Objective To investigate the effects and mechanisms of extracellular-signal regulated protein kinase-motogenactived protein kinase (ERKMAPK) 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 RTPCR. 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% ±0.01%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.
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