Analysis of Secreted Proteins for the Study of Bladder Cancer Cell Aggressiveness

Abstract

Secreted proteins play a key role in cell signaling, communication, and migration. We recently described the development of an aggressive variant (T24M) of the bladder cancer cell line T24. Using this cell line model, the objective of our work was the identification of secreted proteins involved in the acquisition of the aggressive phenotype. Using in vitro assays, we demonstrate that conditioned media of the T24M cells promote motility of the parental less aggressive T24 cells. Proteomic analysis of cell culture conditioned media by the use of 2-dimensional gel electrophoresis coupled to MALDI TOF MS and LC-MS approaches resulted in enrichment and detection of multiple classical extracellular and secreted proteins such as fibronectin, cystatin, fibrillin, fibulin, interleukin 6, etc. Comparison of the secretome of the T24 and T24M cells indicated differences in proteins with potential involvement in the mechanisms of cell aggressiveness including SPARC, tPA, and clusterin. These findings were further confirmed by Western blot analysis. In the case of SPARC, further studies involving transwell assays indicated that blockage of the protein in the presence of SPARC-specific Abs results in decreased cell motility. Collectively, our study provides a 2DE-based comprehensive analysis of bladder cancer cell secretome. The results indicate various secreted proteins with potential involvement in bladder cancer cell aggressiveness and more specifically provide initial evidence for special role of SPARC in bladder cancer cell motility and invasiveness.

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“…Two-dimensional gel electrophoresis was performed as previously described (Makridakis et al, 2010) with the following specifications. Mitochondrial fractions were solubilized in the isoelectric focusing (IEF) sample buffer [7 M urea, 2 M thiourea, 4% CHAPS, 1% dithioerythreitol, 2% ampholytes ( pH range: 3-10)] and resolved (100 μg per sample) on 7-cm strips, pH range 3-10 non-linear (Bio-Rad), using the in gel rehydration method.…”

Profilin 1 is a Potential Biomarker for Bladder Cancer Aggressiveness

“…Transwell Migration Assay-In Vitro Blocking Experiments-Transwell migration assays were performed as previously described (14,16). In brief, T24 cells were cultured for 48 h in Dulbecco's modified Eagle's medium supplemented with 2% fetal bovine serum and then were transferred, at concentration of 5 ϫ 10 4 /100 l, to the 5 m pore size insert of a transwell plate (Corning-Costar, Cambridge, MA).…”

Global secretome analysis identifies novel mediators of bone metastasis

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