Withaferin A targets heat shock protein 90 in pancreatic cancer cells.
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 IC(50)s of 1.24, 2.93 and 2.78 microM) 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 microM) disrupted Hsp90-Cdc37 complexes from 1 to 24h post-treatment, while it neither blocked ATP binding to Hsp90, nor changed Hsp90-P23 association. WA (3, 6mg/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.
Yu
Yanke
Y
Department of Pharmaceutical Sciences, College of Pharmacy, The University of Michigan, 428 Church Street, Ann Arbor, MI 48109, USA.
Hamza
Adel
A
Zhang
Tao
T
Gu
Mancang
M
Zou
Peng
P
Newman
Bryan
B
Li
Yanyan
Y
Gunatilaka
A A Leslie
AA
Zhan
Chang-Guo
CG
Sun
Duxin
D
D000818:Animals; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D045744:Cell Line, Tumor; D018833:Chaperonins / Q000037:antagonists & inhibitors / Q000378:metabolism; D016503:Drug Delivery Systems / Q000379:methods; D004875:Ergosterol / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D005260:Female; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D010190:Pancreatic Neoplasms / Q000188:drug therapy / Q000378:metabolism*; D054358:Withanolides; D023041:Xenograft Model Antitumor Assays / Q000379:methods
C498362:CDC37 protein, human; D018797:Cell Cycle Proteins; D018841:HSP90 Heat-Shock Proteins; D054358:Withanolides; D018833:Chaperonins; C009684:withaferin A; D004875:Ergosterol