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 1–24 hour 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.

Anti-cancer activity of withaferin A in B-cell lymphoma

“…al., we conclude that WA inhibits the growth and survival of DLBCL by interacting and inhibiting the activity of the chaperone Hsp90, resulting in reduced expression of its client proteins. 15 Studies also indicate that WA has no noticeable toxicity on normal lymphocytes 28 , and our results illustrate that WA does not appear to affect proliferating colon epithelial cells in vivo. Further analyses on in vivo stability of WA and its effects on normal tissues need to be completed in order evaluate the clinical potential of WA as a therapeutic for DLBCL but current findings are promising.…”

“…demonstrated that WA treatment leads to degradation of a variety of client proteins of the Hsp90 chaperone, suggesting that WA works by inhibiting heat shock proteins. 15 This mechanism of action has not yet been described in any other cancer cells. The Hsp90/Cdc37 chaperone complex has been described to have functional roles in promoting the maturation of client proteins such as Src, Akt, IKK, Cdc2, and CDK4.…”

“…15 Decrease in total protein levels of several kinases (Akt, Lyn, CDK4, IKKa/b), which have been demonstrated to be Hsp90 substrates led us to postulate that WA may also inhibit Hsp90 function in lymphoma cells. Best indication of decrease in Hsp90 function is rapid induction of Hsp70 which could compensate for Hsp90 partially.…”

“…13 WA, the active compound found in the plant extract, shows promise to be a key anti-cancer therapeutic. 14 WA has been reported to inhibit proliferation and induce cell death in a variety of tumor models such as pancreatic, 15 breast, 16 lung, 17 cervical, 18 and prostate, 19 but its effects on DLBCL are not yet known.…”

Withaferin A inhibits breast cancer invasion and metastasis at sub‐cytotoxic doses by inducing vimentin disassembly and serine 56 phosphorylation

“…25 Moreover, WFA has proapoptotic and anti-tumor activity in breast and prostate cancers. [26][27][28] There are several other documented targets of WFA including NF-Kb, BCL-2, FOXO3A, Hsp90, phosphorylated STAT3 and annexin II 27,[29][30][31][32][33] ; however, the mechanisms by which this leads to anti-tumor activity are not fully understood.…”