Gingerol sensitizes TRAIL-induced apoptotic cell death of glioblastoma cells - PubMed (original) (raw)

Gingerol sensitizes TRAIL-induced apoptotic cell death of glioblastoma cells

Dae-Hee Lee et al. Toxicol Appl Pharmacol. 2014.

Abstract

Glioblastoma multiforme (GBM) is the most lethal and aggressive astrocytoma of primary brain tumors in adults. Although there are many clinical trials to induce the cell death of glioblastoma cells, most glioblastoma cells have been reported to be resistant to TRAIL-induced apoptosis. Here, we showed that gingerol as a major component of ginger can induce TRAIL-mediated apoptosis of glioblastoma. Gingerol increased death receptor (DR) 5 levels in a p53-dependent manner. Furthermore, gingerol decreased the expression level of anti-apoptotic proteins (survivin, c-FLIP, Bcl-2, and XIAP) and increased pro-apoptotic protein, Bax and truncate Bid, by generating reactive oxygen species (ROS). We also found that the sensitizing effects of gingerol in TRAIL-induced cell death were blocked by scavenging ROS or overexpressing anti-apoptotic protein (Bcl-2). Therefore, we showed the functions of gingerol as a sensitizing agent to induce cell death of TRAIL-resistant glioblastoma cells. This study gives rise to the possibility of applying gingerol as an anti-tumor agent that can be used for the purpose of combination treatment with TRAIL in TRAIL-resistant glioblastoma tumor therapy.

Keywords: Apoptosis; Gingerol; Glioblastoma; Reactive oxygen species (ROS); Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL); p53.

Copyright © 2014 Elsevier Inc. All rights reserved.

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Conflict of interest statement

Conflict of interest statement: The authors declare that they have no competing interests.

Figures

Fig. 1

Fig. 1

Sensitizing effect of gingerol in TRAIL-induced apoptosis of U87 glioblastoma cells. (A and B) Cells were treated with DMSO (sham control) or various concentrations (10–100μM) of gingerol for 24 h. (C, D, and E) Cells were incubated in the presence or absence of TRAIL (50 ng/ml) and/or gingerol (25 μM) for 24 h. (F) Microscopic cell morphologies. Scale bar: 100 μm. (G) The cells were stained with annexin V and propidium iodide (PI), followed by FACS analysis. (H) The cell lysates were analyzed by western blotting using indicated antibodies. Data is presented as arbitrary values and results are expressed as the mean ± SEM. The effects of nicotine were determined using a Student's unpaired _t_-test. Asterisk * represents statistically significantly difference between control and gingerol-treated cells at p < 0.05.

Fig. 1

Fig. 1

Sensitizing effect of gingerol in TRAIL-induced apoptosis of U87 glioblastoma cells. (A and B) Cells were treated with DMSO (sham control) or various concentrations (10–100μM) of gingerol for 24 h. (C, D, and E) Cells were incubated in the presence or absence of TRAIL (50 ng/ml) and/or gingerol (25 μM) for 24 h. (F) Microscopic cell morphologies. Scale bar: 100 μm. (G) The cells were stained with annexin V and propidium iodide (PI), followed by FACS analysis. (H) The cell lysates were analyzed by western blotting using indicated antibodies. Data is presented as arbitrary values and results are expressed as the mean ± SEM. The effects of nicotine were determined using a Student's unpaired _t_-test. Asterisk * represents statistically significantly difference between control and gingerol-treated cells at p < 0.05.

Fig. 2

Fig. 2

Gingerol can regulate the expression of death receptors (DR) 5, but not DR4. (A) U87 cells were treated with indicated gingerol doses (0, 10, 25, 50 μM) for 24 h or 25 μM gingerol at an indicated time (0, 4, 8, 16, 24 h). Western blotting analysis was performed with DR4 and DR5 antibodies. (B) U87 cells were incubated with DMSO or gingerol (25 μM) for 24 h and stained with DR4 and DR5 antibodies, followed by FACS analysis (left panels) and immunocytochemistry (right panels). Scale bar: 100 μm. (C) The indicated cell types were incubated with DMSO or gingerol (25 μM) for 24 h. The cell lysates were analyzed by western blotting using DR4 and DR5 antibodies. (D) U87 cells were transfected with Myc-DR5 plasmid or pcDNA (control). After treatment with TRAIL for 4 h, western blot analysis demonstrated that Myc-DR5-induced DR5 overexpression increased apoptosis compared with pcDNA. (E) U87 cells were incubated with indicated gingerol doses (0, 10, 25, 50 μM) for 24 h or 25 μM gingerol at an indicated time (0, 4, 8, 16, 24 h) and then analyzed by western blotting using p53 antibody. (F) HCT116 (p53+/+) and HCT116 (p53−/−) cells were incubated with indicated gingerol (0, 10, 25, 50 μM) for 24 h. The cell lysates were analyzed by western blotting using p53, DR4, and DR5 antibodies. Data is presented as arbitrary values and results are expressed as the mean ± SEM. The effects of nicotine were determined using a Student's unpaired _t_-test. *p < 0.05 vs control. (G) Cell viability was determined using the trypan blue dye exclusion assay. Error bars represent the mean ± SE from six separate experiments. *Significant difference between TRAIL and TRAIL + gingerol-treated cells at p < 0.05. These results are representative of data obtained from at least five independent experiments.

Fig. 2

Fig. 2

Gingerol can regulate the expression of death receptors (DR) 5, but not DR4. (A) U87 cells were treated with indicated gingerol doses (0, 10, 25, 50 μM) for 24 h or 25 μM gingerol at an indicated time (0, 4, 8, 16, 24 h). Western blotting analysis was performed with DR4 and DR5 antibodies. (B) U87 cells were incubated with DMSO or gingerol (25 μM) for 24 h and stained with DR4 and DR5 antibodies, followed by FACS analysis (left panels) and immunocytochemistry (right panels). Scale bar: 100 μm. (C) The indicated cell types were incubated with DMSO or gingerol (25 μM) for 24 h. The cell lysates were analyzed by western blotting using DR4 and DR5 antibodies. (D) U87 cells were transfected with Myc-DR5 plasmid or pcDNA (control). After treatment with TRAIL for 4 h, western blot analysis demonstrated that Myc-DR5-induced DR5 overexpression increased apoptosis compared with pcDNA. (E) U87 cells were incubated with indicated gingerol doses (0, 10, 25, 50 μM) for 24 h or 25 μM gingerol at an indicated time (0, 4, 8, 16, 24 h) and then analyzed by western blotting using p53 antibody. (F) HCT116 (p53+/+) and HCT116 (p53−/−) cells were incubated with indicated gingerol (0, 10, 25, 50 μM) for 24 h. The cell lysates were analyzed by western blotting using p53, DR4, and DR5 antibodies. Data is presented as arbitrary values and results are expressed as the mean ± SEM. The effects of nicotine were determined using a Student's unpaired _t_-test. *p < 0.05 vs control. (G) Cell viability was determined using the trypan blue dye exclusion assay. Error bars represent the mean ± SE from six separate experiments. *Significant difference between TRAIL and TRAIL + gingerol-treated cells at p < 0.05. These results are representative of data obtained from at least five independent experiments.

Fig. 2

Fig. 2

Gingerol can regulate the expression of death receptors (DR) 5, but not DR4. (A) U87 cells were treated with indicated gingerol doses (0, 10, 25, 50 μM) for 24 h or 25 μM gingerol at an indicated time (0, 4, 8, 16, 24 h). Western blotting analysis was performed with DR4 and DR5 antibodies. (B) U87 cells were incubated with DMSO or gingerol (25 μM) for 24 h and stained with DR4 and DR5 antibodies, followed by FACS analysis (left panels) and immunocytochemistry (right panels). Scale bar: 100 μm. (C) The indicated cell types were incubated with DMSO or gingerol (25 μM) for 24 h. The cell lysates were analyzed by western blotting using DR4 and DR5 antibodies. (D) U87 cells were transfected with Myc-DR5 plasmid or pcDNA (control). After treatment with TRAIL for 4 h, western blot analysis demonstrated that Myc-DR5-induced DR5 overexpression increased apoptosis compared with pcDNA. (E) U87 cells were incubated with indicated gingerol doses (0, 10, 25, 50 μM) for 24 h or 25 μM gingerol at an indicated time (0, 4, 8, 16, 24 h) and then analyzed by western blotting using p53 antibody. (F) HCT116 (p53+/+) and HCT116 (p53−/−) cells were incubated with indicated gingerol (0, 10, 25, 50 μM) for 24 h. The cell lysates were analyzed by western blotting using p53, DR4, and DR5 antibodies. Data is presented as arbitrary values and results are expressed as the mean ± SEM. The effects of nicotine were determined using a Student's unpaired _t_-test. *p < 0.05 vs control. (G) Cell viability was determined using the trypan blue dye exclusion assay. Error bars represent the mean ± SE from six separate experiments. *Significant difference between TRAIL and TRAIL + gingerol-treated cells at p < 0.05. These results are representative of data obtained from at least five independent experiments.

Fig. 3

Fig. 3

Modulation of gingerol in the pro-apoptotic and anti-apoptotic signaling pathways in glioblastoma cells. U87 cells were treated with indicated gingerol doses (0, 10, 25, 50 μM) for 24 h. (A) Western blotting analysis was done by using indicated antibodies (XIAP, Survivin, c-FLIP, Bax, Bcl-2, and Bid). (B) Phosphorylation of MAP kinases was analyzed by western blotting using phosphorylation-specific antibodies (p-ERK, p-p38, and p-JNK). (C) U343 and T98G cells were treated with indicated gingerol doses (0, 10, 25, 50 μM) for 24 h, followed by western blotting using Bcl-2 antibody. Data is presented as arbitrary values and results are expressed as the mean ± SEM. The effects of nicotine were determined using a Student's unpaired _t_-test. *p < 0.05 vs control. These results are representative of data obtained from at least five independent experiments.

Fig. 4

Fig. 4

Gingerol can regulate the pro-apoptotic signaling of TRAIL through ROS generation. U87 cells were treated with gingerol (25 μM) in the presence or absence of N-acetylcysteine (Lluis et al., 2010) for 24 h, and then incubated with dihydroethidium (DHE) or 2′,7′-dichlorofluorescein (H2DCF), followed by (A) FACS analysis and (B) immunocytochemical analysis. Scale bar: 100 μm. (C) The cells were incubated with indicated drugs (25 μM gingerol and 5 mM NAC) for 24 h, and then western blotting was performed by using the indicated antibodies (Survivin, c-FLIP, BAX, Bcl-2, and Bid). (D) The cells were treated with indicated reagents (TRAIL, gingerol and NAC) for 24 h. The cell lysates were analyzed by western blotting using indicated antibodies (caspase-3,-8 and PARP-1). Data is presented as arbitrary values and results are expressed as the mean ±SEM. The effects of nicotine were determined using a Student's unpaired _t_-test. *p < 0.05 vs control. These results are representative of data obtained from at least five independent experiments.

Fig. 4

Fig. 4

Gingerol can regulate the pro-apoptotic signaling of TRAIL through ROS generation. U87 cells were treated with gingerol (25 μM) in the presence or absence of N-acetylcysteine (Lluis et al., 2010) for 24 h, and then incubated with dihydroethidium (DHE) or 2′,7′-dichlorofluorescein (H2DCF), followed by (A) FACS analysis and (B) immunocytochemical analysis. Scale bar: 100 μm. (C) The cells were incubated with indicated drugs (25 μM gingerol and 5 mM NAC) for 24 h, and then western blotting was performed by using the indicated antibodies (Survivin, c-FLIP, BAX, Bcl-2, and Bid). (D) The cells were treated with indicated reagents (TRAIL, gingerol and NAC) for 24 h. The cell lysates were analyzed by western blotting using indicated antibodies (caspase-3,-8 and PARP-1). Data is presented as arbitrary values and results are expressed as the mean ±SEM. The effects of nicotine were determined using a Student's unpaired _t_-test. *p < 0.05 vs control. These results are representative of data obtained from at least five independent experiments.

Fig. 5

Fig. 5

Effect of Bcl-2 in the sensitizing function of gingerol. (A) Vector or Bcl-2-overexpressing U87 stable cell lines (upper panel) were treated with TRAIL and gingerol for 24 h. The cell lysates were analyzed by western blotting of Flag antibody. Actin was used to confirm the equal amount of proteins loaded in each lane. All results are representative of the data obtained by five independent experiments. The cells were then analyzed by MTT assay. (B) Bcl-2 was silenced by si-RNA in U87 cells (upper panel). The cells were then treated with TRAIL for 24 h, followed by MTT analysis. Results shown are representative of six independent experiments.

Fig. 6

Fig. 6

Astrocytes are resistant to the combined treatment with gingerol and TRAIL. (A) Human astrocytes were treated with or without gingerol for 30 min and further treated with TRAIL for 16 h at the indicated concentrations. Cellular viability was assessed using trypan blue exclusion assay. (B) Following treatment of astrocytes or U87MG cells with 25 μM gingerol for the indicated times, western blotting of DR5 and Bcl-2 antibodies. Actin was used to confirm the equal amount of proteins loaded in each lane. (C) Astrocytes or U87MG cells were treated with 25 μM gingerol alone, 100 ng/ml TRAIL alone or a combination with gingerol and TRAIL for 16 h. Caspase-3/7 activity is shown. Data is presented as arbitrary values and results are expressed as the mean ± SEM. The effects of nicotine were determined using a Student's unpaired _t_-test. *p < 0.05 vs control. Results shown are representative of six independent experiments.

Fig. 7

Fig. 7

Schematic diagram of working model of gingerol for sensitizing TRAIL-induced apoptosis.

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