title: A targeted proteomics approach to the quantitative analysis of ERK/Bcl-2-mediated anti-apoptosis and multi-drug resistance in breast cancer.
abstract: Apoptosis suppression caused by overexpression of anti-apoptotic proteins is a central factor to the acquisition of multi-drug resistance (MDR) in breast cancer. As a highly conserved anti-apoptotic protein, Bcl-2 can initiate an anti-apoptosis response via an ERK1/2-mediated pathway. However, the details therein are still far from completely understood and a quantitative description of the associated proteins in the biological context may provide more insights into this process. Following our previous attempts in the quantitative analysis of MDR mechanisms, liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based targeted proteomics was continually employed here to describe ERK/Bcl-2-mediated anti-apoptosis. A targeted proteomics assay was developed and validated first for the simultaneous quantification of ERK1/2 and Bcl-2. In particular, ERK isoforms (i.e., ERK1 and ERK2) and their differential phosphorylated forms including isobaric ones were distinguished. Using this assay, differential protein levels and site-specific phosphorylation stoichiometry were observed in parental drug-sensitive MCF-7/WT cancer cells and drug-resistant MCF-7/ADR cancer cells and breast tissue samples from two groups of patients who were either suspected or diagnosed to have drug resistance. In addition, quantitative analysis of the time course of both ERK1/2 and Bcl-2 in doxorubicin (DOX)-treated MCF-7/WT cells confirmed these findings. Overall, we propose that targeted proteomics can be used generally to resolve more complex cellular events.
authors: Department of Pharmacy, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing, Jiangsu, 210029, China.
authors: Ting
authors: T
authors: Yang
authors: School of Pharmacy, Nanjing Medical University, 818 Tian Yuan East Road, Nanjing, Jiangsu, 211166, China.
authors: Feifei
authors: F
authors: Xu
authors: School of Pharmacy, Nanjing Medical University, 818 Tian Yuan East Road, Nanjing, Jiangsu, 211166, China.
authors: Yuan
authors: Y
authors: Sheng
authors: School of Pharmacy, Nanjing Medical University, 818 Tian Yuan East Road, Nanjing, Jiangsu, 211166, China.
authors: Wen
authors: W
authors: Zhang
authors: School of Pharmacy, Nanjing Medical University, 818 Tian Yuan East Road, Nanjing, Jiangsu, 211166, China. ychen@njmu.edu.cn.
authors: Yun
authors: Y
authors: Chen
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods; D004317:Doxorubicin / Q000494:pharmacology; D018432:Drug Resistance, Multiple; D019008:Drug Resistance, Neoplasm; D048049:Extracellular Signal-Regulated MAP Kinases / Q000032:analysis / Q000378:metabolism*; D005260:Female; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects; D061986:MCF-7 Cells; D040901:Proteomics / Q000379:methods; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D000970:Antineoplastic Agents; D019253:Proto-Oncogene Proteins c-bcl-2; D004317:Doxorubicin; D048049:Extracellular Signal-Regulated MAP Kinases
keywords: Anti-apoptosis; Bcl-2; Extracellular signal-regulated protein kinase (ERK); Isoforms; Multi-drug resistance (MDR); Targeted proteomics