Identification of Hypoxia and Metastasis-related Proteins in Neuroblastoma Cells by Comparative Proteomic Analysis

• Main Authors: Ming- Huei Gu, 辜明慧
• Other Authors: Wailap Victor Ng
• Format: Others
• Language: en_US
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/43067253693203693517

碩士 === 國立陽明大學 === 醫學生物技術暨檢驗學系暨研究所 === 98 === Neuroblastoma (NB) is the most common malignant solid tumor with unfortunate outcomes in many infants and children. This tumor arises from the sympathetic nervous system. The primary non-metastatic NB initiates at a localized area and is often resectable with good prognosis, but once metastasized, it is unresectable and resistant to chemotherapy while restraining tumor spread. The major type of NBs is the aggressive metastatic tumor. Furthermore, solid tumor cells have to overcome hypoxia and deficiency of nutrient. To ensure a sufficient supply of oxygen and nutrients, tumor metastasis or angiogenesis is probably a strategy for tumor growing in hypoxia. Therefore, the searching of differentially expressed hypoxia-induced and metastasis-related proteins, which are the aims of my thesis works, are important for the development of therapeutic target or diagnostic biomarker. The first project applied SILAC–based quantitative proteomics approach analyze hypoxia-induced protein in the secretome and cell lysate proteomes of NB cell lines, SH-SY5Y and SK-N-BE(2), cultured in hypoxia or normoxia condition. A total of 228 (6,862 peptides) and 286 (9,601 peptides) SILAC-labeled proteins were identified by LC-MS/MS in the secretomes of SH-SY5Y and SK-N-BE(2) cells, respectively, and 338 (9,154 peptides) and 374 (9,603 peptides) SILAC-labeled proteins in the cell lysates of SH-SY5Y and SK-N-BE(2) cells, respectively. These proteins were further analyzed with the public NV microarray data, in-house consolidated secretome databases, and membrane protein database to narrow down the hypoxia related candidates. In my study, a number of novel hypoxia-related candidates including FN1 (fibronectin 1) and MMP2 (matrix metalloproteinase 2) were identified. The second project used a label-free quantitative proteomics approach to identify metastasis-related candidates by comparing the proteomes of metastatic and non-metastatic NB cell lines, SK-N-BE(2) and IMR-32, respectively. A total of 340 (8,699 peptides) and 377 (9,507 peptides) human proteins were identified in the conditioned media from IMR-32 and SK-N-BE(2) cells, respectively. Approximately 421 (10,291 peptides) and 362 (11,018 peptides) proteins were detected in the cell lysates of IMR-32 and SK-N-BE(2), respectively. To obtain the ideal candidates, the significantly changed proteins were integratively analyzed with public transcriptomic data and secretome and membrane protein databases. This analysis found several potential metastasis candidates including VCAN (versican) and L1CAM (L1 cell adhesion molecule) and many novel neuroblastoma metastasis protein candidates. In this study, we demonstrated the efficiency of our proteomics approach in the identification of hypoxia-induced and metastasis-related candidates which may deserve further investigations to evaluate their potential as neuroblastoma therapeutic targets, diagnostic or prognostic markers