Revealing the epigenetic effect of temozolomide on glioblastoma cell lines in therapeutic conditions

Temozolomide (TMZ) is a drug of choice in glioblastoma treatment. Its therapeutic applications expand also beyond high grade gliomas. However, a significant number of recurrences and resistance to the drug is observed. The key factor in each chemotherapy is to achieve the therapeutic doses of a drug at the pathologic site. Nonetheless, the rate of temozolomide penetration from blood to cerebrospinal fluid is only 20–30%, and even smaller into brain intestinum. That makes a challenge for the therapeutic regimens to obtain effective drug concentrations with minimal toxicity and minor side effects. The aim of our research was to explore a novel epigenetic mechanism of temozolomide action in therapeutic conditions. We analyzed the epigenetic effects of TMZ influence on different glioblastoma cell lines in therapeutically achieved TMZ concentrations through total changes of the level of 5-methylcytosine in DNA, the main epigenetic marker. That was done with classical approach of radioactive nucleotide post-labelling and separation on thin-layer chromatography. In the range of therapeutically achieved temozolomide concentrations we observed total DNA hypomethylation. The significant hypermethylating effect was visible after reaching TMZ concentrations of 10–50 μM (depending on the cell line). Longer exposure time promoted DNA hypomethylation. The demethylated state of the glioblastoma cell lines was overcome by repeated TMZ applications, where dose-dependent increase in DNA 5-methylcytosine contents was observed. Those effects were not seen in non-cancerous cell line. The increase of DNA methylation resulting in global gene silencing and consecutive down regulation of gene expression after TMZ treatment may explain better glioblastoma patients’ survival.

替莫唑胺（Temozolomide, TMZ）是胶质母细胞瘤（glioblastoma）治疗的首选药物，其治疗适应症亦拓展至高级别胶质瘤（high grade gliomas）之外。然而，临床中仍可观察到大量肿瘤复发与药物耐药的情况。化疗的核心目标是让药物在病变部位达到有效治疗剂量。尽管如此，替莫唑胺从血液向脑脊液（cerebrospinal fluid）的穿透率仅为20%~30%，进入脑间质的比例更低，这给治疗方案的优化带来了挑战：需在达成有效药物浓度的同时，最大限度降低药物毒性与不良反应。
本研究旨在探索替莫唑胺在治疗条件下发挥作用的新型表观遗传机制。我们通过检测DNA中主要表观遗传标记物——5-甲基胞嘧啶（5-methylcytosine）的整体水平变化，分析了治疗浓度下TMZ对不同胶质母细胞瘤细胞系的表观遗传影响。实验采用经典的放射性核苷酸后标记法（radioactive nucleotide post-labelling）结合薄层色谱法（thin-layer chromatography）完成检测。
在治疗浓度范围内，我们观察到整体DNA低甲基化现象；而当TMZ浓度达到10~50μM（因细胞系而异）时，则出现显著的高甲基化效应。更长的药物暴露时长可进一步促进DNA低甲基化。反复给予TMZ可逆转胶质母细胞瘤细胞系的去甲基化状态，此时可观察到DNA中5-甲基胞嘧啶含量呈剂量依赖性升高。上述效应在非癌性细胞系中并未出现。
替莫唑胺治疗引发的DNA甲基化水平升高可诱导全局基因沉默，继而导致基因表达持续下调，这一机制或可解释胶质母细胞瘤患者生存期的改善情况。