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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.

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Related in: MedlinePlus

HDACi promote DNA fragmentacion in glioma cell lines, which is dependent on caspase activation. (a) DNA fragmentation analysis on glioma cells treated for 48 h with 10 μM SAHA combined or not with the pan-caspase inhibitor Q-VD-OPh (5 μM) by PFGE (HMWF, upper picture) and ladder (LMWF, lower picture) procedures. As a positive control, LN229 cells were treated with 1 μM STS for 48 h. (b) Analysis of the effect of BCL-X overexpression on DNA fragmentation (HMWF and LMWF) induced by 10 mM VPA or 10 μM SAHA on U251-MG glioma cells. Bcl-xL expression was checked by western blot (upper panel). (c) Downregulation of CAD expression with specific shRNA (4 and 5) blocks the formation of 50- Kb fragments and low-molecular weight fragmentation on U251-MG cells treated for 48 h with 10 μM SAHA. (d) Western blot of CAD on U251-MG cells transduced with lentiviral vectors containing CAD shRNA or scrambled sequence (Scr). Equal loading was verified by GAPDH detection of the same membrane (lower panel). Each DNA integrity assay shown in a, b and c was repeated three times and a representative image is shown. (e) Cell viability analysis by cell counting using trypan blue exclusion on glioma cells treated with 10 μM SAHA or 5 μM Q-VD-OPh or both combined for 48 h. Bars depict mean±S.E.M. from four independent experiments. Statistical analysis was performed by the Student's T-test, no significant differences (n.s.) were obtained by comparing SAHA and SAHA+ Q-VD-treated cells
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fig3: HDACi promote DNA fragmentacion in glioma cell lines, which is dependent on caspase activation. (a) DNA fragmentation analysis on glioma cells treated for 48 h with 10 μM SAHA combined or not with the pan-caspase inhibitor Q-VD-OPh (5 μM) by PFGE (HMWF, upper picture) and ladder (LMWF, lower picture) procedures. As a positive control, LN229 cells were treated with 1 μM STS for 48 h. (b) Analysis of the effect of BCL-X overexpression on DNA fragmentation (HMWF and LMWF) induced by 10 mM VPA or 10 μM SAHA on U251-MG glioma cells. Bcl-xL expression was checked by western blot (upper panel). (c) Downregulation of CAD expression with specific shRNA (4 and 5) blocks the formation of 50- Kb fragments and low-molecular weight fragmentation on U251-MG cells treated for 48 h with 10 μM SAHA. (d) Western blot of CAD on U251-MG cells transduced with lentiviral vectors containing CAD shRNA or scrambled sequence (Scr). Equal loading was verified by GAPDH detection of the same membrane (lower panel). Each DNA integrity assay shown in a, b and c was repeated three times and a representative image is shown. (e) Cell viability analysis by cell counting using trypan blue exclusion on glioma cells treated with 10 μM SAHA or 5 μM Q-VD-OPh or both combined for 48 h. Bars depict mean±S.E.M. from four independent experiments. Statistical analysis was performed by the Student's T-test, no significant differences (n.s.) were obtained by comparing SAHA and SAHA+ Q-VD-treated cells

Mentions: Next, we checked whether the activation of the caspase cascade by HDACi promoted DNA fragmentation. We analyzed DNA integrity by Pulsed-field gel electrophoresis (PFGE) and agarose gel electrophoresis (Figure 3a). SAHA treatment for 48 h caused the formation of 50- kb DNA fragments and the final DNA degradation in a smear pattern (Figure 3a). Interestingly, the addition of a pan-caspase inhibitor (Q-VD-OPh) blocked both high- and low-molecular weight DNA fragmentation, indicating that caspase activation was necessary for DNA degradation. Staurosporine (STS), an apoptosis inducer, was added for 24 h to U251-MG cells as a positive control for DNA fragmentation. The formation of 50- Kbp DNA fragments was observed, but typical oligonucleosomal laddering was not detected (Figure 3a). These results indicated that U251-MG cells exposed to an apoptosis inducer do not perform DNA internucleosomal fragmentation. To further corroborate the implication of the mitochondrial apoptotic pathway in SAHA-induced DNA degradation, we overexpressed the antiapoptotic gene BCL-X in U251-MG glioma cells (Figure 3b). We observed that Bcl-xL-overexpressing cells were protected against the induction of DNA degradation by SAHA, further suggesting the involvement of the intrinsic apoptotic pathway in SAHA effects.


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)

HDACi promote DNA fragmentacion in glioma cell lines, which is dependent on caspase activation. (a) DNA fragmentation analysis on glioma cells treated for 48 h with 10 μM SAHA combined or not with the pan-caspase inhibitor Q-VD-OPh (5 μM) by PFGE (HMWF, upper picture) and ladder (LMWF, lower picture) procedures. As a positive control, LN229 cells were treated with 1 μM STS for 48 h. (b) Analysis of the effect of BCL-X overexpression on DNA fragmentation (HMWF and LMWF) induced by 10 mM VPA or 10 μM SAHA on U251-MG glioma cells. Bcl-xL expression was checked by western blot (upper panel). (c) Downregulation of CAD expression with specific shRNA (4 and 5) blocks the formation of 50- Kb fragments and low-molecular weight fragmentation on U251-MG cells treated for 48 h with 10 μM SAHA. (d) Western blot of CAD on U251-MG cells transduced with lentiviral vectors containing CAD shRNA or scrambled sequence (Scr). Equal loading was verified by GAPDH detection of the same membrane (lower panel). Each DNA integrity assay shown in a, b and c was repeated three times and a representative image is shown. (e) Cell viability analysis by cell counting using trypan blue exclusion on glioma cells treated with 10 μM SAHA or 5 μM Q-VD-OPh or both combined for 48 h. Bars depict mean±S.E.M. from four independent experiments. Statistical analysis was performed by the Student's T-test, no significant differences (n.s.) were obtained by comparing SAHA and SAHA+ Q-VD-treated cells
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig3: HDACi promote DNA fragmentacion in glioma cell lines, which is dependent on caspase activation. (a) DNA fragmentation analysis on glioma cells treated for 48 h with 10 μM SAHA combined or not with the pan-caspase inhibitor Q-VD-OPh (5 μM) by PFGE (HMWF, upper picture) and ladder (LMWF, lower picture) procedures. As a positive control, LN229 cells were treated with 1 μM STS for 48 h. (b) Analysis of the effect of BCL-X overexpression on DNA fragmentation (HMWF and LMWF) induced by 10 mM VPA or 10 μM SAHA on U251-MG glioma cells. Bcl-xL expression was checked by western blot (upper panel). (c) Downregulation of CAD expression with specific shRNA (4 and 5) blocks the formation of 50- Kb fragments and low-molecular weight fragmentation on U251-MG cells treated for 48 h with 10 μM SAHA. (d) Western blot of CAD on U251-MG cells transduced with lentiviral vectors containing CAD shRNA or scrambled sequence (Scr). Equal loading was verified by GAPDH detection of the same membrane (lower panel). Each DNA integrity assay shown in a, b and c was repeated three times and a representative image is shown. (e) Cell viability analysis by cell counting using trypan blue exclusion on glioma cells treated with 10 μM SAHA or 5 μM Q-VD-OPh or both combined for 48 h. Bars depict mean±S.E.M. from four independent experiments. Statistical analysis was performed by the Student's T-test, no significant differences (n.s.) were obtained by comparing SAHA and SAHA+ Q-VD-treated cells
Mentions: Next, we checked whether the activation of the caspase cascade by HDACi promoted DNA fragmentation. We analyzed DNA integrity by Pulsed-field gel electrophoresis (PFGE) and agarose gel electrophoresis (Figure 3a). SAHA treatment for 48 h caused the formation of 50- kb DNA fragments and the final DNA degradation in a smear pattern (Figure 3a). Interestingly, the addition of a pan-caspase inhibitor (Q-VD-OPh) blocked both high- and low-molecular weight DNA fragmentation, indicating that caspase activation was necessary for DNA degradation. Staurosporine (STS), an apoptosis inducer, was added for 24 h to U251-MG cells as a positive control for DNA fragmentation. The formation of 50- Kbp DNA fragments was observed, but typical oligonucleosomal laddering was not detected (Figure 3a). These results indicated that U251-MG cells exposed to an apoptosis inducer do not perform DNA internucleosomal fragmentation. To further corroborate the implication of the mitochondrial apoptotic pathway in SAHA-induced DNA degradation, we overexpressed the antiapoptotic gene BCL-X in U251-MG glioma cells (Figure 3b). We observed that Bcl-xL-overexpressing cells were protected against the induction of DNA degradation by SAHA, further suggesting the involvement of the intrinsic apoptotic pathway in SAHA effects.

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