Analysis of the mechanisms of action of angiogenesis inhibitor, endostatin and its dynamic localization in tumor blood vessels.

内皮抑素是一种内源性血管生成抑制剂，具有很强的抗癌活性。在小鼠肿瘤模型中，内皮抑素抑制肿瘤进展，抑制肿瘤转移，诱导肿瘤消退。本研究的最终目的是在分子水平上阐明内皮抑素的作用机制，为开发新的抗癌药物做出贡献。在2004年至2005年期间，我们取得了以下进展。内皮抑素在类风湿关节炎动物模型中除具有抗癌活性外，还表现出较强的抗炎活性。我们在两种不同的实验模型中证实了内皮抑素的疗效。通过功能研究发现内皮抑素对基质金属蛋白酶MMP-2、9、12、13、14具有抑制活性。对基质金属蛋白酶的抑制活性被认为是其抗炎作用的机制之一。MMP-14在细胞膜上表达，据报道在肿瘤侵袭和转移中起重要作用。因此，内皮抑素可能通过其阻断酶活性结合MMP-14抑制肿瘤侵袭转移。我们分析了内皮抑素暴露于HUVEC后，通过共聚焦显微镜观察到内皮抑素和MMP-14的共定位。然而，我们无法检测到共定位。HUVEC的迁移抑制实验数据表明，一些HUVEC制剂对内皮抑素没有反应。我们假设在应答者和非应答者之间存在基因表达谱的差异。利用基因芯片芯片对基因表达进行估计，对比结果显示，与非应答细胞相比，应答细胞中有1.86%的基因表达加速，2.9%的基因表达下降，95%的基因表达不变。

Endostatin is an endogenous angiogenesis inhibitor and showed quite strong anti-cancer activity. In mouse tumor model, endostatin inhibits tumor progression, tumor metastasis and induces tumor regression. The final goal of this research is to elucidate the functional mechanisms of endostatin on molecular level, then to contribute the development of new anti-cancer drugs. During 2004 to 2005, we achieved those progression described below.In addition to anti-cancer activity, endostatin showed the strong anti-inflammatory activity in animal model for rheumatoid arthritis. We confirmed the efficacy of endostatin in two different experimental models. Also, we discovered that endostatin has inhibitory activity against some kinds of matrix metalloproteinases, MMP-2, 9, 12, 13, 14 through the examination of functional study. Inhibitory activity against matrix metalloproteinases was considered to be one of the mechanisms of anti-inflammatory effects. MMP-14 is expressed on cell membrane and has been reported to participate the considerable contribution for tumor invasion and metastasis. Therefore, it is possible that endostatin binds MMP-14 and inhibits tumor invasion and metastasis through its blocking enzyme activity. We analyzed that the co-localization of endostatin and MMP-14 was observed by confocal microscopy after endostatin was exposed to HUVEC. However, we could not detect the co-localization. The experimental data for HUVEC migration inhibition assay demonstrated that some preparations of HUVEC did not respond to endostatin. We hypothesized that there was the difference of gene expression profile between responder and non-responder. Using gene chip microarray, gene expression was estimated and the results for comparison revealed that accelerated expression was observed in 1.86% of genes, 2.9% of genes showed decreased expression and 95% were unchanged for the responder cells in comparison with the non-responder cells.