PCI-24781, a Novel Hydroxamic Acid HDAC Inhibitor, Exerts Cytotoxicity and Histone Alterations via Caspase-8 and FADD in Leukemia Cells
Figure 3
PCI-24781 induces ROS generation in a caspase-dependent and time-dependent manner. (a) Jurkat cells were treated with 5 M of PCI-24781, incubated between 2 and 22 hours. Intracellular superoxide was measured at each time point using dihydroethidium (HEt) and assessed by flow cytometry. *P < .01 compared to control. (b) Jurkat cells were treated with a range of PCI-24781 doses (0.01 M–10 M), and incubated 16 and 20 hours. The mean HEt fluorescence at each dose was measured by flow cytometry. *P < .05 compared to control. (c) Jurkat cells were treated with 5 M PCI-24781, with or without zVAD-fmk pretreatment, and incubated for 24 hours, intracellular superoxide levels were assessed using HEt. *P < .05. (d) Jurkat cells were treated with 5 M PCI-24781, incubated between 4 and 16 hours, lysed and stained with DEVD-amc to assess caspase-3-like activity. Caspase-3 activity was measured in relative fluorescence unit (RFU). The release of amc was measured on a spectrofluorometer using an excitation of 355 nm and an emission of 460 nm. *P < .05 compared to control. (e) Jurkat cells were pretreated with either inhibitor of caspase 8 (IETD-fmk) or a pan caspase inhibitor (zVAD-fmk) for 30 minutes. After pretreatment, the samples were treated with 0.5 or 5 M PCI-24781 for 16 hours and dyed with PI reagent. DNA fragmentation (% subdiploid) was assessed by flow cytometer. (f) Jurkat and I9.2 cells were treated with or without 0.5 M PCI-24781 and incubated by 16 hours, dyed with PI reagent, and assessed on the flow cytometer. *P < .05 relative to Jurkat 0.5 M PCI-24781. (a)–(f) Error bars represent the means S.D. of three independent experiments. (g) Jurkat and I2.1 cells were treated with or without 0.5 or 5 M PCI-24781 and incubated by 16 hours, dyed with PI reagent, and assessed on the flow cytometer. *P < .05 relative to Jurkat cells treated with PCI-24781.
