were accompanied by obvious alterations of related proteins such as CDKs, Cyclins and PARP etc. Pretreatment with ROS scavenger N-acetyl-L-cysteine could reverse sildenafil-induced ROS accumulation and cell apoptosis. Inhibition of the activity of protein kinase G with KT-5823 could enhance sildenafil-induced apoptosis. Furthermore, sildenafil caused the reduction of xenograft models of human colorectal cancer in nude mice. Overall, these findings suggest that sildenafil has the potential to be used for treatment of human colorectal cancer. PMID:26807313

  13. Inhibition of fibroblast growth factor receptor 3-dependent lung adenocarcinoma with a human monoclonal antibody.

    PubMed

    Yin, Yongjun; Ren, Xiaodi; Smith, Craig; Guo, Qianxu; Malabunga, Maria; Guernah, Ilhem; Zhang, Yiwei; Shen, Juqun; Sun, Haijun; Chehab, Nabil; Loizos, Nick; Ludwig, Dale L; Ornitz, David M

    2016-05-01

    Activating mutations in fibroblast growth factor receptor 3 (FGFR3) have been identified in multiple types of human cancer and in congenital birth defects. In human lung cancer, fibroblast growth factor 9 (FGF9), a high-affinity ligand for FGFR3, is overexpressed in 10% of primary resected non-small cell lung cancer (NSCLC) specimens. Furthermore, in a mouse model where FGF9 can be induced in lung epithelial cells, epithelial proliferation and ensuing tumorigenesis is dependent on FGFR3. To develop new customized therapies for cancers that are dependent on FGFR3 activation, we have used this mouse model to evaluate a human monoclonal antibody (D11) with specificity for the extracellular ligand-binding domain of FGFR3, that recognizes both human and mouse forms of the receptor. Here, we show that D11 effectively inhibits signaling through FGFR3 in vitro, inhibits the growth of FGFR3-dependent FGF9-induced lung adenocarcinoma in mice, and reduces tumor-associated morbidity. Given the potency of FGF9 in this mouse model and the absolute requirement for signaling through FGFR3, this study validates the D11 antibody as a potentially useful and effective reagent for treating human cancers or other pathologies that are dependent on activation of FGFR3. PMID:27056048

  14. A humanized anti-DLL4 antibody promotes dysfunctional angiogenesis and inhibits breast tumor growth

    PubMed Central

    Jia, Xuelian; Wang, Wenyi; Xu, Zhuobin; Wang, Shijing; Wang, Tong; Wang, Min; Wu, Min

    2016-01-01

    Blockage of Delta-like 4 (DLL4)-directed Notch signaling induces excessive tip cell formation and endothelial proliferation resulting in dysfunctional angiogenesis in tumors. MMGZ01, as a murine anti-human DLL4 monoclonal antibody, specifically binds to human DLL4 and blocks Notch pathway. Here, the structure of MMGZ01 variable fragment (Fv) was established and framework region (FR) residues which supported complementarily determining region (CDR) loop conformation were identified. Important residues interactions were also identified through docking MMGZ01 Fv with antigen epitope in DLL4. To humanize the murine antibody, we modified MMGZ01 Fv through CDR grafting and the reconstructed antibody (H3L2) maintained similar structure and binding affinity to parental MMGZ01 after back mutation of 12 canonical murine residues in the FRs. Meanwhile, H3L2 promoted human umbilical vein endothelial cell (HUVEC) proliferation through inhibiting DLL4-directed Notch pathway. Moreover, in MDA-MB-231-bearing nude mice, H3L2 induced dysfunctional angiogenesis and tumor cell apoptosis and showed superior anti-tumor activity. In conclusion, H3L2 is an ideal humanized antibody that inhibits tumor growth through targeting DLL4-Notch pathway and has attracting potentials for clinical applications. PMID:27301650

  15. Inhibition of fibroblast growth factor receptor 3-dependent lung adenocarcinoma with a human monoclonal antibody

    PubMed Central

    Yin, Yongjun; Ren, Xiaodi; Smith, Craig; Guo, Qianxu; Malabunga, Maria; Guernah, Ilhem; Zhang, Yiwei; Shen, Juqun; Sun, Haijun; Chehab, Nabil; Loizos, Nick; Ludwig, Dale L.; Ornitz, David M.

    2016-01-01

    ABSTRACT Activating mutations in fibroblast growth factor receptor 3 (FGFR3) have been identified in multiple types of human cancer and in congenital birth defects. In human lung cancer, fibroblast growth factor 9 (FGF9), a high-affinity ligand for FGFR3, is overexpressed in 10% of primary resected non-small cell lung cancer (NSCLC) specimens. Furthermore, in a mouse model where FGF9 can be induced in lung epithelial cells, epithelial proliferation and ensuing tumorigenesis is dependent on FGFR3. To develop new customized therapies for cancers that are dependent on FGFR3 activation, we have used this mouse model to evaluate a human monoclonal antibody (D11) with specificity for the extracellular ligand-binding domain of FGFR3, that recognizes both human and mouse forms of the receptor. Here, we show that D11 effectively inhibits signaling through FGFR3 in vitro, inhibits the growth of FGFR3-dependent FGF9-induced lung adenocarcinoma in mice, and reduces tumor-associated morbidity. Given the potency of FGF9 in this mouse model and the absolute requirement for signaling through FGFR3, this study validates the D11 antibody as a potentially useful and effective reagent for treating human cancers or other pathologies that are dependent on activation of FGFR3. PMID:27056048

  16. A humanized anti-DLL4 antibody promotes dysfunctional angiogenesis and inhibits breast tumor growth.

    PubMed

    Jia, Xuelian; Wang, Wenyi; Xu, Zhuobin; Wang, Shijing; Wang, Tong; Wang, Min; Wu, Min

    2016-01-01

    Blockage of Delta-like 4 (DLL4)-directed Notch signaling induces excessive tip cell formation and endothelial proliferation resulting in dysfunctional angiogenesis in tumors. MMGZ01, as a murine anti-human DLL4 monoclonal antibody, specifically binds to human DLL4 and blocks Notch pathway. Here, the structure of MMGZ01 variable fragment (Fv) was established and framework region (FR) residues which supported complementarily determining region (CDR) loop conformation were identified. Important residues interactions were also identified through docking MMGZ01 Fv with antigen epitope in DLL4. To humanize the murine antibody, we modified MMGZ01 Fv through CDR grafting and the reconstructed antibody (H3L2) maintained similar structure and binding affinity to parental MMGZ01 after back mutation of 12 canonical murine residues in the FRs. Meanwhile, H3L2 promoted human umbilical vein endothelial cell (HUVEC) proliferation through inhibiting DLL4-directed Notch pathway. Moreover, in MDA-MB-231-bearing nude mice, H3L2 induced dysfunctional angiogenesis and tumor cell apoptosis and showed superior anti-tumor activity. In conclusion, H3L2 is an ideal humanized antibody that inhibits tumor growth through targeting DLL4-Notch pathway and has attracting potentials for clinical applications. PMID:27301650

  17. Mechanism of human telomerase inhibition by BIBR1532, a synthetic, non-nucleosidic drug candidate.

    PubMed

    Pascolo, Emanuelle; Wenz, Christian; Lingner, Joachim; Hauel, Norbert; Priepke, Henning; Kauffmann, Iris; Garin-Chesa, Pilar; Rettig, Wolfgang J; Damm, Klaus; Schnapp, Andreas

    2002-05-01

    Telomerase, a ribonucleoprotein acting as a reverse transcriptase, has been identified as a target for cancer drug discovery. The synthetic, non-nucleosidic compound, BIBR1532, is a potent and selective telomerase inhibitor capable of inducing senescence in human cancer cells (). In the present study, the mode of drug action was characterized. BIBR1532 inhibits the native and recombinant human telomerase, comprising the human telomerase reverse transcriptase and human telomerase RNA components, with similar potency primarily by interfering with the processivity of the enzyme. Enzyme-kinetic experiments show that BIBR1532 is a mixed-type non-competitive inhibitor and suggest a drug binding site distinct from the sites for deoxyribonucleotides and the DNA primer, respectively. Thus, BIBR1532 defines a novel class of telomerase inhibitor with mechanistic similarities to non-nucleosidic inhibitors of HIV1 reverse transcriptase. PMID:11854300

  18. An evaluation of the inhibition of human butyrylcholinesterase and acetylcholinesterase by the organophosphate chlorpyrifos oxon

    SciTech Connect

    Shenouda, Josephine; Green, Paula; Sultatos, Lester

    2009-12-01

    Acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8) are enzymes that belong to the superfamily of alpha/beta-hydrolase fold proteins. While they share many characteristics, they also possess many important differences. For example, whereas they have about 54% amino acid sequence identity, the active site gorge of acetylcholinesterase is considerably smaller than that of butyrylcholinesterase. Moreover, both have been shown to display simple and complex kinetic mechanisms, depending on the particular substrate examined, the substrate concentration, and incubation conditions. In the current study, incubation of butyrylthiocholine in a concentration range of 0.005-3.0 mM, with 317 pM human butyrylcholinesterase in vitro, resulted in rates of production of thiocholine that were accurately described by simple Michaelis-Menten kinetics, with a K{sub m} of 0.10 mM. Similarly, the inhibition of butyrylcholinesterase in vitro by the organophosphate chlorpyrifos oxon was described by simple Michaelis-Menten kinetics, with a k{sub i} of 3048 nM{sup -1} h{sup -1}, and a K{sub D} of 2.02 nM. In contrast to inhibition of butyrylcholinesterase, inhibition of human acetylcholinesterase by chlorpyrifos oxon in vitro followed concentration-dependent inhibition kinetics, with the k{sub i} increasing as the inhibitor concentration decreased. Chlorpyrifos oxon concentrations of 10 and 0.3 nM gave k{sub i}s of 1.2 and 19.3 nM{sup -1} h{sup -1}, respectively. Although the mechanism of concentration-dependent inhibition kinetics is not known, the much smaller, more restrictive active site gorge of acetylcholinesterase almost certainly plays a role. Similarly, the much larger active site gorge of butyrylcholinesterase likely contributes to its much greater reactivity towards chlorpyrifos oxon, compared to acetylcholinesterase.

  19. Tetrandrine suppresses proliferation, induces apoptosis, and inhibits migration and invasion in human prostate cancer cells.

    PubMed

    Liu, Wei; Kou, Bo; Ma, Zhen-Kun; Tang, Xiao-Shuang; Lv, Chuan; Ye, Min; Chen, Jia-Qi; Li, Lei; Wang, Xin-Yang; He, Da-Lin

    2015-01-01

    Tetrandrine (TET), a traditional Chinese medicine, exerts remarkable anticancer activity on various cancer cells. However, little is known about the effect of TET on human prostate cancer cells, and the mechanism of function of TET on prostate cancer has not yet been elucidated. To investigate the effects of TET on the suppression of proliferation, induction of apoptosis, and inhibition of migration and invasion in human prostate cancer cell lines, DU145 and PC-3. Inhibition of growth was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and clone formation assay, and flow cytometry analysis was performed to detect the induction of apoptosis. Activation of poly (ADP-ribose) polymerase, caspase-3, Akt, phospho-Akt, Bcl-2, and Bax was analyzed by Western blotting. Wound healing assay and transwell migration assay were used to evaluate the effect of TET on migration and invasion of cancer cells. TET inhibited the growth of DU145 and PC-3 cells in a dose- and time-dependent manner. Cell cloning was inhibited in the presence of TET in DU145 and PC-3 cells. TET suppressed the migration of DU145 and PC-3 cells. Transwell invasion assay showed that TET significantly weakened invasion capacity of DU145 and PC-3 cells. TET exhibited strong inhibitory effect on proliferation, migration, and invasion of prostate cancer cells. In addition, TET induced apoptosis in a dose-dependent manner by activating the caspase cascade and inhibiting phosphoinositide 3-kinase-Akt signal pathway. The accumulating evidence suggests that TET could be a potential therapeutic candidate against prostate cancer in a clinical setting. PMID:25677131

  20. Inhibition of replicon initiation in human cells following stabilization of topoisomerase-DNA cleavable complexes.

    PubMed Central

    Kaufmann, W K; Boyer, J C; Estabrooks, L L; Wilson, S J

    1991-01-01

    Diploid human fibroblast strains were treated for 10 min with inhibitors of type I and type II DNA topoisomerases, and after removal of the inhibitors, the rate of initiation of DNA synthesis at replicon origins was determined. By alkaline elution chromatography, 4'-(9-acridinylamino)methanesulfon-m-anisidide (amsacrine), an inhibitor of DNA topoisomerase II, was shown to produce DNA strand breaks. These strand breaks are thought to reflect drug-induced stabilization of topoisomerase-DNA cleavable complexes. Removal of the drug led to a rapid resealing of the strand breaks by dissociation of the complexes. Velocity sedimentation analysis was used to quantify the effects of amsacrine treatment on DNA replication. It was demonstrated that transient exposure to low concentrations of amsacrine inhibited replicon initiation but did not substantially affect DNA chainelongation within operating replicons. Maximal inhibition of replicon initiation occurred 20 to 30 min after drug treatment, and the initiation rate recovered 30 to 90 min later. Ataxia telangiectasia cells displayed normal levels of amsacrine-induced DNA strand breaks during stabilization of cleavable complexes but failed to downregulate replicon initiation after exposure to the topoisomerase inhibitor. Thus, inhibition of replicon initiation in response to DNA damage appears to be an active process which requires a gene product which is defective or missing in ataxia telangiectasia cells. In normal human fibroblasts, the inhibition of DNA topoisomerase I by camptothecin produced reversible DNA strand breaks. Transient exposure to this drug also inhibited replicon initiation. These results suggest that the cellular response pathway which downregulates replicon initiation following genotoxic damage may respond to perturbations of chromatin structure which accompany stabilization of topoisomerase-DNA cleavable complexes. PMID:1646393

  1. Tetrandrine suppresses proliferation, induces apoptosis, and inhibits migration and invasion in human prostate cancer cells

    PubMed Central

    Liu, Wei; Kou, Bo; Ma, Zhen-Kun; Tang, Xiao-Shuang; Lv, Chuan; Ye, Min; Chen, Jia-Qi; Li, Lei; Wang, Xin-Yang; He, Da-Lin

    2015-01-01

    Tetrandrine (TET), a traditional Chinese medicine, exerts remarkable anticancer activity on various cancer cells. However, little is known about the effect of TET on human prostate cancer cells, and the mechanism of function of TET on prostate cancer has not yet been elucidated. To investigate the effects of TET on the suppression of proliferation, induction of apoptosis, and inhibition of migration and invasion in human prostate cancer cell lines, DU145 and PC-3. Inhibition of growth was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and clone formation assay, and flow cytometry analysis was performed to detect the induction of apoptosis. Activation of poly (ADP-ribose) polymerase, caspase-3, Akt, phospho-Akt, Bcl-2, and Bax was analyzed by Western blotting. Wound healing assay and transwell migration assay were used to evaluate the effect of TET on migration and invasion of cancer cells. TET inhibited the growth of DU145 and PC–3 cells in a dose- and time-dependent manner. Cell cloning was inhibited in the presence of TET in DU145 and PC-3 cells. TET suppressed the migration of DU145 and PC-3 cells. Transwell invasion assay showed that TET significantly weakened invasion capacity of DU145 and PC-3 cells. TET exhibited strong inhibitory effect on proliferation, migration, and invasion of prostate cancer cells. In addition, TET induced apoptosis in a dose-dependent manner by activating the caspase cascade and inhibiting phosphoinositide 3-kinase-Akt signal pathway. The accumulating evidence suggests that TET could be a potential therapeutic candidate against prostate cancer in a clinical setting. PMID:25677131

  2. (-)-Epigallocatechin gallate inhibits the expression of indoleamine 2,3-dioxygenase in human colorectal cancer cells.

    PubMed

    Ogawa, Kengo; Hara, Takeshi; Shimizu, Masahito; Nagano, Junji; Ohno, Tomohiko; Hoshi, Masato; Ito, Hiroyasu; Tsurumi, Hisashi; Saito, Kuniaki; Seishima, Mitsuru; Moriwaki, Hisataka

    2012-09-01

    Immune escape, the ability of tumor cells to avoid tumor-specific immune responses, occurs during the development and progression of several types of human malignancies, including colorectal cancer (CRC). Indoleamine 2,3-dioxygenase (IDO), the tryptophan catabolic enzyme, plays a significant role in regulating the immune response and provides tumor cells with a potent tool to evade the immune system. In the present study, we examined the effects of (-)-epigallocatechin gallate (EGCG), the major catechin in green tea, on the inhibition of IDO expression induced by interferon (IFN)-γ in human CRC cells. We found that IFN-γ increased the expression levels of IDO protein and mRNA in HT29 and SW837 CRC cell lines. Treatment of SW837 cells with EGCG significantly decreased IFN-γ-induced expression of IDO protein and mRNA in a dose-dependent manner. Enzymatic activity of IDO, determined by the concentration of L-kynurenine in the culture medium, was also significantly inhibited by EGCG treatment. Phosphorylation of signal transducer and activator of transcription 1 (STAT1) induced by IFN-γ was also significantly inhibited by EGCG. Reporter assays indicated that EGCG inhibited the transcriptional activities of IDO promoters, IFN-stimulated response element and IFN-γ activation sequence, activated by STAT1 phosphorylation. These findings suggest that EGCG may exert antitumor effects on CRC, at least in part, by inhibiting the expression and function of IDO through the suppression of STAT1 activation. EGCG may, thus, serve as a potential agent for antitumor immunotherapy and be useful in the chemoprevention and/or treatment of CRC. PMID:23741252

  3. Generation and Characterization of Small Single Domain Antibodies Inhibiting Human Tumor Necrosis Factor Receptor 1*

    PubMed Central

    Steeland, Sophie; Puimège, Leen; Vandenbroucke, Roosmarijn E.; Van Hauwermeiren, Filip; Haustraete, Jurgen; Devoogdt, Nick; Hulpiau, Paco; Leroux-Roels, Geert; Laukens, Debby; Meuleman, Philip; De Vos, Martine; Libert, Claude

    2015-01-01

    The cytokine TNF is a well known drug target for several inflammatory diseases such as Crohn disease. Despite the great success of TNF blockers, therapy could be improved because of high costs and side effects. Selective inhibition of TNF receptor (TNFR) 1 signaling holds the potential to greatly reduce the pro-inflammatory activity of TNF, thereby preserving the advantageous immunomodulatory signals mediated by TNFR2. We generated a selective human TNFR1 inhibitor based on Nanobody (Nb) technology. Two anti-human TNFR1 Nbs were linked with an anti-albumin Nb to generate Nb Alb-70-96 named “TNF Receptor-One Silencer” (TROS). TROS selectively binds and inhibits TNF/TNFR1 and lymphotoxin-α/TNFR1 signaling with good affinity and IC50 values, both of which are in the nanomolar range. Surface plasmon resonance analysis reveals that TROS competes with TNF for binding to human TNFR1. In HEK293T cells, TROS strongly reduces TNF-induced gene expression, like IL8 and TNF, in a dose-dependent manner; and in ex vivo cultured colon biopsies of CD patients, TROS inhibits inflammation. Finally, in liver chimeric humanized mice, TROS antagonizes inflammation in a model of acute TNF-induced liver inflammation, reflected in reduced human IL8 expression in liver and reduced IL6 levels in serum. These results demonstrate the considerable potential of TROS and justify the evaluation of TROS in relevant disease animal models of both acute and chronic inflammation and eventually in patients. PMID:25538244

  4. Adhesion of human bifidobacterial strains to cultured human intestinal epithelial cells and inhibition of enteropathogen-cell interactions.

    PubMed Central

    Bernet, M F; Brassart, D; Neeser, J R; Servin, A L

    1993-01-01

    Thirteen human bifidobacterial strains were tested for their abilities to adhere to human enterocyte-like Caco-2 cells in culture. The adhering strains were also tested for binding to the mucus produced by the human mucus-secreting HT29-MTX cell line in culture. A high level of calcium-independent adherence was observed for Bifidobacterium breve 4, for Bifidobacterium infantis 1, and for three fresh human isolates from adults. As observed by scanning electron microscopy, adhesion occurs to the apical brush border of the enterocytic Caco-2 cells and to the mucus secreted by the HT29-MTX mucus-secreting cells. The bacteria interacted with the well-defined apical microvilli of Caco-2 cells without cell damage. The adhesion to Caco-2 cells of bifidobacteria did not require calcium and was mediated by a proteinaceous adhesion-promoting factor which was present both in the bacterial whole cells and in the spent supernatant of bifidobacterium culture. This adhesion-promoting factor appeared species specific, as are the adhesion-promoting factors of lactobacilli. We investigated the inhibitory effect of adhering human bifidobacterial strains against intestinal cell monolayer colonization by a variety of diarrheagenic bacteria. B. breve 4, B. infantis 1, and fresh human isolates were shown to inhibit cell association of enterotoxigenic, enteropathogenic, diffusely adhering Escherichia coli and Salmonella typhimurium strains to enterocytic Caco-2 cells in a concentration-dependent manner. Moreover, B. breve 4 and B. infantis 1 strains inhibited, dose dependently, Caco-2 cell invasion by enteropathogenic E. coli, Yersinia pseudotuberculosis, and S. typhimurium strains. Images PMID:8285709

  5. The HSP90 inhibitor alvespimycin enhances the potency of telomerase inhibition by imetelstat in human osteosarcoma

    PubMed Central

    Hu, Yafang; Bobb, Daniel; He, Jianping; Hill, D Ashley; Dome, Jeffrey S

    2015-01-01

    The unsatisfactory outcomes for osteosarcoma necessitate novel therapeutic strategies. This study evaluated the effect of the telomerase inhibitor imetelstat in pre-clinical models of human osteosarcoma. Because the chaperone molecule HSP90 facilitates the assembly of telomerase protein, the ability of the HSP90 inhibitor alvespimycin to potentiate the effect of the telomerase inhibitor was assessed. The effect of single or combined treatment with imetelstat and alvespimycin on long-term growth was assessed in osteosarcoma cell lines (143B, HOS and MG-63) and xenografts derived from 143B cells. Results indicated that imetelstat as a single agent inhibited telomerase activity, induced telomere shortening, and inhibited growth in all 3 osteosarcoma cell lines, though the bulk cell cultures did not undergo growth arrest. Combined treatment with imetelstat and alvespimycin resulted in diminished telomerase activity and shorter telomeres compared to either agent alone as well as higher levels of γH2AX and cleaved caspase-3, indicative of increased DNA damage and apoptosis. With dual telomerase and HSP90 inhibition, complete growth arrest of bulk cell cultures was achieved. In xenograft models, all 3 treatment groups significantly inhibited tumor growth compared with the placebo-treated control group, with the greatest effect seen in the combined treatment group (imetelstat, p = 0.045, alvespimycin, p = 0.034; combined treatment, p = 0.004). In conclusion, HSP90 inhibition enhanced the effect of telomerase inhibition in pre-clinical models of osteosarcoma. Dual targeting of telomerase and HSP90 warrants further investigation as a therapeutic strategy. PMID:25920748

  6. The HSP90 inhibitor alvespimycin enhances the potency of telomerase inhibition by imetelstat in human osteosarcoma.

    PubMed

    Hu, Yafang; Bobb, Daniel; He, Jianping; Hill, D Ashley; Dome, Jeffrey S

    2015-01-01

    The unsatisfactory outcomes for osteosarcoma necessitate novel therapeutic strategies. This study evaluated the effect of the telomerase inhibitor imetelstat in pre-clinical models of human osteosarcoma. Because the chaperone molecule HSP90 facilitates the assembly of telomerase protein, the ability of the HSP90 inhibitor alvespimycin to potentiate the effect of the telomerase inhibitor was assessed. The effect of single or combined treatment with imetelstat and alvespimycin on long-term growth was assessed in osteosarcoma cell lines (143B, HOS and MG-63) and xenografts derived from 143B cells. Results indicated that imetelstat as a single agent inhibited telomerase activity, induced telomere shortening, and inhibited growth in all 3 osteosarcoma cell lines, though the bulk cell cultures did not undergo growth arrest. Combined treatment with imetelstat and alvespimycin resulted in diminished telomerase activity and shorter telomeres compared to either agent alone as well as higher levels of γH2AX and cleaved caspase-3, indicative of increased DNA damage and apoptosis. With dual telomerase and HSP90 inhibition, complete growth arrest of bulk cell cultures was achieved. In xenograft models, all 3 treatment groups significantly inhibited tumor growth compared with the placebo-treated control group, with the greatest effect seen in the combined treatment group (imetelstat, p = 0.045, alvespimycin, p = 0.034; combined treatment, p = 0.004). In conclusion, HSP90 inhibition enhanced the effect of telomerase inhibition in pre-clinical models of osteosarcoma. Dual targeting of telomerase and HSP90 warrants further investigation as a therapeutic strategy. PMID:25920748

  7. β-catenin knockdown inhibits the proliferation of human glioma cells in vitro and in vivo

    PubMed Central

    WANG, ZHONG; CHEN, QIANXUE

    2016-01-01

    β-catenin is a crucial oncogene that is capable of regulating cancer progression. The aim of the present study was to clarify whether β-catenin was associated with the proliferation and progress of glioma. In order to knockdown the expression of β-catenin in human U251 glioma cells, three pairs of small interfering (si)RNA were designed and synthesized and the most effective siRNA was selected and used for silencing the endogenous β-catenin, which was detected by western blot analysis and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Proliferation was subsequently detected using a methylthiazolyl-tetrazolium bromide assay and the results demonstrated that knockdown of β-catenin significantly inhibited the proliferation of U251 cells in a time- and dose-dependent manner (P<0.01). Cell apoptosis rate was analyzed using flow cytometry and Annexin V-fluorescein isothiocyanate/propidium iodide staining demonstrated that β-catenin siRNA significantly increased the apoptosis of U251 cells (P<0.01). Furthermore, the results of an in vitro scratch assay demonstrated that β-catenin silencing suppressed the proliferation of U251 cells, as compared with the control group (P<0.01). In vivo, β-catenin expression levels in U251 cells were significantly inhibited (P<0.01) following β-catenin short hairpin (sh)RNA lentiviral-vector transfection, as detected by western blot analysis and RT-qPCR. Tumorigenicity experiments demonstrated that β-catenin inhibition significantly increased the survival rate of nude mice. The results of the present study demonstrated that knockdown of β-catenin expression significantly inhibited the progression of human glioma cancer cells, in vitro and in vivo; thus suggesting that β-catenin silencing may be a novel therapy for the treatment of human glioma. PMID:26998037

  8. Inhibition of Human Neutrophil Responses by Essential Oil of Artemisia kotuchovii and Its Constituents

    PubMed Central

    Schepetkin, Igor A.; Kushnarenko, Svetlana V.; Özek, Gulmira; Kirpotina, Liliya N.; Utegenova, Gulzhakhan A.; Kotukhov, Yuriy A.; Danilova, Alevtina N.; Özek, Temel; Başer, K. Hüsnü Can; Quinn, Mark T.

    2015-01-01

    Essential oils were obtained by hydrodistillation of the flowers+leaves and stems of Artemisia kotuchovii Kupr. (AKEOf+l and AKEOstm, respectively) and analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC/MS). The primary components of the oils were estragole, (E)- and (Z)-β-ocimenes, methyl eugenol, limonene, spathulenol, β-pinene, myrcene, and (E)-methyl cinnamate. Seventy four constituents were present at concentrations from 0.1 to 1.0%, and 34 compounds were identified in trace (<0.1%) amounts in one or both plant components. Screening of the essential oils for biological activity showed that AKEOstm, but not AKEOf+l, inhibited N-formyl-Met-Leu-Phe (fMLF)-stimulated Ca2+ flux and chemotaxis and phorbol-12-myristate-13-acetate (PMA)-induced reactive oxygen species (ROS) production in human neutrophils. Selected pure constituents, representing >96% of the AKEOstm composition, were also tested in human neutrophils and HL-60 cells transfected with N-formyl peptide receptor 1 (FPR1). We found that one component, 6-methyl-3,5-heptadien-2-one (MHDO), inhibited fMLF- and interleukin 8 (IL-8)-stimulated Ca2+ flux, fMLF-induced chemotaxis, and PMA-induced ROS production in human neutrophils. MHDO also inhibited fMLF-induced Ca2+ flux in FPR1-HL60 cells. These results suggest that MHDO may be effective in modulating some innate immune responses, possibly by an inhibition of neutrophil migration and ROS production. PMID:25959257

  9. Deuterium-depleted water inhibits human lung carcinoma cell growth by apoptosis

    PubMed Central

    CONG, FENG-SONG; ZHANG, YA-RU; SHENG, HONG-CAI; AO, ZONG-HUA; ZHANG, SU-YI; WANG, JU-YONG

    2010-01-01

    To investigate the in vivo and in vitro inhibitory effects of deuterium-depleted water (DDW) on human lung cancer and the possible mechanisms underlying these effects, we cultured and treated human lung carcinoma cell line A549 and human embryonic lung fibroblasts HLF-1 with various concentrations of DDW from 2 to 72 h. Cellular growth inhibition rates were determined using the 3-(4, 5-dimethyldiazol-2-yl)-2, 5-diphenyltetrazolium-bromide) (MTT) proliferation assay. A549 cells were treated with 50±5 ppm DDW, and the morphology and structure of cells were observed by scanning electron microscopy (SEM). We observed alterations in the cellular skeleton by transmission electron microscopy (TEM) and changes in cell cycle by flow cytometry. Our data showed that DDW significantly inhibited the proliferation of A549 cells at a specific time point, and cells demonstrated the characteristic morphological changes of apoptosis under SEM and TEM. The length of the S phase increased significantly in cells treated with 50 ppm DDW, whereas the G0 to G1 phase and G2 to M phase were decreased. We observed DDW-induced cellular apoptosis using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and DNA fragment analyses. In addition, we established a tumor transplantion model by injecting H460 tumor cells into subcutaneous tissue of BALB/c mice treated with DDW for 60 days. We determined the tumor inhibition rate of treated and control groups and found that the tumor weight was significantly decreased and the tumor inhibition rate was approximately 30% in the DDW group. We conclude that DDW is a promising new anticancer agent with potential for future clinical application. PMID:22993540

  10. Baicalein inhibits the migration and invasive properties of human hepatoma cells

    SciTech Connect

    Chiu, Yung-Wei; Lin, Tseng-Hsi; Huang, Wen-Shih; Teng, Chun-Yuh; Liou, Yi-Sheng; Kuo, Wu-Hsien; Lin, Wea-Lung; Huang, Hai-I; Tung, Jai-Nien; Huang, Chih-Yang; Liu, Jer-Yuh; Wang, Wen-Hung; Hwang, Jin-Ming

    2011-09-15

    Flavonoids have been demonstrated to exert health benefits in humans. We investigated whether the flavonoid baicalein would inhibit the adhesion, migration, invasion, and growth of human hepatoma cell lines, and we also investigated its mechanism of action. The separate effects of baicalein and baicalin on the viability of HA22T/VGH and SK-Hep1 cells were investigated for 24 h. To evaluate their invasive properties, cells were incubated on matrigel-coated transwell membranes in the presence or absence of baicalein. We examined the effect of baicalein on the adhesion of cells, on the activation of matrix metalloproteinases (MMPs), protein kinase C (PKC), and p38 mitogen-activated protein kinase (MAPK), and on tumor growth in vivo. We observed that baicalein suppresses hepatoma cell growth by 55%, baicalein-treated cells showed lower levels of migration than untreated cells, and cell invasion was significantly reduced to 28%. Incubation of hepatoma cells with baicalein also significantly inhibited cell adhesion to matrigel, collagen I, and gelatin-coated substrate. Baicalein also decreased the gelatinolytic activities of the matrix metalloproteinases MMP-2, MMP-9, and uPA, decreased p50 and p65 nuclear translocation, and decreased phosphorylated I-kappa-B (IKB)-{beta}. In addition, baicalein reduced the phosphorylation levels of PKC{alpha} and p38 proteins, which regulate invasion in poorly differentiated hepatoma cells. Finally, when SK-Hep1 cells were grown as xenografts in nude mice, intraperitoneal (i.p.) injection of baicalein induced a significant dose-dependent decrease in tumor growth. These results demonstrate the anticancer properties of baicalein, which include the inhibition of adhesion, invasion, migration, and proliferation of human hepatoma cells in vivo. - Highlight: > Baicalein inhibits several essential steps in the onset of metastasis.

  11. Human Wharton's jelly stem cells, its conditioned medium and cell-free lysate inhibit the growth of human lymphoma cells.

    PubMed

    Lin, Hao Daniel; Fong, Chui Yee; Biswas, Arijit; Choolani, Mahesh; Bongso, Ariff

    2014-08-01

    Several groups have reported that primitive mesenchymal stem cells from the gelatinous matrix of the Wharton's jelly of the human umbilical cord (hWJSCs) possess tumoricidal properties and inhibit the growth of solid tumours such as human mammary carcinoma, ovarian carcinoma and osteosarcoma. This unique characteristic led to the hypothesis that hWJSCs serve as a natural defence against migrating cancer cells from mother to fetus thus explaining why tumorigenesis in the fetus is rare. However, it is not known whether non-solid malignant hematopoietic cells are also inhibited by hWJSCs and what the exact tumoricidal mechanisms are. We therefore evaluated the influence of hWJSCs and its extracts on Burkitt's lymphoma cells. Cell proliferation (BrdU and Ki67+), viability (MTT) and cell death (Annexin V-Propidium iodide and live/dead) assays showed significant inhibition of lymphoma cell growth after 48 h exposure to hWJSCs or its extracts compared to controls. Increased cell death was observed at sub-G1 and S and decreased proliferation at G2/M phases of the mitotic cycle. Superoxide dismutase and hydrogen peroxide activity were significantly increased and glutathione peroxidase significantly decreased in treated lymphoma cells. Time lapse imaging and confocal z-stack images showed yellow fluorescent in situ hybridization (FISH) signals of lymphoma cell Y chromosomes within the cytoplasm of female red labelled hWJSCs. We hypothesize that the growth of lymphoma cells is inhibited by the molecules secreted by hWJSCs that use oxidative stress pathways to induce cell death followed by engulfment of the apoptotic remains of the lymphoma cells by the hWJSCs. PMID:24789672

  12. Assessment of the inhibition potential of Licochalcone A against human UDP-glucuronosyltransferases.

    PubMed

    Xin, Hong; Qi, Xiao-Yi; Wu, Jing-Jing; Wang, Xin-Xin; Li, Yan; Hong, James Y; He, Wei; Xu, Wei; Ge, Guang-Bo; Yang, Ling

    2016-04-01

    Licochalcone A (LCA) is a major bioactive compound in Licorice, a widely used herbal medicine. In this study, the inhibitory effects of LCA against human UDP-glucuronosyltransferases (UGTs) and LCA associated herb-drug interactions were systematically investigated. Our results demonstrated that LCA displayed broad-spectrum inhibition against human UGTs. LCA exhibited strong inhibitory effects against UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A9, and 2B7 (both IC50 and Ki values lower than 5 μM), while showing moderate inhibitory effects against UGT1A8, 1A10, 2B4, 2B15, and 2B17. The inhibitory effects of LCA against two major UGTs, including UGT1A1 and 1A9, were further investigated in human liver microsomes (HLMs), where the potential risks of LCA via inhibition of UGT1A1 and 1A9 were predicted by combining the in vitro inhibitory data and physiological data. The results from this study also showed that several LCA-containing products were able to increase the area under the curve (AUC) of the substrates that were predominantly metabolized by UGT1A1 or 1A9. These findings together demonstrate that LCA has a potent and broad-spectrum inhibitory effect against most human UGTs and thus suggest that much caution should be exercised when high-dose LCA is co-administered with UGT substrates. PMID:26875642

  13. Arctigenin from Arctium lappa inhibits interleukin-2 and interferon gene expression in primary human T lymphocytes

    PubMed Central

    2011-01-01

    Background Arctium lappa (Niubang), a Chinese herbal medicine, is used to treat tissue inflammation. This study investigates the effects of arctigenin (AC), isolated from A. lappa, on anti-CD3/CD28 Ab-stimulated cell proliferation and cytokine gene expression in primary human T lymphocytes. Methods Cell proliferation was determined with enzyme immunoassays and the tritiated thymidine uptake method. Cytokine production and gene expression were analyzed with reverse transcription-polymerase chain reaction. Results AC inhibited primary human T lymphocytes proliferation activated by anti-CD3/CD28 Ab. Cell viability test indicated that the inhibitory effects of AC on primary human T lymphocyte proliferation were not due to direct cytotoxicity. AC suppressed interleukin-2 (IL-2) and interferon-γ (IFN-γ) production in a concentration-dependent manner. Furthermore, AC decreased the IL-2 and IFN-γ gene expression in primary human T lymphocytes induced by anti-CD3/CD28 Ab. Reporter gene analyses revealed that AC decreased NF-AT-mediated reporter gene expression. Conclusion AC inhibited T lymphocyte proliferation and decreased the gene expression of IL-2, IFN-γ and NF-AT. PMID:21435270

  14. Growth inhibition of BEL-7404 human hepatoma cells by expression of mutant telomerase reverse transcriptase.

    PubMed

    Zhang, Rugang; Wang, Xingwang; Guo, Lixia; Xie, Hong

    2002-01-10

    Human hepatocellular carcinoma (HCC) is one of the most common malignancies in Asia and Africa. Human telomerase reverse transcriptase (hTERT) is expressed in HCC but absent in normal human liver cells, which is consistent with the expression pattern of telomerase. In the present study, expression of a dominant-negative form of hTERT (DN-hTERT) resulted in inhibition of telomerase activity and decreased mean telomeric length of BEL-7404 human hepatoma cells, whereas expression of wild-type hTERT (WT-hTERT) and control vector had no such effects. Cell growth was inhibited by this mutant (DN-hTERT), which was consistent with the changes in telomerase level. Flattened large cells were found in late generations with the DN-hTERT treatment. When mean telomeric length of DN-hTERT-transfected cells reached a critical length (about 1.7 kb), apoptosis was induced. Tumorigenicity of DN-hTERT-expressing cells was eliminated in vivo. These data indicated that hTERT was essential for the growth of hepatoma cells. hTERT can also be used as an important target for anti-HCC drug screening. PMID:11774261

  15. The homing and inhibiting effects of hNSCs-BMP4 on human glioma stem cells

    PubMed Central

    Zhao, Mingming; Zhou, Chunhui; Ren, Junlin; Huang, Qiming; Zhao, Zhongming; Mitra, Ramkrishna; Fan, Wenhong; Fan, Ming

    2016-01-01

    Malignant gliomas patients have a poor survival rate, partially due to the inability in delivering therapeutic agents to the tumors, especially to the metastasis of human glioma stem cells (hGSCs). To explore whether the human neural stem cells (hNSCs) with an over-expression of BMP4 (hNSCs-BMP4) can trace and inhibit hGSCs, in this study, we examined the migration of hNSCs to hGSCs using transwell assay in vitro and performed the fluorescent tracer experiment in vivo. We examined the proliferation, differentiation, apoptosis and migration of hGSCs after co-culturing with hNSCs-BMP4 in vitro and tested the tropism and antitumor effects of hNSCs-BMP4 in the established brain xenograft models of hGSCs. We found that hNSCs-BMP4 could secrete BMP4 and trace hGSCs both in vitro and in vivo. When compared to the normal human astrocytes (NHAs) and hNSCs, hNSCs-BMP4 could significantly inhibit the invasive growth of hGSCs, promote their differentiation and apoptosis by activating Smad1/5/8 signaling, and prolong the survival time of the tumor-bearing nude mice. Collectively, this study suggested that hNSCs-BMP4 may help in developing therapeutic approaches for the treatment of human malignant gliomas. PMID:26908439

  16. LIF-JAK1-STAT3 signaling delays contact inhibition of human corneal endothelial cells.

    PubMed

    Liu, Xin; Tseng, Scheffer C G; Zhang, Ming-Chang; Chen, Szu-Yu; Tighe, Sean; Lu, Wen-Juan; Zhu, Ying-Ting

    2015-01-01

    Human corneal endothelial cells (HCECs) responsible for corneal transparency have limited proliferative capacity in vivo because of "contact-inhibition." This feature has hampered the ability to engineer HCECs for transplantation. Previously we have reported an in vitro model of HCECs in which contact inhibition was re-established at Day 21, even though cell junction and cell matrix interaction were not perturbed during isolation. Herein, we observe that such HCEC monolayers continue to expand and retain a normal phenotype for 2 more weeks if cultured in a leukemia inhibitory factor (LIF)-containing serum-free medium. Such expansion is accompanied initially by upregulation of Cyclin E2 colocalized with nuclear translocation of phosphorylated retinoblastoma tumor suppressor (p-Rb) at Day 21 followed by a delay in contact inhibition through activation of LIF-Janus kinase1 (JAK1)-signal transducer and activator of transcription 3 (STAT3) signaling at Day 35. The LIF-JAK1-STAT3 signaling is coupled with upregulation of E2F2 colocalized with nuclear p-Rb and with concomitant downregulation of p16(INK4a), of which upregulation is linked to senescence. Hence, activation of LIF-JAK1-STAT3 signaling to delay contact inhibition can be used as another strategy to facilitate engineering of HCEC grafts to solve the unmet global shortage of corneal grafts. PMID:25695744

  17. Interfering EZH2 Expression Reverses the Cisplatin Resistance in Human Ovarian Cancer by Inhibiting Autophagy.

    PubMed

    Sun, Yang; Jin, Long; Liu, Jia-Hua; Sui, Yu-Xia; Han, Li-Li; Shen, Xiao-Li

    2016-09-01

    We aimed to determine the effects of the inhibition of enhancer of zeste homolog 2 (EZH2) gene expression on the cisplatin resistance of the human ovarian cancer cell line, SKOV3/DDP, and to identify the underlying mechanisms. SKOV3/DDP cells were stably transfected with pSUPER-EZH2 (EZH2 RNA interference plasmid) or pcDNA3.1-EZH2 (EZH2 gene overexpression plasmid) using the lipofection method. Real-time fluorescence quantitative reverse transcription polymerase chain reaction and western blotting confirmed that EZH2 expression was downregulated in pSUPER-EZH2-transfected cells. Flow cytometry revealed that EZH2 inhibition did not induce apoptosis, but significantly inhibited autophagy. In addition, it significantly increased the expression of the cellular senescence-signaling proteins p14(ARF), p16(INK4a), p53, pRb, and p21, and significantly decreased the expression of cyclin-dependent kinase (CDK)1, CDK2, and H3K27me3. Cellular senescence was characterized by a significant increase in the G0/G1 ratio and the restoration of sensitivity to cisplatin in the drug-resistant cells. These findings suggest that interfering with EZH2 expression can inhibit SKOV3/DDP cell autophagy and reverse resistance to cisplatin. The underlying mechanisms could be associated with the regulation of the cellular senescence-signaling pathway. PMID:27610467

  18. Metabolic inhibition and kinetics of raloxifene by pharmaceutical excipients in human liver microsomes.

    PubMed

    Kim, Ae Ra; Lim, Soo-Jeong; Lee, Beom-Jin

    2009-02-23

    This study was originally undertaken to establish the in vitro metabolic conditions and then evaluate the effect of pharmaceutical excipients (PEs) on drug metabolism in uridine diphosphoglucuronic acid-supplemented human liver microsomes. Poorly bioavailable raloxifene was chosen as a model drug. Intact drug and its two glucuronide metabolites were successfully isolated using gradient HPLC analysis and LC/MS analysis. Formation of raloxifene metabolites was affected by buffer compositions, incubation time and initial raloxifene concentrations. Under optimized metabolic conditions, 41.0% of raloxifene was converted to its metabolites after 2h incubation. This metabolic inhibition of raloxifene by the PEs occurred in a dose-dependent manner and accordingly formed two glucuronide metabolites. In the metabolic kinetics using Lineweaver-Burk analyses, Cremophor EL competitively inhibited formation of metabolites while sodium lauryl sulfate (SLS), polyvinylpyrrolidone K30 (PVP) and Tween 80 significantly inhibited in a mixed competition. Although some PEs showed inhibition on glucuronidation of raloxifene in vitro, their effects on in vivo bioavailability of raloxifene need to be confirmed directly due to the dilution factors and other complicated situations influencing the bioavailability. PMID:18977285

  19. Aminomethylphosphonic acid inhibits growth and metastasis of human prostate cancer in an orthotopic xenograft mouse model.

    PubMed

    Parajuli, Keshab Raj; Zhang, Qiuyang; Liu, Sen; You, Zongbing

    2016-03-01

    Aminomethylphosphonic acid (AMPA) has been shown to inhibit prostate cancer cell growth in vitro. The purpose of the present study was to determine if AMPA could inhibit growth and metastasis of prostate cancer in vivo. Human prostate cancer PC-3-LacZ-luciferase cells were implanted into the ventral lateral lobes of the prostate in 39 athymic Nu/Nu nude male mice. Seven days later, mice were randomized into the control group (n = 14, treated intraperitoneally with phosphate buffered saline), low dose group (n = 10, treated intraperitoneally with AMPA at 400 mg/kg body weight/day), and high dose group (n = 15, treated intraperitoneally with AMPA at 800 mg/kg body weight/day). Tumor growth and metastasis were examined every 4-7 days by bioluminescence imaging of live mice. We found that AMPA treatment significantly inhibited growth and metastasis of orthotopic xenograft prostate tumors and prolonged the survival time of the mice. AMPA treatment decreased expression of BIRC2 and activated caspase 3, leading to increased apoptosis in the prostate tumors. AMPA treatment decreased expression of cyclin D1. AMPA treatment also reduced angiogenesis in the prostate tumors. Taken together, these results demonstrate that AMPA can inhibit prostate cancer growth and metastasis, suggesting that AMPA may be developed into a therapeutic agent for the treatment of prostate cancer. PMID:26840261

  20. Lack of substrate inhibition in a monomeric form of human cytosolic SULT2A1.

    PubMed

    Cook, Ian T; Leyh, Thomas S; Kadlubar, Susan A; Falany, Charles N

    2010-12-01

    Mammalian cytosolic sulfotransferases (SULTs) frequently show substrate inhibition during the sulfation of increasing concentrations of substrates. SULT2A1, a major human liver isoform responsible for the conjugation of hydroxysteroids, bile acids and aliphatic hydroxyl groups in drugs and xenobiotics, is a homodimer and displays substrate inhibition during the conjugation of dehydroepiandrosterone (DHEA). Maltose binding protein (MBP)-SULT2A1 fusion protein, produced as an intermediate step in the purification of the SULT2A1 homodimer, elutes during size exclusion chromatography as a monomer. The initial-rate parameters (Km and Vmax) of the monomer (MBP-SULT2A1) and native SULT2A1 dimer for DHEA sulfation are extremely similar; however, the monomer is not inhibited by DHEA. Intrinsic fluorescence studies show that two DHEA molecules bind each SULT2A1 subunit, one in the catalytic site and one in an apparent allosteric site. Lack of dimerization in the MBP-SULT2A1 fusion protein decreased the Kd for binding of DHEA at the allosteric site. These results suggest that formation of the homodimer is associated with structural re-arrangements leading to increased DHEA binding at an allo-steric site that is associated with substrate inhibition. PMID:25961208

  1. Lack of substrate inhibition in a monomeric form of human cytosolic SULT2A1.

    PubMed

    Cook, Ian T; Leyh, Thomas S; Kadlubar, Susan A; Falany, Charles N

    2010-12-01

    Mammalian cytosolic sulfotransferases (SULTs) frequently show substrate inhibition during the sulfation of increasing concentrations of substrates. SULT2A1, a major human liver isoform responsible for the conjugation of hydroxysteroids, bile acids and aliphatic hydroxyl groups in drugs and xenobiotics, is a homodimer and displays substrate inhibition during the conjugation of dehydroepiandrosterone (DHEA). Maltose binding protein (MBP)-SULT2A1 fusion protein, produced as an intermediate step in the purification of the SULT2A1 homodimer, elutes during size exclusion chromatography as a monomer. The initial-rate parameters (K(m) and V(max)) of the monomer (MBP-SULT2A1) and native SULT2A1 dimer for DHEA sulfation are extremely similar; however, the monomer is not inhibited by DHEA. Intrinsic fluorescence studies show that two DHEA molecules bind each SULT2A1 subunit, one in the catalytic site and one in an apparent allosteric site. Lack of dimerization in the MBP-SULT2A1 fusion protein decreased the K(d) for binding of DHEA at the allosteric site. These results suggest that formation of the homodimer is associated with structural rearrangements leading to increased DHEA binding at an allosteric site that is associated with substrate inhibition. PMID:21822453

  2. Aminomethylphosphonic acid inhibits growth and metastasis of human prostate cancer in an orthotopic xenograft mouse model

    PubMed Central

    Parajuli, Keshab Raj; Zhang, Qiuyang; Liu, Sen; You, Zongbing

    2016-01-01

    Aminomethylphosphonic acid (AMPA) has been shown to inhibit prostate cancer cell growth in vitro. The purpose of the present study was to determine if AMPA could inhibit growth and metastasis of prostate cancer in vivo. Human prostate cancer PC-3-LacZ-luciferase cells were implanted into the ventral lateral lobes of the prostate in 39 athymic Nu/Nu nude male mice. Seven days later, mice were randomized into the control group (n = 14, treated intraperitoneally with phosphate buffered saline), low dose group (n = 10, treated intraperitoneally with AMPA at 400 mg/kg body weight/day), and high dose group (n = 15, treated intraperitoneally with AMPA at 800 mg/kg body weight/day). Tumor growth and metastasis were examined every 4-7 days by bioluminescence imaging of live mice. We found that AMPA treatment significantly inhibited growth and metastasis of orthotopic xenograft prostate tumors and prolonged the survival time of the mice. AMPA treatment decreased expression of BIRC2 and activated caspase 3, leading to increased apoptosis in the prostate tumors. AMPA treatment decreased expression of cyclin D1. AMPA treatment also reduced angiogenesis in the prostate tumors. Taken together, these results demonstrate that AMPA can inhibit prostate cancer growth and metastasis, suggesting that AMPA may be developed into a therapeutic agent for the treatment of prostate cancer. PMID:26840261

  3. AMPK-independent inhibition of human macrophage ER stress response by AICAR.

    PubMed

    Boß, Marcel; Newbatt, Yvette; Gupta, Sahil; Collins, Ian; Brüne, Bernhard; Namgaladze, Dmitry

    2016-01-01

    Obesity-associated insulin resistance is driven by inflammatory processes in response to metabolic overload. Obesity-associated inflammation can be recapitulated in cell culture by exposing macrophages to saturated fatty acids (SFA), and endoplasmic reticulum (ER) stress responses essentially contribute to pro-inflammatory signalling. AMP-activated protein kinase (AMPK) is a central metabolic regulator with established anti-inflammatory actions. Whether pharmacological AMPK activation suppresses SFA-induced inflammation in a human system is unclear. In a setting of hypoxia-potentiated inflammation induced by SFA palmitate, we found that the AMP-mimetic AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) potently suppressed upregulation of ER stress marker mRNAs and pro-inflammatory cytokines. Furthermore, AICAR inhibited macrophage ER stress responses triggered by ER-stressors thapsigargin or tunicamycin. Surprisingly, AICAR acted independent of AMPK or AICAR conversion to 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranosyl monophosphate (ZMP) while requiring intracellular uptake via the equilibrative nucleoside transporter (ENT) ENT1 or the concentrative nucleoside transporter (CNT) CNT3. AICAR did not affect the initiation of the ER stress response, but inhibited the expression of major ER stress transcriptional effectors. Furthermore, AICAR inhibited autophosphorylation of the ER stress sensor inositol-requiring enzyme 1α (IRE1α), while activating its endoribonuclease activity in vitro. Our results suggest that AMPK-independent inhibition of ER stress responses contributes to anti-inflammatory and anti-diabetic effects of AICAR. PMID:27562249

  4. Lactate dehydrogenase-A inhibition induces human glioblastoma multiforme stem cell differentiation and death

    PubMed Central

    Daniele, Simona; Giacomelli, Chiara; Zappelli, Elisa; Granchi, Carlotta; Trincavelli, Maria Letizia; Minutolo, Filippo; Martini, Claudia

    2015-01-01

    Therapies that target the signal transduction and metabolic pathways of cancer stem cells (CSCs) are innovative strategies to effectively reduce the recurrence and significantly improve the outcome of glioblastoma multiforme (GBM). CSCs exhibit an increased rate of glycolysis, thus rendering them intrinsically more sensitive to prospective therapeutic strategies based on the inhibition of the glycolytic pathway. The enzyme lactate dehydrogenase-A (LDH-A), which catalyses the interconversion of pyruvate and lactate, is up-regulated in human cancers, including GBM. Although several papers have explored the benefits of targeting cancer metabolism in GBM, the effects of direct LDH-A inhibition in glial tumours have not yet been investigated, particularly in the stem cell subpopulation. Here, two representative LDH-A inhibitors (NHI-1 and NHI-2) were studied in GBM-derived CSCs and compared to differentiated tumour cells. LDH-A inhibition was particularly effective in CSCs isolated from different GBM cell lines, where the two compounds blocked CSC formation and elicited long-lasting effects by triggering both apoptosis and cellular differentiation. These data demonstrate that GBM, particularly the stem cell subpopulation, is sensitive to glycolytic inhibition and shed light on the therapeutic potential of LDH-A inhibitors in this tumour type. PMID:26494310

  5. Inhibition of human platelet phospholipase A/sub 2/ by mono(2-ethylhexyl)phthalate

    SciTech Connect

    Labow, R.S.; Meek, E.; Adams, G.A.; Rock, G.

    1988-06-01

    There is evidence that the carcinogenic and teratogenic effects attributed to the plasticizer di(2-ethylhexyl)phthalate (DEHP) are due to its major metabolite mono(2-ethylhexyl)phthalate (MEHP). MEHP is also formed ex vivo by a plasma enzyme in blood products stored in polyvinyl chloride (PVC) DEHP plastic containers. People who receive large amounts of blood products, such as hemophiliacs or patients undergoing hemodialysis, cardiopulmonary bypass, or massive transfusion, are exposed to significant levels of plasticizer. In this study, the platelet was used to show that MEHP inhibits phospholipase A/sub 2/ (PLA/sub 2/), one of the enzymes important in the release of arachidonic acid from membrane phospholipids. PLA/sub 2/ was measured by the liberation of /sup 14/C-arachidonic acid from 1-stearoyl-2-(1-/sup 14/C)arachidonyl-L-3-phosphatidylcholine. MEHP inhibits PLA/sub 2/ activity noncompetitively in intact human platelets and lysates with a K/sub i/ of 3.7 x 10/sup -4/ M. DEHP does not inhibit PLA/sub 2/ in whole platelets. Inhibition of PLA/sub 2/ by MEHP occurs at only three times the circulating level of MEHP measured in neonates undergoing exchange transfusion and 20-fold the levels experienced by patients during cardiopulmonary bypass. Therefore, infants and adult patients with multisystem failure who accumulate MEHP in their blood may be at risk for decreased platelet function.

  6. Inhibition of Focal Adhesion Kinase and Src Increases Detachment and Apoptosis in Human Neuroblastoma Cell Lines

    PubMed Central

    Beierle, Elizabeth A.; Ma, Xiaojie; Trujillo, Angelica; Kurenova, Elena V.; Cance, William G.; Golubovskaya, Vita M.

    2010-01-01

    Neuroblastoma is the most common extracranial solid tumor of childhood. Focal adhesion kinase (FAK) is an intracellular kinase that is overexpressed in a number of human tumors including neuroblastoma, and regulates both cellular adhesion and survival. We have studied the effects of FAK inhibition upon neuroblastoma using adenovirus-containing FAK-CD (AdFAK-CD). Utilizing an isogenic MYCN+ / MYCN− neuroblastoma cell line, we found that the MYCN+ cells are more sensitive to FAK inhibition with AdFAK-CD than their MYCN negative counterparts. In addition, we have shown that phosphorylation of Src is increased in the untreated isogenic MYCN− neuroblastoma cells, and that the decreased sensitivity of the MYCN− neuroblastoma cells to FAK inhibition with AdFAK-CD is abrogated by the addition of the Src family kinase inhibitor, PP2. The results of the current study suggest that both FAK and Src play a role in protecting neuroblastoma cells from apoptosis, and that dual inhibition of these kinases may be important when designing therapeutic interventions for this tumor. PMID:19885861

  7. AMPK-independent inhibition of human macrophage ER stress response by AICAR

    PubMed Central

    Boß, Marcel; Newbatt, Yvette; Gupta, Sahil; Collins, Ian; Brüne, Bernhard; Namgaladze, Dmitry

    2016-01-01

    Obesity-associated insulin resistance is driven by inflammatory processes in response to metabolic overload. Obesity-associated inflammation can be recapitulated in cell culture by exposing macrophages to saturated fatty acids (SFA), and endoplasmic reticulum (ER) stress responses essentially contribute to pro-inflammatory signalling. AMP-activated protein kinase (AMPK) is a central metabolic regulator with established anti-inflammatory actions. Whether pharmacological AMPK activation suppresses SFA-induced inflammation in a human system is unclear. In a setting of hypoxia-potentiated inflammation induced by SFA palmitate, we found that the AMP-mimetic AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) potently suppressed upregulation of ER stress marker mRNAs and pro-inflammatory cytokines. Furthermore, AICAR inhibited macrophage ER stress responses triggered by ER-stressors thapsigargin or tunicamycin. Surprisingly, AICAR acted independent of AMPK or AICAR conversion to 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranosyl monophosphate (ZMP) while requiring intracellular uptake via the equilibrative nucleoside transporter (ENT) ENT1 or the concentrative nucleoside transporter (CNT) CNT3. AICAR did not affect the initiation of the ER stress response, but inhibited the expression of major ER stress transcriptional effectors. Furthermore, AICAR inhibited autophosphorylation of the ER stress sensor inositol-requiring enzyme 1α (IRE1α), while activating its endoribonuclease activity in vitro. Our results suggest that AMPK-independent inhibition of ER stress responses contributes to anti-inflammatory and anti-diabetic effects of AICAR. PMID:27562249

  8. beta-Carboline alkaloids in Peganum harmala and inhibition of human monoamine oxidase (MAO).

    PubMed

    Herraiz, T; González, D; Ancín-Azpilicueta, C; Arán, V J; Guillén, H

    2010-03-01

    Peganum harmala L. is a multipurpose medicinal plant increasingly used for psychoactive recreational purposes (Ayahuasca analog). Harmaline, harmine, harmalol, harmol and tetrahydroharmine were identified and quantified as the main beta-carboline alkaloids in P. harmala extracts. Seeds and roots contained the highest levels of alkaloids with low levels in stems and leaves, and absence in flowers. Harmine and harmaline accumulated in dry seeds at 4.3% and 5.6% (w/w), respectively, harmalol at 0.6%, and tetrahydroharmine at 0.1% (w/w). Roots contained harmine and harmol with 2.0% and 1.4% (w/w), respectively. Seed extracts were potent reversible and competitive inhibitors of human monoamine oxidase (MAO-A) with an IC(50) of 27 microg/l whereas root extracts strongly inhibited MAO-A with an IC(50) of 159 microg/l. In contrast, they were poor inhibitors of MAO-B. Inhibition of MAO-A by seed extracts was quantitatively attributed to harmaline and harmine whereas inhibition by root extracts came from harmine with no additional interferences. Stems and leaves extracts were poor inhibitors of MAO. The potent inhibition of MAO-A by seed and root extracts of P. harmala containing beta-carbolines should contribute to the psychopharmacological and toxicological effects of this plant and could be the basis for its purported antidepressant actions. PMID:20036304

  9. Chlorpromazine, clozapine and olanzapine inhibit anionic amino acid transport in cultured human fibroblasts.

    PubMed

    Marchesi, C; Dall'Asta, V; Rotoli, B M; Bianchi, M G; Maggini, C; Gazzola, G C; Bussolati, O

    2006-09-01

    We report here that chlorpromazine, a first generation antipsychotic drug, inhibits anionic amino acid transport mediated by system X(-) (AG) (EAAT transporters) in cultured human fibroblasts. With 30 microM chlorpromazine, transport inhibition is detectable after 3 h of treatment, maximal after 48 h (>60%), and referable to a decrease in V(max). Chlorpromazine effect is not dependent upon changes of membrane potential and is selective for system X(-) (AG) since transport systems A and y(+) are not affected. Among antipsychotic drugs, the inhibitory effect of chlorpromazine is shared by two dibenzodiazepines, clozapine and olanzapine, while other compounds, such as risperidon, zuclopentixol, sertindol and haloperidol, are not effective. Transport inhibition by clozapine and olanzapine, but not by chlorpromazine, is reversible, suggesting that the mechanisms involved are distinct. These results indicate that a subset of antipsychotic drugs inhibits EAAT transporters in non-nervous tissues and prompt further investigation on possible alterations of glutamate transport in peripheral tissues of schizophrenic patients. PMID:16699818

  10. Rat and human colonic mucins bind to and inhibit adherence lectin of Entamoeba histolytica.

    PubMed Central

    Chadee, K; Petri, W A; Innes, D J; Ravdin, J I

    1987-01-01

    Establishment of adherence by Entamoeba histolytica is mediated by a 170-kD Gal/GalNAc inhibitable lectin and is required for cytolysis and phagocytosis of mammalian target cells. We studied the biochemical mechanisms of the in vitro interaction between rat and human colonic mucins and axenic E. histolytica trophozoites. Crude mucus prevented amebic adherence to Chinese hamster ovary (CHO) cells by up to 70%. Purification of the colonic mucins by Sepharose 4B chromatography, nuclease digestion, and cesium chloride gradient centrifugation resulted in a 1,000-fold enrichment of the inhibitory mucins. Purified rat mucin inhibited amebic adherence to and cytolysis of homologous rat colonic epithelial cells. Oxidation and enzymatic cleavage of rat mucin Gal and GalNAc residues completely abrogated mucin inhibition of amebic adherence. The binding of rat 125I-mucin to amebae was galactose specific, saturable, reversible, and pH dependent. A monoclonal antibody specific for the 170-kD amebic Gal/GalNAc lectin completely inhibited the binding of rat 125I-mucin. Rat mucin bound to Affigel affinity purified the amebic lectin from conditioned medium. Colonic mucin glycoproteins act as an important host defense by binding to the parasite's adherence lectin, thus preventing amebic attachment to and cytolysis of host epithelial cells. Images PMID:2890655

  11. Inhibition of human α7 nicotinic acetylcholine receptors by cyclic monoterpene carveol.

    PubMed

    Lozon, Yosra; Sultan, Ahmed; Lansdell, Stuart J; Prytkova, Tatiana; Sadek, Bassem; Yang, Keun-Hang Susan; Howarth, Frank Christopher; Millar, Neil S; Oz, Murat

    2016-04-01

    Cyclic monoterpenes are a group of phytochemicals with antinociceptive, local anesthetic, and anti-inflammatory actions. Effects of cyclic monoterpenes including vanilin, pulegone, eugenole, carvone, carvacrol, carveol, thymol, thymoquinone, menthone, and limonene were investigated on the functional properties of the cloned α7 subunit of the human nicotinic acetylcholine receptor expressed in Xenopus oocytes. Monoterpenes inhibited the α7 nicotinic acetylcholine receptor in the order carveol>thymoquinone>carvacrol>menthone>thymol>limonene>eugenole>pulegone≥carvone≥vanilin. Among the monoterpenes, carveol showed the highest potency on acetylcholine-induced responses, with IC50 of 8.3µM. Carveol-induced inhibition was independent of the membrane potential and could not be reversed by increasing the concentration of acetylcholine. In line with functional experiments, docking studies indicated that cyclic monoterpenes such as carveol may interact with an allosteric site located in the α7 transmembrane domain. Our results indicate that cyclic monoterpenes inhibit the function of human α7 nicotinic acetylcholine receptors, with varying potencies. PMID:26849939

  12. Multicarotenoids at Physiological Levels Inhibit Metastasis in Human Hepatocarcinoma SK-Hep-1 Cells.

    PubMed

    Chen, Huei-Yan; Yang, Chih-Min; Chen, Jen-Yin; Yueh, Te-Cheng; Hu, Miao-Lin

    2015-01-01

    Several studies have demonstrated that single carotenoid, including lycopene, β-carotene, and α-carotene, exhibits antimetastatic effects; however, little is known whether multicarotenoids have similar effects. Herein, we investigated the antimetastatic effect of multicarotenoids at physiological serum levels in Taiwanese (MCT at 1.4 μM) and American (MCA at 1.8 μM) populations using human hepatocarcinoma SK-Hep-1 cells in comparison with single carotenoid, such as lycopene (0.3 or 0.6 μM, respectively), α-carotene (0.1 μM), β-carotene (0.4 μM), lutein (0.4 or 0.5 μM, respectively), and β-cryptoxanthin (0.2 μM). Results reveal that MCA treatment exhibited an additive inhibition on invasion, migration and adhesion at 24 and 48 h of incubation, whereas MCT treatment possessed additive inhibition at 48 h of incubation. The antimetastatic action of MCT and MCA involved additive reduction on activities of matrix metalloproteinase (MMP)-2, -9, and protein expression of Rho and Rac 1 but additive promotion on protein expression of tissue inhibitor of MMP (TIMP)-1 and -2. All of these effects were stronger in MCA than in MCT at 24 and 48 h of incubation. These results demonstrate that multi-carotenoids effectively inhibit metastasis of human hepatocarcinoma SK-Hep-1 cells. More in vivo studies are needed to confirm these findings. PMID:25868689

  13. CTRP6 inhibits fibrogenesis in TGF-β1-stimulated human dermal fibroblasts.

    PubMed

    Fan, Rong-Hui; Zhu, Xiu-Mei; Sun, Yao-Wen; Peng, Hui-Zi; Wu, Hang-Li; Gao, Wen-Jie

    2016-07-01

    Skin fibrosis is characterized by excessive proliferation of fibroblasts and overproduction of extracellular matrix (ECM). C1q/tumor necrosis factor-related protein 6 (CTRP6), a member of CTRPs, has been involved in the development of cardiac fibrosis. However, the function and detailed regulatory mechanism of CTRP6 in skin fibrosis remain unclear. The aim of this study was to investigate the effect of CTRP6 on the activation of human dermal fibroblasts. Our results showed that CTRP6 was lowly expressed in scar tissues and transforming growth factor-β1 (TGF-β1)-treated dermal fibroblasts. CTRP6 overexpression significantly inhibited the proliferation of dermal fibroblasts, as well as suppressed the expression of ECM in TGF-β1-treated dermal fibroblasts. Furthermore, CTRP6 overexpression markedly inhibited TGF-β1-induced phosphorylation of Smad3 in dermal fibroblasts. In conclusion, the data reported here demonstrate that CTRP6 is able to inhibit the proliferation and ECM expression in human dermal fibroblasts through suppressing the TGF-β1/Smad3 signaling pathway. These findings suggest that CTRP6 may be a potential therapeutic target for the prevention of skin fibrosis. PMID:27155158

  14. Inhibition of syk activity and degranulation of human mast cells by flavonoids.

    PubMed

    Shichijo, Michitaka; Yamamoto, Noriyuki; Tsujishita, Hideki; Kimata, Masahiro; Nagai, Hiroichi; Kokubo, Toshio

    2003-12-01

    To investigate the effect of flavonoids on the activation of p72(syk) (Syk) protein tyrosine kinase which plays a pivotal role in the high affinity IgE receptor-mediated degranulation of mast cells, we picked out 10 flavonoids, classified them into 4 series, and examined their effects on the activation of Syk and on the degranulation of human mast cells. Flavones and flavonols showed clear inhibition, whereas flavanones and isoflavones had either weak or no effect on Syk enzymatic activity induced by amino acid peptide corresponding to the activation loop domain and on IgE-dependent degranulation of human cultured mast cells (HCMC). On the basis of calculated logP (ClogP) values as a prediction of compound lipophilicity, some flavonoids were speculated to have low lipophilicity, the reason for poor cell permeability. A significant relationship was observed between the inhibition of Syk activity and HCMC degranulation attributable to flavonoids when the ClogP values of the compounds were taken into account (r(2)=0.89). These results suggested that the impairment of mast cell degranulation by several flavonoids classified into flavones and flavonols might be mediated via inhibition of the intracellular activation of Syk. PMID:14646171

  15. Interleukin 4 inhibits in vitro proliferation of leukemic and normal human B cell precursors.

    PubMed Central

    Pandrau, D; Saeland, S; Duvert, V; Durand, I; Manel, A M; Zabot, M T; Philippe, N; Banchereau, J

    1992-01-01

    In the present study, we have investigated the effects of IL-4 on the proliferation and differentiation of leukemic and normal human B cell precursors (BCP). We have demonstrated that IL-4 significantly inhibited spontaneous [3H]thymidine ([3H]-TdR) incorporation by leukemic blasts from some B lineage acute lymphoblastic leukemia (BCP-ALL) patients (8 of 14). Furthermore, IL-4 was found to suppress the spontaneous and factor-dependent (IL-7 and IL-3) proliferation of normal BCP (CD10+ surface [s] IgM- cells) isolated from fetal bone marrow. Maximum growth inhibition of either leukemic or normal BCP was reached at low IL-4 concentrations (10 U/ml), and the effect was specifically neutralized by anti-IL-4 antibody. IL-4 was further found to induce the expression of CD20 antigen on BCP-ALL cells from a number of the cases examined (5 of 8), but in contrast to leukemic cells, IL-4 failed to induce CD20 antigen on normal BCP. Finally, IL-4 was found to induce neither the expression of cytoplasmic mu chain, nor the appearance of sIgM+ cells in cultures of normal or leukemic BCP. Our data indicate that IL-4 has the potential to inhibit cell proliferation in leukemic and normal human B lymphopoiesis but is unable to drive the transition from BCP to mature B cells. Images PMID:1385474

  16. Extracts of various species of Epilobium inhibit proliferation of human prostate cells.

    PubMed

    Vitalone, Annabella; Guizzetti, Marina; Costa, Lucio G; Tita, Beatrice

    2003-05-01

    This study examined whether various species of Epilobium, a phytotherapeutic agent used in folk medicine as a treatment for benign prostatic hyperplasia, may have an antiproliferative effect in PZ-HPV-7 human prostatic epithelial cells in-vitro. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) test, [methyl-(3)H]thymidine incorporation into DNA and flow cytometry analysis were used to evaluate cell proliferation. Ethanolic extracts of E. spicatum, E. rosmarinifolium and E. tetragonum inhibited DNA synthesis in PZ-HPV-7 cells. While at high concentrations all extracts were cytotoxic, DNA synthesis was also decreased at levels that caused no or little cytotoxicity. Treatment of cells with Epilobium extracts did not result in a formation of DNA fragments (evaluated by the TUNEL assay) or chromatin condensation (assessed by Hoechst staining). Flow cytometry analysis indicated that Epilobium extracts inhibit the progression of the cell cycle from the G(0)/G(1) phase. These results suggest that extracts of Epilobium inhibit proliferation of human PZ-HPV-7 cells in-vitro by affecting progression of the cell cycle. This study provides some initial biological plausibility for the use of Epilobium extracts in benign prostatic hyperplasia. PMID:12831512

  17. Inhibition of Cytochrome P450 by Propolis in Human Liver Microsomes

    PubMed Central

    Ryu, Chang Seon; Oh, Soo Jin; Oh, Jung Min; Lee, Ji-Yoon; Lee, Sang Yoon; Chae, Jung-woo; Kwon, Kwang-il; Kim, Sang Kyum

    2016-01-01

    Although propolis is one of the most popular functional foods for human health, there have been no comprehensive studies of herb-drug interactions through cytochrome P450 (CYP) inhibition. The purpose of this study was to determine the inhibitory effects of propolis on the activities of CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1 and 3A4 using pooled human liver microsomes (HLMs). Propolis inhibited CYP1A2, CYP2E1 and CYP2C19 with an IC50 value of 6.9, 16.8, and 43.1 μg/mL, respectively, whereas CYP2A6, 2B6, 2C9, 2D6, and 3A4 were unaffected. Based on half-maximal inhibitory concentration shifts between microsomes incubated with and without nicotinamide adenine dinucleotide phosphate, propolis-induced CYP1A2, CYP2C19, and CYP2E1 inhibition was metabolism-independent. To evaluate the interaction potential between propolis and therapeutic drugs, the effects of propolis on metabolism of duloxetine, a serotonin-norepinephrine reuptake inhibitor, were determined in HLMs. CYP1A2 and CYP2D6 are involved in hydroxylation of duloxetine to 4-hydroxy duloxetine, the major metabolite, which was decreased following propolis addition in HLMs. These results raise the possibility of interactions between propolis and therapeutic drugs metabolized by CYP1A2. PMID:27437087

  18. Recombinant human vascular endothelial growth factor receptor 1 effectively inhibits angiogenesis in vivo.

    PubMed

    Wang, Jinliang; Shi, Minglei; Xi, Yongyi; Gao, Lihua; Zhang, Guanyi; Shao, Yong; Chen, Huipeng; Hu, Xianwen

    2015-05-01

    Vascular endothelial growth factor (VEGF) plays an important role in both physiological and pathological angiogenesis. VEGF receptor‑1 (VEGFR‑1) acts as a decoy VEGF receptor that enables the regulation of VEGF on the vascular endothelium. In the present study, the recombinant human VEGFR1D1‑3/Fc (rhVEGFR‑1), which contains key domains for VEGF binding, was cloned and expressed in Chinese hamster ovary (CHO) cells. The rhVEGFR‑1 protein was purified using protein‑A affinity chromatography. The molecular weight of rhVEGFR‑1 was found to be ~162 and 81 kD in non‑reducing and reducing SDS‑PAGE, respectively. The majority of the final protein products were in the dimeric conformation. Western blot analysis revealed that rhVEGFR‑1 was only capable of binding to the full glycan form of rhVEGF‑165 and rhVEGF‑121. The dissociation constant for the binding of rhVEGFR‑1 to VEGF‑165, detected using Biacore, was 285 pM. In addition, rhVEGFR‑1 inhibited the proliferation and migration of human microvascular endothelial cells. In vivo experiments also demonstrated that rhVEGFR‑1 inhibited chicken chorioallantoic membrane neovascularization and angiogenesis in nude mice. In conclusion, an anti‑angiogenic recombinant soluble VEGFR was expressed (up to 5 mg/l) in CHO cells and was shown to be capable of inhibiting neovascularization in vivo and in vitro. PMID:25607471

  19. Decreasing lncRNA HOTAIR expression inhibits human colorectal cancer stem cells

    PubMed Central

    Dou, Jun; Ni, Yaoyao; He, Xiangfeng; Wu, Di; Li, Miao; Wu, Songyan; Zhang, Rong; Guo, Mei; Zhao, Fengsu

    2016-01-01

    Research on the relationship between aberrant long non-coding RNA (lncRNA) and cancer stem cell (CSC) biology in cancer patients has been recently gaining attention. The goal of this study was to investigate whether the decreasing lncRNA HOTAIR expression would inhibit human colorectal cancer (CRC) stem cells. CD133+CSCs were isolated from human CRC LoVo cell line by using a magnetic-activated cell sorting system, and were transfected with the expression vector-based small hairpin RNA targeting HOTAIR (shHOTAIR). The ability of cellular proliferation, migration, invasion, colony-forming, and the epithelial-mesenchymal transition (EMT)-associated molecule expression as well as the tumorigenicity of CD133+-shHOTAIR were evaluated by the MTT, wound-healing, cellular invasion, colony formation and Western blot assays, respectively. This study found that, when compared with control cells in vitro, CD133+-shHOTAIR exhibited the decreased HOTAIR expression, suppressed cellular proliferation, migration, invasion, colony-forming, and inhibited the Vimentin expression with increased E-cadherin expression. In particular, the down-regulation of the HOTAIR expression in CD133+CSCs markedly attenuated the tumor growth and lung metastasis in xenograft nude mice. Taken together, this study found that down-regulating the HOTAIR expression in CD133+CSCs could serve as a potential anti-cancer regimen to inhibit the invasiveness and metastasis of CRC CSCs. PMID:27069543

  20. Thymus vulgaris (thyme) inhibits proliferation, adhesion, migration, and invasion of human colorectal cancer cells.

    PubMed

    Al-Menhali, Afnan; Al-Rumaihi, Aisha; Al-Mohammed, Hana; Al-Mazrooey, Hana; Al-Shamlan, Maryam; AlJassim, Meaad; Al-Korbi, Noof; Eid, Ali Hussein

    2015-01-01

    Colorectal cancer (CRC) remains one of the most common malignancies and a leading cause of cancer-related deaths. Its prognosis remains poor for patients with several grades of this disease. This underscores the need for alternative modalities, such as herbal medicines, to treat this disease. A commonly used plant that appears to be of high medicinal value is Thymus vulgaris L. However, the effects of this plant on the malignant behavior of human CRC cells remains poorly investigated. This study was undertaken to determine the anticancer efficacy of T. vulgaris extract (TVE) in CRC cells. Our results show that TVE inhibits proliferation in a concentration- and time-dependent fashion. This decreased proliferation was concomitant with increased apoptotic cell death as evidenced by increased caspase3/7 activity. Moreover, TVE also decreased adhesion to fibronectin in a concentration-dependent manner. The migratory and invasive capacities of HCT116 cells were significantly inhibited by TVE. Taken together, these data suggest that the TVE inhibits malignant phenotype of colon cancer cells. Therefore, T. vulgaris could have an anticancer effect and that some of its bioactive compounds may prove to be effective treatment modalities for human CRC. PMID:25379783

  1. Effects of Mitochondrial Uncoupling Protein 2 Inhibition by Genipin in Human Cumulus Cells

    PubMed Central

    Ge, Hongshan; Zhang, Fan; Shan, Dan; Chen, Hua; Wang, Xiaona; Ling, Chao; Xi, HaiTao; Huang, Jianying; Zhu, ChunFang; Lv, Jeiqiang

    2015-01-01

    UCP2 plays a physiological role by regulating mitochondrial biogenesis, maintaining energy balance, ROS elimination, and regulating cellular autophagy in numerous tissues. But the exact roles of UCP2 in cumulus cells are still not clear. Genipin, a special UCP2 inhibitor, was added into the cultural medium to explore the roles of UCP2 in human cumulus cells. There were no significant differences in ATP and mitochondrial membrane potential levels in cumulus cells from UCP2 inhibiting groups as compared with the control. The levels of ROS and Mn-SOD were markedly elevated after UCP2 inhibited Genipin. However, the ratio of reduced GSH to GSSG significantly declined after treatment with Genipin. UCP2 inhibition by Genipin also resulted in obvious increase in the active caspase-3, which accompanied the decline of caspase-3 mRNA. The level of progesterone in culture medium declined obviously after Genipin treatment. But there was no significant difference in estradiol concentrations. This study indicated that UCP2 is expressed in human cumulus cells and plays important roles on mediate ROS production, apoptotic process, and steroidogenesis, suggesting UCP2 may be involved in regulation of follicle development and oocyte maturation and quality. PMID:26356408

  2. Fully humanized neutralizing antibodies to interleukin-8 (ABX-IL8) inhibit angiogenesis, tumor growth, and metastasis of human melanoma.

    PubMed

    Huang, Suyun; Mills, Lisa; Mian, Badar; Tellez, Carmen; McCarty, Marya; Yang, X-D; Gudas, Jean M; Bar-Eli, Menashe

    2002-07-01

    Interleukin-8 (IL-8) has recently been shown to contribute to human melanoma progression by functioning as a mitogenic and angiogenic factor. In the present study, we investigated whether targeting IL-8 by a fully human anti-IL-8 antibody (ABX-IL8) could be a potential therapeutic strategy to control angiogenesis, growth, and metastasis of melanoma. The human melanoma cells A375SM (high IL-8 producer) and TXM-13 (intermediate IL-8 producer) were injected subcutaneously into nude mice, which were then treated with ABX-IL8 (1 mg/3 times weekly, i.p., for 3 weeks). Tumor growth of both melanomas in ABX-IL8-treated mice was significantly inhibited when compared with control IgG-treated animals. ABX-IL8 treatment also suppressed experimental metastasis when the melanoma cells were injected intravenously. IL-8 blockade by ABX-IL8 significantly inhibited the promoter activity and the collagenase activity of matrix metalloproteinase-2 in human melanoma cells, resulting in decreased invasion through reconstituted basement membrane in vitro. In vivo, ABX-IL8 treatment resulted in decreased expression of matrix metalloproteinase-2, and decreased vascularization (angiogenesis) of tumors concomitant with increased apoptosis of tumor cells. Moreover, in an in vitro vessel formation assay, ABX-IL8 directly interfered with the tubule formation by human umbilical vein endothelial cells. Taken together, these results point to the potential utility of ABX-IL8 as a modality to treat melanoma and other solid tumors either alone or in combination with conventional chemotherapy or other anti-tumor agents. PMID:12107097

  3. Organophosphorothionate pesticides inhibit the bioactivation of imipramine by human hepatic cytochrome P450s

    SciTech Connect

    Di Consiglio, Emma; Meneguz, Annarita; Testai, Emanuela . E-mail: testai@iss.it

    2005-06-15

    The drug-toxicant interaction between the antidepressant imipramine (IMI) and three organophosphorothionate pesticides (OPTs), to which humans may be chronically and simultaneously exposed, has been investigated in vitro. Concentrations of IMI (2-400 {mu}M) and OPTs ({<=}10 {mu}M) representative of actual human exposure have been tested with recombinant human CYPs and human liver microsomes (HLM). The different CYPs involved in IMI demethylation to the pharmacologically active metabolite desipramine (DES) were CYP2C19 > CYP1A2 > CYP3A4. The OPTs significantly inhibited (up to >80%) IMI bioactivation catalyzed by the recombinant CYPs tested, except CYP2D6, and by HLM; the inhibition was dose-dependent and started at low pesticide concentrations (0.25-2.5 {mu}M). The OPTs, having lower K {sub m} values, efficiently competed with IMI for the enzyme active site, as in the case of CYP2C19. However, with CYP1A2 and CYP3A4, a time- and NADPH-dependent mechanism-based inactivation also occurred, consistently with irreversible inhibition due to the release of the sulfur atom, binding to the active CYP during OPT desulfuration. At low IMI and OPT concentrations, lower IC50 values have been obtained with recombinant CYP1A2 (0.7-1.1 {mu}M) or with HLM rich in 1A2-related activity (2-10.8 {mu}M). The K {sub i} values (2-14 {mu}M), independent on substrate concentrations, were quite low and similar for the three pesticides. Exposure to OPTs during IMI therapeutic treatments may lead to decreased DES formation, resulting in high plasma levels of the parent drug, eventual impairment of its pharmacological action and possible onset of adverse drug reactions (ADRs)

  4. Intracortical inhibition and facilitation in different representations of the human motor cortex.

    PubMed

    Chen, R; Tam, A; Bütefisch, C; Corwell, B; Ziemann, U; Rothwell, J C; Cohen, L G

    1998-12-01

    Intracortical inhibition and facilitation in different representations of the human motor cortex. J. Neurophysiol. 80: 2870-2881, 1998. Intracortical inhibition (ICI) and intracortical facilitation (ICF) of the human motor cortex can be studied with paired transcranial magnetic stimulation (TMS). Plastic changes and some neurological disorders in humans are associated with changes in ICI and ICF. Although well characterized in the hand representation, it is not known if ICI and ICF vary across different body part representations. Therefore we studied ICI and ICF in different motor representations of the human motor cortex. The target muscles were rectus abdominus (RA), biceps brachii (BB), abductor pollicis brevis (APB), quadriceps femoris (QF), and abductor hallucis (AH). For each muscle, we measured the rest and active motor thresholds (MTs), the motor-evoked potential (MEP) stimulus-response curve (MEP recruitment), ICI, and ICF. The effects of different interstimulus intervals (ISIs) were studied with a conditioning stimulus (CS) intensity of 80% active MT. The effects of different CS intensities were studied at ISI of 2 ms for ICI and ISI of 15 ms for ICF. MT was lowest for APB, followed by BB, AH, and QF, and was highest for RA. Except for BB, MEP recruitment was generally steeper for muscles with lower MT. ICI and ICF were present in all the motor representations tested. The stimulus intensity necessary to elicit ICI was consistently lower than that required to elicit ICF, suggesting that they are mediated by separate mechanisms. Despite wide differences in MT and MEP recruitment, the absolute CS intensities (expressed as percentage of the stimulator's output) required to elicit ICI and ICF appear unrelated to MT and MEP recruitment in the different muscles tested. These findings suggest that the intracortical mechanisms for inhibition and facilitation in different motor representations are not related to the strength of corticospinal projections. PMID

  5. trans-Resveratrol inhibits calcium influx in thrombin-stimulated human platelets

    PubMed Central

    Dobrydneva, Yuliya; Williams, Roy L; Blackmore, Peter F

    1999-01-01

    The phytoestrogenic compound trans-resveratrol (trans-3,5,4′-trihydroxystilbene) is found in appreciable quantities in grape skins and wine. It has been shown that both products rich in trans-resveratrol and pure trans-resveratrol inhibit platelet aggregation both in vivo and in vitro. However the mechanism of this action still remains unknown. An essential component of the aggregation process in platelets is an increase in intracellular free Ca2+ ([Ca2+]i). Ca2+ must enter the cell from the external media through specific and tightly regulated Ca2+ channels in the plasma membrane. The objective of this study was to characterize what effect trans-resveratrol had on the Ca2+ channels in thrombin stimulated platelets. In this study we showed that trans-resveratrol immediately inhibited Ca2+ influx in thrombin-stimulated platelets with an IC50 of 0.5 μM. trans-Resveratrol at 0.1, 1.0 and 10.0 μM produced 20±6, 37±6 and 57±4% inhibition respectively of the effect of thrombin (0.01 u  ml−1) to increase [Ca2+]i. trans-Resveratrol also inhibited spontaneous Ba2+ entry into Fura-2 loaded platelets, with 0.1, 1.0 and 10.0 μM trans-resveratrol producing 10±5, 30±5 and 50±7% inhibition respectively. This indicated that trans-resveratrol directly inhibited Ca2+ channel activity in the platelets in the absence of agonist stimulation. trans-Resveratrol also inhibited thapsigargin-mediated Ca2+ influx into platelets. This suggests that the store-operated Ca2+ channels are one of the possible targets of trans-resveratrol. These channels rely on the emptying of the internal Ca2+ stores to initiate influx of Ca2+ into the cell. The phytoestrogens genistein, daidzein, apigenin and genistein-glucoside (genistin) produced inhibitory effects against thrombin similar to those seen with trans-resveratrol. We conclude that trans-resveratrol is an inhibitor of store-operated Ca2+ channels in human platelets. This accounts for the ability of trans-resveratrol to

  6. Common Drugs Inhibit Human Organic Cation Transporter 1 (OCT1)-Mediated Neurotransmitter Uptake

    PubMed Central

    Boxberger, Kelli H.; Hagenbuch, Bruno

    2014-01-01

    The human organic cation transporter 1 (OCT1) is a polyspecific transporter involved in the uptake of positively charged and neutral small molecules in the liver. To date, few endogenous compounds have been identified as OCT1 substrates; more importantly, the effect of drugs on endogenous substrate transport has not been examined. In this study, we established monoamine neurotransmitters as substrates for OCT1, specifically characterizing serotonin transport in human embryonic kidney 293 cells. Kinetic analysis yielded a Km of 197 micomolar and a Vmax of 561 pmol/mg protein/minute for serotonin. Furthermore, we demonstrated that serotonin uptake was inhibited by diphenhydramine, fluoxetine, imatinib, and verapamil, with IC50 values in the low micromolar range. These results were recapitulated in primary human hepatocytes, suggesting that OCT1 plays a significant role in hepatic elimination of serotonin and that xenobiotics may alter the elimination of endogenous compounds as a result of interactions at the transporter level. PMID:24688079

  7. Ingested Human Insulin Inhibits the Mosquito NF-κB-Dependent Immune Response to Plasmodium falciparum

    PubMed Central

    Corby-Harris, Vanessa; Green, Gabriel P.; Smithers, Hannah M.; Cheung, Kong W.; Riehle, Michael A.; Luckhart, Shirley

    2012-01-01

    We showed previously that ingested human insulin activates the insulin/IGF-1 signaling pathway in Anopheles stephensi and increases the susceptibility of these mosquitoes to Plasmodium falciparum. In other organisms, insulin can alter immune responsiveness through regulation of NF-κB transcription factors, critical elements for innate immunity that are also central to mosquito immunity. We show here that insulin signaling decreased expression of NF-κB-regulated immune genes in mosquito cells stimulated with either bacterial or malarial soluble products. Further, human insulin suppressed mosquito immunity through sustained phosphatidylinositol 3-kinase activation, since inhibition of this pathway led to decreased parasite development in the mosquito. Together, these data demonstrate that activation of the insulin/IGF-1 signaling pathway by ingested human insulin can alter NF-κB-dependent immunity, and ultimately the susceptibility, of mosquitoes to P. falciparum. PMID:22473605

  8. Cigarette smoke affects posttranslational modifications and inhibits capacitation-induced changes in human sperm proteins.

    PubMed

    Shrivastava, Vibha; Marmor, Hannah; Chernyak, Sholom; Goldstein, Marc; Feliciano, Miriam; Vigodner, Margarita

    2014-01-01

    Sperm are highly dependent on posttranslational modifications of proteins. Massive phosphorylation on tyrosine residue is required for sperm capacitation. Sumoylation has also been recently implicated in spermatogenesis and sperm functions. Cigarette smoke is known to cause oxidative stress in different tissues, and several studies suggest that it causes oxidative stress in sperm. Whether tobacco affects posttranslational modifications in human sperm is currently unknown. In this study, we show that a short exposure of human sperm to physiological concentrations of cigarette smoke extract (CSE) causes the partial de-sumoylation of many sperm proteins. Furthermore, the presence of a low concentration of CSE in the human tubal fluid during an induction of in vitro capacitation inhibits the capacitation-associated increase in protein phosphorylation. Collectively, changes in posttranslational modifications may be one of the mechanisms through which exposure to tobacco can negatively affect sperm functions and cause fertility problems. PMID:24345728

  9. Ranolazine inhibits voltage-gated mechanosensitive sodium channels in human colon circular smooth muscle cells.

    PubMed

    Neshatian, Leila; Strege, Peter R; Rhee, Poong-Lyul; Kraichely, Robert E; Mazzone, Amelia; Bernard, Cheryl E; Cima, Robert R; Larson, David W; Dozois, Eric J; Kline, Crystal F; Mohler, Peter J; Beyder, Arthur; Farrugia, Gianrico

    2015-09-15

    Human jejunum smooth muscle cells (SMCs) and interstitial cells of Cajal (ICCs) express the SCN5A-encoded voltage-gated, mechanosensitive sodium channel NaV1.5. NaV1.5 contributes to small bowel excitability, and NaV1.5 inhibitor ranolazine produces constipation by an unknown mechanism. We aimed to determine the presence and molecular identity of Na(+) current in the human colon smooth muscle and to examine the effects of ranolazine on Na(+) current, mechanosensitivity, and smooth muscle contractility. Inward currents were recorded by whole cell voltage clamp from freshly dissociated human colon SMCs at rest and with shear stress. SCN5A mRNA and NaV1.5 protein were examined by RT-PCR and Western blots, respectively. Ascending human colon strip contractility was examined in a muscle bath preparation. SCN5A mRNA and NaV1.5 protein were identified in human colon circular muscle. Freshly dissociated human colon SMCs had Na(+) currents (-1.36 ± 0.36 pA/pF), shear stress increased Na(+) peaks by 17.8 ± 1.8% and accelerated the time to peak activation by 0.7 ± 0.3 ms. Ranolazine (50 μM) blocked peak Na(+) current by 43.2 ± 9.3% and inhibited shear sensitivity by 25.2 ± 3.2%. In human ascending colon strips, ranolazine decreased resting tension (31%), reduced the frequency of spontaneous events (68%), and decreased the response to smooth muscle electrical field stimulation (61%). In conclusion, SCN5A-encoded NaV1.5 is found in human colonic circular smooth muscle. Ranolazine blocks both peak amplitude and mechanosensitivity of Na(+) current in human colon SMCs and decreases contractility of human colon muscle strips. Our data provide a likely mechanistic explanation for constipation induced by ranolazine. PMID:26185330

  10. Quercetin induces human colon cancer cells apoptosis by inhibiting the nuclear factor-kappa B Pathway

    PubMed Central

    Zhang, Xiang-An; Zhang, Shuangxi; Yin, Qing; Zhang, Jing

    2015-01-01

    Quercetin can inhibit the growth of cancer cells with the ability to act as chemopreventers. Its cancer-preventive effect has been attributed to various mechanisms, including the induction of cell-cycle arrest and/or apoptosis as well as the antioxidant functions. Nuclear factor kappa-B (NF-κB) is a signaling pathway that controls transcriptional activation of genes important for tight regulation of many cellular processes and is aberrantly expressed in many types of cancer. Inhibitors of NF-κB pathway have shown potential anti-tumor activities. However, it is not fully elucidated in colon cancer. In this study, we demonstrate that quercetin induces apoptosis in human colon cancer CACO-2 and SW-620 cells through inhibiting NF-κB pathway, as well as down-regulation of B-cell lymphoma 2 and up-regulation of Bax, thus providing basis for clinical application of quercetin in colon cancer cases. PMID:25829782