内质网应激诱导胶质瘤U87细胞替莫唑胺化疗抵抗

Endoplasmic reticulum stress enhances chemoresistance of glioma cell line U87 to temozolomide

目的探讨内质网应激对胶质瘤U87细胞替莫唑胺（TMZ）化疗敏感性的影响及其机制。方法（1）体外常规培养胶质瘤U87细胞，培养24h后加入0、12．5、25、50、100μmol／LTMZ作用24h，MTT检测细胞增殖抑制率，观察TMZ的半抑制浓度（IC50）；0、2、4、8μmol／L内质网应激诱导剂衣霉素（TM）处理U87细胞6h后加入50μmol／LTMZ作用24h，MTT检测细胞增殖抑制率的变化；（2）将细胞分为对照组、TM组、TMZ组、TM＋TMZ组，TM组、TM＋TMZ组细胞加入4μmol／LTM预处理6h，后2组细胞加入50μmol／LTMZ，对照组加入等量培养基。继续作用24h后流式细胞仪检测各组细胞的凋亡率：Western blotting检测细胞含半胱氨酸的天冬氨酸蛋白水解酶3（caspase-3）、B淋巴细胞瘤-2基因（Bcl-2）相关X蛋白（Bax）、DNA错配修复酶（MGMT）、内质网腔内分子伴侣（GRP78）、磷酸化肌醇需要酶1（IRE-1）的表达。结果（1）MTT检测显示TMZ对U87细胞的IC50为50μmol／L。与0μmol／LTM±50μmol／LTMZ组、2μmol／LTM±50μmol／LTMZ组比较，4μmol／LTM±50μmol／LTMZ组、8μmol／LTM±50μmol／LTMZ组细胞增殖抑制率均降低，差异有统计学意义（P〈0．05）；（2）与TMZ组比较，TM±TMZ组细胞凋亡率[（46．98±4．79）％ vs（35．74±4．09）％1明显减少，凋亡因子caspase-3和Bax蛋白的表达减少，MGMT、GRP78和IRE．1蛋白的表达增加，差异有统计学意义（P〈0．05）。结论内质网应激可增加胶质瘤U87细胞耐药基因MGMT的表达，降低细胞对TMZ的化疗敏感性，诱发细胞产生化疗抵抗。

Objective To investigate the effect of endoplasmic reticulum stress on sensitivity of glioma cell line U87 to temozolomide （TMZ） and underlying mechanism. Methods （1） Glioma U87 cells were routinely cultured for 24 h in vitro; different concentrations of TMZ （0, 12.5, 25, 50, 100 μmol/L） were added to intervene the cell proliferation of U87 cells for 24 h; MTT method was used to detect the cellular proliferation inhibition rate, and half maximal inhibitory concentration （IC50） of TMZ was calculated; tunicamycin （TM） treatment （0, 2, 4, 8μmol/L） was given to the ceils for 6 h, and then, 50μmol/L TMZ was given for 24 h, and cellular proliferation inhibition rate of U87 was detect by MTT method. （2） U87 cells were randomly divided into control group, TM group, TMZ group and TM±TMZ group; pretreatment of TM for 6 h was given to cells from TM group and TM±TMZ group; and 50 μmol/L TMZ was given to cells from TMZ group and TM±TMZ group; same amount of medium was given to cells from control group; 24 h after treatment, the apoptotic rate was examined by flow cytometry; Western blotting was used to detect the protein expressions of caspase-3, B-cell lymphoma 2 associated X protein （Bax）, O-6-methlguanine-DNA methyltransferase （MGMT）, glucose-regulated protein 78 （GRP78）, and inositol-requiring enzyme 1 （IRE-l）. Results （1） MTT showed that IC50 of TMZ was 50 μmol/L. As compared with that of 0μmol/L TM±50 i^mol/L TMZ group or 2μmol/L TM±50μmol/L TMZ group, the cellular proliferation inhibition rate of 4 ptmol/L TM±50μmol/L TMZ group and 8μmol/L TM±50 μmol/L TMZ group was significantly decreased （P〈0.05）. （2） As compared with TMZ group, TM＋TMZ group had significantly decreased apoptotic rate （46.98%±4.79% vs. 35.74% ±4.09%）, significantly decreased caspase-3 and Bax protein expressions, and significantly increased MGMT, GRP78 and IRE-1 protein expressions （P〈0.05）. Conclusion Endoplasmic reticulum stress can increase the resistance gene MGMT expression, decrease the chemotherapy sensitivity to TMZ, and induce chemoresistance ofglioma cell line U87.