新型DNA甲基转移酶抑制剂SGI-1027对尤文肉瘤细胞增殖抑制的实验研究

摘要

目的：探讨新型DNA甲基转移酶（ DNMT）抑制剂SGI-1027对尤文肉瘤SK-N-MC和SK-ES-1细胞系增殖和凋亡的影响。方法：细胞活性分析测定细胞增殖状况。流式细胞计数法测量细胞周期的改变。实时定量PCR和Western blot检测DNMT1、DNMT3a和DNMT3b以及抑癌基因p16、p21的mRNA和蛋白水平的变化以及凋亡剪切产物cleaved PARP的表达。结果：SGI-1027明显抑制尤文肉瘤细胞增殖，诱导G2期细胞增加并可见subG1峰，cleaved PARP表达增加。DNMT1、DNMT3a和DNMT3b的mRNA水平不变，但DN-MT1蛋白水平明显下调；p16和p21的mRNA和蛋白水平上调。结论：新型DNA甲基转移酶抑制剂SGI-1027能够抑制DNMT1活性，激活抑癌基因p16和p21，发挥对尤文肉瘤细胞凋亡诱导作用。%Objective:To investigate the anti-proliferative effect of novel DNA methyltransferase( DNMT)inhibi-tor SGI-1027 in Ewingˊs sarcoma cell lines SK-N-MC and SK-ES-1 in vitro. Methods:The proliferation of SK-N-MC and SK-ES-1 cells was determined by cell viability assay after treatment of SGI-1027. Cell cycle dis-tribution was determined by flow cytometry. The mRNA level and protein expression of DNMT1,DNMT3a,DNMT3b, tumor suppressor p16,p21 and production of cleaved PARP were detected by real-time PCR and Western blot,re-spectively. Results:SGI-1027 significantly inhibited cell proliferation and resulted in G2 arrest as well as appearance of subG1 peaK. Western blot showed the expression of cleaved PARP. Although there were no change of mRNA level in DNMT1,DNMT3a and DNMT3b,protein level of DNMT1 was downregulated. The mRNA and protein level of p21 and p16 was upregulated. Conclusion:Novel DNA methyltransferase inhibitor SGI-1027 can inhibit DNMT1 activity and reactivate tumor suppressor p21 and p16,finally induce apoptosis in Ewingˊs sarcoma cell lines.