A Novel Synthetic Compound That Interrupts Androgen Receptor Signaling in Human Prostate Cancer Cells
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2057 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. Mol Cancer Ther 2007;6(7). July 2007 Downloaded from mct.aacrjournals.org on September 24, 2021. © 2007 American Association for Cancer Research. 2058 A Novel AR Signaling Inhibitor 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  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  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, niluta- mide, and bicalutamide on PSA expression. A, chemical structure of DL3. Androgen-starved LNCaP cells in 96-well plates at 2  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. MolCancerTher2007;6(7).July2007 Downloaded from mct.aacrjournals.org on September 24, 2021. © 2007 American Association for Cancer Research. Molecular Cancer Therapeutics 2059 Quantitative Real-time PCR Cells were seeded in 60-mm dishes (2  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 normal- ized 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  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.