Green Tea Polyphenol Epigallocatechin-3 Gallate Inhibits Her-2/Neu Signaling, Proliferation, and Transformed Phenotype of Breast Cancer Cells¹

The Her-2/neu (or c-erbB-2) oncogene, which is the second member of the EGFR⁴ family (EGFR-2), encodes a transmembrane tyrosine receptor kinase. Overexpression of Her-2/neu, which has been seen in ∼30% of breast cancers, is associated with poor overall survival (1). In particular, it has been found associated with increased metastatic potential and resistance to chemotherapeutic agents. Transgenic mice overexpressing Her-2/neu develop focal mammary tumors (2). Recent work has implicated the PI 3-kinase to serine/threonine kinase Akt/protein kinase B to NF-κB signaling pathway in control of growth and transformed phenotype of Her-2/neu-overexpressing cells (3, 4, 5). Green tea is rich in polyphenols, such as EGCG, that possess antioxidant qualities, which have been shown to have anticarcinogenic activity against a variety of tumor types including breast cancer (reviewed in Ref. 6). For example, we recently showed that female rats given green tea as their drinking fluid displayed a significant decrease in carcinogen-induced mammary tumor burden and invasiveness and significantly increased latency to first tumor (7). Doses of either green tea polyphenol mix or EGCG between ∼40 to 80 μg/ml slowed growth of various estrogen receptor-negative breast cancer cell lines in culture (7). Furthermore, statistics indicate that the incidence of breast cancer in regions in which green tea is consumed in large quantities, including China and Japan, is much lower than in western societies. Here we have examined the effects of EGCG on Her-2/neu-overexpressing breast cancer cells. We report that treatment of MMTV-Her-2/neu mouse mammary tumor NF639 cells with EGCG slows proliferation and reduces growth in soft agar via inhibiting the PI 3- to Akt kinase to NF-κB pathway. EGCG-reduced basal receptor tyrosine phosphorylation in NF639 and two other Her-2/neu-overexpressing lines. These findings suggest the use of EGCG may be beneficial in chemoprevention of breast cancer and represents a possible tool in adjuvant therapy modalities of patients with tumors overexpressing the Her-2/neu receptor.

The MMTV-Her-2/neu cell lines NF639 and SMF (kindly provided by P. Leder, Harvard Medical School, Boston, MA) were derived from mammary gland tumors and cultured as described previously (8). Ba/F3 pro-B cells, which are transfected to express Her-2/neu and EGFR-4 (Ba/F3–2 + 4), were kindly provided by David Stern (Yale University, New Haven, CN; Ref. 9). These cells, which display high constitutive Her-2/neu phosphorylation (5), were grown in RPMI supplemented with 10% fetal bovine serum, conditioned medium from WEHI 3 cells, and antibiotics, as described previously (9). EGCG, purchased from LKT Laboratories Inc. (St Paul, MN), was dissolved in sterile 50% DMSO at a concentration of 100 mg/ml, and diluted with double-distilled H₂O to a 1 mg/ml working-strength solution. Nonradioactive MTS cell proliferation assays (Promega) were performed essentially as we have published previously (7).

Nuclear extracts were prepared from breast cancer cells in extraction buffer [420 mm KCl, 20 mm HEPES (pH 7.9), 1.5 mm MgCl₂, 0.2 mm EDTA, and 20% glycerol] plus protease inhibitors (0.5 mm DTT, 0.5 mm PMSF, and 10 μg/ml LP), as we have published previously (5). The sequence of the URE NF-κB-containing oligonucleotide from the c-myc gene is as follows: 5′-GATCCAAGTCCGGGTTTTCCCCAACC-3′, in which the underlined region indicates the core binding element. The sequence of the Sp1 oligonucleotide is 5′-ATTCGATCGGGGCGGGGCGACC-3′. Oligonucleotides were end labeled with large Klenow fragment of DNA polymerase and [³²P]dNTPs. The EMSA was performed using 5 μg of nuclear extract, essentially as we have published previously (5).

The Akt kinase assay, was performed following the directions of the Akt Kinase Assay kit (New England Biolabs, Beverly, MA). Briefly, samples (100-μg) of WCEs were immunoprecipitated overnight with an agarose conjugated anti-Akt antibody (New England Biolabs) at 4°C. The immunoprecipitate was resuspended in kinase buffer, and the assay performed at 30°C for 45 min, using 1 μg of GSK3α-GST fusion protein as substrate in the presence of 10 μm ATP. The resulting products were resolved in a 10% polyacrylamide-SDS gel and subjected to immunoblotting, as below, using phosphospecific GSK-3α antibody (New England Biolabs).

Cells were rinsed with cold PBS, and harvested in lysis buffer [50 mm Tris-HCl (pH 8.0), 5 mm EDTA (pH 8.0), 150 mm NaCl, 0.5 mm DTT, 2 μg/ml aprotinin, 2 μg/ml LP, 0.5 mm PMSF, 0.5% and NP40]. WCEs were obtained by sonication, followed by centrifugation at 14,000 rpm for 30 min. Samples were subjected to electrophoresis in a 10% polyacrylamide-SDS gel and immunoblotting, as previously described (5). Antibodies used were: phosphorylated Akt Ser473 (Cell Signaling) and tyrosine phosphorylated Her-2/neu (Santa Cruz Biotechnology), and β-actin (Sigma Chemical Co.).

NF639 cells were plated, in triplicate, at 7.5 × 10³/ml in top plugs consisting of complete medium and 0.4% SeaPlaque agarose (FMC Bioproducts, Rockland, ME) with the indicated concentration of EGCG. Plates were subsequently incubated for 2 weeks in humidified incubator at 37°C. Cells were stained with 2 ml of crystal violet solution and washed extensively with water, and colonies were counted using an inverted bright field microscope at a ×2 magnification. Three random fields were counted from each of the triplicate samples, and average values presented ± SD, which was determined in each set of nine values obtained using the Student’s t test.

Twenty-four h after plating at 30% confluence in P150 dishes, NF639 cells were transfected with 5 μg of NF-κB-element-driven luciferase reporter DNA (10), kindly provided by Georges Rawadi (Hoescht-Marion-Roussel, Romainville, France) with 25 μg of pcDNA empty vector in 45 μl of FuGENE Reagent. Three h after transfection, cells were trypsinized and replated in multiple P60 dishes. After overnight incubation, cells were treated in duplicate with either 50 μg/ml EGCG or carrier DMSO solution for 24 h. Cells were harvested and extracts assayed for luciferase activity as described previously (5). Averages values are presented ± SD. SD was obtained using the Student’s t test.

We first tested the ability of EGCG to inhibit growth of the MMTV-Her-2/neu mouse breast tumor derived cell line NF639. Cultures were plated, in triplicate, at a density of 3.9 × 10³ cells/cm². After 24 h, EGCG was added to a final concentration of 20–160 μg/ml, or the volume of carrier DMSO equivalent to the highest dose was added. Cell growth was assessed every 24 h using a nonradioactive MTS cell proliferation assay (Fig. 1). A dose-dependent decrease in the rate of proliferation was seen with 20 and 40 μg/ml EGCG, whereas no cell growth was seen with 80 μg/ml EGCG. On treatment with 160 μg/ml EGCG, a drop in cell numbers was seen. This suggested that extensive death of NF639 cells was occurring only at this higher dose, which was confirmed by trypan blue staining of cells with 0, 40, 80, or 160 μg/ml EGCG (data not shown). Thus, treatment of the NF639 breast tumor cells with 20–80 μg/ml EGCG decreases their rate of proliferation in culture.

Growth in soft agar is a hallmark of transformed phenotype. Hence, we next assessed the ability of EGCG to reduce growth of NF639 cells in soft agar. NF639 cells were plated, in triplicate, at a density of 7.5 × 10³ cells/ml in top plugs consisting of complete medium and 0.4% SeaPlaque agarose containing 0, 20, 40 or 80 μg/ml EGCG. The plates were incubated for two weeks and colonies stained with crystal violet and counted as described in the “Materials and Methods.” A decrease in colony numbers was seen which was more pronounced with increasing doses of EGCG (Fig. 2, inset). Quantitation of the results confirmed that the inhibition of colony formation occurred in a dose-dependent fashion with EGCG treatment (Fig. 2). Thus, EGCG inhibits growth in soft agar, an important property of the transformed phenotype.

To assess the effects of EGCG on Her-2/neu signaling, we first monitored the recently identified downstream target NF-κB (5). NF639 cells were treated with 40 μg/ml EGCG for 24 h and nuclear extracts prepared. These were used in EMSA with an oligonucleotide containing the NF-κB element upstream of the c-myc promoter. Extracts from DMSO-treated cells gave rise to two bands (Fig. 3,A). These have been identified previously as containing complexes composed of p65/p50 and p50 homodimers (p50/p50; 5). Incubation in the presence of 40 μg/ml EGCG reduced formation of both of these two complexes. This decrease was selective, because no change was seen in Sp-1 binding (Fig. 3 A). We next assessed the effects of EGCG on NF-κB activity. A transfection assay was performed using an NF-κB element-driven luciferase reporter. In two experiments, treatment with 50 μg/ml EGCG for 24 h reduced NF-κB activity by 76.3% ± 5.9%. Thus, EGCG inhibits NF-κB binding and activity in NF639 cells.

Previously, we showed that induction of NF-κB in NF639 cells occurs via a PI 3-kinase-to-Akt-kinase pathway (5). To test the effect of EGCG treatment on Akt phosphorylation, the cells were incubated for 24 h in the presence of either 40 μg/ml of EGCG that had been dissolved in DMSO or the equivalent amount of carrier solution. Alternatively, cells were incubated in the presence of 100 nm Wortmannin, a potent specific inhibitor of PI 3-kinase (11). WCEs were prepared and immunoprecipitated with a monoclonal anti-Akt, which preferentially recognizes phosphorylated protein. GSK3α-GST was then used as the substrate for the resulting immunoprecipitated Akt, and the phosphorylated material identified by immunoblot analysis for phosphorylated GSK3α-GST protein (Fig. 3 B). The EGCG treatment resulted in a dramatic decrease in phosphorylated GSK3α-GST. A similar decrease was observed with Wortmannin treatment.

As a test for the effects of EGCG on PI 3-kinase activity, we measured the presence of phosphorylated Akt, which is one of its critical signaling substrates. Immunoblotting was performed using an antibody specific for phospho-Akt (Ser473). Treatment with EGCG caused an ∼50% decrease in phosphorylated Akt protein (Fig. 3 C). A similar level of decrease was observed on treatment with Wortmannin, which suggested that the remaining phosphoprotein detected is attributable to other kinase activities. Immunoblotting for β-actin confirmed equal loading. Together, these results demonstrate the ability of EGCG to reduce the activities of both PI 3-kinase and Akt.

Overexpression of Her-2/neu can lead to constitutive phosphorylation and basal receptor activation and signaling. Thus, we tested whether treatment with EGCG can reduce this basal Her-2/neu phosphorylation. NF639 cells were treated with 0, 20, 40, or 80 μg/ml EGCG for 48 h. WCEs were isolated and subjected to immunoblot analysis using a tyrosine phosphospecific Her-2/neu antibody (Fig. 4,A). A dose-dependent drop in phosphorylated Her-2/neu protein was noted. Densitometry indicated treatment with 20, 40, or 80 μg/ml caused a decrease of 13, 38, and 96%, respectively compared with control cells. Immunoblotting for β-actin confirmed equal loading. To verify that this effect was general, we analyzed two additional cell lines: (a) SMF cells, similarly derived from an MMTV-Her-2/neu mouse mammary tumor (8); and (b) Ba/F3 2 + 4 cells, a pro-B cell line clone stably expressing Her-2/neu + EGFR-4 (5, 9), displaying high constitutive Her-2/neu activity (5). SMF and Ba/F3 2 + 4 cells were treated with 40 μg/ml EGCG for 24 h and analyzed by immunoblotting for phosphorylated Her-2/neu (Fig. 4 B). EGCG treatment similarly reduced basal phosphorylation of Her-2/neu in both cell lines. Thus, EGCG reduces the basal phosphorylation and constitutive activation of the Her-2/neu receptor.

Here we show that the green tea polyphenol EGCG inhibits the signaling by Her-2/neu that promotes cell proliferation, survival, and transformed phenotype. Treatment of MMTV-Her-2/neu mammary gland tumor NF639 cells with doses of EGCG up to 80 μg/ml slowed growth and dramatically reduced colony formation in soft agar with little effect on cell viability. Higher doses (160 μg/ml) reduced cell numbers and induced cell death as judged by trypan blue assays. Previously, we demonstrated that the overexpression of Her-2/neu in NF639 cells leads to the induction of NF-κB via a PI 3-kinase/Akt kinase signaling pathway (5). EGCG inhibited this Her-2/neu signaling as judged by the observed decreases in PI 3-kinase, Akt kinase, and NF-κB activities. Furthermore, EGCG inhibited constitutive Her-2/neu phosphorylation in NF639 cells, as well as in SMF cells, a second mouse MMTV-Her-2/neu tumor-derived cell line, and Ba/F3 2 + 4 cells, a pro-B cell line transfected to express Her-2/neu + EGFR-4. Overall, these studies demonstrate for the first time that EGCG can ablate the Her-2/neu signaling cascade in breast cancer cells, by reducing basal Her-2/neu receptor phosphorylation. These results suggest further study of the potential role of EGCG in adjuvant therapy treatment modalities and of green tea components in chemoprevention of breast cancer are warranted.

In animal models, green tea extracts have been shown to inhibit chemical and photo carcinogenesis. For example, green tea was found to inhibit cancers of the gastrointestinal tract, lung, and skin in mice (reviewed in Ref. 6). It has been reported also that EGCG inhibits the growth of human breast and prostate tumors transplanted into athymic mice (12). We recently demonstrated that green tea extracts given to female Sprague Dawley rats in their drinking fluid significantly decrease DMBA-induced mammary tumor burden and invasiveness and significantly increase latency to first tumor (7). Another study using a diet containing 1% green tea catechins fed to female Sprague Dawley rats showed that tea was effective in reducing mammary gland tumorigenesis in the promotion, but not the progression, stages of carcinogenesis (13). Consistent with these findings, several studies have also found that green tea treatment of breast cancer cells in vitro reduces their rate of proliferation (7, 14, and references therein). Overall, the toxicity of green tea extracts is low, and, thus, they represent potentially useful cancer chemopreventive agents.

Here, EGCG was shown to reduce NF-κB levels and activity in NF639 cells because of its ability to inhibit the Her-2/neu signaling pathway that leads to NF-κB activation. Further insight into the regulatory mechanisms will be provided when the endogenous downstream targets of NF-κB responding to EGCG treatment are elucidated. Our findings are consistent with previous observations on the ability of EGCG to block signaling by the EGFR family (15, 16). For example, Liang et al. (15) demonstrated the ability of EGCG to block EGF signaling via the EGFR in A431 epidermoid carcinoma cells. Interestingly, we observed cooperative inhibition of NF-κB binding levels on EGCG cotreatment with an antibody against the Her-2/neu receptor.⁵ Yang et al. (17) observed that green tea polyphenols, including EGCG, could inhibit activated IκB kinase in the intestinal epithelial cell line IEC-6. Green tea extracts have also been found to block NF-κB activation in cancer cells on tumor necrosis factor-α, lipopolysaccharide, or UV treatment (18, 19). In contrast, EGCG failed to reduce NF-κB levels in Hs578T breast cancer cells⁶ that were derived from a carcinosarcoma and display constitutive IκB kinase activation and elevated levels of protein kinase CK2 (formerly casein kinase II) that promote increased NF-κB activity (20). Thus, different signaling pathways display variable sensitivities to EGCG. Of note, the basal activation of Her-2/neu seems sensitive to EGCG inhibition.

We thank G. Rawadi, and P. Leder, M. Michelman, and D. Stern for generously providing cloned DNAs, and cells, respectively. Darin Sloneker is acknowledged for assistance in preparation of the manuscript.