| Abstract: | Caspases play important roles in cell apoptosie.Measurement of the dynamics of caspase activation in tumor cells not only facilitates understanding of the molecular mechanisms of apoptosis but also contributes to the development,screening,and evaluation of anticancer drugs that target apoptotic pathways.The fluorescence resonance energy transfer (FRET) technique provides a valuable approach for defining the dynamics of apoptosis with high spatio-temporal resolution.However,FRET generally functions in the single-cell level and becomes ineffective when applied in the high throughput detection of caspase activation.In the current study,a FRET sensor was combined with capillary electrophoresis (CE) to achieve a high throughput method for cellular caspase detection.The FRET-based CE system is composed of a homemade CE system and a laser source for detecting the dynamics of caspase-3 in various cells expressing sensors of caspase-3 that have been treated with anticancer drugs,such as cell cycle-independent drug cisplatin and specific cell cycle drugs camptothecin and etoposide,as well as their combination with tumor necrosis factor (TNF).A positive correlation between the caspase-3 activation velocity and drug concentration was observed when the cells were treated with cisplatin,but cells induced by camptothecin and etoposide did not show any apparent correlation with their concentrations.Moreover,different types of cells presented distinct sensitivities under the same drug treatment,and the combination treatment of TNF and anticancer drugs significantly accelerated the caspase-3 activation process.Its high throughput capability and detection sensitivity make the FRET-based CE system a useful tool for investigating the mechanisms of anticancer drugs and anticancer drug screening. |
