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Sample records for alveolar epithelial a549

  1. Trichomonas vaginalis induces cytopathic effect on human lung alveolar basal carcinoma epithelial cell line A549.

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    Salvador-Membreve, Daile Meek C; Jacinto, Sonia D; Rivera, Windell L

    2014-12-01

    Trichomonas vaginalis, the causative agent of trichomoniasis is generally known to inhabit the genitourinary tract. However, several case reports with supporting molecular and immunological identifications have documented its occurrence in the respiratory tract of neonates and adults. In addition, the reports have documented that its occurrence is associated with respiratory failures. The medical significance or consequence of this association is unclear. Thus, to establish the possible outcome from the interaction of T. vaginalis with lung cells, the cytopathic effects of the parasites were evaluated using monolayer cultures of the human lung alveolar basal carcinoma epithelial cell line A549. The possible effect of association of T. vaginalis with A549 epithelial cells was analyzed using phase-contrast, scanning electron microscopy and fluorescence microscopy. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), crystal-violet and TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling) assays were conducted for cytotoxicity testing. The results demonstrate that T. vaginalis: (1) adheres to A549 epithelial cells, suggesting a density-dependent parasite-cell association; (2) adherence on A549 is through flagella, membrane and axostyle; (3) causes cell detachment and cytotoxicity (50-72.4%) to A549 and this effect is a function of parasite density; and (4) induces apoptosis in A549 about 20% after 6 h of incubation. These observations indicate that T. vaginalis causes cytopathic effects on A549 cell. To date, this is the first report showing a possible interaction of T. vaginalis with the lung cells using A549 monolayer cultures. Further studies are recommended to completely elucidate this association. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Ghrelin ameliorates the human alveolar epithelial A549 cell apoptosis induced by lipopolysaccharide

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    Huang, Chunrong; Zheng, Haichong; He, Wanmei; Lu, Guifang; Li, Xia [Department of Medical Intensive Care Unit, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080 (China); Deng, Yubin, E-mail: dengyub@mail.sysu.edu.cn [Research Center of Translational Medicine, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080 (China); Zeng, Mian, E-mail: zengmian2004@163.com [Department of Medical Intensive Care Unit, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080 (China)

    2016-05-20

    Ghrelin is a gastric acyl-peptide that plays an inhibitory role in cell apoptosis. Herein we investigate the protective effects of ghrelin in LPS-induced apoptosis of human alveolar epithelial A549 cells, along with the possible molecular mechanisms. LPS exposure impaired cell viability and increased apoptosis of A549 cells significantly in concentration- and time-dependent manners embodied in increased Bax and cleaved caspase-3 production, coupled with decreased Bcl-2 levels. Simultaneously, LPS remarkably decreased the expression of phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) and extracellular signal-regulated kinas (ERK) in A549 cells. However, ghrelin'pretreatment ameliorated LPS-caused alterations in the ratio of Bax/Bcl-2 and cleaved caspase-3 expression, whereas activated the PI3K/Akt and ERK signaling. These results demonstrate that ghrelin lightens LPS-induced apoptosis of human alveolar epithelial cells partly through activating the PI3K/Akt and ERK pathway and thereby might benefit alleviating septic ALI. -- Graphical abstract: Ghrelin ameliorates the human alveolar epithelial A549 cells apoptosis induced by lipopolysaccharide partly through activating the PI3K/Akt and ERK pathway. Display Omitted -- Highlights: •It has been observed that LPS insult significantly increased apoptosis in A549 cells. •Both Akt and ERK signaling are critical adapter molecules to mediate the ghrelin-mediated proliferative effect. •Ghrelin may have a therapeutic effect in the prevention of LPS-induced apoptosis.

  3. Cytotoxicity and gene array analysis of alveolar epithelial A549 cells exposed to paraquat.

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    Mitsopoulos, Panagiotis; Suntres, Zacharias E

    2010-12-05

    Paraquat (PQ), a commonly used herbicide, is highly toxic to humans and animals. The primary injury occurs in the lung, where PQ is actively taken up by alveolar epithelial cells and consequently produces damaging reactive oxygen species (ROS) via redox cycling. ROS have also been shown to induce expression of several early response genes and to activate transcription factors, which may contribute to the inflammatory response associated with PQ injury. In order to further elucidate the mechanism(s) of PQ injury, we investigated its effects on the cellular status and gene expression profile of immortalized human alveolar epithelial A549 cells in vitro. Incubation of cells with PQ resulted in concentration- and time-dependent PQ uptake, which correlated with increases in intracellular ROS levels and decreases in intracellular glutathione content, mitochondrial membrane potential, and cell viability. Gene array analysis showed differential expression in response to PQ exposure over time, particularly increases in: (i) the expression of growth arrest and cell cycle-related genes (e.g. CDKN1A, DDIT3 GADD45A, GDF15, MDM2, EGR1, CASP10, CASP8) and (ii) the expression of pro-inflammatory genes (e.g. IL1A, IL6, IL18, NFKB1, SERPINE1), which correlated with increases in the secretion of pro-inflammatory cytokines (e.g. IL-8, IL-6). These data suggest that uptake of PQ by A549 cells altered the cellular redox status and the expression of several early response genes, including the inflammatory response, all of which might contribute to the overall cytotoxicity of PQ. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  4. Differential Regulation of Gene Expression of Alveolar Epithelial Cell Markers in Human Lung Adenocarcinoma-Derived A549 Clones

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    Hiroshi Kondo

    2015-01-01

    Full Text Available Stem cell therapy appears to be promising for restoring damaged or irreparable lung tissue. However, establishing a simple and reproducible protocol for preparing lung progenitor populations is difficult because the molecular basis for alveolar epithelial cell differentiation is not fully understood. We investigated an in vitro system to analyze the regulatory mechanisms of alveolus-specific gene expression using a human alveolar epithelial type II (ATII cell line, A549. After cloning A549 subpopulations, each clone was classified into five groups according to cell morphology and marker gene expression. Two clones (B7 and H12 were further analyzed. Under serum-free culture conditions, surfactant protein C (SPC, an ATII marker, was upregulated in both H12 and B7. Aquaporin 5 (AQP5, an ATI marker, was upregulated in H12 and significantly induced in B7. When the RAS/MAPK pathway was inhibited, SPC and thyroid transcription factor-1 (TTF-1 expression levels were enhanced. After treatment with dexamethasone (DEX, 8-bromoadenosine 3′5′-cyclic monophosphate (8-Br-cAMP, 3-isobutyl-1-methylxanthine (IBMX, and keratinocyte growth factor (KGF, surfactant protein B and TTF-1 expression levels were enhanced. We found that A549-derived clones have plasticity in gene expression of alveolar epithelial differentiation markers and could be useful in studying ATII maintenance and differentiation.

  5. Genomic signature and toxicogenomics comparison of polycationic gene delivery nanosystems in human alveolar epithelial A549 cells

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    J Barar

    2009-10-01

    Full Text Available "nBackground and the purpose of the study: Of the gene delivery systems, non-viral polycationic gene delivery nanosystems have been alternatively exploited as a relatively safe delivery reagents compared to viral vectors. However, little is known about the genomic impacts of these delivery systems in target cells/tissues. In this study, the toxicogenomics and genotoxicity potential of some selected polycationic lipid/polymer based nanostructures (i.e., Oligofectamine® (OF, starburst polyamidoamine Polyfect® (PF and diaminobutane (DAB dendrimers were investigated in human alveolar epithelial A549 cells. "nMethods: To study the nature and the ontology of the gene expression changes in A549 cells upon treatment with polycationic nanostructures, MTT assay and microarray gene expression profiling methodology were employed. For microarray analysis, cyanine (Cy3/Cy5 labeled cDNA samples from treated and untreated cells were hybridized on target arrays housing 200 genes. "nResults and major conclusions: The polycationic nanosystems induced significant gene expression changes belonging to different genomic ontologies such as cell defence and apoptosis pathways. These data suggest that polycationic nanosystems can elicit multiple gene expression changes in A549 cells upon their chemical structures and interactions with cellular/subcellular components. Such impacts may interfere with the main goals of the desired genemedicine.

  6. Differential replication of avian influenza H9N2 viruses in human alveolar epithelial A549 cells

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    Peiris Malik

    2010-03-01

    Full Text Available Abstract Avian influenza virus H9N2 isolates cause a mild influenza-like illness in humans. However, the pathogenesis of the H9N2 subtypes in human remains to be investigated. Using a human alveolar epithelial cell line A549 as host, we found that A/Quail/Hong Kong/G1/97 (H9N2/G1, which shares 6 viral "internal genes" with the lethal A/Hong Kong/156/97 (H5N1/97 virus, replicates efficiently whereas other H9N2 viruses, A/Duck/Hong Kong/Y280/97 (H9N2/Y280 and A/Chicken/Hong Kong/G9/97 (H9N2/G9, replicate poorly. Interestingly, we found that there is a difference in the translation of viral protein but not in the infectivity or transcription of viral genes of these H9N2 viruses in the infected cells. This difference may possibly be explained by H9N2/G1 being more efficient on viral protein production in specific cell types. These findings suggest that the H9N2/G1 virus like its counterpart H5N1/97 may be better adapted to the human host and replicates efficiently in human alveolar epithelial cells.

  7. Water-pipe smoke condensate increases the internalization of Mycobacterium Bovis of type II alveolar epithelial cells (A549).

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    Mortaz, Esmaeil; Alipoor, Shamila D; Movassaghi, Masoud; Varahram, Mohammad; Ghorbani, Jahangir; Folkerts, Gert; Garssen, Johan; Adcock, Ian M

    2017-04-21

    Tuberculosis (TB) is a major global health problem, and there is an association between tobacco smoke and TB. Water pipe smoking has become an increasing problem not only in Middle Eastern countries but also globally because users consider it as safer than cigarettes. The presence of high levels of toxic substances in water-pipe smoke may be a predisposing factor that enhances the incidence of pulmonary disorders. For example, uncontrolled macropinocytosis in alveolar epithelial cells following exposure to water-pipe smoke may predispose subjects to pulmonary infection. Here, we studied the effects of water-pipe condense (WPC) on the internalization of Mycobacterium Bovis BCG by macropinocytosis in the alveolar epithelial cell line A549. A549 cells were exposed to WPC (4 mg/ml) for 24, 48, 72 and 96 h. Cell viability was studied using the methyl thiazolyldipenyl-tetrazolium bromide (MTT) reduction assay and proliferation by bromodeoxyUridine (BrdU) incorporation. Cells were exposed to FITC-Dextran (1 mg/ml) (as a control) and FITC-BCG (MOI = 10) for 20 min at 37 °C before cells were collected and the uptake of BCG-FITC determined by flow cytometry. Similar experiments were performed at 4 °C as a control. The Rho-associated protein kinase (ROCK) inhibitor Y-27632 (1 μM) was used to assess the mechanism by which WPC enhanced BCG uptake. WPC (4 mg/ml) increased the uptake of BCG-FITC after 72 (1.3 ± 0.1 fold, p WPC also significantly increased the uptake of FITC-Dextran (2.9 ± 0.3 fold, p WPC significantly decreased cell viability after 24 (84 ± 2%, p WPC. Cell proliferation showed a decreasing trend in a time-dependent manner with WPC exposure. WPC exposure increased epithelial cell endocytosis activity and death as well as enhancing their capacity for macropinocytosis. Our in vitro data indicates possible harmful effects of WPC on the ability of lung epithelial cells to phagocytose mycobacterium.

  8. TNF-α increases Staphylococcus aureus-induced death of human alveolar epithelial cell line A549 associated with RIP3-mediated necroptosis.

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    Wen, Shun-Hang; Lin, Luo-Na; Wu, Hu-Jun; Yu, Lu; Lin, Li; Zhu, Li-Li; Li, Hai-Yan; Zhang, Hai-Lin; Li, Chang-Chong

    2018-02-15

    To explore the role of tumor necrosis factor-alpha (TNF-α) on Staphylococcus aureus-induced necroptosis in alveolar epithelial cells. The A549 alveolar epithelial cell line was pretreated with small interfering RNA (siRNA) against receptor interacting protein-3 (RIP3) and then stimulated by S. aureus, where some cells were pretreated with TNF-α or TNF-α with anti-TNF-α antibody simultaneously. A549 cell death was assessed using lactate dehydrogenase (LDH) leakage and flow cytometry analyses. The protein expressions of RIP1, RIP3, cleaved caspase-1, and cleaved caspase-8 were analyzed by western blot. S. aureus-induced LDH release was increased significantly by TNF-α. In addition, flow cytometry showed that TNF-α increased A549 cell apoptosis and necrosis in S. aureus-infected cell cultures. Levels of RIP3 and cleaved caspase-1 protein in A549 cells infected with S. aureus increased at 12 h post-infection, as shown by western blot. Significant additional increases in RIP3 expression were observed following the addition of TNF-α. Decreasing RIP3 levels by siRNA significantly suppressed the release of LDH induced by TNF-α and S. aureus. RIP3 siRNA also significantly suppressed A549 cell necrosis induced by S. aureus and TNF-α at 6 and 12 h post-infection as shown by flow cytometry analysis. TNF-α enhances the damage of S. aureus on lung epithelial cells, and its mechanism is associated with RIP3 mediated necroptosis. Copyright © 2018 Elsevier Inc. All rights reserved.

  9. Andrographolide antagonizes cigarette smoke extract-induced inflammatory response and oxidative stress in human alveolar epithelial A549 cells through induction of microRNA-218.

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    Li, Ying-jie; Yu, Chang-hai; Li, Jing-bo; Wu, Xi-ya

    2013-12-01

    Andrographolide is a major bioactive labdane diterpenoid isolated from Andrographis paniculata and has protective effects against cigarette smoke (CS)-induced lung injury. This study was done to determine whether such protective effects were mediated through modulation of microRNA (miR)-218 expression. Therefore, we exposed human alveolar epithelial A549 cells to cigarette smoke extract (CSE) with or without andrographolide pretreatment and measured the level of glutathione, nuclear factor-kappaB (NF-κB) activation, proinflammatory cytokine production, and miR-218 expression. We found that andrographolide pretreatment significantly restored the glutathione level in CSE-exposed A549 cells, coupled with reduced inhibitor κB (IκB)-α phosphorylation and p65 nuclear translocation and interleukin (IL)-8 and IL-6 secretion. The miR-218 expression was significantly upregulated by andrographolide pretreatment. To determine the biological role of miR-218, we overexpressed and downregulated its expression using miR-218 mimic and anti-miR-218 inhibitor, respectively. We observed that miR-218 overexpression led to a marked reduction in IκB-α phosphorylation, p65 nuclear accumulation, and NF-κB-dependent transcriptional activity in CSE-treated A549 cells. In contrast, miR-218 silencing enhanced IκB-α phosphorylation and p65 nuclear accumulation in cells with andrographolide pretreatment and reversed andrographolide-mediated reduction of IL-6 and IL-8 production. In addition, depletion of miR-218 significantly reversed the upregulation of glutathione levels in A549 cells by andrographolide. Taken together, our results demonstrate that andrographolide mitigates CSE-induced inflammatory response in A549 cells, largely through inhibition of NF-κB activation via upregulation of miR-218, and thus has preventive benefits in CS-induced inflammatory lung diseases.

  10. Inflammatory effects induced by selected limonene oxidation products: 4-OPA, IPOH, 4-AMCH in human bronchial (16HBE14o-) and alveolar (A549) epithelial cell lines.

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    Lipsa, Dorelia; Leva, Paolo; Barrero-Moreno, Josefa; Coelhan, Mehmet

    2016-11-16

    Limonene, a monoterpene abundantly present in most of the consumer products (due to its pleasant citrus smell), easily undergoes ozonolysis leading to several limonene oxidation products (LOPs) such as 4-acetyl-1-methylcyclohexene (4-AMCH), 4-oxopentanal (4-OPA) and 3-isopropenyl-6-oxoheptanal (IPOH). Toxicological studies have indicated that human exposure to limonene and ozone can cause adverse airway effects. However, little attention has been paid to the potential health impact of specific LOPs, in particular of IPOH, 4-OPA and 4-AMCH. This study evaluates the cytotoxic effects of the selected LOPs on human bronchial epithelial (16HBE14o-) and alveolar epithelial (A549) cell lines by generating concentration-response curves using the neutral red uptake assay and analyzing the inflammatory response with a series of cytokines/chemokines. The cellular viability was mostly reduced by 4-OPA [IC 50 =1.6mM (A549) and 1.45mM (16HBE14o-)] when compared to IPOH [IC 50 =3.5mM (A549) and 3.4mM (16HBE14o-)] and 4-AMCH [IC 50 could not be calculated]. As a result from the inflammatory response, IPOH [50μM] induced an increase of both IL-6 and IL-8 secretion in A549 (1.5-fold change) and in 16HBE14o- (2.8- and 7-fold change respectively). 4-OPA [50μM] treatment of A549 increased IL-6 (1.4-times) and IL-8 (1.3-times) levels, while in 16HBE14o- had an opposite effect. A549 treated with 4-AMCH [50μM] elevate both IL-6 and IL-8 levels by 1.2-times, while in 16HBE14o- had an opposite effect. Based on our results, lung cellular injury characterized by inflammatory cytokine release was observed for both cell lines treated with the selected chemicals at concentrations that did not affect their cellular viability. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  11. Puerarin protects against Staphylococcus aureus-induced injury of human alveolar epithelial A549 cells via downregulating alpha-hemolysin secretion.

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    Tang, Feng; Li, Wen-Hua; Zhou, Xuan; Liu, Yong-Hua; Li, Zhe; Tang, Yu-Shun; Kou, Xu; Wang, Shu-De; Bao, Min; Qu, Lian-Da; Li, Min; Li, Bing

    2014-08-01

    Alpha-hemolysin, a secreted pore-forming toxin, plays an indispensable role in the pathogenicity of Staphylococcus aureus. In this study, the antimicrobial activity of puerarin against S. aureus was investigated; as a result, puerarin showed no influence on the growth of this organism. However, hemolysis and western blotting assays showed that puerarin concentration dependently inhibited the secretion of alpha-hemolysin at low concentrations. Real-time RT-PCR assay was further employed to evaluate the transcriptional level of hla, the gene encoding alpha-hemolysin, and RNAIII, an effector molecule of the agr system. The results indicated that the RNAIII expression and subsequent hla transcription were also inhibited by puerarin in a dose-dependent manner. Furthermore, puerarin significantly prevented human alveolar epithelial A549 cells from S. aureus-induced injury. Thereby, puerarin may be considered as a potential candidate for the development of antivirulence drugs in the treatment of S. aureus-mediated infections.

  12. Toxic Effects of the Major Components of Diesel Exhaust in Human Alveolar Basal Epithelial Cells (A549

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    Pavel Rossner

    2016-08-01

    Full Text Available We investigated the toxicity of benzo[a]pyrene (B[a]P, 1-nitropyrene (1-NP and 3-nitrobenzanthrone (3-NBA in A549 cells. Cells were treated for 4 h and 24 h with: B[a]P (0.1 and 1 μM, 1-NP (1 and 10 μM and 3-NBA (0.5 and 5 μM. Bulky DNA adducts, lipid peroxidation, DNA and protein oxidation and mRNA expression of CYP1A1, CYP1B1, NQO1, POR, AKR1C2 and COX2 were analyzed. Bulky DNA adducts were induced after both treatment periods; the effect of 1-NP was weak. 3-NBA induced high levels of bulky DNA adducts even after 4-h treatment, suggesting rapid metabolic activation. Oxidative DNA damage was not affected. 1-NP caused protein oxidation and weak induction of lipid peroxidation after 4-h incubation. 3-NBA induced lipid peroxidation after 24-h treatment. Unlike B[a]P, induction of the aryl hydrocarbon receptor, measured as mRNA expression levels of CYP1A1 and CYP1B1, was low after treatment with polycyclic aromatic hydrocarbon (PAH nitro-derivatives. All test compounds induced mRNA expression of NQO1, POR, and AKR1C2 after 24-h treatment. AKR1C2 expression indicates involvement of processes associated with reactive oxygen species generation. This was supported further by COX2 expression induced by 24-h treatment with 1-NP. In summary, 3-NBA was the most potent genotoxicant, whereas 1-NP exhibited the strongest oxidative properties.

  13. [Effect of Polydatin on Epithelial-Mesenchymal Transition of Human Alveolar Epithelium A549 Cells Induced by TGF-β1].

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    Yang, Jun-chao; Xu, Lu; Song, Kang; Wang, Yuan; Gao, Run-di; Chen, Rui-lin; Cao, Yu

    2016-04-01

    To explore the effect of polydatin on the growth of TGF-β₁induced humanalveolar epithelium A549 cells and the mechanism of polydatin for inhibiting the process of epithelial-mesenchymal transition (EMT). A549 cells in vitro cultured were randomly divided into five groups, i.e., the blank group, the control group, the low dose polydatin group, the middle dose polydatin group, the high dose polydatin group. Common culture fluid was added in A549 cells of the blank group. Five ng/mLTGF-β₁contained culture fluid was added in A549 cells of the control group. 50, 100, and 150 μmol/mL of polydatin plus 5 ng/mL TGF-β₁contained culture fluid was added in A549 cells of low, middle, and high dosepolydatin groups, respectively. Morphological changes were observed and recorded at different time points. The optimal concentration of polydatin was determined by MTT method. Protein and mRNA expressions of E-cad epithelial cell marker) and Vimentin (mesenchymal cell marker) were detected by Western blot and Real-time PCR. Under inverted phase contrast microscope, A549 cells turned from previous pebble shape to fusiform shape after intervened by polydatin and TGF-β1. The intercellular space was enlargedand the intercellular connection became loose. These phenomena were more obviously seen in the control group. A549 cells were more satiated in low, middle, and high dose polydatin groups than in the control group. The EMT inhibition was most obviously seen in the middle dose polydatin group at 48 h. Protein and mRNA expressions of E-cad showed an overall descending tendency after intervened by polydatin and TGF-β1 (P A549 cells time- and dose-dependently. It also played roles in inhibiting pulmonary fibrosis.

  14. In vitro effects of water-pipe smoke condensate on the endocytic activity of Type II alveolar epithelial cells (A549 with bacillus Calmette–Guérin

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    Ian M Adcock

    2016-01-01

    Conclusion: WPC exposure increased epithelial cells' permeability and death and enhanced their capacity for macropinocytosis. Our in vitro data suggest possible harmful effects of WPC on the ability of lung epithelial cells to phagocytose mycobacteria. Further studies will be conducted to understand the mechanism of action of WPC.

  15. Alveolar epithelial cells (A549) exposed at the air-liquid interface to diesel exhaust: First study in TNO's powertrain test center

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    Kooter, I.M.; Alblas, M.J.; Jedynska, A.D.; Steenhof, M.; Houtzager, M.M.G.; Ras, M.G. van

    2013-01-01

    Air–liquid interface (ALI) exposures enable in vitro testing ofmixtures of gases and particles such as diesel exhaust (DE). The main objective of this study was to investigate the feasibility of exposing human lung epithelial cells at the ALI to complete DE generated by a heavy-duty truck in the

  16. Paracrine control of differentiation in the alveolar carcinoma, A549, by human foetal lung fibroblasts.

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    Speirs, V; Ray, K P; Freshney, R I

    1991-10-01

    Synthesis of pulmonary surfactant (PS) is necessary for normal functioning of the lungs and its production is indicative of normal differentiated lung. The human alveolar carcinoma, A549, has been found to synthesis and secrete PS in vitro. The purpose of this study was to optimise the culture conditions for PS synthesis by A549 as well as to determine the potential role of foetal lung fibroblasts in the induction of PS by glucocorticoids. A549 cells growing in filter wells produced higher levels of PS in response to steroid, a 5-fold increase on the filter well compared to only a 1.5-fold increase when the cells were cultured on a conventional plastic substrate. A549 cells grown in filter wells responded to coculture with fibroblasts whether in direct contact or separated co-culture. A 20-fold increase in PS over control values was observed in separated steroid-treated co-cultures, suggesting the presence of a diffusible factor. A partially purified factor was isolated from fibroblast conditioned medium which was capable of inducing differentiation and other phenotypic changes in A549, namely induction of PS, reduction of plasminogen activator activity and reduction in the in vivo growth of A549 xenografts in nude mice. These results suggest that, under the correct conditions, A549 cells, although transformed, still retain the capacity to respond to differentiation-inducing signals from normal fibroblasts.

  17. High throughput determination of TGFβ1/SMAD3 targets in A549 lung epithelial cells.

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    Yingze Zhang

    Full Text Available BACKGROUND: Transforming growth factor beta 1 (TGFβ1 plays a major role in many lung diseases including lung cancer, pulmonary hypertension, and pulmonary fibrosis. TGFβ1 activates a signal transduction cascade that results in the transcriptional regulation of genes in the nucleus, primarily through the DNA-binding transcription factor SMAD3. The objective of this study is to identify genome-wide scale map of SMAD3 binding targets and the molecular pathways and networks affected by the TGFβ1/SMAD3 signaling in lung epithelial cells. METHODOLOGY: We combined chromatin immunoprecipitation with human promoter region microarrays (ChIP-on-chip along with gene expression microarrays to study global transcriptional regulation of the TGFβ1/SMAD3 pathway in human A549 alveolar epithelial cells. The molecular pathways and networks associated with TGFβ1/SMAD3 signaling were identified using computational approaches. Validation of selected target gene expression and direct binding of SMAD3 to promoters were performed by quantitative real time RT-PCR and electrophoretic mobility shift assay on A549 and human primary lung epithelial cells. RESULTS AND CONCLUSIONS: Known TGFβ1 target genes such as SERPINE1, SMAD6, SMAD7, TGFB1 and LTBP3, were found in both ChIP-on-chip and gene expression analyses as well as some previously unrecognized targets such as FOXA2. SMAD3 binding of FOXA2 promoter and changed expression were confirmed. Computational approaches combining ChIP-on-chip and gene expression microarray revealed multiple target molecular pathways affected by the TGFβ1/SMAD3 signaling. Identification of global targets and molecular pathways and networks associated with TGFβ1/SMAD3 signaling allow for a better understanding of the mechanisms that determine epithelial cell phenotypes in fibrogenesis and carcinogenesis as does the discovery of the direct effect of TGFβ1 on FOXA2.

  18. Interaction of airway epithelial cells (A549) with spores and mycelium of Aspergillus fumigatus

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    Zhang, ZH; Liu, RY; Noordhoek, JA; Kauffman, HF

    2005-01-01

    Objective. To study the interaction of airway epithelial cell line A549 with fragments of mycelium, spores of Aspergitlus fumigatus in vitro and to determine if toll-like receptors (TLRs) are involved in the process. Methods. A549 cells were exposed to fragments of A. fumigatus mycelium, zymosan and

  19. Human lung epithelial cells A549 epithelial-mesenchymal transition induced by PVA/Collagen nanofiber.

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    Li, Xiuchun; Yan, Shanshan; Dai, Jing; Lu, Yi; Wang, Yiqun; Sun, Man; Gong, Jinkang; Yao, Yuan

    2018-02-01

    Epithelial-mesenchymal transition (EMT) is a process by which epithelial cells lose their cell-cell contact to become mesenchymal stem cells, which is important on development and embryogenesis, wound healing, and cancer metastasis. This research aims to investigate the effect of topological cue as modulating factor on the EMT by tuning the diameter of electrospinning nanofiber. The cell-nanofiber interaction between human lung epithelial cell A549 and electrospinning nanofibers composed of polyvinyl alcohol (PVA) and type I collagen were investigated. The electrospinning of regenerated PVA/Collagen nanofibers were performed with water/acetic acid as a spinning solvent and glutaraldehyde as a chemical cross-linker. Parameterization on concentration, applied voltage and feeding rate was finalized to generate smooth nanofibers with good homogeneity. The scanning electron microscopy result demonstrated that A549 cell appropriately achieved extended morphology by the filopodia attaching to the surface of the nanofibrous mats. When the diameter changed from 90nm to 240nm, the A549 cell was correspondingly express varied EMT related genes. Gene expression analysis was conducted by qPCR using three typical markers for detecting EMT: N-cadherin (NCad), Vimentin (Vim), and Fibronectin (Fib). An increasing expression pattern was observed on cell culturing on 170nm sample with respect to cell cultured on 90nm and 240nm. This result indicated the 170nm PVA/Collagen nanofibers induce A549 cells to process epithelial-mesenchymal transition more seriously than those on 90nm or 240nm. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Alveolocapillary model system to study alveolar re-epithelialization

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    Willems, Coen H.M.P.; Zimmermann, Luc J.I.; Sanders, Patricia J.L.T.; Wagendorp, Margot; Kloosterboer, Nico [Department of Paediatrics, School for Oncology and Developmental Biology (GROW), Maastricht University Medical Centre, Maastricht (Netherlands); Cohen Tervaert, Jan Willem [Division of Clinical and Experimental Immunology, Department of Internal Medicine, Maastricht University Medical Centre, Maastricht (Netherlands); Duimel, Hans J.Q.; Verheyen, Fons K.C.P. [Electron Microscopy Unit, Department of Molecular Cell Biology, Maastricht University Medical Centre, Maastricht (Netherlands); Iwaarden, J. Freek van, E-mail: f.vaniwaarden@maastrichtuniversity.nl [Department of Paediatrics, School for Oncology and Developmental Biology (GROW), Maastricht University Medical Centre, Maastricht (Netherlands)

    2013-01-01

    In the present study an in vitro bilayer model system of the pulmonary alveolocapillary barrier was established to investigate the role of the microvascular endothelium on re-epithelialization. The model system, confluent monolayer cultures on opposing sides of a porous membrane, consisted of a human microvascular endothelial cell line (HPMEC-ST1.6R) and an alveolar type II like cell line (A549), stably expressing EGFP and mCherry, respectively. These fluorescent proteins allowed the real time assessment of the integrity of the monolayers and the automated analysis of the wound healing process after a scratch injury. The HPMECs significantly attenuated the speed of re-epithelialization, which was associated with the proximity to the A549 layer. Examination of cross-sectional transmission electron micrographs of the model system revealed protrusions through the membrane pores and close contact between the A549 cells and the HPMECs. Immunohistochemical analysis showed that these close contacts consisted of heterocellular gap-, tight- and adherens-junctions. Additional analysis, using a fluorescent probe to assess gap-junctional communication, revealed that the HPMECs and A549 cells were able to exchange the fluorophore, which could be abrogated by disrupting the gap junctions using connexin mimetic peptides. These data suggest that the pulmonary microvascular endothelium may impact the re-epithelialization process. -- Highlights: ► Model system for vital imaging and high throughput screening. ► Microvascular endothelium influences re-epithelialization. ► A549 cells form protrusions through membrane to contact HPMEC. ► A549 cells and HPMECs form heterocellular tight-, gap- and adherens-junctions.

  1. Proteinase-activated receptor 4 stimulation-induced epithelial-mesenchymal transition in alveolar epithelial cells

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    Araki Hiromasa

    2007-04-01

    Full Text Available Abstract Background Proteinase-activated receptors (PARs; PAR1–4 that can be activated by serine proteinases such as thrombin and neutrophil catepsin G are known to contribute to the pathogenesis of various pulmonary diseases including fibrosis. Among these PARs, especially PAR4, a newly identified subtype, is highly expressed in the lung. Here, we examined whether PAR4 stimulation plays a role in the formation of fibrotic response in the lung, through alveolar epithelial-mesenchymal transition (EMT which contributes to the increase in myofibroblast population. Methods EMT was assessed by measuring the changes in each specific cell markers, E-cadherin for epithelial cell, α-smooth muscle actin (α-SMA for myofibroblast, using primary cultured mouse alveolar epithelial cells and human lung carcinoma-derived alveolar epithelial cell line (A549 cells. Results Stimulation of PAR with thrombin (1 U/ml or a synthetic PAR4 agonist peptide (AYPGKF-NH2, 100 μM for 72 h induced morphological changes from cobblestone-like structure to elongated shape in primary cultured alveolar epithelial cells and A549 cells. In immunocytochemical analyses of these cells, such PAR4 stimulation decreased E-cadherin-like immunoreactivity and increased α-SMA-like immunoreactivity, as observed with a typical EMT-inducer, tumor growth factor-β (TGF-β. Western blot analyses of PAR4-stimulated A549 cells also showed similar changes in expression of these EMT-related marker proteins. Such PAR4-mediated changes were attenuated by inhibitors of epidermal growth factor receptor (EGFR kinase and Src. PAR4-mediated morphological changes in primary cultured alveolar epithelial cells were reduced in the presence of these inhibitors. PAR4 stimulation increased tyrosine phosphorylated EGFR or tyrosine phosphorylated Src level in A549 cells, and the former response being inhibited by Src inhibitor. Conclusion PAR4 stimulation of alveolar epithelial cells induced epithelial

  2. Anti-inflammatory effects of embelin in A549 cells and human asthmatic airway epithelial tissues.

    Science.gov (United States)

    Lee, In-Seung; Cho, Dong-Hyuk; Kim, Ki-Suk; Kim, Kang-Hoon; Park, Jiyoung; Kim, Yumi; Jung, Ji Hoon; Kim, Kwanil; Jung, Hee-Jae; Jang, Hyeung-Jin

    2018-02-01

    Allergic asthma is the most common type in asthma, which is defined as a chronic inflammatory disease of the lung. In this study, we investigated whether embelin (Emb), the major component of Ardisia japonica BL. (AJB), exhibits anti-inflammatory effects on allergic asthma via inhibition of NF-κB activity using A549 cells and asthmatic airway epithelial tissues. Inflammation was induced in A549 cells, a human airway epithelial cell line, by IL-1β (10 ng/ml) treatment for 4 h. The effects of Emb on NF-κB activity and COX-2 protein expression in inflamed airway epithelial cells and human asthmatic airway epithelial tissues were analyzed via western blot. The secretion levels of NF-κB-mediated cytokines/chemokines, including IL-4, 6, 9, 13, TNF-α and eotaxin, were measured by a multiplex assay. Emb significantly blocked NF-κB activity in IL-1β-treated A549 cells and human asthmatic airway epithelial tissues. COX-2 expression was also reduced in both IL-1β-treated A549 cells and asthmatic tissues Emb application. Emb significantly reduced the secretion of IL-4, IL-6 and eotaxin in human asthmatic airway epithelial tissues by inhibiting activity of NF-κB. The results of this study suggest that Emb may be used as an anti-inflammatory agent via inhibition of NF-κB and related cytokines.

  3. Epithelial mesenchymal transition of non-small-cell lung cancer cells A549 induced by SPHK1.

    Science.gov (United States)

    Ni, Min; Shi, Xiao-Lei; Qu, Zhi-Gang; Jiang, Hong; Chen, Zi-Qian; Hu, Jun

    2015-02-01

    To explore the effect and molecular mechanism of SPHK1 in the invasion and metastasis process of non-small-cell lung cancer cells (A549). Recombinant retrovirus was used to mediate the production of A549/vector, A549/SPHK1, A549/scramble, and A549/SPHKl/RNAi that stably expressed or silenced SPHK1. The invasion and migration capacities of A549 cells overexpressing or silencing SPHK1 were determined using Transwell invasion assay and scratch wound repair experiment. The protein and mRNA expression levels of E-cadherin, fibronectin, vimentin in A549/vector, A549/SPHK1, A549/scramble, A549/SPHK1/RNAi were detected with Western blot (WB) and quantitative PCR (QPCR) methods, respectively. Transwell invasion assay and scratch wound repair experiments showed that over-expression of SPHK1 obviously enhanced the invasion and migration capacities of A549 cells. WB and QPCR detection results showed that, the expression of E-cadherin (a molecular marker of epithelial cells) and fibronectin, vimentin (molecular markers of mesenchymal cells) in A549 cells was upregulated after overexpression of SPHK1; while SPHK1 silencing significantly reduced the invasion and metastasis capacities of A549 cells, upregulated the expression of molecular marker of epithelial cells, and downregulated the expression of molecular marker of mesenchymal cells. SPHK1 promotes epithelial mesenchymal transition of non-small-cell lung cancer cells and affects the invasion and metastasis capacities of these cells. Copyright © 2015 Hainan Medical College. Production and hosting by Elsevier B.V. All rights reserved.

  4. Modulation of epithelial sodium channel in human alveolar epithelial ...

    African Journals Online (AJOL)

    Modulation of epithelial sodium channel in human alveolar epithelial cells by lipoxin A4 through AhR-cAMP-dependent pathway. Bi-Huan Cheng, Li-Wei Pan, Sheng-Rong Zhang, Bin-Yu Ying, Ben-Ji Wang, Guo-Liang Lin, Shi-Fang Ding ...

  5. Cell stress induces upregulation of osteopontin via the ERK pathway in type II alveolar epithelial cells.

    Directory of Open Access Journals (Sweden)

    Aki Kato

    Full Text Available Osteopontin (OPN is a multifunctional protein that plays important roles in cell growth, differentiation, migration and tissue fibrosis. In human idiopathic pulmonary fibrosis and murine bleomycin-induced lung fibrosis, OPN is upregulated in type II alveolar epithelial cells (AEC II. However, the mechanism of OPN induction in AEC II is not fully understood. In this study, we demonstrate the molecular mechanism of OPN induction in AEC II and elucidate the functions of OPN in AEC II and lung fibroblasts. Human lung adenocarcinoma cells (A549 and mouse alveolar epithelial cells (MLE12, used as type II alveolar epithelial cell lines for in vitro assays, and human pulmonary alveolar epithelial cells (HPAEpiC were treated with either bleomycin, doxorubicin or tunicamycin. The mechanism of OPN induction in these cells and its function as a pro-fibrotic cytokine on A549 and lung fibroblasts were analyzed. The DNA damaging reagents bleomycin and doxorubicin were found to induce OPN expression in A549, MLE12 and HPAEpiC. OPN expression was induced via activation of the extracellular signal-regulated protein kinase (ERK-dependent signaling pathway in A549 and MLE12. The endoplasmic reticulum (ER stress-inducing reagent tunicamycin induced OPN mRNA expression in A549, MLE12 and HPAEpiC, and OPN mRNA expression was induced via activation of the ERK-dependent signaling pathway in A549 and MLE12. Another ER stress-inducing reagent thapsigargin induced the expression of OPN mRNA as well as the subsequent production of OPN in A549 and MLE12. Furthermore, OPN promoted the proliferation of A549 and the migration of normal human lung fibroblasts. Inhibition of OPN by small interference RNA or neutralizing antibody suppressed both of these responses. The results of this study suggest that cell stress induces the upregulation of OPN in AEC II by signaling through the ERK pathway, and that upregulated OPN may play a role in fibrogenesis of the lung.

  6. Preprocessing with Photoshop Software on Microscopic Images of A549 Cells in Epithelial-Mesenchymal Transition.

    Science.gov (United States)

    Ren, Zhou-Xin; Yu, Hai-Bin; Shen, Jun-Ling; Li, Ya; Li, Jian-Sheng

    2015-06-01

    To establish a preprocessing method for cell morphometry in microscopic images of A549 cells in epithelial-mesenchymal transition (EMT). Adobe Photoshop CS2 (Adobe Systems, Inc.) was used for preprocessing the images. First, all images were processed for size uniformity and high distinguishability between the cell and background area. Then, a blank image with the same size and grids was established and cross points of the grids were added into a distinct color. The blank image was merged into a processed image. In the merged images, the cells with 1 or more cross points were chosen, and then the cell areas were enclosed and were replaced in a distinct color. Except for chosen cellular areas, all areas were changed into a unique hue. Three observers quantified roundness of cells in images with the image preprocess (IPP) or without the method (Controls), respectively. Furthermore, 1 observer measured the roundness 3 times with the 2 methods, respectively. The results between IPPs and Controls were compared for repeatability and reproducibility. As compared with the Control method, among 3 observers, use of the IPP method resulted in a higher number and a higher percentage of same-chosen cells in an image. The relative average deviation values of roundness, either for 3 observers or 1 observer, were significantly higher in Controls than in IPPs (p Photoshop, a chosen cell from an image was more objective, regular, and accurate, creating an increase of reproducibility and repeatability on morphometry of A549 cells in epithelial to mesenchymal transition.

  7. Modulation of epithelial sodium channel in human alveolar epithelial ...

    African Journals Online (AJOL)

    Modulation of epithelial sodium channel in human alveolar epithelial cells by lipoxin A4 through AhR-cAMP-dependent pathway. Bi-Huan Cheng1,2, Li-Wei Pan2, Sheng-Rong Zhang3, Bin-Yu Ying2, Ben-Ji. Wang2, Guo-Liang Lin2 and Shi-Fang Ding1*. 1Department of Critical Care Medicine, Qilu Hospital of Shandong ...

  8. An Optimised Human Cell Culture Model for Alveolar Epithelial Transport

    Science.gov (United States)

    Birch, Nigel P.; Suresh, Vinod

    2016-01-01

    Robust and reproducible in vitro models are required for investigating the pathways involved in fluid homeostasis in the human alveolar epithelium. We performed functional and phenotypic characterisation of ion transport in the human pulmonary epithelial cell lines NCI-H441 and A549 to determine their similarity to primary human alveolar type II cells. NCI-H441 cells exhibited high expression of junctional proteins ZO-1, and E-cadherin, seal-forming claudin-3, -4, -5 and Na+-K+-ATPase while A549 cells exhibited high expression of pore-forming claudin-2. Consistent with this phenotype NCI-H441, but not A549, cells formed a functional barrier with active ion transport characterised by higher electrical resistance (529 ± 178 Ω cm2 vs 28 ± 4 Ω cm2), lower paracellular permeability ((176 ± 42) ×10−8 cm/s vs (738 ± 190) ×10−8 cm/s) and higher transepithelial potential difference (11.9 ± 4 mV vs 0 mV). Phenotypic and functional properties of NCI-H441 cells were tuned by varying cell seeding density and supplement concentrations. The cells formed a polarised monolayer typical of in vivo epithelium at seeding densities of 100,000 cells per 12-well insert while higher densities resulted in multiple cell layers. Dexamethasone and insulin-transferrin-selenium supplements were required for the development of high levels of electrical resistance, potential difference and expression of claudin-3 and Na+-K+-ATPase. Treatment of NCI-H441 cells with inhibitors and agonists of sodium and chloride channels indicated sodium absorption through ENaC under baseline and forskolin-stimulated conditions. Chloride transport was not sensitive to inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) under either condition. Channels inhibited by 5-nitro-1-(3-phenylpropylamino) benzoic acid (NPPB) contributed to chloride secretion following forskolin stimulation, but not at baseline. These data precisely define experimental conditions for the application of NCI

  9. Silica nanoparticles and biological dispersants: genotoxic effects on A549 lung epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Brown, David M., E-mail: d.brown@hw.ac.uk [Heriot-Watt University, Nanosafety Research Group, School of Life Sciences (United Kingdom); Varet, Julia, E-mail: julia.varet@IOM-world.org [Institute of Occupational Medicine (United Kingdom); Johnston, Helinor, E-mail: h.johnston@hw.ac.uk; Chrystie, Alison; Stone, Vicki, E-mail: v.stone@hw.ac.uk [Heriot-Watt University, Nanosafety Research Group, School of Life Sciences (United Kingdom)

    2015-10-15

    Silica nanoparticle exposure could be intentional (e.g. medical application or food) or accidental (e.g. occupational inhalation). On entering the body, particles become coated with specific proteins depending on the route of entry. The ability of silica particles of different size and charge (non-functionalized 50 and 200 nm and aminated 50 and 200 nm) to cause genotoxic effects in A549 lung epithelial cells was investigated. Using the modified comet assay and the micronucleus assay, we examined the effect of suspending the particles in different dispersion media [RPMI or Hanks’ balanced salt solution (HBSS), supplemented with bovine serum albumin (BSA), lung lining fluid (LLF) or serum] to determine if this influenced the particle’s activity. Particle characterisation suggested that the particles were reasonably well dispersed in the different media, with the exception of aminated 50 nm particles which showed evidence of agglomeration. Plain 50, 200 nm and aminated 50 nm particles caused significant genotoxic effects in the presence of formamidopyrimidine-DNA glycosylase when dispersed in HBSS or LLF. These effects were reduced when the particles were dispersed in BSA and serum. There was no significant micronucleus formation produced by any of the particles when suspended in any of the dispersants. The data suggest that silica particles can produce a significant genotoxic effect according to the comet assay in A549 cells, possibly driven by an oxidative stress-dependent mechanism which may be modified depending on the choice of dispersant employed.

  10. Silica nanoparticles and biological dispersants: genotoxic effects on A549 lung epithelial cells

    International Nuclear Information System (INIS)

    Brown, David M.; Varet, Julia; Johnston, Helinor; Chrystie, Alison; Stone, Vicki

    2015-01-01

    Silica nanoparticle exposure could be intentional (e.g. medical application or food) or accidental (e.g. occupational inhalation). On entering the body, particles become coated with specific proteins depending on the route of entry. The ability of silica particles of different size and charge (non-functionalized 50 and 200 nm and aminated 50 and 200 nm) to cause genotoxic effects in A549 lung epithelial cells was investigated. Using the modified comet assay and the micronucleus assay, we examined the effect of suspending the particles in different dispersion media [RPMI or Hanks’ balanced salt solution (HBSS), supplemented with bovine serum albumin (BSA), lung lining fluid (LLF) or serum] to determine if this influenced the particle’s activity. Particle characterisation suggested that the particles were reasonably well dispersed in the different media, with the exception of aminated 50 nm particles which showed evidence of agglomeration. Plain 50, 200 nm and aminated 50 nm particles caused significant genotoxic effects in the presence of formamidopyrimidine-DNA glycosylase when dispersed in HBSS or LLF. These effects were reduced when the particles were dispersed in BSA and serum. There was no significant micronucleus formation produced by any of the particles when suspended in any of the dispersants. The data suggest that silica particles can produce a significant genotoxic effect according to the comet assay in A549 cells, possibly driven by an oxidative stress-dependent mechanism which may be modified depending on the choice of dispersant employed

  11. Induction of Programmed Cell Death in Human Alveolar Epithelial Cells Infected with Influenza Virus

    Directory of Open Access Journals (Sweden)

    Sh Shahsavandi

    2015-11-01

    Full Text Available Introduction: Avian influenza viruses are considered as a serious threat to human and animal health. An increase in expression of proinflammatory cytokines and type I IFN genes, as well as host cell death responses contribute to the pathogenesis of influenza infection. Hence, this study aimed to evaluate the growth dynamics of subacute avian influenza virus in human respiratory alveolar epithelium cells (A549. Methods: The A549 cell cultures were infected at MOIs 0.1 and 2.0 viral doses in the presence and absence of trypsin. The virus growth kinetics were elucidated by the plaque assay and the cell viability was determined by MTT at various times after the infection. The induction quality of programmed cell death as well as the signal transduction pathway of death were assessed by genomic DNA fragmentation and western blotting respectively. Results: The study findings indicated that although the H9N2 virus replication did produce a marked cytopathic effect on the alveolar cells, which led to a reduction in the cell viability, the viral titers were increased in the infected cells. The virus replication of in these cells indicated repression of host defense mechanism as well as activation of cell death. The induction of apoptosis in A549 cells was correlated with the increased virus titers as well as virus replication (p< 0.05. Conclusion: H9N2 avian influenza virus were demonstrated to induce apoptosis in human alveolar epithelial cells via the intrinsic pathway in a dose-dependent manner.

  12. Barrier-protective effects of activated protein C in human alveolar epithelial cells.

    Directory of Open Access Journals (Sweden)

    Ferranda Puig

    Full Text Available Acute lung injury (ALI is a clinical manifestation of respiratory failure, caused by lung inflammation and the disruption of the alveolar-capillary barrier. Preservation of the physical integrity of the alveolar epithelial monolayer is of critical importance to prevent alveolar edema. Barrier integrity depends largely on the balance between physical forces on cell-cell and cell-matrix contacts, and this balance might be affected by alterations in the coagulation cascade in patients with ALI. We aimed to study the effects of activated protein C (APC on mechanical tension and barrier integrity in human alveolar epithelial cells (A549 exposed to thrombin. Cells were pretreated for 3 h with APC (50 µg/ml or vehicle (control. Subsequently, thrombin (50 nM or medium was added to the cell culture. APC significantly reduced thrombin-induced cell monolayer permeability, cell stiffening, and cell contraction, measured by electrical impedance, optical magnetic twisting cytometry, and traction microscopy, respectively, suggesting a barrier-protective response. The dynamics of the barrier integrity was also assessed by western blotting and immunofluorescence analysis of the tight junction ZO-1. Thrombin resulted in more elongated ZO-1 aggregates at cell-cell interface areas and induced an increase in ZO-1 membrane protein content. APC attenuated the length of these ZO-1 aggregates and reduced the ZO-1 membrane protein levels induced by thrombin. In conclusion, pretreatment with APC reduced the disruption of barrier integrity induced by thrombin, thus contributing to alveolar epithelial barrier protection.

  13. Doxycycline decreases production of interleukin-8 in a549 human lung epithelial cells

    Directory of Open Access Journals (Sweden)

    Hoyt JC

    2013-03-01

    Full Text Available Doxycycline is an antibiotic that possess anti-inflammatory properties. These anti-inflammatory properties make doxycycline an attractive candidate for possible treatments for a variety of common chronic obstructive airway diseases. Interleukin-8 (IL-8 is a major inflammatory chemokine and a powerful chemo-attractant for both neutrophils and monocytes. We hypothesized that doxycycline might exert its anti-inflammatory effects, at least in part, by modulating IL-8 production. To test this hypothesis, A549 human lung epithelial cells were stimulated with cytomix (IL-1beta, TNF-alpha and gamma-IFN in the presence or absence of varying concentrations of doxycycline. Doxycycline decreased IL-8 protein production in a concentration- and time-dependent manner. In the presence of 30 microg/ml doxycycline IL-8 protein production was decreased by 63% through out a 30 hr time course. In chemotaxis assays monocyte and neutrophil migration was decreased by 55% and 57% respectively. Reverse transcriptase-polymerase chain reaction (RT-PCR experiments suggest that doxycycline does not decrease expression of IL-8 mRNA and that use of the RNA polymerase II inhibitor DRB indicates that doxycycline does not effect stability of this mRNA. In the presence of doxycycline p38-alpha mitogen-activated protein kinase (MAPK expression is decreased by 36% in cytomix-stimulated cells. These data demonstrate that doxycycline can modulate IL-8 release and suggest that it has potential as an anti-inflammatory in those disorders where IL-8 is an important inflammatory mediator.

  14. Long Term Culture of the A549 Cancer Cell Line Promotes Multilamellar Body Formation and Differentiation towards an Alveolar Type II Pneumocyte Phenotype.

    Science.gov (United States)

    Cooper, James Ross; Abdullatif, Muhammad Bilal; Burnett, Edward C; Kempsell, Karen E; Conforti, Franco; Tolley, Howard; Collins, Jane E; Davies, Donna E

    2016-01-01

    Pulmonary research requires models that represent the physiology of alveolar epithelium but concerns with reproducibility, consistency and the technical and ethical challenges of using primary or stem cells has resulted in widespread use of continuous cancer or other immortalized cell lines. The A549 'alveolar' cell line has been available for over four decades but there is an inconsistent view as to its suitability as an appropriate model for primary alveolar type II (ATII) cells. Since most work with A549 cells involves short term culture of proliferating cells, we postulated that culture conditions that reduced proliferation of the cancer cells would promote a more differentiated ATII cell phenotype. We examined A549 cell growth in different media over long term culture and then used microarray analysis to investigate temporal regulation of pathways involved in cell cycle and ATII differentiation; we also made comparisons with gene expression in freshly isolated human ATII cells. Analyses indicated that long term culture in Ham's F12 resulted in substantial modulation of cell cycle genes to result in a quiescent population of cells with significant up-regulation of autophagic, differentiation and lipidogenic pathways. There were also increased numbers of up- and down-regulated genes shared with primary cells suggesting adoption of ATII characteristics and multilamellar body (MLB) development. Subsequent Oil Red-O staining and Transmission Electron Microscopy confirmed MLB expression in the differentiated A549 cells. This work defines a set of conditions for promoting ATII differentiation characteristics in A549 cells that may be advantageous for studies with this cell line.

  15. AMPK is required for PM2.5-induced autophagy in human lung epithelial A549 cells

    Science.gov (United States)

    Wang, Yahong; Lin, Ziying; Huang, Haili; He, Huijuan; Yang, Lawei; Chen, Ting; Yang, Teng; Ren, Nina; Jiang, Yun; Xu, Wenya; Kamp, David W; Liu, Tie; Liu, Gang

    2015-01-01

    The aim is to investigate the molecular mechanisms underlying the PM2.5-induced autophagy in human lung cancer epithelial cells (A549). The effects of the PM2.5 on morphological and biochemical markers of autophagy in A549 were analyzed by electron microscopy, GFP-LC3 puncta was observed by confocal fluorescence microscope. The effects of phosphorylation of AMPK, mTOR, AKT, ERK, JNK, and p53 on LC3II in A549 were observed following PM2.5 exposure; the role of autophagy in PM2.5-induced apoptosis was examined using 3-methyladenine and rapamycin. PM2.5 induced morphological and biochemical markers of autophagy in A549. Phosphorylation of AMPK and dephosphorylation of mTOR were observed following PM2.5 treatment, and AMPK inhibitor blocked LC3B-II expression. In addition, we demonstrated that PM2.5-induced autophagy confers a pro-survival role in host defense. PMID:25784975

  16. Pneumocystis carinii major surface glycoprotein induces interleukin-8 and monocyte chemoattractant protein-1 release from a human alveolar epithelial cell line

    DEFF Research Database (Denmark)

    Benfield, T L; Lundgren, Bettina; Shelhamer, J H

    1999-01-01

    (IL-8) and monocyte chemoattractant protein-1 (MCP-1) from an alveolar epithelial cell line (A549). RESULTS: Incubation of A549 cells with MSG in concentrations from 0.4 to 10 microg mL-1 for 24 h caused dose-dependent increases in IL-8 release (3.4-fold above control, P ..., suggesting that MSG stimulates A549 cells in part through carbohydrate moieties. Dexamethasone significantly inhibited MSG-induced IL-8 release in concentrations of 10-6-10-8 mol L-1 compared with control experiments (P

  17. Cigarette Smoke Enhances the Expression of Profibrotic Molecules in Alveolar Epithelial Cells.

    Directory of Open Access Journals (Sweden)

    Marco Checa

    Full Text Available Idiopathic pulmonary fibrosis (IPF is a progressive and lethal disease of unknown etiology. A growing body of evidence indicates that it may result from an aberrant activation of alveolar epithelium, which induces the expansion of the fibroblast population, their differentiation to myofibroblasts and the excessive accumulation of extracellular matrix. The mechanisms that activate the alveolar epithelium are unknown, but several studies indicate that smoking is the main environmental risk factor for the development of IPF. In this study we explored the effect of cigarette smoke on the gene expression profile and signaling pathways in alveolar epithelial cells. Lung epithelial cell line from human (A549, was exposed to cigarette smoke extract (CSE for 1, 3, and 5 weeks at 1, 5 and 10% and gene expression was evaluated by complete transcriptome microarrays. Signaling networks were analyzed with the Ingenuity Pathway Analysis software. At 5 weeks of exposure, alveolar epithelial cells acquired a fibroblast-like phenotype. At this time, gene expression profile revealed a significant increase of more than 1000 genes and deregulation of canonical signaling pathways such as TGF-β and Wnt. Several profibrotic genes involved in EMT were over-expressed, and incomplete EMT was observed in these cells, and corroborated in mouse (MLE-12 and rat (RLE-6TN epithelial cells. The secretion of activated TGF-β1 increased in cells exposed to cigarette smoke, which decreased when the integrin alpha v gene was silenced. These findings suggest that the exposure of alveolar epithelial cells to CSE induces the expression and release of a variety of profibrotic genes, and the activation of TGF-β1, which may explain at least partially, the increased risk of developing IPF in smokers.

  18. Electrospinning of PVA/sericin nanofiber and the effect on epithelial-mesenchymal transition of A549 cells.

    Science.gov (United States)

    Yan, Shanshan; Li, Xiuchun; Dai, Jing; Wang, Yiqun; Wang, Binbin; Lu, Yi; Shi, Jianlin; Huang, Pengyu; Gong, Jinkang; Yao, Yuan

    2017-10-01

    This research aims to investigate the cell-nanomaterial interaction between epithelial-mesenchymal transition of A549 cell and electrospinning nanofibers composed of polyvinyl alcohol (PVA)/silk sericin (SS). The electrospinning of regenerated nanofiber was performed with water as a spinning solvent and glutaraldehyde as a chemical cross-linker. Solution concentration, applied voltage and spin distances as well as other parameters were optimized to generate fine nanofibers with smooth surface in good homogeneity. From the scanning electron microscopy (SEM) analysis, the nanofibers had an average diameter of 200nm. Epithelial-mesenchymal transition (EMT) is a process by which epithelial cells lose their cell polarity to become mesenchymal stem cells. This transition is affected by multiple biochemical and physical factors in cell metabolism cascade. Herein, we investigate the biophysical effect on A549 EMT by culturing cells on nanofibrous mats with different topography and composition. The cell viability was evaluated by biochemical assay and its morphology was observed with SEM. The results demonstrate that cells appropriately attached to the surface of the nanofibrous mats with extended morphology by their filopodia. Gene expression analysis was conducted by real-time PCR using multiple markers for detecting EMT: N-cadherin (NCad), Vimentin (Vim), Fibronectin (Fib) and Matrix metallopeptidase (MMP9). An increasing expression pattern was observed on NCad, Vim, Fib, with respect to a negative control as cell cultured on polystyrene dish. This result indicates the 200nm PVA/SS nanofibers may induce A549 cells to process epithelial-mesenchymal transition during the culturing. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. MicroRNA-1 targets Slug and endows lung cancer A549 cells with epithelial and anti-tumorigenic properties.

    Science.gov (United States)

    Tominaga, Eiji; Yuasa, Katsutoshi; Shimazaki, Sho; Hijikata, Takao

    2013-02-01

    MicroRNA-1 (miR-1) has recently been suggested to function as a tumor suppressor. Its functional relevance was assessed by exploring structural and tumorigenic properties of lung cancer A549 cells stably transduced with retrovirus containing pre-miR-1. A549 cells overexpressing miR-1 exhibited a significant morphological change from a mesenchymal to an epithelial phenotype characterized by cell polarization and intercellular junctions. The cells showed increased expression of E-cadherin, which colocalized with cortical actin filaments and vinculin to form typical adherens junction at the apical regions of intercellular borders. Additionally, they exhibited occludin-positive tight junctions at similar apical regions. Moreover, their migratory and invasive activities were inhibited, and their sensitivity to doxorubicin was increased slightly compared to control mock-infected cells. These structural and tumorigenic properties induced by miR-1 were associated with the reduced expression of Slug, which was a transcriptional repressor of E-cadherin or an inducer of epithelial-to-mesenchymal transition. Consistently, Slug was identified as a miR-1 target by bioinformatics and a luciferase reporter assay with plasmids containing luciferase-Slug 3'UTR. Collectively, the data presented here suggest that re-expression of miR-1 may be an effective therapy that prevents cancer malignancy by converting cells from a mesenchymal phenotype to an epithelial phenotype via the downregulation of Slug. Copyright © 2012 Elsevier Inc. All rights reserved.

  20. Evidence for the involvement of cofilin in Aspergillus fumigatus internalization into type II alveolar epithelial cells.

    Science.gov (United States)

    Bao, Zhiyao; Han, Xuelin; Chen, Fangyan; Jia, Xiaodong; Zhao, Jingya; Zhang, Changjian; Yong, Chen; Tian, Shuguang; Zhou, Xin; Han, Li

    2015-08-13

    The internalization of Aspergillus fumigatus into alveolar epithelial cells (AECs) is tightly controlled by host cellular actin dynamics, which require close modulation of the ADF (actin depolymerizing factor)/cofilin family. However, the role of cofilin in A. fumigatus internalization into AECs remains unclear. Here, we demonstrated that germinated A. fumigatus conidia were able to induce phosphorylation of cofilin in A549 cells during the early stage of internalization. The modulation of cofilin activity by overexpression, knockdown, or mutation of the cofilin gene in A549 cells decreased the efficacy of A. fumigatus internalization. Reducing the phosphorylation status of cofilin with BMS-5 (LIM kinase inhibitor) or overexpression of the slingshot phosphatases also impeded A. fumigatus internalization. Both the C. botulimun C3 transferase (a specific RhoA inhibitor) and Y27632 (a specific ROCK inhibitor) reduced the internalization of A. fumigatus and the level of phosphorylated cofilin. β-1,3-glucan (the major component of the conidial cell wall) and its host cell receptor dectin-1 did not seem to be associated with cofilin phosphorylation during A. fumigatus infection. These results indicated that cofilin might be involved in the modulation of A. fumigatus internalization into type II alveolar epithelial cells through the RhoA-ROCK-LIM kinase pathway.

  1. Decreased CXCL12 is associated with impaired alveolar epithelial cell migration and poor lung healing after lung resection.

    Science.gov (United States)

    Kanter, Jacob A; Sun, Haiying; Chiu, Stephen; DeCamp, Malcolm M; Sporn, Peter H S; Sznajder, Jacob I; Bharat, Ankit

    2015-10-01

    Prolonged air leak (PAL) is an important cause of morbidity and mortality after lung resection, but its pathogenesis has not been elucidated. Migration of alveolar type II epithelial cells is essential for lung wound repair. Here we determined the role of C-X-C motif chemokine 12 (CXCL12) on alveolar epithelial cell migration and lung wound healing. CXCL12 in the pleural fluid of patients was analyzed using enzyme-linked immunosorbent assay. Human A549 and murine MLE12 alveolar epithelial cell lines were used for wound closure, cell migration, and proliferation assays. Western blot was used to analyze Rac1 and cofilin. Pleural CXCL12 was decreased in patients with PAL (1,389 ± 192 vs 3,270 ± 247 pg/mL; P alveolar epithelial cell migration by binding to its receptor CXCR4 and may have a role in lung healing. CXCL12-mediated alveolar epithelial cell migration is associated with Rac1 and cofilin activation. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Oxidative stress and inflammatory response to printer toner particles in human epithelial A549 lung cells.

    Science.gov (United States)

    Könczöl, Mathias; Weiß, Adilka; Gminski, Richard; Merfort, Irmgard; Mersch-Sundermann, Volker

    2013-02-04

    Reports on adverse health effects related to occupational exposure to toner powder are still inconclusive. Therefore, we have previously conducted an in vitro-study to characterize the genotoxic potential of three commercially available black printer toner powders in A549 lung cells. In these cell-based assays it was clearly demonstrated that the tested toner powders damage DNA and induce micronucleus (MN) formation. Here, we have studied the cytotoxic and proinflammatory potential of these three types of printer toner particles and the influence of ROS and NF-κB induction in order to unravel the underlying mechanisms. A549 cells were exposed to various concentrations of printer toner particle suspensions for 24 h. The toner particles were observed to exert significant cytotoxic effects in the WST-1 and neutral red (NR)-assays, although to a varying extent. Caspase 3/7 activity increased, while the mitochondrial membrane potential (MMP) was not affected. Particles of all three printer toner powders induced concentration-dependent formation of reactive oxygen species (ROS), as measured in the DCFH-DA assay. Furthermore, toner particle exposure enhanced interleukin-6 and interleukin-8 production, which is in agreement with activation of the transcription factor NF-κB in A549 cells shown by the electrophoretic mobility shift assay (EMSA). Therefore, it can be concluded that exposure of A549 lung cells to three selected printer toner powders caused oxidative stress through induction of ROS. Increased ROS formation may trigger genotoxic effects and activate proinflammatory pathways. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  3. Dexmedetomidine Attenuates Oxidative Stress Induced Lung Alveolar Epithelial Cell Apoptosis In Vitro

    Directory of Open Access Journals (Sweden)

    Jian Cui

    2015-01-01

    Full Text Available Background. Oxidative stress plays a pivotal role in the lung injuries of critical ill patients. This study investigates the protection conferred by α2 adrenoceptor agonist dexmedetomidine (Dex from lung alveolar epithelial cell injury induced by hydrogen peroxide (H2O2 and the underlying mechanisms. Methods. The lung alveolar epithelial cell line, A549, was cultured and then treated with 500 μM H2O2 with or without Dex (1 nM or Dex in combination with atipamezole (10 nM, an antagonist of α2 receptors. Their effect on mitochondrial membrane potential (Δψm, reactive oxygen species (ROS, and the cell cycle was assessed by flow cytometry. Cleaved-caspases 3 and 9, BAX, Bcl-2, phospho-mTOR (p-mTOR, ERK1/2, and E-cadherin expression were also determined with immunocytochemistry. Results. Upregulation of cleaved-caspases 3 and 9 and BAX and downregulation of Bcl-2, p-mTOR, and E-cadherin were found following H2O2 treatment, and all of these were reversed by Dex. Dex also prevented the ROS generation, cytochrome C release, and cell cycle arrest induced by H2O2. The effects of Dex were partially reversed by atipamezole. Conclusion. Our study demonstrated that Dex protected lung alveolar epithelial cells from apoptotic injury, cell cycle arrest, and loss of cell adhesion induced by H2O2 through enhancing the cell survival and proliferation.

  4. Increased survival and proliferation of the epidemic strain Mycobacterium abscessus subsp. massiliense CRM0019 in alveolar epithelial cells.

    Science.gov (United States)

    Ribeiro, Giovanni Monteiro; Matsumoto, Cristianne Kayoko; Real, Fernando; Teixeira, Daniela; Duarte, Rafael Silva; Mortara, Renato Arruda; Leão, Sylvia Cardoso; de Souza Carvalho-Wodarz, Cristiane

    2017-09-13

    Outbreaks of infections caused by rapidly growing mycobacteria have been reported worldwide generally associated with medical procedures. Mycobacterium abscessus subsp. massiliense CRM0019 was obtained during an epidemic of postsurgical infections and was characterized by increased persistence in vivo. To better understand the successful survival strategies of this microorganism, we evaluated its infectivity and proliferation in macrophages (RAW and BMDM) and alveolar epithelial cells (A549). For that, we assessed the following parameters, for both M. abscessus CRM0019 as well as the reference strain M. abscessus ATCC 19977: internalization, intracellular survival for up 3 days, competence to subvert lysosome fusion and the intracellular survival after cell reinfection. CRM0019 and ATCC 19977 strains showed the same internalization rate (approximately 30% after 6 h infection), in both A549 and RAW cells. However, colony forming units data showed that CRM0019 survived better in A549 cells than the ATCC 19977 strain. Phagosomal characteristics of CRM0019 showed the bacteria inside tight phagosomes in A549 cells, contrasting to the loosely phagosomal membrane in macrophages. This observation holds for the ATCC 19977 strain in both cell types. The competence to subvert lysosome fusion was assessed by acidification and acquisition of lysosomal protein. For M. abscessus strains the phagosomes were acidified in all cell lines; nevertheless, the acquisition of lysosomal protein was reduced by CRM0019 compared to the ATCC 19977 strain, in A549 cells. Conversely, in macrophages, both M. abscessus strains were located in mature phagosomes, however without bacterial death. Once recovered from macrophages M. abscessus could establish a new intracellular infection. Nevertheless, only CRM0019 showed a higher growth rate in A549, increasing nearly 10-fold after 48 and 72 h. M. abscessus CRM0019 creates a protective and replicative niche in alveolar epithelial cells mainly by

  5. The surface charge of liposomal adjuvants is decisive for their interactions with the Calu-3 and A549 airway epithelial cell culture models.

    Science.gov (United States)

    Ingvarsson, Pall Thor; Rasmussen, Ida Svahn; Viaene, Michelle; Irlik, Przemyslaw Jedrzej; Nielsen, Hanne Mørck; Foged, Camilla

    2014-08-01

    One of the main reasons for the unmet medical need for mucosal vaccines is the lack of safe and efficacious mucosal adjuvants. The cationic liposome-based adjuvant system composed of dimethyldioctadecylammonium (DDA) bromide and trehalose 6,6'-dibehenate (TDB) is a versatile adjuvant that has shown potential for mucosal vaccination via the airways. The purpose of this study was to investigate the importance of the liposomal surface charge on the interaction with lung epithelial cells. Thus, the cationic DDA in the liposomes was subjected to a step-wise replacement with the zwitterionic distearoylphosphatidylcholine (DSPC). The liposomes were tested with the model protein antigen ovalbumin for the mucosal deposition, the effect on cellular viability and the epithelial integrity by using the two cell lines A549 and Calu-3, representing cells from the alveolar and the bronchiolar epithelium, respectively. The Calu-3 cells were cultured under different conditions, resulting in epithelia with a low and a high mucus secretion, respectively. A significantly larger amount of lipid and ovalbumin was deposited in the epithelial cell layer and in the mucus after incubation with the cationic liposomes, as compared to incubation with the neutral liposomes, which suggests that the cationic charge is important for the delivery. The integrity and the viability of the cells without a surface-lining mucus layer were decreased upon incubation with the cationic formulations, whereas the mucus appeared to retain the integrity and viability of the mucus-covered Calu-3 cells. Our in vitro results thus indicate that DDA/TDB liposomes might be efficiently and safely used as an adjuvant system for vaccines targeting the mucus-covered epithelium of the upper respiratory tract and the conducting airways. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. CXCL9 Regulates TGF-β1-Induced Epithelial to Mesenchymal Transition in Human Alveolar Epithelial Cells.

    Science.gov (United States)

    O'Beirne, Sarah L; Walsh, Sinead M; Fabre, Aurélie; Reviriego, Carlota; Worrell, Julie C; Counihan, Ian P; Lumsden, Robert V; Cramton-Barnes, Jennifer; Belperio, John A; Donnelly, Seamas C; Boylan, Denise; Marchal-Sommé, Joëlle; Kane, Rosemary; Keane, Michael P

    2015-09-15

    Epithelial to mesenchymal cell transition (EMT), whereby fully differentiated epithelial cells transition to a mesenchymal phenotype, has been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF). CXCR3 and its ligands are recognized to play a protective role in pulmonary fibrosis. In this study, we investigated the presence and extent of EMT and CXCR3 expression in human IPF surgical lung biopsies and assessed whether CXCR3 and its ligand CXCL9 modulate EMT in alveolar epithelial cells. Coexpression of the epithelial marker thyroid transcription factor-1 and the mesenchymal marker α-smooth muscle actin and CXCR3 expression was examined by immunohistochemical staining of IPF surgical lung biopsies. Epithelial and mesenchymal marker expression was examined by quantitative real-time PCR, Western blotting, and immunofluorescence in human alveolar epithelial (A549) cells treated with TGF-β1 and CXCL9, with Smad2, Smad3, and Smad7 expression and cellular localization examined by Western blotting. We found that significantly more cells were undergoing EMT in fibrotic versus normal areas of lung in IPF surgical lung biopsy samples. CXCR3 was expressed by type II pneumocytes and fibroblasts in fibrotic areas in close proximity to cells undergoing EMT. In vitro, CXCL9 abrogated TGF-β1-induced EMT. A decrease in TGF-β1-induced phosphorylation of Smad2 and Smad3 occurred with CXCL9 treatment. This was associated with increased shuttling of Smad7 from the nucleus to the cytoplasm where it inhibits Smad phosphorylation. This suggests a role for EMT in the pathogenesis of IPF and provides a novel mechanism for the inhibitory effects of CXCL9 on TGF-β1-induced EMT. Copyright © 2015 by The American Association of Immunologists, Inc.

  7. Toxicity of engineered nanomaterials and their transformation products following wastewater treatment on A549 human lung epithelial cells

    Directory of Open Access Journals (Sweden)

    Yanjun Ma

    2014-01-01

    Full Text Available Here we characterize the toxicity of environmentally-relevant forms of engineered nanomaterials (ENMs, which can transform during wastewater treatment and persist in aqueous effluents and biosolids. In an aerosol exposure scenario, cytotoxicity and genotoxicity of effluents and biosolids from lab-scale sequencing batch reactors (SBRs to A549 human lung epithelial cells were examined. The SBRs were dosed with nanoAg, nano zero-valent iron (NZVI, nanoTiO2 and nanoCeO2 at sequentially increasing concentrations from 0.1 to 20 mg/L. Toxicities were compared to outputs from SBRs dosed with ionic/bulk analogs, undosed SBRs, and pristine ENMs. Pristine nanoAg and NZVI showed significant cytotoxicity to A549 cells in a dose-dependent manner from 1 to 67 μg/mL, while nanoTiO2 and nanoCeO2 only exerted cytotoxicity at 67 μg/mL. Only nanoAg induced a genotoxic response, at 9, 33 and 53 μg/mL. However, no significant cytotoxic or genotoxic effects of the SBR effluents or biosolids containing nanomaterials were observed.

  8. Cathepsin L is involved in cathepsin D processing and regulation of apoptosis in A549 human lung epithelial cells.

    Science.gov (United States)

    Wille, Aline; Gerber, Annegret; Heimburg, Anke; Reisenauer, Anita; Peters, Christoph; Saftig, Paul; Reinheckel, Thomas; Welte, Tobias; Bühling, Frank

    2004-07-01

    Cathepsins are implicated in a multitude of physiological and pathophysiological processes. The aim of the present study was to investigate the function of cathepsin L (catL) in the proteolytic network of human lung epithelial cells and its role in the regulation of apoptosis. We found that catL-deficient A549 cells as well as lung tissue extracts of catL(-/-) mice express increased amounts of single-chain cathepsin D (catD). Degradation experiments indicate that catL specifically degrades the single-chain isoform of catD. Furthermore, we found that catL-deficient cells showed increased sensitivity to apoptosis. Finally, we demonstrate that the inhibition of catD activity by pepstatin A decreased the number of apoptotic cells in catL-deficient A549 cells after anti-Fas treatment. In conclusion, catL is involved in catD processing and the accumulation of catD isoforms in catL-deficient cells is associated with increased rates of spontaneous and anti-Fas-induced apoptosis.

  9. Modeling Alveolar Epithelial Cell Behavior In Spatially Designed Hydrogel Microenvironments

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    Lewis, Katherine Jean Reeder

    The alveolar epithelium consists of two cell phenotypes, elongated alveolar type I cells (AT1) and rounded alveolar type II cells (ATII), and exists in a complex three-dimensional environment as a polarized cell layer attached to a thin basement membrane and enclosing a roughly spherical lumen. Closely surrounding the alveolar cysts are capillary endothelial cells as well as interstitial pulmonary fibroblasts. Many factors are thought to influence alveolar epithelial cell differentiation during lung development and wound repair, including physical and biochemical signals from the extracellular matrix (ECM), and paracrine signals from the surrounding mesenchyme. In particular, disrupted signaling between the alveolar epithelium and local fibroblasts has been implicated in the progression of several pulmonary diseases. However, given the complexity of alveolar tissue architecture and the multitude of signaling pathways involved, designing appropriate experimental platforms for this biological system has been difficult. In order to isolate key factors regulating cellular behavior, the researcher ideally should have control over biophysical properties of the ECM, as well as the ability to organize multiple cell types within the scaffold. This thesis aimed to develop a 3D synthetic hydrogel platform to control alveolar epithelial cyst formation, which could then be used to explore how extracellular cues influence cell behavior in a tissue-relevant cellular arrangement. To accomplish this, a poly(ethylene glycol) (PEG) hydrogel network containing enzymatically-degradable crosslinks and bioadhesive pendant peptides was employed as a base material for encapsulating primary alveolar epithelial cells. First, an array of microwells of various cross-sectional shapes was photopatterned into a PEG gel containing photo-labile crosslinks, and primary ATII cells were seeded into the wells to examine the role of geometric confinement on differentiation and multicellular arrangement

  10. Activity of interferon alpha, interleukin 6 and insulin in the regulation of differentiation in A549 alveolar carcinoma cells.

    Science.gov (United States)

    McCormick, C; Freshney, R I; Speirs, V

    1995-02-01

    The differentiation of A549, a human tumour cell line from type II pneumocytes, can be induced by a crude fibroblast-derived factor (FDF) isolated from the conditioned medium of glucocorticoid-treated lung fibroblasts. In the present report, we have used alkaline phosphatase as a differentiation marker to investigate the activity of a number of growth factors as potential candidates for this paracrine activity. This showed that insulin, interleukin 6 (IL-6), and interferon alpha (IFN-alpha) could simulate the activity of conditioned medium. Their effects were dexamethasone (DX) dependent, additive and reversible with a half-life of 1 week. Transforming growth factor alpha and beta, IL-1 alpha and epidermal growth factor, were all inhibitory, and inhibition was opposed, partially or completely, by DX. The most potent inducer was IL-6, but as DX was shown to decrease the concentration of IL-6 in lung fibroblast-conditioned medium it seems an unlikely candidate for FDF. Unlike FDF, all of the positive-acting factors were shown to induce plasminogen activator. FDF has also been shown to be active in the absence of DX. This suggests that differentiation-inducing activity may be present in several paracrine factors, but that so far a candidate for FDF has not been identified.

  11. Mesenchymal stem cells promote cell invasion and migration and autophagy-induced epithelial-mesenchymal transition in A549 lung adenocarcinoma cells.

    Science.gov (United States)

    Luo, Dan; Hu, Shiyuan; Tang, Chunlan; Liu, Guoxiang

    2018-03-01

    Mesenchymal stem cells (MSCs) are recruited into the tumour microenvironment and promote tumour growth and metastasis. Tumour microenvironment-induced autophagy is considered to suppress primary tumour formation by impairing migration and invasion. Whether these recruited MSCs regulate tumour autophagy and whether autophagy affects tumour growth are controversial. Our data showed that MSCs promote autophagy activation, reactive oxygen species production, and epithelial-mesenchymal transition (EMT) as well as increased migration and invasion in A549 cells. Decreased expression of E-cadherin and increased expression of vimentin and Snail were observed in A549 cells cocultured with MSCs. Conversely, MSC coculture-mediated autophagy positively promoted tumour EMT. Autophagy inhibition suppressed MSC coculture-mediated EMT and reduced A549 cell migration and invasion slightly. Furthermore, the migratory and invasive abilities of A549 cells were additional increased when autophagy was further enhanced by rapamycin treatment. Taken together, this work suggests that microenvironments containing MSCs can promote autophagy activation for enhancing EMT; MSCs also increase the migratory and invasive abilities of A549 lung adenocarcinoma cells. Mesenchymal stem cell-containing microenvironments and MSC-induced autophagy signalling may be potential targets for blocking lung cancer cell migration and invasion. Copyright © 2018 John Wiley & Sons, Ltd.

  12. Blockage of glycolysis by targeting PFKFB3 alleviates sepsis-related acute lung injury via suppressing inflammation and apoptosis of alveolar epithelial cells.

    Science.gov (United States)

    Gong, Yuanqi; Lan, Haibing; Yu, Zhihong; Wang, Meng; Wang, Shu; Chen, Yu; Rao, Haiwei; Li, Jingying; Sheng, Zhiyong; Shao, Jianghua

    2017-09-16

    Sepsis-related acute lung injury (ALI) is characterized by excessive lung inflammation and apoptosis of alveolar epithelial cells resulting in acute hypoxemic respiratory failure. Recent studies indicated that anaerobic glycolysis play an important role in sepsis. However, whether inhibition of aerobic glycolysis exhibits beneficial effect on sepsis-induced ALI is not known. In vivo, a cecal ligation and puncture (CLP)-induced ALI mouse model was set up and mice treated with glycolytic inhibitor 3PO after CLP. The mice treated with the 3PO ameliorated the survival rate, histopathological changes, lung inflammation, lactate increased and lung apoptosis of mice with CLP-induced sepsis. In vitro, the exposure of human alveolar epithelial A549 cells to lipopolysaccharide (LPS) resulted in cell apoptosis, inflammatory cytokine production, enhanced glycolytic flux and reactive oxygen species (ROS) increased. While these changes were attenuated by 3PO treatment. Sequentially, treatment of A549 cells with lactate caused cell apoptosis and enhancement of ROS. Pretreatment with N-acetylcysteine (NAC) significantly lowered LPS and lactate-induced the generation of ROS and cell apoptosis in A549 cells. Therefore, these results indicate that anaerobic glycolysis may be an important contributor in cell apoptosis of sepsis-related ALI. Moreover, LPS specifically induces apoptotic insults to A549 cell through lactate-mediated enhancement of ROS. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Picfeltarraenin IA inhibits lipopolysaccharide-induced inflammatory cytokine production by the nuclear factor-κB pathway in human pulmonary epithelial A549 cells.

    Science.gov (United States)

    Shi, Rong; Wang, Qing; Ouyang, Yang; Wang, Qian; Xiong, Xudong

    2016-02-01

    The present study aimed to investigate the effect of picfeltarraenin IA (IA) on respiratory inflammation by analyzing its effect on interleukin (IL)-8 and prostaglandin E2 (PGE2) production. The expression of cyclooxygenase 2 (COX2) in human pulmonary adenocarcinoma epithelial A549 cells in culture was also examined. Human pulmonary epithelial A549 cells and the human monocytic leukemia THP-1 cell line were used in the current study. Cell viability was measured using a methylthiazol tetrazolium assay. The production of IL-8 and PGE2 was investigated using an enzyme-linked immunosorbent assay. The expression of COX2 and nuclear factor-κB (NF-κB)-p65 was examined using western blot analysis. Treatment with lipopolysaccharide (LPS; 10 µg/ml) resulted in the increased production of IL-8 and PGE2, and the increased expression of COX2 in the A549 cells. Furthermore, IA (0.1-10 µmol/l) significantly inhibited PGE2 production and COX2 expression in cells with LPS-induced IL-8, in a concentration-dependent manner. The results suggested that IA downregulates LPS-induced COX2 expression, and inhibits IL-8 and PGE2 production in pulmonary epithelial cells. Additionally, IA was observed to suppress the expression of COX2 in THP-1 cells, and also to regulate the expression of COX2 via the NF-κB pathway in the A549 cells, but not in the THP-1 cells. These results indicate that IA regulates LPS-induced cytokine release in A549 cells via the NF-κB pathway.

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

    Science.gov (United States)

    Farzin, Hamidreza; Toroghi, Reza; Haghparast, Alireza

    2016-01-01

    Influenza 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 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.

  15. Oxidative Stress, Cell Death, and Other Damage to Alveolar Epithelial Cells Induced by Cigarette Smoke

    Directory of Open Access Journals (Sweden)

    Nagai A

    2003-09-01

    Full Text Available Abstract Cigarette smoking is a major risk factor in the development of various lung diseases, including pulmonary emphysema, pulmonary fibrosis, and lung cancer. The mechanisms of these diseases include alterations in alveolar epithelial cells, which are essential in the maintenance of normal alveolar architecture and function. Following cigarette smoking, alterations in alveolar epithelial cells induce an increase in epithelial permeability, a decrease in surfactant production, the inappropriate production of inflammatory cytokines and growth factors, and an increased risk of lung cancer. However, the most deleterious effect of cigarette smoke on alveolar epithelial cells is cell death, i.e., either apoptosis or necrosis depending on the magnitude of cigarette smoke exposure. Cell death induced by cigarette smoke exposure can largely be accounted for by an enhancement in oxidative stress. In fact, cigarette smoke contains and generates many reactive oxygen species that damage alveolar epithelial cells. Whether apoptosis and/or necrosis in alveolar epithelial cells is enhanced in healthy cigarette smokers is presently unclear. However, recent evidence indicates that the apoptosis of alveolar epithelial cells and alveolar endothelial cells is involved in the pathogenesis of pulmonary emphysema, an important cigarette smoke-induced lung disease characterized by the loss of alveolar structures. This review will discuss oxidative stress, cell death, and other damage to alveolar epithelial cells induced by cigarette smoke.

  16. Ontogeny of pulmonary alveolar epithelial markers of differentiation.

    Science.gov (United States)

    Joyce-Brady, M F; Brody, J S

    1990-02-01

    We studied differentiation of the pulmonary epithelium in the periphery of fetal rat lung in vivo and in vitro by comparing the ontogeny of cell-surface glycoconjugates with that of surfactant phospholipids. Apical surface binding of the lectin Maclura pomifera agglutinin (MPA) and expression of a 200-kDa MPA-binding glycoprotein (MPA-gp200) was evident at 20 days gestation in type 2 cells, but did not correlate with ultrastructural features of type 2 cell differentiation. Epithelial cells isolated from peripheral lung of 18-day gestation fetal rats displayed hormone-sensitive surfactant synthesis prior to the hormone-insensitive expression of MPA-gp200. Expression of MPA-gp200 occurred in association with the appearance of many new apical surface proteins suggesting a hormone-independent process of polar membrane differentiation. Thus membrane and secretory differentiation are discordant and can be dissociated. In vivo binding of Ricinus communis 1 agglutinin (RCA1), an apical marker of the differentiated alveolar type 1 cell occurred in undifferentiated peripheral lung epithelial cells as early as 18 days gestation, disappeared from differentiating type 2 cells and appeared in differentiated type 1 cells. Both undifferentiated fetal epithelial cells at 18 days gestation and fully differentiated type 1 cells express multiple glycoproteins with terminal beta-linked galactose residues which bind RCA1. Some of these RCA1-binding glycoproteins appear to be similar. These observations suggest that alveolar epithelial type 1 cells may derive directly from undifferentiated peripheral lung epithelial cells as well as from fully differentiated type 2 cells. In addition, terminal differentiation of fetal lung peripheral epithelium into type 1 and type 2 cells may involve repression as well as induction of differentiation-related genes.

  17. Activator protein-1 (AP-1): a bridge between life and death in lung epithelial (A549) cells under hypoxia.

    Science.gov (United States)

    Yadav, Seema; Kalra, Namita; Ganju, Lilly; Singh, Mrinalini

    2017-12-01

    Activator protein-1 (AP-1) transcription factor plays a central role in hypoxia to modulate the expression of genes that decides the fate of the cell. The aim of the present study was to explore the role of AP-1 subunits in lung epithelial (A549) cells under hypoxia. Cell cycle studies by flow cytometry indicated that cell viability was unaffected by the initial hypoxia exposure (0.5% O 2 at 37 °C) for 6 and 12 h. However, both transient cell cycle arrest and cell death was detected at 24 and 48 h. Flow cytometry and spectrofluorometry data confirmed the increase in ROS levels. Elevated ROS and calcium levels activated the stress-related MAPK signaling cascade. ERK and JNK were activated in early hypoxic exposure (within 6 h), whereas p38 were activated in 48 h of hypoxia. These subtypes further stimulated the subunits of AP-1 at different times of hypoxia exposure to orchestrate different genes responsible for cell proliferation (6 and 12 h) and apoptosis (24 and 48 h). Our results clearly depict the role of AP-1 heterodimer, i.e., p-c-jun/c-fos, p-c-jun/fosB, junD/c-fos, and junD/fosB in cell proliferation/survival by regulating the expression of Bcl-2 and cyclins (D1 and B1) at 6 h and 12 h of hypoxia, whereas junB/Fra-1 heterodimer have important role in apoptosis by regulating the expression of p53, Bax, and cyclin-dependent kinase inhibitors (p16, p21, p27) at 24 h and 48 h of hypoxia. Also, the cell survival signaling pathway NO-AKT interrupted at 24 h and 48 h of hypoxia indicating cell death. In conclusion, hypoxia for different time points activated different subunits of AP-1 that combined to form different heterodimers. These dimers regulated the expression of genes responsible for cell proliferation and apoptosis. Since, AP-1 plays a role in the decisive phenomenon of the cell to choose between proliferation and apoptosis; thus, its subunits or dimers could be a good therapeutic target for many diseases.

  18. Effect of cigarette smoke and dexamethasone on Hsp72 system of alveolar epithelial cells.

    Science.gov (United States)

    Gál, Krisztina; Cseh, Aron; Szalay, Balázs; Rusai, Krisztina; Vannay, Adám; Lukácsovits, József; Heemann, Uwe; Szabó, Attila J; Losonczy, György; Tamási, Lilla; Müller, Veronika

    2011-07-01

    Smoking is the leading risk factor of chronic obstructive pulmonary disease (COPD) and lung cancer. Corticosteroids are abundantly used in these patients; however, the interaction of smoking and steroid treatment is not fully understood. Heat shock proteins (Hsps) play a central role in the maintenance of cell integrity, apoptosis and cellular steroid action. To better understand cigarette smoke-steroid interaction, we examined the effect of cigarette smoke extract (CSE) and/or dexamethasone (DEX) on changes of intracellular heat shock protein-72 (Hsp72) in lung cells. Alveolar epithelial cells (A549) were exposed to increasing doses (0; 0.1; 1; and 10 μM/μl) of DEX in the medium in the absence(C) and presence of CSE. Apoptosis, necrosis, Hsp72 messenger-ribonucleic acid (mRNA) and protein expression of cells were measured, and the role of Hsp72 on steroid effect examined. CSE reduced the number of viable cells by significantly increasing the number of apoptotic and necrotic cells. DEX dose-dependently decreased the ratio of apoptosis when CSE was administered, without change in necrosis. CSE - DEX co-treatment dose-dependently increased Hsp72 mRNA and protein expression, with the highest level measured in CSE + DEX (10) cells, while significantly lower levels were noted in all respective C groups. Pretreatment with Hsp72 silencing RNA confirmed that increased survival observed following DEX administration in CSE-treated cells was mainly mediated via the Hsp72 system. CSE significantly decreases cell survival by inducing apoptosis and necrosis. DEX significantly increases Hsp72 mRNA and protein expression only in the presence of CSE resulting in increased cellular protection and survival. DEX exerts its cell protective effects by decreasing apoptotic cell death via the Hsp72 system in CSE-treated alveolar epithelial cells.

  19. Mitochondrial DNA-depleted A549 cells are resistant to bleomycin

    Science.gov (United States)

    Brar, Sukhdev S.; Meyer, Joel N.; Bortner, Carl D.; Van Houten, Bennett

    2012-01-01

    Alveolar epithelial cells are considered to be the primary target of bleomycin-induced lung injury, leading to interstitial fibrosis. The molecular mechanisms by which bleomycin causes this damage are poorly understood but are suspected to involve generation of reactive oxygen species and DNA damage. We studied the effect of bleomycin on mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) in human alveolar epithelial A549 cells. Bleomycin caused an increase in reactive oxygen species production, DNA damage, and apoptosis in A549 cells; however, bleomycin induced more mtDNA than nDNA damage. DNA damage was associated with activation of caspase-3, cleavage of poly(ADP-ribose) polymerase, and cleavage and activation of protein kinase D1 (PKD1), a newly identified mitochondrial oxidative stress sensor. These effects appear to be mtDNA-dependent, because no caspase-3 or PKD1 activation was observed in mtDNA-depleted (ρ0) A549 cells. Survival rate after bleomycin treatment was higher for A549 ρ0 than A549 cells. These results suggest that A549 ρ0 cells are more resistant to bleomycin toxicity than are parent A549 cells, likely in part due to the depletion of mtDNA and impairment of mitochondria-dependent apoptotic pathways. PMID:22773697

  20. Effect of SLC34A2 gene mutation on extracellular phosphorus transport in PAM alveolar epithelial cells.

    Science.gov (United States)

    Ma, Tiangang; Qu, Danhua; Yan, Bingdi; Zhang, Qinghua; Ren, Jin; Hu, Yanbing

    2018-01-01

    A mutation in the IIb sodium phosphate transporter SLC34A2 gene has recently been described in pulmonary alveolar microlithiasis (PAM) patients. Experiments in this study were aimed at confirming the role of the gene product in PAM by comparing phosphorylated products in extracellular fluid of alveolar epithelial cells overexpressing the SLC34A2 gene or its mutated version. Eukaryotic expression vectors were constructed and transfected into A549 human alveolar epithelial cells. There were three groups of cells including those transfected with empty vector plasmid pcDNA3.1(+) (plasmid control group), those transfected with normal SLC34A2 gene expressed from pcDNA3.1 (normal control group), and those transfected with a version of the PAM SLC34A2 gene linked to the pcDNA3.1(+) (PAM group). Transfection efficiencies were detected by reverse transcription-polymerase chain reaction (RT-PCR). At 48 h after transfection, the concentration of inorganic phosphorus in the culture medium was detected using an automatic biochemical analyzer. Our results showed the concentration of inorganic phosphorus in the supernatant of the normal control group was significantly lower than that in the plasmid control and PAM groups (PPAM group was significantly lower than that in the plasmid control group (PPAM patients, given that the function of the phosphate transporter seems to be affected and it is conceivable that it would lead to extracellular fluid alterations in vivo .

  1. Simulation of lung alveolar epithelial wound healing in vitro.

    Science.gov (United States)

    Kim, Sean H J; Matthay, Michael A; Mostov, Keith; Hunt, C Anthony

    2010-08-06

    The mechanisms that enable and regulate alveolar type II (AT II) epithelial cell wound healing in vitro and in vivo remain largely unknown and need further elucidation. We used an in silico AT II cell-mimetic analogue to explore and better understand plausible wound healing mechanisms for two conditions: cyst repair in three-dimensional cultures and monolayer wound healing. Starting with the analogue that validated for key features of AT II cystogenesis in vitro, we devised an additional cell rearrangement action enabling cyst repair. Monolayer repair was enabled by providing 'cells' a control mechanism to switch automatically to a repair mode in the presence of a distress signal. In cyst wound simulations, the revised analogue closed wounds by adhering to essentially the same axioms available for alveolar-like cystogenesis. In silico cell proliferation was not needed. The analogue recovered within a few simulation cycles but required a longer recovery time for larger or multiple wounds. In simulated monolayer wound repair, diffusive factor-mediated 'cell' migration led to repair patterns comparable to those of in vitro cultures exposed to different growth factors. Simulations predicted directional cell locomotion to be critical for successful in vitro wound repair. We anticipate that with further use and refinement, the methods used will develop as a rigorous, extensible means of unravelling mechanisms of lung alveolar repair and regeneration.

  2. Differential effects of cigarette smoke on oxidative stress and proinflammatory cytokine release in primary human airway epithelial cells and in a variety of transformed alveolar epithelial cells

    Directory of Open Access Journals (Sweden)

    Rahman Irfan

    2006-10-01

    Full Text Available Abstract Background Cigarette smoke mediated oxidative stress and inflammatory events in the airway and alveolar epithelium are important processes in the pathogenesis of smoking related pulmonary diseases. Previously, individual cell lines were used to assess the oxidative and proinflammatory effects of cigarette smoke with confounding results. In this study, a panel of human and rodent transformed epithelial cell lines were used to determine the effects of cigarette smoke extract (CSE on oxidative stress markers, cell toxicity and proinflammatory cytokine release and compared the effects with that of primary human small airway epithelial cells (SAEC. Methods Primary human SAEC, transformed human (A549, H1299, H441, and rodent (murine MLE-15, rat L2 alveolar epithelial cells were treated with different concentrations of CSE (0.2–10% ranging from 20 min to 24 hr. Cytotoxicity was assessed by lactate dehydrogenase release assay, trypan blue exclusion method and double staining with acridine orange and ethidium bromide. Glutathione concentration was measured by enzymatic recycling assay and 4-hydroxy-2-nonenal levels by using lipid peroxidation assay kit. The levels of proinflammatory cytokines (e.g. IL-8 and IL-6 were measured by ELISA. Nuclear translocation of the transcription factor, NF-κB was assessed by immunocytochemistry and immunoblotting. Results Cigarette smoke extract dose-dependently depleted glutathione concentration, increased 4-hydroxy-2-nonenal (4-HNE levels, and caused necrosis in the transformed cell lines as well as in SAEC. None of the transformed cell lines showed any significant release of cytokines in response to CSE. CSE, however, induced IL-8 and IL-6 release in primary cell lines in a dose-dependent manner, which was associated with the nuclear translocation of NF-κB in SAEC. Conclusion This study suggests that primary, but not transformed, lung epithelial cells are an appropriate model to study the inflammatory

  3. Responses of A549 human lung epithelial cells to cristobalite and α-quartz exposures assessed by toxicoproteomics and gene expression analysis.

    Science.gov (United States)

    Vuong, Ngoc Q; Goegan, Patrick; De Rose, Francesco; Breznan, Dalibor; Thomson, Errol M; O'Brien, Julie S; Karthikeyan, Subramanian; Williams, Andrew; Vincent, Renaud; Kumarathasan, Premkumari

    2017-06-01

    In this study, we used cytotoxicity assays, proteomic and gene expression analyses to examine the difference in response of A549 cells to two silica particles that differ in physical properties, namely cristobalite (CR) and α-quartz (Min-U-Sil 5, MI). Cytotoxicity assays such as lactate dehydrogenase release, 5-bromo-2'-deoxyuridine incorporation and cellular ATP showed that both silica particles could cause cell death, decreased cell proliferation and metabolism in the A549 human lung epithelial cells. While cytotoxicity assays revealed little difference between CR and MI exposures, proteomic and gene expression analyses unveiled both similar and unique molecular changes in A549 cells. For instance, two-dimensional gel electrophoresis data indicated that the expression of proteins in the cell death (e.g., ALDH1A1, HTRA2 and PRDX6) and cell proliferation (e.g., FSCN1, HNRNPAB and PGK1) pathways were significantly different between the two silica particles. Reverse transcription-polymerase chain reaction data provided additional evidence supporting the proteomic findings. Preliminary assessment of the physical differences between CR and MI suggested that the extent of surface interaction between particles and cells could explain some of the observed biological effects. However, the differential dose-response curves for some other genes and proteins suggest that other physical attributes of particulate matter can also contribute to particulate matter-related cellular toxicity. Our results demonstrated that toxicoproteomic and gene expression analyses are sensitive in distinguishing subtle toxicity differences associated with silica particles of varying physical properties compared to traditional cytotoxicity endpoints. Copyright © 2016 Her Majesty the Queen in Right of Canada. Journal of Applied Toxicology published by John Wiley & Sons, Ltd. Copyright © 2016 Her Majesty the Queen in Right of Canada. Journal of Applied Toxicology published by John Wiley & Sons

  4. Differential effects of hypoxic stress in alveolar epithelial cells and microvascular endothelial cells

    NARCIS (Netherlands)

    Signorelli, Sara; Jennings, Paul; Leonard, Martin O; Pfaller, Walter

    2010-01-01

    Under hypoxic conditions eukaryotic cells and tissues undergo adaptive responses involving glycolysis, angiogenesis, vasoconstriction and inflammation. The underlying molecular mechanisms are not yet fully elucidated and are most likely cell and tissue specific. In the lung, alveolar epithelial

  5. HIV-1 transgene expression in rats causes oxidant stress and alveolar epithelial barrier dysfunction

    Directory of Open Access Journals (Sweden)

    Jacob Barbara A

    2009-02-01

    Full Text Available Abstract Background HIV-infected individuals are at increased risk for acute and chronic airway disease even though there is no evidence that the virus can infect the lung epithelium. Although HIV-related proteins including gp120 and Tat can directly cause oxidant stress and cellular dysfunction, their effects in the lung are unknown. The goal of this study was to determine the effects of HIV-1 transgene expression in rats on alveolar epithelial barrier function. Alveolar epithelial barrier function was assessed by determining lung liquid clearance in vivo and alveolar epithelial monolayer permeability in vitro. Oxidant stress in the alveolar space was determined by measuring the glutathione redox couple by high performance liquid chromatography, and the expression and membrane localization of key tight junction proteins were assessed. Finally, the direct effects of the HIV-related proteins gp120 and Tat on alveolar epithelial barrier formation and tight junction protein expression were determined. Results HIV-1 transgene expression caused oxidant stress within the alveolar space and impaired epithelial barrier function even though there was no evidence of overt inflammation within the airways. The expression and membrane localization of the tight junction proteins zonula occludens-1 and occludin were decreased in alveolar epithelial cells from HIV-1 transgenic rats. Further, treating alveolar epithelial monolayers from wild type rats in vitro with recombinant gp120 or Tat for 24 hours reproduced many of the effects on zonula occludens-1 and occludin expression and membrane localization. Conclusion Taken together, these data indicate that HIV-related proteins cause oxidant stress and alter the expression of critical tight junction proteins in the alveolar epithelium, resulting in barrier dysfunction.

  6. Airborne particulate matter in vitro exposure induces cytoskeleton remodeling through activation of the ROCK-MYPT1-MLC pathway in A549 epithelial lung cells.

    Science.gov (United States)

    Chirino, Yolanda I; García-Cuellar, Claudia María; García-García, Carlos; Soto-Reyes, Ernesto; Osornio-Vargas, Álvaro Román; Herrera, Luis A; López-Saavedra, Alejandro; Miranda, Javier; Quintana-Belmares, Raúl; Pérez, Irma Rosas; Sánchez-Pérez, Yesennia

    2017-04-15

    Airborne particulate matter with an aerodynamic diameter ≤10μm (PM 10 ) is considered a risk factor for the development of lung cancer. Little is known about the cellular mechanisms by which PM 10 is associated with cancer, but there is evidence that its exposure can lead to an acquired invasive phenotype, apoptosis evasion, inflammasome activation, and cytoskeleton remodeling in lung epithelial cells. Cytoskeleton remodeling occurs through actin stress fiber formation, which is partially regulated through ROCK kinase activation, we aimed to investigate if this protein was activated in response to PM 10 exposure in A549 lung epithelial cells. Results showed that 10μg/cm 2 of PM 10 had no influence on cell viability but increased actin stress fibers, cytoplasmic ROCK expression, and phosphorylation of myosin phosphatase-targeting 1 (MYPT1) and myosin light chain (MLC) proteins, which are targeted by ROCK. The inhibition of ROCK prevented actin stress fiber formation and the phosphorylation of MYPT1 and MLC, suggesting that PM 10 activated the ROCK-MYPT1-MLC pathway in lung epithelial cells. The activation of ROCK1 has been involved in the acquisition of malignant phenotypes, and its induction by PM 10 exposure could contribute to the understanding of PM 10 as a risk factor for cancer development through the mechanisms associated with invasive phenotype. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. The expression of NADPH oxidases and production of reactive oxygen species by human lung adenocarcinoma epithelial cell line A549

    Czech Academy of Sciences Publication Activity Database

    Kolářová, Hana; Binó, Lucia; Pejchalová, Kateřina; Kubala, Lukáš

    2010-01-01

    Roč. 56, č. 5 (2010), s. 211-217 ISSN 0015-5500 R&D Projects: GA ČR(CZ) GA524/06/1197; GA ČR(CZ) GA524/08/1753 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : lung epithelial cells * reactive oxygen species * NADPH oxidase Subject RIV: BO - Biophysics Impact factor: 0.729, year: 2010

  8. Bile acids induce activation of alveolar epithelial cells and lung fibroblasts through farnesoid X receptor-dependent and independent pathways.

    Science.gov (United States)

    Chen, Bi; Cai, Hou-Rong; Xue, Shan; You, Wen-Jie; Liu, Bin; Jiang, Han-Dong

    2016-08-01

    The roles of bile acid microaspiration and bile acid-activated farnesoid X receptor (FXR) in the pathogenesis of idiopathic pulmonary fibrosis (IPF) remain unclear. We hypothesized that bile acids activate alveolar epithelial cells (AECs) and lung fibroblasts, which may be regulated by FXR activation. Human AECs and normal or IPF-derived lung fibroblast cells were incubated with the three major bile acids: lithocholic acid (LCA), deoxycholic acid (DCA) and chenodeoxycholic acid (CDCA). The AECs injury indices, epithelial-mesenchymal transition (EMT) and lung fibroblast activation were evaluated. FXR expression in IPF lungs and the roles of FXR and FXR-independent pathways in bile acid-induced profibrotic effects were also investigated. LCA, DCA and CDCA reduced cell viability and increased intracellular reactive oxygen species (ROS) production in A549 cells. They all induced EMT, as shown by enhanced α-SMA and vimentin and decreased E-cadherin levels. LCA directly induced differentiation of lung fibroblasts to myofibroblasts. All three bile acids promoted cellular migration but not proliferation of lung fibroblasts. FXR expression was upregulated in IPF lungs, and inhibition of FXR restrained the bile acid-induced EMT and lung fibroblast activation. Differentiation and proliferation were enhanced in lung fibroblasts exposed to conditioned medium from bile acid-stimulated A549 cells, which contained increased levels of profibrotic factors. TGF-β/Smad3 signaling was also involved in the bile acid-induced EMT and lung fibroblast differentiation. Bile acid microaspiration may promote the development of pulmonary fibrosis by inducing activation of AECs and lung fibroblasts via FXR-dependent and independent pathways. © 2016 Asian Pacific Society of Respirology.

  9. A micRNA-200c/cathepsin L feedback loop determines paclitaxel resistance in human lung cancer A549 cells in vitro through regulating epithelial-mesenchymal transition.

    Science.gov (United States)

    Zhao, Yi-Fan; Han, Mei-Ling; Xiong, Ya-Jie; Wang, Long; Fei, Yao; Shen, Xiao; Zhu, Ying; Liang, Zhong-Qin

    2017-12-07

    Cathepsin L (CTSL), a cysteine protease, is closely related to tumor occurrence, development, and metastasis, and possibly regulates cancer cell resistance to chemotherapy. miRNAs, especially the miR-200 family, have been implicated in drug-resistant tumors. In this study we explored the relationship of CTSL, micRNA-200c and drug resistance, and the potential regulatory mechanisms in human lung cancer A549 cells and A549/TAX cells in vitro. A549/TAX cells were paclitaxel-resistant A549 cells overexpressing CTSL and characterized by epithelial-mesenchymal transition (EMT). We showed that micRNA-200c and CTSL were reciprocally linked in a feedback loop in these cancer cells. Overexpression of micRNA-200c in A549/TAX cells decreased the expression of CTSL, and enhanced their sensitivity to paclitaxel and suppressed EMT, whereas knockdown of micRNA-200c in A549 cells significantly increased the expression of CTSL, and decreased their sensitivity to paclitaxel and induced EMT. Overexpression of CTSL in A549 cells significantly decreased the expression of micRNA-200c, and reduced their sensitivity to paclitaxel and induced EMT, but these effects were reversed by micRNA-200c, whereas knockdown of CTSL in A549/TAX cells attenuated paclitaxel resistance and remarkably inhibited EMT, but the inhibition of micRNA-200c could reverse these effects. Therefore, micRNA-200c may be involved in regulating paclitaxel resistance through CTSL-mediated EMT in A549 cells, and CTSL and micRNA-200c are reciprocally linked in a feedback loop.

  10. Subtoxic and toxic concentrations of benzene and toluene induce Nrf2-mediated antioxidative stress response and affect the central carbon metabolism in lung epithelial cells A549.

    Science.gov (United States)

    Murugesan, Kalaimathi; Baumann, Sven; Wissenbach, Dirk K; Kliemt, Stefanie; Kalkhof, Stefan; Otto, Wolfgang; Mögel, Iljana; Kohajda, Tibor; von Bergen, Martin; Tomm, Janina M

    2013-11-01

    Since people in industrialized countries spend most of their time indoors, the effects of indoor contaminants such as volatile organic compounds become more and more relevant. Benzene and toluene are among the most abundant compounds in the highly heterogeneous group of indoor volatile organic compounds. In order to understand their effects on lung epithelial cells (A549) representing lung's first line of defense, we chose a global proteome and a targeted metabolome approach in order to detect adverse outcome pathways caused by exposure to benzene and toluene. Using a DIGE approach, 93 of 469 detected protein spots were found to be differentially expressed after exposure to benzene, and 79 of these spots were identified by MS. Pathway analysis revealed an enrichment of proteins involved in Nrf2-mediated and oxidative stress response glycolysis/gluconeogenesis. The occurrence of oxidative stress at nonacute toxic concentrations of benzene and toluene was confirmed by the upregulation of the stress related proteins NQO1 and SOD1. The changes in metabolism were validated by ion chromatography MS/MS analysis revealing significant changes of glucose-6-phosphate, fructose-6-phosphate, 3-phosphoglycerate, and NADPH. The molecular alterations identified as a result of benzene and toluene exposure demonstrate the detrimental effect of nonacute toxic concentrations on lung epithelial cells. The data provided here will allow for a targeted validation in in vivo models. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Toxic Effects of the Major Components of Diesel Exhaust in Human Alveolar Basal Epithelial Cells (A549)

    Czech Academy of Sciences Publication Activity Database

    Rössner ml., Pavel; Strapáčová, S.; Štolcpartová, Jitka; Schmuczerová, Jana; Milcová, Alena; Neča, J.; Vlková, Veronika; Brzicová, Táňa; Machala, M.; Topinka, Jan

    2016-01-01

    Roč. 17, č. 9 (2016), s. 1393 E-ISSN 1422-0067 R&D Projects: GA ČR(CZ) GA13-01438S; GA ČR(CZ) GA16-14631S; GA MŠk(CZ) LO1508; GA MŠk(CZ) LM2015073 Institutional support: RVO:68378041 Keywords : benzo[a]pyrene * 1-nitropyrene * 3-nitrobenzanthrone Subject RIV: DN - Health Impact of the Environment Quality Impact factor: 3.226, year: 2016

  12. Water-pipe smoke condensate increases the internalization of Mycobacterium Bovis of type II alveolar epithelial cells (A549)

    NARCIS (Netherlands)

    Mortaz, Esmaeil; Alipoor, Shamila D.; Movassaghi, Masoud; Varahram, Mohammad; Ghorbani, Jahangir; Folkerts, Gert; Garssen, Johan; Adcock, Ian M

    2017-01-01

    Background: Tuberculosis (TB) is a major global health problem, and there is an association between tobacco smoke and TB. Water pipe smoking has become an increasing problem not only in Middle Eastern countries but also globally because users consider it as safer than cigarettes. The presence of

  13. Novel functional view of the crocidolite asbestos-treated A549 human lung epithelial transcriptome reveals an intricate network of pathways with opposing functions

    Directory of Open Access Journals (Sweden)

    Stevens John R

    2008-08-01

    Full Text Available Abstract Background Although exposure to asbestos is now regulated, patients continue to be diagnosed with mesothelioma, asbestosis, fibrosis and lung carcinoma because of the long latent period between exposure and clinical disease. Asbestosis is observed in approximately 200,000 patients annually and asbestos-related deaths are estimated at 4,000 annually1. Although advances have been made using single gene/gene product or pathway studies, the complexity of the response to asbestos and the many unanswered questions suggested the need for a systems biology approach. The objective of this study was to generate a comprehensive view of the transcriptional changes induced by crocidolite asbestos in A549 human lung epithelial cells. Results A statistically robust, comprehensive data set documenting the crocidolite-induced changes in the A549 transcriptome was collected. A systems biology approach involving global observations from gene ontological analyses coupled with functional network analyses was used to explore the effects of crocidolite in the context of known molecular interactions. The analyses uniquely document a transcriptome with function-based networks in cell death, cancer, cell cycle, cellular growth, proliferation, and gene expression. These functional modules show signs of a complex interplay between signaling pathways consisting of both novel and previously described asbestos-related genes/gene products. These networks allowed for the identification of novel, putative crocidolite-related genes, leading to several new hypotheses regarding genes that are important for the asbestos response. The global analysis revealed a transcriptome that bears signatures of both apoptosis/cell death and cell survival/proliferation. Conclusion Our analyses demonstrate the power of combining a statistically robust, comprehensive dataset and a functional network genomics approach to 1 identify and explore relationships between genes of known importance

  14. CCR2 and CXCR3 agonistic chemokines are differently expressed and regulated in human alveolar epithelial cells type II

    Directory of Open Access Journals (Sweden)

    Prasse Antje

    2005-07-01

    Full Text Available Abstract The attraction of leukocytes from circulation to inflamed lungs depends on the activation of both the leukocytes and the resident cells within the lung. In this study we determined gene expression and secretion patterns for monocyte chemoattractant protein-1 (MCP-1/CCL2 and T-cell specific CXCR3 agonistic chemokines (Mig/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11 in TNF-α-, IFN-γ-, and IL-1β-stimulated human alveolar epithelial cells type II (AEC-II. AEC-II constitutively expressed high level of CCL2 mRNA in vitro and in situ , and released CCL2 protein in vitro . Treatment of AEC-II with proinflammatory cytokines up-regulated both CCL2 mRNA expression and release of immunoreactive CCL2, whereas IFN-γ had no effect on CCL2 release. In contrast, CXCR3 agonistic chemokines were not detected in freshly isolated AEC-II or in non-stimulated epithelial like cell line A549. IFN-γ, alone or in combination with IL-1β and TNF-α resulted in an increase in CXCL10, CXCL11, and CXCL9 mRNA expression and generation of CXCL10 protein by AEC-II or A549 cells. CXCL10 gene expression and secretion were induced in dose-dependent manner after cytokine-stimulation of AEC-II with an order of potency IFN-γ>>IL-1β ≥ TNF-α. Additionally, we localized the CCL2 and CXCL10 mRNAs in human lung tissue explants by in situ hybridization, and demonstrated the selective effects of cytokines and dexamethasone on CCL2 and CXCL10 expression. These data suggest that the regulation of the CCL2 and CXCL10 expression exhibit significant differences in their mechanisms, and also demonstrate that the alveolar epithelium contributes to the cytokine milieu of the lung, with the ability to respond to locally generated cytokines and to produce potent mediators of the local inflammatory response.

  15. Physicochemical characterization of ambient PM2.5in Tehran air and its potential cytotoxicity in human lung epithelial cells (A549).

    Science.gov (United States)

    MohseniBandpi, Anoushiravan; Eslami, Akbar; Shahsavani, Abbas; Khodagholi, Fariba; Alinejad, Abdolazim

    2017-09-01

    As air pollution is a major problem in Tehran, this study aimed to investigate the physicochemical characterization of the water-soluble and organic contents of ambient PM 2.5 in Tehran and determine its in vitro toxicological impact on human lung epithelial cells (A549). A total of 11 sampling stations were selected, including three categories: traffic, urban, and suburban. All sampling was carried out in the spring and summer of 2015. Ion chromatography (IC), inductively coupled plasma atomic emission spectroscopy (ICP-AES), and GC-MS were used to analyze ionic compounds, heavy metals, and polycyclic aromatic hydrocarbons (PAHs), respectively, and an ELISA reader was used for cytotoxicity analysis. The most prevalent ionic species found for all three categories was SO 4 2- . PAH concentrations were 43.45±32.71, 50.51±37.27, and 29.13±33.29ng/m 3 for traffic, urban, and suburban stations, respectively. For all sampling stations, Al and Fe had the highest values among the investigated heavy metals. Cell viability measurements, carried out using the MTT assay, showed that all three categories of samples cause cytotoxicity, although the urban station samples showed higher cytotoxicity than those from the other stations (p˂0.05). Based on the results of the present study, organic compounds and insoluble particles could be the main causes of cytotoxicity. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Differential regulation of epidermal growth factor receptor by hydrogen peroxide and flagellin in cultured lung alveolar epithelial cells.

    Science.gov (United States)

    Nishi, Hiroyuki; Maeda, Noriko; Izumi, Shunsuke; Higa-Nakamine, Sayomi; Toku, Seikichi; Kakinohana, Manabu; Sugahara, Kazuhiro; Yamamoto, Hideyuki

    2015-02-05

    In previous studies, we found that stimulation of Toll-like receptor 5 (TLR5) by flagellin induced the activation of mitogen-activated protein kinase (MAPK)-activated protein kinase-2 (MAPKAPK-2) through activation of the p38 MAPK pathway in cultured alveolar epithelial A549 cells. Our studies strongly suggested that MAPKAPK-2 phosphorylated epidermal growth factor receptor (EGFR) at Ser1047. It has been reported that phosphorylation of Ser1047 after treatment with tumor necrosis factor α (TNFα) induced the internalization of EGFR. In the present study, we first found that treatment of A549 cells with hydrogen peroxide induced the activation of MAPKAPK-2 and phosphorylation of EGFR at Ser1047 within 30 min. This was different from flagellin treatment because hydrogen peroxide treatment induced the phosphorylation of EGFR at Tyr1173 as well as Ser1047, indicating the activation of EGFR. We also found that KN93, an inhibitor of CaM kinase II, inhibited the hydrogen peroxide-induced phosphorylation of EGFR at Ser1047 through inhibition of the activation of the p38 MAPK pathway. Furthermore, we examined the internalization of EGFR by three different methods. Flow cytometry with an antibody against the extracellular domain of EGFR and biotinylation of cell surface proteins revealed that flagellin, but not hydrogen peroxide, decreased the amount of cell-surface EGFR. In addition, activation of extracellular signal-regulated kinase by EGF treatment was reduced by flagellin pre-treatment. These results strongly suggested that hydrogen peroxide activated the p38 MAPK pathway via activation of CaM kinase II and that flagellin and hydrogen peroxide regulate the functions of EGFR by different mechanisms. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. The Milieu of Damaged Alveolar Epithelial Type 2 Cells Stimulates Alveolar Wound Repair by Endogenous and Exogenous Progenitors

    Science.gov (United States)

    Buckley, Susan; Shi, Wei; Carraro, Gianni; Sedrakyan, Sargis; Da Sacco, Stefano; Driscoll, Barbara A.; Perin, Laura; De Filippo, Roger E.

    2011-01-01

    Alveolar epithelial integrity is dependent upon the alveolar milieu, yet the milieu of the damaged alveolar epithelial cell type 2 (AEC2) has been little studied. Characterization of its components may offer the potential for ex vivo manipulation of stem cells to optimize their therapeutic potential. We examined the cytokine profile of AEC2 damage milieu, hypothesizing that it would promote endogenous epithelial repair while recruiting cells from other locations and instructing their engraftment and differentiation. Bronchoalveolar lavage and lung extract from hyperoxic rats represented AEC2 in vivo damage milieu, and medium from a scratch-damaged AEC2 monolayer represented in vitro damage. CINC-2 and ICAM, the major cytokines detected by proteomic cytokine array in AEC2 damage milieu, were chemoattractive to normoxic AECs and expedited in vitro wound healing, which was blocked by their respective neutralizing antibodies. The AEC2 damage milieu was also chemotactic for exogenous uncommitted human amniotic fluid stem cells (hAFSCs), increasing migration greater than 20-fold. hAFSCs attached within an in vitro AEC2 wound and expedited wound repair by contributing cytokines migration inhibitory factor and plasminogen activator inhibitor 1 to the AEC2 damage milieu, which promoted wound healing. The AEC2 damage milieu also promoted differentiation of a subpopulation of hAFSCs to express SPC, TTF-1, and ABCA3, phenotypic markers of distal alveolar epithelium. Thus, the microenvironment created by AEC2 damage not only promotes autocrine repair but also can attract uncommitted stem cells, which further augment healing through cytokine secretion and differentiation. PMID:21700959

  18. Effect of exogenous surfactants on viability and DNA synthesis in A549, immortalized mouse type II and isolated rat alveolar type II cells

    NARCIS (Netherlands)

    Wemhöner, Andreas; Jennings, Paul; Haller, Thomas; Rüdiger, Mario; Simbruner, Georg

    2011-01-01

    BACKGROUND: In mechanically ventilated preterm infants with respiratory distress syndrome (RDS), exogenous surfactant application has been demonstrated both to decrease DNA-synthesis but also and paradoxically to increase epithelial cell proliferation. However, the effect of exogenous surfactant has

  19. Exposure to Bordetella pertussis adenylate cyclase toxin affects integrin-mediated adhesion and mechanics in alveolar epithelial cells.

    Science.gov (United States)

    Angely, Christelle; Nguyen, Ngoc-Minh; Andre Dias, Sofia; Planus, Emmanuelle; Pelle, Gabriel; Louis, Bruno; Filoche, Marcel; Chenal, Alexandre; Ladant, Daniel; Isabey, Daniel

    2017-08-01

    The adenylate cyclase (CyaA) toxin is a major virulent factor of Bordetella pertussis, the causative agent of whooping cough. CyaA toxin is able to invade eukaryotic cells where it produces high levels of cyclic adenosine monophosphate (cAMP) affecting cellular physiology. Whether CyaA toxin can modulate cell matrix adhesion and mechanics of infected cells remains largely unknown. In this study, we use a recently proposed multiple bond force spectroscopy (MFS) with an atomic force microscope to assess the early phase of cell adhesion (maximal detachment and local rupture forces) and cell rigidity (Young's modulus) in alveolar epithelial cells (A549) for toxin exposure 95%) at CyaA concentration of 0.5 nM, but a significant effect (≈81%) at 10 nM. MFS performed on A549 for three different concentrations (0.5, 5 and 10 nM) demonstrates that CyaA toxin significantly affects both cell adhesion (detachment forces are decreased) and cell mechanics (Young's modulus is increased). CyaA toxin (at 0.5 nM) assessed at three indentation/retraction speeds (2, 5 and 10 μm/s) significantly affects global detachment forces, local rupture events and Young modulus compared with control conditions, while an enzymatically inactive variant CyaAE5 has no effect. These results reveal the loading rate dependence of the multiple bonds newly formed between the cell and integrin-specific coated probe as well as the individual bond kinetics which are only slightly affected by the patho-physiological dose of CyaA toxin. Finally, theory of multiple bond force rupture enables us to deduce the bond number N which is reduced by a factor of 2 upon CyaA exposure (N ≈ 6 versus N ≈ 12 in control conditions). MFS measurements demonstrate that adhesion and mechanical properties of A549 are deeply affected by exposure to the CyaA toxin but not to an enzymatically inactive variant. This indicates that the alteration of cell mechanics triggered by CyaA is a consequence of the increase in

  20. Cultured alveolar epithelial cells from septic rats mimic in vivo septic lung.

    Directory of Open Access Journals (Sweden)

    Taylor S Cohen

    2010-06-01

    Full Text Available Sepsis results in the formation of pulmonary edema by increasing in epithelial permeability. Therefore we hypothesized that alveolar epithelial cells isolated from septic animals develop tight junctions with different protein composition and reduced barrier function relative to alveolar epithelial cells from healthy animals. Male rats (200-300 g were sacrificed 24 hours after cecal ligation and double puncture (2CLP or sham surgery. Alveolar epithelial cells were isolated and plated on fibronectin-coated flexible membranes or permeable, non-flexible transwell substrates. After a 5 day culture period, cells were either lysed for western analysis of tight junction protein expressin (claudin 3, 4, 5, 7, 8, and 18, occludin, ZO-1, and JAM-A and MAPk (JNK, ERK, an p38 signaling activation, or barrier function was examined by measuring transepithelial resistance (TER or the flux of two molecular tracers (5 and 20 A. Inhibitors of JNK (SP600125, 20 microM and ERK (U0126, 10 microM were used to determine the role of these pathways in sepsis induced epithelial barrier dysfunction. Expression of claudin 4, claudin 18, and occludin was significantly lower, and activation of JNK and ERK signaling pathways was significantly increased in 2CLP monolayers, relative to sham monolayers. Transepithelial resistance of the 2CLP monolayers was reduced significantly compared to sham (769 and 1234 ohm-cm(2, respectively, however no significant difference in the flux of either tracer was observed. Inhibition of ERK, not JNK, significantly increased TER and expression of claudin 4 in 2CLP monolayers, and prevented significant differences in claudin 18 expression between 2CLP and sham monolayers. We conclude that alveolar epithelial cells isolated from septic animals form confluent monolayers with impaired barrier function compared to healthy monolayers, and inhibition of ERK signaling partially reverses differences between these monolayers. This model provides a unique

  1. DNA damage and cytotoxicity in type II lung epithelial (A549 cell cultures after exposure to diesel exhaust and urban street particles

    Directory of Open Access Journals (Sweden)

    Møller Peter

    2008-04-01

    Full Text Available Abstract Background Exposure to air pollution particles has been acknowledged to be associated with excess generation of oxidative damage to DNA in experimental model systems and humans. The use of standard reference material (SRM, such as SRM1650 and SRM2975, is advantageous because experiments can be reproduced independently, but exposure to such samples may not mimic the effects observed after exposure to authentic air pollution particles. This study was designed to compare the DNA oxidizing effects of authentic street particles with SRM1650 and SRM2975. The authentic street particles were collected at a traffic intensive road in Copenhagen, Denmark. Results All of the particles generated strand breaks and oxidized purines in A549 lung epithelial cells in a dose-dependent manner and there were no overt differences in their potency. The exposures also yielded dose-dependent increase of cytotoxicity (as lactate dehydrogenase release and reduced colony forming ability with slightly stronger cytotoxicity of SRM1650 than of the other particles. In contrast, only the authentic street particles were able to generate 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG in calf thymus DNA, which might be due to the much higher level of transition metals. Conclusion Authentic street particles and SRMs differ in their ability to oxidize DNA in a cell-free environment, whereas cell culture experiments indicate that the particle preparations elicit a similar alteration of the level of DNA damage and small differences in cytotoxicity. Although it cannot be ruled out that SRMs and authentic street particles might elicit different effects in animal experimental models, this study indicates that on the cellular level, SRM1650 and SRM2975 are suitable surrogate samples for the study of authentic street particles.

  2. The combined effects of physicochemical properties of size-fractionated ambient particulate matter on in vitro toxicity in human A549 lung epithelial cells

    Directory of Open Access Journals (Sweden)

    Umme S. Akhtar

    2014-01-01

    Full Text Available Epidemiological and toxicological studies have suggested that the health effects associated with exposure to particulate matter (PM are related to the different physicochemical properties of PM. These effects occur through the initiation of differential cellular responses including: the induction of antioxidant defenses, proinflammatory responses, and ultimately cell death. The main objective of this study was to investigate the effects of size-fractionated ambient PM on epithelial cells in relation to their physicochemical properties. Concentrated ambient PM was collected on filters for three size fractions: coarse (aerodynamic diameter [AD] 2.5–10 μm, fine (0.15–2.5 μm, and quasi-ultrafine (<0.2 μm, near a busy street in Toronto, Ontario, Canada. Filters were extracted and analyzed for chemical composition and redox activity. Chemical analyses showed that the coarse, fine, and quasi-ultrafine particles were comprised primarily of metals, water-soluble species, and organic compounds, respectively. The highest redox activity was observed for fine PM. After exposure of A549 cells to PM (10–100 μg/ml for 4 h, activation of antioxidant, proinflammatory and cytotoxic responses were assessed by determining the expression of heme oxygenase (HMOX-1, mRNA, interleukin-8 (IL-8, mRNA, and metabolic activity of the cells, respectively. All three size fractions induced mass-dependent antioxidant, proinflammatory, and cytotoxic responses to different degrees. Quasi-ultrafine PM caused significant induction of HMOX-1 at the lowest exposure dose. Correlation analyses with chemical components suggested that the biological responses correlated mainly with transition metals and organic compounds for coarse and fine PM and with organic compounds for quasi-ultrafine PM. Overall, the observed biological responses appeared to be related to the combined effects of size and chemical composition and thus both of these physicochemical properties should be

  3. Alveolar epithelial type II cells induce T cell tolerance to specific antigen

    DEFF Research Database (Denmark)

    Lo, Bernice; Hansen, Søren; Evans, Kathy

    2008-01-01

    The lungs face the immunologic challenge of rapidly eliminating inhaled pathogens while maintaining tolerance to innocuous Ags. A break in this immune homeostasis may result in pulmonary inflammatory diseases, such as allergies or asthma. The observation that alveolar epithelial type II cells (Ty...

  4. Activated alveolar epithelial cells initiate fibrosis through autocrine and paracrine secretion of connective tissue growth factor.

    Science.gov (United States)

    Yang, Jibing; Velikoff, Miranda; Canalis, Ernesto; Horowitz, Jeffrey C; Kim, Kevin K

    2014-04-15

    Fibrogenesis involves a pathological accumulation of activated fibroblasts and extensive matrix remodeling. Profibrotic cytokines, such as TGF-β, stimulate fibroblasts to overexpress fibrotic matrix proteins and induce further expression of profibrotic cytokines, resulting in progressive fibrosis. Connective tissue growth factor (CTGF) is a profibrotic cytokine that is indicative of fibroblast activation. Epithelial cells are abundant in the normal lung, but their contribution to fibrogenesis remains poorly defined. Profibrotic cytokines may activate epithelial cells with protein expression and functions that overlap with the functions of active fibroblasts. We found that alveolar epithelial cells undergoing TGF-β-mediated mesenchymal transition in vitro were also capable of activating lung fibroblasts through production of CTGF. Alveolar epithelial cell expression of CTGF was dramatically reduced by inhibition of Rho signaling. CTGF reporter mice demonstrated increased CTGF promoter activity by lung epithelial cells acutely after bleomycin in vivo. Furthermore, mice with lung epithelial cell-specific deletion of CTGF had an attenuated fibrotic response to bleomycin. These studies provide direct evidence that epithelial cell activation initiates a cycle of fibrogenic effector cell activation during progressive fibrosis. Therapy targeted at epithelial cell production of CTGF offers a novel pathway for abrogating this progressive cycle and limiting tissue fibrosis.

  5. Eukaryotic translation initiation factor 2 subunit α (eIF2α) inhibitor salubrinal attenuates paraquat-induced human lung epithelial-like A549 cell apoptosis by regulating the PERK-eIF2α signaling pathway.

    Science.gov (United States)

    Wang, Rui; Sun, Da-Zhuang; Song, Chun-Qing; Xu, Yong-Min; Liu, Wei; Liu, Zhi; Dong, Xue-Song

    2018-02-01

    Paraquat (PQ), as one of the most widely used herbicides in the world, can cause severe lung damage in humans and animals. This study investigated the underlying molecular mechanism of PQ-induced lung cell damage and the protective role of salubrinal. Human lung epithelial-like A549 cells were treated with PQ for 24h and were pre-incubated with salubrinal for 2h, followed by 500μM of PQ treatment. Silencing eIF2α gene of the A549 cells with siRNA interference method was conducted. Cell morphology, cell viability, apoptosis and caspase-3 activity were assessed by different assays accordingly thereafter. The expression of PERK, p-PERK, ATF6, c-ATF6, IRE1α, p-IRE1α, CHOP, GRP78, p-eIF2α and β-actin was assayed by western blot. The data showed that PQ significantly reduced A549 cell viability, changed cell morphology, induced cell apoptosis and significantly upregulated the levels of GRP78, CHOP, p-PERK, c-ATF6 and p-IRE1α. However, 30μM salubrinal could attenuate the effects of PQ on damages to A549 cells through upregulating p-eIF2α. In contrast, knocking down eIF2α gene inhabited the effects of salubrinal. These results suggest that PQ-induced A549 cell apoptosis involved endoplasmic reticulum (ER) stress, specially the PERK-eIF2α pathway. Salubrinal attenuated A549 cells from PQ-induced damages through regulation of the PERK-eIF2α signaling. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Lipoteichoic acid induces surfactant protein-A biosynthesis in human alveolar type II epithelial cells through activating the MEK1/2-ERK1/2-NF-κB pathway

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    Liu Feng-Lin

    2012-10-01

    Full Text Available Abstract Background Lipoteichoic acid (LTA, a gram-positive bacterial outer membrane component, can cause septic shock. Our previous studies showed that the gram-negative endotoxin, lipopolysaccharide (LPS, could induce surfactant protein-A (SP-A production in human alveolar epithelial (A549 cells. Objectives In this study, we further evaluated the effect of LTA on SP-A biosynthesis and its possible signal-transducing mechanisms. Methods A549 cells were exposed to LTA. Levels of SP-A, nuclear factor (NF-κB, extracellular signal-regulated kinase 1/2 (ERK1/2, and mitogen-activated/extracellular signal-regulated kinase kinase (MEK1 were determined. Results Exposure of A549 cells to 10, 30, and 50 μg/ml LTA for 24 h did not affect cell viability. Meanwhile, when exposed to 30 μg/ml LTA for 1, 6, and 24 h, the biosynthesis of SP-A mRNA and protein in A549 cells significantly increased. As to the mechanism, LTA enhanced cytosolic and nuclear NF-κB levels in time-dependent manners. Pretreatment with BAY 11–7082, an inhibitor of NF-κB activation, significantly inhibited LTA-induced SP-A mRNA expression. Sequentially, LTA time-dependently augmented phosphorylation of ERK1/2. In addition, levels of phosphorylated MEK1 were augmented following treatment with LTA. Conclusions Therefore, this study showed that LTA can increase SP-A synthesis in human alveolar type II epithelial cells through sequentially activating the MEK1-ERK1/2-NF-κB-dependent pathway.

  7. Association of advanced glycation end products with A549 cells, a human pulmonary epithelial cell line, is mediated by a receptor distinct from the scavenger receptor family and RAGE.

    Science.gov (United States)

    Nakano, Nahoko; Fukuhara-Takaki, Kaori; Jono, Tadashi; Nakajou, Keisuke; Eto, Nobuaki; Horiuchi, Seikoh; Takeya, Motohiro; Nagai, Ryoji

    2006-05-01

    Cellular interactions with advanced glycation end products (AGE)-modified proteins are known to induce several biological responses, not only endocytic uptake and degradation, but also the induction of cytokines and growth factors, combined responses that may be linked to the development of diabetic vascular complications. In this study we demonstrate that A549 cells, a human pulmonary epithelial cell line, possess a specific binding site for AGE-modified bovine serum albumin (AGE-BSA) (K(d) = 27.8 nM), and additionally for EN-RAGE (extracellular newly identified RAGE binding protein) (K(d) = 118 nM). Western blot and RT-PCR analysis showed that RAGE (receptor for AGE) is highly expressed on A549 cells, while the expression of other known AGE-receptors such as galectin-3 and SR-A (class A scavenger receptor), are below the level of detection. The binding of (125)I-AGE-BSA to these cells is inhibited by unlabeled AGE-BSA, but not by EN-RAGE. In contrast, the binding of (125)I-EN-RAGE is significantly inhibited by unlabeled EN-RAGE and soluble RAGE, but not by AGE-BSA. Our results indicate that A549 cells possess at least two binding sites, one specific for EN-RAGE and the other specific for AGE-BSA. The latter receptor on A549 cells is distinct from the scavenger receptor family and RAGE.

  8. Complementary roles of KCa3.1 channels and β1-integrin during alveolar epithelial repair.

    Science.gov (United States)

    Girault, Alban; Chebli, Jasmine; Privé, Anik; Trinh, Nguyen Thu Ngan; Maillé, Emilie; Grygorczyk, Ryszard; Brochiero, Emmanuelle

    2015-09-04

    Extensive alveolar epithelial injury and remodelling is a common feature of acute lung injury and acute respiratory distress syndrome (ARDS) and it has been established that epithelial regeneration, and secondary lung oedema resorption, is crucial for ARDS resolution. Much evidence indicates that K(+) channels are regulating epithelial repair processes; however, involvement of the KCa3.1 channels in alveolar repair has never been investigated before. Wound-healing assays demonstrated that the repair rates were increased in primary rat alveolar cell monolayers grown on a fibronectin matrix compared to non-coated supports, whereas an anti-β1-integrin antibody reduced it. KCa3.1 inhibition/silencing impaired the fibronectin-stimulated wound-healing rates, as well as cell migration and proliferation, but had no effect in the absence of coating. We then evaluated a putative relationship between KCa3.1 channel and the migratory machinery protein β1-integrin, which is activated by fibronectin. Co-immunoprecipitation and immunofluorescence experiments indicated a link between the two proteins and revealed their cellular co-distribution. In addition, we demonstrated that KCa3.1 channel and β1-integrin membrane expressions were increased on a fibronectin matrix. We also showed increased intracellular calcium concentrations as well as enhanced expression of TRPC4, a voltage-independent calcium channel belonging to the large TRP channel family, on a fibronectin matrix. Finally, wound-healing assays showed additive effects of KCa3.1 and TRPC4 inhibitors on alveolar epithelial repair. Taken together, our data demonstrate for the first time complementary roles of KCa3.1 and TRPC4 channels with extracellular matrix and β1-integrin in the regulation of alveolar repair processes.

  9. RAGE-induced changes in the proteome of alveolar epithelial cells.

    Science.gov (United States)

    Downs, Charles A; Johnson, Nicholle M; Tsaprailis, George; Helms, My N

    2018-02-12

    The receptor for advanced glycation end-products (RAGE) is a pattern recognition receptor and member of the immunoglobulin superfamily. RAGE is constitutively expressed in the distal lung where it co-localizes with the alveolar epithelium; RAGE expression is otherwise minimal or absent, except with disease. This suggests RAGE plays a role in lung physiology and pathology. We used proteomics to identify and characterize the effects of RAGE on rat alveolar epithelial (R3/1) cells. LC-MS/MS identified 177 differentially expressed proteins and the PANTHER Classification System further segregated proteins. Proteins involved in gene transcription (RNA and mRNA splicing, mRNA processing) and transport (protein, intracellular protein) were overrepresented; genes involved in a response to stimulus were underrepresented. Immune system processes and response to stimuli were downregulated with RAGE knockdown. Western blot confirmed RAGE-dependent changes in protein expression for NFκB and NLRP3 that was functionally supported by a reduction in IL-1β and phosphorylated p65. We also assessed RAGE's effect on redox regulation and report that RAGE knockdown attenuated oxidant production, decreased protein oxidation, and increased reduced thiol pools. Collectively the data suggest that RAGE is a critical regulator of epithelial cell response and has implications for our understanding of lung disease, specifically acute lung injury. In the present study, we undertook the first proteomic evaluation of RAGE-dependent processes in alveolar epithelial cells. The alveolar epithelium is a primary target during acute lung injury, and our data support a role for RAGE in gene transcription, protein transport, and response to stimuli. More over our data suggest that RAGE is a critical driver of redox regulation in the alveolar epithelium. The conclusions of the present work assist to unravel the molecular events that underlie the function of RAGE in alveolar epithelial cells and have

  10. Smad2/3-Regulated Expression of DLX2 Is Associated with Radiation-Induced Epithelial-Mesenchymal Transition and Radioresistance of A549 and MDA-MB-231 Human Cancer Cell Lines

    Science.gov (United States)

    Choi, Yeo-Jin; Baek, Ga-Young; Park, Hae-Ran; Jo, Sung-Kee; Jung, Uhee

    2016-01-01

    The control of radioresistance and metastatic potential of surviving cancer cells is important for improving cancer eradication by radiotheraphy. The distal-less homeobox2 (DLX2) gene encodes for a homeobox transcription factor involved in morphogenesis and its deregulation was found in human solid tumors and hematologic malignancies. Here we investigated the role of DLX2 in association with radiation-induced epithelial to mesenchymal transition (EMT) and stem cell-like properties and its regulation by Smad2/3 signaling in irradiated A549 and MDA-MB-231 human cancer cell lines. In irradiated A549 and MDA-MB-231 cells, EMT was induced as demonstrated by EMT marker expression, phosphorylation of Smad2/3, and migratory and invasive ability. Also, irradiated A549 and MDA-MB-231 cells showed increased cancer stem cells (CSCs) marker. Interestingly, DLX2 was overexpressed upon irradiation. Therefore, we examined the role of DLX2 in radiation-induced EMT and radioresistance. The overexpression of DLX2 alone induced EMT, migration and invasion, and CSC marker expression. The reduced colony-forming ability in irradiated cells was partially restored by DLX2 overexpression. On the other hand, the depletion of DLX2 using si-RNA abolished radiation-induced EMT, CSC marker expression, and phosphorylation of Smad2/3 in irradiated A549 and MDA-MB-231 cells. Also, depletion of DLX2 increased the radiation sensitivity in both cell lines. Moreover, knockdown of Smad2/3, a key activator of TGF-β1 pathway, abrogated the radiation-induced DLX2 expression, indicating that radiation-induced DLX2 expression is dependent on Smad2/3 signaling. These results demonstrated that DLX2 plays a crucial role in radioresistance, radiation-induced EMT and CSC marker expression, and the expression of DLX2 is regulated by Smad2/3 signaling in A549 and MDA-MB-231 cell lines. PMID:26799321

  11. Characterization of the in vitro adhesion of Actinobacillus pleuropneumoniae to swine alveolar epithelial cells.

    Science.gov (United States)

    Van Overbeke, Ingrid; Chiers, Koen; Charlier, Gerard; Vandenberghe, Isabel; Van Beeumen, Jozef; Ducatelle, Richard; Haesebrouck, Freddy

    2002-08-02

    Actinobacillus pleuropneumoniae biovar 1 serotypes 2, 5a, 9 and 10 strains were tested for their ability to adhere to alveolar epithelial cells in culture. For the serotypes 5a, 9 and 10 strains, optimal adherence was observed after growth of bacterial cells in a NAD-restricted medium (0.001% NAD). This condition was also associated with the expression of a 55 kDa outer membrane protein (OMP) and of fimbriae. For the serotype 2 strain, adherence and expression of fimbriae and a 55 kDa OMP was less influenced by the growth conditions. The N-terminal amino acid sequence of the 55 kDa OMP had no homology with any known sequence, suggesting that it is an as yet unknown protein. Adherence capabilities were significantly reduced following treatment of the bacteria with proteolytic enzymes or heat. These findings suggest that proteins are involved in adhesion. The hydrophobic bond-breaking agent tetramethylurea was unable to inhibit the adherence of A. pleuropneumoniae to alveolar epithelial cells. Treatment of the bacteria with sodium metaperiodate resulted in lower adhesion scores for the serotypes 2 and 9 strains but the inhibition of adhesion was clearly lower than after treatment with proteolytic enzymes. This indicates that, besides proteins, carbohydrates might also be involved in adhesion of A. pleuropneumoniae to alveolar epithelial cells. The finding that inhibition of adhesion was very high when bacteria were treated with a combination of sodium metaperiodate and pronase also suggests that more than one adhesin is involved.

  12. In vivo metabolism of pulmonary alveolar epithelial type II pneumonocytes and macrophages from Syrian hamsters

    International Nuclear Information System (INIS)

    Pfleger, R.C.; Waide, J.J.

    1976-01-01

    Young adult Syrian hamsters were injected intraperitoneally with 14 C-glycerol and 3 H-palmitate 17 hr before they were sacrificed and pulmonary alveolar epithelial type II cells and pulmonary alveolar macrophages (PAM) were isolated. Incorporation of the two labeled components into the cellular lipids showed that the 3 H-specific activity of the phospholipids from the type II cells was three times that of the PAM and the utilization of 14 C-glycerol into phosphatidyl choline (PC) was 50% greater than incorporation into the PC from PAMs. The PC from type II cells showed that 30% was disaturated and from PAMs 21% was disaturated. Another phosphatide, phosphatidyl glycerol contained about one-third of the molecules in disaturated form. These data are consistent with the view that both type II cells and PAMs can synthesize surface-active phospholipids but it is generally accepted that only the pulmonary alveolar epithelial type II cells excrete the disaturated phospholipids which comprise the surface-active components of pulmonary surfactant

  13. G4-Tetra DNA Duplex Induce Lung Cancer Cell Apoptosis in A549 Cells

    Science.gov (United States)

    Xu, Xiaobo; Zhao, YiZhuo; Lu, Hu; Fu, Cuiping; Li, Xiao; Jiang, Liyan; Li, Shanqun

    2016-10-01

    The specific DNA is typically impermeable to the plasma membrane due to its natural characters, but DNA tetra structures (DTNs) can be readily uptake by cells in the absence of transfection agents, providing a new strategy to deliver DNA drugs. In this research, the delivery efficiency of tetrahedral DNA nanostructures was measured on adenocarcinomic human alveolar basal epithelial (A549) cells via delivering AS1411 (G4). The DNA tetra-AS1411 complex was rapidly and abundantly uptake by A549 cells, and the induced apoptosis was enhanced. Furthermore, biodistribution in mouse proved the rapid clearance from non-targeted organs in vivo. This study improved the understanding of potential function in DNA-based drug delivery and proved that DTNs-AS1411 could be potentially useful for the treatment of lung cancer.

  14. Establishment and evaluation of a stable cattle type II alveolar epithelial cell line.

    Directory of Open Access Journals (Sweden)

    Feng Su

    Full Text Available Macrophages and dendritic cells are recognized as key players in the defense against mycobacterial infection. Recent research has confirmed that alveolar epithelial cells (AECs also play important roles against mycobacterium infections. Thus, establishing a stable cattle AEC line for future endogenous immune research on bacterial invasion is necessary. In the present study, we first purified and immortalized type II AECs (AEC II cells by transfecting them with a plasmid containing the human telomerase reverse trancriptase gene. We then tested whether or not the immortalized cells retained the basic physiological properties of primary AECs by reverse-transcription polymerase chain reaction and Western blot. Finally, we tested the secretion capacity of immortalized AEC II cells upon stimulation by bacterial invasion. The cattle type II alveolar epithelial cell line (HTERT-AEC II that we established retained lung epithelial cell characteristics: the cells were positive for surfactants A and B, and they secreted tumor necrosis factor-α and interleukin-6 in response to bacterial invasion. Thus, the cell line we established is a potential tool for research on the relationship between AECs and Mycobacterium tuberculosis.

  15. Carbon black nanoparticles induce type II epithelial cells to release chemotaxins for alveolar macrophages

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    Donaldson Ken

    2005-12-01

    Full Text Available Abstract Background Alveolar macrophages are a key cell in dealing with particles deposited in the lungs and in determining the subsequent response to that particle exposure. Nanoparticles are considered a potential threat to the lungs and the mechanism of pulmonary response to nanoparticles is currently under intense scrutiny. The type II alveolar epithelial cell has previously been shown to release chemoattractants which can recruit alveolar macrophages to sites of particle deposition. The aim of this study was to assess the responses of a type II epithelial cell line (L-2 to both fine and nanoparticle exposure in terms of secretion of chemotactic substances capable of inducing macrophage migration. Results Exposure of type II cells to carbon black nanoparticles resulted in significant release of macrophage chemoattractant compared to the negative control and to other dusts tested (fine carbon black and TiO2 and nanoparticle TiO2 as measured by macrophage migration towards type II cell conditioned medium. SDS-PAGE analysis of the conditioned medium from particle treated type II cells revealed that a higher number of protein bands were present in the conditioned medium obtained from type II cells treated with nanoparticle carbon black compared to other dusts tested. Size-fractionation of the chemotaxin-rich supernatant determined that the chemoattractants released from the epithelial cells were between 5 and 30 kDa in size. Conclusion The highly toxic nature and reactive surface chemistry of the carbon black nanoparticles has very likely induced the type II cell line to release pro-inflammatory mediators that can potentially induce migration of macrophages. This could aid in the rapid recruitment of inflammatory cells to sites of particle deposition and the subsequent removal of the particles by phagocytic cells such as macrophages and neutrophils. Future studies in this area could focus on the exact identity of the substance(s released by the

  16. Intracellular dynamics and fate of polystyrene nanoparticles in A549 Lung epithelial cells monitored by image (cross-) correlation spectroscopy and single particle tracking.

    Science.gov (United States)

    Deville, Sarah; Penjweini, Rozhin; Smisdom, Nick; Notelaers, Kristof; Nelissen, Inge; Hooyberghs, Jef; Ameloot, Marcel

    2015-10-01

    Novel insights in nanoparticle (NP) uptake routes of cells, their intracellular trafficking and subcellular targeting can be obtained through the investigation of their temporal and spatial behavior. In this work, we present the application of image (cross-) correlation spectroscopy (IC(C)S) and single particle tracking (SPT) to monitor the intracellular dynamics of polystyrene (PS) NPs in the human lung carcinoma A549 cell line. The ensemble kinetic behavior of NPs inside the cell was characterized by temporal and spatiotemporal image correlation spectroscopy (TICS and STICS). Moreover, a more direct interpretation of the diffusion and flow detected in the NP motion was obtained by SPT by monitoring individual NPs. Both techniques demonstrate that the PS NP transport in A549 cells is mainly dependent on microtubule-assisted transport. By applying spatiotemporal image cross-correlation spectroscopy (STICCS), the correlated motions of NPs with the early endosomes, late endosomes and lysosomes are identified. PS NPs were equally distributed among the endolysosomal compartment during the time interval of the experiments. The cotransport of the NPs with the lysosomes is significantly larger compared to the other cell organelles. In the present study we show that the complementarity of ICS-based techniques and SPT enables a consistent elaborate model of the complex behavior of NPs inside biological systems. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. Alveolar epithelial type II cells induce T cell tolerance to specific antigen

    DEFF Research Database (Denmark)

    Lo, Bernice; Hansen, Søren; Evans, Kathy

    2008-01-01

    The lungs face the immunologic challenge of rapidly eliminating inhaled pathogens while maintaining tolerance to innocuous Ags. A break in this immune homeostasis may result in pulmonary inflammatory diseases, such as allergies or asthma. The observation that alveolar epithelial type II cells (Type...... II) constitutively express the class II MHC led us to hypothesize that Type II cells play a role in the adaptive immune response. Because Type II cells do not express detectable levels of the costimulatory molecules CD80 and CD86, we propose that Type II cells suppress activation of naive T cells...

  18. Silver nanowire interactions with primary human alveolar type-II epithelial cell secretions: contrasting bioreactivity with human alveolar type-I and type-II epithelial cells

    Science.gov (United States)

    Sweeney, Sinbad; Theodorou, Ioannis G.; Zambianchi, Marta; Chen, Shu; Gow, Andrew; Schwander, Stephan; Zhang, Junfeng (Jim); Chung, Kian Fan; Shaffer, Milo S. P.; Ryan, Mary P.; Porter, Alexandra E.; Tetley, Teresa D.

    2015-06-01

    Inhaled nanoparticles have a high deposition rate in the alveolar units of the deep lung. The alveolar epithelium is composed of type-I and type-II epithelial cells (ATI and ATII respectively) and is bathed in pulmonary surfactant. The effect of native human ATII cell secretions on nanoparticle toxicity is not known. We investigated the cellular uptake and toxicity of silver nanowires (AgNWs; 70 nm diameter, 1.5 μm length) with human ATI-like cells (TT1), in the absence or presence of Curosurf® (a natural porcine pulmonary surfactant with a low amount of protein) or harvested primary human ATII cell secretions (HAS; containing both the complete lipid as well as the full protein complement of human pulmonary surfactant i.e. SP-A, SP-B, SP-C and SP-D). We hypothesised that Curosurf® or HAS would confer improved protection for TT1 cells, limiting the toxicity of AgNWs. In agreement with our hypothesis, HAS reduced the inflammatory and reactive oxygen species (ROS)-generating potential of AgNWs with exposed TT1 cells. For example, IL-8 release and ROS generation was reduced by 38% and 29%, respectively, resulting in similar levels to that of the non-treated controls. However in contrast to our hypothesis, Curosurf® had no effect. We found a significant reduction in AgNW uptake by TT1 cells in the presence of HAS but not Curosurf. Furthermore, we show that the SP-A and SP-D are likely to be involved in this process as they were found to be specifically bound to the AgNWs. While ATI cells appear to be protected by HAS, evidence suggested that ATII cells, despite no uptake, were vulnerable to AgNW exposure (indicated by increased IL-8 release and ROS generation and decreased intracellular SP-A levels one day post-exposure). This study provides unique findings that may be important for the study of lung epithelial-endothelial translocation of nanoparticles in general and associated toxicity within the alveolar unit.Inhaled nanoparticles have a high deposition rate in

  19. Expression of functional toll-like receptor-2 and -4 on alveolar epithelial cells.

    Science.gov (United States)

    Armstrong, Lynne; Medford, Andrew R L; Uppington, Kay M; Robertson, John; Witherden, Ian R; Tetley, Teresa D; Millar, Ann B

    2004-08-01

    The recognition of potentially harmful microorganisms involves the specific recognition of pathogen-associated molecular patterns (PAMPs) and the family of Toll-like receptors (TLRs) is known to play a central role in this process. TLR-4 is the major recognition receptor for lipopolysaccharide (LPS), a component of gram-negative bacterial cell walls, whereas TLR-2 responds to bacterial products from gram-positive organisms. Although resident alveolar macrophages are the first line of defense against microbial attack, it is now understood that the alveolar epithelium also plays a pivotal role in the innate immunity of the lung. The purpose of the current study was to determine whether human primary type II alveolar epithelial cells (ATII) express functional TLR-2 and TLR-4 and how they may be regulated by inflammatory mediators. We have used reverse transcriptase-polymerase chain reaction and flow cytometry to determine basal and inducible expression on ATII. We have used highly purified preparations of the gram-positive bacterial product lipoteichoic acid (LTA) and LPS to look at the functional consequences of TLR-2 and TLR-4 ligation, respectively, in terms of interleukin-8 release. We have shown that human primary ATII cells express mRNA and protein for both TLR-2 and TLR-4, which can be modulated by incubation with LPS and tumor necrosis factor. Furthermore, we have demonstrated that these receptors are functional. This suggests that ATII have the potential to contribute significantly to the host defense of the human alveolus against bacteria.

  20. Ultrastructural Study of Alveolar Epithelial Type II Cells by High-Frequency Oscillatory Ventilation

    Directory of Open Access Journals (Sweden)

    Xiaofei Qin

    2013-01-01

    Full Text Available Alveolar epithelial type II cells (AECIIs containing lamellar bodies (LBs are alveolar epithelial stem cells that have important functions in the repair of lung structure and function after lung injury. The ultrastructural changes in AECIIs after high-frequency oscillatory ventilation (HFOV with a high lung volume strategy or conventional ventilation were evaluated in a newborn piglet model with acute lung injury (ALI. After ALI with saline lavage, newborn piglets were randomly assigned into five study groups (three piglets in each group, namely, control (no mechanical ventilation, conventional ventilation for 24 h, conventional ventilation for 48 h, HFOV for 24 h, and HFOV for 48 h. The lower tissues of the right lung were obtained to observe the AECII ultrastructure. AECIIs with reduced numbers of microvilli, decreased LBs electron density, and vacuole-like LBs deformity were commonly observed in all five groups. Compared with conventional ventilation groups, the decrease in numbers of microvilli and LBs electron density, as well as LBs with vacuole-like appearance and polymorphic deformity, was less severe in HFOV with high lung volume strategy groups. AECIIs were injured during mechanical ventilation. HFOV with a high lung volume strategy resulted in less AECII damage than conventional ventilation.

  1. Type IV collagen drives alveolar epithelial-endothelial association and the morphogenetic movements of septation.

    Science.gov (United States)

    Loscertales, Maria; Nicolaou, Fotini; Jeanne, Marion; Longoni, Mauro; Gould, Douglas B; Sun, Yunwei; Maalouf, Faouzi I; Nagy, Nandor; Donahoe, Patricia K

    2016-07-13

    Type IV collagen is the main component of the basement membrane that gives strength to the blood-gas barrier (BGB). In mammals, the formation of a mature BGB occurs primarily after birth during alveologenesis and requires the formation of septa from the walls of the saccule. In contrast, in avians, the formation of the BGB occurs rapidly and prior to hatching. Mutation in basement membrane components results in an abnormal alveolar phenotype; however, the specific role of type IV collagen in regulating alveologenesis remains unknown. We have performed a microarray expression analysis in late chick lung development and found that COL4A1 and COL4A2 were among the most significantly upregulated genes during the formation of the avian BGB. Using mouse models, we discovered that mutations in murine Col4a1 and Col4a2 genes affected the balance between lung epithelial progenitors and differentiated cells. Mutations in Col4a1 derived from the vascular component were sufficient to cause defects in vascular development and the BGB. We also show that Col4a1 and Col4a2 mutants displayed disrupted myofibroblast proliferation, differentiation and migration. Lastly, we revealed that addition of type IV collagen protein induced myofibroblast proliferation and migration in monolayer culture and increased the formation of mesenchymal-epithelial septal-like structures in co-culture. Our study showed that type IV collagen and, therefore the basement membrane, play fundamental roles in coordinating alveolar morphogenesis. In addition to its role in the formation of epithelium and vasculature, type IV collagen appears to be key for alveolar myofibroblast development by inducing their proliferation, differentiation and migration throughout the developing septum.

  2. Gene expression patterns induced at different stages of rhinovirus infection in human alveolar epithelial cells.

    Directory of Open Access Journals (Sweden)

    Mohammad Reza Etemadi

    Full Text Available Human rhinovirus (HRV is the common virus that causes acute respiratory infection (ARI and is frequently associated with lower respiratory tract infections (LRTIs. We aimed to investigate whether HRV infection induces a specific gene expression pattern in airway epithelial cells. Alveolar epithelial cell monolayers were infected with HRV species B (HRV-B. RNA was extracted from both supernatants and infected monolayer cells at 6, 12, 24 and 48 hours post infection (hpi and transcriptional profile was analyzed using Affymetrix GeneChip and the results were subsequently validated using quantitative Real-time PCR method. HRV-B infects alveolar epithelial cells which supports implication of the virus with LRTIs. In total 991 genes were found differentially expressed during the course of infection. Of these, 459 genes were up-regulated whereas 532 genes were down-regulated. Differential gene expression at 6 hpi (187 genes up-regulated vs. 156 down-regulated were significantly represented by gene ontologies related to the chemokines and inflammatory molecules indicating characteristic of viral infection. The 75 up-regulated genes surpassed the down-regulated genes (35 at 12 hpi and their enriched ontologies fell into discrete functional entities such as regulation of apoptosis, anti-apoptosis, and wound healing. At later time points of 24 and 48 hpi, predominated down-regulated genes were enriched for extracellular matrix proteins and airway remodeling events. Our data provides a comprehensive image of host response to HRV infection. The study suggests the underlying molecular regulatory networks genes which might be involved in pathogenicity of the HRV-B and potential targets for further validations and development of effective treatment.

  3. Host gene expression profiling in influenza A virus-infected lung epithelial (A549 cells: a comparative analysis between highly pathogenic and modified H5N1 viruses

    Directory of Open Access Journals (Sweden)

    Chakrabarti Alok K

    2010-09-01

    Full Text Available Abstract Background To understand the molecular mechanism of host responses to highly pathogenic avian influenza virus infection and to get an insight into the means through which virus overcomes host defense mechanism, we studied global gene expression response of human lung carcinoma cells (A549 at early and late stages of infection with highly pathogenic avian Influenza A (H5N1 virus and compared it with a reverse genetics modified recombinant A (H5N1 vaccine virus using microarray platform. Results The response was studied at time points 4, 8, 16 and 24 hours post infection (hpi. Gene ontology analysis revealed that the genes affected by both the viruses were qualitatively similar but quantitatively different. Significant differences were observed in the expression of genes involved in apoptosis and immune responses, specifically at 16 hpi. Conclusion We conclude that subtle differences in the ability to induce specific host responses like apoptotic mechanism and immune responses make the highly pathogenic viruses more virulent.

  4. P2X7R: independent modulation of aquaporin 5 expression in CdCl2-injured alveolar epithelial cells.

    Science.gov (United States)

    Heupel, Julia; Bläsche, Robert; Wesslau, Karl-Philipp; Kasper, Michael; Barth, Kathrin

    2018-03-01

    The expression of aquaporin 5 in alveolar epithelial type I cells under conditions of cadmium-induced injury has not yet been discovered. We investigated the effect of the P2X7R agonist BzATP under this condition, since P2X7R is involved in altered regulation of aquaporin 5 in pulmonary fibrosis. CdCl 2 /TGF-β1 treatment of lung epithelial MLE-12 cells was leading to increasing P2X7R, and aquaporin 5 protein levels. The aquaporin 5 expression was P2X7R-independent in MLE-12 cells under cadmium, as was shown in blocking experiments with oxATP. Further, the expression of both proteins increased after 24 h CdCl 2 /TGF-β1 treatment of precision-cut lung slices, but decreased after 72 h. Using immunohistochemistry, the activation of the P2X7R with the agonist BzATP modulated the aquaporin 5 immunoreactivity in the alveolar epithelium of precision-cut lung slices from wild-type but not from P2X7R knockout mice. Similarly, aquaporin 5 protein was reduced in BzATP-treated immortal lung epithelial E10 cells. Surprisingly, untreated alveolar epithelial type II cells of P2X7R knockouts exhibited a pronounced apical immunoreactivity in addition to the remaining alveolar epithelial type I cells. BzATP exposure did not alter this distribution pattern, but increased the number of apoptotic alveolar epithelial type II cells in wild-type lung slices.

  5. A heteromeric molecular complex regulates the migration of lung alveolar epithelial cells during wound healing.

    Science.gov (United States)

    Ghosh, Manik C; Makena, Patrudu S; Kennedy, Joseph; Teng, Bin; Luellen, Charlean; Sinclair, Scott E; Waters, Christopher M

    2017-05-19

    Alveolar type II epithelial cells (ATII) are instrumental in early wound healing in response to lung injury, restoring epithelial integrity through spreading and migration. We previously reported in separate studies that focal adhesion kinase-1 (FAK) and the chemokine receptor CXCR4 promote epithelial repair mechanisms. However, potential interactions between these two pathways were not previously considered. In the present study, we found that wounding of rat ATII cells promoted increased association between FAK and CXCR4. In addition, protein phosphatase-5 (PP5) increased its association with this heteromeric complex, while apoptosis signal regulating kinase-1 (ASK1) dissociated from the complex. Cell migration following wounding was decreased when PP5 expression was decreased using shRNA, but migration was increased in ATII cells isolated from ASK1 knockout mice. Interactions between FAK and CXCR4 were increased upon depletion of ASK1 using shRNA in MLE-12 cells, but unaffected when PP5 was depleted. Furthermore, we found that wounded rat ATII cells exhibited decreased ASK1 phosphorylation at Serine-966, decreased serine phosphorylation of FAK, and decreased association of phosphorylated ASK1 with FAK. These changes in phosphorylation were dependent upon expression of PP5. These results demonstrate a unique molecular complex comprising CXCR4, FAK, ASK1, and PP5 in ATII cells during wound healing.

  6. Genotoxic effects of three selected black toner powders and their dimethyl sulfoxide extracts in cultured human epithelial A549 lung cells in vitro.

    Science.gov (United States)

    Gminski, Richard; Decker, Katharina; Heinz, Christina; Seidel, Albrecht; Könczöl, Mathias; Goldenberg, Ella; Grobéty, Bernard; Ebner, Winfried; Gieré, Reto; Mersch-Sundermann, Volker

    2011-05-01

    Until now, the adverse effects of toner powders on humans have been considered to be minimal. However, several recent reports have suggested possible significant adverse health effects from toner dust inhalation. The aim of this study was to evaluate the genotoxic potential of black toner powders in vitro. For the study of DNA damage, A549 cells were exposed to toner-powder suspensions and to their DMSO extracts, and then subjected to the comet assay and to the in-vitro cytokinesis block micronucleus test (CB-MNvit). Cytotoxic effects of the toner samples were assessed by the erythrosin B assay. Furthermore, size, shape, and composition of the toner powders were investigated. None of the three toner powders or their DMSO extracts reduced cell viability; however, they did induce DNA damage and formed micronuclei at concentrations from 80 to 400 μg cm(-2) , although to a varying extent. All toner powders contain considerable amounts of the pigments carbon black and magnetite (Fe(3) O(4) ) as well as small amounts of polycyclic aromatic hydrocarbons (PAHs). The overall results of our in-vitro study suggest that the investigated toner-powder samples are not cytotoxic but genotoxic. From the results of the physical and chemical characterization, we conclude that metals and metalloids as components of magnetite, or PAHs as components of the carbon-bearing material, are responsible for the genotoxic effects. Further research is necessary to determine the relevance of these in-vitro observations for private and occupational toner powder exposure. Copyright © 2010 Wiley-Liss, Inc.

  7. Investigations on cytotoxic and genotoxic effects of laser printer emissions in human epithelial A549 lung cells using an air/liquid exposure system.

    Science.gov (United States)

    Tang, Tao; Gminski, Richard; Könczöl, Mathias; Modest, Christoph; Armbruster, Benedikt; Mersch-Sundermann, Volker

    2012-03-01

    Exposure to emissions from laser printers during the printing process is commonplace worldwide, both in the home and workplace environment. In the present study, cytotoxic and genotoxic effects of the emission from five low to medium-throughput laser printers were investigated with respect to the release of ozone (O(3) ), volatile organic compounds (VOC), particulate matter (PM), and submicrometer particles (SMP) during standby and operation. Experiments were conducted in a 1 m(3) emission chamber connected to a Vitrocell® exposure system. Cytotoxicity was determined by the WST-1 assay and genotoxicity by the micronucleus test in human A549 lung cells. The five laser printers emitted varying but generally small amounts of O(3) , VOC, and PM. VOC emissions included 13 compounds with total VOC concentrations ranging from 95 to 280 μg/m(3) (e.g., 2-butanone, hexanal, m,p-xylene, and o-xylene). Mean PM concentrations were below 2.4 μg/m(3). SMP number concentration levels during standby ranged from 9 to 26 particles/cm(3). However, three of the printers generated a 90 to 16 × 10(3) -fold increase of SMP during the printing process (maximum 294,460 particles/cm(3)). Whereas none of the printer emissions were found to cause cytotoxicity, emissions from two printers induced formation of micronuclei (P printers. Because laser printing technology is widely used, studies with additional cytogenetic endpoints are necessary to confirm the DNA-damaging potency and to identify emission components responsible for genotoxicity. Copyright © 2011 Wiley-Liss, Inc.

  8. Influence of the Paracoccidioides brasiliensis 14-3-3 and gp43 proteins on the induction of apoptosis in A549 epithelial cells.

    Science.gov (United States)

    Silva, Julhiany de Fátima da; Vicentim, Juliana; Oliveira, Haroldo Cesar de; Marcos, Caroline Maria; Assato, Patricia Akemi; Andreotti, Patrícia Ferrari; Silva, Juliana Leal Monteiro da; Soares, Christiane Pienna; Benard, Gil; Almeida, Ana Marisa Fusco; Mendes-Giannini, Maria José Soares

    2015-06-01

    The fungal strain Paracoccidioides brasiliensis remains viable inside of epithelial cells and can induce apoptosis in this population. However, until now, the molecules that participate in this process remained unknown. Thus, this study evaluated the contribution of two P. brasiliensis molecules, the 14-3-3 and glycoprotein of 43 kDa proteins, which had been previously described as extracellular matrix adhesins and apoptosis inductors in human pneumocytes. Accordingly, epithelial cells were treated with these molecules for different periods of time and the expression of the apoptosis regulating-proteins Bak, Bax, Bcl-2, p53 and caspases were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling, flow cytometry and real-time polymerase chain reaction analysis. Our results demonstrated that treatment with these molecules induces apoptosis signalling in pulmonary epithelial cells, showing the same pattern of programmed cell-death as that observed during infection with P. brasiliensis. Thus, we could conclude that P. brasiliensis uses these molecules as virulence factors that participate not only in the fungal adhesion process to host cells, but also in other important cellular mechanisms such as apoptosis.

  9. Influence of the Paracoccidioides brasiliensis 14-3-3 and gp43 proteins on the induction of apoptosis in A549 epithelial cells

    Directory of Open Access Journals (Sweden)

    Julhiany de Fátima da Silva

    2015-06-01

    Full Text Available The fungal strain Paracoccidioides brasiliensis remains viable inside of epithelial cells and can induce apoptosis in this population. However, until now, the molecules that participate in this process remained unknown. Thus, this study evaluated the contribution of two P. brasiliensis molecules, the 14-3-3 and glycoprotein of 43 kDa proteins, which had been previously described as extracellular matrix adhesins and apoptosis inductors in human pneumocytes. Accordingly, epithelial cells were treated with these molecules for different periods of time and the expression of the apoptosis regulating-proteins Bak, Bax, Bcl-2, p53 and caspases were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling, flow cytometry and real-time polymerase chain reaction analysis. Our results demonstrated that treatment with these molecules induces apoptosis signalling in pulmonary epithelial cells, showing the same pattern of programmed cell-death as that observed during infection with P. brasiliensis. Thus, we could conclude that P. brasiliensis uses these molecules as virulence factors that participate not only in the fungal adhesion process to host cells, but also in other important cellular mechanisms such as apoptosis.

  10. Lung fibroblasts accelerate wound closure in human alveolar epithelial cells through hepatocyte growth factor/c-Met signaling.

    Science.gov (United States)

    Ito, Yoko; Correll, Kelly; Schiel, John A; Finigan, Jay H; Prekeris, Rytis; Mason, Robert J

    2014-07-01

    There are 190,600 cases of acute lung injury/acute respiratory distress syndrome (ALI/ARDS) each year in the United States, and the incidence and mortality of ALI/ARDS increase dramatically with age. Patients with ALI/ARDS have alveolar epithelial injury, which may be worsened by high-pressure mechanical ventilation. Alveolar type II (ATII) cells are the progenitor cells for the alveolar epithelium and are required to reestablish the alveolar epithelium during the recovery process from ALI/ARDS. Lung fibroblasts (FBs) migrate and proliferate early after lung injury and likely are an important source of growth factors for epithelial repair. However, how lung FBs affect epithelial wound healing in the human adult lung has not been investigated in detail. Hepatocyte growth factor (HGF) is known to be released mainly from FBs and to stimulate both migration and proliferation of primary rat ATII cells. HGF is also increased in lung tissue, bronchoalveolar lavage fluid, and serum in patients with ALI/ARDS. Therefore, we hypothesized that HGF secreted by FBs would enhance wound closure in alveolar epithelial cells (AECs). Wound closure was measured using a scratch wound-healing assay in primary human AEC monolayers and in a coculture system with FBs. We found that wound closure was accelerated by FBs mainly through HGF/c-Met signaling. HGF also restored impaired wound healing in AECs from the elderly subjects and after exposure to cyclic stretch. We conclude that HGF is the critical factor released from FBs to close wounds in human AEC monolayers and suggest that HGF is a potential strategy for hastening alveolar repair in patients with ALI/ARDS. Copyright © 2014 the American Physiological Society.

  11. Mitochondrial quality control in alveolar epithelial cells damaged byS. aureuspneumonia in mice.

    Science.gov (United States)

    Suliman, Hagir B; Kraft, Bryan; Bartz, Raquel; Chen, Lingye; Welty-Wolf, Karen E; Piantadosi, Claude A

    2017-10-01

    Mitochondrial damage is often overlooked in acute lung injury (ALI), yet most of the lung's physiological processes, such as airway tone, mucociliary clearance, ventilation-perfusion (Va/Q) matching, and immune surveillance require aerobic energy provision. Because the cell's mitochondrial quality control (QC) process regulates the elimination and replacement of damaged mitochondria to maintain cell survival, we serially evaluated mitochondrial biogenesis and mitophagy in the alveolar regions of mice in a validated Staphylococcus aureus pneumonia model. We report that apart from cell lysis by direct contact with microbes, modest epithelial cell death was detected despite significant mitochondrial damage. Cell death by TdT-mediated dUTP nick-end labeling staining occurred on days 1 and 2 postinoculation: apoptosis shown by caspase-3 cleavage was present on days 1 and 2, while necroptosis shown by increased levels of phospho- mixed lineage kinase domain-like protein (MLKL) and receptor-interacting serine/threonine-protein kinase 1 (RIPK1) was present on day 1 Cell death in alveolar type I (AT 1 ) cells assessed by bronchoalveolar lavage fluid receptor for advanced glycation end points (RAGE) levels was high, yet AT 2 cell death was limited while both mitochondrial biogenesis and mitophagy were induced. These mitochondrial QC mechanisms were evaluated mainly in AT 2 cells by localizing increases in citrate synthase content, increases in nuclear mitochondrial biogenesis regulators nuclear respiratory factor-1 (NRF-1) and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), and increases in light chain 3B protein (LC3-I)/LC3II ratios. Concomitant changes in p62, Pink 1, and Parkin protein levels indicated activation of mitophagy. By confocal microscopy, mitochondrial biogenesis and mitophagy were often observed on day 1 within the same AT 2 cells. These findings imply that mitochondrial QC activation in pneumonia-damaged AT 2 cells promotes cell

  12. Sustained distribution of aerosolized PEGylated liposomes in epithelial lining fluids on alveolar surfaces.

    Science.gov (United States)

    Kaneko, Keita; Togami, Kohei; Yamamoto, Eri; Wang, Shujun; Morimoto, Kazuhiro; Itagaki, Shirou; Chono, Sumio

    2016-10-01

    The distribution characteristics of aerosolized PEGylated liposomes in alveolar epithelial lining fluid (ELF) were examined in rats, and the ensuing mechanisms were investigated in the in vitro uptake and protein adsorption experiments. Nonmodified or PEGylated liposomes (particle size 100 nm) were aerosolized into rat lungs. PEGylated liposomes were distributed more sustainably in ELFs than nonmodified liposomes. Furthermore, the uptake of PEGylated liposomes by alveolar macrophages (AMs) was less than that of nonmodified liposomes. In further in vitro uptake experiments, nonmodified and PEGylated liposomes were opsonized with rat ELF components and then added to NR8383 cells as cultured rat AMs. The uptake of opsonized PEGylated liposomes by NR8383 cells was lower than that of opsonized nonmodified liposomes. Moreover, the protein absorption levels in opsonized PEGylated liposomes were lower than those in opsonized nonmodified liposomes. These findings suggest that sustained distributions of aerosolized PEGylated liposomes in ELFs reflect evasion of liposomal opsonization with surfactant proteins and consequent reductions in uptake by AMs. These data indicate the potential of PEGylated liposomes as aerosol-based drug delivery system that target ELF for the treatment of respiratory diseases.

  13. Induction of human microsomal prostaglandin E synthase 1 by activated oncogene RhoA GTPase in A549 human epithelial cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Hye Jin [Laboratory of Systems Mucosal Biomodulation, Department of Microbiology and Immunology, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Lee, Dong-Hyung [Department of Obstetrics and Gynecology, Medical Research Institute, Pusan National University, Busan (Korea, Republic of); Park, Seong-Hwan; Kim, Juil; Do, Kee Hun [Laboratory of Systems Mucosal Biomodulation, Department of Microbiology and Immunology, Pusan National University School of Medicine, Yangsan (Korea, Republic of); An, Tae Jin; Ahn, Young Sup; Park, Chung Berm [Department of Herbal Crop Research, NIHHS, RDA, Eumseong (Korea, Republic of); Moon, Yuseok, E-mail: moon@pnu.edu [Laboratory of Systems Mucosal Biomodulation, Department of Microbiology and Immunology, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Medical Research Institute and Research Institute for Basic Sciences, Pusan National University, Busan (Korea, Republic of)

    2011-09-30

    Highlights: {yields} As a target of oncogene RhoA-linked signal, a prostaglandin metabolism is assessed. {yields} RhoA activation increases PGE{sub 2} levels and its metabolic enzyme mPGES-1. {yields} RhoA-activated NF-{kappa}B and EGR-1 are positively involved in mPGES-1 induction. -- Abstract: Oncogenic RhoA GTPase has been investigated as a mediator of pro-inflammatory responses and aggressive carcinogenesis. Among the various targets of RhoA-linked signals, pro-inflammatory prostaglandin E{sub 2} (PGE{sub 2}), a major prostaglandin metabolite, was assessed in epithelial cancer cells. RhoA activation increased PGE{sub 2} levels and gene expression of the rate-limiting PGE{sub 2} producing enzymes, cyclooxygenase-2 and microsomal prostaglandin E synthase 1 (mPGES-1). In particular, human mPGES-1 was induced by RhoA via transcriptional activation in control and interleukin (IL)-1{beta}-activated cancer cells. To address the involvement of potent signaling pathways in RhoA-activated mPGES-1 induction, various signaling inhibitors were screened for their effects on mPGES-1 promoter activity. RhoA activation enhanced basal and IL-1{beta}-mediated phosphorylated nuclear factor-{kappa}B and extracellular signal-regulated kinase1/2 proteins, all of which were positively involved in RhoA-induced gene expression of mPGES-1. As one potent down-stream transcription factor of ERK1/2 signals, early growth response gene 1 product also mediated RhoA-induced gene expression of mPGES-1 by enhancing transcriptional activity. Since oncogene-triggered PGE{sub 2} production is a critical modulator of epithelial tumor cells, RhoA-associated mPGES-1 represents a promising chemo-preventive or therapeutic target for epithelial inflammation and its associated cancers.

  14. The surface charge of liposomal adjuvants is decisive for their interactions with the Calu-3 and A549 airway epithelial cell culture models

    DEFF Research Database (Denmark)

    Ingvarsson, Pall Thor; Rasmussen, Ida Svahn; Viaene, Michelle

    2014-01-01

    One of the main reasons for the unmet medical need for mucosal vaccines is the lack of safe and efficacious mucosal adjuvants. The cationic liposome-based adjuvant system composed of dimethyldioctadecylammonium (DDA) bromide and trehalose 6,6'-dibehenate (TDB) is a versatile adjuvant that has sho...... and viability of the mucus-covered Calu-3 cells. Our in vitro results thus indicate that DDA/TDB liposomes might be efficiently and safely used as an adjuvant system for vaccines targeting the mucus-covered epithelium of the upper respiratory tract and the conducting airways....... potential for mucosal vaccination via the airways. The purpose of this study was to investigate the importance of the liposomal surface charge on the interaction with lung epithelial cells. Thus, the cationic DDA in the liposomes was subjected to a step-wise replacement with the zwitterionic...

  15. Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells

    Directory of Open Access Journals (Sweden)

    Yuen Kit M

    2009-10-01

    Full Text Available Abstract Background Highly pathogenic avian influenza (HPAI H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease. Aim To study influenza A (H5N1 virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease. Methods We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces. Results We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our

  16. Cellular responses of A549 alveolar epithelial cells to serially collected Pseudomonas aeruginosa from cystic fibrosis patients at different stages of pulmonary infection

    DEFF Research Database (Denmark)

    Hawdon, Nicole A; Aval, Pouya Sadeghi; Barnes, Rebecca J

    2010-01-01

    proinflammatory cytokine and adhesion molecule expression. Our findings suggest that despite the loss of virulence factors during the adaptation process in the CF lung by late P. aeruginosa strains, they retain high proinflammatory abilities that likely contribute to the disease pathogenesis....

  17. P2X7R-dependent regulation of glycogen synthase kinase 3β and claudin-18 in alveolar epithelial type I cells of mice lung.

    Science.gov (United States)

    Barth, K; Bläsche, R; Neißer, A; Bramke, S; Frank, J A; Kasper, M

    2016-12-01

    The purinergic receptor P2X7 represents an ATP-gated ionotropic receptor with a selective localization in alveolar epithelial type I cells of the lung. Despite the involvement of the receptor in inflammatory processes of the lung, it is not established whether this receptor plays a specific role in the alveolar epithelial cell biology. There is evidence that P2X7 receptor influences Wnt/β-catenin signalling pathways in alveolar epithelial cells under conditions of injury. Here, we investigated the expression of GSK-3β, a potent protein kinase involved in alveolar epithelial barrier functions, and of tight junction molecules occludin, claudin-4 and claudin-18 in wild-type and P2X7 -/- mice. Western blot analysis, immunohistochemistry and quantitative real-time RT-PCR revealed a remarkable increase in claudin-18 mRNA and protein in lungs of P2X7 -/- mice animals. Furthermore, alveolar epithelial cells from P2X7 -/- animals showed decreased levels of GSK-3β protein and its inactive form GSK-3β (pS9). Conversely, claudin-18 knockout mice exhibited decreased P2X7 mRNA transcript abundance as measured by mRNA expression microarray and quantitative PCR. Our data are consistent with the hypothesis that P2X7R contributes to alveolar epithelial barrier function through effects on GSK-3β. Furthermore, these data suggest a potential reciprocal regulation of claudin-18 and P2X7R in the alveolar epithelium.

  18. Folliculin Controls Lung Alveolar Enlargement and Epithelial Cell Survival through E-Cadherin, LKB1, and AMPK

    Directory of Open Access Journals (Sweden)

    Elena A. Goncharova

    2014-04-01

    Full Text Available Spontaneous pneumothoraces due to lung cyst rupture afflict patients with the rare disease Birt-Hogg-Dubé (BHD syndrome, which is caused by mutations of the tumor suppressor gene folliculin (FLCN. The underlying mechanism of the lung manifestations in BHD is unclear. We show that BHD lungs exhibit increased alveolar epithelial cell apoptosis and that Flcn deletion in mouse lung epithelium leads to cell apoptosis, alveolar enlargement, and an impairment of both epithelial barrier and overall lung function. We find that Flcn-null epithelial cell apoptosis is the result of impaired AMPK activation and increased cleaved caspase-3. AMPK activator LKB1 and E-cadherin are downregulated by Flcn loss and restored by its expression. Correspondingly, Flcn-null cell survival is rescued by the AMPK activator AICAR or constitutively active AMPK. AICAR also improves lung condition of Flcnf/f:SP-C-Cre mice. Our data suggest that lung cysts in BHD may result from an underlying defect in alveolar epithelial cell survival, attributable to FLCN regulation of the E-cadherin-LKB1-AMPK axis.

  19. Spatiotemporal dynamics of actin remodeling and endomembrane trafficking in alveolar epithelial type I cell wound healing.

    Science.gov (United States)

    Godin, Lindsay M; Vergen, Jorge; Prakash, Y S; Pagano, Richard E; Hubmayr, Rolf D

    2011-04-01

    Alveolar epithelial type I cell (ATI) wounding is prevalent in ventilator-injured lungs and likely contributes to pathogenesis of "barotrauma" and "biotrauma." In experimental models most wounded alveolar cells repair plasma membrane (PM) defects and survive insults. Considering the force balance between edge energy at the PM wound margins and adhesive interactions of the lipid bilayer with the underlying cytoskeleton (CSK), we tested the hypothesis that subcortical actin depolymerization is a key facilitator of PM repair. Using real-time fluorescence imaging of primary rat ATI transfected with a live cell actin-green fluorescent protein construct (Lifeact-GFP) and loaded with N-rhodamine phosphatidylethanolamine (PE), we examined the spatial and temporal coordination between cytoskeletal remodeling and PM repair following micropuncture. Membrane integrity was inferred from the fluorescence intensity profiles of the cytosolic label calcein AM. Wounding led to rapid depolymerization of the actin CSK near the wound site, concurrent with accumulation of endomembrane-derived N-rhodamine PE. Both responses were sustained until PM integrity was reestablished, which typically occurs between ∼10 and 40 s after micropuncture. Only thereafter did the actin CSK near the wound begin to repolymerize, while the rate of endomembrane lipid accumulation decreased. Between 60 and 90 s after successful PM repair, after translocation of the actin nucleation factor cortactin, a dense actin fiber network formed. In cells that did not survive micropuncture injury, actin remodeling did not occur. These novel results highlight the importance of actin remodeling in ATI cell repair and suggest molecular targets for modulating the repair process.

  20. Protein Expression Profile of Rat Type Two Alveolar Epithelial Cells During Hyperoxic Stress and Recovery

    Science.gov (United States)

    Bhargava, Maneesh

    Rationale: In rodent model systems, the sequential changes in lung morphology resulting from hyperoxic injury are well characterized, and are similar to changes in human acute respiratory distress syndrome (ARDS). In the injured lung, alveolar type two (AT2) epithelial cells play a critical role restoring the normal alveolar structure. Thus characterizing the changes in AT2 cells will provide insights into the mechanisms underpinning the recovery from lung injury. Methods: We applied an unbiased systems level proteomics approach to elucidate molecular mechanisms contributing to lung repair in a rat hyperoxic lung injury model. AT2 cells were isolated from rat lungs at predetermined intervals during hyperoxic injury and recovery. Protein expression profiles were determined by using iTRAQRTM with tandem mass spectrometry. Results: Of 959 distinct proteins identified, 183 significantly changed in abundance during the injury-recovery cycle. Gene Ontology enrichment analysis identified cell cycle, cell differentiation, cell metabolism, ion homeostasis, programmed cell death, ubiquitination, and cell migration to be significantly enriched by these proteins. Gene Set Enrichment Analysis of data acquired during lung repair revealed differential expression of gene sets that control multicellular organismal development, systems development, organ development, and chemical homeostasis. More detailed analysis identified activity in two regulatory pathways, JNK and miR 374. A Short Time-series Expression Miner (STEM) algorithm identified protein clusters with coherent changes during injury and repair. Conclusion: Coherent changes occur in the AT2 cell proteome in response to hyperoxic stress. These findings offer guidance regarding the specific molecular mechanisms governing repair of the injured lung.

  1. Breakdown of Epithelial Barrier Integrity and Overdrive Activation of Alveolar Epithelial Cells in the Pathogenesis of Acute Respiratory Distress Syndrome and Lung Fibrosis

    Directory of Open Access Journals (Sweden)

    Shigehisa Yanagi

    2015-01-01

    Full Text Available Individual alveolar epithelial cells (AECs collaboratively form a tight barrier between atmosphere and fluid-filled tissue to enable normal gas exchange. The tight junctions of AECs provide intercellular sealing and are integral to the maintenance of the AEC barrier integrity. Disruption and failure of reconstitution of AEC barrier result in catastrophic consequences, leading to alveolar flooding and subsequent devastating fibrotic scarring. Recent evidences reveal that many of the fibrotic lung diseases involve AECs both as a frequent target of injury and as a driver of ongoing pathological processes. Aberrantly activated AECs express most of the growth factors and chemokines responsible for the proliferation, migration, and activation of fibroblasts. Current evidences suggest that AECs may acquire overdrive activation in the initial step of fibrosis by several mechanisms, including abnormal recapitulation of the developmental pathway, defects of the molecules essential for epithelial integrity, and acceleration of aging-related properties. Among these initial triggering events, epithelial Pten, a multiple phosphatase that negatively regulates the PI3K/Akt pathway and is crucial for lung development, is essential for the prevention of alveolar flooding and lung fibrosis through the regulation of AEC barrier integrity after injury. Reestablishment of AEC barrier integrity also involves the deployment of specialized stem/progenitor cells.

  2. Deletion of SMARCA4 impairs alveolar epithelial type II cells proliferation and aggravates pulmonary fibrosis in mice

    Directory of Open Access Journals (Sweden)

    Danyi Peng

    2017-12-01

    Full Text Available Alveolar epithelial cells (AECs injury and failed reconstitution of the AECs barrier are both integral to alveolar flooding and subsequent pulmonary fibrosis (PF. Nevertheless, the exact mechanisms regulating the regeneration of AECs post-injury still remain unclear. SMARCA4 is a part of the large ATP-dependent chromatin remodelling complex SWI/SNF, which is essential for kidney and heart fibrosis. We investigates SMARCA4 function in lung fibrosis by establishing PF mice model with bleomycin firstly and found that the expression of SMARCA4 was mainly enhanced in alveolar type II (ATII cells. Moreover, we established an alveolar epithelium-specific SMARCA4-deleted SP-C-rtTA/(tetO7-Cre/SMARCA4f/f mice (SOSM4Δ/Δ model, as well as a new SMARCA4-deleted alveolar type II (ATII-like mle-12 cell line. We found that the bleomycin-induced PF was more aggressive in SOSM4Δ/Δ mice. Also, the proliferation of ATII cells was decreased with the loss of SMARCA4 in vivo and in vitro. In addition, we observed increased proliferation of ATII cells accompanied by abnormally high expression of SMARCA4 in human PF lung sections. These data uncovered the indispensable role of SMARCA4 in the proliferation of ATII cells, which might affect the progression of PF.

  3. The Role of Mitochondrial DNA in Mediating Alveolar Epithelial Cell Apoptosis and Pulmonary Fibrosis

    Science.gov (United States)

    Kim, Seok-Jo; Cheresh, Paul; Jablonski, Renea P.; Williams, David B.; Kamp, David W.

    2015-01-01

    Convincing evidence has emerged demonstrating that impairment of mitochondrial function is critically important in regulating alveolar epithelial cell (AEC) programmed cell death (apoptosis) that may contribute to aging-related lung diseases, such as idiopathic pulmonary fibrosis (IPF) and asbestosis (pulmonary fibrosis following asbestos exposure). The mammalian mitochondrial DNA (mtDNA) encodes for 13 proteins, including several essential for oxidative phosphorylation. We review the evidence implicating that oxidative stress-induced mtDNA damage promotes AEC apoptosis and pulmonary fibrosis. We focus on the emerging role for AEC mtDNA damage repair by 8-oxoguanine DNA glycosylase (OGG1) and mitochondrial aconitase (ACO-2) in maintaining mtDNA integrity which is important in preventing AEC apoptosis and asbestos-induced pulmonary fibrosis in a murine model. We then review recent studies linking the sirtuin (SIRT) family members, especially SIRT3, to mitochondrial integrity and mtDNA damage repair and aging. We present a conceptual model of how SIRTs modulate reactive oxygen species (ROS)-driven mitochondrial metabolism that may be important for their tumor suppressor function. The emerging insights into the pathobiology underlying AEC mtDNA damage and apoptosis is suggesting novel therapeutic targets that may prove useful for the management of age-related diseases, including pulmonary fibrosis and lung cancer. PMID:26370974

  4. Non-toxic engineered carbon nanodiamond concentrations induce oxidative/nitrosative stress, imbalance of energy metabolism, and mitochondrial dysfunction in microglial and alveolar basal epithelial cells.

    Science.gov (United States)

    Fresta, Claudia G; Chakraborty, Aishik; Wijesinghe, Manjula B; Amorini, Angela M; Lazzarino, Giacomo; Lazzarino, Giuseppe; Tavazzi, Barbara; Lunte, Susan M; Caraci, Filippo; Dhar, Prajnaparamita; Caruso, Giuseppe

    2018-02-14

    Engineered nanoparticles are finding a wide spectrum of biomedical applications, including drug delivery and capacity to trigger cytotoxic phenomena, potentially useful against tumor cells. The full understanding of their biosafety and interactions with cell processes is mandatory. Using microglial (BV-2) and alveolar basal epithelial (A549) cells, in this study we determined the effects of engineered carbon nanodiamonds (ECNs) on cell viability, nitric oxide (NO) and reactive oxygen species (ROS) production, as well as on energy metabolism. Particularly, we initially measured decrease in cell viability as a function of increasing ECNs doses, finding similar cytotoxic ECN effects in the two cell lines. Subsequently, using apparently non-cytotoxic ECN concentrations (2 µg/mL causing decrease in cell number < 5%) we determined NO and ROS production, and measured the concentrations of compounds related to energy metabolism, mitochondrial functions, oxido-reductive reactions, and antioxidant defences. We found that in both cell lines non-cytotoxic ECN concentrations increased NO and ROS production with sustained oxidative/nitrosative stress, and caused energy metabolism imbalance (decrease in high energy phosphates and nicotinic coenzymes) and mitochondrial malfunctioning (decrease in ATP/ADP ratio).These results underline the importance to deeply investigate the molecular and biochemical changes occurring upon the interaction of ECNs (and nanoparticles in general) with living cells, even at apparently non-toxic concentration. Since the use of ECNs in biomedical field is attracting increasing attention the complete evaluation of their biosafety, toxicity and/or possible side effects both in vitro and in vivo is mandatory before these highly promising tools might find the correct application.

  5. Characterisation of cellular adhesion reinforcement by multiple bond force spectroscopy in alveolar epithelial cells.

    Science.gov (United States)

    Nguyen, Ngoc-Minh; Angely, Christelle; Andre Dias, Sofia; Planus, Emmanuelle; Filoche, Marcel; Pelle, Gabriel; Louis, Bruno; Isabey, Daniel

    2017-07-01

    Integrin-mediated adhesion is a key process by which cells physically connect with their environment, and express sensitivity and adaptation through mechanotransduction. A critical step of cell adhesion is the formation of the first bonds which individually generate weak contacts (∼tens pN) but can sustain thousand times higher forces (∼tens nN) when associated. We propose an experimental validation by multiple bond force spectroscopy (MFS) of a stochastic model predicting adhesion reinforcement permitted by non-cooperative, multiple bonds on which force is homogeneously distributed (called parallel bond configuration). To do so, spherical probes (diameter: 6.6 μm), specifically coated by RGD-peptide to bind integrins, are used to statically indent and homogenously stretch the multiple bonds created for short contact times (2 s) between the bead and the surface of epithelial cells (A549). Using different separation speeds (v = 2, 5, 10 μm/s) and measuring cellular Young's modulus as well as the local stiffness preceding local rupture events, we obtain cell-by-cell the effective loading rates both at the global cell level and at the local level of individual constitutive bonds. Local rupture forces are in the range: f*=60-115 pN , whereas global rupture (detachment) forces reach F*=0.8-1.7 nN . Global and local rupture forces both exhibit linear dependencies with the effective loading rate, the slopes of these two linear relationships providing an estimate of the number of independent integrin bonds constituting the tested multiple bond structure (∼12). The MFS method enables to validate the reinforcement of integrin-mediated adhesion induced by the multiple bond configuration in which force is homogeneously distributed amongst parallel bonds. Local rupture events observed in the course of a spectroscopy manoeuver (MFS) lead to rupture force values considered in the literature as single-integrin bonds. Adhesion reinforcement permitted by the parallel

  6. Pirfenidone inhibits TGF-β1-induced over-expression of collagen type I and heat shock protein 47 in A549 cells

    Directory of Open Access Journals (Sweden)

    Hisatomi Keiko

    2012-06-01

    Full Text Available Abstract Background Pirfenidone is a novel anti-fibrotic and anti-inflammatory agent that inhibits the progression of fibrosis in animal models and in patients with idiopathic pulmonary fibrosis (IPF. We previously showed that pirfenidone inhibits the over-expression of collagen type I and of heat shock protein (HSP 47, a collagen-specific molecular chaperone, in human lung fibroblasts stimulated with transforming growth factor (TGF-β1 in vitro. The increased numbers of HSP47-positive type II pneumocytes as well as fibroblasts were also diminished by pirfenidone in an animal model of pulmonary fibrosis induced by bleomycin. The present study evaluates the effects of pirfenidone on collagen type I and HSP47 expression in the human alveolar epithelial cell line, A549 cells in vitro. Methods The expression of collagen type I, HSP47 and E-cadherin mRNAs in A549 cells stimulated with TGF-β1 was evaluated by Northern blotting or real-time PCR. The expression of collagen type I, HSP47 and fibronectin proteins was assessed by immunocytochemical staining. Results TGF-β1 stimulated collagen type I and HSP47 mRNA and protein expression in A549 cells, and pirfenidone significantly inhibited this process. Pirfenidone also inhibited over-expression of the fibroblast phenotypic marker fibronectin in A549 cells induced by TGF-β1. Conclusion We concluded that the anti-fibrotic effects of pirfenidone might be mediated not only through the direct inhibition of collagen type I expression but also through the inhibition of HSP47 expression in alveolar epithelial cells, which results in reduced collagen synthesis in lung fibrosis. Furthermore, pirfenidone might partially inhibit the epithelial-mesenchymal transition.

  7. Kaempferol Suppresses Transforming Growth Factor-β1–Induced Epithelial-to-Mesenchymal Transition and Migration of A549 Lung Cancer Cells by Inhibiting Akt1-Mediated Phosphorylation of Smad3 at Threonine-1791

    Science.gov (United States)

    Jo, Eunji; Park, Seong Ji; Choi, Yu Sun; Jeon, Woo-Kwang; Kim, Byung-Chul

    2015-01-01

    Kaempferol, a natural dietary flavonoid, is well known to possess chemopreventive and therapeutic anticancer efficacy; however, its antimetastatic effects have not been mechanistically studied so far in any cancer model. This study was aimed to investigate the inhibitory effect and accompanying mechanisms of kaempferol on epithelial-to-mesenchymal transition (EMT) and cell migration induced by transforming growth factor-β1 (TGF-β1). In human A549 non–small lung cancer cells, kaempferol strongly blocked the enhancement of cell migration by TGF-β1–induced EMT through recovering the loss of E-cadherin and suppressing the induction of mesenchymal markers as well as the upregulation of TGF-β1–mediated matrix metalloproteinase-2 activity. Interestingly, kaempferol reversed TGF-β1–mediated Snail induction and E-cadherin repression by weakening Smad3 binding to the Snail promoter without affecting its C-terminus phosphorylation, complex formation with Smad4, and nuclear translocation under TGF-β1 stimulation. Mechanism study revealed that the phosphorylation of Smad3 linker region induced by TGF-β1 was required for the induction of EMT and cell migration, and selective downregulation of the phosphorylation of Smad3 at Thr179 residue (not Ser204, Ser208, and Ser213) in the linker region was responsible for the inhibition by kaempferol of TGF-β1–induced EMT and cell migration. Furthermore, Akt1 was required for TGF-β1–mediated induction of EMT and cell migration and directly phosphorylated Smad3 at Thr179, and kaempferol completely abolished TGF-β1–induced Akt1 phosphorylation. In summary, kaempferol blocks TGF-β1–induced EMT and migration of lung cancer cells by inhibiting Akt1-mediated phosphorylation of Smad3 at Thr179 residue, providing the first evidence of a molecular mechanism for the anticancer effect of kaempferol. PMID:26297431

  8. Kaempferol Suppresses Transforming Growth Factor-β1-Induced Epithelial-to-Mesenchymal Transition and Migration of A549 Lung Cancer Cells by Inhibiting Akt1-Mediated Phosphorylation of Smad3 at Threonine-179.

    Science.gov (United States)

    Jo, Eunji; Park, Seong Ji; Choi, Yu Sun; Jeon, Woo-Kwang; Kim, Byung-Chul

    2015-07-01

    Kaempferol, a natural dietary flavonoid, is well known to possess chemopreventive and therapeutic anticancer efficacy; however, its antimetastatic effects have not been mechanistically studied so far in any cancer model. This study was aimed to investigate the inhibitory effect and accompanying mechanisms of kaempferol on epithelial-to-mesenchymal transition (EMT) and cell migration induced by transforming growth factor-β1 (TGF-β1). In human A549 non-small lung cancer cells, kaempferol strongly blocked the enhancement of cell migration by TGF-β1-induced EMT through recovering the loss of E-cadherin and suppressing the induction of mesenchymal markers as well as the upregulation of TGF-β1-mediated matrix metalloproteinase-2 activity. Interestingly, kaempferol reversed TGF-β1-mediated Snail induction and E-cadherin repression by weakening Smad3 binding to the Snail promoter without affecting its C-terminus phosphorylation, complex formation with Smad4, and nuclear translocation under TGF-β1 stimulation. Mechanism study revealed that the phosphorylation of Smad3 linker region induced by TGF-β1 was required for the induction of EMT and cell migration, and selective downregulation of the phosphorylation of Smad3 at Thr179 residue (not Ser204, Ser208, and Ser213) in the linker region was responsible for the inhibition by kaempferol of TGF-β1-induced EMT and cell migration. Furthermore, Akt1 was required for TGF-β1-mediated induction of EMT and cell migration and directly phosphorylated Smad3 at Thr179, and kaempferol completely abolished TGF-β1-induced Akt1 phosphorylation. In summary, kaempferol blocks TGF-β1-induced EMT and migration of lung cancer cells by inhibiting Akt1-mediated phosphorylation of Smad3 at Thr179 residue, providing the first evidence of a molecular mechanism for the anticancer effect of kaempferol. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Essential role for cathepsin D in bleomycin-induced apoptosis of alveolar epithelial cells.

    Science.gov (United States)

    Li, Xiaopeng; Rayford, Heather; Shu, Ruijie; Zhuang, Jiaju; Uhal, Bruce D

    2004-07-01

    Our earlier studies showed that bleomycin-induced apoptosis of type II alveolar epithelial cells (AECs) requires the autocrine synthesis and proteolytic processing of angiotensinogen into ANG II and that inhibitors of ANG-converting enzyme (ACEis) block bleomycin-induced apoptosis (Li X, Zhang H, Soledad-Conrad V, Zhuang J, and Uhal BD. Am J Physiol Lung Cell Mol Physiol 284: L501-L507, 2003). Given the documented role of cathepsin D (CatD) in apoptosis of other cell types, we hypothesized that CatD might be the AEC enzyme responsible for the conversion of angiotensinogen into ANG I, the substrate for ACE. Primary cultures of rat type II AECs challenged with bleomycin in vitro showed upregulation and secretion of CatD enzymatic activity and immunoreactive protein but no increases in CatD mRNA. The aspartyl protease inhibitor pepstatin A, which completely blocked CatD enzymatic activity, inhibited bleomycin-induced nuclear fragmentation by 76% and reduced bleomycin-induced caspase-3 activation by 47%. Antisense oligonucleotides against CatD mRNA reduced CatD-immunoreactive protein and inhibited bleomycin-induced nuclear fragmentation by 48%. A purified fragment of angiotensinogen (F1-14) containing the CatD and ACE cleavage sites, when applied to unchallenged AEC in vitro, yielded mature ANG II peptide and induced apoptosis. The apoptosis induced by F1-14 was inhibited 96% by pepstatin A and 77% by neutralizing antibodies specific for CatD (both P CatD in bleomycin-induced apoptosis of cultured AEC and suggest that the role(s) of CatD in AEC apoptosis include the conversion of newly synthesized angiotensinogen to ANG II.

  10. The influence of volatile anesthetics on alveolar epithelial permeability measured by noninvasive radionuclide lung scan

    International Nuclear Information System (INIS)

    Hung, Chih-Jen; Wu, Rick Sai-Chuen; Lin, Cheng-Chieh; Kao, Albert; Tsai, Jeffrey J.P.

    2003-01-01

    Many volatile anesthetics have long been thought to affect pulmonary functions including lung ventilation (LV) and alveolar epithelial permeability (AEP). The purpose of this study is to examine the influence of volatile anesthetics on LV and AEP by noninvasive radionuclide lung imaging of technetium-99m labeled diethylene triamine pentaacetic acid radioaerosol inhalation lung scan (DTPA lung scan). Twenty patients undergoing surgery and receiving volatile anesthesia with 1% halothane were enrolled as the study group 1. The other 20 patients undergoing surgery and receiving volatile anesthesia with 1.5% isoflurane were enrolled as the study group 2. At the same time, 20 patients undergoing surgery with intravenous anesthesia drugs were included as a control group. Before surgery, 1 hour after surgery, and 1 week after surgery, we investigated the 3 groups of patients with DTPA lung scan to evaluate LV and AEP by 99m Tc DTPA clearance halftime (T1/2). No significant change or abnormality of LV before surgery, 1 hour after surgery, or 1 week after surgery was found among the 3 groups of patients. In the control group, the 99m Tc DTPA clearance T1/2 was 63.5±16.4, 63.1±18.4, and 62.8±17.0 minutes, before surgery, 1 hour after surgery, and 1 week after surgery, respectively. In group 1, it was 65.9±9.3, 62.5±9.1, and 65.8±10.3 minutes, respectively. No significant change in AEP before surgery, 1 hour after surgery, or 1 week after surgery was found. However, in group 2, the 99m Tc DTPA clearance T1/2 was 65.5±13.2, 44.9±10.5, and 66.1±14.0 minutes, respectively. A significant transient change in AEP was found 1 hour after surgery, but it recovered 1 week after surgery. We conclude that volatile anesthesia is safe for LV and AEP, and only isoflurane can induce transient change of AEP. (author)

  11. Mammary alveolar epithelial cells convert to brown adipocytes in post-lactating mice

    DEFF Research Database (Denmark)

    Giordano, Antonio; Perugini, Jessica; Kristensen, David Møbjerg

    2017-01-01

    found close to the mammary alveoli contain milk protein granules. Use of the Cre-loxP recombination system allowed showing that the involuting mammary gland of whey acidic protein-Cre/R26R mice, whose secretory alveolar cells express the lacZ gene during pregnancy, contains some X...

  12. Dual role for plasminogen activator inhibitor type 1 as soluble and as matricellular regulator of epithelial alveolar cell wound healing.

    Science.gov (United States)

    Maquerlot, François; Galiacy, Stephane; Malo, Michel; Guignabert, Christophe; Lawrence, Daniel A; d'Ortho, Maria-Pia; Barlovatz-Meimon, Georgia

    2006-11-01

    Epithelium repair, crucial for restoration of alveolo-capillary barrier integrity, is orchestrated by various cytokines and growth factors. Among them keratinocyte growth factor plays a pivotal role in both cell proliferation and migration. The urokinase plasminogen activator (uPA) system also influences cell migration through proteolysis during epithelial repair. In addition, the complex formed by uPAR-uPA and matrix-bound plasminogen activator inhibitor type-1 (PAI-1) exerts nonproteolytic roles in various cell types. Here we present new evidence about the dual role of PAI-1 under keratinocyte growth factor stimulation using an in vitro repair model of rat alveolar epithelial cells. Besides proteolytic involvement of the uPA system, the availability of matrix-bound-PAI-1 is also required for an efficient healing. An unexpected decrease of healing was shown when PAI-1 activity was blocked. However, the proteolytic action of uPA and plasmin were still required. Moreover, immediately after wounding, PAI-1 was dramatically increased in the newly deposited matrix at the leading edge of wounds. We thus propose a dual role for PAI-1 in epithelial cell wound healing, both as a soluble inhibitor of proteolysis and also as a matrix-bound regulator of cell migration. Matrix-bound PAI-1 could thus be considered as a new member of the matricellular protein family.

  13. Wnt-inducible protein (WISP-1 is a key regulator of alveolar epithelial cell hyperplasia in pulmonary fibrosis

    Directory of Open Access Journals (Sweden)

    Melanie Königshoff

    2006-12-01

    Full Text Available Fibrotic lung disease is characterized by distorted lung architecture and severe loss of respiratory function secondary to alveolar epithelial cell (AEC hyperplasia, enhanced extracellular matrix (ECM deposition and fibroblast proliferation. Repetitive epithelial injuries with impaired alveolar wound healing and altered AEC gene expression represent a trigger mechanism for development of fibrosis. To reveal gene regulatory networks in lung fibrosis, we compared gene expression profiles of freshly isolated AEC obtained from mice 14 days after saline or bleomycin (BM instillation using whole genome microarray analysis. Several genes of the Wnt signaling pathway, in particular WISP-1, a member of the CCN family, were highly regulated. WISP-1 protein expression was demonstrated in proliferating AEC in BM-treated lungs by immunofluorescence. When analyzing all six CCN family members, WISP-1 was upregulated the most 14 days after BM challenge, as analyzed by qRT-PCR. To elucidate WISP-1 function, cultured primary mouse AEC were stimulated with WISP-1 and demonstrated a 230% increase in proliferation, analyzed by 3H-thymidine incorporation. This was mediated through enhanced phosphorylation, but not expression of protein kinase B (PKB/Akt, as detected by immunoblot. Finally, increased expression of WISP-1 was detected in lung homogenates and isolated AEC from IPF patients, using qRT-PCR. Immunohistochemical analysis of WISP-1 and Ki67 verified the existence of hyperplastic and proliferative AEC expressing WISP-1 in vivo. Our study thus identifies WISP-1 as a novel regulator of AEC injury and repair, and suggests that WISP-1 is a key mediator in pulmonary fibrosis.

  14. The reduction of l-cystine to l-cysteine in the supernatant of A549 cell culture causes imipenem inactivation.

    Science.gov (United States)

    Takemura, Hiromu; Terakubo, Shigemi; Okamura, Ninyo; Nakashima, Hideki

    2018-02-26

    In the course of measuring the intracellular antibacterial activity of antibiotics using a human alveolar epithelial cell line A549, we discovered that the antimicrobial activity of several carbapenems (CPs) decreased in the supernatant of the cells cultured with fetal calf serum (FCS)-free RPMI1640 medium (RPMI). Further investigation revealed A549 culture supernatant inhibited the antibacterial activity of CPs but did not inactivate other types of antibiotics. CE-TOFMS and LC-TOFMS metabolomics analysis of the supernatant revealed the presence of l-cysteine (Cys), which is not an original component in RPMI. Cys is known to hydrolyze and inactivate CPs in a time- and concentration-dependent manner. In this study, the inactivating effects of A549 culture supernatant on the imipenem (IPM) were examined. Antimicrobial activity of 100 μg/mL IPM decreased to 25% with two-fold dilution of A549 supernatant incubated for 3 h. l-Cystine (CS), the Cys oxide, and an original component in RPMI did not inactivate IPM. However, the inactivating effects of A549 supernatant on IPM corresponds with the Cys concentration and depends on the CS content of the culture medium. Addition of FCS to the culture medium decreased the Cys concentration and reduced inactivation of IPM in a dose-dependent manner. Our data suggest that IPM were inactivated by Cys reduced from CS, and this CS-to-Cys conversion must be considered when evaluating the antimicrobial activity of CPs in cell culture. Further studies are needed to understand if the same inactivation occurs around the cells in the human body. Copyright © 2017 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  15. Enolase 1 (ENO1 and protein disulfide-isomerase associated 3 (PDIA3 regulate Wnt/β-catenin-driven trans-differentiation of murine alveolar epithelial cells

    Directory of Open Access Journals (Sweden)

    Kathrin Mutze

    2015-08-01

    Full Text Available The alveolar epithelium represents a major site of tissue destruction during lung injury. It consists of alveolar epithelial type I (ATI and type II (ATII cells. ATII cells are capable of self-renewal and exert progenitor function for ATI cells upon alveolar epithelial injury. Cell differentiation pathways enabling this plasticity and allowing for proper repair, however, are poorly understood. Here, we applied proteomics, expression analysis and functional studies in primary murine ATII cells to identify proteins and molecular mechanisms involved in alveolar epithelial plasticity. Mass spectrometry of cultured ATII cells revealed a reduction of carbonyl reductase 2 (CBR2 and an increase in enolase 1 (ENO1 and protein disulfide-isomerase associated 3 (PDIA3 protein expression during ATII-to-ATI cell trans-differentiation. This was accompanied by increased Wnt/β-catenin signaling, as analyzed by qRT-PCR and immunoblotting. Notably, ENO1 and PDIA3, along with T1α (podoplanin; an ATI cell marker, exhibited decreased protein expression upon pharmacological and molecular Wnt/β-catenin inhibition in cultured ATII cells, whereas CBR2 levels were stabilized. Moreover, we analyzed primary ATII cells from mice with bleomycin-induced lung injury, a model exhibiting activated Wnt/β-catenin signaling in vivo. We observed reduced CBR2 significantly correlating with surfactant protein C (SFTPC, whereas ENO1 and PDIA3 along with T1α were increased in injured ATII cells. Finally, siRNA-mediated knockdown of ENO1, as well as PDIA3, in primary ATII cells led to reduced T1α expression, indicating diminished cell trans-differentiation. Our data thus identified proteins involved in ATII-to-ATI cell trans-differentiation and suggest a Wnt/β-catenin-driven functional role of ENO1 and PDIA3 in alveolar epithelial cell plasticity in lung injury and repair.

  16. Airway epithelial cell response to human metapneumovirus infection

    International Nuclear Information System (INIS)

    Bao, X.; Liu, T.; Spetch, L.; Kolli, D.; Garofalo, R.P.; Casola, A.

    2007-01-01

    Human metapneumovirus (hMPV) is a major cause of lower respiratory tract infections (LRTIs) in infants, elderly and immunocompromised patients. In this study, we show that hMPV can infect in a similar manner epithelial cells representative of different tracts of the airways. hMPV-induced expression of chemokines IL-8 and RANTES in primary small alveolar epithelial cells (SAE) and in a human alveolar type II-like epithelial cell line (A549) was similar, suggesting that A549 cells can be used as a model to study lower airway epithelial cell responses to hMPV infection. A549 secreted a variety of CXC and CC chemokines, cytokines and type I interferons, following hMPV infection. hMPV was also a strong inducer of transcription factors belonging to nuclear factor (NF)-κB, interferon regulatory factors (IRFs) and signal transducers and activators of transcription (STATs) families, which are known to orchestrate the expression of inflammatory and immunomodulatory mediators

  17. Balance of life and death in alveolar epithelial type II cells: proliferation, apoptosis, and the effects of cyclic stretch on wound healing.

    Science.gov (United States)

    Crosby, Lynn M; Luellen, Charlean; Zhang, Zhihong; Tague, Larry L; Sinclair, Scott E; Waters, Christopher M

    2011-10-01

    After acute lung injury, repair of the alveolar epithelium occurs on a substrate undergoing cyclic mechanical deformation. While previous studies showed that mechanical stretch increased alveolar epithelial cell necrosis and apoptosis, the impact of cell death during repair was not determined. We examined epithelial repair during cyclic stretch (CS) in a scratch-wound model of primary rat alveolar type II (ATII) cells and found that CS altered the balance between proliferation and cell death. We measured cell migration, size, and density; intercellular gap formation; cell number, proliferation, and apoptosis; cytoskeletal organization; and focal adhesions in response to scratch wounding followed by CS for up to 24 h. Under static conditions, wounds were closed by 24 h, but repair was inhibited by CS. Wounding stimulated cell motility and proliferation, actin and vinculin redistribution, and focal adhesion formation at the wound edge, while CS impeded cell spreading, initiated apoptosis, stimulated cytoskeletal reorganization, and attenuated focal adhesion formation. CS also caused significant intercellular gap formation compared with static cells. Our results suggest that CS alters several mechanisms of epithelial repair and that an imbalance occurs between cell death and proliferation that must be overcome to restore the epithelial barrier.

  18. Environmental particulate (PM2.5 augments stiffness-induced alveolar epithelial cell mechanoactivation of transforming growth factor beta.

    Directory of Open Access Journals (Sweden)

    Marilyn M Dysart

    Full Text Available Dysfunctional pulmonary homeostasis and repair, including diseases such as pulmonary fibrosis (PF, chronic obstructive pulmonary disease (COPD, and tumorigenesis have been increasing over the past decade, a fact that heavily implicates environmental influences. Several investigations have suggested that in response to increased transforming growth factor--beta (TGFβ signaling, the alveolar type II (ATII epithelial cell undergoes phenotypic changes that may contribute to the complex pathobiology of PF. We have previously demonstrated that increased tissue stiffness associated with PF is a potent extracellular matrix (ECM signal for epithelial cell activation of TGFβ. The work reported here explores the relationship between tissue stiffness and exposure to environmental stimuli in the activation of TGFβ. We hypothesized that exposure of ATII cells to fine particulate matter (PM2.5 will result in enhanced cell contractility, TGFβ activation, and subsequent changes to ATII cell phenotype. ATII cells were cultured on increasingly stiff substrates with or without addition of PM2.5. Exposure to PM2.5 resulted in increased activation of TGFβ, increased cell contractility, and elongation of ATII cells. Most notably, on 8 kPa substrates, a stiffness greater than normal but less than established fibrotic lung, addition of PM2.5 resulted in increased cortical cell stiffness, enhanced actin staining and cell elongation; a result not seen in the absence of PM2.5. Our work suggests that PM2.5 exposure additionally enhances the existing interaction between ECM stiffness and TGFβ that has been previously reported. Furthermore, we show that this additional enhancement is likely a consequence of intracellular reactive oxygen species (ROS leading to increased TGFβ signaling events. These results highlight the importance of both the micromechanical and biochemical environment in lung disease initiation and suggest that individuals in early stages of lung

  19. Perfluorocarbon reduces cell damage from blast injury by inhibiting signal paths of NF-κB, MAPK and Bcl-2/Bax signaling pathway in A549 cells.

    Science.gov (United States)

    Zhang, Zhaorui; Liang, Zhixin; Li, Huaidong; Li, Chunsun; Yang, Zhen; Li, Yanqin; She, Danyang; Cao, Lu; Wang, Wenjie; Liu, Changlin; Chen, Liangan

    2017-01-01

    Blast lung injury is a common type of blast injury and has very high mortality. Therefore, research to identify medical therapies for blast injury is important. Perfluorocarbon (PFC) is used to improve gas exchange in diseased lungs and has anti-inflammatory functions in vitro and in vivo. The aim of this study was to determine whether PFC reduces damage to A549 cells caused by blast injury and to elucidate its possible mechanisms of action. A549 alveolar epithelial cells exposed to blast waves were treated with and without PFC. Morphological changes and apoptosis of A549 cells were recorded. PCR and enzyme-linked immunosorbent assay (ELISA) were used to measure the mRNA or protein levels of IL-1β, IL-6 and TNF-α. Malondialdehyde (MDA) levels and superoxide dismutase (SOD) activity levels were detected. Western blot was used to quantify the expression of NF-κB, Bax, Bcl-2, cleaved caspase-3 and MAPK cell signaling proteins. A549 cells exposed to blast wave shrank, with less cell-cell contact. The morphological change of A549 cells exposed to blast waves were alleviated by PFC. PFC significantly inhibited the apoptosis of A549 cells exposed to blast waves. IL-1β, IL-6 and TNF-α cytokine and mRNA expression levels were significantly inhibited by PFC. PFC significantly increased MDA levels and decreased SOD activity levels. Further studies indicated that NF-κB, Bax, caspase-3, phospho-p38, phosphor-ERK and phosphor-JNK proteins were also suppressed by PFC. The quantity of Bcl-2 protein was increased by PFC. Our research showed that PFC reduced A549 cell damage caused by blast injury. The potential mechanism may be associated with the following signaling pathways: 1) the signaling pathways of NF-κB and MAPK, which inhibit inflammation and reactive oxygen species (ROS); and 2) the signaling pathways of Bcl-2/Bax and caspase-3, which inhibit apoptosis.

  20. Overexpression of sICAM-1 in the Alveolar Epithelial Space Results in an Exaggerated Inflammatory Response and Early Death in Gram Negative Pneumonia

    Directory of Open Access Journals (Sweden)

    Curtis Jeffery L

    2011-01-01

    Full Text Available Abstract Background A sizeable body of data demonstrates that membrane ICAM-1 (mICAM-1 plays a significant role in host defense in a site-specific fashion. On the pulmonary vascular endothelium, mICAM-1 is necessary for normal leukocyte recruitment during acute inflammation. On alveolar epithelial cells (AECs, we have shown previously that the presence of normal mICAM-1 is essential for optimal alveolar macrophage (AM function. We have also shown that ICAM-1 is present in the alveolar space as a soluble protein that is likely produced through cleavage of mICAM-1. Soluble intercellular adhesion molecule-1 (sICAM-1 is abundantly present in the alveolar lining fluid of the normal lung and could be generated by proteolytic cleavage of mICAM-1, which is highly expressed on type I AECs. Although a growing body of data suggesting that intravascular sICAM-1 has functional effects, little is known about sICAM-1 in the alveolus. We hypothesized that sICAM-1 in the alveolar space modulates the innate immune response and alters the response to pulmonary infection. Methods Using the surfactant protein C (SPC promoter, we developed a transgenic mouse (SPC-sICAM-1 that constitutively overexpresses sICAM-1 in the distal lung, and compared the responses of wild-type and SPC-sICAM-1 mice following intranasal inoculation with K. pneumoniae. Results SPC-sICAM-1 mice demonstrated increased mortality and increased systemic dissemination of organisms compared with wild-type mice. We also found that inflammatory responses were significantly increased in SPC-sICAM-1 mice compared with wild-type mice but there were no difference in lung CFU between groups. Conclusions We conclude that alveolar sICAM-1 modulates pulmonary inflammation. Manipulating ICAM-1 interactions therapeutically may modulate the host response to Gram negative pulmonary infections.

  1. Comparison of plasma, epithelial lining fluid, and alveolar macrophage concentrations of solithromycin (CEM-101) in healthy adult subjects.

    Science.gov (United States)

    Rodvold, Keith A; Gotfried, Mark H; Still, J Gordon; Clark, Kay; Fernandes, Prabhavathi

    2012-10-01

    The steady-state concentrations of solithromycin in plasma were compared with concomitant concentrations in epithelial lining fluid (ELF) and alveolar macrophages (AM) obtained from intrapulmonary samples during bronchoscopy and bronchoalveolar lavage (BAL) in 30 healthy adult subjects. Subjects received oral solithromycin at 400 mg once daily for five consecutive days. Bronchoscopy and BAL were carried out once in each subject at either 3, 6, 9, 12, or 24 h after the last administered dose of solithromycin. Drug concentrations in plasma, ELF, and AM were assayed by a high-performance liquid chromatography-tandem mass spectrometry method. Solithromycin was concentrated extensively in ELF (range of mean [± standard deviation] concentrations, 1.02 ± 0.83 to 7.58 ± 6.69 mg/liter) and AM (25.9 ± 20.3 to 101.7 ± 52.6 mg/liter) in comparison with simultaneous plasma concentrations (0.086 ± 0.070 to 0.730 ± 0.692 mg/liter). The values for the area under the concentration-time curve from 0 to 24 h (AUC(0-24) values) based on mean and median ELF concentrations were 80.3 and 63.2 mg · h/liter, respectively. The ratio of ELF to plasma concentrations based on the mean and median AUC(0-24) values were 10.3 and 10.0, respectively. The AUC(0-24) values based on mean and median concentrations in AM were 1,498 and 1,282 mg · h/L, respectively. The ratio of AM to plasma concentrations based on the mean and median AUC(0-24) values were 193 and 202, respectively. Once-daily oral dosing of solithromycin at 400 mg produced steady-state concentrations that were significantly (P solithromycin administration.

  2. The role of alveolar epithelial cells in initiating and shaping pulmonary immune responses: communication between innate and adaptive immune systems.

    Directory of Open Access Journals (Sweden)

    Olga D Chuquimia

    Full Text Available Macrophages and dendritic cells have been recognized as key players in the defense against mycobacterial infection. However, more recently, other cells in the lungs such as alveolar epithelial cells (AEC have been found to play important roles in the defense and pathogenesis of infection. In the present study we first compared AEC with pulmonary macrophages (PuM isolated from mice in their ability to internalize and control Bacillus Calmette-Guérin (BCG growth and their capacity as APCs. AEC were able to internalize and control bacterial growth as well as present antigen to primed T cells. Secondly, we compared both cell types in their capacity to secrete cytokines and chemokines upon stimulation with various molecules including mycobacterial products. Activated PuM and AEC displayed different patterns of secretion. Finally, we analyzed the profile of response of AEC to diverse stimuli. AEC responded to both microbial and internal stimuli exemplified by TLR ligands and IFNs, respectively. The response included synthesis by AEC of several factors, known to have various effects in other cells. Interestingly, TNF could stimulate the production of CCL2/MCP-1. Since MCP-1 plays a role in the recruitment of monocytes and macrophages to sites of infection and macrophages are the main producers of TNF, we speculate that both cell types can stimulate each other. Also, another cell-cell interaction was suggested when IFNs (produced mainly by lymphocytes were able to induce expression of chemokines (IP-10 and RANTES by AEC involved in the recruitment of circulating lymphocytes to areas of injury, inflammation, or viral infection. In the current paper we confirm previous data on the capacity of AEC regarding internalization of mycobacteria and their role as APC, and extend the knowledge of AEC as a multifunctional cell type by assessing the secretion of a broad array of factors in response to several different types of stimuli.

  3. Ethanol alters alveolar fluid balance via Nadph oxidase (NOX signaling to epithelial sodium channels (ENaC in the lung.

    Directory of Open Access Journals (Sweden)

    Charles A Downs

    Full Text Available Chronic alcohol consumption is associated with increased incidence of ICU-related morbidity and mortality, primarily from acute respiratory distress syndrome (ARDS. However, the mechanisms involved are unknown. One explanation is that alcohol regulates epithelial sodium channels (ENaC via oxidant signaling to promote a pro- injury environment. We used small rodent models to mimic acute and chronic alcohol consumption and tested the hypothesis that ethanol (EtOH would affect lung fluid clearance by up-regulating ENaC activity in the lung. Fluorescence labeling of rat lung slices and in vivo mouse lung revealed an increase in ROS production in response to acute EtOH exposure. Using western blots and fluorescein-5-maleimide labeling, we conclude that EtOH exposure modifies cysteines of α-ENaC while data from single channel patch clamp analysis confirm that 0.16% EtOH increased ENaC activity in rat alveolar cells. In vivo lung fluid clearance demonstrated a latent increase in fluid clearance in mice receiving EtOH diet. Ethanol mice given a tracheal instillation of LPS demonstrated early lung fluid clearance compared to caloric control mice and C57Bl/6 mice. Standard biochemical techniques reveal that chronic EtOH consumption resulted in greater protein expression of the catalytic gp91(phox subunit and the obligate Rac1 protein. Collectively these data suggest that chronic EtOH consumption may lead to altered regulation of ENaC, contributing to a 'pro-injury' environment in the alcohol lung.

  4. Development of a lung slice preparation for recording ion channel activity in alveolar epithelial type I cells

    Directory of Open Access Journals (Sweden)

    Crandall Edward D

    2005-04-01

    Full Text Available Abstract Background Lung fluid balance in the healthy lung is dependent upon finely regulated vectorial transport of ions across the alveolar epithelium. Classically, the cellular locus of the major ion transport processes has been widely accepted to be the alveolar type II cell. Although evidence is now emerging to suggest that the alveolar type I cell might significantly contribute to the overall ion and fluid homeostasis of the lung, direct assessment of functional ion channels in type I cells has remained elusive. Methods Here we describe a development of a lung slice preparation that has allowed positive identification of alveolar type I cells within an intact and viable alveolar epithelium using living cell immunohistochemistry. Results This technique has allowed, for the first time, single ion channels of identified alveolar type I cells to be recorded using the cell-attached configuration of the patch-clamp technique. Conclusion This exciting new development should facilitate the ascription of function to alveolar type I cells and allow us to integrate this cell type into the general model of alveolar ion and fluid balance in health and disease.

  5. Runx3 is a key modulator during the epithelial-mesenchymal transition of alveolar type II cells in animal models of BPD.

    Science.gov (United States)

    Yang, Haiping; Fu, Jianhua; Yao, Li; Hou, Ana; Xue, Xindong

    2017-11-01

    Bronchopulmonary dysplasia (BPD) is a major challenge for premature infants; however, the underlying mechanisms remain unclear. We previously reported that epithelial-mesenchymal transition (EMT) in alveolar type II (AT2) epithelial cells influences the normal alveolar development process. In this study, we wished to examine whether Runx3 is an important factor for BPD by regulating EMT in AT2 cells. In vivo, animal models of BPD were established by placing newborn rats in hyperoxia tanks. Lung tissue and isolated AT2 cells were collected on different days following exposure to oxygen. The pathological changes in lung tissue, alveolar development and Runx3 expression were then investigated. In vitro, RLE-6TN cells were divided into 5 groups as follows: the cont-rol, Runx3, siRunx3, transforming growth factor-β1 (TGF-β1) and Runx3 + TGF-β1 groups, and the biomarkers of EMT were investigated. In the newborn rat model of BPD, Runx3 protein and mRNA levels in both lung tissue and BPD-derived AT2 cells were significantly lower than those in the control group. The correlation between Runx3 protein expression and pulmonary development indicators was analyzed; Runx3 expression positively correlated with the radial alveolar count (RAC) and the percentage of smooth muscle actin-positive secondary septa, but negatively correlated with alveolar wall thickness. EMT was observed in the RLE-6TN cells in which the Runx3 gene was knocked down and follwoing TGF-β1‑induced EMT stimulation; however, TGF-β1 failed to induce EMT in the RLE-6TN cells overexpressing Runx3. On the whole, our data indicte that low Runx3 levels may promote EMT, while high Runx3 levels inhibit TGF-β1-induced EMT. Therefore, we predict that low levels of Runx3 in BPD lung tissue may promote EMT in AT2 cells, thus affecting alveolar development.

  6. Gefitinib and Erlotinib Lead to Phosphorylation of Eukaryotic Initiation Factor 2 Alpha Independent of Epidermal Growth Factor Receptor in A549 Cells.

    Science.gov (United States)

    Koyama, Satoshi; Omura, Tomohiro; Yonezawa, Atsushi; Imai, Satoshi; Nakagawa, Shunsaku; Nakagawa, Takayuki; Yano, Ikuko; Matsubara, Kazuo

    2015-01-01

    Gefitinib and erlotinib are anticancer agents, which inhibit epidermal growth factor receptor (EGFR) tyrosine kinase. Interstitial lung disease (ILD) occurs in patients with non-small cell lung cancer receiving EGFR inhibitors. In the present study, we examined whether gefitinib- and erlotinib-induced lung injury related to ILD through endoplasmic reticulum (ER) stress, which is a causative intracellular mechanism in cytotoxicity caused by various chemicals in adenocarcinomic human alveolar basal epithelial cells. These two EGFR inhibitors increased Parkinson juvenile disease protein 2 and C/EBP homologous protein mRNA expressions, and activated the eukaryotic initiation factor (eIF) 2α/activating transcription factor 4 pathway without protein kinase R-like ER kinase activation in A549 cells. Gefitinib and erlotinib caused neither ER stress nor cell death; however, these agents inhibited cell growth via the reduction of cyclin-D1 expression. Tauroursodeoxycholic acid, which is known to suppress eIF2α phosphorylation, cancelled the effects of EGFR inhibitors on cyclin-D1 expression and cell proliferation in a concentration-dependent manner. The results of an EGFR-silencing study using siRNA showed that gefitinib and erlotinib affected eIF2α phosphorylation and cyclin-D1 expression independent of EGFR inhibition. Therefore, the inhibition of cell growth by these EGFR inhibitors might equate to impairment of the alveolar epithelial cell repair system via eIF2α phosphorylation and reduced cyclin-D1 expression.

  7. Mitochondrial catalase overexpressed transgenic mice are protected against lung fibrosis in part via preventing alveolar epithelial cell mitochondrial DNA damage.

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    Kim, Seok-Jo; Cheresh, Paul; Jablonski, Renea P; Morales-Nebreda, Luisa; Cheng, Yuan; Hogan, Erin; Yeldandi, Anjana; Chi, Monica; Piseaux, Raul; Ridge, Karen; Michael Hart, C; Chandel, Navdeep; Scott Budinger, G R; Kamp, David W

    2016-12-01

    Alveolar epithelial cell (AEC) injury and mitochondrial dysfunction are important in the development of lung fibrosis. Our group has shown that in the asbestos exposed lung, the generation of mitochondrial reactive oxygen species (ROS) in AEC mediate mitochondrial DNA (mtDNA) damage and apoptosis which are necessary for lung fibrosis. These data suggest that mitochondrial-targeted antioxidants should ameliorate asbestos-induced lung. To determine whether transgenic mice that express mitochondrial-targeted catalase (MCAT) have reduced lung fibrosis following exposure to asbestos or bleomycin and, if so, whether this occurs in association with reduced AEC mtDNA damage and apoptosis. Crocidolite asbestos (100µg/50µL), TiO 2 (negative control), bleomycin (0.025 units/50µL), or PBS was instilled intratracheally in 8-10 week-old wild-type (WT - C57Bl/6J) or MCAT mice. The lungs were harvested at 21d. Lung fibrosis was quantified by collagen levels (Sircol) and lung fibrosis scores. AEC apoptosis was assessed by cleaved caspase-3 (CC-3)/Surfactant protein C (SFTPC) immunohistochemistry (IHC) and semi-quantitative analysis. AEC (primary AT2 cells from WT and MCAT mice and MLE-12 cells) mtDNA damage was assessed by a quantitative PCR-based assay, apoptosis was assessed by DNA fragmentation, and ROS production was assessed by a Mito-Sox assay. Compared to WT, crocidolite-exposed MCAT mice exhibit reduced pulmonary fibrosis as measured by lung collagen levels and lung fibrosis score. The protective effects in MCAT mice were accompanied by reduced AEC mtDNA damage and apoptosis. Similar findings were noted following bleomycin exposure. Euk-134, a mitochondrial SOD/catalase mimetic, attenuated MLE-12 cell DNA damage and apoptosis. Finally, compared to WT, asbestos-induced MCAT AT2 cell ROS production was reduced. Our finding that MCAT mice have reduced pulmonary fibrosis, AEC mtDNA damage and apoptosis following exposure to asbestos or bleomycin suggests an important role

  8. Quantitative Analysis of Proteome Modulations in Alveolar Epithelial Type II Cells in Response to PulmonaryAspergillus fumigatusInfection.

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    Seddigh, Pegah; Bracht, Thilo; Molinier-Frenkel, Válerie; Castellano, Flavia; Kniemeyer, Olaf; Schuster, Marc; Weski, Juliane; Hasenberg, Anja; Kraus, Andreas; Poschet, Gernot; Hager, Thomas; Theegarten, Dirk; Opitz, Christiane A; Brakhage, Axel A; Sitek, Barbara; Hasenberg, Mike; Gunzer, Matthias

    2017-12-01

    The ubiquitous mold Aspergillus fumigatus threatens immunosuppressed patients as inducer of lethal invasive aspergillosis. A. fumigatus conidia are airborne and reach the alveoli, where they encounter alveolar epithelial cells (AEC). Previous studies reported the importance of the surfactant-producing AEC II during A. fumigatus infection via in vitro experiments using cell lines. We established a negative isolation protocol yielding untouched primary murine AEC II with a purity >90%, allowing ex vivo analyses of the cells, which encountered the mold in vivo By label-free proteome analysis of AEC II isolated from mice 24h after A. fumigatus or mock infection we quantified 2256 proteins and found 154 proteins to be significantly differentially abundant between both groups (ANOVA p value ≤ 0.01, ratio of means ≥1.5 or ≤0.67, quantified with ≥2 peptides). Most of these proteins were higher abundant in the infected condition and reflected a comprehensive activation of AEC II on interaction with A. fumigatus This was especially represented by proteins related to oxidative phosphorylation, hence energy production. However, the most strongly induced protein was the l-amino acid oxidase (LAAO) Interleukin 4 induced 1 (IL4I1) with a 42.9 fold higher abundance (ANOVA p value 2.91 -10 ). IL4I1 has previously been found in B cells, macrophages, dendritic cells and rare neurons. Increased IL4I1 abundance in AEC II was confirmed by qPCR, Western blot and immunohistology. Furthermore, A. fumigatus infected lungs showed high levels of IL4I1 metabolic products. Importantly, higher IL4I1 abundance was also confirmed in lung tissue from human aspergilloma. Because LAAO are key enzymes for bactericidal product generation, AEC II might actively participate in pathogen defense. We provide insights into proteome changes of primary AEC II thereby opening new avenues to analyze the molecular changes of this central lung cell on infectious threats. Data are available via Proteome

  9. Decreased Inflammatory Responses of Human Lung Epithelial Cells after Ethanol Exposure Are Mimicked by Ethyl Pyruvate

    Directory of Open Access Journals (Sweden)

    B. Relja

    2014-01-01

    Full Text Available Background and Purpose. Leukocyte migration into alveolar space plays a critical role in pulmonary inflammation resulting in lung injury. Acute ethanol (EtOH exposure exerts anti-inflammatory effects. The clinical use of EtOH is critical due to its side effects. Here, we compared effects of EtOH and ethyl pyruvate (EtP on neutrophil adhesion and activation of cultured alveolar epithelial cells (A549. Experimental Approach. Time course and dose-dependent release of interleukin- (IL- 6 and IL-8 from A549 were measured after pretreatment of A549 with EtP (2.5–10 mM, sodium pyruvate (NaP, 10 mM, or EtOH (85–170 mM, and subsequent lipopolysaccharide or IL-1beta stimulation. Neutrophil adhesion to pretreated and stimulated A549 monolayers and CD54 surface expression were determined. Key Results. Treating A549 with EtOH or EtP reduced substantially the cytokine-induced release of IL-8 and IL-6. EtOH and EtP (but not NaP reduced the adhesion of neutrophils to monolayers in a dose- and time-dependent fashion. CD54 expression on A549 decreased after EtOH or EtP treatment before IL-1beta stimulation. Conclusions and Implications. EtP reduces secretory and adhesive potential of lung epithelial cells under inflammatory conditions. These findings suggest EtP as a potential treatment alternative that mimics the anti-inflammatory effects of EtOH in early inflammatory response in lungs.

  10. Mesenchymal Stem Cell Conditioned Medium Promotes Proliferation and Migration of Alveolar Epithelial Cells under Septic Conditions In Vitro via the JNK-P38 Signaling Pathway

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    Jie Chen

    2015-11-01

    Full Text Available Background/Aims: Mesenchymal stem cell (MSC based therapies may be useful for treating acute respiratory distress syndrome (ARDS, but the underlying mechanisms are incompletely understood. We investigated the impact of human umbilical cord Wharton's jelly-derived MSC (hUC-MSC secreted factors on alveolar epithelial cells under septic conditions and determined the relevant intracellular signaling pathways. Methods: Human alveolar epithelial cells (AEC and primary human small airway epithelial cells (SAEC were subjected to lipopolysaccharide (LPS with or without the presence of hUC-MSC-conditioned medium (CM. Proliferation and migration of AEC and SAEC were determined via an MTT assay, a wound healing assay and a transwell migration assay (only for AEC. Protein phosphorylation was determined by western blot and the experiments were repeated in presence of small-molecule inhibitors. The hMSC-secretory proteins were identified by LC-MS/MS mass spectrometry. Results: MSC-CM enhanced proliferation and migration. Activation of JNK and P38, but not ERK, was required for the proliferation and migration of AEC and SAEC. Pretreatment of AEC or SAEC with SP600125, an inhibitor of JNK1 or SB200358, an inhibitor of P38, significantly reduced cell proliferation and migration. An array of proteins including TGF-beta receptor type-1, TGF-beta receptor type-2, Ras-related C3 botulinum toxin substrate 1 and Ras-related C3 botulinum toxin substrate 2 which influencing the proliferation and migration of AEC and SAEC were detected in MSC-CM. Conclusion: Our data suggest MSC promote epithelial cell repair through releasing a repertoire of paracrine factors via activation of JNK and P38 MAPK.

  11. Alveolar epithelial and endothelial cell apoptosis in emphysema: What we know and what we need to know

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    Mathieu C Morissette

    2008-12-01

    Full Text Available Mathieu C Morissette, Julie Parent, Julie MilotCentre de Recherche de l’Hôpital Laval, Institut Universitaire de Cardiologie et de Pneumologie de l’Université Laval, Québec, CanadaAbstract: Emphysema is mainly caused by cigarette smoking and is characterized by the loss of alveolar integrity and an enlargement of the alveolar space. However, mechanisms involved in its development are not fully understood. Alveolar cell apoptosis has been previously investigated in the lung of emphysematous subjects as a potential contributor to the loss of alveolar cell and has been found abnormally elevated. Though, mechanisms involved in the increased alveolar apoptosis that occurs in emphysema have now become a prolific field of research. Those mechanisms are reviewed here with special focus on how they affect cell viability and how they may be implicated in emphysema. Moreover, we suggest a model that integrates all those mechanisms to explain the increased alveolar apoptosis observed in emphysema. This review also includes some reflections and suggestions on the research to come.Keywords: emphysema, apoptosis, proteases, VEGF, oxidative stress, TRAIL, autoimmunity

  12. Syndecan-1 mediates the coupling of positively charged submicrometer amorphous silica particles with actin filaments across the alveolar epithelial cell membrane.

    Science.gov (United States)

    Orr, Galya; Panther, David J; Cassens, Kaylyn J; Phillips, Jaclyn L; Tarasevich, Barbara J; Pounds, Joel G

    2009-04-15

    The cellular interactions and pathways of engineered submicro- and nano-scale particles dictate the cellular response and ultimately determine the level of toxicity or biocompatibility of the particles. Positive surface charge can increase particle internalization, and in some cases can also increase particle toxicity, but the underlying mechanisms are largely unknown. Here we identify the cellular interaction and pathway of positively charged submicrometer synthetic amorphous silica particles, which are used extensively in a wide range of industrial applications, and are explored for drug delivery and medical imaging and sensing. Using time lapse fluorescence imaging in living cells and other quantitative imaging approaches, it is found that heparan sulfate proteoglycans play a critical role in the attachment and internalization of the particles in alveolar type II epithelial cell line (C10), a potential target cell type bearing apical microvilli. Specifically, the transmembrane heparan sulfate proteoglycan, syndecan-1, is found to mediate the initial interactions of the particles at the cell surface, their coupling with actin filaments across the cell membrane, and their subsequent internalization via macropinocytosis. The observed interaction of syndecan molecules with the particle prior to their engagement with actin filaments suggests that the particles initiate their own internalization by facilitating the clustering of the molecules, which is required for the actin coupling and subsequent internalization of syndecan. Our observations identify a new role for syndecan-1 in mediating the cellular interactions and fate of positively charged submicrometer amorphous silica particles in the alveolar type II epithelial cell, a target cell for inhaled particles.

  13. Syndecan-1 mediates the coupling of positively charged submicrometer amorphous silica particles with actin filaments across the alveolar epithelial cell membrane

    International Nuclear Information System (INIS)

    Orr, Galya; Panther, David J.; Cassens, Kaylyn J.; Phillips, Jaclyn L.; Tarasevich, Barbara J.; Pounds, Joel G.

    2009-01-01

    The cellular interactions and pathways of engineered submicro- and nano-scale particles dictate the cellular response and ultimately determine the level of toxicity or biocompatibility of the particles. Positive surface charge can increase particle internalization, and in some cases can also increase particle toxicity, but the underlying mechanisms are largely unknown. Here we identify the cellular interaction and pathway of positively charged submicrometer synthetic amorphous silica particles, which are used extensively in a wide range of industrial applications, and are explored for drug delivery and medical imaging and sensing. Using time lapse fluorescence imaging in living cells and other quantitative imaging approaches, it is found that heparan sulfate proteoglycans play a critical role in the attachment and internalization of the particles in alveolar type II epithelial cell line (C10), a potential target cell type bearing apical microvilli. Specifically, the transmembrane heparan sulfate proteoglycan, syndecan-1, is found to mediate the initial interactions of the particles at the cell surface, their coupling with actin filaments across the cell membrane, and their subsequent internalization via macropinocytosis. The observed interaction of syndecan molecules with the particle prior to their engagement with actin filaments suggests that the particles initiate their own internalization by facilitating the clustering of the molecules, which is required for the actin coupling and subsequent internalization of syndecan. Our observations identify a new role for syndecan-1 in mediating the cellular interactions and fate of positively charged submicrometer amorphous silica particles in the alveolar type II epithelial cell, a target cell for inhaled particles.

  14. Properties and inflammatory effects of various size fractions of ambient particulate matter from Beijing on A549 and J774A.1 cells.

    Science.gov (United States)

    Wang, Bin; Li, Kexin; Jin, Wenjie; Lu, Yan; Zhang, Yuzhong; Shen, Guofeng; Wang, Rong; Shen, Huizhong; Li, Wei; Huang, Ye; Zhang, Yanyan; Wang, Xilong; Li, Xiqing; Liu, Wenxin; Cao, Hongying; Tao, Shu

    2013-09-17

    Particulate matter (PM) is a major ambient air pollutant causing millions of premature deaths each year in China. The toxicity of PM is property and size dependent. In this study, ambient PM samples collected in Beijing were divided into five size fractions with nominal aerodynamic ranges of properties including particle size distribution, specific surface area, zeta potential, dithiothreitol (DTT)-based redox ability, and contents of water-soluble organic carbon (WSOC), polycyclic aromatic hydrocarbons (PAHs), selected metals, and endotoxin. Human adenocarcinomic alveolar epithelial cell line A549 and small mouse monocyte-macrophage cell line J774A.1 were tested for their relative viabilities and inflammatory effects (interleukine-8 for A549 and tumor necrosis factor-α for J774A.1) after exposure to PM of various sizes. It was found that PM specific area was positively correlated with WSOC, high molecular weight PAHs, DTT-based redox ability, negatively correlated with surface zeta potential and lithophile metals. Several trace metals from combustion sources were enriched in intermediate size fractions. For both endotoxin concentrations of the PM and PM induced inflammatory cytokine expressions by the two cell lines, there were general increasing trends as PM size increased with an exception of the finest fraction, which induced the highest inflammatory effects. It seems that the size dependence of cytokine expression was associated with a number of properties including endotoxin content, zeta potential, settling velocity, metal content, and DTT-based redox ability.

  15. Different endocytotic uptake mechanisms for nanoparticles in epithelial cells and macrophages

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    Dagmar A. Kuhn

    2014-09-01

    Full Text Available Precise knowledge regarding cellular uptake of nanoparticles is of great importance for future biomedical applications. Four different endocytotic uptake mechanisms, that is, phagocytosis, macropinocytosis, clathrin- and caveolin-mediated endocytosis, were investigated using a mouse macrophage (J774A.1 and a human alveolar epithelial type II cell line (A549. In order to deduce the involved pathway in nanoparticle uptake, selected inhibitors specific for one of the endocytotic pathways were optimized regarding concentration and incubation time in combination with fluorescently tagged marker proteins. Qualitative immunolocalization showed that J774A.1 cells highly expressed the lipid raft-related protein flotillin-1 and clathrin heavy chain, however, no caveolin-1. A549 cells expressed clathrin heavy chain and caveolin-1, but no flotillin-1 uptake-related proteins. Our data revealed an impeded uptake of 40 nm polystyrene nanoparticles by J774A.1 macrophages when actin polymerization and clathrin-coated pit formation was blocked. From this result, it is suggested that macropinocytosis and phagocytosis, as well as clathrin-mediated endocytosis, play a crucial role. The uptake of 40 nm nanoparticles in alveolar epithelial A549 cells was inhibited after depletion of cholesterol in the plasma membrane (preventing caveolin-mediated endocytosis and inhibition of clathrin-coated vesicles (preventing clathrin-mediated endocytosis. Our data showed that a combination of several distinguishable endocytotic uptake mechanisms are involved in the uptake of 40 nm polystyrene nanoparticles in both the macrophage and epithelial cell line.

  16. Small airway epithelial cells exposure to printer-emitted engineered nanoparticles induces cellular effects on human microvascular endothelial cells in an alveolar-capillary co-culture model.

    Science.gov (United States)

    Sisler, Jennifer D; Pirela, Sandra V; Friend, Sherri; Farcas, Mariana; Schwegler-Berry, Diane; Shvedova, Anna; Castranova, Vincent; Demokritou, Philip; Qian, Yong

    2015-01-01

    The printer is one of the most common office equipment. Recently, it was reported that toner formulations for printing equipment constitute nano-enabled products (NEPs) and contain engineered nanomaterials (ENMs) that become airborne during printing. To date, insufficient research has been performed to understand the potential toxicological properties of printer-emitted particles (PEPs) with several studies using bulk toner particles as test particles. These studies demonstrated the ability of toner particles to cause chronic inflammation and fibrosis in animal models. However, the toxicological implications of inhalation exposures to ENMs emitted from laser printing equipment remain largely unknown. The present study investigates the toxicological effects of PEPs using an in vitro alveolar-capillary co-culture model with Human Small Airway Epithelial Cells (SAEC) and Human Microvascular Endothelial Cells (HMVEC). Our data demonstrate that direct exposure of SAEC to low concentrations of PEPs (0.5 and 1.0 µg/mL) caused morphological changes of actin remodeling and gap formations within the endothelial monolayer. Furthermore, increased production of reactive oxygen species (ROS) and angiogenesis were observed in the HMVEC. Analysis of cytokine and chemokine levels demonstrates that interleukin (IL)-6 and MCP-1 may play a major role in the cellular communication observed between SAEC and HMVEC and the resultant responses in HMVEC. These data indicate that PEPs at low, non-cytotoxic exposure levels are bioactive and affect cellular responses in an alveolar-capillary co-culture model, which raises concerns for potential adverse health effects.

  17. Hydrogen protects against hyperoxia-induced apoptosis in type II alveolar epithelial cells via activation of PI3K/Akt/Foxo3a signaling pathway.

    Science.gov (United States)

    Wu, Dan; Liang, Mulin; Dang, Hongxing; Fang, Fang; Xu, Feng; Liu, Chengjun

    2018-01-08

    Oxidative stress is regarded as a key regulator in the pathogenesis of prolonged hyperoxia-induced lung injury, which causes injury to alveolar epithelial cells and eventually leads to development of bronchopulmonary dysplasia (BPD). Many studies have shown that hydrogen has a protective effect in a variety of cells. However, the mechanisms by which hydrogen rescues cells from damage due to oxidative stress in BPD remains to be fully elucidated. This study sought to evaluate the effects of hydrogen on hyperoxia-induced lung injury and to investigate the underlying mechanism. Primary type II alveolar epithelial cells (AECIIs) were divided into four groups: control (21% oxygen), hyperoxia (95% oxygen), hyperoxia + hydrogen, and hyperoxia + hydrogen + LY294002 (a PI3K/Akt inhibitor). Proliferation and apoptosis of AECIIs were assessed using MTS assay and flow cytometry (FCM), respectively. Gene and protein expression were detected by quantitative polymerase chain reaction (q-PCR) and western blot analysis. Stimulation with hyperoxia decreased the expression of P-Akt, P- FoxO3a, cyclinD1 and Bcl-2. Hyperoxic conditions increased levels of Bim, Bax, and Foxo3a, which induced proliferation restriction and apoptosis of AECIIs. These effects of hyperoxia were reversed with hydrogen pretreatment. Furthermore, the protective effects of hydrogen were abrogated by PI3K/Akt inhibitor LY294002. The results indicate that hydrogen protects AECIIs from hyperoxia-induced apoptosis by inhibiting apoptosis factors and promoting the expression of anti-apoptosis factors. These effects were associated with activation of the PI3K/Akt/FoxO3a pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Cathepsin L upregulation-induced EMT phenotype is associated with the acquisition of cisplatin or paclitaxel resistance in A549 cells.

    Science.gov (United States)

    Han, Mei-Ling; Zhao, Yi-Fan; Tan, Cai-Hong; Xiong, Ya-Jie; Wang, Wen-Juan; Wu, Feng; Fei, Yao; Wang, Long; Liang, Zhong-Qin

    2016-12-01

    Cathepsin L (CTSL), a lysosomal acid cysteine protease, is known to play important roles in tumor metastasis and chemotherapy resistance. In this study we investigated the molecular mechanisms underlying the regulation of chemoresistance by CTSL in human lung cancer cells. Human lung cancer A549 cells, A549/PTX (paclitaxel-resistant) cells and A549/DDP (cisplatin-resistant) cells were tested. The resistance to cisplatin or paclitaxel was detected using MTT and the colony-formation assays. Actin remodeling was observed with FITC-Phalloidin fluorescent staining or immunofluorescence. A wound-healing assay or Transwell assay was used to assess the migration or invasion ability. The expression of CTSL and epithelial and mesenchymal markers was analyzed with Western blotting and immunofluorescence. The expression of EMT-associated transcription factors was measured with Western blotting or q-PCR. BALB/c nude mice were implanted subcutaneously with A549 cells overexpressing CTSL, and the mice were administered paclitaxel (10, 15 mg/kg, ip) every 3 d for 5 times. Cisplatin or paclitaxel treatment (10-80 ng/mL) induced CTSL expression in A549 cells. CTSL levels were much higher in A549/PTX and A549/DDP cells than in A549 cells. Silencing of CTSL reversed the chemoresistance in A549/DDP and A549/TAX cells, whereas overexpression of CTSL attenuated the sensitivity of A549 cells to cisplatin or paclitaxel. Furthermore, A549/DDP and A549/TAX cells underwent morphological and cytoskeletal changes with increased cell invasion and migration abilities, accompanied by decreased expression of epithelial markers (E-cadherin and cytokeratin-18) and increased expression of mesenchymal markers (N-cadherin and vimentin), as well as upregulation of EMT-associated transcription factors Snail, Slug, ZEB1 and ZEB2. Silencing of CTSL reversed EMT in A549/DDP and A549/TAX cells; In contrast, overexpression of CTSL induced EMT in A549 cells. In xenograft nude mouse model, the mice implanted

  19. Toxicity of ZnO nanoparticles (NPs) to A549 cells and A549 epithelium in vitro: Interactions with dipalmitoyl phosphatidylcholine (DPPC).

    Science.gov (United States)

    He, Tong; Long, Jimin; Li, Juan; Liu, Liangliang; Cao, Yi

    2017-12-01

    Once inhaled, nanoparticles (NPs) will first interact with lung surfactant system, which may influence the colloidal aspects of NPs and consequently the toxic potential of NPs to pulmonary cells. In this study, we investigated the effects of dipalmitoyl phosphatidylcholine (DPPC), the major component in lung surfactant, on stability and toxicity of ZnO NPs. The presence of DPPC increased the UV-vis spectra, hydrodynamic size, Zeta potential and dissolution rate of ZnO NPs, which indicates that DPPC might interact with NPs and affect the colloidal stability of NPs. Exposure to ZnO NPs induced cytotoxicity associated with increased intracellular Zn ions but not superoxide in A549 cells. In A549 epithelium model, exposure to ZnO NPs induced cytotoxicity and decreased the release of interleukin 6 (IL-6) without a significant effect on epithelial permeability rate. Co-exposure of A549 cells or A549 epithelium model to DPPC and ZnO NPs induced a higher release of lactate dehydrogenase (LDH) and interleukin-6 (IL-6) compared with the exposure of ZnO NPs alone. We concluded that the presence of DPPC could influence the colloidal stability of ZnO NPs and increase the damage of NPs to membrane probably due to the increased positive surface charge. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Potential in vitro model for testing the effect of exposure to nanoparticles on the lung alveolar epithelial barrier

    Directory of Open Access Journals (Sweden)

    Raymond Derk

    2015-03-01

    Full Text Available Pulmonary barrier function plays a pivotal role in protection from inhaled particles. However, some nano-scaled particles, such as carbon nanotubes (CNT, have demonstrated the ability to penetrate this barrier in animal models, resulting in an unusual, rapid interstitial fibrosis. To delineate the underlying mechanism and specific bio-effect of inhaled nanoparticles in respiratory toxicity, models of lung epithelial barriers are required that allow accurate representation of in vivo systems; however, there is currently a lack of consistent methods to do so. Thus, this work demonstrates a well-characterized in vitro model of pulmonary barrier function using Calu-3 cells, and provides the experimental conditions required for achieving tight junction complexes in cell culture, with trans-epithelial electrical resistance measurement used as a biosensor for proper barrier formation and integrity. The effects of cell number and serum constituents have been examined and we found that changes in each of these parameters can greatly affect barrier formation. Our data demonstrate that use of 5.0 × 104 Calu-3 cells/well in the Transwell cell culture system, with 10% serum concentrations in culture media is optimal for assessing epithelial barrier function. In addition, we have utilized CNT exposure to analyze the dose-, time-, and nanoparticle property-dependent alterations of epithelial barrier permeability as a means to validate this model. Such high throughput in vitro cell models of the epithelium could be used to predict the interaction of other nanoparticles with lung epithelial barriers to mimic respiratory behavior in vivo, thus providing essential tools and bio-sensing techniques that can be uniformly employed.

  1. The concentrations of clinafloxacin in alveolar macrophages, epithelial lining fluid, bronchial mucosa and serum after administration of single 200 mg oral doses to patients undergoing fibre-optic bronchoscopy.

    Science.gov (United States)

    Honeybourne, D; Andrews, J M; Cunningham, B; Jevons, G; Wise, R

    1999-01-01

    The concentrations of clinafloxacin were measured in serum, bronchial mucosa, alveolar macrophages and epithelial lining fluid after single 200 mg oral doses of clinafloxacin had been administered to 15 subjects who were undergoing bronchoscopy. Concentrations were measured using a microbiological assay method. Mean concentrations in serum, bronchial mucosa, alveolar macrophages and epithelial lining fluid at a mean of 1.27 h post-dose were 1.54, 2.65, 15.60 and 2.71 mg/L respectively. These site concentrations exceeded the MIC90 for common respiratory pathogens and indicate that clinafloxacin is likely to be effective in the treatment of a wide range of respiratory tract infections.

  2. Elevated expression of SLC34A2 inhibits the viability and invasion of A549 cells

    Science.gov (United States)

    YANG, WEIHAN; WANG, YU; PU, QIANG; YE, SUJUAN; MA, QINGPING; REN, JIANG; ZHONG, GUOXING; LIU, LUNXU; ZHU, WEN

    2014-01-01

    Abnormal expression of solute carrier family 34 (sodium phosphate), member 2 (SLC34A2) in the lung may induce abnormal alveolar type II (AT II) cells to transform into lung adenocarcinoma cells, and may also be important in biological process of lung adenocarcinoma. However, at present, the effects and molecular mechanisms of SLC34A2 in the initiation and progression of lung cancer remain to be elucidated. To the best of our knowledge, the present study revealed for the first time that the expression levels of SLC34A2 were downregulated in the A549 and H1299 lung adenocarcinoma cell lines. Further investigation demonstrated that the elevated expression of SLC34A2 in A549 cells was able to significantly inhibit cell viability and invasion in vitro. In addition, 10 upregulated genes between the A549-P-S cell line stably expressing SLC34A2 and the control cell line A549-P were identified by microarray analysis and quantitative polymerase chain reaction, including seven tumor suppressor genes and three complement genes. Furthermore, the upregulation of complement gene C3 and complement 4B preproprotein (C4b) in A549-P-S cells was confirmed by ELISA analysis and was identified to be correlated with recovering Pi absorption in A549 cells by the phosphomolybdic acid method by enhancing the expression of SLC34A2. Therefore, it was hypothesized that the mechanisms underlying the effect of SLC34A2 on A549 cells might be associated with the activation of the complement alternative pathway (C3 and C4b) and upregulation of the expression of selenium binding protein 1, thioredoxin-interacting protein, PDZK1-interacting protein 1 and dual specificity protein phosphatase 6. Downregulation of SLC34A2 may primarily cause abnormal AT II cells to escape from complement-associated immunosurveillance and abnormally express certain tumor-suppressor genes inducing AT II cells to develop into lung adenocarcinoma. The present study further elucidated the effects and mechanisms of SLC34A2 in

  3. Transforming growth factor-β impairs glucocorticoid activity in the A549 lung adenocarcinoma cell line.

    Science.gov (United States)

    Salem, S; Harris, T; Mok, J S L; Li, M Y S; Keenan, C R; Schuliga, M J; Stewart, A G

    2012-08-01

    The lung adenocarcinoma cell line, A549, undergoes epithelial-mesenchymal cell transition (EMT) in response to TGF-β. Glucocorticoids do not prevent the EMT response, but TGF-β induced resistance to the cytokine-regulatory action of glucocorticoids. We sought to characterize the impairment of glucocorticoid response in A549 cells. A549 cells were exposed to TGF-β for up to 96 h before glucocorticoid treatment and challenge with IL-1α to assess glucocorticoid regulation of IL-6 and CXCL8 production. Nuclear localization of the glucocorticoid receptor α (GRα) was ascertained by immunofluorescence and Western blotting. Transactivation of the glucocorticoid response element (GRE) was measured with a transfected GRE-secreted human placental alkaline phosphatase reporter. TGF-β (40-400 pM) reduced the maximum inhibitory effect of dexamethasone on IL-1α-induced IL-6 and CXCL8 production. The impaired glucocorticoid response was detected with 4 h of TGF-β (40 pM) exposure (and 4 h IL-1α to induce CXCL8 expression) and therefore was not secondary to EMT, a process that requires longer incubation periods and higher concentrations of TGF-β. TGF-β also impaired dexamethasone regulation of granulocyte-macrophage colony-stimulating factor in thrombin-stimulated BEAS-2B epithelial cells. Impaired regulation of CXCL8 was associated with markedly reduced GRE transactivation and reduced induction of mRNA for IκBα, the glucocorticoid-inducible leucine zipper and the epithelial sodium channel (SCNN1A). The expression, cellular levels and nuclear localization of GRα were reduced by TGF-β. We have identified mechanisms underlying the impairment of responses to glucocorticoids by TGF-β in the A549 and BEAS-2B cell lines. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

  4. Tropomyosin-1 protects transformed alveolar epithelial cells against cigaret smoke extract through the stabilization of F-actin-dependent cell-cell junctions.

    Science.gov (United States)

    Gagat, Maciej; Grzanka, Dariusz; Izdebska, Magdalena; Sroka, Wiktor Dariusz; Hałas-Wiśniewska, Marta; Grzanka, Alina

    2016-04-01

    The aim of the study was to estimate the effect of tropomyosin-1-based structural stabilization of F-actin in transformed human alveolar epithelial line H1299 cells subjected to high oxidative stress induced by cigaret smoke extract. We demonstrated here that cigaret smoke extract induces cell shrinking and detachment as a consequence of F-actin cytoskeleton degradation in H1299 cells not overexpressing tropomyosin-1. Furthermore, the treatment of these cells with cigaret smoke extract resulted in the loss of peripheral localization of ZO-1 and initiated apoptosis. In contrast, structural stabilization of F-actin, by overexpression of tropomyosin-1, preserved cell to cell interactions through the attenuation of cortical actin organization into thin fibers and thus protected these cells against oxidative stress-induced degradation of actin cytoskeleton and cell death. In conclusion, we suggest that structural stabilization of thin cortical F-actin fibers increases link between tight junctions proteins and actin cytoskeleton and thus protects H1299 cells against cigaret smoke extract. Copyright © 2016 Elsevier GmbH. All rights reserved.

  5. Aerosol-based efficient delivery of telithromycin, a ketolide antimicrobial agent, to lung epithelial lining fluid and alveolar macrophages for treatment of respiratory infections.

    Science.gov (United States)

    Togami, Kohei; Chono, Sumio; Seki, Toshinobu; Morimoto, Kazuhiro

    2010-07-01

    The efficacy of aerosol-based delivery of telithromycin (TEL), as a model antimicrobial agent, for the treatment of respiratory infections was evaluated by comparison with oral administration. The aerosol formulation (0.2 mg/kg) was administered to rat lungs using a Liquid MicroSprayer. The time courses of the concentration of TEL in lung epithelial lining fluid (ELF) and alveolar macrophages (AMs) following administration of an aerosol formulation to rat lungs were markedly higher than that following the administration of an oral formulation (50 mg/kg). The time course of the concentrations of TEL in plasma following administration of the aerosol formulation was markedly lower than that in ELF and AMs. These results indicate that the aerosol formulation is more effective in delivering TEL to ELF and AMs, compared to the oral formulation, despite a low dose and it avoids distribution of TEL to the blood. In addition, the antibacterial effects of TEL in ELF and AMs following administration of the aerosol formulation were estimated by pharmacokinetics/pharmacodynamics analysis. The concentrations of TEL in ELF and the AMs time curve/minimum inhibitory concentration of TEL ratio were markedly higher than the effective values. This study indicates that an antibiotic aerosol formulation may be an effective pulmonary drug delivery system for the treatment of respiratory infections.

  6. Comparative physicochemical and biological characterization of NIST Interim Reference Material PM2.5 and SRM 1648 in human A549 and mouse RAW264.7 cells.

    Science.gov (United States)

    Mitkus, Robert J; Powell, Jan L; Zeisler, Rolf; Squibb, Katherine S

    2013-12-01

    The epidemiological association between exposure to fine particulate matter (PM2.5) and adverse health effects is well-known. Here we report the size distribution, metals content, endotoxin content, and biological activity of National Institute of Standards and Technology (NIST) Interim Reference Material (RM) PM2.5. Biological activity was measured in vitro by effects on cell viability and the release of four inflammatory immune mediators, from human A549 alveolar epithelial cells or murine RAW264.7 monocytes. A dose range covering three orders of magnitude (1-1000μg/mL) was tested, and biological activity was compared to an existing Standard Reference Material (SRM) for urban PM (NIST SRM 1648). Robust release of IL-8 and MCP-1 from A549 cells was observed in response to IRM PM2.5 exposures. Significant TNF-α, but not IL-6, secretion from RAW264.7 cells was observed in response to both IRM PM2.5 and SRM 1648 particle types. Cytokine or chemokine release at high doses often occurred in the presence of cytotoxicity, likely as a result of externalization of preformed mediator. Our results are consistent with a local cytotoxic and pro-inflammatory mechanism of response to exposure to inhaled ambient PM2.5 and reinforce the continued relevance of in vitro assays for mechanistic research in PM toxicology. Our study furthers the goal of developing reference samples of environmentally relevant particulate matter of various sizes that can be used for hypothesis testing by multiple investigators. Published by Elsevier Ltd.

  7. MicroRNA-9 and Cell Proliferation in Lipopolysaccharide and Dexamethasone-Treated Naïve and Desialylated A549 Cells Grown in Cigarette Smoke Conditioned Medium.

    Science.gov (United States)

    Holownia, A; Wielgat, P; Eljaszewicz, A

    2018-03-01

    In this study we assessed microRNA-9 (miR-9) levels (RT-PCR) and cell proliferation (flow cytometry) in naïve and desialylated human alveolar epithelial cells (A549 cells), grown for 24 h in cigarette smoke-conditioned medium. Cells were additionally treated with lipopolysaccharide (LPS) and/or dexamethasone. Proliferation positively correlated with miR-9 levels in both naïve and desialylated cells. Cigarette smoke decreased miR-9 levels in both cell types by about three-fold but there was no significant correlation between both parameters. Dexamethasone was without substantial effect on cigarette smoke-induced changes in proliferation of naïve cells, but some normalization was observed in desialylated cells. Dexamethasone increased miR-9 levels in both cell types grown in cigarette smoke-medium but the effect was stronger in desialylated cells. LPS increased cell proliferation and miR-9 by more than six-fold only in naïve cells, while correlation coefficient for both parameters in cigarette smoke-LPS group was 0.41. Herein we identify miR-9 as the cigarette smoke (decrease) and LPS-responsive but dexamethasone-unresponsive microRNA. It is possible that increased miR-9 levels in naïve A549 cells treated with LPS may be related to the activation of Toll-like receptor 4. Moreover, differences in cell response (both miR-9 and proliferation) to dexamethasone in naïve and desialylated cells may point to non-genomic dexamethasone effects.

  8. Protective Effects of Hydrogen-Rich Saline Against Lipopolysaccharide-Induced Alveolar Epithelial-to-Mesenchymal Transition and Pulmonary Fibrosis.

    Science.gov (United States)

    Dong, Wen-Wen; Zhang, Yun-Qian; Zhu, Xiao-Yan; Mao, Yan-Fei; Sun, Xue-Jun; Liu, Yu-Jian; Jiang, Lai

    2017-05-19

    BACKGROUND Fibrotic change is one of the important reasons for the poor prognosis of patients with acute respiratory distress syndrome (ARDS). The present study investigated the effects of hydrogen-rich saline, a selective hydroxyl radical scavenger, on lipopolysaccharide (LPS)-induced pulmonary fibrosis. MATERIAL AND METHODS Male ICR mice were divided randomly into 5 groups: Control, LPS-treated plus vehicle treatment, and LPS-treated plus hydrogen-rich saline (2.5, 5, or 10 ml/kg) treatment. Twenty-eight days later, fibrosis was assessed by determination of collagen deposition, hydroxyproline, and type I collagen levels. Development of epithelial-to-mesenchymal transition (EMT) was identified by examining protein expressions of E-cadherin and α-smooth muscle actin (α-SMA). Transforming growth factor (TGF)-β1 content, total antioxidant capacity (T-AOC), malondialdehyde (MDA) content, catalase (CAT), and superoxide dismutase (SOD) activity were determined. RESULTS Mice exhibited increases in collagen deposition, hydroxyproline, type I collagen contents, and TGF-β1 production in lung tissues after LPS treatment. LPS-induced lung fibrosis was associated with increased expression of α-SMA, as well as decreased expression of E-cadherin. In addition, LPS treatment increased MDA levels but decreased T-AOC, CAT, and SOD activities in lung tissues, indicating that LPS induced pulmonary oxidative stress. Hydrogen-rich saline treatment at doses of 2.5, 5, or 10 ml/kg significantly attenuated LPS-induced pulmonary fibrosis. LPS-induced loss of E-cadherin in lung tissues was largely reversed, whereas the acquisition of α-SMA was dramatically decreased by hydrogen-rich saline treatment. In addition, hydrogen-rich saline treatment significantly attenuated LPS-induced oxidative stress. CONCLUSIONS Hydrogen-rich saline may protect against LPS-induced EMT and pulmonary fibrosis through suppressing oxidative stress.

  9. Curcumin modulates the effect of histone modification on the expression of chemokines by type II alveolar epithelial cells in a rat COPD model

    Directory of Open Access Journals (Sweden)

    Gan L

    2016-11-01

    Full Text Available Lixing Gan,1 Chengye Li,2 Jian Wang,1 Xuejun Guo3 1Department of Respiratory Medicine, Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 2Department of Respiratory Medicine, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 3Department of Respiratory Medicine, Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, People’s Republic of China Background: Studies have suggested that histone modification has a positive impact on various aspects associated with the progression of COPD. Histone deacetylase 2 (HDAC2 suppresses proinflammatory gene expression through deacetylation of core histones.Objective: To investigate the effect of histone modification on the expression of chemokines in type II alveolar epithelial cells (AEC II in a rat COPD model and regulation of HDAC2 expression by curcumin in comparison with corticosteroid.Materials and methods: The rat COPD model was established by cigarette smoke exposure and confirmed by histology and pathophysioloy. AEC II were isolated and cultured in vitro from the COPD models and control animals. The cells were treated with curcumin, corticosteroid, or trichostatin A, and messenger RNA (mRNA expression of interleukin-8 (IL-8, monocyte chemoattractant protein-1 (MCP-1, and macrophage inflammatory protein-2α (MIP-2α was assessed by quantitative real-time polymerase chain reaction (RT-PCR. The expression of HDAC2 was measured by Western blot. Chromatin immunoprecipitation was used to detect H3/H4 acetylation and H3K9 methylation in the promoter region of three kinds of chemokine genes (IL-8, MCP-1, and MIP-2α. Results: Compared to the control group, the mRNAs of MCP-1, IL-8, and MIP-2α were upregulated 4.48-fold, 3.14-fold, and 2.83-fold, respectively, in the AEC II from COPD model. The protein expression of HDAC2 in the AEC II from COPD model was significantly lower than from the control group (P<0

  10. Aerosol-based efficient delivery of clarithromycin, a macrolide antimicrobial agent, to lung epithelial lining fluid and alveolar macrophages for treatment of respiratory infections.

    Science.gov (United States)

    Togami, Kohei; Chono, Sumio; Morimoto, Kazuhiro

    2012-04-01

    Macrolide antimicrobial agents are generally given by the oral route for the treatment of respiratory infections caused by pathogenic microorganisms infected in lung epithelial lining fluid (ELF) and alveolar macrophages (AMs). However, because macrolides distribute to many different tissues via the blood after oral administration, systemic side effects are frequently induced. In contrast with oral administration, aerosolization may be an efficient method for delivering macrolides directly to ELF and AMs. In this study, the efficacy of aerosol-based delivery of clarithromycin (CAM), as a model macrolide, for the treatment of respiratory infections was evaluated by comparison with oral administration. The aerosol formulation of CAM (0.2 mg/kg) was administered to rat lungs using a Liquid MicroSprayer(®). The oral formulation of CAM (50 mg/kg) was used for comparison. Time courses of concentrations of CAM in ELF and AMs following administration were obtained, and then the bioavailability (BA) was calculated. In addition, the area under the concentrations of CAM in ELF and AMs-time curve/minimum inhibitory concentration at which 90% of isolates ratio [area under the curve (AUC/MIC(90))] were calculated to estimate the antibacterial effects in ELF and AMs. The BA of CAM in ELF and AMs following administration of aerosol formulation were markedly greater than that following administration of oral formulation. This indicates that the aerosol formulation is more effective in delivering CAM to ELF and AMs, compared with the oral formulation, despite a low dose. The AUC/MIC(90) of CAM in ELF and AMs were markedly higher than the effective values. This indicates that the aerosol formulation could be useful for the treatment of respiratory infections caused by pathogenic microorganisms infected in ELF and AMs. This study suggests that aerosol formulation of macrolides is an effective pulmonary drug delivery system for the treatment of respiratory infections.

  11. Effect of irradiation/bone marrow transplantation on alveolar epithelial type II cells is aggravated in surfactant protein D deficient mice.

    Science.gov (United States)

    Mühlfeld, Christian; Madsen, Jens; Mackay, Rose-Marie; Schneider, Jan Philipp; Schipke, Julia; Lutz, Dennis; Birkelbach, Bastian; Knudsen, Lars; Botto, Marina; Ochs, Matthias; Clark, Howard

    2017-01-01

    Irradiation followed by bone marrow transplantation (BM-Tx) is a frequent therapeutic intervention causing pathology to the lung. Although alveolar epithelial type II (AE2) cells are essential for lung function and are damaged by irradiation, the long-term consequences of irradiation and BM-Tx are not well characterized. In addition, it is unknown whether surfactant protein D (SP-D) influences the response of AE2 cells to the injurious events. Therefore, wildtype (WT) and SP-D -/- mice were subjected to a myeloablative whole body irradiation dose of 8 Gy and subsequent BM-Tx and compared with age- and sex-matched untreated controls. AE2 cell changes were investigated quantitatively by design-based stereology. Compared with WT, untreated SP-D -/- mice showed a higher number of larger sized AE2 cells and a greater amount of surfactant-storing lamellar bodies. Irradiation and BM-Tx induced hyperplasia and hypertrophy in WT and SP-D -/- mice as well as the formation of giant lamellar bodies. The experimentally induced alterations were more severe in the SP-D -/- than in the WT mice, particularly with respect to the surfactant-storing lamellar bodies which were sometimes extremely enlarged in SP-D -/- mice. In conclusion, irradiation and BM-Tx have profound long-term effects on AE2 cells and their lamellar bodies. These data may explain some of the clinical pulmonary consequences of this procedure. The data should also be taken into account when BM-Tx is used as an experimental procedure to investigate the impact of bone marrow-derived cells for the phenotype of a specific genotype in the mouse.

  12. Assessing the survival of MRC5 and a549 cell lines upon exposure to pyruvic Acid, sodium citrate and sodium bicarbonate - biomed 2013.

    Science.gov (United States)

    Farah, Ibrahim O; Lewis, Veshell L; Ayensu, Wellington K; Cameron, Joseph A

    2013-01-01

    Lung cancer is among the most prevalent and deadly cancers in United States. In general, cancer cells are known to exhibit higher rates of glycolysis in comparison to normal cells. In attempting to exploit this unique cancer-dependent ATP generation phenomenon, it was our hypothesis that upon exposure to organic inhibitors of glycolysis, cancer cells would not survive normally and that their growth and viability would be vastly decreased; essential glycolytic ATP production will be exhausted to the point of collapsing energy utilization. Furthermore, we hypothesize that no negative effect would be seen with exposures to organic inhibitors for normal lung cells. The human lung fibroblast MRC-5 and the human A549 alveolar epithelial cell lines were used as in vitro models of normal lung and lung cancers respectively. Using standard methods, both cell lines were maintained and exposed to pyruvic acid, sodium citrate and sodium bicarbonate reagents at concentration levels ranging from 31.3-2,000 µg/ml in 96 well plates in quadruplets and experiments repeated at least three times using MTT, and cell counting (T4 Cellometer) assays as well as phase-contrast photo-imaging for parallel morphological displays of any changes in the course of their vitality and metabolic activities. Our results indicate that exposure of both cell lines to these organics resulted in concentration dependent cell destruction/cell survival depending on the cell line exposed. Pyruvic acid, sodium citrate and sodium bicarbonate showed statistically significant (pcancer biotherapeutics.

  13. Abberent expression of oncogenic and tumor-suppressive microRNAs and their target genes in human adenocarcinoma alveolar basal epithelial cells

    Directory of Open Access Journals (Sweden)

    Elham Tafsiri

    2016-01-01

    Conclusion: The significant differential expression level of these miRNAs made them as candidate biomarkers in NSCLC tumor tissues of patients. Perhaps Bcl-2 down-regulation and Akt-3 up-regulation can be linked with survival signals in A549 cell line. We can conclude that Bcl-2 and Akt-3 might be therapeutic targets to inhibit cell proliferation in NSCLC.

  14. Activation of Focal Adhesion Kinase and Src Mediates Acquired Sorafenib Resistance in A549 Human Lung Adenocarcinoma Xenografts.

    Science.gov (United States)

    Zhou, Qingyu; Guo, Xiaofang; Choksi, Riya

    2017-12-01

    Despite encouraging clinical results with sorafenib monotherapy in patients with KRAS- mutant non-small-cell lung cancer (NSCLC), the overall survival benefit of this drug is limited by the inevitable development of acquired resistance. The exact mechanism underlying acquired sorafenib resistance in KRAS -mutant NSCLC is unclear. In this study, the mechanism of acquired sorafenib resistance was explored using a biologically relevant xenograft model, which was established by using the A549 human lung adenocarcinoma cell line and an in vivo-derived, sorafenib-resistant A549 subline (A549/SRFres). Results from the initial study demonstrated that sorafenib treatment significantly decreased E-cadherin ( P A549/SRFres tumors, whereas expression levels of phospho-protein kinase B (AKT), phospho-focal adhesion kinase (FAK), and phospho-Src were elevated in sorafenib-treated A549 and A549/SRFres tumors. We next examined whether concomitant dasatinib treatment could overcome acquired sorafenib resistance by blocking the FAK/Src escape route that mediates resistance. Despite the observed in vitro synergy between sorafenib and dasatinib, the in vivo antitumor effect of half-dose sorafenib-dasatinib combination therapy was inferior to that of the full-dose sorafenib treatment. Although the sorafenib-dasatinib combination effectively inhibited Src and AKT phosphorylation, it did not block the Y576/577-FAK phosphorylation, nor did it decrease vimentin protein expression; unexpectedly, it increased Y397-FAK phosphorylation and MMP9 protein expression in tumors. These results suggest that acquired sorafenib resistance in KRAS -mutant A549 xenografts involves the compensatory activation of FAK and Src, and Src inhibition alone is insufficient to diminish sorafenib-promoted epithelial-mesenchymal transition process and invasive potentials in tumors. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  15. Gene expression profiles of human dendritic cells interacting with Aspergillus fumigatus in a bilayer model of the alveolar epithelium/endothelium interface.

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    Charles Oliver Morton

    Full Text Available The initial stages of the interaction between the host and Aspergillus fumigatus at the alveolar surface of the human lung are critical in the establishment of aspergillosis. Using an in vitro bilayer model of the alveolus, including both the epithelium (human lung adenocarcinoma epithelial cell line, A549 and endothelium (human pulmonary artery epithelial cells, HPAEC on transwell membranes, it was possible to closely replicate the in vivo conditions. Two distinct sub-groups of dendritic cells (DC, monocyte-derived DC (moDC and myeloid DC (mDC, were included in the model to examine immune responses to fungal infection at the alveolar surface. RNA in high quantity and quality was extracted from the cell layers on the transwell membrane to allow gene expression analysis using tailored custom-made microarrays, containing probes for 117 immune-relevant genes. This microarray data indicated minimal induction of immune gene expression in A549 alveolar epithelial cells in response to germ tubes of A. fumigatus. In contrast, the addition of DC to the system greatly increased the number of differentially expressed immune genes. moDC exhibited increased expression of genes including CLEC7A, CD209 and CCL18 in the absence of A. fumigatus compared to mDC. In the presence of A. fumigatus, both DC subgroups exhibited up-regulation of genes identified in previous studies as being associated with the exposure of DC to A. fumigatus and exhibiting chemotactic properties for neutrophils, including CXCL2, CXCL5, CCL20, and IL1B. This model closely approximated the human alveolus allowing for an analysis of the host pathogen interface that complements existing animal models of IA.

  16. Bulky PAH-DNA induced by exposure of a co-culture model of human alveolar macrophages and embryonic epithelial cells to atmospheric particulate pollution

    International Nuclear Information System (INIS)

    Abbas, Imane; Garcon, Guillaume; Billet, Sylvain; Shirali, Pirouz; Andre, Veronique; Le Goff, Jeremie; Sichel, Francois; Roy Saint-Georges, Francoise; Mulliez, Philippe

    2012-01-01

    Because of their deep penetration in human lungs, fine airborne particulate matter were described as mainly responsible for the deleterious effects of exposure to air pollution on health. Organic constituents are adsorbed on particles surface and, after inhalation, some (polycyclic aromatic hydrocarbons, PAHs) can be activated into reactive metabolites and can bind to DNA. The formation of bulky DNA adducts has been researched after exposure of mono-and co-cultures of alveolar macrophages (AM) and human embryonic human lung epithelial (L132), to fine air pollution particulate matter Air samples have been collected with cascade impactor and characterized: size distribution (92.15% 2 /g), inorganic (Fe, AI, Ca, Na, K, Mg, Pb, etc.) and organic compounds (PAHs, etc.). 32 P post-labeling method was applied to detect bulky DNA adducts in AM and L132, in mono-and co-cultures, 72 h after their exposure to atmospheric particles at their Lethals and Effects concentrations or (LC or CE) to 50% (i.e. MA: EC 50 = 74.63 μg/mL and L132: LC-5-0 = 75.36 μg/mL). Exposure to desorbed particles (MA: C1= 61.11 μg/mL and L132 : C2 = 61.71 μg/mL) and B[a]P (1 μM) were included. Bulky PAH-DNA adducts were detected in AM in mono-culture after exposure to total particles (Pt), to B[a]P and desorbed particles (Pd). Whatever the exposure, no DNA adduct was detected in L132 in mono-culture. These results are coherent with the enzymatic activities of cytochrome P450 l Al in AM and L132. Exposure of co-culture to Pt, or Pd induced bulky adducts to DNA in AM but not in L132. Exposure to B[a]P alone has altered the DNA of AM and L132, in co-culture. Exposure to Pt is closer to the environmental conditions, but conferred an exposure to amounts of genotoxic agents compared to studies using organic extracts. The formation of bulky DNA adducts was nevertheless observed in AM exposed to Pt, in mono- or co-culture, indicating that they were competent in terms of metabolic activation of PAHs. The

  17. Concentrations of garenoxacin in plasma, bronchial mucosa, alveolar macrophages and epithelial lining fluid following a single oral 600 mg dose in healthy adult subjects.

    Science.gov (United States)

    Andrews, J; Honeybourne, D; Jevons, G; Boyce, M; Wise, R; Bello, A; Gajjar, D

    2003-03-01

    A microbiological assay was used to measure concentrations of garenoxacin (BMS-284756) in plasma, bronchial mucosa (BM), alveolar macrophages (AM) and epithelial lining fluid (ELF), following a single 600 mg oral dose. Twenty-four healthy subjects were allocated into four nominal time intervals after the dose, 2.5-3.5, 4.5-5.5, 10.5-11.5 and 23.5-24.5 h. Mean concentrations in plasma, BM, AM and ELF, respectively, for the four nominal time windows were for 2.5-3.5 h 10.0 mg/L (S.D. 2.8), 7.0 mg/kg (S.D. 1.3), 106.1 mg/L (S.D. 60.3) and 9.2 mg/L (S.D. 3.6); 4.5-5.5 h 8.7 mg/L (S.D. 2.2), 6.0 mg/kg (S.D. 1.9), 158.6 mg/L (S.D. 137.4) and 14.3 mg/L (S.D. 8.2); 10.5-11.5 h 6.1 mg/L (S.D. 1.9), 4.0 mg/kg (S.D. 1.4), 76.0 mg/L (S.D. 47.7) and 7.9 mg/L (S.D. 4.6); and 23.5-24.5 h 2.1 mg/L (S.D. 0.5), 1.7 mg/kg (S.D. 0.7), 30.7 mg/L (S.D. 12.9) and 3.3 mg/L (S.D. 2.3). Concentrations at all sites exceeded MIC(90)s for the common respiratory pathogens Haemophilus influenzae (0.03 mg/L), Moraxella catarrhalis (0.015 mg/L) and Streptococcus pneumoniae (0.06 mg/L). These data suggest that garenoxacin should be effective in the treatment of community-acquired pneumonia and chronic obstructive pulmonary disease.

  18. Nicotine transport in lung and non-lung epithelial cells.

    Science.gov (United States)

    Takano, Mikihisa; Kamei, Hidetaka; Nagahiro, Machi; Kawami, Masashi; Yumoto, Ryoko

    2017-11-01

    Nicotine is rapidly absorbed from the lung alveoli into systemic circulation during cigarette smoking. However, mechanism underlying nicotine transport in alveolar epithelial cells is not well understood to date. In the present study, we characterized nicotine uptake in lung epithelial cell lines A549 and NCI-H441 and in non-lung epithelial cell lines HepG2 and MCF-7. Characteristics of [ 3 H]nicotine uptake was studied using these cell lines. Nicotine uptake in A549 cells occurred in a time- and temperature-dependent manner and showed saturation kinetics, with a Km value of 0.31mM. Treatment with some organic cations such as diphenhydramine and pyrilamine inhibited nicotine uptake, whereas treatment with organic cations such as carnitine and tetraethylammonium did not affect nicotine uptake. Extracellular pH markedly affected nicotine uptake, with high nicotine uptake being observed at high pH up to 11.0. Modulation of intracellular pH with ammonium chloride also affected nicotine uptake. Treatment with valinomycin, a potassium ionophore, did not significantly affect nicotine uptake, indicating that nicotine uptake is an electroneutral process. For comparison, we assessed the characteristics of nicotine uptake in another lung epithelial cell line NCI-H441 and in non-lung epithelial cell lines HepG2 and MCF-7. Interestingly, these cell lines showed similar characteristics of nicotine uptake with respect to pH dependency and inhibition by various organic cations. The present findings suggest that a similar or the same pH-dependent transport system is involved in nicotine uptake in these cell lines. A novel molecular mechanism of nicotine transport is proposed. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Expression and Significance of IKBKB in Pulmonary Adenocarcinoma A549 Cells and Its Cisplatin-resistant Variant A549/DDP

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    Kang QI

    2014-05-01

    Full Text Available Background and objective Cisplatin-resistance in Lung cancer cells is widespread in the clinical treatment, seriously affecting the effects of the treatment of lung cancer. Therefore, the research of mechanisms of cisplain-resistance has significant meaning for developing new chemotherapeutic drug and solving the cisplain-resistance in clinic treatment. IKBKB is one of the most important catalytic subunits of IKK complexes. It plays an important regulatory role in activation of NF-κB. The aim of this study is to investigate the differential expression of IKBKB gene in human lung adenocarcinoma cells line A549 and the cisplatin-resistant variant A549/DDP and the mechanisms of cisplain-resistance induced by IKBKB gene. Methods MTT assay was employed to determine the sensitivity of A549 and A549/DDP cells line to cisplatin and the effect of IKBKB gene on A549 cell lines’ sensitivity to cisplatin. The mRNA level of IKBKB was determined by real-time PCR. Dual luciferase reporter gene experiment was employed to determine the activity of the NF-κB. Apoptosis rate of lung adenocarcinoma cells was determined by flow cytometry. Results Apoptosis rate and IC50 were significantly different in A549 and A549/DDP cells, the expression of mRNA level of IKBKB gene in A549/DDP was significantly higher than that in A549. Compared with control group, IKBKB gene was able to reduce the cisplain sensitivity of A549 cells. After A549 was transfected with pcDNA3.1/IKBKB plasmid, mRNA level of IKBKB was significantly increased, the sensitivity of cisplain was decreased, the IC50 was increased 2.85 fold, the apoptosis rate was decreased 59%, the activity of NF-κB has been greatly increased. Conclusion IKBKB inhibits cisplatin-induced apoptosis via the activation of NF-κB pathway. It will be helpful in the development of new anticancer drug and solving the challenge of cisplatin-resistance.

  20. [Expression and significance of IKBKB in pulmonary adenocarcinoma A549 cells and its cisplatin-resistant variant A549/DDP].

    Science.gov (United States)

    Qi, Kang; Li, Yang; Li, Xuebing; Zhang, Fang; Shao, Yi; Zhou, Qinghua

    2014-05-01

    Cisplatin-resistance in Lung cancer cells is widespread in the clinical treatment, seriously affecting the effects of the treatment of lung cancer. Therefore, the research of mechanisms of cisplain-resistance has significant meaning for developing new chemotherapeutic drug and solving the cisplain-resistance in clinic treatment. IKBKB is one of the most important catalytic subunits of IKK complexes. It plays an important regulatory role in activation of NF-κB. The aim of this study is to investigate the differential expression of IKBKB gene in human lung adenocarcinoma cells line A549 and the cisplatin-resistant variant A549/DDP and the mechanisms of cisplain-resistance induced by IKBKB gene. MTT assay was employed to determine the sensitivity of A549 and A549/DDP cells line to cisplatin and the effect of IKBKB gene on A549 cell lines' sensitivity to cisplatin. The mRNA level of IKBKB was determined by real-time PCR. Dual luciferase reporter gene experiment was employed to determine the activity of the NF-κB. Apoptosis rate of lung adenocarcinoma cells was determined by flow cytometry. Apoptosis rate and IC50 were significantly different in A549 and A549/DDP cells, the expression of mRNA level of IKBKB gene in A549/DDP was significantly higher than that in A549. Compared with control group, IKBKB gene was able to reduce the cisplain sensitivity of A549 cells. After A549 was transfected with pcDNA3.1/IKBKB plasmid, mRNA level of IKBKB was significantly increased, the sensitivity of cisplain was decreased, the IC50 was increased 2.85 fold, the apoptosis rate was decreased 59%, the activity of NF-κB has been greatly increased. IKBKB inhibits cisplatin-induced apoptosis via the activation of NF-κB pathway. It will be helpful in the development of new anticancer drug and solving the challenge of cisplatin-resistance.

  1. Effects of cigarette smoke extract on A549 cells and human lung fibroblasts treated with transforming growth factor-beta1 in a coculture system.

    Science.gov (United States)

    Liu, Yin; Gao, Wei; Zhang, Deping

    2010-09-01

    Smoking is a risk factor for idiopathic pulmonary fibrosis (IPF), but the mechanism of the association remains unknown. The aim of this study was to investigate the effects of cigarette smoke extract (CSE) on A549 cells and human lung fibroblasts treated with transforming growth factor-beta1. A transwell two-chamber coculture system was used to study the proliferation, differentiation, morphologic changes and soluble factors production of A549 cells and myofibroblasts. Low concentrations of CSE promoted myofibroblasts proliferation; however, high concentrations of CSE inhibited their proliferation. Low concentrations of CSE also markedly increased extracellular secretion of hydrogen peroxide, inhibited proliferation, induced apoptosis and produced epithelial-mesenchymal transition (EMT) in cocultured A549 cells. This cigarette smoke-induced A549 cells EMT may become a new pathophysiological concept in the development of IPF. CSE possibly takes part in the development and progress of IPF by increasing oxidative stress.

  2. Cytotoxicity of semiconductor nanoparticles in A549 cells is attributable to their intrinsic oxidant activity

    Science.gov (United States)

    Escamilla-Rivera, Vicente; Uribe-Ramirez, Marisela; Gonzalez-Pozos, Sirenia; Velumani, Subramaniam; Arreola-Mendoza, Laura; De Vizcaya-Ruiz, Andrea

    2016-04-01

    Copper indium gallium diselenide (CIGS) and cadmium sulfide (CdS) nanoparticles (NP) are next generation semiconductors used in photovoltaic cells (PV). They possess high quantum efficiency, absorption coefficient, and cheaper manufacturing costs compared to silicon. Due to their potential for an industrial development and the lack of information about the risk associated in their use, we investigated the influence of the physicochemical characteristics of CIGS (9 nm) and CdS (20 nm) in relation to the induction of cytotoxicity in human alveolar A549 cells through ROS generation and mitochondrial dysfunction. CIGS induced cytotoxicity in a dose dependent manner in lower concentrations than CdS; both NP were able to induce ROS in A549. Moreover, CIGS interact directly with mitochondria inducing depolarization that leads to the induction of apoptosis compared to CdS. Antioxidant pretreatment significantly prevented the loss of mitochondrial membrane potential and cytotoxicity, suggesting ROS generation as the main cytotoxic mechanism. These results demonstrate that semiconductor characteristics of NP are crucial for the type and intensity of the cytotoxic effects. Our work provides relevant information that may help guide the production of a safer NP-based PV technologies, and would be a valuable resource on future risk assessment for a safer use of nanotechnology in the development of clean sources of renewable energy.

  3. Cytotoxicity of semiconductor nanoparticles in A549 cells is attributable to their intrinsic oxidant activity

    Energy Technology Data Exchange (ETDEWEB)

    Escamilla-Rivera, Vicente; Uribe-Ramirez, Marisela [Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN), Departamento de Toxicología (Mexico); Gonzalez-Pozos, Sirenia [CINVESTAV-IPN, Unidad de Microscopia Electrónica (LaNSE) (Mexico); Velumani, Subramaniam [CINVESTAV-IPN, Departamento de Ingeniería Eléctrica (Mexico); Arreola-Mendoza, Laura [Centro Interdisciplinario de Investigaciones y Estudios sobre Medio Ambiente y Desarrollo del Instituto Politécnico Nacional (CIIEMAD-IPN), Departamento de Biociencias e Ingeniería (Mexico); Vizcaya-Ruiz, Andrea De, E-mail: avizcaya@cinvestav.mx [Centro de Investigación y de Estudios Avanzados del IPN (CINVESTAV-IPN), Departamento de Toxicología (Mexico)

    2016-04-15

    Copper indium gallium diselenide (CIGS) and cadmium sulfide (CdS) nanoparticles (NP) are next generation semiconductors used in photovoltaic cells (PV). They possess high quantum efficiency, absorption coefficient, and cheaper manufacturing costs compared to silicon. Due to their potential for an industrial development and the lack of information about the risk associated in their use, we investigated the influence of the physicochemical characteristics of CIGS (9 nm) and CdS (20 nm) in relation to the induction of cytotoxicity in human alveolar A549 cells through ROS generation and mitochondrial dysfunction. CIGS induced cytotoxicity in a dose dependent manner in lower concentrations than CdS; both NP were able to induce ROS in A549. Moreover, CIGS interact directly with mitochondria inducing depolarization that leads to the induction of apoptosis compared to CdS. Antioxidant pretreatment significantly prevented the loss of mitochondrial membrane potential and cytotoxicity, suggesting ROS generation as the main cytotoxic mechanism. These results demonstrate that semiconductor characteristics of NP are crucial for the type and intensity of the cytotoxic effects. Our work provides relevant information that may help guide the production of a safer NP-based PV technologies, and would be a valuable resource on future risk assessment for a safer use of nanotechnology in the development of clean sources of renewable energy.

  4. Comparative study of the effects of PM1-induced oxidative stress on autophagy and surfactant protein B and C expressions in lung alveolar type II epithelial MLE-12 cells.

    Science.gov (United States)

    Bai, Ru; Guan, Longfei; Zhang, Wei; Xu, Jinxia; Rui, Wei; Zhang, Fang; Ding, Wenjun

    2016-12-01

    There is a strong link between smaller air pollution particles and a range of serious health conditions. Thus, there is a need for understanding the impacts of airborne fine particulate matter (PM) with an aerodynamic diameter of PM1) on lung alveolar epithelial cells. In the present study, mouse lung epithelial type II cell MLE-12 cells were used to examine the intracellular oxidative responses and the surfactant protein expressions after exposure to various concentrations of PM1 collected from an urban site and a steel-factory site (referred as uPM1 and sPM1 hereafter, respectively). Physicochemical characterization of PM1 was performed by using scanning electron microscopy and transmission electron microscopy. Cytotoxicity and autophagy induced by PM1 were assessed by using comprehensive approaches after MLE-12 cells were exposed to different concentrations of PM1 for various times. Expression of surfactant proteins B and C in MLE-12 cells was determined by Western blotting. All of the tested PM1 induced cytotoxicity evidenced by significant decrease of cell viability and increase of lactate dehydrogenase (LDH) release in a time- and concentration-dependent manner in the exposed cells compared with the unexposed cells. A similar pattern of increase of intercellular reactive oxygen species (ROS) generation and decrease of superoxide dismutase (SOD) and catalase (CAT) activities was also observed. PM1-induced autophagy was evidenced by an increase in microtubule-associated protein light chain-3 (LC3) puncta, accumulation of LC3II, and increased levels of beclin1. Data from Western blotting showed significant decrease of surfactant protein B and C expressions. Relatively high concentrations of transition metals, including Fe, Cu and Mn, may be responsible for the higher toxicity of sPM1 compared with uPM1. Moreover, pretreatment with N-acetylcysteine (NAC) or Chelex (a metal chelating agent, which removes a large suite of metals from PM1) prevented the increase of

  5. Extracts of Magnoliae flos inhibit inducible nitric oxide synthase via ERK in human respiratory epithelial cells.

    Science.gov (United States)

    Baek, Jin Ah; Lee, Yang Deok; Lee, Chan Bog; Go, Hyeon Kyu; Kim, Jin Pyo; Seo, Jeong Ju; Rhee, Yang Keun; Kim, A Mi; Na, Dong Jib

    2009-03-01

    Nitric oxide (NO) is a marker of pulmonary inflammation. In asthma, the levels of exhaled NO are elevated and the source of this increased NO is inducible nitric oxide synthase (iNOS) within airway epithelial cells. Epimagnolin and fargesin are compounds isolated from the ethanol extract of Magnoliae flos, the seed of the Magnolia plant and are used to treat nasal congestion, headache and sinusitis in Asian countries. This study investigated whether epimagnolin and fargesin inhibit extracellular signal-regulated kinase (ERK) activation and decrease iNOS expression and NO production in stimulated human respiratory epithelial cells. An immortal Type II alveolar cell line of human origin (A549) was stimulated by cytomix (CM), composed of IL-1beta, TNF-alpha and IFN-gamma, with or without concurrent exposure to M. flos extract (epimagnolin or fargesin). CM-induced levels of NO production, iNOS expression and ERK activation were evaluated. A549 cells stimulated with CM showed increases in iNOS mRNA and protein expression, and NO synthesis. However, treatment with epimagnolin or fargesin decreased levels of iNOS mRNA and protein expression, and NO synthesis. CM stimulated a rapid increase in the activity of ERK, whereas epimagnolin and fargesin inhibited ERK phosphorylation. Epimagnolin and fargesin inhibit iNOS expression and decrease production of NO via ERK pathway in cytokine-stimulated human respiratory epithelial cells.

  6. Effects of gene F10 over-expression on the tumorigenicity of A549 cells

    OpenAIRE

    Ya-li SONG; Gong ZHANG; Zhan-jun PANG; Xiu-lan ZHU; Xiao-ping YANG; Ya-fang LI; Song QUAN; Fu-qi XING

    2012-01-01

    Objective To explore the effects and mechanism of gene F10 over-expression on the tumorigenicity of A549 cells in nude mice. Methods Eighteen SPF nude mice (4-5weeks of age) were randomly equally divided into the three groups: A549-WT (vaccination with wild-type strain A549), Mock-A549 (vaccination with controlled cells Mock-A549 transfected by blank vectors) and F10+A549 (vaccination with F10+A549 cells which overexpressed F10 gene) according to their vaccination and then revaccinated into t...

  7. Pulmonary alveolar proteinosis

    Science.gov (United States)

    PAP; Alveolar proteinosis; Pulmonary alveolar phospholipoproteinosis; Alveolar lipoproteinosis phospholipidosis ... PAP is unknown. In others, it occurs with lung infection or an immune problem. It also can ...

  8. Inhibition of lung cancer cells A549 and H460 by curcuminoid extracts and nanoemulsions prepared from Curcuma longa Linnaeus

    Science.gov (United States)

    Chang, Hong-Bin; Chen, Bing-Huei

    2015-01-01

    The objectives of this study were to explore the inhibition mechanism of lung cancer cells A549 and H460 by curcuminoid extracts and nanoemulsions prepared from Curcuma longa Linnaeus. In addition, human bronchus epithelial cell line BEAS-2B (normal cell) was selected for comparison. A high-performance liquid chromatography (HPLC) method was developed to separate and quantify the various curcuminoids in C. longa extract, including curcumin (1,714.5 μg/mL), demethoxycurcumin (1,147.4 μg/mL), and bisdemethoxycurcumin (190.2 μg/mL). A high-stability nanoemulsion composed of Tween 80, water, and curcuminoid extract was prepared, with mean particle size being 12.6 nm. The cell cycle was retarded at G2/M for both the curcuminoid extract and nanoemulsion treatments; however, the inhibition pathway may be different. H460 cells were more susceptible to apoptosis than A549 cells for both curcuminoid extract and nanoemulsion treatments. Growth of BEAS-2B remained unaffected for both the curcuminoid extract and nanoemulsion treatments, with a concentration range from 1 to 4 μg/mL. Also, the activities of caspase-3, caspase-8, and caspase-9 followed a dose-dependent increase for both A549 and H460 cells for both the treatments, accompanied by a dose-dependent increase in cytochrome C expression and a dose-dependent decrease in CDK1 expression. Interestingly, a dose-dependent increase in cyclin B expression was shown for A549 cells for both the treatments, while a reversed trend was found for H460 cells. Both mitochondria and death receptor pathways may be responsible for apoptosis of both A549 and H460 cells. PMID:26345201

  9. Inhibition of lung cancer cells A549 and H460 by curcuminoid extracts and nanoemulsions prepared from Curcuma longa Linnaeus.

    Science.gov (United States)

    Chang, Hong-Bin; Chen, Bing-Huei

    2015-01-01

    The objectives of this study were to explore the inhibition mechanism of lung cancer cells A549 and H460 by curcuminoid extracts and nanoemulsions prepared from Curcuma longa Linnaeus. In addition, human bronchus epithelial cell line BEAS-2B (normal cell) was selected for comparison. A high-performance liquid chromatography (HPLC) method was developed to separate and quantify the various curcuminoids in C. longa extract, including curcumin (1,714.5 μg/mL), demethoxycurcumin (1,147.4 μg/mL), and bisdemethoxycurcumin (190.2 μg/mL). A high-stability nanoemulsion composed of Tween 80, water, and curcuminoid extract was prepared, with mean particle size being 12.6 nm. The cell cycle was retarded at G2/M for both the curcuminoid extract and nanoemulsion treatments; however, the inhibition pathway may be different. H460 cells were more susceptible to apoptosis than A549 cells for both curcuminoid extract and nanoemulsion treatments. Growth of BEAS-2B remained unaffected for both the curcuminoid extract and nanoemulsion treatments, with a concentration range from 1 to 4 μg/mL. Also, the activities of caspase-3, caspase-8, and caspase-9 followed a dose-dependent increase for both A549 and H460 cells for both the treatments, accompanied by a dose-dependent increase in cytochrome C expression and a dose-dependent decrease in CDK1 expression. Interestingly, a dose-dependent increase in cyclin B expression was shown for A549 cells for both the treatments, while a reversed trend was found for H460 cells. Both mitochondria and death receptor pathways may be responsible for apoptosis of both A549 and H460 cells.

  10. Cimicifuga foetida L. inhibited human respiratory syncytial virus in HEp-2 and A549 cell lines.

    Science.gov (United States)

    Wang, Kuo Chih; Chang, Jung San; Chiang, Lien Chai; Lin, Chun Ching

    2012-01-01

    Human respiratory syncytial virus (HRSV) causes serious pediatric infection of the lower respiratory tract without effective therapeutic modality. Sheng-Ma-Ge-Gen-Tang (SMGGT; Shoma-kakkon-to) has been proven to be effective at inhibiting HRSV-induced plaque formation, and Cimicifuga foetida is the major constituent of SMGGT. We tested the hypothesis that C. foetida effectively inhibited the cytopathic effects of HRSV by a plaque reduction assay in both human upper (HEp2) and lower (A549) respiratory tract cell lines. Its ability to stimulate anti-viral cytokines was evaluated by an enzyme-linked immunosorbent assay (ELISA). C. foetida dose-dependently inhibited HRSV-induced plaque formation (p < 0.0001) before and after viral inoculation, especially in A549 cells (p < 0.0001). C. foetida dose-dependently inhibited viral attachment (p < 0.0001) and could increase heparins effect on viral attachment. In addition, C. foetida time-dependently and dose-dependently (p < 0.0001) inhibited HRSV internalization. C. foetida could stimulate epithelial cells to secrete IFN-β to counteract viral infection. However, C. foetida did not stimulate TNF-α secretion. Therefore, C. foetida could be useful in managing HRSV infection. This is the first evidence to support that C. foetida possesses antiviral activity.

  11. [Effects of heme oxygenase-1/carbon monoxide pathway on the mitochondrial fusion in rat alveolar epithelial type II cells stimulated by lipopolysaccharide].

    Science.gov (United States)

    Jia, Haojuan; Shi, Jia; Dong, Shu'an; Zhang, Yuan; Yu, Jianbo

    2018-03-01

    To investigate the effects of heme oxygenase-1/carbon monoxide (HO-1/CO) pathway on mitochondrial fusion in rat alveolar epithelial type II cells (AEC II) stimulated by lipopolysaccharide (LPS). Once the cultured in vitro rat AEC II cells line RLE-6TN reached confluency of 85%, they were subcultured and randomly divided into seven groups (n = 5 each). RLE-6TN cells were routinely cultured in control group. The cells in LPS group was stimulated with 10 mg/L LPS to reproduce the model of endotoxin challenge in AECII cells. The cells in carbon monoxide-releasing molecule-2 (CORM-2, in vitro CO release agent) + LPS group (CL group) and Hemin (HO-1 inducer) + LPS group (HL group) were pretreated with 100 μmol/L CORM-2 or 20 μmol/L Hemin for 1 hour, respectively, followed by 10 mg/L LPS stimulation. The cells in zinc protoporphyrin-IX (ZnPP-IX, HO-1 inhibitor) + LPS group (ZL group) was pretreated with 10 μmol/L ZnPP-IX for 0.5 hour followed by 10 mg/L LPS stimulation. The cells in CORM-2 + ZnPP-IX + LPS group (CZL group) and Hemin + ZnPP-IX + LPS group (HZL group) were pretreated with 100 μmol/L CORM-2 or 20 μmol/L Hemin respectively for 1 hour, and other treatments were similar to those previously described in ZL group. At 24 hours after LPS stimulation, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the supernatant were determined by enzyme linked immunosorbent assay (ELISA), the protein expressions of HO-1, mitochondrial fusion related proteins 1 and 2 (Mfn1, Mfn2) and optic atrophy 1 (OPA1) were determined by Western Blot. Compared with control group, IL-6 and TNF-α contents in the supernatant were increased, HO-1 protein expression was up-regulated, Mfn1, Mfn2 and OPA1 protein expressions were down-regulated in all treatment groups. Compared with LPS group, IL-6 and TNF-α contents were significantly decreased after CORM-2 or Hemin pretreatment [IL-6 (ng/L): 48.6±3.7, 48.4±3.1 vs. 58.7±2.5; TNF-α (ng/L): 40.7±5.3, 39.4±4.3 vs. 51.8±5

  12. Glucocorticoids and erythropoietin in chronic lung disease of prematurity: Proliferative potential in lung fibroblast and epithelial cells exposed to tracheal aspirates.

    Science.gov (United States)

    Ohnishi, Satoshi; Ichiba, Hiroyuki; Saito, Mika; Hamazaki, Takashi; Matsumura, Hisako; Shintaku, Haruo

    2016-11-01

    We investigated the effects of glucocorticoids, erythropoietin (EPO) and spironolactone (SPL) n human fetal lung fibroblasts and human alveolar epithelial cells exposed to tracheal aspirate fluid (TAF) from extremely premature infants with chronic lung disease (CLD), characterized by fibrosis and changes in the alveolar epithelium. Fibroblasts and epithelial cells (FHs 738Lu and A549, respectively) were treated with different concentrations of hydrocortisone (HDC), dexamethasone (DEX), betamethasone (BET), SPL, and EPO in the absence or presence of TAF from infants with CLD (gestational age, 25.3 ± 0.8 weeks; birthweight, 658 ± 77 g; postnatal age, 0-28 days) and assayed for proliferation. Exposure to TAF resulted in a concentration-dependent proliferation of fibroblasts and epithelial cells. Proliferation of TAF-exposed fibroblasts was suppressed most significantly by 100 μmol/L DEX (21%, P = 0.046) and 300 mIU/mL EPO (18%, P = 0.02) and promoted most significantly by 0.4 μmol/L HDC (10%, P = 0.04). Epithelial proliferation was promoted by 4 μmol/L HDC (15%, P = 0.04), 10 μmol/L DEX (53%, P glucocorticoids alone did not significantly affect fibroblast proliferation. Glucocorticoids and EPO reduced fibroproliferation while promoting epithelial cell growth in vitro within certain dose ranges. Appropriate doses of glucocorticoids and EPO may be useful in the prevention and resolution of CLD in extremely premature infants. © 2016 Japan Pediatric Society.

  13. Alveolar development and disease.

    Science.gov (United States)

    Whitsett, Jeffrey A; Weaver, Timothy E

    2015-07-01

    Gas exchange after birth is entirely dependent on the remarkable architecture of the alveolus, its formation and function being mediated by the interactions of numerous cell types whose precise positions and activities are controlled by a diversity of signaling and transcriptional networks. In the later stages of gestation, alveolar epithelial cells lining the peripheral lung saccules produce increasing amounts of surfactant lipids and proteins that are secreted into the airspaces at birth. The lack of lung maturation and the associated lack of pulmonary surfactant in preterm infants causes respiratory distress syndrome, a common cause of morbidity and mortality associated with premature birth. At the time of birth, surfactant homeostasis begins to be established by balanced processes involved in surfactant production, storage, secretion, recycling, and catabolism. Insights from physiology and engineering made in the 20th century enabled survival of newborn infants requiring mechanical ventilation for the first time. Thereafter, advances in biochemistry, biophysics, and molecular biology led to an understanding of the pulmonary surfactant system that made possible exogenous surfactant replacement for the treatment of preterm infants. Identification of surfactant proteins, cloning of the genes encoding them, and elucidation of their roles in the regulation of surfactant synthesis, structure, and function have provided increasing understanding of alveolar homeostasis in health and disease. This Perspective seeks to consider developmental aspects of the pulmonary surfactant system and its importance in the pathogenesis of acute and chronic lung diseases related to alveolar homeostasis.

  14. Novel synthetic chalcones induce apoptosis in the A549 non-small cell lung cancer cells harboring a KRAS mutation.

    Science.gov (United States)

    Wang, Yiqiang; Hedblom, Andreas; Koerner, Steffi K; Li, Mailin; Jernigan, Finith E; Wegiel, Barbara; Sun, Lijun

    2016-12-01

    A series of novel chalcones were synthesized by the Claisen-Schmidt condensation reaction of tetralones and 5-/6-indolecarboxaldehydes. Treatment of human lung cancer cell line harboring KRAS mutation (A549) with the chalcones induced dose-dependent apoptosis. Cell cycle analyses and Western blotting suggested the critical role of the chalcones in interrupting G2/M transition of cell cycle. SAR study demonstrated that substituent on the indole N atom significantly affects the anticancer activity of the chalcones, with methyl and ethyl providing the more active compounds (EC 50 : 110-200nM), Compound 1g was found to be >4-fold more active in the A549 cells (EC 50 : 110nM) than in prostate (PC3) or pancreatic cancer (CLR2119, PAN02) cells. Furthermore, compound 1l selectively induced apoptosis of lung cancer cells A549 (EC 50 : 0.55μM) but did not show measurable toxicity in the normal lung bronchial epithelial cells (hBEC) at doses as high as 10μM, indicating specificity towards cancer cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Effects of the Notch1 signaling pathway on human lung cancer A549 cells.

    Science.gov (United States)

    Zeng, Yun; Yin, Bijian; Wang, Xinwei; Xia, Guohao; Shen, Zhengjie; Gu, Wenzhe; Wu, Mianhua

    To evaluate the effects of the Notch1 signaling pathway on human lung cancer A549 cells. A549 cells were transfected with recombinant plasmids. Cell proliferation was detected by MTT assay. A tumor-bearing mouse model was established for intratumoral gene injection. Apoptosis-related factors were detected by immunohistochemical assay. Caspase-8, caspase-3, caspase-9, PI3K, pAkt and pSTAT3 expressions were detected by Western blotting. Compared with A549-GFP and A549 cells, A549-ICN cell growth in mice decelerated, tumor volume significantly reduced (p A549 cell proliferation decelerated, growth was significantly inhibited (p A549-ICN cell growth time- and dose-dependently. After treatment for 24 h or longer, TRAIL induced apoptosis of more A549-ICN cells. Cleaved caspase-3 and cleaved caspase-9 were detected only in A549-ICN cells after 6 h of 40 ng/mL TRAIL treatment, but cleaved caspase-8 was not detected. Combining Notch1 signal with TRAIL inhibited PI3K, phosphorylated Akt and phosphorylated STAT3 expressions. The Notch1 signaling pathway may inhibit A549 cell growth in vitro and in vivo by regulating cell cycle-related and anti-apoptotic protein expressions. Notch1 activation also suppressed A549 cell apoptosis by inhibiting the PI3K/pAkt pathway and activating the caspase-3 pathway in cooperation with TRAIL.

  16. Anti-Inflammatory Effects of Ginsenoside Rg3 via NF-κB Pathway in A549 Cells and Human Asthmatic Lung Tissue

    Directory of Open Access Journals (Sweden)

    In-Seung Lee

    2016-01-01

    Full Text Available Objective. There is limited information of the anti-inflammatory effects of Rg3 on inflamed lung cells and tissues. Therefore, we confirmed the anti-inflammatory mechanism of ginsenoside Rg3 in inflamed human airway epithelial cells (A549 and tissues whether Rg3 regulates nuclear factor kappa B (NF-κB activity. Methods. To induce the inflammation, IL-1β (10 ng/ml was treated to A549 cells for 4 h. The effects of Rg3 on NF-κB activity and COX-2 expression were evaluated by western blotting analysis in both IL-1β-induced inflamed A549 cell and human asthmatic airway epithelial tissues. Using multiplex cytokines assay, the secretion levels of NF-κB-mediated cytokines/chemokines were measured. Result. Rg3 showed the significant inhibition of NF-κB activity thereby reduced COX-2 expression was determined in both IL-1β-induced inflamed A549 cell and human asthmatic airway epithelial tissues. In addition, among NF-κB-mediated cytokines, the secretion levels of IL-4, TNF-α, and eotaxin were significantly decreased by Rg3 in asthma tissues. Even though there was no significant difference, IL-6, IL-9, and IL-13 secretion showed a lower tendency compared to saline-treated human asthmatic airway epithelial tissues. Conclusion. The results from this study demonstrate the potential of Rg3 as an anti-inflammatory agent through regulating NF-κB activity and reducing the secretion of NF-κB-mediated cytokines/chemokines.

  17. Activation of paracrine growth factors by heparan sulphate induced by glucocorticoid in A549 lung carcinoma cells.

    Science.gov (United States)

    Yevdokimova, N; Freshney, R I

    1997-01-01

    Alkaline phosphatase, a marker of differentiation in the human alveolar adenocarcinoma cell line A549, is inducible by conditioned medium from lung fibroblasts and by cytokines including oncostatin M and interleukin 6, but only in the presence of a glucocorticoid, dexamethasone. Dexamethasone was shown to induce incorporation of [3H]glucosamine into three fractions of medium and cell trypsinate from subconfluent A549 cells, eluting from DEAE ion-exchange chromatography. The first peak did not correspond to any of the unlabelled glycosaminoglycans and was not characterized further. Induction was seen in two other peaks, corresponding to hyaluronic acid and heparan sulphate. Of these, heparan sulphate, eluting as one well-defined peak (referred to as HS1) and another of lower activity and less well defined (HS2), was selected as the most likely to interact with growth factors and cytokines and was isolated from the eluate, concentrated and desalted, and used in alkaline phosphatase induction experiments in place of dexamethasone. HS1 isolated from the medium (HS1m) of subconfluent A549 cells was shown to replace dexamethasone in induction experiments with fibroblast-conditioned medium, oncostatin M and interleukin 6. HS1 from the cell trypsinate and HS2 from the medium and trypsinate were inactive. As the activity of HS1m could be abolished by heparinase and heparitinase but not by chondroitinase ABC, it was concluded that HS1m was a fraction of heparan sulphate involved in the regulation of paracrine growth factor activity in lung fibroblast-conditioned medium, and in the regulation of other growth factors with potential roles in the paracrine control of cell differentiation.

  18. MicroRNA-223 Promotes Tumor Progression in Lung Cancer A549 Cells via Activation of the NF-κB Signaling Pathway.

    Science.gov (United States)

    Huang, Li; Li, Fang; Deng, Pengbo; Hu, Chengping

    2016-10-27

    Our study aimed to investigate the role of microRNA-223 (miR-223) in lung cancer A549 cells and to further elucidate its possible regulatory mechanism. The expression levels of normal human lung epithelial cell line BEAS-2B and human lung cancer cell line A549 were investigated by quantitative real-time PCR. The A549 cells were transfected with miR-223 inhibitor and miR-223 scramble. Afterward, the effects of miR-223 inhibition on cell viability, invasion, and apoptosis, as well as the expression levels of nuclear factor-κB (NF-κB) and its downstream proteins, were detected. In addition, the NF-κB inhibitor JSH-23 was used to detect the relationship between NF-κB and miR-223. miR-223 was upregulated in human lung cancer A549 cells when compared with BEAS-2B cells. In addition, miR-223 expression was successfully inhibited by the miR-223 inhibitor. Suppression of miR-223 significantly decreased cell viability, inhibited invasion, and induced apoptosis of lung cancer A549 cells. Suppression of miR-223 resulted in a significant decrease in the expression levels of NF-κB and its downstream proteins P-IKBα and P-IKKα/β. After treatment with the NF-κB inhibitor, the inhibitory effects of miR-233 inhibitor on cell invasion, as well as the expression levels of NF-κB and p-p65, were enhanced. Our findings indicate that miR-223 may increase proliferation, promote invasion, and inhibit apoptosis of lung cancer A549 cells via activation of the NF-κB signaling pathway. miR-223 may serve as a potential therapeutic target in lung cancer.

  19. A new look at the pathogenesis of the meconium aspiration syndrome: a role for fetal pancreatic proteolytic enzymes in epithelial cell detachment.

    Science.gov (United States)

    Ivanov, Vadim A; Gewolb, Ira H; Uhal, Bruce D

    2010-09-01

    We hypothesized that fetal pancreatic digestive enzymes play a role in the lung damage after meconium aspiration. We studied the effect of meconium on the A549 alveolar epithelial cell line. The exposure of the cells to 0.5 to 5% meconium resulted in significant disruption of connections between A549 cells and caused dose-dependent cell detachment, without signs of cell death. A protease inhibitor cocktail prevented the A549 cell detachment induced by meconium. After the exposure to 2.5% meconium, a protective effect was quantified by measuring light absorbance by gentian violet stain of still attached cells. The protease inhibitor cocktail and chymostatin showed significant protective effects, increasing the number of attached cells by 135 and 123%, respectively (p meconium aspiration syndrome (MAS). We speculate that disruption of intercellular connections and cell detachment from the basement membrane are key events in the pathology associated with MAS. The observed protective effects of protease inhibitors suggest that they may be useful in the treatment and/or prophylaxis of MAS.

  20. Protection of Meconium-Induced Lung Epithelial Injury by Protease Inhibitors

    Science.gov (United States)

    Ota, C; Gopallawa, I; Ivanov, V; Gewolb, IH; Uhal, BD

    2017-01-01

    Earlier work form this laboratory showed that exposure of alveolar epithelial cells (AECs) to meconium caused significant cell detachment and that meconium-induced detachment of cells was prevented by a protease inhibitor cocktail. Therefore, it was hypothesized that protease inhibitors might protect AEC monolayers against meconium-induced collapse of epithelial barrier function both in vitro and in vivo. To investigate this theory in vitro, albumin flux was measured across cultured, confluent monolayers of human type II derived cell line A549 on microporous filter inserts. Human meconium was collected from seven healthy full-term neonates and the samples were pooled and diluted prior to analysis. Exposure of AECs to 5% human meconium increased albumin flux across the cultured AEC monolayers, but the increase was significantly blocked by protease inhibitors (Pmeconium increased the passage of Evans Blue Dye (EBD) from the vascular compartment into the alveolar spaces, measured in bronchoalveolar lavage (BAL) fluid after intravenous injection of EBD. Moreover, intratrachial coinstillation of protease inhibitors prevented the meconium-induced increase in EBD passage into BAL fluid (Pmeconium-induced injury, and suggest the future possibility of using protease inhibitors in the treatment of meconium aspiration syndrome. PMID:29218325

  1. Silencing of TGIF attenuates the tumorigenicity of A549 cells in vitro and in vivo.

    Science.gov (United States)

    Wang, Yadong; Pan, Teng; Wang, Haiyu; Li, Li; Li, Jiangmin; Zhang, Congke; Yang, Haiyan

    2016-09-01

    The aim of this study was to investigate the effects of the silencing of the TG-interacting factor (TGIF) on the tumorigenicity of A549 cells in vitro and in vivo. Stable TGIF-silenced A549 cells were established by infecting shRNA lentiviral particles. Western blotting analysis was used to detect the expression of proteins. Cell cycle was detected by flow cytometry. Soft agar assay and tumor formation assay in nude mice were applied. The silencing of TGIF inhibited A549 cell proliferation, colony formation in vitro, growth of tumor xenograft in vivo, and arrested the cell cycle in the G1 phase. The expression of CDK4, cyclin D1, and phospho-Rb was markedly decreased in the A549-shTGIF cells compared with the A549-shcon cells, and p21 was markedly increased in the A549-shTGIF cells compared with the A549-shcon cells. A lower level of β-Catenin protein expression was observed in the A549-shTGIF cells than that in the A549-shcon cells. The silencing of TGIF attenuates the tumorigenicity of A549 cells in vitro and in vivo.

  2. Increased alveolar soluble Annexin V promotes lung inflammation and fibrosis

    OpenAIRE

    Buckley, S.; Shi, W.; Xu, W.; Frey, M.R.; Moats, R.; Pardo, A.; Selman, M.; Warburton, D.

    2015-01-01

    The causes underlying the self-perpetuating nature of idiopathic pulmonary fibrosis (IPF), a progressive and usually lethal disease, remain unknown. We hypothesized that alveolar soluble Annexin V contributes to lung fibrosis, based on the observation that human IPF BALF containing high Annexin V levels promoted fibroblast involvement in alveolar epithelial wound healing that was reduced when Annexin V was depleted from the BALF.

  3. [Knockdown of SIRT1 enhances the sensitivity to cisplatin by inhibiting autophagy in A549 cells].

    Science.gov (United States)

    Peng, Wenhong; Mei, Chunxia; Wang, Bing; Liang, Zhengmin

    2017-12-01

    Objective To inhibit cisplatin-induced autophagy and improve the cisplatin sensitivity of A549 cells by knockdown the silent information regulator of transcription 1 (SIRT1). Methods Both mRNA and protein levels of SIRT1 in BEAS-2B, A549 and A549/DDP cells were detected by real-time quantitative PCR and Western blotting. After cisplatin treatment, the protein levels of SIRT1, LC3, P62 and beclin-1 in A549 cells were detected by Western blotting. A549 cells were transfected by siRNA to silence SIRT1 expression. Then, the apoptotic morphology was observed by fluorescence microscopy with Hoechst33258 staining. The apoptotic rate was analyzed by flow cytometry. The expressions of SIRT1, LC3, P62, cleaved caspase-3 and poly(ADP-ribose)polymerase (PARP) were measured by Western blotting. Results Both mRNA and protein levels of SIRT1 in A549 cells and A549/DDP cells were significantly higher than those in BEAS-2B cells, and they were higher in A549/DDP cells than in A549 cells. After cisplatin treatment, the protein levels of SIRT1, LC3 and beclin-1 in A549 cells increased, while P62 decreased. After transfected with SIRT1-siRNA, the expression of SIRT1 in A549 cells decreased. Compared with cisplatin group, the number of the apoptotic cells increased with the obvious occurrence of pyknosis and nuclear fragmentation in cisplatin plus SIRT1-siRNA group. Moreover, the expressions of P62, cleaved caspase-3 and PARP were up-regulated accompanied with LC3 decrease. Conclusion SIRT1 is highly expressed in A549 cells. The sensitivity of A549 cells to cisplatin can be improved by inhibiting the cisplatin-induced autophagy through knockdown of SIRT1.

  4. Inhibition of lung cancer cells A549 and H460 by curcuminoid extracts and nanoemulsions prepared from Curcuma longa Linnaeus

    Directory of Open Access Journals (Sweden)

    Chang HB

    2015-08-01

    Full Text Available Hong-Bin Chang,1 Bing-Huei Chen1,21Department of Food Science, 2Graduate Institute of Medicine, Fu Jen Catholic University, Taipei, TaiwanAbstract: The objectives of this study were to explore the inhibition mechanism of lung cancer cells A549 and H460 by curcuminoid extracts and nanoemulsions prepared from Curcuma longa Linnaeus. In addition, human bronchus epithelial cell line BEAS-2B (normal cell was selected for comparison. A high-performance liquid chromatography (HPLC method was developed to separate and quantify the various curcuminoids in C. longa extract, including curcumin (1,714.5 µg/mL, demethoxycurcumin (1,147.4 µg/mL, and bisdemethoxycurcumin (190.2 µg/mL. A high-stability nanoemulsion composed of Tween 80, water, and curcuminoid extract was prepared, with mean particle size being 12.6 nm. The cell cycle was retarded at G2/M for both the curcuminoid extract and nanoemulsion treatments; however, the inhibition pathway may be different. H460 cells were more susceptible to apoptosis than A549 cells for both curcuminoid extract and nanoemulsion treatments. Growth of BEAS-2B remained unaffected for both the curcuminoid extract and nanoemulsion treatments, with a concentration range from 1 to 4 µg/mL. Also, the activities of caspase-3, caspase-8, and caspase-9 followed a dose-dependent increase for both A549 and H460 cells for both the treatments, accompanied by a dose-dependent increase in cytochrome C expression and a dose-dependent decrease in CDK1 expression. Interestingly, a dose-dependent increase in cyclin B expression was shown for A549 cells for both the treatments, while a reversed trend was found for H460 cells. Both mitochondria and death receptor pathways may be responsible for apoptosis of both A549 and H460 cells.Keywords: curcuminoid extract, curcuminoid nanoemulsion, Curcuma longa Linnaeus, lung cancer cell, cell cycle, apoptosis mechanism

  5. Measurement of Regional and Global Pulmonary Clearance of 99mTc-DTPA (Demethylamitriptylene-Acetate): An Index of Alveolar Epithelial Permeability

    International Nuclear Information System (INIS)

    Vaskova, Olivija

    1996-01-01

    The main purpose of this study has been introduction of a new method for alveoli-capillary permeability evaluation. Many reports pointed out to the altered transit of soluble particles through this barrier. From pathophysiological aspect the main interest is the elucidation of permeability's alteration in different pulmonary pathology. We decided to use for lung epithelial permeability measurements 99m Tc-DTPA inhaled aerosols and sequential assessment of its lung clearance. The aerosols were obtained using oxygen flow nebulizers with aerosols' generators Ultra Vent (Malinkrodt) and Venticis II (CIS bio international) that enabled as to get submicron particles. Oxygen flow between 9 and 11 liters per minute was used. Optimum images were obtained with 1480 MBq of inhaled aerosols at least 2 to 3 minutes. DTPA that was used for aerosols labeling had been produced in our Department and the results were compared with DTPA provided by CIS bio international. High correlation between both agents was proven. During the whole study ex tamper prepared radiopharmaceuticals were used and quality control was done using paper chromatography method. Acquisition was done in sitting position with gamma camera interfaced to a ADAC and Scintiview. The measurements lasted for 20 minutes. Data were stored on 64x64 matrices. Regions of interest over both lungs were drown and each one was divided in three segments: apical, medial, and basal. Using computer program curves of 99m Tc-DTPA lung clearance were derived. From the obtained time activity curves half-time of the global and the regional lung clearance was assessed. In the control group comprised of 32 healthy volunteers (non-smokers) we had got values, used after works as reference range. Our normal values for global clearance are: 68±5,5 min. for left whole lung, 68,1±6,5 min for right whole lung, and 49±7,7 min for apical, 66,9±8 min for middle, and 75,9±6,4 min basal regional lung clearance, and they are in keeping with the

  6. Inflammatory response and barrier properties of a new alveolar type 1-like cell line (TT1).

    NARCIS (Netherlands)

    Bogaard, E.H.J. van den; Dailey, L.A.; Thorley, A.J.; Tetley, T.D.; Forbes, B.

    2009-01-01

    PURPOSE: To evaluate the inflammatory response and barrier formation of a new alveolar type 1-like (transformed type I; TT1) cell line to establish its suitability for toxicity and drug transport studies. METHODS: TT1 and A549 cells were challenged with lipopolysaccharide (LPS). Secretion of

  7. Potent proapoptotic actions of dihydroartemisinin in gemcitabine-resistant A549 cells.

    Science.gov (United States)

    Zhao, Chubiao; Qin, Guiqi; Gao, Weijie; Chen, Jingqin; Liu, Hongyu; Xi, Gaina; Li, Tan; Wu, Shengnan; Chen, Tongsheng

    2014-10-01

    Our recent studies have demonstrated the key roles of reactive oxygen species (ROS)-mediated caspase-8- and Bax-dependent apoptotic pathways in dihydroartemisinin (DHA)-induced apoptosis of A549 cells. This report is designed to investigate the proapoptotic mechanisms of DHA in gemcitabine (Gem)-resistant A549 (A549GR) cells. A549GR cells exhibited lower basal antioxidant capacity, higher level of basal ROS and intracellular Fe(2+) than Gem-sensitive A549 (A549) cells. In contrast to the sluggish ROS generation induced by Gem, DHA induced a rapid ROS generation within 30min. Moreover, Gem induced similar ROS generation in both cell lines, while DHA induced more ROS generation in A549GR cells than in A549 cells. More importantly, after treatment with DHA, A549GR cells showed more potent induction in Bax activation, loss of mitochondrial membrane potential (ΔΨm), caspase activation and apoptosis than A549 cells. Furthermore, NAC pretreatment potently prevented DHA-induced ROS generation and loss of ΔΨm as well as apoptosis, and silencing Bax by shRNA or inhibition of one of caspase-3, -8 and -9 also significantly prevented DHA-induced apoptosis in both cell lines, indicating the key roles of ROS and Bax as well as the caspases. Collectively, DHA presents more potent proapoptotic actions in A549GR cells preferentially over normal A549 cells via ROS-dependent apoptotic pathway, in which Bax and caspases are involved. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Proteome data from a host-pathogen interaction study with Staphylococcus aureus and human lung epithelial cells

    Directory of Open Access Journals (Sweden)

    Kristin Surmann

    2016-06-01

    Full Text Available To simultaneously obtain proteome data of host and pathogen from an internalization experiment, human alveolar epithelial A549 cells were infected with Staphylococcus aureus HG001 which carried a plasmid (pMV158GFP encoding a continuously expressed green fluorescent protein (GFP. Samples were taken hourly between 1.5 h and 6.5 h post infection. By fluorescence activated cell sorting GFP-expressing bacteria could be enriched from host cell debris, but also infected host cells could be separated from those which did not carry bacteria after contact (exposed. Additionally, proteome data of A549 cells which were not exposed to S. aureus but underwent the same sample processing steps are provided as a control. Time-resolved changes in bacterial protein abundance were quantified in a label-free approach. Proteome adaptations of host cells were monitored by comparative analysis to a stable isotope labeled cell culture (SILAC standard. Proteins were extracted from the cells, digested proteolytically, measured by nanoLC–MS/MS, and subsequently identified by database search and then quantified. The data presented here are related to a previously published research article describing the interplay of S. aureus HG001 and human epithelial cells (Surmann et al., 2015 [1]. They have been deposited to the ProteomeXchange platform with the identifiers PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD002384 for the S. aureus HG001 proteome dataset and PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD002388 for the A549 proteome dataset.

  9. [Synergistic Antitumor Effect of Amorphigenin Combined with Cisplatin in Human Lung Adenocarcinoma A549/DDP Cells].

    Science.gov (United States)

    Zhong, Hongzhen; Zuo, Yufang; Wu, Xin; Peng, Yan; He, Huiping; Yang, Jun; Guan, Chengnong; Xu, Zumin

    2016-12-20

    Amorphigenin, a rotenoid compouns, from seeds of Amorpha fruticosa, has been shown to possess anti-proliferation activities in several cancer cells. To explore the antitumor effects of amorphigenin on cisplatin-resistant human lung adenocarcinoma A549/DDP cells and explore the underlying mechanisms. CCK-8 assay was used to measure the proliferation of A549/DDP cells; Colony formation assay was used to measure the colony formation of A549/DDP cells; Flow cytometry assay was used to detect the apoptosis rates; Western blot analysis was used to explore the expression of apoptosis-related proteins (caspase-3 protein, PARP protein) and lung resistance protein (LRP). Our results demonstrated that amorphigenin could inhibit the proliferation of A549/DDP cells with a inhibition concentration of 50% cell growth (IC50) at 48 h of (2.19±0.92) μmol/L. Amorphigenin could inhibit the colony formation ability and induce apoptosis of A549/DDP cells; Furthermore, amorphigenin combined with cisplatin showed synergistic proliferation-inhibitory effect and apoptosis-promoting effect in A549/DDP cells; reduced the expression of LRP of A549/DDP cells. Amorphigenin remarkably inhibits the proliferation and induces apoptosis in A549/DDP cells. Combination of amorphigenin with cisplatin had the synergistic inhibitory effect on A549/DDP cells by downregulating the expression of LRP.
.

  10. Cytokine-Induced Killer Cells Modulates Resistance to Cisplatin in the A549/DDP Cell Line.

    Science.gov (United States)

    Yang, Lili; Du, Chunjuan; Wu, Lei; Yu, Jinpu; An, Xiumei; Yu, Wenwen; Cao, Shui; Li, Hui; Ren, Xiubao

    2017-01-01

    Background Cytokine-induced killer (CIK) cells can potentially enhance the tumor-killing activity of chemotherapy. Objective This study aimed to evaluate the effects of CIK cells on cisplatin (DDP) resistance in the human lung adenocarcinoma cell line A549/DDP. Methods The detect resistance index, drug resistance related-genes and cytokine secretion of A549/DDP co-cultured with CIK cells were assayed in vitro . Results After A549/DDP co-culture with CIK cells, the DDP resistance of A549/DDP significantly decreased in a time-dependent manner. The DDP resistance of A549/DDP co-cultured with CIK cells for 20 h decreased 4.93-fold compared with that of A549/DDP cells cultured alone ( P A549/DDP significantly decreased after co-culture with CIK cells ( P A549/DDP with CIK cells. The expression of GST-π was restored by adding the neutralizing IFN-γ. Conclusion CIK cells can reverse the drug resistance of A549/DDP in a time-dependent manner by reducing GST-π expression to increase the accumulation of DDP. The effect of CIK cells on re-sensitizing lung cancer cells to the chemotherapy drug was partially dependent on the secretion of IFN-γ.

  11. Biosynthesis of gold nanoparticles and related cytotoxicity evaluation using A549 cells.

    Science.gov (United States)

    Sathishkumar, M; Pavagadhi, S; Mahadevan, A; Balasubramanian, R

    2015-04-01

    Biosynthesis of gold nanoparticles (AuNPs) has become an attractive area of research as it is environmentally benign. The toxicity of AuNPs synthesized by chemical routes has been widely studied. However, little is known about the toxicity associated with the biological synthesis of AuNPs. The present study was carried out to synthesize AuNPs using star anise (Illicium verum; a commercially available spice in abundance)and evaluate its toxicity using human epithelial lung cells (A549) in comparison with AuNPs synthesized by the traditional chemical methods (using sodium citrate and sodium borohydride). Apart from cell viability, markers of oxidative stress (reduced glutathione) and cell death (caspases) were also evaluated to understand the mechanisms of toxicity. Cell viability was observed to be 65.7 percent and 72.3 percent in cells exposed to chemically synthesized AuNPs at the highest dose (200nM) as compared to 80.2 percent for biologically synthesized AuNPs. Protective coating/capping of AuNPs by various polyphenolic compounds present in star anise extract appears to be a major contributor to lower toxicity observed in biologically synthesized AuNPs. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Characterization of indoor dust from Brazil and evaluation of the cytotoxicity in A549 lung cells.

    Science.gov (United States)

    Deschamps, E; Weidler, P G; Friedrich, F; Weiss, C; Diabaté, S

    2014-04-01

    Over the past decade, ambient air particulate matter (PM) has been clearly associated with adverse health effects. In Brazil, small and poor communities are exposed to indoor dust derived from both natural sources, identified as blowing soil dust, and anthropogenic particles from mining activities. This study investigates the physicochemical and mineralogical composition of indoor PM10 dust samples collected in Minas Gerais, Brazil, and evaluates its cytotoxicity and inflammatory potential. The mean PM10 mass concentration was 206 μg/m(3). The high dust concentration in the interior of the residences is strongly related to blowing soil dust. The chemical and mineralogical compositions were determined by ICP-OES and XRD, and the most prominent minerals were clays, Fe-oxide, quartz, feldspars, Al(hydr)oxides, zeolites, and anatase, containing the transition metals Fe, Cr, V, Ni, Cu, Zn, Ti, and Mn as well as the metalloid As. The indoor dust samples presented a low water solubility of about 6 %. In vitro experiments were carried out with human lung alveolar carcinoma cells (A549) to study the toxicological effects. The influence of the PM10 dust samples on cell viability, intracellular formation of reactive oxygen species (ROS), and release of the pro-inflammatory cytokine IL-8 was analysed. The indoor dust showed little effects on alamarBlue reduction indicating unaltered mitochondrial activity. However, significant cell membrane damage, ROS production, and IL-8 release were detected in dependence of dose and time. This study will support the implementation of mitigation actions in the investigated area in Brazil.

  13. Mechanism of cigarette smoke condensate-induced acute inflammatory response in human bronchial epithelial cells

    Directory of Open Access Journals (Sweden)

    Mohapatra Shyam S

    2002-07-01

    Full Text Available Abstract Background To demonstrate the involvement of tobacco smoking in the pathophysiology of lung disease, the responses of pulmonary epithelial cells to cigarette smoke condensate (CSC — the particulate fraction of tobacco smoke — were examined. Methods The human alveolar epithelial cell line A549 and normal human bronchial epithelial cells (NHBEs were exposed to 0.4 μg/ml CSC, a concentration that resulted in >90% cell survival and Results NHBEs exposed to CSC showed increased expression of the inflammatory mediators sICAM-1, IL-1β, IL-8 and GM-CSF, as determined by RT-PCR. CSC-induced IL-1β expression was reduced by PD98059, a blocker of mitogen-actived protein kinase (MAPK kinase (MEK, and by PDTC, a NFκB inhibitor. Analysis of intracellular signaling pathways, using antibodies specific for phosphorylated MAPKs (extracellular signal-regulated kinase [ERK]-1/2, demonstrated an increased level of phosphorylated ERK1/2 with increasing CSC concentration. Nuclear localization of phosphorylated ERK1/2 was seen within 30 min of CSC exposure and was inhibited by PD98059. Increased phosphorylation and nuclear translocation of IκB was also seen after CSC exposure. A549 cells transfected with a luciferase reporter plasmid containing a NFκB-inducible promoter sequence and exposed to CSC (0.4 μg/ml or TNF-α (50 ng/ml had an increased reporter activity of approximately 2-fold for CSC and 3.5-fold for TNF-α relative to untreated controls. Conclusion The acute phase response of NHBEs to cigarette smoke involves activation of both MAPK and NFκB.

  14. Reversal of galectin-1 gene silencing on resistance to cisplatin in human lung adenocarcinoma A549 cells.

    Science.gov (United States)

    Zhang, Lei; Liu, Xuegang; Tang, Zhen; Li, Xiaojun; Wang, Gengming

    2016-10-01

    This study aims to investigate reversal of Galectin-1 gene silencing on resistance to cisplatin in human lung adenocarcinoma A549 (or A549/DDP) in vivo and in vitro. The stably transfected lentivirus vector was used to silence Galectin-1 in human lung adenocarcinoma cell line A549 and A549/DDP cells and the cell lines were cultured and passaged. RT-PCR and western blot assay were used to test A549, A549/DDP cells, silenced Galectin-1A549 (A549/I) cells, Galectin-1 mRNA and protein expression levels, respectively, in A549/DDP (A549/DDP/I) cells. CCK8 assay was used to measure median inhibitory concentration (IC50) in each group and resistant index of A549/DDP cells and A549/DDP/I cells. Tumor model in nude mice was established by armpit injection of A549, A549/DDP, A549/I, A549/DDP/I cells. Cisplatin was injected intraperitoneally in tumor models and growth of tumor was observed in vivo model. Four weeks later, nude mice were killed and tumor weight and diameter was measured. mRNA and protein expression of Galectin-1 in A549/DDP cells was higher than that in A549 cells. mRNA and protein expression of Galectin-1 in A549/DDP/I cells was lower than that in A549/DDP cells. Moreover, IC50 values ​​and resistance index in A549/DDP cells was higher than that in A549 cells group and IC50 values ​​and resistance index A549/DDP/I cell group were lower than that in A549/DDP cells. Additionally, tumor weight and volume in A549/DDP/I cell group were lower than that in A549/DDP. In conclusion, Galectin-1 gene silencing would improve the sensitivity of A549/DDP cells to cisplatin in vivo and in vitro. Copyright © 2016. Published by Elsevier Masson SAS.

  15. DKK4-knockdown enhances chemosensitivity of A549/DTX cells to docetaxel.

    Science.gov (United States)

    Yang, Xueliang; Liu, Yang; Li, Weina; Li, Aimin; Sun, Quan

    2017-10-01

    Drug resistance greatly limits docetaxel efficiency in the treatment of non-small cell lung cancer (NSCLC). Dickkopf 4 (DKK4), a negative regulator of Wnt/β-catenin pathway, is believed to be involved in various human cancers; whereas the association of DKK4 with acquired docetaxel resistance in NSCLC remains unclear. In the present study, we investigated the involvement of DKK4 in the docetaxel-resistant human lung adenocarcinoma A549 (A549/DTX) cells. Our results showed that DKK4 expression was significantly increased in the A549/DTX cells compared with in the A549 cells, as well as in the culture supernatant of A549/DTX cells. DKK4 overexpression increased the resistance of A549 cells to docetaxel. DKK4-knockdown promoted inhibition of A549/DTX cell growth, and reduced the colony formation and invasion capacity of A549/DTX cells. Moreover, DKK4-knockdown promoted the pro-apoptotic effect of docetaxel characterized with caspase 3 activation and inhibition of BCL-2 expression in A549/DTX cells, which was possibly mediated by inducing the activation of c-Jun N-terminal kinase (JNK)-related signaling pathway. Thus, our results indicated that DKK4-knockdown promoted the cytotoxic and pro-apoptotic activity of A549/DTX cells, which suggests a critical role of DKK4 in docetaxel resistance of the A549 cells and provides the potential to combine docetaxel therapy with DKK4 depletion in treating NSCLC. © The Author 2017. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. An imaging flow cytometry method to assess ricin trafficking in A549 human lung epithelial cells.

    Science.gov (United States)

    Jenner, Dominic; Chong, Damien; Walker, Nicola; Green, A Christopher

    2018-02-01

    The endocytosis and trafficking of ricin in mammalian cells is an important area of research for those producing ricin anti-toxins and other ricin therapeutics. Ricin trafficking is usually observed by fluorescence microscopy techniques. This gives good resolution and leads to a detailed understanding of the internal movement of ricin within cells. However, microscopy techniques are often hampered by complex analysis and quantification techniques, and the inability to look at ricin trafficking in large populations of cells. In these studies we have directly labelled ricin and assessed if its trafficking can be observed using Imaging Flow Cytometry (IFC) both to the cytoplasmic region of cells and specifically to the Golgi apparatus. Using IDEAS® data analysis software the specific fluorescence location of the ricin within the cells was analysed. Then, using cytoplasmic masking techniques to quantify the number of cells with endocytosed cytoplasmic ricin or cells with Golgi-associated ricin, kinetic endocytosis curves were generated. Here we present, to the authors' knowledge, the first example of using imaging flow cytometry for evaluating the subcellular transport of protein cargo, using the trafficking of ricin toxin in lung cells as a model. Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.

  17. An in vitro cytotoxicity assessment of graphene nanosheets on alveolar cells

    Science.gov (United States)

    Dervin, Saoirse; Murphy, James; Aviles, Ruth; Pillai, Suresh C.; Garvey, Mary

    2018-03-01

    The collection of intrinsic properties possessed by graphene family nanomaterials (GFNs) results in their continuous exploitation for biomedical applications. The materials biomedical potential has motivated an upsurge in green preparation routes for the production of graphene like materials with limited toxicity. A number of bio-friendly reducing agents have been utilized for the preparation of chemically reduced graphene oxide (GO), and their resulting cytotoxic effects examined. However, the toxicology effects of one of the first biomolecules implemented for the reduction of GO, ascorbic acid (AA) has yet to be investigated. Herein, the toxicity of three distinct GFNs; GO, hydrazine reduced GO (H.rGO) and AA.rGO, prepared through diverse chemical routes are studied, to demonstrate the cytotoxic activity of a green reducer, in comparison to an established reduction method using hydrazine hydrate. The variation in atomic structure of GO, H.rGO and AA.rGO resulting from different synthesis techniques demonstrates the dependence of toxicity on particle shape and size. All GFNs induced high levels of alveolar cell toxicity. Interaction of AA.rGO with the A549 human lung epithelial carcinoma cell line resulted in increased leakage of lactate dehydrogenase, indicative of diminished cell membrane integrity. The uncharacteristic shape of the AA.rGO may be responsible for this proliferated release of the essential protein. The presented data therefore demonstrates that modification of synthetic processes significantly alter the biological activities of GFNs.

  18. KDR gene silencing inhibits proliferation of A549 cells and enhances their sensitivity to docetaxel.

    Science.gov (United States)

    Wei, R; Zang, J-P

    2015-11-23

    We investigated the effects of kinase-domain insert containing receptor (KDR) gene silencing on the proliferation of A549 cells and their sensitivity to docetaxel. After designing and synthesizing the KDR siRNA sequence, the sequence was transfected into A549 cells using Lipofectamine 2000. The expression of KDR mRNA and protein after KDR gene silencing was detected by reverse transcription-polymerase chain reaction and western blotting; A549 cell cycle was detected by flow cytometry. An MTT assay and colony formation was performed to determine the sensitivity of A549 cells to docetaxel after KDR gene silencing. After 48-h KDR gene silencing, KDR gene and protein expression significantly decreased (P A549 cell cycle was significantly arrested in G0/G1 phase, and the number of cells in S phase was reduced; the difference was statistically significant (P A549 cells to docetaxel showed a significant enhancement (P A549 cells, inhibit the proliferation of A549 cells, and enhance their sensitivity to docetaxel.

  19. Per2 participates in AKT-mediated drug resistance in A549/DDP lung adenocarcinoma cells.

    Science.gov (United States)

    Chen, Bo; Tan, Yaoxi; Liang, Yan; Li, Yan; Chen, Lei; Wu, Shuangshuang; Xu, Wei; Wang, Yan; Zhao, Weihong; Wu, Jianqing

    2017-01-01

    Period2 (Per2) is a key mammalian circadian clock protein, and additionally has a tumor suppressive function. The present study aimed to investigate its role in drug resistance in A549/cisplatin (DDP) lung adenocarcinoma cells. Per2 knockdown and overexpression in A549/DDP cells were used to compare cell proliferation (by MTT assay), apoptosis (active-caspase 3 western blot) and clone forming assay. The activation of AKT/mechanistic target of rapamycin (mTOR) was investigated by a western blot assay. The Per2 expression level was decreased in A549/DDP cells compared with A549 cells. Per2 knockdown by short hairpin RNA protects A549/DDP cells from apoptosis, and promotes proliferation and migration. Per2 knockdown results in increased activation of the phosphoinositide 3-kinase (PI3K)/AKT/mTOR signaling pathway. Overexpression of Per2 in A549/DDP cells may reduce the activity of the PI3K/AKT/mTOR signaling pathway, and promote apoptosis of A549 cells. The results of the present study suggest that Per2 participates in AKT-mediated drug resistance in A549/DDP lung adenocarcinoma cells.

  20. MicroRNA-146 protects A549 and H1975 cells from LPS-induced ...

    Indian Academy of Sciences (India)

    The presentstudy explored the protective effects of miR-146 overexpression on lipopolysaccharide (LPS)-mediated injury in A549 andH1975 cells. In this study, A549 and H1975 cells were transfected with miR-146 mimic or inhibitor, and then weresubjected with LPS. Thereafter, cell viability, colony formation capacity, ...

  1. Nitric oxide gas phase release in human small airway epithelial cells

    Directory of Open Access Journals (Sweden)

    Suresh Vinod

    2009-01-01

    Full Text Available Abstract Background Asthma is a chronic airway inflammatory disease characterized by an imbalance in both Th1 and Th2 cytokines. Exhaled nitric oxide (NO is elevated in asthma, and is a potentially useful non-invasive marker of airway inflammation. However, the origin and underlying mechanisms of intersubject variability of exhaled NO are not yet fully understood. We have previously described NO gas phase release from normal human bronchial epithelial cells (NHBEs, tracheal origin. However, smaller airways are the major site of morbidity in asthma. We hypothesized that IL-13 or cytomix (IL-1β, TNF-α, and IFN-γ stimulation of differentiated small airway epithelial cells (SAECs, generation 10–12 and A549 cells (model cell line of alveolar type II cells in culture would enhance NO gas phase release. Methods Confluent monolayers of SAECs and A549 cells were cultured in Transwell plates and SAECs were allowed to differentiate into ciliated and mucus producing cells at an air-liquid interface. The cells were then stimulated with IL-13 (10 ng/mL or cytomix (10 ng/mL for each cytokine. Gas phase NO release in the headspace air over the cells was measured for 48 hours using a chemiluminescence analyzer. Results In contrast to our previous result in NHBE, baseline NO release from SAECs and A549 is negligible. However, NO release is significantly increased by cytomix (0.51 ± 0.18 and 0.29 ± 0.20 pl.s-1.cm-2, respectively reaching a peak at approximately 10 hours. iNOS protein expression increases in a consistent pattern both temporally and in magnitude. In contrast, IL-13 only modestly increases NO release in SAECs reaching a peak (0.06 ± 0.03 pl.s-1.cm-2 more slowly (30 to 48 hours, and does not alter NO release in A549 cells. Conclusion We conclude that the airway epithelium is a probable source of NO in the exhaled breath, and intersubject variability may be due, in part, to variability in the type (Th1 vs Th2 and location (large vs small airway

  2. Cigarette smoke extract (CSE) induces transient receptor potential ankyrin 1(TRPA1) expression via activation of HIF1αin A549 cells.

    Science.gov (United States)

    Nie, Yichu; Huang, Chuqin; Zhong, Shan; Wortley, Michael A; Luo, Yulong; Luo, Wei; Xie, Yanqing; Lai, Kefang; Zhong, Nanshan

    2016-10-01

    We previously found that transient receptor potential ankyrin 1 (TRPA1) in guinea pig tracheal epithelial cells was elevated after 14 days of cigarette smoke (CS) exposure. However, the mechanism underlying CS-induced TRPA1 expression remains unknown. Here, we explored whether cigarette smoke extract (CSE)-induced TRPA1 expression is related with modulation of HIF1α in A549 cells. Our results showed that CSE increased TRPA1 expression in A549 cells, decreased Iκ B, PHD2, and HDAC2, and increased ROS release and nuclear translocation of NF-κ B and HIF1α. Moreover, HIF1α siRNA and/or MG132 (a proteasome inhibitor) pretreatment significantly inhibited CSE-induced TRPA1 expression and HIF1α nuclear translocation in A549 cells. However, HIF1α siRNA pretreatment did not affect CSE-induced NF-κ B nuclear translocation, suggesting that CSE-induced TRPA1 expression in A549 cells is directly mediated by HIF1α, but not by NF-κ B. Similar to CSE treatment, treatment of A549 cells with LPS caused significant increases in nuclear translocation of NF-κ B and HIF1α mRNA expression, but did not alter TRPA1 mRNA expression. However, pretreatment with PHD2 siRNA did result in increased TRPA1 mRNA expression in LPS-treated A549 cells; an effect that was inhibited by SN50 (a NF-κ B inhibitor). It suggests a role for NF-κ B to indirectly regulate TRPA1 mRNA expression via modulating HIF1α mRNA transcription. In addition, treatment cells with HDAC2 siRNA plus 2%CSE resulted in increased HIF1α nuclear translocation and TRPA1 expression, which was significantly inhibited by MG132 and HIF1α siRNA. These results suggest that HDAC2 indirectly modulates TRPA1 expression by promoting the DNA-binding activity of HIF1α. These findings show that CSE increases TRPA1 expression in airway epithelial cells by directly activating HIF1α, and that this increase in TRPA1 expression is indirectly regulated via NF-κ B, PHD2 and HDAC2 modulation of HIF1α activity. Copyright © 2016

  3. Fibroblast α11β1 Integrin Regulates Tensional Homeostasis in Fibroblast/A549 Carcinoma Heterospheroids

    Science.gov (United States)

    Lu, Ning; Karlsen, Tine V.; Reed, Rolf K.; Kusche-Gullberg, Marion; Gullberg, Donald

    2014-01-01

    We have previously shown that fibroblast expression of α11β1 integrin stimulates A549 carcinoma cell growth in a xenograft tumor model. To understand the molecular mechanisms whereby a collagen receptor on fibroblast can regulate tumor growth we have used a 3D heterospheroid system composed of A549 tumor cells and fibroblasts without (α11+/+) or with a deletion (α11-/-) in integrin α11 gene. Our data show that α11-/-/A549 spheroids are larger than α11+/+/A549 spheroids, and that A549 cell number, cell migration and cell invasion in a collagen I gel are decreased in α11-/-/A549 spheroids. Gene expression profiling of differentially expressed genes in fibroblast/A549 spheroids identified CXCL5 as one molecule down-regulated in A549 cells in the absence of α11 on the fibroblasts. Blocking CXCL5 function with the CXCR2 inhibitor SB225002 reduced cell proliferation and cell migration of A549 cells within spheroids, demonstrating that the fibroblast integrin α11β1 in a 3D heterospheroid context affects carcinoma cell growth and invasion by stimulating autocrine secretion of CXCL5. We furthermore suggest that fibroblast α11β1 in fibroblast/A549 spheroids regulates interstitial fluid pressure by compacting the collagen matrix, in turn implying a role for stromal collagen receptors in regulating tensional hemostasis in tumors. In summary, blocking stromal α11β1 integrin function might thus be a stroma-targeted therapeutic strategy to increase the efficacy of chemotherapy. PMID:25076207

  4. Concentrations in plasma, epithelial lining fluid, alveolar macrophages and bronchial mucosa after a single intravenous dose of 1.6 mg/kg of iclaprim (AR-100) in healthy men.

    Science.gov (United States)

    Andrews, J; Honeybourne, D; Ashby, J; Jevons, G; Fraise, A; Fry, P; Warrington, S; Hawser, S; Wise, R

    2007-09-01

    A validated microbiological assay was used to measure concentrations of iclaprim (AR-100) in plasma, bronchial mucosa (BM), alveolar macrophages (AM) and epithelial lining fluid (ELF) after a single 1.6 mg/kg intravenous 60 min iv infusion of iclaprim. Male volunteers were randomly allocated to three nominal sampling time intervals 1-2 h (Group A), 3-4 h (Group B) and 5.5-7.0 h (Group C) after the start of the drug infusion. Mean iclaprim concentrations in plasma, BM, AM and ELF, respectively, were for Group A 0.59 mg/L (SD 0.18), 0.51 mg/kg (SD 0.17), 24.51 mg/L (SD 21.22) and 12.61 mg/L (SD 7.33); Group B 0.24 mg/L (SD 0.05), 0.35 mg/kg (SD 0.17), 7.16 mg/L (SD 1.91) and 6.38 mg/L (SD 5.17); and Group C 0.14 mg/L (SD 0.05), no detectable level in BM, 5.28 mg/L (SD 2.30) and 2.66 mg/L (SD 2.08). Iclaprim concentrations in ELF and AM exceeded the MIC(90) for penicillin-susceptible Streptococcus pneumoniae (MIC90 0.06 mg/L), penicillin-intermediate S. pneumoniae (MIC90 2 mg/L), penicillin-resistant S. pneumoniae (MIC90 4 mg/L) for 7, 7 and 4 h, respectively, and Chlamydia pneumoniae (MIC90 0.5 mg/L) for 7 h. Mean iclaprim concentrations in ELF exceeded the MIC90 for Haemophilus influenzae (MIC90 4 mg/L) and Moraxella catarrhalis (MIC90 8 mg/L) for up to 4 and 2 h, respectively; in AM the MIC90 was exceeded for up to 7 h. Furthermore, the MIC90 for methicillin-resistant Staphylococcus aureus of 0.12 mg/L was exceeded at all sites for up to 7 h. These data suggest that iclaprim reaches lung concentrations that should be effective in the treatment of community-acquired pneumonia.

  5. TNF-α-Induced cPLA2 Expression via NADPH Oxidase/Reactive Oxygen Species-Dependent NF-κB Cascade on Human Pulmonary Alveolar Epithelial Cells

    Science.gov (United States)

    Lin, Chih-Chung; Lin, Wei-Ning; Cho, Rou-Ling; Wang, Chen-yu; Hsiao, Li-Der; Yang, Chuen-Mao

    2016-01-01

    Tumor necrosis factor-α (TNF-α) triggers activation of cytosolic phospholipase A2 (cPLA2) and then enhancing the synthesis of prostaglandin (PG) in inflammatory diseases. However, the detailed mechanisms of TNF-α induced cPLA2 expression were not fully defined in human pulmonary alveolar epithelial cells (HPAEpiCs). We found that TNF-α-stimulated increases in cPLA2 mRNA (5.2 folds) and protein (3.9 folds) expression, promoter activity (4.3 folds), and PGE2 secretion (4.7 folds) in HPAEpiCs, determined by Western blot, real-time PCR, promoter activity assay and PGE2 ELISA kit. These TNF-α-mediated responses were abrogated by the inhibitors of NADPH oxidase [apocynin (APO) and diphenyleneiodonium chloride (DPI)], ROS [N-acetyl cysteine, (NAC)], NF-κB (Bay11-7082) and transfection with siRNA of ASK1, p47phox, TRAF2, NIK, IKKα, IKKβ, or p65. TNF-α markedly stimulated NADPH oxidase activation and ROS including superoxide and hydrogen peroxide production which were inhibited by pretreatment with a TNFR1 neutralizing antibody, APO, DPI or transfection with siRNA of TRAF2, ASK1, or p47phox. In addition, TNF-α also stimulated p47phox phosphorylation and translocation in a time-dependent manner. On the other hand, TNF-α induced TNFR1, TRAF2, ASK1, and p47phox complex formation in HPAEpiCs, which were attenuated by a TNF-α neutralizing antibody. We found that pretreatment with NAC, DPI, or APO also attenuated the TNF-α-stimulated IKKα/β and NF-κB p65 phosphorylation, NF-κB (p65) translocation, and NF-κB promoter activity in HPAEpiCs. Finally, we observed that TNF-α-stimulated NADPH oxidase activation and ROS generation activates NF-κB through the NIK/IKKα/β pathway. Taken together, our results demonstrated that in HPAEpiCs, up-regulation of cPLA2 by TNF-α is, at least in part, mediated through the cooperation of TNFR1, TRAF2, ASK1, and NADPH oxidase leading to ROS generation and ultimately activates NF-κB pathway. PMID:27932980

  6. Zebularine inhibits the growth of A549 lung cancer cells via cell cycle arrest and apoptosis.

    Science.gov (United States)

    You, Bo Ra; Park, Woo Hyun

    2014-11-01

    Zebularine (Zeb) is a DNA methyltransferase (DNMT) inhibitor to that has an anti-tumor effect. Here, we evaluated the anti-growth effect of Zeb on A549 lung cancer cells in relation to reactive oxygen species (ROS) levels. Zeb inhibited the growth of A549 cells with an IC50 of approximately 70 µM at 72 h. Cell cycle analysis indicated that Zeb induced an S phase arrest in A549 cells. Zeb also induced A549 cell death, which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨm ), Bcl-2 decrease, Bax increase, p53 increase and activation of caspase-3 and -8. In contrast, Zeb mildly inhibited the growth of human pulmonary fibroblast (HPF) normal cells and lead to a G1 phase arrest. Zeb did not induce apoptosis in HPF cells. In relation to ROS level, Zeb increased ROS level in A549 cells and induced glutathione (GSH) depletion. The well-known antioxidant, N-acetyl cysteine (NAC) prevented the death of Zeb-treated A549 cells. Moreover, Zeb increased the level of thioredoxin reductase 1 (TrxR1) in A549 cells. While the overexpression of TrxR1 attenuated death and ROS level in Zeb-treated A549 cells, the downregulation of TrxR1 intensified death and ROS level in these cells. In conclusion, Zeb inhibited the growth of A549 lung cancer cells via cell cycle arrest and apoptosis. The inhibition was influenced by ROS and TrxR1 levels. © 2013 Wiley Periodicals, Inc.

  7. Suitable parameter choice on quantitative morphology of A549 cell in epithelial–mesenchymal transition

    Science.gov (United States)

    Ren, Zhou-Xin; Yu, Hai-Bin; Li, Jian-Sheng; Shen, Jun-Ling; Du, Wen-Sen

    2015-01-01

    Evaluation of morphological changes in cells is an integral part of study on epithelial to mesenchymal transition (EMT), however, only a few papers reported the changes in quantitative parameters and no article compared different parameters for demanding better parameters. In the study, the purpose was to investigate suitable parameters for quantitative evaluation of EMT morphological changes. A549 human lung adenocarcinoma cell line was selected for the study. Some cells were stimulated by transforming growth factor-β1 (TGF-β1) for EMT, and other cells were as control without TGF-β1 stimulation. Subsequently, cells were placed in phase contrast microscope and three arbitrary fields were captured and saved with a personal computer. Using the tools of Photoshop software, some cells in an image were selected, segmented out and exchanged into unique hue, and other part in the image was shifted into another unique hue. The cells were calculated with 29 morphological parameters by Image Pro Plus software. A parameter between cells with or without TGF-β1 stimulation was compared statistically and nine parameters were significantly different between them. Receiver operating characteristic curve (ROC curve) of a parameter was described with SPSS software and F-test was used to compare two areas under the curves (AUCs) in Excel. Among them, roundness and radius ratio were the most AUCs and were significant higher than the other parameters. The results provided a new method with quantitative assessment of cell morphology during EMT, and found out two parameters, roundness and radius ratio, as suitable for quantification. PMID:26182364

  8. Differentiated bronchiolar epithelium in alveolar ducts of rats exposed to ozone for 20 months

    Energy Technology Data Exchange (ETDEWEB)

    Pinkerton, K.E.; Dodge, D.E.; Cederdahl-Demmler, J.; Wong, V.J.; Peake, J.; Haselton, C.J.; Mellick, P.W.; Singh, G.; Plopper, C.G. (Univ. of California, Davis (United States))

    1993-03-01

    The effects of exposure to 1.0 ppm of ozone for twenty months were studied in male Fischer 344 rats. Light microscopic, morphometric, and immunohistological approaches were used to determine the distribution and degree of differentiation of ciliated and nonciliated bronchiolar epithelial (Clara) cells lining alveolar ducts of the central acinus, a primary target of ozone-induced lung injury. Alveolar duct pathways extending beyond the level of the most proximal alveolar outpocketing of terminal bronchioles were isolated in longitudinal profile. The distance that ciliated and nonciliated bronchiolar epithelial (Clara) cells projected down each alveolar duct pathway was determined by placing concentric arcs radiating outward from a single reference point at the level of the first alveolar outpocketing. A high degree of heterogeneity in the magnitude of bronchiolar epithelial cell extension into alveolar ducts was noted for each isolation and animal. Age-matched control animals also demonstrated variation in the degree of bronchiolar epithelial cell extension down alveolar ducts. In animals exposed to ozone, a striking similarity was noted by scanning electron microscopy in the surface characteristics of cells lining both terminal bronchioles and alveolar ducts. The presence of Clara cell secretory protein in cells of bronchioles and alveolar ducts was also detected immunohistochemically and visualized using confocal laser scanning microscopy in the reflectance mode. Well-differentiated ciliated and nonciliated bronchiolar epithelial cells were found lining alveolar septal tips and alveoli up to a depth of 1,000 mu into the pulmonary acinus after 20 months of exposure to ozone. No evidence of inflammation was present in alveolar ducts, suggesting that epithelial cell transformations in alveolar ducts is a natural consequence of lifetime exposures to oxidant gases.

  9. [Role of SOX4 on DDP Resistance in Non-small Cell Lung Cancer Cell of A549].

    Science.gov (United States)

    Li, Wei; Liu, Xu; Zhang, Guoqian; Zhang, Linlin

    2017-05-20

    Lung cancer is one of the most serious disease and the incidence of non-small cell lung cancer (NSCLC) is the highest in lung cancer. The main reason for the failure of chemotherapy is the tolerance to cisplatin. Transcriptional regulator SOX4 plays an important role in the occurrence and development of many tumors, and regulates Wnt signaling pathway by regulating the expression of β-catenin. We aimed to investigate the role of SOX4 on cisplatin-resistance in NSCLC cell A549 cell. The cisplatin-resistance lung cancer cell line A549/DDP was constructed by induction method in vitro, and cisplatin-resistance detected by CCK8 assay. Growth curves of A549 and A549/DDP was calculated. The expression level of SOX4 in A549 and A549/DDP cells were detected by Western blot. A549/DDP were knockdown of SOX4 by siRNA transfection, and the cisplatin-resistance of detected by CCK-8 assay, the expression level of β-catenin and Survivin were detected by real-time PCR and Western blot. The cisplatin-resistance cell line A549/DDP was constructed successfully, and its cisplatin-resistance is 13.7 times higher than in A549. There was no significance difference between A549 and A549/DDP in cell proliferation. The expression level of SOX4 is higher in A549/DDP than in A549. The cisplatin-resistance significantly decreased in A549/DDP cells after knockdown of SOX4 by siRNA transfection. The expression level of β-catenin and Survivin significantly decreased in A549/DDP cells after knockdown of SOX4. SOX4 can strengthen cisplatin-resistance of non-small cell lung cancer cell A549.
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  10. MiR-200c increases the radiosensitivity of non-small-cell lung cancer cell line A549 by targeting VEGF-VEGFR2 pathway.

    Directory of Open Access Journals (Sweden)

    Liangliang Shi

    Full Text Available MicroRNAs (miRNAs have been demonstrated to participate in many important cellular processes including radiosensitization. VEGF family, an important regulator of angiogenesis, also plays a crucial role in the regulation of cancer cell radiosensitivity. VEGFR2 mediates the major growth and permeability actions of VEGF in a paracrine/autocrine manner. MiR-200c, at the nexus of epithelial-mesenchymal transition (EMT, is predicted to target VEGFR2. The purpose of this study is to test the hypothesis that regulation of VEGFR2 pathway by miR-200c could modulate the radiosensitivity of cancer cells. Bioinformatic analysis, luciferase reporter assays and biochemical assays were carried out to validate VEGFR2 as a direct target of miR-200c. The radiosensitizing effects of miR-200c on A549 cells were determined by clonogenic assays. The downstream regulating mechanism of miR-200c was explored with western blotting assays, FCM, tube formation assays and migration assays. We identified VEGFR2 as a novel target of miR-200c. The ectopic miR-200c increased the radiosensitivity of A549 while miR-200c down-regulation decreased it. Besides, we proved that miR-200c radiosensitized A549 cells by targeting VEGF-VEGFR2 pathway specifically, thus leading to inhibition of its downstream pro-survival signaling transduction and angiogenesis, and serves as a potential target for radiosensitizition research.

  11. Lung epithelial tip progenitors integrate glucocorticoid- and STAT3-mediated signals to control progeny fate

    Science.gov (United States)

    Laresgoiti, Usua; Rao, Chandrika; Brady, Jane L.; Richardson, Rachel V.; Batchen, Emma J.; Chapman, Karen E.

    2016-01-01

    Insufficient alveolar gas exchange capacity is a major contributor to lung disease. During lung development, a population of distal epithelial progenitors first produce bronchiolar-fated and subsequently alveolar-fated progeny. The mechanisms controlling this bronchiolar-to-alveolar developmental transition remain largely unknown. We developed a novel grafting assay to test if lung epithelial progenitors are intrinsically programmed or if alveolar cell identity is determined by environmental factors. These experiments revealed that embryonic lung epithelial identity is extrinsically determined. We show that both glucocorticoid and STAT3 signalling can control the timing of alveolar initiation, but that neither pathway is absolutely required for alveolar fate specification; rather, glucocorticoid receptor and STAT3 work in parallel to promote alveolar differentiation. Thus, developmental acquisition of lung alveolar fate is a robust process controlled by at least two independent extrinsic signalling inputs. Further elucidation of these pathways might provide therapeutic opportunities for restoring alveolar capacity. PMID:27578791

  12. MiR-9 enhances the sensitivity of A549 cells to cisplatin by inhibiting autophagy.

    Science.gov (United States)

    Zhang, Yan; Meng, Xia; Li, Cheng; Tan, Zhoulin; Guo, Xinwei; Zhang, Zhiting; Xi, Tao

    2017-07-01

    To demonstrate that miR-9 inhibits autophagy by down-regulating Beclin1 and thus enhances the sensitivity of A549 cells to cisplatin. MiR-9 inhibited Beclin1 expression by binding to its 3'UTR. The inhibition decreased the cisplatin-induced autophagy in A549 cells, evidenced by the decreased expression of LC3II and GFP-LC3 puncta and the increased expression of P62. Upregulation of miR-9 level enhanced the sensibility of A549 cells to cisplatin and increased the cisplatin-induced apoptosis. Overexpression of Beclin1 reversed above effects of miR-9 mimics, cisplatin-induced autophagy was increased and apoptosis was decreased. MiR-9 inhibits autophagy via targeting Beclin1 3'UTR and thus enhances cisplatin sensitivity in A549 cells.

  13. CD18-mediated adhesion is required for the induction of a proinflammatory phenotype in lung epithelial cells by mononuclear cell-derived extracellular vesicles.

    Science.gov (United States)

    Neri, Tommaso; Scalise, Valentina; Passalacqua, Ilaria; Giusti, Ilaria; Lombardi, Stefania; Balia, Cristina; D'Alessandro, Delfo; Berrettini, Stefano; Pedrinelli, Roberto; Paggiaro, Pierluigi; Dolo, Vincenza; Celi, Alessandro

    2018-04-01

    Extracellular vesicles are submicron vesicles that upregulate the synthesis of proinflammatory mediators by lung epithelial cells. We investigated whether these structures adhere to lung epithelial cells, and whether adhesion is a prerequisite for their proinflammatory activity. Extracellular vesicles were generated by stimulation of normal human mononuclear cells with the calcium ionophore A23187, and labelled with carboxyfluorescein diacetate succinimidyl ester. Adhesion of vesicles to monolayers of immortalized bronchial epithelial (16HBE) and alveolar (A549) cells was analyzed by fluorescence microscopy. The role of candidate adhesion receptors was evaluated with inhibitory monoclonal antibodies and soluble peptides. The synthesis of proinflammatory mediators was assessed by ELISA. Transmission electron microscopy confirmed the generation of closed vesicles with an approximate size range between 50 and 600 nm. Adhesion of extracellular vesicles to epithelial cells was upregulated upon stimulation of the latter with tumor necrosis factor-α. Adhesion was blocked by an anti-CD18 antibody, by peptides containing the sequence RGD and, to a lesser extent, by an antibody to ICAM-1. The same molecules also blocked the upregulation of the synthesis of interleukin-8 and monocyte chemotactic protein-1 induced by extracellular vesicles. CD18-mediated adhesion of extracellular vesicles is a prerequisite for their proinflammatory activity. Copyright © 2018 Elsevier Inc. All rights reserved.

  14. CCL20 and Beta-Defensin 2 Production by Human Lung Epithelial Cells and Macrophages in Response to Brucella abortus Infection

    Science.gov (United States)

    Fernández, Andrea G.; Bonetto, Josefina; Giambartolomei, Guillermo H.; Fossati, Carlos A.; Baldi, Pablo C.

    2015-01-01

    Both CCL20 and human β-defensin 2 (hBD2) interact with the same membrane receptor and display chemotactic and antimicrobial activities. They are produced by airway epithelia in response to infectious agents and proinflammatory cytokines. Whereas Brucella spp. can infect humans through inhalation, their ability to induce CCL20 and hBD2 in lung cells is unknown. Here we show that B. abortus induces CCL20 expression in human alveolar (A549) or bronchial (Calu-6) epithelial cell lines, primary alveolar epithelial cells, primary human monocytes, monocyte-derived macrophages and the monocytic cell line THP-1. CCL20 expression was mainly mediated by JNK1/2 and NF-kB in both Calu-6 and THP-1 cells. CCL20 secretion was markedly induced in A549, Calu-6 and THP-1 cells by heat-killed B. abortus or a model Brucella lipoprotein (L-Omp19) but not by the B. abortus lipopolysaccharide (LPS). Accordingly, CCL20 production by B. abortus-infected cells was strongly TLR2-dependent. Whereas hBD2 expression was not induced by B. abortus infection, it was significantly induced in A549 cells by conditioned media from B. abortus-infected THP-1 monocytes (CMB). A similar inducing effect was observed on CCL20 secretion. Experiments using blocking agents revealed that IL-1β, but not TNF-α, was involved in the induction of hBD2 and CCL20 secretion by CMB. In the in vitro antimicrobial assay, the lethal dose (LD) 50 of CCL20 for B. abortus (>50 μg/ml) was markedly higher than that against E. coli (1.5 μg/ml) or a B. abortus mutant lacking the O polysaccharide in its LPS (8.7 ug/ml). hBD2 did not kill any of the B. abortus strains at the tested concentrations. These results show that human lung epithelial cells secrete CCL20 and hBD2 in response to B. abortus and/or to cytokines produced by infected monocytes. Whereas these molecules do not seem to exert antimicrobial activity against this pathogen, they could recruit immune cells to the infection site. PMID:26448160

  15. Antiproliferative and apoptotic effects of diffractaic acid in A549 and AGS cancer cells

    Science.gov (United States)

    Kızıl, Hamit Emre; Aǧar, Güleray

    2017-04-01

    In this study, we determined the antiproliferative and apoptotic effects of diffractaic acid by measuring the gene expression changes of topo II α, caspase-3 and p53 on A549 and AGS cancer cells. Real time PCR assay was used to measure the change folds. It was determined that concentrations of 12,5, 50 and 100 µg / ml were antiproliferative and apoptotic for the A549 cancer cell line and 50 µg / ml for the AGS cell line.

  16. [Effect of paraquat on the expression of a disintegrin and metalloproteinase-17 in A549 cells].

    Science.gov (United States)

    Fang, W Y; Lin, C W; Wang, B F; Feng, S G

    2018-01-20

    Objective: Construct a paraquat (PQ) cell fibrosis model in vitro, observe the effect of PQ on the expression of a disintegrin and metalloproteinase-17 (ADAM17) in A549 cells, and explore the role of ADAM17 in the pulmonary fibrosis induced by PQ poisoning. Methods: A549 cells are divided into normal control group, different concentration of PQ groups, CCK-8 is used to detect cell viability, screening concentration and time of PQ, cell morphology is observed under microscope; Enzyme-linked immunosorbent assay (ELISA) detectes fibrosis markers of collagen type I (Col I) and fibronectin (FN) expression. Establishment of cell model of fibrosis; distribution by immunocytochemical detection of ADAM17 in A549 cells, Reverse transcription-polymerase chain reaction and Western blot are used to detect the expression of ADAM17 mRNA and protein. Results: 1. With the increase of PQ concentration and the prolongation of the action time, the activity of A549 cells decreased ( P A549 cells fusion is paving stone growth and arranged more closely. After PQ induction, the cell arrangement was loose, the intercellular connection became loose, and some cells dissolved and died. 3.ELISA showed that with the increase of PQ concentration, the expression of Col I and FN increased ( P A549 cells. 5. RT-PCR and Western blot showed that the expression of ADAM17 mRNA and protein increased significantly with the increase of PQ concentration ( P A549 cells. ADAM17 is overexpressed in the A549 cells induced by PQ and may be involved in the process of pulmonary fibrosis induced by paraquat.

  17. MicroRNA-146 protects A549 and H1975 cells from LPS-induced ...

    Indian Academy of Sciences (India)

    Qiang Wang

    2017-10-26

    Oct 26, 2017 ... nicotine adenine dinucleotide (NAD)-dependent deacetylase. Figure 4. Expression of miR-146 and the release of pro-inflammatory cytokines in A549 and H1975 cells. A549 and H1975 cells were transfected with miR-146 mimic, miR-146 inhibitor, or negative control (NC) and were subjected with 10 μg/mL ...

  18. Acrylamide-derived cytotoxic, anti-proliferative, and apoptotic effects on A549 cells.

    Science.gov (United States)

    Kacar, S; Vejselova, D; Kutlu, H M; Sahinturk, V

    2017-01-01

    Acrylamide is a very common compound even reaching up to our daily foods. It has been studied in a wealth of cell lines on which it proved to have various toxic effects. Among these cell lines, human lung adenocarcinoma cell line (A549) is one of that on which acrylamide's toxicity has not been studied well yet. We intended to determine the half maximal inhibitory concentration (IC 50 ) dose of acrylamide and to investigate its cytotoxic, anti-proliferative and apoptotic effects on A549 cells. We determined the IC 50 dose by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Then, the mode of cell death was evaluated by flow cytometry using Annexin-V fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. Next, we performed transmission electron microscopy (TEM) and confocal microscopy analyses for morphological alterations and apoptotic indices. According to the MTT assay results, A549 cell viability decreases proportionally with increasing acrylamide concentrations and IC 50 for A549 was 4.6 mM for 24 h. Annexin-V FITC/PI assay results indicated that acrylamide induces apoptosis in 64% of the A549 cells. TEM and confocal microscopy analyses showed nuclear condensations, fragmentations, cytoskeleton laceration, and membrane blebbing, which are morphological characteristics of apoptosis. Our research suggests that acrylamide causes cytotoxic, anti-proliferative, and apoptotic effects on A549 cells at 4.6 mM IC 50 dose in 24 h.

  19. TGF-β Suppresses COX-2 Expression by Tristetraprolin-Mediated RNA Destabilization in A549 Human Lung Cancer Cells

    Science.gov (United States)

    Kang, Soyeong; Min, Ahrum; Im, Seock-Ah; Song, Sang-Hyun; Kim, Sang Gyun; Kim, Hyun-Ah; Kim, Hee-Jun; Oh, Do-Youn; Jong, Hyun-Soon; Kim, Tae-You; Bang, Yung-Jue

    2015-01-01

    Purpose Overexpression of cyclooxygenase 2 (COX-2) is thought to promote survival of transformed cells. Transforming growth factor β (TGF-β) exerts anti-proliferative effects on a broad range of epithelial cells. In the current study, we investigated whether TGF-β can regulate COX-2 expression in A549 human lung adenocarcinoma cells, which are TGF-β-responsive and overexpress COX-2. Materials and Methods Western blotting, Northern blotting, and mRNA stability assays were performed to demonstrate that COX-2 protein and mRNA expression were suppressed by TGF-β. We also evaluated the effects of tristetraprolin (TTP) on COX-2 mRNA using RNA interference. Results We demonstrated that COX-2 mRNA and protein expression were both significantly suppressed by TGF-β. An actinomycin D chase experiment demonstrated that COX-2 mRNA was more rapidly degraded in the presence of TGF-β, suggesting that TGF-β–induced inhibition of COX-2 expression is achieved via decreased mRNA stability. We also found that TGF-β rapidly and transiently induced the expression of TTP, a well-known mRNA destabilizing factor, before suppression of COX-2 mRNA expression was observed. Using RNA interference, we confirmed that increased TTP levels play a pivotal role in the destabilization of COX-2 mRNA by TGF-β. Furthermore, we showed that Smad3 is essential to TTP-dependent down-regulation of COX-2 expression in response to TGF-β. Conclusion The results of this study show that TGF-β down-regulated COX-2 expression via mRNA destabilization mediated by Smad3/TTP in A549 cells. PMID:25544576

  20. Antitumor activity of acriflavine in lung adenocarcinoma cell line A549.

    Science.gov (United States)

    Lee, Chia-Jen; Yue, Chia-Herng; Lin, Yu-Jie; Lin, Yu-Yu; Kao, Shao-Hsuan; Liu, Jer-Yuh; Chen, Yieng-How

    2014-11-01

    Aim/Materials and Methods: In order to develop better drugs against non-small cell lung cancer (NSCLC), we screened a variety of compounds and treated the human lung adenocarcinoma cell line A549 with different drug concentrations. We then examined the cell viability using the MTT assay. Data show that a new candidate drug, acriflavine (ACF), suppresses the viability of A549 cells in a concentration- and time-dependent manner. Flow cytometry analysis revealed that ACF significantly caused cell growth arrest in the G2/M phase on A549 cells. Moreover, ACF decreased Bcl-2 expression and increased Bax expression. The content of cleaved poly(ADP-ribose)polymerase-1 (PARP-1) and caspase-3 are significantly increased. These findings suggest that ACF is cytotoxic against A549 cells and suppresses A549 cells growth through the caspase-3 activation pathway. In the in vivo test, nude mice bearing A549 cells xenografts by intravenous injection were randomly assigned into two groups: control and experimental group. Treatment was initiated 10 days after implantation and intraperitoneal injection of 0.9% normal saline or 2 mg/kg of ACF was continued daily for five weeks. ACF treatment significantly decreased tumor size and tumor spots on lung surface of tumor-bearing mice. ACF can inhibit cell growth in A549 cells. Our results may assist on the delineation of the mechanism(s) leading to NSCLC cell growth inhibition and provide a new antitumor strategy against NSCLC. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  1. Radiation-Induced Bystander Effects in A549 Cells Exposed to 6 MV X-rays.

    Science.gov (United States)

    Yang, Shuning; Xu, Jing; Shao, Weixian; Geng, Chong; Li, Jia; Guo, Feng; Miao, Hui; Shen, Wenbin; Ye, Tao; Liu, Yazhou; Xu, Haiting; Zhang, Xuguang

    2015-07-01

    The aim of the study is to explore the bystander effects in A549 cells that have been exposed to 6MV X-ray. Control group, irradiated group, irradiated conditioned medium (ICM)-received group, and fresh medium group were designed in this study. A549 cells in the logarithmic growth phase were irradiated with 6MV X-ray at 0, 0.5, 1, 1.5, and 2. In ICM-received group, post-irradiation A549 cells were cultured for 3 h and were transferred into non-irradiated A549 cells for further cultivation. Clone forming test was applied to detect the survival fraction of cells. Annexin V-FITC/PI double-staining assay was used to detect the apoptosis of A549 cells 24, 48, 72, and 96 h after 2-Gy 6MV X-ray irradiation, and the curves of apoptosis were drawn. The changes in the cell cycles 4, 48, 72, and 96 h after 2-Gy 6MV X-ray irradiation were detected using PI staining flow cytometry. With the increase of irradiation dose, the survival fraction of A549 cells after the application of 0.5 Gy irradiation was decreasing continuously. In comparison to the control group, the apoptosis rate of the ICM-received group was increased in a time-dependent pattern, with the highest apoptosis rate observed at 72 h (p X-ray irradiation can induce bystander effect on A549 cells, which reaches a peak at 72 h.

  2. An important role for peroxiredoxin II in survival of A549 lung cancer cells resistant to gefitinib

    Science.gov (United States)

    Kwon, Taeho; Kyung Rho, Jin; Cheol Lee, Jae; Park, Young-Ho; Shin, Hye-Jun; Cho, Sunwha; Kang, Yong-Kook; Kim, Bo-Yeon; Yoon, Do-Young; Yu, Dae-Yeul

    2015-01-01

    Redox adaptation is an important concept that explains the mechanisms by which cancer cells survive under persistent endogenous oxidative stress and become resistant to certain anticancer agents. To investigate this concept, we determined the expression levels of peroxiredoxins (Prxs), antioxidant enzymes in drug-resistant non-small cell lung carcinoma cells. Prx II was remarkably increased only in A549/GR (gefitinib-resistant) cells compared with A549 cells, consistent with methylation/demethylation. Prx II was highly methylated in the A549 cells but was demethylated in the A549/GR cells. The elevated expression of Prx II resulted in the downregulation of reactive oxygen species (ROS) and cell death and upregulation of cell cycle progression in the A549/GR cells. When Prx II mRNA in the A549/GR cells was knocked down, the levels of ROS and apoptosis were significantly recovered to the levels of the controls. In addition, signaling molecules involved in apoptosis were increased in the A549/GR-shPrx II cells. There was no difference in the expression of MAPK/ERK between the A549/GR cells and A549/GR-shPrx II cells, but the phosphorylation of JNK was increased in the A549/GR cells and was markedly decreased in the A549/GR-shPrx II cells. Colony number and tumor growth were significantly decreased in the A549/GR-shPrx II cells compared with the A549/GR cells. Our findings suggest that Prx II has an important role in cancer cell survival via the modulation of signaling molecules involved in apoptosis and the phosphorylation of JNK by the downregulation of ROS levels in A549/GR cells. PMID:26021759

  3. Repopulation of denuded tracheal grafts with alveolar type II cells

    International Nuclear Information System (INIS)

    Johnson, N.F.

    1988-01-01

    Repopulation of denuded heterotopic tracheal grafts with populations of specific epithelial cell types is one approach to study the differentiation potential of various cell types. This technique has been adopted to delineate the differentiation pathways of alveolar type II cells isolated from rat lungs. Under the conditions of this experiment, the reestablished epithelial lining was alveolar-like, however, ultrastructural analysis of the cells showed them to be like Clara cells. These preliminary results suggest that the secretary cells of the lung parenchyma and terminal airways may share a common ancestry. (author)

  4. [Metabotropic glutamate receptor 8 activation promotes the apoptosis of lung carcinoma A549 cells in vitro].

    Science.gov (United States)

    Li, Tian-Jiao; Huang, Yan-Hong; Chen, Xi; Zhou, Zhou; Luo, Si-Wei; Feng, Dan-Dan; Han, Jian-Zhong; Luo, Zi-Qiang

    2015-10-25

    This study aims to detect the expression of metabotropic glutamate receptors (mGluRs) in lung carcinoma A549 cells, and to investigate the effects of mGluR8 and mGluR4 activation on the growth of A549 cells in vitro. The mRNA expression levels of the 8 subtypes of mGluRs in A549 cells were determined by real-time PCR. Immunohistochemistry was used to analyze the protein expression of mGluR4 and mGluR8 in A549 cells and lung tissue sections obtained from lung adenocarcinoma patients. To observe the effects of mGluR8 and mGluR4 activation on the growth of A549 cells, the cultured cells were treated with (S)-3,4-DCPG (an agonist of mGluR8) and VU0155041 (an agonist of mGluR4), respectively, and then the cell viability was analyzed by CCK-8 kit, the percentage of DNA synthesis was detected by EdU incorporation, and the apoptosis of the cells was measured by hoechst 33258 staining and flow cytometry. The results showed that there were low expressions of mGluR1, mGluR5, mGluR6, mGluR7 mRNA, no expression of mGluR2 and mGluR3 mRNA, and high expressions of mGluR8 and mGluR4 mRNA in A549 cells. Accordingly, there were also mGluR4 and mGluR8 protein expressions in the A549 cells and the lung adenocarcinoma tissue sections. VU0155041 had no effect on the growth of A549 cells, but (S)-3,4-DCPG significantly decreased the cells' growth in a dose-dependent manner and increased the apoptosis of the cells. The results revealed a role of mGluR8 in the growth and apoptosis of A549 cells and suggested a potential target for clinical treatment of lung cancer.

  5. Oxymatrine inhibited cell proliferation by inducing apoptosis in human lung cancer A549 cells.

    Science.gov (United States)

    Wang, Baiyan; Han, Qianqian; Zhu, Yanqin

    2015-01-01

    To investigate the inhibition effect of oxymatrine induces human lung cancer A549 cells apoptosis. The A549 cells were cultured for 24 h, than the various concentration of oxymatrine (2 mmol/L, 4 mmol/L, 8 mmol/L, 15 mmol/L) were added into different experimental group cells, and 5-fluorouracil were added into the positive control group cells for 12 h, 24 h, 36 h, 48 h respectively. The A549 cells inhibition rate, apoptosis, and the expression of Bcl-2 and Bax were examined by MTT method, Annexin V/PI double staining method, real-time quantitative PCR and western blot, respectively. At same time, the morphological changes of A549 cells were observed with an inverted microscope. In the range of 2 mmol/L~15 mmol/L, oxymatrine had obvious inhibition effects on the proliferation of A549 cells. Compared with the negative control group, it has significantly different (PA549 cells were treated with 8 mmol/L oxymatrine for 24 h, the morphological change of cell apoptosis was observed and the extent of apoptosis was quantified by flow cytometry. Furthermore, the expression of Bcl-2 was reduced and the expression of Bax was increased remarkably (PA549 cells by regulating the expression of Bcl-2 and Bax.

  6. Inhibition of Plasminogen Activator Inhibitor-1 Attenuates Transforming Growth Factor-β-Dependent Epithelial Mesenchymal Transition and Differentiation of Fibroblasts to Myofibroblasts.

    Directory of Open Access Journals (Sweden)

    Keitaro Omori

    Full Text Available Transforming growth factor-β (TGF-β is central during the pathogenesis of pulmonary fibrosis, in which the plasminogen activator inhibitor-1 (PAI-1 also has an established role. TGF-β is also known to be the strongest inducer of PAI-1. To investigate the link between PAI-1 and TGF-β in fibrotic processes, we evaluated the effect of SK-216, a PAI-1-specific inhibitor, in TGF-β-dependent epithelial-mesenchymal transition (EMT and fibroblast to myofibroblast differentiation. In human alveolar epithelial A549 cells, treatment with TGF-β induced EMT, whereas co-treatment with SK-216 attenuated the occurrence of EMT. The inhibition of TGF-β-induced EMT by SK-216 was also confirmed in the experiment using murine epithelial LA-4 cells. Blocking EMT by SK-216 inhibited TGF-β-induced endogenous production of PAI-1 and TGF-β in A549 cells as well. These effects of SK-216 were not likely mediated by suppressing either Smad or ERK pathways. Using human lung fibroblast MRC-5 cells, we demonstrated that SK-216 inhibited TGF-β-dependent differentiation of fibroblasts to myofibroblasts. We also observed this inhibition by SK-216 in human primary lung fibroblasts. Following these in vitro results, we tested oral administration of SK-216 into mice injected intratracheally with bleomycin. We found that SK-216 reduced the degree of bleomycin-induced pulmonary fibrosis in mice. Although the precise mechanisms underlying the link between TGF-β and PAI-1 regarding fibrotic process were not determined, PAI-1 seems to act as a potent downstream effector on the pro-fibrotic property of TGF-β. In addition, inhibition of PAI-1 activity by a PAI-1 inhibitor exerts an antifibrotic effect even in vivo. These data suggest that targeting PAI-1 as a downstream effector of TGF-β could be a promising therapeutic strategy for pulmonary fibrosis.

  7. [Cancer-associated-fibroblasts regulate the chemoresistance of lung cancer cell line A549 via SDF-1 secretion].

    Science.gov (United States)

    Zou, F; Zhang, Z H; Zhang, Y T; Zhao, J Q; Zhang, X L; Wen, C L; Song, X Y; Zhou, W M

    2017-05-23

    Objective: To investigate whether cancer-associated- fibroblasts (CAF), the key component of tumor microenvironment, regulate the chemoresistant capacity of lung cancer cell line A549 through SDF-1 secretion. Methods: Primary cell isolation techniques was used to isolate cancer-associated-fibroblasts from lung cancer patients. MTT assay was applied to determine the proliferation and chemoresistance of A549 cells. Quantative PCR was used to detect the mRNA changes of Bcl-xL. Western blotting was used to detect the protein expression of Bcl-xL. ELISA was applied to detect the SDF-1 secretion from normal fibroblasts (NF) and CAF. Results: CAF promoted the proliferation of A549 cells, while NF had no significant effect on them. After 72 hrs incubation, the absorbance value of A549+ CAF medium group was 0.814±0.006, significantly different from the 0.753±0.006 of the A549+ NF medium group ( P A549 group, A549+ NF medium group and A549+ CAF medium group were 1.00±0.11, 1.10±0.09 and 3.50±0.30, respectively, showing a significant difference between the A549+ NF medium group and A549+ CAF medium group ( P A549 group, A549+ NF medium group and A549+ CAF medium group were 1.00±0.08, 1.10±0.12 and 3.10±0.25, respectively, with a significant difference between the A549+ NF medium group and A549+ CAF medium group ( P A549+ NF medium group and A549+ CAF medium group were 3.23±0.02 and 9.53±0.10, respectively, significantly different from each other ( P A549 group, A549+ AMD3100 group, A549+ NF medium group, A549+ NF medium+ AMD3100 group, A549+ CAF medium and A549+ CA Fmedium+ AMD3100 group were 0.43±0.03, 0.25±0.02, 0.48±0.03, 0.31±0.03, 0.72±0.06 and 0.45±0.03, respectively. The data of A549+ NF medium group was significantly different from that of A549+ CAF medium group ( P A549 cells through SDF-1 secretion, upregulating the expression level of Bcl-xL through interaction with CXCR4. Our study not only illustrates that tumor microenvironment is able to enhance

  8. Proteinosis alveolar pulmonar Pulmonary alveolar proteinosis

    Directory of Open Access Journals (Sweden)

    Concepción Sánchez Infante

    2011-12-01

    Full Text Available La proteinosis alveolar pulmonar es una enfermedad respiratoria crónica, caracterizada por alteración en el metabolismo del surfactante, lo que determina su acumulación anormal en el espacio alveolar. Es una enfermedad extremadamente rara. Se han reportado solamente 500 casos en la literatura. Se describió por primera vez en 1958. Se presenta un caso de proteinosis alveolar pulmonar en un lactante de 2 meses, con desnutrición proteico energética, que ingresa por dificultad respiratoria e hipoxemia, y, con imágenes radiológicas de tipo retículo-nodulillar, en vidrio deslustrado, en el cual se plantea inicialmente el diagnóstico de bronconeumonía. Ante la evolución desfavorable y no respuesta al tratamiento, se realizó un estudio para descartar enfermedades pulmonares crónicas. El paciente fallece y se confirma el diagnóstico por anatomía patológica. Se realiza una revisión del tema.The pulmonary alveolar proteinosis is a chronic respiratory disease characterized by surfactant metabolism alteration determining its abnormal accumulation in the alveolar space. It is a disease very rare and in literature only 500 cases have been reported; it was described for the first time in 1958. This is a case presentation of pulmonary alveolar proteinosis in an infant aged 2 months with energetic protein malnutrition admitted due to respiratory difficulty and hypoxemia and with radiologic images of the reticulonodulillary, in frosting glass, where initially is made the diagnosis of bronchopneumonia. In the face of unfavorable evolution and no response to treatment, a study was conducted to rule out chronic pulmonary diseases. Patient died confirming the diagnosis according to the pathologic anatomy. A review on subject is carried out.

  9. MiR-1244 sensitizes the resistance of non-small cell lung cancer A549 cell to cisplatin.

    Science.gov (United States)

    Li, Weili; Wang, Wenzhe; Ding, Mingjian; Zheng, Xiaoliang; Ma, Shenglin; Wang, Xiaoju

    2016-01-01

    Cisplatin (DDP)-based chemotherapy is the mainstay of first-line therapy for lung cancer. However, their efficacy is often limited by the existence or development of chemoresistance. The aim of this study was to find and investigate the function of miRNAs in cisplatin (DDP)-resistant non-small cell lung cancer (NSCLC) A549 cell. Quantitative real-time PCR assay was employed to compare the differences of miRNA expression in both cisplatin-resistant A549 (A549/DDP) cell and the parental A549 cell. The dysregulated miRNAs were then corrected by transfecting oligonucleotides into A549/DDP cells. The cellular sensitivity to cisplatin, cell apoptosis and migration were conducted by MTT, flow cytometry and cell wound healing assay, respectively. Both miR-589 and miR-1244 were significantly down-regulated in A549/DDP cell compared to the parental A549, while the expression of miR-182 and miR-224 were increased in A549/DDP cell (P A549/DDP cell. The study indicates a crucial role of miR-1244 in the progress of cisplatin resistance of A549. Further understanding of miR-1244-mediated signaling pathways may promote the clinical use of miR-1244 in lung cancer therapy.

  10. Xylitol induces cell death in lung cancer A549 cells by autophagy.

    Science.gov (United States)

    Park, Eunjoo; Park, Mi Hee; Na, Hee Sam; Chung, Jin

    2015-05-01

    Xylitol is a widely used anti-caries agent that has anti-inflammatory effects. We have evaluated the potential of xylitol in cancer treatment. It's effects on cell proliferation and cytotoxicity were measured by MTT assay and LDH assay. Cell morphology and autophagy were examined by immunostaining and immunoblotting. Xylitol inhibited cell proliferation in a dose-dependent manner in these cancer cells: A549, Caki, NCI-H23, HCT-15, HL-60, K562, and SK MEL-2. The IC50 of xylitol in human gingival fibroblast cells was higher than in cancer cells, indicating that it is more specific for cancer cells. Moreover, xylitol induced autophagy in A549 cells that was inhibited by 3-methyladenine, an autophagy inhibitor. These results indicate that xylitol has potential in therapy against lung cancer by inhibiting cell proliferation and inducing autophagy of A549 cells.

  11. Chemosensitivity of irradiated resistant cells of multicellular spheroids in A549 lung adenocarcinoma

    International Nuclear Information System (INIS)

    Shi Degang; Shi Genming; Huang Gang

    2006-01-01

    Objective: To investigate the chemosensitivity of irradiated resistant cells of multicellular spheroids in A549 lung adenocarcinoma. Methods: The A549 irradiated resistant cells were the 10th regrowth generations after irradiated with 2.5 Gy of 6 MV X-ray, the control groups were A549 parent cells and MCFY/VCR resistant cells. The 6 kinds of chemotherapeutic drugs were DDP, VDS, 5-FU, HCP, MMC and ADM respectively, with verapamil (VPL) as reverse agent. The treatment effect was compared with MTT assay, and the multidrug resistant gene expressions of mdrl and MRP were measured with RT-PCR method. Results: A549 cells and irradiated resistant cells were resistant to DDP, but sensitivity to VDS,5-FU, HCP, MMC and ADM. The inhibitory rates of VPL to the above two cells were 98% and 25% respectively(P 2 -MG and MRP/β 2 -MG of all A549 cells were about 0 and 0.7 respectively, and those of MCFT/VCR cells were 35 and 4.36. Conclusion: The chemosensitivity of A549 irradiated resistant cells had not changed markedly, the decreased sensitivity to VPL could not be explained by the gene expression of mdrl and MRP. It is conferred that some kinds of changes in the cell membrane and decreased regrowth ability to result in resistance. Unlike multidrug resistance induced by chemotherapy, VPL may be not an ideal reverser to irradiated resistant cells. The new kinds of biological preparation should be sought to combine chemotherapy to treat recurring tumor with irradiated resistance. (authors)

  12. STIM1 silencing inhibits the migration and invasion of A549 cells

    OpenAIRE

    Wang, Yadong; Wang, Haiyu; Pan, Teng; Li, Li; Li, Jiangmin; Yang, Haiyan

    2017-01-01

    The present study aimed to explore the effects of stromal interaction molecule 1 (STIM1) knockdown on the migration, invasion and metastasis of A549 cells in vitro and in vivo. Western blotting and immunohistochemistry were used to detect protein expression levels. Wound healing and Transwell invasion assays were used to assess the migratory and invasive abilities of A549 cells transfected with STIM1-specific short hairpin (sh)RNA (shSTIM1). In addition, a tail vein metastatic assay was perfo...

  13. Isolation and characterization of erlotinib-resistant human non-small cell lung cancer A549 cells

    OpenAIRE

    IKEDA, RYUJI; VERMEULEN, LEE C.; LAU, ELIM; JIANG, ZHISHENG; KAVANAUGH, SHANNON M.; YAMADA, KATSUSHI; KOLESAR, JILL M.

    2010-01-01

    Erlotinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, is an effective therapy for non-small cell lung cancer (NSCLC). However, resistance to erlotinib reduces its efficacy. To investigate the basis of erlotinib resistance, we isolated erlotinib-resistant human NSCLC A549 cells, termed A549/ER cells. The A549/ER cells were found to be resistant to erlotinib, as well as paclitaxel and gemcitabine. We then performed a PCR array to investigate the resistance to erlotini...

  14. Knockdown of c‑Myc activates Fas-mediated apoptosis and sensitizes A549 cells to radiation.

    Science.gov (United States)

    Zhang, Jing; Zhou, Ling; Nan, Zhaodi; Yuan, Qing; Wen, Jie; Xu, Maolei; Li, Youjie; Li, Baosheng; Wang, Pingyu; Liu, Changmin; Ma, Ying; Chen, Shaoshui; Xie, Shuyang

    2017-10-01

    Several studies have demonstrated that cancer radiosensitivity is associated with the deregulation of c‑Myc, but the relationship between c‑Myc and Fas in radioresistance of lung adenocarcinoma remains unclear. In this study, we established radiation-resistant A549 cell model (A549/R), and investigated the roles of c‑Myc and Fas in radiation-induced cytotoxicity of A549 cells. Apoptosis detection showed that there were fewer apoptotic cells in A549/R cells treated with radiation than in A549 cells. Western blotting results demonstrated the inverse expression pattern of c‑Myc and Fas in A549 and A549/R cells. Suppression of c‑Myc expression by small interfering RNA (siRNA) displayed enhancement of Fas-mediated apoptosis in A549/R cells, accompanying a significant decrease of Bid, Bcl‑2, pro‑caspase‑8, -9 and -3 and increase of Bax. In contrast, Fas-mediated apoptosis was attenuated while Fas expression was suppressed by ectopic expression of c‑Myc in A549 cells. Moreover, decreased cell viability and increased induction of apoptosis were observed in A549/R cells followed by combinational treatment of c‑Myc siRNA and irradiation, whereas, upregulation of c‑Myc reduced the sensitivity of A549 cells to irradiation. These results indicated that c‑Myc and Fas regulated the sensitivity of A549 cells to irradiation by regulating caspase‑8-mediated Bid activation and the subsequent association with the mitochondrial pathway of apoptosis.

  15. Overexpression of the hydatidiform mole-related gene F10 inhibits apoptosis in A549 cells through downregulation of BCL2-associated X protein and caspase-3

    OpenAIRE

    SONG, YALI; ZHANG, GONG; ZHU, XIULAN; PANG, ZHANJUN; XING, FUQI; QUAN, SONG

    2012-01-01

    The aim of this study was to investigate how the overexpression of the hydatidiform mole-related gene F10 affects apoptosis in human lung cancer A549 cells. A549 cells were transfected with pEGFP-N1-F10 (A549-F10) or pEGFP-N1 empty vector (A549-empty). Untransfected A549, A549-F10 or A549-empty cells were examined using the MTT cell proliferation assay and the TUNEL-FITC/Hoechst 33258 apoptosis assay. Western blotting was used to examine the expression levels of the pro-apoptotic genes, BCL2-...

  16. Enhanced Replication of Hepatitis E Virus Strain 47832c in an A549-Derived Subclonal Cell Line.

    Science.gov (United States)

    Schemmerer, Mathias; Apelt, Silke; Trojnar, Eva; Ulrich, Rainer G; Wenzel, Jürgen J; Johne, Reimar

    2016-09-29

    Hepatitis E virus (HEV) is a human pathogen with increasing importance. The lack of efficient cell culture systems hampers systematic studies on its replication cycle, virus neutralization and inactivation. Here, several cell lines were inoculated with the HEV genotype 3c strain 47832c, previously isolated from a chronically infected transplant patient. At 14 days after inoculation the highest HEV genome copy numbers were found in A549 cells, followed by PLC/PRF/5 cells, whereas HepG2/C3A, Huh-7 Lunet BLR and MRC-5 cells only weakly supported virus replication. Inoculation of A549-derived subclone cell lines resulted in most cases in reduced HEV replication. However, the subclone A549/D3 was susceptible to lower virus concentrations and resulted in higher virus yields as compared to parental A549 cells. Transcriptome analysis indicated a downregulation of genes for carcinoembryonic antigen-related cell adhesion molecules ( CEACAM ) 5 and 6 , and an upregulation of the syndecan 2 ( SDC2 ) gene in A549/D3 cells compared to A549 cells. However, treatment of A549/D3 cells or A549 cells with CEACAM- or syndecan 2-specific antisera did not influence HEV replication. The results show that cells supporting more efficient HEV replication can be selected from the A549 cell line. The specific mechanisms responsible for the enhanced replication remain unknown.

  17. Tamarind seed coat ameliorates fluoride induced cytotoxicity, oxidative stress, mitochondrial dysfunction and apoptosis in A549 cells.

    Science.gov (United States)

    Ameeramja, Jaishabanu; Panneerselvam, Lakshmikanthan; Govindarajan, Vimal; Jeyachandran, Sivakamavalli; Baskaralingam, Vaseeharan; Perumal, Ekambaram

    2016-01-15

    Fluoride (F) is an environmental contaminant and industrial pollutant. Molecular mechanisms remain unclear in F induced pulmonary toxicity even after numerous studies. Tamarind fruits act as defluoridating agents, but no study was conducted in in vitro systems. Hence, we aimed to assess the ameliorative impact of the tamarind seed coat extract (TSCE) against F toxicity utilizing lung epithelial cells, A549. Cells were exposed to sodium fluoride (NaF-5 mM) alone and in combination with TSCE (750 ng/ml) or Vitamin C (positive control) for 24 h and analyzed for F content, intracellular calcium ([Ca(2+)]i) level, oxidative stress, mitochondrial integrity and apoptotic markers. TSCE treatment prevented the F induced alterations in [Ca(2+)]i overload, F content, oxidant (reactive oxygen species generation, lipid peroxidation, protein carbonyl content and nitric oxide) and antioxidant (superoxide dismutase, catalase, glutathione peroxidase and glutathione) parameters. Further, TSCE modulates F activated changes in mitochondrial membrane potential, permeability transition pore opening, cytochrome-C release, Bax/Bcl-2 ratio, caspase-3 and PARP-1 expressions. In conclusion, our study demonstrated that TSCE as a potential protective agent against F toxicity, which can be utilized as a neutraceutical. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Cyto- and genotoxicity assessment of Gold nanoparticles obtained by laser ablation in A549 lung adenocarcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Bucchianico, Sebastiano Di [Karolinska Institutet, Institute of Environmental Medicine (Sweden); Migliore, Lucia [University of Pisa, Department of Translational Research and New Technologies in Medicine and Surgery, Division of Medical Genetics (Italy); Marsili, Paolo [Institute of Complex Systems (ISC-CNR) (Italy); Vergari, Chiara [Plasma Diagnostics and Technologies s.r.l. (Italy); Giammanco, Francesco [University of Pisa, Department of Physics “E. Fermi” (Italy); Giorgetti, Emilia, E-mail: emilia.giorgetti@fi.isc.cnr.it [Institute of Complex Systems (ISC-CNR) (Italy)

    2015-05-15

    Gold nanoparticles have attracted enormous interest in biomedical applications, based on their unique optical properties. However, their toxicity on human tissues is still an open issue. Beyond the potential intrinsic toxicity of nanostructured gold, a non-negligible contribution of stabilizers or reaction by-products related to current wet chemical synthesis procedures can be expected. Aimed at isolating gold contribution from that of any other contaminant, we produced colloidal suspensions of Gold nanoparticles having average size <10 nm in deionized water or acetone by pulsed laser ablation, that permits preparation of uncoated and highly stable Gold nanoparticles in pure solvents. Subsequently, we investigated the role of surface chemistry, size, and dispersivity of synthesized Gold nanoparticles in exerting toxicity in a cell model system of deep respiratory tract, representing the main route of exposure to NPs, namely adenocarcinoma epithelial A549 cells. Gold nanoparticles prepared in water showed no particular signs of cytotoxicity, cytostasis, and/or genotoxicity as assessed by MTT colorimetric viability test and Cytokinesis-block micronucleus cytome assay up to concentrations of the order of 5 μg/mL. In contrast, Gold nanoparticles produced in pure acetone and then transferred into deionized water showed impaired cell viability, apoptosis responses, micronuclei, and dicentric chromosomes induction as well as nuclear budding, as a function of the amount of surface contaminants like amorphous carbon and enolate ions.

  19. Cyto- and genotoxicity assessment of Gold nanoparticles obtained by laser ablation in A549 lung adenocarcinoma cells

    International Nuclear Information System (INIS)

    Bucchianico, Sebastiano Di; Migliore, Lucia; Marsili, Paolo; Vergari, Chiara; Giammanco, Francesco; Giorgetti, Emilia

    2015-01-01

    Gold nanoparticles have attracted enormous interest in biomedical applications, based on their unique optical properties. However, their toxicity on human tissues is still an open issue. Beyond the potential intrinsic toxicity of nanostructured gold, a non-negligible contribution of stabilizers or reaction by-products related to current wet chemical synthesis procedures can be expected. Aimed at isolating gold contribution from that of any other contaminant, we produced colloidal suspensions of Gold nanoparticles having average size <10 nm in deionized water or acetone by pulsed laser ablation, that permits preparation of uncoated and highly stable Gold nanoparticles in pure solvents. Subsequently, we investigated the role of surface chemistry, size, and dispersivity of synthesized Gold nanoparticles in exerting toxicity in a cell model system of deep respiratory tract, representing the main route of exposure to NPs, namely adenocarcinoma epithelial A549 cells. Gold nanoparticles prepared in water showed no particular signs of cytotoxicity, cytostasis, and/or genotoxicity as assessed by MTT colorimetric viability test and Cytokinesis-block micronucleus cytome assay up to concentrations of the order of 5 μg/mL. In contrast, Gold nanoparticles produced in pure acetone and then transferred into deionized water showed impaired cell viability, apoptosis responses, micronuclei, and dicentric chromosomes induction as well as nuclear budding, as a function of the amount of surface contaminants like amorphous carbon and enolate ions

  20. In vitro effects of nicotine on the non-small-cell lung cancer line A549.

    Science.gov (United States)

    Gao, Tao; Zhou, Xue-Liang; Liu, Sheng; Rao, Chang-Xiu; Shi, Wen; Liu, Ji-Chun

    2016-04-01

    To investigate in vitro effects of nicotine on the non-small-cell lung cancer line A549. The case-control study was conducted at the First Affiliated Hospital of Nanchang University from 1st January to 30th June, 2014 and comprised A549 cells which were treated with a series of concentrations of nicotine (0.01 µM, 0.1 µM, 1 µM and 10 µM) for 24 hours. Control cells were incubated under the same conditions without the addition of nicotine. Cell growth was detected by monotetrazolium salt [3-(4, 5-dimethyl-2-thiazolyl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay. Cell apoptosis was detected by Haematoxylin and Eosin staining, immunofluorescence analysis of Filamentous actin and electron microscope observation. Nicotine had no significant effect on A549 cell growth at the dose of 0.01µM (p>0.05), but had significant growth inhibitory effects at the doses of 0.1µM, 1µM and 10µM (pA549 cells were found to be dose-dependent.

  1. Cold stress increases reactive oxygen species formation via TRPA1 activation in A549 cells.

    Science.gov (United States)

    Sun, Wenwu; Wang, Zhonghua; Cao, Jianping; Cui, Haiyang; Ma, Zhuang

    2016-03-01

    Reactive oxygen species (ROS) are responsible for lung damage during inhalation of cold air. However, the mechanism of the ROS production induced by cold stress in the lung is still unclear. In this work, we measured the changes of ROS and the cytosolic Ca(2+) concentration ([Ca(2+)]c) in A549 cell. We observed that cold stress (from 20 to 5 °C) exposure of A549 cell resulted in an increase of ROS and [Ca(2+)]c, which was completely attenuated by removing Ca(2+) from medium. Further experiments showed that cold-sensing transient receptor potential subfamily member 1 (TRPA1) agonist (allyl isothiocyanate, AITC) increased the production of ROS and the level of [Ca(2+)]c in A549 cell. Moreover, HC-030031, a TRPA1 selective antagonist, significantly inhibited the enhanced ROS and [Ca(2+)]c induced by AITC or cold stimulation, respectively. Taken together, these data demonstrated that TRPA1 activation played an important role in the enhanced production of ROS induced by cold stress in A549 cell.

  2. Exogenous p53 upregulated modulator of apoptosis (PUMA) decreases growth of lung cancer A549 cells.

    Science.gov (United States)

    Liu, Chun-Ju; Zhang, Xia-Li; Luo, Da-Ya; Zhu, Wei-Feng; Wan, Hui-Fang; Yang, Jun-Ping; Yang, Xiao-Jun; Wan, Fu-Sheng

    2015-01-01

    To investigate the influence of exogenous p53 upregulated modulator of apoptosis (PUMA) expression on cell proliferation and apoptosis in human non-small cell lung cancer A549 cells and transplanted tumor cell growth in nude mice. A549 cells were divided into the following groups: control, non- carrier (NC), PUMA (transfected with pCEP4- (HA) 2-PUMA plasmid), DDP (10 μg/mL cisplatin treatment) and PUMA+DDP (transfected with pCEP4-(HA)2-PUMA plasmid and 10 μg/mL cisplatin treatment). The MTT method was used to detect the cell survival rate. Cell apoptosis rates were measured by flow cytometry, and PUMA, Bax and Bcl-2 protein expression levels were measured by Western blotting. Compared to the control group, the PUMA, DDP and PUMA+DDP groups all had significantly decreased A549 cell proliferation (pPUMA+DDP group. Conversely, the apoptosis rates of the three groups were significantly increased (PPUMA and DDP treatments were synergistic. Moreover, Bax protein levels significantly increased (pPUMA+DDP group was significantly higher than in the single DDP or PUMA groups. Exogenous PUMA effectively inhibited lung cancer A549 cell proliferation and transplanted tumor growth by increasing Bax protein levels and reducing Bcl-2 protein levels.

  3. Antitumor and apoptotic effects of cucurbitacin a in A-549 lung ...

    African Journals Online (AJOL)

    Background: The main aim of this study was to demonstrate the antitumor potential of cucurbitacin A on A-549 NSCLC (non-small cell lung cancer cells). The effects of Cucurbitacin A on apoptotic induction, cell physic, cell cycle failure and m-TOR/PI3K/Akt signalling pathway were also investigated in the present study.

  4. Evaluation of a549 as a new vaccine cell substrate: digging deeper with massively parallel sequencing.

    Science.gov (United States)

    Shabram, Paul; Kolman, John L

    2014-01-01

    In the past three decades, the use of tumorigenic cell substrates has been the topic of five Vaccine and Related Biological Products Advisory Committee (VRBPAC) meetings, including a review of the A549 cell line in September 2012. Over that period of time, major technological advances in biotechnology have improved our ability to assess the risk associated with using a tumorigenic cell line. As part of the September 2012 review, we assessed the history of A549 cells and evaluated the probable transforming event based on patterns of mutations to cancer genes. In addition, massively parallel sequencing was used to first screen then augment the characterization of A549 cells by searching for the presence of hidden viral threats using sequencing of the entire cellular transcriptome and comparing sequences to a curated viral sequence database. Based upon the combined results of next-generation sequencing technology along with standard cell characterization as outlined in published regulatory guidances, we believe that A549 cells pose no more risk than any other cell substrate for the manufacture of vaccines. © PDA, Inc. 2014.

  5. Α-MMC and MAP30, two ribosome-inactivating proteins extracted from Momordica charantia, induce cell cycle arrest and apoptosis in A549 human lung carcinoma cells.

    Science.gov (United States)

    Fan, Xiang; He, Lingli; Meng, Yao; Li, Gangrui; Li, Linli; Meng, Yanfa

    2015-05-01

    α‑Momorcharin (α‑MMC) and momordica anti‑human immunodeficiency virus protein (MAP30), produced by Momordica charantia, are ribosome‑inactivating proteins, which have been reported to exert inhibitory effects on cultured tumor cells. In order to further elucidate the functions of these agents, the present study aimed to investigate the effects of α‑MMC and MAP30 on cell viability, the induction of apoptosis, cell cycle arrest, DNA integrity and superoxide dismutase (SOD) activity. α‑MMC and MAP30 were purified from bitter melon seeds using ammonium sulfate precipitation in combination with sulfopropyl (SP)‑sepharose fast flow, sephacryl S‑100 and macro‑Cap‑SP chromatography. MTT, flow cytometric and DNA fragmentation analyses were then used to determine the effects of α‑MMC and MAP30 on human lung adenocarcinoma epithelial A549 cells. The results revealed that A549 cells were sensitive to α‑MMC and MAP30 cytotoxicity assays in vitro. Cell proliferation was significantly suppressed following α‑MMC and MAP30 treatment in a dose‑ and time‑dependent manner; in addition, the results indicated that MAP30 had a more potent anti‑tumor activity compared with that of α‑MMC. Cell cycle arrest in S phase and a significantly increased apoptotic rate were observed following treatment with α‑MMC and MAP30. Furthermore, DNA integrity analysis revealed that the DNA of A549 cells was degraded following treatment with α‑MMC and MAP30 for 48 h. The pyrogallol autoxidation method and nitrotetrazolium blue chloride staining were used to determine SOD activity, the results of which indicated that α‑MMC and MAP30 did not possess SOD activity. In conclusion, the results of the present study indicated that α‑MMC and MAP30 may have potential as novel therapeutic agents for the prophylaxis and treatment of cancer.

  6. Alveolar but not intravenous S-ketamine inhibits alveolar sodium transport and lung fluid clearance in rats

    NARCIS (Netherlands)

    Berger, Marc M.; Pitzer, Bernhard; Zügel, Stefanie; Wieland, Catharina W.; Vlaar, Alexander P.; Schultz, Marcus J.; Dahan, Albert; Bärtsch, Peter; Hollmann, Markus W.; Mairbäurl, Heimo

    2010-01-01

    BACKGROUND: S-ketamine is frequently used for analgosedation, especially during sepsis and cardiovascular instability. Because S-ketamine blocks voltage-gated sodium (Na+) channels in neurons and skeletal muscle, it is conceivable that S-ketamine also blocks alveolar epithelial Na+ channels that are

  7. Role of gambogic acid and NaI131in A549/DDP cells.

    Science.gov (United States)

    Huang, Jing; Zhu, Xiaoli; Wang, Huan; Han, Shuhua; Liu, Lu; Xie, Yan; Chen, Daozhen; Zhang, Qiang; Zhang, Li; Hu, Yue

    2017-01-01

    Resistance to platinum in tumor tissue is a considerable barrier against effective lung cancer treatment. Radionuclide therapy is the primary adjuvant treatment, however, the toxic side effects limit its dosage in the clinical setting. Therefore, the present study aimed to determine whether an NaI 131 radiosensitizer could help reduce the toxic side effects of radionuclide therapy. In vitro experiments were conducted to determine whether NaI 131 can inhibit platinum resistance in A549/DDP cells, which are cisplatin-resistant non-small cell lung cancer cells, and whether gambogic acid (GA) is an effective NaI 131 radiosensitizer. Cell proliferation following drug intervention was analyzed using MTT and isobolographic analysis. Apoptosis was assessed by flow cytometry. In addition, the mechanisms of drug intervention were analyzed by measuring the expression of P-glycoprotein (P-gP), B cell lymphoma 2 (Bcl-2), Bcl2-associated X protein (Bax) and P53 using western blot analysis and immunocytochemistry. According to isobolographic analysis, a low concentration of NaI 131 combined with GA had a synergistic effect on the inhibition of A549/DDP cell proliferation, which was consistent with an increased rate of apoptosis. Furthermore, the overexpression of Bax, and the downregulation of P-gP, P53 and Bcl-2 observed demonstrated the potential mechanism(s) of NaI 131 and GA intervention. NaI 131 may induce apoptosis in A549/DDP cells by regulating apoptosis-related proteins. A low concentration combination of NaI 131 and GA was able to significantly inhibit A549/DDP cell proliferation and induce cell apoptosis. Thus, the two drugs appear to have a synergistic effect on apoptosis of A549/DDP cells.

  8. Curcumin induced autophagy anticancer effects on human lung adenocarcinoma cell line A549.

    Science.gov (United States)

    Liu, Furong; Gao, Song; Yang, Yuxuan; Zhao, Xiaodan; Fan, Yameng; Ma, Wenxia; Yang, Danrong; Yang, Aimin; Yu, Yan

    2017-09-01

    To investigate the anticancer effects of curcumin-induced autophagy and its effects on the human lung adenocarcinoma A549 cell line, inverted phase contrast microscopy was used to observe alterations to the cytomorphology of cells. An MTT assay was used to measure cell viability. Autophagy was detected using acridine orange (AO) staining and 3-methyladenine (3-MA) was used as an autophagy-specific inhibitor. Dose- and time-dependent A549 cell viability inhibition was observed following curcumin treatment. A dose-dependent increase in the red fluorescent structures in A549 cells was identified following curcumin treatment for 48 h through AO staining. In addition, the activation of autophagy was determined through changes in the number of autophagic vesicles (AVs; fluorescent particles) infected with monodansylcadaverine (MDC). The fluorescence intensity and density of AVs in the curcumin-treated groups were higher at 48 h compared with the control group. Finally, the MTT assay demonstrated that the survival rates of the curcumin-treated cells were increased when pretreated with 3-MA for 3 h, indicating that the inhibitory effect of curcumin on A549 cells is reduced following the inhibition of autophagy. Furthermore, AO and MDC staining confirmed that 3-MA does inhibit the induction of autophagy. Thus, it was hypothesized that the induction of autophagy is partially involved in the reduction of cell viability observed following curcumin treatment. The anticancer effects of curcumin on A549 cells can be reduced using autophagy inhibitors. This suggests a possible cancer therapeutic application of curcumin through the activation of autophagy. These findings have improved the understanding of the mechanism underlying the anticancer property of curcumin.

  9. Resveratrol raisesin vitroanticancer effects of paclitaxel in NSCLC cell line A549 through COX-2 expression.

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    Kong, Fanhua; Zhang, Runqi; Zhao, Xudong; Zheng, Guanlin; Wang, Zhou; Wang, Peng

    2017-09-01

    The aim of this study was to determine the raising anticancer effects of resveratrol (Res) on paclitaxel (PA) in non-small cell lung cancer (NSCLC) cell line A549. The 10 µg/ml of Res had no effect on human fetal lung fibroblast MRC-5 cells or on A549 cancer cells and the 5 or 10 µg/ml of PA also had no effect on MRC-5 normal cells. PA-L (5 µg/ml) and PA-H (10 µg/ml) had the growth inhibitory effects in NSCLC cell line A549, and Res increased these growth inhibitory effects. By flow cytometry experiment, after Res (5 µg/ml)+PA-H (10 µg/ml) treatment, the A549 cells showed the most apoptosic cells compared to other group treatments, and after additional treatment with Res, the apoptosic cells of both two PA concentrations were raised. Res+PA could reduce the mRNA and protein expressions of COX-2, and Res+PA could reduce the COX-2 related genes of VEGF, MMP-1, MMP-2, MMP-9, NF-κB, Bcl-2, Bcl-xL, procollagen I, collagen I, collagen III and CTGF, TNF-α, IL-1β, iNOS and raise the TIMP-1, TIMP-2, TIMP-3, IκB-α, p53, p21, caspase-3, caspase-8, caspase-9, Bax genes compared to the control cells and the PA treated cells. From these results, it can be suggested that Res could raise the anticancer effects of PA in A549 cells, thus Res might be used as a good sensitizing agent for PA.

  10. Synergistic anticancer effects of andrographolide and paclitaxel against A549 NSCLC cells.

    Science.gov (United States)

    Yuan, Huihui; Sun, Bo; Gao, Feng; Lan, Minbo

    2016-11-01

    Paclitaxel (PTX) is widely used in chemotherapy for cancer treatment; however, it has some serious side effects. Andrographolide (Andro) is a potential cancer therapeutic agent isolated from Andrographis paniculata (Burm. f.) Nees (Acanthaceae). The objective of this study is to evaluate the effects of PTX combined with Andro against A549 cells. The effects of 24-48 h treatment with 0.48-60.75 nM PTX and 5.10-328.0 μM Andro on cellular proliferation, apoptosis, cell cycle and intracellular reactive oxygen species (ROS) were determined by sulphorhodamine B assay, Annexin V-FITC/PI apoptosis detection, PI staining and ROS assay, respectively. Synergy was determined using combination index. The antitumour efficacy of 20 mg/kg PTX with 100 mg/kg Andro was studied in a xenograft murine model. IC 50 value of the PTX combined with Andro against A549 cells was 0.5-7.4 nM, which was significantly lower than that of PTX (15.9 nM). PTX with 10 μM Andro caused (1.22-1.27)-fold apoptosis and 1.7-fold ROS accumulation compared with PTX alone. N-Acetylcysteine, a ROS scavenger, blocked this synergy in vitro. In contrast, G2/M phase cell cycle arrest resulting from PTX was not potentiated by Andro. Moreover, PTX in combination with Andro inhibited the growth of A549 transplanted tumours by 98%. The results indicate that the combination of PTX and Andro exert significant synergistic anticancer effect on A549 cells in vitro and in vivo. The synergy may be the result of the accumulation of ROS. The combination of Andro and PTX represents a potential strategy for the treatment of A549 cells.

  11. Green tea extract induces protective autophagy in A549 non-small lung cancer cell line

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    Magdalena Izdebska

    2015-12-01

    Full Text Available Background and objectives: For many decades, polyphenols, including green tea extract catechins, have been reported to exert multiple anti-tumor activities. However, to date the mechanisms of their action have not been completely elucidated. Thus, the aim of this study was to assess the effect of green tea extract on non-small lung cancer A549 cells. Material and methods: A549 cells following treatment with GTE were analyzed using the inverted light and fluorescence microscope. In order to evaluate cell sensitivity and cell death, the MTT assay and Tali image-based cytometer were used, respectively. Ultrastructural alterations were assessed using a transmission electron microscope.Results: The obtained data suggested that GTE, even at the highest dose employed (150 μM, was not toxic to A549 cells. Likewise, the treatment with GTE resulted in only a very small dose-dependent increase in the population of apoptotic cells. However, enhanced accumulation of vacuole-like structures in response to GTE was seen at the light and electron microscopic level. Furthermore, an increase in the acidic vesicular organelles and LC3-II puncta formation was observed under the fluorescence microscope, following GTE treatment. The analysis of the functional status of autophagy revealed that GTE-induced autophagy may provide self-protection against its own cytotoxicity, since we observed that the blockage of autophagy by bafilomycin A1 decreased the viability of A549 cells and potentiated necrotic cell death induction in response to GTE treatment.Conclusion: Collectively, our results revealed that A549 cells are insensitive to both low and high concentrations of the green tea extract, probably due to the induction of cytoprotective autophagy. These data suggest that a potential utility of GTE in lung cancer therapy may lie in its synergistic combinations with drugs or small molecules that target autophagy, rather than in monotherapy.

  12. ATM activation accompanies histone H2AX phosphorylation in A549 cells upon exposure to tobacco smoke

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    Traganos Frank

    2007-06-01

    Full Text Available Abstract Background In response to DNA damage or structural alterations of chromatin, histone H2AX may be phosphorylated on Ser139 by phosphoinositide 3-kinase related protein kinases (PIKKs such as ataxia telangiectasia mutated (ATM, ATM-and Rad-3 related (ATR kinase, or by DNA dependent protein kinase (DNA-PKcs. When DNA damage primarily involves formation of DNA double-strand breaks (DSBs, H2AX is preferentially phosphorylated by ATM rather than by the other PIKKs. We have recently reported that brief exposure of human pulmonary adenocarcinoma A549 cells or normal human bronchial epithelial cells (NHBE to cigarette smoke (CS induced phosphorylation of H2AX. Results We report here that H2AX phosphorylation in A549 cells induced by CS was accompanied by activation of ATM, as revealed by ATM phosphorylation on Ser1981 (ATM-S1981P detected immunocytochemically and by Western blotting. No cell cycle-phase specific differences in kinetics of ATM activation and H2AX phosphorylation were observed. When cells were exposed to CS from cigarettes with different tobacco and filter combinations, the expression levels of ATM-S1981P correlated well with the increase in expression of phosphorylated H2AX (γH2AX (R = 0.89. In addition, we note that while CS-induced γH2AX expression was localized within discrete foci, the activated ATM was distributed throughout the nucleoplasm. Conclusion These data implicate ATM as the PIKK that phosphorylates H2AX in response to DNA damage caused by CS. Based on current understanding of ATM activation, expression and localization, these data would suggest that, in addition to inducing potentially carcinogenic DSB lesions, CS may also trigger other types of DNA lesions and cause chromatin alterations. As checkpoint kinase (Chk 1, Chk2 and the p53 tumor suppressor gene are known to be phosphorylated by ATM, the present data indicate that exposure to CS may lead to their phosphorylation, with the downstream consequences

  13. Effects of kinase insert domain receptor (KDR) gene silencing on the sensitivity of A549 cells to erlotinib.

    Science.gov (United States)

    Zhu, W L; Liu, Y H

    2015-11-25

    We investigated the effects of kinase insert domain receptor (KDR) gene silencing on the proliferation of A549 cells and their sensitivity to erlotinib. A KDR small interfering RNA (siRNA) sequence was designed and synthesized; then, it was transfected into A549 cells using Lipofectamine(TM) 2000. KDR mRNA and protein expression after KDR gene silencing was detected by reverse transcription polymerase chain reaction and western blotting; the A549 cell cycle was detected by flow cytometry. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and colony formation assay were performed to determine the sensitivity of A549 cells to erlotinib after KDR gene silencing. After 48h of KDR gene silencing, there was a significant decrease in KDR gene and protein expression (P A549 cell cycle was arrested at the G0/G1 phase, and the number of cells in the S phase decreased; the difference was statistically significant (P A549 cells to erlotinib was significantly enhanced (P A549 cells, inhibit the proliferation of A549 cells, and enhance their sensitivity to erlotinib.

  14. Melatonin inhibits the migration of human lung adenocarcinoma A549 cell lines involving JNK/MAPK pathway.

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    Qiaoyun Zhou

    Full Text Available OBJECTIVE: Melatonin, an indolamine produced and secreted predominately by the pineal gland, exhibits a variety of physiological functions, possesses antioxidant and antitumor properties. But, the mechanisms for the anti-cancer effects are unknown. The present study explored the effects of melatonin on the migration of human lung adenocarcinoma A549 cells and its mechanism. METHODS: MTT assay was employed to measure the viability of A549 cells treated with different concentrations of melatonin. The effect of melatonin on the migration of A549 cells was analyzed by wound healing assay. Occludin location was observed by immunofluorescence. The expression of occludin, osteopontin (OPN, myosin light chain kinase (MLCK and phosphorylation of myosin light chain (MLC, JNK were detected by western blots. RESULTS: After A549 cells were treated with melatonin, the viability and migration of the cells were inhibited significantly. The relative migration rate of A549 cells treated with melatonin was only about 20% at 24 h. The expression level of OPN, MLCK and phosphorylation of MLC of A549 cells were reduced, while the expression of occludin was conversely elevated, and occludin located on the cell surface was obviously increased. The phosphorylation status of JNK in A549 cells was also reduced when cells were treated by melatonin. CONCLUSIONS: Melatonin significantly inhibits the migration of A549 cells, and this may be associated with the down-regulation of the expression of OPN, MLCK, phosphorylation of MLC, and up-regulation of the expression of occludin involving JNK/MAPK pathway.

  15. ITRAQ-Based Proteomics Analysis of Triptolide On Human A549 Lung Adenocarcinoma Cells.

    Science.gov (United States)

    Li, Fangqiong; Zhao, Dongxiao; Yang, Suwen; Wang, Juan; Liu, Qin; Jin, Xin; Wang, Wei

    2018-01-01

    Triptolide (TP) is a diterpenoid triepoxide extracted from the traditional Chinese medical herb Tripterygium wilfordii that exerts prominent broad-spectrum anticancer activity to repress proliferation and induce cancer cell apoptosis through various molecular pathways. We previously observed that TP inhibits the progression of A549 cells and pancreatic cancer cells (PNCA-1) in vitro. However, the complex molecular mechanism underlying the anticancer activity of TP is not well understood. To explore the molecular mechanisms by which TP induces lung cancer cell apoptosis, we investigated changes in the protein profile of A549 cells treated with TP using a proteomics approach (iTRAQ [isobaric tags for relative and absolute quantitation] combined with NanoLC-MS/MS [nano liquid chromatography-mass spectrometry]). Changes in the profiles of the expressed proteins were analyzed using the bioinformatics tools OmicsBean and the Kyoto Encyclopedia of Genes and Genomes (KEGG) and were verified using western blotting. Apoptosis and cell cycle effects were analyzed using flow cytometry. TP induced apoptosis in A549 cells and blocked A549 cells at the G2/M phase. Using iTRAQ technology, we observed 312 differentially expressed proteins associated in networks and implicated in different KEGG pathways. Gene Ontology (GO) analysis showed the overviews of dysregulated proteins in the biological process (BP), cell component (CC), and molecular function (MF) categories. Moreover, some candidate proteins involved in PARP1/AIF and nuclear Akt signaling pathways or metastasis processes were validated by western blotting. TP exerted anti-tumor activity on non-small cell lung cancer (NSCLC) A549 lung adenocarcinoma cells by dysregulating tumor-related protein expression. Herein, we provide a preliminary study of TP-related cytotoxicity on A549 cells using proteomics tools. These findings may improve the current understanding of the anti-tumor effects of TP on lung cancer cells and may

  16. The axonal guidance cue semaphorin 3C contributes to alveolar growth and repair.

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    Arul Vadivel

    Full Text Available Lung diseases characterized by alveolar damage such as bronchopulmonary dysplasia (BPD in premature infants and emphysema lack efficient treatments. Understanding the mechanisms contributing to normal and impaired alveolar growth and repair may identify new therapeutic targets for these lung diseases. Axonal guidance cues are molecules that guide the outgrowth of axons. Amongst these axonal guidance cues, members of the Semaphorin family, in particular Semaphorin 3C (Sema3C, contribute to early lung branching morphogenesis. The role of Sema3C during alveolar growth and repair is unknown. We hypothesized that Sema3C promotes alveolar development and repair. In vivo Sema3C knock down using intranasal siRNA during the postnatal stage of alveolar development in rats caused significant air space enlargement reminiscent of BPD. Sema3C knock down was associated with increased TLR3 expression and lung inflammatory cells influx. In a model of O2-induced arrested alveolar growth in newborn rats mimicking BPD, air space enlargement was associated with decreased lung Sema3C mRNA expression. In vitro, Sema3C treatment preserved alveolar epithelial cell viability in hyperoxia and accelerated alveolar epithelial cell wound healing. Sema3C preserved lung microvascular endothelial cell vascular network formation in vitro under hyperoxic conditions. In vivo, Sema3C treatment of hyperoxic rats decreased lung neutrophil influx and preserved alveolar and lung vascular growth. Sema3C also preserved lung plexinA2 and Sema3C expression, alveolar epithelial cell proliferation and decreased lung apoptosis. In conclusion, the axonal guidance cue Sema3C promotes normal alveolar growth and may be worthwhile further investigating as a potential therapeutic target for lung repair.

  17. Oxidant-mediated epithelial cell injury in idiopathic pulmonary fibrosis.

    OpenAIRE

    Cantin, A M; North, S L; Fells, G A; Hubbard, R C; Crystal, R G

    1987-01-01

    Lung inflammatory cells of patients with idiopathic pulmonary fibrosis (IPF) were evaluated for their ability to injure 51Cr-labeled AKD alveolar epithelial cells in the presence and absence of IPF alveolar epithelial lining fluid (ELF). The IPF cells were spontaneously releasing exaggerated amounts of superoxide (O.2) and hydrogen peroxide (H2O2) compared with normal (P less than 0.02). Cytotoxicity of the AKD cells was markedly increased when the IPF inflammatory cells were incubated with a...

  18. Overexpression of Telomerase Protects Human and Murine Lung Epithelial Cells from Fas- and Bleomycin-Induced Apoptosis via FLIP Upregulation.

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    Nissim Arish

    Full Text Available High doses of bleomycin administered to patients with lymphomas and other tumors lead to significant lung toxicity in general, and to apoptosis of epithelial cells, in particular. Apoptosis of alveolar epithelium is an important step in the pathogenesis of bleomycin-induced pulmonary fibrosis. The Fas-FasL pathway is one of the main apoptotic pathways involved. Telomerase is a ribonucleoprotein RNA-dependent DNA polymerase complex consisting of an RNA template and a catalytic protein, telomerase reverse transcriptase (TERT. Telomerase also possess extra-telomeric roles, including modulation of transcription of anti-apoptotic genes, differentiation signals, and more. We hypothesized that telomerase overexpression affects Fas-induced epithelial cell apoptosis by an extra-telomeric role such as regulation of anti-apoptotic genes, specifically FLICE-like inhibitory protein (FLIP. Telomerase in mouse (MLE and human (A549 lung epithelial cell lines was upregulated by transient transfection using cDNA hTERT expression vector. Telomerase activity was detected using a real-time PCR-based system. Bleomycin, and bleomycin-induced Fas-mediated apoptosis following treatment with anti-Fas activating mAb or control IgG, were assessed by Annexin V staining, FACS analysis, and confocal microscopy; caspase cleavage by Western blot; FLIP or Fas molecule detection by Western blot and flow cytometry. hTERT transfection of lung epithelial cells resulted in a 100% increase in their telomerase activity. Fas-induced lung epithelial cell apoptosis was significantly reduced in hTERT-transfected cells compared to controls in all experiments. Lung epithelial cells with increased telomerase activity had higher levels of FLIP expression but membrane Fas expression was unchanged. Upregulation of hTERT+ in human lung epithelial cells and subsequent downregulation of FLIP by shFLIP-RNA annulled hTERT-mediated resistance to apoptosis. Telomerase-mediated FLIP overexpression may

  19. Pulmonary alveolar microlithiasis

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    Surender Kashyap

    2013-01-01

    Full Text Available Pulmonary alveolar microlithiasis (PAM is a rare, chronic lung disease with bilateral intra-alveolar calcium and phosphate deposition throughout the lung parenchyma with predominance to lower and midzone. Although, etiology and pathogenesis of PAM is not fully understood, the mutation in SLC34A2 gene that encodes a sodium-phosphate co-transporter in alveolar type II cells resulting in the accumulation and forming of microliths rich in calcium phosphate (due to impaired clearance are considered to be the cause of the disease. Chest radiograph and high-resolution CT of thorax are nearly pathognomonic for diagnosing PAM. HRCT demonstrates diffuse micronodules showing slight perilobular predominance resulting in calcification of interlobular septa. Patients with PAM are asymptomatic till development of hypoxemia and cor-pulmonale. No therapy has been proven to be beneficial except lung transplantation.

  20. Staphylococcus aureus hemolysin A disrupts cell-matrix adhesions in human airway epithelial cells.

    Science.gov (United States)

    Hermann, Ina; Räth, Susann; Ziesemer, Sabine; Volksdorf, Thomas; Dress, Regine J; Gutjahr, Melanie; Müller, Christian; Beule, Achim G; Hildebrandt, Jan-Peter

    2015-01-01

    Treatment of primary or immortalized human airway epithelial cells (16HBE14o-, S9) or alveolar cancer cells (A549) with recombinant hemolysin A (rHla), a major virulence-associated factor of Staphylococcus aureus, induces alterations in cell shape and formation of paracellular gaps in the cell layer. Semiquantitative Western blotting using extracts of freshly isolated airway tissue (nasal epithelium) or 16HBE14o- model cells revealed that phosphorylation levels of focal adhesion kinase (Fak) and paxillin were altered upon treatment of tissue or cells with rHla. Immune fluorescence analyses showed that rHla treatment of 16HBE14o- cells results in losses of vinculin and paxillin from focal contacts and a net reduction in the number of focal contacts. The actin cytoskeleton was strongly remodeled. We concluded that treatment of cells with rHla activates Fak signaling, which accelerates focal contact turnover and prevents newly formed focal contacts (focal complexes) from maturation to focal adhesions. The inability of rHla-treated cells to form stable focal adhesions may be one factor that contributes to gap formation in the cell layer. In vivo, such changes may disturb the defensive barrier function of the airway epithelium and may facilitate lung infections by S. aureus.

  1. Effects of tanshinone nanoemulsion and extract on inhibition of lung cancer cells A549

    Science.gov (United States)

    Lee, W. D.; Liang, Y. J.; Chen, B. H.

    2016-12-01

    Danshen (Salvia miltiorrhiza), a Chinese medicinal herb, consists of several functional components including tanshinones responsible for prevention of several chronic diseases. This study intends to prepare tanshinone extract and nanoemulsion from danshen and determine their inhibition effect on lung cancer cells A549. A highly stable tanshinone nanoemulsion composed of Capryol 90, Tween 80, ethanol and deionized water with the mean particle size of 14.2 nm was successfully prepared. Tanshinone nanoemulsion was found to be more effective in inhibiting A549 proliferation than tanshinone extract. Both nanoemulsion and extract could penetrate into cytoplasm through endocytosis, with the former being more susceptible than the latter. A dose-dependent response in up-regulation of p-JNK, p53 and p21 and down-regulation of CDK2, cyclin D1 and cyclin E1 expressions was observed with the cell cycle arrested at G0/G1 phase. The cellular microcompartment change of A549 was also investigated. The study demonstrated that tanshinone nanoemulsion may be used as a botanic drug for treatment of lung cancer.

  2. Wnt/β-catenin signaling regulates cancer stem cells in lung cancer A549 cells

    International Nuclear Information System (INIS)

    Teng, Ying; Wang, Xiuwen; Wang, Yawei; Ma, Daoxin

    2010-01-01

    Wnt/β-catenin signaling plays an important role not only in cancer, but also in cancer stem cells. In this study, we found that β-catenin and OCT-4 was highly expressed in cisplatin (DDP) selected A549 cells. Stimulating A549 cells with lithium chloride (LiCl) resulted in accumulation of β-catenin and up-regulation of a typical Wnt target gene cyclin D1. This stimulation also significantly enhanced proliferation, clone formation, migration and drug resistance abilities in A549 cells. Moreover, the up-regulation of OCT-4, a stem cell marker, was observed through real-time PCR and Western blotting. In a reverse approach, we inhibited Wnt signaling by knocking down the expression of β-catenin using RNA interference technology. This inhibition resulted in down-regulation of the Wnt target gene cyclin D1 as well as the proliferation, clone formation, migration and drug resistance abilities. Meanwhile, the expression of OCT-4 was reduced after the inhibition of Wnt/β-catenin signaling. Taken together, our study provides strong evidence that canonical Wnt signaling plays an important role in lung cancer stem cell properties, and it also regulates OCT-4, a lung cancer stem cell marker.

  3. [Nickel exposure to A549 cell damage and L-ascorbic acid interference effect].

    Science.gov (United States)

    Fu, Yao; Wang, Yue; Dan, Han; Zhang, Lin; Ma, Wenhan; Pan, Yulin; Wu, Yonghui

    2015-05-01

    Studying different concentrations of nickel smelting smoke subjects of human lung adenocarcinoma cells (A549) carcinogenic effects, discusses the influence of L-ascorbic acid protection. The A549 cells were divided into experimental and L-ascorbic acid in the intervention group. Plus exposure group concentration of nickel refining dusts were formulated 0.00, 6.25, 12.50, 25.00, 50.00, 100.00 µg/ml suspension, the intervention group on the basis of the added exposure group containing L-ascorbic acid (100 mmol/L), contact 24 h. Detection of cell viability by MTT assay. When the test substance concentration select 0.00, 25.00, 50.00, 100.00 µg/ml experiment for internal Flou-3 fluorescent probe to detect cell Ca²⁺ concentration, within DCFH-DA detect intracellular reactive oxygen (ROS) content, real-time quantitative PCR (real time, in the RT-PCR) was used to detect cell HIF-1α gene expression. With the increase of concentration, subjects increased cell growth inhibition rate, intracellular Ca²⁺ concentration increases, ROS content increased, HIF-1α gene expression increased, differences were statistically significant (P nickel exposure damage to cells. With subjects following exposure to nickel concentration increased, its effect on A549 cell damage increases, L-ascorbic acid cell damage caused by nickel has certain protective effect.

  4. Cordycepin regulates the malignant biological behaviors of lung cancer cell lines A549

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    Ning Shi

    2017-03-01

    Full Text Available Objective: To study the effect of cordycepin on proliferation, apoptosis and invasion-related molecule expression in lung cancer cell lines A549. Methods: Lung cancer cell lines A549 were cultured and treated with different doses of cordycepin (0, 0.25, 0.5, 1.0, 2.0 and 4.0 ng/mL for 24 h, and then the proliferation, apoptosis and invasion-related molecule mRNA expression in cells were detected. Results: After 0.25, 0.5, 1.0, 2.0 and 4.0 ng/mL cordycepin treatment, Caspase-3, Caspase-8, NOX1 and LATS1 mRNA expression were significantly higher than those after 0 ng/mL cordycepin treatment (P<0.05 while CyclinD1, Bcl-2, c-Myc, c-FLIP, TRAF6, N-cadherin and Vimentin mRNA expression were significantly lower than those after 0 ng/mL cordycepin treatment (P<0.05. The greater the cordycepin dosage, the higher the Caspase-3, Caspase-8, NOX1 and LATS1 mRNA expression, and the lower the CyclinD1, Bcl-2, c-Myc, c-FLIP, TRAF6, N-cadherin and Vimentin mRNA expression. Conclusions: Cordycepin can promote pro-apoptosis gene expression and inhibit proproliferation and pro-invasion gene expression in lung cancer cell lines A549.

  5. A549 and MRC-5 cell aggregation in a microfluidic Lab-on-a-chip system.

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    Zuchowska, A; Jastrzebska, E; Zukowski, K; Chudy, M; Dybko, A; Brzozka, Z

    2017-03-01

    In this paper, we present a culture of A549 and MRC-5 spheroids in a microfluidic system. The aim of our work was to develop a good lung cancer model for the evaluation of drug cytotoxicity. Our research was focused on determining the progress of cell aggregation depending on such factors as the depth of culture microwells in the microdevices, a different flow rate of the introduced cell suspensions, and the addition of collagen to cell suspensions. We showed that these factors had a significant influence on spheroid formation. It was found that both MRC-5 and A549 cells exhibited higher aggregation in 500  μ m microwells. We also noticed that collagen needs to be added to A549 cells to form the spheroids. Optimizing the mentioned parameters allowed us to form 3D lung tissue models in the microfluidic system during the 10-day culture. This study indicates how important an appropriate selection of the specified parameters is (e.g., geometry of the microwells in the microsystem) to obtain the spheroids characterized by high viability in the microfluidic system.

  6. Asymmetric [14C]albumin transport across bullfrog alveolar epithelium

    International Nuclear Information System (INIS)

    Kim, K.J.; LeBon, T.R.; Shinbane, J.S.; Crandall, E.D.

    1985-01-01

    Bullfrog lungs were prepared as planar sheets and bathed with Ringer solution in Ussing chambers. In the presence of a constant electrical gradient (20, 0, or -20 mV) across the tissue, 14 C-labeled bovine serum albumin or inulin was instilled into the upstream reservoir and the rate of appearance of the tracer in the downstream reservoir was monitored. Two lungs from the same animal were used to determine any directional difference in tracer fluxes. An apparent permeability coefficient was estimated from a relationship between normalized downstream radioactivities and time. Results showed that the apparent permeability of albumin in the alveolar to pleural direction across the alveolar epithelial barrier is 2.3 X 10(-7) cm/s, significantly greater (P less than 0.0005) than that in the pleural to alveolar direction (5.3 X 10(-8) cm/s) when the tissue was short circuited. Permeability of inulin, on the other hand, did not show any directional dependence and averaged 3.1 X 10(-8) cm/s in both directions. There was no effect on radiotracer fluxes permeabilities of different electrical gradients across the tissue. Gel electrophoretograms and corresponding radiochromatograms suggest that the large and asymmetric isotope fluxes are not primarily due to digestion or degradation of labeled molecules. Inulin appears to traverse the alveolar epithelial barrier by simple diffusion through hydrated paracellular pathways. On the other hand, [ 14 C]albumin crosses the alveolar epithelium more rapidly than would be expected by simple diffusion. These asymmetric and large tracer fluxes suggest that a specialized mechanism is present in alveolar epithelium that may be capable of helping to remove albumin from the alveolar space

  7. Knocking down Dp71 expression in A549 cells reduces its malignancy in vivo and in vitro.

    Science.gov (United States)

    Tan, Sichuang; Tan, Sipin; Chen, Zhikang; Cheng, Ke; Chen, Zhicao; Wang, Wenmei; Wen, Qiaocheng; Zhang, Weilin

    2016-01-01

    Dp71 is one of the most ubiquitously expressed isoforms of dystrophin, the pathological genes of DMD. In order to find whether the alteration of Dp71 can affect the phenotypes of cell other than PC12, an A549 cell line with stably transfected Dp71 siRNA plasmids was set up and named A549-Dp71AS cell. It is demonstrated for the first time that the A549-Dp71AS cell line displayed decreased invasion capabilities, reduced migration ability, decreased proliferation rate, and lessened clonogenic formation. Cisplatin-induced apoptosis was also increased in A549-Dp71AS cell line via enhancing the Caspase 3, Caspase 8, and Caspase 9 activities. Knocking down Dp71 expression can significantly inhibit the A549 xenograft tumor growth in nude mice. The A549-Dp71AS cells and xenograft tumor tissues displayed reduced lamin B1, Bcl-2, and MMP2 protein expression, which accounts for the reduced malignancy of A549-Dp71AS cells in vivo and in vitro.

  8. Microfluidic wound-healing assay to assess the regenerative effect of HGF on wounded alveolar epithelium.

    OpenAIRE

    Felder Marcel; Sallin Pauline; Barbe Laurent; Haenni Beat; Gazdhar Amiq; Geiser Thomas; Guenat Olivier

    2012-01-01

    We present a microfluidic epithelial wound healing assay that allows characterization of the effect of hepatocyte growth factor (HGF) on the regeneration of alveolar epithelium using a flow focusing technique to create a regular wound in the epithelial monolayer. The phenotype of the epithelial cell was characterized using immunostaining for tight junction (TJ) proteins and transmission electron micrographs (TEMs) of cells cultured in the microfluidic system a technique that is reported here ...

  9. Oxidative injury induced by cadmium sulfide nanoparticles in A549 cells and rat lungs.

    Science.gov (United States)

    Wang, Junfeng; Jiang, Chunyang; Alattar, Mohamed; Hu, Xiaoli; Ma, Dong; Liu, Huibin; Meng, Chunyan; Cao, Fuyuan; Li, Weihong; Li, Qingzhao

    2015-01-01

    Rod-shaped cadmium sulfide nanoparticles (CdS NPs) are becoming increasingly important in many industrial fields, but their potential hazards remain unknown. This study aimed to explore the patterns and mechanisms of lung injury induced by CdS NPs. A549 cells and rats were exposed to two types of CdS NPs with a same diameter of 20-30 nm but different lengths, CdS1 (80-100 nm) and CdS2 (110-130 nm). The using doses were included 10 μg/ml and 20 μg/ml two types of CdS NPs for cellular experiments and five times dose of 20 mg/kg body weight for rats' exposure. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) and trypan blue staining were used to detect the A549 cell mortality percentage. The levels of reactive oxygen species (ROS) were determined in A549 cell. The vigor of superoxide dismutase (SOD) and the contents of catalase (CAT) and malondialdehyde (MDA) were detected both in A549 cells and in rats' serum and lung tissues. The cellular morphological changes were observed under transmission electron microscopy (TEM) and the pathological changes were observed in rats' lung tissue. CdS NPs significantly increased A549 cell mortality percentage. The CdS NPs also increased the levels of ROS and MDA content, whereas they decreased SOD and CAT activities. In parallel, similar changes of the contents of MDA, SOD and CAT were also observed in the sera and lung tissues of CdS NP-treated rats. The cellular TEM detection revealed that two types of CdS nanorods appeared as orderly arranged rounded fat droplets separately and leading to nucleus condensation (CdS1). These cellular and rats' tissues changes in the group treated with CdS1 were more significant than the CdS2 groups. Furthermore, CdS NPs induced many pathological changes, including emphysematous changes in rat lung tissue. Especially visible lung consolidation can be observed in the CdS1 group. CdS NPs induce oxidative injury in the respiratory system, and their toxic effects may be related to grain length.

  10. Intracellular calcium promotes radioresistance of non-small cell lung cancer A549 cells through activating Akt signaling.

    Science.gov (United States)

    Wang, Yiling; He, Jiantao; Zhang, Shenghui; Yang, Qingbo

    2017-03-01

    Radiotherapy is a major therapeutic approach in non-small cell lung cancer but is restricted by radioresistance. Although Akt signaling promotes radioresistance in non-small cell lung cancer, it is not well understood how Akt signaling is activated. Since intracellular calcium (Ca 2+ ) could activate Akt in A549 cells, we investigated the relationship between intracellular calcium (Ca 2+ ) and Akt signaling in radioresistant A549 cells by establishing radioresistant non-small cell lung cancer A549 cells. The radioresistant cell line A549 was generated by dose-gradient irradiation of the parental A549 cells. The cell viability, proliferation, and apoptosis were, respectively, assessed using the cell counting kit-8, EdU labeling, and flow cytometry analysis. The phosphorylation of Akt was evaluated by Western blotting, and the intracellular Ca 2+ concentration was assessed by Fluo 4-AM. The radioresistant A549 cells displayed mesenchymal morphology. After additional irradiation, the radioresistant A549 cells showed decreased cell viability and proliferation but increased apoptosis. Moreover, the intracellular Ca 2+ concentration and the phosphorylation level on the Akt473 site in radioresistant A549 cells were higher than those in original cells, whereas the percentage of apoptosis in radioresistant A549 cells was less. All these results could be reversed by verapamil. In conclusion, our study found that intracellular Ca 2+ could promote radioresistance of non-small cell lung cancer cells through phosphorylating of Akt on the 473 site, which contributes to a better understanding on the non-small cell lung cancer radioresistance, and may provide a new target for radioresistance management.

  11. [Effects of 17-AAG on the proliferation and apoptosis of human lung cancer A549 and H446 cells].

    Science.gov (United States)

    Niu, Ben; Lin, Jingshuang; Feng, Tao

    2015-04-01

    To observe the effect of 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) on the apoptosis of human lung cancer cell lines A549 and H446, and to investigate the potential mechanisms. Proliferation inhibition and apoptosis assays, and the cell cycles were detected by MTT and flow cytometry respectively. Western blot was used to determine the expression level of proteins such as Hsp90, Hsp70, AKt, Her-2, Bcl-2 and Bax. After treated with 17-AAG, the proliferation of both A549 and H446 cells was inhibited significantly in a dose-dependent manner; as the concentration of 17-AAG was from 50 to 500 nmol/L, the IC₅₀ values to A549 and H446 cell lines were (222 ± 13) nmol/L and (189 ± 7) nmol/L respectively at 48 h. Cell cycle assays showed that 17-AAG was able to arrest cell cycles of A549 and H446 cell lines at the G₂/M phase. Apoptosis assay showed that 17-AAG was capable of inducing apoptosis in A549 and H446 cell lines. After treated with 17-AAG for 48 h, there were significant differences between the 400 nmol/L groups(46.3% for A549 cell line and 56.9% for H446 cell line) and the control group (11.9% for A549 cell line and 6.9% for H446 cell line, P AAG treatment: Akt and Her-2 decreased significantly while the expression of Hsp70 increased. Meanwhile, the expression of Bcl-2 decreased but that of Bax increased, indicating that 17-AAG was able to promote apoptosis mode in A549 and H446 cells. 17-AAG can regulate the expression level of apoptosis-related proteins such as Bax and Bcl-2 by Hsp90 signaling pathway in A549 and H446 cells, and ultimately inhibit cell proliferation and induce apoptosis.

  12. Paraquat induces epithelial-mesenchymal transition-like cellular response resulting in fibrogenesis and the prevention of apoptosis in human pulmonary epithelial cells.

    Directory of Open Access Journals (Sweden)

    Atsushi Yamada

    Full Text Available The aim of this study is to investigate the molecular mechanisms underlying delayed progressive pulmonary fibrosis, a characteristic of subacute paraquat (PQ poisoning. Epithelial-mesenchymal transition (EMT has been proposed as a cause of organ fibrosis, and transforming growth factor-β (TGF-β is suggested to be a powerful mediator of EMT. We thus examined the possibility that EMT is involved in pulmonary fibrosis during PQ poisoning using A549 human alveolar epithelial cells in vitro. The cells were treated with various concentrations of PQ (0-500 μM for 2-12 days. Short-term (2 days high-dose (>100 μM treatments with PQ induced cell death accompanied by the activation of caspase9 as well as a decrease in E-cadherin (an epithelial cell marker, suggesting apoptotic cell death with the features of anoikis (cell death due to the loss of cell-cell adhesion. In contrast, long-term (6-12 days low-dose (30 μM treatments with PQ resulted in a transformation into spindle-shaped mesenchymal-like cells with a decrease of E-cadherin as well as an increase of α-smooth muscle actin (α-SMA. The mesenchymal-like cells also secreted the extracellular matrix (ECM protein fibronectin into the culture medium. The administration of a TGF-β1 receptor antagonist, SB431542, almost completely attenuated the mesenchymal transformation as well as fibronectin secretion, suggesting a crucial role of TGF-β1 in EMT-like cellular response and subsequent fibrogenesis. It is noteworthy that despite the suppression of EMT-fibrogenesis, apoptotic death was observed in cells treated with PQ+SB431542. EMT-like cellular response and subsequent fibrogenesis were also observed in normal human bronchial epithelial (NHBE cells exposed to PQ in a TGF-β1-dependent manner. Taken together, our experimental model reflects well the etiology of PQ poisoning in human and shows the involvement of EMT-like cellular response in both fibrogenesis and resistance to cell death during

  13. Decrease in paracellular permeability and chemosensitivity to doxorubicin by claudin-1 in spheroid culture models of human lung adenocarcinoma A549 cells.

    Science.gov (United States)

    Akizuki, Risa; Maruhashi, Ryohei; Eguchi, Hiroaki; Kitabatake, Kazuki; Tsukimoto, Mitsutoshi; Furuta, Takumi; Matsunaga, Toshiyuki; Endo, Satoshi; Ikari, Akira

    2018-03-07

    Chemotherapy resistance is a major problem in the treatment of cancer, but the underlying mechanisms are not fully understood. We found that the expression levels of claudin-1 (CLDN1) and 3, tight junctional proteins, are upregulated in cisplatin (CDDP)-resistant human lung adenocarcinoma A549 (A549R) cells. A549R cells showed cross-resistance to doxorubicin (DXR). Here, the expression mechanism and function of CLDN1 and 3 were examined. CLDN1 and 3 were mainly localized at tight junctions concomitant with zonula occludens (ZO)-1, a scaffolding protein, in A549 and A549R cells. The phosphorylation levels of Src, MEK, ERK, c-Fos, and Akt in A549R cells were higher than those in A549 cells. The expression levels of CLDN1 and 3 were decreased by LY-294002, a phosphoinositide 3-kinase (PI3K) inhibitor, and BAY 11-7082, an NF-κB inhibitor. The overexpression of CLDN1 and 3 decreased the paracellular permeability of DXR in A549 cells. Hypoxia levels in A549R and CLDN1-overexpressing cells (CLDN1/A549) were greater than those in A549, mock/A549, and CLDN3/A549 cells in a spheroid culture model. In contrast, accumulation in the region inside the spheroids and the toxicity of DXR in A549R and CLDN1/A549 cells were lower than those in other cells. Furthermore, the accumulation and toxicity of DXR were rescued by CLDN1 siRNA in A549R cells. We suggest that CLDN1 is upregulated by CDDP resistance through activation of a PI3K/Akt/NF-κB pathway, resulting in the inhibition of penetration of anticancer drugs into the inner area of spheroids. Copyright © 2018 Elsevier B.V. All rights reserved.

  14. Meconium is a source of pro-inflammatory substances and can induce cytokine production in cultured A549 epithelial cells

    NARCIS (Netherlands)

    de Beaufort, Arnout Jan; Bakker, Astrid C.; van Tol, Maarten J. D.; Poorthuis, Ben J.; Schrama, Alexandra J.; Berger, Howard M.

    2003-01-01

    Inflammation plays an important role in the pathogenesis of meconium aspiration syndrome, and pneumonitis is one of the major characteristics. We have previously shown that meconium has chemotactic properties because of the presence of IL-8. We hypothesize that IL-8 and other proinflammatory

  15. [Impact of Cystic Fibrosis Transmembrane Conductance Regulator on Malignant
 Properties of KRAS Mutant Lung Adenocarcinoma A549 Cells].

    Science.gov (United States)

    Li, Hui; Wang, Ying; Yang, Jiali; Liu, Xiaoming; Shi, Juan

    2018-02-20

    The incidence of lung cancer is gradually increased, and the cystic fibrosis transmembrane conductance regulator (CFTR) has recently demonstrated to have an implication in the deoncogenesis and malignant transformation of many types of cancers. The aim of this study is to investigate impacts of CFTR on the malignant features of lung adenocarcinoma A549 cells. The capacity of cell proliferation, migration, invasion and clonogenicity of non-small cell lung cancer A549 cells were detected by CCK8 cell proliferation assay, cell scratch assay, Transwell cell invasion assay and clone formation assay, respectively. Meanwhile, the effect of CFTR gene on the expression of cancer stem cell related transcriptional factors was also detected by immunoblotting (Western blot) assay. An overexpression of CFTR gene in A549 cells significantly inhibited the malignant capacity of A549 cells, including potencies of cell proliferation, migration, invasion and colony formation; while knockdown of CFTR gene expression by RNA interference in A549 cells resulted in an opposite effect seen in above cells overexpressing CFTR gene. Mechanistically, immunoblotting assay further revealed that the ectopic expression of CFTR gene led an inhibitory expression of stem cell-related transcriptional factors SOX2 and OCT3/4, and cancer stem cell surface marker CD133 in A549 cells, while a knockdown of CFTR expression yielded a moderately increased expression of these gene. However, an alteration of CFTR gene expression had neither effect on the expression of putative lung cancer stem cell marker aldehyde dehydrogenase1 (ALDH1), nor the frequency of ALDH1A-positive cells in A549 cells, as ascertained by the immunoblotting assay and cytometry analysis, respectively. The CFTR exhibited an inhibitory role in the malignancy of lung adenocarcinoma A549 cells, suggesting that it may be a novel potential target for lung cancer treatment. However, its functions in other lung adenocarcinoma cell lines and its

  16. MiR-200a enhances the migrations of A549 and SK-MES-1 cells by ...

    Indian Academy of Sciences (India)

    2013-07-14

    Jul 14, 2013 ... The relative levels of mature miR-200a in different lung cancer cell lines and normal lung cells. A549, SK-MES-1: non- small cell lung cancer (NSCLC) cells, HELF: normal lung cells. The value of miRNA-200a in HELF cells was designated as 1. MiR-200a enhances migrations of A549 and SK-MES-1 cells.

  17. Diffuse bronchiolo-alveolar carcinoma in a dog.

    Science.gov (United States)

    Bertazzolo, W; Zuliani, D; Pogliani, E; Caniatti, M; Bussadori, C

    2002-06-01

    An eight-year-old female German wirehaired pointer was presented with signs of respiratory distress. Clinical examination, laboratory results, thoracic radiography and echocardiography indicated the presence of a diffuse interstitial lung disease with secondary appropriate erythrocytosis, pulmonary hypertension and cor pulmonale. Transthoracic fine needle aspiration biopsy of the lung suggested malignant epithelial neoplasia. A primary lung cancer with an unusually diffuse distribution of miliary/micronodular lesions was found at postmortem examination. Histological diagnosis was bronchiolo-alveolar carcinoma. Bronchiolo-alveolar carcinoma can occasionally occur in a diffuse fashion involving most or all of the lung parenchyma. In man, diffuse bronchiolo-alveolar carcinoma is considered a great imitator of other, more common diffuse interstitial forms of lung disease. This case report indicates that it is also a differential diagnosis to consider in dogs.

  18. Proteinosis alveolar pulmonar

    Directory of Open Access Journals (Sweden)

    Concepción Sánchez Infante

    2011-12-01

    Full Text Available La proteinosis alveolar pulmonar es una enfermedad respiratoria crónica, caracterizada por alteración en el metabolismo del surfactante, lo que determina su acumulación anormal en el espacio alveolar. Es una enfermedad extremadamente rara. Se han reportado solamente 500 casos en la literatura. Se describió por primera vez en 1958. Se presenta un caso de proteinosis alveolar pulmonar en un lactante de 2 meses, con desnutrición proteico energética, que ingresa por dificultad respiratoria e hipoxemia, y, con imágenes radiológicas de tipo retículo-nodulillar, en vidrio deslustrado, en el cual se plantea inicialmente el diagnóstico de bronconeumonía. Ante la evolución desfavorable y no respuesta al tratamiento, se realizó un estudio para descartar enfermedades pulmonares crónicas. El paciente fallece y se confirma el diagnóstico por anatomía patológica. Se realiza una revisión del tema.

  19. The radiation sensitization effect of miRNA-34c on A549 cell

    International Nuclear Information System (INIS)

    Xiao Hainan; Duan Guangxin; Zhang Yanjuan; Zhou Xinwen

    2012-01-01

    Objective: To study the radiation sensitization effect of miRNA-34c on A549 cell. Methods: The cells were divided into transfection, transfection-irradiation and irradiation groups. The transfection group was transfected with miRNA-34c series. The transfection-irradiation and irradiation groups were irradiated with or without miRNA-34c transfection respectively. Then cell growth inhibition ratio was measured by MTT, cell cycle was detected by FCM, cell apoptosis ratio was observed by Erythro- sin B staining and p53 expression was visualized by Western blot. Results: The inhibition ratio and apoptosis ratio of the group with transfection-irradiation combined treatment was apparently higher than those treated by transfection or irradiation and rose as the transfection density increases (P<0.05). The cell cycle analysis showed that the cell was obviously blocked in G 1 /G 0 Phase. Compared with the irradiation group, the p53 protein expression of the cell with combined treatment increased in a dose dependent manner. Conclusion: The miRNA-34c has the radiation sensitization effect on A549 adenocarcinoma cell, through enhancing the expression of p53 protein and inducing the cell cycle blockade and apoptosis. (authors)

  20. Role of Smac in apoptosis of lung cancer cells A549 induced by Taxol.

    Science.gov (United States)

    Zhang, Ying; Hao, Yingtao; Sun, Qifeng; Peng, Chuanliang

    2015-01-01

    A series of structurally unique second mitochondria-derived activators of caspase (Smac) that act as antagonists of inhibitor of apoptosis proteins (IAPs) directly have been discovered and have been shown to promote chemotherapy-induced apoptosis. In this study, we investigate the role of Smac in Taxol-induced apoptosis of lung cancer cell in vitro. PcDNA3.1/Smac recombinants were transfected into the non-small cell lung cancer cell line A549. Smac expression was detected by RT-PCR and Western blot. The invasive ability of cells was examined. Flow cytometry was used to analyze apoptosis of cells induced by Taxol with Annexin V/PI double staining technique. Smac expression was significantly higher in the PcDNA3.1/Smac recombinant group than in the untransfected group at mRNA and protein level (p Smac group. There were significant differences compared to the empty vector group and control group. The apoptosis rate was significantly higher in PcDNA3.1/Smac + Taxol group than in other groups (p Smac can enhance the chemosensitivity of the non-small cell lung cancer cell line A549 to Taxol.

  1. DNA damage response signaling in lung adenocarcinoma A549 cells following gamma and carbon beam irradiation.

    Science.gov (United States)

    Ghosh, Somnath; Narang, Himanshi; Sarma, Asitikantha; Krishna, Malini

    2011-11-01

    Carbon beams (5.16MeV/u, LET=290keV/μm) are high linear energy transfer (LET) radiation characterized by higher relative biological effectiveness than low LET radiation. The aim of the current study was to determine the signaling differences between γ-rays and carbon ion-irradiation. A549 cells were irradiated with 1Gy carbon or γ-rays. Carbon beam was found to be three times more cytotoxic than γ-rays despite the fact that the numbers of γ-H2AX foci were same. Percentage of cells showing ATM/ATR foci were more with γ-rays however number of foci per cell were more in case of carbon irradiation. Large BRCA1 foci were found in all carbon irradiated cells unlike γ-rays irradiated cells and prosurvival ERK pathway was activated after γ-rays irradiation but not carbon. The noteworthy finding of this study is the early phase apoptosis induction by carbon ions. In the present study in A549 lung adenocarcinoma, authors conclude that despite activation of same repair molecules such as ATM and BRCA1, differences in low and high LET damage responses might be due to their distinct macromolecular complexes rather than their individual activation and the activation of cytoplasmic pathways such as ERK, whether it applies to all the cell lines need to be further explored. Copyright © 2011 Elsevier B.V. All rights reserved.

  2. High-throughput quantitative proteomic analysis of dengue virus type 2 infected A549 cells.

    Directory of Open Access Journals (Sweden)

    Han-Chen Chiu

    Full Text Available Disease caused by dengue virus is a global health concern with up to 390 million individuals infected annually worldwide. There are no vaccines or antiviral compounds available to either prevent or treat dengue disease which may be fatal. To increase our understanding of the interaction of dengue virus with the host cell, we analyzed changes in the proteome of human A549 cells in response to dengue virus type 2 infection using stable isotope labelling in cell culture (SILAC in combination with high-throughput mass spectrometry (MS. Mock and infected A549 cells were fractionated into nuclear and cytoplasmic extracts before analysis to identify proteins that redistribute between cellular compartments during infection and reduce the complexity of the analysis. We identified and quantified 3098 and 2115 proteins in the cytoplasmic and nuclear fractions respectively. Proteins that showed a significant alteration in amount during infection were examined using gene enrichment, pathway and network analysis tools. The analyses revealed that dengue virus infection modulated the amounts of proteins involved in the interferon and unfolded protein responses, lipid metabolism and the cell cycle. The SILAC-MS results were validated for a select number of proteins over a time course of infection by Western blotting and immunofluorescence microscopy. Our study demonstrates for the first time the power of SILAC-MS for identifying and quantifying novel changes in cellular protein amounts in response to dengue virus infection.

  3. Oxidative stress, DNA damage, and inflammation induced by ambient air and wood smoke particulate matter in human A549 and THP-1 cell lines.

    Science.gov (United States)

    Danielsen, Pernille Høgh; Møller, Peter; Jensen, Keld Alstrup; Sharma, Anoop Kumar; Wallin, Håkan; Bossi, Rossana; Autrup, Herman; Mølhave, Lars; Ravanat, Jean-Luc; Briedé, Jacob Jan; de Kok, Theo Martinus; Loft, Steffen

    2011-02-18

    Combustion of biomass and wood for residential heating and/or cooking contributes substantially to both ambient air and indoor levels of particulate matter (PM). Toxicological characterization of ambient air PM, especially related to traffic, is well advanced, whereas the toxicology of wood smoke PM (WSPM) is poorly assessed. We assessed a wide spectrum of toxicity end points in human A549 lung epithelial and THP-1 monocytic cell lines comparing WSPM from high or low oxygen combustion and ambient PM collected in a village with many operating wood stoves and from a rural background area. In both cell types, all extensively characterized PM samples (1.25-100 μg/mL) induced dose-dependent formation of reactive oxygen species and DNA damage in terms of strand breaks and formamidopyrimidine DNA glycosylase sites assessed by the comet assay with WSPM being most potent. The WSPM contained more polycyclic aromatic hydrocarbons (PAH), less soluble metals, and expectedly also had a smaller particle size than PM collected from ambient air. All four types of PM combined increased the levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine dose-dependently in A549 cells, whereas there was no change in the levels of etheno-adducts or bulky DNA adducts. Furthermore, mRNA expression of the proinflammatory genes monocyte chemoattractant protein-1, interleukin-8, and tumor necrosis factor-α as well as the oxidative stress gene heme oxygenase-1 was upregulated in the THP-1 cells especially by WSPM and ambient PM sampled from the wood stove area. Expression of oxoguanine glycosylase 1, lymphocyte function-associated antigen-1, and interleukin-6 did not change. We conclude that WSPM has small particle size, high level of PAH, low level of water-soluble metals, and produces high levels of free radicals, DNA damage as well as inflammatory and oxidative stress response gene expression in cultured human cells.

  4. Amiodarone increases the accumulation of DEA in a human alveolar epithelium-derived cell line.

    Science.gov (United States)

    Seki, Satoru; Itagaki, Shirou; Kobayashi, Masaki; Hirano, Takeshi; Iseki, Ken

    2008-07-01

    Amiodarone (AMD)-induced pulmonary toxicity (AIPT) is the most life-threatening side-effect of AMD treatment. N-Monodesethylamiodarone (DEA), an active metabolite of AMD, also exhibits cytotoxicity and tends to accumulate in the lung more intensively than AMD. In this study, we characterized the mechanism of DEA accumulation using A549 cells as a model of the alveolar epithelium. Typical ATP-depletion compounds caused an approximately 30% increase in the accumulation of DEA in A549 cells, although these effects were less than those in Caco-2 cells. Triiodothyronine (T(3)), which exhibited an inhibitory effect on DEA efflux in Caco-2 cells, did not affect the accumulation of DEA in A549 cells. On the other hand, 100 microM AMD caused an approximately 200% increase in DEA content in A549 cells, although AMD accumulation was not affected by 100 microM DEA. Since the reducing effect of AMD on cellular ATP levels and that of FCCP were similar, the mechanism by which DEA accumulation is increased by AMD might be different from the ATP-dependent DEA efflux mechanism. The decrease in cell viability by DEA in the presence of AMD (IC(50) value of DEA for A549 cell viability: 25.4+/-2.4 microM) was more pronounced than that by DEA alone (IC(50) value: 11.5+/-3.0 microM). This further DEA accumulation by AMD might be a factor responsible for the greater accumulation of DEA than that of AMD in the lung in long-term AMD-treated patients.

  5. Jolkinolide A and Jolkinolide B Inhibit Proliferation of A549 Cells and Activity of Human Umbilical Vein Endothelial Cells.

    Science.gov (United States)

    Shen, Lei; Zhang, Shan-Qiang; Liu, Lei; Sun, Yu; Wu, Yu-Xuan; Xie, Li-Ping; Liu, Ji-Cheng

    2017-01-14

    BACKGROUND Jolkinolide A (JA) and Jolkinolide B (JB) are diterpenoids extracted from the roots of Euphorbia fischeriana Steud and have been shown to have anti-tumor activity. However, their effects on the ability of tumor cells to invade blood vessels and metastasize remain largely unknown. Investigations into the effects of JA and JB on the angiogenesis of tumor tissues may facilitate the identification of new natural drugs with anti-tumor growth and metastasis activities. MATERIAL AND METHODS We used different concentrations of JA and JB (20 μg/ml, 40 μg/ml, 60 μg/ml, 80 μg/ml, and 100 μg/ml) to stimulate A549 cells and then studied the effects on the growth and metastasis of lung cancers. In addition, we used conditional media from A549 cells (A549-CM) stimulated by either JA or JB in different concentrations to culture human umbilical vein endothelial cells (HUVECs). RESULTS We found that both JA and JB significantly inhibited the Akt-STAT3-mTOR signaling pathway and reduced the expression of VEGF in A549 cells, but JB exhibited more significant inhibitory effects than JA. The JB-stimulated A549 cell conditional media had a greater inhibitory effect on the proliferation and migration of HUVECs than did the conditional media of JA-stimulated A549 cells. This effect gradually increased with increasing concentrations of either type of Jolkinolide. CONCLUSIONS Our results suggest that JA and JB inhibited VEGF expression in A549 cells through the inhibition of the Akt-STAT3-mTOR signaling pathway, and directly inhibited the proliferation and migration of HUVECs. These findings are of great significance for the development of new plant-derived chemotherapy agents for the treatment of cancer.

  6. High pemetrexed sensitivity of docetaxel-resistant A549 cells is mediated by TP53 status and downregulated thymidylate synthase.

    Science.gov (United States)

    Kuo, Wei-Ting; Tu, Dom-Gene; Chiu, Ling-Yen; Sheu, Gwo-Tarng; Wu, Ming-Fang

    2017-11-01

    The chemoresistance of non-small cell lung cancer (NSCLC) that occurs in docetaxel (DOC) chemotherapy substantially decreases the survival of patients. To overcome DOC-induced chemoresistance, we established DOC-selected A549 lung cancer sublines (A549/D16 and A549/D32) and revealed that both sublines were cross-resistant to vincristine (VCR) and doxorubicin (DXR). Notably, both sublines were more sensitive to pemetrexed (PEM) than parental cells according to MTT and clonogenic assays. The expression levels of thymidylate synthase (TS) and γ-glutamyl hydrolase (GGH) were downregulated in DOC-resistant sublines. When exogenous TS was overexpressed in A549/D16 cells, PEM sensitivity was significantly decreased, however it was not decreased by overexpression of exogenous GGH. PEM treatment induced more apoptotic sub-G1 cells in both DOC-resistant sublines and in the in vivo PEM sensitivities of A549/D16 cells. These findings were further confirmed by a xenografted tumor model. To unmask the mediator of TS downregulation, we investigated human lung cancer cell lines that have various TP53 statuses using DOC treatment. The level of TS protein was significantly decreased in wild-type TP53-containing cells with DOC treatment; TS expression levels were not affected in mutant-TP53 and TP53‑null cells under the same conditions. Furthermore, when the expression of TP53 was inhibited in A549 cells, the expression level of TS was increased. Our data indicated that DOC activated wild-type TP53 and suppressed TS expression under continuous DOC exposure. Therefore, the expression of TS remained at low levels in DOC-resistant A549 cancer cells. Our data revealed that for lung cancer with DOC resistance and wild‑type TP53 status, the administration of PEM as a second-line agent to overcome DOC-resistance may benefit patients.

  7. Genistein inhibits A549 human lung cancer cell proliferation via miR-27a and MET signaling.

    Science.gov (United States)

    Yang, Yang; Zang, Aimin; Jia, Youchao; Shang, Yanhong; Zhang, Zhuoqi; Ge, Kun; Zhang, Jinchao; Fan, Wufang; Wang, Bei

    2016-09-01

    Genistein is a soybean isoflavone; in its aglycone it has various biological activities. Animal experiments, clinical studies and epidemiological investigations suggest that genistein has preventative and curative functions for a number of diseases, particularly in cancer. The present study explored the potential anti-cancer effect of genistein by observing its role in inhibiting A549 human lung cancer cell proliferation and investigating the possible mechanism. A549 cells were exposed to various concentrations of genistein (0, 10, 25, 50, 100 and 200 µM; dissolved in physiological saline) for 1, 2 and 3 days. Subsequently, the viability of A549 cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, cell apoptosis was examined using a flow cytometer, caspase 3/9 activity was measured using commercial kits, reverse transcription quantitative polymerase chain reaction was used to analyze the miR-27a expression and western blotting was used to investigate MET protein expression. The results suggested a significant inhibition of A549 cell growth following treatment with genistein in a time- and dose-dependent manner. The current study also indicated that treatment with genistein significantly induces cell apoptosis and promotes caspase-3/9 activation of A549 cells in a dose-dependent manner. Further functional assays revealed that the anti-cancer effect of genistein activated microRNA-27a (miR-27a) expression levels and reduced MET protein expression in A549 cells. In conclusion, the present study demonstrates that genistein inhibits A549 human lung cancer cell proliferation. Furthermore, this study reports, for the first time, a correlation between the anti-cancer effect of genistein and miR-27a-mediated MET signaling.

  8. Effect of inhibition proliferation in human lung adenocarcinoma A549 cells by cytokine-induced killer cells.

    Science.gov (United States)

    Li, Dengrui; Guo, Sumin; Li, Hui; Zhu, Guiyun; Gao, Li; Xin, Xin; Yan, Dandan; Li, Xiuwu; Geng, Shujun; Hou, Hongwei; Yang, Yonghui

    2015-07-01

    Adenocarcinoma, the most common form of lung cancer, is one of main human malignant tumors. In this paper, we focus on the effect of antitumor activity of cytokine-induced killer (CIK) cells on human lung adenocarcinoma cell line A549. CIK cells were obtained by inducing peripheral blood mononuclear cells with recombinant human (rh) interferon-gamma, monoclonal anti-CD3 antibody, rh interleukin (IL)-1alpha, and rhIL-2, which were added into the culture. A549 cell viability of CIK cells was determined using MTS assay. Flow cytometry (FCM) experiments were performed to detect cell cycle changes. The expression of P27 in A549 cells treated by CIK cells was evaluated by Western blot. The percentage of CD3+CD16+CD56+ T cells in a representative peripheral blood mononucleated cell sample was 33.7 ± 1.3%. CIK cells, in dose and time dependent manners, inhibited the proliferation of A549. FCM demonstrated that A549 cells were accumulated in G2/M and G0/G1 phases when treated with CIK cells. FCM was used to analyze whether A549 cells treated with CIK cells induced apotosis or necrosis at 10:1 or 20:1. Compared to the control group, P27 was prominently upregulated in the CIK treated group. We propose that the pharmacological mechanisms of A549 cells inhibited by CIK cells can be estimated to possibly elicit different biological significance, which, in part, can be ascribed to a different mass transport rate in vitro.

  9. Enhanced DNA double-strand break repair of microbeam targeted A549 lung carcinoma cells by adjacent WI38 normal lung fibroblast cells via bi-directional signaling.

    Science.gov (United States)

    Kobayashi, Alisa; Tengku Ahmad, Tengku Ahbrizal Farizal; Autsavapromporn, Narongchai; Oikawa, Masakazu; Homma-Takeda, Shino; Furusawa, Yoshiya; Wang, Jun; Konishi, Teruaki

    2017-10-01

    Understanding the mechanisms underlying the radiation-induced bystander effect (RIBE) and bi-directional signaling between irradiated carcinoma cells and their surrounding non-irradiated normal cells is relevant to cancer radiotherapy. The present study investigated propagation of RIBE signals between human lung carcinoma A549 cells and normal lung fibroblast WI38 cells in bystander cells, either directly or indirectly contacting irradiated A549 cells. We prepared A549-GFP/WI38 co-cultures and A549-GFP/A549 co-cultures, in which A549-GFP cells stably expressing H2BGFP were co-cultured with either A549 cells or WI38 cells, respectively. Using the SPICE-NIRS microbeam, only the A549-GFP cells were irradiated with 500 protons per cell. The level of γ-H2AX, a marker for DNA double-strand breaks (DSB), was subsequently measured for up to 24h post-irradiation in three categories of cells: (1) "targeted"/irradiated A549-GFP cells; (2) "neighboring"/non-irradiated cells directly contacting the "targeted" cells; and (3) "distant"/non-irradiated cells, which were not in direct contact with the "targeted" cells. We found that DSB repair in targeted A549-GFP cells was enhanced by co-cultured WI38 cells. The bystander response in A549-GFP/A549 cell co-cultures, as marked by γ-H2AX levels at 8h post-irradiation, showed a decrease to non-irradiated control level when approaching 24h, while the neighboring/distant bystander WI38 cells in A549-GFP/WI38 co-cultures was maintained at a similar level until 24h post-irradiation. Surprisingly, distant A549-GFP cells in A549-GFP/WI38 co-cultures showed time dependency similar to bystander WI38 cells, but not to distant cells in A549-GFP/A549 co-cultures. These observations indicate that γ-H2AX was induced in WI38 cells as a result of RIBE. WI38 cells were not only involved in rescue of targeted A549, but also in the modification of RIBE against distant A549-GFP cells. The present results demonstrate that radiation-induced bi

  10. TGF-β1 downregulates COX-2 expression leading to decrease of PGE2 production in human lung cancer A549 cells, which is involved in fibrotic response to TGF-β1.

    Directory of Open Access Journals (Sweden)

    Erina Takai

    Full Text Available Transforming growth factor-ß1 (TGF-β1 is a multifunctional cytokine that is involved in various pathophysiological processes, including cancer progression and fibrotic disorders. Here, we show that treatment with TGF-β1 (5 ng/mL induced downregulation of cyclooxygenase-2 (COX-2, leading to reduced synthesis of prostaglandin E2 (PGE2, in human lung cancer A549 cells. Treatment of cells with specific inhibitors of COX-2 or PGE2 receptor resulted in growth inhibition, indicating that the COX-2/PGE2 pathway contributes to proliferation in an autocrine manner. TGF-β1 treatment induced growth inhibition, which was attenuated by exogenous PGE2. TGF-β1 is also a potent inducer of epithelial mesenchymal transition (EMT, a phenotype change in which epithelial cells differentiate into fibroblastoid cells. Supplementation with PGE2 or PGE2 receptor EP4 agonist PGE1-alcohol, as compared with EP1/3 agonist sulprostone, inhibited TGF-β1-induced expression of fibronectin and collagen I (extracellular matrix components. Exogenous PGE2 or PGE2 receptor agonists also suppressed actin remodeling induced by TGF-β1. These results suggest that PGE2 has an anti-fibrotic effect. We conclude that TGF-β1-induced downregulation of COX-2/PGE2 signaling is involved in facilitation of fibrotic EMT response in A549 cells.

  11. Antitumor activity of cobrotoxin in human lung adenocarcinoma A549 cells and following transplantation in nude mice.

    Science.gov (United States)

    Shen, Jian; Xie, Yan; Sun, Mei-Lin; Han, Rong; Qin, Zheng-Hong; He, Jing-Kang

    2014-11-01

    The aim of the present study was to investigate cobra neurotoxin (cobrotoxin) activity in A549 cell lines transplanted into nude mice, and to explore its molecular mechanism. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method was used to detect the growth inhibition rate of cobrotoxin in human lung A549 adenocarcinoma cells and HFL1 lung fibroblasts. Cell colony formation assays were performed to determine the effect of cobrotoxin on A549 cell colony formation, and transmission electron microscopy was used to detect cobrotoxin autophagy. In addition, western blot analysis was performed to determine the effect of 3-methyl adenine (3-MA) activity on the inhibition of autophagy, SB203580 inhibition of the p38-mitogen-activated protein kinase (MAPK) pathway, and Beclin 1, LC3, p62, p38 and phosphorylated (p)-p38 protein expression. Nude mice were injected with human lung A549 cells, and intervention and control groups were compared with regard to tumor suppression. The MTT assay revealed that various concentrations of cobrotoxin inhibited growth of A549 cells, but not HFL1 cells. A549 cell colony formation decreased and autophagosome activity was significantly increased compared with the controls. Following 3-MA administration, SB203580 autophagosome activity decreased, and following cobrotoxin administration, Beclin 1, p-p38, and LC3-II protein expression significantly increased, whereas p62 expression significantly decreased. Following 3-MA inhibition of autophagy, Beclin 1, LC3-II and p62 expression increased. Furthermore, following SB203580 inhibition of the p38-MAPK pathway, Beclin 1, p-p38, LC3-II and p62 protein expression increased. Cobrotoxin exhibited inhibitory activity on the human lung cancer A549 cells transplanted into the nude mice, suppressing the tumor growth rate by 43.4% (cobrotoxin 40 μg/kg group). However, following the addition of 3-MA (10 mmol/kg) and SB203580 (5 mg/kg), the suppression of the tumor growth rate

  12. Ten-eleven translocation 1 functions as a mediator of SOD3 expression in human lung cancer A549 cells.

    Science.gov (United States)

    Kamiya, Tetsuro; Nakahara, Risa; Mori, Namiki; Hara, Hirokazu; Adachi, Tetsuo

    2017-03-01

    Superoxide dismutase (SOD) 3, one of the SOD isozymes, plays a pivotal role in extracellular redox homeostasis. The expression of SOD3 is regulated by epigenetics in human lung cancer A549 cells and human monocytic THP-1 cells; however, the molecular mechanisms governing SOD3 expression have not been elucidated in detail. Ten-eleven translocation (TET), a dioxygenase of 5-methylcytosine (5mC), plays a central role in DNA demethylation processes and induces target gene expression. In the present study, TET1 expression was abundant in U937 cells, but its expression was weakly expressed in A549 and THP-1 cells. These results are consistent with the expression pattern of SOD3 and its DNA methylation status in these cells. Moreover, above relationship was also observed in human breast cancer cells, human prostate cancer cells, and human skin fibroblasts. The overexpression of TET1-catalytic domain (TET1-CD) induced the expression of SOD3 in A549 cells, and this was accompanied by the direct binding of TET1-CD to the SOD3 promoter region. Furthermore, in TET1-CD-transfected A549 cells, the level of 5-hydroxymethylcytosine within that region was significantly increased, whereas the level of 5mC was decreased. The results of the present study demonstrate that TET1 might function as one of the key molecules in SOD3 expression through its 5mC hydroxylation in A549 cells.

  13. The Effect of dcEFs on migration behavior of A549 cells and Integrin beta1 expression

    Directory of Open Access Journals (Sweden)

    Yunjie WANG

    2008-04-01

    Full Text Available Background and objective The effect of direct-current electric fields (dcEFs on cells attracted extensive attention. Moreover the metastasis and its potential are considered to be related to dcEFs. The aim is to study the effect of dcEFs on migration behavior of A549 cells, Integrin ?1 and its signal pathways. Methods According to exposure to 5 V/cm dcEFs or not and the time of exposure, the A549 cells were divided into 4 groups. Images were taken per 5 min within 2 h to recode the migration of the cells. The data of results were analyzed statistically. Results Most of A549cells exposed to the dcEFs aligned and elongated perpendicularly to the electric field lines and migrated to the cathode continually during 2 h. On the contrary, cells unexposed to dcEFs showed slightly random movements. Immunofluorescence showed that Integrin ?1 on plasma membrane polarized to the cathode of the dcEFs. Western blot showed that Integrin beta1 downstream signal pathways p-FAK and p-ERK were overexpressed in the dcEFs. Conclusion A549 cells have a galvanotatic feature of cathodal directed migration while exposed to the dcEFs. The polarization of Integrin beta1 and the promotion of its downstream signal pathways may play an important roles in the galvanotaxis of A549 cells.

  14. Effects of X-ray irradiation on expression of Pokemon gene in A549 cells of human lung adenocarcinoma

    International Nuclear Information System (INIS)

    Wang Lu; Zou Yue; Jiang Qisheng; Li Wei; Song Xiujun; Zhou Xiangyan; Wang Cuilan

    2011-01-01

    Objective: To study the dose-and time-effects of X-ray irradiation on the expression of Pokemon gene in A549 cells of human lung adenocarcinoma. Methods: A549 cells were cultured in vitro and exposed to X-rays with the doses of 2, 4, 6 and 8 Gy, respectively. Untreated A549 cells were used as control group. The relative levels of Pokemon mRNA expression in the cells were detected by using quantitative real-time PCR at 2, 4, 8, 12, 24, 48 and 72 h after irradiation. Results: The Pokemon mRNA expression levels decreased in the early period after irradiation (except 2 and 4 h after irradiation in 2 Gy group) and then increased in the later stage (48 h after irradiation) with significant statistical differences at the most time points in comparison with the control group (t=3.40-154.76, P<0.05). Conclusions: Higher doses of X-rays may degrade the expression of Pokemon mRNA in the human A549 cells and induce apoptosis in the early period, hut also may upgrade its expression in the later period, which might be correlated with the cell cycle regulation and DNA damage repair in the A549 cells. (authors)

  15. Induction of interleukin-6 by coal containing bioavailable iron is ...

    Indian Academy of Sciences (India)

    Unknown

    occupational analogues, also appear ... ered as one of the human lung pathologies related to oxi- dative stress (Castranova and Vallyathan 2000). ... Human lung epithelial cell line, A549, with characteris- tics of alveolar epithelial type II cells (ATCC ...

  16. Apoptosis inducing ability of silver decorated highly reduced graphene oxide nanocomposites in A549 lung cancer

    Directory of Open Access Journals (Sweden)

    Khan M

    2016-03-01

    Full Text Available Merajuddin Khan,1 Mujeeb Khan,1 Abdulhadi H Al-Marri,1 Abdulrahman Al-Warthan,1 Hamad Z Alkhathlan,1 Mohammed Rafiq H Siddiqui,1 Vadithe Lakshma Nayak,2 Ahmed Kamal,2 Syed F Adil1 1Department of Chemistry, College of Science, King Saud University, Riyadh, Kingdom of Saudi Arabia; 2Department of Medicinal Chemistry and Pharmacology, CSIR – Indian Institute of Chemical Technology, Hyderabad, India Abstract: Recently, graphene and graphene-based materials have been increasingly used for various biological applications due to their extraordinary physicochemical properties. Here, we demonstrate the anticancer properties and apoptosis-inducing ability of silver doped highly reduced graphene oxide nanocomposites synthesized by employing green approach. These nano­composites (PGE-HRG-Ag were synthesized by using Pulicaria glutinosa extract (PGE as a reducing agent and were evaluated for their anticancer properties against various human cancer cell lines with tamoxifen as the reference drug. A correlation between the amount of Ag nanoparticles on the surface of highly reduced graphene oxide (HRG and the anticancer activity of nanocomposite was observed, wherein an increase in the concentration of Ag nanoparticles on the surface of HRG led to the enhanced anticancer activity of the nanocomposite. The nanocomposite PGE-HRG-Ag-2 exhibited more potent cytotoxicity than standard drug in A549 cells, a human lung cancer cell line. A detailed investigation was undertaken and Fluorescence activated cell sorting (FACS analysis demonstrated that the nanocomposite PGE-HRG-Ag-2 showed G0/G1 phase cell cycle arrest and induced apoptosis in A549 cells. Studies such as, measurement of mitochondrial membrane potential, generation of reactive oxygen species (ROS and Annexin V-FITC staining assay suggested that this compound induced apoptosis in human lung cancer cells. Keywords: plant extract, graphene/silver nanocomposites, anticancer, apoptosis

  17. Adhesion of MRC-5 and A549 cells on poly(dimethylsiloxane) surface modified by proteins.

    Science.gov (United States)

    Zuchowska, Agnieszka; Kwiatkowski, Piotr; Jastrzebska, Elzbieta; Chudy, Michal; Dybko, Artur; Brzozka, Zbigniew

    2016-02-01

    PDMS is a very popular material used for fabrication of Lab-on-a-Chip systems for biological applications. Although PDMS has numerous advantages, it is a highly hydrophobic material, which inhibits adhesion and proliferation of the cells. PDMS surface modifications are used to enrich growth of the cells. However, due to the fact that each cell type has specific adhesion, it is necessary to optimize the parameters of these modifications. In this paper, we present an investigation of normal (MRC-5) and carcinoma (A549) human lung cell adhesion and proliferation on modified PDMS surfaces. We have chosen these cell types because often they are used as models for basic cancer research. To the best of our knowledge, this is the first presentation of this type of investigation. The combination of a gas-phase processing (oxygen plasma or ultraviolet irradiation) and wet chemical methods based on proteins' adsorption was used in our experiments. Different proteins such as poly-l-lysine, fibronectin, laminin, gelatin, and collagen were incubated with the activated PDMS samples. To compare with other works, here, we also examined how ratio of prepolymer to curing agent (5:1, 10:1, and 20:1) influences PDMS hydrophilicity during further modifications. The highest adhesion of the tested cells was observed for the usage of collagen, regardless of PDMS ratio. However, the MRC-5 cell line demonstrated better adhesion than A549 cells. This is probably due to the difference in their morphology and type (normal/cancer). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Overcoming resistance to mitochondrial apoptosis by BZML-induced mitotic catastrophe is enhanced by inhibition of autophagy in A549/Taxol cells.

    Science.gov (United States)

    Bai, Zhaoshi; Gao, Meiqi; Xu, Xiaobo; Zhang, Huijuan; Xu, Jingwen; Guan, Qi; Wang, Qing; Du, Jianan; Li, Zhengqiang; Zuo, Daiying; Zhang, Weige; Wu, Yingliang

    2018-03-01

    Our previous in vitro study showed that 5-(3, 4, 5-trimethoxybenzoyl)-4-methyl-2-(p-tolyl) imidazol (BZML) is a novel colchicine binding site inhibitor with potent anti-cancer activity against apoptosis resistance in A549/Taxol cells through mitotic catastrophe (MC). However, the mechanisms underlying apoptosis resistance in A549/Taxol cells remain unknown. To clarify these mechanisms, in the present study, we investigated the molecular mechanisms of apoptosis and autophagy, which are closely associated with MC in BZML-treated A549 and A549/Taxol cells. Xenograft NSCLC models induced by A549 and A549/Taxol cells were used to evaluate the efficacy of BZML in vivo. The activation of the mitochondrial apoptotic pathway was assessed using JC-1 staining, Annexin V-FITC/PI double-staining, a caspase-9 fluorescence metric assay kit and western blot. The different functional forms of autophagy were distinguished by determining the impact of autophagy inhibition on drug sensitivity. Our data showed that BZML also exhibited desirable anti-cancer activity against drug-resistant NSCLC in vivo. Moreover, BZML caused ROS generation and MMP loss followed by the release of cytochrome c from mitochondria to cytosol in both A549 and A549/Taxol cells. However, the ROS-mediated apoptotic pathway involving the mitochondria that is induced by BZML was only fully activated in A549 cells but not in A549/Taxol cells. Importantly, we found that autophagy acted as a non-protective type of autophagy during BZML-induced apoptosis in A549 cells, whereas it acted as a type of cytoprotective autophagy against BZML-induced MC in A549/Taxol cells. Our data suggest that the anti-apoptosis property of A549/Taxol cells originates from a defect in activation of the mitochondrial apoptotic pathway, and autophagy inhibitors can potentiate BZML-induced MC to overcome resistance to mitochondrial apoptosis. © 2018 John Wiley & Sons Ltd.

  19. Podophyllotoxin acetate blocks IR-induced invasion of non-small cell lung cancer cell, A549

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Jeong Hyun; Choi, Jae Yeon; Hwang, Sang-Gu; Um, Hong-Duck; Park, Jong Kuk [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2015-05-15

    Some research result presented that local radiotherapy administered to primary tumors speeds their metastatic growth in vivo (4-6), thereby suggesting that besides its therapeutic effects, IR promotes the malignant behaviors of surviving cancer cells. Our findings demonstrate podophyllotoxin acetate (PA), one of new natural products, prevented side effects of IR such as invasion or metastasis promotion for improve the efficacy of radiotherapy. In this study, we demonstrated that PA inhibits IR-induced invasion and migration of A549 cells. We also observed that IR stimulates several intracellular pathway involving EMT and MAPKinses; EMT-associated events including an increase of vimentin levels and increased phosphorylation of p38 ERK, JNK in A549 cells. PA could decrease these activations of several intracellular signaling molecules. Therefore, PA might inhibit IRinduced invasion and migration via blocking EMT and MAPKiase pathway of A549 cells.

  20. Okadaic acid inhibits cell multiplication and induces apoptosis in a549 cells, a human lung adenocarcinoma cell line.

    Science.gov (United States)

    Wang, Renjun; Lv, Lili; Zhao, Yunfeng; Yang, Nana

    2014-01-01

    This essay aims to research the effect of okadaic acid (OA) on A549 cell multiplication, and cell apoptosis induced by OA was observed by cell morphology. MTT assay, trypan blue exclusion test (TBET), Giemsa staining method and acridine orange (AO) fluorescence staining assay were applied. The results of cell survival evaluated by TBET and colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) showed: The number of A549 cells was decreased in a dose-dependent manner. Cytomorphology observation of okadaic acid-treated cells showed that cells became shrinkage and turned round, some cells floated in the nutrient medium with nucleus agglutination broken, resulting in apoptotic bodies. Above-mentioned results indicated that OA exerted significantly inhibitory effect on A549 cell multiplication due to the apoptosis induced by OA.

  1. Microfluidic wound-healing assay to assess the regenerative effect of HGF on wounded alveolar epithelium.

    Science.gov (United States)

    Felder, Marcel; Sallin, Pauline; Barbe, Laurent; Haenni, Beat; Gazdhar, Amiq; Geiser, Thomas; Guenat, Olivier

    2012-02-07

    We present a microfluidic epithelial wound-healing assay that allows characterization of the effect of hepatocyte growth factor (HGF) on the regeneration of alveolar epithelium using a flow-focusing technique to create a regular wound in the epithelial monolayer. The phenotype of the epithelial cell was characterized using immunostaining for tight junction (TJ) proteins and transmission electron micrographs (TEMs) of cells cultured in the microfluidic system, a technique that is reported here for the first time. We demonstrate that alveolar epithelial cells cultured in a microfluidic environment preserve their phenotype before and after wounding. In addition, we report a wound-healing benefit induced by addition of HGF to the cell culture medium (19.2 vs. 13.5 μm h(-1) healing rate).

  2. Acid-sensing ion channels contribute to the effect of extracellular acidosis on proliferation and migration of A549 cells.

    Science.gov (United States)

    Wu, Yu; Gao, Bo; Xiong, Qiu-Ju; Wang, Yu-Chan; Huang, Da-Ke; Wu, Wen-Ning

    2017-06-01

    Acid-sensing ion channels, a proton-gated cation channel, can be activated by low extracellular pH and involved in pathogenesis of some tumors such as glioma and breast cancer. However, the role of acid-sensing ion channels in the growth of lung cancer cell is unclear. In this study, we investigated the expression of acid-sensing ion channels in human lung cancer cell line A549 and their possible role in proliferation and migration of A549 cells. The results show that acid-sensing ion channel 1, acid-sensing ion channel 2, and acid-sensing ion channel 3 are expressed in A549 cells at the messenger RNA and protein levels, and acid-sensing ion channel-like currents were elicited by extracellular acid stimuli. Moreover, we found that acidic extracellular medium or overexpressing acid-sensing ion channel 1a promotes proliferation and migration of A549 cells. In addition psalmotoxin 1, a specific acid-sensing ion channel 1a inhibitor, or acid-sensing ion channel 1a knockdown can abolish the effect of acid stimuli on A549 cells. In addition, acid-sensing ion channels mediate increase of [Ca 2+ ] i induced by low extracellular pH in A549 cells. All these results indicate that acid-sensing ion channel-calcium signal mediate lung cancer cell proliferation and migration induced by extracellular acidosis, and acid-sensing ion channels may serve as a prognostic marker and a therapeutic target for lung cancer.

  3. TXNIP mediates the differential responses of A549 cells to sodium butyrate and sodium 4-phenylbutyrate treatment.

    Science.gov (United States)

    Jin, Xuefang; Wu, Nana; Dai, Juji; Li, Qiuxia; Xiao, XiaoQiang

    2017-02-01

    Sodium butyrate (NaBu) and sodium 4-phenylbutyrate (4PBA) have promising futures in cancer treatment; however, their underlying molecular mechanisms are not clearly understood. Here, we show A549 cell death induced by NaBu and 4PBA are not the same. NaBu treatment induces a significantly higher level of A549 cell death than 4PBA. A gene expression microarray identified more than 5000 transcripts that were altered (>1.5-fold) in NaBu-treated A549 cells, but fewer than 2000 transcripts that were altered in 4PBA. Moreover, more than 100 cell cycle-associated genes were greatly repressed by NaBu, but slightly repressed by 4PBA; few genes were significantly upregulated only in 4PBA-treated cells. Gene expression was further validated by other experiments. Additionally, A549 cells that were treated with these showed changes in glucose consumption, caspase 3/7 activation and histone modifications, as well as enhanced mitochondrial superoxide production. TXNIP was strongly induced by NaBu (30- to 40-fold mRNA) but was only slightly induced by 4PBA (two to fivefold) in A549 cells. TXNIP knockdown by shRNA in A549 cells significantly attenuated caspase 3/7 activation and restored cell viability, while TXNIP overexpression significantly increased caspase 3/7 activation and cell death only in NaBu-treated cells. Moreover, TXNIP also regulated NaBu- but not 4PBA-induced H4K5 acetylation and H3K4 trimethylation, possibly by increasing WDR5 expression. Finally, we demonstrated that 4PBA induced a mitochondrial superoxide-associated cell death, while NaBu did so mainly through a TXNIP-mediated pathway. The above data might benefit the future clinic application. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

  4. Curcumin protects against cytotoxic and inflammatory effects of quartz particles but causes oxidative DNA damage in a rat lung epithelial cell line

    International Nuclear Information System (INIS)

    Li Hui; Berlo, Damien van; Shi Tingming; Speit, Guenter; Knaapen, Ad M.; Borm, Paul J.A.; Albrecht, Catrin; Schins, Roel P.F.

    2008-01-01

    Chronic inhalation of high concentrations of respirable quartz particles has been implicated in various lung diseases including lung fibrosis and cancer. Generation of reactive oxygen species (ROS) and oxidative stress is considered a major mechanism of quartz toxicity. Curcumin, a yellow pigment from Curcuma longa, has been considered as nutraceutical because of its strong anti-inflammatory, antitumour and antioxidant properties. The aim of our present study was to investigate whether curcumin can protect lung epithelial cells from the cytotoxic, genotoxic and inflammatory effects associated with quartz (DQ12) exposure. Electron paramagnetic resonance (EPR) measurements using the spin-trap DMPO demonstrated that curcumin reduces hydrogen peroxide-dependent hydroxyl-radical formation by quartz. Curcumin was also found to reduce quartz-induced cytotoxicity and cyclooxygenase 2 (COX-2) mRNA expression in RLE-6TN rat lung epithelial cells (RLE). Curcumin also inhibited the release of macrophage inflammatory protein-2 (MIP-2) from RLE cells as observed upon treatment with interleukin-1 beta (IL-1β) and tumour necrosis factor-alpha (TNFα). However, curcumin failed to protect the RLE cells from oxidative DNA damage induced by quartz, as shown by formamidopyrimidine glycosylase (FPG)-modified comet assay and by immunocytochemistry for 8-hydroxydeoxyguanosine. In contrast, curcumin was found to be a strong inducer of oxidative DNA damage itself at non-cytotoxic and anti-inflammatory concentrations. In line with this, curcumin also enhanced the mRNA expression of the oxidative stress response gene heme oxygenase-1 (ho-1). Curcumin also caused oxidative DNA damage in NR8383 rat alveolar macrophages and A549 human lung epithelial cells. Taken together, these observations indicate that one should be cautious in considering the potential use of curcumin in the prevention or treatment of lung diseases associated with quartz exposure

  5. F10, a novel hydatidiform mole-associated gene, inhibits the paclitaxel sensitivity of A549 lung cancer cells by downregulating BAX and caspase-3.

    Science.gov (United States)

    Song, Yali; Cao, Wei; Zhu, Xi; Qiu, Zhuolin; Yang, Xiaoping; Liu, Jing; Xu, Ruoting; Yuan, Weizhuang; Quan, Song

    2017-04-01

    F10 is a novel hydatidiform mole (HM)-associated gene that was initially identified during a study into the pathogenesis of HMs. However, the role of the F10 gene requires further investigation. Our, previous studies have indicated that F10 may be involved in the malignant transformation of HMs and the development of certain types of adenocarcinoma, and that the overexpression of F10 may lead to excessive proliferation and decreased apoptosis of A549 cells. The present study aimed to investigate whether F10 may suppress the sensitivity of A549 lung cancer cells to paclitaxel therapy. A previously established F10-overexpressing A549 cell line (A549-F10) was treated with paclitaxel, using untransfected A549 cells and A549-mock cells (non-carrier A549) as the controls. These three groups of cells were subsequently examined by an MTT cell proliferation assay and a TUNEL-fluorescein isothiocyanate/Hoechst 33258 apoptosis assay. A western blot analysis was used to determine the expression levels of the pro-apoptotic genes B-cell lymphoma-2-associated X protein (BAX) and caspase-3. The effects of paclitaxel treatment on the proliferation and apoptosis of A549 cells were compared between the aforementioned cell lines. It was revealed that F10 inhibited the chemosensitivity of A549 cells to paclitaxel, as demonstrated by the decreased rates of growth inhibition and apoptosis in the A549-F10 group compared with the two control groups. Furthermore, the A549-F10 cells treated with paclitaxel exhibited significantly lower expression levels of the pro-apoptotic genes. The results of the current study demonstrate that F10 may inhibit the chemosensitivity of A549 cells to paclitaxel and that this inhibitory effect may be mediated by the downregulation of BAX and caspase-3 expression, which subsequently inhibits cell apoptosis.

  6. [SIRT1 Influences the Sensitivity of A549 Non-small Cell Lung Cancer Cell Line to 
Cisplatin via Modulating the Noxa Expression].

    Science.gov (United States)

    Cao, Bin; He, Xiaofeng; Wang, Wengong; Shi, Minke

    2016-02-01

    The resistance of non-small cell lung cancer cells to cisplant is a common clinical phenomenon which could induce a poor therapeutic effect and should be difficult problem to be solved. SIRT1 and Noxa expression are associated with the chemotherapy for tumors. The present study focused on how SIRT1 expression influence the senstivity of non-small cell lung cancer cells and dissected the potential mechanism involved with Noxa. The difference of SIRT1 and Noxa expression between A549 cells and A549/DDP cells was detected by real-time quantitative PCR (qRT-PCR) and Western blot. SIRT1 targeted siRNA was uesed to inhibit the SIRT1 expression in A549/DDP, after transfection, Cell Titer Blue assay, flow cytometry were performed to analyze the cell viability, cell cycle and cell apoptosis in order to reveal the effect of inhibition of SIRT1 on sensitivity of A549/DDP cells to cisplant. Moreover, the expression changes of Noxa in A549/DDP cells after siRNA treatment were detected by qRT-PCR and Western blot. There was a significant difference in senstivity to cisplant between A549 and A549/DDP cells. Compared with A549 cells, the A549/DDP cells showed a higher SIRT1 expression and lower Noxa expression. After transfected with SIRT1 targeted siRNA, the cell viability decreased accompanied with a increasing apoptosis rate, meanwhile, higher percent of G2/M phase was detected after the 4 μg/mL cisplant treatment. Further more, inhibition of SIRT1 could induce the Noxa expression in A549/DDP cells. Higher SIRT1 expression may induce resistance to cisplant in A549 cells. SIRT1 inhibition may improve the sensitivity of A549/DDP cells to cisplantin though modulating the Noxa expression.
.

  7. [Downregulation of miR-18a or miR-328 inhibits the invasion and migration of lung adenocarcinoma A549 cells].

    Science.gov (United States)

    DU, Chuanchong; Zheng, Jinxu; Lu, Xiaowei; Wang, Yi

    2016-08-01

    Objective To investigate the expressions of miR-18a and miR-328 in lung adenocarcinoma A549 cells, and explore the effect of miR-18a or miR-328 on invasion and migration of A549 cells. Methods The expressions of miR-18a and miR-328 in A549 cells were detected by real-time quantitative PCR. Then the specific miR-18a or miR-328 inhibitor sequences were transfected into A549 cells to downregulate the expression of miR-18a or miR-328. The invasion and migration abilities of A549 cells were evaluated by Transwell(TM) assay. Results The miR-18a and miR-328 were overexpressed in A549 cells. And with the corresponding inhibitors being transfected, the expressions of miR-18a and miR-328 in A549 cells were downregulated. In addition, TranswellTM assay showed that decreased expression of miR-18a or miR-328 significantly inhibited the invasion and migration of A549 cells. Conclusion Downregulation of miR-18a or miR-328 can inhibit the invasion and migration abilities of A549 cells effectively.

  8. Effects of RNAi-mediated TUSC3 silencing on radiation-induced autophagy and radiation sensitivity of human lung adenocarcinoma cell line A549 under hypoxic condition.

    Science.gov (United States)

    Li, Ya-Guang; Liang, Nai-Xin; Qin, Ying-Zhi; Ma, Dong-Jie; Huang, Chang-Jin; Liu, Lei; Li, Shan-Qing

    2016-11-30

    This study examined the effects of RNAi-mediated TUSC3 silencing on radiation-induced autophagy and radiation sensitivity of human lung adenocarcinoma cell line A549 under hypoxic condition. Different CoCl 2 concentrations were used to treat A549 cells and establish a CoCl 2 -induced hypoxic model of A549 cells. MTT and clone formation assays were used to determine the effects of different concentrations of CoCl 2 on the growth and proliferation of A549 cells treated by different doses of X-ray irradiation. The siRNA-expressing vector was transfected by liposomes and for silencing of TUSC3. Flow cytometry was used to measure cell cycle changes and apoptosis rate. Real-time quantitative polymerase chain reaction (qRT-PCR) assay was performed to detect the expression of TUSC3 mRNA. Western blotting was applied to detect the changes of TUSC3, LC3, and p62 proteins under different CoCl 2 concentrations and after siRNA silencing of TUSC3. The TUSC3 levels in A549 cells increased under hypoxic conditions in a dose-dependent manner (P A549 cells and promoted apoptosis (P A549 cells showed significantly increased growth and proliferation and decreased apoptosis (P A549 cell growth and proliferation after radiotherapy under hypoxic condition, promoted apoptosis, increased G0/G1 phase cells, and reduced S phase cells (all P A549 lung adenocarcinoma cells.

  9. Interleukin-4-mediated 15-lipoxygenase-1 trans-activation requires UTX recruitment and H3K27me3 demethylation at the promoter in A549 cells.

    Directory of Open Access Journals (Sweden)

    Hongya Han

    Full Text Available Arachidonate 15-lipoxygenase-1 (ALOX15 oxygenates polyunsaturated fatty acids and bio-membranes, generating multiple lipid signalling mediators involved in inflammation. Several lines of evidence indicate that ALOX15 activation in the respiratory tract contributes to asthma progression. Recent experimental data reveals that histone modification at the promoter plays a critical role in ALOX15 gene transcription. In the present study, we examined the status of histone H3 trimethyl-lysine 27 (H3K27me3 at the ALOX15 promoter by chromatin immunoprecipitation assay in human lung epithelial carcinoma A549 cells incubated with or without interleukin (IL-4. We identified demethylation of H3K27me3 at the ALOX15 promoter after IL-4 treatment. Furthermore, we found that the H3K27me2/3-specific demethylase, ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX, mediates the H3K27me3 demethylation during ALOX15 transcriptional activation. When UTX expression was knocked down using siRNA, IL-4-mediated H3K27me3 demethylation and ALOX15 induction were significantly attenuated. The critical role of UTX in ALOX15 expression was confirmed in human monocytes and the Hodgkin lymphoma (HL cell line L1236, but was in these cells not related to H3K27me3-demethylase activity. These results demonstrate that UTX is implicated in IL-4 mediated transcriptional activation of the ALOX15 gene.

  10. Inhibition of heme oxygenase-1 enhances the radiosensitivity in human nonsmall cell lung cancer a549 cells.

    Science.gov (United States)

    Zhang, Wenyi; Qiao, Tiankui; Zha, Lin

    2011-10-01

    Abstract undergoing radiotherapy or chemotherapy failed to respond. The aim of this study was to evaluate whether Inhibitor of HO-1, zinc protoporphyrin IX (Znpp), enhances the radiosensitivity in human nonsmall cell lung cancer (NSCLC) A549 Cells. A549 cells were induced by Znpp and irradiated by X-rays. Then, expression of HO-1 was measured by real-time polymerase chain reaction. Cell survival was evaluated using the MTS assay and the clonogenic survival assay; apoptosis and cell cycle distribution were monitored by flow cytometry. First, overexpression of the HO-1 mRNA was found in treatment with irradiation alone in A549 cells, and expression of the HO-1 mRNA was reduced after combined treatments with 12 μmol/L of Znpp and irradiation. Second, diminished cell viability percentage, decreased cell clonogenic survival fraction, enhanced cell apoptotic index, and increased percentage of cells in the G1 phase were found after combined treatments with 12 μmol/L of Znpp and irradiation compared to either treatment alone (pZnpp, can increase the radiosensitivity of human NSCLC A549 cells.

  11. Enhanced production of nitric oxide in A549 cells through activation of TRPA1 ion channel by cold stress.

    Science.gov (United States)

    Sun, Wenwu; Wang, Zhonghua; Cao, Jianping; Wang, Xu; Han, Yaling; Ma, Zhuang

    2014-08-31

    The respiratory epithelium is exposed to the external environment, and inhalation of cold air is common during the season of winter. In addition, the lung is a major source of nitric oxide (NO). However, the effect of cold stress on the production of NO is still unclear. In the present work, We measured the change of NO in single cell with DACF-DA and the change in cytosolic Ca(2+) concentration ([Ca(2+)]c) in A549 cell. We observed that cold stress (from 20 °C to 5 °C) induced an increase of NO in A549 cell, which was completely abolished by applying an extracellular Ca(2+) free medium. Further experiments showed that cold-sensing transient receptor potential subfamily member 1 (TRPA1) channel agonist (allyl isothiocyanate, AITC) increased the production of NO and the level of [Ca(2+)]c in A549 cell. Additionally, TRPA1 inhibitor, Ruthenium red (RR) and camphor, significantly blocked the enhanced production of NO and the rise of [Ca(2+)]c induced by AITC or cold stimulation, respectively. Taken together, these data indicated that cold-induced TRPA1 activation was responsible for the enhanced production of NO in A549 cell. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. The development and plasticity of alveolar type 1 cells

    Science.gov (United States)

    Yang, Jun; Hernandez, Belinda J.; Martinez Alanis, Denise; Narvaez del Pilar, Odemaris; Vila-Ellis, Lisandra; Akiyama, Haruhiko; Evans, Scott E.; Ostrin, Edwin J.; Chen, Jichao

    2016-01-01

    Alveolar type 1 (AT1) cells cover >95% of the gas exchange surface and are extremely thin to facilitate passive gas diffusion. The development of these highly specialized cells and its coordination with the formation of the honeycomb-like alveolar structure are poorly understood. Using new marker-based stereology and single-cell imaging methods, we show that AT1 cells in the mouse lung form expansive thin cellular extensions via a non-proliferative two-step process while retaining cellular plasticity. In the flattening step, AT1 cells undergo molecular specification and remodel cell junctions while remaining connected to their epithelial neighbors. In the folding step, AT1 cells increase in size by more than 10-fold and undergo cellular morphogenesis that matches capillary and secondary septa formation, resulting in a single AT1 cell spanning multiple alveoli. Furthermore, AT1 cells are an unexpected source of VEGFA and their normal development is required for alveolar angiogenesis. Notably, a majority of AT1 cells proliferate upon ectopic SOX2 expression and undergo stage-dependent cell fate reprogramming. These results provide evidence that AT1 cells have both structural and signaling roles in alveolar maturation and can exit their terminally differentiated non-proliferative state. Our findings suggest that AT1 cells might be a new target in the pathogenesis and treatment of lung diseases associated with premature birth. PMID:26586225

  13. [Effect of Nm23-H1 Nuclear Localization on Proliferation of 
Human Lung Adenocarcinoma Cell Line A549].

    Science.gov (United States)

    Sheng, Ya; Xiong, Yanli; Xu, Mingfang; Kuang, Xunjie; Wang, Dong; Yang, Xueqin

    2017-04-20

    Recent studies have indicated that Nm23-H1 is found in the nucleus, but previous studies have been based on the overexpression or suppression of Nm23-H1 in the cytoplasm. Due to the lacking nuclear localization signal of Nm23-H1, these results cannot reflect or repeat cells in which Nm23-H1 mainly positioned in nuclei and whether they cause clinical biological effects. Therefore, to explore the effects of transposing Nm23-H1 from the cytoplasm to the nucleus during lung cancer cell proliferation, a vector with a nuclear localization signal of Nm23-H1 was constructed and A549 cells were transfected. Gene recombination technology was used to construct pLentis-CMV-NME1-IRES2-PURO lentiviral vectors using a nuclear localization signal sequence, and the recombinant plasmid was verified using restriction enzyme analysis and sequencing. Nm23-H1 positioning and expression were performed after the stably transfected A549 cells were assessed by Western blot and confocal laser scanning microscope. The A549 cell proliferation was assessed using a cell counting kit-8. Flow cytometry was performed to assess the cell cycle distribution of A549 cells. The directional Nm23-H1 lentiviral vector was successfully constructed within the nucleus. Compared with that of the empty vector group, the proliferation rates of the transfection groups at 72 h, 96 h, and 120 h were remarkably increased (PA549 cells in the G0/G1 phase proportion was 35.69%, which was higher than the 28.28% of the transfection group (t=1.461, P=0.217); furthermore, the transfection group of A549 cells in the G2/M phase proportion was 58.7% and that of the empty vector group was 31.30% (t=4.560, P=0.010). Human lung adenocarcinoma cell line A549 cells of Nm23-H1 nuclear localized mainly in the G2/M phase and the nuclear Nm23-H1 promoted A549 cell proliferation in vitro.

  14. Down-regulated βIII-tubulin Expression Can Reverse Paclitaxel Resistance in A549/Taxol Cells Lines

    Directory of Open Access Journals (Sweden)

    Yinling ZHUO

    2014-08-01

    Full Text Available Background and objective Chemotherapy drug resistance is the primary causes of death in patients with pulmonary carcinoma which make tumor recurrence or metastasis. β-tubulin is the main cell targets of anti-microtubule drug. Increased expression of βIII-tubulin has been implicated in non-small cell lung cancer (NSCLC cell lines. To explore the relationship among the expression level of βIII-tubulin and the sensitivity of A549/Taxolcell lines to Taxol and cell cycles and cell apoptosis by RNA interference-mediated inhibition of βIII-tubulin in A549/Taxol cells. Methods Three pairs of siRNA targetd βIII-tubulin were designed and prepared, which were transfected into A549/Taxol cells using LipofectamineTM 2000. We detected the expression of βIII-tubulin mRNA using Real-time fluorescence qRT-PCR. Tedhen we selected the most efficient siRNA by the expression of βIII-tubulin mRNA in transfected group. βIII-tubulin protein level were mesured by Western blot. The taxol sensitivity in transfected group were evaluated by MTT assay. And the cell apoptosis and cell cycles were determined by flow cytometry. Results βIII-tubulin mRNA levels in A549/Taxol cells were significantly decreased in transfected grop by Real-time qRT-PCR than control groups. And βIII-tubulin siRNA-1 sequence showed the highest transfection efficiency, which was (87.73±4.87% (P<0.01; Western blot results showed that the expressional level of BIII tublin protein was significantly down-reulated in the transfectant cells than thant in the control cells. By MTT assay, we showed that the inhibition ratio of Taxol to A549/Taxol cells transfeced was higher than that of control group (51.77±4.60% (P<0.01. The early apoptosis rate of A549/Taxol cells in transfected group were significantly higher than that of control group (P<0.01; G2-M content in taxol group obviously increased than untreated samples by the cell cycle (P<0.05. Conclusion βIII-tubulin down-regulated significantly

  15. p53 alterations in atypical alveolar hyperplasia of the human lung

    NARCIS (Netherlands)

    Slebos, R. J.; Baas, I. O.; Clement, M. J.; Offerhaus, G. J.; Askin, F. B.; Hruban, R. H.; Westra, W. H.

    1998-01-01

    Atypical alveolar hyperplasia (AAH) is a potential precursor lesion from which lung adenocarcinomas arise and may be a good target for studying the early events of lung tumorigenesis. We have previously shown that AAHs are neoplastic epithelial proliferations that often harbor activating mutations

  16. Nanoparticles of Selaginella doederleinii leaf extract inhibit human lung cancer cells A549

    Science.gov (United States)

    Syaefudin; Juniarti, A.; Rosiyana, L.; Setyani, A.; Khodijah, S.

    2016-01-01

    The aim of the present study is to evaluate cytotoxicity effect of nanoparticles of Selaginella doederleinii (S. doederleinii) leaves extract. S. doederleinii was extracted by maceration method using 70%(v/v) ethanol as solvent. Phytochemical content was analyzed qualitatively by using Harborne and Thin Layer Chromatography (TLC) methods. Nanoparticle extract was prepared by ionic gelation using chitosan as encapsulant agent. Anticancer activity was performed by using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The results showed that S. doederleinii contains of flavonoids. Nanoparticle of S. doederleinii leaves extract greatly inhibited A549 cells growth (cancer cells), with IC50 of 3% or 1020 μg/ml. These nanoparticles extract also inhibited the growth of Chang cells (normal cells), with IC50 of 4% or 1442 μg/ml. The effective concentration of nanoparticles extract which inhibits cancer cells without harming the normal cells is 0.5% or 167 μg/ml. Further studies are needed to obtain the concentration of nanoparticles extract which can selectively suppress cancer cells.

  17. Nimesulide has a role of radio-sensitizer against lung carcinoma A549 cells

    Energy Technology Data Exchange (ETDEWEB)

    Won, Joo Yoon; Park, Jong Kuk; Hong, Sung Hee [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2006-07-01

    Cyclooxygenases (COX) are key enzymes in the prostaglandin synthesis. There are two isoforms of the COX enzyme, COX-1 and COX-2. COX-2 expression is associated with carcinogenesis in variety of cancers and to render cells resistant to apoptotic stimuli. Increased expression of COX-2 is shown in non-small cell lung cancer (NSCLC), specifically in adenocarcinomas. Radiotherapy has been the important treatment for NSCLC. In recent studies, newer molecules that target specific pathophysiology or molecular pathways have been tested for the radiation sensitizers. COX-2 inhibitors are shown to enhanced radioresponse of cultured human cancer cell lines and immunodeficient mice. However, little is known about the molecular and biochemical mechanisms how NSAIDs enhance radioresponse of tumor cells. Nimesulide (methanesulfonamide, N-(4-nitro-2- phenoxyphenyl)), selective COX-2 inhibitors, is a drug with anti-inflammatory, anti-pyretic and analgesic properties. Nimesulide has the specific affinity to inhibit the inducible form of cyclooxygenase (COX-2) rather than the constitutive form (COX-1), and is well tolerated by adult, elderly and pediatric patients. Nimesulide was found also to have a chemopreventive activity against colon, urinary bladder, breast, tongue, and liver carcinogenesis. In this study, we examined whether nimesulide can increase radiation induced cell death and its mechanism in NSCLC cells A549.

  18. The effects of dietary phenolic compounds on cytokine and antioxidant production by A549 cells.

    Science.gov (United States)

    Gauliard, Benoit; Grieve, Douglas; Wilson, Rhoda; Crozier, Alan; Jenkins, Carol; Mullen, William D; Lean, Michael

    2008-06-01

    Levels of inflammatory cytokines are raised in chronic obstructive pulmonary disease (COPD). A diet rich in antioxidant vitamins may protect against the development of COPD. This study examined the effects of phenolic compounds and food sources on cytokine and antioxidant production by A549 cells. The effects of the following phenolic compounds on basal and interleukin (IL)-1-stimulated release of IL-8, IL-6, and reduced glutathione (GSH) were examined: resveratrol; Bouvrage, a commercially available raspberry juice (Ella Drinks Ltd., Alloa, Clacksmannanshire, UK); and quercetin 3'-sulfate. Purification of the raspberry juice by high-performance liquid chromatography gave three fractions: Fraction 1 contained phenolic acid and vitamin C, Fraction 2 contained flavonoids and ellagic acid, and Fraction 3 contained anthocyanins and ellagitannins. IL-8 production was increased in the presence of IL-1 (165 vs. 6,011 pg/mL, P or =50 micromol/mL significantly inhibited IL-8 and IL-6 production. Similar findings were made with raspberry juice at concentrations > or =25 microL/mL, and Fractions 1 and 3 were best able to inhibit IL-8 production. Quercetin 3'-sulfate, at 25 micromol/mL, inhibited IL-8 and IL-6 production. The changes observed in IL-8 were paralleled by changes in tumor necrosis factor-alpha. Thus, phenolic compounds can significantly alter cytokine and antioxidant production.

  19. Cytotoxic Effect of a Novel Synthesized Carbazole Compound on A549 Lung Cancer Cell Line.

    Directory of Open Access Journals (Sweden)

    Refilwe P Molatlhegi

    Full Text Available Increased death rates due to lung cancer have necessitated the search for potential novel anticancer compounds such as carbazole derivatives. Carbazoles are aromatic heterocyclic compounds with anticancer, antibacterial and anti-inflammatory activity. The study investigated the ability of the novel carbazole compound (Z-4-[9-ethyl-9aH-carbazol-3-yl amino] pent-3-en-2-one (ECAP to induce cytotoxicity of lung cancer cells and its mechanism of action. ECAP was synthesized as a yellow powder with melting point of 240-247 °C. The 3-(4,5-dimethythiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT, lipid peroxidation and comet assays were used to assess the cytotoxic effect of the compound on A549 lung cancer cells. Protein expression was determined using western blots, apoptosis was measured by luminometry (caspase-3/7, -8 and -9 assay and flow cytometry was used to measure phosphatidylserine (PS externalisation. ECAP induced a p53 mediated apoptosis of lung cancer cells due to a significant reduction in the expression of antioxidant defence proteins (Nrf2 and SOD, Hsp70 (p < 0.02 and Bcl-2 (p < 0.0006, thereby up-regulating reactive oxygen species (ROS production. This resulted in DNA damage (p < 0.0001, up-regulation of Bax expression and caspase activity and induction of apoptosis in lung cancer cells. The results show the anticancer potential of ECAP on lung cancer.

  20. Combined toxic effect of airborne heavy metals on human lung cell line A549.

    Science.gov (United States)

    Choi, Yeowool; Park, Kihong; Kim, Injeong; Kim, Sang D

    2018-02-01

    Many studies have demonstrated that heavy metals existing as a mixture in the atmospheric environment cause adverse effects on human health and are important key factors of cytotoxicity; however, little investigation has been conducted on a toxicological study of a metal mixture from atmospheric fine particulate matter. The objective of this study was to predict the combined effects of heavy metals in aerosol by using in vitro human cells and obtain a suitable mixture toxicity model. Arsenic, nickel, and lead were selected for mixtures exposed to A549 human lung cancer cells. Cell proliferation (WST-1), glutathione (GSH), and interleukin (IL)-8 inhibition were observed and applied to the prediction models of mixture toxicity, concentration addition (CA) and independent action (IA). The total mixture concentrations were set by an IC 10 -fixed ratio of individual toxicity to be more realistic for mortality and enzyme inhibition tests. The results showed that the IA model was statistically closer to the observed results than the CA model in mortality, indicating dissimilar modes of action. For the GSH inhibition, the results predicted by the IA and CA models were highly overestimated relative to mortality. Meanwhile, the IL-8 results were stable with no significant change in immune reaction related to inflammation. In conclusion, the IA model is a rapid prediction model in heavy metals mixtures; mortality, as a total outcome of cell response, is a good tool for demonstrating the combined toxicity rather than other biochemical responses.

  1. Tissue factor deficiency increases alveolar hemorrhage and death in influenza A virus-infected mice.

    Science.gov (United States)

    Antoniak, S; Tatsumi, K; Hisada, Y; Milner, J J; Neidich, S D; Shaver, C M; Pawlinski, R; Beck, M A; Bastarache, J A; Mackman, N

    2016-06-01

    Essentials H1N1 Influenza A virus (IAV) infection is a hemostatic challenge for the lung. Tissue factor (TF) on lung epithelial cells maintains lung hemostasis after IAV infection. Reduced TF-dependent activation of coagulation leads to alveolar hemorrhage. Anticoagulation might increase the risk for hemorrhages into the lung during severe IAV infection. Background Influenza A virus (IAV) infection is a common respiratory tract infection that causes considerable morbidity and mortality worldwide. Objective To investigate the effect of genetic deficiency of tissue factor (TF) in a mouse model of IAV infection. Methods Wild-type mice, low-TF (LTF) mice and mice with the TF gene deleted in different cell types were infected with a mouse-adapted A/Puerto Rico/8/34 H1N1 strain of IAV. TF expression was measured in the lungs, and bronchoalveolar lavage fluid (BALF) was collected to measure extracellular vesicle TF, activation of coagulation, alveolar hemorrhage, and inflammation. Results IAV infection of wild-type mice increased lung TF expression, activation of coagulation and inflammation in BALF, but also led to alveolar hemorrhage. LTF mice and mice with selective deficiency of TF in lung epithelial cells had low basal levels of TF and failed to increase TF expression after infection; these two strains of mice had more alveolar hemorrhage and death than controls. In contrast, deletion of TF in either myeloid cells or endothelial cells and hematopoietic cells did not increase alveolar hemorrhage or death after IAV infection. These results indicate that TF expression in the lung, particularly in epithelial cells, is required to maintain alveolar hemostasis after IAV infection. Conclusion Our study indicates that TF-dependent activation of coagulation is required to limit alveolar hemorrhage and death after IAV infection. © 2016 International Society on Thrombosis and Haemostasis.

  2. Bulky PAH-DNA induced by exposure of a co-culture model of human alveolar macrophages and embryonic epithelial cells to atmospheric particulate pollution; Adduits encombrants a l'ADN dans des cocultures de cellules pulmonaires humaines exposees a une pollution atmospherique particulaire

    Energy Technology Data Exchange (ETDEWEB)

    Abbas, Imane; Garcon, Guillaume; Billet, Sylvain; Shirali, Pirouz [Universite Lille Nord de France - Lille (France); Unite de Chimie Environnementale et Interactions sur le Vivant, MREI, Universite du Littoral Cote d' Opale, Dunkerque (France); Andre, Veronique; Le Goff, Jeremie; Sichel, Francois [GRECAN, Universite de Caen Basse-Normandie et centre Francois Baclesse, Caen (France); Roy Saint-Georges, Francoise; Mulliez, Philippe [Service de Pneumologie, Hopital Saint-Philibert, GHICL, Lille (France)

    2012-01-15

    Because of their deep penetration in human lungs, fine airborne particulate matter were described as mainly responsible for the deleterious effects of exposure to air pollution on health. Organic constituents are adsorbed on particles surface and, after inhalation, some (polycyclic aromatic hydrocarbons, PAHs) can be activated into reactive metabolites and can bind to DNA. The formation of bulky DNA adducts has been researched after exposure of mono-and co-cultures of alveolar macrophages (AM) and human embryonic human lung epithelial (L132), to fine air pollution particulate matter Air samples have been collected with cascade impactor and characterized: size distribution (92.15% < 2.5{mu}.m), specific surface area (1 m{sup 2}/g), inorganic (Fe, AI, Ca, Na, K, Mg, Pb, etc.) and organic compounds (PAHs, etc.). {sup 32}P post-labeling method was applied to detect bulky DNA adducts in AM and L132, in mono-and co-cultures, 72 h after their exposure to atmospheric particles at their Lethals and Effects concentrations or (LC or CE) to 50% (i.e. MA: EC{sub 50} = 74.63 {mu}g/mL and L132: LC-5-0 = 75.36 {mu}g/mL). Exposure to desorbed particles (MA: C1= 61.11 {mu}g/mL and L132 : C2 = 61.71 {mu}g/mL) and B[a]P (1 {mu}M) were included. Bulky PAH-DNA adducts were detected in AM in mono-culture after exposure to total particles (Pt), to B[a]P and desorbed particles (Pd). Whatever the exposure, no DNA adduct was detected in L132 in mono-culture. These results are coherent with the enzymatic activities of cytochrome P450 l Al in AM and L132. Exposure of co-culture to Pt, or Pd induced bulky adducts to DNA in AM but not in L132. Exposure to B[a]P alone has altered the DNA of AM and L132, in co-culture. Exposure to Pt is closer to the environmental conditions, but conferred an exposure to amounts of genotoxic agents compared to studies using organic extracts. The formation of bulky DNA adducts was nevertheless observed in AM exposed to Pt, in mono- or co-culture, indicating that

  3. Tripchlorolide induces autophagy in lung cancer cells by inhibiting the PI3K/AKT/mTOR pathway and improves cisplatin sensitivity in A549/DDP cells.

    Science.gov (United States)

    Chen, Li-Min; Song, Tian-Jiao; Xiao, Jian-Hong; Huang, Zheng-Hui; Li, Yong; Lin, Ting-Yan

    2017-09-08

    Tripchlorolide (T4) has been shown to induce A549 lung cancer cell death predominantly by activating an autophagy pathway. However, the underlying mechanism remains unclear. Herein, we demonstrated that compared with T4 treatment alone, pretreatment with wortmannin (an inhibitor of phosphatidylinositol 3-kinase), perifosine (an inhibitor of AKT) or rapamycin (an inhibitor of mTOR) combined with a subsequent T4 treatment significantly impaired the cell viability of A549 and A549/DDP lung cancer cells. We found that either treatment scheme markedly reduced the activity of P13K and AKT. Expression of LC3II increased in parallel to the increase of the T4 concentration in both A549 and A549/DDP cells and was repressed by overexpression of AKT. The expression levels of PI3-K, PI3-P, AKT, TSC2, mTOR, p70S6K and 4E-BP1 were minimally affected by the wortmannin, perifosine, or rapamycin plus T4 treatments, but their phosphorylated products were greatly affected in A549 lung cancer cells and slightly affected in A549/DDP lung cancer cells. These results indicate that T4 induces autophagy in lung cancer cells by inhibiting the PI3K/AKT/mTOR signaling pathway. We further found that T4 decreased expression of MDR1 and improved cisplatin sensitivity of A549/DDP cells. Altogether, these results have meaningful implications for tumor therapy in the future.

  4. Elevated pressure, a novel cancer therapeutic tool for sensitizing cisplatin-mediated apoptosis in A549

    International Nuclear Information System (INIS)

    Oh, Sangnam; Kim, Yanghee; Kim, Joonhee; Kwon, Daeho; Lee, Eunil

    2010-01-01

    Research highlights: → Sensitized apoptosis in cancer cells stimulated by EP precondition with p53 dependence. → EP attenuates several CDDP-resistance mechanisms. → No harmful effect of EP on normal fibroblasts. -- Abstract: Intensive cancer therapy strategies have thus far focused on sensitizing cancer cells to anticancer drug-mediated apoptosis to overcome drug resistance, and this strategy has led to more effective cancer therapeutics. Cisplatin (cis-diamminedichloroplatinum(II), CDDP) is an effective anticancer drug used to treat many types of cancer, including non-small cell lung carcinoma (NSCLC), and can be used in combination with various chemicals to enhance cancer cell apoptosis. Here, we introduce the use of elevated pressure (EP) in combination with CDDP for cancer treatment and explore the effects of EP on CDDP-mediated apoptosis in NSCLC cells. Our findings demonstrate that preconditioning NSCLC cells with EP sensitizes cells for CDDP-induced apoptosis. Enhanced apoptosis was dependent on p53 and HO-1 expression, and was associated with increased DNA damage and down-regulation of genes involved in nucleotide excision repair. The transcriptional levels of transporter proteins indicated that the mechanism by which EP-induced CDDP sensitization was intracellular drug accumulation. The protein levels of some antioxidants, such as hemeoxygenase-1 (HO-1), glutathione (GSH) and glutathione peroxidase (Gpx), were decreased in A549 cells exposed to EP via the down-regulation of the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf-2). Furthermore, normal human fibroblasts were resistant to EP treatment, with no elevated DNA damage or apoptosis. Collectively, these data show that administration of EP is a potential adjuvant tool for CDDP-based chemosensitivity of lung cancer cells that may reduce drug resistance.

  5. Upregulation of miR-146a increases cisplatin sensitivity of the non-small cell lung cancer A549 cell line by targeting JNK-2.

    Science.gov (United States)

    Pang, Linrong; Lu, Jinger; Huang, Jia; Xu, Caihong; Li, Hui; Yuan, Guangbo; Cheng, Xiaochun; Chen, Jun

    2017-12-01

    The aim of the present study was to investigate the effects of microRNA (miR-)146a on the cisplatin sensitivity of the non-small cell lung cancer (NSCLC) A549 cell line and study the underlying molecular mechanism. The differences in expression of miRNAs between A549 and A549/cisplatin (A549/DDP) cells were determined, and miR-146a was selected to study its effect on cisplatin sensitivity of A549/DDP cells. miR-146a mimic and inhibitor transient transfection systems were constructed using vectors, and A549/DDP cells were infected with miR-146a mimic and inhibitor to investigate growth, apoptosis and migration. The directed target of miR-146a was determined and the underlying molecular mechanism was validated in the present study. The results of the present study demonstrated that miR-146a was downregulated in NSCLC A549/DDP cells, compared with A549 cells. The overexpression of miR-146a induced apoptosis and inhibited the growth and invasion of A549/DDP cells, which resulted in increased cisplatin sensitivity in NSCLC cells. The JNK2 gene was determined as the direct target of miR-146a, and may be activated by the overexpression of miR-146a. Additionally, JNK2 activated the expression of p53 and inhibited B cell lymphoma 2. The upregulation of miR-146a increased cisplatin sensitivity of the A549 cell line by targeting JNK2, which may provide a novel method for treating NSCLC cisplatin resistance.

  6. Inhibition of microRNA-196a might reverse cisplatin resistance of A549/DDP non-small-cell lung cancer cell line.

    Science.gov (United States)

    Li, Jian-Huang; Luo, Ning; Zhong, Mei-Zuo; Xiao, Zhi-Qiang; Wang, Jian-Xin; Yao, Xiao-Yi; Peng, Yun; Cao, Jun

    2016-02-01

    We aimed to explore the possible mechanism of microRNA-196a (miR-196a) inhibition and reversion of drug resistance to cisplatin (DDP) of the A549/DDP non-small-cell lung cancer (NSCLC) cell line. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect expression differences of miR-196a in the drug-resistant A549/DDP NLCLC cell line and the parental A549 cell line, and expressions of miR-196a in the A549/DDP NLCLC cell line transfected with miR-196a inhibitor (anti-miR-196a group) and the miR-196a negative control (miR-NC) group and blank group (without transfection). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was applied in examining the cell viability of A549/DDP cell line before and after transfection. Clonogenic assay was used to detect cell proliferation ability. Flow cytometry was applied in detecting apoptosis rate of assayed tumor cell and rhodamine-123 changes in cells. Western blot was applied in detecting proteins of drug-resistant related gene in A549/DDP cell line. Significantly higher expression of miR-196a was detected in the drug-resistant A549/DDP cell line than that in the parental A549 cell line (P A549/DDP cell line in the early stage were found among the three groups (all P > 0.05), but the late-stage apoptosis rate in the anti-miR-196a group was significantly higher than that in the blank group and the miR-NC group (both P A549/DDP cell lines, which might relate with inhibition of drug efflux, down-regulation of drug-resistant protein expression, cell apoptosis, and cell proliferation suppression.

  7. NMR studies of the relationship between the changes of membrane lipids and the cisplatin-resistance of A549/DDP cells

    Directory of Open Access Journals (Sweden)

    Huang Youguo

    2003-04-01

    Full Text Available Abstract Changes of membrane lipids in cisplatin-sensitive A549 and cisplatin-resistant A549/DDP cells during the apoptotic process induced by a clinical dose of cisplatin (30 μM were detected by 1H and 31P-NMR spectroscopy and by membrane fluidity measurement. The apoptotic phenotypes of the two cell lines were monitored with flow cytometry. The assays of apoptosis showed that significant apoptotic characteristics of the A549 cells were induced when the cells were cultured for 24 hours after treatment with cisplatin, while no apoptotic characteristic could be detected for the resistant A549/DDP cells even after 48 hours. The results of 1H-NMR spectroscopy demonstrated that the CH2/CH3 and Glu/Ct ratios of the membrane of A549 cells increased significantly, but those in A549/DDP cell membranes decreased. In addition, the Chol/CH3 and Eth/Ct ratios decreased for the former but increased for the latter cells under the same conditions. 31P-NMR spectroscopy indicated levels of phosphomonoesters (PME and ATP decreased in A549 but increased in A549/DDP cells after being treated with cisplatin. These results were supported with the data obtained from 1H-NMR measurements. The results clearly indicated that components and properties of membrane phospholipids of the two cell lines were significantly different during the apoptotic process when they were treated with a clinical dose of cisplatin. Plasma membrane fluidity changes during cisplatin treatment as detected with the fluorescence probe TMA-DPH also indicate marked difference between the two cell lines. We provided evidence that there are significant differences in plasma membrane changes during treatment of cisplatin sensitive A549 and resistant A549/DDP cells.

  8. Grape Seed Proanthocyanidin Inhibits Mucin Synthesis and Viral Replication by Suppression of AP-1 and NF-κB via p38 MAPKs/JNK Signaling Pathways in Respiratory Syncytial Virus-Infected A549 Cells.

    Science.gov (United States)

    Lee, Jin-Woo; Kim, Young Il; Im, Chang-Nim; Kim, Sung Wan; Kim, Su Jin; Min, Seoyeon; Joo, Yong Hoon; Yim, Sung-Vin; Chung, Namhyun

    2017-06-07

    Airway epithelial cells are often infected by respiratory syncytial virus (RSV), one of the most common causes of asthma, bronchiolitis, chronic obstructive pulmonary disease, and pneumonia. During the infection process, excessive mucins instigate airway inflammation. However, the mechanism underlying RSV-induced airway hyper-responsiveness and inflammation is poorly understood. Furthermore, no reliable vaccines or drugs for antiviral therapy are available. In this study, the effect of the natural compound grape seed proanthocyanidin (GSP) on RSV-infected human airway epithelial cells A549 was evaluated. After pretreatment of the cells with or without exposure to RSV with 5-10 μg GSP/mL, the expression of various mucins (MUC1, MUC2, MUC5AC, MUC5B, and MUC8) was evaluated by real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blotting, as well as confocal microscopy. We found that GSP significantly decreased RSV-induced mucin synthesis at the mRNA and protein levels. In addition, GSP suppressed the RSV-induced signaling pathways, including extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38, together with nuclear factor kappa B (NF-κB) and activating protein-1 family members (c-Jun and c-Fos). Concomitantly, GSP inhibited the replication of RSV within A549 cells. Taken together, all our results suggest that GSP could be a potent therapeutic agent to suppress excessive mucus production and viral replication in RSV-induced airway inflammatory disorders.

  9. Oxidative stress induced apoptosis of human lung carcinoma (A549) cells by a novel copper nanorod formulation.

    Science.gov (United States)

    Thounaojam, Menaka C; Jadeja, Ravirajsinh N; Valodkar, Mayur; Nagar, Padamanabhi S; Devkar, Ranjitsinh V; Thakore, Sonal

    2011-11-01

    This study elucidates the process of synthesis of copper (Cu) nanorods using almond skin extract as stabilizing cum capping agent. These nanorods were (about 200 nm long and 40 nm wide) characterized by transmission electron microscopy (TEM). Further, cytotoxicity potential of these nanorods was evaluated in A549 cells (Human lung carcinoma cell line) via cell viability assay and extracellular lactate dehydrogenase (LDH) release. Also, reduced glutathione (GSH), lipid peroxidation (LPO), cellular oxidative stress (Rhodamine 123 florescence) and apoptosis (Annexin V FITC/Propidium iodide staining) were also investigated in control and treated cells. Results indicated that Cu nanorods induced apoptotic death of cancer cells by induction of oxidative stress, depletion of cellular antioxidants and mitochondrial dysfunction. This study reports a novel process of synthesis of almond skin extract capped Cu nanorods and its potential as an anticancer agent against A549 lung carcinoma cells. Copyright © 2011 Elsevier Ltd. All rights reserved.

  10. Oligomeric proanthocyanidins protects A549 cells against H2O2-induced oxidative stress via the Nrf2-ARE pathway.

    Science.gov (United States)

    Sun, Chao; Jin, Weiguo; Shi, Hongcan

    2017-06-01

    Oxidative signaling and oxidative stress contribute to aging, cancer and diseases resulting from lung fibrosis. In this study, we explored the anti-oxidative potential of oligomeric proanthocyanidins (OPCs), natural flavonoid compounds. We examined the protective effects of OPCs against hydrogen peroxide (H2O2)-induced oxidative stress in non-small cell lung cancer cells (A549). We demonstrated that OPC markedly attenuated H2O2-induced A549 cell viability, as shown by by 3-[4,5-dimethylthiazol-2-yl)]-2,5-diphenyl-tetrazolium bromide (MTT) assay. At the same time, OPC inhibited H2O2-induced oxidative stress by significantly increasing the activities of superoxide dismutase, catalase and glutathione, and reducing the levels of reactive oxygen species (ROS) and malondialdehyde (MDA). Treatment of the A549 cells with OPC significantly promoted the nuclear translocation of NF-E2-related factor 2 (Nrf2) and significantly enhanced the expression of its target genes [heme oxygenase-1 (HO-1), NAD(P)H quinone dehydrogenase 1 (NQO1) and thioredoxin reductase 1 (TXNRD1)] with different fold change values at both the mRNA and protein level. The knockout of Nrf2 using CRISPR/Cas9 technology attenuated OPC-mediated ARE gene transcription, and almost abolished the OPC-mediated protective effects against H2O2-induced oxidative stress. On the whole, our study suggests that OPC plays an important role in controlling the antioxidant response of A549 cells via the Nrf2-ARE pathway.

  11. Analysis of gene expression changes in A549 cells induced by organic compounds from respirable air particles

    Czech Academy of Sciences Publication Activity Database

    Líbalová, Helena; Krčková, S.; Uhlířová, Kateřina; Kléma, J.; Ciganek, M.; Rössner ml., Pavel; Šrám, Radim; Vondráček, J.; Machala, M.; Topinka, Jan

    2014-01-01

    Roč. 770, DEC 2014 (2014), s. 94-105 ISSN 0027-5107 R&D Projects: GA ČR GAP503/11/0142; GA ČR(CZ) GBP503/12/G147 Institutional support: RVO:68378041 Keywords : Ah receptor * gene expression profile * A549 cells Subject RIV: DN - Health Impact of the Environment Quality Impact factor: 3.680, year: 2014

  12. [Overexpression of Keap1 inhibits the cell proliferation and metastasis and overcomes the drug resistance in human lung cancer A549 cells].

    Science.gov (United States)

    Weng, X; Yan, Y Y; Tong, Y H; Fan, Y; Zeng, J M; Wang, L L; Lin, N M

    2016-06-23

    To investigate the effect of Keap1-Nrf2 pathway on cell proliferation, metastasis and drug resistance of human lung cancer A549 cell line. A549-Keap1 cell line, constantly expressing wild type Keap1, was established by lentiviral transfection. Real-time RT-PCR and western blot were used to determine the expression of Nrf2 and its target gene in A549 cells. Sulforhodamine B (SRB) assay, flow cytometry, colony formation assay, transwell assay, and cell wound-healing assay were performed to explore the effect of wild type Keap1 expression on the proliferation, invasion, migration and drug resistance of A549 cells. Over-expressed Keap1 decreased the expression of Nrf2 protein and the mRNA level of its downstream target genes and inhibited the ability of cell proliferation and clone formation of A549 cells. Keap1 overexpression induced G0/G1 phase arrest. The percentage of A549-Keap1 cells in G0/G1 phase was significantly higher than that of A549-GFP cells (80.2±5.9)% vs. (67.1±0.9%)(PA549-Keap1 cells (156.33±17.37), the number of invasive A549-GFP cells was significantly higher (306.67±22.19) in a high power field. Keap1 overexpression significantly enhanced the sensitivity of A549 cells to carboplatin and gemcitabine (PA549-Keap1 and A549-GFP cells were (52.1±3.3) μmol/L and (107.8±12.9) μmol/L, respectively. The IC50s of gemcitabine in A549-Keap1 and A549-GFP cells were (6.8±1.2) μmol/L and (9.9±0.5) μmol/L, respectively. Keap1 overexpression significantly inhibits the expression of Nrf2 and its downstream target genes, suppresses tumor cell proliferation and metastasis, and enhances the sensitivity of A549 cells to anticancer drugs.

  13. Functional interaction between human papillomavirus type 16 E6 and E7 oncoproteins and cigarette smoke components in lung epithelial cells.

    Directory of Open Access Journals (Sweden)

    Juan Pablo Muñoz

    Full Text Available The smoking habit is the most important, but not a sufficient cause for lung cancer development. Several studies have reported the human papillomavirus type 16 (HPV16 presence and E6 and E7 transcripts expression in lung carcinoma cases from different geographical regions. The possible interaction between HPV infection and smoke carcinogens, however, remains unclear. In this study we address a potential cooperation between tobacco smoke and HPV16 E6 and E7 oncoproteins for alterations in proliferative and tumorigenic properties of lung epithelial cells. A549 (alveolar, tumoral and BEAS-2B (bronchial, non-tumoral cell lines were stably transfected with recombinant pLXSN vectors expressing HPV16 E6 and E7 oncoproteins and exposed to cigarette smoke condensate (CSC at different concentrations. HPV16 E6 and E7 expression was associated with loss of p53 stability, telomerase (hTERT and p16(INK4A overexpression in BEAS-2B cells as demonstrated by quantitative real-time polymerase chain reaction (qRT-PCR and western blotting (WB. In A549 cells we observed downregulation of p53 but not a significant increase of hTERT transcripts. In addition, the HPV16 E6/E7 transfected cell lines showed an increased proliferation rate and anchorage-independent growth in a HPV16 E6 and E7 expression-dependent manner. Moreover, both HPV16 E6/E7 and mock transfected cells showed an increased proliferation rate and anchorage-independent growth in the presence of 0.1 and 10 µg/mL CSC. However, this increase was significantly greater in HPV16 E6/E7 transfected cells (p<0.001. Data were confirmed by FCSE proliferation assay. The results obtained in this study are suggestive of a functional interaction between tobacco smoke and HPV16 E6/E7 oncoproteins for malignant transformation and tumorigenesis of lung epithelial cells. More studies are warranted in order to dissect the molecular mechanisms involved in this cooperation.

  14. The Effects of Davallic Acid from Davallia divaricata Blume on Apoptosis Induction in A549 Lung Cancer Cells

    Directory of Open Access Journals (Sweden)

    Tsu-Liang Chang

    2012-11-01

    Full Text Available Traditional or folk medicinal herbs continue to be prescribed in the treatment of various diseases and conditions in many cultures. Recent scientific efforts have focused on the potential roles of extracts of traditional herbs as alternative and complementary medications for cancer treatment. In Taiwan, Davallia divaricata Blume has been traditionally employed in folk medicine for therapy of lung cancer, davallic acid being the major active compound of D. divaricata Blume. In this study, we investigated the inhibitory activity of davallic acid on the proliferation of A549 lung cancer cells. Davallic acid was extracted from D. divaricata Blume, and its effects on cell viability, cell cycle distribution, ROS level, and apoptotic protein expression in A549 cells were determined. Davallic acid significantly induced reactive oxygen species (ROS generation as well as caspase-3, -8, and -9 activation, thereby repressing A549 cell growth and elevating apoptotic activity. Since lung cancer has a high incidence of recurrence, these results indicate that davallic acid may have the potential to be a natural anti-lung cancer compound, and may provide a basis for further study of its use in combating cancer.

  15. Vitamin D Analogs Potentiate the Antitumor Effect of Imatinib Mesylate in a Human A549 Lung Tumor Model

    Science.gov (United States)

    Maj, Ewa; Filip-Psurska, Beata; Świtalska, Marta; Kutner, Andrzej; Wietrzyk, Joanna

    2015-01-01

    In previous papers, we presented data on studies on the anticancer activity of the vitamin D3 analogs, named PRI-2191 and PRI-2205, in different cancer models. In this study, we showed the improved antiproliferative activity of a combination of imatinib mesylate (Gleevec, GV) and cytostatic agents in in vitro studies, when used with a third compound, namely PRI-2191, in an A549 human lung cancer model. Furthermore, we analyzed the influence of both PRI-2191, as well as PRI-2205 on the anticancer activity of GV in mice bearing A549 tumors. The route of PRI-2191 analog administration showed a significant impact on the outcome of GV treatment: subcutaneous injection was more efficient and less toxic than oral gavage. Moreover, both vitamin D compounds increased the anticancer activity of GV; however, they might also potentiate some adverse effects. We also evaluated in tumor tissue the expression of VEGF, PDGF-BB, vitamin D receptor, CYP27B1, CYP24, p53 and Bcl-2, as well as PDGF receptors: α and β. We observed the upregulation of p53 expression and the downregulation of Bcl-2, as well as VEGF in A549 tumors as a result of the tested treatment. However, vitamin D analogs did not significantly influence the expression of these proteins. PMID:26580599

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

    Science.gov (United States)

    Maza, Paloma K; Suzuki, Erika

    2016-01-01

    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.

  17. Monocrotaline pyrrole-induced megalocytosis of lung and breast epithelial cells: Disruption of plasma membrane and Golgi dynamics and an enhanced unfolded protein response

    International Nuclear Information System (INIS)

    Mukhopadhyay, Somshuvra; Shah, Mehul; Patel, Kirit; Sehgal, Pravin B.

    2006-01-01

    The pyrrolizidine alkaloid monocrotaline (MCT) initiates pulmonary hypertension by inducing a 'megalocytosis' phenotype in target pulmonary arterial endothelial, smooth muscle and Type II alveolar epithelial cells. In cultured endothelial cells, a single exposure to the pyrrolic derivative of monocrotaline (MCTP) results in large cells with enlarged endoplasmic reticulum (ER) and Golgi and increased vacuoles. However, these cells fail to enter mitosis. Largely based upon data from endothelial cells, we proposed earlier that a disruption of the trafficking and mitosis-sensor functions of the Golgi (the 'Golgi blockade' hypothesis) may represent the subcellular mechanism leading to MCTP-induced megalocytosis. In the present study, we investigated the applicability of the Golgi blockade hypothesis to epithelial cells. MCTP induced marked megalocytosis in cultures of lung A549 and breast MCF-7 cells. This was associated with a change in the distribution of the cis-Golgi scaffolding protein GM130 from a discrete juxtanuclear localization to a circumnuclear distribution consistent with an anterograde block of GM130 trafficking to/through the Golgi. There was also a loss of plasma membrane caveolin-1 and E-cadherin, cortical actin together with a circumnuclear accumulation of clathrin heavy chain (CHC) and α-tubulin. Flotation analyses revealed losses/alterations in the association of caveolin-1, E-cadherin and CHC with raft microdomains. Moreover, megalocytosis was accompanied by an enhanced unfolded protein response (UPR) as evidenced by nuclear translocation of Ire1α and glucose regulated protein 58 (GRP58/ER-60/ERp57) and a circumnuclear accumulation of PERK kinase and protein disulfide isomerase (PDI). These data further support the hypothesis that an MCTP-induced Golgi blockade and enhanced UPR may represent the subcellular mechanism leading to enlargement of ER and Golgi and subsequent megalocytosis

  18. BZML, a novel colchicine binding site inhibitor, overcomes multidrug resistance in A549/Taxol cells by inhibiting P-gp function and inducing mitotic catastrophe.

    Science.gov (United States)

    Bai, Zhaoshi; Gao, Meiqi; Zhang, Huijuan; Guan, Qi; Xu, Jingwen; Li, Yao; Qi, Huan; Li, Zhengqiang; Zuo, Daiying; Zhang, Weige; Wu, Yingliang

    2017-08-28

    Multidrug resistance (MDR) interferes with the efficiency of chemotherapy. Therefore, developing novel anti-cancer agents that can overcome MDR is necessary. Here, we screened a series of colchicine binding site inhibitors (CBSIs) and found that 5-(3, 4, 5-trimethoxybenzoyl)-4-methyl-2-(p-tolyl) imidazol (BZML) displayed potent cytotoxic activity against both A549 and A549/Taxol cells. We further explored the underlying mechanisms and found that BZML caused mitosis phase arrest by inhibiting tubulin polymerization in A549 and A549/Taxol cells. Importantly, BZML was a poor substrate for P-glycoprotein (P-gp) and inhibited P-gp function by decreasing P-gp expression at the protein and mRNA levels. Cell morphology changes and the expression of cycle- or apoptosis-related proteins indicated that BZML mainly drove A549/Taxol cells to die by mitotic catastrophe (MC), a p53-independent apoptotic-like cell death, whereas induced A549 cells to die by apoptosis. Taken together, our data suggest that BZML is a novel colchicine binding site inhibitor and overcomes MDR in A549/Taxol cells by inhibiting P-gp function and inducing MC. Our study also offers a new strategy to solve the problem of apoptosis-resistance. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Curcumin promotes apoptosis in A549/DDP multidrug-resistant human lung adenocarcinoma cells through an miRNA signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Jian, E-mail: zhangjian197011@yahoo.com [Department of Respiratory Medicine, Xijing Hospital, The Fourth Military Medical University, Xi' an 710032 (China); Zhang, Tao [Department of Thoracic Surgery, Tangdu Hospital, The Fourth Military Medical University, Xi' an 710038 (China); Ti, Xinyu; Shi, Jieran; Wu, Changgui; Ren, Xinling [Department of Respiratory Medicine, Xijing Hospital, The Fourth Military Medical University, Xi' an 710032 (China); Yin, Hong, E-mail: yinnhong@yahoo.com [The Medical Image Center, Xijing Hospital, The Fourth Military Medical University, Xi' an 710032 (China)

    2010-08-13

    Research highlights: {yields} Curcumin had anti-cancer effects on A549/DDP multidrug-resistant human lung adenocarcinoma cells {yields} Curcumin promotes apoptosis in A549/DDP cells through a miRNA signaling pathway {yields} Curcumin induces A549/DDP cell apoptosis by downregulating miR-186* {yields} miR-186* may serve as a potential gene therapy target for refractory lung cancer that is sensitive to curcumin -- Abstract: Curcumin extracted from the rhizomes of Curcuma longa L. has been shown to have inhibitory effects on cancers through its anti-proliferative and pro-apoptotic activities. Emerging evidence demonstrates that curcumin can overcome drug resistance to classical chemotherapies. Thus, the mechanisms underlying the anti-tumor activities of curcumin require further study. In our study, we first demonstrated that curcumin had anti-cancer effects on A549/DDP multidrug-resistant human lung adenocarcinoma cells. Further studies showed that curcumin altered miRNA expression; in particular, significantly downregulated the expression of miR-186* in A549/DDP. In addition, transfection of cells with a miR-186* inhibitor promoted A549/DDP apoptosis, and overexpression of miR-186* significantly inhibited curcumin-induced apoptosis in A549/DDP cells. These observations suggest that miR-186* may serve as a potential gene therapy target for refractory lung cancer that is sensitive to curcumin.

  20. Curcumin promotes apoptosis in A549/DDP multidrug-resistant human lung adenocarcinoma cells through an miRNA signaling pathway

    International Nuclear Information System (INIS)

    Zhang, Jian; Zhang, Tao; Ti, Xinyu; Shi, Jieran; Wu, Changgui; Ren, Xinling; Yin, Hong

    2010-01-01

    Research highlights: → Curcumin had anti-cancer effects on A549/DDP multidrug-resistant human lung adenocarcinoma cells → Curcumin promotes apoptosis in A549/DDP cells through a miRNA signaling pathway → Curcumin induces A549/DDP cell apoptosis by downregulating miR-186* → miR-186* may serve as a potential gene therapy target for refractory lung cancer that is sensitive to curcumin -- Abstract: Curcumin extracted from the rhizomes of Curcuma longa L. has been shown to have inhibitory effects on cancers through its anti-proliferative and pro-apoptotic activities. Emerging evidence demonstrates that curcumin can overcome drug resistance to classical chemotherapies. Thus, the mechanisms underlying the anti-tumor activities of curcumin require further study. In our study, we first demonstrated that curcumin had anti-cancer effects on A549/DDP multidrug-resistant human lung adenocarcinoma cells. Further studies showed that curcumin altered miRNA expression; in particular, significantly downregulated the expression of miR-186* in A549/DDP. In addition, transfection of cells with a miR-186* inhibitor promoted A549/DDP apoptosis, and overexpression of miR-186* significantly inhibited curcumin-induced apoptosis in A549/DDP cells. These observations suggest that miR-186* may serve as a potential gene therapy target for refractory lung cancer that is sensitive to curcumin.

  1. Moringa oleifera Gold Nanoparticles Modulate Oncogenes, Tumor Suppressor Genes, and Caspase-9 Splice Variants in A549 Cells.

    Science.gov (United States)

    Tiloke, Charlette; Phulukdaree, Alisa; Anand, Krishnan; Gengan, Robert M; Chuturgoon, Anil A

    2016-10-01

    Gold nanoparticles (AuNP's) facilitate cancer cell recognition and can be manufactured by green synthesis using nutrient rich medicinal plants such as Moringa oleifera (MO). Targeting dysregulated oncogenes and tumor suppressor genes is crucial for cancer therapeutics. We investigated the antiproliferative effects of AuNP synthesized from MO aqueous leaf extracts (MLAuNP ) in A549 lung and SNO oesophageal cancer cells. A one-pot green synthesis technique was used to synthesise MLAuNP . A549, SNO cancer cells and normal peripheral blood mononuclear cells (PBMCs) were exposed to MLAuNP and CAuNP to evaluate cytotoxicity (MTT assay); apoptosis was measured by phosphatidylserine (PS) externalization, mitochondrial depolarization (ΔΨm) (flow cytometry), caspase-3/7, -9 activity, and ATP levels (luminometry). The mRNA expression of c-myc, p53, Skp2, Fbw7α, and caspase-9 splice variants was determined using qPCR, while relative protein expression of c-myc, p53, SRp30a, Bax, Bcl-2, Smac/DIABLO, Hsp70, and PARP-1 were determined by Western blotting. MLAuNP and CAuNP were not cytotoxic to PBMCs, whilst its pro-apoptotic properties were confirmed in A549 and SNO cells. MLAuNP significantly increased caspase activity in SNO cells while MLAuNP significantly increased PS externalization, ΔΨm, caspase-9, caspase-3/7 activities, and decreased ATP levels in A549 cells. Also, p53 mRNA and protein levels, SRp30a (P = 0.428), Bax, Smac/DIABLO and PARP-1 24 kDa fragment levels were significantly increased. Conversely, MLAuNP significantly decreased Bcl-2, Hsp70, Skp2, Fbw7α, c-myc mRNA, and protein levels and activated alternate splicing with caspase-9a splice variant being significantly increased. MLAuNP possesses antiproliferative properties and induced apoptosis in A549 cells by activating alternate splicing of caspase-9. J. Cell. Biochem. 117: 2302-2314, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  2. Effects of miR-424 on Proliferation and Migration Abilities in Non-small Cell Lung Cancer A549 Cells and Its Molecular Mechanism

    Directory of Open Access Journals (Sweden)

    Hongmin LI

    2016-09-01

    Full Text Available Background and objective The inhibitory ability of miR-424 on the proliferation of renal carcinoma cell and the migration and invasion of cancer cells has been widely explored and demonstrated. However, the effects of miR-424 on non-small cell lung cancer (NSCLC have not been systematically examined. In this study, detected the growth and invasion effect of miR-424 in NSCLC A549 cell. The migration and molecular mechanism of this cell are also detected. Methods NSCLC A549 cell was transfected with miR-424 and its inhibitor. After transfection, the proliferation ability of A549 cell was detectedby CCK8 assay. Then, the migration ability in A549 cell was detected by migration assays. Furthermore, the expression level of MMP2 and MMP9 in A549 was detected by Western blot and immune fluorescence. The 3'UTR of E2F6 was cloned into luciferase reporter vector and its enzymatic activitywas detected to verify whether miR-424 can target E2F6. The expression level of E2F6 in a549 cell after transfecing with miR-424 was detected by Western blot. Results After transfection of miR-424, the proliferation and migration abilities were remarkably decreased and the expression level of MMP-2 and MMP-9 were down-regulated in A549. Moreover, MiR-424 inhibited the enzymatic activity of luviferase reporter vector of E2F6. Specifically, the expression level of E2F6 was down-regulated in A549. Conclusion miR-424 can inhibit the proliferation and migration abilities of A549 by negatively regulating the expression of E2F6.

  3. Beauvericin-induced cell apoptosis through the mitogen-activated protein kinase pathway in human nonsmall cell lung cancer A549 cells.

    Science.gov (United States)

    Lu, Chien-Lin; Lin, Hen-I; Chen, Bing-Fang; Jow, Guey-Mei

    2016-01-01

    Beauvericin (BEA) is a cyclic hexadepsipeptide that derives from Codyceps cicadae. Our previous study results indicated that the cytotoxic effects of BEA on human A549 lung cancer cells BEA occur through an apoptotic pathway, which involves the up-regulation of cytochrome c release from mitochondria, upregulation of caspase 3 activity, and cellular and morphological changes. In this study, we identified that the mitogen-activated protein kinase (MAPK) inhibitor U0126 inhibits the cytotoxic effects of BEA on A549 cells. After exposing human A549 cells to 10 μM BEA, we observed a significant and dose-dependent increase in the percentage of hypoploid (sub-G1) phase cells in the A549 population. Following the pretreatment of the A549 cells with 25 μM U0126, the distribution of A549 cells in the sub-G1 phase decreased significantly. The BEA treatment resulted in a significant increase apoptosis in A549 cells by in situ terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Moreover, the MEK1/2 (mitogen-activated protein kinase kinase)-ERK42/44 (extracellular signal-regulated kinases)-90RSK (ribosomal s6 kinase) signaling pathway was activated in BEA-induced apoptotic A549 cells. Furthermore, treatment with MEK1/2 inhibitor U0126 was capable to attenuate the BEA induced typical apoptotic morphological change, apoptotic cells, and MEK1/2-ERK42/44-90RSK signaling pathway. These results suggested that MEK1/2-ERK42/44-90RSK signaling pathway may play a important role in BEA-induced apoptosis in human NSCLC A549 cancer cells.

  4. [Effects of miR-424 on Proliferation and Migration Abilities in Non-small Cell Lung Cancer A549 Cells and Its Molecular Mechanism].

    Science.gov (United States)

    Li, Hongmin; Lan, Haitao; Zhang, Ming; An, Ning; Yu, Ruilian; He, Yangke; Gan, Chongzhi

    2016-09-20

    The inhibitory ability of miR-424 on the proliferation of renal carcinoma cell and the migration and invasion of cancer cells has been widely explored and demonstrated. However, the effects of miR-424 on non-small cell lung cancer (NSCLC) have not been systematically examined. In this study, detected the growth and invasion effect of miR-424 in NSCLC A549 cell. The migration and molecular mechanism of this cell are also detected. NSCLC A549 cell was transfected with miR-424 and its inhibitor. After transfection, the proliferation ability of A549 cell was detectedby CCK8 assay. Then, the migration ability in A549 cell was detected by migration assays. Furthermore, the expression level of MMP2 and MMP9 in A549 was detected by Western blot and immune fluorescence. The 3'UTR of E2F6 was cloned into luciferase reporter vector and its enzymatic activitywas detected to verify whether miR-424 can target E2F6. The expression level of E2F6 in a549 cell after transfecing with miR-424 was detected by Western blot. After transfection of miR-424, the proliferation and migration abilities were remarkably decreased and the expression level of MMP-2 and MMP-9 were down-regulated in A549. Moreover, MiR-424 inhibited the enzymatic activity of luviferase reporter vector of E2F6. Specifically, the expression level of E2F6 was down-regulated in A549. miR-424 can inhibit the proliferation and migration abilities of A549 by negatively regulating the expression of E2F6.
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  5. Alveolar bone resorption after tooth extraction

    OpenAIRE

    Dimova, Cena; Popovski, Stipica

    2012-01-01

    Alveolar ridge resorption has long been considered an unavoidable consequence of tooth extraction. Atrophy of the alveolar bone may cause significant esthetic and surgical problems in implantation, as well as at prosthetic and restorative dentistry. Alveolar ridge prophylaxis immediately upon tooth extraction may reduce such sequelae for both, the treating dentist and the patient. Attempts to reduce alveolar bone resorption have included the placement of natural roots, root analogues, and...

  6. Nitrative DNA damage induced by multi-walled carbon nanotube via endocytosis in human lung epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Feiye, E-mail: zhizi0269@doc.medic.mie-u.ac.jp [Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie, 514-8507 (Japan); Ma, Ning, E-mail: maning@suzuka-u.ac.jp [Faculty of Health Science, Suzuka University of Medical Science, 1001-1 Kishioka-cho, Suzuka, Mie, 510-0293 (Japan); Horibe, Yoshiteru, E-mail: violinteru@yahoo.co.jp [Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie, 514-8507 (Japan); Kawanishi, Shosuke, E-mail: kawanisi@suzuka-u.ac.jp [Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, 3500-3 Minami-Tamagaki-cho, Suzuka, Mie, 513-8670 (Japan); Murata, Mariko, E-mail: mmurata@doc.medic.mie-u.ac.jp [Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie, 514-8507 (Japan); Hiraku, Yusuke, E-mail: y-hiraku@doc.medic.mie-u.ac.jp [Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie, 514-8507 (Japan)

    2012-04-15

    Carbon nanotube (CNT) has a promising usage in the field of material science for industrial purposes because of its unique physicochemical property. However, intraperitoneal administration of CNT was reported to cause mesothelioma in experimental animals. Chronic inflammation may contribute to carcinogenesis induced by fibrous materials. 8-Nitroguanine is a mutagenic DNA lesion formed during inflammation and may play a role in CNT-induced carcinogenesis. In this study, we examined 8-nitroguanine formation in A549 human lung alveolar epithelial cells treated with multi-walled CNT (MWCNT) by fluorescent immunocytochemistry. Both MWCNTs with diameter of 20–30 nm (CNT20) and 40–70 nm (CNT40) significantly induced 8-nitroguanine formation at 5 and 10 μg/ml (p < 0.05), which persisted for 24 h, although there was no significant difference in DNA-damaging abilities of these MWCNTs. MWCNTs significantly induced the expression of inducible nitric oxide synthase (iNOS) for 24 h (p < 0.05). MWCNTs also significantly increased the level of nitrite, a hydrolysis product of oxidized NO, in the culture supernatant at 4 and 8 h (p < 0.05). MWCNT-induced 8-nitroguanine formation and iNOS expression were largely suppressed by inhibitors of iNOS (1400 W), nuclear factor-κB (Bay11-7082), actin polymerization (cytochalasin D), caveolae-mediated endocytosis (methyl-β-cyclodextrin, MBCD) and clathrin-mediated endocytosis (monodansylcadaverine, MDC). Electron microscopy revealed that MWCNT was mainly located in vesicular structures in the cytoplasm, and its cellular internalization was reduced by MBCD and MDC. These results suggest that MWCNT is internalized into cells via clathrin- and caveolae-mediated endocytosis, leading to inflammatory reactions including iNOS expression and resulting nitrative DNA damage, which may contribute to carcinogenesis. Highlights: ►Multi-walled carbon nanotube (MWCNT) caused DNA damage in A549 cells. ►MWCNT formed 8-nitroguanine, a DNA lesion

  7. Intravascular bronchio-alveolar tumor

    International Nuclear Information System (INIS)

    Mata, J.M.; Caceres, J.; Prat, J.; Lopez, J.I.; Velilla, O.

    1991-01-01

    In 1975 Dail and Liebow described the clinical and pathological characteristics of a pulmonary tumor which they dominated intravascular bronchio-alveolar tumor (IVBAT). Our aim is to acquaint radiologists with the existence of this tumor by describing the radiologic findings in 2 patients with IVBAT, 1 with hepatic involvement ant the other with pulmonary osteoarthropathy. (author). 7 refs.; 2 figs

  8. Human alveolar echinococcosis in Kyrgyzstan.

    Science.gov (United States)

    Usubalieva, Jumagul; Minbaeva, Gulnara; Ziadinov, Iskender; Deplazes, Peter; Torgerson, Paul R

    2013-07-01

    Human echinococcosis is a reportable disease in Kyrgyzstan. Between 1995 and 2011, human alveolar echinococcosis increased from 60 cases per year. The origins of this epidemic, which started in 2004, may be linked to the socioeconomic changes that followed the dissolution of the former Soviet Union.

  9. True Fibroma of Alveolar Mucosa

    Directory of Open Access Journals (Sweden)

    Shankargouda Patil

    2014-01-01

    Full Text Available Benign fibrous overgrowths are often found in the oral cavity, almost always being reactive/irritational in nature. However, benign mesenchymal neoplasms of the fibroblasts are extremely uncommon. Here we report a case of “True Fibroma of Alveolar Mucosa” for its rarity.

  10. Alveolar macrophage dysregulation in Hermansky-Pudlak syndrome type 1.

    Science.gov (United States)

    Rouhani, Farshid N; Brantly, Mark L; Markello, Thomas C; Helip-Wooley, Amanda; O'Brien, Kevin; Hess, Richard; Huizing, Marjan; Gahl, William A; Gochuico, Bernadette R

    2009-12-01

    Individuals with Hermansky-Pudlak syndrome type 1 (HPS-1), an autosomal recessive disorder characterized by defective biogenesis of lysosome-related organelles, develop an accelerated form of progressive fibrotic lung disease. The etiology of pulmonary fibrosis associated with HPS-1 is unknown. To investigate the potential pathogenesis of pulmonary fibrosis in HPS-1, lung cells and proteins from individuals with HPS-1 were studied. Forty-one subjects with HPS-1 with and without pulmonary fibrosis were evaluated with pulmonary function tests, high-resolution computed tomography scan, and bronchoscopy. Bronchoalveolar lavage cells and analytes were analyzed. Concentrations of total bronchoalveolar lavage cells and alveolar macrophages were significantly higher in epithelial lining fluid from subjects with HPS-1 with and without pulmonary fibrosis compared with healthy research volunteers. Concentrations of cytokines and chemokines (i.e., monocyte chemoattractant protein-1, macrophage inflammatory protein-1alpha, and granulocyte-macrophage colony-stimulating factor) in alveolar epithelial lining fluid were significantly higher in subjects with HPS-1 with and without pulmonary fibrosis compared with healthy research volunteers (P system in which to study the pathogenesis and treatment of HPS pulmonary fibrosis.

  11. Curcumin inhibits interferon-α induced NF-κB and COX-2 in human A549 non-small cell lung cancer cells

    International Nuclear Information System (INIS)

    Lee, Jeeyun; Im, Young-Hyuck; Jung, Hae Hyun; Kim, Joo Hyun; Park, Joon Oh; Kim, Kihyun; Kim, Won Seog; Ahn, Jin Seok; Jung, Chul Won; Park, Young Suk; Kang, Won Ki; Park, Keunchil

    2005-01-01

    The A549 cells, non-small cell lung cancer cell line from human, were resistant to interferon (IFN)-α treatment. The IFN-α-treated A549 cells showed increase in protein expression levels of NF-κB and COX-2. IFN-α induced NF-κB binding activity within 30 min and this increased binding activity was markedly suppressed with inclusion of curcumin. Curcumin also inhibited IFN-α-induced COX-2 expression in A549 cells. Within 10 min, IFN-α rapidly induced the binding activity of a γ- 32 P-labeled consensus GAS oligonucleotide probe, which was profoundly reversed by curcumin. Taken together, IFN-α-induced activations of NF-κB and COX-2 were inhibited by the addition of curcumin in A549 cells

  12. Stromal-epithelial interactions in aging and cancer: Senescent fibroblasts alter epithelial cell differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Parrinello, Simona; Coppe, Jean-Philippe; Krtolica, Ana; Campisi, Judith

    2004-07-14

    Cellular senescence suppresses cancer by arresting cells at risk for malignant tumorigenesis. However, senescent cells also secrete molecules that can stimulate premalignant cells to proliferate and form tumors, suggesting the senescence response is antagonistically pleiotropic. We show that premalignant mammary epithelial cells exposed to senescent human fibroblasts in mice irreversibly lose differentiated properties, become invasive and undergo full malignant transformation. Moreover, using cultured mouse or human fibroblasts and non-malignant breast epithelial cells, we show that senescent fibroblasts disrupt epithelial alveolar morphogenesis, functional differentiation, and branching morphogenesis. Further, we identify MMP-3 as the major factor responsible for the effects of senescent fibroblasts on branching morphogenesis. Our findings support the idea that senescent cells contribute to age-related pathology, including cancer, and describe a new property of senescent fibroblasts--the ability to alter epithelial differentiation--that might also explain the loss of tissue function and organization that is a hallmark of aging.

  13. Brazilian green propolis induced apoptosis in human lung cancer A549 cells through mitochondrial-mediated pathway.

    Science.gov (United States)

    Frión-Herrera, Yahima; Díaz-García, Alexis; Ruiz-Fuentes, Jenny; Rodríguez-Sánchez, Hermis; Sforcin, José Maurício

    2015-10-01

    Propolis effect on the growth and apoptosis of human lung adenocarcinoma (A549 cells) was investigated as well as its mechanisms. Cells were incubated with propolis for 72 h, and 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays were employed to assess cell viability and the inhibitory concentration (IC). Apoptosis was detected by Acridine Orange/Ethidium Bromide and 4',6-diamidino-2-phenylindole staining after 24 and 48 h of incubation with ¼ IC50 of propolis by testing the mitochondrial membrane potential (ΔΨm) and the expression of apoptosis-related genes (p53, Caspase-3, Bax, Bcl-2, Bcl-XL , Noxa, Puma and p21) by reverse transcription polymerase chain reaction. Propolis displayed antiproliferative and cytotoxic effects on A549 cells in a dose- and time-dependent manner, but it did not suppress the growth of normal Vero cells. An enhanced apoptosis was seen in A549 propolis-treated cells after 48 h compared with the control cells. Propolis decreased mitochondrial membrane potential by overexpression of pro-apoptotic genes (Bax and Noxa) and reduction of the antiapoptotic gene Bcl-XL . The expression level of other genes remained unchanged (p53, Caspse-3 and Bax), whereas p21 expression was increased. Propolis induced caspase-independent apoptosis through a p53-independent mitochondrial pathway, and cell cycle arrest by upregulation of p21. Although propolis induces apoptosis mainly by p53-independent manner, it may be induced by another pathway, and new insights may arise for preventing or treating lung cancer. © 2015 Royal Pharmaceutical Society.

  14. Nickel decreases cellular iron level and converts cytosolic aconitase to iron-regulatory protein 1 in A549 cells

    International Nuclear Information System (INIS)

    Chen Haobin; Davidson, Todd; Singleton, Steven; Garrick, Michael D.; Costa, Max

    2005-01-01

    Nickel (Ni) compounds are well-established carcinogens and are known to initiate a hypoxic response in cells via the stabilization and transactivation of hypoxia-inducible factor-1 alpha (HIF-1α). This change may be the consequence of nickel's interference with the function of several Fe(II)-dependent enzymes. In this study, the effects of soluble nickel exposure on cellular iron homeostasis were investigated. Nickel treatment decreased both mitochondrial and cytosolic aconitase (c-aconitase) activity in A549 cells. Cytosolic aconitase was converted to iron-regulatory protein 1, a form critical for the regulation of cellular iron homeostasis. The increased activity of iron-regulatory protein 1 after nickel exposure stabilized and increased transferrin receptor (Tfr) mRNA and antagonized the iron-induced ferritin light chain protein synthesis. The decrease of aconitase activity after nickel treatment reflected neither direct interference with aconitase function nor obstruction of [4Fe-4S] cluster reconstitution by nickel. Exposure of A549 cells to soluble nickel decreased total cellular iron by about 40%, a decrease that likely caused the observed decrease in aconitase activity and the increase of iron-regulatory protein 1 activity. Iron treatment reversed the effect of nickel on cytosolic aconitase and iron-regulatory protein 1. To assess the mechanism for the observed effects, human embryonic kidney (HEK) cells over expressing divalent metal transporter-1 (DMT1) were compared to A549 cells expressing only endogenous transporters for inhibition of iron uptake by nickel. The inhibition data suggest that nickel can enter via DMT1 and compete with iron for entry into the cell. This disturbance of cellular iron homeostasis by nickel may have a great impact on the ability of the cell to regulate a variety of cell functions, as well as create a state of hypoxia in cells under normal oxygen tension. These effects may be very important in how nickel exerts phenotypic

  15. The effects of combined treatment with sevoflurane and cisplatin on growth and invasion of human adenocarcinoma cell line A549.

    Science.gov (United States)

    Liang, Hua; Wang, Han Bing; Liu, Hong Zhen; Wen, Xian Jie; Zhou, Qiao Ling; Yang, Cheng Xiang

    2013-07-01

    Sevoflurane, an inhalational anesthetic, and cisplatin (DDP)-based chemotherapy have been widely used during lung cancer surgery. However, the effect of sevoflurane on the sensitivity of lung cancer cells to DDP chemotherapy remains unclear. In this study, the effects of combined treatment with sevoflurane and cisplatin on the growth and invasion of human lung adenocarcinoma A549 cell line have been investigated. The underlying mechanism has also been explored. In our experiment, A549 cells were treated with 2.5% sevoflurane, 10μmol/L DDP, or the co-treatment of sevoflurane and DDP for 4h, respectively. Cell proliferation was evaluated by the MTT assay and colony formation assay. Apoptosis was assessed by flow cytometry. Cell invasion was detected by Transwell assay. The expressions of X-linked inhibitor of apoptosis protein (XIAP), Survivin, matrix metalloproteinase (MMP)-2 and MMP-9 were determined by western blotting. Our results showed that sevoflurane combined with DDP resulted in a more pronounced inhibition of tumor cells growth and invasion as compared with either drug alone. Besides, XIAP, Survivin, MMP-2, and MMP-9 were downregulated more significantly by the co-treatment of the two drugs as compared to sevoflurane treatment or DDP treatment alone. Taken together, the growth-inhibitory and invasion-inhibitory synergy between sevoflurane and DDP in human adenocarcinoma A549 cell line was found in this study. Furthermore, we showed that the growth-inhibitory synergy between sevoflurane and DDP might be associated with the downregulation of XIAP and Survivin, and the invasion-inhibitory synergy between sevoflurane and DDP might be involved in the downregulation of MMP-2 and MMP-9. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  16. Apatinib resensitizes cisplatin-resistant non-small cell lung carcinoma A549 cell through reversing multidrug resistance and suppressing ERK signaling pathway.

    Science.gov (United States)

    Liu, Z-L; Jin, B-J; Cheng, C-G; Zhang, F-X; Wang, S-W; Wang, Y; Wu, B

    2017-12-01

    To observe the reversal effect of apatinib on the resistance to cisplatin (DDP) of A549/cisplatin (A549/DDP) cells and its relevant mechanism. A549/DDP cells were treated with the control method, apatinib alone, DDP alone and DDP combined with apatinib. The cell proliferation was detected by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the cell clone formation assay. The cell apoptosis was detected by Hoechst 33258 staining and annexin V and propidium iodide (PI) double labeling. The changes in apoptotic proteins, multidrug resistance protein 1 (MDR1) and extracellular signal-regulated kinase (ERK) signaling pathway proteins in each group after treatment were detected by Western blotting. MTT assay results showed that compared with A549 cells, A549/DDP cells had obvious resistance to DDP. MTT assay and cell clone formation assay revealed that the tumor inhibition rate of the sub-lethal dose of apatinib (10 μM) combined with DDP was higher than that of DDP alone. The apoptosis detection results indicated that the proportion of apoptotic cells in the apatinib (10 μM) combined with DDP group was significantly increased. Western blotting results revealed that compared with that in parental A549 cells, the expression level of MDR1 in A549/DDP cells was significantly increased, and the ERK signaling pathway was activated. In the apatinib combined with DDP group, the levels of cleaved caspase-3, cleaved caspase-9 and B-cell lymphoma-2 (Bcl-2)-associated X (BAX) proteins were significantly upregulated, while the level of Bcl-2 proteins was downregulated. Apatinib could inhibit the expression of MDR1 and the activity of the ERK signaling pathway in a dose-dependent manner. Apatinib can restore the sensitivity of A549/DDP cells to DDP by down-regulating the expression level of MDR1 and inhibiting the activity of the ERK signaling pathway.

  17. Genistein mediates the selective radiosensitizing effect in NSCLC A549 cells via inhibiting methylation of the keap1 gene promoter region.

    Science.gov (United States)

    Liu, Xiongxiong; Sun, Chao; Liu, Bingtao; Jin, Xiaodong; Li, Ping; Zheng, Xiaogang; Zhao, Ting; Li, Feifei; Li, Qiang

    2016-05-10

    Non-small cell lung cancer (NSCLC) cells often possess a hypermethylated Keap1 promoter, which decreases Keap1 mRNA and protein expression levels, thus impairing the Nrf2-Keap1 pathway and thereby leading to chemo- or radio-resistance. In this study, we showed that genistein selectively exhibited a radiosensitizing effect on NSCLC A549 cells but not on normal lung fibroblast MRC-5 cells. Genistein caused oxidative stress in A549 cells rather than MRC-5 cells, as determined by the oxidation of the ROS-sensitive probe DCFH-DA and oxidative damage marked by MDA, PCO or 8-OHdG content. In A549 instead of MRC-5 cells, genistein reduced the level of methylation in the Keap1 promoter region, leading to an increased mRNA expression, thus effectively inhibited the transcription of Nrf2 to the nucleus, which suppressed the Nrf2-dependent antioxidant and resulted in the upregulation of ROS. Importantly, when combined with radiation, genistein further increased the ROS levels in A549 cells whereas decreasing the radiation-induced oxidative stress in MRC-5 cells, possibly via increasing the expression levels of Nrf2, GSH and HO-1. Moreover, radiation combined with genistein significantly increased cell apoptosis in A549 but not MRC-5 cells. Together, the results herein show that the intrinsic difference in the redox status of A549 and MRC-5 cells could be the target for genistein to selectively sensitize A549 cells to radiation, thereby leading to an increase in radiosensitivity for A549 cells.

  18. [Construction of A eukaryotic expression vector carrying the iNOS gene and its effect on A549 lung cancer cells].

    Science.gov (United States)

    Ye, Sujuan; Yang, Weihan; Wang, Yu; Ou, Wenjing; Ma, Qingping; Zhu, Wen

    2012-05-01

    The iNOS gene is associated with NO-mediated antitumor effects. The aims of this study are to construct a eukaryotic expression plasmid that carries the iNOS gene and to detect the expression levels and antitumor effects of the iNOS gene on A549 lung cancer cells. A DNA fragment of the human iNOS coding sequence was amplified using reverse transcription polymerase chain reaction (RT-PCR). The DNA fragment was subsequently cloned into the multiple cloning sites of the eukaryotic expression vector pVAX. The recombinant plasmid was confirmed using restriction enzyme treatment, PCR, and sequencing and was then transfected into A549 lung cancer cells. The expression of the iNOS gene in the A549 lung cancer cells after transfection was verified by RT-PCR and Western blot analysis. The effects of iNOS on cell apoptosis, proliferation, and migration were identified by staining with Hoechst 3235, an MTT assay, and a scratch assay, respectively. The results of the restriction enzyme digestion, PCR, and sequencing verified the successful construction of the eukaryotic expression plasmid pVAX-iNOS. The iNOS gene expression level was increased in the transfected A549 cells. Further experiments also showed increased cell apoptosis among the A549 lung cancer cells transfected with pVAX-iNOS. Meanwhile, the proliferation and migration of A549 cells were significantly inhibited by the enhanced iNOS gene expression. The recombinant eukaryotic expression vector pVAX-iNOS was successfully constructed and transfected into A549 cells. The enhanced iNOS gene expression significantly promoted cell apoptosis, whereas the proliferation and migration of A549 cells were inhibited. These findings contribute to the development of novel and effective gene therapies for lung cancer.

  19. Enhanced Replication of Hepatitis E Virus Strain 47832c in an A549-Derived Subclonal Cell Line

    OpenAIRE

    Schemmerer, Mathias; Apelt, Silke; Trojnar, Eva; Ulrich, Rainer G.; Wenzel, J?rgen J.; Johne, Reimar

    2016-01-01

    Hepatitis E virus (HEV) is a human pathogen with increasing importance. The lack of efficient cell culture systems hampers systematic studies on its replication cycle, virus neutralization and inactivation. Here, several cell lines were inoculated with the HEV genotype 3c strain 47832c, previously isolated from a chronically infected transplant patient. At 14 days after inoculation the highest HEV genome copy numbers were found in A549 cells, followed by PLC/PRF/5 cells, whereas HepG2/C3A, Hu...

  20. Seleno-short-chain chitosan induces apoptosis in human non-small-cell lung cancer A549 cells through ROS-mediated mitochondrial pathway.

    Science.gov (United States)

    Zhao, Yana; Zhang, Shaojing; Wang, Pengfei; Fu, Shengnan; Wu, Di; Liu, Anjun

    2017-12-01

    Seleno-short-chain chitosan (SSCC) is a synthesized chitosan derivative. In this study, antitumor activity and underlying mechanism of SSCC on human non-small-cell lung cancer A549 cells were investigated in vitro. The MTT assay showed that SSCC could inhibit cell viability in a dose- and time-dependent manner, and 200 μg/ml SSCC exhibited significantly toxic effects on A549 cells. The cell cycle assay showed that SSCC triggered S phase cell cycle arrest in a dose- and time-dependent manner, which was related to a downregulation of S phase associated cyclin A. The DAPI staining and Annexin V-FITC/PI double staining identified that the SSCC could induce A549 cells apoptosis. Further studies found that SSCC led to the generation of reactive oxygen species (ROS) and the disruption of mitochondrial membrane potential (MMP) by DCFH-DA and Rhodamin 123 staining, respectively. Meanwhile, free radical scavengers N-acetyl-L-cysteine (NAC) pretreatment confirmed that SSCC-induced A549 cells apoptosis was associated with ROS generation. Furthermore, real-time PCR and western blot assay showed that SSCC up-regulated Bax and down-regulated Bcl-2, subsequently incited the release of cytochrome c from mitochondria to cytoplasm, activated the increase of cleaved-caspase 3 and finally induced A549 cells apoptosis in vitro. In general, the present study demonstrated that SSCC induced A549 cells apoptosis via ROS-mediated mitochondrial apoptosis pathway.

  1. [Effects of bufalin combined with doxorubicin on the proliferation and apoptosis of human lung cancer cell line A549 in vitro].

    Science.gov (United States)

    Zhang, Cuili; Fu, Li'na

    2017-07-28

    To explore the effects of bufalin (BUF) combined with doxorubicin (DOX) on the proliferation and apoptosis in human lung cancer cell line A549 in vitro.
 Methods: Methyl thiazolyl tetrazolium (MTT) assay was used to measure the inhibitory effects of BUF, DOX and their combination on the growth of A549 cells. Hoechst 33342 staining was used to observe the changes of nucleus. Flow cytometry was used to investigate the apoptosis and cell cycle distribution of A549 cells. Western blot was used to examine the expression of apoptotic protein.
 Results: BUF and DOX showed inhibitory effect on the A549 cells in a dose and time-dependent manner. Compared with BUF or DOX alone, combination of BUF (1, 20, 100 nmol/L) with DOX (1.0 μg/mL) could significantly increase the growth inhibition rate of A549 cells at 24, 36, 72 h, respectively (all PA549 cells, keep the cell stage stay in S stage and up-regulate the expression of caspase-3.
 Conclusion: BUF combined with DOX can significantly inhibit the proliferation of A549 cells, which might be related to the induction of apoptosis, cell cycle S phase arrest and caspase-3 up-regulation.

  2. Flavonoids isolated fromCitrus platymammainduced G2/M cell cycle arrest and apoptosis in A549 human lung cancer cells.

    Science.gov (United States)

    Nagappan, Arulkumar; Lee, Ho Jeong; Saralamma, Venu Venkatarame Gowda; Park, Hyeon Soo; Hong, Gyeong Eun; Yumnam, Silvia; Raha, Suchismita; Charles, Shobana Nancy; Shin, Sung Chul; Kim, Eun Hee; Lee, Won Sup; Kim, Gon Sup

    2016-08-01

    Citrus platymamma hort. ex Tanaka belongs to the Rutaceae family and is widely used in folk medicines in Korea due to its anti-proliferative, anti-cancer, anti-oxidant, anti-inflammatory and anti-diabetic activities. However, the molecular mechanism of its anti-cancer effect is not well understood. The present study was conducted to elucidate the anti-cancer effect and molecular mechanism of flavonoids from Citrus platymamma (FCP) on A549 cells. FCP displayed concentration-dependent inhibition on A549 cells proliferation. Further, flow cytometry revealed that FCP significantly increased the sub-G1 (apoptotic cell population) and G2/M phase population, and the total number of apoptotic cells, in a dose-dependent manner. Nuclear condensation and fragmentation were also observed upon staining with Hoechst 33342 in FCP-treated A549 cells. Immunoblotting demonstrated a dose-dependent downregulation of cyclin B1, cyclin-dependent kinase 1, cell division cycle 25c, pro-caspases -3, -6, -8 and -9, and poly (adenosine diphosphate-ribose) polymerase (PARP) in FCP-treated A549 cells. In addition, FCP induced caspase-3 activation and subsequent PARP cleavage, and increased the B-cell lymphoma (Bcl)-2-associated X protein/Bcl-extra large ratio in A549 cells. These findings suggest that FCP induced G2/M arrest and apoptosis of A549 cells. The present study provides evidence that FCP may be useful in the treatment of human lung cancer.

  3. FoxM1 inhibition enhances chemosensitivity of docetaxel-resistant A549 cells to docetaxel via activation of JNK/mitochondrial pathway.

    Science.gov (United States)

    Wang, Ke; Zhu, Xue; Zhang, Kai; Zhu, Ling; Zhou, Fanfan

    2016-09-01

    Docetaxel is recommended as a second-line chemotherapy agent for the non-small-cell lung cancer (NSCLC); however, drug resistance greatly limits its efficiency. Forkhead box M1 (FoxM1), an oncogenic transcription factor, is believed to be involved in the chemoresistance of various human cancers; whereas the association of FoxM1 with acquired docetaxel-resistance in NSCLC remains unclear. In the present study, we investigated the involvement of FoxM1 in the docetaxel-resistant human lung adenocarcinoma A549 cells (A549/DTX). Our results showed that FoxM1 expression was significantly increased in the A549/DTX cells compared with that in the parental A549 cells. FoxM1 siRNA silencing promoted the cytotoxic and pro-apoptotic effect of docetaxel in A549/DTX cells, which was possibly mediated through inducing the activation of c-Jun N-terminal kinases/mitochondrial signaling pathway. Our results suggest a critical role of FoxM1 in docetaxel-resistance of the A549 cells and form the basis for the development of combined therapy of docetaxel and FoxM1 depletion in treating NSCLC. © The Author 2016. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  4. Salvianolic acid A reverses cisplatin resistance in lung cancer A549 cells by targeting c-met and attenuating Akt/mTOR pathway.

    Science.gov (United States)

    Tang, Xia-Li; Yan, Li; Zhu, Ling; Jiao, De-Min; Chen, Jun; Chen, Qing-Yong

    2017-09-01

    Drug resistance is one of the leading causes of chemotherapy failure in non-small cell lung cancer (NSCLC) treatment. The purpose of this study was to investigate the role of c-met in human lung cancer cisplatin resistance cell line (A549/DDP) and the reversal mechanism of salvianolic acid A (SAA), a phenolic active compound extracted from Salvia miltiorrhiza. In this study, we found that A549/DDP cells exert up-regulation of c-met by activating the Akt/mTOR signaling pathway. We also show that SAA could increase the chemotherapeutic efficacy of cisplatin, suggesting a synergistic effect of SAA and cisplatin. Moreover, we revealed that SAA enhanced sensitivity to cisplatin in A549/DDP cells mainly through suppression of the c-met/AKT/mTOR signaling pathway. Knockdown of c-met revealed similar effects as that of SAA in A549/DDP cells. In addition, SAA effectively prevented multidrug resistance associated protein1 (MDR1) up-regulation in A549/DDP cells. Taken together, our results indicated that SAA suppressed c-met expression and enhanced the sensitivity of lung adenocarcinoma A549 cells to cisplatin through AKT/mTOR signaling pathway. Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  5. Polyurethane dispersion containing quaternized ammonium groups: An efficient nanosize gene delivery carrier for A549 cancer cell line transfection.

    Science.gov (United States)

    Yousefpour Marzbali, Mahsa; Yari Khosroushahi, Ahmad; Movassaghpour, AliAkbar; Yeganeh, Hamid

    2016-01-25

    A novel polyurethane containing cationic ammonium groups (QPU) was synthesized and used as vector for gene therapy and cancer gene targeting. The synthesized QPU was characterized by Fourier transform infrared and nuclear magnetic resonance spectroscopy methods. An agarose gel retardation electrophoresis assay was conducted to verify the complete complex formation between QPU and pDNA. The particles size and zeta potential of neat polymers, plasmid DNA, polymers/DNA polyplexes were determined by the dynamic light scattering technique. The polyplexes cytotoxicity was determined using [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and its transfection efficiency was examined qualitatively by fluorescent microscopy and quantitatively by flow cytometery methods. The gel retardation assay, particle size and zeta potential measurements were confirmed that the synthesized cationic polymer could condense DNA efficiently in the physiologic condition. QPU polyplexes showed a significantly lower cytotoxicity compared to Polyfect polyplexes in the examined human cancerous (A549) or normal cells (KDR). Based on our findings, the transfection efficiency by QPU was 2.2 fold higher than Polyfect in the A549 cells whereas in the KDR cells, the cell transfection by Polyfect was 18.1 fold higher than QPU. Due to low cytotoxicity for normal cells and high transfection efficiency in cancer cells, the potential applicability of designed QPU as a non-viral gene carrier for targeting of cancer gene therapy was confirmed. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  6. Phorbol ester potentiates the growth inhibitory effects of troglitazone via up-regulation of PPARγ in A549 cells

    International Nuclear Information System (INIS)

    Kim, Hyo Jung; Woo, Im Sun; Kang, Eun Sil; Eun, So Young; Kim, Gil Hyeong; Ham, Sun Ah; Kim, Hye Jung; Lee, Jae Heun; Chang, Ki Churl; Kim, Jin-Hoi; Lee, Hoon Taek; Seo, Han Geuk

    2006-01-01

    The activation of peroxisome proliferator-activated receptor gamma (PPARγ) has been shown to induce growth arrest and differentiation of various cancer cells. In the current study, we investigated the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the expression of PPARγ and proliferation of A549 cells. TPA elicited a dose- and time-dependent increase in PPARγ mRNA and protein levels. PPARγ expression in response to TPA was attenuated by pretreatment with bisindolylmaleimide I, N-acetyl-L-cysteine (NAC) and PD98059. TPA-induced protein kinase C (PKC) activation was linked to the generation of reactive oxygen species (ROS), both of which were indispensable for PPARγ expression in A549 cells. Pretreatment with bisindolylmaleimide I or NAC blocked TPA-induced phosphorylation of extracellular signal-regulated kinase (ERK), suggesting that ERK-mediated signaling is also involved in the induction of PPARγ. Furthermore, the growth inhibitory effect of troglitazone was significantly potentiated by prolonged incubation with TPA and was attenuated in the presence of GW9662, a specific inhibitor of PPARγ. These effects were associated with an induction of cell cycle arrest at G /G 1 phase, which was accompanied by the induction of p21 Waf1/Cip1 expression and decreased cyclin D1 expression. Taken together, these observations indicate that TPA synergizes with PPARγ ligand to inhibit cell growth through up-regulation of PPARγ expression

  7. Shikonin Induces Apoptosis, Necrosis, and Premature Senescence of Human A549 Lung Cancer Cells through Upregulation of p53 Expression

    Directory of Open Access Journals (Sweden)

    Yueh-Chiao Yeh

    2015-01-01

    Full Text Available Shikonin, a natural naphthoquinone pigment isolated from Lithospermum erythrorhizon, has been reported to suppress growth of various cancer cells. This study was aimed to investigate whether this chemical could also inhibit cell growth of lung cancer cells and, if so, works via what molecular mechanism. To fulfill this, A549 lung cancer cells were treated with shikonin and then subjected to microscopic, biochemical, flow cytometric, and molecular analyses. Compared with the controls, shikonin significantly induced cell apoptosis and reduced proliferation in a dose-dependent manner. Specially, lower concentrations of shikonin (1–2.5 μg/mL cause viability reduction; apoptosis and cellular senescence induction is associated with upregulated expressions of cell cycle- and apoptotic signaling-regulatory proteins, while higher concentrations (5–10 μg/mL precipitate both apoptosis and necrosis. Treatment of cells with pifithrin-α, a specific inhibitor of p53, suppressed shikonin-induced apoptosis and premature senescence, suggesting the role of p53 in mediating the actions of shikonin on regulation of lung cancer cell proliferation. These results indicate the potential and dose-related cytotoxic actions of shikonin on A549 lung cancer cells via p53-mediated cell fate pathways and raise shikonin a promising adjuvant chemotherapeutic agent for treatment of lung cancer in clinical practice.

  8. Development of drug-loaded chitosan hollow nanoparticles for delivery of paclitaxel to human lung cancer A549 cells.

    Science.gov (United States)

    Jiang, Jie; Liu, Ying; Wu, Chao; Qiu, Yang; Xu, Xiaoyan; Lv, Huiling; Bai, Andi; Liu, Xuan

    2017-08-01

    In this study, biodegradable chitosan hollow nanospheres (CHN) were fabricated using polystyrene nanospheres (PS) as templates. CHN were applied to increase the solubility of poorly water-soluble drugs. The lung cancer drug paclitaxel (PTX), which is used as a model drug, was loaded into CHN by the adsorption equilibrium method. The drug-loaded sample (PTX-CHN) offered sustained PTX release and good bioavailability. The state characterization of PTX by differential scanning calorimetry (DSC), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) showed that the PTX absorbed into CHN existed in an amorphous state. An in vitro toxicity experiment indicated that CHN were nontoxic as carriers of poorly water-soluble drugs. The PTX-CHN produced a marked inhibition of lung cancer A549 cells proliferation and encouraged apoptosis. A cell uptake experiment indicated that PTX-CHN was successfully taken up by lung cancer A549 cells. Furthermore, a degradation experiment revealed that CHN were readily biodegradable. These findings state clearly that CHN can be regarded as promising biomaterials for lung cancer treatment.

  9. Proteomic analysis of cellular response induced by multi-walled carbon nanotubes exposure in A549 cells.

    Directory of Open Access Journals (Sweden)

    Li Ju

    Full Text Available The wide application of multi-walled carbon nanotubes (MWCNT has raised serious concerns about their safety on human health and the environment. However, the potential harmful effects of MWCNT remain unclear and contradictory. To clarify the potentially toxic effects of MWCNT and to elucidate the associated underlying mechanisms, the effects of MWCNT on human lung adenocarcinoma A549 cells were examined at both the cellular and the protein level. Cytotoxicity and genotoxicity were examined, followed by a proteomic analysis (2-DE coupled with LC-MS/MS of the cellular response to MWCNT. Our results demonstrate that MWCNT induces cytotoxicity in A549 cells only at relatively high concentrations and longer exposure time. Within a relatively low dosage range (30 µg/ml and short time period (24 h, MWCNT treatment does not induce significant cytotoxicity, cell cycle changes, apoptosis, or DNA damage. However, at these low doses and times, MWCNT treatment causes significant changes in protein expression. A total of 106 proteins show altered expression at various time points and dosages, and of these, 52 proteins were further identified by MS. Identified proteins are involved in several cellular processes including proliferation, stress, and cellular skeleton organization. In particular, MWCNT treatment causes increases in actin expression. This increase has the potential to contribute to increased migration capacity and may be mediated by reactive oxygen species (ROS.

  10. Screening of Stat3 inhibitory effects of Korean herbal medicines in the A549 human lung cancer cell line.

    Science.gov (United States)

    Park, Jong-Shik; Bang, Ok-Sun; Kim, Jinhee

    2014-06-01

    The transcription factor signal transducer and activator of transcription 3 (Stat3) is constitutively activated in many human cancers. It promotes tumor cell proliferation, inhibits apoptosis, induces angiogenesis and metastasis, and suppresses antitumor host immune responses. Therefore, Stat3 has emerged as a promising molecular target for cancer therapies. In this study, we evaluated the Stat3-suppressive activity of 38 herbal medicines traditionally used in Korea. Medicinal herb extracts in 70% ethanol were screened for their ability to suppress Stat3 in the A549 human lung cancer cell line. A Stat3-responsive reporter assay system was used to detect intracellular Stat3 activity in extract-treated cells, and Western blot analyses were performed to measure the expression profiles of Stat3-regulated proteins. Fifty percent of the 38 extracts possessed at least mild Stat3-suppressive activities (i.e., activity less than 75% of the vehicle control). Ethanol extracts of Bupleurum falcatum L., Taraxacum officinale Weber, Solanum nigrum L., Ulmus macrocarpa Hance, Euonymus alatus Sieb., Artemisia capillaris Thunb., and Saururus chinensis (Lour.) Baill inhibited up to 75% of the vehicle control Stat3 activity level. A549 cells treated with these extracts also had reduced Bcl-xL, Survivin, c-Myc, and Mcl-1 expression. Many medicinal herbs traditionally used in Korea contain Stat3 activity-suppressing substances. Because of the therapeutic impact of Stat3 inhibition, these results could be useful when developing novel cancer therapeutics from medicinal herbs.

  11. The influence of Hurricanes Katrina and Rita on the inflammatory cytokine response and protein expression in A549 cells exposed to PM2.5 collected in the Baton Rouge-Port Allen industrial corridor of Southeastern Louisiana in 2005.

    Science.gov (United States)

    Bourgeois, Brian; Owens, John Wesley

    2014-03-01

    Hurricanes Katrina and Rita hit the coast of Louisiana in 2005 and killed more than 2000 people. The two storms resulted in a significant spike in particulate matter (PM2.5) levels across the state of Louisiana. This report focuses on PM2.5 samples collected in 2005 from two monitoring sites in the neighboring cities of Baton Rouge and Port Allen, Louisiana. Inductively coupled plasma (ICP) revealed the presence of PM2.5-adsorbed representative and Fenton-active transition metals. Gas chromatography/mass spectrometry (GC-MS) analyses revealed the presence of 23 PAH compounds. Endotoxins were also detected. Metals and endotoxins were extracted with water. PAH were extracted with dichloromethane. In order to assess cytotoxicity, aqueous PM2.5 extracts were introduced to A549 Human Epithelial Lung Carcinoma Cells. Results indicated decreased cell viability in a dose-dependent manner, with an LC50 of 235 µg/ml and 250 µg/ml, respectively, for the two sites featured here. Endotoxins alone were not cytotoxic. The concentration of reactive oxygen species (ROS) and released LDH activity increased following exposure of A549 cells to aqueous PM2.5 extracts. Fluorescence microscopy revealed apoptotic and necrotic cell death mechanisms. ELISA revealed increased secretion of primary pro-inflammatory cytokines, IL-6, IL-8, and TNF-α. Global PCR gene expression revealed up-regulation of proteins associated with the cytokine storm; e.g. interleukins, chemokines, and TNF-α. Global antibody microarray was consistent with an inflammatory response, with up-regulation of cytokines involved in the down-field activation of the caspase cascade and kinase pathways. The up-regulation of metal-redox sensitive transcription factors, NF-κβ and AP-1, is consistent with a cell death mechanism initiated by Fenton-active transition metal redox catalysis.

  12. Tanshinone IIA combined with adriamycin inhibited malignant biological behaviors of NSCLC A549 cell line in a synergistic way.

    Science.gov (United States)

    Xie, Jun; Liu, Jia-Hui; Liu, Heng; Liao, Xiao-Zhong; Chen, Yuling; Lin, Mei-Gui; Gu, Yue-Yu; Liu, Tao-Li; Wang, Dong-Mei; Ge, Hui; Mo, Sui-Lin

    2016-11-18

    The study was designed to develop a platform to verify whether the extract of herbs combined with chemotherapy drugs play a synergistic role in anti-tumor effects, and to provide experimental evidence and theoretical reference for finding new effective sensitizers. Inhibition of tanshinone IIA and adriamycin on the proliferation of A549, PC9 and HLF cells were assessed by CCK8 assays. The combination index (CI) was calculated with the Chou-Talalay method, based on the median-effect principle. Migration and invasion ability of A549 cells were determined by wound healing assay and transwell assay. Flow cytometry was used to detect the cell apoptosis and the distribution of cell cycles. TUNEL staining was used to detect the apoptotic cells. Immunofluorescence staining was used to detect the expression of Cleaved Caspase-3. Western blotting was used to detect the proteins expression of relative apoptotic signal pathways. CDOCKER module in DS 2.5 was used to detect the binding modes of the drugs and the proteins. Both tanshinone IIA and adriamycin could inhibit the growth of A549, PC9, and HLF cells in a dose- and time-dependent manner, while the proliferative inhibition effect of tanshinone IIA on cells was much weaker than that of adriamycin. Different from the cancer cells, HLF cells displayed a stronger sensitivity to adriamycin, and a weaker sensitivity to tanshinone IIA. When tanshinone IIA combined with adriamycin at a ratio of 20:1, they exhibited a synergistic anti-proliferation effect on A549 and PC9 cells, but not in HLF cells. Tanshinone IIA combined with adriamycin could synergistically inhibit migration, induce apoptosis and arrest cell cycle at the S and G2 phases in A549 cells. Both groups of the single drug treatment and the drug combination up-regulated the expressions of Cleaved Caspase-3 and Bax, but down-regulated the expressions of VEGF, VEGFR2, p-PI3K, p-Akt, Bcl-2, and Caspase-3 protein. Compared with the single drug treatment groups, the drug

  13. Alternaria extract activates autophagy that induces IL-18 release from airway epithelial cells.

    Science.gov (United States)

    Murai, Hiroki; Okazaki, Shintaro; Hayashi, Hisako; Kawakita, Akiko; Hosoki, Koa; Yasutomi, Motoko; Sur, Sanjiv; Ohshima, Yusei

    2015-09-04

    Alternaria alternata is a major outdoor allergen that causes allergic airway diseases. Alternaria extract (ALT-E) has been shown to induce airway epithelial cells to release IL-18 and thereby initiate Th2-type responses. We investigated the underlying mechanisms involved in IL-18 release from ALT-E-stimulated airway epithelial cells. Normal human bronchial epithelial cells and A549 human lung adenocarcinoma cells were stimulated with ALT-E in the presence of different inhibitors of autophagy or caspases. IL-18 levels in culture supernatants were measured by ELISA. The numbers of autophagosomes, an LC3-I to LC3-II conversion, and p62 degradation were determined by immunofluorescence staining and immunoblotting. 3-methyladenine and bafilomycin, which inhibit the formation of preautophagosomal structures and autolysosomes, respectively, suppressed ALT-E-induced IL-18 release by cells, whereas caspase 1 and 8 inhibitors did not. ALT-E-stimulation increased autophagosome formation, LC-3 conversion, and p62 degradation in airway epithelial cells. LPS-stimulation induced the LC3 conversion in A549 cells, but did not induce IL-18 release or p62 degradation. Unlike LPS, ALT-E induced airway epithelial cells to release IL-18 via an autophagy dependent, caspase 1 and 8 independent pathway. Although autophagy has been shown to negatively regulate canonical inflammasome activity in TLR-stimulated macrophages, our data indicates that this process is an unconventional mechanism of IL-18 secretion by airway epithelial cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Novel CHOP activator LGH00168 induces necroptosis in A549 human lung cancer cells via ROS-mediated ER stress and NF-κB inhibition.

    Science.gov (United States)

    Ma, Yi-Ming; Peng, Yan-Min; Zhu, Qiong-Hua; Gao, An-Hui; Chao, Bo; He, Qiao-Jun; Li, Jia; Hu, You-Hong; Zhou, Yu-Bo

    2016-09-01

    C/EBP homologous protein (CHOP) is a transcription factor that is activated at multiple levels during ER stress and plays an important role in ER stress-induced apoptosis. In this study we identified a novel CHOP activator, and further investigated its potential to be a therapeutic agent for human lung cancer. HEK293-CHOP-luc reporter cells were used in high-throughput screening (HTS) to identify CHOP activators. The cytotoxicity against cancer cells in vitro was measured with MTT assay. The anticancer effects were further examined in A549 human non-small cell lung cancer xenograft mice. The mechanisms underlying CHOP activation were analyzed using luciferase assays, and the anticancer mechanisms were elucidated in A549 cells. From chemical libraries of 50 000 compounds, LGH00168 was identified as a CHOP activator, which showed cytotoxic activities against a panel of 9 cancer cell lines with an average IC 50 value of 3.26 μmol/L. Moreover, administration of LGH00168 significantly suppressed tumor growth in A549 xenograft bearing mice. LGH00168 activated CHOP promoter via AARE1 and AP1 elements, increased DR5 expression, decreased Bcl-2 expression, and inhibited the NF-κB pathway. Treatment of A549 cells with LGH00168 (10 μmol/L) did not induce apoptosis, but lead to RIP1-dependent necroptosis, accompanied by cell swelling, plasma membrane rupture, lysosomal membrane permeabilization, MMP collapse and caspase 8 inhibition. Furthermore, LGH00168 (10 and 20 μmol/L) dose-dependently induced mito-ROS production in A549 cells, which was reversed by the ROS scavenger N-acetyl-L-cysteine (NAC, 10 mmol/L). Moreover, NAC significantly diminished LGH00168-induced CHOP activation, NF-κB inhibition and necroptosis in A549 cells. LGH00168 is a CHOP activator that inhibits A549 cell growth in vitro and lung tumor growth in vivo.

  15. Citotoxicidad de extractos de plantas medicinales sobre la línea celular de carcinoma de pulmón humano A549 Cytotoxicity of medicinal plant extracts on the human lung carcinoma cell line A549

    OpenAIRE

    Alexis Díaz García; Hermis Rodríguez Sánchez; Ramón Scull Lizama

    2011-01-01

    OBJETIVO: evaluar el efecto de 10 extractos de plantas medicinales sobre el crecimiento de la línea celular humana de carcinoma de pulmón A549. METODOS: el efecto de los extractos sobre la células tumorales se midió a través de un ensayo colorimétrico mediante el empleo del bromuro de 3-(4,5-dimetil-tiazol-2-yl)-2,5-difenil tetrazolio a concentraciones entre 3,9-250 µg/mL durante 72 h y se calculó la concentración citotóxica media para cada uno. RESULTADOS: del total de los extractos evaluado...

  16. Human bronchial epithelial cells express PAR-2 with different sensitivity to thermolysin.

    Science.gov (United States)

    Ubl, Joachim J; Grishina, Zoryana V; Sukhomlin, Tatiana K; Welte, Tobias; Sedehizade, Fariba; Reiser, Georg

    2002-06-01

    Protease-activated receptor-2 (PAR-2) plays a role in inflammatory reactions in airway physiology. Proteases cleaving the extracellular NH(2) terminus of receptors activate or inactivate PAR, thus possessing a therapeutic potential. Using RT-PCR and immunocytochemistry, we show PAR-2 in human airway epithelial cell lines human bronchial epithelial (HBE) and A549. Functional expression of PAR-2 was confirmed by Ca(2+) imaging studies using the receptor agonist protease trypsin. The effect was abolished by soybean trypsin inhibitor and mimicked by the specific PAR-2 peptide agonist SLIGKV. Amplitude and duration of PAR-2-elicited Ca(2+) response in HBE and A549 cells depend on concentration and time of agonist superfusion. The response is partially pertussis toxin (PTX) insensitive, abolished by the phospholipase C inhibitor U-73122, and diminished by the inositol 1,4,5-trisphosphate receptor antagonist 2-aminoethoxydiphenyl borate. Cathepsin G altered neither the resting Ca(2+) level nor PAR-2-elicited Ca(2+) response. Thermolysin, a prototypic bacterial metalloprotease, induced a dose-dependent Ca(2+) response in HBE, but not A549, cells. In both cell lines, thermolysin abolished the response to a subsequent trypsin challenge but not to SLIGKV. Thus different epithelial cell types express different PAR-2 with identical responses to physiological stimuli (trypsin, SLIGKV) but different sensitivity to modifying proteases, such as thermolysin.

  17. Depletion of hepatoma-derived growth factor-related protein-3 induces apoptotic sensitization of radioresistant A549 cells via reactive oxygen species-dependent p53 activation

    International Nuclear Information System (INIS)

    Yun, Hong Shik; Hong, Eun-Hee; Lee, Su-Jae; Baek, Jeong-Hwa; Lee, Chang-Woo; Yim, Ji-Hye; Um, Hong-Duck; Hwang, Sang-Gu

    2013-01-01

    Highlights: •HRP-3 is a radiation- and anticancer drug-responsive protein in A549 cells. •Depletion of HRP-3 induces apoptosis of radio- and chemoresistant A549 cells. •Depletion of HRP-3 promotes ROS generation via inhibition of the Nrf2/HO-1 pathway. •Depletion of HRP-3 enhances ROS-dependent p53 activation and PUMA expression. -- Abstract: Biomarkers based on functional signaling have the potential to provide greater insight into the pathogenesis of cancer and may offer additional targets for anticancer therapeutics. Here, we identified hepatoma-derived growth factor-related protein-3 (HRP-3) as a radioresistance-related gene and characterized the molecular mechanism by which its encoded protein regulates the radio- and chemoresistant phenotype of lung cancer-derived A549 cells. Knockdown of HRP-3 promoted apoptosis of A549 cells and potentiated the apoptosis-inducing action of radio- and chemotherapy. This increase in apoptosis was associated with a substantial generation of reactive oxygen species (ROS) that was attributable to inhibition of the Nrf2/HO-1 antioxidant pathway and resulted in enhanced ROS-dependent p53 activation and p53-dependent expression of PUMA (p53 upregulated modulator of apoptosis). Therefore, the HRP-3/Nrf2/HO-1/ROS/p53/PUMA cascade is an essential feature of the A549 cell phenotype and a potential radiotherapy target, extending the range of targets in multimodal therapies against lung cancer

  18. ROS and NF-κB are involved in upregulation of IL-8 in A549 cells exposed to multi-walled carbon nanotubes

    International Nuclear Information System (INIS)

    Ye Shefang; Wu Yihui; Hou Zhenqing; Zhang Qiqing

    2009-01-01

    Carbon nanotubes (CNTs) have potential applications in biosensors, tissue engineering, and biomedical devices because of their unique physico-chemical, electronic and mechanical properties. However, there is limited literature data available concerning the biological properties and toxicity of CNTs. This study aimed to assess the toxicity exhibited by multi-walled CNTs (MWCNTs) and to elucidate possible molecular mechanisms underlying the biological effects of MWCNTs in A549 cells. Exposing A549 cells to MWCNTs led to cell death, changes in cell size and complexity, reactive oxygen species (ROS) production, interleukin-8 (IL-8) gene expression and nuclear factor (NF)-κB activation. Treatment of A549 cells with antioxidants prior to adding MWCNTs decreased ROS production and abrogated expression of IL-8 mRNA. Pretreatment of A549 cells with NF-κB inhibitors suppressed MWCNTs-induced IL-8 mRNA expression. These results indicate that MWCNTs are able to induce expression of IL-8 in A549 cells, at least in part, mediated by oxidative stress and NF-κB activation.

  19. MicroRNA-126 Targeting PIK3R2 Inhibits NSCLC A549 Cell Proliferation, Migration, and Invasion by Regulation of PTEN/PI3K/AKT Pathway.

    Science.gov (United States)

    Song, Lei; Li, Dan; Gu, Yue; Wen, Zhong-Mei; Jie, Jing; Zhao, Dan; Peng, Li-Ping

    2016-09-01

    Our study explored whether the microRNA-126 (miR-126)-mediated PTEN/PI3K/AKT (phosphatase and tensin homology deleted on chromosome 10/phosphatidylinositol 3-kinase regulatory subunit-β/AKT) signaling pathway by targeting PIK3R2 affects the proliferation, migration, and invasion of non-small-cell lung cancer (NSCLC) A549 cells. Quantitative real-time polymerase chain reaction was used to measure the expression of miR-126 in A549 cells. The MTT (methyl thiazolyl tetrazolium) assay, cell scratch test, Transwell assay, and Western blot were used to detect the proliferation, migration, and invasion of A549 cells and protein expression in A549 cells, respectively. The expression of miR-126 decreased and the expression of PIK3R2 increased in A549 cells (P A549 cells, the downregulation of the expression of PIK3R2, PI3K, and phosphorylated Akt (p-Akt) protein, and the upregulation of PTEN expression (P A549 cells increased, and the expression of these 3 proteins was upregulated with downregulation of miR-126 (P A549 cells (P A549 cells can reduce the expression of the target gene PIK3R2 and influence the PTEN/PI3K/AKT signaling pathway, suppressing the proliferation, migration, and invasive abilities of A549 cells. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Repeated Aurora-A siRNA Transfection Results in Effective Apoptosis of A549 Cells Compared to Single Transfection.

    Science.gov (United States)

    Wang, Zhonghua; Sun, Wenwu; Cao, Jianping; Cui, Haiyang; Ma, Zhuang

    2016-01-01

    Suppression of Aurora kinase A (Aurora-A, AURKA) by Aurora-A siRNA has been proposed for lung tumor treatment. However, protocols using single administration have shown little benefit in some types of lung tumor. Given that transfection efficiency of Aurora-A siRNA is low due to tightly packed cells in the tumor, we hypothesized that repeated administration would result in efficient cell apoptosis. We compared single vs. repeated transfection (thrice) in A549 cells by transfecting Aurora-A siRNA (siA) on the 1st or 1st, 2nd and 3rd day after cell seeding. A random sequence was used as the negative siRNA control (siC). Cells in the single transfection group received only transfection reagent without siRNAs on the 2nd and 3rd day. Two days after the third transfection, both single and repeated siA administration decreased mRNA expression of Aurora-A and cell viability compared to no administration and siC single administration. However, the decrease in these two indices with repeated transfection was more obvious than that following single administration: cell viability decreased to 72.8 ± 3.05% (p transfection and to 64.2 ± 1.99% (p transfection, compared with normal control cells, respectively. Gene expression decreased to 17 ± 16.6% (p transfection and to 43.2 ± 13.0% (p transfection. Compared to single transfection, repeated Aurora-A siRNA transfection decreased Aurora-A, which, in turn, resulted in effective apoptosis of A549 cells.

  1. Activities of ten essential oils towards Propionibacterium acnes and PC-3, A-549 and MCF-7 cancer cells.

    Science.gov (United States)

    Zu, Yuangang; Yu, Huimin; Liang, Lu; Fu, Yujie; Efferth, Thomas; Liu, Xia; Wu, Nan

    2010-04-30

    Ten essential oils, namely, mint (Mentha spicata L., Lamiaceae), ginger (Zingiber officinale Rosc., Zingiberaceae), lemon (Citrus limon Burm.f., Rutaceae), grapefruit (Citrus paradisi Macf., Rutaceae), jasmine (Jasminum grandiflora L., Oleaceae), lavender (Mill., Lamiaceae), chamomile (Matricaria chamomilla L., Compositae), thyme (Thymus vulgaris L., Lamiaceae), rose (Rosa damascena Mill., Rosaceae) and cinnamon (Cinnamomum zeylanicum N. Lauraceae) were tested for their antibacterial activities towards Propionibacterium acnes and in vitro toxicology against three human cancer cell lines. Thyme, cinnamon and rose essential oils exhibited the best antibacterial activities towards P. acnes, with inhibition diameters of 40 +/- 1.2 mm, 33.5 +/- 1.5 mm and 16.5 +/- 0.7 mm, and minimal inhibitory concentrations of 0.016% (v/v), 0.016% (v/v) and 0.031% (v/v), respectively. Time-kill dynamic procedures showed that thyme, cinnamon, rose, and lavender essential oils exhibited the strongest bactericidal activities at a concentration of 0.25% (v/v), and P. acnes was completely killed after 5 min. The thyme essential oil exhibited the strongest cytotoxicity towards three human cancer cells. Its inhibition concentration 50% (IC(50)) values on PC-3, A549 and MCF-7 tumor cell lines were 0.010% (v/v), 0.011% (v/v) and 0.030% (v/v), respectively. The cytotoxicity of 10 essential oils on human prostate carcinoma cell (PC-3) was significantly stronger than on human lung carcinoma (A549) and human breast cancer (MCF-7) cell lines.

  2. Activities of Ten Essential Oils towards Propionibacterium acnes and PC-3, A-549 and MCF-7 Cancer Cells

    Directory of Open Access Journals (Sweden)

    Yuangang Zu

    2010-04-01

    Full Text Available Ten essential oils, namely, mint (Mentha spicata L.,Lamiaceae, ginger (Zingiber officinaleRosc.,Zingiberaceae, lemon (Citrus limon Burm.f.,Rutaceae, grapefruit (Citrus paradisi Macf., Rutaceae, jasmine (Jasminum grandiflora L.,Oleaceae, lavender (Mill.,Lamiaceae, chamomile (Matricaria chamomilla L., Compositae, thyme (Thymus vulgaris L., Lamiaceae, rose (Rosa damascena Mill.,Rosaceae and cinnamon (Cinnamomum zeylanicumN. Lauraceae were tested for their antibacterial activities towards Propionibacterium acnes and in vitro toxicology against three human cancer cell lines. Thyme, cinnamon and rose essential oils exhibited the best antibacterial activities towards P. acnes, with inhibition diameters of 40 ± 1.2 mm, 33.5 ± 1.5 mm and 16.5 ± 0.7 mm, and minimal inhibitory concentrations of 0.016% (v/v, 0.016% (v/v and 0.031% (v/v, respectively. Time-kill dynamic procedures showed that thyme, cinnamon, rose, and lavender essential oils exhibited the strongest bactericidal activities at a concentration of 0.25% (v/v, and P. acnes was completely killed after 5 min. The thyme essential oil exhibited the strongest cytotoxicity towards three human cancer cells. Its inhibition concentration 50% (IC50 values on PC-3, A549 and MCF-7 tumor cell lines were 0.010% (v/v, 0.011% (v/v and 0.030% (v/v, respectively. The cytotoxicity of 10 essential oils on human prostate carcinoma cell (PC-3 was significantly stronger than on human lung carcinoma (A549 and human breast cancer (MCF-7 cell lines.

  3. Pulmonary alveolar microlithiasis in children

    International Nuclear Information System (INIS)

    Schmidt, H.; Loercher, U.; Kitz, R.; Zielen, S.; Ahrens, P.; Koenig, R.

    1996-01-01

    Two asymptomatic Turkish sibs are presented, a 4-year-old boy and his 7-year-old sister, with pulmonary alveolar microlithiasis (PAM) confirmed by transbronchial lung biopsy and bronchoalveolar lavage. Chest radiographs and high resolution CT demonstrated wide-spread intra-alveolar calcifications in both lungs. The lesions were sharply defined and less than 1 mm in diameter. CT documented a high concentration of microliths along the bronchovascular bundles, the intralobular fissue and the (sub)pleural lung parenchyma. The combination of bronchoalveolar lavage and roentgenographic appearance in high resolution CT are characteristic and pathognomonic, and can confirm the diagnosis. The more severe changes in the elder sib and the radiographic controls suggest that the pulmonary disease may be progressive in our patients. The described family of consanguineous, unaffected parents with two affected and one healthy child confirmed the autosomal recessive inheritance of PAM (McKusick 265100). In addition, the affected girl had autosomal recessive Waardenburg-anophthalmia syndrome (McKusick 206920), raising the question of whether this is a chance occurrence or possibly a contiguous gene syndrome. (orig.)

  4. Enhanced rifampicin delivery to alveolar macrophages by solid lipid nanoparticles

    International Nuclear Information System (INIS)

    Chuan Junlan; Li Yanzhen; Yang Likai; Sun Xun; Zhang Qiang; Gong Tao; Zhang Zhirong

    2013-01-01

    The present study aimed at developing a drug delivery system targeting the densest site of tuberculosis infection, the alveolar macrophages (AMs). Rifampicin (RFP)-loaded solid lipid nanoparticles (RFP-SLNs) with an average size of 829.6 ± 16.1 nm were prepared by a modified lipid film hydration method. The cytotoxicity of RFP-SLNs to AMs and alveolar epithelial type II cells (AECs) was examined using MTT assays. The viability of AMs and AECs was above 80 % after treatment with RFP-SLNs, which showed low toxicity to both AMs and AECs. Confocal Laser Scanning Microscopy was employed to observe the interaction between RFP-SLNs and both AMs and AECs. After incubating the cells with RFP-SLNs for 2 h, the fluorescent intensity in AMs was more and remained longer (from 0.5 to 12 h) when compared with that in AECs (from 0.5 to 8 h). In vitro uptake characteristics of RFP-SLNs in AMs and AECs were also investigated by detection of intracellular RFP by High performance liquid chromatography. Results showed that RFP-SLNs delivered markedly higher RFP into AMs (691.7 ng/mg in cultured AMs, 662.6 ng/mg in primary AMs) than that into AECs (319.2 ng/mg in cultured AECs, 287.2 ng/mg in primary AECs). Subsequently, in vivo delivery efficiency and the selectivity of RFP-SLNs were further verified in Sprague–Dawley rats. Under pulmonary administration of RFP-SLNs, the amount of RFP in AMs was significantly higher than that in AECs at each time point. Our results demonstrated that solid lipid nanoparticles are a promising strategy for the delivery of rifampicin to alveolar macrophages selectively.

  5. ENVIRONMENTAL PARTICULATE-MEDIATED CYTOKINE PRODUCTION IN LUNG EPITHELIAL CELLS (A549): ROLE OF PREEXISTING INFLAMMATION AND OXIDANT STRESS. (R824790)

    Science.gov (United States)

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  6. LUNG EPITHELIAL CELL (A549) INTERACTION WITH UNOPSONIZED ENVIRONMENTAL PARTICULATES: QUANTITATION OF PARTICLE-SPECIFIC BINDING AND IL-8 PRODUCTION. (R824790)

    Science.gov (United States)

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  7. DNA damage and cytotoxicity in type II lung epithelial (A549) cell cultures after exposure to diesel exhaust and urban street particles

    DEFF Research Database (Denmark)

    Danielsen, Pernille Høgh; Loft, Steffen; Møller, Peter

    2008-01-01

    can be reproduced independently, but exposure to such samples may not mimic the effects observed after exposure to authentic air pollution particles. This study was designed to compare the DNA oxidizing effects of authentic street particles with SRM1650 and SRM2975. The authentic street particles were...... be due to the much higher level of transition metals. CONCLUSION: Authentic street particles and SRMs differ in their ability to oxidize DNA in a cell-free environment, whereas cell culture experiments indicate that the particle preparations elicit a similar alteration of the level of DNA damage...... and small differences in cytotoxicity. Although it cannot be ruled out that SRMs and authentic street particles might elicit different effects in animal experimental models, this study indicates that on the cellular level, SRM1650 and SRM2975 are suitable surrogate samples for the study of authentic street...

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

    DEFF Research Database (Denmark)

    Roursgaard, Martin; Knudsen, Kristina Bram; Northeved, Helle

    2016-01-01

    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...

  9. Growth inhibitory and apoptosis-inducing effects of allergen-free Rhus verniciflua Stokes extract on A549 human lung cancer cells.

    Science.gov (United States)

    Jang, Ik-Soon; Park, Jae-Woo; Jo, Eun-Bi; Cho, Chong-Kwan; Lee, Yeon-Weol; Yoo, Hwa-Seung; Park, Junsoo; Kim, Jihye; Jang, Byeong-Churl; Choi, Jong-Soon

    2016-11-01

    Evidence suggests that Rhus verniciflua Stokes (RVS) or its extract has the potential to be used for the treatment of inflammatory and neoplastic diseases. However, direct use of RVS or its extract as a herbal medicine has been limited due to the presence of urushiol, an allergenic toxin. In the present study, we prepared an extract of the allergen‑removed RVS (aRVS) based on a traditional method and investigated its inhibitory effect on the growth of various types of human cancer cells, including lung (A549), breast (MCF-7) and prostate (DU-145) cancer cell lines. Notably, among the cell lines tested, treatment with the aRVS extract strongly inhibited proliferation of the A549 cells at a 0.5 mg/ml concentration for 24 h that was not cytotoxic to normal human dermal fibroblasts. Furthermore, aRVS extract treatment largely reduced the survival and induced apoptosis of the A549 cells. At the mechanistic levels, treatment with the aRVS extract led to the downregulation of Bcl-2 and Mcl-1 proteins, the activation of caspase-9/-3 proteins, an increase in cytosolic cytochrome c levels, the upregulation of Bax protein, an increase in phosphorylated p53 protein but a decrease in phosphorylated S6 protein in the A549 cells. Importantly, treatment with z-VAD‑fmk, a pan-caspase inhibitor attenuated aRVS extract-induced apoptosis in the A549 cells. These results demonstrate firstly that aRVS extract has growth inhibitory and apoptosis-inducing effects on A549 human lung cancer cells through modulation of the expression levels and/or activities of caspases, Bcl-2, Mcl-1, Bax, p53 and S6.

  10. Targeted interfering DEP domain containing 1 protein induces apoptosis in A549 lung adenocarcinoma cells through the NF-κB signaling pathway.

    Science.gov (United States)

    Wang, Qingqing; Li, Aili; Jin, Junfei; Huang, Guojin

    2017-01-01

    Ectopic expression of DEP domain containing 1 (DEPDC1) in lung adenocarcinomas is associated with poor prognosis, but its role and the underlying mechanism remain unknown. In this study, DEPDC1 expression in lung cancer cell lines was examined with Western blot assay, and DEPDC1-positive cell A549 was selected for further experiments. DEPDC1 inhibitor miR-130a was overexpressed in A549 cells, and the proliferation and apoptosis of these cells were analyzed with cell counting and flow cytometry assay. Interfering peptide 11R-DEP:611-628 and JNK inhibitor SP600125 were used alone or in combination to treat A549 cells, and the cell proliferation and apoptosis were assessed by flow cytometry assay; caspase 3 and cleaved caspase 3, phosphor-JNK, and total JNK were detected by Western blotting; and nuclear factor kappa B (NF-κB) localization was determined by immunofluorescence staining. We found that miR-130a and 11R-DEP:611-628 peptides (5 μM) both inhibited A549 proliferation and induced apoptosis. We observed that 11R-DEP:611-628 peptide treatment resulted in elevated A20 expression, dramatically reduced nuclear NF-κB, and increased phosphor-JNK. These findings indicate that DEPDC1 inhibits apoptosis of A549 cell by suppressing A20 expression to regulate NF-κB activity, and that JNK plays a protective role upon 11R-DEP:611-628 peptide treatment. In conclusion, DEPDC1 might be a novel therapeutic target for lung cancer, and the 11R-DEP:611-628 peptide is a potent apoptosis inducer in A549 cells.

  11. Benzopyrene promotes lung cancer A549 cell migration and invasion through up-regulating cytokine IL8 and chemokines CCL2 and CCL3 expression.

    Science.gov (United States)

    Zhang, Jin; Chang, Li; Jin, Hanyu; Xia, Yaoxiong; Wang, Li; He, Wenjie; Li, Wenhui; Chen, Hong

    2016-08-01

    Tobacco-sourced carcinogen including benzopyrene (B[a]P) in lung cancer metastasis has not been fully reported. In this study, lung carcinoma A549 cell line was used to investigate the potential roles of tobacco-sourced B[a]P on cell metastasis and invasion and to assess its underlying mechanism. Effects of tobacco-sourced carcinogen on A549 cell proliferation, metastasis, and invasion were analyzed using MTT assay, Transwell assay, and scratch method, respectively. The effects of tobacco-sourced carcinogen on cytokines and chemokines secretion were detected using enzyme-linked immunosorbent assay. Moreover, correlation between inflammatory factor expression and cancer cell migration and invasion was assessed using siRNA-mediated gene silencing. Data showed that both B[a]P and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone either at high or low dose performed no significant difference on A549 cell proliferation with time increasing. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone performed no significant difference on A549 cell migration and invasion while B[a]P significantly increased A549 cell migration and invasion compared to the control group (P A549 cells were significantly decreased compared to the control, respectively (P < 0.05), while silenced IL-8 drastically decreased the migrated and invasive cells compared to the control (P < 0.01). Taken together, this study illustrated that there may be significant correlation between smoking and lung cancer metastasis. B[a]P maybe an excellent contributor for lung cancer metastasis through up-regulating IL-8, CCL-2, and CCL-3 expression. © 2016 by the Society for Experimental Biology and Medicine.

  12. β-Escin sodium inhibits inducible nitric oxide synthase expression via downregulation of the JAK/STAT pathway in A549 cells.

    Science.gov (United States)

    Ji, Deng Bo; Xu, Bo; Liu, Jing Tao; Ran, Fu Xiang; Cui, Jing Rong

    2011-12-01

    β-escin, a triterpene saponin, is one of the major active compounds extracted from horse chestnut (Aesculus hippocastanum) seed. Previous work has found that β-escin sodium has antiinflammatory and antitumor effects. In the present study, we investigated its effect on cell proliferation and inducible nitric-oxide synthase (iNOS) expression in human lung carcinoma A549 cells. β-escin sodium (5-40 µg/mL) inhibited cytokine mixture (CM)-induced nitric oxide (NO) production in A549 cells by reducing the expression of iNOS. β-escin sodium suppressed phosphorylation and nuclear translocation of STAT1 (Tyr701) and STAT3 (Tyr705) induced by CM but did not affect the activation of c-Jun and NF-κB. β-escin sodium inhibited the activation of protein tyrosine kinase JAK2. Pervanadate treatment reversed the β-escin sodium-induced downregulation of STAT3 and STAT1. β-escin sodium treatment enhanced an activating phosphorylation of the phosphatase SHP2. Small interfering RNA-mediated knockdown of SHP2 inhibited β-escin sodium-induced phospho-STAT dephosphorylation. Moreover β-escin sodium reduced the activation of p38 MAPK. Finally, β-escin sodium inhibited the proliferation of A549 cells, did not change the cell membrane's permeability, nuclear morphology and size and the mitochondria's transmembrane potential of A549 cells. Taken together, these results demonstrate that β-escin sodium could downregulate iNOS expression through inhibiting JAK/STAT signaling and p38 MAPK activation in A549 cells. β-escin sodium has a marked antiproliferative effect on A549 cells at least in part by inhibiting the JAK/STAT signaling pathway, but not by a cytotoxic effect. β-escin sodium would be useful as a chemopreventive agent or a therapeutic against inflammatory-associated tumor. © 2011 Wiley Periodicals, Inc. Copyright © 2011 Wiley Periodicals, Inc.

  13. Curcumin Promoted the Apoptosis of Cisplain-resistant Human Lung Carcinoma Cells A549/DDP through Down-regulating miR-186*

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    Jian ZHANG

    2010-04-01

    Full Text Available Background and objective Curcumin, a natural compound, is derived from the rthizom of Curcuma longa. In vitro and in vivo preclinical studies have shown its anti-inflammatory, antioxidant, anticancer activities and so on. miR-186*, which was found by microarray technology, was highly expressed in lung carcinoma cells A549/DDP. The aim of this study is to illustrate whether Curcumin could promote the apoptosis of A549/DDP cells through regulating the expression of miR-186*. Methods An oligonucleotide microarray chip was used to profile microRNA (miRNA expressions in A549/DDP cells treated with and without Curcumin. The significantly differentially expressed miRNA, which was selected from microarray chip, validated by quantitative real-time PCR. Ultimately, the remarkably expressed miRNA modulated the apoptosis assaying by flow cytometry expriments and the survival rate was measured by MTT method. Results The microarray chip results demonstrated: Curcumin altered the expression level of miRNAs compared with untreated control in A549/DDP cell line, miR-186* was significantly down-regulated after Curcumin treatment, which confirmed by quantitative real-time PCR. Downregulation of miR-186* expression by curcumin elevated the apoptosis, and the survival rate of A549/DDP cells decreased; but up-regulation of miR-186* expression by transfection its mimics restrained the apoptosis, the survival rate of A549/DDP cells increased, which were assayed by flow cytometry expriments and MTT method. Conclusion Modulation of miRNAs expression may be an important mechanism underlying the biological roles of Curcumin.

  14. The total flavonoids of Clerodendrum bungei suppress A549 cells proliferation, migration, and invasion by impacting Wnt/β-Catenin signaling

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    Na Yu

    2017-01-01

    Full Text Available Objectives: The objective of this study is to evaluate the effect of the total flavonoids of Clerodendrum bungei (TFCB on the proliferation, invasion, and metastasis of A549 lung cancer cells through the Wnt signaling pathway. Materials and Methods: A549 cells were transfected with a β-catenin overexpression plasmid and the empty vector pcDNA3.1. The A549 cells were divided into six groups: normal A549 cell group, normal A549 cells with TFCB group, vector control group, vector with TFCB group, β-catenin overexpression group, and β-catenin with TFCB group. We used the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay to detect cell proliferation, a scratch test was used to observe cell migration, and a transwell experiment was employed to evaluate cell invasion. Proteins related to the Wnt pathway were detected with Western blot analysis, including β-catenin, GSK-3 β, P-GSK-3 β, c-Myc, and CyclinD1. Results: The proliferation, invasion, and metastasis of A549 cells were significantly enhanced after being transfected with the β-catenin overexpression plasmid (P < 0.05 or 0.01, accompanied by increased expression of β-catenin, C-Myc, CyclinD1 and reduced expression of Gsk-3 β and P-GSK-3 β. Treatment of cells with TFCB resulted in inhibition of cell proliferation, migration, and invasion; downregulated expression of β-catenin, C-Myc, and CyclinD1; and upregulated expression of GSK-3 β and P-GSK-3 β, especially in the β-catenin overexpression group. Conclusion: TFCB has the potential to inhibit the Wnt/β-catenin pathway by prohibiting the overexpression of β-catenin and regulating its downstream factors.

  15. Diffuse Alveolar Hemorrhage Associated with Warfarin Therapy

    OpenAIRE

    Kaya, Bülent; Yildiz, Ibrahim; Baha, Reshat Mehmet; Zeytun, Neslihan Ebru Eryaşar; Yetisgen, Azize

    2015-01-01

    Diffuse alveolar hemorrhage (DAH) is a life-threatening clinical pathologic syndrome caused by a variety of diseases. We report a case of DAH related to therapy of warfarin use. In this case report, we present the diffuse alveolar hemorrhage case as a rare and life-threatening complication of warfarin.

  16. Diffuse Alveolar Hemorrhage Associated with Warfarin Therapy.

    Science.gov (United States)

    Kaya, Bülent; Yildiz, Ibrahim; Baha, Reshat Mehmet; Zeytun, Neslihan Ebru Eryaşar; Yetisgen, Azize

    2015-01-01

    Diffuse alveolar hemorrhage (DAH) is a life-threatening clinical pathologic syndrome caused by a variety of diseases. We report a case of DAH related to therapy of warfarin use. In this case report, we present the diffuse alveolar hemorrhage case as a rare and life-threatening complication of warfarin.

  17. Diffuse Alveolar Hemorrhage Associated with Warfarin Therapy

    Directory of Open Access Journals (Sweden)

    Bülent Kaya

    2015-01-01

    Full Text Available Diffuse alveolar hemorrhage (DAH is a life-threatening clinical pathologic syndrome caused by a variety of diseases. We report a case of DAH related to therapy of warfarin use. In this case report, we present the diffuse alveolar hemorrhage case as a rare and life-threatening complication of warfarin.

  18. CT quantification of pulmonary alveolar microlithiasis

    International Nuclear Information System (INIS)

    Wurche, K.D.; Kubale, R.; Vallee, D.; Ostertag, H.

    1987-01-01

    Pulmonary alveolar microlithiasis is a rare, familial disease with massive symmetrical intra-alveolar calcium deposition. Conventional CT findings and CT measurements with a dual energy technique were carried out in a 26-year-old patient suffering from this disease. The importance of the findings in the differential diagnosis and for estimating the progression and prognosis of the disease is discussed. (orig.) [de

  19. Antioxidant macromolecules in the epithelial lining fluid of the normal human lower respiratory tract.

    OpenAIRE

    Cantin, A M; Fells, G A; Hubbard, R C; Crystal, R G

    1990-01-01

    We hypothesized that the alveolar structures may contain extracellular macromolecules with antioxidant properties to defend against oxidants. To evaluate this 51Cr-labeled human lung fibroblasts (HFL-1) and cat lung epithelial cells (AKD) were exposed to a H2O2-generating system and alveolar epithelial lining fluid (ELF) from healthy nonsmokers was tested for its ability to protect the lung cells from H2O2-mediated injury. The ELF provided marked antioxidant protection, with most from a H2O-s...

  20. Citotoxicidad de extractos de plantas medicinales sobre la línea celular de carcinoma de pulmón humano A549 Cytotoxicity of medicinal plant extracts on the human lung carcinoma cell line A549

    Directory of Open Access Journals (Sweden)

    Alexis Díaz García

    2011-03-01

    Full Text Available OBJETIVO: evaluar el efecto de 10 extractos de plantas medicinales sobre el crecimiento de la línea celular humana de carcinoma de pulmón A549. METODOS: el efecto de los extractos sobre la células tumorales se midió a través de un ensayo colorimétrico mediante el empleo del bromuro de 3-(4,5-dimetil-tiazol-2-yl-2,5-difenil tetrazolio a concentraciones entre 3,9-250 µg/mL durante 72 h y se calculó la concentración citotóxica media para cada uno. RESULTADOS: del total de los extractos evaluados solo cuatro (Parthenium hysterophorus, Bixa orellana, Momordica charantia y Cucurbita maxima evidenciaron concentraciones citotóxicas medias inferiores a 100 µg/mL. Excepto Parthenium hysterophorus, las restantes se emplean en la medicina tradicional para el tratamiento del cáncer. Los extractos de Cecrop