P2Et fraction induction of MMP loss, activation of caspase 3 and DNA fragmentation. (A) K562 cells treated with the P2Et fraction for 4, 8 or 12 h, stained with JC-1 and analyzed by flow cytometry. Ethanol was used as negative and valinomycin as positive controls (*** p < 0.001). (B) Caspase 3 activity was measured using the enzyme-linked immunosorbent assay (ELISA) after cell treatment with the P2Et fraction or doxorubicin (positive control) for 48 h (** p < 0.005). (C) PS externalization was estimated in K562 cells using flow cytometry after cell treatment for 48 h with the P2Et fraction, doxorubicin (positive control) or ethanol (negative control). (D) DNA was stained with 4',6-diamidino-2-phenylindole (DAPI) after cell treatment for 48 h. Apoptotic cells or fragmented nuclei were observed under fluorescent microscope (40X). (E) Apoptotic cells were estimated by random observation in six fields and by counting cells with strong fluorescence or fragmented nuclei.