Figure 1.
Effect of pterostilbene (PTER) on the cell proliferation of acute myeoloid leukemia (AML) cell lines.
(A) The chemical structure of PTER. (B) Five AML cell lines were treated with the vehicle (DMSO) or PTER (12.5∼150 µM) in serum-containing medium for 24 h. Cell proliferation was determined by an MTS assay. Results are expressed as multiples of cell proliferation rate. Values represent the mean ± SE of 3 independent experiments. *, #, &, @, ∧ p<0.05, compared to the vehicle groups. (C) HL-60 cells were treated with different concentrations of PTER (0∼150 µM) for 24 and 48 h and analyzed by a trypan blue exclusion assay. Quantitative assessment of the mean number of cells is expressed as the mean ± SE.
Figure 2.
Effect of pterostilbene (PTER) on HL-60 cell-cycle regulation and apoptosis.
(A) HL-60 cells were treated with different concentrations of PTER (0∼100 µM) for 24 h, and flow cytometry was used to detect the cell cycle phase distribution and cell death in the sub-G1 phase. Data are presented as the mean ± SE of three independent experiments. Results were analyzed using one-way ANOVA with Tukey’s post hoc tests at 95% confidence intervals. Different letters represent significantly different, p<0.05. (B) Quantitative analysis of cell apoptosis by Annexin-V and propidium iodide (PI) double-staining flow cytometry. Values represent the mean ± SE of three independent experiments. *p<0.05, compared to the vehicle group. (C) HL-60 cells were treated with 100 µM PTER for 24 h and analyzed by fluorescence microscopy after DAPI staining. White arrows indicate apoptotic HL-60 cells.
Figure 3.
Effect of pterostilbene (PTER) on alterations of cell-cycle regulatory proteins in HL-60 cells.
Proteins were extracted from cultured HL-60 cells at 24 h after PTER treatment and probed with proper dilutions of specific antibodies. (A and B) PTER at a concentration of 100 µM induced significant decreases in protein levels of cyclin D3, CDK2, and CDK6. Upper panels: Representative results of cyclins and cyclin-dependent kinase (CDK) protein levels as determined by a Western blot analysis. Lower panels: Quantitative results of cyclin and CDK protein levels, which were adjusted to the β-actin protein level and expressed as multiples of induction beyond its own control. Values are presented as the mean ± SE of three independent experiments. *p<0.05, compared to the vehicle control group. (C) There were no significant differences in protein levels of p15 INK4B, p21 Cip1, or p27 Kip1 between control and PTER-treated HL-60 cells. Upper panel: Representative results of p15, p21, and p27 protein levels as determined by a Western blot analysis. Lower panel: Quantitative results of p15, p21, and p27 protein levels, which were adjusted with the β-actin protein level and expressed as multiples of induction beyond its own control. (D) Cyclin D3, CDK2, and CDK6 peotein expression were downregulated in a concentration-dependent fashion after PTER treatment in HL-60 cells. Left panel: Representative results of cyclin D3, CDK2, and CDK6 protein levels as determined by a Western blot analysis. Right panel: Quantitative results of cyclin D3, CDK2, and CDK6 protein levels, which were adjusted with the β-actin protein level and expressed as multiples of induction beyond its own control. *p<0.05, compared to the vehicle control group.
Figure 4.
Effect of pterostilbene (PTER) on caspase activation and mitochondrial membrane permeability (MMP) in HL-60 cells.
(A) Expression levels of cleaved caspases-3, -8, and -9, and poly (ADP-ribose polymerase (PARP) were assessed by a Western blot analysis after treatment with various concentrations of PTER (0∼150 µM) for 24 h. (B) Quantitative results of cleaved caspase-3, -8, and -9, and PARP protein levels, which were adjusted to the α-tubulin protein level and expressed as multiples of induction beyond each respective control. Values are presented as the mean ± SE of three independent experiments. *, #, &, $p<0.05, compared to the vehicle control groups. (C) Activated caspase-9, -8, and -3 protein expression were upregulated in a time-dependent fashion after PTER 100 µM) treatment, peaking at 24 h in HL-60 cells. (D) Effect of a caspase-3 inhibitor on PTER-induced cell death. Cells were treated with 100 µM PTER for 24 h in the presence or absence of 2 µM Z-DEVE-FMK. Cell proliferation was determined by an MTS assay. Data are presented as the mean ± SE of three independent experiments performed in triplicate. *p<0.05, control vs. PTER; #p<0.05, PTER vs. Z-DEVE-FMK plus PTER. (E and F) Loss of the MMP after 24 h of treatment with PTER as determined by FACS and immunofluorescence analyses of JC-1 staining. (E) From the FACS analysis, increased percentages of green fluorescent apoptotic (FL 1) populations of HL-60 at the indicated drug concentrations (cells in the lower right field) are indicated (upper panel). The red-to-green fluorescence ratio indicates functional mitochondria with membrane potential. Data are presented as the mean ± SE of three independent experiments performed in triplicate. *p<0.05, control vs. PTER (F) Immunofluorescence analysis showed that green-fluorescent monomeric form increases in HL-60 cells after treatment with 100 µM PTER for 24 h. Original magnification, 200×.
Figure 5.
Role of mitogen-activated protein kinase (MAPKs) in pterostilbene (PTER)-induced activation of caspases-8,-9, and -3.
(A–C, upper panel) Phosphorylation levels of extracellular signal-regulated kinase (ERK)1/2, p38, and c-Jun N-terminal kinase (JNK)1/2 were assessed by a Western blot analysis after treatment with various concentrations of PTER (0∼150 µM M) for 24 h. (A–C, lower panel) Quantitative results of phopho-ERK1/2, p38, and JNK1/2 protein levels, which were adjusted with the total ERK1/2, p38, and JNK1/2 protein levels and expressed as multiples of induction beyond each respective control. Values represent the mean ± SE of three independent experiments. *p<0.05, compared to the vehicle control group. (D, upper panel) HL-60 cells were pretreated with or without 20 µM U0126, SP600125, or SB202190 for 1 h followed by PTER (100 µM) treatment for an additional 24 h. Expression levels of cleaved caspase-3, -8, and -9 were determined by a Western blot analysis. (D, lower panel) Quantitative results of cleaved caspase-3, -8, and -9 protein levels, which were adjusted to the α-tubulin protein level and expressed as multiples of induction beyond each respective control. Values represent 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 different levels of significance. Different letters represent significantly different, p<0.05.
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.
Figure 7.
Proposed signal transduction pathways by which pterostilbene (PTER) inhibits the growth of HL-60 cells.
‘Bold solid lines’ indicate pathways affected by PTER. ‘Bold dashed lines’ indicate hypothetical pathways which might be affected by PTER. LMP, lysosomal membrane permeabilization; MMP, mitochondrial membrane permeabilization; cyto C, cytochrome C.