Abstract
HeLa cells treated with celastrol, a natural compound with inhibitive effect on proteasome, exhibited increase in apoptotic rate and characteristics of apoptosis. To clarify the signal network activated by celastrol to induce apoptosis, both the direct target proteins and undirect target proteins of celastrol were searched in the present study. Proteasome catalytic subunit β1 was predicted by computational analysis to be a possible direct target of celastrol and confirmed by checking direct effect of celastrol on the activity of recombinant human proteasome subunit β1 in vitro. Undirect target-related proteins of celastrol were searched using proteomic studies including two-dimensional electrophoresis (2-DE) analysis and iTRAQ-based LC-MS analysis. Possible target-related proteins of celastrol such as endoplasmic reticulum protein 29 (ERP29) and mitochondrial import receptor Tom22 (TOM22) were found by 2-DE analysis of total cellular protein expression profiles. Further study showed that celastrol induced ER stress and ER stress inhibitor could ameliorate cell death induced by celastrol. Celastrol induced translocation of Bax into the mitochondria, which might be related to the upregulation of BH-3-only proteins such as BIM and the increase in the expression level of TOM22. To further search possible target-related proteins of celastrol in ER and ER-related fractions, iTRAQ-based LC-MS method was use to analyze protein expression profiles of ER/microsomal vesicles-riched fraction of cells with or without celastrol treatment. Based on possible target-related proteins found in both 2-DE analysis and iTRAQ-based LC-MS analysis, protein–protein interaction (PPI) network was established using bioinformatic analysis. The important role of glycogen synthase kinase-3β (GSK3β) in the signal cascades of celastrol was suggested. Pretreatment of LiCL, an inhibitor of GSK3β, could significantly ameliorate apoptosis induced by celastrol. On the basis of the results of the present study, possible signal network of celastrol activated by celastrol leading to apoptosis was predicted.
Highlights
Many reports have shown that celastrol, as a cytotoxic agent, induced apoptosis in cancer cells.[6,7,8,9,10,11,12,13,14,15] In our previous report, we found that celastrol could induce different kinds of cell death including apoptosis, autophagy and paraptosis in cancer cells.[6]
The results indicated that celastrol induced apoptosis and caused an increase in the percentage of apoptotic cells
Consistent with previous reports, apoptosis was induced in HeLa cells treated with celastrol
Summary
Many reports have shown that celastrol, as a cytotoxic agent, induced apoptosis in cancer cells.[6,7,8,9,10,11,12,13,14,15] In our previous report, we found that celastrol could induce different kinds of cell death including apoptosis, autophagy and paraptosis in cancer cells.[6]. We tried to clarify the signal network by which celastrol induce apoptosis in HeLa cells. As a multi-target compound, might have different direct targets and Received 08.11.12; revised 03.5.13; accepted 06.5.13; Edited by H-U Simon present study to provide relatively comprehensive and unbiased information about the target-related proteins involved in the effects of celastrol. Based on the results of proteomic analysis and in depth study of the effects of celastrol on the ER and mitochondria, signal cascades activated by celastrol leading to apoptosis were sequentially clarified. Direct effect of celastrol on the activity of recombinant proteasome b1 subunit protein in vitro was checked to confirm the prediction that proteasome b1 subunit might be a direct target of celastrol. Undirect target-related proteins of celastrol were searched in the present study using proteomic methods.
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