Immunoblot analysis showed J7-DKK4 cells secreted more DKK4 into the culture medium than did J7-control cells (Fig.

4A). The overexpression of DKK4 was significantly decreased cellular proliferation compared with J7-control cells (Fig. 4B). To test the effect of the DKK4 gene on cell invasiveness was measured in vitro using Matrigel Transwell invasion assays. Further, the AG-14699 invasiveness of J7-DKK4#1 and #2 cells was inhibited by 75%-80% in J7 cells (Fig. 4C). Images of cell density were shown for two control and two overexpressing cell lines (Fig. 4C). The quantified results are shown in Supporting Fig. 2A. To determine the effect of DKK4 on the Wnt-canonical signaling pathway, the expression of several proteins involved in this pathway was measured in J7 cells. β-Catenin was significantly down-regulated by 35%-40% in the two DKK4-overexpressing cell lines compared with control cell lines. In contrast, phosphorylated β-catenin protein was up-regulated by almost 1.6-fold in

the two DKK4-overexpressing cell lines. The expression levels of CD44, cyclin D1, and c-Jun significantly decreased in DKK4-overexpressing cell lines in immunoblot analysis (Fig. 4D; Supporting Fig. 2B). This is consistent Metabolism inhibitor with a previous study indicating that c-Jun is a target gene of the Wnt/β-catenin pathway in human colorectal carcinomas.18 Overexpression of DKK4 decreased the activity of pro-matrix metallopeptidase (pro-MMP)-9 (92 kDa) and pro-MMP-2 (72 kDa) by 65%-70% and 18%-30% in J7 cells, respectively (Fig. 4E; Supporting Fig. 2C). To confirm the effect of DKK4 on β-catenin-ubiquitin complex formation, we performed immunoprecipitation assays. The data indicate the DKK4-mediated ubiquitination of β-catenin

is involved in the degradation of β-catenin (Fig. 4F). To investigate the effect of DKK4 and TR on tumor growth in vivo, we established a xenograft of J7 cells in BALB/c nude mice. Three J7 isogenic cell lines, J7-control, J7-DKK4, and J7-TRα1 were established. To verify the levels of expression of the DKK4 and TR proteins in the three J7 cell lines, cells were incubated with 1 nM T3 for 24 hours. The levels of the DKK4 protein were increased in J7-DKK4 selleck and J7-TRα1 cells compared with J7-control cells (Fig. 5A). The three J7 cell lines were injected subcutaneously and monitored continuously for 21 days. Figure 4B shows the average tumor volume observed in each of the three groups (n = 4). J7-DKK4 and J7-TRα1-induced tumors grew significantly slower than did control tumors. On average, after 21 days, tumors detected in mice injected with J7-DKK4 and J7-TRα1 cells were 45%-90% smaller compared with the tumors observed in control mice (Fig. 5B). To determine whether the in vitro results (Fig. 4C) could be reproduced in vivo, we investigated the effect of DKK4 on lung colony-forming ability in SCID mice (Fig. 5C).