Background
Calcium (Ca
2+) signalling is essential for regulating physiological functions such as cell proliferation and differentiation [
1]. Prostate cancer is the second most lethal tumour among men, and Ca
2+ has been shown to be essential for increased cell proliferation in advanced prostate cancer (PCa) cells [
2‐
4]. However, the ion channel(s) involved in Ca
2+ entry are not fully understood and the mechanism that leads to alteration of Ca
2+ handling in PCa is still poorly defined. Understanding the factors that drive PCa towards increased cell proliferation is crucial for the development of new therapies that can prevent and/or inhibit the initiation and/or progression of PCa.
Among the transient receptor potential (TRP) channel proteins, human TRP vanilloid type 1 (TRPV1) is expressed in normal prostate epithelial cells, PCa tissues and in PC3 as well as LNCaP cells [
5]. The expression of TRPV1 has been found to be significantly up-regulated in PCa compared with benign prostate hyperplasia (BPH) tissues, and the increased expression of TRPV1 correlates with increasing PCa tumour grade [
6].
Alpha1-Adrenoceptors (α
1-ARs) mediate actions of the endogenous adrenaline and noradrenaline (NA) in several target cells. On the basis of pharmacological and binding studies α
1-ARs have been subdivided into three subtypes, namely α
1A (α
1a), α
1B (α
1b) and α
1D (α
1d) [
7]. α
1-AR subtypes show different cellular localization: α
1B-AR is mainly expressed on cell surface [
8,
9], α
1A-AR is evidenced on the cell surface and intracellularly [
10,
11] and α
1D-AR appears to be localized primarily perinuclearly [
8,
9]. Moreover, α
1-ARs are expressed in a variety of human tissues, including liver, kidneys, blood vessels, heart and prostate. In the human prostate α
1A-AR and α
1B-AR subtypes are expressed in BHP [
12,
13], PCa tissues as well as in PC3 and DU145 PCa cell lines [
14,
15] whereas the α
1D-AR subtype in the PC3 cell line [
16,
17].
The α
1-ARs are G-protein-coupled receptors (GPCRs) that are linked to the heterotrimeric G-protein containing the Gαq/11/14/16 subunits. The Gαq subunit is a primary activator of phospholipase C (PLC), which promotes the cleavage of inositol 4,5-bisphosphate (PIP
2) to yield diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP
3). DAG and IP
3 then promote the activation of protein kinase C (PKC). Growing evidence supports the role for α
1-ARs in the direct mitogenic effect of catecholamines on prostate growth [
18]. We previously reported the expression of the α
1B- and α
1D-AR subtypes in PC3 cells and the ability of NA to stimulate PC3 cell proliferation in a α
1D-AR-dependent manner [
17]. Therefore, the aim of the present study was to evaluate the correlation between α
1D-AR and TRPV1 expression levels in patients with PCa and to demonstrate the role of TRPV1 in the regulation of NA-induced α
1D-AR-dependent PC3 cell proliferation.
Methods
Prostate cancer cell line
Human prostate cancer cell lines PC3, and DU145 were purchased from American Type Culture Collection (ATCC, Rockville, MD). Cell lines were maintained in DMEM and RPMI medium (Lonza Group Ltd, Basel Switzerland), respectively, supplemented with 10% heat-inactivated fetal bovine serum (FBS, Lonza), 100 IU/ml of penicillin, 100 μg/ml of streptomycin at 37°C, 5% CO2 and 95% of humidity. The androgen sensitive cell line LNCaP was purchased from Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna (IZSLER, Brescia, Italy) and maintained in RPMI supplemented as above described at 37°C, 5% CO2 and 95% of humidity.
PCa tissues
Specimens (n = 37), from adult patients with high-risk clinically localized adenocarcinoma of the prostate underwent radical prostectomy at the Urology Operative Unit, ASUR 9 Macerata, were collected for qRT-PCR analysis (Additional file
1: Table S1). As control, 5 samples of BPH tissues removed by transurethral resection were used. All the specimens were embedded in paraffin and 5–7 μm-thick sections were collected on slides. Patients, giving their informed written consent, that covered the use of their tissues for research purposes, were included in the prostate cancer database of the Pathology Unit, ASUR 9. The study was approved by the Ethics committee Ospedale Civile Macerata and ASUR 9 and all procedures were conducted in accordance with the Declaration of Helsinki.
Antibodies and reagents
The following anti-human polyclonal antibodies (Abs) were used: goat anti-TRPV1, rabbit anti-α
1D-AR from Santa Cruz Biotechnology (Heidelberg, Germany), rabbit anti-ERK, rabbit anti-phospho p38 (anti-pp38), rabbit anti-p(Ser)-PKC substrate and rabbit anti-p38 from Cell Technology (Danvers, MA). The following anti-human mouse monoclonal Abs (mAbs) were used: anti-GAPDH (Sigma Aldrich, St. Louis, MO), anti-pERK (Cell) and anti-Bromodeoxyuridine (BrdU) fluorescein isothiocyanate (FITC)-conjugated (Prodotti Gianni, Italy). The horseradish peroxidise (HRP)-conjugated donkey anti-goat and donkey anti-mouse from Santa Cruz Biotechnology, HRP-conjugated goat anti-rabbit Ab from Cell Signaling. Purified FITC-conjugated rabbit anti-goat (RAG)IgG (EMD Chemicals, Inc. San Diego, CA), phycoerythrin (PE)-conjugated goat anti-rabbit (GARB) IgG (BD Biosciences, San Jose, CA), Alexa Fluor 488-conjugated rabbit anti-goat and Alexa Fluor 594-conjugated goat anti-rabbit Abs (Invitrogen, Carlsbad, CA) were used as secondary Abs. {2-[2-(4-Chlorobenzyloxy)phenoxy]ethyl}-[2-(2,6-dimethoxyphenoxy)ethyl]amine (clopenphendioxan, WS433, α
1D-AR antagonist) was provided by Prof. Wilma Quaglia, School of Pharmacy, University of Camerino [
17]. NA, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), BrdU and dimethyl sulfoxide (DMSO, used as vehicle) from Sigma Aldrich. Chelerythrine chloride (PKC inhibitor), U73122 (PLC inhibitor), PD98059 (MEK inhibitor) and the TRPV1 antagonist, capsazepine (CPZ) [
19] were purchased from Tocris Bioscience (Bristol, UK).
Double immunofluorescence and flow cytometry
To determine the co-expression of TRPV1 and α1D-AR, 3×105 PC3, DU145 and LNCaP cells were fixed with 4% paraformaldheyde in PBS for 10 min at room temperature, washed with permeabilizing solution (1% FBS, 0.1% saponin and 0.1% sodium azide in PBS) and stained for 30 min at 4°C first with anti-TRPV1 Ab (1:25) followed by FITC-RAG (1:40) and then with anti-α1D-AR (1:25) followed by PE-GARB (1:40). Normal goat and rabbit serum were used as negative control. In some experiments double immunofluorescence and FACS analysis were performed in PC3 cells double silenced for α1D-AR and TRPV1 genes. The percentage of positive cells determined over 10,000 events was analyzed on a FACScan cytofluorimeter (BD Bioscience) and fluorescent intensity was expressed in arbitrary units on a logarithmic scale.
Confocal laser scanning microscopy analysis
2 × 105/mL PC3 cells grown for 24 h at 37°C in poly-L-lysine coated slides, were permeabilized using 2% of paraformaldehyde with 0.5% of Triton X-100 in PBS and fixed by 4% of paraformaldehyde in PBS. After washes in PBS, cells were incubated with 3% of bovine serum albumin (BSA) and 0.1% of Tween-20 in PBS for 1 h at room temperature and then double stained with anti-TRPV1 (1:25) and anti-α1D-AR (1:25) Abs overnight at 4°C. Finally, samples were washed with 0.3% of Triton X-100 in PBS, incubated with Alexa Fluor 488-conjugated and Alexa Fluor 594-conjugated secondary Abs (1:100) for 1 h at 37°C and analysed with MRC600 confocal laser scanning microscope (BioRad, Hercules, CA) equipped with a Nikon (Diaphot-TMD) inverted microscope. Fluorochrome was excited with the 600 line of an argon-kripton laser. Serial optical sections were taken at 1-μm intervals through the cells. Images were processed using Jacs Paint Shop Pro (Jacs Sotfware Inc).
Fluorescence microscopy analysis
The co-expression of α1D-AR and TRPV1 in tissue specimens from patients with adenocarcinoma or BHP, used as control, was evaluated by double immunofluorescence. Briefly fixed paraffin-embedded tissue slices were deparaffinized, rehydrated and washed with 0.3% Triton X-100 in PBS. After incubation with 3% of BSA and 0.3% of Triton X-100 in PBS for 1 h at room temperature, sections were first stained with anti-α1D-AR (1:25) Ab overnight at 4°C, washed with 0.3% of Triton X-100 in PBS and then labelled with Alexa Fluor 594-conjugated secondary Ab (1:100) for 1 h at 37°C. Subsequently, samples were washed with 0.3% of Triton X-100 in PBS and incubated with anti-TRPV1 (1:25) overnight at 4°C followed by Alexa Fluor 488-conjugated Ab (1:100) for 1 h at 37°C. Sections were analyzed using a BX51 fluorescence microscope at 10× magnification (Olympus, Milan, Italy). Merge images were obtained by using the DP controller software (Olympus).
Western blot analysis
PC3 cells were lysed in lysis buffer (10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.5% deoxycholate, 1 mM phenylmethylsulfonylfluoride) containing protease inhibitor cocktail (SigmaAldrich) by using the Mixer Mill MM300 (Qiagen GmbH, Hilden, Germany). Lysates from PC3 cells treated for different times with NA (100 μM), WS433 (1 μM) and CPZ (1 μM) alone or in combination, were separated on 8 and 12% SDS-polyacrylamide gels, transferred and blotted with anti-pERK (1:1000) mAb followed by HRP-conjugated anti-mouse (1:1000) Ab, anti-ERK (1:1000), anti-p(Ser)-PKC substrate (1:1000), anti-pp38 (1:2000) and anti-p38 (1:2000) Abs followed by HRP-conjugated anti-rabbit (1:2000) Ab. Anti-GAPDH mAb was used as protein loading control. To verify silencing efficiency, lysates from siTRPV1 or siα1D-AR PC3 cells were immunoblotted with anti-TRPV1 (1:100) or anti-α1D-AR (1:1000) Abs followed by HRP-conjugated anti-goat (1:1000) and anti-rabbit (1:2000) Abs respectively. Immunoreactivity was detected using the LiteAblot ®PLUS (EuroClone, Milan, Italy) kits and densitometric analysis was carried out by evaluating three independent experiments by a Chemidoc using the Quantity One software (BioRad, Hèrcules, CA).
The eight-channel CytosensorTM microphysiometer (Molecular Devices Corp., Sunnyvale, CA, USA) was used as previously described [
20] to evaluate small changes in the extracellular release of protons induced by NA in the culture medium surrounding PC3 cells. Briefly, 3 × 10
5 PC3 cells were seeded into 12-mm capsule cups and cultured for 24 h. Then the capsule cups were loaded into the sensor chambers and the chambers were perfused with running medium (bicarbonate-free DMEM with 0.584 g/L glutamine and 2.59 g/L NaCl) at a flow rate of 100 μL /min. PC3 cells were stimulated for 30 sec with vehicle, NA (100 μM), WS433 (1 μM), CPZ (1 μM), alone or in combination, diluted into running medium and perfused through either fluid path. ECAR rate data were expressed as percentages of response respect to the baseline value.
Gene silencing
siGENOME SMARTpools for TRPV1 (siTRPV1) and for α1D-AR (siα1D-AR) consisting of four RNA duplex targeting respectively TRPV1 and α1D-AR genes, and a siCONTROL non-targeting small interfering RNA (siGLO) with at least four mismatches to any human gene used as negative control were purchased from Dharmacon (Lafayette, CO). Briefly, 4×104/ml PC3 cells were plated and after an overnight incubation, 20 nM of siTRPV1, siα1D-AR or siGLO was added to the wells, following METAFECTENE SI (Biontex Laboratories GmbH, Martinsried/Planegg, Germany) transfection protocol. Cells were harvested at day 2 post-transfection for analysis.
Total RNA extraction and complementary DNA synthesis
Total RNA from siGLO, siTRPV1 and siα1D-AR PC3 cells was extracted with the RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany). Total RNA from fixed paraffin-embedded tissue slices (5–7 μm-thick) was isolated by Absolutely RNA® formalin-fixed, paraffin-embedded (FFPE) kit (Stratagene, Austin, TX, USA). Five hundred ng of extracted RNA were subjected to reverse transcription using the High-Capacity cDNA Archive Kit (Life Technologies Corporation, Carlsbad, CA). One μL of the resulting cDNA products was used as template for qRT-PCR quantification.
qRT-PCR analysis
qRT-PCR was performed using the iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad). The reaction mixture contained the Syber Green Master Mix (Bio-Rad) and primer sets. Human β-actin, TRPV1 and α1D-AR primers designed with Primer Premier 5 (Bio-Rad, Hèrcules, CA) and purchased from SigmaAldrich. Primers sequences were: β-actin: forward 5′-ATCAGCAAGCAGGAGTATGACG -3′; reverse 5′-AAAGCCATGCCAATCTCATCTG-3′; TRPV1: forward 5′ -CTGATGGCAAGGACGACTACC-3′; reverse: 5′ -TTGACCGCAGGGAGAAGCTC-3′; α1D-AR: forward 5′ -GGTCGTAGCCCTGGTGGTG -3′; reverse: 5′ -CGGAGGAGAAGACAGCGTAGC -3′. Each amplification consisted of heat activation for 15 min at 95°C followed by 40 cycles at 95°C for 10 sec and 60°C for 50 sec. All samples were assayed in triplicate in the same plate and in three different experiments. Measurement of β-actin levels was used to normalize mRNA contents. TRPV1 and α1D-AR levels were calculated by the 2-ΔΔCt method and expressed as relative fold respect to control levels.
[Ca2+]imeasurement
3×106/mL PC3 cells were washed in calcium and magnesium free PBS supplemented with 4.5 g/L of glucose used as experimental medium. Cells were resuspended in the medium supplemented with 7 μmol/L FLUO 3-AM and 1 μg/mL Pluronic F-127 (Life Technologies Corporation, Carlsbad, CA) and incubated in the dark for 30 min at 37°C, 5% CO2. After washing, cells were resuspended in the medium containing or not 2 mmol/L Ca2+ and stimulated with vehicle, NA (100 μM), WS433 (1 μM), CPZ (1 μM), alone or in combination. Fluorescence was measured by FACScan; not stimulated cells were analyzed for 2 min to establish baseline fluorescence levels.
Proliferation assay
BrdU incorporation was determined in PC3 cells treated for 24 h with NA (100 μM), WS433 (1 μM) and CPZ (1 μM), alone or in combination and in siGLO, siTRPV1 and siα1D-AR PC3 cells treated with NA (100 μM) for 24 h. Cells were labelled by adding 20 μL/well of BrdU. After trypsinization and washing in PBS supplemented with 0.5% BSA and 2 mM ethylenediaminetetraacetic acid, cells were fixed for 30 min in PBS containing 30% methanol and 0.4% paraformaldheyde, permealized with PBS containing 1% paraformaldheide and 0.01% Tween-20, and then incubated for 15 min in DNAse buffer containing 500 KU/mL of DNAse (SigmaAldrich). Thereafter, cells were stained with anti-BrdU FITC-conjugated Ab (1:10) incubated for l h at room temperature and washed in PBS containing 0.5% BSA and 2 mM ethylenediaminetetraacetic acid. Samples were analyzed by a FACScan cytofluorimeter as above described.
MTT assay
Cell growth was measured by MTT assay. 4 × 10
4 PC3 cells/mL were plated in a 96-well microtiter plate, treated for 24 h with NA (100 μM), WS433 (1 μM) and CPZ (1 μM), alone or in combination and then incubated with 0.8 mg/ml of MTT for the last 3 h. Four replicates were used for each treatment. The supernatants were discarded and colored formazan crystals, dissolved with 100 μL/well of DMSO, were read at 570 nm wavelength by an ELISA reader (BioTek Instruments, Bad Friedrichshall, Germany). In some experiments, MTT assay was performed in PC3 cells treated for 24 h with NA (100 μM), chelerythrine (0.5 μM), U73122 (5 μM) and PD98059 (50 μM), alone or in combination. Dose response curves have been performed after 24 h treatments for chelerythrine, U73122 and PD98059 compounds (Additional file
2: Figure S1F). The highest doses that did not affect cell viability were used.
Inositol-1,4,5-trisphosphate (IP3) measurement
IP
3 was measured using the inositol-1,4,5-trisphosphate [
3H] radioreceptor assay kit (PerkinElmer Life Sciences, Inc., Waltham, MA) [
21]. Briefly, 1.5×10
5/mL PC3 cells were treated with NA (100 μM), WS433 (1 μM) and CPZ (1 μM), alone or in combination for different times in DMEM supplemented with 1% FBS. After treatment IP
3 was read by β scintillation counter in 5 ml of Atomlight scintillation cocktail (PerkinElmer).
Statistical analysis
The statistical significance was determined by one-way Anova or by 2-way Anova with Bonferroni post-test. Unpaired t tests and Spearman’s rank correlation tests were performed with GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA, USA). No differences were found comparing vehicle-treated with untreated PC3 cells (control).
Discussion
TRP channels may participate in Ca
2+ homeostasis in PCa cells [
24]. It has been hypothesised that abnormal Ca
2+ signalling may be an essential step in the α
1-AR-mediated increased proliferation in PCa cells [
3].
Herein, we firstly demonstrated that prostate cancer cell lines co-expressed α
1D-AR and TRPV1 proteins and these receptors co-localized mainly in the plasma membrane, perinuclear region and intracellular vesicles. Therefore, we investigated whether these receptors functionally interact each other. Previously, cross-sensitisation between TRPV1 and other members of the GPCR family, such as P2X3 and CCR1 in DRG neurons [
25] and CCR1/TRPV1-transfected human embryonic kidney (HEK293) cells, respectively, have been demonstrated [
26].
We also evaluated the α
1D-AR and TRPV1 co-expression at mRNA and protein levels in advanced PCa tissues. As previously reported [
6], TRPV1 mRNA expression is higher in PCa than in BPH tissues. Our data demonstrated that α
1D-AR mRNA and protein levels were markedly increased in PCa samples compared with BPH specimens and, more importantly, that there is a great correlation between α
1D-AR and TRPV1 expression levels. Moreover, a strong reduction of the α
1D-AR and TRPV1 mRNAs was observed in 5/37 PCa patients who received neoadjuvant androgen deprivation therapy compared with untreated PCa patients. This result suggests that the transcriptional activity of TRPV1 and α
1D-AR may be androgen dependent. Chromatin immunoprecipitation analysis revealed that TRPV1 is a novel androgen receptor target gene in castration-resistant C4-2 PCa cells [
27]. Similarly, decreased TRPV6 levels were detected in androgen-insensitive tumours after androgen deprivation therapy [
2]. No data on the relationship between α
1D-AR and androgen receptors has been published to date; however, the expression of β
2-AR, a well-known activator of the androgen receptors, was transiently down-regulated in hormone-sensitive LNCaP cells treated with the anti-androgen compound bicalutamide [
28,
29]. Thus, there may be a similar effect on α
1D-AR in PCa patients treated with anti-androgen drugs.
α
1D-AR has a 10- to 100-fold higher affinity for endogenous catecholamines than the α
1A- and α
1B-AR subtypes [
30]. Therefore, we studied whether the binding of NA to α
1D-AR sensitises TRPV1 in PC3 cells. By protons release and Ca
2+ flux analysis NA resulted in a rapid response, which was inhibited by WS433 and CPZ. Moreover, NA stimulates a cross-talk between α
1D-AR and TRPV1 in PC3 cells that involves the PLC-PKC-ERK pathways. In particular, NA sensitises TRPV1, but not α
1D-AR, to activate the PLC pathway. This result is in agreement with previous findings demonstrating a weaker coupling between PLC and α
1D-AR than between PLC and other α
1-AR subtypes [
9,
31]. Calcium flowing through TRPV1 activates PLC, and the resulting depletion of PIP
2 plays a role in PKC-dependent TRPV1 sensitisation [
32]. Moreover, recently TRPV1 has been demonstrated to be either inhibited or activated by PIP
2[
33]. In this regard, we found that the IP
3 production is a TRPV1-dependent event; WS433 in combination with CPZ significantly inhibited the IP
3 production to a greater extent than CPZ alone, suggesting a co-stimulatory effect of α
1D-AR signalling in sustained TRPV1-dependent PLC activation. Moreover, the CPZ and WS433 drug combination significantly reduced the NA-induced phospho-(Ser) PKC substrate phosphorylation. Finally, both CPZ and WS433 antagonists alone markedly inhibited the ERK1/2 phosphorylation, although the maximal effect was evidenced by their use in combination, suggesting that ERK1/2 represents a downstream component of NA-induced TRPV1 and α
1D-AR signalling pathway. In this regard, in human HEK293 cell line transfected with α
1D-AR, a constitutive ERK1/2 phosphorylation, which was reduced by incubation with the selective α
1D-AR antagonist BMY7378, was evidenced; in addition a rapid and transient ERK1/2 phosphorylation, that was not inhibited by the PKC inhibitor, Ro-8425, following α
1D-AR activation was demonstrated [
34]. Moreover, α
1D-AR activation stimulated the ERK pathway in CHO cells [
35] and in lacrimal gland epithelial cells [
36].
Previous reports have indicated the contribute of α
1D-AR in NA-induced proliferation of PC3 cells [
17], however no data on the potential effects of TRPV channels in PCa proliferation were reported. Herein we found that complete abrogation of NA-induced increase in PC3 cell proliferation was reached only in double-silenced α
1D-AR/TRPV1 PC3 cells. The silencing of the α
1D-AR or TRPV1 gene or the use of WS433 or CPZ alone, partially but not completely, inhibited the NA-induced effects suggesting that these receptors act cooperatively.
Coupling of α
1-AR to Ca
2+-permeable TRPCs channels has been reported in LNCaP cells [
24,
37,
38] and WB4101 and the TRP channel blockers 2-ABP and SK&F 96365 [
24]. Furthermore, naftopidil, an α
1D-AR selective antagonist, has been also reported to affect the proliferation of human prostate epithelial cells [
39], and labedipinedilol-A, which shows high selectivity for α
1A- and α
1D-AR, to inhibit the NA-stimulated proliferation and ERK phosphorylation in LNCaP and PC3 cells [
40]. Thus, because of the lack of potent and selective subtype-specific α
1-AR antagonists, experimental or clinical trials using these compounds are few [
41,
42].
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
MBM and CA carried-out all experiments, and analyzed data with the assistance of MN, SL, CC, VF, DT, AF, AG. GS conceived the study, participated in its design with all authors, coordinated and helped to draft the manuscript with the assistance of all authors. Critical revision was performed by WQ, AP, AB, FDB, LS, MS and GM. All authors read and approved the final manuscript.