Figure 4
Agents accumulating NOXA protein efficiently induce NOXA-dependent apoptosis in MCL cells. (a) Screening for substances able to induce NOXA protein expression. MCL cell lines Mino and Rec1 were treated with Cisplatin (10 μM), Doxorubicin (100 nM), Bortezomib (10 nM), Orlistat (15 μM) or MLN4924 (0.5 μM) for 16 h and NOXA protein expression was analyzed by western blot. (b) Accumulation of NOXA efficiently induces cell death. MCL cells were treated with Cisplatin (10 μM), Doxorubicin (100 nM), Bortezomib (10 nM), Orlistat (15 μM) or MLN4924 (0.5 μM) for 24 h and cell viability was analyzed by Annexin V staining and flow cytometry. (c) Dose-response curves of MCL cell lines Mino, Jeko1, Rec1, Jvm2 and Granta519 compared with PBMCs and fibroblasts of healthy donors. Cells were treated with increasing concentrations of Bortezomib, Orlistat or MLN4924 and cell viability measured by MTT. (d) NOXA siRNA rescues from Bortezomib, Orlistat and MLN4924 induced apoptosis. Cells were transfected with control siRNA or siRNA targeting NOXA. At 24 h after transfection, cells were treated with Bortezomib (10 nM), Orlistat (15 μM) or MLN4924 (0.5 μM) and further cultivated for 24 h. Cells were then harvested for western blot analysis and quantification of cell death by Annexin V staining and flow cytometry (**P<0.01, ***P<0.001). Data represent means±S.D. from three experiments. (e) Active PI3K/AKT/mTOR signaling is needed for effective accumulation of NOXA protein and induction of cell death upon treatment with Bortezomib, Orlistat or MLN4924. MCL cell lines Mino and Rec1 were pre-treated with 1 μM Bez235 (6 h) before treatment with Bortezomib (10 nM), Orlistat (15 μM) or MLN4924 (0.5 μM). After 16 h, cells were harvested for western blot analysis (lower panel). Quantification of cell death by Annexin V staining and flow cytometry was determined 24 h upon treatment (*P<0.05). Data represent means±S.D. from three experiments
