人结肠癌细胞胃泌素-黏着斑激酶通路的研究

A study of gastrin-focal adhesion kinase signal pathway in human colon cancer cells

目的研究胃泌素对人结肠癌细胞信号分子黏着斑激酶(FAK)酪氨酸磷酸化和蛋白质表达的影响。方法使用胆囊收缩素2受体(CCK2R)的真核表达载体pCR3.1/CCK2R,转染人结肠癌细胞株Colo320,上调胃泌素作用;使用胃泌素拮抗剂下调胃泌素作用。使用胃泌素按浓度和时间梯度刺激细胞,以免疫沉淀和蛋白质印迹法检测FAK酪氨酸磷酸化和蛋白质表达情况。结果胃泌素能够引起FAK酪氨酸磷酸化,具有时间和剂量依赖性;CCK2R表达上调可以增强此作用;胃泌素拮抗剂具有相反作用。结论FAK是胃泌素发挥效应的下游信号分子,以酪氨酸磷酸化的形式发挥作用。胃泌素CCKRFAK信号通路在胃泌素引起的肿瘤细胞增殖过程中发挥重要作用。

Objective The study investigated the effect of gastrin on tyrosine phosphorylation and protein expression of focal adhesion kinase (FAK) in human colon cancer cells.Methods The eukaryotic plasmid that expresses cholecystokinin 2 receptor(CCK_2R) stably, named pCR3.1/CCK_2R,was transfected into Colo320 to construct an up-regulated gastrin-CCK_2R signal pathway; the gastrin antagonist was used to down-regulate the signal pathway. Thus, including the normal control, there were three signal pathways with different CCK_2R levels. Different doses of gastrin were used to stimulate FAK in the different time. FAK tyrosine phosphorylation and FAK expression in different groups were detected by immunoprecipitation and Western-blotting assays, and analyzed with Labimage software. Results RT-PCR result showed that Colo320 transfected with CCK_2R had a mRNA level four times higher than that normal Colo320 did, and which suggested that up-regulated signal transduction pathway was constructed. After stimulated by gastrin with 0, 0.1, 1, 10 and 100 nmol/L, tyrosine phosphorylation levels of Colo320 were 24.0%,39.7%, 46.2%, 50.4% and 44.5%, and those of Colo320 transfected with CCK_2R were 24.6%, 70.7%, 90.1%, 100% and 88.6%. When incubated with gastrin at 0, 2.5, 5, 10 and 20 min, the tyrosine phosphorylation levels of Colo320 were 23.9%, 63.6%, 58.6%, 45.5% and 40.9%, and those of Colo320 transfected with CCK_2R were 24.5%, 84.6%, 100%, 98.6% and 97.9%. The increases of tyrosine phosphorylation in both Colo320 and Colo320 transfected with CCK_2R were dose dependence of gastrin. In Colo320, the time of phosphorylation had a tendency of exhaustion at 2.5 min; but in Colo320 transfected with CCK_2R, it was at 10 min. Gastrin had no effects on FAK protein expression in different cell groups. An up-regulated level of CCK_2R could enhance the effect of gastrin on FAK tyrosine phosphorylation. The gastrin antagonist showed an effect of competitive inhibition on tyrosine phosphorylation of FAK. Conclusions FAK is a signal transducer in downstream of CCK_2R; FAK exerts its functions by tyrosine phosphorylation, but dose not increase FAK protein.Gastrin-CCK_2R-FAK signal pathway is a pivotal one in the cell growth and proliferation caused by gastrin.