Normal Human Bronchial Epithelial Cells Cells Research Articles

Proteomic characteristics of PM2.5-induced differentially expressed proteins in k-ras-silenced HBE cells

The human bronchial epithelial cells (HBE) and K-ras-silenced HBE cells were treated with fine particulate matter (PM2.5) samples from Taiyuan for 24 h. To screen the proteomic characteristics of PM2.5-induced differentially expressed proteins (DEPs), the Q Exactive mass spectrometer was used. Gene ontology (GO) analysis, Kyoto encyclopedia of genes and genomes (KEGG) analysis, functional prediction, protein–protein interaction (PPI) network analysis, and visualization of differential protein interactions were performed. 251 DEPs in K-ras silenced cells and 535 DEPs in normal HBE cells were identified, respectively. KEGG analysis showed that the differentially expressed proteins of PM2.5-treated cells were related to the biosynthesis of ribosomes, antibiotics, and amino acids. On the other hand, K-ras silenced cells were related to metabolic pathways, RNA transport, and DNA replication. Through the construction of a PPI network, the top 10 hub proteins were screened from the two cell groups, among which MRPL13, RPS20, and EIF1AX were of great significance. Our results indicated that the K-ras gene plays an important role in PM2.5-induced DEPs, and the findings provide a scientific basis for the further study of PM2.5 toxic mechanisms and biomarkers.

Protein Coding and Long Noncoding RNA (lncRNA) Transcriptional Landscape in SARS-CoV-2 Infected Bronchial Epithelial Cells Highlight a Role for Interferon and Inflammatory Response.

The global spread of COVID-19, caused by pathogenic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscores the need for an imminent response from medical research communities to better understand this rapidly spreading infection. Employing multiple bioinformatics and computational pipelines on transcriptome data from primary normal human bronchial epithelial cells (NHBE) during SARS-CoV-2 infection revealed activation of several mechanistic networks, including those involved in immunoglobulin G (IgG) and interferon lambda (IFNL) in host cells. Induction of acute inflammatory response and activation of tumor necrosis factor (TNF) was prominent in SARS-CoV-2 infected NHBE cells. Additionally, disease and functional analysis employing ingenuity pathway analysis (IPA) revealed activation of functional categories related to cell death, while those associated with viral infection and replication were suppressed. Several interferon (IFN) responsive gene targets (IRF9, IFIT1, IFIT2, IFIT3, IFITM1, MX1, OAS2, OAS3, IFI44 and IFI44L) were highly upregulated in SARS-CoV-2 infected NBHE cell, implying activation of antiviral IFN innate response. Gene ontology and functional annotation of differently expressed genes in patient lung tissues with COVID-19 revealed activation of antiviral response as the hallmark. Mechanistic network analysis in IPA identified 14 common activated, and 9 common suppressed networks in patient tissue, as well as in the NHBE cell model, suggesting a plausible role for these upstream regulator networks in the pathogenesis of COVID-19. Our data revealed expression of several viral proteins in vitro and in patient-derived tissue, while several host-derived long noncoding RNAs (lncRNAs) were identified. Our data highlights activation of IFN response as the main hallmark associated with SARS-CoV-2 infection in vitro and in human, and identified several differentially expressed lncRNAs during the course of infection, which could serve as disease biomarkers, while their precise role in the host response to SARS-CoV-2 remains to be investigated.

Human airway epithelial extracellular vesicle miRNA signature is altered upon asthma development.

miRNAs are master regulators of signaling pathways critically involved in asthma and are transferred between cells in extracellular vesicles (EV). We aimed to investigate whether the miRNA content of EV secreted by primary normal human bronchial epithelial cells (NHBE) is altered upon asthma development. NHBE cells were cultured at air-liquid interface and treated with interleukin (IL)-13 to induce an asthma-like phenotype. EV isolations by precipitation from basal culture medium or apical surface wash were characterized by nanoparticle tracking analysis, transmission electron microscopy, and Western blot, and EV-associated miRNAs were identified by a RT-qPCR-based profiling. Significant candidates were confirmed in EVs isolated by size-exclusion chromatography from nasal lavages of children with mild-to-moderate (n=8) or severe asthma (n=9), and healthy controls (n=9). NHBE cells secrete EVs to the apical and basal side. 47 miRNAs were expressed in EVs and 16 thereof were significantly altered in basal EV upon IL-13 treatment. Expression of miRNAs could be confirmed in EVs from human nasal lavages. Of note, levels of miR-92b, miR-210, and miR-34a significantly correlated with lung function parameters in children (FEV1 FVC%pred and FEF25-75%pred ), thus lower sEV-miRNA levels in nasal lavages associated with airway obstruction. Subsequent ingenuity pathway analysis predicted the miRNAs to regulate Th2 polarization and dendritic cell maturation. Our data indicate that secretion of miRNAs in EVs from the airway epithelium, in particular miR-34a, miR-92b, and miR-210, might be involved in the early development of a Th2 response in the airways and asthma.

Optimization of Normal Human Bronchial Epithelial (NHBE) Cell 3D Cultures for in vitro Lung Model Studies

Robust in vitro lung models are required for risk assessment to measure key events leading to respiratory diseases. Primary normal human bronchial epithelial cells (NHBE) represent a good lung model but obtaining well-differentiated 3D cultures can be challenging. Here, we evaluated the ability to expand primary NHBE cells in different culture conditions while maintaining their 3D culture characteristics such as ciliated and goblet cells, and ion channel function. Differentiated cultures were optimally obtained with PneumaCult-Ex Plus (expansion medium)/PneumaCult-ALI (differentiation medium). Primary cells passaged up to four times maintained airway epithelial characteristics as evidenced by ciliated pseudostratified columnar epithelium with goblet cells, trans-epithelial electrical resistance (TEER) (>400 Ohms.cm2), and cystic fibrosis transmembrane conductance regulator-mediated short-circuit currents (>3 µA/cm2). No change in ciliary beat frequency (CBF) or airway surface liquid (ASL) meniscus length was observed up to passage six. For the first time, this study demonstrates that CFTR ion channel function and normal epithelial phenotypic characteristics are maintained in passaged primary NHBE cells. Furthermore, this study highlights the criticality of evaluating expansion and differentiation conditions for achieving optimal phenotypic and functional endpoints (CBF, ASL, ion channel function, presence of differentiated cells, TEER) when developing in vitro lung models.

Pro-inflammatory effects of extracellular Hsp70 and cigarette smoke in primary airway epithelial cells from COPD patients

Extracellular Hsp70 (eHsp70) can activate immune cells via Toll-like receptors (TLR) 2 and 4, and induce cytokine synthesis. The aim of this study was to explore inflammation-associated effects of eHsp70 alone and in combination with cigarette smoke extract (CSE) in primary bronchial epithelial cells.We assessed IL-6 and IL-8 concentrations, TLR2, TLR4 and Hsp70 mRNA expressions, and mitogen-activated protein kinases (MAPKs) activation induced by recombinant human (rh) Hsp70, CSE or their combinations in normal human bronchial epithelial cells (NHBE) obtained commercially, and primary bronchial epithelial cells isolated from non-COPD lung donors (PBEC) or COPD patients (PBEC COPD).Baseline levels of IL-6 and IL-8 were significantly higher in PBEC COPD than in non-COPD PBECs. Upon rhHsp70 stimulation, IL-6 and IL-8 were significantly increased, with the strongest response in COPD-derived PBECs. CSE alone elevated cytokine secretion in all examined cells. rhHsp70 and CSE had antagonistic interactions on IL-8 release in PBECs from COPD patients, while the addition of rhHsp70 further increased CSE-induced IL-6 secretion in NHBE cells. rhHsp70 and CSE alone decreased TLR2 and TLR4 mRNA expression in COPD-derived PBECs. In non-COPD PBECs, combined treatments decreased only TLR2 mRNA expression. Hsp70 mRNA expression, as indicator of intracellular Hsp70, which may have anti-inflammatory effects, was reduced in COPD-derived cells upon exposure to CSE and rhHsp70 alone, but not with their combinations. Contrary to this, in PBECs from lung donors only combined treatments supressed Hsp70 gene expression. CSE activated JNK and p38 MAPKs, while rhHsp70 increased activation of c-Jun kinase in NHBE cells.Collectively, both eHsp70 and CSE induce pro-inflammatory responses in PBECs from non-COPD as well as COPD donors, but in combination antagonistic effects were observed in COPD-derived cells. These effects may be related to the regulation of TLR2/4 and might lead to modulation of inflammation with possible deleterious consequences for COPD patients.

IL-1β upregulates Muc5ac expression via NF-κB-induced HIF-1α in asthma

The manifest and important feature in respiratory diseases, including asthma and COPD (chronic obstructive pulmonary disease), is the increased numbers and hypersecretion of goblet cells and overexpression of mucins, especially Muc5ac. Many proinflammatory cytokines play important roles in goblet cell metaplasia and overproduction of Muc5ac. However, the effect of IL-1β on Muc5ac expression in asthma remains unknown. Here, we detected the correlation between IL-1β and Muc5ac in asthma patients and further explored the mechanism of IL-1β-induced Muc5ac overexpression. Our results showed that Muc5ac and IL-1β were up-regulated in 41 patients with asthma and that Muc5ac overexpression was related with IL-1β in asthma (R2=0.668, p≪0.001). Furthermore, the correlation between IL-1β and Muc5ac is higher in severe group than that in moderate group. In vitro experiments with normal human bronchial epithelial cells (NHBECs) showed that IL-1β up-regulated Muc5ac expression in NHBEC in a time- and dosage-dependent manner. Hypoxia-induced HIF-1α was responsible for Muc5ac expression mediated by IL-1β. Knocking down HIF-1α by siRNA decreased Muc5ac expression under hypoxia even in IL-1β-treated NHBEC cells. Luciferase reporter assay showed that HIF-1α enhanced Muc5ac promoter activity in HEK293T cells. HIF-1α could specifically bind to the promoter of Muc5ac by EMSA. The correlation among IL-1β, HIF-1α and Muc5ac was observed in patients with asthma. Mechanically, NF-κB activation was essential to IL-1β-induced HIF-1α upregulation via the canonical pathway of NF-κB. The level of nuclear p65, a subunit of NF-κB, was obviously increased in NHBEC cells under IL-1β treatment. IL-1β did not change either HIF-1α or Muc5ac expression when inhibiting NF-κB signaling with Bay11-7082, an inhibitor of NF-κB. Collectively, we concluded that IL-1β up-regulated Muc5ac expression via NF-κB-induced HIF-1α in asthma and provided a potential therapeutic target for asthma.

Particulate metal bioaccessibility in physiological fluids and cell culture media: Toxicological perspectives

According to the literature, tiny amounts of transition metals in airborne fine particles (PM2.5) may induce proinflammatory cell response through reactive oxygen species production. The solubility of particle-bound metals in physiological fluids, i.e. the metal bioaccessibility is driven by factors such as the solution chemical composition, the contact time with the particles, and the solid-to-liquid phase ratio (S/L). In this work, PM2.5-bound metal bioaccessibility was assessed in various physiological-like solutions including cell culture media in order to evidence the potential impact on normal human bronchial epithelial cells (NHBE) when studying the cytotoxicity and inflammatory responses of PM2.5 towards the target bronchial compartment. Different fluids (H2O, PBS, LHC-9 culture medium, Gamble and human respiratory mucus collected from COPD patients), various S/L conditions (from 1/6000 to 1/100,000) and exposure times (6, 24 and 72h) were tested on urban PM2.5 samples. In addition, metals’ total, soluble and insoluble fractions from PM2.5 in LHC-9 were deposited on NHBE cells (BEAS-2B) to measure their cytotoxicity and inflammatory potential (i.e., G6PDH activity, secretion of IL-6 and IL-8). The bioaccessibility is solution-dependent. A higher salinity or organic content may increase or inhibit the bioaccessibiliy according to the element, as observed in the complex mucus matrix. Decreasing the S/L ratio also affect the bioaccessibility depending on the solution tested while the exposure time appears less critical. The LHC-9 culture medium appears to be a good physiological proxy as it induces metal bioaccessibilities close to the mucus values and is little affected by S/L ratios or exposure time. Only the insoluble fraction can be linked to the PM2.5-induced cytotoxicity. By contrast, both soluble and insoluble fractions can be related to the secretion of cytokines. The metal bioaccessibility in LHC-9 of the total, soluble, and insoluble fractions of the PM2.5 under study did not explain alone, the cytotoxicity nor the inflammatory response observed in BEAS-2B cells. These findings confirm the urgent need to perform further toxicological studies to better evaluate the synergistic effect of both bioaccessible particle-bound metals and organic species.

Hydrocortisone Affects the Transport Phenotype of Differentiated Normal Human Bronchial Epithelial Cells

Glucocorticoids play a crucial role in transitioning the developing lung epithelium from a secretory to an absorptive tissue. The objective of this study was to evaluate the effects of hydrocortisone (HC) on the transport phenotype of the bronchial epithelium during differentiation. Normal human bronchial epithelial cells (NHBE) were differentiated on polyester membranes and harvested at four time points over a 24 day time period. NHBE cells differentiated in the presence and absence of HC (HC0) along with cells withdrawn from HC after 8 days (HC8) all formed pseudostratified layers, as confirmed by confocal microscopy, and developed transepithelial resistances (TER) greater than 1000 Ωcm2 by day 8. mRNAs associated with bronchial basal cells (p63, NKX2.1) were detected at days 0, 4, 8 and 24 while airway surface markers (FOXJ1, MUC5ac, MUC5b) were not detected until day 4. HC0 and HC8 NHBE cells expressed lower levels of mucin mRNA than control cells at day 24. At day 8, Ussing chamber experiments showed that the total and benzamil sensitive currents were significantly decreased under HC0 conditions (4.0±0.13, 0.46±0.14, respectively) but not under HC8 conditions (8.8±1.2, 5.7±1.3) compared to control (7.1±0.44, 3.9±0.53). At day 24, total and benzamil sensitive currents were significantly decreased under HC0 and HC8 conditions. HC had no effect on CFTRinh‐172 sensitive current, indicating that there was no detectable effect on CFTR‐dependent anion secretion. ENaCα and γ subunits as well as CFTR co‐localized at the apical membrane in control and HC8 cells at day 24 as observed by immunofluorescence. HC0 cells had noticeably less apical fluorescence at day 24 corresponding to reduced ENaCα and γ expression compared to control cells. Interestingly, ENaC subunits and CFTR appeared to localize within the cilia. Control and HC0 NHBE cells stimulated with the selective β2‐AR agonist salbutamol produced outward currents which resulted in increased benzamil sensitive current in control but not HC0 cells. Additionally, NHBE cells stimulated with UTP initially exhibitedan inward, paxilline‐sensitive current characteristic of KCa1.1 channel activation, followed by an outward, DIDS‐sensitive current (TMEM16a/ANO1) in control and HC8 cells at days 8 and 24. HC0 cells stimulated with UTP at day 8 produced only outward currents consistent with reduced KCNMA1 mRNA expression and K+ channel activity. There was a significantly reduced UTP‐induced inward current at day 24 for both HC0 and HC8 cells. Expression of TMEM16a and P2RY2 receptor mRNA was not affected by HC, whereas P2RY4 receptor expression was not detectable at days 8 and 24 in HC0 cells. Immunofluorescence indicated co‐localization of TMEM16a, KCa1.1, and P2Y2 at the apical membrane in control cells suggesting that these proteins form a signaling complex at the apical membrane that may also include P2Y4 receptors. These results demonstrate an important role for glucocorticoids in development of the transport phenotype of bronchial epithelial cells during differentiation.Support or Funding InformationF31EB018707

ADAM10 mediates the house dust mite-induced release of chemokine ligand CCL20 by airway epithelium.

House dust mite (HDM) acts on the airway epithelium to induce airway inflammation in asthma. We previously showed that the ability of HDM to induce allergic sensitization in mice is related to airway epithelial CCL20 secretion. As a disintegrin and metalloprotease (ADAM)s have been implicated in chemokine shedding, we sought to determine their involvement in HDM-induced release of chemokines, including CCL20, by airway epithelial cells. We studied the effects of pharmacological ADAM inhibitors as well as ADAM10 and ADAM17 siRNA downregulation on chemokine release using (multiplex) ELISA in supernatants from HDM-exposed human bronchial epithelial 16HBE cells and primary normal human bronchial epithelial cells (NHBE) at 4-24h. House dust) mite markedly increased CCL20 levels in both 16HBE and NHBE cells (16-24h). In 16HBE cells, the HDM-induced increase was observed as early as 4h upon exposure and the use of specific inhibitors indicated the involvement of ADAM10/17-mediated shedding. siRNA knockdown of ADAM10, but not of ADAM17, significantly reduced the HDM-induced release of CCL20 in both 16HBE and NHBE cells. A similar effect was observed for HDM-induced CCL2, CCL5, and CXCL8 release in NHBE cells. The HDM-induced increase in CCL20 levels was not affected by protein synthesis inhibitor cycloheximide nor protein transport inhibitor monensin, indicating that HDM induces surface shedding of chemokines. Our data show for the first time that ADAM10 activity contributes to HDM-induced shedding of chemokines, including CCL20. The ADAM10/CCL20 axis may be a target for novel therapeutic strategies in asthma.

Fibroblast-epithelial cell interactions drive epithelial-mesenchymal transition differently in cells from normal and COPD patients.

BackgroundEpithelial-to-mesenchymal transition (EMT), which involves changes in cellular morphology of highly polarized epithelial cells and the gain of mesenchymal cell phenotype with migratory and invasive capacities, is implicated in smoking-related chronic obstructive pulmonary disease (COPD). However, the interactions of fibroblasts and epithelial cells and the participation of fibroblasts in the EMT processes in COPD are poorly understood. Here, we investigated the hypothesis that EMT is active in human bronchial epithelial (HBE) cells of COPD patients, and that mediators secreted by lung fibroblasts from COPD patients induce EMT.MethodsPrimary HBE cells from normal subjects and COPD patients were purchased from LONZA. HLFs were derived from resected lung obtained from normal (N) and COPD (D) subjects and their conditioned medium (CM) was collected after 2-day culture in serum-free medium. The expression of epithelial and mesenchymal markers as well as EMT-related transcription factors in lung biopsies, and in HBE cells following stimulation with CM from both normal human lung fibroblasts (NHLF) and COPD human lung fibroblasts (DHLF) was evaluated by immunohistochemistry, qRT-PCR and western blot.ResultsBasal mRNA expression of mesenchymal markers and EMT-related transcription factors were increased in DHBE cells compared to normal human bronchial epithelial cells (NHBE) cells as well as in COPD lungs. CM from NHLF significantly induced vimentin expression in both NHBE and COPD human bronchial epithelial cells (DHBE) cells, but only increased N-cadherin expression in DHBE cells. CM from NHLF significantly induced Twist1 and Twist2 expression in NHBE cells and increased Snai2 (Slug) expression in DHBE cells. While CM from NHLF had no effect on such EMT markers, CM from DHLF significantly increased the protein expression of E-cadherin and vimentin in NHBE cells compared to control. N-cadherin expression was upregulated to a greater degree in NHBE cells than DHBE cells. Only CM from DHLF significantly increased E-/N-cadherin ratio in DHBE cells.ConclusionsOur results suggest that DHBE cells have partially undergone EMT under baseline conditions. DHLF-CM promoted EMT in NHBE, suggesting that interactions between fibroblast and epithelial cells may play an important role in the EMT process in COPD.

Tight Junction Protein Expression and Junction Development During Differentiation of Normal Human Bronchial Epithelial Cells

Normal human bronchial epithelial cells (NHBE) possessing a basal cell phenotype (p63+, KRT5/6a+) were differentiated on Transwell polyester membranes under air‐liquid interface (ALI) conditions. After 24 days of differentiation, NHBE cells had developed sufficient transepithelial resistance (TER) to perform voltage clamp experiments and expressed mRNAs associated with airway surface cells (Muc5b, FOXJ1). Additionally, mRNA transcripts for ENaC, CFTR and ATP1A1 all increased during differentiation and the corresponding proteins were appropriately locatized as confirmed by immunohistochemistry. Transcripts associated with epithelial adhesions (adherens and tight junctions) were also measured. Upregulation of Cldn3, Cldn8, and Cldn10 correlated with high TER, while ZO1/2, Jam1 did not exhibit differentiation‐dependent increases in mRNA expression. Examination of paracellular ionic permeability demonstrated sodium selectivity over chloride (PCl/PNa ∼ 83%). Furthermore, comparison of NHBE mRNA to an immortalized human airway cell line (HBEC) unable to develop TER revealed loss of claudin, ZO1, and E‐Cad transcripts as measured by qRT‐PCR. These results suggest that TER development in NHBE cells requires constitutive expression of ZO1, E‐Cad and differentiation induced increases in expression of Cldn3, Cldn8, and Cldn10.

Evaluation of the Calu-3 cell line as a model of in vitro respiratory syncytial virus infection

Respiratory syncytial virus (RSV) replication is primarily limited to the upper respiratory tract epithelium and primary, differentiated normal human bronchial epithelial cells (NHBE) have, therefore, been considered a good system for in vitro analysis of lung tissue response to respiratory virus infection and virus–host interactions. However, NHBE cells are expensive, difficult to culture, and vary with the source patient. An alternate approach is to use a continuous cell line that has features of bronchial epithelial cells such as Calu-3, an epithelial cell line derived from human lung adenocarcinoma, as an in vitro model of respiratory virus infection. The results show that Calu-3 fully polarize when grown on permeable supports as liquid-covered cultures. Polarized Calu-3 are susceptible to RSV infection and release infectious virus primarily from the apical surface, consistent with studies in NHBE cells. The data demonstrate that polarized Calu-3 may serve as a useful in vitro model to study host responses to RSV infection.

Reactive Oxygen Species and Hyaluronidase 2 Regulate Airway Epithelial Hyaluronan Fragmentation

Hyaluronidase 2 (Hyal2) is a hyaluronan (HA)-degrading enzyme found intracellularly or/and anchored to the plasma membrane through glycosylphosphatidylinositol (GPI). Normal human bronchial epithelial cells (NHBE) grown at the air-liquid interphase (ALI), treated with PI-specific phospholipase C (PI-PLC), exhibited increased Hyal activity in secretions and decreased protein and activity on the apical membrane, confirming that GPI-anchored Hyal2 is expressed in NHBE cells and it remains active in its soluble form. We have reported that HA degradation was mediated by reactive oxygen species (ROS) in human airways. Here we show that ROS increase Hyal2 expression and activity in NHBE cells and that the p38MAPK signaling pathway is involved in this effect. Hyal2 induction was confirmed by using small interfering RNA (siRNA) expressing lentivirus. These in vitro findings correlated in vivo with smokers, where increased Hyal2 immunoreactivity in the epithelium was associated with augmented levels of HA and the appearance of low molecular mass HA species in bronchial secretions. In summary, this work provides evidence that ROS induce Hyal2, suggesting that Hyal2 is likely responsible for the sustained HA fragmentation in the airway lumen observed in inflammatory conditions associated with oxidative stress.

Candidate Dietary Phytochemicals Modulate Expression of Phase II Enzymes GSTP1 and NQO1 in Human Lung Cells , ,

Many phytochemicals possess cancer-preventive properties, some putatively through phase II metabolism-mediated mutagen/oxidant quenching. We applied human lung cells in vitro to investigate the effects of several candidate phytopreventive agents, including green tea extracts (GTE), broccoli sprout extracts (BSE), epigallocatechin gallate (EGCG), sulforaphane (SFN), phenethyl isothiocyanate (PEITC), and benzyl isothiocyanate (BITC), on inducing phase II enzymes glutathione S-transferase P1 (GSTP1) and NAD(P)H:quinone oxidoreductase 1 (NQO1) at mRNA and protein levels. Primary normal human bronchial epithelial cells (NHBE), immortalized human bronchial epithelial cells (HBEC), and lung adenocarcinoma cells (A549) were exposed to diet-achievable levels of GTE and BSE (0.5, 1.0, 2.0 mg/L), or individual index components EGCG, SFN, PEITC, BITC (0.5, 1.0, 2.0 μmol/L) for 24 h, 48 h, and 6 d, respectively. mRNA assays employed RNA-specific quantitative RT-PCR and protein assays employed Western blotting. We found that in NHBE cells, while GSTP1 mRNA levels were slightly but significantly increased after exposure to GTE or BSE, NQO1 mRNA increased to 2- to 4-fold that of control when exposed to GTE, BSE, or SFN. Effects on NQO1 mRNA expression in HBEC cells were similar. NQO1 protein expression increased up to 11.8-fold in SFN-treated NHBE cells. Both GSTP1 and NQO1 protein expression in A549 cells were constitutively high but not induced under any condition. Our results suggest that NQO1 is more responsive to the studied chemopreventive agents than GSTP1 in human lung cells and there is discordance between single agent and complex mixture effects. We conclude that modulation of lung cell phase II metabolism by chemopreventive agents requires cell- and agent-specific discovery and testing.

Abstract 589: Vascular endothelial growth factor facilitates vaccinia virus tropism and oncolysis in human pancreatic ductal adenocarcinoma

Abstract Background: Oncolytic virotherapy is a promising approach to treat cancers resistant to conventional therapies. Vaccinia virus, an enveloped DNA virus from the poxvirus family, has been used as a vector for cancer vaccines and oncolytic viral gene therapy. Wild type vaccinia virus has a natural tropism for cancer cells both in vitro and in vivo due to their increased proliferation, abnormalities in epidermal growth factor and interferon signalling, in addition to other unknown mechanisms. We have recently demonstrated that the anti-tumor efficacy of the Lister strain vaccinia virus, unlike some replicating oncolytic vectors, is not attenuated by hypoxia and even augmented 20 fold in some pancreatic ductal adenocarcinoma (PDAC) cells lines (CT Hiley et al. Gene Therapy 2009. In press). The underlying mechanism is not fully understood. Methods: Vascular Endothelial Growth Factor (VEGF) was detected by an enzyme-linked immunosorbent assay. The effect of VEGF on vaccinia virus infection in human cancer cells was investigated by manipulation of VEGF expression using siRNAs or gene over-expression. Normal human bronchial epithelial cells (NHBE) were used to assess the effect of VEGF on non-transformed cells. Results: Induction of VEGF by hypoxia was found in the supernatants of cancer cell lines showing sensitisation to vaccinia virus under hypoxia. Replication of vaccinia virus was correlated with the level of VEGF expression in a panel of human cancer cell lines. Knockdown of VEGF expression in Suit2 cells by siRNAs reduced transgene expression over 7 fold at 24 and 48 hours post infection (PI) (P<0.05 & P<0.01 respectively) and viral replication 2 fold (P<0.01) at 72 hours PI. Over-expression of VEGF in cell line MiaPaca2, which has a lower level of VEGF expression, enhanced its sensitivity to vaccinia virus. Four fold over-expression of VEGF in MiaPaca2 resulted in levels of only half that seen in Suit2 cells. However a threefold increase in viral replication was observed at 72 hours PI (P<0.01) together with significant increases in transgene expression at 24 and 48 hours and a 40% increase in cytotoxicity at 6 days PI (p<0.05) compared to control. No similar effect on vaccinia virus tropism was seen in NHBE cells at levels of VEGF expression seen in PDAC cell lines. VEGF also augmented vaccinia virus tumour targeting in vivo after systemic administration (P<0.01 at 60 hours post intravenous injection). Conclusion: This novel finding suggests that VEGF expression by cancer cells is at least partly responsible for some of the tumour tropism of wild type Lister vaccinia virus and highlights its suitability for use in poor prognosis tumour types, notably those with high levels of hypoxia and VEGF expression. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 589.

Identification of Long stress-induced non-coding transcripts that have altered expression in cancer

It has recently become clear that the transcriptional output of the human genome is far more abundant than previously anticipated, with the vast majority of transcripts not coding for protein. Utilizing whole-genome tiling arrays, we analyzed the transcription across the entire genome in both normal human bronchial epithelial cells (NHBE) and NHBE cells exposed to the tobacco carcinogen NNK. Our efforts focused on the characterization of non-coding transcripts that were greater than 300 nucleotides in length and whose expression was increased in response to NNK. We identified 12 Long Stress-Induced Non-coding Transcripts that we term LSINCTs. Northern blot analysis revealed that these transcripts were larger than predicted from the tiling array data. Quantitative real-time RT-PCR performed across a panel of normal cell lines indicates that these transcripts are more abundantly expressed in rapidly growing tissues or in tissues that are more prone to cellular stress. These transcripts that have increased expression after exposure to NNK also had increased expression in a number of lung cancer cell lines and also in many breast cancer cell lines. Collectively, our results identified a new class of long stress responsive non-coding transcripts, LSINCTs, which have increased expression in response to DNA damage induced by NNK. LSINCTs interestingly also have increased expression in a number of cancer-derived cell lines, indicating that the expression is increased in both, correlating cellular stress and cancer.

Leukotriene D4‐induced, epithelial cell‐derived transforming growth factor β1 in human bronchial smooth muscle cell proliferation

Cysteinyl-leukotrienes (cys-LTs) orchestrate many pathognomonic features of asthma in animal models of allergic airway inflammation, including bronchial smooth muscle cell (BSMC) hyperplasia. However, because cys-LTs alone do not induce mitogenesis in monocultures of human BSMC, the effect observed in vivo seemingly involves indirect mechanisms, which are still undefined. This study aims to investigate the regulatory role of leukotriene (LT)D(4) on TGF-beta1 expression in airway epithelial cells and the consequence of this interplay on BSMC proliferation. HEK293 cells stably transfected with cys-LT receptor 1 (CysLT1) (293LT1) were stimulated with LTD(4) and TGF-beta1 mRNA and protein expression was measured using Northern blot and ELISA, respectively. Conditioned medium (CM) harvested from LTD(4)-treated cells was then assayed for its proliferative effect on primary human BSMC. TGF-beta1 mRNA expression was also determined in tumoural type II pneumocytes A549 and in normal human bronchial epithelial cells (NHBE) following LTD(4) stimulation. The results demonstrated that LTD(4)-induced TGF-beta1 mRNA production in a time- and concentration-dependent manner in 293LT1. TGF-beta1 secretion was also up-regulated and CM from LTD(4)-treated 293LT1 was shown to increase BSMC proliferation in a TGF-beta1-dependent manner. The increased expression of TGF-beta1 mRNA by LTD(4) also occured in A549 and NHBE cells via a CysLT1-dependent mechanism. In conclusion, elevated expression of cys-LTs in asthmatic airways might contribute to BSMC hyperplasia and concomitant clinical features of asthma such as airway hyperresponsiveness via a paracrine loop involving TGF-beta1 production by airway epithelial cells.

Peroxisome proliferator‐activated receptor‐γ agonists inhibit respiratory syncytial virus‐induced expression of intercellular adhesion molecule‐1 in human lung epithelial cells

Respiratory syncytial virus (RSV) is the major causative agent of severe lower respiratory tract disease and death in infants worldwide. The epithelial cells of the airways are the target cells for RSV infection and the site of the majority of the inflammation associated with the disease. However, despite five decades of intensive RSV research there exist neither an effective active vaccine nor a promising antiviral and anti-inflammatory therapy. Recently, peroxisome proliferator-activated receptor-gamma (PPAR-gamma), a member of the nuclear hormone receptor superfamily, has been shown to possess anti-inflammatory properties. Therefore, we hypothesized whether the detrimental increase of intercellular adhesion molecule-1 (ICAM-1) on RSV-infected lung epithelial cells (A549 and primary normal human bronchial epithelial cells (NHBE)) might be modulated by natural and synthetic PPAR-gamma agonists (15d-PGJ2, ciglitazone, troglitazone, Fmoc-Leu). Our data show that all PPAR-gamma agonists under study significantly down-regulated the RSV-induced expression of ICAM-1 on A549- and NHBE cells in a dose-dependent manner resulting in a reduced beta2 integrin-mediated adhesion of monocytic effector cells (U937) to RSV-infected A549 cell monolayers. In contrast, the PPAR-alpha agonist bezafibrate had no impact on the RSV-induced ICAM-1 expression. The reduced ICAM-1 expression was associated with a diminished ICAM-1 mRNA level and binding activity of nuclear factor-kappaB (p65/p50) in A549 cells. These findings suggest that PPARgamma agonists have beneficial effects in the suppression of the inflammatory response during RSV infection and therefore might have clinical efficacy in the course of severe RSV-infection.

The Anti-Inflammatory Effect of Ciglitazone is Partially Mediated by Glucocorticoid Induced Leucine Zipper in Human Airway Epithelial Cells

RATIONALE: PPARγ activation has been shown to inhibit airway inflammation but the mechanisms are not fully understood; we therefore assessed whether the anti-inflammatory protein Glucocorticoid-Induced Leucine Zipper (GILZ), which we have shown is upregulated by the PPARγ agonist ciglitazone, is involved in this process. METHODS: The expression of GILZ was assessed by immunoblotting in A549 and normal human bronchial epithelial cells (NHBE) at baseline and following ciglitazone treatment. RNA silencing was used to knock down GILZ protein levels in A549 cells in order to determine whether GILZ mediates the anti-inflammatory effects of ciglitazone. GILZ siRNA- and Control siRNA-treated A549 cells were stimulated for 20 hours with ciglitazone followed by IL-1beta for 4 hours. Total RNA was isolated and IL-8 mRNA levels were assessed by quantitative real-time PCR. RESULTS: GILZ levels were upregulated by ciglitazone in A549 and NHBE cells. Ciglitazone potently inhibited the induction of IL-8 mRNA by IL-1beta in control siRNA cells, whereas in GILZ siRNA cells this effect was partially abrogated. CONCLUSION: Here we demonstrate the induction of GILZ, an anti-inflammatory protein known to inhibit NF-κB and AP-1, by ciglitazone in human airway epithelial cells. Further we demonstrate that GILZ in part mediates the anti-inflammatory effects of agonist-bound PPARγ. These findings help elucidate a mechanism via which PPARγ agonists reduce airway inflammation, and suggest the potential of the thiazolidinediones in the treatment of asthma.

Pomegranate fruit extract inhibits prosurvival pathways in human A549 lung carcinoma cells and tumor growth in athymic nude mice

Developing novel mechanism-based chemopreventive approaches for lung cancer through the use of dietary substances which humans can accept has become an important goal. In the present study, employing normal human bronchial epithelial cells (NHBE) and human lung carcinoma A549 cells, we first compared the growth inhibitory effects of pomegranate fruit extract (PFE). Treatment of PFE (50-150 microg/ml) for 72 h was found to result in a decrease in the viability of A549 cells but had only minimal effects on NHBE cells as assessed by the MTT and Trypan blue assays. PFE treatment of A549 cells also resulted in dose-dependent arrest of cells in G0-G1 phase of the cell cycle (as assessed by DNA cell cycle analysis). We further found that PFE treatment also resulted in (i) induction of WAF1/p21 and KIP1/p27, (ii) decrease in the protein expressions of cyclins D1, D2 and E, and (iii) decrease in cyclin-dependent kinase (cdk) 2, cdk4 and cdk6 expression. The treatment of cells with PFE inhibited (i) phosphorylation of MAPK proteins, (ii) inhibition of PI3K, (iii) phosphorylation of Akt at Thr308, (iv) NF-kappaB and IKKalpha, (v) degradation and phosphorylation of IkappaBalpha, and (vi) Ki-67 and PCNA. We also found that PFE treatment to A549 cells resulted in inhibition of NF-kappaB DNA-binding activity. Oral administration of PFE (0.1 and 0.2%, wt/vol) to athymic nude mice implanted with A549 cells resulted in a significant inhibition in tumor growth. Our results provide a suggestion that PFE can be a useful chemopreventive/chemotherapeutic agent against human lung cancer.