Activation of the Y1 Receptor by Neuropeptide Y Regulates the Growth of Prostate Cancer Cells
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《内分泌学杂志》
Center for Endocrinological Oncology (M.R., E.D., S.B., V.M.R., M.M., P.M.), Istituto di Endocrinologia, Universita degli Studi di Milano, 20133 Milan, Italy
Unita Operativa di Anatomia Patologica (G.B.), Azienda Ospedaliera San Gerardo, 20052 Monza (Milan), Italy
Abstract
This study deals with the role of neuropeptide Y (NPY) in the regulation of cell proliferation. NPY is expressed in the normal and tumoral prostate, but no data on its possible role in prostate cancer (PCa) progression are available. Therefore, we evaluated the direct effect of NPY on the growth of the human PCa cell lines LNCaP (androgen dependent) and DU145 and PC3 (androgen independent). All PCa cell lines expressed Y1-R gene and protein. NPY treatment reduced the proliferation of LNCaP and DU145 cells and increased that of PC3 cells. The Y1-R antagonist BIBP3226 abolished such effects, suggesting a mandatory role of Y1-R in this process. LNCaP cells showed elevated constitutive levels of phosphorylated ERK1/2, which were not affected by NPY. In DU145 cells, NPY stimulated a long-lasting ERK1/2 activation, whereas, in PC3 cells, this effect was rapid and transient and required activation of protein kinase C. Moreover, in both cell lines, pretreatment with BIBP3226 prevented the NPY-induced ERK1/2 phosphorylation, further supporting Y1-R involvement. NPY treatment reduced forskolin-stimulated cAMP accumulation only in PC3 cells and did not change intracellular calcium concentration in any PCa cell line. These data indicate that NPY may directly regulate PCa cell growth via Y1-R. The direction of this effect appears to be related to the time kinetics of MAPK activation, i.e. long-lasting vs. transient, and to the clone-specific involvement of other intracellular signals. These findings suggest that NPY-related mechanisms might play a relevant role in the progression of PCa, at both androgen dependent and independent stages.
Introduction
PROSTATE CANCER (PCa) represents one of the most common malignant diseases among men in the Western world, where there is a 10% chance of developing PCa and a 3–4% chance of dying of causes directly related to it (1). PCa is initially androgen dependent, and it may later progress to androgen independence (2). This latter stage is associated with a lack of efficacy of hormonal therapy and appears to be promoted at least in part by several growth factors and neurohormones (1). Therefore, the study of neuroendocrine regulatory peptides might be of relevance in tumors that express their respective receptors in high amounts, as suggested by Reubi et al. (3) for breast cancer. Scant data are presently available on the possible role exerted by neuropeptide Y (NPY) and its receptors on PCa progression. NPY is a highly conserved 36-amino acid residue peptide, belonging to the pancreatic polypeptide family. NPY is widely distributed throughout the central and peripheral nervous systems and is involved in the regulation of several physiological functions (neuroendocrine mechanisms, cognitive functions, eating behavior, cardiovascular activity, etc.) (4, 5). More recently NPY has been shown to be involved in prostate physiology and pathophysiology, including PCa growth and progression. In the human prostate, particularly in the smooth muscle layer, NPY is mainly localized in nerve fibers and neuroendocrine cells (6, 7). Moreover, immunopositivity for NPY has been shown in 75% of the PCa specimens obtained from a series of patients (8). NPY activates specific G protein-coupled receptors, present in humans in at least five subtypes, named Y1-R to Y5-R. Moreover, NPY has been demonstrated to be involved in mitogenic pathways and stimulate cell proliferation via the Y1-R (9). The activation of Y1-R is generally associated with reduction of cAMP accumulation, increase of intracellular free calcium concentration ([Ca2+]i), and modulation of the MAPK pathway via several signaling molecules, including the protein kinase C (PKC) (4, 9). The aim of the present study was to elucidate the role of NPY in PCa biology by investigating the expression of Y1-R in three commonly used human PCa cell lines (the androgen-dependent LNCaP and the androgen-independent DU145 and PC3) as well as the activation of intracellular signals and the possible direct proliferative effects of NPY. Our data suggest that Y1-R activation by NPY represents an important regulator of cell proliferation, with a peculiar intracellular signaling pattern according to each of the PCa cell lines used.
Materials and Methods
Cell cultures
LNCaP, DU145, and PC3 human PCa and SK-N-MC human neuroblastoma cell lines (American Type Culture Collection, Manassas, VA) were grown at 37 C in a humidified CO2 incubator in monolayer. The culture medium for LNCaP, DU145, and PC3 cells was RPMI 1640, with 10 mg/liter phenol red (Biochrom, Berlin, Germany), and 5 or 10% fetal calf serum (FCS; Life Technologies, Inc., Grand Island, NY); for SK-N-MC cells, the medium was MEM containing nonessential amino acids, 1 mM sodium pyruvate, 10 mg/liter phenol red, and 10% FCS. Confluent cells were harvested with 0.05% trypsin/0.02% EDTA (Biochrom) and were seeded in petri dishes (Becton Dickinson, Plymouth, UK) for all the experiments, which were performed using subconfluent cell cultures.
RNA extraction and RT-PCR analysis of NPY and Y1-R gene expression
For RNA studies, cells were washed with cold PBS, collected, snap frozen in liquid nitrogen, and stored at –80 C until RNA extraction. Total cellular RNA was extracted with the phenol-chloroform method using the Tri-Reagent solution (Sigma-Aldrich, Milan, Italy). RT-PCR analysis of the expression of the genes coding for NPY and Y1-R was performed on total RNA samples, quantified after an initial DNAase digestion step using the Deoxyribonuclease I kit (Sigma-Aldrich). The reverse transcription reaction was carried out in a 50-μl volume, using a commercially available GeneAmp kit (Applied Biosystems, Milan, Italy). A control RNA (pAW 109), provided with the RT-PCR kit, was used as a positive control of the RT-PCR. Negative control reactions omitting reverse transcriptase were carried out in parallel, confirming that no sample contamination by genomic DNA occurred (not shown). The primers used for Y1-R subtype, NPY and -actin (housekeeping gene) were as follows: Y1-R forward (5'-TATACCACTCTTCTCTTggTgCTg-3') and Y1-R reverse (5'-CTggAAgTTTTTgTTCAggAACCCA-3') (10); NPY forward (5'-CCAgATACTACTCggCgCTgCgACACTACA-3') and NPY reverse (5'-gAATgCATgATACTTTATTTAAACACACATATATACAA-3') (11); -actin forward (5'-TgACggggTCACCCACACTgTgCCCATCTA-3') and -actin reverse (5'-CTAgAAgCATTTgCggTggACgATggAggg-3') (12). Amplification products were separated by electrophoresis in a 2% agarose/1x Tris-borate EDTA gel and were detected by ethidium bromide fluorescence on an UV transilluminator. The identity of the PCR products was confirmed by DNA sequencing (data not shown).
Western blot analysis
PCa cells were collected in rapid immunoprecipitation assay lysis buffer (13) containing 1% protease inhibitor mix (Sigma-Aldrich). Protein concentration was quantified by the BCA protein assay (Pierce, Rockford, IL). Fifty milligrams protein cell extracts were resuspended in Laemmli sample buffer (Sigma-Aldrich) and were separated on a SDS-PAGE, using Rainbow molecular mass markers (Amersham Biosciences Europe, Milan, Italy). Proteins were transferred to nitrocellulose membranes (Bio-Rad Laboratories, Milan, Italy) overnight. After wash and blockade steps, the blot was incubated overnight at 4 C with a diluted solution of the specific primary antibody [anti-Y1-R (1:700), which is an affinity-purified antiserum generated in rabbit against a C-terminal peptide (amino acids 355–382) of this receptor and was kindly donated by Dr. B. Bunnemann (GlaxoSmithKline, Verona, Italy) (14); anti-ERK 1/2 (1:1000); and anti-pERK1/2 (1:150), both from Santa Cruz Biotechnology, Santa Cruz, CA]. The incubation with the secondary antibody conjugated with horseradish-peroxidase (Santa Cruz Biotechnology) was performed at room temperature for 2 h. The immunoreactivity was detected by the SuperSignal West Pico substrate working solution (Pierce) and exposure of the membrane to radiographic film at room temperature. A negative control carried out by incubating the membrane with the secondary antibody alone did not result in any band on the blot.
Cell proliferation studies
For cell count experiments, LNCaP, DU145, and PC3 cells were seeded in 60-mm-diameter petri dishes. After 72 h, the culture medium was replaced by the experimental medium (RPMI 1640/0.1% BSA or 2% FCS) containing 10–10 to 10–7 M human NPY (Bachem, Bubendorf, Switzerland) and, in selected experiments, the specific Y1-R antagonist BIBP3226 (Bachem). The experimental incubation was carried on for 96 h, and then cells were harvested with 0.05% trypsin/0.02% EDTA and resuspended in RPMI 1640. Cell viability was assessed by the trypan-blue exclusion method, and then cells were counted. For [3H]thymidine incorporation studies, LNCaP, DU145, and PC3 cells were plated in 24-well plates at 1.5–3 x 104 cells/well. After 24 h, the culture medium was replaced by the experimental medium (RPMI 1640/0.1% BSA or 2% FCS) containing the MAPK kinase (MEK) inhibitor U0126 (10–6 M; Biomol, Plymouth Meeting, PA) or the specific PKC inhibitor GF109203X (10–7 M; Sigma-Aldrich). Thirty minutes later, 10–8 M human NPY (Bachem) was added and the incubation was carried on for 48 h. Two microcuries per well [3H]thymidine (Amersham Biosciences Europe) were added 6 h before the end of the incubation. Cells were harvested and the incorporated [3H]thymidine counted in the presence of scintillation fluid using a -counter. Experiments were repeated at least three times.
Effect of NPY treatment on cAMP concentration
To study the NPY modulation of cAMP levels, LNCaP, DU145, and PC3 cells were seeded in 24-well plates. Subconfluent cell cultures were washed with RPMI 1640 and incubated in 200 μl RPMI 1640 containing 0.5 mM isobutylmethylxanthine (Sigma-Aldrich) for 15 min at 37 C. Subsequently 200 μl RPMI 1640 containing the test substances (forskolin, Sigma-Aldrich; human NPY, BIBP3226) were added to each well and the incubation was carried on for another 15 min at 37 C. After removal of the medium, cells were collected in 500 μl cold 75% ethanol; membranes were precipitated by centrifugation at 10,000 rpm for 3 min. The supernatant was freeze dried and resuspended in RIA buffer. The cAMP content was quantified by a commercial RIA kit (Amersham Biosciences Europe).
Effect of NPY treatment on [Ca2+]i
The effect of NPY treatment on [Ca2+]i in PCa cells was determined by using the fluorescent probe fura-2 acetoxymethyl ester (fura-2; Molecular Probes, Eugene, OR), according to Grynkiewicz et al. (15). PCa cells were grown on 12-mm-diameter glass slides in petri dishes. Subconfluent cells adherent to glass slides were loaded with fura-2 by incubation in 1 mM fura-2 solution at 37 C for 60 min; control cells were incubated in the same conditions without fura-2 solution. Cells were washed with HEPES-saline solution (pH 7.4). The fluorescence of control cells, which served as background level, was first measured by means of a fluorometer (Fast Filter polarizer, model LS 50B; PerkinElmer, Norwalk, CT). Fura-2-loaded cells were inserted into the fluorometer, and when the fluorescence level appeared to be stabilized, the test substances (NPY, ATP, and the calcium ionophore A23187; Sigma-Aldrich) were added and the fluorescence was measured. [Ca2+]i corresponding to measured fluorescence was calculated with the Grynkiewicz equation. Ionomycin (final concentration: 2.7 μM; Sigma-Aldrich) and digitonin (final concentration: 100 μM; Merck, Darmstadt, Germany) were added to establish the maximum amount of Ca2+ bound to fura-2. EGTA (final concentration: 5 mM) and Tris-base (final concentration: 60 mM; both from Sigma-Aldrich) were then added to establish the minimum levels of Ca2+ bound to fura-2.
Analysis of the data
Statistical analysis was performed using the Systat statistical analysis package (Systat Software, Erkrath, Germany). Data are presented as mean ± SEM; n = number of replicates within an experiment. Significance of differences between treatment groups was evaluated by ANOVA, followed by Tukey test, and was considered significant when P < 0.05.
Results
Expression of NPY and Y1-R in PCa cells
The expression of NPY and Y1-R isoform was initially evaluated in PCa cell lines at the gene level by RT-PCR analysis. The NPY gene was not found to be expressed in any of the PCa cell lines tested (data not shown). A 354-bp PCR product corresponding to Y1-R, together with a 97-bp-longer additional band, was detected in all PCa cells and SK-N-MC human neuroblastoma cells (positive control) (16) (Fig. 1A). Western blot analysis using an anti-Y1-R antibody showed a specific immunoreactive band with a size of 70 kDa in all PCa cell lines (Fig. 1B).
Effect of NPY on PCa cell proliferation: involvement of Y1-R
A 96-h treatment with 10–10 to 10–7 M NPY resulted in a significant reduction of LNCaP and DU145 cell proliferation, with a maximal inhibition at 10–8 M NPY (–45–63%) for the LNCaP cell line and at 5 x 10–8 M NPY (–33–35%) for the DU145 cell line. On the contrary, a marked proliferative effect was observed for the androgen-independent cell line PC3 (+36–60% at 10–8 M NPY) (Fig. 2A). We then examined whether NPY effects on cell proliferation were mediated through Y1-R, which is expressed in all PCa cell lines (Fig. 1A). The addition of 1 μM BIBP3226, a specific Y1-R antagonist (17), abolished both the proliferative (on PC3) and antiproliferative (on LNCaP and DU145) effects of NPY (Fig. 2B). Treatment with any concentration of NPY and BIBP3226 did not modify the survival rate of the cells (viable cells > 95% according to trypan blue exclusion) or induce any morphological change (data not shown).
Effect of NPY on intracellular signaling in PCa cells
To dissect out the intracellular mechanisms underlying the above-described effects of NPY on cell proliferation via Y1-R, we first studied the involvement of MAPK/ERK1/2 signaling. ERK1/2 phosphorylation was evaluated after treatment of PCa cell lines with 10–8 M NPY. LNCaP cells showed a constitutive activation of the ERK1/2 pathway, with no further modulation by NPY (Fig. 3). In DU145 cells, low basal pERK1/2 amounts were present, and NPY treatment resulted in a marked increase of ERK1/2 phosphorylation, which occurred within 5–15 min and remained elevated up to at least 6 h (Fig. 3). In PC3 cells, very low basal pERK1/2 levels were detected, and NPY treatment resulted in an early (5–15 min) and transient increase of ERK1/2 phosphorylation (Fig. 3). Moreover, in DU145 and PC3 cell lines, the NPY-mediated ERK1/2 phosphorylation was markedly reduced by a pretreatment (30 min) with 10–6 M BIBP3226 (Fig. 4).
Because it has been proposed that PKC activation is an upstream event for ERK1/2 phosphorylation in some systems (18), we tested this hypothesis also in DU145 and PC3 cells, in which, as reported above, NPY has been shown to activate this process. It was found that a pretreatment with 10–7 M GF109203X, a specific PKC inhibitor, abolished NPY-induced ERK1/2 phosphorylation in PC3 cells but not in DU145 cells (Fig. 5). The role of NPY-mediated activation of MAPK/ERK1/2 in the proliferation of PCa cells was then studied by using the specific MEK inhibitor U0126 in combination with the [3H]thymidine incorporation assay (Fig. 6). In LNCaP cells, a pretreatment with 10–6 M U0126 plus 10–8 M NPY determined an additional inhibitory effect on cell proliferation, compared with NPY alone, suggesting that NPY, which does not substantially affect ERK1/2 phosphorylation (Fig. 3), reduces LNCaP cell proliferation by mechanisms that are at least in part independent of ERK1/2 activation. In DU145 cells, a pretreatment with U0126, a specific MEK inhibitor, did not affect the NPY-associated reduction of cell proliferation (Fig. 6). The efficacy of NPY in stimulating PC3 cell proliferation was, however, prevented by U0126 pretreatment, indicating the involvement of NPY-activated ERK1/2 in this effect (Fig. 6).
According to previous studies (19), another early event coupled to Y1-R activation is the involvement of PKC. In all PCa cell lines, exposure to 10–7 M GF109203X significantly reduced [3H]thymidine incorporation (Fig. 6). In LNCaP and DU145 cells, treatment with NPY alone or NPY plus GF109203X reduced [3H]thymidine incorporation to the same extent, whereas in PC3 cells, the addition of GF109203X abolished the proliferative action of NPY, suggesting a role for PKC activity in this effect (Fig. 6).
The short-term effect of NPY on other relevant intracellular signals known to be coupled to Y1-R (namely, cAMP accumulation and [Ca2+]i change) was then assessed. PCa cells were treated for 15 min with 10–8 M NPY in the presence or absence of 5 μM forskolin. NPY treatment alone did not modify basal cAMP levels, and, as expected, exposure to forskolin induced a marked increase of cAMP concentrations. NPY treatment significantly reduced forskolin-induced cAMP accumulation only in PC3 cells (–52%), but it was ineffective in LNCaP and DU145 cells (Fig. 7). Treatment with 10–8 M NPY did not induce any change of [Ca2+]i in the three PCa cell lines (Fig. 8). The viability of the cells in this assay was confirmed by their correct responsiveness to agents known to increase [Ca2+]i [10–4 M ATP in DU145 and PC3 cells (20); 10–6 M A23187 calcium ionophore in LNCaP cells (21)].
Discussion
The present study indicates that NPY-mediated activation of the Y1-R, which is expressed in human PCa cell lines, either stimulated (in PC3) or reduced (in LNCaP and DU145) PCa cell proliferation. The transduction systems associated with the effects on cell growth appear to differ according to each clone: in LNCaP cells, no apparent involvement of ERK1/2 was found, whereas in DU145 cells, NPY rapidly induced a long-lasting phosphorylation of ERK1/2. At variance, in PC3 cells, the proliferative effect of NPY/Y1-R appeared to be mediated by a signaling cascade, which includes PKC and ERK1/2 activation as well as inhibition of forskolin-induced cAMP production. The analysis of the expression of Y1-R mRNA, along with the main product of 354 bp, showed an additional product of 451 bp containing a small intron (97 bp) with a stop codon after the region coding for the fifth transmembrane domain (22, 23). It has been proposed that such intron-retention may play some role in Y1-R production through posttranscriptional mechanisms (24); it is less likely that it may act as a transcriptional enhancer or a putative Y1-R-related 5TM accessory protein encoded by this nonspliced Y1-R mRNA, which would facilitate Y1-R production at a posttranslational level (24).
Our findings provide the first evidence that the Y1-R protein is expressed in the three human PCa cell lines tested. In agreement with previous studies (25, 26), the size of this Y1-R protein was found to be about 70 kDa. Exposure of PCa cells to NPY treatment reduced the proliferation of LNCaP and DU145 cell lines, whereas it stimulated that of PC3 cells. The maximally effective concentration of NPY (about 10–8 M) for each clone was in agreement with the expected range for this peptide (3) as well as the affinity of NPY to binding sites present on PC3 cell membranes (affinity constant: 30 nM) (27). As shown by experiments using the selective Y1-R inhibitor BIBP3226, these effects appeared promoted by activation of Y1-R, which has been shown to mediate proliferative or antiproliferative effects of NPY in different tissues and cells, including vascular smooth muscle and endothelial cells (28, 29) and injured glial cells (30), and in neuroproliferation in rodent olfactory epithelium (31). Moreover, the Y1-R has been identified in proliferating tissues such as primary and metastatic breast carcinoma (3). Y1-R seems very important in tumor cell proliferation because NPY can inhibit the growth of the Y1-expressing SK-N-MC cell line (3). NPY actions on PCa cells appear also quite specific for cell proliferation because this peptide has been reported to have no effect on invasion and migration of PC3 (32), LNCaP, and DU145 cells (33).
Some possible explanations regarding the mechanisms underlying the proliferative or antiproliferative response of PCa cells to Y1-R activation have emerged from our findings on the intracellular signaling recruited in this process. Our results on NPY-associated MAPK/ERK1/2 activation have revealed marked differences among the human PCa cell lines. LNCaP cells display high constitutive activation of this pathway, as also found in other cell lines (34, 35), with no further modulation by NPY, which appears to reduce LNCaP cell proliferation by mechanisms that might be at least in part independent of ERK1/2 activation. The apparent lack of involvement of ERK1/2 in the control of LNCaP cell growth does not appear exclusive for NPY because it has been shown even for dihydrotestosterone, the main prostatic trophic factor (36). Thus, it is possible that NPY may affect cell growth through alternative mechanisms, like the phosphatidyl-inositol-3 kinase pathway (37, 38). In both androgen-independent clones, DU145 and PC3, NPY treatment rapidly stimulated ERK1/2 phosphorylation, which quickly returned to low basal levels in PC3 cells but showed a persistent elevation in DU145 cells. This different pattern of MAPK/ERK1/2 activation is associated with an opposite effect on cell proliferation exerted by the NPY/Y1-R system.
It is interesting to note that, according to our experiments using the Y1-R antagonist BIBP3226, both patterns of ERK1/2 activation are mediated by Y1-R. These data agree with previous studies conducted in different cancers or immortalized cells, showing that a long-lasting stimulation of this pathway resulted in a reduced proliferation rate, as we observed in DU145 cells, whereas rapid and transient activation of MAPK leads to enhanced cell growth (as found in PC3 cells) (39, 40, 41). The involvement of MAPK in the growth-modulating action of NPY via Y1-R has been previously reported in other cell systems (9, 17, 42), and in some studies, it has been shown to require an earlier activation of PKC (19), although this pathway has also been reported to be PKC independent (43). In our systems, PKC activation is required for NPY-induced ERK1/2 phosphorylation only in PC3 cells, whereas in DU145 cells, this process is clearly PKC independent. In addition, the present findings indicate that NPY treatment inhibits forskolin-induced cAMP accumulation only in PC3 but not in LNCaP and DU145 cells. Differently from neural cells expressing NPY receptors, exposure to NPY did not affect [Ca2+]i in any PCa cell line.
The present study indicates that the Y1-R plays a pivotal role in NPY-modulated cell proliferation and the related intracellular signaling, thus ruling out any effect through other NPY-R isoforms, whose gene expression was found in PC3 (Y2-R) and DU145 and LNCaP (Y4-R) cells (Ruscica, M., E. Dozio, and P. Magni, unpublished observation).
The present observations, underlining the role of the NPY/Y1-R system in the regulation of PCa cell growth, add novel insights into the complex molecular basis of PCa progression. Future studies should extend the evaluation of the role of the NPY/Y1-R system to the clinical setting to assess the potential involvement of this neuropeptidergic system in PCa pathophysiology.
Acknowledgments
The authors thank Dr. Maria Rosa Accomazzo for advice in the experiments evaluating [Ca2+]i. The expert technical collaboration of Ms. Paola Assi and Ms. Giovanna Micciche is also gratefully acknowledged.
Footnotes
This work was supported by Universita degli Studi di Milano (Fondo Interno Ricerca Scientifica e Tecnologica), Center for Endocrinological Oncology, Milan, and Ministero dell’Istruzione, dell’Universita e della Ricerca (Fondo per gli Investimenti della Ricerca di Base). S.B. is the recipient of a Marie Curie Training Fellowship (no. QLGA 1999-51238).
The authors have no conflict of interest.
First Published Online December 8, 2005
Abbreviations: [Ca2+]i, Intracellular free calcium concentration; FCS, fetal calf serum; MEK, MAPK kinase; NPY, neuropeptide Y, PCa, prostate cancer; PKC, protein kinase C.
Accepted for publication November 30, 2005.
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Unita Operativa di Anatomia Patologica (G.B.), Azienda Ospedaliera San Gerardo, 20052 Monza (Milan), Italy
Abstract
This study deals with the role of neuropeptide Y (NPY) in the regulation of cell proliferation. NPY is expressed in the normal and tumoral prostate, but no data on its possible role in prostate cancer (PCa) progression are available. Therefore, we evaluated the direct effect of NPY on the growth of the human PCa cell lines LNCaP (androgen dependent) and DU145 and PC3 (androgen independent). All PCa cell lines expressed Y1-R gene and protein. NPY treatment reduced the proliferation of LNCaP and DU145 cells and increased that of PC3 cells. The Y1-R antagonist BIBP3226 abolished such effects, suggesting a mandatory role of Y1-R in this process. LNCaP cells showed elevated constitutive levels of phosphorylated ERK1/2, which were not affected by NPY. In DU145 cells, NPY stimulated a long-lasting ERK1/2 activation, whereas, in PC3 cells, this effect was rapid and transient and required activation of protein kinase C. Moreover, in both cell lines, pretreatment with BIBP3226 prevented the NPY-induced ERK1/2 phosphorylation, further supporting Y1-R involvement. NPY treatment reduced forskolin-stimulated cAMP accumulation only in PC3 cells and did not change intracellular calcium concentration in any PCa cell line. These data indicate that NPY may directly regulate PCa cell growth via Y1-R. The direction of this effect appears to be related to the time kinetics of MAPK activation, i.e. long-lasting vs. transient, and to the clone-specific involvement of other intracellular signals. These findings suggest that NPY-related mechanisms might play a relevant role in the progression of PCa, at both androgen dependent and independent stages.
Introduction
PROSTATE CANCER (PCa) represents one of the most common malignant diseases among men in the Western world, where there is a 10% chance of developing PCa and a 3–4% chance of dying of causes directly related to it (1). PCa is initially androgen dependent, and it may later progress to androgen independence (2). This latter stage is associated with a lack of efficacy of hormonal therapy and appears to be promoted at least in part by several growth factors and neurohormones (1). Therefore, the study of neuroendocrine regulatory peptides might be of relevance in tumors that express their respective receptors in high amounts, as suggested by Reubi et al. (3) for breast cancer. Scant data are presently available on the possible role exerted by neuropeptide Y (NPY) and its receptors on PCa progression. NPY is a highly conserved 36-amino acid residue peptide, belonging to the pancreatic polypeptide family. NPY is widely distributed throughout the central and peripheral nervous systems and is involved in the regulation of several physiological functions (neuroendocrine mechanisms, cognitive functions, eating behavior, cardiovascular activity, etc.) (4, 5). More recently NPY has been shown to be involved in prostate physiology and pathophysiology, including PCa growth and progression. In the human prostate, particularly in the smooth muscle layer, NPY is mainly localized in nerve fibers and neuroendocrine cells (6, 7). Moreover, immunopositivity for NPY has been shown in 75% of the PCa specimens obtained from a series of patients (8). NPY activates specific G protein-coupled receptors, present in humans in at least five subtypes, named Y1-R to Y5-R. Moreover, NPY has been demonstrated to be involved in mitogenic pathways and stimulate cell proliferation via the Y1-R (9). The activation of Y1-R is generally associated with reduction of cAMP accumulation, increase of intracellular free calcium concentration ([Ca2+]i), and modulation of the MAPK pathway via several signaling molecules, including the protein kinase C (PKC) (4, 9). The aim of the present study was to elucidate the role of NPY in PCa biology by investigating the expression of Y1-R in three commonly used human PCa cell lines (the androgen-dependent LNCaP and the androgen-independent DU145 and PC3) as well as the activation of intracellular signals and the possible direct proliferative effects of NPY. Our data suggest that Y1-R activation by NPY represents an important regulator of cell proliferation, with a peculiar intracellular signaling pattern according to each of the PCa cell lines used.
Materials and Methods
Cell cultures
LNCaP, DU145, and PC3 human PCa and SK-N-MC human neuroblastoma cell lines (American Type Culture Collection, Manassas, VA) were grown at 37 C in a humidified CO2 incubator in monolayer. The culture medium for LNCaP, DU145, and PC3 cells was RPMI 1640, with 10 mg/liter phenol red (Biochrom, Berlin, Germany), and 5 or 10% fetal calf serum (FCS; Life Technologies, Inc., Grand Island, NY); for SK-N-MC cells, the medium was MEM containing nonessential amino acids, 1 mM sodium pyruvate, 10 mg/liter phenol red, and 10% FCS. Confluent cells were harvested with 0.05% trypsin/0.02% EDTA (Biochrom) and were seeded in petri dishes (Becton Dickinson, Plymouth, UK) for all the experiments, which were performed using subconfluent cell cultures.
RNA extraction and RT-PCR analysis of NPY and Y1-R gene expression
For RNA studies, cells were washed with cold PBS, collected, snap frozen in liquid nitrogen, and stored at –80 C until RNA extraction. Total cellular RNA was extracted with the phenol-chloroform method using the Tri-Reagent solution (Sigma-Aldrich, Milan, Italy). RT-PCR analysis of the expression of the genes coding for NPY and Y1-R was performed on total RNA samples, quantified after an initial DNAase digestion step using the Deoxyribonuclease I kit (Sigma-Aldrich). The reverse transcription reaction was carried out in a 50-μl volume, using a commercially available GeneAmp kit (Applied Biosystems, Milan, Italy). A control RNA (pAW 109), provided with the RT-PCR kit, was used as a positive control of the RT-PCR. Negative control reactions omitting reverse transcriptase were carried out in parallel, confirming that no sample contamination by genomic DNA occurred (not shown). The primers used for Y1-R subtype, NPY and -actin (housekeeping gene) were as follows: Y1-R forward (5'-TATACCACTCTTCTCTTggTgCTg-3') and Y1-R reverse (5'-CTggAAgTTTTTgTTCAggAACCCA-3') (10); NPY forward (5'-CCAgATACTACTCggCgCTgCgACACTACA-3') and NPY reverse (5'-gAATgCATgATACTTTATTTAAACACACATATATACAA-3') (11); -actin forward (5'-TgACggggTCACCCACACTgTgCCCATCTA-3') and -actin reverse (5'-CTAgAAgCATTTgCggTggACgATggAggg-3') (12). Amplification products were separated by electrophoresis in a 2% agarose/1x Tris-borate EDTA gel and were detected by ethidium bromide fluorescence on an UV transilluminator. The identity of the PCR products was confirmed by DNA sequencing (data not shown).
Western blot analysis
PCa cells were collected in rapid immunoprecipitation assay lysis buffer (13) containing 1% protease inhibitor mix (Sigma-Aldrich). Protein concentration was quantified by the BCA protein assay (Pierce, Rockford, IL). Fifty milligrams protein cell extracts were resuspended in Laemmli sample buffer (Sigma-Aldrich) and were separated on a SDS-PAGE, using Rainbow molecular mass markers (Amersham Biosciences Europe, Milan, Italy). Proteins were transferred to nitrocellulose membranes (Bio-Rad Laboratories, Milan, Italy) overnight. After wash and blockade steps, the blot was incubated overnight at 4 C with a diluted solution of the specific primary antibody [anti-Y1-R (1:700), which is an affinity-purified antiserum generated in rabbit against a C-terminal peptide (amino acids 355–382) of this receptor and was kindly donated by Dr. B. Bunnemann (GlaxoSmithKline, Verona, Italy) (14); anti-ERK 1/2 (1:1000); and anti-pERK1/2 (1:150), both from Santa Cruz Biotechnology, Santa Cruz, CA]. The incubation with the secondary antibody conjugated with horseradish-peroxidase (Santa Cruz Biotechnology) was performed at room temperature for 2 h. The immunoreactivity was detected by the SuperSignal West Pico substrate working solution (Pierce) and exposure of the membrane to radiographic film at room temperature. A negative control carried out by incubating the membrane with the secondary antibody alone did not result in any band on the blot.
Cell proliferation studies
For cell count experiments, LNCaP, DU145, and PC3 cells were seeded in 60-mm-diameter petri dishes. After 72 h, the culture medium was replaced by the experimental medium (RPMI 1640/0.1% BSA or 2% FCS) containing 10–10 to 10–7 M human NPY (Bachem, Bubendorf, Switzerland) and, in selected experiments, the specific Y1-R antagonist BIBP3226 (Bachem). The experimental incubation was carried on for 96 h, and then cells were harvested with 0.05% trypsin/0.02% EDTA and resuspended in RPMI 1640. Cell viability was assessed by the trypan-blue exclusion method, and then cells were counted. For [3H]thymidine incorporation studies, LNCaP, DU145, and PC3 cells were plated in 24-well plates at 1.5–3 x 104 cells/well. After 24 h, the culture medium was replaced by the experimental medium (RPMI 1640/0.1% BSA or 2% FCS) containing the MAPK kinase (MEK) inhibitor U0126 (10–6 M; Biomol, Plymouth Meeting, PA) or the specific PKC inhibitor GF109203X (10–7 M; Sigma-Aldrich). Thirty minutes later, 10–8 M human NPY (Bachem) was added and the incubation was carried on for 48 h. Two microcuries per well [3H]thymidine (Amersham Biosciences Europe) were added 6 h before the end of the incubation. Cells were harvested and the incorporated [3H]thymidine counted in the presence of scintillation fluid using a -counter. Experiments were repeated at least three times.
Effect of NPY treatment on cAMP concentration
To study the NPY modulation of cAMP levels, LNCaP, DU145, and PC3 cells were seeded in 24-well plates. Subconfluent cell cultures were washed with RPMI 1640 and incubated in 200 μl RPMI 1640 containing 0.5 mM isobutylmethylxanthine (Sigma-Aldrich) for 15 min at 37 C. Subsequently 200 μl RPMI 1640 containing the test substances (forskolin, Sigma-Aldrich; human NPY, BIBP3226) were added to each well and the incubation was carried on for another 15 min at 37 C. After removal of the medium, cells were collected in 500 μl cold 75% ethanol; membranes were precipitated by centrifugation at 10,000 rpm for 3 min. The supernatant was freeze dried and resuspended in RIA buffer. The cAMP content was quantified by a commercial RIA kit (Amersham Biosciences Europe).
Effect of NPY treatment on [Ca2+]i
The effect of NPY treatment on [Ca2+]i in PCa cells was determined by using the fluorescent probe fura-2 acetoxymethyl ester (fura-2; Molecular Probes, Eugene, OR), according to Grynkiewicz et al. (15). PCa cells were grown on 12-mm-diameter glass slides in petri dishes. Subconfluent cells adherent to glass slides were loaded with fura-2 by incubation in 1 mM fura-2 solution at 37 C for 60 min; control cells were incubated in the same conditions without fura-2 solution. Cells were washed with HEPES-saline solution (pH 7.4). The fluorescence of control cells, which served as background level, was first measured by means of a fluorometer (Fast Filter polarizer, model LS 50B; PerkinElmer, Norwalk, CT). Fura-2-loaded cells were inserted into the fluorometer, and when the fluorescence level appeared to be stabilized, the test substances (NPY, ATP, and the calcium ionophore A23187; Sigma-Aldrich) were added and the fluorescence was measured. [Ca2+]i corresponding to measured fluorescence was calculated with the Grynkiewicz equation. Ionomycin (final concentration: 2.7 μM; Sigma-Aldrich) and digitonin (final concentration: 100 μM; Merck, Darmstadt, Germany) were added to establish the maximum amount of Ca2+ bound to fura-2. EGTA (final concentration: 5 mM) and Tris-base (final concentration: 60 mM; both from Sigma-Aldrich) were then added to establish the minimum levels of Ca2+ bound to fura-2.
Analysis of the data
Statistical analysis was performed using the Systat statistical analysis package (Systat Software, Erkrath, Germany). Data are presented as mean ± SEM; n = number of replicates within an experiment. Significance of differences between treatment groups was evaluated by ANOVA, followed by Tukey test, and was considered significant when P < 0.05.
Results
Expression of NPY and Y1-R in PCa cells
The expression of NPY and Y1-R isoform was initially evaluated in PCa cell lines at the gene level by RT-PCR analysis. The NPY gene was not found to be expressed in any of the PCa cell lines tested (data not shown). A 354-bp PCR product corresponding to Y1-R, together with a 97-bp-longer additional band, was detected in all PCa cells and SK-N-MC human neuroblastoma cells (positive control) (16) (Fig. 1A). Western blot analysis using an anti-Y1-R antibody showed a specific immunoreactive band with a size of 70 kDa in all PCa cell lines (Fig. 1B).
Effect of NPY on PCa cell proliferation: involvement of Y1-R
A 96-h treatment with 10–10 to 10–7 M NPY resulted in a significant reduction of LNCaP and DU145 cell proliferation, with a maximal inhibition at 10–8 M NPY (–45–63%) for the LNCaP cell line and at 5 x 10–8 M NPY (–33–35%) for the DU145 cell line. On the contrary, a marked proliferative effect was observed for the androgen-independent cell line PC3 (+36–60% at 10–8 M NPY) (Fig. 2A). We then examined whether NPY effects on cell proliferation were mediated through Y1-R, which is expressed in all PCa cell lines (Fig. 1A). The addition of 1 μM BIBP3226, a specific Y1-R antagonist (17), abolished both the proliferative (on PC3) and antiproliferative (on LNCaP and DU145) effects of NPY (Fig. 2B). Treatment with any concentration of NPY and BIBP3226 did not modify the survival rate of the cells (viable cells > 95% according to trypan blue exclusion) or induce any morphological change (data not shown).
Effect of NPY on intracellular signaling in PCa cells
To dissect out the intracellular mechanisms underlying the above-described effects of NPY on cell proliferation via Y1-R, we first studied the involvement of MAPK/ERK1/2 signaling. ERK1/2 phosphorylation was evaluated after treatment of PCa cell lines with 10–8 M NPY. LNCaP cells showed a constitutive activation of the ERK1/2 pathway, with no further modulation by NPY (Fig. 3). In DU145 cells, low basal pERK1/2 amounts were present, and NPY treatment resulted in a marked increase of ERK1/2 phosphorylation, which occurred within 5–15 min and remained elevated up to at least 6 h (Fig. 3). In PC3 cells, very low basal pERK1/2 levels were detected, and NPY treatment resulted in an early (5–15 min) and transient increase of ERK1/2 phosphorylation (Fig. 3). Moreover, in DU145 and PC3 cell lines, the NPY-mediated ERK1/2 phosphorylation was markedly reduced by a pretreatment (30 min) with 10–6 M BIBP3226 (Fig. 4).
Because it has been proposed that PKC activation is an upstream event for ERK1/2 phosphorylation in some systems (18), we tested this hypothesis also in DU145 and PC3 cells, in which, as reported above, NPY has been shown to activate this process. It was found that a pretreatment with 10–7 M GF109203X, a specific PKC inhibitor, abolished NPY-induced ERK1/2 phosphorylation in PC3 cells but not in DU145 cells (Fig. 5). The role of NPY-mediated activation of MAPK/ERK1/2 in the proliferation of PCa cells was then studied by using the specific MEK inhibitor U0126 in combination with the [3H]thymidine incorporation assay (Fig. 6). In LNCaP cells, a pretreatment with 10–6 M U0126 plus 10–8 M NPY determined an additional inhibitory effect on cell proliferation, compared with NPY alone, suggesting that NPY, which does not substantially affect ERK1/2 phosphorylation (Fig. 3), reduces LNCaP cell proliferation by mechanisms that are at least in part independent of ERK1/2 activation. In DU145 cells, a pretreatment with U0126, a specific MEK inhibitor, did not affect the NPY-associated reduction of cell proliferation (Fig. 6). The efficacy of NPY in stimulating PC3 cell proliferation was, however, prevented by U0126 pretreatment, indicating the involvement of NPY-activated ERK1/2 in this effect (Fig. 6).
According to previous studies (19), another early event coupled to Y1-R activation is the involvement of PKC. In all PCa cell lines, exposure to 10–7 M GF109203X significantly reduced [3H]thymidine incorporation (Fig. 6). In LNCaP and DU145 cells, treatment with NPY alone or NPY plus GF109203X reduced [3H]thymidine incorporation to the same extent, whereas in PC3 cells, the addition of GF109203X abolished the proliferative action of NPY, suggesting a role for PKC activity in this effect (Fig. 6).
The short-term effect of NPY on other relevant intracellular signals known to be coupled to Y1-R (namely, cAMP accumulation and [Ca2+]i change) was then assessed. PCa cells were treated for 15 min with 10–8 M NPY in the presence or absence of 5 μM forskolin. NPY treatment alone did not modify basal cAMP levels, and, as expected, exposure to forskolin induced a marked increase of cAMP concentrations. NPY treatment significantly reduced forskolin-induced cAMP accumulation only in PC3 cells (–52%), but it was ineffective in LNCaP and DU145 cells (Fig. 7). Treatment with 10–8 M NPY did not induce any change of [Ca2+]i in the three PCa cell lines (Fig. 8). The viability of the cells in this assay was confirmed by their correct responsiveness to agents known to increase [Ca2+]i [10–4 M ATP in DU145 and PC3 cells (20); 10–6 M A23187 calcium ionophore in LNCaP cells (21)].
Discussion
The present study indicates that NPY-mediated activation of the Y1-R, which is expressed in human PCa cell lines, either stimulated (in PC3) or reduced (in LNCaP and DU145) PCa cell proliferation. The transduction systems associated with the effects on cell growth appear to differ according to each clone: in LNCaP cells, no apparent involvement of ERK1/2 was found, whereas in DU145 cells, NPY rapidly induced a long-lasting phosphorylation of ERK1/2. At variance, in PC3 cells, the proliferative effect of NPY/Y1-R appeared to be mediated by a signaling cascade, which includes PKC and ERK1/2 activation as well as inhibition of forskolin-induced cAMP production. The analysis of the expression of Y1-R mRNA, along with the main product of 354 bp, showed an additional product of 451 bp containing a small intron (97 bp) with a stop codon after the region coding for the fifth transmembrane domain (22, 23). It has been proposed that such intron-retention may play some role in Y1-R production through posttranscriptional mechanisms (24); it is less likely that it may act as a transcriptional enhancer or a putative Y1-R-related 5TM accessory protein encoded by this nonspliced Y1-R mRNA, which would facilitate Y1-R production at a posttranslational level (24).
Our findings provide the first evidence that the Y1-R protein is expressed in the three human PCa cell lines tested. In agreement with previous studies (25, 26), the size of this Y1-R protein was found to be about 70 kDa. Exposure of PCa cells to NPY treatment reduced the proliferation of LNCaP and DU145 cell lines, whereas it stimulated that of PC3 cells. The maximally effective concentration of NPY (about 10–8 M) for each clone was in agreement with the expected range for this peptide (3) as well as the affinity of NPY to binding sites present on PC3 cell membranes (affinity constant: 30 nM) (27). As shown by experiments using the selective Y1-R inhibitor BIBP3226, these effects appeared promoted by activation of Y1-R, which has been shown to mediate proliferative or antiproliferative effects of NPY in different tissues and cells, including vascular smooth muscle and endothelial cells (28, 29) and injured glial cells (30), and in neuroproliferation in rodent olfactory epithelium (31). Moreover, the Y1-R has been identified in proliferating tissues such as primary and metastatic breast carcinoma (3). Y1-R seems very important in tumor cell proliferation because NPY can inhibit the growth of the Y1-expressing SK-N-MC cell line (3). NPY actions on PCa cells appear also quite specific for cell proliferation because this peptide has been reported to have no effect on invasion and migration of PC3 (32), LNCaP, and DU145 cells (33).
Some possible explanations regarding the mechanisms underlying the proliferative or antiproliferative response of PCa cells to Y1-R activation have emerged from our findings on the intracellular signaling recruited in this process. Our results on NPY-associated MAPK/ERK1/2 activation have revealed marked differences among the human PCa cell lines. LNCaP cells display high constitutive activation of this pathway, as also found in other cell lines (34, 35), with no further modulation by NPY, which appears to reduce LNCaP cell proliferation by mechanisms that might be at least in part independent of ERK1/2 activation. The apparent lack of involvement of ERK1/2 in the control of LNCaP cell growth does not appear exclusive for NPY because it has been shown even for dihydrotestosterone, the main prostatic trophic factor (36). Thus, it is possible that NPY may affect cell growth through alternative mechanisms, like the phosphatidyl-inositol-3 kinase pathway (37, 38). In both androgen-independent clones, DU145 and PC3, NPY treatment rapidly stimulated ERK1/2 phosphorylation, which quickly returned to low basal levels in PC3 cells but showed a persistent elevation in DU145 cells. This different pattern of MAPK/ERK1/2 activation is associated with an opposite effect on cell proliferation exerted by the NPY/Y1-R system.
It is interesting to note that, according to our experiments using the Y1-R antagonist BIBP3226, both patterns of ERK1/2 activation are mediated by Y1-R. These data agree with previous studies conducted in different cancers or immortalized cells, showing that a long-lasting stimulation of this pathway resulted in a reduced proliferation rate, as we observed in DU145 cells, whereas rapid and transient activation of MAPK leads to enhanced cell growth (as found in PC3 cells) (39, 40, 41). The involvement of MAPK in the growth-modulating action of NPY via Y1-R has been previously reported in other cell systems (9, 17, 42), and in some studies, it has been shown to require an earlier activation of PKC (19), although this pathway has also been reported to be PKC independent (43). In our systems, PKC activation is required for NPY-induced ERK1/2 phosphorylation only in PC3 cells, whereas in DU145 cells, this process is clearly PKC independent. In addition, the present findings indicate that NPY treatment inhibits forskolin-induced cAMP accumulation only in PC3 but not in LNCaP and DU145 cells. Differently from neural cells expressing NPY receptors, exposure to NPY did not affect [Ca2+]i in any PCa cell line.
The present study indicates that the Y1-R plays a pivotal role in NPY-modulated cell proliferation and the related intracellular signaling, thus ruling out any effect through other NPY-R isoforms, whose gene expression was found in PC3 (Y2-R) and DU145 and LNCaP (Y4-R) cells (Ruscica, M., E. Dozio, and P. Magni, unpublished observation).
The present observations, underlining the role of the NPY/Y1-R system in the regulation of PCa cell growth, add novel insights into the complex molecular basis of PCa progression. Future studies should extend the evaluation of the role of the NPY/Y1-R system to the clinical setting to assess the potential involvement of this neuropeptidergic system in PCa pathophysiology.
Acknowledgments
The authors thank Dr. Maria Rosa Accomazzo for advice in the experiments evaluating [Ca2+]i. The expert technical collaboration of Ms. Paola Assi and Ms. Giovanna Micciche is also gratefully acknowledged.
Footnotes
This work was supported by Universita degli Studi di Milano (Fondo Interno Ricerca Scientifica e Tecnologica), Center for Endocrinological Oncology, Milan, and Ministero dell’Istruzione, dell’Universita e della Ricerca (Fondo per gli Investimenti della Ricerca di Base). S.B. is the recipient of a Marie Curie Training Fellowship (no. QLGA 1999-51238).
The authors have no conflict of interest.
First Published Online December 8, 2005
Abbreviations: [Ca2+]i, Intracellular free calcium concentration; FCS, fetal calf serum; MEK, MAPK kinase; NPY, neuropeptide Y, PCa, prostate cancer; PKC, protein kinase C.
Accepted for publication November 30, 2005.
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