A Novel Screening System For Apoptotic Inducer Drugs Based On FRET Technology And Flow Cytometric Analysis

Currently, the screening of apoptotic anti-tumor drug candidates from natural or synthetic compounds is a process of time-consuming and complicated experimental process; furthermore, the process needs experimental equipments of high standards. In this thesis, combined with the high-throughput measurement ability of flow cytometry and FRET probe for caspases, we design a novel FRET probe which can be used to screen apoptotic anti-tumor drug candidates more convenient and high throughput. The design principle of the FRET probe is to measure the change of two FRETs when cells are treated with apoptotic agents to activate either caspase-8 or caspase-2 activity. Caspase-8 and caspase-2 are two critical proteases in two separate apoptosis pathways. By this approach, we can judge the apoptotic pathway by which the tested agent induces apoptosis. Combining the FRET technology with the high-throughput screening ability of flow cytometry, our methodology makes the drug screening more efficient and accurate. In this thesis, we test and validate our designed tri-color dual FRET probe, EBFP2-C8-EGFP-C2-TagRFP, by flow cytometric method. We validate our method with two apoptosis inducers, RGD-TRAIL and CDDP, whose apoptotic mechanism are clearly known. The results show that our FRET probe can sensitize the activity change from either of caspase-8 or caspase-2 or both of them by the measurement of change of FRETs. In addition, the experiment with caspase inhibitors further demonstrates the correlation between FRET efficiency and caspase activity. In summary, the change of FRET efficiency can describe the change of caspases’activities sensitively. This study provides a solid foundation for development FRET probe to screen apoptotic inducer based on flow cytometric system.