胃泌素对胃癌细胞粘着斑激酶酪氨酸磷酸化的影响

Effect of Gastrin on Tyrosine Phosphorylation of Focal Adhesion Kinase in Human Gastric Cancer Cell Line SGC7901

背景与目的:胃泌素能够刺激胃癌细胞的生长和增殖,这一作用同酪氨酸激酶有关。本研究旨在阐明胃泌素对人胃癌细胞内粘着斑激酶(focaladhesion kinase,FAK)酪氨酸磷酸化的影响。方法:用人胃泌素受体(gastrinreceptor,GR)的真核表达载体pCR3.1/GR,转染人胃癌细胞株SGC7901,增强胃泌素受体表达;用胃泌素受体拮抗剂L-365260,抑制胃泌素和其受体结合;以不同浓度和作用时间的胃泌素刺激胃癌细胞,利用免疫沉淀和蛋白质印迹法检测上述情况下,FAK酪氨酸磷酸化的变化。结果:分别用0.1nmol/L、1nmol/L和10nmol/L的胃泌素作用后,转染pCR3.1/GR的SGC7901细胞内FAK酪氨酸磷酸化表达量分别为0.64±0.06、0.91±0.10和1.00±0.10,高于SGC7901细胞的0.40±0.05、0.52±0.07和0.62±0.06(P<0.01);转染pCR3.1/GR的SGC7901细胞内FAK酪氨酸磷酸化表达量分别为0.72±0.08、0.83±0.05、0.88±0.06和1.00±0.08,高于SGC7901细胞的0.59±0.05、0.65±0.07、0.58±0.03和0.47±0.10(P<0.01或P<0.05)。胃泌素受体拮抗剂L-365260使转染pCR3.1/GR的SGC7901细胞内FAK酪氨酸磷酸化表达量从1.00±0.07降至0.72±0.07(P<0.01),使SGC7901细胞内表达量由0.62±0.06降至0.45±0.05(P<0.01)。在此过程中,FAK蛋白表达量差异无统计学意义(P>0.05)。结论:FAK是胃泌素和其受体结合后发挥效应的关键下游信号分子,酪氨酸磷酸化是其活性形式。

BACKGROUND ＆ OBJECTIVE： Gastrin contributes to the growth and proliferation of gastric cancer cells and it is related to the effect of tyrosine kinase. This study was to investigate the effect of gastrin on tyrosine phosphorylation of focal adhesion kinase （FAK） in human gastric cancer cell line SGC7901. METHODS： The vector pCR3.1/GR,, that expresses human gastrin receptor （GR） stably, was transfected into SGC7901 cells （SGC7901- GR）. The expression of GR was tested by reverse transcription-polymerase chain reaction （RT-PCR）. SGC7901-GR and SGC7901 cells were treated with L-365260, an antagonist of GR, and stimulated with gastrin at different concentrations for different time. The tyrosine phosphorylation level of FAK was detected by immunoprecipitation and Western blot. RESULTS： When treated with different concentrations of gastrin for 1 min, the tyrosine phosphorylation levels of FAK were significantly higher in SGC7901-GR cells than in SGC7901 cells （0.64±0.06 vs. 0.40±0.05 at 0.1 nmol/L, 0.91 ±0.10 vs. 0.52±0.07 at 1 nmol/L, and 1.00±0.10 vs. 0.62±0,06 at 10 nmol/L, P〈 0.01）. When treated with 10 nmol/L gastrin for different time, the tyrosine phosphorylation levels of FAK were also significantly higher in SGC7901-GR cells than in SGC7901 cells （0.72±0.08 vs. 0.59±0.05 at 1 min, 0.83±0.05 vs. 0.65±0.07 at 5 min, 0.88±0.06 vs. 0.58±0.03 at 10 min, and 1.00±0.08 vs. 0.47±0.10 at 20 min, P〈0.05）. L-365260 decreased the tyrosine phosphorylation levels of FAK from 1.00±0.07 to 0.72±0.07 in SGC7901-GR cells （P〈0.01）, and from 0.62±0.06 to 0.45±0.05 in SGC7901 cells （P〈 0.01）. The protein levels of FAK in different ceils remained unchanged during these experiments （P〉0.05）, CONCLUSIONS： FAK is a pivotal signal transducer in downstream of gastrin with GR. Tyrosine phosphorylation is the symbol of FAK activation.