Novel Aspect Secretome analysis is a feasible and efficient method to find, identify and characterize clinical biomarkers Introduction Secretome (proteins secreted by cells into the extracellular environment) can reflect a large variety of pathological conditions and can be a useful source of biomarkers. It is increasingly clear that a key role in carcinogenesis is played by secreted molecules either by tumor and surrounding stromal cells. During the last years the incidence and mortality of melanoma have rapidly increased. Metastatic spread of malignant melanoma is often associated to cancer progression with poor prognosis and survival. Deregulation of dynamic interactions between tumor melanocytes and neighbouring stromal cells leads to the acquisition of cell proliferation capabilities and invasiveness. Proteomic profiling of secretomes of human metastatic cell lines derived from the same patient was performed by a shotgun LC-MS/MS-based approach. Methods Cell lines PES 41-43-47 were isolated from different metastases of the same patient (PES 41 and 47 from sub cutaneous metastases and PES 43 from lung metastases). For secretome analysis, cells were seeded in 30 cm plates in 10% FCS DMEM. After 24 h, culture media were replaced with 0.5% FCS DMEM and after 24h culture media were replaced with serum-free media. Media were collected after 24 h, lyophilized and resuspended in MilliQ water. Proteins were precipitated with TCA, resuspended in AMBIC buffer, reduced, alkylated and digested with trypsin. Aliquots of the supernatant were analyzed by LC-MS/MS. For Western Blot analyses proteins were separated by SDS-PAGE, transferred onto nitrocellulose membranes and incubated with SPARC, ECM1 and OPN antibodies. Preliminary Data Results provide a list of candidates associated to the metastatic potential of PES human melanoma cell lines. Among them, several matricellular proteins previously reported as involved in melanoma aggressiveness were identified (i.e. SPARC, osteopontin, galectins). In addition, the extracellular matrix protein 1 that stimulates the proliferation and angiogenesis of endothelial cells and fibronectin that is involved in cell adhesion and motility were also identified. The secretion of selected proteins (i.e. SPARC, ECM1 and OPN) was further validated by Western Blot analysis. Equal amounts of SPARC and ECM1 were detected in the three cell lines analyzed, whereas OPN secretion was found to be less pronounced in PES 47 cells secretome. Bioinformatic approaches can support the experimental work by providing algorithms for pathway analysis that aims at connecting proteins identified in "omics" surveys. To better understand the mutual interactions of identified proteins, network maps were constructed using the Ingenuity Pathway Analysis software. The graphical view of the resulting network revealed that several secreted proteins were clustered on the basis of their reciprocal interactions. The two pathways obtained for PES secretomes show that several proteins differentially expressed were found to converge on the JNK, ERK, and NF-?? molecular complexes. In addition, a second network was found to converge on Myc and FOS signaling pathways. Overall, proteins identified in PES secretomes converge on pathways involved in the activation of cancer signaling cascades. Although network data need to be further investigated, information derived from the analysis of these pathways will provide useful suggestions for future experimental works.
Analysis of human melanoma cell lines secretomes
R Cozzolino;G Palmieri;C Rozzo;
2011
Abstract
Novel Aspect Secretome analysis is a feasible and efficient method to find, identify and characterize clinical biomarkers Introduction Secretome (proteins secreted by cells into the extracellular environment) can reflect a large variety of pathological conditions and can be a useful source of biomarkers. It is increasingly clear that a key role in carcinogenesis is played by secreted molecules either by tumor and surrounding stromal cells. During the last years the incidence and mortality of melanoma have rapidly increased. Metastatic spread of malignant melanoma is often associated to cancer progression with poor prognosis and survival. Deregulation of dynamic interactions between tumor melanocytes and neighbouring stromal cells leads to the acquisition of cell proliferation capabilities and invasiveness. Proteomic profiling of secretomes of human metastatic cell lines derived from the same patient was performed by a shotgun LC-MS/MS-based approach. Methods Cell lines PES 41-43-47 were isolated from different metastases of the same patient (PES 41 and 47 from sub cutaneous metastases and PES 43 from lung metastases). For secretome analysis, cells were seeded in 30 cm plates in 10% FCS DMEM. After 24 h, culture media were replaced with 0.5% FCS DMEM and after 24h culture media were replaced with serum-free media. Media were collected after 24 h, lyophilized and resuspended in MilliQ water. Proteins were precipitated with TCA, resuspended in AMBIC buffer, reduced, alkylated and digested with trypsin. Aliquots of the supernatant were analyzed by LC-MS/MS. For Western Blot analyses proteins were separated by SDS-PAGE, transferred onto nitrocellulose membranes and incubated with SPARC, ECM1 and OPN antibodies. Preliminary Data Results provide a list of candidates associated to the metastatic potential of PES human melanoma cell lines. Among them, several matricellular proteins previously reported as involved in melanoma aggressiveness were identified (i.e. SPARC, osteopontin, galectins). In addition, the extracellular matrix protein 1 that stimulates the proliferation and angiogenesis of endothelial cells and fibronectin that is involved in cell adhesion and motility were also identified. The secretion of selected proteins (i.e. SPARC, ECM1 and OPN) was further validated by Western Blot analysis. Equal amounts of SPARC and ECM1 were detected in the three cell lines analyzed, whereas OPN secretion was found to be less pronounced in PES 47 cells secretome. Bioinformatic approaches can support the experimental work by providing algorithms for pathway analysis that aims at connecting proteins identified in "omics" surveys. To better understand the mutual interactions of identified proteins, network maps were constructed using the Ingenuity Pathway Analysis software. The graphical view of the resulting network revealed that several secreted proteins were clustered on the basis of their reciprocal interactions. The two pathways obtained for PES secretomes show that several proteins differentially expressed were found to converge on the JNK, ERK, and NF-?? molecular complexes. In addition, a second network was found to converge on Myc and FOS signaling pathways. Overall, proteins identified in PES secretomes converge on pathways involved in the activation of cancer signaling cascades. Although network data need to be further investigated, information derived from the analysis of these pathways will provide useful suggestions for future experimental works.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
