胃泌素干预影响人结肠癌细胞P190RhoGAP磷酸化的机制

目的:研究胃泌素对人结肠癌细胞P190RhoGAP的酪氨酸磷酸化水平的影响。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,上调胃泌素表达;使用胃泌素拮抗剂下调胃泌素表达。使用相同浓度的胃泌素(1×10-7mol/L)按时间梯度刺激细胞;采用蛋白质印迹法检测总FAK、酪氨酸磷酸化的FAK的表达情况,并确定FAK最大磷酸化时间点;以免疫沉淀和蛋白质印迹法检测FAK最大磷酸化时间点的P190RhoGAP的酪氨酸磷酸化水平。结果:胃泌素引起FAK最大酪氨酸磷酸化作用时间点为12 h,胃泌素刺激12 h较0 h、胃泌素拮抗剂+胃泌素刺激12 h后P190RhoGAP的酪氨酸磷酸化水平明显增加。结论:P190RhoGAP为FAK的下游信号分子,酪氨酸磷酸化的P190RhoGAP在胃泌素-CCK2R-FAK信号通路引起肿瘤细胞的侵袭、浸润、转移过程中发挥重要作用。

Mechanisms of the Increased Tyrosine Phosphorylation Level of P190RhoGAP in Human Colon Cancer Cells Treated by Gastrin

Objective: To investigate the effect of gastrin on the tyrosine phosphorylation level of P190RhoGAP in human colon cancer cells and to elucidate its mechanisms.Methods: The eukaryotic plasmid that expresses cholecystokinin 2 receptor(CCK_(2)R) stably,named pCR3.1 /CCK_(2)R,was transfected into Colo320 to up-regulate gastrin-CCK_(2)R signal pathway.This new cell line was named Colo320WT.Then the gastrin antagonist(L365,260) was used to down-regulate the signal pathway.The same dose of gastrin(1×10~(-7) mol/L) was used to stimulate the Colo320WT cells in different time.FAK tyrosine phosphorylation and FAK expression in different time was detected by immunoprecipitation and Western blotting assays.The tyrosine phosphorylation level of P190RhoGAP at 0 h and at the time of max FAK tyrosine phophorylation level,and incubated with L365,260 and gastrin was respectively detected by immunoprecipitation and Western blotting assays.Results: The RT-PCR result showed that Colo320 transfected with CCK_(2)R had a mRNA level four times higher than that of normal Colo320,which suggested that up-regulated signal transduction pathway was constructed.After incubated with 1×10~(-7)mol/L~()gastrin at 0,1,6,12,24,and 48 h,the tyrosine phosphorylation level of FAK and FAK expression in Colo320WT cells was detected.The max tyrosine phosphorylation level of FAK in Colo320WT cells was incubated with gastrin at 12 h.Then,Colo320WT cells were incubated with 1×10~(-7) mol/L gastrin at 0,12 h and with 1×10~(-6) mol/L~(-1)L365,260 + 1×10~(-7) mol/L gastrin at 12 h.The tyrosine phosphorylation level of P190RhoGAP was detected.The tyrosine phosphorylation level of P190RhoGAP at 12 h was significantly increased compared with that at 0 h that with and L365,260 + gastrin at 12 h.The gastrin antagonist showed competitive inhibition on tyrosine phosphorylation of P190RhoGAP.(Conclusion:)P190RhoGAP is a downstream signaling molecule of FAK and exerts its functions by tyrosine phosphorylation.Tyrosine phosphorylated P190RhoGAP is a critical one in the process of malignant cell invasion,infiltration and metastasis through gastrin-CCK_(2)(R-FAK) signal pathway.