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Effects of Estrogen on Voltage-Gated Sodium Channel Expression and Function in Human Breast Cancer Cell Lines

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Effects of Estrogen on Voltage-Gated Sodium Channel Expression and Function in Human Breast Cancer Cell Lines EFFECTS OF ESTROGEN ON VOLTAGE-GATED SODIUM CHANNEL EXPRESSION AND FUNCTION IN HUMAN BREAST CANCER CELL LINES ILEY OZERLAT Thesis submitted to the University of London for the Degree of Doctor of Philosophy January, 2009 Imperial College London Division of Cell and Molecular Biology ABSTRACT This PhD Thesis aimed to elucidate a possible functional interaction between estrogen receptor signaling and voltage-gated sodium channel (VGSC) expression/activity in human breast cancer (BCa) cell lines. In particular, a novel estrogen receptor mechanism (G-protein coupled receptor 30, GPR30) was evaluated in metastatic human BCa cells, and short-term (non-genomic) and long-term (genomic) mechanisms regulating the expression of 'neonatal' Nav1.5 (nNav1.5), the predominant isoform expressed in these cells, were investigated. The Introduction chapter gives general information about cancer, estrogen and its role in BCa, VGSCs and regulation of ion channels by steroid hormones. This is followed by the Materials and Methods chapter where detailed information is given on the methodologies, cell lines and pharmacological treatments, used. The Thesis includes 3 main Results chapters, presenting data as follows: (1) Short-term effects of 170-estradiol on MDA-MB-231 cell adhesion: Evaluating the role of GPR30: Acute (up to 10 minutes) and short-term (10-20 minutes) treatments of strongly metastatic human BCa cell line (MDA-MB-231) with 1713-estradiol (E2) reduced cellular adhesion and co-application of (i) VGSC blocker tetrodotoxin (TTX) or (ii) a protein kinase A (PICA) inhibitor inhibited this effect. A range of experiments suggested that GPR30 was involved in the non-genomic effects of E2 on adhesion. (2) Long-term effects of E2 on MDA-MB-231 cells: VGSC expression and adhesion: Long-term (24 h) treatments of MDA-MB-231 cells with E2 increased nNav1.5 mRNA expression without affecting total protein expression. A VGSC-dependent reduction in adhesion was shown by use of TTX, and over-expression of GPR30 increased the effect of E2 on adhesion. (3) Long-term effects of E2 on VGSC expression and adhesion of ERa-expressing MDA-MB-231 cells: ERa transfection in MDA-MB-231 cells (giving MDA-ERa cells) reduced nNav1.5 mRNA expression. Long-term (24 h) treatment of MDA-MB-231 cells with E2 reduced nNav1.5 mRNA and protein expression, and decreased VGSC- dependent adhesion. Long-term (> 24 h) treatment of MDA-ERa cells with estrogen antagonist ICI-182,780 did not affect cell adhesion, but increased lateral motility and transverse migration. 2 The overall conclusion is that functional expression of nNav1.5 in MDA-MB-23 1 cells is controlled by estrogen and this includes a non-genomic receptor mechanism. The Thesis concludes with a General Discussion chapter in which the basic findings are discussed more broadly and as regards potential clinical significance. 3 ACKNOWLEDGEMENTS First of all, I would like to express my gratitude to Professor Mustafa Djamgoz who has given me the chance to pursue this degree of Doctor of Philosophy in his laboratory and who has been my supervisor for the past four years. He has been extremely helpful during the construction of my project as well as throughout the whole process. His wisdom and grand experience has guided me till the end. Especially for his patience to read the whole Thesis several times and to discuss it in depth, I am extremely thankful! I can not express how thankful I am to Dr. Scott Fraser for being such a wonderful person he is and for helping me with any problem I had during my time in the lab. He has been a great supervisor and a friend throughout these four years. I have benefited immensely from his technical and practical experiences in the laboratory, and I would like to thank him for his time and patience in helping me and answering my (sometimes endless) questions. I have enjoyed working with him a lot. Another wonderful person I can not thank enough is Dr. James Diss. I really appreciate his time and patience. He spent one summer to teach me everything about RNA extraction and real-time PCR and he spent much more time answering all my questions about primers and molecular techniques very patiently! He has been a great teacher and a great friend. I would like to also express my appreciation to Dr. Dongmin Shao for all his time and help with plasmid extractions, transfections and siRNA experiments, as well as with my Thesis. Also many thanks to Dr. Martin Spitaler and Dr. Peter Clark for their help with confocal microscopy. I have to say that I find myself extremely lucky for having many more great people to help me and befriend me throughout the four years I spent in the laboratory. For their support, guidance and friendship I would like to thank to Dr. Chris Palmer, Dr. Ebru Aydar, Dr. Emma Williams, Dr. Myrto Chioni and Dr. Pinar Uysal-Onganer. I would like to thank to my advisors Dr. Kenji Okuse and Prof. Simak Ali for their help and time during the first years of the project. Also many thanks to Prof. Eric Prossnitz for providing the GPR30 antibody, to Prof. Ronald Weigel, Prof. Edward Filardo and Prof. Martin Allday, for providing the plasmids and to Prof. Craig Jordan for providing the MDA-ERa, VC5 and MDA-10A cell lines. I would like to also thank to my lovely friends here in UK: Adil Seytanoglu for his friendship and also helping me with EndNote, Fatma Mokhtar, Mine Guzel, Nahit 4 Rizaner, Rustem Onkal, Seden Cigizoglu Grippon and Sophie Pageon for their friendship and constant support. Also many thanks to my friends Dr. Banu Isbilen and Dr. Umut Fahrioglu, and my family in Cyprus: my aunt Hatice, uncle Sener, cousins Nazan and Hasan Yorgozlu, my grandmother Ismet Ozerlat, my mother- father- and sister-in-law Mebruke, Sonmez and Meryem Gunduz, my dear English teacher Gulten Fettahoglu and dear Hulya Bingollu for their love and support. There are many people without whom this PhD would not have been possible. My uncle, Prof. Ihsan Gursel has always been an inspiration for me since when I first chose to study biology and later when I decided to do a PhD. I can not express how thankful I am to him for his help during my initial communication with Prof. Djamgoz and his support throughout this journey. I would like to also thank to my aunt Dr. Mayda Gursel and my cousin Devrim Gursel for their love and support. I would like to thank to my uncle, Zeki Gursel, for his constant support and love. His wisdom and love of education has always inspired me and I have always admired and been proud of his level of knowledge and intelligence. This has given me a way of guidance and self-confidence since my childhood. My dear aunt Neslihan and my cousins Arda and Deha Gursel: Thank you very much for your love and support! I would like to express my love and thanks to my beloved grandmother Faize Gursel. Thank you for your deepest love and prayers that helped me throughout my PhD! My parents, Imren and Dervis Ozerlat, and my beloved sisters, Idil and Ilke: Without you I could not have done any of this! Mom and dad, thank you very much for your love, and financial and psychological support during this rollercoaster-like journey. Idil and Ilke, your love and support kept me going, thank you! One important person I saved for the last but definitely not the least is my beloved, other-half, Asim Gunduz. He has been the best throughout these four years. He was the one whom I shared my laughter with when I got a nice result, the shoulder I cried on when the experiments did not work, whom I discussed my project with and got constructive criticism, who cooked for me and looked after me when I was in the lab till late or at home working on my Thesis. He is the one without whom none of this would have been possible. For all of this and his love, I am very grateful! Finally, I would like to thank to Pro-Cancer Research Fund (PCRF) for supporting this project; and Kanser Arastirma Vakfi (KAV) and North Cyprus Ministry of Education for the financial aid they have provided. 5 Dedicated to my mom (imren), dad (Dervi), sisters (idil & ilke) and fiancée (Asian). 6 TABLE OF CONTENTS ABSTRACT 2 ACKNOWLEDGEMENTS 4 TABLE OF CONTENTS 7 LIST OF FIGURES AND TABLES 12 ABBREVIATIONS 18 Chapter 1 GENERAL INTRODUCTION 26 1.1 Cancer basics 27 1.1.1 MicroRNAs 28 1.2 Metastasis 29 1.2.1 Epithelial-to-mesenchymal transition and invasion 31 1.2.2 Angiogenesis and tumour cell dissemination 34 1.2.3 Adhesion and extravasation 35 1.3 Breast cancer (BCa) 36 1.4 Estrogen 38 1.4.1 Genomic pathway 38 1.4.2 Non-genomic pathway 41 1.4.2.1 Classical ERs 41 1.4.2.2 Non-classical ERs and other receptors 42 1.4.3 Role of estrogen in BCa 45 1.5 Voltage-gated sodium channels (VGSCs) 47 1.5.1 Regulation of VGSCs 49 1.5.1.1 Transcriptional regulation 49 1.5.1.2 Post-transcriptional regulation 50 1.5.1.3 Post-translational regulation 51 1.6 Steroid hormone regulation of ion channels 53 1.6.1 Regulation of VGSCs 53 1.7 Ion channels and disease 54 1.7.1 Role of VGSCs in metastatic cell behaviours 55 1.8 Aims and scope of the Thesis 57 Chapter 2 MATERIALS AND METHODS 58 2.1 Cell culture 59 2.1.1 Cell lines 59 2.1.2 Culture conditions 59 2.1.3 Passaging and trypsinizing the cells 60 2.1.4 Freezing and thawing cells 60 2.2 Pharmacological agents and treatments 61 2.2.1 1713-estradiol 61 7 2.2.2 BSA-conjugated E2 61 2.2.3 G1 compound 63 2.2.4 ICI-182,780 63 2.2.5 Tetrodotoxin treatment 63 2.2.6 PKA inhibitor treatment 63 2.3 In vitro functional assays 64 2.3.1 Single-cell adhesion measurement apparatus (SCAMA) 64 2.3.2 Cell motility 64 2.3.3 Cell migration 66 2.3.4 MTT assay 66 2.3.5 Data analysis 68 2.4 Molecular biology 68 2.4.1 Total RNA extraction and purification 68 2.4.2 Determination of RNA quality 69 2.4.3 Quantification of total RNA 69 2.4.4 Synthesis of single-stranded complementary DNA (cDNA) 69 2.4.5 Polymerase chain reaction (PCR) 71 2.4.5.1 Analyzing the quality of PCR products 75 2.4.5.2 Data analysis 75 2.5.
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