Caspase-3 inhibitor Z-DEVD-fmk blocks CDDO-Me–induced annexin V positivity and caspase-3 cleavage in leukemic cells.

(A) Evidence of apoptosis was identified by staining with annexin V (x-axis) and PI (y-axis) after 6 hours of 1 μM CDDO-Me treatment. DMSO, which was used as a solvent, was used as the control treatment. Cells binding annexin V and retaining PI were apoptotic (lower right quadrant); double-positive cells underwent secondary necrosis (upper right quadrant). In panel 3, U937 and HL-60 cells were pretreated with 25 μM Z-DEVD-fmk (a caspase-3 inhibitor) for 1 hour followed by 6 hours of exposure to CDDO-Me (1 μM). (B) Western blot analysis shows the appearance of the cleaved caspase-3 band at 2, 4, and 6 hours of CDDO-Me exposure (detected with antibody that recognizes only cleaved caspase-3, IDUN) and absence of the cleaved product in DEVD-pretreated cells.