Nonsteroidal anti-inflammatory drug-activated gene-1 expression inhibits urethane-induced pulmonary tumorigenesis in transgenic mice

Abstract

The expression of nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1) inhibits gastrointestinal tumorigenesis in NAG-1 transgenic mice (C57/BL6 background). In the present study, we investigated whether the NAG-1 protein would alter urethane-induced pulmonary lesions in NAG-1 transgenic mice on an FVB background (NAG-1(Tg+/FVB)). NAG-1(Tg+/FVB) mice had both decreased number and size of urethane-induced tumors, compared with control littermates (NAG-1(Tg+/FVB) = 16 +/- 4 per mouse versus control = 20 +/- 7 per mouse, P < 0.05). Urethane-induced pulmonary adenomas and adenocarcinomas were observed in control mice; however, only pulmonary adenomas were observed in NAG-1(Tg+/FVB) mice. Urethane-induced tumors from control littermates and NAG-1(Tg+/FVB) mice highly expressed proteins in the arachidonic acid pathway (cyclooxygenases 1/2, prostaglandin E synthase, and prostaglandin E(2) receptor) and highly activated several kinases (phospho-Raf-1 and phosphorylated extracellular signal-regulated kinase 1/2). However, only urethane-induced p38 mitogen-activated protein kinase (MAPK) phosphorylation was decreased in NAG-1(Tg+/FVB) mice. Furthermore, significantly increased apoptosis in tumors of NAG-1(Tg+/FVB) mice compared with control mice was observed as assessed by caspase-3/7 activity. In addition, fewer inflammatory cells were observed in the lung tissue isolated from urethane-treated NAG-1(Tg+/FVB) mice compared with control mice. These results paralleled in vitro assays using human A549 pulmonary carcinoma cells. Less phosphorylated p38 MAPK was observed in cells overexpressing NAG-1 compared with control cells. Overall, our study revealed for the first time that the NAG-1 protein inhibits urethane-induced tumor formation, probably mediated by the p38 MAPK pathway, and is a possible new target for lung cancer chemoprevention.

Figure 1. Characterization of NAG-1^Tg+/FVB mice

(A) Schematic diagram of experimental design. (B) Body weight for control and NAG-1^Tg+/FVB mice (**, p<0.01; ***, p<0.001). (C) Human NAG-1 expression in lung tissue of NAG-1^Tg+/FVB mice was confirmed by RT-PCR and WB. Human HCT-116 colorectal cancer cell lysate was used as the standard (STD) for human NAG-1 expression.

Figure 2. Suppression of urethane-induced pulmonary neoplasia in NAG-1^Tg+/FVB mice

(A) Top, images of dissected lungs from control (left) and NAG-1^Tg+/FVB (right) mice. The bottom graph shows that NAG-1^Tg+/FVB mice had a significantly lower gross incidence of urethane-induced pulmonary neoplasia compared to control littermates. The graph represents mean ± S.D. of surface tumors from each treatment group, and the student t test was used to analyze the data (*p<0.05). (B) Top, a representative lung section stained by H&E with pulmonary tumors from control (left) and NAG-1^Tg+/FVB (right) mice. Magnification 20×, scale bar 1.0 mm. The bottom graph shows tumor numbers per lung as mean ± S.D., and the student t test was used to analyze the data (*p<0.05). (C) Representative images of foci of hyperplasia (HP, asterisk, left top panel), atypical adenomatous hyperplasia (AAH, asterisk, right top panel), multiple adenomas (AD, asterisk, left bottom panel) characterized as well-differentiated and compressing the surrounding tissues (arrows in AD), and adenocarcinomas (AC, asterisk, right bottom panel) with localized fingering invasion into surrounding parenchyma or airways (arrows in AC). Magnification 400× and 200×, scale bars 50 and 100 µm, respectively. Graph at right shows evaluation of each type of lesion per lung section of each treatment group as mean ± S.D., and the student t test was used to analyze the data. (D) The distribution of tumor numbers as percentage of total numbers n=33 of control and n=29 of NAG-1^Tg+/FVB mice as related to size (diameter, mm). The tumor diameters were divided into four groups according to size (<0.5mm, 0.5–1.0mm, 1.1–1.5mm, >1.5mm).

Figure 3. p38 MAPK signaling pathway is affected by NAG-1 in urethane-treated lung tissue

(A) Lung tumors (T) and adjacent normal tissues (N) were isolated from three representative control and NAG-1^Tg+/FVB mice and were subjected to WB analysis using the antibodies for COX-1, COX-2, PGES, and EP2 expression. (B) Expression of phospho-Raf-1, phospho-ERK1/2 and phospho-p38 MAPK in lung tissues. Actin and total p38 MAPK antibodies were used as loading controls. (C) The effect of NAG-1 expression on the cell cycle proteins and tumor suppressor protein p53.

Figure 4. NAG-1 protein activates apoptosis in urethane-induced pulmonary tumors

(A) Normal (N) and tumor (T) tissue from control and NAG-1^Tg+/FVB mice were subjected to caspase 3/7 activity assay. RLU, relative luciferase units. (mean ± S.D. from three different mice, **, p<0.01). (B) TUNEL assay of control (left panel) and NAG-1^Tg+/FVB (right panel) mice. Positive control is shown as an insert in left panel. Asterisks indicate pulmonary adenomas; arrows indicate positively brown stained cells. Magnification 400×, scale bar 50 µm.

Figure 5. NAG-1 inhibits urethane-induced inflammation

(A) Immunohistochemical analysis of lysozyme marker in lung tissues from control and NAG-1^Tg+/FVB mice. Hematoxylin was used as counterstain. Magnification 400×. (B) The graph represents the semi-quantization of scored intensity. Statistical analyses were perform using Student t test, *p<0.05.

Figure 6. NAG-1 inhibits phosphorylation of p38 MAPK in A549 cells

A549 cells were grown and transiently transfected with empty or pcDNA3.1/NAG-1 expression vector. After transfection, A549 cells were treated with 20 nM TPA and 40 µg/ml CSC (A), 100 ng/ml EGF and 100 ng/ml IGF-1 (B), or 50 nM PGE₂ (C) for 30 and 120 min. The phosphorylation of p38 MAPK, NAG-1 and actin expression was evaluated by WB analysis. The numbers below represent densitometry analysis of phospho-p38 normalized to actin, and then compared to the controls (0 min set as 1.0 in each transfection). (D) Schematic diagram of NAG-1’s role in lung tumorigenesis.