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ROS-dependent phosphorylation of Bax by wortmannin sensitizes melanoma cells for TRAIL-induced apoptosis.

Quast SA, Berger A, Eberle J - Cell Death Dis (2013)

Bottom Line: Thus, wortmannin resulted in early reduction of the Bax-inactivating phosphorylation at serine-184, whereas the Bax-activating phosphorylation at threonine-167 was enhanced.Proving the responsibility of the pathway, comparable effects were obtained with an Akt inhibitor (MK-2206); while suppressed phosphorylation of serine-184 may be attributed to reduced Akt activity itself, the causes of enhanced threonine-167 phosphorylation were addressed here.This correlated with reduced Bax phosphorylation at threonine-167.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Dermatology and Allergy, Skin Cancer Center, University Medical Center Charité, Berlin, Germany [2] Institute for Chemistry and Biochemistry, Free University of Berlin, Berlin, Germany.

ABSTRACT
The pathways of reactive oxygen species (ROS)-mediated apoptosis induction, of Bax activation and the sensitization of tumor cells for TRAIL (TNF-related apoptosis-inducing ligand)-induced apoptosis are still largely elusive. Here, sensitization of melanoma cells for TRAIL by the PI3-kinase inhibitor wortmannin correlated to the activation of mitochondrial apoptosis pathways. Apoptosis was dependent on Bax and abrogated by Bcl-2 overexpression. The synergistic enhancement was explained by Bax activation through wortmannin, which tightly correlated to the characteristic Bax phosphorylation patterns. Thus, wortmannin resulted in early reduction of the Bax-inactivating phosphorylation at serine-184, whereas the Bax-activating phosphorylation at threonine-167 was enhanced. Proving the responsibility of the pathway, comparable effects were obtained with an Akt inhibitor (MK-2206); while suppressed phosphorylation of serine-184 may be attributed to reduced Akt activity itself, the causes of enhanced threonine-167 phosphorylation were addressed here. Characteristically, production of ROS was seen early in response to wortmannin and MK-2206. Providing the link between ROS and Bax, we show that abrogated ROS production by α-tocopherol or by NADPH oxidase 4 (NOX4) siRNA suppressed apoptosis and Bax activation. This correlated with reduced Bax phosphorylation at threonine-167. The data unraveled a mechanism by which NOX4-dependent ROS production controls apoptosis via Bax phosphorylation. The pathway may be considered for proapoptotic, anticancer strategies.

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Role of Bax. (a) Apoptosis by wortmannin/TRAIL was investigated in HCT-116 parental cells (Bax+, Bak+) and in subclones with knockdown for Bax and/or Bak. Statistical significance of reduced apoptosis in treated subclones is indicated (*P<0.005). Expression of Bax and Bak in the respective cell clones is shown in the inset. (b) Apoptosis (% of sub-G1 cells) and cytotoxicity (% LDH release) are shown after siRNA-mediated Bax knockdown (24 h) in A-375 and Mel-HO treated for 24 h with TRAIL (20 ng/ml)+/− wortmannin (4 μM). The respective mock control (Si-Ctrl) is shown for comparison (Mean values and SDs of two independent experiments, each one with triplicates). Statistical significance is indicated (*P<0.005), when comparing si-Bax and si-Ctrl-transfected cells. Knockdown of Bax is shown by western blotting in insets. Equal protein loading (30 μg per lane) was proven by GAPDH. (c) The mitochondrial Δψm was determined in indicated cell clones treated with wortmannin (4 μM) or wortmannin/TRAIL for 2 h. Treated cells (open graphs) were compared to DMSO controls (gray). (d) ROS levels were determined in HCT-116 parental cells and in subclones with knockdown for Bax and/or Bak treated with wortmannin (4 μM) or wortmannin/TRAIL for 2 h. In each case, three independent experiments with triplicates showed highly comparable results. (e) The mitochondrial membrane potential (Δψm) was determined in A-375, A-375-TS and Mel-HO treated with TRAIL (20 ng/ml) and/or wortmannin (4 μM) for 2 h. Treated cells (open graphs) were compared to DMSO controls (gray). Before, cells were transfected with Bax siRNA (Si-Bax) or the respective control RNA (Si-Ctrl). (f) Mitochondrial extracts (Mito) of A-375 and A-375-TS treated for 2 h with TRAIL +/− wortmannin (4 μM) were analyzed for Bax translocation by western blotting. Equal loading was proven by the mitochondrial protein VDAC; a cytosolic extract is shown for control (Cyto). (g) A-375 and A-375-TS cells were treated for 1–8 h with wortmannin (4 μM) or with wortmannin/TRAIL followed by flow cytometry analysis for Bax conformational changes (Bax-NT antibody; two independent experiments with triplicates). (h) A-375-pIRES and A-375-Bcl-2 cells were treated for 2 h with wortmannin (4 μM)+/− TRAIL (20 ng/ml) followed by flow cytometry analysis for Bax conformational changes (Bax-NT antibody; two independent experiments with triplicates)
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fig5: Role of Bax. (a) Apoptosis by wortmannin/TRAIL was investigated in HCT-116 parental cells (Bax+, Bak+) and in subclones with knockdown for Bax and/or Bak. Statistical significance of reduced apoptosis in treated subclones is indicated (*P<0.005). Expression of Bax and Bak in the respective cell clones is shown in the inset. (b) Apoptosis (% of sub-G1 cells) and cytotoxicity (% LDH release) are shown after siRNA-mediated Bax knockdown (24 h) in A-375 and Mel-HO treated for 24 h with TRAIL (20 ng/ml)+/− wortmannin (4 μM). The respective mock control (Si-Ctrl) is shown for comparison (Mean values and SDs of two independent experiments, each one with triplicates). Statistical significance is indicated (*P<0.005), when comparing si-Bax and si-Ctrl-transfected cells. Knockdown of Bax is shown by western blotting in insets. Equal protein loading (30 μg per lane) was proven by GAPDH. (c) The mitochondrial Δψm was determined in indicated cell clones treated with wortmannin (4 μM) or wortmannin/TRAIL for 2 h. Treated cells (open graphs) were compared to DMSO controls (gray). (d) ROS levels were determined in HCT-116 parental cells and in subclones with knockdown for Bax and/or Bak treated with wortmannin (4 μM) or wortmannin/TRAIL for 2 h. In each case, three independent experiments with triplicates showed highly comparable results. (e) The mitochondrial membrane potential (Δψm) was determined in A-375, A-375-TS and Mel-HO treated with TRAIL (20 ng/ml) and/or wortmannin (4 μM) for 2 h. Treated cells (open graphs) were compared to DMSO controls (gray). Before, cells were transfected with Bax siRNA (Si-Bax) or the respective control RNA (Si-Ctrl). (f) Mitochondrial extracts (Mito) of A-375 and A-375-TS treated for 2 h with TRAIL +/− wortmannin (4 μM) were analyzed for Bax translocation by western blotting. Equal loading was proven by the mitochondrial protein VDAC; a cytosolic extract is shown for control (Cyto). (g) A-375 and A-375-TS cells were treated for 1–8 h with wortmannin (4 μM) or with wortmannin/TRAIL followed by flow cytometry analysis for Bax conformational changes (Bax-NT antibody; two independent experiments with triplicates). (h) A-375-pIRES and A-375-Bcl-2 cells were treated for 2 h with wortmannin (4 μM)+/− TRAIL (20 ng/ml) followed by flow cytometry analysis for Bax conformational changes (Bax-NT antibody; two independent experiments with triplicates)

Mentions: The roles of Bax and Bak were distinguished in a HCT-116 cell culture model that consisted of Bax+Bak+ parental, Bax knockout, Bak knockdown and double knockdown cells. TRAIL/wortmannin significantly induced apoptosis at 24 h in parental and Bak knockdown cells, demonstrating that Bak played no role in this setting. In clear contrast, Bax knockout completely abrogated TRAIL- and TRAIL/wortmannin-induced apoptosis (Figure 5a). Also, in A-375 and Mel-HO melanoma cells, Bax knockdown by siRNA abrogated TRAIL- and TRAIL/wortmannin-induced apoptosis. Again, cytotoxicity played no role (Figure 5b).


ROS-dependent phosphorylation of Bax by wortmannin sensitizes melanoma cells for TRAIL-induced apoptosis.

Quast SA, Berger A, Eberle J - Cell Death Dis (2013)

Role of Bax. (a) Apoptosis by wortmannin/TRAIL was investigated in HCT-116 parental cells (Bax+, Bak+) and in subclones with knockdown for Bax and/or Bak. Statistical significance of reduced apoptosis in treated subclones is indicated (*P<0.005). Expression of Bax and Bak in the respective cell clones is shown in the inset. (b) Apoptosis (% of sub-G1 cells) and cytotoxicity (% LDH release) are shown after siRNA-mediated Bax knockdown (24 h) in A-375 and Mel-HO treated for 24 h with TRAIL (20 ng/ml)+/− wortmannin (4 μM). The respective mock control (Si-Ctrl) is shown for comparison (Mean values and SDs of two independent experiments, each one with triplicates). Statistical significance is indicated (*P<0.005), when comparing si-Bax and si-Ctrl-transfected cells. Knockdown of Bax is shown by western blotting in insets. Equal protein loading (30 μg per lane) was proven by GAPDH. (c) The mitochondrial Δψm was determined in indicated cell clones treated with wortmannin (4 μM) or wortmannin/TRAIL for 2 h. Treated cells (open graphs) were compared to DMSO controls (gray). (d) ROS levels were determined in HCT-116 parental cells and in subclones with knockdown for Bax and/or Bak treated with wortmannin (4 μM) or wortmannin/TRAIL for 2 h. In each case, three independent experiments with triplicates showed highly comparable results. (e) The mitochondrial membrane potential (Δψm) was determined in A-375, A-375-TS and Mel-HO treated with TRAIL (20 ng/ml) and/or wortmannin (4 μM) for 2 h. Treated cells (open graphs) were compared to DMSO controls (gray). Before, cells were transfected with Bax siRNA (Si-Bax) or the respective control RNA (Si-Ctrl). (f) Mitochondrial extracts (Mito) of A-375 and A-375-TS treated for 2 h with TRAIL +/− wortmannin (4 μM) were analyzed for Bax translocation by western blotting. Equal loading was proven by the mitochondrial protein VDAC; a cytosolic extract is shown for control (Cyto). (g) A-375 and A-375-TS cells were treated for 1–8 h with wortmannin (4 μM) or with wortmannin/TRAIL followed by flow cytometry analysis for Bax conformational changes (Bax-NT antibody; two independent experiments with triplicates). (h) A-375-pIRES and A-375-Bcl-2 cells were treated for 2 h with wortmannin (4 μM)+/− TRAIL (20 ng/ml) followed by flow cytometry analysis for Bax conformational changes (Bax-NT antibody; two independent experiments with triplicates)
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fig5: Role of Bax. (a) Apoptosis by wortmannin/TRAIL was investigated in HCT-116 parental cells (Bax+, Bak+) and in subclones with knockdown for Bax and/or Bak. Statistical significance of reduced apoptosis in treated subclones is indicated (*P<0.005). Expression of Bax and Bak in the respective cell clones is shown in the inset. (b) Apoptosis (% of sub-G1 cells) and cytotoxicity (% LDH release) are shown after siRNA-mediated Bax knockdown (24 h) in A-375 and Mel-HO treated for 24 h with TRAIL (20 ng/ml)+/− wortmannin (4 μM). The respective mock control (Si-Ctrl) is shown for comparison (Mean values and SDs of two independent experiments, each one with triplicates). Statistical significance is indicated (*P<0.005), when comparing si-Bax and si-Ctrl-transfected cells. Knockdown of Bax is shown by western blotting in insets. Equal protein loading (30 μg per lane) was proven by GAPDH. (c) The mitochondrial Δψm was determined in indicated cell clones treated with wortmannin (4 μM) or wortmannin/TRAIL for 2 h. Treated cells (open graphs) were compared to DMSO controls (gray). (d) ROS levels were determined in HCT-116 parental cells and in subclones with knockdown for Bax and/or Bak treated with wortmannin (4 μM) or wortmannin/TRAIL for 2 h. In each case, three independent experiments with triplicates showed highly comparable results. (e) The mitochondrial membrane potential (Δψm) was determined in A-375, A-375-TS and Mel-HO treated with TRAIL (20 ng/ml) and/or wortmannin (4 μM) for 2 h. Treated cells (open graphs) were compared to DMSO controls (gray). Before, cells were transfected with Bax siRNA (Si-Bax) or the respective control RNA (Si-Ctrl). (f) Mitochondrial extracts (Mito) of A-375 and A-375-TS treated for 2 h with TRAIL +/− wortmannin (4 μM) were analyzed for Bax translocation by western blotting. Equal loading was proven by the mitochondrial protein VDAC; a cytosolic extract is shown for control (Cyto). (g) A-375 and A-375-TS cells were treated for 1–8 h with wortmannin (4 μM) or with wortmannin/TRAIL followed by flow cytometry analysis for Bax conformational changes (Bax-NT antibody; two independent experiments with triplicates). (h) A-375-pIRES and A-375-Bcl-2 cells were treated for 2 h with wortmannin (4 μM)+/− TRAIL (20 ng/ml) followed by flow cytometry analysis for Bax conformational changes (Bax-NT antibody; two independent experiments with triplicates)
Mentions: The roles of Bax and Bak were distinguished in a HCT-116 cell culture model that consisted of Bax+Bak+ parental, Bax knockout, Bak knockdown and double knockdown cells. TRAIL/wortmannin significantly induced apoptosis at 24 h in parental and Bak knockdown cells, demonstrating that Bak played no role in this setting. In clear contrast, Bax knockout completely abrogated TRAIL- and TRAIL/wortmannin-induced apoptosis (Figure 5a). Also, in A-375 and Mel-HO melanoma cells, Bax knockdown by siRNA abrogated TRAIL- and TRAIL/wortmannin-induced apoptosis. Again, cytotoxicity played no role (Figure 5b).

Bottom Line: Thus, wortmannin resulted in early reduction of the Bax-inactivating phosphorylation at serine-184, whereas the Bax-activating phosphorylation at threonine-167 was enhanced.Proving the responsibility of the pathway, comparable effects were obtained with an Akt inhibitor (MK-2206); while suppressed phosphorylation of serine-184 may be attributed to reduced Akt activity itself, the causes of enhanced threonine-167 phosphorylation were addressed here.This correlated with reduced Bax phosphorylation at threonine-167.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Dermatology and Allergy, Skin Cancer Center, University Medical Center Charité, Berlin, Germany [2] Institute for Chemistry and Biochemistry, Free University of Berlin, Berlin, Germany.

ABSTRACT
The pathways of reactive oxygen species (ROS)-mediated apoptosis induction, of Bax activation and the sensitization of tumor cells for TRAIL (TNF-related apoptosis-inducing ligand)-induced apoptosis are still largely elusive. Here, sensitization of melanoma cells for TRAIL by the PI3-kinase inhibitor wortmannin correlated to the activation of mitochondrial apoptosis pathways. Apoptosis was dependent on Bax and abrogated by Bcl-2 overexpression. The synergistic enhancement was explained by Bax activation through wortmannin, which tightly correlated to the characteristic Bax phosphorylation patterns. Thus, wortmannin resulted in early reduction of the Bax-inactivating phosphorylation at serine-184, whereas the Bax-activating phosphorylation at threonine-167 was enhanced. Proving the responsibility of the pathway, comparable effects were obtained with an Akt inhibitor (MK-2206); while suppressed phosphorylation of serine-184 may be attributed to reduced Akt activity itself, the causes of enhanced threonine-167 phosphorylation were addressed here. Characteristically, production of ROS was seen early in response to wortmannin and MK-2206. Providing the link between ROS and Bax, we show that abrogated ROS production by α-tocopherol or by NADPH oxidase 4 (NOX4) siRNA suppressed apoptosis and Bax activation. This correlated with reduced Bax phosphorylation at threonine-167. The data unraveled a mechanism by which NOX4-dependent ROS production controls apoptosis via Bax phosphorylation. The pathway may be considered for proapoptotic, anticancer strategies.

Show MeSH
Related in: MedlinePlus