Zhang Jian-Feng, Zhang Jian-Guo, Kuai Xiao-Ling, Zhang Hong, Jiang Wei, Ding Wei-Feng, Li Zeng-Li, Zhu Hui-Jun, Mao Zhen-Biao
Department of Gastroenterology, Affiliated Hospital of Nantong University, Nantong, Jiangsu 226001, P.R. China.
Exp Ther Med. 2013 Mar;5(3):735-741. doi: 10.3892/etm.2013.901. Epub 2013 Jan 17.
The DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR) is widely used as an anticancer drug for the treatment of leukemia and solid tumors. Gastric cancer (GC) patients who were positive for caudal type homeobox transcription factor 2 (CDX2) expression showed a higher survival rate compared with those who were CDX2 negative, which suggests that CDX2 performs a tumor suppressor role. However, the molecular mechanisms leading to the inactivation of CDX2 remain unclear. In the present study we demonstrated that the expression levels of CDX2 and DNA methyltransferase enzyme 1 (DNMT1) mRNA were significantly higher in GC compared with distal non-cancerous tissue. The expression of CDX2 mRNA was significantly correlated with Lauren classification, TNM stage and lymph node metastasis. DNMT1 mRNA expression was significantly correlated with TNM stage, pathological differentiation and lymph node metastasis. The expression of CDX2 mRNA was inversely correlated with that of DNMT1 mRNA in GC. Hypermethylation of the CDX2 gene promoter region, extremely low expression levels of CDX2 mRNA and no expression of CDX2 protein were the characteristics observed in MKN-45 and SGC-7901 GC cell lines. Following the treatment of MKN-45 cells with 5-aza-CdR, the hypermethylated CDX2 gene promoter region was demethylated and expression of CDX2 was upregulated, while DNMT1 expression was downregulated. Furthermore, a concentration- and time-dependent growth inhibition as well as increased apoptosis were observed. Caspase-3, -8 and -9 activities increased in a concentration-dependent manner following exposure to different concentrations of 5-aza-CdR. Therefore, our data show that the overexpression of DNMT1 and methylation of the CDX2 gene promoter region is likely to be responsible for CDX2 silencing in GC. 5-Aza-CdR may effectively induce re-expression of the CDX2 gene, inhibit cell proliferation and enhance the caspase-independent apoptosis of MKN-45 cells in vitro.
DNA甲基化抑制剂5-氮杂-2'-脱氧胞苷（5-aza-CdR）作为一种抗癌药物被广泛用于治疗白血病和实体瘤。尾型同源盒转录因子2（CDX2）表达呈阳性的胃癌（GC）患者的生存率高于CDX2阴性患者，这表明CDX2发挥肿瘤抑制作用。然而，导致CDX2失活的分子机制仍不清楚。在本研究中，我们证明与远端非癌组织相比，GC中CDX2和DNA甲基转移酶1（DNMT1）mRNA的表达水平显著更高。CDX2 mRNA的表达与Lauren分类、TNM分期和淋巴结转移显著相关。DNMT1 mRNA表达与TNM分期、病理分化和淋巴结转移显著相关。GC中CDX2 mRNA的表达与DNMT1 mRNA的表达呈负相关。CDX2基因启动子区域的高甲基化、CDX2 mRNA极低的表达水平以及CDX2蛋白无表达是在MKN-45和SGC-7901 GC细胞系中观察到的特征。用5-aza-CdR处理MKN-45细胞后，高甲基化的CDX2基因启动子区域去甲基化，CDX2的表达上调，而DNMT1的表达下调。此外，观察到浓度和时间依赖性的生长抑制以及凋亡增加。暴露于不同浓度的5-aza-CdR后，半胱天冬酶-3、-8和-9的活性以浓度依赖性方式增加。因此，我们的数据表明，DNMT1的过表达和CDX2基因启动子区域的甲基化可能是GC中CDX2沉默的原因。5-aza-CdR可能有效地诱导CDX2基因的重新表达，抑制细胞增殖并增强体外MKN-45细胞的非半胱天冬酶依赖性凋亡。
