Figure 5.

Involvement of ASK-1 and JNK in helenalin-induced apoptosis. (A) HCT116 cells were treated with 2 μM helenalin for 16 hours and ASK-1 activity (upper panel) or JNK activity (lower panel) was determined by immunokinase assays. (B) Cells were transfected with a dominant negative mutant of ASK-1 (black bars) or a control plasmid (open bars) and active caspase-3 was determined. The ASK-1 construct was HA-tagged and cells were stained with antibodies to HA and active caspase-3 followed by analysis by flow cytometry; (C) HCT116 cells were treated with 2 μM helenalin. SP600125 (5 μM) was added at the times indicated and apoptosis was assessed at 24 hours of drug treatment. The experiment was repeated with similar results. (D) Activation of an AP-1 reporter construct by helenalin. The CellSensor AP-1-bla HCT116 cell line (Invitrogen) was treated with 2 μM helenalin in the presence or absence of the indicated inhibitors. Activity was determined after 18 hours. Statistical significance is indicated relative to helenalin-only-treated cells (* p < 0.02; ** p < 0.002; Student's t-test).