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標題: Clarithromycin抑制非小型肺腺癌細胞轉移作用之研究
To study the anti-metastatic effects of clarithromycin on non-small lung cancer cells
作者: 楊呈琦
Yang, Cheng-Chi
關鍵字: clarithromycin;micorarray;NSCLC
出版社: 生命科學系
Clarithromycin is a new 14-member macrolide antibiotic, it has been used as anti-inflammatory drug to treat diffuse panbronchiolitis and to restraining Helicobacter Pylori since 1990. Clinically mainly in long term-low dose; in 1997, Yasunami indicated that clarithromycin is a potent angiogenesis inhibitor for anti-tumor activity, the research later indicated clarithromycin can suppress tumor-associated growth factor expression by tumor, such as TNF-α and IL-6, result in suppressed tumor growth and metastasis, but its detail mechanism is unclear. And in our study, clarithromycin can not obviously suppress the proliferation of non-small-cell lung cancer (NSCLC) cell line, CL1-5, but remarkably decreases the invasion ability of CL1-5. Metastasis means that the malignant cancer cell moves from original position to the second place. Metastasis contains several important stages; including cell migration, invasion, and angiogenesis. Tumor macrophage infiltrating can promote metastasis, it is a normal immune response, but in tumor infiltrating process macrophages can secrete many kinds of inflammatory factors, such as TNF-α, that increases the IL-8 expression and angiogenesis of tumor, makes malignly to tumor and negatively to patient prognosis. In this study, we used the CL1-5/macrophage co-culture system to promote angiogenesis and metastasis of tumor, and then treated with clarithromycin. Furthermore, microarray analysis is used to find out the effect of clarithromycin on interaction between macrophage-CL1-5. And confirm the difference of gene expression probably by quantitative real-time RT-PCR, we confirmed several genes by quantitative real-time RT-PCR, the genes that could be increased by macrophage co-cultured and decreased by clarithromycin include PlGF, DnaJ, and JunB, and other genes that could be decreased by macrophage co-cultured and increased by clarithromycin include profilin 2, INF-PK, and archain-1. PlGF is a VEGF family protein, can promote angiogenesis; JunB is a subunit of AP-1 that is an important transcription increasing expression factor of IL-8; DnaJ and profilin 2 may be involved in cell migration. Both are highly expressed in cell with strongly invasion and migration activity. In addition, previous study indicated that clarithromycin has inhibitor activity on IL-8 produced by inflammatory cells. IL-8 is an important angiogenesis factor, and it was highly expressed under macrophage co-culture condition, suggest that clarithromycin suppress tumor angiogenesis and metastasis through its inhibitor activity of IL-8. The results of quantitative real-time RT-PCR indicated that IL-8 mRNA expression in CL1-5 was reduced under treatment with clarithromycin, and the IL-8 mRNA expression in macrophage co-culture induced CL1-5 is less reduced under treatment with clarithromycin; furthermore, the result of luciferase reporter gene assay indicated that IL-8 promoter activity is upregulated by macrophage co-culture and down-regulated by treatment with clarithromycin. It is reporter that IL-8 is regulated by NF-κB pathway, when the inhibitor of NF-κB, IκBα was phosphorylated, NF-κB would translocate into nucleus and bind to IL-8 promoter. In this study, western blotting was used to analyze NF-κB translocation, the result indicated that NF-κB translocation activity is upregulated by macrophage co-culture and down-regulated by treatment with clarithromycin, but in macrophage co-culture induced CL1-5 NF-κB translocation activity had not significantly suppressed under treatment with clarithromycin, and the phosphorylated IκBα analyzed by western blotting had the same trend. We think that clarithromycin inhibits the secretion of IL-8 through other pathway, such as AP-1 pathway. In microarray analysis, we could modified the gene variation and putatively influenced signal transduction pathway under treatment with clarithromycin; clarithromycin inhibits tumor angiogenesis and metastasis also through NF-κB pathway, and the NF-κB pathway is generally regulated by TNF-α and IL-1α, whether is clarithromycin begins to influence or there is further characterization of pathway identified will be in progress in the future.
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