INHIBITION OF LUNG CANCER INVASION AND METASTASIS BY NOVEL ANGIOGENESIS INHIBITORS

内皮抑素（ES）是XVIII型胶原的羧基末端片段，具有很强的抗血管生成活性，已被引入多项临床研究。与最初的预期相反，ES的疗效并不一致，这表明需要使用动物模型进行更详细的研究。本研究的目的是确定ES基因转移对小鼠模型体内肿瘤生长的影响。用ES基因转染肺癌细胞株Lewis Lung Carcinoma (LLC)，通过脂质体蛋白表达和分泌ES。通过ELISA筛选ES基因的克隆后，建立了含有ES基因的稳定转染株（LLC/ES）和对照转染株（LLC/mock）。用MTT法对这些转染物进行体外增殖，结果显示出相似的生长速度。与之前的报道相反，LLC/ES转染的体内皮下肿瘤发生率明显高于LLC/模拟转染。使用抗cd31抗体进行免疫组化染色分析表明，ES基因的转移诱导了血管生成，这表明另一个基因参与了血管生成。正如预期的那样，LLC/ES转染物不仅分泌ES，而且分泌血管内皮生长因子（VEGF）的量远高于LLC/模拟转染物。有趣的是，LLC/ES细胞培养上清液对人脐静脉内皮细胞（HUVEC）体外增殖的促进作用远大于LLC/模拟细胞。这些结果表明，ES基因在小鼠肺癌细胞中的转移可诱导VEGE分泌，从而增强小鼠模型的体内致瘤性。ES基因治疗肺癌细胞可能会影响其他基因的表达，从而上调血管生成，因此应引起更多的关注。

Endostatin (ES), a carboxyl-terminal fragment of type XVIII collagen, which shows a strong anti-angiogenic activity, has been introduced to several clinical studies. Opposite to the initial expectation, the efficacy of ES is inconsistent, suggesting more detail investigations using animal model are required. The purpose of this study is to determine the effect of ES gene transfer on in vivo tumor growth in a murine model. A ung cancer cell line, Lewis Lung Carcinoma (LLC), was transfected with ES gene to, express and secrete ES by lipofectin. After clones were selected to secrete ES by ELISA, several stable transfectants with ES gene (LLC/ES) and control transfectants (LLC/mock) were established. In vitro proliferation using MTT assay of these transfectants demonstrated similar growing speed. In contrast to previous reports, in vivo subcutaneous tumorignecity of LLC/ES transfectants are significantly greater than that of LLC/mock transfectants. Immunohistochemical staining analysis using anti-CD31 antibody demonstrated that ES gene transfer induced angiogenesis, suggesting coinduction of another gene implicated in neovascularization. As expected, LLC/ES transfectants secreted not only ES but also vascular endothelial growth factor (VEGF) to much greater than LLC/mock transfectants. Interestingly, culture supernatants of LLC/ES cells enhanced in vitro proliferation of human umbilical vein endothelial cells (HUVEC) to much greater than those of LLC/mock cells. These results indicate that ES gene transfer in murine lung carcinoma cells induces VEGE secretion, resulting in enhanced in vivo tumorigenecity in a murine model. More attention should be paid for ES gene therapy into lung cancer cells because it may affect other genes expression, which upregulates angiogenesis.