ANTI-INFLAMMATORY PHARMACOLOGY OF HISTONE DEACETYLASE INHIBITORS IN PRE-CLINICAL MODELS OF RHEUMATOID ARTHRITIS CHOO QIUYI (B.Sc.(Pharmacy)(Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACY NATIONAL UNIVERSITY OF SINGAPORE 2010 Dedication This thesis is dedicated to my parents for nurturing me, without whom I would not be who I am this day. In addition, I would like to thank my parents and siblings for their unconditional support. Every little step that I take, I know you will be there for me as I will be there for you, to rejoice together when I succeed and to pick me up when I fall. It is also dedicated to my significant other for his love. Your presence has given me the strength and confidence to things I never thought I could. Thank you for being there for me whenever I needed you, even when I added on to the competing demands of a budding career, study and personal development. Acknowledgements I am grateful to NUS for providing the Research Scholarship so that I can be financially independent. Experiments had been undertaken at NUS with the support from the Department of Pharmacy. In particular, my gratification extends to my mentors A/P Paul Ho Chi Lui and Dr Lin Haishu, for allowing the study to be executed in the laboratories, their guidance along the way and many stimulating discussions. Publications and Conference Abstracts Publications The following manuscripts arose based on this thesis: 1. Choo QY1, Ho PC1, Lin HS1. Histone Deacetylase Inhibitors: New Hope for Rheumatoid Arthritis. Current Pharmaceutical Design 2008;14:803-820. 2. Choo QY1, Ho PC1, Tanaka Y2, Lin HS1. Histone Deacetylase Inhibitors MS-275 and SAHA Induced Growth Arrest and Suppressed Lipopolysaccharide-Stimulated NF-κB p65 Nuclear Accumulation in Human Rheumatoid Arthritis Synovial Fibroblastic E11 Cells. Rheumatology 2010. DOI:10.1093/rheumatology/keq108 3. Choo QY1, Ho PC1, Tanaka Y2, Lin HS1. Histone Deacetylase Inhibitors MS-275 and SAHA Suppress p38 Mitogen Activated Protein Kinase Signaling Pathway and Chemotaxis in Rheumatoid Arthritic Synovial Fibroblastic E11 Cells. (Manuscript submitted for review). 4. Choo QY1, Ho PC1, Tanaka Y2, Lin HS1. Anti-inflammatory mechanisms of Belinostat: Suppression of Nuclear Factor-kappa B and Mitogen-Activated Protein Kinase Signaling Pathways. (Manuscript in preparation). 5. Choo QY1, Ho PC1, Lin HS1. Anti-inflammatory Mechanisms of Action of Histone Deacetylase Inhibitors in Auto-Immune Diseases: A Recent Update. (Manuscript in preparation). Contribution to publications for other research projects during candidature: 1. Lin HS1, Zhang W1, Go ML1, Choo QY1, Ho PC1. Determination of Z-3,5,4’trimethoxystilbene in rat plasma by a simple HPLC method: application in a pre-clinical pharmacokinetic study. Journal of Pharmaceutical and Biomedical Analysis. DOI: 10.1016/j.jpba.2010.03.028. 2. Lin HS1, Choo QY1, Ho PC1. Quantification of oxyresveratrol analog trans-2,4,3’,5’tetramethoxystilbene in rat plasma by a rapid HPLC method: application in a pre-clinical pharmacokinetic study. Biomedical Chromatography. DOI: 10.1002/bmc.1454 Conference Abstracts The following conference abstracts were presented based on this thesis: 1. Choo QY1, Yang J1, Ho PC1, Chan SY1, Lin HS1. In-Vitro Anti-Inflammatory Activities of PXD-101. 7th Biennial Globalization of Pharmaceutics Education Network (GPEN) Conference. Katholieke Universiteit, Utrecht University, Leiden University, Leuven, Belgium. 9th to 12th September, 2008. 2. Choo QY1, Yang J1, Ho PC1, Chan SY1, Lin HS1. Suppressive Effects of Histone Deacetylase Inhibitiors PXD-101 and TSA on Pro-Inflammatory Cytokines and Nitric Oxide Secretion. Chromatin Conference: Histones, Nucleosomes, Chromosomes and Genomes by Abcam Inc. Singapore. 9th February, 2009. 3. Choo QY1, Tanaka Y2, Ho PC1, Lin HS1. Anti-Inflammatory Mechanisms of Histone Deacetylase Inhibitor – Trichostatin A. PharmSci@Asia. China Pharmaceutical University, Nanjing, China. 27th May, 2009. 4. Choo QY1, Ho PC1, Tanaka Y2, Lin HS1. Histone Deacetylase Inhibitors SAHA and MS-275 Induced Growth Arrest, Suppressed NF-κB Activation, Down-Regulated the Secretions of Nitric Oxide, IL-6, IL-18, VEGF and MMPs in Rheumatoid Arthritis Synovial Fibroblast-Like Cells. 73rd Annual Scientific Meeting of the American College of Rheumatology by American College of Rheumatology. Pennsylvania, USA. 16th to 21st October, 2009. 5. Choo QY1, Ho PC1, Lin HS1. Anti-Inflammatory Mechanisms of Histone Deacetylase Inhibitor Trichostatin A: Suppression of NF-κB Activation. AAPS Annual Meeting and Exposition by American Association of Pharmaceutical Scientists. Los Angeles, USA. 8th to 12th November, 2009. 6. Choo QY1, Ho PC1, Tanaka Y2, Lin HS1. Anti-Inflammatory Activities of Histone Deacetylase Inhibitors MS-275 and SAHA: Suppression of NF-κB Activation. BioPharma Asia Convention 2010 by Terrapin. Singapore. 17th to 18th March, 2010. Department of Pharmacy, National University of Singapore, Singapore and 2First Department of Internal Medicine, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan. Table of Contents Chapter Page 1. INTRODUCTION 1.1 Rheumatoid Arthritis 1.2 The Histone Code and HDAC Inhibitors 1.3 Pharmacological Activities of HDAC Inhibitors in Inflammation 14 1.4 Anti-Rheumatic Activities of HDAC Inhibitors in Pre-Clinical RA Models 18 2. HYPOTHESES AND OBJECTIVES 47 3. EFFECTS OF HDAC INHIBITORS ON RASF PROLIFERATION 50 3.1 Materials and Methods 3.1.1 Materials 50 50 A. Cell Line and Cell Culture 50 B. HDAC Inhibitors and Methotrexate (MTX) 50 C. Cell Proliferation Assay 50 D. Cytoxicity Assay 50 E. Flow Cytometry Analysis 51 F. Western Blot Analysis 51 3.1.2 Methods 52 A. Cell Proliferation Assay 52 B. Cytoxicity Assay 52 C. Interaction between HDAC Inhibitors and MTX 53 D. Flow Cytometry Analysis 54 E. Western Blot Analysis for p16, p21 and p27 Proteins 55 F. Calculations 56 3.2 Results 58 A. HDAC Inhibitors Induced Growth Arrest of E11 RASF-like Cells in a Concentration-Dependent but Non-Cytotoxic Manner 57 B. Combination of HDAC Inhibitors with MTX can be Synergistic for E11 Growth Arrest 61 C. HDAC Inhibitors cause E11 Gowth Arrest by Inducing Cell Cycle Arrest D. HDAC Inhibitors Up-Regulated p21Expression 3.3 Discussions 63 66 67 4. GENE PROFILING AFTER TREATMENT WITH HDAC INHIBITORS 4.1 Materials and Methods 4.1.1 Materials 70 70 70 A. Cell Line and Cell Culture 70 B. RNA Extraction 70 4.1.2 Methods 71 A. RNA Extraction and Quality Assessment 71 B. Microarray Hybridization and Image Acquisition 72 C. Microarray Data Mining 73 D. Pathway Analysis for Differentially Expressed Genes in E11 Cells 4.2 Results 73 75 A. HDAC Inhibitors Induced Gene Expression Changes in E11 RASF-like Cells 75 B. Network and Pathway Analysis of Differentially Expressed Genes 83 4.3 Discussions 88 5. EFFECTS OF HDAC INHIBITORS ON NF-κB AND MAPK SIGNALING PATHWAYS 91 5.1 Materials and Methods 5.1.1 Materials 91 91 A. Cell Lines and Cell Culture 91 B. NF-κB Assay 91 C. Co-IP Analysis 91 D. Western Blot Analysis 91 5.1.2 Methods A. NF-κB Assay 93 93 B. Co-IP Analysis for the Association between NF-κB p65 and p300 as well as yje Association between MKP-1 and p38α 93 C. Western Blot Analysis for the Distribution of Acetylated NF-κB p65 as well as for MKP-1, p38α and p-p38 expression D. Calculations 5.2 Results A. HDAC Inhibitors Inhibited LPS-induced NF-κB p65 94 94 95 Nuclear Accumulation in E11 RASF-like and THP-1 Monocyte-like Cells 95 B. HDAC Inhibitors Increased the Association between NF-κB p65 and p300 98 C. HDAC Inhibitor Increased Acetylated NF-κB p65 Accumulation in the Cytoplasm 99 D. HDAC Inhibitor-Treated Cells Expressed more MKP-1 and less p38α 101 E. HDAC Inhibitors Increased the Association between MKP-1 and p38α 5.3 Discussions 103 104 6. EFFECTS OF HDAC INHIBITORS ON PRO-INFLAMMATORY CYTOKINE AND NO SECRETION 6.1 Materials and Methods 6.1.1 Materials 111 111 111 A. Cell Lines and Cell Culture 111 B. Hydrocortisone (HYD) and BAY 11-7082 111 C. ELISA Sets 111 D. NO Assay 111 E. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) 6.1.2 Methods 111 113 A. Cytokine Secretion 113 B. NO Assay 113 C. Interaction between HDAC Inhibitors and MTX 114 D. RT-PCR analysis for COX-2 and iNOS Expression 115 E. Calculations 116 6.2 Results 117 A. HDAC Inhibitors Suppressed Pro-Inflammatory Cytokines in E11 RASF-like and THP-1 Monocyte-like Cells in a Concentration-Dependent Manner 117 B. HDAC Inhibitors Suppressed NO in E11 RASF-like and RAW264.7 Macrophage-like Cells in a ConcentrationDependent Manner 127 C. Combination of HDAC Inhibitor with MTX can be Synergistic for NO Suppression 131 D. HDAC Inhibitors Reduced COX-2 and iNOS Expression in E11 Cells 6.3 Discussions 134 135 7. EFFECTS OF HDAC INHIBITORS ON OTHER DOWNSTREAM EFFECTORS OF NF-κB AND MAPK SIGNALING PATHWAYS 7.1 Materials and Methods 7.1.1 Materials 140 140 140 A. Cell Lines and Cell Culture 140 B. VEGF Assay 140 C. HUVEC Angiogenesis Assay 140 D. Chemotaxis under Agarose 140 E. Chemokine ELISA 141 F. Substrate Gel Zymography 7.1.2 Methods 141 142 A. VEGF Assay 142 B. HUVEC Angiogenesis Assay 142 C. Chemotaxis ELISA 143 D. Chemotaxis under Agarose 143 E. Substrate Gel Zymography for MMP-2 and MMP-9 in E11 cells 7.2 Results 144 146 A. HDAC Inhibitors Down-Regulated VEGF in E11 RASF-like Cells in a concentration-dependent manner 147 B. 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Annals of Oncology. 2008; 19:161-161. 250. http://www.curis.com/AACR2008CUDC101liver.pdf - last accessed on 16 June 2010. 251. http://www.4sc.de/en/news/pdf/Ecco_Presentation_17_09_09_eng.pdf - last accessed on 16 June 2010. 211 [...]... of Histone Deacetylase Inhibitors in Pre- clinical Models of Rheumatoid Arthritis HDAC inhibitors have emerged as a novel class of anti- cancer agents Their antirheumatic activities had been documented in various pre- clinical RA models However, their anti- rheumatic mechanisms of action are not well elucidated The work that was carried out for this thesis aimed to elucidate the anti- inflammatory and anti- rheumatic... values of HDAC inhibitors on NF-κB p65 nuclear accumulation inhibition in E11 cells 5.2 IC50 values of HDAC inhibitors on NF-κB p65 nuclear accumulation inhibition in THP-1 cells 6.1 97 IC50 values of HDAC inhibitors on pro -inflammatory cytokine inhibition in THP-1 cells 6.2 96 122 IC50 values of HDAC inhibitors on pro -inflammatory cytokine inhibition in E11 cells 125 6.3 IC50 values of HDAC inhibitors. .. NO inhibition in RAW264.7 cells 129 6.4 IC50 values of HDAC inhibitors on NO inhibition in E11 cells 130 6.5 Combination indices for the drug combinations in RAW264.7 cells 133 6.6 Combination indices for the drug combinations in E11 cells 134 6.7 Relative densitometry readings 135 7.1 IC50 values of HDAC inhibitors on VEGF inhibition in E11 cells 147 7.2 HDAC inhibitors totally abrogated VEGF-induced... effects of HDAC inhibitors 44 2.1 Possible anti- inflammatory and anti- rheumatic mechanisms of HDAC inhibitors 48 3.1 HDAC inhibitors and MTX inhibited E11 cell proliferation in a concentrationdependent manner 57 3.2 Effects of HDAC inhibitors and MTX on LDH release from E11 cells 59 3.3 Median-drug-effect plots for combinations of HDAC inhibitors and MTX 62 3.4 A representative DNA histogram and HDAC inhibitors. .. values of HDAC inhibitors on GCP-2 inhibition in THP-1 cells 151 7.4 IC50 values of HDAC inhibitors on MCP-2 inhibition in THP-1 cells 152 7.5 IC50 values of HDAC inhibitors on MIF inhibition in THP-1 cells 153 List of Figures Figure Page 1.1 The integrated immune response and pathogenesis of RA 2 1.2 The histone acetylation status controls gene expression 5 1.3 Effects of IL-1 in RA 18 1.4 Effects of. .. 1.5 HDAC inhibitors can inhibit cell proliferation by modulating CDK inhibitors 26 1.6 Involvement of the NF-κB signaling pathway in RA 30 1.7 HDAC2 as an important co-repressor molecule for glucocorticoid-mediated suppression of NF-κB-driven inflammatory gene expression 33 1.8 The MAPK signaling pathway 35 1.9 Inhibition of cartilage damage and bone destruction by HDAC inhibitors 41 1.10 Anti- angiogenic... combinations of HDAC inhibitors with MTX in RAW264.7 cells 6.9 130 SB203580 suppressed NO in RAW264.7 and E11 cells in a concentrationdependent manner 6.8 128 HDAC inhibitors and MTX suppressed NO in E11 cells in a concentrationdependent manner 6.7 126 HDAC inhibitors and MTX suppressed NO in RAW264.7 cells in a concentration-dependent manner 6.6 124 BAY 11-7082 suppressed IL-6 and IL-18 in E11 cells in. .. HDAC inhibitors as innovative anti- cancer modalities In October 2006, US Food and Drug Administration approved the first HDAC inhibitor – vorinostat (suberoylanilide hydroxamic acid or SAHA) It is indicated for cutaneous T-cell lymphoma (CTCL) [14-16] At least twenty other HDAC inhibitors had entered Phase I clinical trials (Table 1.2) Besides being anti- neoplastic, HDAC inhibitors are promising antiinflammatory... mechanisms of action of HDAC inhibitors Inhibition of RASF proliferation, suppression of proinflammatory cytokines, chemokines and NO as well as down-regulation of angiogenesis, chemotaxis and MMPs may provide beneficial effects in RA The aforementioned effects may be a result of CDK inhibitor p21 up-regulation as well as MAPK and NF-κB inhibition Hence, HDAC inhibitors appear to be an innovative strategy... 6.5 123 HDAC inhibitors suppressed IL-6 and IL-18 in E11 cells in a concentrationdependent manner 6.4 119 BAY 11-7082 suppressed IL-1β, IL-6, IL-18 and TNF-α in THP-1 cells in a concentration-dependent manner 6.3 103 133 Median-drug-effect plots for the combinations of HDAC inhibitors with MTX in E11 cells 134 6.10 HDAC inhibitors reduced COX-2 and iNOS expression in E11 cells 135 7.1 HDAC inhibitors . Summary Anti-inflammatory Pharmacology of Histone Deacetylase Inhibitors in Pre-clinical Models of Rheumatoid Arthritis HDAC inhibitors have emerged as a novel class of anti-cancer. ANTI-INFLAMMATORY PHARMACOLOGY OF HISTONE DEACETYLASE INHIBITORS IN PRE-CLINICAL MODELS OF RHEUMATOID ARTHRITIS CHOO QIUYI (B.Sc.(Pharmacy)(Hons.),. 6.1 IC 50 values of HDAC inhibitors on pro-inflammatory cytokine inhibition in THP-1 cells 122 6.2 IC 50 values of HDAC inhibitors on pro-inflammatory cytokine inhibition in E11 cells 125