Proteomic Analysis Of Esophageal Squamous Cell Carcinoma Cells Between Cancer Tissue And Metastatic Lymph Nodes

Esophageal squamous cell carcinoma (ESCC), the predominant histological subtype of esophageal cancer, is the fourth most common malignancy and still represents a great health concern in China. The aggressive behavior of this tumor is often associated with systemic spread of the disease at the time of diagnosis. One of the main reasons for the low survival rate is that neoplasms in esophagus are not detected until they have invaded into surrounding tissues or spread throughout the body at advanced stages, such as regional lymph node metastasis. It is very important for studing the mechanism of lymphatic metastasis in ESCC.Laser capture microdissection (LCM) is a rapid, reliable method to procure pure populations of targeted cells from specific microscopic regions of tissue sections for subsequent analysis. Two-dimensional electrophoresis (2-DE) is a highly resolving technique for arraying proteins by isoelectric point and molecular mass. To contribute to the development and improvement of molecular markers for diagnostics and prognostics and of therapeutic targets for the disease, we have largely expanded the currently available cancer tissue and metastatic lymph node maps and studied the differential expression of proteins in cancer tissues and metastatic lymph nodes. Protein identification was done by peptide mass fingerprinting with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS), validated by western blotting and immunolohistochemical analysis.PART I Application of laser capture microdissection in esophageal squamous cell carcinoma proteomic analysisObjective: To establish the technology for proteomic analysis of ESCC with LCM. The LCM is a rapid, reliable method to procure pure populations of ESCC cells from specific microscopic regions of tissue sections for subsequent analysis.Methods: Fresh frozen normal esophageal tissue, cancer tissue and metastatic lymph node samples were cut into 10μm, the slices were made hematoxylin's staining. Targeted cells were transferred to the cap surface, and the cap was placed directly onto a vial for molecular processing. Single cells could be individually targeted and transferred.Results: Single cells of normal esophageal tissue and cancer tissue have been obtained by LCM, as were the samples of cancer tissue and metastatic lymph nodes. We got representative 2-DE images with good resolution and reproducibility differently. Conclusion: LCM is a key technique in proteomic study which can effectively resolve the problem of irrelevant tissues.PART II Proteomic analysis of esophageal squamous cell carcinoma cells between normal esophageal tissue and cancer tissueObjective: To obtain and analyze the protein spots with differential expression of 2-DE maps.Methods: Single ESCC cells of normal esophageal tissue and cancer tissue have been obtained by LCM. We used 2-DE and Matrix-assisted laser desorption/ionization mass spectrometry-time of flight (MALDI-TOF) based proteomics to compare the protein profiles between normal esophageal tissue and cancer tissue and to identify differently expressed proteins in the ESCC.Results: 38-55 protein spots were discovered differently and 20 were identified by PMF,including 11 proteins as galectin 7,14-3-3 proteinεwere up-regulated and 9 proteins as MTCBP-1 were down-regulated in ESCC cells of cancer tissue. Conclusion: 2-DE maps of cells between normal esophageal tissue and cancer tissue showed different protein expression. The variety of proteins may be involved in the carcinogenesis of ESCC.PART III Proteomic analysis of esophageal squamous cell carcinoma cells between cancer tissue and metastatic lymph nodesObjective: To analyze the 2-DE protein separation and identification by PMF in ESCC cells of cancer tissue and metastatic lymph nodes Methods: Single ESCC cells of cancer tissue and metastatic lymph nodes have been obtained by LCM. We used 2-DE and MALDI-TOF based proteomics to compare the protein profiles between cancer tissue and metastatic lymph nodes and to identify differently expressed proteins in the ESCC.Results: 20-29 protein spots were displayed differently and 6 were identified by PMF,including 5 proteins as peroxiredoxin 1,gelsolin family-capping proteins were up-regulated and 1 protein as MTCBP-1 was down-regulated in ESCC cells of metastatic lymph nodes. Conclusion: 2-D gel images of ESCC cells between cancer tissue and metastatic lymph nodes showed different protein expression. The variety of proteins may be concerned with metastatic mechanism of lymph node in ESCC possibly.PART IV Protein identification by Western blotting and immunolohistochemical analysisObjective: To identify the confidence of some potential diagnostic marker proteins and detection of therapeutic medicine. To validate differently expressed proteins among the normal esophageal tissue, cancer tissue and metastatic lymph nodes.Methods: Protein identification was validated by Western blotting and immunolohistochemical analysis.Results: MnSOD,14-3-3 proteinεwere up-regulated in ESCC cells of cancer tissue comparing with normal epithelial cells and validated by western blotting and immunolo- histochemical analysis. Gelsolin family-capping proteins was down-regulated in ESCC cells of metastatic lymph nodes comparing with that of cancer tissue . Conclusion: Western blotting and immunolohistochemical analysis is comfortable to proteomic analysis for protein identification.