Pilch Jan, Franzin Carla M, Knowles Lynn M, Ferrer Fernando J, Marassi Francesca M, Ruoslahti Erkki
Burnham Institute for Medical Research, 10901 N. Torrey Pines Rd., La Jolla, CA 92037, USA.
J Mol Biol. 2006 Mar 3;356(4):876-85. doi: 10.1016/j.jmb.2005.12.006. Epub 2005 Dec 20.
Anginex is a synthetic beta-sheet peptide with anti-angiogenic and anti-tumor activity. When added to cultured endothelial cells at concentrations ranging from 2.5 microM to 25 microM, anginex induced cell death, which was reflected by a strong increase of subdiploid cells and fragments, loss of cellular ATP, and LDH release. Cytotoxicity remained the same whether cells were treated with anginex at 4 degrees C or at 37 degrees C. At low temperatures, fluorescein-conjugated anginex accumulated on the endothelial surface, but did not reach into the cytoplasm, indicating that the cell membrane is the primary target for the peptide. Within minutes of treatment, anginex caused endothelial cells to take up propidium iodide and undergo depolarization, both parameters characteristic for permeabilization of the cell membrane. This process was amplified when cells were activated with hydrogen peroxide. Red blood cell membranes were essentially unaffected by anginex. Anginex bound lipid bilayers with high affinity and with a clear preference for anionic over zwitterionic phospholipids. Structural studies by circular dichroism and solid-state nuclear magnetic resonance showed that anginex forms a beta-sheet and adopts a unique and highly ordered conformation upon binding to lipid membranes. This is consistent with lipid micellization or the formation of pore-forming beta-barrels. The data suggest that the cytotoxicity of anginex stems from its ability to target and disrupt the endothelial cell membrane, providing a possible explanation for the angiostatic activity of the peptide.
血管抑制素是一种具有抗血管生成和抗肿瘤活性的合成β-折叠肽。当以2.5微摩尔至25微摩尔的浓度添加到培养的内皮细胞中时，血管抑制素会诱导细胞死亡，这表现为亚二倍体细胞和碎片的大量增加、细胞内三磷酸腺苷（ATP）的丧失以及乳酸脱氢酶（LDH）的释放。无论细胞在4℃还是37℃下用血管抑制素处理，细胞毒性都保持不变。在低温下，荧光素偶联的血管抑制素在内皮表面积累，但未进入细胞质，这表明细胞膜是该肽的主要作用靶点。在处理后的几分钟内，血管抑制素使内皮细胞摄取碘化丙啶并发生去极化，这两个参数都是细胞膜通透性的特征。当细胞用过氧化氢激活时，这个过程会被放大。红细胞膜基本上不受血管抑制素的影响。血管抑制素以高亲和力结合脂质双层，并且明显偏好阴离子磷脂而非两性离子磷脂。通过圆二色性和固态核磁共振进行的结构研究表明，血管抑制素形成β-折叠，并在与脂质膜结合时采用独特且高度有序的构象。这与脂质胶束化或形成成孔β-桶一致。数据表明，血管抑制素的细胞毒性源于其靶向并破坏内皮细胞膜的能力，这为该肽的血管生成抑制活性提供了一种可能的解释。
