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Comparative proteomic analysis of secreted proteins from nasopharyngeal carcinoma-associated stromal fibroblasts and normal fibroblasts.

ABSTRACT: Since the concept of the secretome (ensemble of proteins secreted and/or shed from cells) was proposed, it has become an attractive and challenging proteomic technology in recent years. However, secretome analysis still faces some difficulties mainly related to sample collection and preparation. In the present study, we established a reliable method for extracting secreted proteins by ultrafiltration centrifugation and conducting secretomic analysis. Accumulating evidence suggests that carcinoma-associated stromal fibroblasts (CAFs) play an important role in cancer initiation and progression. To investigate the expression patterns of secreted proteins from fibroblasts and to identify the secreted proteins involved in nasopharyngeal carcinoma (NPC) carcinogenesis, we conducted comparative secretome analysis between CAFs and normal fibroblasts. After two-dimensional gel electrophoresis (2-D PAGE), 11 significant spots were differentially expressed and identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). These proteins may take part in the regulation of the tumor micro-environment through different processes. The expression level of galectin-1 in the CAF supernatant was also determined by ELISA. This study provides useful information and new clues for the further understanding of the regulatory mechanisms of CAFs in the NPC microenvironment.

SUBMITTER: Ge S

PROVIDER: S-EPMC3438594 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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Comparative proteomic analysis of secreted proteins from nasopharyngeal carcinoma-associated stromal fibroblasts and normal fibroblasts.

Ge Shenglei S Mao Yitao Y Yi Yan Y Xie Dinghua D Chen Zhuchu Z Xiao Zhiqiang Z

Experimental and therapeutic medicine 20120213 5

Since the concept of the secretome (ensemble of proteins secreted and/or shed from cells) was proposed, it has become an attractive and challenging proteomic technology in recent years. However, secretome analysis still faces some difficulties mainly related to sample collection and preparation. In the present study, we established a reliable method for extracting secreted proteins by ultrafiltration centrifugation and conducting secretomic analysis. Accumulating evidence suggests that carcinoma ...[more]

PMID: 22969981

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Project description:BACKGROUND: The anaerobic Gram-positive bacterium Propionibacterium acnes is a human skin commensal that resides preferentially within sebaceous follicles; however, it also exhibits many traits of an opportunistic pathogen, playing roles in a variety of inflammatory diseases such as acne vulgaris. To date, the underlying disease-causing mechanisms remain ill-defined and knowledge of P. acnes virulence factors remains scarce. Here, we identified proteins secreted during anaerobic cultivation of a range of skin and clinical P. acnes isolates, spanning the four known phylogenetic groups. RESULTS: Culture supernatant proteins of P. acnes were separated by two-dimensional electrophoresis (2-DE) and all Coomassie-stained spots were subsequently identified by MALDI mass spectrometry (MALDI-MS). A set of 20 proteins was secreted in the mid-exponential growth phase by the majority of strains tested. Functional annotation revealed that many of these common proteins possess degrading activities, including glycoside hydrolases with similarities to endoglycoceramidase, ?-N-acetylglucosaminidase and muramidase; esterases such as lysophospholipase and triacylglycerol lipase; and several proteases. Other secreted factors included Christie-Atkins-Munch-Petersen (CAMP) factors, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and several hypothetical proteins, a few of which are unique to P. acnes. Strain-specific differences were apparent, mostly in the secretion of putative adhesins, whose genes exhibit variable phase variation-like sequence signatures. CONCLUSIONS: Our proteomic investigations have revealed that the P. acnes secretome harbors several proteins likely to play a role in host-tissue degradation and inflammation. Despite a large overlap between the secretomes of all four P. acnes phylotypes, distinct differences between predicted host-tissue interacting proteins were identified, providing potential insight into the differential virulence properties of P. acnes isolates. Thus, our data presents a rich resource for guiding much-needed investigations on P. acnes virulence factors and host interacting properties.

Project description:BackgroundThe extensional signals in cross-talk between stromal cells and tumor cells generated from extracellular matrix molecules, soluble factor, and cell-cell adhesion complexes cooperate at the extra- and intracellular level in the tumor microenvironment. CAFs are the primary type of stromal cells in the tumor microenvironment and play a pivotal role in tumorigenesis and development. Hitherto, there is hardly any systematic analysis of the intrinsic relationship between CAFs function and its abnormal signaling pathway. The extreme complexity of CAFs' features and their role in tumor development are needed to be further investigated.Methodology/principal findingsWe primary cultured CAFs and NFs from early stages of breast cancer tissue and identified them using their biomarker by immunohistochemistry for Fibronectin, ?-SMA and FAP. Microarray was applied to analyze gene expression profiles of human breast CAFs and the paired NFs. The Up-regulated genes classified by Gene Ontology, signal pathways enriched by DAVID pathway analysis. Abnormal signaling pathways in breast cancer CAFs are involved in cell cycle, cell adhesion, signal transduction and protein transport being reported in CAFs derived from other tumors. Significantly, the altered ATM signaling pathway, a set of cell cycle regulated signaling, and immune associated signaling are identified to be changed in CAFs.Conclusions/significanceCAFs have the vigorous ability of proliferation and potential of invasion and migration comparing with NFs. CAFs could promote breast cancer cell invasion under co-culture conditions through up-regulated CCL18 and CXCL12. Consistently with its biologic behavior, the gene expression profiling analyzed by microarray shows that some of key signaling pathways, such as cell cycle, cell adhesion, and secreting factors play an important role in CAFs. The altered ATM signaling pathway is abnormally active in the early stage of breast cancer. The set of immune associated signaling may be involved in tumor cell immune evasion.

Dataset Information

Comparative proteomic analysis of secreted proteins from nasopharyngeal carcinoma-associated stromal fibroblasts and normal fibroblasts.

Publications

Comparative proteomic analysis of secreted proteins from nasopharyngeal carcinoma-associated stromal fibroblasts and normal fibroblasts.

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