Limits...
Histone deacetylase inhibitors promote glioma cell death by G2 checkpoint abrogation leading to mitotic catastrophe.

Cornago M, Garcia-Alberich C, Blasco-Angulo N, Vall-Llaura N, Nager M, Herreros J, Comella JX, Sanchis D, Llovera M - Cell Death Dis (2014)

Bottom Line: They have multiple effects, such as inducing the production of reactive oxygen species (ROS) and activating the mitochondrial apoptotic pathway, nevertheless cell death is not prevented by the pan-caspase inhibitor Q-VD-OPh.Importantly, we found that HDACi alter cell cycle progression by decreasing the expression of G2 checkpoint kinases Wee1 and checkpoint kinase 1 (Chk1).In addition, HDACi reduce the expression of proteins involved in DNA repair (Rad51), mitotic spindle formation (TPX2) and chromosome segregation (Survivin) in glioma cells and in human glioblastoma multiforme primary cultures.

View Article: PubMed Central - PubMed

Affiliation: Cell Signaling and Apoptosis Group, Institut de Recerca Biomèdica de Lleida (IRBLleida), Universitat de Lleida, Lleida, Spain.

ABSTRACT
Glioblastoma multiforme is resistant to conventional anti-tumoral treatments due to its infiltrative nature and capability of relapse; therefore, research efforts focus on characterizing gliomagenesis and identifying molecular targets useful on therapy. New therapeutic strategies are being tested in patients, such as Histone deacetylase inhibitors (HDACi) either alone or in combination with other therapies. Here two HDACi included in clinical trials have been tested, suberanilohydroxamic acid (SAHA) and valproic acid (VPA), to characterize their effects on glioma cell growth in vitro and to determine the molecular changes that promote cancer cell death. We found that both HDACi reduce glioma cell viability, proliferation and clonogenicity. They have multiple effects, such as inducing the production of reactive oxygen species (ROS) and activating the mitochondrial apoptotic pathway, nevertheless cell death is not prevented by the pan-caspase inhibitor Q-VD-OPh. Importantly, we found that HDACi alter cell cycle progression by decreasing the expression of G2 checkpoint kinases Wee1 and checkpoint kinase 1 (Chk1). In addition, HDACi reduce the expression of proteins involved in DNA repair (Rad51), mitotic spindle formation (TPX2) and chromosome segregation (Survivin) in glioma cells and in human glioblastoma multiforme primary cultures. Therefore, HDACi treatment causes glioma cell entry into mitosis before DNA damage could be repaired and to the formation of an aberrant mitotic spindle that results in glioma cell death through mitotic catastrophe-induced apoptosis.

Show MeSH

Related in: MedlinePlus

VPA and SAHA induce nuclear condensation and caspase activation in glioma cell lines. (a) Nuclear morphology of U87-MG, U373-MG and LN229 after 24-h treatment with 10 mM VPA, 10 μM SAHA or 100 ng/ml TRAIL. After treatment, cells were fixed in 4% PFA and nuclei were stained with Hoechst 33342. Condensed and abnormal nuclei are indicated with yellow arrowheads. Scale bar= 50 μm. (b) Analysis of caspase-9, caspase-3 and fodrin expression and cleavage on glioma cell lines after 24-h treatment with 10 mM VPA and 10 μM SAHA by western blot. Membrane was reprobed with an anti-α-tubulin antibody to verify equal loading. A representative blot from three independent experiments is shown. (c) Caspase-3 activity in HDACi-treated cells. Glioma cells were treated with 10 mM VPA, 10 μM SAHA alone or in combination with the caspase inhibitor Q-VD-OPh (5 μM) for 24 h. Cell lysates were incubated with Ac-DEVD-AFC fluorogenic substrate up to 8 h, and its cleavage was measured hourly using a fluorometer. Bars depict mean±S.E.M. from four independent experiments. Statistical analysis was performed using the Student's T-test by comparing treated versus non-treated (NT) cells (*P<0.05, **P< 0.01)
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4237242&req=5

fig2: VPA and SAHA induce nuclear condensation and caspase activation in glioma cell lines. (a) Nuclear morphology of U87-MG, U373-MG and LN229 after 24-h treatment with 10 mM VPA, 10 μM SAHA or 100 ng/ml TRAIL. After treatment, cells were fixed in 4% PFA and nuclei were stained with Hoechst 33342. Condensed and abnormal nuclei are indicated with yellow arrowheads. Scale bar= 50 μm. (b) Analysis of caspase-9, caspase-3 and fodrin expression and cleavage on glioma cell lines after 24-h treatment with 10 mM VPA and 10 μM SAHA by western blot. Membrane was reprobed with an anti-α-tubulin antibody to verify equal loading. A representative blot from three independent experiments is shown. (c) Caspase-3 activity in HDACi-treated cells. Glioma cells were treated with 10 mM VPA, 10 μM SAHA alone or in combination with the caspase inhibitor Q-VD-OPh (5 μM) for 24 h. Cell lysates were incubated with Ac-DEVD-AFC fluorogenic substrate up to 8 h, and its cleavage was measured hourly using a fluorometer. Bars depict mean±S.E.M. from four independent experiments. Statistical analysis was performed using the Student's T-test by comparing treated versus non-treated (NT) cells (*P<0.05, **P< 0.01)

Mentions: Our results showed that SAHA and VPA decrease cell viability dose dependently. Therefore, to assess the participation of the apoptotic cell death pathway on this effect, we analyzed the nuclear morphology of glioma cells after 24 h of treatment. As a reference, apoptosis inducer TRAIL was added to cells at 100 ng/ml. Nuclear staining with Hoechst showed that HDACi cause nuclear condensation and abnormal nuclear morphology in some cells (Figure 2a). However, nuclear fragmentation was absent and it was very infrequent in TRAIL-treated cells. Therefore, glioma cells do not show a typical apoptotic nuclear fragmentation after VPA and SAHA treatment. In addition, HDACi induced an increase in nuclear size in U87-MG and LN229 cells, indicating that chromatin was remodeled due to histone acetylation (Supplementary Figure 1).


Histone deacetylase inhibitors promote glioma cell death by G2 checkpoint abrogation leading to mitotic catastrophe.

Cornago M, Garcia-Alberich C, Blasco-Angulo N, Vall-Llaura N, Nager M, Herreros J, Comella JX, Sanchis D, Llovera M - Cell Death Dis (2014)

VPA and SAHA induce nuclear condensation and caspase activation in glioma cell lines. (a) Nuclear morphology of U87-MG, U373-MG and LN229 after 24-h treatment with 10 mM VPA, 10 μM SAHA or 100 ng/ml TRAIL. After treatment, cells were fixed in 4% PFA and nuclei were stained with Hoechst 33342. Condensed and abnormal nuclei are indicated with yellow arrowheads. Scale bar= 50 μm. (b) Analysis of caspase-9, caspase-3 and fodrin expression and cleavage on glioma cell lines after 24-h treatment with 10 mM VPA and 10 μM SAHA by western blot. Membrane was reprobed with an anti-α-tubulin antibody to verify equal loading. A representative blot from three independent experiments is shown. (c) Caspase-3 activity in HDACi-treated cells. Glioma cells were treated with 10 mM VPA, 10 μM SAHA alone or in combination with the caspase inhibitor Q-VD-OPh (5 μM) for 24 h. Cell lysates were incubated with Ac-DEVD-AFC fluorogenic substrate up to 8 h, and its cleavage was measured hourly using a fluorometer. Bars depict mean±S.E.M. from four independent experiments. Statistical analysis was performed using the Student's T-test by comparing treated versus non-treated (NT) cells (*P<0.05, **P< 0.01)
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4237242&req=5

fig2: VPA and SAHA induce nuclear condensation and caspase activation in glioma cell lines. (a) Nuclear morphology of U87-MG, U373-MG and LN229 after 24-h treatment with 10 mM VPA, 10 μM SAHA or 100 ng/ml TRAIL. After treatment, cells were fixed in 4% PFA and nuclei were stained with Hoechst 33342. Condensed and abnormal nuclei are indicated with yellow arrowheads. Scale bar= 50 μm. (b) Analysis of caspase-9, caspase-3 and fodrin expression and cleavage on glioma cell lines after 24-h treatment with 10 mM VPA and 10 μM SAHA by western blot. Membrane was reprobed with an anti-α-tubulin antibody to verify equal loading. A representative blot from three independent experiments is shown. (c) Caspase-3 activity in HDACi-treated cells. Glioma cells were treated with 10 mM VPA, 10 μM SAHA alone or in combination with the caspase inhibitor Q-VD-OPh (5 μM) for 24 h. Cell lysates were incubated with Ac-DEVD-AFC fluorogenic substrate up to 8 h, and its cleavage was measured hourly using a fluorometer. Bars depict mean±S.E.M. from four independent experiments. Statistical analysis was performed using the Student's T-test by comparing treated versus non-treated (NT) cells (*P<0.05, **P< 0.01)
Mentions: Our results showed that SAHA and VPA decrease cell viability dose dependently. Therefore, to assess the participation of the apoptotic cell death pathway on this effect, we analyzed the nuclear morphology of glioma cells after 24 h of treatment. As a reference, apoptosis inducer TRAIL was added to cells at 100 ng/ml. Nuclear staining with Hoechst showed that HDACi cause nuclear condensation and abnormal nuclear morphology in some cells (Figure 2a). However, nuclear fragmentation was absent and it was very infrequent in TRAIL-treated cells. Therefore, glioma cells do not show a typical apoptotic nuclear fragmentation after VPA and SAHA treatment. In addition, HDACi induced an increase in nuclear size in U87-MG and LN229 cells, indicating that chromatin was remodeled due to histone acetylation (Supplementary Figure 1).

Bottom Line: They have multiple effects, such as inducing the production of reactive oxygen species (ROS) and activating the mitochondrial apoptotic pathway, nevertheless cell death is not prevented by the pan-caspase inhibitor Q-VD-OPh.Importantly, we found that HDACi alter cell cycle progression by decreasing the expression of G2 checkpoint kinases Wee1 and checkpoint kinase 1 (Chk1).In addition, HDACi reduce the expression of proteins involved in DNA repair (Rad51), mitotic spindle formation (TPX2) and chromosome segregation (Survivin) in glioma cells and in human glioblastoma multiforme primary cultures.

View Article: PubMed Central - PubMed

Affiliation: Cell Signaling and Apoptosis Group, Institut de Recerca Biomèdica de Lleida (IRBLleida), Universitat de Lleida, Lleida, Spain.

ABSTRACT
Glioblastoma multiforme is resistant to conventional anti-tumoral treatments due to its infiltrative nature and capability of relapse; therefore, research efforts focus on characterizing gliomagenesis and identifying molecular targets useful on therapy. New therapeutic strategies are being tested in patients, such as Histone deacetylase inhibitors (HDACi) either alone or in combination with other therapies. Here two HDACi included in clinical trials have been tested, suberanilohydroxamic acid (SAHA) and valproic acid (VPA), to characterize their effects on glioma cell growth in vitro and to determine the molecular changes that promote cancer cell death. We found that both HDACi reduce glioma cell viability, proliferation and clonogenicity. They have multiple effects, such as inducing the production of reactive oxygen species (ROS) and activating the mitochondrial apoptotic pathway, nevertheless cell death is not prevented by the pan-caspase inhibitor Q-VD-OPh. Importantly, we found that HDACi alter cell cycle progression by decreasing the expression of G2 checkpoint kinases Wee1 and checkpoint kinase 1 (Chk1). In addition, HDACi reduce the expression of proteins involved in DNA repair (Rad51), mitotic spindle formation (TPX2) and chromosome segregation (Survivin) in glioma cells and in human glioblastoma multiforme primary cultures. Therefore, HDACi treatment causes glioma cell entry into mitosis before DNA damage could be repaired and to the formation of an aberrant mitotic spindle that results in glioma cell death through mitotic catastrophe-induced apoptosis.

Show MeSH
Related in: MedlinePlus