Exisulind and Related Compounds Inhibit Expression and Function of the Androgen Receptor in Human Prostate Cancer Cells

4972 Vol. 9, 4972–4982, October 15, 2003 Clinical Cancer Research

Jin T. E. Lim, Gary A. Piazza, Rifat Pamukcu, nosed, and 30,200 men died from this disease (1). Although the W. Joseph Thompson, and I. Bernard Weinstein1 precise mechanism by which steroid hormones affect the development of prostate cancer is not known, it is believed that Herbert Irving Comprehensive Cancer Center, Columbia University College of Physicians and Surgeons, New York, New York 10021 prostate cancer develops only in the presence of androgens (2). [J. T. E. L., I. B. W.]; Cancer Therapy and Research Center, San The AR2 is the most important mediator of androgen action and Antonio, Texas 78229 [G. A. P.]; and Cell Pathways, Inc., Horsham, functions as a ligand-dependent transcription factor (3). Muta- Pennsylvania 19044 [R. P., W. J. T.] tions in the AR gene and amplification of this gene are thought to be two possible causes for the more aggressive hormone- ABSTRACT independent forms of prostate cancer, but other unknown mechanisms also appear to play a role (4). It has been demonstrated In recent studies, we found that sulindac sulfide (SS), that although castration-induced androgen deprivation causes a exisulind, CP248, and CP461 induce growth inhibition and 95% loss in serum testosterone, the concentration of DHT, the apoptosis in a series of human prostate cancer cell lines, active form of testosterone in prostate tissue is only reduced by irrespective of cyclooxygenase expression, p53 mutations, or 60% (5). Unfortunately, some clinically available antiandrogens bcl-2 overexpression. Exisulind also inhibited the growth of have an increased affinity for mutant forms of the AR (6). the androgen-dependent LNCaP human prostate cancer cell Therefore, compounds that can down-regulate the expression of line when grown as a xenograft in nude mice. This study the AR gene, thereby bypassing AR gene mutations and AR gene demonstrates that doses of these compounds that induce amplification, might provide a more effective therapy for ag- growth inhibition and apoptosis in LNCaP cells also cause gressive hormone independent prostate cancers. Few studies decreased prostate-specific antigen (PSA) secretion and de- have examined pharmaceutical compounds that act at this level. creased cellular levels of PSA. These effects appear to be a Sulindac is a non-steroidal anti-inflammatory drug that has result, at least in part, of inhibition of the androgen receptor been used predominantly to treat patients with chronic inflam- (AR) signaling pathway because the treated cells also display matory diseases and for its analgesic, antipyretic, and platelet decreases in the level of the AR protein and mRNA and inhibitory activities (7). The drug has been used for the treat- inhibition of transcription of an AR promoter luciferase ment of patients with familial adenomatous polyposis of the reporter in transient transfection assays. SS and exisulind colon. Sulindac sulfone (exisulind), a normal oxidative metab- were more effective in inhibiting the expression of PSA and olite of sulindac, has been shown to also induce polyp regression the AR than CP248 or CP461, apparently because of differ- and prevent polyp recurrence in familial adenomatous polyposis ential effects of these compounds on specific transcription patients. In recent studies, we found that the sulindac metabo- factors. These findings suggest that the growth inhibition by lites SS and exisulind, as well as two more potent analogues of these compounds in human prostate cancer cells may be exisulind, CP248 and CP461 (8), induce growth inhibition and mediated, in part, by inhibition of AR signaling. Thus, these apoptosis in several human prostate cancer cell lines, irrespec- compounds may provide a novel approach to the prevention tive of COX-1/-2 expression, bcl-2 overexpression, or androgen and treatment of human prostate cancer. dependence (9). Although the growth of normal prostate epithe- lial cells was also inhibited, these cells did not display apoptosis, INTRODUCTION even at high doses of these compounds (9). Related studies Prostate cancer is currently the most frequently diagnosed showed that oral administration of exisulind inhibited growth cancer and the second leading cause of cancer deaths in men in and induced apoptosis in xenografts of LNCaP cells in nude the United States. In 2002, ϳ189,000 new cases were diag- mice (10). During the course of our cell culture studies (9), we noted that the LNCaP androgen-sensitive prostate cancer cell line was more sensitive to both growth inhibition and induction of ap- Received 3/20/03; revised 6/6/03; accepted 6/11/03. optosis than the PC3 cells, an androgen-insensitive prostate The costs of publication of this article were defrayed in part by the cancer cell line. This study addresses whether this increased payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. This study was supported by Cell Pathways, Inc., and awards to I. B. W. from the T. J. Martell Foundation and the National Foundation for 2 The abbreviations used are: AR, androgen receptor; DHT, dihydrotes- Cancer Research. terone; ARE, androgen-response element; SS, sulindac sulfide; ␤-gal. 1 To whom requests for reprints should be addressed, at Herbert Irving ␤-galactosidase; IGFBP, insulin-like growth factor binding protein; Comprehensive Cancer Center, Columbia University College of Physi- CREB, cAMP-responsive element binding protein; PSA, prostate- th cians and Surgeons, 701 West 168 Street, New York, NY 10032. specific antigen; Hsp, heat shock protein; JNK-1, c-Jun NH2-terminal Phone: (212) 305-6921; Fax: (212) 305-6889; E-mail: Weinstein@ kinase 1; WCE, whole cell extract; COX, cyclooxygenase; CMV, cuccfa.ccc.columbia.edu. cytomegalovirus; NF-1, nuclear factor-1; SP-1, sequence-specific–1.

sensitivity is related to the androgen-dependent nature of these mega) was also assayed using the same whole cell extracts to cells. We found that treatment of LNCaP cells with concentra- normalize transfections. Each assay was done in triplicate and tions of SS, exisulind, CP461, or CP248 that induce growth the average relative luciferase activity calculated. inhibition and apoptosis also inhibited expression of the andro- Electrophoretic-Mobility Gel Shift Assays. The GT- gen-responsive gene PSA. PSA is a secreted serine protease that ARE gel shift oligonucleotide probe was formed by annealing augments cleavage of IGFBP3-IGF-1 and activates tumor 5Ј-CTAGAAGTCTGGTACAGGGTGTTCTTTTTGCA-3Ј and growth factor ␤ and other growth factors in the extracellular 5Ј-TGCAAAAAGAACACCC TGTATCCAGACTTCTAG-3Ј. matrix (11). Blood levels of PSA provide a convenient diagnos- Ten pmol of double-stranded AR oligonucleotide were radioac- tic marker for prostate cancer (11). In this study, we obtained tively labeled by mixing and incubation with 1 ␮lof␥P32[ATP], ␮ ϫ evidence that the down-regulation of PSA is attributable to the 1 lofT4 polynucleotide kinase, and 1 T4 kinase buffer and ability of these compounds to inhibit expression of the AR gene incubation at 37°C for 30 min. The mixture was then brought up ␮ itself. These novel effects provide additional justification for the to a total volume of 200 l with dH2O, and labeled probe was use of exisulind and CP461 in the chemoprevention and treat- cleared through a tightly packed G50 Sephadex column. The ment of prostate cancer. column purified radiolabeled probe was precipitated overnight in 100% ethanol at Ϫ20°C. The recovered DNA pellet was ␮ resuspended in 50 lofdH2O. Twenty-thousand cpm of each MATERIALS AND METHODS probe were added to a final concentration of 0.5 mM spermidine, Drugs and Plasmids. SS, exisulind, CP248 and CP461 10 ␮g LNCaP lysate extract, and 1ϫ gel-shift binding buffer [10 were obtained from Cell Pathways, Inc. (Horsham, PA). We mM HEPES-KOH (pH 7.9), 50 mM NaCl, 5 mM Tris-HCl (pH refer to these four agents as sulindac-related compounds. Ex- 7.5), 15 mM EDTA, 1 mM DTT, 10% glycerol, and 1 mM isulind, CP461, and CP248 have no analgesic or antipyretic ZnSO4]. The final concentrations of one of the following drugs: properties, and the latter two drugs are clinically distinct from 0.1% DMSO; 0.2 mM SS; 0.5 mM exisulind; 0.5 ␮M CP248; 5 SS or sulindac sulfone. Aspirin (acetyl salicylic acid) was pur- ␮M CP461; or 5 mM aspirin were also added to the reaction chased from Sigma Chemical Co. (St. Louis, MO). The drugs mixture, as indicated (Fig. 2B). The samples were placed un- were dissolved in 100% DMSO and added to the cell culture disturbed at room temperature for 45 min and then subjected to medium at a final vehicle concentration of 0.1% DMSO. The gel electrophoresis using a 4% nondenaturing gel [4% acrylam- (ARR)3-luciferase, ARPN1-luciferase, and PSAP-luciferase re- ide, 1% bis-acryl, 1.4% glycine, 1 mM EDTA (pH 8.0), 25 mM porter plasmids were gifts from Drs. Robert Matusik, Donald Tris-HCl (pH 8.5)]. The gel was run at 150 V for3hinglycine Tindall, and Marian Sadar, respectively. The pSVAR expression buffer [1.4% glycine, 1 mM EDTA (pH 8.0), 25 mM Tris-HCl vector was generously provided by Dr. Albert O. Brinkmann. (pH 8.5)], dried, and autoradiographed. 32 The (AP-1)4-luciferase reporter plasmid was a gift from Dr. Northern Blot Analysis. The P-labeled AR cDNA Zigang Dong. probe was constructed from a PstI/NcoI restriction digest of the Cell Culture. The LNCaP and PC3 human prostate can- pSVAR plasmid. In brief, the DNA sequence encoding 1549– cer cell lines were purchased from the American Type Culture 2782 bp of the pSVAR plasmid was used as a template for Collection. The cells were grown in RPMI 1640 supplemented random priming according to the manufacturer’s protocol (Strat- with 10% FBS, 100 units/ml penicillin G, and 100 mg/ml agene, La Jolla, CA). Cells were plated onto 150-mm diameter streptomycin (Life Technologies, Inc.) and maintained at 37°C plates at a density of 106 cells/plate for 24 h. The cells were in a humidified atmosphere containing 5% CO2. treated with 0.1% DMSO, 0.2 mM SS, 0.5 mM exisulind, 0.5 ␮M Immunoblotting. Immunoblotting techniques were de- CP248, 5 ␮M CP461, or 5 mM aspirin and harvested 10 and 24 h scribed previously (9). The primary antibodies used were: PSA later. Total RNA was extracted and then subjected to Northern (1:1000; Dako Corp., Carpinteria, CA); AR (1:2000; Santa Cruz blot analyses, as described previously (12). Biotechnology, Inc., Santa Cruz, CA); Hsp70 (1:2000; Phar- Statistical Analysis. Statistical analysis of the data were Mingen, San Diego, CA); NF-1 (1:500; Santa Cruz Biotechnol- performed using student t test (P Ͻ 0.001) with the computer ogy, Inc.); SP-1 (1:2000; Santa Cruz Biotechnology, Inc.); software SigmaPlot version 8.0 (SigmaPlot, Golden, CO). CREB (1:1000; UBI, Lake Placid, NY); ␤-actin (1:500; Sigma Densitometry. Densitometry of the data were quantified Chemical Co.); c-jun (1:1000; Santa Cruz Biotechnology, Inc.); using ImageQuant software version 1.2 (Molecular Dynamics, c-fos (1:500; Santa Cruz Biotechnology, Inc.); and IGFBP-2 Sunnyvale, CA). (1:2000; UBI). Transient Transfections and Reporter Assays. Cells were plated at a density of 1 ϫ 104 cells/plate in 60-mm RESULTS diameter plates. Twenty-four h later, the cells were transiently Sulindac-Related Compounds Decrease Secreted and transfected with 2 ␮g of the luciferase reporter plasmid and 1 ␮g Intracellular Levels of PSA. The well-characterized LNCaP of pcmv-␤-gal expression vector (internal control), using the human prostate cancer cell line was used in these studies (13). manufacturer’s protocol (Life Technologies, Inc., Frederick, LNCaP cells express the endogenous AR and various androgen- MD). After 20 h, the cells were treated with 0.1% DMSO, 0.2 inducible genes, including human glandular kallikrein-2 and mM SS, 0.5 mM exisulind, 10 ␮M CP248, 50 ␮M CP461, or 5 mM PSA. PSA, a biomarker for prostate cancer, is a secreted serine aspirin. Twenty h later, whole cell extracts were prepared, and protease produced by both normal and malignant prostate epi- luciferase assays were performed according to the manufactur- thelial cells. Several clinical studies have shown a direct corre- er’s instructions (Promega, Madison, WI). ␤-Gal activity (Pro- lation between increased serum levels of PSA and the progres-

sion of prostate cancer (14, 15). In cell culture, LNCaP cells were transfected with the probasin reporter plasmid, together secrete PSA into the medium, mimicking the secretion of PSA with a ␤-gal plasmid as an internal control, and the cells were by prostate cancers into the circulation. Therefore, it was of incubated in charcoal-stripped RPMI 1640 containing 20 nM interest to compare the levels of secreted PSA in the growth DHT, together with DMSO (vehicle control) or each of the medium of LNCaP cells after treating the cells with sulindac- drugs, for 20 h. Cell extracts were then analyzed for luciferase related compounds by performing Western blot analysis of the activity. On the basis of previous studies (18–20), we used 20 cell-free medium, with an anti-PSA antibody that detects a nM DHT to maximize the activation of the ARE promoter. We

characteristic Mr 33,000–34,000 protein band. In these studies, found that 0.2 mM SS and 0.5 mM exisulind caused a dramatic we used concentrations of the sulindac-related compounds that decrease in luciferase activity when compared with the 0.1% induce growth inhibition and apoptosis (8, 9). Fig. 1A (left DMSO-treated cells. However, treatment with 0.5 ␮M CP248 or panel) indicates that the vehicle (0.1% DMSO)-treated cells 5 ␮M CP461 showed only a partial decrease in luciferase activity showed a time-dependent increase of PSA in the growth me- (Fig. 2A), and cells treated with 5 mM aspirin showed no dium. LNCaP cells treated with 0.2 mM SS or 0.5 mM exisulind inhibition of luciferase activity. Similar results were obtained in showed decreases in the levels of secreted PSA, within 10–20 h, four independent studies. Negligible activity was obtained when and the PSA levels remained low even after 48 h when com- the DMSO control studies were done in the absence of DHT, pared with the vehicle (0.1% DMSO) control. The secreted demonstrating that the activity of this reporter was androgen- levels of PSA in cultures of LNCaP cells treated with 0.5 ␮M dependent (Fig. 2A, third lane). The relatively low activity of CP248 or 5 ␮M CP461 were partially inhibited when compared CP248 and CP461, although they were tested at high concen- with the vehicle control (0.1% DMSO)-treated cells. Treatment trations with respect to growth inhibition and apoptosis, is with a high concentration of aspirin (5 mM) did not inhibit the consistent with our studies above on PSA secretion (Fig. 1). secreted level of PSA (Fig. 1A, left panel). LNCaP cells also Therefore, we also tested CP248 and CP461 in probasin-lucif- secrete the protein IGFBP-2 into the growth medium (11), but erase reporter assays using very high concentrations of CP248 none of these drugs caused an appreciable inhibition of the and CP461, i.e., 10 and 50 ␮M, respectively. Again, we found secretion of this protein (Fig. 1A, right panel). These results only partial inhibition of CP248 and CP461, compared with the indicate that sulindac derivatives, but not aspirin, can decrease marked inhibition seen with exisulind and SS (Fig. 2B). We the levels of PSA secreted into the medium of LNCaP cells. It obtained qualitatively similar results with these sulindac-related is curious that SS and exisulind were much more active than compounds when we assayed for ARE activity using a PSA CP248 and CP461, although all four drugs were tested at con- promoter-luciferase reporter (Fig. 2C). Results were statistically centrations that induce growth inhibition and apoptosis (8, 9). significant using student t test with P Ͻ 0.001 (SigmaPlot, SPSS To determine whether the decreased levels of secreted PSA United Kingdom). seen in the above studies reflected a specific effect on intracel- Sulindac-Related Compounds Do Not Directly Block lular levels of PSA, we next examined intracellular levels of Binding of the AR to an ARE. The above described inhibi- PSA in the above described cells by Western blot analysis. The tion of the transcriptional activity of an ARE by these com- cells were washed repeatedly with PBS before lysis to reduce pounds might be attributable to direct inhibition of the binding the secreted PSA adherent to the LNCaP cells. Within 10 h of of the AR protein to the ARE DNA sequence. We tested this treatment of LNCaP cells with 0.2 mM SS or 0.5 mM exisulind, possibility by incubating LNCaP WCEs together with a radio- there was a marked and persistent decrease in the intracellular labelled ARE probe in the absence or presence of each of the level of PSA when compared with the DMSO control cells (Fig. sulindac derivatives and then carried out electrophoretic mobil- 1B). LNCaP cells treated with 0.5 ␮M CP248 or 5 ␮M CP461 ity gel shift assays (Fig. 3). Fig. 3, Lane 3, shows that binding displayed decreased levels of PSA within 20–24 h. However, of the LNCaP WCE to the ARE produced three major com- aspirin did not cause a decrease in the intracellular levels of PSA plexes. The addition of a 10-fold excess of the nonradioactive (Fig. 1B, left panel). In contrast to their inhibitory effects on ARE sequence markedly inhibited the formation of these three PSA, SS, exisulind, CP248, and CP461 did not cause an appre- bands Fig. 3, Lane 2, indicating that the binding of the labeled ciable inhibition of intracellular levels of the IGFBP-2 protein oligonucleotide was saturable. We then added to the reaction (Fig. 1B, right panel). These data suggest that the decrease in mixture containing the labeled ARE DNA sequence and WCE secreted levels of PSA, described in Fig. 1A, is secondary to a the following compounds: 0.1% DMSO (Fig. 3, Lane 4); 0.2 mM decrease in the intracellular levels of this protein. SS (Fig. 3, Lane 5); 0.5 mM exisulind (Fig. 3, Lane 6); 0.5 ␮M Sulindac-Related Compounds Inhibit the Transcrip- CP248 (Fig. 3, Lane 7); 5 ␮M CP461 (Fig. 3, Lane 8); or 5 mM tional Activity of an Androgen-Responsive Promoter. The aspirin (Fig. 3, Lane 9). None of these drugs significantly above results raised the possibility that these drugs might inhibit inhibited the binding of the ARE to cellular proteins (compare transcription of the PSA gene. The transcription of this gene is with the control Fig. 3, Lanes 2 and 3). These results suggest known to be controlled by an ARE. Therefore, we performed that none of these compounds directly inhibit the formation of transient transfection luciferase reporter assays using a probasin the AR-ARE complex.

(ARE)3-tk-luciferase reporter plasmid, composed of three con- Sulindac-Related Compounds Decrease AR Protein secutive cassettes of the probasin ARE region (nucleotides Levels but Do Not Alter HSP70 Expression. Another pos- Ϫ244 to Ϫ96) adjacent to the thymidine kinase promoter (16). sible explanation for the ability of these compounds to inhibit The probasin gene encodes a protein secreted by the rat dorso- the transcriptional activity of the ARE is that they might cause lateral prostate (17). The expression of this gene is specific to a decrease in cellular levels of the AR protein. We investigated the prostate and is highly androgen responsive. LNCaP cells this possibility using Western blot analysis of the AR protein.

Fig. 1 A, effects of sulindac-related compounds on secretion of PSA by LNCaP cells. A total of 0.1% DMSO (vehicle control), 0.2 mM SS, 0.5 mM exisulind, 0.5 ␮M CP248, 5 ␮M CP461, or 5 mM aspirin was added in fresh growth medium to cultures of LNCaP cells and the cell-free medium collected at the indicated time (h). Fifty ␮l of this medium were subjected to 10% SDS-PAGE and immunoblotted using an anti-PSA (left panel) or anti-IGFBP-2 (right panel) antibody. Similar results were obtained in two additional studies. Nu- merical values below each band indicate relative image density with respect to the 10-h DMSO control sample. B, the effect of sulindac- related compounds on intracellular levels of PSA. A total of 0.1% DMSO, 0.2 mM SS, 0.5 mM exisulind, 0.5 ␮M CP248, 5 ␮M CP461, or 5 mM aspirin were added to the culture medium for the indicated time (h). The cells were collected, washed in PBS, and WCEs prepared as described in “Materials and Methods.” Fifty ␮gof LNCaP WCE were run on 10% SDS- PAGE and immunoblotted for intracellular PSA levels (left panel) and intracellular IGFBP-2 levels (right panel) with respective antibody. Sim- ilar results were obtained in two additional studies. Numerical values below each band indicate relative image density with respect to the 10-h DMSO control sample.

When compared with the 0.1% DMSO control cells, 0.2 mM SS within 20–24 h. However, no inhibition was seen with 5 mM or 0.5 mM exisulind caused a decrease in AR levels as early as aspirin. Similar results were obtained in three independent ex- 10 h after treatment (Fig. 4, left panel). LNCaP cells treated with periments. These findings are consistent with our earlier results 0.5 ␮M CP248 or 5 ␮M CP461 displayed a decrease in AR levels on the relative effects of these compounds in inhibiting secreted

Fig. 2 A, effects of sulindac- related compounds on androgen- responsive probasin promoter- luciferase reporter activity. LNCaP cells were transiently transfected with a pCMV-␤-gal and the probasin-luciferase reporter (left panel) or the PSA-luciferase reporter (right panel). The cells were grown in RPMI medium containing charcoal-stripped serum plus either 0.1% ethanol (vehicle control) or 20 nM DHT. The cells were treated with one of the following drugs: 0.1% DMSO; 0.2 mM SS; 0.5 mM exisulind; 5 ␮M CP461; 0.5 ␮M CP248; or 5 mM aspirin, for 20 h. Extracts were then assayed for luciferase activity and for ␤-gal activity as an internal control for efficiency of transfection, and the relative luciferase activity was calculated. Each sample was done in triplicate and the results as mean values. B, effects at higher concentrations of CP248 and CP461 on probasin promoter-luciferase reporter activity. Reporter assays were conducted as in Fig. 2A but with the following concentrations of drugs: 0.1% DMSO; 0.2 mM SS; 0.5 mM exisulind; 50 ␮M CP461; 10 ␮M CP248; or 5 mM aspirin. C, sulindac-related compounds inhibit androgen-responsive PSA promoter-luciferase reporter activity. Re- porter assays were conducted with the same concentrations of drugs as in Fig. 2B but using the PSA promoter sequence linked to the luciferase gene reporter.

in AR mRNA levels, whereas 5 ␮M CP461 produced a moderate decrease. However, 0.5 ␮M CP248 caused no decrease at 10 h and only a slight decrease in AR mRNA at 20 h. There was no inhibition of AR mRNA levels in the cells treated with 5 mM Aspirin (Fig. 5). Agarose gel staining with ethidium bromide (data not shown) indicates equal loading and no mRNA degradation. Sulindac-Related Compounds Do Not Decrease the Levels of an Exogenously Expressed AR Protein. We transiently transfected an exogenous AR expression vector driven by the CMV promoter into PC3 prostate cancer cells that do not otherwise express endogenous AR. We found that none of the sulindac-related compounds altered the level of this exogenously expressed AR protein (Fig. 6). These results suggest that the inhibitory effects of these compounds on the AR are specific to the promoter of the AR gene and do not affect the general transcriptional machinery or simply cause proteolytic degradation of the AR protein. Sulindac-Related Compounds Inhibit Transcriptional Activity of the Promoter of the AR Gene. We next examined the possibility that the sulindac-related compounds might inhibit Fig. 3 Sulindac-related compounds do not directly inhibit binding of the transcriptional activity of the promoter of the AR gene. There- the AR to an ARE. Electrophoretic mobility gel shift assays were done fore, we performed transient transfection luciferase reporter assays using LNCaP WCEs incubated with a radiolabelled ARE probe (GT- ARE) in gel-shift binding buffer and 0.5 mM spermidine (Lane 1). A 1ϫ using two AR promoter-luciferase reporter plasmids, ARPN1 and labeled GT-ARE minus WCE (Lane 2)1ϫ labeled GT-ARE and 10 ␮g ARPS1. ARPN1 contains a Ϫ1500 to ϩ580 bp sequence of the AR of LNCaP WCE (Lane 3)1ϫ labeled GT-ARE, 10ϫ GT-ARE cold, gene linked to luciferase, and ARPS1 contains a Ϫ1250 to ϩ580 ␮ ϫ ␮ and 10 g of LNCaP WCE (Lane 4)1 labeled GT-ARE, 10 gof bp sequence linked to luciferase. LNCaP cells were transfected LNCaP WCE, and 0.1% DMSO (Lane 5)1ϫ labeled GT-ARE, 10 ␮g with the respective plasmids, and the cells were then incubated for of LNCaP WCE, and 0.2 mM SS (Lane 6)1ϫ labeled GT-ARE, 10 ␮g of LNCaP WCE, and 0.5 mM exisulind (Lane 7)1ϫ labeled 20 h in charcoal-stripped RPMI 1640 containing 20 nM DHT, GT-ARE, 10 ␮g of LNCaP WCE, and 0.5 ␮M CP248 (Lane 8)1ϫ together with 0.1% DMSO (vehicle control) or each of the sulin- labeled GT-ARE, 10 ␮g of LNCaP WCE, and 5 ␮M CP461 (Lane 9) dac-related compounds, and cell extracts were analyzed for lucif- ϫ ␮ 1 labeled GT-ARE, 10 g of LNCaP WCE, and 5 mM aspirin. erase activity. We found that treatment of the ARPN1-transfected cells with 0.2 mM SS or 0.5 mM exisulind caused about a 90% inhibition of luciferase activity when compared with the 0.1% and intracellular levels of PSA (Fig. 1). Therefore, SS, exisu- DMSO-treated cells. However, treatment with high doses of CP248 lind, CP248, and CP461 cause a decrease in AR protein levels and CP461, 10 and 50 ␮M, respectively, caused only partial inhi- at concentrations similar to those that induce apoptosis (8, 9) bition of luciferase activity. Cells treated with 5 mM aspirin showed and that cause a decrease in PSA levels in LNCaP cells (Fig. 1). no significant inhibition of luciferase activity (Fig. 7, left panel). Hsps are often bound to the inactive AR in the nucleus and Hsps Similar results were obtained with the ARPS1 plasmid (Fig. 7, 90, 70, and 56 have been shown to coimmunoprecipitate with right panel). These results were reproducible and statistically sig- the AR protein (21). It has been previously reported that the nificant using the student t test with P Ͻ 0.001 (SigmaPlot, SPSS promoter element of Hsp70B is activated in human monocytic United Kingdom). They are consistent with the above-described cells treated with sodium salicylate and sulindac sulfoxide (7). ARE luciferase reporter assays (Fig. 2) and suggest that the de- However, we found that none of the sulindac-related com- creased expression of PSA observed in Fig. 1, A and B, and the pounds, nor aspirin, altered the level of the Hsp70 protein (Fig. decrease in cellular levels of the AR mRNA and protein (Figs. 4 4, right panel). Thus, although the levels of the AR protein are and 5), after treating LNCaP cells with these compounds may be decreased after treatment with these compounds, the levels of due, at least in part, to inhibition of transcription of the AR gene. It the Hsp70 protein, a partner of the AR within the nucleus, is also apparent that CP248 and CP461 are relatively weak inhib- remain unaffected. These findings indicate that the sulindac- itors of all of these effects when compared with cytotoxic doses of related compounds can preferentially down-regulate the AR SS and exisulind. protein and that the loss of the AR protein induced by these Sulindac-Related Compounds Down-Regulate the drugs is not simply because of nonspecific proteolysis. Transcription Factors SP-1, NF-1, and CREB but CP248 Sulindac-Related Compounds Down-Regulate the Level and CP461 Up-Regulate AP-1. The AR gene promoter con- of AR mRNA. We next determined the effects of these com- tains DNA binding sequences for several transcription factors pounds on AR mRNA levels using Northern blot analyses. (22). The above studies indicated that the sulindac compounds When compared with the DMSO control treated cells, 0.2 mM inhibited the transcriptional activity of a Ϫ1250 to ϩ580 bp SS, 0.5 mM exisulind, and 5 ␮M CP461 caused a partial decrease sequence of the AR gene (Fig. 7, right panel). To additionally in AR mRNA levels within 10 h of treatment (Fig. 5). After explore the mechanism responsible for this effect, we examined 24 h, 0.2 mM SS and 0.5 mM exisulind caused a marked decrease the levels of expression of several proteins known to bind to

Fig. 4 The effects of sulindac-related compounds on cellular levels of the AR and Hsp70 proteins. LNCaP cells were treated with 0.1% DMSO, 0.2 mM SS, 0.5 mM exisulind, 0.5 ␮M CP248, 5 ␮M CP461, or 5 mM aspirin, for the indicated time (h). Fifty ␮g of WCE were run on 10% SDS-PAGE and immunoblotted with an anti-AR antibody (left panel) or with anti HSP70 antibody (right panel). Numerical values below each band indicate relative image density with respect to the 10-h DMSO control sample.

Fig. 5 Sulindac-related compounds cause a decrease in AR mRNA. LNCaP cells were treated with 0.1% DMSO, 0.2 mM SS, 0.5 mM exisulind, 0.5 ␮M CP248, 5 ␮M CP461, or 5 mM aspirin for 10 or 24 h, and RNA extracts were prepared. Twenty ␮g of total mRNA was subjected to Northern blot analysis using a 32P-labeled AR cDNA probe encoding the 1549–2782-bp sequence of the AR gene.

specific transcription control elements in this region of the AR found that 0.2 mM SS and 0.5 mM exisulind caused no change in gene promoter (22). We found that when LNCaP cells were luciferase activity when compared with the 0.1% DMSO-treated treated with either SS, exisulind, CP248, or CP461 for 24 or cells. However, treatment with CP248 or CP461 caused a marked 48 h, there was a marked decrease in the transcription factors increase in luciferase activity (Fig. 9). Similar results were obtained SP-1, NF-1, and CREB (Fig. 8). The transcription factor AP-1 in three independent studies. These results are consistent with the contains the proteins c-jun and c-fos. Treatment with SS and ability of CP248 and CP461 to induce increased expression of both exisulind caused a slight increase in c-jun, but treatment with c-Jun and c-Fos (Fig. 8) and previous evidence that these two CP248 or CP461 caused a greater increase in c-jun and also a compounds are very potent activators of JNK-1, which phospho- marked increase in c-fos (Fig. 8). rylates c-jun, thereby activating its transcriptional activity (24). In view of the above results, we transfected LNCaP cells with a reporter plasmid containing four cassettes of the AP-1 promoter sequence linked to a luciferase reporter (23). The cells were incu- DISCUSSION bated in charcoal-stripped RPMI 1640 containing 20 nM DHT, In previous studies, we demonstrated that exisulind, SS, together with DMSO (vehicle control) or each of the drugs, for CP248, and CP461 inhibit growth and induce apoptosis in a 20 h. Cell extracts were then analyzed for luciferase activity. We series of human prostate cancer cell lines, irrespective of bcl-2

that the sulindac-related compounds do not act simply by caus- ing proteolysis of the AR protein. In addition to inducing parallel decreases in the PSA and AR proteins and their respective mRNAs (Fig. 3B and data not shown), these compounds directly inhibited the transcriptional activity of the AR in transient transfection reporter assays using ARE-containing promoters (Fig. 2A) and also inhibited the transcriptional activity of the promoter region of the AR gene Fig. 6 Sulindac-related compounds do not inhibit cellular levels of an (Fig. 7). Electrophoretic mobility gel shift assays indicate that exogenously expressed AR protein. PC-3 cells were transiently trans- these drugs do not act by directly inhibiting binding of the AR fected with the pSVAR plasmid that encodes the AR driven by a CMV promoter, and the cells were treated with 0.1% DMSO, 0.2 mM SS, 0.5 protein to ARE DNA sequences (Fig. 2B). Additional studies mM exisulind, 0.5 ␮M CP248, 5 ␮M CP461, or 5 mM aspirin were added are required to determine whether these drugs act by altering the for 20 h. Fifty ␮g of WCE were then run on 10% SDS-PAGE and state of phosphorylation of the AR, inhibiting the function of immunoblotted with an anti-AR antibody. cofactors that bind to the AR, or altering the levels or activities of one or more transcription factors required for mediating the transcriptional activity of the AR. With respect to the latter possibility, we found that treatment of LNCaP cells with SS, overexpression, androgen dependence, or COX expression. exisulind, CP248, or CP461 led to a marked decrease in cellular However, exisulind did not induce apoptosis in prostate epithe- levels of the transcription factors SP-1, NF-1, and CREB (Fig. lial cells derived from normal prostate tissue (9). In this study, 8), and all three of these factors play a role in transcriptional we found that when the androgen-dependent prostate cancer cell activation of the promoter sequence of the AR gene (22). How- line LNCaP was treated with concentrations of SS, exisulind, ever, CP248 and CP461, but not SS and exisulind, induced CP248, or CP461 that induce growth inhibition and apoptosis, increased expression of c-jun and c-fos and caused marked within 24 h there was a decrease in both secreted and intracel- activation of AP-1 reporter activity (Figs. 8 and 9). Therefore, lular levels of the PSA (Fig. 1). Furthermore, reverse transcrip- we postulate that these compounds impair the transcriptional tion-PCR analysis indicated that the mRNA levels of PSA were activity of the promoter of the AR gene by depleting cells of also decreased in the drug-treated cells (data not shown). In critical transcription factors. The significance of the induction of transient transfection reporter assays using promoter sequences AP-1 activity by CP248 and CP461 is discussed below. derived from either the PSA or the probasin genes, which Recent studies indicate that SS, exisulind, CP248, and contain AREs, SS, exisulind, CP248, and CP461 inhibited the CP461 inhibit the activity of the cyclic GMP-specific phos- transcriptional activities of these promoters (Fig. 2). Treatment phodiesterases of gene families, 2 and 1 (8), and that the with SS, exisulind, CP248, and CP461 also led, within 24 h, to subsequent activation of protein kinase G mediates the apoptosis a decrease in cellular levels of the AR protein and AR mRNA, induced by these compound via both its inhibitory effects on and these compounds also inhibited the transcriptional activity ␤-catenin (8) and activation of the JNK-1 pathway (24, 29). It is of an AR promoter sequence in transient transfection reporter curious that on a molar basis, CP461 and CP248 are ϳ100- and assays (Fig. 7). However, treatment of LNCaP cells with 5 mM 1000-fold more potent, respectively, than exisulind with respect aspirin, which induces other biological effects in cell cultures to cyclic GMP phosphodiesterase inhibition and induction of (25), did not affect cellular levels of the PSA and AR proteins, apoptosis (8, 9), and yet in the present study, we found that AR mRNA, or the above-described reporter assays (Figs. when CP461 and CP248 were tested at relatively high concen- 1–3, 7). trations, these drugs were considerably less active than SS or These results indicate that SS, exisulind, CP248, and exisulind in inhibiting PSA or AR expression or in inhibiting CP461 can cause a decrease in cellular levels of the PSA and AR-related transcriptional activity (Figs. 2 and 7). As discussed AR proteins in human prostate cancer cells by inhibiting tran- above, we found that CP248 and CP461, but not SS or exisulind, scription of both the PSA and AR genes. The down-regulation caused marked activation of AP-1 activity (Fig. 9). This may be of PSA by sulindac derivatives may be due, at least in part, to the because these two compounds also induced increased expres- decrease in the AR caused by these drugs (Fig. 4). Both the PSA sion of c-jun and c-fos (Fig. 8), and both compounds are highly and the AR genes contain AREs in their upstream promoter potent activators of JNK-1, which phorphorylates c-jun, and regions (26, 27). The decreased levels of the PSA and AR thereby activates its transcriptional activity (24). Because the proteins in the treated cells do not appear to be simply because promoter sequence of the AR gene also contains an AP-1 ele- of an increase in proteolytic activity, associated with the apo- ment (22), we postulate that the increased AP-1 activity induced ptotic response induced by these compounds, because we did by CP248 and CP461 may partially compensate for the decrease not see a parallel decrease in the proteins IGFBP-2, HSP70 or in other transcription factors, thus blunting the inhibitory effects actin (Figs. 1, A and B, and 3A; data not shown). The PC3 of these two compounds on AR-related transcription. Additional prostate cancer cell line does not express the AR protein (28). studies are required to determine how the activation of protein When we expressed an exogenous AR protein in PC3 cells by kinase G might lead to differential effects on the expression of transfection with an AR cDNA under the control of a CMV specific transcription factors. Additional studies are also re- promoter, treatment of the transfected cells with the above quired to exclude other possible mechanisms by which these compounds did not cause a decrease in the cellular level of the compounds inhibit AR-mediated functions. AR protein (Fig. 3C). These results provide additional evidence Finally, it is of interest to discuss possible clinical signif-

Fig. 7 Sulindac-related compounds inhibit AR promoter activity. LNCaP cells were cotransfected with an AR promoter (Ϫ1500 to ϩ580 bp)-luciferase reporter (left panel) or an AR promoter (Ϫ1250 to ϩ580 bp)-luciferase reporter (right panel). The cells were grown in RPMI medium containing charcoal-stripped serum plus either 0.1% ethanol (vehicle control) or 20 nM DHT. The cells were also treated with 0.1% DMSO, 0.2 mM SS, 0.5 mM exisulind, 50 mM CP461, 10 mM CP248, or 5 mM aspirin for 20 h in the presence of 20 nM DHT. Extracts were then assayed for luciferase activity and relative luciferase activity calculated. Each sample was assayed in triplicate, and the results presented are mean values.

Fig. 8 Sulindac-related compounds down-regulate the transcriptional factors SP-1, NF-1, and CREB, but CP248 and CP461 up-regulate c-fos and c-jun. LNCaP cells were treated with 0.1% DMSO, 0.2 mM SS, 0.5 mM exisulind, 10 ␮M CP248, 50 ␮M CP461, or 5 mM aspirin for the indicated number of hours. Fifty ␮g of WCE were examined by Western blot analysis using anti-c-Jun, anti-c-Fos, anti-SP-1, anti-NF-1, anti-CREB, or anti-␤-actin (control) antibodies.

icance of this study. Normal prostate development and mainte- desirable to have antiprostate cancer drugs that bypass the nance depend on androgens, and at least 75% of the tumors in function of mutant or amplified forms of the AR gene by directly men with metastatic prostate cancer are androgen dependent at inhibiting transcription of the AR gene in prostate cancer cells. the initial time of diagnosis (30). Several therapeutic drugs In this context, the results obtained with sulindac-related com- target the steroid-AR interaction, whereas other drugs such as pounds in this study may have considerable clinical signifi- finasteride directly inhibit 5-␣-reductase, the enzyme that con- cance. verts testosterone to its biologically active form DHT. Unfortu- Other investigators reported that the nonsteroidal anti- nately, agents like flutamide can also act as agonists when they inflammatory drug flufenamic acid, but not piroxican or bind to mutant forms of the AR, which are frequently observed sulindac sulfoxide, also inhibits expression of the AR in in prostate cancers (9). Thus, flutamide binds to the 868 LNCaP cells (31). Our results using exisulind (sulindac sul- (Thra Ala) mutant of the AR, and this induces dissociation of the fone) and SS, which are metabolites of sulindac sulfoxide, AR-Hsp complex, and thereby activates the transcription of indicate that these compounds inhibit expression of the AR in androgen responsive genes (11). Moreover, there is increasing LNCaP cells. Furthermore, in contrast to flufenamic acid, evidence that the overexpression of the AR gene might be a exisulind lacks COX inhibitory activity (9). In addition, precursor to androgen independence (30). Therefore, it would be although aspirin inhibits COX activity, we found that it did

Fig. 9 CP248 or CP461, but not SS or exisulind, activate AP-1 promoter-luciferase reporter activity. LNCaP cells were transfected with an AP-1 promoter luciferase reporter and a pCMV-␤-gal reporter. The cells were grown in RPMI medium containing charcoal-stripped serum plus either 0.1% ethanol (vehicle control) or 20 nM DHT. The cells were also treated with 0.1% DMSO, 0.2 mM SS, 0.5 mM exisulind, 10 ␮M CP248, or 50 ␮M CP461 for 20 h in the presence of 20 nM DHT. Extracts were then assayed for luciferase and ␤-gal activities and relative luciferase activity calculated. Each sample was assayed in triplicate and the results presented are mean values.

not inhibit AR-related activity in LNCaP cells. Therefore, our effects in rodents (10, 35, 37). Furthermore, the administration results provide evidence that sulindac-related compounds of exisulind to postprostectomy prostate cancer patients with inhibit AR-related activity by a COX-independent mecha- rising levels of serum PSA caused a significant decrease in nism. We should emphasize that exisulind and related com- serum levels of PSA when compared with placebo controls (38). pounds also induce apoptosis in androgen-independent prostate cancer cell lines (9). Therefore, the clinical use of these compounds would not be limited to patients with androgen- ACKNOWLEDGMENTS dependent prostate tumors. At the same time, we hypothesize We thank Drs. Robert Matusik, Donald Tindall, A. O. Brinkmann, and Marian Sadar for providing the (ARR) -luciferase, ARPN1-lucifer- that inhibition of the AR could augment the antiproliferative 3 ase, pSVAR, and PSAP-luciferase reporter constructs, respectively. We effects of these compounds in androgen-dependent tumors also thank Drs. Masumi Suzui, Muneyuki Masuda, and Gerhard Sperl and precursor lesions. This may be of particular importance for their very helpful advice. in the chemoprevention of prostate cancer. In contrast to the drug suramin, which can decrease PSA secretion in the absence of growth inhibition (32), exisulind, REFERENCES CP248, and CP461 inhibit PSA and AR expression in LNCaP 1. Zerhouni, E. Prostate Cancer Res. Plan; FY 2003–FY 2008. pp. cells at concentrations similar to or higher than those needed for 1–50. Bethesda: NIH, 2002. growth inhibition and apoptosis induction. Thus, it seems un- 2. Kantoff, P., Giovannucci, E., and Brown, M. The androgen receptor CAG repeat polymorphism and its relationship to prostate cancer. Bio- likely that a decrease in PSA levels in a prostate cancer patient chim. Biophys. Acta, 1378: C1–C5, 1998. treated with exisulind or related compounds will represent a 3. Evans, R. M. The steroid and thyroid hormone receptor superfamily. false positive for tumor antiproliferative activity. Therefore, Science (Wash. DC), 240: 889–895, 1988. PSA levels should remain useful as biomarker for the efficacy of 4. Koivisto, P., Kononen, J., Palmberg, C., Tammela, T., Hyytinen, E., these compounds in clinical trials on prostate cancer therapy or Isola, J., Trapman, J., Cleutjens, K., Noordzij, A., Visakorpi, T., and chemoprevention. Although as emphasized above, the inhibitory Kallioniemi, O. P. Androgen receptor gene amplification: a possible effects on transcription of the AR may potentiate antitumor molecular mechanism for androgen deprivation therapy failure in prostate cancer. Cancer Res., 57: 314–319, 1997. activity. 5. Labrie, F., Dupont, A., Belanger, A., St-Arnaud, R., Giguere, M., We should emphasize that in this study, SS and exisulind Lacourciere, Y., Emond, J., and Monfette, G. Treatment of prostate were used at relatively high concentrations, 0.2 and 0.5 mM, cancer with gonadotropin-releasing hormone agonists. Endocr. Rev., 7: respectively. These concentrations are in the range used in 67–74, 1986. previous cell culture studies with these compounds (8, 9, 33– 6. Kallioniemi, O. P., and Visakorpi, T. Genetic basis and clonal 36). However, they probably exceed maximum in vivo blood evolution of human prostate cancer. Adv. Cancer Res., 68: 225–255, 1996. levels. On the other hand, during chronic administration to 7. Housby, J. N., Cahill, C. M., Chu, B., Prevelige, R., Bickford, K., patients, these drugs may be concentrated in tumor tissue and Stevenson, M. A., and Calderwood, S. K. Non-steroidal anti-inflamma- also exert cumulative effects. Indeed, despite relative low blood tory drugs inhibit the expression of cytokines and induce HSP70 in levels sulindac, indomethacin, and exisulind do exert antitumor human monocytes. Cytokine, 11: 347–358, 1999.

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