Figure 1.
Contribution of death receptors DR4 and DR5 in TRAIL-mediated cell death in HCT116 p53+/+ and HCT116 p53−/− cells.
(A) Levels of constitutive surface expression of the death and the decoy receptors in HCT116 cells as determined by flow cytometry. (B) Cells were pre-incubated with 20 µg/ml antagonistic antibodies to death and decoy receptors or IgG1 control for 1 h following 4 h treatment with TRAIL or DR5-B (1 µg/ml) and cell death was determined by MTT test. Values are mean ± SD of at least three independent experiments.
Figure 2.
Time-depended influence of bortezomib on TRAIL or DR5-B mediated cell death in HCT116 p53+/+ and HCT116 p53−/− cells.
(A) Viability of the cells treated with different concentrations of TRAIL or DR5-B during 8, 16, 20 and 24 h of incubation. (B) Viability of the cells treated with 1 nM bortezomib and different concentrations of TRAIL or DR5-B during 8, 16, 20 and 24 h of incubation. (C) Cells were incubated with different concentrations of bortezomib and cell death was measured after 24 h of incubation. (D) Calculation of effective concentrations of TRAIL variants after 24 h treatment of cells with 1 nM bortezomib. Values are mean ± SD of at least three independent experiments.
Figure 3.
Time-dependent expression of the death and the decoy receptors at the surface of HCT116 p53+/+ and p53 null cells treated by bortezomib and TRAIL (or DR5-B).
In all experiments, cells were treated with 1 nM bortezomib and 1 ng/ml TRAIL variants alone or in combination. (A) Profiles of the death receptors at the cell surface. (B) Profiles of the decoy receptors at the cell surface. (C) Relative level of death and decoy receptors membrane expression in cells during combined treatment with TRAIL variants and bortezomib. Values in all experiments are mean ± SD of at least three independent experiments.
Figure 4.
The content of death and decoy receptors in total cell extracts of HCT116 p53+/+ and HCT116 p53−/− cells treated by TRAIL, bortezomib or by combination of both drugs.
HCT116 p53+/+ (A) and HCT116 p53−/− (B) cells were treated with TRAIL (1 ng/ml), bortezomib (1 nM), or by combination of both drugs for various time periods and the content of receptors in total cell extract was determined by Western blot analysis using appropriate biotinylated antibodies to each receptor. Densitometric analysis of three independent experiments was performed using ImageJ software. Values in all experiments are mean ± SD of at least three independent experiments.
Figure 5.
Confocal microscopic analysis of death and decoy receptors localization in HCT116 p53−/− cells treated with TRAIL and bortezomib for indicated periods. Scale bar = 10 µm.
Cells were grown for different periods in the presence of indicated reagents, washed with PBS, fixed in 3% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100 in PBS for 10 min and blocked in 3% BSA for 30 min. FITC-conjugated antibodies to DR4, DR5, DcR1 and DcR2 receptors (Abnova) were added at dilution 1∶100 and cells were incubated for 1 h in the presence of Hoerst 33342 for visualization of cell nuclei. Then non-specific bound antibodies were washed with blocking buffer containing 0.1% of Triton X-100. Cells were visualized in 0.6- µm sections using an inverted Nikon Eclipse TE2000-E laser scanning confocal microscope under a ×60 oil immersion objective.
Figure 6.
Inhibition of bortezomib and TRAIL induced DR4 internalization by z-VAD-FMK.
DR4 receptor cell surface expression and viability of the HCT116 p53+/+ cells were analyzed after incubation with 10 µM z-VAD-FMK for 1 h following treatments with TRAIL (1 ng/ml) and bortezomib (1 nM) or in combination of the two drugs.
Figure 7.
Inhibition of bortezomib and TRAIL induced DR4 internalization by E-64 in HCT116 p53−/− cells.
(A) DR4 and DR5 receptors cell surface expression was analyzed after incubation of the cells with E-64 (25 µM) for 1 h following medium change and treatment with TRAIL (1 ng/ml), bortezomib (1 nM) or in combination of the two drugs for 24 h. (B) Cells were treated as in (A) and the expression of DR4 and DR5 receptors in total cell extract was determined by Western blot analysis using appropriate biotinylated antibodies. Densitometric analysis of three independent experiments was performed using ImageJ software. (C) Inhibition of bortezomib and TRAIL induced cell death by E-64 in the cells was determined by MTT test. Values in all experiments are mean ± SD of at least three independent experiments.
Figure 8.
Inhibition of lysosomal activity by Baf-A1 prevented TRAIL and bortezomib induced DR4 internalization.
(A) DR4 and DR5 receptors cell surface expression was analyzed in HCT116 p53−/− cells pretreated with Baf-A1 (25 µM) for 1 h following bortezomib (1 nM) and TRAIL (1 ng/ml) treatment alone or in combination for 24 h. (B) Viability of the cells treated as in (A) was determined by MTT test. Values in all experiments are mean ± SD of at least three independent experiments.