5-氮杂-2-脱氧胞苷和曲古抑菌素A对胃癌MGMT基因表达及NF-κB活性的影响  

Effects of 5-Aza-dC combined with trichostatin A on MGMT expression and NF-κB activity in gastric carcinoma cells

作　　者：孟兰 程民 沈国栋 徐婷娟 刘林青 唐杨琛 韩晓青 沈干 胡世莲 

机构地区：[1]安徽医科大学附属省立医院老年医学科安徽省老年医学研究所,合肥230001 [2]安徽医科大学附属省立医院肿瘤免疫与营养治疗安徽省重点实验室,合肥230001

出　　处：《第三军医大学学报》2014年第13期1370-1375,共6页Journal of Third Military Medical University

基　　金：国家自然科学基金(81071808);安徽省国际科技合作计划项目(1303063017);安徽省科技厅科技攻关项目(12010402126);安徽省自然科学基金(H1610)~~

摘　　要：目的探讨5-氮杂-2-脱氧胞苷(5-Aza-2'-deoxycytidine,5-Aza-dC)和曲古抑菌素A(trichostatin A,TSA)对不同分化程度的人胃癌MKN-45和MGC-803细胞系增殖、凋亡,抑癌基因O6-甲基鸟嘌呤-DNA甲基转移酶(O6-Methylguanine-DNA Methyltransferase,MGMT)甲基化水平及其核转录因子NF-κB磷酸化程度影响。方法利用CCK-8、流式细胞术、MSP、RT-PCR、Western blot等方法检测5-Aza-dC(10μmol/L)、TSA(1μmol/L)单独或联合刺激后,MKN-45和MGC-803细胞增殖、凋亡,MGMT甲基化、mRNA、蛋白表达水平及NF-κB p65蛋白的磷酸化程度。结果①5-Aza-dC、TSA单独或联合使用均可抑制MKN-45和MGC-803细胞的生长,与单药组相比,联合用药组抑制作用更明显(P<0.05);②5-Aza-dC及TSA均可促进MKN-45和MGC-803细胞的凋亡,联合用药组凋亡率[(8.43±1.80)%和(21.75±3.06)%]均明显高于5-Aza-dc组[(7.33±1.18)%、(15.22±1.03)%]和TSA组[(3.53±1.18)%、(12.66±1.10)%](P<0.05);③5-AzadC、TSA单独或联合刺激均可逆转MGMT基因甲基化,使其mRNA及蛋白表达水平上调,联合用药作用更加明显(P<0.05);④NF-κB活性高表达的MKN-45和MGC-803经5-Aza-dC和TSA处理后细胞的NF-κB活性降低,且联合用药组[(12.81±4.18)%和(9.08±2.66)%]抑制程度较5-Aza-dc组[(39.13±3.38)%、(34.90±4.89)%]和TSA组[(27.55±5.00)%、(22.11±3.07)%]更加明显(P<0.05)。结论 5-Aza-dC与TSA均可抑制胃癌细胞生长和促进细胞凋亡,逆转MGMT基因甲基化反应,提高基因表达和抑制NF-κB的活性,二药联合作用提示效果更加明显。Objective To determine the effects of 5-Aza-2′-deoxycytidine （5-Aza-dC） and trichostatin A （TSA） alone, or in combination on the proliferation, apoptosis, the methylation level of O6-methylguanine-DNA methyltransferase （MGMT） and the phosphorylation of NF-κB in human gastric carcinoma cell lines with different differentiation grades （MKN-45 and MGC-803）. Methods After MKN-45 and MGC-803 cells were treated by 5-Aza-dC, TSA and combination respectively, the cell viability was assayed by CCK-8 kit, and apoptosis by Annexin V-FITC/PI staining. The methylation level of MGMT promoter was determined by methylation specific PCR （MSP）, and mRNA expression by RT-PCR. Western blotting was employed to detect the expression of MGMT, NF-κB p65 and p-NF-κB p65 in the 2 cell lines. Results 5-Aza-dC or TSA monotherapy and combination significantly inhibited cell growth in a dose- and time- dependent manner in MKN45 and MGC-803 cells, and the combination treatment was more significant in the inhibition （P〈0.05）. The monotherapies and combination treatment both improved cell apoptosis, and the combination group [（8.43±1.80）% and （21.75±1.06）%] had higher apoptotic rate than the 5-Aza-dC [（7.33±1.18）% and （15.22±1.03）%] and TSA [（3.53±1.18）% and （12.66±1.10）%] monotherapies（P〈0.05）. The monotherapies and combination treatment all reversed MGMT promoter methylation, and increased its mRNA and protein levels, but the combination treatment showed more significant effects （P〈0.05）. The expression levels of NF-κB p65 were significantly reduced in the MKN-45 and MGC-803 cells after monotherapies and combination treatment, and the combination treatment [（12.81±4.18）% and （9.08±2.66）%] showed stronger inhibitory effects than the 5-Aza-dC [（39.13±3.38）% and （34.90±4.89）%] and TSA [（27.55±5.00）% and （22.11±3.07）%] monotherapies（P〈0.05）. Conclusion Both 5-Aza-dC and TSA inhibit the proliferation, induce the apoptosis, and enhance MGMT

关 键 词：胃癌 5-氮杂-2-脱氧胞苷 曲古抑菌素A MGMT基因 核转录因子-ΚB 

分 类 号：R73-361[医药卫生—肿瘤] R735.2[医药卫生—临床医学]