Cao Jun, Yu Jie-ping, Zhou Lan, Song Wen-chong, Luo He-sheng, Yu Hong-gang
Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan 430060, China.
Zhonghua Yi Xue Za Zhi. 2007 Jun 26;87(24):1704-8.
To explore the molecular mechanism of increasing the invasion of colon cancer cells by gastrin 17.
The plasmid pCR 3.1/GR expressing the gastrin receptor cholecystokinin-2 receptor (CCK-2R) was transfected into colonic carcinoma cells of the line Colo320 by Lipofectamine 2000. The clones expressing stably CCK-2R were screened by G418 and named as Colo320WT cells. The expression levels of gastrin receptor of the Colo320 and Colo320WT cells were assayed by RT-PCR and Western blotting. The Colo320WT cells were treated by gastrin-17, and the expression levels of phosphorylated FAKTyr397 and total focal adhesion kinase (FAK) in the Colo320WT cells at the time points 0, 1, 6, 12, 24, and 48 h were detected by Western blotting. Another Colo320WT cells were treated by L365, 260, gastrin17 receptor blocker, for 30 minutes firstly and then treated by gastrin17 again for 12 hours, and then Western blotting was used to detect the expression levels of phosphorylated FAKTyr397 and total focal adhesion kinase (FAK) at the time points 0, 1, 6, 12, 24, and 48 h. Confocal microscopy was used to observe the phosphorylated FAKTyr397 localizing in the lamellipodia. The information of FAK-Src-p130(Cas)-Dock180 signaling complex was assayed by coimmuniprecipation and immunity blotting. The level of Rac-GTPase was tested by pull down assay.
The level of phosphorylated FAKTyr397 expression in the Colo320WT cells after the gastrin17 intervention increased time-dependently and peaked at the time point of 12 h, and the phosphorylated FAKTyr397 expression in the Colo320WT cells treated by L365, 260 decreased remarkably, but the level of total FAK remained unchanged. The phosphorylated FAKTyr397/FAK levels were 2.82%, 9.28%, 22.62%, 38.59%, 28.41%, and 14.94%, 0, 1, 6, 12, 24, and 48 h after the gastrin17 treatment respectively, and the level was 7.21% after L365, 260 treatment. The amount of phosphorylated FAKTyr397 localizing in the lamellipodia of the Colo320WT cells that were treated by gastrin17 increased time-dependently and peaked at the time-point 12 h. FAK-Src-p130(Cas)-Dock180 signaling complex was formed in the Colo320WT cells stimulated with gastrin17. Gastrin17 activated Rac, but did not affect the total Rac expression.
The mechanism of increasing the colon cancer cells' invasion by gastrin17 is probably that gastrin17 makes FAK-Tyr397 phosphorylated and be localized to lamellipodia, causes the forming of FAK-Src-p130(Cas)-Dock180 signaling complex when it is bound to its receptor CCK-2, and activation of Rac.
探讨胃泌素17增加结肠癌细胞侵袭能力的分子机制。
采用脂质体2000将表达胃泌素受体胆囊收缩素-2受体（CCK-2R）的质粒pCR 3.1/GR转染至Colo320结肠癌细胞系。通过G418筛选稳定表达CCK-2R的克隆，命名为Colo320WT细胞。采用RT-PCR和蛋白质免疫印迹法检测Colo320细胞和Colo320WT细胞中胃泌素受体的表达水平。用胃泌素-17处理Colo320WT细胞，采用蛋白质免疫印迹法检测处理后0、1、6、12、24和48小时Colo320WT细胞中磷酸化FAKTyr397和总粘着斑激酶（FAK）的表达水平。另取Colo320WT细胞，先用胃泌素17受体阻断剂L365,260处理30分钟，再用胃泌素17处理12小时，然后采用蛋白质免疫印迹法检测处理后0、1、6、12、24和48小时磷酸化FAKTyr397和总粘着斑激酶（FAK）的表达水平。采用共聚焦显微镜观察磷酸化FAKTyr397在片状伪足中的定位。通过免疫共沉淀和免疫印迹分析FAK-Src-p130（Cas）-Dock180信号复合物的情况。采用下拉实验检测Rac-GTP酶的水平。
胃泌素17干预后，Colo320WT细胞中磷酸化FAKTyr397的表达水平呈时间依赖性增加，在12小时达到峰值，L365,260处理的Colo320WT细胞中磷酸化FAKTyr397的表达显著降低，但总FAK水平保持不变。胃泌素17处理后0、1、6、12、24和48小时磷酸化FAKTyr397/FAK水平分别为2.82%、9.28%、2
