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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 initial 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 ...
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 initial 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 in vitro 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 a special role of SPARC in bladder cancer cell motility and invasiveness.
The identification of biomarkers for disease aggressiveness is one of the most important clinical needs for bladder cancer management. Urine is a “gold mine” for biomarker discovery, nevertheless, with multiple proteins having low concentration, urine proteomics becomes challenging. In this part of the study we developed a fractionation strategy of urinary proteins based on the use of immobilized metal (nickel) affinity chromatography (IMAC). Urine samples from patients with bladder cancer and healthy or benign controls were submitted to IMAC fractionation and eluates analyzed by 1D-SDS PAGE, band excision and LC-MS/MS. Among the identified proteins in the former, multiple corresponded to proteins with affinity for metals and/or reported to be phosphorylated and included proteins with demonstrated association with bladder cancer such as MMP9, fibrinogen isoforms, and clusterin. Among the detected proteins, Aminopeptidase N was further found to be at decreased levels in urine from patients with invasive compared to non invasive bladder cancer and benign controls, in a pilot western blot analysis of a total of 108 samples. Additionally, profilin 1 detected in the IMAC eluates, by tissue microarray analysis had a
SUMMARY
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marked decrease of expression in the epithelial cells of the invasive (T2+) versus high risk non invasive (T1G3) tumors. Profilin also exhibited occasionally increased expression in stroma of high risk non invasive (T1G3) tumors and this pattern strongly correlated with poor prognosis and disease specific survival. The functional relevance of profilin 1 was investigated in the T24 bladder cancer cells where blockage of the protein by the use of antibodies resulted in decrease in cell motility with concomitant decrease in actin polymerization. Collectively, our in vivo part of the study provides a fractionation method of urinary proteins and as a main result of this analysis reveals the association of profilin with bladder cancer paving the way for its further investigation in bladder cancer stratification and therapeutic
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