NPI-0052 induced apoptosis through caspase activation and mitochondrial perturbations. (A) A caspase-8 inhibitor blocks NPI-0052–induced caspase-3 activation. Jurkat cells were treated with 200 nM NPI-0052 and 10 μM IETD-fmk, 10 μM LEHD-fmk, or 50 μM zVAD-fmk for 6 hours. Cells were lysed and caspase-3 activity was measured by the cleavage of DEVD-amc fluorescent peptide as described in “Materials and methods”. ** P < .001, significantly different from NPI-0052–treated cells. (B) NPI-0052 induces activation and cleavage of caspase-3. Cells were treated with 200 nM or 1 μM NPI-0052 over a period of 24 hours. Cells were lysed at indicated time points, and caspase-3 activity was measured as previously described. *P > 0.01 for 8 hour time point compared with 0 hour time point. (B) Inset: Jurkat cell lysates from cells treated with 200 nM or 1 μM NPI-0052 for 8 hours were subjected to SDS-PAGE. Membranes were probed with caspase-3–specific antibody to detect pro–caspase-3 and/or cleaved products or actin-specific antibody. (C) NPI-0052 induces cleavage of caspase-8 in Jurkat cells. Cells were stimulated with 200 nM NPI-0052 for indicated times and subjected to SDS-PAGE. Immunoblotting was performed using caspase-8–specific antibody that detects full and cleaved caspase-8 or actin-specific antibody. (D) A caspase-8 inhibitor protects from NPI-0052–induced apoptosis. DNA fragmentation by PI staining was assessed after exposure of Jurkat cells for 24 hours to 200 nM NPI-0052 and 10 μM caspase-8 inhibitor (IETD-fmk), or 10 μM caspase-9 inhibitor (LEHD-fmk). * P > .001. (E) NPI-0052 induces cleavage of Bid. Jurkat cells were preincubated with 24 mM NAC for 15 minutes followed by treatment with 1 μM NPI-0052 or treated with1 μM NPI-0052 alone or 1 μM staurosporine (STS) as a positive control for 8 hours. Western blot detected full-length and cleaved Bid. (F) NPI-0052 triggers mitochondrial injury. Drops in mitochondrial membrane potential (ΔΨm) were assessed by TMRE staining of Jurkat cells treated with NPI-0052 alone or with pretreatment with 24 mM NAC for 15 minutes prior to NPI-0052 exposure. Samples were analyzed by flow cytometry. *P = .004, compared with control. (G) NPI-0052 induces cytochrome c release and caspase-9 activation. After exposure to 200 nM NPI-0052, Western blots detected cytochrome c in cytosolic fractions after 4 hours and reduction of pro–caspase-9 after 6 hours in lysates from Jurkat cells. (H) NPI-0052 increases intracellular superoxide and hydrogen peroxide levels. Jurkat cells were exposed to 200 nM NPI-0052 for 14 hours and stained with HEt or CM-H2DCF-DA followed by FACS analysis. Experiments A, B, D, and F were from 3 independent experiments and presented as mean ± SD.
