VIP and PACAP are autocrine factors that protect the androgen-independent prostate cancer cell line PC-3 from apoptosis induced by serum withdrawal

Authors


Biochemistry and Molecular Biology, University of Alcala, Campus Universitario, Alcala de Henares, Madrid 28871, Spain. E-mail: juancarlos.prieto@uah.es

Abstract

  • In the present study, we describe the expression of the neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) as well as their receptors in PC-3 cells, a human prostate cancer cell line. In addition, we have investigated their role in apoptosis induced by serum starvation.

  • By RT–PCR and immunocytochemistry assays, we have demonstrated the production of VIP and PACAP in PC-3 cells.

  • We have demonstrated by RT–PCR and binding assays the expression of common PACAP/VIP (VPAC1 and VPAC2) receptors, but not PACAP-specific (PAC1) receptors. The pharmacological profile of [125I]-VIP binding assays was as follows: VPAC1 antagonist=VPAC1 agonist>VIP>VPAC2 agonist (IC50=1.2, 1.5, 2.3 and 30 nM, respectively). In addition, both receptor subtypes are functional since VIP, PACAP-27 or VPAC1 and VPAC2 agonists all increased the intracellular levels of cAMP.

  • The expression of both peptides and their receptors is similar in serum-cultured and serum-deprived PC-3 cells. The treatment of serum-deprived PC-3 cells with exogenous VIP or PACAP-27 increases cell number and viability in a dose-dependent manner, as demonstrated by cellular counting and MTT assays. The increased cell survival is exerted through the VPAC1 receptor, since a VPAC1, but not VPAC2, receptor agonist, mimics the effects and a VPAC1 receptor antagonist blocks it. Moreover, VIP and PACAP-27 inhibit genomic DNA fragmentation in PC-3 cells triggered by serum starvation, and increase the immunoreactivity of the antiapoptotic protein bcl-2.

  • Our results suggest that VIP and PACAP are autocrine/paracrine factors that protect PC-3 cells from apoptosis through VPAC1 receptors.

British Journal of Pharmacology (2003) 139, 1050–1058. doi:10.1038/sj.bjp.0705317

Abbreviations:
IC50

concentration of ligand required for 50% inhibition of tracer binding

PACAP

pituitary adenylate cyclase-activating polypeptide

VIP

vasoactive intestinal peptide

VPAC1 agonist

[K15, R16, L27] VIP (1–7)/GFR (8–27)

VPAC1 antagonist

acetyl-His1[DPhe2, K15, R16, L27] VIP (1–7)/GFR (8–27)

VPAC2 agonist

RO 25-1553: acetyl-His1[E8, K12, Nle17, A19, D25, L26, K27, K28, G29, G30, T31] cyclo 21-25 VIP (2–24)

VPAC2 antagonist

myristoyl-[K12]VIP (1–26)KKGGT

Introduction

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) are two pleiotropic and structurally related neuropeptides widely distributed in the central and peripheral nervous systems, including the male genital tract (Said, 1992; Vaudry et al., 2000). Both neuropeptides are involved in the proliferation and/or differentiation of various normal and cancer cell lines, and several studies have reported an effect of VIP and PACAP analogues on tumour growth in animal models, mediated by specific receptors (Reubi et al., 2000). The biological effects of VIP are mediated by at least two receptors, designated as VPAC1 and VPAC2 receptors (Harmar et al., 1998). Both VIP receptors can also be activated by the parent peptide PACAP, which recognises PACAP-selective receptors known as PAC1 receptors. PAC1, VPAC1 and VPAC2 receptors are coupled to adenylate cyclase (AC) via a Gs protein. They display distinct pharmacological characteristics and a different tissue distribution (Harmar et al., 1998). Recently, different selective agonists and antagonists discriminating these receptors have been described. The VPAC1 agonist [K15, R16, L27] VIP (1–7)/ GFR (8–27) and the VPAC1 antagonist Acetyl-His1[DPhe2, K15, R16, L27] VIP (1–7)/ GFR (8–27) are a highly selective for VPAC1 receptors (Gourlet et al., 1997a, 1997c). RO 25–1553 and myristoyl-[K12]VIP (1–26)KKGGT are highly selective agonist and antagonist for VPAC2 receptors, respectively (Gourlet et al., 1997d; Moreno et al., 2000). VIP receptors coupled to AC are present in rat ventral prostate epithelial cells (Carmena & Prieto, 1983; Prieto & Carmena, 1983; Juarranz et al., 1994) and correspond mainly to the VPAC1 receptor subtype (Juarranz et al., 1999). This receptor subtype is also predominant in human prostate cancer tissue (Reubi et al., 2000) and in the androgen-dependent prostate cancer cell line LNCaP (Juarranz et al., 2001b).

Prostate cancer is composed of androgen-dependent and androgen-independent cells. It is well established that androgen ablation eliminates most androgen-dependent cancer cells by inducing apoptosis, but can rarely cure the patients because of the presence of androgen-independent cells and the emergence of apoptosis-resistant clones (Denmeade et al., 1996). Quite a large number of factors have now been identified in such tumours that may individually contribute to resistance to apoptosis in prostate cancer, including neuropeptides such as calcitonin, neurotensin and bombesin, which regulate the growth of prostate cancer cells, as well as their progression to hormone independence (Sehgal et al., 1994; Shah et al., 1994; Markwalder and Reubi, 1999). Furthermore, some of these neuropeptides have been described to modulate cell survival pathways in prostate cancer (Salido et al., 2000). In this regard, VIP stimulates rat prostatic epithelial cell proliferation and induces neuroendocrine differentiation in LNCaP cells (Juarranz et al., 2001a, 2001b).

The aim of this study was to investigate whether VIP and PACAP act as autocrine/paracrine factors in PC-3 cells, to establish the expression pattern of VIP/PACAP receptors in these cells and to analyse the role of these two neuropeptides in apoptosis triggered by serum starvation of PC-3 cells during 4 days.

Methods

Cell culture and membrane preparation

The androgen-independent human prostate cancer cell line PC-3 was obtained from American Type Culture Collection (ATCC) and routinely cultured in RPMI-1640 medium (Life Technologies, Barcelona, Spain) supplemented with 10% heat-inactivated foetal bovine serum (FBS) and 1% penicillin/streptomycin/amphotericin B (Life Technologies) and seeded at a density of 30,000–40,000 cells cm−2. The culture medium was changed every 3 days. For PC-3-induced apoptosis, cells were allowed to attach to plates for 24 h and were subsequently washed twice with PBS. The medium was changed to serum-free RPMI-1640 and maintained in culture for 4 days. For membrane preparation, cells were harvested with a rubber policeman and pelleted by low-speed centrifugation. The supernatant was discarded and the cells lysed in 1 mM NaHCO3 and immediately frozen in liquid nitrogen. After thawing, the lysate was first centrifuged at 800 × g for 10 min. The supernatant was further centrifuged at 20,000 × g for 15 min. The pellet was resuspended in 1 mM NaHCO3 and used immediately as a crude membrane preparation. Membrane protein concentration was measured according to the method of Bradford.

RT–PCR assays

Total RNA was prepared from PC-3 cells using standard techniques. Total RNA (5 μg) was reverse-transcribed using 6 μg hexamer random primers and 200 U M-MLV retrotranscriptase (Life Technologies) in the buffer supplied with the enzyme supplemented with 10 mM dithiothreitol, 40 U RNasin (Promega, Madison, WI, U.S.A.), and 0.5 mM of deoxyribonucleotides (dNTPs). RT reaction (2 μl) then PCR-amplified with specific primers for VIP, PACAP and each VIP/PACAP receptor subtype. Primers for VIP were: sense, 5′-TAAAAGAAGACATTGACATGTTG-3′ and antisense, 5′-GAAGTTGTTTTCTTGAATTACTT-3′, which should give a PCR product of 470 bp; for PACAP: sense, 5′-AAACAAAGGACGACGCCGATAG-3′ and antisense, 5′-AGACTCACTGGGAAAGAATGC-3′, which should give a PCR product of 576 bp; for VPAC1 receptor: sense, 5′-ATGTGCAGATGATCGAGGTG and antisense, 5′-TGTAGCCGGTCTTCACAGAA, which should give a PCR product of 324 bp; for VPAC2 receptor: sense, 5′-TACAGAGCTTCTGAGGTCTC and antisense, 5′-TACTGCAGGAAGACCAGGC, which yield a PCR product of 529 bp; for PAC1 receptor: sense, 5′-GCCTGTACCTCTTCACTCTGC and antisense, 5′-CTTTCCCTTTTGCTGACATTC, which would be expected to produce a PCR product size of 450 bp for the null variant. The PCR conditions for VIP were: denaturation at 94°C for 3 min followed by 40 cycles of 50 s at 94°C, 50 s at 56°C and 1 min at 72°C and then a final cycle of 10 min at 72°C. The PCR conditions for PACAP were similar to those for VIP, except for the cycles: 1 min at 94°C, 1 min at 60°C, and 1 min at 72°C. In the case of VIP/PACAP receptors, the PCR conditions were: denaturation at 94°C for 3 min, and 45 cycles of 30 s at 94°C, 45 s at 58°C and 45 s at 72°C and then a final cycle of 10 min at 72°C. The PCR products were analysed in 2% agarose gels.

Immunocytochemical staining

Cells were detached with trypsin/EDTA and pelleted by low-speed centrifugation. The resulting supernatant was discarded and the cells resuspended in PBS. Cell suspensions were centrifuged onto glass slides (9 × 104 cells per slide), dried overnight and then fixed for 10 min in acetone at –20°C. After drying for 2 h, slides were rinsed in PBS and then treated for 5 min with methanol/water/H2O2 in order to block endogenous peroxidase. Slides were again rinsed again in PBS and treated with normal rabbit and goat serum (for VIP or PACAP antibodies, respectively) to block background staining. Immunocytochemical demonstration of immunoreactive VIP (IR-VIP) and PACAP (IR-PACAP) was carried out by successive incubation with a monoclonal anti-VIP antibody (dilution 1 : 1000) or rabbit anti-PACAP antibody (dilution 1 : 4000) for 1 h, biotine-conjugated rabbit anti-mouse or goat anti-rabbit IgG (for IR-VIP and IR-PACAP, respectively), and subsequently incubated with streptavidin–peroxidase for 1 h followed by addition of the 3,3′-diaminobenzidine-tetrachloride (DAB) substrate and hydrogen peroxide in PBS. Methylene blue was used for counterstaining. Controls for the immunocytochemical studies were carried out by replacing the mouse anti-VIP or rabbit anti-PACAP with PBS and staining with the secondary antibodies according to the above protocol. The specificity for these antibodies was previously demonstrated (Wong et al., 1996; Abad et al., 2002). The mouse anti-VIP antibody was kindly supplied by Dr Helen Wong. The rabbit anti-PACAP antibody was a generous gift from Dr A. Arimura.

Binding assays

For identification of VIP/PACAP receptors, VIP and PACAP-27 were iodinated as described previously (Juarranz et al., 2001b). Membranes (10–20 μg protein) were incubated with [125I]VIP or [125I]PACAP-27 and increasing concentrations of unlabelled peptides. Nonspecific binding was defined as the residual binding in the presence of 1 μM VIP or PACAP-27, respectively (Neosystem, Strasburg, France). The selective agonists and antagonists for VPAC1 and VPAC2 receptors were kindly supplied by Dr P. Robberecht. [K15, R16, L27] VIP (1–7)/ GFR (8–27) and acetyl-His1[DPhe2, K15, R16, L27] VIP (1–7)/ GFR (8–27) were used as a VPAC1 selective agonist and antagonist, respectively. RO 25–1553 and myristoyl-[K12]VIP(1–26)KKGGT were used as VPAC2 selective agonist and antagonist, respectively.

Measurement of cAMP accumulation

PC-3 cells were seeded in 24-multiwell plates (60,000 cells per well). After 24 h, cells were washed with 1 ml of serum-free medium and treated with 1 μCi of [2-H3]adenine for 2 h in a serum-free medium. The cells were then incubated for 10 min at 37°C in HBS buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 4.2 mM CaCl2, 0.5 mM MgCl2, 0.1% glucose, 1 mM IBMX and 0.1 mg ml−1 BSA) and subsequently for 15 min with increasing concentrations of the peptides tested. After incubation period, the medium was aspirated and the reaction was stopped by the addition of 1 ml of 5% trichloroacetic acid at 4°C. After scraping and centrifugation, cAMP levels in the supernatant were measured as described previously (Salomon et al., 1974).

Cell growth assays

PC-3 cells were seeded in 24-well plates at 1.5 × 104. cells per well and allowed to attach for 24 h in RPMI-1640 medium with 10% FBS and 1% antibiotic. The following day, the medium was removed, and the cells were cultured in serum-free medium with or without increasing concentrations of VIP, PACAP-27, the VPAC1 selective agonist or VPAC2 selective antagonist. The medium and peptide were renewed every 48 h. After 4 days, cells in each well were trypsinised, resuspended in Fast-Flow Beckton Dickinson Isotone™ solution and counted in a Casy® Model DT counter.

Cell viability measurement: MTT assay

Cell survival was estimated using a microculture tetrazolium assay. This assay measures the reduction of substrate [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] (MTT) to a dark blue formazan product by mitochondrial dehydrogenases in living cells. Briefly, 50 μl of 2 mg ml−1 MTT (Sigma, Alcobendas, Madrid) was added to each well. After 3 h at 37°C in darkness, 0.01 N HCl containing 10% SDS was added to dissolve the formazan product. Absorbance at 540 nm was measured in the plate reader (Bio-Tek Instruments, ELX 800).

Genomic DNA fragmentation

PC-3 cells were seeded in 10 cm diameter dishes with complete medium. After 24 h, the medium was replaced by complete medium (control cells), or serum-free medium with or without 0.1 μM VIP. The medium and peptide were renewed every 48 h. After 4 days, cells were detached and centrifuged. The pellet was resuspended in 450 μl of ice-cold lysis buffer (5 mM Tris-HCl, pH 7.4, 20 mM EDTA, 0.5% Triton X-100) for 20 min at 4°C. The chromatin was pelleted by centrifugation at 20,000 × g for 30 min. The supernatant (fragmented DNA) was removed and incubated with 4.5 μl of 20 mg ml−1 Proteinase K (Sigma) at 60°C for 2 h and then at 37°C overnight. DNA was extracted with phenol/chloroform/isoamylic alcohol. DNA was precipitated with ethanol, adding glycogen as a carrier, and then resuspended in TE buffer (10 mM Tris-HCl, pH=8.0, 1 mM EDTA) supplemented with 2 μg ml−1 RNase A, and incubated overnight at 37°C. Samples (20 μl) were electrophoresed through a 1.2% agarose gel.

Western blotting of Bcl-2 and caspase-3 proteins

For Western blotting of Bcl-2 and caspase-3, cells were collected, rinsed twice with cold PBS and pelleted. Cells were then briefly sonicated in lysis buffer A (100 mM Tris-HCl, pH 7.4, 300 mM NaCl, 2 mM EDTA, 2 mM phenylmethylsuflonyl fluoride, 10 μg ml−1 aprotinin, and 10 μg ml−1 leupeptin). Total fraction was used for Bcl-2 immunodetection, whereas cytosolic fractions were separated by centrifugation of cell extracts for 30 min at 50,000 × g and used for procaspase-3 immunodetection. Equal amounts of protein (30 μg for caspase-3 and 80 μg for Bcl-2) were subjected to SDS–PAGE and blotted on a nitrocellulose membrane (BioTrace® NT, Pall Corporation, VWR International). Membranes were blocked with Tris-buffered saline (pH 7.6) containing 5% nonfat dry milk and 0.05% Tween-20 and then incubated with mouse anti-caspase-3 (1 : 1000; Transduction Laboratorie, BD Biosciences), or rabbit anti-Bcl-2 (1 : 250; Calbiochem). For detection, horseradish peroxidase conjugated secondary antibody was used. Proteins were detected using an enhanced chemiluminescence Western blotting analysis system (Pierce). The membranes were then stripped and reprobed with mouse antiactin (1 : 10,000; Oncogene), used as a control for loading. Densitometric analyses of protein bands from scanned ray films were performed using Scion Image software (Scion Corporation, MD, U.S.A.) and the values were normalised against the intensity of actin.

Statistical analysis

The results are expressed either as the mean±s.e.m. or as representative experiments. When appropriate, statistical significance was assessed comparing data from those obtained with starved cells using the Student's t-test. The level of significance was regarded as P<0.05.

Results

Presence of VIP and PACAP in PC-3 cells

The expression of mRNA coding for VIP and PACAP in PC-3 cells was measured by RT–PCR amplification. A single DNA band was observed in both control and starved cells, at 470 bp for VIP and 576 bp for PACAP (Figure 1). Similar PCR products amplification was observed for both peptides in all situations. These PCR products correspond to the predicted size for PCR amplification of VIP and PACAP. Furthermore, both sequences were verified by sequencing the PCR products. Having demonstrated the expression of VIP and PACAP mRNAs in PC-3 cells, we next studied the production of these neuropeptides in cell suspensions by immunocytochemical methods. Immunoreactive VIP and PACAP were detected in cytocentrifuge preparations from control and serum-deprived PC-3 cells (Figure 2b, d, f and h). In both situations, the reaction products were spread throughout the cytoplasm and appeared in secretory vesicles as well. In addition, negative controls were performed by treating the cytocentrifuge preparations with PBS instead of the primary antibody (Figure 2a, c, e and g).

Figure 1.

VIP and PACAP mRNA expression in control and serum-deprived PC-3 cells. RT–PCR was performed with total RNA from control (+FBS) and serumdeprived (-FBS) PC-3 cells as described in the Methods section. Reverse transcriptase was omitted in the control experiments (RT−). The sise of the amplified fragments were 470 bp for VIP (a) and 576 bp for PACAP (b). Lane M: 100 bp ladder. Lane C: PCR performed without cDNA. A representative experiment of three others is shown.

Figure 2.

Immunocytochemical analysis of VIP and PACAP expression in control and serum-deprived PC-3 cells. VIP immunoreactive cells were detected in control (b) (× 40) and serum-deprived (d) (× 40) PC-3 cells. Negative controls of immunostaining were performed in the absence of monoclonal anti-VIP antibody (a, c). PACAP immunoreactive cells were detected in control (f) (× 40) and serumdeprived (h) (× 40) PC-3 cells. Negative controls of immunostaining were performed in the absence of polyclonal anti-PACAP antibody (e, g). A representative experiment of three others is shown.

Expression of VIP/PACAP receptors in PC-3 cells

Figure 3 shows the expression of mRNA for PAC1, VPAC1 and VPAC2 receptors in control and serum-deprived PC-3 cells. As a control, the corresponding pcDNA3-cDNAs for each VIP/PACAP receptor subtype were amplified. Amplification of the RT reactions exhibited bands of 324 and 529 bp corresponding to VPAC1 and VPAC2, receptors, whereas no product amplification was found for the PAC1 receptor. This pattern of VIP/PACAP receptor expression was similar in serum-cultured (Figure 3a) and serum-deprived (Figure 3b) PC-3 cells. In order to discard genomic DNA amplification, the same PCR reactions were carried out omitting the RT reaction. In this case, no product amplification was detected. We next identified the VIP/PACAP receptor proteins by analysing the binding of [125I]PACAP-27 and [125I]VIP to PC-3 membranes (Figure 4). Competition curves for [125I]PACAP-27 binding by PACAP-27 and VIP showed IC50 values of 3.0 and 4.5 nM, respectively, indicating that common VIP/PACAP receptors are expressed in PC-3 membranes (Figure 4a). This result is in accordance with the absence of expression of PAC1 receptor mRNA in PC-3 cells, demonstrated by the RT–PCR approach. VPAC1 and VPAC2 receptor expression was evaluated by the capacity of selected agonists to compete with [125I]VIP for binding. The order of potency of each peptide tested was as follows: VPAC1 antagonist (IC50= 1.2 nM)=VPAC1 agonist (IC50=1.5 nM)>VIP (IC50=2.3 nM) >VPAC2 agonist (IC50=30 nM). These results suggest that the majority of binding sites expressed in PC-3 cell membranes were VPAC1 receptors, although VPAC2 receptors are also present (Figure 4b).

Figure 3.

VIP/PACAP receptor mRNA expression in PC-3 cells. RT–PCR was performed with total RNA from control (a) and serum-deprived (b) PC-3 cells as described in the Methods section. Reverse transcriptase was omitted in the control experiments (RT−). As positive controls for PAC1, VPAC1 and VPAC2 receptor amplification, a PCR was performed with the corresponding pcDNA3-cDNAs. The amplified fragment sises were 450 bp for PAC1, 324 bp for VPAC1 and 529 bp for VPAC2. Lane M: 100 bp ladder. Lane C: PCR performed without DNA. A representative experiment of three others is shown.

Figure 4.

Competitive inhibition of [125I]PACAP-27 (a) and [125I]-VIP (b) binding to control PC-3 membranes. Binding competitors were VIP, PACAP-27, VPAC1 agonist, VPAC1 antagonist and VPAC2 agonist. Nonspecific binding was determined in the presence of 1 μM unlabelled PACAP-27 (a) and VIP (b). Results are the mean of four separate experiments performed in duplicate and the standard deviation (s.d.) was less than 0.1 log unit in all cases (±25% of the mean value, or less).

Functionality of VIP/PACAP receptors in PC-3 cells

The functional properties of VIP/PACAP receptors were assayed by measuring the intracellular cAMP levels in PC-3 cells (Table 1). VIP and PACAP-27 increased cAMP levels with the same potency (ED50=5 nM) and efficacy, which indicates a common receptor for both peptides. VPAC1 and VPAC2 agonists were also able to stimulate the enzyme with an ED50=23.7 and 1.4 nM, respectively, indicating that both VIP receptors present in these cells are coupled to the adenylate cyclase system. In serum-deprived cells, intracellular cAMP levels were also measured (Table 1). VIP, PACAP-27, VPAC1 and VPAC2 agonists showed a similar potency (ED50=3.3, 1.5, 1.3 and 6.2 nM, respectively). These results indicate that both receptors (VPAC1 and VPAC2) expressed in PC3 cells are also functional in the serum-deprived cells.

Table 1. cAMP production in PC3 cells
 Control cells (+FBS)Serum-deprived cells (FBS)
 cAMP productionaED50 (nM)cAMP productionaED50 (nM)
  • a

    Results are the mean of two to five separate experiments performed in triplicate and the standard deviation (s.d.) was always lower than ±10% for agonists cAMP production and below 0.1 log units for ED50 values. Data are expressed as percentage of maximum: that produced by (1 μM) VIP corresponding to two-fold basal value.

VIP (1 μM)100.05.3100.03.3
PACAP-27 (1 μM)97.35.2100.01.5
VPAC1 agonist (1 μM)60.523.765.71.3
VPAC2 agonist (1 μM)81.21.482.96.2

Effect of VIP, PACAP-27, VPAC1 and VPAC2 agonists on the survival of PC-3 cells after serum withdrawal

Figure 5 shows the effect of VIP, PACAP-27, the VPAC1 agonist or the VPAC2 agonist on the growth of starved PC-3 cells. Cells grown in the absence of trophic factors reduced their survival to 59% compared with controls. However, VIP, PACAP-27 and the VPAC1 agonist (but not the VPAC2 agonist) were able to increase starved PC-3 survival up to 77%. The effects of VIP, PACAP-27 and the VPAC1 agonist were dose dependent, with ED50 values of 5, 1 and 1 nM, respectively, differences being significant from starved PC-3 cells for doses of 100 nM neuropeptide or VPAC1 agonist. None of the VPAC2 agonist concentrations used had any effect on PC-3 cell survival. This observation is consistent with MTT measurement (Figure 6) showing that VIP, PACAP-27 and the VPAC1 agonist (but not VPAC2 agonist) are able to increase the survival of PC-3 cells grown in the absence of trophic factors from 30 to 45% (Figure 6a). When the serum-deprived PC-3 cells were grown in the presence of VIP and the specific VPAC1 receptor antagonist, no increase in survival was detected, whereas the specific VPAC2 receptor antagonist was unable to block the effect produced by VIP (Figure 6b).

Figure 5.

Effect of increasing concentrations of VIP, PACAP-27, VPAC1 agonist and VPAC2 agonist on the growth of PC-3 cells. Quantitation of cell number present in the well after 4 days of treatment was carried out as described in the Methods section, in the presence or absence of FBS. Results are the percentage cell number compared to control (cells grown in the presence of FBS, 158.000 cells per well), and derived from four independent experiments performed in triplicate. *P< 0.05.

Figure 6.

Effect of VIP, PACAP-27, VPAC1 agonist, VPAC2 agonist, VPAC1 antagonist and VPAC2 antagonist on the survival of PC-3 cells grown in the absence of trophic factors. Quantitation of live cells using MTT assays was performed as described in the Methods section. Results are the percentage of the absorbance (570–630 nm) compared to controls (cells grown in the presence of FBS, A570–630=0.485±0.023) and represent the mean of three separate experiments performed in triplicate. (a) Effect of increasing concentrations of VIP, PACAP-27, VPAC1 agonist and VPAC2 agonist on PC-3 survival in the absence of trophic factors. (b) Effect of 0.1 μM of VPAC1 or VPAC2 antagonists on VIP-increased survival of PC-3 cells grown in the absence of trophic factors. *P< 0.05.

Effect of VIP on DNA fragmentation in PC-3 cells

The increase in the survival of serum-deprived PC-3 cells treated with VIP may be due to a decrease of apoptosis in the cells. Apoptosis is characterised by a number of morphological and biochemical events such as nuclear DNA fragmentation, membrane blebbing or increase in the expression of some proapoptotic proteins. PC-3 cells showed nuclear DNA fragmentation after 4 days of growth in the absence of trophic factors (Figure 7). When 0.1 μM of VIP was added to the culture medium, a decreased nuclear DNA fragmentation was observed, suggesting a protective role for VIP from apoptosis induced by serum withdrawal in PC-3 cells.

Figure 7.

Effect of VIP on genomic DNA fragmentation in PC-3 cells. Fragmented DNA from control and serum-deprived PC-3 cells were isolated as described in the Methods section. DNA fragmentation was observed after growth factor withdrawal. Cells treated with 0.1 μM VIP show a reduction in DNA fragmentation. A representative experiment of three others is shown.

Effect of VIP on the expression of caspase-3 and Bcl-2 in PC-3 cells

Figure 8 shown the Western blot analysis for caspase-3 and Bcl-2 analysis in PC-3 cells. Results revealed the presence of a 32-kDa band corresponding to the zymogen form of caspase-3 in control PC-3 cells that decreases after serum withdrawal. The treatment of PC-3 cells with 0.1 μM VIP or PACAP-27, increased the procaspase-3 immunoreactive band (Figure 8a). Diminished levels of procaspase-3 (which has a low proteolytic activity) have been associated with increased activity of caspase-3 (Munshi et al., 2001). Western blot analysis of bcl-2 revealed a 26-kDa band whose intensity is reduced in serum-deprived cells, whereas treatment with 0.1 μM VIP or PACAP-27 also increased the levels of this antiapoptotic protein (Figure 8b). Although neither VIP nor PACAP-27 was able to increase the levels of procaspase-3 and Bcl-2 to control values, we observed that both peptides increased the intensity of the immunoreactive band for procaspase- 3 and Bcl-2 to about 30 and 50%, respectively.

Figure 8.

Western blot analysis of procaspase-3 (a) and Bcl-2 (b) protein expression in PC-3 cells. Cell lysates from control and serum-deprived cells treated or not with 0.1 μM VIP or PACAP were prepared and electrophoresed as described in the Methods section. A decrease in the procaspase 3 (a) and bcl-2 (b) proteins were observed in serum-deprived PC-3 cells. VIP and PACAP treatment of serum-deprived PC-3 cells increase the expression of procaspase 3 and bcl-2. Actin was used as a loading control and the ratio of procaspase-3 to bcl-2/actin is shown. A representative experiment of three others is shown.

Discussion

In the prostate, androgens play a major role in supporting the maintenance of normal growth and function. However, this gland also contains neuropeptides located either in nerve terminals or in neuroendocrine cells, which can also play a regulatory role in the pathophysiology of the prostate (Gkonos et al., 1995). This is of great importance regarding prostate cancer, since neuropeptides may influence the behaviour of the tumours. In this sense, the neuroendocrine differentiation is a marker of poor prognosis in prostate cancer and may correlate with development of an androgen-resistant state (Bonkhoff et al., 1995). Prostate cancer is composed of androgen-dependent and -independent cancer cells and the emergence of apoptosis-resistant clones (Denmeade et al., 1996). One of the best studied neuropeptides present in the prostate is VIP, which stimulates rat prostatic epithelial cell proliferation and induces neuroendocrine cell differentiation in LNCaP cells (Juarranz et al., 2001a, 2001b). The first aim of this study was to identify whether VIP and PACAP act as autocrine and/or paracrine factors in an androgen-independent prostate cancer cell line, PC-3. We have demonstrated the presence of both peptides by RT–PCR studies and immunocytochemical analysis. These results are in line with a previous study on nude mice bearing PC-3 human androgen-independent prostate carcinoma (Plonowski et al., 2002). The presence of VIP has been described in autonomic nerves surrounding the human prostate acini (Tainio, 1995) and, very recently, we have described the expression and distribution of PACAP in the epithelium layer of normal and carcinomatous human prostate (García-Fernández et al., 2002). On the other hand, VPAC1 and VPAC2 receptors, but not PAC1 receptors, are present in PC-3 cells. RT–PCR studies as well as functional studies using a specific agonist and antagonist for each receptor subtype, peptide binding and adenylate cyclase stimulation, show that both VIP receptors are present in PC-3 cells. This is in accord with previous studies in rat prostate, human cancer prostate, prostate LNCaP cells and PC-3 cells (Juarranz et al., 1999; Reubi et al., 2000; Juarranz et al., 2001b; Plonowski et al., 2002). In contrast with the present results, Gkonos et al. have reported that PC-3 cells do not express VPAC1 receptors, whereas PC-3 cells stably expressing the androgen receptor (PC-3/AR) do, although no regulation of the VPAC1 receptor by androgens was observed (Gkonos et al., 2000). This discrepancy could be due to differences related to cell batch. Altogether, these results suggest that VIP and PACAP act, in addition with the well-described paracrine mechanism, as an autocrine factor in human prostate cancer.

The second aim of this study was to establish the role of both neuropeptides in this androgen-independent prostatic cancer cell line, since VIP is related to proliferation, neuroendocrine differentiation and migration in other prostatic cancer cell lines (Jongsma et al., 2000; Juarranz et al., 2001b; Nagakawa et al., 2001). In order to test this, PC-3 cells were grown in the absence of trophic factors in order to induce an apoptotic process (Tang et al., 1998). This cell status did not modify either the production of VIP and PACAP in PC-3 cells or the expression and functionality of their receptors. VIP, PACAP-27 and [K15, R16, L27] VIP (1–7)/ GFR (8–27), the specific VPAC1 agonist, were able to increase the survival of PC-3 cells grown in the absence of trophic factors, whereas RO-25-1553, the specific VPAC2 agonist, was unable to do so, which suggests that the receptor involved in this effect is the VPAC1 receptor. These results are in agreement with MTT studies since the specific VPAC1 antagonist, but not the specific VPAC2 antagonist, was able to block the increased cell survival induced by VIP. In this regard, another VIP antagonist (JV-1-53, a GH-RH analogue) can inhibit the growth of androgen-independent prostate tumours in nude mice (Plonowski et al., 2002).

The increased survival of PC-3 cells (grown in medium lacking trophic factors) induced by VIP, PACAP and the VPAC1 agonist, may be associated with a decrease in the apoptotic process of the cells. The present results show that VIP reduces DNA fragmentation, increases the expression levels of some antiapoptotic proteins, such as Bcl-2, and decreases the expression of proapoptotic proteins such as caspase-3 in this androgen-independent prostate cancer cell line. The resistance to apoptosis is clearly related with prostate cancer progression, since androgen ablation in patients with prostate cancer eliminates most androgen-dependent cancer cells by inducing apoptosis, but can rarely cure the patients due to the presence of androgen-independent cells and the emergence of apoptosis-resistant clones (Denmeade et al., 1996). In this regard, our results are interesting since PC-3, an androgen-independent cancer cell line, is characterised by a high expression of antiapoptotic proteins and a low expression of proapoptotic proteins (Tang et al., 1998); when we induced apoptosis in PC-3 cells by serum withdrawal, VIP and PACAP-27 treatments were able to modulate the expression levels of antiapoptotic and proapoptotic proteins (Bcl-2 and caspase-3) in addition to inhibiting DNA fragmentation. Caspase-3 makes an essential contribution to cell death; all cell types examined from caspase-3-defective mice fail to display some typical hallmarks of apoptosis (i.e., DNA fragmentation); moreover, activation of procaspase-3 is triggered, in part, by activated upstream caspases such as caspase-9, whose activity is regulated by the bcl-2 protein (Budihardjo et al., 1999). Thus, the increased levels of inactive caspase-3 and the partial inhibition from apoptosis exerted by VIP and PACAP could be associated with the observed increase of Bcl-2 immunoreactivity induced by both peptides in serum-deprived PC-3 cells. The protective effect of VIP and PACAP from cell death has been described in other systems such as cerebellar granule cells or pituitary adenoma cells (Vaudry et al., 2000). This effect has been reported for other neuropeptides as well, such as bombesin or calcitonin in a prostate cancer cell line (Salido et al., 2000). Recently, Sumitomo et al. (2001) have described a mechanism that could explain the survival effect exerted by G-protein-coupled receptor agonists, such as bombesin and endothelin-1. They observed that these neuropeptides stimulate ligand-independent activation of the IGF-I receptor, which results in the activation of Akt, a serine-threonin kinase that mediates cell survival. Moreover, they observed that neutral neuroendopeptidase 24.11 inhibits the survival effect exerted by bombesin and endothelin-1 in prostate cancer cells. Interestingly, VIP and PACAP-27 are substrates of this metalloprotease (Gourlet et al., 1997b), which is absent in PC-3 cells.

Our results extend previous observations showing that the neuropeptides VIP and PACAP play an important role in prostate cancer development, especially in the androgen-independent status, since they are able to stimulate the neurocrine differentiation of LNCaP cells grown in the absence of androgens (Juarranz et al., 2001b) and inhibit induced cell death in PC-3 cells (present study). Our data suggest that VIP/PACAP antagonists can inhibit the growth of androgen-independent prostate cancer cells by abrogating the autocrine/paracrine mitogenic stimuli of VIP and PACAP, which could be potentially beneficial for prostate cancer therapy.

Acknowledgments

We thank Dr Patrick Robberecht (Université Libre de Bruxelles, Belgium) for the VPAC1 and VPAC2 agonists and antagonists, Dr. Helen C. Wong (UCLA School of Medicine, USA) for the anti-VIP antibody, Dr Akira Arimura (Tulane University School of Medicine, New Orleans, U.S.A.) for the anti-PACAP antibody and Dr Rosa P. Gomariz (Universidad Complutense de Madrid, Spain) for technical assistance in immunocytochemical assays. This work was supported by the Ministerio de Ciencia y Tecnología (Grant SAF2001-1025). We thank Professor L. Puebla for English assistance.

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