丝裂原激活蛋白激酶阻断剂与DNA甲基化酶抑制剂对人结肠癌细胞的协同影响

The co-effect of extracellular-signal regulated kinase mitogen-activated protein kinase signaling pathway inhibitor and DNA methyltransferase inhibitor on human colon cancer cells

目的研究细胞外信号调节激酶-丝裂原激活蛋白激酶途径(ERK-MAPK)与DNA甲基化间的关系及对结肠癌细胞生物学行为的协同影响。方法培养人结肠癌细胞SW1116,分别以PBS、二甲基亚砜(DMSO)为对照组,PD 98059 50μmol／L、5-氮脱氧胞苷(5-aza-dC)5μmol／L、PD 98059 50 μmol／L+5-aza-dC 5μmol／L进行药物干预,以定量RT-PCR检测DNA甲基化酶(Dnmt)1、3a和3b基因转录水平;流式细胞仪分析细胞周期;MTT测定细胞活力;光学显微镜下观察细胞形态学变化。结果 ERK-MAPK途径阻断剂PD 98059下调Dnmt 1和Dnmt 3b,Dnmt抑制剂5 aza-dC下调Dnmt 1、 Dnmt 3a和Dnmt 3b,日5-aza-dC联合PD98059对Dnmt1及Dnmt 3a mRNA的表达下调更为显著。5- aza-dC明显降低G0／G1期细胞百分比(P<0．05),G2／M期细胞百分比明显增加(P<0．05);PD98059 使G0／G1期细胞百分比降低(P<0．05),G2／M期增加(P<0．05)。PD98059明显抑制细胞生长。PD 98059促进细胞分化,呈上皮样改变,细胞变狭长,胞质减少,细胞排列开始出现相对整齐;5-aza-dC干预组细胞大小不一,出现较多多倍体细胞(多个核分裂相)。结论 ERK-MAPK途径阻断剂及Dnmt抑制剂均能抑制结肠癌SW1116细胞分裂、增殖,并诱导细胞分化;两者有协同作,ERK-MAPK信号转导途径能调控DNA甲基化水平。

Objective To evaluate the relationship between extracellular-signal regulated kinase mitogen-aetivated protein kinase （ERK MAPK） signal transduetion pathway and DNA methylation and their biological cooperative effect on human colon cancer cells. Methods Human colon cancer cells SW1116 were treated with PD98059, 5-azA-2＇-deoxyeytidine （5 aza-dC）, or PD 98059 plus 5-aza-dC, respectively. The transcriptional level of DNA methyltransferase （Dnmt）1, Dnmt 3a and Dnmt 3b was detected by real-time RT-PCR. Cell cycle distribution was evaluated by flow cytometry . Cell proliferation was analyzed by MTT assay. In addition, cellular morphology changes were observed with optical microscopy. Results ERK MAPK inhibitor down regulated the expression of Dnmtl and Dnmt 3b mRNA. Dnmt inhibitor down-regulated the expression of Dnmt 1, Dnmt 3a and Dnmt 3b . The down regulation of Dnmt 1 and Dnmt 3a mRNA was more stronger in cells treated with 5-aza-dC plus PD98059 than that with 5-aza-dC or PD98059 seperately. Both 5-aza-dC and PD98059 decreased the percentage of phase G0/G1 eells（P〈0. 05） and increased the percentage of phase G2/M eells（P〈0. 05）. The cell proliferation was significantly inhibited by the treatment of PD98059. There was a remarkable change of cellular morphology under optical microscopy. The cells differentiated to analogous epithelium after treatment with PD98059, the profile of cells was slender, the endoehylema was reduced, and the array of cells was relatively regular. The size of cells was various and the polyploid cell was increased in the group treated with 5-aza-dC. Conclusions ERK MAPK inhibitor and DNA methylation inhibitor show an inhibitory effect on mitochysis, proliferation and differentiation of human colon cancer cell line, SW1116. ERK-MAPK signal transduction pathway might regulate DNA methylation level, and there is a cooperation effect of ERK MAPK inhibitor and DNA methylation inhibitor on cellular morphology and cell cycle in SW1116 cell line.