The inhibitory effects of (-)-Epigallocatechin Gallate (EGCG) on epidermal growth factor receptor (EGFR) activity are associated with changes in membrane domain organization

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We previously reported that EGCG inhibits growth and activation of the EGF receptor (EGFR) and downstream signaling pathways in human colon cancer cells (Shimizu et al., Clin. Cancer Res. 11, 2735-2746, 2005), but the precise mechanism is not known. Several proteins, including EGFR, are believed to partition into detergent-insoluble liquid ordered membrane domains enriched in cholesterol, sphingomyelin, and gangliosides, so-called “lipid rafts”. Therefore, we examined the effects of EGCG on ordered membrane domains in HT29 colon cancer cells. When the cells were first treated with the fluorescent lipid analog DiIC_16,, which preferentially incorporates into ordered domains in the plasma membrane, subsequent treatment with EGCG caused a marked increase the sensitivity of the plasma membrane to extraction by cold Triton X-100, indicating that liquid ordered domains are decreased. Pretreatment with EGCG also inhibited subsequent incorporation of DiIC₁₆ into the plasma membrane, indicating that EGCG caused a change in membrane organization. This effect was detected with as little as 2 μg/ml of EGCG, and within 5 minutes. Polyphenon E (PolyE), a mixture of catechins, also inhibited incorporation of DiIC₁₆, but epicatechin (EC) itself, which is a very weak inhibitor of the growth of HT29 cell, was inactive. Phosphorylation of EGFR, ERK1/2 and AKT were suppressed in cells treated with methyl-β-cyclodextrin, which depletes plasma membrane cholesterol. However, filipin staining indicated that the effects of EGCG on plasma membrane properties are not associated with cholesterol depletion. In addition, the inhibitory effect of EGCG on phosphorylation of EGFR was not blocked by the addition of cholesterol to the growth medium. These findings provide evidence that EGCG can alter membrane domain organization in colon cancer cells and that this is not due to cholesterol depletion. Furthermore, we found that pre-treatment of the cells with EGCG or PolyE, but not EC, inhibited the binding of Alexa 488-labeled EGF to the cell surface receptor EGFR. Taken together, these data suggest that EGCG inhibits EGF binding, subsequent activation of the EGFR and downstream signaling pathway by disrupting membrane domain organization. These effects on membrane domain organization may also explain the ability of EGCG to inhibit activation of other membrane-associated tyrosine kinase receptors. Further studies are in progress to confirm this mechanism.