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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

Centrosome dysregulation by GSK-3β inhibitors.(a) Frequency of abnormal centrosomes. Cells were treated with 20 μM AR-A014418 for 48 h and then stained with an anti-γ-tubulin antibody. At least 100 cells were examined in each sample. CIs and p-values were calculated by Fisher’s exact test. Error bars indicate 95% CIs. (*p < 0.01) (b) Representative examples of centrosome abnormalities in interphase cells. Arrows indicate aberrant centrosomes. (Scale bar = 10 μm) (c) Representative examples of centrosome abnormalities in mitotic cells. Arrows indicate extra-pole. (Scale bar = 10 μm) (d) Centrosome localization of phospho-GSK-3β. MDA-MB-435S cells were stained with an anti-γ-tubulin antibody and an anti-GSK-3β or anti-phospho-GSK-3β antibody. The right column shows higher power images of the boxed areas in the left column images. Arrows indicate centrosomes. (Scale bar = 10 μm) (e) Frequency of centrosome abnormalities induced by three distinct GSK-3β inhibitors. Cells were examined after 48 h treatment with AR-A014418 (20 μM), GSK-3β inhibitor XXVI (Inhibitor XXVI, 10 μM), or lithium chloride (LiCl, 50 mM). The data was obtained and analysed by the same way as (a). Error bars indicate 95% CIs (*p < 0.01). (f) Frequency of centrosome abnormalities after docetaxel treatment. Cells were treated with docetaxel at their IC50s (0.5 nM for HCT 116 and 0.25 nM for RKO) or higher concentration for 48 h. The data was obtained and analysed by the same way as (a). (g) Frequency of centrosome abnormalities after GSK-3β knock-down. Centrosomes were counted 72 h after transfection of siRNA. The data was obtained and analysed by the same way as (a). (*p < 0.001, †p < 0.005, ‡p = 0.02).
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f5: Centrosome dysregulation by GSK-3β inhibitors.(a) Frequency of abnormal centrosomes. Cells were treated with 20 μM AR-A014418 for 48 h and then stained with an anti-γ-tubulin antibody. At least 100 cells were examined in each sample. CIs and p-values were calculated by Fisher’s exact test. Error bars indicate 95% CIs. (*p < 0.01) (b) Representative examples of centrosome abnormalities in interphase cells. Arrows indicate aberrant centrosomes. (Scale bar = 10 μm) (c) Representative examples of centrosome abnormalities in mitotic cells. Arrows indicate extra-pole. (Scale bar = 10 μm) (d) Centrosome localization of phospho-GSK-3β. MDA-MB-435S cells were stained with an anti-γ-tubulin antibody and an anti-GSK-3β or anti-phospho-GSK-3β antibody. The right column shows higher power images of the boxed areas in the left column images. Arrows indicate centrosomes. (Scale bar = 10 μm) (e) Frequency of centrosome abnormalities induced by three distinct GSK-3β inhibitors. Cells were examined after 48 h treatment with AR-A014418 (20 μM), GSK-3β inhibitor XXVI (Inhibitor XXVI, 10 μM), or lithium chloride (LiCl, 50 mM). The data was obtained and analysed by the same way as (a). Error bars indicate 95% CIs (*p < 0.01). (f) Frequency of centrosome abnormalities after docetaxel treatment. Cells were treated with docetaxel at their IC50s (0.5 nM for HCT 116 and 0.25 nM for RKO) or higher concentration for 48 h. The data was obtained and analysed by the same way as (a). (g) Frequency of centrosome abnormalities after GSK-3β knock-down. Centrosomes were counted 72 h after transfection of siRNA. The data was obtained and analysed by the same way as (a). (*p < 0.001, †p < 0.005, ‡p = 0.02).

Mentions: Centrosomes participate in the formation of mitotic spindles31. To investigate any changes in centrosomes induced by AR-A014418, we used immunocytofluorescent staining for γ-tubulin, one of the major components of centrosomes. Treatment with 20 μM AR-A0114418 for 48 h significantly increased the numbers of cells with more than two centrosomes or with morphologically abnormal centrosomes in all cell lines, except for KPK13 cells (Fig. 5a). Representative images of these centrosome abnormalities are shown in Fig. 5b. In addition, pseudo-dipole formation, which one of the sequelae of centrosome amplification32, was found in RKO and MDA-MB-435S cells treated with AR-A0114418 (Fig. 5c).


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)

Centrosome dysregulation by GSK-3β inhibitors.(a) Frequency of abnormal centrosomes. Cells were treated with 20 μM AR-A014418 for 48 h and then stained with an anti-γ-tubulin antibody. At least 100 cells were examined in each sample. CIs and p-values were calculated by Fisher’s exact test. Error bars indicate 95% CIs. (*p < 0.01) (b) Representative examples of centrosome abnormalities in interphase cells. Arrows indicate aberrant centrosomes. (Scale bar = 10 μm) (c) Representative examples of centrosome abnormalities in mitotic cells. Arrows indicate extra-pole. (Scale bar = 10 μm) (d) Centrosome localization of phospho-GSK-3β. MDA-MB-435S cells were stained with an anti-γ-tubulin antibody and an anti-GSK-3β or anti-phospho-GSK-3β antibody. The right column shows higher power images of the boxed areas in the left column images. Arrows indicate centrosomes. (Scale bar = 10 μm) (e) Frequency of centrosome abnormalities induced by three distinct GSK-3β inhibitors. Cells were examined after 48 h treatment with AR-A014418 (20 μM), GSK-3β inhibitor XXVI (Inhibitor XXVI, 10 μM), or lithium chloride (LiCl, 50 mM). The data was obtained and analysed by the same way as (a). Error bars indicate 95% CIs (*p < 0.01). (f) Frequency of centrosome abnormalities after docetaxel treatment. Cells were treated with docetaxel at their IC50s (0.5 nM for HCT 116 and 0.25 nM for RKO) or higher concentration for 48 h. The data was obtained and analysed by the same way as (a). (g) Frequency of centrosome abnormalities after GSK-3β knock-down. Centrosomes were counted 72 h after transfection of siRNA. The data was obtained and analysed by the same way as (a). (*p < 0.001, †p < 0.005, ‡p = 0.02).
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Related In: Results  -  Collection

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f5: Centrosome dysregulation by GSK-3β inhibitors.(a) Frequency of abnormal centrosomes. Cells were treated with 20 μM AR-A014418 for 48 h and then stained with an anti-γ-tubulin antibody. At least 100 cells were examined in each sample. CIs and p-values were calculated by Fisher’s exact test. Error bars indicate 95% CIs. (*p < 0.01) (b) Representative examples of centrosome abnormalities in interphase cells. Arrows indicate aberrant centrosomes. (Scale bar = 10 μm) (c) Representative examples of centrosome abnormalities in mitotic cells. Arrows indicate extra-pole. (Scale bar = 10 μm) (d) Centrosome localization of phospho-GSK-3β. MDA-MB-435S cells were stained with an anti-γ-tubulin antibody and an anti-GSK-3β or anti-phospho-GSK-3β antibody. The right column shows higher power images of the boxed areas in the left column images. Arrows indicate centrosomes. (Scale bar = 10 μm) (e) Frequency of centrosome abnormalities induced by three distinct GSK-3β inhibitors. Cells were examined after 48 h treatment with AR-A014418 (20 μM), GSK-3β inhibitor XXVI (Inhibitor XXVI, 10 μM), or lithium chloride (LiCl, 50 mM). The data was obtained and analysed by the same way as (a). Error bars indicate 95% CIs (*p < 0.01). (f) Frequency of centrosome abnormalities after docetaxel treatment. Cells were treated with docetaxel at their IC50s (0.5 nM for HCT 116 and 0.25 nM for RKO) or higher concentration for 48 h. The data was obtained and analysed by the same way as (a). (g) Frequency of centrosome abnormalities after GSK-3β knock-down. Centrosomes were counted 72 h after transfection of siRNA. The data was obtained and analysed by the same way as (a). (*p < 0.001, †p < 0.005, ‡p = 0.02).
Mentions: Centrosomes participate in the formation of mitotic spindles31. To investigate any changes in centrosomes induced by AR-A014418, we used immunocytofluorescent staining for γ-tubulin, one of the major components of centrosomes. Treatment with 20 μM AR-A0114418 for 48 h significantly increased the numbers of cells with more than two centrosomes or with morphologically abnormal centrosomes in all cell lines, except for KPK13 cells (Fig. 5a). Representative images of these centrosome abnormalities are shown in Fig. 5b. In addition, pseudo-dipole formation, which one of the sequelae of centrosome amplification32, was found in RKO and MDA-MB-435S cells treated with AR-A0114418 (Fig. 5c).

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