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OpenAIR@RGU The Open Access Institutional Repository at Robert Gordon University http://openair.rgu.ac.uk Citation Details Citation for the version of the work held in ‘OpenAIR@RGU’: WARASIHA, B., 2008. Cytochrome P450 mRNA profile in human breast cancer cell lines. Available from OpenAIR@RGU. [online]. Available from: http://openair.rgu.ac.uk Copyright Items in ‘OpenAIR@RGU’, Robert Gordon University Open Access Institutional Repository, are protected by copyright and intellectual property law. If you believe that any material held in ‘OpenAIR@RGU’ infringes copyright, please contact [email protected] with details. The item will be removed from the repository while the claim is investigated. CYTOCHROME P450 mRNA PROFILE IN HUMAN BREAST CANCER CELL LINES BENJAMART WARASIHA A thesis submitted in partial fulfilment of the requirements of The Robert Gordon University for the degree of Doctor of Philosophy School of Pharmacy and Life Sciences, The Robert Gordon University, Aberdeen. United Kingdom. May 2008 Abstract Abstract Cytochrome P450 enzymes (P450s) are involved in cancer development and treatment due to their roles in the oxidative metabolism of various endogenous (e.g. oestrogen) and exogenous (e.g. tamoxifen) compounds. It is well-known that intermediate P450 metabolites derived from oestrogen metabolism are associated with breast carcinogenesis. The main aim of this project was to profile the cytochrome P450 and P450-regulatory nuclear receptor mRNAs in a series of breast cancer cell lines (BCCs) and compare this profile with normal breast cells. This study used the qualitative reverse transcriptase- polymerase chain reaction (RT-PCR) to detect mRNA expression of target genes. Results showed CYP1B1, CYP2D6, CYP2J2, CYP2R1, CYP2U1 and CYP4X1 mRNA to be present in all cell lines. CYP2A6, CYP2C8, CYP2C18, CYP2F1 and CYP4Z1 mRNA were expressed in oestrogen receptor (ER)-positive Caucasian and ER-negative Afro- Caribbean BCCs. Although no differences in P450 mRNA were observed between the different ethnic groups, these preliminary findings suggest potential similarities in the ER- positive Caucasian and ER-negative Afro-Caribbean BCCs which warrant further investigation The CYP4Z1 PCR product was identified as two distinct bands. Specific primer sets were used to demonstrate potential intron retention in CYP4Z1. Using established in vitro models for the study of regulatory mechanisms of CYP4Z1, T47D and ZR-75-1 breast cancer cell lines were used to determine the appropriate nuclear receptors (i.e. progesterone receptor, glucocorticoid receptor or peroxisome proliferator-activated receptor alpha ). These findings suggest that there may be an alternative receptor mechanism involved in CYP4Z1 mRNA induction in these cells. In conjunction, pre-treatment of these two cell lines with the RNA synthesis inhibitor actinomycin D followed by the agonists showed a significant reduction ( p < 0.05) of CYP4Z1 mRNA levels and inhibited CYP4Z1 induction by either progesterone, dexamethasone or pirinixic acid, indicating that these agonists have effects on CYP4Z1 mRNA transcription or stability. In contrast, cycloheximide differentially affected the level of CYP4Z1 mRNA induction by these agonists. Taken together, these results suggest that CYP4Z1 mRNA induction in T47D and ZR-75-1 is mediated through differential cell type specific regulatory mechanisms and there is evidence for differential regulation of the splice variants. ii Abstract Keywords: Cytochrome P450 mRNA, cytochrome P450-regulatory nuclear receptor mRNA, Oestrogen receptor (ER)-positive and negative breast cancer cell lines (BCCs), Caucasian and Afro-Caribbean BCCs, CYP4Z1 alternative splicing, regulation of CYP4Z1 iii Acknowledgements Acknowledgements I wish to express my sincere gratitude to the following people for their help, support, guidance and encouragement during the course of my studies. My Director of Studies, Dr Morag McFadyen, for introducing me to the world of cytochrome P450s, her valuable suggestions, patience and encouragement throughout my PhD studies. Dr Rachel Knott, for her constant guidance, support and participating as my supervisor. Professor Angela Drake-Holland, for her helpful suggestions and discussion on the project. Mrs Dorothy Moir, for helping me out with practical support in the molecular biology laboratory, her wonderful friendship and motivation. Many thanks to Professor Mark Noble, Dr Graeme Kay and Dr Kennedy Nelson for taking the time to critically evaluate my thesis. Professor Terry Healey, Head of the School of Pharmacy and Life Sciences, for giving me the opportunity to pursue my studies. Dr Susanne Boyle, for her support and advice in the first year of my studies. Mr Brian De Jonckheere, for information technology assistance. All of the technicians in the School of Pharmacy who have helped with my project and been wonderful friends. Mrs Elizabeth Tomchak, my English tutor, for helping me improve my English skills. Mr Martin Simpson, the Research Degrees officer, for his support and assistance. Associate Professor Veerapol Kukongwiriyapan from the Department of Pharmacology, Faculty of Medicine, Khon Kaen University, Thailand, for his support and guidance. Sarah Garry, Michelle Frazier, Eimear Molloy and Ian Lonergan, Honours students at the School of Pharmacy and Life Sciences, for the results of the preliminary work in Chapter 4. Dr Colin Thompson, Mr Ray Mulrooney, Dr Bridgeen McCaughan, Mrs Julie Young, Miss Anita Weidman, Miss Nicola Silva, Mr Vibhu Paudyal and all other colleagues for sharing their time and friendship both in and out of the laboratory. To Anusorn Thiangsungnoen, Pattarin Kittiboonyakun, Sansanee Wangvoralak, Sirichai Mongkolkiatsri and all of the other Thai students in Aberdeen for their wonderful friendship and encouragement and for making me feel less homesick. To everyone in the Taekwondo and Badminton clubs at The Robert Gordon University, for their friendship. Special thanks to my Taekwondo instructor Miss Lyndzie Jefferey, whose classes always helped me to unwind and relax, no matter how stressful my day in the lab had been. iv Acknowledgements To my wonderful parents, Samruay and Sudjai Warasiha who have taught me never to give up, for their constant care, unconditional love, continuous support and encouragement throughout my life. My two brothers, Taweesit and Supakit Warasiha, and two sisters Somjit Nampha and Jurairat Sriwicha including their families for their understanding, encouragement, support and love. My boyfriend, Tim Cushnie, for his help and encouragement and for sharing my frustration and happy moments during the studies-you have helped keep me smiling. The Royal Thai Government Scholarship, for financial support throughout my studies. The School of Pharmacy and Life Sciences at The Robert Gordon University, for financial aid during the writing up period. The British Association for Cancer Research (BACR), for the travel grants to attend and present my research in the 2006 and 2007 National Cancer Research Institute (NCRI) Conference in Birmingham. v List of Publications List of Publications Abstract: 1. Warasiha B , Knott R, Boyle S. Investigation of the role of xenobiotic metabolising enzymes in breast cancer development. The Journal of Nutrition. 2004 December; 134 (12 Suppl): 3525S. (Poster presented as part of the International Research Conference on Food, Nutrition and Cancer held in Washington, DC, USA, July, 2004). Conference proceedings: 1. Warasiha B, Knott R, McFadyen M. (2007) Comparative analysis of Cytochrome P450 mRNA Expression between Human Caucasian and Afro-Caribbean Breast Cancer Cell Lines. In the proceedings of National Cancer Research Institute (NCRI) Cancer Conference October 2007 at the ICC in Birmingham, UK. (Oral and poster presentation) 2. Warasiha B, Knott R, McFadyen M. (2007) Differential Cytochrome P450 mRNA Profile between Non-invasive and Invasive Breast Cancer Phenotypes. In the proceedings of National Cancer Research Institute (NCRI) Cancer Conference October 2007 at the ICC in Birmingham, UK. (Poster presentation) 3. Kay G, Cairns D, McCaughan B, Warasiha B. (2007) The Design, Synthesis and Biological Evaluation of Novel Prodrugs for the Treatment of Cystinosis. In the proceedings of British Conference Pharmaceutical (BCP) September 2007 at the Manchester central in Manchester, UK. (Poster presentation) 4. Kay G, Warasiha B, Melville M, Mincher DJ. (2007) Design, Synthesis and Biological Evaluation of Novel DNA Binding Agents. In the proceedings of British Conference Pharmaceutical (BCP) September 2007 at the Manchester central in Manchester, UK. (Poster presentation) 5. Warasiha B, Knott R, McFadyen M. (2006) Profile of cytochrome P450 mRNA expression in oestrogen positive and negative breast cancer cell lines. In the Proceedings of National Cancer Research Institute (NCRI) Cancer Conference October 2006 at the ICC in Birmingham, UK. (Poster presentation) vi Table of Contents Table of Contents Title page-------------------------------------------------------------------------------------- i Abstract --------------------------------------------------------------------------------------- ii Acknowledgements -------------------------------------------------------------------------- iv List of Publications -------------------------------------------------------------------------- vi Table of Contents ----------------------------------------------------------------------------- vii List of Abbreviations ------------------------------------------------------------------------