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）是用于治疗胶质母细胞瘤的化学疗法。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（IFN）可以下调MGMT表达，从而改善未甲基化MGMT启动子患者的生存率。HeberFERON是I型和II型IFN的共同制剂，对胶质母细胞瘤细胞系的抗增殖作用高于单个IFN。我们调查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启动子甲基化和HeberFERON治疗后MGMT转录调控的关系。根据TCGA和MGMT启动子甲基化对11种胶质母细胞瘤培养物进行分子分类，使用CellTiter-Glo发光细胞生存力测定法（Promega），以低剂量（1–25 IU / mL）[单独或与TMZ组合]或更高剂量（50–200 IU / mL）的HeberFERON进行增殖抑制试验。将八个培养物进一步用100 IU / mL的HeberFERON处理72小时，纯化总RNA（Qiagen），并转化为cDNA（Superscript III试剂盒，Invitrogen）作为定量PCR模板。监测MGMT和P53转录物水平的变化。根据八种经典文化数据，文化对HeberFERON的反应是可变的，剂量依赖性的，并且显然与TCGA分类和MGMT甲基化状态无关。当将HeberFERON与TMZ结合使用时，培养物的细胞死亡增加，甲基化的MGMT启动子增加2/4，甲基化的5MT启动子增加5/5。五种文化中有两种具有未甲基化的MGMT状态，我们观察到了MGMT基因水平的下降和P53编码基因水平的上升。HeberFERON和TMZ组合应在胶质母细胞瘤中进一步测定，主要用于那些未甲基化MGMT启动子的患者。