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A molecular study of pathways involved in the inhibition of cell proliferation in neuroblastoma B65 cells by the GSK-3 inhibitors lithium and SB-415286.

Pizarro JG, Folch J, Esparza JL, Jordan J, Pallàs M, Camins A - J. Cell. Mol. Med. (2008)

Bottom Line: Treatment of B65 cells with either drug administered separately caused a decrease in cell proliferation that was associated with G(2)/M cell cycle arrest.Likewise, GSK-3 inhibitors did not affect the mitochondrial release of apoptosis inducing factor (AIF).We conclude that inhibitors of GSK-3 induced cell-cycle arrest, mediated by the phosphorylation of cdc2 and, in the case of SB-415286, SIRT2 expression, which induced apoptosis in a caspase-independent manner.

View Article: PubMed Central - PubMed

Affiliation: Unitat de Farmacologia i Farmacognòsia, Facultat de Farmàcia, Institut de Biomedicina and Centros de Investigación Biomédica en Red de Enfermedades Neurodegenerativas, Universitat de Barcelona, Nucli Universitari de Pedralbes, Barcelona, Spain.

ABSTRACT
Pharmacological GSK-3 inhibitors are potential drugs for the treatment of neurodegenerative diseases, cancer and diabetes. We examined the antiproliferative effects of two GSK-3 inhibitors, lithium and SB-415286, on B65 neuroblastoma cell line. Treatment of B65 cells with either drug administered separately caused a decrease in cell proliferation that was associated with G(2)/M cell cycle arrest. Cell-cycle proteins such as cyclins D, E, A, cdk4 and cdk2 were up-regulated. Since lithium and SB-415286-induced G(2)/M arrest we studied changes in the expression of proteins involved in this phase, specifically cyclin B, cdc2 and the phosphorylated form of this protein (tyr15-cdc2). Both drugs increased the expression of tyr15-cdc2, thus inhibiting mitosis. On the other hand, SB-415286 increased the expression of SIRT2, involved in the regulation of proliferation. Moreover, cell-cycle arrest mediated by SB-415286 was accompanied by apoptosis that was not prevented by 100 microM of zVAD-fmk (benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone), a pan-caspase inhibitor. Likewise, GSK-3 inhibitors did not affect the mitochondrial release of apoptosis inducing factor (AIF). We conclude that inhibitors of GSK-3 induced cell-cycle arrest, mediated by the phosphorylation of cdc2 and, in the case of SB-415286, SIRT2 expression, which induced apoptosis in a caspase-independent manner.

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Representative flow cytometric analysis of cell cycle inhibition by Li+ and SB-415286.
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fig06: Representative flow cytometric analysis of cell cycle inhibition by Li+ and SB-415286.

Mentions: To examine the phase of the cell-cycle in which cells were arrested after treatment with Li+ (1–15 mM) and SB-415286 (1–44 μM), DNA was stained by propidium iodide and analysed by flow cytometry (Fig. 6). Lithium increased the fraction of cells in the S phase of the cell cycle at 15 mM (about 50%; ***P < 0.001) (Table 2). The number of cells arrested in the G2/M phase increased at a concentration of 15 mM (about 38% at the highest concentration evaluated). On the other hand, when B65 cells were treated with SB-415286, the fraction of cells in the G2/M phase increased in a dose-dependent manner. For example, when cells were exposed to 20 μM SB-415286, the fraction of cells in G2/M was about 42% (***P < 0.001), at 30 μM about 73% (***P < 0.001) and at 44 μM about 93%*** (P < 0.001) (Fig. 6).


A molecular study of pathways involved in the inhibition of cell proliferation in neuroblastoma B65 cells by the GSK-3 inhibitors lithium and SB-415286.

Pizarro JG, Folch J, Esparza JL, Jordan J, Pallàs M, Camins A - J. Cell. Mol. Med. (2008)

Representative flow cytometric analysis of cell cycle inhibition by Li+ and SB-415286.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4516538&req=5

fig06: Representative flow cytometric analysis of cell cycle inhibition by Li+ and SB-415286.
Mentions: To examine the phase of the cell-cycle in which cells were arrested after treatment with Li+ (1–15 mM) and SB-415286 (1–44 μM), DNA was stained by propidium iodide and analysed by flow cytometry (Fig. 6). Lithium increased the fraction of cells in the S phase of the cell cycle at 15 mM (about 50%; ***P < 0.001) (Table 2). The number of cells arrested in the G2/M phase increased at a concentration of 15 mM (about 38% at the highest concentration evaluated). On the other hand, when B65 cells were treated with SB-415286, the fraction of cells in the G2/M phase increased in a dose-dependent manner. For example, when cells were exposed to 20 μM SB-415286, the fraction of cells in G2/M was about 42% (***P < 0.001), at 30 μM about 73% (***P < 0.001) and at 44 μM about 93%*** (P < 0.001) (Fig. 6).

Bottom Line: Treatment of B65 cells with either drug administered separately caused a decrease in cell proliferation that was associated with G(2)/M cell cycle arrest.Likewise, GSK-3 inhibitors did not affect the mitochondrial release of apoptosis inducing factor (AIF).We conclude that inhibitors of GSK-3 induced cell-cycle arrest, mediated by the phosphorylation of cdc2 and, in the case of SB-415286, SIRT2 expression, which induced apoptosis in a caspase-independent manner.

View Article: PubMed Central - PubMed

Affiliation: Unitat de Farmacologia i Farmacognòsia, Facultat de Farmàcia, Institut de Biomedicina and Centros de Investigación Biomédica en Red de Enfermedades Neurodegenerativas, Universitat de Barcelona, Nucli Universitari de Pedralbes, Barcelona, Spain.

ABSTRACT
Pharmacological GSK-3 inhibitors are potential drugs for the treatment of neurodegenerative diseases, cancer and diabetes. We examined the antiproliferative effects of two GSK-3 inhibitors, lithium and SB-415286, on B65 neuroblastoma cell line. Treatment of B65 cells with either drug administered separately caused a decrease in cell proliferation that was associated with G(2)/M cell cycle arrest. Cell-cycle proteins such as cyclins D, E, A, cdk4 and cdk2 were up-regulated. Since lithium and SB-415286-induced G(2)/M arrest we studied changes in the expression of proteins involved in this phase, specifically cyclin B, cdc2 and the phosphorylated form of this protein (tyr15-cdc2). Both drugs increased the expression of tyr15-cdc2, thus inhibiting mitosis. On the other hand, SB-415286 increased the expression of SIRT2, involved in the regulation of proliferation. Moreover, cell-cycle arrest mediated by SB-415286 was accompanied by apoptosis that was not prevented by 100 microM of zVAD-fmk (benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone), a pan-caspase inhibitor. Likewise, GSK-3 inhibitors did not affect the mitochondrial release of apoptosis inducing factor (AIF). We conclude that inhibitors of GSK-3 induced cell-cycle arrest, mediated by the phosphorylation of cdc2 and, in the case of SB-415286, SIRT2 expression, which induced apoptosis in a caspase-independent manner.

Show MeSH
Related in: MedlinePlus