Role of ERK-MAPK signaling pathway in pentagastrin-regulated growth of large intestinal carcinoma
Jia-Ding Mao;Pei Wu;Jian-Xiong Huang;Jian Wu;Guang Yang;Department of General Surgery,the First Affiliated Yijishan Hospital of Wannan Medical College;
AIM:To explore the role and mechanisms of extracellular signal-regulated protein kinase-mitogen-activated protein kinase(ERK-MAPK) signaling in pentagastrinregulated growth of large intestinal carcinoma.METHODS:HT-29 cells were incubated in different media and divided into the control group,pentagastrin group,proglumide group,and pentagastrin + proglumide group.No reagent was added to the control group,and other groups were incubated with reagent at different concentrations.Changes in proliferation of HT-29 cells were detected by MTT assay,and the optimal concentrations of pentagastrin and proglumide were determined.The changes in proliferation index(PI) and apoptosis rate(AR) of HT-29 cells were detected by Annexin V-fluorescein isothiocyanate flow cytometry.mRNA expression of pentagastrin receptor/cholecystokinin-B receptor(CCK-BR),ERK1/2 and K-ras were detected by reverse transcriptase polymerase chain reaction.The protein and phosphorylation level of ERK1/2 and K-ras were detected by western blotting.All data were analyzed by analysis of variance and SNK-q test.RESULTS:The proliferation of HT-29 cells was stimulated by pentagastrin at a concentration of 6.25-100 mg/L,and the optimal concentration of pentagastrin was 25.0 mg/L(F = 31.36,P 0.05).Proglumide had no obvious effect on the proliferation of HT-29 cells,while it significantly inhibited the proliferation of HT-29 cells stimulated by pentagastrin when the concentration of proglumide was 8.0-128.0 mg/L,and the optimal concentration was 32.0 mg/L(F = 24.31,P 0.05).The PI of the pentagastrin(25.0 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 pentagastrin(25.0 mg/L) + proglumide(32.0 mg/L) group(Q = 4.56-4.75,P 0.05).The AR of the pentagastrin(25.0 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 pentagastrin(25.0 mg/L) + proglumide(32.0 mg/L) group(Q = 4.23-4.06,P 0.05).mRNA expression of CCK-BR was detected in HT-29 cells.The phosphorylation levels of ERK1/2 protein and phosphorylated K-ras protein of the pentagastrin group 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 0.36% ± 0.01% and 0.35% ± 0.04% of the pentagastrin + proglumide group(Q = 5.72-4.08,P 0.05).There were no significant differences in the mRNA and protein expression of ERK1/2 and K-ras among the control,pentagastrin,proglumide and pentagastrin + proglumide groups(F = 0.52,0.72,0.78,0.28;P 0.05).CONCLUSION:Gastrin stimulates proliferation of HT-29 cells and inhibits apoptosis by upregulating phosphorylation of ERK and K-ras through the Ras-Raf-MEK1/2-ERK1/2 pathway,and this is restrained by proglumide.
【Fund】： Supported by Natural Science Foundation of Anhui Province No.1408085MH148;; Natural Science Fund of Education Bureau of Anhui Province No.kj2010b242;; Natural Science Fund of Wannan Medical College No.wk2012zf02;; the key science and technology project of Wuhu City No.health-2-4
【CateGory Index】： R735.34
【CateGory Index】： R735.34