Figure 5
Orlistat and MLN4924 kill Bortezomib-resistant MCL cells by stabilizing NOXA protein in a proteasome-independent manner. (a) Bortezomib, Orlistat and MLN4924 are stabilizing NOXA protein by inhibiting rapid turnover. MCL cell line Mino was treated with Bortezomib (10 nM), Orlistat (15 μM) or MLN4924 (0.5 μM) for 8 h and then exposed to 20 μg/ml cycloheximide and NOXA half-life determined by western blot. (b) Orlistat and MLN4924 stabilize NOXA by inhibiting ubiquitination of NOXA. Cells were cultivated in absence or presence of Bortezomib (10 nM), Orlistat (15 μM) or MLN4924 (0.5 μM) for 8 h then harvest and lysed. Total polyubiquitinated proteins were isolated from cell lysates using agarose-TUBE2 beads. Protein expression and polyubiquitination state of NOXA were analyzed by western blot analysis using an NOXA-specific antibody. (c) Treatment with Orlistat and MLN4924 can kill Bortezomib-resistant MCL cells. Bortetomib-sensitive (Jeko1-BS, left panel) and Bortezomib-resistant (Jeko1-BR) MCL cells were treated for 24 h with Bortezomib (10 nM), Orlistat (15 μM) or MLN4924 (0.5 μM) and cell viability measured by Annexin V staining and flow cytometry (upper panel). Data represent means±S.D. from three experiments. NOXA accumulation was determined after 8 h of treatment by western blot analysis (lower panel)
