Effects of soluble forms of recombinant tumor necrosis factor-related apoptosis-inducing ligand:iron superoxide dismutase (sTRAIL:FeSOD) on the levels of phosphorylated Akt and antiapoptotic proteins. (A) and (B) Dose response of Akt dephosphorylation and cellular FLICE-inhibitory protein (c-FLIPL) downregulation by sTRAIL:FeSOD in human promyelocytic leukemia (HL-60) cells or erythroleukemia (K562) cells. Before they were disrupted, cells expressing phosphorylated Akt (p-Akt), c-FLIPL and c-FLIPS were treated with sTRAIL:FeSOD for 30 minutes, 1 hour and 1 hour, respectively. Bcl-xl, Bcl-2, Bax and Bid levels were detected after treatment with sTRAIL:FeSOD for 6 hours. (C) After treatment of LO2 cells with sTRAIL:FeSOD (500 μg/ml) or LY294002 (10 μM) for 4 hours, cell lysates were probed for p-Akt and c-FLIPL. (D) Enhancement of TRAIL-induced cytotoxicity caused by LY294002 (10 μM) in LO2 cells. Cells were pretreated with LY294002 for 30 minutes and then treated with sTRAIL:mFeSOD (1,000 ng/ml) or left untreated for 8 hours. Cell survival was determined by staining cells with anti-annexin V and propidium iodide. (E) In the presence or absence of the specific inhibitor of total caspase 50 μM (Z-VAD-FMK, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone), cells were treated with 1,000 ng/ml sTRAIL:FeSOD for 1 hour, and then c-FLIPL levels were determined by Western blot analysis.