Background In castration-resistant prostate cancer (CRPC), recent evidence has demonstrated the persistence of the intratumoral androgens. 48?hours. Dose-dependent responses to Simvastatin were analyzed using cell proliferation and cytotoxicity assays. Cellular growth curve was generated using haemocytometer. HMGCR activity was assessed using 14C-acetic acid detected by thin layer chromatography, and the protein expression was quantified using western blot analysis. Intracellular cholesterol and prostate specific antigen (PSA) levels were quantified using enzyme-linked immunosorbent assays (ELISA). Results Significant decrease in cell viability and growth curve observed at 75?M of Simvastatin compared to no treatment group in the castration-resistant C4-2 cells. HMGCR activity was significantly decreased up to 50% and 70% at 50?M and 75?M of Simvastatin respectively compared to the vehicle control in C4-2 cells. Simvastatin did not affect the protein expression. 80% decrease in the amount of total intracellular cholesterol levels was observed in 75?M Simvastatin treatment group compared to vehicle control. PSA secretion levels were significantly reduced in the C4-2 cell line at 50?M and 75?M of Simvastatin compared to vehicle control. Conclusion The inhibition of HMGCR via Simvastatin lowered the viability of castration-resistant C4-2 cells. Simvastatins ability to limit the endogenous supply of cholesterol contributes to the effects seen in cell viability. steroidogenesis [5,6]. Androgen levels within metastastic tumors of castrated men are found to be higher than the levels within the primary prostate cancer tumors in untreated men . In addition, CRPC tumors are shown to continuously express the necessary enzymes to create androgens intracellularly [8,9]. While the steps towards androgen production consist of multiple pathways, all the steps originate from a common upstream precursor molecule, cholesterol . Cellular cholesterol homeostasis is comprised of complex and multiple regulatory pathways as cholesterol has important functions in humans including regulating membrane fluidity, influencing cellular signaling, and being a precursor for PIK3R1 bile and androgens . Cells obtain cholesterol from two major sources: exogenous and endogenous supplies. Exogenous cholesterol supply involves uptake of cholesterol from circulating lipoproteins via membrane transporters such as Scavenger Receptor Class B Type I (SR-BI) and low density lipoprotein receptor (LDLr). Once in the cell, cholesterol is stored as buy CRT0044876 cholesteryl esters in lipid droplets and metabolized accordingly to cells demand for cholesterol via acetyl-CoA acyltransferase (ACAT) and hormone sensitive lipase (HSL). Endogenously, cholesterol is synthesized from acetyl-CoA in the endoplasmic reticulum through is the mevalonate pathway, in which the rate-limiting is step 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) that is responsible for converting 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) into mevalonate [11,12]. Mevalonate molecule further undergoes multiple reactions to be converted into cholesterol downstream. While normal physiological cholesterol homeostasis is tightly regulated, it has been shown that this process is dysregulated in CRPC, suggesting a constant, unregulated supply of cholesterol to meet cellular requirements including supply for steroidogenesis [6,13,14]. A potential site of dysregulation has shown to be at site of cholesterol uptake via SR-BI; our group has shown that SR-BI protein expression was significantly increased upon progression to castration-resistance in the LNCaP xenograft model . Also, upon inhibition of cholesterol uptake via SR-BI silencing as well as studies despite the androgen castrated environment, supporting the existence of an intracellular androgen synthesis pathway rather than the tumors ability to sustain itself despite the lack of androgens [24-27]. HMGCR has buy CRT0044876 been shown to be essential regulator for provision of cholesterol to the androgen synthesis pathway in the steroidogenic tissues of the body . Upon progression to castration-resistance in LNCaP xenograft model, an increase HMGCR activity has been observed . Also, in castration resistant cell model upon the knockdown of SR-BI transporter a significant increase in HMGCR activity was observed . These finding suggests that castration-resistant cells may adapt to the low androgen environment by increasing cholesterol synthesis to supply cellular needs as well as providing the precursor for androgen synthesis. While suggesting an important role of this enzyme in intracellular androgen synthesis and potentially suggesting common HMGCR inhibitors such as statins as possible therapeutic agent, the effect of inhibiting HMGCR in a castration-resistant model has not been investigated. The buy CRT0044876 current study aimed to investigate the physiological relevance of one source of cholesterol to the cell, HMGCR, a rate-limiting enzyme in the cholesterol synthesis-mevalonate pathway. Simvastatin is a commonly used drug to control hypercholesterolemia to prevent cardiovascular disease,.