1 THE ROLES OF PROSTAGLANDIN E2, PROSTAGLANDIN F2α AND ALDO-KETO REDUCTASE 1C ISOENZYMES IN ENDOMETRIOSIS AND BREAST CANCER February 2017 A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Biology, Medicine and Health Osman Zarroug School of Health Sciences 2 Contents List of figures and tables .......................................................................................................... 6 Abstract .................................................................................................................................... 9 Declaration ............................................................................................................................. 11 Copyright statement .............................................................................................................. 12 Published work from thesis ................................................................................................... 13 Acknowledgements ................................................................................................................ 14 List of abbreviations ............................................................................................................... 15 1. Introduction ................................................................................................................... 19 1.1. The human uterus .................................................................................................. 20 1.1.1. The menstrual cycle ....................................................................................... 21 1.2. An overview of endometriosis ............................................................................... 23 1.2.1. Background .................................................................................................... 23 1.2.2. Symptoms of endometriosis .......................................................................... 24 1.2.3. Diagnosis of endometriosis ............................................................................ 24 1.2.4. Treatment of endometriosis .......................................................................... 25 1.2.5. The aetiology of endometriosis ..................................................................... 26 1.2.6. Pathophysiology of endometriosis ................................................................ 28 1.3. The aldo-keto reductase (AKR) Superfamily .......................................................... 32 1.3.1. The role of AKR1C isoenzymes in humans ..................................................... 34 1.3.2. The role of AKR1C isoenzymes and prostaglandin biosynthesis .................... 37 1.3.3. AKR1C isoenzymes contribute to aberrations of oestrogen biosynthesis ..... 42 1.3.4. AKR1C isoenzymes contribute to the disruption of progesterone metabolism and functions ................................................................................................................. 45 1.3.5. The role of sex steroid hormones, prostaglandins and AKR1C isoenzymes in breast cancer .................................................................................................................. 47 1.4. The role of adipose tissue in hormone dependent diseases: endometriosis and breast cancer ...................................................................................................................... 50 1.5. Aim ......................................................................................................................... 55 2. General Methodology .................................................................................................... 57 2.1. Human endometrial cell culture ............................................................................ 58 2.1.1. Materials ........................................................................................................ 58 2.1.2. Methods ......................................................................................................... 60 2.2. Explant Culture ....................................................................................................... 68 3 2.2.1. Materials ........................................................................................................ 68 2.2.2. Method .......................................................................................................... 69 2.3. Enzyme-linked Immunosorbent Assay (ELISA) ....................................................... 70 2.3.1. Materials ........................................................................................................ 70 2.3.2. Methods ......................................................................................................... 70 2.4. Real time quantitative polymerase chain reaction (qRT PCR) ............................... 76 2.4.1. RNA isolation .................................................................................................. 76 2.4.2. Purification of RNA ......................................................................................... 77 2.4.3. Quantification of RNA .................................................................................... 78 2.4.4. Checking RNA integrity .................................................................................. 79 2.4.5. cDNA synthesis using QuaniTect Reverse Transcription kit ........................... 80 2.4.6. Primer design ................................................................................................. 81 2.4.7. Quantitative Real-Time PCR ........................................................................... 85 2.4.8. Determining the efficiency of different primers ............................................ 86 2.4.9. Checking product specificity .......................................................................... 90 2.4.10. Data analysis of relative mRNA expression .................................................... 93 2.5. The Aldo-keto reductase (AKR) 1C3 kinetics .......................................................... 95 2.5.1. Materials ........................................................................................................ 95 2.5.2. Method: ......................................................................................................... 96 2.6. Extracellular Flux Analysis ...................................................................................... 98 2.7. Statistical analysis of data .................................................................................... 100 3. The gene expression of Aldo-keto reductases (1C1-1C3), prostaglandin E receptors (1- 4) and prostaglandin F receptor in the human endometrium, endometrial lesion, omental fat and breast fat ................................................................................................................. 101 3.1. Introduction ......................................................................................................... 102 3.1.1. Background .................................................................................................. 102 3.1.2. Patient data .................................................................................................. 104 3.2. Results .................................................................................................................. 106 3.2.1. The gene expression of AKRs in the human endometrium ......................... 106 3.2.2. The gene expression of AKRs in the omental adipose tissue ....................... 109 3.2.3. The gene expression of AKRs in the breast adipose tissue from breast cancer patients 111 3.2.4. The gene expression of EP1-4 and FP receptors in the human endometrium 116 4 3.2.5. The gene expression of EP1-4 and FP receptors in the human omental adipose tissue .............................................................................................................. 120 3.2.6. The gene expression of EP1-4 and FP receptors in the human breast adipose tissue from breast cancer patients .............................................................................. 123 3.3. Discussion ............................................................................................................. 129 3.3.1. The role of AKR1C (1-3) in hormone dependent diseases ........................... 129 3.3.2. The role of EP1-4 and FP receptors in hormone dependent diseases ........... 135 4. Bimatoprost as a potential aldo-keto reductase 1C3 inhibitor ................................... 139 4.1. Introduction ......................................................................................................... 140 4.1.1. Background .................................................................................................. 140 4.1.2. Bimatoprost mechanism of action ............................................................... 140 4.1.3. Prostamides or prostaglandin ethanolamides ............................................. 141 4.1.4. Bimatoprost: a prostamide F2α or prostaglandin F2α analogue? ................. 144 4.1.5. Bimatoprost: An AKR1C3 inhibitor ............................................................... 145 4.2. Results .................................................................................................................. 146 4.2.1. AKR1C3 kinetic assay .................................................................................... 146 4.2.2. Patient data .................................................................................................