Pterostilbene Simultaneously Induced G0/G1-Phase Arrest and MAPK-Mediated Mitochondrial-Derived Apoptosis in Human Acute Myeloid Leukemia Cell Lines
Figure 6
Effect of pterostilbene (PTER) on lysosomal membrane alterations in HL-60 cells.
(A) PTER enhanced lysosome permeability. HL-60 cells were treated with 100 µM PTER for 12 h, then stained with acridine orange (5 µg/ml) for 15 min, and examined under a fluorescence microscope (×200 magnification). Representative images of three independent experiments are shown. (B) PTER concentration-dependently induced translocation of cathepsin B from lysosomes to the cytosol. Cytosolic cathepsin B levels were assessed by a Western blot analysis after treatment with various concentrations of PTER (0∼100 µM) for 24 h. Protein levels of both the 37-kDa pro-cathepsin and 25-kDa activated cathepsin B are shown. β-actin and C23 were respectively used as positive and negative cytosolic internal controls. (C) HL-60 cells were treated with 100 µM PTER for 1 h and stained with H2DCFDA; then total ROS level was analyzed by FACS, and data are presented as the mean multiples of increase in fluorescence compared to the control ± SE. *p<0.05, compared to the control. (D and E) The reactive oxygen species (ROS) scavenger, N-acetyl cysteine (NAC; 10 mM) was added 1 h prior to the addition of 100 µM PTER. Lysosome permeability (D) and cathepsin B cytosolic translocation (E) were analyzed 24 h later. (F) The cathepsin B inhibitor, CA074-Me (50 and 100 µM), was added 1 h prior to the addition of 100 µM PTER. Cell proliferation was determined by an MTS assay. Values are presented as the mean ± SE of three independent experiments. Data were analyzed using a one-way ANOVA with Tukey’s post-hoc tests at 95% confidence intervals; Different letters represent significantly different, p<0.05.