Celastrol targets mitochondrial respiratory chain complex I to induce reactive oxygen species-dependent cytotoxicity in tumor cells

Guozhu Chen,Xuhui Zhang,Zhiyan Du,Di Wang,Xiang Cheng,Ming Zhao,Xiaodan Yu,Yuanji Xu,Yan Wang

doi:10.1186/1471-2407-11-170

Abstract

BackgroundCelastrol 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.MethodsThe 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.ResultsCelastrol 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.ConclusionWe 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.

Highlights

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; the lack of information on the target and mechanism of action of this compound have impeded its clinical application
Celastrol initiates reactive oxygen species (ROS) accumulation and mediates cytotoxicity in a dose-dependent manner To determine the role of ROS in mediating celastrolinduced cytotoxicity, we first measured ROS levels in H1299 and HepG2 cells after celastrol exposure
The results of annexin V-FITC and propidium iodide (PI) staining showed that celastrol induced apoptotic and necrotic cell death in a dose-dependent manner, and the percent of cell death was 41 ± 4.1% in H1299 cells and 22 ± 2.5% in HepG2 cells treated with 6 μM celastrol for 24 h (Figure 1D)

Summary

Introduction

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; the lack of information on the target and mechanism of action of this compound have impeded its clinical application. Several other molecular targets have been proposed to explain the anticancer effects of celastrol, including NFB [10,18,19], topoisomerase II [5], and xcCystine/Glutamate antiporter [20]. These targets positively correlate with celastrol-induced cytotoxicity, it is not clear which, if any, is the principal mediator of the antitumor activity of celastrol. As celastrol is moved into clinical studies, it is important to gain a better understanding of its target and mechanism

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Conclusion