| Summary: | 碩士 === 國立中興大學 === 生命科學系所 === 99 === Gelsolin (GSN) is one of actin binding proteins (ABPs) that regulate dynamic actin filament organization by severing and capping. In recent years, many studies have devoted to establish the role of dynamic actin filament formation by ABPs in controlling the proliferation of malignant tumors. These studies showed that GSN might act as a tumor activator or as a tumor suppressor, and that is related to tumor stage and its ability of metastasis. Apparently, the effect of GSN on tumor cell proliferation is still not clear yet. In this study, GSN-overexpressed MDA-MB-231 human breast cancer cell line was utilized as a model to examine the effect of GSN overexpression on actin filament reorganization and cell proliferation in breast cancer cells. Firstly, a pcDNA6-GSN that contains full-length GSN cDNA was transfected into MDA-MB-231, and then the stable GSN overexpression cell lines of MDA-MB-231 confirmed by real time q-PCR and western bolt were cloned subsequently. Trypan blue exclusion assay was used to estimate the population doubling time (PDT) for cell proliferation of GSN-overexpressed MDA-MB-231, and then the cell morphology and intracellular actin filament formation were observed by the phase microscopy and the epi-fluorescent microscopy, respectively. The results showed, GSN overexpression would inhibit cell proliferation and make cell morphology wider as well as longer, and reduce cell aggregation, and promote actin filament depolymerization. A futher study was performed to investigate how GSN-overexpression could inhibit cell proliferation. The methods and results were as follows: (1) Measuring fura-2 F340/F380 fluorescence ratio in the cells indicated 1.56 folds of increases in Ca2+ levels for GSN-overexpressed cells. (2) Detecting lipid-related genes by q-PCR showed GSN-overexpression facilitates the fatty acid synthase (FAS) but attenuates the peroxisome proliferator-activated receptor-γ2 (PPAR-γ2). (3) Detecting by atomic force microscopy (AFM) indicated GSN-overexpression increased cell surface adhesion force. (4) Detecting cell cycle by cytometry showed GSN-overexpression cell increased percentage of the cell population at G1 phase. In addition, GSN-overexpressed cells will cause Tropomyosin-1 (Tm1) gene up-regulation. Using siRNA to silence Tm1 and detecting cell adhesion force showed that silenced Tm1 gene increased the cell adhesion to the extracellular matrix. Taken together, results obtained in this study suggested that GSN overexpression might affect cell proliferation through (1) actin filament depolymerization, directly or indirectly increased intracellular calcium concentration, and then affecting intracellular calcium signal; (2) changing the lipid-related gene expression to affect lipid metabolism; (3) modulating actin filament polymerization/ depolymerization to arrest cells in G1 phase by inhibiting the β-catenin/cyclin D1 signal pathway; (4) upregulating Tm1 gene that might affect cell adhesion to the extracellular force, inhibited cell growth.
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