(-)-Epigallocatechin-3-gallate Inhibits Hsp90 Function by Impairing Hsp90 Association with Cochaperones in Pancreatic Cancer Cell Line Mia Paca-2

(-)-Epigallocatechin-3-gallate [(-)-EGCG], the most abundant polyphenolic catechin in green tea, showed chemoprevention and anticancer activities. (-)-EGCG was reported to bind to the C-terminal domain of heat shock protein 90 (Hsp90). The purpose of this study is to investigate (-)-EGCG as a novel Hsp90 inhibitor to impair Hsp90 superchaperone complex for simultaneous downregulation of oncogenic proteins in pancreatic cancer cells. MTS assay showed that (-)-EGCG exhibited antiproliferative activity against pancreatic cancer cell line Mia Paca-2 in vitro with IC50 below 50 mu M. (-)-EGCG increased caspase-3 activity up to 3-fold in a time- and concentration-dependent manner. Western blotting analysis demonstrated that (-)-EGCG induced downregulation of oncogenic Hsp90 client proteins by approximately 70-95%, including Akt, Cdk4, Raf-1, Her-2, and pERK. Co-immunoprecipitation showed that (-)-EGCG decreased the association of cochaperones p23 and Hsc70 with Hsp90 by more than 50%, while it had little effect on the ATP binding to Hsp90. Proteolytic fingerprinting assay confirmed direct binding between (-)-EGCG and the Hsp90 C-terminal domain. These data suggest that the binding of (-)-EGCG to Hsp90 impairs the association of Hsp90 with its cochaperones, thereby inducing degradation of Hsp90 client proteins, resulting antiproliferating effects in pancreatic cancer cells.