(-)-Epigallocatechin-3-gallate inhibits Met signaling, proliferation, and invasiveness in human colon cancer cells

Abstract

The Met receptor tyrosine kinase is deregulated in a variety of cancers and is correlated with advanced stage and poor prognosis. Thus, Met has been identified as an attractive candidate for targeted therapy. We compared the tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) and a specific Met inhibitor, SU11274, as suppressing agents of Met signaling in HCT116 human colon cancer cells. Treatment with hepatocyte growth factor increased phospho-Met levels, and this was inhibited in a concentration-dependent manner by EGCG and SU11274 (IC(50) 3.0 vs. 0.05muM, respectively). Downstream activation of Erk and Akt signaling pathways also was suppressed. Both compounds at a concentration of 5muM lowered cell viability and proliferation, with EGCG being more effective than SU11274, and the invasion of colon cancer cells in Matrigel assays was strongly inhibited. These findings are discussed in the context of the pleiotropic effects of tea catechins, their tissue metabolite levels, and the potential to inhibit colon cancer metastasis and invasion.

Fig. 1

EGCG and SU11274 inhibit Met activation in human colon cancer cells. (A) Chemical structures of SU11274 and EGCG. (B) HCT116 cells were serum-starved for 4 h and then treated with various concentrations of EGCG or SU11274 for 30 min. HGF (30 ng/ml) was added to the cell culture media, followed by incubation for an additional 15 min. Immunoblotting was performed on cell lysates using primary antibodies to phosphorylated-Met (p-Met), total Met, or β-actin (loading control). (C) Results from STAR phospho-Met ELISA kit (Upstate), using cell lysates from (B). Data (mean±SD, n=3) are representative of the findings from three separate experiments.

Fig. 2

Inhibitory effects of EGCG on MAPK and PI3K signaling pathways. (A) HCT116 cells were serum-starved for 4 h and treated with or without 5 µM EGCG for 30 min. HGF (30 ng/ml) was added and incubations were continued for the times indicated. Immunoblotting was performed on cell lysates using primary antibodies to phosphorylated-Met (p-Met), total Met, phosphorylated-Akt (p-Akt), total Akt, phosphorylated-Erk (p-Erk), total Erk, or β-actin (loading control). (B) Quantification of immunoblots by densitometry, showing phosphorylated protein normalized to corresponding total protein. Open bars −EGCG, solid bars +EGCG. (C) Using the same approach as above, HCT116 cells were serum-starved for 4 h, treated for 30 min with 5 µM SU11274, 5 µM EGCG, or 5 µM SU11274 + 5 µM EGCG in combination, followed by HGF or media alone (-HGF). Immunoblots (not shown) were quantified from three separate experiments. In (B) and (C), data=mean±SD, n=3; *P<0.05; **P<0.01.

Fig. 2

Inhibitory effects of EGCG on MAPK and PI3K signaling pathways. (A) HCT116 cells were serum-starved for 4 h and treated with or without 5 µM EGCG for 30 min. HGF (30 ng/ml) was added and incubations were continued for the times indicated. Immunoblotting was performed on cell lysates using primary antibodies to phosphorylated-Met (p-Met), total Met, phosphorylated-Akt (p-Akt), total Akt, phosphorylated-Erk (p-Erk), total Erk, or β-actin (loading control). (B) Quantification of immunoblots by densitometry, showing phosphorylated protein normalized to corresponding total protein. Open bars −EGCG, solid bars +EGCG. (C) Using the same approach as above, HCT116 cells were serum-starved for 4 h, treated for 30 min with 5 µM SU11274, 5 µM EGCG, or 5 µM SU11274 + 5 µM EGCG in combination, followed by HGF or media alone (-HGF). Immunoblots (not shown) were quantified from three separate experiments. In (B) and (C), data=mean±SD, n=3; *P<0.05; **P<0.01.

Fig. 3

Inhibition of cell viability and proliferation by EGCG and SU11274. (A) Viability of HCT116 cells treated with EGCG, SU11274, or vehicle control in the presence and absence of HGF. (B) Proliferation of HCT116 cells treated with EGCG, SU11274, or vehicle, in the presence and absence of HGF. Data shown in (A) and (B) are representative findings from three separate experiments (mean±SD, n=3); *P<0.05; **P<0.01; ***P<0.001.

Fig. 4

Suppression of invasion by ECGC and SU11274. (A) Representative images showing invasion of HCT116 cells through matrigel-coated Transwell inserts, under the assays conditions indicated. (B) Quantification of three independent in vitro invasion assays (mean±SD, n=3). *P<0.05; **P<0.01; n/s = not significant.