c-Src plays a role in internalization of epidermal growth factor receptors (EGFR) induced by (-)-epigallocatechin gallate (EGCG) in SW480 colon cancer cells.

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EGCG, the major biologically active constituent of green tea, inhibits activation of the EGFR and its downstream signaling pathways in several types of human cancer. We recently discovered that this inhibition is associated with internalization of the EGFR. Although the precise mechanism of ligand-induced EGFR internalization is still unclear, some reports suggest that a src family non-receptor tyrosine kinase may play a role in this process. Therefore, we hypothesized that a src family kinase may also play a role in EGCG-induced EGFR internalization. Using SW480 cells, we conducted a time course study and found that EGCG can activate c-Src in a dose- and time-dependent manner. Activation was detected with as low as 1μM of EGCG and within 10min. This time course is consistent with our previous findings that EGCG can induce EGFR internalization within 30min. We examined whether EGCG can bind to the c-Src protein by affinity chromatography using EGCG-coupled Sepharose beads. SW480 total cell lysates were applied to the EGCG affinity column and eluted fractions were analyzed by western blot analysis. c-Src was detected in the fraction containing proteins that bind to EGCG with high affinity. Next, we investigated the effect of EGCG on the level of cell surface EGFR using a quantitative ELISA assay, and found that EGCG 40μM causes a significant decrease in the amount of cell surface EGFR. This reduction was partially rescued by the src kinase specific inhibitor PP1. We confirmed these findings using fluorescence microscopy by demonstrating that combined treatment with EGCG and PP1 decreased the amount of internalized vesicles containing EGFR. Src family kinases have been shown to bind to and laterally activate EGFR, leading to stimulation of specific downstream signaling pathway and DNA synthesis. Therefore, we examined the phosphorylation status of EGFR by immunoblotting using site-specific and non-specific anti-phospho-tyrosine antibodies. In these studies, treatment with EGCG did not cause phosphorylation of tyrosine residues or increase total tyrosine phosphorylation of EGFR. Taken together, our studies provide the first evidence that EGCG can bind to and preferentially activate c-Src and that this activation may play a role in EGCG-induced internalization of the EGFR in SW480 cells. However, this activation of c-Src does not activate the EGFR and its downstream signaling pathways. Our findings suggest a novel mechanism by which EGCG inhibits the EGFR pathway. Further studies will be required to determine the significance of c-Src activation by EGCG with respect to its anti-cancer effects.