Induction of apoptosis by [8]-shogaol via reactive oxygen species generation, glutathione depletion, and caspase activation in human leukemia cells

Abstract

Ginger, the rhizome of Zingiber officinale , is a traditional medicine with a carminative effect and antinausea, anti-inflammatory, and anticarcinogenic properties. This study examined the growth inhibitory effects of [8]-shogaol, one of the pungent phenolic compounds in ginger, on human leukemia HL-60 cells. It demonstrated that [8]-shogaol was able to induce apoptosis in a time- and concentration-dependent manner. Treatment with [8]-shogaol caused a rapid loss of mitochondrial transmembrane potential, stimulation of reactive oxygen species (ROS) production, release of mitochondrial cytochrome c into cytosol, and subsequent induction of procaspase-9 and procaspase-3 processing. Taken together, these results suggest for the first time that ROS production and depletion of glutathione that contributed to [8]-shogaol-induced apoptosis in HL-60 cells.

Figure 1. Effect of [8]-shogaol on cell cytotoxicity

(A) Chemical structure of [8]-shogaol. (B) HL-60 cells were treated with different concentration of [8]-shogaol (0, 10, 20, 30, 40, and 50 µM) for 24 h. Cell viability then was determined by the MTT assay. The values are expressed as means ± SD of triplicates tests. *P < 0.001 indicate statistically significant difference from control.

Figure 2. Determination of sub-G1 cells in [8]-shogaol-treated HL-60 cells by flow cytometry

HL-60 cells were treated with (A) different concentration of [8]-shogaol (0, 10, 20, 30, 40, and 50 µM) for 24 h or (B) treated with 30 µM of [8]-shogaol for indicated time. The method of flow cytometry used is descried under Materials and Methods. AP (apoptotic peak) represents apoptotic cells with a lower DNA content. The data presented are representative of three independent experiments.

Figure 2. Determination of sub-G1 cells in [8]-shogaol-treated HL-60 cells by flow cytometry

HL-60 cells were treated with (A) different concentration of [8]-shogaol (0, 10, 20, 30, 40, and 50 µM) for 24 h or (B) treated with 30 µM of [8]-shogaol for indicated time. The method of flow cytometry used is descried under Materials and Methods. AP (apoptotic peak) represents apoptotic cells with a lower DNA content. The data presented are representative of three independent experiments.

Figure 3. Induction of mitochondrial dysfunction, reactive oxygen species (ROS) generation, GSH depletion, and cytochrome c release in [8]-shogaol-induced apoptosis

(A) HL-60 cells were treated with 30 µM [8]-shogaol for indicated times and were then incubated with 3, 3'-dihexyloxacarbocyanine (40 nM), DCFH-DA (20 µM), DHE (20 µM), CMFDA (20 µM) respectively and analyzed by flow cytometry. Data are presented as log fluorescence intensity. C: control. (B) Cells were treated with 30 µM [8]-shogaol for 15 min. Subcellular fractions were prepared and cytosolic cytochrome c was analyzed by Western blotting as described in the Material and Methods section. These experiments were performed at least three times, and a representative experiment is presented. *P < 0.05 and **P < 0.01 indicate statistically significant difference from control.

Figure 4. Induction of caspase activities, PARP cleavage, DFF-45 degradation in [8]-shogaol-induced apoptosis in HL-60 cells

(A) [8]-Shogaol induced caspase-9 processing and further caused caspase-3 activation. Total cell lysates were prepared from HL-60 cells treated with 30 µM [8]-shogaol in a time-dependent manner and analyzed by Western blotting. Degradation of pro-caspase protein represents its activation. (B) Kinetics of caspase activation in HL-60 cells. Cells were treated with 30 µM [8]-shogaol for different times. Caspase activities were analyzed as described in the Materials and Methods section. Data represent means ± SD for three determinations. (C) Cleavage of PARP, DFF-45, and D4-GDI induced by [8]-shogaol was time-dependent. HL-60 cells were treated as indicated and analyzed by Western blotting as described in the Materials and Methods. These experiments were performed at least three times, and a representative experiment is presented.

Figure 4. Induction of caspase activities, PARP cleavage, DFF-45 degradation in [8]-shogaol-induced apoptosis in HL-60 cells

(A) [8]-Shogaol induced caspase-9 processing and further caused caspase-3 activation. Total cell lysates were prepared from HL-60 cells treated with 30 µM [8]-shogaol in a time-dependent manner and analyzed by Western blotting. Degradation of pro-caspase protein represents its activation. (B) Kinetics of caspase activation in HL-60 cells. Cells were treated with 30 µM [8]-shogaol for different times. Caspase activities were analyzed as described in the Materials and Methods section. Data represent means ± SD for three determinations. (C) Cleavage of PARP, DFF-45, and D4-GDI induced by [8]-shogaol was time-dependent. HL-60 cells were treated as indicated and analyzed by Western blotting as described in the Materials and Methods. These experiments were performed at least three times, and a representative experiment is presented.

Figure 5. Effect of [8]-shogaol on Bcl-2 family protein, Fas, Fas L, and Bid protein expression in [8]-shogaol-treated HL-60 cells

HL-60 cells were treated with 30 µM [8]-shogaol for indicated time. The expression of Bcl-X_L, Bcl-2, MCl-1, Bag-1, Bad, and Bax (A), Fas, FasL, caspase-8 and Bid (B) were analyzed by Western blotting as described in the Material and Methods. This experiment was repeated three times with similar results.

Figure 6. Schematic representation of mechanisms of action by which [8]-shogaol-induced apoptosis in HL-60 cells