The apoptotic effect of AVO is mediated by activation of caspase cascades. (A) Whole cell extracts (50 μg), from HL-60 cells after treatment with varying concentrations of AVO-l or AVO-b for 24, were analyzed by Western blotting with antibodies against caspase-8 and -9 and caspase–3, respectively. The unprocessed forms of procaspase-8, -9 and -3, and their respective cleavage products of the active enzymes are indicated. The same membranes were also probed with an antibody against β-actin as a loading control. (B) Extracts (30 μg proteins) from cells treated with varying concentrations of AVO-l or AVO-b were analyzed for caspase-8, -9 and -3 catalytic activities using specific colorimetric tetrapeptide substrates. The specific enzyme activities, which represent the mean ± SD of three independent experiments, were measured as described in the methods. HL-60 cells were treated with 4.0 and 8.0 μM etoposide as positive controls. Statistical analysis showed that all samples are significantly different (p < 0.05), when compared to their untreated control. (C) HL-60 cells were incubated with either 35 μM of Caspase-8 inhibitor (z-IETD-fmk), caspase-9 inhibitor (z-LEHD-fmk), caspase-3 inhibitor (z-DEVD-fmk), or 50 μM of the general caspase inhibitor (z-VAD-fmk) for 6 h prior to addition of 1.0 μg/mL AVO-l or AVO-b for 24 h. After incubation, cell viability was assessed by the MTT assay. The cell viability for each sample treated with the specific caspase inhibitor was expressed as percentages relative to the parallel control cells incubated with the same caspase inhibitor alone, without the essential oil, which was considered 100%. The results shown represent the means ± SD of three independent experiments. Statistical analysis showed that all samples treated with caspase inhibitors and AVO are significantly different (p < 0.05), when compared to samples treated with the specific caspase inhibitor alone, AVO alone or untreated control.