Low doses of NPI-0052 and HDAC inhibitors induce synergistic apoptosis. (A) NPI-0052 synergizes with MS-275 to induce cytotoxicity. Jurkat cells were exposed to increasing doses of MS-275 (1-5 μM) and 10 nM NPI-0052 for 24 hours. Cells were assessed for apoptosis by measuring DNA fragmentation as previously described. The combination of 2.5 mM or 5 mM MS-275 with 10 nM NPI-0052 was statistically significant, P > .05, when compared with either single agent alone. (B) ROS and caspase-8 involvement in NPI-0052– and MS-275–induced apoptosis. Caspase-3 activity in wild-type Jurkat and their caspase-8–deficient counterparts exposed to 10 nM NPI-0052 and 5 μM MS-275 for 6 hours. *P > .001 comparing NPI-0052 treatment versus combination treatment in Jurkat. **P > .001 comparing Jurkat treated with NPI-0052 and MS-275 versus I9.2 cells also treated with both agents. Inset: Superoxide levels measured by HEt staining in Jurkat cells incubated with MS-275 and NPI-0052. Shown is a representative histogram of 3 separate experiments and mean fluorescent (MF) values. (C) NPI-0052 synergizes with MS-275 more effectively than bortezomib. Cells were treated with indicated doses of MS-275 and 10 nM NPI-0052 or 10 nM bortezomib for 24 hours. PI staining determined the percentage of cells undergoing DNA fragmentation. Shown is the percent increase in the subdiploid population; *P > .05. Isobologram analysis determined the combination index (CI) values, calculated by Calcusyn software. CI > 0.1 indicates synergism, where a CI range from 0.1-0.3 indicates strong synergism, and from 0.3-0.7 synergism. CI values for 10 nM bortezomib and MS-275 were CI = 0.48 for 2.5 μM MS-275 and CI = 0.47 for 5 μM MS-275. CI values for 10 nM NPI-0052 and MS-275 were CI = 0.27 for 2.5 μM MS-275 and CI = 0.21 for 5 μM MS-275. (D) Low-dose NPI-0052 and Valproic acid synergistically enhance apoptosis. DNA fragmentation was assessed in cells treated with 1 mM or 2.5 mM VPA and 5 nM NPI-0052 for 24 hours. *P > .05. Inset: Jurkat cells were incubated with 2.5 mM VPA and 10 nM NPI-0052 or 10 nM bortezomib. After 24 hours, cells were stained with PI to determine DNA fragmentation. Shown is the percent increase in the subdiploid population. Analysis of synergism by isobologram determined the CI values. CI > 0.1 indicates synergism. CI = 1.0 indicates additive effects.