Piceatannol was purchased from Tocris (St. Louis, MO, USA) and dissolved in DMSO (vehicle). Antibodies against p65, p50, β-actin, phospho (p)-Akt and Akt were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). A specific NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) and a specific Akt inhibitor LY294002 were purchased from Calbiochem (San Diego, CA, USA). MMP-9 inhibitor I was obtained from Merck (Darmstadt, Germany). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and propidium iodine (PI) were obtained from Sigma (St. Louis, MO, USA).

Human prostate cancer DU145 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were maintained in DMEM (WelGENE, Inc., Daegu, Korea) supplemented with 10% heat-inactivated FBS (WelGENE, Inc.) and 1% penicillin-streptomycin (WelGENE, Inc.) in 5% CO₂ at 37°C. Cells were seeded at 1×10⁵ cells/ml and treated with the indicated concentrations of piceatannol. Following a 24-h incubation, the viability was determined by an MTT assay.

Cell cycle distribution was analyzed by PI-stained cells. Briefly, cells (1×10⁶) were fixed in 70% ethanol overnight at 4°C. The cells were washed in phosphate-buffered saline (PBS) with 0.1% BSA. Cells were incubated with 1 U/ml RNase A (DNase free, Sigma) and 10 μg/ml PI overnight at room temperature in the dark. Cells were analyzed using a FACS Calibur flow cytometer (Becton Dickenson, San Jose, CA, USA). The levels of apoptotic cells with sub-G1 DNA were determined as a percentage of the total number of cells.

Cells were treated with the indicated chemicals and then lysed on ice in a buffer containing 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA and 0.5% Triton X-100 for 30 min. Lysates were vortexed and cleared by centrifugation at 10,000 x g for 20 min. Fragmented DNA in the supernatant was extracted with an equal volume of neutral phenol:chloroform:isoamylalcohol (25:24:1, v/v/v) and analyzed electrophoretically on a 1.5% agarose gel containing ethidium bromide.

DU145 cells were grown to 90% confluence in a 6-well plate at 37°C in a 5% CO₂ incubator. A wound was created by scratching cells with a sterile 200-μl pipette tip, cells were washed twice with PBS to remove floating cells and then added to a medium without serum. Images of the wound were captured under an ×100 magnitude microscope.

Cultured DU145 cells were harvested and washed with serum-free DMEM three times and incubated for 24 h at 5×10⁵ cells/ml of serum-free DMEM. The cells were stimulated with piceatannol in the presence of TNF-α (20 ng/ml). MMP-9 activity was determined by gelatin zymography using 0.1% gelatin as a substrate. The conditioned medium was mixed with SDS-PAGE sample buffer in the absence of reducing agent and electrophoresed in 8% polyacrylamide gel. Following electrophoresis, the gels were washed three times with 2.5% Triton X-100 in water and then incubated overnight in a closed container at 37°C in 0.2% Brij 35.5 mM CaCl₂, 1 mM NaCl and 50 mM Tris, pH 7.4. The gels were stained for 30 min with 0.25% Coomassie Blue R-250 in 10% acetic acid and 45% methanol and then destained for 30 min using an aqueous mix of 20% acetic acid, 20% methanol and 17% ethanol. Areas of protease activity appeared as clear bands.

Total RNA was isolated using TRIzol reagent (GIBCO-BRL, Gaithersburg, MD, USA) according to the manufacturer’s recommendations. Genes of interest were amplified from cDNA that was reverse-transcribed from 1 μg of total RNA using the One-Step RT-PCR Premix (iNtRON Biotechnology, Sungnam, Korea). Primers for MMP-9 sense (5′-CCTGGAGACCTGAGAACCAATCT-3′) and antisense (5′-CCACCCGAGTGTAACCATAGC-3′) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sense (5′-CCACCCATGGCAAATTCCATGGCA-3′) and antisense (5′-TCTAGACGGCAGGTCAGGTCCACC-3′) were used. The PCR was initiated at 94°C for 2 min followed by 31 cycles of 94°C for 30 min, 30-min annealing temperature, 72°C for 30 min followed by final extension at 72°C for 5 min. The annealing temperatures for MMP-9 and GAPDH were 63°C and 62°C, respectively. Following amplification, PCR products were separated on 1.5% agarose gels and visualized by ethidium bromide fluorescence.

Cells (5×10⁴/chamber) were used for each invasion assy. Invasion assays were performed using modified Boyden chambers with polycarbonate nucleopore membrane (Corning, Corning, NY, USA). Precoated filters (6.5 mm in diameter, 8 μm pore-size, Matrigel 100 μg/cm²) were rehydrated and 5×10⁴ cells in medium with or without piceatannol or MMP-9 inhibitor I (5 nM) in the presence of TNF-α were seeded into the upper part of each chamber. Following 24 h incubation, nonmigratory cells on the upper surface of the filter were wiped with a cotton swab and migrated cells on the lower surface of the filter were fixed and stained with 0.125% Commassie Blue in a methanol:acetic acid:water mixture (45:10:45, v/v/v). Random fields were counted under a light microscope.

NF-κB reporter construct was purchased from Clonetech (Palo Alto, CA, USA) and MMP-9 promoter was obtained from Professor Y.H. Choi (Oriental Medicine, Dongeui University, Busan, Korea). Briefly, DU145 cells were plated onto six-well plates at a density of 5×10⁵ cells/well and grown overnight. Cells were transfected with 2 μg of each plasmid construct for 6 h by the Lipofectamine method. Following transfection, the cells were cultured in 10% FBS containing DMEM with the indicated concentrations of piceatannol in the presence of 20 ng/ml TNF-α for 24 h. Cells were lysed with lysis buffer (20 mM Tris-HCl, pH 7.8, 1% Triton X-100, 150 mM NaCl and 2 mM DTT). The cell lysate (5 μl) was mixed with luciferase activity assay reagent (25 μl) and luminescence produced for 5 sec was measured using GLOMAX luminometer (Promega, Madison, WI, USA).

The preparation of cytoplasmic and nuclear extracts was conducted using the NE-PER nuclear and cytoplasmic extraction reagents (Pierce, Rockford, IL, USA). DNA-protein binding assays were carried out with nuclear extract. Synthetic complementary NF-κB-binding oligonucleotides (5′-AGTTGAGGGGACTTTCCCAGGC-30′; Santa Cruz Biotechnology) were biotinylated using the biotin 30-end DNA labeling kit (Pierce) according to the manufacturer’s instructions and annealed for 1 h at room temperature. Binding reactions were carried out for 20 min at room temperature in the presence of 50 ng/ml poly(dI-dC), 0.05% Nonidet P-40, 5 mM MgCl_2, 10 mM EDTA and 2.5% glycerol in 1X binding buffer (LightShift™ chemiluminescent EMSA kit) with 20 fmol of biotin-end-labeled target DNA and 10 μg of nuclear extract. Assays were loaded onto native 4% polyacrylamide gels pre-electrophoresed for 60 min in 0.5X Tris borate/EDTA and transferred onto a positively charged nylon membrane (HybondTM-N+) in 0.5X Tris borate/EDTA at 100 V for 30 min. Transferred DNAs were cross-linked to the membrane at 120 mJ/cm² and detected using horseradish peroxidase-conjugated streptavidin according to the manufacturer’s instructions.

Total cell extracts were prepared using PRO-PREP protein extraction solution (iNtRON Biotechnology). Proteins were separated by SDS-PAGE and electrotransferred to nitrocellulose membranes (Amersham, Arlington Heights, IL, USA). The detection of specific proteins was carried out with an ECL western blotting kit (Amersham) according to the manufacturer’s instructions.

All data were derived from at least three independent experiments. The images were visualized with Chemi-Smart 2000 (Vilber Lourmat, Cedex, France). Images were captured using Chemi-Capt (Vilber Lourmat) and transported into Adobe Photoshop (version 8.0). All data are presented as mean ± SE. Significant differences between the groups were determined using one-way ANOVA test. P<0.05 was considered to indicate a statistically significant difference.

In order to determine the cytotoxic potential of piceatannol on DU145 cells, an MTT assay was performed at 24 h following treatment with various concentrations (0–40 μM) of piceatannol in the presence or absence of TNF-α, regardless of the presence of FBS. Piceatannol alone, or in conjunction with TNF-α, was not cytotoxic at any of the concentrations tested in this study (Fig. 1A). Therefore, piceatannol levels of <40 μM were selected for subsequent experiments. Subsequently, the effect of piceatannol on cell viability and cytotoxicity was analyzed in detail. According to the percentages of sub-G₁ DNA content as measured by flow cytometry, no apoptotic cell death was observed compared with the positive H₂O₂-treated group (Fig. 1B). Furthermore, DNA fragmentation was analyzed to determine the level of fragmented DNA. No fragmented DNA was identified in the piceatannol alone or the TNF-α-treated groups compared with the positive H₂O₂-treated group (Fig. C). To determine whether piceatannol also inhibited TNF-α-induced migration, a scratch wound healing assay was performed. Cell migration was significantly increased by TNF-α treatment and was inhibited by the presence of piceatannol (Fig. 1D). Overall, these data indicate that piceatannol inhibited TNF-α-induced migration, and hence tumor invasion, without any cytotoxicity.

Zymography, RT-PCR and measurement of luciferase reporter activity were conducted to assess whether piceatannol regulates TNF-α-induced MMP-9 expression. The zymography data revealed that the secretion of MMP-9 was significantly increased by TNF-α treatment compared with the untreated control group. Piceatannol decreased TNF-α-induced MMP-9 activity in a dose-dependent manner (Fig. 2A). In a subsequent experiment, the luciferase activity of MMP-9 was assayed using transient transfection with reporter vectors that included MMP-9 promoters. Treatment with TNF-α significantly increased MMP-9 luciferase activity, whereas piceatannol downregulated TNF-α-induced MMP-9 luciferase reporter activity (Fig. 2B). In addition, RT-PCR analysis revealed that steady-state MMP-9 mRNA levels are undetectable in piceatannol-treated cells compared with the untreated control group (Fig. 2C). TNF-α stimulation of cells resulted in a significant increase in MMP-9 mRNA expression compared with the untreated control; however, piceatannol completely reversed TNF-α-induced MMP-9 mRNA levels to the levels of the untreated control. Since MMP-9 is thought to be critically involved in the processes of tumor invasion (25), the effects of piceatannol on the invasion of DU145 cells was examined. In the invasion assay, when the cells were treated with TNF-α alone, a marked 4-fold increase in cell invasion was observed compared with the untreated control. However, the addition of piceatannol resulted in an ∼50% reduction in penetration through a Matrigel-coated membrane when compared with the TNF-α-treated group (Fig. 2D). Addition of MMP-9 inhibitor I significantly blocked TNF-α-induced cell invasion. These results confirm that piceatannol suppressed the upregulation of TNF-α-stimulated MMP-9 expression at the transcription level and inhibited TNF-α-induced invasion of DU145 carcinoma cells.

In order to determine whether MMP-9 mRNA expression was linked to NF-κB activity, a promoter assay was conducted using DU145 cells that had been transiently transfected with a luciferase reporter vector, which included the NF-κB-binding sites. NF-κB luciferase activity was significantly increased, ∼7.4-fold, in TNF-α-stimulated DU145 cells compared with the untreated control group (Fig. 3A). However, the TNF-α-stimulated luciferase activity in the cells containing the NF-κB construct was significantly reduced to ∼50%, by treatment with piceatannol. An EMSA was conducted to evaluate the effect of piceatannol on the DNA-binding activity of NF-κB to assess its inhibitory effect on the transcriptional activity of MMP-9. Nuclear extracts from control and piceatannol-treated DU145 cells were subjected to analysis for the DNA-binding activity of NF-κB. This reached a maximum at 30 min and was sustained for 1 h following treatment with TNF-α (data not shown). EMSA data revealed that treatment with piceatannol downregulated the TNF-α-induced DNA-binding activity of NF-κB at 30 min (Fig. 3B). As shown in the western blot analysis, treatment with TNF-α decreased the levels of p65 and p50 in the cytosolic extract, whereas the addition of piceatannol resulted in sustainment of the levels of p65 and p50. This suggests that piceatannol inhibits the nuclear translocation of NF-κB subunits p65 and p50 (Fig. 3C). In addition, our data indirectly demonstrated that a specific NF-κB inhibitor PDTC has the ability to inhibit TNF-α-induced expression of MMP-9 mRNA (Fig. 3D). These results indicate that treatment with piceatannol suppresses MMP-9 gene expression in TNF-α-induced DU145 cells via inhibition of NF-κB activity.

As Akt is known to be an upstream regulator of NF-κB, we investigated whether NF-κB activity is regulated by piceatannol-induced Akt phosphorylation or dephosphorylation. Western blot analysis revealed that TNF-α stimulation induced the phosphorylation of Akt at 15 min, whereas treatment with piceatannol suppressed the phosphorylation of Akt (Ser 473, Fig. 4A). It was also demonstrated that a specific Akt inhibitor LY294002 completely eliminated the TNF-α-induced NF-κB promoter activity, suggesting that inactivation of Akt by piceatannol regulates TNF-α-induced NF-κB (Fig. 4B). Moreover, RT-PCR analysis demonstrated that pretreatment of DU145 cells with LY294002 suppressed TNF-α-induced MMP-9 activity at the transcription level (Fig. 4C). These results confirmed that piceatannol regulated TNF-α-induced MMP-9 gene expression by blocking Akt phosphorylation-dependent NF-κB activity.