Glycogen synthase kinase 3β (GSK3β) is a
serine/threonine protein kinase involved in human
cancers including
glioblastoma. We have previously demonstrated that GSK3β inhibition enhances
temozolomide effect in
glioma cells. In this report, we investigated the molecular mechanisms of sensitization of
glioblastoma cells to
temozolomide by GSK3β inhibition, focusing on
O(6)-methylguanine DNA methyltransferase (MGMT) gene silencing.
Glioblastoma tissues from patients treated with the GSK3β-inhibiting drugs were subjected to immunohistochemistry and methylation-specific PCR assay. Human
glioblastoma cell lines T98G, U138, U251 and U87 were treated with a small-molecule GSK3β inhibitor,
AR-A014418 or GSK3β-specific
small interfering RNA. The combined effect of
temozolomide and
AR-A014418 on cell proliferation was determined by
AlamarBlue assay and an isobologram method. MGMT promoter methylation was estimated by methylation-specific PCR and MethyLight assay. MGMT gene expression was evaluated by real-time quantitative
reverse transcriptase-PCR. c-Myc and
DNA (cytosine-5)-methyltransferase 3A binding to the MGMT promoter was estimated by
chromatin immunoprecipitation assay. GSK3β inhibition decreased phosphorylation of
glycogen synthase and reduced MGMT expression and increased MGMT promoter methylation in clinical
tumors. In
glioblastoma cell lines, GSK3β inhibition decreased cell viability, enhanced
temozolomide effect and downregulated MGMT expression with relevant changes in the methylation levels of the MGMT promoter. Here, we showed for the first time that c-Myc binds to the MGMT promoter with consequent recruitment of
DNA (cytosine-5)-methyltransferase 3A, regulating the levels of MGMT promoter methylation. The results of this study suggest that GSK3β inhibition enhances
temozolomide effect by silencing MGMT expression via c-Myc-mediated promoter methylation.