RESUMO
Triptolide (TL) is a potent anti-tumor, anti-inflammatory and immunosuppressive natural compound. Mechanistic studies revealed that TL inhibits tumor growth and triggers programmed cell death. Studies further suggested that TL inhibits heat shock response in cancer cells to induce apoptosis. HSP90ß is the major component of heat shock response and is overexpressed in different types of cancers. Given almost all identified HSP90ß inhibitors are either N or C-terminal inhibitors, small molecules attacking cysteine(s) in the middle domain might represent a new class of inhibitors. In the current study, we showed that TL inhibits HSP90ß in triple manner. Characterization suggests that TL inhibits ATPase activity by preventing ATP binding thus blunts the chaperone activity. TL disrupts HSP90ß-CDC37 (co-chaperone) complex through middle domain Cys366 of HSP90ß and causes kinase client protein degradation. At the cellular level, the TL-mediated decrease in CDK4 protein levels in HeLa cells causes reduced phosphorylation of Rb resulting in cell cycle arrest at the G1 phase. Furthermore, our results demonstrated that TL triggers programmed cell death in an HSP90ß-dependent manner as knockdown of HSP90ß further sensitized TL-mediated cell cycle arrest and apoptotic effect. Surprisingly, our data showed that TL is the first drug to be reported to induce site-specific phosphorylation of HSP90ß to drive apoptosome formation in the early phase of the treatment. In summary, our study established that TL is a novel middle domain HSP90ß inhibitor with bi-phasic multi-mechanistic inhibition. The unique regulatory mechanism of TL on HSP90ß makes it an effective inhibitor.
