Withaferin A targets heat shock protein 90 in pancreatic cancer cells

Yanke Yu; Adel Hamza; Tao Zhang; Mancang Gu; Peng Zou; Bryan Newman; Yanyan Li; A. A.Leslie Gunatilaka; Chang Guo Zhan; Duxin Sun

doi:10.1016/j.bcp.2009.09.017

Withaferin A targets heat shock protein 90 in pancreatic cancer cells

Yanke Yu, Adel Hamza, Tao Zhang, Mancang Gu, Peng Zou, Bryan Newman, Yanyan Li, A. A.Leslie Gunatilaka, Chang Guo Zhan, Duxin Sun

Pharmaceutical Sciences

Research output: Contribution to journal › Article › peer-review

262 Scopus citations

Abstract

The purpose of this study is to investigate the efficacy and the mechanism of Hsp90 inhibition of Withaferin A (WA), a steroidal lactone occurring in Withania somnifera, in pancreatic cancer in vitro and in vivo. Withaferin A exhibited potent antiproliferative activity against pancreatic cancer cells in vitro (with IC₅₀s of 1.24, 2.93 and 2.78 μM) in pancreatic cancer cell lines Panc-1, MiaPaCa2 and BxPc3, respectively. Annexin V staining showed that WA induced significant apoptosis in Panc-1 cells in a dose-dependent manner. Western blotting demonstrated that WA inhibited Hsp90 chaperone activity to induce degradation of Hsp90 client proteins (Akt, Cdk4 and glucocorticoid receptor), which was reversed by the proteasomal inhibitor, MG132. WA-biotin pull down assay of Hsp90 using Panc-1 cancer cell lysates and purified Hsp90 showed that WA-biotin binds to C-terminus of Hsp90 which was competitively blocked by unlabeled WA. Co-immunoprecipitation exhibited that WA (10 μM) disrupted Hsp90-Cdc37 complexes from 1 to 24 h post-treatment, while it neither blocked ATP binding to Hsp90, nor changed Hsp90-P23 association. WA (3, 6 mg/kg) inhibited tumor growth in pancreatic Panc-1 xenografts by 30% and 58%, respectively. These data demonstrate that Withaferin A binds Hsp90, inhibits Hsp90 chaperone activity through an ATP-independent mechanism, results in Hsp90 client protein degradation, and exhibits in vivo anticancer activity against pancreatic cancer.

Original language	English
Pages (from-to)	542-551
Number of pages	10
Journal	Biochemical Pharmacology
Volume	79
Issue number	4
DOIs	https://doi.org/10.1016/j.bcp.2009.09.017
State	Published - Feb 15 2010

Bibliographical note

Funding Information:
This study is partially supported by NIH funding RO1 CA 120023, University of Michigan Cancer Center Research Grant (Munn), University of Michigan Cancer Center Core Grant to DS. We thank Dr. Thomas Ratajczak (University of Western Australia, Australia) for the generous gifts of the purified N-Hsp90β and expression plasmids, pET15b-hHsp90β and pET28a(+)-hHsp90β (530-724), respectively. We also thank Dr. Dan Bolon (University of Massachusetts, Massachusetts) for the kind gifts of the purified yeast Hsp90, human full length Hsp90, N-terminus Hsp90 and C-terminus Hsp90. We thank Luke Whitesell (Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142) for reagents and insightful suggestions.

Keywords

Cdc37
Client protein
Hsp90
Pancreatic cancer
Reactive cysteine
Withaferin A

ASJC Scopus subject areas

Biochemistry
Pharmacology

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1016/j.bcp.2009.09.017

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title = "Withaferin A targets heat shock protein 90 in pancreatic cancer cells",

abstract = "The purpose of this study is to investigate the efficacy and the mechanism of Hsp90 inhibition of Withaferin A (WA), a steroidal lactone occurring in Withania somnifera, in pancreatic cancer in vitro and in vivo. Withaferin A exhibited potent antiproliferative activity against pancreatic cancer cells in vitro (with IC50s of 1.24, 2.93 and 2.78 μM) in pancreatic cancer cell lines Panc-1, MiaPaCa2 and BxPc3, respectively. Annexin V staining showed that WA induced significant apoptosis in Panc-1 cells in a dose-dependent manner. Western blotting demonstrated that WA inhibited Hsp90 chaperone activity to induce degradation of Hsp90 client proteins (Akt, Cdk4 and glucocorticoid receptor), which was reversed by the proteasomal inhibitor, MG132. WA-biotin pull down assay of Hsp90 using Panc-1 cancer cell lysates and purified Hsp90 showed that WA-biotin binds to C-terminus of Hsp90 which was competitively blocked by unlabeled WA. Co-immunoprecipitation exhibited that WA (10 μM) disrupted Hsp90-Cdc37 complexes from 1 to 24 h post-treatment, while it neither blocked ATP binding to Hsp90, nor changed Hsp90-P23 association. WA (3, 6 mg/kg) inhibited tumor growth in pancreatic Panc-1 xenografts by 30% and 58%, respectively. These data demonstrate that Withaferin A binds Hsp90, inhibits Hsp90 chaperone activity through an ATP-independent mechanism, results in Hsp90 client protein degradation, and exhibits in vivo anticancer activity against pancreatic cancer.",

keywords = "Cdc37, Client protein, Hsp90, Pancreatic cancer, Reactive cysteine, Withaferin A",

author = "Yanke Yu and Adel Hamza and Tao Zhang and Mancang Gu and Peng Zou and Bryan Newman and Yanyan Li and Gunatilaka, {A. A.Leslie} and Zhan, {Chang Guo} and Duxin Sun",

note = "Funding Information: This study is partially supported by NIH funding RO1 CA 120023, University of Michigan Cancer Center Research Grant (Munn), University of Michigan Cancer Center Core Grant to DS. We thank Dr. Thomas Ratajczak (University of Western Australia, Australia) for the generous gifts of the purified N-Hsp90β and expression plasmids, pET15b-hHsp90β and pET28a(+)-hHsp90β (530-724), respectively. We also thank Dr. Dan Bolon (University of Massachusetts, Massachusetts) for the kind gifts of the purified yeast Hsp90, human full length Hsp90, N-terminus Hsp90 and C-terminus Hsp90. We thank Luke Whitesell (Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142) for reagents and insightful suggestions.",

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AU - Yu, Yanke

AU - Hamza, Adel

AU - Zhang, Tao

AU - Gu, Mancang

AU - Zou, Peng

AU - Newman, Bryan

AU - Li, Yanyan

AU - Gunatilaka, A. A.Leslie

AU - Zhan, Chang Guo

AU - Sun, Duxin

N1 - Funding Information: This study is partially supported by NIH funding RO1 CA 120023, University of Michigan Cancer Center Research Grant (Munn), University of Michigan Cancer Center Core Grant to DS. We thank Dr. Thomas Ratajczak (University of Western Australia, Australia) for the generous gifts of the purified N-Hsp90β and expression plasmids, pET15b-hHsp90β and pET28a(+)-hHsp90β (530-724), respectively. We also thank Dr. Dan Bolon (University of Massachusetts, Massachusetts) for the kind gifts of the purified yeast Hsp90, human full length Hsp90, N-terminus Hsp90 and C-terminus Hsp90. We thank Luke Whitesell (Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142) for reagents and insightful suggestions.

PY - 2010/2/15

Y1 - 2010/2/15

N2 - The purpose of this study is to investigate the efficacy and the mechanism of Hsp90 inhibition of Withaferin A (WA), a steroidal lactone occurring in Withania somnifera, in pancreatic cancer in vitro and in vivo. Withaferin A exhibited potent antiproliferative activity against pancreatic cancer cells in vitro (with IC50s of 1.24, 2.93 and 2.78 μM) in pancreatic cancer cell lines Panc-1, MiaPaCa2 and BxPc3, respectively. Annexin V staining showed that WA induced significant apoptosis in Panc-1 cells in a dose-dependent manner. Western blotting demonstrated that WA inhibited Hsp90 chaperone activity to induce degradation of Hsp90 client proteins (Akt, Cdk4 and glucocorticoid receptor), which was reversed by the proteasomal inhibitor, MG132. WA-biotin pull down assay of Hsp90 using Panc-1 cancer cell lysates and purified Hsp90 showed that WA-biotin binds to C-terminus of Hsp90 which was competitively blocked by unlabeled WA. Co-immunoprecipitation exhibited that WA (10 μM) disrupted Hsp90-Cdc37 complexes from 1 to 24 h post-treatment, while it neither blocked ATP binding to Hsp90, nor changed Hsp90-P23 association. WA (3, 6 mg/kg) inhibited tumor growth in pancreatic Panc-1 xenografts by 30% and 58%, respectively. These data demonstrate that Withaferin A binds Hsp90, inhibits Hsp90 chaperone activity through an ATP-independent mechanism, results in Hsp90 client protein degradation, and exhibits in vivo anticancer activity against pancreatic cancer.

AB - The purpose of this study is to investigate the efficacy and the mechanism of Hsp90 inhibition of Withaferin A (WA), a steroidal lactone occurring in Withania somnifera, in pancreatic cancer in vitro and in vivo. Withaferin A exhibited potent antiproliferative activity against pancreatic cancer cells in vitro (with IC50s of 1.24, 2.93 and 2.78 μM) in pancreatic cancer cell lines Panc-1, MiaPaCa2 and BxPc3, respectively. Annexin V staining showed that WA induced significant apoptosis in Panc-1 cells in a dose-dependent manner. Western blotting demonstrated that WA inhibited Hsp90 chaperone activity to induce degradation of Hsp90 client proteins (Akt, Cdk4 and glucocorticoid receptor), which was reversed by the proteasomal inhibitor, MG132. WA-biotin pull down assay of Hsp90 using Panc-1 cancer cell lysates and purified Hsp90 showed that WA-biotin binds to C-terminus of Hsp90 which was competitively blocked by unlabeled WA. Co-immunoprecipitation exhibited that WA (10 μM) disrupted Hsp90-Cdc37 complexes from 1 to 24 h post-treatment, while it neither blocked ATP binding to Hsp90, nor changed Hsp90-P23 association. WA (3, 6 mg/kg) inhibited tumor growth in pancreatic Panc-1 xenografts by 30% and 58%, respectively. These data demonstrate that Withaferin A binds Hsp90, inhibits Hsp90 chaperone activity through an ATP-independent mechanism, results in Hsp90 client protein degradation, and exhibits in vivo anticancer activity against pancreatic cancer.

KW - Cdc37

KW - Client protein

KW - Hsp90

KW - Pancreatic cancer

KW - Reactive cysteine

KW - Withaferin A

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JF - Biochemical Pharmacology

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Withaferin A targets heat shock protein 90 in pancreatic cancer cells

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

UN SDGs

Access to Document

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