Perillyl alcohol inhibits the expression and function of the androgen receptor in human prostate cancer cells

Cancer Letters 236 (2006) 222–228 www.elsevier.com/locate/canlet

Perillyl alcohol inhibits the expression and function of the androgen receptor in human prostate cancer cells* Byung Ha Chunga,*, Hye-young Leea, Jae Seok Leea, Charles Y.F. Youngb a

Department of Urology and the Urological Science Institute, College of Medicine, Yonsei University, Yongdong P.O. Box 1217, Seoul, Korea Departments of Biochemistry and Molecular Biology and Urology, Mayo Graduate School, Mayo Clinic/Foundation, Rochester, MN, USA

b

Received 3 March 2005; received in revised form 15 May 2005; accepted 16 May 2005

Abstract Perillyl alcohol is a hydroxylated monocyclic monoterpene. In animal study, monoterpene has shown to have an anti-tumor effect. The aim of this study is to evaluate whether POH plays an important role in the development and progression of prostate cancer (pCa). We treated LNCaP cells with different concentrations of perillyl alcohol (POH). First of all, we performed cell proliferation assay and prostate-specific antigen (PSA) and human glandular kallikrein (hK2) quantification assays. LNCaP cells were treated with or without POH for Western blot analysis of androgen receptor (AR) and c-Jun. Finally, we performed transient transfection assay by transfecting LNCaP cells—which were treated with or without POH—with pGL-3 luciferase vector containing PSA promoter and AR promoter. We observed inhibition of the expression and function of the AR by POH, through inhibition of androgen-induced cell growth and androgen-stimulated secretion of prostate-specific antigen and hK2, in human pCa cell line LNCaP. In addition, we demonstrated, for the first time, that POH inhibits the transcription activities of the AR gene promoter by over-expression of c-Jun protein. These novel properties of POH strongly suggest that POH could be highly useful for intervention of pCa. q 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Perillyl alcohol; Androgen receptor; Prostate carcinoma cell line LNCaP

1. Introduction Perillyl alcohol (POH), a naturally occurring hydroxylated monocyclic monoterpene, is produced by plants and found in many commonly consumed *

Supported by NIH grants NCI/DK89000 and Army Defense grant DAMD17-98-1-8523. * Corresponding author. Tel.: C82 2 3497 3474; fax: C82 2 3462 8887. E-mail address: [email protected] (B.H. Chung).

fruits and vegetables including citrus and food flavorings (peppermint, spearmint, sage, cherries, orange and tangerine, etc.). In animal studies, monoterpenes have shown an anti-tumor effect and proved to be preventive against a wide range of tumors with low toxicity. Several mechanisms of monoterpene action have been previously explored including activation of transforming growth factor-b (TGF-b) pathway via receptor for mannose-6-phosphate/insulin-like growth factor type II (M6P/IGF2R) and inhibition of isoprenylation of Mr 21,000–26,000

0304-3835/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2005.05.023

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proteins, including p21ras, involved in signal transduction and growth regulation [1]. However, the actual mechanism of the anticancer action of monoterpene is far from clear. Moreover, its activity on prostate cancer (pCa) remains to be discovered. LNCaP is an androgen responsive pCa cell line that expresses androgen receptor (AR) and a number of androgen inducible genes such as prostate-specific antigen (PSA) and human glandular kallikrein (hK2) [2–4]. AR, the key nuclear transcription factor in the prostate, is expressed in all histological types and stages of pCa including hormone refractory tumors. Discovery of new drugs with properties that can modulate the function of AR are thus necessary for preventing the development and progression of pCa. Our study, for the first time, focused on the relationship between POH and AR gene in LNCaP cells, in other words between POH and pCa.

2. Material and methods 2.1. Cell culture LNCaP was obtained from The American Type Culture Collection (Rochville, MD, USA). Cells were propagated in 24-well, 60 or 100 mm culture dishes at the desired density in RPIM 1640 medium (Celox, St Paul, MN, USA) supplemented with 5% fetal bovine serum (FBS) (Biofluids, Rockville, MD, USA) and 5% CO2 at 37 8C until reaching approximately 50– 70% confluence. Cells were treated with POH at designated concentrations with or without Mibolerone (Mib, a non-metabolizable synthetic androgen, from New England Nuclear). POH (Sigma) dissolved in 100% ethanol at 100 mM were used as stock solutions and were stored in a K20 8C freezer. A 32 mM Mib dissolved in 100% ethanol was also stored in a K20 8C freezer and used as a stock solution. Cells treated with equivalent amounts (1–2 ml per ml of media) of solvent (100% ethanol) were included as controls. 2.2. Cell proliferation assay and PSA and hK2 quantification assays LNCaP cells were seeded at 2!104/well in 24well dishes for 48 h. Cells were incubated in

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serum-free RPIM 1640 medium for further 24 h to deplete endogenous steroid hormones prior to experiments. Cells were then treated with different concentrations of POH for 6 days in the presence or absence of 1 nM Mib. Spent media were collected and the protein levels for PSA and hK2 were measured by an immunometric assay as described previously [5,6]. The number of viable cells was measured by MTS assay (Promega, Madison, WI, USA). MTS (a tetrazolium salt) is a reagent that would be converted to a color compound (formazan) which was then measured at 490 nm of absorbance when applied to living cells. The protein levels of PSA and hK2 were normalized by the number of viable cells. 2.3. Western blot analysis For immunoblotting of the AR and c-Jun, LNCaP cells were grown in 100 mm culture dishes. After depleting endogenous steroids, cells were treated with or without POH in the presence or absence of 1 nM Mib for 24 h. Whole cell lysates were prepared using RIPA lysis buffer containing 1!PBS, 1% NP40, 0. 5% sodium deoxycholate and 0.1% SDS. Freshly prepared protease inhibitors (100 mg/ml PMSF, 30 ml/ml aprotinin, and 1 mM sodium orthovanadate) were also included. Total protein content was quantified by the Bio-Rad DC protein assay (BioRad, Hercules, CA, USA). Proteins were run with a Reducing SDS polyacrylamide gel (4–12%) and electrotransferred onto nitrocellulose membrane (Bio-Rad). The nitrocellulose membranes were immediately stained by a working solution of Ponceau S (0.1% Ponceau S, 5% acetic acid) to show loading and transfer efficiency of proteins and were photographed. The blots were blocked overnight at 4 8C with 5% non-fat milk in TBST buffer (20 mM Tris– HCl, 137 mM NaCl, and 0.1% Tween 20, pH 8.0) prior to incubation with a monoclonal antibody against AR (1:2000 dilution; PharMingen, San Diego, CA, USA) or c-Jun/AP-1 (a rabbit antibody, 1:500, Oncogene Research Products, Darmstadt, Germany) for 1 h at room temperature. An antimouse or anti-rabbit IgG secondary antibody conjugated to horseradish peroxidase (1:10,000 dilution; Amersham Pharmacia Biotech UK limited, Buckinghamshire, England) was applied to the blots for 1 h at room temperature. Blots were washed between each

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2.4. Transient transfection assay LNCaP cells were seeded at 4!105 cells in 60-mm dishes and grown under the conditions described above. Cells in triplicate plates were transfected with pGL-3 luciferase vector containing 6 kb PSA promoter (K5824/C12) in front of the luciferase reporter gene (2–4 mg/plate) using GenePORTORe Transfection Reagent (GST, Inc., CA, USA). Transient transfection experiment was also performed with the pGL-3 luciferase vector containing the AR promoter (K1380/C577) or the pGL-3 SV 40 promoter luciferase vector containing three copies of ARE [4,7,8]. The parental vector pGL3 or pGL3SV40 promoter luciferase vector (Promega) was used as a control. In a co-transfection experiment, LNCaP cells were transfected with various amounts (0.5, 1.0, 2.0 mg/plate) of a c-Jun expression construct [9] and the pGL-3 luciferase vector containing the AR promoter (2 mg/plate). A plasmid containing a CMV promoter and b galactosidase gene (b-gal expression vector, 0.3 mg/plate) was co-transfected to every dish to normalize transfection efficiency. Twenty-four hours after transfection, cells were treated with or without POH in the presence or absence of 1 nM Mib for 24 h. Cell extracts were prepared and used for b-gal assay according to published methods [4] and for luciferase assays (Promega, Madison, WI, USA). At least three independent transfections were performed. 2.5. Statistics Results were analyzed by two-tailed Student’s ttest. A P!0.05 was accepted as the level of significance.

3. Results 3.1. Androgen-induced cell growth in LNCaP cell was inhibited by POH To evaluate the effects of POH on androgeninduced cell growth, 1 nM of Mib and various concentrations of POH were added into the cultures. The number of viable cells was measured by an MTS assay 6 days later. As shown in Fig. 1a, LNCaP cells treated with Mib alone showed an increase in growth compared to cells without Mib treatment. When Mib was present cell growth was significantly inhibited with the treatment of POH. 3.2. Androgen-stimulated secretion of PSA and hK2 protein in LNCaP cell was reduced by POH Our result showed that cells treated with Mib increased the secretion of PSA (Fig. 1b) and hK2 (Fig. 1c) dramatically. However, this (a) Luc/β-gal activity

step with TBST and visualized by enhanced chemiluminescence substrate (ECL, Amersham Corporation, Arlington Heights, IL, USA) by exposure to X-ray film. A mouse anti-human b-tubulin antibody (1:10,000 dilution, Santa Cruz, CA, USA) was also applied to the same blots after stripping (Re-blot Plus Mild, Chemicon International, CA, USA). b-Tubulin was used to normalize the quantity of the protein on the blot.

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Fig. 1. Effects of POH on androgen stimulated growth responses and expression of PSA and hK2 protein in LNCaP Cells. LNCaP cells were incubated with various concentrations of POH for 6 days with or without Mib (1 nM) followed by an MTS assay performed in quadruplicate and SD bars are shown (a). Total PSA (b) and hK2 (c) quantification were performed on the spent media from cells treated with POH. These protein levels were normalized to the MTS measurements shown in (a). *Depicts significant (P!0.05) inhibition compared to the no POH controls.

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To determine the mechanism by which POH affects AR’s function, a luciferase reporter gene containing the PSA promoter and an empty vector were transfected into LNCaP cells. Cells were then treated with or without Mib and various concentrations of POH for 24 h. As shown in Fig. 2a, in Mibtreated cells, PSA promoter gave a strong androgenic induction of luciferase activity. However, POH treatments inhibited this androgenic response. To further demonstrate the specific site of the effect of POH, a luciferase reporter construct containing a minimum SV-40 promoter and three copies of ARE was transfected into LNCaP cells. As shown in Fig. 2b, again, in Mib-treated cells the ARE gave a strong androgenic induction of luciferase activity and POH inhibited such an effect significantly. Also, the results from Fig. 2a and b appear to be consistent with that in Fig. 1b and c, suggesting that the PSA gene is more sensitive to POH than the hK2 gene, perhaps at the transcriptional level. 3.4. The expression of AR protein in LNCaP cell was repressed by POH It has been demonstrated that a reduced AR protein expression may result in a decrease of AR function [10]. To determine whether POH has an effect on the expression of AR protein, we performed Western blot analysis. As shown in Fig. 3a and b, the protein levels of the AR were increased in Mib-treated cells but reduced by POH treatment for 24 h. 3.5. The expression of c-Jun protein in LNCaP cell was induced by POH We also examined the level of c-Jun protein in POH-treated cells by using Western blot analysis. As shown in Fig. 3c and d, c-Jun protein was strongly induced by POH at 24 h treatment in a dose dependent

3.6. Transcriptional activity of the AR promoter was inhibited by POH To test whether the transcriptional activity of the AR gene was also affected by POH, a gene transfer experiment was performed in LNCaP cells using a luciferase reporter plasmid containing the AR promoter. Compared to cells transfected with (a)

AR

β-tubulin (b) AR/Tubulin Expression

3.3. AR-mediated transactivation on androgen-regulated genes was inhibited by POH

manner. The greatest increase was about 15 times the control levels at the highest concentration (1 mM) of POH tested.

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androgen-stimulated secretion was decreased significantly with the treatment of POH (Fig. 1b and c). Note, the androgen-stimulated secretion of PSA seemed to be more sensitive to POH induced inhibition than that of hK2.

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Fig. 2. Effects of POH on the AR mediated transcription of a heterologous reporter gene with the PSA promoter or hK2 ARE. A transient transfection in LNCaP cells with (a) PSA promoter/pGL3, or (b) pGL3 SV40-3 ARE/pGL3 SV40 plasmid DNA was performed. After 24 h, cells were treated with POH at designated concentrations in the presence or absence of Mib (1 nM). Cell extracts were prepared for luciferase and b-gal assays. Luciferase activities were normalized by b-gal activities and presented as relative light units/mU b-gal. The transfection was repeated three times with SD bars shown. *Depicts significant (P!0.05) inhibition compared to the no POH but treated with Mib controls.

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4. Discussion

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Our data indicated that androgen-induced cell growth in LNCaP cell was inhibited by POH. This result suggested an impaired function of the AR in LNCaP cells as a result of POH treatment which was exhibited by reduction of androgen-stimulated secretion of PSA and hK2 protein in LNCaP cells by POH.

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Fig. 3. Effects of POH on the expression of the AR protein and c-jun protein. (a) A representative western blot analysis of the androgen receptor in LNCaP cells treated with varying amounts of POH for 24 h was performed. b-Tubulin was used as an internal control. (b) Results of densitometry measurement for AR protein levels. (c) A representative Western blot analysis shows c-jun protein levels in LNCaP cells treated with varying amounts of POH for 24 h. bTubulin was used as an internal control. (d) The graph shows the protein levels normalized by b-tubulin.

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the empty vector, the luciferase activity was strongly increased by the AR promoter (Fig. 4a). However, POH treatment inhibited this effect (Fig. 4a). 3.7. The expression of the AR gene was inhibited by overexpression of c-Jun in LNCaP cell To determine whether overexpression of c-Jun may affect the expression of the AR gene, we cotransfected a c-Jun expression construct at various concentrations with the plasmid containing the AR promoter with a luciferase-reporter gene into LNCaP cells. The b-gal expression vector was also cotransfected to normalize the transfection efficiency. Our results showed that the AR promoter-induced luciferase activity was gradually but significantly inhibited by the increasing amount of c-Jun (Fig. 4b).

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Fig. 4. Effects of POH on the expression of the AR gene and effects of c-Jun on the transcription activity of the AR promoter. (a) POH inhibits the expression of the AR gene at the transcription level. A transient transfection was performed in LNCaP cells using a 2-kb AR promoter or the control vector (pGL3) and treated with or without POH for 24 h. The resulting luciferase activities were normalized with b-gal activities. The transfections were performed three times and presented as an average; bars denote one SD. (b) cJun inhibits the transcription activity of the AR promoter. A transient transfection was performed in LNCaP cells using a 2-kb AR promoter or the control vector (pGL3) and with increasing amount of c-Jun expression construct DNA. The b-gal expression plasmid was also co-transfected into the LNCaP cells. The resulting luciferase activities were normalized with b-gal activities. The transfections were performed three times and presented as an average; bars denote one SD. *Depicts significant (P!0.05) inhibition compared to the controls.

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AR is a phosphoprotein that mediates the actions of androgens as a transcription factor. Once androgens are bound to AR, AR interacts with coactivators and binds as a homodimer to a specific DNA site, the androgen response element (ARE), in the promoter region of androgen-responsive genes such as PSA and hK2, to activate transcription of these genes. The quantity of PSA and hK2 protein secreted into the spent media thus represents the function of the AR in LNCaP [3]. Our results show that cells treated with Mib increased the secretion of PSA (Fig. 1b) and hK2 (Fig. 1c) dramatically. However, this androgenstimulated secretion was decreased significantly with the treatment of POH (Fig. 1b and c). In Mib-treated cells, the ARE gave a strong androgenic induction of luciferase activity and POH inhibited such an effect significantly (Fig. 2b). Taken together, these data suggested that AR-mediated transactivation on androgen-regulated genes was inhibited by POH. It has been demonstrated that a reduced AR protein expression may result in a decrease of AR function [10]. The protein levels of the AR were increased in Mib-treated cells but reduced by 24 h of POH treatment (Fig. 3a and b). Our data indicated that the expression of AR protein in LNCaP cells was repressed by POH. Previous studies by several groups including ours have shown that stimulated over-expression of c-Jun protein can inhibit the function of AR [9,11–13]. Sato et al. [14] further demonstrated that there is a direct protein to protein interaction between the DNA and ligand-binding domains of AR and the leucine zipper region of c-Jun that might affect the function of the DNA binding domain of the AR. Wang et al. [15] showed that the interaction may also involve in the hinge region of the AR. In addition, at least two studies indicated that the transcriptional interference between AR and AP-1 is partly mediated through competition for intracellular CBP that serves as an integrator/coactivator for AR transactivation activities [16,17]. Compared to cells transfected with the empty vector, the luciferase activity was strongly increased by the AR promoter (Fig. 4a). However, POH treatment inhibited this effect (Fig. 4a). Our data suggested that the transcriptional activity of the AR promoter is inhibited by POH.

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Our results showed that the AR promoter-induced luciferase activity was gradually but significantly inhibited by the increasing amount of c-Jun (Fig. 4b). These data suggested, for the first time, that c-Jun has an inhibitory effect on the expression of the AR gene at the transcription level.

5. Conclusion In this study we have shown a novel aspect of POH in that it can attenuate AR-mediated action in androgen responsive prostate cells by inhibiting AR gene expression and stimulating the expression of c-Jun, which in turn represses the expression and function of the AR. Our study will warrant a further investigation into the mechanism by which c-Jun regulates the AR promoter activities. Importantly, since AR plays a critical role in the development and progression of pCa, this study has provided an additional mechanism for anti-pCa activities of POH and possibly other monoterpenes and their analogs which may potentially be developed into chemopreventive and/or chemotherapeutic agents for pCa.

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