Inhibition of NF-κB induces apoptosis in prostate cancer cells and inhibits tumor formation in SCID mice. (A) Apoptosis assay. DU145 cells were infected with AdCMVIκBα or AdCMVβ-gal and apoptosis was measured 24, 48, and 72 h after infection. Data are means ± SD of three independent infections for each virus at each time point. (B and C) Inhibition of tumor formation by IκBα expression. A quantity of 2 × 10⁶ DU145 cells infected with AdCMVIκBα or AdCMVβ-gal were implanted orthotopically into the prostate of SCID mice. The IL-6 expression (B) and the size of the tumors and tumor weight (C) were measured 2 months after implantation.

Expression of GADD45 family members is regulated by the NF-κB/IκB signaling pathway through c-Myc expression. (A) Real-time PCR analysis of GADD45α, β, and γ after inhibition of NF-κB in different cancer cell lines. Total RNA was collected from DU145, SKBR3, LNCaP, Caki, and UOK cells infected with Ad5CMVβ-gal or Ad5CMVIκB. Each RNA was normalized to GAPDH. (B) Real-time PCR analysis of GADD45α, β, and γ upon activation of NF-κB. Total RNA was collected from U138 MG cells, treated with IL-1β for 6 h, and infected with Ad5CMVβ-gal or Ad5CMVIκB or without Ad infection (control). Each RNA was normalized to GAPDH. (C) Real-time PCR analysis of GADD45α and γ in prostate cancer cells transfected with NF-κB. Total RNA was collected from LNCaP cells transfected with p50 and p65 NF-κB expression vectors or pCI vector as a control. Each RNA was normalized to GAPDH. (D) Western blot analysis of GADD45α, β, and γ after inhibition of NF-κB. Protein extracts were obtained 24, 48, and 72 h after infection with Ad5CMVβ-gal or Ad5CMVIκB. (E) Western blot analysis of c-Myc after inhibition of NF-κB. Protein extracts were obtained 24, 48, and 72 h after infection with Ad5CMVβ-gal or Ad5CMVIκB. (F) Transcriptional activity of the GADD45α promoter. LNCaP cells were transfected with the GADD45α promoter-luciferase construct with the NF-κB p50 and p65 expression vectors or c-Myc expression vector. Luciferase activity was determined 16 h later. Data are means ± SD of two results of one representative transfection normalized to the amount of β-gal expression. Luciferase activity of the GADD45α promoter is shown as percentage of control of the pCI parental vector as indicated on the left. (G) Transcriptional activity of the GADD45α promoter in DU145 cells after infection with Ad5CMVβ-gal or Ad5CMVIκB and transfection of c-Myc expression vector or parental vector. Luciferase activity of the GADD45α promoter is shown as fold induction over the β-gal control.

IκBα-induced GADD45 expression is essential for apoptosis induction. (A) Apoptosis of prostate cancer cells after transfection with GADD45α, β, and γ. DU145 and PC-3 cells were transfected with GADD45α, β, and γ expression vectors (1μg) and apoptosis was measured after 48 h. Data are means ± SD of three independent transfections for each vector. (B) Apoptosis assay of DU145 prostate cancer cells and MDA-231 breast cancer cells after infection with Ad5CMVIκB or AdCMVβ-gal and infections with lentiviruses encoding GADD45α, β, and γ siRNA duplexes. Data are means ± SD of three independent transfections.

JNK activation in response to blockage of NF-κB due to induction of GADD45α and γ. (A) JNK kinase assays after transfection with GADD45α, β, and γ. DU145 cells were transfected with GADD45α, β, and γ expression vectors (1 μg) and analyzed by using the SAPK/JNK assay kit (Cell Signaling Technology). (B) DU145 cells were infected with AdCMVIκBα or AdCMVβ-gal and protein extracts were collected 48 h after infection. The proteins were analyzed by Western blots (Top and Middle) by using anti-MKK4 antibody (Cell Signaling Technology), anti-phospho-JNK (Cell Signaling Technology), and anti-total JNK (Cell Signaling Technology), and c-jun phosphorylation (Bottom) showed MKK4 and JNK activity in prostate cancer cells at 24, 48, and 72 h after infection with each virus.

Induction of GADD45 expression is essential for JNK activation and apoptosis induction. (A) Apoptosis of prostate and breast cancer cells 48 h after infection with Ad5CMVIκB or AdCMVβ-gal and treatment with the JNK inhibitor JNKII SP600125 (100 nM, Calbiochem). Data are means ± SD of three independent infections. (B) Apoptosis of two prostate cancer cell lines after transfection with expression vectors (1 μg) for the JNK family members, JNK1 and 2, and its upstream regulator MKK4. Data are means ± SD of three independent transfections. (C) Apoptosis of prostate cancer cells after transfection with the GADD45 family members (1 μg) and treatment with the JNK inhibitor, SP600125 (100 nM). Data are means ± SD of three independent transfections. Apoptosis is shown as the percentage of apoptosis for each GADD45 family gene in the absence of the JNK inhibitor. (D) Kinase assay showing inhibition of JNK kinase activity by GADD45 siRNA duplexes (50 nM) in prostate cancer cells infected with Ad5CMVIκB or AdCMVβ-gal.