Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 4 (5), e640

Antitumor Activity of IL-32β Through the Activation of Lymphocytes, and the Inactivation of NF-κB and STAT3 Signals

Affiliations

Antitumor Activity of IL-32β Through the Activation of Lymphocytes, and the Inactivation of NF-κB and STAT3 Signals

H-M Yun et al. Cell Death Dis.

Abstract

Cytokine and activation of lymphocytes are critical for tumor growth. We investigated whether interleukin (IL)-32β overexpression changes other cytokine levels and activates cytotoxic lymphocyte, and thus modify tumor growth. Herein, IL-32β inhibited B16 melanoma growth in IL-32β-overexpressing transgenic mice (IL-32β mice), and downregulated the expressions of anti-apoptotic proteins (bcl-2, IAP, and XIAP) and cell growth regulatory proteins (Ki-67 antigen (Ki-67) and proliferating cell nuclear antigen (PCNA)), but upregulated the expressions of pro-apoptotic proteins (bax, cleaved caspase-3, and cleaved caspase-9). IL-32β also inhibited colon and prostate tumor growth in athymic nude mice inoculated with IL-32β-transfected SW620 colon or PC3 prostate cancer cells. The forced expression of IL-32β also inhibited cell growth in cultured colon and prostate cancer cells, and these inhibitory effects were abolished by IL-32 small interfering RNA (siRNA). IL-10 levels were elevated, but IL-1β, IL-6, and tumor necrosis factor-alpha (TNF-α) levels were reduced in the tumor tissues and spleens of IL-32β mice, and athymic nude mice. The number of cytotoxic T (CD8(+)) and natural killer (NK) cells in tumor tissues, spleen, and blood was significantly elevated in IL-32β mice and athymic nude mice inoculated with IL-32β-transfected cancer cells. Constituted activated NF-κB and STAT3 levels were reduced in the tumor tissues of IL-32β mice and athymic nude mice, as well as in IL-32β-transfected cultured cancer cells. These findings suggest that IL-32β inhibits tumor growth by increasing cytotoxic lymphocyte numbers, and by inactivating the NF-κB and STAT3 pathways through changing of cytokine levels in tumor tissues.

Figures

Figure 1
Figure 1
Generation of IL-32β transgenic mice. (a) Scheme for IL-32β transgenic generation. (b) PCR analysis (genotyping) was performed to analyze the existence of IL-32β gene in transgenic mice, as described in Materials and Methods. (c) RT-PCR and western blott analyses for IL-32β in the tissues of IL-32β transgenic and nontransgenic mice. (d) Detection of IL-32 in the sera of transgenic or nontransgenic mice. The results are expressed as mean±SD of three mice. * Significant difference from nontransgenic mice (*P<0.05)
Figure 2
Figure 2
Effect of IL-32β on tumor growth in IL-32β transgenic mice. (a) Tumor images (upper panel), volumes (middle panel), and weights (lower panel) were measured at study termination (on day 24 in transgenic mice (a) as described in Materials and Methods section. The results are expressed as mean±SD from 10 mice; *P<0.05 compared with the nontransgenic mice. (b) tumor sections were analyzed by immunohistochemistry. (c) Tumor extracts were analyzed by western blotting as described in Materials and Methods section. Each images and band are representative of three independent mice. (d) Apoptotic cells were examined by TUNEL staining. (e) Tumor sections were analyzed by immunohistochemistry for detection of IL-32β expression in tumor tissues. Each image is representative of three independent mice. The values on the right or bottom of panels are average fold difference from three independent nontransgenic mice *Significant difference from nontransgenic mice (*P < 0.05)
Figure 3
Figure 3
Effect of IL-32β on cytokine levels in tumor and spleen tissues. The level of IL-6, IL-1β, TNF-α, and IL-10 was determined by qRT-PCR in the tumor (a) and spleen (b) of tumor-bearing IL-32β-transgenic mice. The cytokine levels of tumor (c and e) or spleen (d and f) of athymic nude mice inoculated with IL-32β-transfected colon cancer cells (c and d) or prostate cancer cells (e and f). The results are expressed as mean±SD of five mice. *Significant difference from nontransgenic mice or athymic nude mice inoculated with vector-transfected cancer cells (*P<0.05)
Figure 4
Figure 4
Effect of IL-32β on CD8+ cytotoxic T cell and CD57+ NK cell number in tumor and spleen tissues, and blood of athymic nude mice inoculated with IL-32β-trasfected colon and prostate cancer cells. (a) Effect of IL-32β on the number of CD8+ cytotoxic T cells and CD57+ NK cells in tumor and spleen tissue section of IL-32β-transgenic mice, as determined in immunohistochemistry. (b) Effect of IL-32β on the number of CD8+ cytotoxic T cells and CD57+ NK cells in tumor and spleen tissue sections of athymic nude mice inoculated with IL-32β-trasfected colon and prostate cancer cells, as determined in immunohistochemistry. The images shown in (a) and (b) are representative of three sections from each mouse (n=3). (c and d) Subpopulation of immune cells determined after FACS analysis, as described in Materials and Methods section. The values in each area are the average subpopulation of immune cells (NK and CD8+ cells) *Significant difference from nontransgenic mice (*P < 0.05).
Figure 5
Figure 5
Effect of IL-32β on the activation of NF-κB and STAT3 in tumor tissues, and cultured colon and prostate cancer cells. (a) The DNA-binding activity of NF-κB was determined by EMSA in the nuclear extracts of tumor tissues of IL-32β-overexpressed transgenic mice or the athymic nude mice (a, upper panel). Expression of p50 and p65 in nuclear extracts (NE, middle panel of a), IκB phosphorylation in the cytosol extracts (CE, lower panel of a), and DNA-binding activity and STAT3 phosphorylation (c) in total lysates of tumors were determined by EMSA (upper panel) or by western blotting (lower panel). (b and d) Expression of p65 (b), and phosphorylated STAT3 (d) in murine tumors were determined by immunohistochemistry. (e) Cellular localization p-STAT3 (green) and p65 (red) in tumor tissues. IL-32β transgenic and athymic nude mice were observed by fluorescence microscopy after Immunofluorescence staining, as described in Materials and Methods section. Each image and band are representative of three independent mice. (f) Colon and prostate cancer cells were transfected with the vector or the IL-32β and cultured for 24 h, and then NF-κB was determined by EMSA (f, upper panel). Expression of p50 and p65 in nuclear extracts (NE, middle panel) and IκB phosphorylation in the cytosol extracts (CE, lower panel) were determined by western blotting. (g) Phosphorylation of STAT3 in total cell extracts was analyzed by western blotting (g, left panel). Cellular localization of p-STAT3 (green) and p65 (red) in IL-32β-transfected colon and prostate cancer cells was determined with confocal microscopy, as described in Materials and Methods (f, right panel). Each band is representative of three independent experiments

Similar articles

See all similar articles

Cited by 21 PubMed Central articles

See all "Cited by" articles

References

    1. Danese S, Mantovani A. Inflammatory bowel disease and intestinal cancer: a paradigm of the Yin-Yang interplay between inflammation and cancer. Oncogene. 2010;29:3313–3323. - PubMed
    1. Lewis CE, Pollard JW. Distinct role of macrophages in different tumor microenvironments. Cancer Res. 2006;66:605–612. - PubMed
    1. Lin WW, Karin M. A cytokine-mediated link between innate immunity, inflammation, and cancer. J Clin Invest. 2007;117:1175–1183. - PMC - PubMed
    1. Jee SH, Shen SC, Chiu HC, Tsai WL, Kuo ML. Overexpression of interleukin-6 in human basal cell carcinoma cell lines increases anti-apoptotic activity and tumorigenic potency. Oncogene. 2001;20:198–208. - PubMed
    1. Ishiguro H, Akimoto K, Nagashima Y, Kojima Y, Sasaki T, Ishiguro-Imagawa Y, et al. aPKClambda/iota promotes growth of prostate cancer cells in an autocrine manner through transcriptional activation of interleukin-6. Proc Natl Acad Sci USA. 2009;106:16369–16374. - PMC - PubMed

Publication types

MeSH terms

Feedback