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

Figure 1 Expression and receptor binding characteristics of CCK2 receptor in Colo320 and Colo320 WT cells. A: RT-PCR using RNA extracted from Colo320 and Colo320WT cells; B: Immunoblotting using protein extracted from Colo320 and Colo320WT cells. Blotting result of β-actin was used to show the equal loading.

Figure 2 Immunoprecipitation of β-catenin in TX-100 solution fraction and the insoluble fraction of Colo320WT cells treated with G17 (10^-8 mol/L). TX-100 solution fraction was immunoprecipitated with anti-E-cadherin antibody, while TX-100 insoluble fraction was immunoprecipitated with anti-β-catenin antibody. E-cadherin immunoprecipitate and β-catenin immunoprecipite were separated on 100 g/L SDS/PAGE gel and probed with β-catenin antibody. Stimulation with G17 induced an increase in the level of β-catenin protein extracted from TX-100 -insoluble fraction and reached the highest at 12 h, while a reduction in the level of β-catenin protein extracted from TX-100 solution fraction. But the responses stimulated by G17 were blocked by L365,260 (Right lane: L).

Figure 3 Immunoprecipitation of β-catenin/Tcf-4 transcription complex in cell protein fraction treated with G17 (10^-8 mol/L). Cells were stimulated with G17 (10^-8 mol/L) for the times indicated and lysed. Whole cell lysates were immunoprecipitated with anti-Tcf-4 antibody and Tcf-4 immunoprecipitate was probed separately with anti-β-catenin or anti-Tcf-4 antibody. The results showed that the level of Tcf-4 and β-catenin from immunoprecipitation with Tcf-4 increased under G17 stimulation.

Figure 4 Immuno-cytochemical staining of β-catenin in Colo320WT cells treated with G17. A: Expression of β-catenin in Colo320WT cells stimulated for 0 h by G17 (10^-8 mol/L); B: Expression of β-catenin in Colo320WT cells stimulated for 12 h by G17 (10^-8 mol/L); C: Expression of β-catenin in Colo320WT cells stimulated for 12 h by L365,260 (10^-6 mol/L); D: Expression of β-catenin in Colo320WT cells stimulated for 30 min by 10^-6 mol/L L365,260 and then for 12 h with G17 (10^-8 mol/L).

Figure 5 Over-expression of c-myc and cyclin D1 in Colo320WT cells stimulated by G17. Expression levels of c-myc and cyclin D1 were determined by immunoblotting. The results showed that expression levels of both proteins increased markedly under G17 stimulation for 12 h. But L365,260 could abrogate the responses stimulated by G17.