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Sample records for alveolar epithelial a549

  1. 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.

  2. 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.

  3. 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.

  4. Oncostatin M, but not interleukin-6 or leukemia inhibitory factor, stimulates expression of alpha1-proteinase inhibitor in A549 human alveolar epithelial cells.

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    Sallenave, J M; Tremblay, G M; Gauldie, J; Richards, C D

    1997-06-01

    Alpha-1 proteinase inhibitor (A1-Pi) is the main serine proteinase inhibitor found in human plasma and is a potent elastase inhibitor in various tissues, including lung. A1-Pi is expressed and induced in liver during inflammatory responses but can also be produced by epithelial cells. Since hepatocyte A1-Pi production is stimulated by interleukin-6 (IL-6) and other gp130-cytokines, such as leukemia inhibitory factor (LIF) and oncostatin M (OM), we investigated the role of these cytokines in regulating A1-Pi in lung epithelial cells. We show that OM, a monocyte and T cell product, can specifically and potently induce A1-Pi production in lung-derived A549 alveolar (epithelial) cells, as well as in liver-derived HepG2 cells. Both A1-Pi protein (as detected by ELISA and Western blots) and mRNA levels were enhanced 20-fold to 30-fold in A549 cells. OM was also able to stimulate the expression of tissue inhibitor of metalloproteinase-1 in these cells. Interestingly, other members of the IL-6 family (IL-6 and LIF) had little or no effect on A549 cells, and proinflammatory cytokines, such as IL-1 beta and tumor necrosis factor-alpha (TNF-alpha) also had no stimulatory effect on A1-Pi synthesis in A549 cells. Costimulation with IL-1 beta resulted in a decrease in A1-Pi production from OM-stimulated A549 cells. However, IL-6 production was synergistically enhanced. OM was also able to stimulate A1-Pi production from a bronchial epithelial primary cell line, whereas an intestinal epithelial cell line HT29 responded to IL-6 but not OM. These results suggest that lung levels A1-Pi could be derived not only from liver and inflammatory cells but also from epithelial cells, which can be upregulated on stimulation by OM. This may have implications for regulation of local activity of human neutrophil elastase (HNE) in such diseases as emphysema and cystic fibrosis. PMID:9198001

  5. Legionella pneumophila infection induces programmed cell death, caspase activation, and release of high-mobility group box 1 protein in A549 alveolar epithelial cells: inhibition by methyl prednisolone

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    Koide Michio

    2008-05-01

    Full Text Available Abstract Background Legionella pneumophila pneumonia often exacerbates acute lung injury (ALI and acute respiratory distress syndrome (ARDS. Apoptosis of alveolar epithelial cells is considered to play an important role in the pathogenesis of ALI and ARDS. In this study, we investigated the precise mechanism by which A549 alveolar epithelial cells induced by L. pneumophila undergo apoptosis. We also studied the effect of methyl prednisolone on apoptosis in these cells. Methods Nuclear deoxyribonucleic acid (DNA fragmentation and caspase activation in L. pneumophila-infected A549 alveolar epithelial cells were assessed using the terminal deoxyribonucleotidyl transferase-mediated triphosphate (dUTP-biotin nick end labeling method (TUNEL method and colorimetric caspase activity assays. The virulent L. pneumophila strain AA100jm and the avirulent dotO mutant were used and compared in this study. In addition, we investigated whether methyl prednisolone has any influence on nuclear DNA fragmentation and caspase activation in A549 alveolar epithelial cells infected with L. pneumophila. Results The virulent strain of L. pneumophila grew within A549 alveolar epithelial cells and induced subsequent cell death in a dose-dependent manner. The avirulent strain dotO mutant showed no such effect. The virulent strains of L. pneumophila induced DNA fragmentation (shown by TUNEL staining and activation of caspases 3, 8, 9, and 1 in A549 cells, while the avirulent strain did not. High-mobility group box 1 (HMGB1 protein was released from A549 cells infected with virulent Legionella. Methyl prednisolone (53.4 μM did not influence the intracellular growth of L. pneumophila within alveolar epithelial cells, but affected DNA fragmentation and caspase activation of infected A549 cells. Conclusion Infection of A549 alveolar epithelial cells with L. pneumophila caused programmed cell death, activation of various caspases, and release of HMGB1. The dot/icm system, a

  6. Reactive oxygen species mediated DNA damage in human lung alveolar epithelial (A549) cells from exposure to non-cytotoxic MFI-type zeolite nanoparticles.

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    Bhattacharya, Kunal; Naha, Pratap C; Naydenova, Izabela; Mintova, Svetlana; Byrne, Hugh J

    2012-12-17

    Increasing utilization of engineered nanoparticles in the field of electronics and biomedical applications demands an assessment of risk associated with deliberate or accidental exposure. Metal based nanoparticles are potentially most important of all the nanoparticles in terms of health risks. Microporous alumino-silicates and pure silicates named as zeolites and zeo-type materials with variety of structures, chemical compositions, particle sizes and morphologies have a significant number of industrial uses such as in catalysis, sorption and ion-exchange processes. In particular, the nanosized particles due to their unique properties are used in hybrid organic-inorganic materials for photography, photonics, electronics, labeling, imaging, and sensing. The aim of the current study is to investigate pure silica MFI-type zeolites nanoparticles with sizes of 50nm and 100nm (samples MFI-50 and MFI-100) under suspended conditions and their toxicological effects on human lung alveolar (A549) cells under in vitro conditions. Live cell imaging showed that the nanoparticles precipitated from the colloidal suspension of cell culture media as large agglomerates, coming in contact with the cell surface through sedimentation. A cellular proliferative capacity test showed the zeolite nanoparticles to exhibit no significant cytotoxicity below a concentration of 100μg/ml. However, both the MFI-50 and MFI-100 nanoparticles induced high intracellular reactive oxygen species (ROS) generation and elevated mitochondrial membrane potential in the A549 cells over the measured time period of 12h and at concentrations up to ≤50μg/ml. DNA fragmentation analysis using the comet assay showed that the MFI-50 and MFI-100 nanoparticles cause genotoxicity in a concentration dependent manner. Furthermore, the rate at which maximum genomic damage was caused by MFI-100 nanoparticles in the A549 cells was found to be high as compared to the MFI-50 nanoparticles. However, the damage caused by the

  7. Dexmedetomidine Attenuates Bilirubin-Induced Lung Alveolar Epithelial Cell Death In Vitro and In Vivo*

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    Cui, Jian; Zhao, Hailin; Yi, Bin; Zeng, Jing; Lu, Kaizhi; Ma, Daqing

    2015-01-01

    Objective: To investigate bilirubin-induced lung alveolar epithelial cell injury together with the protection afforded by dexmedetomidine. Design: Prospective, randomized, controlled study. Setting: Research laboratory. Subjects: Sprague Dawley rats. Interventions: Alveolar epithelial A549 cell lines were cultured and received bilirubin (from 0 to 160 μM) to explore the protective pathway of dexmedetomidine on bilirubin-induced alveolar epithelial cell injury assessed by immunochemistry and f...

  8. 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.

  9. 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.

  10. 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

  11. Caffeic acid phenethyl ester protects lung alveolar epithelial cellsfrom cigarette smoke-induced damage

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    BARLAS, FIRAT BARIŞ; ERDOGAN, SUAT

    2015-01-01

    Background/aim: To evaluate the influence of caffeic acid phenethyl ester (CAPE) on cigarette smoke (CS)-induced cell damage, oxidative stress, and inflammation in human alveolar epithelial cells. Materials and methods: A549 alveolar epithelial cells were divided into control, CS exposure, CAPE, and CS+CAPE treatment groups. Undiluted CS-exposed medium (100%) and three dilutions (50%, 25%, and 10%) of CS-exposed media were applied to cultured A549 cells, which were analyzed after 3 h of inc...

  12. Interactions of 3 nm, 8 nm, and 15 nm gold particles with human alveolar epithelial cells : a microscopy study

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    Böse, Katharina

    2013-01-01

    The inhalation of nanoparticles can cause interactions with pulmonary structures. Human alveolar epithelial cells type II organize the alveolar epithelium and thus can be regarded as barrier against pulmonary nanoparticle uptake. Within the present work, interactions of differently sized gold nanoparticles with A549 cells, a model for type II human alveolar epithelial cells, were studied. The intracellular location of the fluorescently labeled gold particles was analyzed by STED (stimulated e...

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

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    Haller Thomas

    2011-02-01

    Full Text Available Abstract 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 not been studied directly on alveolar type II cells (ATII cells, a key cell type responsible for alveolar function and repair. Objective The aim of this study was to investigate the effects of two commercially available surfactant preparations on ATII cell viability and DNA synthesis. Methods Curosurf® and Alveofact® were applied to two ATII cell lines (human A549 and mouse iMATII cells and to primary rat ATII cells for periods of up to 24 h. Cell viability was measured using the redox indicator resazurin and DNA synthesis was measured using BrdU incorporation. Results Curosurf® resulted in slightly decreased cell viability in all cell culture models. However, DNA synthesis was increased in A549 and rat ATII cells but decreased in iMATII cells. Alveofact® exhibited the opposite effects on A549 cells and had very mild effects on the other two cell models. Conclusion This study showed that commercially available exogenous surfactants used to treat preterm infants with RDS can have profound effects on cell viability and DNA synthesis.

  14. Human lung epithelial cell A549 proteome data after treatment with titanium dioxide and carbon black.

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    Vuong, Ngoc Q; Goegan, Patrick; Mohottalage, Susantha; Breznan, Dalibor; Ariganello, Marianne; Williams, Andrew; Elisma, Fred; Karthikeyan, Subramanian; Vincent, Renaud; Kumarathasan, Premkumari

    2016-09-01

    Here, we have described the dataset relevant to the A549 cellular proteome changes after exposure to either titanium dioxide or carbon black particles as compared to the non-exposed controls, "Proteomic changes in human lung epithelial cells (A549) in response to carbon black and titanium dioxide exposures" (Vuong et al., 2016) [1]. Detailed methodologies on the separation of cellular proteins by 2D-GE and the subsequent mass spectrometry analyses using MALDI-TOF-TOF-MS are documented. Particle exposure-specific protein expression changes were measured via 2D-GE spot volume analysis. Protein identification was done by querying mass spectrometry data against SwissProt and RefSeq protein databases using Mascot search engine. Two-way ANOVA analysis data provided information on statistically significant A549 protein expression changes associated with particle exposures. PMID:27508218

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

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

    2015-01-01

    Objective:To explore the effect and molecular mechanism ofSPHK1 in the invasion and metastasis process of non-small-cell lung cancer cells(A549).Methods:Recombinant retrovirus was used to mediate the production ofA549/vector,A549/SPHK1,A549/scramble, andA549/SPHK1/RNAi that stably expressed or silencedSPHK1.The invasion and migration capacities of A549 cells overexpressing or silencingSPHK1 were determined usingTranswell invasion assay and scratch wound repair experiment.The protein and mRNA expression levels ofE-cadherin, fibronectin, vimentin inA549/vector,A549/SPHK1,A549/scramble,A549/SPHK1/RNAi were detected withWestern blot(WB) and quantitativePCR(QPCR) methods, respectively.Results:Transwell invasion assay and scratch wound repair experiments showed that over-expression of SPHK1 obviously enhanced the invasion and migration capacities ofA549 cells.WB andQPCR detection results showed that, the expression ofE-cadherin(a molecular marker of epithelial cells) and fibronectin, vimentin(molecular markers of mesenchymal cells) inA549 cells was upregulated after overexpression ofSPHK1; whileSPHK1 silencing significantly reduced the invasion and metastasis capacities ofA549cells, upregulated the expression of molecular marker of epithelial cells, and downregulated the expression of molecular marker of mesenchymal cells. Conclusions:SPHK1 promotes epithelial mesenchymal transition of non-small-cell lung cancer cells and affects the invasion and metastasis capacities of these cells.

  16. Human Vγ9Vδ2-T cells efficiently kill influenza virus-infected lung alveolar epithelial cells

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    LI Hong; Xiang, Zheng; Feng, Ting; Li, Jinrong; Liu, Yinping; Fan, Yingying; Lu, Qiao; Yin, Zhongwei; Yu, Meixing; Shen, Chongyang; Tu, Wenwei

    2013-01-01

    γδ-T cells play an indispensable role in host defense against different viruses, including influenza A virus. However, whether these cells have cytotoxic activity against influenza virus-infected lung alveolar epithelial cells and subsequently contribute to virus clearance remains unknown. Using influenza virus-infected A549 cells, human lung alveolar epithelial cells, we investigated the cytotoxic activity of aminobisphosphonate pamidronate (PAM)-expanded human Vγ9Vδ2-T cells and their under...

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

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    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.

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

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    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

  19. Direct and in vitro observation of growth hormone receptor molecules in A549 human lung epithelial cells by nanodiamond labeling

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    Cheng, C.-Y.; Perevedentseva, E.; Tu, J.-S.; Chung, P.-H.; Cheng, C.-L.; Liu, K.-K.; Chao, J.-I.; Chen, P.-H.; Chang, C.-C.

    2007-04-01

    This letter presents direct observation of growth hormone receptor in one single cancer cell using nanodiamond-growth hormone complex as a specific probe. The interaction of surface growth hormone receptor of A549 human lung epithelial cells with growth hormone was observed using nanodiamond's unique spectroscopic signal via confocal Raman mapping. The growth hormone molecules were covalent conjugated to 100nm diameter carboxylated nanodiamonds, which can be recognized specifically by the growth hormone receptors of A549 cell. The Raman spectroscopic signal of diamond provides direct and in vitro observation of growth hormone receptors in physiology condition in a single cell level.

  20. Reactive oxygen species involved in apoptosis induction of human respiratory epithelial (A549) cells by Streptococcus agalactiae.

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    da Costa, Andréia Ferreira Eduardo; Moraes, João Alfredo; de Oliveira, Jessica Silva Santos; dos Santos, Michelle Hanthequeste Bittencourt; Santos, Gabriela da Silva; Barja-Fidalgo, Christina; Mattos-Guaraldi, Ana Luiza; Nagao, Prescilla Emy

    2016-01-01

    Streptococcus agalactiae (Group B Streptococcus; GBS) is an important pathogen and is associated with pneumonia, sepsis and meningitis in neonates and adults. GBS infections induce cytotoxicity of respiratory epithelial cells (A549) with generation of reactive oxygen species (ROS) and loss of mitochondrial membrane potential (ψm). The apoptosis of A549 cells by GBS was dependent on the activation of caspase-3 and caspase-9 with increased pro-apoptotic Bim and Bax molecules and decreased Bcl-2 pro-survival protein. Treatment of infected A549 cells with ROS inhibitors (diphenyleniodonium chloride or apocynin) prevented intracellular ROS production and apoptosis. Consequently, oxidative stress is included among the cellular events leading to apoptosis during GBS human invasive infections. PMID:26490153

  1. TGF-β1 induces human alveolar epithelial to mesenchymal cell transition (EMT

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    Kamimura Takashi

    2005-06-01

    Full Text Available Abstract Background Fibroblastic foci are characteristic features in lung parenchyma of patients with idiopathic pulmonary fibrosis (IPF. They comprise aggregates of mesenchymal cells which underlie sites of unresolved epithelial injury and are associated with progression of fibrosis. However, the cellular origins of these mesenchymal phenotypes remain unclear. We examined whether the potent fibrogenic cytokine TGF-β1 could induce epithelial mesenchymal transition (EMT in the human alveolar epithelial cell line, A549, and investigated the signaling pathway of TGF-β1-mediated EMT. Methods A549 cells were examined for evidence of EMT after treatment with TGF-β1. EMT was assessed by: morphology under phase-contrast microscopy; Western analysis of cell lysates for expression of mesenchymal phenotypic markers including fibronectin EDA (Fn-EDA, and expression of epithelial phenotypic markers including E-cadherin (E-cad. Markers of fibrogenesis, including collagens and connective tissue growth factor (CTGF were also evaluated by measuring mRNA level using RT-PCR, and protein by immunofluorescence or Western blotting. Signaling pathways for EMT were characterized by Western analysis of cell lysates using monoclonal antibodies to detect phosphorylated Erk1/2 and Smad2 after TGF-β1 treatment in the presence or absence of MEK inhibitors. The role of Smad2 in TGF-β1-mediated EMT was investigated using siRNA. Results The data showed that TGF-β1, but not TNF-α or IL-1β, induced A549 cells with an alveolar epithelial type II cell phenotype to undergo EMT in a time-and concentration-dependent manner. The process of EMT was accompanied by morphological alteration and expression of the fibroblast phenotypic markers Fn-EDA and vimentin, concomitant with a downregulation of the epithelial phenotype marker E-cad. Furthermore, cells that had undergone EMT showed enhanced expression of markers of fibrogenesis including collagens type I and III and CTGF. MMP-2

  2. Regulation of epithelial sodium channel a-subunit expression by adenosine receptor A2a in alveolar epithelial cells

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    DENG Wang; WANG Dao-xin; ZHANG Wei; LI Chang-yi

    2011-01-01

    Background The amiloride-sensitive epithelial sodium channel a-subunit (a-ENaC) is an important factor for alveolar fluid clearance during acute lung injury. The relationship between adenosine receptor A2a (A2aAR) expressed in alveolar epithelial cells and aα-ENaC is poorly understood. We targeted the A2aAR in this study to investigate its role in the expression of αa-ENaC and in acute lung injury.Methods A549 cells were incubated with different concentrations of A2aAR agonist CGS-21680 and with 100 μmol/L CGS-21680 for various times. Rats were treated with lipopolysaccharide (LPS) after CGS-21680 was injected. Animals were sacrificed and tissue was harvested for evaluation of lung injury by analysis of the lung wet-to-dry weight ratio, lung permeability and myeloperoxidase activity. RT-PCR and Western blotting were used to determine the mRNA and protein expression levels of α-ENaC in A549 cells and alveolar type II epithelial cells.Results Both mRNA and protein levels of α-ENaC were markedly higher from 4 hours to 24 hours after exposure to 100μmol/L CGS-21680. There were significant changes from 0.1 umol/L to 100 μmol/L CGS-21680, with a positive correlation between increased concentrations of CGS-21680 and expression of α-ENaC. Treatment with CGS-21680during LPS induced lung injury protected the lung and promoted α-ENaC expression in the alveolar epithelial cells.Conclusion Activation of A2aAR has a protective effect during the lung injury, which may be beneficial to the prognosis of acute lung injury.

  3. 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.

  4. Cigarette Smoke Extract Inhibits the Proliferation of Alveolar Epithelial Cells and Augments the Expression of P21WAF1

    Institute of Scientific and Technical Information of China (English)

    Zongxian JIAO; Qilin AO; Xiaona GE; Mi XIONG

    2008-01-01

    Cigarette smoking is intimately related with the development of chronic obstructive pulmonary diseases, and alveolar epithelium is a major target for the exposure of cigarette smoke ex- tract. In order to investigate the effect of cigarette smoke extract on the proliferation of alveolar epithelial cell type Ⅱand its relationship with P21WAF1, the alveolar epithelial type Ⅱ cell line (A549) cells were chosen as surrogate cells to represent alveolar epithelial type Ⅱ cells. MTT assay was used to detect cell viability after interfered with different concentrations of cigarette smoke ex-tract. It was observed cigarette smoke extract inhibited the growth of A549 cells in a dose- and time-dependent manner. The morphological changes, involving the condensation and margination of nuclear chromatin, even karyorrhexis, were observed by both Hoechst staining and electronic mi-croscopy. Flow cytometry analysis demonstrated the increased cell percentages in G1 and subG1phases after the cells were incubated with cigarette smoke extract. The expression of p21WAF1 protein and mRNA was also significantly increased as detected by the methods of Western blot or reverse transcription-polymerase chain reaction respectively. In conclusion, cigarette smoke extract inhibits the proliferation of alveolar epithelial cell type Ⅱ and blocks them in G1/S phase. The intracellular accumulation of P21WAF1 may be one of the mechanisms which contribute to cigarette smoke ex-tract-induced inhibition of cell proliferation.

  5. Construction of p66Shc gene interfering lentivirus vectors and its effects on alveolar epithelial cells apoptosis induced by hyperoxia

    Science.gov (United States)

    Zhang, Chan; Dong, Wen-Bin; Zhao, Shuai; Li, Qing-Ping; Kang, Lan; Lei, Xiao-Ping; Guo, Lin; Zhai, Xue-Song

    2016-01-01

    Background The aim of this study is to observe the inhibitive effects of p66Shc gene interfering lentivirus vectors on the expression of p66Shc, and to explore its effects on alveolar epithelial cells apoptosis induced by hyperoxia. Methods The gene sequences were cloned into the pLenR-GPH-shRNA lentiviral vector, which was selected by Genebank searches. The pLenR-GPH-shRNA and lentiviral vector packaging plasmid mix were cotransfected into 293T cells to package lentiviral particles. Culture virus supernatant was harvested, and then the virus titer was determined by serial dilution assay. A549 cells were transduced with the constructed lentiviral vectors, and real-time polymerase chain reaction (RT-PCR) and Western blot were used to evaluate p66Shc expression. This study is divided into a control group, a hyperoxia group, an A549-p66ShcshRNA hyperoxia group, and a negative lentivirus group. Cell apoptosis was detected by flow cytometry after 24 hours; the expression of X-linked inhibitor of apoptosis protein (XIAP) and caspase-9 were detected by immunohistochemistry assay. The production of reactive oxygen species and cellular mitochondria membrane potential (ΔΨm) were determined by fluorescence microscopy. Results We successfully established the p66Shc gene interfering lentivirus vectors, A549-p66ShcshRNA. The A549-p66ShcshRNA was transfected into alveolar epithelial cells, and the inhibitive effects on the expression of p66Shc were observed. Both RT-PCR and Western blot demonstrated downregulation of p66Shc expression in A549 cells. In the A549-p66ShcshRNA hyperoxia group, we found dampened oxidative stress. A549-p66ShcshRNA can cause p66Shc gene silencing, reduce mitochondrial reactive oxygen species generation, reduce membrane potential decrease, reduce the apoptosis of A549 cells, and reduce alveolar epithelial cell injury, while the lentiviral empty vector group had no such changes. Conclusion p66Shc gene interfering lentivirus vector can affect the

  6. Inflammatory response and genotoxicity of seven wood dusts in the human epithelial cell line A549.

    Science.gov (United States)

    Bornholdt, Jette; Saber, Anne T; Sharma, Anoop K; Savolainen, Kai; Vogel, Ulla; Wallin, Håkan

    2007-08-15

    Exposure to wood dust is common in many workplaces. Epidemiological studies indicate that occupational exposure to hardwood dusts is more harmful than to softwood dusts. In this study, human epithelial cell line A549 was incubated with well-characterized dusts from six commonly used wood species and from medium density fibreboard (MDF), at concentrations between 10 and 300microg/ml. After 3 and 6h of incubation, genotoxicity was assessed by measurement of DNA damage with the single-cell gel electrophoresis (comet) assay and inflammation was measured by the expression of IL-6 and IL-8 mRNA and by the amount of IL-8 protein. There was a 1.2-1.4-fold increase in DNA strand breaks after incubation with beech, teak, pine and MDF dusts compared with the levels in untreated cells, but after 6h only the increase induced by the MDF dust remained. Increased expression of cellular IL-6 and IL-8 mRNA was induced by all of the wood dusts at both times. Similar to IL-8 mRNA expression, the amounts of secreted IL-8 protein were elevated, except after incubation with oak dust, where a marginal reduction was seen. On the basis of the effects on IL-8 mRNA expression, the wood dusts could be divided into three groups, with teak dust being the most potent, MDF, birch, spruce and pine being intermediate, and beech and oak being the least potent. The induction of DNA strand breaks did not correlate well with the interleukin response. In conclusion, all wood dusts induced cytokine responses, and some dusts induced detectable DNA damage. The inflammatory potency seemed intermediate for dusts from the typical softwoods spruce and pine, whereas the dusts from species linked to cancer, beech and oak, were the least inflammatory. The variation of the effects induced by different wood dusts over time indicates that the DNA damage was not secondary to the cytokine response. Although hardwoods are often considered more harmful than softwoods by regulatory agencies, the current experiments do not

  7. 应用A549细胞单层模型研究蛋白多肽类药物肺部吸收的特性%Transport of proteins and peptides across human cultured alveolar A549 cell monolayers

    Institute of Scientific and Technical Information of China (English)

    王智瑛; 张悦; 张强

    2004-01-01

    Aim An in vitro cultured monolayer system of alveolar epithelial cells was used as a model to investigate the transport pathway peptides or proteins, salmon calcitonin (sCT), insulin (INS), recombinant hirudin (rHAV2), and recombinant human growth hormone (rhGH), in pulmonary epithelium in vivo. Methods Human lung adenocareinoma A549 cells formed continuous monolayers with growing polycarbonate filters of Transwell plate. Transport studies of macromolecules in the monolayer system were carried out after 6 days in culture. The transport of peptides or proteins with MW 3 400 - 22 000 was studied in cultured human lung adenocareinoma A549 cell monolayers at different conditions. Results The results showed that the apparent permeability coefficients (Papp) of these macromolecules across A549 cell monolayers ranged from 2×10-6 to 5×10-6 cm·s-1 and exhibited good inverse correlation with molecule weight. No concentration, direction and temperature dependence were observed in the permeation of sCT, INS and rHAV2. While the Papp of rhGH in the BA direction (2.25×10-6 cm·s-1) was significantly less than that in the reverse direction. ThePapp values of rhGH were concentration and temperature independent in the AB direction. Conclusion These findings suggest that the hydrophilic peptides and proteins, salmon calcitonin, insulin, recombinant hirudin, and recombinant human growth hormone used in this study, appeared to penetrate the A549 cell monolayers via a paracellular pathway by passive diffusion mechanism.

  8. 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.

  9. Effect of fucoidan from Turbinaria conoides on human lung adenocarcinoma epithelial (A549) cells.

    Science.gov (United States)

    Alwarsamy, Madhavarani; Gooneratne, Ravi; Ravichandran, Ramanibai

    2016-11-01

    Fucoidan was purified from seaweed, Turbinaria conoides. Isolated fragments were characterized with NMR ((13)C, (1)H), Gas Chromatography-Mass Spectronomy (GC-MS) and HPLC analysis. The autohydrolysate of fucoidans consisted of sulfated fuco-oligosaccharides having the backbone of α-(1, 3)-linked fuco-pyranose derivatives and minor components of galactose, glucose, mannose and xylose sugars. Fucoidan induced a dose-dependent reduction in cell survival of lung cancer A549 cells by MTT assay (GI50, 75μg/mL). However, it was not cytotoxic to a non-tumorigenic human keratinocyte cell line of skin tissue (HaCaT) (GI50>1.0mg/mL). The apoptotic cells in fucoidan-treated A549 cells were visualized by laser confocal microscopy and cell cycle analysis showed induction of G0/G1 phase arrest of the cell progression cycle. Further, CFSE labeling and flow cytometry highlighted that fucoidan significantly (P<0.05) inhibited the proliferation rate of A549 cells by up to 2-fold compared with the control cells. It is concluded that fucoidan has the potential to act as an anti-proliferative agent on lung carcinoma (A549) cells. PMID:27516266

  10. Claudins and alveolar epithelial barrier function in the lung

    OpenAIRE

    Frank, James A.

    2012-01-01

    The alveolar epithelium of the lung constitutes a unique interface with the outside environment. This thin barrier must maintain a surface for gas transfer while being continuously exposed to potentially hazardous environmental stimuli. Small differences in alveolar epithelial barrier properties could therefore have a large impact on disease susceptibility or outcome. Moreover, recent work has focused attention on the alveolar epithelium as central to several lung diseases, including acute lu...

  11. Alveolar epithelial type II cell: defender of the alveolus revisited

    OpenAIRE

    Fehrenbach Heinz

    2001-01-01

    Abstract In 1977, Mason and Williams developed the concept of the alveolar epithelial type II (AE2) cell as a defender of the alveolus. It is well known that AE2 cells synthesise, secrete, and recycle all components of the surfactant that regulates alveolar surface tension in mammalian lungs. AE2 cells influence extracellular surfactant transformation by regulating, for example, pH and [Ca2+] of the hypophase. AE2 cells play various roles in alveolar fluid balance, coagulation/fibrinolysis, a...

  12. 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.

  13. Thrombin stimulation of synthesis and secretion of fibronectin by human A549 epithelial cells and mouse LB fibroblasts

    International Nuclear Information System (INIS)

    Thrombin, a serine protease generated at wound sites, takes part in multiple biological functions, including wound healing. The present report elucidates the effect of thrombin on fibronectin (FN) synthesis and secretion in fibroblasts and epithelial cells. Subconfluent cultures of mouse LB fibroblasts and human A549 epithelial cells were exposed to various concentrations of bovine plasma thrombin at 37 degrees C for 16 hr. After exposure, cells were processed for determination of cell-associated and secreted FN by metabolic labeling, immunoprecipitation, immunofluorescence, and peroxidase immunocytochemistry. The correlation of FN production with cell growth was studied by a combined procedure of peroxidase immunocytochemistry and light microscopic autoradiography. The amounts of cell-associated and secreted FN were significantly increased with dose increments of thrombin. The increases were most evident in secreted FN. The increase of cell-associated FN was also evidenced by results from immunofluorescence and immunocytochemical studies. Ultrastructurally, the intracellular FN was localized in rough endoplasmic reticulum, Golgi complexes, and secretory granules, whereas non-released extracellular FN was localized in the plasma membrane of cell-to-cell contacts and in the extracellular fibrils. More intense cytoplasmic FN staining was observed in cells that were not labeled with [3H]-thymidine, indicating that FN production may vary with different phases of cell growth. The results imply that thrombin may play an important role in the early phases of wound healing

  14. Autophagy protects type II alveolar epithelial cells from Mycobacterium tuberculosis infection

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Xu-Guang [Center for Clinical Laboratory Medicine of PLA, Xijing Hospital, Fourth Military Medical University, Xi' an (China); Department of Laboratory Medicine, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou (China); Ji, Tian-Xing [Department of Laboratory Medicine, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou (China); Xia, Yong, E-mail: gysyxy@gmail.com [Center for Clinical Laboratory Medicine of PLA, Xijing Hospital, Fourth Military Medical University, Xi' an (China); Ma, Yue-Yun, E-mail: cmbmayy@fmmu.edu.cn [Center for Clinical Laboratory Medicine of PLA, Xijing Hospital, Fourth Military Medical University, Xi' an (China)

    2013-03-08

    Highlights: ► We investigated the protective effect of autophagy pathway against MTB infection. ► MTB-infected A549 cells had higher LDH release. ► Inhibition of autophagy signaling significantly enhanced the MTB-induced necrosis. ► Autophagy prevents apoptosis and promotes cell survival in infected cells. -- Abstract: This study was designed to investigate the protective effect of the autophagy signaling pathway against Mycobacterium tuberculosis infection in type II alveolar epithelial cells. An in vitro M. tuberculosis system was established using human A549 cells. Infection-induced changes in the expression of the autophagic marker LC3 were assessed by reverse transcription-PCR and Western blotting. Morphological changes in autophagosomes were detected by transmission electron microscopy (TEM). The function of the autophagy signaling pathway during infection was assessed by measuring the level of cell death and the amount of lactate dehydrogenase (LDH) released in the presence or absence of the inhibitor 3-methyladenine (3-MA). In addition, effects on LDH release were assessed after the siRNA-mediated knockdown of the essential autophagosomal structural membrane protein Atg5. LC3 mRNA expression was significantly reduced in M.tuberculosis-infected A549 cells (16888.76 ± 1576.34 vs. uninfected: 12744.29 ± 1089.37; P < 0.05). TEM revealed M.tuberculosis bacilli-containing compartments that were surrounded by double membranes characteristic of the autophagic process. M.tuberculosis-infected A549 cells released more LDH (1.45 ± 0.12 vs. uninfected: 0.45 ± 0.04; P < 0.05). The inhibition of autophagy signaling significantly enhanced M.tuberculosis-induced necrosis (3-MA: 75 ± 5% vs. untreated: 15 ± 1%; P < 0.05) and LDH release (3-MA: 2.50 ± 0.24 vs. untreated: 0.45 ± 0.04; Atg5 knockdown: 3.19 ± 0.29 vs. untreated: 1.28 ± 0.11; P < 0.05). Our results indicate that autophagy signaling pathway prevents apoptosis in type II alveolar epithelial cells

  15. Autophagy protects type II alveolar epithelial cells from Mycobacterium tuberculosis infection

    International Nuclear Information System (INIS)

    Highlights: ► We investigated the protective effect of autophagy pathway against MTB infection. ► MTB-infected A549 cells had higher LDH release. ► Inhibition of autophagy signaling significantly enhanced the MTB-induced necrosis. ► Autophagy prevents apoptosis and promotes cell survival in infected cells. -- Abstract: This study was designed to investigate the protective effect of the autophagy signaling pathway against Mycobacterium tuberculosis infection in type II alveolar epithelial cells. An in vitro M. tuberculosis system was established using human A549 cells. Infection-induced changes in the expression of the autophagic marker LC3 were assessed by reverse transcription-PCR and Western blotting. Morphological changes in autophagosomes were detected by transmission electron microscopy (TEM). The function of the autophagy signaling pathway during infection was assessed by measuring the level of cell death and the amount of lactate dehydrogenase (LDH) released in the presence or absence of the inhibitor 3-methyladenine (3-MA). In addition, effects on LDH release were assessed after the siRNA-mediated knockdown of the essential autophagosomal structural membrane protein Atg5. LC3 mRNA expression was significantly reduced in M.tuberculosis-infected A549 cells (16888.76 ± 1576.34 vs. uninfected: 12744.29 ± 1089.37; P < 0.05). TEM revealed M.tuberculosis bacilli-containing compartments that were surrounded by double membranes characteristic of the autophagic process. M.tuberculosis-infected A549 cells released more LDH (1.45 ± 0.12 vs. uninfected: 0.45 ± 0.04; P < 0.05). The inhibition of autophagy signaling significantly enhanced M.tuberculosis-induced necrosis (3-MA: 75 ± 5% vs. untreated: 15 ± 1%; P < 0.05) and LDH release (3-MA: 2.50 ± 0.24 vs. untreated: 0.45 ± 0.04; Atg5 knockdown: 3.19 ± 0.29 vs. untreated: 1.28 ± 0.11; P < 0.05). Our results indicate that autophagy signaling pathway prevents apoptosis in type II alveolar epithelial cells

  16. Cytotoxicity of carbon nanotube variants: a comparative in vitro exposure study with A549 epithelial and J774 macrophage cells.

    Science.gov (United States)

    Kumarathasan, Prem; Breznan, Dalibor; Das, Dharani; Salam, Mohamed A; Siddiqui, Yunus; MacKinnon-Roy, Christine; Guan, Jingwen; de Silva, Nimal; Simard, Benoit; Vincent, Renaud

    2015-03-01

    While production of engineered carbon nanotubes (CNTs) has escalated in recent years, knowledge of risk associated with exposure to these materials remains unclear. We report on the cytotoxicity of four CNT variants in human lung epithelial cells (A549) and murine macrophages (J774). Morphology, metal content, aggregation/agglomeration state, pore volume, surface area and modifications were determined for the pristine and oxidized single-walled (SW) and multi-walled (MW) CNTs. Cytotoxicity was evaluated by cellular ATP content, BrdU incorporation, lactate dehydrogenase (LDH) release, and CellTiter-Blue (CTB) reduction assays. All CNTs were more cytotoxic than respirable TiO2 and SiO2 reference particles. Oxidation of CNTs removed most metallic impurities but introduced surface polar functionalities. Although slopes of fold changes for cytotoxicity endpoints were steeper with J774 compared to A549 cells, CNT cytotoxicity ranking in both cell types was assay-dependent. Based on CTB reduction and BrdU incorporation, the cytotoxicity of the polar oxidized CNTs was higher compared to the pristine CNTs. In contrast, pristine CNTs were more cytotoxic than oxidized CNTs when assessed for cellular ATP and LDH. Correlation analyses between CNTs' physico-chemical properties and average relative potency revealed the impact of metal content and surface area on the potency values estimated using ATP and LDH assays, while surface polarity affected the potency values estimated from CTB and BrdU assays. We show that in order to reliably estimate the risk posed by these materials, in vitro toxicity assessment of CNTs should be conducted with well characterized materials, in multiple cellular models using several cytotoxicity assays that report on distinct cellular processes. PMID:24713075

  17. FXYD1 negatively regulates Na(+)/K(+)-ATPase activity in lung alveolar epithelial cells.

    Science.gov (United States)

    Wujak, Łukasz A; Blume, Anna; Baloğlu, Emel; Wygrecka, Małgorzata; Wygowski, Jegor; Herold, Susanne; Mayer, Konstantin; Vadász, István; Besuch, Petra; Mairbäurl, Heimo; Seeger, Werner; Morty, Rory E

    2016-01-01

    Acute respiratory distress syndrome (ARDS) is clinical syndrome characterized by decreased lung fluid reabsorption, causing alveolar edema. Defective alveolar ion transport undertaken in part by the Na(+)/K(+)-ATPase underlies this compromised fluid balance, although the molecular mechanisms at play are not understood. We describe here increased expression of FXYD1, FXYD3 and FXYD5, three regulatory subunits of the Na(+)/K(+)-ATPase, in the lungs of ARDS patients. Transforming growth factor (TGF)-β, a pathogenic mediator of ARDS, drove increased FXYD1 expression in A549 human lung alveolar epithelial cells, suggesting that pathogenic TGF-β signaling altered Na(+)/K(+)-ATPase activity in affected lungs. Lentivirus-mediated delivery of FXYD1 and FXYD3 allowed for overexpression of both regulatory subunits in polarized H441 cell monolayers on an air/liquid interface. FXYD1 but not FXYD3 overexpression inhibited amphotericin B-sensitive equivalent short-circuit current in Ussing chamber studies. Thus, we speculate that FXYD1 overexpression in ARDS patient lungs may limit Na(+)/K(+)-ATPase activity, and contribute to edema persistence. PMID:26410457

  18. Effects of PPARγ ligands on TGF-β1-induced epithelial-mesenchymal transition in alveolar epithelial cells

    Directory of Open Access Journals (Sweden)

    Dagher Hayat

    2010-02-01

    Full Text Available Abstract Background Transforming growth factor β1 (TGF-β1-mediated epithelial mesenchymal transition (EMT of alveolar epithelial cells (AEC may contribute to lung fibrosis. Since PPARγ ligands have been shown to inhibit fibroblast activation by TGF-β1, we assessed the ability of the thiazolidinediones rosiglitazone (RGZ and ciglitazone (CGZ to regulate TGF-β1-mediated EMT of A549 cells, assessing changes in cell morphology, and expression of cell adhesion molecules E-cadherin (epithelial cell marker and N-cadherin (mesenchymal cell marker, and collagen 1α1 (COL1A1, CTGF and MMP-2 mRNA. Methods Serum-deprived A549 cells (human AEC cell line were pre-incubated with RGZ and CGZ (1 - 30 μM in the absence or presence of the PPARγ antagonist GW9662 (10 μM before TGFβ-1 (0.075-7.5 ng/ml treatment for up to 72 hrs. Changes in E-cadherin, N-cadherin and phosphorylated Smad2 and Smad3 levels were analysed by Western blot, and changes in mRNA levels including COL1A1 assessed by RT-PCR. Results TGFβ-1 (2.5 ng/ml-induced reductions in E-cadherin expression were associated with a loss of epithelial morphology and cell-cell contact. Concomitant increases in N-cadherin, MMP-2, CTGF and COL1A1 were evident in predominantly elongated fibroblast-like cells. Neither RGZ nor CGZ prevented TGFβ1-induced changes in cell morphology, and PPARγ-dependent inhibitory effects of both ligands on changes in E-cadherin were only evident at submaximal TGF-β1 (0.25 ng/ml. However, both RGZ and CGZ inhibited the marked elevation of N-cadherin and COL1A1 induced by TGF-β1 (2.5 ng/ml, with effects on COL1A1 prevented by GW9662. Phosphorylation of Smad2 and Smad3 by TGF-β1 was not inhibited by RGZ or CGZ. Conclusions RGZ and CGZ inhibited profibrotic changes in TGF-β1-stimulated A549 cells independently of inhibition of Smad phosphorylation. Their inhibitory effects on changes in collagen I and E-cadherin, but not N-cadherin or CTGF, appeared to be PPAR

  19. Lysosome-associated membrane glycoprotein 3 is involved in influenza A virus replication in human lung epithelial (A549 cells

    Directory of Open Access Journals (Sweden)

    Wang Jianwei

    2011-08-01

    Full Text Available Abstract Background Influenza A virus mutates rapidly, rendering antiviral therapies and vaccines directed against virus-encoded targets ineffective. Knowledge of the host factors and molecular pathways exploited by influenza virus will provide further targets for novel antiviral strategies. However, the critical host factors involved in influenza virus infection have not been fully defined. Results We demonstrated that LAMP3, a member of lysosome-associated membrane glycoprotein (LAMP family, was significantly induced in human lung epithelial (A549 cells upon influenza A virus infection. Knockdown of LAMP3 expression by RNA interference attenuated production of viral nucleoprotein (NP as well as virus titers. Confocal microscopy results demonstrated that viral NP is colocalized within LAMP3 positive vesicles at early stages of virus infection. Furthermore, knockdown of LAMP3 expression led to a reduction in nuclear accumulation of viral NP and impeded virus replication. Conclusions LAMP3 is an influenza A virus inducible gene, and plays an important role in viral post-entry steps. Our observations may provide insights into the mechanism of influenza virus replication and potential targets for novel anti-influenza therapeutics.

  20. Oxidative Stress Facilitates IFN-γ-Induced Mimic Extracellular Trap Cell Death in A549 Lung Epithelial Cancer Cells.

    Science.gov (United States)

    Lin, Chiou-Feng; Chen, Chia-Ling; Chien, Shun-Yi; Tseng, Po-Chun; Wang, Yu-Chih; Tsai, Tsung-Ting

    2016-01-01

    We previously demonstrated that IFN-γ induces an autophagy-regulated mimic extracellular trap cell death (ETosis) in A549 human lung cancer cells. Regarding reactive oxygen species (ROS) are involved in ETosis, this study investigated the role of oxidative stress. After IFN-γ stimulation, a necrosis-like cell death mimic ETosis occurred accompanied by the inhibition of cell growth, aberrant nuclear staining, and nucleosome release. ROS were generated in a time-dependent manner with an increase in NADPH oxidase component protein expression. STAT1-mediated IFN regulatory factor-1 activation was essential for upregulating ROS production. By genetically silencing p47phox, IFN-γ-induced ROS and mimic ETosis were significantly attenuated. This mechanistic study indicated that ROS may mediate DNA damage followed by histone H3 citrullination. Furthermore, ROS promoted IFN-γ-induced mimic ETosis in cooperation with autophagy. These findings further demonstrate that ROS regulates IFN-γ-induced mimic ETosis in lung epithelial malignancy. PMID:27575372

  1. Alveolar epithelial permeability in bronchial asthma in children

    International Nuclear Information System (INIS)

    To evaluate alveolar epithelial permeability (kep) in children with bronchial asthma, 99mTc-DTPA (diethylene triamine penta acetate) aerosol lung inhalation scintigraphies were performed. There was no correlation between the kep value and the severity of asthma. On the other hand, out of 10 cases which had no aerosol deposition defect in the lung field, 4 showed high kep values on the whole lung field and 7 had high kep value areas, particularly apparent in the upper lung field. These results suggest that even when the central airway lesions are mild, severe damage exists in the alveolar region of the peripheral airway. (author)

  2. Human Vγ9Vδ2-T cells efficiently kill influenza virus-infected lung alveolar epithelial cells

    Science.gov (United States)

    Li, Hong; Xiang, Zheng; Feng, Ting; Li, Jinrong; Liu, Yinping; Fan, Yingying; Lu, Qiao; Yin, Zhongwei; Yu, Meixing; Shen, Chongyang; Tu, Wenwei

    2013-01-01

    γδ-T cells play an indispensable role in host defense against different viruses, including influenza A virus. However, whether these cells have cytotoxic activity against influenza virus-infected lung alveolar epithelial cells and subsequently contribute to virus clearance remains unknown. Using influenza virus-infected A549 cells, human lung alveolar epithelial cells, we investigated the cytotoxic activity of aminobisphosphonate pamidronate (PAM)-expanded human Vγ9Vδ2-T cells and their underlying mechanisms. We found that PAM could selectively activate and expand human Vγ9Vδ2-T cells. PAM-expanded human Vγ9Vδ2-T cells efficiently killed influenza virus-infected lung alveolar epithelial cells and inhibited virus replication. The cytotoxic activity of PAM-expanded Vγ9Vδ2-T cells was dependent on cell-to-cell contact and required NKG2D activation. Perforin–granzyme B, tumor-necrosis factor-related apoptosis-inducing ligand (TRAIL) and Fas–Fas ligand (FasL) pathways were involved in their cytotoxicity. Our study suggests that targeting γδ-T cells by PAM can potentially offer an alternative option for the treatment of influenza virus. PMID:23353835

  3. Cell mechanics of alveolar epithelial cells (AECs) and macrophages (AMs).

    OpenAIRE

    Féréol, Sophie; Fodil, Redouane; Pelle, Gabriel; Louis, Bruno; Isabey, Daniel

    2008-01-01

    Cell mechanics provides an integrated view of many biological phenomena which are intimately related to cell structure and function. Because breathing constitutes a sustained motion synonymous with life, pulmonary cells are normally designed to support permanent cyclic stretch without breaking, while receiving mechanical cues from their environment. The authors study the mechanical responses of alveolar cells, namely epithelial cells and macrophages, exposed to well-controlled mechanical stre...

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

    OpenAIRE

    Aoshiba K; Nagai A

    2003-01-01

    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 inapprop...

  5. 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.

  6. The South Pacific epidemic strain of Zika virus replicates efficiently in human epithelial A549 cells leading to IFN-β production and apoptosis induction.

    Science.gov (United States)

    Frumence, Etienne; Roche, Marjolaine; Krejbich-Trotot, Pascale; El-Kalamouni, Chaker; Nativel, Brice; Rondeau, Philippe; Missé, Dorothée; Gadea, Gilles; Viranaicken, Wildriss; Desprès, Philippe

    2016-06-01

    Zika virus (ZIKV) is an emerging flavivirus since the first epidemics in South Pacific in 2007. The recent finding that ZIKV is now circulating in Western Hemisphere and can be associated to severe human diseases, warrants the need for its study. Here we evaluate the susceptibility of human lung epithelial A549 cells to South Pacific epidemic strain of ZIKV isolated in 2013. We showed that ZIKV growth in A549 cells is greatly efficient. ZIKV infection resulted in the secretion of IFN-β followed by the expression of pro-inflammatory cytokines such as IL-1β, and transcriptional activity of IFIT genes. At the maximum of virus progeny production, ZIKV triggers mitochondrial apoptosis through activation of caspases-3 and -9. Whereas at early infection times, the rapid release of IFN-β which exerts an antiviral effect against ZIKV might delay apoptosis in infected cells. PMID:27060565

  7. Alveolar epithelial type II cell: defender of the alveolus revisited

    Directory of Open Access Journals (Sweden)

    Fehrenbach Heinz

    2001-01-01

    Full Text Available Abstract In 1977, Mason and Williams developed the concept of the alveolar epithelial type II (AE2 cell as a defender of the alveolus. It is well known that AE2 cells synthesise, secrete, and recycle all components of the surfactant that regulates alveolar surface tension in mammalian lungs. AE2 cells influence extracellular surfactant transformation by regulating, for example, pH and [Ca2+] of the hypophase. AE2 cells play various roles in alveolar fluid balance, coagulation/fibrinolysis, and host defence. AE2 cells proliferate, differentiate into AE1 cells, and remove apoptotic AE2 cells by phagocytosis, thus contributing to epithelial repair. AE2 cells may act as immunoregulatory cells. AE2 cells interact with resident and mobile cells, either directly by membrane contact or indirectly via cytokines/growth factors and their receptors, thus representing an integrative unit within the alveolus. Although most data support the concept, the controversy about the character of hyperplastic AE2 cells, reported to synthesise profibrotic factors, proscribes drawing a definite conclusion today.

  8. Effects of elastase and cigarette smoke on alveolar epithelial permeability

    International Nuclear Information System (INIS)

    To determine whether instilled porcine pancreatic elastase (PPE) increases alveolar epithelial permeability, the authors measured alveolar epithelium permeability X surface area (PS) for [14C]sucrose and 125I-bovine serum albumin in isolated perfused lungs from hamsters previously exposed to PPE and/or cigarette smoke. Saline (0.5 ml) with 0, 5, or 20 units PPE was instilled intratracheally in anesthetized hamsters. Those exposed to smoke for 4-6 wk received 0 or 5 units; PS was measured 3 h later. Nonsmokers received 0, 5, or 20 units; PS was measured 3 h, 24 h, or 5 days later. Control PS values were (cm3/s X 10(-4), +/- SE) 0.84 +/- 0.11 for sucrose and 0.030 +/- 0.006 for BSA. Three and 24 h following 20 units PPE, (PS)sucrose was twice the control valve. (PS)BSA was four times control at 3 h but not significantly increased at 24 h. Five days after PPE both were back to control levels. Five units PPE or smoke exposure alone caused no PS changes. Smoke exposure and 5 units PPE caused (PS)sucrose to increase markedly (1.85 +/- 0.32); (PS)BSA was not significantly increased (0.076 +/- 0.026). Thus, instilled PPE causes reversible increases in alveolar epithelial PS; cigarette smoking potentiates this effect

  9. Hemoglobin is Expressed in Alveolar Epithelial Type II Cells

    OpenAIRE

    Bhaskaran, Manoj; Chen, Haifeng; Chen, Zhongmong; Liu, Lin

    2005-01-01

    Hemoglobin is the main oxygen carrying heme protein in erythrocytes. In an effort to study the differential gene expression of alveolar epithelial type I and type II cells using DNA microarray technique, we found that the mRNAs of hemoglobin α- and β-chains were expressed in type II cells, but not in type I cells. The microarray data were confirmed by RT-PCR. The mRNA expression of both chains decreased when type II cells trans-differentiated into type I-like cells. Immunocyto/histochemistry ...

  10. Microarray identifies ADAM family members as key responders to TGF-β1 in alveolar epithelial cells

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    Walls Dermot

    2006-09-01

    Full Text Available Abstract The molecular mechanisms of Idiopathic Pulmonary Fibrosis (IPF remain elusive. Transforming Growth Factor beta 1(TGF-β1 is a key effector cytokine in the development of lung fibrosis. We used microarray and computational biology strategies to identify genes whose expression is significantly altered in alveolar epithelial cells (A549 in response to TGF-β1, IL-4 and IL-13 and Epstein Barr virus. A549 cells were exposed to 10 ng/ml TGF-β1, IL-4 and IL-13 at serial time points. Total RNA was used for hybridisation to Affymetrix Human Genome U133A microarrays. Each in vitro time-point was studied in duplicate and an average RMA value computed. Expression data for each time point was compared to control and a signal log ratio of 0.6 or greater taken to identify significant differential regulation. Using normalised RMA values and unsupervised Average Linkage Hierarchical Cluster Analysis, a list of 312 extracellular matrix (ECM proteins or modulators of matrix turnover was curated via Onto-Compare and Gene-Ontology (GO databases for baited cluster analysis of ECM associated genes. Interrogation of the dataset using ontological classification focused cluster analysis revealed coordinate differential expression of a large cohort of extracellular matrix associated genes. Of this grouping members of the ADAM (A disintegrin and Metalloproteinase domain containing family of genes were differentially expressed. ADAM gene expression was also identified in EBV infected A549 cells as well as IL-13 and IL-4 stimulated cells. We probed pathologenomic activities (activation and functional activity of ADAM19 and ADAMTS9 using siRNA and collagen assays. Knockdown of these genes resulted in diminished production of collagen in A549 cells exposed to TGF-β1, suggesting a potential role for these molecules in ECM accumulation in IPF.

  11. The potential of microfluidic lung epithelial wounding: towards in vivo-like alveolar microinjuries

    OpenAIRE

    Felder, Marcel; Stucki, Andreas; Stucki, Janick; Geiser, Thomas; Guenat, Olivier Thierry

    2014-01-01

    Idiopathic pulmonary fibrosis (IPF) remains a major clinical challenge to date. Repeated alveolar epithelial microinjuries are considered as the starting point and the key event in both the development and the progression of IPF. Various pro-fibrotic agents have been identified and shown to cause alveolar damage. In IPF, however, no leading cause of alveolar epithelial microinjuries can be identified and the exact etiology remains elusive. New results from epidemiologic studies suggest a caus...

  12. A pure population of lung alveolar epithelial type II cells derived from human embryonic stem cells

    OpenAIRE

    Wang, Dachun; Haviland, David L.; Burns, Alan R.; Zsigmond, Eva; Wetsel, Rick A.

    2007-01-01

    Alveolar epithelial type II (ATII) cells are small, cuboidal cells that constitute ≈60% of the pulmonary alveolar epithelium. These cells are crucial for repair of the injured alveolus by differentiating into alveolar epithelial type I cells. ATII cells derived from human ES (hES) cells are a promising source of cells that could be used therapeutically to treat distal lung diseases. We have developed a reliable transfection and culture procedure, which facilitates, via genetic selection, the ...

  13. Biophysical determinants of alveolar epithelial plasma membrane wounding associated with mechanical ventilation

    OpenAIRE

    Hussein, Omar; Walters, Bruce; Stroetz, Randolph; Valencia, Paul; McCall, Deborah; Hubmayr, Rolf D.

    2013-01-01

    Mechanical ventilation may cause harm by straining lungs at a time they are particularly prone to injury from deforming stress. The objective of this study was to define the relative contributions of alveolar overdistension and cyclic recruitment and “collapse” of unstable lung units to membrane wounding of alveolar epithelial cells. We measured the interactive effects of tidal volume (VT), transpulmonary pressure (PTP), and of airspace liquid on the number of alveolar epithelial cells with p...

  14. CK2 inhibitor CX-4945 blocks TGF-β1-induced epithelial-to-mesenchymal transition in A549 human lung adenocarcinoma cells.

    Directory of Open Access Journals (Sweden)

    Jiyeon Kim

    Full Text Available BACKGROUND: The epithelial-to-mesenchymal transition (EMT is a major phenotype of cancer metastasis and invasion. As a druggable cancer target, the inhibition of protein kinase CK2 (formally named to casein kinase 2 has been suggested as a promising therapeutic strategy to treat EMT-controlled cancer metastasis. This study aimed to evaluate the effect of the CK2 inhibitor CX-4945 on the processes of cancer migration and invasion during the EMT in A549 human lung adenocarcinoma cells. MATERIALS AND METHODS: The effect of CX-4945 on TGF-β1-induced EMT was evaluated in A549 cells treated with TGF-β1 (5 ng/ml and CX-4945. The effect of CX-4945 on TGF-β1-induced cadherin switch and activation of key signaling molecules involved in Smad, non-Smad, Wnt and focal adhesion signaling pathways were investigated by Western blot analysis, immunocytochemistry and reporter assay. Additionally, the effect of CX-4945 on TGF-β1-induced migration and invasion was investigated by wound healing assay, Boyden chamber assay, gelatin zymography, and the quantitative real-time PCR. RESULTS: CX-4945 inhibits the TGF-β1-induced cadherin switch and the activation of key signaling molecules involved in Smad (Smad2/3, Twist and Snail, non-Smad (Akt and Erk, Wnt (β-catenin and focal adhesion signaling pathways (FAK, Src and paxillin that cooperatively regulate the overall process of EMT. As a result, CX-4945 inhibits the migration and invasion of A549 cells accompanied with the downregulation of MMP-2 and 9. CONCLUSIONS: Clinical evaluation of CX-4945 in humans as a single agent in solid tumors and multiple myeloma has established its promising pharmacokinetic, pharmacodynamic, and safety profiles. Beyond regression of tumor mass, CX-4945 may be advanced as a new therapy for cancer metastasis and EMT-related disorders.

  15. 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

  16. Cytotoxicity evaluation of nanoclays in human epithelial cell line A549 using high content screening and real-time impedance analysis

    International Nuclear Information System (INIS)

    Continuously expanding use of products containing nanoclays for wide range of applications have raised public concerns about health and safety. Although the products containing nanoclays may not be toxic, it is possible that nanomaterials may come in contact with humans during handling, manufacture, or disposal, and cause adverse health impact. This necessitates biocompatibility evaluation of the commonly used nanoclays. Here, we investigated the cytotoxic effects of platelet (Bentone MA, ME-100, Cloisite Na+, Nanomer PGV, and Delite LVF) and tubular (Halloysite, and Halloysite MP1) type nanoclays on cultured human lung epithelial cells A549. For the first time with this aim, we employed a cell-based automated high content screening in combination with real-time impedance sensing. We demonstrate varying degree of dose- and time-dependent cytotoxic effects of both nanoclay types. Overall, platelet structured nanoclays were more cytotoxic than tubular type. A low but significant level of cytotoxicity was observed at 25 μg/mL of the platelet-type nanoclays. A549 cells exposed to high concentration (250 μg/mL) of tubular structured nanoclays showed inhibited cell growth. Confocal microscopy indicated intracellular accumulation of nanoclays with perinuclear localization. Results indicate a potential hazard of nanoclay-containing products at significantly higher concentrations, which warrant their further biohazard assessment on the actual exposure in humans.

  17. Human alveolar epithelial type II cells in primary culture.

    Science.gov (United States)

    Mao, Pu; Wu, Songling; Li, Jianchun; Fu, Wei; He, Weiqun; Liu, Xiaoqing; Slutsky, Arthur S; Zhang, Haibo; Li, Yimin

    2015-02-01

    Alveolar epithelial type II (AEII) cells are a key structure and defender in the lung but also are the targets in many lung diseases, including acute respiratory distress syndrome, ventilator-induced lung injury, and pulmonary fibrosis. We sought to establish an optimized method for high yielding and long maintenance of characteristics of primary human AEII cells to facilitate the investigation of the mechanisms of lung diseases at the cellular and molecular levels. Adult human peripheral normal lung tissues of oncologic patients undergoing lung resection were collected. The AEII cells were isolated and identified by the expression of pro-surfactant protein (SP)C, epithelial sodium channel (αENaC) and cytokeratin (CK)-8, the lamellar bodies specific for AEII cells, and confirmed by the histology using electron microscopy. The phenotype of AEII cells was characterized by the expression of surfactant proteins (SP-A, SP-B, SP-C, SP-D), CK-8, KL-6, αENaC, and aquaporin (AQP)-3, which was maintained over 20 days. The biological activity of the primary human AEII cells producing SP-C, cytokines, and intercellular adhesion molecule-1 was vigorous in response to stimulation with tumor necrosis factor-α. We have modified previous methods and optimized a method for isolation of high purity and long maintenance of the human AEII cell phenotype in primary culture. This method provides an important tool for studies aiming at elucidating the molecular mechanisms of lung diseases exclusively in AEII cells. PMID:25677546

  18. The Study on Anti-cancer Effects of Distilling Fresh-ginseng Herbal acupuncture against implanted Sarcoma-180 in vivo and A549 human epithelial lung cancer cells in vitro

    OpenAIRE

    Hae-Young Jang; Ki-Rok Kwon; Hee-Soo Park

    2004-01-01

    Objectives : This study was to investigate the anti-cancer effects of herbal acupuncture with distilled fresh ginseng. The herbal acupuncture was injected to Chung-wan(C.V12) and Wisu(BL21) of mice that were subjected to Sarcoma-180 abdominal cancer cell and A549 human epithelial lung cancer cells in vitro. Methods : Anti-cancer effects of distilled fresh ginseng herbal acupuncture were tested by measruing Cox, Bcl-2, and Bax by using RT-PCR in A549 human epithelial lung cancer cells in v...

  19. Exosome cargo reflects TGF-β1-mediated epithelial-to-mesenchymal transition (EMT) status in A549 human lung adenocarcinoma cells.

    Science.gov (United States)

    Kim, Jiyeon; Kim, Tae Yeon; Lee, Myung Shin; Mun, Ji Young; Ihm, Chunhwa; Kim, Soon Ae

    2016-09-16

    It has been suggested that tumor cells secrete exosomes to modify the local microenvironment, which then promotes intercellular communication and metastasis. Although exosomes derived from cancer cells may contribute to the epithelial-mesenchymal transition (EMT) in untransformed cells, few studies have defined exosome cargo upon induction of EMT. In this study, we investigated the changes in exosomal cargo from the epithelial to mesenchymal cell phenotype by inducing EMT with transforming growth factor (TGF)-β1 in A549 human lung adenocarcinoma cells. The protein content of the exosomes reflects the change in the cell phenotype. In addition, miR-23a was significantly enriched in the exosomes after mesenchymal transition. Following treatment of exosomes from mesenchymal cells via EMT induction with TGF-β1 to the epithelial cell type, phenotypic changes in protein expression level and cell morphology were observed. Autologous treatment of exosomes enhanced the transcriptional activity and abundance of β-catenin. Our results suggest that the exosomal protein and miRNA content reflects the physiological condition of its source and that exosomes induce phenotypic changes via autocrine signaling. PMID:27492069

  20. Targeted Injury of Type II Alveolar Epithelial Cells Induces Pulmonary Fibrosis

    OpenAIRE

    Sisson, Thomas H.; Mendez, Michael; Choi, Karen; Subbotina, Natalya; Courey, Anthony; Cunningham, Andrew; Dave, Aditi; Engelhardt, John F.; Liu, Xiaoming; White, Eric S.; Thannickal, Victor J.; Bethany B Moore; Christensen, Paul J; Simon, Richard H

    2009-01-01

    Rationale: Ineffective repair of a damaged alveolar epithelium has been postulated to cause pulmonary fibrosis. In support of this theory, epithelial cell abnormalities, including hyperplasia, apoptosis, and persistent denudation of the alveolar basement membrane, are found in the lungs of humans with idiopathic pulmonary fibrosis and in animal models of fibrotic lung disease. Furthermore, mutations in genes that affect regenerative capacity or that cause injury/apoptosis of type II alveolar ...

  1. Cytotoxicity and inflammation in human alveolar epithelial cells following exposure to occupational levels of gold and silver nanoparticles

    International Nuclear Information System (INIS)

    While inhalation represents one of the most likely routes of exposure, the toxicity and response of nanoparticles at concentrations expected from such an exposure are not well understood. Here we characterized the in vitro response of human A549 adenocarcinomic alveolar epithelial cells following exposure to gold (AuNP) and silver (AgNP) nanoparticles at levels approximating an occupational exposure. Changes in neither oxidative stress nor cytotoxicity were significantly affected by exposure to AgNPs and AuNPs, regardless of NP type (Ag vs. Au), concentration, surface ligand (citrate or tannic acid), or size. An inflammatory response was, however, observed in response to 20 nm AgNPs and 20 nm AuNPs, where significant differences in the release of interleukin (IL)-8 but not IL-6 were observed. Additional data demonstrated that increased IL-8 secretion was strongly dependent on both nanoparticle size and concentration. Overall these data suggest that, while not acutely toxic, occupational exposure to AuNPs and AgNPs may trigger a significant inflammatory response in alveolar epithelium. Moreover, the differential responses in IL-8 and IL-6 secretion suggest that NPs may induce a response pathway that is distinct from those commonly elicited by allergens and pathogens.

  2. Shape matters: effects of silver nanospheres and wires on human alveolar epithelial cells

    Directory of Open Access Journals (Sweden)

    Stoehr Linda C

    2011-12-01

    Full Text Available Abstract Background In nanotoxicology, the exact role of particle shape, in relation to the composition, on the capacity to induce toxicity is largely unknown. We investigated the toxic and immunotoxic effects of silver wires (length: 1.5 - 25 μm; diameter 100 - 160 nm, spherical silver nanoparticles (30 nm and silver microparticles ( Methods Wires and nanoparticles were synthesized by wet-chemistry methods and extensively characterized. Cell viability and cytotoxicity were assessed and potential immunotoxic effects were investigated. To compare the effects on an activated and a resting immune system, cells were stimulated with rhTNF-α or left untreated. Changes in intracellular free calcium levels were determined using calcium imaging. Finally, ion release from the particles was assessed by ICP-MS and the effects of released ions on cell viability and cytotoxicity were tested. Results No effects were observed for the spherical particles, whereas the silver wires significantly reduced cell viability and increased LDH release from A549 cells. Cytokine promoter induction and NF-κB activation decreased in a concentration dependent manner similar to the decrease seen in cell viability. In addition, a strong increase of intracellular calcium levels within minutes after addition of wires was observed. This toxicity was not due to free silver ions, since the samples with the highest ion release did not induce toxicity and ion release control experiments with cells treated with pre-incubated medium did not show any effects either. Conclusions These data showed that silver wires strongly affect the alveolar epithelial cells, whereas spherical silver particles had no effect. This supports the hypothesis that shape is one of the important factors that determine particle toxicity.

  3. Water soluble and insoluble components of urban PM2.5 and their cytotoxic effects on epithelial cells (A549) in vitro.

    Science.gov (United States)

    Zou, Yajuan; Jin, Chengyu; Su, Yue; Li, Jiaru; Zhu, Bangshang

    2016-05-01

    When PM2.5 enters human bodies, the water soluble (WS-PM2.5) and insoluble components (WIS-PM2.5) of PM2.5 would interact with cells and cause adverse effects. However, the knowledge about the individual toxicity contribution of these two components is limited. In this study, the physiochemical properties of PM2.5 were well characterized. The toxic effects of WS-PM2.5 and WIS-PM2.5, which include the cell viability, cell membrane damage, reactive oxygen species (ROS) generation and morphological changes, were examined with human lung epithelial A549 cells in vitro. The results indicated that WS-PM2.5 could induce the early response of ROS generation, multiplied mitochondria and multi-lamellar bodies in A549 cells, which might cause cell damage through oxidative stress. Meanwhile, WIS-PM2.5 was predominantly associated with the cell membrane disruption, which might lead to the cell damage through cell-particle interactions. Moreover, the synergistic cytotoxic effects of WS-PM2.5 and WIS-PM2.5 were observed at longer exposure time. These findings demonstrate the different cytotoxicity mechanisms of WS-PM2.5 and WIS-PM2.5, which suggest that not only the size and dosage of PM2.5 but also the solubility of PM2.5 should be taken into consideration when evaluating the toxicity of PM2.5. PMID:27039898

  4. 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

  5. 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.

  6. Type I Alveolar Epithelial Cells Mount Innate Immune Responses during Pneumococcal Pneumonia

    OpenAIRE

    Yamamoto, Kazuko; Ferrari, Joseph D.; Cao, Yuxia; Ramirez, Maria I.; Jones, Matthew R.; Quinton, Lee J.; Mizgerd, Joseph P.

    2012-01-01

    Pneumonia results from bacteria in the alveoli. The alveolar epithelium consists of type II cells, which secrete surfactant and associated proteins, and type I cells, which constitute 95% of the surface area and met anatomic and structural needs. Other than constitutively expressed surfactant proteins, it is unknown whether alveolar epithelial cells have distinct roles in innate immunity. Since innate immunity gene induction depends on NF-κB RelA (also known as p65) during pneumonia, we gener...

  7. 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.

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

    Directory of Open Access Journals (Sweden)

    Taylor S Cohen

    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

  9. Clinical value of the alveolar epithelial permeability in various pulmonary diseases

    International Nuclear Information System (INIS)

    The authors have measured the pulmonary epithelial permeability in normals, smokers, ex-smokers and in various pulmonary diseases, using the sup(99m)Tc-DTPA monodisperse radioaerosol delivered by a newly designed nebulizer. Reference values for alveolar epithelial permeability were those of their own laboratory. Accelerated clearance of small idrophylic solutes from the lungs to the blood was found in smokers and in all the patients with idiopathic diffuse pulmonary fibrosis, chronic obstructive lung disease, congestive heart failure, acute viral pneumonia and adult respiratory distress syndrome. The greatest increase of alveolar epithelial clearance was found in the lung zone affected by the viral infection. The normal upper-lover lobe gradient of epithelial clearance was lost only in some patients. The increased permeability of the alveolar wall, although not specific, is characteristic and early feature of many acute and chronic pulmonary disease. For practical purposes, this parameter, rather than diagnostic, should be considered as a sensitive index of alveolar damage and repair, especially suitable for the follow-up of patients with spontaneous or therapeutic reversibility of parenchimal lung diseases. (orig.)

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

    DEFF Research Database (Denmark)

    Lo, Bernice; Hansen, Søren; Evans, Kathy; Heath, John K; Wright, Jo Rae

    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...

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

    Directory of Open Access Journals (Sweden)

    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.

  12. Endothelial-monocyte activating polypeptide II disrupts alveolar epithelial type II to type I cell transdifferentiation

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    Chen Yao

    2012-01-01

    Full Text Available Abstract Background Distal alveolar morphogenesis is marked by differentiation of alveolar type (AT-II to AT-I cells that give rise to the primary site of gas exchange, the alveolar/vascular interface. Endothelial-Monocyte Activating Polypeptide (EMAP II, an endogenous protein with anti-angiogenic properties, profoundly disrupts distal lung neovascularization and alveolar formation during lung morphogenesis, and is robustly expressed in the dysplastic alveolar regions of infants with Bronchopulmonary dysplasia. Determination as to whether EMAP II has a direct or indirect affect on ATII→ATI trans-differentiation has not been explored. Method In a controlled nonvascular environment, an in vitro model of ATII→ATI cell trans-differentiation was utilized to demonstrate the contribution that one vascular mediator has on distal epithelial cell differentiation. Results Here, we show that EMAP II significantly blocked ATII→ATI cell transdifferentiation by increasing cellular apoptosis and inhibiting expression of ATI markers. Moreover, EMAP II-treated ATII cells displayed myofibroblast characteristics, including elevated cellular proliferation, increased actin cytoskeleton stress fibers and Rho-GTPase activity, and increased nuclear:cytoplasmic volume. However, EMAP II-treated cells did not express the myofibroblast markers desmin or αSMA. Conclusion Our findings demonstrate that EMAP II interferes with ATII → ATI transdifferentiation resulting in a proliferating non-myofibroblast cell. These data identify the transdifferentiating alveolar cell as a possible target for EMAP II's induction of alveolar dysplasia.

  13. 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.

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    Yeo-Jin Choi

    Full Text Available 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.

  14. 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.

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    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

  15. Direct and indirect air particle cytotoxicity in human alveolar epithelial cells.

    Science.gov (United States)

    Orona, N S; Astort, F; Maglione, G A; Saldiva, P H N; Yakisich, J S; Tasat, D R

    2014-08-01

    Air particulate matter has been associated with adverse impact on the respiratory system leading to cytotoxic and proinflammatory effects. The biological mechanisms behind these associations may be initiated by inhaled small size particles, particle components (soluble fraction) and/or mediators released by particle-exposed cells (conditioned media). The effect of Urban Air Particles from Buenos Aires (UAP-BA) and Residual Oil Fly Ash (ROFA) a surrogate of ambient air pollution, their Soluble Fractions (SF) and Conditioned Media (CM) on A549 lung epithelial cells was examined. After 24 h exposure to TP (10 and 100 μg/ml), SF or CM, several biological parameters were assayed on cultured A549 cells. We tested cell viability by MTT, superoxide anion (O₂(-)) generation by NBT and proinflammatory cytokine (TNFα, IL-6 and IL-8) production by ELISA. UAP-BA particles or its SF (direct effect) did not modify cell viability and generation of O₂(-) for any of the doses tested. On the contrary, UAP-BA CM (indirect effect) reduced cell viability and increased both generation of O₂(-) and IL-8 production. Exposure to ROFA particles, SF or ROFA CM reduced proliferation and O₂(-) but, stimulated IL-8. It is worth to note that UAP-BA and ROFA depicted distinct effects on particle-exposed A549 cells implicating morphochemical dependence. These in vitro findings support the hypothesis that particle-induced lung inflammation and disease may involve lung-derived mediators. PMID:24590061

  16. Alveolar epithelial stem and progenitor cells: emerging evidence for their role in lung regeneration.

    Science.gov (United States)

    Hoffman, A M; Ingenito, E P

    2012-01-01

    Lung injuries that impact the alveolus, such as emphysema, pulmonary fibrosis, and acute lung injury, are costly and prevalent problems. Moreover, the extent of alveolar injury and impairment of gas exchange is strongly associated with prognosis and survival. Thus, mechanisms of repair and regeneration of the lung alveolar compartment have received mounting attention as newer approaches to the study of stem and progenitor cells in this region unfold. The role of type II alveolar epithelial as the sole source of type I (AECI) and II (AECII) alveolar epithelial cells following lung injury has been recently challenged; recently, investigators have described stemprogenitor cells that function like precursors to AECII either in vitro or in vivo, both in mice and humans. Techniques to explore selfrenewal and multipotency have been rigorously applied to these putative stem-progenitor cell populations and the data thus far is compelling. This review provides background to the study of alveolar regeneration with the aim to provide context to the recent discoveries of putative stem-progenitor cells that may contribute to this process. PMID:23016551

  17. SchA-p85-FAK complex dictates isoform-specific activation of Akt2 and subsequent PCBP1-mediated post-transcriptional regulation of TGFβ-mediated epithelial to mesenchymal transition in human lung cancer cell line A549.

    Science.gov (United States)

    Xue, Xinying; Wang, Xin; Liu, Yuxia; Teng, Guigen; Wang, Yong; Zang, Xuefeng; Wang, Kaifei; Zhang, Jinghui; Xu, Yali; Wang, Jianxin; Pan, Lei

    2014-08-01

    A post-transcriptional pathway by which TGF-β modulates expression of specific proteins, Disabled-2 (Dab2) and Interleukin-like EMT Inducer (ILEI), inherent to epithelial to mesenchymal transition (EMT) in murine epithelial cells through Akt2-mediated phosphorylation of poly r(C) binding protein (PCBP1), has been previously elucidated. The aims of the current study were to determine if the same mechanism is operative in the non-small cell lung cancer (NSCLC) cell line, A549, and to delineate the underlying mechanism. Steady-state transcript and protein expression levels of Dab2 and ILEI were examined in A549 cells treated with TGF-β for up to 48 h. Induction of translational de-repression in this model was quantified by polysomal fractionation followed by qRT-PCR. The underlying mechanism of isoform-specific activation of Akt2 was elucidated through a combination of co-immunoprecipitation studies. TGF-β induced EMT in A549 cells concomitant with translational upregulation of Dab2 and ILEI proteins through isoform-specific activation of Akt2 followed by phosphorylation of PCBP1 at serine-43. Our experiments further elucidated that the adaptor protein SchA is phosphorylated at tyrosine residues following TGF-β treatment, which initiated a signaling cascade resulting in the sequential recruitment of p85 subunit of PI3K and focal adhesion kinase (FAK). The SchA-FAK-p85 complex subsequently selectively recruited and activated Akt2, not Akt1. Inhibition of the p85 subunit through phosphorylated 1257 peptide completely attenuated EMT in these cells. We have defined the underlying mechanism responsible for isoform-specific recruitment and activation of Akt2, not Akt1, during TGF-β-mediated EMT in A549 cells. Inhibition of the formation of this complex thus represents an important and novel therapeutic target in metastatic lung carcinoma. PMID:24819169

  18. NO2 decreases paracellular resistance to ion and solute flow in alveolar epithelial monolayers

    International Nuclear Information System (INIS)

    Primary cultured monolayers of rat alveolar epithelial cells grown on tissue culture-treated Nuclepore filters were exposed to 2.5 ppm nitrogen dioxide NO2 for 2-20 min. Changes in monolayer bioelectric properties and solute permeabilities were subsequently measured. Exposure to NO2 produced a dose-dependent decrease in monolayer transepithelial electrical resistance (Rt), whereas monolayer short-circuit current was unaffected. Post-exposure monolayer permeability to 14C-sucrose (which primarily crosses alveolar epithelium via the paracellular pathway) increased markedly. That for 3H-glycerol (which permeates through both paracellular and transcellular pathways) increased to a lesser extent. Partial recovery of Rt and solute permeabilities was noted by 48-h post-exposure. The time courses of the decrease in Rt and increase in solute permeabilities were similar. These results suggest that NO2 primarily impairs passive alveolar epithelial barrier functions in vitro, probably by altering intercellular junctions, and does not appear to directly affect cell membrane active ion transport processes. When correlated with results obtained from experimental approaches, studies of in vitro alveolar epithelial monolayers may facilitate investigations of dosimetry, sites, and mechanisms of oxidant injury in the lung

  19. Glucose-6-phosphate dehydrogenase in rat lung alveolar epithelial cells. An ultrastructural enzyme-cytochemical study

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    S Matsubara

    2010-01-01

    Full Text Available Glucose-6-phosphate dehydrogenase (G6PD is the key enzyme of the pentose phosphate pathway in carbohydrate metabolism, and it plays an important role in cell proliferation and antioxidant regulation within cells in various organs. Although marked cell proliferation and oxidant/antioxidant metabolism occur in lung alveolar epithelial cells, definite data has been lacking as to whether cytochemically detectable G6PD is present in alveolar epithelial cells. The distribution pattern of G6PD within these cells, if it is present, is also unknown. The purpose of the present study was to investigate the subcellular localization of G6PD in alveolar cells in the rat lung using a newly- developed enzyme-cytochemistry (copper-ferrocyanide method. Type I cells and stromal endothelia and fibroblasts showed no activities. Electron-dense precipitates indicating G6PD activity were clearly visible in the cytoplasm and on the cytosolic side of the endoplasmic reticulum of type II alveolar epithelial cells. The cytochemical controls ensured specific detection of enzyme activity. This enzyme may play a role in airway defense by delivering substances for cell proliferation and antioxidant forces, thus maintaining the airway architecture.

  20. Pulmonary surfactant mitigates silver nanoparticle toxicity in human alveolar type-I-like epithelial cells.

    Science.gov (United States)

    Sweeney, Sinbad; Leo, Bey Fen; Chen, Shu; Abraham-Thomas, Nisha; Thorley, Andrew J; Gow, Andrew; Schwander, Stephan; Zhang, Junfeng Jim; Shaffer, Milo S P; Chung, Kian Fan; Ryan, Mary P; Porter, Alexandra E; Tetley, Teresa D

    2016-09-01

    Accompanying increased commercial applications and production of silver nanomaterials is an increased probability of human exposure, with inhalation a key route. Nanomaterials that deposit in the pulmonary alveolar region following inhalation will interact firstly with pulmonary surfactant before they interact with the alveolar epithelium. It is therefore critical to understand the effects of human pulmonary surfactant when evaluating the inhalation toxicity of silver nanoparticles. In this study, we evaluated the toxicity of AgNPs on human alveolar type-I-like epithelial (TT1) cells in the absence and presence of Curosurf(®) (a natural pulmonary surfactant substitute), hypothesising that the pulmonary surfactant would act to modify toxicity. We demonstrated that 20nm citrate-capped AgNPs induce toxicity in human alveolar type I-like epithelial cells and, in agreement with our hypothesis, that pulmonary surfactant acts to mitigate this toxicity, possibly through reducing AgNP dissolution into cytotoxic Ag(+) ions. For example, IL-6 and IL-8 release by TT1 cells significantly increased 10.7- and 35-fold, respectively (P<0.01), 24h after treatment with 25μg/ml AgNPs. In contrast, following pre-incubation of AgNPs with Curosurf(®), this effect was almost completely abolished. We further determined that the mechanism of this toxicity is likely associated with Ag(+) ion release and lysosomal disruption, but not with increased reactive oxygen species generation. This study provides a critical understanding of the toxicity of AgNPs in target human alveolar type-I-like epithelial cells and the role of pulmonary surfactant in mitigating this toxicity. The observations reported have important implications for the manufacture and application of AgNPs, in particular for applications involving use of aerosolised AgNPs. PMID:27182651

  1. Expression and function of aquaporin-1 in hyperoxia-exposed alveolar epithelial type II cells

    OpenAIRE

    ZHANG, QIU-YUE; Fu, Jian-Hua(Department of Physics, Henan University of Technology, Zhengzhou 450001, China); XUE, XIN-DONG

    2014-01-01

    The aim of the present study was to investigate water transport dysfunction in alveolar epithelial type II cells (AECII), which were exposed to hyperoxia, and to investigate the mechanism of pulmonary edema resulting from hyperoxic lung injury. The lung cells of newborn rats were isolated for primary cell culture and divided into control and experimental groups. The control and experimental group cells were placed into a normoxic incubator (oxygen volume fraction, 0.21) or hyperoxic incubator...

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

    OpenAIRE

    Seok-Jo Kim; Paul Cheresh; 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 evidenc...

  3. Cell Stress Induces Upregulation of Osteopontin via the ERK Pathway in Type II Alveolar Epithelial Cells

    OpenAIRE

    Aki Kato; Takafumi Okura; Chizuru Hamada; Seigo Miyoshi; Hitoshi Katayama; Jitsuo Higaki; Ryoji Ito

    2014-01-01

    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 ...

  4. Biophysical determinants of alveolar epithelial plasma membrane wounding associated with mechanical ventilation.

    Science.gov (United States)

    Hussein, Omar; Walters, Bruce; Stroetz, Randolph; Valencia, Paul; McCall, Deborah; Hubmayr, Rolf D

    2013-10-01

    Mechanical ventilation may cause harm by straining lungs at a time they are particularly prone to injury from deforming stress. The objective of this study was to define the relative contributions of alveolar overdistension and cyclic recruitment and "collapse" of unstable lung units to membrane wounding of alveolar epithelial cells. We measured the interactive effects of tidal volume (VT), transpulmonary pressure (PTP), and of airspace liquid on the number of alveolar epithelial cells with plasma membrane wounds in ex vivo mechanically ventilated rat lungs. Plasma membrane integrity was assessed by propidium iodide (PI) exclusion in confocal images of subpleural alveoli. Cyclic inflations of normal lungs from zero end-expiratory pressure to 40 cmH2O produced VT values of 56.9 ± 3.1 ml/kg and were associated with 0.12 ± 0.12 PI-positive cells/alveolus. A preceding tracheal instillation of normal saline (3 ml) reduced VT to 49.1 ± 6 ml/kg but was associated with a significantly greater number of wounded alveolar epithelial cells (0.52 ± 0.16 cells/alveolus; P < 0.01). Mechanical ventilation of completely saline-filled lungs with saline (VT = 52 ml/kg) to pressures between 10 and 15 cmH2O was associated with the least number of wounded epithelial cells (0.02 ± 0.02 cells/alveolus; P < 0.01). In mechanically ventilated, partially saline-filled lungs, the number of wounded cells increased substantially with VT, but, once VT was accounted for, wounding was independent of maximal PTP. We found that interfacial stress associated with the generation and destruction of liquid bridges in airspaces is the primary biophysical cell injury mechanism in mechanically ventilated lungs. PMID:23997173

  5. Generation of Alveolar Epithelial Spheroids via Isolated Progenitor Cells from Human Pluripotent Stem Cells

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    Shimpei Gotoh

    2014-09-01

    Full Text Available No methods for isolating induced alveolar epithelial progenitor cells (AEPCs from human embryonic stem cells (hESCs and induced pluripotent stem cells (hiPSCs have been reported. Based on a study of the stepwise induction of alveolar epithelial cells (AECs, we identified carboxypeptidase M (CPM as a surface marker of NKX2-1+ “ventralized” anterior foregut endoderm cells (VAFECs in vitro and in fetal human and murine lungs. Using SFTPC-GFP reporter hPSCs and a 3D coculture system with fetal human lung fibroblasts, we showed that CPM+ cells isolated from VAFECs differentiate into AECs, demonstrating that CPM is a marker of AEPCs. Moreover, 3D coculture differentiation of CPM+ cells formed spheroids with lamellar-body-like structures and an increased expression of surfactant proteins compared with 2D differentiation. Methods to induce and isolate AEPCs using CPM and consequently generate alveolar epithelial spheroids would aid human pulmonary disease modeling and regenerative medicine.

  6. A pure population of lung alveolar epithelial type II cells derived from human embryonic stem cells.

    Science.gov (United States)

    Wang, Dachun; Haviland, David L; Burns, Alan R; Zsigmond, Eva; Wetsel, Rick A

    2007-03-13

    Alveolar epithelial type II (ATII) cells are small, cuboidal cells that constitute approximately 60% of the pulmonary alveolar epithelium. These cells are crucial for repair of the injured alveolus by differentiating into alveolar epithelial type I cells. ATII cells derived from human ES (hES) cells are a promising source of cells that could be used therapeutically to treat distal lung diseases. We have developed a reliable transfection and culture procedure, which facilitates, via genetic selection, the differentiation of hES cells into an essentially pure (>99%) population of ATII cells (hES-ATII). Purity, as well as biological features and morphological characteristics of normal ATII cells, was demonstrated for the hES-ATII cells, including lamellar body formation, expression of surfactant proteins A, B, and C, alpha-1-antitrypsin, and the cystic fibrosis transmembrane conductance receptor, as well as the synthesis and secretion of complement proteins C3 and C5. Collectively, these data document the successful generation of a pure population of ATII cells derived from hES cells, providing a practical source of ATII cells to explore in disease models their potential in the regeneration and repair of the injured alveolus and in the therapeutic treatment of genetic diseases affecting the lung. PMID:17360544

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

    International Nuclear Information System (INIS)

    Young adult Syrian hamsters were injected intraperitoneally with 14C-glycerol and 3H-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 3H-specific activity of the phospholipids from the type II cells was three times that of the PAM and the utilization of 14C-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

  8. Conditional deletion of epithelial IKKβ impairs alveolar formation through apoptosis and decreased VEGF expression during early mouse lung morphogenesis

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    Li Changgong

    2011-10-01

    Full Text Available Abstract Background Alveolar septation marks the beginning of the transition from the saccular to alveolar stage of lung development. Inflammation can disrupt this process and permanently impair alveolar formation resulting in alveolar hypoplasia as seen in bronchopulmonary dysplasia in preterm newborns. NF-κB is a transcription factor central to multiple inflammatory and developmental pathways including dorsal-ventral patterning in fruit flies; limb, mammary and submandibular gland development in mice; and branching morphogenesis in chick lungs. We have previously shown that epithelial overexpression of NF-κB accelerates lung maturity using transgenic mice. The purpose of this study was to test our hypothesis that targeted deletion of NF-κB signaling in lung epithelium would impair alveolar formation. Methods We generated double transgenic mice with lung epithelium-specific deletion of IKKβ, a known activating kinase upstream of NF-κB, using a cre-loxP transgenic recombination strategy. Lungs of resulting progeny were analyzed at embryonic and early postnatal stages to determine specific effects on lung histology, and mRNA and protein expression of relevant lung morphoreulatory genes. Lastly, results measuring expression of the angiogenic factor, VEGF, were confirmed in vitro using a siRNA-knockdown strategy in cultured mouse lung epithelial cells. Results Our results showed that IKKβ deletion in the lung epithelium transiently decreased alveolar type I and type II cells and myofibroblasts and delayed alveolar formation. These effects were mediated through increased alveolar type II cell apoptosis and decreased epithelial VEGF expression. Conclusions These results suggest that epithelial NF-κB plays a critical role in early alveolar development possibly through regulation of VEGF.

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

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    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.

  10. The Study on Anti-cancer Effects of Distilling Fresh-ginseng Herbal acupuncture against implanted Sarcoma-180 in vivo and A549 human epithelial lung cancer cells in vitro

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    Hae-Young Jang

    2004-12-01

    Full Text Available Objectives : This study was to investigate the anti-cancer effects of herbal acupuncture with distilled fresh ginseng. The herbal acupuncture was injected to Chung-wan(C.V12 and Wisu(BL21 of mice that were subjected to Sarcoma-180 abdominal cancer cell and A549 human epithelial lung cancer cells in vitro. Methods : Anti-cancer effects of distilled fresh ginseng herbal acupuncture were tested by measruing Cox, Bcl-2, and Bax by using RT-PCR in A549 human epithelial lung cancer cells in vitro. And four weeks old Balb/c line male mice weighing around 20±3g were used to measure survival rate and anti-cancer effect to outputs of interleukin-2 and interleukin-4 using flow cytometry, possibility of mRNA menifestation using RT-PCR, and Cox mRNA. The results are as follows. Results : 1. In measuring mRNA menifestation in Cox, Bcl-2, and Bax by using RT-PCR in A549 human epithelial lung cancer cells in vitro, the result showed that fresh ginseng decreased Cox-2 which is directly involved in inflammation process. 2. Survival rate was measured in an anti-cancer effect experiment against Sarcoma-180 abdorminal cancer. Median survival time of controlled group was 27 days, of experiment group I was 21 days, and of experiment group II was 27 days. Therefore, experiment group I showed -22.2% increase in survival rate and experiment group II showed no difference compare to controlled group. 3. There was no difference between condition group and controlled and experiment group in measuring outputs of interleukin-2 and interleukin 4 by using flow cytometry 4. In measuring outputs of interleukin-2 by using ELISA, there was no significant difference between condition group and controlled group and there was decrease in experiment group II compared to conditioned and controlled group. 5. In measuring cytokine mRNA menifestation by using RT-PCR, experiment group I showed increase of mRNA menifestation in interleukin-2,4 and interferon-γ and experiment group II showed

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

    OpenAIRE

    Shigehisa Yanagi; Hironobu Tsubouchi; Ayako Miura; Nobuhiro Matsumoto; Masamitsu Nakazato

    2015-01-01

    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 ...

  12. Effect of Napsin A transfection into type Ⅱ alveolar epithelial cells on pulmonary fibrosis%Ⅱ型肺泡上皮细胞转染Napsin A基因对肺纤维化的干预作用

    Institute of Scientific and Technical Information of China (English)

    郑金旭; 管淑红; 许清; 汤艳; 刘继柱; 吕晓婷

    2010-01-01

    信号传导通路有关.%Objective To study the in vitro effect and mechanism of Napsin A gene transfection into type Ⅱ alveolar epithelial cells on pulmonary fibrosis. Methods A recombinant lentiviral plasmid PLJM1Napsin A was constructed and transfected into human type Ⅱ alveolar epithelial cell line A549. The model of pulmonary fibrosis was established by the in vitro stimulation of A549 cells by transforming growth factor beta-1 (TGF-β1). The morphological changes were observed continuously under inverted microscopy. The proliferation of transgenic and non-transgenic cells was detected by MTT. To observe the degree of epithelialmesenchymal transition ( EMT ) by TGF-β1 intervening A549 cells, the expressions of E-cadherin and fibronectin were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot.Lastly the protein expression of focal adhesion kinase (FAK) was detected by Western blot to investigate the mechanism. Results The result of sequencing the recombinant lentiviral plasmid PLJM1-Napsin A was the same as the design sequence. Napsin A mRNA and protein were expressed in transgenic A549 cells( P <0. 01 ). The model of pulmonary fibrosis was established successfully based on the morphology of transformed interstitial cell. As compared with the control group, the proliferation rate of transgenic cells decreased significantly (P <0. 05 ). The mRNA and protein expression of E-cadherin significantly decreased in the model of pulmonary fibrosis ( P < 0. 01 ), while the expression of fibronectin markedly increased ( P < 0. 01 ).But the change rate of transgenic cells decreased ( P < 0. 01, P < 0. 05 ). The expression of FAK was significantly elevated after the stimulation of TGF-β1 ( P < 0. 01 ). But the upward trend of the transgenic cells was smaller as compared with the control group (P < 0. 01 ). Conclusion Pulmonary fibrosis may be suppressed by the transfection of Napsin A gene into type Ⅱ alveolar epithelial cells. And the mechanism may

  13. 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

  14. Prognostic value of immunohistochemical surfactant protein A expression in regenerative/hyperplastic alveolar epithelial cells in idiopathic interstitial pneumonias

    Directory of Open Access Journals (Sweden)

    Kajiki Akira

    2011-03-01

    Full Text Available Abstract Background It is difficult to predict survival in patients with idiopathic pulmonary fibrosis. Recently, several proteins, such as surfactant protein (SP and KL-6, have been reported to be useful biologic markers for prediction of prognosis for interstitial pneumonias. It is not clear whether there is any relationship between expression of these proteins in regenerative/hyperplastic alveolar epithelial cells and prognosis of idiopathic interstitial pneumonias (IIPs. Objectives This study aimed to elucidate the clinical significance of the expression of such lung secretory proteins as SP-A and KL-6 in lung tissues of patients with IIPs. Methods We retrospectively investigated the immunohistochemical expression of SP-A, KL-6, cytokeratin (CK, and epithelial membrane antigen (EMA in alveolar epithelial cells in lung tissues obtained from surgical lung biopsy in 43 patients with IIPs, and analyzed the correlation between expression of these markers and the prognosis of each IIP patient. CK and EMA were used as general markers for epithelial cells. Results In patients with usual interstitial pneumonia (UIP, the ratio of SP-A positive epithelial cells to all alveolar epithelial cells (SP-A positive ratio in the collapsed and mural fibrosis areas varied, ranging from cases where almost all alveolar epithelial cells expressed SP-A to cases where only a few did. On the other hand, in many patients with nonspecific interstitial pneumonia (NSIP, many of the alveolar epithelial cells in the diseased areas expressed SP-A. The SP-A positive ratio was significantly lower in patients who died from progression of UIP than in patients with UIP who remained stable or deteriorated but did not die. In NSIP patients, a similar tendency was noted between the SP-A positive ratio and prognosis. Conclusions The results suggest that the paucity of immunohistochemical SP-A expression in alveolar epithelial cells in diseased areas (i.e. regenerative

  15. Electron microscopic studies of various cells in the alveolar wall of mice with special reference to spheroid alveolar epithelial cells after intravenous injection of squid-ink (sepia-melanin solution

    Directory of Open Access Journals (Sweden)

    Suwa,Kiichi

    1977-02-01

    Full Text Available The effect of an intravenous injection of squid-ink (sepia-melanin solution on adult mouse spheroid alveolar epithelial cells was observed by the electron microscope. Sepia-melanin particles were seen in all alveolar wall cells examined that seems to suggest the entrance of sepia-melanin particles into the spheroid alveolar epithlial cells from the alveolar blood capillary. In cases of large penetrations of sepia-melanin particles into spheroid alveolar epithelial cells, a greater increase was found in the intramitochondrial granules. In addition, the so-called inclusion body believed to be formed by the degeneration of mitochondria had very high electron density and its quantity was abundant. On the contrary, in cases where the quantity of sepia-melanin entrance into the spheroid alveolar epithelial cell was small, neither an increase of intramitochondrial granules, an increase of the electron density nor an increase in the quantity of specific inclusion body was found.

  16. Role of cytoskeleton in cytokine production from lung alveolar epithelial cells

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Cytokines are involved in both host defense and inflammatory lung injury. Recent work from our laboratory and others has demonstrated that in addition to classical immune cells, lung alveolar epithelial cells (or pneumocytes) can also produce cytokines in response to various stimuli. This new knowledge has advanced our view of the host defense system in the lung. The regulatory mechanisms of cytokine production have been studied in great detail at various cellular and molecular levels, but the mechanisms of intracellular cytokine transport are largely unknown. Our recent studies suggest that the cytoskeleton could play an important role in mediating intracellular cytokine trafficking. This could be an important regulatory step for cytokine production. For example, lipopolyssacharide (LPS) induced tumor necrosis factor-α (TNF-α) from rat pneumocytes, which was further enhanced by a microfilament-disrupting agent. LPS also induced macrophage inflammatory protein-2(MIP-2), a chemokine for neutrophil recruitment and activation, from rat pneumocytes. This effect was enhanced by microtubule-disrupting agents. We speculate that both microfilaments and microtubules are involved in regulating cytokine transportation in pneumocytes through different mechanisms. Further investigation in on going in my laboratory. From a clinical perspective, if we understand the mechanisms regulating cytokine production and release from lung alveolar epithelial cells, we may be able to enhance or inhibit release of crucial cytokines depending on the clinical situation.

  17. Effect of functionalized and non-functionalized nanodiamond on the morphology and activities of antioxidant enzymes of lung epithelial cells (A549).

    Science.gov (United States)

    Solarska-Ściuk, Katarzyna; Gajewska, Agnieszka; Glińska, Sława; Michlewska, Sylwia; Balcerzak, Łucja; Jamrozik, Agnieszka; Skolimowski, Janusz; Burda, Květoslava; Bartosz, Grzegorz

    2014-10-01

    The development of nanotechnology opens up new ways for biomedical applications of unmodified and modified diamond nanoparticles which are one of the most popular nanomaterials used in biology, biotechnology, medicine, cosmetics and engineering. They have been applied as diagnostic and therapeutic agents because they can be targeted to and localized in cells causing apoptosis and necrosis. The problem of biocompatibility of nanodiamonds at higher concentrations is thus of primary importance. The first step in the modification of DNPs is usually the introduction of hydrogen groups, which can bind other functional groups. The basic method to introduce -OH groups onto nanoparticles is the Fenton reaction. The aim of this study was to compare the effect of unmodified nanodiamond particles and nanoparticles modified by introduction of -OH groups and etoposide onto their surface reaction on human non-small lung cancer cells. A549 cells were incubated with 2-100μg/ml nanopowders and at 0.6-24μg/ml etoposide in the DMEM medium. We observed a decrease of cells viability and generation of reactive oxygen/ nitrogen species in the cells after incubation, estimated by oxidation of H2DCF-DA and DAF-FM-DA. Modified detonation nanoparticles affected also the cellular content of glutathione and activities of main antioxidant enzymes (glutathione peroxidase, glutathione reductase, glutathione S-transferase, superoxide dismutase and catalase). The results of TEM microscopy show changes in cell morphology. These data demonstrate that modified nanoparticles induce oxidative stress in the target cells. PMID:25451571

  18. 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; Irlik, Przemyslaw Jedrzej; Nielsen, Hanne Mørck; Foged, Camilla

    2014-01-01

    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...

  19. MicroRNA 16 Modulates Epithelial Sodium Channel in Human Alveolar Epithelial Cells

    OpenAIRE

    Parthasarathy, Prasanna Tamarapu; Galam, Lakshmi; Huynh, Bao; Yunus, Asfiya; Abuelenen, Toaa; Castillo, Annie; Ramanathan, Gurukumar Kollongod; Ruan, Cox; Kolliputi, Narasaiah

    2012-01-01

    Acute lung injury (ALI) is a devastating disease characterized by pulmonary edema. Removal of edema from the air spaces is a critical function of the epithelial sodium channel (ENaC) in ALI. The molecular mechanisms behind resolution of pulmonary edema are incompletely understood. MicroRNA’s (miRNA) are crucial gene regulators and are dysregulated in various diseases including ALI. Recent studies suggest that microRNA-16 (miR-16) targets serotonin transporter (SERT) involved in the serotonin ...

  20. Mesenchymal stem cells protect from hypoxia-induced alveolar epithelial-mesenchymal transition.

    Science.gov (United States)

    Uzunhan, Yurdagül; Bernard, Olivier; Marchant, Dominique; Dard, Nicolas; Vanneaux, Valérie; Larghero, Jérôme; Gille, Thomas; Clerici, Christine; Valeyre, Dominique; Nunes, Hilario; Boncoeur, Emilie; Planès, Carole

    2016-03-01

    Administration of bone marrow-derived human mesenchymal stem cells (hMSC) reduces lung inflammation, fibrosis, and mortality in animal models of lung injury, by a mechanism not completely understood. We investigated whether hMSC would prevent epithelial-mesenchymal transition (EMT) induced by hypoxia in primary rat alveolar epithelial cell (AEC). In AEC cultured on semipermeable filters, prolonged hypoxic exposure (1.5% O2 for up to 12 days) induced phenotypic changes consistent with EMT, i.e., a change in cell morphology, a decrease in transepithelial resistance (Rte) and in the expression of epithelial markers [zonula occludens-1 (ZO-1), E-cadherin, AQP-5, TTF-1], together with an increase in mesenchymal markers [vimentin, α-smooth muscle actin (α-SMA)]. Expression of transcription factors driving EMT such as SNAIL1, ZEB1, and TWIST1 increased after 2, 24, and 48 h of hypoxia, respectively. Hypoxia also induced TGF-β1 mRNA expression and the secretion of active TGF-β1 in apical medium, and the expression of connective tissue growth factor (CTGF), two inducers of EMT. Coculture of AEC with hMSC partially prevented the decrease in Rte and in ZO-1, E-cadherin, and TTF-1 expression, and the increase in vimentin expression induced by hypoxia. It also abolished the increase in TGF-β1 expression and in TGF-β1-induced genes ZEB1, TWIST1, and CTGF. Finally, incubation with human recombinant KGF at a concentration similar to what was measured in hMSC-conditioned media restored the expression of TTF-1 and prevented the increase in TWIST1, TGF-β1, and CTGF in hypoxic AEC. Our results indicate that hMSC prevent hypoxia-induced alveolar EMT through the paracrine modulation of EMT signaling pathways and suggest that this effect is partly mediated by KGF. PMID:26702148

  1. Hypoxia-induced alveolar epithelial-mesenchymal transition requires mitochondrial ROS and hypoxia-inducible factor 1

    OpenAIRE

    Zhou, Guofei; Dada, Laura A.; Wu, Minghua; Kelly, Aileen; Trejo, Humberto; Zhou, Qiyuan; Varga, John; Sznajder, Jacob I.

    2009-01-01

    Patients with acute lung injury develop hypoxia, which may lead to lung dysfunction and aberrant tissue repair. Recent studies have suggested that epithelial-mesenchymal transition (EMT) contributes to pulmonary fibrosis. We sought to determine whether hypoxia induces EMT in alveolar epithelial cells (AEC). We found that hypoxia induced the expression of α-smooth muscle actin (α-SMA) and vimentin and decreased the expression of E-cadherin in transformed and primary human, rat, and mouse AEC, ...

  2. CXCR4 regulates migration of lung alveolar epithelial cells through activation of Rac1 and matrix metalloproteinase-2

    OpenAIRE

    Ghosh, Manik C.; Makena, Patrudu S.; Gorantla, Vijay; Sinclair, Scott E.; Waters, Christopher M.

    2012-01-01

    Restoration of the epithelial barrier following acute lung injury is critical for recovery of lung homeostasis. After injury, alveolar type II epithelial (ATII) cells spread and migrate to cover the denuded surface and, eventually, proliferate and differentiate into type I cells. The chemokine CXCL12, also known as stromal cell-derived factor 1α, has well-recognized roles in organogenesis, hematopoiesis, and immune responses through its binding to the chemokine receptor CXCR4. While CXCL12/CX...

  3. 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.

  4. Saponins from the roots of Platycodon grandiflorum suppresses TGFβ1-induced epithelial-mesenchymal transition via repression of PI3K/Akt, ERK1/2 and Smad2/3 pathway in human lung carcinoma A549 cells.

    Science.gov (United States)

    Choi, Jae Ho; Hwang, Yong Pil; Kim, Hyung Gyun; Khanal, Tilak; Do, Minh Truong; Jin, Sun Woo; Han, Hwa Jeong; Lee, Hyun Sun; Lee, Young Chun; Chung, Young Chul; Jeong, Tae Cheon; Jeong, Hye Gwang

    2014-01-01

    Transforming growth factor β (TGFβ) is a multifunctional cytokine that induces growth arrest, tissue fibrosis, and epithelial-mesenchymal transition (EMT) through activation of Smad and non-Smad signaling pathways. EMT is the differentiation switch by which polarized epithelial cells differentiate into contractile and motile mesenchymal cells. Our previous studies have shown that saponins from the roots of Platycodon grandiflorum (CKS) have antiinflammatory, antioxidant, antimetastatic, and hepatoprotective effects. In this study, we investigated the inhibitory effect of CKS on TGFβ1-induced alterations characteristic of EMT in human lung carcinoma A549 cells. We found that CKS-treated cells displayed inhibited TGFβ1-mediated E-cadherin downregulation and Vimentin upregulation and also retained epithelial morphology. Furthermore, TGFβ1-increased Snail expression, a repressor of E-cadherin and an inducer of the EMT, was reduced by CKS. CKS inhibited TGFβ1-induced phosphorylation of Akt, ERK1/2, and glycogen synthase kinase-3β (GSK-3β). Inhibition of PI3K/Akt and ERK1/2 also blocked TGFβ1-induced GSK-3β phosphorylation and Snail activation. Furthermore, TGFβ1-increased Snail expression was reduced by selective inhibitors of Akt and ERK1/2. Moreover, CKS treatment attenuated TGFβ1-induced Smad2/3 phosphorylation and upregulated Smad7 expression. These results indicate that pretreatment with the CKS inhibits the TGFβ1-induced EMT through PI3K/Akt, ERK1/2, GSK-3β and Smad2/3 in human lung carcinoma cells. PMID:24341702

  5. Induction of IL-6 and CCL5 (RANTES in human respiratory epithelial (A549 cells by clinical isolates of respiratory syncytial virus is strain specific

    Directory of Open Access Journals (Sweden)

    Levitz Ruth

    2012-09-01

    Full Text Available Abstract Background Respiratory syncytial virus (RSV is the major respiratory pathogen of infants and young children. During each seasonal epidemic, multiple strains of both subgroup A and B viruses circulate in the community. Like other RNA viruses, RSV genome replication is prone to errors that results in a heterogeneous population of viral strains some of which may possess differences in virulence. We sought to determine whether clinical isolates of RSV differ in their capacity to induce inflammatory cytokines IL-6 and CCL5 (previously known as RANTES [regulated upon activation, normal T-cell expressed and secreted protein], which are known to be induced in vitro and in vivo in response to RSV, during infection of A549 cells. Results Screening of subgroup A and B isolates revealed heterogeneity among strains to induce IL-6 and CCL5. We chose two subgroup B strains, New Haven (NH1067 and NH1125, for further analysis because of their marked differences in cytokine inducing properties and because subgroup B strains, in general, are less genetically heterogeneous as compared to subgroup A strains. At 12 and 24 hours post infection RSV strains, NH1067 and NH1125 differed in their capacity to induce IL-6 by an order of magnitude or more. The concentrations of IL-6 and CCL5 were dependent on the dose of infectious virus and the concentration of these cytokines induced by NH1125 was greater than that of those induced by NH1067 when the multiplicity of infection of NH1067 used was as much as 10-fold higher than that of NH1125. The induction of IL-6 was dependent on viable virus as infection with UV-inactivated virus did not induce IL-6. The difference in IL-6 induction most likely could not be explained by differences in viral replication kinetics. The intracellular level of RSV RNA, as determined by quantitative RT-PCR, was indistinguishable between the 2 strains though the titer of progeny virus produced by NH1125 was greater than that produced by

  6. Isolation and characterization of alveolar epithelial type II cells derived from mouse embryonic stem cells.

    Science.gov (United States)

    Sun, Huanhuan; Quan, Yuan; Yan, Qing; Peng, Xinmiao; Mao, Zhengmei; Wetsel, Rick A; Wang, Dachun

    2014-06-01

    The use of embryonic stem cells (ESCs) to regenerate distal lung epithelia damaged by injuries or diseases requires development of safe and efficient methodologies that direct ESC differentiation into transplantable distal lung epithelial progenitors. Time-consuming culture procedure and low differentiation efficiency are major problems that are associated with conventional differentiation approaches via embryoid body formation. The use of a growth factor cocktail or a lung-specific cell-conditioned medium to enrich definitive endoderm for efficient differentiation of mouse ESCs (mESC) into alveolar epithelial progenitor type II cells (ATIICs) has been reported, but not yet successful for generating a homogenous population of ATIICs for tissue regeneration purpose, and it remains unclear whether or not those mESC-derived ATIICs possess normal biological functions. Here, we report a novel method using a genetically modified mESC line harboring an ATIIC-specific neomycin(R) transgene in Rosa 26 locus. We showed that ATIICs can be efficiently differentiated from mESCs as early as day 7 by culturing them directly on Matrigel-coated plates in DMEM containing 15% knockout serum replacement. With this culture condition, the genetically modified mESCs can be selectively differentiated into a homogenous population (>99%) of ATIICs. Importantly, the mESC-derived ATIICs (mESC-ATIICs) exhibited typical lamellar bodies and expressed surfactant protein A, B, and C as normal control ATIICs. When cultured with an air-liquid-interface culture system in Small Airway Epithelial Cell Growth Medium, the mESC-ATIICs can be induced to secrete surfactant proteins after being treated with dibutyryl cAMP+dexamethasone. These mESC-ATIICs can synthesize and secrete surfactant lipid in response to secretagogue, demonstrating active surfactant metabolism in mESC-ATIICs as that seen in normal control ATIICs. In addition, we demonstrated that the selected mESC-ATIICs can be maintained on Matrigel

  7. Alveolar macrophages modulate the epithelial cell response to coal dust in vitro.

    Science.gov (United States)

    Lee, Y C; Rannels, D E

    1996-01-01

    The response of the alveolar epithelium to coal dust exposure is poorly understood. Coal or other dusts may act on the epithelium directly or indirectly through nearby alveolar macrophages (AM) that produce cytokines and other soluble products. AM and type II pneumocytes (T2P) were thus exposed to dust in coculture to evaluate their possible interactions. Anthracite coal dust PSOC 867 increased synthesis of extracellular matrix (ECM) components by T2P. AM alone did not produce ECM. Similarly, coculture of T2P with AM (3.75:1) had little effect on epithelial ECM synthesis. In contrast, coculture of T2P with AM significantly increased PSOC 867 effects on T2P rates of ECM synthesis, ECM fibronectin content, and T2P levels of fibronectin mRNA. AM-conditioned medium did not change the PSOC 867 effect on T2P. Neither control nor PSOC 867-treated AM on Falcon culture inserts (0.45-micron pore size) over T2P stimulated ECM synthesis by either untreated or dust-exposed epithelium. Thus AM-mediated changes in ECM synthesis by PSOC 867-treated T2P require close cell-cell interactions, suggesting a role for cell-cell contact or for short-lived soluble mediators of the AM effects. PMID:8772535

  8. Role of primary human alveolar epithelial cells in host defense against Francisella tularensis infection.

    Science.gov (United States)

    Gentry, Megan; Taormina, Joanna; Pyles, Richard B; Yeager, Linsey; Kirtley, Michelle; Popov, Vsevolod L; Klimpel, Gary; Eaves-Pyles, Tonyia

    2007-08-01

    Francisella tularensis, an intracellular pathogen, is highly virulent when inhaled. Alveolar epithelial type I (ATI) and type II (ATII) cells line the majority of the alveolar surface and respond to inhaled pathogenic bacteria via cytokine secretion. We hypothesized that these cells contribute to the lung innate immune response to F. tularensis. Results demonstrated that the live vaccine strain (LVS) contacted ATI and ATII cells by 2 h following intranasal inoculation of mice. In culture, primary human ATI or ATII cells, grown on transwell filters, were stimulated on the apical (AP) surface with virulent F. tularensis Schu 4 or LVS. Basolateral (BL) conditioned medium (CM), collected 6 and 24 h later, was added to the BL surfaces of transwell cultures of primary human pulmonary microvasculature endothelial cells (HPMEC) prior to the addition of polymorphonuclear leukocytes (PMNs) or dendritic cells (DCs) to the AP surface. HPMEC responded to S4- or LVS-stimulated ATII, but not ATI, CM as evidenced by PMN and DC migration. Analysis of the AP and BL ATII CM revealed that both F. tularensis strains induced various levels of a variety of cytokines via NF-kappaB activation. ATII cells pretreated with an NF-kappaB inhibitor prior to F. tularensis stimulation substantially decreased interleukin-8 secretion, which did not occur through Toll-like receptor 2, 2/6, 4, or 5 stimulation. These data indicate a crucial role for ATII cells in the innate immune response to F. tularensis. PMID:17502386

  9. Role of Primary Human Alveolar Epithelial Cells in Host Defense against Francisella tularensis Infection▿

    Science.gov (United States)

    Gentry, Megan; Taormina, Joanna; Pyles, Richard B.; Yeager, Linsey; Kirtley, Michelle; Popov, Vsevolod L.; Klimpel, Gary; Eaves-Pyles, Tonyia

    2007-01-01

    Francisella tularensis, an intracellular pathogen, is highly virulent when inhaled. Alveolar epithelial type I (ATI) and type II (ATII) cells line the majority of the alveolar surface and respond to inhaled pathogenic bacteria via cytokine secretion. We hypothesized that these cells contribute to the lung innate immune response to F. tularensis. Results demonstrated that the live vaccine strain (LVS) contacted ATI and ATII cells by 2 h following intranasal inoculation of mice. In culture, primary human ATI or ATII cells, grown on transwell filters, were stimulated on the apical (AP) surface with virulent F. tularensis Schu 4 or LVS. Basolateral (BL) conditioned medium (CM), collected 6 and 24 h later, was added to the BL surfaces of transwell cultures of primary human pulmonary microvasculature endothelial cells (HPMEC) prior to the addition of polymorphonuclear leukocytes (PMNs) or dendritic cells (DCs) to the AP surface. HPMEC responded to S4- or LVS-stimulated ATII, but not ATI, CM as evidenced by PMN and DC migration. Analysis of the AP and BL ATII CM revealed that both F. tularensis strains induced various levels of a variety of cytokines via NF-κB activation. ATII cells pretreated with an NF-κB inhibitor prior to F. tularensis stimulation substantially decreased interleukin-8 secretion, which did not occur through Toll-like receptor 2, 2/6, 4, or 5 stimulation. These data indicate a crucial role for ATII cells in the innate immune response to F. tularensis. PMID:17502386

  10. 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. PMID:27334278

  11. Male Sex is Associated with a Reduced Alveolar Epithelial Sodium Transport

    Science.gov (United States)

    Kaltofen, Till; Haase, Melanie; Thome, Ulrich H.; Laube, Mandy

    2015-01-01

    Respiratory distress syndrome (RDS) is the most frequent pulmonary complication in preterm infants. RDS incidence differs between genders, which has been called the male disadvantage. Besides maturation of the surfactant system, Na+ transport driven alveolar fluid clearance is crucial for the prevention of RDS. Na+ transport is mediated by the epithelial Na+ channel (ENaC) and the Na,K-ATPase, therefore potential differences in their expression or activity possibly contribute to the gender imbalance observed in RDS. Fetal distal lung epithelial (FDLE) cells of rat fetuses were separated by sex and analyzed regarding expression and activity of the Na+ transporters. Ussing chamber experiments showed a higher baseline short-circuit current (ISC) and amiloride-sensitive ΔISC in FDLE cells of female origin. In addition, maximal amiloride-sensitive ΔISC and maximal ouabain-sensitive ΔISC of female cells were higher when measured in the presence of a permeabilized basolateral or apical membrane, respectively. The number of FDLE cells per fetus recoverable during cell isolation was also significantly higher in females. In addition, lung wet-to-dry weight ratio was lower in fetal and newborn female pups. Female derived FDLE cells had higher mRNA levels of the ENaC- and Na,K-ATPase subunits. Furthermore, estrogen (ER) and progesterone receptor (PR) mRNA levels were higher in female cells, which might render female cells more responsive, while concentrations of placenta-derived sex steroids do not differ between both genders during fetal life. Inhibition of ER-β abolished the sex differences in Na+ transport and female cells were more responsive to estradiol stimulation. In conclusion, a higher alveolar Na+ transport, possibly attributable to a higher expression of hormone receptors in female FDLE cells, provides an explanation for the well known sex-related difference in RDS occurrence and outcome. PMID:26291531

  12. Male Sex is Associated with a Reduced Alveolar Epithelial Sodium Transport.

    Directory of Open Access Journals (Sweden)

    Till Kaltofen

    Full Text Available Respiratory distress syndrome (RDS is the most frequent pulmonary complication in preterm infants. RDS incidence differs between genders, which has been called the male disadvantage. Besides maturation of the surfactant system, Na+ transport driven alveolar fluid clearance is crucial for the prevention of RDS. Na+ transport is mediated by the epithelial Na+ channel (ENaC and the Na,K-ATPase, therefore potential differences in their expression or activity possibly contribute to the gender imbalance observed in RDS. Fetal distal lung epithelial (FDLE cells of rat fetuses were separated by sex and analyzed regarding expression and activity of the Na+ transporters. Ussing chamber experiments showed a higher baseline short-circuit current (ISC and amiloride-sensitive ΔISC in FDLE cells of female origin. In addition, maximal amiloride-sensitive ΔISC and maximal ouabain-sensitive ΔISC of female cells were higher when measured in the presence of a permeabilized basolateral or apical membrane, respectively. The number of FDLE cells per fetus recoverable during cell isolation was also significantly higher in females. In addition, lung wet-to-dry weight ratio was lower in fetal and newborn female pups. Female derived FDLE cells had higher mRNA levels of the ENaC- and Na,K-ATPase subunits. Furthermore, estrogen (ER and progesterone receptor (PR mRNA levels were higher in female cells, which might render female cells more responsive, while concentrations of placenta-derived sex steroids do not differ between both genders during fetal life. Inhibition of ER-β abolished the sex differences in Na+ transport and female cells were more responsive to estradiol stimulation. In conclusion, a higher alveolar Na+ transport, possibly attributable to a higher expression of hormone receptors in female FDLE cells, provides an explanation for the well known sex-related difference in RDS occurrence and outcome.

  13. Effects of Pseudomonas aeruginosa elastase on alveolar epithelial permeability in guinea pigs

    International Nuclear Information System (INIS)

    Elastase-deficient mutants of Pseudomonas aeruginosa are less virulent than the wild type and are easily cleared from the lungs of guinea pigs. The effect of P. aeruginosa elastase on lung epithelium, however, is not yet understood. We addressed the hypothesis that breach of the epithelial barrier by elastase from P. aeruginosa allows invading organisms and toxic substances to penetrate the interstitium. We measured the clearance of aerosolized technetium-labeled albumin (molecular weight, 69,000) from the lungs of anesthetized guinea pigs with the aid of a gamma camera and a dedicated computer. Aerosols of the elastase (0.1 to 5 micrograms) increased the rate of clearance of labeled albumin from the lungs in proportion to the elastase dose. Electron microscopic studies using horseradish peroxidase as a tracer revealed that elastase interrupts intercellular tight junctions of the epithelial lining, thereby increasing the permeability to macromolecules. The amounts of elastase used in this report did not cause interstitial or alveolar edema, as determined by both postmortem extravascular lung water volume measurement and morphological examination. The data indicate that the elastase is a potentially important virulence factor in acute lung infection

  14. Translocation of PEGylated quantum dots across rat alveolar epithelial cell monolayers

    Directory of Open Access Journals (Sweden)

    Fazlollahi F

    2011-11-01

    Full Text Available Farnoosh Fazlollahi1,8, Arnold Sipos1,2, Yong Ho Kim1,2, Sarah F Hamm-Alvarez6, Zea Borok1–3, Kwang-Jin Kim1,2,5–7, Edward D Crandall1,2,4,8 1Will Rogers Institute Pulmonary Research Center, 2Department of Medicine, 3Department of Biochemistry and Molecular Biology, 4Department of Pathology, 5Department of Physiology and Biophysics, 6Department of Pharmacology and Pharmaceutical Sciences, 7Department of Biomedical Engineering, 8Mork Family Department of Chemical Engineering and Materials Science, University of Southern California, Los Angeles, CA, USA Background: In this study, primary rat alveolar epithelial cell monolayers (RAECM were used to investigate transalveolar epithelial quantum dot trafficking rates and underlying transport mechanisms. Methods: Trafficking rates of quantum dots (PEGylated CdSe/ZnS, core size 5.3 nm, hydrodynamic size 25 nm in the apical-to-basolateral direction across RAECM were determined. Changes in bioelectric properties (ie, transmonolayer resistance and equivalent active ion transport rate of RAECM in the presence or absence of quantum dots were measured. Involvement of endocytic pathways in quantum dot trafficking across RAECM was assessed using specific inhibitors (eg, methyl-ß-cyclodextrin, chlorpromazine, and dynasore for caveolin-, clathrin-, and dynamin-mediated endocytosis, respectively. The effects of lowering tight junctional resistance on quantum dot trafficking were determined by depleting Ca2+ in apical and basolateral bathing fluids of RAECM using 2 mM EGTA. Effects of temperature on quantum dot trafficking were studied by lowering temperature from 37°C to 4°C. Results: Apical exposure of RAECM to quantum dots did not elicit changes in transmonolayer resistance or ion transport rate for up to 24 hours; quantum dot trafficking rates were not surface charge-dependent; methyl-ß-cyclodextrin, chlorpromazine, and dynasore did not decrease quantum dot trafficking rates; lowering of temperature

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

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    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

    Pseudomonas aeruginosa is the major cause of chronic pulmonary disease in cystic fibrosis (CF) patients. During chronic infection, P. aeruginosa lose certain virulence factors, transform into a mucoid phenotype, and develop antibiotic resistance. We hypothesized that these genetic and phenotypic...

  17. The alveolar epithelial differentiation of glandular inner lining cells in a mucoepidermoid carcinoma of the lung: a case report

    Directory of Open Access Journals (Sweden)

    Xu Hong-Tao

    2012-10-01

    Full Text Available Abstract Mucoepidermoid carcinoma is a common malignant epithelial tumor of salivary glands, but relatively rare in lung. The histological features of mucoepidermoid carcinoma of the lung are similar to its counterpart arising from the salivary glands. Here, we reported a special tumor that occurred in the medial segment of the right lower lobe in a 22-year-old man. This tumor exhibited typical features of mucoepidermoid carcinoma with 3 cell types: squamoid cells, mucin-secreting cells and cells of intermediate type. These 3 types of cells organized into cysts, nests, glands and solid patterns. Specially, the inner lining cells of some glandular structures were uniform cuboidal and hobnail-like, similar to the alveolar epithelial cells. Immunohistochemistry staining revealed that the inner lining cells of glandular structures were positive for thyroid transcription factor-1 and surfactant protein-B, used as markers of alveolar epithelial cells, and were negative for p63. These findings for the first time demonstrated a rare alveolar epithelial differentiation of glandular inner lining cells in a mucoepidermoid carcinoma of the lung. Virtual Slides The virtual slide(s for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/7095988968057804

  18. 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.

  19. 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

    OpenAIRE

    Kode, Aruna; Yang, Se-Ran; Rahman, Irfan

    2006-01-01

    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 ...

  20. 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

    OpenAIRE

    Rahman Irfan; Yang Se-Ran; Kode Aruna

    2006-01-01

    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 o...

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

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    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.

  3. Macrophage-expressed IFN-β contributes to apoptotic alveolar epithelial cell injury in severe influenza virus pneumonia.

    Directory of Open Access Journals (Sweden)

    Katrin Högner

    2013-02-01

    Full Text Available Influenza viruses (IV cause pneumonia in humans with progression to lung failure and fatal outcome. Dysregulated release of cytokines including type I interferons (IFNs has been attributed a crucial role in immune-mediated pulmonary injury during severe IV infection. Using ex vivo and in vivo IV infection models, we demonstrate that alveolar macrophage (AM-expressed IFN-β significantly contributes to IV-induced alveolar epithelial cell (AEC injury by autocrine induction of the pro-apoptotic factor TNF-related apoptosis-inducing ligand (TRAIL. Of note, TRAIL was highly upregulated in and released from AM of patients with pandemic H1N1 IV-induced acute lung injury. Elucidating the cell-specific underlying signalling pathways revealed that IV infection induced IFN-β release in AM in a protein kinase R- (PKR- and NF-κB-dependent way. Bone marrow chimeric mice lacking these signalling mediators in resident and lung-recruited AM and mice subjected to alveolar neutralization of IFN-β and TRAIL displayed reduced alveolar epithelial cell apoptosis and attenuated lung injury during severe IV pneumonia. Together, we demonstrate that macrophage-released type I IFNs, apart from their well-known anti-viral properties, contribute to IV-induced AEC damage and lung injury by autocrine induction of the pro-apoptotic factor TRAIL. Our data suggest that therapeutic targeting of the macrophage IFN-β-TRAIL axis might represent a promising strategy to attenuate IV-induced acute lung injury.

  4. Prognostic value of immunohistochemical surfactant protein A expression in regenerative/hyperplastic alveolar epithelial cells in idiopathic interstitial pneumonias

    OpenAIRE

    Kajiki Akira; Fukushima Kazuo; Kawabata Masaharu; Wakamatsu Kentaro; Kitasato Yasuhiko; Nagata Nobuhiko; Kitahara Yoshinari; Watanabe Kentaro

    2011-01-01

    Abstract Background It is difficult to predict survival in patients with idiopathic pulmonary fibrosis. Recently, several proteins, such as surfactant protein (SP) and KL-6, have been reported to be useful biologic markers for prediction of prognosis for interstitial pneumonias. It is not clear whether there is any relationship between expression of these proteins in regenerative/hyperplastic alveolar epithelial cells and prognosis of idiopathic interstitial pneumonias (IIPs). Objectives This...

  5. Prognostic value of immunohistochemical surfactant protein A expression in regenerative/hyperplastic alveolar epithelial cells in idiopathic interstitial pneumonias

    OpenAIRE

    Nagata, Nobuhiko; Kitasato, Yasuhiko; Wakamatsu, Kentaro; Kawabata, Masaharu; Fukushima, Kazuo; Kajiki, Akira; Kitahara, Yoshinari; Watanabe, Kentaro

    2011-01-01

    Background It is difficult to predict survival in patients with idiopathic pulmonary fibrosis. Recently, several proteins, such as surfactant protein (SP) and KL-6, have been reported to be useful biologic markers for prediction of prognosis for interstitial pneumonias. It is not clear whether there is any relationship between expression of these proteins in regenerative/hyperplastic alveolar epithelial cells and prognosis of idiopathic interstitial pneumonias (IIPs). Objectives This study ai...

  6. Regulation of alveolar procoagulant activity and permeability in direct acute lung injury by lung epithelial tissue factor.

    Science.gov (United States)

    Shaver, Ciara M; Grove, Brandon S; Putz, Nathan D; Clune, Jennifer K; Lawson, William E; Carnahan, Robert H; Mackman, Nigel; Ware, Lorraine B; Bastarache, Julie A

    2015-11-01

    Tissue factor (TF) initiates the extrinsic coagulation cascade in response to tissue injury, leading to local fibrin deposition. Low levels of TF in mice are associated with increased severity of acute lung injury (ALI) after intratracheal LPS administration. However, the cellular sources of the TF required for protection from LPS-induced ALI remain unknown. In the current study, transgenic mice with cell-specific deletions of TF in the lung epithelium or myeloid cells were treated with intratracheal LPS to determine the cellular sources of TF important in direct ALI. Cell-specific deletion of TF in the lung epithelium reduced total lung TF expression to 39% of wild-type (WT) levels at baseline and to 29% of WT levels after intratracheal LPS. In contrast, there was no reduction of TF with myeloid cell TF deletion. Mice lacking myeloid cell TF did not differ from WT mice in coagulation, inflammation, permeability, or hemorrhage. However, mice lacking lung epithelial TF had increased tissue injury, impaired activation of coagulation in the airspace, disrupted alveolar permeability, and increased alveolar hemorrhage after intratracheal LPS. Deletion of epithelial TF did not affect alveolar permeability in an indirect model of ALI caused by systemic LPS infusion. These studies demonstrate that the lung epithelium is the primary source of TF in the lung, contributing 60-70% of total lung TF, and that lung epithelial, but not myeloid, TF may be protective in direct ALI. PMID:25884207

  7. Incorporation of tritiated thymidine by epithelial and interstitial cells in bronchiolar-alveolar regions of asbestos-exposed rats

    International Nuclear Information System (INIS)

    Inhaled asbestos causes progressive interstitial lung disease. The authors have performed a series of studies to elucidate early pathogenetic events at sites of fiber deposition in asbestos-exposed rats. This study reports that a single 5-hour exposure to chrysotile asbestos induces significant increases in incorporation of tritiated thymidine (3HTdR) into nuclei of epithelial and interstitial cells of bronchiolar-alveolar regions. No cell populations in air-exposed or carbonyl iron-exposed control animals exhibited more than 1% labeling at any point in time. Immediately after the 5-hour asbestos exposure, incorporation was normal. By 19 hours after exposure there was a significant increase in incorporation of 3HTdR, particularly by Type II epithelial cells of the first alveolar duct bifurcations. The greatest increase in degree of incorporation (up to 18-fold) was observed 24 hours after exposure, and increased percentages of 3HTdR-labeled cells were maintained through the 48 hours postexposure period. Normal labeling was present by 8 days after exposure, and this level remained through the 1-month period studied. This apparent mitogenic response correlates with increased numbers of bronchiolar-alveolar epithelial and interstitial cells demonstrated by ultrastructural morphometry in correlative studies. The authors speculate that the incorporation of 3HTdR could be induced by the direct effects of inhaled fibers or by mitogenic factors released from macrophages attracted to the inhaled asbestos

  8. Effect of P2X7 receptor knockout on AQP-5 expression of type I alveolar epithelial cells.

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    Georg Ebeling

    Full Text Available P2X7 receptors, ATP-gated cation channels, are specifically expressed in alveolar epithelial cells. The pathophysiological function of this lung cell type, except a recently reported putative involvement in surfactant secretion, is unknown. In addition, P2X7 receptor-deficient mice show reduced inflammation and lung fibrosis after exposure with bleomycin. To elucidate the role of the P2X7 receptor in alveolar epithelial type I cells we characterized the pulmonary phenotype of P2X7 receptor knockout mice by using immunohistochemistry, western blot analysis and real-time RT PCR. No pathomorphological signs of fibrosis were found. Results revealed, however, a remarkable loss of aquaporin-5 protein and mRNA in young knockout animals. Additional in vitro experiments with bleomycin treated precision cut lung slices showed a greater sensitivity of the P2X7 receptor knockout mice in terms of aquaporin-5 reduction as wild type animals. Finally, P2X7 receptor function was examined by using the alveolar epithelial cell lines E10 and MLE-12 for stimulation experiments with bleomycin. The in vitro activation of P2X7 receptor was connected with an increase of aquaporin-5, whereas the inhibition of the receptor with oxidized ATP resulted in down regulation of aquaporin-5. The early loss of aquaporin-5 which can be found in different pulmonary fibrosis models does not implicate a specific pathogenetic role during fibrogenesis.

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

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    Seok-Jo Kim

    2015-09-01

    Full Text Available 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.

  10. A novel aminothiazole KY-05009 with potential to inhibit Traf2- and Nck-interacting kinase (TNIK) attenuates TGF-β1-mediated epithelial-to-mesenchymal transition in human lung adenocarcinoma A549 cells.

    Science.gov (United States)

    Kim, Jiyeon; Moon, Seong-Hee; Kim, Bum Tae; Chae, Chong Hak; Lee, Joo Yun; Kim, Seong Hwan

    2014-01-01

    Transforming growth factor (TGF)-β triggers the epithelial-to-mesenchymal transition (EMT) of cancer cells via well-orchestrated crosstalk between Smad and non-Smad signaling pathways, including Wnt/β-catenin. Since EMT-induced motility and invasion play a critical role in cancer metastasis, EMT-related molecules are emerging as novel targets of anti-cancer therapies. Traf2- and Nck-interacting kinase (TNIK) has recently been considered as a first-in-class anti-cancer target molecule to regulate Wnt signaling pathway, but pharmacologic inhibition of its EMT activity has not yet been studied. Here, using 5-(4-methylbenzamido)-2-(phenylamino)thiazole-4-carboxamide (KY-05009) with TNIK-inhibitory activity, its efficacy to inhibit EMT in cancer cells was validated. The molecular docking/binding study revealed the binding of KY-05009 in the hinge region of TNIK, and the inhibitory activity of KY-05009 against TNIK was confirmed by an ATP competition assay (Ki, 100 nM). In A549 cells, KY-05009 significantly and strongly inhibited the TGF-β-activated EMT through the attenuation of Smad and non-Smad signaling pathways, including the Wnt, NF-κB, FAK-Src-paxillin-related focal adhesion, and MAP kinases (ERK and JNK) signaling pathways. Continuing efforts to identify and validate potential therapeutic targets associated with EMT, such as TNIK, provide new and improved therapies for treating and/or preventing EMT-based disorders, such as cancer metastasis and fibrosis. PMID:25337707

  11. A novel aminothiazole KY-05009 with potential to inhibit Traf2- and Nck-interacting kinase (TNIK attenuates TGF-β1-mediated epithelial-to-mesenchymal transition in human lung adenocarcinoma A549 cells.

    Directory of Open Access Journals (Sweden)

    Jiyeon Kim

    Full Text Available Transforming growth factor (TGF-β triggers the epithelial-to-mesenchymal transition (EMT of cancer cells via well-orchestrated crosstalk between Smad and non-Smad signaling pathways, including Wnt/β-catenin. Since EMT-induced motility and invasion play a critical role in cancer metastasis, EMT-related molecules are emerging as novel targets of anti-cancer therapies. Traf2- and Nck-interacting kinase (TNIK has recently been considered as a first-in-class anti-cancer target molecule to regulate Wnt signaling pathway, but pharmacologic inhibition of its EMT activity has not yet been studied. Here, using 5-(4-methylbenzamido-2-(phenylaminothiazole-4-carboxamide (KY-05009 with TNIK-inhibitory activity, its efficacy to inhibit EMT in cancer cells was validated. The molecular docking/binding study revealed the binding of KY-05009 in the hinge region of TNIK, and the inhibitory activity of KY-05009 against TNIK was confirmed by an ATP competition assay (Ki, 100 nM. In A549 cells, KY-05009 significantly and strongly inhibited the TGF-β-activated EMT through the attenuation of Smad and non-Smad signaling pathways, including the Wnt, NF-κB, FAK-Src-paxillin-related focal adhesion, and MAP kinases (ERK and JNK signaling pathways. Continuing efforts to identify and validate potential therapeutic targets associated with EMT, such as TNIK, provide new and improved therapies for treating and/or preventing EMT-based disorders, such as cancer metastasis and fibrosis.

  12. Transforming Growth Factor β1 Inhibits Cystic Fibrosis Transmembrane Conductance Regulator-dependent cAMP-stimulated Alveolar Epithelial Fluid Transport via a Phosphatidylinositol 3-Kinase-dependent Mechanism*

    OpenAIRE

    Roux, Jérémie; Carles, Michel; Koh, Hidefumi; Goolaerts, Arnaud; Ganter, Michael T.; Chesebro, Brian B; Howard, Marybeth; Houseman, Benjamin T.; Finkbeiner, Walter; Shokat, Kevan M.; Paquet, Agnès C.; Matthay, Michael A; Pittet, Jean-François

    2009-01-01

    Exogenous or endogenous β2-adrenergic receptor agonists enhance alveolar epithelial fluid transport via a cAMP-dependent mechanism that protects the lungs from alveolar flooding in acute lung injury. However, impaired alveolar fluid clearance is present in most of the patients with acute lung injury and is associated with increased mortality, although the mechanisms responsible for this inhibition of the alveolar epithelial fluid transport are not completely understood. Here, we found that tr...

  13. Combination of fluid and solid mechanical stresses contribute to cell death and detachment in a microfluidic alveolar model.

    Science.gov (United States)

    Douville, Nicholas J; Zamankhan, Parsa; Tung, Yi-Chung; Li, Ran; Vaughan, Benjamin L; Tai, Cheng-Feng; White, Joshua; Christensen, Paul J; Grotberg, James B; Takayama, Shuichi

    2011-02-21

    Studies using this micro-system demonstrated significant morphological differences between alveolar epithelial cells (transformed human alveolar epithelial cell line, A549 and primary murine alveolar epithelial cells, AECs) exposed to combination of solid mechanical and surface-tension stresses (cyclic propagation of air-liquid interface and wall stretch) compared to cell populations exposed solely to cyclic stretch. We have also measured significant differences in both cell death and cell detachment rates in cell monolayers experiencing combination of stresses. This research describes new tools for studying the combined effects of fluid mechanical and solid mechanical stress on alveolar cells. It also highlights the role that surface tension forces may play in the development of clinical pathology, especially under conditions of surfactant dysfunction. The results support the need for further research and improved understanding on techniques to reduce and eliminate fluid stresses in clinical settings. PMID:21152526

  14. Comparison of Cytotoxic Activity of L778123 as a Farnesyltranferase Inhibitor and Doxorubicin against A549 and HT-29 Cell Lines

    Directory of Open Access Journals (Sweden)

    Ali Abdollahi

    2013-02-01

    Full Text Available Purpose: Farnesyltransferase (FTase is a zinc-dependent enzyme that adds a farnesyl group to the Ras proteins. L778, 123 is a potent peptidomimetic imidazole-containing FTase inhibitor. Methods: L778123 was synthesized according to known methods and evaluated alone and in combination with doxorubicin against A549 (adenocarcinomic human alveolar basal epithelial cells and HT29 (human colonic adenocarcinoma cell lines by MTT assay. Results: L778123 showed weak cytotoxic activity with IC50 of 100 and 125 for A549 and HT-29 cell lines, respectively. The combination of doxorubicin and L778123 can decrease IC50 of doxorubicin in both cell lines significantly. Conclusion: It can be concluded that L778, 123 can be a good agent for combination therapy.

  15. Detection of alveolar epithelial injury by 99mTc-DTPA radioaerosol inhalation lung scan following blunt chest trauma

    International Nuclear Information System (INIS)

    DTPA clearance rate is a reliable index of alveolar epithelial permeability, and is a highly sensitive marker of pulmonary epithelial damage, even of mild degree. In this study, 99mTc-DTPA aerosol inhalation scintigraphy was used to assess the pulmonary epithelial membrane permeability and to investigate the possible application of this permeability value as an indicator of early alveolar or interstitial changes in patients with blunt chest trauma. A total of 26 patients was chest trauma (4 female, 22 male, 31-80 yrs, mean age; 53±13 yrs) who were referred to the emergency department in our hospital participated in this study. Technetium-99m diethylene triamine pentaacetic acid (DTPA) aerosol inhalation scintigraphy was performed on the first and thirtieth days after trauma. Clearance half times (T1/2) were calculated by placing a mono-exponential fit on the curves. Penetration index (PI) was calculated on the first-minute image. On the first day, mean T1/2 value of the whole lung was 63±19 minutes (min), and thirtieth day mean T1/2 value was 67±21 min. On the first day, mean PI values of the lung and 30th day mean PI value were 0.60±0.05, and 0.63 ±0.05, respectively. Significant changes were observed in radioaerosol clearance and penetration indices. Following chest trauma, clearance of 99mTc-DTPA increased owing to breakdown of the alveolar-capillary barrier. This increase in the epithelial permeability of the lung appears to be an early manifestation of lung disease that may lead to efficient therapy in the early phase. (author)

  16. The vitronectin RGD motif regulates TGF-β-induced alveolar epithelial cell apoptosis.

    Science.gov (United States)

    Wheaton, Amanda K; Velikoff, Miranda; Agarwal, Manisha; Loo, Tiffany T; Horowitz, Jeffrey C; Sisson, Thomas H; Kim, Kevin K

    2016-06-01

    Transforming growth factor-β (TGF-β) is a critical driver of acute lung injury and fibrosis. Injury leads to activation of TGF-β, which regulates changes in the cellular and matrix makeup of the lung during the repair and fibrosis phase. TGF-β can also initiate alveolar epithelial cell (AEC) apoptosis. Injury leads to destruction of the laminin-rich basement membrane, which is replaced by a provisional matrix composed of arginine-glycine-aspartate (RGD) motif-containing plasma matrix proteins, including vitronectin and fibronectin. To determine the role of specific matrix proteins on TGF-β-induced apoptosis, we studied primary AECs cultured on different matrix conditions and utilized mice with deletion of vitronectin (Vtn(-/-)) or mice in which the vitronectin RGD motif is mutated to nonintegrin-binding arginine-glycine-glutamate (RGE) (Vtn(RGE/RGE)). We found that AECs cultured on fibronectin and vitronectin or in wild-type mouse serum are resistant to TGF-β-induced apoptosis. In contrast, AECs cultured on laminin or in serum from Vtn(-/-) or Vtn(RGE/RGE) mice undergo robust TGF-β-induced apoptosis. Plasminogen activator inhibitor-1 (PAI-1) sensitizes AECs to greater apoptosis by disrupting AEC engagement to vitronectin. Inhibition of integrin-associated signaling proteins augments AEC apoptosis. Mice with transgenic deletion of PAI-1 have less apoptosis after bleomycin, but deletion of vitronectin or disruption of the vitronectin RGD motif reverses this protection, suggesting that the proapoptotic function of PAI-1 is mediated through vitronectin inhibition. Collectively, these data suggest that integrin-matrix signaling is an important regulator of TGF-β-mediated AEC apoptosis and that PAI-1 functions as a natural regulator of this interaction. PMID:27106291

  17. Canonical pathways, networks and transcriptional factor regulation by clinical strains of Mycobacterium tuberculosis in pulmonary alveolar epithelial cells.

    Science.gov (United States)

    Mvubu, Nontobeko E; Pillay, Balakrishna; Gamieldien, Junaid; Bishai, William; Pillay, Manormoney

    2016-03-01

    Limited knowledge exists on pathways, networks and transcriptional factors regulated within epithelial cells by diverse Mycobacterium tuberculosis genotypes. This study aimed to elucidate these mechanisms induced in A549 epithelial cells by dominant clinical strains in KwaZulu-Natal, South Africa. RNA for sequencing was extracted from epithelial cells at 48 h post-infection with 5 strains at a multiplicity of infection of approximately 10:1. Bioinformatics analysis performed with the RNA-Seq Tuxedo pipeline identified differentially expressed genes. Changes in pathways, networks and transcriptional factors were identified using Ingenuity Pathway Analysis (IPA). The interferon signalling and hepatic fibrosis/hepatic stellate cell activation pathways were among the top 5 canonical pathways in all strains. Hierarchical clustering for enrichment of cholesterol biosynthesis and immune associated pathways revealed similar patterns for Beijing and Unique; F15/LAM4/KZN and F11; and, F28 and H37Rv strains, respectively. However, the induction of top scoring networks varied among the strains. Among the transcriptional factors, only EHL, IRF7, PML, STAT1, STAT2 and VDR were induced by all clinical strains. Activation of the different pathways, networks and transcriptional factors revealed in the current study may be an underlying mechanism that results in the differential host response by clinical strains of M. tuberculosis. PMID:26980499

  18. Linking progression of fibrotic lung remodeling and ultrastructural alterations of alveolar epithelial type II cells in the amiodarone mouse model.

    Science.gov (United States)

    Birkelbach, Bastian; Lutz, Dennis; Ruppert, Clemens; Henneke, Ingrid; Lopez-Rodriguez, Elena; Günther, Andreas; Ochs, Matthias; Mahavadi, Poornima; Knudsen, Lars

    2015-07-01

    Chronic injury of alveolar epithelial type II cells (AE2 cells) represents a key event in the development of lung fibrosis in animal models and in humans, such as idiopathic pulmonary fibrosis (IPF). Intratracheal delivery of amiodarone to mice results in a profound injury and macroautophagy-dependent apoptosis of AE2 cells. Increased autophagy manifested in AE2 cells by disturbances of the intracellular surfactant. Hence, we hypothesized that ultrastructural alterations of the intracellular surfactant pool are signs of epithelial stress correlating with the severity of fibrotic remodeling. With the use of design-based stereology, the amiodarone model of pulmonary fibrosis in mice was characterized at the light and ultrastructural level during progression. Mean volume of AE2 cells, volume of lamellar bodies per AE2 cell, and mean size of lamellar bodies were correlated to structural parameters reflecting severity of fibrosis like collagen content. Within 2 wk amiodarone leads to an increase in septal wall thickness and a decrease in alveolar numbers due to irreversible alveolar collapse associated with alveolar surfactant dysfunction. Progressive hypertrophy of AE2 cells and increase in mean individual size and total volume of lamellar bodies per AE2 cell were observed. A high positive correlation of these AE2 cell-related ultrastructural changes and the deposition of collagen fibrils within septal walls were established. Qualitatively, similar alterations could be found in IPF samples with mild to moderate fibrosis. We conclude that ultrastructural alterations of AE2 cells including the surfactant system are tightly correlated with the progression of fibrotic remodeling. PMID:25957292

  19. Open reading frame 3 of genotype 1 hepatitis E virus inhibits nuclear factor-κappa B signaling induced by tumor necrosis factor-α in human A549 lung epithelial cells.

    Directory of Open Access Journals (Sweden)

    Jian Xu

    Full Text Available Hepatitis E virus (HEV is one of the primary causative agents of acute hepatitis, and represents a major cause of severe public health problems in developing countries. The pathogenesis of HEV is not well characterized, however, primarily due to the lack of well-defined cell and animal models. Here, we investigated the effects of genotype 1 HEV open reading frame 3 (ORF3 on TNF-α-induced nucleus factor-κappa B (NF-κB signaling. Human lung epithelial cells (A549 were transiently transfected with ORF3 containing plasmids. These cells were then stimulated with TNF-α and the nucleus translocation of the p65 NF-κB subunit was assessed using western blot and laser confocal microscopy. DNA-binding activity of p65 was also examined using electrophoretic mobility shift assay (EMSA, and the suppression of NF-κB target genes were detected using real-time RT-PCR and ELISA. These results enabled us to identify the decreased phosphorylation levels of IKBα. We focused on the gene of negative regulation of NF-κB, represented by TNF-α-induced protein 3 (TNFAIP3, also known as A20. Reducing the levels of A20 with siRNAs significantly enhances luciferase activation of NF-κB. Furthermore, HEV ORF3 regulated A20 primarily via activating transcription factor 6 (ATF6, involved in unfolded protein response (UPR, resulting in the degradation or inactivation of the receptor interacting protein 1 (RIP1, a major upstream activator of IKB kinase compounds (IKKs. Consequently, the phosphorylation of IKBα and the nucleus translocation of p65 are blocked, which contributes to diminished NF-κB DNA-binding activation and NF-κB-dependent gene expression. The findings suggest that genotype 1 HEV, through ORF3, may transiently activate NF-κB through UPR in early stage, and subsequently inhibit TNF-α-induced NF-κB signaling in late phase so as to create a favorable virus replication environment.

  20. Macrophages programmed by apoptotic cells inhibit epithelial-mesenchymal transition in lung alveolar epithelial cells via PGE2, PGD2, and HGF

    Science.gov (United States)

    Yoon, Young-So; Lee, Ye-Ji; Choi, Youn-Hee; Park, Young Mi; Kang, Jihee Lee

    2016-01-01

    Apoptotic cell clearance results in the release of growth factors and the action of signaling molecules involved in tissue homeostasis maintenance. Here, we investigated whether and how macrophages programmed by apoptotic cells inhibit the TGF-β1-induced Epithelial-mesenchymal transition (EMT) process in lung alveolar epithelial cells. Treatment with conditioned medium derived from macrophages exposed to apoptotic cells, but not viable or necrotic cells, inhibited TGF-β1-induced EMT, including loss of E-cadherin, synthesis of N-cadherin and α-smooth muscle actin, and induction of EMT-activating transcription factors, such as Snail1/2, Zeb1/2, and Twist1. Exposure of macrophages to cyclooxygenase (COX-2) inhibitors (NS-398 and COX-2 siRNA) or RhoA/Rho kinase inhibitors (Y-27632 and RhoA siRNA) and LA-4 cells to antagonists of prostaglandin E2 (PGE2) receptor (EP4 [AH-23848]), PGD2 receptors (DP1 [BW-A868C] and DP2 [BAY-u3405]), or the hepatocyte growth factor (HGF) receptor c-Met (PHA-665752), reversed EMT inhibition by the conditioned medium. Additionally, we found that apoptotic cell instillation inhibited bleomycin-mediated EMT in primary mouse alveolar type II epithelial cells in vivo. Our data suggest a new model for epithelial cell homeostasis, by which the anti-EMT programming of macrophages by apoptotic cells may control the progressive fibrotic reaction via the production of potent paracrine EMT inhibitors. PMID:26875548

  1. Macrophages programmed by apoptotic cells inhibit epithelial-mesenchymal transition in lung alveolar epithelial cells via PGE2, PGD2, and HGF.

    Science.gov (United States)

    Yoon, Young-So; Lee, Ye-Ji; Choi, Youn-Hee; Park, Young Mi; Kang, Jihee Lee

    2016-01-01

    Apoptotic cell clearance results in the release of growth factors and the action of signaling molecules involved in tissue homeostasis maintenance. Here, we investigated whether and how macrophages programmed by apoptotic cells inhibit the TGF-β1-induced Epithelial-mesenchymal transition (EMT) process in lung alveolar epithelial cells. Treatment with conditioned medium derived from macrophages exposed to apoptotic cells, but not viable or necrotic cells, inhibited TGF-β1-induced EMT, including loss of E-cadherin, synthesis of N-cadherin and α-smooth muscle actin, and induction of EMT-activating transcription factors, such as Snail1/2, Zeb1/2, and Twist1. Exposure of macrophages to cyclooxygenase (COX-2) inhibitors (NS-398 and COX-2 siRNA) or RhoA/Rho kinase inhibitors (Y-27632 and RhoA siRNA) and LA-4 cells to antagonists of prostaglandin E2 (PGE2) receptor (EP4 [AH-23848]), PGD2 receptors (DP1 [BW-A868C] and DP2 [BAY-u3405]), or the hepatocyte growth factor (HGF) receptor c-Met (PHA-665752), reversed EMT inhibition by the conditioned medium. Additionally, we found that apoptotic cell instillation inhibited bleomycin-mediated EMT in primary mouse alveolar type II epithelial cells in vivo. Our data suggest a new model for epithelial cell homeostasis, by which the anti-EMT programming of macrophages by apoptotic cells may control the progressive fibrotic reaction via the production of potent paracrine EMT inhibitors. PMID:26875548

  2. Caracterización química del material particulado PM 10 de la zona urbana de Cuenca- Ecuador e investigación de su genotoxicidad e inducción de estrés oxidativo en células epiteliales alveolares A549

    Directory of Open Access Journals (Sweden)

    AL Astudillo Alemán

    2015-01-01

    Full Text Available La exposición a partículas amb ientales es un factor de riesgo que ocasiona daños en la s alud humana, como trastornos respiratorios, cardiovasculares y cáncer. La toxicidad y el efe cto inflamatorio de estas partículas están relacionados con su tam año y características químicas. El objetivo de este estudio fue deter minar las características químicas de la fracción hidrosoluble de Mat erial Particulado PM 10 , recolectado en tres sitios de monitoreo de la ciudad de Cuenca-Ecuador, y eval uar su actividad genotóxica e inducción de especies r eactivas de oxígeno (ROS en la línea c elular epitelial alveolar humana A-549. Las muestras fueron recolectad as empleando un equipo de bajo volum en. Las concentraciones de material particulado determinadas por análisis gravimétrico superaron en los tres puntos de estudio los 50 μg/m 3 , límite estipulado en la Legislación Ecuatoriana. En la caracterización de la solución acuosa se determinó la presencia de aniones (Cl - , NO 3 - , SO 4 -2 y metales pesados (Cr, Fe, Ni, Zn, Cu, Mn, mediante técnicas de cromatografía iónica y espe ctroscopía de absorción atómica respectivamente; SO 4 -2 y Fe presentaron las mayores concentraciones. Las células A-549 fueron expuestas a diferen tes concentraciones (0,82; 1,25 y 1,63 m3/mL de la fracción hidrosoluble de PM 10 , con la finalidad de observar el posible efecto genotóxico mediante el ensayo del cometa y la inducción de especies reactivas de oxígeno mediante fluorimetría. Finalmente se determinó que los extractos acuosolubles de PM 10 inducen daño celular bajo (tipo I, e incrementan la producción de ROS en cé lulas A-549, lo que pudiera constituir un riesgo en la salud de la po blación expuesta.

  3. Comparative toxicity of 24 manufactured nanoparticles in human alveolar epithelial and macrophage cell lines

    OpenAIRE

    Boczkowski Jorge; Maillot-Marechal Emmanuelle; Dupont Aurélie; Rogerieux Françoise; Geys Jorina; Lanone Sophie; Lacroix Ghislaine; Hoet Peter

    2009-01-01

    Abstract Background A critical issue with nanomaterials is the clear understanding of their potential toxicity. We evaluated the toxic effect of 24 nanoparticles of similar equivalent spherical diameter and various elemental compositions on 2 human pulmonary cell lines: A549 and THP-1. A secondary aim was to elaborate a generic experimental set-up that would allow the rapid screening of cytotoxic effect of nanoparticles. We therefore compared 2 cytotoxicity assays (MTT and Neutral Red) and an...

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

    International Nuclear Information System (INIS)

    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 99mTc 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 99mTc 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, the99mTc 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)

  5. Interactions of Francisella tularensis with Alveolar Type II Epithelial Cells and the Murine Respiratory Epithelium.

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    Matthew Faron

    Full Text Available Francisella tularensis is classified as a Tier 1 select agent by the CDC due to its low infectious dose and the possibility that the organism can be used as a bioweapon. The low dose of infection suggests that Francisella is unusually efficient at evading host defenses. Although ~50 cfu are necessary to cause human respiratory infection, the early interactions of virulent Francisella with the lung environment are not well understood. To provide additional insights into these interactions during early Francisella infection of mice, we performed TEM analysis on mouse lungs infected with F. tularensis strains Schu S4, LVS and the O-antigen mutant Schu S4 waaY::TrgTn. For all three strains, the majority of the bacteria that we could detect were observed within alveolar type II epithelial cells at 16 hours post infection. Although there were no detectable differences in the amount of bacteria within an infected cell between the three strains, there was a significant increase in the amount of cellular debris observed in the air spaces of the lungs in the Schu S4 waaY::TrgTn mutant compared to either the Schu S4 or LVS strain. We also studied the interactions of Francisella strains with human AT-II cells in vitro by characterizing the ability of these three strains to invade and replicate within these cells. Gentamicin assay and confocal microscopy both confirmed that F. tularensis Schu S4 replicated robustly within these cells while F. tularensis LVS displayed significantly lower levels of growth over 24 hours, although the strain was able to enter these cells at about the same level as Schu S4 (1 organism per cell, as determined by confocal imaging. The Schu S4 waaY::TrgTn mutant that we have previously described as attenuated for growth in macrophages and mouse virulence displayed interesting properties as well. This mutant induced significant airway inflammation (cell debris and had an attenuated growth phenotype in the human AT-II cells. These

  6. Exacerbation of benzene pneumotoxicity in connexin 32 knockout mice: enhanced proliferation of CYP2E1-immunoreactive alveolar epithelial cells

    International Nuclear Information System (INIS)

    The pulmonary pathogenesis triggered by benzene exposure was studied. Since the role of the connexin 32 (Cx32) gap junction protein in mouse pulmonary pathogenesis has been suggested, in the present study, we explored a possible role of Cx32 in benzene-induced pulmonary pathogenesis using the wild-type (WT) and Cx32 knockout (KO) mice. The mice were exposed to 300 ppm benzene by inhalation for 6 h per day, 5 days per week for a total of 26 weeks, and then sacrificed to evaluate the pneumotoxicity or allowed to live out their life span to evaluate the reversibility of the lesions and tumor incidence. Our results clearly revealed exacerbated pneumotoxicity in the benzene-exposed Cx32 KO mice, characterized by diffuse granulomatous interstitial pneumonia, markedly increased mucin secretion of bronchial/bronchiolar and alveolar epithelial cells, and hyperplastic alveolar epithelial cells positive for CYP2E1. But the results did not indicate any enhancement of pulmonary tumorigenesis in the Cx32 KO mice though the number of animals was small

  7. Elastolytic activity and alveolar epithelial type-1 cell damage after chronic LPS inhalation: Effects of dexamethasone and rolipram

    International Nuclear Information System (INIS)

    This study investigated whether a correlation between leukocyte-derived elastolytic activity, alveolar epithelial type-1 cell damage, and leukocyte infiltration of the airways existed in guinea-pigs chronically exposed to inhaled lipopolysaccharide (LPS). The airway pathology of this model, notably the neutrophilia, resembles chronic obstructive pulmonary disease (COPD). The effect of the corticosteroid, dexamethasone, or the phosphodiesterase-4 (PDE4)-inhibitor, rolipram, on these features was studied. Conscious guinea-pigs were exposed for 1 h to single or repeated (nine) doses of LPS (30 μg ml-1). Dexamethasone (20 mg kg-1, ip) or rolipram (1 mg kg-1, ip) was administered 24 and 0.5 h before the first exposure and daily thereafter. Bronchoalveolar lavage fluid (BALF) was removed and elastolytic activity determined as the elastase-like release of Congo Red from impregnated elastin. The presence of the specific epithelial cell type-1 protein (40-42 kDa) RT140 in BALF was identified by Western blotting using a rat monoclonal antibody and semi-quantified by dot-blot analysis. The antibody was found to identify guinea-pig RT140. BALF inflammatory cells, particularly neutrophils and macrophages, and elastolytic activity were increased in chronic LPS-exposed guinea-pigs, the latter by 90%. Chronic LPS exposure also increased (10.5-fold) RT140 levels, indicating significant alveolar epithelial type-1 cell damage. Dexamethasone or rolipram treatment reduced the influx of inflammatory cells, the elastolytic activity (by 40% and 38%, respectively), and RT140 levels (by 50% and 57%, respectively). In conclusion, chronic LPS-exposed guinea-pigs, like COPD, exhibit elastolytic lung damage. This was prevented by a PDE4 inhibitor and supports their development for suppressing this leukocyte-mediated pathology

  8. Inhibitory effect of receptor for advanced glycation end products (RAGE) on the TGF-β-induced alveolar epithelial to mesenchymal transition

    OpenAIRE

    Song, Jeong Sup; Kang, Chun Mi; Park, Chan Kwon; Yoon, Hyung Kyu; Lee, Sook Young; Ahn, Joong Hyun; Moon, Hwa-Sik

    2011-01-01

    Idiopathic pulmonary fibrosis (IPF) is a lethal parenchymal lung disease characterized by myofibroblast proliferation. Alveolar epithelial cells (AECs) are thought to produce myofibroblasts through the epithelial to mesenchymal transition (EMT). Receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily of cell surface receptors whose activation is associated with renal fibrosis during diabetes and liver fibrosis. RAGE is expressed at low basal levels in...

  9. Activated Alveolar Epithelial Cells Initiate Fibrosis through Secretion of Mesenchymal Proteins

    OpenAIRE

    Yang, Jibing; Wheeler, Sarah E.; Velikoff, Miranda; Kleaveland, Kathryn R.; LaFemina, Michael J.; Frank, James A.; Chapman, Harold A.; Christensen, Paul J; Kim, Kevin K.

    2013-01-01

    Fibrosis is characterized by accumulation of activated fibroblasts and pathological deposition of fibrillar collagens. Activated fibroblasts overexpress matrix proteins and release factors that promote further recruitment of activated fibroblasts, leading to progressive fibrosis. The contribution of epithelial cells to this process remains unknown. Epithelium-directed injury may lead to activation of epithelial cells with phenotypes and functions similar to activated fibroblasts. Prior report...

  10. AIRWAY EPITHELIAL CELL RESPONSE TO HUMAN METAPNEUMOVIRUS INFECTION

    Science.gov (United States)

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

    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 immuno-modulatory mediators. PMID:17655903

  11. Human neutrophil elastase regulates the expression and secretion of elafin (elastase-specific inhibitor) in type II alveolar epithelial cells.

    Science.gov (United States)

    Reid, P T; Marsden, M E; Cunningham, G A; Haslett, C; Sallenave, J M

    1999-08-20

    Elafin is a low molecular weight antiproteinase believed to be important in the regulation of elastase mediated tissue damage. The expression of elafin is known to be regulated by proinflammatory cytokines such as interleukin-1 beta and tumour necrosis factor but little was known regarding the effect of human neutrophil elastase (HNE). Employing a chloramphenicol acetyltransferase reporter construct of the human elafin gene, reverse transcription PCR from total cellular RNA and ELISA techniques, we have examined the effect of human neutrophil elastase on the transcription and secretion of human elafin in the pulmonary epithelial A549 cell line. Stimulation with HNE at concentrations of 10(-10) and 10(-11) M resulted in a significant upregulation of elafin promoter activity. Similarly, transcription of the endogenous human elafin gene was upregulated with HNE concentrations ranging from 10(-10) to 10(-12) M. In addition, we demonstrate that stimulation with HNE at concentrations ranging from 10(-9) and 10(-12) M resulted in a significant reduction in the secreted elafin protein as measured in the cell supernatant. These results provide further evidence for a role of elafin in the regulation of HNE driven proteolysis of the extracellular matrix. PMID:10486558

  12. Electron microscope study on the relationship between macrophages of the alevolar space and spheroid alveolar epithelial cells on mice after injection of squid-ink (sepia-melanin solution into the trachea

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    Suwa,Kiichi

    1977-02-01

    Full Text Available The relationship between alveolar macrophages and spheroid alveolar epithelial cells was studied with the electron microscope after injection of squid-ink solution into the trachea of the mouse. At 20 hours after injection of squid-ink solution slight degeneration was evident in alveolar macrophages with sepia-melanin particles being phagocytized with partial digestion by lysosmes. Furthermore, hardly any changes were seen in mitochondria and inclusion bodies of the spheroid alveolar epithelial cells. In contrast, at one week after injection of squid-ink solution, almost all alveolar macrophages were degenerated with destruction of the ectoplasm in which the ingested sepia-melanin particles were digested by lysosomes into fine particles, and the mitochondria of spheroid alveolar epithelial cells were degenerated and the inclusion bodies were hardly formed. At three weeks after injection of squid-ink solution, alveolar macrophages as well as speroid alveolar epithelial cells showed almost complete recovery of functional structure. As the phagocyte in the alveolar space, neutrophile leucocytes were also observed in addition to the so-called alveolar macrophage.

  13. Intratracheal Administration of Recombinant Human Keratinocyte Growth Factor Promotes Alveolar Epithelial Cell Proliferation during Compensatory Lung Growth in Rat

    International Nuclear Information System (INIS)

    Keratinocyte growth factor (KGF) is considered to be one of the most important mitogens for lung epithelial cells. The objectives of this study were to confirm the effectiveness of intratracheal injection of recombinant human KGF (rhKGF) during compensatory lung growth and to optimize the instillation protocol. Here, trilobectomy in adult rat was performed, followed by intratracheal rhKGF instillation with low (0.4 mg/kg) and high (4 mg/kg) doses at various time-points. The proliferation of alveolar cells was assessed by the immunostaining for proliferating cell nuclear antigen (PCNA) in the residual lung. We also investigated other immunohistochemical parameters such as KGF, KGF receptor and surfactant protein A as well as terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. Consequently, intratracheal single injection of rhKGF in high dose group significantly increased PCNA labeling index (LI) of alveolar cells in the remaining lung. Surprisingly, there was no difference in PCNA LI between low and high doses of rhKGF with daily injection, and PCNA LI reached a plateau level with 2 days-consecutive administration (about 60%). Our results indicate that even at low dose, daily intratracheal injection is effective to maintain high proliferative states during the early phase of compensatory lung growth

  14. Detection of alveolar epithelial injury by Tc-99m DTPA radioaerosol inhalation lung scan in rheumatoid arthritis patients

    International Nuclear Information System (INIS)

    Rheumatoid arthritis (RA) is a systemic autoimmune disorder primarily involving the joints. Lung alterations in RA may be primary or secondary to pharmacological treatments and may involve the alveoli, interstitium, airways and/or pleura. Technetium-99 m diethylenetriaminepentaacetic acid (Tc-99m DTPA) aerosol inhalation scintigraphy is a sensitive and noninvasive test commonly employed to assess pulmonary epithelial membrane permeability. The purpose of this study was to investigate the changes of pulmonary alveolar epithelial permeability in patients with RA, to determine the relationship between the clearance rate of Tc-99m DTPA and pulmonary function test (PFT) results, and to determine the relationship between the clearance rate of Tc-99m DTPA and clinical parameters of disease. Twenty-five patients with RA but without lung alterations were included in the study. The patients were 22 females, and 3 males; mean age 53.6±8.7 years. Technetium-99m DTPA aerosol inhalation scintigraphy was performed on the study and healthy control groups. Clearance half times (T1/2) were calculated by placing a mono-exponential fit on the curves. Penetration index (PI) was calculated on the first-minute image. There were no significant differences in the mean T1/2 or mean PI values between the RA patients and control subjects. No correlation was found between the mean T1/2 values of Tc-99m DTPA clearance and activity of RA, clinical values, or the spirometric measurements except FEV1/FVC and functional status in RA patients (p=0.02, p=0.01, respectively). However, a weak correlation was found between duration of disease and T1/2 values of Tc-99m DTPA clearance (p=0.006). PI values tended to correlate with FEF25-75, although, this was not statistically significant (p=0.057). This study shows that no changes occur in alveolar-capillary permeability in RA patients without lung alterations. (author)

  15. Isolation of alveolar epithelial type II progenitor cells from adult human lungs

    OpenAIRE

    Fujino, Naoya; Kubo, Hiroshi; Suzuki, Takaya; Ota, Chiharu; Hegab, Ahmed E.; He, Mei; Suzuki, Satoshi; Suzuki, Takashi; Yamada, Mitsuhiro; Kondo, Takashi; Kato, Hidemasa; Yamaya, Mutsuo

    2010-01-01

    Resident stem/progenitor cells in the lung are important for tissue homeostasis and repair. However, a progenitor population for alveolar type II (ATII) cells in adult human lungs has not been identified. The aim of this study is to isolate progenitor cells from adult human lungs with the ability to differentiate into ATII cells. We isolated colony-forming cells that had the capability for self-renewal and the potential to generate ATII cells in vitro. These undifferentiated progenitor cells ...

  16. 石棉暴露下内质网应激在肺泡上皮细胞凋亡中的作用机制%THE MECHANISM OF ENDOPLASMIC RETICULUM STRESS INDUCING ALVEOLAR EPI-THELIAL CELLS APOPTOSIS UNDERLYING ASBESTOS EXPOSURE

    Institute of Scientific and Technical Information of China (English)

    周煦; 刘刚

    2014-01-01

    Objective To investigate the role of endoplasmic reticulum stress ( ERS) in asbestos-induced Alveolar epithelial cell apoptosis.Methods A549 cells were treated with asbestos to observe the expression of ERS proteins and apop-tosis gene by immunofluorescence staining and western blotting.Results Under asbestos exposure, the expression of Bip, GRP94, IRE-1α, BAX and BAK are up-regulated.There is a positive correlativity of the expression of proteins to the ex-posure time of asbestos.Conclusion Endoplasmic reticulum stress takes part in asbestos-induced apoptosis.%目的:探讨石棉暴露下内质网应激在细胞凋亡中的作用机制。方法应用石棉处理A549细胞,免疫荧光染色免疫法和免疫印迹法观察内质网应激( ERS)相关蛋白与促凋亡基因的变化。结果石棉暴露下,ERS相关蛋白Bip、IRE-1α和GRP94,以及促凋亡基因BAX、BAK蛋白表达均上调,且与石棉暴露时间呈正相关。结论内质网应激参与了石棉诱导的细胞凋亡。

  17. 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.

  18. IL-8 inhibits cAMP-stimulated alveolar epithelial fluid transport via a GRK2/PI3K-dependent mechanism

    Science.gov (United States)

    Roux, Jérémie; McNicholas, Carmel M.; Carles, Michel; Goolaerts, Arnaud; Houseman, Benjamin T.; Dickinson, Dale A.; Iles, Karen E.; Ware, Lorraine B.; Matthay, Michael A.; Pittet, Jean-François

    2013-01-01

    Patients with acute lung injury (ALI) who retain maximal alveolar fluid clearance (AFC) have better clinical outcomes. Experimental and small clinical studies have shown that β2-adrenergic receptor (β2AR) agonists enhance AFC via a cAMP-dependent mechanism. However, two multicenter phase 3 clinical trials failed to show that β2AR agonists provide a survival advantage in patients with ALI. We hypothesized that IL-8, an important mediator of ALI, directly antagonizes the alveolar epithelial response to β2AR agonists. Short-circuit current and whole-cell patch-clamping experiments revealed that IL-8 or its rat analog CINC-1 decreases by 50% β2AR agonist-stimulated vectorial Cl− and net fluid transport across rat and human alveolar epithelial type II cells via a reduction in the cystic fibrosis transmembrane conductance regulator activity and biosynthesis. This reduction was mediated by heterologous β2AR desensitization and down-regulation (50%) via the G-protein-coupled receptor kinase 2 (GRK2)/PI3K signaling pathway. Inhibition of CINC-1 restored β2AR agonist-stimulated AFC in an experimental model of ALI in rats. Finally, consistent with the experimental results, high pulmonary edema fluid levels of IL-8 (>4000 pg/ml) were associated with impaired AFC in patients with ALI. These results demonstrate a novel role for IL-8 in inhibiting β2AR agonist-stimulated alveolar epithelial fluid transport via GRK2/PI3K-dependent mechanisms.—Roux, J., McNicholas, C. M., Carles, M., Goolaerts, A., Houseman, B. T., Dickinson, D. A., Iles, K. E., Ware, L. B., Matthay, M. A., Pittet, J.-F. IL-8 inhibits cAMP-stimulated alveolar epithelial fluid transport via a GRK2/PI3K-dependent mechanism. PMID:23221335

  19. [Grape seed proanthocyanidins inhibits the invasion and migration of A549 lung cancer cells].

    Science.gov (United States)

    Zhou, Yehan; Ye, Xiufeng; Shi, Yao; Wang, Ke; Wan, Dan

    2016-02-01

    Objective To explore the effect of grape seed proanthocyanidins (GSPs) on the invasion and migration of A549 lung cancer cells and the underlying mechanism. Methods Trypan blue dye exclusion assay was used to determine the cytotoxic effect of varying doses of GSPs on the BEAS-2B normal human pulmonary epithelial cells. After treated with 0, 10, 20, 40, 80 μg/mL GSP, the proliferation of A549 cells was detected by MTT assay; the invasion and migration of A549 cells were determined by Transwell(TM) assay and scratch wound assay, respectively. The levels of epithelial growth factor receptor (EGFR), E-cadherin, N-cadherin in A549 cells treated with GSPs were detected by Western blotting. Results (0-40) μg/mL GSPs had no significant toxic effect on BEAS-2B cells, while 80 μg/mL GSPs had significant cytotoxicity to BEAS-2B cells. The proliferation of A549 cells was significantly inhibited within limited dosage in a dose-dependent manner, and the abilities of invasion and migration of A549 cells were also inhibited. Western blotting showed that the expression of EGFR and N-cadherin decreased, while E-cadherin increased after GSPs treatment. Conclusion GSPs could inhibit the abilities of proliferation, invasion and migration of A549 cells, which might be related to the dow-regulation of EGFR and N-cadherin and the up-regulation of E-cadherin. PMID:26927375

  20. Comparative toxicity of 24 manufactured nanoparticles in human alveolar epithelial and macrophage cell lines

    Directory of Open Access Journals (Sweden)

    Boczkowski Jorge

    2009-04-01

    Full Text Available Abstract Background A critical issue with nanomaterials is the clear understanding of their potential toxicity. We evaluated the toxic effect of 24 nanoparticles of similar equivalent spherical diameter and various elemental compositions on 2 human pulmonary cell lines: A549 and THP-1. A secondary aim was to elaborate a generic experimental set-up that would allow the rapid screening of cytotoxic effect of nanoparticles. We therefore compared 2 cytotoxicity assays (MTT and Neutral Red and analyzed 2 time points (3 and 24 hours for each cell type and nanoparticle. When possible, TC50 (Toxic Concentration 50 i.e. nanoparticle concentration inducing 50% cell mortality was calculated. Results The use of MTT assay on THP-1 cells exposed for 24 hours appears to be the most sensitive experimental design to assess the cytotoxic effect of one nanoparticle. With this experimental set-up, Copper- and Zinc-based nanoparticles appear to be the most toxic. Titania, Alumina, Ceria and Zirconia-based nanoparticles show moderate toxicity, and no toxicity was observed for Tungsten Carbide. No correlation between cytotoxicity and equivalent spherical diameter or specific surface area was found. Conclusion Our study clearly highlights the difference of sensitivity between cell types and cytotoxicity assays that has to be carefully taken into account when assessing nanoparticles toxicity.

  1. Cadmium exposure down-regulates 8-oxoguanine DNA glycosylase expression in rat lung and alveolar epithelial cells

    International Nuclear Information System (INIS)

    The current study tested the hypothesis that the pulmonary carcinogenic potential of cadmium (Cd) is related to its ability to inhibit the expression (mRNA and protein) and activity of 8-oxoguanine-DNA glycosylase (OGG1), a base excision repair (BER) enzyme that functions to preferentially excise pre-mutagenic 7,8-dihydro-8-oxoguanine (8-oxoG) from DNA. We demonstrate that a single Cd aerosol exposure of adult male Lewis rats causes time- and dose-dependent down-regulation in the pulmonary levels of rOGG1 mRNA and OGG1 protein, quantified by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assays and western analyses, respectively. Immunohistochemical studies confirmed that Cd inhalation reduces the relative amount of OGG1 in lungs of exposed animals without altering its over-all distribution within the lung, which appears to be more prominent within the alveolar epithelium. In agreement with our in vivo studies, we show that OGG1 expression is also attenuated in alveolar epithelial cell cultures exposed to CdCl2 either acutely or by repeated passaging in Cd-containing medium. The effects caused by Cd were observed in cells that show no loss in viability, as assessed by colony forming ability, the MTT assay, and propidium iodide membrane permeability studies. Nuclear extracts prepared from Cd-treated cells also exhibit a reduction in the ability to nick a synthetic oligonucleotide containing 8-oxoG. We conclude from these studies that Cd causes suppression of OGG1 in the lung and that this mechanism may, in part, play a role in the Cd carcinogenic process

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

    OpenAIRE

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

    2014-01-01

    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...

  3. A Novel Approach for Ovine Primary Alveolar Epithelial Type II Cell Isolation and Culture from Fresh and Cryopreserved Tissue Obtained from Premature and Juvenile Animals

    OpenAIRE

    Marcinkiewicz, Mariola M.; Baker, Sandy T.; Jichuan Wu; Terrence L Hubert; Wolfson, Marla R.

    2016-01-01

    The in vivo ovine model provides a clinically relevant platform to study cardiopulmonary mechanisms and treatments of disease; however, a robust ovine primary alveolar epithelial type II (ATII) cell culture model is lacking. The objective of this study was to develop and optimize ovine lung tissue cryopreservation and primary ATII cell culture methodologies for the purposes of dissecting mechanisms at the cellular level to elucidate responses observed in vivo. To address this, we established ...

  4. Increased ectodomain shedding of cell adhesion molecule 1 as a cause of type II alveolar epithelial cell apoptosis in patients with idiopathic interstitial pneumonia

    OpenAIRE

    Yoneshige, Azusa; Hagiyama, Man; Inoue, Takao; Mimae, Takahiro; Kato, Takashi; Okada, Morihito; Enoki, Eisuke; Ito, Akihiko

    2015-01-01

    Background Lung alveolar epithelial cell (AEC) apoptosis has attracted attention as an early pathogenic event in the development of idiopathic interstitial pneumonia (IIP); however, the causative mechanism remains unclear. Cell adhesion molecule 1 (CADM1) is an AEC adhesion molecule in the immunoglobulin superfamily. It generates a membrane-associated C-terminal fragment, αCTF, through protease-mediated ectodomain shedding, termed α-shedding. Increased CADM1 α-shedding contributes to AEC apop...

  5. Alveolar epithelial permeability in patients with primary spontaneous pneumothorax as determined by Tc-99m DTPA aerosol scintigraphy

    International Nuclear Information System (INIS)

    Primary spontaneous pneumothorax (PSP) occurs subsequent to a disruption in the continuity of visceral pleura and escape of air into the pleural space. The cause of PSP is most often the rupture of subpleural blebs or bullae. It is usually difficult to detect evidence of pulmonary pathology. The purposes of the present study were to investigate the changes of pulmonary alveolar epithelial permeability in patients with PSP as determined by Tc-99m DTPA aerosol lung scintigraphy, to assess whether or not some differences exist between apical and basal parts of the lungs, and to determine the relationship between the clearance rate of Tc-99m DTPA and the pulmonary function test (PFT) results, the recurrence rate of PSP, and the percentage of pneumothorax in affected lung. Thirteen PSP patients (two females, 11 males; mean age 32.5±11.8 years) with normal chest X-ray were studied. Thirteen healthy non-smoking volunteers (1 female, 12 males; mean age, 35.8±10 years) were selected as a control group. Tc-99m DTPA aerosol lung scintigraphy and PFT were performed in all patients and controls. Clearance rates (%/min) of Tc-99m DTPA aerosol in right and left lung field, and apical and basal parts of each lung were calculated from dynamic images for 15 min. There was no significant difference (p>0.05) between patients and controls, or between apical and basal parts of each lung. No correlation was found between the clearance rate of Tc-99m DTPA and PFT results, the recurrence rate of PSP, or the percentage of pneumothorax. This study demonstrates that pulmonary epithelial permeability is not altered in PSP patients; the clearance rate of Tc-99m DTPA shows no difference between apical and basal parts of each lung. (author)

  6. 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

  7. Assessment of alveolar epithelial permeability in Behcet's disease with 99mTc-DTPA aerosol scintigraphy

    International Nuclear Information System (INIS)

    Behcet's disease (BD) is a multisystem disorder characterized by vasculitis, and consists of a triad of recurrent ulcers of the oral and genital mucosa with relapsing uveitis. The prevalance of pulmonary involvement varies in the range of 1-10% in various studies and its complications are severe and life threatening. In this study, we investigated the changes of pulmonary epithelial permeability of patients with BD using technetium-99m diethylene triamine penta-acetic acid (99mTc-DTPA) aerosol scintigraphy, so as to begin the therapy regimen as soon as possible. Twenty-one nonsmoking patients with BD (8 women, 13 men; mean age 38.67±8.86 years) and 15 healthy volunteer nonsmoking controls (8 women, 7 men; mean age 50.87±12.45 years) underwent 99mTc-DTPA aerosol inhalation scintigraphy and pulmonary function tests (PFTs). Subjects inhaled 1480 MBq of 99mTc-DTPA for 4 min in the supine position. Scintigraphic data were recorded dynamically (1 frame/min) in the posterior projection on a 64 x 64 matrix for a 30-min period using a double-headed gamma camera (Infinia, GE, Tirat Hacarmel, Israel) equipped with a low-energy all-purpose parallel hole collimator. Half time of 99mTc-DTPA clearance (T1/2) was calculated by placing a mono-exponential fit on the curves. Penetration index (PI) was also calculated by dividing the peripheral total counts by the sum of the peripheral and central total counts on the first minute image, in order to quantify the distribution of the inhaled aerosol. The clearance half time of 99mTc-DTPA radioaerosols in the BD patients (24.81±6.22 min) was faster than in the normal control group (46.53±22.41 min) (P=0.004). There was also a significant difference between PI of the patients with BD (0.15±0.03) and that of the controls (0.21±0.06) (P=0.002). No correlation was found between the mean T1/2 values of 99mTc-DTPA clearance or the spirometric measurements in the BD patients. Penetration indices were not correlated with PET in the BD

  8. 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.

  9. Hypoxia inducible factor 3α plays a critical role in alveolarization and distal epithelial cell differentiation during mouse lung development.

    Directory of Open Access Journals (Sweden)

    Yadi Huang

    Full Text Available Lung development occurs under relative hypoxia and the most important oxygen-sensitive response pathway is driven by Hypoxia Inducible Factors (HIF. HIFs are heterodimeric transcription factors of an oxygen-sensitive subunit, HIFα, and a constitutively expressed subunit, HIF1β. HIF1α and HIF2α, encoded by two separate genes, contribute to the activation of hypoxia inducible genes. A third HIFα gene, HIF3α, is subject to alternative promoter usage and splicing, leading to three major isoforms, HIF3α, NEPAS and IPAS. HIF3α gene products add to the complexity of the hypoxia response as they function as dominant negative inhibitors (IPAS or weak transcriptional activators (HIF3α/NEPAS. Previously, we and others have shown the importance of the Hif1α and Hif2α factors in lung development, and here we investigated the role of Hif3α during pulmonary development. Therefore, HIF3α was conditionally expressed in airway epithelial cells during gestation and although HIF3α transgenic mice were born alive and appeared normal, their lungs showed clear abnormalities, including a post-pseudoglandular branching defect and a decreased number of alveoli. The HIF3α expressing lungs displayed reduced numbers of Clara cells, alveolar epithelial type I and type II cells. As a result of HIF3α expression, the level of Hif2α was reduced, but that of Hif1α was not affected. Two regulatory genes, Rarβ, involved in alveologenesis, and Foxp2, a transcriptional repressor of the Clara cell specific Ccsp gene, were significantly upregulated in the HIF3α expressing lungs. In addition, aberrant basal cells were observed distally as determined by the expression of Sox2 and p63. We show that Hif3α binds a conserved HRE site in the Sox2 promoter and weakly transactivated a reporter construct containing the Sox2 promoter region. Moreover, Hif3α affected the expression of genes not typically involved in the hypoxia response, providing evidence for a novel

  10. Macrophage control of phagocytosed mycobacteria is increased by factors secreted by alveolar epithelial cells through nitric oxide independent mechanisms.

    Directory of Open Access Journals (Sweden)

    Dagbjort H Petursdottir

    Full Text Available Tissue-resident macrophages are heterogeneous with tissue-specific and niche-specific functions. Thus, simplified models of macrophage activation do not explain the extent of heterogeneity seen in vivo. We focus here on the respiratory tract and ask whether factors secreted by alveolar epithelial cells (AEC can influence the functionality of resident pulmonary macrophages (PuM. We have previously reported that factors secreted by AEC increase control of intracellular growth of BCG in macrophages. In the current study, we also aimed to investigate possible mechanisms by which AEC-derived factors increase intracellular control of BCG in both primary murine interstitial macrophages, and bone marrow-derived macrophages and characterize further the effect of these factors on macrophage differentiation. We show that; a in contrast to other macrophage types, IFN-γ did not increase intracellular growth control of Mycobacterium bovis, Bacillus Calmette-Guérin (BCG by interstitial pulmonary macrophages although the same macrophages could be activated by factors secreted by AEC; b the lack of response of pulmonary macrophages to IFN-γ was apparently regulated by suppressor of cytokine signaling (SOCS1; c AEC-derived factors did not induce pro-inflammatory pathways induced by IFN-γ e.g. expression of inducible nitric oxide synthase (iNOS, secretion of nitric oxide (NO, or IL-12, d in contrast to IFN-γ, intracellular bacterial destruction induced by AEC-derived factors was not dependent on iNOS transcription and NO production. Collectively, our data show that PuM were restricted in inflammatory responses mediated by IFN-γ through SOCS1 and that factors secreted by AEC- enhanced the microbicidal capacities of macrophages by iNOS independent mechanisms.

  11. cDNA microarray analysis of rat alveolar epithelial cells following exposure to organic extract of diesel exhaust particles

    International Nuclear Information System (INIS)

    Diesel exhaust particles (DEP) induce pulmonary diseases including asthma and chronic bronchitis. Comprehensive evaluation is required to know the mechanisms underlying the effects of air pollutants including DEP on lung diseases. Using a cDNA microarray, we examined changes in gene expression in SV40T2 cells, a rat alveolar type II epithelial cell line, following exposure to an organic extract of DEP. We identified candidate sensitive genes that were up- or down-regulated in response to DEP. The cDNA microarray analysis revealed that a 6-h exposure to the DEP extract (30 μg/ml) increased (>2-fold) the expression of 51 genes associated with drug metabolism, antioxidation, cell cycle/proliferation/apoptosis, coagulation/fibrinolysis, and expressed sequence tags (ESTs), and decreased (<0.5-fold) that of 20 genes. In the present study, heme oxygenase (HO)-1, an antioxidative enzyme, showed the maximum increase in gene expression; and type II transglutaminase (TGM-2), a regulator of coagulation, showed the most prominent decrease among the genes. We confirmed the change in the HO-1 protein level by Western blot analysis and that in the enzyme activity of TGM-2. The organic extract of DEP increased the expression of HO-1 protein and decreased the enzyme activity of TGM-2. Furthermore, these effects of DEP on either HO-1 or TGM-2 were reduced by N-acetyl-L-cysteine (NAC), thus suggesting that oxidative stress caused by this organic fraction of DEP may have induced these cellular responses. Therefore, an increase in HO-1 and a decrease in TGM-2 might be good markers of the biological response to organic compounds of airborne particulate substances

  12. Assessment of alveolar epithelial permeability in progressive systemic sclerosis (PSS) using 99mTc-DTPA (diethylene triamine penta acetate) aerosol inhalation

    International Nuclear Information System (INIS)

    To evaluate alveolar epithelial damage in PSS, we studied pulmonary epithelial permeability by measuring the clearance of inhaled 99mTc-DTPA aerosol and performing thin slice CT scan, pulmonary function tests and right heart catheterization in 28 patients with PSS. The 99mTc-DTPA clearance rate (kep value) in PSS was greater than in 11 non-smoking normal subjects (18.2±7.63x10-3/min vs. 9.12±0.77x10-3/min, p2. In contrast, the kep value showed significant correlations with %DLco (diffusing capacity for carbon monoxide), extent of interstitial lesions evaluated by CT scan (CT score), and mean pulmonary artery pressure. On the other hand, the kep value was high in some patients with normal CT scan and normal %DLco. These findings indicate that pulmonary interstitial lesions in PSS are accompanied by alveolar epithelial damage, and that the clearance of 99mTc-DTPA may be an early predictor of interstitial change. (author)

  13. Microrheology of human lung epithelial cells measured by atomic force microscopy

    OpenAIRE

    Alcaraz, Jordi; Buscemi, Lara; Grabulosa, Mireia; Trepat, Xavier; Fabry, Ben; Farré, Ramon; Navajas, Daniel

    2003-01-01

    Lung epithelial cells are subjected to large cyclic forces from breathing. However, their response to dynamic stresses is poorly defined. We measured the complex shear modulus (G*(ω)) of human alveolar (A549) and bronchial (BEAS-2B) epithelial cells over three frequency decades (0.1–100 Hz) and at different loading forces (0.1–0.9 nN) with atomic force microscopy. G*(ω) was computed by correcting force-indentation oscillatory data for the tip-cell contact geometry and for the hydrodynamic vis...

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

    Directory of Open Access Journals (Sweden)

    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.

  15. Streptococcus pneumoniae induces autophagy through the inhibition of the PI3K-I/Akt/mTOR pathway and ROS hypergeneration in A549 cells.

    Directory of Open Access Journals (Sweden)

    Pu Li

    Full Text Available The present study focused on the action mechanism of S. pneumoniae (Sp in inducing autophagy in human alveolar epithelial cells. Sp, a gram-positive extracellular bacterium, activates autophagy with considerably increased microtuble-associated protein light chain 3 (LC3 punctation in A549 cells. The accumulation of typical autophagosomes and conjugation of LC3 to phosphatidylethanolamine were observed in Sp-infected cells as an indication of autophagy. Using the pneumolysin (PLY mutant, we successfully demonstrated that PLY is involved in initiating autophagy without affecting the expression levels of PI3K-III and Beclin1. PLY-mediated autophagy depends on the inhibition of the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR pathway. Furthermore, Sp could also lead to the reactive oxygen species (ROS hypergeneration in A549 cells. Taken together, Sp infection-induced autophagy is PLY-mediated through ROS hypergeneration and mTOR inhibition. PI3K-I and rapamycin (autophagy inducers enhanced bacterial clearance, whereas wortmannin (autophagy inhibitor and acetylcysteine (ROS inhibitor reduced intracellular bacteria clearance. Thus, Sp-induced autophagy represents a host-protective mechanism, providing new insight into the pathogenesis of respiratory tract Sp infection.

  16. Effects of Tumor Suppressor Gene TCF21 on the Proliferation, Migration and Apoptosis of A549 Cells

    Directory of Open Access Journals (Sweden)

    Song HU

    2014-04-01

    Full Text Available Background and objective TCF21, a newly discovered gene, exhibits tumor suppressor function in a variety of tumors. This study aims to observe the effects of TCF21 on the proliferation, apoptosis and migration of A549 human lung adenocarcinoma epithelial cells. Methods TCF21 was overexpressed in A549 cells via lentiviral transfection. Fluorescence-based quantitative polymerase chain reaction and Western blot analysis were used to analyze the expression of the target gene. Transwell, proliferation assay, and flow cytometry were applied to detect the effect of TCF21 overexpression on the migration, proliferation, and apoptosis of A549 cells after transfection. Results The proliferation and migration of A549 cells were inhibited, and the apoptotic rate was increased by overexpressing TCF21. Conclusion The tumor suppressor gene, TCF21, significantly inhibits the proliferation and migration, as well as facilitates early apoptosis of A549 cells.

  17. 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

    BACKGROUND: The major surface glycoprotein (MSG) is an abundant, immunogenic glycoprotein located on the surface of Pneumocystis carinii. Little is known about the proinflammatory effects of MSG. DESIGN: We have investigated the effect of human MSG on the secretion of the chemokines interleukin 8...... in response to MSG stimulation occurred by 4 h and persisted throughout 8 h of stimulation. CONCLUSION: These findings suggest that MSG can alter alveolar epithelial cytokine release and may be capable of modulating the local inflammatory response in this manner....

  18. Alteration in Intrapulmonary Pharmacokinetics of Aerosolized Model Compounds Due to Disruption of the Alveolar Epithelial Barriers Following Bleomycin-Induced Pulmonary Fibrosis in Rats.

    Science.gov (United States)

    Togami, Kohei; Chono, Sumio; Tada, Hitoshi

    2016-03-01

    Idiopathic pulmonary fibrosis is a lethal lung disease that is characterized by the accumulation of extracellular matrix and a change in lung structure. In this study, intrapulmonary pharmacokinetics of aerosolized model compounds were evaluated using rats with bleomycin-induced pulmonary fibrosis. Aerosol formulations of indocyanine green, 6-carboxyfluorescein (6-CF), and fluorescein isothiocyanate dextrans (FD; 4.4, 10, 70, and 250 kDa) were administered to rat lungs using a MicroSprayer. Indocyanine green fluorescence signals were significantly weaker in fibrotic lungs than in control lungs and 6-CF and FD concentrations in the plasma of pulmonary fibrotic animals were markedly higher than in the plasma of control animals. Moreover, disrupted epithelial tight junctions, including claudins-1, -3, and -5, were observed in pulmonary fibrotic lesions using immunofluorescence microscopy. In addition, destruction of tight junctions on model alveolar epithelial cells (NCI-H441) by transforming growth factor-β1 treatment enhanced the permeability of 6-CF and FDs through NCI-H441 cell monolayers. These results indicate that aerosolized drugs are easily distributed into the plasma after leakage through damaged tight junctions of alveolar epithelium. Therefore, the development of delivery systems for anti-fibrotic agents to improve intrapulmonary pharmacokinetics may be necessary for effective idiopathic pulmonary fibrosis therapy. PMID:26886341

  19. Modeling pulmonary alveolar microlithiasis by epithelial deletion of the Npt2b sodium phosphate cotransporter reveals putative biomarkers and strategies for treatment.

    Science.gov (United States)

    Saito, Atsushi; Nikolaidis, Nikolaos M; Amlal, Hassane; Uehara, Yasuaki; Gardner, Jason C; LaSance, Kathleen; Pitstick, Lori B; Bridges, James P; Wikenheiser-Brokamp, Kathryn A; McGraw, Dennis W; Woods, Jason C; Sabbagh, Yves; Schiavi, Susan C; Altinişik, Göksel; Jakopović, Marko; Inoue, Yoshikazu; McCormack, Francis X

    2015-11-11

    Pulmonary alveolar microlithiasis (PAM) is a rare, autosomal recessive lung disorder associated with progressive accumulation of calcium phosphate microliths. Inactivating mutations in SLC34A2, which encodes the NPT2b sodium-dependent phosphate cotransporter, has been proposed as a cause of PAM. We show that epithelial deletion of Npt2b in mice results in a progressive pulmonary process characterized by diffuse alveolar microlith accumulation, radiographic opacification, restrictive physiology, inflammation, fibrosis, and an unexpected alveolar phospholipidosis. Cytokine and surfactant protein elevations in the alveolar lavage and serum of PAM mice and confirmed in serum from PAM patients identify serum MCP-1 (monocyte chemotactic protein 1) and SP-D (surfactant protein D) as potential biomarkers. Microliths introduced by adoptive transfer into the lungs of wild-type mice produce marked macrophage-rich inflammation and elevation of serum MCP-1 that peaks at 1 week and resolves at 1 month, concomitant with clearance of stones. Microliths isolated by bronchoalveolar lavage readily dissolve in EDTA, and therapeutic whole-lung EDTA lavage reduces the burden of stones in the lungs. A low-phosphate diet prevents microlith formation in young animals and reduces lung injury on the basis of reduction in serum SP-D. The burden of pulmonary calcium deposits in established PAM is also diminished within 4 weeks by a low-phosphate diet challenge. These data support a causative role for Npt2b in the pathogenesis of PAM and the use of the PAM mouse model as a preclinical platform for the development of biomarkers and therapeutic strategies. PMID:26560359

  20. Inhibitory Effect of Cantharidin on Proliferation of A549 Cells

    Institute of Scientific and Technical Information of China (English)

    WANG Xiao-hua; YIN Yuan-qin; SUI Cheng-guang; MENG Fan-dong; MA Ping; JIANG You-hong

    2007-01-01

    Objective: To study the inhibition of Cantharidin against the proliferation of human lung cancer A549 cells and its mechanism. Methods: MTT assay was employed to determine the inhibition of Cantharidin against proliferation of A549 cells and flow Cytometry was applied to analyze A549 cell cycle and the effect of Cantharidin on cell cycle. Results: Cantharidin showed inhibition against the proliferation of A549 cells, and the inhibition was mediated by blocking A549 cell cycle at G2/M phase significantly. Conclusion: Cantharidin exhibits inhibition against the proliferation of human lung cancer A549 cells.

  1. Adiponectin ameliorates the apoptotic effects of paraquat on alveolar type II cells via improvements in mitochondrial function

    Science.gov (United States)

    HE, YARONG; ZOU, LIQUN; ZHOU, YAXIONG; HU, HAI; YAO, RONG; JIANG, YAOWEN; LAU, WAYNE BOND; YUAN, TUN; HUANG, WEN; ZENG, ZHI; CAO, YU

    2016-01-01

    Previous studies have demonstrated that excessive reactive oxygen/nitrogen species (ROS/RNS)-induced apoptosis is an important feature of the injury to the lung epithelium in paraquat (PQ) poisoning. However the precise mechanisms of PQ-induced dysfunction of the mitochondria, where ROS/RNS are predominantly produced, remain to be fully elucidated. Whether globular adiponectin (gAd), a potent molecule protective to mitochondria, regulates the mitochondrial function of alveolar type II cells to reduce PQ-induced ROS/RNS production remains to be investigated. The current study aimed to investigate the precise mechanisms of PQ poisoning in the mitochondria of alveolar type II cells, and to elucidate the role of gAd in protecting against PQ-induced lung epithelium injury. Therefore, lung epithelial injury was induced by PQ co-culture of alveolar type II A549 cells for 24 h. gAd was administrated to and removed from the injured cells in after 24 h. PQ was observed to reduce cell viability and increase apoptosis by ~1.5 fold in A549 cells. The oxidative/nitrative stress, resulting from ROS/RNS and disordered mitochondrial function were evidenced by increased O2−., NO production and reduced mitochondrial membrane potential (ΔΨ), adenosine 5′-triphosphate (ATP) content in PQ-poisoned A549 cells. gAd treatment significantly reversed the PQ-induced cell injury and mitochondrial dysfunction in A549 cells. The protective effects of gAd were partly abrogated by an adenosine 5′-monophosphate-activated protein kinase (AMPK) inhibitor, compound C. The results suggest that reduced ΔΨ and ATP content may result in PQ-induced mitochondrial dysfunction of the lung epithelium, which constitutes a novel mechanism for gAd exerting pulmonary protection against PQ poisoning via AMPK activation. PMID:27220901

  2. Mycoplasma bovis isolates recovered from cattle and bison (Bison bison) show differential in vitro effects on PBMC proliferation, alveolar macrophage apoptosis and invasion of epithelial and immune cells.

    Science.gov (United States)

    Suleman, Muhammad; Prysliak, Tracy; Clarke, Kyle; Burrage, Pat; Windeyer, Claire; Perez-Casal, Jose

    2016-04-15

    In the last few years, several outbreaks of pneumonia, systemically disseminated infection, and high mortality associated with Mycoplasma bovis (M. bovis) in North American bison (Bison bison) have been reported in Alberta, Manitoba, Saskatchewan, Nebraska, New Mexico, Montana, North Dakota, and Kansas. M. bovis causes Chronic Pneumonia and Polyarthritis Syndrome (CPPS) in young, stressed calves in intensively-managed feedlots. M. bovis is not classified as a primary pathogen in cattle, but in bison it appears to be a primary causative agent with rapid progression of disease with fatal outcomes and an average 20% mature herd mortality. Thus, there is a possibility that M. bovis isolates from cattle and bison differ in their pathogenicity. Hence, we decided to compare selected cattle isolates to several bison isolates obtained from clinical cases. We show differences in modulation of PBMC proliferation, invasion of trachea and lung epithelial cells, along with modulation of apoptosis and survival in alveolar macrophages. We concluded that some bison isolates showed less inhibition of cattle and bison PBMC proliferation, were not able to suppress alveolar macrophage apoptosis as efficiently as cattle isolates, and were more or less invasive than the cattle isolate in various cells. These findings provide evidence about the differential properties of M. bovis isolated from the two species and has helped in the selection of bison isolates for genomic sequencing. PMID:27016754

  3. Regulation and function of the two-pore-domain (K2P) potassium channel Trek-1 in alveolar epithelial cells

    OpenAIRE

    Schwingshackl, Andreas; Teng, Bin; Ghosh, Manik; West, Alina Nico; Makena, Patrudu; Gorantla, Vijay; Sinclair, Scott E.; Waters, Christopher M.

    2011-01-01

    Hyperoxia can lead to a myriad of deleterious effects in the lung including epithelial damage and diffuse inflammation. The specific mechanisms by which hyperoxia promotes these pathological changes are not completely understood. Activation of ion channels has been proposed as one of the mechanisms required for cell activation and mediator secretion. The two-pore-domain K+ channel (K2P) Trek-1 has recently been described in lung epithelial cells, but its function remains elusive. In this stud...

  4. Fas Activation in Alveolar Epithelial Cells Induces KC (CXCL1) Release by a MyD88-Dependent Mechanism

    OpenAIRE

    Farnand, Alex W.; Eastman, Alison J.; Herrero, Raquel; Hanson, Josiah F.; Mongovin, Steve; Altemeier, William A.; Matute-Bello, Gustavo

    2011-01-01

    Activation of the Fas/Fas ligand (FasL) system is associated with activation of apoptotic and proinflammatory pathways that lead to the development of acute lung injury. Previous studies in chimeric mice and macrophage-depleted mice suggested that the main effector cell in Fas-mediated lung injury is not a myeloid cell, but likely an epithelial cell. The goal of this study was to determine whether epithelial cells release proinflammatory cytokines after Fas activation, and to identify the rel...

  5. In vivo autoradiographic demonstration of β-adrenergic binding sites in adult rat type II alveolar epithelial cells

    International Nuclear Information System (INIS)

    Adult male rats were injected intravenously with the muscarinic binding probe 3H-Quinuclidinyl benzilate (QNB) or the β-adrenergic probe 3H-dihydroalprenolol (DHA). Other rats were pre-treated with an intraperitoneal injection of a 500-fold excess of L-isoproterenol prior to the DHA. Light microscopic autoradiography of 0.5 μm sections of lung from the QNB group demonstrated very little labelling even after 6 months of exposure. In constrast, trachealis smooth muscle from these animals contained substantial labelling. Autoradiographs of lung from rats injected with DHA demonstrated labelling which was well localized over alveolar septa and concentrated over the cytoplasm of type II cells. Quantitative analysis of labelling in the DHA groups indicated a significant reduction of labelling in animals treated with L-isoproterenol prior to DHA, in both the alveolar parenchyma in general and over type II cells. The results of this study provide morphologic evidence for the uptake and specific binding of β-adrenergic antagonists by the adult lung in vivo, while failing to demonstrate similar binding of a muscarinic probe. In addition, the results demonstrate specific β-adrenergic receptors on type II cells in vivo and substantiate the view of a direct effect of β-adrenergic agonists on alveolar type II cells

  6. Internalization of SiO₂ nanoparticles by alveolar macrophages and lung epithelial cells and its modulation by the lung surfactant substitute Curosurf.

    Science.gov (United States)

    Vranic, Sandra; Garcia-Verdugo, Ignacio; Darnis, Cécile; Sallenave, Jean-Michel; Boggetto, Nicole; Marano, Francelyne; Boland, Sonja; Baeza-Squiban, Armelle

    2013-05-01

    Because of an increasing exposure to environmental and occupational nanoparticles (NPs), the potential risk of these materials for human health should be better assessed. Since one of the main routes of entry of NPs is via the lungs, it is of paramount importance to further characterize their impact on the respiratory system. Here, we have studied the uptake of fluorescently labeled SiO₂ NPs (50 and 100 nm) by epithelial cells (NCI-H292) and alveolar macrophages (MHS) in the presence or absence of pulmonary surfactant. The quantification of NP uptake was performed by measuring cell-associated fluorescence using flow cytometry and spectrometric techniques in order to identify the most suitable methodology. Internalization was shown to be time and dose dependent, and differences in terms of uptake were noted between epithelial cells and macrophages. In the light of our observations, we conclude that flow cytometry is a more reliable technique for the study of NP internalization, and importantly, that the hydrophobic fraction of lung surfactant is critical for downregulating NP uptake in both cell types. PMID:23288678

  7. Long-term exposure of A549 cells to titanium dioxide nanoparticles induces DNA damage and sensitizes cells towards genotoxic agents.

    Science.gov (United States)

    Armand, Lucie; Tarantini, Adeline; Beal, David; Biola-Clier, Mathilde; Bobyk, Laure; Sorieul, Sephanie; Pernet-Gallay, Karin; Marie-Desvergne, Caroline; Lynch, Iseult; Herlin-Boime, Nathalie; Carriere, Marie

    2016-09-01

    Titanium dioxide nanoparticles (TiO2-NPs) are one of the most produced NPs in the world. Their toxicity has been studied for a decade using acute exposure scenarios, i.e. high exposure concentrations and short exposure times. In the present study, we evaluated their genotoxic impact using long-term and low concentration exposure conditions. A549 alveolar epithelial cells were continuously exposed to 1-50 μg/mL TiO2-NPs, 86% anatase/14% rutile, 24 ± 6 nm average primary diameter, for up to two months. Their cytotoxicity, oxidative potential and intracellular accumulation were evaluated using MTT assay and reactive oxygen species measurement, transmission electron microscopy observation, micro-particle-induced X-ray emission and inductively-coupled plasma mass spectroscopy. Genotoxic impact was assessed using alkaline and Fpg-modified comet assay, immunostaining of 53BP1 foci and the cytokinesis-blocked micronucleus assay. Finally, we evaluated the impact of a subsequent exposure of these cells to the alkylating agent methyl methanesulfonate. We demonstrate that long-term exposure to TiO2-NPs does not affect cell viability but causes DNA damage, particularly oxidative damage to DNA and increased 53BP1 foci counts, correlated with increased intracellular accumulation of NPs. In addition, exposure over 2 months causes cellular responses suggestive of adaptation, characterized by decreased proliferation rate and stabilization of TiO2-NP intracellular accumulation, as well as sensitization to MMS. Taken together, these data underline the genotoxic impact and sensitization effect of long-term exposure of lung alveolar epithelial cells to low levels of TiO2-NPs. PMID:26785166

  8. Nemo-like kinase regulates the expression of vascular endothelial growth factor (VEGF) in alveolar epithelial cells.

    Science.gov (United States)

    Ke, Hengning; Masoumi, Katarzyna Chmielarska; Ahlqvist, Kristofer; Seckl, Michael J; Rydell-Törmänen, Kristina; Massoumi, Ramin

    2016-01-01

    The canonical Wnt signaling can be silenced either through β-catenin-mediated ubiquitination and degradation or through phosphorylation of Tcf and Lef by nemo-like kinase (NLK). In the present study, we generated NLK deficient animals and found that these mice become cyanotic shortly before death because of lung maturation defects. NLK-/- lungs exhibited smaller and compressed alveoli and the mesenchyme remained thick and hyperplastic. This phenotype was caused by epithelial activation of vascular endothelial growth factor (VEGF) via recruitment of Lef1 to the promoter of VEGF. Elevated expression of VEGF and activation of the VEGF receptor through phosphorylation promoted an increase in the proliferation rate of epithelial and endothelial cells. In summary, our study identifies NLK as a novel signaling molecule for proper lung development through the interconnection between epithelial and endothelial cells during lung morphogenesis. PMID:27035511

  9. Deleted in malignant brain tumors 1 (DMBT1) elicits increased VEGF and decreased IL-6 production in type II lung epithelial cells

    DEFF Research Database (Denmark)

    Müller, Hanna; Nagel, Christian; Weiss, Christel;

    2015-01-01

    between VEGF and IL-6 levels to DMBT1 expression in the lungs of preterm and term infants and in lung epithelial cells in vitro. METHODS: We examined by ELISA VEGF levels in 120 tracheal aspirates of 57 preterm and term infants and tested for correlation with different perinatal factors as well as with...... DMBT1 levels. To examine the effect of DMBT1 on VEGF and IL-6 expression we compared type II lung epithelial A549 cells stably transfected with a DMBT1 expression plasmid (DMBT1+ cells) to A549 cells stably transfected with an empty expression plasmid (DMBT1- cells). The concentrations of VEGF and IL-6...... that DMBT1 promotes VEGF and suppresses IL-6 production in alveolar tissues, which could point to DMBT1 having a possible role in the transition from inflammation to regeneration and being a potentially useful clinical marker....

  10. Recruited alveolar macrophages, in response to airway epithelial-derived monocyte chemoattractant protein 1/CCl2, regulate airway inflammation and remodeling in allergic asthma.

    Science.gov (United States)

    Lee, Yong Gyu; Jeong, Jong Jin; Nyenhuis, Sharmilee; Berdyshev, Evgeny; Chung, Sangwoon; Ranjan, Ravi; Karpurapu, Manjula; Deng, Jing; Qian, Feng; Kelly, Elizabeth A B; Jarjour, Nizar N; Ackerman, Steven J; Natarajan, Viswanathan; Christman, John W; Park, Gye Young

    2015-06-01

    Although alveolar macrophages (AMs) from patients with asthma are known to be functionally different from those of healthy individuals, the mechanism by which this transformation occurs has not been fully elucidated in asthma. The goal of this study was to define the mechanisms that control AM phenotypic and functional transformation in response to acute allergic airway inflammation. The phenotype and functional characteristics of AMs obtained from human subjects with asthma after subsegmental bronchoprovocation with allergen was studied. Using macrophage-depleted mice, the role and trafficking of AM populations was determined using an acute allergic lung inflammation model. We observed that depletion of AMs in a mouse allergic asthma model attenuates Th2-type allergic lung inflammation and its consequent airway remodeling. In both human and mouse, endobronchial challenge with allergen induced a marked increase in monocyte chemotactic proteins (MCPs) in bronchoalveolar fluid, concomitant with the rapid appearance of a monocyte-derived population of AMs. Furthermore, airway allergen challenge of allergic subjects with mild asthma skewed the pattern of AM gene expression toward high levels of the receptor for MCP1 (CCR2/MCP1R) and expression of M2 phenotypic proteins, whereas most proinflammatory genes were highly suppressed. CCL2/MCP-1 gene expression was prominent in bronchial epithelial cells in a mouse allergic asthma model, and in vitro studies indicate that bronchial epithelial cells produced abundant MCP-1 in response to house dust mite allergen. Thus, our study indicates that bronchial allergen challenge induces the recruitment of blood monocytes along a chemotactic gradient generated by allergen-exposed bronchial epithelial cells. PMID:25360868

  11. 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.

  12. Preliminary Proteomic Analysis of A549 Cells Infected with Avian Influenza Virus H7N9 and Influenza A Virus H1N1

    Science.gov (United States)

    Ding, Xiaoman; Lu, Jiahai; Yu, Ruoxi; Wang, Xin; Wang, Ting; Dong, Fangyuan; Peng, Bo; Wu, Weihua; Liu, Hui; Geng, Yijie; Zhang, Renli; Ma, Hanwu; Cheng, Jinquan; Yu, Muhua; Fang, Shisong

    2016-01-01

    A newly emerged H7N9 influenza virus poses high risk to human beings. However, the pathogenic mechanism of the virus remains unclear. The temporal response of primary human alveolar adenocarcinoma epithelial cells (A549) infected with H7N9 influenza virus and H1N1 influenza A virus (H1N1, pdm09) were evaluated using the proteomics approaches (2D-DIGE combined with MALDI-TOF-MS/MS) at 24, 48 and 72 hours post of the infection (hpi). There were 11, 12 and 33 proteins with significant different expressions (P<0.05) at 24, 48 and 72hpi, especially F-actin-capping protein subunit alpha-1 (CAPZA1), Ornithine aminotransferase (OAT), Poly(rC)-binding protein 1 (PCBP1), Eukaryotic translation initiation factor 5A-1 (EIF5A) and Platelet-activating factor acetylhydrolaseⅠb subunit beta (PAFAH1B2) were validated by western-blot analysis. The functional analysis revealed that the differential proteins in A549 cells involved in regulating cytopathic effect. Among them, the down-regulation of CAPZA1, OAT, PCBP1, EIF5A are related to the death of cells infected by H7N9 influenza virus. This is the first time show that the down-regulation of PAFAH1B2 is related to the later clinical symptoms of patients infected by H7N9 influenza virus. These findings may improve our understanding of pathogenic mechanism of H7N9 influenza virus in proteomics. PMID:27223893

  13. Intracellular accumulation dynamics and fate of zinc ions in alveolar epithelial cells exposed to airborne ZnO nanoparticles at the air-liquid interface

    Energy Technology Data Exchange (ETDEWEB)

    Mihai, Cosmin; Chrisler, William B.; Xie, Yumei; Hu, Dehong; Szymanski, Craig J.; Tolic, Ana; Klein, Jessica; Smith, Jordan N.; Tarasevich, Barbara J.; Orr, Galya

    2015-02-01

    Airborne nanoparticles (NPs) that enter the respiratory tract are likely to reach the alveolar region. Accumulating observations support a role for zinc oxide (ZnO) NP dissolution in toxicity, but the majority of in vitro studies were conducted in cells exposed to NPs in growth media, where large doses of dissolved ions are shed into the exposure solution. To determine the precise intracellular accumulation dynamics and fate of zinc ions (Zn2+) shed by airborne NPs in the cellular environment, we exposed alveolar epithelial cells to aerosolized NPs at the air-liquid interface (ALI). Using a fluorescent indicator for Zn2+, together with organelle-specific fluorescent proteins, we quantified Zn2+ in single cells and organelles over time. We found that at the ALI, intracellular Zn2+ values peaked 3 h post exposure and decayed to normal values by 12 h, while in submersed cultures, intracellular Zn2+ values continued to increase over time. The lowest toxic NP dose at the ALI generated peak intracellular Zn2+ values that were nearly 3 folds lower than the peak values generated by the lowest toxic dose of NPs in submersed cultures, and 8 folds lower than the peak values generated by the lowest toxic dose of ZnSO4 or Zn2+. At the ALI, the majority of intracellular Zn2+ was found in endosomes and lysosomes as early as 1 h post exposure. In contrast, the majority of intracellular Zn2+ following exposures to ZnSO4 was found in other larger vesicles, with less than 10% in endosomes and lysosomes. Together, our observations indicate that low but critical levels of intracellular Zn2+ have to be reached, concentrated specifically in endosomes and lysosomes, for toxicity to occur, and point to the focal dissolution of the NPs in the cellular environment and the accumulation of the ions specifically in endosomes and lysosomes as the processes underlying the potent toxicity of airborne ZnO NPs.

  14. Hyperoxia-mediated LC3B activation contributes to the impaired transdifferentiation of type II alveolar epithelial cells (AECIIs) to type I cells (AECIs).

    Science.gov (United States)

    Zhang, Liang; Zhao, Shuang; Yuan, Lijie; Wu, Hongmin; Jiang, Hong; Luo, Gang

    2016-09-01

    Life-saving mechanical ventilation can also cause lung injury through the overproduction of reactive oxygen species (ROS), leading to bronchopulmonary dysplasia (BPD)-like symptoms in preterm infants. It is reported that the autophagic protein microtubule-associated protein-1 light chain (LC)-3B can confer protection against hyperoxia-induced DNA damage in lung alveolar epithelium. However, its role in the transdifferentiation of type II alveolar epithelial cells (AECIIs) to type I cells (AECIs) is unclear and requires further investigation. In this study, newborn Sprague-Dawley rats were exposed to 90% oxygen for up to 14 days to mimic BPD in human infants, with neonatal pups exposed to room air (21% oxygen) as controls. Primary rat AECIIs were cultured under hyperoxic conditions for up to 24 hours to further investigate the underlying mechanisms. This study found that hyperoxia promoted a significant and time-dependent increase of AECII marker surfactant protein (SP)-C in the lung. The increase of AECI marker T1α was repressed by hyperoxia during lung development. These results indicated an impaired AECII transdifferentiation. Pulmonary ROS concentration and expression of autophagic protein LC-3B were increased gradually in response to hyperoxia exposure. Furthermore, AECIIs produced more ROS when cultured under hyperoxic conditions in vitro. Both the LC3B expression and the conversion from LC3BI to LC3BII were enhanced in hyperoxic AECs. Interestingly, inhibition of LC3B either by ROS inhibitor N-acetyl-l-cysteine (NAC) or adenovirus-mediated LC3B shRNA could partly restore AECII transdifferentiation under hyperoxia condition. In summary, the current study reveals a novel role of activated LC3B induced by hyperoxia in AECII transdifferentiation. PMID:27187184

  15. Evaluation of pulmonary alveolar epithelial integrity by the detection of restriction to diffusion of hydrophilic solutes of different molecular sizes.

    Science.gov (United States)

    Mason, G R; Peters, A M; Bagdades, E; Myers, M J; Snook, D; Hughes, J M

    2001-03-01

    The rate of transfer of a hydrophilic solute from the alveoli to pulmonary blood following inhalation as an aerosol depends on the molecular size of the solute and the permeability of the alveolar epithelium. The value of this measurement for assessing damage to the epithelium in lung disease is compromised by cigarette smoking, which accelerates clearance by unknown mechanisms. The rates of clearance of (99m)Tc-labelled diethylenetriaminepenta-acetic acid (DTPA) (molecular mass 492 Da) and (113m)In-labelled biotinylated DTPA (B-DTPA) (molecular mass 1215 Da) were monitored simultaneously by dynamic gamma-radiation camera imaging following simultaneous inhalation, and compared between eight normal non-smoking subjects and nine habitual cigarette smokers. The clearance rates of DTPA were 0.95 (S.D. 0.39)%/min in non-smokers and 4.13 (1.06) %/min in smokers. These were about twice the clearance rates of B-DTPA, which in the corresponding groups were 0.41 (0.26) and 2.12 (0.72)%/min respectively. The ratio of the B-DTPA/DTPA clearance rates was, in all subjects, less than the ratio (0.74) of the cube roots of the molecular masses of the solutes, assumed to correspond to the ratio of their free diffusion coefficients in water, and was not significantly different between smokers and non-smokers. As alveolar permeability increased, the ratio of clearance rates in the entire population showed a significant trend to increase in a non-linear fashion towards the value corresponding to the ratio of the free diffusion coefficients. We conclude that the diffusion of at least the larger of these two solutes through the pulmonary alveolar epithelium is restricted (i.e. associated with a reflection coefficient greater than zero). Cigarette smoking, however, does not appear to cause a loss of this restriction, and may increase solute clearance by other mechanisms, such as reducing fluid volume within the alveolus, thereby raising the local radiotracer concentration, or increasing

  16. Regulation of cytokine production in human alveolar macrophages and airway epithelial cells in response to ambient air pollution particles: Further mechanistic studies

    International Nuclear Information System (INIS)

    In order to better understand how ambient air particulate matter (PM) affect lung health, the two main airway cell types likely to interact with inhaled particles, alveolar macrophages (AM) and airway epithelial cells have been exposed to particles in vitro and followed for endpoints of inflammation, and oxidant stress. Separation of Chapel Hill PM 10 into fine and coarse size particles revealed that the main proinflammatory response (TNF, IL-6, COX-2) in AM was driven by material present in the coarse PM, containing 90-95% of the stimulatory material in PM10. The particles did not affect expression of hemoxygenase-1 (HO-1), a sensitive marker of oxidant stress. Primary cultures of normal human bronchial epithelial cells (NHBE) also responded to the coarse fraction with higher levels of IL-8 and COX-2, than induced by fine or ultrafine PM. All size PM induced oxidant stress in NHBE, while fine PM induced the highest levels of HO-1 expression. The production of cytokines in AM by both coarse and fine particles was blocked by the toll like receptor 4 (TLR4) antagonist E5531 involved in the recognition of LPS and Gram negative bacteria. The NHBE were found to recognize coarse and fine PM through TLR2, a receptor with preference for recognition of Gram positive bacteria. Compared to ambient PM, diesel PM induced only a minimal cytokine response in both AM and NHBE. Instead, diesel suppressed LPS-induced TNF and IL-8 release in AM. Both coarse and fine ambient air PM were also found to inhibit LPS-induced TNF release while silica, volcanic ash or carbon black had no inhibitory effect. Diesel particles did not affect cytokine mRNA induction nor protein accumulation but interfered with the release of cytokine from the cells. Ambient coarse and fine PM, on the other hand, inhibited both mRNA induction and protein production. Exposure to coarse and fine PM decreased the expression of TLR4 in the macrophages. Particle-induced decrease in TLR4 and hyporesponsiveness to LPS

  17. A Novel Approach for Ovine Primary Alveolar Epithelial Type II Cell Isolation and Culture from Fresh and Cryopreserved Tissue Obtained from Premature and Juvenile Animals.

    Directory of Open Access Journals (Sweden)

    Mariola M Marcinkiewicz

    Full Text Available The in vivo ovine model provides a clinically relevant platform to study cardiopulmonary mechanisms and treatments of disease; however, a robust ovine primary alveolar epithelial type II (ATII cell culture model is lacking. The objective of this study was to develop and optimize ovine lung tissue cryopreservation and primary ATII cell culture methodologies for the purposes of dissecting mechanisms at the cellular level to elucidate responses observed in vivo. To address this, we established in vitro submerged and air-liquid interface cultures of primary ovine ATII cells isolated from fresh or cryopreserved lung tissues obtained from mechanically ventilated sheep (128 days gestation-6 months of age. Presence, abundance, and mRNA expression of surfactant proteins was assessed by immunocytochemistry, Western Blot, and quantitative PCR respectively on the day of isolation, and throughout the 7 day cell culture study period. All biomarkers were significantly greater from cells isolated from fresh than cryopreserved tissue, and those cultured in air-liquid interface as compared to submerged culture conditions at all time points. Surfactant protein expression remained in the air-liquid interface culture system while that of cells cultured in the submerged system dissipated over time. Despite differences in biomarker magnitude between cells isolated from fresh and cryopreserved tissue, cells isolated from cryopreserved tissue remained metabolically active and demonstrated a similar response as cells from fresh tissue through 72 hr period of hyperoxia. These data demonstrate a cell culture methodology using fresh or cryopreserved tissue to support study of ovine primary ATII cell function and responses, to support expanded use of biobanked tissues, and to further understanding of mechanisms that contribute to in vivo function of the lung.

  18. A Novel Approach for Ovine Primary Alveolar Epithelial Type II Cell Isolation and Culture from Fresh and Cryopreserved Tissue Obtained from Premature and Juvenile Animals.

    Science.gov (United States)

    Marcinkiewicz, Mariola M; Baker, Sandy T; Wu, Jichuan; Hubert, Terrence L; Wolfson, Marla R

    2016-01-01

    The in vivo ovine model provides a clinically relevant platform to study cardiopulmonary mechanisms and treatments of disease; however, a robust ovine primary alveolar epithelial type II (ATII) cell culture model is lacking. The objective of this study was to develop and optimize ovine lung tissue cryopreservation and primary ATII cell culture methodologies for the purposes of dissecting mechanisms at the cellular level to elucidate responses observed in vivo. To address this, we established in vitro submerged and air-liquid interface cultures of primary ovine ATII cells isolated from fresh or cryopreserved lung tissues obtained from mechanically ventilated sheep (128 days gestation-6 months of age). Presence, abundance, and mRNA expression of surfactant proteins was assessed by immunocytochemistry, Western Blot, and quantitative PCR respectively on the day of isolation, and throughout the 7 day cell culture study period. All biomarkers were significantly greater from cells isolated from fresh than cryopreserved tissue, and those cultured in air-liquid interface as compared to submerged culture conditions at all time points. Surfactant protein expression remained in the air-liquid interface culture system while that of cells cultured in the submerged system dissipated over time. Despite differences in biomarker magnitude between cells isolated from fresh and cryopreserved tissue, cells isolated from cryopreserved tissue remained metabolically active and demonstrated a similar response as cells from fresh tissue through 72 hr period of hyperoxia. These data demonstrate a cell culture methodology using fresh or cryopreserved tissue to support study of ovine primary ATII cell function and responses, to support expanded use of biobanked tissues, and to further understanding of mechanisms that contribute to in vivo function of the lung. PMID:26999050

  19. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  1. The mechanism of CpG ODN enhancing the radiosensitivity of lung cancer cell line A549%CpG ODN增强人肺癌细胞株A549放射增敏作用的研究

    Institute of Scientific and Technical Information of China (English)

    颜伟; 孙梯业; 杨春敏; 贾敏; 李静; 史蕊; 唐和兰; 杜斌; 韩全利

    2011-01-01

    Objective To investigate the effect of CpG ODN on the radiosensitivity of lung epithelial cell line A549.Methods The TNF-α,IL-12 and INF-γ secretion by A549 were detected by ELISA;NO level was tested by Griess method ,AP-1 activation within A549 cells was observed using electrophoretic mobility shift assay.Results The inhibitory role was enhanced when CpG ODN 1826(10μg/ml)were combined with β-ray irradiation ,with the increase of TNF-α,IL-2 and INF - γ secretion by cells.CpG ODN1826 combined with β-ray irradiation increased NO leve in A549 cells and inhibited the AP-1 activation within A549 cells.Conclusions CpG ODN1826 can increase the radiosensitivity of lung epithelial cell line A549 and may be tightly related to increasing secretions of IL-12,IFN-γ,TNF-α and NO from cells and the inhibition of AP-1 activation.%目的 初步探讨CpG ODN增强人肺腺上皮细胞株A549放射增敏作用.方法 ELISA法检测细胞TNF-α、IL-12和INF-γ的分泌水平,Griess检测细胞NO的含量并观察CpGODN1826与β射线诱导A549细胞AP-1活化的抑制作用.结果 CpG ODN增加了人肺癌细胞株A549 TNF-α、IL-12、INF-γ和NO的分泌,在联合β射线照射后对A549细胞的杀伤作用更加显著,并显著抑制A549细胞AP-1的活化.结论 CpG ODN对A549有明显的放射增敏作用,其机制可能与CpG ODN增强+4549细胞分泌TNF-α、IL-L2、INF-γ、NO和抑制A549细胞AP-1的活化有关.

  2. Alveolar inflammation in cystic fibrosis

    DEFF Research Database (Denmark)

    Ulrich, Martina; Worlitzsch, Dieter; Viglio, Simona;

    2010-01-01

    BACKGROUND: In infected lungs of the cystic fibrosis (CF) patients, opportunistic pathogens and mutated cystic fibrosis transmembrane conductance regulator protein (CFTR) contribute to chronic airway inflammation that is characterized by neutrophil/macrophage infiltration, cytokine release and...... accumulated in type II alveolar epithelial cells, lacking CFTR. P. aeruginosa organisms were rarely present in inflamed alveoli. CONCLUSIONS: Chronic inflammation and remodeling is present in alveolar tissues of the CF lung and needs to be addressed by anti-inflammatory therapies....

  3. MicroRNA-490-3p inhibits proliferation of A549 lung cancer cells by targeting CCND1

    International Nuclear Information System (INIS)

    Highlights: • We examined the level of miR-490-3p in A549 lung cancer cells compared with normal bronchial epithelial cell line. • We are the first to show the function of miR-490-3p in A549 lung cancer cells. • We demonstrate CCND1 may be one of the targets of miR-490-3p. - Abstract: MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate the translation of messenger RNAs by binding their 3′-untranslated region (3′UTR). In this study, we found that miR-490-3p is significantly down-regulated in A549 lung cancer cells compared with the normal bronchial epithelial cell line. To better characterize the role of miR-490-3p in A549 cells, we performed a gain-of-function analysis by transfecting the A549 cells with chemically synthesized miR-490-3P mimics. Overexpression of miR-490-3P evidently inhibits cell proliferation via G1-phase arrest. We also found that forced expression of miR-490-3P decreased both mRNA and protein levels of CCND1, which plays a key role in G1/S phase transition. In addition, the dual-luciferase reporter assays indicated that miR-490-3P directly targets CCND1 through binding its 3′UTR. These findings indicated miR-490-3P could be a potential suppressor of cellular proliferation

  4. Alveolar Development and Disease

    OpenAIRE

    Jeffrey A Whitsett; Weaver, Timothy E.

    2015-01-01

    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. T...

  5. 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)

    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.). 32P 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: EC50 = 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 DNA

  6. A method to measure mechanical properties of pulmonary epithelial cell layers.

    Science.gov (United States)

    Dassow, Constanze; Armbruster, Caroline; Friedrich, Christian; Smudde, Eva; Guttmann, Josef; Schumann, Stefan

    2013-10-01

    The lung has a huge inner alveolar surface composed of epithelial cell layers. The knowledge about mechanical properties of lung epithelia is helpful to understand the complex lung mechanics and biomechanical interactions. Methods have been developed to determine mechanical indices (e.g., tissue elasticity) which are both very complex and in need of costly equipment. Therefore, in this study, a mechanostimulator is presented to dynamically stimulate lung epithelial cell monolayers in order to determine their mechanical properties based on a simple mathematical model. First, the method was evaluated by comparison to classical tensile testing using silicone membranes as substitute for biological tissue. Second, human pulmonary epithelial cells (A549 cell line) were grown on flexible silicone membranes and stretched at a defined magnitude. Equal secant moduli were determined in the mechanostimulator and in a conventional tension testing machine (0.49 ± 0.05 MPa and 0.51 ± 0.03 MPa, respectively). The elasticity of the cell monolayer could be calculated by the volume-pressure relationship resulting from inflation of the membrane-cell construct. The secant modulus of the A549 cell layer was calculated as 0.04 ± 0.008 MPa. These findings suggest that the mechanostimulator may represent an adequate device to determine mechanical properties of cell layers. PMID:23564730

  7. Development of an in vitro model of human bronchial epithelial barrier to study nanoparticle translocation.

    Science.gov (United States)

    George, Isabelle; Vranic, Sandra; Boland, Sonja; Courtois, Arnaud; Baeza-Squiban, Armelle

    2015-02-01

    Inhalation is the most frequent route of unintentional exposure to nanoparticles (NPs). Our aim was to compare different in vitro models of human lung epithelial monolayers for their suitability to assess the translocation of 50 nm fluorescently labelled silica NPs (50 nm-SiO(2)-FITC-NPs). Human bronchial epithelial cell lines NCI-H292 and Calu-3 as well as human alveolar cell line A549 were seeded onto Transwell filters (TF) separating the well into an apical and a basal compartment. Measurements of the transepithelial electric resistance and monitoring the paracellular transport of a fluorescent marker (Lucifer Yellow) have shown that only Calu-3 cells formed a tight epithelium. In the absence of cells 4% of the initially applied NP concentration was found to cross the TF but the majority remained trapped inside the filter. After 24 h of treatment, 50 nm-SiO(2)-FITC-NPs were taken up by all cell types but their translocation was inversely correlated to the efficiency to prevent LY passage: translocation represented 3% of the initially apically applied NP concentration for Calu-3 cells, 9% for NCI-H292 cells and 35% for A549 cells. In conclusion, 50 nm-SiO(2)-FITC-NPs can cross different bronchial epithelial barriers, but the Calu-3 cell line appears to be the most relevant model for studying NP translocation. PMID:25197033

  8. Depletion of resident alveolar macrophages does not prevent Fas-mediated lung injury in mice

    OpenAIRE

    Bem, R. A.; Farnand, A. W.; Wong, V; Koski, A; Rosenfeld, M. E.; Van Rooijen, N.; C. W. Frevert; Martin, T R; Matute-Bello, G.

    2008-01-01

    Activation of the Fas/Fas ligand (FasL) system in the lungs results in a form of injury characterized by alveolar epithelial apoptosis and neutrophilic inflammation. Studies in vitro show that Fas activation induces apoptosis in alveolar epithelial cells and cytokine production in alveolar macrophages. The main goal of this study was to determine the contribution of alveolar macrophages to Fas-induced lung inflammation in mice, by depleting alveolar macrophages using clodronate-containing lip...

  9. Regulation of secretory leukocyte proteinase inhibitor (SLPI) and elastase-specific inhibitor (ESI/elafin) in human airway epithelial cells by cytokines and neutrophilic enzymes.

    Science.gov (United States)

    Sallenave, J M; Shulmann, J; Crossley, J; Jordana, M; Gauldie, J

    1994-12-01

    The regulation of the activity of potentially harmful proteinases secreted by neutrophils during inflammation is important for the prevention of excessive tissue injury. Secretory leukocyte proteinase inhibitor (SLPI), also called antileukoprotease (ALP) or mucus proteinase inhibitor (MPI), is a serine proteinase inhibitor that has been found in a variety of mucous secretions and that is secreted by bronchial epithelial cells. We recently reported the presence of SLPI and of an elastase-specific inhibitor (ESI), also called elafin, in the supernatants of two cell lines, NCI-H322 and A549, which have features of Clara cells and type II alveolar cells, respectively. We showed in addition that epithelial cell lines produce the elastase-specific inhibitor as a 12 to 16 kD precursor of the elafin molecule (6 kD) called pre-elafin. In the present study, we show that NCI-H322 cells produced higher amounts of both inhibitors than A549 cells and that basal production of SLPI in both cell lines is higher than the production of elafin/pre-elafin. In addition, we show that interleukin-1 beta and tumor necrosis factor induce significant SLPI expression and are major inducers of elafin/pre-elafin expression. Moreover, induction is greater in A549 cells than in NCI-H322 cells. The implications of these findings for the peripheral airways are twofold: (1) alveolar epithelial cells may respond to cytokines secreted during the onset of inflammation by increasing their antiprotease shield; (2) elafin/pre-elafin seems to be a true local "acute phase reactant" whereas SLPI, in comparison, may be less responsive to local inflammatory mediators. PMID:7946401

  10. The effects of disodium cromoglycate on enhanced adherence of Haemophilus influenzae to A549 cells infected with respiratory syncytial virus.

    Science.gov (United States)

    Fukasawa, Chie; Ishiwada, Naruhiko; Ogita, Junko; Hishiki, Haruka; Kohno, Yoichi

    2009-08-01

    Nontypeable Haemophilus influenzae (NTHi) secondary infection often complicates respiratory syncytial virus (RSV) infections. Previous studies have revealed that RSV infections enhance NTHi adherence to airway epithelial cells. In this study, we investigated the effects of disodium cromoglycate (DSCG) and corticosteroids, which are frequently used for the treatment of wheezing often related to RSV infections, on the adherence of NTHi to RSV-infected A549 cells. DSCG inhibited enhanced adherence of NTHi to RSV-infected A549 cells, whereas dexamethasone (Dex) and fluticasone propionate (Fp) did not. DSCG suppressed the expression of ICAM-1, which is one of the NTHi receptors. Furthermore, DSCG exhibited an inhibitory effect on RSV infections. It is suggested that DSCG exerts an anti-RSV effect, and consequently attenuates the expression of NTHi receptors. PMID:19390482

  11. Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry.

    Science.gov (United States)

    Pohl, Marie O; Stertz, Silke

    2015-01-01

    Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed protocol for measuring relative changes in the amount of viral particles that attach to A549 cells, a human lung epithelial cell line, as well as in the amount of particles that are internalized into the cell. We use biotinylated virus which can be easily detected following staining with Cy3-labeled streptavidin (STV-Cy3). We describe the growth, purification and biotinylation of A/WSN/33, a widely used IAV laboratory strain. Cold-bound biotinylated IAV particles on A549 cells are stained with STV-Cy3 and measured using flow cytometry. To investigate uptake of viral particles, cold-bound virus is allowed to internalize at 37 °C. In order to differentiate between external and internalized viral particles, a blocking step is applied: Free binding spots on the biotin of attached virus on the cell surface are bound by unlabeled streptavidin (STV). Subsequent cell permeabilization and staining with STV-Cy3 then enables detection of internalized viral particles. We present a calculation to determine the relative amount of internalized virus. This assay is suitable to measure effects of drug-treatments or other manipulations on attachment or internalization of IAV. PMID:26575457

  12. Napsin A transfection interferes the ephethlial-mesenchymal transition of A549 cells%Napsin A基因转染干预A549细胞的上皮-间质转化

    Institute of Scientific and Technical Information of China (English)

    管淑红; 郑金旭; 汤艳; 宋萍; 许清; 刘继柱

    2011-01-01

    the mechanism, the protein expression of focal adhesion kinase( FAK ) was detected by Western blotting. Results The result of sequencing the recomhinant lentiviral plasmid PLJM1-Napsin A was predicted as designed. The expression of Napsin A protein in transgenic A549 cells was more than that in A549 cells and A549-PLJM1 cells( P < 0. 01 ). After TGF-β1 treatment, the cells changed from epithelial-shaped into interstitial-shaped; TGF-β1 induced EMT in A549 cells, as demonstrated by significant reduction of E-cadherin mRNA and protein levels( P < 0. 01 ) as well as up-regulation of collagen type Ⅰ ( P < 0. 01 ). Transfection of Napsin A in A549 cells could not only restrain the TGF-β1-induced morphological changes, but also partially block the TGF-β1-induced expression of E-caherin( P < 0. 01 ) and collagen type Ⅰ ( P < 0. 05 ) expression. In EMT model in vitro , TGF-β1 treatment significantly induced the expression of FAK( P <0. 01 ).but this induction was significantly blocked by Napsin A overexpression( P < 0. 01 ). Conclusion It could partially block EMT by Napsin A gene transfection into A549 cells, and the mechanism might be associated with the inhibition of integrin signaling pathway.

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

    Directory of Open Access Journals (Sweden)

    Ya-li SONG

    2012-07-01

    Full Text Available 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 the subcutaneous tissue of the back with cell suspension (0.1ml with 5×106 cells in each mouse. After vaccination, the mice were observed and weighed once every 3-4 days, then the tumor formation time was recorded, the tumor growth curve drawn and tumor formation rate calculated. The mice were sacrificed and the subcutaneous tumor tissues were paraffin embedded and sectioned, and stained with HE for histopathological examinati on 5 weeks after the vaccination. Meanwhile, the expressions of Caspase-3, Bax and Bcl-2 in the tumor tissue were detected by immunohistochemistry. Results The tumor formation time was significantly different (F=13.523, P=0.000 between the groups of A549-WT (12.0±1.4d, Mock-A549 (11.7±1.0d and F10+A549 (8.5±1.4d, and it was longer in the groups A549-WT and Mock-A549 than that in F10+A549 group (P 0.05. Pathological examination revealed that the tumor tissues in the groups A549-WT and Mock-A549 harbored more necrotic cells, with structure of homogeneously red stained and amorphous material. In F10+A549 group, the number of necrotic cell was less, and cell proliferation was obvious in the edge of tumor tissues, in which, microvessels were found. In groups A549-WT and Mock-A549, the expression of Bcl-2 was rarely observed, while it was obvious in F10+A549 group, with positive cells diffusely distributed. In groups A549-WT and Mock-A549, there was strong the expression of both Bax and Caspase-3, especially in the A549-WT group. But the

  14. Epithelial hyperplasia, alveoli —

    Science.gov (United States)

    Solitary or multiple foci of increased cellularity distal to terminal bronchioles. The background of broncho-alveolar architecture remains detectable, and epithelial cells are usually single layered. Round to oval hypertrophic type II pneumocytes with abundant eosinophilic cytoplasm line alveolar walls. In bronchiolar subvariant, also called bronchiolization of alveoli, alveolar walls are lined by cuboidal to columnar cells with features of bronchiolar differentiation, such as formation of cilia, Clara cell resemblance, and presence of mucous granules. Foci of consolidation may indicate early stages of adenoma formation. Macrophages may be present in the alveolar lumens.

  15. 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. PMID:25932959

  16. Cytochrome c oxidase is activated by the oncoprotein Ras and is required for A549 lung adenocarcinoma growth

    Directory of Open Access Journals (Sweden)

    Telang Sucheta

    2012-08-01

    Full Text Available Abstract Background Constitutive activation of Ras in immortalized bronchial epithelial cells increases electron transport chain activity, oxygen consumption and tricarboxylic acid cycling through unknown mechanisms. We hypothesized that members of the Ras family may stimulate respiration by enhancing the expression of the Vb regulatory subunit of cytochrome c oxidase (COX. Results We found that the introduction of activated H-RasV12 into immortalized human bronchial epithelial cells increased eIF4E-dependent COX Vb protein expression simultaneously with an increase in COX activity and oxygen consumption. In support of the regulation of COX Vb expression by the Ras family, we also found that selective siRNA-mediated inhibition of K-Ras expression in A549 lung adenocarcinoma cells reduced COX Vb protein expression, COX activity, oxygen consumption and the steady-state concentration of ATP. We postulated that COX Vb-mediated activation of COX activity may be required for the anchorage-independent growth of A549 cells as soft agar colonies or as lung xenografts. We transfected the A549 cells with COX Vb small interfering or shRNA and observed a significant reduction of their COX activity, oxygen consumption, ATP and ability to grow in soft agar and as poorly differentiated tumors in athymic mice. Conclusion Taken together, our findings indicate that the activation of Ras increases COX activity and mitochondrial respiration in part via up-regulation of COX Vb and that this regulatory subunit of COX may have utility as a Ras effector target for the development of anti-neoplastic agents.

  17. Investigation of radiation-induced transcriptome profile of radioresistant non-small cell lung cancer A549 cells using RNA-seq.

    Directory of Open Access Journals (Sweden)

    Hee Jung Yang

    Full Text Available Radioresistance is a main impediment to effective radiotherapy for non-small cell lung cancer (NSCLC. Despite several experimental and clinical studies of resistance to radiation, the precise mechanism of radioresistance in NSCLC cells and tissues still remains unclear. This result could be explained by limitation of previous researches such as a partial understanding of the cellular radioresistance mechanism at a single molecule level. In this study, we aimed to investigate extensive radiation responses in radioresistant NSCLC cells and to identify radioresistance-associating factors. For the first time, using RNA-seq, a massive sequencing-based approach, we examined whole-transcriptome alteration in radioresistant NSCLC A549 cells under irradiation, and verified significant radiation-altered genes and their chromosome distribution patterns. Also, bioinformatic approaches (GO analysis and IPA were performed to characterize the radiation responses in radioresistant A549 cells. We found that epithelial-mesenchymal transition (EMT, migration and inflammatory processes could be meaningfully related to regulation of radiation responses in radioresistant A549 cells. Based on the results of bioinformatic analysis for the radiation-induced transcriptome alteration, we selected seven significant radiation-altered genes (SESN2, FN1, TRAF4, CDKN1A, COX-2, DDB2 and FDXR and then compared radiation effects in two types of NSCLC cells with different radiosensitivity (radioresistant A549 cells and radiosensitive NCI-H460 cells. Interestingly, under irradiation, COX-2 showed the most significant difference in mRNA and protein expression between A549 and NCI-H460 cells. IR-induced increase of COX-2 expression was appeared only in radioresistant A549 cells. Collectively, we suggest that COX-2 (also known as prostaglandin-endoperoxide synthase 2 (PTGS2 could have possibility as a putative biomarker for radioresistance in NSCLC cells.

  18. Protective effect of Ac-SDKP on alveolar epithelial cells through inhibition of EMT via TGF-β1/ROCK1 pathway in silicosis in rat.

    Science.gov (United States)

    Deng, Haijing; Xu, Hong; Zhang, Xianghong; Sun, Yue; Wang, Ruimin; Brann, Darrell; Yang, Fang

    2016-03-01

    The epithelial-mesenchymal transition (EMT) is a critical stage during the development of silicosis fibrosis. In the current study, we hypothesized that the anti-fibrotic tetrapeptide, N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) may exert its anti-fibrotic effects via activation of the TGF-β1/ROCK1 pathway, leading to inhibition of EMT. To address this hypothesis, we first examined the effect of Ac-SDKP upon EMT using an in vivo rat silicosis model, as well as in an in vitro model of TGF-β1-induced EMT. Confocal laser scanning microscopy was used to examine colocalization of surfactant protein A (SP-A), fibroblast specific protein-1 (FSP-1) and α-smooth muscle actin (α-SMA) in vivo. Western blot analysis was used to examine for changes in the protein levels of E-cadherin (E-cad) and SP-A (epithelial cell markers), vimentin (mesenchymal cell marker), α-SMA (active myofibroblast marker), and collagen I and III in both in vivo and in vitro experiments. Secondly, we utilized Western blot analysis and confocal laser scanning microscopy to examine the protein expression of TGF-β1 and ROCK1 in in vivo and in vitro studies. The results revealed that Ac-SDKP treatment prevented increases in the expression of mesenchymal markers as well as TGF-β1, ROCK1, collagen I and III. Furthermore, Ac-SDKP treatment prevented decreases in the expression of epithelial cell markers in both in vivo and in vitro experiments. Based on the results, we conclude that Ac-SDKP inhibits the transition of epithelial cell-myofibroblast in silicosis via activation of the TGF-β1/ROCK1 signaling pathway, which may serve as a novel mechanism by which it exerts its anti-fibrosis properties. PMID:26785300

  19. Sessile alveolar macrophages communicate with alveolar epithelium to modulate immunity

    Science.gov (United States)

    Westphalen, Kristin; Gusarova, Galina A.; Islam, Mohammad N.; Subramanian, Manikandan; Cohen, Taylor S.; Prince, Alice S.; Bhattacharya, Jahar

    2014-02-01

    The tissue-resident macrophages of barrier organs constitute the first line of defence against pathogens at the systemic interface with the ambient environment. In the lung, resident alveolar macrophages (AMs) provide a sentinel function against inhaled pathogens. Bacterial constituents ligate Toll-like receptors (TLRs) on AMs, causing AMs to secrete proinflammatory cytokines that activate alveolar epithelial receptors, leading to recruitment of neutrophils that engulf pathogens. Because the AM-induced response could itself cause tissue injury, it is unclear how AMs modulate the response to prevent injury. Here, using real-time alveolar imaging in situ, we show that a subset of AMs attached to the alveolar wall form connexin 43 (Cx43)-containing gap junction channels with the epithelium. During lipopolysaccharide-induced inflammation, the AMs remained sessile and attached to the alveoli, and they established intercommunication through synchronized Ca2+ waves, using the epithelium as the conducting pathway. The intercommunication was immunosuppressive, involving Ca2+-dependent activation of Akt, because AM-specific knockout of Cx43 enhanced alveolar neutrophil recruitment and secretion of proinflammatory cytokines in the bronchoalveolar lavage. A picture emerges of a novel immunomodulatory process in which a subset of alveolus-attached AMs intercommunicates immunosuppressive signals to reduce endotoxin-induced lung inflammation.

  20. 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

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

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    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.

  2. Exosomes: Decreased Sensitivity of Lung Cancer A549 Cells to Cisplatin

    OpenAIRE

    Xia Xiao; Shaorong Yu; Shuchun Li; Jianzhong Wu; Rong Ma; Haixia Cao; Yanliang Zhu; Jifeng Feng

    2014-01-01

    Exosomes are small extracellular membrane vesicles of endocytic origin released by many cells that could be found in most body fluids. The main functions of exosomes are cellular communication and cellular waste clean-up. This study was conducted to determine the involvement of exosomes in the regulation of sensitivity of the lung cancer cell line A549 to cisplatin (DDP). When DDP was added to A549 cells, exosomes secretion was strengthened. Addition of the secreted exosomes to other A549 cel...

  3. Pulmonary alveolar proteinosis

    Science.gov (United States)

    Alveolar proteinosis; Pulmonary alveolar phospholipoproteinosis ... In some cases, the cause of pulmonary alveolar proteinosis is unknown. In others, it occurs with lung infection or an immune problem. It also can occur with cancers of the blood system, ...

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

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    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.

  5. Dexamethasone enhances invasiveness of Aspergillus fumigatus conidia and fibronectin expression in A549 cells

    Institute of Scientific and Technical Information of China (English)

    LI Tao; LI Jing-chao; QI Qian; LI Yu

    2013-01-01

    Background The efficacies of current treatments for invasive aspergillus (IA) are unsatisfactory and new therapeutic targets or regimens to treat IA are urgently needed.Previous studies have indicated that the ability of conidia to invade host cells is critical in IA development and fibronectin has a hand in the conidia adherence process.In the clinical setting,many patients who receive glucocorticoid for extended periods are susceptible to Aspergillus fumigatus (A.fumigatus) infection,for this reason we investigated the effect of glucocorticoid on conidia invasiveness by comparing the invasiveness of A.fumigatus conidia in the type Ⅱ human alveolar cell line (A549) cultured with different concentrations of dexamethasone.We also explored the relationships between dexamethasone and fibronectin expression.Methods Following culture with anti-fibronectin antibodies and/or dexamethasone,type Ⅱ human alveolar A549 cells were infected with conidia of A.fumigatus.After 4 hours,the extracellular free conidia were washed away and the remaining immobilized conidia were released using Triton-X 100 and quantified by counting the colony-forming units.The invasiveness of conidia was measured by calculating the invasion rate (%).The transcription of the fibronectin gene in cells cultured with different concentrations of dexamethasone for 24 hours was tested by fiuorogenic quantitative RT-PCR while the expression of fibronectinin cells cultured for 48 hours was tested by Western blotting and immunocytochemistry.Results A significant reduction in the invasiveness of conidia was seen in the cells cultured with anti-fibronectin antibody ((14.42±1.68)% vs.(19.17±2.53)%,P <0.05),but no significant difference was observed in cells cultured with a combination of anti-fibronectin antibody and dexamethasone (6.37×10-5 mol/L).There was no correlation between the dexamethasone concentration and the invasiveness of conidia after dexamethasone pretreatment of cells for 4 hours

  6. 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. PMID:24835429

  7. 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. PMID:23990125

  8. Dispersion medium modulates oxidative stress response of human lung epithelial cells upon exposure to carbon nanomaterial samples

    International Nuclear Information System (INIS)

    Due to their large specific surface area, the potential of nanoparticles to be highly reactive and to induce oxidative stress is particularly high. In addition, some types of nanoparticles contain transition metals as trace impurities which are known to generate reactive oxygen species (ROS) in biological systems. This study investigates the potential of two types of single-walled carbon nanotube samples, nanoparticulate carbon black and crocidolite asbestos to induce ROS in lung epithelial cells in vitro. Carbon nanotube and carbon black samples were used as produced, without further purification or processing, in order to best mimic occupational exposure by inhalation of airborne dust particles derived from carbon nanomaterial production. Intracellular ROS were measured following short-term exposure of primary bronchial epithelial cells (NHBE) and A549 alveolar epithelial carcinoma cells using the redox sensitive probe carboxydichlorofluorescin (carboxy-DCFDA). The oxidative potential of agglomerated nanomaterial samples was compared following dispersion in cell culture medium with and without foetal calf serum (FCS) supplement. In addition, samples were dispersed in dipalmitoylphosphatidylcholine (DPPC), the major component of lung surfactant. It could be illustrated that in vitro exposure of lung epithelial cells to carbon nanomaterial samples results only in moderate or low oxidative stress under the exposure conditions employed. However, cell responses are strongly dependent on the vehicle used for dispersion. Whereas the presence of DPPC increased intracellular ROS formation, FCS seemed to protect the cells from oxidative insult.

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

    International Nuclear Information System (INIS)

    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 99mTc-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 99mTc-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

  10. 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

  11. Interleukin 23 Promotes Lung Adenocarcinoma A549 Cell Migration and Invasion

    Directory of Open Access Journals (Sweden)

    Sen ZHANG

    2012-05-01

    Full Text Available Background and objective Interleukin 23 (IL-23 is a pro-inflammatory cytokine that plays an important role in inflammatory disease and tumor microenvironment. The IL-23 receptor is expressed in colon, lung, and oral carcinomas. We performed this study to investigate whether IL-23 promotes directly carcinoma cell migration and invasion as well as further explore its mechanism. Methods The migration and invasion of human lung adenocarcinoma A549 cells induced by IL-23 were detected by a scratch test and Transwell experiment. MMP-9 expression of the mRNA and protein levels of A549 cells cultured with and without IL-23 was respectively detected by Real-time PCR and ELISA. The effect of IL-23 on A549 cells was further verified using anti-IL-23p19 neutralization antibody (Ab IL-23p19 to eliminate IL-23. Results IL-23 remarkably promoted A549 cell migration and invasion. MMP-9 expression in A549 cells was upregulated by IL-23 stimulation. In addition, the effect of IL-23 on the migration and invasion of A549, as well as the MMP-9 expression in A549 cells induced by IL-23, was eliminated by Ab IL-23p19. Conclusion IL-23 promotes the migration and invasion of A549 cells by inducing MMP-9 expression.

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

    OpenAIRE

    Hoyt JC; Ballering JG; Hayden JM; Robbins RA

    2013-01-01

    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 ...

  13. Src激酶在机械通气性牵张致肺泡上皮细胞损伤中的作用%Role of Src tyrosine kinase in damage to alveolar epithelial cells caused by mechanical stretch

    Institute of Scientific and Technical Information of China (English)

    赵涛; 刘孟洁; 谷长平; 王月兰

    2014-01-01

    Objective To evaluate the role of Src tyrosine kinase in damage to alveolar epithelial cells caused by mechanical stretch.Methods MLE-12 cells cultured in vitro were randomly divided into 3 groups using a random number table:mechanical stretch group (group S),dimethyl sulfoxide control group (group D),and Src tyrosine kinase inhibitor PP2 group (group P).In D and P groups,dimethyl sulfoxide 30 μl/ml and PP2 100 μmol/L were added to the culture medium,respectively,and the cells were then cultured for 30 min.The cells underwent mechanical stretch for 8 h with frequency of0.5 Hz and amplitude of 20% in the three groups.At 0,2,4 and 8 h of mechanical stretch,MLE-12 cells in 3 wells of each group were collected for determination of cell apoptosis with flow cytometry and expression of occludin using Western blot.The apoptosis rate was calculated.Results Compared with S group,no significant changes were found in the apoptosis rate and expression of occludin at each time point in group D,and the apoptosis rate was significantly decreased,and the expression of occludin was up-regulated at 2,4 and 8 h of mechanical stretch in group P.Conclusion The activation of Src tyrosine kinase is involved in damage to alveolar epithelial cells caused by mechanical stretch.%目的 评价Src激酶在机械通气性牵张致肺泡上皮细胞损伤中的作用.方法 采用随机数字表法,将体外培养的MEL12细胞分为3组:机械通气性牵张组(S组)、二甲基亚砜对照组(D组)和Src激酶抑制剂PP2组(P组).D组和P组分别加入二甲基亚砜30 μl/ml和Src激酶抑制剂PP2100 μmol/L(用二甲基亚砜溶解)孵育30 min,然后3组给予20%应变率的机械牵张,频率0.5 Hz.分别于机械牵张即刻、2、4和8h时,每组取3孔 MLE-12细胞,测定细胞凋亡情况,计算细胞凋亡率,采用Western blot法测定Occludin蛋白的表达.结果 与S组比较,D组各时点细胞凋亡率和Occludin蛋白表达差异无统计学意义(P>0.05),P

  14. The effect of autophagy on Streptococcus pneumoniae infections of lung epithelial cells and its mechanism%自噬体在肺炎链球菌感染过程中的保护作用机制探讨

    Institute of Scientific and Technical Information of China (English)

    郭旭光; 唐希才; 夏勇

    2012-01-01

    目的:检测微管相关蛋白轻链-3(LC3)在肺泡Ⅱ型上皮细胞A549上的表达情况,及3-甲基腺嘌呤(3MA)刺激后对其表达的影响,为研究自噬体在抵抗肺炎链球菌感染过程中的保护作用奠定基础.方法:体外培养肺泡Ⅱ型上皮细胞A549,在肺炎链球菌感染A549细胞12h时用倒置显微镜拍照并提取RNA,采用RT-PCR的方法检测LC3 mRNA的表达情况.同时检测对照组、3MA组、Spn组和3MA+ Spn组上清液乳酸脱氢酶(LDH)的OD值.结果:RT-PCR检测LC3 mRNA有明显表达,3MA可以抑制LC3的表达.LDH检测显示Spn组和3MA+ Spn组上清液LDH的OD值数据两两比较差异有统计学意义(P<0.05).结论:LC3在肺泡Ⅱ型上皮细胞中显著表达,3MA可以抑制细胞的自噬作用.加3MA后,肺炎链球菌感染肺泡Ⅱ型上皮细胞坏死增加,提示自噬体在抵抗肺炎链球菌的感染过程中起一定的保护作用.%Objective To detect the expression of LC3 in human alveolar type Ⅱ epithelial cells A549 and the effect of Streptococcus pneumoniae and 3MA on it, lay the foundation for studying on autophagosome resistance in the process of Streptococcus pneumoniae infection. Methods Human pulmonary type Ⅱ epithelial cells cultured in vitro and stimulated with Streptococcus pneumoniae. Extract the RNA of A549 cells on 12 h and detect LC3 mRNA expression by RT-PCR. The necrosis of control group, 3-MA group, Spn group and 3-MA + Spn group were detected by the necrosis kit after 12h by Non-Radioactive cytotoxicity assay respectively. Results The expression of LC3 mRNA stimulated of 3MA deteced by RT-PCR was different. The necrosis test showed the blank group and 3-MA group was not significantly different (P > 0.05), Spn group and 3-MA + Spn group significantly different (P < 0.05). Conclusions The expression of LC3 mRNA in alveolar type Ⅱ epithelial cells stimulated with Streptococcus pneumoniae was significantly different. The necrosis number of alveolar type

  15. Influence of suppressor gene p16 on retinoic acid inducing cancer cell A549 differentiation

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective To investigate the role of suppressor gene p16 in the process of differential regulation of retinoic acid (RA) on the A549 lung cancer cells.Methods Tumor suppressor gene p16 was transferred into A549 cells and the cells were treated with all-trans retinoic acid (ATR) at the dosage of 5×10-6 mol/L for 4 d. After that, the proliferation and differentiation of A549 cells were examined by growth curve and cytometry analysis, the change of lung lineage-specific marker MUC1 was tested by immunohistochemical staining. Meanwhile, Western blot was used to observe the change of p16 protein expression in A549 cells treated with ATRA.Results ATRA could obviously inhibit the growth and induce the differentiation of A549 Cells that were transferred with p16 gene. There were more cells arrested in G1/G0 phase and the expression of MUG1 was markedly down-regulated than in control cells. The expression of p16 protein was up-regulated in A549 cells treated with ATRA.Conclusion Suppressor gene p16 could enhance the effects of RA and proliferated suppression and differential induction of A549 cells.

  16. Klotho inhibits growth and promotes apoptosis in human lung cancer cell line A549

    Directory of Open Access Journals (Sweden)

    Zhao Weihong

    2010-07-01

    Full Text Available Abstract Background Klotho, as a new anti-aging gene, can shed into circulation and act as a multi-functional humoral factor that influences multiple biological processes. Recently, published studies suggest that klotho can also serve as a potential tumor suppressor. The aim of this study is to investigate the effects and possible mechanisms of action of klotho in human lung cancer cell line A549. Methods In this study, plasmids encoding klotho or klotho specific shRNAs were constructed to overexpress or knockdown klotho in vitro. A549 cells were respectively treated with pCMV6-MYC-KL or klotho specific shRNAs. The MTT assay was used to evaluate the cytotoxic effects of klotho and flow cytometry was utilized to observe and detect the apoptosis of A549 cells induced by klotho. The activation of IGF-1/insulin signal pathways in A549 cells treated by pCMV6-MYC-KL or shRNAs were evaluated by western blotting. The expression levels of bcl-2 and bax transcripts were evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR. Results Overexpression of klotho reduced the proliferation of lung cancer A549 cells, whereas klotho silencing in A549 cells enhanced proliferation. Klotho did not show any effects on HEK-293 cells. Klotho overexpression in A549 cells was associated with reduced IGF-1/insulin-induced phosphorylation of IGF-1R (IGF-1 receptor/IR (insulin receptor (P P P P P Conclusions Klotho can inhibit proliferation and increase apoptosis of A549 cells, this may be partly due to the inhibition of IGF-1/insulin pathways and involving regulating the expression of the apoptosis-related genes bax/bcl-2. Thus, klotho can serve as a potential tumor suppressor in A549 cells.

  17. Phospholipid flippase associates with cisplatin resistance in plasma membrane of lung adenocarcinoma A549 cells

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The fusion of the liposomes containing N-(7-nitro-2, 1, 3-benzoxadiazol-4-yl)-i ,2-hexadecanoylSn-glycero-3-1abeled phosphatidylethanolamine (NBD-PE) with A549 and A549/DDP cells was performed, and the activity of the phospholipid flippase in the plasma membrane of the cells was measured by fluorescence intensity change of NBDPE in the outer membrane. When A549 or A549/DDP cells containing N BD-PE were incubated at 37 C for 0, 30, 60 and 90 min, the fluorescence intensities in the outer membrane of the cells were 0%, 1.4%, 2.9% and 7.8% for A59cells, and 0%, 10.5 %, 15. 5 % and 18.3 % for A549/DDP cells respectively, demonstrating that the phospholipid flippase was distributed in the plasma membrane of As49 cells, but its activity in the drug-resistant A549/DDP cells was much higher than that in the A549 cells. When the A549/DDP cells were incubated with a multidrug resistance reverse agent, verapamil, for 60 min at 37C, the results showed that the NBD-PE in outer membrane decreased by 25.0% compared with the control's. Furthermore, when A549/DDP cells were incubated with 25 μmol/L cisplatin, which is a specific anticancer drug, the flippase activity decreased by 31.6%, and it further decreased with the increase of cisplatin concentration, suggesting that phospholipid flippase in the membrane might be related to the cisplatin-resistance of human lung adenocarcinoma cancer cells.

  18. Some ABCA3 mutations elevate ER stress and initiate apoptosis of lung epithelial cells

    Directory of Open Access Journals (Sweden)

    Holzinger Andreas

    2011-01-01

    Full Text Available Abstract Background ABCA3 transporter (ATP-binding cassette transporter of the A subfamily is localized to the limiting membrane of lamellar bodies, organelles for assembly and storage of pulmonary surfactant in alveolar epithelial type II cells (AECII. It transports surfactant phospholipids into lamellar bodies and absence of ABCA3 function disrupts lamellar body biogenesis. Mutations of the ABCA3 gene lead to fatal neonatal surfactant deficiency and chronic interstitial lung disease (ILD of children. ABCA3 mutations can result in either functional defects of the correctly localized ABCA3 or trafficking/folding defects where mutated ABCA3 remains in the endoplasmic reticulum (ER. Methods Human alveolar epithelial A549 cells were transfected with vectors expressing wild-type ABCA3 or one of the three ABCA3 mutant forms, R43L, R280C and L101P, C-terminally tagged with YFP or hemagglutinin-tag. Localization/trafficking properties were analyzed by immunofluorescence and ABCA3 deglycosylation. Uptake of fluorescent NBD-labeled lipids into lamellar bodies was used as a functional assay. ER stress and apoptotic signaling were examined through RT-PCR based analyses of XBP1 splicing, immunoblotting or FACS analyses of stress/apoptosis proteins, Annexin V surface staining and determination of the intracellular glutathion level. Results We demonstrate that two ABCA3 mutations, which affect ABCA3 protein trafficking/folding and lead to partial (R280C or complete (L101P retention of ABCA3 in the ER compartment, can elevate ER stress and susceptibility to it and induce apoptotic markers in the cultured lung epithelial A549 cells. R43L mutation, resulting in a functional defect of the properly localized ABCA3, had no effect on intracellular stress and apoptotic signaling. Conclusion Our data suggest that expression of partially or completely ER localized ABCA3 mutant proteins can increase the apoptotic cell death of the affected cells, which are factors that

  19. Adenosine regulation of alveolar fluid clearance

    OpenAIRE

    Factor, Phillip; Mutlu, Göskhan M.; Chen, Lan; Mohameed, Jameel; Akhmedov, Alexander T.; Meng, Fan Jing; Jilling, Tamas; Lewis, Erin Rachel; Johnson, Meshell D.; Xu, Anna; Kass, Daniel; Martino, Janice M.; Bellmeyer, Amy; Albazi, John S.; Emala, Charles

    2007-01-01

    Adenosine is a purine nucleoside that regulates cell function through G protein-coupled receptors that activate or inhibit adenylyl cyclase. Based on the understanding that cAMP regulates alveolar epithelial active Na+ transport, we hypothesized that adenosine and its receptors have the potential to regulate alveolar ion transport and airspace fluid content. Herein, we report that type 1 (A1R), 2a (A2aR), 2b (A2bR), and 3 (A3R) adenosine receptors are present in rat and mouse lungs and alveol...

  20. Effects of siRNA-silenced CHD6 on radiosensitivity in A549

    International Nuclear Information System (INIS)

    Objective To observe the effects of siRNA-silenced chromatin reconstitution protein CHD6 on the proliferation and radiosensitivity in A549 cells. Methods: CHD6-silenced cellular model was constructed by plasmid mediated siRNA technique. RT-PCR was used to detect the expression of mRNA. Growth curve, flow cytometry and fluorescent staining method were used to measure changes of cell proliferation, cell cycle and apoptosis, respectively. The radiosensitivity of A549 cells was determined with cell cloning efficiency test. Results siRNA-asilence CHD6 increased the growth of A549 cells and enhanced the radiation resistance of A549 cells when gamma irradiation dose was lesser than two grays, whereas it had no significant effect on apoptosis in cells being irradiated with larger doses. Conclusion: Inhibition of CHD6 could enhance cell proliferation and cell radiosensitivity. (authors)

  1. 高氧及TGF-β1对肺泡Ⅱ型细胞上皮间质转化的影响%Effect of hyperoxia and TGF-β1 on epithelial-mesenchymal transition of type Ⅱ alveolar epithelial cells

    Institute of Scientific and Technical Information of China (English)

    刘芳君; 邓春; 郭春宝; 符州

    2012-01-01

    AIM: To investigate the effect of hyperoxia and TGF-pi on epithelial-mesenchymal transition (EMT)of type II alveolar epithelial cells (AEC-Ⅱ ) of mice. METHODS; AEC-Ⅱ cells (MLE-12 lines) were randomly divided into following groups: air exposure group, hyperoxia exposure group, air exposure combined with TGF-pi treatment group, hyperoxia exposure combined with TGF-pi treatment group. The morphological changes of cells in each group were observed at 6, 12, 24, 48 hours. The protein and mR-NA expressions of AEC Ⅱ specific marker lung surfactant protein B ( SP-B) and fibroblast specific marker fibroblast specific protein ( FSP1) were detected by double-labeled immunoflu orescence and real time-PCR at the same time point, respectively. RESULTS: Along with the time of exposure to hyperoxia and TGF-pi, AEC Ⅱ cells gradually changed from pebble-like shape to spindle shape, and showed some fibroblast appearances. Synchronously, the protein expression of SP-B in AEC Ⅱ cells decreased, whereas the expression of FSP1 increased. The co-expressed were observed at 24 hours. Comparing with that of the air exposure group, the mRNA expression of SP-B in the hyperoxia exposure group, air exposure combined with TGF-pi treatment group, hyperoxia exposure combined with TGF-pi treatment group decreaseed significantly, whereas the mRNA expression of FSPl increased significantly at 24 hours and 48 hours (P<0.01). CONCLUSION; Hyperoxia and TGF-β1 can induce EMT of type II alveolar epithelial cells in a time-dependent manner.%目的:探讨高氧及TGF-β1干预小鼠肺泡Ⅱ型细胞(AECⅡ)后,是否发生上皮间质转化(EMT)及其影响.方法:小鼠肺泡Ⅱ型细胞系MLE-12,随机分为空气暴露组、高氧暴露组、TGF-β1干预空气暴露组、TGF-β1干预高氧暴露组.观察各组6、12、24、48 h细胞形态变化.应用细胞免疫荧光双标法及荧光定量PCR法检测各组各时间点肺表面活性物质B (SP-B)及成纤维细胞特异性蛋白1(FSP

  2. 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 A549 cell damage, indicating that 6MV X-ray irradiation can induce bystander effect on A549 cells, which reaches a peak at 72 h. PMID:25686868

  3. Pulmonary alveolar proteinosis

    OpenAIRE

    Crestani, B; Epaud, R.; Aubier, M.; M-C. Dombret; Taille, C.; M-P. Debray; Danel, C.; R. Borie

    2011-01-01

    Pulmonary alveolar proteinosis (PAP) is a rare pulmonary disease characterised by alveolar accumulation of surfactant. It may result from mutations in surfactant proteins or granulocyte macrophage-colony stimulating factor (GM-CSF) receptor genes, it may be secondary to toxic inhalation or haematological disorders, or it may be auto-immune, with anti-GM-CSF antibodies blocking activation of alveolar macrophages. Auto-immune alveolar proteinosis is the most frequent form of PAP, representing 9...

  4. Effcts of Vitamin C on A549 Cell Proliferation, Apoptosis and Expressions of Caspase, Survivin

    Directory of Open Access Journals (Sweden)

    Lanzhen HUANG

    2010-02-01

    Full Text Available Background and objective It was proven that Vitamin C could inhibit the growth of many types of tumors as an antioxidant. The aim of this study is to explore role of Vitamin C in proliferation and apoptosis of lung carcinoma cell line A549 and the underlying mechanism. Methods A549 cells were cultured in vitro and incubated with Vitamin C. The cell viability was measured by growth curve and clonogentic assay. Flow cytometry was used to analyze cell cycle and detect apoptosis. The levels of expression of Caspase-3 mRNA and Survivin mRNA were detected by RT-PCR. Results Vitamin C of 400 μg/mL, 4 mg/mL significantly inhibited the growth of A549 cell lines (P=0.024, P=0.015, respectively. Flow cytometry showed that the cells major stagnation stayed in the G0/G1 and S phase and the apoptotic rate increased with time prolonged. Vitamin C signifiantly up-ragulated the expression of Caspase-3 mRNA, but had no effect on Survivin mRNA. Conclusion Vitamin C can inhibit the proliferation of A549, block A549 cells in G0/G1 and S phase, and induce apoptosis of A549 cells. Apotosis occurred by up-ragulated the expressionof Caspase-3.

  5. 烹调油烟中细颗粒物PM2.5对A-549细胞存活率和IL-10水平的影响%Effects of PM2.5 in cooking oil fume on cell survival and IL-10 levels in A-549 cells

    Institute of Scientific and Technical Information of China (English)

    冯哲伟; 梁春梅; 操基玉; 郭冬梅; 王勇; 王绩凯

    2011-01-01

    Objective To explore fine particle in cooking oil fume ( FPCOF, PM2. 5 ) on the level of anti-inflammatory factor IL-10 in human lung adenocarcinoma epithelial cell (A-549 cells). Methods A-549 cells were exposed to different concentrations of FPCOF, for 12 h, 24 h and 36 h, respectively, then detecting the cell survival rate with MTT assay and IL-10 level in media supernatant by enzyme linked immunosorbent assay. Results ( 1 ) The survival rate of A-549 cells was reduced time-and dose-dependently, but the difference at 12 h among different doses was not significant, meanwhile the rate in 100 μg/ml PM2.5 + 10 mmol/L NAC group was significantly higher than that in 100 μg/ml PM2.5 group after 24 h or 36 h exposure. (2) There was no obvious effect on IL-10 after 24 h exposure to FPCOF, the rise of IL-10 level was only seen in all the exposed groups after 36 h exposure to FPCOF; additionally, IL-10 level in 100 μg/ml PM2.5 + 10 mmol/L NAC group was significantly lower than that in 100 μg/ml PM2.5 group. Conclusion FPCOF might promote the secretion of anti-inflammatory factor IL-10 in A-549 cells, but its expression was later than the inhibition of cell proliferation caused by FPCOF; and the antioxidant NAC might protect A-549 cells from the adverse effects induced by FPCOF.%探讨烹调油烟中大气细颗粒物PM2.5(FPCOF)对人肺癌上皮细胞(A-549细胞)抗炎因子IL-10分泌的影响.分别用不同浓度的FPCOF对细胞进行染毒,于12 h、24 h和36 h后用MTT法测细胞存活率;用酶联免疫吸附法测染毒24 h和36 h的细胞上清中IL-10的浓度.结果:(1)FPCOF作用于A-549细胞24 h和36 h后,细胞存活率随染毒浓度升高而降低,100μg/ml PM2.5+10 mmol/L NAC组存活率高于100μg/ml PM2.5染毒组.(2)FPCOF对A-549细胞作用24 h后,对抗炎因子IL-10的影响不明显;作用36 h后均可见IL-10水平增高,100 μg/ml PM2.5+10mmol/L NAC组IL-10浓度低于100 μg/ml PM2.5染毒组.提示FPCOF促进A-549

  6. 淫羊藿苷逆转耐甲氨蝶呤肺癌A549细胞转移表型%Icariin reversed metastatic phenotype of methotrexate-resistant lung cancer A549 cells

    Institute of Scientific and Technical Information of China (English)

    吴剑锋; 何晓东; 许卫东; 李道静; 孙利; 沈佐君

    2009-01-01

    目的:研究中药淫羊藿苷(icariin,ICA)作用甲氨蝶呤(methotrexate,MTX)耐药肺癌A549细胞后对细胞转移表型的影响,初步探讨ICA逆转A549/MTX耐药细胞转移表型的作用机制及对肺癌的治疗价值.方法:采用MTT法检测ICA对A549/MTX耐药细胞的半数抑制浓度(half inhibition concentration,IC_(50)).采用双层软琼脂克隆形成实验检测A549/MTX 组和A549/MTX+ICA组细胞的克隆形成率,并观察其集落形态.细胞划痕实验检测A549/MTX组和A549/MTX+ICA组细胞的迁移能力.Transwell小室实验检测细胞侵袭能力的变化.结果:MTT结果显示,无毒剂量的ICA与MTX联合应用后A549/MTX细胞的IC_(50)值为35.50±1.85 μmol/L,比单独应用MTX(同等剂量)后A549/MTX细胞的IC50值(52.17±2.25 μmol/L)有了一定程度的下降.软琼脂实验发现,A549/MTX+ICA组细胞克隆形成率为0.94±0.09,小于A549/MTX组细胞的1.56±1.07(P<0.05).划痕实验显示,72 h后A549/MTX组细胞的迁移能力大于A549/MTX+ICA组细胞(P<0.05).Transwell实验显示,A549/MTX组细胞的穿膜细胞数明显多于A549/MTX+ICA组细胞(P<0.05),说明A549/MTX+ICA组细胞的侵袭浸润能力小于A549/MTX组细胞.结论:中药ICA具有逆转A549/MTX耐药细胞转移表型的作用.

  7. Isolation and Culture of Human Alveolar Type II Pneumocytes.

    Science.gov (United States)

    Witherden, I R; Tetley, T D

    2001-01-01

    Alveolar type II pneumocytes (alveolar type II cells; TII cells) play an important role in the homeostasis of the alveolar unit. They are the progenitor cells to the type I pneumocyte and are therefore responsible for regeneration of alveolar epithelium following alveolar epithelial cell damage. The type I cell covers over 90% of the alveolar surface, reflecting its capacity to stretch into a flattened cell with very little depth (approx. 0.1 µm), but with a large surface area, to facilitate gas exchange. Nevertheless, the type II cell outnumbers type I cells, estimated to be by 2:1 in rodents. Most of the type II cell lies buried in the interstitium of the alveolus, with only the apical tip of the cell reaching into the airspace, through which another crucial function, provision of alveolar surfactant, occurs. Surfactant synthesis and secretion is a unique feature of type II cells; surfactant consists of a high proportion of phospholipids (approx. 90%) and a small proportion of protein (approx. 10%), which contains surfactant apoprotein (SP), of which four have so far been described, SP-A, SP-B, SP-C, and SP-D (1,2). Surfactant is highly surface active and is essential to prevent alveolar collapse. In addition, surfactant has many other roles, including pulmonary host defense. Compromised surfactant synthesis and function are believed to be a feature of numerous disease states (1,2), including infant respiratory distress syndrome, adult respiratory distress syndrome, alveolar proteinosis, and microbial infection. PMID:21336897

  8. Differential activation of inflammatory pathways in A549 type II pneumocytes by Streptococcus pneumoniae strains with different adherence properties

    Directory of Open Access Journals (Sweden)

    Horvat Rebecca T

    2006-04-01

    Full Text Available Abstract Background Adherence of Streptococcus pneumoniae bacteria to lung cells is a first step in the progression from asymptomatic carriage to pneumonia. Adherence abilities vary widely among S. pneumoniae patient isolates. In this study, the binding properties of S. pneumoniae isolates and the effects of binding on activation of the Nuclear Factor-Kappa-B (NFκB pathway and cytokine secretion by type II pneumocytes were measured. Methods Mechanisms of high- and low-binding S. pneumoniae adherence to A549 cells were investigated by blocking putative receptors on bacteria and host cells with antibody and by eluting choline-binding proteins off of bacterial surfaces. NFκB activation was measured by western blot and immunocytochemistry and cytokine secretion was detected by a protein array. Results This study shows that S. pneumoniae isolates from pneumonia patients (n = 298 can vary by as much as 1000-fold in their ability to bind to human lung epithelial cells. This difference resulted in differential activation of the NFκB pathway. High-, but not low-binding S. pneumoniae used Choline-binding protein A (CbpA to bind to complement component C3 on epithelial cell surfaces. Interleukin-8 (IL-8 was the only cytokine secreted by cells treated with either low- or high-binding S. pneumoniae. Conclusion These results indicate that S. pneumoniae clinical isolates are not homogeneous in their interaction with host epithelial cells. The differential activation of host cells by high- and low-binding S. pneumoniae strains could have implications for the treatment of pneumococcal pneumonia and for vaccine development.

  9. Alveolar macrophages regulate neutrophil recruitment in endotoxin-induced lung injury

    Directory of Open Access Journals (Sweden)

    Reyes Livia

    2005-06-01

    Full Text Available Abstract Background Alveolar macrophages play an important role during the development of acute inflammatory lung injury. In the present study, in vivo alveolar macrophage depletion was performed by intratracheal application of dichloromethylene diphosphonate-liposomes in order to study the role of these effector cells in the early endotoxin-induced lung injury. Methods Lipopolysaccharide was applied intratracheally and the inflammatory reaction was assessed 4 hours later. Neutrophil accumulation and expression of inflammatory mediators were determined. To further analyze in vivo observations, in vitro experiments with alveolar epithelial cells and alveolar macrophages were performed. Results A 320% increase of polymorphonuclear leukocytes in bronchoalveolar lavage fluid was observed in macrophage-depleted compared to macrophage-competent lipopolysaccharide-animals. This neutrophil recruitment was also confirmed in the interstitial space. Monocyte chemoattractant protein-1 concentration in bronchoalveolar lavage fluid was significantly increased in the absence of alveolar macrophages. This phenomenon was underlined by in vitro experiments with alveolar epithelial cells and alveolar macrophages. Neutralizing monocyte chemoattractant protein-1 in the airways diminished neutrophil accumulation. Conclusion These data suggest that alveolar macorphages play an important role in early endotoxin-induced lung injury. They prevent neutrophil influx by controlling monocyte chemoattractant protein-1 production through alveolar epithelial cells. Alveolar macrophages might therefore possess robust anti-inflammatory effects.

  10. Phagocytic properties of lung alveolar wall cells

    Directory of Open Access Journals (Sweden)

    Tanaka,Akisuke

    1974-04-01

    Full Text Available For the purpose to define the mechanism of heavy metal intoxication by inhalation, morphologic observations were made on rat lungs after nasal instillation of iron colloid particles of positive and negative electric charges. Histochemical observation was also made on the liver and spleen of these animals. The instilled iron colloid particles reach the alveolar cavity easily, as can be seen in the tissue sections stained by Prussian blue reaction. Alveolar macrophages do take up them avidly both of positive and negative charges, though much less the positive particles than negative ones. In contrast, the alveolar epithelial cells take up solely positive particles by phagocytosis but not negative ones. Electron microscope observation revealed that the positive particles are ingested by Type I epithelial cells by pinocytosis and by Type II cells by phagocytosis as well. Then the iron colloid particles are transferred into the basement membrane by exocytosis. Travelling through the basement membrane they are again taken up by capillary endothelial cells by phagocytosis. Some particles were found in the intercellular clefts of capillary endothelial cells but not any iron colloid particles in the intercellular spaces of epithelial cells and in the capillary lumen. However, the liver and spleen tissues of the animals given iron colloid showed a strong positive iron reaction. On the basis of these observations, the mechanism of acute intoxication by inhaling heavy metal dusts like lead fume is discussed from the view point of selective uptake of alveolar epithelial and capillary endothelial cells for the particles of the positive electric cha'rge.

  11. Identification and Isolation of Cancer Stem Cells from A549 Cells

    Directory of Open Access Journals (Sweden)

    Hui XIA

    2013-08-01

    Full Text Available Background and objective Lung cancer stem cells are the root causes of lung cancer malignant phenotype and potential therapeutic target, the aim of this study is to isolate and characterize the cancer stem cells in the lung adenoearcinomas cell line A549, so as to provide an experimental basis for further stem cell research. Methods The cancer stem cells were isolated from the lung adenoearcinomas cell line A549 using FACS. And the difference of colony formation, cell proliferation and tumorigenicity in vitro were also tested. The expression of CD133 and ABCG2 were evaluated by RT-PCR and Western blot. Results The percentage of SP cells was 5.93% of A549 and 0.32% of A549 after incubation with verapamil. The results showed that there were significantly higher expression of CD133 and ABCG2 on SP cells than that of non-SP cells. And the ability of colony formation, cell proliferation and tumorigenicity in SP cell group were remarkably higher than that in non-SP cell group. Conclusion Our results suggested that the cancer stem cells with higher expression of CD133 and ABCG2 can be isolated from the lung adenoearcinomas cell line A549 using FACS and be used in the further research experiments.

  12. Hypoxia Upregulates the Expression of Annexin A1 in Lung Adenocarcinoma A549 Cells

    Directory of Open Access Journals (Sweden)

    Zhenhong HU

    2012-05-01

    Full Text Available Background and objective The growth of tumor often faced up with lackness of blood and oxygen, and it has been reported that Annexin A1 may be involved in tumor. The aim of this investigation is to explore the characteristics of expression of Annexin A1 in lung adenocarcinoma A549 cells after hypoxia. Methods A549 cells were exposured to either normoxia (21%O2 or hypoxia (1%O2 condition for 4 h, 12 h, 24 h. The expressions of Annexin A1 mRNA levels were measured by RT-PCR. The expressions of Annexin 1 protein were investigaged by Western blot. The relative content of reactive oxygen species (ROS were assayed by special kit. The expressions of nuclear translocation of NF-κB was assayed by Western blot; After been treated with ROS scavenger NAC and PDTC, the levels of Annexin 1 protein of A549 cells were measured by Western blot. Results Compared with normoxia group, the Annexin A1 mRNA in hypoxia group increased after 4 h, and then decreased gradually; Moreover, Annexin 1 protein levels of A549 cells were also increased when treated with hypoxia. An increaing of ROS production in cells exprosed to hypoxia was detected. NAC and PDTC inhibited hypoxia-induced Annexin A1 increase. Conclusion Hypoxia upregulates the expression of Annexin A1 in lung adenocarcinoma A549 cells, in which process ROS-NF-κB may paticipate in.

  13. Transcription Activity of Ectogenic Human Carcinoembryonic Antigen Promoter in Lung Adenocarcinoma Cells A549

    Institute of Scientific and Technical Information of China (English)

    XIONG Weining; FANG Huijuan; XU Yongjian; XIONG Shendao; CAO Yong; SONG Qingfeng; ZENG Daxiong; ZHANG Huilan

    2006-01-01

    The transcription activity of ectogenic human carcinoembryonic antigen (CEA) promoter in lung adenocarcinoma cells A549 was investigated for the further gene-targeting therapy. The reporter gene green fluorescent protein (GFP) driven by CEA promoter and human cytomegalovirus (CMV) promoter were relatively constructed and named plasmid pCEA-EGFP and pCMV-GFP respectively. The intensity of fluorescence was detected by fluorescence microscope and flow cytometry analysis after the pCEA-GFP and pSNAV-GFP plasmids were transfected into A549 cells through liposome respectively. The results showed (4.08±0.63) % of the A549 cells transfected with pCEA-AFP plasmid expressed, significantly lower than that of the A549 cells transfected with pCMV-GFP [(43.27±3.54) %]. It was suggested that ectogenic human CEA promoter in lung adenocarcinoma cells A549 was weakly expressed. The distinct specificity of CEA promoter in CEA high expression cells was regarded as a tool in selective gene therapy, but the transcription activity of ectogenic human CEA promoter was needed to increase in the future.

  14. Role of Rad52 in fractionated irradiation induced signaling in A549 lung adenocarcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Ghosh, Somnath, E-mail: ghosh.barc@gmail.com [Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400085 (India); Krishna, Malini, E-mail: malinik00@gmail.com [Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400085 (India)

    2012-01-03

    The effect of fractionated doses of {gamma}-irradiation (2 Gy per fraction over 5 days), as delivered in cancer radiotherapy, was compared with acute doses of 10 and 2 Gy, in A549 cells. A549 cells were found to be relatively more radioresistant if the 10 Gy dose was delivered as a fractionated regimen. Microarray analysis showed upregulation of DNA repair and cell cycle arrest genes in the cells exposed to fractionated irradiation. There was intense activation of DNA repair pathway-associated genes (DNA-PK, ATM, Rad52, MLH1 and BRCA1), efficient DNA repair and phospho-p53 was found to be translocated to the nucleus of A549 cells exposed to fractionated irradiation. MCF-7 cells responded differently in fractionated regimen. Silencing of the Rad52 gene in fractionated group of A549 cells made the cells radiosensitive. The above result indicated increased radioresistance in A549 cells due to the activation of Rad52 gene.

  15. Role of Rad52 in fractionated irradiation induced signaling in A549 lung adenocarcinoma cells.

    Science.gov (United States)

    Ghosh, Somnath; Krishna, Malini

    2012-01-01

    The effect of fractionated doses of γ-irradiation (2Gy per fraction over 5 days), as delivered in cancer radiotherapy, was compared with acute doses of 10 and 2Gy, in A549 cells. A549 cells were found to be relatively more radioresistant if the 10Gy dose was delivered as a fractionated regimen. Microarray analysis showed upregulation of DNA repair and cell cycle arrest genes in the cells exposed to fractionated irradiation. There was intense activation of DNA repair pathway-associated genes (DNA-PK, ATM, Rad52, MLH1 and BRCA1), efficient DNA repair and phospho-p53 was found to be translocated to the nucleus of A549 cells exposed to fractionated irradiation. MCF-7 cells responded differently in fractionated regimen. Silencing of the Rad52 gene in fractionated group of A549 cells made the cells radiosensitive. The above result indicated increased radioresistance in A549 cells due to the activation of Rad52 gene. PMID:22001234

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

    International Nuclear Information System (INIS)

    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)

  17. 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. PMID:25650339

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

    International Nuclear Information System (INIS)

    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(P2-MG and MRP/β2-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)

  19. Pulmonary alveolar proteinosis

    OpenAIRE

    Patel, Sandeep M; Sekiguchi, Hiroshi; Jordan P Reynolds; Krowka, Michael J.

    2012-01-01

    Pulmonary alveolar proteinosis (PAP) is a disease of alveolar accumulation of phospholipoproteinaceous material that results in gas exchange impairment leading to dyspnea and alveolar infiltrates. There are three forms of PAP: congenital, acquired and idiopathic; of which the latter two are predominant in the adult population. Previous case studies have found that the acquired form can be secondary to various autoimmune, infectious, malignant and environmental etiologies. Recent advances in t...

  20. Pulmonary Alveolar Proteinosis

    OpenAIRE

    Patel, Sandeep M; Hiroshi Sekiguchi; Jordan P Reynolds; Krowka, Michael J.

    2012-01-01

    Pulmonary alveolar proteinosis (PAP) is a disease of alveolar accumulation of phospholipoproteinaceous material that results in gas exchange impairment leading to dyspnea and alveolar infiltrates. There are three forms of PAP: congenital, acquired and idiopathic; of which the latter two are predominant in the adult population. Previous case studies have found that the acquired form can be secondary to various autoimmune, infectious, malignant and environmental etiologies. Recent advances in t...

  1. Primary pulmonary alveolar proteinosis

    OpenAIRE

    Šarac Sanja; Milić Rade; Zolotarevski Lidija; Aćimović Slobodan; Tomić Ilija; Plavec Goran

    2012-01-01

    Introduction. Pulmonary alveolar proteinosis is an uncommon disease characterized by the accumulation of surfactant proteins and phospholipids within the alveolar spaces. Acquired disease can be idiopathic (primary) and secondary. The prevalence of acquired pulmonary alveolar proteinosis is about 0.37 per 100,000 persons. Common symptoms are dyspnea and cough. Chest X-ray shows bilateral perihilar infiltrates. Open-lung biopsy is the gold standard for the diagnosis. Treatment includes w...

  2. Extensive pulmonary alveolar microlithiasis

    OpenAIRE

    Rittayamai, Nuttapol; Muangman, Nisa; Ruangchira-Urai, Ruchira

    2013-01-01

    Pulmonary alveolar microlithiasis is a rare pulmonary disorder that is caused by abnormal sodium-dependent phosphate co-transporter from the mutation of SLC34A2 gene, leading to accumulation of microliths in the alveoli. We report the extensive pulmonary alveolar microlithiasis in an elderly woman who presented with progressive dyspnea for 2 months. Chest radiograph revealed diffuse pulmonary calcification. Tissue histopathology from open lung biopsy demonstrated widespread intra-alveolar lam...

  3. MicroRNA-126 inhibits the proliferation of lung cancer cell line A549

    Institute of Scientific and Technical Information of China (English)

    Xun Yang; Bei-Bei Chen; Ming-Hua Zhang; Xin-Rong Wang

    2015-01-01

    Objective:To study the role of microRNA-126 in the development of lung cancer.Methods:The biological function of microRNA-126 was detected using EdU assay and CCK-8 assay;the target gene of microRNA-126 was analyzed using real time RT-PCR and Western blot assay.Results: In A549 cell line, overexpression of microRNA-126 inhibits the proliferation rate; VEGF is the target gene of microRNA-126; microRNA-126 exerts its function via regulating VEGF protein level.Conclusions: microRNA-126 inhibits the proliferation in A549 cell line.

  4. Rapid elevation of sodium transport through insulin is mediated by AKT in alveolar cells

    OpenAIRE

    Mattes, Charlott; Laube, Mandy; Thome, Ulrich H.

    2014-01-01

    Abstract Alveolar fluid clearance is driven by vectorial Na+ transport and promotes postnatal lung adaptation. The effect of insulin on alveolar epithelial Na+ transport was studied in isolated alveolar cells from 18–19‐day gestational age rat fetuses. Equivalent short‐circuit currents (I SC) were measured in Ussing chambers and different kinase inhibitors were used to determine the pathway of insulin stimulation. In Western Blot measurements the activation of mediators stimulated by insulin ...

  5. Rapid elevation of sodium transport through insulin is mediated by AKT in alveolar cells

    OpenAIRE

    Mattes, Charlott; Thome, Ulrich H.

    2014-01-01

    Alveolar fluid clearance is driven by vectorial Na+ transport and promotes postnatal lung adaptation. The effect of insulin on alveolar epithelial Na+ transport was studied in isolated alveolar cells from 18–19-day gestational age rat fetuses. Equivalent short-circuit currents (ISC) were measured in Ussing chambers and different kinase inhibitors were used to determine the pathway of insulin stimulation. In Western Blot measurements the activation of mediators stimulated by ...

  6. Experimental Study on A549 Cell Death Mediated by Xenoantigen α-gal 
in Human Serum

    Directory of Open Access Journals (Sweden)

    Shengming ZHU

    2012-11-01

    Full Text Available Background and objective The absence of α-gal in humans is caused by the inactivity of α-1,3GT gene. However, humans have pre-existing and abundant anti-gal antibodies. Xenotransplantation procedures have indicated the high potential of introducing α-1,3GT gene to synthesize α-gal for cancer gene therapy by mimicking hyper-acute rejection. The aim of this study is to construct a lung cancer A549 cell line that expressed α-gal, and to observe the antitumor mechanisms mediated by human serum. Methods A549 cells were transfected with pEGFP-N1-GT plasmids constructed in a previous study. A stable transgenic cell line, A549-GT, was then selected and cultivated. The biological characteristics of A549-GT cells, including morphology and proliferation, were examined. α-1,3GT mRNA expression was detected by RT-PCR. Direct immunofluorescence staining and flow cytometry (FCM were used to analyze the synthesis of α-gal in A549-GT. The binding of human serum IgM and C3 with A549-GT were also detected. Results α-1,3GT mRNA was expressed in A549-GT. Direct immunofluorescence staining and FCM indicated a high and stable α-gal expression rate in A549-GT. Compared with parental A549 cells, the biological characteristics of A549-GT were unaltered. α-Gal expression was not detected in the human fetal lung fibroblast cell line MRC-5 even though A549-GT and its culture medium were cultivated with the enzyme. Immunofluorescence staining and FCM also indicated abundant binding between A549-GT treated with human serum and IgM/C3. Conclusion α-Gal expression in tumor cells by gene transduction can induce complement-dependent cytototic antitumor effects.

  7. Alterations in the alveolar epithelium after injury leading to pulmonary fibrosis

    OpenAIRE

    Kasper, M; Haroske, G.

    1996-01-01

    This review discusses current knowledge of the involvement of the alveolar epithelium in tissue remodelling during fibrogenesis. The purpose of the present paper is to give an overview, including the authors' own results, of knowledge of ultrastructural alterations, proliferation kinetics and phenotypic changes of pneumocytes in experimental and clinical pathology of pulmonary fibrosis. After lung injury, the alveolar epithelial cells show ultrastructural alter...

  8. Jamming dynamics of stretch-induced surfactant release by alveolar type II cells

    OpenAIRE

    Majumdar, Arnab; Arold, Stephen P.; Bartolák-Suki, Erzsébet; Parameswaran, Harikrishnan; Suki, Béla

    2011-01-01

    Secretion of pulmonary surfactant by alveolar epithelial type II cells is vital for the reduction of interfacial surface tension, thus preventing lung collapse. To study secretion dynamics, rat alveolar epithelial type II cells were cultured on elastic membranes and cyclically stretched. The amounts of phosphatidylcholine, the primary lipid component of surfactant, inside and outside the cells, were measured using radiolabeled choline. During and immediately after stretch, cells secreted less...

  9. Pro-inflammatory effects and oxidative stress in lung macrophages and epithelial cells induced by ambient particulate matter

    International Nuclear Information System (INIS)

    The objective of this study was to compare the toxicological effects of different source-related ambient PM10 samples in regard to their chemical composition. In this context we investigated airborne PM from different sites in Aachen, Germany. For the toxicological investigation human alveolar epithelial cells (A549) and murine macrophages (RAW264.7) were exposed from 0 to 96 h to increasing PM concentrations (0–100 μg/ml) followed by analyses of cell viability, pro-inflammatory and oxidative stress responses. The chemical analysis of these particles indicated the presence of 21 elements, water-soluble ions and PAHs. The toxicological investigations of the PM10 samples demonstrated a concentration- and time-dependent decrease in cell viability and an increase in pro-inflammatory and oxidative stress markers. -- Highlights: ► The study compares the toxicological effects of different source-related particles with regard to their chemical composition. ► The chemical characterization of the coarse particles revealed clear differences in elemental, TC and PAH composition. ► Equal mass concentrations of urban traffic and rural PM caused different toxicological responses. ► The observations confirm the hypothesis that particle composition, as well as origin, influence the PM-induced toxicity. -- The toxicological responses of lung epithelial cells and macrophages differ significantly after an exposure to equal mass concentrations of urban traffic and rural PM

  10. 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.

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

    International Nuclear Information System (INIS)

    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

  12. Alveolar Surfactant Homeostasis and the Pathogenesis of Pulmonary Disease

    OpenAIRE

    Whitsett, Jeffrey A.; Wert, Susan E.; Weaver, Timothy E.

    2010-01-01

    The alveolar region of the lung creates an extensive epithelial surface that mediates the transfer of oxygen and carbon dioxide required for respiration after birth. Maintenance of pulmonary function depends on the function of type II epithelial cells that synthesize and secrete pulmonary surfactant lipids and proteins, reducing the collapsing forces created at the air-liquid interface in the alveoli. Genetic and acquired disorders associated with the surfactant system cause both acute and ch...

  13. 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. PMID:25573293

  14. Pulmonary alveolar microlithiasis

    OpenAIRE

    Gayathri Devi, H.J.; K N Mohan Rao; Prathima, K. M.; Das, Jayanth K.

    2011-01-01

    Pulmonary alveolar microlithiasis is a rare disease of unknown cause. We report a case in a young boy who presented with history of failure to thrive and chest X-ray finding suggestive of miliary mottling. Open lung biopsy revealed pulmonary alveolar microlithiasis.

  15. Expressions and Significances of PRL-3 and RhoC in A549 Cell

    Directory of Open Access Journals (Sweden)

    Ping ZHANG

    2010-12-01

    Full Text Available Background and objective The expression of phosphatase of regenerating liver-3 (PRL-3 is correlated with Ras homologue C (RhoC in non-small cell lung cancer (NSCLC, suggesting that they have interactions. The aim of this study is to investigate the functions of PRL-3 and RhoC in the migration of A549 cell and the potential mechanism of PRL-3 and RhoC in carcinogenesis and cancer development. Methods PRL-3Ab and RhoCAb were used to block the functions of PRL-3 and RhoC respectively. Wound healing assay was applied to detect the migration of A549 cell and the expression levels of PRL-3 and RhoC were detected by RT-PCR. Results The migration of A549 cell decreased after blockage of PRL-3 and RhoC. The expression of RhoC decreased when PRL-3 was blocked without any changes on the expression of PRL-3. Conclusion PRL-3, RhoC could increase cell migration in A549 cells.

  16. Effects of EPO Gene on Growth and Apoptosis of Lung Adenocarcinoma Cell Line A549

    Directory of Open Access Journals (Sweden)

    Jianqing WU

    2009-09-01

    Full Text Available Background and objective Published data on the association between erythropoietin (EPO and cancer cell are inconclusive. The aim of this study is to investigate the effect of erythropoietin (EPO on the growth and survival of lung adenocarcinoma cell line A549. Methods The recombinant plasmid pcDNA3.1(--hEPO was constructed and transfected into A549 cells by liposome protoco1. The Levels of EPO in culture supernatant were detected by ELISA. Effects of EPO gene on growth and survival of the transfected cells were evaluated by MTT assay and flow cytometry (FCM . Levels of vascular endothelial growth factor (VEGF were also evaluated by ELISA. Results The recombinant eukaryotic expression vector pcDNA3.1(--hEPO was successfully constructed. The growth of cells in hEPO transfected cells was significantly inhibited after transfection (P < 0.01. More cells were blocked in S phase in hEPO transfected group compared with control group (P < 0.05, and the apoptotic rate were also significantly higher than those of their controls (P < 0.01. Levels of VEGF in hEPO transfected cells were significantly lower than controls (P < 0.01. Conclusion Exogenous EPO gene expression in A549 cells can induce cell growth inhibition and apoptosis of A549 cells, and expression of VEGF can also be inhibited.

  17. Chemosensitivity of radioresistant cells in the multicellular spheroids of A549 lung adenocarcinoma

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    Huang Gang

    2009-06-01

    Full Text Available Abstract Background The relapse of cancer after radiotherapy is a clinical knotty problem. Previous studies have demonstrated that the elevation of several factors is likely in some way to lead to the development of treatment tolerance, so it is necessary to further explore the problem of re-proliferated radioresistant cells to chemotherapeutic agents. In the present study, we aimed to investigate the chemosensitivity of radioresistant cells originated from the multicellular spheroids of A549 lung adenocarcinoma. Methods After irradiated with 25 Gy of 6 MV X-ray to A549 multicellular spheroids, whose 10th re-proliferated generations were employed as radioresistant cells, and the control groups were A549 parental cells and MCF7/VCR resistant cells. The chemo-sensitivity test was made by six kinds of chemotherapeutic drugs which were DDP, VDS, 5-Fu, HCP, MMC and ADM respectively, while verapamil (VPL was used as the reversal agent. Then the treatment effect was evaluated by MTT assay, and the multidrug resistant gene expressions of mdr1 and MRP were measured by RT-PCR. Results Both A549 parental cells and A549 derived radioresistant cells were resistant to DDP, but sensitive to VDS, 5-Fu, HCP, MMC and ADM. The inhibitory rates of VPL to these two types of cell were 98% and 25% respectively (P Mdr1/β2-MG and MRP/β2-MG of all A549 cells were about 0 and 0.7 respectively, and those of MCF7/VCR cells were 35 and 4.36. Conclusion The chemosensitivity of A549 radioresistant cells had not changed markedly, and the decreased sensitivity to VPL could not be explained by the gene expression of mdr1 and MRP. It is possible that the changes in the cell membrane and decreased proliferate ability might be attributed to the resistance. Unlike multidrug resistance induced by chemotherapy, VPL may be not an ideal reverser to radioresistant cells. Therefore, the new biological strategy needs to be developed to treat recurring radioresistant tumor in combination

  18. Biomimetics of fetal alveolar flow phenomena using microfluidics.

    Science.gov (United States)

    Tenenbaum-Katan, Janna; Fishler, Rami; Rothen-Rutishauser, Barbara; Sznitman, Josué

    2015-01-01

    At the onset of life in utero, the respiratory system begins as a liquid-filled tubular organ and undergoes significant morphological changes during fetal development towards establishing a respiratory organ optimized for gas exchange. As airspace morphology evolves, respiratory alveolar flows have been hypothesized to exhibit evolving flow patterns. In the present study, we have investigated flow topologies during increasing phases of embryonic life within an anatomically inspired microfluidic device, reproducing real-scale features of fetal airways representative of three distinct phases of in utero gestation. Micro-particle image velocimetry measurements, supported by computational fluid dynamics simulations, reveal distinct respiratory alveolar flow patterns throughout different stages of fetal life. While attached, streamlined flows characterize the shallow structures of premature alveoli indicative of the onset of saccular stage, separated recirculating vortex flows become the signature of developed and extruded alveoli characteristic of the advanced stages of fetal development. To further mimic physiological aspects of the cellular environment of developing airways, our biomimetic devices integrate an alveolar epithelium using the A549 cell line, recreating a confluent monolayer that produces pulmonary surfactant. Overall, our in vitro biomimetic fetal airways model delivers a robust and reliable platform combining key features of alveolar morphology, flow patterns, and physiological aspects of fetal lungs developing in utero. PMID:25759753

  19. Apoptosis of Alveolar Epithelial Cells Induced by Extraction of the Second Stage Larvae of Ascaris lumbricoides%人蛔虫Ⅱ期幼虫提取物诱导人肺上皮细胞凋亡

    Institute of Scientific and Technical Information of China (English)

    彭国华; 袁铿; 周宪民; 彭卫东

    2004-01-01

    目的探讨人蛔虫Ⅱ期幼虫提取物诱导体外培养的人肺上皮细胞A549凋亡,以及提取物浓度和作用时间与细胞凋亡的相互关系.方法根据四氮唑盐酶还原法(MTT)结果,选用5种不同浓度的提取物诱导人肺上皮细胞凋亡.分别在诱导后的5个时间段,采用苏木素-伊红(HE)染色盲法计数和二苯胺法检测DNA断裂率,观察肺上皮细胞凋亡情况.同时对某个时间和浓度组的样本,用DNA琼脂糖凝胶电泳和流式细胞仪检测细胞凋亡.结果不同浓度人蛔虫Ⅱ期幼虫提取物诱导A549细胞凋亡,在5 h之内细胞凋亡率随提取物浓度增加而增加,两者呈正相关关系,且细胞凋亡率显著高于对照组(P<0.05),5 h细胞凋亡率达高峰(65.2%).结论人蛔虫Ⅱ期幼虫提取物可诱导人肺上皮细胞凋亡,细胞凋亡与其提取物浓度呈明显的相关关系.在时间上表现为双向变化关系,其变化程度同时受提取物浓度的影响.

  20. Radix Tetrastigma hemsleyani flavone inhibits proliferation, migration, and invasion of human lung carcinoma A549 cells

    Directory of Open Access Journals (Sweden)

    Zhong LR

    2016-02-01

    Full Text Available Liangrui Zhong,1 Junxian Zheng,2 Qianqian Sun,3 Kemin Wei,2 Yijuan Hu2 1Department of Oncology, Tongde Hospital of Zhejiang Province, Affiliated to Zhejiang Chinese Medical University, 2Department of Chinese Medicine, Zhejiang Academy of Traditional Chinese Medicine, 3Department of Chinese Medicine, Zhejiang Chinese Medical University, Hangzhou, Zhejiang, People’s Republic of China Abstract: Radix Tetrastigma hemsleyani flavone (RTHF is widely used as a traditional herb and has detoxification and anti-inflammatory effects. In this study, we investigated the potential effects of RTHF on the growth and metastasis of human lung adenocarcinoma A549 cells and evaluated its mechanisms. A549 cells were treated with RTHF at various concentrations for different periods. In vitro Cell Counting Kit-8 assay and colony formation methods showed that RTHF had dose- and time-dependent antiproliferation effects on A549 cells. A cell adhesion assay showed that RTHF decreased A549 cell adhesion in a dose-dependent manner. Cell invasion and migration were investigated using the Transwell assay and observed using an inverted microscope; the results showed that cell metastasis was significantly lower in the treatment group than that in the control group (P<0.01. Expression of metastasis-related matrix metalloproteinases (MMPs and tissue inhibitors of metalloproteinases (TIMPs was detected by real-time polymerase chain reaction and Western blotting. The results showed that the expression of MMP-2, MMP-9, and TIMP-1 decreased, while that of TIMP-2 increased significantly in the RTHF group when compared with the results of the control group. These results show that RTHF exhibits antigrowth and antimetastasis activity in lung cancer A549 cells by decreasing the expression of MMP-2/-9 and TIMP-1 and increasing that of TIMP-2. Keywords: flavone, radix Tetrastigma hemsleyani, metastasis, lung cancer

  1. Anti-tumor activity of CrTX in human lung adenocarcinoma cell line A549

    Institute of Scientific and Technical Information of China (English)

    Bin YE; Yan XIE; Zheng-hong QIN; Jun-chao WU; Rong HAN; Jing-kang HE

    2011-01-01

    Aim:To assess the cytotoxic effect of crotoxin (CrTX),a potent neurotoxin extracted from the venom of the pit viper Crotalus durissus terrificus,in human lung adenocarcinoma A549 cells and investigated the underlying mechanisms.Methods:A549 cells were treated with gradient concentrations of CrTX,and the cell cycle and apoptosis were analyzed using a flow cytometric assay.The changes of cellular effectors p53,caspase-3 and cleaved caspase-3,total P38MAPK and pP38MAPK were investigated using Western blot assays.A549 xenograft model was used to examine the inhibition of CrTX on tumor growth in vivo.Results:Treatment of A549 cells with CrTX (25-200 μg/mL) for 48 h significantly inhibited the cell growth in a dose-dependent manner (IC50=78 μg/mL).Treatment with CrTX (25 iJg/mL) for 24 h caused G1 arrest and induced cell apoptosis.CrTX (25 μg/mL) significantly increased the expression of wt p53,cleaved caspase-3 and phospho-P38MAPK.Pretreatment with the specific P38MAPK inhibitor SB203580 (5 μmol/L) significantly reduced CrTX-induced apoptosis and cleaved caspase-3 level,but G1 arrest remained unchanged and highly expressed p53 sustained.Intraperitoneal injection of CrTX (10 μg/kg,twice a week for 4 weeks) significantly inhibited A549 tumor xenograft growth,and decreased MVD and VEGF levels.Conclusion:CrTX produced significant anti-tumor effects by inducing cell apoptosis probably due to activation of P38MAPK and caspase-3,and by cell cycle arrest mediated by increased wt p53 expression.In addition,CrTX displayed anti-angiogenic effects in vivo.

  2. Effect of ionizing radiation on invasiveness of pulmonary adenocarcinoma cells A549 and its mechanism

    International Nuclear Information System (INIS)

    Objective: To investigate the effect of ionizing radiation on the invasion of the pulmonary adenocarcinoma cell line A549. Methods: The invasiveness of A549 cells irradiated with 2 and 4 Gy doses of γ-rays was detected by using transwell invasion assay. The expression levels of matrix metalloproteinase (MMP)-2 mRNA and protein and phosphorylated signal transducers and activators of transcription 3 (STAT3) protein were detected by reverse transcription PCR and Western blot. Results: After irradiation with 2 or 4 Gy, the invasiveness of A549 cells increased by 200.0% (F=111.7, P<0.01) and 390.9% (F=593.7, P<0.01), respectively, compared with that in untreated A549 cells.Furthermore, the transcription and protein expression of MMP-2 24 h after irradiation and the phosphorylation of STAT3 12 h after irradiation were promoted. The irradiation-induced elevation of MMP-2 protein expression was suppressed using STAT3 phosphorylation specific inhibitor (AG490). Moreover, compared with 4 Gy of irradiation alone, treatment with 4 Gy of irradiation plus AG490 decreased the number of invasive cells by 76.1% (F=555.9, P<0.01), and the number of invasive cells in 4 Gy of irradiation plus AG490 group made up only 117.8% of that in untreated group (F=3.6, P>0.05). Conclusions: Ionizing radiation could activate STAT3, which triggers the transcription of MMP-2, and then promote the invasiveness of A549 cells. (authors)

  3. Induction of COX-2 protein expression by vanadate in A549 human lung carcinoma cell line through EGF receptor and p38 MAPK-mediated pathway

    International Nuclear Information System (INIS)

    Vanadate is a transition metal widely distributed in the environment. It has been reported that vanadate associated with air pollution particles can modify DNA synthesis, causing cell growth arrest, and apoptosis. Moreover, vanadium exposure was also found to cause the synthesis of inflammatory cytokines, such as interleukin-1, tumor necrosis factor-α, and prostaglandin E2. Here, we found that exposure of A549 human lung carcinoma cells to vanadate led to extracellular signal-regulated kinase, c-Jun NH2-terminal protein kinases (JNKs), p38 mitogen-activated protein kinase (p38) activation, and COX-2 protein expression in a dose-dependent manner. SB203580, a p38 MAPK inhibitor, but not PD098059 and SP600125, specific inhibitor of MKK1 and selective inhibitor of JNK, respectively, suppressed COX-2 expression. Furthermore, the epithelial growth factor (EGF) receptor specific inhibitor (PD153035) reduced vanadate-induced COX-2 expression. However, scavenging of vanadate-induced reactive oxygen species by catalase, a specific H2O2 inhibitor, or DPI, an NADPH oxidase inhibitor, resulted in no inhibition on COX-2 expression. Together, we suggested that EGF receptor and p38 MAPK signaling pathway may be involved in vanadate-induced COX-2 protein expression in A549 human lung carcinoma cell line

  4. PLAG (1-Palmitoyl-2-Linoleoyl-3-Acetyl-rac-Glycerol) Modulates Eosinophil Chemotaxis by Regulating CCL26 Expression from Epithelial Cells.

    Science.gov (United States)

    Jeong, Jinseon; Kim, Young-Jun; Yoon, Sun Young; Kim, Yong-Jae; Kim, Joo Heon; Sohn, Ki-Young; Kim, Heung-Jae; Han, Yong-Hae; Chong, Saeho; Kim, Jae Wha

    2016-01-01

    Increased number of eosinophils in the circulation and sputum is associated with the severity of asthma. The respiratory epithelium produces chemokine (C-C motif) ligands (CCL) which recruits and activates eosinophils. A chemically synthesized monoacetyl-diglyceride, PLAG (1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol) is a major constituent in the antlers of Sika deer (Cervus nippon Temminck) which has been used in oriental medicine. This study was aimed to investigate the molecular mechanism of PLAG effect on the alleviation of asthma phenotypes. A549, a human alveolar basal epithelial cell, and HaCaT, a human keratinocyte, were activated by the treatment of interleukin-4 (IL-4), and the expression of chemokines, known to be effective on the induction of eosinophil migration was analyzed by RT-PCR. The expression of IL-4 induced genes was modulated by the co-treatment of PLAG. Especially, CCL26 expression from the stimulated epithelial cells was significantly blocked by PLAG, which was confirmed by ELISA. The transcriptional activity of signal transducer and activator of transcription 6 (STAT6), activated by IL-4 mediated phosphorylation and nuclear translocation, was down-regulated by PLAG in a concentration-dependent manner. In ovalbumin-induced mouse model, the infiltration of immune cells into the respiratory tract was decreased by PLAG administration. Cytological analysis of the isolated bronchoalveolar lavage fluid (BALF) cells proved the infiltration of eosinophils was significantly reduced by PLAG. In addition, PLAG inhibited the migration of murine bone marrow-derived eosinophils, and human eosinophil cell line, EoL-1, which was induced by the addition of A549 culture medium. PMID:27010397

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

    Directory of Open Access Journals (Sweden)

    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.

  6. Capsaicinoids cause inflammation and epithelial cell death through activation of vanilloid receptors.

    Science.gov (United States)

    Reilly, Christopher A; Taylor, Jack L; Lanza, Diane L; Carr, Brian A; Crouch, Dennis J; Yost, Garold S

    2003-05-01

    Capsaicinoids, found in less-than-lethal self-defense weapons, have been associated with respiratory failure and death in exposed animals and people. The studies described herein provide evidence for acute respiratory inflammation and damage to epithelial cells in experimental animals, and provide precise molecular mechanisms that mediate these effects using human bronchiolar and alveolar epithelial cells. Inhalation exposure of rats to pepper sprays (capsaicinoids) produced acute inflammation and damage to nasal, tracheal, bronchiolar, and alveolar cells in a dose-related manner. In vitro cytotoxicity assays demonstrated that cultured human lung cells (BEAS-2B and A549) were more susceptible to necrotic cell death than liver (HepG2) cells. Transcription of the human vanilloid receptor type-1, VR1 or TRPV1, was demonstrated by RT-PCR in all of these cells, and the relative transcript levels were correlated to cellular susceptibility. TRPV1 receptor activation was presumably responsible for cellular cytotoxicity, but prototypical functional antagonists of this receptor were cytotoxic themselves, and did not ameliorate capsaicinoid-induced damage. Conversely, the TRPV1 antagonist capsazepine, as well as calcium chelation by EGTA ablated cytokine (IL-6) production after capsaicin exposure. To address these seemingly contradictory results, recombinant human TRPV1 was cloned and overexpressed in BEAS-2B cells. These cells exhibited dramatically increased cellular susceptibility to capsaicinoids, measured using IL-6 production and cytotoxicity, and an apoptotic mechanism of cell death. Surprisingly, the cytotoxic effects of capsaicin in TRPV1 overexpressing cells were also not inhibited by TRPV1 antagonists or by treatments that modified extracellular calcium. Thus, capsaicin interacted with TRPV1 expressed by BEAS-2B and other airway epithelial cells to cause the calcium-dependent production of cytokines and, conversely, calcium-independent cell death. These results

  7. Epithelial cell invasion and survival of Bordetella bronchiseptica.

    OpenAIRE

    SCHIPPER, H; Krohne, G F; R. Gross

    1994-01-01

    Wild-type Bordetella bronchiseptica and a bvg mutant strain were used for invasion and survival experiments in human Caco-2 and A549 epithelial cells. Both bacterial strains were able to enter and persist within the host cells for at least a week. A significant proportion of the bacteria from both B. bronchiseptica strains but not from Bordetella pertussis were found free in the cytoplasm, suggesting different invasion and survival strategies of the two species in epithelial cells.

  8. Liposome-mediated IL-28 and IL-29 expression in A549 cells and antiviral effect of IL-28 and IL-29 on WISH cells

    Institute of Scientific and Technical Information of China (English)

    Ming-cai LI; Hao-yang WANG; Hai-yan WANG; Tao LI; Shao-heng HE

    2006-01-01

    Aim:To construct the recombinant vectors carrying interleukin (IL) -28A,IL-28B and IL-29 cDNAs and express them in human A549 cells,and analyze their antiviral activity in vesicular stomatitis virus (VSV)-infected human immortalized amnion epithelial cell line (WISH cells).Methods:Total cell RNA was extracted from human peripheral blood mononuclear cells (PBMC) activated with poly I:C.The cDNAs encoding human IL-28A.IL-28B.and IL-29 were amplified by reversetranscription polymerase chain reaction (RT-PCR) and inserted into pcDNA3.1/V5-His-TOPO vectors.These recombinant vectors were transfected into human A549 cells by a liposome-mediated gene transfer method.Semiquantitative RT-PCR and Westem blotting were used to detect the mRNA and protein expression of IL-28A,IL-28B,and IL-29.The antiviral activity of IL-28A,IL.28B,and IL-29 was determined by a cytopathic eflfect reduction assay on WISH cells using VSV as a challenge virus.Results:The DNA sequences of the inserts were identical to the published sequences encoding IL-28A,IL-28B,and IL-29 in GenBank.It was transfected cells.Expression of all 3 interleukins in A549 cells was confirmed by Wlestem blot analysis.IL-28 and IL-29 expressed by A549 cells.1ike interferon (IFN)α-2b,were able to protect WISH cells against VSV infection.Conclusion:IL-28 and IL-29 cDNAs were successfully cloned and expressed in eukaryotic cells via transfection with pcDNA3.1/V5-His-TOPO-IL-28/IL-29.Transfection with this vector produced a specific antiviral activity similar to that of IFN-α.which will provide a new tool for the functional study of these cytokines in humans.

  9. 氨甲蝶呤对映体对肺癌A549细胞的生长抑制作用研究%Inhibitory effects of methotrexate enantiomers on the growth of human lung cancer A549 cells

    Institute of Scientific and Technical Information of China (English)

    陶绍能; 王莹莹; 周建国; 程光华; 张梦莹; 钟民; 吕坤

    2015-01-01

    目的:研究氨甲喋呤(MTX)对映体对肺癌A549细胞的生长抑制作用.方法:倒置相差显微镜观察加入MTX对映体后细胞形态变化,应用MTT法检测MTX对映体对A549细胞的生长抑制作用,应用流式细胞术分析细胞周期分布及凋亡率的变化.结果:倒置相差显微镜观察加入MTX对映体后细胞形态发生明显变化,MTT法检测表明两种MTX对映体抑制A549细胞的生长呈剂量与时间依赖性,L-(+)-MTX对A549细胞的抑制作用明显强于D-(-)-MTX.流式细胞检测发现两种MTX对映体药物对A549细胞的细胞周期和凋亡具有明显干扰作用.结论:MTX对映体对A549细胞的作用明显具有手性差异,L-(+)-MTX对A549细胞的抑制作用明显强于D-(-)-MTX.

  10. 低氧对人肺腺癌A549细胞迁移和黏附的影响%Effect of hypoxia on migration, invasion and adhesion to endothelium of human pulmonary adenocarcinoma A549 cells

    Institute of Scientific and Technical Information of China (English)

    Weigan Shen; Jun Zhu; Zhiyong Yu; Qingyu Xue

    2008-01-01

    Objective:To evaluate the effect of hypoxia on migration,invasion and adhesion to endothelium of human pulmonary adenocarcinoma A549 cells.Methods:Wound-healing and Transwell invasion assays were performed to study the effect of hypoxia on migration and invasion of A549 cells,and A549 cells were added to a monolayer of human umbilical vein endothelial cells (HUVECs) to test the ability to adhere to endothelium.Immunofluorescence assay and luciferase reporter gene assay were also used to evaluate the effect of hypoxia on distribution of E-cadherin,β-catenin,and actin,and hypoxia-inducible factor-1 (HIF-1)-dependent transcription,respectively.Results:Hypoxia facilitated A549 cell migration,invasion,and A549 cell-endothelial cells adhesion,and modulated the distribution of E-cadherin and β-catenin,and actin cytoskeleton rearrangement,and up-regulated HIF-1-dependent reporter gene expression in A549 cells.Conclusion:Promotion of A549 cell migration,invasion,and adhesion on endothelium by hypoxia might be modulated through its up-regulating HIF-l-dependent gene expression,which then induced the redistribution of E-cadherin and β-catenin,and the actin cytoskeletal reorganization.

  11. Congenital Pulmonary Alveolar Proteinosis

    OpenAIRE

    Saber Hammami; Khaled Harrathi; Khaled Lajmi; Samir Hadded; Chebil Ben Meriem; Mohamed Néji Guédiche

    2013-01-01

    Pulmonary alveolar proteinosis (PAP) is a rare syndrome characterized by pulmonary surfactant accumulation within the alveolar spaces. It occurs with a reported prevalence of 0.1 per 100,000 individuals. Two clinically different pediatric types have been defined as congenital PAP which is fatal and a late-onset PAP which is similar to the adult form and less severe. The clinical course of PAP is variable, ranging from spontaneous remission to respiratory failure. Whole-lung lavage is the curr...

  12. Apoptotic Effect of Coix Polysaccharides on A549 Lung Cancer Cells in Vitro

    OpenAIRE

    Luo, Cheng; Liu, Wei; Lu, Xiangyi

    2012-01-01

    Background and objective Coix seeds are commonly used in Traditional Chinese Medicine and ingested through daily diet. The aim of this study is to analyze the apoptotic effect of coix polysaccharides on A549 cells. Methods A fraction of polysaccharides was isolated from coix seeds and extracted by ethanol precipitation. The extract was then purified by dialysis and DEAE-52 ion-exchange chromatography. Cell viability was determined by the MTT assay. Cell morphology was observed by scanning ele...

  13. Brusatol Enhances the Radiosensitivity of A549 Cells by Promoting ROS Production and Enhancing DNA Damage

    Directory of Open Access Journals (Sweden)

    Xiaohui Sun

    2016-06-01

    Full Text Available NF-E2-related factor 2 (Nrf2 has been identified as a master regulatory factor in the protection of cells from oxidative and electrophilic stress. However, overexpression of Nrf2 in lung cancer may cause chemoresistance, as well as radioresistance. In this study, we examined the relationship between radioresistance and Nrf2 protein levels in H1299, A549, and H460 cells, and finally chose the A549 cell line to continue with due to its strong radioresistance and high Nrf2 protein levels. We found that the Nrf2 inhibitor, brusatol, could prevent the increase and accumulation of Nrf2 after exposure to irradiation. Additionally, following treatment with 80 nM brusatol, A549 cells became sensitive to irradiation, suffering severe DNA damage. Combination treatment with brusatol and ionizing radiation (IR can distinctly increase the level of reactive oxygen species in A549 cells, causing a 1.8-fold increase compared with the control, and a 1.4-fold increase compared with IR alone. In fact, in the treatment with both brusatol and IR, lung cancer cell proliferation is halted, gradually leading to cell death. Because Nrf2 is closely linked to DNA damage repair, inhibiting the function of Nrf2, as in brusatol treatment, may increase the DNA damage caused by radiotherapy or chemotherapy, possibly enhancing the efficacy of chemotherapeutic drugs. Our study is the first to demonstrate brusatol’s ability to enhance the responsiveness of lung cancer cells to irradiation, and its potential application as a natural sensitizer in radiotherapy.

  14. Brusatol Enhances the Radiosensitivity of A549 Cells by Promoting ROS Production and Enhancing DNA Damage.

    Science.gov (United States)

    Sun, Xiaohui; Wang, Qin; Wang, Yan; Du, Liqing; Xu, Chang; Liu, Qiang

    2016-01-01

    NF-E2-related factor 2 (Nrf2) has been identified as a master regulatory factor in the protection of cells from oxidative and electrophilic stress. However, overexpression of Nrf2 in lung cancer may cause chemoresistance, as well as radioresistance. In this study, we examined the relationship between radioresistance and Nrf2 protein levels in H1299, A549, and H460 cells, and finally chose the A549 cell line to continue with due to its strong radioresistance and high Nrf2 protein levels. We found that the Nrf2 inhibitor, brusatol, could prevent the increase and accumulation of Nrf2 after exposure to irradiation. Additionally, following treatment with 80 nM brusatol, A549 cells became sensitive to irradiation, suffering severe DNA damage. Combination treatment with brusatol and ionizing radiation (IR) can distinctly increase the level of reactive oxygen species in A549 cells, causing a 1.8-fold increase compared with the control, and a 1.4-fold increase compared with IR alone. In fact, in the treatment with both brusatol and IR, lung cancer cell proliferation is halted, gradually leading to cell death. Because Nrf2 is closely linked to DNA damage repair, inhibiting the function of Nrf2, as in brusatol treatment, may increase the DNA damage caused by radiotherapy or chemotherapy, possibly enhancing the efficacy of chemotherapeutic drugs. Our study is the first to demonstrate brusatol's ability to enhance the responsiveness of lung cancer cells to irradiation, and its potential application as a natural sensitizer in radiotherapy. PMID:27347930

  15. The biophysical property of A549 cells transferred by VEGF-D.

    Science.gov (United States)

    Wang, Zhen; Wu, Xiu-Li; Wang, Xu; Tian, Hong-Xia; Chen, Zhi-Hong; Li, Yang-Qiu

    2014-01-01

    Vascular endothelial growth factor-D (VEGF-D) together with VEGF-C is considered to be associated with lymphangiogenesis and angiogenesis and involve in tumorization. This study aims to investigate the influence of exogenous VEGF-D gene on the biophysical property of cell surface of lung adenocarcinoma cell line. A panel of lung adenocarcinoma cell lines were examined the expression of VEGF-D and VEGF-C by real-time PCR. The VEGF-D recombinant plasmid containing enhanced green fluorescence protein (EGFP) was constructed and transfected to the cell line with no expression of VEGF-D and confirmed by real-time PCR and Western blot analysis. Topographic images of cells were obtained by using atomic force microscope (AFM) in contact mode. Unlike VEGF-C, VEGF-D was found to have a very low expression or undetectable expression in lung adenocarcinoma cell lines. The VEGF-D recombinant plasmid had been constructed successfully and was transferred into the human lung adenocarcinoma cell line A549 cells which had no endogenous expression of VEGF-D, and exogenous VEGF-D could be detected in mRNA and protein expression levels in the gene modified cells, while the VEGF-C gene expression had no change after VEGF-D transfection. After transfection, the irregular microspikes or nano clusters could observe on the surface of A549 cells, and VEGF-D transfected A549 cells became more rigid. The exogenous VEGF-D gene might cause the remarkable biophysical architectural changes in the A549 cells, which might as a novel biomarker for evaluation of its biological function. PMID:23526563

  16. Apoptotic Effect of Coix Polysaccharides on A549 Lung Cancer Cells in Vitro

    Directory of Open Access Journals (Sweden)

    Cheng LUO

    2012-11-01

    Full Text Available Background and objective Coix seeds are commonly used in Traditional Chinese Medicine and ingested through daily diet. The aim of this study is to analyze the apoptotic effect of coix polysaccharides on A549 cells. Methods A fraction of polysaccharides was isolated from coix seeds and extracted by ethanol precipitation. The extract was then purified by dialysis and DEAE-52 ion-exchange chromatography. Cell viability was determined by the MTT assay. Cell morphology was observed by scanning electronic microscopy (SEM, and cell cycle was detected by flow cytometry (FCM. The relative quantities of caspase-3 and caspase-9 were determined by RT-PCR. Results Coix polysaccharides exerted remarkable inhibitory effects on A549 cell proliferation. Apoptotic bodies were observed by SEM. Apoptotic induction was also verified by DNA accumulation using propidium iodide nucleus staining in the S phase by flow cytometry, as well as by DNA fragmentation using the comet assay. Regarding the molecular mechanism of apoptosis induction, the gene expression of caspase-3 and caspase-9 increased after coix polysaccharide treatment. Conclusion Polysaccharide fraction CP-1 induced A549 cell apoptosis.

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

    International Nuclear Information System (INIS)

    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.

  18. Effect of evodiamine on the proliferation and apoptosis of A549 human lung cancer cells.

    Science.gov (United States)

    Lin, Li; Ren, Li; Wen, Liujing; Wang, Yu; Qi, Jin

    2016-09-01

    Evodia rutaecarpa is a plant, which has antitumor activity. Evodiamine is an alkaloid with antitumor activity present in E. rutaecarpa and has potential to be developed into a therapeutic antitumor agent. The present study investigated the effect of evodiamine on the proliferation of A549 human lung cancer cells and the mechanism underlying these effects. The results indicated that evodiamine significantly inhibited proliferation, induced apoptosis and the expression of reactive oxygen species, arrested the cell cycle, regulated the expression of Survivin, Bcl-2 and Cyclin B1, regulated the activity of caspase-3/8 and glutathione in tumor cells, and decreased the activity of AKT/nuclear factor‑κB (NF‑κB) and Sonic hedgehog/GLI family zinc finger 1 (SHH/GLI1) signaling pathways in A549 cells. In conclusion, the evodiamine-induced inhibition of the proliferation of A549 lung cancer cells may be attributable to its ability to promote oxidative injury in the cells, induce apoptosis, arrest the cell cycle and regulate the AKT/NF‑κB and SHH/GLI1 signaling pathways, subsequently controlling the expression of tumor‑associated genes. PMID:27485202

  19. MicroRNA-1228(*) inhibit apoptosis in A549 cells exposed to fine particulate matter.

    Science.gov (United States)

    Li, Xiaobo; Ding, Zhen; Zhang, Chengcheng; Zhang, Xin; Meng, Qingtao; Wu, Shenshen; Wang, Shizhi; Yin, Lihong; Pu, Yuepu; Chen, Rui

    2016-05-01

    Studies have reported associations between fine particulate matter (PM2.5) and respiratory disorders; however, the underlying mechanism is not completely clear owing to the complex components of PM2.5. microRNAs (miRNAs) demonstrate tremendous regulation to target genes, which are sensitive to exogenous stimulation, and facilitate the integrative understood of biological responses. Here, significantly modulated miRNA were profiled by miRNA microarray, coupled with bioinformatic analysis; the potential biological function of modulated miRNA were predicted and subsequently validated by cell-based assays. Downregulation of miR-1228-5p (miR-1228(*)) expression in human A549 cells were associated with PM2.5-induced cellular apoptosis through a mitochondria-dependent pathway. Further, overexpression of miR-1228(*) rescued the cellular damages induced by PM2.5. Thus, our results demonstrate that PM2.5-induced A549 apoptosis is initiated by mitochondrial dysfunction and miR-1228(*) could protect A549 cells against apoptosis. The involved pathways and target genes might be used for future mechanistic studies. PMID:26867688

  20. Factors influencing recovery from potentially lethal radiation damage in A549 human lung carcinoma cells

    International Nuclear Information System (INIS)

    Plateau phase A549 cells exhibit potentially lethal radiation damage recovery (PLDR) that is dependent upon both the pH and the glucose content of the spent medium. At 9-10 days after plating, unfed A549 plateau cultures are acidic (pH 6.5 - 6.7), contain 2 to 4 mM glucose, and exhibit an approximately 40-fold increase in survival when held for 6 hrs in spent medium vs being subcultured immediately after 10 Gy aerobic irradiation. PLDR is maximal 24 hrs. post-irradiation. Adjustment of the pH of the spent medium to 7.5, by NaOH addition, either prior to or immediately post irradiation, nearly completely inhibits PLDR in this cell line. The authors found that medium acidity inhibits glucose utilization, and that alkalinization of spent medium, to pH 7.5, results in stimulation of glucose consumption. Plateau phase cultures depleted of glucose, as a result of medium alkalinization, are not capable of PLDR. In addition to pH effects, they observed that several agents, including nicotinamide, 3-aminobenzamide, caffeine, 2-deoxyglucose and glucosamine, partially inhibit PLDR in A549 plateau phase cultures

  1. Effect of Juglone in qinglongyi on cell cycle status and apoptosis in A-549 cells

    Institute of Scientific and Technical Information of China (English)

    ZOU Xiang; KONG Ling-sheng; JI Yu-bin

    2008-01-01

    Objective To explore the inhibition of juglone in Qinglongyi on A-549 cells in vitro. Methods MTT assay was used. Laser confocal scanning microscope was used to observe apoptotic morphology.Changes of cell cycle are studied by flow cytometry analysis. Results MTT assay showed that juglone had a marked growth inhibition in A-549 cells and the IC50 is respectively 3.4×10-5 mol·L-1, 1.8×10-5 mol·L-1 and 2.6×10-6 mol·L-1 after treatment for 24, 48 and 72 h by juglone. Through Laser confocal scanning microscope, we can see that juglone can induce the apoptosis. Cell cycle changes are analyzed by flow cytometry with cells at G1 phase significantly less than those of control and ceils at G2 phase significantly more than those of control. Conclusions It suggests that juglone could apoptosis of A-549 cells with the cell cycle arrest on G2 phase in distinct dose-dependent manner.

  2. Effect of Long Non-coding RNA SPRY4-IT1 on Invasion and Migration of A549 Cells

    Directory of Open Access Journals (Sweden)

    Song CHAI

    2015-08-01

    Full Text Available Background and objective The abnormal expression of human long chain non encoding RNA gene is related to many kinds of tumors. The aim of this study is to investigate the expression of long non-coding RNA maternally expressed gene 3 (SPRY4-IT1 in lung cancer (A549 cells, and to observe the effect of SPRY4-IT1 on the invasion and migration of A549 cells. Methods The levels of SPRY4-IT1 in A549 was detected by Real-time PCR. The effects of SPRY4-IT1 on the invasion and migration of A549 cell were analyzed by MTT and Transwell assay. The expression of matrix metalloproteinase (MMP family proteins was determined by Western blot. Results The invasion and migration of A549 cells were increased after SPRY4-IT1 over-expression. The cell spaces were narrower after SPRY4-IT1 over expression in the wound healing assay. Transwell assays showed that the numbers of transmembrane A549 cells were significantly higher in SPRY4-IT1 over expression group than that in control group (P<0.05. Meanwhile, over expression of SPRY4-IT1 reduced the expression of MMP-2 and MMP-9. Conclusion Over expression of SPRY4-IT1 enhanced the invasion and migration of A549 cells. MMP-2 and MMP-9 might play an important role in this regulation.

  3. Enrichment and identification of lung adenocarcinoma initiating cells from A 549%A549肺腺癌始动细胞的富集和鉴定

    Institute of Scientific and Technical Information of China (English)

    林盛; 张振华; 饶明月; 吴敬波

    2013-01-01

    Objective To obtain the lung adenocarcinoma initiating cells from the A 549 cell line based on paclitaxel treatment combination with serum-free cultivation and to validate spared cells can represent tumor initiating cells (TICs) .Methods After dis-sociated by trypsogen ,about 106 /mL cells were suspended in serum-free medium supplemented with 0 .4% bovine serum albumin (BSA) ,insulin ,basic fibroblast growth factor (bFGF) ,human recombinant epidermal growth factor (EGF) and obtained spheroid cells .At the second passage ,paclitaxel was added at a concentration of 100 nmol/L for 48 h and then replaced with completely fresh medium once or twice per week until new spheroids emerged .Results The subpopulation of cells that survived serum-free cultiva-tion and paclitaxel treatment could highly express the cluster of differentiation 133/cluster of differentiation (CD133/CD326) mo-lecular markers and have features of stemness including differentiation ,high expression of cancer stem cells (CSCs)-associated genes and stronger capability of tumorigenesis .Conclusion The survived subpopulation that highly express the CD 133/CD326 molecu-lar markers presenting the characteristics of stemness in vitro and in vivo ,and could be used in future researches of biological functions .%目的:利用紫杉醇联合无血清培养完成对 A549肺腺癌始动细胞的富集并鉴定富集亚群的干细胞特性。方法对数生长期的 A549细胞经胰酶消化,干细胞培养基重悬,得到成球状生长的细胞;传至第2代时加入紫杉醇作用48 h ,离心去除死细胞和紫杉醇,换新鲜干细胞培养基培养,至存活细胞恢复克隆生长后鉴定其干细胞相关特性。结果紫杉醇联合无血清培养方式成功从 A549细胞中富集得到肿瘤干细胞,该群细胞高表达分化抗原簇蛋白133/人上皮细胞黏附分子(CD133/CD326),具有多向分化潜能、高表达干细胞相关基因及更强的致瘤能力,具备

  4. 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

  5. Macrophage-epithelial interactions in pulmonary alveoli.

    Science.gov (United States)

    Bhattacharya, Jahar; Westphalen, Kristin

    2016-07-01

    Alveolar macrophages have been investigated for years by approaches involving macrophage extraction from the lung by bronchoalveolar lavage, or by cell removal from lung tissue. Since extracted macrophages are studied outside their natural milieu, there is little understanding of the extent to which alveolar macrophages interact with the epithelium, or with one another to generate the lung's innate immune response to pathogen challenge. Here, we review new evidence of macrophage-epithelial interactions in the lung, and we address the emerging understanding that the alveolar epithelium plays an important role in orchestrating the macrophage-driven immune response. PMID:27170185

  6. 莪术油对人肺腺癌细胞A549增殖的影响%Effect of Zedoary Turmeric Oil on Proliferation in Human Lung Adenocarcinoma Cell Line A549

    Institute of Scientific and Technical Information of China (English)

    王晓波; 牛建昭; 崔巍; 刘飒; 杨长福; 赵丕文; 唐炳华

    2011-01-01

    目的 探讨莪术油对人肺腺癌细胞A549增殖的抑制作用.方法 体外培养肺腺癌细胞A549,MTT比色法测定莪术油对A549细胞作用24、48、72 h后抑制率;流式细胞术分析莪术油对A549细胞作用24 h后细胞周期的变化;Annexin V-FITC/PI双染检测莪术油对A549细胞作用24 h后细胞凋亡与坏死情况.结果 莪术油对A549细胞增殖的抑制率随时间延长明显升高,随药物浓度增加抑制作用增强;莪术油对A549细胞作用24 h后,细胞周期停滞在G0/G1期,阻止其进入S期;细胞的早期凋亡、晚期凋亡和坏死比例随着莪术油浓度的增加而增加,且坏死细胞的比例高于凋亡细胞.结论 莪术油对A549细胞的增殖具有抑制作用,并呈时间、浓度依赖,其作用是通过阻滞细胞周期及诱导凋亡和坏死采实现的.%Objective To explore the inhibiting effect of Zedoary turmeric oil on the proliferation of A549 cell line. Methods Lung adenocarcinoma cell line A549 was cultured in vitro. The inhibition rate of Zedoary turmeric oil on the proliferation of lung adenocarcinoma cell line A549 for 24, 48, 72 h were determined by MTT colorimetric assay. The cell cycle of lung adenocarcinoma cell line A549 stimulated by Zedoary turmeric oil for 24 h was analyzed by flow cytometry. The apoptosis and necrosis of lung adenocarcinoma cell line A549 stimulated by Zedoary turmeric oil for 24 h was tested by Annexin V-FITC/PI assay. Results MTT assay indicated that the inhibition rate of Zedoary turmeric oil on the proliferation of lung adenocarcinoma cell line A549 increased significantly with the growing of time and concentration. Further analysis by flow cytometry indicated that Zedoary turmeric oil stimulating the A549 cells for 24 h led to Go/Gi phase arrest and blocked S phase entry. Meanwhile cells in early apoptosis, late apoptosis and necrosis were increased, and the percentage of necrotic cells was more than apoptotic cells with the increase of

  7. Pulmonary alveolar proteinosis

    Directory of Open Access Journals (Sweden)

    B. Crestani

    2011-06-01

    Full Text Available Pulmonary alveolar proteinosis (PAP is a rare pulmonary disease characterised by alveolar accumulation of surfactant. It may result from mutations in surfactant proteins or granulocyte macrophage-colony stimulating factor (GM-CSF receptor genes, it may be secondary to toxic inhalation or haematological disorders, or it may be auto-immune, with anti-GM-CSF antibodies blocking activation of alveolar macrophages. Auto-immune alveolar proteinosis is the most frequent form of PAP, representing 90% of cases. Although not specific, high-resolution computed tomography shows a characteristic “crazy paving” pattern. In most cases, bronchoalveolar lavage findings establish the diagnosis. Whole lung lavage is the most effective therapy, especially for auto-immune disease. Novel therapies targeting alveolar macrophages (recombinant GM-CSF therapy or anti-GM-CSF antibodies (rituximab and plasmapheresis are being investigated. Our knowledge of the pathophysiology of PAP has improved in the past 20 yrs, but therapy for PAP still needs improvement.

  8. Pulmonary Alveolar Proteinosis

    Directory of Open Access Journals (Sweden)

    Sandeep M Patel

    2012-01-01

    Full Text Available Pulmonary alveolar proteinosis (PAP is a disease of alveolar accumulation of phospholipoproteinaceous material that results in gas exchange impairment leading to dyspnea and alveolar infiltrates. There are three forms of PAP: congenital, acquired and idiopathic; of which the latter two are predominant in the adult population. Previous case studies have found that the acquired form can be secondary to various autoimmune, infectious, malignant and environmental etiologies. Recent advances in the understanding of the pathophysiology of PAP demonstrate that the idiopathic form is due to antigranulocyte macrophage-colony stimulating factor antibodies. Therapeutic targets that replace granulocyte macrophage colony stimulating factor or remove these antibodies are being actively developed. The current standard of care is to perform whole lung lavage on these patients to clear the alveolar space to help improve respiratory physiology. A case of PAP is reported, followed by a literature review on the diagnosis and management of this rare condition with the aim of increasing awareness among physicians when treating patients who present with alveolar infiltrates.

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

    International Nuclear Information System (INIS)

    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, 14C-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, [14C]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

  10. An estimation of mechanical stress on alveolar walls during repetitive alveolar reopening and closure.

    Science.gov (United States)

    Chen, Zheng-Long; Song, Yuan-Lin; Hu, Zhao-Yan; Zhang, Su; Chen, Ya-Zhu

    2015-08-01

    Alveolar overdistension and mechanical stresses generated by repetitive opening and closing of small airways and alveoli have been widely recognized as two primary mechanistic factors that may contribute to the development of ventilator-induced lung injury. A long-duration exposure of alveolar epithelial cells to even small, shear stresses could lead to the changes in cytoskeleton and the production of inflammatory mediators. In this paper, we have made an attempt to estimate in situ the magnitudes of mechanical stresses exerted on the alveolar walls during repetitive alveolar reopening by using a tape-peeling model of McEwan and Taylor (35). To this end, we first speculate the possible ranges of capillary number (Ca) ≡ μU/γ (a dimensionless combination of surface tension γ, fluid viscosity μ, and alveolar opening velocity U) during in vivo alveolar opening. Subsequent calculations show that increasing respiratory rate or inflation rate serves to increase the values of mechanical stresses. For a normal lung, the predicted maximum shear stresses are surface tension or viscosity, the maximum shear stress will notably increase, even at a slow respiratory rate. Similarly, the increased pressure gradients in the case of elevated surface or viscosity may lead to a pressure drop >300 dyn/cm(2) across a cell, possibly inducing epithelial hydraulic cracks. In addition, we have conceived of a geometrical model of alveolar opening to make a prediction of the positive end-expiratory pressure (PEEP) required to splint open a collapsed alveolus, which as shown by our results, covers a wide range of pressures, from several centimeters to dozens of centimeters of water, strongly depending on the underlying pulmonary conditions. The establishment of adequate regional ventilation-to-perfusion ratios may prevent recruited alveoli from reabsorption atelectasis and accordingly, reduce the required levels of PEEP. The present study and several recent animal experiments likewise

  11. 去甲斑蝥素对人肺腺癌A549细胞的抑制作用%The inhibition of norcantharidin on human lung adenocarcinoma A549 cells

    Institute of Scientific and Technical Information of China (English)

    崔宝弟; 王敏; 孙震晓

    2015-01-01

    OBJECTIVE:To study the effects of norcantharidin (NCTD) on human lung cancer cells,and investigate the mechanisms.METHODS:The growth inhibition of A549 cells treated with 0-240µmol/L NCTD for 0-72 hours was analyzed by MTT assay. The recovery growth and proliferation of A549 cells treated with 0-120µmol/L NCTD for 24 h was evaluated by MTT assay. The morphological changes of A549 cells treated with 40,50 and 60µmol/L NCTD for 0-72 h were examined under inverted microscope. The apoptosis and cell cycle changes of A549 cells treated with 40-60µmol/L NCTD were detected by flow cytometry.RESULTS:NCTD inhibited the growth of A549 cells in 30-240 µmol/L(P0.05)。结论:40~60µmol/L NCTD主要通过诱导A549细胞G2~M期阻滞而抑制细胞生长。

  12. Alveolar damage in AIDS-related Pneumocystis carinii pneumonia

    DEFF Research Database (Denmark)

    Benfield, T L; Prentø, P; Junge, Jette;

    1997-01-01

    P carinii pneumonia, whereas none without P carinii pneumonia had this finding (p < 0.05). Erosion of the type II pneumocyte was not observed. CONCLUSION: Inflammation, interstitial fibrosis, and alveolar epithelial erosion are characteristic features of P carinii pneumonia. The changes may form the......, exudate, fibrosts, type II pneumocyte proliferation, and cellular infiltration of the alveolar wall when compared with other lung diseases (all p < 0.05). Electron microscopy showed apposition of the trophozoite to the type I pneumocyte. Erosion of type I pneumocytes was observed in 13 of 15 patients with...

  13. Effect of antisense transfecting of monocarboxylate transporter gene on biological characteristics of lung adenocarcinoma A549 cells

    Institute of Scientific and Technical Information of China (English)

    ZHANG Gui-zhi; HUANG Gui-jun; GUO Xian-jian; QIAN Gui-sheng

    2002-01-01

    Objective: To study the influence of transfecting antisense expression vector of the first subtype of the monocarboxylate transporter (MCT1) gene into lung cancer cells on pHi regulation, lactate transportation and cell growth, Methods: MCT1 antisense gene recombinant vector was introduced into human lung cancer cell line A549 by electroporation. The transfected A549 cells resistant to G418 were selected. Positive clones were examined by using PCR. The changes of intracellular pH and lactate were examined with spectrophotometric method. Cell growth was studied with cell growth curve. Results: Intracellular pH and lactate were remarkably decreased in the cells transfected pLXSN-MCT1 in comparison with A549 cells without transfection (P<0. 001). The growth of A549 cells transfected pLXSN-MCT1 was also inhibited remarkably. Conclusion: MCT1 gene may play an important role in pHi regulation, lactate transportation and cell growth in tumor cells.

  14. Roles of neuropeptide substance P in transdifferentiation of premature rat type Ⅱ alveolar epithelial cells induced by hyperxia%神经肽P物质促进高氧诱导的胎鼠肺泡Ⅱ型上皮细胞转分化

    Institute of Scientific and Technical Information of China (English)

    黄波; 付红敏; 杨鸣; 方芳; 匡凤梧; 许峰

    2009-01-01

    目的 探讨感觉神经肽P物质(substance P,SP)对高氧诱导的胎鼠Ⅱ型肺泡上皮细胞(type Ⅱ alveolar epithelial cells,AECⅡ)转分化的影响.方法 剖官取出孕21 d(足月为22 d)SD胎鼠,分离纯化原代培养AECⅡ,采用随机分组法分为:空气暴露组(氧体积分数为0.21)、高氧暴露组(氧体积分数为0.95)、SP干预组,SP干预组于暴露前加入SP 1×10-6 mol/L,在置于氧体积分数为0.21和0.95中分别暴露12、24、和48 h,电镜观察AECⅡ的形态变化;免疫细胞化学染色法和流式细胞仪检测AECⅡ特异性肺泡表而活性蛋白-C(surfactant protein C,sp-C)及Ⅰ型肺泡上皮细胞(type Ⅰ alveolar epithelial cells,AEC Ⅰ)特异性水通道蛋5(aquaporin5,AQP5)的表达.结果 与空气组比较,高氧暴露后,AECⅡ SP-C的表达、SP-C细胞的表达率及荧光指数(fluorescence index,FI)明显降低,AQP5表达明显增加;而SP干预后,SP-C、AQP5表达都明显增加,形态学的损伤也有明显的改善.结论 SP可促进高氧诱导的胎鼠AECⅡ的转分化,在肺损伤修复中可能起重要作用.

  15. Diffuse alveolar hemorrhages

    International Nuclear Information System (INIS)

    The Diffuse Alveolar Hemorrhage (DAH) it is a clinical syndrome that generally manifests with hemoptysis, anemia and infiltrated in the thorax x-ray. From the anatomical point of view, the DAH is defined as the presence of blood in the distal alveolar spaces without it can identify any endobronchial abnormality. The radiological presentation of the DAH is characterized by the presence of having infiltrated of alveolar occupation in the Rx of thorax of prevalence perihiliar and bilateral that goes converging to configure an image of complete consolidation of the air space, the apexes and the periphery of the lungs are generally respected. These infiltrated are solved in one to two weeks, but with the repeated episodes of having bled it can develop interstitial fibrosis

  16. Influence of Tamoxifen or the combination of Tamoxifen and Cisplatin on the growth of human lung adenocarcinoma A549 cells

    Institute of Scientific and Technical Information of China (English)

    Yuxuan Che; Xiuhua Sun; Chaomei Huang; Jinbo Zhao 

    2014-01-01

    Objective:The experiment aims to investigate the influence of Tamoxifen and the combination of Tamoxifen and Cisplatin (DDP) on the growth of human lung adenocarcinoma A549 cel s. Methods:We treated human lung adenocarcinoma A549 cel s with dif erent concentrations of Tamoxifen, DDP and combination of DDP and Tamoxifen with non-toxicity for 72 h. Then we calculated the inhibition rate through MTT approach and detected the apoptosis rate by flow cytometry. The statistical analysis was performed with SPSS 13.0 software and statistical dif erences were determined by one-way ANOVA. The data were expressed as the mean ± standard deviation and al experiments were performed in three times. The value of P0.05). 2. As the increase concentration of Tamoxifen, the S stage and G2/M of the A549 cel s decreased while the G0/G1 increased. The apoptosis rate of Tamoxifen with 0 µmol/L, 0.1 µmol/L, 1 µmol/L and 10 µmol/L on the A549 cel s were 6.51%, 8.91%, 17.97%and 42.7%, respectively. 3. The inhibition rates of combination of Tamoxifen with 1 µmol/L and DDP with 1.25 µg/mL, 2.5 µg/mL, 5 µg/mL, 10 µg/mL and 20 µg/mL on the A549 cel s were 40.4%, 54.4%, 72.9%, 86.1%and 92.4%, respectively (P<0.05). Conclusion:Tamoxifen can inhibit the proliferation of human lung adenocarcinoma A549 cel s and induce the apoptosis of the A549 cel s. The combination of Tamoxifen with non-toxicity and DDP can improve the sensitivity of chemotherapy on the A549 cel s.

  17. In vitro and in vivo studies on radiobiological effects of prolonged fraction delivery time in A549 cells

    OpenAIRE

    Jiang, Ling; Xiong, Xiao-Peng; Hu, Chao-su; Ou, Zhou-Luo; Zhu, Guo-Pei; Ying,, Hong-Mei

    2012-01-01

    Intensity-modulated radiation therapy, when used in the clinic, prolongs fraction delivery time. Here we investigated both the in vivoand in vitroradiobiological effects on the A549 cell line, including the effect of different delivery times with the same dose on A549 tumor growth in nude mice. The in vitroeffects were studied with clonogenic assays, using linear-quadratic and incomplete repair models to fit the dose-survival curves. Fractionated irradiation of different doses was given at on...

  18. Lentinan exerts synergistic apoptotic effects with paclitaxel in A549 cells via activating ROS-TXNIP-NLRP3 inflammasome

    OpenAIRE

    Liu, Wei; Gu, Jun; Qi, Jun; Zeng, Xiao-Ning; Ji, Juan; Chen, Zheng-Zhen; Sun, Xiu-Lan

    2015-01-01

    Paclitaxel is generally used to treat cancers in clinic as an inhibitor of cell division. However, the acquired resistance in tumours limits its clinical efficacy. Therefore, the aim of this study was to detect whether co-treatment with lentinan enhanced the anti-cancer effects of paclitaxel in A549 cells. We found that the combination of paclitaxel and lentinan resulted in a significantly stronger inhibition on A549 cell proliferation than paclitaxel treatment alone. Co-treatment with paclit...

  19. Congenital Pulmonary Alveolar Proteinosis

    Directory of Open Access Journals (Sweden)

    Saber Hammami

    2013-01-01

    Full Text Available Pulmonary alveolar proteinosis (PAP is a rare syndrome characterized by pulmonary surfactant accumulation within the alveolar spaces. It occurs with a reported prevalence of 0.1 per 100,000 individuals. Two clinically different pediatric types have been defined as congenital PAP which is fatal and a late-onset PAP which is similar to the adult form and less severe. The clinical course of PAP is variable, ranging from spontaneous remission to respiratory failure. Whole-lung lavage is the current standard treatment for PAP patients. We report a new congenital case of PAP.

  20. The Effect of 5-FU and Radiation on A549 Cells In Vitro

    International Nuclear Information System (INIS)

    Effects of ionizing radiation alone and combined with chemotherapy on tumor growth and it clonal specificity Monitored by changes in distribution of chromosome number were studies in A549 cell line originated from human adenocarcinoma of the lung. Radiation (300 rad, 600 rad and 900 rad) were delivered with or without 5-FU. Forty eight hours later, 57.5% of growth inhibition of cell was Seen in cells treated with 5-FU concentration of 0.47g/ml for 24 hr exposure. Cell survival carves after radiation with and without 5-FU were made. Chromosomal analysis of cells in metaphase in control, and in cells treated with 300 rad of radiation, or 0.47g/ml of 5-FU treatment, and combined treatment of cloth were 77ne to examine the changes in ploidy and number of chromosome. Radiation combined with 5-FU enhanced growth inhibition of A549 cells. However, no evidence of synergetic effects in growth inhibition was observed in the cells treated with the combination therapy. Pattern of chromosomal distribution of survived cells were shifted from hyperploidy to hypoploidy by single dose of radiation(300 rad). As radiation dose increased a large number of hypoploidy cells were observed. Following treatment of cells with 5-FU, chomosomal distribution of survived cells were also shifted to hypodiploidy, which were seen in cells treated with radiation. The cell treated with 5-FU and followed by radiation within 24 hrs had cell with increased number of hypodiploidy cells. Almost same type of chromosomal changes were reproduced in cells treated with combined treatment with radiation and 5-FU. Minor differences were that cells with fewer number of chromosome were more frequent in cells treated with combined therapy. Further increase in cells of hypoploidy(93%) having 1-10 chromosome were induced by additional radiation. Therefore, the enhanced therapeutic effect of 5-FU combined with radiation of A549 cells appeared to be additive rather than synergistic

  1. Enhancement of radiation sensitivity by erlotinib and celecoxib in A549 human lung cancer cell line

    International Nuclear Information System (INIS)

    Objective: To investigate the role of epidermal growth factor receptor and cyclooxygenase-2 pathways in the erlotinib and celecoxib enhanced radiation sensitivity in A549 human lung cancer cell line. Methods: IC20 of erlotinib and celecoxib on in A549 human lung cancer cells was measured by MTT assay, Clonogenic assays were used to evaluate the antitumor effects of the drugs and X-irradiation. Flow cytometry was used to assess the apoptosis and cell cycle alteration, and Western blot was used for the detection of Akt and phospho-Akt.Results Both erlotinib and celecoxib could inhibit the proliferation of A549 cells in vitro in a dose-dependent manner and their values of IC20 were (5.15 ± 0.14) and (40.32 ± 1.26) μmol/L, respectively. For radiation survival,the values of Dq, D0, SF2 of the combination of two drugs were lower than those of either drug (t=6.62, P<0.05). The SER of celecoxib, erlotinib and their combination were 1.299, 1.503 and 2.217, respectively. Flow cytometry assay showed that both celecoxib and erlotinib could enhance radiation-induced G0/G1 arrest, reduce the cell number in S phase, and enhance radiation-induced apoptosis, especially for the combination of drugs. Western blot assay showed that the expressions of Akt protein were similar in all groups. However, pAkt expression was suppressed by erlotinib and celecoxib, but promoted by radiation. pAkt had the lowest expression in the radiated cells with the treatment of two drugs (t=4.89, P<0.05). Conclusions: The erlotinib and/or celecoxib could enhance radiosensitivity probably by increasing cell apoptosis and reducing the number of S-phase cells with low radiosensitivity. (authors)

  2. Effect of elemene on radiosensitivity of A549 cells and its possible molecular mechanism

    International Nuclear Information System (INIS)

    Objective: To investigate the effect of elemene on the radiosensitivity of A549 cells and its possible molecular mechanism. Methods: The effect of radiosensitivity was detected by colony forming assay. The protein expressions of DNA-PKcs, Bcl-2 and P53 were detected with Western blot. The correlation between the protein expression of DNA-PKcs and Bcl-2, DNA-PKcs and P53 was analyzed. Results: Elemene had radiosensitizing effect on A549 cells, with the SERD0 and SERDq 1.54 ± 0.20 and 1.43±0.15, respectively for 10 μg/ml elemene, and 1.63 ±0.32 and 1.75 ±0.19, respectively for 20 μg/ml elemene. Compared with irradiation group, the expression of DNA-PKcs was reduced significantly in 10, 20 μg/ml elemene combined with radiation group (t=7.52, 8.33, P<0.05), so was for Bcl-2 (t=10.74, 11.33, P<0.05). The expression of P53 protein increased significantly (t=-9.25, 7.66, P<0.05). There was a remarkable negative correlation between the expression of DNA-PKcs and P53 (r=-0.569, P<0.05), and a remarkable positive correlation between DNA-PKcs and Bcl-2 (r=0.755, P<0.05 ). Conclusions: Elemene has radiosensitizing effect on A549 cells, which might be related to down-regulation of DNA-PKcs gene expression, up-regulation of P53 and down-regulation of Bcl-2. (authors)

  3. Human epithelial cells exposed to functionalized multiwalled carbon nanotubes: interactions and cell surface modifications.

    Science.gov (United States)

    Fanizza, C; Casciardi, S; Incoronato, F; Cavallo, D; Ursini, C L; Ciervo, A; Maiello, R; Fresegna, A M; Marcelloni, A M; Lega, D; Alvino, A; Baiguera, S

    2015-09-01

    With the expansion of the production and applications of multiwalled carbon nanotubes (MWCNTs) in several industrial and science branches, the potential adverse effects on human health have attracted attention. Numerous studies have been conducted to evaluate how chemical functionalization may affect MWCNT effects; however, controversial data have been reported, showing either increased or reduced toxicity. In particular, the impact of carboxylation on MWCNT cytotoxicity is far from being completely understood. The aim of this work was the evaluation of the modifications induced by carboxylated-MWCNTs (MWCNTs-COOH) on cell surface and the study of cell-MWCNT-COOH interactions by means of field emission scanning electron microscope (FESEM). Human pulmonary epithelial cells (A549) were incubated with MWCNTs-COOH for different exposure times and concentrations (10 μg/mL for 1, 2, 4 h; 5, 10, 20 μg/mL for 24 h). At short incubation time, MWCNTs-COOH were easily observed associated with plasma membrane and in contact with microvilli. After 24 h exposure, FESEM analysis revealed that MWCNTs-COOH induced evident changes in the cellular surface in comparison to control cells: treated cells showed blebs, holes and a depletion of the microvilli density in association with structure modifications, such as widening and/or lengthening. In particular, an increase of cells showing holes and microvilli structure alterations was observed at 20 μg/mL concentration. FESEM analysis showed nanotube agglomerates, of different sizes, entering into the cell with two different mechanisms: inward bending of the membrane followed by nanotube sinking, and nanotube internalization directly through holes. The observed morphological microvilli modifications, induced by MWCNTs-COOH, could affect epithelial functions, such as the control of surfactant production and secretion, leading to pathological conditions, such as alveolar proteinosis. More detailed studies will be, however, necessary to

  4. Effect of XPA expression on the chemotherapy sensitivity of A549/DDP cells%着色性干皮病A基因表达对A549/DDP化疗敏感性的影响

    Institute of Scientific and Technical Information of China (English)

    张强; 吴金香; 魏玉平; 郝俊青; 黄山英; 董亮

    2012-01-01

    目的:探讨沉默着色性干皮病A(XPA)基因表达在非小细胞肺癌耐药细胞株顺铂化疗敏感性的影响.方法:采用免疫组化法、实时定量PCR(qPCR)及Western blot方法检测非小细胞肺癌患者肿瘤组织中XPA的表达情况.应用qPCR及Western blot方法检测A549/DDP细胞经XPA-shRNA转染后XPA-mRNA及其蛋白表达.通过MTT法检测沉默XPA基因后A549/DDP细胞凋亡情况及其对顺铂的敏感性.结果:肺癌组织XPA表达水平明显高于癌旁组织;沉默XPA基因能够促进A549/DDP细胞凋亡,并能提高A549/DDP对顺铂的药物敏感性.结论:沉默XPA基因表达能够逆转肺癌A549/DDP细胞对顺铂的耐药性.%AIM; To investigate the influence on platinum-based chemotherapy sensitivity by silencing xeroderma pigmentosum group A (XPA) gene expression in non-small cell lung cancer (NSCLC) drug resistance cell lines (A549/ DDP). METHODS; We detected the expression of XPA in lung normal and tumor tissues by immunohistochemistry, quantitative real-time PCR (qPCR) and Western blotting. We silenced XPA expression in A549/DDP cells by XPA-shRNA transfection, and detected the expression of XPA by qPCR and Western blotting. The cell sensitivity to cisplatin and the apoptosis of A549/DDP cells transfected with XPA-shRNA were determined by MTT assay. RESULTS: The expression of XPA was higher in NSCLC tissues than that in normal lung tissues. Silencing XPA gene increased the apoptosis and sensitivity of A549/DDP cells to cisplatin. CONCLUSION: Silencing XPA gene can partly reverse the cisplatin resistance in human cisplatin-resistant NSCLC cell line A549/DDP.

  5. Nitric oxide-induced activation of NF-κB-mediated NMDA-induced CTP:phosphocholine cytidylyltransferase alpha expression inhibition in A549 cells.

    Science.gov (United States)

    Li, Lian; Shen, Li; She, Hua; Yue, Shaojie; Feng, Dandan; Luo, Ziqiang

    2011-02-01

    Pulmonary surfactant is a lipoprotein complex on the alveolar surface. It reduces the surface tension at the air-water interface and stabilizes the alveoli during expiration. Surfactant deficiency or dysfunction is associated with occurrence and development of many pulmonary diseases. Family members of CTP:phosphocholine cytidylyltransferase are rate-limiting enzymes for surfactant phospholipid synthesis. We had reported recently that the expression of CTP:phosphocholine cytidylyltransferase alpha (CCT-α) was inhibited during N-methyl-D: -aspartic acid (NMDA)-induced lung injury. But the molecular mechanism underlining remains elusive. In this work, we reported that NMDA induced nitric oxide synthase (NOS) activation and nuclear factor-kB (NF-κB) subunit p65 nuclear translocation in A549 cells, which were responsible for decreased (CCT-α) expression. Furthermore, NOS activation and elevated NO production are upstream regulators for p65 nuclear translocation and (CCT-α) expression inhibition. Our results provided important clues for further elucidating the mechanisms underlying glutamate-induced lung injury. PMID:20661636

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

    Science.gov (United States)

    Song, Yali; Zhang, Gong; Zhu, Xiulan; Pang, Zhanjun; Xing, Fuqi; Quan, Song

    2012-09-01

    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-associated X protein (BAX) and caspase-3. F10 was stably expressed in A549 cells. From 12 h, A549-F10 cells proliferated markedly faster than the untransfected and A549-empty cells. F10 overexpression also significantly inhibited apoptosis, as shown by the reduced number of TUNEL and Hoechst 33258 double-positive cells. This inhibition was likely due to an F10-induced reduction in the BAX and caspase-3 levels. The results of this study indicate that F10 overexpression inhibits apoptosis in A549 cells through the downregulation of the pro-apoptotic genes BAX and caspase-3. PMID:23741243

  7. Effects of herpes simplex virus thymidine kinase/acyclovir system on growth of human pulmonary adenocarcinoma A549 cell line in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    HE Xiang-liang; HE Dong-hua; GUO Xian-jian; QIAN Gui-sheng; HUANG Gui-jun; CHEN Wei-zhong; LI Shu-ping

    2002-01-01

    Objective: To observe the effect of anciclovir (ACV) treatment on tumors induced by inoculation of TK gene-transfected human pulmonary adenocarcinoma A549 cells in nude mice. Methods: A recombinant plasmid containing TK gene was constructed and transfected into A549 cells by electroporation. The sensitivity of the transgenic cells (A549-TK) to ACV was examined by MTT assay in vitro and for in vivo observation, inoculation of A549-TK and A-549 cells into nude mice was separately performed to induce tumor growth, the response of which to ACV treatment was observed, and the tumor tissues were pathologically examined. Results: A recombinant plasmid containing TK gene was successfully constructed and transfected into A549 cells. The sensitivity of A549-TK cells to ACV was 43 times higher than that of A549 cells. The tumors induced by A549-TK cells showed no significant increase in size after ACV treatment (P>0. 05), and light microscopy revealed local tissue necrosis, karyoklasis, and nuclei disappearance. Conclusion: A549-TK cells acquires sensitivity to ACV both in vitro and in vivo, and ACV can inhibit the growth of tumors induced by A549-TK cell inoculation in nude mice.

  8. Primary pulmonary alveolar proteinosis

    Directory of Open Access Journals (Sweden)

    Šarac Sanja

    2012-01-01

    Full Text Available Introduction. Pulmonary alveolar proteinosis is an uncommon disease characterized by the accumulation of surfactant proteins and phospholipids within the alveolar spaces. Acquired disease can be idiopathic (primary and secondary. The prevalence of acquired pulmonary alveolar proteinosis is about 0.37 per 100,000 persons. Common symptoms are dyspnea and cough. Chest X-ray shows bilateral perihilar infiltrates. Open-lung biopsy is the gold standard for the diagnosis. Treatment includes whole-lung lavage, application of granulocyte-macrophage colonystimulating factor and lung transplantation. Case report. We reported a 51 year-old man with primary form of the disease. It was the second case of this extremely rare disease in the past 30 years in our clinic. The symptoms were longlasting dry cough, fever and physical deterioration. Chest Xray revealed bilateral pulmonary infiltrates; computed tomography showed patchy ground-glass opacification with interlobular thickening. The diagnosis was established by open lung biopsy. Additional tests were performed to exclude secondary form of the disease. Conclusion. We presented a rare clinical entity with typical clinical features and clinical and radiological course of the disease, in order to improve differential diagnostic approach to patients with bilateral lung infiltrations. In patients with pulmonary alveolar proteinosis timely diagnosis and adequate treatment can improve a prognosis.

  9. Pulmonary alveolar proteinosis

    OpenAIRE

    Chan, Adrian Kwok Wai; Takano, Angela; Hsu, Ann Ling; Low, Su Ying

    2014-01-01

    We report a 68-year-old female who presented with chronic cough and progressive dyspnoea. Computed tomography of the thorax and subsequent bronchoscopy confirmed the diagnosis of pulmonary alveolar proteinosis (PAP), which was treated with whole lung lavage. This case is reported in view of the low incidence of PAP.

  10. 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.

  11. Anti-Tumor Effect of Heat Shock Protein 70-Peptide Complexes on A-549 Cells

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective: To investigate the anti-tumor immunity in vitro of heat shock protein 70-peptide complexes (HSP70-PC) from human lung cancer tissue. Methods: HSP70-PC was purified from lung tumor tissues and corresponding non-tumor lung samples with the methods of ADP-affinity chromatography, DEAE ion-exchange chromatography and Western-blot. The activation and proliferation of PBMC induced by different HSP70-PC and tumor cytotoxic reactivity to A549 cells in vitro were measured by the MTT cell proliferation assay. Results: The purified HSP70-PC had a very high purity found by SDS-PAGE and Western-blot. Human lymphocytes were sensitized efficiently by HSP70 preparation purified from lung cancer tissues and a definite cytotoxicity to A-549 cells was observed. There was significant difference with HSP70-PC purified from lung cancer, compared with the control group (P<0.001). Conclusion: High purity of HSP70-PC could be achieved from tumor tissues in this study. HSP70-PC purified from human tumor tissues can induce anti-tumor immunity in vitro mainly implemented by eliciting CTL immunity.

  12. Diallyl trisnlfide induces apoptosis and inhibits proliferation of A549 cells in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    Wenjun Li; Bin Hao; Cun Gao; Libo Si; Fei Gao; Hui Tian; Lin Li; Shuhai Li; Weiming Yue; Zhitao Chen; Lei Qi; Wensi Hu; Yingchao Zhu

    2012-01-01

    Lung cancer is the leading cause of cancer-related mortality all over the world.In recent years,pulmonary adenocarcinoma has surpassed squamous cell carcinoma in frequency and is the predominant form of lung cancer in many countries.Epidemiological investigations have shown an inverse relationship between garlic (Allium sativum) consumption and death rate from many cancers.Diallyl trisulfide (DATS) is one of the garlic-derived compounds (also known as:organosulfer compounds,OSC).DATS can induce apoptosis and inhibit the growth of many cancer cell lines.Our study demonstrated that the apoptotic incidents induced by DATS were a mitochondriadependent caspase cascade through a significant decrease of the anti-apoptotic Bcl-2 that resulted in up-regulation of the ratio of Bax/Bcl-2 and the activity of caspase-3,-8,and -9.Eventually,DATS induced the apoptosis and inhibitedthe proliferation in a concentration- and time-dependentmanner.Furthermore,by establishing an animal model of female BALB/c nude mice with A549 xenografts,we found that oral gavage of DATS significantly retarded growth of A549 xenografts in nude mice without causing weight loss or any other side effects compared with the control group.All the evidence both in vitro and in vivo suggested that DATS could be an ideal anti-cancer drug.

  13. Iron stimulates plasma-activated medium-induced A549 cell injury.

    Science.gov (United States)

    Adachi, Tetsuo; Nonomura, Saho; Horiba, Minori; Hirayama, Tasuku; Kamiya, Tetsuro; Nagasawa, Hideko; Hara, Hirokazu

    2016-01-01

    Non-thermal atmospheric pressure plasma is applicable to living cells and has emerged as a novel technology for cancer therapy. Plasma has recently been shown to affect cells not only by direct irradiation, but also by indirect treatments with previously prepared plasma-activated medium (PAM). Iron is an indispensable element but is also potentially toxic because it generates the hydroxyl radical (•OH) in the presence of hydrogen peroxide (H2O2) via the Fenton reaction. The aim of the present study was to demonstrate the contribution of iron to PAM-induced A549 adenocarcinoma cell apoptosis. We detected the generation of •OH and elevation of intracellular ferrous ions in PAM-treated cells and found that they were inhibited by iron chelator. The elevations observed in ferrous ions may have been due to their release from the intracellular iron store, ferritin. Hydroxyl radical-induced DNA injury was followed by the activation of poly(ADP-ribose) polymerase-1, depletion of NAD(+) and ATP, and elevations in intracellular Ca(2+). The sensitivities of normal cells such as smooth muscle cells and keratinocytes to PAM were less than that of A549 cells. These results demonstrated that H2O2 in PAM and/or •OH generated in the presence of iron ions disturbed the mitochondrial-nuclear network in cancer cells. PMID:26865334

  14. 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). PMID:26311334

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

    Energy Technology Data Exchange (ETDEWEB)

    Ghosh, Somnath [Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085 (India); Narang, Himanshi, E-mail: himinarang@gmail.com [Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085 (India); Sarma, Asitikantha [Radiation Biology Laboratory, Inter University Accelerator Centre, Aruna Asaf Ali Marg, New Delhi 110 067 (India); Krishna, Malini [Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085 (India)

    2011-11-01

    Carbon beams (5.16 MeV/u, LET = 290 keV/{mu}m) 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 {gamma}-rays and carbon ion-irradiation. A549 cells were irradiated with 1 Gy carbon or {gamma}-rays. Carbon beam was found to be three times more cytotoxic than {gamma}-rays despite the fact that the numbers of {gamma}-H2AX foci were same. Percentage of cells showing ATM/ATR foci were more with {gamma}-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 {gamma}-rays irradiated cells and prosurvival ERK pathway was activated after {gamma}-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.

  16. Ethanol, ethyl and sodium pyruvate decrease the inflammatory responses of human lung epithelial cells via Akt and NF-κB in vitro but have a low impact on hepatocellular cells.

    Science.gov (United States)

    Relja, B; Omid, N; Wagner, N; Mörs, K; Werner, I; Juengel, E; Perl, M; Marzi, I

    2016-02-01

    Increases in pro-inflammatory cytokine levels and tissue-infiltrating leukocytes have been closely linked to increased systemic and local inflammation, which result in organ injury. Previously, we demonstrated the beneficial and hepatoprotective anti-inflammatory effects of acute ethanol (EtOH) ingestion in an in vivo model of acute inflammation. Due to its undesirable side-effects, however, EtOH does not represent a therapeutic option for treatment of acute inflammation. Therefore, in this study, we compared the effects of acute EtOH exposure with ethyl pyruvate (EtP) as an alternative anti-inflammatory drug in an in vitro model of hepatic and pulmonary inflammation. Human hepatocellular carcinoma cells Huh7 and alveolar epithelial cells A549 were stimulated with either interleukin (IL) IL-1β (1 ng/ml, 24 h) or tumor necrosis factor (TNF) (10 ng/ml, 4 h), and then treated with EtP (2.5-10 mM), sodium pyruvate (NaP, 10 mM) or EtOH (85-170 mM) for 1 h. IL-6 or IL-8 release from Huh7 or A549 cells, respectively, was measured by an enzyme‑linked immunosorbent assay. Neutrophil adhesion to cell monolayers and CD54 expression were also analyzed. Bcl-2 and Bax gene expression was determined by RT-qPCR, and western blot analysis was performed to determine the mechanisms involved. Treating A549 cells with either EtOH or EtP significantly reduced the IL-1β- or TNF‑induced IL-8 release, whereas treating Huh7 cells did not significantly alter IL-6 release. Similarly, neutrophil adhesion to stimulated A549 cells was significantly reduced by EtOH or EtP, whereas for Huh7 cells the tendency for reduced neutrophil adhesion rates by EtOH or EtP was not significant. CD54 expression was noticeably reduced in A549 cells, but this was not the case in Huh7 cells after treatment. The Bax/Bcl-2 ratio was dose‑dependently decreased by EtOH and by high-dose EtP in A549 cells, indicating a reduction in apoptosis, whereas this effect was not observed in Huh7 cells. The underlying

  17. 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.

  18. 透明质酸寡糖调节A549/DDP多药耐药作用的研究%Effects of reversing drug resistant of hyaluronate oligomers on A549/DDP cell line of human lung cancer

    Institute of Scientific and Technical Information of China (English)

    张宪真; 王宝中

    2009-01-01

    Objective:To investigate the effects of hyaluronate oligomers on the multiple drug resistance of lung cancer cell lines A549/DDP. Methods: After co-culturing A549/DDP and CD44 monoclonal antibody or hyaluronan oligomers for 48 hours,to detect the following parameters:Hyaluronate contents of the medium by radioimmunoassay,MDR1,MRP,LRP expressions by flow cytometry,survival rate of cells under different concentrations of cisplatin by MTT tests. Results: Hyaluronan oligomers can decrease hyaluronan expression,and MDR1,MRP,LRP expression of A549/DDP.In addition,apoptosis level of cells treated by hyaluronan oligomers increased significantly in higher concentration cisplatin. Conclution: In vitro,hyaluronan oligomers can reverse drug resistance of A549/DDP.%目的:通过研究透明质酸寡糖对人肺腺癌细胞系A549/DDP的P糖蛋白和多药耐药相关蛋白(MRP)、肺耐药蛋白(LRP)表达的影响,探讨透明质酸在引起肿瘤细胞多药耐药中的作用.方法: 将CD44单抗或透明质酸寡糖与A549/DDP细胞共培养48小时,放免法检测培养基中细胞所分泌透明质酸的含量,流式细胞仪检测经上述处理的A549/DDP表面MDR1 、MRP、LRP的表达率,MTT法检测在不同浓度顺铂作用下,各组细胞的存活率.结果: 经透明质酸寡糖处理后A549/DDP,分泌透明质酸较未处理组明显减少(P<0.05);且细胞表面与多药耐药相关的MDR1 、MRP、LRP的表达率均降低(P<0.05).另外,处理后的细胞,在不同浓度顺铂作用时,细胞凋亡率明显增加.结论: 体外条件下,透明质酸寡糖能逆转A549/DDP的耐药.

  19. Screening radiosensitizing-related genes mediated by elemene in lung adenocarcinoma A549 cells by using gene chip

    International Nuclear Information System (INIS)

    Objective: To screen radiosensitizing-related genes mediated by elemene in lung adenocarcinoma A549 cells by using gene chip. Methods: MTT test was used to calculate the IC50 of elemene. (1) The effect of radiosensitivity was detected by colony forming assay. A549 cells were divided into 2 groups: radiation group and radiation + elemene group. Oligonucleotide chip was used to screen the gene expression changes of A549 cells from these 2 groups. The up-regulated gene Egr-1 and the down-regulated gene CyclinD1 were selected to undergo RT-PCR so as to confirm the reliability of the result. Results: MTT test showed the elemene inhibited the proliferation of the A549 cells dose-dependently. The IC50 value of elemene on the A549 cells was 120 mg/L. (2) 10 mg/L elemene had radiosensitising effect on A549 cells.The values of SERD0 and SERDq obtained from the survival curve were (1.54±0. 20) and (1.43±0.15) respectively. Gene chip screened 122 differentially-expressed genes, including 89 up-regulated genes and 33 down-regulated genes. (3) These altered genes could be related to cell structure, substance metabolism,cell proliferation, cell differentiation, signal transduction, material transport, DNA repair, apoptosis, immune response and so forth. The RT-PCR results of Egr-1 and Cyclin D1 were consistent with the gene chip analysis. Conclusions: The mechanism of elemene enhancing the radiosensitivity of lung adenocarcinoma A549 cells is the result of participation and collaboration of multiple genes. Further study of the newly-discovered differentially-expressed gene helps find out new radiosensitizational targets of elemene. (authors)

  20. Atomic force microscope elastography reveals phenotypic differences in alveolar cell stiffness

    OpenAIRE

    Azeloglu, Evren U.; Bhattacharya, Jahar; COSTA, KEVIN D.

    2008-01-01

    To understand the connection between alveolar mechanics and key biochemical events such as surfactant secretion, one first needs to characterize the underlying mechanical properties of the lung parenchyma and its cellular constituents. In this study, the mechanics of three major cell types from the neonatal rat lung were studied; primary alveolar type I (AT1) and type II (AT2) epithelial cells and lung fibroblasts were isolated using enzymatic digestion. Atomic force microscopy indentation wa...

  1. Permanent alveolar collapse is the predominant mechanism in idiopathic pulmonary fibrosis.

    Science.gov (United States)

    Todd, Nevins W; Atamas, Sergei P; Luzina, Irina G; Galvin, Jeffrey R

    2015-08-01

    Alveolar epithelial cell loss and impaired epithelial cell regeneration are currently accepted as central initiating events in idiopathic pulmonary fibrosis (IPF), but subsequent downstream effects remain uncertain. The most accepted downstream effect is aberrant and dysregulated mesenchymal cell proliferation and excess extracellular matrix (ECM) accumulation. However, biochemical and imaging studies have perhaps somewhat surprisingly indicated little increase in total lung collagen and lung tissue, and have rather shown a substantial decrease in lung aeration and lung air volume. Loss of tissue aeration is a consequence of alveolar collapse, which occurs in IPF as a result of apposition and septal incorporation of denuded basal lamina. Permanent alveolar collapse is well-documented following epithelial injury, has the ability to mimic interstitial fibrosis radiologically and histologically, and is a better supported explanation than dysregulated fibroblast proliferation and excess ECM accumulation for the constellation of findings in patients with IPF. PMID:26165208

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

    Science.gov (United States)

    Buckley, Susan; Shi, Wei; Xu, Wei; Frey, Mark R; Moats, Rex; Pardo, Annie; Selman, Moises; Warburton, David

    2015-11-01

    The causes underlying the self-perpetuating nature of idiopathic pulmonary fibrosis (IPF), a progressive and usually lethal disease, remain unknown. We hypothesised that alveolar soluble annexin V contributes to lung fibrosis, based on the observation that human IPF bronchoalveolar lavage fluid (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. Conditioned medium from annexin V-treated alveolar epithelial type 2 cells (AEC2), but not annexin V per se, induced proliferation of human fibroblasts and contained pro-fibrotic, IPF-associated proteins, as well as pro-inflammatory cytokines that were found to correlate tightly (r>0.95) with annexin V levels in human BALF. ErbB2 receptor tyrosine kinase in AECs was activated by annexin V, and blockade reduced the fibrotic potential of annexin V-treated AEC-conditioned medium. In vivo, aerosol delivery of annexin V to mouse lung induced inflammation, fibrosis and increased hydroxyproline, with activation of Wnt, transforming growth factor-β, mitogen-activated protein kinase and nuclear factor-κB signalling pathways, as seen in IPF. Chronically increased alveolar annexin V levels, as reflected in increased IPF BALF levels, may contribute to the progression of IPF by inducing the release of pro-fibrotic mediators. PMID:26160872

  3. Alveolar Ridge Augmentation with Titanium Mesh. A Retrospective Clinical Study

    OpenAIRE

    Poli, Pier P; Beretta, Mario; Cicciù, Marco; Maiorana, Carlo

    2014-01-01

    An adequate amount of bone all around the implant surface is essential in order to obtain long-term success of implant restoration. Several techniques have been described to augment alveolar bone volume in critical clinical situations, including guided bone regeneration, based on the use of barrier membranes to prevent ingrowth of the epithelial and gingival connective tissue cells. To achieve this goal, the use of barriers made of titanium micromesh has been advocated. A total of 13 patients...

  4. Effects of Magnetic Fluid Hyperthermia Induced by An Alternative Magnetic Field on Human Carcinoma A549 Cell in vitro

    Directory of Open Access Journals (Sweden)

    Guoqing WANG

    2011-03-01

    Full Text Available Background and objective Magnetic fluid hyperthermia (MFH is a method of heat therapy using nanometer techniques and hyperthermia. It has the advantage of high specificity of targeting. The aim of this study is to detect the effects of MFH induced by an alternating magnetic field on human being carcinoma A549 cells in vitro. Methods A human adenocarcinoma cell line A549 was cultured with various concentrations of ferroferric oxide (Fe3O4 magnetic fluid (1.5-6.0 mg/mL and exposed to an alternative magnetic field (AMF for 30 min. And then the optical density (OD of viable cell, cytotocixity index, growth curve of cells, morphologic changes of cell, cell cycle and aposptosis were measured. Results The proliferation of the A549 cells were remarkably inhibited, the OD value of viable cells decreased and cytotoxity index (CI increased; Apoptosis of the A549 cells were observed to have cell shrinkage, chromatin condensation, margination, unclear fragmentation and intact cell membrane by light and electron microscopy; The cells were inhibited in the stage S. Conclusion MFH induced by AMF could inhibit the proliferation, which promotes apoptosis and arrest at S stage of the A549 cells.

  5. 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.

  6. Screening Metastasis-associated Genes from Anoikis Resistant A549 Lung Cancer Cells by Human Genome Array

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    Xiaoping WANG

    2010-01-01

    Full Text Available Background and objective As a barrier to metastases, cells normally undergo apoptosis after they lose contact with their extra cellular matrix (ECM. This process has been termed “anoikis”. Tumour cells that acquire malignant potential have developed mechanisms to resist anoikis and thereby survive after detachment from their primary site while traveling through the lymphatic and circulatory systems. This “anoikis resistance” is considered the first step to tumor metastases. The aim of this study was to screen metastasis-associated genes from anoikis resistant and adherent growth A549 lung cancer cell by Human Genome Array. Methods Establish anoikis resistant A549 lung cancer cell lines by using poly-hydroxyethyl methacrylate resin processed petri dishes, which causes cell free from adherent. The different expressed gene between anoikis resistant A549 cell and adherent growth A549 cell was tested using human V2.0 whole-genome oligonucleotide microarray, a product of Capitalbio Corporation, Beijing. Screen metastasis-associated genes. Results 745 different expressed genes were screened, including 63 highly metastasis-associated genes. Conclusion The successfully established anoikis resistant A549 cell lines and screened different expressed genes provide us basis for further research on metastasis of lung cancer.

  7. Panduratin A, a Possible Inhibitor in Metastasized A549 Cells through Inhibition of NF-Kappa B Translocation and Chemoinvasion

    Directory of Open Access Journals (Sweden)

    Mohd. Rais Mustafa

    2013-07-01

    Full Text Available In the present study, we investigated the effects of panduratin A (PA, isolated from Boesenbergia rotunda, on apoptosis and chemoinvasion in A549 human non-small cell lung cancer cells. Activation of the executioner procaspase-3 by PA was found to be dose-dependent. Caspase-3 activity was significantly elevated at the 5 µg/mL level of PA treatment and progressed to a maximal level. However, no significant elevated level was detected on procaspase-8. These findings suggest that PA activated caspase-3 but not caspase-8. Numerous nuclei of PA treated A549 cells stained brightly by anti-cleaved PARP antibody through High Content Screening. This result further confirmed that PA induced apoptotic cell death was mediated through activation of caspase-3 and eventually led to PARP cleavage. Treatment of A549 cells with PA resulted in a strong inhibition of NF-κB activation, which was consistent with a decrease in nuclear levels of NF-κB/p65 and NF-κB/p50 and the elevation of p53 and p21. Besides that, we also showed that PA significantly inhibited the invasion of A549 cells in a dose-dependent manner through reducing the secretion of MMP-2 of A549 cells gelatin zymography assay. Our findings not only provide the effects of PA, but may also be important in the design of therapeutic protocols that involve targeting of either p53 or NF-κB.

  8. Transfection of gene Livin α/β into A549 cells and separate effect on the cell growth

    Institute of Scientific and Technical Information of China (English)

    SUN Jian-guo; LIAO Rong-xia; CHEN Zheng-tang; WANG Zhi-xin; ZHANG Qing; HU Yi-de; WANG Dong-lin

    2005-01-01

    Objective:To express two Livin isoforms (Livin α & β genes) with transfection techniques in A549 cell line respectively in order to observe their effect on growth of cell line. Methods:Two eukaryotic expression vectors of Livin, pcDNA3.1-Livin α & β, were transfected into A549 cell line by electroporation. Then G418-resistant clones were screened. RT-PCR, Northern blot and immunofluorescence cytochemistry were used to detect Livin α & β expression level in the transfected cells. Finally, observation of cell morphology, growth curve assay and colony formation analysis were performed to explore the effect of Livin on growth of the cells. Results:Livin α & β were expressed in transfected A549 cells, and induced a faster cell growth, shorter doubling time and stronger cell colony forming ability, yet had no morphology change.Conclusion:Both isoforms can accelerate the growth of A549 cells, indicating a close relationship between Livin expression and the genesis and development of lung cancer. The expression of Livin α & β in A549 cells provides basis for further study of their different biological functions of anti-apoptosis and of their role in lung cancer cell resistance to radiotherapy and chemotherapy.

  9. Effects of X-ray irradiation on expression of Pokemon gene in A549 cells of human lung adenocarcinoma

    International Nuclear Information System (INIS)

    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)

  10. Ophiopogonin B induces apoptosis, mitotic catastrophe and autophagy in A549 cells.

    Science.gov (United States)

    Chen, Meijuan; Guo, Yuanyuan; Zhao, Ruolin; Wang, Xiaoxia; Jiang, Miao; Fu, Haian; Zhang, Xu

    2016-07-01

    Ophiopogonin B (OP-B), a saponin compound isolated from Radix Ophiopogon japonicus, was verified to inhibit cell proliferation in numerous non-small cell lung cancer (NSCLC) cells in our previous study. However, the precise mechanisms of action have remained unclear. In the present study, we mainly investigated the effects of OP-B on adenocarcinoma A549 cells to further elaborate the underlying mechanisms of OP-B in different NSCLC cell lines. Detection by high content screening (HCS) and TUNEL assay verified that OP-B induced apoptosis in this cell line, while detection of Caspase-3, Bcl-2 and Bax showed that OP-B induced cell death was caspase and mitochondrial independent. Further experiments showed that OP-B induced cell cycle arrest in the S and G2/M phases by inhibiting the expression of Myt1 and phosphorylation of Histone H3 (Ser10), which resulted in mitotic catastrophe in the cells. Transmission electron microscopy (TEM) observation of cell micro-morphology combined with detection of Atgs by western blot analysis showed that OP-B induced autophagy in this cell line. Autophagy inhibition by the lysosome inhibitor CQ or Beclin1-siRNA knockdown both attenuated cell viability, demonstrated that autophagy also being the vital reason resulted in cell death. More importantly, the xenograft model using A549 cells provided further evidence of the inhibition of OP-B on tumor proliferation. Immunohistochemistry detection of LC3 and Tunel assay both verified that high dose of OP-B (75 mg/kg) induced autophagy and apoptosis in vivo, and western blot detection of p-Histone H3 (Ser10), Survivin and XIAP further indicated the molecular mechanism of OP-B in vivo. As our findings revealed, multiple types of cell death overlapped in OP-B treated A549 cells, it displayed multitarget characteristics of the compounds extracted from the Chinese herbal, which may be used as candidate anticancer medicine in clinic. PMID:27175570

  11. Pulmonary alveolar proteinosis

    International Nuclear Information System (INIS)

    Since the original description of pulmonary alveolar proteinosis (PAP) as bilaterally symmetric, confluent, perihilar infiltrates, no large series evaluating the relative frequency of radiographic findings has been published. The authors reviewed 53 histologically proved cases and found a predominantly confluent pattern in 77.4%; of these, 61% had a nodular component. A predominantly nodular pattern was seen in 22.6% often with some coalescence or reticular shadows. Distribution was perhilar 56.6%, diffuse in 26.4%, basilar in 26.4%, and asymmetric in 35.8%. The classic pattern was seen in 18.9%. The authors conclude that the spectrum of patterns in pulmonary alveolar proteinosis is wider than generally appreciated

  12. Nontypeable Haemophilus influenzae induces COX-2 and PGE2 expression in lung epithelial cells via activation of p38 MAPK and NF-kappa B

    Directory of Open Access Journals (Sweden)

    Koga Tomoaki

    2008-01-01

    Full Text Available Abstract Background Nontypeable Haemophilus influenzae (NTHi is an important respiratory pathogen implicated as an infectious trigger in chronic obstructive pulmonary disease, but its molecular interaction with human lung epithelial cells remains unclear. Herein, we tested that the hypothesis that NTHi induces the expression of cyclooxygenase (COX-2 and prostaglandin E2 (PGE2 via activation of p38 mitogen-activated protein kinase (MAPK and nuclear factor (NF-kappa B in pulmonary alveolar epithelial cells. Methods Human alveolar epithelial A549 cells were infected with different concentrations of NTHi. The phosphorylation of p38 MAPK was detected by Western blot analysis, the DNA binding activity of NF-kappa B was assessed by electrophoretic mobility shift assay (EMSA, and the expressions of COX-1 and 2 mRNA and PGE2 protein were measured by reverse transcription-polymerase chain reaction (RT-PCR and enzyme linked immunosorbent assay (ELISA, respectively. The roles of Toll-like receptor (TLR 2 and TLR4, well known NTHi recognizing receptor in lung epithelial cell and gram-negative bacteria receptor, respectively, on the NTHi-induced COX-2 expression were investigated in the HEK293 cells overexpressing TLR2 and TLR4 in vitro and in the mouse model of NTHi-induced pneumonia by using TLR2 and TLR4 knock-out mice in vivo. In addition, the role of p38 MAPK and NF-kappa B on the NTHi-induced COX-2 and PGE2 expression was investigated by using their specific chemical inhibitors. Results NTHi induced COX-2 mRNA expression in a dose-dependent manner, but not COX-1 mRNA expression in A549 cells. The enhanced expression of PGE2 by NTHi infection was significantly decreased by pre-treatment of COX-2 specific inhibitor, but not by COX-1 inhibitor. NTHi induced COX-2 expression was mediated by TLR2 in the epithelial cell in vitro and in the lungs of mice in vivo. NTHi induced phosphorylation of p38 MAPK and up-regulated DNA binding activity of NF-kappa B

  13. The alveolar macrophage.

    OpenAIRE

    Bowden, D. H.

    1984-01-01

    The pulmonary macrophagic system is critical to the defense of the lung, keeping the alveoli clean and sterile and responding on demand with an adaptive outpouring of new cells into the air sacs. Under basal conditions alveolar macrophages, in common with other mononuclear phagocytes, are derived from the bone marrow. A population of macrophage precursors within the pulmonary interstitium provides a reserve pool capable of proliferation and delivery of phagocytes in response to unusually heav...

  14. Pulmonary alveolar microlithiasis

    International Nuclear Information System (INIS)

    Pulmonary alveolar microlithiasis (PAM) is a rare disease characterized by the diffuse and bilateral presence of calcium phosphate microlite in the alveolar spaces. The progression of this potentially lethal disease is show and most of the patients remain asymptomatic during years or decades, resulting in a show deterioration of the pulmonary function. The typical finding of the sand storm in the chest X-ray is characteristic of this entity. Mutations in the SLC34A2 gene that does the coding for the type II co-transporter of sodium phosphate were identified as responsible for this disease. Of the almost 600 cases, only 6 have been reported in Colombia. We are presenting a case of pulmonary alveolar microlite in a 27 year old man, with progressive respiratory distress whose diagnosis was made by the X-ray findings and confirmed by trans bronchial biopsy. In the 2 years follow-up, shows evolution towards deterioration of his respiratory function making him a candidate for lung transplantation.

  15. ANTICANCER ACTIVITY OF OSCILLATORIA TEREBRIFORMIS CYANOBACTERIA IN HUMAN LUNG CANCER CELL LINE A549

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    S.Mukund

    2014-04-01

    Full Text Available Purpose: To evaluate the anti-cancer properties of the cyanobacterial extract of Oscillatoria terebriformis Methods: The extract was tested in Human lung cancer cell lines and examined for its effect on cell viability, nuclear morphology and sub-G1 formation. Cell viability was determined by micro culture tetrazolium technique (MTT, nuclear morphology investigated using 4’-6-diamidino-2-phenylindole (DAPI staining technique, and apoptosis assay using DNA fragmentation. Results: The results showed decreasing cell viability in a concentration-dependent manner. Altered cell morphology after treatment with the extract demonstrated that cells experienced apoptosis. Conclusion: The data demonstrate that Oscillatoria Terebriformis extract induced apoptosis in Human lung cancer A549 cells, and therefore, has a potential as an anti-cancer agent.

  16. Oxidative damage to DNA by diesel exhaust particle exposure in co-cultures of human lung epithelial cells and macrophages

    DEFF Research Database (Denmark)

    Jantzen, Kim; Roursgaard, Martin; Madsen, Claus Desler; Loft, Steffen; Rasmussen, Lene Juel; Møller, Peter

    2012-01-01

    epithelium and immune cells. Macrophages have important immune defence functions by engulfing insoluble foreign materials, including particles, although they might also promote or enhance inflammation. We investigated the effect of co-culturing type II lung epithelial A549 cells with macrophages upon...... relationship between levels of respiration and ROS production. In conclusion, exposure of mono-cultured cells to DEPs generated oxidative stress to DNA, whereas co-cultures with macrophages had lower levels of oxidatively damaged DNA than A549 epithelial cells....

  17. Synthesis, characterization and anticancer activity studies of ruthenium(II) polypyridyl complexes on A549 cells.

    Science.gov (United States)

    Zeng, Chuan-Chuan; Jiang, Guang-Bin; Lai, Shang-Hai; Zhang, Cheng; Yin, Hui; Tang, Bing; Wan, Dan; Liu, Yun-Jun

    2016-08-01

    Four new ruthenium(II) polypyridyl complexes [Ru(N-N)2(bddp)](ClO4)21-4 (N-N=dmb: 4,4'-dimethyl-2,2'-bipyridine 1, bpy: 2,2'-bipyridine 2, phen: 1,10-phenanthroline 3 and dmp: 2,9-dimethyl-1,10-phenanthroline 4, bddp=benzilo[2,3-b]-1,4-diazabenzo[i]dipyrido[3,2-a:2',3'-c]phenazine) were synthesized and characterized by elemental analysis, ESI-MS and (1)H NMR. The cytotoxicity in vitro of the complexes against BEL-7402, HeLa, MG-63 and A549 cell lines was investigated by MTT method. The complexes show high cytotoxic activity toward the selected cell lines with an IC50 value ranging from 5.3±0.6 to 15.7±3.6μM. The apoptosis was studied with acridine orange (AO)/ethdium bromide (EB) and Hoechst 33258 staining methods. The cellular uptake was investigated with DAPI staining method. The reactive oxygen species (ROS) and mitochondrial membrane potential were performed under fluorescent microscope and flow cytometry. The complexes can induce an increase in the ROS levels and a decrease in the mitochondrial membrane potential. The comet assay was studied with fluorescent microscope. The percentage in apoptotic and necrotic cells and cell cycle arrest were assayed by flow cytometry. The effects of the complexes on the expression of caspases and Bcl-2 family proteins were studied by western blot analysis. The results show that the complexes induce apoptosis in A549 cells through an ROS-mediated mitochondrial dysfunction pathway, which was accompanied by regulating the expression of Bcl-2 family proteins. PMID:27288660

  18. Effect of Inhibiting NGAL Gene Expression on A549 Lung Cancer Cell Migration and Invasion

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    Jian TANG

    2015-04-01

    Full Text Available Background and objective To detect the expression of neutrophil gelatinase-assoeiated lipocalin (NGAL in the different differentiations of lung cancer tissues and to study the mechanism of invasion of A549 cells affected by NGAL. Methods The expression of NGAL was detected by immunochemistry in lung cancer tissue and the tissue around edge of the cancer. The effect of NGAL expression on A549 cells was observed by using qRT-PCR and Western blot. The abilities of invasion and metastasis were evaluated by transwell invasion and migration assay, and cell scratch assay in vitro. The protein expression of E-cadherin, Vimentin was measured by immunofluoresence and Western blot. Results The positive expression rate of NGAL was 76.32% (58/76 in the lung cancer, 13.3% (4/30 in adjacent tissue by immunochemistry. NGAL expression levels in the lung cancer tissues were significantly higher than that in adjacent tissues. The rate of migration and invasion in NGAL-siRNA group was 60.4%±6.4% compared to 50.5%±4.4% in the control group, there was a significant difference (P<0.05. Vimentin was suppressed, and E-cadherin was upregulated when NGAL was inhibited. MMP-2 and MMP-9 decreased when NGAL was knocked down. Conclusion The expression level of NGAL is highly expressed in lung cancer. NGAL may be one of important indicators involved in lung cancer infiltrated and transferred. NGAL might be one of potential targets for lung cancer treatment.

  19. COPD promotes migration of A549 lung cancer cells: the role of chemokine CCL21

    Directory of Open Access Journals (Sweden)

    Kuźnar-Kamińska B

    2016-05-01

    Full Text Available Barbara Kuźnar-Kamińska,1 Justyna Mikuła-Pietrasik,2 Patrycja Sosińska,2 Krzysztof Książek,2 Halina Batura-Gabryel1 1Department of Pulmonology, Allergology and Respiratory Oncology, 2Department of Pathophysiology, Poznań University of Medical Sciences, Poznań, Poland Abstract: Patients with COPD develop lung cancer more frequently than healthy smokers. At the same time, molecular mediators promoting various aspects of cancer cell progression are still elusive. In this report, we examined whether COPD can be coupled with increased migration of non-small-cell lung cancer cells A549 and, if so, whether this effect may be related to altered production and activity of chemokines CCL21, CXCL5, and CXCL12. The study showed that the migration of A549 cells through the polycarbonate membrane and basement membrane extract toward a chemotactic gradient elicited by serum from patients with COPD was markedly higher as compared with serum from healthy donors. The concentration of CCL21 and CXCL12, but not CXCL5, in serum from patients with COPD was also increased. Experiments in which CCL21- and CXCL12-dependent signaling was blocked revealed that increased migration of the cancer cells upon treatment with serum from patients with COPD was mediated exclusively by CCL21. Collectively, our results indicate that COPD may contribute to the progression of lung cancer via CCL21-dependent intensification of cancer cell migration. Keywords: chemokines, COPD, lung cancer, migration

  20. 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

  1. New insights of P2X7 receptor signaling pathway in alveolar functions

    OpenAIRE

    Mishra, Amarjit

    2013-01-01

    Purinergic P2X7 receptor (P2X7R), an ATP-gated cation channel, is unique among all other family members because of its ability to respond to various stimuli and to modulate pro-inflammatory signaling. The activation of P2X7R in immune cells is absolutely required for mature interleukin -1beta (IL-1beta) and IL-18 production and release. Lung alveoli are lined by the structural alveolar epithelial type I (AEC I) and alveolar epithelial type II cells (AEC II). AEC I plays important roles in alv...

  2. Inhibitory Effects of Natural Compound Alantolactone on Human Non-small Cell Lung Cancer A549 Cells

    Institute of Scientific and Technical Information of China (English)

    ZONG Min-ru; ZHAO Ying-hao; ZHANG Kun; YANG Long-fei; ZHENG Yong-chen; HE Cheng-yan

    2011-01-01

    Alantolactone is a natural compound identified from the roots of Inula helenium L. that has multiple bio-activities. We examined its inhibitory effects on human non-small cell lung cancer(NSCLC) A549 cells. The antiproliferative effect of alantolactone on A549 cells was investigated via MTT[3'-(4,5dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide]assay and its apoptosis-inducing effect was determined by Hoechst staining and flow cytometry. We found that alantolactone significantly inhibited the proliferation of A549 cells and induced morphological changes typical for apoptosis. Flow cytometry analysis indicates dose-dependent cell cycle retardation at G0/G1 and S stages. The results indicate that alantolactone could be an attractive small-molecular natural compound for further development as a therapeutic drug against NSCLC.

  3. 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)

    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

  4. 5-allyl-7-gen-difluoromethoxychrysin enhances TRAIL-induced apoptosis in human lung carcinoma A549 cells

    International Nuclear Information System (INIS)

    5-allyl-7-gen-difluoromethoxychrysin (AFMC) is a novel synthetic analogue of chrysin that has been reported to inhibit proliferation in various cancer cell lines. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-cancer agent. The cytotoxicity of A549 and WI-38 cells were determined using colorimetry. Apoptosis was detected by flow cytometry (FCM) after propidium iodide (PI) fluorescence staining and agarose gel electrophoresis. Caspase activities were evaluated using enzyme-linked immunosorbent assay (ELISA).The expressions of DR4 and DR5 were analyzed using FCM and western blot. Subtoxic concentrations of AFMC sensitize human non-small cell lung cancer (NSCLC) A549 cells to TRAIL-mediated apoptosis. Combined treatment of A549 cells with AFMC and TRAIL significantly activated caspase-3, -8 and -9. The caspase-3 inhibitor zDEVD-fmk and the caspase-8 inhibitor zIETD-fmk blocked the apoptosis of A549 cells induced by co-treatment with AFMC and TRAIL. In addition, we found that treatment of A549 cells with AFMC significantly induced the expression of death receptor 5 (DR5). AFMC-mediated sensitization of A549 cells to TRAIL was efficiently reduced by administration of a blocking antibody or small interfering RNAs against DR5. AFMC also caused increase of the Sub-G1 cells by TRAIL treatment and increased the expression levels of DR5 in other NSCLC H460 and H157 cell lines. In contrast, AFMC-mediated induction of DR5 expression was not observed in human embryo lung WI-38 cells, and AFMC did not sensitize WI-38 cells to TRAIL-induced apoptosis. AFMC synergistically enhances TRAIL-mediated apoptosis in NSCLC cells through up-regulating DR5 expression

  5. Theoretical basis of alveolar sampling.

    OpenAIRE

    Kelman, G R

    1982-01-01

    The conditions under which the partial pressure of a solvent in the alveolar gas is likely to provide a valid index of its partial pressure in the mixed venous blood, and thus of whole-body exposure, is explored on a theoretical basis. Under steady-state conditions, providing the solvent's blood/gas partition coefficient exceeds 10, its mixed venous and alveolar pressures will agree within 10% and become virtually identical during the prolonged expiration necessary to obtain an alveolar sampl...

  6. Effect of type III group B streptococcal capsular polysaccharide on invasion of respiratory epithelial cells.

    OpenAIRE

    Hulse, M L; Smith, S; Chi, E Y; Pham, A; Rubens, C E

    1993-01-01

    Group B streptococcal (GBS) capsular polysaccharide is an important virulence factor, and its role in invasion of cultured respiratory epithelial cells was investigated. A type III GBS clinical isolate, COH1, and asialo and unencapsulated isogenic transposon capsule mutants of it were compared in an in vitro invasion assay. The results demonstrated that capsule attenuated the invasion process. Invasion was not affected when the A549 epithelial cells were preincubated with purified type III GB...

  7. Okadaic acid inhibits cell multiplication and induces apoptosis in a549 cells, a human lung adenocarcinoma cell line

    OpenAIRE

    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-depende...

  8. Effect of staurosporine on the mobility and invasiveness of lung adenocarcinoma A549 cells: an in vitro study

    International Nuclear Information System (INIS)

    Lung cancer is one of the most malignant tumors, representing a significant threat to human health. Lung cancer patients often exhibit tumor cell invasion and metastasis before diagnosis which often render current treatments ineffective. Here, we investigated the effect of staurosporine, a potent protein kinase C (PKC) inhibitor on the mobility and invasiveness of human lung adenocarcinoma A549 cells. All experiments were conducted using human lung adenocarcinoma A549 cells that were either untreated or treated with 1 nmol/L, 10 nmol/L, or 100 nmol/L staurosporine. Electron microscopy analyses were performed to study ultrastructural differences between untreated A549 cells and A549 cells treated with staurosporine. The effect of staurosporine on the mobility and invasiveness of A549 was tested using Transwell chambers. Western blot analyses were performed to study the effect of staurosporine on the levels of PKC-α, integrin β1, E-cadherin, and LnR. Changes in MMP-9 and uPA levels were identified by fluorescence microscopy. We demonstrated that treatment of A549 cells with staurosporine caused alterations in the cell shape and morphology. Untreated cells were primarily short spindle- and triangle-shaped in contrast to staurosporine treated cells which were retracted and round-shaped. The latter showed signs of apoptosis, including vacuole fragmentation, chromatin degeneration, and a decrease in the number of microvilli at the surface of the cells. The A549 cell adhesion, mobility, and invasiveness significantly decreased with higher staurosporine concentrations. E-cadherin, integrin β1, and LnR levels changed by a factor of 1.5, 0.74, and 0.73, respectively compared to untreated cells. In addition, the levels of MMP-9 and uPA decreased in cells treated with staurosporine. In summary, this study demonstrates that staurosporine inhibits cell adhesion, mobility, and invasion of A549 cells. The staurosporine-mediated inhibition of PKC-α, induction of E

  9. Synthesis and cytotoxicity evaluation of 4-amino-4-dehydroxylarctigenin derivatives in glucose-starved A549 tumor cells.

    Science.gov (United States)

    Lei, Min; Gan, Xianwen; Zhao, Kun; Yu, Qiang; Hu, Lihong

    2015-02-01

    The natural product arctigenin (ATG) demonstrated preferential cytotoxicity to cancer cells under glucose starvation. A series of 4-amino-4-dehydroxylarctigenin derivatives based on lead compound ATG were designed and synthesized by bioisosteric modifications. Their cytotoxicities were evaluated in glucose-starved A549 tumor cells and the results indicated that the 4-amino-4-dehydroxylarctigenin showed more potent cytotoxicity than arctigenin, and the further substituent group on 4-amino would result in the cytotoxicities decreased significantly. 4-Substituted-arctigenin could selectively target on glucose-starved A549 tumor cells which provide an alternative strategy for anticancer drug development with minimal normal tissue toxicity. PMID:25571795

  10. Cell deformation at the air-liquid interface induces Ca2+-dependent ATP release from lung epithelial cells.

    Science.gov (United States)

    Ramsingh, Ronaldo; Grygorczyk, Alexandra; Solecki, Anna; Cherkaoui, Lalla Siham; Berthiaume, Yves; Grygorczyk, Ryszard

    2011-04-01

    Extracellular nucleotides regulate mucociliary clearance in the airways and surfactant secretion in alveoli. Their release is exquisitely mechanosensitive and may be induced by stretch as well as airflow shear stress acting on lung epithelia. We hypothesized that, in addition, tension forces at the air-liquid interface (ALI) may contribute to mechanosensitive ATP release in the lungs. Local depletion of airway surface liquid, mucins, and surfactants, which normally protect epithelial surfaces, facilitate such release and trigger compensatory mucin and fluid secretion processes. In this study, human bronchial epithelial 16HBE14o(-) and alveolar A549 cells were subjected to tension forces at the ALI by passing an air bubble over the cell monolayer in a flow-through chamber, or by air exposure while tilting the cell culture dish. Such stimulation induced significant ATP release not involving cell lysis, as verified by ethidium bromide staining. Confocal fluorescence microscopy disclosed reversible cell deformation in the monolayer part in contact with the ALI. Fura 2 fluorescence imaging revealed transient intracellular Ca(2+) elevation evoked by the ALI, which did not entail nonspecific Ca(2+) influx from the extracellular space. ATP release was reduced by ∼40 to ∼90% from cells loaded with the Ca(2+) chelator BAPTA-AM and was completely abolished by N-ethylmalemide (1 mM). These experiments demonstrate that in close proximity to the ALI, surface tension forces are transmitted directly on cells, causing their mechanical deformation and Ca(2+)-dependent exocytotic ATP release. Such a signaling mechanism may contribute to the detection of local deficiency of airway surface liquid and surfactants on the lung surface. PMID:21239538

  11. 油烟中细颗粒物致胎鼠肺泡Ⅱ型上皮细胞DNA损伤的研究%Assessment of DNA Damage Induced by Cooking Oil Fumes Particulate in the Mice Alveolar Type Ⅱ Epithelial Cells

    Institute of Scientific and Technical Information of China (English)

    梁春梅; 操基玉; 王勇; 冯哲伟; 汪磊

    2011-01-01

    目的 探讨油烟中的细颗粒物(PM2.5)对原代培养的胎鼠肺泡Ⅱ型上皮细胞(AECⅡ)DNA的损伤效应.方法 将1只妊娠18d的SPF级ICR小鼠体内的胎鼠肺组织制成AECⅡ细胞悬液,取对数生长期细胞,调整细胞密度为1×106/ml,分别加入终浓度为0(溶剂对照,含10% FBS的DMEM)、12.5、25、50、100μg/ml的PM2.5(来源于烹调油烟)溶液,培养6、12h后进行MTT试验和彗星试验,并检测AECⅡ细胞的尾长、尾部DNA百分比、尾矩和Olive尾矩.结果与溶剂对照组比较,50、100 μg/ml PM2.5染毒6、12 h时胎鼠AECⅡ存活率下降,差异均有统计学意义(P<0.05);12.5、25、50μg/mlPM2.5染毒6、12h时胎鼠AECⅡ的尾长、尾部DNA百分比、尾矩和Olive尾矩升高,差异均有统计学意义(P<0.05).随着PM2.5染毒浓度的升高,AECⅡ细胞存活率呈下降趋势,尾长、尾部DNA百分比、尾矩和Olive尾矩均呈上升趋势.结论油烟中的PM2.5可降低AECⅡ的细胞活性,对AECⅡ的DNA具有损伤效应.%Objective To investigate DNA damage induced by cooking oil fume participate (PM2.5) in the mice alveolar type II epithelial cells in primary cultured. Methods The mice alveolar type Ⅱ epithelial cells were isolated from 18 days old fetuses of ICR mice. The cells in exponential phase were cultured at a density of 1×106 cells/ml,AEC II cells were treated with PM2.5 at the doses of 0 μg/ml (the solvent control, 10% FBS of DMEM), 12.5,25,50 and 100 μg/ml for 6 and 12 h. AEC Ⅱ cell proliferation were detected with MTT assays and the comet assay was used to detect the Olive tail moment,tail moment,tail length and tail intensity. Results The survival rate of AEC II was decreased with a dose-dependent manner; Olive tail moment, tail moment, tail length and tail intensity were increased with a dose-dependent manner. Conclusion Cooking oil fume participate may decrease the viability of AEC II cells and produce evident DNA damage.

  12. Inhibition of Oridonin on Human Lung Adenocarcinoma A549 Cells and Its Mechanisms%冬凌草甲素诱导人肺腺癌细胞株A549凋亡及其机制研究

    Institute of Scientific and Technical Information of China (English)

    彭蕾; 顾振纶; 薛仁宇; 周颖; 蒋小岗; 郭次仪

    2010-01-01

    目的:探讨冬凌草甲素(oridnin)对人肺腺癌细胞株A549细胞凋亡的影响.方法:利用MTT法检测oridnin对A549细胞增殖作用的影响;Hoechst 33258染色观察给药后细胞形态改变;透射电镜观察给药后细胞超微结构改变;FITC-AnnexinV/PI双标记检测细胞凋亡率.结果:Hoechst 33258染色和透射电镜观察,oridonin给药后A549细胞出现空泡变性,染色质高度凝集;FTTC-AnnexinV/PI双标记检测oridnin(25,50,100μmol/L)作用细胞48 h后凋亡率分别为1.5%,6.2%,59.7%.结论:Oridonin对A549细胞具有抑制增殖和诱导凋亡作用.

  13. Immune Evasion of Human Lung Carcinoma Cell A549 Suppressed by Human Lymphoma Cell Jurkat via Fas/FasL Pathway

    Directory of Open Access Journals (Sweden)

    Hongmei WANG

    2010-07-01

    Full Text Available Background and objective Tumor escape from the host immune system has been a major problem in immunotherapy of human malignancies. FasL/Fas-induced apoptosis plays an important role in various immunological processes. The aim of this study is to investigate the immune evasion in human lung carcinoma cell A549 induced by human lymphoma cell Jurkat via Fas/FasL pathway. Methods Jurkat cells and A549 cells were co-cultured at different proportions. The apoptotic rates of A549 cells were determined by flow cytometry (FCM. Protein levels of Fas, FasL and Caspase-8 in A549 cells were detected by Western blot. Results Survival rates of A549 cells gradually decreased and apoptotic rates of A549 cells were significantly enhanced along with ratio increasing between Jurkat and A549. Meanwhile, the protein levels of Fas and Caspase-8 gradually increased in A549 cells, and the protein levels of FasL had no significant difference in all groups. Conclusion The Jurkat cells might decrease the survival rates of A549 cells and enhanced its apoptosis and immune evasion partly via Fas/FasL pathway.

  14. The mRNA and protein expression of folylpolyglutamate synthetase in methotrexate enantiomer-resistant A549 cell lines%信息动态

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    Objective To study the expression of folylpolyglutamate synthetase ( FPGS ) in methotrexate ( MTX ) enantiomer-resistant A549 cell lines [ L-( + )-MTX and D-( - )-MTX ]. Methods The expression of FPGS on genetic and protein level was determined by FQ-PCR and Western blot in lung cancer A549 cells, and MTX enantiomer-resistant A549 cells [ L-( + )-MTX and D-( - )-MTX ], with the concentration of drug resistance was 15 μmol/L. Results The genetic expression level of FPGS was ( 0.80 ± 0. 09 ) and ( 2. 04 ± 0. 34 ) folds in L-( + )- MTX/A549 cells and D-( - )-MTX/A549 cells compared with lung cancer A549 cells, there was statistical difference between two groups ( P < 0.05 ). The protein expression level of FPGS was ( 0. 85 ± 0. 12 ) and( 1.62 ± 0. 24 ) folds in L-( + )-MTX/A549 cells and D-( - )-MTX/A549 cells compared with lung cancer A549 cells,there was statistical difference ( P < 0. 05 ). Conclusion The expression level of FPGS on genetic and protein level in drug resistant cells have been changed, and significant difference in two enantiomer-resistant cells are appeared.

  15. Protective effects of amygdalin on hyperoxia-exposed type Ⅱ alveolar epithelial cells isolated from premature rat lungs in vitro%苦杏仁甙对高氧暴露早产鼠肺泡Ⅱ型细胞的保护作用

    Institute of Scientific and Technical Information of China (English)

    常立文; 祝华平; 李文斌; 刘汉楚; 张谦慎; 陈红兵

    2005-01-01

    目的探讨苦杏仁甙对体外高氧暴露早产鼠肺泡Ⅱ型细胞( type 2 alveolar epithelial cell,AECⅡ)的保护作用机制.方法原代培养早产鼠AECⅡ,建立高氧细胞模型,采用MTT比色法、流式细胞术、免疫印迹(Western blot)、逆转录聚合酶链反应(RT-PCR)等方法,观察苦杏仁甙对高氧暴露早产鼠AECⅡ增殖及表面活性物质蛋白(surfactant associated protein, SP)mRNA表达的影响.结果高氧暴露导致早产鼠AECⅡ增殖抑制,AECⅡSPs mRNA表达降低.MTT试验显示,苦杏仁甙50~200μmol/L时呈剂量依赖方式促进早产鼠AECⅡ细胞增殖,200 μmol/L浓度时,其作用最强,400 μmol/L浓度时反而呈抑制作用.200 μmol/L苦杏仁甙可显著促进体外高氧暴露AECⅡ增殖,提高其SP mRNA表达水平.结论高氧暴露导致早产鼠AECⅡ增殖抑制及SP mRNA表达降低,200 μmol/L苦杏仁甙对体外高氧暴露的早产鼠AECⅡ有一定保护作用.

  16. X射线对人肺腺癌A549细胞Pokemon基因表达的影响%Effects of X-ray irradiation on expression of Pokemon gene in A549 cells of human lung adenocarcinoma

    Institute of Scientific and Technical Information of China (English)

    王璐; 邹跃; 江其生; 李伟; 宋秀军; 周湘艳; 王翠兰

    2011-01-01

    目的 研究不同剂量X射线照射及照射后不同时间点对人肺腺癌A549细胞Pokemon基因表达的影响.方法 用吸收剂量分别为2、4、6和8 Gy的X射线照射体外堵养的人肺腺癌A549细胞,2、4、8、12、24、48和72 ha,用实时定量PCR技术检测其中的Pokemon mRNA表达水平,以未照射组为对照.结果 在2、4、6、8 Gy X射线照射后的早期(除2 Gy照射后的2和4 h外)Pokemon mRNA的表达降低,但在晚期(48 h以后)呈升高趋势,在大部分时间点实验组与对照组的差异有统计学意义(t=3.40~154.76,P<0.05).结论 较大剂量的X射线在早期可下调A549细胞Pokemon基因mRNA的表达,诱导肿瘤细胞凋亡;但在晚期又可诱导A549细胞高表达PokemonmRNA,这可能与辐射所致A549细胞的DNA损伤修复和细胞周期调控有关.%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.

  17. CT in alveolar proteinosis

    International Nuclear Information System (INIS)

    Nine patients with pulmonary alveolar proteinosis (PAP) were scanned with CT. Results were not specific for PAP but may be help0ful in the differential diagnosis is visualization of pulmonary vessels is maintained and air bronchograms are scarce in diseased areas. CT is better than conventional radiography in identifying infectious complications and evaluating disease extent and may help in deciding whether or not therapeutic lavage is indicated. CT has also identified an interstitial component of PAP that correlates histologically with an inflammatory cell infiltrate. The cellular infiltrate may play a role in the development of the interstitial fibrosis seen in some cases of PAP

  18. Effects of simvastatin on the transdifferentiation of hypoxia and reoxygenation injury of alveolar epithelial type Ⅱ cells%辛伐他汀促进缺氧复氧损伤后肺泡Ⅱ型上皮细胞转分化的研究

    Institute of Scientific and Technical Information of China (English)

    武雅琴; 冯冬杰; 蒋峰; 张治; 黄建峰; 张帅; 尹荣; 许林

    2012-01-01

    目的 观察辛伐他汀是否能在体外促进缺氧复氧损伤后肺泡Ⅱ型上皮细胞向Ⅰ型细胞转分化,并探讨其作用机制.方法 体外培养小鼠肺泡上皮细胞MLE-12,建立缺氧复氧损伤模型,分为空白对照组(Blank)、辛伐他汀组(Sim)及缺氧复氧组(H/R),分别于缺氧2h后复氧0h、1d、3d和7d共4个时间点获取细胞,流式细胞仪检测肺泡Ⅰ型/Ⅱ型细胞表面特异性标志Caveolin- 1/Pro-SP-C 阳性细胞数百分比,Western blot法测定各组Pro-SP-C和Caveolin-1蛋白水平,最后行甲羟戊酸通路竞争实验观察左旋甲羟戊酸( L-meva)对辛伐他汀作用的影响.结果 流式细胞术结果显示:在缺氧复氧早期(d0及d1),Sim组较H/R组Caveolin-1/Pro-SP-C百分比显著降低;至d3和d7百分比则显著升高;Western blot结果显示:与H/R组比较,Sim组Pro-SP-C蛋白水平在d0及d1最高,至d3和d7则显著下降,Caveolin-1蛋白水平在d1最低,至d3和d7则逐渐升高,两者比较均有显著统计学差异(P<0.01).L-meva竞争试验显示:与Sim组比较,Sim+ L-meva组在各个时间点Pro-SP-C和Caveolin-1蛋白水平差异无统计学意义(P>0.05).结论 辛伐他汀可以促进缺氧复氧损伤后肺泡Ⅱ型上皮细胞向Ⅰ型细胞的转分化,但其作用机制不依赖于甲羟戊酸通路.%Objective To investigate the role of simvastatin in the transdifferentiation of hypoxia and reoxygenation injury of type Ⅱ alveolar epithelial cells and explore the underlying mechanisms.Methods Mouse AT Ⅱ cell line MLE-12 were subjected to hypoxia reoxygenation (H/R) injury.Blank,control and simvastatin-treated groups (20 μmol/L) were designed in the present study.The percentage of Caveolin-1/Pro-SP-C expression in the different points was assessed by flow cytometry double-staining.The protein levels of pro-surfactant protein-C (Pro-SP-C) and Caveolin-1 in AT Ⅱ cells was determined by Western blotting.And the protein levels of Pro-SP-C and Caveolin-1 was

  19. Natural iron chelators: Protective role in A549 cells of flavonoids-rich extracts of Citrus juices in Fe(3+)-induced oxidative stress.

    Science.gov (United States)

    Ferlazzo, Nadia; Visalli, Giuseppa; Cirmi, Santa; Lombardo, Giovanni Enrico; Laganà, Pasqualina; Di Pietro, Angela; Navarra, Michele

    2016-04-01

    Exogenous iron in particulate matter and imbalanced iron homeostasis cause deleterious effects on health. Natural and synthetic iron chelators may be of therapeutic benefit, therefore we evaluated the protective effect of Citrus flavonoids-rich extracts from bergamot and orange juices in iron overloaded human lung epithelial cells. Cytofluorimetric, biochemical and genotoxic analyses were performed in Fe2(SO4)3 exposed A549, pretreated with each extract whose chemical composition was previously detected. Chelating activity was assessed in cells by a calcein ester. Both extracts reduced the generation of reactive oxygen species and membrane lipid peroxidation, improved mitochondrial functionality, and prevented DNA-oxidative damage in iron-exposed cells. Antioxidant effects were attributed to the chelating property, blocking upstream the redox activity of iron. Flavonoid-rich extracts also induced antioxidant catalase. The bergamot and orange juice extracts had a broad-spectrum protective effect. Their use prevents iron oxidative injury and these natural iron chelators could be used as therapeutic agents. PMID:27037654

  20. Genetics Home Reference: pulmonary alveolar microlithiasis

    Science.gov (United States)

    ... Genetics Home Health Conditions pulmonary alveolar microlithiasis pulmonary alveolar microlithiasis Enable Javascript to view the expand/collapse boxes. Print All Open All Close All Description ...

  1. Inhibiting Effect and Its Mechanism of Ibandronate on the Proliferation of Humanized NSCLC A549 Cells in Vitro

    Institute of Scientific and Technical Information of China (English)

    YAO Qiang; HUA Dong

    2014-01-01

    Objective:To explore the effect of ibandronate on the proliferation and the expression of human telomerase reverse transcriptase (hTERT) of non-small cell lung cancer (NSCLC) A549 cell line in vitro. Methods: Methyl thiazolyl tetrazolium (MTT) assay, microscope, flow cytometry (FCM) and semi-quantitative RT-PCR were employed to detect the cell proliferation, cell cycle as well as the morphological change and the expression of hTERT mRNA of A549 cell line. Results:The data showed that ibandronate could effectively inhibit the proliferation of A549 cell line in time-and concentration-dependent. Under the microscope, the lfoating cells increased gradually as the drug concentration increasing. FCM detection showed that ibandronate could induce the cell cycle stopped in G0/G1 phase and downregulation expression of hTERT. Conclusion:Ibandronate can inhibit the proliferation of A549 cell line in vitro, whose mechanism may be associated with cell cycle arrestted in phase G0/G1 and downregulation expression of hTERT.

  2. Epithelial fibroblast triggering and interactions in pulmonary fibrosis

    OpenAIRE

    Noble, P W

    2008-01-01

    Idiopathic pulmonary fibrosis (IPF) is characterised by repeated injury to the alveolar epithelium with loss of lung epithelial cells and abnormal tissue repair, resulting in excessive accumulation of fibroblasts and myofibroblasts, deposition of extracellular matrix components and distortion of lung architecture, eventually leading to respiratory failure. There is growing circumstantial evidence to suggest that in IPF the alveolar epithelium is prone to undergoing programmed cell death follo...

  3. Mechanism of Thymosin Beta 10 Inhibiting the Apoptosis 
and Prompting Proliferation in A549 Cells

    Directory of Open Access Journals (Sweden)

    Zixuan LI

    2014-11-01

    Full Text Available Background and objective Thymosin beta 10 (Tβ10 is one of β-thymosin family members, has a highly conserved polar 5 kDa peptides. This peptide is now regarded to be a small actin-binding protein and thereby induce depolymerization of the intracellular F-actin networks. Alteration of Tβ10 expression may alter the balance of cell growth, cell death, cell attachment and cell migration. Tβ10 also affects cell metastasis as well as proliferation, apoptosis and vascularization of cancer cells. But function of Tβ10 appear to be rather different between cancer cells, and the molecular mechanisms of β-thymosins to regulate cell apoptosis and proliferation in NSCLC (non-small cell lung cancer cell lines are unclear. In this study, we used lung adenocarcinoma cell line A549, added Tβ10 or down-regulated the expression of Tβ10. We observed the change of apoptosis, proliferation and cell cyclin ability in A549 and the mechanisms underline them were also identified. Methods After A549 was treated with 100 ng/mL recombinant human Tβ10 or siTβ10, apoptosis rate of A549 and cell cycle distribution were detected by flow cytometry (FCM. CCK-8 assay was employed to determine the proliferation of A549. The mRNA level of P53, Caspase-3, Cyclin A and Cyclin E were determined by real-time PCR. The protein level of P53, Caspase-3, Cyclin A and Cyclin E were detected by Western blot. Results Add Tβ10 can inhibit the apoptosis and prompt the proliferation of A549. It can also increase the cell rates of S-phrase and G2/M-phrase, decrease the expression of P53 and Caspase-3, but increase the expression of Cyclin A and Cyclin E. Interferance of Tβ10 can prompt the apoptosis and inhibit the proliferation of A549. It can also increase the cell rates of G0/G1-phrase, increase the expression of P53 and Caspase-3, but decrease the expression of Cyclin A and Cyclin E. Conclusion In lung cancer cell line, Tβ10 can inhibit the apoptosis by increase P53, drive cells into

  4. 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

  5. β-elemene reverses the drug resistance of lung cancer A549/DDP cells via the mitochondrial apoptosis pathway.

    Science.gov (United States)

    Yao, Cheng-Cai; Tu, Yuan-Rong; Jiang, Jie; Ye, Sheng-Fang; Du, Hao-Xin; Zhang, Yi

    2014-05-01

    β-elemene (β-ELE) is a new anticancer drug extracted from Curcuma zedoaria Roscoe and has been widely used to treat malignant tumors. Recent studies have demonstrated that β-ELE reverses the drug resistance of tumor cells. To explore the possible mechanisms of action of β-ELE, we investigated its effects on cisplatin-resistant human lung adenocarcinoma A549/DDP cells. The effects of β-ELE on the growth of A549/DDP cells in vitro were determined by MTT assay. Apoptosis was assessed by fluorescence microscopy with Hoechst 33258 staining and flow cytometry with Annexin V-FITC/PI double staining. Mitochondrial membrane potential was assessed using JC-1 fluorescence probe and laser confocal scanning microscopy, and intracellular reactive oxygen species levels were measured by 2',7'-dichlorofluorescein-diacetate staining and flow cytometry. Cytosolic glutathione content was determined using GSH kits. The expression of cytochrome c, caspase-3, procaspase-3 and the Bcl-2 family proteins was assessed by western blotting. The results demonstrated that β-ELE inhibited the proliferation of A549/DDP cells in a time- and dose-dependent manner. Furthermore, β-ELE enhanced the sensitivity of A549/DDP cells to cisplatin and reversed the drug resistance of A549/DDP cells. Consistent with a role in activating apoptosis, β-ELE decreased mitochondrial membrane potential, increased intracellular reactive oxygen species concentration and decreased the cytoplasmic glutathione levels in a time- and dose-dependent manner. The combination of β-ELE and cisplatin enhanced the protein expression of cytochrome c, caspase-3 and Bad, and reduced protein levels of Bcl-2 and procaspase-3 in the A549/DDP lung cancer cells. These results define a pathway of procaspase‑3-β-ELE function that involves decreased mitochondrial membrane potential, leading to apoptosis triggered by the release of cytochrome c into the cytoplasm and the modulation of apoptosis-related genes. The reversal of drug

  6. Antineoplastic effects of deoxyelephantopin,a sesquiterpene lactone from Elephantopus scaber, on lung adenocarcinoma (A549) cells

    Institute of Scientific and Technical Information of China (English)

    Farha A.Kabeer; Geetha B.Sreedevi; Mangalam S.Nair; Dhanya S.Rajalekshmi; Latha P.Gopalakrishnan; Sujathan Kunjuraman; Remani Prathapan

    2013-01-01

    OBJECTIVE:Deoxyelephantopin,a sesquiterpene lactone from Elephantopus scaber,showed inhibition of the growth of various tumor cells in vitro.In the present study,we investigated the cytotoxicity and apoptosis-inducing capacity of deoxyelephantopin on lung adenocarcinoma (A549) cells.METHODS:The cytotoxic effect of deoxyelephantopin on A549 cells and normal lymphocytes was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and 50% inhibitory concentration (IC50) value was determined.The self-renewal and proliferating potential of A549 cells after treatment with deoxyelephantopin were examined by colony formation assay.Cellular morphology of deoxyelephantopin-treated cells was observed using phasecontrast microscopy.The induction of apoptosis was evaluated using acridine orange and ethidium bromide staining,Hoechst 33342 staining,terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end-labeling (TUNEL) assay,DNA fragmentation analysis and Annexin V-fluorescein isothiocyanate staining by flow cytometry.Activation of caspases was detected using fluorogenic substrate specific to caspases 2,3,8 and 9 and flow cytometric analysis.The total cellular DNA content and expression of cleaved poly (ADP-ribose) polymerase was also analyzed.RESULTS:Deoxyelephantopin exhibited cytotoxicity to A549 cells (IC5o =12.287 μg/mL),however,there was no toxicity towards normal human lymphocytes.Deoxyelephantopin suppressed the colony-forming ability of A549 cells in a dose-dependent manner.Acridine orange,ethidium bromide and Hoechst 33342 staining showed cell shrinkage,chromosomal condensation and nuclear fragmentation,indicating induction of apoptosis.Deoxyelephantopin increased apoptosis of A549 cells,as evidenced by more TUNEL-positive cells.DNA fragmentation and Annexin V staining revealed late-stage apoptotic cell population.Deoxyelephantopin inhibited A549 cell growth by cell cycle arrest at G2/M phase and induced apoptosis through

  7. Pulmonary alveolar proteinosis

    Directory of Open Access Journals (Sweden)

    Hadice Selimoğlu Şen

    2011-06-01

    Full Text Available Pulmonary alveolar proteinosis is a rare disease characterized by the accumulation of lipoproteinosis material within the alveoli. A 36 age male patient applied to our hospital with dyspnea that began 5 months ago. Bilateral diffuse infiltration on postero-anterior chest graphy was seen. Arterial blood gas measurements (ABG was: pH:7.44, pCO2: 36 mmHg, pO2: 49 mmHg, SaO2: 85%, HCO3: 24 mmol/L. High resolution computed tomography (HRCT reported as bilateral diffuse ground glass areas and interlobuler septal thickening on this areas. Asido resistant bacillus was negative in sputum at three times and was also negative in post bronchoscopic sputum and bronchoalveoler lavage material. Wedge resection was made by video-assisted thoracoscopic surgery. Histopathology report of biopsy material revealed “Pulmonary alveolar proteinosis (PAP”. Massive lung lavage under general anesthesia was planned for patient. Clinical improvement was seen in patient beginning from admission and ABG measurements entered the recovery trend in follow-up. Partial pressure of oxygen was increased to 65 mmHg and patient was followed without lung lavage. Control chest radiography and HRCT showed significant radiological improvement. After three months, radiological lesions had completely regressed at control HRCT. As a result, in patients with symptoms and radiologically bilateral diffuse infiltration physicians should consider PAP as a rare disease in the differential diagnosis.

  8. 人肺癌A549细胞系肿瘤干细胞的筛选和鉴定%Identiifcation and Isolation of Cancer Stem Cells from A549 Cells

    Institute of Scientific and Technical Information of China (English)

    夏晖; 于长海; 张文; 张宝石; 赵英男; 方芳

    2013-01-01

    Background and objective Lung cancer stem cells are the root causes of lung cancer malignant phe-notype and potential therapeutic target, the aim of this study is to isolate and characterize the cancer stem cells in the lung adenoearcinomas cell line A549, so as to provide an experimental basis for further stem cell research. Methods hTe cancer stem cells were isolated from the lung adenoearcinomas cell line A549 using FACS. And the difference of colony formation, cell proliferation and tumorigenicity in vitro were also tested. hTe expression of CD133 and ABCG2 were evaluated by RT-PCR and Western blot. Results hTe percentage of SP cells was 5.93%of A549 and 0.32%of A549 atfer incubation with verapamil. hTe results showed that there were signiifcantly higher expression of CD133 and ABCG2 on SP cells than that of non-SP cells. And the ability of colony formation, cell proliferation and tumorigenicity in SP cell group were remarkably higher than that in non-SP cell group. Conclusion Our results suggested that the cancer stem cells with higher expression of CD133 and ABCG2 can be isolated from the lung adenoearcinomas cell line A549 using FACS and be used in the further research experiments.%背景与目的肺癌干细胞是肺癌恶性表型的根源和潜在的治疗靶点,从人肺癌A549细胞株中分离肺癌干细胞,观察特异性干细胞标志物分子的表达,为进一步的干细胞研究提供试验基础。方法接种肺癌A549细胞株,经流式细胞术,特异性筛选分离肺癌干细胞,观察克隆形成能力、细胞增殖能力和体外致瘤能力的差别,并分别用RT-PCR和Western blot的方法分析干细胞标志物分子CD133和ABCG2的表达。结果经过流式细胞仪成功分选了人肺腺癌A549细胞系SP细胞亚群,结果表明此SP细胞亚群约占A549细胞总数的5.93%,经维拉帕米处理后Hoechest33342阴性/弱阳性细胞含量下降为0.32%,SP细胞克隆形成能力,细胞增殖能力和

  9. 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.

  10. Role of cytoskeleton network in anisosmotic volume changes of intact and permeabilized A549 cells.

    Science.gov (United States)

    Platonova, Alexandra; Ponomarchuk, Olga; Boudreault, Francis; Kapilevich, Leonid V; Maksimov, Georgy V; Grygorczyk, Ryszard; Orlov, Sergei N

    2015-10-01

    Recently we found that cytoplasm of permeabilized mammalian cells behaves as a hydrogel displaying intrinsic osmosensitivity. This study examined the role of microfilaments and microtubules in the regulation of hydrogel osmosensitivity, volume-sensitive ion transporters, and their contribution to volume modulation of intact cells. We found that intact and digitonin-permeabilized A549 cells displayed similar rate of shrinkage triggered by hyperosmotic medium. It was significantly slowed-down in both cell preparations after disruption of actin microfilaments by cytochalasin B, suggesting that rapid water release by intact cytoplasmic hydrogel contributes to hyperosmotic shrinkage. In hyposmotic swelling experiments, disruption of microtubules by vinblastine attenuated the maximal amplitude of swelling in intact cells and completely abolished it in permeabilized cells. The swelling of intact cells also triggered ~10-fold elevation of furosemide-resistant (86)Rb+ (K+) permeability and the regulatory volume decrease (RVD), both of which were abolished by Ba2+. Interestingly, RVD and K+ permeability remained unaffected in cytocholasin/vinblastine treated cells demonstrating that cytoskeleton disruption has no direct impact on Ba2+-sensitive K+-channels involved in RVD. Our results show, for the first time, that the cytoskeleton network contributes directly to passive cell volume adjustments in anisosmotic media via the modulation of the water retained by the cytoplasmic hydrogel. PMID:26171817

  11. Effect of glutathione depletion on the aerobic radiation response of A549 human lung carcinoma cells

    International Nuclear Information System (INIS)

    The authors demonstrated that depletion of glutathione (GSH) from cultured A549 cells to non-detectable levels, using L-buthionine sulfoximine (L-BSO), results in an increased aerobic radiation response. This response can be further increased if dimethylfumarate (DMF) is added concurrently with L-BSO. L-BSO is a relatively slow depletor of GSH compared to DMF, which acts by both spontaneous and enzyme catalysed reactions. The authors have studied: 1. the effect of continuous long-term exposure to 0.1 mM L-BSO on GSH levels and the subsequent radiation response and 2. the effect of GSH depletion on enzymes essential for radical detoxification. The results show an enhanced aerobic radiation response that increases with the time of exposure to L-BSO. For example surviving fraction (S.F.) after 5 Gy for cells exposed to L-BSO for 24 hrs is 0.004 and 0.08 for control cultures. Cells washed free of medium and irradiated in Hanks' show 0.0007 S.F. after 120 hr exposure to L-BSO and S.F. of 0.075 for the control cultures. The relationship between the chronic GSH depleted state, GSH peroxidase, and radiation induced lipid peroxidation is being investigated

  12. 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.

  13. Nimesulide has a role of radio-sensitizer against lung carcinoma A549 cells

    International Nuclear Information System (INIS)

    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

  14. In vitro photodynamic effect by phthalocyanine in A549 cell line

    Science.gov (United States)

    Nevrelova, Pavla; Kolarova, Hana; Bajgar, Robert; Strnad, Miroslav

    2007-03-01

    Photodynamic therapy (PDT) utilizes a combination of sensitizer, visible light and molecular oxygen to generate singlet oxygen and reactive oxygen species (ROS) such as hydrogen peroxide, hydroxyl radical and superoxid anion. Photochemical reactions lead to damage and destruction of cancer cells. The most suitable and effective source of radiation used in PDT is a laser. For this study, a semiconductor laser with output power of 50 mW and 675 nm was selected. In this paper we report a generation of ROS using chloroaluminium disulphonated phthalocyanine (ClAlPcS II) in A549 bronchogenic carcinoma cell line after PDT in vitro. Phthalocyanines, belonging to a new generation of substances for PDT, exhibit effective tissue penetration because of their proper light absorption region, chemical stability and photodynamic stability. The fluorescence measurement with molecular probes, CM-H IIDCFDA and Amplex Red, was performed for detection of ROS generation and hydrogen peroxide release from cells. Our results demonstrated, that irradiation of cells by laser dose of 10 J.cm -2 induces higher rates of fluorescence in cells loaded with phthalocyanine compared to 20 J.cm -2. Furthermore, the production of ROS increases up to sensitizer concentration of 10 μM. The highest ROS generation was observed at laser dose of 10 J.cm -2 and 10 μM ClAlPcS II. The rates of fluorescence for hydrogen peroxid measurements were almost identical with all chosen concentrations at laser doses of 10 and 20 J.cm -2.

  15. In vitro anticancer activity of fucoidan from Turbinaria conoides against A549 cell lines.

    Science.gov (United States)

    Marudhupandi, Thangapandi; Ajith Kumar, Thipramalai Thankappan; Lakshmanasenthil, Shanmugaasokan; Suja, Gunasekaran; Vinothkumar, Thirumalairaj

    2015-01-01

    The present study was conducted to evaluate the anticancer activity of fucoidan isolated from brown seaweed Turbinaria conoides. Extracted fucoidan contained 53 ± 0.69% of fucose and 38 ± 0.42% of sulphate, respectively. Functional groups and structural characteristics of the fucoidan were analyzed by FT-IR and NMR. In vitro anticancer effect was studied on A549 cell line. Fucoidan inhibited the growth of cancer cells in a dose-dependent manner and potent anticancer activities were 24.9-73.5% in the concentrations of 31.25-500 μg/ml. The CTC50 value against the cancer cell was found to be 45 μg/ml and the CTC50 value of normal Vero cell line is 325 μg/ml. This study suggests that the fucoidan from T. conoides could be significantly improved if the active component is further purified and tested for further investigation in various cancer cell lines. PMID:25451746

  16. CUX1/Wnt signaling regulates Epithelial Mesenchymal Transition in EBV infected epithelial cells

    International Nuclear Information System (INIS)

    Idiopathic pulmonary fibrosis (IPF) is a refractory and lethal interstitial lung disease characterized by alveolar epithelial cells apoptosis, fibroblast proliferation and extra-cellular matrix protein deposition. EBV, localised to alveolar epithelial cells of pulmonary fibrosis patients is associated with a poor prognosis. A strategy based on microarray-differential gene expression analysis to identify molecular drivers of EBV-associated lung fibrosis was utilized. Alveolar epithelial cells were infected with EBV to identify genes whose expression was altered following TGFβ1-mediated lytic phase. EBV lytic reactivation by TGFβ1 drives a selective alteration in CUX1 variant (a) (NCBI accession number NM181552) expression, inducing activation of non-canonical Wnt pathway mediators, implicating it in Epithelial Mesenchymal Transition (EMT), the molecular event underpinning scar production in tissue fibrosis. The role of EBV in EMT can be attenuated by antiviral strategies and inhibition of Wnt signaling by using All-Trans Retinoic Acids (ATRA). Activation of non-canonical Wnt signaling pathway by EBV in epithelial cells suggests a novel mechanism of EMT via CUX1 signaling. These data present a framework for further description of the link between infectious agents and fibrosis, a significant disease burden.

  17. CUX1/Wnt signaling regulates Epithelial Mesenchymal Transition in EBV infected epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Malizia, Andrea P.; Lacey, Noreen [Clinical Research Centre, School of Medicine and Medical Science, University College Dublin. 21, Nelson Street. Dublin, 7. Ireland (Ireland); Walls, Dermot [School of Biotechnology, Dublin City University. Dublin, 9. Ireland (Ireland); Egan, Jim J. [Advanced Lung Disease and Lung Transplant Program, Mater Misericordiae University Hospital. 44, Eccles Street. Dublin, 7. Ireland (Ireland); Doran, Peter P., E-mail: peter.doran@ucd.ie [Clinical Research Centre, School of Medicine and Medical Science, University College Dublin. 21, Nelson Street. Dublin, 7. Ireland (Ireland)

    2009-07-01

    Idiopathic pulmonary fibrosis (IPF) is a refractory and lethal interstitial lung disease characterized by alveolar epithelial cells apoptosis, fibroblast proliferation and extra-cellular matrix protein deposition. EBV, localised to alveolar epithelial cells of pulmonary fibrosis patients is associated with a poor prognosis. A strategy based on microarray-differential gene expression analysis to identify molecular drivers of EBV-associated lung fibrosis was utilized. Alveolar epithelial cells were infected with EBV to identify genes whose expression was altered following TGF{beta}1-mediated lytic phase. EBV lytic reactivation by TGF{beta}1 drives a selective alteration in CUX1 variant (a) (NCBI accession number NM{sub 1}81552) expression, inducing activation of non-canonical Wnt pathway mediators, implicating it in Epithelial Mesenchymal Transition (EMT), the molecular event underpinning scar production in tissue fibrosis. The role of EBV in EMT can be attenuated by antiviral strategies and inhibition of Wnt signaling by using All-Trans Retinoic Acids (ATRA). Activation of non-canonical Wnt signaling pathway by EBV in epithelial cells suggests a novel mechanism of EMT via CUX1 signaling. These data present a framework for further description of the link between infectious agents and fibrosis, a significant disease burden.

  18. Effect of artemether on the poliferation of human lung adenocarcinoma cell line A549%蒿甲醚对人肺腺癌A549细胞体外生长的影响

    Institute of Scientific and Technical Information of China (English)

    郭燕; 王俊; 章必成; 陈正堂

    2007-01-01

    目的:研究抗疟疾药物蒿甲醚(Artemether)对人肺腺癌A549细胞株体外生长的影响,为蒿甲醚治疗肺癌提供实验依据. 方法:采用单四唑(MTT)比色法检测蒿甲醚对体外培养的人肺腺癌A549细胞的生长抑制作用;用细胞计数法绘制细胞生长曲线,计算对数生长期群体倍增时间;用流式细胞术研究蒿甲醚对细胞周期的影响;采用苏木精-伊红(H-E)染色在光镜下观察凋亡细胞的形态学特征. 结果:蒿甲醚对A549细胞株的半数抑制浓度(IC50)为1.34 mg/L.A549肺腺癌细胞对数生长期群体倍增时间在蒿甲醚作用后(20.7±0.5)h,对照组为(32.2±0.3 )h,两组比较有显著性差异(P< 0.01).A549细胞经蒿甲醚作用后G1期细胞百分比增加(P<0.01),G2或S期细胞减少(P<0.01),凋亡率明显增加(P<0.01). 结论:蒿甲醚对人肺腺癌A549细胞株生长具有显著抑制作用;蒿甲醚的细胞毒作用与其诱导肿瘤细胞凋亡有关.

  19. Dehydroepiandrosterone inhibits the spontaneous release of superoxide radical by alveolar macrophages in vitro in asbestosis

    Energy Technology Data Exchange (ETDEWEB)

    Rom, W.N.; Harkin, T. (New York Univ. Medical Center, New York (United States))

    1991-08-01

    Asbestosis is characterized by an alveolar macrophage alveolitis with injury and fibrosis of the lower respiratory tract. Alveolar macrophages recovered by bronchoalveolar lavage spontaneously release exaggerated amounts of oxidants including superoxide anion and hydrogen peroxide that may mediate alveolar epithelial cell injury. Dehydroepiandrosterone (DHEA) is a normally occurring adrenal androgen that inhibits glucose-6-phosphate dehydrogenase, the initial enzyme in the pentose phosphate shunt necessary for NADPH generation and superoxide anion formation. In this regard, the authors hypothesized that DHEA may reduce asbestos-induced oxidant release. DHEA added in vitro to alveolar macrophages lavaged from 11 nonsmoking asbestos workers significantly reduced superoxide anion release. DHEA is an antioxidant and potential anticarcinogenic agent that may have a therapeutic role in reducing the increased oxidant burden in asbestos-induced alveolitis of the lower respiratory tract.

  20. 荞麦七提取物对肺癌A549细胞增殖及凋亡的影响%Effects of Fagopyrum cymosum extracts on proliferation and apoptosis of lung cancer cell line A549

    Institute of Scientific and Technical Information of China (English)

    李健; 王晓梅; 杨春娟; 刘帆

    2015-01-01

    Objective To investigate the effects of Fagopyrum cymosum extracts on proliferation and apoptosis of human lung cancer cell line A549. Methods A549 lung cancer cells were processed with aqueous extracts and anthraquinone of Fagopyrum cymosum. Cell viability was detected by trypan blue staining. The inhibition rate of cell proliferation was detected by MTT. The protein expression levels of Csapase 9 and P53 were detected by immunohis-tochemical method. Results The inhibition effects of Fagopyrum cymosum aqueous extracts on lung cancer cell line A549 increased along with higher concentration of the extracts. The inhibition rate at 72 h was significantly higher than the rates at 24 h and 48 h, while there were no significant differences in inhibition rates among the three con-centrations of Fagopyrum cymosum anthraquinone. The induction on Csapase 9 and inhibition on P53 by both extracts were enhanced with the increase of concentration. Conclusion The aqueous extracts and anthraquinone of Fagopy-rum cymosum can inhibit the proliferation of human lung cancer cell line A549 and induce their apoptosis, with the underlying mechanism possibly related to the up-regulation of Caspase 9 and down-regulation of P53.%目的:研究荞麦七提取物对人肺癌A549细胞增殖及凋亡的影响。方法应用荞麦七水提物及荞麦七蒽醌处理肺癌A549细胞,锥虫蓝染色法检测细胞存活率,MTT法检测细胞增殖抑制率,免疫细胞化学法检测Caspase 9和P53蛋白表达水平。结果荞麦七水提取物对肺癌A549细胞增殖的抑制作用随浓度而增强,72 h的抑制率明显较24 h及48 h强,荞麦七蒽醌3种浓度的抑制率之间差异不大。2种提取物对Caspase 9的诱导作用均随着浓度的增大而增强,对P53的抑制作用也随着浓度的增大而增强。结论荞麦七水提物及蒽醌能抑制人肺癌A549细胞的增殖,并诱导其凋亡,其机制可能与上调Caspase 9的表达及下调P53的表达有关。

  1. 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.

  2. Particle-induced indentation of the alveolar epithelium caused by surface tension forces.

    Science.gov (United States)

    Mijailovich, S M; Kojic, M; Tsuda, A

    2010-10-01

    Physical contact between an inhaled particle and alveolar epithelium at the moment of particle deposition must have substantial effects on subsequent cellular functions of neighboring cells, such as alveolar type-I, type-II pneumocytes, alveolar macrophage, as well as afferent sensory nerve cells, extending their dendrites toward the alveolar septal surface. The forces driving this physical insult are born at the surface of the alveolar air-liquid layer. The role of alveolar surfactant submerging a hydrophilic particle has been suggested by Gehr and Schürch's group (e.g., Respir Physiol 80: 17-32, 1990). In this paper, we extended their studies by developing a further comprehensive and mechanistic analysis. The analysis reveals that the mechanics operating in the particle-tissue interaction phenomena can be explained on the basis of a balance between surface tension force and tissue resistance force; the former tend to move a particle toward alveolar epithelial cell surface, the latter to resist the cell deformation. As a result, the submerged particle deforms the tissue and makes a noticeable indentation, which creates unphysiological stress and strain fields in tissue around the particle. This particle-induced microdeformation could likely trigger adverse mechanotransduction and mechanosensing pathways, as well as potentially enhancing particle uptake by the cells. PMID:20634359

  3. Application of linear integration in the morphometric study of mild and severe pulmonary alveolar injury.

    Science.gov (United States)

    Coulombe, P A; Côté, M G

    1988-02-01

    In this study we applied linear integration morphometry to characterize the pulmonary alveolar reaction to toxic injury and to study possible relationships between the major tissue and cell compartments of alveolar tissue, normal and injured. Acute alveolar injury of mild and severe intensity was induced in Swiss-Webster mice by the ip administration of the chemicals diquat (4 mg/kg) and butylated hydroxytoluene (BHT; 400 mg/kg). Animals were sacrificed at Days 1 and 2 after diquat treatment and at Days 1, 3, and 5 after BHT treatment. Sampling and analysis of alveolar tissue were conducted at both levels of light and electron microscopy. Thickness distributions of arithmetic and reciprocal intercepts, as well as the arithmetic (tau) and harmonic (tau h) mean thicknesses, were established for the following alveolar compartments: septum, alveolo-capillary barrier (ACB), type I and total epithelia, capillary endothelium, and interstitium. A relative measure of the pulmonary diffusion capacity and the capillary load of alveolar septa were also determined. The parameters calculated from these thickness distributions, such as their slopes, percentages of thin intercepts, and tau/tau h ratios, proved very sensitive and useful in the detection and characterization of morphological alterations in the type I epithelial and capillary endothelial cells following either mild or severe alveolar injury. The epithelial, endothelial, and interstitial layers of pulmonary septa were all characterized by their own pattern of structural changes, so that it proved impossible to relate them in a simple way to the tissue reaction, which can be easily studied at the light microscopic level. Linear integration morphometry thus proved very useful as a morphometric approach to the study of pulmonary alveolar injury and repair. PMID:3335253

  4. Expression of epidermal growth factor receptor gene in methotrexate enantiomers-resistant A549 cells and influence on cellular migration ability%氨甲蝶呤对映体耐药A549细胞的表皮生长因子受体基因表达及迁移能力

    Institute of Scientific and Technical Information of China (English)

    张白银; 何晓东; 孙余婕; 张永娟; 嵇金陵; 沈佐君

    2012-01-01

    目的 研究氨甲蝶呤(MTX)对映体耐药人非小细胞肺癌A549细胞的迁移能力以及表皮生长因子受体(EGFR)mRNA的表达.方法 用细胞划痕试验检测L-(+)-MTX/A549细胞和D-(-)-MTX/A549细胞的迁移能力;双层软琼脂克隆试验检测L-(+ )-MTX/A549细胞和D-(-)-MTX/A549细胞的克隆形成率并观察集落的形态;用RT-PCR检测亲本A549细胞、L-(+ )-MTX/A549细胞和D-(-)-MTX/A549细胞中EGFR mRNA的表达.结果 加入MTX 72 h后D-(-)-MTX/A549细胞的迁移能力(1 230.1±40.2)高于L-(+ )-MTX/A549细胞(530.3±25.4);D-(-)-MTX/A549细胞、L-(+ )-MTX/A549细胞和亲本A549细胞的克隆形成率(%)分别为(1.38±0.17)、(1.36±0.13)和(1.37±0.15),差异无统计学意义(P>0.05);亲本A549细胞、L(+)-MTX/A549细胞均有EGFR mRNA表达,其光密度值(IDV)分别为(6 630±64)、(3 697±27),差异有统计学意义(t=103.42,P<0.01).而D-(-)-MTX/A549细胞不表达EGFR.结论 D-(-)-MTX诱导的A549细胞的迁移能力大于L-(+ )-MTX.EGFR基因表达具有手性差异.%Objective To investigate the migration ability of methotrexate (MTX) enantiomers -resistant non-small cell lung cancer ( NSCLC) cell line A549 and the expression of epidermal growth factor receptor (EGFR) in the cells. Methods The migration ability of L-( + )-MTX/A549 and D-( -) -MTX/A549 cells were evaluated by cell scratch assay. The colony formation rates and the morphology of cell cluster of L-( + ) ,MTX/A549 and D-(-) -MTX/A549 were determined by double-layer soft agar colony formation assay. The mRNA expression of EGFR in parental A549 cells, L-( + )-MTX/A549 cells, D-(-)-MTX/A549 cells were detected by RT-PCR. Results The migration ability of D-(-)-MTX/A549 cells (1 230. 1 ±40. 2) was stronger than that of L-( + )-MTX/A549 cells (530.3 ±25.4) at72 h after adding MTT. The rate of colony formation in D-(-)-MTX/A549, L-( + }-MTX/A549 and parental A549 cells was (1.38 ±0.17), (1.36±0.13) and (1.37 ±0. 15) respectively. There was

  5. PPARs in Alveolar Macrophage Biology

    Directory of Open Access Journals (Sweden)

    Monica R. Smith

    2007-01-01

    Full Text Available PPARs, most notably PPAR-γ, play a crucial role in regulating the activation of alveolar macrophages, which in turn occupy a pivotal place in the immune response to pathogens and particulates drawn in with inspired air. In this review, we describe the dual role of the alveolar macrophage as both a first-line defender through its phagocytotic activity and a regulator of the immune response. Depending on its state of activation, the alveolar macrophage may either enhance or suppress different aspects of immune function in the lung. We then review the role of PPAR-γ and its ligands in deactivating alveolar macrophages—thus limiting the inflammatory response that, if unchecked, could threaten the essential respiratory function of the alveolus—while upregulating the cell's phagocytotic activity. Finally, we examine the role that inadequate or inappropriate PPAR-γ responses play in specific lung diseases.

  6. 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

  7. Claudins: Gatekeepers of lung epithelial function.

    Science.gov (United States)

    Schlingmann, Barbara; Molina, Samuel A; Koval, Michael

    2015-06-01

    The lung must maintain a proper barrier between airspaces and fluid filled tissues in order to maintain lung fluid balance. Central to maintaining lung fluid balance are epithelial cells which create a barrier to water and solutes. The barrier function of these cells is mainly provided by tight junction proteins known as claudins. Epithelial barrier function varies depending on the different needs within the segments of the respiratory tree. In the lower airways, fluid is required to maintain mucociliary clearance, whereas in the terminal alveolar airspaces a thin layer of surfactant enriched fluid lowers surface tension to prevent airspace collapse and is critical for gas exchange. As the epithelial cells within the segments of the respiratory tree differ, the composition of claudins found in these epithelial cells is also different. Among these differences is claudin-18 which is uniquely expressed by the alveolar epithelial cells. Other claudins, notably claudin-4 and claudin-7, are more ubiquitously expressed throughout the respiratory epithelium. Claudin-5 is expressed by both pulmonary epithelial and endothelial cells. Based on in vitro and in vivo model systems and histologic analysis of lungs from human patients, roles for specific claudins in maintaining barrier function and protecting the lung from the effects of acute injury and disease are being identified. One surprising finding is that claudin-18 and claudin-4 control lung cell phenotype and inflammation beyond simply maintaining a selective paracellular permeability barrier. This suggests claudins have more nuanced roles for the control of airway and alveolar physiology in the healthy and diseased lung. PMID:25951797

  8. Abses Alveolar Akut Pada Anak

    OpenAIRE

    Delfitri, Meri

    2008-01-01

    Abses alveolar akut adalah suatu kumpulan nanah yang terlokalisasi dalam tulang alveolar sekitar apeks gigi setelah kematian pulpa yang merupakan kelanjutan proses infeksi yang mulai di pulpa dan berkembang ke jaringan periapikal. Perluasan infeksi dari pulpa non vital dapat melalui foramen apikal menuju jaringan periapikal yang mengakibatkan inflamasi akut, bila disertai dengan meningkatnya virulensi bakteri streptokokus alfa, disertai staffilokokus dan sejumlah anaerob lainnya serta r...

  9. Micronuclei in human alveolar macrophages.

    Science.gov (United States)

    D'Agostini, F; Bonatti, S; Oddera, S; De Flora, S

    1992-01-01

    Occurrence of micronuclei was monitored in pulmonary alveolar macrophages collected from 31 individuals undergoing diagnostic bronchoalveolar lavage. The overall frequency of micronucleated cells was 3.88 +/- 1.84/1000, without any significant difference attributable to sex, age, pathology, occupation, or smoking habits. The lack of influence of cigarette smoke on this clastogenicity index presumably reflects the very low rate of mitoses of macrophages in the alveolar lumen. PMID:1579732

  10. 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

  11. Effect of silencing of ATM expression by siRNA on radiosensitivity of human lung adenocarcinoma A549 cells

    International Nuclear Information System (INIS)

    Objective: To investigate the effect of silencing of ataxia-telangiectasia mutated (ATM) expression by plasmid-mediated RNA interference on the radiosensitivity of human lung adenocarcinoma A549 cells. Methods: Eukaryotic expression plasmid containing ATM small interfering RNA (siRNA) (pSilencer2.1-ATM), as well as pSilencer2.1-nonspecific, was constructed.Lung adenocarcinoma A549 cells were divided into positive group, negative group,and control group to be transfected with pSilencer2.1-ATM, pSilencer2.1-nonspecific, and no plasmid, respectively. The mRNA and protein expression of ATM was measured by RT-PCR and Western blot, respectively. The change in cell radiosensitivity was observed by colony-forming assay. Cell cycle and cell apoptosis were analyzed by flow cytometry. Results: The eukaryotic expression plasmid containing ATM siRNA was successfully constructed. The RT-PCR and Western blot demonstrated that the expression of ATM was down-regulated in the positive group. The sensitization enhancement ratios (D0 ratios) for the positive group and negative group were 1.50 and 1.01, respectively. The flow cytometry revealed that the proportions of A549 cells in G1 and G2/M phases were significantly lower in the positive group than in the control group (51.27% vs 61.85%, P = 0.012; 6.34% vs 10.91%, P = 0.008) and that the apoptosis rate was significantly higher in the positive group than in the control group and negative group (49.31% vs 13.58%, P = 0.000; 49.31% vs 13.17%, P = 0.000). Conclusions: Silencing of ATM expression may increase the radiosensitivity of human lung adenocarcinoma A549 cells, probably by affecting the cell cycle and promoting cell apoptosis. (authors)

  12. Effects of paclitaxel on cell proliferation and apoptosis and its mechanism in human lung adenocarcinoma A549 cells

    Institute of Scientific and Technical Information of China (English)

    Baoan Gao; Chunling Du; Wenbo Ding; Shixiong Chen; Jun Yang

    2006-01-01

    Objective: To investigate the effect of paclitaxel on cell proliferation and apoptosis of human lung adenocarcinoma A549 cells line and its mechanism in vitro. Methods: Cell growth inhibition of paclitaxel on A549 cells was analyzed by MTT assay. Cell apoptosis was detected by DNA cytofluorometry, Hoechst33258 staining when treated with paclitaxel for 48hours. Meanwhile, Cell cycle and apoptotic rate were analyzed by flow cytometry. The protein expressions of Bax and Bcl-2 were studied by Western Blot. Results: Paclitaxel inhibited the proliferation of A549 cells in a time-and dose-dependant manner.Hoechst33258 staining indicated that apoptosis was induced by paclitaxel. After treated for 48 hours, cell apoptosis rates of 25nmol/L, 50 nmol/L and 100 nmol/L paclitaxel groups were 11.52 ± 1.94% ,17.73 ± 2.53%, and 29.32 ± 5.51% respectively,which were significantly higher than those of control group 5.88 ± 1.07%(all P < 0.01 ), and apoptosis rate increased in dose-dependant manner. Meanwhile, G2/M stage cell percentage of 25 nmol/L, 50 nmol/L and 100 nmol/L paclitaxel groups were 42.52± 6.25%, 40.46 ± 5.81%, and 35.34 ± 6.17% respectively,which were significantly higher than that of control group 22.32 ±3.30%(all P < 0.01 ); Western blot showed that paclitaxel increased the expression of Bax and decreased the expression of Bcl-2 in dose-dependant manner. Conclusion: Paclitaxel can inhibit A549 cell proliferation in a time- and dose-dependant manner. Its mechanism may be related to arresting cell cycle in G2/M stage and induce cell apoptosis by up-modulating Bax expression and down-modulating Bcl-2 expression.

  13. Growth arrest and apoptosis via caspase activation of dioscoreanone in human non-small-cell lung cancer A549 cells

    OpenAIRE

    Hansakul, Pintusorn; Aree, Kalaya; Tanuchit, Sermkiat; Itharat, Arunporn

    2014-01-01

    Background Dioscoreanone (DN) isolated from Dioscorea membranacea Pierre has been reported to exert potent cytotoxic effects against particular types of cancer. The present study was carried out to investigate the cytotoxicity of DN against a panel of different human lung cancer cell lines. The study further examined the underlying mechanisms of its anticancer activity in the human lung adenocarcinoma cell line A549. Methods Antiproliferative effects of DN were determined by SRB and CFSE assa...

  14. 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)

    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

  15. Functional ion channels in pulmonary alveolar type I cells support a role for type I cells in lung ion transport

    OpenAIRE

    Johnson, Meshell D.; Bao, Hui-Fang; Helms, My N.; Chen, Xi-Juan; Tigue, Zac; Jain, Lucky; Dobbs, Leland G.; Eaton, Douglas C.

    2006-01-01

    Efficient gas exchange in the lungs depends on regulation of the amount of fluid in the thin (average 0.2 μm) liquid layer lining the alveolar epithelium. Fluid fluxes are regulated by ion transport across the alveolar epithelium, which is composed of alveolar type I (TI) and type II (TII) cells. The accepted paradigm has been that TII cells, which cover 95% of the surface area, provide a route for water absorption. Here we present data that TI cells contain functional epithelial Na+ channels...

  16. 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.

  17. 流感病毒NS1蛋白稳定表达的A549细胞系建立%Establishment of A549 Cell Line Stably Expressing NS1 Protein of Influenza Virus

    Institute of Scientific and Technical Information of China (English)

    李志辉; 曾琳姣; 王慧煜; 梅琳; 刘永飞; 韩雪清

    2013-01-01

    NS1 of influenza A virus is a key multifunctional protein that plays various roles in regulating viral replication mechanisms, disease pathogenesis. In order to establish stable A549 cell line expressing NS1 protein of influenza A Virus, NSl cDNA was obtained by RT-PCR using 2009 A(H1N1) influenza virus total RNA as template. The fragment was cloned in the pMD19-T vector, then the fragment was obtained by BamHI and NdeI digestion, and ligated with pCMV-HA. Linearized pCMV-HA-NS1 and neo were transfect-ed into A549 cells. The stable expressing NSl protein cell line was screened by G418. DNA, RNA, protein levels of NS1 were detected in A549 cells by PCR, RT-PCR and Western blot, the location of the NSl protein in cells was observed by immunofluorescence. The result indicated that NS1 protein was stable expressed in A549 cell line, suggesting that NSl stable expression A549 cell line was successfully constructed, and the NS1 protein is located in nucleus. This stable cell line can be used for further study of biological functions of NS1.%A型流感病毒的NSl(Nonstructurol 1 protein,NSl)蛋白是病毒复制、毒力等的重要调节蛋白.运用RT-PCR方法扩增A/Beijing/501/2009 (H1N1)流感病毒NS1基因,克隆至真核表达载体pCMV-HA,用Lipofectamine 2000将线性化pCMV-HA-NS1与neo基因共同转染A549细胞,通过G418筛选获得阳性重组细胞,并采用PCR、RT-PCR、Western blot技术检测重组细胞中NS1蛋白的表达,通过免疫荧光技术观察NS1蛋白在细胞中的定位.PCR、RT-PCR检测显示NS1基因成功整合进入细胞基因组,并转录为mRNA;Western blot检测显示重组细胞系稳定表达NS1蛋白,免疫荧光显示NS1蛋白定位于细胞核内.表明通过G418筛选,成功构建稳定表达NS1蛋白的重组A549-HA-NS1细胞系,且NS1蛋白定位于细胞核内,为进一步研究NS1蛋白的生物学功能奠定基础.

  18. Pulmonary epithelial cancer cells and their exosomes metabolize myeloid cell-derived leukotriene C4 to leukotriene D4.

    Science.gov (United States)

    Lukic, Ana; Ji, Jie; Idborg, Helena; Samuelsson, Bengt; Palmberg, Lena; Gabrielsson, Susanne; Rådmark, Olof

    2016-09-01

    Leukotrienes (LTs) play major roles in lung immune responses, and LTD4 is the most potent agonist for cysteinyl LT1, leading to bronchoconstriction and tissue remodeling. Here, we studied LT crosstalk between myeloid cells and pulmonary epithelial cells. Monocytic cells (Mono Mac 6 cell line, primary dendritic cells) and eosinophils produced primarily LTC4 In coincubations of these myeloid cells and epithelial cells, LTD4 became a prominent product. LTC4 released from the myeloid cells was further transformed by the epithelial cells in a transcellular manner. Formation of LTD4 was rapid when catalyzed by γ-glutamyl transpeptidase (GGT)1 in the A549 epithelial lung cancer cell line, but considerably slower when catalyzed by GGT5 in primary bronchial epithelial cells. When A549 cells were cultured in the presence of IL-1β, GGT1 expression increased about 2-fold. Also exosomes from A549 cells contained GGT1 and augmented LTD4 formation. Serine-borate complex (SBC), an inhibitor of GGT, inhibited conversion of LTC4 to LTD4 Unexpectedly, SBC also upregulated translocation of 5-lipoxygenase (LO) to the nucleus in Mono Mac 6 cells, and 5-LO activity. Our results demonstrate an active role for epithelial cells in biosynthesis of LTD4, which may be of particular relevance in the lung. PMID:27436590

  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. Effects of Coptis extract combined with chemotherapeutic agents on ROS production, multidrug resistance, and cell growth in A549 human lung cancer cells

    Directory of Open Access Journals (Sweden)

    He Chengwei

    2012-04-01

    Full Text Available Abstract Background Non–small cell lung cancer is associated with high expression of multidrug resistance (MDR proteins and low production of reactive oxygen species (ROS. Coptis extract (COP, a Chinese medicinal herb, and its major constituent, berberine (BER, have anticancer properties. This study aims to investigate the effects of COP and BER combined with chemotherapeutic agents, including fluorouracil (5-FU, camptothecin (CPT, and paclitaxel (TAX, on cell proliferation, ROS production, and MDR in A549 human non-small cell lung cancer cells. Methods A549 cells were treated with different doses of COP and BER, combined with 5-FU, CPT, and TAX. Cell viability was measured by an XTT (2,3-bis-(2-methoxy-4- nitro-5-sulfophenyl-2 H-tetrazolium-5-carboxanilide assay. Intracellular ROS levels were determined by measuring the oxidative conversion of cell permeable 2′,7′-dichlorofluorescein diacetate to fluorescent dichlorofluorescein. MDR of A549 cells was assessed by rhodamine 123 retention assay. Results Both COP and BER significantly inhibited A549 cell growth in a dose-dependent manner. Combinations of COP or BER with chemotherapeutic agents (5-FU, CPT, and TAX exhibited a stronger inhibitory effect on A549 cell growth. In addition, COP and BER increased ROS production and reduced MDR in A549 cells. Conclusion As potential adjuvants to chemotherapy for non–small cell lung cancer, COP and BER increase ROS production, reduce MDR, and enhance the inhibitory effects of chemotherapeutic agents on A549 cell growth.

  1. Curcumin promotes apoptosis in A549/DDP multidrug-resistant human lung adenocarcinoma cells through an miRNA signaling pathway

    International Nuclear Information System (INIS)

    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.

  2. 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

  3. Nitrative DNA damage induced by multi-walled carbon nanotube via endocytosis in human lung epithelial cells

    International Nuclear Information System (INIS)

    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

  4. 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. PMID:26923760

  5. The effect of β-elemene combined with irradiation on DNA damage and repair in A549 cells

    International Nuclear Information System (INIS)

    Objective: To study if β-elemene can increase radiation-induced deoxyribonucleic acid (DNA) damage and decrease the damage repair. Methods: Exponentially growing human lung adenocarcinoma cells (A549) were exposed to 10 or 20 μg/ml β-elemene for 24 h before irradiation.The effect of β-elemene on the in vitro radiosensitivity of A549 cells was evaluated using clonogenic assay. DNA damage and repair were evaluated using comet assay. Results: Exposure to β-elemene before irradiation increased the radiosensitivity of A549 cells. The SERD0 for 10 μg/ml and 20 μg/ml β-elemene was 1.55 and 1.64, respectively. The SERDq for 10 μg/ml and 20 μg/ml β-elemene was 1.43 and 1.75, respectively. Combined treatment, comparing to irradiation or β-elemene treatment alone, induced higher levels of DNA damage and slower rate of damage repair. A549 cells exposed to 20 μg/ml β-elemene followed by irradiation showed a higher levels of tail moment (TM) than those exposed to irradiation or β-elemene alone at 0 h, 2 h, 6 h and 24 h after irradiation. The TM of the three groups at 0 h, 2 h, 6 h and 24 h after irradiation was 7.16±2.61, 0.95±0.65 and 1.81±1.23 (F=231.24, P<0.01), 3.65±2.06, 0.11±0.07 and 1.58±1.40(F=90.22, P<0.01), 2.09±0.83, 0.1±0.05 and 0.45±0.25 (F=238.44, P<0.01), 1.45±1.37, 0.11±0.08 and 0.60±0.40 (F=38.94, P<0.01), respectively. Conclusions: β-elemene can enhance the radiosensitivity of A549 cells through the enhancement of DNA damage and the inhibition of DNA damage repair. (authors)

  6. Mechanisms of silica-induced IL-8 release from A549 cells: Initial kinase-activation does not require EGFR activation or particle uptake

    International Nuclear Information System (INIS)

    Understanding how mineral particles trigger cellular responses is crucial in order to elucidate what characteristics determine their harmful effects. It is not clear whether cellular effects are triggered through the cell membrane or require particle uptake. However, studies with asbestos suggest that activation of the epidermal growth factor receptor (EGFR) may be important. We have previously reported that crystalline silica-induced interleukin (IL)-8 release from human lung epithelial cells (A549) was regulated through Src family kinases (SFKs) and the mitogen-activated protein kinases (MAPKs) p38 and extracellular signal-regulated kinase (ERK)-1 and -2. The present study shows that SFK and p38 phosphorylation increased almost immediately upon crystalline silica exposure, whereas ERK1/2 phosphorylation increased after 10 min of exposure. The p38 inhibitor SB202190 increased the silica-induced ERK1/2 phosphorylation suggesting that p38 activity may attenuate activation of ERK1/2. Scanning electron microscopy showed that some silica particles were phagocytosed between 1 and 4 h of exposure, but that the majority remained bound by microvilli on the cell surface. The EGFR inhibitor AG1478 attenuated both silica-induced IL-8 release and phosphorylation of SFKs and ERK1/2. However, AG1478 also inhibited the respective background levels, and the EGFR was not phosphorylated at the onset of silica exposure. The results suggest that crystalline silica triggers p38 and SFK-ERK1/2 signaling through interactions with membrane components as both pathways were rapidly activated prior to particle internalization. However, the silica-induced up-regulation of IL-8 release through the SFK-ERK1/2 pathway does not appear to be initiated through activation of the EGFR, although basal EGFR activity may affect the magnitude of the responses

  7. New insights of P2X7 receptor signaling pathway in alveolar functions.

    Science.gov (United States)

    Mishra, Amarjit

    2013-01-01

    Purinergic P2X7 receptor (P2X7R), an ATP-gated cation channel, is unique among all other family members because of its ability to respond to various stimuli and to modulate pro-inflammatory signaling. The activation of P2X7R in immune cells is absolutely required for mature interleukin -1beta (IL-1beta) and IL-18 production and release. Lung alveoli are lined by the structural alveolar epithelial type I (AEC I) and alveolar epithelial type II cells (AEC II). AEC I plays important roles in alveolar barrier protection and fluid homeostasis whereas AEC II synthesizes and secrete surfactant and prevents alveoli from collapse. Earlier studies indicated that purinergic P2X7 receptors were specifically expressed in AEC I. However, their implication in alveolar functions has not been explored. This paper reviews two important signaling pathways of P2X7 receptors in surfactant homeostatsis and Acute Lung Injury (ALI). Thus, P2X7R resides at the critical nexus of alveolar pathophysiology. PMID:23634990

  8. Displasia alveolar congénita Congenital alveolar dysplasia

    OpenAIRE

    D.P. Sánchez Hernández; M.M. Suárez Galvis; M.T. García Vélez; A. Uribe Molina

    2012-01-01

    La displasia alveolar congénita es una patología congénita poco frecuente caracterizada por disminución de las unidades alveolares y anormalidades del desarrollo de la vasculatura pulmonar. Esta entidad produce hipertensión pulmonar severa e hipoxemia persistente con una evolución mortal en la mayoría de los casos. Afecta generalmente a recién nacidos a término, con un test de Apgar normal, que posteriormente desarrollan dificultad respiratoria, habitualmente en el primer día de vida. La enfe...

  9. Reversal Effects of Piperlongumine on Drug Resistance of Human Lung Caner A549/DDP Cell to Cisplatin%荜茇酰胺对人肺癌A549/DDP细胞耐药性的逆转作用

    Institute of Scientific and Technical Information of China (English)

    钱钧强; 孙蓓; 房志仲

    2014-01-01

    目的:研究荜茇酰胺对人肺癌A549/顺铂(DDP)细胞耐药性的逆转作用.方法:A549/DDP细胞经0、20、30 μmol/L荜茇酰胺作用48h后,用MTS法检测肿瘤细胞抑制率;流式细胞术检测肿瘤细胞凋亡、细胞周期、P-糖蛋白(P-gp)表达和肿瘤细胞内罗丹明Rht123含量的变化;Western blotting法检测多药耐药基因(MDR)1、多药耐药相关蛋白(MRP)1、DNA拓扑异构酶(Top)Ⅱ、谷胱甘肽S-转移酶(GST)-π、凋亡抑制蛋白Survivin、周期蛋白依赖性蛋白激酶(CDK)1和蛋白激酶(PK)Cζ蛋白表达;实时荧光聚合酶链反应(RT-PCR)法检测MDR1、MRP1、Top-Ⅱ、GST-π、Survivin和CDKl mRNA表达;酶标仪检测含半胱氨酸的天冬氨酸蛋白水解酶(Caspase)-3、8活性.结果:A549/DDP细胞经0、20、30 μmol/L荜茇酰胺作用48 h后,DDP对肿瘤细胞增殖的抑制率明显升高;与0 μmol/L比较,20、30μmol/L荜茇酰胺作用48 h后,DDP导致的细胞凋亡率和G2期/M期明显升高,P-gp表达明显减弱,Rh-123浓度明显增加,MDR1、MRP1、Top-Ⅱ、GST-π、Survivin、CDK1和PKCζ蛋白表达明显减弱,MDR1、MRP1、Top-Ⅱ、GST-π、Survivin、CDKmRNA表达明显减弱,Caspase-3、8的活性明显增强.结论:荜茇酰胺可逆转人肺癌A549/DDP细胞DDP耐药性,可能与其调节多药耐药相关基因表达有关.

  10. Construction of A Eukaryotic Expression Vector Carrying the iNOS Gene and Its Effect on A549 Lung Cancer Cells

    Directory of Open Access Journals (Sweden)

    Sujuan YE

    2012-05-01

    Full Text Available Background and objective 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. Methods 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. Results 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. Conclusion 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.

  11. CDK-associated Cullin 1 promotes cell proliferation with activation of ERK1/2 in human lung cancer A549 cells

    International Nuclear Information System (INIS)

    Highlights: •CDK-associated Cullin 1 (CAC1) expression increases in human lung carcinoma. •CAC1 promotes the proliferation of lung cancer A549 cells. •CAC1 promotes human lung cancer A549 cell proliferation with activation of ERK1/2. -- Abstract: Lung cancer is one of the most common causes of cancer-related death in the world, but the mechanisms remain unknown. In this study, we investigated the expression of CDK-associated Cullin 1 (CAC1) in lung cancer, the effect of CAC1 on the proliferation of human lung cancer A549 cells, and the activation of signaling pathways of mitogen-activated protein kinases (MAPKs). Results showed that CAC1 expression was higher levels in human lung carcinoma than normal lung tissue, and CAC1 siRNA reduced the proliferation of lung cancer A549 cells by decreasing cell activity and cell division in vitro. The proportion of cells treated with CAC1 siRNA increased in the G1 phase and decreased in the S and G2/M phase, indicative of G1 cell cycle arrest. Furthermore, the proportions of early/late apoptosis in lung cancer A549 cells were enhanced with CAC1 siRNA treatment. It was also found that activation of extracellular signal-regulated protein kinase (ERK) and p38 signaling pathways were involved in the proliferation of A549 cells. After CAC1 siRNA treatment, p-ERK1/2 levels decreased, and meanwhile p-p38 level increased, A549 cell proliferation increased when ERK1/2 signaling is activated by PMA. Our findings demonstrated that CAC1 promoted the proliferation of human lung cancer A549 cells with activation of ERK1/2 signaling pathways, suggesting a potential cure target for treatment of human lung cancer

  12. Enhancement of radiosensitivity by topoisomerase II inhibitor, amrubicin and amrubicinol, in human lung adenocarcinoma A549 cells and kinetics of apoptosis and necrosis induction

    OpenAIRE

    Hayashi, Sachiko; Hatashita, Masanori; Matsumoto, Hideki; Shioura, Hiroki; KITAI, Ryuhei; Kano, Eiichi

    2006-01-01

    The effects of amrubicin (AMR) and its activemetabolite, amrubicinol (AMROH), on the sensitivity ofhuman lung adenocarcinoma A549 cells to ionizing radiationwere investigated in vitro. Further, the kinetics of apoptosisand necrosis induction were also analyzed. The cytocidalefftcts of X-ray irradiation on A549 cells resulted in a lowlevel of radiosensitivity with a D value of 12 Gy. The slopesof the survival curves in the exponential phase were plottedon semilogarithmic paper for radiation co...

  13. CDK-associated Cullin 1 promotes cell proliferation with activation of ERK1/2 in human lung cancer A549 cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Tian Jun [Respiratory Department, The First Affiliated Hospital, Xi’an Jiaotong University College of Medicine, Xi’an 710061 (China); Gao, Fei [Hua-shan Central Hospital of Xi’an, Xi’an 710043 (China); Yang, Tian; Thakur, Asmitanand; Ren, Hui; Li, Yang; Zhang, Shuo; Wang, Ting [Respiratory Department, The First Affiliated Hospital, Xi’an Jiaotong University College of Medicine, Xi’an 710061 (China); Chen, Ming Wei, E-mail: xjtucmw@163.com [Respiratory Department, The First Affiliated Hospital, Xi’an Jiaotong University College of Medicine, Xi’an 710061 (China)

    2013-07-19

    Highlights: •CDK-associated Cullin 1 (CAC1) expression increases in human lung carcinoma. •CAC1 promotes the proliferation of lung cancer A549 cells. •CAC1 promotes human lung cancer A549 cell proliferation with activation of ERK1/2. -- Abstract: Lung cancer is one of the most common causes of cancer-related death in the world, but the mechanisms remain unknown. In this study, we investigated the expression of CDK-associated Cullin 1 (CAC1) in lung cancer, the effect of CAC1 on the proliferation of human lung cancer A549 cells, and the activation of signaling pathways of mitogen-activated protein kinases (MAPKs). Results showed that CAC1 expression was higher levels in human lung carcinoma than normal lung tissue, and CAC1 siRNA reduced the proliferation of lung cancer A549 cells by decreasing cell activity and cell division in vitro. The proportion of cells treated with CAC1 siRNA increased in the G1 phase and decreased in the S and G2/M phase, indicative of G1 cell cycle arrest. Furthermore, the proportions of early/late apoptosis in lung cancer A549 cells were enhanced with CAC1 siRNA treatment. It was also found that activation of extracellular signal-regulated protein kinase (ERK) and p38 signaling pathways were involved in the proliferation of A549 cells. After CAC1 siRNA treatment, p-ERK1/2 levels decreased, and meanwhile p-p38 level increased, A549 cell proliferation increased when ERK1/2 signaling is activated by PMA. Our findings demonstrated that CAC1 promoted the proliferation of human lung cancer A549 cells with activation of ERK1/2 signaling pathways, suggesting a potential cure target for treatment of human lung cancer.

  14. Monocyte adhesion induced by multi-walled carbon nanotubes and palmitic acid in endothelial cells and alveolar-endothelial co-cultures

    DEFF Research Database (Denmark)

    Cao, Yi; Roursgaard, Martin; Jacobsen, Nicklas Raun;

    2016-01-01

    Free palmitic acid (PA) is a potential pro-atherogenic stimulus that may aggravate particle-mediated cardiovascular health effects. We hypothesized that the presence of PA can aggravate oxidative stress and endothelial activation induced by multi-walled carbon nanotube (MWCNT) exposure in vitro. We...... investigated the interaction between direct exposure to MWCNTs and PA on THP-1 monocyte adhesion to human umbilical vein endothelial cells (HUVECs), as well as on indirect exposure in an alveolar-endothelial co-culture model with A549 cells and THP-1-derived macrophages exposed in inserts and the effect...... monocultures. Both effects were found to be independent of the presence of PA. MWCNT exposure significantly increased THP-1 monocyte adhesion to HUVECs, and co-exposure to PA aggravated the NM400-mediated adhesion but decreased the NM402-mediated adhesion. For the co-cultures, the exposure of A549 cells did...

  15. Platinum(II) phenanthroimidazole G-quadruplex ligand induces selective telomere shortening in A549 cancer cells.

    Science.gov (United States)

    Mancini, Johanna; Rousseau, Philippe; Castor, Katherine J; Sleiman, Hanadi F; Autexier, Chantal

    2016-02-01

    Telomere maintenance, achieved by the binding of protective shelterin capping proteins to telomeres and by either telomerase or a recombination-based alternative lengthening of telomere (ALT) mechanism, is critical for cell proliferation and survival. Extensive telomere shortening or loss of telomere integrity activates DNA damage checkpoints, leading to cell senescence or death. Although telomerase upregulation is an attractive target for anti-cancer therapy, the lag associated with telomere shortening and the potential activation of ALT pose a challenge. An alternative approach is to modify telomere interactions with binding proteins (telomere uncapping). G-quadruplex ligands stabilize structures generated from single-stranded G-rich 3'-telomere end (G-quadruplex) folding, which in principle, cannot be elongated by telomerase, thus leading to telomere shortening. Ligands can also mediate rapid anti-proliferative effects by telomere uncapping. We previously reported that the G-quadruplex ligand, phenylphenanthroimidazole ethylenediamine platinum(II) (PIP), inhibits telomerase activity in vitro[47]. In the current study, a long-term seeding assay showed that PIP significantly inhibited the seeding capacity of A549 lung cancer cells and to a lesser extent primary MRC5 fibroblast cells. Importantly, treatment with PIP caused a significant dose- and time-dependent decrease in average telomere length of A549 but not MRC5 cells. Moreover, cell cycle analysis revealed a significant increase in G1 arrest upon treatment of A549 cells, but not MRC5 cells. Both apoptosis and cellular senescence may contribute to the anti-proliferative effects of PIP. Our studies validate the development of novel and specific therapeutic ligands targeting telomeric G-quadruplex structures in cancer cells. PMID:26724375

  16. In vitro and in vivo studies on radiobiological effects of prolonged fraction delivery time in A549 cells.

    Science.gov (United States)

    Jiang, Ling; Xiong, Xiao-Peng; Hu, Chao-Su; Ou, Zhou-Luo; Zhu, Guo-Pei; Ying, Hong-Mei

    2013-03-01

    Intensity-modulated radiation therapy, when used in the clinic, prolongs fraction delivery time. Here we investigated both the in vivoand in vitroradiobiological effects on the A549 cell line, including the effect of different delivery times with the same dose on A549 tumor growth in nude mice. The in vitroeffects were studied with clonogenic assays, using linear-quadratic and incomplete repair models to fit the dose-survival curves. Fractionated irradiation of different doses was given at one fraction per day, simulating a clinical dose-time-fractionation pattern. The longer the interval between the exposures, the more cells survived. To investigate the in vivoeffect, we used sixty-four nude mice implanted with A549 cells in the back legs, randomly assigned into eight groups. A 15 Gy radiation dose was divided into different subfractions. The maximum and minimum tumor diameters were recorded to determine tumor growth. Tumor growth was delayed for groups with prolonged delivery time (40 min) compared to the group receiving a single dose of 15 Gy (P< 0.05), and tumors with a 20 min delivery time had delayed growth compared to those with a 40 min delivery time [20' (7.5 Gy × 2 F) vs 40' (7.5 Gy × 2 F), P= 0.035; 20' (3 Gy × 5 F) vs 40' (3 Gy × 5 F); P= 0.054; 20' (1.67 Gy × 9 F) vs 40' (1.67 Gy × 9 F), P= 0.028]. A prolonged delivery time decreased the radiobiological effects, so we strongly recommend keeping the delivery time as short as possible. PMID:23090953

  17. Quercetin metabolites inhibit MMP-2 expression in A549 lung cancer cells by PPAR-γ associated mechanisms.

    Science.gov (United States)

    Chuang, Cheng-Hung; Yeh, Chiao-Lin; Yeh, Shu-Lan; Lin, En-Shyh; Wang, Li-Yu; Wang, Ying-Hsuna

    2016-07-01

    Our previous study demonstrated that quercetin-metabolite-enriched plasma (QP) but not quercetin itself upregulates peroxisome proliferator-activated receptor gamma (PPAR-γ) expression to induce G2/M arrest in A549 cells. In the present study, we incubated A549 cells with QP as well as quercetin-3-glucuronide (Q3G) and quercetin-3'-sulfate (Q3'S), two major metabolites of quercetin, to investigate the effects of quercetin metabolites on cell invasion and migration, the possible mechanisms and the role of PPAR-γ. We also compared the effects of QP with those of quercetin and troglitazone (TGZ), a PPAR-γ ligand. The results showed that QP significantly suppressed cell invasion and migration, as well as matrix metalloproteinases (MMPs)-2 activity and expression in a dose-dependent manner. The effects of 10% QP on those parameters were similar to those of 10μM quercetin and 20μM TGZ. However, QP and TGZ rather than quercetin itself increased the expressions of nm23-H1 and tissue inhibitor of metalloproteinase (TIMP-2). Furthermore, we demonstrated that Q3G and Q3'S also inhibited the protein expression of MMP-2. GW9662, a PPAR-γ antagonist, significantly diminished such an effect of Q3G and Q3'S. Silencing PPAR-γ expression in A549 cells also significantly diminished the suppression effect of Q3G and Q3'S on MMP-2 expression. Taken together, our study demonstrated that QP inhibited cell invasion and migration through nm23-H1/TIMP-2/MMP-2 associated mechanisms. The upregulation of PPAR-γ by quercetin metabolites such as Q3G and Q3'S could play an important role in the effects of QP. PMID:27260467

  18. 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.

  19. Pulmonary alveolar proteinosis in a cat

    OpenAIRE

    Szatmári, Viktor; Teske, Erik; Peter G. J. Nikkels; Griese, Matthias; de Jong, Pim A.; Grinwis, Guy; Theegarten, Dirk; Veraa, Stefanie; van Steenbeek, Frank G.; Drent, Marjolein; Bonella, Francesco

    2015-01-01

    BACKGROUND: Pulmonary alveolar proteinosis is an extremely rare lung disease in animals and humans. It is characterized by the deposition of a large amount of phospholipoproteinaceous material in the alveoli. There are several possible etiologies, both congenital and acquired. Alveolar macrophages play an important role in the clearance of surfactant. This is the first report of pulmonary alveolar proteinosis in the feline species. CASE PRESENTATION: Pulmonary alveolar proteinosis was diagnos...

  20. Effect of RNAi targeting survivin gene combined with X-rays radiation on apoptosis of lung adenocarcinoma A549 cells

    International Nuclear Information System (INIS)

    Objective: To construct the vector of RNA interference (RNAi) targeting survivin gene and observe its effect combined with X-rays radiation on lung adenocarcinoma A549 cell apoptosis. Methods: One pair of RNAi sequence targeting survivin gene were designed according to its cDNA sequence reported in GenBank, the recombinant RNAi plasmid pGenesil2-survivin was constructed. After identified by enzyme digestion and sequencing, the pGenesil2-survivin plasmid was trasfeced into A549 cells.In the experiment, normal group,pGenesil2 group, pGenesil2-survivin group,5 Gy irradiation group and pGenesil2-survivin + 5 Gy irradiation group were set up.The apoptosis of A549 cells was measured by flow cytometry with PI/Annexin V and TUNEL,the survivin and caspase-3 expressions were measured by Western blotting. Results: Two fragments about 389 bp and 4 206 bp were gotten by Kpn I and EcoR I enzyme digestion, they are the same to expected result, the sequencing result was compared to oligonucleotide chain with DNAssist 2.0, they were equal, these indicated the identification of pGenesil2-survivin vector was right; pGenesil2-survivin was transfected into A549 cells for 48 h, the apoptotic percentage in pGenesil2-survivin and 5 Gy X-rays groups increased obviously (P< 0.05), when the both were combined, the effect was more obvious;the Western blotting results appeared that the survivin gray scale/β-actin gray scale in pGenesil2-survivin group was lower than that in normal group(P< 0.01), and the caspase-3 gray scale/β-actin gray scale was higher than that in normal group,and that ratio in pGenesil2-survivin+5 Gy irradiation group was more high(P< 0.01). Conclusion: RNAi targeting surviving gene could inhibit survivin protein expression,but enhance caspase-3 protein expression, and promote apoptosis. When it is combined with 5 Gy X-rays irradiation, the promotion of apoptosis is enhanced. (authors)

  1. Cytoskeletal changes as an early event in hydrogen peroxide-induced cell injury: a study in A549 cells.

    OpenAIRE

    Raghu, G.; Striker, L.; Harlan, J; Gown, A.; Striker, G

    1986-01-01

    Hydrogen peroxide (H2O2) and other oxygen metabolites have been implicated in the pathogenesis of cell and tissue injury. The nature of the injury occurring in cells exposed to oxygen metabolites is unknown. A549 cells, derived from human lung carcinoma, were exposed to glucose-glucose oxidase or hydrogen peroxide in vitro. The distribution of actin and cytokeratin filaments, as well as 51chromium (51Cr) release and trypan blue dye exclusion were assessed. Both glucose-glucose oxidase and H2O...

  2. Pulmonary alveolar microlithiasis with calcified pleural plaques

    OpenAIRE

    Malhotra Balbir; Sabharwal Raghu; Singh Mandeep; Singh Amarjeet

    2010-01-01

    Pulmonary alveolar microlithiasis (PAM) is a rare disease. Herein we report a case of pulmonary alveolar microlithiasis who was suspected to have the disease on chest X-ray and was confirmed on high resolution CT and transbronchial lung biopsy. These investigations showed characteristic features of pulmonary alveolar microlithiasis with diffuse interstitial pulmonary fibrosis.

  3. Pulmonary alveolar microlithiasis with calcified pleural plaques

    Directory of Open Access Journals (Sweden)

    Malhotra Balbir

    2010-01-01

    Full Text Available Pulmonary alveolar microlithiasis (PAM is a rare disease. Herein we report a case of pulmonary alveolar microlithiasis who was suspected to have the disease on chest X-ray and was confirmed on high resolution CT and transbronchial lung biopsy. These investigations showed characteristic features of pulmonary alveolar microlithiasis with diffuse interstitial pulmonary fibrosis.

  4. Biological effects of heavy ion and X-ray irradiation on lung cancer cells A549%重离子与X射线照射肺癌细胞A549的生物学效应比较

    Institute of Scientific and Technical Information of China (English)

    杨立娜; 冉俊涛; 张红; 刘圆圆; 孙超; 张秋宁; 王新宇; 王小虎

    2014-01-01

    Objective To compare the effects of carbon heavy ion and X-ray irradiation on survival fraction,cell cycle,cell apoptosis and expression of DNA-PKcs of A549 lung cancer cells.Methods A549 cells were irradiated by carbon heavy ion and X-ray.Cell survival fraction,cell cycle and apoptosis were analyzed by clonogenic formation assay,flow cytometry and Hoechst 33258 staining,respectively.Real time-PCR was performed to detect the expressions of DNA-PKcs and H2AX mRNA.Results Lower cell survival fraction,more G2/M phase arrest and higher apoptosis rate were detected in the A549 cells exposed to carbon heavy ion than X-ray(t =4.77,14.53,14.54,P < 0.05).Expression of DNA-PKcs was up-regulated after irradiation to carbon heavy ion and X-ray(t =10.91,5.05,P < 0.05).Conclusions Both heavy ion and X-ray irradiations enhance the expression of DNA-PKcs,induce apoptosis through regulating cell cycle arrest,and hence reduce survival of A549 cells.Heavy ion irradiation shows more stronger biological effects than X-ray irradiation.%目的 比较碳重离子与X射线对肺癌细胞的生物学效应.方法 对A549细胞分别进行碳重离子和X射线照射,通过克隆形成实验检测照射后细胞存活情况;流式细胞术检测细胞周期分布;通过Hoechst 33258荧光染料对照射后固定的细胞进行染色,计算凋亡率;采用实时荧光定量PCR方法检测照射后48 h细胞内DNA依赖性蛋白激酶催化亚单位(DNA-PKcs)和H2AX的mRNA表达水平.结果 细胞存活曲线显示,碳重离子造成的细胞存活分数远低于X射线,并将细胞周期阻滞于G2/M期(t=4.77、14.53、14.54,P<0.05),导致大部分细胞进入凋亡途径.碳重离子与X射线辐照后DNA-PKcs的表达上调(t=10.91、5.05,P<0.05).结论 碳重离子照射对肺癌细胞造成生物学效应远高于X射线.

  5. The effect of phospholipid transfer protein on cigarette smoke extract induced epithelial-mesenchymal transition of rat alveolar type Ⅱ cells%磷脂转运蛋白在烟草诱导RLE-6TN细胞株发生上皮间质转化中的作用

    Institute of Scientific and Technical Information of China (English)

    巫凤苹; 陈亚娟; 余秀英; 廖科; 李丹丹; 陈虹

    2016-01-01

    Objective To investigate the effect of phospholipid transfer protein(PLTP) on cigarette smoke extract(CSE) induced epithelial-mesenchymal transition(EMT) in rat alveolar Type Ⅱ cells (RLE-6TN).Methods CSE of different concentrations (0%,0.25%,0.5% and 1%) was co-cultured for 2 or 3days with RLE-6TN,either pre-treated or not pre-treated with siRNA-PLTP for 6 h.Expression levels of E-cadherin mRNA and Vimentin mRNA were examined by RT-PCR,while expression levels of PLTP,E-cadherin,N-cadherin and Vimentin were examined by Western blot.Results Our results showed that the expression of E-cadherin mRNA decreased in CSE-treated groups:1.01 ± 0.05,0.74 ± 0.05,0.65 ± 0.03,0.30 ±0.08 respectively at different concentrations of CSE (0 %,0.25%,0.5 %,and 1.0%);while the level of Vimentin mRNA increased significantly in 1% CSE treated cells (1.88 ± O.49),compared with control cells (1.01 ±0.20).Treatment with CSE at different concentrations (0%,0.25%,0.5% and 1%) showed that the protein levels of PLTP were 0.42 ± 0.02,0.89 ± 0.25,1.08 ± 0.18,1.61 ± 0.06 respectively;those of E-cadherin were 1.61 ± 0.04,1.08 ± 0.10,0.62 ± 0.08,0.68 ± 0.17,respectively;those of N-cadherin were 0.60 ± 0.14,0.57 ± 0.26,0.88 ± 0.30,1.94 ± 0.54,respectively;and those of Vimentin were 0.61 ± 0.05,0.98 ± 0.16,1.07 ± 0.14,1.34 ± 0.19,respectively;all P < 0.05 when the 1% CSE group was compared with the control group.EMT induced by CSE was significantly inhibited by siRNA-PLTP.Conclusion PLTP may be involved in CSE induced EMT of rat alveolar cells.%目的 探讨磷脂转运蛋白(PLTP)在烟草提取物(CSE)诱导大鼠Ⅱ型肺泡上皮细胞株RLE-6TN发生上皮间质转化(EMT)中的作用.方法 体外培养RLE-6TN细胞株24 h,分为4组,每组3孔,分别加入0%、0.25%、0.5%和1% CSE培养2d,检测E-钙黏蛋白和波形蛋白mRNA表达以及细胞和CSE共培养3d检测PLTP、EMT相关蛋白(E-钙黏蛋白、N-钙黏蛋白和波形

  6. miRNA-200c对非小细胞肺癌A549细胞甲氨蝶呤耐药性的影响%Impact of miRNA-200c on Methotrexate Resistance of Non-small Cell Lung Cancer Cells A549

    Institute of Scientific and Technical Information of China (English)

    单武林; 邓芳; 张晓蕾; 张婧; 韩丹丹; 万玲玲; 李明

    2016-01-01

    目的 探讨miRNA-200c(miR-200c)在非小细胞肺癌A549细胞耐甲氨蝶呤(MTX)(A549/MTX)中的影响及可能的作用机制.方法 通过实时荧光定量(qRT-PCR)法检测人肺癌亲本细胞NA549细胞、转染miR-200c模拟物(mimic)的A549/MTX细胞(A549/MTX-M)及转染miR-阴性对照(NC) A549/MTX细胞(A549/MTX-N)中miR-200c的表达水平.分别采用MTT法、锥虫兰染色及流式细胞术检测三组细胞对MTX的药物敏感度、细胞增殖能力及细胞凋亡变化,并采用qRT-PCR检测其P53和P21基因表达的变化.结果 miR-200c在A549细胞中的表达水平显著高于A549/MTX-N细胞;A549/MTX-M细胞miR-200c水平高于A549/MTX-N细胞;用不同浓度MTX刺激细胞,与A549/MTX-N细胞比较,A549/MTX-M细胞的增殖能力减弱、凋亡细胞增多,并呈剂量依赖性,差异均有统计学意义.转染miR-200c mimic后,P53和P21基因表达水平上升,与转染miR-NC细胞比较,差异有统计学意义.结论 miR-200c能够逆转A549/MTX细胞对MTX的耐药性,其作用机制可能是通过P53/P21信号转导通路诱导细胞凋亡来实现的.

  7. 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.

  8. Pretreatment of Epithelial Cells with Rifaximin Alters Bacterial Attachment and Internalization Profiles▿

    OpenAIRE

    Brown, Eric L.; Xue, Qiong; Jiang, Zhi-Dong; Xu, Yi; Herbert L. DuPont

    2009-01-01

    Rifaximin is a poorly absorbed semisynthetic antibiotic derivative of rifampin licensed for use in the treatment of traveler's diarrhea. Rifaximin reduces the symptoms of enteric infection, often without pathogen eradication and with limited effects on intestinal flora. Epithelial cells (HEp-2 [laryngeal], HCT-8 [ileocecal], A549 [lung], and HeLa [cervical]) were pretreated with rifaximin (or control antibiotics) prior to the addition of enteroaggregative Escherichia coli (EAEC). EAEC adheren...

  9. Streptococcus pyogenes Serotype M1 Encodes Multiple Pathways for Entry into Human Epithelial Cells

    OpenAIRE

    Cue, D; Dombek, P. E.; Lam, H; Cleary, P P

    1998-01-01

    The ability of a serotype M1 strain of Streptococcus pyogenes to efficiently invade A549 human lung epithelial cells was previously shown to be dependent on bacterial exposure to human or bovine serum proteins or synthetic peptides containing the sequence RGD. In this study, stimulation by invasion agonists was determined to be dependent on expression of the streptococcal cell surface protein, M1. Fetal bovine serum (FBS), fibronectin (Fn), the extracellular matrix protein laminin (Lm), and R...

  10. Illustration of Pneumococcal Polysaccharide Capsule during Adherence and Invasion of Epithelial Cells

    OpenAIRE

    Hammerschmidt, Sven; Wolff, Sonja; Hocke, Andreas; Rosseau, Simone; Müller, Ellruth; Rohde, Manfred

    2005-01-01

    The capsular polysaccharide of Streptococcus pneumoniae represents an important virulence factor and protects against phagocytosis. In this study the amount of capsular polysaccharide present on the bacterial surface during the infection process was illustrated by electron microscopic studies. After infection of A549 cells (type II pneumocytes) and HEp-2 epithelial cells a modified fixation method was used that allowed visualization of the state of capsule expression. This modified fixation p...

  11. Direct electric current treatment modifies mitochondrial function and lipid body content in the A549 cancer cell line.

    Science.gov (United States)

    Holandino, Carla; Teixeira, Cesar Augusto Antunes; de Oliveira, Felipe Alves Gomes; Barbosa, Gleyce Moreno; Siqueira, Camila Monteiro; Messeder, Douglas Jardim; de Aguiar, Fernanda Silva; da Veiga, Venicio Feo; Girard-Dias, Wendell; Miranda, Kildare; Galina, Antonio; Capella, Marcia Alves Marques; Morales, Marcelo Marcos

    2016-10-01

    Electrochemical therapy (EChT) entails treatment of solid tumors with direct electric current (DC). This work evaluated the specific effects of anodic flow generated by DC on biochemical and metabolic features of the A549 human lung cancer cell line. Apoptosis was evaluated on the basis of caspase-3 activity and mitochondrial transmembrane potential dissipation. Cell morphology was analyzed using transmission electron microscopy, and lipid droplets were studied through morphometric analysis and X-ray qualitative elemental microanalysis. High-resolution respirometry was used to assess mitochondrial respiratory parameters. Results indicated A549 viability decreased in a dose-dependent manner with a prominent drop between 18 and 24h after treatment (pbodies' number, quantified by morphometrical analyses. Conversely, 24h after treatment, the cells presented a two-fold increase of residual oxygen consumption, accounting for 45.3% of basal oxygen consumption. These results show remarkable alterations promoted by anodic flow on human lung cancer cells which are possibly involved with the antitumoral effects of EChT. PMID:27243447

  12. Auto-synthesis of 11C-gefitinib and micro PET/CT imaging for A549

    International Nuclear Information System (INIS)

    Gefitinib is an approved anticancer drug inhibiting epidermal growth factor receptor/tyrosine kinase (EGFR/TK) in non-small cell lung cancer (NSCLC). In this work, we aimed at developing an auto-synthesis method for 11C-Gefitinib with 11C-CH3-Triflate as methylation agent, and evaluating 11C-Gefinitib to explore its specific uptake with micro PET imaging, 11C-Gefitinib was synthesized by the reaction of 7-demethyl precursor with 11C-CH3-Triflate under base condition, and purified by HPLC. Micro PET/CT imaging was performed in A549 tumor-bearing mice. The results showed that it took 28 min for synthesis of 11C-Gefitinib with radiochemical yields of 30%-35% (with decay correction, n>5). The radiochemical purity was over 99%, and the specific activity was 55.5 GBq/μmol, respectively. Micro PET/CT imaging showed that the A549 tumor uptake of 11C-Gefitinib could be blocked by co-injection of non-radioactive Gefitinib (50 mg/kg), demonstrating the tumor specific accumulation of 11C-Gefitinib. It is concluded that 11C-Gefitinib can be served as PET imaging agent for the evaluation of Gefinitib therapy in NSCLC. (authors)

  13. The cytotoxicity of organophosphate flame retardants on HepG2, A549 and Caco-2 cells.

    Science.gov (United States)

    An, Jing; Hu, Jingwen; Shang, Yu; Zhong, Yufang; Zhang, Xinyu; Yu, Zhiqiang

    2016-09-18

    In order to elucidate the cytotoxicity of organophosphate flame retardants (OPFRs), three human in vitro models, namely the HepG2 hepatoma cells, the A549 lung cancer cells and the Caco-2 colon cancer cells, were chosen to investigate the toxicity of triphenyl phosphate (TPP), tributylphosphate (TBP), tris(2-butoxyexthyl) phosphate (TBEP) and tris (2-chloroisopropyl) phosphate (TCPP). Cytotoxicity was assayed in terms of cell viability, DNA damage status, reactive oxygen species (ROS) level and lactate dehydrogenase (LDH) leakage. The results showed that all these four OPFRs could inhibit cell viability, overproduce ROS level, induce DNA lesions and increase the LDH leakage. In addition, the toxic effects of OPFRs in Caco-2 cells were relatively severer than those in HepG2 and A549 cells, which might result from some possible mechanisms apart from oxidative stress pathway. In conclusion, TBP, TPP, TBEP and TCPP could induce cell toxicity in various cell lines at relatively high concentrations as evidenced by suppression of cell viability, overproduction of ROS, induction of DNA lesions and increase of LDH leakage. Different cell types seemed to have different sensitivities and responses to OPFRs exposure, as well as the underlying potential molecular mechanisms. PMID:27336727

  14. 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.

  15. 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. PMID:26658031

  16. Predicting the clonogenic survival of A549 cells after modulated x-ray irradiation using the linear quadratic model

    Energy Technology Data Exchange (ETDEWEB)

    Bromley, Regina; Oliver, Lyn [Northern Sydney Cancer Centre, Radiation Oncology, Royal North Shore Hospital, Sydney, NSW 2065 (Australia); Davey, Ross; Harvie, Rozelle [Department of Medical Oncology, Bill Walsh Cancer Research Laboratories, Royal North Shore Hospital, Sydney, NSW 2065 (Australia); Baldock, Clive [Institute of Medical Physics, School of Physics, Sydney University, NSW 2006 (Australia)

    2009-01-21

    In this study we present two prediction methods, mean dose and summed dose, for predicting the number of A549 cells that will survive after modulated x-ray irradiation. The prediction methods incorporate the dose profile from the modulated x-ray fluence map applied across the cell sample and the linear quadratic (LQ) model. We investigated the clonogenic survival of A549 cells when irradiated using two different modulated x-ray fluence maps. Differences between the measured and predicted surviving fraction were observed for modulated x-ray irradiation. When the x-ray fluence map produced a steep dose gradient across the sample, fewer cells survived in the unirradiated region than expected. When the x-ray fluence map produced a less steep dose gradient across the sample, more cells survived in the unirradiated region than expected. Regardless of the steepness of the dose gradient, more cells survived in the irradiated region than expected for the reference dose range of 1-10 Gy. The change in the cell survival for the unirradiated regions of the two different dose gradients may be an important factor to consider when predicting the number of cells that will survive at the edge of modulated x-ray fields. This investigation provides an improved method of predicting cell survival for modulated x-ray radiation treatment. It highlights the limitations of the LQ model, particularly in its ability to describe the biological response of cells irradiated under these conditions.

  17. Predicting the clonogenic survival of A549 cells after modulated x-ray irradiation using the linear quadratic model

    Science.gov (United States)

    Bromley, Regina; Oliver, Lyn; Davey, Ross; Harvie, Rozelle; Baldock, Clive

    2009-01-01

    In this study we present two prediction methods, mean dose and summed dose, for predicting the number of A549 cells that will survive after modulated x-ray irradiation. The prediction methods incorporate the dose profile from the modulated x-ray fluence map applied across the cell sample and the linear quadratic (LQ) model. We investigated the clonogenic survival of A549 cells when irradiated using two different modulated x-ray fluence maps. Differences between the measured and predicted surviving fraction were observed for modulated x-ray irradiation. When the x-ray fluence map produced a steep dose gradient across the sample, fewer cells survived in the unirradiated region than expected. When the x-ray fluence map produced a less steep dose gradient across the sample, more cells survived in the unirradiated region than expected. Regardless of the steepness of the dose gradient, more cells survived in the irradiated region than expected for the reference dose range of 1-10 Gy. The change in the cell survival for the unirradiated regions of the two different dose gradients may be an important factor to consider when predicting the number of cells that will survive at the edge of modulated x-ray fields. This investigation provides an improved method of predicting cell survival for modulated x-ray radiation treatment. It highlights the limitations of the LQ model, particularly in its ability to describe the biological response of cells irradiated under these conditions.

  18. Diallyl trisulfide inhibits naphthalene-induced oxidative injury and the production of inflammatory responses in A549 cells and mice.

    Science.gov (United States)

    Zhang, Fang; Zhang, Yongchun; Wang, Kaiming; Zhu, Xiaosong; Lin, Guimei; Zhao, Zhongxi; Li, Shanzhong; Cai, Jianhua; Cao, Jimin

    2015-12-01

    Diallyl trisulfide (DATS) is a garlic organosulfide that may have a therapeutic potential in the treatment of some diseases. We sought to determine whether DATS could inhibit naphthalene-induced oxidative injury and the production of inflammatory responses in vitro and in vivo. A549 cells were either pre-treated (PreTx, prevention) or concurrently treated (CoTx, treatment) with 20μM naphthalene and either 5 or 10μM DATS. PreTx and CoTx showed the prevention and the treatment potential of DATS to inhibit the generation of naphthalene-induced reactive oxygen species (ROS) in the A549 cells. DATS showed antioxidative activity by elevating the SOD activities in the low dose groups. The mechanistic study showed that the DATS-mediated inhibition of naphthalene-induced oxidative injury and the production of inflammatory responses (i.e., TNF-α, IL-6, and IL-8) were attributed to inhibiting the activity of nuclear factor-kappa B (NF-κB). In addition, DATS inhibited the production of serum nitric oxide NO and myeloperoxidase (MPO) in the lungs of Kunming mice. The histological analysis results indicate that DATS inhibited the naphthalene-induced lung damage, which is consistent with the in vitro study results. The in vivo and in vitro results suggest that DATS may be an effective attenuator of naphthalene-induced lung damage. PMID:26548347

  19. Chemosensitization and radiosensitization of a lung cancer cell line A549 induced by a composite polymer micelle.

    Science.gov (United States)

    Xu, Jing; Zhang, Bi-Cheng; Li, Xiang-Long; Xu, Wen-Hong; Zhou, Juan; Shen, Li; Wei, Qi-Chun

    2016-08-01

    Multidrug resistance (MDR) to Doxorubicin (DOX) remains a major obstacle to successful cancer treatment. The present study sought to overcome the MDR of lung cancer cells and achieve radiosensitization by developing a composite DOX-loaded micelle (M-DOX). M-DOX containing PEG-PCL/Pluronic P105 was prepared by the solvent evaporation method. Lung cancer cell line A549 was adopted in this study. In vitro cytotoxicity, cellular uptake behavior, subcellular distribution, and radiosensitivity were evaluated by the treatment with M-DOX, and free DOX was used as a control. A549 cells treated with M-DOX as opposed to free DOX showed greater cellular uptake as well as greater cytotoxicity. Furthermore, M-DOX reached the mitochondria and lysosome effectively after cellular uptake, and fluorescence used to track M-DOX was found to be surrounding the nucleus. Finally, colony-forming assays demonstrated that M-DOX treatment improved radiosensitization when compared to free DOX. Based on the increased cytotoxicity and radiosensitization, M-DOX could be considered as a promising drug delivery system to overcome MDR in lung cancer therapy. PMID:27585226

  20. Protective effects of pulmonary epithelial lining fluid on oxidative stress and DNA single-strand breaks caused by ultrafine carbon black, ferrous sulphate and organic extract of diesel exhaust particles.

    Science.gov (United States)

    Chuang, Hsiao-Chi; Cheng, Yi-Ling; Lei, Yu-Chen; Chang, Hui-Hsien; Cheng, Tsun-Jen

    2013-02-01

    Pulmonary epithelial lining fluid (ELF) is the first substance to make contact with inhaled particulate matter (PM) and interacts chemically with PM components. The objective of this study was to determine the role of ELF in oxidative stress, DNA damage and the production of proinflammatory cytokines following physicochemical exposure to PM. Ultrafine carbon black (ufCB, 15 nm; a model carbonaceous core), ferrous sulphate (FeSO(4); a model transition metal) and a diesel exhaust particle (DEP) extract (a model organic compound) were used to examine the acellular oxidative potential of synthetic ELF and non-ELF systems. We compared the effects of exposure to ufCB, FeSO(4) and DEP extract on human alveolar epithelial Type II (A549) cells to determine the levels of oxidative stress, DNA single-strand breaks and interleukin-8 (IL-8) production in ELF and non-ELF systems. The effects of ufCB and FeSO(4) on the acellular oxidative potential, cellular oxidative stress and DNA single-strand breakage were mitigated significantly by the addition of ELF, whereas there was no decrease following treatment with the DEP extract. There was no significant effect on IL-8 production following exposure to samples that were suspended in ELF/non-ELF systems. The results of the present study indicate that ELF plays an important role in the initial defence against PM in the pulmonary environment. Experimental components, such as ufCB and FeSO(4), induced the production of oxidative stress and led to DNA single-strand breaks, which were moderately prevented by the addition of ELF. These findings suggest that ELF plays a protective role against PM-driven oxidative stress and DNA damage. PMID:23261976

  1. 三种缝线材料对人肺腺癌细胞A549增殖和细胞周期的影响%Effect of three suture lines on the proliferation and cell cycle of lung adenocarcinoma cell A549 in vitro

    Institute of Scientific and Technical Information of China (English)

    Lianhua Ye; Yunchao Huang; Qilin Jin; Feng Hua; Guangqiang Zhao

    2011-01-01

    Objective: The interaction of cell and medical biomaterial is one of the significant factors to affect clinical application of medical biomaterial. This research is to investigate three of suture lines how to affect the proliferation and cell cycle of lung adenocarcinoma cell A549 in vitro. Methods: Three of suture lines were respectively cultivated with lung adenocarcinoma cell A549, after of 72 hours, we detected absorptions of each group by MTT method in order to reflect the proliferation of lung adenocarcinoma cell A549, and also examined percentage of G1 period cells and S period cells of each group by flow cytometry. Results: Different of suture lines had different effects on the proliferation and cell cycle of lung adenocarcinoma cell A549 (P < 0.05). The effect of absorbent suture line was the strongest on the proliferation and cell cycle of lung adenocarcinoma cell A549, the effect of chorda serica chirurgicalis was medium, and the effect of slide wire was poor. Different length of each suture line had different effects on the proliferation and cell cycle of lung adenocarcinoma cell A549 (P < 0.05).Conclusion: Three of suture line materials have different effects on the proliferation and cell cycle of lung adenocarcinoma cell A549, with dose-effect relationship.

  2. Effect of Integrin α5β1-mediated ERK Signal Pathway on Proliferation 
and Migration of A549 Cells

    Directory of Open Access Journals (Sweden)

    Jing BAI

    2011-07-01

    Full Text Available Background and objective Recent studies have shown that integrin α5β1 as a core in the integrin family plays an important role in metastasis, invasion and poor difference of non-small cell lung cancer. In this study, A549 cells were cultured and treated with integrin α5β1 small interfering RNA (siRNA and extracelluar signal-regulated protein kinase (ERK inhibitor PD98059 to investigate the effect of integrin α5β1 on proliferation and migration of A549 cells and explore its signal transduction mechanism. Methods A549 cells were divided into four groups: Untransfection, Lipofectamine, Integrin α5β1 siRNA-transfected group and PD98059 group. The protein expression levels o