Human prostate tumor cell lines PC-3, DU-145 and LNCaP were obtained from DSMZ (Braunschweig, Germany). Normal adult prostatic epithelial PNT-2 cells were purchased from Sigma-Aldrich, München, Germany. Tumor and normal cells were grown and subcultured in RPMI 1640 (Gibco/Invitrogen; Karlsruhe, Germany). The medium contained 10% fetal calf serum (FCS), 2% HEPES-buffer (1 M, pH 7.4), 2% glutamine and 1% penicillin/streptomycin. Subcultures from passages 7-11 were selected for experimental use.
Human endothelial cells (HUVEC) were isolated from human umbilical veins and harvested by enzymatic treatment with chymotrypsin. HUVEC were grown in Medium 199 (M199; Biozol, Munich, Germany), supplemented with 10% FCS, 10% pooled human serum, 20 μg/ml endothelial cell growth factor (Boehringer, Mannheim, Germany), 0.1% heparin, 100 ng/ml gentamycin and 20 mM HEPES-buffer (pH 7.4). Subcultures from passages 2-6 were selected for experimental use.
AEE788 (provided by Novartis Pharma AG, Basel, Switzerland) was dissolved in DMSO as a 10 mM stock solution and stored in aliquots at -20°C. Prior to the experiments, AEE788 was diluted in cell culture medium to 1 μM. RAD001 (provided by Novartis Pharma AG, Basel, Switzerland) was dissolved in DMSO as a 10 mM stock solution and stored in aliquots at -20°C. Prior to the experiments, RAD001 was diluted in cell culture medium to 1 nM. VPA (gift from G. L. Pharma GmbH, Lannach, Austria) was used at a final concentration of 1 mM. Prostate carcinoma cells were treated either with 1 μM AEE788 or 1 nM RAD001 for 24 h or with 1 mM VPA for 3 days, or with all compounds in combination, AEE788+RAD001+VPA. AEE788 and RAD001 were then added for the final 24 h. Controls remained untreated. To exclude toxic effects of the compounds, cell viability was determined by trypan blue (Gibco/Invitrogen). For apoptosis detection the expression of Annexin V/propidium iodide (PI) was evaluated using the Annexin V-FITC Apoptosis Detection kit (BD Pharmingen, Heidelberg, Germany). Tumor cells were washed twice with PBS, and then incubated with 5 μl of Annexin V-FITC and 5 μl of PI in the dark for 15 min at RT. Cells were analyzed on a FACScalibur (BD Biosciences, Heidelberg, Germany). The percentage of apoptotic cells (early and late) in each quadrant was calculated using CellQuest software (BD Biosciences).
Tumor cell adhesion
To analyze tumor cell adhesion, HUVEC were transferred to 6-well multiplates (Falcon Primaria; BD Biosciences) in complete HUVEC-medium. When confluency was reached, PC-3, DU-145 or LNCaP cells were detached from the culture flasks by accutase treatment (PAA Laboratories, Cölbe, Germany) and 0.5 × 106 cells were then added to the HUVEC monolayer for 1 h, 2 h or 4 h. Subsequently, non-adherent tumor cells were washed off using warmed (37°C) Medium 199. The remaining cells were fixed with 1% glutaraldehyde. Adherent tumor cells, which appeared translucent with a rounded morphology, were counted in five different fields of a defined size (5 × 0.25 mm2) using a phase contrast microscope and the mean cellular adhesion rate was calculated.
Attachment to extracellular matrix components
6-well plates were coated with collagen G (extracted from calfskin, consisting of 90% collagen type I and 10% collagen type III; Seromed; diluted to 400 μg/ml in PBS), laminin (derived from the Engelbreth-Holm-Swarm mouse tumor; BD Biosciences; diluted to 50 μg/ml in PBS), or fibronectin (derived from human plasma; BD Biosciences; diluted to 50 μg/ml in PBS) overnight. Unspecific cell binding was evaluated by culture plates treated with Poly-D-Lysin (Nunc, Wiesbaden, Germany). Plastic dishes served as the background control. Plates were washed with 1% BSA (bovine serum albumin) in PBS to block nonspecific cell adhesion. Thereafter, 0.5 × 106 tumor cells were added to each well for 60 min. Subsequently, non-adherent tumor cells were washed off, the remaining adherent cells were fixed with 1% glutaraldehyde and counted microscopically. The mean cellular adhesion rate, defined by adherent cellscoated well - adherent cellsbackground, was calculated from five different observation fields.
Cell migration and invasion
Serum induced cell migration was examined using 6-well Transwell chambers (Greiner, Frickenhausen, Germany) with 8- μm pores, precoated with collagen (400 μg/ml). 0.5 × 106 PC-3 or LNCaP cells/ml were incubated with VPA, AEE788, RAD001, or the drug combination. Controls remained untreated. To evaluate cell migration, cells were then placed in the upper chamber for 20 h in serum-free medium. The lower chamber contained 10% serum. After incubation, the upper surface of the Transwell membrane was wiped gently with a cotton swab to remove non-migrating cells. Cells which migrated to the lower surface of the membrane were stained using hematoxylin and counted. Graphical results are shown as % inhibition as compared to the 100% untreated control.
Measurement of tumor cell growth
Cell proliferation was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye reduction assay (Roche Diagnostics, Penzberg, Germany). Treated versus non-treated PC-3, DU-145 or LNCaP cells (100 μl, 1 × 104 cells/ml) were seeded onto 96-well tissue culture plates. After 24, 48 and 72 h, MTT (0.5 mg/ml) was added for an additional 4 h. Thereafter, cells were lysed in a buffer containing 10% SDS in 0.01 M HCl. The plates were allowed to stand overnight at 37°C, 5% CO2. Absorbance at 570 nm was determined for each well using a microplate ELISA reader. Each experiment was done in triplicate. After subtracting background absorbance, results were expressed as mean cell number.
Cell cycle analysis
PC-3, DU-145 or LNCaP cells were grown to 70% confluency and then treated with AEE788, RAD001 or with VPA or with all compounds in combination (controls remained untreated). Cell cycle analyses were carried out after 24 h. After 24 h tumor cell populations were stained with propidium iodide using a Cycle TEST PLUS DNA Reagent Kit (Becton Dickinson) and then subjected to flow cytometry with a FACScan flow cytometer (Becton Dickinson). 10,000 events were collected from each sample. Data acquisition was carried out using Cell-Quest software and cell cycle distribution calculated using the ModFit software (Becton Dickinson). The number of gated cells in G1, G2/M or S-phase was presented as %.
Integrin surface expression
PC-3 or LNCaP cells were washed in blocking solution (PBS, 0.5% BSA) and then incubated for 60 min at 4 C with phycoerythrin (PE)-conjugated monoclonal antibodies directed against the following integrin subtypes: Anti-α1 (IgG1; clone SR84), anti-α2 (IgG2a; clone 12F1-H6), anti-α3 (IgG1; clone C3II.1), anti-α4 (IgG1; clone 9F10), anti-α5 (IgG1; clone IIA1), anti-α6 (IgG2a; clone GoH3), anti-β1 (IgG1; clone MAR4), anti-β3 (IgG1; clone VI-PL2) or anti-β4 (IgG2a; clone 439-9B; all: BD Biosciences). Integrin expression of tumor cells was then measured using a FACscan (Becton Dickinson; FL-2H (log) channel histogram analysis; 1 × 104 cells/scan) and expressed as mean fluorescence units. A mouse IgG1-PE (MOPC-21) or IgG2a-PE (G155-178; all: BD Biosciences) was used as an isotype control.
Real Time (RT) qPCR
RT qPCR was also done in triplicate. cDNA-synthesis was performed using 3 μg of total RNA per sample according to the manufacturer's protocol by AffinityScript QPCR cDNA Synthesis Kit (Stratagene, Amsterdam, Netherlands). Quantitative gene expression analysis by Real Time PCR was performed by the M × 3005 p (Stratagene, Amsterdam, Netherlands) using SYBR-Green SuperArray (SABioscience Corporation, USA) and SuperArray primer sets: GAPDH (NM_002046.3, Hs.592355), integrin α1 (ITGA1, NM_181501, Hs.644352), integrin α2 (ITGA2, NM_002203, Hs.482077), integrin α3 (ITGA3, NM_002204, Hs.265829), integrin α5 (ITGA5, NM_002205, Hs.505654), integrin α6 (ITGA6, NM_000210, Hs.133397), integrin β1 (ITGB1, NM_002211, Hs.643813), integrin β3 (ITGB3, NM_000212, Hs.218040), integrin β4 (ITGB4, NM_000213, Hs.632226; all: SABioscience Corporation). Calculation of the relative expression of each gene was done by the ΔΔCt method in the analysis program of SABioscience Corporation. The housekeeping gene GAPDH was used for normalisation.
Western blot analysis
To explore cell cycle regulating proteins as well as the whole cellular integrin level, tumor cell lysates were applied to a 7% polyacrylamide gel and electrophoresed for 90 min at 100 V. The protein was then transferred to nitrocellulose membranes. After blocking with non-fat dry milk for 1 h, the membranes were incubated overnight with monoclonal antibodies directed against cell cycle proteins: Cdk1 (IgG1, clone 1), cdk2 (IgG2a, clone 55), cdk4 (IgG1, clone 97), cyclin B (IgG1, clone 18), cyclin D1 (IgG1, clone G124-326), cyclin E (IgG1, clone HE12), Rb (IgG2a, clone 2), Rb2 (IgG2a, clone 10), p21 (IgG1, clone 2G12), p27 (IgG1, clone 57; all: BD Biosciences). Integrins were analyzed using the monoclonal antibodies listed above. Additionally, integrin-related signaling was explored by anti-integrin-linked kinase (ILK; clone 3), anti-focal adhesion kinase (FAK; clone 77) and anti-phospho-specific FAK (pFAK; pY397; clone 18) antibodies (all: BD Biosciences). HRP-conjugated goat-anti-mouse IgG (Upstate Biotechnology, Lake Placid, NY, USA; dilution 1:5.000) served as the secondary antibody. The membranes were briefly incubated with ECL detection reagent (ECL™, Amersham/GE Healthcare, München, Germany) to visualize the proteins and exposed to an x-ray-film (Hyperfilm™ EC™, Amersham/GE Healthcare). β-actin (1:1.000; Sigma, Taufenkirchen, Germany) served as the internal control.
For control purposes, EGF receptor (EGFr) and mTOR signaling were evaluated. Prostate carcinoma cells were treated with each drug alone or with the triple drug combination as indicated above. Cells were then kept for 2 h in serum-free cell culture medium and subsequently stimulated for 30 min with EGF (100 ng/ml). The following monoclonal antibodies were used: Akt (IgG1, clone 55, dilution 1:500), phospho Akt (pAkt; IgG1, clone 104A282, dilution 1:500), ERK1 (IgG1, clone MK12, dilution 1:5000), ERK2 (IgG2b, clone 33, dilution 1:5000), phospho ERK1/2 (pERK; IgG1, clone 20A, dilution 1:1000), EGFr (IgG1, clone 13/EGFR, dilution 1:500), phospho EGFr (pEGFr; IgG1, clone 74, dilution 1:1000; all: BD Biosciences), p70S6k (IgG, clone 49D7, dilution 1:1000), phospho p70S6k (pp70S6k; IgG, clone 108D2, dilution 1:1000; all: New England Biolabs, Frankfurt, Germany).
All experiments were performed 3-6 times. Statistical significance was investigated by the Wilcoxon-Mann-Whitney-U-test. Differences were considered statistically significant at a p value less than 0.05.