Limits...
Inhibition of SIRT2 in merlin/NF2-mutant Schwann cells triggers necrosis.

Petrilli A, Bott M, Fernández-Valle C - Oncotarget (2013)

Bottom Line: Pharmacological inhibition of SIRT2 decreases merlin-mutant MSC viability in a dose dependent manner without substantially reducing wild-type MSC viability.Inhibition of SIRT2 activity in merlin-mutant MSC is accompanied by release of lactate dehydrogenase and high mobility group box 1 protein into the medium in the absence of significant apoptosis, autophagy, or cell cycle arrest.These findings suggest that SIRT2 inhibition triggers necrosis of merlin-mutant MSCs and that SIRT2 is a potential NF2 drug target.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Science, College of Medicine, University of Central Florida, Lake Nona-Orlando, Florida, USA.

ABSTRACT
Mutations in the NF2 gene cause Neurofibromatosis Type 2 (NF2), a disorder characterized by the development of schwannomas, meningiomas and ependymomas in the nervous system. Merlin, a tumor suppressor encoded by the NF2 gene, modulates activity of many essential signaling pathways. Yet despite increasing knowledge of merlin function, there are no NF2 drug therapies. In a pilot high-throughput screen of the Library of Pharmacologically Active Compounds, we assayed for compounds capable of reducing viability of mouse Schwann cells (MSC) with Nf2 inactivation as a cellular model for human NF2 schwannomas. AGK2, a SIRT2 (sirtuin 2) inhibitor, was identified as a candidate compound. SIRT2 is one of seven mammalian sirtuins that are NAD+-dependent protein deacetylases. We show that merlin-mutant MSC have higher expression levels of SIRT2 and lower levels of overall lysine acetylation than wild-type control MSC. Pharmacological inhibition of SIRT2 decreases merlin-mutant MSC viability in a dose dependent manner without substantially reducing wild-type MSC viability. Inhibition of SIRT2 activity in merlin-mutant MSC is accompanied by release of lactate dehydrogenase and high mobility group box 1 protein into the medium in the absence of significant apoptosis, autophagy, or cell cycle arrest. These findings suggest that SIRT2 inhibition triggers necrosis of merlin-mutant MSCs and that SIRT2 is a potential NF2 drug target.

Show MeSH

Related in: MedlinePlus

SIRT2 Inhibition With AGK2 and AK1 Selectively Decreases Merlin-Mutant MSC Viabilitya) AGK2 dose response curve for cell viability. Merlin-mutant MSC were seeded at 5,000 cells/well in 384-well dishes and were incubated with increasing concentrations of AGK2 for 24 hours. Cell viability was assessed with the CellTiter-Fluor assay. DMSO control was considered 100% viability. Rapamycin (RM) (50 μM) was used as positive control for cell death. Graph represents the mean ± SEM of 3 independent experiments analyzed together (n=96), IC50=9.01μM, log [inhibitor] vs. response, variable slope (four parameters). b) AGK2 dose response for control MSC viability. Cell viability was measured as in (a). Graph represents the mean ± SEM (n=36). **P<0.01; ***P<0.001;****P<0.0001 determined by one-way ANOVA using Turkey's multiple comparison test. c) AK1 dose response curve for merlin-mutant MSC viability. Cell viability assessed as in (a). Graph represents the mean ± SEM of 3 independent experiments analyzed together (n=96), IC50=26.1μM, log [inhibitor] vs. response, variable slope (four parameters). d) AK1 dose viability response for control MSC. Cell viability was measured as in (a). Graph represents the mean ± SEM (n=20). ***P<0.001; ****P<0.0001 determined by one-way ANOVA using Turkey's multiple comparison test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3926832&req=5

Figure 2: SIRT2 Inhibition With AGK2 and AK1 Selectively Decreases Merlin-Mutant MSC Viabilitya) AGK2 dose response curve for cell viability. Merlin-mutant MSC were seeded at 5,000 cells/well in 384-well dishes and were incubated with increasing concentrations of AGK2 for 24 hours. Cell viability was assessed with the CellTiter-Fluor assay. DMSO control was considered 100% viability. Rapamycin (RM) (50 μM) was used as positive control for cell death. Graph represents the mean ± SEM of 3 independent experiments analyzed together (n=96), IC50=9.01μM, log [inhibitor] vs. response, variable slope (four parameters). b) AGK2 dose response for control MSC viability. Cell viability was measured as in (a). Graph represents the mean ± SEM (n=36). **P<0.01; ***P<0.001;****P<0.0001 determined by one-way ANOVA using Turkey's multiple comparison test. c) AK1 dose response curve for merlin-mutant MSC viability. Cell viability assessed as in (a). Graph represents the mean ± SEM of 3 independent experiments analyzed together (n=96), IC50=26.1μM, log [inhibitor] vs. response, variable slope (four parameters). d) AK1 dose viability response for control MSC. Cell viability was measured as in (a). Graph represents the mean ± SEM (n=20). ***P<0.001; ****P<0.0001 determined by one-way ANOVA using Turkey's multiple comparison test.

Mentions: To evaluate selectivity of SIRT2 inhibition for merlin-mutant MSC viability, we conducted a dose response study of AGK2 using CellTiter-Fluor assay. We found that a 24 hour exposure to AGK2 decreased merlin-mutant MSC viability in a dose-dependent manner with an IC50= 9.0 μM (Fig. 2a). In contrast, AGK2 did not decrease control MSC viability as effectively as for merlin-mutant cells (Fig. 2b). At 10 μM AGK2, merlin-mutant cells retained 45.8 ± 0.7 % viability compared to control MSC that retained 70.9 ± 1.8 % viability.


Inhibition of SIRT2 in merlin/NF2-mutant Schwann cells triggers necrosis.

Petrilli A, Bott M, Fernández-Valle C - Oncotarget (2013)

SIRT2 Inhibition With AGK2 and AK1 Selectively Decreases Merlin-Mutant MSC Viabilitya) AGK2 dose response curve for cell viability. Merlin-mutant MSC were seeded at 5,000 cells/well in 384-well dishes and were incubated with increasing concentrations of AGK2 for 24 hours. Cell viability was assessed with the CellTiter-Fluor assay. DMSO control was considered 100% viability. Rapamycin (RM) (50 μM) was used as positive control for cell death. Graph represents the mean ± SEM of 3 independent experiments analyzed together (n=96), IC50=9.01μM, log [inhibitor] vs. response, variable slope (four parameters). b) AGK2 dose response for control MSC viability. Cell viability was measured as in (a). Graph represents the mean ± SEM (n=36). **P<0.01; ***P<0.001;****P<0.0001 determined by one-way ANOVA using Turkey's multiple comparison test. c) AK1 dose response curve for merlin-mutant MSC viability. Cell viability assessed as in (a). Graph represents the mean ± SEM of 3 independent experiments analyzed together (n=96), IC50=26.1μM, log [inhibitor] vs. response, variable slope (four parameters). d) AK1 dose viability response for control MSC. Cell viability was measured as in (a). Graph represents the mean ± SEM (n=20). ***P<0.001; ****P<0.0001 determined by one-way ANOVA using Turkey's multiple comparison test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3926832&req=5

Figure 2: SIRT2 Inhibition With AGK2 and AK1 Selectively Decreases Merlin-Mutant MSC Viabilitya) AGK2 dose response curve for cell viability. Merlin-mutant MSC were seeded at 5,000 cells/well in 384-well dishes and were incubated with increasing concentrations of AGK2 for 24 hours. Cell viability was assessed with the CellTiter-Fluor assay. DMSO control was considered 100% viability. Rapamycin (RM) (50 μM) was used as positive control for cell death. Graph represents the mean ± SEM of 3 independent experiments analyzed together (n=96), IC50=9.01μM, log [inhibitor] vs. response, variable slope (four parameters). b) AGK2 dose response for control MSC viability. Cell viability was measured as in (a). Graph represents the mean ± SEM (n=36). **P<0.01; ***P<0.001;****P<0.0001 determined by one-way ANOVA using Turkey's multiple comparison test. c) AK1 dose response curve for merlin-mutant MSC viability. Cell viability assessed as in (a). Graph represents the mean ± SEM of 3 independent experiments analyzed together (n=96), IC50=26.1μM, log [inhibitor] vs. response, variable slope (four parameters). d) AK1 dose viability response for control MSC. Cell viability was measured as in (a). Graph represents the mean ± SEM (n=20). ***P<0.001; ****P<0.0001 determined by one-way ANOVA using Turkey's multiple comparison test.
Mentions: To evaluate selectivity of SIRT2 inhibition for merlin-mutant MSC viability, we conducted a dose response study of AGK2 using CellTiter-Fluor assay. We found that a 24 hour exposure to AGK2 decreased merlin-mutant MSC viability in a dose-dependent manner with an IC50= 9.0 μM (Fig. 2a). In contrast, AGK2 did not decrease control MSC viability as effectively as for merlin-mutant cells (Fig. 2b). At 10 μM AGK2, merlin-mutant cells retained 45.8 ± 0.7 % viability compared to control MSC that retained 70.9 ± 1.8 % viability.

Bottom Line: Pharmacological inhibition of SIRT2 decreases merlin-mutant MSC viability in a dose dependent manner without substantially reducing wild-type MSC viability.Inhibition of SIRT2 activity in merlin-mutant MSC is accompanied by release of lactate dehydrogenase and high mobility group box 1 protein into the medium in the absence of significant apoptosis, autophagy, or cell cycle arrest.These findings suggest that SIRT2 inhibition triggers necrosis of merlin-mutant MSCs and that SIRT2 is a potential NF2 drug target.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Science, College of Medicine, University of Central Florida, Lake Nona-Orlando, Florida, USA.

ABSTRACT
Mutations in the NF2 gene cause Neurofibromatosis Type 2 (NF2), a disorder characterized by the development of schwannomas, meningiomas and ependymomas in the nervous system. Merlin, a tumor suppressor encoded by the NF2 gene, modulates activity of many essential signaling pathways. Yet despite increasing knowledge of merlin function, there are no NF2 drug therapies. In a pilot high-throughput screen of the Library of Pharmacologically Active Compounds, we assayed for compounds capable of reducing viability of mouse Schwann cells (MSC) with Nf2 inactivation as a cellular model for human NF2 schwannomas. AGK2, a SIRT2 (sirtuin 2) inhibitor, was identified as a candidate compound. SIRT2 is one of seven mammalian sirtuins that are NAD+-dependent protein deacetylases. We show that merlin-mutant MSC have higher expression levels of SIRT2 and lower levels of overall lysine acetylation than wild-type control MSC. Pharmacological inhibition of SIRT2 decreases merlin-mutant MSC viability in a dose dependent manner without substantially reducing wild-type MSC viability. Inhibition of SIRT2 activity in merlin-mutant MSC is accompanied by release of lactate dehydrogenase and high mobility group box 1 protein into the medium in the absence of significant apoptosis, autophagy, or cell cycle arrest. These findings suggest that SIRT2 inhibition triggers necrosis of merlin-mutant MSCs and that SIRT2 is a potential NF2 drug target.

Show MeSH
Related in: MedlinePlus