Anti-angiogenic actions of taxotere

描述（由申请人提供）：紫杉烷类是治疗晚期癌症最有效的药物之一，其对肿瘤细胞的直接细胞毒性作用已得到充分证明，并被认为是其临床活性的主要机制。在我们最近发表的初步研究中，我们发现这些微管破坏剂在体外阻断了内皮细胞迁移，在体内阻断了血管生成。特别是泰索帝，在极低的浓度（10 pM）下，在体外产生了一些这些作用，对内皮细胞增殖或总微管结构没有影响，这表明泰索帝具有以前没有描述过的作用机制，并且与其对有丝分裂，凋亡和细胞增殖的作用不同。本提案的总体目标是确定泰索帝抑制内皮细胞迁移的分子机制，并获得将提供其作为抗血管生成剂的最佳临床用途的信息。该申请提出了泰索帝通过调节介导定向细胞迁移的信号传导途径中的关键早期步骤来抑制血管生成的假设。为了验证这一假设，我们将记录刺激内皮细胞迁移的信号转导事件，以及泰索帝对这些事件的影响，包括：a）整合素的上调和表面聚集; B）粘着斑激酶（FAK）、桩蛋白和p130 cas的特异性酪氨酸残基的磷酸化; c）这些分子的空间和时间关联形成粘着斑; d）这些分子定位在中心体（也称为微管组织中心，MOC）的程度; e）MOC在定向细胞迁移中的作用（使用活细胞中MOC重新定位的延时测量）;和f）酪氨酸化微管蛋白的程度和细胞内定位（即在迁移前沿）的变化。这些实验的基本原理是先前的研究，这些研究表明这些信号传导途径的关键组分，特别是FAK和桩蛋白，与微管、7-微管蛋白和MOC在细胞内相关。因此，我们将在无细胞系统和完整细胞中测试泰索帝特异性破坏细胞信号传导/细胞骨架途径组分与微管和/或微管蛋白的结合，最终导致观察到的细胞迁移抑制的假设。另一个假设，即泰索帝影响微管依赖性激活的Rho家族的GTP酶，也将进行检查。除了了解其抗血管生成作用的机制外，我们还证明了泰索帝对内皮细胞迁移的有效作用。有必要进一步探索泰索帝的抗血管生成作用有助于其临床抗肿瘤活性的可能性，并确定泰索帝最佳抑制血管生成的条件。在这些研究中，我们将测试泰索帝的“节拍式”给药方案将增加其体内疗效的假设。我们还将使用对泰索帝的直接细胞毒性作用敏感或耐药的乳腺和卵巢肿瘤的体内异种移植物，以便区分泰索帝对内皮细胞和血管生成的作用与其对肿瘤细胞的直接作用。

DESCRIPTION (provided by applicant): The taxanes are among the most effective agents for the treatment of advanced cancers, and their direct cytotoxic effects on tumor cells have been well documented and are presumed to be the primary mechanism for their clinical activity. In our recently published Preliminary Studies, we found that these microtubule-disrupting agents blocked endothelial cell migration in vitro and angiogenesis in vivo. Taxotere in particular, produced some of these actions in vitro at extremely low concentrations (10 pM) that had no effect on endothelial cell proliferation or on gross microtubule structure, suggesting that taxotere has a mechanism of action that has not been previously described and that differs from its well-described effects on mitosis, apoptosis, and cell proliferation. The overall objectives of this proposal are to determine the molecular mechanism(s) by which taxotere inhibits endothelial cell migration, and obtain information which will provide for its optimal clinical use as an anti-angiogenic agent. This application proposes the hypothesis that taxotere inhibits angiogenesis by modulating a critical early step in the signaling pathways which mediate directed cell migration. To test this hypothesis, we will document signal transduction events in endothelial cells stimulated to undergo migration, and the effect of taxotere on these events, including: a) upregulation and surface clustering of integrins; b) phosphorylation of specific tyrosine residues of focal adhesion kinase (FAK), paxillin, and p130cas; c) spatial and temporal association of these molecules to form focal adhesions; d) the extent to which these molecules localize at the centrosome (also known as the microtubule-organizing center, MOC); e) the role of the MOC in directed cell migration (using time-lapse measurements of MOC repositioning in live cells); and f) changes in the extent and intracellular localization of (i.e. in the migratory front) of tyrosinated tubulin. The rationale for these experiments are previous studies which demonstrated that key components of these signaling pathways, in particular FAK and paxillin, were associated intracellularly with microtubules, 7-tubulin, and the MOC. Therefore we will test, both in cell-free systems and in intact cells, the hypothesis that taxotere specifically disrupts the binding of components of the cell signaling/cell skeleton pathways to microtubules and/or tubulin, ultimately leading to the observed inhibition of cell migration. An additional hypothesis, that taxotere affects the microtubule-dependent activation of the Rho family of GTPases, will also be examined. In addition to understanding its mechanism of anti-angiogenic action, there are therapeutic implications to our demonstration of a potent effect of taxotere on endothelial cell migration. There is a need for further exploration of the possibility that taxotere's anti-angiogenic actions contribute to its clinical anti-tumor activity, and for the determination of the conditions under which taxotere optimally inhibits angiogenesis. In these studies, we will test the hypothesis that a "metronomic" dosing schedule of taxotere will increase its efficacy in vivo. We will also use in vivo xenografts of breast and ovarian tumors which are either sensitive or resistant to taxotere's direct cytotoxic actions, so as to distinguish between taxotere's effect on endothelial cells and angiogenesis from its direct effects on the tumor cells.