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, 9 (3), 306

IL-32 Gamma Reduces Lung Tumor Development Through Upregulation of TIMP-3 Overexpression and Hypomethylation

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IL-32 Gamma Reduces Lung Tumor Development Through Upregulation of TIMP-3 Overexpression and Hypomethylation

Jaesuk Yun et al. Cell Death Dis.

Abstract

The low expression of tissue inhibitor of metalloproteinase 3 (TIMP-3) is important in inflammatory responses. Therefore, inhibition of TIMP-3 may promote tumor development. Our study showed that expression of TIMP-3 was elevated in lL-32γ mice lung tissues. In this study, we investigated whether IL-32γ mice inhibited lung tumor development through overexpression of TIMP-3 and its methylation. To explore the possible underlying mechanism, lung cancer cells were transfected with IL-32γ cDNA plasmid. A marked increase in TIMP-3 expression was caused by promoter methylation. Mechanistic studies indicated that TIMP-3 overexpression reduced NF-κB activity, which led to cell growth inhibition in IL-32γ transfected lung cancer cells. We also showed that IL-32γ inhibits expression of DNA (cytosine-5-)-methyltransferase 1 (DNMT1). Moreover, IL-32γ inhibits the binding of DNMT1 to TIMP-3 promoter, but this effect was reversed by the treatment of DNA methyltransferase inhibitor (5-Aza-CdR) and NF-κB inhibitor (PS1145), suggesting that a marked increase in TIMP-3 expression was caused by inhibition of promoter hypermethylation via decreased DNMT1 expression through the NF-κB pathway. In an in vivo carcinogen induced lung tumor model, tumor growth was inhibited in IL-32γ overexpressed mice with elevated TIMP-3 expression and hypomethylation accompanied with reduced NF-κB activity. Moreover, in the lung cancer patient tissue, the expression of IL-32 and TIMP-3 was dramatically decreased at a grade-dependent manner compared to normal lung tissue. In summary, IL-32γ may increase TIMP-3 expression via hypomethylation through inactivation of NF-κB activity, and thereby reduce lung tumor growth.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. Effect of IL-32γ on lung tumor development.
a Effect of IL-32γ on lung tumorigenesis in transgenic mice. The results are expressed as mean ± SD. *P < 0.05 compared with non-transgenic mice (n = 10). b Effect of IL-32γ on the expression of proliferation marker proteins and IL-32 and PCNA in tumor tissues determined by immunohistochemistry. c Effect of IL-32γ on the expression of IL-32 and PCNA in tumor tissues determined by Western blotting. d Effect of IL-32γ on apoptosis was determined by DAPI/TUNEL assay. Total number of cells in a given area was determined using a DAPI nuclear staining (fluorescent microscope). The green color in the fixed cells marks TUNEL-labeled cells. e Effect of IL-32γ on the expression of apoptotic proteins in tumor tissues determined by Western blotting with specific antibodies. β-actin protein was used as an internal control. Each band is representative of three independent experiments
Fig. 2
Fig. 2. Effect of IL-32γ on cell viability and apoptosis in lung cancer cell lines.
a Effect of IL-32γ on growth rates and apoptotic cell death in IL-32γ-transfected lung cancer cells. Lung cancer cells (1 × 104) were transfected with pcDNA or IL-32γ plasmid for 24, 48 or 72 h and cell proliferation was determined by MTT assay. 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 lung cancer cells. b Effects of IL-32γ on apoptotic cell death. Lung cancer cell lines were transfected with IL-32γ and then labeled with DAPI and TUNEL solutions. Total number of cells in a given area was determined using a DAPI nuclear staining (fluorescent microscope). The green color in the fixed cells marks TUNEL-labeled cells. c Lung cancer 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
Fig. 3
Fig. 3. Effect of IL-32γ on the expression and promoter methylation of TIMP-3 in lung tumors and lung cancer cell lines
a Expression of TIMP-3 and IL-32 is lower in human lung cancer tissues (n = 4) compared with normal lung tissues. Immunohistochemical staining was performed on a lung cancer tissue array using a TIMP-3 or IL-32 antibodies. The figure representative of 4 tissue samples. b Effect of IL-32γ on the expression of TIMP-3 in tumor tissues determined by immunohistochemistry. c Effect of IL-32γ on the expression of TIMP-3 in tumor tissues determined by Western blotting. d Effect of IL-32γ on the methylation of TIMP-3 in tumor tissues determined by qPCR. Scale bar: 50 μm or 200 μm. Each band is representative of three independent experiments. e Lung cancer cells were transfected with pcDNA or IL-32γ for 24 h. Cell extracts were examined for expression of IL-32 and TIMP-3 by Western blotting. f, g TIMP-3 promoter methylation levels were determined by methylation-specific PCR (f) or quantitative methylation-specific PCR by qPCR (g) in cell extracts from pcDNA or IL-32γ transfected lung cancer cells
Fig. 4
Fig. 4. Effect of IL-32γ on NF-κB signaling and on the binding of DNMT1 in TIMP-3 promoter.
Effect of IL-32γ on NF-κB activation in tumor tissues and lung cancer cells. a DNA-binding activity of NF-κB was determined by electromobility shift assay in the nuclear extracts of non-tg or IL-32γ-tg mice tumor samples. b, c Nuclear translocation of p50 and p65 of murine tumors was determined by immunohistochemistry (b) and Western blotting (c). Each image and band is representative of three independent experiments. d DNA-binding activity of NF-κB was investigated using electromobility shift assay in nuclear extracts of lung cancer cells that were transfected by IL-32γ. e Nuclear translocation of p50 and p65 was determined by Western blotting
Fig. 5
Fig. 5. Effect of IL-32γ on the binding of DNMT1 in TIMP-3 promoter.
a Expression of DNMT1 of murine tumors was determined by Western blotting. b Expression of DNMT1 in nuclear extracts of lung cancer cells transfected by IL-32γ was determined by Western blotting. c A549 lung cancer cells were transfected with pcDNA or IL-32γ plasmid for 24 h. Reduced binding of DNMT1 by IL-32γ in TIMP-3 promoter was determined by ChiP assay. d Effect of DNA methyltransferase inhibitor (5-Aza-CdR; 5 μM) on reduced binding of DNMT1 by IL-32γ in TIMP-3 promoter was determined by ChiP assay. Effect of NF-κB inhibitor (PS1145; 10 μM) on reduced binding of DNMT1 by IL-32γ in TIMP-3 promoter was determined by chip assay. TIMP-3 promoter was determined by ChiP assay. Each band is representative of three independent experiments. e, f Effect of DNA methyltransferase inhibitor (5-Aza-CdR; 5 μM) (e) or NF-κB inhibitor (PS1145; 10 μM) (f) on cell viability was determined by MTT assay. g Effect of NF-κB inhibitor (PS1145; 10 μM) on TIMP-3 methylation and Expression of DNMT1. h Effect of recombinant protein (rP) of p50 (50 ng/ml) on TIMP-3 methylation and Expression of DNMT1 in A549 cells. Allthe experiment were performed three times with duplicates. *P < 0.05 compared with the lung cancer cells transfected with vector. #P < 0.05 compared with the lung cancer cells transfected with IL-32γ alone
Fig. 6
Fig. 6. Effect of IL-32γ on the expression of metastasis related proteins.
a Lung cancer cells were transfected with pcDNA or IL-32γ plasmid for 48 h. Cell lysates were analyzed by Western blotting for detection of MMP2, MMP3, MMP9 and MMP13 expression. β-actin protein was used as an internal control. Each band is representative for three experiments. b-d Lung cancer cells were co-transfected with the IL-32γ and TIMP-3 siRNA for up to 48 h. Effect of siTIMP-3 on the expression of metastatic proteins (b) and apoptotic protein (c) and was determined by Western blotting. Cell growth was measured by MTT assay (d). The results are expressed as mean ± s.d. of three experiments with triplicate tests in each experiment. *P < 0.05 compared with the lung cancer cells transfected with vector. #P < 0.05 compared with the lung cancer cells transfected with IL-32γ alone. The values are average percentages of vector control over three independent experiments

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