Fig. 5.
Blocking NF-κB activation increases colon carcinoma metastatic potential in vivo. (A) Blocking NF-κB did not significantly alter CT26 cell growth in vitro. Left panel: CT26.pcDNA and CT26.IκBα-AA cells were cultured in the presence of IgG control mAb and ACH6, respectively, and NF-κB activation was analyzed by NF-κB reporter assay. Right panel: CT26.pcDNA and CT26.IκBα-AA cells were cultured as in the right panel for 5 days and analyzed by MTT assay. (B) Blocking NF-κB activation significantly increased the metastatic potential of colon carcinoma cells in vivo. CT26 cells were stably transfected with the pcDNA plasmid (Vector) and pcDNA-containing IκBα-AA (IκBα-AA) (0.5×105 cells/mouse). Cells were injected i.v. into syngeneic mice. Lung metastasis was analyzed 21 days after tumor injection. Left panel: images of tumor-bearing lungs. Right panel: the number of lung tumor nodules in each mouse was enumerated. Shown are results of one representative experiment of five independent experiments. (C) Left panel: RT–PCR analysis of IKKβ expression. CT26 cells were stably transfected with scramble shRNA (Scramble) or IKKβ-specific shRNA (IKKβ.shRNA) plasmid and analyzed for IKKβ mRNA expression. β-Actin was used as normalization control. Right panel: NF-κB activation in CT26 cells. CT26.Scramble and CT26.IKKβ.shRNA cells were transiently transfected with a NF-κB-luciferase reporter overnight and then treated with ACH6 mAb for 8h for luciferase reporter assay. (D) Silencing IKKβ significantly increased the metastatic potential of colon carcinoma cells in vivo. CT26.Scramble and CT26.IKKβ.shRNA cells were injected i.v. into syngeneic mice. Lung metastasis was analyzed 21 days after tumor injection. Left panel: images of tumor-bearing lungs. Right panel: the number of lung tumor nodules in each mouse was enumerated. Each dot represents the number of tumor nodules of a single mouse.