Proteomic Profiling of the Epithelial-Mesenchymal Transition Using 2D DIGE
Metastasis remains the primary cause of cancer patient death. Although the precise molecular mechanisms at play remain largely unknown, tumor progression is currently hypothesized to follow a series of sequential steps known as the metastatic cascade. An important component, thought to be involved early in this cascade, is the process known as epithelial-mesenchymal transition (EMT), whereby epithelial cells undergo morphogenetic alterations and acquire properties typical of mesenchymal cells. EMT confers a metastatic advantage to the cancer cells through the loss of cell-cell adhesion, enhanced proteolytic activity, and increased cell migration and invasiveness. This chapter describes the experimental workflow for the secretome analysis of MDCK cells undergoing oncogenic Ras, and Ras/TGF-β-mediated EMT. To enable this comparison, serum-free cell culture conditions were optimized, and a secretome purification methodology established. Secretome samples were then subjected to DIGE analysis to reveal and quantify proteins that are differentially expressed during EMT. The proteomic strategy detailed within successfully identified several EMT modulators and broadens our understanding of the extracellular facets of the EMT process.
Available from: PubMed Central
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ABSTRACT: The aim of this study was to explore whether alendronate sodium regulates tissue remodeling by controlling the transforming growth factor (TGF)-β1-induced epithelial-mesenchymal transition (EMT) and bone morphogenetic protein (BMP)-7-induced mesenchymal-epithelial transition (MET) in CCl(4)-induced hepatic fibrosis in mice. A mouse model of CCl(4)-induced hepatic fibrosis was evaluated using the hematoxylin and eosin (HE) and Masson's trichrome staining histological methods. The activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using an automated biochemical analyzer. The expression of TGF-β1, α-smooth muscle actin (α-SMA), BMP-7 and E-cadherin in the hepatic tissue was detected using immunohistochemistry. The mRNA and protein levels of TGF-β1, α-SMA, BMP-7, fibroblast-specific protein 1 (FSP1), E-cadherin and N-cadherin were detected using RT-PCR and western blot analysis. Immunohistochemical and molecular biochemical examination revealed that alendronate sodium significantly arrested the progression of hepatic fibrosis. Alendronate sodium caused significant amelioration of liver injury and reduced the activities of serum ALT and AST (P<0.001). Furthermore, alendronate sodium markedly reduced TGF-β1 and α-SMA mRNA expression and increased BMP-7 and E-cadherin in the mouse liver tissue (P<0.001). Alendronate sodium significantly arrested the progression of hepatic fibrosis. The underlying mechanism was associated with changes in the redox state, which remains variable in liver fibrosis, and depends on the balance between TGF-β/smad- and BMP-7-modulated mechanisms which regulate EMT and MET in multifunctional progenitors.
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ABSTRACT: Most biological processes including growth, proliferation, differentiation, and apoptosis are coordinated by tightly regulated signaling pathways, which also involve secreted proteins acting in an autocrine and/or paracrine manner. In addition, extracellular signaling molecules affect local niche biology and influence the cross-talking with the surrounding tissues. The understanding of this molecular language may provide an integrated and broader view of cellular regulatory networks under physiological and pathological conditions. In this context, the profiling at a global level of cell secretomes (i.e., the subpopulations of a proteome secreted from the cell) has become an active area of research. The current interest in secretome research also deals with its high potential for the biomarker discovery and the identification of new targets for therapeutic strategies. Several proteomic and mass spectrometry platforms and methodologies have been applied to secretome profiling of conditioned media of cultured cell lines and primary cells. Nevertheless, the analysis of secreted proteins is still a very challenging task, because of the technical difficulties that may hamper the subsequent mass spectrometry analysis. This chapter describes a typical workflow for the analysis of proteins secreted by cultured cells. Crucial issues related to cell culture conditions for the collection of conditioned media, secretome preparation, and mass spectrometry analysis are discussed. Furthermore, an overview of quantitative LC-MS-based approaches, computational tools for data analysis, and strategies for validation of potential secretome biomarkers is also presented.
Available from: Germain Gillet
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Disease phenotype reorganizations are the consequences of signaling pathway perturbations and protein abundance modulations. Characterizing the protein signature of a biological event allows the identification of new candidate biomarkers, new targets for treatments and selective patient therapy. The combination of discovery LC-MS/MS analyses and targeted mass spectrometry using selected reaction monitoring (SRM) mode has emerged as a powerful technology for biomarker identification and quantification owing to faster development time and multiplexing capability. The epithelial-mesenchymal transition (EMT) is a process that controls local invasion and metastasis generation by stimulating changes in adhesion and migration of cells but also in metabolic pathways. In this study, the non-transformed human breast epithelial cell line MCF10A, treated by TGFβ or overexpressing mutant K-Ras(v12), two EMT inducers frequently involved in cancer progression, was used to characterize protein abundance changes during an EMT event. The LC-MS/MS analysis and label-free quantification revealed that TGFβ and K-Ras(v12) induce a similar pattern of protein regulation and that besides the expected cytoskeletal changes, a strong increase in the anabolism and energy production machinery was observed.
To our knowledge, this is the first proteomic analysis combining a label-free quantification with an SRM validation of proteins regulated by TGFβ and K-Rasv12. This study reveals new insights in the characterization of the changes occurring during an epithelial-mesenchymal transition (EMT) event. Notably, a strong increase in the anabolism and energy production machinery was observed upon both EMT inducers.
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