Anti-angiogenic actions of taxotere

说明(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作用已有很好的文献记载，并被推定为其临床活性的主要机制。在我们最近发表的初步研究中，我们发现这些微管干扰剂在体外阻止内皮细胞迁移，在体内阻止血管生成。尤其是泰索帝，在体外极低浓度(10 PM)下产生了一些对内皮细胞增殖或毛细微管结构没有影响的作用，这表明泰索帝具有以前没有描述过的作用机制，不同于其对有丝分裂、细胞凋亡和细胞增殖的已有描述的作用。这项建议的总体目标是确定紫杉醇抑制内皮细胞迁移的分子机制(S)，并获得将为其作为抗血管生成剂的最佳临床应用提供信息。这一应用提出的假设是，紫杉醇通过调节介导细胞定向迁移的信号通路中的关键早期步骤来抑制血管生成。为了验证这一假设，我们将记录受刺激进行迁移的内皮细胞中的信号转导事件，以及紫杉醇对这些事件的影响，包括：a)整合素的上调和表面聚集；b)粘着斑激酶(FAK)、Paxlin和p130Cas的特定酪氨酸残基的磷酸化；c)这些分子的时空结合形成焦点粘连；d)这些分子定位于中心体(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范围和细胞内定位的变化(即在迁移前沿)。这些实验的理论基础是以前的研究，这些研究表明这些信号通路的关键成分，特别是FAK和PXLIN，与微管、7-微管蛋白和MOC在细胞内联系在一起。因此，我们将在无细胞系统和完整细胞中测试这一假设，即紫杉醇专门破坏细胞信号/细胞骨架通路的组件与微管和/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，即紫杉醇影响Rho家族GTP酶的微管依赖的激活，也将被检验。除了了解其抗血管生成作用的机制外，我们还展示了紫杉醇对内皮细胞迁移的有效作用，这对我们的治疗具有重要意义。有必要进一步探讨紫杉醇的抗血管生成作用是否有助于其临床抗肿瘤活性，以及确定紫杉醇最佳抑制血管生成的条件。在这些研究中，我们将检验这样一种假设，即紫杉醇的“节律”给药程序将增加其体内疗效。我们还将使用对紫杉醇直接细胞毒作用敏感或耐药的乳腺和卵巢肿瘤体内移植瘤，以区分紫杉醇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.