THE DIETARY PHYTOCHEMICAL MYRICETIN INDUCES ROS-DEPENDENT BREAST CANCER CELL DEATH by Allison F. Knickle Submitted in partial fulfilment of the requirements for the degree of Master of Science at Dalhousie University Halifax, Nova Scotia December 2014 © Copyright by Allison F. Knickle, 2014 Table of Contents List of Figures ....................................................................................................... iv List of Tables ........................................................................................................ vi Abstract ................................................................................................................ vii List of Abbreviations and Symbols Used ............................................................. viii Acknowledgements ............................................................................................. xiii CHAPTER 1 INTRODUCTION ............................................................................. 1 1.1. Cancer ...................................................................................................................... 1 1.2 Breast Cancer ........................................................................................................... 1 1.2.1 Triple-negative breast cancer .............................................................................. 3 1.3 Cell death .................................................................................................................. 4 1.3.1 Apoptosis ............................................................................................................. 4 1.3.2 Necrosis/Necroptosis ........................................................................................... 6 1.3.3 Caspase-independent cell death involving the mitochondria .............................. 8 1.4 ROS ........................................................................................................................... 9 1.4.1 The role of ROS in cell signaling ...................................................................... 10 1.4.2 ROS and cancer ................................................................................................. 12 1.5 Phytochemicals ...................................................................................................... 14 1.6 Phytochemicals and Cancer .................................................................................. 17 1.7 Myricetin and cancer ............................................................................................ 20 1.8 Rationale and Objectives ...................................................................................... 22 CHAPTER 2 MATERIALS AND METHODS ........................................................ 33 2.1. Cell lines and primary cells ................................................................................. 33 2.2. Reagents ................................................................................................................. 33 2.3. Antibodies .............................................................................................................. 34 2.4. Culture medium and conditions .......................................................................... 35 2.5. Cell seeding ............................................................................................................ 35 2.5.1. Human breast carcinoma cells ......................................................................... 36 2.5.2. Mouse mammary carcinoma cells .................................................................... 36 2.5.3. Human dermal fibroblasts ................................................................................ 36 ii 2.5.4. Human mammary epithelial cells (HMEpiC) .................................................. 37 2.6. Acid Phosphatase Assay ....................................................................................... 37 2.7. Amplex® Red Assay ............................................................................................. 38 2.8. Flow Cytometry .................................................................................................... 38 2.8.1. Annexin-V-FLUOS/PI staining ....................................................................... 39 2.8.2. DiOC6 staining ................................................................................................. 40 2.8.3. DCFDA assay ................................................................................................... 41 2.8.4. Intracellular antibody staining for γ-H2AX ..................................................... 41 2.9. Protein isolation .................................................................................................... 42 2.10. Protein quantification ........................................................................................ 43 2.11. Western blotting ................................................................................................. 43 2.12. Statistical analysis ............................................................................................... 45 CHAPTER 3 RESULTS ...................................................................................... 46 3.1. Myricetin reduces mammary carcinoma cell phosphatase activity ................. 46 3.2. Myricetin is cytotoxic to mammary carcinoma cells ......................................... 47 3.3. Myricetin causes a loss of mammary carcinoma cell mitochondrial membrane integrity. ....................................................................................................................... 48 3.4. Myricetin induces accumulation of intracellular reactive oxygen species (ROS), which promotes mammary carcinoma cell death. ....................................... 50 3.5. Myricetin causes ROS-dependent DNA damage in mammary carcinoma cells. ....................................................................................................................................... 51 3.6. Myricetin forms extracellular ROS in cell culture medium. ............................ 52 3.7. Myricetin causes p38 MAPK activation in mammary carcinoma cells. .......... 53 CHAPTER 4 DISCUSSION ............................................................................... 103 4.1. Myricetin is cytotoxic for breast cancer cells ................................................... 103 4.2 Myricetin-induced mammary carcinoma cell death is ROS-dependent. ....... 107 4.3 Myricetin forms ROS in the extracellular environment. ................................. 108 4.4 Summary of the effects of myricetin on mammary carcinoma cells. .............. 111 4.5. Limitations of this study .................................................................................... 112 4.6 Future Directions ................................................................................................. 113 4.7 Concluding remarks ............................................................................................ 115 APPENDIX ......................................................................................................... 120 REFERENCES .................................................................................................. 128 iii LIST OF FIGURES Figure 1.1 The apoptotic and necroptotic cell death pathways ........................... 24 Figure 1.2 The effects of ROS on cancer ............................................................ 26 Figure 1.3 Classification of phytochemicals ........................................................ 28 Figure 1.4 The chemical structure of myricetin .................................................... 30 Figure 3.1 Myricetin reduces mammary carcinoma cell phosphatase activity ..... 55 Figure 3.2 Myricetin reduces human mammary epithelial cell, but not human dermal fibroblast cell phosphatase activity .......................................................... 57 Figure 3.3 The effect of myricetin on mammary carcinoma cell phosphatase activity is similar to the effects on phosphatase activity seen with EGCG, and greater than the effect of resveratrol ................................................................... 59 Figure 3.4 The dose-dependent inhibitory effect of myricetin on mammary carcinoma cell phosphatase activity is similar to the effects of conventional chemotherapeutic drugs ...................................................................................... 61 Figure 3.5 Myricetin reduces mammary carcinoma cell number and size ........... 63 Figure 3.6 Myricetin induces human mammary carcinoma cell death ................. 65 Figure 3.7 Myricetin induces mouse mammary carcinoma cell death ................. 68 Figure 3.8 The stability of the mitochondrial membrane in human mammary carcinoma cells is decreased by myricetin .......................................................... 70 Figure 3.9 The stability of the mitochondrial membrane in mouse mammary carcinoma cells is decreased by myricetin .......................................................... 73 Figure 3.10 Myricetin-induced mammary carcinoma cell death is caspase- independent and RIPK1-independent ................................................................. 75 Figure 3.11 Intracellular ROS levels are increased in mammary carcinoma cells treated with myricetin ........................................................................................... 77 Figure 3.12 NAC prevents myricetin-induced human mammary carcinoma cell death