铁皮石斛宁 A 靶向胰腺癌细胞热休克蛋白 90。
Withaferin A targets heat shock protein 90 in pancreatic cancer cells.
机构信息
Department of Pharmaceutical Sciences, College of Pharmacy, The University of Michigan, 428 Church Street, Ann Arbor, MI 48109, USA.
出版信息
Biochem Pharmacol. 2010 Feb 15;79(4):542-51. doi: 10.1016/j.bcp.2009.09.017.
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.
本研究旨在探讨 Hsp90 抑制剂 Withaferin A（WA）——一种存在于印度人参中的甾体内酯——对胰腺癌的体内外疗效及其作用机制。WA 在体外对胰腺癌细胞系 Panc-1、MiaPaCa2 和 BxPc3 具有显著的增殖抑制活性（IC50 分别为 1.24、2.93 和 2.78 μM）。Annexin V 染色显示 WA 以剂量依赖性方式诱导 Panc-1 细胞发生显著凋亡。Western blot 分析表明，WA 通过抑制 Hsp90 伴侣活性诱导 Hsp90 客户蛋白（Akt、Cdk4 和糖皮质激素受体）降解，这种作用可被蛋白酶体抑制剂 MG132 逆转。用 Panc-1 癌细胞裂解液和纯化的 Hsp90 进行的 WA-生物素下拉实验表明，WA-生物素结合 Hsp90 的 C 端，未标记的 WA 可竞争性阻断其结合。免疫共沉淀实验显示，WA（10 μM）在处理后 1 至 24 小时内破坏 Hsp90-Cdc37 复合物，而不阻断 ATP 与 Hsp90 的结合，也不改变 Hsp90-P23 结合。WA（3、6mg/kg）分别使胰腺 Panc-1 异种移植瘤的生长抑制率达到 30%和 58%。这些数据表明，Withaferin A 与 Hsp90 结合，通过一种不依赖于 ATP 的机制抑制 Hsp90 伴侣活性，导致 Hsp90 客户蛋白降解，并在体内显示出对胰腺癌的抗癌活性。
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