(-)-Epigallocatechin-3-gallate, Found in the Green Tea Extract, is a Novel Hsp90 Inhibitor

Thesis (Ph. D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Environmental Medicine, 2009.Numerous animal studies have shown that epigallocatechin-3-gallate (EGCG), a major component of green tea, protects against certain types of cancers, although the mechanism has not yet been determined. Our laboratory previously demonstrated that EGCG blocks aryl hydrocarbon receptor (AhR)-mediated transcription induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Unlike other AhR antagonists that directly bind to the AhR, EGCG inhibits AhR-mediated transcription by binding to hsp90. I hypothesize that EGCG exerts anti-AhR and anti-cancer effects by acting as an hsp90 inhibitor. Using proteolytic footprinting, immunoprecipitation and an ATP-agarose pull-down assay, I determined that EGCG directly modulates the conformation of hsp90 and binds at or near to the C-terminal ATP binding site of hsp90. Direct binding of 3H-EGCG to purified human hsp90 was observed by competitive binding assay suggesting a Kd of ~780nM. Novobiocin (a C-terminal hsp90 inhibitor) and ATP compete with 3H-EGCG for binding to hsp90. Hsp90 chaperone function, as demonstrated by the ability to mediate refolding of denatured luciferase, is inhibited by EGCG treatment. Hsp90 dimerization, which occurs at the C-terminal end, is also inhibited by EGCG treatment. EGCG also modified the association of hsp90 with several cochaperones. Co-immunoprecipitation studies show that EGCG stabilizes an AhR complex that includes hsp90 and XAP2 (hepatitis B virus X-associated protein 2), and decreases the association of aryl hydrocarbon nuclear translocator (Arnt) to ligand-activated AhR. Thus, EGCG, through its ability to bind to hsp90, decreases the association of AhR, an hsp90 client protein, with Arnt and blocks AhR response element (AhRE) recognition. These studies indicate a novel mechanism whereby EGCG inhibits ligand-induced AhRE binding and AhR-mediated transcriptional activity by acting as an hsp90 inhibitor. In EGCG-treated human ovarian carcinoma SKOV3 cells, decreased levels of several cancer-related hsp90 client proteins, such as ErbB2, Raf-1 and phospho-AKT were observed. Overall, these data indicate that EGCG is a novel hsp90 inhibitor. Further studies are needed to determine if this has a role in the anti-tumor actions of EGCG. Since hsp90 inhibitors are being intensively studied for cancer therapy, this investigation will advance further research that may ultimately lead to the use of EGCG and/or its derivatives for chemotherapeutic purposes