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, 30 (30), 3345-59

IL-32γ Inhibits Cancer Cell Growth Through Inactivation of NF-κB and STAT3 Signals

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IL-32γ Inhibits Cancer Cell Growth Through Inactivation of NF-κB and STAT3 Signals

J H Oh et al. Oncogene.

Abstract

Several studies have shown physiological functions of interleukin (IL)-32, a novel cytokine. However, the role of IL-32 in cancer development has not been reported. In this study, we showed that IL-32γ inhibited tumor growth in IL-32γ-overexpressing transgenic mice inoculated with melanoma as well as colon tumor growth in xenograft nude mice inoculated with IL-32γ-transfected colon cancer cells (SW620). The inhibitory effect of IL-32γ on tumor growth was associated with the inhibition of constitutive activated nuclear transcription factor-κB (NF-κB) and of signal transducer and activator of transcription 3 (STAT3). The expression of antiapoptotic, cell proliferation and tumor-promoting genes (bcl-2, X-chromosome inhibitor of apoptosis protein (IAP), cellular IAP and cellular FADD-like IL-1β-converting enzyme-inhibitory protein, cyclin D), cyclin-dependent kinase 4, cycolooxygenase-2 and inducible nitric oxide synthase was decreased, whereas the expression of apoptotic target genes (caspase-3 and -9, bax) increased. In tumor, spleen and blood, the number of cytotoxic CD8(+) T cells and CD57(+) natural killer cells and the levels of IL-10 increased, but that of tumor necrosis factor-α (TNF-α), IL-1β and IL-6 decreased. We also found that forced overexpression of IL-32γ inhibited colon cancer cell (SW620 and HCT116) growth accompanied with the inhibition of activated NF-κB and STAT3 in vitro. In addition, when IL-32γ was knocked down by small interfering RNA (siRNA) or neutralized with an anti-IL-32γ antibody, IL-32γ-induced colon cancer cell growth inhibition, the IL-32γ-induced decrease of TNF-α, IL-1 and IL-6 production, and the increase of IL-10 production were abolished. However, siRNA of NF-κB and STAT3 augmented IL-32γ-induced colon cancer cell growth inhibition. These findings indicate significant pathophysiological roles of IL-32γ in cancer development.

Figures

Figure 1
Figure 1
Generation of IL-32γ transgenic mice and detection of IL-32γ. (a) Scheme for IL-32γ transgenic generation. (b) PCR analysis was performed to analyze IL-32γ gene expression, as described in Materials and methods. (c) RT–PCR and western blotting analyses for IL-32γ in the spleen, thymus, liver, lung, kidney, colon and brain tissues of IL-32γ transgenic and non-transgenic mice. (d) Detection of IL-32 in the sera of transgenic mice or non-transgenic mice. The results are expressed as mean±s.d. of three mice. *P<0.05 compared with non-transgenic mice. A full colour version of this figure is available at the Oncogene journal online.
Figure 2
Figure 2
Effect of IL-32γ on tumor growth in IL-32γ-overexpressing transgenic mice. (a) Tumor images, volumes and weights. The results are expressed as mean±s.d. *P<0.05 compared with the non-transgenic mice. (b) Tumor sections were analyzed by immunohistochemistry. (c) Apoptotic cells were examined by fluorescence microscopy after TUNEL staining. (d) Tumor extracts were analyzed by western blotting. Each image and band is representative of three independent experiments. The values on the right of panels b and d are average percentages of vector control over three independent experiments.
Figure 3
Figure 3
Effect of IL-32γ on tumor growth in the SW620 xenograft in vivo model. (a) Tumor images, volumes and weights. The results are expressed as mean±s.d. *P<0.05 compared with the mice inoculated with vector-transfected colon cancer cells. (b) Tumor sections were analyzed by immunohistochemistry. (c) Apoptotic cells were examined by fluorescence microscopy after TUNEL staining. (d) Tumor extracts were analyzed by western blotting. Each image and band is representative of three independent experiments. The values on the right of panels b and d are average percentages of vector control over three independent experiments.
Figure 4
Figure 4
Expression of IL-32γ, growth rates and apoptotic cell death in IL-32γ-transfected colon cancer cells. (a) Colon cancer cells (1 × 106) were transfected with various amounts of the IL-32γ plasmid (1.5–6.0 μg) for 24 h and harvested. IL-32γ expression was detected by western blotting using monoclonal antibody KU32-52. To determine the effects of different IL-32γ levels on colon cancer cell growth, the cells were inoculated into 24-well plates (5 × 104 cells per well) and transfected with the IL-32γ plasmid (0.1–0.4 μg per well) for 72 h. Cell growth was measured by direct counting after Trypan blue staining. (b) Colon cancer cells were inoculated into 24-well plates (5 × 104 cells per well) and transfected with the vector or IL-32γ plasmid (0.4 μg per well). At 24, 48 and 72 h post-transfection, the cells were harvested by trypsinization and counted after Trypan blue staining. Control: untransfected cells. (c) Colon cancer cells were transfected with the vector or the IL-32γ for 72 h. Apoptotic cells were examined under a fluorescence microscope after TUNEL staining. The total number of cells in a given area was determined by 4′,6-diamidino-2-phenylindole nuclear staining. The results are expressed as mean±s.d. of three experiments with each experiment performed in triplicate. *P<0.05 compared with the vector-transfected colon cancer cells. (d) Cells were transfected with the vector or the IL-32γ for 24 h. Cell extracts were analyzed by western blotting. Each band is representative of three independent experiments.
Figure 5
Figure 5
Effect of silencing endogenous IL-32γ expression on colon cancer cell growth. (a) Colon cancer cells were inoculated into 24-well plates (5 × 104 cells per well) and co-transfected with the IL-32γ plasmid (0.4 μg per well) and the IL-32γ-specific siRNA (siIL-32; 50–100 n) or anti-IL-32 (5 or 10 μg/ml) for up to 72 h. The expression of IL-32γ was determined as described above. Thereafter, cell growth was measured by direct counting after Trypan blue staining. siCON: non-targeting control siRNA. (b) Colon cancer cells were treated with either recombinant IL-32γ alone or with its antibody and cultured for 72 h. (c) Colon cancer cells were inoculated into 24-well plates (5 × 104 cells per well) and transfected with the IL-32γ plasmid (0.4 μg per well) with/without anti-IL-32 (5 or 10 μg/ml) for up to 72 h. All results are expressed as mean±s.d. of three experiments with triplicate tests in each experiment. #P<0.05 compared with the colon cancer cells transfected with vector alone (a, c) or untreated control (b). *P<0.05 compared with the colon cancer cells transfected with IL-32γ alone (a, c) or with recombination IL-32γ protein (b). A full colour version of this figure is available at the Oncogene journal online.
Figure 6
Figure 6
Effect of IL-32γ on NF-κB activation in tumor tissues and colon cancer cells. (a) The DNA-binding activity of NF-κB was determined by electromobility shift assay in the nuclear extracts of the xenograft mouse or IL-32γ transgenic mice tumor samples. (b, c) Expression of p50 and p65 in nuclear extracts and IκB phosphorylation in the cytosol of murine tumors, as determined by western blotting (b) and immunohistochemistry (c). Each image and band is representative of three independent experiments. (d) The DNA-binding activity of NF-κB was investigated using electromobility shift assay in nuclear extracts of colon cancer cells that were transfected by IL-32γ. (e, f) Expression of p50 and p65 in nuclear extracts and IκB phosphorylation in the cytosol, as determined by western blotting (e), and fluorescence microscopy (f). (g) Colon cancer cells were co-transfected with the IL-32γ and p50/p65 siRNA for up to 72 h. Cell growth was measured by direct counting of cells stained with Trypan blue. The results are expressed as mean±s.d. of three experiments with triplicate tests in each experiment. #P<0.05 compared with the colon cancer cells transfected with vector. *P<0.05 compared with the colon cancer cells transfected with IL-32γ alone. The values on the left of panels b and e are average percentages of vector control over three independent experiments.
Figure 7
Figure 7
Effect of IL-32γ on STAT3 activation in tumor tissues and colon cancer cells. (a, b) Phosphorylation of STAT3 in whole extracts of murine tumors, as determined by western blotting (a) and immunohistochemistry (b). (c, d) Cellular localization p-STAT3 (green) and p65 (red) in tumor tissues of xenograft mice (c) and IL-32γ transgenic mice (d). Each image and band is representative of three independent experiments. (e) Colon cancer cells were transfected with the vector or the IL-32γ for 24 h. Whole-cell extracts were prepared and analyzed for phosphorylated STAT3 by western blotting. Each band is representative of three independent experiments. (f) Cellular localization of p-STAT3 (green) and p65 (red) was observed by confocal microscopy after immunofluorescence staining of colon cancer cells transfected with IL-32γ. (g) Colon cancer cells were co-transfected with the IL-32γ and STAT3 siRNA for up to 72 h. Cell growth was measured by direct counting of cells stained with Trypan blue. The results are expressed as mean±s.d. of three experiments with triplicate tests in each experiment. #P<0.05 compared with the colon cancer cells transfected with vector. *P<0.05 compared with the colon cancer cells transfected with IL-32γ alone.
Figure 8
Figure 8
Effect of IL-32γ on the infiltration of CD8+ T cells and NK cells into tumor tissues and immune organ tissues and on cytokine levels in tumor tissues. (ad) Immunohistochemistry was used to determine the level of CD8+- and CD57-reactive cell number in tumor sections and immunity-related organs, as described in Materials and methods. Shown are the expression patterns of CD8+ and CD57 in the tumor sections of nude mouse xenografts (a) and IL-32γ transgenic mice (b), as well as in the spleens (c) and thymuses (d) of IL-32γ transgenic mice. The images shown are representative of three sections from each mouse (n=3). Each image and band is representative of three independent experiments. (e) Subpopulation of immune cells determined after FACS analysis, as described in detail in Materials and methods. The values in each area are the average subpopulation of immune cells (NK cells and CD8+ cells). Each image is representative of three independent experiments. (f) The levels of IL-10, TNF-α, IL-1β and IL-6 in the tumor tissues of transgenic mice (n=3) were measured using quantitative real-time PCR, as described in Materials and methods. *P<0.05 compared with non-transgenic mice tumor tissues.

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