ERK-MAPK信号转导通路在胃泌素促进人结直肠癌细胞增殖中的作用及其机制

茆家定; 钱海鑫; 吴佩; 黄剑雄; 武健

doi:10.3760/cma.j.issn.1673-9752.2013.02.013

ERK-MAPK信号转导通路在胃泌素促进人结直肠癌细胞增殖中的作用及其机制

Effects and mechanisms of extracellular signal regulated protein kinase motogenactived protein kinase signaling pathway in gastrin induced cell proliferation and apoptosis of colorectal cancer cells

摘要

摘要: 目的探讨细胞外信号调节蛋白激酶(ERK)-丝裂素活化蛋白激酶(MAPK)信号转导通路在胃泌素促进人结直肠癌细胞株HT-29增殖与凋亡中的作用及其机制｡方法体外培养结直肠癌HT-29细胞株,将细胞分为对照组､胃泌素组､丙谷胺组和胃泌素+丙谷胺组,胃泌素组设6.25､12.50､25.00､50.00､100.00mg/L5个浓度梯度,丙谷胺组设8.00､16.00､32.00､64.00､128.00mg/L5个浓度梯度,并设胃泌素(25.00mg/L)+丙谷胺组(8.00､16.00､32.00､64.00､128.00mg/L)浓度梯度｡对照组不加药,其余各组加不同浓度的药物处理｡运用MTT法观察细胞增殖活力改变情况,确立5-肽胃泌素和丙谷胺处理HT-29细胞的最佳浓度;流式细胞仪检测各组细胞增殖指数及凋亡率的变化;RT-PCR检测结直肠癌HT-29细胞株胃泌素/胆囊收缩素B受体(CCK-BR)mRNA表达情况以及各组细胞ERK1/2､K-rasmRNA表达水平的变化;Westernblot检测ERK1/2和K-ras蛋白表达及其磷酸化水平｡采用方差分析和SNK-q检验进行统计学分析｡结果胃泌素浓度在6.25~100.00mg/L能促进结直肠癌HT-29细胞的增殖,且当浓度达25.00mg/L时为最佳浓度(F=31.36,P<0.05);单独丙谷胺对结直肠癌HT-29细胞增殖无明显影响,但丙谷胺浓度在8.00~128.00mg/L能明显拮抗胃泌素促进HT29细胞的增殖作用,其最佳浓度为32.00mg/L(F=24.31,P<0.05)｡胃泌素(25.00mg/L)组细胞增殖指数为37.5%±5.2%,显著高于对照组的27.7%±5.0%和胃泌素(25.00mg/L)+丙谷胺(32.00mg/L)组的27.3%±5.8%(q=4.56,4.75,P<0.05);胃泌素(25.00mg/L)组细胞凋亡率为1.9%±0.4%,低于对照组的2.5%±0.4%和胃泌素(25.00mg/L)+丙谷胺(32.00mg/L)组的2.4%±0.3%(q=4.23,4.06,P<0.05)｡HT29细胞中存在明显的CCK-BRmRNA表达｡胃泌素(25.00mg/L)组磷酸化ERK1/2､磷酸化K-ras蛋白相对表达量分别为0.43%±0.04%和0.45%±0.06%,高于对照组的0.32%±0.02%和0.31%±0.05%(q=7.78,4.95,P<0.05),也高于胃泌素(25.00mg/L)+丙谷胺(32.00mg/L)组的0.36%±0.01%和0.35%±0.04%(q=5.72,4.08,P<0.05);但对照组､胃泌素(25.00mg/L)组､丙谷胺(32.00mg/L)组､胃泌素(25.00mg/L)+丙谷胺(32.00mg/L)组间ERK1/2mRNA和蛋白表达水平以及K-rasmRNA和蛋白表达水平比较,差异均无统计学意义(F=0.52,0.72,0.78,0.28,P>0.05)｡结论胃泌素可以促进结直肠癌细胞株HT-29的增殖,抑制细胞凋亡,其作用可能是通过ERK-MAPK信号通路中Ras-Raf-MEK1/2-ERK1/2途径上调ERK和K-ras的磷酸化水平来实现的,但能够被其受体拮抗剂丙谷胺抑制｡

Abstract:
Objective To investigate the effects and mechanisms of extracellular-signal regulated protein kinase-motogenactived protein kinase (ERKMAPK) signaling pathway in gastrin-induced cell proliferation and apoptosis of colorectal cancer cells.
Methods HT-29 cells were incubated in different media, and then were divided into the control group, gastrin group, proglumide group and gastrin+proglumide group. No reagent was added in the control group, and other groups were dealed with reagent in different concentrations. The changes of proliferation of the HT-29 cells were detected by MTT assay, and the optimal concentration of gastrin and proglumide were determined. The changes of proliferation index and apoptotic rates of HT-29 cells were detected by cell cytometry. The mRNA expressions of gastrin receptor/cholecystokinin-B receptor (CCK-BR), ERK1/2 and K-ras were detected by RTPCR. The protein of ERK1/2, K-ras protein and phosphorylation levels were detected by Western blot. All data were analyzed by analysis of variance and SNK-q test.
Results The proliferation of HT-29 was stimulated by gastrin when the concentration of the gastrin was 6.25-100.00 mg/L, and the optimal concentration of gastrin was 25.00 mg/L ( F=31.36, P<0.05). Proglumide had no obvious effects on the proliferation of HT-29 cells, while it significantly inhibited the proliferation of HT-29 cells stimulated by gastrin when the concentration of proglumide was 8.00-128.00 mg/L, and the optimal concentration was 32.00 mg/L (F=24.31, P<0.05). The proliferation index of the gastrin (25.00 mg/L) group was 37.5%±5.2%, which was significantly higher than 27.7%±5.0% of the control group and 27.3%±5.8% of the gastrin (25.00 mg/L)+proglumide (32.00 mg/L) group (q=4.56, 4.75, P<0.05). The apoptotic index of the gastrin (25.00 mg/L) group was 1.9% ±0.01%and 0.35%±0.04% of the gastrin (25.00 mg/L)+proglumide (32.00 mg/L) group (q=5.72, 4.08, P<0.05). There were no significant differences in the mRNA and protein expressions of ERK1/2 and K-ras among the control group, gastrin (25.00 mg/L) group, proglumide (32.00 mg/L) group and gastrin (25.00 mg/L)+proglumide (32.00 mg/L) group (F=0.52, 0.72, 0.78, 0.28, P>0.05).
Conclusion Gastrin could stimulate the proliferation of HT-29 cells and inhibit their apoptosis by upregulate the phosphorylation levels of ERK and K-ras through the Ras Raf MEK1/2 ERK1/2 pathway, while the effect can be restrained by gastrin receptor antagonist proglumide.

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