二十碳五烯酸通过抑制MGMT表达提高胶质瘤U87细胞对替莫唑胺的化疗敏感性  

Eicosapentaenoic acid enhances sensitivity of glioma cell line U87 to temozolomide via inhibiting MGMT expression

作　　者：黄天造[1] 陈祥荣[1] 刘楚斌 高延磊 胡伟鹏[1] Huang Tianzao;Chen Xiangrong;Liu Chubin;Gao Yanlei;Hu Weipeng(Department of Neurosurgery,Second Affiliated Hospital,Fujian Medical University,Quanzhou 362000,China)

机构地区：[1]福建医科大学附属第二医院神经外科,泉州362000

出　　处：《中华神经医学杂志》2018年第9期879-885,共7页Chinese Journal of Neuromedicine

基　　金：福建省自然科学基金（2015J01443）;福建省科技创新联合资金项目（2017Y9201）

摘　　要：目的探讨二十碳五烯酸（EPA）对胶质瘤U87细胞替莫唑胺（TMZ）化疗敏感性的影响及其机制。方法（11体外常规培养U87细胞，加入0、25、50、100、200μmol／LEPA分别作用12、24、48h后MTT检测细胞增殖率；（21将U87细胞分为对照组、EPA组、TMZ组、EPA＋TMZ组（EPA、TMZ的浓度分别为25、100mol／L，EPA预处理时间为12、24或48h，TMZ作用时间为24h），MTT检测4组细胞的增殖抑制率；（3）将U87细胞分为对照组、EPA组、TMZ组、EPA＋TMZ组（EPA、TMZ的浓度分别为25、100mol／L，作用时间分别为12、24h）。流式细胞术检测4组细胞凋亡率：Westernblotting检测细胞活化半胱氨酸蛋白酶．3（caspase-3）、B淋巴细胞瘤．2相关X蛋白（Bax）蛋白表达，DNA错配修复酶fMGMT）及核因子κB（NF-κB）p65、NF-κB抑制蛋白（IKBa）的表达；免疫荧光染色检测细胞MGMT和NF．KBp65的表达：甲基化特异性PCR（MSP）法检测细胞MGMT基因启动子甲基化水平。结果（1）50、100、200μmol／LEPA作用12、24、48h时对U87细胞的增殖抑制呈浓度和时间依赖性；（2）与同样预处理时间的TMZ组比较，EPA＋TMZ组U87细胞的增殖抑制率均明显升高，差异有统计学意义（P〈0．05）；（3）流式细胞术检测显示：与TMZ组【（34．58±4．35）％】比较，EPA＋TMZ组U87细胞的凋亡率[（53．28±5．05）％]明显升高，差异有统计学意义（P〈0．05）；Westemblotting检测显示：与TMZ组比较，EPA＋TMZ组U87细胞活化caspase．3、Bax和IKBd蛋白表达升高，MGMT和NF-κBp65蛋白表达降低，差异有统计学意义（P〈0．05）；免疫荧光染色显示EPA＋TMZ组细胞MGMT和NF-κBp65蛋白的表达低于TMZ组；MSP检测显示EPA＋TMZ组U87细胞MGMT基因启动子甲基化水平高于TMZ组。结论EPA预处理可提高胶质瘤U87细胞对TMZ的化疗敏感性，其机制可能是通过下调NF-κB信号通路抑制MGMT表达来实现。Objective To investigate the effect of eicosapentaenoic acid （EPA） on sensitivity of glioma cell line U87 to temozolomide （TMZ） and its underlying mechanism. Methods （1） U87 cells were routinely cultured in vitro, and 0, 25, 50, 100 and 200 μmol/L EPA was given to these cells for 12, 24, and 48 h; MTT assay was used to detect the cell viability. （2） U87 cells were randomly divided into control group, EPA group, TMZ group and EPA＋TMZ group （concentrations of EPA and TMZ were 25 and 100 mol/L; EPA pretreatment for 12, 24 and 48 h was given; TMZ was given for 24 h）; MTT assay was used to evaluate the inhibition ratio of cell proliferation. （3） U87 cells were randomly divided into control group I, EPA group I, TMZ group I and EPA＋TMZ group I （concentrations of EPA and TMZ were 25 and 100 mol/L; EPA pretreatment for 6 h was given; TMZ was given for 24 h）; the apoptoticratio was examined by flow eytometry （FCM）; Western blotting was used to detect the protein expressions ofcleavcdcaspase-3, Bax, O-6-methlguanine-DNAmethyltransferase （MGMT）, nuclear factor （NF）-KB signaling pathway related protein p65, and NF-KB inhibitor IKBa; immunofluorescent staining was employed to detect the MGMT and NF-KB p65 expressions; methylated specific PCR （MSP） was used to detect the MGMT gene promoter methylation. Results （1） The 50, 100 and 200 μmol/L EPA caused concentration-dependent and time-dependent proliferation inhibition of U87 ceils. （2） The inhibition ratio of cell proliferation in EPA＋TMZ group was significantly higher as compared with that in the TMZ group （P〈0.05）. （3） As compared with that in the TMZ group [ （34.58%±4.35%）, the apoptotic ratio of U87 cells in the EPA＋TMZ group I was significantly increased （53.28%±5.05%, P〈0.05）; Western blotting showed that as compared with those in TMZ group I, the protein expressions of activated caspase-3, Bax and IKBα were significantly increased, and MGMT and NF-KB p65 protein expressions were signifi

关 键 词：神经胶质瘤 二十碳五烯酸 替莫唑胺 DNA错配修复酶 核因子ΚB 

分 类 号：R730.264[医药卫生—肿瘤]