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

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Abstract
(−)-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 μM. (−)-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.