Triptolide, a HSP90 middle domain inhibitor, induces apoptosis in triple manner
Frederick Zhehao Zhang1,2, Derek Hoi-Hang Ho1, Roger Hoi-Fung Wong1
1Department of Biology, Hong Kong Baptist University, Hong Kong SAR, Hong Kong.
Abstract:
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. HSP90β is the major component of heat shock response and is overexpressed in different types of cancers. Given almost all identified HSP90β 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 HSP90β in triple manner. Characterization suggests that TL inhibits ATPase activity by preventing ATP binding thus blunts the chaperone activity. TL disrupts HSP90β-CDC37 (co-chaperone) complex through middle domain Cys366 of HSP90β 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 HSP90β-dependent manner as knockdown of HSP90β 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 HSP90β to drive apoptosome formation in the early phase of the treatment. In summary, our study established that TL is a novel middle domain HSP90β inhibitor with bi-phasic multi-mechanistic inhibition. The unique regulatory mechanism of TL on HSP90β makes it an effective inhibitor.
Insights
Triptolide (TL) is a novel middle domain inhibitor of Heat Shock Protein 90 beta (HSP90β). It effectively induces cancer cell death through multiple mechanisms, including inhibiting ATPase activity and disrupting protein complexes.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Triptolide (TL) is a natural compound with anti-tumor, anti-inflammatory, and immunosuppressive properties.
- Heat Shock Protein 90 beta (HSP90β) is overexpressed in cancers and plays a role in tumor growth.
- Targeting HSP90β offers a potential therapeutic strategy for cancer treatment.
Purpose of the Study:
- To investigate Triptolide (TL) as a novel inhibitor of HSP90β.
- To elucidate the multi-mechanistic inhibition of HSP90β by TL.
- To explore the therapeutic potential of TL in cancer treatment.
Main Methods:
- Biochemical assays to characterize TL's inhibition of HSP90β ATPase activity.
- Co-immunoprecipitation to analyze the disruption of HSP90β-CDC37 complex.
- Western blotting to assess protein levels (e.g., CDK4, pRb) and cell cycle analysis.
- Cell viability assays and apoptosis assays in cancer cell lines.
- HSP90β knockdown experiments to confirm its role in TL's effects.
Main Results:
- TL inhibits HSP90β ATPase activity by preventing ATP binding, thus reducing its chaperone function.
- TL disrupts the HSP90β-CDC37 complex via Cys366 in HSP90β's middle domain, leading to client protein degradation.
- TL treatment reduces CDK4 levels, causing hypophosphorylation of Rb and G1 cell cycle arrest in HeLa cells.
- TL induces HSP90β-dependent apoptosis.
- TL is the first reported drug to induce site-specific phosphorylation of HSP90β, promoting apoptosome formation.
Conclusions:
- TL is a novel middle domain inhibitor of HSP90β with bi-phasic, multi-mechanistic inhibitory effects.
- TL's unique mechanism of action on HSP90β positions it as a promising anti-cancer agent.
- Targeting HSP90β through novel inhibitors like TL warrants further investigation for cancer therapy.
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