Role of O6-methylguanine-DNA methyltransferase and p53 in response of neuroblastoma cells to an alkylating agent temozolomide

摘要

Aim. To study the role of p53 and O6-methylguanine-DNA methyltransferase in sensitivity of neuroblastoma cells to temozolomide. Methods. The study was performed on SK.N.SH neuroblastoma cell line cultured in DMEM medium supplemented with fetal bovine serum and antibiotics penicillin-streptomycin in the standard conditions (37°C and 5% СО2). The cells were cultured with an alkylating agent temozolomide for 48-72 h. For particular experiments, cells were pre-cultured for 2 hours with O6-benzylguanine (a competitive inhibitor of O6-methylguanine-DNA methyltransferase) or nutlin-3a (reactivator of p53). Proliferative activity was evaluated by using a system of multiparametric analysis of cell cultures (RTCA iCELLigence) as well as MTS-based colorimetric assay. Protein expression was measured by Western blotting by using the corresponding monoclonal antibodies. Results. Reactivation of p53 protein substantially inhibited proliferation rate of SK.N.SH cells. Cytotoxic effect of a medication was more significant compared to temozolomide considered as an agent of choice for chemotherapy for patients with glioblastoma multiform or neuroblastoma. Inhibition of O6-methylguanine-DNA methyltransferase also enhanced the cytotoxic effect of temozolomide, however, cytotoxic effect of a chemotherapeutic agent was less expressed compared to temozolomide, along with p53 reactivation. Conclusion. Functional state of p53 protein in tumor cells is a more important prognostic marker of neuroblastoma cells’ sensitivity to an alkylating agent temozolomide compared to O6-methylguanine-DNA methyltransferase expression; in addition, reactivation of p53 protein induces the decrease of proliferation rate of neuroblastoma SK.N.SH cells and their death via apoptosis.