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Effects of gastrin 17 on β-catenin/Tcf-4 pathway in Colo320WT colon cancer cells

Jun Cao, Jie-Ping Yu, Hong-Gang Yu, Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China Chao-Hong Liu, State Key Laboratory of Virology, Wuhan institute of Virology, China Adcamy of Science, Wuhan 430060, Hubei Province, China Lan Zhou, Traditional Chinese Medicine Experiment Center, Hubei College of Traditional Chinese Medicine, Wuhan 4300061, Hubei Province, China  
AIM: To explore the effect of gastrin 17 (G17) on β-catenin/T cell factor-4 (Tcf-4) signaling in colonic cancer cell line Colo320WT. METHODS: The pCR3.1/GR plasmid, which expresses gastrin receptor, cholecystokinin-2 receptor (CCK-2R), was transfected into a colonic cancer cell line Colo320 by Lipofectamine TM2000 and the stably expressing CCK-2R clones were screened by G418. The expression levels of gastrin receptor in the Colo320 and the transfected Colo320WT cell line were assayed by RT- PCR. Colo320WT cells were treated with G17 in a time- dependent manner (0, 1, 6, 12, 24 and 48 h), then with L365,260 (Gastrin17 receptor blocker) for 30 min, and with G17 again for 12 h or L365,260 for 12 h. Expression levels of β-catenin in a TX-100 soluble fraction and TX-100 insoluble fraction of Colo320WT cells treated with G17 were detected by co-immuniprecipation and Western blot. Immunocytochemistry was used to examine the distribution of β-catenin in CoLoWT320 cells. Expression levels of c-myc and cyclin D1 in Colo320WT cells treated with G17 were assayed by Western blot. RESULTS: Expression levels of β-catenin in the TX-100 solution fraction decreased apparently in a time- dependent fashion and reached the highest level after G17 treatment for 12 h, while expression levels of β-catenin in the TX-100 insoluble fraction were just on the contrary. Immunocytochemistry showed that β-catenin was translocated from the cell membranes into the cytoplasm and nucleus under G17 treatment.Expression levels of c-myc and cyclin D1 in the G17- treated Colo320WT cells were markedly higher compared to the untreated Colo320WT cells. In addition, the aforementioned G17-stimulated responses were blocked by L365,260. CONCLUSION: Gastrin17 activates β-catenin/Tcf-4 signaling in Colo320WT cells, thereby leading to over- expression of c-myc and cyclin D1.
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