Ebselen attenuates oxidative stress-induced apoptosis via the inhibition of the c-Jun N-terminal kinase and activator protein-1 signalling pathway in PC12 cells

Abstract

1: Ebselen (2-phenyl-1,2-benzisoselenazol-3[2H]-one) is a selenoorganic compound exhibiting both glutathione peroxidase activity and antioxidant activity. Although it has been reported that ebselen is effective for oxidative stress-induced neuronal damage both in vivo and clinically, the precise mechanisms of the efficacy have not yet been elucidated. Thus, we hypothesized that ebselen may affect reactive oxygen species-induced mitogen-activated protein (MAP) kinase activation in cultured PC12 cells. 2: Our findings showed that hydrogen peroxide (H(2)O(2)) stimulated rapid and significant activation of extracellular signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) and p38 in PC12 cells, which is a model of catecholamine-containing neurons. 3: H(2)O(2)-induced JNK activation was inhibited by ebselen, whereas ERK1/2 and p38 activation by H(2)O(2) were not affected by ebselen. 4: Inhibition by ebselen of H(2)O(2)-induced hydroxyl radical generation in PC12 cells was observed using electron paramagnetic resonance measurements. Ebselen also inhibited H(2)O(2)-induced increases in DNA binding activity of activator protein-1 (AP-1), a downstream transcription factor of JNK, composed of the c-Jun homo/heterodimer. 5: Finally, pretreatment of cells with ebselen resulted in a significant recovery from cell death including apoptosis by H(2)O(2) in PC12 cells. 6 These findings suggest that ebselen attenuates oxidative stress-induced neuronal cell death through the inhibition of the JNK and AP-1 signalling pathway. Thus, inhibition of JNK by ebselen may imply its usefulness for treatment of ischaemic cerebral diseases relevant to neuronal cell death.

Figure 1

Time course of H₂O₂-induced ERK 1/2, JNK and p38 activation in PC12 cells. Cells were stimulated with 300 μM H₂O₂ for the indicated periods of time. Cells were harvested, lysed, and used for subsequent analysis. The activities of ERK1/2, JNK and p38 were measured as described in Methods. (A) Representative blots are shown. (B) Densitometric analysis of ERK 1/2 (sum of the ERK1 and ERK2 bands), JNK and p38 activation. Values were normalized by arbitrarily setting the densitometry of control cells (time=0) to 1.0 (values are the means±s.d., n=5). No obvious MAP kinase activation was observed in vehicle-treated samples. Two-way ANOVA detected significant differences between groups with or without H₂O₂ treatment: ERK1/2 (F=65.859, P<0.0001, d.f.=5), JNK (F=109.645, P<0.0001, d.f.=5) and p38 (F=68.570, P<0.0001, d.f.=5). *P<0.05 when compared with vehicle-treated groups at each time point (ANOVA followed by Scheffe's test).

Figure 2

H₂O₂ stimulates a concentration-dependent increase in ERK 1/2, JNK and p38 activities in PC12 cells. Cells were stimulated with the indicated concentrations of H₂O₂ for 5 min for ERK1/2 activation and for 10 min for JNK and p38 activation. Cells were harvested, lysed, and used for subsequent analysis. The activities of ERK1/2, JNK and p38 were measured as described in Methods. (A) Representative blots are shown. (B) Densitometric analysis of ERK1/2 (sum of the ERK1 and ERK2 bands), JNK and p38 activation. Values were normalized by arbitrarily setting the densitometry of control cells (without H₂O₂) to 1.0 (values are the means±s.d., n=5). No obvious MAP kinase activation was observed in vehicle-treated samples. Two-way ANOVA detected significant differences between groups with or without H₂O₂ treatment: ERK1/2 (F=129.504, P<0.0001, d.f.=5), JNK (F=107.909, P<0.0001, d.f.=5) and p38 (F=74.18, P<0.0001, d.f.=5). *P<0.05 when compared with vehicle-treated groups at each concentration (ANOVA followed by Scheffe's test).

Figure 3

Inhibition by ebselen of H₂O₂-induced JNK activation in a concentration-dependent manner, but not ERK1/2 and p38 activation in PC12 cells. Cells were pretreated with ebselen at the indicated concentrations for 30 min. Controls with 0 μM ebselen represent vehicle-treated samples. Then the cells were stimulated with 300 μM H₂O₂ for 5 min for ERK activity and 10 min for JNK and p38 activities. Cells were harvested, lysed, and used for subsequent analysis. The activities of ERK1/2, JNK and p38 were measured as described in Methods. (A) Representative blots are shown. (B) Densitometric analysis of the effect of ebselen on ERK1/2, JNK and p38 activation. Values are expressed as % of controls which was defined from each MAP kinase activity stimulated by 300 μM H₂O₂ (values are the means±s.d., n=5). Two-way ANOVA detected significant differences between groups with or without H₂O₂ treatment for JNK (F=77.746, P<0.0001, d.f.=3), but not for ERK1/2 (F=3.104, P=0.0562, d.f.=3) and p38 (F=0.849, P=0.4873, d.f.=3). *P<0.05 when compared with H₂O₂-treated groups (ANOVA followed by Scheffe's test).

Figure 4

EPR spectra of DMPO/^·OH adduct from PC12 cells by H₂O₂ stimulation and its dismutation by ebselen. (A) a: computer simulation of DMPO/^·OH adduct EPR spectra; b: PC12 cells (1×10⁶ cells ml⁻¹) with 0.9 M DMPO; c: same as b, but with ebselen (10 μM); d: same as b, but with H₂O₂ (300 μM) and e: same as b, but with ebselen (10 μM) pretreatment for 5 min and following H₂O₂ (300 μM) stimulation. To avoid interference from the extracellular EPR signal of H₂O₂, an excess amount of catalase (100 units) was added to the incubation medium immediately before measurements. (B) Relative signal intensity was defined as a relative peak height of the second EPR signal of the DMPO/^·OH adduct with the first EPR signal of the external standard, MnO. Values are the means±s.d. of five separate experiments done in duplicate. Two-way ANOVA detected significant differences among groups (F=148.879, P<0.0001, d.f.=3). *P<0.05 when compared with H₂O₂-treated groups (ANOVA followed by Scheffe's test).

Figure 5

Time course for the activation of AP-1 DNA binding from PC12 cell nuclear extracts stimulated with 300 μM H₂O₂ (A) and its inhibition by ebselen (B). (A) The bracket in the panel indicates PC12 cell nuclear extract AP-1 DNA binding complexes induced by H₂O₂ (300 μM) stimulation at the indicated time points. A competition assay for AP-1 was carried out in the presence of a 100 fold molar excess of unlabelled AP-1 oligonucleotide (competitor). Supershift analysis was performed with specific pan-c-Jun antibody (SS). F, free probe. (B) Effects of ebselen on H₂O₂-induced increase in AP-1 DNA binding activity from PC12 cell nuclear extracts. For the experiments of ebselen against H₂O₂-induced increase in AP-1 DNA binding activity, different concentrations of ebselen were added to the incubation medium 30 min prior to H₂O₂ (300 μM) stimulation for 4 h. Representative blots from three separate experiments are shown.

Figure 6

H₂O₂-induced PC12 cell death and its inhibition by ebselen. Cell viability was evaluated with MTT reduction as described in Methods. Cells were treated with or without 300 μM H₂O₂ for 24 h, then assayed. Ebselen at the indicated concentrations was added to the incubation medium 30 min prior to H₂O₂ stimulation. Values are the means±s.d. of five experiments preformed in triplicate. Two-way ANOVA detected significant differences among groups (F=46.247, P<0.0001, d.f.=5). The asterisks represent significant differences between the groups (*P<0.05, ANOVA followed by Scheffe's test).

Figure 7

Ebselen inhibits H₂O₂-induced PC12 cell apoptosis assessed by apoptotic nuclei staining with Hoechst 33248 dye (A) and agarose gel electrophoretic analysis of DNA fragmentation (B) as described in Methods. Arrowheads indicate the typical apoptotic nuclei and the bar represents 20 μm in (A). Cells were treated with or without 300 μM H₂O₂ for 24 h. Ebselen (10 μM) was added to the incubation medium 30 min prior to H₂O₂ stimulation. M, marker (B). Representative photographs from three separate experiments are shown in both (A) and (B).