The Cyclooxygenase 2-Specific Nonsteroidal Anti-Inflammatory

Vol. 12, 769–774, August 2003 Cancer Epidemiology, Biomarkers & Prevention 769

The Cyclooxygenase 2-specific Nonsteroidal Anti-inflammatory Drugs Celecoxib and Nimesulide Inhibit Androgen Receptor Activity via Induction of c-Jun in Prostate Cancer Cells1

Yunqian Pan, Jin-San Zhang, Mozammel H. Gazi, and cancer of the breast, ovary, rectum, and esophagus (1, 2). For Charles Y. F. Young2 prostate cancer, there are a few studies showing a weak (but not Departments of Urology and Biochemistry and Molecular Biology, Mayo statistically significant) inverse association between NSAIDs Graduate School, Mayo Clinic/Foundation, Rochester, Minnesota 55905 and the cancer (3). However, a recent report by Nelson and Harris (4) indicated a 70% reduction in the risk of prostate cancer among NSAID users. A more recent, community pop- Abstract ulation-based study (3) showed that there is a significant re- Nonsteroidal anti-inflammatory drugs (NSAIDs) play duction in prostate cancer risk among men aged 60 years or potential roles in cancer chemoprevention. In this study, older who were daily users of NSAIDs. Interestingly, the study we investigated the effects of NSAIDs on androgen found that the strongest inverse association with an 83% re- receptor (AR)-mediated functions in prostate cancer cells. duction in risk was among men of age 70 years or older. The We found that two cyclooxygenase 2-specific NSAIDs, authors attributed this to the highest use of NSAIDs in this celecoxib and nimesulide, dramatically reduced the group of men studied and suggested NSAIDs as potential expression of androgen-inducible genes, such as prostate- chemopreventive agents for prostate cancer. However, the mo- specific antigen, hK2, and the FK506-binding protein 51 lecular mechanism(s) supporting this hypothesis remains to be (FKBP51). We demonstrated that both NSAIDs repressed elucidated. AR-mediated activation of prostate-specific antigen and The AR, a ligand-dependent transcription factor and a hK2 promoter activity as well as AR protein expression. member of nuclear receptor superfamily, plays a central role in Finally, our findings suggested that overexpressed c-Jun androgen action in the prostate (5, 6). Androgens and the AR by the NSAIDs not only inhibited the function of AR but have been strongly suggested as risk factors for prostate cancer also directly repressed AR expression at the transcription development (5–7). Both epidemiological and biochemical level. Our findings provide a strong rationale for studies strongly support androgen deprivation-based chemopre- celecoxib and nimesulide as potential agents for prostate vention and chemotherapy for prostate neoplastic diseases (5– cancer prevention and/or treatment. 7). The AR could be one of the important targets for intervention at all stages of development and progression of prostate cancer. Introduction We reported previously (8) that flufenamic acid could Epidemiological studies, laboratory bioassays, and human clin- inhibit AR-regulated gene expression. In this study, we further ical intervention trials strongly support a protective role of describe our novel findings on the regulation of AR-mediated 3 NSAIDs against cancer development (1, 2). For example, function by the two COX-2-specific NSAIDs, celecoxib and several epidemiological studies showed a significant inverse nimesulide. Our data suggested that NSAIDS strongly induced association between the intake of aspirin and other NSAIDs and expression and phosphorylation of c-Jun, which inhibited the the risk of colorectal cancer in the general population (1, 2). expression of AR-regulated genes as well as AR itself. Both NSAIDs are effective in reducing existing polyps in familial mechanisms seem to contribute to their potent inhibitory effect adenomatous polyposis patients and reduce the tumor burden in on the function of the AR. Our study provides a potential several animal models of colorectal cancer. A similar inverse molecular mechanism linking the down-regulation of AR- association has been described for other cancers including mediated function in prostate cancer cells by celecoxib and nimesulide to their anti-prostate cancer activity.

Received 1/6/03; revised 4/17/03; accepted 5/9/03. Materials and Methods The costs of publication of this article were defrayed in part by the payment of Cell Culture. Human prostate cancer cell lines LNCaP (Amer- page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ican Type Culture Collection, Manassas, VA) and LAPC-4 1 Supported in part by NIH Grant NCI/DK89000 and Army Defense Grant (kindly provided by Dr. Charles L. Sawyers; Ref. 9) were DAMID17-98-1-8523. maintained in RPMI 1640 (Mediatech, Hercules, CA) contain- 2 To whom requests for reprints should be addressed, at Departments of Urology ing 5% FBS (Biofluids, Rockville, MD) at 37°C and 5% CO . and Biochemistry/Molecular Biology, Mayo Clinic/Foundation, Guggenheim 2 Building 502, 200 First Street SW, Rochester, MN 55905. Phone: (507) 284- To avoid potential interference of existing steroids in FBS, the 8336; Fax: (507) 284-3757; E-mail: [email protected]. media were first replaced by serum-free RPMI 1640 for 24 h. 3 The abbreviations used are: NSAID, nonsteroidal anti-inflammatory drug; Cells were then cultured in RPMI 1640 with 5% charcoal- COX, cyclooxygenase; AR, androgen receptor; PSA, prostate-specific antigen; stripped FBS supplemented with or without 1 nM Mib (New ␤-gal, ␤-galactosidase; Mib, mibolerone; FBS, fetal bovine serum; CMV, cyto- megalovirus; ARE, androgen response element; AP-1, activator protein 1; PG, England Nuclear, Boston, MA), a nonmetabolizable, synthetic prostaglandin; FKBP51, FK506-binding protein 51. androgen.

Table 1 Effects of selected NSAIDs on growth responses and expression of androgen-regulated genes in androgen-responsive human prostate cancer cell lines

a a LNCaP (IC50) LAPC-4 (IC50) NSAIDs Selective inhibitor to Growth PSA hK2 Growth PSA hK2 Aspirin COX-1 and -2 Ͼ1000 Ͼ1000 Ͼ1000 Ͼ1000 Ͼ1000 Ͼ1000 Ibuprofen COX-1 and -2 Ͼ1000 783.3 860 740 870 803 Meloxicam COX 2 193.1 300 377 92 Ͼ300 Ͼ300 Ketoprofen COX-1 and -2 Ͼ300 Ͼ300 Ͼ300 Ͼ300 Ͼ300 Ͼ300 Flurbiprofen COX-1 and -2 Ͼ300 206 281 347 234 221 Nimesulide COX-2 38.2 27 23 104 76.5 61 Sulindac COX-1 and -2 Ͼ300 Ͼ300 Ͼ300 Ͼ300 Ͼ300 Ͼ300 Sulindac sulfone b Ͼ300 206 97.6 253 193.8 Ͼ300 Celecoxib COX-2 32.6 20 29.6 44.5 28.7 43.7 Fenoprofen COX-1 and -2 Ͼ300 210.4 228.6 N/Dc N/D N/D Indomethacin COX-1 and -2 Ͼ300 224 272 170 203 207 a ␮ IC50 is given in M. b A metabolite of sulindac loses NSAID activities. c N/D, not determined.

Growth Response and PSA and hK2 Levels. Cells were Statistics. The data were analyzed by Student’s t test. P Ͻ 0.05 plated in 24-well plates at 2 ϫ 104 cells/well. Forty-eight h after was accepted as the level of significance. plating, cells were treated with celecoxib or nimesulide (LKT Lab, St. Paul, MN) and other NSAIDs as shown in Table 1 at Results different doses in the presence or absence of Mib. MTS assay, Celecoxib and Nimesulide Inhibited the Expression of An- a non-radioactive cell proliferation assay, composed of [3-(4,5- drogen Up-Regulated Genes. We were interested in finding dimethylthiazol-2-yl)-5-(3-carboxymethoxphenyl)-2-(4-sulfo- out whether commonly used NSAIDs have the ability to reg- phenyl)-2H-tetrazolium] (Promega, Madison, WI) was performed ulate the function of the AR in prostate cells. Thus we studied to determine cell proliferation 6 days after the treatment. To the effects of several NSAIDs on inhibition of androgen action measure secreted PSA and hK2 levels, 400 ␮l of spent medium and growth in prostate cancer cells. Using PSA and hK2, two from cells treated for 6 days were collected. PSA and hK2 well-established AR target genes, as markers, we tested the proteins levels were determined using specific immunoassays effects of a panel of 11 NSAIDs on androgen action in two (Mayo Immunochemical Core Facility). These measurements androgen-responsive human prostate cancer cell lines, LNCaP were used to the calculate 50% inhibitory concentration (IC50) and LAPC-4, respectively. Among the NSAIDs tested, COX- of each of the NSAIDs. 2-specific inhibitors seem to have a higher potency than other Western Blot Analysis. Cells were seeded at 1 ϫ 105 cells/ NSAIDs in inhibiting androgen action. Celecoxib and nimesu-

plate in 100-mm dishes. Cells grown in log phase were co- lide showed the lowest IC50 concentrations in both cell lines treated with 1 nM Mib and different concentrations of celecoxib (Table 1). Because of their highest potency on inhibition of cell or nimesulide for 15 or 24 h. The cells were collected by growth and androgen function, celecoxib and nimesulide were centrifugation and washed with cold PBS. Cell lysates were chosen for additional studies. (Fig. 1) illustrates that expression prepared in radioimmunoprecipitation assay buffer (PBS con- of PSA and hK2 was suppressed by celecoxib and nimesulide taining 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS plus in a dose-dependent manner in the two cell lines. Significant freshly added protease inhibitors, 100 ␮g/ml phenylmethylsul- inhibitory activity was observed for celecoxib at a concentra- fonyl fluoride, 30 ␮l/ml aprotinin, and 1 mM sodium orthovana- tion of 10 ␮M for both PSA and hK2. Nimesulide at 10 ␮M date) and used for Western blot analysis. The sample filters resulted in a similar inhibition of PSA, although a higher were immunoblotted with c-Jun, phospho-c-Jun (Cell Signal- concentration was required to achieve significant down-regu- ing, Beverly, MA), AR (PharMingen, San Diego, CA), lation in LAPC-4 cells. Recently, we discovered that FKBP51, FKBP51 (a gift from Dr. D. O. Toft; Mayo Clinic) specific an immunophilin, is up-regulated by androgens (11). Similarly, primary antibodies and horseradish peroxidase-conjugated sec- it was found that androgen-up-regulated FKBP51 protein ex- ondary antibodies and visualized by enhanced chemilumines- pression was alleviated by celecoxib and nimesulide treatment cence (Amersham Pharmacia, Piscataway, NJ). as measured by Western analysis using specific antibody (Fig. Transfections and Transcriptional Reporter Assays. 2A). These results suggest these NSAIDs are potent inhibitors LNCaP cells were plated into 60-mm dishes. Cells at 50–70% of AR-induced gene expression. confluence were transfected with the appropriate constructs Celecoxib and Nimesulide Inhibited AR-induced Gene Ex- [6-kb PSA promoter-pGL3, AR promoter (Ϫ74/ϩ87)-pGL3, pression and AR Promoter Activity at the Transcription AR promoter (Ϫ1380/ϩ577)-pGL3, hK2 3xARE-SV40 mini- Level. To test whether celecoxib and nimesulide can directly mal promoter pGL3, or empty pGL3 vectors] by using the repress the promoters of AR-dependent genes, we performed method described previously (10). Twenty-four h after trans- reporter assays using a PSA promoter-luciferase construct. As fection, cells were treated with celecoxib or nimesulide in can be seen in Fig. 2B, celecoxib and nimesulide significantly combination with Mib or ethanol. Whole cell lysate was pre- reduced the androgen-induced PSA promoter activity at a con- pared for luciferase assay according to the manufacturer’s in- centration as low as 25 ␮M. Because the AR binds directly to structions (Promega). CMV-␤-gal expression vector was also the ARE of target genes for androgen action, we also tested cotransfected for normalization of transfection efficiency. Each another reporter construct containing three tandem repeats of transfection was done three times, and SDs were calculated. ARE derived from the hK2 promoter (pGL3 hK2 3xARE SV40

Fig. 1. Effects of NSAIDs on the expression of PSA and hK2 proteins in prostate cancer cells Ϯ 1nM Mib. LNCaP cells (A) and LAPC-4 cells (B) were treated with or without the indicated concentrations of celecoxib and nimesulide for 7 days. Spent media were collected and used for PSA (Hybritech) or hK2 (sandwich) im- munoassay. PSA and hK2 values were normalized to growth response measured by a 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide assay and expressed as a percentage of that of groups treated with Mib only. Error bars indicate the SE of four sep- arate experiments.

minimal; Ref. 12). The result shown in Fig. 2B demonstrated plasmid. We hypothesized that c-Jun may potentially be in- that both celecoxib and nimesulide significantly reduced AR/ volved in NSAID-mediated inhibition of AR. As shown in Fig. ARE-mediated gene expression (P Ͻ 0.05). Thus, celecoxib 2C, c-Jun protein was strongly induced by celecoxib and nime- and nimesulide acted as potent inhibitors of AR-mediated gene sulide at 24 h of treatment. Several studies (14–16), including transcription. ours, have shown that the transactivation functions of the AR as To determine whether celecoxib and nimesulide may di- well as other steroid receptors can be affected by c-Jun. There- rectly affect the transcriptional activity of the AR gene, tran- fore, these results strongly suggest that overexpressed c-Jun scriptional reporter assay was performed in LNCaP cells using induced by celecoxib and nimesulide could interfere with AR- a luciferase reporter plasmid containing the AR promoter mediated up-regulation of PSA and hK2. Note that celecoxib at (Ϫ1380/ϩ577). Compared with control vector alone, cells a relatively low concentration of 25 ␮M may not have an transfected with the AR promoter revealed significantly higher observable inhibitory effect on AR protein expression (Fig. 2A), luciferase activities, as expected (Fig. 3A). However, celecoxib but low concentrations of the NSAIDs could still increase c-Jun and nimesulide, at the concentrations used, repressed the tran- protein expression (Fig. 2C) and subsequently reduced the scription activities of the promoter (Fig. 3A). Furthermore, function of the AR, as evident in the transfections of Fig. 2B. Western analysis using AR-specific antibody indicated that AR c-Jun is usually a short-lived protein, and it can be induced protein expression was reduced by celecoxib and nimesulide by many extracellular stimuli (17). In most cases, the induction (Fig. 2A) at concentrations used in the transfections. Taken is transient at early time of stimulation. However, the results in together, these results suggest that celecoxib and nimesulide are Fig. 2C show that c-Jun protein levels were elevated with 15 potent inhibitors of AR function at least partially through down- and 24 h of treatments, implying that the NSAIDs induced a regulation of AR expression. prolonged overexpression of c-Jun. Enhanced Expression and Phosphorylation of c-Jun by Overexpression of c-Jun Inhibited the AR Promoter. To Celecoxib and Nimesulide in LNCaP Cells. To further dis- determine whether overexpression of c-Jun can affect the ex- sect the molecular mechanisms underlying NSAID-mediated pression of the AR gene, we cotransfected a c-Jun expression inhibition of AR function, we next examined the expression of construct with the two AR promoter reporter plasmids, AR c-Jun in celecoxib- and nimesulide-treated LNCaP cells by promoter (Ϫ1380/ϩ577)-pGL3 and AR promoter (Ϫ77/ϩ84)- Western blot analysis. We have shown previously (13) that pGL3, respectively, in LNCaP cells. The result shown in Fig. androgen-induced PSA promoter activity is inhibited in a dose- 3B suggests that overexpression of c-Jun significantly inhibited dependent manner by cotransfection with c-Jun expression the activity of both tested AR promoters.

Fig. 2. A, effects of NSAIDs on the expression of AR protein and FKBP51 in LNCaP cells by Western blot analysis. B, LNCaP cells were transfected with a luciferase reporter plasmid containing the 6-kb PSA promoter or three copies of ARE or control plasmid (pGL3) and a CMV-␤-gal expression vector and treated with NSAIDs Ϯ P Ͻ 0.05 for PSA promoter and hK2–3ARE promoter. After normalization with ␤-gal, luciferase activities were expressed as a percentage of that ,ء .1nM Mib for 24 h of groups treated with Mib only. C, effects of NSAIDs on the expression of c-Jun and phospho-c-Jun (p-c-Jun) in LNCaP cells by Western blot analysis. Whole cell lysates were prepared from cells treated with or without celecoxib and nimesulide at the indicated concentrations for 15 or 24 h. ␤-Tubulin was used as an internal control for protein loading and transfer efficiency in A and C.

Discussion much stronger activities than the other NSAIDs tested. The data Recent studies (18, 19) have shown that AR expression appears strongly suggest that these two NSAIDs are potent inhibitors of in all stages of prostate cancer, regardless of the androgen- AR function and that the induction of c-Jun expression/phos- responsive status of the cancer. It has been shown that overex- phorylation (Fig. 2C) plays an important role in mediating their pression of many cofactors (e.g., caveolin-1, signal transducers effects. Clearly, our previous studies and those of others (13– and activators of transcription 3, and ␤-catenin) or coactivators 16) demonstrated that c-Jun exhibits an inhibitory effect on AR (e.g., TIF2 and SRC-1) for the AR may enhance the function of protein activity. In addition, this study delineated that c-Jun can the AR by low levels of androgens or by other non-androgen inhibit the transcription activities of the AR promoter. In the ligands (20–25). The presence of the AR and/or overexpression future, small interference RNA targeting c-Jun mRNA (26) or of cofactors or coactivators may provide a growth advantage for dominant negative c-Jun (27) might be used to further address prostate cancer cells and represents one of the central targets for the contribution of c-Jun to the function and expression of AR preventing/treating prostate cancer. in prostate cancer cells. Many studies support a potential role of NSAIDs as che- c-Jun is a component of AP-1 protein complex and a mopreventive agents (1–3). The molecular mechanism(s) by member of the basic leucine zipper family of sequence-specific which NSAIDs exhibit anticancer activities is not completely dimeric DNA-binding proteins (17). Previously, we and others understood. In the present study, we examined a panel of 11 have shown that stimulated overexpression of c-Jun protein can commonly used NSAIDs, focusing on their effects on AR- inhibit the function of the AR (13–16). Several studies have mediated action in two androgen-responsive prostate cancer shown that the transactivation functions of the AR as well as cell lines. We found that celecoxib and nimesulide exhibit other steroid receptors can be affected by c-Jun. The interaction

has been suggested that the anticarcinogenic properties of NSAIDs may depend on their ability to inhibit PG synthesis. However, some reports indicate that this mechanism can only partially explain the anticarcinogenic properties of NSAIDs (31–33). Song et al. (32) showed that in COX-2 antisense prostate cancer cell clones, celecoxib still exhibits its antipro- liferative activity independent of COX-2 expression. Our studies here have clearly shown that even at concentrations of approximately 25 ␮M, these two NSAIDs can significantly inhibit the function of AR. The low concentrations used (Table 1 and Figs. 1 and 2) are physiologically achievable for nimesulide or nearly achievable for celecoxib (32–34). However, note that these concentrations are far higher than that (at approximately 1 ␮M or lower levels) needed to inhibit COX-2 activity (33, 34). Therefore, this and our data seem to suggest that the AR-inhibiting effect mediated by these two NSAIDs is independent of their COX-2 inhibitory effects, although more direct evidence may be required to prove this point in the future study. Moreover, the effective concentrations of these two NSAIDs for inhibiting AR shown in the present study might be able to inhibit COX-1 activities; this is another issue that will need to be resolved in the future study. In summary, our study shows strong evidence that celecoxib and nimesulide exhibit a novel property by which androgen action is blocked in prostate cancer cells. Traditionally, AR antagonists and the inhibitors of AR agonist synthesis are used to repress or reduce the function of the AR in hormone therapy for prostate cancer. However, both celecoxib and nimesulide can enhance the expression of c-Jun and further activate it by phosphorylation, through which the expression and function of the AR were inhibited. Our findings provide a strong rationale Fig. 3. A, LNCaP cells were cotransfected with AR promoter-luciferase reporter that celecoxib and nimesulide be potential agents for prostate (AR-pGL3) or the parental vector (pGL3) and CMV-␤-gal and treated with 1 nM cancer prevention and/or treatment. Mib and NSAIDs at the indicated concentrations for 24 h. B, LNCaP cells were cotransfected with AR promoter (Ϫ74/ϩ87)-pGL3, AR promoter (Ϫ1380/ ϩ577)-pGL3, or the parental vector (pGL3) plus CMV-␤-gal and different References amounts of c-jun expression vector for 24 h. Cell extracts were prepared from the above-mentioned transfections for luciferase and ␤-gal assays. The resulting 1. Levy, G. N. Prostaglandin H synthases, nonsteroidal anti-inflammatory drugs, activities of both AR-pGL3 were further normalized to ␤-gal and expressed as a and colon cancer. FASEB J., 11: 234–247, 1997. P Ͻ 2. Husain, S. S., Szabo, I. L., and Tarnawski, A. S. 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Downloaded from cebp.aacrjournals.org on September 29, 2021. © 2003 American Association for Cancer Research. The Cyclooxygenase 2-specific Nonsteroidal Anti-inflammatory Drugs Celecoxib and Nimesulide Inhibit Androgen Receptor Activity via Induction of c-Jun in Prostate Cancer Cells

Yunqian Pan, Jin-San Zhang, Mozammel H. Gazi, et al.

Cancer Epidemiol Biomarkers Prev 2003;12:769-774.

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