The Establishment Of Molecular Models That Taxane Binds To Microtubules And The Application For Tumor Drug Resistance And Alzheimer's Disease Research

Taxol (Paclitaxel) is a mitotic inhibitor which is widely used in tumor chemotherapy. In recent years, Taxol has been successfully used in the treatment of a variety of human malignancies, including breast, lung, ovarian, gastric cancers, Kaposis's sarcoma, etc. The mechanism for paclitaxel's antimitotic effects is attributed to its binding to microtubule protofilaments, subsequently promoting tubulin polymerization, stabilizing microtubules and inhibiting their depolymerization. As a consequence, spindle fibers can not connect with microtubule organizing centers, which induces cell cycle arrest in G2/M phase. The cell cycle arrest results in an aberrant mitosis or mitotic catastrophe, after which the apoptotic pathway is activated and tumor cells die.The study of the interaction of Taxol and tubulin/microtubule is very important to deeply understand its mechanism of action and structure activity relationship of taxanes, to discover new taxanes effective against tumor drug resistances, and to extend their uses in the treatment of other diseases.My thesis is committed to build a series of in vitro models which will appropriately reflect the interaction of taxanes and tubulin, and apply them to the discovery of compounds effective for drug resistance tumor cells and Alzheimer's disease (AD). In our experiments we prepared some high purity tubulin (W-tubulin) and microtubule proteins (MTP, i.e. tubulin with MAPs) with two methods. And then we established fluorescence intensity and fluorescence polarization based methods, in the presence of probes derivatized from paclitaxel, to determinate the Kbs (binding constants) of taxanes to microtubules. For the compounds with high affinity with microtubule, HPLC was used to determinate Kbs.The determination of Kbs was applied to evaluate the binding ability of compounds designed for the study of tumor drug resistance. We have also det up a method, with fluorescent DAPI as fluorescent indicator, to show the tubulin polymerization extents in the presence of taxanes, and to measure the EC50 of taxanes. The model was applied to find anti-AD drug from taxanes. Its characteristic is high throughput and sensitivity, compared to traditional turbidity method. In addition, we established the methods to determinate the critical concentration of tubulin polymerization to evaluate the ability of compounds inducing microtubule dissembly. These methods will play an important role in the study of drugs interacting with tubulin/microtubule.