5-杂氮-2’-脱氧胞苷抑制人喉癌细胞生长机制的研究

Inhibition of 5-aza-2-deoxycytidine on Human Laryngeal Cancer Cells

目的观察5-杂氮-2’-脱氧胞苷对Hep-2人喉癌细胞系生长增殖的影响,探讨其抗肿瘤效应及可能的机制。方法用5- 杂氮-2’-脱氧胞苷对Hep-2人喉癌细胞系处理后,甲基化特异性PCR(MSP),T-A克隆测序分析死亡相关蛋白激酶(death-associ- ated protein kinase,DAPK)基因甲基化状态,逆转录聚合酶链反应、免疫细胞化学、流式细胞仪检测用药前后DAPK mRNA和蛋白的表达情况及细胞凋亡和周期的变化。结果未经5-杂氮-2’-脱氧胞苷处理的Hep-2细胞中DAPK基因CpG岛甲基化,且 DAPK mRNA不表达。经5-杂氮-2’-脱氧胞苷处理后,甲基化的DAPK基因部分去甲基化,大于或等于10-7 mol·L-1组的细胞中有DAPK mRNA表达,且表达随浓度增高而增强。免疫细胞化学染色显示,经处理后的肿瘤细胞DAPK蛋白表达呈阳性,而对照组不表达。流式分析结果为处理后凋亡率明显增加,且G1期细胞增加,G2／M期减少。结论 5-杂氮-2’-脱氧胞苷能抑制喉癌细胞的生长和增殖,可能的机制是其能使因甲基化而失活的DAPK基因再转录,诱导该基因的重新表达。

OBJECITVE To observe the effect of 5-aza-2＇-deoxycytidine on the growth of Hep-2 cell line. To explore it＇s antitumor effect and mechanism.METHOdS Hep-2 cell line was treated by 5-aza-2＇-deoxycytidine. Methytation status was evaluated by methylation specific PCR,T-A cloning and sequence analysis. Cell apoptosis,the expression of DAPK mRNA and protein were determined by flow cytometer, reverse transcription polymerase chain reaction （RT-PCR） and immunocytochemistry. RESULTS Promoter hypennethylation of DAPK gene was obtained and no expression of DAPK mRNA was found in Hep-2 cell without being treated with 5-aza-2＇-deoxycytidine. Hypermethylated DAPK gene was partly demethylated. Induced DAPK expression in a dose-dependent manner was observed in Hep-2 cell treated with 5-aza-2＇-deoxycytidine（ 〉 10^-7 mol·L^-1）. Immunneytnehemistry indicated that the expression of DAPK protein was positive in treatment group; it was negative in control group. Flow cytometer showed that the proportion of apoptosis cells and G1 stage cells was significantly increased; while the proportion of G2/M stages cells was reduced in Hep-2 cells after being treated with 5-aza-2＇-deoxycytidine, CONCLUSION 5-Aza-2＇-deoxycytidine may slow the growth of Hep-2 cells by reactivating DAPK genes silenced by promoter methylation in vitro.