Figure 3

OPG attenuates TRAIL-induced apoptosis in an Akt-dependent manner. CaOV3 cells were treated with increasing concentrations (0–100 ng/ml) of OPG (A) or with 25 ng/ml OPG for various times (0–180 min) (B). Cells were lysed, and the levels of total and phosphorylated Akt were determined by immunoblot. Densitometric quantification of phosphorylated Akt from three separate experiments normalized to total Akt was done. (C) OVCAR3 and OVC238A cells were treated with 25 ng/ml OPG and 60 min later, cells were lysed and immunoblot was performed to determine the levels of total and phosphorylated Akt. (D) CaOV3 cells were treated with increasing concentrations (0–25 ng/ml) of OPG and total and phosphorylated ERK1/2 were determined by immunoblot. The levels of phosphorylated ERK1/2 were determined by densitometric quantification. (E) CaOV3 cells were preincubated with LY294002 (5 uM) or Akt inhibitor (10 uM) for 1 h. OPG (25 ng/ml) was then added for 90 min. Cells were washed and TRAIL (50 ng/ml) was added for 48 h. Viable colonies were counted after 14 days and data were expressed at fold increase relative to control (untreated) cells. Results are from three independent experiments and express as mean fold increase ± SD. (F) CaOV3 cells were preincubated for 1 h with either Akt or ERK1/2 inhibitor and OPG (25 ng/ml) was added for 90 min. Cells were washed and incubated with TRAIL (50 ng/ml) for 24 h, and apoptosis was assessed. Apoptosis is expressed as fold increase relative to control (untreated) cells with the mean of triplicates from three independent experiments ± SD. * P < 0.01 compared to TRAIL + OPG treated cells; ** P < 0.001.