Human prostate cancer cell line hormone-independent DU145, was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and grown in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with glutamine, essential and non-essential amino acids, vitamins, antibiotics, and 10% heat-inactivated fetal bovine serum (Gibco/Invitrogen, Grand Island, NY, USA). The cells were maintained at 37°C in a humidified atmosphere of 5% CO₂. All experiments were performed with cultures grown for no longer than 6 weeks after recovery from frozen stocks.

Thirty-eight-week-old male Foxn^nu/nu mice were purchased by Harlan, San Pietro al Natisone (Italy). Mice were housed 5 per cage and maintained on a 12-h light:12-h dark cycle (lights on at 7.00 a.m.) in a temperature-controlled room (22±2°C) and with food and water ad libitum. The experimental protocols were in compliance with the European Community Council directive (86/609/EEC). After 1-week of acclimation to the housing conditions, mice were injected subcutaneously (s.c.) with a suspension of DU145 cells (1×10⁶ cells/mouse in the right hind limb).

After 1 week, mice were randomized for tumor volume and weight according to Fisher’s exact test. Tumor growth was measured every 2–3 days with a digital caliper 2BIOL (Besozzo, Italy) and expressed as volume, according to the formula: V = (a × b2)/2 (a = the largest superficial diameter and b = the smallest superficial diameter). Animals, were sacrificed with cervical dislocation when they reached the cut-off of 1500 mm³ or when presenting signs of pain.

When the tumor mass was palpable and measurable, mice were treated with S-propranolol hydrochloride, a non-selective β-adrenoreceptor antagonist (Sigma-Aldrich, St. Louis, MO, USA) (2 mg/kg/day), and with 28 μM of norepinephrine tartrate (Sigma-Aldrich) via intra-peritoneal injection, or either combination every day until they reached the cut-off.

DU145 cells were seeded at the density of 40×10³ cells per well into a 24-multiwell plate, and cultured in their respective culture medium supplemented with 1% FBS. At the time of confluence, the cells were incubated in the absence or presence of norepinephrine and propranolol and their combination at same concentration (10 μM) for 24 and 48 h. Then, a slit was made horizontally with a white tip at the center of each confluent well, the medium was changed after gentle rinse and cells were cultured for 24 h with or without norepinephrine and propranolol. Cell invasion on the slit of the confluent well, was assessed at time 0, 24 and 48 h in each condition, by light microscopy.

In vitro cell migration was assayed in 24-well cell culture plates using inserts with 8-μm pore membranes (Costar, San Diego, CA, USA) according to the method described by Katayama et al (11). In brief, membrane was precoated with 20 μl per insert of Matrigel (BD Pharmingen, Bedford, MA, USA) diluted 1:1 by RPMI-1640. DU145 cell line (1×10⁵ per ml) were suspended in 500 μl migration buffer (DMEM/0.5% bovine serum albumin) per well and loaded into the upper compartment of the chamber. The lower compartment of the chamber was loaded with 10–400 ng/ml norepinephrine (Sigma, St. Louis, MO, USA) in 500 μl migration buffer. After incubation at 37°C for 24 h, the cells on the lower surface of membrane were fixed in 70% ethanol, stained with hematoxylin, and counted under a microscope (Olympus, Tokyo, Japan). Each value was expressed as a mean number of five different fields. Three experiments were performed independently and the results were compared.

Cell viability was evaluated using 3-(4, 5-dimethyl (thia zol-2-yl)-2, 5-diphenyltetrazolium bromide) (MTT), the reagent that measures the metabolic activity of cells. The stock solution of MTT (5 mg/ml) was prepared in phosphate-buffered saline (PBS) and stored in the dark at 4°C. A 100-μl aliquot of a dilution prepared in culture medium (1 mg/ml final) was filtered (0.22 μm) and added to the cells growing with/without CBGM. After 3 h of incubation the supernatant was removed. Formazan crystals in viable cells were dissolved in ethanol (96% v/v) and absorbance was measured by the Multiskan^® FC microplate photometer (Thermo Scientific, USA) at 540 nm. After the treatment described above, MTT (Sigma) was added to a final concentration of 0.5 mg/ml and incubated for 4 h at 37 °C. The culture medium was then removed, and the remaining blue precipitate was solubilized in DMSO followed by an absorbance reading at 570 nm in a plate reader using 630 nm as a reference (Spectra Max 340; Molecular Devices, Sunnyvale, CA, USA). This reading was divided by the adjusted absorbance reading of untreated cells in control wells to obtain the percentage of cell survival. All experiments were carried out in triplicates.

Sections were deparaffinized in xylene for 10 min each and then were rehydrated through graded alcohols. To inhibit endogenous peroxide activity, sections were rinsed in mixture of 100% methanol and 0.3% hydrogen peroxide for 40 min. Then, they were put in a microwave oven in a jar filled with 10 mmol/l sodium citrate buffer (pH 6.0) for 10 min. Following this treatment, sections were allowed to cool at room temperature. Sections were incubated with normal goat serum (Zymed, San Diego, CA, USA) at RT for 20 min and then incubated with chicken anti-MMP-2, MMP-9 5–15 μg/ml (R&D Systems, Minneapolis, MN, USA) and pan cytokeratin antibody 1:50 (Dako, Italy) at room temperature for 1 h. A negative control was used in all experiments without the primary antibody. After the period of incubation, sections were washed three times with PBS. Then, they were incubated with the linking reagent (biotin-labeled affinity purified antibody; KPL, Gaithersburg, MD, USA) at room temperature for 1 h. After three washes with PBS, the sections were incubated with a complex of avidin DH and biotinylated enzyme (Zymed) at room temperature for 30 min. Then, sections were washed three times with PBS again and incubated with a mixture of an equal volume of 0.02% hydrogen peroxide and diaminobenzidine tetrahydrochloride (Dako, Italy) for 1 min in the dark. After chromogenic development, sections were washed in water and counter-stained with hematoxylin. The stained slides were investigated independently by two pathologists who had no knowledge of the clinical parameters and outcomes. For microscope analysis of the slides, they each selected five high-powered fields whereby each field contained >200 tumor cells, and counted both positive and negative cancer cells. In total, >1,000 tumor cells were counted. We calculated the average of 10 readings of positive cells as a percentage of all the cells to determine staining scores. We judged the expression of MMP2 and MMP9 and Pan cytokeratin. The cases were graded as either positive of marker expression (MMP-2, MMP-9, Pan cytokeratin >30%) or negative cases (<30% positive tumor cells) for the subsequent statistical analyses.

DU145 cells were treated with 10 μM norepinephrine (NE10), 10 μM propranolol (P10) and N10-P10 combination. After 48 h, DU145 cells were fixed in PBS 4% paraformaldehyde then permeabilized for 5 min with PBS 1% Triton. Immunostaining was carried out by incubation with anti-E-cadherin and anti-vimentin antibodies 1:1,000 followed by revelation using Alexa Fluor 633-conjugated anti-rabbit immunoglobulin (Ig)G antibodies and Alexa Fluor 488-conjugated anti-rabbit IgG antibodies, respectively (Jackson Immunoresearch Laboratories, West Grove, PA, USA) at a dilution of 1:1,000 for 1 h. The cells were analyzed by an LSM-410 Zeiss confocal microscope.

Cells (DU145) grown in monolayers were harvested at early confluence. Total RNA was extracted from cultured cells using TRIzol reagent. The RNA concentration was measured by Biophotometer (Eppendorf) at 260 nm. Then 1 μl of the product was subjected to PCR amplification using Mastercycler Personal (Eppendorf, Italy) according to the manufacturer’s instructions. Semiquantitative RT-PCR was performed starting with 1cDNA, followed by specific gene product amplification with One-Step RT-PCR kit (Invitrogen, Burlington, ON, Canada). PCR cycling conditions for b2-AR mRNA were 95°C for 5 min; then 32 cycles of 94°C for 1 min, 54°C for 1 min, 72°C for 1 min; and finally extension at 72°C for 7 min. The primer sequences of human b2-AR were designed as follows: forward 5′-ACGCAGCAAAGGGACGAG-3′ and reverse 5′-CACACCATCAGAATGATCAC-3′. Experiments were performed three times, for densitometric analysis of each set of data. PCR products were resolved by electrophoresis in 1–2% agarose gels and visualized by ethidium bromide staining. The densities of the b2-AR bands were divided by those of the GAPDH bands.

Cell lysates were prepared by adding ice-cold lysis buffer [0.5% Triton X-100, 50 mmol/l Tris (pH 7.2), 140 mmol/l NaCl, 10 mmol/l EDTA, 50 mmol/lNaF, 1 mmol/l Na₃VO₄] containing the protease inhibitor cocktail Complete Mini (Roche, Mannheim, Germany). Protein samples were mixed with an equal volume of 2% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, boiled for 5 min, and then separated using 10% SDS-PAGE. After electrophoresis, proteins were transferred to PVDF membranes by semi-dry electrophoretic transfer. The membranes were blocked in 5% dry milk, rinsed and then incubated with primary antibody of p53 (Sigma) overnight at 4°C. The primary antibody was removed, membranes were washed four times and followed by HRP-conjugated secondary antibody. Detection was then performed using an enhanced chemiluminescence kit and exposed to X-ray film. Anti α-actin was used as loading control.

Normally distributed data are presented as mean ± SEM. Two-way ANOVA and Bonferroni post hoc test, were used to examine the significance of differences among groups (Graph pad Prism 5.0).

We first determined whether norepinephrine increases the proliferation of human prostate cancer cells DU145 by performing in vitro assays. Proliferation test was performed by wound healing assay on cells treated with norepinephrine at 10 μM (NE10), propranolol at 10 μM (P10) and their combination at the same concentration (NE+P10). Our results indicate that norepinephrine (Fig. 1B, F and J) increased the proliferation of DU145 at 48 h compared to controls (Fig. 1A, E and I), to propranolol (Fig. 1C, G and K) and the combination (Fig. 1D, H and L). These results were also confirmed by MTT assay (data not shown).

Since it has been demonstrated that the expression of β2-adrenoreceptor (β2-AR) in DU145 cell line is overexpressed when these cells are treated with norepinephrine, we performed expression studies of β2-AR on RNAs and proteins extracted from DU145 cells treated with 10 μM norepinephrine (NE10), 10 μM propranolol (P10) and their combination (NE+P10). RT-PCR analysis and western blotting results (Fig. 2A and B) showed that norepinephrine causes approximately 3-fold increase of β2-AR protein expression paralleled by enhanced expression of its mRNA. Propanolol almost completely antagonized this effect in DU145 cells.

We investigated the mechanisms of the in vivo acquisition of tumor cell migratory properties induced by norepinephrine treatment and its possible correlation to EMT occurrence. Therefore, we evaluated both cell expression and localization of E-cadherin and vimentin in prostate cancer DU145 cells. Confocal microscopy showed that 10 μM norepinephrine (NE10) (Fig. 3B) reduced E-cadherin expression and caused a delocalization of E-cadherin from cell membrane to the cytoplasm. The addition of 10 μM propranolol (P10) 30 min before NE10 treatment completely antagonized this effect (Fig. 3D). Moreover, NE10 increased the expression of vimentin and P10 antagonized again this effect together with polar localization of vimentin on the inner side of cell membrane (Fig. 3D). Interestingly, P10 alone had slight effects on E-cadherin expression and localization if compared to untreated cells while inducing a decreased expression and delocalization of vimentin (Fig. 3C). These effects suggest that P10 antagonized the EMT occurrence induced by NE10 that mimics an in vivo stress condition.

In order to study the role of norepinephrine on in vivo tumor growth, we generated a mouse model of prostate cancer by injection of DU145 cells subcutaneously into the right flank of mice. When tumors reached ~30–60 mm³, 2 weeks following cell injection, the mice were randomized into four groups: a) normal saline (control); b) 28 mM NE; c) 2 mg/kg propranolol; d) 28 mM NE and 2 mg/kg propanol. Tumor volumes were monitored once a week by using a digital caliper. Therapy was continued for 3 weeks. We also monitored the body weight of mice twice a week until the end of treatment. No difference was observed between the body weight of the different animal groups, indicating that treatments of mice with drugs is not associated with toxic effects. Mice were sacrificed at the end of treatment. Differently from that demonstrated by in vitro assays, in vivo experiments showed that norepinephrine is not able to influence the tumor growth of mice (P>0.05) (Fig. 4). In fact, NE induced an apparent but not significant decrease of tumor volume and this effect was almost completely antagonized again by the treatment with propranolol.

In order to show whether norepinephrine in vivo treatment induces the formation of metastasis foci in mice we performed immunostaining for pan-cytokeratin in inguinal lymph nodes in order to find epithelial cells that normally are absent. Our results show a significant increase of the positivity of lymph nodes for cells expressing CK in all the NE-treated animals suggesting a strong increase of metastatic cells and this effect was potently antagonized by propranolol in the combination-treated animals (Fig. 5G, H and I). Moreover, overexpression of MMP2 and MMP9 in NE treated tumor samples (Fig. 5B and E) was observed compared to those of controls (Fig. 5A and D) and of mice treated with combination of the two substances (Fig. 5C and F). These data suggest an in vivo EMT effect induced by NE, with consequent increase of the metastatic processes, is not paralleled by tumor growth promotion.