Caspase-8 mediates ROS production, hyperacetylation of histone-H3, and caspase-3 activation by NPI-0052/HDACi. (A) Decreased superoxide levels in a caspase-8–deficient cell line. Jurkat cells and caspase-8–deficient Jurkat cells (I9.2) were treated with 5 μM MS-275, 10 nM NPI-0052, or a combination for 12 hours. Superoxide levels were measured by staining cells with HEt and subsequent flow cytometric analysis on the FL-3 channel. Shown are mean fluorescence values for 4 independent experiments. (B) Lower superoxide levels are detected in caspase-8–deficient cells with vorinostat/NPI-0052 treatment. I9.2 and Jurkat cells were exposed to 500 nM vorinostat, 10 nM NPI-0052, or a combination for 12 hours. Dihydroethidium staining was used to detect superoxide levels. Shown are mean fluorescence values for 4 independent experiments. (C) Caspase-8 plays a role in the hyperacetylation of histone-H3 by NPI-0052. Top panel, the I9.2 (caspase-8–deficient) cells were incubated with 24 mM NAC for 30 minutes, before 5 μM MS-275, 10 nM NPI-0052, or a combination for a total of 6 hours. Cell lysates were analyzed by Western blot analysis for Ac-H3 and actin. Bottom left panel, protein lysates from Jurkat and I9.2 cells treated with 10 nM NPI-0052, 5 mM MS-275, or diluent for 6 hours were analyzed for Ac-H3 and histone H3 protein expression by Western blot analysis. Bottom right panel, protein lysates from I9.2 cells and I9.2/caspase-8/EGFP-transfected cells exposed to DMSO or 10 nM NPI-0052 for 6 hours were evaluated for Ac-H3, caspase-8, and actin protein expression by Western blot analysis. (D) Caspase-8 mediates caspase-3 activation by NPI-0052/vorinostat regimen. Caspase-3 activity was measured in Jurkat and I9.2 cells exposed to DMSO, 500 nM vorinostat, 10 nM NPI-0052, or the combination for 8 hours. *P < .001 compared control or either agent alone; †P < .001 compared with Jurkat cells incubated with NPI-0052/vorinostat.