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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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NOX4 is essential for wortmannin-induced ROS production. (a) Apoptosis (% of sub-G1 cells) and cytotoxicity (% LDH release) in response to 24 h treatment with TRAIL (20 ng/ml) and/or wortmannin (4 μM) was monitored in A-375 and Mel-HO after siRNA-mediated knockdown of NOX4. The respective mock control (Si-Ctrl) is shown for comparison. Mean values and SDs of two independent experiments, each with triplicates, are shown, and statistical significance is indicated (*P<0.005) when comparing NOX4 and mock-transfected cells after TRAIL/wortmannin treatment. Knockdown of NOX4 (70 kDa), as determined by western blotting, is shown in insets; equal protein loading (30 μg per lane) was proven by GAPDH. (b) ROS levels were determined by H2DCFDA staining and flow cytometry in A-375 and Mel-HO after transfection with siRNA for NOX4 (Si-NOX4) and control siRNA (Si-Ctrl). Treatments with 20 ng/ml TRAIL +/− 4 μM wortmannin were for 2 h. Treated cells (open graphs) are compared to DMSO controls (gray). Two independent experiments (triplicates) revealed comparable results. (c) 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 transfected with NOX4 siRNA or control siRNA (Si-Ctrl). Treatments with TRAIL (20 ng/ml) +/− wortmannin (4 μM) were for 2 h. Treated cells (open graphs) are shown in overlays with DMSO controls (gray). Two complete and independent experiments with triplicates revealed comparable results. (d) The suggested pathways induced by wortmannin/TRAIL are explaned within the text
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fig8: NOX4 is essential for wortmannin-induced ROS production. (a) Apoptosis (% of sub-G1 cells) and cytotoxicity (% LDH release) in response to 24 h treatment with TRAIL (20 ng/ml) and/or wortmannin (4 μM) was monitored in A-375 and Mel-HO after siRNA-mediated knockdown of NOX4. The respective mock control (Si-Ctrl) is shown for comparison. Mean values and SDs of two independent experiments, each with triplicates, are shown, and statistical significance is indicated (*P<0.005) when comparing NOX4 and mock-transfected cells after TRAIL/wortmannin treatment. Knockdown of NOX4 (70 kDa), as determined by western blotting, is shown in insets; equal protein loading (30 μg per lane) was proven by GAPDH. (b) ROS levels were determined by H2DCFDA staining and flow cytometry in A-375 and Mel-HO after transfection with siRNA for NOX4 (Si-NOX4) and control siRNA (Si-Ctrl). Treatments with 20 ng/ml TRAIL +/− 4 μM wortmannin were for 2 h. Treated cells (open graphs) are compared to DMSO controls (gray). Two independent experiments (triplicates) revealed comparable results. (c) 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 transfected with NOX4 siRNA or control siRNA (Si-Ctrl). Treatments with TRAIL (20 ng/ml) +/− wortmannin (4 μM) were for 2 h. Treated cells (open graphs) are shown in overlays with DMSO controls (gray). Two complete and independent experiments with triplicates revealed comparable results. (d) The suggested pathways induced by wortmannin/TRAIL are explaned within the text

Mentions: For unraveling the ROS pathway in this setting, the function of NADPH oxidase 4 (NOX4) was investigated. Downregulation of NOX4 by siRNA significantly reduced apoptosis by wortmannin/TRAIL, as shown in A-375 (44%→19%) and Mel-HO (28%→11%). In contrast, TRAIL-induced apoptosis in A-375 was not affected by NOX4 siRNA (Figure 8a). Proving the critical role of NOX4 for wortmannin-induced ROS, NOX4 siRNA completely suppressed ROS production in A-375 and Mel-HO (Figure 8b).


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

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

NOX4 is essential for wortmannin-induced ROS production. (a) Apoptosis (% of sub-G1 cells) and cytotoxicity (% LDH release) in response to 24 h treatment with TRAIL (20 ng/ml) and/or wortmannin (4 μM) was monitored in A-375 and Mel-HO after siRNA-mediated knockdown of NOX4. The respective mock control (Si-Ctrl) is shown for comparison. Mean values and SDs of two independent experiments, each with triplicates, are shown, and statistical significance is indicated (*P<0.005) when comparing NOX4 and mock-transfected cells after TRAIL/wortmannin treatment. Knockdown of NOX4 (70 kDa), as determined by western blotting, is shown in insets; equal protein loading (30 μg per lane) was proven by GAPDH. (b) ROS levels were determined by H2DCFDA staining and flow cytometry in A-375 and Mel-HO after transfection with siRNA for NOX4 (Si-NOX4) and control siRNA (Si-Ctrl). Treatments with 20 ng/ml TRAIL +/− 4 μM wortmannin were for 2 h. Treated cells (open graphs) are compared to DMSO controls (gray). Two independent experiments (triplicates) revealed comparable results. (c) 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 transfected with NOX4 siRNA or control siRNA (Si-Ctrl). Treatments with TRAIL (20 ng/ml) +/− wortmannin (4 μM) were for 2 h. Treated cells (open graphs) are shown in overlays with DMSO controls (gray). Two complete and independent experiments with triplicates revealed comparable results. (d) The suggested pathways induced by wortmannin/TRAIL are explaned within the text
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fig8: NOX4 is essential for wortmannin-induced ROS production. (a) Apoptosis (% of sub-G1 cells) and cytotoxicity (% LDH release) in response to 24 h treatment with TRAIL (20 ng/ml) and/or wortmannin (4 μM) was monitored in A-375 and Mel-HO after siRNA-mediated knockdown of NOX4. The respective mock control (Si-Ctrl) is shown for comparison. Mean values and SDs of two independent experiments, each with triplicates, are shown, and statistical significance is indicated (*P<0.005) when comparing NOX4 and mock-transfected cells after TRAIL/wortmannin treatment. Knockdown of NOX4 (70 kDa), as determined by western blotting, is shown in insets; equal protein loading (30 μg per lane) was proven by GAPDH. (b) ROS levels were determined by H2DCFDA staining and flow cytometry in A-375 and Mel-HO after transfection with siRNA for NOX4 (Si-NOX4) and control siRNA (Si-Ctrl). Treatments with 20 ng/ml TRAIL +/− 4 μM wortmannin were for 2 h. Treated cells (open graphs) are compared to DMSO controls (gray). Two independent experiments (triplicates) revealed comparable results. (c) 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 transfected with NOX4 siRNA or control siRNA (Si-Ctrl). Treatments with TRAIL (20 ng/ml) +/− wortmannin (4 μM) were for 2 h. Treated cells (open graphs) are shown in overlays with DMSO controls (gray). Two complete and independent experiments with triplicates revealed comparable results. (d) The suggested pathways induced by wortmannin/TRAIL are explaned within the text
Mentions: For unraveling the ROS pathway in this setting, the function of NADPH oxidase 4 (NOX4) was investigated. Downregulation of NOX4 by siRNA significantly reduced apoptosis by wortmannin/TRAIL, as shown in A-375 (44%→19%) and Mel-HO (28%→11%). In contrast, TRAIL-induced apoptosis in A-375 was not affected by NOX4 siRNA (Figure 8a). Proving the critical role of NOX4 for wortmannin-induced ROS, NOX4 siRNA completely suppressed ROS production in A-375 and Mel-HO (Figure 8b).

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