Metabolomics Reveals Cysteine Metabolism Plays a Role in Celastrol-Induced Mitochondrial Apoptosis of Acute Promyelocytic Leukemia Cells

Background: Recently, celastrol shows great potential for inducing apoptosis in the treatment of leukemia, especially acute promyelocytic leukemia. However, the mechanism is poorly understood. Metabolomics provides overall understanding of biological mechanisms by surveying small molecules, which contributes to the illustration of celastrol's action mode.   Methods: We treated nude mice bearing xenografts of HL-60 cell in vivo and HL-60 cell as well as NB-4 cell in vitro with celastrol. UPLC coupled to an Orbitrap mass spectrometer was used for a hypothesis free metabolomics analysis of HL-60 in vivo, and was used for targeted L-cysteine analysis of HL-60 and NB-4 cells. Flow cytometry analysis was performed to detect the mitochondrial membrane potential, production of reactive oxygen species (ROS) and percentage of apoptosis. Western blot was conducted to detect the protein level of p53, Bax, cleaved caspase 9 and cleaved caspase 3.   Findings: Celastrol inhibited tumor growth, induced apoptosis, and up-regulated pro-apoptotic proteins in xenograft tumor mice model. Metabolomics profiles delineated cysteine metabolism was the key changed metabolism after celastrol treatment in HL-60 cells in vivo. Celastrol decreased L-cysteine in HL-60 cells. The supplementation of acetylcysteine reversed the ROS and apoptosis induced by celastrol, and reversed the dramatic decline of mitochondrial membrane potential and the up-regulation of pro-apoptotic proteins in HL-60 cells. Celastrol decreased L-cysteine, and acetylcysteine reversed ROS and apoptosis induced by celastrol in NB-4 cells.   Interpretation: We firstly identified cysteine metabolism/ROS/p53/Bax/caspase 9/caspase 3 pathway in celastrol-triggered apoptosis of APL cells, providing a novel mechanism underlying which celastrol might be used for the treatment of acute promyelocytic leukemia.   Funding: This study was supported by grants from the National Natural Science Foundation of China (81872650, 81503134, 81573182); the Key Natural Science Foundation of the Jiangsu Higher Education Institutions of China (18KJA320003); the Key Project of the Science and Technology Development Foundation of Nanjing Medical University (2014NJMUZD005); the Key Research & Development Plan of Jiangsu Province (BE2017628); the Southeast University & Nanjing Medical University Collaborative Research Project (2242018K3DN25); the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).   Declaration of Interest: The authors declare that they have no competing interests. Ethical Approval: All procedures performed in studies involving animals were in accordance with the ethical standards of Nanjing Medical 419 University Institutional Animal Care and Use Committee.