Zerumbone suppresses IKKα, Akt, and FOXO1 activation, resulting in apoptosis of GBM 8401 cells

Abstract

Background: Zerumbone, a sesquiterpene compound isolated from subtropical ginger, Zingiber zerumbet Smith, has been documented to exert antitumoral and anti- inflammatory activities. In this study, we demonstrate that zerumbone induces apoptosis in human glioblastoma multiforme (GBM8401) cells and investigate the apoptotic mechanism.

Methods: We added a caspase inhibitor and transfected wild-type (WT) IKK and Akt into GBM 8401 cells, and measured cell viability and apoptosis by MTT assay and flow cytometry. By western blotting, we evaluated activation of caspase-3, dephosphorylation of IKK, Akt, FOXO1 with time, and change of IKK, Akt, and FOXO1 phosphorylation after transfection of WT IKK and Akt.

Results: Zerumbone (10~50 μM) induced death of GBM8401 cells in a dose-dependent manner. Flow cytometry studies showed that zerumbone increased the percentage of apoptotic GBM cells. Zerumbone also caused caspase-3 activation and poly (ADP-ribose) polymerase (PARP) production. N-benzyloxycarbonyl -Val-Ala-Asp- fluoromethylketone (zVAD-fmk), a broad-spectrum caspase inhibitor, hindered zerumbone-induced cell death. Transfection of GBM 8401 cells with WT IKKα inhibited zerumbone-induced apoptosis, and zerumbone significantly decreased IKKα phosphorylation levels in a time-dependent manner. Similarly, transfection of GBM8401 cells with Akt suppressed zerumbone-induced apoptosis, and zerumbone also diminished Akt phosphorylation levels remarkably and time-dependently. Moreover, transfection of GBM8401 cells with WT IKKα reduced the zerumbone-induced decrease in Akt and FOXO1 phosphorylation. However, transfection with WT Akt decreased FOXO1, but not IKKα, phosphorylation.

Conclusion: The results suggest that inactivation of IKKα, followed by Akt and FOXO1 phosphorylation and caspase-3 activation, contributes to zerumbone-induced GBM cell apoptosis.

Figure 1

Zerumbone induced GBM cell death. (A) U87MG and GBM 8401 cells were treated with DMSO or zerumbone at indicated concentrations for 24 h. Cell viability was then determined by the MTT assay. We used GBM8401 cells for further studies, since zerumbone had a greater effect on cell viability in GBM8401 cells. * p < 0.05, compared with the control group. (B) Cells were treated with DMSO, or zerumbone at indicated concentrations, for 24 h. After treatment, the percentage of sub-G0/G1 contentetric analysis of PI-stained cells as described in Materials and methods. Each column represents the mean ± S.E.M. of at least 3 independent experiments. * p < 0.05, compared with the control group.

Figure 2

Zerumbone evoked caspase-3 activation in GBM cells. (A) GBM8401 cells were pretreated with DMSO or zVAD-fmk (50 or 100 μM) for 30 min before the addition of zerumbone (50 μM) for another 24 h. Cell viability was then determined by the MTT assay. *p < 0.05, compared with the group treated with zerumbone alone. Cells were treated with DMSO or zerumbone (50 μM) for indicated time intervals. Protein levels of procaspase-3 and caspase-3 (B) and PARP (C) were then determined by immunoblotting. Typical traces, representive of data from 3 independent experiments with similar results, are shown.

Figure 3

Zerumbone suppressed IKKα phosphorylation in GBM8401 cells. (A) Cells were transiently transfected with pcDNA (control vector), IKKα or IKKβ and HA for 24 h and then were treated with zerumbone (50 μM) for another 24 h before harvesting. Cell viability was then determined by the MTT assay and western blotting. * p < 0.05, compared with the pc DNA-transfected group in the presence of zerumbone. (B) Cells were treated with 50 μM zerumbone for the indicated time intervals. IKKα/βphosphorylation was then determined by immunoblotting. Each column represents the mean ± S.E.M. of at least three independent experiments. *p < 0.05, compared with the control group.

Figure 4

Akt in zerumbone-induced GBM cell apoptosis. (A) Cells were transfected with empty vector (mock) or wild-type Akt (WT-Akt) for 24 h. Following transfection, cells were treated with vehicle or 50 μM zerumbone for 24 h. Cell viability was then determined by the MTT assay and immunoblotting. Under overexpression of Akt, the phosphorylation level of Akt also increased compared to the mock group, suggesting Akt is functional in GBM8401 cells. Each column represents the mean ± S.E.M. of at least 3 independent experiments. *p < 0.05, compared with the group with trasfected with the empty vector, in the presence of zerumbone. (B) Cells were treated with vehicle or zerumbone (50 μM) for indicated time intervals. Phosphorylation status of Akt was then determined by immunoblotting. Each of the columns represents the mean ± S.E.M. of at least three independent experiments. * p < 0.05, compared with the control group.

Figure 5

The link between IKK and Akt in zerumbone-induced apoptosis. (A) Cells were transfected with pcDNA or IKKα forκ 48 h. After transfection, cells were treated with vehicle or 50 μM zerumbone for another 1 h. The phosphorylation of Akt was then determined by immunoblotting. Each column represents the mean ± S.E.M. of at least 3 independent experiments. *p < 0.05, compared with the group trasfected with pcDNA, in the presence of zerumbone. (B) GBM cells then were transfected with WT Akt for 48 h. Then the cells were treated with zerumbone for 1 h and the phosphorylation of IKKα and IKKβwas measured by immunoblotting. There was no significant difference of phosphorylation of IKKα and IKKβ between cells transfected with empty vector or with WT Akt before treatment with zerumbone.

Figure 6

Zerumbone- induced FOXO1 dephosphorylation in GBM cells. (A) Cells were treated with 50 μM zerumbone for indicated time intervals. FOXO1 phosphorylation status was then evaluated by immunoblotting. Each column represents the mean ± S.E.M. of at least 3 independent experiments. *p <0.05, compared with the control group. (B) Cells were transfected with pcDNA or WT IKK for 48 h. After transfection, cells were treated with vehicle or 50 μM zerumbone for 1 h. The phosphorylation status of FOXO1 was then determined by immunoblotting. Each column represents the mean ± S.E.M. of at least three independent experiments. *p < 0.05, compared with the group tranfected with pcDNA, in the presence of zerumbone. (C) Cells were transfected with empty vector (mock) and WT Akt. Then cells were treated with vehicle or 50 μM zerumbone and FOXO1 phosphorylation level was measured by immunoblotting. Each column represents the mean ± S.E.M. of at least 3 independent experiments. *p < 0.05, compared with empty vector, in the presence of zerumbone.

Figure 7

Schematic summary of apoptotic pathway invoked in zerumbone-induced apoptosis of GBM8401 cell. Zerumbone-induced inactivation of IKKα leads to FOXO1 dephosphorylation, via Akt dephosphorylation or not, then causing caspase-3 activation, and subsequent cell apoptosis.