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Introduction

Colorectal cancer is one of the most common types of cancer and is responsible for 10% of all cancer-related mortality worldwide (1). The liver is the most common site of distant metastasis in colorectal cancer. Approximately 15% of patients have liver metastases when the primary tumor is diagnosed (2), and half of patients ultimately develop liver metastases during the course of colorectal cancer. Colorectal cancer is unique among solid tumors in that the surgical resection of distant metastases, such as those in the liver and lung, can offer long-term survival in selected patients (3). Indeed, hepatic resection is the only curative treatment and is the standard therapy for liver metastases of colorectal cancer. However, only 25% of patients with liver metastases are candidates for liver resection based on tumor number, size and location (4), and one-third of patients treated with curative liver resection experience a recurrence in the liver (5,6). Systemic chemotherapy is the treatment of choice for patients with unresectable liver metastases (7). Although long-term survival is rare among patients treated only with chemotherapy (8), recent advances in systemic chemotherapy have prolonged the survival of patients with unresectable metastases (9). The development of new agents and the shift from monotherapy to combination chemotherapy have been key in improving the clinical outcome for patients with unresectable metastases.

Green tea, a common beverage worldwide, has been studied extensively for its health benefits, including its anticancer effects (10). Epidemiological studies have shown that green tea has a potential preventive effect against colorectal cancer (11,12). The antiproliferative effect of green tea has been attributed to the biological activities of its polyphenol components. The principal polyphenols in green tea include (−)-epicatechin (EC), (−)-epicatechin-3-gallate (ECG), (−)-epigallocatechin (EGC) and (−)-epigallocatechin-3-gallate (EGCG). EGCG is the most abundant and accounts for half of tea polyphenols (13); it is also the major biologically active component that inhibits cell proliferation and induces apoptosis in colorectal cancer cells (14). Recent studies have revealed that EGCG exerts its antiproliferative effect, at least in part, by modulating the activities of various receptor tyrosine kinases and their multiple downstream signaling pathways, including the Akt signaling pathway, which controls the expression of the multiple target genes involved in cell proliferation and apoptosis (15). Additionally, EGCG activates stress signals, such as c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), and induces apoptosis in colorectal cancer cell lines (16). EGCG has also been reported to inhibit the growth of human colorectal cancer cells in subcutaneous xenograft models (17–19). However, no previous studies have reported the ability of EGCG to suppress liver metastases of human colorectal cancer. The aim of the present study was to extend the investigation of the potential use of EGCG to treat liver metastases of human colorectal cancer. We examined the effect of EGCG on cell proliferation and apoptosis in the human colorectal cancer cell lines, RKO and HCT116. Subsequently, we investigated the effect of EGCG on liver metastases of RKO tumors in vivo.

Materials and methods

EGCG

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.

Cell culture

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.

Cell viability assay

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.

Cell proliferation assay

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.

Apoptosis assay

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.

Western blot analysis

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.

Total RNA extraction

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.

Quantitative real-time RT-PCR

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′.

Animal model of liver metastasis

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.

Histological 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).

Immunohistological examination

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).

Serum parameters

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).

Statistical analysis

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.

Results

Inhibition of colorectal cancer cell proliferation by EGCG

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).

Figure 1 EGCG inhibits cell proliferation in colorectal cancer cells. Colorectal cancer cells were treated with 0–100 μM EGCG. (A) CCK-8 of RKO; (B) CCK-8 of HCT116; (C) BrdU of RKO; and (D) BrdU of HCT116. Data are expressed as the means ± standard deviation with n=8 for each group. The values indicate the ratio compared to 0 μM EGCG as 100%. *P<0.01 compared to 0 μM EGCG.

Induction of apoptosis by EGCG

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.

Figure 2 EGCG induces apoptosis in colorectal cancer cells. TUNEL staining of RKO cells treated with (A) EGCG 0 μM, (B) 25 μM, (C) 50 μM and (D) 100 μM. The staining of HCT116 cells treated with (E) EGCG 0 μM, (F) 25 μM, (G) 50 μM and (H) 100 μM. Arrows indicate apoptotic colorectal cancer cells. Scale bars indicate 100 μm. Original magnification, ×400.

The dephosphorylation of Akt and the phosphorylation of p38 by EGCG

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).

Figure 3 EGCG induces the dephosphorylation of Akt and the phosphorylation of p38. Colorectal cancer cells were treated with 0, 25, 50 and 100 μM of EGCG. After cell lysis, equal amounts of proteins were separated by SDS-PAGE, transferred to an Immobilon membrane and immunoblotted with antibodies against phospho-Akt, Akt, phospho-p38, p38 and β-actin. Left, RKO; right, HCT116.

Decrease of VEGFR2 mRNA by EGCG

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).

Figure 4 EGCG decreases the expression of VEGFR2 mRNA. RKO cells were treated with 0, 25, 50 and 100 μM EGCG. Total RNA was isolated from these cells, and the expression of (A) VEGFR2, (B) EGFR, (C) HER2 and (D) c-Met mRNAs was examined by quantitative real-time RT-PCR. Data are expressed as the means ± standard deviation with n=4 for each group. *P<0.01 compared to 0 μM EGCG.

Suppression of liver metastases of human colorectal cancer by EGCG

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).

Figure 5 EGCG suppresses liver metastases of human colorectal cancer. (A) Representative livers from the control and EGCG groups. (B) Histological cross-sections of livers from the control and EGCG groups. Scale bars indicate 1 mm. Original magnification, ×40. (C) Liver metastatic area in the livers of all mice in the control and EGCG groups. Data are expressed as the means ± standard deviation with n=12 for each group. *P<0.05 compared to the control group. (D) TUNEL staining of tumor tissues. Many TUNEL-positive apoptotic cells were observed in the EGCG group compared to the control group. Scale bars indicate 100 μm. Original magnification, ×400. (E) Angiogenesis of liver metastases. Fewer tumor blood vessels were observed in the EGCG group than in the control group. Arrows indicate blood vessels stained with an anti-CD31 antibody. Scale bars indicate 100 μm. Original magnification, ×400.

Figure 6 EGCG has no side-effects, such as weight loss or hepatotoxicity. (A) Body weight, (B) serum aspartate aminotransferase (AST) and (C) alanine aminotransferase (ALT) levels in the control and EGCG groups. Data are expressed as the means ± standard deviation with n=12 for each group.

Discussion

The most common site of distant metastases in colorectal cancer is the liver. We extended the investigation of EGCG for the treatment of liver metastases of human colorectal cancer. In the present study, we revealed that EGCG inhibits cell proliferation and induces apoptosis in human colorectal cancer cells in vitro, and suppresses the growth of liver metastases of human colorectal cancer in vivo. Several studies of EGCG as a treatment of ectopic cancer models, particularly subcutaneous models, using various cancer cell lines, including colorectal cancer, have been reported (17–19). The major advantage of the ectopic model is that it allows the rapid screening of new cytotoxic agents. A variety of host organ environment factors have profound influences on the biological behavior of tumor cells, including the production of degradative enzymes, angiogenesis, the induction of differentiation and the transcriptional properties of genes. Studies using ectopic inoculation do not account for the organ-specific factors that influence the growth of tumor cells, and it is necessary to further examine the antitumor activity of agents in the appropriate organ-specific environment (20). This is the first study on inhibiting liver metastases of human colorectal cancer by administering EGCG.

Angiogenesis is the process of generating new blood vessels, and it plays a critical role in the growth and metastasis of solid tumors by supplying nutrients and oxygen and removing waste products from the tumor (21). The inhibition of angiogenesis using chemotherapeutic agents, such as bevacizumab, is an attractive strategy for antitumor treatment (22). VEGFR2 activates various downstream signal transduction proteins, including Akt (23), which is a critical regulator of cell survival, proliferation, migration and angiogenesis (24). The interruption of VEGFR2 signaling is considered necessary for inhibition of tumor angiogenesis and tumor growth. In the present study, we demonstrated that EGCG inhibited the expression of VEGFR2 and the phosphorylation of Akt in vitro and that it suppressed angiogenesis and the growth of liver metastases in vivo.

p38 is a member of the MAPK family that is activated in response to a variety of cellular stresses including osmotic shock, inflammatory cytokines, lipopolysaccharide and UV light (25). Once phosphorylated, p38 interacts with a wide range of substrates and acts on inflammatory responses, cell differentiation, cell cycle arrest, senescence, apoptosis and other cell pathways (26). Anticancer drugs, such as irinotecan and oxaliplatin, have been reported to activate p38 in colon cancer cells (27,28). EGCG can induce apoptosis in human colon adenocarcinoma cells by activating JNK and p38 MAPK (16). In the present study, we demonstrated that EGCG promoted the phosphorylation of p38 and induced apoptosis.

Cases of hepatotoxicity have been associated with the consumption of high doses of dietary supplements containing green tea (29), and a high oral dose of EGCG has been reported to induce hepatotoxicity in mice (30). In the present study, EGCG administration did not induce hepatotoxicity and weight loss. EGCG preferentially inhibits the growth of human colorectal and liver cancer cells in comparison to normal colon epithelial cells and hepatocytes (31,32). Taken together, the administration of an appropriate dose of EGCG has little influence on normal cells and effectively controls liver metastases of colorectal cancer with few side-effects.

In the present study, EGCG inhibited liver metastases of human colorectal cancer in a mouse model. EGCG was recently reported to have a synergistic antitumor effect in combination with existing anticancer drugs, such as 5-fluorouracil (5-FU), capecitabine and oxaliplatin (33–35). Both 5-FU and oxaliplatin are key drugs for treating patients with liver metastases of colorectal cancer (9), and EGCG treatment in combination with these drugs has the potential to prolong survival in patients with liver metastases of colorectal cancer. Green tea, which contains abundant EGCG, is a commonly consumed beverage worldwide, and the oral intake of EGCG is reported to inhibit the growth of colorectal cancer in vivo (17). In a pilot study, supplementation with green tea extract prevented the development of metachronous colorectal adenomas (36). Collectively, EGCG may be suitable not only for chemotherapy for patients with liver metastases of colorectal cancer in combination with existing anticancer drugs, but also for oral adjuvant therapy for patients after colorectal cancer surgery.

In conclusion, EGCG inhibits cell proliferation and induces apoptosis in human colorectal cancer through Akt, p38 and VEGFR2, and it suppresses angiogenesis and liver metastases of colorectal cancer. EGCG may be useful in the treatment of liver metastases of human colorectal cancer.

Acknowledgements

The present study was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) and Public Trust Surgery Research Fund. The authors thank Ako Takahashi for providing technical assistance.

References