5-氮杂-2-脱氧胞苷通过上调MGMT表达增加5-氟尿嘧啶联合紫杉醇的化疗敏感性

摘要

目的 研究5-氮杂-2-脱氧胞苷(5-Aza-dC)与传统化疗药物5-氟尿嘧啶联合紫杉醇(5-FU+ PTX)对人胃癌细胞生长的影响,探讨5-Aza-dC抗肿瘤的机制.方法 培养人胃癌细胞株MKN-45,分为对照组、5-Aza-dC组、5-FU+PTX组和5-Aza-dC+ 5-FU+ PTX组,利用CCK-8细胞增殖检测试剂盒、流式细胞术检测各组细胞的增殖和凋亡情况,利用RT-PCR和Western blot方法分别检测各组胃癌细胞的MGMT基因mRNA与蛋白表达,及凋亡蛋白cleaved caspase-3的表达.结果 5-Aza-dC及5-FU联合紫杉醇均可抑制MKN-45细胞的生长,48 h细胞存活率分别为(71.60±5.77,57.00±6.09)％,与5-FU+ PTX组比较,三药联合使用后效果更明显(42.85±2.29)％,P=0.02;5-Aza-dC及5-FU联合紫杉醇组细胞凋亡率[(7.33±0.34)％,(13.06±1.67)％]及凋亡蛋白cleaved caspase-3水平[(43.00±4.00)％,(45.67±4.50)％]均增加,三药联合用药组凋亡率[(17.31±1.05)％]和cleaved caspase-3水平[(174.67±6.50)％]的表达更高.与对照组抑癌基因MGMT mRNA和蛋白表达量[(23.10±2.15)％、(10.47±1.39)％]比较,5-Aza-dC单独处理的MKN-45细胞MGMT基因mRNA(56±5.57)％及蛋白表达水平(20.33±5.50)％均升高,三药联合用药组MGMT升高程度更加明显[(75.67±6.50)％,(174.67±6.50)％],P＜0.001,而5-FU联合紫杉醇组未发现明显变化.结论 5-Aza-dC可以有效抑制胃癌细胞的生长,它与化疗药物5-FU和紫杉醇联合应用时抗肿瘤效应更加明显.%Objective To explore the potential effects of 5-Aza-2'-deoxycitydine (5-Aza-dC),5-FU combined Paclitaxel (PTX) for the treatment of patients with gastric cancer and its mechanism.Methods The inhibitory effects of 5-Aza-dC and/or 5-FU combined PTX on MKN-45 cell proliferation were assayed by using CCK-8 kit.The apoptosis of MKN-45 cell was determined by Annexin V-FITC/PI staining method.MGMT mRNA in vitro was determined by RTPCR,and the cleaved caspase-3 and MGMT protein expression were detected by western blotting.Results The cell growth of human gastric carcinoma cell line MKN-45 was significantly inhibited by 5-Aza-dC(71.60 ± 5.77)％ or 5-FU combined PTX (57.00 ± 6.09) ％,the combination of 5-Aza-dC and 5-FU + PTX (42.85 ± 2.29) ％ resulted in a better inhibition.Compared with the control group,5-Aza-dC and 5-FU combined PTX group induced the apoptosis of MKN-45 cells [(7.33 ± 0.34) ％,(13.06 ± 1.67) ％],with the apoptotic rate significantly higher in the combination group than that in single drug treatment group(17.31 ± 1.05)％,as well as indicated in cleaved caspase-3 protein expression.Exposure of MKN-45cell to 5-Aza-dC alone resulted in a significant increase in the expression levels of MGMT mRNA and protein [(56.00 ± 5.57) ％,(20.33 ± 5.50) ％],and the effects were consequently more pronounced in the combined group[(75.67 ±6.50)％,(174.67 ±6.50)％],P ＜0.001.Conclusion 5-Aza-dC can inhibit cell proliferation and induce apoptosis in human gastric carcinoma.Moreover,5-Aza-dC combined with 5-FU and PTX is more efficient than either 5-Aza-dC or 5-FU combined PTX alone.