Galectin-3 Is Implicated in Tumor Progression and Resistance to Anti-androgen Drug Through Regulation of Androgen Receptor Signaling in Prostate Cancer

Materials and Methods

Cell culture. The prostate cancer androgen-dependent cell line LNCaP and LNCaP cells transfected with Gal-3 (LNCaP-Gal-3) (10, 11), and the metastatic type of the androgen-independent cell lines DU145 and PC3 that were used in this study were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). All cells were maintained in RPMI-1640 medium containing 2 mmol/l glutamine, penicillin, and streptomycin (Life Technologies, Carlsbad, CA, USA) and 10% (v/v) fetal bovine serum in 5% CO₂ at 37°C using standard cell culture procedures (10). At 24 h before treatment with the anti-androgen drug MDV3100 (Santa Cruz Biotechnology, Dallas, TX, USA) or with bicalutamide (Wako Pure Chemical, Osaka, Japan), the cells were transferred to RPMI-1640 phenol red-free medium for 24 h. 10 μM MDV3100 and 10 μM bicalutamide were dissolved in dimethyl sulfoxide (Kanto Chemical Co, Inc, Tokyo, Japan) and 1 nM dihydrotestosterone (DHT) (Sigma Aldrich, St. Louis, MO, USA) in methanol.

Primary antibodies. The primary antibodies used in this study were: anti-androgen receptor (AR) (clone number AR441i DAKO North America, Inc. Carpinteria, CA, USA) and TIB166 monoclonal rat anti-gal-3 from the ATCC (10, 11), and anti-human β-actin antibodies (Clone number A5441); (Sigma-Aldrich).

Real-time quantitative polymerase chain reaction (qPCR). Total RNA was extracted from cultured cells using the RNeasy micro kit (Qiagen, Hilden, Germany), as previously described (12). The appropriate dilutions of each single-stranded cDNA were prepared for subsequent PCR amplification, and reactions using β-actin were monitored as a quantitative control. Each sample was analyzed in triplicate for each primer pair. The relative expression levels of prostate-specific antigen (PSA), transmembrane protease, serine 2 (TMPRSS2), and galectin-3 (LGALS3) normalized to β-actin (ACTB) were calculated using the relative standard curve method and Light Cycler Software Ver.3.5 (Roche Diagnostics, Madison, WI, USA). The primer sequences were as follows: PSA: F-GCATGGGATGGGGATGAAGTAAG, R-CATCAAATCTGAGGGTTGTCTGGA; TMPRSS2: F-CCATTTGCAGGATCTGTCTG, R-GGATGTGTCTTGGGGAGCAA; LGALS3: F-GCCTTCCACTTTAACCCACG, R-ACTGCAACCTTGAAGTGGTCA; and ACTB: F-GAGGTGATAGCATTGCTTTCG, R-CAAGTCAGTGTACAGGTAAGC.

Western blot analysis. Cell lysates were prepared using the Mammalian Cell Extraction kit (Bio Vision, Mountain View, CA, USA). After centrifugation at 180 × g for 10 min, the supernatant was collected as the total protein extract and stored at −80°C. Protein concentration was then measured using a protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and electrophoretically transferred to a polyvinylidene difluoride membrane. The membrane was then blocked with Block Ace (Dainippon Pharmaceutical, Osaka, Japan) for 60 min and incubated with primary antibody at 4°C overnight. Blots were developed by chemiluminescence using a horseradish peroxidase-coupled secondary antibody (Invitrogen) and the ECL-Plus Kit (GE Healthcare, Piscataway, NJ, USA) (10).

Cell migration and invasion assays. Invasion and migration assays of LNCaP with and without galectin-3 expression were performed using the xCELLigence RTCA Analyzer (Roche Applied Science, Mannheim, Germany) in a humidified atmosphere of 5% CO₂ and 95% air at 37°C (13). The medium of cells in a cell invasion and migration (CIM) plate (ACEA Biosciences, San Diego, CA, USA) containing cells at a density of 4×10⁴ cells per well in transfection solution was replaced with fresh RPMI 1640 phenol red-free medium supplemented with 5% FBS. The lower well was filled with 160 μl of phenol red-free with 10% FBS. The invasion assay was tested on a tumor invasion system in Matrigel (BD Biosciences, Bedford, MA, USA), diluted 1:40 with phenol red-free medium (as detailed by Roche in the application notes) coated onto 16-well plates. Cells were further incubated for measurement of changes in the cell index over time intervals between 0 to 60 h. The cell index shows cell invasion or migration status based on the measured electrical impedance.

Luciferase assay. LNCaP and LNCaP-Gal-3 cells were seeded in 12-well plates (2×10⁵ cells/well) after counting. After 24 h, the medium was changed to phenol red-free RPMI-1640 with 5% charcoal-stripped FBS. Cells were then co-transfected with pGL4.74 firefly luciferase reporter plasmid and Renilla luciferase PSA plasmid (14) using Lipofectamine® 2000 (Invitrogen). After 24 h of transfection, the medium was changed to phenol red-free RPMI-1640 supplemented with DHT and MDV3100 for 24 h. Subsequently, cell lysates were prepared, and luciferase activity was measured using the Dual-Luciferase Reporter Assay system (Promega, Madison, WI, USA). Firefly luciferase activity was normalized to Renilla luciferase activity (15, 16).

Spheroid production. Spheroids were prepared from single-cell suspensions of prostate cancer cell lines, as previously described (17-19). In brief, cells at a density of 0.5×10⁴ cells per ml in RPMI-1640 phenol red-free medium supplemented with 5% (v/v) charcoal-stripped FBS were placed in the wells of a 96-well ultra-low attachment (ULA) plate (17-19). The ULA plate was then centrifuged at 100 × g for 3 min at room temperature. After 72-h incubation, when spheroids had formed, the medium was exchanged with fresh RPMI phenol red-free medium supplemented with DHT and MDV3100 (150 μl/well) followed by medium exchange every 48 h with care not to disturb the spheroids. Spheroids were cultured for 7 days before final analysis (20, 21). Images of all spheroids were taken daily for growth determination and cytotoxicity experiments using an Olympus IX53 microscope with a ×10 objective and an attached Olympus DP22/27 (Olympus Corporation, Tokyo, Japan) camera. Spheroid size was measured by open-source software ImageJ (ImageJ, version 1.48; NIH, Bethesda, MD, USA). We obtained descriptive data by Microsoft Excel (Microsoft Corporation, Redmond, WA, USA). and statistical analysis was performed by StatView 5.0 (SAS Institute, Inc., Cary, NC, USA).

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Figure 1.

Endogenous galectin-3 expression in prostate cancer cell lines. Endogenous expressions of galectin-3 mRNA and protein in the androgen-dependent LNCaP and the androgen-independent DU145 and PC3 cell lines were examined using quantitative real-time polymerase chain reaction (A) and western blot analysis (B). Data in (A) are the means±SE.

Mouse xenograft model. BALB/c nude mice that lack a thymus and are unable to produce T-cells were used (Charles River Laboratory, Wilmington, DE, USA). LNCaP and LNCaP-Gal3 cells (8×10⁶) suspended in 200 μl of Dulbecco's phosphate-buffered saline with 50% Matrigel (BD Biosciences) were subcutaneously inoculated into the right flank of all mice. We then divided mice into four groups: LNCaP and LNCaP-gal-3 for non-castrated groups, LNCaP and LNCaP-gal-3 for castrated groups. When each xenograft tumor reached a volume of approximately 200 mm³, the mice of the castrated groups were castrated by orchiectomy. Further growth of the tumors was monitored in all groups (22-24).

Microarray analysis. Total RNA from LNCaP and LNCaP-Gal-3 cells exposed to DHT, MDV3100, or bicalutamide for 24 h was extracted using an RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. RNA was quantified using a NanoDrop-1000 spectrophotometer, and quality was monitored with an Agilent 2100 Bioanalyzer (G2938C; Agilent Technologies, Palo Alto, CA, USA). Cyanine-3 (Cy3)-labeled cRNA was prepared from 0.2 μg RNA using the Low Input Quick Amp One-Color Labeling Kit (Agilent) according to the manufacturer's instructions. Dye incorporation and cRNA yield were checked with a NanoDrop ND-1000 spectrophotometer. Cy3-labelled cRNA (specific activity >15.0 pmol Cy3/μg cRNA) was fragmented at 60°C for 30 min and hybridized to Agilent SurePrint G3 Human GE Oligo Microarrays Ver3.0 (G4858A#072363) for 17 h at 65°C in a rotating Agilent hybridization oven. After hybridization, the microarrays were washed for 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and for 1 min at 37°C with GE Wash buffer 2 (Agilent). After washing, slides were scanned immediately on an Agilent DNA Microarray Scanner (G2505C) using a one-color scan setting for 8×60 k array slides (scan area 61 mm × 21.6 mm, scan resolution 3 μm, dye channel set to green). The scanned images were analyzed with Feature Extraction Software 10.7 (Agilent) using default parameters (protocol GE1-107_Sep09 and Grid: 072363_D_F_20150612). The microarray data across all chips and genes were analyzed using GeneSpring (version 13.0) and normalized by 75 percentile shift (25, 26).

Statistical analysis. All statistical analyses were performed using StatView 5.0 (SAS Institute, Inc., Cary, NC, USA). Parameters were compared by Student's t-test, and ANOVA was used to compare the groups with more than two parameters. A difference of p<0.05 was considered significant.