Abstract
Epithelial-to-mesenchymal transition (EMT) is an essential biological process involved in embryonic development, cancer progression, and metastatic diseases. EMT has often been used as a model for elucidating the mechanisms that underlie bladder cancer progression. However, no study to date has addressed the quantitative global variation of proteins in EMT using normal and non-malignant bladder cells. We treated normal bladder epithelial HCV29 cells and low grade nonmuscle invasive bladder cancer KK47 cells with transforming growth factor-beta (TGF-β) to establish an EMT model, and studied non-treated and treated HCV29 and KK47 cells by the stable isotope labeling amino acids in cell culture (SILAC) method. Labeled proteins were analyzed by 2D ultrahigh-resolution liquid chromatography/LTQ Orbitrap mass spectrometry. Among a total of 2994 unique identified and annotated proteins in HCV29 and KK47 cells undergoing EMT, 48 and 56 proteins, respectively, were significantly upregulated, and 106 and 24 proteins were significantly downregulated. Gene ontology (GO) term analysis and pathways analysis indicated that the differentially regulated proteins were involved mainly in enhancement of DNA maintenance and inhibition of cell-cell adhesion. Proteomes were compared for bladder cell EMT vs. bladder cancer cells, revealing 16 proteins that displayed similar changes in the two situations. Studies are in progress to further characterize these 16 proteins and their biological functions in EMT.
Highlights
The process of epithelial-to-mesenchymal transition (EMT) plays an important role in the invasion and metastasis of solid cancer cells
Strategies developed for this purpose include 2-dimensional difference gel electrophoresis (2D-DIGE), isotope-coded affinity tagging (ICAT), the similar isobaric tag for relative and absolute quantitation, and stable isotope labeling by amino acids in cell culture (SILAC) [11,12,13]
Proteins isolated from HCV29 and KK47 cells with and without TGF-β treatment were mixed and digested
Summary
The process of epithelial-to-mesenchymal transition (EMT) plays an important role in the invasion and metastasis of solid cancer cells. In-depth analysis of large numbers of proteins has been facilitated by recent advances in proteomics using mass spectrometry (MS) for identification and quantification Strategies developed for this purpose include 2-dimensional difference gel electrophoresis (2D-DIGE), isotope-coded affinity tagging (ICAT), the similar isobaric tag for relative and absolute quantitation (iTRAQ), and stable isotope labeling by amino acids in cell culture (SILAC) [11,12,13]. We previously established a model of TGF-β-induced EMT in normal bladder epithelial HCV29 cells [18], and applied a SILAC method for differential proteomic analysis of HCV29, low grade nonmuscle invasive bladder cancer cell line KK47, and metastatic BC cell line YTS1 [19]. Global proteome levels in the two cell lines under the two conditions were quantitatively analyzed and compared
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