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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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Changes at the level of Bax phosphorylation and relation to ROS. (a) Flow cytometry signals for Bax were compared in HCT-116 parental cells (Bax+) and in HCT-116 Bax knockout cells (Bax−/−). Antibodies for total Bax were used, as well as those for Bax phosphorylated at serine-184 and at threonine-167, respectively. Non-treated cells (gray) are shown in overlays with IgG1-stained controls (open graphs). Two independent experiments with triplicates revealed identical results. (b–e) A-375 and A-375-TS were treated for 2 h with wortmannin (4 μM), TRAIL (20 ng/ml) and/or α-tocopherol (200 μM, 1 h pretreatment). The drugs were applied together at and for the same time. For subsequent flow cytometry, cells were stained with antibodies specific for (b) pBax(Ser-184), (c) pBax(Thr-167), (d) activated Bax (Bax-NT) and (e) for total Bax as control. Treated cells (open graphs) are shown in overlays with DMSO controls (gray) and IgG1-stained controls (dashed line). Two complete and independent experiments with triplicates revealed comparable results
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fig6: Changes at the level of Bax phosphorylation and relation to ROS. (a) Flow cytometry signals for Bax were compared in HCT-116 parental cells (Bax+) and in HCT-116 Bax knockout cells (Bax−/−). Antibodies for total Bax were used, as well as those for Bax phosphorylated at serine-184 and at threonine-167, respectively. Non-treated cells (gray) are shown in overlays with IgG1-stained controls (open graphs). Two independent experiments with triplicates revealed identical results. (b–e) A-375 and A-375-TS were treated for 2 h with wortmannin (4 μM), TRAIL (20 ng/ml) and/or α-tocopherol (200 μM, 1 h pretreatment). The drugs were applied together at and for the same time. For subsequent flow cytometry, cells were stained with antibodies specific for (b) pBax(Ser-184), (c) pBax(Thr-167), (d) activated Bax (Bax-NT) and (e) for total Bax as control. Treated cells (open graphs) are shown in overlays with DMSO controls (gray) and IgG1-stained controls (dashed line). Two complete and independent experiments with triplicates revealed comparable results

Mentions: Addressing the relation between kinase inhibition and Bax activation, assays were applied for monitoring Bax phosphorylation at serine-184 and threonine-167. Assays were based on cell permeabilization, staining with phosphorylation-specific antibodies and flow cytometry. Bax phosphorylation at serine-184 had been described as an inactivating step, while phosphorylation at threonine-167 was described as an activating step.12 Reliability of the used antibodies was checked in the HCT-116 Bax knockout cells. Whereas Bax antibodies resulted in significant signals in the HCT-116 parental cells, Bax knockout cells revealed no staining, thus excluding non-specific binding (Figure 6a).


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

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

Changes at the level of Bax phosphorylation and relation to ROS. (a) Flow cytometry signals for Bax were compared in HCT-116 parental cells (Bax+) and in HCT-116 Bax knockout cells (Bax−/−). Antibodies for total Bax were used, as well as those for Bax phosphorylated at serine-184 and at threonine-167, respectively. Non-treated cells (gray) are shown in overlays with IgG1-stained controls (open graphs). Two independent experiments with triplicates revealed identical results. (b–e) A-375 and A-375-TS were treated for 2 h with wortmannin (4 μM), TRAIL (20 ng/ml) and/or α-tocopherol (200 μM, 1 h pretreatment). The drugs were applied together at and for the same time. For subsequent flow cytometry, cells were stained with antibodies specific for (b) pBax(Ser-184), (c) pBax(Thr-167), (d) activated Bax (Bax-NT) and (e) for total Bax as control. Treated cells (open graphs) are shown in overlays with DMSO controls (gray) and IgG1-stained controls (dashed line). Two complete and independent experiments with triplicates revealed comparable results
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3824654&req=5

fig6: Changes at the level of Bax phosphorylation and relation to ROS. (a) Flow cytometry signals for Bax were compared in HCT-116 parental cells (Bax+) and in HCT-116 Bax knockout cells (Bax−/−). Antibodies for total Bax were used, as well as those for Bax phosphorylated at serine-184 and at threonine-167, respectively. Non-treated cells (gray) are shown in overlays with IgG1-stained controls (open graphs). Two independent experiments with triplicates revealed identical results. (b–e) A-375 and A-375-TS were treated for 2 h with wortmannin (4 μM), TRAIL (20 ng/ml) and/or α-tocopherol (200 μM, 1 h pretreatment). The drugs were applied together at and for the same time. For subsequent flow cytometry, cells were stained with antibodies specific for (b) pBax(Ser-184), (c) pBax(Thr-167), (d) activated Bax (Bax-NT) and (e) for total Bax as control. Treated cells (open graphs) are shown in overlays with DMSO controls (gray) and IgG1-stained controls (dashed line). Two complete and independent experiments with triplicates revealed comparable results
Mentions: Addressing the relation between kinase inhibition and Bax activation, assays were applied for monitoring Bax phosphorylation at serine-184 and threonine-167. Assays were based on cell permeabilization, staining with phosphorylation-specific antibodies and flow cytometry. Bax phosphorylation at serine-184 had been described as an inactivating step, while phosphorylation at threonine-167 was described as an activating step.12 Reliability of the used antibodies was checked in the HCT-116 Bax knockout cells. Whereas Bax antibodies resulted in significant signals in the HCT-116 parental cells, Bax knockout cells revealed no staining, thus excluding non-specific binding (Figure 6a).

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