Proteomics Analysis And Related Mechanism Study On Adriamycin Resistance In Breast Cancer Cells

Background and PurposeThe incidence of breast cancer has become the highest for female malignant tumors all over the world.Almost 1.3 million women were diagnosed breast cancer,and more than 450,000 people died of breast cancer per year.Chemotherapy is the irreplaceable treatment for breast cancer due to high sensitivity,but 1/4 patients have to face the question of multiple-drug resistant(MDR).Thus,chemoresistance is a poor prognostic factor in many cancers and is a primary problem to resolve by doctors.Up to now,the mechanism of drug resistance in breast cancer is still unclear.Although it has became a hot field,breast cancer chemotherapy effect is difficult to break through.Deficiency in reverseing resistance methods,breast cancer has been a threat to health and brings about the economic burden on the family and society.DNA chip can afford genetic information,but it doesn't explain the progress of protein synthesis.Protein is the real practitioner rather than gene.There are many types of proteins,which can interact with each other and compise of a huge and complex net.Understanding the mechanisms behind the perspective of proteomic profiling will ultimately facilitate treatments with enhanced tumor responses in the clinic.Therefore,it is important to investigate the effects of proteins on tumor drug resistance.It is necessary to apply proteomics technology to elucidate the molecular mechanism of the cancer MDR.The aim of this study is to conduct an in vitro investigation into adriamycin-resistance mechanisms in breast cancer cells using proteomic strategies,to increase our understanding of the molecular processes involved,and to identifypotentially valuable diagnostic or therapeutic resistance biomarkers.MethodsMass spectrometry(MS)-based proteomics has greatly benefitted from enormous advance in high resolution instrumentation.A novel mass spectrometer,the Q Exactive,couples a mass selective quadrupole to the Orbitrap analyzer,which has been proven to be a popular instrument configuration.Consequently,the Q Exactive offers the potential to analyze many more peptides in a given time,with very high MS/MS data quality.We wanted to combine these benefits with a compact Ultra High Pressure Liquid Chromatography(UHPLC)system,known as the EASY-nLC 1000,which was not available to us in previous single-run analysis.To quantify protein expression changes,we applied TMT labeled samples together with tandem MS to determine differences between the drug sensitive and drug resistant cells.Protein intensities resulted from the average of the single TMT reporter ion intensities.We have run a minimalistic proteomic workflow and presented the quantitative proteomic profiles of two breast cancer cells: MCF-7 and MCF-7/ADR,which are of the same breast cancer origin,but are characterized by different reactions to adriamycin,and therefore,could possibly represent differences in the active molecular networks.The core of this work lies in the identification of cancer up/down regulated proteins externalized by cells,based on the combined exploitation of MS and bioinformatics tools.Furthermore,the candidate proteins can be validate by bioinformatics and Western Blot methods in order to acquire the whole proteins of resistance and construct a drug resistance proteins network,which can afford valuable evidence for reservsing resistance and target therapy.ResultsWe obtained 719 proteins which were identified and quantified in the mixed cell sample.Using the DAVID network analysis tool,we analyzed the molecular functions/localizations of the protein data sets according to GO functional annotationsand categories.With a threshold of 1.5 fold-change(?1.5 or ?0.67),we found 80 differentially expressed proteins(DEPs)showing 49 up-regulated and 31down-regulated proteins between MCF-7 and MCF-7/ADR cells.Basing on the human protein-protein interactions(PPI),we have retrieved the associated functional interaction networks for the DEPs and analyzed the biological functions.Six different signaling pathways and most of the DEPs strongly linked to chemoresistance,invasion,metastasis development,proliferation,and apoptosis.The identified proteins in biological networks served to resistant drug and to select critical candidates for validation analyses by western blot.The glucose-6-phosphate dehydrogenase(G6PD),gamma-glutamyl cyclotransferase(GGCT),isocitrate dehydrogenase 1(NADP+,soluble)(IDH1),isocitrate dehydrogenase 2(NADP+,mitochondrial)(IDH2)and glutathione S-transferase pi 1(GSTP1),five of the critical components of GSH pathway,contribute to chemoresistance.Human protein interacting data sets were downloaded from HPRD,which including 9617 proteins and 39240 interacting relations.In the HPRD database,we found 66 DEPs and used as seed proteins for further network research.From literature,we collected scientific publications of DEPs and one-step interacting proteins that have been experimentally discovered and verified.64(97%)DEPs had been reported to have correlation with drug resistance.ConclusionsThe novelty of our study deals with the application of this proposed quantitative proteomic approach to dissect the DEPs association with drug-resistance mechanisms in breast cancer cells.This proteomics technique is a powerful method to reach very large coverage of the cell proteome and allow systems wide analysis,and to discovery of DEPs aiming to address potentially valuable diagnostic or therapeutic resistance biomarkers,which may led to a better characterization of the MCF-7/ADR cell line and to a better understanding of its drug resistant phenotype.GGCT,G6 PD,IDH1,IDH2 and GSTP1,five of the critical components of GSH pathway,contribute to chemoresistance.97% DEPs had been reported to have correlation with drug resistance.These results were highly in accordance with the proteomics data,which further validate the accuracy of our proteomic experimental method.