MULTIDRUG RESISTANCE PROTEIN 1 (MDR1) AND GLYCOSPHINGOLIPIDS BIOSYNTHESIS: ADVANTAGES FOR THERAPEUTICS by María Fabiana De Rosa A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Laboratory Medicine and Pathobiology University of Toronto © Copyright by María Fabiana De Rosa (2009) Multidrug Resistance Protein 1 (MDR1) And Glycosphingolipids Biosynthesis: Advantages for Therapeutics Doctor of Philosophy, 2009 María Fabiana De Rosa Department of Laboratory Medicine and Pathobiology University of Toronto ABSTRACT ABC drug transporter, MDR1, is a drug flippase that moves a variety of hydrophobic molecules from the inner to the outer leaflet of the plasma membrane. We have previously reported that MDR1 can function as a glycolipid flippase, being one of the mechanisms responsible for the translocation of glucosylceramide into the Golgi for neutral, but not acidic, glycosphingolipids (GSLs) synthesis. The interplay between GSLs and MDR1 could provide a whole new spectrum of innovative therapeutic options. We found that cell surface MDR1 partially co-localized with globotriaosyl ceramide (Gb3) in MDR1 transfected cells. Inhibition of GSL biosynthesis results in the loss of drug resistance and of cell surface MDR1. We speculated that an association of MDR1 and cell surface GSLs, in particular Gb3, may be functional at the cell surface, as MDR1 partitions into plasma membrane lipid rafts regulating MDR1 function. We therefore tested adamantyl Gb3 (adaGb3), a water soluble analog of Gb3, on MDR1 functions. AdaGb3 was able to inhibit MDR1-mediated rhodamine 123 drug efflux from MDR1 expressing cells, like cyclosporin A (CsA), a classical MDR1 inhibitor. AdaGb3 was also ii able to reverse vinblastine drug resistance in cell culture, whereas adamantyl galactosylceramide had no effect on drug resistance. The strong MDR1 reversal effects of adaGb3, as well as its favourable in vivo features make it a possible choice for inhibition of MDR1 to increase bioavailability of drugs across the intestinal epithelium (De Rosa et al., 2008). Thus, specific GSL analogs provide a new approach to MDR reversal. We have previously shown that MDR1 inhibitor CsA depletes Fabry cell lines of Gb3, the characteristic GSL accumulated in this disease, by preventing its de novo synthesis, and can also deplete Gaucher lymphoid cell lines of accumulated GlcCer (Mattocks et al., 2006). Liver and heart sections of Fabry mice treated with third generation MDR1 inhibitors showed significantly less Gb3 than liver and heart sections of untreated Fabry mice. Thus, MDR1 inhibition offers a potential alternative therapeutic approach not only for Fabry disease given the extraordinary cost of conventional enzyme replacement therapy, but also for other neutral GSL storage diseases, such as Gaucher disease. iii ACKNOWLEDGEMENTS I would like to specially thank my supervisor, Dr. Clifford Lingwood, for his firm and warmth valuable guidance through the fascinating world of cell biology, his constant and unconditional support, and his patience along this project. I would like to thank all my committee members, Dr. David Clarke, Dr. Peter Dirks, and Dr. Reinhart Reithmeier for their support and their brilliant input in this project along these years. I would also like to specially thank my external examine, Dr. Inka Brockhausen, for her valuable appraisal of this thesis. I would also like to thanks all the collaborators in this project, Dr. Shinya Ito, Dr Jeffrey Medin, Bernice Wang, Cameron Ackerley, Aina Tulips, Vanessa Rassaiah, and Xin Fan that bring their invaluable expertise on specific aspects of this project. And finally, I specially would like to thank all past and present members from Dr. Lingwood’s laboratory, especially Dr. Mylvaganam, Beth Binnington and Shirley Thompson that not only bring me their unconditional help but also their constant support in good and not so good times along this project. iv To the beloved memory of my parents, Emma and Jose Maria, for their loving encouragement and support in the pursuit of my career’s dream. To the beloved memory of my parents-in-law, Gloria and specially Ricardo, who always believed in my scientific capabilities and for his support in my decision to pursue a PhD degree. To the beloved memory of my aunt, Maria Elvira, who initiates me in the intriguing and challenging principles of biology. To my beloved husband, for his unconditional love, his loving support and strength in weakest times, for his comforting hugs that hold me tightly and do not let me fall, for sharing side by side the precious and not the so precious moments of this difficult path of pursuing a PhD, and for being that wonderful dad, always but even more when mommy had those abominable deadlines. To my children, Ricardo, Thomas, and my little princess, Gloria that enlighten every minute of my life with their smiles and laughs, and for their big hugs and loving kisses that kept me going even in the most difficult times along this project. To my brothers-in-law, Francisco and Juan Manuel, that make me feel as their special sister and friend and in whom they believe and encourage in the pursuit of her dreams. v TABLE OF CONTENTS ABSTRACT ii ACKNOWLEDGEMENTS iv TABLE OF CONTENTS vi LIST OF ABBREVIATIONS xii LIST OF TABLES xvi LIST OF FIGURES xvii LIST OF SCHEMAS xix CHAPTER ONE. INTRODUCTION 1 1.1. Multidrug Resistance 1 1.1.1. Multidrug Resistance in Cancer 3 1.1.2. Cellular Mechanisms of Multidrug Resistance 4 1.2. ATP-Binding Cassette Transporters 6 1.2.1. Structural Organization and Mechanism of ABC Transporters 8 1.2.2. ABC Transporters in Normal Cells 13 1.2.3. ABC Transporters in Cancer Cells 14 1.2.4. ABC Lipid Transporters 15 1.3. P-glycoprotein – MDR1 16 1.3.1 Structure of P-glycoprotein 17 1.3.2 Mechanism of Action 21 1.3.3 P-glycoprotein Substrates and Inhibitors 25 1.3.4 P-glycoprotein Silent Polymorphisms 30 1.3.5 P-glycoprotein Regulation 32 1.3.6 P-glycoprotein Knockout Mice 35 vi 1.3.7 Other Approaches in Modulation of Multidrug Resistance 37 1.3.8 P-glycoprotein and Stem Cells 39 1.4. Glycosphingolipids 41 1.4.1. Structure of Glycosphingolipids 42 1.4.2. Classification of Glycosphingolipids 43 1.4.3. Biosynthesis and Degradation of Glycosphingolipids 44 1.4.4. Inhibitors of Glycosphingolipids Biosynthesis 48 1.4.5. Lipid Rafts 50 1.4.6. Biological Functions of Glycosphingolipids 52 1.4.7. Globotriaosylceramide 54 1.4.7.1. Structure of Globotriaosylceramide 54 1.4.7.2. Biological Functions of Globotriaosylceramide 55 1.4.7.2.1. Antineoplastic Potential of Verotoxin 62 1.4.8. Lysosomal Storage Diseases 63 1.4.8.1. Pathogenesis 64 1.4.8.2. Fabry and Gaucher Diseases 66 1.4.8.3. Therapeutic Approaches 69 1.5. Multidrug Resistance and Glycosphingolipids 71 1.5.1. MDR1 and Ceramide Metabolism 72 1.5.2. MDR1 and Lipid Flippase 75 1.5.2.1.Alternative Mechanisms for GlcCer Transport into the Golgi 78 1.5.3. MDR1, Cholesterol and Lipid Rafts 79 CHAPTER TWO. HYPOTHESIS AND SPECIFIC AIMS 82 vii 2.1. Rationale 82 2.2. Hypothesis 84 2.2.1. Specific Aims 85 CHAPTER THREE. INHIBITION OF MULTIDRUG RESISTANCE BY ADAMANTYLGb3, A GLOBOTRIAOSYLCERAMIDE ANALOG 86 3.1. Abstract 86 3.2. Introduction 87 3.3. Materials and Methods 89 3.3.1. Materials 89 3.3.2. Cell Culture 89 3.3.3. Immunostaining of MDR1 90 3.3.4. Post-embedding Immunogold Cryolectron Microscopy 91 3.3.5. Neutral Glycolipids Extraction and Analysis 92 3.3.6. Verotoxin 1 Thin Layer Chromatography Overlay 93 3.3.7. Cytotoxicity Assay 93 3.3.8. MDR1-MDCK Raft Isolation 94 3.3.9. Western Blot Analysis of MDR1 95 3.3.10. Rhodamine 123 Efflux Assay 96 3.3.11. Drug Transport 96 3.3.12. Disulfide Cross-Linking Analysis 97 3.4. Results 98 3.4.1. MDR1 in Part Co-localizes with Gb3 98 3.4.2. AdaGb3 Prevents Multidrug Resistance in MDR1-MDCK and SK VLB Cells 102 viii 3.4.3. AdaGb3 Inhibits Cell Surface MDR1 Expression in the Long Term in MDR1-MDCK and SK VLB Cells but Increases Intracellular MDR1 as PPMP, VT1B, and CsA 105 3.4.4. Effect of Adamantyl Analogs on GSL Levels 109 3.4.5. MDR1 Distribution in Lipid Rafts 112 3.4.6. AdaGb3 Inhibits the Efflux of Rhodamine 123 in MDR1-MDCK and SK VLB Cells 113 3.4.7. Verotoxin Treatment Inhibits MDR1-mediated Rhodamine 123 Efflux 114 3.4.8. AdaGb3 Inhibits MDR1-mediated Vinblastine Efflux in Polarized Gastrointestinal Epithelial Cells 115 3.4.9. AdaGb3 Differentially Binds the MDR1 Drug Binding Pocket as Compared with Verapamil and Cyclosporin A 118 3.5. Conclusions 120 CHAPTER FOUR. INHIBITION OF ABC DRUG TRANSPORTER, MDR1, AS POSSIBLE THERAPY FOR FABRY DISEASE 122 4.1. Introduction 122 4.2. Materials and Methods 125 4.2.1. Materials 125 4.2.2. Cell Culture 126 4.2.3. Neutral Glycolipids Extraction and Analysis 126 4.2.4. Gangliosides Extraction and Analysis 126 4.2.5. Verotoxin 1 Thin Layer Chromatography Overlay 127 4.2.6. Metabolic Labeling of MDR1-MDCK Glycosphingolipids 128 ix 4.2.7. Cytotoxicity Assay 128 4.2.8. Treatment of Neonatal Fabry Mice 129 4.2.9. Tissue Extraction of Neutral GSL 130 4.2.10. VT1 Tissue Staining 130 4.2.11. Epitope Unmasking Treatment 131 4.2.12. Anti-Gb3 Tissue Staining 131 4.2.13. HPLC Quantitation of Gb3 132 4.3. Results 133 4.3.1. MDR1 Inhibition in LSD Cell Lines 133 4.3.2. Accmulation of Gb3 in Fabry Mice by 10 Weeks 135 4.3.3.