Differedtial Proteins In Esophageal Squamous Cell Line EC9706/CDDP Identified By Silac Quantitative Proteomic Approach

BackgroundsMultidrug resistance (MDR) is a generic term for cancers or tumor cells to evade thecytotoxic effects of anticancer drugs. In clinical scenario, cancers or tumor cells frequentlyacquire MDR in the process of chemotherapy, leading to poor response to chemotherapeutictreatment. In basic research of medicine,lots of researchers have investigated the molecularmechanisms of MDR..MDR may result from structural and functional changes at the plasmamembrane or the cytoplasm, cellular compartments, or nucleus. MDR includes (i) alteredcomposition of plasma membrane;(ii) binding and cellular accumulation;(iii) changes ofexpression and activity of plasma membrane;(iv) altered of endocytosis and subsequenttargeting of endosomes;(v) alter and extent of exocytosis;(vi) modified ionic environments,such as pH, Ca2+;(vii) alterations in the expression of proteins necessary for drugdetoxification and DNA replication.Quantitative proteomics is a hot research field in post-genomic era, it has become animportant tool for biomarker identification of disease diagnosis. The findings derived fromsuch exploration can improve our understanding of biological processes. In particular, it isuseful for clarifying the molecular mechanisms of diseases. Two-dimensional gelelectrophoresis (2-DE) and stable isotope labeling are two commonly used methods inquantitative proteomics. In previous studies, we have identified more than30proteinsassociated with the development of esophageal cancer using esophageal cancer and adjacentnon-cancer tissue samples. However, defects of2-DE limit the identification of moleculeswhich are too small or too large, too alkaline or too acidic and insoluble protein. Further more,it is impossible for2-DE automation. Stable isotope labeling with amino acids in cellculture(SILAC) is a new technique for quantitative analysis of proteome in living cells usingstable isotope labeled amino acids combined with mass spectrometry. SILAC is able to achievequalitative analysis and quantitative comparison by using a small amount of sample and it is adirect, simple and effective technology. SILAC combined with mass spectrometry is graduallyreplacing the2-DE.Among the most common cancers worldwide, esophageal carcinoma (EC) is characterizedby the remarked regional distribution evidenced by a large body of epidemiological studies.The incidence rate between high and low incidence area for EC can be as high as500-fold difference. Long time survival after confirmed diagnosis of EC is very poor and the five-yearsurvival is approximately10%. In northern China, Linzhou has been known for its highestincidence and mortality of EC across world. In the past decades, scientists believed that EC is amulti-stage process involved a variety of abnormalities after comprehensive andmulti-displinary investigations. The incidence and mortality rate of EC in this high area,however, haven’t ameliorated yet and EC still persists one of the main causes of cancer-relateddeaths. CDDP and5-FU are the major drugs for EC chemotherapy, but, treatment effectivenessis very poor, which is attribulated to MDR. Therefore, it is important to identify proteinsassociated with MDR, which can provide a rational plan for chemotherapy and understand themolecular mechanism of MDR.AimsThe purpose of this study was to quantitatively identify proteomic fingerprints associatedwith MDR of esophageal cancer by SILAC and HPLC-ESI-MS/MS. It is helpful to institute aplan for chemotherapy of EC and understand the molecular mechanisms of MDR.Methods1EC9706/CDDP was generated by increasing concentration of CDDP during EC9706cultivation；2SILAC was used to label EC9706and EC9706/CDDP with heavy and light mediumrespectively. Subsequently, mixed peptides derived from EC9706and EC9706/CDDP wereanalyzed by HPLC-ESI-MS/MS to identify differential proteins between EC9706andEC9706/CDDP；3Western blotting was used to validate proteins associated with MDR.ResultsCompared to parental EC9706, EC9706/CDDP manifested slow proliferation, cellpleomorphology, atypia and increased resistant-index3.23. Seventy-four differential proteinsbelong to various families by means of function, such as energy metabolism (11%),cytoskeleton (20%), transcription regulation and DNA repair (11%), nucleosome assembly(2.7%), protein biosynthesis and mRNA processing (12%), intracellular transport (5.4%),molecular chaperone (8.1%), immunity/inflammation (5.4%), ribosome constituent (8.1%) andredox homeostasis (9.5%).ConclusionsDevelopment of MDR is a complicated process involving dysregulation of multiplemolecules and pathways. The data is of great value for in-depth elucidation of molecular mechanisms of the MDR implicated in ESCC and may represent potential molecular targets forfuture therapeutic development.