Figure 7
Cellular FLICE-inhibitory protein (c-FLIP L ) is involved in soluble forms of recombinant tumor necrosis factor-related apoptosis-inducing ligand:iron superoxide dismutase (sTRAIL:FeSOD)-induced apoptosis, and the lack of responsiveness to sTRAIL:FeSOD in LO2 cells results from low levels of H 2 O 2 . (A) through (C) Effects of c-FLIPL overexpression on apoptosis and caspase-8 activities. After determination of c-FLIPL overexpression (A), cells were treated with sTRAIL:FeSOD (1,000 ng/ml) or sTRAIL:mFeSOD (1,000 ng/ml), and apoptosis (B) and caspase-8 activity (C) were subsequently assayed. (D) Caspase-9 small interfering RNA (siRNA) was transfected into LO2 cells for 24 hours. After determining the inhibition of caspase-9 expression, cells were treated with sTRAIL:FeSOD (1,000 ng/ml) or sTRAIL:mFeSOD (1,000 ng/ml) for 8 hours, and cell apoptosis was assayed by flow cytometry. (E) through (G) Effect of H2O2 burst on cell death and caspase-8 activity in LO2 cells. After catalase siRNA transfection for 24 hours, cells were treated with 1,000 ng/ml sTRAIL:FeSOD for 0, 1, 2 or 3 hours, and then intracellular H2O2 was measured using dihydrorhodamine 123 (E). Cells were treated with sTRAIL:FeSOD (1,000 ng/ml) or sTRAIL:mFeSOD (1,000 ng/ml), and apoptosis (F) and caspase-8 activity (G) were subsequently assayed. Data represent the mean ± SD of three independent experiments (*P < 0.05 vs. untreated control).