Pancreatic cancer is one of the most challenging cancers to treat. This study was aimed to examine the in vitro and in vivo anticancer efficacy and mechanism of Hsp90 inhibition of withaferin A (WA), a natural product purified from the Indian medicinal plant Withania somnifera, in pancreatic cancer cells.. Withaferin A exhibited potent antiproliferative activity against pancreatic cancer cells in vitro with IC50 values below 3 microM in the three pancreatic cancer cell lines Panc-1, MiaPaca-2 and BxPc-3. Withaferin A induced Hsp90 client protein (Akt, Cdk4 and glucocorticoid receptor) degradation mediated by proteasome. WA-Biotin pull-down assay showed that WA-biotin bound directly to the C-terminus of Hsp90, which could be competitively blocked by unlabeled WA. Co-immunoprecipitation demonstrated that WA disrupted the Hsp90-Cdc37 interaction, but not the Hsp90-P23 association and ATP binding to Hsp90. Finally, WA significantly decreased tumor growth in a mouse xenograft model of pancreatic cancer.. As WA induced Hsp70 upregulation, which has potent anti-apoptotic activity, we further examined whether inhibition of Hsp70 by myricetin (MY) would sensitize pancreatic cancer cells to WA. Combined treatment of MY and WA exhibited a much higher anticancer effect against pancreatic cancer cells in vitro compared to MY or WA treatment alone. . MY co-treatment (10 microM) with 1 microM WA decreased WA-induced Hsp70 expression by 3-fold compared to WA treatment alone. In addition, WA and MY acted synergistically in downregulating Hsp90 client proteins, including mutated P53, Akt, and Cdk4. Combined WA and MY treatment also showed significant advantage in tumor growth inhibition in pancreatic cancer mouse xenografts compared to a single treatment. Finally, the structure-activity relationship (SAR) of withanolides for inhibition of Hsp90 and anti-proliferative activities in pancreatic cancer cells was investigated. WA and the three analogues withanolide E (WE), 4β-hydroxywithanolide E (HWE), 3-aziridinylwithaferin A (AzWA) were examined for their anticancer and Hsp90 inhibitory activities in pancreatic cancer cells. SAR analysis showed that the C-5, 6 epoxy functional group was responsible for Hsp90 inhibition whereas, the hydroxyl group at C-4 might enhance the Hsp90 inhibitory activity of withanolide. In contrast, the steric bulk substitution at C-3 may reduce activity.Ph.D.Pharmaceutical SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/89629/1/yorkyu_1.pd
