抑制组蛋白去乙酰化酶与DNA甲基转移酶活性对胶质瘤恶性表型的影响

摘要

目的:探讨阻断组蛋白去乙酰化酶与DNA甲基转移酶的活性后对胶质瘤恶性表型的抑制作用.方法:选择丙戊酸(VPA)为组蛋白去乙酰化酶抑制剂,5-氮杂-2'-脱氧胞苷(Aza)为DNA甲基转移酶抑制剂.将U251胶质瘤细胞分为对照组、VPA治疗组、Aza治疗组和VPA+Aza联合治疗组.采用四唑盐(MTT)比色法分析肿瘤细胞增殖活性,流式细胞术分析细胞周期,Annexin V-FITC染色检测细胞凋亡,2D Matrigel、3D Matrigel、Transwell法检测胶质瘤细胞侵袭能力,并建立U251细胞裸鼠皮下移植瘤模型,进行肿瘤局部多点注射上述药物治疗,治疗24 d,每4天测量肿瘤体积.结果:与对照组比较,在治疗2d后各治疗组肿瘤细胞的增殖均出现明显抑制,S期和G2/M期细胞比例减少,诱导细胞周期阻滞于G0/G1期,治疗组细胞凋亡率明显下降,侵袭和运动迁移能力均明显下降,各治疗组鼠移植瘤体积增长较对照组明显减缓,与对照组相比差异均有统计学意义(P＜0.05).以上结果均以VPA+Aza联合治疗组最为显著.结论:联合阻断DNA甲基转移酶和组蛋白去乙酰化酶活性可抑制胶质瘤表观遗传学特征,抑制胶质瘤恶性表型.%Objective: To investigate the suppressive effect of histone deacetylase and DNA methyltransferase on the malignant phenotype of human glioma cells. Methods: Valproic acid (VPA) was used as histone deacetylase inhibitor and 5-aza-deoxycytidine (Aza) as DNA methyltransferase inhibitor. For in vitro study, U251 glioma cells were divided into four groups: control group, VPA treatment group, Aza treatment group and VPA+Aza combined treatment group. The proliferation activity of tumor cells was detected by MTT assay, cell cycle analysis by flow cytometry, apoptotic rate by Annexin V-FTTC assay and the invasion ability of glioma cells by 2D Matrigel, 3D Matrigel and transwell assay. The subcutaneous U251 tumor model was established in nude mice for in vivo study, xenografts were treated with VPA, Aza and VPA+Aza by multi-point local injection into tumor bed every 4 days within the observation period for 28 days. The gross tumor volume was measured. Results: Compared with control group, the tumor cell proliferation activity was significantly decreased in VPA, Aza and VPA + Aza combined treatment groups (P < 0.05). The cells were arrested in G0/G1 phase. The apoptotic cell rate was increased and cell invasion ability decreased significantly (P < 0.05). The tumor volume was much smaller in all treatment groups than that in control group. The most significant effect on inhibition of malignant biological behavior of glioma cells was observed in VPA +Aza combined treatment group. Conclusion: To treat glioma cells with DNA methyltransferase and histone deacetylase inhibitors can reverse the malignant phenotype by inhibiting their epigenetic alterations.