(–)‐Epigallocatechin gallate suppresses the growth of human hepatocellular carcinoma cells by inhibiting activation of the vascular endothelial growth factor–vascular endothelial growth factor receptor axis

Cell lines and cell culture conditions.  Six human HCC cell lines, HLF, PLC/PRF/5, HepG2, HuH7, HLE, and Hep3B, were obtained from the Japanese Cancer Research Resources Bank (Tokyo, Japan) and maintained in DF10 medium containing DMEM (Invitrogen, San Diego, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen). The Hc human normal hepatocyte cell line was purchased from Applied Cell Biology Research Institute (Kirkland, WA, USA) and maintained in CS‐C complete medium (Cell Systems Biotechnologie Vertrieb, St Katharinen, Germany). The cells were cultured in an incubator with humidified air with 5% CO₂ at 37°C.

Cell viability assays.  Cell viability assays were conducted using the MTT cell proliferation kit I (Roche Diagnosis Co., Indianapolis, IN, USA), according to the manufacturer's instructions, as described previously.^{( 23 )} Three thousand HuH7 or Hc cells were seeded into 96‐well plates. Twenty‐four hours later, the cells were treated with the indicated concentrations of EGCG (0–100 µg/mL) for 48 h in DF10 medium, and cell viability was examined. All assays were carried out in triplicate.

VEGF production assays.  HuH7 cells were plated into six‐well plates and grown to 70% confluence. After washing with PBS, the cells were treated with the indicated concentrations of EGCG (0–100 µg/mL) in serum‐minus medium for 24 h. The cell‐free medium was then collected and the amounts of VEGF secreted by the cells into the medium were measured using a VEGF ELISA kit (R&D Systems, Minneapolis, MN, USA), according to the manufacturer's instructions.

Protein extraction and western blot analysis.  Total protein was extracted from the cell lines or xenografts of HuH7 cells and equivalent amounts of protein (20 µg/lane) were subjected to a western blot analysis, as described previously.^{( 24} , ^{25 )} The primary antibodies for ERK, p‐ERK, Akt, and p‐Akt were described previously.^{( 23 )} The primary antibodies for VEGFR‐2 (#2479), p‐VEGFR‐2 (#2478), Bcl‐x_L (#2762), and GAPDH (#2118) were purchased from Cell Signaling Technology (Beverly, MA, USA). An antibody to GAPDH served as a loading control.

RNA extraction and semiquantitative RT‐PCR analysis.  A semiquantitative RT‐PCR analysis was carried out, as described previously.^{( 26 )} Total RNA was isolated from the xenopgrafts of HuH7 cells using ISOGEN reagent (Nippon Gene Co., Tokyo, Japan), according to the manufacturer's instructions. The cDNA was amplified from 1 µg of total RNA using SuperScript one‐step RT‐PCR with the platinum Taq system (Invitrogen). The primers used for amplification of VEGF and GAPDH specific genes were as follows: VEGF forward, 5′‐CTA CCT CCA CCA TGC CAA GT‐3′; VEGF reverse, 5′‐AAA TGC TTT CTC CGC TCT GA‐3′; GAPDH forward, 5′‐CGA GAT CCC TCC AAA ATC AA‐3′; and GAPDH reverse, 5′‐TTC AGC TCA GGG ATG ACC TT‐3′. Using a thermal controller (Programmable Thermal Controller; MJ Research, Watertown, MA, USA), 35‐cycle rounds of PCR were chosen for data analysis of the mRNA expression as a semiquantitative assessment indicated that the reaction had not reached a plateau and was still in log phase. The amplified products obtained with GAPDH‐specific primers served as an internal control. The intensities of the PCR products stained with ethidium bromide were quantified using the NIH Image software program version 1.62 (URL: http://rsb.info.nih.gov/nih‐image/index.html).

In vivo experimental protocol.  Twenty‐four male BALB/c nude mice (5 weeks of age) were obtained from Charles River Japan (Tokyo, Japan). All mice were maintained at Gifu University Life Science Research Center, according to the Institutional Animal Care Guidelines, and were housed in plastic cages with free access to drinking water (tap water supplemented with or without EGCG) and the pelleted basal diet CRF‐1 (Oriental Yeast Co., Tokyo, Japan). Xenograft tumors were made by subcutaneous injection of HuH7 cells, at a concentration of 5 × 10⁶ cells per 200 µL, into the flanks of these mice. One week after tumor cell injection, the mice were randomly divided into three groups (eight mice per group) and then treated with (groups 2 and 3) or without (group 1) EGCG for 5 weeks. The mice in groups 2 and 3 were given tap water containing 0.01 or 0.1% EGCG, respectively. The concentrations of EGCG (0.01 and 0.1%) were established according to the findings of previous reports^{( 27} , ^{28 )} because these doses could exert anticancer properties without causing various side effects in any organs. The mice in group 1 were given tap water and served as an untreated control. A freshly prepared solution of EGCG in tap water was supplied to the experimental mice three times a week. The tumor size and bodyweight were measured once a week and the tumor volume was calculated using the formula: largest diameter × (smallest diameter)² × 0.5.

Statistical analysis.  The data were expressed as the mean ± SD. The statistical significance of the difference in mean values was tested using a one‐way analysis of variance (ANOVA) and the unpaired t‐test. Significance was defined as a P‐value less than 0.05. All analyses were carried out using the StatView ver. 5.0 software program (SAS Institute, Cary, NC, USA).

Effects of EGCG on the growth of HuH7 and Hc cells.  We then examined the growth‐inhibitory effects of EGCG on HuH7 and Hc cell lines using MTT assays. As shown in Figure 2, EGCG inhibited the growth of HuH7 cells with an IC₅₀ value of approximately 25 µg/mL. However, the Hc cells were more resistant to EGCG because the IC₅₀ value with this agent was approximately 84 µg/mL (Fig. 2). These findings suggest that EGCG preferentially inhibits the growth of HuH7 HCC cells, which express higher levels of the VEGFR‐2 and p‐VEGFR‐2 proteins, when compared with Hc cells that do not express these proteins (Fig. 1).

Effects of EGCG on the expression and activation of VEGFR‐2 in HuH7 cells.  We next examined whether EGCG alters the expression and activation of VEGFR‐2 in HuH7 cells. A time‐course study indicated that when the cells were treated with 25 µg/mL EGCG, which is the same as the IC₅₀ concentration determined by the MTT assays (Fig. 2), a marked decrease was observed in the expression levels of both VEGFR‐2 and p‐VEGFR‐2 proteins within 6 h of the addition of this agent (Fig. 3a). When the cells were treated with the indicated concentrations of EGCG (0–100 µg/mL) for 6 h, the expression levels of VEGFR‐2 as well as p‐VEGFR‐2 proteins were also inhibited in a dose‐dependent manner (Fig. 3b).

Effects of EGCG on VEGF production by HuH7 cells.  VEGF, which is produced by cancer cells, has been reported to play a critical role in tumor angiogenesis.^{( 7} , ^{8 )} We therefore next examined the effects of EGCG on production of VEGF by HuH7 cells using an ELISA system. As shown in Figure 4, HuH7 cells secreted an abundant amount of VEGF into the growth medium when the cells were cultured in serum‐free medium for 24 h and, interestingly, a low (10 µg/mL, less than IC₅₀ value) and high (100 µg/mL) concentration of EGCG significantly reduced the production of VEGF from these cancer cells.

Effects of EGCG on the growth of HCC xenografts in nude mice.  We next examined whether the growth inhibition of the treatment with EGCG in HuH7 cells was also observed in vivo using a nude mouse xenograft model. Figure 5 shows that drinking water with not only a high concentration (0.1%), but also a low concentration (0.01%) of EGCG strongly inhibited the growth of the HuH7 xenograft during treatment with this agent (5 weeks). The tumor volume of the mice treated with 0.1% EGCG was less than that of tumors in 0.01% EGCG‐drinking mice, but the difference was not significant (Fig. 5). All of the treatments were well tolerated and the bodyweights remained stable in all groups during the experiment (data not shown). There were no pathological alterations suggesting toxicity of EGCG in the liver, spleen, and kidneys of mice (data not shown).

Effects of EGCG on the activation of VEGFR‐2 and its downstream signaling molecules and on the cellular levels of Bcl‐x_L in xenografts of HuH7 cells.  We next examined whether treatment with EGCG inhibits the activation of VEGFR‐2 and its multiple downstream signaling pathways in the HuH7 xenografts. Drinking both 0.01 and 0.1% EGCG decreased the total levels of VEGFR‐2 and Akt proteins in these xenografts (Fig. 6a). There was also a marked decrease in the levels of p‐VEGFR‐2, p‐ERK, and p‐Akt proteins by treatment with both concentrations of EGCG (Fig. 6a). In addition, EGCG caused a decrease in the levels of Bcl‐x_L, an anti‐apoptotic Bcl‐2 family member, in HuH7 xenogrfts (Fig. 6a).

Effects of EGCG on the expression of VEGF mRNA in xenografts of HuH7 cells.  A semiquantitative RT‐PCR study showed that there was a significant decrease in the level of VEGF mRNA in the xenografts of mice treated with 0.1% EGCG compared to that of the tumors in control mice (Fig. 6b). Therefore, the inhibitory effect of EGCG on the production of VEGF was not only observed in vitro (Fig. 4) but also in vivo.