Human Bronchial Epithelial Cells Cells Research Articles

METTL3 mediates SOX5 m6A methylation in bronchial epithelial cells to attenuate Th2 cell differentiation in T2 asthma

ObjectiveAsthma, a chronic inflammatory disease in which type 2 T helper cells (Th2) play a causative role in the development of T2 asthma. N6-methyladenosine (m6A) modification, an mRNA modification, and methyltransferase-like 3 (METTL3) is involved in the development of T2 asthma by inhibiting Th2 cell differentiation. Sex determining region Y-box protein 5 (SOX5) is involved in regulating T cell differentiation, but its role in T2 asthma was unclear. The objective of this study was to explore the role of METTL3 and SOX5 in T2 asthma and whether there is an interaction between the two. Materials and methodsAdults diagnosed with T2 asthma (n = 14) underwent clinical information collection and pulmonary function tests. In vivo and in vitro T2 asthma models were established using female C57BL/6 mice and human bronchial epithelial cells (HBE). The expressions of METTL3 and SOX5 were detected by Western blot and qRT-PCR and Western blot. Th2 cell differentiation was determined by flow cytometry and IL-4 level was detected by ELISA. m6A methylation level was determined by m6A quantitative assay. The relationship between METTL3 expression and clinical parameters was determined by Spearman rank correlation analysis. The function of METTL3 and SOX5 genes in asthma was investigated in vitro and in vivo. The RNA immunoprecipitation assay detected the specific interaction between METTL3 and SOX5. ResultsPatients with T2 asthma displayed lower METTL3 levels compared to healthy controls. Within this group, a negative correlation was observed between METTL3 and Th2 cells, while a positive correlation was noted between METTL3 and clinical parameters as well as Th1 cells. In both in vitro and in vivo models representing T2 asthma, METTL3 levels decreased significantly, while SOX5 levels showed the opposite trend. Overexpression of METTL3 gene in HBE cells significantly inhibited Th2 cell differentiation and increased m6A methylation activity. From a mechanism perspective, low METTL3 negatively regulates SOX5 expression through m6A modification dependence, while high SOX5 expression is positively associated with T2 asthma severity. Cell transfection experiments confirmed that METTL3 regulates Th2 cell differentiation and IL-4 release through SOX5. ConclusionsOverall, our results indicate that METTL3 alleviates Th2 cell differentiation in T2 asthma by modulating the m6A methylation activity of SOX5 in bronchial epithelial cells. This mechanism could potentially serve as a target for the prevention and management of T2 asthma.

PM2.5 Causes Increased Bacterial Invasion by Affecting HBD1 Expression in the Lung.

Our research addresses the critical environmental issue of a fine particulate matter (PM2.5), focusing on its association with the increased infection risks. We explored the influence of PM2.5 on human beta-defensin 1 (HBD1), an essential peptide in mucosal immunity found in the airway epithelium. Using C57BL/6J mice and human bronchial epithelial cells (HBE), we examined the effects of PM2.5 exposure followed by Pseudomonas aeruginosa (P. aeruginosa) infection on HBD1 expression at both mRNA and protein levels. The study revealed that PM2.5's toxicity to epithelial cells and animals varies with time and concentration. Notably, HBE cells exposed to PM2.5 and P. aeruginosa showed increased bacterial invasion and decreased HBD1 expression compared to the cells exposed to P. aeruginosa alone. Similarly, mice studies indicated that combined exposure to PM2.5 and P. aeruginosa significantly reduced survival rates and increased bacterial invasion. These harmful effects, however, were alleviated by administering exogenous HBD1. Furthermore, our findings highlight the activation of MAPK and NF-κB pathways following PM2.5 exposure. Inhibiting these pathways effectively increased HBD1 expression and diminished bacterial invasion. In summary, our study establishes that PM2.5 exposure intensifies P. aeruginosa invasion in both HBE cells and mouse models, primarily by suppressing HBD1 expression. This effect can be counteracted with exogenous HBD1, with the downregulation mechanism involving the MAPK and NF-κB pathways. Our study endeavors to elucidate the pathogenesis of lung infections associated with PM2.5 exposure, providing a novel theoretical basis for the development of prevention and treatment strategies, with substantial clinical significance.

Hypoxia-induced activation of NDR2 underlies brain metastases from Non-Small Cell Lung Cancer

The molecular mechanisms induced by hypoxia are misunderstood in non-small cell lung cancer (NSCLC), and above all the hypoxia and RASSF1A/Hippo signaling relationship. We confirmed that human NSCLC (n = 45) as their brain metastases (BM) counterpart are hypoxic since positive with CAIX-antibody (target gene of Hypoxia-inducible factor (HIF)). A severe and prolonged hypoxia (0.2% O2, 48 h) activated YAP (but not TAZ) in Human Bronchial Epithelial Cells (HBEC) lines by downregulating RASSF1A/kinases Hippo (except for NDR2) regardless their promoter methylation status. Subsequently, the NDR2-overactived HBEC cells exacerbated a HIF-1A, YAP and C-Jun-dependent-amoeboid migration, and mainly, support BM formation. Indeed, NDR2 is more expressed in human tumor of metastatic NSCLC than in human localized NSCLC while NDR2 silencing in HBEC lines (by shRNA) prevented the xenograft formation and growth in a lung cancer-derived BM model in mice. Collectively, our results indicated that NDR2 kinase is over-active in NSCLC by hypoxia and supports BM formation. NDR2 expression is thus a useful biomarker to predict the metastases risk in patients with NSCLC, easily measurable routinely by immunohistochemistry on tumor specimens.

Airway epithelial Paraoxonase-2 in obese asthma.

BackgroundObesity in asthmatics has been associated with higher airway oxidative stress in which dysfunctional mitochondria are a potential contributing source of excess free radicals. Paraoxonase 2 (PON2) plays an important role in reducing mitochondrial-derived oxidative stress and could, therefore, have therapeutic potential in these patients.ObjectivesWe used primary human bronchial epithelial cells (HBECs) from asthmatics and healthy controls to evaluate: a) protein levels of Paraoxonase 2 and b) to test the potential protective effect of quercetin supplementation in cells under oxidative stress conditions.ResultsCompared to lean controls, obese asthmatics had significantly lower PON2 airway epithelial levels (respectively, 1.08 vs. 0.47 relative units normalized by GAPDH) (p-value < 0.006). Treating HBECs in vitro for 24 hrs. with 25μM quercetin significantly increased PON2 protein levels: 15.5 treated cells vs. 9.8 untreated cells (relative units normalized by GAPDH) (p value = 0.004). Notably, compared to untreated cells, quercetin supplementation reduces mitochondrial superoxide and hydrogen peroxide production on HBECs cells exposed to different oxidative stress triggers such as 1–2 Naphthoquinone (1–2 NQ) and hydrogen peroxide, suggesting that PON2 might play a protective role ameliorating oxidative injury on human airway epithelium.ConclusionCompared to lean controls, obese asthmatics have significantly reduced PON2 levels in airway epithelial cells. Treatment with quercetin in vitro increased PON2 protein levels and prevented oxidative stress from different types of stimuli. Hence, quercetin supplementation may be a potential therapeutic strategy to prevent obesity-mediated airway oxidative stress in obese asthmatics.

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.

Toxicity evaluation of particles formed during 3D-printing: Cytotoxic, genotoxic, and inflammatory response in lung and macrophage models

Additive manufacturing (AM) or “3D-printing” is a ground-breaking technology that enables the production of complex 3D parts. Its rapid growth calls for immediate toxicological investigations of possible human exposures in order to estimate occupational health risks. Several laser-based powder bed fusion AM techniques are available of which many use metal powder in the micrometer range as feedstock. Large energy input from the laser on metal powders generates several by-products, like spatter and condensate particles. Due to often altered physicochemical properties and composition, spatter and condensate particles can result in different toxicological responses compared to the original powder particles. The toxicity of such particles has, however, not yet been investigated. The aim of the present study was to investigate the toxicity of condensate/spatter particles formed and collected upon selective laser melting (SLM) printing of metal alloy powders, including a nickel-chromium-based superalloy (IN939), a nickel-based alloy (Hastelloy X, HX), a high-strength maraging steel (18Ni300), a stainless steel (316L), and a titanium alloy (Ti6Al4V). Toxicological endpoints investigated included cytotoxicity, generation of reactive oxygen species (ROS), genotoxicity (comet and micronucleus formation), and inflammatory response (cytokine/chemokine profiling) following exposure of human bronchial epithelial cells (HBEC) or monocytes/macrophages (THP-1). The results showed no or minor cytotoxicity in the doses tested (10−100 μg/mL). Furthermore, no ROS generation or formation of micronucleus was observed in the HBEC cells. However, an increase in DNA strand breaks (detected by comet assay) was noted in cells exposed to HX, IN939, and Ti6Al4V, whereas no evident release of pro-inflammatory cytokine was observed from macrophages. Particle and surface characterization showed agglomeration in solution and different surface oxide compositions compared to the nominal bulk content. The extent of released nickel was small and related to the nickel content of the surface oxides, which was largely different from the bulk content. This may explain the limited toxicity found despite the high Ni bulk content of several powders. Taken together, this study suggests relatively low acute toxicity of condensates/spatter particles formed during SLM-printing using IN939, HX, 18Ni300, 316L, and Ti6Al4V as original metal powders.

Abstract 2434: Transcriptional crosstalk between YAP, TEAD and TP63 is associated with early lung carcinogenesis

Abstract Introduction: The Hippo pathway effector YAP contributes to carcinogenesis by directing stem cell activity, cell proliferation and cell survival signals. In the lung, aberrant nuclear YAP activity promotes bronchial epithelial growth, which in animal models can drive pathology that resembles lung premalignant lesions (PMLs). However, the molecular mechanisms by which YAP contributes to aberrant lung epithelial pathology are poorly understood. In this project, we investigated the DNA binding pattern and gene expression program regulated by YAP and associated TEAD and TP63 transcriptional factors in primary normal human bronchial epithelial cells (HBECs) and lung cancer cell lines. Methods: RNA-seq experiments were conducted using HBEC, H2170 and SW900 cells transfected with siRNA targeting YAP, TEADs or TP63, and expression profiles were compared to corresponded controls to derive gene signatures. In parallel, ChIP-seq experiments were carried out using antibodies specific for the respective proteins to identify genome binding peaks. Direct target genes were inferred with Binding and Expression Target Analysis (BETA) using gene signature and peak profile of each factor in each cell line respectively. The metagene score for target genes in longitudinally collected endobronchial biopsies from patients with PMLs was calculated using GSVA and the association with PML phenotypes was tested with a mixed-effects model adjusting for batch and patient as random effect (GSE109743). Results: Chromatin binding analyses revealed that YAP co-occupies genomic regions with TEAD and TP63 in normal bronchial epithelial cells aligning with H3K27ac chromatin modifications. Integrating genomic binding and gene expression profiling, we identified large overlaps in target genes directly regulated by YAP, TEAD and TP63 in HBECs, demonstrating cooperation between these factors in the control of bronchial epithelial transcription. YAP-TEAD-TP63 induced targets in HBECs are enriched for genes involved in cell proliferation and extracellular matrix alteration, while repressed targets are enriched for genes involved in cell fate specification. Notably, cooperatively regulated genes are strongly associated with gene expression profile identified in progressive PML, and in particular with genes linked to early immune responses that are observed with PML pathology progression. Similar analyses of YAP, TEAD and TP63 in lung cancer cells revealed an altered transcriptional program for YAP with a divergence of YAP-TEAD away from TP63, suggesting contextual changes in YAP activity with cancer progression. Conclusion: We found that YAP, TP63 and TEADs co-regulate lung epithelial gene expression related to PML pathology progression. These findings provide insight into potential YAP function in the early stages of lung carcinogenesis and offer potential new avenues to develop lung cancer interception strategies. Citation Format: Boting Ning, Andrew Tilston-Lunel, Julia Hicks-Berthet, Sarah A. Mazzilli, Jennifer E. Beane, Xaralabos Varelas, Marc E. Lenburg. Transcriptional crosstalk between YAP, TEAD and TP63 is associated with early lung carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2434.

HSPA2 Chaperone Contributes to the Maintenance of Epithelial Phenotype of Human Bronchial Epithelial Cells but Has Non-Essential Role in Supporting Malignant Features of Non-Small Cell Lung Carcinoma, MCF7, and HeLa Cancer Cells.

Simple SummaryHeat shock proteins A (HSPA) are molecular chaperones that play a central role in the cellular protein quality control. One of them is HSPA2 which recently was indicated as a novel cancer-related protein due to its elevated expression in various tumors and reported prognostic significance. Several previous in vitro studies have shown significant role of HSPA2 in supporting cancer cells growth and invasiveness. Our data presented in this article contradict the current belief of the essential role of HSPA2 chaperone and show that HSPA2 is not crucial for maintenance of the malignant phenotype of lung, breast, and cervical cancer cells. Instead, we revealed HSPA2’s role in supporting clonogenic potential and adhesive ability of bronchial epithelial cells. Therefore, further research should concentrate on elucidating HSPA2 roles in epithelial cells.Heat Shock Protein A2 (HSPA2) is a member of the HSPA (HSP70) chaperone family and has a critical role for male fertility. HSPA2 is present in a number of somatic organs. Limited evidence suggests that HSPA2 may be involved in regulating epithelial cell differentiation. HSPA2 also emerged as a cancer-related chaperone; however, no consensus on its functional significance has been reached so far. In this study, we compared the phenotypic effects of HSPA2 deficit in non-transformed human bronchial epithelial cells (HBEC), and in lung, breast, and cervical cancer cells. We used various techniques to inhibit the HSPA2 gene expression in order to examine the impact of HSPA2 deficiency on cell growth, migration, adhesion, and invasion. Our results show that HBEC but not cancer cells are sensitive to HSPA2 deficit. HSPA2 knockdown in HBEC cells impaired their clone-forming ability and adhesiveness. Thus, our results indicate that epithelial cells can rely on a specific activity of HSPA2, but such dependence can be lost in epithelial cells that have undergone malignant transformation.

Effects of stomatin protein expression on proliferation and apoptosis of lung cancer cells

Objective: To investigate the effect of stomatin protein expression on the proliferation and apoptosis of lung cancer cells. Methods: The expressions of stomatin mRNA in human bronchial epithelial cells (HBE) and lung cancer cells (H520, A549, 95D, H460, Glc-82, 973 and H1299) were detected by Real-time PCR. Immunohistochemistry (IHC) was used to detect stomatin protein expression in 4 lung cancer tissue microarrays with 259 cases of lung cancer and adjacent normal tissues. After knocking down the expression of stomatin in A549 cells, the proliferation was detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, the apoptosis was detected by flow cytometry, the expression levels of total protein kinase B (AKT) and phosphorylated AKT at Ser473 were detected by Western blot. BALB/c nude mice were used to detect the tumorigenic ability of stomatin downregulated A549 cells (3 mice) and control cells (3 mice), and the protein expressions of stomatin, Ki67 and CD31 in tumor tissues were detected by IHC. Results: The M (range) of stomatin mRNA expression level in H520, A549, 95D, H460, Glc-82, 973, H1299 and HBE cells were 2.71 (2.66), 3.55 (3.16), 0.26 (0.22), 2.08 (1.98), 0.87 (0.35), 1.72 (2.53), 1.10 (1.82) and 0.01 (0.02), respectively. The mRNA expression levels of stomatin in H520, A549 and H460 cells were higher than that of HBE cells (all P<0.05), whereas there was no statistical difference among 95D, Glc-82, 973, H1299 and HBE cells (all P>0.05). IHC of lung tissue microarrays showed that the positive rate of stomatin expression in human lung cancer tissues was 34.7% (90/259), which was significantly higher than that in adjacent normal tissues [1.9% (5/259)] (P<0.05). In stomatin positive lung cancer tissues, the M (IQR) of tumor size for lower stomatin expression tissues (67 cases) was [41.22 (2 761.50) cm], which was smaller than that of higher stomatin expression tissues [(23 cases, 57.98(1 333.50) cm) (P<0.05). After knocking down stomatin expression, the fourth day absorbance value of stomatin-downregulated A549 cells was 0.55±0.07, which was lower than that of control cells (0.79±0.16) (P=0.012). The proportion of early apoptotic cells of stomatin-downregulated A549 cells [8.83 (53.00)] was higher than that of control cells [4.17 (25.00)] (P=0.026). The Ser473 phosphorylated AKT protein expression level in stomatin-downregulated A549 cells was 0.68±0.16, which was decreased compared with control cells (1.16±0.39) (P<0.05). The M (IQR) of total AKT expression level in stomatin-downregulated A549 cells was 4.25 (17.00), without statistically significant difference from control cells [4.75 (19.00)] (P>0.05). After inoculation of stomatin-downregulated A549 cells in nude mice for 43 days, the tumor volume was (37.93±3.12) mm(3), which was significantly smaller than that of the control group [(454.04±32.39) mm(3)] (P<0.001). And the expression levels of stomatin, nuclear proliferation antigen Ki67, and platelet-endothelial cell adhesion molecule CD31 were 1.78±0.69, 5.19±3.84, and 10.77±1.67, respectively, which were all decreased compared with control group (17.52±8.76, 54.14±41.02, and 19.72±6.97, respectively) (all P<0.05). Conclusion: Stomatin promotes lung cancer cell proliferation and inhibits cell early apoptosis by regulating AKT signaling pathway.

B24 The Role of Cigarette Smoke and miR520a in Pulmonary Frizzled 9 Expression

Lung cancer is the deadliest cancer, and for this reason treatment is highly researched. Alternatively, chemoprevention can be used to combat lung cancer in individuals who are at high risk of diagnosis, such as cigarette smokers. Frizzled 9 (Fzd9) is required in vitro for chemopreventive effects of iloprost, a prostacyclin analogue, in the lung. In non-small cell lung cancer (NSCLC) cell lines, Fzd9 activates PPARγ, leading to inhibition of transformed growth. Cigarette smoke exposure decreases Fzd9 expression. The goal of this study is to elucidate the relationship between cigarette smoke and miRNA regulation of Fzd9 expression. NSCLC cells exposed to cigarette smoke condensate (CSC) showed decreased Fzd9 3’ UTR activity, suggesting CSC regulates Fzd9 expression through miRNA. miRNA database analysis suggested miR-95, miR-106b, and miR-520a as potential regulators of Fzd9. Immortalized human bronchial epithelial cells (HBEC) and an Fzd9-positive NSCLC cell line (A549) transfected with miR-520a oligonucleotide mimic showed decreased Fzd9 3’UTR luciferase activity. miR-520a expression increased in HBEC and NSCLC cells after CSC exposure. We have tested a miR-520a inhibitor to use for future rescue experiments in an Fzd9-negative cell line (H322). Transient overexpression of miR-520a in HBEC and A549 did not affect cell viability or proliferation, so we made a stable miR-520a expressing HBEC line that we will use for longer-duration cell assays. miR-520a may play an important role in the regulation of Fzd9 by cigarette smoke, and future experiments will characterize this relationship and potentially impact the application of iloprost chemoprevention.

Kappa-carrageenan sinus rinses reduce inflammation and intracellular Staphylococcus aureus infection in airway epithelial cells.

Chronic rhinosinusitis (CRS) is a common disease, often refractory to conventional antimicrobial treatment. In this study we investigate the antimicrobial and anti-inflammatory effects of adding kappa-carrageenan to a commercially available sinus rinse. Kappa-carrageenan was added to Flo CRS and Flo Sinus Care sinus rinses and applied directly to air-liquid interface cultured primary human nasal epithelial cells (HNECs) from 10 CRS patients. Inflammatory markers were measured using enzyme-linked immunosorbent assay. Kappa-carrageenan-supplemented sinus rinses were applied to human bronchial epithelial cells (HBEs) in the presence of different Staphylococcus aureus strains to observe the effect on intracellular infection rates. Flo Sinus Care with kappa-carrageenan rinse solutions resulted in a marked reduction of interleukin-6 (IL-6) production by HNECs from CRS patients (p = 0.007). Both Flo CRS and Flo Sinus Care rinses significantly reduced the S aureus intracellular infection of HBEs (p < 0.0001). The addition of kappa-carrageenan to both Flo CRS and Flo Sinus Care rinses further reduced the intracellular infection rate by an average of 2%. The commonly used sinus irrigation product Flo Sinus Care with added kappa-carrageenan reduces IL-6 production by HNECs in vitro. Flo CRS and Flo Sinus Care rinses significantly reduced S aureus intracellular infection rates of HBE cells. Our findings may have clinical relevance for CRS patient management.

Interferon gamma induces inflammatory responses through the interaction of CEACAM1 and PI3K in airway epithelial cells

BackgroundInterferon gamma (IFNγ) plays an important role in the development of chronic lung diseases via the production of inflammatory mediators, although the exact mechanism remains unclear. The present study aimed at investigating the potential mechanisms by which IFNγ induced over-production of interleukins through the interaction between carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) pathway.MethodsIFN-γ induced over-production of interleukin (IL) 6 and IL8, and RNA expression of CEACAM1 and its subtypes or PI3K and its subtypes in human bronchial epithelial cells (HBE). The production of IL6 and IL8 or cell proliferation and movement were also evaluated in cellCEACAM1− or cellCEACAM1+ after the induction of IFN-γ. Roles of PI3K subtype proteins, e.g. PI3Kp110α/δ, Akt, p110α/γ/δ/β/mTOR, PI3Kp110α/δ/β, PI3Kp110δ, or pan-PI3K in IFN-γ-induced CEACAM1 subtype alterations were furthermore validated using those proteins of PI3K subtypes.ResultsCEACAM1, especially CEACAM1-S isoforms, was significantly up-regulated in HBE cells after treatment with IFN-γ. CEACAM1 played roles in expression of IL-6 and IL-8, and facilitated cellular proliferation and migration. IFN-γ up-regulated the expression of CEACAM1 in airway epithelial cells, especially CEACAM1-S isoforms, promoting cellular proliferation, migration, and the production of inflammatory factors. PI3K (p110δ)/Akt/mTOR pathway was involved in the process of IFN-γ-upregulated CEACAM1, especially CEACAM1-S. On the other hand, CEACAM1 could promote the activation of PI3K/Akt/mTOR pathway.ConclusionIFN-γ could induce inflammatory responses, cellular growth and proliferation through the interaction of CEACAM1 (especially CEACAM1-S isoforms) and PI3K(p110δ)/Akt/mTOR in airway epithelial cells, which might be new alternative of future therapies against epithelial transition from inflammation to cancer.

Airway epithelial cells increase macrophage chemotaxis and inflammatory cytokine secretion under hypoxic conditions

To investigate the effects of airway epithelial cells on macrophages chemotaxis and inflammatory cytokine expression under hypoxic conditions. Methods: Human bronchial epithelial cells (HBE) treated with different concentrations (0, 100, 200, 400, 800 μmol/L) of CoCl2 or transfected with HIF-1α siRNA were co-cultured with THP-1-derived M1 macrophages or M2 macrophages. The chemotactic effects on macrophages were analyzed by Transwell assay. The levels of TNF-α, IFN-γ, IL-4, IL-13 and IL-10 in the supernatants of macrophages were detected by ELISA, and HIF-1α or Cav-1 mRNA expression in HBE or macrophages was detected by RT-qPCR. Results: HBE cells promoted macrophages chemotaxis in a time- and concentration-dependent manner. Compared to un-transfected group, the chemotactic ability of HBE transfected with HIF-1α siRNA was significantly weakened (P<0.01). Under the same culture conditions, the chemotaxis of M2 macrophages was greater than that in THP1-derived M1 macrophages. The concentrations of TNF-α, IFN-γ, IL-4, IL-13 and IL-10 in the supernatants of macrophages were increased in a time-and concentration-dependent manner. The concentrations of TNF-α and IFN-γ were increased further after co-culturing for 8 and 12 h; while IL-4, IL-13 and IL-10 concentrations were increased further during 24 h of co-culture. The levels of cytokines in the supernatants of macrophages co-cultured with HBE and transfected with HIF-1α siRNA were significantly lower than those in un-transfected cells (P<0.05 or P<0.01). The reduction of TNF-α or IFN-γ was more obvious. The expression of HIF-1α or Cav-1 mRNA in HBE or macrophages was increased in a concentration-dependent manner after 8 or 12 h co-culture, which was significantly reduced when HBE was transfected with HIF-1α siRNA. Conclusion: Airway epithelial cells can enhance macrophages chemotaxis and pro-inflammatory cytokines expressions under hypoxic condition. HIF-1α and Cav-1 may be the important mediators in these processes.

In vitro and in vivo genotoxicity of oxygenated polycyclic aromatic hydrocarbons

Oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs) are a group of environmental pollutants found in complex mixtures together with PAHs. In contrast to the extensively studied PAHs, which have been established to have mutagenic and carcinogenic properties, much less is known about the effects of oxy-PAHs. The present work aimed to investigate the genotoxic potency of a set of environmentally relevant oxy-PAHs along with environmental soil samples in human bronchial epithelial cells (HBEC). We found that all oxy-PAHs tested induced DNA strand breaks in a dose-dependent manner and some of the oxy-PAHs further induced micronuclei formation. Our results showed weak effects in response to the oxy-PAH containing subfraction of the soil sample. The genotoxic potency was confirmed in both HBEC and HepG2 cells following exposure to oxy-PAHs by an increased level of phospho-Chk1, a biomarker used to estimate the carcinogenic potency of PAHs in vitro. We further exposed zebrafish embryos to single oxy-PAHs or a binary mixture with PAH benzo[a]pyrene (B[a]P) and found the mixture to induce comparable or greater effects on the induction of DNA strand breaks compared to the sum of that induced by B[a]P and oxy-PAHs alone. In conclusion, oxy-PAHs were found to elicit genotoxic effects at similar or higher levels to that of B[a]P which indicates that oxy-PAHs may contribute significantly to the total carcinogenic potency of environmental PAH mixtures. This emphasizes further investigations of these compounds as well as the need to include oxy-PAHs in environmental monitoring programs in order to improve health risk assessment.

Abstract 2015: Exosomes secreted by highly migratory premalignant lung epithelial cells promote epithelial mesenchymal transition and migration

Abstract Lung cancer is a highly metastatic disease. Contrary to the common belief that metastasis is a late event, recent findings have suggested that micrometastasis may also occur as an early phenomenon. An increasing body of evidence supports a role for epithelial-mesenchymal transition (EMT) in inducing field cancerization. Reports suggest that cancer-derived exosomes can act via intra-cellular communication to induce EMT and migration. However, the role of premalignant cell-derived exosomes in EMT induction, cellular migration and establishment of field cancerization in unknown. Herein, we describe the role of premalignant cell-derived exosomes on early metastatic behavior. Our laboratory has recently discovered a unique highly motile (HM) subpopulation of human bronchial epithelial cells (HBECs), using a novel “constricted migration” selection strategy, with enhanced metastatic potential in vivo. The HBECs used for selection were modified with changes reciprocating premalignancy (p53null, activated Kras-G12D). Comparative RNA-seq datasets illustrate increased expression of key EMT genes in HM-HBECs. This unique subpopulation of HM-HBECs offer a unique model to investigate premalignant cell migration. We used exosomes derived from HM, SM (slow motile) and unselected (UN) HBECs to evaluate EMT induction, migration, and invasion in vitro and in vivo. Exosomes were isolated by ultracentrifugation and characterized for size distribution using dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), electron microscopy, and characterized by western blotting with antibodies specific for Alix, Flotillin, CD9 and CD63. Exosome labeling for cell uptake studies was performed using Dil dye entrapment and by transient transfection with CD63-GFP, releasing GFP+ exosomes. 6X108 HBEC cells produced approximately 6 x 108 exosomes with size ranging from 50 to 130 nm. The exosome signature of HM-, SM-, and UN-HBECs were characterized by mass spectroscopy and micro RNA sequencing. Exosomes from HM-HBECs induce UN-HBECs to obtain a HM phenotype with enhanced EMT, faster migration and increased invasive capacity. Western blot and qPCR data suggest a time-dependent activation of an EMT signature. Further in vivo experiments are in progress to evaluate the role of HM-HBEC derived exosomes on metastatic homing, tumor formation and spread of premalignant HBEC cells and tumor cells. Our data suggest that premalignant cell-derived exosomes possess distinct molecular signatures and could potentially mediate early migration, resulting in the creation of a premalignant field. As such, exosomes afford novel targets in premalignancy for early intervention and cancer interception. Citation Format: Manash K. Paul, Suman Dutta, Bharti Bisht, Salehi-Rad Ramin, Paul Pagano, Gal Bitan, John D. Minna, Steven M. Dubinett. Exosomes secreted by highly migratory premalignant lung epithelial cells promote epithelial mesenchymal transition and migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2015.

Human bronchial epithelial cells as a good control for evaluation potential therapeutic Notch signaling in non-small cell lung cancer

Purpose: Notch signaling is often deregulated in non-small cell lung cancer (NSCLC), but little is known about the initial endogenous mRNA status of Notch ligands and receptors. Therefore, the aim of this study was to evaluate expression level of NOTCH1 receptor and Notch ligands, such as delta like ligands (DLL1, DLL3, DLL4), and jagged ligands (JAG1, JAG2), as well as target gene-hes family bHLH transcription factor 1 (HES1) in diverse NSCLC cell lines. Materials and Methods: We have investigated the mRNA expression of chosen genes by using quantitative real time method (RT-PCR). We compared the results from NSCLC cells with results obtained in non-cancerous human bronchial epithelial cells (HBEpC). We also measured NOTCH1 expression in A549 cells, before and after treatment with γ-secretase inhibitor (GSI). Results: The expression level of NOTCH1, HES1, JAG1 and JAG2 was downregulated when compared to HBEpC. The expression of Notch ligand DLL1 was lower in all cancer cell lines, but mRNA level of DLL3 was elevated in H1299 and A549 cells when related to HBEpC. The mRNA level of DLL4 was higher in H520 and in A549 cell lines. Moreover, the mRNA level of NOTCH1 dropped down after GSI treatment, in addition A549 cells proliferated slower after drug implementation. Conclusions: We conclude that non-cancerous HBEpC cells could serve as a good control for Notch mRNAs expression analysis in NSCLC. Moreover, GSI-treated cells could inhibit proliferation through suppressing NOTCH1 in A549 cells.

Overexpression of the base excision repair NTHL1 glycosylase causes genomic instability and early cellular hallmarks of cancer.

Base excision repair (BER), which is initiated by DNA N-glycosylase proteins, is the frontline for repairing potentially mutagenic DNA base damage. The NTHL1 glycosylase, which excises DNA base damage caused by reactive oxygen species, is thought to be a tumor suppressor. However, in addition to NTHL1 loss-of-function mutations, our analysis of cancer genomic datasets reveals that NTHL1 frequently undergoes amplification or upregulation in some cancers. Whether NTHL1 overexpression could contribute to cancer phenotypes has not yet been explored. To address the functional consequences of NTHL1 overexpression, we employed transient overexpression. Both NTHL1 and a catalytically-dead NTHL1 (CATmut) induce DNA damage and genomic instability in non-transformed human bronchial epithelial cells (HBEC) when overexpressed. Strikingly, overexpression of either NTHL1 or CATmut causes replication stress signaling and a decrease in homologous recombination (HR). HBEC cells that overexpress NTHL1 or CATmut acquire the ability to grow in soft agar and exhibit loss of contact inhibition, suggesting that a mechanism independent of NTHL1 catalytic activity contributes to acquisition of cancer-related cellular phenotypes. We provide evidence that NTHL1 interacts with the multifunctional DNA repair protein XPG suggesting that interference with HR is a possible mechanism that contributes to acquisition of early cellular hallmarks of cancer.

Genotoxic and mutagenic properties of Ni and NiO nanoparticles investigated by comet assay, γ-H2AX staining, Hprt mutation assay and ToxTracker reporter cell lines.

Nickel (Ni) compounds are classified as carcinogenic to humans but the underlying mechanisms are still poorly understood. Furthermore, effects related to nanoparticles (NPs) of Ni have not been fully elucidated. The aim of this study was to investigate genotoxicity and mutagenicity of Ni and NiO NPs and compare the effect to soluble Ni from NiCl2. We employed different models; i.e., exposure of (1) human bronchial epithelial cells (HBEC) followed by DNA strand break analysis (comet assay and γ‐H2AX staining); (2) six different mouse embryonic stem (mES) reporter cell lines (ToxTracker) that are constructed to exhibit fluorescence upon the induction of various pathways of relevance for (geno)toxicity and cancer; and (3) mES cells followed by mutagenicity testing (Hprt assay). The results showed increased DNA strand breaks (comet assay) for the NiO NPs and at higher doses also for the Ni NPs whereas no effects were observed for Ni ions/complexes from NiCl2. By employing the reporter cell lines, oxidative stress was observed as the main toxic mechanism and protein unfolding occurred at cytotoxic doses for all three Ni‐containing materials. Oxidative stress was also detected in the HBEC cells following NP‐exposure. None of these materials induced the reporter related to direct DNA damage and stalled replication forks. A small but statistically significant increase in Hprt mutations was observed for NiO but only at one dose. We conclude that Ni and NiO NPs show more pronounced (geno)toxic effects compared to Ni ions/complexes, indicating more serious health concerns. Environ. Mol. Mutagen. 59:211–222, 2018. © 2017 The Authors Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society

Flavone protects HBE cells from DNA double-strand breaks caused by PM2.5.

Ambient air particulate matter 2.5 (PM2.5) contains many harmful components that can enter the circulatory system and produce reactive oxygen species (ROS) in body. Oxidative stress and DNA damage induced by ROS may affect any cellular macromolecule and lead to DNA double-strand breaks (DSBs). Flavonoids, widely distributed in some herbs and berries, have been proved having anti-oxidative or anti-cancer efficacy. In this study, we investigated whether Flavone, a kind of flavonoids, can protect human bronchial epithelial cells (HBE) from DSBs caused by PM2.5 and how this function is probably implemented. We found that cells exposed to PM2.5 obviously induced viability inhibition, DNA damage and part of apoptosis. However, Flavone treatment prior to PM2.5 apparently improved cell viability, and mitigated the formation of 8-hydroxy-2-deoxyguanosine, the expression of DNA damage-relative protein and cell apoptosis. Our studies demonstrated that PM2.5 induced oxidative DSBs while Flavone ameliorated the DNA damage and increased cell viability probably through influencing DNA repair mechanism of cells.

Human primary airway epithelial cells isolated from active smokers have epigenetically impaired antiviral responses.

BackgroundCigarette smoking (CS) is the main risk factor for the development of chronic obstructive pulmonary disease (COPD) and most COPD exacerbations are caused by respiratory infections including influenza. Influenza infections are more severe in smokers. The mechanism of the increased risk and severity of infections in smokers is likely multifactorial, but certainly includes changes in immunologic host defenses.MethodsWe investigated retinoic acid-inducible protein I (RIG-I) and interferon (IFN) induction by influenza A virus (IAV) in human bronchial epithelial cells (HBEC) isolated from smokers or nonsmokers. Subcultured HBEC cells were infected with A/Puerto Rico/8/1934 (PR8) IAV at an MOI of 1. After 24 h of infection, cells and supernatants were collected for qRT-PCR, immunoblot or ELISA to determine RIG-I, Toll-like receptor3 (TLR3) and IFN expression levels.ResultsIAV exposure induced a vigorous IFN-β, IFN-λ 1 and IFN-λ 2/3 antiviral response in HBEC from nonsmokers and significant induction of RIG-I and TLR3. In cells from smokers, viral RIG-I and TLR3 mRNA induction was reduced 87 and 79 % compared to the response from nonsmokers. CS exposure history was associated with inhibition of viral induction of the IFN-β, IFN-λ1 and IFN-λ 2/3 mRNA response by 85, 96 and 95 %, respectively, from that seen in HBEC from nonsmokers. The demethylating agent 5-Aza-2-deoxycytidine reversed the immunosuppressive effects of CS exposure in HBEC since viral induction of all three IFNs was restored. IFN-β induction of RIG-I and TLR3 was also suppressed in the cells from smokers.ConclusionOur results suggest that active smoking reduces expression of antiviral cytokines in primary HBEC cells. This effect likely occurs via downregulation of RIG-I and TLR3 due to smoke-induced epigenetic modifications. Reduction in lung epithelial cell RIG-I and TLR3 responses may be a major mechanism contributing to the increased risk and severity of viral respiratory infections in smokers and to viral-mediated acute exacerbations of COPD.