| Summary: | 碩士 === 高雄醫學大學 === 醫學研究所 === 89 === Abstract
Anti-angiogenesis strategy has been actively investigated for cancer treatment in recent years. One the latest identified angiogenesis inhibitors, vasostatin, was isolated from conditioned media of EBV-immortalized cells and identified as NH2-terminal fragment (amino acids 1-180) of a calcium-binding protein calreticulin. In this study, we have cloned vasostatin cDNA from lymphoma cells by RT-PCR. After verification by DNA sequencing, vasostatin cDNA was subcloned into E.coli expression vector to express and generate large quantities of recombinant 6xHis-tagged vasostatin. The 6xHis-tagged is soluble and capable of inhibiting proliferation of bovine aortic endothelial cells (BAEC) with IC50~ 40 nM without significant adverse effect on 3T3 cells. Flow cytometry and fluorescence microscope analysis by Hoechst 33258 indicated that vasostatin induced apoptosis in BAE cells. Besides, injection of vasostatin (200 mg/egg) inhibited in vivo angiogenesis in chicken chorioallantoic membrane (CAM) and suppressed tumor growth in mice bearing Lewis lung carcinoma cells. Moreover, potency of vasostatin in inhibition of neovascularization in CAM and tumor size in mice is significantly stronger than that of endostatin at the same dose. This finding seems to be consistent with previous study which reported the effective dose of vasostatin is 4-10 fold lower than that of endostatin and angiostatin. Therefore, this potent anti-angiogenesis molecule may be useful for the prevention and treatment of human cancers.
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