Ciechomska Iwona Anna, Marciniak Marta Patrycja, Jackl Judyta, Kaminska Bozena
Laboratory of Molecular Neurobiology, Neurobiology Center, Nencki Institute of Experimental Biology, Warsaw, Poland.
Front Pharmacol. 2018 Nov 2;9:1271. doi: 10.3389/fphar.2018.01271. eCollection 2018.
Glioblastoma (GBM) is a malignant, primary brain tumor, highly resistant to conventional therapies. Temozolomide (TMZ) is a first line therapeutic agent in GBM patients, however, survival of such patients is poor. High level of DNA repair protein, O-methylguanine-DNA-methyltransferase (MGMT) and occurrence of glioma stem-like cells contribute to GBM resistance to the drug. Here, we explored a possibility of epigenetic reprograming of glioma cells to increase sensitivity to TMZ and restore apoptosis competence. We combined TMZ treatment with BIX01294, an inhibitor of histone methyltransferase G9a, known to be involved in cancerogenesis. Two treatment combinations were tested: BIX01294 was administered to human LN18 and U251 glioma cell cultures 48 h before TMZ or 48 h after TMZ treatment. Despite their different status of the gene promoter, there was no correlation with the response to TMZ. The analyses of cell viability, appearance of apoptotic alterations in morphology of cells and nuclei, and markers of apoptosis, such as levels of cleaved caspase 3, caspase 7 and PARP, revealed that both pre-treatment and post-treatment with BIX01294 sensitize glioma cells to TMZ. The additive effect was stronger in LN18 cells. Moreover, BIX01294 enhanced the cytotoxic effect of TMZ on glioma stem-like cells, although it was not associated with modulation of the pluripotency markers () expression or methylation of and gene promoters. Accordingly, knockdown of methyltransferase G9a augments TMZ-induced cell death in LN18 cells. We found the significant increases of the LC3-II levels in LN18 cells treated with BIX01294 alone and with drug combination that suggests involvement of autophagy in enhancement of anti-tumor effect of TMZ. Treatment with BIX01294 did not affect methylation of the gene promoter. Altogether, our results suggest that G9a is a potential therapeutic target in malignant glioma and the treatment with the G9a inhibitor reprograms glioma cells and glioma stem-like cells to increase sensitivity to TMZ and restore apoptosis competence.
胶质母细胞瘤（GBM）是一种恶性原发性脑肿瘤，对传统疗法具有高度抗性。替莫唑胺（TMZ）是GBM患者的一线治疗药物，然而，此类患者的生存率较低。高水平的DNA修复蛋白O-甲基鸟嘌呤-DNA甲基转移酶（MGMT）以及胶质瘤干细胞样细胞的出现导致GBM对该药物产生抗性。在此，我们探讨了对胶质瘤细胞进行表观遗传重编程以增加对TMZ的敏感性并恢复凋亡能力的可能性。我们将TMZ治疗与BIX01294（一种已知参与肿瘤发生的组蛋白甲基转移酶G9a抑制剂）联合使用。测试了两种治疗组合：在TMZ治疗前48小时或TMZ治疗后48小时将BIX01294施用于人LN18和U251胶质瘤细胞培养物。尽管它们的基因启动子状态不同，但与对TMZ的反应无关。对细胞活力、细胞和细胞核形态学中凋亡改变的出现以及凋亡标志物（如裂解的半胱天冬酶3、半胱天冬酶7和PARP的水平）的分析表明，BIX01294预处理和后处理均使胶质瘤细胞对TMZ敏感。LN18细胞中的相加效应更强。此外，BIX01294增强了TMZ对胶质瘤干细胞样细胞的细胞毒性作用，尽管它与多能性标志物（）表达的调节或和基因启动子的甲基化无关。因此，甲基转移酶G9a的敲低增强了TMZ诱导的LN18细胞死亡。我们发现单独用BIX01294处理以及药物联合处理的LN18细胞中LC3-II水平显著增加，这表明自噬参与增强TMZ的抗肿瘤作用。用BIX01294处理不影响基因启动子的甲基化。总之，我们的结果表明G9a是恶性胶质瘤的潜在治疗靶点，用G9a抑制剂治疗可对胶质瘤细胞和胶质瘤干细胞样细胞进行重编程，以增加对TMZ的敏感性并恢复凋亡能力。
