THE CYTOTOXIC EFFECTS OF MORINDA CITRIFOLIA THROUGH TLR4 IN HUMAN BREAST CANCER CELLS A DISSERTATION SUBMITTED TO THE FACULTY OF CLARK ATLANTA UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY BY SABRENIA M. PARKER DEPARTMENT OF CHEMISTRY ATLANTA, GEORGIA MAY 2009 © 2009 SABRENIA M. PARKER All Rights Reserved Contents ACKNOWLEDGMENTS.. 11 LIST OF FIGURES x LIST OF TABLES xii LIST OF ABBREVIATIONS xlii CHAPTER 1: INTRODUCTION 1.1 Background and Review of Literature 1 1.1.1 What is Complementary and Alternative Medicine9 1 1.1.2 What is Morinda citr~folia9 3 1.1.2.1 Origin 3 1.1.2.2 Traditional and Modern Uses 3 1.1.2.3 ProfihingM. citr~folia 6 1.1.2.4 Evidence of Bio logical Activity 12 1.1.3 Breast Cancer 15 1.1.3.1 Prevalence 15 1.1.3.2 Risk Factors 17 1.1.3.3 Etiology 17 1.1.4 Immunology: Innate and Adaptive Immunity 19 1.1.4.1 Immunity 19 1.1.4.2 Toll-like Receptors 20 1.1.4.2.1 Summary of the TLR Signaling Pathway. 22 1.1.4.2.2 Toll-like Receptor 4 25 1.1.5 Recent Patents in the TLR Pathway 28 1.1.5.1 Toll-like Receptor Patents. 28 1.1.5.2 TLR variants 28 1.1.5.3 Adaptor proteins 30 1.1.6 Cancer Cells and Immune Surveillance. 31 1.2 Specific Aims 33 CHAPTER 2: RESEARCH DESIGN AND METHODOLOGY 2.1 Subject Stock 37 2.1.1 Cell lines and culture 37 2.1.2 Morindacitrifolia 37 2.2 Empirical Data and Collection 38 2.2.1 Small-scale extractions of noni powder 38 2.2.2 Large-scale extractions of noni powder in BuOH 41 2.2.3 Analysis of cell cytotoxicity 41 2.2.3.1 Single treatment with noni extraction dilutions 41 2.2.3.2 Multiple treatments with noni extraction dilutions . 42 2.2.3.3 Treatment with BuNoni dilutions plus LPS-RS 42 2.2.4 GeneChip Target Preparation and Hybridization 43 2.2.5 Microarray Data Analysis 44 2.2.6 Semiquantitative Reverse Transcription PCR 44 2.2.6.1 RT-PCR verification 47 2.2.6.2 Western blot analysis 48 vii 2.2.7 CytoTox~ONETM Homogeneous Membrane Integrity Assay. 50 2.2.8 Wound-healing Assay 51 2.2.9 Colony formation Assay 51 2.2.10 FACS analysis and Annexin V/PE Assay 52 2.2.10.1 Analysis of cellular DNA content by flow cytometry. 52 2.2.10.2 Assessment ofApoptosis 53 2.2.11 IC50 (Dose-response curve) Assay 53 2.3 Statistical Analysis 54 CHAPTER 3: RESULTS AND DISCUSSION 3.1. Noni Characterized 55 3.2 Results Observed 55 3.2.1 Extracts ofNoni display cytotoxic effects 55 3.2.2 BuNoni’s IC50 value in cell proliferation 62 3.2.3 Gene expression profiles identify cytotoxic pathways • 63 3.2.3.1 Microarray results were confirmed 78 3.2.3.2 Signaling pathway was analyzed 80 3.2.4 TLR4 agonist induces cell death in human BrCa cells • 86 3.2.5 Viability assays used to show the cytotoxic effect of BuNoni. 91 3.2.5.1 BuNoni extract caused a decrease in LDH 91 3.2.5.2 FACS shows BuNoni extract caused apoptotic effect. 93 3.2.6 BuNoni inhibits cell migration and alters cell structure 98 viii . 3.2.7 BuNoni prevents tumor formation • . • • 102 3.3 Discussion of Results • . • 104 3.3.1 Effect of extraction on cell proliferation • .111 3.3.2 Clinical Impact of the Research • . 112 3.4 Conclusion • . .118 3.5 Future Directions and Impact 121 4.0 References 125 LIST OF FIGURES Figure Page 1. Morinda citr~folia plant 4 2. Structures of glycosidic sugars isolated from M. citrifolia 11 3. Toll-like Receptor Pathway 24 4. Multi-solvent Survival Curves 59 5. BuOH Survival Curve with MDA-MB-231 cells 62 6. Dose Response Curve of BuNoni on MDA-MB-23 1 cells 63 7. RT-PCR Confirmations using BuNoniILPS 80 8. Ingenuity Pathway 82 9. RT-PCR Gene Confirmation 85 10. TLR4 Expression on Breast Cancer Cell Line Panel 86 11. Comparison of Glycosides from BuNoni extract 87 12. Effect of BuNoni on Proliferation ofMDA-JvfB-231 Cells 89 13. Effect of BuNoni on Proliferation of T47-D Cells 89 14. Effect of LPS-RS on MDA-MB-231 Cell Survival 91 15. Effect of LPS-RS on MDA-MB-23 1 Cells % Recovery 91 16. Lactate Dehydrogenase Measurement 94 17. Flow Cytometric Analysis 96 18. IRAK-1 Analysis 97 19. Caspase-7 Analysis 98 20. PARP/IRAK-4/IRAK-2 Analysis 98 21. Wound Healing ~a1ysis ~ 100 22. Morphology Observation 103 23. Colony Formation Assay Results 105 24. TLR4 Pathway Leading to Apoptosis 111 25. Toll-like Receptor Pathway with BuNoni 116 LIST OF TABLES Table Page 1. List of Compounds Found in Morinda citr~folia. 7 2. List of Toll-like Receptors and Their Mutants 29 3. List of Adaptor Proteins 31 4. List of Solvent Abbreviations 39 5. Microscale Extraction Solvents 40 6. List of RT-PCR Primers 45 7. List of 1°Antibodies 49 8. List of 2° Antibodies 50 9. Microarray List of Regulated Genes 65 10. List of Select Regulated Genes 77 11. Description of Human BrCa Cell Lines. 84 xii LIST OF ABBREVIATIONS AM Alternative Medicine AMPK AMP-activated protein kinase AP-l Activator protein-i BMI Body Mass Index BrCa Breast Cancer BuOH Butanol BuNoni Butanol Noni extract C. albicans Candida albicans CAM Complementary and Alternative Medicine CD14 Cluster of differentiation 14 cRNA Copy ribonucleic acid CHC13 Chloroform CM Complementary Medicine CpG Cytosine—phosphate-—-Guanine DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid cDNA Copy deoxyribonucleic acid DPPH 1,1 -Diphenyl-2-Picrylhydrazyl DTT Dithiotbreitol DXR Doxorubicin EDTA Ethylenedinitrile tetra-acetic acid EGFR Epidermal growth factor receptor xiii EGTA Ethylene glycol tetra-acetic acid ER Estrogen Receptor Era Estrogen receptor-a EtBr Ethidium bromide EtOH Ethanol FAAD Fas-Associated Death Domain Protein FACS Fluorescence-activated cell sorter FBS Fetal Bovine Serum Fyn Human gene ofprotein-tyrosine kinase oncogene family GAPDH Glyceraldehyde 3-phosphate dehydrogenase GPR3O G Protein-coupled Receptor 30 HC1 Hydrochloric acid HepOH Heptanol HER2 Human epidermal growth factor receptor-2 HexOH Hexanol HIV Human immunodeficiency virus HRP Horseradish peroxidase-conjugated HRT Hormone replacement therapy 1C50 Half maximal inhibitory concentration IFN-~3 Interferon-Beta IgG Immunoglobulin G IHC Immunohistochemistry IKK Inhibitor of Kappa Light Polypeptide Gene Enhancer in B Cells Kinase xiv IL-i Interleukin- 1 IL-i R Interleukin- 1 Receptor IRAK Interleukin (IL)- 1 Receptor—associated Kinase IRF Interferon regulatory factor JNK c-Jun N-terminal Kinase Lck Leukocyte-specific protein tyrosine kinase LDH Lactate dehydrogenase LLC Lewis Lung Carcinoma LPS Lipopolysaccharide LPS-RS Rhodobacter sphaeroides lipopolysaccaride luc Luciferase-positive Lyn V-yes-i Yamaguchi sarcoma viral related oncogene homolog M. citrifolia Morinda citrifolia MAPK Mitogen-activated protein kinases MD-2 Lymphocyte antigen 96 MeOH Methanol MgC12 Magnesium Chloride MKK Mitogen-Activated Protein Kinase Kinase mRNA Messenger ribonucleic acid MS Mass Spectometry MSDS Material safety data sheet MyD88 Myeloid differentiation primary response gene (88) NaC1 Sodium Chloride NF-icB Nuclear Factor-Kappa B NIH National Institutes of Health NMR Nuclear magnetic resonance OctOH Octanol OXL Oxaliplatin PBS Phosphate buffered saline PE Phycoerythrin PenOH Pentanol P1 Propidium iodide PR Progesterone Receptor PrOH Propanol PVDF Polyvinylidene Fluoride membrane RNA Ribonucleic acid RPMI Roswell Park Memorial Institute RT Reverse transcriptase RTPCR Reverse transcription-polymerase chain reaction SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis Ser/Thr Serine/Threonine shRNA Short hairpin RNA Src A family of proto-oncogenic tyrosine kinases TBE Tris-Borate-EDTA buffer TBK-1 TANK-binding kinase 1 TBST Tris-Buffered Saline Tween-20 xvi TC Cytotoxic T cell TIR Toll-interleukin 1 receptor TIRAP Toll-interleukin 1 receptor (TIR) domain-containing adaptor protein TLR Toll-like Receptor TNF Tumor necrosis factor TNJ Tahitian Noni Juice® TRAF TNF Receptor Associated Factor TRAM TRIF-related adaptor molecule Treg Thymus regulatory cells TRIF TIR-domain-containing adapter-inducing interferon-~3 Tris—HC1 Tris hydrochloric acid UVB-induced Ultraviolet radiation-induced at wavelength 290 to 320 nm xvii ACKNOWLEDGMENTS This is dedicated to Reagan and Riley and the future Parker children to come. I would like to thank my family and friends for their constant encouragement and support throughout the course of this dissertation when it seemed this day would never come. More directly, I would like to thank my children for their understanding and unconditional love during this time of hard work and for believing in me and praying that I achieved my education goals to leave a new legacy for generations to come. I would like to thank the National Institutes of Health (NIH), National Center for Complementary and Alternative Medicine (NCCAM) for support from grant #5R03AT003935-02. I would also like to thank the Minority Biomedical Research Support (MBRS) Research Initiative for Scientific Enhancement (RISE) Program-NIH! National Institute of General Medical Sciences (NIGMS) for support from grant #5R25GM060414. I would like to thank the laboratories at Emory Winship Cancer Center for working with me to ensure that I completed my task at hand. I would also like to thank my professors for their patience and explanations throughout my research and this dissertation writing process. And, I would like to especially thank Dr. Paul McGeady (deceased) for giving me this opportunity and encouraging me to finish the task he started. CHAPTER 1 INTRODUCTION 1.1 Background and Review ofLiterature 1.1.1 What is Complementary and Alternative Medicine? Complementary and alternative medicine (CAM), as used in the modern western world, covers an extensive range of medical approaches that do not fall within the realm of conventional medicine.