Diverse Natural Products That Stabilize Microtubules

描述（由申请人提供）：我们研究的长期目标是全面了解紫杉醇的作用机制和耐药性，紫杉醇是一种已知在治疗人类癌症方面有效的抗肿瘤药物。对药物开发的强烈兴趣现在集中在埃泊霉素和迪德莫内酯上，这两种天然产物的化学结构与紫杉醇不同，但其作用机制和耐药性与紫杉醇有相似之处，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，埃泊霉素和/或迪德莫内酯可能具有更好的临床活性，特别是在对紫杉醇耐药的肿瘤中。这项拨款申请的首要主题是低剂量的紫杉醇、埃泊霉素和迪地莫胺在杀死癌细胞中的作用。随着紫杉醇在低剂量药物作用下导致细胞死亡，而不阻断细胞有丝分裂，这种对低剂量药物的强调是我们实验室的一个新重点。毫无疑问，低剂量紫杉醇杀死癌细胞的途径多种多样。临床前数据的可用性是至关重要的，必须有助于决定将药物转移到临床试验。本建议的具体目的是：1。确定用低浓度紫杉醇处理的细胞逃避纺锤体检查点的机制。将用检查点成分MAD2转染HeLa细胞，以确定其过表达是否可以拯救那些逃离有丝分裂检查点的细胞免于死亡。有丝分裂检查点组成部分CENP-E蛋白的过表达与耐药性之间的关系也将被研究。2. 研究p53依赖性凋亡在低浓度紫杉醇引起的细胞毒性中的作用。确定异常有丝分裂是否导致p53依赖性细胞凋亡。DNA微阵列将用于调查和比较不同浓度的微管稳定药物处理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差异敏感性相关，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.