Nanoliposomal C₆ ceramide induces caspase-dependent apoptosis in leukemic NK cells but not normal PBMCs. (A) The PBMC from NK-LGL leukemia patients (CD3⁻CD56⁺ cells > 80%; patients #1 and #2 with aggressive NK-LGL leukemia, patients #3 and #4 with chronic NK-LGL leukemia) or 2 normal donors #1 and #2 were treated with 12.5, 25, and 50μM ghost or C₆-ceramide nanoliposome or dihydro-C₆-ceramide or ghost nanoliposome (patients #5 and #6 with chronic NK-LGL leukemia), respectively for 18 hours; then cells were assayed for apoptosis by flow cytometry. *P < .05, **P < .005 indicate significant differences of C₆-treated cells versus ghost nanoliposome–treated cells (Student t test). (B) The PBMCs from individual NK-LGL leukemia patients (CD3⁻CD56⁺ cells > 80%; patients #1 and #2 with aggressive NK-LGL leukemia, and patients #7 and #8 with chronic NK-LGL leukemia) or 2 normal donors #3 and #4 were treated with 25μM C₆-ceramide or ghost nanoliposome for 2, 6, and 24 hours; then cells were assayed for apoptosis by flow cytometry. *P < .05, ***P < .0005 indicate significant difference of ghost nanoliposome–treated cells (Student t test). (C) Western blot analysis was performed for caspase-3 after treatment of PBMCs from patient #8, which were treated with 25μM C₆-ceramide or ghost nanoliposome for 2, 6, and 24 hours. Data are representative of 4 independent experiments on PBMCs from 4 NK-LGL leukemia patients. (D) NKL cells were exposed to 25μM C₆-ceramide nanoliposome, in the absence or presence of various concentrations of z-VAD-fmk, a pan caspase inhibitor. z-VAD-fmk was added 2 hours before ceramide treatment at the indicated concentration. Cell survival was determined 18 hours later by the MTT assay. *P < .05 indicates significant differences of each dose of z-VAD-fmk–treated cells compared with z-VAD-fmk–untreated cells, respectively (Student t test).