Figure 5
Involvement of caspase-8 and the role of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor and H 2 O 2 in soluble forms of recombinant TRAIL:iron superoxide dismutase (sTRAIL:FeSOD)-induced apoptosis. (A) and (B) Involvement of caspase-8 in sTRAIL:FeSOD-induced apoptosis. Caspase-8 or caspase-9 small interfering RNA was transfected into the indicated cells for 24 hours. After determining the inhibition of caspase-8 or caspase-9 expression (A), cells were treated with sTRAIL:FeSOD (1,000 ng/ml) for 8 hours, and cell apoptosis was assayed by flow cytometry (B). (C) Cells were treated with sTRAIL:FeSOD (1,000 ng/ml) for 6 hours. Cell lysates were tested for protease activity by the addition of caspase-specific peptides. Cleavage of the peptide by the caspase releases a chromophore, which was quantified using a fluorometer at 505 nm. (D) and (E) Cells were treated with sTRAIL:FeSOD at the indicated concentrations for 6 hours. Cell lysates were subjected to Western blot analysis using cleaved caspase-8, caspase-9 or caspase-3 antibody. (F) The DR5 and DR4 from untreated cells or cells treated with sTRAIL:FeSOD (for 6 hours) were quantified by Western blot analysis. (G) Cells were incubated with DR5 antibody and/or DR4 antibody for 1.5 hours prior to exposure to sTRAIL:FeSOD (1,000 ng/ml). (H) After cells were pretreated with sTRAIL:FeSOD for 0, 1, 2, 3 or 4 hours, 10 mM N-acetylcysteine (NAC) was added to the medium, and apoptosis was detected when cells had been treated with sTRAIL:FeSOD for 8 hours. (I) through (K) After pretreatment with 10 mM NAC for 24 hours, cells were incubated in fresh medium with 1,000 ng/ml sTRAIL:FeSOD or sTRAIL:mFeSOD for 8 hours. Apoptosis was determined by staining cells with anti-annexin V antibody and propidium iodide. H2O2 and reactive oxygen species levels were also detected after NAC-pretreated cells were treated with sTRAIL:FeSOD for 0, 2, 4 and 6 hours as described above. The untreated cells served as control. Data represent the mean ± SD of three independent experiments (*P < 0.05 vs. untreated control).