EGCG was obtained from Sigma (St. Louis, MO, USA). For use in cell culture, EGCG was dissolved in sterile water at a concentration of 1 mg/ml and stored at −20°C. For use in intraperitoneal (i.p.) injections, EGCG was dissolved in saline at a concentration of 6 mg/ml and stored at 4°C.

The human colorectal cancer cell lines, RKO and HCT116, were tested for mycoplasma prior to use. All cancer cells were aliquoted and frozen in liquid nitrogen immediately upon receipt and used for the present experiments within 6 months of thawing. All cancer cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Wako Pure Chemical Industries, Ltd., Osaka, Japan) supplemented with 10% fetal bovine serum (FBS) (HyClone, Logan, UT, USA) and 1% penicillin and streptomycin (Invitrogen, Grand Island, NY, USA). All cultures were maintained at 37°C in a humidified atmosphere containing 5% CO₂.

To measure the cytotoxicity of EGCG against these cancer cells, a total of 3×10³ cells in 100 μl of DMEM supplemented with 10% FBS were seeded into each well of a 96-well plate. Following overnight culture, EGCG was added at the specified concentrations. After 24 h of incubation, cell viability was measured by the level of mitochondrial activity in reducing 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5- (2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8) to formazan using a Cell Counting kit-8 (CCK-8) (Dojindo Laboratories, Kumamoto, Japan). The cells were incubated with the reagent as specified by the manufacturer’s instructions. The plates were read at A₄₅₀ on a spectrometer.

To measure the effect of EGCG on the proliferative activity of these cancer cells, a total of 3×10³ cells in 100 μl of DMEM supplemented with 10% FBS were seeded in each well of a 96-well plate. Following overnight culture, EGCG was added at the specified concentrations. After 24 h of incubation, cell proliferation was measured with a Cell Proliferation ELISA, BrdU (colorimetric) (Roche Diagnostics, Penzberg, Germany). The cells were incubated with the reagent as specified by the manufacturer and the plates were read at A₃₅₀ on a spectrometer.

To detect apoptosis in these cancer cells, a total of 6×10⁴ cells in 1 ml of DMEM supplemented with 10% FBS were seeded in each well of a Lab-Tek II Chamber Slide (Nalge Nunc International, Tokyo, Japan). Following overnight culture, EGCG was added at the specified concentrations, and the cells were incubated for a further 4 h. Apoptosis in treated cells and paraffin-embedded liver samples was evaluated with a DeadEnd™ Colorimetric TUNEL System (Promega, Madison, WI, USA) according to the manufacturer’s recommendations.

For western blot analysis of these cancer cells, a total of 3×10⁵ cells in 5 ml of DMEM supplemented with 10% FBS were seeded into each well of a 6-cm dish. After 48 h of culture, EGCG was added at the specified concentrations, and the cells were harvested 12 h later. Cell lysates were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Millipore, Bedford, MA, USA). The following antibodies were used as primary antibodies: Akt (9272), phospho-Akt (9271), p38 (9212) and phospho-p38 (9211) (Cell Signaling Technology, Beverly, MA, USA). β-actin (4970) (Cell Signaling Technology) was used as the endogenous control. An anti-rabbit IgG horseradish peroxidase-conjugated antibody (7074) (Cell Signaling Technology) was used as the secondary antibody. Immunoblots were analyzed by enhanced chemiluminescence.

For reverse transcription-polymerase chain reaction (RT-PCR), a total of 3×10⁵ cells in 5 ml of DMEM supplemented with 10% FBS were seeded into each well of a 6-cm dish and cultured for 48 h. EGCG was added at the specified concentrations, and after 2 h of incubation, total RNA was isolated from whole cells using a NucleoSpin^® RNA II kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions. RNA concentrations were determined by measuring the absorbance at 260/280 nm with a NanoDrop Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). The synthesis of complementary DNA was performed using AMV reverse transcriptase (Promega) and random primers (Takara Bio Inc., Shiga, Japan). Briefly, a mixture of 1 mM dNTPs (Fermentas Life Sciences, Burlingame, ON, Canada), 0.025 μg/ml random primers, 0.25 U/ml reverse transcriptase, and 500 ng of total RNA was incubated at 30°C for 10 min, 37°C for 60 min, 95°C for 5 min and at 4°C before storage at −80°C.

Primers for RT-PCR were designed using Primer Express^® software for Real-Time PCR ver. 3.0 (Applied Biosystems, Foster City, CA, USA) based on sequences available in GenBank. Primers were purchased from Hokkaido System Science (Hokkaido, Japan). We examined 4 receptor tyrosine kinases: vascular endothelial growth factor receptor 2 (VEGFR2), epidermal growth factor receptor (EGFR), human EGFR-related 2 (HER2) and c-Met. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control. RT-PCR was performed using SYBR-Green real-time PCR Master Mix-Plus (Toyobo, Osaka, Japan) and an Applied Biosystems 7300 real-time PCR system (Applied Biosystems) as recommended by the manufacturers. The following primers were used for amplification: VEGFR2, 5′-TTGCCCTTGTTCTGTCCTTTTT-3′ and 5′-GTCATTGTTCCCAGCATTTCAC-3′; EGFR, 5′-GGCTGCCTCCTGGACTATGT-3′ and 5′-AGTTCATGCCCTTTGCATCT-3′; HER2, 5′-TCCCCCAAAGCCAACAAAG-3′ and 5′-CCGTGGATGTCAGGCAGAT-3′; c-Met, 5′-CTCTCTGCCCCACCCTTTG-3′ and 5′-TGTCCCGCTCAGGCATTC-3′; and GAPDH, 5′-GGAGTCCACTGGCGTCTTCA-3′ and 5′-TTCACACCCATGACGAACATG-3′.

All animal experiments were conducted in a humane manner in accordance with the Regulation for Animal Experiments of the University and Fundamental Guidelines for Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology of Japan, and were approved by the Institutional Animal Experiment Committee of the University of Tsukuba. All surgery was performed under isoflurane anesthesia and all efforts were made to minimize suffering.

Eight-week-old male severe combined immunodeficiency (SCID) mice weighing 21–26 g (CLEA Japan, Tokyo, Japan) were utilized in all experiments. The mice were housed in a temperature-controlled room with a 12-h light-dark cycle. They had free access to water and standard chow throughout the experiment. After an acclimation period of at least 7 days, laparotomy was performed with midline incision, and hepatic ischemia was induced by clamping the portal triad (hepatic artery, portal vein and bile duct) with a microclip (Aesculap, Tuttlingen, Germany) for 1 min. At 1 min after reperfusion of the liver, 2×10⁶ RKO cells were injected into the lower splenic pole with a 27-gauge needle, and splenectomy was performed to prevent peritoneal dissemination from the spleen tumor. The mice were separated into two groups; the control group remained untreated (n=12), and the EGCG group was treated with EGCG (n=12). In the EGCG group, we administered EGCG (30 mg/kg body weight) by i.p. injection every other day over a 2-week period beginning 1 week after inoculation. At 21 days after inoculation, the mice were sacrificed and their livers were removed for examination.

To assess the liver metastatic area, all lobes of the liver were divided into 2 pieces, fixed with 10% neutral buffered formaldehyde. The tissues were embedded in paraffin and cut into 4 μm sections. They were stained with hematoxylin-eosin, and the percentage of liver metastatic area was calculated using the image-processing software WinROOF (Mitani Co., Fukui, Japan).

The paraffin-embedded tissues were deparaffinized with xylene, rehydrated with ethanol, immersed in 0.03% hydrogen peroxidase to block endogenous peroxidase activity, and then blocked with 3% bovine serum albumin (BSA) in phosphate-buffered saline to reduce background staining. An anti-CD31 antibody (LifeSpan Biosciences, Seattle, WA, USA) diluted 1:100 in 3% BSA was added to the sections and incubated for 1 h at room temperature. They were then incubated with a peroxidase-labeled polymer conjugated to goat anti-rabbit immunoglobulins (Dako, Tokyo, Japan) for 30 min at room temperature, followed by diaminobenzidine chromogen (Dako) for 5 min. The sections were counterstained with hematoxylin, dehydrated with ethanol, made transparent with xylene and mounted with Multi Mount 480 (Matsunami Glass, Osaka, Japan).

Blood was collected from the retro-orbital sinuses of the mice before sacrifice. The blood was centrifuged at 1,200 × g for 10 min at 4°C. Supernatants were collected and stored at −20°C until testing using a FUJI DRI-CHEM serum multiple biochemical analyzer (Fujifilm, Tokyo, Japan) to measure liver function, including aspartate aminotransferase (AST) and alanine aminotransferase (ALT).

All data are expressed as the means ± standard deviation of samples. Unpaired t-test was used to compare two groups. Comparisons among more than three groups were performed using one-way analysis of variance with the Bonferroni post-test. In all cases, P<0.05 was considered to indicate a statistically significant difference.

We initially determined whether EGCG treatment led to the inhibition of colorectal cancer cell proliferation. Colorectal cancer cells were treated with various doses of EGCG for 24 h. Cell viability was assayed using the CCK-8 assay (Fig. 1A and B), and DNA synthesis was assessed using the BrdU assay (Fig. 1C and D). As shown in Fig. 1, EGCG at 50 and 100 μM inhibited the proliferation of both colorectal cancer cells significantly compared to no EGCG treatment (P<0.01).

The cancer cells were treated with various doses of EGCG for 4 h, and cell apoptosis was detected by TUNEL staining. As shown in Fig. 2, TUNEL staining revealed that EGCG induced apoptosis in both colorectal cancer cells at 50 and 100 μM.

In subsequent experiments, we examined the effects of EGCG on the expression of the signal transduction pathways in colorectal cancer cells. When RKO and HCT116 cells were treated with 25, 50 or 100 μM EGCG, there was the dephosphorylation of constitutive phosphorylation of Akt. In RKO cells, there was a marked increase in phospho-p38 at 50 and 100 μM EGCG. In HCT116 cells, the activation of p38 occurred at 100 μM EGCG (Fig. 3).

We examined the effects of EGCG on the expression of VEGFR2, EGFR, HER2 and c-Met mRNAs in RKO cells. Quantitative real-time RT-PCR indicated that treatment with 25, 50 or 100 μM EGCG reduced the level of VEGFR2 mRNA significantly compared to no treatment; EGFR, HER2 and c-Met mRNAs were not affected by EGCG treatment (Fig. 4).

To evaluate the effect of EGCG on liver metastases, we administered EGCG (30 mg/kg body weight) by i.p. injection every other day over a 2-week period beginning 1 week after RKO cell inoculation. Fig. 5A and B shows macroscopic findings and histological cross-sections of the livers removed from representative control and EGCG mice. A reduction in the growth of liver metastases was observed in the EGCG group. Fig. 5C shows the tumor areas throughout the liver in both groups. Tumor growth was significantly reduced by EGCG administration. Fig. 5D shows the TUNEL staining of liver sections in the control and EGCG groups. In the EGCG group, apoptotic tumor cells were observed within the liver metastases. To further investigate whether EGCG inhibited angiogenesis, we used an anti-CD31 antibody to stain liver metastases. As shown in Fig. 5E, fewer tumor blood vessels were observed in the EGCG group than in the control group. No signs of toxicity, such as weight loss or elevated AST and ALT, were observed in mice treated with EGCG compared to the control mice (Fig. 6).