GLI1抑制剂FL18对胶质母细胞瘤的抑制作用及其机制  

The anti-tumor activity and mechanisms of a GLI1 inhibitor FL18 in suppression of glioblastoma growth

作　　者：刘春霞[1] 黄璐璐 闫辰 杜倩倩 李铁钢[1] 冯志强[1] 李燕[1] 李学记 LIU Chun-xia;HUANG Lu-lu;YAN Chen;DU Qian-qian;LI Tie-gang;FENG Zhi-qiang;LI Yan;LI Xue-ji(Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China;National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China)

机构地区：[1]中国医学科学院北京协和医学院药物研究所,北京100050 [2]中国医学科学院北京协和医学院肿瘤医院国家癌症中心,北京100021

出　　处：《药学学报》2018年第7期1113-1121,共9页Acta Pharmaceutica Sinica

基　　金：中国医学科学院医学与健康科技创新工程-重大协同创新项目(2016-I2M-1-008)

摘　　要：Hedgehog(HH)信号通路的异常激活在胶质母细胞瘤的发生发展中起着重要作用。胶质瘤相关的致癌基因同族体-1(glioma-associated oncogene homoglog-1,GLI1)蛋白作为HH通路末端的转录因子,是该通路最终的功能执行者,被认为是胶质母细胞瘤的重要治疗靶点。本研究旨在探究新型GLI1抑制剂FL18的抗胶质母细胞瘤活性及其作用机制。MTT实验和集落形成实验研究FL18对脑瘤细胞的生长抑制作用;运用流式细胞术分析FL18对细胞凋亡的影响;Transwell实验探究FL18对细胞侵袭能力的抑制作用;通过体内移植瘤实验研究FL18对胶质母细胞瘤的生长抑制作用;建立及优化双荧光素酶报告基因的检测体系,并检测FL18对GLI1转录活性的影响;采用Western blot方法对FL18的作用机制进行初步探讨。FL18对体外培养的胶质母细胞瘤的半数抑制浓度(IC_(50))在纳摩尔水平。体内实验结果显示,22.5和45 mg·kg^(-1) FL18对裸鼠原位移植瘤的抑制率分别为55.4%和89.8%。FL18能够抑制GLI1的转录活性,IC_(50)为3.32×10^(-11) mol·L^(-1)。Western blot实验显示,在FL18作用后的胶质母细胞瘤细胞和肿瘤组织中,其GLI1表达明显降低,而SMO表达无明显变化,调控GLI1上游的ERK和AKT等蛋白的磷酸化水平变化均不显著;FL18还可升高凋亡相关蛋白切割型的半胱氨酸天冬氨酸蛋白酶-3(c-caspase 3)和聚腺苷二磷酸-核糖聚合酶(c-PARP)以及Bax的表达,降低Bcl-2的表达;降低侵袭相关蛋白金属基质蛋白酶(MMP2和MMP9)的水平。以上研究结果表明,FL18通过抑制GLI1而对胶质母细胞瘤的生长起到一定的抑制作用,其抗肿瘤活性可能与抑制GLI1的表达水平有关。The abnormal activation of hedgehog（HH） signaling pathway plays an important role in the development and progression of glioblastoma（GBM）. As a transcription factor at the end of the HH pathway, the final effector of glioma-associated oncogene homoglog-1（GLI1） is an important target in the treatment of GBM. The study was designed to evaluate the anti-tumor activities and mechanisms of a novel GLI1 inhibitor FL18 in GBM. MTT and colony formation assay were performed to determine anti-proliferation activity of FL18 in vitro. The effect of FL18 on cell apoptosis was measured by flow cytometry（FCM） analysis. Transwell experiment was used to explore the inhibitory activity of FL18 in cell invasion. In vivo experiments, the subcutaneously transplanted and orthotopic U-87 MG GBM xenograft model were used to study the activity of FL18 on tumor growth. The optimized dual report gene screening model was used to detect the effect of FL18 on the transcriptional activity of GLI1. Western blot assay was used to study the mechanisms of action of FL18. The results showed that the IC50 of FL18 in glioblastoma was in the nanomole level in vitro. It was observed that 22.5 and 45 mg·kg^-1 FL18 reduced the tumor volume with the rate of 55.4% and 89.8% in xenograft model in mice in situ. The IC50 of FL18 on the inhibition of GLI1 transcriptional activity was 3.32×10～（-11） mol·L^-1 analyzed by the optimized dual report gene screening model. By the Western blot experiments, it was proved that FL18 inhibited expression of GLI1 without influencing the upstream canonical HH/SMO signaling and cross-talk oncogenic pathway, such as ERK and AKT signaling. The results also demonstrated that FL18 significantly downregulated GLI1 target genes such as Bcl-2, MMP2 and MMP9 and increased the expression of c-caspase3, c-PARP and Bax. These data suggest that FL18 may generate the anti-glioma activity by inhibition of GLI1.

关 键 词：胶质瘤相关的致癌基因同族体-1 胶质母细胞瘤 抗肿瘤 侵袭 

分 类 号：R966[医药卫生—药理学]