摘要
目的:研究组蛋白去乙酰化酶抑制剂RGFP109逆转胶质母细胞瘤U251细胞的耐药性机制。方法:建立对替莫唑胺(TMZ)耐药的TR/U251细胞,试验分为正常对照组、TMZ组(40μmol/L)和TMZ(40μmol/L)+RGFP109(0~120μmol/L)不同浓度组,加入相应药物作用24 h后,CCK-8法检测细胞存活率,计算半数抑制浓度(IC_(50))。TUNEL法和Annexin V/PI法检测正常对照组、TMZ组和TMZ+RGFP109(42μmol/L)组细胞的凋亡情况,免疫印迹法检测3组细胞中O6-甲基鸟嘌呤-DNA甲基转移酶(MGMT)、存活蛋白(Survivin)、B淋巴细胞瘤2(Bcl-2)、B淋巴细胞瘤x L(Bcl-x L)蛋白表达,凝胶迁移实验检测3组细胞中p65乙酰化水平及其与κB-DNA的结合能力。结果:与正常对照组和TMZ组比较,TMZ+RGFP109不同浓度组细胞存活率明显降低(P<0.05),且呈浓度依赖性。当RGFP109浓度为42μmol/L时,TMZ对TR/U251细胞的敏感性与U251细胞相同。与正常对照组比较,TMZ组细胞中MGMT、Survivin、Bcl-2、Bcl-x L蛋白表达均增强(P<0.01),p65乙酰化水平无明显变化,但p65与κB-DNA的结合能力增强(P<0.01)。与TMZ组比较,TMZ+RGFP109组细胞中MGMT、Survivin、Bcl-2、Bcl-x L蛋白表达均减弱(P<0.01),p65乙酰化水平增强(P<0.01),p65与κB-DNA的结合能力减弱(P<0.01)。结论:RGFP109可通过下调转录因子κB调节的抗凋亡蛋白表达,减弱p65与κB-DNA的结合来逆转U251细胞对TMZ耐药。
OBJECTIVE:To study the mechanism of histone deacetylase inhibitor RGFP109 in reversing resistance of glioma U251 cells. METHODS:TR/U251 cells resistance to temozolomide(TMZ)was extrablished. The test was divided into normal control group,TMZ group(40 μmol/L)and TMZ(40 μmol/L)+RGFP109(0-120 μmol/L)different concentrations groups. After 24 h of adding into related medicines,CCK-8 was used to detect the cell survival rate and calculate the half inhibitory concentration(IC_(50)). TUNEL and Annexin V/PI were used to detect the cell apoptosis in normal control group,TMZ group and TMZ+RGFP109(42 μmol/L)group. Immunoblotting was used to detect the O6-methyl guanine-DNA methyltransferase(MGMT),Survivin,B lymphoma 2(Bcl-2),B lymphoma xL(Bcl-xL)protein expression;and gel migration test was used to detect the p65 acetylation level and its binding capacity with κB-DNA. RESULTS:Compared with normal control group,cell survival rate in TMZ+RGFP109 different concentrations groups was obviously decreased(P〈0.05),showing a concentration-dependent manner. When the RGFP109 concentration was 42 μmol/L,the sensitivity of TMZ to TR/U251 cells was the same with U251 cells. Compared with normal control group,MGMT,Survivin,Bcl-2,Bcl-xL protein expressions in cells of TMZ groups were enhanced(P〈0.01);p65 acetylation level had no obvious changes,while the binding capacity of p65 and κB-DNA was strengthened(P〈0.01). Compared with TMZ group,MGMT,Survivin,Bcl-2,Bcl-xL protein expressions in cells of TMZ groups were weakened(P〈0.01);p65 acetylation level was enhanced(P〈0.01);and the binding capacity of p65 and κB-DNA was weakened(P〈0.01). CONCLUSIONS:RGFP109 can reverse the resistance of U251 cells to TMZ by down-regulating the anti-apoptotic protein expressions adjusted by transcription factor κB(NF-κB)and weakening the binding of p65 and κB-DNA.
出处
《中国药房》
CAS
北大核心
2017年第22期3091-3095,共5页
China Pharmacy