| Breast cancer has become the world's highest incidence of women malignant tumors,in which the efficacy of chemotherapy is limited by the multi-drug resistance(MDR).It was reported that multiple siganling pathways and proteins are involved in the development of breast cancer multi-drug resistance,including P-glycoprotein(P-gp)regulated by Heat shock protein 27(HSP27)and anti-apoptotic protein B-cell lymphoma-2(Bcl-2)regulated by Extracellular regulated kinase 1/2(ERK1/2).Liquid chromatography –tandem mass spectrometry(LC/MS/MS)-based targeted proteomics turns out to be a promising quantification approach for the study of proteins in the preclinical and clinical environment.Unfortunately,rare studies applied this technology to detect multiple associated proteins or protein modification in one experiment.This study demonstrated the potential of LC-MS/MS-based targeted proteomics to understand multiple associated proteins or protein modification of the breast cancer multi-drug resistance cell events in a more accurate context of biological system.One missing puzzle piece to study HSP27 in P-gp mediated MDR was the amount of HSP27 and the extent of its phosphorylation in the biological context.LC-MS/MS-based targeted proteomics allows researchers to monitor associated proteins and their modification simultaneously and quantitatively.In this study,a targeted proteomics assay was first developed and validated for the quantification of HSP27 and its phosphorylated forms.Using this assay,the level of HSP27 was determined in non-tumoral cells MCF-10 A,parental drug-sensitive cancer cells MCF-7/WT and drug-resistant cancer cells MCF-7/ADR.A decrease of HSP27 expression was observed in P-gp overexpressed MCF-7/ADR cells.A quantitative time-course analysis of both HSP27 and P-gp in Doxorubicin(DOX)treated MCF-7/WT cells also implied that HSP27 may participate in the P-gp modulation.By the quantitative time-course analysis of HSP27 and its phosphorylated forms at sites of Ser15 and Ser82,the possible role of HSP27 in P-gp mediated MDR was suggested.Furthermore,stoichiometry of site-specific HSP27 phosphorylation indicated that DOX treatment rapidly induced the HSP27 phosphorylation at Ser82.Moreover,conventional analytical methods including Confocal Microscopy,Western blotting,Flow cytometry were also performed for a comparison.On the other hand,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 success 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 first developed and validated for the simultaneous quantification of ERK1/2 and Bcl-2.In particular,ERK isoforms(i.e.,ERK1 and ERK2)and their site-specific phosphorylated forms(i.e.,non-,isobaric mono-and diphosphorylated ERK1/2)can be distinguished.Using this assay,differential protein levels and phosphorylation stoichiometry were observed in drug-sensitive MCF-7/WT cells and drug resistant MCF-7/ADR cells.In addition,a quantitative time-course analysis of both ERK1/2 and Bcl-2 in Doxorubicin-treated MCF-7/WT cells confirmed these findings.For a comparison,Confocal microscopy and Western blotting were performed.Finally,the interplay between ERK/Bcl-2 and HSP27/P-gp mediated pathways in MDR acquisition was first highlighted in quantitative values.In our study,we use LC-MS/MS based targeted proteomics to quantitative analyse HSP27/P-gp and ERK/Bcl-2 mediated multi-drug resistance in breast cancer.Further development of targeted proteomics in future may provide more insight into signal transduction pathways upon perturbation of a protein network or changes to a panel of proposed biomarkers in a given disease state. |
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