Reagents and antibodies
Pinocembrin (purity≧95%) was purchased from Extrasynthese (Genay, France). Poly-D-lysine, DMSO, Tris–HCl, EDTA, SDS, phenylmethylsulfonyl fluoride, bovine serum albumin (BSA), gelatin, crystal violet, leupeptin, Nonidet P-40, deoxycholic acid, sodium orthovanadate, mouse monoclonal antibody specific for αvβ3 integrin, and a selective αvβ3 integrin antagonist cyclic RGD (cycloRGDfV) peptide were purchased from Sigma-Aldrich (St. Louis, MO, USA); the protein assay kit was obtained from Bio-Rad Laboratories (Hercules, CA, USA). Dulbecco’s phosphate buffer solution (PBS), trypsin-EDTA, and RPMI 1640 medium were purchased from Life Technologies, Inc. (Gibco/BRL, Gaithersburg, MD). Matrigel was purchased from BD Biosciences (Bedford, MA). Antibodies against E-cadherin, N-cadherin, vimentin, FAK, FAK (Tyr397, Tyr576, Tyr925), ERK1/2, ERK1/2 (Thr202/Tyr204), JNK1/2, JNK1/2 (Thr183/Tyr185), p38α, and p38α (Thr180/Tyr182) were purchased from Cell Signaling Tech. (Beverly, MA, USA). NF-κB (p50 and p65), β-actin, and C23 antibodies were purchased from BD Transduction Laboratories (San Diego, CA, USA). An enhanced chemiluminescence (ECL) kit was purchased from Amersham GE Healthcare UK Ltd (Buckinghamshire, England).
Cell culture and pinocembrin treatment
The human retinoblastoma Y-79 cell line was obtained from BCRC (Bioresource Collection and Research Center in Hsin-Chu, Taiwan). Cells were cultured at 37°C in a humidified atmosphere of 5% CO2-95% air. In medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 1 mM sodium pyruvate, and antibiotics (100 U/ml of penicillin and 100 mg/ml of streptomycin). Y-79 cells were grown in suspension at a concentration of 105-106 cells/ml. For all anti-metastatic examinations, cells were cultured in poly-D-lysine-coated culture plates. The stock solution of pinocembrin was dissolved in dimethyl sulfoxide (DMSO) and sterilized by filtration through 0.2 μm disc filters. Appropriate amounts of stock solution (1 mg/ml in DMSO) of pinocembrin were added to the cultured medium to achieve the indicated concentrations (the final DMSO concentration was less than 0.2%).
Cell viability assay
To evaluate the cytotoxicity of pinocembrin, a MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide] assay was performed to determine the cell viability [26]. Briefly, cells were seeded at a density of 4 × 104 cells/ml in a 24-well plate for 24 h. Then, the cells were treated with or without pinocembrin under various concentrations for 24 h. Each concentration was repeated three times. Also, to further investigate whether pinocembrin and/or TGF-β1 influence cell viability, Y-79 cells were treated with the presence or absence of drugs (10 ng/ml TGF-β1 and 5 μM pinocembrin) for 24 h. After the exposure period, the medium was removed and followed by washing of cells with PBS. Then, the medium was changed and incubated with MTT solution (5 mg/ml)/well for 4 h. The medium was removed, and formazan was solubilized in isopropanol and measured spectrophotometrically at 563 nm. The percentage of viable cells was estimated by comparing with the untreated control cells.
Flow cytometric assay
The effects of pinocembrin on Y-79 cell-cycle progression and TGF-β1-induced avβ3 integrin expression were determined by flow cytometry using FACScan (Becton Dickinson Immunocytometry Systems, UK). Firstly, to analyze the cell-cycle distribution, the cells were first treated with various concentrations of pinocembrin for 24 h, and then were collected by trypsinization, fixed in 75% absolute ethanol, washed in PBS, and resuspended in 1 ml of PBS containing 0.5 mg/ml RNase A and 0.01 mg/ml propidium iodide (PI) in the dark for 30 min at room temperature. The cell-cycle profiles were analyzed by a flow cytometer. The percentage of cells in the sub-G1, G0/G1, S, and G2/M phases of the cell cycle was analyzed by the ModFit LT 3.0 software (Verity Software, Topsham, ME). Furthermore, the cell surface expression of avβ3 integrin was determined using flow cytometry. Y-79 cells were plated in six-well dishes. The cells were then washed with PBS and detached with trypsin at 37°C. Cells were fixed for 10 min in PBS containing 1% paraformaldehyde. After rinsing in PBS, the cells were incubated with a mouse anti-human antibody against avβ3 integrin (1:100) for 1 h at 4°C. Cells were then washed again and incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit secondary IgG (1:100; Leinco Tec. Inc., St. Louis, MO, USA) for 45 min and analyzed by flow cytometry.
Cell-matrix adhesion assay
Y-79 cells were pretreated with 5 μg/ml anti-αvβ3 monoclonal antibody (mAb) or 80 nM cyclic RGD peptide (cyclo-RGDfV) for 30 min and then stimulated with 10 ng/ml TGF-β1 in the presence or absence of 5 μM pinocembrin for 24 h. Subsequently, cells were seeded at a density of 1 × 105 cells/ml in a 24-well plate and coated with 500 μl type IV collagen (10 μg/ml); then they were cultured for 30 min. Then, non-adherent cells were removed by PBS washes, and adherent cells were fixed in ethanol. After staining with 0.1% crystal violet, fixed cells were lysed in 0.2% Triton X-100, and measured spectrophotometrically at 550 nm.
Transwell invasion and migration assay
Invasion and migration assay were performed by using Hanging Cell Culture-inserts (BD Biosciences, San Jose, CA; pore size, 8-μm) in 6-well dishes. The ability of Y-79 cells to pass through filters coated with Matrigel (BD Biosciences) was measured by invasion assay. Matrigel was diluted to 200 μg/ml with filtered distill water and applied to the upper surface of the filter inserts. Briefly, Y-79 cells were pretreated with 5 μg/ml anti-αvβ3 monoclonal antibody (mAb) or 80 nM cyclic RGD peptide (cyclo-RGDfV) for 30 min and then stimulated with 10 ng/ml TGF-β1 in the presence or absence of 5 μM pinocembrin. After 24 h, cells were detached by trypsin and resuspended in serum-free medium. Medium containing 10% fetal bovine serum was applied to the lower chamber as a chemoattractant, and then cells were seeded on the upper filter at a density of 1 × 105 cells/ml in the serum-free medium. The plates were incubated for 24 h at 37°C in 5% CO2, filter inserts were removed from the wells and the cells on the upper surface of the filter were wiped with a cotton-tipped swab. Filters were fixed with methanol for 10 min and stained with Giemsa dye for 1 h, and then the cells that had invaded the lower surface of the filter were counted under a light microscope. The data are presented as the average number of cells attached to the bottom surface from randomly chosen fields. Each experiment was carried out in triplicate. To measure the migration ability of Y-79 cells, cells were seeded into a transwell with 8 μm pore polycarbonate filters which were not coated with matrigel. Migrating cells were treated with the presence or absence of drugs (TGF-β1 and pinocembrin). Migration assay was measured as described in the invasion assay.
Gelatin zymography assay
Cells (4 × 105 cells/ml) were seeded into the culture and stimulated with 10 ng/ml TGF-β1 for 2 h and then incubated in different concentrations of pinocembrin (0, 1, 2.5, and 5 μM) for 24 h. Subsequently, the conditioned medium was collected and gelatin zymography was performed to examine the activities of MMP-2 and MMP-9. Samples were mixed with loading buffer and electrophoresed on 8% SDS-polyacrylamide gel containing 0.1% gelatin. Electrophoresis was performed at 140 and 110 V for 3 h. Gels were then washed twice in zymography washing buffer (2.5% Triton X-100 in double-distilled H2O) at room temperature to remove SDS, followed by incubation at 37°C for 12–16 h in zymography reaction buffer (40 mM Tris–HCl, 10 mM CaCl2, 0.02% NaN3), stained with Coomassie blue R-250 (0.125% Coomassie brilliant blue R-250, 0.1% amino black, 50% methanol, 10% acetic acid) for 1 h and destained with destaining solution (20% methanol, 10% acetic acid, 70% double-distilled H2O). Nonstaining bands representing the levels of the latent forms of MMP-2 and MMP-9 were quantified by densitometer measurement using a digital imaging analysis system.
Isolation of total RNA, reverse transcriptase polymerase chain reaction (RT-PCR) and DNA electrophoresis
Total RNA was isolated from human Y-79 cells using the total RNA Extraction Midiprep System (Viogene BioTek Corporation, Taiwan). Total RNA (2 μg) was transcribed to 20 μl cDNA with 1 μl dNTPs (2.5 mM), 1 μl oligo dT (10 pmole/μl), 1 μl RTase (200 U), 1 μl RNase inhibitor and 5 X reaction buffer. The appropriate primers (sense of MMP-2, 5′-GGCCCTGTCACTCCTGAGAT-3′ nt 1337–1356; antisense of MMP-2, 5′-GGCATCCGGTTATCGGGGA-3′, nt 2026–2007; sense of MMP-9, 5′-AGGCCTCTACAGAGTCTTTG-3′ nt 1201–1220; antisense of MMP-9, 5′-CAGTCCAACAAGAAAGGACG-3′, nt 1700–1683; sense of GADPH, 5′-CGGAGTCAACGGATTGGTGTT-3′, nt 94–126; antisense of 5′-AGCCTTCTCCATGGTTGGTGAAGAC-3′, nt 399–375) were used for PCR amplifications. PCR was performed with Platinum Taq polymerase (Invitrogen) under the following conditions: 30 cycles of 94°C for 1 min, 59°C (MMP-2) or 60°C (MMP-9 and GAPDH) for 1 min, 72°C for 1 min followed by 10 min at 72°C.
Western blotting assay
The preparation of cytosolic and nuclear fractions of the cells was performed as described previously [27]. Western blotting was performed as follows. The denatured samples (50 μg purified protein) were resolved on 10-12% SDS-PAGE gels. The proteins were then transferred onto nitrocellulose membranes. Non-specific binding of the membranes was blocked with Tris-buffered saline (TBS) containing 1% (w/v) nonfat dry milk and 0.1% (v/v) Tween-20 (TBST) for more than 2 h. Membranes were washed with TBST three times for 10 min and incubated with an appropriate dilution of specific primary antibodies in TBST overnight at 4°C. Subsequently, membranes were washed with TBST and incubated with an appropriate secondary antibody (horseradish peroxidase-conjugated goat antimouse or antirabbit IgG) for 1 h. After washing the membrane three times for 10 min in TBST, the bands detection was carried out by enhanced chemiluminescence using ECL Western blotting detection reagents and exposed ECL hyperfilm in FUJFILM Las-3000 mini (Tokyo, Japan). Then proteins were quantitatively determined by densitometry using FUJFILM-Multi Gauge V3.0 software.
Electrophoretic mobility shift assay (EMSA)
Cell nuclear proteins were extracted with a nuclear extract buffer and measured by electrophoretic mobility shift assay (EMSA) [28]. Cells (1 × 105/ml) were collected in PBS buffer (pH 7.4) and centrifuged at 2000 × g for 5 min at 4°C. Cells were lysed with buffer A (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, and 0.5 mM PMSF (pH 7.9) containing 5% NP-40) for 10 min on ice, and this was followed by vortexing to shear the cytoplasmic membranes. The lysates were centrifuged at 2000 × g for 10 min at 4°C. The pellet containing the nuclei was extracted with high salt buffer B (20 mM HEPES, 420 mM NaCl, 1.5 mM MgCl2, 0.5 mM DTT, 0.5 mM PMSF, 0.2 mM EDTA, and 25% glycerol) for 15 min on ice. The lysates were centrifugated at 13000 × g for 10 min at 4°C. The supernatant containing the nuclear proteins was collected and frozen at -80°C until use. The protein content of nuclear fractions was determined with Bio-Rad protein assay. Synthetic double-strand oligonucleotides of the consensus NF-κB binding sequence, 5′-AGTTGAGG GGACTTTCCCAGGC-3′ and 3′-TCAACTCCCCTGAAAGGGTCCG-5′, were 5′end- labeled with biotin. Binding reactions containing 5 μg of nuclear proteins, double-distilled H2O, 2 μl 10-fold binding buffer, 2 μg poly (dI · dC) and 2 pmol oligonucleotide probe were incubated for 15 min at room temperature. Specific competition binding assays were performed by adding a 200-fold excess of an unlabeled probe as a specific competitor. Following formation of protein-DNA complexes, samples were loaded on a 6% nondenaturing polyacrylamide gel in 0.5 × TBE buffer and then transferred to positively charged nitrocellulose membranes (Millipore, Bedford, MA, USA) by a transfer blotting apparatus and cross-linked in a Stratagene crosslinker. Gel shifts were visualized with streptavidin-horseradish peroxidase followed by chemiluminescent detection.
Transient transfection and luciferase report gene assays
NF-κB transcriptional activity was measured by NF-κB-luciferase report gene expression. Y-79 cells (4 × 104 cells/well) were plated in six-well plates. The cells were transiently co-transfected with the plasmids, pGL3-NF-κB, pCMV-β-gal and pcDNA3.1 using Lipofectamine Plus according to the manufacturer’s protocol. Briefly, a transfection mixture containing 0.5 μg pGL3-NF-κB and 0.2 μg pCMV-β-gal was mixed with the Lipofectamine Plus reagent and added to the cells. After 8 h, the cells were stimulated with 10 ng/ml TGF-β1 for 2 h and then incubated in 5 μM pinocembrin for 0, 1, 3, 6, and 9 h. Then the NF-κB-luciferase activity was measured according to the manufacturer’s instructions (Promega). Briefly, cells were washed twice with cold PBS and lysed by adding 100 μl 1X reporter lysis buffer (24 mM Tris–HCl (pH 7.8), 2 mM dithiotreitol, 2 mM EDTA, 10% glycerol, and 1% Triton X-100) (Promega, Madison, WI, USA). After centrifugation (13000 × g, 2 min), samples were measured for luciferase activity by using a Sirus Luminometer (Berthold Detection System; OAK Ridge, TN) using 10 μl of cell lysate and 100 μl of luciferase assay reagent (Promega). Luciferase activity was measured with a 10 s delay and 30 s integration time and was normalized to β-galactosidase or Renilla luciferase activity to determine transfection efficiency. The values shown represent an average of three independent transfections and each transfection was carried out in triplicate.
Statistical analysis
Data were expressed as means ± standard deviation of three independent experiments. Statistical comparisons of the results were made using analysis of variance (ANOVA). Significant differences were established at p ≤ 0.05.