Anti-angiogenic actions of taxotere

描述（由申请人提供）：紫杉烷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被充分记录，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内皮细胞迁移和体内血管生成。特别是，在体外极低浓度（10pm）下，他索帝产生了一些对内皮细胞增殖或总体微管结构没有影响的这些作用，这表明他索帝的作用机制以前没有被描述过，不同于它对有丝分裂、细胞凋亡和细胞增殖的作用。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，并获得其作为抗血管生成药物的最佳临床应用信息。这一应用提出了一个假设，即泰索帝通过调节介导定向细胞迁移的信号通路的关键早期步骤来抑制血管生成。为了验证这一假设，我们将记录刺激内皮细胞进行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类；b)局灶黏附激酶（FAK）、paxillin和p130cas特异性酪氨酸残基的磷酸化；C)这些分子在空间和时间上形成黏附；d)这些分子在中心体（也称为微管组织中心，MOC）上的定位程度；e) MOC在细胞定向迁移中的作用（使用活细胞中MOC重新定位的延时测量）；f)酪氨酸化微管蛋白的范围和细胞内定位（即迁移前沿）的变化。这些实验的基本原理是先前的研究表明，这些信号通路的关键成分，特别是FAK和paxillin，与细胞内微管、7-微管蛋白和MOC相关。因此，我们将在无细胞系统和完整细胞中测试这样一种假设，即泰索帝会特异性地破坏细胞信号通路/细胞骨架通路与微管和/或微管蛋白的结合，最终导致观察到的细胞迁移抑制。另一个假设，即泰索帝影响GTPases的Rho家族的微管依赖性激活，也将被检查。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抗血管生成作用对其临床抗肿瘤活性的影响，并确定泰索帝抑制血管生成的最佳条件。在这些研究中，我们将验证一个假设，即一个“节拍”给药计划将增加其体内疗效。我们还将使用对taxoere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，以便将taxoere对内皮细胞和血管生成的影响与对肿瘤细胞的直接作用区分开来。

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.