Glycogen synthase kinase 3β inhibition sensitizes human glioblastoma cells to temozolomide by affecting O 6 -methylguanine DNA methyltransferase promoter methylation via c-Myc signaling

Abstract

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.

Glycogen Synthase Kinase 3β Sustains Invasion of Glioblastoma via the Focal Adhesion Kinase, Rac1, and c-Jun N-Terminal Kinase-Mediated Pathway

“…In addition, changes were observed in the cellular microstructures of lamellipodia and invadopodia, which coordinate to influence the motility and invasion of glioblastoma cells, respectively. We have previously reported that deregulated GSK3b is important for the survival and proliferation of glioblastoma cells and confers them with resistance to chemotherapeutic agents and radiation (12,13). Taken together, these results establish GSK3b as a therapeutic target with multiple functional roles in glioblastoma.…”

“…We have also demonstrated that a specific GSK3b inhibitor (AR-A014418; ref. 18) synergizes with temozolomide against glioblastoma cells by silencing O 6 -methylguanine DNA methyltransferase expression via c-Myc-mediated promoter methylation (13). GSK3b is, therefore, heavily implicated in the two major pathologic characteristics of glioblastoma cells, invasive activity and therapy resistance, through pivotal oncogenic pathways.…”

“…We and others have demonstrated that inhibition of GSK3b attenuates the survival and proliferation of glioblastoma cells by modulating specific molecular pathways (11,12), thus sensitizing them to chemotherapeutic agents and radiation (12,13). It has also been reported that GSK3b inhibition with lithium and indirubins reduces the invasive potential of glioblastoma cells (14,15).…”

Quercetin sensitizes human glioblastoma cells to temozolomide in vitro via inhibition of Hsp27

“…O6-methylguanine DNA methyltransferase (MGMT) expression and MGMT gene promoter methylation are considered to be implicated in chemosensitivity to TMZ [5] . Studies have revealed that MGMT is not the only factor that regulates chemosensitivity to TMZ [6][7][8][9] .…”

KLRC3, a Natural Killer receptor gene, is a key factor involved in glioblastoma tumourigenesis and aggressiveness

“…The decrease in DAP12 expression level in shKLRC3 cells associated with the significant decrease in the GSK3β protein strongly supports the hypothesis of a link between KLRC3 and DAP12 in the regulation of the glioblastoma aggressiveness, particularly in the radioresistance phenotype. GSK3β has been linked to glioma invasion ability 26 and its inhibition leads to an increase in glioma sensitivity to Temozolomide 27 and to radiotherapy 28. It has been also demonstrated that inhibition of GSK3β in GSC isolated from GBM patient samples attenuates cell proliferation, suggesting GSK3β as a potential therapeutical target in GSC 29.…”