PEITC induces CLL cell death through oxidative damage to mitochondria. (A) Induction of oxidative damage to cardiolipin in CLL cells by PEITC (5 μM). Cardiolipin oxidation was measured by flow cytometry using NAO staining.17 M1 indicates the gating of the subpopulation of CLL cells that lost cardiolipin signal due to oxidation. Representative histograms of the time course experiments in a CLL patient sample are shown. Similar results were obtained using another sample. (B) Loss of mitochondrial cytochrome c induced by 5 μM PEITC in CLL cells. The overlays of the control (gray shade) and PEITC-treated (black line) samples show the distribution of mitochondrial cytochrome c fluorescent intensity of each cell population, with the mean value of the relative intensity indicated. Representative histograms of a CLL patient sample are shown. Similar results were obtained using another sample. (C) Caspase-3 activation in CLL cells treated with 5 μM PEITC, measured by flow cytometry using FITC-conjugated antibody specific for active caspase-3. M1 indicates the gating of subpopulation of cells with positive caspase-3 activation. Representative histograms of a CLL patient sample are shown. Similar results were obtained using 2 other different patient samples. (D) Partial suppression of PEITC-induced cell death by Z-VAD-fmk. CLL cells were preincubated with 20 μM Z-VAD-fmk for 30 minutes before incubation with 5 μM PEITC for 24 hours. Cell death was detected by annexin V/PI assay. Each bar represents the mean and 95% CI (n = 5 CLL samples). (E) Suppression of PEITC-induced caspase-3 activation by NAC. CLL cells were preincubated with 1 mM NAC for 1 hour before incubation with 5 μM PEITC for 5 hours. Procaspase-3 was detected by Western blot and quantified by densitometry and normalized with β-actin. Each bar represents the mean and 95% CI of 3 different CLL samples. (F) No effect of Z-VAD-fmk on PEITC-induced glutathione depletion was found. CLL cells were preincubated with 20 μM Z-VAD-fmk for 30 minutes before incubation with 5 μM PEITC for 5 hours. Each bar represents the mean and 95% CI of 3 different CLL samples.
