Reduction of the antiapoptotic protein cFLIP enhances the susceptibility of human renal cancer cells to TRAIL apoptosis

Abstract

Human renal carcinoma cells (RCCs) were sensitized to the apoptotic effects of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), by treatment with cycloheximide (CHX). In contrast to a previous study, a rapid and dramatic decrease in levels of cellular FLICE (Fas-associated death domain-like IL-1beta-converting enzyme) inhibitory protein (cFLIP) following cycloheximide treatment was observed in all RCCs studied. The unambiguous detection of this decrease in cFLIP was dependent on the quality of the particular antibody preparation used to detect cFLIP. Cycloheximide treatment caused no major change in levels of pro-caspase-8 or cell surface expression of TRAIL receptors. Therefore, cycloheximide treatment resulted in an increase in the pro-caspase-8 to cFLIP ratio, which correlated with sensitization to TRAIL-mediated apoptosis. Furthermore, treatment of human RCCs with small interfering oligoribonucleotides (siRNA) for cFLIP caused a reduction of cFLIP protein and sensitized cells to TRAIL-mediated apoptosis. We concluded that in the presence of an intact TRAIL signaling pathway, a significant reduction of cFLIP alone is sufficient to sensitize human RCCs to TRAIL apoptosis.

Fig. 1

Four RCCs tested for the effect of cycloheximide on TRAIL-mediated apoptosis, in the presence or absence of caspase inhibition. RCCs were incubated overnight with TRAIL alone (open squares) or in the presence of CHX (solid squares), and growth inhibition was determined. To determine the effects of caspase inhibition, RCCs were pretreated with caspase inhibitors Z-VAD-FMK (solid diamonds), Z-LEHD-FMK (solid triangles),or Z-IEDT-FMK (solid circles) for 2 h prior to the addition of CHX, followed 2 h later with TRAIL and then overnight incubation

Fig. 2

Reduction of cFLIP in RCCs by CHX and siRNA-FLIP. a Western blotting of ACHN cells treated with (1) control siRNA, (2) siRNA-cFLIP, (3) media, (4) CHX, and (5) 293 cells, and (6) 293 cells, transfected with cFLIP using Ab1 (monoclonal anti-cFLIP, Dave-2), Ab2 (monoclonal anti-cFLIP, NF-6), Ab3 (rabbit polyclonal anti-cFLIP?, ABR), or anti-actin as a loading control as described in “Materials and methods.” b Western blotting of various human RCCs after overnight treatment with CHX using Ab1 (Dave-2). c Kinetics of cFLIP disappearance on Western blotting of ACHN cells using Ab1 (Dave-2) at indicated times following CHX treatment

Fig. 3

Sensitization of three RCCs to TRAIL by siRNA-cFLIP. RCCs untreated (solid diamonds), treated with siRNA-control (solid triangles) or siRNA-cFLIP (open circles) were prepared, then treated overnight with TRAIL, and growth inhibition was determined

Fig. 4

Lack of an effect of CHX on TRAIL receptor levels. ACHN cells were incubated overnight in the presence or absence of CHX. Cell surface levels of TRAIL-R1 (a), TRAIL-R2 (b), TRAIL-R3 (c), and TRAIL-R4 (d) were then determined by FACS analysis. Untreated ACHN with isotope control (dotted histograms), anti-TRAIL-R (shaded histograms), or CHX-treated ACHN with anti-TRAIL-receptors (solid line histograms) are shown