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Inhibition of glycogen synthase kinase-3 beta induces apoptosis and mitotic catastrophe by disrupting centrosome regulation in cancer cells.

Yoshino Y, Ishioka C - Sci Rep (2015)

Bottom Line: After GSK-3β inhibitor treatment, these cells exhibited characteristic features of mitotic catastrophe, including distended and multivesiculated nuclei and inappropriate reductions in cyclin B1 expression.From these data, GSK-3β seems to regulate centrosome function.Thus, we propose that centrosome dysregulation is an important mechanism for the anticancer effects of GSK-3β inhibitors and that mitotic catastrophe serves as a safe-guard system to remove cells with any mitotic abnormalities induced by GSK-3β inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Oncology, Institute of Development, Aging and Cancer, Tohoku University, Seiryo-machi 4-1, Aoba-ku, Sendai 980-8575, Japan.

ABSTRACT
Glycogen synthase kinase-3 beta (GSK-3β) has been investigated as a therapeutic target for numerous human diseases including cancer because of their diverse cellular functions. Although GSK-3β inhibitors have been investigated as anticancer reagents, precise biological mechanisms remain to be determined. In this study, we investigated the anticancer effects of GSK-3β inhibitors on cancer cell lines and observed centrosome dysregulation which resulted in abnormal mitosis. Mitotic checkpoints sensed the mitotic abnormalities and induced apoptosis. For cells that were inherently resistant to apoptosis, cell death distinct from apoptosis was induced. After GSK-3β inhibitor treatment, these cells exhibited characteristic features of mitotic catastrophe, including distended and multivesiculated nuclei and inappropriate reductions in cyclin B1 expression. This suggested that mitotic catastrophe was an alternative mechanism in cells resistant to apoptosis. Although the role of GSK-3β in centrosomes has not yet been clarified, phosphorylated GSK-3β was localised in centrosomes. From these data, GSK-3β seems to regulate centrosome function. Thus, we propose that centrosome dysregulation is an important mechanism for the anticancer effects of GSK-3β inhibitors and that mitotic catastrophe serves as a safe-guard system to remove cells with any mitotic abnormalities induced by GSK-3β inhibition.

No MeSH data available.


Related in: MedlinePlus

AR-A014418 effects on the Wnt/β-catenin, NF-κB, MAPK, and PI3K/AKT signalling pathways.(a) β-catenin accumulation by AR-A0114418 in RKO and HCT 116. Cells were harvested for immunoblotting after treatment with 20 μM AR-A014418 for indicated periods. (b) Comparisons of DNA histograms between β-catenin-overexpressing and AR-A014418-treated HeLa cells. A CDK4 inhibitor (1 μM) was added at 24 h after transfection with a mutant (S33Y) β-catenin expression vector or after adding 20 μM AR-A014418. After another 48 h, cells were harvested for cell cycle analysis. (c) Cell cycle distributions of HeLa cells subjected to mutant β–catenin overexpression or AR-A014418 treatment with or without a CDK4 inhibitor. Cells were treated in the same way as (b) Average of three independent experiments is shown. Error bars indicate 95% CIs (*p < 0.05). (d) NF-κB reporter activities with or without AR-A014418 treatment. Cells were pre-treated with DMSO or 20 μM AR-A0114418 for 48 h from the next day of reporter plasmid transfection. After the treatment, cells were allowed to release MMPs into fresh medium for 24 h. (*p < 0.01) (e) AKT and ERK phosphorylation after AR-A014418 treatment. Cells were cultured in serum-free media with AR-A014418 at the indicated concentrations for 48 h. After serum starvation, cells were stimulated with 10% serum for 30 min and then harvested for western blot.
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f3: AR-A014418 effects on the Wnt/β-catenin, NF-κB, MAPK, and PI3K/AKT signalling pathways.(a) β-catenin accumulation by AR-A0114418 in RKO and HCT 116. Cells were harvested for immunoblotting after treatment with 20 μM AR-A014418 for indicated periods. (b) Comparisons of DNA histograms between β-catenin-overexpressing and AR-A014418-treated HeLa cells. A CDK4 inhibitor (1 μM) was added at 24 h after transfection with a mutant (S33Y) β-catenin expression vector or after adding 20 μM AR-A014418. After another 48 h, cells were harvested for cell cycle analysis. (c) Cell cycle distributions of HeLa cells subjected to mutant β–catenin overexpression or AR-A014418 treatment with or without a CDK4 inhibitor. Cells were treated in the same way as (b) Average of three independent experiments is shown. Error bars indicate 95% CIs (*p < 0.05). (d) NF-κB reporter activities with or without AR-A014418 treatment. Cells were pre-treated with DMSO or 20 μM AR-A0114418 for 48 h from the next day of reporter plasmid transfection. After the treatment, cells were allowed to release MMPs into fresh medium for 24 h. (*p < 0.01) (e) AKT and ERK phosphorylation after AR-A014418 treatment. Cells were cultured in serum-free media with AR-A014418 at the indicated concentrations for 48 h. After serum starvation, cells were stimulated with 10% serum for 30 min and then harvested for western blot.

Mentions: To investigate the possible involvement of the Wnt/β-catenin signalling pathway, we assessed changes in β-catenin expression induced by AR-A014418 treatment. β-catenin levels began to increase at 48 h after adding 20 μM AR-A014418 to RKO cells with wild-type β-catenin29 but not in HCT 116 cells with mutant β-catenin which has deletion of serine 45 residue, target of GSK-3β29 (Fig. 3a). However, accelerated rather than suppressed cell proliferation was observed after forced expression of wild-type or constitutively active mutant β-catenin (S33Y) in RKO and HCT 116 (Supplementary Fig. S2a). To clarify the difference of overexpression and AR-A0114418 induced upregulation of β-catenin, we utilized HeLa, which harbours no mutation in Wnt/β-catenin pathway. The DNA histogram of cells with forced β-catenin expression was distinct from that of cells treated with 20 μM AR-A0114418 (Fig. 3b). Administering a cdk4 inhibitor in conjunction with β-catenin overexpression reversed these changes in cell cycle distribution, although the cdk4 inhibitor could not counteract the effects of AR-A014418 (Fig. 3c).


Inhibition of glycogen synthase kinase-3 beta induces apoptosis and mitotic catastrophe by disrupting centrosome regulation in cancer cells.

Yoshino Y, Ishioka C - Sci Rep (2015)

AR-A014418 effects on the Wnt/β-catenin, NF-κB, MAPK, and PI3K/AKT signalling pathways.(a) β-catenin accumulation by AR-A0114418 in RKO and HCT 116. Cells were harvested for immunoblotting after treatment with 20 μM AR-A014418 for indicated periods. (b) Comparisons of DNA histograms between β-catenin-overexpressing and AR-A014418-treated HeLa cells. A CDK4 inhibitor (1 μM) was added at 24 h after transfection with a mutant (S33Y) β-catenin expression vector or after adding 20 μM AR-A014418. After another 48 h, cells were harvested for cell cycle analysis. (c) Cell cycle distributions of HeLa cells subjected to mutant β–catenin overexpression or AR-A014418 treatment with or without a CDK4 inhibitor. Cells were treated in the same way as (b) Average of three independent experiments is shown. Error bars indicate 95% CIs (*p < 0.05). (d) NF-κB reporter activities with or without AR-A014418 treatment. Cells were pre-treated with DMSO or 20 μM AR-A0114418 for 48 h from the next day of reporter plasmid transfection. After the treatment, cells were allowed to release MMPs into fresh medium for 24 h. (*p < 0.01) (e) AKT and ERK phosphorylation after AR-A014418 treatment. Cells were cultured in serum-free media with AR-A014418 at the indicated concentrations for 48 h. After serum starvation, cells were stimulated with 10% serum for 30 min and then harvested for western blot.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4543981&req=5

f3: AR-A014418 effects on the Wnt/β-catenin, NF-κB, MAPK, and PI3K/AKT signalling pathways.(a) β-catenin accumulation by AR-A0114418 in RKO and HCT 116. Cells were harvested for immunoblotting after treatment with 20 μM AR-A014418 for indicated periods. (b) Comparisons of DNA histograms between β-catenin-overexpressing and AR-A014418-treated HeLa cells. A CDK4 inhibitor (1 μM) was added at 24 h after transfection with a mutant (S33Y) β-catenin expression vector or after adding 20 μM AR-A014418. After another 48 h, cells were harvested for cell cycle analysis. (c) Cell cycle distributions of HeLa cells subjected to mutant β–catenin overexpression or AR-A014418 treatment with or without a CDK4 inhibitor. Cells were treated in the same way as (b) Average of three independent experiments is shown. Error bars indicate 95% CIs (*p < 0.05). (d) NF-κB reporter activities with or without AR-A014418 treatment. Cells were pre-treated with DMSO or 20 μM AR-A0114418 for 48 h from the next day of reporter plasmid transfection. After the treatment, cells were allowed to release MMPs into fresh medium for 24 h. (*p < 0.01) (e) AKT and ERK phosphorylation after AR-A014418 treatment. Cells were cultured in serum-free media with AR-A014418 at the indicated concentrations for 48 h. After serum starvation, cells were stimulated with 10% serum for 30 min and then harvested for western blot.
Mentions: To investigate the possible involvement of the Wnt/β-catenin signalling pathway, we assessed changes in β-catenin expression induced by AR-A014418 treatment. β-catenin levels began to increase at 48 h after adding 20 μM AR-A014418 to RKO cells with wild-type β-catenin29 but not in HCT 116 cells with mutant β-catenin which has deletion of serine 45 residue, target of GSK-3β29 (Fig. 3a). However, accelerated rather than suppressed cell proliferation was observed after forced expression of wild-type or constitutively active mutant β-catenin (S33Y) in RKO and HCT 116 (Supplementary Fig. S2a). To clarify the difference of overexpression and AR-A0114418 induced upregulation of β-catenin, we utilized HeLa, which harbours no mutation in Wnt/β-catenin pathway. The DNA histogram of cells with forced β-catenin expression was distinct from that of cells treated with 20 μM AR-A0114418 (Fig. 3b). Administering a cdk4 inhibitor in conjunction with β-catenin overexpression reversed these changes in cell cycle distribution, although the cdk4 inhibitor could not counteract the effects of AR-A014418 (Fig. 3c).

Bottom Line: After GSK-3β inhibitor treatment, these cells exhibited characteristic features of mitotic catastrophe, including distended and multivesiculated nuclei and inappropriate reductions in cyclin B1 expression.From these data, GSK-3β seems to regulate centrosome function.Thus, we propose that centrosome dysregulation is an important mechanism for the anticancer effects of GSK-3β inhibitors and that mitotic catastrophe serves as a safe-guard system to remove cells with any mitotic abnormalities induced by GSK-3β inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Oncology, Institute of Development, Aging and Cancer, Tohoku University, Seiryo-machi 4-1, Aoba-ku, Sendai 980-8575, Japan.

ABSTRACT
Glycogen synthase kinase-3 beta (GSK-3β) has been investigated as a therapeutic target for numerous human diseases including cancer because of their diverse cellular functions. Although GSK-3β inhibitors have been investigated as anticancer reagents, precise biological mechanisms remain to be determined. In this study, we investigated the anticancer effects of GSK-3β inhibitors on cancer cell lines and observed centrosome dysregulation which resulted in abnormal mitosis. Mitotic checkpoints sensed the mitotic abnormalities and induced apoptosis. For cells that were inherently resistant to apoptosis, cell death distinct from apoptosis was induced. After GSK-3β inhibitor treatment, these cells exhibited characteristic features of mitotic catastrophe, including distended and multivesiculated nuclei and inappropriate reductions in cyclin B1 expression. This suggested that mitotic catastrophe was an alternative mechanism in cells resistant to apoptosis. Although the role of GSK-3β in centrosomes has not yet been clarified, phosphorylated GSK-3β was localised in centrosomes. From these data, GSK-3β seems to regulate centrosome function. Thus, we propose that centrosome dysregulation is an important mechanism for the anticancer effects of GSK-3β inhibitors and that mitotic catastrophe serves as a safe-guard system to remove cells with any mitotic abnormalities induced by GSK-3β inhibition.

No MeSH data available.


Related in: MedlinePlus