Human Lung Epithelial Cell Line Research Articles

P3.02c-004 SBI0206965, a Novel Inhibitor of Ulk1, Suppresses Non-Small Cell Lung Cancer Cell Growth via Modulating Both Autophagy and Apoptosis Pathways: Topic: Targeted Therapy

Autophagy is a catabolic process that regulates the lysosomal turnover of damaged proteins and organelles in order to maintain cellular homeostasis. Dysregulation of autophagy is often observed in lung cancer. Kinase inhibitors have proved successful in the clinic. The fact that uncoordinated (Unc) 51-like kinase (Ulk1) is the only conserved serine/threonine kinase in the autophagy cascade makes it a very attractive target for therapeutic development. Up-regulation of Ulk1 has been shown to promote cell survival of several cancer cell lines. Moreover, overexpression of Ulk1 has been shown to be negatively correlated with the prognosis of several types of human cancer. However, the role of Ulk1 in NSCLC is largely unknown. We evaluated Ulk1 expression levels in human normal lung epithelial cell line BEAS-2B and five NSCLC cell lines, A549, H460, H1299, H292 and HCC827. We analyzed the correlation between Ulk1 expression levels and the prognosis of NSCLC patients. We evaluated the effect of SBI0206965 alone or in combination with cisplatin on cell proliferation, apoptosis and autophagy in two human NSCLC cell lines, A549 and H460. Cell proliferation of untreated or drug-treated cells was measured by CCK8 assay. Percentage of apoptotic cells was measured using PE-conjugated Annexin V with a flow cytometer. Autophagy was determined by conversion of LC3I to LC3II and p62 degradation using Western blot. Ulk1 is overexpressed in NSCLC cell lines and negatively correlated with the prognosis of NSCLC patients. The inhibition of Ulk1 by a selective inhibitor, SBI0206965, blocks the proliferation of NSCLC cells and induces apoptosis. SBI0206965 treatment augments the efficacy of cisplatin to kill NSCLC cells by inhibiting cisplatin induced cell protective autophagy. SBI0206965 can also destabilize Bcl2/Bclxl to promote cisplatin induced apoptosis in NSCLC cell lines. Our results suggest that the inhibition of Ulk1 may be a promising strategy for the treatment of NSCLC. SBI0206965 may be a promising agent for the treatment of NSCLC by modulating autophagy and apoptosis pathways. Furthermore, the combination of SBI0206965 with classical chemotherapy agents represents a promising therapeutic strategy that warrants further clinical evaluation in NSCLC.

Characteristics, sources, and cytotoxicity of atmospheric polycyclic aromatic hydrocarbons in urban roadside areas of Hangzhou, China

ABSTRACTThe primary objective of this study is to understand the profiles, sources and cytotoxic effects of atmospheric polycyclic aromatic hydrocarbons (PAHs), which are closely related to urban air contamination and public health, in urban roadside environments. On-road sampling campaigns were conducted from 2014 to 2015 at three urban road sites in Hangzhou, China. Sixteen gaseous and particulate matter (PM) 2.5-bound PAHs were identified and quantified using gas chromatography-mass spectrometry (GC-MS). The total PAH concentrations at the three sites ranged from 750 to1142 ng/m3 and 1050 to 1483 ng/m3 in summer and winter, respectively. Low molecular weight PAHs were the most abundant compounds (77–86%) and primarily existed in gas phase. The concentrations and phase distributions of high molecular weight PAHs were varied at three sites due to the differences in traffic volume, vehicle composition, engine loading, and nearby artificial activity. Diagnostic ratios of the principal mass (m/z,178, 202, 228 and 276) parent PAHs were statistically described to determine the PAH sources to urban roadsides; principal component analysis (PCA) was applied to apportion the sources. The results indicated that high- and low-temperature fuel processes, as well as residential and industrial emissions, were major contributors to roadside PAHs. The cytotoxic potential of the roadside PAHs was evaluated using a human epithelial lung cell line (A549). Cell viability was measured after a direct exposure to PAH extract. The results reflected the profiles of roadside PAHs at the three sites. The cytotoxicity of reference PAHs was evaluated to provide further insights into the cytotoxic potential of PAHs. We found that low molecular weight PAHs, which are less cytotoxic compounds, synergistically promoted the lethal effect of cytotoxic compounds, posing a potential threat to public health.

Release of copper-amended particles from micronized copper-pressure-treated wood during mechanical abrasion.

BackgroundWe investigated the particles released due to abrasion of wood surfaces pressure-treated with micronized copper azole (MCA) wood preservative and we gathered preliminary data on its in vitro cytotoxicity for lung cells. The data were compared with particles released after abrasion of untreated, water (0% MCA)-pressure-treated, chromated copper (CC)-pressure-treated wood, and varnished wood. Size, morphology, and composition of the released particles were analyzed.ResultsOur results indicate that the abrasion of MCA-pressure-treated wood does not cause an additional release of nanoparticles from the unreacted copper (Cu) carbonate nanoparticles from of the MCA formulation. However, a small amount of released Cu was detected in the nanosized fraction of wood dust, which could penetrate the deep lungs. The acute cytotoxicity studies were performed on a human lung epithelial cell line and human macrophages derived from a monocytic cell line. These cell types are likely to encounter the released wood particles after inhalation.ConclusionsOur findings indicate that under the experimental conditions chosen, MCA does not pose a specific additional nano-risk, i.e. there is no additional release of nanoparticles and no specific nano-toxicity for lung epithelial cells and macrophages.Electronic supplementary materialThe online version of this article (doi:10.1186/s12951-016-0232-7) contains supplementary material, which is available to authorized users.

Procyanidin A2 Modulates IL-4-Induced CCL26 Production in Human Alveolar Epithelial Cells.

Allergic asthma is an inflammatory lung disease that is partly sustained by the chemokine eotaxin-3 (CCL26), which extends eosinophil migration into tissues long after allergen exposure. Modulation of CCL26 could represent a means to mitigate airway inflammation. Here we evaluated procyanidin A2 as a means of modulating CCL26 production and investigated interactions with the known inflammation modulator, Interferon γ (IFNγ). We used the human lung epithelial cell line A549 and optimized the conditions for inducing CCL26. Cells were exposed to a range of procyanidin A2 or IFNγ concentrations for varied lengths of time prior to an inflammatory insult of interleukin-4 (IL-4) for 24 h. An enzyme-linked immunosorbent assay was used to measure CCL26 production. Exposing cells to 5 μM procyanidin A2 (prior to IL-4) reduced CCL26 production by 35% compared with control. Greatest inhibition by procyanidin A2 was seen with a 2 h exposure prior to IL-4, whereas IFNγ inhibition was greatest at 24 h. Concomitant incubation of procyanidin A2 and IFNγ did not extend the inhibitory efficacy of procyanidin A2. These data provide evidence that procyanidin A2 can modulate IL-4-induced CCL26 production by A549 lung epithelial cells and that it does so in a manner that is different from IFNγ.

Wild-Type U2AF1 Antagonizes the Splicing Program Characteristic of U2AF1-Mutant Tumors and Is Required for Cell Survival.

We have asked how the common S34F mutation in the splicing factor U2AF1 regulates alternative splicing in lung cancer, and why wild-type U2AF1 is retained in cancers with this mutation. A human lung epithelial cell line was genetically modified so that U2AF1S34F is expressed from one of the two endogenous U2AF1 loci. By altering levels of mutant or wild-type U2AF1 in this cell line and by analyzing published data on human lung adenocarcinomas, we show that S34F-associated changes in alternative splicing are proportional to the ratio of S34F:wild-type gene products and not to absolute levels of either the mutant or wild-type factor. Preferential recognition of specific 3′ splice sites in S34F-expressing cells is largely explained by differential in vitro RNA-binding affinities of mutant versus wild-type U2AF1 for those same 3′ splice sites. Finally, we show that lung adenocarcinoma cell lines bearing U2AF1 mutations do not require the mutant protein for growth in vitro or in vivo. In contrast, wild-type U2AF1 is required for survival, regardless of whether cells carry the U2AF1S34F allele. Our results provide mechanistic explanations of the magnitude of splicing changes observed in U2AF1-mutant cells and why tumors harboring U2AF1 mutations always retain an expressed copy of the wild-type allele.

GL-9 peptide regulates gene expression of CD44 cancer marker and pro-inflammatory cytokine TNF-α in human lung epithelial adenocarcinoma cell line (A549).

In this study, we examined cytotoxic effect of GL-9 peptide on A459 cell line through studying the changes in TNF-α and CD44 gene expression and ROS production. Real-time PCR analysis showed that the treated A549 cells highly over expressed TNF-α, which was associated with a significant reduction of CD44 gene expression levels (p < 0.05). ROS production rate was measured through the usage of DCFH-DA primer. Results demonstrated that GL-9 peptide could also induce cell death via ROS production. The effect of GL-9 peptide on human erythrocytes and leukocytes was analyzed. GL-9 peptide showed no significant toxic effect on human blood cells. Our results suggested that the GL-9 peptide as a potent natural agent could modulate gene expression of cancer cell markers.

Particulate emissions from the combustion of birch, beech, and spruce logs cause different cytotoxic responses in A549 cells.

According to the World Health Organization particulate emissions from the combustion of solid fuels caused more than 110,000 premature deaths worldwide in 2010. Log wood combustion is the most prevalent form of residential biomass heating in developed countries, but it is unknown how the type of wood logs used in furnaces influences the chemical composition of the particulate emissions and their toxicological potential. We burned logs of birch, beech and spruce, which are used commonly as firewood in Central and Northern Europe in a modern masonry heater, and compared them to the particulate emissions from an automated pellet boiler fired with softwood pellets. We determined the chemical composition (elements, ions, and carbonaceous compounds) of the particulate emissions with a diameter of less than 1 µm and tested their cytotoxicity, genotoxicity, inflammatory potential, and ability to induce oxidative stress in a human lung epithelial cell line. The chemical composition of the samples differed significantly, especially with regard to the carbonaceous and metal contents. Also the toxic effects in our tested endpoints varied considerably between each of the three log wood combustion samples, as well as between the log wood combustion samples and the pellet combustion sample. The difference in the toxicological potential of the samples in the various endpoints indicates the involvement of different pathways of toxicity depending on the chemical composition. All three emission samples from the log wood combustions were considerably more toxic in all endpoints than the emissions from the pellet combustion. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1487-1499, 2017.

Polycyclic aromatic hydrocarbons are associated with increased risk of chronic obstructive pulmonary disease during haze events in China

Although exposure to particulate matter with a diameter of <2.5μm (PM2.5) is associated with chronic obstructive pulmonary disease (COPD), the major components of PM2.5 in COPD pathogenesis are controversial. Here we employed the human lung epithelial cell line BEAS-2B to elucidate the association between COPD and the organic and water-soluble components of PM2.5. We found that the PM2.5 organic extract was a potential major risk factor for pulmonary epithelial barrier dysfunction through the depletion of proteins from the zonula occludens. This extract induced severe oxidative stress that increased DNA damage and the production of proinflammatory cytokines by BEAS-2B cells as well as decreased α1-antitrypsin expression, suggesting a mechanism that increases the risk of COPD. These effects were mainly mediated by polycyclic aromatic hydrocarbons (PAHs) through the aryl hydrocarbon receptor pathway. PAHs with high benzo(a)pyrene (BaP)-equivalent concentrations, but not major PAH components, have an increased risk of causing COPD, suggesting that BaP-equivalent concentrations represent a PM2.5-induced COPD risk metric, which may contribute to provide a rationale for the remediation of air pollution.

Development of novel biocompatible hybrid nanocomposites based on polyurethane-silica prepared by sol gel process

Due to high biocompatibility, polyurethane has found many applications, particularly in development of biomedical devices. A new nanocomposite based on thermoset polyurethane and silica nanoparticles was synthesized using sol-gel method. Sol-gel process was fulfilled in two acidic and basic conditions by using tetraethylorthosilicate (TEOS) and trimethoxyisocyanatesilane as precursors. The hybrid films characterized for mechanical and surface properties using tensile strength, contact angle, ATR-FTIR and scanning electron microscopy. Biocompatibility and cytotoxicity of the hybrids were assessed using standard MTT, LDH and TUNEL assays. The results revealed that incorporation of silica nanoparticles was significantly improved tensile strength and mechanical properties of the hybrids. Based on the contact angle results, silica nanoparticles increased hydrophilicity of the hybrids. Biocompatibility by using human lung epithelial cell line (MRC-5) demonstrated that the hybrids were significantly less cytotoxic compared to pristine polymer as tested by MTT and LDH assays. TUNEL assay revealed no signs of apoptosis in all tested samples. The results of this study demonstrated that incorporation of silica nanoparticles into polyurethane lead to the enhancement of biocompatibility, indicating that these hybrids could potentially be used in biomedical field in particular as a new coating for medical implants.

In vitro toxicity of cationic micelles and liposomes in cultured human hepatocyte (HepG2) and lung epithelial (A549) cell lines

The aim of this study was to compare the effects of cationic micelle and liposome drug delivery systems on liver and lung cells in a toxicological in vitro screening model, with observations on cytotoxicity and genotoxicity. A screening battery was established for assessment of a broad range of parameters related to adverse effects. Clear concentration response effects were observed related to impairment of mitochondrial function, membrane integrity and oxidative stress markers, but no effect was observed on genotoxicity. The adverse effects were highest for the liposomes. The High Content Screening seems optimal for initial screening of adverse effects, and combined with standard cytotoxicity measurements initial screening can be performed for predictive toxicological screening.

Clarithromycin prevents human respiratory syncytial virus-induced airway epithelial responses by modulating activation of interferon regulatory factor-3

Macrolide antibiotics exert immunomodulatory activity by reducing pro-inflammatory cytokine production by airway epithelial cells, fibroblasts, vascular endothelial cells, and immune cells. However, the underlying mechanism of action remains unclear. Here, we examined the effect of clarithromycin (CAM) on pro-inflammatory cytokine production, including interferons (IFNs), by primary human nasal epithelial cells and lung epithelial cell lines (A549 and BEAS-2B cells) after stimulation by Toll-like receptor (TLR) and RIG-I-like receptor (RLR) agonists and after infection by human respiratory syncytial virus (RSV). CAM treatment led to a significant reduction in poly I:C- and RSV-mediated IL-8, CCL5, IFN-β and -λ production. Furthermore, IFN-β promoter activity (activated by poly I:C and RSV infection) was significantly reduced after treatment with CAM. CAM also inhibited IRF-3 dimerization and subsequent translocation to the nucleus. We conclude that CAM acts a crucial modulator of the innate immune response, particularly IFN production, by modulating IRF-3 dimerization and subsequent translocation to the nucleus of airway epithelial cells. This newly identified immunomodulatory action of CAM will facilitate the discovery of new macrolides with an anti-inflammatory role.

MiR-200b inhibits migration and invasion in non-small cell lung cancer cells via targeting FSCN1.

Distant metastasis of non-small cell lung cancer (NSCLC) leads to high postoperative recurrence and low long‑term survival rates. Deregulation of microRNA (miR)-200b has been demonstrated to be associated with NSCLC metastasis. However, the underlying molecular mechanism of miR‑200b in mediating NSCLC cell migration and invasion remains to be fully elucidated. In the current study, reverse transcription‑quantitative polymerase chain reaction data indicated that miR‑200b was significantly downregulated in several NSCLC cell lines, including A549, L78, H1229, H358 and H1650, compared with a normal human lung epithelial cell line, BEAS‑2B. Overexpression of miR‑200b significantly inhibited NSCLC cell migration and invasion. Bioinformatics analysis and a luciferase reporter assay were additionally conducted, which identified fascin actin‑bundling protein1 (FSCN1) as a novel target of miR‑200b. In addition, miR‑200b negatively mediated the protein expression of FSCN1 in NSCLC H1229 cells. siRNA‑mediated FSCN1 inhibition also significantly inhibited the migration and invasion of H1229 cells. In addition, overexpression of FSCN1 effectively reversed the suppressive effect of miR‑200b overexpression on NSCLC cell migration and invasion. Accordingly, it is suggested that miR‑200b is able to inhibit the migration and invasion of NSCLC cells, partly at least, via targeting FSCN1. The current study provides novel insight into miR‑200 regulation in NSCLC metastasis.

Histoplasma capsulatum-Induced Cytokine Secretion in Lung Epithelial Cells Is Dependent on Host Integrins, Src-Family Kinase Activation, and Membrane Raft Recruitment.

Histoplasma capsulatum var. capsulatum is a dimorphic fungus that causes histoplasmosis, a human systemic mycosis with worldwide distribution. In the present work, we demonstrate that H. capsulatum yeasts are able to induce cytokine secretion by the human lung epithelial cell line A549 in integrin- and Src-family kinase (SFK)-dependent manners. This conclusion is supported by small interfering RNA (siRNA) directed to α3 and α5 integrins, and PP2, an inhibitor of SFK activation. siRNA and PP2 reduced IL-6 and IL-8 secretion in H. capsulatum-infected A549 cell cultures. In addition, α3 and α5 integrins from A549 cells were capable of associating with H. capsulatum yeasts, and this fungus promotes recruitment of these integrins and SFKs to A549 cell membrane rafts. Corroborating this finding, membrane raft disruption with the cholesterol-chelator methyl-β-cyclodextrin reduced the levels of integrins and SFKs in these cell membrane domains. Finally, pretreatment of A549 cells with the cholesterol-binding compound, and also a membrane raft disruptor, filipin, significantly reduced IL-6 and IL-8 levels in A549-H.capsulatum cultures. Taken together, these results indicate that H. capsulatum yeasts induce secretion of IL-6 and IL-8 in human lung epithelial cells by interacting with α3 and α5 integrins, recruiting these integrins to membrane rafts, and promoting SFK activation.

Evaluation of a Gelatin-Modified Poly(ε-Caprolactone) Film as a Scaffold for Lung Disease

ABSTRACTLung transplantation is a necessary step for the patients with the end stage of chronic obstructive pulmonary disease. The use of artificial lungs is a promising alternative to natural lung transplantation which is complicated and is restricted by low organ donations. For successful lung engineering, it is important to choose the correct combination of specific biological cells and a synthetic carrier polymer. The focus of this study was to investigate the interactions of human lung epithelial cell line NCl-H292 that is involved in lung tissue development with the biodegradable poly(ε-caprolactone) before and after its chemical modification to evaluate potential for use in artificial lung formation. Also, the effect of polymer chemical modification on its mechanical and surface properties has been investigated. The poly(ε-caprolactone) surface was modified using aminolysis followed by immobilization of gelatin. The unmodified and modified polymer surfaces were characterized for roughness, tensile strength, and NCl-H292 metabolic cell activity. The results showed for the first time the possibility for NCI-H292 cells to adhere on this polymeric material. The resazurin assay showed that the metabolic activity at 24 h postseeding of 80% in the presence of the unmodified and greater than 100% in the presence of the modified polymer was observed. The roughness of the poly(ε-caprolactone) increased from 4 to 26 nm and the film strength increased from 0.01 to 0.045 kN when the material was chemically modified. The results obtained to date show potential for using modified poly(ε-caprolactone) as a scaffold for lung tissue engineering.

Analysis of the activation routes induced by different metal oxide nanoparticles on human lung epithelial cells.

Nanoparticles (Nps) can induce toxicity in the lung by accidental or intentional exposure. The main objective of the study reported here was to characterize the effect that four metal oxide Nps (CeO2, TiO2, Al2O3 and ZnO) had at the cellular level on a human lung epithelial cell line. This goal was achieved by studying the capacity of the Nps to activate the main mitogen-activated protein kinases (MAPKs) and the nuclear factor NFκB. Only ZnO Nps were able to activate all of the MAPKs and the release of Zn2+ ions was the main cause of activation. ZnO and Al2O3 Nps activated the NFκB pathway and induced the release of inflammatory cytokines. CeO2 and TiO2 Nps were found to have safer profiles. The graphical abstract was obtained using Servier Medical Art.

Proteomic changes in human lung epithelial cells (A549) in response to carbon black and titanium dioxide exposures

This study combined cytotoxicity assays with proteomic analysis to characterize the unique biological responses of the A549 human lung epithelial cell line to two physicochemically distinct respirable particles titanium dioxide (TiO2) and carbon black (CB). Cellular LDH, ATP, BrdU incorporation and resazurin reduction indicated that CB was more potent than TiO2. Proteomic analysis was done using 2D-GE and MALDI-TOF-TOF-MS. Proteomic changes reflected common and particle-specific responses. Particle-specific proteomic responses were associated with cell death (necrosis and apoptosis), viability and proliferation pathways. Our results suggested that these pathways were consistent with the cytotoxicity data. For instance, increased expressions of anti-proliferative proteins LMNA and PA2G4 were in agreement with the decreased BrdU incorporation in A549 cells after exposure to CB. Similarly, increased expression of HSPA5 that is associated with ATPase activity was consistent with decreased cellular ATP levels in these cells. These findings reveal that proteomic changes can explain the cellular cytotoxicity characteristics of the particles. In essence, our results demonstrate that the in vitro toxicoproteomic approach is a promising tool to gain insight into molecular mechanisms underlying particle exposure-specific cytotoxicity. Biological significanceIn this study we have shown that toxicoproteomics is a sensitive and informative method to resolve the toxicity characteristics of particles with different physicochemical properties. This approach can be useful in the investigation of molecular mechanisms underpinning cellular cytotoxic responses elicited by particle exposures. Thus, the toxicoproteomic approach can be valuable in assessing the risk associated with particle exposures in vitro.

Reconstitution of ST2 (IL-1R4) specific for IL-33 activity; no suppression by IL-1Ra though a common chain IL-1R3 (IL-1RAcP) shared with IL-1

Interleukin-33 (IL-33) receptors are composed of ST2 (also known as IL-1R4), a ligand binding chain, and IL-1 receptor accessory protein (IL-1RAcP, also known as IL-1R3), a signal transducing chain. IL-1R3 is a common receptor for IL-1α, and IL-1β, IL-33, and three IL-36 isoforms. A549 human lung epithelial cells are highly sensitive to IL-1α and IL-1β but not respond to IL-33. The lack of responsiveness to IL-33 is due to ST2 expression. ST2 was stably transfected into A549 cells to reconstitute its activity. RT-PCR and FACS analysis confirmed ST2 expression on the cell surface of A549/ST2 cells. Upon IL-33 stimulation, A549/ST2 cells induced IL-8 and IL-6 production in a dose dependent manner while A549/mock cells remained unresponsive. There was no difference in IL-1α and IL-1β activity in A549/ST2 cells compared to A549/mock cells despite the fact that IL-33 shares IL-1R3 with IL-1α/β. IL-33 activated inflammatory signaling molecules in a time- and dose-dependent manner. Anti-ST2 antibody and soluble recombinant ST2-Fc abolished IL-33-induced IL-6 and IL-8 production in A549/ST2 cells but the IL-1 receptor antagonist failed to block IL-33-induced cytokines. This result demonstrates for the first time the reconstitution of ST2 in A549 human lung epithelial cell line and verified its function in IL-33-mediated cytokine production and signal transduction.

Deinococcus radiodurans pprI expression enhances the radioresistance of eukaryotes.

PprI accelerates radiation-induced DNA damage repair via regulating the expression of DNA repair genes and enhances antioxidative enzyme activity in Deinococcus radiodurans after radiation. The main aim of our study was to determine whether the expression of pprI gene could fulfil its DNA repair function in eukaryotes and enhance the radioresistance of eukaryotic organism or not. In this study, we constructed pEGFP-c1-pprI eukaryotic expression vector and established a human lung epithelial cell line BEAS-2B with stable integration of pprI gene. We found that pprIexpression enhanced radioresistance of BEAS-2B cells, decreased γ-H2AX foci formation and apoptosis in irradiated BEAS-2B cells and alleviated radiation induced G2/M arrest of BEAS-2B cells. Moreover, we transferred pEGFP-c1-pprI vector into muscle of BALB/c mice by in vivo electroporation and studied the protective effect of prokaryotic pprI gene in irradiated mice. We found that pprI expression alleviated acute radiation induced hematopoietic system, lung, small intestine and testis damage and increased survival rate of irradiated mice via regulating Rad51 expression in different organs. These findings suggest that prokaryotic pprI gene expression in mammalian cells could enhance radioresistance in vitro and in vivo.

Epiregulin is required for lung tumor promotion in a murine two-stage carcinogenesis model.

Adenocarcinoma accounts for ∼40% of lung cancer, equating to ∼88 500 new patients in 2015, most of who will succumb to this disease, thus, the public health burden is evident. Unfortunately, few early biomarkers as well as effective therapies exist, hence the need for novel targets in lung cancer treatment. We previously identified epiregulin (Ereg), an EGF-like ligand, as a biomarker in several mouse lung cancer models. In the present investigation we used a primary two-stage initiation/promotion model to test our hypothesis that Ereg deficiency would reduce lung tumor promotion in mice. We used 3-methylcholanthrene (initiator) or oil vehicle followed by multiple weekly exposures to butylated hydroxytoluene (BHT; promoter) in mice lacking Ereg (Ereg-/- ) and wildtype controls (BALB/ByJ; Ereg+/+ ) and examined multiple time points and endpoints (bronchoalveolar lavage analysis, tumor analysis, mRNA expression, ELISA, wound assay) during tumor promotion. At the early time points (4 and 12 wk), we observed significantly reduced amounts of inflammation (macrophages, PMNs) in the Ereg-/- mice compared to controls (Ereg+/+ ). At 20 wk, tumor multiplicity was also significantly decreased in the Ereg-/- mice versus controls (Ereg+/+ ). IL10 expression, an anti-inflammatory mediator, and downstream signaling events (Stat3) were significantly increased in the Ereg-/- mice in response to BHT, supporting both reduced inflammation and tumorigenesis. Lastly, wound healing was significantly increased with recombinant Ereg in both human and mouse lung epithelial cell lines. These results indicate that Ereg has proliferative potential and may be utilized as an early cancer biomarker as well as a novel potential therapeutic target. © 2016 Wiley Periodicals, Inc.

Up-Regulation of Pro-Inflammatory Cytokines and Chemokine Production in Avian Influenza H9N2 Virus-Infected Human Lung Epithelial Cell Line (A549)

ABSTRACTInfluenza H9N2 virus mostly infects avian species but poses a potential health risk to humans. Little is known about the mammalian host immune responses to H9N2 virus. To obtain insight into the innate immune responses of human lung epithelial cells to the avian H9N2 virus, the expressions of pro-inflammatory cytokines and chemokine in the human airway epithelial cells infected with avian H9N2 virus were examined by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). H9N2 virus was able to cultivate in the human lung epithelial cell line (A549) and stimulate production of pro-inflammatory cytokines (IL-1β, IL-6) and chemokine (IL-8). Expressions of cytokine genes were up-regulated to a significantly higher level for IL-1β (p < 0.01), IL-6 (p < 0.01 after 12 hours and p < 0.05 after 24 hours) and IL-8 (p < 0.01 after 12 hours and p < 0.001 after 24 hours) in virus-cultured A549 cells as compared with non-virus-cultured cells. The amount of IL-6 and IL-1β proteins secreted into the culture medium was also increased after virus culture infection of A549 cell line compared to non-virus-cultured A549 cells and were significant in both IL-1β (p < 0.05 in 18 hours and p < 0.001 in 24–48 hours harvested supernatant) and IL-6 (p < 0.001). Silencing the p65 component of NF-κB in A549 cells suppressed the stimulatory effects of influenza virus on secretion of pro-inflammatory cytokines and chemokine. The findings in this study will broaden our understanding of host innate immune mechanisms and the pathogenesis of H9N2 influenza viruses in human respiratory epithelium.