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

Abstract

To explore the effect of gastrin 17 (G17) on beta-catenin/T cell factor-4 (Tcf-4) signaling in colonic cancer cell line Colo320WT. 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 (TM)2000 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 (Gastrin(17) receptor blocker) for 30 min, and with G17 again for 12 h or L365,260 for 12 h. Expression levels of beta-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 beta-catenin in CoLoWT320 cells. Expression levels of c-myc and cyclin D1 in Colo320WT cells treated with G17 were assayed by Western blot. Expression levels of beta-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 beta-catenin in the TX-100 insoluble fraction were just on the contrary. Immunocytochemistry showed that beta-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. Gastrin17 activates beta-catenin/Tcf-4 signaling in Colo320WT cells, thereby leading to over-expression of c-myc and cyclin D1.