Effects and mechanisms of extracellular-signal regulated protein kinase-motogenactived protein kinase signaling pathway in gastrin-induced cell proliferation and apoptosis of colorectal cancer cells / 中华消化外科杂志

ABSTRACT

Objective To investigate the effects and mechanisms of extracellular-signal regulated protein kinase-motogenactived protein kinase(ERK-MAPK)signaling pathway in gastrin-induced cell proliferation and apoptosis of colorectal cancer cells.Methods HT-29 cells were incubated in different media,and then were divided into the control group,gastrin group,proglumide group and gastrin + proglumide group.No reagent was added in the control group,and other groups were dealed with reagent in different concentrations.The changes of proliferation of the HT-29 cells were detected by MTT assay,and the optimal concentration of gastrin and proglumide were determined.The changes of proliferation index and apoptotic rates of HT-29 cells were detected by cell cytometry.The mRNA expressions of gastrin receptor/cholecystokinin-B receptor(CCK-BR),ERK1/2 and K-ras were detected by RT-PCR.The protein of ERK1/2,K-ras protein and phosphorylation levels were detected by Western blot.All data were analyzed by analysis of variance and SNK-q test.Results The proliferation of HT-29 was stimulated by gastrin when the concentration of the gastrin was 6.25-100.00 mg/L,and the optimal concentration of gastrin was 25.00 mg/L(F =31.36,P ＜ 0.05).Proglumide had no obvious effects on the proliferation of HT-29 cells,while it significantly inhibited the proliferation of HT-29 cells stimulated by gastrin when the concentration of proglumide was 8.00-128.00 mg/L,and the optimal concentration was 32.00 mg/L(F =24.31,P ＜ 0.05).The proliferation index of the gastrin(25.00 mg/L)group was 37.5 ％ ± 5.2％,which was significantly higher than 27.7％ ± 5.0％ of the control group and 27.3％ ± 5.8％ of the gastrin(25.00 mg/L)+ proglumide(32.00 mg/L)group(q =4.56,4.75,P ＜ 0.05).The apoptotic index of the gastrin(25.00 mg/L)group was 1.9％ ± 0.4％,which was significantly lower than 2.5％ ± 0.4％ of the control group and 2.4％ ± 0.3％ of the gastrin(25.00 mg/L)+ proglumide(32.00 mg/L)group(q =4.23,4.06,P＜0.05).The mRNA expression of CCK-BR was detected in the HT-29 cells.The levels of phosphorylated ERK1/2 protein and phosphorylated K-ras protein were 0.43％ ± 0.04％ and 0.45％ ± 0.06％,which were significantly higher than 0.32％ ± 0.02％ and 0.31％ ± 0.05 ％ of the control group(q =7.78,4.95,P ＜ 0.05),and they were also higher than 0.36％ ± 0.01％ and 0.35 ％ ± 0.04％ of the gastrin(25.00 mg/L)+ proglumide(32.00 mg/L)group(q =5.72,4.08,P ＜0.05).There were no significant differences in the mRNA and protein expressions of ERK1/2 and K-ras among the control group,gastrin(25.00 mg/L)group,proglumide(32.00 mg/L)group and gastrin (25.00 mg/L)+ proglumide(32.00 mg/L)group(F =0.52,0.72,0.78,0.28,P ＞0.05).Conclusion Gastrin could stimulate the proliferation of HT-29 cells and inhibit their apoptosis by upregulate the phosphorylation levels of ERK and K-ras through the Ras→Raf→ MEK1/2→ ERK1/2 pathway,while the effect can be restrained by gastrin receptor antagonist proglumide.