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Endocrinology Vol. 141, No. 5 1699-1704
Copyright © 2000 by The Endocrine Society

ARTICLES

Androgen-Induced Growth Inhibition of Androgen Receptor Expressing Androgen-Independent Prostate Cancer Cells Is Mediated by Increased Levels of Neutral Endopeptidase1

Ruoqian Shen, Makoto Sumitomo, Jie Dai2, Adam Harris3, David Kaminetzky, Min Gao, Kerry L. Burnstein and David M. Nanus

Urologic Oncology Research Laboratory (R.S., M.S., J.D., A.H., D.K., D.M.N.), Department of Urology, the Division of Hematology and Medical Oncology (D.M.N.), Department of Medicine, and the Department of Physiology (M.G.), Joan and Stanford I. Weill Medical College of Cornell University, New York, New York 10021; and the Department of Molecular & Cellular Pharmacology (K.L.B.), University of Miami School of Medicine, Miami, Florida 33101

Address all correspondence and requests for reprints to: Dr. David M. Nanus, The New York Presbyterian Hospital-Weill Medical College, 520 E. 70th Street, ST-341, New York, New York 10021. E-mail: dnanus{at}mail.med.cornell.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Androgen-mediated growth repression of androgen-independent prostate cancer (AIPC) cells has been reported in androgen-independent PC-3 cells overexpressing the androgen receptor, and in androgen-independent derivatives of LNCaP cells that develop following prolonged culture in androgen-free media. Using two models of AIPC, PC3/AR cells and LNCaP-OM1 cells, a subclone of LNCaP cells derived by prolonged culturing in charcoal-stripped media, we investigated whether expression of neutral endopeptidase 24.11 (NEP), a cell-surface peptidase that cleaves and inactivates neuropeptides implicated in the growth of AIPC, is induced by androgen, and whether NEP contributes to the observed androgen-mediated growth repression. These cell lines each express high levels of androgen receptor. Culturing in dihyrotestosterone (DHT) resulted in a 30–56% (PC3) and 35–43% (LNCaP-OM1) decrease in cell number over 7 days concomitant with a significant increase in NEP enzyme specific activity. Northern analysis detected an increase in NEP transcripts following DHT treatment in PC3/AR cells. The addition of the NEP enzyme inhibitor phosphoramidon to PC3 and LNCaP-OM1 or the NEP competitive inhibitor CGS 24592 to LNCaP-OM1 blocked the increase in NEP enzyme activity and reversed the DHT-induced growth inhibition. Neither phosphoramidon or CGS 24592 alone inhibited cell growth. Furthermore, the reversal of growth inhibition in LNCaP-OM1 cells was dose dependent on the concentration of CGS 24592. These data indicate that androgen-induced growth repression of AIPC cells PC3 and LNCaP-OM1 results in part from androgen-induced expression of NEP in these cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE MOLECULAR events involved in the development of androgen-independent prostate cancer (PC) are not well defined. Potential explanations include increased expression of the bcl-2 proto-oncogene (1), mutations of the p53 tumor suppressor gene (2), increased expression of polypeptide growth factors including epidermal growth factor (EGF), fibroblast growth factors and insulin-like growth factors (IGF) (3), increased expression of neuropeptide growth factors (4, 5), and alterations in the androgen receptor (AR) or AR signaling pathways (6, 7, 8). Numerous investigators have examined androgen-independent PC cell lines or androgen-independent sublines of androgen-sensitive LNCaP cells to decipher the mechanisms of androgen-independent growth. PC-3 cells are commonly used as a model for androgen-independent PC. One deficiency of this model is that PC-3 cells express no AR or low levels of nonfunctioning AR (9), in contrast to androgen-independent PC cells in vivo in which expression of AR is present and often amplified (7). To study AR function in androgen-independent PCs, researchers have stably introduced a full-length human AR complementary DNA (cDNA) into PC-3 cells (PC3/AR) (10, 11, 12, 13). In contrast to LNCaP cells that express a functioning AR and proliferate in vitro following treatment with androgen, Yaun et al. and Heisler et al. each reported a paradoxical inhibition of cell growth in PC3/AR cells cultured in androgen (10, 13). A similar androgen-mediated growth repression has also been reported in androgen-independent derivatives of LNCaP cells that developed following prolonged culture in androgen-free media (14, 15, 16). The exact cause of this paradoxical growth inhibition has not been fully explained.

We reported that expression of neutral endopeptidase 24.11 (NEP), a cell-surface peptidase that inactivates neuropeptide growth factors through hydrolysis, is decreased in androgen-independent PC cell lines including PC-3 cells but strongly expressed in androgen-sensitive LNCaP cells (17). Expression of NEP is androgen regulated in PC cells, with expression increasing following DHT treatment and decreasing with androgen withdrawal. Furthermore, overexpression of NEP in androgen-independent PC cells using an inducible vector construct significantly inhibits PC cell growth (17). We considered whether the DHT-induced growth inhibition observed in PC3/AR cells or in androgen-independent sublines of LNCaP cells resulted from induction of expression of NEP in these cells following treatment with androgen. We report here that androgen-mediated growth repression of these cell types is accompanied by an increase in NEP-specific enzyme activity, and that this growth inhibition can be reversed by cocultivation with an NEP enzyme inhibitor. These data suggest that androgen-induced growth inhibition in AR-expressing, androgen-independent PC cells is mediated in part by NEP.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
LNCaP cells were maintained in RPMI containing 5% FBS. PC3/AR and PC3/neo cells were cultured in RPMI 1640 without phenol red containing 10% charcoal stripped FBS (HyClone Laboratories, Inc.). LNCaP-OM1 cells are a subclone of LNCaP-OM cells (18) and were maintained in RPMI 1640 without phenol red containing 10% charcoal stripped FBS. NEP enzyme inhibitor phosphoramidon [N-({alpha}-rhamnopyranosyloxy-hydroxyphosphinyl)-leu-trp] and dihydrotestosterone (DHT) were purchased from Sigma (St. Louis, MO). CGS 24592, a competitive inhibitor of NEP, was supplied by Novartis Pharmaceuticals.

Enzyme assays
Cells in logarithmic phase of cell growth were rinsed in cold lysis buffer (50 mM Tris/150 mM NaCl) and lysed in lysis buffer containing 0.5% CHAPS (3-[3-cholamidopropyl-dimethylammonio]-1-propane-sulfonate), which did not affect NEP enzyme specific activity. Protein concentrations were measured using the Bio-Rad DC protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA). NEP activity was assayed using Suc-Ala-Ala-Ala-Phe-para-aminobenzoate (pAB) (Bachem Bioscience, Inc.) as substrate. Thirty microliters of cell membrane suspension was added to a mixture of 200 µl of 100 mM Tris-HCl, pH.7.6, 10 µl of 20 mM substrate (dissolved in dimethyl sulfoxide), and 10 µl of aminopeptidase N enzyme solution (EC 3.4.11.2; Roche Molecular Biochemicals, Indianapolis, IN), and incubated at 37 C for 10 min. The reaction was stopped by adding 10% trichloroacetic acid, centrifuged at 2500 rpm x 5 min, and 250 µl of supernatant was removed for colorimetric analysis. The absorbance of the chromogen was immediately read at 540 nm against a reaction mixture without cell membrane as blank. Specific activities were expressed as pmol/µg protein/minute and represent an average of at least two separate measurements. The SE of measurement of duplicate experiments was approximately 10 to 20% of the mean value.

Growth assays
Approximately 4,000 cells/well were plated in 12-well tissue culture plates or 10,000 cells/well were plated in six well tissue culture plates (Falcon Division, Becton Dickinson and Co., Cockeysville, MD) in RPMI 1640 10% charcoal-stripped serum for 18 h, counted using a Coulter Counter ZM (Coulter Electronics, Hialeah, FL) (Day 1), and refed with RPMI 1640 10% charcoal-stripped media containing either 30 nM DHT, 10 µM phosphoramidon, or 30 nM DHT plus 10 µM phosphoramidon. DHT was maintained as a 3.4 µM stock dissolved in ethanol, and all cells received an equal concentration of ethanol. Cells were refed on day 3 and counted on day 6. Results represent an average of two independent experiments performed in triplicate. P values were determined using a Student’s t test.

Northern analysis
Total RNA was extracted from logarithmically growing cells using RNazol B (Cinna/Biotecx Laboratories, Houston, TX) according to the manufacturer’s recommendations. Twenty micrograms of RNA per lane were electrophoresed in 1.2% agarose/formaldehyde gels, transferred to nitrocellulose membranes, and hybridized with a 0.9 kb NEP specific probe fragment containing the 5' end of the NEP cDNA, a PstI/XbaI cut 0.78 kb glyceraldehyde 3 phosphate dehydrogenase (GAPDH) cDNA which were random prime radiolabeled with 32P-dCTP using PrimeIt II (Stratagene Cloning Systems, La Jolla, CA) as per the manufacturer’s recommendations.

Protein extraction and Western blot analysis
Protein was extracted from exponentially growing cells and analyzed by Western blotting as previously described (19) using 0.1 µg/ml of anti-AR polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Blots were incubated with enhanced chemiluminescent (ECL) detection reagents (Amersham Pharmacia Biotech, Arlington Heights, IL) and AR protein was detected by autoradiography by exposure of blots to Kodak XAR film for 2–15 min.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Androgen effect on PC3/AR proliferation and NEP enzyme activity
PC3/AR cells contain a stably transfected AR and express AR protein, whereas PC3/neo cells contain the identical vector without the AR and do not express AR protein (12). Northern analysis confirmed that PC3/AR cells express high levels of AR transcripts, whereas PC3/neo cells do not (Fig. 1Go). We first determined the effects of 30 nM DHT on growth of PC3/AR and PC3/neo cells. In three separate experiments performed in triplicate, PC3/AR cells were 30%, 34% and 56% growth inhibited (P values < 0.001, 0.003 and 0.005, respectively), whereas PC3/neo cells were not significantly inhibited (P value > 0.2 in all three experiments; representative data illustrated in Fig. 2AGo). To determine if growth inhibition correlated with a change in NEP-specific enzyme activity, PC3/AR and control PC3/neo cells were simultaneously assayed for cell growth and NEP enzyme activity. In all three experiments, NEP enzyme activity increased in cell lysates derived from PC3/AR cultured in DHT but not in lysates from and PC3/neo cells (Table 1Go, columns Control and DHT). To determine if growth inhibition resulted from an increase in NEP enzyme activity, 10 µM of the NEP enzyme inhibitor phosphoramidon (20) was added to the media on day 3 of a 6-day growth assay. Phosphoramidon alone did not significantly affect growth of PC3/AR or PC3/neo cells. Measurement of NEP enzyme activity confirmed that phosphoramidon blocked the increase in enzyme activity observed following incubation with DHT in PC3/AR cells (Table 1Go). Analysis of cell number indicated that incubation with DHT and phosphoramidon resulted in complete abrogation of the DHT-induced growth inhibition observed in PC3/AR cells (Fig. 2AGo). These data suggest that DHT-induced growth inhibition in PC3/AR cells results from an increase in NEP enzyme activity.



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  		Figure 1. Northern analysis of androgen receptor expression in PC3/AR and PC3/neo cells. Twenty micrograms of RNA extracted from PC3/AR and PC3/neo cells were separated on an agarose gel, transferred to nitrocellulose, and probed with a cDNA probe for the androgen receptor. Note abundant androgen receptor transcripts in PC3/AR cells but not PC3/neo cells. Membrane was stripped and reprobed with a cDNA for GAPDH to confirm equal loading (bottom panel).

 


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  		Figure 2. A, Effect of DHT and phosphoramidon on PC3/AR and PC3/neo cell growth. PC3/AR and PC3/neo cells were seeded in 12-well plates overnight and then refed with RPMI 1640 containing 10% charcoal-stripped serum containing 30 nM DHT. Cell number was determined on Day 6. Data representative of one experiment performed in triplicate on three separate occasions. P value <= 0.01 for DHT growth inhibition vs. control, or DHT growth inhibition vs. DHT + phosphoramidon in all three experiments. Enzyme analysis performed simultaneously for all three replicate experiments shown in Table 1Go. B, Northern analysis of NEP expression in PC3/AR and PC3/neo cells following DHT treatment. RNA was extracted from PC3/AR and PC3/neo cells incubated for 24 h in RPMI containing 10% charcoal stripped serum without (-) or with (+) 30 nm DHT. Twenty micrograms of RNA were separated on an agarose gel, transferred to nitrocellulose, and probed with a cDNA probe for NEP. RNA extracted from LNCaP cells which constitutively express high levels of NEP transcripts was used as control to illustrate NEP transcripts. Note low levels of NEP transcripts in PC3/AR cells treated with DHT in contrast to other cell lines. Membrane was stripped and reprobed with a cDNA for GAPDH to confirm equal loading (bottom panel). Arrow, NEP transcript. Band above arrow in all lanes represents cross-hybridization of the NEP probe to 28s ribosomal RNA.

 

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  		Table 1. NEP enzymatic activities for PC3/AR and PC3/neo incubated in 30 nM DHT

 
DHT induces NEP transcripts in PC3/AR cells
LNCaP cells constitutively express NEP transcripts (Fig. 2BGo, lane 5), while PC3/AR and PC3/neo cells grown in charcoal-stripped media do not express detectable NEP transcripts by Northern analysis (Fig. 2BGo, lanes 1 and 3). However, incubation of PC3/AR and PC3/neo cells in 30 nM of DHT for 24 h resulted in the expression of low levels of detectable NEP transcripts in PC3/AR cells but not PC3/neo cells (Fig. 2BGo, lanes 2 and 4).

DHT inhibits growth of LNCaP-OM1 cells and induces NEP enzyme activity
Our data on PC3/AR cells suggested that DHT-mediated expression of NEP resulted in growth inhibition. We next examined a second model of AR expressing androgen-independent PC in which DHT-mediated growth repression had previously been reported. LNCaP-OM cells were originally derived by culturing parental LNCaP cells in media containing charcoal stripped serum for over 12 months (18). LNCaP-OM1 cells, which express high levels of AR protein (Fig. 3AGo), are a subline obtained by dilutional cloning of LNCaP-OM cells.



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  		Figure 3. A, Western Analysis of AR expression in LNCaP-OM1 and LNCaP cells. Cell lysates derived from LNCaP-OM1 and LNCaP cells grown in media containing charcoal-stripped serum (CS), charcoal strip serum plus 30 nM DHT (CS plus DHT), or FCS were separated on SDS-DAGE gel transferred to nitrocellulose and immunoblotted with a monoclonal antibody that recognizes AR. Note the high levels of AR that are expressed by both cell lines under all three conditions. B, Effect of increasing concentrations of DHT on growth of LNCaP-OM1 cells. LNCaP-OM1 cells were seeded in 12-well plates overnight and refed with RPMI containing 10% charcoal strip serum with increasing concentrations of DHT. Cell number was determined on day 6. All experiments were performed in triplicate on three separate occasions. The data are representative of one experiment. C and D, Effect of DHT on NEP Specific enzyme activity in LNCaP-OM1 cells. LNCaP-OM1 cells were cultured RPMI containing 10% charcoal-stripped serum with the addition of DHT. C, Cell cultured in 1 nM DHT and NEP-specific enzyme activity measured every 24 h for 4 days. Note increase in enzyme activity over time. D, Enzyme activity measured with increasing concentrations of DHT following 4-day incubation.

 
Incubation of LNCaP-OM1 cells in media containing DHT for 7 days resulted in a dose-dependent decrease in cell number (Fig. 3BGo). This was accompanied by a significant increase in NEP specific enzyme activity measured over 96 h (Fig. 3CGo). Furthermore, the increase in NEP enzyme activity was dependent on the concentration of DHT (Fig. 3DGo). These results were similar to those observed with PC3/AR cells.

NEP inhibitors reverse DHT-induced growth inhibition of LNCaP-OM1 cells
As illustrated in Fig. 4AGo, the addition of 10 µM phosphoramidon to the media reversed the DHT-induced growth inhibition of LNCaP-OM1 cells, suggesting that growth inhibition results from an increase in NEP enzyme activity. This was accompanied by a significant decrease in NEP enzyme activity (data not shown). Phosphoramidon can inhibit other peptidases in addition to NEP. Therefore, to confirm that NEP inhibition reversed DHT induced growth inhibition in LNCaP-OM1 cells, we obtained the NEP competitive inhibitor CGS 24592 (21). While incubation in media containing 10 nM CGS 24592 had no effect on cell growth, 10 nM of CGS 24592 reversed the growth inhibition resulting from 1 nM DHT (Fig. 4BGo). NEP specific enzyme activity in LNCaP-OM1 cells grown in 1 nM DHT decreased from 27.7 pmol/µg protein/min to 0.3 pmol/µg protein/min (P value < 0.005) following incubation with 10 nM CGS 24952 concomitant with the reversal of DHT-induced growth inhibition. This reversal of DHT-induced growth inhibition was dose dependent on the concentration of CGS 24592 (Fig. 4CGo).



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  		Figure 4. Effect of NEP inhibitors phosporamidon and CGS 24592 on LNCaP-OM1 cell growth. A, LNCaP-OM1 cells were seeded in 12-well plates overnight and then refed with RPMI 1640 containing 10% charcoal-stripped serum (Control), charcoal-stripped serum plus 1 nM DHT, charcoal-stripped serum plus 10 µM phosphoramidon (Phosp), or both DHT and phosporamidon. Note that growth inhibition induced by DHT is reversed by the addition of phosporamidon (P < 0.005), whereas phosphoramidon alone has no effect on cell growth. Data representative of one experiment performed in triplicate on three separate occasions. B, Identical experiment as panel A except 10 nM CGS 24592 used instead of phosphoramidon (P < 0.005). C, LNCaP-OM1 cells were cultured RPMI containing 10% charcoal strip serum with the addition of 10 nM DHT and increasing concentrations of CGS 24592. Note reversal of DHT growth inhibition is more pronounced with higher concentrations of CGS 24592.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The objective of this study was to determine if an increase in NEP expression and associated catalytic activity could account for the paradoxical growth inhibition observed following incubation in androgen in PC-3 cells expressing AR and in an androgen-independent derivative of LNCaP cells. PC3/AR cells were generated to better delineate the role of the AR in the development and progression of hormone-refractory PC. Androgen may increase the proliferation of these cells because androgen is a growth and survival factor for PC cells. However, as previously reported in PC3/AR cells (12, 13), the addition of androgen results in significant growth inhibition. Our data implicate an androgen-induced increase in NEP enzyme activity as a cause for androgen-induced growth inhibition in PC3/AR cells. Similar results implicating androgen-induced expression of NEP as a contributing factor to growth inhibition were obtained with a second androgen-independent, AR expressing PC cell line derivative of LNCaP cells, LNCaP-OM1. In both of these cell lines, androgen-induced growth inhibition was reversed with the NEP enzyme inhibitor phosphoramidon. Furthermore, a similar reversal of growth inhibition was observed in LNCaP-OM1 cells using a second, more specific NEP inhibitor CGS 24592. The effects of CGS 24592 were dose dependent as would be expected with a competitive enzyme inhibitor. Of interest, parental LNCaP cells exhibit a biphasic growth response to androgens, with growth stimulation at lower, physiologic concentrations of androgens and a reduction in growth stimulation as the concentration of androgen is increased in the media (22). The etiology of this response to androgen in LNCaP cells is unknown, but our preliminary data indicate NEP inhibitors do not reverse the reduction in LNCaP growth at high androgen concentrations, suggesting this phenomenon may not be mediated by NEP.

The increase in NEP expression following androgen treatment results from the fact that the NEP gene is transcriptionally activated by androgen (17). Progesterone also increases NEP messenger RNA and protein expression in human endometrial stromal cells (23), and glucocorticoids increase NEP expression in human bronchial epithelial BEAS-2B cells (24) and in human vascular smooth muscle cells (25), indicating that the NEP gene is regulated by this family of steroid hormones. We have identified an androgen response element (ARE) located in exon 24 of the NEP gene and a second androgen response region located in the NEP promoter that bind AR and activate transcription of a reporter gene in response to androgen treatment (26). Thus, the introduction of DHT into the media of these cells stimulates NEP transcription. The less pronounced increase in NEP activity in PC-3 cells compared with LNCaP-OM1 may result from the fact that the NEP promoter contains a 5' CpG island spanning the transcriptional regulatory region (27, 28), and this region that contains the androgen response region is hypermethylated in PC-3 cells (29). Therefore, transcriptional activation of the NEP gene in response to steroid induction may be limited.

The mechanism of NEP growth inhibition in PC cells involves hydrolysis of neuropeptides such as bombesin and endothelin-1, or other unknown peptides important in androgen-independent growth (30, 31). PC-3 and LNCaP cells each express cell-surface receptors for the NEP substrates bombesin (32) and endothelin-1 (29, 33). An increase in NEP expression in PC3/AR or LNCaP-OM1 cells following DHT treatment would inactivate these neuropeptides. Sudden loss of these neuropeptides may have an immediate adverse effect on cell proliferation. The resulting growth inhibition may be moderate because neuropeptides are not strong mitogens but appear to interact with other polypeptide growth factors such as epidermal growth factor and insulin-like growth factors to stimulate cell growth (5, 33).

Other explanations have been implicated as a cause for androgen-mediated growth repression of androgen-independent PC cells. These include increased number of cells in G1 and an increase in the number of cells undergoing apoptosis in DHT treatment of PC3/AR cells (13) and induced expression of p27Kip1 in another androgen-independent LNCaP subline 104-R1 (15). Our results are compatible with these explanations because overexpression of NEP can result in growth arrest, apoptosis and altered expression of cyclin-dependent kinases (unpublished data).

In conclusion, these experiments further implicate NEP in the development and progression of androgen-independent PC. NEP is normally expressed by prostate epithelial cells, in vitro by androgen-sensitive LNCaP cells and in vivo in metastatic PC cells from patients with androgen-dependent disease. NEP expression is diminished in androgen-independent PC cell lines and in the majority of metastatic androgen-independent PCs in vivo. Overexpression of NEP in another androgen-independent PC cell line, Tsu-Pr1 cells, inhibits growth (17). Similarly, induction of NEP expression in PC3/AR and LNCaP-OM1 cells inhibits growth. Taken together, these independent experiments firmly establish a link between increased NEP activity and inhibition of androgen-independent PC cellular proliferation.


   Acknowledgments
 
The authors thank Dr. O. Platica for supplying LNCaP-OM cells and Ms. Lana Winter for secretarial support.


   Footnotes
 
1 This work was supported by NIH Grant CA-80240, the Association for the Cure of Cancer of the Prostate (CaP CURE), and the Dorothy Rodbell Foundation for Sarcoma Research. Back

2 Recipient of a Department of Defense Prostate Cancer Research Program Post-doctoral Traineeship Award. Back

3 New York Academy of Science Summer Student. Back

Received November 12, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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