BACKGROUND Glioblastoma (GBM) is the most lethal and aggressive malignant primary brain tumor in adults. After surgical resection of the tumor, the patient typically should be subjected to chemotherapy (temozolomide, TMZ) and concomitant radiotherapy. Since the TMZ treatment does not lead to complete remission and often develops resistance, the identification of efficacious therapeutics is strongly to pursue. Among the epigenetic players, the H3K27 methyltransferase (MT) EZH2 (enhancer of zeste homologue 2) has been found overexpressed or mutated in several human cancers including gliomas, and its overexpression is associated with poor outcome in GBM. Two EZH2 inhibitors (EZH2i), UNC1999 and GSK343, suppressed GBM growth in vitro and in vivo indicating that EZH2i can be potential drugs against GBM. RESULTS Two new EZH2i, MC4040 and MC4041, were designed, prepared, and tested by us to determine their effects in primary GBM cell cultures. MC4040 and MC4041 displayed single-digit micromolar inhibition of EZH2, 10-fold less potency against EZH1, and no activity towards other MTs. In primary GBM cells as well as in U-87 GBM cells, the two compounds reduced H3K27me3 levels, and dose- and time-dependently impaired GBM cell viability without inducing apoptosis and arresting the cell cycle in the G0/G1 phase, with increased p21 and p27 levels. In combination with TMZ, MC4040 and MC4041 displayed stronger, but not additive, effects on cell viability. The potent clinical candidate as EZH2i tazemetostat, alone or in combination with TMZ, exhibited a similar potency of inhibition of GBM cell growth when compared to MC4040 and MC4041. At the molecular level, MC4040 and MC4041 reduced the VEGFR1/VEGF expression, reversed the epithelial-mesenchymal transition (EMT), and hampered cell migration and invasion attenuating the cancer malignant phenotype. Treatment of GBM cells with MC4040 and MC4041 also impaired the GBM pro-inflammatory phenotype, with a significant decrease of TGF-β, TNF-α, and IL-6, joined to an increase of the anti-inflammatory cytokine IL-10. CONCLUSIONS The two novel EZH2i MC4040 and MC4041 impaired primary GBM cell viability, showing even stronger effects in combination with TMZ. They also weakened the aggressive malignant phenotype by reducing angiogenesis, EMT, cell migration/invasion and inflammation, thus they may be considered potential candidates against GBM also for combination therapies.

中文翻译：

---

剖析新型 EZH2 抑制剂在原代胶质母细胞瘤细胞培养中的作用：对增殖、上皮间质转化、迁移和促炎表型的影响。

背景胶质母细胞瘤（GBM）是成人中最致命和最具侵袭性的恶性原发性脑肿瘤。手术切除肿瘤后，患者通常应接受化疗（替莫唑胺，TMZ）和伴随的放疗。由于 TMZ 治疗不会导致完全缓解，并且经常产生耐药性，因此迫切需要寻找有效的治疗方法。在表观遗传因素中，H3K27 甲基转移酶 (MT) EZH2（zeste 同源物 2 的增强子）被发现在包括神经胶质瘤在内的几种人类癌症中过度表达或突变，并且其过度表达与 GBM 的不良预后相关。两种 EZH2 抑制剂 (EZH2i) UNC1999 和 GSK343 在体外和体内抑制 GBM 生长，表明 EZH2i 可以成为抗 GBM 的潜在药物。结果 我们设计、制备和测试了两种新的 EZH2i：MC4040 和 MC4041，以确定它们在原代 GBM 细胞培养物中的作用。MC4040 和 MC4041 对 EZH2 的抑制作用为个位数微摩尔，对 EZH1 的抑制作用低 10 倍，对其他 MT 没有活性。在原代 GBM 细胞和 U-87 GBM 细胞中，这两种化合物降低了 H3K27me3 水平，并且剂量和时间依赖性地损害 GBM 细胞活力，但不诱导细胞凋亡并将细胞周期阻滞在 G0/G1 期，同时增加 p21和p27水平。与 TMZ 联合使用时，MC4040 和 MC4041 对细胞活力表现出更强的影响，但不是相加的。有效的临床候选药物 EZH2i tazemetostat，单独或与 TMZ 联合使用，与 MC4040 和 MC4041 相比，表现出相似的 GBM 细胞生长抑制效力。在分子水平上，MC4040和MC4041降低了VEGFR1/VEGF表达，逆转上皮间质转化（EMT），并阻碍细胞迁移和侵袭，从而减弱癌症恶性表型。用 MC4040 和 MC4041 处理 GBM 细胞也会损害 GBM 促炎表型，TGF-β、TNF-α 和 IL-6 显着减少，同时抗炎细胞因子 IL-10 增加。结论 两种新型 EZH2i MC4040 和 MC4041 损害了原代 GBM 细胞的活力，与 TMZ 联合使用时显示出更强的效果。它们还通过减少血管生成、EMT、细胞迁移/侵袭和炎症来削弱侵袭性恶性表型，因此它们可能被认为是针对 GBM 的联合疗法的潜在候选者。