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(AAD-1566) As a Potential Anticancer Agent in Ovarian Cancer

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(AAD-1566) As a Potential Anticancer Agent in Ovarian Cancer Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Farnaz Bahrami-Budlalu A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) Cancer Research Laboratories, Department of Surgery, St George Clinical School, University of New South Wales 2015 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Acknowledgments I am extremely grateful to my principal supervisor, Professor David L. Morris, for directing this project and supporting me throughout the PhD. I am very privileged to have been able to work under the guidance of someone so inspirational. I would also very much like to thank my research mentor and co- supervisor, Dr Mohammad H. Pourgholami, for his invaluable guidance and support during the last four years. His dedication to students covers not only scientific advice and methods, but also all the other elements required for being a qualified scientist. More than a mentor for research, he is also a great teacher who aided my personal growth during my PhD. I am indebted to him for the scientific training I have received. This thesis would not have been possible without the help from many individuals. Firstly, I would like to thank Dr Parvin Ataie-Kachoie and Dr. Ahmed H. Mekkawy for being the best colleagues and friends I could have ever wished. I would also like to thank Dr. Javed Akhtar, Mrs. Samina Badar, Dr. Zohra Ahmadi and Ms. Stephanie Chu for the enormous technical support they have offered me. I apologize to many of my colleagues and friends whose names cannot be all listed here. I want you to know that I would never have made it this far without your help and encouragement. Finally, I express my deepest gratitude to my father’s soul, my mother, my lovely sister, and my dear brother, for their support, encouragement, and unconditional love on which I have always relied. Moreover, I would like to thank my husband, Dr. Vahid Toloui and my daughter, Sara who have provided unwavering support and motivation to complete this thesis. Page 1 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer Table of Contents Acknowledgments ............................................................................................ 1 List of Figures ................................................................................................... 6 List of Tables ..................................................................................................... 9 List of Abbreviations ...................................................................................... 10 List of Publications ......................................................................................... 14 Journal articles in preparation: ...................................................................... 15 Conference poster presentations .................................................................. 15 Chapter 1. Literature Review ....................................................................... 17 1.1 Ovarian cancer ................................................................................... 17 1.1.1 Ovarian cancer cause and origin ................................................. 18 1.2 mTOR ................................................................................................. 19 1.2.1 The mTOR proteins ..................................................................... 19 1.2.2 Regulation of mTORC1 Activity .................................................. 22 1.2.3 Downstream pathway of mTORC1 ..................................................... 26 1.2.4 Cellular function of mTORC1 ......................................................... 28 1.2.5 Regulator of mTORC2 Activity ...................................................... 30 1.2.6 Downstream pathway of mTORC2 ................................................ 30 1.2.7 Cellular function of mTORC2 ......................................................... 32 1.2.8 mTOR, diseases and the challenges associated with targeting mTOR ………………………………………………………………………….35 1.2.8.1 mTOR signalling and cancer ...................................................... 35 1.2.8.2 Other mTOR-related disorders ................................................... 39 1.2.9 mTOR and autophagy ................................................................... 39 1.2.10 mTOR and apoptosis ................................................................... 40 1.2.11 mTOR inhibitors........................................................................... 41 1.2.11.1 First generation mTOR inhibitors: Rapamycin and its analogues……………………………………………………………………..41 1.2.11.2 Second generation mTOR inhibitors: Catalytic site (ATP- competitive) inhibitors ............................................................................. 44 1.3 Autophagy .......................................................................................... 48 1.3.1 Molecular machinery of autophagy .............................................. 49 1.3.2 Regulation of autophagy .............................................................. 53 1.3.3 Role of autophagy in physiology .................................................. 54 1.3.3.1 Autophagy-mediated protein quality control ............................. 54 1.3.3.2 Autophagy-mediated organelle quality control ......................... 55 1.3.3.3 Autophagy in cellular remodelling and development ................ 55 Page 2 Evaluation of monepantel (AAD-1566) as a potential anticancer agent in ovarian cancer 1.3.3.4 Autophagy in metabolic homeostasis ....................................... 56 1.3.3.5 Autophagy confronts stress and environmental insults ............ 57 1.3.3.6 The importance of controlling p62/SQSTM1 levels by autophagy58 1.3.4 Autophagy and tumorigenesis ..................................................... 60 1.3.4.1 Autophagy suppresses tumor initiation by limiting genome mutation ……………………………………………………………………….60 1.3.4.2 Autophagy suppresses tumor initiation and progression by limiting chronic inflammation .................................................................. 61 1.3.4.3 Tumor cells with oncogenic mutations may be more dependent on autophagy for survival ....................................................................... 61 1.3.4.4 Autophagy inhibition sensitizes tumor cells to cell death ......... 62 1.4 Monepantel ......................................................................................... 63 1.4.1 History ......................................................................................... 63 1.4.2 Chemical structure ....................................................................... 64 1.4.3 Physicochemical properties ......................................................... 65 1.4.4 Mode of action ............................................................................. 65 1.4.5 Pharmacokinetics of monepantel................................................. 66 1.4.6 Toxicology ................................................................................... 67 1.5 Aim of the study and hypothesis ......................................................... 68 Chapter 2: Material and Methods ................................................................ 70 2.1 Material ............................................................................................... 70 2.2 Cell lines ............................................................................................. 71 2.3 General methods ................................................................................ 72 2.3.1 Dissociation of cells from the culture flask ................................... 72 2.3.2 Cell counting ................................................................................ 72 2.3.3 Freezing adherent cells ............................................................... 73 2.3.4 Thawing of cryopreserved cells ................................................... 73 2.3.5 BCA protein assay ....................................................................... 73 2.4 In-vitro Methods .................................................................................. 74 2.4.1 Drug preparation .......................................................................... 74 2.4.2 Morphology .................................................................................. 75 2.4.3 Cell viability ................................................................................. 75 2.4.4 Cell proliferation assay (Sulforhodamine B, SRB) ....................... 76 2.4.5 Cell proliferation assay (MTT) ..................................................... 76 2.4.6 Colonogenic assay (colony formation) ......................................... 77 2.4.7 Cell cycle analysis ....................................................................... 77 2.4.8 Western blot analysis .................................................................. 78 3 2.4.9 H-thymidine incorporation assay ................................................ 79 2.4.10 Caspases activity assay .............................................................. 80 2.4.11 Annexin V / 7-AAD staining ......................................................... 81 2.4.12 Analysis of inter-nucleosomal DNA fragmentation (DNA Ladder) 81 2.4.13 Quantification of acidic vesicular organelles (AVO) by acridine orange (AO) staining .............................................................................. 82 2.4.14 Immuno-fluorescence,
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