ET-1 was purchased from Sigma-Aldrich; Merck KGaA. ET-1 receptor antagonists, 2-[(3R,6R,9S,12R,15S)-6-(1H-indol-3-ylmethyl)-9-(2-methylpropyl)-2,5,8,11,14-pentaoxo-12-propan-2-yl-1,4,7,10,13-pentazabicyclo[13.3.0]octadecan-3-yl] acetic acid or cyclo(D-tryptamine-D-aspartic acid-L-proline-D-valine-L-leucine) (BQ123) and 2,6-Dimethylpiperidinecarbonyl-γ-Methyl-Leu-N_in-(Methoxycarbonyl)-D-Trp-D-Nle,-N-[N-[N-[(2,6-Dimethyl-1-piperidinyl)carbonyl]-4-methyl-L-leucyl]-1-(methoxycarbonyl)-D-tryptophyl]-D-norleucine sodium salt (BQ788) were purchased from Cayman Chemical Company and bosentan was purchased from Sigma-Aldrich; Merck KGaA. The salts, chloroform and alcohols were obtained from MilliporeSigma and Hank's buffer was obtained from Thermo Fisher Scientific, Inc.

Cells were incubated with BQ123 (ET_AR selective antagonist; 1 µM), BQ788 [endothelin B receptor (ET_BR) selective antagonist; 1 µM] or bosentan (ET_AR and ET_BR dual antagonist; 1 µM) for 30 min at 37°C with 5% CO₂. In all experiments, DMSO 0.1% was used.

PCa cells from androgen-dependent lymph nodal metastases (LNCaP) and PCa cells from bone metastases (PC3) were obtained from American Type Culture Collection. LNCaP cells, clone FDG (CRL1740), were maintained in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) and PC3 cells (CRL1435) were maintained in DMEM F12 medium (Gibco; Thermo Fisher Scientific, Inc.). Both media were supplemented with 10% FBS (Thermo Fisher Scientific, Inc.), 1% penicillin, 1% streptomycin and 0.05% amphotericin B (Corning, Inc.). All cell cultures were maintained at 37°C in a humidified atmosphere with 5% CO₂. The media of PC3 cells were replaced with phenol red-free RPMI (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% activated charcoal (Sigma-Aldrich; Merck KGaA)-treated FBS, 1% streptomycin, 0.05% amphotericin B and 1% penicillin 24 h before the different treatments. Afterwards, cells were washed with PBS three times and harvested.

A TMA was constructed with 5-µm sections of formalin (10% v/v)-fixed for 24 h at room temperature and paraffin-embedded PCa samples from male patients (age range, 47–80 years; mean age, 63.7 years) with different Gleason Score (GS) (21) from the biopsy archive of our institutional Pathology Department (Clinical Hospital of the University of Chile, Santiago, Chile). All samples were diagnosis biopsies collected in different years (March 2016-January 2018) with a confirmed diagnosis and GS. The sample inclusion criterion was: PCa confirmed. All protocols for tissue archive use and processing have been approved by the institutional Ethical Committee of Faculty of Medicine, University of Chile (approval nos. 135-2015 and 083-2020). For diagnosis biopsies, patients were asked to sign a general consent when biopsies were obtained. For the use of the archive of biopsies from the Pathology Service, a signed authorization of the director of the Department of Pathology, University of Chile (authorization 03012016; Santiago, Chile) is required. TMAs with cores of 1 mm in diameter, including 7 samples with a low GS (GS<7), 14 samples with an intermediate GS (GS=7) and 11 samples with a high GS (GS>7) were obtained and evaluated by a pathologist.

TMA tissue sections were deparaffinized and rehydrated in decreasing concentrations of ethanol and distilled water [xylene (×2), ethanol 100% (×2), ethanol 95%, ethanol 70% and distilled water], and incubated for 40 min at 95°C in antigen recovery buffer (10 mM citrate buffer, pH 6.0). After cooling, endogenous peroxidase was inhibited by incubation with 0.3% H₂O₂ for 30 min and samples were blocked with 2.5% horse serum (Vector Laboratories, Inc.) for 30 min at room temperature. Then, sections were incubated with primary antibody against ET_AR (dilution, 1:300; cat. no. PA3-065; Thermo Fisher Scientific, Inc.) or ET_BR (dilution, 1:800; cat. no. PA3-066; Thermo Fisher Scientific, Inc.) overnight at 4°C. Subsequently, the samples were washed and incubated with secondary antibody conjugated to the HRP enzyme for 1 h at room temperature (ready to use; anti-rabbit-mouse-IgG; cat. no. PK-7200; Vector Laboratories, Inc.). Then, samples were washed and incubated with ABC amplification system for 30 min at room temperature (ready to use; cat. no. PK-7 200; Vector Laboratories, Inc.). Finally, the chromogenic substrate 3,3′-diaminobenzidine (DAB) was added. Furthermore, the nuclei were stained with hematoxylin (ScyTek Laboratories, Inc.) for 1 min at room temperature. Images were obtained using a Leica DM2500 light microscope (Leica Microsystems GmbH), digitized and the DAB signal was quantified by ImageJ 1.51w software (National Institutes of Health), using the IHC toolbox plugin (https://imagej.nih.gov/ij/plugins/ihc-toolbox/index.html) and plotted using GraphPad Prism version 7.1 (GraphPad Software, Inc.). For semi-quantitative analysis of immunohistochemistry results, the gray level transformation method was implemented, using the logarithmic transformation technique. The digitized images were processed through gray level transformation techniques, since it operates directly on the pixels. The image involves 256 levels of gray, so in the histogram on the horizontal axis it ranges from 0 to 255 levels of gray. On the other hand, the vertical axis differs from the number of pixels in the image. The Log (255/average color/grays in the selected area) was determined as previously described (22).

Total RNA was isolated from cells using TRIzol^® (Thermo Fisher Scientific, Inc.), chloroform, isopropanol and 75% ethanol. Subsequently, nuclease free water (diethylpyrocarbonate-treated) was used to re-suspend extracted RNA. The purity and concentration of RNA were determined by spectrophotometry at 260/280 nm using a BioTeK Synergy HT plate reader (BioTek Instruments, Inc.).

A total of 1,000 ng total RNA from each sample was used for cDNA synthesis using the 5X All-In-One RT MasterMix kit (25°C for 10 min, 42°C for 15 min and 85°C for 5 min, 1 cycle; Applied Biological Materials Inc.). Subsequently, 50 ng/ml were amplified by qPCR using the Brilliant II SYBR Green qPCR Master Mix kit (95°C for 10 min, 1 cycle; 95°C for 15 min, 60°C for 15 min and 72°C for 15 min, 40 cycles; 95°C for 15 min, 65°C for 15 min and 95°C for 15 min, 1 cycle; Agilent Technologies, Inc.). RT-qPCR oligonucleotides are shown in Table I. The housekeeping gene Pumilio was used to normalize data and the results were analyzed using the ΔΔCq method (23). To facilitate comparison, data were referred to those cells that express the highest levels of the receptor. PC3 cells expressed the highest levels of ET_AR mRNA and protein, so the expression levels of ET_AR of LNCaP cells were compared with PC3 cells. By contrast, LNCaP cells expressed the highest levels of ET_BR mRNA and protein, so ET_BR expression levels of PC3 cells were compared with LNCaP cells.

PC3 cells were grown on 25-mm diameter glass coverslips and kept in DMEM F12 medium for 24 h. Subsequently, the cells were washed with PBS and loaded with 2 µM Fluor-4 acetoxymethyl ester (Fluo4/AM; Thermo Fisher Scientific, Inc.) at room temperature for 15 min. Subsequently, the cells were maintained in Hank's Balanced Salt Solution at a final volume of 500 µl containing 142 mM NaCl, 5.6 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 0.34 mM Na₂HPO₄, 0.44 mM KH₂PO₄, 4.2 mM NaHCO₃, 10 mM HEPES and 5.6 mM glucose. On the one hand, 100 nM ET-1 was added at 60 sec after reaction was initiated with Fluo4/AM, at room temperature, to cells previously incubated with DMSO or with selective antagonists for endothelin receptors, at 37°C for 30 min. In all experiments DMSO (0.1%) was used. On the other hand, 17 mM carbachol (positive control) was added at 245 sec after reaction was initiated with Fluo4AM, at room temperature, to cells preincubated with BQ123 at 37°C for 30 min. The kinetics recording was carried out for 300 sec and the fluorescence intensity was measured in an AxioCam MRm (Carl Zeiss AG) coupled to a Carl Zeiss AG inverted fluorescence microscope AxioVert. A1 equipped for epifluorescence and the ImageJ 1.51w software (National Institutes of Health) was used.

PC3 and LNCaP cells were grown on 12-mm diameter glass coverslips seeded at a confluence of 60–70%. After 24–48 h, the cells were fixed with a fixing solution (4% paraformaldehyde) for 30 min at room temperature, washed and blocked with 3% BSA (Winkler, Ltd.) in PBS-glycine for 30 min at room temperature. Cells were incubated overnight at 4°C with the primary antibodies against ET_AR (dilution, 1:200; cat. no. PA3-065; Thermo Fisher Scientific, Inc.) and ET_BR (dilution, 1:200; cat. no. PA3-066; Thermo Fisher Scientific, Inc.), washed and incubated for 30 min at 37°C with the secondary antibody Alexa Fluor 594 (dilution, 1:500; cat. no. A21207; Thermo Fisher Scientific, Inc.). DAPI (dilution, 1:10,000; cat. no. sc3598; Santa Cruz Biotechnology, Inc.) was used for nuclear staining for 7 min at room temperature. The images were obtained using a Leica D2500 fluorescence microscope (Leica Microsystems GmbH).

Proteins obtained from cell lines were extracted using RIPA buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% v/v NP-40, 1% w/v sodium deoxycholate, 2.5 mM Na₃PO₄, 1 mM b-glycerophosphate and 1 mM Na₃VO₄, pH 7.4) with a protease inhibitor cocktail (Roche Diagnostics). The homogenate was centrifuged at 16,708 × g for 15 min at 4°C. Finally, the supernatant was quantified using a Bradford assay. A total of 25 µg of protein was loaded per lane, separated by SDS-PAGE (10 or 12% polyacrylamide for ET_AR and ET_BR), and transferred to a nitrocellulose membrane, except for detection of 3β HSD2, for which protein was transferred to a PVDF membrane. The membranes were blocked at room temperature with 5% BSA (Winkler, Ltd.) or milk of 0.2% TBS-Tween for 90 min and exposed to the primary antibody overnight at 4°C. After washing, the binding of the primary antibodies was detected with secondary antibodies conjugated with the HRP enzyme for 90 min at room temperature, and detected using EZ-ECL chemiluminescence kit (Biological Industries) and a Vilber Lourmat equipment (Fusion FX5-XT 826.WL/superbright serial number 15200393; Vilber). Primary and secondary antibodies for western blotting are shown in Tables II and III, respectively. The densitometric analysis of western blot bands was performed using ImageJ v1.51 software (National Institutes of Health).

PC3 cells were grown on 6-well plates in RPMI medium free of phenol red with 10% androgen-free FBS. After 24 h, DMSO (control) or endothelin receptor antagonist was added for 30 min at 37°C, and then 100 nM ET-1 was added every 24 h at 37°C. In all experiments, DMSO (0.1%) was used. After 96 h, the culture medium was removed and centrifuged at 1,000 × g for 5 min at room temperature. The number of viable cells was quantified in each condition. Parameter Testosterone Assay (cat. no. KGE010; R&D Systems, Inc.) was used for T determination in culture supernatant according to the manufacturer's protocols. The assay was based on a competitive binding technique. A total of 50 µl of a monoclonal antibody specific for T (excluding non-specific binding wells) was added for binding the anti-mouse antibody coated microplate and incubated with shaking for 1 h at room temperature. After three washes with wash buffer (400 µl), 100 µl calibrator (non-specific binding wells and B0, zero standard), 100 µl standard, control or sample (remaining wells) were added. Subsequently, 50 µl conjugated T was incorporated into each well, and incubated for 3 h at room temperature on a horizontal orbital microplate shaker at 37 × g After three washes with wash buffer, 200 µl of the substrate were added for 30 min at room temperature. Afterwards, 50 µl of the stop solution was added to stop the reaction. Finally, the optical density was determined within 30 min by spectrophotometry at 450 nm using the BioTeK Synergy HT plate reader (BioTek Instruments, Inc.).

Plots were obtained using the GraphPad Prism 7.1 software (GraphPad Software, Inc.). Data are presented as the mean ± SD of at least three independent experiments for RT-qPCR and western blotting. Mann Whitney or Kruskal-Wallis (Dunn's multiple comparisons test) test was carried out. The box plots represent the signal intensity of ET_AR and ET_BR in each group. The points outside the box represent the outliers. The number of samples analyzed was 32 for low GS (<7), 14 for intermediate GS (=7) and 11 for high GS (>7). The bar shows the mean ± SD. P≤0.05 was considered to indicate a statistically significant difference.

In the present study, quantitative immunohistochemistry for ET_AR and ET_BR was performed on constructed TMAs. Intracellular staining of ET_AR and ET_BR was observed in PCa samples (Fig. 1A and C). Furthermore, semi-quantification analysis indicated that the intensity of ET_AR immunostaining was significantly higher in samples with low GS (Kruskal-Wallis test; P<0.05) compared with in samples with intermediate GS (Fig. 1A and B). No changes in ET_BR were observed to be associated with GS in primary tumor samples (Fig. 1C and D). However, in benign prostatic hyperplasia samples, used as a non-neoplastic control, ET_AR was revealed to be located in epithelial and stromal cells (Fig. S1A), and ET_BR was revealed to be primarily located in epithelial cells (Fig. S1B).

Basal expression levels of ET-1, ET_AR and ET_BR in LNCaP and PC3 cells were determined using RT-qPCR and western blotting. In addition, ET_AR and ET_BR levels were also determined by immunofluorescence. PC3 cells were demonstrated to exhibit the highest levels of ET_AR and ET-1 expression at the mRNA (ET_AR; Mann-Whitney test; P=0.05; Fig. 2A; ET-1; Mann-Whitney test; P=0.05; Fig. 3A) and protein levels (ET_AR; Mann-Whitney test; P<0.05; Fig. 2B and E; ET-1 Mann-Whitney; P=0.05; Fig. 3B). By contrast, LNCaP cells were revealed to express increased levels of ET_BR mRNA (ET_BR; Mann-Whitney test; P=0.05; Fig. 2C) and protein (ET_BR; Mann-Whitney test; P=0.05; Fig. 2D and F) compared with PC3 cells. Negative controls are shown in Fig. 2G.

Since ET_AR is a Gq protein-coupled receptor (15,17), the activity of this receptor was evaluated via intracellular calcium measurement. The results in Fig. 3C indicated that activation of ET_AR by ET-1 induced an intracellular Ca²⁺ transient signal, which was reflected as an increase in relative fluorescence intensity (F/F0) when adding ET-1 at 60 sec compared with carbachol (positive control; Fig. 3D), suggesting the exit of calcium from the endoplasmic reticulum and/or the subsequent entry of calcium from the extracellular medium. This effect was inhibited by a selective antagonist for ET_AR, BQ123 (Fig. 3D), and the non-selective antagonist Bosentan (Fig. 3E).

In the present study, the effect of ET-1 on the enzymes of the steroidogenic pathway in PC3 cells was investigated. The effect was not determined in LNCaP cells since PC3 cells expressed higher levels of ET-1 and ET_AR. Therefore, by using PC3 cells, the effects could be observed more clearly. It was demonstrated that ET-1 treatment increased the expression levels of CyP11A1 (Kruskal-Wallis test; P<0.05; Fig. 4A and B-D), 3β HSD2 (Kruskal-Wallis test; P<0.05; Fig. 4A and H-J) and AR (Kruskal-Wallis test; P<0.05; Fig. 5A-C) in PC3 cells compared with the control without ET-1. Blocking ET-1 receptors with BQ123 and/or Bosentan prevented the stimulatory effect of ET-1 on the expression of CyP11A1 (ET-1/BQ123 + ET-1; Kruskal-Wallis test; P<0.001; Fig. 4B), 3β HSD2 (ET-1/Bosentan + ET-1; Kruskal-Wallis test; P<0.05; Fig. 4J) and AR (ET-1/BQ123 + ET-1; Kruskal-Wallis test; P<0.01; Fig. 5B; ET-1/Bosentan + ET-1; Kruskal-Wallis test; P<0.05; Fig. 5D). Additionally, blocking ET_BR with BQ788 decreased, with respect to ET-1, the expression levels of 3β HSD2 (ET-1/BQ788 + ET-1; Kruskal-Wallis test; P<0.05; Fig. 4I). There were no changes observed in CyP17A1 and aldo-keto reductase family member C2 (AKR1C2) protein expression in the three antagonist treatment groups, with respect to ET-1 (Fig. 4E-G and K-M). The aforementioned results demonstrated that ET-1 may regulate the protein expression levels of CyP11A1, 3β HSD2 and AR via ET_AR or ET_BR.

To determine if the increase in the expression levels of steroidogenic enzymes was associated with the steroidogenic process, T production was evaluated in PC3 cells. Blocking of endothelin receptors was indicated to induce a decrease in T concentration (ET-1/BQ123 + ET-1; Kruskal-Wallis test; P<0.05; Fig. 5E; ET-1/BQ788 + ET-1; Kruskal-Wallis test; P<0.05; Fig. 5F).