A Novel Synthetic Compound That Interrupts Androgen Receptor Signaling in Human Prostate Cancer Cells

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A novel synthetic compound that interrupts androgen receptor signaling in human prostate cancer cells

Shan Lu,1 Amy Wang,1 Shan Lu,2 Introduction 1 and Zhongyun Dong Prostate cancer is the most common cancer and the second most common cause of cancer death among men in the Departments of 1Internal Medicine and 2Pathology, University of Cincinnati College of Medicine, Cincinnati, Ohio United States (1). As detection techniques improve, more patients are diagnosed with localized disease and can be cured byeither surgeryor radiation therapy. Abstract Metastasis in manypatients, however, still occurs before The purpose of this study was to determine the effects of 6- the initial diagnosis. Hormonal therapies, commonlywith amino-2-[2-(4-tert-butyl-phenoxy)-ethylsulfonyl]-1H-py- combinations of antiandrogens (flutamide, nilutamide, rimidine-4-one (DL3), a novel synthetic compound with or bicalutamide) and androgen deprivation, are the main- small-molecule drug properties, on androgen-regulated staytreatment for advanced diseases. These therapies, gene expression and cell growth in human prostate cancer however, onlydelaytumor progression byan average of cells. LNCaP, 22Rv1, and LAPC-4 cells were used in the <18 months, followed bythe development of hormone- studies. Expression of prostate-specific antigen (PSA) and refractorydisease (2). A discontinuation of an antian- androgen receptor (AR) was determined by ELISA, Western drogen therapyoften results in clinical improvement and blotting, real-time reverse transcription-PCR, nuclear run- a decrease in serum prostate-specific antigen (PSA) in on, and/or promoter luciferase reporter assays. Effects of manypatients with hormone-refractorydisease (i.e., DL3 on cell growth were determined by the 3-(4,5-dime- antiandrogen withdrawal syndrome), which is partially thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide staining. caused bythe intrinsic androgenic activityof the anti- DL3 inhibited dihydrotestosterone (DHT)-induced PSA ex- androgens (3). Therefore, there is an urgent need to iden- pression in a dose-dependent fashion. The inhibitory effects tifyand develop more effective therapeutic agents for of DL3 were more potent than those of flutamide, nilu- prostate cancer. tamide, and bicalutamide. Moreover, DL3 blocked the sti- Androgen receptor (AR), a member of the steroid receptor mulatory effects of nilutamide on PSA expression in LNCaP superfamily, is a ligand-dependent transcription factor cells. Unlike the three classic antiandrogens, DL3 did not that mediates androgen action in cells. AR is composed show intrinsic AR agonist activity. Nuclear run-on and PSA of three major domains: an NH2-terminal transcriptional promoter reporter assays revealed that DL3 blocked DHT- activation domain (NTD), a central DNA-binding domain, induced PSA gene transcription. Consistent with its effects and a COOH-terminal ligand-binding domain (LBD; 4, 5). on PSA expression, DL3 inhibited DHT-stimulated cell AR is associated with cellular chaperones in the cytosol growth with a potency significantly superior to flutamide, in its inactive state (6). After binding to androgens, such nilutamide, or bicalutamide. Furthermore, cells resistant to as testosterone and, more potently, dihydrotestosterone flutamide or nilutamide were as susceptible as their parental (DHT), AR translocates to the nucleus, binds to andro- counterparts to the inhibitory effects of DL3 on both PSA gen response elements of AR target gene promoter, and expression and cell growth. DL3 did not inhibit AR nuclear regulates expression of AR target genes (4, 7). AR hyper- sensitivity, as a result of AR gene mutation and/or localization and the NH2- and COOH-terminal interaction of AR induced by DHT. These data show that DL3 is a novel amplification, overexpression of coactivators, and AR inhibitor of the AR signaling axis and a potentially potent cross-talking with other signal transduction pathways, therapeutic agent for the management of advanced human often occurs and plays crucial roles in prostate cancer prostate cancer. [Mol Cancer Ther 2007;6(7):2057–64] development, progression, and androgen-independent growth (7–9). Therefore, AR and its signaling axis are the most important targets for therapies against advanced prostate cancer (7–9). Received 11/28/06; revised 5/17/07; accepted 5/23/07. In the present report, we identified 6-amino-2-[2-(4-tert- Grant support: University of Cincinnati Cancer Center (S. Lu2 and Z. Dong) butyl-phenoxy)-ethylsulfonyl]-1H-pyrimidine-4-one (DL3), and NIH/National Cancer Institute grant CA97099-01 A1 (Z. Dong). a novel synthetic small molecule with potent anti-AR The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked signaling activities. DL3 inhibited expression of PSA, a advertisement in accordance with 18 U.S.C. Section 1734 solely to widelyused serologic marker for prostate cancer burdens indicate this fact. and an indicator of therapeutic efficacyand recurrence Requests for reprints: Zhongyun Dong, Department of Internal Medicine, (10, 11). DL3 also inhibited androgen-stimulated growth University of Cincinnati College of Medicine, Room 1308, 3125 Eden Avenue, Cincinnati, OH 45267. Phone: 513-558-2176; in prostate cancer cells. Moreover, DL3 did not show any Fax: 513-558-6703. E-mail: [email protected] detectable intrinsic AR agonist activityand inhibited PSA Copyright C 2007 American Association for Cancer Research. expression and in vitro growth of prostate cancer cells doi:10.1158/1535-7163.MCT-06-0735 resistant to flutamide or nilutamide.

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Materials and Methods and the cell suspension was gentlyagitated to produce a DL3 Compound single-cell suspension. The cells were washed in HBSS and During a high-throughput screening of a chemical library resuspended in growth medium. Onlysuspensions of (ChemBridge Co.), we identified the novel compound DL3 single cells with viabilityexceeding 95% (ascertained by (Fig. 1A), which selectivelyinhibited expression of genes trypan blue exclusion) were used. regulated byandrogen response element–driven promoter. PSA Detection Â 4 Tu m o r C e l l s a n d C u l t u r e LNCaP cells were plated into 96-well plates at 2 10 The well-characterized LNCaP (androgen responsive; per well in S-MEM (MEM medium supplemented with refs. 12, 13) and 22Rv1 (androgen weaklyresponsive; 10% charcoal-dextran treated fetal bovine serum) or refs. 13, 14) cell lines were purchased from American Type C-MEM. After 24-h incubation, the cells were treated for Culture Collection. HeLa cells were also obtained from 48 h as detailed in Results. PSA in the culture superna- American Type Culture Collection. The cells were main- tants was quantified with an ELISA kit (United Biotech, tained as a monolayer culture in MEM supplemented with Inc.) following the manufacturer’s instruction. Intracel- 10% fetal bovine serum, nonessential amino acids, sodium lular PSA was detected byWestern blotting as described pyruvate, vitamin A, and glutamine (C-MEM). LAPC-4 below. cells, which express the wild-type AR and are androgen Western Blot Analysis 6 responsive (13, 15), were generouslyprovided byDr. Karen Cells (2 Â 10 per 60-mm dish) were washed and scraped Knudson, with the approval of Dr. Robert Reiter. LAPC-4 into a lysis buffer and analyzed by Western blotting, as cells were maintained in Iscove’s modified Dulbecco’s described in our previous study(16), with PSA and AR- medium supplemented with 10% fetal bovine serum and specific antibodies obtained from DakoCytomation and 10 nmol/L DHT. LNCaP cells were cultured in C-MEM Santa Cruz Biotechnology, Inc., respectively. The immuno- containing 20 Amol/L of flutamide or nilutamide for >2 reactive signals were revealed with the enhanced chemilu- months to derive LNCaP-Flut-R and LNCaP-Nilut-R cells, minescence Western blotting detection system (Michigan respectively. Cells in exponential growth phase were Diagnostic LLC) and visualized in a KODAK Image Station harvested bya 1- to 3-min treatment with a 0.25% IS4000MM Digital Imaging System (Eastman Kodak Co.). trypsin-0.02% EDTA solution. The flasks were tapped to Expression of PSA, AR, and h-actin was quantitated in the detach the cells, MEM-10% fetal bovine serum was added, linear range of exposure.

Figure 1. Effects of DL3, flutamide, nilutamide, and bicalutamide on PSA expression. A, chemical structure of DL3. Androgen-starved LNCaP cells in 96-well plates at 2 Â 104 per well in S-MEM were incubated for 48 h with 1 nmol/L DHT and/or the four drugs (B), with DL3 and/or DHT (C), or with DL3 and/or nilutamide (D). PSA levels, normalized by cell density with the MTT staining, in the culture supernatant were determined by ELISA. *, P < 0.05; **, P < 0.01; ***, P < 0.001, in comparison with the cultures in the absence of inhibitors or DHT.

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Quantitative Real-time PCR Cells were seeded in 60-mm dishes (2 Â 106 per dish). After treatment as desired, the cells were washed and total RNA was extracted with TRIzol reagent. Expression of PSA and AR was analyzed by real-time reverse transcription- PCR (RT-PCR) as detailed in our previous studies (17). The cycle threshold values were used to calculate the normalized gene expression against h-actin using the Q-Gene software. Nuclear Run-On Assay RT-PCR-based nuclear run-on assaywas done as detailed in our previous study(17). Briefly,cells in 15-cm dishes were rinsed with cold PBS and scraped in lysis buffer on ice, followed byaddition of the same volume of 0.6 mol/L sucrose buffer. Cell nucleus pellets, obtained bycentrifu- gation at 500 Â g for 10 min, were incubated for 30 min at 30jC in 500 AL of transcription reaction buffer (pH 7.9) containing 50 mmol/L Tris, 5 mmol/L MgCl2, 0.1 mmol/L EDTA, and 0.5 mmol/L nucleotide triphosphate. RNA was extracted and followed byDNase treatment and real-time RT-PCR. Samples without transcription reaction were used as controls. Transient Transfection and Luciferase Assays LNCaP cells (5 Â 104 per well in 48-well plates) were cotransfected for 24 h with PSA promoter luciferase reporter plasmid (pGL3-PSA-luc) and internal control Renilla luciferase vector (pRL-CMV-luc) using the Lip- ofectamine 2000 transfection reagent (Invitrogen). Lucifer- Figure 2. Effects of DL3, flutamide, nilutamide, and bicalutamide on ase activities were measured byusing the dual-luciferase PSA and AR expression. A, androgen-starved LNCaP cells in S-MEM were reporter gene assaysystem(Promega) following the treated for 48 h with DL3. B, LAPC-4 (in S-MEM) or 22Rv1 cells (in C- manufacturer’s instruction. Final results were normalized MEM) were treated for 48 h with 10 nmol/L DHT, 20 Amol/L DL3, or 10 nmol/L DHT plus 20 Amol/L DL3 (LAPC-4 cells) or with DL3 (22Rv1 for transfection efficiencies using the Renilla luciferase cells). C, androgen-starved LNCaP cells in S-MEM were treated for 48 h with assayvalue (16, 17). 20 Amol/L DL3, flutamide, nilutamide, or bicalutamide. Cellular PSA and AR The mammalian two-hybrid assay was done to evaluate were analyzed by Western blotting. Representative of five experiments. ligand-induced AR NH2- and COOH-terminal interaction (18–20). Briefly, HeLa cells (105 per well in 12-well plates) Immunofluorescent Staining of AR were cotransfected for 24 h with GAL4-luc (a luciferase Control and treated LNCaP cells were permeabilized with reporter driven bythe GAL4 binding site), GAL4-LBD (a 0.25% NP40 in PBS, fixed in 3.7% formaldehyde in PBS for plasmid encoding GAL4 DNA-binding domain fused with 10 min, blocked in PBS containing 5 mg/mL bovine serum the LBD of AR), and VP16-NTD [a plasmid encoding albumin and 0.1% Tween 20 for 30 min, and incubated with etoposide (VP16) transactivation domain fused with the the antibodyagainst AR in the blocking buffer for 2 h; then, NTD of AR]. GAL4-LBD and VP16-NTD were generously cells were washed in PBS-0.1% Tween 20 and incubated with provided byDr. Karen Knudson. The cells were treated Texas red–conjugated antirabbit antibodyin the blocking with DHT, flutamide, or DL3 in S-MEM for 24 h, followed buffer for 1 h. After wash in PBS-0.1% Tween 20, the slides byluciferase as detailed above. were mounted and examined under a fluorescent micro- Cell DensityAssay scope. The images were captured with a Spot cooled charge- LNCaP cells were plated into 96-well plates at 1,000 per coupled device camera and digitized to a computer. well. After an overnight incubation, the cells were treated, Statistical Analysis as detailed in Results, for 4 days and viable cells in the All experiments were done at least twice. Data shown are wells were stained with the 3-(4,5-dimethylthiazol-2-yl)- the mean F SE. Differences between means were compared 2,5-diphenyltetrazolium bromide (MTT) assay as previous- bythe two-tailed Student t test and were considered lydescribed (17, 21). During the final 2 h, 0.4 mg/mL MTT significantlydifferent at P < 0.05. (Sigma) was added. After removing the medium, dark-blue formazan was dissolved in DMSO and the absorbance at 570 nm was measured with a FLUOstar Optima microplate Results reader (BMG LABTECH, Inc.). Inhibition of cell growth Inhibitory Effects of DL3 on PSA Expression was calculated bythe following formula: growth inhibition After starvation for 48 h in S-MEM, LNCaP cells were À Â (%) = (1 A570 of treated / A570 of control) 100. treated for 48 h with DHT in the absence or presence of

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DL3, flutamide, nilutamide, or bicalutamide. The normal- wild-type AR (Fig. 2B, left) and 22Rv1 cells that express a ized data in Fig. 1B show that LNCaP cells secreted very mutant AR (Fig. 2B, right). Note that both LAPC-4 and low basal levels of PSA in the absence of DHT, which was 22Rv1 cells expressed much lower levels of PSA. The expo- significantlyelevated in cells incubated with 1 nmol/L sure of the membranes in Fig. 2B was five to six times DHT (Fig. 1B). Treatment with 20 Amol/L DL3 did not longer than that in Fig. 2A. induce PSA production and inhibited the DHT-induced Because down-regulation of AR could be one of the PSA production by f90% (Fig. 1B). Unlike DL3, treatment potential mechanisms bywhich DL3 interrupts AR with 20 Amol/L of flutamide or bicalutamide and, much signaling, we examined the effects of DL3 on AR more potently, with nilutamide stimulated PSA production expression. As shown in Fig. 2A and B, the expression of under androgen-free conditions (Fig. 1B), which is consis- AR in both LNCaP and 22Rv1 cells was not significantly tent with the observations reported previouslybyothers altered in cells treated with 5 and 10 Amol/L DL3, but (3, 22–26). Flutamide and bicalutamide, but not niluta- reduced in cells treated with 20 and 40 Amol/L DL3. mide, reduced DHT-induced PSA production (Fig. 1B). Similarly, AR expression was reduced in LAPC-4 cells The inhibitoryeffects of DL3 on DHT-induced PSA pro- treated with 20 Amol/L DL3 (Fig. 2B). Next, we compared duction were dose dependent, with a 50% inhibition at the effects of DL3, flutamide, nilutamide, and bicalutamide f5to10Amol/L DL3 and were partiallyreversed by on PSA and AR expression in LNCaP cells (Fig. 2C). increasing concentrations of DHT, suggesting that the Consistent with the data from ELISA, flutamide and bica- inhibition was due to AR blockade rather than nonspecific lutamide moderatelyenhanced PSA expression under toxicity(Fig. 1C). Data in Fig. 1D show that stimulatory androgen-free culture conditions but inhibited DHT- effects of nilutamide on PSA production were dose induced PSA expression. On the other hand, nilutamide dependent and were also blocked byDL3. enhanced PSA expression in LNCaP cells regardless of To determine whether the reduction of PSA in the culture the absence or presence of DHT. AR expression was down- supernatant of DL3-treated cells was due to a blockade in regulated in cells treated with DL3 or bicalutamide, not PSA secretion, we investigated intracellular accumula- altered byflutamide, and enhanced bynilutamide. tion of PSA byWestern blotting. Data in Fig. 2A show DL3 Inhibited PSA Gene Transcription that androgen-starved LNCaP cells expressed a low level As shown in Fig. 3A, PSA mRNA level in LNCaP cells of PSA, which was significantlyincreased bytreatment was f20-fold higher than that in 22Rv1 cells, which was with 1 nmol/L DHT (Fig. 2A). The DHT-induced PSA in agreement with PSA protein levels in the two cell expression was reduced byDL3 in a dose-dependent lines. DL3 inhibited the expression of PSA mRNA in both manner and diminished in cells treated with 40 Amol/L LNCaP and 22Rv1 cells in a dose-dependent fashion DL3 (Fig. 2A). The similar inhibitoryeffects of DL3 on PSA (Fig.3A).TreatmentofLNCaPcellsfor24hwith expression were observed in LAPC-4 cells that express a 20 Amol/L DL3 reduced basal PSA mRNA level and

Figure 3. Effects of DL3 and bicalutamide on expression of PSA mRNA and transcription of PSA gene. A, LNCaP or 22Rv1 cells were incubated for 24 h in C-MEM with DL3. Total cellular RNA was analyzed by real-time RT-PCR for PSA expression with h- actin as loading control. B and C, androgen-starved LNCaP cells in S- MEM were treated with 20 Amol/L of DL3 or bicalutamide and/or 1 nmol/L DHT. Total cellular RNA was analyzed by real-time RT-PCR for PSA (B)orAR (C) mRNA expression. D, androgen- starved LNCaP cells were incubated with 20 Amol/L DL3 and/or 1 nmol/L DHT. Cell nucleus pellets were incubated in transcription reaction buffer to synthesize RNA followed by real-time RT-PCR analysis. Representative of two experiments. D, LNCaP cells in S-MEM were cotransfected for 24 h with pGL3-PSA-luc and control Renilla luciferase vector pRL-CMV-luc, followed by treatment for 24 h with 1 nmol/L DHT and/or 20 Amol/L DL3. Luciferase activity in the lysates was assessed. Representative of five experiments.

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Figure 4. Effects of DL3, flutamide, nilutamide, and bicalutamide on cell growth. Androgen-starved LNCaP cells in S-MEM were treated for 4 d with DL3 (A), flutamide (B), nilutamide (C), or bicalutamide (D) in the presence or absence of 1 nmol/L DHT. Viable cells were stained with MTT and effects of treatments on cell growth were calculated. Repre- sentative of three to five experiments. Points, mean; bars, SD. *, P < 0.05; **, P < 0.01; ***; P < 0.001, in comparison with the cultures in the absence of the drugs.

blocked DHT-stimulated PSA mRNA expression (Fig. 3B). cell growth were, however, much more potent than those In contrast, incubation with the same concentration of on cell growth in the absence of DHT. As shown in Fig. 4A, bicalutamide had no significant effects on the basal level of the growth of LNCaP cells in the presence and absence of PSA mRNA and onlypartiallyinhibited DHT-stimulated DHT was inhibited by82% and 27%, respectively,in cells PSA mRNA expression (Fig. 3B). Expression of AR mRNA treated with 10 Amol/L DL3. Similar inhibitoryeffects were in LNCaP cells was partiallydown-regulated by1 nmol/L observed in LAPC-4 and 22Rv1 cells (data not shown). DHT, 20 Amol/L DL3 or bicalutamide, or a combination of Flutamide (Fig. 4B), nilutamide (Fig. 4C), and bicalutamide either DHT and DL3 or bicalutamide (Fig. 3C). (Fig. 4D) also inhibited growth of LNCaP cells in a dose- Next, we carried out nuclear run-on assayto determine dependent manner; the magnitude of inhibition was whether DL3 decreased the transcription of PSA. LNCaP much inferior to that of DL3. Moreover, although both cells were starved and treated for 24 h with 1 nmol/L DHT. flutamide and nilutamide at 40 Amol/L inhibited cell Nuclei from control and treated cells were isolated and growth under DHT-free conditions, theypromoted cell in vitro PSA mRNA transcription bythe nuclear extracts growth in S-MEM (the absence of androgen) at concen- was assessed. As shown in Fig. 3D, the in vitro transcription trations up to 20 Amol/L (Fig. 4B and C). of PSA mRNA was increased by8-fold in DHT-treated Effects of DL3 on Cells Resistant to Flutamide and cells. Consistent with its effects on PSA protein and mRNA Nilutamide expression, DL3 abolished the DHT-induced transcription We have established LNCaP-Flut-R and LNCaP-Nilut-R of PSA. To further determine the inhibitoryeffects of DL3 cells resistant to flutamide and nilutamide, respectively. on PSA gene transcription, the PSA promoter–driven AR expression level was moderatelyreduced in the two luciferase reporter activitywas assessed in LNCaP cells sublines of drug-resistant cell (Fig. 5A). Next, we investi- transfected with pGL3-PSA-luc in which expression of gated effects of DL3 on PSA expression (Fig. 5B) and cell luciferase gene was driven by4.3 kb of human PSA gene growth (Fig. 5C) in cells refractoryto antiandrogens fluta- promoter. Data in Fig. 3D show that DHT enhanced PSA mide and nilutamide. LNCaP, LNCaP-Flut-R, and LNCaP- promoter activityby f5-fold, which was abolished when Nilut-R cells in C-MEM were treated with 10 Amol/L the cells were treated with DL3. of DL3, flutamide, or nilutamide as described above for Inhibitory Effects of DL3 on Androgen-Stimulated PSA production and cell growth. As shown in Fig. 5A Cell Growth and B, PSA production was elevated in the resistant cells DL3 inhibited growth of LNCaP cells in a dose- compared with that in their parental cells. DL3 abolished dependent manner regardless of the presence or absence PSA production in all three sublines of LNCaP cells. of DHT (Fig. 4A). The inhibitoryeffects on DHT-stimulated In contrast, the inhibitoryeffect of flutamide on PSA

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production was reduced in the drug-resistant cells com- cells but promoted growth of LNCaP-Flut-R cells (Fig. 5C), pared with that in parental cells (Fig. 5B). Nilutamide whereas nilutamide moderatelyinhibited growth of paren- enhanced PSA production byboth parental and drug- tal cells but promoted growth of the two lines of resistant cells (Fig. 5B). Similar to its effect on PSA antiandrogen-resistant cells (Fig. 5C). production, DL3 inhibited growth of all three sublines of DL3 Did Not Alter the Interaction of ARand DHT cell to a similar extent (Fig. 5C). In contrast, flutamide To determine whether DL3 interfered with the interaction moderatelyinhibited growth of parental and LNCaP-Nilut of DHT and AR, we examined DHT-induced nuclear localization of AR. LNCaP cells in S-MEM were plated into eight-well chamber slides at 2 Â 103 per well. Three days later, the cells were treated for 1 h with 20 Amol/L of DL3 or flutamide, followed byincubation for 2 h with 1 nmol/L DHT. As shown in Fig. 6A, AR in untreated LNCaP cells distributed in both cytoplasm and nucleus. AR was translocated into the nucleus after treatment with DHT. DL3 treatment alone did not significantlyalter the AR localization and had no inhibitoryeffects on DHT- induced AR translocation. In contrast, treatment with flutamide alone induced nuclear translocation of AR. TheassociationofandrogenwithARleadstoan interaction between the NH2-terminal domain and the COOH-terminal LBD of AR (i.e., the AR NH2- and COOH- terminal interaction; refs. 18, 19), which enhances the sensitivityof AR to low concentrations of androgen by increasing the stabilityof AR and the recruitment of coactivators (20). HeLa cells do not express AR. To further determine whether DL3 alters the interaction between DHT and AR, we investigated the effects of DL3 on the AR NH2- and COOH-terminal interaction in HeLa cells cotransfected with GAL4-luc, GAL4-LBD, and VP16-NTD. Data in Fig. 6B show that the luciferase activitywas not significantlyaltered byeither DL3 or flutamide and was increased by f40- to 50-fold in cells treated with 1 nmol/L DHT. Flutamide inhibited DHT-stimulated NH2-and COOH-terminal interaction in a dose-dependent manner. In contrast, the DHT-induced NH2- and COOH-terminal interaction was not significantlyaltered byDL3 (Fig. 6B).

Discussion AR signaling axis is crucial for growth of both androgen- dependent and androgen-independent prostate cancers and a keytarget in treatment of advanced prostate cancer (7–9). The goal of this research was to characterize the inhibitoryeffects of DL3, a novel syntheticsmall-molecule compound, on AR signaling. Among numerous androgen- regulated genes, we have chosen PSA because it is primarilyregulated byAR signaling at the transcriptional level and is a well-established marker for prostate cancer (10, 11). As an initial step toward exploring therapeutic potentials of this novel compound against prostate cancer, we investigated its effects on cell growth in culture. Our Figure 5. Effects of DL3, flutamide, or nilutamide on PSA expression data clearlyshow that DL3 is a potent and unique inhibitor and cell growth in androgen-refractory cells. A, immunoblotting analysis of AR and PSA expression in LNCaP (lane 1), LNCaP-Flut-R (lane 2), and of AR signaling axis. Not onlyis DL3 more potent than LNCaP-Nilut-R (lane 3) cells. LNCaP, LNCaP-Flut-R, or LNCaP-Nilut-R cells flutamide, nilutamide, and bicalutamide in suppressing in C-MEM were treated for 48 h (B)or4d(C) with 10 Amol/L of DL3, both DHT-induced PSA expression and cell growth, but it flutamide, or nilutamide. PSA in the culture supernatants was measured by also blocks the AR agonist activityof nilutamide on PSA ELISA (B) and viable cells were stained with MTT (C). Representative of five experiments. Columns, mean; bars, SD. *, P < 0.05; **, P < 0.01, expression. Moreover, DL3 inhibits PSA expression in and in comparison with untreated cells. growth of cells resistant to flutamide and nilutamide.

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Figure 6. Effects of DL3 on the interaction of AR and DHT. A, androgen-starved LNCaP cells in S-MEM were treated for 2 h with 20 Amol/L of DL3 or flutamide and/or 1 nmol/L DHT. The cells were then fixed for immunofluorescent staining. B, HeLa cells were plated into 12-well plates and cotransfected for 24 h with GAL4-luc, GAL4- LBD, and VP16-NTD. The cells were then treated for 24 h with DL3 or flutamide in the presence or absence of 1 nmol/L DHT. Luciferase activity in cell lysates was determined. **, P < 0.01, in comparison with cells treated with DHT alone.

Furthermore, DL3 has no detectable AR agonist activityin were used at similar concentrations. Fourth, cells resistant either stimulating cell growth or inducing PSA expression. to either flutamide or nilutamide are still susceptible to Taken together, these data indicate that DL3 is a unique AR the inhibitoryeffects of DL3 on both PSA expression and signaling inhibitor. cell growth. Whether DL3 alters the binding of DHT to the The data presented clearlyshow that both cellular and LBD of AR remains to be determined in a ligand-binding secreted PSA are significantlyreduced in cells exposed to assay. DL3, suggesting that the treatment with this compound In summary, the data presented in this study reveal that down-regulates PSA production. Whether this reduction of DL3 is a novel AR signaling inhibitor, which can block AR- PSA production in DL3-treated cells is caused bya regulated gene expression and cell growth in human reduction of PSA synthesis, its stability, or both remains prostate cancer cells expressing both mutant and wild- to be further determined. However, given that PSA type AR and in both parental and antiandrogen-resistant expression is regulated mainlyat transcription level cells. The most unique propertyof this novel compound is (10, 11) and that DL3 down-regulates PSA mRNA, PSA that it displays no detectable intrinsic AR agonist activity. gene transcription in vitro, and PSA promoter activity, the The inhibitoryeffects of DL3 seem to be due not to blockade of PSA gene transcription is probablyof major interference with the interaction of AR and its ligand, but importance in DL3-regulated PSA expression. to novel mechanisms remaining to be further elucidated The dose-response analysis reveals the differential effects and to the down-regulation of AR. The molecular mecha- of DL3 on AR expression and the AR-induced responses nisms, such as its effects on expression of cyclin-dependent (i.e., DHT-induced PSA expression and DHT-stimulated kinases and cyclins, by which DL3 inhibits cell growth, cell growth). At concentrations of 5 to 10 Amol/L, DL3 particularlythe growth in the absence of androgen, remain significantlyinhibits both PSA expression and cell growth to be elucidated byin-depth analysis.Moreover, further but has no significant effects on AR expression. In contrast, studies on the in vivo antitumor effect in prostate cancer DL3, at concentrations >20 Amol/L, down-regulates AR xenografts or mouse models of prostate cancer progression expression at both protein and mRNA levels. These data are required to determine therapeutic potential of DL3 in suggest that the inhibition of AR signaling byDL3 maybe treatment of human prostate cancer. mediated byboth direct interruption of AR function and down-regulation of AR expression. It is noteworthythat Acknowledgments DL3, at concentrations that abolish DHT-stimulated cell We thank Dr. Zhengxin Wang (Department of Cancer Biology, University growth or PSA expression, has minimal effects on both of Texas M. D. Anderson Cancer Center, Houston) for providing the plasmid pGL3-PSA-luc, Dr. Robert Reiter (Department of Urology, basal and DHT-stimulated AR NH2- and COOH-terminal University of California at Los Angeles, Los Angeles, CA) for the approval interaction (19, 22, 27). Because the AR NH2- and COOH- to use LAPC-4 cells in our laboratory, Dr. Karen Knudson (Department of terminal interaction relies on the association of androgen Cell and Cancer Biology, University of Cincinnati College of Medicine, with the LBD of AR, these data suggest that the inhibition Cincinnati, OH) for providing LAPC-4 cells and the plasmids GAL4-LBD and VP16-NTD, and Dr. Robert Franco (Department of Internal Medicine, of AR signaling byDL3 maynot be mediated byinterfering University of Cincinnati College of Medicine) for critical reading of this with the interaction of AR with its ligand. This conclusion manuscript. is supported byseveral lines of evidence. First, DL3 does not significantlyalter the AR localization and has no References inhibitoryeffects on DHT-induced AR nuclear transloca- 1. Jemal A, Murray T, Ward E, et al. Cancer statistics, 2005. CA Cancer J Clin 2005;55:10 – 30. tion. Second, unlike the three AR antagonists (flutamide, 2. Crawford ED. Challenges in the management of prostate cancer. Br J nilutamide, and bicalutamide), DL3 has no detectable Urol 1992;70 Suppl 1:33 – 8. intrinsic AR agonist activity. Third, DL3 inhibits the agonist 3. Miyamoto H, Rahman MM, Chang C. Molecular basis for the activityof nilutamide on PSA expression even when they antiandrogen withdrawal syndrome. J Cell Biochem 2004;91:3 – 12.

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