Withanolides cause thiol oxidation and aggregation of Hsp90 in MDA-MB-231 cells.

<p>(A) Cells were treated with WA (5 µM), TA (5 µM), HW (20 µM), AA (20 µM) or PH (20 µM) for 12 h. The Triton-insoluble fractions of cell lysates were prepared as described under “Experimental Procedures.” The protein levels of the disulfide-linked-Hsp 90 were analyzed by nonreducing SDS-PAGE. (B, C) The thiol-reducing agent NAC prevents the degradation of Hsp90 client proteins and cell death caused by withanolides. MDA-MB-231 cells were pretreated with or without NAC (2.5 mM) for 1 h before challenged with withanolides for 12 h (B) or 48 h (C). The protein levels of heat shock proteins and client proteins were analyzed by western blot (B). The viability of MDA-MB-231 cells was determined by the MTT assay (C). Results are presented as means ± S.E.M. (n = 3). **<i>P</i><0.01, ***<i>P</i><0.001 as compared with each group without NAC pretreated.