title: Glycogen synthase kinase 3β inhibition sensitizes human glioblastoma cells to temozolomide by affecting O6-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.
authors.affiliation: Department of Neurosurgery, Division of Neuroscience, Graduate School of Medical Science, Kanazawa University, 13-1 Takara-Machi, 920-8641, Kanazawa, Ishikawa, Japan.
authors.forename: Ilya V
authors.initials: IV
authors.lastname: Pyko
authors.forename: Mitsutoshi
authors.initials: M
authors.lastname: Nakada
authors.forename: Hemragul
authors.initials: H
authors.lastname: Sabit
authors.forename: Lei
authors.initials: L
authors.lastname: Teng
authors.forename: Natsuki
authors.initials: N
authors.lastname: Furuyama
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Hayashi
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Kawakami
authors.forename: Toshinari
authors.initials: T
authors.lastname: Minamoto
authors.forename: Aliaksandr S
authors.initials: AS
authors.lastname: Fedulau
authors.forename: Jun-ichiro
authors.initials: J
authors.lastname: Hamada
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology / Q000627:therapeutic use; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism; D019175:DNA Methylation; D000090205:DNA Methyltransferase 3A; D003606:Dacarbazine / Q000031:analogs & derivatives / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D004357:Drug Synergism; D015972:Gene Expression Regulation, Neoplastic; D005909:Glioblastoma / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism*; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors*; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics*; D010766:Phosphorylation / Q000187:drug effects; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000494:pharmacology / Q000627:therapeutic use; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D015398:Signal Transduction; D000077204:Temozolomide
chemical_list: D000970:Antineoplastic Agents; C000717967:DNMT3A protein, human; D047428:Protein Kinase Inhibitors; D016271:Proto-Oncogene Proteins c-myc; D003606:Dacarbazine; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; D019853:O(6)-Methylguanine-DNA Methyltransferase; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3; D000077204:Temozolomide