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Effect of simvastatin on castration-resistant prostate cancer cells Kim, Jenny H; Cox, Michael E; Wasan, Kishor M Mar 26, 2014

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RESEARCH Open AccessEffect of simvastatin on castration-resistantprostate cancer cellsJenny Hanbi Kim1, Michael E Cox2 and Kishor M Wasan1*AbstractBackground: In castration-resistant prostate cancer (CRPC), recent evidence has demonstrated the persistenceof the intratumoral androgens. The multi-step androgen synthesis pathway originates from cholesterol, whichcan be obtained by cells from several major sources including intracellular synthesis through an enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR). The inhibition of this enzyme by the use of statins has beeninvestigated in prostate cancer as a possible therapeutic target for blocking the de novo androgen synthesisresulting in decreased tumor growth. However, the effectiveness of statins in CRPC has not been investigated.Methods: Castration-resistant C4-2 and androgen-sensitive LNCaP cells were treated with Simvastatin for 48 hours.Dose-dependent responses to Simvastatin were analyzed using cell proliferation and cytotoxicity assays. Cellulargrowth curve was generated using haemocytometer. HMGCR activity was assessed using 14C-acetic acid detectedby thin layer chromatography, and the protein expression was quantified using western blot analysis. Intracellularcholesterol 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 μM of Simvastatin compared to notreatment group in the castration-resistant C4-2 cells. HMGCR activity was significantly decreased up to 50% and70% at 50 μM and 75 μM of Simvastatin respectively compared to the vehicle control in C4-2 cells. Simvastatin didnot affect the protein expression. 80% decrease in the amount of total intracellular cholesterol levels was observedin 75 μM Simvastatin treatment group compared to vehicle control. PSA secretion levels were significantly reducedin the C4-2 cell line at 50 μM and 75 μM of Simvastatin compared to vehicle control.Conclusion: The inhibition of HMGCR via Simvastatin lowered the viability of castration-resistant C4-2 cells.Simvastatin’s ability to limit the endogenous supply of cholesterol contributes to the effects seen in cell viability.Keywords: Simvastatin, Castration-resistant prostate cancer, HMGCR, Cholesterol synthesisBackgroundProstate cancer (PCa) occurs in the form of an unregu-lated cellular growth in the prostate, a gland in the malereproductive system, leading to symptoms such as erect-ile dysfunction, hematuria, and pain. PCa is commonlydiagnosed in men over the age of 50, after which inci-dence rates increase with age; it is the most commoncancer diagnosed among North American men, account-ing for 28% of all cancers diagnosed, and is the secondmost common cause of cancer death among the samecohort (10%) in 2012 [1,2]. Upon diagnosis, local PCa istreated with surgical removal of the tumor, radiationtherapy, and/or androgen deprivation therapy (ADT).Within the prostate gland, androgens such as testoster-one and dihydrotestosterone (DHT) promote cell growthand proliferation via androgen receptor (AR), a ligand-responsive transcription factor. Upon ADT, 99% ofprostate cancer patients reach 5-year survival due toapoptotic regression of tumor from the lack of androgengrowth stimuli [3,4]. However, patients with metastaticprostate cancer are subject to temporary remission.Eventually the tumor regrows despite the depleted levelof circulating androgens that is insufficient to supportthe prostate tumor growth; this is referred to ascastration-resistant prostate cancer (CRPC) [3,4].* Correspondence: kwasan@mail.ubc.ca1Faculty of Pharmaceutical Sciences, University of British Columbia, 2405Wesbrook Mall, Vancouver, British Columbia, CanadaFull list of author information is available at the end of the article© 2014 Kim et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly credited. The Creative Commons Public DomainDedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,unless otherwise stated.Kim et al. Lipids in Health and Disease 2014, 13:56http://www.lipidworld.com/content/13/1/56Previously CRPC has been known as an ‘androgenindependent’ cancer due to its ability to grow andmetastasize despite the low androgen environment. Re-cent findings by multiple groups however have shownthat CRPC cells have the ability to produce their ownintracellular androgens that promote growth via AR acti-vation without having to rely on exogenous androgensupply – known as intratumoral de novo steroidogenesis[5,6]. Androgen levels within metastastic tumors of cas-trated men are found to be higher than the levels withinthe primary prostate cancer tumors in untreated men[7]. In addition, CRPC tumors are shown to continu-ously express the necessary enzymes to create androgensintracellularly [8,9]. While the steps towards androgenproduction consist of multiple pathways, all the stepsoriginate from a common upstream precursor molecule,cholesterol [10].Cellular cholesterol homeostasis is comprised of com-plex and multiple regulatory pathways as cholesterol hasimportant functions in humans including regulatingmembrane fluidity, influencing cellular signaling, andbeing a precursor for bile and androgens [11]. Cells ob-tain cholesterol from two major sources: exogenous andendogenous supplies. Exogenous cholesterol supply in-volves uptake of cholesterol from circulating lipoproteinsvia membrane transporters such as Scavenger ReceptorClass B Type I (SR-BI) and low density lipoprotein re-ceptor (LDLr). Once in the cell, cholesterol is stored ascholesteryl esters in lipid droplets and metabolized ac-cordingly to cell’s demand for cholesterol via acetyl-CoAacyltransferase (ACAT) and hormone sensitive lipase(HSL). Endogenously, cholesterol is synthesized fromacetyl-CoA in the endoplasmic reticulum through isthe mevalonate pathway, in which the rate-limiting isstep 3-hydroxy-3-methylglutaryl-coenzyme A reductase(HMGCR) that is responsible for converting 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) into mevalo-nate [11,12]. Mevalonate molecule further undergoesmultiple reactions to be converted into cholesteroldownstream.While normal physiological cholesterol homeostasis istightly regulated, it has been shown that this process isdysregulated in CRPC, suggesting a constant, unregu-lated supply of cholesterol to meet cellular requirementsincluding supply for de novo steroidogenesis [6,13,14]. Apotential site of dysregulation has shown to be at site ofcholesterol uptake via SR-BI; our group has shown thatSR-BI protein expression was significantly increasedupon progression to castration-resistance in the LNCaPxenograft model [8]. Also, upon inhibition of cholesteroluptake via SR-BI silencing in vitro, a compensatory chol-esterol synthesis via increased HMGCR activity wasidentified and significant decreases in prostate specificantigen (PSA) and cell viability of CaP cells wereobserved [15]. However, relevant changes in total choles-terol concentration and androgen levels were not seen,most likely due to the activation of compensatory chol-esterol synthesis via HMGCR [15]. Recently, our grouphas demonstrated that HMGCR expression and activity,and thereby cholesterol synthesis, was increased duringCRPC progression in LNCaP xenografts [6,16]. Statins,inhibitors of HMGCR, have been the subject of manyPCa studies with mixed results [17-19]. Recently, anassociation between statin users and a reduction in theonset of the aggressive, late-stage disease state was re-ported and has been paired with decreased serum andro-gens [20]. However to date, only a handful of researchhas looked directly at the effect of statin as a treatmentoption for CRPC; it has been shown that inhibition ofcholesterol synthesis via statin prevents cell proliferationby inducing apoptosis through reduction in nuclearfactor-κB activity [21]. However there are limitations tothe studies with regards to cell line specificity, and thelack of cholesterol metabolism data.The present study sought to determine the role ofHMGCR in castration-resistant prostate cancer cells interms of cell viability, cholesterol synthesis, and PSAproduction when the cholesterol synthesis via HMGCRis blocked with Simvastatin. LNCaP and C4-2 cells sharesimilar features by expressing AR and producing PSA;C4-2 cells are lineage-derived second generation sublineof LNCaP cells that were derived by subcutaneous co-inoculation of LNCaP and osteosarcoma cells in cas-trated mice [22]. Therefore, LNCaP cells were selectedas the appropriate control cell line.ResultsC4-2: castration-resistant prostate cancer cell lineCytotoxicity of simvastatin treated C4-2 cellsThe cytotoxicity of cells was measured based on thepresence of lactate dehydrogenase (LDH) in the media,which indicates poor cellular membrane integrity andcell lysis. LDH in media causes the conversion of a tetra-zolium salt reagent into a red formazan product, whichthe absorbance can be measured at 492 nm. Results areexpressed as values normalized to a 100% death controlwhich represents untreated cells lysed with a 1% TritonX-100 solution. The means of the cytotoxicity measuredat different Simvastatin doses were compared to theVehicle Control, consisting of 0.5% DMSO. The meanswere found to be significantly different at 60 μM,75 μM, 80 μM, 100 μM, and 250 μM in C4-2 cells(Figure 1).Cell viability and growth curve of simvastatin treatedC4-2 cellsThe cell viability was measured based on the presence isof dehydrogenase enzymes in metabolically active cellsKim et al. Lipids in Health and Disease 2014, 13:56 Page 2 of 12http://www.lipidworld.com/content/13/1/56which are able to bioreduce the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) tetrazolium reagent into a coloredformazan product that can be measured at 490 nm, pro-ducing signal measurements that are directly propor-tional to the number of living cells in culture. Theviability of cells was normalized to protein levels (μg) inthe respective cell samples. As shown in Figure 2, cellu-lar viability, expressed as fold change from negative con-trol, decreased significantly at 75 μM, 80 μM, 100 μM,250 μM, and Triton-X 1% control for C4-2 cells in adose-dependent manner.The cumulative growth curve was generated to ob-serve the long-term cell growth patterns over 8 dayspost-seeding by measuring the total cell count usinghaemocytometer at 48 hours post-seeding (Day 2), be-fore SV treatment on Day 3, 48 hours post-treatment(Day 5), and 72 hours post-Simvastatin removal (Day 8).As shown in Figure 3, the only significant difference inthe growth pattern of C4-2 cells upon Simvastatin treat-ments compared to vehicle control was at 75 μM ofSimvastatin at Day 8. Despite the difference in thegrowth rate of C4-2 cells at 75 μM compared to vehiclecontrol at Day 5, the difference was not statisticallysignificant.HMGCR activity and protein expressionβ-scintillation readings of silica gel at a Rf value of ap-proximately 0.25 indicated the location of cholesterol onthe stationary phase as compared to positive cold chol-esterol control sample. The measurements were used toassess the cholesterol synthesis activity via HMGCR en-zyme by the incorporation of radiolabeled acetate pre-cursor molecule into cholesterol synthesis pathway. TheFigure 1 Cell cytotoxicity is demonstrated as % cytotoxicity based on a 100% cytotoxic control, representing the amount of LDH inthe media of C4-2 cells treated with increasing dose of Simvastatin. Columns, mean (n = 5); bars, ±SEM. *P < 0.05, Vehicle control versus μM,80 μM, 100 μM, 250 μM.Figure 2 Cell viability is demonstrated as fold change in viability from the negative control in C4-2 cells treated with increasing doseof Simvastatin. Columns, mean (n = 5); bars, ±SEM. *P < 0.05, Vehicle control versus 60 μM, 75 μM, 80 μM, 100 μM, 250 μM and TX1%.Kim et al. Lipids in Health and Disease 2014, 13:56 Page 3 of 12http://www.lipidworld.com/content/13/1/56β-scintillation readings (14C DPM/3H DPM) were nor-malized to protein levels (μg) as measured by Lowryassay. The mean cholesterol synthesis activities in C4-2cells significantly decreased up to 50% and 70% upon50 μM and 75 μM of Simvastatin respectively comparedto vehicle control group (Figure 4).Western blot analysis of C4-2 cell lysates showed thatthere is no significant decrease in HMGCR expressionupon Simvastatin treatments when compared to the ve-hicle control (Figure 5).Cellular cholesterol of simvastatin treated C4-2 cellsThe total intracellular cholesterol concentrations weremeasured in C4-2 cells treated with Simvastatin for48 hours, and the values were normalized to protein(mg) in the cell samples from the respective wells ofwhole cell lysates. Significant decrease in the amount oftotal cholesterol was observed at 75 μM of Simvastatincompared to vehicle control in C4-2 cells (Figure 6).The decrease in cholesterol levels corresponded to thedecrease in HMGCR protein activity at 75 μM Simva-statin treatment results.PSA secretion is reduced in C4-2 cells treated withsimvastatinThe levels of PSA secretion of C4-2 cells were quantifiedin the supernatant or treated cells. The amount of PSAis expressed as concentration (ng/mL) and was thennormalized to the amount of protein (mg) in the cellsamples from the respective wells of the analyzedFigure 3 Cumulative cell growth curve expressed as total cell count of C4-2 cells over 8 days. Curve, mean (n = 5); error bars, ± SEM.*P < 0.05, Vehicle control versus TX1% in C4-2 at Day 5; Vehicle control versus 75 μM and TX1% at Day 8.Figure 4 Cholesterol synthesis measurement by incorporation of 14C-acetic acid into cellular cholesterol as measured by β-scintillationcounter in C4-2 after 48 hour Simvastatin treatment followed by 3 hour 14C-acetic acid treatment and separation by thin layerchromatography. Columns, mean (n = 5); bars, ±SEM. *P < 0.05, Vehicle Control versus 50 μM, and 75 μM.Kim et al. Lipids in Health and Disease 2014, 13:56 Page 4 of 12http://www.lipidworld.com/content/13/1/56supernatant. The PSA secretion levels were significantlyless at 50 μM and 75 μM of Simvastatin treatmentscompared to vehicle control in C4-2 cells; vehicle con-trol had secreted 192.1 ± 52.0 ηg/mL per mg protein ascompared to 34.4 ± 15.4 ng/mL per mg at 50 μM and41.32 ± 14.0 ng/mL per mg at 75 μM of Simvastatintreatments (Figure 7).LNCaP: androgen-sensitive prostate cancer cell lineCytotoxicity results expressed as mean values normal-ized to a 100% death control were found to be signifi-cantly different at 60 μM, 75 μM, 80 μM, 100 μM, and250 μM when compared to vehicle control group’scytotoxicity mean value (Figure 8). Corresponding cellviability results were observed where cellular viability,expressed as fold change from negative control, de-creased significantly at 75 μM, 80 μM, 100 μM, and250 μM, and Triton-X 1% control (Figure 9). Thecontrol LNCaP cells showed a cumulative growth curvethat was showed a significant difference in the growthpattern of upon 75 μM of Simvastatin treatment com-pared to vehicle control at Days 5 and 8 (Figure 10).Upon Simvastatin treatments, LNCaP cells displayed asignificant reduction in cholesterol synthesis activity is at75 μM of Simvastatin treatment as shown in Figure 11;however, no significant changes were observed inthe HMGCR expression across the Simvastatin doses(Figure 12). The total intracellular cholesterol concen-trations, normalized to protein levels (mg), showeda significant reduction in the total cholesterol concen-tration at 75 μM of Simvastatin compared to vehiclecontrol (Figure 13). The decrease in cholesterol levelscorresponded to the decrease in HMGCR proteinactivity at 75 μM Simvastatin treatment results. Despitethe change in cholesterol synthesis activity and totalintracellular cholesterol concentration, there were noFigure 5 Relative protein expression in C4-2 cells to vehicle control expression after 48 hour Simvastatin treatment, normalized toactin expression. Columns, mean (n = 4); bars, ±SEM.Figure 6 Total cholesterol concentration in whole cell lysates of C4-2 cells after 48 hour Simvastatin treatment. Columns, mean (n = 6);bars, ±SEM. *P < 0.05, Vehicle control versus 75 μM.Kim et al. Lipids in Health and Disease 2014, 13:56 Page 5 of 12http://www.lipidworld.com/content/13/1/56significant changes observed in the levels of PSAsecretion (ng/mL) of LNCaP cells, normalized to theamount of protein (mg), upon Simvastatin treatments(Figure 14).DiscussionIn the past castration-resistant prostate cancer, now witha median overall survival of 18.4 months with combin-ation therapy of docetaxel and AR blocker MDV3100,was misunderstood as an ‘androgen independent’ formof prostate cancer based on the observations that it is ableto recur and more importantly aggressively metastasize inpatients treated with androgen deprivation therapy [23].As mentioned however, many groups have reported thecontinuous expression of AR and activation of the AR-androgen signaling pathway in castration-resistant pros-tate cancer through both in vitro as well as in vivo studiesdespite the androgen castrated environment, supportingthe existence of an intracellular de novo androgen synthe-sis pathway rather than the tumor’s ability to sustain itselfdespite the lack of androgens [24-27].HMGCR has been shown to be essential regulator forprovision of cholesterol to the androgen synthesis path-way in the steroidogenic tissues of the body [15]. Uponprogression to castration-resistance in LNCaP xenograftmodel, an increase ex vivo HMGCR activity has beenobserved [6]. Also, in castration resistant cell modelupon the knockdown of SR-BI transporter a significantFigure 7 PSA production demonstrated as secreted PSA levels in the media of C4-2 after 48 hour Simvastatin treatment. Columns,mean (n = 6); bars, ±SEM. *P < 0.05, Vehicle control versus 50 μM and 75 μM.Figure 8 Cell cytotoxicity is demonstrated as % cytotoxicity based on a100 % cytotoxic control, representing the amount of LDH inthe media of LNCaP cells treated with increasing dose of Simvastatin. Columns, mean (n = 5); bars, ±SEM. *P < 0.05, Vehicle control versus75 μM, 80 μM, 100 μM, 250 μM and TX1%.Kim et al. Lipids in Health and Disease 2014, 13:56 Page 6 of 12http://www.lipidworld.com/content/13/1/56increase in HMGCR activity was observed [15]. Thesefinding suggests that castration-resistant cells mayadapt to the low androgen environment by increasingcholesterol synthesis to supply cellular needs as wellas providing the precursor for de novo androgensynthesis. While suggesting an important role of thisenzyme in intracellular de novo androgen synthesisand potentially suggesting common HMGCR inhibi-tors such as statins as possible therapeutic agent, theeffect of inhibiting HMGCR in a castration-resistantmodel has not been investigated. The current studyaimed to investigate the physiological relevance ofone source of cholesterol to the cell, HMGCR, arate-limiting enzyme in the cholesterol synthesis-mevalonate pathway.Simvastatin is a commonly used drug to control hyper-cholesterolemia to prevent cardiovascular disease, and assuch epidemiological studies have shown significantlylower serum PSA, tumor volume, and percentage ofcancer in radical prostatectomy samples in PCa patientson preoperative statins compared to non-users [28,29].As mentioned, the effects of statins in prostate cancermodels have been extensively studied in cell and animalmodels as well, reporting a decrease in tumor viabilityand proliferation in PC-3 in vitro model as well as re-duction in PSA production in mice in vivo model; how-ever, the effects have not been thoroughly investigated inCRPC models [24-27]. The treatment of Simvastatin inandrogen-sensitive LNCaP cells confirmed the cytotoxiceffects as seen in previous studies (Figure 1). In addition,Figure 9 Cell viability is demonstrated as fold change in viability from the negative control in LNCaP cells treated with increasing doseof Simvastatin. Columns, mean (n = 5); bars, ±SEM. *P < 0.05, Vehicle control versus 60 μM, 75 μM, 80 μM, 100 μM, 250 μM and TX1%.Figure 10 Cumulative cell growth curve expressed as total cell count of LNCaP cells over 8 days. Curve, mean (n = 5); error bars, ± SEM.*P < 0.05, Vehicle control versus 75 μM and TX1% in at Day 5 and 8.Kim et al. Lipids in Health and Disease 2014, 13:56 Page 7 of 12http://www.lipidworld.com/content/13/1/56the inhibition of HMGCR activity via Simvastatin signifi-cantly stunted the cellular growth of castration-resistantC4-2 cells in a similar dose-dependent manner as thecontrol LNCaP cells (Figures 1 and 8). The two cell lineswere shown to display cellular cytotoxicity, viability, andgrowth curve patterns that are not significantly differentfrom each other; both cell lines showed nearly 50%reduction in cell viability after 48 hour incubation of75 μM of Simvastatin (Figures 1, 2 and 3).Cholesterol is an important precursor molecule in thepathways involved in the production of androgens,which stimulate prostate growth; the changes in growthand proliferation seen in Figures 1, 2 and 3 may be a re-sult of cholesterol synthesis alteration caused by Simva-statin. Upon Simvastatin treatments, C4-2 cells showedsignificant changes in the HMGCR activity, normalizedto protein amount (μg), at 50 μM and 75 μM of Simva-statin (Figure 4). Corresponding to past studies whichreported an increase in ex vivo HMGCR activity uponprogression to castration-resistance in a LNCaP xeno-graft model, the current study also reported a five-foldincrease in basal cholesterol synthesis activity in the notreatment groups of C4-2 cells compared to LNCaP cellsas shown in Figures 4 and 11 [6]. Upon examining theHMGCR protein expression levels, Simvastatin treat-ment did not significantly alter the expression of theHMGCR protein in C4-2 and LNCaP cells (Figures 5and 12); as a competitive inhibitor of HMGCR, statinsare not known to affect the enzyme function via silen-cing mechanism. Corresponding to the HMGCR activityresults, at 75 μM of Simvastatin both cell lines showeda significant decrease in the intracellular cholesterolFigure 11 Cholesterol synthesis measurement by incorporation of 14C-acetic acid into cellular cholesterol as measured by β-scintillationcounter in LNCaP after 48 hour Simvastatin treatment followed by 3 hour 14C-acetic acid treatment and separation by thin layerchromatography. Columns, mean (n = 5); bars, ±SEM. *P < 0.05, Vehicle control versus 75 μM.Figure 12 Relative protein expression in LNCaP cells to vehicle control expression after 48 hour Simvastatin treatment, normalized toactin expression. Columns, mean (n = 4); bars, ±SEM.Kim et al. Lipids in Health and Disease 2014, 13:56 Page 8 of 12http://www.lipidworld.com/content/13/1/56content, normalized to protein levels (mg) (Figures 6and 13). However in C4-2 cells, upon 50 μM of Simva-statin, the intracellular cholesterol quantity remained thesame despite the decrease in HMGCR activity, whichmay be an indication of compensatory pathways activa-tion to supply the cell with the necessary cholesterolsource via other cholesterol homeostasis pathways des-pite the inhibition of HMGCR (Figures 4 and 6).An important indication of AR-androgen pathway acti-vation is the production of PSA, which is produced in re-sponse to transcription of genes that contain androgenresponse elements (AREs). Similar to the PSA observa-tions seen in previously published papers, the basal PSAsecretion levels in C4-2 cells was significantly higher; thecurrent study showed an increase that is approximatelyfive magnitude greater in C4-2 cells than the amount se-creted by LNCaP cells (Figures 7 and 14) [30]. Whileprevious studies have reported a lack of change in the levelof AR protein expression between LNCaP and C4-2 cells,statin use has been shown to decrease the expression andactivity of the AR via suppression of the Akt/mTOR sig-naling pathway [15,30] These finding indicates the exist-ence of a continuous androgen supply, and whether thereis a greater total amount of androgens to activate the ARor the ligand-receptor binding interaction is stronger inC4-2 cells is unknown. Furthermore, the inhibition ofcholesterol synthesis significantly reduced the secretedPSA levels in C4-2 cells at 50 μM and 75 μM of Simva-statin compared to vehicle control, while no differenceswere observed in LNCaP cells across the treatment groupsimplicating that the decrease in cholesterol synthesis has agreater effect in castration-resistant state and supportingthe hypothesis of existence and greater reliance on intra-cellular de novo steroidogenesis in CRPC.Figure 13 Total cholesterol concentration in whole cell lysates of LNCaP cells after 48 hour Simvastatin treatment. Columns, mean(n = 6); bars, ±SEM. *P < 0.05, Vehicle control versus 75 μM.Figure 14 PSA production demonstrated as secreted PSA levels in the media of LNCaP after 48 hour Simvastatin treatment.Columns, mean (n = 6); bars, ±SEM.Kim et al. Lipids in Health and Disease 2014, 13:56 Page 9 of 12http://www.lipidworld.com/content/13/1/56In addition to the competitive inhibition of HMGCR,Simvastatin like most other statins may exert choles-terol-independent or pleiotropic effects through directinhibition of small signaling molecules; by inhibitingHMGCR, statins inhibit cholesterol synthesis as well asthe synthesis of isoprenoids which are important lipidattachments for intracellular signaling molecules such asRho, Rac, and Cdc42 [31,32]. Despite the decreases inHMGCR activity, total intracellular cholesterol, andPSA, if a constant intracellular testosterone level acrossthe Simvastatin treatment groups were observed, it mayindicate that the signaling processes are being affected.In the current study, the decrease in PSA secretion maybe due to the direct effect of Simvastatin on cholesterol-androgen production and androgen receptor activationand/or indirect effect on the signaling pathways.The effect of regulating the cholesterol homeostasispathway to alter the growth of CRPC cells has also beenseen in cholesterol influx regulation [15]. Recentlyscavenger-receptor class B type I (SR-BI) receptor hasalso emerged as a potential therapeutic target of interestin investigating PCa progression as well as de novo an-drogen synthesis due to the discovery of a significant in-crease in the SR-BI protein expression in CRPC cellscompared to non-castrated LNCaP cells [15]. SR-BI re-ceptor is one of the two major cholesterol influx trans-porters, mainly uptake of cholesterol esters bound tohigh density lipoprotein (HDL) via a docking mechan-ism. It has been shown that down-regulation of SR-BIexpression via gene silencing significantly reduced theviability of C4-2 cells, and at a greater extent comparedto LNCaP cells [15]. Furthermore, a significant reductionin the amount of PSA secreted by both C4-2 and LNCaPcells was observed when comparing SR-BI knockdownto negative control cells [15]. However, the limitations ofthe study were that no corresponding differences inintracellular cholesterol and androgen levels were ob-served. The results from the current study looking at in-hibition of cholesterol synthesis via use of Simvastatinreported a significant reduction in cell viability, intracellu-lar cholesterol levels, PSA secretion, and cholesterol syn-thesis activity in C4-2 cells upon Simvastatin treatments.It would be worthwhile to investigate the inhibition ofmultiple cholesterol regulatory pathways including choles-terol influx and synthesis using combination treatment ofSR-BI silencing and Simvastatin to assess the effect of acombination treatment in CRPC models.ConclusionIn summary, the inhibition of HMGCR via Simvastatinlowered the viability of castration-resistant C4-2 cells.Simvastatin’s ability to limit the endogenous supply ofcholesterol likely contributes to the effects seen in cellviability.Materials and methodsMaterialsPoly-L-lysine (0.01% solution), cholesterol, Triton® X-100,dimethyl sulphoxide, phenylmethylsulfonyl fluoride, prote-ase inhibitor cocktail, Trizma®-hydrochloride, Trizma®base, glycine, lyophilized bovine serum albumin, sodiumdodecyl sulfate, ammonium persulfate, tetramethylethyle-nediamine, sodium hydroxide, sodium chloride, sodiumdeoxycholate, nonyl phenoxypolyethenyl ethanol (NP-40)and ethylenediamininetetraacetic acid (Sigma-Aldrich, St.Louis, MO, USA), acetic acid [1-14C]- (American Radiola-beled Chemicals, Inc., Saint Louis, MO, USA), cholesterol[1, 2-3H(N)]- (PerkinElmer Inc. Waltham, MA, USA).Scintillation fluid was purchased from MP Biomedicals(Solon OH, USA). RPMI-1640 without phenol red, Hank’sbalanced salt solution, 0.25% trypsin-EDTA, penicillin-streptomycin liquid, fetal bovine serum, charcoal-strippedfetal bovine serum, were purchased from Invitrogen(Life Technologies, Invitrogen, Burlington, Ontario).Chloroform, ethyl acetate, hexanes, ethanol, methanoland isopropanol were purchased from Fisher Scientific(Waltham, MA, USA). SuperSubstrate® West Pico Solu-tion was obtained from Thermo Scientific (Rockford IL,USA).Cell cultureC4-2 and LNCaP cells were obtained from the VancouverProstate Centre. LNCaP cells were used for experimentbetween the passage numbers of 42-52. The cells werecultured in RPMI-1640 medium without phenol redcontaining 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in 5% CO2 environment. 72 hoursprior to Simvastatin treatment, the cells were seeded inplates in 10% fetal bovine serum supplemented RPMImedium. After the initial 24 hours, the medium wasreplaced with RPMI medium supplemented with 5%dextran-charcoal stripped medium with restricted andro-gens to mimic an androgen-castrated environment. Cellswere seeded in plates to include vehicle control, Simva-statin treatment, no treatment (NT) and 100% cell lysis(Triton X100, 1%) control wells.Cell cytotoxicity and viabilityCells were plated in 96-well plates at a density of 6 × 103cells per well 72 hours prior to Simvastatin treatment.Treatments and controls were assigned in triplicates ineach plate. 50 μL of media was taken from above thecells 48 hours post Simvastatin incubation for LDHcytotoxicity assay. The remaining cells were rinsed withHank’s Balanced Salt Solution (HBSS) and MTS assaywas performed to detect cell viability. Protein determin-ation was performed using BSA assay to normalize theMTS values to protein concentration.Kim et al. Lipids in Health and Disease 2014, 13:56 Page 10 of 12http://www.lipidworld.com/content/13/1/56Cumulative cellular growthCells were plated in 6-well plates at a density of 1.8 × 105cells per well 72 hours prior to Simvastatin treatment.48 hour post-seeding (Day 0), cells were carefully detachedfrom the plates using 0.25% Trypsin-EDTA, and countedusing haemocytometer. The procedure was repeated at72 hour post-seeding (Day 1), 48 hours post-Simvastatintreatment (Day 3), and 72 hours post-Simvastatin treat-ment removal (Day 6).Cholesterol synthesis (HMGCR activity) assayC4-2 and LNCaP cells were seeded in 12-well plates at adensity of 8 × 105 cells per well 72 hours prior to Simva-statin treatment. Following the Simvastatin treatmentfor 48 hours, the cells were washed twice with HBSSand labeled with 0.5 μCi of 14C-acetic acid prepared infresh Simvastatin solutions for 3 hours. Cells werewashed three times with HBSS following incubation.The lipid content in the samples was extracted using theBligh Dyer lipid extraction method as previously de-scribed, dried under nitrogen, and reconstituted inchloroform [33]. The chloroform samples were sepa-rated using thin layer chromatography on silica gel platein 9:1 hexane to ethyl acetate solution. A 10 mg/mL coldcholesterol sample and an internal extraction control,3H [1,2]-cholesterol loaded at 1 uCi/mL, were includedin the thin layer chromatography procedure as controls.After the solution has reached the top, the plate was air-dried and exposed to iodine crystals. The yellow choles-terol bands were excised and reconstituted in scintilla-tion counter fluid to be measured in beta scintillationcounter (Perkin Elmer).Protein expressionProtein levels were measured in the collected supernatantsand known amounts of protein were separated using4-15% SDS gels. Protein transfer, membranes wereblocked and incubated overnight with HMGCR (UpstateBiotech®) and β-actin (Santa Cruz®) 1° antibodies at 1:1000dilutions. Membranes were then incubated with HRP-labeled 2° antibodies at a 1:3000 dilution to detect the pro-teins using chemiluminescence detection method.Quantification of cholesterol and prostate specific antigenThe Amplex Red Cholesterol assay (Invitrogen) was per-formed as per manufacturer’s instructions to measurethe total intracellular cholesterol amount. 50 μL of cellextracts and cholesterol reference standards were ali-quoted in triplicates into a 96-well plate and AmplexRed reaction buffer and working solution were added.Plates were incubated for thirty minutes at 37°C in thedark. Fluorescence was measured using an excitationwavelength of 560 nm and an emission wavelength of590 nm. Cholesterol concentration was normalized tothe amount of protein present in the lysates.The amount of PSA secreted by the cancer cells wasquantified using the PSA ELISA assay (ProClin Inter-national). 50uL of reference standards and media fromeach treatment and control wells were aliquoted in trip-licates into a 96-well plate. Following incubations withreaction buffer, enzyme conjugate reagent, and TMB re-agent, the optical density was measured at 450 nm andthe PSA concentration was normalized to the amount ofprotein present in the media.Statistical analysisAll data are presented as mean + standard error of themean. Paired t-tests and one-way ANOVA tests (Sigma-Stat 3.5) were performed to compare the normalized ex-pressions and concentrations between treatment groups.Tukey post hoc tests were done to determine the criticaldifferences, and a difference was considered to be signifi-cant if the probability of chance explaining the differencewas less than 5% (p < 0.05).AbbreviationsACAT: Acetyl-CoA acyltransferase; ADT: Androgen deprivation therapy;AR: Androgen receptor; CRPC: Castration-resistant prostate cancer;DHT: Dihydrotestosterone; ELISA: Enzyme-linked immunosorbent assay;HMG-CoA: 3-hydroxy-3-methylglutaryl-coenzyme A; HMGCR: 3-hydroxy-3-methylglutaryl-coenzyme A reductase; HSL: Hormone sensitive lipase;LDH: Lactate dehydrogenase; LDLr: Low density lipoprotein receptor;MTS: (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; NT: No Treatment; PCa: Prostate cancer;PSA: Prostate specific antigen; SR-BI: Scavenger receptor Class B Type I;SV: Simvastatin.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsJK carried out the studies, performed the statistical analysis, and drafted themanuscript. MC and KW conceived of the study, participated in the designof the study, and helped to draft the manuscript. All authors read andapproved the final manuscript.AcknowledgementsThe research was supported by funding from Prostate Cancer Canada andCanadian Institutes of Health Research. We would also like to thank theVancouver Prostate Centre for all their support.Author details1Faculty of Pharmaceutical Sciences, University of British Columbia, 2405Wesbrook Mall, Vancouver, British Columbia, Canada. 2Department ofUrologic Sciences, The Prostate Centre at Vancouver General Hospital, 2660Oak Street, Vancouver, British Columbia, Canada.Received: 26 December 2013 Accepted: 11 March 2014Published: 26 March 2014References1. Siegel R, Naishadham D, Jemal A: Cancer statistics 2012. Cancer J Clin 2012,62:10–29.2. Baade PD, Youlden DR, Krnjacki LJ: International epidemiology of prostatecancer: geographical distribution and secular trends. Mol Nutr Food Res2009, 53:171–184.3. 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Pharmacol Res 1995, 31(1):9–27.33. Bligh EG, Dyer WJ: A rapid method of total lipid extraction andpurification. Can J Biochem Physiol 1959, 37(8):911–917.doi:10.1186/1476-511X-13-56Cite this article as: Kim et al.: Effect of simvastatin on castration-resistantprostate cancer cells. Lipids in Health and Disease 2014 13:56.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionSubmit your manuscript at www.biomedcentral.com/submitKim et al. Lipids in Health and Disease 2014, 13:56 Page 12 of 12http://www.lipidworld.com/content/13/1/56

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