Identifying Aryl Hydrocarbon Receptor Modulators from a Natural Source
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UNIVERSITY OF ALBERTA IDENTIFYING ARYL HYDROCARBON RECEPTOR MODULATORS FROM A NATURAL SOURCE By MOHAMED ABDEL MEGUID MOHAMED EL GENDY A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY IN PHARMACEUTICAL SCIENCES FACULTY OF PHARMACY AND PHARMACEUTICAL SCIENCES © MOHAMED ABDEL MEGUID MOHAMED EL GENDY FALL 2012 EDMONTON, ALBERTA Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The authors reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author’s prior written permission. This work is dedicated to my family ABSTRACT Dioxins are widespread environmental contaminants that have been linked to a variety of deleterious effects on human health including increased cancer rates via aryl hydrocarbon receptor (AhR)-dependent mechanism. AhR is a transcription factor that regulates the expression of the carcinogen-activating enzyme, cytochrome P450 1A1 (CYP1A1). Activation of AhR and its regulated gene, CYP1A1, have been correlated with the incidence of several cancers. Therefore, the use of AhR antagonists has been proposed as a promising chemopreventative approach. Nonetheless, most of the currently used AhR antagonists are not specific to AhR and some of them act as partial agonists. Therefore, the search for new AhR antagonists is still in progress. The specific objectives of the present work were to identify new AhR modulators from a natural source. In this regard, first we demonstrated that Peganum harmala, a common traditional plant in Middle East, and North Africa, significantly inhibited the dioxin-mediated induction of CYP1A1 at mRNA, protein and activity levels using human and mouse hepatoma cells. The role of AhR was confirmed using AhR-dependent luciferase assay and electrophoretic mobility shift assay. Additionally, we identified two β-carboline alkaloids (harmine and harmaline) as the active constituents of the plant extract. Second, we demonstrated that harman, a common β-carboline in several foods and drinks and the parent structure of harmine, significantly induced CYP1A1 mainly through an AhR-dependent mechanism. Third, the active constituents of Peganum harmala extract, harmine and harmaline, and their metabolites, harmol and harmalol, significantly decreased the dioxin-mediated induction of CYP1A1 at mRNA, protein and activity levels via transcriptional (through AhR) and post-translational (through ubiquitin-proteasomal pathway as well as a direct inhibitory effect on CYP1A1 enzyme). Additionally, we demonstrated that harmine, harmol, and harmalol can act as direct antagonists for AhR, whereas harmalol affected AhR activation without a direct interfering with AhR binding to its ligands. Finally, we confirmed the effect of harmine and harmaline on dioxin-mediated induction of Cyp1a1 in vivo using the responsive C57BL/6 mouse strain. In conclusion, our data clearly demonstrate the promising effects of Peganum harmala, harmine, harmol, harmaline, and harmalol to prevent the toxicity and carcinogenicity of several AhR ligands. ACKNOWLEDGEMENTS I would like to express my deepest appreciation and sincere gratitude to my supervisor Dr. Ayman El-Kadi, Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, AB, Canada, for his continuous guidance, endless support and invaluable mentorship through this research project. I am particularly grateful to my supervisory committee members; Dr. Dion Brocks and Dr. Arno Siraki, Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, AB, Canada, for their helpful suggestions and valuable advice. I wish to thank Dr. Michael Denison and Dr. Anatoly Soshilov, Department of Environmental Toxicology, University of California, CA, USA, for performing the Competitive Ligand Binding Assay and for providing us with pGudLuc 6.1 and pGudLuc 1.1 plasmids. My gratitude goes to Dr. Vishwa Somayaji, Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, AB, Canada, for his technical assistance with the LC-ESI-MS. I wish to thank Dr. Loren Kline, Department of Physiology, University of Alberta, AB, Canada, for providing us with guinea pig livers. I am grateful to the reviewers of my publications for their invaluable comments that helped me to improve my work. My sincere appreciation goes to all my Lab members that helped me throughout my research project. I am profoundly grateful to the Egyptian Government, University of Alberta, and Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, AB, Canada, for their financial support. I am grateful to the Natural Sciences and Engineering Council of Canada (NSERC) for the financial support of this research. And I wish to thank all administrative and support staff in the Faculty of Pharmacy and Pharmaceutical Sciences for their endless help, with special thanks to Mrs. Joyce Johnson and Mr. Jeff Turchinsky. TABLE OF CONTENTS Page No. 1. INTRODUCTION…………………………………………… 1 1.1. Aryl Hydrocarbon Receptor (AhR)…………………………… 2 1.1.1. Background and Historical Perspective.................................. 2 1.1.2. Characterization of the AhR.................................................... 3 1.1.3. Species, Strain, Tissue and Cellular Expression of AhR........ 6 1.2. AhR-Regulated Genes………………………………................ 8 1.2.1. The Phase I AhR-Regulated Genes........………………......... 8 1.2.1.1. CYP1A1............................................................................... 10 1.2.1.2. Other Phase I AhR-Regulated Genes................................... 14 1.2.2. The Phase II AhR-Regulated Genes........…………………… 16 1.3. AhR Activation………………………………………….......... 18 1.3.1. Ligand-Dependent Activation…………………………......... 18 1.3.2. Exogenous Ligand-Independent Activation………………… 21 1.3.3. AhR Ligands........................................................................... 22 1.3.3.1. Classical AhR Ligands......................................................... 23 1.3.3.1.1. Genotoxic AhR Ligands.................................................... 23 1.3.3.1.2. Non-Genotoxic AhR Ligands............................................ 24 1.3.3.2. Non-Classical AhR Ligands.......………………….............. 25 1.3.3.3. AhR Antagonists.................................................................. 27 1.3.4. Regulatory Proteins of AhR Activation/Deactivation............. 30 1.4. Mechanisms Participating in Regulation of AhR-Regulated Genes......................................................................................... 34 1.4.1. Transcriptional Mechanisms………………………………... 34 1.4.2. Post-Transcriptional Mechanisms…………………………... 35 1.4.3. Post-Translational Mechanisms…………………………….. 36 1.4.3.1. Phosphorylation-Dependent Mechanism………………….. 37 1.4.3.2. 26S Proteasome-Dependent Mechanism…………………... 37 1.5. Role of AhR inToxicity and Carcinogenicity……………….... 39 1.6. Herbs and Herbal Components (HC) as Potential Therapeutic Agents ……………………………………................................ 42 1.6.1. HC and Cancer………………………………….................... 42 1.6.1.1. HC and Cancer Treatment………………………................ 42 1.6.1.2. HC and Cancer Chemoprevention………………………… 45 1.6.2. AhR Modulators from Natural Sources…………………….. 46 1.6.3. Peganum harmala……………............................................... 47 1.6.4. β-Carbolines…………………………………........................ 48 1.7. Rationale, Hypothesis, and Objectives....................................... 52 1.7.1. Rationale.........…………………………………..................... 52 1.7.2. Hypothesis...............………………………………………… 53 1.7.3. Objectives......…………………………………...................... 54 2. CHAPTER 2-MATERIALS AND METHODS……............. 56 2.1. Chemicals……………………………………………………... 57 2.2. Methods………………………………………………….......... 59 2.2.1. Cell Model…………………………………………………... 59 2.2.2. Cell Culture and Treatments………………………………... 60 2.2.3. Plant Material……………………………………………….. 61 2.2.4. Extraction and Isolation of Plant Extract………………........ 61 2.2.5. Characterization and Quantification of Active Components of the Extract ……………………………………………….. 62 2.2.6. Animals …………………………………………………….. 63 2.2.7. Determination of Cell Viability …………………………….. 63 2.2.8. RNA Extraction from Cell Cultures ……………………....... 64 2.2.9. Reverse Transcription (RT)…………………………………. 65 2.2.10. Quantification of mRNA Expression by Real-time Polymerase Chain Reaction (Real-time PCR)……………… 65 2.2.11. Protein Extraction and Western Blot Analysis …………....... 68 2.2.12. Determination of CYP1A1 Enzymatic Activity ……………. 69 2.2.13. Transfecting HepG2 cells with AhR siRNA ……………….. 70 2.2.14. Transient Transfection and Luciferase Assay ……………… 70 2.2.15. Electrophoretic Mobility Shift Assay (EMSA) ……………. 71 2.2.16. Competitive Ligand Binding Assay ……………………….. 72 2.2.17. CYP1A1 mRNA Stability ………………………………….. 73 2.2.18. CYP1A1 Protein Stability ………………………………….. 74 2.2.19. Direct Inhibitory Study ……………………………………... 74 2.2.20. Mice Treatment And Tissues Isolation …………………….. 75 2.2.21. RNA Isolation and cDNA Synthesis from Tissues ………… 75 2.2.22. Microsomal Incubation