Diverse Natural Products That Stabilize Microtubules

描述(申请人提供)：我们研究的长期目标是彻底了解紫杉醇的作用机制和对紫杉醇的耐药性，紫杉醇是一种已知在治疗人类癌症中有效的抗肿瘤药物。人们对药物开发的浓厚兴趣现在集中在环磷酰胺类和盘状分子，这两种天然产物的化学结构与紫杉醇不同，但其作用和耐药机制与紫杉醇相似，但肯定不完全相同。我们的计划是剖析这三种微管稳定药物之间的异同。与紫杉醇相比，Epothilone和/或Discodermolide可能具有更好的临床活性，特别是在对紫杉烷耐药的肿瘤中。这项拨款申请的一个压倒一切的主题是低剂量紫杉醇、埃博西酮和盘状核苷在杀死癌细胞方面所起的作用。这种对低药物剂量的强调是我们实验室的一个新焦点，随着紫杉醇在不阻止细胞有丝分裂的低剂量药物中导致细胞死亡的事实变得清晰起来。毫无疑问，低剂量紫杉醇杀死癌细胞的途径多种多样。临床前数据的可获得性是至关重要的，必须有助于决定将药物投入临床试验。这一建议的具体目的是：1.确定低浓度紫杉醇处理的细胞逃避纺锤体检查点的机制(S)。HeLa细胞将被转染MAD2，一种检查点成分，以确定其过度表达是否可以拯救那些逃脱有丝分裂检查点的细胞死亡。CENP-E蛋白的过度表达是有丝分裂检查点的一个组成部分，它的过度表达与耐药性之间的关系也将被调查。2.检测P53依赖性细胞凋亡在低浓度紫杉醇细胞毒作用中的作用。确定异常有丝分裂是否导致P53依赖的细胞凋亡。DNA微阵列将被用来调查和比较不同浓度的微管稳定药物处理的A549细胞的基因表达谱。3.研究微管调节蛋白stathmin和MAP4在癌细胞中的表达水平和磷酸化状态如何与微管稳定剂的不同敏感性有关，以及这些药物如何影响这些蛋白的表达谱。

DESCRIPTION (provided by applicant): The long-term objectives of our research are to acquire a thorough understanding of the mechanisms of action and of resistance to Taxol, an antitumor agent that is known to be efficacious in the treatment of human cancer. An intense interest in drug development is now concentrated on the epothilones and discodermolide, natural products whose chemical structures are distinct from Taxol but whose mechanisms of action and resistance have similarities, but are definitely not identical, to those of Taxol. Our plan is to dissect the similarities and differences between these three microtubule stabilizing drugs. The epothilones and/or discodermolide may have superior clinical activity compared to Taxol, particularly in tumors resistant to taxanes. An overriding theme of this grant application is the role that low doses of Taxol, the epothilones and discodermolide play in the killing of cancer cells. This emphasis on low drug dosage is a new focus for our laboratory that developed as it became clear that Taxol was responsible for cell death at low doses of drug that did not block cells in mitosis. There are undoubtedly diverse pathways by which low doses of Taxol kill cancer cells. The availability of preclinical data is essential and must contribute to the decision to move drugs into clinical trials. The specific aims of this proposal are to: 1. Determine the mechanism(s) by which cells treated with low concentrations of Taxol escape the spindle checkpoint. HeLa cells will be transfected with MAD2, a checkpoint component, to determine if its overexpression can rescue from cell death, those cells that escape from the mitotic checkpoint. The relationship between overexpression of the protein CENP-E, a component of the mitotic checkpoint, and drug resistance will also be investigated. 2. Examine the contribution of p53-dependent apoptosis to cytotoxicity caused by low concentrations of Taxol. Determine if aberrant mitosis leads to p53-dependent apoptosis. DNA microarrays will be used to survey and compare gene expression profiles of A549 cells treated with different concentrations of microtubule stabilizing drugs. 3. Investigate how the expression level and the phosphorylation status of the microtubule regulatory proteins stathmin and MAP4 in cancer cells relate to differential sensitivity to microtubule-stabilizing agents, and how these agents affect the expression profile of these proteins.