Mao Jia-Ding, Wu Pei, Huang Jian-Xiong, Wu Jian, Yang Guang
Jia-Ding Mao, Pei Wu, Jian-Xiong Huang, Jian Wu, Guang Yang, Department of General Surgery, the First Affiliated Yijishan Hospital of Wannan Medical College, Wuhu 241001, Anhui Province, China.
World J Gastroenterol. 2014 Sep 21;20(35):12542-50. doi: 10.3748/wjg.v20.i35.12542.
To explore the role and mechanisms of extracellular signal-regulated protein kinase-mitogen-activated protein kinase (ERK-MAPK) signaling in pentagastrin-regulated growth of large intestinal carcinoma.
HT-29 cells were incubated in different media and divided into the control group, pentagastrin group, proglumide group, and pentagastrin + proglumide group. No reagent was added to the control group, and other groups were incubated with reagent at different concentrations. Changes in proliferation of HT-29 cells were detected by MTT assay, and the optimal concentrations of pentagastrin and proglumide were determined. The changes in proliferation index (PI) and apoptosis rate (AR) of HT-29 cells were detected by Annexin V-fluorescein isothiocyanate flow cytometry. mRNA expression of pentagastrin receptor/cholecystokinin-B receptor (CCK-BR), ERK1/2 and K-ras were detected by reverse transcriptase polymerase chain reaction. The protein and phosphorylation level of ERK1/2 and K-ras were detected by western blotting. All data were analyzed by analysis of variance and SNK-q test.
The proliferation of HT-29 cells was stimulated by pentagastrin at a concentration of 6.25-100 mg/L, and the optimal concentration of pentagastrin was 25.0 mg/L (F = 31.36, P < 0.05). Proglumide had no obvious effect on the proliferation of HT-29 cells, while it significantly inhibited the proliferation of HT-29 cells stimulated by pentagastrin when the concentration of proglumide was 8.0-128.0 mg/L, and the optimal concentration was 32.0 mg/L (F = 24.31, P < 0.05). The PI of the pentagastrin (25.0 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 pentagastrin (25.0 mg/L) + proglumide (32.0 mg/L) group (Q = 4.56-4.75, P < 0.05). The AR of the pentagastrin (25.0 mg/L) group was 1.9% ± 0.4%, which was significantly lower than 2.5% ± 0.4% of the control group and 2.4% ± 0.3% of the pentagastrin (25.0 mg/L) + proglumide (32.0 mg/L) group (Q = 4.23-4.06, P < 0.05). mRNA expression of CCK-BR was detected in HT-29 cells. The phosphorylation levels of ERK1/2 protein and phosphorylated K-ras protein of the pentagastrin group were 0.43% ± 0.04% and 0.45% ± 0.06%, which were significantly higher than 0.32% ± 0.02% and 0.31% ± 0.05% of the control group (Q = 7.78-4.95, P < 0.05), and 0.36% ± 0.01% and 0.35% ± 0.04% of the pentagastrin + proglumide group (Q = 5.72-4.08, P < 0.05). There were no significant differences in the mRNA and protein expression of ERK1/2 and K-ras among the control, pentagastrin, proglumide and pentagastrin + proglumide groups (F = 0.52, 0.72, 0.78, 0.28; P > 0.05).
Gastrin stimulates proliferation of HT-29 cells and inhibits apoptosis by upregulating phosphorylation of ERK and K-ras through the Ras-Raf-MEK1/2-ERK1/2 pathway, and this is restrained by proglumide.
探讨细胞外信号调节蛋白激酶-丝裂原活化蛋白激酶（ERK-MAPK）信号通路在五肽胃泌素调节大肠癌细胞生长中的作用及机制。
将HT-29细胞置于不同培养基中，分为对照组、五肽胃泌素组、丙谷胺组和五肽胃泌素+丙谷胺组。对照组不添加任何试剂，其他组用不同浓度的试剂孵育。采用MTT法检测HT-29细胞增殖变化，确定五肽胃泌素和丙谷胺的最佳浓度。采用膜联蛋白V-异硫氰酸荧光素流式细胞术检测HT-29细胞增殖指数（PI）和凋亡率（AR）变化。采用逆转录聚合酶链反应检测五肽胃泌素受体/胆囊收缩素B受体（CCK-BR）、ERK1/2和K-ras的mRNA表达。采用蛋白质印迹法检测ERK1/2和K-ras的蛋白及磷酸化水平。所有数据采用方差分析和SNK-q检验进行分析。
五肽胃泌素在6.25～100mg/L浓度范围内可刺激HT-29细胞增殖，最佳浓度为25.0mg/L（F=31.36，P<0.05）。丙谷胺对HT-29细胞增殖无明显影响，但当丙谷胺浓度为8.0～128.0mg/L时，可显著抑制五肽胃泌素刺激的HT-29细胞增殖，最佳浓度为32.0mg/L（F=24.31，P<0.05）。五肽胃泌素（25.0mg/L）组的PI为37.5%±5.2%，显著高于对照组的27.7%±5.0%和五肽胃泌素（25.0mg/L）+丙谷胺（32.0mg/L）组的27.3%±5.8%（Q=4.56～4.75，P<0.05）。五肽胃泌素（25.0mg/L）组的AR为1.9%±0.4%，显著低于对照组的2.5%±0.4%和五肽胃泌素（25.0mg/L）+丙谷胺（32.0mg/L）组的2.4%±0.3%（Q=4.23～4.06，P<0.05）。在HT-29细胞中检测到CCK-BR的mRNA表达。五肽胃泌素组ERK1/2蛋白和磷酸化K-ras蛋白的磷酸化水平分别为0.43%±0.04%和0.45%±0.06%，显著高于对照组的0.32%±0.02%和0.31%±0.05%（Q=7.78～4.95，P<0.05），以及五肽胃泌素+丙谷胺组的0.36%±0.01%和0.35%±0.04%（Q=5.72～4.08，P<0.05）。对照组、五肽胃泌素组、丙谷胺组和五肽胃泌素+丙谷胺组之间ERK1/2和K-ras的mRNA和蛋白表达无显著差异（F=0.52、0.72、0.78、0.28；P>0.05）。
胃泌素通过Ras-Raf-MEK1/2-ERK1/2途径上调ERK和K-ras的磷酸化，刺激HT-29细胞增殖并抑制凋亡，而丙谷胺可抑制这一过程。
