Figure 1.
Effect of TRAIL on cell proliferation.
Human breast cancer cell lines T47D, MDA231, and MCF7 and ovarian cancer cell lines DOV13, E52, OV90, OV433, OVCA432, RMG-1, TOV-21G, TOV-112D, CAOV3, OVCA420, and SKOV3 were treated with various doses of TRAIL for 24 h. Cell proliferation was determined by MTT assays. Cell proliferation data are expressed as percentage of untreated cells. Representative of three independent experiments.
Figure 2.
Activation of Akt/mTOR pathway by TRAIL in resistant cell lines but not in sensitive cell lines.
T47D (A), SKOV3 (B), OVCA432 (C), OV90 (D), MCF7 (E) and OVCA420 (F) cells were treated with 100 ng/ml TRAIL for 0, 2, 4 and 8 h (in T47D cells, 6 h treatment was included), and total protein was extracted. The levels of total and phosphorylated Akt (p-AKT), and phosphorylated mTOR (p-mTOR) were determined by Western blot analysis. In T47D (A) and OV90 (D) cells, the levels of phosphorylated p-p70S6K and phosphorylated elF4E (p-elF4E) were also examined. β-actin was used as a loading control. Of note, there were two bands for Akt (Akt1 and Akt2) in both OVCA432 and OV90 cells while only one band for Akt was detected in T47D and SKOV3 cells.
Figure 3.
Inhibition of Akt activity sensitizes resistant cancer cells to TRAIL.
A and C, effect of the PI3K inhibitor LY294002 on TRAIL-induced apoptosis. Breast cancer cell T47D (A) and ovarian cancer cell OVCA432 (C) were left untreated or pretreated with 10 µM LY294002 (LY) for 30 min, and then treated with or without 100 ng/ml TRAIL for 6 h. Total protein was extracted, and cleaved PARP and total and phosphorylated Akt were determined by Western blotting. B and D, effect of LY294002 on TRAIL-induced growth inhibition. T47D (B) and OVCA432 cells (D) were left untreated or pretreated with 10 µM LY294002 for 30 min, and then treated with or without 100 ng/ml TRAIL for 24 h. Cell proliferation was determined by MTT assays and expressed as percentage of untreated cells. Representative of three independent experiments. **, P<0.001, statistical significance; *, P<0.01, statistical significance.
Figure 4.
Knockdown of Akt sensitizes breast cancer T47D cells to TRAIL.
A, effect of Akt knockdown on cell proliferation. T47D cells were plated at 6×105 per well in six-well plates. The next day, cells were transfected with Akt or control siRNAs using Oligofectamine. After 2 d, cells were left untreated or treated with TRAIL (100 ng/ml) for 24 h, and cell proliferation was determined by MTT assays. Cell proliferation data are expressed as percentage of untreated cells. Representative of three independent experiments. *, P<0.01, statistical significance. B, effect of Akt knockdown on TRAIL-induced apoptosis. T47D cells were transfected with Akt or control siRNAs and then treated with or without TRAIL (100 ng/ml) for 24 h as described in (A). Total protein was extracted for assaying total and phosphorylated Akt (p-AKT) and PARP by Western blot analysis. β-actin was used as a loading control.
Figure 5.
The role of PTEN in TRAIL sensitivity. A, effect of loss of PTEN in TRAIL-induced growth inhibition.
Mouse prostate epithelial cells with wild type PTEN (PTEN+/+), PTEN knockout (PTEN+/−) and heterozygous (PTEN−/−) were plated in 96-well plates, and then treated with 100 ng/ml TRAIL for 48 h. Cells proliferation was determined by MTT assays and expressed as percentage of untreated cells. Representative of three independent experiments. *, P<0.01, statistical significance. B, effect of PTEN loss on TRAIL-induced apoptosis. PTEN+/+, PTEN+/−, and PTEN−/− cells were treated with 100 ng/ml TRAIL for 48 h as described in (A). Total protein was extracted for assaying total and phosphorylated Akt (p-AKT), PTEN and PARP by Western blot analysis. β-actin was used as a loading control. C, restored PTEN expression sensitizes PTEN−/− cells to TRAIL. PTEN−/− cells (5×105) were infected with adenoviruses expressing wild-type PTEN (Ad-PTEN wt) or expressing LacZ (Ad-lacZ). At 72 h after infection, cells were trypsinized and plated in 60 mm dish at 5×105 cells. The next day, cells were left untreated or treated with TRAIL (100 ng/ml) for 48 h. Cells were harvested for MTT assays (Upper panel) and Western blot analysis was employed for detecting the levels of PARP and PTEN proteins (lower panel), respectively. Cell proliferation data were expressed as percentage of untreated cells. Representative of three independent experiments. **, P<0.001, statistical significance. D, inhibition of PTEN function by a dominant negative form of PTEN confers TRAIL resistance. PTEN+/+ and PTEN+/− cells were infected with adenoviruses expressing a dominant negative form of PTEN for 72 h and then treated with TRAIL (100 ng/ml) for 48 h. Cells were either used for growth inhibition by MTT assays (upper panel) or harvested for detecting the levels of PARP, PTEN, and total and phosphorylated Akt by Western blot analysis (lower panel). Cell proliferation data were expressed as percentage of untreated cells. Representative of three independent experiments. *, P<0.01, statistical significance. **, P<0.001, statistical significance.
Figure 6.
Inhibition of PTEN function renders breast cancer T47D cells more resistant to TRAIL.
A, effect of inhibition of PTEN function on cell proliferation. T47D cells (8×105) were infected with adenovirus expressing a dominant negative form of PTEN or adenoviruses expressing LacZ for 72 h and then treated with TRAIL (100 ng/ml) for 48 h. Cell proliferation was determined by MTT assays. Cell proliferation data are expressed as percentage of untreated cells. Representative of three independent experiments. *, P<0.01, statistical significance. B, effect of inhibition of PTEN function on apoptosis. T47D cells infected with adenoviruses as described in A were treated with TRAIL (100 ng/ml) for 48 h and then harvested for analyzing the levels of PARP, PTEN, total and phosphorylated Akt proteins by Western blot analysis (lower panel). β-actin was used as a loading control.