Spermidine-induced apoptosis via reactive oxygen species generation and caspase 3 activation in mouse P19 embryonal carcinoma cells

Abstract

The present study was to investigate the effects of spermidine-induced apoptosis in mouse PI 9 embryonal carcinoma cells. The cell viability using MTT (3-[4,5-dimefhyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) assay showed that spermidine decreased cell viability after 24-h incubation in a concentration-dependent manner with an IC50 value of approximately 20 μM. Using Hoechst33342 staining, chromatin condensation and apoptotic bodies in PI 9 cells treated with spermidine were observed. Moreover, flow cytometric analysis stained with propidium iodide increased the accumulation of DNA fragmentation in the pre GJGi phase, indicating apoptotic cell death, which was accompanied by the generation of reactive oxygen species (ROS) as detected with the fluorescence probe 2',7'-dichlorofluorescin diacetate (DCFH-DA). Western blot analysis revealed that spermidine-induced apoptosis was associated with an increase of Bax and caspase 3 expression, meanwhile the expression of Bcl-2 was decreased. The results suggest that spermidine-induced PI 9 apoptotic cell death is involving ROS, Bcl-2 family as well as caspase 3. © 2010 Asian Network for Scientific Information.