In this FLIM–FRET approach we study six different constructs where the BSA is covalently linked to one (BSA-F1) or five (BSA-F5) fluorescein molecules, one (BSA-T1) or seven (BSA-T7) rhodamine molecules, and hetero labeled with both (BSA-F4-T2 and BSA-F6-T1). Here we apply a series of multi-fluorophore labeled Bovine Serum Albumin (BSA) proteins as model probes to investigate surface-induced conformational changes of BSA by the use of a confocal Fluorescence Lifetime Imaging Microscopy and Förster Resonance Energy Transfer (FLIM–FRET) method. Reliably measuring the physicochemical properties of protein thin layers deposited on surfaces is critical to understanding the surface chemistry, biocompatibility, and performance of implanted biomaterials. The overall results suggest that ESB induces mitochondria-mediated intrinsic apoptotic pathway in HL60 cells and might have therapeutic value against human leukemia. DNA fragmentation analysis revealed typical ladders as early as 18 h indicative of caspase-3 role in the apoptotic pathway. Dissipation of mitochondrial membrane potential of intact cells accompanied by increase in cytosolic cytochrome c was observed, which was followed by activation of caspase-9 and -3 but not caspase-8. Increase in intracellular ROS production up to sixfold was detected in ESB-treated HL60 cells by DCFH-DA assay. ESB treatment resulted in a dose-dependent increase in annexin V positive cells. Cell cycle analysis showed a significant increase in Sub G0 population of cells above 50 μg/ml. The gold standard scanning electron micrographs showed apoptotic bodies and formation of blebs. A series of well-documented morphological changes, such as cell shrinkage, condensation of nuclear chromatin, and nuclear fragmentation, were observed by fluorescence microscopy. We found that the ESB inhibited cell proliferation in a dose- and time-dependent manner. Cell viability was estimated by MTT assay. In this study, the apoptosis-inducing effect of an alcoholic extract from Erythrina suberosa stem bark (ESB) was investigated using human promyelocytic leukemia HL60 cells.
0 Comments
Leave a Reply. |
Details
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |