Pharmacogenomic Group, Biomedical Research Direction, Center for Genetic Engineering and Biotechnology, Havana, Cuba.
Neurosurgical Department, Erasmus MC, Rotterdam, The Netherlands.
Mol Biol Rep. 2020 Jul;47(7):5263-5271. doi: 10.1007/s11033-020-05604-2. Epub 2020 Jun 30.
Temozolomide (TMZ) is a chemotherapeutic used for the treatment of glioblastoma. The MGMT repair enzyme (O'-(6)-methyl guanine-DNA-methyltransferase) promoter methylation is a predictive biomarker to TMZ response; interferons (IFNs) type I can downregulate MGMT expression improving survival in patients with unmethylated MGMT promoter. HeberFERON is a co-formulation of IFNs type I and II with higher antiproliferative effect over glioblastoma cell lines than individual IFNs. We investigated the proliferative response of patient-derived glioblastoma cultures to HeberFERON and its combination with TMZ in relation to MGMT promoter methylation and the regulation of MGMT transcript after HeberFERON treatment. Eleven glioblastoma-derived cultures, molecularly classified according to TCGA and MGMT promoter methylation, were assayed for proliferation inhibition with HeberFERON at low doses (1-25 IU/mL) [alone or combined with TMZ] or at higher doses (50-200 IU/mL) using CellTiter-Glo Luminescent Cell Viability Assay (Promega). Eight cultures were further treated with 100 IU/mL of HeberFERON for 72 h, total RNA purified (Qiagen) and converted to cDNA (Superscript III kit, Invitrogen) as quantitative PCR templates. Changes of MGMT&P53 transcripts level were monitored. Response of cultures to HeberFERON is variable, dose-dependent and apparently independent from TCGA classification and MGMT methylation status, based on the eight Classical cultures data. When combining HeberFERON with TMZ there was an increase in cell death for cultures, 2/4 with methylated and 5/5 with unmethylated MGMT promoter. In two out five cultures with unmethylated MGMT status, we observed a decrease of MGMT gene levels and an increase in P53 encoding gene levels. HeberFERON and TMZ combination should be further assayed in glioblastoma, mainly for those with unmethylated MGMT promoter.
替莫唑胺（TMZ）是一种用于治疗胶质母细胞瘤的化疗药物。MGMT 修复酶（O'-(6)-甲基鸟嘌呤-DNA-甲基转移酶）启动子甲基化是 TMZ 反应的预测生物标志物；I 型干扰素（IFNs）可下调 MGMT 表达，提高未甲基化 MGMT 启动子患者的生存率。HeberFERON 是 I 型和 II 型 IFN 的联合制剂，对胶质母细胞瘤细胞系的增殖抑制作用高于单独的 IFN。我们研究了患者来源的胶质母细胞瘤培养物对 HeberFERON 的增殖反应及其与 TMZ 的组合与 MGMT 启动子甲基化的关系，以及 HeberFERON 治疗后 MGMT 转录本的调节。根据 TCGA 和 MGMT 启动子甲基化，对 11 种源自胶质母细胞瘤的培养物进行了低剂量（1-25 IU/mL）[单独或与 TMZ 联合]或高剂量（50-200 IU/mL）HeberFERON 抑制增殖的测定，使用 CellTiter-Glo 发光细胞活力测定法（Promega）。进一步用 100 IU/mL 的 HeberFERON 处理 8 种培养物 72 小时，用 Qiagen 纯化总 RNA，并将其转化为 cDNA（Superscript III 试剂盒，Invitrogen）作为定量 PCR 模板。监测 MGMT 和 P53 转录本水平的变化。根据 8 种经典培养物的数据，培养物对 HeberFERON 的反应是可变的、剂量依赖性的，且显然与 TCGA 分类和 MGMT 甲基化状态无关。当 HeberFERON 与 TMZ 联合使用时，培养物的细胞死亡增加，4 个甲基化和 5 个未甲基化 MGMT 启动子的培养物中有 2 个。在 5 个未甲基化 MGMT 状态的培养物中，有 2 个观察到 MGMT 基因水平降低，P53 编码基因水平升高。应进一步研究 HeberFERON 和 TMZ 联合治疗胶质母细胞瘤，特别是那些未甲基化 MGMT 启动子的患者。
