NCS 613, a Potent PDE4 Inhibitor, Displays Anti-Inflammatory and Anti-Proliferative Properties on A549 Lung Epithelial Cells and Human Lung Adenocarcinoma Explants

Abstract

Chronic inflammation is a deleterious process occurring in several pulmonary diseases; it is a driving force promoting tumorigenesis. By regulating local cyclic nucleotide concentration, cyclic nucleotide phosphodiesterases (PDE) govern important biological processes, including inflammation and proliferation. The aim of this study was to investigate the anti-inflammatory and anti-proliferative effects of NCS 613, a specific PDE4 inhibitor, on TNFα-treated human lung adenocarcinoma cell line (A549) and on human lung adenocarcinoma explants. PDE4 isoforms and inflammatory pathways mediated by p38 MAPK, ERK1/2, and IκBα were analyzed by Western blot and immunostainings. Proliferation were performed using [³H]-thymidine incorporation under different experimental conditions. TNFα-stimulation increased p38 MAPK phosphorylation and NF-κB translocation into the nucleus, which was abolished by NCS 613 treatment. Concomitantly, NCS 613 restores IκBα detection level in human adenocarcinoma. An IC₅₀ value of 8.5 μM was determined for NCS 613 on anti-proliferative properties while ERK1/2 signaling was down-regulated in A549 cells and lung adenocarcinoma explants. These findings shed light on PDE4 signaling as a key regulator of chronic inflammation and cancer epithelial cell proliferation. It suggests that PDE4 inhibition by NCS 613 represent potential and interesting strategy for therapeutic intervention in tackling chronic inflammation and cell proliferation.

Keywords: PDE4 inhibitor; cAMP signaling; human lung adenocarcinoma; inflammation; proliferation.

Figure 1

Detection of PDE4A, PDE4B, and PDE4C expressions in control and TNFα-stimulated A549 cells following NCS 613 treatment. A549 cells were stimulated with TNFα to induce inflammation and treated with NCS 613 for 48 h under the following experimental conditions: control, +10 μM NCS 613, + 10 ng/ml TNFα, and +TNFα + NCS 613. Relative protein expression levels were calculated following digitalization and image analysis from 3 independent experiments. (A) NCS 613 prevents and TNFα increases PDE4A 70 kDa overexpression; (B) NCS 613 induces PDE4B 96 kDa expression in A549 cell line; (C) TNFα stimulation induces PDE4B 66 kDa upregulation which is decreased with NCS 613 treatment; (D) PDE4C 66 kDa increases upon NCS 613 treatment; (E) representative immuno-reactive bands revealed by Western blot analysis (n=3). Unpaired Student’s t-test. Mean ± SEM. **p < 0.01 and ***p < 0.001.

Figure 2

PDE4B and PDE4C immuno-cyto-staining. (A–E) Detection of PDE4B isoforms in A549 cells under control and TNFα treated conditions, in the absence or presence of NCS 613. (A) Negative control showing anti-PDE4B specificity; (B) PDE4B basal level in A549 cells; (C) NCS 613 increases PDE4B expression; (D) TNFα stimulation greatly increases PDE4B expression which is reduce by NCS 613 (E). (F–I) Detection of PDE4C in A549 cells. (F) Negative control and (G) Basal PDE4C location; (H) and (I) NCS 613 treatments increase nuclear PDE4C in A549 cells. Data are representative of 3 independent experiments. Shown are merged images [x60 (A–E) and x40 (F–I) magnification] of DAPI staining (red) and anti-rabbit-Alexa Fluor 488 (green) recognizing primary anti-PDE4B or anti-PDE4C antibody (green).

Figure 3

Effect of NCS 613 on p38 MAPK phosphorylation and NF-κB nuclear translocation upon TNFα stimulation. (A) TNFα increases and NCS 613 reduces p38 MAPK phosphorylation in A549 cells; (B) NCS 613 prevents NF-κB translocation into nucleus while TNFα stimulation greatly increases NF-κB p65-subunit translocation into nucleus. Shown are merged images (60x magnification) of DAPI staining and anti-mouse Alexa 654 recognizing primary p65-NF-κB antibody (n=3). Unpaired Student’s t-test. Mean ± SEM. **p < 0.01 and ***p < 0.001.

Figure 4

Concentration dependent effect of NCS 613 treatment on IκBα detection in adenocarcinoma explants. IκBα was detected in fresh human lung parenchyma. After a 72 h culture period, IκBα was undetectable in control adenocarcinoma explants suggesting a high inflammation level. Three and 10 µM NCS 613 treatment reestablished IκBα detection level (n= 4). Unpaired Student’s t-test. Mean ± SEM. **p < 0.01 and ***p < 0.001.

Figure 5

Anti-proliferative effect of NCS 613 on A549 cells. (A) Control: A549 cells display rapid proliferation rate toward confluency. (B, C) A549 cells treated with either 5 µM or 10 µM NCS 613 for 48 h results in lower density and higher apoptosis. Plates are representative of three independent experiments; (D) Bar histogram showing [³H]-thymidine incorporation into DNA. Gradual (2.5–30 µM) NCS 613 treatment significantly decreases A549 cells proliferation; (E) Concentration dependent inhibition curve displaying the antiproliferative effects of NCS 613 from which an IC₅₀ value of 8.5 µM was calculated. Unpaired Student’s t-test. Mean ± SEM. ***p < 0.001.

Figure 6

Effect of NCS 613 treatment on ERK1/2 phosphorylation. (A, B) Concentration dependent effect of NCS 613 on the phosphorylation level of ERK1/2 as a function of experimental conditions (n=3); (C, D) Concentration dependent effect of NCS 613 on phospho-ERK1/2 detection in cultured lung adenocarcinoma explants (n=4). Unpaired Student’s t-test. Mean ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001.