Figure 1.
CDH17 expression in gastric carcinoma tumor samples.
IHC staining in gastric carcinoma tissue (original magnification ×200). A: Negative, B: Weak, C: Positive(+), D: Positive(2+), E: Positive(3+).
Table 1.
Association Between Clinicopathologic Features and CDH17 expression in 156 patients with gastric cancer.
Figure 2.
Relationship between CDH17 expression and outcome of patients with gastric cancer.
Kaplan-Meier survival analysis with different CDH17 expression. A. Overall survival in 156 patients with gastric carcinoma (p<0.01) B. Disease-Free Survival in 131 gastric carcinoma patients who underwent R0 resection (p<0.01). C. Overall survival in 156 patients with gastric carcinoma according to CDH17 status (IHC 1+, 2+ and 3+)(p<0.01).
Figure 3.
Targeting CDH17 reduced the malignant phenotype in gastric carcinoma cells.
The expression of CDH17 was suppressed by lentiviral-mediated CDH17 shRNA (shCDH17); cells infected with non-targeted vector (Mock) and parental AGS or MKN-45 cells were used as controls. (A) qRT-PCR(Left) and western blot (Middle) show the mRNA and protein level of CDH17 in different experimental groups after puromycin selection. β-Actin was used as aloading control. (A) (Right) Growth curves of shCDH17 cells were compared with Mock, AGS cells by XTT assay. (Points, mean of at least three independent experiments; bars, SD). (B) Representative inhibition of foci formation in monolayer culture(Left) and colony formation in soft agar culture(Right) by CDH17 knockdown. Column chart shows quantitative analyses of foci or colonies numbers (columns, mean of at least three independent experiments; bars, SD). (C) (Left) A wound was created on a subconfluent culture of parental AGS, Mock cells and shCDH17 cells, and the rate of wound closure was monitored at 0, 24 and 48 hours. A representative photograph from three independent experiments is shown. Original magnification, ×200. (C) (Right) Representative images show the shCDH17, Mock and AGS cells that invaded through the matrigel. The number of invading tumor cells was quantified. Columns, mean of triplicate experiments; bars, SD. Original magnification, ×200. (D) Cell cycle analysis by way of flow cytometry. Knockdown CDH17 significantly impaired the tumorigenic and invasive properties of AGS cells, while inducing cell cycle G0/G1 arrest. (*P<0.05; **p<0.01; ***, P<0.001).
Figure 4.
CDH17 knockdown in AGS cells reduced their tumorigenicity in vivo and were used for targeting CDH17 for gastric cancer therapy.
A. Representative examples of tumors formed in nude mice following injection of shCDH17 cells in the left dorsal flanks. AGS and Mock cells were injected into the right, respectively. Line chart illustrates that the changes in volume of the subcutaneous tumors, shRNA vs. Mock,*p<0.05. B. Effect of CDH17 shRNA suppression on the growth of subcutaneous tumors in nude mice. Subcutaneous tumors were induced in nude mice by injection of AGS cells; treatment involved an intratumoral injection of Mock reagents (non-targeted RNAi vector) or shCDH17 regimen at a dose of 109 viral particle/injection at the tumor site of animals (n = 3). Photographs illustrate the subcutaneous tumor produced by Mock and shCDH17 in nude mice. The volume of tumor induced in nude mice was measured among the three experimental groups after 35 days. IHC pictures demonstrate that CDH17 expression decreased in shRNA treated mice. Original magnification, ×400. Line chart illustrates that the changes in volume of the subcutaneous tumors. shRNA vs. Mock, ***p<0.001.
Figure 5.
CDH17 activates multiple signal transduction pathways.
A. Expression of β-catenin, GSK-3β, p- GSK-3β, Rb, Cyclin D1were compared between shCDH17 and Mock both in AGS and MKN-45 cells by Western blot analysis. MDM2, p53 and p21 also were evaluated. β-Actin was used as a loading control. B. TOPflash/FOPflash reporter assay shows that Wnt signaling re-activated after CDH17 restoration in AGS and MKN-45. (* P<0.05). C. Increase of both the total and nuclear β-catenin protein after restoration of CDH17 in AGS and MKN-45, when compared with the shCDH17.