c-FLIP Degradation Mediates Sensitization of Pancreatic Cancer Cells to TRAIL-Induced Apoptosis by the Histone Deacetylase Inhibitor LBH589
Figure 6
LBH589 reduces c-FLIP levels through ubiquitin/proteasome-mediated protein degradation.
A, Panc-1 cells were treated with DMSO or 50 nM LBH589 for 4 h. The cells were then washed with PBS 3 times and refed with fresh medium containing 10 µg/ml CHX. At the indicated times post CHX, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis. Protein levels were quantitated with NIH Image J software (Bethesda, MA) and were normalized to GAPGH. The results were plotted as the relative c-FLIP levels compared to those at the time 0 of CHX treatment (lower panel). B, Panc-1 cells were pretreated with 20 µM MG132 for 30 minutes prior to the addition of 50 nM LBH589. After co-treatment for 4 h, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis. C, Panc-1/FLIPL-5 cells which stably express ectopic flag-FLIPL were transfected with HA-ubiquitin plasmid using FuGENE 6 transfection reagent for 24 h. The cells were then pretreated with 20 µM MG132 for 30 minutes and then co-treated with 50 nM LBH589 for 4 h. Whole-cell protein lysates were then prepared for immunoprecipitation (IP) using anti-Flag antibody followed by Western blotting (WB) using anti-HA antibody for detection of ubiquitinated FLIPL (Ub-FLIPL) and anti-Flag antibody for detection of ectopic FLIPL.