A novel method to detect intracellular metabolite alterations in MCF-7 cells by doxorubicin induced cell death
- 3 January 2021
- journal article
- research article
- Published by Springer Science and Business Media LLC in Metabolomics
- Vol. 17 (1), 1-16
- https://doi.org/10.1007/s11306-020-01755-2
Abstract
Background Metabolic reprogramming within cancer cells has been recognized as a potential barrier to chemotherapy. Additionally, metabolic tumor heterogeneity is the one of factors behind discernible hallmarks such as drug resistance, relapse of the tumor and the formation of secondary tumors. Methods In this paper, cell-based assays including PI/annexin V staining and immunoblot assay were performed to show the apoptotic cell death in MCF-7 cells treated with DOX. Further, MCF-7 cells were lysed in a hypotonic buffer and the whole cell lysate was purified by a novel and specifically designed metabolite (~ 100 to 1000 Da) fractionation system called vertical tube gel electrophoresis (VTGE). Further, purified intracellular metabolites were subjected to identification by LC-HRMS technique. Results Cleaved PARP 1 in MCF-7 cells treated with DOX was observed in the present study. Concomitantly, data showed the absence of active caspase 3 in MCF-7 cells. Novel findings are to identify key intracellular metabolites assisted by VTGE system that include lipid (CDP-DG, phytosphingosine, dodecanamide), non-lipid (N-acetyl-D-glucosamine, N1-acetylspermidine and gamma-L-glutamyl-L-cysteine) and tripeptide metabolites in MCF-7 cells treated by DOX. Interestingly, we reported the first evidence of doxorubicinone, an aglycone form of DOX in MCF-7 cells that are potentially linked to the mechanism of cell death in MCF-7 cells. Conclusion This paper reported novel methods and processes that involve VTGE system based purification of hypotonically lysed novel intracellular metabolites of MCF-7 cells treated by DOX. Here, these identified intracellular metabolites corroborate to caspase 3 independent and mitochondria induced apoptotic cell death in MCF-7 cells. Finally, these findings validate a proof of concept on the applications of novel VTGE assisted purification and analysis of intracellular metabolites from various cell culture models.Funding Information
- DPU, Pune (DPU(2016))
This publication has 51 references indexed in Scilit:
- Metabolomic approach to evaluating adriamycin pharmacodynamics and resistance in breast cancer cellsMetabolomics, 2013
- Caspase-7 uses an exosite to promote poly(ADP ribose) polymerase 1 proteolysisProceedings of the National Academy of Sciences of the United States of America, 2012
- γ-Glutamylcysteine detoxifies reactive oxygen species by acting as glutathione peroxidase-1 cofactorNature Communications, 2012
- Metformin Induces Both Caspase-Dependent and Poly(ADP-ribose) Polymerase-Dependent Cell Death in Breast Cancer CellsMolecular Cancer Research, 2011
- Hallmarks of Cancer: The Next GenerationCell, 2011
- Phytosphingosine induced mitochondria‐involved apoptosisCancer Science, 2005
- Impairment of myocardial contractility by anticancer anthracyclines: role of secondary alcohol metabolites and evidence of reduced toxicity by a novel disaccharide analogueBritish Journal of Pharmacology, 2001
- Inhibition of Phosphatidylcholine Biosynthesis following Induction of Apoptosis in HL-60 CellsOnline Journal of Public Health Informatics, 1999
- Species pattern of phosphatidic acid, diacylglycerol, CDP-diacylglycerol and phosphatidylglycerol synthesized de novo in rat liver mitochondriaBiochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1989
- Aspects of the degradation kinetics of doxorubicin in aqueous solutionInternational Journal of Pharmaceutics, 1986