Abstract
Recently, celastrol has shown great potential for inducing apoptosis in acute myeloid leukemia cells, especially acute promyelocytic leukaemia cells. However, the mechanism is poorly understood. Metabolomics provides an overall understanding of metabolic mechanisms to illustrate celastrol's mechanism of action. We treated both nude mice bearing HL-60 cell xenografts in vivo and HL-60 cells as well as NB-4 cells in vitro with celastrol. Ultra-performance liquid chromatography coupled with mass spectrometry was used for metabolomics analysis of HL-60 cells in vivo and for targeted L-cysteine analysis in HL-60 and NB-4 cells in vitro. Flow cytometric analysis was performed to assess mitochondrial membrane potential, reactive oxygen species and apoptosis. Western blotting was conducted to detect the p53, Bax, cleaved caspase 9 and cleaved caspase 3 proteins. Celastrol inhibited tumour growth, induced apoptosis, and upregulated pro-apoptotic proteins in the xenograft tumour mouse model. Metabolomics showed that cysteine metabolism was the key metabolic alteration after celastrol treatment in HL-60 cells in vivo. Celastrol decreased L-cysteine in HL-60 cells. Acetylcysteine supplementation reversed reactive oxygen species accumulation and apoptosis induced by celastrol and reversed the dramatic decrease in the mitochondrial membrane potential and upregulation of pro-apoptotic proteins in HL-60 cells. In NB-4 cells, celastrol decreased L-cysteine, and acetylcysteine reversed celastrol-induced reactive oxygen species accumulation and apoptosis. We are the first to identify the involvement of a cysteine metabolism/reactive oxygen species/p53/Bax/caspase 9/caspase 3 pathway in celastrol-triggered mitochondrial apoptosis in HL-60 and NB-4 cells, providing a novel underlying mechanism through which celastrol could be used to treat acute myeloid leukaemia, especially acute promyelocytic leukaemia.
Highlights
Acute leukemia is the most common malignant hematological malignancy, of which acute myeloid leukemia (AML) accounts for over 80% of all acute leukemias in individuals aged >18 years[1]
Metabolomics profiles showed that cysteine metabolism was the key metabolic alteration after celastrol treatment in HL-60 cells in vivo
To further identify key metabolic changes, the changed metabolites were imported into the “enrichment analysis” module, and protein biosynthesis and glutathione metabolism were identified as the key changed pathways (Fig. 2b, Supplementary Material: Table S2)
Summary
Acute leukemia is the most common malignant hematological malignancy, of which acute myeloid leukemia (AML) accounts for over 80% of all acute leukemias in individuals aged >18 years[1]. ATRA and ATO can lead to the development of differentiation syndrome and multiorgan toxicity, and a few patients do not respond favourably to the drugs[5,6]. Traditional Chinese medicine, as an important source of therapeutically effective drugs, has attracted increasing attention in cancer therapy[8]. In fresh cells from patients of various types of AML, celastrol showed effect for the treatment of leukemia[13,15]. The previous study has demonstrated that celastrol showed stronger anti-tumour effect than ATRA in leukemia cells[13]. The anti-tumour effects of celastrol have been consistently attributed to its ability to induce apoptosis in AML and APL NB-4 cells[15,19,20,21], but the mechanism is poorly understood
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