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Effect of simvastatin on castration-resistant prostate cancer cells Kim, Jenny Hanbi 2014

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EFFECT OF SIMVASTATIN ON CASTRATION-RESISTANT PROSTATE CANCER CELLS  by Jenny Hanbi Kim  BMLSc., The University of British Columbia, 2011  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Pharmaceutical Sciences)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  May 2014  © Jenny Hanbi Kim, 2014 ii  Abstract  Background In castration-resistant prostate cancer (CRPC), recent evidence has demonstrated the persistence of intratumoral androgens through de novo steroidogenesis despite androgen deprivation therapy. The multi-step androgen synthesis pathway originates from common precursor cholesterol molecule, which can be obtained by cells from several major sources including intracellular synthesis through 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) enzyme.  The inhibition of this enzyme by the use of statins has been investigated in various prostate cancer models with inconclusive results. In addition, the role of statins as a possible therapeutic agent for blocking de novo androgen synthesis in established CRPC models has not been thoroughly investigated.  Methods Castration-resistant C4-2 and androgen-sensitive LNCaP cells were treated with simvastatin (SV) for 48 hours. Cellular responses to SV were analyzed using cell proliferation and cytotoxicity assays. Cellular growth curve was generated by measuring the total cell count using haemocytometer over the course of 8 days. HMGCR activity was assessed by the incorporation of 14C-acetic acid into cholesterol that was detected by thin layer chromatography. Western blotting technique was used to determine the HMGCR protein expression in the cell lines. Intracellular cholesterol, prostate specific antigen (PSA) secretion, and intracellular testosterone levels were quantified using enzyme-linked immunosorbent assays (ELISA).  iii  Results % cytotoxicity of castration-resistant C4-2 cells following 48-hour SV treatment significantly increased at SV concentration greater than 60 μM compared to vehicle control. Corresponding results were observed in cell viability data where a significant decrease in cell viability occurred in C4-2 cells treated with SV concentration greater than 75 μM compared to vehicle control. A significant difference in the total cell count compared to vehicle control occurred in C4-2 cells treated with 75 μM SV on Day 8, 72 hours after the removal of SV. HMGCR activity was significantly decreased up to 50% and 70% at 50 μM and 75 μM SV, respectively, compared to vehicle control in C4-2 cells; on Day 8 continuous inhibition of HMGCR activity was observed in cells treated with 75 μM SV. SV did not affect HMGCR protein expression in C4-2 cells, while 80% decrease in total intracellular cholesterol concentration was observed in the cells treated with 75 μM SV compared to vehicle control. Prostate specific antigen concentration in media of C4-2 cells treated with 50 μM and 75 μM SV was significantly different compared to PSA level in vehicle control, whereas intracellular testosterone concentration was not statistically different in C4-2 cells following 48-hour SV treatment compared to vehicle control.  Conclusion Inhibition of the HMGCR using SV lowered the viability of castration-resistant C4-2 cells. The ability of SV to limit the intracellular cholesterol synthesis and concentration may contribute to the effect seen in cell viability. Clinically, SV may be considered as primary therapy in patients with established CRPC; however further tests must be conducted to support the findings in this study.   iv  Preface  At the time of writing, part of the work presented in this thesis has been published:  Parts of data, methodology and discussion found in Chapter 2, 3, and 4 have been published. Kim JH, Cox ME, Wasan KM. Effect of simvastatin on castration-resistant prostate cancer cells. Lipids in Health and Disease. 2014. I conducted all the experiments presented in this paper and wrote the article. Dr. Michael Cox is the advisor and collaborator who supplied the cells and technical support. Dr. Kishor Wasan is the advisor and principal investigator on the grant that funded this work.  I conducted all the experiments presented in this thesis and wrote the manuscript. Dr. Kishor Wasan is the advisor and principal investigator on the grant that funded this work.   v  Table of Contents  Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iv Table of Contents ...........................................................................................................................v List of Tables ................................................................................................................................ ix List of Figures .................................................................................................................................x List of Abbreviations .................................................................................................................. xii Acknowledgements ......................................................................................................................xv Dedication ................................................................................................................................... xvi Chapter 1: Introduction ..............................................................................................................17 1.1 Prostate and Introduction to Prostate Cancer ................................................................ 17 1.2 Androgens and Androgen Receptor .............................................................................. 19 1.2.1 Androgens ................................................................................................................. 19 1.2.2 Androgen-dependent AR Activation ........................................................................ 21 1.2.3 AR Variants and Androgen Resistance..................................................................... 23 1.3 Prostate Cancer and CRPC ........................................................................................... 24 1.3.1 Experimental Models ................................................................................................ 25 1.3.1.1 In vitro Models .................................................................................................. 25 1.3.1.2 In vivo Models................................................................................................... 26 1.3.2 Current Treatments ................................................................................................... 27 1.3.2.1 AR-targeting Treatments .................................................................................. 28 1.3.2.2 AR-independent Treatments ............................................................................. 29 vi  1.3.2.3 Implications of  Current Treatments ................................................................. 30 1.4 Cellular Cholesterol Homeostasis ................................................................................. 31 1.4.1 Exogenous Cholesterol Supply and Uptake .............................................................. 33 1.4.2 Storage of Intracellular Cholesterol .......................................................................... 35 1.4.3 Efflux of Intracellular Cholesterol ............................................................................ 36 1.4.4 Synthesis of Intracellular de novo Cholesterol ......................................................... 37 1.4.4.1 Statins ................................................................................................................ 38 1.4.4.2 Previous Research ............................................................................................. 41 1.5 Research Hypothesis ..................................................................................................... 43 1.6 Aims of the Thesis ........................................................................................................ 44 1.7 Rationale and Significance ........................................................................................... 44 Chapter 2: Materials and Methods ............................................................................................45 2.1 Materials and Reagents ................................................................................................. 45 2.2 Cell Culture ................................................................................................................... 46 2.3 Simvastatin Preparation ................................................................................................ 46 2.4 Protein Quantification ................................................................................................... 47 2.5 Cytotoxicity and Cell Viability ..................................................................................... 48 2.5.1 LDH Assay................................................................................................................ 48 2.5.2 MTS Assay................................................................................................................ 49 2.6 Growth Curve................................................................................................................ 50 2.7 HMGCR Activity Assay ............................................................................................... 50 2.8 Western Blotting ........................................................................................................... 51 2.9 Cholesterol Quanitification ........................................................................................... 53 vii  2.10 Prostate Specific Antigen Assay ................................................................................... 54 2.11 Testosterone Assay ....................................................................................................... 55 2.12 Stastistical Analyses...................................................................................................... 56 Chapter 3: Results........................................................................................................................57 3.1 Aim 1 Experiments ....................................................................................................... 57 3.1.1 Cytotoxicity of Simvastatin-Treated Cells................................................................ 57  3.1.2 Cell Viability of Simvastatin-Treated Cells .............................................................. 59 3.1.3 Growth Curve of Simvastatin-Treated Cells............................................................. 61 3.2 Aim 2 Experiments ....................................................................................................... 64 3.2.1 HMGCR Activity of Simvastatin-Treated Cells ....................................................... 64 3.2.2 Protein Expression of Simvastatin-Treated Cells ..................................................... 68 3.2.3 Intracellular Cholesterol of Simvastatin-Treated Cells............................................. 70 3.3 Aim3 Experiments ........................................................................................................ 72 3.3.1 Intracellular Testosterone of Simvastatin-Treated Cells........................................... 72 3.3.2 PSA Secretion from Simvastatin-Treated Cells ........................................................ 73 Chapter 4: Discussion ..................................................................................................................76 4.1 Time-Dependent Effect of Simvastatin on Cell Viability............................................. 76 4.2 Simvastatin Reduces Cell Viability .............................................................................. 77 4.3 Simvastatin Reduces Cholesterol Synthesis ................................................................. 79 4.4 Reduced PSA Secretion in Simvastatin-Treated C4-2 Cells ........................................ 82 4.5 Effect of Simvastatin on Testosterome ......................................................................... 85 4.6 A Focus on Castration-Resistant State .......................................................................... 86  viii  4.7 Limitations and Future Directions ................................................................................ 87 4.8 Conclusion .................................................................................................................... 90 4.9 Significance of Findings ............................................................................................... 91 References .....................................................................................................................................92 Appendices ..................................................................................................................................105 Appendix A ............................................................................................................................. 105 A.1 C4-2 Cytotoxicity following 6-Hour Simvastatin Treatment ................................. 105 A.2 C4-2 Cytotoxicity following 24-Hour Simvastatin Treatment ............................... 106 Appendix B ............................................................................................................................. 107 B.1 LD50, IC50 Calcuation Equation and Coefficients ................................................... 107  Appendix C ............................................................................................................................ 108 C.1 Western Blot Representation of HMGCR .............................................................. 108 Appendix D ............................................................................................................................. 109 D.1 Basal Intracellular Total Cholesterol Concentration in cell lysates of the C4-2 and LNCaP Cell Lines .................................................................................................. 109    ix  List of Tables  Table 2.1: Preparation of simvastatin stock solutions .................................................................. 47 Table 2.2: Characteristics of primary and secondary antibodies used for the detection of HMGCR in C4-2 and LNCaP cells. ............................................................................................................. 53 Appendix Table B.1: LD50, IC50 Calcuation Equation and Coefficients  ................................... 107   x  List of Figures  Figure 1.1: Cellular androgen synthesis pathways ....................................................................... 20  Figure 1.2: Androgen-dependent androgen receptor signaling pathway ...................................... 22  Figure 1.3: Diagram of cellular cholesterol homeostasis including influx, metabolism, synthesis, and efflux pathways ...................................................................................................................... 32  Figure 1.4: Domain structure of HMGCR (121) .......................................................................... 38  Figure 1.5: (A) Increase in HMGCR activity measured in SR-BI knocked-down C4-2 cells 6 days post-siRNA transfection  (modified from (107)) (B) Increase in HMGCR activity measured in CRPC state that has progressed from LNCaP xenograft model (53). ...................................... 42  Figure 3.1: C4-2 cell cytotoxicity following 48-hour SV treatment (Day 5) ............................... 58 Figure 3.2: LNCaP cell cytotoxicity following 48-hour SV treatment (Day 5) ........................... 59 Figure 3.3: C4-2 cell viability following 48-hour SV treatment (Day 5) ..................................... 60 Figure 3.4: LNCaP cell viability following 48-hour SV treatment (Day 5) ................................. 61 Figure 3.5: Cumulative cell growth curve of C4-2 cells over 8 days. .......................................... 62 Figure 3.6: Cumulative cell growth curve of LNCaP cells over 8 days. ...................................... 63 Figure 3.7: Intracellular cholesterol synthesis of C4-2 cells following 48-hour SV treatment (Day 5) ................................................................................................................................................... 65 Figure 3.8: Intracellular cholesterol synthesis of C4-2 cells on Day 8 ......................................... 66 Figure 3.9: Intracellular cholesterol synthesis of LNCaP cells following 48-hour SV treatment (Day 5) .......................................................................................................................................... 67 xi  Figure 3.10: (A) Baseline (NT) HMGCR activity of C4-2 and LNCaP cells (B) baseline HMGCR activity of C4-2 cell and LNCaP cells (NT) and HMGCR activity of the two cell lines following 75μM SV treatment (Day 5). ........................................................................................ 68 Figure 3.11: HMGCR protein expression of C4-2 cells following 48-hour SV treatment (Day 5)....................................................................................................................................................... 69 Figure 3.12: HMGCR protein expression of LNCaP cells following 48-hour SV treatment (Day 5)....................................................................................................................................................... 69 Figure 3.13: Total intracellular cholesterol concentration in C4-2 cell lysates following 48-hour SV treatment (Day 5) .................................................................................................................... 70 Figure 3.14: Total intracellular cholesterol concentration in LNCaP cell lysates following 48-hour SV treatment (Day 5)  ........................................................................................................... 71 Figure 3.15: Intracellular testosterone concentration of C4-2 cells following 48-hour SV treatment (Day 5) .......................................................................................................................... 72 Figure 3.16: PSA secretion from C4-2 cells following 48-hour SV treatment (Day 5) ............... 73 Figure 3.17: PSA secretion from LNCaP cells following 48-hour SV treatment (Day 5) ........... 74 Figure 3.18: (A) Baseline (NT) PSA concentration in media of C4-2 and LNCaP cells (B) baseline PSA concentration in media of C4-2 and LNCaP cells (NT) and PSA secretion levels from the two cell lines following 75M SV treatment (Day 5) .................................................... 75 Figure A.1: C4-2 Cytotoxicity following 6 Hour of simvastatin Treatment .............................. 105  Figure A.2: C4-2 Cytotoxicity following 24 Hour of simvastatin Treatment ............................ 106  Figure C.1: Western Blot Representation ................................................................................... 108 Figure D.1: Basal Intracellular Total Cholesterol Concentration in cell lysates of the C4-2 and LNCaP Cell Lines ....................................................................................................................... 109  xii  List of Abbreviations ABC ATP-binding cassette transporter superfamily ACAT Acetyl-CoA acyltransferase ACTH Adrenocorticotropic hormone ADT Androgen deprivation therapy AR Androgen receptor ARE Androgen response element BSA Bovine serum albumin BLT-1 Block lipid transporter-1 C4-2 LNCaP-derived castration-resistant cell line CEs Cholesteryl esters CVD Cardiovascular disease CRPC Castration-resistant prostate cancer CSS Charcoal-stripped serum DHEA Dehydroepiandrosterone DHT Dihydrotestosterone ELISA Enzyme-linked immunosorbent assay ER Endoplasmic reticulum ERK Extracellular-signal-regulated kinases FBS Fetal bovine serum FSH Follicle stimulating hormone GEMM Genetically engineered mouse model GnRH Gonadotropin-releasing hormone xiii  HBSS Hanks balanced salt solution HDL High-density lipoprotein HMG-CoA       3-hydroxy-3-methylglutaryl-coenzyme A HMGCR 3-hydroxy-3-methylglutaryl-coenzyme A reductase HSL Hormone sensitive lipase HSP Heat shock protein IDL Intermediate-density lipoprotein IGF-1R Insulin-like growth factor 1 receptor INSIGs Insulin-induced gene proteins  LBD Ligand binding domain LDH lactate dehydrogenase LDL Low-density lipoprotein LDLr Low density lipoprotein receptor LH Luteinizing hormone LHRH Luteinizing hormone-releasing hormone LNCaP Lymph node metastatic prostate adenocarcinoma cell-line, androgen-sensitive LRP-1 Low density lipoprotein receptor-related protein 1 MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium NT No treatment; control group of cells PCa Prostate cancer PKB Protein kinase B PSA Prostate specific antigen  xiv  PTEN Phosphatase and tensin homolog RIPA Radioimmunoprecipitation assay  RT-PCR Real-time polymerase chain reaction S1P Site-1 protease S2P Site-2 protease SCAP SREBP cleave activating protein  SEM Standard error of the mean SHBG Sex hormone-binding globulin SR-BI Scavenger receptor Class B Type I SRE Sterol response element SREBP Sterol response element binding protein StAR Steroidogenic acute regulatory protein SV Simvastatin  TBS-T Tris-buffered saline with 0.01% (v/v) Tween-20 TLC Thin layer chromatography  xv  Acknowledgements  First and foremost, I would like to thank my supervisor Dr. Kishor Wasan for giving me the chance to explore the world of scientific research. Through this invaluable experience, I have grown intellectually and emotionally thanks to Dr. Wasan’s guidance, patience, and contagious passion for research.  I would like to thank my committee members, Dr. Michael Cox, Dr. Marc Levine, Dr. Urs Hafeli, Dr. John Hill, Dr. Brian Rodrigues, Dr. Ujendra Kumar, and Dr. Frank Abbott for their support and guidance. Each of you has inspired me to think logically and creatively during the course of this project.  I would like to thank the members of Dr. Wasan’s laboratory past and present: Dr. Kristina Sachs-Barrable, Ms. Alexis Twiddy, Ms. Jo-Ann Osei-Twum, Mr. Ankur Midha, Mr. Jacob Gordon, Dr. Stephen Lee, Dr. Fady Ibrahim, Ms. Olena Sivak, Ms. Jinying Zhao, and Dr. Pavel Gershvokich for their constructive feedbacks, words of encouragement and mostly for their company.   Finally, special thanks to my parents and brother John for their support, and to those who made my graduate student life unforgettable.   xvi  Dedication  This work is dedicated to 25% of male cancer patients suffering from prostate cancer. 17  Chapter 1: Introduction  1.1 Prostate and Introduction to Prostate Cancer The prostate is a compound tubuloalveolar exocrine gland in the male reproductive system that produces alkaline fluid as part of the seminal vesicle fluid; this fluid helps to neutralize the acidity in the vaginal tract once secreted, prolonging the viability of sperm. The gland is located at the base of the bladder, and in front of the rectum with the urethra running through the gland. Similar to the other organs in the male reproductive system, prostate gland growth is regulated by androgens such as testosterone and 5α-dihydrotestosterone (DHT). Histologically, it is divided into four distinct glandular regions: peripheral, central, transition, and anterior fibromuscular zone. Prostate cancer (PCa) rising from the peripheral zone accounts for 70-80% of the occurrences, while PCa in the central zone tends to be more aggressive (1,2).   PCa occurs in the form of an unregulated cellular growth in the prostate. It is commonly diagnosed in men over the age of 50, after which incidence rates increase with age; the median age of diagnosis is 66. PCa is the most common cancer diagnosed among North American men, accounting for 28% of all cancers diagnosed, and it is the second most common cause of cancer deaths among the same cohort accounting for 10% of all cancer related deaths (3,4). In 2013, more than 23,600 new PCa patients will be diagnosed and 3,900 will die of PCa (3). Some of the common symptoms of PCa include erectile dysfunction, urinary dysfunction, hematuria, and pain. Therefore, men are encouraged to go through PCa screening starting at the age of 50. Other factors that are correlated with having a higher risk of developing PCa include obesity, diet, and 18  familial history of PCa, and men with a familial history of PCa are recommended to start the screening as early as age 40 (5,6).   Prostate specific antigen (PSA) is a protein produced and secreted by the prostate cells, and serum PSA levels are often elevated in PCa patients (7-9). PSA test that measures PSA level in patient’s serum has been used since 1986 to monitor the progression of PCa in men who had already been diagnosed with the disease. In 1994, the PSA test was approved by the FDA to be used in conjunction with a digital rectal exam (DRE) as a diagnostic tool to test asymptomatic men for PCa (10,11). A serum PSA level that is greater than 4 ng/mL combined with abnormal findings from DRE including enlargement of the prostate gland and growths in the gland as well as surrounding areas would lead to a prostate biopsy to confirm the presence of PCa (12). Upon a positive diagnosis, PCa can be treated by androgen deprivation therapy (ADT). ADT, which is achieved through surgical removal of the gland, radiation therapy, and/or chemotherapy, depletes androgen sources in the body including the testes and the adrenal cortex (13). Upon ADT, up to 90% of prostate cancer patients reach 5-year survival due to apoptotic regression of tumor from the lack of androgen growth stimuli (14). However, patients with metastatic prostate cancer only achieve a temporary remission and eventually the tumor regrows despite the depleted level of circulating androgens that is insufficient to support the prostate tumor growth; this is referred to as castration-resistant prostate cancer (CRPC; 15,16). In the past, patients with the recurrent disease had a poor prognosis with a median survival of 10-13 months. However, since 2010, new treatments for CRPC have significantly prolonged the median survival to 16-18 months (17-21).  19  1.2 Androgens and Androgen Receptor 1.2.1 Androgens Androgens are a subclass of steroid hormone derived from a common precursor cholesterol molecule and serve to regulate the development and maintenance of male characteristics and accessory reproductive organs. Testosterone is mainly synthesized in the Leydig cells of the testes while the synthesis of precursor androgen molecules such as dehydroepiandrosterone (DHEA), androstenedione and a lesser amount of testosterone occurs in the zona reticularis of the adrenal cortex (22). In addition, the synthesis of androgens in these steroidogenic organs is regulated by the hypothalamus-pituitary axis; lack of circulating androgens prevents the negative feedback on the axis, allowing the release of gonadotropin-releasing hormone (GnRH) and luteinizing hormone-releasing hormone (LHRH) from the hypothalamus. These hormones promote the release of luteinizing hormone (LH), follicle stimulating hormone (FSH) and adrenocorticotropic hormone (ACTH) from the anterior pituitary gland to stimulate the production of androgens in the testes and adrenal cortex (23,24). Upon stimulation, androgen synthesis is initiated with the transport of cholesterol into the mitochondrial matrix via steroidogenic acute regulatory protein (StAR) as shown in Figure 1.1; the transport via StAR is the rate-limiting step in the androgen synthesis pathway (25-27). Inside the mitochondrial matrix, cholesterol is converted into pregnenolone by a CYP11A1 enzyme (27,28). Pregnenolone further undergoes multiple reactions to be converted into testosterone and dihydrotestosterone (DHT) downstream. Although the conventional route for DHT production involves the intermediates androstenedione and testosterone through the classical pathway, recent findings suggest the existence and activation of an alternative backdoor pathway (Figure 1.1) that also contributes to androgen synthesis in normal and pathological conditions (29,30). 20    Figure 1.1: Cellular androgen synthesis pathways. Dihydrotesterone (DHT) synthesis is initiated with the transport of cholesterol into the mitochondrial matrix via steroidogenic acute regulatory protein (StAR). Once cholesterol is converted into pregnenolone by a CYP11A1 enzyme, pregnenolone is converted into 17-OH pregnenolone and progesterone by CYP11A1 and HSD3B2 enzymes respectively. Progesterone molecule either follows through the classical or the backdoor pathway, both which can eventually lead to the production of DHT.      StAR 21  1.2.2 Androgen-dependent AR Activation Once the androgens are produced by the steroidogenic cells, they are secreted into the extracellular compartment and transported in the blood circulation as free testosterone or mostly by binding to sex hormone-binding globulin (SHBG) and albumin as shown in Figure 1.2 (31). Inside the target cells, the androgens exert their effects by binding to the androgen receptor (AR), a ligand-activated nuclear receptor (32). While the main androgens that interact with the AR include both testosterone and DHT, DHT that is produced through a reduction process of testosterone by the 5α-reductase enzyme has nearly a 10-fold higher AR binding affinity than testosterone; the dissociation constant (Kd) for DHT is 0.1 nM vs 1.0 nM for testosterone (32-35). Inside the cytosol, DHT binds to AR that is sequestered by linkage to a stabilizing chaperone complex composed of tetratricopeptide repeat (TPR), p23 protein, and heat shock proteins (HSP) (34). The binding of DHT to AR causes a conformational change that releases the chaperone complex, and allows the DHT-AR to form a homodimer with another DHT-AR complex (34). The homodimer then translocates into the nucleus where it binds to a region in the DNA containing the androgen response elements (ARE). These AREs are short sequences of DNA within a gene promoter region that are recognized by several steroid receptors, including AR. The binding of the homodimer complex to ARE region initiates the transcription of genes responsible for cellular growth and survival, and one of the products of the activation is PSA.  22   Figure 1.2: Androgen-dependent androgen receptor activation pathway. Testosterone (T) is transported in the blood circulation by binding to sex hormone-binding globulin (SHBG). Once T is transported into the cytoplasm of a prostate cell via diffusion and endocytosis, it can be converted into dihydrotestosterone (DHT) by the 5α-reductase enzyme. Phosphorylated (P) androgen receptor (AR) in the cytoplasm is sequestered by chaperone complex composed of tetratricopeptide repeat (TPR), heat shock proteins (HSP), and p23 protein. When DHT binds to AR, the chaperone complex is released. The AR-DHT complex forms a homodimer with another AR-DHT complex, and the homodimer translocates into the nucleus where it binds to a region in the DNA containing the androgen response element (ARE). The binding of AR-DHT homodimer activates the transcription of factors responsible for cell growth, survival, and PSA secretion.     23  1.2.3 AR Variants and Androgen Resistance In normal physiological conditions, AR is expressed in many parts of the body including the organs of the reproductive system, cardiac muscle, hepatocytes, and epidermal glands in the form of a full length 110 kDa protein (32,34,35,36). Similar to other nuclear receptors, AR is modular in structure and is composed of multiple domains: N-terminus domain (NTD), DNA binding domain, a hinge region, ligand binding domain (LBD), and a C-terminal domain (34). Other naturally occurring isoforms such as 45 kDa and 80 kDa ARs with truncated N-terminus domain have been identified in many tissues in lesser amounts (36,37). The AR has been extensively studied in PCa models as alternative splicing of the AR could cause a dysregulation of cellular growth processes leading to cancer; it has been shown that in PCa, various constitutively active splice variants of AR without LBD and other excised domains have been identified, rendering the requirement for ligand binding to AR dispensable (38-39). Thus, targeting the activated AR with flutamide, nilutamide, and bicalutamide AR antagonists to inhibit the constant ligand-independent activation has been a first-line therapy (39-42). Currently, pre-clinical evidence also suggests that targeting the AR NTD may be a new approach in treating PCa patients with AR variants lacking LBD (43,44). AR antagonists should effectively inhibit AR transcriptional activity in PCa and reduces the expression of AR target genes (45,46). However, the heterogeneous mixture of AR splice variants in PCa patients and the presence of AR splice variants sensitized to AR antagonists (discussed further in section 1.3.2.1) can render the ADT treatment ineffective (47,48). In such case, the regrowth of PCa is promoted and the disease can recur as a highly metastatic castration-resistant prostate cancer.  24  1.3 Prostate Cancer and CRPC When PCa progresses to castration resistance, AR activation is maintained through a variety of mechanisms including AR amplification, gain-of-function mutation, and ligand-independent AR activation (49). However, non-castration resistant PCa may also exhibit AR mutations (49). To determine the characteristics of CRPC state and exact mechanisms that cause PCa to progress to a castration resistance, molecular mechanisms of PCa initiation and progression have been investigated by multiple groups to find mechanistical causes that lead to CRPC. CRPC state has been correlated with a loss allele copy number of phosphatase and tensin homolog (PTEN), a tumor suppressor gene, and a reduction in the PTEN protein activity (49). Also, simultaneous activation of Akt/mTOR and MAPK signaling pathways, responsible for cell proliferation, has been shown to promote castration resistance in prostate cancer cell lines and mouse models (49). Another proposed mechanism through which PCa progresses to castration resistance is via intratumoral de novo steroidogenesis. Previously, CRPC has been known as an androgen independent prostate cancer due to its ability to grow and metastasize in an environment deprived of the androgen growth stimuli (15,49,50). Contrary to this belief, recent findings by multiple groups have shown that CRPC cells have the ability to produce their own androgens intracellularly and are able to sustain growth via AR activation without having to rely on exogenous androgen supply – known as intratumoral de novo steroidogenesis (51-55). The hypothesis of de novo steroidogenesis was also supported through the findings in an epidemiological study in which the androgen levels within metastatic tumors of castrated men were found to be higher than the levels within the primary prostate cancer tumors in untreated men (54). In addition, CRPC tumors are shown to continuously express the necessary enzymes involved in the synthesis of androgens intracellularly (Figure 1.1); in vitro studies have shown 25  an increased expression of HSD3B1, CYP17A1, and AKR1C3 enzymes in CRPC state compared to benign and androgen-sensitive prostate cancer states (52,53).   1.3.1 Experimental models 1.3.1.1 In Vitro Model In order to determine the disease characteristics and mechanisms through which PCa progress to CRPC, various cell lines including LNCaP, C4-2, C4-2B, DU145, PC-3, DuCaP, and VCaP have been cultured to represent various stages of PCa for in vitro research (56-60). One of the most commonly used cell lines in PCa research is the androgen-sensitive LNCaP cells derived from lymph node metastases. With a doubling time of 60 hours, LNCaP cells closely mimic the PCa disease state by expressing AR-mRNA and protein, as well as PSA (56,57). In addition, LNCaP cells possess a T877A point mutation in the AR that causes the replacement of a threonine with an alanine amino acid at position 877. T877A-mutant AR is able to bind to other ligands including flutamide, estradiol, and progesterone; therefore, even in the presence of low testosterone and DHT concentrations, the T877A-mutant AR is activated and promotes cell proliferation (61). Other characteristics of LNCaP cells including mutations in the phosphatase and tensin homolog (PTEN) tumor suppressor gene, reduction in apoptosis mediated by p53 activation, and presence of insulin-like growth factor 1 receptor (IGF-1R), which mediates tumor cell growth, adhesion, and evasion from apoptosis (63). A lineage-derived second generation subline of LNCaP cells, C4-2, has been derived by subcutaneous co-inoculation of LNCaP cells and human osteosarcoma cells in a castrated mouse, resulting in a castration-resistant prostate cancer cell line (64). With a shorter doubling time of 40 hours, C4-2 cells also express T877A-AR and PSA similar to LNCaP cells (64,65). Since the establishment of the C4-2 cell line, recent 26  CRPC studies have commonly used C4-2 and LNCaP cell lines to compare the castration resistance to androgen sensitive state as these two cell lines share same lineage and express AR-mRNA and PSA protein (64-65). The co-inoculation of LNCaP cells with osteosarcoma cells supports the need of a cell line that reflects the tendency of PCa to metastasize to bone tissue, and allows the investigation of the effect of bone stromal environment on the progression to the castration-resistant state (65,66). The clinical treatment for patients with metastatic bone disease normally involves an arduous process and can result in multiple skeletal complications, significantly impairing the patient’s quality of life even more (67). In order to investigate the bone metastatic disease originating from PCa, C4-2 cells were injected into castrated mice and resulting C4-2B osseous tumor cells were collected (58). C4-2B cells are commonly used in to examine the mechanism through which PCa produce osteoblastic lesions and mineralization (58,66). DU145 and PC-3 cells are also common in vitro models for CRPC studies, derived from brain and bone metastases respectively (68-70). However, AR mechanism studies are limited in these cells as they do not express AR and PSA. In order to assess the AR mechanisms, DuCaP and VCaP cell lines expressing wild type AR, derived from dura mater and vertebral metastases respectively, are commonly used as in PCa studies (59,60).  1.3.1.2 In Vivo Model In addition to cell models, immunodeficient and genetically engineered mouse models have been used to mimic the changes observed in PCa and to investigate the mechanisms of the altered genetic pathways (71). These animal models have the benefit of having genomes that are very similar to humans as well as the benefit of being able to modify their genetics easily. However, due to the differences in prostate anatomy and drug pharmacokinetics between the two species 27  there are always limitations in extrapolating the findings from mice model studies to humans (71).   A common in vivo model is a xenograft model that has been developed by inoculating PCa cells into immunodeficient mice. Various types of immunodeficient mice, which are unable to mount an immune response to the foreign human tissue, allow the human PCa cells to grow and progress into castration resistant state in the animal. Although human PCa cells would be able to proliferate in the immunocompromised mice, lack of normal immune response to tumor cells due to dysfunctional immune system may limit the accuracy of PCa progression in humans. Other than the immunodeficient mouse models, genetically engineered mouse models (GEMMs) allow the mouse to carry genetic modifications present in human PCa tumors (71). Some examples of GEMM models include C3-Tag, FG-Tag, and TRAMP genetic modifications to induce the expression of SV40 tumor antigens and fetal globin-γ (71). While these induction studies help to characterize different forms of PCa, GEMMs studies are limited because aggressive forms of PCa and CRPC likely result from multiple genetic pathways (71).  1.3.2 Current Treatments Standard treatment for patients diagnosed with PCa includes surgery for those in good health with localized tumor, radiation therapy with high-energy x-rays, hormone therapy to remove hormones or block their action and stop cancer cells from growing, and chemotherapy with drugs to kill cancer cells or stop cell division (72). Despite the initial response to treatments, due to drug resistance, AR mutations, and de novo steroidogenesis, prostate cancer that regrows as CRPC has poor prognosis with median survival of less than 2 years (72-74). Due to the 28  importance of AR activation in the proliferation of prostate cells and tumor cells, development of novel treatments that target various pathways of androgen-AR axis including androgen synthesis, AR activation, and AR translocation is ongoing. In recent years, there have been advances in developing new treatment options for managing CRPC patients including FDA-approved use of abiraterone, a CYP17A1 inhibitor, and enzalutamide an AR antagonist (75-83). Also in the last three years, AR-independent therapies including radium-223 and cabazitaxel have shown to improve the survival of CRPC patients (84-86).   1.3.2.1 AR-targeting Treatments Abiraterone sold under the trade name ZytigaTM (Johnson & Johnson) is an inhibitor of CYP17A1, which is an enzyme that catalyzes two sequential reactions: the conversion of pregnenolone and progesterone into their 17-α-hydroxy derivatives, and the formation of dehydroepiandrosterone (DHEA) and androstenedione respectively (81,87). Thus, abiraterone elicits anti-tumor effects by limiting the precursor molecules involved in the synthesis of testosterone and DHT that can bind to the AR (81). Phase III clinical trials have shown an overall increase in survival by 4 months in CRPC patients given both abiraterone with prednisone compared to the placebo group treated with only prednisone (87). Although abiraterone was approved by the FDA in 2011 for the treatment of CRPC, resistance to the drug induced by AR splice variants and presence of multiple adverse effects including urinary tract infection, hypertension, hypokalemia, and diarrhea have been observed in a few patient cases (83,87).  Enzalutamide (XtandiTM) developed by Medivation pharmaceutical company is an AR antagonist with a binding affinity that is five-fold greater than that of conventional AR antagonist 29  bicalutamide (88). Upon binding, it prevents the nuclear translocation of AR and the binding of AR to coactivator proteins (88). In vitro work around enzalutamide has shown decreased expression of PSA and induction of apoptosis in multiple PCa models (88). Also, clinical trials have shown greater than 50% reduction in PSA levels in 62% of chemotherapy-naïve patients and in 50% of chemotherapy-treated patients (89, 90) and an increase in the survival of CRPC patients by approximately 5 months compared to those assigned to the placebo group (91). In 2012, enzalutamide was approved by the FDA for the treatment of CRPC. A limitation of enzalutamide can be hypothesized from observations seen in patients treated with its predecessor AR antagonist drugs such as bicalutamide and flutamide. In the past, it has been shown that these drugs have been rendered ineffective in treating CRPC due to drug resistance and a switch in the function of these drugs from acting as AR antagonist to AR agonist (40, 41, 80, 88). Therefore, in CRPC patients with increased AR expression, these AR agonists bind to the AR and induce a change in the LBD (80, 88). Alteration in the LBD further promotes AR mutation, AR expression and various cofactor expressions (80,88). While enzalutamide significantly improves the survival period of CRPC patients by 4 months, similar to its predecessors, a group of CRPC patients showing resistance to enzalutamide has already been discovered (92).    1.3.2.2 AR-independent Treatments In addition to abiraterone and enzalutamide, treatments independent of the AR pathway have also been approved for the treatment of CRPC. Radium-233 dichloride is a novel treatment agent that specifically targets the minerals in the bone and delivers radiation directly to bone tumors while minimizing the damage to surrounding normal tissues (93). Radium-233 emits α-radiation upon uptake, which results in DNA breakage and apoptosis of bone-metastasized PCa cells (93). 30  In Phase III trials where patients were treated once a month by an intravenous injection, an increase in the median overall survival of 3 months was observed in the test patients compared to patients on a control standard treatment (94). Although common adverse effects included nausea, diarrhea, vomiting, and leg swelling, in 2013 FDA approved the use of Radium-233 for treating CRPC that has metastasized to the bone due to the ability of therapy to specifically target the bone tissue and minerals (94). Another AR-independent therapy that was approved by the FDA in 2010 is cabazitaxel, a microtubule inhibitor. Clinically, a combination of cabazitaxel and prednisone is administered to patients who no longer respond to docetaxel-based regimen (84,85,95).  1.3.2.3 Implications of Current Treatments Despite the improvements in the median survival of CRPC patients treated with the approved novel therapies, the investigation into novel targets must continue as there is a definite need for stable therapies with longer survival benefits. The discovery of de novo steroidogenesis as briefly described in the introduction of section 1.3 has placed a focus on targeting CRPC through the androgen synthesis pathways. Due to the limitations of the current drugs that target various pathways in the androgen synthesis pathways, upstream reactions of the androgen synthesis pathways involving cholesterol molecule have also been investigated in PCa models as a potential therapeutic target in treating CRPC.   31  1.4 Cellular Cholesterol Homeostasis Cholesterol metabolism is comprised of complex and multiple regulatory pathways as cholesterol is one of the major components of membrane lipids. It also influences cellular signaling and is a precursor for bile and androgen production. Thus, regulation of cholesterol balance is crucial in the maintenance of cellular homeostasis (96). Cells obtain cholesterol from two major sources: exogenous dietary and endogenously synthesized supplies. Figure 1.3 illustrates the main pathways involved in cellular cholesterol homeostasis.   Expression of enzymes involved in cellular cholesterol homeostasis is regulated by sterols and Sterol Regulatory Element-Binding Proteins (SREBPs). SREBPs are transcription factors that bind to sterol response element (SRE) in the DNA and induce the transcription of enzymes responsible for cholesterol biosynthesis and uptake (97). In the presence of high cholesterol content, inactivated SREBPs bound to SREBP cleave activating protein (SCAP) can be found on the nuclear membrane and the ER membranes. When sterol levels are low, insulin-induced gene proteins (INSIGs) on ER membrane protein no longer bind to SCAP, and the SREBP: SCAP complex is transported from the ER to the Golgi apparatus where site-1 protease (S1P) and site-2 protease (S2P) cleave the complex, releasing the cytoplasmic component of SREBP. The component then translocates to the nucleus to activate the transcription of target genes containing SRE that will induce sterol synthesis and uptake (97). While normal physiological cholesterol metabolic pathways are tightly regulated through the SREBP pathway and other regulatory proteins, it has been shown that many processes involved in cholesterol regulation are modified in PCa and CRPC, possibly contributing to the observed de novo steroidogenesis and to a 32  constant unregulated supply of cholesterol to meet cellular requirements for tumor growth (98,99).  Figure 1.3: Diagram of cellular cholesterol (     ) homeostasis including influx, metabolism, synthesis, and efflux pathways. Cholesterol inhibits the transport of SREBP-SCAP (          ) complex from the ER. In the presence of low cholesterol concentration, SREBP (      ) would be cleaved from SCAP. SREBP translocates to the nucleus and up-regulates proteins involved in cholesterol uptake and synthesis. Cholesterol can move to mitochondria to initiate androgen (DHT) synthesis.     33  1.4.1 Exogenous Cholesterol Supply and Uptake Exogenous cholesterol supply is provided by dietary consumptions of meat and dairy products.  In the past, multiple epidemiological studies have been conducted to examine the relationship between dietary cholesterol intake and the risk of PCa (100-102). While many studies reported that low serum cholesterol was associated with lower risk of developing PCa, a few studies contradicted this finding by concluding that cholesterol intake was inversely associated with PCa and that low HDL-cholesterol was associated to increased PCa risk (100-102). Although the results are inconclusive on whether the dietary cholesterol and which dietary cholesterol has an effect on PCa, patients with advanced PCa or CRPC commonly adhere to a diet plan that avoids excess cholesterol consumption and limits cholesterol supply from an exogenous source (103,104). Thus, targeting the dysfunction in cholesterol regulation at cellular level may be more appropriate. Consumed cholesterol is transported within lipoproteins, mainly high-density lipoproteins (HDL) and low-density lipoproteins (LDL). The contents of these lipoproteins are taken up by the cells via lipoprotein influx membrane transporters such as scavenger receptor class B type I (SR-BI), low density lipoprotein receptor-related protein1 (LRP1), and low density lipoprotein receptor (LDLr).  Scavenger receptor class B type I (SR-BI) is an 82-kDa integral membrane protein found throughout the body, but mostly concentrated in the liver, adrenal gland, and other steroidogenic tissues (105). While it can interact with a variety of ligands including anionic phospholipids, LDL, and VLDL, SR-BI interacts with HDL with the highest affinity (105). Even among HDL particles, SR-BI binds with greater affinity to apoA-I/II and cholesterol-ester enriched α-HDL particles than pre-β-HDL particles (105,106). In the liver, this selective lipid uptake drives the 34  reverse cholesterol transport, a movement of cholesterol from peripheral tissues to liver for excretion, and acts as a protective mechanism against the development of atherosclerosis and cardiovascular disease (105). Previously in PCa models, it has been shown that SR-BI expression was significantly increased in castration-resistant cells compared to androgen-sensitive cells, and down-regulation of SR-BI resulted in significant decrease in the cell viability and PSA secretion in the castration-resistant cells (107,108). These observations suggest that SR-BI and its modifications may play an important role in the regulation and growth of CRPC. Commercially available SR-BI drug inhibitors such as Block Lipid Transporter-1 (BLT-1) and its derivatives are available for research use; however, there are no FDA approved chemical SR-BI antagonists for the treatment of CRPC (106).  Low density lipoprotein receptor-related protein 1 (LRP1), also known as apolipoprotein E receptor, is another membrane protein involved in receptor-mediated endocytosis of cholesterol in mainly apoE-rich intermediate-density lipoprotein (IDL) particles. Due to the fast turn-over of IDL particles into LDL in circulation and in the liver, LRP1 has a minor role in the uptake of cholesterol in steroidogenic tissues (109).  Low-density lipoprotein receptor (LDLr) mainly interacts with LDL as it recognizes the apolipoprotein B100 embedded in LDL particles; it can also interact with intermediate-density lipoprotein (IDL) particles with apoE protein (110). Cholesterol uptake through LDLr occurs through clathrin-mediated endocytosis of LDL particles. The expression of LDLr is mediated by sterols or sterol-containing LDL; excess sterols inhibit the activation of SREBPs and down-regulates the expression of LDLr in normal tissue (111,112). In normal prostate cells, presence 35  of exogenous LDL and cholesterol decreased the expression of LDLr. However, in PCa cells, despite the presence of exogenous LDL and cholesterol, LDLr expression was not down-regulated, suggesting that a continuous influx of cholesterol may be an important function in PCa cells (112).  1.4.2 Storage of Intracellular Cholesterol Once inside the cell, cholesterol is converted into cholesteryl esters (CE) facilitated by the acetyl-coA acyltransferase (ACAT) enzyme on the membrane of endoplasmic reticulum (ER), and these are stored in lipid droplets due to pro-apoptotic, toxic characteristics of free cholesterol in the cytosol (113). There are two ACAT genes found in humans: ACAT-1 and ACAT-2 (113). Structurally, the two isoforms differ in their N-terminus and number of transmembrane domains. While ACAT-1 controls the conversion of cholesterol in the brain, kidney, adrenal glands, and macrophages under the regulation of SREBPs and STAT1α, ACAT-2 regulates the process in the liver and intestine and is regulated by cholesterol concentration (114). Both isoforms have been detected in castration-resistant cell lines (114). Upon the progression of an LNCaP xenograft model to the CRPC state, a significant reduction in ACAT-2 expression was observed, suggesting that alterations in the cholesterol storage processes may play a role in the survival of CRPC cells (53).  When the cell requires cholesterol supply, stored cholesteryl esters are hydrolyzed back into free cholesterol by the hormone sensitive lipase (HSL) enzyme. Similar to ACAT, HSL exists in two isoforms (115). The long form is expressed in steroidogenic tissues such as testes and the adrenal gland, and is responsible for the conversion of cholesteryl esters to free cholesterol for steroid 36  hormone production (115). In adipose tissue, the short form of HSL hydrolyzes stored triglycerides to free fatty acids (115). Hormones such as epinephrine, catecholamines, and ACTH can activate the HSL enzyme activity whereas insulin has been observed to inhibit HSL activity in adipose tissues (115). An in vitro study looking at cholesterol metabolism in the androgen-sensitive LNCaP cell line and the castration-resistant C4-2 cell line has shown an increased expression of HSL protein in C4-2 cells (106,107). This observation supports de novo steroidogenesis in castration-resistant cells where greater free cholesterol supply may be needed to produce androgens intracellularly for tumor proliferation (106,107).  1.4.3 Efflux of Intracellular Cholesterol In the presence of an abundant intracellular cholesterol concentration, in addition to ACAT activation, efflux pathways are activated to remove cholesterol from the cell. The major cholesterol efflux transporters belong to the ATP-binding cassette transporter superfamily (ABCs) with ABC-A and ABC-G major families. 200 kDa ABCA1 is expressed ubiquitously and in higher concentration in tissues that regulate lipid content such as the liver, small intestine, and adipose tissue (116). It mediates the efflux of cholesterol to lipid-poor apolipoproteins such as apo-A1 and apoE (116). In PCa cells, as well as intermediate- to high-grade radical prostatectomy specimens, CpG island hypermethylation at the ABCA1 promoter region was observed; hypermethylation of ABCA1 renders the promoter to be unresponsive to transactivation, resulting in elevated levels of cholesterol in the PCa cells (117,118). This suggests that high intracellular cholesterol levels may contribute to tumor progression and proliferation. Similar to ABCA1, ABCG1 transporter is found throughout the body whereas ABCG4 is localized to the central nervous system (119,120). Contrary to ABCA1, these two 37  efflux transporters mediate the transfer of cellular cholesterol to HDL particles but not to lipid-poor apolipoproteins (119,120).  1.4.4 Synthesis of Intracellular de novo Cholesterol Intracellularly, cholesterol is synthesized from acetyl-CoA in the endoplasmic reticulum through the mevalonate pathway, in which the rate-limiting step is the conversion of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) into mevalonate, mediated by 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) (96,113). Mevalonate further undergoes multiple reactions to be converted into cholesterol downstream. HMGCR, which share similar structure in mammals, is a localized to the membrane of the ER with the major site of cholesterol synthesis is in the liver and intestine (121,122). The 118 kDa HMGCR is commonly depicted as having two domains as shown in Figure 1.4: trans-membranous N-terminal domain and cytosolic C-terminal domain (121). The N-terminal domain consists of eight membrane-spanning segments that anchor the enzyme to the ER membrane, whereas the C-terminal domain contains the active site. HMGCR is one of the most highly regulated enzymes in the body; the transcription rate of HMGCR mRNA is regulated by the SREBP and the translation rate of HMGCR mRNA is regulated by intracellular non-sterol metabolites and cholesterol levels through a negative feedback loop (122). In the presence of an elevated cholesterol level, HMGCR is degraded or inactivated to maintain cholesterol homeostasis. The degradation of HMGCR is mediated by ER-associated signals through which the cytosolic and membrane domains of HMGCR undergo conformational changes to induce proteolysis (122). The inactivation of the enzyme is mediated by cAMP-activated protein kinases that phosphorylate the enzyme and renders it inactive (121,122). In addition to the physiological regulation of this 38  enzyme, a class of drugs known as statins has been successfully used to target the human HMGCR; the competitive inhibition of HMGCR induces the expression of LDLr in the liver, lowering circulating LDL and serum cholesterol levels (123,124).   Figure 1.4: Domain structure of HMGCR. HMGCR is anchored in the memrane of the ER with eight transmembrane domains (121). The catalytic domain of the enzyme faces the cytosol and reduces HMG-CoA to mevalonate. Permission granted for reproduction of this image by the Nature Publishing Group.  1.4.4.1 Statins As briefly described in the previous section, statins are competitive chemical inhibitors of HMGCR. Since the early 1970s, a lipid hypothesis was described as a correlation between increased cholesterol and increased cardiovascular disease (CVD) risk (125). Since then, accumulation of research evidence has supported the acceptance of the lipid hypothesis as a scientific fact by majority of researchers. With the discovery of cholesterol synthesis being driven by the HMGCR, several groups were successful in isolating inhibitors of HMGCR from 39  fungal agents (126,127). While the first statin ever isolated, mevastatin, was never marketed due to severe adverse effects including muscle deterioration and death, lovastatin became the first statin to be marketed for clinical use by Merck & Co in the late 1980s (126,127).  Statins are commonly used as secondary prevention therapies in patients with pre-existing cardiovascular diseases to achieve low serum LDL and total cholesterol profile (128). Some of the common adverse effects include liver and kidney toxicities with rare occurrences of on-target muscle toxicity including rhabdomyolysis and myositis (129,130). The coprescription of contraindicated drugs not compatible with statins such as fibrates, macrolides, and niacin should be intervened to ensure that the risk of adverse effects is avoided (131). Statins primarily function by competitively inhibiting HMGCR, as statins share structural similarities to HMG-CoA. Also, in response to the decreased cellular cholesterol synthesis, statins can up-regulate LDLr, which increases LDL-cholesterol uptake and reduces serum cholesterol levels (124). Lastly, statins are well known to exert cholesterol-independent effects in improving endothelial function, maintaining plaque stability, preventing thrombus formulation, and stimulating inflammatory responses (132). Due to the significant impact these effects could have on various disease conditions, the potential benefits from the use of statins to treat cholesterol-independent diseases has also been explored; patients with elevated C-reactive protein without elevated cholesterol or heart problems treated with rosuvastatin showed benefit in the reduction of vascular events (133). The effects of statins on various cancers have also been explored as well, including breast, colorectal, and lung cancer, often with mixed results between various studies (134-136). A prospective study over an average follow-up of 6.7 years looked at statin use and breast cancer incidence (135). While overall statin use was not associated with invasive breast 40  cancer incidence, the study concluded that use of hydrophobic statins such as simvastatin and lovastatin may be associated with lower breast cancer incidence, suggesting possible within-class differences (135). A large cohort study with a mean follow-up of 2.9 years compared incidence rates of colorectal, lung, and breast cancers in elderly statin users and glaucoma-medication users with mean age of 76 and 80 respectively; the findings indicate that it is unlikely that statin use decrease or increase the risk of colorectal, lung, or breast cancer (134). However, the study did not separate different statin users into separate cohorts, but rather grouped all statin users in a single cohort (134). As described in the earlier prospective study, statins may exert within-class differences as they differ in their structure, metabolism, and potency (135). Therefore, correlating cancer incidence rates to overall statin use may not accurately represent the effect of individual statin drugs on cancer incidence rates.    Currently, numerous statins are commercially available for treatment use with the most popular ones being atorvastatin, lovastatin, pravastatin, rosuvastatin, and simvastatin (128). These statins differ in their structure, origin, enzymes involved in metabolic pathways, as well as potency, which is described as the average decrease in serum LDL for a specific dose. Currently, simvastatin has one of the highest serum LDL-reduction potency with 29% serum LDL reduction with a 10 mg dose compared to 22%, 21% with 10 mg of pravastatin and lovastatin respectively (125). In addition, brand and generic simvastatin are more affordable compared to other statin drugs: $20 for 20 mg SV compared to $171 for 5 mg rosuvastatin (125).   41  1.4.4.2 Previous Research Due to the importance of androgens in regulating PCa growth and cholesterol being a common precursor molecule in androgen synthesis pathways, the effect of statins in various PCa models have been widely investigated. In the past, a correlation between statin users and a reduction in risk of aggressive prostate cancer state was reported and has been paired with decreased serum androgens (137-140). Other studies have concluded that statin use is not associated with prostate cancer risk and that the use of statins does not provide therapeutic benefits to PCa patients compared to placebo (141-142). The mix of results from epidemiological studies has limited the potential use of statins as treatment for PCa patients, and has grounded statin research in PCa as a well-worn topic, rendering further statin research in PCa models unintriguing. However it is important to recognize that these studies encompass a large variety of PCa patients, and the significance of statin use may only be applicable in PCa states where cholesterol dysregulation plays a role in driving tumor growth through de novo steroidogenesis.   Although in an androgen-castrated environment, CRPC cells would be able to survive and proliferate through de novo steroidogenesis pathways. This idea was supported by observations from epidemiological studies in which serum testosterone levels within metastatic tumors of castrated men were found to be significantly higher than those within primary prostate cancer tumors in untreated men (54,55). In addition, CRPC cells were shown to continuously express enzymes involved in androgen synthesis and cellular cholesterol regulation, and in fact many of the proteins were modified in various PCa stages to maintain the supply and synthesis of intracellular cholesterol as described in previous sections; SR-BI influx transporter expression was significantly increased in CRPC cell line compared to androgen-sensitive cell line. When the 42  SR-BI expression was down-regulated up to 80% compared to basal expression using siRNA, a significant increase in HMGCR activity was observed (Figure 1.5A) together with decrease in cell viability and PSA secretion in the castration-resistant C4-2 cell line (107). Similarly in a xenograft LNCaP mouse model (Figure 1.6B), when androgen-dependent (AD) tumors progressed to a castration-resistant state, newly synthesized cholesterol expressed as fold change from AD nearly doubled, suggesting a role of HMGCR in the development of the castration-resistant state (53).     Figure 1.5: (A) Increase in HMGCR activity measured in SR-BI knocked-down C4-2 cells 6 days post-siRNA transfection; n = 3 *, p <0.05 SRBI-KD vs NC (modified from (107)) (B) Increase in HMGCR activity measured in CRPC state that has progressed from LNCaP xenograft model; n = 3 *, p <0.05 SRBI-KD vs AD and N (53). Permission granted for reproduction of these images by John Wiley and Sons)  Although the use of statins have been extensively studied in various PCa models and studies have looked at cholesterol metabolism in CRPC state, very little research has looked at the effect of statins on established CRPC models (73,143).  One epidemiological study in 2010 showed improvement in prevention of biochemical failure upon ADT in CRPC patients treated with A B 43  statins (139). However, these patients were on statins prior to developing CRPC and the results of the study were limited in making an inference regarding the use and effect of statin as primary therapy after the recurrence of PCa as CRPC is observed (139). An in vitro study that looked at the effect of SV in established castration-resistant PC-3 cells concluded that inhibition of cholesterol synthesis via statin prevents cell proliferation by inhibiting proliferation through a reduction in nuclear factor-κB (NF- κB) activity (143). However there were limitations to the studies regarding the use of cell lines that do not express PSA and AR, and the lack of cholesterol metabolism data. Thus, studies that examine the effect of statin in appropriate CRPC models and which that explore the cholesterol and androgen metabolism pathways are much needed to come to any conclusions.  1.5 Research Hypothesis Intracellular cholesterol synthesis feeding into the de novo steroidogenesis pathway plays a contributing role in the proliferation of castration-resistant prostate cancer cells; therefore, the inhibition of HMGCR using simvastatin will decrease intracellular cholesterol concentration, testosterone concentration, and viability in castration-resistant C4-2 cells.     44  1.6  Aims of the Thesis The overall aim of this thesis was to study the contribution of HMGCR, the rate-limiting enzyme in the intracellular cholesterol synthesis pathway, to the viability of castration-resistant C4-2 cells. The specific aims were to: 1) To examine the effect of different concentrations of simvastatin on viability of C4-2 cells 2) To assess the effect of simvastatin on cholesterol synthesis of C4-2 cells 3) To determine the effect of simvastatin on intracellular testosterone levels and PSA secretion of C4-2 cells  1.7 Rationale and Significance As discussed in the sections above, very few studies have investigated the cholesterol regulation pathways for de novo steroidogenesis in the castration-resistant state. While the role of modified expression of SR-BI influx transporter has been studied in a castration resistant prostate cancer cell model, the HMGCR responsible for cellular cholesterol synthesis has not been thoroughly examined in CRPC models with regard to its potential contribution to de novo steroidogenesis despite the evidence that the activity of enzyme is modified in castration-resistant states. Thus, this study sought to determine the effect of inhibiting HMGCR with simvastatin in the CRPC cell model. The results of this thesis may indicate the possibility of a new CRPC treatment regimen and could have important clinical implications if the results in this thesis support the research hypothesis as statins are commonly accessible and administered as inhibitors of HMGCR. 45  Chapter 2: Materials and Methods  2.1   Materials and Reagents Poly-L-lysine (0.01% solution), cholesterol, Triton® X-100, dimethyl sulphoxide (DMSO), simvastatin (SV), phenylmethylsulfonyl fluoride (PMSF), protease inhibitor cocktail, Trizma®-hydrochloride, Trizma® base, glycine, lyophilized bovine serum albumin (BSA), ammonium persulfate, tetramethylethylenediamine, sodium hydroxide, sodium chloride, sodium deoxycholate, nonyl phenoxypolyethenyl ethanol (NP-40), β-mercaptoethanol, Kodak® processing chemicals for autoradiography films, and Kodak® fixer and replenisher were purchased from Sigma-Aldrich (St. Louis, MO, USA). Classic single-emulsion autoradiography films were purchased from Mandel Scientific (St. Louise, MO, USA). Acetic acid [1-14C] was obtained from American Radiolabeled Chemicals, Inc. (Saint Louis, MO, USA) and cholesterol [1, 2-3H(N)] was purchsed from PerkinElmer Inc. (Waltham, MA, USA). Scintillation fluid was purchased from MP Biomedicals (Solon OH, USA). RPMI-1640 without phenol red, Hank’s balanced salt solution (HBSS), 0.25% trypsin-EDTA,  penicillin-streptomycin (5,000U/mL), fetal bovine serum (FBS), charcoal-stripped fetal bovine serum (CSS), were purchased from Invitrogen (Life Technologies, Invitrogen, Burlington, Ontario). Chloroform, ethyl acetate, hexanes, ethanol, methanol, isopropanol, and PageRuler Plus Prestrained Protein Ladder were purchased from Fisher Scientific (Waltham, MA, USA). Bio-Safe Coomassie G-250 Stain, Ready Gel® Tris-HCl Precast Gels, and 4X Laemmli buffer were purchased from Bio-Rad Laboratories (Hercules, CA, USA). SuperSubstrate® West Pico Solution was obtained from Thermo Scientific (Rockford IL, USA).  46  2.2 Cell Culture C4-2 and LNCaP cells were obtained from the Prostate Centre at Vancouver General Hospital. LNCaP cells were used for experiments between the passage numbers of 42-52. Both cell lines were cultured in RPMI-1640 medium without phenol red containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37 °C in 5% CO2 environment. Seventy-two (72) hours prior to the 48-hour simvastatin incubation, the cells were seeded in poly-L-lysine coated plates in 10% fetal bovine serum supplemented RPMI medium. After the initial 24 hours, the medium was replaced with RPMI medium supplemented with 5% CSS with restricted androgens to mimic an androgen-castrated environment. Cells were seeded in plates to include the necessary blank, vehicle (0.5% DMSO), SV treatments, no treatment (NT) and 100% cell lysis Triton-X 1% (TX 1%) controls.  2.3 Simvastatin Preparation SV powder (25 mg) was dissolved in filtered 2.9864 mL of DMSO to yield a 20 mM solution. As shown in Table 2.1, stock solutions were prepared from the 20 mM solution in order to achieve a final concentration of 0.5% DMSO (v/v) in each of the wells treated with different concentrations of SV. The stock solutions were aliquoted into microcentrifuge tubes and stored in 4 ℃ until use. A range of SV concentrations from 10-100 μM were tested using cell viability and toxicity assays to determine if there is a concentration-dependent effect and to determine the appropriate concentrations for cholesterol and androgen synthesis experiments.   47  Table 2.1. Preparation of simvastatin stock solutions. Individual stock solutions were prepared to achieve a final concentration of 0.5% DMSO (v/v) in the wells. Amount of 20 mM SV (prepared in DMSO) added (μL) Amount of DMSO (μL) Stock SV Solution Concentration (mM) Final SV Concentration (μM; 1:200 dilution in media) 50 450 2 10 100 400 4 20 200 300 8 40 250 250 10 50 300 200 12 60 375 225 15 75 400 100 16 80 500 0 20 100  2.4 Protein Quantification Protein quantification was performed using a DC Protein Assay (BioRad) to calculate the protein concentration for gel electrophoresis loading. The quantification was also done to normalize data to the amount of cells present in the wells. After the removal of SV, cells in the culture plates were rinsed twice with HBSS and were lysed on ice with cold modified RIPA buffer containing 1% PMSF and protease inhibitor cocktail for an hour. The cells were scraped off from the bottom and transferred into individual 1.5-mL microcentrifuge tubes. The tubes were centrifuged at 12,000 rpm for 20 minutes at 4 ℃ to remove the nuclear fraction. After the fractions have been separated, the supernatant was transferred to a new 1.5-mL microcentrifuge tube. BSA standards 48  in the range of 0-1 mg/mL were prepared by diluting 1 mg/mL BSA standard stock solution in distilled water. Supernatant (5 μL) from each sample and BSA standards (5 μL) were added in duplicates to a 96-well plate. Reaction mixture (25 μL) containing 50:1 Reagent A (alkaline copper tartrate): Reagent S (mild detergent) was added to each well followed by 200 μL of Reagent B (Folin reagent). The plate was incubated for 15 minutes in room temperature, after which the absorbance was measured at 650 nm using a Labsystems, Multiskan plate reader (Fisher Scientific). The protein concentration in each cell sample was extrapolated from the linear regression line of the BSA standard curve.   2.5 Cytotoxicity and Cell Viability Cells were plated in 96-well plates at a density of 8x103 cells per well on Day 0, 72 hours prior to SV treatment. Treatment and control wells were assigned in triplicate in each plate. Media (50 μL) was taken from above the cells following 48-hour SV treatment (Day 5) for the cytotoxicity assay. The remaining cells were rinsed with HBSS and the cell viability assay was performed.  2.5.1 LDH Assay The lactate dehydrogenase (LDH) assay was used to determine cytotoxic effects of the SV concentrations on C4-2 and LNCaP cells. The presence of LDH in the medium indicates poor cell membrane integrity and cell lysis. CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega) uses the conversion of a tetrazolium salt reagent into a formazan product in the presence of LDH. After the cells were treated with SV for 48 hours, 50 μL of medium was removed from each sample well and placed in a new 96-well plate. Substrate Mix (50 μL) was added to each well, and the plate was incubated for 30 minutes at room temperature, covered and 49  protected from light. After the incubation, 50 μL of the Stop Solution was added to each well and the absorbance was measured at 492 nm using a Labsystems, Multiskan plate reader (Fisher Scientific). A 100% cell death control was included by treating the cells with 1% Triton-X diluted in media at the same time as SV treatment. The background absorbance of the media was measured and subtracted from the sample measurements.  To decide the duration of SV incubation, LDH cytotoxicity assays were performed on media samples of C4-2 cells incubated with SV for 6, 24, and 48 hours as preliminary experiments to determine the effect of different concentrations of SV at various incubation time points (Appendix Figure A.1, Appendix Figure A.2, Figure 3.1 respectively).   2.5.2 MTS Assay MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay was used to determine the effect of different SV concentrations on cell viability. CellTitre 96® Aqueous One Solution Cell Proliferation MTS assay (Promega) measures the absorbance of the formazan product that is produced in the presence of NADH and NADPH in metabolically active cells. The cells remaining in the plates from the LDH assay were rinsed once with HBSS. A 50μL volume of 2:3 MTS reagent:media was added to the cells. The plate was protected from light and incubated for 2 hours at 37 ℃, after which the absorbance was measured at 492 nm using a Labsystems, Multiskan plate reader (Fisher Scientific). The background absorbance of the reagent mixture was measured and subtracted from the sample measurements. Protein determination as described in section 2.4 was performed to normalize the MTS values to the protein concentration in each well. 50  2.6 Growth Curve Cells were plated in 6-well plates at a density of 1.5x105 cells per well (Day 0). 24 hours after seeding, the media was switched to 5% CSS-RPMI. On Day 2, 48 hours post-seeding, cells were carefully detached from the plates using 0.25% Trypsin-EDTA, and the total cell count was calculated using a haemocytometer. The procedure was repeated on Day 3, before SV treatment at 72 hours post-seeding, after the removal of SV treatment on Day 5, and on Day 8, 72 hours after the removal of SV.  2.7 HMGCR Activity Assay C4-2 and LNCaP cells were seeded in 12-well plates at a density of 8 x 105 cells per well. The cells were grown according to the protocol described in section 2.2. Following SV treatment, the cells were washed twice with HBSS and incubated with 5% CSS-RPMI media containing 0.5 μCi of 14C-acetic acid and respective SV concentrations for 3 hours. Cells were carefully washed with HBSS three times following the incubation. Distilled water (500 μL) was added to each well and cells were scraped off carefully from the bottom surface. The distilled water containing detached cells was transferred to a 16 x 100mm glass tube. A volume of 10 μL of 1 μCi/mL 3H-cholsterol prepared in 100% ethanol was added to the tubes as an internal control for the lipid extraction procedure. The lipid content in the cells was extracted using the Bligh & Dyer extraction method (144): 3.75 mL of 2:1 chloroform:methanol, 1.25 mL of chloroform, and 1.25 mL of distilled water were added sequentially to the tubes, each followed by 1 minute of vortexing. Then the tubes were centrifuged at 425 g for 2 minutes. The upper aqueous layer was discarded and the lower layer containing the lipids was transferred to a clean 16 x 100 mm glass tube. The samples were dried under nitrogen gas and reconstituted in 100 μL of chloroform. The 51  chloroform samples were separated using thin layer chromatography (TLC) on silica gel plate in 200 mL of 9:1 hexane: ethyl acetate solution. A volume of 10 μL of cold cholesterol (10 mg/mL) and 3H-cholesterol samples were included onto the gel as controls. After 1.5 hours when the solution had reached the top edge, the plate was air-dried and exposed to iodine crystals that caused lipid spots to appear yellow. The yellow cholesterol bands located on the same plane as the cold cholesterol control band were excised and reconstituted in 6mL of scintillation counter fluid to be measured in a beta scintillation counter (Perkin Elmer). Distintegrations per minute (dpm) for both 14C and 3H were measured, and the values reflecting cholesterol synthesis for the samples were obtained by dividing 14C dpm by 3H dpm and normalizing to protein content in each cell sample.  To assess the residual effect of a high concentration of simvastatin on C4-2 cells on Day 8, the cells were grown according to the protocol described in section 2.2. Following the SV treatment, the cells were washed twice with HBSS and incubated with the 14C-acetic acid as described above. After 3 hours of incubation, the cells were carefully washed twice with HBSS and 1 mL of fresh 5% CSS-RPMI was added to each well. After 72 hours, the lipid content was extracted from the cells and measurements were read as described in the protocol above.  2.8 Western Blotting Whole cell lysates (25 μg protein per lane) were prepared in 4 X Laemmli buffer containing 5% v/v β-mercaptoethanol according to the protein concentration calculated in each sample as described in section 2.4. Six μL (6 μL) of PageRuler Plus Prestained Protein Ladder molecular weight marker and 50 μL of protein samples were loaded on to Ready Gel® Tris-HCl Precast 52  Gels (4-15% gradient gels). The Mini Transwell Protean system was run at a constant voltage of 100 V for approximately 75 minutes until the molecular weight marker band reached the bottom of the gels. The gels were removed from the cassette, rinsed with distilled water, and the separated protein bands were transferred onto the nitrocellulose membranes in 1 X Towbin’s buffer at a constant voltage of 100 V for 60 minutes. After the transfer, the gels were stained with Bio-Safe Coomassie G-250 Stain for 30 minutes to assess the transfer efficiency. The nitrocellulose membranes were cut at approximately 50 kDa for separate detection of HMGCR and β-actin protein bands. Membranes containing HMGCR band were blocked with 3:1 ratio of 3% milk (w/v) in Tris-buffered saline with 0.01% (v/v) Tween-T (TSB-T): 3% BSA (w/v) in TBS-T blocking buffer while β-actin containing membranes were blocked with 3% BSA (w/v) in TBS-T blocking buffer for 2 hours at room temperature. After blocking, the membranes were incubated with primary antibodies diluted in respective blocking buffers overnight at 4 ℃. The next day, the primary antibodies were removed, and the membranes were rinsed 4 times for 5 minutes with TBS-T. Respective secondary antibodies prepared in TBS-T were added onto the membranes for 1hour in room temperature. The specific dilutions and information of the primary and secondary antibodies used are outlined in Table 2.2. After the secondary antibody incubation, the membranes were washed 4 times for 5 minutes with TBS-T, and then treated with SuperSubstrate® West Pico Solution at 1 mL per 1 cm2 of membrane for 5 minutes. After 5 minutes, the chemiluminescent reagent was poured off and the membranes were developed in the darkroom by exposing the membrane onto an autoradiography film for 1 minute. The film was developed in Kodak® processing solution for 5 minutes, briefly rinsed in water, and fixed using Kodak® fixer and replenisher solution for 1 minute. The fixed film was scanned and the band density was analyzed using ImageJ® software. The HMGCR band was normalized to the β-actin 53  band in the same lane.   Table 2.2. Characteristics of primary and secondary antibodies used for the detection of HMGCR in C4-2 and LNCaP cells. Protein Molecular Weight Antibody Dilution Manufacturer HMGCR Primary 97 kDa Rabbit polyclonal 1:500 Millipore (ABS229 ) β-actin Primary 43 kDa Goat polyclonal 1:1000 Santa Cruz (sc-1616 ) Anti-rabbit IgG Secondary NA Goat anti-rabbit IgG-HRP 1:3000 Novus Biological (NB730-H ) Anti-goat Secondary NA Bovine anti-goat IgG-HRP 1:3000 Santa Cruz (sc-2378 )  2.9 Cholesterol Quantification C4-2 and LNCaP cells were seeded in 48-well plates at a density of 2 x 104 cells per well. The cells were grown according to the protocol described in section 2.2. Whole cell lysates were extracted from cells treated with SV using cold modified RIPA buffer as described in section 2.4. The intracellular cytosolic cholesterol content remaining in the supernatant was analyzed using Amplex®Red Cholesterol assay (Invitrogen). 50 μL of each lysate samples and cholesterol standards ranging from 0-8 μg/mL, were added in triplicate to a 96-well plate. Hydrogen peroxide (10 μM) was used as a positive control for the assay procedure. As described by the manufacturer’s instructions, 300 μM of Amplex Red reagent containing 2 U/mL horse radish 54  peroxidase, 2 U/mL cholesterol oxidase, 0.2 U/mL cholesterol esterase was prepared in 1 X reaction buffer. Amplex Red (50 μL) reagent was added to each well containing sample or standard. The plate was incubated for 30 minutes at 37 ℃, protected from light. The Amplex Red reagent reacts with hydrogen peroxide, produced during the oxidation of cholesterol into cholesterol oxidase, in a 1:1 stoichiometry to produce highly fluorescent resorufin. The fluorescence readings were measured using a SynergyTM HI Hybrid Multi-Mode Microplate Reader (BioTek Instruments) at excitation and emission wavelength of 530 nm and 590 nm respectively. The cholesterol concentration in each sample was extrapolated using the standard curve that was generated. The concentration results were normalized to protein concentration in each sample.  2.10 Prostate Specific Antigen Assay C4-2 and LNCaP cells were seeded in 48-well plates at a density of 2 x 104 cells per well. The cells were grown according to the protocol described in section 2.2. The amount of prostate specific antigen (PSA) secreted by the cells was quantified using an ELISA kit (ProClin. International). The standards and assay were prepared according to the manufacturer’s guidelines. A volume of 50 uL of reference standards (0-120 ng/mL) and sample media fractions were added in triplicate to the provided antibody-coated 96-well plate. Zero Buffer (50 μL) was added to the wells and incubated for an hour at room temperature. Following the incubation, the wells were rinsed five times with distilled water. Then, 100 μL of Enzyme Conjugate Reagent was added for an hour, followed by another set of rinsing with distilled water. TMB Reagent (100 μL)was added to the wells for 20 minutes, after which 100 μL of Stop Solution was added and mixed for 30 seconds. In the presence of PSA, the TMB molecule is converted into colored 55  tetralmethylbenzidine diimine; the absorbance was measured at 450 nm using a Labsystems, Multiskan plate reader (Fisher Scientific). PSA concentration in each sample was extrapolated using the standard curve that was generated. The concentration results were normalized to the protein concentration in each sample.  2.11 Testosterone Assay The cells were seeded in 6-well plates at a density of 1.5x105 cells per well and grown according to the protocol described in section 2.2. The intracellular testosterone concentration was measured using an ACETM competitive EIA kit (Cayman Chem.). After the removal of SV, 300 μL of distilled water was added to each well. The cells were gently scrapped off and transferred into thin-walled glass ampoules. In order to lyse the cells without using chemical reagents (as recommended by manufacturer), sonication was performed on the transferred samples with a bath-type Branson Sonifier 450 for 30 minutes at 25 ℃. A volume of 50 μL of sample and provided standards ranging from 0-0.5 ηg/mL were added in duplicate to the provided antibody-coated 96-well plate. In addition, wells were allocated for blank, total activity, non-specific binding, and maximum binding controls. Testosterone acetylcholinesterase (AChE) tracer and testosterone EIA antiserum (50 μL each) were added sequentially to the wells. The plate was covered with a plastic film and incubated for 2 hours at room temperature on an orbital shaker. After incubation, the plate was rinsed five times with the wash buffer followed by the addition of Ellman’s reagent (200 μL) to each well. The plate was incubated in the dark at room temperature for 75minutes on an orbital shaker after which the absorbance was measured at 410 nm using a Labsystems, Multiskan plate reader (Fisher Scientific). Testosterone concentration in each sample was extrapolated using the standard curve that was generated by plotting the data as logit 56  (B/Bo; sample of standard bound/ maximum bound) vs log concentration and performing a linear regression fit. Logit (B/Bo) can be calculated using the formula: logit (B/Bo) = ln [B/Bo/(1-B/Bo)] The final testosterone concentration results were normalized to the protein concentration in each sample.  2.12 Statistical Analyses The statistical analyses on all data were performed using SigmaStat 3.5 software. All data are presented as mean values of independent experiments ± standard error of the mean (SEM). Independent experiments represent assays conducted on different days under the same experimental conditions. Prior to statistical analysis, the significance criterion was set a priori at 0.05. Unpaired student’s t-tests were used to determine differences between mean values of basal HMGCR activity and PSA concentration of C4-2 and LNCaP cell lines. A one-way analysis of variance (ANOVA) was used to analyze differences in mean values of all other experimental parameters in each of the two cell lines separately. Tukey post-hoc test was used when the overall ANOVA result was significant.  57  Chapter 3: Results  3.1 Aim 1: Effect of Different Concentrations of SV on C4-2 and LNCaP Cells 3.1.1 Cytotoxicity of Simvastatin-Treated Cells The cytotoxicity of castration-resistant C4-2 and androgen-sensitive LNCaP cells following 48-hour SV treatment (Day 5) was assessed as described in section 2.5.1 to determine if there are any effects on the two cell lines over a range of SV concentrations. Results are expressed as values normalized to a 100% cell-death control in which untreated cells were lysed with a 1% Triton X-100 (TX 1%) solution. The mean % cytotoxicity measured at different SV concentrations was compared to vehicle control. The mean was found to be significantly different at SV concentration ≥ 60 μM in C4-2 cells (Figure 3.1).  The LD50 value of 60.2 μM was estimated using online sigmoidal curve fitting software (Zunzun®; Appendix Table B.1).     58   Figure 3.1: C4-2 cell cytotoxicity following 48-hour SV treatment (Day 5). Results are  demonstrated as % cytotoxicity based on a 100% cytotoxic control. The % cytotoxicity represents the amount of LDH in the media of C4-2 cells. Columns, mean (n = 6); bars, ± SEM. *, p < 0.05 vehicle vs 60 M, 75 M, 80 M, and 100 M SV.   In LNCaP cells, % cytotoxicity results expressed as mean values normalized to a 100% cell-death control were found to be significantly different at SV concentration ≥ 60 μM when compared to vehicle control (Figure 3.2). The estimated LD50 value of SV from a sigmoidal curve fit in LNCaP cells was 60.2 μM.  0102030405060708090100NT Vehicle 10μM 20μM 40μM 50μM 60μM 75μM 80μM 100μM TX1%% Cytotoxicity (Abs @ 492nm/ Abs of 100% cytotoxicity control) Treatment C4-2 * * * * 59   Figure 3.2: LNCaP cell cytotoxicity following 48-hour SV treatment (Day 5). Results are demonstrated as % cytotoxicity based on a 100% cytotoxic control. The % cytotoxicity represents the amount of LDH in the media of LNCaP cells. Columns, mean (n = 6); bars, ± SEM. *, p < 0.05 vehicle vs 60 M, 75 M, 80 M, and 100 M SV.   3.1.2 Cell Viability of Simvastatin-Treated Cells The viability of castration-resistant C4-2 and androgen-sensitive LNCaP cells following 48-hour SV treatment (Day 5) was assessed as described in section 2.5.2. The results expressed as values relative to NT control were normalized to protein levels (µg) in the respective cell samples. As shown in Figure 3.3, cellular viability significantly decreased at SV concentrations ≥ 75μM and in TX 1% control when compared to vehicle control. The estimated IC50 value of SV for C4-2 cells was 74.9 μM.  0102030405060708090100NT Vehicle 10μM 20μM 40μM 50μM 60μM 75μM 80μM 100μM TX1%% Cytotoxicity (Abs @ 492nm/ Abs of 100% cytotoxicity control) Treatment LNCaP 60   Figure 3.3: C4-2 cell viability following 48-hour SV treatment (Day 5). Results are demonstrated as values relative to vehicle control cell viability. Columns, mean (n = 5); bars, ± SEM. *, p < 0.05 vehicle vs 75 M, 80 M, 100 M SV and TX 1%.   In LNCaP cells, cell viability results were found to be significantly different at SV concentrations ≥ 75μM and in TX 1% control when compared to vehicle control (Figure 3.4). The estimated IC50 value of SV for C4-2 cells was 74.9 μM.  00.20.40.60.811.2NT Vehicle 10μM 20μM 40μM 50μM 60μM 75μM 80μM 100μM TX 1%Cell viability (relative to NT control) Treatment C4-2 61   Figure 3.4: LNCaP cell viability following 48-hour SV treatment (Day 5). Results are demonstrated as values relative to vehicle control cell viability. Columns, mean (n = 5); bars, ± SEM. *, p < 0.05 vehicle vs 75 M, 80 M, 100 M SV and TX 1%.   3.1.3 Growth Curve of Simvastatin-Treated Cells The cumulative growth curve was generated as described in section 2.6 to observe the long-term cellular growth patterns over 8 days post-seeding by measuring the total cell count using a haemocytometer at five different time points. The total cell count was measured prior to seeding on Day 0, 48 hours post-seeding on Day 2, before SV treatment on Day 3, following 48 hour SV treatment on Day 5, and 72 hours post-SV removal on Day 8. As shown in Figure 3.5, a significant difference in the total number of C4-2 cells compared to vehicle control occurred on Day 8, 72 hours post-SV removal, in cells treated with 75 μM SV. In addition, absence of a significant change in the total cell count of cells treated with 75 μM SV between Day 3 and Day 5 was observed. In contrast, a significant difference in total cell counts in vehicle control 00.20.40.60.811.21.41.6NT Vehicle 10μM 20μM 40μM 50μM 60μM 75μM 80μM 100μM TX 1%Cell viability (relative to NT control) Treatment LNCaP 62  between Day 3 and Day 5 was measured. Despite the lack of cell accumulation from Day 3 to Day 5 in cells treated with 75 μM SV, unlike vehicle control, no significant difference in total cell count was observed between vehicle control and cells treated with 75 μM SV on Day 5. Due to the highly cytotoxic effects of TX 1% on cells, dashed line was used to represent the immediate cell death following the addition of TX 1% to cells on Day 3.   Figure 3.5: Cumulative growth curve of C4-2 cells over 8 days. Results are expressed as total cell count. Curve, mean (n = 5); error bars, ± SEM. *, p < 0.05 vehicle vs 75 M SV on Day 5; vehicle vs TX 1% on Day 5 and Day 8.   The growth curve of LNCaP cells showed a significant difference in the total number of cells on Day 5 and Day 8 following 75 μM SV treatment compared to vehicle control (Figure 3.6). As expected, the total cell count of LNCaP cells with a slower doubling time of 60 hours was significantly lower on Day 8 in all treatment groups except for TX 1% compared to C4-2 cells 0200,000400,000600,000800,0001,000,0000 2 4 6 8Total Cell Count (cells/ mL) Days Post-Seeding C4-2 NTVehicle10μM 50μM 75μM TX1%* * * 63  with a doubling time of 40 hours. However, unlike the castration-resistant C4-2 cells in which no significant difference in total cell count was observed between vehicle control and 75 μM SV on Day 5 a significant difference was observed in LNCaP cells between the two groups on Day 5. The dashed line represents immediate cell death following the addition of TX 1% to LNCaP cells on Day 3.   Figure 3.6: Cumulative growth curve of LNCaP cells over 8 days. Results are expressed as total cell count. Curve, mean (n = 5); error bars, ± SEM. *, p < 0.05 vehicle vs 75 M SV on Day 5 and Day 8; vehicle vs TX 1% on Day 5 and Day 8.      0200,000400,000600,000800,0001,000,0000 2 4 6 8Total Cell Count (cells/ mL) Days Post-Seeding LNCaP NTVehicle10μM 50μM 75μM TX1%* * * * 64  3.2 Aim 2: Effect of SV on Cholesterol Synthesis in C4-2 and LNCaP Cells 3.2.1 HMGCR Activity of Simvastatin-Treated Cells The activity of HMGCR, the rate-limiting step enzyme in the intracellular cholesterol synthesis pathway, was inhibited by the use of SV. The inhibition of HMGCR activity with different concentrations of SV was assessed by the incorporation of 14C-acetic acid precursor molecule into the cholesterol synthesis pathway as described in section 2.7. β-scintillation values expressed as 14C disintegrations per minute (dpm) were divided by dpm of 3H which was used as an internal control for the lipid extraction procedure. 14C dpm by 3H dpm values were normalized to protein concentration in each sample. As shown in Figure 3.7, HMGCR activity was significantly lower in C4-2 cells (Day 5) following treatment with 50 μM (0.24 ± 0.02 14C dpm/ 3H dpm/ μg) and 75 μM (0.13 ± 0.05 14C dpm/ 3H dpm/μg) SV for 48 hours compared to vehicle control (0.45 ± 0.05 14Cdpm/ 3H dpm/ μg); 46% reduction in HMGCR activity in cells treated with 50 μM SV and 69% reduction in cells treated with 75 μM SV compared to vehicle control were observed.   65   Figure 3.7: Intracellular cholesterol synthesis of C4-2 cells following 48-hour SV treatment. Cholesterol synthesis was measured by following the incorporation of 14C-acetic acid into cholesterol synthesis pathway and detected by β-scintillation counting after separation by thin layer chromatography in C4-2 cells on Day 5. Columns, mean (n = 5); bars, ± SEM. *, p < 0.05 vehicle vs 50 M and 75 M SV.   In addition, HMGCR activity was analyzed on Day 8 to assess the residual effect of high SV treatment (75 μM) on the enzyme activity 3 days after the removal of SV (Figure 3.8). 14C dpm by 3H dpm values were normalized to protein concentration in each sample. On Day 8, HMGCR activity continued to be inhibited by 75 μM SV; compared to vehicle control, the difference was statistically significant and showed approximately 67% reduction in HMGCR activity. 00.10.20.30.40.50.6NT Vehicle 10µM 50µM 75µMCholesterol Synthesis Activity (14C dpm/ 3H dpm per μg protein) Treatment C4-2 * * *  * 66   Figure 3.8: Intracellular cholesterol synthesis of C4-2 cells on Day 8, 72 hours after the removal of SV. Columns, mean (n = 4); bars, ± SEM. *, p < 0.05 vehicle vs 75 M SV.    Unlike C4-2 cells, no significant change in HMGCR activity was observed in LNCaP cells treated with 50 μM SV. However, a 58% reduction in the enzyme activity was observed in LNCaP cells treated with 75 μM SV compared to vehicle control; 0.06 ± 0.02 14C dpm/ 3H dpm/ μg vs 0.15 ± 0.02 14C dpm/ 3H dpm/ μg in 75 μM SV and vehicle control respectively (Figure 3.9).  00.020.040.060.080.10.120.140.16NT Vehicle 75μM Cholesterol Synthesis Activity (14C dpm/ 3H dpm per ug protein) Treatment C4-2 67   Figure 3.9: Intracellular cholesterol synthesis of LNCaP cells following 48-hour SV treatment. Cholesterol synthesis was measured by following the incorporation of 14C-acetic acid into cholesterol synthesis pathway and detected by β-scintillation counting after separation by thin layer chromatography in LNCaP cells on Day 5. Columns, mean (n = 5); bars, ± SEM. *, p < 0.05 vehicle vs 75 M SV.   Analysis of baseline (NT) HMGCR activity of C4-2 and LNCaP cells (Day 5) using unpaired t-test (SigmaStat 3.5®) showed a significant, nearly five-fold difference between the two cell lines; 0.49 ± 0.06 14C dpm/ 3H dpm/ μg vs 0.13 ± 0.01 14C dpm/ 3H dpm/ μg in C4-2 and LNCaP cells respectively (Figure 3.10A). As shown in Figure 3.10B, HMGCR activity in C4-2 cells following 75 M SV treatment for 48 hours was not statistically different from baseline HMGCR activity in LNCaP cells.    00.050.10.150.2NT Vehicle 10µM 50µM 75µMCholesterol Synthesis Activity (14C dpm/3H dpm per μg protein) Treatment LNCaP   * 68        Figure 3.10: (A) Baseline (NT) HMGCR activity of C4-2 and LNCaP cells. Columns, mean (n = 5); bars, ± SEM. *, p < 0.05 C4-2 NT vs LNCaP NT (B) baseline HMGCR activity of C4-2 and LNCaP cells (NT) and HMGCR activity of the two cell lines following 75 μM SV treatment (Day 5). Columns, mean (n = 5); bars, ± SEM.   3.2.2 HMGCR Expression of Simvastatin-Treated Cells The effect of SV treatment on HMGCR protein expression was examined in the two cell lines (Day 5) using western blotting technique as described in section 2.8. The results are expressed as fold change from NT control. HMGCR protein expression in both C4-2 and LNCaP cell lines, although displaying a decreasing tendency in the higher concentrations of SV, was not statistically different from the respective vehicle control groups (Figure 3.11 and Figure 3.12; C4-2 and LNCaP respectively). Representative blots are presented in Appendix Figure C.1.  00.10.20.30.40.50.6BaselineCholesterol Synthesis Activity (14C dpm/3H dpm per ug protein) C4-2LNCaP* 00.10.20.30.40.50.6NT 75µMCholesterol Synthesis Activity (14C dpm/3H dpm per ug protein) Treatment C4-2LNCaPA B 69   Figure 3.11: HMGCR protein expression of C4-2 cells following 48-hour SV treatment (Day 5). Results expressed as fold change in HMGCR expression from the expression in NT control. Columns, mean (n = 4); bars, ± SEM.   Figure 3.12: HMGCR protein expression of LNCaP cells following 48-hour SV treatment (Day 5). Results expressed as fold change in HMGCR expression from the expression in NT control. Columns, mean (n = 4); bars, ± SEM.  00.20.40.60.811.2NT Vehicle 10μM 50μM 75μM HMGCR Protein Expression (HMGCR/ actin band density) Treatment C4-2 00.20.40.60.811.2NT Vehicle 10μM 50μM 75μM HMGCR Protein Expression     (HMGCR/ actin band density) Treatment LNCaP 70  3.2.3 Intracellular Cholesterol Concentration of Simvastatin-Treated Cells The total intracellular cholesterol concentration was measured in C4-2 and LNCaP cell lines following 48-hour SV treatment (Day 5) as described in section 2.9. Figure 3.13 shows total intracellular cholesterol concentration expressed as μg/mL of cholesterol in cell lysates. The concentration values were normalized to the amount of protein in the cell samples from the respective wells. A statistically significant difference was observed in C4-2 cell lysates treated with 75 μM SV compared to vehicle control; 1.58 ± 0.59 μg/mL per mg protein vs 9.10 ± 2.21 μg/mL per mg protein in 75 μM SV and vehicle control respectively.   Figure 3.13: Total intracellular cholesterol concentration in C4-2 cell lysates following 48-hour SV treatment (Day 5). The results are shown as μg/mL cholesterol per mg protein in whole cell lysates. Columns, mean (n = 6); bars, ± SEM. *, p < 0.05 vehicle vs 75 M SV.     024681012NT Vehicle 10μM 50μM 75μM Total Intracellular Cholesterol Concentration (ug/mL per mg protein) Treatment C4-2 * 71  In LNCaP cells (Figure 3.14), a significant reduction in the total intracellular cholesterol concentration was observed in cell lysates treated with 75 μM SV compared to vehicle control. In section 3.2.1, a significant difference in the basal HMGCR activity was observed between C4-2 and LNCaP cell lines. To see if this observation translates into a difference in total intracellular cholesterol concentration between the two cell lines, an unpaired t-test analysis was conducted to compare the baseline (NT) cholesterol concentrations (Appendix Figure D.1).  The analysis showed that the basal testosterone concentrations of the two cell lines are not statistically different.   Figure 3.14: Total intracellular cholesterol concentration in LNCaP cell lysates following 48-hour SV treatment (Day 5). The results are shown as μg/mL cholesterol per mg protein in whole cell lysates. Columns, mean (n = 6); bars, ± SEM. *, p < 0.05 vehicle vs 75 M SV.    012345678NT Vehicle 10μM 50μM 75μM Total Intracellular  Cholesterol Concentration (ug/mL per mg protein)  Treatment LNCaP 72  3.3 Aim 3: Effect of SV on Intracellular Testosterone and PSA Secretion in C4-2 and LNCaP Cells 3.3.1 Intracellular Testosterone of Simvastatin-Treated Cells Intracellular testosterone concentration was measured in the cell lysates of C4-2 cells as described in the protocol (section 2.10). The concentration results are expressed as pg/mL per μg of protein, normalized to the protein content in the respective cell sample. The testosterone level was not significantly different in C4-2 cells treated with SV compared to vehicle control (Figure 3.15).   Figure 3.15: Intracellular testosterone concentration of C4-2 cells following 48-hour SV treatment (Day 5). Columns, mean (n = 6); bars, ± SEM.   00.20.40.60.811.21.41.61.8NT Vehicle 10μM 50μM 75μM Intracellular Testosterone Concentration (pg/mL per μg protein) Treatment C4-2 73  3.3.2 PSA Secretion from Simvastatin-Treated Cells The prostate specific antigen (PSA) secretion levels from the two cell lines following 48-hour SV treatment (Day 5) was measured in the respective cell sample medium as described in section 2.11. The concentration results are expressed as ng/mL per μg of protein, normalized to the protein content in the respective cell sample. The PSA secretion level in C4-2 cells was significantly different in the medium of cells treated with 50 μM and 75 μM SV compared to vehicle control (Figure 3.16). At 50 μM and 75 μM SV, C4-2 cells secreted into the medium 34.4 ± 15.4 ng/mL PSA per mg protein and 41.3 ± 14.0 ng/mL PSA per mg protein respectively compared to PSA level of 192.1 ± 52.0 ng/mL per mg protein in vehicle control.     Figure 3.16: PSA secretion from C4-2 cells following 48-hour SV treatment (Day 5). Results are expressed as concentration in the media, normalized to the amount of protein in the respective samples. Columns, mean (n = 6); bars, ± SEM. *, p < 0.05 vehicle vs 50 M and 75 M SV. 050100150200250300NT Vehicle 10μM 50μM 75μM Prostate Specific Antigen Concentration in Media                   (ng/mL per mg protein) Treatment C4-2   *    * 74  PSA level within the media of LNCaP cells treated with SV was not statistically different from the PSA secretion levels from the cells of vehicle control (Figure 3.17).     Figure 3.17: PSA secretion from LNCaP cells following 48-hour SV treatment (Day 5). Results are expressed as concentration in the media, normalized to the amount of protein in the respective samples. Columns, mean (n = 6); bars, ± SEM.   Analysis of baseline (NT) PSA concentration in media of C4-2 and LNCaP cells (Day 5) using unpaired t-test (SigmaStat 3.5®) showed a significant, four-fold difference between the two cell lines; 226.4 ± 46.1 ng/mL per mg protein from C4-2 cells vs 54.1 ± 21.4 ng/mL per mg protein from LNCaP cells (Figure 3.18A). As shown in Figure 3.18B, PSA concentration in media of C4-2 cells following 75 M SV treatment for 48 hours was not statistically different from baseline PSA concentration in media of LNCaP cells. A similar trend between the two cell lines was observed in the HMGCR activity as discussed earlier in section 3.2.1.  020406080100NT Vehicle 10μM 50μM 75μM Prostate Specific Antigen Concentration in Media              (ng/mL per mg protein) Treatment LNCaP 75        Figure 3.18: (A) Baseline (NT) PSA concentration in media of C4-2 and LNCaP cells. Columns, mean (n = 6); bars, ± SEM. *, p < 0.05 C4-2 NT vs LNCaP NT (B) baseline PSA concentration in media of C4-2 and LNCaP cells (NT) and PSA secretion levels from the two cell lines following 75 μM SV treatment (Day 5). Columns, mean (n = 6); bars, ± SEM.  050100150200250300BaselinePSA Concentration in Media (ng/mL per mg protein) C4-2LNCaP* 050100150200250300NT 75uMPSA Concentration in Media (ng/mL per mg protein) Treatment C4-2LNCaPA B 76  Chapter 4: Discussion  The common use and availability of statins to lower hypercholesterolemia has led multiple groups to conduct in vitro, in vivo, and epidemiological studies looking at the effect of statins on cancers in which cholesterol-derived androgens may impact the disease progression (134-143). Although PCa studies looking for an association between statin use and risk of developing PCa have resulted in mixed observations, it is difficult to reach a definitive conclusion based on PCa epidemiological studies, as these studies differed in their patient selection criteria. As described earlier in section 1.4.4.1, PCa patients may be on a different statin drug for various pre-existing CVD diagnoses that could have confound the results. Although the majority of these results suggested that either no reduction or improved reduction in the risk of developing PCa was observed in statin users compared to control patients, some studies have shown a significant reduction in serum PSA, tumor volume, and percentage of cancer in radically removed prostate samples of PCa patients on preoperative statins compared to non-users (145,146). Additional PCa studies done in in vitro and in vivo animal models have reported similar decreases in cell proliferation, tumor viability, and PSA production (137,140,143). However, there are very few mechanistic studies that serve to link the effect of statin treatment on cholesterol metabolism with the observed growth inhibition of CRPC cells. This study was conducted to address this undefined link, and the findings are discussed below.   4.1 Time-Dependent Effect of Simvastatin on Cell Viability In order to determine the appropriate incubation period of SV that would elicit an effect on C4-2 cells, preliminary studies were carried out as described in section 2.5.1. Figures in Appendix A 77  illustrate the time-dependent cytotoxic effects of SV on C4-2 cells. Cytotoxicity results following 6-hour SV treatment did not show any significant difference, even at 100 μM SV compared to vehicle control (Appendix Figure A.1). Similar results were observed in C4-2 cells following 24-hour SV treatment in which a significant difference in cytotoxicity values only occurred in 100 μM SV treated cells compared to vehicle control (Appendix Figure A.2). Due to the lack of an effect in the cytotoxicity results even at high concentrations of SV after 6 and 12 hours of incubation, a 48 hour SV incubation period was chosen for further studies.  The three concentrations of SV used in most of the assays were chosen after assessing a range of concentrations on cell viability and toxicity following 48-hour incubation period, and the three concentrations represent values well below, near, and at the estimated IC50 values.  4.2 Simvastatin Reduces Cell Viability To confirm whether castration-resistant C4-2 and androgen-sensitive LNCaP cells show an effect when treated with SV, cell viability and toxicity were assessed across a range of SV concentrations (0-100 μM). The cell viability results showed a significant abrupt reduction in cell viability in the two cell lines at concentrations of SV greater than 75 μM, indicating that the effect of SV does not follow a concentration-dependent manner (Figures 3.3, 3.4). An IC50 value, the concentration needed to inhibit biological process by half, was estimated to be 74.9 μM SV in both cell lines.  Similar results were observed in cell toxicity data in which significant differences of % cytotoxicity in the C4-2 and LNCaP cell lines occurred at SV concentrations greater than 60 μM compared to vehicle control (Figures 3.1, 3.2). An estimated LD50 value representing the 78  concentration required to kill half of the cell population was 60.2 μM SV in both cell lines. These findings indicate that the effect of SV on the metabolic activity of these cells correlates with the maintenance of the cell membrane integrity. However, the difference in IC50 andLD50 values indicates that while half of the cell population displays poor membrane integrity and LDH may start to leak out of the cells into the media starting at 60.2 μM SV, more than half of the cell population is still metabolically active at this SV concentration.  The long-term effect of a single 48-hour SV treatment was assessed in the two cell lines by measuring the total number of living cells at various time points over 8 days post-seeding (Figures 3.5, 3.6). As expected in androgen-sensitive LNCaP cells, the overall growth of the cells was slower compared to the castration-resistant C4-2 cells. The LNCaP cells showed a significant difference in the total cell count compared to vehicle control following the removal of 75 μM SV (Day 3) and at 72 hours after the removal of 75 μM SV treatment (Day 8). Although C4-2 cytotoxicity and cell viability data on Day 5 showed significant increase and decrease respectively in the cells treated with 75 μM SV, the total cell count was not significantly different compared to vehicle control on the day SV was removed (Day 5). The expected statistical difference in total number of living cells was not observed on Day 5; however, increasing the sample size to improve the accuracy of the mean as well as conducting additional cell-cycle analysis using a flow cytometer to distinguish cells in different cell cycle phases following SV treatment may provide insight on the observed differences between cell viability and growth curve findings.  On Day 8 however, a significant difference in the total number of living cells was observed in both C4-2 and LNCaP cell lines following 75 μM SV treatment. The 79  observed effects on Day 8 in both cell lines likely suggest a continuous influence of SV treatment on total cell count even 3 days after the removal of the drug.   4.3 Simvastatin Reduces Cholesterol Synthesis To provide an explanation for the observed difference in total cell count on Day 8, HMGCR activity in C4-2 cells treated with 75μM SV was assessed on Day 8 (Figure 3.8). Following SV removal, the cells were washed with HBSS twice prior to the addition of fresh CSS-media. Then the cells were allowed to grow for 72 hours before being detached from the plates for cell count analysis. Clinically, simvastatin (ZocorTM) has a half-life of 2 hours, and patients are given the drug daily in an oral formulation, which undergoes extensive first-pass extraction by the liver (123). Although the experimental design of this study did not follow a clinically-relevant SV treatment regimen, HMGCR activity on Day 8 was assessed for any residual effect of 75 μM SV on HMGCR activity 72 hours post-removal of the drug, a period long enough to allow clinically significant decay of any unwashed SV. Interestingly, a significant difference of nearly 66% in HMGCR activity was observed on Day 8 in C4-2 cells treated with 75μM SV compared to vehicle control: 0.03 ± 0.01 14C dpm/ 3H dpm/ μg vs 0.09 ± 0.02 14C dpm/ 3H dpm/ μg in 75μM SV and vehicle control respectively. Although 72 hours has passed since the removal of the drug, the activity of HMGCR was continuously inhibited in these cells. In addition, the indirect measurement of baseline (NT) HMGCR activity in C4-2 cells showed a significant difference in the β-scintillation readings between Day 8 and Day 5: 0.50 ± 0.06 14C dpm/ 3H dpm/ μg in NT group on Day 5 vs 0.12 ± 0.03 14C dpm/ 3H dpm/μg in NT group on Day 8. These findings suggest an unexpected effect of SV, which has a dissociation constant of an enzyme-inhibitor complex (Ki) value of 0.1-0.2 nM (147), that allows a continuous inhibition of HMGCR activity 80  even long after the removal of SV; this residual effect corresponds and may contribute to the decrease in total cell count on Day 8 (Figure 3.5).  Corresponding to the past observations that reported an increase in ex vivo HMGCR activity in the castration resistant state derived from a LNCaP xenograft model, the current study also showed a five-fold increase in basal (NT) cholesterol synthesis activity of C4-2 cells compared to LNCaP cells as shown in Figures 3.10A (53). This observation suggests that castration-resistant state may be requiring a higher cholesterol synthesis activity to maintain cellular functions compared to the androgen-sensitive state; although this study did not reveal a definitive purpose of the increase in basal HMGCR activity of C4-2 cells, higher basal cholesterol synthesis activity supports de novo steroidogenesis pathway in the castration-resistant state. As the prostate cells that previously did not possess steroidogenic properties start to produce androgens intracellularly, more cholesterol precursor molecule may be required to carry out this gained function.   As shown in Figure 3.7, C4-2 cells following 50μM and 75μM SV treatment showed approximately 50% and 70% reduction in HMGCR activity compared to vehicle control. Similarly in LNCaP cells, approximately 50% HMGCR activity was reduced following 75 μM SV compared to vehicle control (Figures 3.9). Comparing the results from the two cell lines using unpaired t-test analysis showed that HMGCR activity level of C4-2 cells following 75 μM SV was not statistically difference, hence comparable to basal HMGCR activity level of LNCaP cells (Figure 3.10B). If the observed inhibitory effect of HMGCR activity translates into similar observations in other parameters, such as in testosterone and PSA concentrations, a novel ability 81  of SV that allows the castration resistant state to display androgen-sensitive-state-like behaviors may be hypothesized.  SV treatment did not result in significant differences in the HMGCR protein expression in both cell lines (Figures 3.11, 3.12). Although HMGCR protein blots (Appendix Figure C.1) are qualitative measurements of protein expression, as a competitive inhibitor of HMGCR, there is no known evidence that SV affects the HMGCR protein expression. Thus, the effects seen on intracellular cholesterol concentration are likely due to inhibition of the enzyme activity rather than through changes in HMGCR expression.  Cholesterol is an important precursor molecule in the production of androgens and is necessary for growth of normal and cancerous prostate. Thus, multiple groups have investigated the effect of statins on PCa models; however, of these studies, only a few focused on examining the effect of statin on established CRPC models. Of these few studies, none of these studies investigated the effect of statin on castration resistant cells by exploring the effects on de novo steroidogenesis and intracellular cholesterol homeostasis pathways (143). Therefore, it was crucial to quantify intracellular cholesterol concentration to confirm that the inhibitory effects observed in HMGCR activity translated into changes in intracellular cholesterol concentration. Corresponding to the HMGCR activity results, both cell lines following 75 μM SV treatment showed a significant decrease in intracellular cholesterol concentration. Cholesterol concentration, expressed in values normalized to the protein levels present in the sample cells was significantly different in the two cell lines treated with 75 μM SV compared to vehicle control;  nearly 80% reduction in intracellular cholesterol concentration was observed with 75 82  μM SV compared to vehicle control (Figures 3.13, 3.14). Contrary to the HMGCR activity results, in C4-2 cells following 50 μM SV treatment, intracellular cholesterol concentration was not statistically significant compared to the concentration measured in vehicle control (Figures 3.7, 3.13). Although a decrease of 50% in HMGCR activity following 50 μM SV treatment may not translate into a significant difference in intracellular total cholesterol concentration of cells also treated with 50 μM SV, this observation may be an indication that compensatory pathways are activated in response to partial HMGCR inhibition to supply the cell with necessary cholesterol supply via modifications in other cholesterol homeostasis pathways to maintain cellular growth and function. This hypothesis can be tested as future studies by examining the expression of other major proteins that regulate cholesterol homeostasis, such as SR-BI influx transporter, ACAT-1, HSL, and ABCA1 efflux transporter through qualitative western blotting technique or through quantitative measurement of mRNA using real-time polymerase chain reaction (RT-PCR).  4.4 Reduced PSA Secretion in Simvastatin-Treated C4-2 Cells PSA is often used as an important indicator of AR-androgen pathway activation, as it is produced in response to the activation of transcription of genes containing AREs; in an AR-dependent activation pathway, activated wild or mutated AR binds to AREs to initiate the transcription of factors responsible for cell function, growth and survival. In this study, PSA was measured to assess the effect of SV on gene transcription activation and to examine whether the observed changes in C4-2 cell viability following SV treatment (Figure 3.3) translate to changes in secreted PSA concentrations.   83  As shown in Figure 3.16, castration-resistant C4-2 cells following 50 μM and 75 μM SV treatment showed significant difference in PSA secretion concentration; 75% reduction was observed for both SV concentrations compared to vehicle control. Although the PSA observations corresponded to the decrease in cell viability, the magnitude of reduction of PSA secretion concentration following SV treatment did not proportionally translate into changes in the cell viability. To elaborate, cell viability in C4-2 cells following 50 μM SV treatment was not significantly different compared to vehicle control; however, PSA secretion concentration in C4-2 cells following 50 μM SV decreased by 75%. To summarize, at 50 μM SV, although PSA secretion is reduced, cell viability is not significantly changed, contrary to 75 μM SV in which significant difference was observed in both PSA secretion concentration and cell viability. 50 μM SV treatment may be inducing an environment in C4-2 cells in which reduced PSA secretion and related gene transcription of factors responsible for cellular growth and survival does not pose an immediate effect on cell viability.   In androgen-sensitive LNCaP cells, SV treatment did not significantly alter PSA secretion concentration compared to vehicle control (Figure 3.17) despite the observed significant changes in HMGCR activity (Figure 3.9) and intracellular total cholesterol concentration (Figure 3.14) following 75 μM SV treatment. The observed difference on PSA secretion concentration in the two cells following SV treatment is interesting and suggests that LNCaP cells may be able to continuously secret the same concentration of PSA despite the reduction in cholesterol synthesis activity and intracellular cholesterol concentration following 75μM SV treatment. To confirm the findings, it would be useful to conduct quantitative RT-PCR analysis on PSA mRNA levels in LNCaP cells following SV treatment.  84  A way that LNCaP cells may achieve gene transcription and PSA production without relying on cholesterol-derived androgens is through cholesterol-independent pathways. There is no known evidence that SV itself can bind to any AR isoforms. Statins are known to affect multiple cholesterol-independent signaling pathways that could potentially induce the gene transcription without the binding of activated AR to ARE-containing genetic sites (132,140,143). Although this observation opens up the possibility of off-target effects that could sustain PSA secretion, the large error bars of the mean at higher SV concentrations raise a concern regarding the accuracy of the LNCaP PSA data, despite the similarity in the basal PSA secretion concentrations to past research studies (148).   Corresponding to PSA secretion concentrations observed in previously published papers, mean basal PSA secretion concentration from C4-2 cells was significantly higher compared to basal concentration from LNCaP cells; the current study showed a four-fold increase in PSA secretion concentration from C4-2 cells compared to PSA concentration secreted by LNCaP cells (Figures 18A) (148). Interestingly, similar magnitude of difference in the basal HMGCR activity was observed between the two cell lines and was discussed in the previous section 4.3. This finding suggests a potential correlation between reduction of HMGCR activity and reduction in PSA secretion across the two lines. In addition, C4-2 cells following 75 μM SV treatment showed a similar magnitude of decrease from their respective vehicle controls in HMGCR activity, intracellular cholesterol concentration, and PSA secretion concentration: 70%, 80%, and 75% reduction compared to vehicle control in HMGCR activity, intracellular cholesterol concentration, and PSA secretion concentration respectively (Figures 3.7, 3.13, 3.16). Although there is a lack of corresponding intracellular testosterone difference, as described in next section, 85  the observed decrease in cholesterol and PSA concentrations of similar magnitude suggests that there may be a proportional correlation between the two parameters in C4-2 cells treated with 75 μM SV.  Similar to the comparable HMGCR activity observed in C4-2 cells following 75 uM SV and LNCaP NT cells (Figure 3.10B), PSA secretion concentration in C4-2 cells following 75 uM SV treatment was comparable to basal (NT) PSA secretion concentration in LNCaP cells (Figure 3.18B). The recurring display of androgen-sensitive-like behaviors in castration-resistant C4-2 cells is a novel finding, and opens up a new area of CRPC research. If C4-2 cells treated with 75 μM SV display characteristics comparable to basal levels of LNCaP cells, it can be hypothesized that the castration-resistant cells may be transforming back to the androgen-sensitive state. While the results in this study do not provide a definitive answer to this hypothesis, a potential mechanism of SV to produce androgen-sensitive-state-like behaviors in castration-resistant may instigate a new CRPC treatment research area.   4.5 Effect of Simvastatin on Testosterone Although a decrease in intracellular cholesterol concentration and PSA secretion concentration was observed in castration-resistant cells following 75 μM SV treatment, corresponding changes in intracellular testosterone concentration, which could link the two observations, was not detected (Figure 3.15). Testosterone was the androgen-of-interest in this study, as previous studies have reported higher concentrations of intracellular testosterone compared to DHT in castration-resistant cells (149,150). Therefore, if a decrease in intracellular cholesterol concentration was observed, together with reduced cell viability and PSA secretion concentration 86  due to a cholesterol-dependent effect of SV, a corresponding decrease in intracellular testosterone concentration was anticipated. The lack of change in intracellular testosterone concentration suggested that a decrease in cholesterol precursor supply may alter the dynamic androgen synthesis pathways. As shown in Figure 1.1, conversion of cholesterol to testosterone is comprised of multiple reactions through various enzymes. Although there may be a decrease in cholesterol concentration, existing supply of progesterone and pregnenolone may be sufficient to produce testosterone through the classical androgenesis pathway and supply the cells with required, constant testosterone concentration. Also, independent of the steroidogenesis pathway, SV may simply not affect intracellular concentrations of any androgens despite observed reduction in precursor cholesterol concentration; whereas, the observed reduction in cell viability and PSA secretion concentration in C4-2 cells may be from alterations in the cholesterol-independent signaling pathways induced by SV. Conducting a LC-MS/MS analysis to quantify multiple androgens and their precursor molecules following SV treatment would be useful in clarifying whether SV affects the androgen synthesis pathways.  4.6 A Focus on Castration-Resistant State While the results obtained from the two cell lines were compared in the discussion sections above, the main objective of this study was not to examine potential differential outcomes of SV on the androgen-sensitive and castration-resistant cell lines, but rather to explore the effect of inhibiting HMGCR on the castration-resistant state. As discussed earlier in the introduction, the effect of statins on various androgen-sensitive models has been investigated by multiple groups, whereas this effect has not been explored in established castration-resistant models. The current study showed that cell viability, HMGCR activity, intracellular cholesterol concentration, and 87  PSA secretion concentrations were significantly changed in castration-resistant C4-2 cells following SV treatment (75 μM) compared to vehicle control.  Although multiple groups have shown that de novo steroidogenesis has a significant impact on the progression of the androgen-sensitive state to the castration-resistant state, SV is expected to affect cell viability and cholesterol-mediated pathways in LNCaP cells as well since cholesterol is an integral component in cellular structure and function.  While comparing the results of the two cell lines has provided a novel finding, it is important to recognize that focusing on the differential effects of SV in the C4-2 and LNCaP cell lines may not be beneficial in determining whether SV affects cell viability of castration-resistant C4-2 cells through altering intracellular cholesterol concentration and hence, de novo steroidogenesis.  4.7 Limitation and Future Directions As mentioned in section 4.5, although a decrease in HMGCR activity was observed along with a decrease in intracellular cholesterol concentration and PSA secretion, corresponding decrease in testosterone concentration following SV treatment in C4-2 cells was not observed. However, it is difficult to reach a definitive conclusion about the effect of SV on de novo androgenesis based on the testosterone data alone, as androgen synthesis pathway is dynamic and composed of multiple reactions and intermediate molecules (Figure 1.1). Although there was no statistically significant difference in intracellular testosterone concentration, further investigation that examines other hormones including more potent DHT and T877A-AR substrate progesterone may provide a more complete explanation as to whether SV affects cell viability through acting on de novo androgen synthesis and AR activation pathways. In addition, inhibition of HMGCR using SV 88  may alter the activity and expression of multiple enzymes involved in the androgen synthesis pathways. Thus, it would also be helpful to investigate if there are changes in the expression and activity of these enzymes following SV treatment. If future research does confirm the effect of SV on cell viability through de novo steroidogenesis pathway, it may be useful to confirm the findings in PC-3 castration-resistant control cell line that lack AR to provide further support to the findings.   In addition, inhibition of HMGCR may alter other pathways involved in the cellular cholesterol homeostasis and potentially activate compensatory pathways to supply cells with necessary cholesterol supply to maintain cellular structure and function. In the case of SR-BI knockdown castration-resistant C4-2 cells, an increase in cholesterol synthesis activity through HMGCR was observed in response to the silencing of SR-BI cholesterol influx transporter (107,108). Similar effects might be observed in cells treated with SV; although a significant decrease in total intracellular cholesterol concentration following 75 μM SV treatment in C4-2 cells was observed, potential activation of compensatory pathways may contribute to the measured concentrations of cholesterol that was not statistically different compared to vehicle control at the lower SV concentrations. Future studies that look at changes in expression and activity of major enzymes involved in cholesterol homeostasis using western blot analysis or RT-PCR would provide an insight to possible off-target effect of SV on other cholesterol regulatory pathways.  A key limitation of this study involves the off-target effects of statins; statins may exert cholesterol-independent effects by interacting with inflammatory response and signaling pathways (132). Multiple studies have shown that statins are shown to affect various signaling 89  molecules in prostate cancer models (140,143,151). In a PC-3 xenograft nude mouse model, SV treatment exhibited reduced tumor growth that was associated with decreased Akt/PKB activity (140). In vitro studies by multiple groups have reported that SV reduces viability of castration-resistant PC-3 cells by reducing NF-κB, IGF-1R, ERK, and Akt/PKB signaling pathways (140,143,151). Although the results of the current study suggest that SV decreases cell viability and PSA secretion through a reduction in intracellular cholesterol concentration, the missing link of corresponding testosterone data and previous findings that SV affects CRPC cells through various signaling pathways indicate that the observed effects of SV in C4-2 cells may be due to a combination of cholesterol-dependent and cholesterol-independent action of statin.  Clinically, statins are orally administered daily over a period of years. Another limitation of the current study was that the findings were based on SV concentration that is extremely high (75 μM; 31 μg/mL). In patients given a single 40-mg dose of simvastatin, the peak concentration of simvastatin acid measured up to 12 hours was 6.78 ± 4.67 ng/mL, which represents concentration that is roughly five-thousand-fold less compared to SV concentration used in the current study (152). Therefore, the observed effect of SV on cell viability, cholesterol concentration, and PSA concentration could be due to off-target effects of abundant SV concentration in the wells. An analysis of intracellular SV concentration using LC-MS/MS would be beneficial in determining bioavailable concentration of SV that is responsible for exerting the inhibitory effect on HMGCR inside the cell.   Whether simvastatin exerts observed effects through limiting the precursor cholesterol molecule for de novo steroidogenesis pathway and AR activation or through AR-independent signaling 90  pathways, reduction in cell viability of castration-resistant C4-2 cells following SV treatment is still a significant finding. Although further studies should be continued to understand the mechanism behind the observed effects, in vivo studies using a mouse model, specifically using the LNCaP xenograft mouse that has progressed into CRPC state, would be interesting to conduct to see whether a similar inhibition in tumor cell growth can be confirmed in a model given clinically relevant SV dosing regimen with heterogeneous cellular environment. In addition, in vivo experiments designed to test whether CRPC animal models treated with SV show androgen-sensitive-state-like behaviors would be an interesting and a novel research area; an experiment comparing groups within an CRPC animal model, in which one group is treated with simvastatin followed by treatment for androgen-sensitive state and others that are either treated with only simvastatin or treatment for androgen-sensitive state, may provide significant implications to CRPC treatment applications should the prior group have better prognosis.  4.8 Conclusion In conclusion, it seems that inhibiting HMGCR activity using SV in C4-2 cells reduces viability of these cells. A decrease in the level of HMGCR activity and a corresponding change in intracellular cholesterol concentration were observed in C4-2 cells following 75 μM SV treatment. Reduction in PSA secretion concentrations were observed from C4-2 cells following  50 μM and 75 μM SV treatment. The observations from HMGCR activity, intracellular cholesterol concentration, and PSA secretion concentrations suggest that SV may affect cell viability through altering these parameters. However, the lack of corresponding change in the intracellular testosterone concentration in C4-2 cells treated with SV makes it difficult to conclude that observed decrease in cell viability is due to the effect of SV in reducing 91  intracellular cholesterol concentration that feeds into the de novo steroidogenesis pathway. While these findings support the hypothesis that simvastatin does affect cell viability, the mechanism through which SV affects the growth of castration-resistant cells needs to be investigated further to reach a conclusive statement.  4.9 Significance of Findings The findings suggest that HMGCR inhibition using simvastatin disturbs cell viability and cholesterol synthesis in the castration-resistant and androgen-sensitive cell lines. 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Columns, mean (n = 4); bars, ± SEM.        0102030405060708090100NT Vehicle 10μM 20μM 40μM 50μM 60μM 75μM 80μM 100μM %Cytotoxicity (Abs @ 492nm/ Abs of 100% cytotoxicity control) Treatment C4-2 107  Appendix B    Equation y = b + (a-b)/(1+10x-c)  C4-2 LD50 a b c 19.290305496879299 75.5215533333233300 60.117796943835145 LNCaP LD50 a b c 22.322873516652809 69.540066666441305 60.006492669457721 C4-2 IC50 a b c 0.81024981770360838 0.085794512295636199 85.052775021452192 LNCaP IC50 a b c 0.86849985830034893 0.094694872909435512 74.996421619901326  Table B.1. LD50 and IC50 calculation equation and coefficients. The coefficients were derived from online curve fitting software (Zunzun®). LD50 and IC50 values were estimated by solving for x when y equals 50. Variable a represents y-value at the bottom of the asymptote that is generated. Variable b represents y-value at the top of the asymptote and variable c is derived from the center of asymptote and the hill slope.         108  Appendix C    A.                                                                   B.       Figure C.1: Western blot representation of HMGCR expression in C4-2 (A) and LNCaP (B) cells following SV treatment for 48 hours.               HMGCR (97 kDa)    β-actin (42 kDa)  Treatment    NT        V    10 μM  50 μM  75 μM                NT        V    10 μM  50 μM  75 μM               Treatment 109  Appendix D      Figure D.1: Basal intracellular total cholesterol concentrations in cell lysates of C4-2 and LNCaP cell lines. Columns, mean (n = 6); bars, ± SEM. 012345678910BaselineTotal Intracellular Cholesterol Concentration (ug/mL per mg protein) C4-2LNCaP

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