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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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Enhanced TRAIL-induced apoptosis and Bax activation by the Akt inhibitor MK-2206. (a) A-375, A-375-TS and Mel-HO cells were treated with increasing concentrations of the allosteric Akt inhibitor MK-2206 (0.5–8 μM), and the effects on Akt phosphorylation (serine-473) were monitored by western blotting after 24 h. Signals of an antibody for total Akt (Akt) served as controls. (b) Apoptosis (percentage of sub-G1 cells) and cytotoxicity (LDH release) were determined in these cells after 24 h of treatment (means and SDs of two independent experiments, each one with triplicates). Statistical significance (*P<0.005) is indicated, when comparing treatments with MK-2206/TRAIL and TRAIL alone. (c) ROS levels were determined by H2DCFDA staining and flow cytometry in A-375 and Mel-HO at 2 h after starting treatment with 20 ng/ml TRAIL, 4 μM MK-2206 and/or α-tocopherol (200 μM, 1 h pretreatment). Treated cells (open graphs) were compared to DMSO controls (gray). Two independent experiments (each with triplicates) revealed comparable results. (d) Flow cytometry signals for Bax phosphorylation (Ser-184; Thr-167) and Bax activation (Bax-NT) were compared in A-375, A-375-TS and Mel-HO cells after 2 h treatment with TRAIL (20 ng/ml), MK-2206 (4 μM) and MK-2206/TRAIL. Treated cells (open graphs) are shown in overlays with DMSO controls (gray). Two complete and independent experiments with triplicates revealed the same results
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fig7: Enhanced TRAIL-induced apoptosis and Bax activation by the Akt inhibitor MK-2206. (a) A-375, A-375-TS and Mel-HO cells were treated with increasing concentrations of the allosteric Akt inhibitor MK-2206 (0.5–8 μM), and the effects on Akt phosphorylation (serine-473) were monitored by western blotting after 24 h. Signals of an antibody for total Akt (Akt) served as controls. (b) Apoptosis (percentage of sub-G1 cells) and cytotoxicity (LDH release) were determined in these cells after 24 h of treatment (means and SDs of two independent experiments, each one with triplicates). Statistical significance (*P<0.005) is indicated, when comparing treatments with MK-2206/TRAIL and TRAIL alone. (c) ROS levels were determined by H2DCFDA staining and flow cytometry in A-375 and Mel-HO at 2 h after starting treatment with 20 ng/ml TRAIL, 4 μM MK-2206 and/or α-tocopherol (200 μM, 1 h pretreatment). Treated cells (open graphs) were compared to DMSO controls (gray). Two independent experiments (each with triplicates) revealed comparable results. (d) Flow cytometry signals for Bax phosphorylation (Ser-184; Thr-167) and Bax activation (Bax-NT) were compared in A-375, A-375-TS and Mel-HO cells after 2 h treatment with TRAIL (20 ng/ml), MK-2206 (4 μM) and MK-2206/TRAIL. Treated cells (open graphs) are shown in overlays with DMSO controls (gray). Two complete and independent experiments with triplicates revealed the same results

Mentions: For addressing the relation of PI3K and Akt inhibition in this setting, some experiments were repeated with the allosteric Akt inhibitor MK-2206. It resulted in a concentration-dependent and long-lasting (24 h) downregulation of Akt phosphorylation, as shown for serine-473 in A-375, A-375-TS and Mel-HO (Figure 7a). Comparable to wortmannin, MK-2206 resulted in a significant and dose-dependent enhancement of TRAIL-induced apoptosis in TRAIL-sensitive and in resistant melanoma cells, as determined at 24 h by cell cycle analyses (A-375, 48% Mel-HO, 24% A-375-TS, 28%). Again, cytotoxicity played no role (Figure 7b). In parallel to wortmannin, ROS levels were significantly enhanced by MK-2206, as shown in A-375 and Mel-HO at 2 h of treatment, and ROS production was interrupted by α-tocopherol (Figure 7c).


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

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

Enhanced TRAIL-induced apoptosis and Bax activation by the Akt inhibitor MK-2206. (a) A-375, A-375-TS and Mel-HO cells were treated with increasing concentrations of the allosteric Akt inhibitor MK-2206 (0.5–8 μM), and the effects on Akt phosphorylation (serine-473) were monitored by western blotting after 24 h. Signals of an antibody for total Akt (Akt) served as controls. (b) Apoptosis (percentage of sub-G1 cells) and cytotoxicity (LDH release) were determined in these cells after 24 h of treatment (means and SDs of two independent experiments, each one with triplicates). Statistical significance (*P<0.005) is indicated, when comparing treatments with MK-2206/TRAIL and TRAIL alone. (c) ROS levels were determined by H2DCFDA staining and flow cytometry in A-375 and Mel-HO at 2 h after starting treatment with 20 ng/ml TRAIL, 4 μM MK-2206 and/or α-tocopherol (200 μM, 1 h pretreatment). Treated cells (open graphs) were compared to DMSO controls (gray). Two independent experiments (each with triplicates) revealed comparable results. (d) Flow cytometry signals for Bax phosphorylation (Ser-184; Thr-167) and Bax activation (Bax-NT) were compared in A-375, A-375-TS and Mel-HO cells after 2 h treatment with TRAIL (20 ng/ml), MK-2206 (4 μM) and MK-2206/TRAIL. Treated cells (open graphs) are shown in overlays with DMSO controls (gray). Two complete and independent experiments with triplicates revealed the same results
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fig7: Enhanced TRAIL-induced apoptosis and Bax activation by the Akt inhibitor MK-2206. (a) A-375, A-375-TS and Mel-HO cells were treated with increasing concentrations of the allosteric Akt inhibitor MK-2206 (0.5–8 μM), and the effects on Akt phosphorylation (serine-473) were monitored by western blotting after 24 h. Signals of an antibody for total Akt (Akt) served as controls. (b) Apoptosis (percentage of sub-G1 cells) and cytotoxicity (LDH release) were determined in these cells after 24 h of treatment (means and SDs of two independent experiments, each one with triplicates). Statistical significance (*P<0.005) is indicated, when comparing treatments with MK-2206/TRAIL and TRAIL alone. (c) ROS levels were determined by H2DCFDA staining and flow cytometry in A-375 and Mel-HO at 2 h after starting treatment with 20 ng/ml TRAIL, 4 μM MK-2206 and/or α-tocopherol (200 μM, 1 h pretreatment). Treated cells (open graphs) were compared to DMSO controls (gray). Two independent experiments (each with triplicates) revealed comparable results. (d) Flow cytometry signals for Bax phosphorylation (Ser-184; Thr-167) and Bax activation (Bax-NT) were compared in A-375, A-375-TS and Mel-HO cells after 2 h treatment with TRAIL (20 ng/ml), MK-2206 (4 μM) and MK-2206/TRAIL. Treated cells (open graphs) are shown in overlays with DMSO controls (gray). Two complete and independent experiments with triplicates revealed the same results
Mentions: For addressing the relation of PI3K and Akt inhibition in this setting, some experiments were repeated with the allosteric Akt inhibitor MK-2206. It resulted in a concentration-dependent and long-lasting (24 h) downregulation of Akt phosphorylation, as shown for serine-473 in A-375, A-375-TS and Mel-HO (Figure 7a). Comparable to wortmannin, MK-2206 resulted in a significant and dose-dependent enhancement of TRAIL-induced apoptosis in TRAIL-sensitive and in resistant melanoma cells, as determined at 24 h by cell cycle analyses (A-375, 48% Mel-HO, 24% A-375-TS, 28%). Again, cytotoxicity played no role (Figure 7b). In parallel to wortmannin, ROS levels were significantly enhanced by MK-2206, as shown in A-375 and Mel-HO at 2 h of treatment, and ROS production was interrupted by α-tocopherol (Figure 7c).

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