A Pre-Clinical Investigation of the Anti-Cancer Effects of the Frankincense Constituent AKBA on Ovarian Cancer Cells Thesis submitted for the degree of Doctor of Philosophy at the University of Leicester by Kamla Khalfan Said Al-Salmani BSc (Hons), MSc Department of Cancer Studies University of Leicester 2016 Abstract A pre-clinical investigation of the anti-cancer effects of the frankincense constituent AKBA on ovarian cancer cells This study examines the biologically active component of frankincense, 3-O-acetyl-11- keto-β-boswellic acid (AKBA), in ovarian cancer cells to evaluate its potential cytotoxicity towards high grade serous ovarian cancer and its potential ability to address the cancer’s known resistance to cisplatin. Ovarian cancer causes significant mortality, the five-year survival rate being very low compared to other cancers as most of the cases are diagnosed late, typically at stages IIIa- IIIc and IV. Most of these cases relapse and develop resistance to first line chemotherapy; therefore, new strategies are urgently needed to overcome resistance. Extracts from Boswellia sp., used for centuries as herbal medicine in Asia, have known anti- inflammatory properties and anti-cancer potential alone or in combination with other chemotherapies. The active ingredients of Boswellia sp., boswellic acids, have many effects on various cancer cells including induction of apoptosis. In this study, several approaches and analyses were performed in examining the effect of AKBA on four ovarian cancer cell lines, including a cisplatin resistant line. These investigations included analysis of cell proliferation and viability, cell cycle distribution, apoptosis, DNA damage formation, production of reactive oxygen species and gene expression. The results obtained from this work suggest that AKBA induces apoptosis in ovarian cancer cell lines either directly through extrinsic and intrinsic pathways, or indirectly by affecting other cellular mechanisms such as inhibiting cell proliferation/viability, inducing DNA damage and decreasing production of reactive oxygen species. Furthermore, AKBA exposure alters the expression of multiple genes, potentially impacting on several cellular processes. The key findings of this project are that AKBA is cytotoxic to ovarian cancer cells, at pharmacologically achievable concentrations. AKBA exposure also induces multiple gene expression changes that would impact on many different biological pathways in ovarian cancer cells including genes related to DNA damage and repair, cell cycle, cell metabolism, and cell adhesion and metastasis. Consequently, AKBA may form the basis of a novel anticancer treatment for ovarian cancer potentially alongside conventional therapy. I Acknowledgements Firstly, I would like to express my thanks to the University of Leicester for accepting this project proposal for my PhD. I would like to express my gratitude to my supervisors, Prof George DD Jones, Dr Mark Evans and Dr Esther Moss for their guidance, advice, support, and encouragement during this project. I also thank Dr Raj Patel for his advice and support during this project and I thank Dr Salvador Macip for his help in using the flow cytometry machine. I would like to thank my co-supervisor Dr Ikram Burney from Sultan Qaboos University for all his support throughout the project time. My thanks also extends to our departmental postgraduate tutor Dr Don Jones for his support, encouragement and overseeing of this project and also to Dr Nicolas Sylvius of Nucleus Genomics, Genetics Department for running the microarray samples and for his assistance in analysing the results. I would like to thank all the people in other laboratories who gave me help and who allowed me to use their facilities. I am grateful to the Ministry of Health, Ministry of Higher Education and my colleagues and staff at the National genetic centre in Oman. Lastly, huge thanks to my family, words cannot express how grateful I am to my parents, my in-laws for their support and encouragement. Finally, I would like to acknowledge my deepest gratitude to my beloved husband and my children. II List of contents Abstract ....................................................................................................................... I Acknowledgements ..................................................................................................... II List of table’s and figures ........................................................................................ VII List of Equations ........................................................................................................ X List of Abbreviations: ............................................................................................... XI Chapter one ................................................................................................................... 15 Thesis Introduction ....................................................................................................... 15 1.1 Frankincense ....................................................................................................... 16 1.1.1 Boswellic acids .......................................................................................... 18 1.1.2 Medicinal uses of Frankincense ................................................................. 20 1.1.3 Acetyl-11-keto-beta-boswellic acid (AKBA): ........................................... 22 1.1.4 Cytotoxic properties of Boswellic acid and mainly AKBA: ..................... 23 1.1.5 Cell line studies and cytotoxicity ............................................................... 23 1.1.6 Metabolic activity of Boswellic acids and AKBA ..................................... 30 1.2 Ovarian Cancer ................................................................................................... 31 1.2.1 Ovarian cancer subtype .............................................................................. 33 1.2.2 Aetiology and Pathophysiology of Ovarian Cancer .................................. 33 1.2.3 Current treatment of ovarian cancer. ......................................................... 35 1.2.4 Ovarian cancer cell-lines for in vitro studies ............................................. 37 1.2.4.1 A2780 and A2780cis .............................................................................. 37 1.2.4.2 OVCAR-4 ............................................................................................... 38 1.2.4.3 UWB1.289 .............................................................................................. 39 1.3 DNA damage & repair ........................................................................................ 40 1.4 The Cell Cycle. ................................................................................................... 41 1.5 Aims and objectives ............................................................................................ 42 1.5.1 Hypothesis ................................................................................................. 43 III Chapter 2 ....................................................................................................................... 44 Materials and Methods ................................................................................................. 44 2. Materials and Methods: ........................................................................................ 45 2.1 Cell-lines for in vitro studies ........................................................................ 45 2.2 Cell lines used in this research ...................................................................... 45 2.4 Summary of the techniques/approach ........................................................... 46 2.5 Cell culture medium and supplements .......................................................... 46 2.6 Chemicals and Reagents ............................................................................... 46 2.7 Kits and antibodies ........................................................................................ 47 2.8 Preparation of buffers and working reagents ................................................ 48 2.8.1 The test compound AKBA ........................................................................ 49 2.9 Cell culture .................................................................................................... 49 2.10 Cell Culture: ................................................................................................ 50 2.11 Methods ...................................................................................................... 51 2.11.9 Gene expression ....................................................................................... 66 2.11.10 Quantitative-PCR (Q-PCR) ................................................................... 70 2.11.11 Statistical Analysis ................................................................................. 73 Chapter 3 ....................................................................................................................... 74 Investigations for the ability of AKBA to address cisplatin resistance in ovarian cancer cell lines .............................................................................................................. 74 3.1 Introduction ......................................................................................................... 75 3.1.1 Ovarian Cancer: ........................................................................................