CHEMICAL AND BIOLOGICAL CHARACTERIZATION OF TRACE AMOUNT OF SEX HORMONE RECEPTOR ACTIVE COMPOUNDS IN PROSTATE CANCER-RELATED BIOLOGICAL SAMPLES SOH SHU FANG, FLORA (M.Sc., National University of Singapore) A THESIS SUBMITTED FOR DEGREE OF DOCTOR OF PHILOSOPHY YONG LOO LIN SCHOOL OF MEDICINE DEPARTMENT OF OBSTETRICS & GYNAECOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2015 Declaration I hereby declare that the thesis is my original work and it has been written by me in its entirety I have duly acknowledged all the sources of information which have been used in the thesis This thesis has also not been submitted for any degree in any university previously ii Acknowledgements I would like to express my utmost appreciation to several groups of people whom have helped me in one way or another in the process of completion of this study Firstly, I am very thankful to my supervisor, Assistant Professor Gong Yinhan for his guidance and support during the course of the study He has been very nurturing and taught me a lot in work, as well as invaluable life lessons Without his resolute to make me endure till the last lap, this thesis would not have been possible And I would like to show my appreciation to Prof Yong Eu Leong and Dr Li Jun for making their laboratory amenities easily available to me throughout my research My deepest appreciation to Miss Lee Baohui, Mr Ryan Lim, Dr Sun Feng and Ms Vanessa Lim who provided me with insights to the biological studies and also showed me the ropes to doing bioassays And to Dr Terry Tong, Dr Inthrani, Miss Chua Seok Eng, Mr Zhang Zhiwei, Miss Tan Huey Min, Ms Wang Xiaochong, Mr Zhao Jia, Mr Shanker and the rest of the lab members who have helped and given me lots of moral support during the course of this project Most importantly, to my grandma, parents and siblings, for showing me great support and patience these years when I spent most of my time in the lab with my beloved LC-MS machine and my favourite BSC and fumehood and had very little time with them iii Table of Contents Page List of Tables iii xii-xiii xiv-xv List of Figures xvi-xx Acknowledgements Summary List of Abbreviations and Symbols xxi-xxiv List of Publications and Manuscripts from this Study xxv-xxvi List of Other Publications Published During Candidature xxvi-xxvii Chapter Introduction 1.1 Common Occurrence of Prostate Cancer (PCa) in Men 2-3 1.2 Prostate and its Importance 1.3 Signs & Symptoms and Detection of Prostate Cancers 1.4 Treatment Options for Prostate Cancer 1.4.1 Hormone or Androgen Deprivation Therapy (ADT) for Prostate Cancer 3-6 7 1.4.1.1 Orchiectomy or Surgical Castration 1.4.1.2 Luteinizing Hormone-releasing Hormone (LHRH) Agonists 1.4.1.3 Luteinizing Hormone-releasing Hormone (LHRH) Antagonists 1.4.1.4 Anti-androgens 1.4.1.5 Recently Developed Hormone Therapy 10 1.5 Occurrence of Castration Resistant Prostate Cancer (CRPC) 1.5.1 Androgens - Growth Factor for Prostate Cancer (PCa) iv 11 12-14 1.5.2 Possible Mechanisms behind Occurrence of Castration Resistant Prostate Cancer (CRPC) 1.5.2.1 Hypersensitive AR 14 15-16 1.5.2.2 Promiscuous AR 17-18 1.5.2.3 Outlaw AR 18-19 1.5.2.4 Bypass AR 20 1.5.2.5 Lurker Cells 21-22 1.6 AKR1C3 Enzyme Involvement in CRPC 23-25 1.7 Dimethoxycurcumin 26-29 1.8 Liquid Chromatography Tandem Mass Spectrometry (LCMS/MS) 29 1.8.1 Brief History behind Development of ChromatographicMass Spectrometric Techniques 30-31 1.8.2 Pros and Cons of Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) and Method of Choice for Detection for Steroids 31-33 1.9 Sensitive Cell-based Bioassay for Hormone Measurements 34-35 36 1.10 Hypotheses and Objectives 1.10.1 Hypotheses 36 1.10.2 Objectives 36-38 v Chapter Establishment of Reference Levels of Three Main Intracellular Androgens in Prostate Cells through LC-MS/MS Analysis and Human Cell-based Androgen-driven Reporter Gene Bioassay 39-40 Introduction 2.1 LC-MS/MS Method Development and Validation for Simultaneous Detection and Quantitation of A4, T and DHT 40-41 2.1.1 Materials 2.1.2 Experimental 2.1.2.1 Preparation and Extraction of Calibration Standards and Quality Controls 42 2.1.2.2 Preparation of Stability Tests Samples 43 2.1.2.3 Cell Culture under Different Treatments 43-45 2.1.2.4 Preparation and Extraction from Cell Samples 45-46 2.1.2.5 LC-MS/MS Analysis 46-48 48 2.1.2.6 Method Validation 2.2 Correlation of Intracellular A4, T and DHT under Different Treatments using Stable Human Cell-based AR-Driven Reporter Gene Assay 2.3 MTS Cell Proliferation Assay with Cell Lysates after Treatment 48-50 50 2.4 Results and Discussions 2.4.1 LC-MS/MS Method Development for Simultaneous Detection of A4, T and DHT 51-52 2.4.2 Validation of LC-MS/MS Method 53-56 2.4.3 Stabilities of A4, T and DHT in PBS/BSA 57-59 2.4.4 Measurement of Intracellular A4, T and DHT in Prostate Cells under Different Treatments and Correlation with Stable Human Cell-based AR-Driven Reporter Gene Assay 60-71 2.4.5 MTS Proliferation Assay using the PCa Cell Extracts under Different Treatments 71-72 2.5 Summary of Results 73-74 vi Chapter Establishment of Reference Levels of Two Intracellular Estrogens in Prostate Cells through LC-MS/MS Analysis and Human Cell-based Estrogen-driven Reporter Gene Bioassay 75-76 Introduction 3.1 LC-MS/MS Method Development and Validation Simultaneous Detection and Quantitation of E1 and E2 for 77-78 3.1.1 Materials 3.1.2 Experimental 3.1.2.1 Preparation and Extraction of Calibration Standards and Quality Controls 3.1.2.2 Preparation of Stability Tests Samples 3.1.2.3 Cell Culture under Different Treatments 3.1.2.4 Preparation and Extraction from Cell Samples 78-79 79 79-80 80 80-82 3.1.2.5 LC-MS/MS Analysis 82 3.1.2.6 Method Validation 3.2 Correlation of Intracellular E1 and E2 under Different Treatments using Stable Human Cell-based ERα- and ERβ-Driven Reporter Gene Assay 82-83 3.3 MTS Cell Proliferation Assay with Cell Lysates after Treatment 83-84 3.4 Results and Discussions 3.4.1 LC-MS/MS Method Development for Simultaneous Detection of E1 and E2 84-86 3.4.2 Validation of LC-MS/MS Method 87-89 3.4.3 Stabilities of E1 and E2 in PBS/BSA 90-91 3.4.4 Measurement of Intracellular E1 and E2 levels in Prostate Cells under Different Treatments and Correlation with Stable Human Cell-based ERα- and ERβ-Driven Reporter Gene Assay 3.4.5 MTS Proliferation Assays using the PCa Cell Extracts under Different Treatments 92-99 3.5 Summary of Results 100 101 vii Chapter Dimethoxycurcumin as a Potential AKR1C3 Enzyme Inhibitor for Treatment of Castration Resistant Prostate Cancer (CRPC) Introduction 102-105 106-107 4.1 Materials 4.2 Experimental 4.2.1 Determination of the Presence of AKR1C3 Enzyme in Various PCa Cell Lines using Western Blot Analysis 4.2.1.1 Cell culture for Western Blot Analysis 108 108 4.2.1.2 Cell Lysis 108-109 4.2.1.3 Bradford Protein Measurement Assay 109-110 4.2.1.4 Western Blot Analysis 4.2.1.4.1 Gel Casting 110-111 4.2.1.4.2 Denaturation of Proteins and Sample Loading 4.2.1.4.3 Transfer of Bands from Gel to Membrane and Addition of AKR1C3 Antibody 4.2.1.4.4 Addition of Secondary Antibody for AKR1C3 111-112 112-113 113 4.2.1.4.5 Film Development 113 4.2.1.4.6 Standardisation via checking on β-actin 114 4.2.2 Determination of Saturating Doses of Known AKR1C3 Inhibitor and Dimethoxycurcumin and its Potential as AKR1C3 Enzyme Inhibitor 4.2.2.1 Preparation of Drugs in Different Concentrations 114 4.2.2.2 Preparation of Drugs in Different Doses in Media 115 4.2.2.3 MTS Cell Proliferation Assay on CRPC Cell containing AKR1C3 enzyme 115-116 viii 4.2.3 Investigation of Dimethoxycurcumin using LC-MS/MS as a Potential Drug Treatment for CRPC via Multiple Enzymatic Pathways 4.2.3.1 Preparation of Different Doses of Drugs 116 4.2.3.2 Culture of CWR22Rv1 for Dose Dependent Curve 116-117 4.2.3.3 Culture of CWR22Rv1 under Saturating Doses of Drugs 4.2.3.4 Preparation and Extraction of Calibration Standards and Quality Controls 118 4.2.3.5 Preparation of Stability Tests Samples 120 119 4.2.3.6 Preparation and Extraction from Cell Samples 120-121 4.2.3.7 LC-MS/MS Analysis 121-123 123 4.2.3.8 Method Validation 4.2.4 Western Blot Analysis for Detection of Changes to AKR1C3 and AKR1C2 after Drug Treatment 124 4.3 Results and Discussions 4.3.1 Determination of the Presence of AKR1C3 Enzyme in Various PCa Cell Lines using Western Blot Analysis 125 4.3.2 Determination of Saturating Doses of Known AKR1C3 Inhibitor and Dimethoxycurcumin and its Potential as AKR1C3 Enzyme Inhibitor 126 4.3.3 Investigation of Dimethoxycurcumin using LC-MS/MS as a Potential Drug Treatment for CRPC via Multiple Enzymatic Pathways 4.3.3.1 LC-MS/MS Method Development for Simultaneous Detection of Six Key Androgens 4.3.3.2 Validation of LC-MS/MS Method 4.3.3.2 Stabilities of DHEA, A4, T, DHT, 3α-Diol and 3β-Diol in PBS/BSA 4.3.3.3 Dimethoxycurcumin as a Selective Inhibitor of AKR1C3 enzyme 4.3.3.4 Changes to Intracellular Androgen Levels under Different Treatments using Dimethoxycurcumin and Known AKR1C3 Inhibitor 4.4 Summary of Results 127-129 129-136 137-141 142-147 147-152 152-154 ix Chapter Investigation of Pharmacokinetics and Pharmacodistribution of Dimethoxycurcumin in Mice Sera and Organs using LC-MS/MS Introduction 155-156 157 5.1 Materials 5.2 Experimental 5.2.1 Preparation and Extraction of Calibration Standards and Quality Controls 5.2.2 Preparation of Stability Tests Samples 157-158 158 5.2.3 Sample Preparation for Mice Sera and Organ Samples 5.2.3.1 Mice Sera Samples 159 5.2.3.2 Mice Organ Samples 159 160-162 5.2.4 LC-MS/MS Analysis 162 5.2.5 Method Validation 5.3 Results and Discussions 5.3.1 Validation of LC-MS/MS Method 163-165 5.3.2 Stability of Dimethoxycurcumin in Serum and PBS/BSA Matrix 166-167 5.3.3 Pharmacokinetic Study of Dimethoxycurcumin in Mice Sera 168-169 5.3.4 Distribution of Dimethoxycurcumin in Mice Organs at and 24 hrs after Single Dose Injection 169-170 171 5.4 Summary of Results Chapter Conclusions and Limitations & Future Works 6.1 Conclusions 172-176 6.2 Limitations & Future Works 177-178 References 179-205 x LnCap (P10) PCa Cells (Androgen dpt) A4 T DHT C4-2 (P11&13) PCa Cells (Androgen Indpt) A4 T DHT C4-2B (P7&9) PCa Cells (Androgen Indpt) A4 T DHT CWR22Rv1 PCa Cells (P11&12) (Androgen Indpt) A4 T DHT 0.0219 0.0024 0.0079 0.0014 0.1010 0.1480 0.0213 0.0039 0.0285 0.0020 0.1495 0.2275 0.0266 0.0060 0.0307 0.0012 0.0405 0.0011 0.0131 0.0022 0.0178 0.0018 0.0574 0.0917 0.0153 0.0039 0.0120 0.0072 0.1034 0.0975 0.0188 0.0025 0.0317 0.0081 0.1657 0.0335 0.0177 0.0040 0.0351 0.0021 0.3739 0.2245 0.0139 0.0016 0.0279 0.0075 0.1288 0.1254 227 0.0132 0.0013 0.0125 0.0093 0.3020 0.0602 0.0147 0.0042 0.0075 0.0022 0.2872 0.2667 0.0107 0.0033 0.0084 0.0015 0.2535 0.2218 0.0106 0.0056 0.0077 0.0008 0.2435 0.3443 0.0158 0.0062 0.0053 0.0022 0.2140 0.1889 0.0141 0.0038 0.0068 0.0006 0.4062 0.2880 0.0173 0.0091 0.0094 0.0020 0.4337 0.1687 0.0324 0.0413 0.0073 0.0009 0.0407 0.0215 Appendix VIII Chromatograms of Blank matrices detecting E1 and E2 in (e) PBS/BSA Blank Matrix (f) RPMI media (g) DMEM media 229 (h) KSFM media 230 Appendix IX Partial Validation Data for Detection of E1 and E2 in (a) RPMI-1640 media matrix Summary of Validation of E1 for Interday Quality Controls in RPMI-1640 Matrix Estimated Interday Concentration (nM) Interday Validation Summary Nominal Concentration Day Day Day Mean Conc Stdev CV % Accuracy % (nM) (nM) 0.049 0.048 0.053 0.050 0.003 5.1 100.0 0.05 0.465 0.428 0.463 0.452 0.021 4.6 90.4 0.2 4.990 4.847 4.708 4.848 0.141 2.9 97.0 Summary of Validation of E1 for Intraday Quality Controls in RPMI-1640 Matrix Estimated Intraday Concentration (nM) Intraday Validation Summary Nominal Concentration 1st Run 2nd Run 3rd Run 4th Run 5th Run 6th Run Mean Stdev CV % Accuracy (nM) Conc % (nM) 0.052 0.055 0.052 0.053 0.060 0.053 0.054 0.003 5.7 108.0 0.05 0.488 0.430 0.472 0.523 0.458 0.449 0.470 0.033 7.0 94.0 0.2 4.730 4.613 4.782 4.821 4.439 4.382 4.628 0.183 4.0 92.6 Summary of Validation of E2 for Interday Quality Controls in RPMI-1640 Matrix Estimated Interday Concentration (nM) Interday Validation Summary Nominal Concentration Day Day Day Mean Conc Stdev CV % Accuracy % (nM) (nM) 0.052 0.051 0.046 0.049 0.003 6.5 98.5 0.05 0.504 0.458 0.440 0.467 0.033 7.1 93.5 0.2 5.120 4.933 4.573 4.875 0.278 5.7 97.5 Summary of Validation of E2 for Intraday Quality Controls in RPMI-1640 Matrix Estimated Intraday Concentration (nM) Intraday Validation Summary Nominal st nd rd th th th Concentration Run Run Run Run Run Run Mean Stdev CV % Accuracy (nM) Conc % (nM) 0.041 0.052 0.044 0.054 0.049 0.052 0.049 0.005 10.5 97.0 0.05 0.442 0.452 0.425 0.464 0.475 0.457 0.453 0.018 3.9 90.6 0.2 4.742 4.111 4.867 4.878 4.589 4.472 4.610 0.291 6.3 92.2 231 (b) DMEM media matrix Summary of Validation of E1 for Interday Quality Controls in DMEM Matrix Estimated Interday Concentration (nM) Interday Validation Summary Nominal Concentration Day Day Day Mean Conc Stdev CV % Accuracy % (nM) (nM) 0.052 0.044 0.052 0.049 0.005 9.4 98.8 0.05 0.556 0.471 0.522 0.516 0.042 8.2 103.3 0.2 5.552 5.200 4.536 5.096 0.516 10.1 101.9 Summary of Validation of E1 for Intraday Quality Controls in DMEM Matrix Estimated Intraday Concentration (nM) Intraday Validation Summary Nominal Concentration 1st Run 2nd Run 3rd Run 4th Run 5th Run 6th Run Mean Stdev CV % Accuracy (nM) Conc % (nM) 0.050 0.046 0.060 0.056 0.048 0.055 0.052 0.005 10.0 104.8 0.05 0.474 0.554 0.540 0.428 0.432 0.545 0.495 0.058 11.7 99.1 0.2 4.524 4.707 4.376 5.132 5.013 4.952 4.784 0.297 6.2 95.7 Summary of Validation of E2 for Interday Quality Controls in DMEM Matrix Estimated Interday Concentration (nM) Interday Validation Summary Nominal Concentration Day Day Day Mean Conc Stdev CV % Accuracy % (nM) (nM) 0.056 0.049 0.053 0.053 0.004 7.5 105.5 0.05 0.513 0.520 0.494 0.509 0.013 2.6 101.8 0.2 5.685 5.210 5.070 5.322 0.322 6.1 106.4 Summary of Validation of E2 for Intraday Quality Controls in DMEM Matrix Estimated Intraday Concentration (nM) Intraday Validation Summary Nominal Concentration 1st Run 2nd Run 3rd Run 4th Run 5th Run 6th Run Mean Stdev CV % Accuracy (nM) Conc % (nM) 0.052 0.051 0.056 0.046 0.055 0.050 0.052 0.004 7.4 103.6 0.05 0.501 0.491 0.490 0.452 0.469 0.487 0.482 0.018 3.7 96.3 0.2 5.036 5.239 4.936 5.378 5.716 5.524 5.305 0.295 5.6 106.1 232 (c) KSFM media matrix Summary of Validation of E1 for Interday Quality Controls in KSFM Matrix Estimated Interday Concentration (nM) Interday Validation Summary Nominal Concentration Day Day Day Mean Conc Stdev CV % Accuracy % (nM) (nM) 0.047 0.050 0.048 0.048 0.002 3.8 96.7 0.05 0.540 0.532 0.507 0.526 0.017 3.3 105.3 0.2 5.394 5.122 5.603 5.373 0.241 4.5 107.5 Summary of Validation of E1 for Intraday Quality Controls in KSFM Matrix Estimated Intraday Concentration (nM) Intraday Validation Summary Nominal Concentration 1st Run 2nd Run 3rd Run 4th Run 5th Run 6th Run Mean Stdev CV % Accuracy (nM) Conc % (nM) 0.043 0.053 0.046 0.050 0.042 0.054 0.048 0.005 10.7 96.6 0.05 0.521 0.539 0.461 0.543 0.555 0.573 0.532 0.039 7.4 106.4 0.2 5.651 5.855 5.304 4.528 5.366 5.389 5.349 0.453 8.5 107.0 Summary of Validation of E2 for Interday Quality Controls in KSFM Matrix Estimated Interday Concentration (nM) Interday Validation Summary Nominal Concentration Day Day Day Mean Conc Stdev CV % Accuracy % (nM) (nM) 0.048 0.050 0.045 0.048 0.003 5.3 95.6 0.05 0.561 0.554 0.503 0.539 0.031 5.8 107.8 0.2 5.398 5.345 5.220 5.321 0.091 1.7 106.4 Summary of Validation of E2 for Intraday Quality Controls in KSFM Matrix Estimated Intraday Concentration (nM) Intraday Validation Summary Nominal Concentration 1st Run 2nd Run 3rd Run 4th Run 5th Run 6th Run Mean Stdev CV % Accuracy (nM) Conc % (nM) 0.047 0.048 0.040 0.044 0.042 0.047 0.045 0.003 6.9 89.2 0.05 0.556 0.548 0.405 0.546 0.540 0.546 0.524 0.058 11.1 104.7 0.2 5.384 5.269 5.008 4.743 5.535 5.594 5.255 0.326 6.2 105.1 233 Appendix X Cell Line Table showing the Intracellular Estrogens Ratios Type of Cell Average Ratio btw passages Line Ratio 10 % 10 % 10 % FBS FBS + 10 CD-FBS nM E2* Av Ratio Std Dev RWPE-1 (P2 & 3) Normal Prostate cells (Epithelial) Normal Prostate Cells (Stroma) Benign Prostate Hyperplasia Cells PCa Cells (Androgen dpt) Av Ratio Std Dev Av Ratio Std Dev 0.66 0.65 0.85 0.34 0.25 0.51 NA T:E2 0.10 0.17 0.01 0.11 WPMY-1 (P4 E1:E2 1.20 0.64 1.44 0.48 0.25 1.08 & 6) NA T:E2 2.45 0.53 1.05 0.47 BPH-1 E1:E2 0.88 0.92 0.93 0.62 0.29 0.42 NA T:E2 0.07 1.16 0.04 1.51 LnCap (P10) E1:E2 0.76 0.04 1.27 NA NA NA NA T:E2 1.27 0.69 NA NA C4-2 PCa Cells E1:E2 1.30 0.13 1.17 0.54 0.00 0.18 (P11&13) (Androgen Indpt) NA T:E2 1.99 1.40 0.62 0.33 C4-2B (P7&9) PCa Cells E1:E2 1.38 0.19 1.30 0.61 0.07 0.01 (Androgen Indpt) NA T:E2 1.52 1.07 0.05 0.43 CWR22Rv1 PCa Cells E1:E2 1.22 0.16 1.41 0.58 0.06 0.58 (P11&12) (Androgen Indpt) NA T:E2 2.13 0.89 0.59 0.16 *As E2 was artificially added here, so T:E2 ratio was not applicable here E1:E2 234 10 % CD-FBS + 10 nM E2* Av Ratio Std Dev 0.39 0.49 NA 0.63 0.40 NA 0.71 0.34 NA 0.36 NA NA 0.12 0.02 NA 0.16 0.08 NA 0.12 0.00 NA Appendix XI Basal Concentrations of the Respective Estrogens of Interest in Media Types of Media Used for Cell Lines RPMI (10% FBS) RPMI (10 % CDFBS) DMEM (10% FBS) DMEM (10 % CD-FBS) KSFM (w growth factors) KSFM (w/o growth factors) Av Basal Conc Of Estrogens in Different Treatments nM Std Dev E1 E2 0.4878 0.3785 0.0502 0.1133 0.1743 0.0518 0.1885 0.0071 0.0646 0.0242 0.0038 0.0086 0.0631 0.1348 0.0081 0.1932 0.0551 0.0352 0.0064 0.0127 0.0497 0.0240 0.0041 0.0015 235 Appendix XII Average Concentrations of Respective Estrogens in Conditioned Media after Different Treatments Cell Line Type of Cell Av Conc Of Estrogens in Different Treatments (Passage Line nM Std Dev No.) (1) 10 % FBS RWPE-1 (P2 & 3) WPMY-1 (P4 & 6) BPH-1 (P26 &27) LnCap (P10) C4-2 (P11&13) Normal Prostate cells (Epithelial) Normal Prostate Cells (Stroma) Benign Prostate Hyperplasia Cells PCa Cells (Androgen dpt) E1 PCa Cells (Androgen E1 E2 E1 E2 E1 E2 E1 E2 0.10855 0.10967 0.29980 0.35808 0.06627 0.00197 0.23860 0.28137 0.08067 0.00653 0.09363 0.01053 0.04180 0.00621 1.1110 0.1142 0.13343 0.03538 (2) 10 % FBS + nM DHT 0.07127 0.05605 0.38540 0.25395 0.26057 0.01685 3.86333 0.05506 0.13380 0.01545 0.24343 0.08016 5.46550 0.79799 7.49507 0.16715 4.38390 0.36588 (3) 10 % CD-FBS 0.03440 0.00769 0.06090 0.05020 0.03593 0.00660 0.04163 0.03439 0.03827 0.00967 0.04897 0.03792 0.02910 0.00135 0.06817 0.00764 0.08030 0.01464 (4) 10 % CD-FBS + nM DHT 0.0406 0.0045 0.12797 0.00816 0.22250 0.01794 4.67317 0.06932 0.07257 0.01476 0.28157 0.05044 7.10130 0.46629 14.8564 1.58306 1.52673 0.07763 Indpt) E2 PCa Cells (Androgen Indpt) E1 CWR22Rv1 PCa Cells (P11&12) (Androgen Indpt) E1 C4-2B (P7&9) E2 E2 1.6440 2.7340 0.15030 0.03805 1.72497 0.09157 0.05957 0.01316 0.7212 0.0937 15.6206 1.03915 3.12230 0.59021 5.5401 1.2911 2.87910 0.09510 11.5851 0.61580 237 0.19653 0.13005 0.03553 0.00207 0.1680 0.0405 0.02117 0.00072 0.03647 0.01165 16.8126 1.03474 8.18720 1.63069 12.7564 2.65586 2.74210 0.11065 15.8067 0.99707 Appendix XIII Solutions for Tris/Glycine SDS-Polyacrylamide Gel Electrophoresis 6% H 2O 30% Acrylamide Mixa 1.5 M Tris (pH 8.8)b 10% SDSc 10% APS TEMEDd 8% H 2O 30% Acrylamide Mixa 1.5 M Tris (pH 8.8)b 10% SDSc 10% APS TEMEDd 10% H 2O 30% Acrylamide Mixa 1.5 M Tris (pH 8.8)b 10% SDSc 10% APS TEMEDd 12% H 2O 30% Acrylamide Mixa 1.5 M Tris (pH 8.8)b 10% SDSc 10% APS TEMEDd 15% H 2O 30% Acrylamide Mixa 1.5 M Tris (pH 8.8)b 10% SDSc 10% APS TEMEDd STACK 5% H 2O 30% Acrylamide Mixa 1.0 M Tris (pH 6.8)b 10% SDSc 10% APS TEMEDd mL 2.7 1.3 0.005 0.005 0.004 mL 2.3 1.3 1.3 0.05 0.05 0.003 mL 1.7 1.3 0.005 0.005 0.002 mL 1.7 1.3 0.005 0.005 0.002 mL 1.2 2.5 1.3 0.005 0.005 0.002 mL 0.68 0.17 0.13 0.01 0.01 0.001 10 mL 5.3 2.5 0.1 0.1 0.008 10 mL 4.6 2.7 2.5 0.1 0.1 0.006 10 mL 3.3 2.5 0.1 0.1 0.004 10 mL 3.3 2.5 0.1 0.1 0.004 10 mL 2.3 2.5 0.1 0.1 0.004 10 mL 1.4 0.33 0.25 0.02 0.02 0.002 15 mL 3.8 0.15 0.15 0.012 15 mL 3.8 0.15 0.15 0.009 15 mL 5.9 3.8 0.15 0.15 0.006 15 mL 3.8 0.15 0.15 0.006 15 mL 3.5 7.5 3.8 0.15 0.15 0.006 15 mL 2.1 0.5 0.38 0.03 0.03 0.003 20 mL 10.6 0.2 0.2 0.016 20 mL 9.3 5.3 0.2 0.2 0.012 20 mL 7.9 6.7 0.2 0.2 0.008 20 mL 6.6 0.2 0.2 0.008 20 mL 4.6 10 0.2 0.2 0.008 20 mL 2.7 0.67 0.5 0.04 0.04 0.004 25 mL 13.2 6.3 0.25 0.25 0.02 25 mL 11.6 6.7 6.3 0.25 0.25 0.015 25 mL 9.9 8.3 6.3 0.25 0.25 0.01 25 mL 8.3 10 6.3 0.25 0.25 0.01 25 mL 5.7 12.5 6.3 0.25 0.25 0.01 25 mL 3.4 0.83 0.63 0.05 0.05 0.005 30 mL 15.9 7.5 0.3 0.3 0.024 30 mL 13.9 7.5 0.3 0.3 0.018 30 mL 11.9 10 7.5 0.3 0.3 0.012 30 mL 9.9 12 7.5 0.3 0.3 0.012 30 mL 6.9 15 7.5 0.3 0.3 0.012 30 mL 4.1 0.75 0.06 0.06 0.006 40 mL 21.1 10 0.4 0.4 0.032 40 mL 18.6 10.7 10 0.4 0.4 0.024 40 mL 15.8 13.3 10 0.4 0.4 0.016 40 mL 13.2 14 10 0.4 0.4 0.016 40 mL 9.2 20 10 0.4 0.4 0.016 40 mL 5.5 1.3 0.08 0.08 0.008 50 mL 26.5 10 12.5 0.5 0.5 0.04 50 mL 23.2 13.4 12.5 0.5 0.5 0.03 50 mL 20 16.6 12.5 0.5 0.5 0.02 50 mL 16.4 20 12.5 0.5 0.5 0.02 50 mL 11.4 25 12.5 0.5 0.5 0.02 50 mL 6.8 1.7 1.25 0.1 0.1 0.01 Appendix XIV Formulation for 4x SDS Loading buffer For 10 ml of loading buffer:  ml 100% glycerol  2.4 ml 1M Tris/HCl pH 6.8  0.8 g SDS  mg bromophenol blue  0.5 ml beta-mercaptoethanol  3.1 ml H2O 239 Appendix XV Chromatograms of Blank matrices detecting DHEA, A4, T, DHT, 3α-Diol and 3β-Diol in (d) PBS/BSA Blank Matrix 240 241 ... Validation of A4 in g/L of PBS/BSA Table 5a Interday Validation of T in g/L of PBS/BSA Table 5b Intraday Validation of T in g/L of PBS/BSA Table 6a Interday Validation of DHT in g/L of PBS/BSA... 4.3.2 Determination of Saturating Doses of Known AKR1C3 Inhibitor and Dimethoxycurcumin and its Potential as AKR1C3 Enzyme Inhibitor 126 4.3.3 Investigation of Dimethoxycurcumin using LC-MS/MS... Mechanisms Behind Development of Castration Resistant Prostate Cancer Table Analysis of LC-MS/MS Pros and Cons in Clinical Diagnostics Table 4a Interday Validation of A4 in g/L of PBS/BSA Table 4b Intraday