5-脱氧杂氮胞苷抑制肝癌细胞株生物学行为的机制

Effects of 5-Aza-2' -deoxycytidine on growth of human hepatocellular carcinoma cell lines

目的 DNA甲基化模式的改变伴随着永生性细胞系的确立,生长调控基因CpG岛的重新甲基化可能导致了其转录的不活跃,在体外培养的情况下,使得肿瘤细胞选择了有利于生长的条件,我们用DNA甲基转移酶抑制剂5-脱氧杂氮胞苷处理两株肝癌细胞,研究其对肝癌细胞的影响,探讨DNA甲基化异常与肝细胞癌间的相关性及5-脱氧杂氮胞苷对肝癌细胞株恶性生物学行为的影响及其机制。 方法 用5-脱氧杂氮胞苷处理肝癌细胞株SMMC-7721和HePG2,然后采用相差显微镜观察药物处理前后细胞的形态变化,采用MTT法观察细胞的生长速度变化,采用流式细胞仪检测细胞周期、细胞凋亡率、P16蛋白表达的变化,采用RT-PCR法比较p16和甲基转移酶mRNA表达量的变化,比较裸鼠致瘤性的大小。 结果 5-脱氧杂氮胞苷处理后细胞形态趋于规则,生长速度减慢,SMMC-7721细胞,G1期细胞增加了8.5％,而S期和C_2/M朗细胞分别减少了47.8％和10.4％,HePG2细胞,G_1期细胞增加了3.5％,S期减少了46.2％,G_2/M期增加了23.7％,两细胞凋亡率分别增加了91.6％和133.3％,P16蛋白表达分别增加了23.4％和20.9％,p16 mRNA表达增高,而DNA甲基转移酶mRNA表达明显降低,裸鼠皮下移植瘤生长减慢。 结论 肝细胞癌的发生与DNA甲基化异常有关,甲基化酶抑制剂5-脱氧杂氮胞苷可能通过抑制甲?

AIM In order to analyse the correlation between DNA methylation alternation and hepatocellular carcinoma cell lines, two hepatoma cell lines HePG2 and SMMC-7721 were exposed to the agent 5-Aza-2'-deoxycytidine to study the effects of 5-Aza-2'-deoxycytidine on the growth of two cell lines.METHODS Cell morphology, growth speed, cell cycle distribution, apoptotic rate, expression of p16 mRNA and protein, expression of methyltransferase mRNA and tumorigenicity in nude mice of SMMC-7721 and HepG2 cells after treatment with 5-Aza-2'-deoxycytidine were observed under phase contrast microscope, MTT assay, flow cytometer and RT-PCR.RESULTS HepG2 and SMMC-7721 cells after treatment with 5-Aza-2 -deoxycytidine displayed that supermicro structure inclined to normal and a slowed growth. In SMMC-7721 cells, G1 increased by 8.5%, S and G2 +M decreased by 47.8% and 10.6%, and apoptotic rate increased by 91.4%. In HePG2 cells, GI increased by 3.5%, S decreased 46.2%, G2+M increased 31%, and apoptotic rate increased 123.1%. The expression of p16 mRNA and protein increased, but the expression of methyltransferase mRNA decreased in two cell lines. Tumorigenicities in nude mice were low. CONCLUSION Hepatocellular carcinoma is associated with alternation of DNA methylation. 5-Aza-2' -deoxycytidine, as a well-established inhibitor of DNA methylation, may slow the growth of tumor cells by inhibited DNA methyltransferase and reactivating growth-regulatory genes silenced by de novo methylation and genes silenced by promoter hypermathylation (such as tumor suppressor p16 gene). This provides a new approach for tumor therapy by inhibition of methlyltransferase.