molecular merchanisms underlying inhibitory effect of fuciodan on cytokine-induced inflammation in glioma cells

Molecular Mechanisms underlying Inhibitory Effect of Fucoidan on Cytokine-Induced Inflammation in Glioma Cells DO THI THU HANG The Graduate School Sungkyunkwan University Department of Pharmacy Molecular Mechanisms underlying Inhibitory Effect of Fucoidan on Cytokine-Induced Inflammation in Glioma Cells 2008 DO THI THU HANG Molecular Mechanisms nderlying Inhibitory Effect of Fucoidan on Cytokine-Induced Inflammation in Glioma Cells DO THI THU HANG The Graduate School Sungkyunkwan University Department of Pharmacy Molecular Mechanisms underlying Inhibitory Effect of Fucoidan on Cytokine-Induced Inflammation in Glioma Cells DO THI THU HANG A Dissertation Submitted to the Department of Pharmacy and the Graduate School of Sungkyunkwan University in partial fulfillment of the requirements for the degree of Doctor in Philosophy in Pharmacy October 2008 Approved by Professor Suhkneung Pyo Major Advisor CONTENTS I Introduction Alzheimer’s disease 1.1 Overview of Alzheimer’s disease ·································································· 1.2 Etiology of Alzheimer’s disease ··································································· 1.3 Mechanism of Alzheimer’s disease ······························································· 1.4 Inflammation in Alzheimer’s disease ···························································· Fucoidan 2.1 Structure of fucoidans ··················································································· 2.2 Biological properties of fucoidans ······························································ 11 2.3 Scavenger receptors ···················································································· 14 iNOS 3.1 Overview of iNOS ······················································································ 16 3.2 Regulation of iNOS expression in glial cells ················································ 17 Aim of study ·································································································································· 25 II Materials and methods Materials ············································································································ 27 i Cell culture ········································································································ 28 Preparation of beta-amyloid aggregation ·························································· 29 MTT assay for cell viability ············································································· 29 Nitric oxide measurement··················································································· 30 ELISA ·············································································································· 30 RNA extraction ································································································· 31 Reverse transcription-polymerase chain reaction analysis ································ 32 Western blotting analysis ·················································································· 35 10 Nuclear extraction ···························································································· 36 11 Electrophoretic mobility shift assay ································································ 36 12 Cell transfection and RNA interference ·························································· 38 13 Statistical analysis ·························································································· 38 III Results The inhibitory effect of fucoidan on TNF-α and IFN-γ (T/I) - stimulated inflammation in glioma cells and neuroblastoma cells ········································ 39 1.1 TNF-α and IFN-γ induces NO/iNOS, ICAM-1 and AD’s related genes production and increases beta-amyloid -induced NO production ·················· 39 1.2 Fucoidan inhibits TNF-α and IFN-γ (T/I) – induced ICAM-1 expression ···· 47 ii 1.3 Fucoidan inhibits NO and iNOS production in TNF-α and IFN-γ (T/I) stimulated C6 cells ························································································ 47 The mechanism underlying inhibitory effect of fucoidan on TNF-α and IFN-γ (T/I) – induced iNOS/NO production in C6 glioma cells ································ 53 2.1 Fucoidan suppresses the activation of p38 MAPK in TNF-α and IFN-γ (T/I) - stimulated iNOS/NO production ························································ 53 2.2 Fucoidan inhibits the activation of AP-1, STAT1, and IRF-1 in TNF-α and IFN-γ (T/I) - stimulated C6 cells ··································································· 57 2.3 SR-B1 signaling is not involved in the inhibitory effect of fucoidan on TNF-α and IFN-γ-stimulated iNOS expression ············································· 59 2.4 SR-B1 expression is required for the inhibitory effect of fucoidan on iNOS expression and might be related to p38 phosphorylation ······························· 62 Mechanism underlying inhibitory effect of fucoidan on IFN-γ - induced iNOS/NO production, comparing its effect between C6 glioma cells and Raw264.7 macrophages ····················································································· 67 3.1 Fucoidan regulates IFN-γ – induced NO production and iNOS expression differently in C6 glioma cells and Raw264.7 macrophages·························· … 67 3.2 Fucoidan regulates IFN-γ – induced JAK/STAT and p38 phosphorylation differently in C6 glioma cells and RAW264.7 macrophages ························· 72 iii 3.3 Involvement of p38 and JAK/STAT in IFN-γ – induced iNOS expression in C6 glioma cells and Raw264.7 macrophages ·············································· 77 3.4 The stimulatory effect of fucoidan on IFN-γ - induced iNOS expression is mediated by TNF-α production which is negatively regulated by p38 in Raw264.7 macrophages ················································································ 79 3.5 SR-A1 is not involved in effect of fucoidan on IFN-γ-induced – iNOS in Raw264.7 macrophages ·················································································· 85 IV Discussion Fucoidan could be a potential candidate for neurological diseases treatment via suppression neuro-inflammation ········································································· 87 Suppression of iNOS expression by fucoidan is mediated by regulation of p38 MAPK, AP-1, and IRF-1, and depends on up-regulation of SR-B1 expression in TNF-α and IFN-γ-stimulated C6 glioma cells ················································· 89 Fucoidan regulates IFN-γ – induced iNOS/NO production differently in glial cells and macrophages: the involvement of JAK/STAT, p38 activation and TNF-α production ································································································ 95 iv nitric oxide synthase expression in astroglia: role of Erk mitogen-activated protein kinase and NF-kappaB Glia 2003 41:152–60 Martin E, Nathan C, Xie QW Role of interferon regulatory factor in induction of nitric oxide synthase J Exp Med 1994 180:977–84 Maruyama H, Nakajima J, Yamamoto IA Study on the anticoagulant and fibrinolytic activities of a crude fucoidan from the edible brown seaweed Laminaria religiosa, with special reference to its inhibitory effect on the growth of sarcoma-180 ascites cells subcutaneously implanted into mice Kitasato Arch Exp Med 1987 60:105– 121 Matsui MS, Muizzuddin N, Arad S, Marenus K Sulfated polysaccharides from red microalgae have anti-inflammatory properties in vitro and in vivo Appl Biochem Biotechnol 2003 104:13–22 Mattioli AV Heparin-induced thrombocytopenia: Implictions for cardiologist G Ital Cardiol (Rome) 2006 7: 675–83 Meldrum KK, Meldrum DR, Hile KL, Yerkes EB, Ayala A, Cain MP, Rink RC, Casale 122 AJ, Kaefer MA p38 MAPK mediates renal tubular cell TNF-alpha production and TNF-alpha-dependent apoptosis during simulated ischemia Am J Physiol Cell Physiol 2001 281(2):C563-70 Mulder M, Terwel D Possible link between lipid metabolism and cerebral amyloid angiopathy in Alzheimer's disease: A role for high-density lipoproteins? Haemostasis 1998 28(3-4):174-94 Mulloy B, Ribeiro AC, Alves AP, Vieira RP, and Mourao PAS Sulfated fucans from echinoderms have a regular tetrasaccharide repeating unit defined by specific patterns of sulfation at the O-2 and O-4 positions J Biol Chem 1994 269: 22113–23 Mytar B, Gawlicka M, Szatanek R, Woloszyn M Induction of intracellular cytokine production in human monocytes/macrophages stimulated with ligands of pattern recognition receptors Inflamm Res 2004 53:100–106 Nakamura T, Suzuki H, Wada Y, Kodama T, Doi T Fucoidan induces nitric oxide production via p38 mitogen-activated protein kinase and NF-kappaB-dependent signaling pathways through macrophage scavenger receptors Biochem Biophys Res Commun 2006 343:286-94 123 Nathan C, Xie QW Nitric oxide synthases: roles, tolls, and controls Cell 1994 78:915–8 Nelson S, Summer WR Innate immunity, cytokines, and pulmonary host defense Infect Dis Clin North Am 1998 12:555-67 Nezgovorov DV, Dobrodeeva LK, Mokrousova EV, Menshikova EA Influence of fucoidan on cells of peritoneal exudates at intraperitoneal introduction to outbred white mice Med Immunol 2004 3-5:242–3 Obluchinskaya ED, Voskoboynikov GM, Galinkin VA Content of alginic acid and fucoidan in the Fucales of Barentsev sea Prikl Biokhim Microbiol 2002 38: 213–6 Okai Y, Higashi-Okai K, Ishizaka S, Yamashita U Enhancing effect of polysaccharides from an edible brown alga, Hijikia fusiforme (Hijiki), on release of tumor necrosis factor-α from macrophages of endotoxin-nonresponder C3H/HeJ mice Nutr Cancer 1997 27:74–9 Oomizu S, Yanase Y, Suzuki H, Kameyoshi Y, Hide M Fucoidan prevents C epsilon 124 germline transcription and NFkappaB p52 translocation for IgE production in B cells Biochem Biophys Res Commun 2006 350:501-7 Ordija CM, Freeman MW Activation of signaling pathways by putative scavenger receptor class A (SR-A) ligands requires CD14 but not SR-A Biochem Biophys Res Commun 2003 310:542-9 Pahan K, Sheikh FG, Namboodiri AM, Singh I N-acetyl cysteine inhibits induction of no production by endotoxin or cytokine stimulated rat peritoneal macrophages, C6 glial cells and astrocytes Free Radic Biol Med 1998 24:39–48 Paresce DM, Ghosh RN, and Maxfield FR Microglial Cells Internalize Aggregates of the AD Amyloid β-Protein Via a Scavenger Receptor Neuron 1996 17: 553-65 Park SY, Lee H, Hur J, Kim SY, Kim H, Park JH, Cha S, Kang SS, Cho GJ, Choi WS, Suk K Hypoxia induces nitric oxide production in mouse microglia via p38 mitogenactivated protein kinase pathway Brain Res Mol Brain Res 2002 107:9–16 Pei JJ, Sjögren M, Winblad B Neurofibrillary degeneration in Alzheimer's disease: from molecular mechanisms to identification of drug targets Curr Opin Psychiatry 125 2008 21(6):555-61 Peiser L, Mukhopadhyay S, Gordon S Scavenger receptors in innate immunity Current Opinion in Immunology 2002 14:123-8 Perneczky R, Pohl C, Sorg C, Hartmann J, Komossa K, Alexopoulos P, Wagenpfeil S, Kurz A Complex activities of daily living in mild cognitive impairment: conceptual and diagnostic issues Age Ageing 2006 35(3):240-5 Piet R, Vargová L, Syková E, Poulain DA, Oliet SH Physiological contribution of the astrocytic environment of neurons to intersynaptic crosstalk Proc Natl Acad Sci U S A 2004 101(7):2151-5 Pollaud-Chérion C, Vandaele J, Quartulli F, Séguélas MH, Decerprit J, Pipy B 1998 Involvement of calcium and arachidonate metabolism in acetylated-low-densitylipoprotein-stimulated tumor-necrosis-factor-alpha production by rat peritoneal macrophages Eur J Biochem 1998 253:345-53 Polvikoski T, Sulkava R, Haltia M, Kainulainen K, Vuorio A, Verkkoniemi A, Niinistö 126 L, Halonen P, Kontula K Apolipoprotein E, dementia, and cortical deposition of betaamyloid protein N Engl J Med 1995 333(19):1242-7 Ramsauer K, Sadzak I, Porras A, Pilz A, Nebreda AR, Decker T, Kovarik P p38 MAPK enhances STAT1-dependent transcription independently of Ser-727 phosphorylation Proc Natl Acad Sci U S A 2002 99:12859-64 Rapp MA, Schnaider-Beeri M, Grossman HT, Sano M, Perl DP, Purohit DP, Gorman JM, Haroutunian V Increased hippocampal plaques and tangles in patients with Alzheimer disease with a lifetime history of major depression Arch Gen Psychiatry 2006 63(2):161-7 Rentzos M, Michalopoulou M, Nikolaou C, Cambouri C, Rombos A, Dimitrakopoulos A, Vassilopoulos D The role of soluble intercellular adhesion molecules in neurodegenerative disorders J Neurol Sci 2005 228:129-35 Rossner S, Lange-Dohna C, Zeitschel U, Perez-Polo JR Alzheimer’s disease betasecretase BACE1 is not a neuron-specific enzyme J Neurochem 2005 92:226–34 Saha RN, Pahan K Regulation of inducible nitric oxide synthase gene in glial cells 127 Antioxid Redox Signal 2006 8(5-6):929-47 Santello M, Volterra A Synaptic modulation by astrocytes via Ca(2+)-dependent glutamate release Neuroscience 2008 [Epub ahead of print] Saura M, Zaragoza C, Bao C, McMillan A, Lowenstein CJ Interaction of interferon regulatory factor-1 and nuclear factor kappaB during activation of inducible nitric oxide synthase transcription J Mol Biol 1999 289:459–71 Schwartz C, Beck K, Mink S, Schmolke M, Budde B, Wenning D, Klempnauer KH Recruitment of p300 by C/EBPbeta triggers phosphorylation of p300 and modulates coactivator activity EMBO J 2003 22:882–92 Schwenger P, Alpert D, Skolnik EY, Vilcek J Activation of p38 mitogen-activated protein kinase by sodium salicylate leads to inhibition of tumor necrosis factorinduced IkappaB alpha phosphorylation and degradation Mol Cell Biol 1998 18(1):78-84 Semenov AV, Mazurov AV, Preobrazhenskaya ME Sulfated polysaccharides as 128 inhibitors of receptor activity of Pselectin and P-selectin-dependent inflammation Quest Med Chem 1999 2:135–43 Shibata H, Kimura-Takagi I, Nagaoka M, Hashimoto S Properties of fucoidan from Cladosiphon okamuranus tokida in gastric mucosal protection Biofactors 2000 11:235–45 Shinoda J, Whittle IR Nitric oxide and glioma: a target for novel therapy? Br J Neurosurg 2001 15:213-220 Shrestha B, Zhang B, Purtha WE, Klein RS, Diamond MS Tumor necrosis factor{alpha} protects against lethal West Nile virus infection by promoting trafficking of mononuclear leukocytes into the central nervous system J Virol 2008 Epub ahead of print Spitsin SV, Koprowski H, Michaels FH Characterization and functional analysis of the human inducible nitric oxide synthase gene promoter Mol Med 1996 2:226–35 Springer GF, Wurzel HA, McNeal GMJ, Ansell NJ Isolation of anticoagulant fractions from crude fucoidan Proc Soc Biol Med 1957 94:404–9 129 Starke R, Mackie ID, rummond O, MacGregor I Prion protein in patients with renal failure Transfus Med 2006 16:165–8 Steiner H, Capell A, Leimer U, Haass C Genes and mechanisms involved in betaamyloid generation and Alzheimer's disease Eur Arch Psychiatry Clin Neurosci 1999 249(6):266-70 Taylor BS, De Vera ME, Ganster RW, Wang Q, Shapiro RA, Morris SM Jr, Billiar TR, Geller DA Multiple NF-kappaB enhancer elements regulate cytokine induction of the human inducible nitric oxide synthase gene J Biol Chem 1998 273:15148–56 Taylor BS, Geller DA Molecular regulation of the human inducible nitric oxide synthase (iNOS) gene Shock 2000 13:413–24 Tiraboschi P, Hansen LA, Thal LJ, Corey-Bloom J The importance of neuritic plaques and tangles to the development and evolution of AD Neurology 2004 62(11):1984-9 Wada H, Nakajoh K, Satoh-Nakagawa T, Suzuki T, Ohrui T, Arai H, Sasaki H Risk 130 factors of aspiration pneumonia in Alzheimer's disease patients Gerontology 2001 47(5):271-6 Wang MJ, Jeng KC, Kuo JS, Chen HL, Huang HY, Chen WF, Lin SZ c-Jun Nterminal kinase and, to a lesser extent, p38 mitogen-activated protein kinase regulate inducible nitric oxide synthase expression in hyaluronan fragments-stimulated BV-2 microglia J Neuroimmunol 2004 146:50–62 Waring SC, Rosenberg RN Genome-wide association studies in Alzheimer disease Arch Neurol 2008 65(3):329-34 Williams GM Antitumor necrosis factor-alpha therapy and potential cancer inhibition Eur J Cancer Prev 2008 17:169-77 Won JS, Im YB, Key L, Singh I, Singh AK The involvement of glucose metabolism in the regulation of inducible nitric oxide synthase gene expression in glial cells: possible role of glucose-6-phosphate dehydrogenase and CCAAT/enhancing binding protein J Neurosci 2003 23:7470–8 Won JS, Im YB, Singh AK, Singh I Dual role of cAMP in iNOS expression in glial 131 cells and macrophages is mediated by differential regulation of p38-MAPK/ATF-2 activation and iNOS stability Free Radic Biol Med 2004 37:1834-44 Wyss-Coray T, Loike JD, Brionne TC, Lu E, Anankov R, Yan F, Silverstein SC, Husemann J Adult mouse astrocytes degrade amyloid-β in vitro and in situ Nature Medicine 2003 9(4):453-45 Xie QW, Whisnant R and Nathan C Promoter of the mouse gene encoding calciumindependent nitric oxide synthase confers inducibility by interferon γ and bacterial lipopolysaccharide J Exp Med 1993 177:1779–84 Yamada Y, Doi T, Hamakubo T, Kodama T Scavenger receptor family proteins: roles for atherosclerosis, host defence and disorders of the central nervous system Cell Mol Life Sci 1998 54:628-40 Yamamoto I, Takahashi M, Suzuki T, Seino H Antitumor effect of seaweeds IV Enhancement of antitumor activity by sulfation of a crude fucoidan fraction from Sargassum kjellmanianum Jpn J Exp Med 1984 54:143–51 132 Yoon SJ, Pyun YR, Hwang JK, Mourão PAS A sulfated fucan from the brown alga Laminaria cichorioides has mainly heparin cofactor II-dependent anticoagulant activity Carbohydr Res 2007 342: 2326–30 Yuhanna IS, Zhu Y, Cox BE, Hahner LD, Osborne-Lawrence S, Lu P, Marcel YL, Anderson RG, Mendelsohn ME, Hobbs HH, Shaul PW High-density lipoprotein binding to scavenger receptor-BI activates endothelial nitric oxide synthase Nat Med 2001 7:853-7 Zaporozhets TS, Ivanushko LA, Zvyagintseva TA, Molchanova VI Cytokine inducing activity of biopolymers from sea hydrobionts Med Immunol 2004 6:89–96 Zen K, Liu Y, Cairo D, Parkos CA CD11b/CD18-dependent interactions of neutrophils with intestinal epithelium are mediated by fucosylated proteoglycans J Immunol 2002 169:5270–8 Zhang XW, Liu Q, Thorlacius H Inhibition of selectin function and leukocyte rolling protects against dextran sodium sulfate-induced murine colitis Scand J Gastroenterol 2001 36: 270–25 133 Zhang C, Baumgartner RA, Yamada K, Beaven MA Mitogen-activated protein (MAP) kinase regulates production of tumor necrosis factor-alpha and release of arachidonic acid in mast cells Indications of communication between p38 and p42 MAP kinases J Biol Chem 1997 272(20):13397-402 Zhang H, Chen X, Teng X, Snead C, Catravas JD Molecular cloning and analysis of the rat inducible nitric oxide synthase gene promoter in aortic smooth muscle cells Biochem Pharmacol 1998 55:1873–80 Zvyagintseva TN, Shevchenko NM, Nazarova IV, Scobun AS Inhibition of complement activation by water-soluble polysaccharides of some far-eastern brown seaweeds Comp Biochem Physiol C Toxicol Pharmacol 2000 126: 209–15 Zvyagintseva T, Shevchenko N, Nazarenko E, Gorbach V Water-soluble polysaccharides of some brown algae of the Russian Far-East Structure and biological action of lowmolecular mass polyuronans J Exp Mar Biol Ecol 2005 320:123–31 134 국문 초록 알츠하이머병은 신경계 염증과 관련된 전형적인 질환이다 beta-amyloid (bA) 의 형성은 신경계 세포사멸의 유도와 신경교세포에 의해 매개되는 염증반응 으로 인한 신경 세포의 죽음, 이 두 가지 경로를 통해 알츠하이머병의 발병 을 가속화 시킨다 본 실험에서는, 고분자의 황산화 다당류인 fucoidan이 사 이토카인에 의한 신경계 염증에 미치는 영향을 관찰하였다 In vitro상에서 TNF-α 와 IFN-γ (T/I)를 이용하여 염증반응을 유도한 결과, fucoidan이 T/I에 의해 활성화 된 신경교세포의 iNOS-NO 시스템을 억제 시켰으며, T/I에 의해 활성화 된 신경아세포종의 경우 fucoidan에 의해 ICAM-1의 발현이 감소하였 다 Fucoidan이 T/I에 의한 신경교세포의 NO 생성 및 iNOS의 발현을 억제하는 과정에서 p38 MAPK, AP-1, 그리고 IRF-1이 fucoidan의 억제기능에 중요하게 작용함을 확인하였으며, 세포내 SR-B1의 발현이 p38의 인산화 과정을 통한 fucoidan의 iNOS/NO 생성 억제기능과 관련이 있으리라 사료된다 이러한 결 과는 fucoidan이 알츠하이머병 등, 신경계 염증질환을 치료하는데 가능성이 있음을 보여준다 TNF-α와 IFN-γ에 의한 염증 반응 및 염증 부위에 따른 fucoidan의 효과를 확인한 결과, 신경교세포(C6) 및 대식세포(Raw264.7) 모두에서 IFN-γ에 의한 135 iNOS/NO 생성에만 영향을 미치며, TNF-α에 의한 iNOS/NO 생성에는 효과가 없었다 C6 세포의 경우, fucoidan은 JAK/STAT 및 p38의 활성을 억제시키고 이로 인해 IFN-γ에 의한 iNOS/NO의 생성이 억제된 반면, Raw264.7 세포에 서는 fucoidan이 p38에 의해 억제되는 TNF-α의 생성을 촉진시킴으로써 IFNγ에 의한 iNOS/NO의 생성이 증가하였다 본 연구를 통해 신경교세포와 대 식세포에서의 fucoidan의 효과의 차이가 대조적임을 확인하였으며 이는 fucoidan의 임상적인 사용에 있어서 고려되어야 할 것이다 136 .. .Molecular Mechanisms underlying Inhibitory Effect of Fucoidan on Cytokine-Induced Inflammation in Glioma Cells 2008 DO THI THU HANG Molecular Mechanisms nderlying Inhibitory Effect of Fucoidan... fucoidan on systemic inflammation and neuro -inflammation, I found the effect of fucoidan only on IFN-γ-induced iNOS/NO production but not on TNF-αinduced iNOS/NO production in both C6 and Raw264.7 cells. .. the involvement of AP-1, p38, and STAT1/IRF-1 pathways in the inhibitory effects of fucoidan In addition, the increasing SR-B1 expression may contribute to the inhibitory effect of fucoidan on iNOS