title: Celastrol targets mitochondrial respiratory chain complex I to induce reactive oxygen species-dependent cytotoxicity in tumor cells.
abstract: BACKGROUND
  Celastrol is an active ingredient of the traditional Chinese medicinal plant Tripterygium Wilfordii, which exhibits significant antitumor activity in different cancer models in vitro and in vivo; however, the lack of information on the target and mechanism of action of this compound have impeded its clinical application. In this study, we sought to determine the mode of action of celastrol by focusing on the processes that mediate its anticancer activity.
  
  
  METHODS
  The downregulation of heat shock protein 90 (HSP90) client proteins, phosphorylation of c-Jun NH2-terminal kinase (JNK), and cleavage of PARP, caspase 9 and caspase 3 were detected by western blotting. The accumulation of reactive oxygen species (ROS) was analyzed by flow cytometry and fluorescence microscopy. Cell cycle progression, mitochondrial membrane potential (MMP) and apoptosis were determined by flow cytometry. Absorption spectroscopy was used to determine the activity of mitochondrial respiratory chain (MRC) complexes.
  
  
  RESULTS
  Celastrol induced ROS accumulation, G2-M phase blockage, apoptosis and necrosis in H1299 and HepG2 cells in a dose-dependent manner. N-acetylcysteine (NAC), an antioxidative agent, inhibited celastrol-induced ROS accumulation and cytotoxicity. JNK phosphorylation induced by celastrol was suppressed by NAC and JNK inhibitor SP600125 (SP). Moreover, SP significantly inhibited celastrol-induced loss of MMP, cleavage of PARP, caspase 9 and caspase 3, mitochondrial translocation of Bad, cytoplasmic release of cytochrome c, and cell death. However, SP did not inhibit celastrol-induced ROS accumulation. Celastrol downregulated HSP90 client proteins but did not disrupt the interaction between HSP90 and cdc37. NAC completely inhibited celastrol-induced decrease of HSP90 client proteins, catalase and thioredoxin. The activity of MRC complex I was completely inhibited in H1299 cells treated with 6 μM celastrol in the absence and presence of NAC. Moreover, the inhibition of MRC complex I activity preceded ROS accumulation in H1299 cells after celastrol treatment.
  
  
  CONCLUSIONS
  We identified ROS as the key intermediate for celastrol-induced cytotoxicity. JNK was activated by celastrol-induced ROS accumulation and then initiated mitochondrial-mediated apoptosis. Celastrol induced the downregulation of HSP90 client proteins through ROS accumulation and facilitated ROS accumulation by inhibiting MRC complex I activity. These results identify a novel target for celastrol-induced anticancer activity and define its mode of action.
authors.affiliation: Department of Pathology, Beijing Institute of Basic Medical Sciences, Beijing 100850, China.
authors.forename: Guozhu
authors.initials: G
authors.lastname: Chen
authors.forename: Xuhui
authors.initials: X
authors.lastname: Zhang
authors.forename: Ming
authors.initials: M
authors.lastname: Zhao
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.forename: Xiang
authors.initials: X
authors.lastname: Cheng
authors.forename: Di
authors.initials: D
authors.lastname: Wang
authors.forename: Yuanji
authors.initials: Y
authors.lastname: Xu
authors.forename: Zhiyan
authors.initials: Z
authors.lastname: Du
authors.forename: Xiaodan
authors.initials: X
authors.lastname: Yu
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D042967:Electron Transport Complex I / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D048670:MAP Kinase Kinase 4 / Q000378:metabolism; D009369:Neoplasms / Q000378:metabolism*; D053978:Pentacyclic Triterpenes; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D014315:Triterpenes / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; D018841:HSP90 Heat-Shock Proteins; D053978:Pentacyclic Triterpenes; D017382:Reactive Oxygen Species; D014315:Triterpenes; D048670:MAP Kinase Kinase 4; D042967:Electron Transport Complex I; C050414:celastrol