探索替莫唑胺联合amlexanox对胶质瘤细胞的作用效果及机制  

Effect and mechanism of temozolomide combined with amlexanox on human glioblastoma cells

作　　者：熊金彪 郭高超 曹艺耀 陈旨娟[1] 黄强[1] 杨卫东[1] XIONG Jin-biao;GUO Gao-chao;CAO Yi-yao;CHEN Zhi-juan;HAUNG Qiang;YANG Wei-dong(Department of Neurosurgery,General Hospital,Tianjin Medical University,Tianjin 300052,China)

机构地区：[1]天津医科大学总医院神经外科,天津300052

出　　处：《天津医科大学学报》2020年第5期401-407,共7页Journal of Tianjin Medical University

基　　金：国家自然科学基金资助项目(81572490)。

摘　　要：目的:探索替莫唑胺(TMZ)联合amlexanox对胶质瘤细胞的作用效果及机制。方法:本研究分为体外和体内实验,其中体外实验分为4组:对照(NC)组、amlexanox组、TMZ组和TMZ+amlexanox组,包括检测细胞增殖能力的cell counting kit-8(CCK-8)实验、检测细胞侵袭和迁移的细胞划痕和Transwell实验及检测核因子κ-B激酶抑制剂(IKBKE)及蛋白激酶B(Akt)通路蛋白质变化的Western印迹实验;体内实验分为3组:NC组、TMZ组和TMZ+amlexanox组,包括颅内成瘤实验和免疫组织化学染色(IHC)实验。结果:与amlexanox组、TMZ组相比,TMZ联合amlexanox组U87 MG和原代细胞增殖能力明显受抑制、划痕愈合面积显著降低(P<0.01)。Transwell侵袭和迁移实验表明,TMZ+amlexanox组穿出小室细胞数量显著降低(P<0.01)。Western印迹实验结果显示,TMZ联合amlexanox组Akt和哺乳动物雷帕霉素靶蛋白(mTOR)活性明显受抑制。动物实验表明,TMZ联合amlexanox组裸鼠颅内肿瘤生长显著抑制(P<0.01),颅内肿瘤动物模型的生存时间延长(由21 d延长至31 d);IHC结果显示,TMZ联合amlexanox组肿瘤标本中磷酸化Akt(p-Akt)、磷酸化mTOR(p-mTOR)表达量明显下降。结论:TMZ联合amlexanox能显著抑制U87 MG和原代细胞增殖、迁移和侵袭能力,而且二者联合能显著抑制裸鼠颅内肿瘤生长并延长裸鼠生存时间。该作用机制可能是amlexanox在一定程度上通过抑制IKBKE活性逆转TMZ诱导Akt激活,进而降低胶质瘤细胞对TMZ的耐药性。Objective:To evaluate the efficacy of combined therapy with temozolomide(TMZ)and amlexanox for human glioblastoma cells.Methods:This study was divided as experiments in vitro and in vivo.In vitro,all assays were divided into four groups:negative control(NC)group,amlexanox group,TMZ group and TMZ+amlexanox group,including cell counting kit-8(CCK-8)for determining glioblastoma cells viability,cell wound scratch and Transwell assays for assessing ability of glioblastoma cells on migration and invasion,Western-blot for detecting changes of inhibitor of nuclear factor kappa-B kinase subunit epsilon(IKBKE)and relative proteins in protein kinase B(Akt)signal pathway.In vivo,all assays were divided into three groups:NC group,TMZ group and TMZ+amlexanox group,including establishment of intracranial tumor models and immunohistochemistry(IHC)assay.Results:The treatment combined with TMZ and amlexanox obviously inhibited the proliferation of U87MG and primary glioma cell compared with single agent treatment.Moreover,the treatment with TMZ and amlexanox significantly reduced the scratch healing area(P<0.01).The results of transwell invasion and migration assays showed that TMZ combined amlexanox significantly reduced the number of cells penetrated out of the chamber(P<0.01).Western-blot showed that TMZ combined amlexanox could significantly inhibit the activity of Akt and mammalian rapamycin target protein(mTOR).Animal experiments showed that TMZ combined amlexanox significantly inhibited intracranial tumor growth(P<0.01)and prolonged survival of animalswith intracranial tumors(from 21 d to 31 d).IHC results showed that in the group treated with combined drugs,the expression of phosphorylation of Akt(p-Akt)and phosphorylation of mTOR(p-mTOR)were obviously decreased.Conclusion:Combined treatment with TMZ and amlexanox results in significant inhibition of cell viability,migration and invasion in primary glioma cells and in the human glioma cell line,U87 MG.Moreover,combined treatment significantly inhibits the growth of intracr

关 键 词：AMLEXANOX 替莫唑胺 胶质瘤 P-AKT IKBKE 化学耐药性 

分 类 号：R730.264[医药卫生—肿瘤]