Molecular mechanism of gastrin increasing colon cancer cells'invasion
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
Objective To explore the molecular mechanism of increasing the invasion of colon cancer cells by gastrin 17.Methods 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~(TM)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 gastrinl7 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.Cofoeal microscopy was used to observe the phosphorylated FAKTyr397 localizing in the lamellipoda. 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.Results 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 I265,260 treatment.The amount of phosphorylated FAKTyr397 localizing in the lamellipoda of the Colo320WT cells that were treated by gastrinl7 increased time-dependently and peaked at the time-point 12 h.FAK-Src-pl30~(Cas)-Dockl80 signaling complex was formed in the Colo320WT cells stimulated with gastrin17.Gastrin17 activated Rac,but did not affect the total Rac expression.Conclusion The mechanism of increasing the colon cancer cells' invasion by gastrin17 is probably that gastrin17 makes FAK-Tyr397 phophorylated and be localized to lamellipoda,causes the forming of FAK-Src-p130~(Cas)-Dock180 signaling complex when it is bound to its receptor CCK-2,and activation of Rac.
【CateGory Index】： R735.34
【CateGory Index】： R735.34