EGCG was obtained from Catch Bio-Science & Technology Ltd. (Jiangsu, China) with a purity of >99.99%. All cell culture reagents were purchased from Biowest (USA). MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] detection kit, Annexin V-FITC apoptosis detection kit and cell cycle detection kit were obtained from BestBio (Shanghai, China). Western blotting antibodies specific to p-Stat3, total-Stat3 and β-actin were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Cell-based ELISA kit [human/mouse phospho-Stat3 (Y705) immunoassay] was purchased from R&D Systems (Minneapolis, MN, USA).

The BEL-7402 and QGY-7703 human HCC cell lines were provided by Shanghai Institute of Biochemistry and Cell Biology. They were cultured in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum, 1X penicillin-streptomycin solution in a humidified 5% CO₂ atmosphere at 37°C.

Stat3 binding assays were performed at 25°C with a BIAcore 3000 biosensor using 20 mM Tris-buffer pH 8.0 that contained 2 mM β-mercaptoethanol and 5% DMSO as running buffer. Phosphorylated and non-phosphorylated control biotinylated EGFR-derived dodecapeptides based on the sequence surrounding Y1068 were immobilized on a streptavidin-coated sensor chip (BIAcore Inc., Piscataway, NJ, USA). The binding of Stat3 was conducted in 20 mM Tris-buffer pH 8.0 containing 2 mM β-mercaptoethanol at a flow rate of 10 μl/min for 1–2 min. Aliquots of Stat3 at 500 nM were premixed with compound to achieve a final concentration of 1–1,000 μM, and incubated at 4°C prior to being injected onto the sensor chip. The chip was regenerated by injecting 10 μl of 100 mM glycine at pH 1.5 after each sample injection. A control (Stat3 with DMSO but without compound) was run at the beginning and the end of each cycle (40 sample injections in total) to maintain the integrity of the sensor chip throughout the cycle run. The average of the 2 controls was normalized to 100% and used to evaluate the effect of each compound on Stat3 binding. Responses were normalized by dividing the value at 2 min by the response obtained in the absence of compounds at 2 min and multiplying by 100. IC₅₀ values were determined by plotting the percentage of the maximum response as a function of the log concentration of the compound, and fitting the experimental points to a competitive binding model using a four-parameter logistic equation: R = R_high − (R_high − R_low)/(1 + conc/A₁)A₂, where R is the percent response at the inhibitor concentration; R_high is the percent response with no compound; R_low is the percent response at the highest compound concentration; A₂ is the fitting parameter (slope); and A₁ is the IC₅₀ (BIAevaluation software version 4.1).

Both molecular structures of Stat3 and EGCG were retrieved from the Protein Data Bank (PDB Code: 1BG1 and ENG5) and optimized in the implicit solvent for docking preparation. The docking region mainly constructed by the residues of Arg-609 and K-591 was localized based on the interface between the STAT3 SH2 domain and the phosphopeptide. The docking procedure was executed using CHARMm force field on the CDOCKER module platform in Discovery Studio (Accelrys Inc.). The conformation search space was limited to a spherical region with a center of 104.8, 74.3, 63.3 and a radius of 13 Å. The other parameters were determined based on the default setting of the module, with a grid extension of 8.0. The ligand partial charge method was performed by CHARMm. Ten top hits were obtained from the docking simulation. Simulated annealing method was employed for the final conformation treatment (the system was heated to 700 K with 2,000 steps and then cooled to 300 K with 5,000 steps). Finally, the best conformation was selected as the analysis object according to the values of the scores.

Cell proliferation was determined using MTT assay according to the manufacturer’s instructions. Briefly, BEL-7402 and QGY-7703 cells were then seeded into 96-well plates at a density of 5×10³/well (100 μl). After 24 h, the indicated concentrations of EGCG were added. After incubation for 24 and 48 h, respectively, cells were washed twice with PBS. Ten microliters of MTT medium was then added into each well, at which time cells were incubated for another 3 h. The medium was removed, and 150 μl of dissolution was added into each well. The plate was gently rotated on an orbital shaker for 10 min to dissolve the precipitate completely. The absorbance was detected at 492 nm with a microplate reader.

QGY-7703 cells were treated with EGCG in complete medium for 48 h as previously described. Following treatment, the cells were harvested by trypsin (not containing EDTA) and rinsed twice with PBS at 4°C. Cells were then resuspended in 1X Annexin V binding buffer. Five microliters of Annexin V-FITC solution was added to each tube. All tubes were incubated for 15 min at 4°C in darkness. Fifteen microliters of PI solution was added to each tube. All tubes were incubated for another 5 min at 4°C in darkness. Cells were then analyzed using flow cytometry (Accuri C6; BD Biosciences, USA).

To detect protein expression and modification in response to treatment with EGCG, HCC cells, which were treated with various concentrations of EGCG, were plated onto 6-well plates at a density of 2×10⁵ cells/ml. After incubation for 24 h, cells were lysed in cold RIPA lysis buffer. Total protein was extracted with high-salt buffer (0.5% sodium deoxycholate, 1% SDS, 1 mM sodium orthovanadate, 1 mM β-glycerol phosphate, 1 mM sodium fluoride, 2.5 mM sodium pyrophosphate) containing a protease inhibitor cocktail (Roche, Nutley, NJ, USA). Protein samples were separated by SDS-PAGE, transferred onto PVDF membranes, and immunoblotted with the corresponding antibodies. The signals were visualized with Enhanced Chemiluminescence Plus (ECL Plus) detection system (Dingguo, China). The ELISA procedure was used according to the manufacturer’s instructions (Cell Signaling Technology, Inc.). Briefly, 100 μl of 15,000 BEL-7402 cells was seeded into each well of a black 96-well microplate with a clear bottom, and incubated overnight at 37°C. Cells were then treated with different concentrations of EGCG in complete medium for 24 h. Subsequently, cells were stimulated with interleukin 6 (IL-6) (50 ng/ml) to induce Stat3 phosphorylation. Following the treatments, cells were treated and tested with the cell-based ELISA kit.

The BEL-7402 and QGY-7703 cells were treated with EGCG at 40, 80 and 160 μM for 48 h as previously described. Total RNAs were extracted from the cells using a commercially available RNA-Bee isolation kit (Tel-Test). Standard reverse transcription was performed with 500 ng of total RNA using TIANScriptRT kit (Tiangen Beijing, China). Reverse transcription-PCR was performed using 1 μl of cDNA template, 10 pmol of primers, and a PCR premix (1 U Taq DNA polymerase, 250 mM dNTPs, 10 mM Tris-HCl, 40 mM KCl and 1.5 mM MgCl₂; Tiangen). The following primers were used in the PCR reactions: Bcl-xL forward, 5′-agctggtggt tgactttctctc-3′ and reverse, 5′-ccggaagagttcattcactacc-3′; c-Myc forward, 5′-ctaccctctcaacgacagcag-3′ and reverse, 5′-gtgtgtt cgcctcttgacatt-3′; VEGF forward, 5′-gcagaatcatcacgaagtggt-3′ and reverse, 5′-catttgttgtgctgtaggaagc-3′; cyclin D1 forward, 5′-atctacaccgacaactccatcc-3′ and reverse, 5′-gcattttggaga ggaagtgttc-3′; β-actin forward, 5′-agagctacgagctgcctgctg-3′ and reverse, 5′-agtacttgcgctcaggagga-3′.

The amplified products obtained from the β-actin-specific primers served as internal controls. PCR was conducted using Bio-Rad T-100 (Bio-Rad, Hercules, CA, USA) with a 5-min denaturation step at 94°C; 30 cycles of 94°C for 30 sec, 62°C for 30 sec and 72°C for 30 sec; and a final extension at 72°C for 10 min. PCR amplifications were verified to be in the linear range.

Data are presented as means ± SD for 3 separate experiments. One-way ANOVA was employed for statistical analysis using SPSS 17.0. P<0.05 was considered to indicate a statistically significant result.

EGCG was tested for its ability to block Stat3 binding to its phosphopeptide ligand using SPR binding assay (15). SPR experiments showed that EGCG was able to directly compete with pY-peptide for binding with Stat3 at an IC₅₀ value of 10–30 μM (Fig. 1).

Fig. 2A is a computer model image of EGCG on Stat3. According to the figure, EGCG is located in a phosphopeptide binding pocket formed by the STAT3 SH2 fold. Fig. 2B shows the spatial matching results of EGCG and Stat3; the 3-D structure of EGCG matches perfectly with the phosphopeptide binding site of STAT3 SH2. Fig. 2C and D depict the specific interactions between EGCG and STAT3 SH2. According to these two figures, the -NH3 group of LYS591 is located between the 2 aromatic rings of EGCG and the 2 hydrogen atoms from the -NH3 group, resulting in the formation of cation-π bonds. The other hydrogen atom in the -NH3 group forms a hydrogen bond with the -O- in EGCG. On the other hand, the -C=O group functions as a hydrogen receptor in EGCG, forming hydrogen bonds with -NH in GLU612 and -OH in SER613, respectively. While the 2 aromatic rings both function as hydrogen donors in EGCG, one also forms hydrogen bonds with the -NH2 group in ARG60, while the other forms hydrogen bonds with the -NH2 group in Glu638. The other part of EGCG forms a van der Waals interaction with the phosphopeptide binding pocket of STAT3 SH2 (LYS591, GLU594, ARG609, SER611, GLU612, SER613, THR620, VAL637, GLU638, PRO639) (Fig. 2C). These multiple interactions result in a steady locked relationship (15–18).

To determine the potential cytotoxic and anti-proliferative effects of EGCG, the human HCC cell lines BEL-7402 and QGY-7703 were cultured with EGCG at various concentrations (0–320 μM). Cell viability was then determined by MTT assay. Results showed that treatment with EGCG led to a significant dose-dependent inhibition of HCC cell growth in vitro (Fig. 3A). The half maximal (50%) inhibitory concentrations (IC₅₀) for BEL-7402 and QGY-7703 cells were ~55 and 35 μM, respectively. Induction of cell apoptosis was confirmed by Annexin V-FITC staining in QGY-7703 cells. Results showed that treatment with EGCG led to significant dose-dependent apoptosis-inducing effects on HCC cell growth in vitro. According to Fig. 3B, the upper right quadrant represents late apoptosis, while the lower right quadrant represents early apoptosis. Increasing concentrations of EGCG at 20, 40, 80 and 160 μM, respectively, were added to the QGY-7703 cell line for 48 h. As a result, the rates of cell apoptosis were 11.7, 18.7, 42.6 and 73.6%, respectively. Thus, as the concentration of EGCG increased, the rate of apoptosis of the QGY-7703 cells also increased. The standard deviations were calculated based on 3 independent experiments.

To examine whether EGCG has inhibitory effects on IL-6-induced Stat3 phosphorylation, QGY-7703 cells were cultured and pretreated with different concentrations of EGCG for 48 h, and were then treated with 50 ng/ml of IL-6 stimulation for 30 min. After treatment, the phosphorylated Stat3 and total Stat3 were analyzed by western blotting and cell-based ELISA. EGCG inhibited Stat3 phosphorylation on tyrosine 705 in a dose-dependent manner (Fig. 4A). The p-Stat3 relative florescence intensity was significantly reduced following EGCG treatment. When QGY-7703 cells were treated with EGCG at concentrations of 10, 20, 40, 80 and 160 μM, respectively, the p-Stat3 relative average fluorescence intensities were 7,400, 6,600, 4,500, 3,400 and 2,200, respectively. Statistical analysis showed a P-value of <0.05 for EGCG at 10, 20 and 40 μM in relation to their corresponding fluorescence intensity; EGCG at 80 and 160 μM had a P-value of <0.001 in relation to their corresponding fluorescence intensity (Fig. 4B).

Exposure to EGCG resulted in a dose-dependent decrease in cyclin D1 mRNA expression in both BEL-7402 and QGY-7703 cells, as demonstrated by RT-PCR analysis. Furthermore, the expression levels of Bcl-xL, c-Myc and VEGF were also significantly reduced at the transcriptional levels (Fig. 4C).