| Summary: | 碩士 === 中山醫學大學 === 醫學分子毒理學研究所 === 93 === A large number of polysaccharide and triterpenoids of Ganoderma lucidum has been reported to inhibit the growth of tumors. Fungal immunomodulatory proteins, FIP-gts, were found in Ganoderma tsugae. In this study, we expressed and purified the fungal immunomodulatory protein (FIP-gts) in E. coli. However, the effect of FIP-gts on cancer cells has not been characterized clearly. Metastasis is one of the most difficult problems in cancer therapy. In wound healing assay, the level of motility have been decreased by the increasing concentration of FIP-gts in the previous study. In order to confirm the effect of FIP-gts, the Modified Boyden chamber assay was applied. Treatment of A549 cells with up to 0.07 mM of FIP-gts decreased cell migration by 11% for 8 h. 0.075 mM of FIP-gts decreased cell invasion significantly. In addition, Nm23 protein and mRNA was increased by reFIP-gts addition on A549 cells. We also observed the inhibitor of cell migration by overexpression of Nm23 protein in A549 cells. The secreted MMP-2 activity was notably inhibited measured by gelatin-zymography. Inhibitioty effect the FIP-gts on the MMP-2 activity of A549 cells in gelatin-zymography assay. In RT-PCR, the level of MMP-2 and MMP-9 were decreased but the MMP inhibitor of TIMP-2 had no effect and TIMP-1 increased after treated with FIP-gts. To further ascertain the antimetastatic activity of FIP-gts, we assessed the counteraction of FIP-gts with monoclonal FIP-gts antibody. Monoclonal FIP-gts antibody could reverse cell migration by 55%. In gelatin-zymography assay, MMP-2 activity was also slightly increased. We also observed differentproteins that changed between untreated or treated with FIP-gts in A549 cells using 2-DE gel analysis. We are going to identify these proteins in the future. Our results highlight the potential of FIP-gts in the treatment of clinical cancer metastasis.
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