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Sample records for epithelial beas-2b cells

  1. Human bronchial epithelial BEAS-2B cells, an appropriate in vitro model to study heavy metals induced carcinogenesis.

    PubMed

    Park, Youn-Hee; Kim, Donghern; Dai, Jin; Zhang, Zhuo

    2015-09-15

    Occupational and environmental exposure to arsenic (III) and chromium VI (Cr(VI)) have been confirmed to cause lung cancer. Mechanisms of these metals carcinogenesis are still under investigation. Selection of cell lines to be used is essential for the studies. Human bronchial epithelial BEAS-2B cells are the cells to be utilized by most of scientists. However, due to p53 missense mutation (CCG→TCG) at codon 47 and the codon 72 polymorphism (CGC→CCC) in BEAS-2B cells, its usage has frequently been questioned. The present study has examined activity and expression of 53 and its downstream target protein p21 upon acute or chronic exposure of BEAS-2B cells to arsenic and Cr(VI). The results show that short-term exposure of BEAS-2B cells to arsenic or Cr(VI) was able to activate both p53 and p21. Chronic exposure of BEAS-2B cells to these two metals caused malignant cell transformation and tumorigenesis. In arsenic-transformed BEAS-2B cells reductions in p53 promoter activity, mRNA expression, and phosphorylation of p53 at Ser392 were observed, while the total p53 protein level remained the same compared to those in passage-matched parent ones. p21 promoter activity and expression were decreased in arsenic-transformed cells. Cr(VI)-transformed cells exhibit elevated p53 promoter activity, mRNA expression, and phosphorylation at Ser15, but reduced phosphorylation at Ser392 and total p53 protein level compared to passage-matched parent ones. p21 promoter activity and expression were elevated in Cr(VI)-transformed cells. These results demonstrate that p53 is able to respond to exposure of arsenic or Cr(VI), suggesting that BEAS-2B cells are an appropriate in vitro model to investigate arsenic or Cr(VI) induced lung cancer. PMID:26091798

  2. Human bronchial epithelial BEAS-2B cells, an appropriate in vitro model to study heavy metals induced carcinogenesis

    SciTech Connect

    Park, Youn-hee; Kim, Donghern; Dai, Jin; Zhang, Zhuo

    2015-09-15

    Occupational and environmental exposure to arsenic (III) and chromium VI (Cr(VI)) have been confirmed to cause lung cancer. Mechanisms of these metals carcinogenesis are still under investigation. Selection of cell lines to be used is essential for the studies. Human bronchial epithelial BEAS-2B cells are the cells to be utilized by most of scientists. However, due to p53 missense mutation (CCG → TCG) at codon 47 and the codon 72 polymorphism (CGC → CCC) in BEAS-2B cells, its usage has frequently been questioned. The present study has examined activity and expression of 53 and its downstream target protein p21 upon acute or chronic exposure of BEAS-2B cells to arsenic and Cr(VI). The results show that short-term exposure of BEAS-2B cells to arsenic or Cr(VI) was able to activate both p53 and p21. Chronic exposure of BEAS-2B cells to these two metals caused malignant cell transformation and tumorigenesis. In arsenic-transformed BEAS-2B cells reductions in p53 promoter activity, mRNA expression, and phosphorylation of p53 at Ser392 were observed, while the total p53 protein level remained the same compared to those in passage-matched parent ones. p21 promoter activity and expression were decreased in arsenic-transformed cells. Cr(VI)-transformed cells exhibit elevated p53 promoter activity, mRNA expression, and phosphorylation at Ser15, but reduced phosphorylation at Ser392 and total p53 protein level compared to passage-matched parent ones. p21 promoter activity and expression were elevated in Cr(VI)-transformed cells. These results demonstrate that p53 is able to respond to exposure of arsenic or Cr(VI), suggesting that BEAS-2B cells are an appropriate in vitro model to investigate arsenic or Cr(VI) induced lung cancer. - Highlights: • Short-term exposure of BEAS-2B cells to arsenic or Cr(VI) activates p53 and p21. • Chronic exposure of BEAS-2B cells to arsenic or Cr(VI) causes cell transformation and tumorigenesis. • Arsenic-transformed cells exhibit

  3. Effects of Size-Fractionated Particulate Matter on Cellular Oxidant Radical Generation in Human Bronchial Epithelial BEAS-2B Cells.

    PubMed

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

    2016-01-01

    The aim of the present study was to investigate the effects of size-fractionated (i.e., <1; 1-2.5, and 2.5-10 µm in an aerodynamic diameter) ambient particulate matter (PM) on reactive oxygen species (ROS) activity and cell viability in human bronchial epithelial cells (BEAS-2B). The PM samples were collected from an urban site (uPM) in Beijing and a steel factory site (sPM) in Anshan, China, from March 2013 to December 2014. Metal elements, organic and elemental carbon, and water-soluble inorganic ions in the uPM and sPM were analyzed. The cell viability and ROS generation in PM-exposed BEAS-2B cells were measured by MTS and DCFH-DA. The results showed that both uPM and sPM caused a decrease in the cell viability and an increase in ROS generation. The level of ROS measured in sPM1.0 was approximately triple that in uPM1.0. The results of correlation analysis showed that the ROS activity and cytotoxicity were related to different PM composition. Moreover, deferoxamine (DFO) significantly prevented the increase of ROS generation and the decrease of cell viability. Taken together, our results suggest that the metals absorbed on PM induced oxidant radical generation in BEAS-2B cells that could lead to impairment of pulmonary function. PMID:27171105

  4. Effects of Size-Fractionated Particulate Matter on Cellular Oxidant Radical Generation in Human Bronchial Epithelial BEAS-2B Cells

    PubMed Central

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

    2016-01-01

    The aim of the present study was to investigate the effects of size-fractionated (i.e., <1; 1–2.5, and 2.5–10 µm in an aerodynamic diameter) ambient particulate matter (PM) on reactive oxygen species (ROS) activity and cell viability in human bronchial epithelial cells (BEAS-2B). The PM samples were collected from an urban site (uPM) in Beijing and a steel factory site (sPM) in Anshan, China, from March 2013 to December 2014. Metal elements, organic and elemental carbon, and water-soluble inorganic ions in the uPM and sPM were analyzed. The cell viability and ROS generation in PM-exposed BEAS-2B cells were measured by MTS and DCFH-DA. The results showed that both uPM and sPM caused a decrease in the cell viability and an increase in ROS generation. The level of ROS measured in sPM1.0 was approximately triple that in uPM1.0. The results of correlation analysis showed that the ROS activity and cytotoxicity were related to different PM composition. Moreover, deferoxamine (DFO) significantly prevented the increase of ROS generation and the decrease of cell viability. Taken together, our results suggest that the metals absorbed on PM induced oxidant radical generation in BEAS-2B cells that could lead to impairment of pulmonary function. PMID:27171105

  5. Identification of PM10 characteristics involved in cellular responses in human bronchial epithelial cells (Beas-2B).

    PubMed

    Van Den Heuvel, Rosette; Den Hond, Elly; Govarts, Eva; Colles, Ann; Koppen, Gudrun; Staelens, Jeroen; Mampaey, Maja; Janssen, Nicole; Schoeters, Greet

    2016-08-01

    Notwithstanding evidence is present that physicochemical characteristics of ambient particles attribute to adverse health effects, there is still some lack of understanding in this complex relationship. At this moment it is not clear which properties (such as particle size, chemical composition) or sources of the particles are most relevant for health effects. This study investigates the in vitro toxicity of PM10 in relation to PM chemical composition, black carbon (BC), endotoxin content and oxidative potential (OP). In 2013-2014 PM10 was sampled (24h sampling, 108 sampling days) in ambient air at three sites in Flanders (Belgium) with different pollution characteristics: an urban traffic site (Borgerhout), an industrial area (Zelzate) and a rural background location (Houtem). To characterize the toxic potential of PM10, airway epithelial cells (Beas-2B cells) have been exposed to particles in vitro. Different endpoints were studied including cell damage and death (cell viability) using the Neutral red Uptake assay, the production of pro-inflammatory molecules by interleukin 8 (IL-8) induction and DNA-damaging activity using the FPG-modified Comet assay. The endotoxin levels in the collected samples were analysed and the capacity of PM10 particles to produce reactive oxygen species (OP) was evaluated by electron paramagnetic resonance (EPR) spectroscopy. Chemical characteristics of PM10 (BC, As, Cd, Cr, Cu, Mn, Ni, Pb, Zn) and meteorological conditions were recorded on the sampling days. PM10 particles exhibited dose-dependent cytotoxicity in Beas-2B cells and were found to significantly induce the release of IL-8 in samples from the three locations. Oxidatively damaged DNA was observed in exposed Beas-2B cells. Endotoxin levels above the detection limit were detected in half of the samples. OP was measurable in all samples. Associations between PM10 characteristics and biological effects of PM10 were assessed by single and multiple regression analyses. The

  6. Nickel compounds induce apoptosis in human bronchial epithelial Beas-2B cells by activation of c-Myc through ERK pathway

    SciTech Connect

    Li Qin; Suen, T.-C.; Sun Hong; Arita, Adriana; Costa, Max

    2009-03-01

    Nickel compounds are carcinogenic to humans and have been shown to alter epigenetic homeostasis. The c-Myc protein controls 15% of human genes and it has been shown that fluctuations of c-Myc protein alter global epigenetic marks. Therefore, the regulation of c-Myc by nickel ions in immortalized but not tumorigenic human bronchial epithelial Beas-2B cells was examined in this study. It was found that c-Myc protein expression was increased by nickel ions in non-tumorigenic Beas-2B and human keratinocyte HaCaT cells. The results also indicated that nickel ions induced apoptosis in Beas-2B cells. Knockout of c-Myc and its restoration in a rat cell system confirmed the essential role of c-Myc in nickel ion-induced apoptosis. Further studies in Beas-2B cells showed that nickel ion increased the c-Myc mRNA level and c-Myc promoter activity, but did not increase c-Myc mRNA and protein stability. Moreover, nickel ion upregulated c-Myc in Beas-2B cells through the MEK/ERK pathway. Collectively, the results demonstrate that c-Myc induction by nickel ions occurs via an ERK-dependent pathway and plays a crucial role in nickel-induced apoptosis in Beas-2B cells.

  7. Role of reactive oxygen species in arsenic-induced transformation of human lung bronchial epithelial (BEAS-2B) cells

    SciTech Connect

    Zhang, Zhuo; Pratheeshkumar, Poyil; Budhraja, Amit; Son, Young-Ok; Kim, Donghern; Shi, Xianglin

    2015-01-09

    Highlights: • Short term exposure of cells to arsenic causes ROS generation. • Chronical exposure of cells to arsenic causes malignant cell transformation. • Inhibition of ROS generation reduces cell transformation by arsenic. • Arsenic-transformed cells exhibit reduced capacity of generating ROS. • Arsenic-transformed cells exhibit increased levels of antioxidants. - Abstract: Arsenic is an environmental carcinogen, its mechanisms of carcinogenesis remain to be investigated. Reactive oxygen species (ROS) are considered to be important. A previous study (Carpenter et al., 2011) has measured ROS level in human lung bronchial epithelial (BEAS-2B) cells and arsenic-transformed BEAS-2B cells and found that ROS levels were higher in transformed cells than that in parent normal cells. Based on these observations, the authors concluded that cell transformation induced by arsenic is mediated by increased cellular levels of ROS. This conclusion is problematic because this study only measured the basal ROS levels in transformed and parent cells and did not investigate the role of ROS in the process of arsenic-induced cell transformation. The levels of ROS in arsenic-transformed cells represent the result and not the cause of cell transformation. Thus question concerning whether ROS are important in arsenic-induced cell transformation remains to be answered. In the present study, we used expressions of catalase (antioxidant against H{sub 2}O{sub 2}) and superoxide dismutase 2 (SOD2, antioxidant against O{sub 2}{sup ·−}) to decrease ROS level and investigated their role in the process of arsenic-induced cell transformation. Our results show that inhibition of ROS by antioxidant enzymes decreased arsenic-induced cell transformation, demonstrating that ROS are important in this process. We have also shown that in arsenic-transformed cells, ROS generation was lower and levels of antioxidants are higher than those in parent cells, in a disagreement with the previous

  8. CULTURE CONDITIONS PROFOUNDLY IMPACT PHENOTYPE IN BEAS-2B, A HUMAN PULMONARY EPITHELIAL MODEL

    PubMed Central

    Zhao, Fei; Klimecki, Walter T.

    2015-01-01

    BEAS-2B, an immortalized, human lung epithelial cell line, has been used to model pulmonary epithelial function for over 30 years. The BEAS-2B phenotype can be modulated by culture conditions that include the presence or absence of fetal bovine serum (FBS). The popularity of BEAS-2B as a model of arsenic toxicology, and the common use of BEAS-2B cultured both with and without FBS, led us to investigate the impact of FBS on BEAS-2B in the context of arsenic toxicology. Comparison of genome-wide gene expression in BEAS-2B cultured with or without FBS revealed altered expression in several biological pathways, including those related to carcinogenesis and energy metabolism. Real-time measurements of oxygen consumption and glycolysis in BEAS-2B demonstrated that FBS culture conditions were associated with a 1.4-fold increase in total glycolytic capacity, a 1.9-fold increase in basal respiration, a 2.0-fold increase in oxygen consumed for ATP production, and a 2.8-fold increase in maximal respiration, compared to BEAS-2B cultured without FBS. Comparisons of the transcriptome changes in BEAS-2B resulting from FBS exposure to the transcriptome changes resulting from exposure to 1 μM sodium arsenite revealed that mRNA levels of 43% of the arsenite-modulated genes were also modulated by FBS. Cytotoxicity studies revealed that BEAS-2B cells exposed to 5% FBS for 8 weeks were almost 5 times more sensitive to arsenite cytotoxicity than non-FBS-exposed BEAS-2B cells. Phenotype changes induced in BEAS-2B by FBS suggest that culture conditions should be carefully considered when using BEAS-2B as an experimental model of arsenic toxicity. PMID:25524072

  9. Use of human bronchial epithelial cells (BEAS-2B) to study immunological markers resulting from exposure to PM{sub 2.5} organic extract from Puerto Rico

    SciTech Connect

    Fuentes-Mattei, Enrique; Rivera, Evasomary; Gioda, Adriana; Sanchez-Rivera, Diana; Roman-Velazquez, Felix R.; Jimenez-Velez, Braulio D.

    2010-03-15

    Fine particulate air pollutants, mainly their organic fraction, have been demonstrated to be associated with cardiovascular and respiratory health problems. Puerto Rico has been reported to have the highest prevalence of pulmonary diseases (e.g., asthma) in the United States. The aim of this study was to assess, for the first time, the immunological response of human bronchial epithelial cells (BEAS-2B) to organic extracts isolated from airborne particulate matter (PM{sub 2.5}) in Puerto Rico. Organic extracts from PM{sub 2.5} collected throughout an 8-month period (2000-2001) were pooled (composite) in order to perform chemical analysis and biological activity testing. BEAS-2B cells were exposed to PM{sub 2.5} organic extract to assess cytotoxicity, levels of cytokines and relative gene expression of MHC-II, hPXR and CYP3A5. Our findings show that organic PM{sub 2.5} consist of toxic as well as bioactive components that can regulate the secretion of cytokines in BEAS-2B, which could modulate inflammatory response in the lung. Trace element analyses confirmed the presence of metals in organic extracts highlighting the relative high abundance of Cu and Zn in polar organic extracts. Polar organic extracts exhibited dose-dependant toxicity and were found to significantly induce the release of interleukin 6 (IL-6), IL-1beta and IL-7 while significantly inhibiting the secretion of IL-8, G-CSF and MCP-1. Moreover, MHC-II transcriptional activity was up-regulated after 24 h of exposure, whereas PXR and CYP3A5 were down-regulated. This research provides a new insight into the effects of PM{sub 2.5} organic fractions on specific effectors and their possible role in the development of respiratory inflammatory diseases in Puerto Rico.

  10. PI3K-delta mediates double-stranded RNA-induced upregulation of B7-H1 in BEAS-2B airway epithelial cells

    SciTech Connect

    Kan-o, Keiko; Matsumoto, Koichiro; Asai-Tajiri, Yukari; Fukuyama, Satoru; Hamano, Saaka; Seki, Nanae; Nakanishi, Yoichi; Inoue, Hiromasa

    2013-05-31

    Highlights: •Double-stranded RNA upregulates B7-H1 on BEAS-2B airway epithelial cells. •The upregulation of B7-H1 is attenuated by inhibition of PI3Kδ isoform. •PI3Kδ-mediated upregulation of B7-H1 is independent of NF-κB activation. •Inhibition of PI3Kδ may prevent persistent viral infection induced by B7-H1. -- Abstract: Airway viral infection disturbs the health-related quality of life. B7-H1 (also known as PD-L1) is a coinhibitory molecule associated with the escape of viruses from the mucosal immunity, leading to persistent infection. Most respiratory viruses generate double-stranded (ds) RNA during replication. The stimulation of cultured airway epithelial cells with an analog of viral dsRNA, polyinosinic-polycytidylic acid (poly IC) upregulates the expression of B7-H1 via activation of the nuclear factor κB(NF-κB). The mechanism of upregulation was investigated in association with phosphatidylinositol 3-kinases (PI3Ks). Poly IC-induced upregulation of B7-H1 was profoundly suppressed by a pan-PI3K inhibitor and partially by an inhibitor or a small interfering (si)RNA for PI3Kδ in BEAS-2B cells. Similar results were observed in the respiratory syncytial virus-infected cells. The expression of p110δ was detected by Western blot and suppressed by pretreatment with PI3Kδ siRNA. The activation of PI3Kδ is typically induced by oxidative stress. The generation of reactive oxygen species was increased by poly IC. Poly IC-induced upregulation of B7-H1 was attenuated by N-acetyl-L-cysteine, an antioxidant, or by oxypurinol, an inhibitor of xanthine oxidase. Poly IC-induced activation of NF-κB was suppressed by a pan-PI3K inhibitor but not by a PI3Kδ inhibitor. These results suggest that PI3Kδ mediates dsRNA-induced upregulation of B7-H1 without affecting the activation of NF-κB.

  11. Differential effects of nitro-PAHs and amino-PAHs on cytokine and chemokine responses in human bronchial epithelial BEAS-2B cells

    SciTech Connect

    Ovrevik, J.; Arlt, V.M.; Oya, E.; Nagy, E.; Mollerup, S.; Phillips, D.H.; Lag, M.; Holme, J.A.

    2010-02-01

    Nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) are found in diesel exhaust and air pollution particles. Along with other PAHs, many nitro-PAHs possess mutagenic and carcinogenic properties, but their effects on pro-inflammatory processes and cell death are less known. In the present study we examined the effects of 1-nitropyrene (1-NP), 3-nitrofluoranthene (3-NF) and 3-nitrobenzanthrone (3-NBA) and their corresponding amino forms, 1-AP, 3-AF and 3-ABA, in human bronchial epithelial BEAS-2B cells. The effects of the different nitro- and amino-PAHs were compared to the well-characterized PAH benzo[a]pyrene (B[a]P). Expression of 17 cytokine and chemokine genes, measured by real-time PCR, showed that 1-NP and 3-NF induced a completely different cytokine/chemokine gene expression pattern to that of their amino analogues. 1-NP/3-NF-induced responses were dominated by maximum effects on CXCL8 (IL-8) and TNF-alpha expression, while 1-AP-/3-AF-induced responses were dominated by CCL5 (RANTES) and CXCL10 (IP-10) expression. 3-NBA and 3-ABA induced only marginal cytokine/chemokine responses. However, 3-NBA exposure induced considerable DNA damage resulting in accumulation of cells in S-phase and a marked increase in apoptosis. B[a]P was the only compound to induce expression of aryl hydrocarbon receptor (AhR)-regulated genes, such as CYP1A1 and CYP1B1, but did not induce cytokine/chemokine responses in BEAS-2B cells. Importantly, nitro-PAHs and amino-PAHs induced both qualitatively and quantitatively different effects on cytokine/chemokine expression, DNA damage, cell cycle alterations and cytotoxicity. The cytokine/chemokine responses appeared to be triggered, at least partly, through mechanisms separate from the other examined endpoints. These results confirm and extend previous studies indicating that certain nitro-PAHs have a considerable pro-inflammatory potential.

  12. Genome-wide analysis of HIF-2α chromatin binding sites under normoxia in human bronchial epithelial cells (BEAS-2B) suggests its diverse functions

    PubMed Central

    Lee, Meng-Chang; Huang, Hsin-Ju; Chang, Tzu-Hao; Huang, Hsieh-Chou; Hsieh, Shen-Yuan; Chen, Yi-Siou; Chou, Wei-Yuan; Chiang, Chiao-Hsi; Lai, Ching-Huang; Shiau, Chia-Yang

    2016-01-01

    Constitutive functional HIF-2α was recently identified in cancer and stem cell lines under normoxia. In this study, BEAS-2B, a bronchial epithelial cell line, was shown to constitutively express active HIF-2α under normoxia and exhibit markers of pluripotency including Oct-4, Nanog, and sphere formation. Oct-4 expression was reduced after knockdown of HIF-2α under normoxia. Global enrichment analysis of HIF-2α demonstrated the diverse functions of HIF-2α under normoxia. Bioinformatics analysis of the enriched loci revealed an enhancer role of HIF-2α binding sites, involvement of HIF-2α interacting proteins, and enriched de novo motifs which suggest the diverse role of HIF-2α in pseudohypoxia. The low ratio of the discovered loci overlapping with those revealed in cancer cell lines 786-O (16.1%) and MCF-7 (15.9%) under hypoxia indicated a prevailing non-canonical mechanism. Hypoxia had positive, marginal or adverse effects on the enrichment of the selected loci in ChIP-PCR assays. Deletion of the N-terminal activation domain (N-TAD) of HIF-2α disrupted the reporting activity of two of the loci annotated to ELN and ANKRD31. Hypoxia incurring abundance variation of HIF-2α may misrepresent the N-TAD functions as canonical hypoxia inducible features via C-TAD activation. Elucidation of the pseudohypoxia functions of constitutive HIF-2α is useful for resolving its role in malignancy and pluripotency. PMID:27373565

  13. Genome-wide analysis of HIF-2α chromatin binding sites under normoxia in human bronchial epithelial cells (BEAS-2B) suggests its diverse functions.

    PubMed

    Lee, Meng-Chang; Huang, Hsin-Ju; Chang, Tzu-Hao; Huang, Hsieh-Chou; Hsieh, Shen-Yuan; Chen, Yi-Siou; Chou, Wei-Yuan; Chiang, Chiao-Hsi; Lai, Ching-Huang; Shiau, Chia-Yang

    2016-01-01

    Constitutive functional HIF-2α was recently identified in cancer and stem cell lines under normoxia. In this study, BEAS-2B, a bronchial epithelial cell line, was shown to constitutively express active HIF-2α under normoxia and exhibit markers of pluripotency including Oct-4, Nanog, and sphere formation. Oct-4 expression was reduced after knockdown of HIF-2α under normoxia. Global enrichment analysis of HIF-2α demonstrated the diverse functions of HIF-2α under normoxia. Bioinformatics analysis of the enriched loci revealed an enhancer role of HIF-2α binding sites, involvement of HIF-2α interacting proteins, and enriched de novo motifs which suggest the diverse role of HIF-2α in pseudohypoxia. The low ratio of the discovered loci overlapping with those revealed in cancer cell lines 786-O (16.1%) and MCF-7 (15.9%) under hypoxia indicated a prevailing non-canonical mechanism. Hypoxia had positive, marginal or adverse effects on the enrichment of the selected loci in ChIP-PCR assays. Deletion of the N-terminal activation domain (N-TAD) of HIF-2α disrupted the reporting activity of two of the loci annotated to ELN and ANKRD31. Hypoxia incurring abundance variation of HIF-2α may misrepresent the N-TAD functions as canonical hypoxia inducible features via C-TAD activation. Elucidation of the pseudohypoxia functions of constitutive HIF-2α is useful for resolving its role in malignancy and pluripotency. PMID:27373565

  14. Surface reactivity and in vitro toxicity on human bronchial epithelial cells (BEAS-2B) of nanomaterials intermediates of the production of titania-based composites.

    PubMed

    Vergaro, Viviana; Aldieri, Elisabetta; Fenoglio, Ivana; Marucco, Arianna; Carlucci, Claudia; Ciccarella, Giuseppe

    2016-08-01

    Titanium dioxide (TiO2) nanoparticles (NPs) are manufactured worldwide in large quantities for use in a wide range of applications. Evaluating the hazards associated with TiO2 NPs is crucial as it enables risk assessment related to human and environmental exposure. In this study the in vitro human toxicity of a set of TiO2 NPs modified with acetic, oleic and boric acids were studied in order to assess the hazard in view of a future scale-up of the synthesis. The surface reactivity of the powders under simulated solar illumination and in the dark has been evaluated by means of EPR spectroscopy. Human bronchial epithelial cells (BEAS-2B) have been chosen as a model for lung epithelium. Cytotoxicity has been assessed by measuring the cells membrane integrity by lactate dehydrogenase (LDH) assay, and the inflammatory response evaluated as nitric oxide (NO) and TNF-α production, and oxidative stress measured as intracellular reduced glutathione (GSH) levels, and induced lipoperoxidation. Aeroxide P25 was used for comparison. The results demonstrated a low photoreactivity and toxic effects lower than Aeroxide P25 of the nano-TiO2 powders, probably as a consequence of the presence of acidic moieties at the surface. PMID:27075777

  15. Organic extracts of urban air pollution particulate matter (PM2.5)-induced genotoxicity and oxidative stress in human lung bronchial epithelial cells (BEAS-2B cells).

    PubMed

    Oh, Seung Min; Kim, Ha Ryong; Park, Yong Joo; Lee, Soo Yeun; Chung, Kyu Hyuck

    2011-08-16

    Traffic is a major source of particulate matter (PM), and ultrafine particulates and traffic intensity probably contribute significantly to PM-related health effects. As a strong relationship between air pollution and motor vehicle-originated pollutants has been shown to exist, air pollution genotoxicity studies of urban cities are steadily increasing. In Korea, the death rate caused by lung cancer is the most rapidly increased cancer death rate in the past 10 years. In this study, genotoxicity of PM2.5 (<2.5μm in aerodynamic diameter particles) collected from the traffic area in Suwon City, Korea, was studied using cultured human lung bronchial epithelial cells (BEAS-2B) as a model system for the potential inhalation health effects. Organic extract of PM2.5 (CE) generated significant DNA breakage and micronucleus formation in a dose-dependent manner (1μg/cm(3)-50μg/cm(3)). In the acid-base-neutral fractionation of PM2.5, neutral samples including the aliphatic (F3), aromatic (F4) and slightly polar (F5) fractions generated significant DNA breakage and micronucleus formation. These genotoxic effects were significantly blocked by scavenging agents [superoxide dismutase (SOD), sodium selenite (SS), mannitol (M), catalase (CAT)]. In addition, in the modified Comet assay using endonucleases (FPG and ENDOIII), CE and its fractions (F3, F4, and F5) increased DNA breakage compared with control groups, indicating that CE and fractions of PM2.5 induced oxidative DNA damage. These results clearly suggest that PM2.5 collected in the Suwon traffic area has genotoxic effects and that reactive oxygen species may play a distinct role in these effects. In addition, aliphatic/chlorinated hydrocarbons, PAH/alkylderivatives, and nitro-PAH/ketones/quinones may be important causative agents of the genotoxic effects. PMID:21524716

  16. DNA damage and DNA damage response in human bronchial epithelial BEAS-2B cells following exposure to 2-nitrobenzanthrone and 3-nitrobenzanthrone: role in apoptosis.

    PubMed

    Oya, Elisabeth; Ovrevik, Johan; Arlt, Volker M; Nagy, Eszter; Phillips, David H; Holme, Jørn A

    2011-11-01

    Nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) are mutagenic and carcinogenic environmental pollutants found in diesel exhaust and on urban air pollution particles. In the present study, human bronchial epithelial BEAS-2B cells were exposed to 2-nitrobenzanthrone (2-NBA) and 3-nitrobenzanthrone (3-NBA). DNA damage responses were compared to those observed after exposure to 1-nitropyrene (1-NP) and benzo[a]pyrene (B[a]P). Examination by microscopy revealed that 3-NBA was the most potent toxic compound while weaker responses were observed with 1-NP and B[a]P. Most interestingly, 2-NBA did not induce cell death or any other stress-related responses. 3-NBA induced a typical apoptotic cell death judged by nuclear condensation and little plasma membrane damage as well as cleavage of caspase 3 and poly-(ADP-ribose) polymerase (PARP). Exposure to 3-NBA resulted in an accumulation of cells in S-phase, and further analysis by Western blotting, immunocytochemistry and flow cytometry revealed that 3-NBA induced a DNA damage response characterized by phosphorylation of ATM (ataxia-telangiectasia mutated), checkpoint kinase (Chk) 2/Chk1, H2AX and p53. The p53 inhibitor pifithrin-α inhibited 3-NBA-induced apoptosis while small effects were seen using pifithrin-μ, suggesting that 3-NBA-induced cell death is a result of transcriptional activation of p53. In conclusion, 3-NBA is a potent inducer of apoptosis, which seemed to be triggered by the DNA damage response. Furthermore, a change of the nitro-group to the second position (i.e. 2-NBA) dramatically changed the cellular reactivity of the compound. PMID:21715570

  17. Perilla frutescens Leaf Extract Inhibits Mite Major Allergen Der p 2-induced Gene Expression of Pro-Allergic and Pro-Inflammatory Cytokines in Human Bronchial Epithelial Cell BEAS-2B

    PubMed Central

    Liu, Jer-Yuh; Chen, Yi-Ching; Lin, Chun-Hsiang; Kao, Shao-Hsuan

    2013-01-01

    Perilla frutescens has been used in traditional medicine for respiratory diseases due to its anti-bacterial and anti-inflammatory activity. This study aimed to investigate effects of Perilla frutescens leaf extract (PFE) on expression of pro-allergic and pro-inflammatory cytokines in airway epithelial cells exposed to mite major allergen Der p 2 (DP2) and the underlying mechanisms. Our results showed that PFE up to 100 µg/mL had no cytotoxic effect on human bronchial epithelial cell BEAS-2B. Further investigations revealed that PFE dose-dependently diminished mRNA expression of pro-allergic cytokine IL-4, IL-5, IL-13 and GM-CSF, as well as pro-inflammatory cytokine IL-6, IL-8 and MCP-1 in BEAS-2B cells treated with DP2. In parallel to mRNA, the DP-2-elevated levels of the tested cytokines were decreased. Further investigation showed that DP2-indued phosphorylation of p38 MAPK (P38) and JNK, but not Erk1/2, was also suppressed by PFE. In addition, PFE elevated cytosolic IκBα level and decreased nuclear NF-κB level in DP2-stimulated BEAS-2B cells. Taken together, these findings revealed that PFE significantly diminished both mRNA expression and protein levels of pro-allergic and pro-inflammatory cytokines in response to DP2 through inhibition of P38/JNK and NK-κB activation. These findings suggest that PFE should be beneficial to alleviate both allergic and inflammatory responses on airway epithelium in response to aeroallergens. PMID:24204835

  18. African Dust Storms Reaching Puerto Rican Coast Stimulate the Secretion of IL-6 and IL-8 and Cause Cytotoxicity to Human Bronchial Epithelial Cells (BEAS-2B).

    PubMed

    Rodríguez-Cotto, Rosa I; Ortiz-Martínez, Mario G; Rivera-Ramírez, Evasomary; Méndez, Loyda B; Dávila, Julio C; Jiménez-Vélez, Braulio D

    2013-10-01

    African dust storm events (ADE) travel across the Atlantic Ocean (ADEAO) and reach the Puerto Rican coast (ADEPRC), potentially impacting air quality and human health. To what extent seasonal variations in atmospheric particulate matter (PM) size fractions, composition and sources trigger respiratory-adverse effects to Puerto Ricans is still unclear. In the present study, we investigated the pro-inflammatory and cytotoxic effects of PM samples harvested during ADEAO (PM10), ADEPRC (PM2.5 and PM10) and Non-ADE (Preand Post-ADEAO and Non-ADEPRC), using BEAS-2B cells. Endotoxins (ENX) in PM2.5 and PM10 extracts and traces of metals (TMET) in PM2.5 extracts were also examined. IL-6 and IL-8 secretion and cytotoxicity were used as endpoints. ADEAO and ADEPRC extracts were found to be more cytotoxic than Non-ADE and ADEAO were more toxic than ADEPRC extracts. PM10 extracts from ADEAO and Post-ADEAO caused significant secretion of IL-8. IL-6 and IL-8 secretion was higher following treatment with PM10 and PM2.5 ADEPRC than with Non-ADEPRC extracts. ENX levels were found to be higher in PM10 ADEAO than in the rest of the samples tested. TMET levels were higher in PM2.5 ADEPRC than in Non-ADEPRC extracts. Deferoxamine significantly reduced cytotoxicity and IL-6 and IL-8 secretion whereas Polymyxin B did not. TMET in PM2.5 fractions is a major determinant in ADEPRC-induced toxicity and work in conjunction with ENX to cause toxicity to lung cells in vitro. ENX and TMET may be responsible, in part, for triggering PM-respiratory adverse responses in susceptible and predisposed individuals. PMID:25002916

  19. African Dust Storms Reaching Puerto Rican Coast Stimulate the Secretion of IL-6 and IL-8 and Cause Cytotoxicity to Human Bronchial Epithelial Cells (BEAS-2B)

    PubMed Central

    Rodríguez-Cotto, Rosa I.; Ortiz-Martínez, Mario G.; Rivera- Ramírez, Evasomary; Méndez, Loyda B.; Dávila, Julio C.; Jiménez-Vélez, Braulio D.

    2014-01-01

    African dust storm events (ADE) travel across the Atlantic Ocean (ADEAO) and reach the Puerto Rican coast (ADEPRC), potentially impacting air quality and human health. To what extent seasonal variations in atmospheric particulate matter (PM) size fractions, composition and sources trigger respiratory-adverse effects to Puerto Ricans is still unclear. In the present study, we investigated the pro-inflammatory and cytotoxic effects of PM samples harvested during ADEAO (PM10), ADEPRC (PM2.5 and PM10) and Non-ADE (Preand Post-ADEAO and Non-ADEPRC), using BEAS-2B cells. Endotoxins (ENX) in PM2.5 and PM10 extracts and traces of metals (TMET) in PM2.5 extracts were also examined. IL-6 and IL-8 secretion and cytotoxicity were used as endpoints. ADEAO and ADEPRC extracts were found to be more cytotoxic than Non-ADE and ADEAO were more toxic than ADEPRC extracts. PM10 extracts from ADEAO and Post-ADEAO caused significant secretion of IL-8. IL-6 and IL-8 secretion was higher following treatment with PM10 and PM2.5 ADEPRC than with Non-ADEPRC extracts. ENX levels were found to be higher in PM10 ADEAO than in the rest of the samples tested. TMET levels were higher in PM2.5 ADEPRC than in Non-ADEPRC extracts. Deferoxamine significantly reduced cytotoxicity and IL-6 and IL-8 secretion whereas Polymyxin B did not. TMET in PM2.5 fractions is a major determinant in ADEPRC-induced toxicity and work in conjunction with ENX to cause toxicity to lung cells in vitro. ENX and TMET may be responsible, in part, for triggering PM-respiratory adverse responses in susceptible and predisposed individuals. PMID:25002916

  20. Signaling factors and pathways of α-particle irradiation induced bilateral bystander responses between Beas-2B and U937 cells.

    PubMed

    Fu, Jiamei; Wang, Juan; Wang, Xiangdong; Wang, Ping; Xu, Jinping; Zhou, Cuiping; Bai, Yang; Shao, Chunlin

    2016-07-01

    Although radiation induced bystander effects (RIBE) have been investigated for decades for their potential health risk, the underlying gene regulation is still largely unclear, especially the roles of immune system and inflammatory response in RIBE. In the present study, macrophage U937 cells and epithelial Beas-2B cells were co-cultured to disclose the cascades of bystander signaling factors and intercellular communications. After α-particle irradiation, both ERK and p38 pathways were activated in Beas-2B cells and were associated with the autocrine and paracrine signaling of TNF-α and IL-8, resulting in direct damage to the irradiated cells. Similar upregulation of TNF-α and IL-8 was induced in the bystander U937 cells after co-culture with α-irradiated Beas-2B cells. This upregulation was dependent on the activation of NF-κB pathway and was responsible for the enhanced damage of α-irradiated Beas-2B cells. Interestingly, the increased expressions of TNF-α and IL-8 mRNAs in the bystander U937 cells were clearly relayed on the activated ERK and p38 pathways in the irradiated Beas-2B cells, and the upregulation of TNF-α and IL-8 mRNAs in co-cultured Beas-2B cells was also partly due to the activated NF-κB pathway in the bystander U937 cells. With the pretreatment of U0126 (MEK1/2 inhibitor), SB203580 (p38 inhibitor) or BAY 11-7082 (NF-κB inhibitor), the aggravated damage in the α-irradiated Beas-2B cells could be largely alleviated. Our results disclosed novel signaling cascades of macrophage-mediated bilateral bystander responses that the release of TNF-α and IL-8 regulated by MAPK and NF-κB pathways synergistically increased cellular injury after α-particle irradiation. PMID:27155559

  1. An analysis of glucocorticoid receptor-mediated gene expression in BEAS-2B human airway epithelial cells identifies distinct, ligand-directed, transcription profiles with implications for asthma therapeutics

    PubMed Central

    Joshi, T; Johnson, M; Newton, R; Giembycz, M

    2015-01-01

    Background and Purpose International asthma guidelines recommend that inhaled glucocorticoids be used as a monotherapy in all patients with mild to moderate disease because of their ability to suppress airways inflammation. Current evidence suggests that the therapeutic benefit of glucocorticoids is due to the transactivation and transrepression of anti-inflammatory and pro-inflammatory genes respectively. However, the extent to which clinically relevant glucocorticoids are equivalent in their ability to modulate gene expression is unclear. Experimental Approach A pharmacodynamics investigation of glucocorticoid receptor (GR)-mediated gene transactivation in BEAS-2B human airway epithelial cells was performed using a glucocorticoid response element luciferase reporter coupled with an analysis of glucocorticoid-inducible genes encoding proteins with anti-inflammatory and adverse-effect potential. Key Results Using transactivation as a functionally relevant output, a given glucocorticoid displayed a unique, gene expression ‘fingerprint’ where intrinsic efficacy and GR density were essential determinants. We showed that depending on the gene selected for analysis, a given glucocorticoid can behave as an antagonist, partial agonist, full agonist or even ‘super agonist’. In the likely event that different, tissue-dependent gene expression profiles are reproduced in vivo, then the anti-inflammatory and adverse-effect potential of many glucocorticoids currently available as asthma therapeutics may not be equivalent. Conclusions and Implications The generation of gene expression ‘fingerprints’ in target and off-target human tissues could assist the rational design of GR agonists with improved therapeutic ratios. This approach could identify compounds that are useful in the management of severe asthma and other inflammatory disorders where systemic exposure is desirable. PMID:25393397

  2. Genotoxicity of polyvinylpyrrolidone-coated silver nanoparticles in BEAS 2B cells.

    PubMed

    Nymark, Penny; Catalán, Julia; Suhonen, Satu; Järventaus, Hilkka; Birkedal, Renie; Clausen, Per Axel; Jensen, Keld Alstrup; Vippola, Minnamari; Savolainen, Kai; Norppa, Hannu

    2013-11-01

    Silver nanoparticles (AgNPs) are widely utilized in various consumer products and medical devices, especially due to their antimicrobial properties. However, several studies have associated these particles with toxic effects, such as inflammation and oxidative stress in vivo and cytotoxic and genotoxic effects in vitro. Here, we assessed the genotoxic effects of AgNPs coated with polyvinylpyrrolidone (PVP) (average diameter 42.5±14.5 nm) on human bronchial epithelial BEAS 2B cells in vitro. AgNPs were dispersed in bronchial epithelial growth medium (BEGM) with 0.6 mg/ml bovine serum albumin (BSA). The AgNP were partially well-dispersed in the medium and only limited amounts (ca. 0.02 μg Ag(+) ion/l) could be dissolved after 24h. The zeta-potential of the AgNPs was found to be highly negative in pure water but was at least partially neutralized in BEGM with 0.6 mg BSA/ml. Cytotoxicity was measured by cell number count utilizing Trypan Blue exclusion and by an ATP-based luminescence cell viability assay. Genotoxicity was assessed by the alkaline single cell gel electrophoresis (comet) assay, the cytokinesis-block micronucleus (MN) assay, and the chromosomal aberration (CA) assay. The cells were exposed to various doses (0.5-48 μg/cm(2) corresponding to 2.5-240 μg/ml) of AgNPs for 4 and 24 h in the comet assay, for 48 h in the MN assay, and for 24 and 48 h in the CA assay. DNA damage measured by the percent of DNA in comet tail was induced in a dose-dependent manner after both the 4-h and the 24-h exposures to AgNPs, with a statistically significant increase starting at 16 μg/cm(2) (corresponding to 60.8 μg/ml) and doubling of the percentage of DNA in tail at 48 μg/cm(2). However, no induction of MN or CAs was observed at any of the doses or time points. The lack of induction of chromosome damage by the PVP-coated AgNPs is possibly due to the coating which may protect the cells from direct interaction with the AgNPs, either by reducing ion leaching from the

  3. Genome-wide analysis of BEAS-2B cells exposed to trivalent arsenicals and dimethylthioarsinic acid.

    PubMed

    Chilakapati, Jaya; Wallace, Kathleen; Ren, Hongzu; Fricke, Michael; Bailey, Kathryn; Ward, William; Creed, Jack; Kitchin, Kirk

    2010-01-31

    Lung is a major target for arsenic carcinogenesis in humans by both oral and inhalation routes. However, the carcinogenic mode of action of arsenicals is unknown. We investigated the effects of inorganic arsenic (iAsIII), monomethylarsonous acid (MMAIII), dimethylarsinous acid (DMAIII) and dimethylthioarsinic acid (DMTA), a sulfur containing dimethyl arsenic metabolite, in human bronchial epithelial (BEAS-2B) cells. Cells were exposed to 3, 15 microM-iAsIII; 0.3, 1 microM-MMAIII; 0.2, 1 microM-DMAIII; 0.2, 0.9 microM-DMTA as non-cytotoxic and minimally cytotoxic ( approximately 20%) concentrations based on Neutral Red uptake assays after 24h of culture. Total RNA was isolated and gene expression analysis conducted using Affymetrix Human Genome 133 Plus 2.0 arrays. Differentially expressed genes (DEGs) were determined using a one-way ANOVA (p < or =0.05) by Rosetta Resolver, a Benjamini-Hochberg FDR (false discovery rate) multiple testing correction (< 0.05) followed by a Scheffe's post hoc test. For all compounds except DMTA, > 90% of DEG altered in the low concentration were also changed at the high concentration. There was a clear dose-response seen in the number of DEGs for all four compounds. iAsIII showed the highest number of DEG at both concentrations (2708 and 123, high and low, respectively). 1749, 420 and 120 DEGs were unique to the high concentrations of iAsIII, MMAIII and DMAIII, respectively. Transferrin receptor is a common DEG in low concentration arsenical treated cells. Ingenuity Pathway Analysis revealed p53 signaling (E2F1 and 2, SERPIN), and cell cycle related genes (cyclin D1) were altered by the high concentrations of DMTA, MMAIII and iAsIII. Oxidative stress (DUSP1, GPX2, NQO1, GCLC) and NF-kappaB signaling (TLR4, NF-kappaB) pathways were changed by the high concentrations of MMAIII and iAsIII. The genes identified in this study can be a valuable tool to determine the mechanism of arsenic toxicity and cancer formation. A number of

  4. Effects of corexit oil dispersants and the WAF of dispersed oil on DNA damage and repair in cultured human bronchial airway cells, BEAS-2B

    PubMed Central

    Major, Danielle; Derbes, Rebecca S.; Wang, He; Roy-Engel, Astrid M.

    2016-01-01

    Large quantities of dispersants were used as a method to disperse the roughly 210 million gallons of spilled crude oil that consumed the Gulf of Mexico. Little is known if the oil-dispersant and oil-dispersant mixtures on human airway BEAS-2B epithelial cells. Here we present the cytotoxic and genotoxic in vitro effects on the human lung cells BEAS-2B following exposure to and oil-dispersant mixtures on human airway BEAS-2B epithelial cells. Here we present the cytotoxic and genotoxic in vitro effects on the human lung cells BEAS-2B following exposure to Corexit dispersants EC9500 and EC9527, Water Accommodated Fraction (WAF) -crude, WAF-9500 + Oil, and WAF-9527 + Oil. Cellular cytotoxicity to WAF-dispersed oil samples was observed at concentrations greater than 1000 ppm with over 70% of observed cellular death. At low concentration exposures (100 and 300 ppm) DNA damage was evidenced by the detection of single strand breaks (SSBs) and double strand breaks (DSBs) as measured by alkaline and neutral comet assay analyses. Immunoblot analyses of the phosphorylated histone H2A.X (ɣ-H2A.X) and tumor suppressor p53 protein confirmed activation of the DNA damage response due to the exposure-induced DNA breaks. Although, many xenobiotics interfere with DNA repair pathways, in vitro evaluation of the nucleotide excision repair (NER) and DSB repair pathways appear to be unaffected by the oil-dispersant mixtures tested. Overall, this study supports that oil-dispersant mixtures induce genotoxic effects in culture.

  5. Analysis of the miRNA-mRNA networks in malignant transformation BEAS-2B cells induced by alpha-particles.

    PubMed

    Nie, Ji-Hua; Chen, Zhi-Hai; Shao, Chun-Lin; Pei, Wei-Wei; Zhang, Jie; Zhang, Shu-Yu; Jiao, Yang; Tong, Jian

    2016-01-01

    The aim of this study was to determine the toxicity induced by irradiation with alpha-particles on malignant transformation of immortalized human bronchial epithelial cells (BEAS-2B) using miRNA-mRNA networks. The expression of BEAS-2B cells was determined by measuring colony formation, mtDNA, mitochondrial membrane potential (MMP), and ROS levels. Changes in BEAS-2B cell gene expression were observed and quantified using microarrays that included an increase in 157 mRNA and 20 miRNA expression and a decrease in 77 mRNA and 48 miRNA. Bioinformatic software was used to analyze these different mRNA and miRNA, which indicated that miR-107 and miR-494 play an important role in alpha-particles-mediated cellular malignant transformation processes. The pathways related to systemic lupus erythematosus, cytokine-cytokine receptor interaction, MAPK signaling pathway, regulation of actin cytoskeleton, and cell adhesion molecules (CAMs) were stimulated, while those of ribosome, transforming growth factor (TGF)-beta signaling pathway, and metabolic pathways were inhibited. Data suggest that miRNA and mRNA play a crucial role in alpha-particles-mediated malignant transformation processes. It is worth noting that three target genes associated with lung cancer were identified and upregulated PEG 10 (paternally expressed gene 10), ARHGAP26, and IRS1. PMID:27267825

  6. RNA-binding motif protein 5 inhibits the proliferation of cigarette smoke-transformed BEAS-2B cells through cell cycle arrest and apoptosis.

    PubMed

    Lv, Xue-Jiao; Du, Yan-Wei; Hao, Yu-Qiu; Su, Zhen-Zhong; Zhang, Lin; Zhao, Li-Jing; Zhang, Jie

    2016-04-01

    Cigarette smoking has been shown to be the most significant risk factor for lung cancer. Recent studies have also indicated that RNA-binding motif protein 5 (RBM5) can modulate apoptosis and suppress tumor growth. The present study focused on the role of RBM5 in the regulation of cigarette smoke extract (CSE)-induced transformation of bronchial epithelial cells into the cancerous phenotype and its mechanism of action. Herein, we exposed normal BEAS-2B cells for 8 days to varying concentrations of CSE or dimethylsulfoxide (DMSO), followed by a recovery period of 2 weeks. Next, the RBM5 protein was overexpressed in these transformed BEAS-2B cells though lentiviral infection. Later, the morphological changes, cell proliferation, cell cycle, apoptosis, invasion and migration were assessed. In addition, we analyzed the role of RBM5 in xenograft growth. The expression of RBM5 along with the genes related to cell cycle regulation, apoptosis and invasion were also examined. Finally, our results revealed that BEAS-2B cells exposed to 100 µg/ml CSE acquired phenotypic changes and formed tumors in nude mice, indicative of their cancerous transformation and had reduced RBM5 expression. Subsequent overexpression of RBM5 in these cells significantly inhibited their proliferation, induced G1/S arrest, triggered apoptosis and inhibited their invasion and migration, including xenograft growth. Thus, we established an in vitro model of CSE-induced cancerous transformation and concluded that RBM5 overexpression inhibited the growth of these transformed cells through cell cycle arrest and induction of apoptosis. Therefore, our study suggests the importance of RBM5 in the pathogenesis of smoking-related cancer. PMID:26782095

  7. EFFECTS OF COREXIT DISPERSANTS ON CYTOTOXICITY PARAMETERS IN A CULTURED HUMAN BRONCHIAL AIRWAY CELLS, BEAS-2B

    PubMed Central

    Shi, Yongli; Roy-Engel, Astrid M.; Wang, He

    2013-01-01

    The objective of this study was to assess the cytotoxicity of COREXIT dispersants EC9500A, EC9527A, and EC9580A on human airway BEAS-2B epithelial cells. Cells were exposed to dispersants for 2 or 24 h at concentrations ranging from 0 to 300 ppm. COREXIT EC9527 at 100 ppm produced 50% viability loss as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at 24 h. COREXIT 9527 at 200 ppm produced 50% cell death at 2 h and 100% at 24 h. At 300 ppm COREXIT 9527 induced 100% cell death at 2 or 24 h. In the case of COREXIT 9500A 50% cell viability was noted with 200 ppm at 2 or 24 h, with a significant decrease in cell survival to 2% at 300 ppm. In contrast, no marked change in cell viability was observed in cells treated at any COREXIT 9580A concentration examined. Western blot analysis showed an increase in expression of LC3B, a marker of autophagy, in cells treated for 2 h with 300 ppm COREXIT EC9527A as well as 100 or 300 ppm Corexit EC9500A. No marked effect on LC3B expression was observed for any COREXIT 9580A concentration. Apoptosis markers as measured by cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP) were detectable only in cells incubated with 300 ppm COREXIT EC9527A. Although all three dispersants induced enhanced generation of reactive oxygen species (ROS) after 2-h treatment at 300 ppm, Western blot analysis revealed that 2-h incubation was not sufficient to induce a significant change in the protein expression of superoxide dismutases SOD1, SOD2, and SOD3. Data thus indicate exposure to certain dispersants may be harmful to human airway epithelial cells in a concentration-dependent manner. PMID:24028667

  8. Effects of COREXIT dispersants on cytotoxicity parameters in a cultured human bronchial airway cells, BEAS-2B.

    PubMed

    Shi, Yongli; Roy-Engel, Astrid M; Wang, He

    2013-01-01

    The objective of this study was to assess the cytotoxicity of COREXIT dispersants EC9500A, EC9527A, and EC9580A on human airway BEAS-2B epithelial cells. Cells were exposed to dispersants for 2 or 24 h at concentrations ranging from 0 to 300 ppm. COREXIT EC9527 at 100 ppm produced 50% viability loss as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at 24 h. COREXIT 9527 at 200 ppm produced 50% cell death at 2 h and 100% at 24 h. At 300 ppm COREXIT 9527 induced 100% cell death at 2 or 24 h. In the case of COREXIT 9500A 50% cell viability was noted with 200 ppm at 2 or 24 h, with a significant decrease in cell survival to 2% at 300 ppm. In contrast, no marked change in cell viability was observed in cells treated at any COREXIT 9580A concentration examined. Western blot analysis showed an increase in expression of LC3B, a marker of autophagy, in cells treated for 2 h with 300 ppm COREXIT EC9527A as well as 100 or 300 ppm Corexit EC9500A. No marked effect on LC3B expression was observed for any COREXIT 9580A concentration. Apoptosis markers as measured by cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP) were detectable only in cells incubated with 300 ppm COREXIT EC9527A. Although all three dispersants induced enhanced generation of reactive oxygen species (ROS) after 2-h treatment at 300 ppm, Western blot analysis revealed that 2-h incubation was not sufficient to induce a significant change in the protein expression of superoxide dismutases SOD1, SOD2, and SOD3. Data thus indicate exposure to certain dispersants may be harmful to human airway epithelial cells in a concentration-dependent manner. PMID:24028667

  9. Proliferative activity of a blend of Echinacea angustifolia and Echinacea purpurea root extracts in human vein epithelial, HeLa, and QBC-939 cell lines, but not in Beas-2b cell lines.

    PubMed

    Cichello, Simon Angelo; Yao, Qian; He, Xiao Qiong

    2016-04-01

    Echinacea is used for its immunostimulating properties and may have a role in modulating adverse immune effects of chemotherapy (i.e., use of 5-fluorouracil (5-FU); fluorouracil and its immunosuppressive effect). Patients may seek herbal remedies such as Echinacea (Echinacea angustifolia and Echinacea purpurea) for immune stimulation. Echinacea extracts have been prescribed to supplement cancer chemotherapy for their immune-supportive effects; however, the extracts may also influence tumourgenesis. Our study aimed to determine the proliferative effect of the ethanolic blend of E. angustifolia and E. purpurea on various cancer cervical and bile duct cell lines, including HELA and QBC-939. Various cancer cells (HeLa and QBC-939) and human vein epithelial cells (HUVEC) were treated with the Echinacea blend sample that was evaporated and reconstituted in Dimethyl sulfoxide (DMSO). As the extract concentration of Echinacea was increased from 12.5 μg/mL to 25 μg/mL, there was an increase in cell inhibition up to 100%, which then reduced to 90% over the next three concentrations, 50 μg/mL, 100 μg/mL, and 200 μg/mL, in HeLa cells; further inhibitory effects were observed in QBC-939 cells, from 9% inhibition at a concentration of 25 μg/mL up to 37.96% inhibition at 100 μg/mL concentration. Moreover, this is the first study to report the growth-promoting effects of this Echinacea blend in HUVEC, up to 800% at a dose concentration of 200 μg/mL. Previous studies have suggested that chicoric acid of Echinacea spp. is responsible for the increased cell growth. The results of this study show that the hydroethanolic extract of Echinacea herbal medicine promotes the growth of HeLa cells and QBC-939 cancer cell proliferation, and may interfere with cancer treatment (i.e., chemotherapy drugs such as 5-fluorouracil and Cisplatin (DDP)). However, the Echinacea blend shows potential in neurodegenerative diseases with growth-promoting effects in HUVEC. Further animal

  10. Proliferative activity of a blend of Echinacea angustifolia and Echinacea purpurea root extracts in human vein epithelial, HeLa, and QBC-939 cell lines, but not in Beas-2b cell lines

    PubMed Central

    Cichello, Simon Angelo; Yao, Qian; He, Xiao Qiong

    2015-01-01

    Echinacea is used for its immunostimulating properties and may have a role in modulating adverse immune effects of chemotherapy (i.e., use of 5-fluorouracil (5-FU); fluorouracil and its immunosuppressive effect). Patients may seek herbal remedies such as Echinacea (Echinacea angustifolia and Echinacea purpurea) for immune stimulation. Echinacea extracts have been prescribed to supplement cancer chemotherapy for their immune-supportive effects; however, the extracts may also influence tumourgenesis. Our study aimed to determine the proliferative effect of the ethanolic blend of E. angustifolia and E. purpurea on various cancer cervical and bile duct cell lines, including HELA and QBC-939. Various cancer cells (HeLa and QBC-939) and human vein epithelial cells (HUVEC) were treated with the Echinacea blend sample that was evaporated and reconstituted in Dimethyl sulfoxide (DMSO). As the extract concentration of Echinacea was increased from 12.5 μg/mL to 25 μg/mL, there was an increase in cell inhibition up to 100%, which then reduced to 90% over the next three concentrations, 50 μg/mL, 100 μg/mL, and 200 μg/mL, in HeLa cells; further inhibitory effects were observed in QBC-939 cells, from 9% inhibition at a concentration of 25 μg/mL up to 37.96% inhibition at 100 μg/mL concentration. Moreover, this is the first study to report the growth-promoting effects of this Echinacea blend in HUVEC, up to 800% at a dose concentration of 200 μg/mL. Previous studies have suggested that chicoric acid of Echinacea spp. is responsible for the increased cell growth. The results of this study show that the hydroethanolic extract of Echinacea herbal medicine promotes the growth of HeLa cells and QBC-939 cancer cell proliferation, and may interfere with cancer treatment (i.e., chemotherapy drugs such as 5-fluorouracil and Cisplatin (DDP)). However, the Echinacea blend shows potential in neurodegenerative diseases with growth-promoting effects in HUVEC. Further animal

  11. Glyoxalase I drives epithelial-to-mesenchymal transition via argpyrimidine-modified Hsp70, miR-21 and SMAD signalling in human bronchial cells BEAS-2B chronically exposed to crystalline silica Min-U-Sil 5: Transformation into a neoplastic-like phenotype.

    PubMed

    Antognelli, Cinzia; Gambelunghe, Angela; Muzi, Giacomo; Talesa, Vincenzo Nicola

    2016-03-01

    Glyoxalase I (Glo1) is the main scavenging enzyme of methylglyoxal (MG), a potent precursor of advanced glycation end products (AGEs). AGEs are known to control multiple biological processes, including epithelial to mesenchymal transition (EMT), a multistep phenomenon associated with cell transformation, playing a major role in a variety of diseases, including cancer. Crystalline silica is a well-known occupational health hazard, responsible for a great number of human pulmonary diseases, such as silicosis. There is still much debate concerning the carcinogenic role of crystalline silica, mainly due to the lack of a causal demonstration between silica exposure and carcinogenesis. It has been suggested that EMT might play a role in crystalline silica-induced lung neoplastic transformation. The aim of this study was to investigate whether, and by means of which mechanism, the antiglycation defence Glo1 is involved in Min-U-Sil 5 (MS5) crystalline silica-induced EMT in BEAS-2B human bronchial epithelial cells chronically exposed, and whether this is associated with the beginning of a neoplastic-like transformation process. By using gene silencing/overexpression and scavenging/inhibitory agents, we demonstrated that MS5 induced hydrogen peroxide-mediated c-Jun-dependent Glo1 up-regulation which resulted in a decrease in the Argpyrimidine-modified Hsp70 protein level which triggered EMT in a novel mechanism involving miR-21 and SMAD signalling. The observed EMT was associated with a neoplastic-like phenotype. The results obtained provide a causal in vitro demonstration of the MS5 pro-carcinogenic transforming role and more importantly they provide new insights into the mechanisms involved in this process, thus opening new paths in research concerning the in vivo study of the carcinogenic potential of crystalline silica. PMID:26784015

  12. Cadmium induces carcinogenesis in BEAS-2B cells through ROS-dependent activation of PI3K/AKT/GSK-3β/β-catenin signaling

    SciTech Connect

    Son, Young-Ok; Wang, Lei; Poyil, Pratheeshkumar; Budhraja, Amit; Hitron, J. Andrew; Zhang, Zhuo; Lee, Jeong-Chae; Shi, Xianglin

    2012-10-15

    Cadmium has been widely used in industry and is known to be carcinogenic to humans. Although it is widely accepted that chronic exposure to cadmium increases the incidence of cancer, the mechanisms underlying cadmium-induced carcinogenesis are unclear. The main aim of this study was to investigate the role of reactive oxygen species (ROS) in cadmium-induced carcinogenesis and the signal transduction pathways involved. Chronic exposure of human bronchial epithelial BEAS-2B cells to cadmium induced cell transformation, as evidenced by anchorage-independent growth in soft agar and clonogenic assays. Chronic cadmium treatment also increased the potential of these cells to invade and migrate. Injection of cadmium-stimulated cells into nude mice resulted in the formation of tumors. In contrast, the cadmium-mediated increases in colony formation, cell invasion and migration were prevented by transfection with catalase, superoxide dismutase-1 (SOD1), or SOD2. In particular, chronic cadmium exposure led to activation of signaling cascades involving PI3K, AKT, GSK-3β, and β-catenin and transfection with each of the above antioxidant enzymes markedly inhibited cadmium-mediated activation of these signaling proteins. Inhibitors specific for AKT or β-catenin almost completely suppressed the cadmium-mediated increase in total and active β-catenin proteins and colony formation. Moreover, there was a marked induction of AKT, GSK-3β, β-catenin, and carcinogenic markers in tumor tissues formed in mice after injection with cadmium-stimulated cells. Collectively, our findings suggest a direct involvement of ROS in cadmium-induced carcinogenesis and implicate a role of AKT/GSK-3β/β-catenin signaling in this process. -- Highlights: ► Chronic exposure to cadmium induces carcinogenic properties in BEAS-2B cells. ► ROS involved in cadmium-induced tumorigenicity of BEAS-2B cells. ► Cadmium activates ROS-dependent AKT/GSK-3β/β-catenin-mediated signaling. ► ROS

  13. Roles of MAPK Pathway Activation During Cytokine Induction in BEAS-2B Cells Exposed to Fine World Trade Center (WTC) Dust

    PubMed Central

    Wang, Shang; Prophete, Colette; Soukup, Joleen M.; Chen, Lung-chi; Costa, Max; Ghio, Andrew; Qu, Qingshan; Chen, Haobin

    2014-01-01

    The World Trade Center (WTC) collapse on September 11, 2001 released copious amounts of particulate matter into the atmosphere of New York City. Follow-up studies on persons exposed to the dusts have revealed a severely increased rate for asthma and other respiratory illnesses. There have only been a few studies that have sought to discern the possible mechanisms underlying these untoward pathologies. In one study, an increased cytokine release was detected in cells exposed to WTC fine dusts (PM2.5 fraction or WTC2.5). However, the mechanism(s) for these increases has yet to be fully defined. Because activation of the MAPK signaling pathways is known to cause cytokine induction, the current study was undertaken to analyze the possible involvement of these pathways in any increased cytokine formation by lung epithelial cells (as BEAS-2B cells) exposed to WTC2.5. Our results showed that exposure to WTC2.5 for 5 hr increased IL-6 mRNA expression in BEAS-2B cells, as well as its protein levels in the culture media, in a dose-dependent manner. Besides IL-6, Cytokine Multiplex analyses revealed that formation of IL-8 and -10 was also elevated by the exposure. Both ERK and p38, but not JNK, signaling pathways were found to be activated in cells exposed to WTC2.5. Inactivation of ERK signaling pathways by PD98059 effectively blocked IL-6, -8, and -10 induction by WTC2.5; the p38 kinase inhibitor SB203580 significantly decreased induction of IL-8 and -10. Together, our data demonstrated activation of MAPK signaling pathway(s) likely played an important role in the WTC2.5-induced formation of several inflammatory (and, subsequently, anti-inflammatory) cytokines. The results are important in that they help to define one mechanism via which the WTC dusts may have acted to cause the documented increases in asthma and other inflammation-associated respiratory dysfunctions in the individuals exposed to the dusts released from the WTC collapse. PMID:20731619

  14. Repeated PM2.5 exposure inhibits BEAS-2B cell P53 expression through ROS-Akt-DNMT3B pathway-mediated promoter hypermethylation

    PubMed Central

    Zhou, Wei; Tian, Dongdong; He, Jun; Wang, Yimei; Zhang, Lijun; Cui, Lan; jia, Li; Zhang, Li; Li, Lizhong; Shu, Yulei; Yu, Shouzhong; Zhao, Jun; Yuan, Xiaoyan; Peng, Shuangqing

    2016-01-01

    Long-term exposure to fine particulate matter (PM2.5) has been reported to be closely associated with the increased lung cancer risk in populations, but the mechanisms underlying PM-associated carcinogenesis are not yet clear. Previous studies have indicated that aberrant epigenetic alterations, such as genome-wide DNA hypomethylation and gene-specific DNA hypermethylation contribute to lung carcinogenesis. And silence or mutation of P53 tumor suppressor gene is the most prevalent oncogenic driver in lung cancer development. To explore the effects of PM2.5 on global and P53 promoter methylation changes and the mechanisms involved, we exposed human bronchial epithelial cells (BEAS-2B) to low concentrations of PM2.5 for 10 days. Our results indicated that PM2.5-induced global DNA hypomethylation was accompanied by reduced DNMT1 expression. PM2.5 also induced hypermethylation of P53 promoter and inhibited its expression by increasing DNMT3B protein level. Furthermore, ROS-induced activation of Akt was involved in PM2.5-induced increase in DNMT3B. In conclusion, our results strongly suggest that repeated exposure to PM2.5 induces epigenetic silencing of P53 through ROS-Akt-DNMT3B pathway-mediated promoter hypermethylation, which not only provides a possible explanation for PM-induced lung cancer, but also may help to identify specific interventions to prevent PM-induced lung carcinogenesis. PMID:26942697

  15. PLD1 activation mediates Amb a 1-induced Th2-associated cytokine expression via the JNK/ATF-2 pathway in BEAS-2B cells.

    PubMed

    Kim, Joo-Hwa; Choi, Hye-Jin; Oh, Cheong-Hae; Oh, Jae-Won; Han, Joong-Soo

    2015-01-01

    The purpose of this study was to identify the role of phospholipase D1 (PLD1) in Amb a 1-induced IL-5 and IL-13 expression. When BEAS-2B cells were stimulated with Amb a 1, PLD activity increased, and knockdown of PLD1 decreased Amb a 1-induced IL-5 and IL-13 expression. Amb a 1 also activated the PLCγ/p70S6K/JNK pathway. Furthermore, Amb a 1-induced PLD activation was also attenuated by PLCγ inhibition, and knockdown of PLD1 decreased Amb a 1-induced activation of P70S6K and JNK. When ATF-2 activity was blocked with ATF-2 siRNA, Amb a 1-induced IL-5 and IL-13 expression was completely abolished, indicating that ATF-2 is a transcriptional factor required for the expression of IL-5 and IL-13 in response to Amb a 1. Taken together, we suggest that PLD1 acts as an important regulator in Amb a 1-induced expression of IL-5 and IL-13 via a PLCγ/p70S6K/JNK/ATF-2 pathway in BEAS-2B cells. PMID:26302934

  16. Downregulation of B-cell lymphoma/leukemia-2 by overexpressed microRNA 34a enhanced titanium dioxide nanoparticle-induced autophagy in BEAS-2B cells

    PubMed Central

    Bai, Wenlin; Chen, Yujiao; Sun, Pengling; Gao, Ai

    2016-01-01

    Titanium dioxide (TiO2) nanoparticles (TNPs) are manufactured worldwide for a wide range of applications and the toxic effect of TNPs on biological systems is gaining attention. Autophagy is recognized as an emerging toxicity mechanism triggered by nanomaterials. MicroRNA 34a (miR34a) acts as a tumor suppressor gene by targeting many oncogenes, but how it affects autophagy induced by TNPs is not completely understood. Here, we observed the activation of TNP-induced autophagy through monodansylcadaverine staining and LC3-I/LC3-II conversion. Meanwhile, the transmission electron microscope ultrastructural analysis showed typical morphological characteristics in autophagy process. We detected the expression of miR34a and B-cell lymphoma/leukemia-2 (Bcl-2). In addition, the underlying mechanism of TNP-induced autophagy was performed using overexpression of miR34a by lentivirus vector transfection. Results showed that TNPs induced autophagy generation evidently. Typical morphological changes in the process of autophagy were observed by the transmission electron microscope ultrastructural analysis and LC3-I/LC3-II conversion increased significantly in TNP-treated cells. Meanwhile, TNPs induced the downregulation of miR34a and increased the expression of Bcl-2. Furthermore, overexpressed miR34a decreased the expression of Bcl-2 both in messenger RNA and protein level, following which the level of autophagy and cell death rate increased after the transfected cells were incubated with TNPs for 24 hours. These findings provide the first evidence that overexpressed miR34a enhanced TNP-induced autophagy and cell death through targeted downregulation of Bcl-2 in BEAS-2B cells. PMID:27226226

  17. DNA Hypermethylation of CREB3L1 and Bcl-2 Associated with the Mitochondrial-Mediated Apoptosis via PI3K/Akt Pathway in Human BEAS-2B Cells Exposure to Silica Nanoparticles

    PubMed Central

    Zou, Yang; Li, Qiuling; Jiang, Lizhen; Guo, Caixia; Li, Yanbo; Yu, Yang; Li, Yang; Duan, Junchao; Sun, Zhiwei

    2016-01-01

    The toxic effects of silica nanoparticles (SiNPs) are raising concerns due to its widely applications in biomedicine. However, current information about the epigenetic toxicity of SiNPs is insufficient. In this study, the epigenetic regulation of low-dose exposure to SiNPs was evaluated in human bronchial epithelial BEAS-2B cells over 30 passages. Cell viability was decreased in a dose- and passage-dependent manner. The apoptotic rate, the expression of caspase-9 and caspase-3, were significantly increased induced by SiNPs. HumanMethylation450 BeadChip analysis identified that the PI3K/Akt as the primary apoptosis-related pathway among the 25 significant altered processes. The differentially methylated sites of PI3K/Akt pathway involved 32 differential genes promoters, in which the CREB3L1 and Bcl-2 were significant hypermethylated. The methyltransferase inhibitor, 5-aza, further verified that the DNA hypermethylation status of CREB3L1 and Bcl-2 were associated with downregulation of their mRNA levels. In addition, mitochondrial-mediated apoptosis was triggered by SiNPs via the downregulation of PI3K/Akt/CREB/Bcl-2 signaling pathway. Our findings suggest that long-term low-dose exposure to SiNPs could lead to epigenetic alterations. PMID:27362941

  18. Cellular Interactions and Biological Responses to Titanium Dioxide Nanoparticles in HepG2 and BEAS-2B Cells: Role of Cell Culture Media

    EPA Science Inventory

    ABSTRACT We have shown previously that the composition of the biological medium used in vitro can affect the cellular interaction and biological response of titanium dioxide nanoparticles (nano-TiO2) in human lung epithelial cells. However, it is unclear if these effects are co...

  19. Sphingosine 1-Phosphate (S1P) Induced Interleukin-8 (IL-8) Release Is Mediated by S1P Receptor 2 and Nuclear Factor κB in BEAS-2B Cells

    PubMed Central

    O’Sullivan, Michael J.; Hirota, Nobuaki; Martin, James G.

    2014-01-01

    The airway epithelium may release pro-inflammatory cytokines and chemokines in the asthmatic airway. Sphingosine 1-phosphate (S1P) is a bioactive lipid, increased in the airways of asthmatics, that may trigger the release of the potent neutrophil chemoattractant Interleukin-8 (IL-8) by epithelial cells. S1P is a ligand for 5 G protein-coupled receptors, S1PR1-5. We wished to explore the mechanisms of S1P induced IL-8 secretion with regard to the receptor(s) and downstream signaling events involved. Our results indicate that S1P induced IL-8 release is mediated by S1PR2 and the transcription factor NF-κB. Since the Epidermal Growth Factor Receptor (EGFR) and reactive oxygen species (ROS) have been implicated in IL-8 release in response to activation of other G protein-coupled receptors, we examined their importance in S1P induced IL-8 release and established that they are not involved. This study reveals S1PR2 and NF-κB as potential therapeutic targets in neutrophilic airway diseases such as severe asthma. PMID:24743449

  20. Luteolin inhibits Cr(VI)-induced malignant cell transformation of human lung epithelial cells by targeting ROS mediated multiple cell signaling pathways

    PubMed Central

    Pratheeshkumar, Poyil; Son, Young-Ok; Divya, Sasidharan Padmaja; Roy, Ram Vinod; Hitron, John Andrew; Wang, Lei; Kim, Donghern; Dai, Jin; Asha, Padmaja; Zhang, Zhuo; Wang, Yitao; Shi, Xianglin

    2015-01-01

    Hexavalent chromium [Cr(VI)] is a well-known human carcinogen associated with the incidence of lung cancer. Inhibition of metal induced carcinogenesis by a dietary antioxidant is a novel approach. Luteolin, a natural dietary flavonoid found in fruits and vegetables, possesses potent antioxidant and anti-inflammatory activity. We found that short term exposure of human bronchial epithelial cells (BEAS-2B) to Cr(VI) (5 μM) showed a drastic increase in ROS generation, NADPH oxidase (NOX) activation, lipid peroxidation, and glutathione depletion, which were significantly inhibited by the treatment with luteolin in a dose dependent manner. Treatment with luteolin decreased AP-1, HIF-1α, COX-2, and iNOS promoter activity induced by Cr(VI) in BEAS-2B cells. In addition, luteolin protected BEAS-2B cells from malignant transformation induced by chronic Cr(VI) exposure. Moreover, luteolin also inhibited the production of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α) and VEGF in chronic Cr(VI) exposed BEAS-2B cells. Western blot analysis showed that luteolin inhibited multiple gene products linked to survival (Akt, Fak, Bcl-2, Bcl-xL), inflammation (MAPK, NF-κB, COX-2, STAT-3, iNOS, TNF-α) and angiogenesis (HIF-1α, VEGF, MMP-9) in chronic Cr(VI) exposed BEAS-2B cells. Nude mice injected with BEAS-2B cells chronically exposed to Cr(VI) in the presence of luteolin showed reduced tumor incidence compared to Cr(VI) alone treated group. Overexpression of catalase (CAT) or SOD2, eliminated Cr(VI)-induced malignant transformation. Overall, our results indicate that luteolin protects BEAS-2B cells from Cr(VI)-induced carcinogenesis by scavenging ROS and modulating multiple cell signaling mechanisms that are linked to ROS. Luteolin, therefore, serves as a potential chemopreventive agent against Cr(VI)-induced carcinogenesis. PMID:25448439

  1. Macrophages Facilitate Coal Tar Pitch Extract-Induced Tumorigenic Transformation of Human Bronchial Epithelial Cells Mediated by NF-κB

    PubMed Central

    Feng, Feifei; Wu, Yiming; Zhang, Shaofeng; Liu, Yu; Qin, Lijuan; Wu, Yongjun; Yan, Zhen; Wu, Weidong

    2012-01-01

    Objective Chronic respiratory inflammation has been associated with lung cancer. Tumor-associated macrophages (TAMs) play a critical role in the formation of inflammation microenvironment. We sought to characterize the role of TAMs in coal tar pitch extract (CTPE)-induced tumorigenic transformation of human bronchial epithelial cells and the underlying mechanisms. Methods The expression of TAMs-specific CD68 in lung cancer tissues and paired adjacent tissues from cancer patients was determined using immunostaining. Co-culture of human bronchial epithelial cells (BEAS-2B) and macrophage-like THP-1 cells were conducted to evaluate the promotive effect of macrophages on CTPE-induced tumorigenic transformation of BEAS-2B cells. BEAS-2B cells were first treated with 2.4 µg/mL CTPE for 72 hours. After removal of CTPE, the cells were continuously cultured either with or without THP-1 cells and passaged using trypsin-EDTA. Alterations of cell cycle, karyotype, colony formation in soft agar and tumor xenograft growth in nude mice of BEAS-2B cells at passages 10, 20 and 30, indicative of tumorigenecity, were determined, respectively. In addition, mRNA and protein levels of NF-κB in BEAS-2B cells were measured with RT-PCR and western blot, respectively. B(a)P was used as the positive control. Results The over-expression of TAMs-specific CD68 around lung tumor tissues was detected and associated with lung cancer progression. The tumorigenic alterations of BEAS-2B cells including increase in cell growth rate, number of cells with aneuploidy, clonogenicity in soft agar, and tumor size in nude mice in vivo occurred at passage 10, becoming significant at passages 20 and 30 of the co-culture following CTPE removal in compared to BEAS-2B cells alone. In addition, the expression levels of NF-κB in BEAS-2B cells were positively correlated to the malignancy of BEAS-2B cells under different conditions of treatment. Conclusion The presence of macrophages facilitated CTPE

  2. GENE EXPRESSION PROFILING OF NORMAL HUMAN BRONCHIAL EPITHELIAL CELLS EXPOSED TO TRIVALENT ARSENICALS AND DIMETHYLTHIOARSINIC ACID

    EPA Science Inventory

    Lung is a major target for arsenic carcinogenesis in humans. However, the carcinogenic mode of action of arsenicals is unknown. We investigated, in human bronchial epithelial (BEAS2B) cells, the effects of inorganic arsenic (iAsIII), monomethylarsonous acid (MMAIII), dimethylarsi...

  3. Uptake and cytotoxic effects of multi-walled carbon nanotubes in human bronchial epithelial cells

    SciTech Connect

    Hirano, Seishiro; Fujitani, Yuji; Furuyama, Akiko; Kanno, Sanae

    2010-11-15

    Carbon nanotubes (CNT) are cytotoxic to several cell types. However, the mechanism of CNT toxicity has not been fully studied, and dosimetric analyses of CNT in the cell culture system are lacking. Here, we describe a novel, high throughput method to measure cellular uptake of CNT using turbimetry. BEAS-2B, a human bronchial epithelial cell line, was used to investigate cellular uptake, cytotoxicity, and inflammatory effects of multi-walled CNT (MWCNT). The cytotoxicity of MWCNT was higher than that of crocidolite asbestos in BEAS-2B cells. The IC{sub 50} of MWCNT was 12 {mu}g/ml, whereas that of asbestos (crocidolite) was 678 {mu}g/ml. Over the course of 5 to 8 h, BEAS-2B cells took up 17-18% of the MWCNT when they were added to the culture medium at a concentration of 10 {mu}g/ml. BEAS-2B cells were exposed to 2, 5, or 10 {mu}g/ml of MWCNT, and total RNA was extracted for cytokine cDNA primer array assays. The culture supernatant was collected for cytokine antibody array assays. Cytokines IL-6 and IL-8 increased in a dose dependent manner at both the mRNA and protein levels. Migration inhibitory factor (MIF) also increased in the culture supernatant in response to MWCNT. A phosphokinase array study using lysates from BEAS-2B cells exposed to MWCNT indicated that phosphorylation of p38, ERK1, and HSP27 increased significantly in response to MWCNT. Results from a reporter gene assays using the NF-{kappa}B or AP-1 promoter linked to the luciferase gene in transiently transfected CHO-KI cells revealed that NF-{kappa}B was activated following MWCNT exposure, while AP-1 was not changed. Collectively, MWCNT activated NF-{kappa}B, enhanced phosphorylation of MAP kinase pathway components, and increased production of proinflammatory cytokines in human bronchial epithelial cells.

  4. Gene expression analysis uncovers novel Hedgehog interacting protein (HHIP) effects in human bronchial epithelial cells

    PubMed Central

    Zhou, Xiaobo; Qiu, Weiliang; Sathirapongsasuti, J. Fah.; Cho, Michael H.; Mancini, John D.; Lao, Taotao; Thibault, Derek M.; Litonjua, Gus; Bakke, Per S.; Gulsvik, Amund; Lomas, David A.; Beaty, Terri H.; Hersh, Craig P.; Anderson, Christopher; Geigenmuller, Ute; Raby, Benjamin A.; Rennard, Stephen I.; Perrella, Mark A.; Choi, Augustine M.K.; Quackenbush, John; Silverman, Edwin K.

    2013-01-01

    Hedgehog Interacting Protein (HHIP) was implicated in chronic obstructive pulmonary disease (COPD) by genome-wide association studies (GWAS). However, it remains unclear how HHIP contributes to COPD pathogenesis. To identify genes regulated by HHIP, we performed gene expression microarray analysis in a human bronchial epithelial cell line (Beas-2B) stably infected with HHIP shRNAs. HHIP silencing led to differential expression of 296 genes; enrichment for variants nominally associated with COPD was found. Eighteen of the differentially expressed genes were validated by real-time PCR in Beas-2B cells. Seven of 11 validated genes tested in human COPD and control lung tissues demonstrated significant gene expression differences. Functional annotation indicated enrichment for extracellular matrix and cell growth genes. Network modeling demonstrated that the extracellular matrix and cell proliferation genes influenced by HHIP tended to be interconnected. Thus, we identified potential HHIP targets in human bronchial epithelial cells that may contribute to COPD pathogenesis. PMID:23459001

  5. Luteolin inhibits Cr(VI)-induced malignant cell transformation of human lung epithelial cells by targeting ROS mediated multiple cell signaling pathways

    SciTech Connect

    Pratheeshkumar, Poyil; Son, Young-Ok; Divya, Sasidharan Padmaja; Roy, Ram Vinod; Hitron, John Andrew; Wang, Lei; Kim, Donghern; Dai, Jin; Asha, Padmaja; Zhang, Zhuo; Wang, Yitao; Shi, Xianglin

    2014-12-01

    Hexavalent chromium [Cr(VI)] is a well-known human carcinogen associated with the incidence of lung cancer. Inhibition of metal induced carcinogenesis by a dietary antioxidant is a novel approach. Luteolin, a natural dietary flavonoid found in fruits and vegetables, possesses potent antioxidant and anti-inflammatory activity. We found that short term exposure of human bronchial epithelial cells (BEAS-2B) to Cr(VI) (5 μM) showed a drastic increase in ROS generation, NADPH oxidase (NOX) activation, lipid peroxidation, and glutathione depletion, which were significantly inhibited by the treatment with luteolin in a dose dependent manner. Treatment with luteolin decreased AP-1, HIF-1α, COX-2, and iNOS promoter activity induced by Cr(VI) in BEAS-2B cells. In addition, luteolin protected BEAS-2B cells from malignant transformation induced by chronic Cr(VI) exposure. Moreover, luteolin also inhibited the production of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α) and VEGF in chronic Cr(VI) exposed BEAS-2B cells. Western blot analysis showed that luteolin inhibited multiple gene products linked to survival (Akt, Fak, Bcl-2, Bcl-xL), inflammation (MAPK, NF-κB, COX-2, STAT-3, iNOS, TNF-α) and angiogenesis (HIF-1α, VEGF, MMP-9) in chronic Cr(VI) exposed BEAS-2B cells. Nude mice injected with BEAS-2B cells chronically exposed to Cr(VI) in the presence of luteolin showed reduced tumor incidence compared to Cr(VI) alone treated group. Overexpression of catalase (CAT) or SOD2, eliminated Cr(VI)-induced malignant transformation. Overall, our results indicate that luteolin protects BEAS-2B cells from Cr(VI)-induced carcinogenesis by scavenging ROS and modulating multiple cell signaling mechanisms that are linked to ROS. Luteolin, therefore, serves as a potential chemopreventive agent against Cr(VI)-induced carcinogenesis. - Highlights: • Luteolin inhibited Cr(VI)-induced oxidative stress. • Luteolin inhibited chronic Cr(VI)-induced malignant transformation.

  6. Evaluation of Differentiated Human Bronchial Epithelial Cell Culture Systems for Asthma Research

    PubMed Central

    Stewart, Ceri E.; Torr, Elizabeth E.; Mohd Jamili, Nur H.; Bosquillon, Cynthia; Sayers, Ian

    2012-01-01

    The aim of the current study was to evaluate primary (human bronchial epithelial cells, HBEC) and non-primary (Calu-3, BEAS-2B, BEAS-2B R1) bronchial epithelial cell culture systems as air-liquid interface- (ALI-) differentiated models for asthma research. Ability to differentiate into goblet (MUC5AC+) and ciliated (β-Tubulin IV+) cells was evaluated by confocal imaging and qPCR. Expression of tight junction/adhesion proteins (ZO-1, E-Cadherin) and development of transepithelial electrical resistance (TEER) were assessed. Primary cells showed localised MUC5AC, β-Tubulin IV, ZO-1, and E-Cadherin and developed TEER with, however, a large degree of inter- and intradonor variation. Calu-3 cells developed a more reproducible TEER and a phenotype similar to primary cells although with diffuse β-Tubulin IV staining. BEAS-2B cells did not differentiate or develop tight junctions. These data highlight the challenges in working with primary cell models and the need for careful characterisation and selection of systems to answer specific research questions. PMID:22287976

  7. Arsenic promotes centrosome abnormalities and cell colony formation in p53 compromised human lung cells

    SciTech Connect

    Liao Weiting; Lin Pinpin; Cheng, T.-S.; Yu, H.-S.; Chang, Louis W.

    2007-12-01

    Epidemiological evidence indicated that residents, especially cigarette smokers, in arseniasis areas had significantly higher lung cancer risk than those living in non-arseniasis areas. Thus, an interaction between arsenic and cigarette smoking in lung carcinogenesis was suspected. p53 dysfunction or mutation in lung epithelial cells was frequently observed in cigarette smokers. Our present study was to explore the differential effects by arsenic on H1355 cells (human lung adenocarcinoma cell line with mutation in p53), BEAS-2B (immortalized lung epithelial cell with functional p53) and pifithrin-{alpha}-treated BEAS-2B cells (p53-inhibited cells). These cells were treated with different doses of sodium arsenite (0, 0.1, 1, 5 and 10 {mu}M) for 48 h. A greater reduction in cell viability was observed in the BEAS-2B cells vs. p53 compromised cells (H1355 or p53-inhibited BEAS-2B). Similar observation was also made on 7-day cell survival (growth) study. TUNEL analysis confirmed that there was indeed a significantly reduced arsenite-induced apoptosis found in p53-compromised cells. Centrosomal abnormality has been attributed to eventual chromosomal missegregation, aneuploidy and tumorigenesis. In our present study, reduced p21 and Gadd45a expressions and increased centrosomal abnormality (atopic and multiple centrosomes) were observed in both arsenite-treated H1355 and p53-inhibited BEAS-2B cells as compared with similarly treated BEAS-2B cells. Increased anchorage-independent growth (colony formation) of BEAS-2B cells co-treated with pifithrin-{alpha} and 5 {mu}M sodium arsenite was also observed in soft agar. Our present investigation demonstrated that arsenic would act specifically on p53 compromised cells (either with p53 dysfunction or inhibited) to induce centrosomal abnormality and colony formation. These findings provided strong evidence on the carcinogenic promotional role of arsenic, especially under the condition of p53 dysfunction.

  8. Identification of gene biomarkers for respiratory synctial virus infection in a bronchical epithelial cell line

    EPA Science Inventory

    Abstract: Respiratory syncytial virus (RSV) infection involves complex virus-host interplay. In this study, we analyzed gene expression in RSV-infected BEAS-2B cells to discover novel signaling pathways and biomarkers. We hybridized RNAs from RSV- or vehicle-treated BEAS-2B to ...

  9. Clara Cell 10-kDa Protein Gene Transfection Inhibits NF-κB Activity in Airway Epithelial Cells

    PubMed Central

    Long, Xiao-Bo; Hu, Shuang; Wang, Nan; Zhen, Hong-Tao; Cui, Yong-Hua; Liu, Zheng

    2012-01-01

    Background Clara cell 10-kDa protein (CC10) is a multifunctional protein with anti-inflammatory and immunomodulatory effects. Induction of CC10 expression by gene transfection may possess potential therapeutic effect. Nuclear factor κB (NF-κB) plays a key role in the inflammatory processes of airway diseases. Method/Results To investigate potential therapeutic effect of CC10 gene transfection in controlling airway inflammation and the underlying intracellular mechanisms, in this study, we constructed CC10 plasmid and transfected it into bronchial epithelial cell line BEAS-2B cells and CC10 knockout mice. In BEAS-2B cells, CC10's effect on interleukin (IL)-1β induced IL-8 expression was explored by means of RT-PCR and ELISA and its effect on NF-κB classical signaling pathway was studied by luciferase reporter, western blot, and immunoprecipitation assay. The effect of endogenous CC10 on IL-1β evoked IL-8 expression was studied by means of nasal explant culture. In mice, CC10's effect on IL-1β induced IL-8 and nuclear p65 expression was examined by immunohistochemistry. First, we found that the CC10 gene transfer could inhibit IL-1β induced IL-8 expression in BEAS-2B cells. Furthermore, we found that CC10 repressed IL-1β induced NF-κB activation by inhibiting the phosphorylation of IκB-α but not IκB kinase-α/β in BEAS-2B cells. Nevertheless, we did not observe a direct interaction between CC10 and p65 subunit in BEAS-2B cells. In nasal explant culture, we found that IL-1β induced IL-8 expression was inversely correlated with CC10 levels in human sinonasal mucosa. In vivo study revealed that CC10 gene transfer could attenuate the increase of IL-8 and nuclear p65 staining in nasal epithelial cells in CC10 knockout mice evoked by IL-1β administration. Conclusion These results indicate that CC10 gene transfer may inhibit airway inflammation through suppressing the activation of NF-κB, which may provide us a new consideration in the therapy of airway

  10. Leptin enhances ICAM-1 expression, induces migration and cytokine synthesis, and prolongs survival of human airway epithelial cells.

    PubMed

    Suzukawa, Maho; Koketsu, Rikiya; Baba, Shintaro; Igarashi, Sayaka; Nagase, Hiroyuki; Yamaguchi, Masao; Matsutani, Noriyuki; Kawamura, Masafumi; Shoji, Shunsuke; Hebisawa, Akira; Ohta, Ken

    2015-10-15

    There is rising interest in how obesity affects respiratory diseases, since epidemiological findings indicate a strong relationship between the two conditions. Leptin is a potent adipokine produced mainly by adipocytes. It regulates energy storage and expenditure and also induces inflammation. Previous studies have shown that leptin is able to activate inflammatory cells such as lymphocytes and granulocytes, but little is known about its effect on lung structural cells. The present study investigated the effects of leptin on human airway epithelial cells by using human primary airway epithelial cells and a human airway epithelial cell line, BEAS-2B. Flow cytometry showed enhanced ICAM-1 expression by both of those cells in response to leptin, and that effect was abrogated by dexamethasone or NF-κB inhibitor. Flow cytometry and quantitative PCR showed that airway epithelial cells expressed leptin receptor (Ob-R), whose expression level was downregulated by leptin itself. Multiplex cytokine analysis demonstrated enhanced production of CCL11, G-CSF, VEGF, and IL-6 by BEAS-2B cells stimulated with leptin. Furthermore, transfection of Ob-R small interference RNA decreased the effect of leptin on CCL11 production as assessed by quantitative PCR. Finally, leptin induced migration of primary airway epithelial cells toward leptin, suppressed BEAS-2B apoptosis induced with TNF-α and IFN-γ, and enhanced proliferation of primary airway epithelial cells. In summary, leptin was able to directly activate human airway epithelial cells by binding to Ob-R and by NF-κB activation, resulting in upregulation of ICAM-1 expression, induction of CCL11, VEGF, G-CSF, and IL-6 synthesis, induction of migration, inhibition of apoptosis, and enhancement of proliferation. PMID:26276826

  11. Pro-inflammatory response and oxidative stress induced by specific components in ambient particulate matter in human bronchial epithelial cells.

    PubMed

    Yang, Lawei; Liu, Gang; Lin, Ziying; Wang, Yahong; He, Huijuan; Liu, Tie; Kamp, David W

    2016-08-01

    Previous studies have shown that biological effect of particulate matter (PM2.5) is involved in including chemical composition and mass concentration, but the precise components and biological action on human bronchial epithelial cell line (BEAS-2B) are still unclear. The aim of this study was to evaluate the in vitro toxicity of PM2.5 collected at six urban sites in China, and to investigate how particle composition affects cytotoxicity. We used human bronchial epithelial (BEAS-2B) cell lines as model in vitro to expose to PM2.5 from different source, and then reactive oxygen species (ROS), superoxide dismutase activity and total antioxidant capacity were analyzed. Furthermore, we estimated the polycyclic aromatic hydrocarbon (PAH) and transition metal and the endotoxin contents. The mRNA expression of IL-1β and IL-10 following exposure to PM2.5 was measured by QRT-PCR. We also observed the mitochondrial membrane potential (MMP) using JC-1 staining, and apoptosis of BEAS-2B using flow cytometry. In addition, double-stranded DNA breaks (DSBs) were assessed using γ-H2AX immunofluorescence. Our results show that high concentrations of PAHs and elemental Ni were strongly associated with high apoptosis rates and high expression of IL-1β, in addition, Fe element was associated with the ROS level, furthermore, Fe and Cr element were associated with DNA damage in BEAS-2B cells. The cytotoxic effects of urban PM2.5 derived from six different cities in China appear dependent on the specific components in each. Our results indicate that air quality standards based on PM2.5 components may be more relevant than concentration-response functions (CRF). © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 923-936, 2016. PMID:25533354

  12. Cigarette Smoke Extract Induces a Phenotypic Shift in Epithelial Cells; Involvement of HIF1α in Mesenchymal Transition

    PubMed Central

    Eurlings, Irene M. J.; Reynaert, Niki L.; van den Beucken, Twan; Gosker, Harry R.; de Theije, C. C.; Verhamme, Fien M.; Bracke, Ken R.; Wouters, Emiel F. M.; Dentener, Mieke A.

    2014-01-01

    In COPD, matrix remodeling contributes to airflow limitation. Recent evidence suggests that next to fibroblasts, the process of epithelial-mesenchymal transition can contribute to matrix remodeling. CSE has been shown to induce EMT in lung epithelial cells, but the signaling mechanisms involved are largely unknown and subject of this study. EMT was assessed in A549 and BEAS2B cells stimulated with CSE by qPCR, Western blotting and immunofluorescence for epithelial and mesenchymal markers, as were collagen production, cell adhesion and barrier integrity as functional endpoints. Involvement of TGF-β and HIF1α signaling pathways were investigated. In addition, mouse models were used to examine the effects of CS on hypoxia signaling and of hypoxia per se on mesenchymal expression. CSE induced EMT characteristics in A549 and BEAS2B cells, evidenced by decreased expression of epithelial markers and a concomitant increase in mesenchymal marker expression after CSE exposure. Furthermore cells that underwent EMT showed increased production of collagen, decreased adhesion and disrupted barrier integrity. The induction of EMT was found to be independent of TGF-β signaling. On the contrary, CS was able to induce hypoxic signaling in A549 and BEAS2B cells as well as in mice lung tissue. Importantly, HIF1α knock-down prevented induction of mesenchymal markers, increased collagen production and decreased adhesion after CSE exposure, data that are in line with the observed induction of mesenchymal marker expression by hypoxia in vitro and in vivo. Together these data provide evidence that both bronchial and alveolar epithelial cells undergo a functional phenotypic shift in response to CSE exposure which can contribute to increased collagen deposition in COPD lungs. Moreover, HIF1α signaling appears to play an important role in this process. PMID:25329389

  13. Radical-containing ultrafine particulate matter initiates epithelial-to-mesenchymal transitions in airway epithelial cells.

    PubMed

    Thevenot, Paul T; Saravia, Jordy; Jin, Nili; Giaimo, Joseph D; Chustz, Regina E; Mahne, Sarah; Kelley, Matthew A; Hebert, Valeria Y; Dellinger, Barry; Dugas, Tammy R; Demayo, Francesco J; Cormier, Stephania A

    2013-02-01

    Environmentally persistent free radicals (EPFRs) in combustion-generated particulate matter (PM) are capable of inducing pulmonary pathologies and contributing to the development of environmental asthma. In vivo exposure of infant rats to EPFRs demonstrates their ability to induce airway hyperresponsiveness to methacholine, a hallmark of asthma. However, the mechanisms by which combustion-derived EPFRs elicit in vivo responses remain elusive. In this study, we used a chemically defined EPFR consisting of approximately 0.2 μm amorphrous silica containing 3% cupric oxide with the organic pollutant 1,2-dichlorobenzene (DCB-230). DCB-230 possesses similar radical content to urban-collected EPFRs but offers several advantages, including lack of contaminants and chemical uniformity. DCB-230 was readily taken up by BEAS-2B and at high doses (200 μg/cm(2)) caused substantial necrosis. At low doses (20 μg/cm(2)), DCB-230 particles caused lysosomal membrane permeabilization, oxidative stress, and lipid peroxidation within 24 hours of exposure. During this period, BEAS-2B underwent epithelial-to-mesenchymal transition (EMT), including loss of epithelial cell morphology, decreased E-cadherin expression, and increased α-smooth muscle actin (α-SMA) and collagen I production. Similar results were observed in neonatal air-liquid interface culture (i.e., disruption of epithelial integrity and EMT). Acute exposure of infant mice to DCB-230 resulted in EMT, as confirmed by lineage tracing studies and evidenced by coexpression of epithelial E-cadherin and mesenchymal α-SMA proteins in airway cells and increased SNAI1 expression in the lungs. EMT in neonatal mouse lungs after EPFR exposure may provide an explanation for epidemiological evidence supporting PM exposure and increased risk of asthma. PMID:23087054

  14. Cross-Talk between Human Mast Cells and Bronchial Epithelial Cells in Plasminogen Activator Inhibitor-1 Production via Transforming Growth Factor-β1

    PubMed Central

    Lee, Sun H.; Kato, Atsushi; Takabayashi, Tetsuji; Kulka, Marianna; Shin, Soon C.; Schleimer, Robert P.

    2015-01-01

    Previous reports suggest that plasminogen activator inhibitor-1 (PAI-1) promotes airway remodeling and that human and mouse mast cells (MCs) are an important source of PAI-1. In the present study we investigated MC–epithelial cell (EC) interactions in the production of PAI-1. We stimulated the human MC line LAD2 with IgE-receptor cross-linking and collected the supernatants. We incubated the human bronchial EC line BEAS-2B with the LAD2 supernatants and measured the level of PAI-1. When the supernatants from IgE-stimulated LAD2 were added to BEAS-2B, there was a significant enhancement of PAI-1 production by BEAS-2B. When we treated the MC supernatants with a transforming growth factor (TGF)-β1 neutralizing antibody, the MC-derived induction of PAI-1 from BEAS-2B was completely abrogated. Although TGF-β1 mRNA was constitutively expressed in resting LAD2, it was not highly induced by IgE-mediated stimulation. Nonetheless, active TGF-β1 protein was significantly increased in LAD2 after IgE-mediated stimulation. Active TGF-β1 produced by primary cultured human MCs was significantly reduced in the presence of a chymase inhibitor, suggesting a role of MC chymase as an activator of latent TGF-β1. This study indicates that stimulation of human MCs by IgE receptor cross-linking triggers activation of TGF-β1, at least in part via chymase, which in turn induces the production of PAI-1 by bronchial ECs. Our data suggest that human MCs may play an important role in airway remodeling in asthma as a direct source of PAI-1 and by activating bronchial ECs to produce further PAI-1 via a TGF-β1–mediated activation pathway. PMID:24987792

  15. Cross-talk between human mast cells and bronchial epithelial cells in plasminogen activator inhibitor-1 production via transforming growth factor-β1.

    PubMed

    Cho, Seong H; Lee, Sun H; Kato, Atsushi; Takabayashi, Tetsuji; Kulka, Marianna; Shin, Soon C; Schleimer, Robert P

    2015-01-01

    Previous reports suggest that plasminogen activator inhibitor-1 (PAI-1) promotes airway remodeling and that human and mouse mast cells (MCs) are an important source of PAI-1. In the present study we investigated MC-epithelial cell (EC) interactions in the production of PAI-1. We stimulated the human MC line LAD2 with IgE-receptor cross-linking and collected the supernatants. We incubated the human bronchial EC line BEAS-2B with the LAD2 supernatants and measured the level of PAI-1. When the supernatants from IgE-stimulated LAD2 were added to BEAS-2B, there was a significant enhancement of PAI-1 production by BEAS-2B. When we treated the MC supernatants with a transforming growth factor (TGF)-β1 neutralizing antibody, the MC-derived induction of PAI-1 from BEAS-2B was completely abrogated. Although TGF-β1 mRNA was constitutively expressed in resting LAD2, it was not highly induced by IgE-mediated stimulation. Nonetheless, active TGF-β1 protein was significantly increased in LAD2 after IgE-mediated stimulation. Active TGF-β1 produced by primary cultured human MCs was significantly reduced in the presence of a chymase inhibitor, suggesting a role of MC chymase as an activator of latent TGF-β1. This study indicates that stimulation of human MCs by IgE receptor cross-linking triggers activation of TGF-β1, at least in part via chymase, which in turn induces the production of PAI-1 by bronchial ECs. Our data suggest that human MCs may play an important role in airway remodeling in asthma as a direct source of PAI-1 and by activating bronchial ECs to produce further PAI-1 via a TGF-β1-mediated activation pathway. PMID:24987792

  16. Gene 33/Mig6 inhibits hexavalent chromium-induced DNA damage and cell transformation in human lung epithelial cells

    PubMed Central

    Park, Soyoung; Li, Cen; Zhao, Hong; Darzynkiewicz, Zbigniew; Xu, Dazhong

    2016-01-01

    Hexavalent Chromium [Cr(VI)] compounds are human lung carcinogens and environmental/occupational hazards. The molecular mechanisms of Cr(VI) carcinogenesis appear to be complex and are poorly defined. In this study, we investigated the potential role of Gene 33 (ERRFI1, Mig6), a multifunctional adaptor protein, in Cr(VI)-mediated lung carcinogenesis. We show that the level of Gene 33 protein is suppressed by both acute and chronic Cr(VI) treatments in a dose- and time-dependent fashion in BEAS-2B lung epithelial cells. The inhibition also occurs in A549 lung bronchial carcinoma cells. Cr(VI) suppresses Gene 33 expression mainly through post-transcriptional mechanisms, although the mRNA level of gene 33 also tends to be lower upon Cr(VI) treatments. Cr(VI)-induced DNA damage appears primarily in the S phases of the cell cycle despite the high basal DNA damage signals at the G2M phase. Knockdown of Gene 33 with siRNA significantly elevates Cr(VI)-induced DNA damage in both BEAS-2B and A549 cells. Depletion of Gene 33 also promotes Cr(VI)-induced micronucleus (MN) formation and cell transformation in BEAS-2B cells. Our results reveal a novel function of Gene 33 in Cr(VI)-induced DNA damage and lung epithelial cell transformation. We propose that in addition to its role in the canonical EGFR signaling pathway and other signaling pathways, Gene 33 may also inhibit Cr(VI)-induced lung carcinogenesis by reducing DNA damage triggered by Cr(VI). PMID:26760771

  17. Malignant human cell transformation of Marcellus Shale gas drilling flow back water.

    PubMed

    Yao, Yixin; Chen, Tingting; Shen, Steven S; Niu, Yingmei; DesMarais, Thomas L; Linn, Reka; Saunders, Eric; Fan, Zhihua; Lioy, Paul; Kluz, Thomas; Chen, Lung-Chi; Wu, Zhuangchun; Costa, Max; Zelikoff, Judith

    2015-10-01

    The rapid development of high-volume horizontal hydraulic fracturing for mining natural gas from shale has posed potential impacts on human health and biodiversity. The produced flow back waters after hydraulic stimulation are known to carry high levels of saline and total dissolved solids. To understand the toxicity and potential carcinogenic effects of these wastewaters, flow back waters from five Marcellus hydraulic fracturing oil and gas wells were analyzed. The physicochemical nature of these samples was analyzed by inductively coupled plasma mass spectrometry and scanning electron microscopy/energy dispersive X-ray spectroscopy. A cytotoxicity study using colony formation as the endpoint was carried out to define the LC50 values of test samples using human bronchial epithelial cells (BEAS-2B). The BEAS-2B cell transformation assay was employed to assess the carcinogenic potential of the samples. Barium and strontium were among the most abundant metals in these samples and the same metals were found to be elevated in BEAS-2B cells after long-term treatment. BEAS-2B cells treated for 6weeks with flow back waters produced colony formation in soft agar that was concentration dependent. In addition, flow back water-transformed BEAS-2B cells show better migration capability when compared to control cells. This study provides information needed to assess the potential health impact of post-hydraulic fracturing flow back waters from Marcellus Shale natural gas mining. PMID:26210350

  18. Regulation of cyclooxygenase-2 expression by cAMP response element and mRNA stability in a human airway epithelial cell line exposed to zinc

    SciTech Connect

    Wu Weidong Silbajoris, Robert A.; Cao Dongsun; Bromberg, Philip A.; Zhang Qiao; Peden, David B.; Samet, James M.

    2008-09-01

    Exposure to zinc-laden particulate matter in ambient and occupational settings has been associated with proinflammatory responses in the lung. Cyclooxygenase 2-derived eicosanoids are important modulators of airway inflammation. In this study, we characterized the transcriptional and posttranscriptional events that regulate COX-2 expression in a human bronchial epithelial cell line BEAS-2B exposed to Zn{sup 2+}. Zn{sup 2+} exposure resulted in pronounced increases in COX-2 mRNA and protein expression, which were prevented by pretreatment with the transcription inhibitor actinomycin D, implying the involvement of transcriptional regulation. This was supported by the observation of increased COX-2 promoter activity in Zn{sup 2+}-treated BEAS-2B cells. Mutation of the cAMP response element (CRE), but not the {kappa}B-binding sites in the COX-2 promoter markedly reduced COX-2 promoter activity induced by Zn{sup 2+}. Inhibition of NF{kappa}B activation did not block Zn{sup 2+}-induced COX-2 expression. Measurement of mRNA stability demonstrated that Zn{sup 2+} exposure impaired the degradation of COX-2 mRNA in BEAS-2B cells. This message stabilization effect of Zn{sup 2+} exposure was shown to be dependent on the integrity of the 3'-untranslated region found in the COX-2 transcript. Taken together, these data demonstrate that the CRE and mRNA stability regulates COX-2 expression induced in BEAS-2B cells exposed to extracellular Zn{sup 2+}.

  19. Haemophilus influenzae increases the susceptibility and inflammatory response of airway epithelial cells to viral infections.

    PubMed

    Gulraiz, Fahad; Bellinghausen, Carla; Bruggeman, Cathrien A; Stassen, Frank R

    2015-03-01

    Nontypeable Haemophilus influenzae (NTHI), a common colonizer of lungs of patients with chronic obstructive pulmonary disease (COPD), can enhance expression of the cellular receptor intercellular adhesion molecule 1 (ICAM-1), which in turn can be used by major group human rhinoviruses (HRVs) for attachment. Here, we evaluated the effect of NTHI-induced up-regulation of ICAM-1 on viral replication and inflammatory responses toward different respiratory viruses. Therefore, human bronchial epithelial cells were pretreated with heat-inactivated NTHI (hi-NTHI) and subsequently infected with either HRV16 (major group), HRV1B (minor group), or respiratory syncytial virus (RSV). Pretreatment with hi-NTHI significantly up-regulated ICAM-1 in BEAS-2B cells and primary bronchial epithelial cells. Concomitantly, release of infectious HRV16 particles was increased in cells pretreated with hi-NTHI. Pretreatment with hi-NTHI also caused a significant increase in HRV16 RNA, whereas replication of HRV1B and RSV were increased to a far lesser extent and only at later time points. Interestingly, release of IL-6 and IL-8 after RSV, but not HRV, infection was synergistically increased in hi-NTHI-pretreated BEAS-2B cells. In summary, exposure to hi-NTHI significantly enhanced sensitivity toward HRV16 but not HRV1B or RSV, probably through ICAM-1 up-regulation. Furthermore, hi-NTHI pretreatment may enhance the inflammatory response to RSV infection, suggesting that preexisting bacterial infections might exaggerate inflammation during secondary viral infection. PMID:25411435

  20. Genomic-wide analysis of BEAS-2B cells exposed to Trivalent Arsenicals and Dimethylthioarsinic acid

    EPA Science Inventory

    Lung is a major target for arsenic carcinogenesis in humans by both oral and inhalation routes. However, the carcinogenic mode of action of arsenicals is unknown. We investigated the effects of inorganic arsenic (iAsIII), monomethylarsonous acid (MMAIII), dimethylarsinous acid (D...

  1. OXIDANT GENERATION PROMOTES IRON SEQUESTRATION IN BEAS-2B CELLS EXPOSED TO ASBESTOS

    EPA Science Inventory

    Lung injury following asbestos exposure is associated with an oxidative stress that is catalyzed by iron in the fiber matrix, complexed to the surface, or both. We tested the hypothesis that the cellular response to asbestos includes the transport and sequestration of this iron ...

  2. *OXIDANT GENERATION PROMOTES IRON SEQUESTRATION IN BEAS-2B CELLS EXPOSED TO ASBESTOS

    EPA Science Inventory

    Lung injury after asbestos exposure is associated with an oxidative stress that is catalyzed by iron in the fiber matrix, complexed to the surface, or both. We tested the hypothesis that the cellular response to asbestos includes the transport and sequestration of this iron throu...

  3. OXIDANTT GENERATION PROMOTES IRON SEQUESTRATION IN BEAS-2B CELLS EXPOSED TO ASBESTOS

    EPA Science Inventory

    Lung injury following asbestos exposure is associated with an oxidative stress that is catalyzed by iron in the matrix, complexed to the surface, or both. We hypothesized that the cellular response to asbestos includes the transport and sequestration of iron by 1) generation of s...

  4. Nickel oxide nanoparticles induce inflammation and genotoxic effect in lung epithelial cells.

    PubMed

    Capasso, Laura; Camatini, Marina; Gualtieri, Maurizio

    2014-04-01

    Nickel oxide nanoparticles (NiONPs) toxicity has been evaluated in the human pulmonary epithelial cell lines: BEAS-2B and A549. The nanoparticles, used at the doses of 20, 40, 60, 80, 100 μg/ml, induced a significant reduction of cell viability and an increase of apoptotic and necrotic cells at 24h. A significant release of interleukin-6 and -8 was assessed after 24h of treatment, even intracellular ROS increased already at 45 min after exposure. The results obtained evidenced that the cytokines release was dependent on mitogen activated protein kinases (MAPK) cascade through the induction of NF-kB pathway. NiONPs induced cell cycle alteration in both the cell lines even in different phases and these modifications may be induced by the NPs genotoxic effect, suggested by the nuclear translocation of phospho-ATM and phospho-ATR. Our results confirm the cytotoxic and pro-inflammatory potential of NiONPs. Moreover their ability in inducing DNA damage responses has been demonstrated. Such effects were present in A549 cells which internalize the NPs and BEAS-2B cells in which endocytosis has not been observed. PMID:24503009

  5. Capsaicinoids Cause Inflammation and Epithelial Cell Death through Activation of Vanilloid Receptors

    PubMed Central

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

    2008-01-01

    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

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

    PubMed

    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. Penta- and octa-bromodiphenyl ethers promote proinflammatory protein expression in human bronchial epithelial cells in vitro.

    PubMed

    Koike, Eiko; Yanagisawa, Rie; Takigami, Hidetaka; Takano, Hirohisa

    2014-03-01

    Polybrominated diphenyl ethers (PBDEs) are widely used as flame retardants in consumer products. Humans can be exposed to PBDEs mainly through the inhalation of air or dust. Thus, PBDEs can affect respiratory and immune systems. In the present study, we investigated whether PBDEs stimulate bronchial epithelial cells. We examined commercial penta-BDE (DE-71), octa-BDE (DE-79), and deca-BDE (DE-83R). Human bronchial epithelial cells (BEAS-2B) were exposed to each PBDE for 24h. Subsequently, the expression of intercellular adhesion molecule-1 (ICAM-1) and proinflammatory cytokines were investigated. DE-71 and DE-79, but not DE-83R, significantly increased the expression of ICAM-1, interleukin-6 (IL-6), and IL-8 in BEAS-2B. Because these remarkable effects were observed with DE-71, we further investigated the underlying intracellular mechanisms. DE-71 promoted epidermal growth factor receptor (EGFR) phosphorylation. Inhibitors of EGFR-selective tyrosine kinase and p38 mitogen-activated protein kinase effectively blocked the increase of IL-6 and IL-8. Furthermore, antagonists of thyroid hormone receptor and aryl hydrocarbon receptor significantly suppressed the increase in IL-6 and/or IL-8 production. In conclusion, penta- and octa-BDE, but not deca-BDE, might promote the expression of proinflammatory proteins in bronchial epithelial cells possibly by activating protein kinases and/or stimulating nuclear receptors related to subsequent activation of transcriptional factors. PMID:24184330

  8. A Cross-Study Biomarker Signature of Human Bronchial Epithelial Cells Infected with Respiratory Syncytial Virus

    PubMed Central

    Gardinassi, Luiz Gustavo

    2016-01-01

    Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in children, elderly, and immunocompromised individuals. Despite of advances in diagnosis and treatment, biomarkers of RSV infection are still unclear. To understand the host response and propose signatures of RSV infection, previous studies evaluated the transcriptional profile of the human bronchial epithelial cell line—BEAS-2B—infected with different strains of this virus. However, the evolution of statistical methods and functional analysis together with the large amount of expression data provide opportunities to uncover novel biomarkers of inflammation and infections. In view of those facts publicly available microarray datasets from RSV-infected BEAS-2B cells were analyzed with linear model-based statistics and the platform for functional analysis InnateDB. The results from those analyses argue for the reevaluation of previously reported transcription patterns and biological pathways in BEAS-2B cell lines infected with RSV. Importantly, this study revealed a biosignature constituted by genes such as ABCC4, ARMC8, BCLAF1, EZH1, FAM118A, FAM208B, FUS, HSPH1, KAZN, MAP3K2, N6AMT1, PRMT2, S100PBP, SERPINA1, TLK2, ZNF322, and ZNF337 which should be considered in the development of new molecular diagnosis tools. PMID:27274726

  9. Ex Vivo Chemical Cytometric Analysis of Protein Tyrosine Phosphatase Activity in Single Human Airway Epithelial Cells

    PubMed Central

    Phillips, Ryan M.; Dailey, Lisa A.; Bair, Eric; Samet, James M.; Allbritton, Nancy L.

    2014-01-01

    We describe a novel method for the measurement of protein tyrosine phosphatase (PTP) activity in single human airway epithelial cells (hAECs) using capillary electrophoresis. This technique involved the microinjection of a fluorescent phosphopeptide that is hydrolyzed specifically by PTPs. Analyses in BEAS-2B immortalized bronchial epithelial cells showed rapid PTP-mediated dephosphorylation of the substrate (2.2 pmol min−1 mg−1) that was blocked by pretreatment of the cells with the PTP inhibitors pervanadate, Zn2+, and 1,2-naphthoquinone (76%, 69%, 100% inhibition relative to PTP activity in untreated controls, respectively). These studies were then extended to a more physiologically relevant model system: primary hAECs cultured from bronchial brushings of living human subjects. In primary hAECs, dephosphorylation of the substrate occurred at a rate of 2.2 pmol min−1 mg−1, and was also effectively inhibited by pre-incubation of the cells with the inhibitors pervanadate, Zn2+, and 1,2- naphthoquinone (91%, 88%, and 87% median PTP inhibition, respectively). Reporter proteolysis in single BEAS-2B cells occurred at a median rate of 43 fmol min−1 mg−1 resulting in a mean half-life of 20 min. The reporter displayed a similar median half-life of 28 min in these single primary cells. Finally, single viable epithelial cells (which were assayed for PTP activity immediately after collection by bronchial brushing of a human volunteer) showed dephosphorylation rates ranging from 0.34–36 pmol min−1 mg−1 (n = 6). These results demonstrate the utility and applicability of this technique for the ex vivo quantification of PTP activity in small, heterogeneous, human cells and tissues. PMID:24380370

  10. Interleukin-17A and Toll-Like Receptor 3 Ligand Poly(I:C) Synergistically Induced Neutrophil Chemoattractant Production by Bronchial Epithelial Cells

    PubMed Central

    Matsuzaki, Hirotaka; Mikami, Yu; Makita, Kousuke; Takeshima, Hideyuki; Horie, Masafumi; Noguchi, Satoshi; Jo, Taisuke; Narumoto, Osamu; Kohyama, Tadashi; Takizawa, Hajime; Nagase, Takahide; Yamauchi, Yasuhiro

    2015-01-01

    Chronic inflammatory airway diseases, such as bronchial asthma and chronic obstructive pulmonary disease, are common respiratory disorders worldwide. Exacerbations of these diseases are frequent and worsen patients’ respiratory condition and overall health. However, the mechanisms of exacerbation have not been fully elucidated. Recently, it was reported that interleukin (IL)-17A might play an important role in neutrophilic inflammation, which is characteristic of such exacerbations, through increased production of neutrophil chemoattractants. Therefore, we hypothesized that IL-17A was involved in the pathogenesis of acute exacerbation, due to viral infection in chronic inflammatory airway diseases. In this study, we assessed chemokine production by bronchial epithelial cells and investigated the underlying mechanisms. Comprehensive chemokine analysis showed that, compared with poly(I:C) alone, co-stimulation of BEAS-2B cells with IL-17A and poly(I:C) strongly induced production of such neutrophil chemoattractants as CXC chemokine ligand (CXCL)8, growth-related oncogene (GRO), and CXCL1. Co-stimulation synergistically induced CXCL8 and CXCL1 mRNA and protein production by BEAS-2B cells and normal human bronchial epithelial cells. Poly(I:C) induced chemokine expression by BEAS-2B cells mainly via Toll-like receptor 3/TIR-domain-containing adapter-inducing interferon-β–mediated signals. The co-stimulation with IL-17A and poly(I:C) markedly activated the p38 and extracellular-signal-regulated kinase 1/2 pathway, compared with poly(I:C), although there was little change in nuclear factor-κB translocation into the nucleus or the transcriptional activities of nuclear factor-κB and activator protein 1. IL-17A promoted stabilization of CXCL8 mRNA in BEAS-2B cells treated with poly(I:C). In conclusion, IL-17A appears to be involved in the pathogenesis of chronic inflammatory airway disease exacerbation, due to viral infection by promoting release of neutrophil

  11. Cadmium induces cytotoxicity in human bronchial epithelial cells through upregulation of eIF5A1 and NF-kappaB

    SciTech Connect

    Chen, De-Ju; Xu, Yan-Ming; Du, Ji-Ying; Huang, Dong-Yang; Lau, Andy T.Y.

    2014-02-28

    Highlights: • Normal human bronchial epithelial cells (BEAS-2B) were dosed with cadmium (Cd). • A low level (2 μM) of Cd treatment for 36 h elicited negligible cytotoxicity. • High levels (20 or 30 μM) of Cd treatment for 36 h induced cell death. • High levels of Cd can upregulate the protein levels of eIF5A1 and NF-κB p65. • We suggest that eIF5A1 level is possibly modulated by NF-κB. - Abstract: Cadmium (Cd) and Cd compounds are widely-distributed in the environment and well-known carcinogens. Here, we report that in CdCl{sub 2}-exposed human bronchial epithelial cells (BEAS-2B), the level of p53 is dramatically decreased in a time- and dose-dependent manner, suggesting that the observed Cd-induced cytotoxicity is not likely due to the pro-apoptotic function of p53. Therefore, this prompted us to further study the responsive pro-apoptotic factors by proteomic approaches. Interestingly, we identified that high levels (20 or 30 μM) of Cd can significantly upregulate the protein levels of eukaryotic translation initiation factor 5A1 (eIF5A1) and redox-sensitive transcription factor NF-κB p65. Moreover, there is an enhanced NF-κB nuclear translocation as well as chromatin-binding in Cd-treated BEAS-2B cells. We also show that small interfering RNA-specific knockdown of eIF5A1 in Cd-exposed cells attenuated the Cd cytotoxicity, indicating the potential role of eIF5A1 in Cd cytotoxicity. As eIF5A1 is reported to be related with cell apoptosis but little is known about its transcriptional control, we hypothesize that NF-κB might likely modulate eIF5A1 gene expression. Notably, by bioinformatic analysis, several potential NF-κB binding sites on the upstream promoter region of eIF5A1 gene can be found. Subsequent chromatin immunoprecipitation assay revealed that indeed there is enhanced NF-κB binding on eIF5A1 promoter region of Cd-treated BEAS-2B cells. Taken together, our findings suggest for the first time a regulatory mechanism for the pro

  12. Senescent bronchial fibroblasts induced to senescence by Cr(VI) promote epithelial-mesenchymal transition when co-cultured with bronchial epithelial cells in the presence of Cr(VI).

    PubMed

    Val, Mariana Monteiro; Mendes, Luís André; Alarcão, Ana; Carvalho, Lina; Carreira, Isabel; Rodrigues, Carlos Fernando D; Alpoim, Maria Carmen

    2015-03-01

    Cellular senescence is a physiological process that serves as a powerful barrier for tumorigenesis. However, senescent cells can be deleterious for the tissue microenvironment. Such is the case of senescent fibroblasts that release several pro-tumorigenic factors that promote malignant transformation in the nearby epithelial cells. Occupational exposure to hexavalent chromium [Cr(VI)] compounds is a cause of respiratory cancers. Although Cr(VI) is known to induce senescence in human foreskin fibroblasts, the role of senescent fibroblasts in the Cr(VI)-induced malignant transformation of human bronchial epithelial cells was never assessed. Thus, to study the evolutionary dynamics generated by the interaction between human bronchial epithelial cells and senescent bronchial fibroblasts, the non-tumorigenic human bronchial epithelial BEAS-2B cells were co-cultured with Cr(VI)-induced senescent human bronchial fibroblasts for 4 weeks. Under the pressure of 0.5 µM Cr(VI), senescent fibroblasts promoted the acquisition of mesenchymal features on BEAS-2B cells, e.g. the fusiform shape and increased Vimentin expression, consistent with the occurrence of an epithelial-mesenchymal transition-like process. Features of transformed cells including larger nuclei, as well as nuclei with heterogeneous size, were also observed. Altogether the results obtained demonstrate that besides acting over the epithelium, Cr(VI) also affects bronchial fibroblasts driving them senescent. As a consequence, a paracrine communication loop is established with the above-placed epithelium prompting the epithelial cells for malignant transformation and thus facilitating the initial steps of tumorigenesis. PMID:25406472

  13. Nicotine exposure induces bronchial epithelial cell apoptosis and senescence via ROS mediated autophagy-impairment.

    PubMed

    Bodas, Manish; Van Westphal, Colin; Carpenter-Thompson, Rhett; K Mohanty, Dillip; Vij, Neeraj

    2016-08-01

    Waterpipe smoking and e-cigarette vaping, the non-combustible sources of inhaled nicotine exposure are increasingly becoming popular and marketed as safer alternative to cigarette smoking. Hence, this study was designed to investigate the impact of inhaled nicotine exposure on disease causing COPD-emphysema mechanisms. For in vitro studies, human bronchial epithelial cells (Beas2b) were treated with waterpipe smoke extract (WPSE, 5%), nicotine (5mM), and/or cysteamine (250μM, an autophagy inducer and anti-oxidant drug), for 6hrs. We observed significantly (p<0.05) increased ubiquitinated protein-accumulation in the insoluble protein fractions of Beas2b cells treated with WPSE or nicotine that could be rescued by cysteamine treatment, suggesting aggresome-formation and autophagy-impairment. Moreover, our data also demonstrate that both WPSE and nicotine exposure significantly (p<0.05) elevates Ub-LC3β co-localization to aggresome-bodies while inducing Ub-p62 co-expression/accumulation, verifying autophagy-impairment. We also found that WPSE and nicotine exposure impacts Beas2b cell viability by significantly (p<0.05) inducing cellular apoptosis/senescence via ROS-activation, as it could be controlled by cysteamine, which is known to have an anti-oxidant property. For murine studies, C57BL/6 mice were administered with inhaled nicotine (intranasal, 500μg/mouse/day for 5 days), as an experimental model of non-combustible nicotine exposure. The inhaled nicotine exposure mediated oxidative-stress induces autophagy-impairment in the murine lungs as seen by significant (p<0.05, n=4) increase in the expression levels of nitrotyrosine protein-adduct (oxidative-stress marker, soluble-fraction) and Ub/p62/VCP (impaired-autophagy marker, insoluble-fraction). Overall, our data shows that nicotine, a common component of WPS, e-cigarette vapor and cigarette smoke, induces bronchial epithelial cell apoptosis and senescence via ROS mediated autophagy-impairment as a potential

  14. Genotoxic and epigenotoxic effects of fine particulate matter from rural and urban sites in Lebanon on human bronchial epithelial cells.

    PubMed

    Borgie, Mireille; Ledoux, Frédéric; Verdin, Anthony; Cazier, Fabrice; Greige, Hélène; Shirali, Pirouz; Courcot, Dominique; Dagher, Zeina

    2015-01-01

    Assessment of air pollution by particulate matter (PM) is strongly required in Lebanon in the absence of an air quality law including updated air quality standards. Using two different PM2.5-0.3 samples collected at an urban and a rural site, we examined genotoxic/epigenotoxic effects of PM exposure within a human bronchial epithelial cell line (BEAS-2B). Inorganic and organic contents evidence the major contribution of traffic and generating sets in the PM2.5-0.3 composition. Urban PM2.5-0.3 sample increased the phosphorylation of H2AX, the telomerase activity and the miR-21 up-regulation in BEAS-2B cells in a dose-dependent manner. Furthermore, urban PM2.5-0.3 induced a significant increase in CYP1A1, CYP1B1 and AhRR genes expression. The variable concentrations of transition metals and organic compounds detected in the collected PM2.5-0.3 samples might be the active agents leading to a cumulative DNA damage, critical for carcinogenesis. PMID:25460656

  15. Inhibition of the interactions between eosinophil cationic protein and airway epithelial cells by traditional Chinese herbs

    PubMed Central

    2010-01-01

    Background The eosinophil cationic protein (ECP) is cytotoxic to bacteria, viruses, parasites and mammalian cells. Cells are damaged via processes of pore formation, permeability alteration and membrane leaking. Some clinical studies indicate that ECP gathers in the bronchial tract of asthma sufferers, damages bronchial and airway epithelial cells, and leads to in breathing tract inflammation; therefore, prevention of the cytotoxicity caused by ECP may serve as an approach to treat airway inflammation. To achieve the purpose, reduction of the ECP-cell interactions is rational. In this work, the Chinese herbal combinative network was generated to predict and identify the functional herbs from the pools of prescriptions. It was useful to select the node herbs and to demonstrate the relative binding ability between ECP and Beas-2B cells with or withour herb treatments. Results Eighty three Chinese herbs and prescriptions were tested and five effective herbs and six prescription candidates were selected. On the basis of effective single-herbal drugs and prescriptions, a combinative network was generated. We found that a single herb, Gan-cao, served as a node connecting five prescriptions. In addition, Sheng-di-huang, Dang-guei and Mu-tong also appeared in five, four and three kinds of prescriptions, respectively. The extracts of these three herbs indeed effectively inhibited the interactions between ECP and Beas-2B cells. According to the Chinese herbal combinative network, eight of the effective herbal extracts showed inhibitory effects for ECP internalizing into Beas-2B cells. The major components of Gang-cao and Sheng-di-huang, glycyrrhizic acid and verbascose, respectively, reduced the binding affinity between ECP and cells effectively. Conclusions Since these Chinese herbs reduced the binding affinity between ECP and cells and inhibited subsequent ECP entrance into cells, they were potential for mitigating the airway inflammation symptoms. Additionally, we

  16. Marijuana smoke condensate induces p53-mediated apoptosis in human lung epithelial cells.

    PubMed

    Kim, Ha Ryong; Jung, Mi Hyun; Lee, Soo Yeun; Oh, Seung Min; Chung, Kyu Hyuck

    2013-01-01

    Since the largely abused worldwide used of marijuana, there have been many ongoing debates regarding the adverse health effects of marijuana smoking. Marijuana smoking was recently proved to cause pulmonary toxicity by inducing genotoxic effects or generating reactive oxygen species. Because p53, a tumor suppressor gene, has an important pathophysiologic role in the regulation of lung epithelial cell DNA damage responses, we hypothesized that p53 may be involved in the oxidative stress-mediated apoptosis induced by marijuana smoking. First, we confirmed that marijuana smoke condensate (MSC) induces oxidative stress in BEAS-2B cells. We observed that reactive oxygen species (ROS) generation was increased by MSC in the DCFH-DA assay. Also, antioxidant enzyme (superoxide dismutase, catalase) activity and their mRNA expressions were up-regulated by MSC. Second, we investigated p53 involvement in the MSC-induced apoptotic pathway in BEAS-2B cells. The results showed that MSC increased caspase-3 activation and DNA fragmentation as markers of apoptosis. In addition, the mRNA levels of apoptosis-related genes (p53 and Bax) were increased by MSC and phospho-p53, along with the increase of Bax protein expression by MSC. Apoptosis and apoptosis-related gene expression were partially blocked by an inhibitor of p53-dependent transcriptional activation (pifithrin-α). The results indicate that p53 plays a role in MSC-induced apoptosis. Taken together, the findings of the present study suggest that MSC partially induces p53-mediated apoptosis through ROS generation in human lung epithelial cells and this may have broader implications for our understanding of pulmonary diseases. PMID:23665932

  17. Sulforaphane inhibits de novo synthesis of IL-8 and MCP-1 in human epithelial cells generated by cigarette smoke extract.

    PubMed

    Starrett, Warren; Blake, David J

    2011-06-01

    Chronic obstructive pulmonary disease (COPD) is currently the fifth leading cause of death worldwide. Exposure to cigarette smoke (CS) is the primary factor associated with the COPD development. CS activates epithelial cells to secrete chemokines such as interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) that recruit neutrophils and macrophages to the lung. These inflammatory cells then release additional chemokines and cytokines leading to chronic inflammation that initiates apoptosis in epithelial and endothelial cells and destruction of alveolar structure. Pulmonary epithelium responds to oxidative stress mediated by CS through activating NRF2-dependent pathways, leading to an increased expression of antioxidant and cytoprotective enzymes thereby providing a protective response against CS-induced lung injury. We hypothesized that activating NRF2-dependent cytoprotective gene expression with sulforaphane (SFN) affords protection against CS-induced lung damage by inhibiting chemokine production. Results indicate that in the human BEAS-2B epithelial cell line, 5 μM SFN activated NRF2-dependent gene expression by triggering the translocation of NRF2 to the nucleus and significantly increased the expression of NRF2-dependent genes such as NADPH quinone oxidoreductase-1, heme oxygenase-1, and glutamate cysteine ligase modulatory subunit. Cigarette smoke extract (CSE) exposure of BEAS-2B cells significantly increased production of both IL-8 and MCP-1. Production of both chemokines was significantly reduced with SFN given prior to CSE; SFN inhibited IL-8 and MCP-1 gene expression at the transcription level. Our results indicate that activating NRF2 pathways with SFN inhibits CSE-induced chemokine production in human epithelial cells. However, the mechanism by which the production of chemokines is inhibited through SFN still remains to be elucidated. SFN may enhance NRF2 transcriptional activity resulting in the inhibition of proinflammatory pathways such

  18. Conjugated linoleic acids suppress inflammatory response and ICAM-1 expression through inhibition of NF-κB and MAPK signaling in human bronchial epithelial cells.

    PubMed

    Huang, Wen-Chung; Tu, Rong-Syuan; Chen, Ya-Ling; Tsai, Yun-Yun; Lin, Chwan-Fwu; Liou, Chian-Jiun

    2016-04-20

    Conjugated linoleic acids (CLAs) comprise a group of natural unsaturated fatty acids. CLA was reported to have anti-asthma, anti-adiposity, and anti-tumor effects. The present study aimed to evaluate the suppressive effects of cis-9, trans-11-CLA (c9,t11-CLA) on the expression of proinflammatory cytokines and intercellular adhesion molecule 1 (ICAM-1) in TNF-α-stimulated human bronchial epithelial (BEAS-2B) cells. After treating with various doses of c9,t11-CLA (12.5-100 μg ml(-1)), BEAS-2B cells were induced into an inflamed state by adding TNF-α or TNF-α/IL-4. The presence of c9,t11-CLA significantly suppressed the secretion of cytokines IL-6, IL-8, CCL5, and MCP-1. We also found that c9,t11-CLA inhibited ICAM-1 expression, and decreased monocyte adhesion to inflamed bronchial epithelial cells. Interestingly, c9,t11-CLA attenuated the phosphorylation of mitogen-activated protein kinase (MAPK) and down-regulated the activation of nuclear factor-κB (NF-κB). These results suggested that the anti-inflammatory effects of c9,t11-CLA were mediated by inhibiting proinflammatory cytokines, chemokines, and ICAM-1 expression by blocking NF-κB transcription regulation and by attenuating MAPK signaling pathways. PMID:27007063

  19. Absence of inflammatory response from upper airway epithelial cells after X irradiation.

    PubMed

    Reiter, R; Deutschle, T; Wiegel, T; Riechelmann, H; Bartkowiak, D

    2009-03-01

    Radiotherapy of head and neck tumors causes adverse reactions in normal tissue, especially mucositis. The dose- and time-dependent response of upper airway cells to X radiation should be analyzed in terms of the pro-inflammatory potential. Immortalized BEAS-2B lung epithelial cells were treated with 2, 5 and 8 Gy. Out of 1232 genes, those that were transcribed differentially after 2, 6 and 24 h were assigned to biological themes according to the Gene Ontology Consortium. Enrichment of differentially regulated gene clusters was determined with GOTree ( http://bioinfo.vanderbilt.edu/gotm ). Eleven cytokines were measured in culture supernatants. The cell cycle response up to 24 h and induction of apoptosis up to 4 days after exposure were determined by flow cytometry. A significant dose- and time-dependent gene activation was observed for the categories response to DNA damage, oxidative stress, cell cycle arrest and cell death/apoptosis but not for immune/inflammatory response. This correlated with functional G(2) arrest and apoptosis. Pro-inflammatory cytokines accumulated in supernatants of control cells but not of X-irradiated cells. The complex gene expression pattern of X-irradiated airway epithelial cells is accompanied by cell cycle arrest and induction of apoptosis. In vivo, this may impair the epithelial barrier. mRNA and protein expression suggest at most an indirect contribution of epithelial cells to early radiogenic mucositis. PMID:19267554

  20. Trans, trans-2,4-decadienal induced cell proliferation via p27 pathway in human bronchial epithelial cells

    SciTech Connect

    Chang, Y.-C.; Lin Pinpin

    2008-04-01

    Lung cancer is the leading cause of cancer deaths worldwide. Epidemiological studies have shown that exposure to cooking oil fumes (COF) is a risk factor for lung cancer. Trans, trans-2,4-decadienal (tt-DDE), a dienaldehyde, is abundant in heated oils and COF. Previously, we found that long-term exposure (45 days) to a sub-lethal dose (1 {mu}M) of tt-DDE significantly increased growth of human bronchial epithelial cells (BEAS-2B). Aims of this study are to understand the mechanism of tt-DDE-induced cell proliferation and possible protective effects of antioxidant, vitamin C and N-acetylcysteine (NAC) in BEAS-2B cells. Utilizing the real-time RT-PCR and Western immunoblotting, we found that p27 mRNA and protein levels were significantly increased by 1 {mu}M tt-DDE treatment. Co-treatment with vitamin C or NAC partially prevented tt-DDE-induced cell proliferation. In addition, the downstream targets of p27, including CDK4, cyclin D{sub 1} and phosphorylated-Rb proteins, increased in 1 {mu}M tt-DDE-treated cells and these changes were prevented by NAC co-treatment. Therefore, these results suggest that tt-DDE increased cell proliferation via inhibition of p27 expression, increase in CDK4/cyclin D{sub 1} protein accumulation and enhancement of Rb phosphorylation. Increased cell proliferation is considered as the early stages of lung carcinogenesis. Administration of antioxidants may prevent COF-associated lung carcinogenesis.

  1. Inhibition of endotoxin-induced airway epithelial cell injury by a novel family of pyrrol derivates.

    PubMed

    Cabrera-Benítez, Nuria E; Pérez-Roth, Eduardo; Ramos-Nuez, Ángela; Sologuren, Ithaisa; Padrón, José M; Slutsky, Arthur S; Villar, Jesús

    2016-06-01

    Inflammation and apoptosis are crucial mechanisms for the development of the acute respiratory distress syndrome (ARDS). Currently, there is no specific pharmacological therapy for ARDS. We have evaluated the ability of a new family of 1,2,3,5-tetrasubstituted pyrrol compounds for attenuating lipopolysaccharide (LPS)-induced inflammation and apoptosis in an in vitro LPS-induced airway epithelial cell injury model based on the first steps of the development of sepsis-induced ARDS. Human alveolar A549 and human bronchial BEAS-2B cells were exposed to LPS, either alone or in combination with the pyrrol derivatives. Rhein and emodin, two representative compounds with proven activity against the effects of LPS, were used as reference compounds. The pyrrol compound that was termed DTA0118 had the strongest inhibitory activity and was selected as the lead compound to further explore its properties. Exposure to LPS caused an intense inflammatory response and apoptosis in both A549 and BEAS-2B cells. DTA0118 treatment downregulated Toll-like receptor-4 expression and upregulated nuclear factor-κB inhibitor-α expression in cells exposed to LPS. These anti-inflammatory effects were accompanied by a significantly lower secretion of interleukin-6 (IL-6), IL-8, and IL-1β. The observed antiapoptotic effect of DTA0118 was associated with the upregulation of antiapoptotic Bcl-2 and downregulation of proapoptotic Bax and active caspase-3 protein levels. Our findings demonstrate the potent anti-inflammatory and antiapoptotic properties of the pyrrol DTA0118 compound and suggest that it could be considered as a potential drug therapy for the acute phase of sepsis and septic ARDS. Further investigations are needed to examine and validate these mechanisms and effects in a clinically relevant animal model of sepsis and sepsis-induced ARDS. PMID:26999659

  2. Chromium(VI) stimulates Fyn to initiate innate immune gene induction in human airway epithelial cells

    PubMed Central

    Nemec, Antonia A.; Zubritsky, Lindsey M.; Barchowsky, Aaron

    2009-01-01

    Mechanisms for pathogenic metal signaling in airway injury or disease promotion are poorly understood. It is widely believed that one mechanism for pathogenic and possible carcinogenic effects of inhaled chromium (Cr(VI)) is inhibition of inducible gene transactivation. However, we recently reported that Cr(VI) inhibition of Sp1-dependent transactivation required signal transducer and activator of transcription 1 (STAT1)-dependent expression of an inhibitory protein in airway epithelium. Thus, Cr(VI) exposures can induce genes and we hypothesized this induction resulted from Cr(VI) signaling through an innate immune-like STAT1-dependent pathway initiated by Fyn. Exposure of human airway epithelial (BEAS-2B) cells to Cr(VI) selectively transactivated STAT-responsive interferon-stimulated response element (ISRE) and induced ISRE-driven transactivation of interferon regulatory factor 7 (IRF7), without affecting the gamma interferon-activated site (GAS)-driven IRF1 expression. Cr(VI)-induced IRF7 was absent or greatly reduced in cells that lacked STAT1, were treated with the Src family kinase inhibitor, PP2, or lacked Fyn. Expressing Fyn, but not Src, in mouse embryonic fibroblasts cells null for Src, Yes, and Fyn restored Cr(VI)-stimulated STAT1 tyrosine phosphorylation and IRF7 expression. Finally, shRNA knockdown of Fyn in BEAS-2B cells prevented Cr(VI)-activated STAT1 transactivation of IRF7. These data support a novel mechanism through which Cr(VI) stimulates Fyn to initiate interferon-like signaling for STAT1-dependent gene transactivation. PMID:19994902

  3. Tungsten-induced carcinogenesis in human bronchial epithelial cells.

    PubMed

    Laulicht, Freda; Brocato, Jason; Cartularo, Laura; Vaughan, Joshua; Wu, Feng; Kluz, Thomas; Sun, Hong; Oksuz, Betul Akgol; Shen, Steven; Peana, Massimiliano; Medici, Serenella; Zoroddu, Maria Antonietta; Costa, Max

    2015-10-01

    Metals such as arsenic, cadmium, beryllium, and nickel are known human carcinogens; however, other transition metals, such as tungsten (W), remain relatively uninvestigated with regard to their potential carcinogenic activity. Tungsten production for industrial and military applications has almost doubled over the past decade and continues to increase. Here, for the first time, we demonstrate tungsten's ability to induce carcinogenic related endpoints including cell transformation, increased migration, xenograft growth in nude mice, and the activation of multiple cancer-related pathways in transformed clones as determined by RNA sequencing. Human bronchial epithelial cell line (Beas-2B) exposed to tungsten developed carcinogenic properties. In a soft agar assay, tungsten-treated cells formed more colonies than controls and the tungsten-transformed clones formed tumors in nude mice. RNA-sequencing data revealed that the tungsten-transformed clones altered the expression of many cancer-associated genes when compared to control clones. Genes involved in lung cancer, leukemia, and general cancer genes were deregulated by tungsten. Taken together, our data show the carcinogenic potential of tungsten. Further tests are needed, including in vivo and human studies, in order to validate tungsten as a carcinogen to humans. PMID:26164860

  4. Aryl hydrocarbon receptor-dependent up-regulation of the heterodimeric amino acid transporter LAT1 (SLC7A5)/CD98hc (SLC3A2) by diesel exhaust particle extract in human bronchial epithelial cells.

    PubMed

    Le Vee, Marc; Jouan, Elodie; Lecureur, Valérie; Fardel, Olivier

    2016-01-01

    The heterodimeric L-type amino acid transporter (LAT) 1/CD98hc is overexpressed in lung cancers with a poor prognosis factor. Factors that contribute to LAT1/CD98hc overexpression in lung cells remain however to be determined, but the implication of atmospheric pollution can be suspected. The present study was therefore designed to analyze the effects of diesel exhaust particle (DEP) extract (DEPe) on LAT1/CD98hc expression in bronchial epithelial BEAS-2B cells. Exposure to DEPe up-regulated LAT1 and CD98hc mRNA levels in a concentration-dependent manner, with DEPe EC50 values (around 0.2 μg/mL) relevant to environmental situations. DEPe concomitantly induced LAT1/CD98hc protein expression and LAT1-mediated leucine accumulation in BEAS-2B cells. Inhibition of the aryl hydrocarbon receptor (AhR) pathway through the use of a chemical AhR antagonist or the siRNA-mediated silencing of AhR expression was next found to prevent DEPe-mediated induction of LAT1/CD98hc, indicating that this regulation depends on AhR, known to be activated by major chemical DEP components like polycyclic aromatic hydrocarbons. DEPe exposure was finally shown to induce mRNA expression and activity of matrix metalloproteinase (MMP)-2 in BEAS-2B cells, in a CD98hc/focal adhesion kinase (FAK)/extracellular regulated kinase (ERK) manner, thus suggesting that DEPe-mediated induction of CD98hc triggers activation of the integrin/FAK/ERK signaling pathway known to be involved in MMP-2 regulation. Taken together, these data demonstrate that exposure to DEPe induces functional overexpression of the amino acid transporter LAT1/CD98hc in lung cells. Such a regulation may participate to pulmonary carcinogenic effects of DEPs, owing to the well-documented contribution of LAT1 and CD98hc to cancer development. PMID:26621329

  5. Cr(VI)-stimulated STAT3 tyrosine phosphorylation and nuclear translocation in human airway epithelial cells requires Lck

    PubMed Central

    O'hara, Kimberley A.; Vaghjiani, Rasilaben J.; Nemec, Antonia A.; Klei, Linda R.; Barchowsky, Aaron

    2006-01-01

    Chronic inhalation of low amounts of Cr(VI) promotes pulmonary diseases and cancers through poorly defined mechanisms. SFKs (Src family kinases) in pulmonary airway cells may mediate Cr(VI) signalling for lung injury, although the downstream effectors of Cr(VI)-stimulated SFKs and how they relate to pathogenic gene induction are unknown. Therefore SFK-dependent activation of transcription factors by non-cytotoxic exposure of human bronchial epithelial cells to Cr(VI) was determined. Protein–DNA binding arrays demonstrated that exposing BEAS 2B cells to 5 μM Cr(VI) for 4 and 24 h resulted in increased protein binding to 25 and 43 cis-elements respectively, while binding to 12 and 16 cis-elements decreased. Of note, Cr(VI) increased protein binding to several STAT (signal transducer and activator of transcription) cis-elements. Cr(VI) stimulated acute tyrosine phosphorylation and nuclear translocation of STAT1 over a 4 h period and a prolonged activation of STAT3 that reached a peak between 48 and 72 h. This prolonged activation was observed for both STAT3α and STAT3β. Immunofluorescent confocal microscopy confirmed that Cr(VI) increased nuclear localization of phosphorylated STAT3 for more than 72 h in both primary and BEAS 2B human airway cells. Cr(VI) induced transactivation of both a STAT3-driven luciferase reporter construct and the endogenous inflammatory gene IL-6 (interleukin-6). Inhibition with siRNA (small interfering RNA) targeting the SFK Lck, but not dominant-negative JAK (Janus kinase), prevented Cr(VI)-stimulated phosphorylation of both STAT3 isoforms and induction of IL-6. The results suggest that Cr(VI) activates epithelial cell Lck to signal for prolonged STAT3 activation and transactivation of IL-6, an important immunomodulator of lung disease progression. PMID:17078813

  6. RELEASE OF IL-8 AND IL-6 BY BEAS-2B CELLS FOLLOWING IN VITRO EXPOSURE TO BIODIESEL PM EXTRACTS

    EPA Science Inventory

    Abstract Body: Biodiesel, an alkyl ester of plant oils that can be used in an unmodified diesel engine, is a renewable fuel alternative which show signs of becoming a commercially accepted part of our nation¿s energy infrastructure. Biodiesel exhaust has been physicochemically ch...

  7. Proteome Profiling of BEAS-2B Cells Treated with Titanium Dioxide Reveals Potential Toxicity of and Detoxification Pathways for Nanomaterial

    EPA Science Inventory

    Oxidative stress is known to play important roles in nanomaterial-induced toxicities. However, the proteins and signaling pathways associated with nanomaterial-mediated oxidative stress and toxicity are largely unknown. To identify oxidative stress-responding toxicity pathways an...

  8. Proteome Profiling Reveals Potential Toxicity and Detoxification Pathways Following Exposure of BEAS-2B Cells to Engineered Nanoparticle Titanium Dioxide

    EPA Science Inventory

    Identification of toxicity pathways linked to chemical -exposure is critical for a better understanding of biological effects of the exposure, toxic mechanisms, and for enhancement of the prediction of chemical toxicity and adverse health outcomes. To identify toxicity pathways a...

  9. Proteome Profiling Reveals Potential Toxicity and Detoxification Pathways Following Exposure of BEAS-2B Cells to Engineered Titanium Dioxide Nanoparticles

    EPA Science Inventory

    Oxidative stress is known to play important roles in engineered nanomaterial induced cellular toxicity. However, the proteins and signaling pathways associated with the engineered nanomaterial mediated oxidative stress and toxicity are largely unknown. To identify these toxicity ...

  10. Chronic occupational exposure to arsenic induces carcinogenic gene signaling networks and neoplastic transformation in human lung epithelial cells

    PubMed Central

    Stueckle, Todd A.; Lu, Yongju; Davis, Mary E.; Wang, Liying; Jiang, Bing-Hua; Holaskova, Ida; Schafer, Rosana; Barnett, John B.; Rojanasakul, Yon

    2012-01-01

    Chronic arsenic exposure remains a human health risk; however a clear mode of action to understand gene signaling-driven arsenic carcinogenesis is currently lacking. This study chronically exposed human lung epithelial BEAS-2B cells to low-dose arsenic trioxide to elucidate cancer promoting gene signaling networks associated with arsenic-transformed (B-As) cells. Following a six month exposure, exposed cells were assessed for enhanced cell proliferation, colony formation, invasion ability and in vivo tumor formation compared to control cell lines. Collected mRNA was subjected to whole genome expression microarray profiling followed by in silico Ingenuity Pathway Analysis (IPA) to identify lung carcinogenesis modes of action. B-As cells displayed significant increases in proliferation, colony formation and invasion ability compared to BEAS-2B cells. B-As injections into nude mice resulted in development of primary and secondary metastatic tumors. Arsenic exposure resulted in widespread up-regulation of genes associated with mitochondrial metabolism and increased reactive oxygen species protection suggesting mitochondrial dysfunction. Carcinogenic initiation via reactive oxygen species and epigenetic mechanisms was further supported by altered DNA repair, histone, and ROS-sensitive signaling. NF-κB, MAPK and NCOR1 signaling disrupted PPARα/δ-mediated lipid homeostasis. A ‘pro-cancer’ gene signaling network identified increased survival, proliferation, inflammation, metabolism, anti-apoptosis and mobility signaling. IPA-ranked signaling networks identified altered p21, EF1α, Akt, MAPK, and NF-κB signaling networks promoting genetic disorder, altered cell cycle, cancer and changes in nucleic acid and energy metabolism. In conclusion, transformed B-As cells with their whole genome expression profile provide an in vitro arsenic model for future lung cancer signaling research and data for chronic arsenic exposure risk assessment. PMID:22521957

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

    PubMed

    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

  12. An antagonist of the platelet-activating factor receptor inhibits adherence of both nontypeable Haemophilus influenzae and Streptococcus pneumoniae to cultured human bronchial epithelial cells exposed to cigarette smoke

    PubMed Central

    Shukla, Shakti D; Fairbairn, Rory L; Gell, David A; Latham, Roger D; Sohal, Sukhwinder S; Walters, Eugene H; O’Toole, Ronan F

    2016-01-01

    Background COPD is emerging as the third largest cause of human mortality worldwide after heart disease and stroke. Tobacco smoking, the primary risk factor for the development of COPD, induces increased expression of platelet-activating factor receptor (PAFr) in the lung epithelium. Nontypeable Haemophilus influenzae (NTHi) and Streptococcus pneumoniae adhere to PAFr on the luminal surface of human respiratory tract epithelial cells. Objective To investigate PAFr as a potential drug target for the prevention of infections caused by the main bacterial drivers of acute exacerbations in COPD patients, NTHi and S. pneumoniae. Methods Human bronchial epithelial BEAS-2B cells were exposed to cigarette smoke extract (CSE). PAFr expression levels were determined using immunocytochemistry and quantitative polymerase chain reaction. The epithelial cells were challenged with either NTHi or S. pneumoniae labeled with fluorescein isothiocyanate, and bacterial adhesion was measured using immunofluorescence. The effect of a well-evaluated antagonist of PAFr, WEB-2086, on binding of the bacterial pathogens to BEAS-2B cells was then assessed. In silico studies of the tertiary structure of PAFr and the binding pocket for PAF and its antagonist WEB-2086 were undertaken. Results PAFr expression by bronchial epithelial cells was upregulated by CSE, and significantly associated with increased bacterial adhesion. WEB-2086 reduced the epithelial adhesion by both NTHi and S. pneumoniae to levels observed for non-CSE-exposed cells. Furthermore, it was nontoxic toward the bronchial epithelial cells. In silico analyses identified a binding pocket for PAF/WEB-2086 in the predicted PAFr structure. Conclusion WEB-2086 represents an innovative class of candidate drugs for inhibiting PAFr-dependent lung infections caused by the main bacterial drivers of smoking-related COPD. PMID:27524890

  13. SILAC-Based Quantitative Proteomic Analysis of Human Lung Cell Response to Copper Oxide Nanoparticles

    PubMed Central

    Edelmann, Mariola J.; Shack, Leslie A.; Naske, Caitlin D.; Walters, Keisha B.; Nanduri, Bindu

    2014-01-01

    Copper (II) oxide (CuO) nanoparticles (NP) are widely used in industry and medicine. In our study we evaluated the response of BEAS-2B human lung cells to CuO NP, using Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics and phosphoproteomics. Pathway modeling of the protein differential expression showed that CuO NP affect proteins relevant in cellular function and maintenance, protein synthesis, cell death and survival, cell cycle and cell morphology. Some of the signaling pathways represented by BEAS-2B proteins responsive to the NP included mTOR signaling, protein ubiquitination pathway, actin cytoskeleton signaling and epithelial adherens junction signaling. Follow-up experiments showed that CuO NP altered actin cytoskeleton, protein phosphorylation and protein ubiquitination level. PMID:25470785

  14. Human bronchial epithelial cells injury and cytokine production induced by Tityus serrulatus scorpion venom: An in vitro study.

    PubMed

    Rigoni, Vera Lucia Silva; Kwasniewski, Fabio H; Vieira, Rodolfo Paula; Linhares, Ingrid Sestrem; da Silva, Joelmir Lucena Veiga; Nogueira-Pedro, Amanda; Zamuner, Stella Regina

    2016-09-15

    Tityus serrulatus is the scorpion specie responsible for the majority of scorpion sting accidents in Brazil. Symptoms of envenomation by Tityus serrulatus range from local pain to severe systemic reactions such as cardiac dysfunction and pulmonary edema. Thus, this study has evaluated the participation of bronchial epithelial cells in the pulmonary effects of Tityus serrulatus scorpion venom (Tsv). Human bronchial epithelial cell line BEAS-2B were utilized as a model target and were incubated with Tsv (10 or 50 μg/mL) for 1, 3, 6 and 24 h. Effects on cellular response of venom-induce cytotoxicity were examined including cell viability, cell integrity, cell morphology, apoptosis/necrosis as well as cell activation through the release of pro-inflammatory cytokines IL-1β, IL-6 and IL-8. Tsv caused a decrease in cell viability at 10 and 50 μg/mL, which was confirmed by lactate dehydrogenase (LDH) measurement. Flow cytometry analyses revealed necrosis as the main cell death pathway caused by Tsv. Furthermore, Tsv induced the release of IL-1β, IL-6 and IL-8. Altogether, these results demonstrate that Tsv induces cytotoxic effects on bronchial epithelial cells, involving necrosis and release of pro-inflammatory cytokines, suggesting that bronchial epithelial cells may play a role in the pulmonary injury caused by Tsv. PMID:27452928

  15. Gene expression profiling of the effects of organic dust in lung epithelial and THP-1 cells reveals inductive effects on inflammatory and immune response genes.

    PubMed

    Boggaram, Vijay; Loose, David S; Gottipati, Koteswara R; Natarajan, Kartiga; Mitchell, Courtney T

    2016-04-01

    The intensification and concentration of animal production operations expose workers to high levels of organic dusts in the work environment. Exposure to organic dusts is a risk factor for the development of acute and chronic respiratory symptoms and diseases. Lung epithelium plays important roles in the control of immune and inflammatory responses to environmental agents to maintain lung health. To better understand the effects of organic dust on lung inflammatory responses, we characterized the gene expression profiles of A549 alveolar and Beas2B bronchial epithelial and THP-1 monocytic cells influenced by exposure to poultry dust extract by DNA microarray analysis using Illumina Human HT-12 v4 Expression BeadChip. We found that A549 alveolar and Beas2B bronchial epithelial and THP-1 cells responded with unique changes in the gene expression profiles with regulation of genes encoding inflammatory cytokines, chemokines, and other inflammatory proteins being common to all the three cells. Significantly induced genes included IL-8, IL-6, IL-1β, ICAM-1, CCL2, CCL5, TLR4, and PTGS2. Validation by real-time qRT-PCR, ELISA, Western immunoblotting, and immunohistochemical staining of lung sections from mice exposed to dust extract validated DNA microarray results. Pathway analysis indicated that dust extract induced changes in gene expression influenced functions related to cellular growth and proliferation, cell death and survival, and cellular development. These data show that a broad range of inflammatory mediators produced in response to poultry dust exposure can modulate lung immune and inflammatory responses. This is the first report on organic dust induced changes in expression profiles in lung epithelial and THP-1 monocytic cells. PMID:26884459

  16. Chromium (VI) Inhibits Heme Oxygenase-1 Expression In Vivo and in Arsenic-Exposed Human Airway Epithelial Cells

    PubMed Central

    O’HARA, KIMBERLEY A.; NEMEC, ANTONIA A.; ALAM, JAWED; KLEI, LINDA R.; MOSSMAN, BROOKE T.; BARCHOWSKY, AARON

    2015-01-01

    Inhaled hexavalent chromium (Cr(VI)) promotes lung injury and pulmonary diseases through poorly defined mechanisms. One hypothesis for this lung pathogenesis is that Cr(VI) silences induction of cytoprotective genes, such as heme oxygenase-1 (HO-1), whose total lung mRNA levels were reduced 21 days after nasal instillation of potassium dichromate in C57BL/6 mice. To investigate the mechanisms for this inhibition, Cr(VI) effects on basal and arsenic (As(III))-induced HO-1 expression were examined in cultured human bronchial epithelial (BEAS-2B) cells. An effect of Cr(VI) on the low basal HO-1 mRNA and protein levels in BEAS-2B cells was not detectible. In contrast, Cr(VI) added to the cells before As(III), but not simultaneously with As(III), attenuated As(III)-induced HO-1 expression. Transient transfection with luciferase reporter gene constructs controlled by the full length ho-1 promoter or deletion mutants demonstrated that this inhibition occurred in the E1 enhancer region containing critical antioxidant response elements (ARE). Cr(VI) pretreatment inhibited As(III)-induced activity of a transiently expressed reporter construct regulated by three ARE tandem repeats. The mechanism for this Cr(VI)-attenuated transactivation appeared to be Cr(VI) reduction of the nuclear levels of the transcription factor Nrf2 and As(III)-stimulated Nrf2 transcriptional complex binding to the ARE cis element. Finally, exposing cells to Cr(VI) prior to co-exposure with As(III) synergized for apoptosis and loss of membrane integrity. These data suggest that Cr(VI) silences induction of ARE-driven genes required for protection from secondary insults. The data also have important implications for understanding the toxic mechanisms of low level, mixed metal exposures in the lung. PMID:16775837

  17. The response of a human bronchial epithelial cell line to histamine: Intracellular calcium changes and extracellular release of inflammatory mediators

    SciTech Connect

    Noah, T.L.; Paradiso, A.M.; Madden, M.C.; McKinnon, K.P.; Devlin, R.B. )

    1991-11-01

    Epithelial cells are likely to modulate inflammation and tissue repair in the airways, but the factors responsible for these processes remain unclear. Because human airway epithelia are infrequently available for in vitro studies, transformed epithelial cell lines are of interest as models. The authors therefore investigated the response of an SV-40/adenovirus-transformed human bronchial epithelial cell line (BEAS-2B) to histamine, a mediator with relevance for airway diseases. The intracellular calcium response to histamine (10(-4) M) was measured, using Fura-2 and microspectrofluorimetry. Histamine induced a transient increase in intracellular calcium that originated from intracellular sources; this effect was inhibited by the H1 receptor antagonist diphenhydramine, suggesting that BEAS cells retain functioning histamine receptors. BEAS cells were grown to confluence on microporous, collagen-coated filters, allowing measurement of vectorial release of soluble mediators. Monolayers exposed to histamine for 30 min released interleukin-6 and fibronectin in the apical direction, in a dose-dependent manner. Little eicosanoid production was induced by histamine, either in the apical or the basolateral direction, although BEAS cells constitutively produced small amounts of prostaglandin E2 and 15-HETE. However, these cells formed large amounts of eicosanoids in response to ozone exposure as a positive control. Comparison of their data with published reports for human airway epithelia in primary culture suggests that the BEAS cell line is, in a number of respects, a relevant model for the study of airway epithelial responses to a variety of stimuli.

  18. Cytochrome P450 2A13 enhances the sensitivity of human bronchial epithelial cells to aflatoxin B1-induced DNA damage

    SciTech Connect

    Yang, Xuejiao; Zhang, Zhan; Wang, Xichen; Wang, Yun; Zhang, Xiaoming; Lu, Huiyuan; Wang, Shou-Lin

    2013-07-15

    Cytochrome P450 2A13 (CYP2A13) mainly expresses in human respiratory system and mediates the metabolic activation of aflatoxin B1 (AFB1). Our previous study suggested that CYP2A13 could increase the cytotoxic and apoptotic effects of AFB1 in immortalized human bronchial epithelial cells (BEAS-2B). However, the role of CYP2A13 in AFB1-induced DNA damage is unclear. Using BEAS-2B cells that stably express CYP2A13 (B-2A13), CYP1A2 (B-1A2), and CYP2A6 (B-2A6), we compared their effects in AFB1-induced DNA adducts, DNA damage, and cell cycle changes. BEAS-2B cells that were transfected with vector (B-vector) were used as a control. The results showed that AFB1 (5–80 nM) dose- and time-dependently induced DNA damage in B-2A13 cells. AFB1 at 10 and 80 nM significantly augmented this effect in B-2A13 and B-1A2 cells, respectively. B-2A6 cells showed no obvious DNA damage, similar to B-vector cells and the vehicle control. Similarly, compared with B-vector, B-1A2 or B-2A6 cells, B-2A13 cells showed more sensitivity in AFB1-induced γH2AX expression, DNA adduct 8-hydroxy-deoxyguanosine formation, and S-phase cell-cycle arrest. Furthermore, AFB1 activated the proteins related to DNA damage responses, such as ATM, ATR, Chk2, p53, BRCA1, and H2AX, rather than the proteins related to DNA repair. These effects could be almost completely inhibited by 100 μM nicotine (a substrate of CYP2A13) or 1 μM 8-methoxypsoralen (8-MOP; an inhibitor of CYP enzyme). Collectively, these findings suggest that CYP2A13 plays an important role in low-concentration AFB1-induced DNA damage, possibly linking environmental airborne AFB1 to genetic injury in human respiratory system. - Highlights: • CYP2A13 plays a critical role in low concentration of AFB1-induced DNA damage. • B-2A13 cells were more sensitive to AFB1 than B-1A2 cells and B-2A6 cells. • AFB1 dose- and time-dependently induced DNA damage in B-2A13 cells • AFB1-induced DNA adducts and damage can be inhibited by nicotine and 8

  19. TRPV1 Antagonists Elevate Cell Surface Populations of Receptor Protein and Exacerbate TRPV1-Mediated Toxicities in Human Lung Epithelial Cells

    PubMed Central

    Johansen, Mark E.; Reilly, Christopher A.; Yost, Garold S.

    2008-01-01

    TRPV1 mediates cell death and pro-inflammatory cytokine production in lung epithelial cells exposed to prototypical receptor agonists. This study shows that NHBE, BEAS-2B and TRPV1 over-expressing BEAS-2B cells pre-treated with various TRPV1 antagonists become sensitized to the prototypical TRPV1 agonist, nonivamide, via a mechanism that involves translocation of existing receptor from the endoplasmic reticulum to the plasma membrane. As such, typical cellular responses to agonist treatment, as measured by calcium flux, inflammatory cytokine gene induction, and cytotoxicity were exacerbated. These data were in contrast to the results obtained when TRPV1 antagonists were co-administered with nonivamide; conditions which inhibited TRPV1-mediated effects. The antagonists LJO-328, SC0030, and capsazepine increased the cytotoxicity of nonivamide by ~20-fold and agonist-induced calcium flux by ~6-fold. Inflammatory-cytokine gene induction by nonivamide was also increased significantly by pre-treatment with the antagonists. The enhanced responses were inhibited by the co-administration of antagonists with nonivamide, confirming that increases in sensitivity were attributable to increased TRPV1-associated activity. Sensitization was attenuated by brefeldin A (a golgi transport inhibitor), but not cycloheximide (a protein synthesis inhibitor), or actinomycin D (a transcription inhibitor). Sensitized cells exhibited increased calcium flux from extracellular calcium sources, while unsensitized cells exhibited calcium flux originating primarily from intracellular stores. These results demonstrate the presence of a novel mechanism for regulating the sub-cellular distribution of TRPV1 and subsequent control of cellular sensitivity to TRPV1 agonists. PMID:16120755

  20. Highly purified, multi-wall carbon nanotubes induce light-chain 3B expression in human lung cells

    SciTech Connect

    Tsukahara, Tamotsu; Matsuda, Yoshikazu; Usui, Yuki; Haniu, Hisao

    2013-10-18

    Highlights: •HTT2800-treated BEAS-2B cells induced LC3B in a time-dependent manner. •HTT2800-treated BEAS-2B cells showed decreased cell proliferation that was both time- and dose-dependent. •Addition of 3-MA, LC3B-II protein and mRNA levels were significantly decreased. •3-MA and E64-d + pepstatin A, but not brefeldin A, provided protection against HTT2800-induced cell death. •These results suggest that HTT2800 predominantly causes autophagy rather than apoptotic cell death in BEAS-2B cells. -- Abstract: Bronchial epithelial cells are targets of inhalation and play a critical role in the maintenance of mucosal integrity as mechanical barriers against various particles. Our previous result suggest that vapor-grown carbon fiber, HTT2800, which is one of the most highly purified multi-wall carbon nanotubes (MWCNT) showed cellular uptake of the carbon nanotube, increased cell death, enhanced DNA damage, and induced cytokine release. Increasing evidence suggests that autophagy may critically influence vital cellular processes such as apoptosis, cell proliferation and inflammation and thereby may play a critical role in pulmonary diseases. Autophagy was recently recognized as a critical cell death pathway, and autophagosome accumulation has been found to be associated with the exposure of various nanoparticles. In this study, the authors focus on the autophagic responses of HTT2800 exposure. The HTT2800-exposed cells induced LC3B expression and induced cell growth inhibition.

  1. Involvement of the MAPK and PI3K pathways in chitinase 3-like 1-regulated hyperoxia-induced airway epithelial cell death

    SciTech Connect

    Kim, Mi Na; Lee, Kyung Eun; Hong, Jung Yeon; Heo, Won Il; Kim, Kyung Won; Kim, Kyu Earn; Sohn, Myung Hyun

    2012-05-18

    Highlights: Black-Right-Pointing-Pointer Hyperoxia induces apoptosis and chitinase 3-like 1 expression in human airway epithelial cells. Black-Right-Pointing-Pointer Presence of chitinase 3-like 1 affects airway epithelial cell death after hyperoxic exposure. Black-Right-Pointing-Pointer Silencing chitinase 3-like 1 manipulate the phosphorylation of ERK, p38 and Akt. -- Abstract: Background: Exposure to 100% oxygen causes hyperoxic acute lung injury characterized by cell death and injury of alveolar epithelial cells. Recently, the role of chitinase 3-like 1 (CHI3L1), a member of the glycosyl hydrolase 18 family that lacks chitinase activity, in oxidative stress was demonstrated in murine models. High levels of serum CHI3L1 have been associated with various diseases of the lung, such as asthma, chronic obstructive pulmonary disease, and cancer. However, the role of CHI3L1 in human airway epithelial cells undergoing oxidative stress remains unknown. In addition, the signaling pathways associated with CHI3L1 in this process are poorly understood. Purpose: In this study, we demonstrate the role of CHI3L1, along with the MAPK and PI3K signaling pathways, in hyperoxia-exposed airway epithelial cells. Method: The human airway epithelial cell line, BEAS-2B, was exposed to >95% oxygen (hyperoxia) for up to 72 h. Hyperoxia-induced cell death was determined by assessing cell viability, Annexin-V FITC staining, caspase-3 and -7 expression, and electron microscopy. CHI3L1 knockdown and overexpression studies were conducted in BEAS-2B cells to examine the role of CHI3L1 in hyperoxia-induced apoptosis. Activation of the MAPK and PI3K pathways was also investigated to determine the role of these signaling cascades in this process. Results: Hyperoxia exposure increased CHI3L1 expression and apoptosis in a time-dependent manner. CHI3L1 knockdown protected cells from hyperoxia-induced apoptosis. In contrast, CHI3L1 overexpression promoted cell death after hyperoxia exposure. Finally

  2. Brd4 Is Essential for IL-1β-Induced Inflammation in Human Airway Epithelial Cells

    PubMed Central

    Khan, Younis M.; Kirkham, Paul; Barnes, Peter J.; Adcock, Ian M.

    2014-01-01

    Background Chronic inflammation and oxidative stress are key features of chronic obstructive pulmonary disease (COPD). Oxidative stress enhances COPD inflammation under the control of the pro-inflammatory redox-sensitive transcription factor nuclear factor-kappaB (NF-κB). Histone acetylation plays a critical role in chronic inflammation and bromodomain and extra terminal (BET) proteins act as “readers” of acetylated histones. Therefore, we examined the role of BET proteins in particular Brd2 and Brd4 and their inhibitors (JQ1 and PFI-1) in oxidative stress- enhanced inflammation in human bronchial epithelial cells. Methods Human primary epithelial (NHBE) cells and BEAS-2B cell lines were stimulated with IL-1β (inflammatory stimulus) in the presence or absence of H2O2 (oxidative stress) and the effect of pre-treatment with bromodomain inhibitors (JQ1 and PFI-1) was investigated. Pro-inflammatory mediators (CXCL8 and IL-6) were measured by ELISA and transcripts by RT-PCR. H3 and H4 acetylation and recruitment of p65 and Brd4 to the native IL-8 and IL-6 promoters was investigated using chromatin immunoprecipitation (ChIP). The impact of Brd2 and Brd4 siRNA knockdown on inflammatory mediators was also investigated. Result H2O2 enhanced IL1β-induced IL-6 and CXCL8 expression in NHBE and BEAS-2B cells whereas H2O2 alone did not have any affect. H3 acetylation at the IL-6 and IL-8 promoters was associated with recruitment of p65 and Brd4 proteins. Although p65 acetylation was increased this was not directly targeted by Brd4. The BET inhibitors JQ1 and PFI-1 significantly reduced IL-6 and CXCL8 expression whereas no effect was seen with the inactive enantiomer JQ1(-). Brd4, but not Brd2, knockdown markedly reduced IL-6 and CXCL8 release. JQ1 also inhibited p65 and Brd4 recruitment to the IL-6 and IL-8 promoters. Conclusion Oxidative stress enhanced IL1β-induced IL-6 and CXCL8 expression was significantly reduced by Brd4 inhibition. Brd4 plays an important role in

  3. Induction of regulator of G-protein signaling 2 expression by long-acting β2-adrenoceptor agonists and glucocorticoids in human airway epithelial cells.

    PubMed

    Holden, Neil S; George, Tresa; Rider, Christopher F; Chandrasekhar, Ambika; Shah, Suharsh; Kaur, Manminder; Johnson, Malcolm; Siderovski, David P; Leigh, Richard; Giembycz, Mark A; Newton, Robert

    2014-01-01

    In asthma and chronic obstructive pulmonary disease (COPD) multiple mediators act on Gαq-linked G-protein-coupled receptors (GPCRs) to cause bronchoconstriction. However, acting on the airway epithelium, such mediators may also elicit inflammatory responses. In human bronchial epithelial BEAS-2B cells (bronchial epithelium + adenovirus 12-SV40 hybrid), regulator of G-protein signaling (RGS) 2 mRNA and protein were synergistically induced in response to combinations of long-acting β2-adrenoceptor agonist (LABA) (salmeterol, formoterol) plus glucocorticoid (dexamethasone, fluticasone propionate, budesonide). Equivalent responses occurred in primary human bronchial epithelial cells. Concentrations of glucocorticoid plus LABA required to induce RGS2 expression in BEAS-2B cells were consistent with the levels achieved therapeutically in the lungs. As RGS2 is a GTPase-activating protein that switches off Gαq, intracellular free calcium ([Ca(2+)]i) flux was used as a surrogate of responses induced by histamine, methacholine, and the thromboxane receptor agonist U46619 [(Z)-7-[(1S,4R,5R,6S)-5-[(E,3S)-3-hydroxyoct-1-enyl]-3-oxabicyclo[2.2.1]heptan-6-yl]hept-5-enoic acid]. This was significantly attenuated by salmeterol plus dexamethasone pretreatment, or RGS2 overexpression, and the protective effect of salmeterol plus dexamethasone was abolished by RGS2 RNA silencing. Although methacholine and U46619 induced interleukin-8 (IL-8) release and this was inhibited by RGS2 overexpression, the repression of U46619-induced IL-8 release by salmeterol plus dexamethasone was unaffected by RGS2 knockdown. Given a role for Gαq-mediated pathways in inducing IL-8 release, we propose that RGS2 acts redundantly with other effector processes to repress IL-8 expression. Thus, RGS2 expression is a novel effector mechanism in the airway epithelium that is induced by glucocorticoid/LABA combinations. This could contribute to the efficacy of glucocorticoid/LABA combinations in asthma and

  4. Inhibition of airway epithelial-to-mesenchymal transition and fibrosis by kaempferol in endotoxin-induced epithelial cells and ovalbumin-sensitized mice.

    PubMed

    Gong, Ju-Hyun; Cho, In-Hee; Shin, Daekeun; Han, Seon-Young; Park, Sin-Hye; Kang, Young-Hee

    2014-03-01

    Chronic airway remodeling is characterized by structural changes within the airway wall, including smooth muscle hypertrophy, submucosal fibrosis and epithelial shedding. Epithelial-to-mesenchymal transition (EMT) is a fundamental mechanism of organ fibrosis, which can be induced by TGF-β. In the in vitro study, we investigated whether 1-20 μM kaempferol inhibited lipopolysaccharide (LPS)-induced bronchial EMT in BEAS-2B cells. The in vivo study explored demoting effects of 10-20 mg/kg kaempferol on airway fibrosis in BALB/c mice sensitized with ovalbumin (OVA). LPS induced airway epithelial TGF-β1 signaling that promoted EMT with concurrent loss of E-cadherin and induction of α-smooth muscle actin (α-SMA). Nontoxic kaempferol significantly inhibited TGF-β-induced EMT process through reversing E-cadherin expression and retarding the induction of N-cadherin and α-SMA. Consistently, OVA inhalation resulted in a striking loss of epithelial morphology by displaying myofibroblast appearance, which led to bronchial fibrosis with submucosal accumulation of collagen fibers. Oral administration of kaempferol suppressed collagen deposition, epithelial excrescency and goblet hyperplasia observed in the lung of OVA-challenged mice. The specific inhibition of TGF-β entailed epithelial protease-activated receptor-1 (PAR-1) as with 20 μM kaempferol. The epithelial PAR-1 inhibition by SCH-79797 restored E-cadherin induction and deterred α-SMA induction, indicating that epithelial PAR-1 localization was responsible for resulting in airway EMT. These results demonstrate that dietary kaempferol alleviated fibrotic airway remodeling via bronchial EMT by modulating PAR1 activation. Therefore, kaempferol may be a potential therapeutic agent targeting asthmatic airway constriction. PMID:24378645

  5. Signal Transducer and Activator of Transcription 1 (STAT1) is Essential for Chromium Silencing of Gene Induction in Human Airway Epithelial Cells

    PubMed Central

    Nemec, Antonia A.; Barchowsky, Aaron

    2009-01-01

    Hexavalent chromium (Cr(VI)) promotes lung injury and pulmonary diseases through poorly defined mechanisms that may involve the silencing of inducible protective genes. The current study investigated the hypothesis that Cr(VI) actively signals through a signal transducer and activator of transcription 1 (STAT1)–dependent pathway to silence nickel (Ni)–induced expression of vascular endothelial cell growth factor A (VEGFA), an important mediator of lung injury and repair. In human bronchial airway epithelial (BEAS-2B) cells, Ni-induced VEGFA transcription by stimulating an extracellular regulated kinase (ERK) signaling cascade that involved Src kinase–activated Sp1 transactivation, as well as increased hypoxia-inducible factor-1α (HIF-1α) stabilization and DNA binding. Ni-stimulated ERK, Src, and HIF-1α activities, as well as Ni-induced VEGFA transcript levels were inhibited in Cr(VI)-exposed cells. We previously demonstrated that Cr(VI) stimulates STAT1 to suppress VEGFA expression. In BEAS-2B cells stably expressing STAT1 short hairpin RNA, Cr(VI) increased VEGFA transcript levels and Sp1 transactivation. Moreover, in the absence of STAT1, Cr(VI), and Ni coexposures positively interacted to further increase VEGFA transcripts. This study demonstrates that metal-stimulated signaling cascades interact to regulate transcription and induction of adaptive or repair responses in airway cells. In addition, the data implicate STAT1 as a rate limiting mediator of Cr(VI)-stimulated gene regulation and suggest that cells lacking STAT1, such as many tumor cell lines, have opposite responses to Cr(VI) relative to normal cells. PMID:19403854

  6. Phosphorylation of p65 Is Required for Zinc Oxide Nanoparticle–Induced Interleukin 8 Expression in Human Bronchial Epithelial Cells

    PubMed Central

    Wu, Weidong; Samet, James M.; Peden, David B.; Bromberg, Philip A.

    2010-01-01

    Background Exposure to zinc oxide (ZnO) in environmental and occupational settings causes acute pulmonary responses through the induction of proinflammatory mediators such as interleukin-8 (IL-8). Objective We investigated the effect of ZnO nanoparticles on IL-8 expression and the underlying mechanisms in human bronchial epithelial cells. Methods We determined IL-8 mRNA and protein expression in primary human bronchial epithelial cells and the BEAS-2B human bronchial epithelial cell line using reverse-transcriptase polymerase chain reaction and the enzyme-linked immunosorbent assay, respectively. Transcriptional activity of IL-8 promoter and nuclear factor kappa B (NFκB) in ZnO-treated BEAS-2B cells was measured using transient gene transfection of the luciferase reporter construct with or without p65 constructs. Phosphorylation and degradation of IκBα, an inhibitor of NF-κB, and phosphorylation of p65 were detected using immunoblotting. Binding of p65 to the IL-8 promoter was examined using the chromatin immunoprecipitation assay. Results ZnO exposure (2–8 μg/mL) increased IL-8 mRNA and protein expression. Inhibition of transcription with actinomycin D blocked ZnO-induced IL-8 expression, which was consistent with the observation that ZnO exposure increased IL-8 promoter reporter activity. Further study demonstrated that the κB-binding site in the IL-8 promoter was required for ZnO-induced IL-8 transcriptional activation. ZnO stimulation modestly elevated IκBα phosphorylation and degradation. Moreover, ZnO exposure also increased the binding of p65 to the IL-8 promoter and p65 phosphorylation at serines 276 and 536. Overexpression of p65 constructs mutated at serines 276 or 536 significantly reduced ZnO-induced increase in IL-8 promoter reporter activity. Conclusion p65 phosphorylation and IκBα phosphorylation and degradation are the primary mechanisms involved in ZnO nanoparticle-induced IL-8 expression in human bronchial epithelial cells. PMID

  7. The Fate of ZnO Nanoparticles Administered to Human Bronchial Epithelial Cells

    PubMed Central

    Gilbert, Benjamin; Fakra, Sirine C.; Xia, Tian; Pokhrel, Suman; Mädler, Lutz; Nel, André E.

    2014-01-01

    A particular challenge for nanotoxicology is the evaluation of the biological fate and toxicity of nanomaterials that dissolve in aqueous fluids. Zinc oxide nanomaterials are of particular concern because dissolution leads to release of the toxic divalent zinc ion. Although dissolved zinc ions have been implicated in ZnO cytotoxicity, direct identification of the chemical form of zinc taken up by cells exposed to ZnO nanoparticles, and its intracellular fate, has not yet been achieved. We combined high resolution X-ray spectromicroscopy and high elemental sensitivity X-ray microprobe analyses to determine the fate of ZnO and less soluble iron-doped ZnO nanoparticles following exposure to cultures of human bronchial epithelial cells, BEAS-2B. We complemented two-dimensional X-ray imaging methods with atomic force microscopy of cell surfaces to distinguish between nanoparticles that were transported inside the cells from those that adhered to the cell exterior. The data suggest cellular uptake of ZnO nanoparticles is a mechanism of zinc accumulation in cells. Following uptake, ZnO nanoparticles dissolved completely generating intracellular Zn2+ complexed by molecular ligands. These results corroborate a model for ZnO nanoparticle toxicity that is based on nanoparticle uptake followed by intracellular dissolution. PMID:22646753

  8. Effects of Chrysotile Exposure in Human Bronchial Epithelial Cells: Insights into the Pathogenic Mechanisms of Asbestos-Related Diseases

    PubMed Central

    Gulino, Giulia Rossana; Polimeni, Manuela; Prato, Mauro; Gazzano, Elena; Kopecka, Joanna; Colombatto, Sebastiano; Ghigo, Dario; Aldieri, Elisabetta

    2015-01-01

    Background: Chrysotile asbestos accounts for > 90% of the asbestos used worldwide, and exposure is associated with asbestosis (asbestos-related fibrosis) and other malignancies; however, the molecular mechanisms involved are not fully understood. A common pathogenic mechanism for these malignancies is represented by epithelial–mesenchymal transition (EMT), through which epithelial cells undergo a morphological transformation to assume a mesenchymal phenotype. In the present work, we propose that chrysotile asbestos induces EMT through a mechanism involving a signaling pathway mediated by tranforming growth factor beta (TGF-β). Objectives: We investigated the role of chrysotile asbestos in inducing EMT in order to elucidate the molecular mechanisms involved in this event. Methods: Human bronchial epithelial cells (BEAS-2B) were incubated with 1 μg/cm2 chrysotile asbestos for ≤ 72 hr, and several markers of EMT were investigated. Experiments with specific inhibitors for TGF-β, glycogen synthase kinase–3β (GSK-3β), and Akt were performed to confirm their involvement in asbestos-induced EMT. Real-time polymerase chain reaction (PCR), Western blotting, and gelatin zymography were performed to detect mRNA and protein level changes for these markers. Results: Chrysotile asbestos activated a TGF-β–mediated signaling pathway, implicating the contributions of Akt, GSK-3β, and SNAIL-1. The activation of this pathway in BEAS-2B cells was associated with a decrease in epithelial markers (E-cadherin and β-catenin) and an increase in mesenchymal markers (α-smooth muscle actin, vimentin, metalloproteinases, and fibronectin). Conclusions: Our findings suggest that chrysotile asbestos induces EMT, a common event in asbestos-related diseases, at least in part by eliciting the TGF-β–mediated Akt/GSK-3β/SNAIL-1 pathway. Citation: Gulino GR, Polimeni M, Prato M, Gazzano E, Kopecka J, Colombatto S, Ghigo D, Aldieri E. 2016. Effects of chrysotile exposure in human

  9. Flotillin-2 Modulates Fas Signaling Mediated Apoptosis after Hyperoxia in Lung Epithelial Cells

    PubMed Central

    Wei, Shuquan; Moon, Hyung-Geun; Zheng, Yijie; Liang, Xiaoliang; An, Chang Hyeok; Jin, Yang

    2013-01-01

    Lipid rafts are subdomains of the cell membrane with distinct protein composition and high concentrations of cholesterol and glycosphingolipids. Raft proteins are thought to mediate diverse cellular processes including signal transduction. However, its cellular mechanisms remain unclear. Caveolin-1 (cav-1, marker protein of caveolae) has been thought as a switchboard between extracellular matrix (ECM) stimuli and intracellular signals. Flotillin-2/reggie-1(Flot-2) is another ubiquitously expressed raft protein which defines non-caveolar raft microdomains (planar raft). Its cellular function is largely uncharacterized. Our novel studies demonstrated that Flot-2, in conjunction with cav-1, played important functions on controlling cell death via regulating Fas pathways. Using Beas2B epithelial cells, we found that in contrast to cav-1, Flot-2 conferred cytoprotection via preventing Fas mediated death-inducing signaling complex (DISC) formation, subsequently suppressed caspase-8 mediated extrinsic apoptosis. Moreover, Flot-2 reduced the mitochondria mediated intrinsic apoptosis by regulating the Bcl-2 family and suppressing cytochrome C release from mitochondria to cytosol. Flot-2 further modulated the common apoptosis pathway and inhibited caspase-3 activation via up-regulating the members in the inhibitor of apoptosis (IAP) family. Last, Flot-2 interacted with cav-1 and limited its expression. Taken together, we found that Flot-2 protected cells from Fas induced apoptosis and counterbalanced the pro-apoptotic effects of cav-1. Thus, Flot-2 played crucial functions in cellular homeostasis and cell survival, suggesting a differential role of individual raft proteins. PMID:24204853

  10. Silica nanoparticles induce cytokine responses in lung epithelial cells through activation of a p38/TACE/TGF-α/EGFR-pathway and NF-κΒ signalling

    SciTech Connect

    Skuland, Tonje Øvrevik, Johan; Låg, Marit; Schwarze, Per; Refsnes, Magne

    2014-08-15

    Amorphous silica nanoparticles (SiNPs) have previously been shown to induce marked cytokine (interleukin-6; IL-6 and interleukin-8; CXCL8/IL-8) responses independently of particle uptake in human bronchial epithelial BEAS-2B cells. In this study the involvement of the mitogen-activated protein kinases (MAP-kinases), nuclear factor-kappa Β (NF-κΒ) and in particular tumour necrosis factor-α converting enzyme (TACE) and—epidermal growth factor receptor (EGFR) signalling pathways were examined in triggering of IL-6 and CXCL8 release after exposure to a 50 nm silica nanoparticle (Si50). Exposure to Si50 increased phosphorylation of NF-κΒ p65 and MAP-kinases p38 and JUN-N-terminal protein kinase pathways (JNK), but not extracellular signal regulated kinases (ERK). Inhibition of NF-κΒ and p38 reduced the cytokine responses to Si50, whereas neither JNK- nor ERK-inhibition exerted any significant effect on the responses to Si50. Increases in membrane-bound transforming growth factor-α (TGF-α) release and EGFR phosphorylation were also observed after Si50 exposure, and pre-treatment with inhibitors of these pathways reduced the release of IL-6 and CXCL8, but did not affect the Si50-induced phosphorylation of p38 and p65. In contrast, p38-inhibition partially reduced Si50-induced TGF-α release, while the p65-inhibition was without effect. Overall, our results indicate that Si50-induced IL-6 and CXCL8 responses in BEAS-2B cells were regulated through combined activation of several pathways, including NF-κΒ and p38/TACE/TGF-α/EGFR signalling. The study identifies critical, initial events in the triggering of pro-inflammatory responses by nanoparticles. - Highlights: • Silica nanoparticles induce IL-6 and CXCL8 via NFκB and MAPKinase p38 in BEAS-2B • Silica nanoparticles induce release of the EGF-receptor ligand TGF-α • TGF-α release contributes to the IL-6 and CXCL8 release • Phosphorylation of p38 is involved in release of TGF-α.

  11. NiO nanoparticles induce apoptosis through repressing SIRT1 in human bronchial epithelial cells

    SciTech Connect

    Duan, Wei-Xia; He, Min-Di; Mao, Lin; Qian, Feng-Hua; Li, Yu-Ming; Pi, Hui-Feng; Liu, Chuan; Chen, Chun-Hai; Lu, Yong-Hui; Cao, Zheng-Wang; Zhang, Lei; Yu, Zheng-Ping; Zhou, Zhou

    2015-07-15

    With application of nano-sized nickel-containing particles (Nano-Ni) expanding, the health concerns about their adverse effects on the pulmonary system are increasing. However, the mechanisms for the pulmonary toxicity of these materials remain unclear. In the present study, we focused on the impacts of NiO nanoparticles (NiONPs) on sirtuin1 (SIRT1), a NAD-dependent deacetylase, and investigated whether SIRT1 was involved in NiONPs-induced apoptosis. Although the NiONPs tended to agglomerate in fluid medium, they still entered into the human bronchial epithelial cells (BEAS-2B) and released Ni{sup 2+} inside the cells. NiONPs at doses of 5, 10, and 20 μg/cm{sup 2} inhibited the cell viability. NiONPs' produced cytotoxicity was demonstrated through an apoptotic process, indicated by increased numbers of Annexin V positive cells and caspase-3 activation. The expression of SIRT1 was markedly down-regulated by the NiONPs, accompanied by the hyperacetylation of p53 (tumor protein 53) and overexpression of Bax (Bcl-2-associated X protein). However, overexpression of SIRT1 through resveratrol treatment or transfection clearly attenuated the NiONPs-induced apoptosis and activation of p53 and Bax. Our results suggest that the repression of SIRT1 may underlie the NiONPs-induced apoptosis via p53 hyperacetylation and subsequent Bax activation. Because SIRT1 participates in multiple biologic processes by deacetylation of dozens of substrates, this knowledge of the impact of NiONPs on SIRT1 may lead to an improved understanding of the toxic mechanisms of Nano-Ni and provide a molecular target to antagonize Nano-Ni toxicity. - Highlights: • NiONPs were taken up by BEAS-2B cells and released Ni{sup 2+}. • NiONPs produced cytotoxicity was demonstrated through an apoptotic process. • NiONPs repressed SIRT1 expression and activated p53 and Bax. • Overexpression of SIRT1 attenuated NiONPs-induced apoptosis via deacetylation p53.

  12. Silica induces NLRP3 inflammasome activation in human lung epithelial cells

    PubMed Central

    2013-01-01

    Background In myeloid cells the inflammasome plays a crucial role in innate immune defenses against pathogen- and danger-associated patterns such as crystalline silica. Respirable mineral particles impinge upon the lung epithelium causing irreversible damage, sustained inflammation and silicosis. In this study we investigated lung epithelial cells as a target for silica-induced inflammasome activation. Methods A human bronchial epithelial cell line (BEAS-2B) and primary normal human bronchial epithelial cells (NHBE) were exposed to toxic but nonlethal doses of crystalline silica over time to perform functional characterization of NLRP3, caspase-1, IL-1β, bFGF and HMGB1. Quantitative RT-PCR, caspase-1 enzyme activity assay, Western blot techniques, cytokine-specific ELISA and fibroblast (MRC-5 cells) proliferation assays were performed. Results We were able to show transcriptional and translational upregulation of the components of the NLRP3 intracellular platform, as well as activation of caspase-1. NLRP3 activation led to maturation of pro-IL-1β to secreted IL-1β, and a significant increase in the unconventional release of the alarmins bFGF and HMGB1. Moreover, release of bFGF and HMGB1 was shown to be dependent on particle uptake. Small interfering RNA experiments using siNLRP3 revealed the pivotal role of the inflammasome in diminished release of pro-inflammatory cytokines, danger molecules and growth factors, and fibroblast proliferation. Conclusion Our novel data indicate the presence and functional activation of the NLRP3 inflammasome by crystalline silica in human lung epithelial cells, which prolongs an inflammatory signal and affects fibroblast proliferation, mediating a cadre of lung diseases. PMID:23402370

  13. Molecular Mechanisms of Malignant Transformation by Low Dose Cadmium in Normal Human Bronchial Epithelial Cells

    PubMed Central

    Kluz, Thomas; Cohen, Lisa; Shen, Steven S.; Costa, Max

    2016-01-01

    Cadmium is a carcinogenic metal, the mechanisms of which are not fully understood. In this study, human bronchial epithelial cells were transformed with sub-toxic doses of cadmium (0.01, 0.05, and 0.1 μM) and transformed clones were characterized for gene expression changes using RNA-seq, as well as other molecular measurements. 440 genes were upregulated and 47 genes were downregulated in cadmium clones relative to control clones over 1.25-fold. Upregulated genes were associated mostly with gene ontology terms related to embryonic development, immune response, and cell movement, while downregulated genes were associated with RNA metabolism and regulation of transcription. Several embryonic genes were upregulated, including the transcription regulator SATB2. SATB2 is critical for normal skeletal development and has roles in gene expression regulation and chromatin remodeling. Small hairpin RNA knockdown of SATB2 significantly inhibited growth in soft agar, indicating its potential as a driver of metal-induced carcinogenesis. An increase in oxidative stress and autophagy was observed in cadmium clones. In addition, the DNA repair protein O6-methylguanine-DNA-methyltransferase was depleted by transformation with cadmium. MGMT loss caused significant decrease in cell viability after treatment with the alkylating agent temozolomide, demonstrating diminished capacity to repair such damage. Results reveal various mechanisms of cadmium-induced malignant transformation in BEAS-2B cells including upregulation of SATB2, downregulation of MGMT, and increased oxidative stress. PMID:27186882

  14. Activation of transcription factor IL-6 (NF-IL-6) and nuclear factor-kappaB (NF-kappaB) by lipid ozonation products is crucial to interleukin-8 gene expression in human airway epithelial cells.

    PubMed

    Kafoury, Ramzi M; Hernandez, Jazmir M; Lasky, Joseph A; Toscano, William A; Friedman, Mitchell

    2007-04-01

    Ozone (O(3)) is a major component of smog and an inhaled toxicant to the lung. O(3) rapidly reacts with the airway epithelial cell membrane phospholipids to generate lipid ozonation products (LOP). 1-Hydroxy-1-hydroperoxynonane (HHP-C9) is an important LOP, produced from the ozonation of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine. This LOP, at a biologically relevant concentration (100 microM), increases the activity of phospholipase C, nuclear factors-kappaB (NF-kappaB), and interleukin-6 (NF-IL-6) and the expression of the inflammatory gene, interleukin-8 (IL-8) in a cultured human bronchial epithelial cell line (BEAS-2B). The signaling pathways of ozone and its biologically-active products are as yet undefined. In the present study, we report that the HHP LOP, HHP-C9 (100 microM x 4 h), activated the expression of IL-8 (218 +/- 26% increase over control, n = 4, P < 0.01) through an apparent interaction between the two transcription factors, NF-kappaB and NF-IL-6. Transfection studies using luciferase reporter assays demonstrated that HHP-C9 induced a significant increase in NF-kappaB-DNA binding activity (37 +/- 7% increase over control, n = 6, P < 0.05). Inhibition of NF-kappaB showed a statistically significant but modest decrease in IL-8 release, which suggested a role for another transcription factor, NF-IL-6. Exposure of BEAS-2B cells to HHP-C9 induced a significant increase in the DNA binding activity of NF-IL-6 (45 +/- 11% increase over control, n = 6, P < 0.05). The results of the present study indicate that NF-IL-6 interacts with NF-kappaB in regulating the expression of IL-8 in cultured human airway epithelial cells exposed to LOP, the biological products of ozone in the lung. PMID:17366569

  15. Chronic occupational exposure to arsenic induces carcinogenic gene signaling networks and neoplastic transformation in human lung epithelial cells

    SciTech Connect

    Stueckle, Todd A.; Lu, Yongju; Davis, Mary E.; Wang, Liying; Jiang, Bing-Hua; Holaskova, Ida; Schafer, Rosana; Barnett, John B.; Rojanasakul, Yon

    2012-06-01

    Chronic arsenic exposure remains a human health risk; however a clear mode of action to understand gene signaling-driven arsenic carcinogenesis is currently lacking. This study chronically exposed human lung epithelial BEAS-2B cells to low-dose arsenic trioxide to elucidate cancer promoting gene signaling networks associated with arsenic-transformed (B-As) cells. Following a 6 month exposure, exposed cells were assessed for enhanced cell proliferation, colony formation, invasion ability and in vivo tumor formation compared to control cell lines. Collected mRNA was subjected to whole genome expression microarray profiling followed by in silico Ingenuity Pathway Analysis (IPA) to identify lung carcinogenesis modes of action. B-As cells displayed significant increases in proliferation, colony formation and invasion ability compared to BEAS-2B cells. B-As injections into nude mice resulted in development of primary and secondary metastatic tumors. Arsenic exposure resulted in widespread up-regulation of genes associated with mitochondrial metabolism and increased reactive oxygen species protection suggesting mitochondrial dysfunction. Carcinogenic initiation via reactive oxygen species and epigenetic mechanisms was further supported by altered DNA repair, histone, and ROS-sensitive signaling. NF-κB, MAPK and NCOR1 signaling disrupted PPARα/δ-mediated lipid homeostasis. A ‘pro-cancer’ gene signaling network identified increased survival, proliferation, inflammation, metabolism, anti-apoptosis and mobility signaling. IPA-ranked signaling networks identified altered p21, EF1α, Akt, MAPK, and NF-κB signaling networks promoting genetic disorder, altered cell cycle, cancer and changes in nucleic acid and energy metabolism. In conclusion, transformed B-As cells with their whole genome expression profile provide an in vitro arsenic model for future lung cancer signaling research and data for chronic arsenic exposure risk assessment. Highlights: ► Chronic As{sub 2}O

  16. Suppression of Epithelial Signal Transducer and Activator of Transcription 1 Activation by Extracts of Aspergillus fumigatus

    PubMed Central

    Bhushan, Bharat; Homma, Tetsuya; Norton, James E.; Sha, Quan; Siebert, Jason; Gupta, Dave S.; Schroeder, James W.

    2015-01-01

    Aspergillus fumigatus (AF) is often pathogenic in immune-deficient individuals and can cause life-threatening infections such as invasive aspergillosis. The pulmonary epithelial response to AF infection and the signaling pathways associated with it have not been completely studied. BEAS-2B cells or primary human bronchial epithelial cells were exposed to extracts of AF and challenged with IFN-β or the Toll-like receptor 3 agonist double-stranded RNA (dsRNA). Cytokine release (B-cell activating factor of the TNF family [BAFF], IFN-γ–induced protein-10 [IP-10], etc.) was assessed. AF extract was separated into low-molecular-weight (LMW) and high-molecular-weight (HMW) fractions using ultra 4 centrifugal force filters to characterize the activity. Real-time PCR was performed with a TaqMan method, and protein estimation was performed using ELISA techniques. Western blot was performed to assess phosphorylation of signal transducer and activator of transcription 1 (STAT1). IFN-β and dsRNA induced messenger RNA (mRNA) expression of BAFF (350- and 452-fold, respectively [n = 3]) and IP-10 (1,081- and 3,044-fold, respectively [n = 3]) in BEAS-2B cells. When cells were pretreated with AF extract for 1 hour and then stimulated with IFN-β or dsRNA for 6 hours, induction of BAFF and IP-10 mRNA was strongly suppressed relative to levels produced by IFN-β and dsRNA alone. When compared with control, soluble BAFF and IP-10 protein levels were maximally suppressed in dsRNA-stimulated wells treated with 1:320 wt/vol AF extract (P < 0.005). Upon molecular size fractionation, a LMW fraction of AF extract had no measurable suppressive effect on IP-10 mRNA expression. However, a HMW fraction of the AF extract significantly suppressed IP-10 expression in BEAS-2B cells that were stimulated with dsRNA or IFN-β. When BEAS-2B cells were pretreated with AF extract and then stimulated with IFN-β, reduced levels of pSTAT1 were observed, with maximum suppression at 4 and 6

  17. Suppression of epithelial signal transducer and activator of transcription 1 activation by extracts of Aspergillus fumigatus.

    PubMed

    Bhushan, Bharat; Homma, Tetsuya; Norton, James E; Sha, Quan; Siebert, Jason; Gupta, Dave S; Schroeder, James W; Schleimer, Robert P

    2015-07-01

    Aspergillus fumigatus (AF) is often pathogenic in immune-deficient individuals and can cause life-threatening infections such as invasive aspergillosis. The pulmonary epithelial response to AF infection and the signaling pathways associated with it have not been completely studied. BEAS-2B cells or primary human bronchial epithelial cells were exposed to extracts of AF and challenged with IFN-β or the Toll-like receptor 3 agonist double-stranded RNA (dsRNA). Cytokine release (B-cell activating factor of the TNF family [BAFF], IFN-γ-induced protein-10 [IP-10], etc.) was assessed. AF extract was separated into low-molecular-weight (LMW) and high-molecular-weight (HMW) fractions using ultra 4 centrifugal force filters to characterize the activity. Real-time PCR was performed with a TaqMan method, and protein estimation was performed using ELISA techniques. Western blot was performed to assess phosphorylation of signal transducer and activator of transcription 1 (STAT1). IFN-β and dsRNA induced messenger RNA (mRNA) expression of BAFF (350- and 452-fold, respectively [n = 3]) and IP-10 (1,081- and 3,044-fold, respectively [n = 3]) in BEAS-2B cells. When cells were pretreated with AF extract for 1 hour and then stimulated with IFN-β or dsRNA for 6 hours, induction of BAFF and IP-10 mRNA was strongly suppressed relative to levels produced by IFN-β and dsRNA alone. When compared with control, soluble BAFF and IP-10 protein levels were maximally suppressed in dsRNA-stimulated wells treated with 1:320 wt/vol AF extract (P < 0.005). Upon molecular size fractionation, a LMW fraction of AF extract had no measurable suppressive effect on IP-10 mRNA expression. However, a HMW fraction of the AF extract significantly suppressed IP-10 expression in BEAS-2B cells that were stimulated with dsRNA or IFN-β. When BEAS-2B cells were pretreated with AF extract and then stimulated with IFN-β, reduced levels of pSTAT1 were observed, with maximum suppression at 4 and 6

  18. Abiotic stress of ambient cold temperature regulates the host receptivity to pathogens by cell surfaced sialic acids.

    PubMed

    Moon, Seong-Cheol; Joo, Su-Yeon; Chung, Tae-Wook; Choi, Hee-Jung; Park, Mi-Ju; Choi, Hee-Jin; Bae, Sung-Jin; Kim, Keuk-Jun; Kim, Cheorl-Ho; Joo, Myungsoo; Ha, Ki-Tae

    2016-07-29

    Ambient cold temperature, as an abiotic stress, regulates the survival, stability, transmission, and infection of pathogens. However, the effect of cold temperature on the host receptivity to the pathogens has not been fully studied. In this study, the expression of terminal α-2,3- and α-2,6-sialic acids were increased in murine lung tissues, especially bronchial epithelium, by exposure to cold condition. The expression of several sialyltransferases were also increased by exposure to cold temperature. Furthermore, in human bronchial epithelial BEAS-2B cells, the expressions of α-2,3- and α-2,6-sialic acids, and mRNA levels of sialyltransferases were increased in the low temperature condition at 33 °C. On the other hand, the treatment of Lith-Gly, a sialyltransferase inhibitor, blocked the cold-induced expression of sialic acids on surface of BEAS-2B cells. The binding of influenza H1N1 hemagglutinin (HA) toward BEAS-2B cells cultured at low temperature condition was increased, compared to 37 °C. In contrast, the cold-increased HA binding was blocked by treatment of lithocholicglycine and sialyl-N-acetyl-D-lactosamines harboring α-2,3- and α-2,6-sialyl motive. These results suggest that the host receptivity to virus at cold temperature results from the expressions of α-2,3- and α-2,6-sialic acids through the regulation of sialyltransferase expression. PMID:27181350

  19. Particle complexation of mitochondrial iron produces superoxide generation and activates MAP kinases, NF-kappa B, nrf-2 in human respiratory epithelial cell

    EPA Science Inventory

    The biological effect of particles is associated with a disruption in cell iron homeostasis. We tested the postulate that complexation of cell iron by silica (Si02) results in both an oxidative stress and biological effect. BEAS-2B cells were exposed to either media or 100 ug/ml....

  20. Modulation of bronchial epithelial cell barrier function by in vitro jet propulsion fuel 8 exposure.

    PubMed

    Robledo, R F; Barber, D S; Witten, M L

    1999-09-01

    The loss of epithelial barrier integrity in bronchial and bronchiolar airways may be an initiating factor in the observed onset of toxicant-induced lung injuries. Acute 1-h inhalation exposures to aerosolized jet propulsion fuel 8 (JP-8) have been shown to induce cellular and morphological indications of pulmonary toxicity that was associated with increased respiratory permeability to 99mTc-DTPA. To address the hypothesis that JP-8 jet fuel-induced lung injury is initiated through a disruption in the airway epithelial barrier function, paracellular mannitol flux of BEAS-2B human bronchial epithelial cells was measured. Incubation of confluent cell cultures with non-cytotoxic concentrations of JP-8 or n-tetradecane (C14), a primary constituent of JP-8, for a 1-h exposure period resulted in dose-dependent increases of paracellular flux. Following exposures of 0.17, 0.33, 0.50, or 0.67 mg/ml, mannitol flux increased above vehicle controls by 10, 14, 29, and 52%, respectively, during a 2-h incubation period immediately after each JP-8 exposure. C14 caused greater mannitol flux increases of 37, 42, 63, and 78%, respectively, following identical exposure conditions. The effect on transepithelial mannitol flux reached a maximum at 12 h and spontaneously reversed to control values over a 48-h recovery period, for both JP-8 and C14 exposure. These data indicate that non-cytotoxic exposures to JP-8 or C14 exert a noxious effect on bronchial epithelial barrier function that may preclude pathological lung injury. PMID:10496683

  1. Nuclear Factor κB1/RelA Mediates Inflammation in Human Lung Epithelial Cells at Atmospheric Oxygen Levels.

    PubMed

    Jagannathan, Lakshmanan; Jose, Cynthia C; Arita, Adriana; Kluz, Thomas; Sun, Hong; Zhang, Xiaoru; Yao, Yixin; Kartashov, Andrey V; Barski, Artem; Costa, Max; Cuddapah, Suresh

    2016-07-01

    Oxygen levels range from 2% to 9% in vivo. Atmospheric O2 levels (21%) are known to induce cell proliferation defects and cellular senescence in primary cell cultures. However, the mechanistic basis of the deleterious effects of higher O2 levels is not fully understood. On the other hand, immortalized cells including cancer cell lines, which evade cellular senescence are normally cultured at 21% O2 and the effects of higher O2 on these cells are understudied. Here, we addressed this problem by culturing immortalized human bronchial epithelial (BEAS-2B) cells at ambient atmospheric, 21% O2 and lower, 10% O2. Our results show increased inflammatory response at 21% O2 but not at 10% O2. We found higher RelA binding at the NF-κB1/RelA target gene promoters as well as upregulation of several pro-inflammatory cytokines in cells cultured at 21% O2. RelA knockdown prevented the upregulation of the pro-inflammatory cytokines at 21% O2, suggesting NF-κB1/RelA as a major mediator of inflammatory response in cells cultured at 21% O2. Interestingly, unlike the 21% O2 cultured cells, exposure of 10% O2 cultured cells to H2O2 did not elicit inflammatory response, suggesting increased ability to tolerate oxidative stress in cells cultured at lower O2 levels. PMID:26588041

  2. Nicotine mediates oxidative stress and apoptosis through cross talk between NOX1 and Bcl-2 in lung epithelial cells.

    PubMed

    Zanetti, Filippo; Giacomello, Marta; Donati, Yves; Carnesecchi, Stephanie; Frieden, Maud; Barazzone-Argiroffo, Constance

    2014-11-01

    Nicotine contributes to the onset and progression of several pulmonary diseases. Among the various pathophysiological mechanisms triggered by nicotine, oxidative stress and cell death are reported in several cell types. We found that chronic exposure to nicotine (48h) induced NOX1-dependent oxidative stress and apoptosis in primary pulmonary cells. In murine (MLE-12) and human (BEAS-2B) lung epithelial cell lines, nicotine acted as a sensitizer to cell death and synergistically enhanced apoptosis when cells were concomitantly exposed to hyperoxia. The precise signaling pathway was investigated in MLE-12 cells in which NOX1 was abrogated by a specific inhibitor or stably silenced by shRNA. In the early phase of exposure (1h), nicotine mediated intracellular Ca(2+) fluxes and activation of protein kinase C, which in its turn activated NOX1, leading to cellular and mitochondrial oxidative stress. The latter triggered the intrinsic apoptotic machinery by modulating the expression of Bcl-2 and Bax. Overexpression of Bcl-2 completely prevented nicotine's detrimental effects, suggesting Bcl-2as a downstream key regulator in nicotine/NOX1-induced cell damage. These results suggest that NOX1 is a major contributor to the generation of intracellular oxidative stress induced by nicotine and might be an important molecule to target in nicotine-related lung pathologies. PMID:25151121

  3. Alteration of canonical and non-canonical WNT-signaling by crystalline silica in human lung epithelial cells.

    PubMed

    Perkins, Timothy N; Dentener, Mieke A; Stassen, Frank R; Rohde, Gernot G; Mossman, Brooke T; Wouters, Emiel F M; Reynaert, Niki L

    2016-06-15

    Growth and development of the mature lung is a complex process orchestrated by a number of intricate developmental signaling pathways. Wingless-type MMTV-integration site (WNT) signaling plays critical roles in controlling branching morphogenesis cell differentiation, and formation of the conducting and respiratory airways. In addition, WNT pathways are often re-activated in mature lungs during repair and regeneration. WNT- signaling has been elucidated as a crucial contributor to the development of idiopathic pulmonary fibrosis as well as other hyper-proliferative lung diseases. Silicosis, a detrimental occupational lung disease caused by excessive inhalation of crystalline silica dust, is hallmarked by repeated cycles of damaging inflammation, epithelial hyperplasia, and formation of dense, hyalinized nodules of whorled collagen. However, mechanisms of epithelial cell hyperplasia and matrix deposition are not well understood, as most research efforts have focused on the pronounced inflammatory response. Microarray data from our previous studies has revealed a number of WNT-signaling and WNT-target genes altered by crystalline silica in human lung epithelial cells. In the present study, we utilize pathway analysis to designate connections between genes altered by silica in WNT-signaling networks. Furthermore, we confirm microarray findings by QRT-PCR and demonstrate both activation of canonical (β-catenin) and down-regulation of non-canonical (WNT5A) signaling in immortalized (BEAS-2B) and primary (PBEC) human bronchial epithelial cells. These findings suggest that WNT-signaling and cross-talk with other pathways (e.g. Notch), may contribute to proliferative, fibrogenic and inflammatory responses to silica in lung epithelial cells. PMID:27095093

  4. Evaluation of uptake, cytotoxicity and inflammatory effects in respiratory cells exposed to pristine and -OH and -COOH functionalized multi-wall carbon nanotubes.

    PubMed

    Ursini, Cinzia Lucia; Maiello, Raffaele; Ciervo, Aureliano; Fresegna, Anna Maria; Buresti, Giuliana; Superti, Fabiana; Marchetti, Magda; Iavicoli, Sergio; Cavallo, Delia

    2016-03-01

    Toxic effects were reported for pristine-multi-wall carbon nanotubes (p-MWCNTs) while the role of the functionalization on MWCNT-induced toxicity is not yet well defined. We evaluated on human alveolar (A549) epithelial cells and normal bronchial (BEAS-2B) cells exposed to p-MWCNTs, MWCNTs-OH and MWCNTs-COOH: uptake by TEM, cell viability by different assays, membrane damage by the LDH assay and cytokine release by ELISA. The aims of the present study were to: (i) confirm MWCNT cytotoxicity mechanisms hypothesized in our previous studies; (ii) identify the most reliable viability assay to screen MWCNT toxicity; and (iii) to test our model to clarify the role of functionalization on MWCNT-induced toxicity. In A549 cells, p-MWCNTs and MWCNTs-OH were localized free in the cytoplasm and inside vacuoles whereas MWCNTs-COOH were confined inside filled cytoplasmic vesicles. WST-1 and Trypan blue assays showed in A549 cells a similar slight viability reduction for all MWCNTs whereas in BEAS-2B cells WST1 showed a high viability reduction at the highest concentrations, particularly for MWCNTs-COOH. The MTT assay showed a false cytotoxicity as a result of MWCNTs-interference. Pristine and MWCNTs-COOH induced membrane damage, particularly in BEAS-2B cells. MWCNTs-COOH induced interleukin-6 (IL-6) and IL-8 release in A549 cells whereas p-MWCNTs induced IL-8 release in BEAS-2B cells. MWCNTs intracellular localization in A549 cells confirms the toxicity mechanisms previously hypothesized, with p-MWCNTs disrupting the membrane and vesicle-confined MWCNTs-COOH inducing inflammation. WST-1 was more reliable than MTT to test MWCNT-toxicity. BEAS-2B cells were more susceptible then A549 cells, particularly to MWCNT-COOH cytotoxicity. Our results confirm the toxicity of p-MWCNTs and demonstrate, also for the two kinds of tested functionalized MWCNTs toxic effects with a different mechanism of action. PMID:26370214

  5. ADAM28 is expressed by epithelial cells in human normal tissues and protects from C1q-induced cell death.

    PubMed

    Miyamae, Yuka; Mochizuki, Satsuki; Shimoda, Masayuki; Ohara, Kentaro; Abe, Hitoshi; Yamashita, Shuji; Kazuno, Saiko; Ohtsuka, Takashi; Ochiai, Hiroki; Kitagawa, Yuko; Okada, Yasunori

    2016-05-01

    ADAM28 (disintegrin and metalloproteinase 28), which was originally reported to be lymphocyte-specific, is over-expressed by carcinoma cells and plays a key role in cell proliferation and progression in human lung and breast carcinomas. We studied ADAM28 expression in human normal tissues and examined its biological function. By using antibodies specific to ADAM28, ADAM28 was immunolocalized mainly to epithelial cells in several tissues, including epididymis, bronchus and stomach, whereas lymphocytes in lymph nodes and spleen were negligibly immunostained. RT-PCR, immunoblotting and ELISA analyses confirmed the expression in these tissues, and low or negligible expression by lymphocytes was found in the lymph node and spleen. C1q was identified as a candidate ADAM28-binding protein from a human lung cDNA library by yeast two-hybrid system, and specific binding was demonstrated by binding assays, immunoprecipitation and surface plasmon resonance. C1q treatment of normal bronchial epithelial BEAS-2B and NHBE cells, both of which showed low-level expression of ADAM28, caused apoptosis through activation of p38 and caspase-3, and cell death with autophagy through accumulation of LC3-II and autophagosomes, respectively. C1q-induced cell death was attenuated by treatment of the cells with antibodies against the C1q receptor gC1qR/p33 or cC1qR/calreticulin. Treatment of C1q with recombinant ADAM28 prior to addition to culture media reduced C1q-induced cell death, and knockdown of ADAM28 using siRNAs increased cell death. These data demonstrate that ADAM28 is expressed by epithelial cells of several normal organs, and suggest that ADAM28 plays a role in cell survival by suppression of C1q-induced cytotoxicity in bronchial epithelial cells. PMID:26918856

  6. The Effect of Therapeutic Blockades of Dust Particles-Induced Ca²⁺ Signaling and Proinflammatory Cytokine IL-8 in Human Bronchial Epithelial Cells.

    PubMed

    Yoon, Ju Hee; Jeong, Sung Hwan; Hong, Jeong Hee

    2015-01-01

    Bronchial epithelial cells are the first barrier of defense against respiratory pathogens. Dust particles as extracellular stimuli are associated with inflammatory reactions after inhalation. It has been reported that dust particles induce intracellular Ca(2+) signal, which subsequently increases cytokines production such as interleukin- (IL-) 8. However, the study of therapeutic blockades of Ca(2+) signaling induced by dust particles in human bronchial epithelial cells is poorly understood. We investigated how to modulate dust particles-induced Ca(2+) signaling and proinflammatory cytokine IL-8 expression. Bronchial epithelial BEAS-2B cells were exposed to PM10 dust particles and subsequent mediated intracellular Ca(2+) signaling and reactive oxygen species signal. Our results show that exposure to several inhibitors of Ca(2+) pathway attenuated the PM10-induced Ca(2+) response and subsequent IL-8 mRNA expression. PM10-mediated Ca(2+) signal and IL-8 expression were attenuated by several pharmacological blockades such as antioxidants, IP3-PLC blockers, and TRPM2 inhibitors. Our results show that blockades of PLC or TRPM2 reduced both of PM10-mediated Ca(2+) signal and IL-8 expression, suggesting that treatment with these blockades should be considered for potential therapeutic trials in pulmonary epithelium for inflammation caused by environmental events such as seasonal dust storm. PMID:26640326

  7. Chitin-Induced Airway Epithelial Cell Innate Immune Responses Are Inhibited by Carvacrol/Thymol.

    PubMed

    Khosravi, Ali Reza; Erle, David J

    2016-01-01

    Chitin is produced in large amounts by fungi, insects, and other organisms and has been implicated in the pathogenesis of asthma. Airway epithelial cells are in direct contact with environmental particles and serve as the first line of defense against inhaled allergens and pathogens. The potential contributions of airway epithelial cells to chitin-induced asthma remain poorly understood. We hypothesized that chitin directly stimulates airway epithelial cells to release cytokines that promote type 2 immune responses and to induce expression of molecules which are important in innate immune responses. We found that chitin exposure rapidly induced the expression of three key type 2-promoting cytokines, IL-25, IL-33 and TSLP, in BEAS-2B transformed human bronchial epithelial cells and in A549 and H292 lung carcinoma cells. Chitin also induced the expression of the key pattern recognition receptors TLR2 and TLR4. Chitin induced the expression of miR-155, miR-146a and miR-21, each of which is known to up-regulate the expression of pro-inflammatory cytokines. Also the expression of SOCS1 and SHIP1 which are known targets of miR-155 was repressed by chitin treatment. The monoterpene phenol carvacrol (Car) and its isomer thymol (Thy) are found in herbal essential oils and have been shown to inhibit allergic inflammation in asthma models. We found that Car/Thy inhibited the effects of chitin on type 2-promoting cytokine release and on the expression of TLRs, SOCS1, SHIP1, and miRNAs. Car/Thy could also efficiently reduce the protein levels of TLR4, inhibit the increase in TLR2 protein levels in chitin plus Car/Thy-treated cells and increase the protein levels of SHIP1 and SOCS1, which are negative regulators of TLR-mediated inflammatory responses. We conclude that direct effects of chitin on airway epithelial cells are likely to contribute to allergic airway diseases like asthma, and that Car/Thy directly inhibits epithelial cell pro-inflammatory responses to chitin. PMID

  8. Chitin-Induced Airway Epithelial Cell Innate Immune Responses Are Inhibited by Carvacrol/Thymol

    PubMed Central

    Erle, David J.

    2016-01-01

    Chitin is produced in large amounts by fungi, insects, and other organisms and has been implicated in the pathogenesis of asthma. Airway epithelial cells are in direct contact with environmental particles and serve as the first line of defense against inhaled allergens and pathogens. The potential contributions of airway epithelial cells to chitin-induced asthma remain poorly understood. We hypothesized that chitin directly stimulates airway epithelial cells to release cytokines that promote type 2 immune responses and to induce expression of molecules which are important in innate immune responses. We found that chitin exposure rapidly induced the expression of three key type 2-promoting cytokines, IL-25, IL-33 and TSLP, in BEAS-2B transformed human bronchial epithelial cells and in A549 and H292 lung carcinoma cells. Chitin also induced the expression of the key pattern recognition receptors TLR2 and TLR4. Chitin induced the expression of miR-155, miR-146a and miR-21, each of which is known to up-regulate the expression of pro-inflammatory cytokines. Also the expression of SOCS1 and SHIP1 which are known targets of miR-155 was repressed by chitin treatment. The monoterpene phenol carvacrol (Car) and its isomer thymol (Thy) are found in herbal essential oils and have been shown to inhibit allergic inflammation in asthma models. We found that Car/Thy inhibited the effects of chitin on type 2-promoting cytokine release and on the expression of TLRs, SOCS1, SHIP1, and miRNAs. Car/Thy could also efficiently reduce the protein levels of TLR4, inhibit the increase in TLR2 protein levels in chitin plus Car/Thy-treated cells and increase the protein levels of SHIP1 and SOCS1, which are negative regulators of TLR-mediated inflammatory responses. We conclude that direct effects of chitin on airway epithelial cells are likely to contribute to allergic airway diseases like asthma, and that Car/Thy directly inhibits epithelial cell pro-inflammatory responses to chitin. PMID

  9. Indications for distinct pathogenic mechanisms of asbestos and silica through gene expression profiling of the response of lung epithelial cells

    PubMed Central

    Perkins, Timothy N.; Peeters, Paul M.; Shukla, Arti; Arijs, Ingrid; Dragon, Julie; Wouters, Emiel F.M.; Reynaert, Niki L.; Mossman, Brooke T.

    2015-01-01

    Occupational and environmental exposures to airborne asbestos and silica are associated with the development of lung fibrosis in the forms of asbestosis and silicosis, respectively. However, both diseases display distinct pathologic presentations, likely associated with differences in gene expression induced by different mineral structures, composition and bio-persistent properties. We hypothesized that effects of mineral exposure in the airway epithelium may dictate deviating molecular events that may explain the different pathologies of asbestosis versus silicosis. Using robust gene expression-profiling in conjunction with in-depth pathway analysis, we assessed early (24 h) alterations in gene expression associated with crocidolite asbestos or cristobalite silica exposures in primary human bronchial epithelial cells (NHBEs). Observations were confirmed in an immortalized line (BEAS-2B) by QRT-PCR and protein assays. Utilization of overall gene expression, unsupervised hierarchical cluster analysis and integrated pathway analysis revealed gene alterations that were common to both minerals or unique to either mineral. Our findings reveal that both minerals had potent effects on genes governing cell adhesion/migration, inflammation, and cellular stress, key features of fibrosis. Asbestos exposure was most specifically associated with aberrant cell proliferation and carcinogenesis, whereas silica exposure was highly associated with additional inflammatory responses, as well as pattern recognition, and fibrogenesis. These findings illustrate the use of gene-profiling as a means to determine early molecular events that may dictate pathological processes induced by exogenous cellular insults. In addition, it is a useful approach for predicting the pathogenicity of potentially harmful materials. PMID:25351596

  10. Potentiation of NF-κB-dependent transcription and inflammatory mediator release by histamine in human airway epithelial cells

    PubMed Central

    Holden, N S; Gong, W; King, E M; Kaur, M; Giembycz, M A; Newton, R

    2007-01-01

    Background and purpose: In asthma, histamine contributes to bronchoconstriction, vasodilatation and oedema, and is associated with the late phase response. The current study investigates possible inflammatory effects of histamine acting on nuclear factor κB (NF-κB)-dependent transcription and cytokine release. Experimental approach: Using BEAS-2B bronchial epithelial cells, NF-κB-dependent transcription and both release and mRNA expression of IL-6 and IL-8 were examined by reporter assay, ELISA and quantitative RT-PCR. Histamine receptors were detected using qualitative RT-PCR and function examined using selective agonists and antagonists. Key results: Addition of histamine to TNFα-stimulated BEAS-2B cells maximally potentiated NF-κB-dependent transcription 1.8 fold, whereas IL-6 and IL-8 protein release were enhanced 7.3- and 2.7-fold respectively. These responses were, in part, NF-κB-dependent and were associated with 2.6- and 1.7-fold enhancements of IL-6 and IL-8 mRNA expression. The H1 receptor antagonist, mepyramine, caused a rightward shift in the concentration-response curves of TNFα-induced NF-κB-dependent transcription (pA2=9.91) and release of IL-6 (pA2=8.78) and IL-8 (pA2=8.99). Antagonists of histamine H2, H3 and H4 receptors were without effect. Similarly, H3 and H4 receptor agonists did not affect TNFα-induced NF-κB-dependent transcription, or IL-6 and IL-8 release at concentrations below 10 μM. The anti-inflammatory glucocorticoid, dexamethasone, inhibited the histamine enhanced NF-κB-dependent transcription and IL-6 and IL-8 release. Conclusions and implications: Potentiation of NF-κB-dependent transcription and inflammatory cytokine release by histamine predominantly involves receptors of the H1 receptor subtype. These data support an anti-inflammatory role for H1 receptor antagonists by preventing the transcription and release of pro-inflammatory cytokines. PMID:17891168

  11. Influence of platinum, palladium and rhodium as compared with cadmium, nickel and chromium on cell viability and oxidative stress in human bronchial epithelial cells.

    PubMed

    Schmid, Michael; Zimmermann, Sonja; Krug, Harald F; Sures, Bernd

    2007-04-01

    The platinum group elements (PGE) Pt, Pd and Rh are increasingly emitted into the environment by automobile catalytic converters. Whereas the biological availability of PGE to plants and animals has been demonstrated, only few studies concentrate on the influence of PGE on a cellular level. The effects of Pt, Pd and Rh compared with Cd, Ni and Cr on cell viability and oxidative stress response using soluble metal salts were studied in the human bronchial epithelial cell line BEAS-2B. Whilst Rh(III) showed little influence, both Pt(II) and Pt(IV) as well as Pd(II) had significant effects on cell viability at levels comparable to Cd(II) and Cr(VI). Arranging metal species in order of increasing toxicity as determined by LC50 yields: Rh(III)=1.2 mmol/Lcell stress. The maximum relative increase in ROS production for Pt(IV) (1134%) was more than twice as high as for Cr(VI) (560%), with Pt(II) still resulting in an increase of 238%. These findings underline the strong effects of PGE on cell metabolism and emphasize the need for further studies of their toxic properties. PMID:17250893

  12. Effects of air pollution-related heavy metals on the viability and inflammatory responses of human airway epithelial cells.

    PubMed

    Honda, Akiko; Tsuji, Kenshi; Matsuda, Yugo; Hayashi, Tomohiro; Fukushima, Wataru; Sawahara, Takahiro; Kudo, Hitomi; Murayama, Rumiko; Takano, Hirohisa

    2015-01-01

    Various metals produced from human activity are ubiquitously detected in ambient air. The metals may lead to induction and/or exacerbation of respiratory diseases, but the significant metals and factors contributing to such diseases have not been identified. To compare the effects of each metal and different oxidation states of metals on human airway, we examined the viability and production of interleukin (IL)-6 and IL-8 using BEAS-2B cell line, derived from human airway epithelial cells. Airway epithelial cells were exposed to Mn(2+), V(4+), V(5+), Cr(3+), Cr(6+), Zn(2+), Ni(2+), and Pb(2+) at a concentration of 0.5, 5, 50, or 500 μmol/L for 24 hours. Mn and V decreased the cell viability in a concentration-dependent manner, and V(5+) tended to have a greater effect than V(4+). The Cr decreased the cell viability, and (Cr(+6)) at concentrations of 50 and 500 μmol/L was more toxic than (Cr(+3)). Zn at a concentration of 500 μmol/L greatly decreased the cell viability, whereas Ni at the same concentration increased it. Pb produced fewer changes. Mn and Ni at a concentration of 500 μmol/L induced the significant production of IL-6 and IL-8. However, most of the metals including (V(+4), V(+5)), (Cr(+3), Cr(+6)), Zn, and Pb inhibited the production of both IL-6 and IL-8. The present results indicate that various heavy metals have different effects on toxicity and the proinflammatory responses of airway epithelial cells, and those influences also depend on the oxidation states of the metals. PMID:25808165

  13. Kaempferol Inhibits Endoplasmic Reticulum Stress-Associated Mucus Hypersecretion in Airway Epithelial Cells And Ovalbumin-Sensitized Mice

    PubMed Central

    Choi, Yean-Jung; Kang, Min-Kyung; Kim, Yun-Ho; Kang, Young-Hee

    2015-01-01

    Mucus hypersecretion is an important pathological feature of chronic airway diseases, such as asthma and pulmonary diseases. MUC5AC is a major component of the mucus matrix forming family of mucins in the airways. The initiation of endoplasmic reticulum (ER)-mediated stress responses contributes to the pathogenesis of airway diseases. The present study investigated that ER stress was responsible for airway mucus production and this effect was blocked by the flavonoid kaempferol. Oral administration of ≥10 mg/kg kaempferol suppressed mucus secretion and goblet cell hyperplasia observed in the bronchial airway and lung of BALB/c mice sensitized with ovalbumin (OVA). TGF-β and tunicamycin promoted MUC5AC induction after 72 h in human bronchial airway epithelial BEAS-2B cells, which was dampened by 20 μM kaempferol. Kaempferol inhibited tunicamycin-induced ER stress of airway epithelial cells through disturbing the activation of the ER transmembrane sensor ATF6 and IRE1α. Additionally, this compound demoted the induction of ER chaperones such as GRP78 and HSP70 and the splicing of XBP-1 mRNA by tunicamycin. The in vivo study further revealed that kaempferol attenuated the induction of XBP-1 and IRE1α in epithelial tissues of OVA-challenged mice. TGF-β and tunicamycin induced TRAF2 with JNK activation and such induction was deterred by kaempferol. The inhibition of JNK activation encumbered the XBP-1 mRNA splicing and MUC5AC induction by tunicamycin and TGF-β. These results demonstrate that kaempferol alleviated asthmatic mucus hypersecretion through blocking bronchial epithelial ER stress via the inhibition of IRE1α-TRAF2-JNK activation. Therefore, kaempferol may be a potential therapeutic agent targeting mucus hypersecretion-associated pulmonary diseases. PMID:26599511

  14. Epithelial Cell Adhesion Molecule

    PubMed Central

    Trzpis, Monika; McLaughlin, Pamela M.J.; de Leij, Lou M.F.H.; Harmsen, Martin C.

    2007-01-01

    The epithelial cell adhesion molecule (EpCAM, CD326) is a glycoprotein of ∼40 kd that was originally identified as a marker for carcinoma, attributable to its high expression on rapidly proliferating tumors of epithelial origin. Normal epithelia express EpCAM at a variable but generally lower level than carcinoma cells. In early studies, EpCAM was proposed to be a cell-cell adhesion molecule. However, recent insights revealed a more versatile role for EpCAM that is not limited only to cell adhesion but includes diverse processes such as signaling, cell migration, proliferation, and differentiation. Cell surface expression of EpCAM may actually prevent cell-cell adhesion. Here, we provide a comprehensive review of the current knowledge on EpCAM biology in relation to other cell adhesion molecules. We discuss the implications of the newly identified functions of EpCAM in view of its prognostic relevance in carcinoma, inflammatory pathophysiology, and tissue development and regeneration as well as its role in normal epithelial homeostasis. PMID:17600130

  15. 4-Methoxyestradiol-induced oxidative injuries in human lung epithelial cells

    SciTech Connect

    Cheng Yahsin; Chang, Louis W.; Cheng Lichuan; Tsai, M.-H.; Lin Pinpin . E-mail: pplin@nhri.org.tw

    2007-05-01

    Epidemiological studies indicated that people exposed to dioxins were prone to the development of lung diseases including lung cancer. Animal studies demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increased liver tumors and promoted lung metaplasia in females. Metabolic changes in 17{beta}-estradiol (E{sub 2}) resulted from an interaction between TCDD and E{sub 2} could be associated with gender difference. Previously, we reported that methoxylestradiols (MeOE{sub 2}), especially 4-MeOE{sub 2}, accumulated in human lung cells (BEAS-2B) co-treated with TCDD and E{sub 2}. In the present study, we demonstrate unique accumulation of 4-MeOE{sub 2}, as a result of TCDD/E{sub 2} interaction and revealed its bioactivity in human lung epithelial cell line (H1355). 4-Methoxyestradiol treatment significantly decreased cell growth and increased mitotic index. Elevation of ROS and SOD activity, with a concomitant decrease in the intracellular GSH/GSSG ratio, was also detected in 4-MeOE{sub 2}-treated cells. Quantitative comet assay showed increased oxidative DNA damage in the 4-MeOE{sub 2}-treated H1355 cells, which could be significantly reduced by the anti-oxidant N-acetylcysteine (NAC). However, inhibition of cell growth and increase in mitotic arrest induced by 4-MeOE{sub 2} were unaffected by NAC. We concluded that 4-MeOE{sub 2} accumulation resulting from TCDD and E{sub 2} interaction would contribute to the higher vulnerability on lung pathogenesis in females when exposed to TCDD.

  16. Proteomic Assessment of Biochemical Pathways That Are Critical to Nickel-Induced Toxicity Responses in Human Epithelial Cells.

    PubMed

    Ge, Yue; Bruno, Maribel; Haykal-Coates, Najwa; Wallace, Kathleen; Andrews, Debora; Swank, Adam; Winnik, Witold; Ross, Jeffrey A

    2016-01-01

    Understanding the mechanisms underlying toxicity initiated by nickel, a ubiquitous environmental contaminant and known human carcinogen is necessary for proper assessment of its risks to human and environment. Among a variety of toxic mechanisms, disruption of protein responses and protein response-based biochemical pathways represents a key mechanism through which nickel induces cytotoxicity and carcinogenesis. To identify protein responses and biochemical pathways that are critical to nickel-induced toxicity responses, we measured cytotoxicity and changes in expression and phosphorylation status of 14 critical biochemical pathway regulators in human BEAS-2B cells exposed to four concentrations of nickel using an integrated proteomic approach. A subset of the pathway regulators, including interleukin-6, and JNK, were found to be linearly correlated with cell viability, and may function as molecular determinants of cytotoxic responses of BEAS-2B cells to nickel exposures. In addition, 128 differentially expressed proteins were identified by two dimensional electrophoresis (2-DE) and mass spectrometry. Principal component analysis, hierarchical cluster analyses, and ingenuity signaling pathway analysis (IPA) identified putative nickel toxicity pathways. Some of the proteins and pathways identified have not previously been linked to nickel toxicity. Based on the consistent results obtained from both ELISA and 2-DE proteomic analysis, we propose a core signaling pathway regulating cytotoxic responses of human BEAS-2B cells to nickel exposures, which integrates a small set of proteins involved in glycolysis and gluconeogenesis pathways, apoptosis, protein degradation, and stress responses including inflammation and oxidative stress. PMID:27626938

  17. Proteome profiling of cadmium-induced apoptosis by antibody array analyses in human bronchial epithelial cells

    PubMed Central

    Xu, Yan-Ming; Yu, Fei-Yuan; Yang, Feng; Yao, Yue; Zhou, Yuan; Ching, Yick-Pang; Lau, Andy T. Y.

    2016-01-01

    Protein array technology is a powerful platform for the simultaneous determination of the expression levels of a number of proteins as well as post-translational modifications such as phosphorylation. Here, we screen and report for the first time, the dominant signaling cascades and apoptotic mediators during the course of cadmium (Cd)-induced cytotoxicity in human bronchial epithelial cells (BEAS-2B) by antibody array analyses. Proteins from control and Cd-treated cells were captured on Proteome Profiler™ Arrays for the parallel determination of the relative levels of protein phosphorylation and proteins associated with apoptosis. Our results indicated that the p38 MAPK- and JNK-related signal transduction pathways were dramatically activated by Cd treatment. Cd potently stimulates the phosphorylations of p38α (MAPK14), JNK1/2 (MAPK8/9), and JUN; while the phosphorylations of Akt1, ERK1/2 (MAPK3/1), GSK3β, and mTOR were suppressed. Moreover, there was an induction of proapoptotic protein BAX, release of cytochrome c (CYCS) from mitochondria, activation of caspase-3/9 (CASP3/9); as well as decreased expression of cell cycle checkpoint proteins (TP53, p21, and p27) and several inhibitors of apoptosis proteins (IAPs) [including cIAP-1/2 (BIRC2/3), XIAP (BIRC4), and survivin (BIRC5)]. Pretreatment of cells with the thiol antioxidant glutathione or p38 MAPK/JNK inhibitors before Cd treatment effectively abrogated ROS activation of p38 MAPK/JNK pathways and apoptosis-related proteins. Taken together, our results demonstrate that Cd causes oxidative stress-induced apoptosis; and the p38 MAPK/JNK and mitochondrial pathways are more importantly participated for signal transduction and the induction of apoptosis in Cd-exposed human lung cells. PMID:26716417

  18. Comparative in vitro studies on the fibrogenic effects of two samples of silica on epithelial bronchial cells.

    PubMed

    Bodo, M; Muzi, G; Bellucci, C; Lilli, C; Calvitti, M; Lumare, A; Dell'Omo, M; Gambelunghe, A; Baroni, T; Murgia, N

    2007-01-01

    The small dimension and particle shape of silica in gypsum used to prepare moulds for lost wax casting might be responsible for the high prevalence of silicosis in gold jewellery. To test this hypothesis, human pulmonary epithelial cell (BEAS-2B) cultures were exposed to two samples of silica with different crystal micro-morphologies: Silica Powder (Silica P) which is used in casting gold jewellery, and no powder Silica (Silica F). Extracellular matrix (ECM) production was evaluated using radio-labelled precursors and quantified by RT-PCR analysis. Expression of basic fibroblast growth factor (FGF2) and its receptor (FGFR2) was also evaluated. The results demonstrated Silica P particles had a very fine lamellar crystalline structure while Silica F was characterized by larger rounded crystals. Silica P stimulated collagen production significantly more than Silica F and downregulated laminin and metalloprotease expression. Both silica samples down-regulated FGF2 but only Silica F enhanced FGF2 receptor expression. In conclusion each Silica sample promoted a profibrotic lung microenvironment in a different manner and also elicited different FGF2 signalling pathways. The data confirm that different micromorphology of Silica particles affects the fibrogenic potential and the molecular mechanisms of dust pathogenicity. PMID:18261261

  19. Carbon Nanotube Uptake Changes the Biomechanical Properties of Human Lung Epithelial Cells in a Time-dependent Manner

    PubMed Central

    Dong, Chenbo; Eldawud, Reem; Sargent, Linda M.; Kashon, Michael L.; Lowry, David; Rojanasakul, Yon

    2015-01-01

    The toxicity of engineered nanomaterials in biological systems depends on both the nanomaterial properties and the exposure duration. Herein we used a multi-tier strategy to investigate the relationship between user-characterized multi-walled carbon nanotubes (MWCNTs) exposure duration and their induced biochemical and biomechanical effects on model human lung epithelial cells (BEAS-2B). Our results showed that exposure to MWCNTs leads to time-dependent intracellular uptake and generation of reactive oxygen species (ROS), along with time-dependent gradual changes in cellular biomechanical properties. In particular, the amount of internalized MWCNTs followed a sigmoidal curve with the majority of the MWCNTs being internalized within 6h of exposure; further, the sigmoidal uptake correlated with the changes in the oxidative levels and cellular biomechanical properties respectively. Our study provides new insights into the time-dependent induced toxicity caused by exposure to occupationally relevant doses of MWCNTs and could potentially help establish bases for early risk assessments of other nanomaterials toxicological profiles. PMID:26146559

  20. Nickel Mobilizes Intracellular Zinc to Induce Metallothionein in Human Airway Epithelial Cells

    PubMed Central

    Nemec, Antonia A.; Leikauf, George D.; Pitt, Bruce R.; Wasserloos, Karla J.; Barchowsky, Aaron

    2009-01-01

    We recently reported that induction of metallothionein (MT) was critical in limiting nickel (Ni)-induced lung injury in intact mice. Nonetheless, the mechanism by which Ni induces MT expression is unclear. We hypothesized that the ability of Ni to mobilize zinc (Zn) may contribute to such regulation and therefore, we examined the mechanism for Ni-induced MT2A expression in human airway epithelial (BEAS-2B) cells. Ni induced MT2A transcript levels and protein expression by 4 hours. Ni also increased the activity of a metal response element (MRE) promoter luciferase reporter construct, suggesting that Ni induces MRE binding of the metal transcription factor (MTF-1). Exposure to Ni resulted in the nuclear translocation of MTF-1, and Ni failed to induce MT in mouse embryonic fibroblasts lacking MTF-1. As Zn is the only metal known to directly bind MTF-1, we then showed that Ni increased a labile pool of intracellular Zn in cells as revealed by fluorescence-activated cell sorter using the Zn-sensitive fluorophore, FluoZin-3. Ni-induced increases in MT2A mRNA and MRE-luciferase activity were sensitive to the Zn chelator, TPEN, supporting an important role for Zn in mediating the effect of Ni. Although neither the source of labile Zn nor the mechanism by which Ni liberates labile Zn was apparent, it was noteworthy that Ni increased intracellular reactive oxygen species (ROS). Although both N-acetyl cysteine (NAC) and ascorbic acid (AA) decreased Ni-induced increases in ROS, only NAC prevented Ni-induced increases in MT2A mRNA, suggesting a special role for interactions of Ni, thiols, and Zn release. PMID:19097988

  1. Transcriptional mechanisms and protein kinase signaling mediate organic dust induction of IL-8 expression in lung epithelial and THP-1 cells

    PubMed Central

    Gottipati, Koteswara R.; Bandari, Shiva Kumar; Nonnenmann, Matthew W.; Levin, Jeffrey L.; Dooley, Gregory P.; Reynolds, Stephen J.

    2014-01-01

    Exposure to the agricultural work environment is a risk factor for the development of respiratory symptoms and chronic lung diseases. Inflammation is an important contributor to the pathogenesis of tissue injury and disease. Cellular and molecular mechanisms mediating lung inflammatory responses to agricultural dust are not yet fully understood. We studied the effects of poultry dust extract on molecular regulation of interleukin-8 (IL-8), a proinflammatory cytokine, in A549 and Beas2B lung epithelial and THP-1 monocytic cells. Our findings indicate that poultry dust extract potently induces IL-8 levels by increasing IL-8 gene transcription without altering IL-8 mRNA stability. Increase in IL-8 promoter activity was due to enhanced binding of activator protein 1 and NF-κB. IL-8 induction was associated with protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) activation and inhibited by PKC and MAPK inhibitors. IL-8 increase was not inhibited by polymyxin B or l-nitroarginine methyl ester, indicating lack of involvement of lipopolysaccharide and nitric oxide in the induction. Lung epithelial and THP-1 cells share common mechanisms for induction of IL-8 levels. Our findings identify key roles for transcriptional mechanisms and protein kinase signaling pathways for IL-8 induction and provide insights into the mechanisms regulating lung inflammatory responses to organic dust exposure. PMID:25398986

  2. Human bronchial epithelial cell injuries induced by fine particulate matter from sandstorm and non-sandstorm periods: Association with particle constituents.

    PubMed

    Wang, Bin; Li, Ning; Deng, Furong; Buglak, Nicholas; Park, George; Su, Shu; Ren, Aiguo; Shen, Guofeng; Tao, Shu; Guo, Xinbiao

    2016-09-01

    Epidemiological studies have demonstrated the exacerbation of respiratory diseases following sandstorm-derived particulate matter (PM) exposure. The presence of anthropogenic and biological agents on the sandstorm PM and the escalation of PM<2.5μm (PM2.5) pollution in China have led to serious concerns regarding the health effects of PM2.5 during Asian sandstorms. We investigated how changes in PM2.5 composition, as the weather transitioned towards a sandstorm, affected human airway epithelial cells. Six PM2.5 samples covering two sandstorm events and their respective background and transition periods were collected in Baotou, an industrial city near the Gobi Desert in China. PM samples from all three periods had mild cytotoxicity in human bronchial epithelial cell line BEAS-2B, which was positively correlated with the contents of polycyclic aromatic hydrocarbons and several metals. All PM samples potently increased the release of interleukin-6 (IL-6) and interleukin-8 (IL-8). Endotoxin in all samples contributed significantly to the IL-6 response, with only a minor effect on IL-8. Cr was positively correlated with both IL-6 and IL-8 release, while Si was only associated with the increase of IL-6. Our study suggests that local agricultural and industrial surroundings in addition to the sandstorm play important roles in the respiratory effects of sandstorm-derived PM. PMID:27593287

  3. Brominated flame retardants, hexabromocyclododecane and tetrabromobisphenol A, affect proinflammatory protein expression in human bronchial epithelial cells via disruption of intracellular signaling.

    PubMed

    Koike, Eiko; Yanagisawa, Rie; Takano, Hirohisa

    2016-04-01

    Hexabromocyclododecane (HBCD) and tetrabromobisphenol A (TBBPA) are widely used as brominated flame retardants (BFRs) in consumer products. Because humans can be exposed to BFRs mainly through air or dust, the effects of the BFRs on the respiratory system and the underlying mechanisms were investigated. HBCD exposure significantly increased the expression of intercellular adhesion molecule (ICAM)-1 and the production of interleukin (IL)-6 and -8 in human bronchial epithelial cells (BEAS-2B). TBBPA exposure significantly increased the expression of ICAM-1 and IL-6, but not IL-8. HBCD and TBBPA stimulated epidermal growth factor (EGF) production and EGF receptor (EGFR) phosphorylation. Inhibitors of EGFR-selective tyrosine kinase and the subsequent mitogen-activated protein kinase effectively blocked the increase in the expression of proinflammatory proteins. The activation of nuclear factor-kappa B (p50, p65) and activator protein 1 (c-Jun) was also observed following HBCD exposure. Furthermore, the modulation for nuclear receptors was investigated. TBBPA but not HBCD showed ligand activity for thyroid hormone receptor (TR) and TR antagonist significantly suppressed the TBBPA-induced increase of the expression of ICAM-1 and IL-6. In conclusion, HBCD and TBBPA can disrupt the expression of proinflammatory proteins in bronchial epithelial cells, possibly via the modulation of EGFR-related pathways and/or nuclear receptors. PMID:26718265

  4. Three-Dimensional Human Bronchial-Tracheal Epithelial Tissue-Like Assemblies (TLAs) as Hosts For Severe Acute Respiratory Syndrome (SARS)-CoV Infection

    NASA Technical Reports Server (NTRS)

    Suderman, M. T.; McCarthy, M.; Mossell, E.; Watts, D. M.; Peters, C. J.; Shope, R.; Goodwin, T. J.

    2006-01-01

    A three-dimensional (3-D) tissue-like assembly (TLA) of human bronchial-tracheal mesenchymal (HBTC) cells with an overlay of human bronchial epithelial (BEAS-2B) cells was constructed using a NASA Bioreactor to survey the infectivity of SARS-CoV. This TLA was inoculated with a low passage number Urbani strain of SARS-CoV. At selected intervals over a 10-day period, media and cell aliquots of the 3-D TLA were harvested for viral titer assay and for light and electron microscopy examination. All viral titer assays were negative in both BEAS-2B two-dimensional monolayer and TLA. Light microscopy immunohistochemistry demonstrated antigen-antibody reactivity with anti-SARS-CoV polyclonal antibody to spike and nuclear proteins on cell membranes and cytoplasm. Coronavirus Group 2 cross-reactivity was demonstrated by positive reaction to anti-FIPV 1 and anti-FIPV 1 and 2 antibodies. TLA examination by transmission electron microscopy indicated increasing cytoplasmic vacuolation with numerous electron-dense bodies measuring 45 to 270 nm from days 4 through 10. There was no evidence of membrane blebbing, membrane duplication, or fragmentation of organelles in the TLAs. However, progressive disruption of endoplasmic reticulum was observed throughout the cells. Antibody response to SARS-CoV specific spike and nucleocapsid glycoproteins, cross-reactivity with FIPV antibodies, and the cytoplasmic pathology suggests this HBTE TLA model is permissive to SARS-CoV infection.

  5. Epithelial stem cells.

    PubMed

    Draheim, Kyle M; Lyle, Stephen

    2011-01-01

    It is likely that adult epithelial stem cells will be useful in the treatment of diseases, such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa, and alopecias. Additionally, other skin problems such as burn wounds, chronic wounds, and ulcers will benefit from stem cell-related therapies. However, there are many questions that need to be answered before this goal can be realized. The most important of these questions is what regulates the adhesion of stem cells to the niche versus migration to the site of injury. We have started to identify the mechanisms involved in this decision-making process. PMID:21618097

  6. Enriched inorganic compounds in diesel exhaust particles induce mitogen-activated protein kinase activation, cytoskeleton instability, and cytotoxicity in human bronchial epithelial cells.

    PubMed

    Seriani, Robson; Junqueira, Mara S; Carvalho-Sousa, Claudia E; Arruda, Alessandra C T; Martinez, Diana; Alencar, Adriano M; Garippo, Ana L; Brito, Jôse Mara; Martins, Milton A; Saldiva, Paulo H N; Negri, Elnara M; Mauad, Thais; Macchione, Mariangela

    2015-04-01

    This study assessed the effects of the diesel exhaust particles on ERK and JNK MAPKs activation, cell rheology (viscoelasticity), and cytotoxicity in bronchial epithelial airway cells (BEAS-2B). Crude DEP and DEP after extraction with hexane (DEP/HEX) were utilized. The partial reduction of some DEP/HEX organics increased the biodisponibility of many metallic elements. JNK and ERK were activated simultaneously by crude DEP with no alterations in viscoelasticity of the cells. Mitochondrial activity, however, revealed a decrease through the MTT assay. DEP/HEX treatment increased viscoelasticity and cytotoxicity (membrane damage), and also activated JNK. Our data suggest that the greater bioavailability of metals could be involved in JNK activation and, consequently, in the reduction of fiber coherence and increase in the viscoelasticity and cytotoxicity of BEAS cells. The adverse findings detected after exposure to crude DEP and to DEP/HEX reflect the toxic potential of diesel compounds. Considering the fact that the cells of the respiratory epithelium are the first line of defense between the body and the environment, our data contribute to a better understanding of the pathways leading to respiratory cell injury and provide evidence for the onset of or worsening of respiratory diseases caused by inorganic compounds present in DEP. PMID:25769681

  7. Cationic polystyrene nanosphere toxicity depends on cell-specific endocytic and mitochondrial injury pathways.

    PubMed

    Xia, Tian; Kovochich, Michael; Liong, Monty; Zink, Jeffrey I; Nel, Andre E

    2008-01-01

    The exponential increase in the number of new nanomaterials that are being produced increases the likelihood of adverse biological effects in humans and the environment. In this study we compared the effects of cationic nanoparticles in five different cell lines that represent portal-of-entry or systemic cellular targets for engineered nanoparticles. Although 60 nm NH(2)-labeled polystyrene (PS) nanospheres were highly toxic in macrophage (RAW 264.7) and epithelial (BEAS-2B) cells, human microvascular endothelial (HMEC), hepatoma (HEPA-1), and pheochromocytoma (PC-12) cells were relatively resistant to particle injury. While the death pathway in RAW 264.7 cells involves caspase activation, the cytotoxic response in BEAS-2B cells is more necrotic in nature. Using fluorescent-labeled NH(2)-PS, we followed the routes of particle uptake. Confocal microscopy showed that the cationic particles entered a LAMP-1 positive lysosomal compartment in RAW 264.7 cells from where the particles could escape by lysosomal rupture. A proton pump inhibitor interfered in this pathway. Subsequent deposition of the particles in the cytosol induced an increase in mitochondrial Ca(2+) uptake and cell death that could be suppressed by cyclosporin A (CsA). In contrast, NH(2)-PS toxicity in BEAS-2B cells did not involve the LAMP-1 endosomal compartment, stimulation of proton pump activity, or an increase in mitochondrial Ca(2+). Particles were taken up by caveolae, and their toxicity could be disrupted by cholesterol extraction from the surface membrane. Although the particles induced mitochondrial damage and ATP depletion, CsA did not affect cytotoxicity. Cationic particles were taken up into HEPA-1, HMEC, and PC-12 cells, but this did not lead to lysosomal permeabilization, increased Ca(2+) flux, or mitochondrial damage. Taken together, the results of this study demonstrate the importance of cell-specific uptake mechanisms and pathways that could lead to sensitivity or resistance to cationic

  8. Reactive oxygen species contribute to arsenic-induced EZH2 phosphorylation in human bronchial epithelial cells and lung cancer cells

    SciTech Connect

    Li, Lingzhi; Qiu, Ping; Chen, Bailing; Lu, Yongju; Wu, Kai; Thakur, Chitra; Chang, Qingshan; Sun, Jiaying; Chen, Fei

    2014-05-01

    Our previous studies suggested that arsenic is able to induce serine 21 phosphorylation of the EZH2 protein through activation of JNK, STAT3, and Akt signaling pathways in the bronchial epithelial cell line, BEAS-2B. In the present report, we further demonstrated that reactive oxygen species (ROS) were involved in the arsenic-induced protein kinase activation that leads to EZH2 phosphorylation. Several lines of evidence supported this notion. First, the pretreatment of the cells with N-acetyl-L-cysteine (NAC), a potent antioxidant, abolishes arsenic-induced EZH2 phosphorylation along with the inhibition of JNK, STAT3, and Akt. Second, H{sub 2}O{sub 2}, the most important form of ROS in the cells in response to extracellular stress signals, can induce phosphorylation of the EZH2 protein and the activation of JNK, STAT3, and Akt. By ectopic expression of the myc-tagged EZH2, we additionally identified direct interaction and phosphorylation of the EZH2 protein by Akt in response to arsenic and H{sub 2}O{sub 2}. Furthermore, both arsenic and H{sub 2}O{sub 2} were able to induce the translocation of ectopically expressed or endogenous EZH2 from nucleus to cytoplasm. In summary, the data presented in this report indicate that oxidative stress due to ROS generation plays an important role in the arsenic-induced EZH2 phosphorylation. - Highlights:: • Arsenic (As{sup 3+}) induces EZH phosphorylation. • JNK, STAT3, and Akt contribute to EZH2 phosphorylation. • Oxidative stress is involved in As{sup 3+}-induced EZH2 phosphorylation. • As{sup 3+} induces direct interaction of Akt and EZH2. • Phosphorylated EZH2 localized in cytoplasm.

  9. cAMP-dependent protein kinase activation decreases cytokine release in bronchial epithelial cells

    PubMed Central

    Poole, Jill A.; Nordgren, Tara M.; DeVasure, Jane M.; Heires, Art J.; Bailey, Kristina L.; Romberger, Debra J.

    2014-01-01

    Lung injury caused by inhalation of dust from swine-concentrated animal-feeding operations (CAFO) involves the release of inflammatory cytokine interleukin 8 (IL-8), which is mediated by protein kinase C-ε (PKC-ε) in airway epithelial cells. Once activated by CAFO dust, PKC-ε is responsible for slowing cilia beating and reducing cell migration for wound repair. Conversely, the cAMP-dependent protein kinase (PKA) stimulates contrasting effects, such as increased cilia beating and an acceleration of cell migration for wound repair. We hypothesized that a bidirectional mechanism involving PKA and PKC regulates epithelial airway inflammatory responses. To test this hypothesis, primary human bronchial epithelial cells and BEAS-2B cells were treated with hog dust extract (HDE) in the presence or absence of cAMP. PKC-ε activity was significantly reduced in cells that were pretreated for 1 h with 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP) before exposure to HDE (P < 0.05). HDE-induced IL-6, and IL-8 release was significantly lower in cells that were pretreated with 8-Br-cAMP (P < 0.05). To exclude exchange protein activated by cAMP (EPAC) involvement, cells were pretreated with either 8-Br-cAMP or 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-CPT-2Me-cAMP) (EPAC agonist). 8-CPT-2Me-cAMP did not activate PKA and did not reduce HDE-stimulated IL-6 release. In contrast, 8-Br-cAMP decreased HDE-stimulated tumor necrosis factor (TNF)-α-converting enzyme (TACE; ADAM-17) activity and subsequent TNF-α release (P < 0.001). 8-Br-cAMP also blocked HDE-stimulated IL-6 and keratinocyte-derived chemokine release in precision-cut mouse lung slices (P < 0.05). These data show bidirectional regulation of PKC-ε via a PKA-mediated inhibition of TACE activity resulting in reduced PKC-ε-mediated release of IL-6 and IL-8. PMID:25150062

  10. cAMP-dependent protein kinase activation decreases cytokine release in bronchial epithelial cells.

    PubMed

    Wyatt, Todd A; Poole, Jill A; Nordgren, Tara M; DeVasure, Jane M; Heires, Art J; Bailey, Kristina L; Romberger, Debra J

    2014-10-15

    Lung injury caused by inhalation of dust from swine-concentrated animal-feeding operations (CAFO) involves the release of inflammatory cytokine interleukin 8 (IL-8), which is mediated by protein kinase C-ε (PKC-ε) in airway epithelial cells. Once activated by CAFO dust, PKC-ε is responsible for slowing cilia beating and reducing cell migration for wound repair. Conversely, the cAMP-dependent protein kinase (PKA) stimulates contrasting effects, such as increased cilia beating and an acceleration of cell migration for wound repair. We hypothesized that a bidirectional mechanism involving PKA and PKC regulates epithelial airway inflammatory responses. To test this hypothesis, primary human bronchial epithelial cells and BEAS-2B cells were treated with hog dust extract (HDE) in the presence or absence of cAMP. PKC-ε activity was significantly reduced in cells that were pretreated for 1 h with 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) before exposure to HDE (P < 0.05). HDE-induced IL-6, and IL-8 release was significantly lower in cells that were pretreated with 8-Br-cAMP (P < 0.05). To exclude exchange protein activated by cAMP (EPAC) involvement, cells were pretreated with either 8-Br-cAMP or 8-(4-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-CPT-2Me-cAMP) (EPAC agonist). 8-CPT-2Me-cAMP did not activate PKA and did not reduce HDE-stimulated IL-6 release. In contrast, 8-Br-cAMP decreased HDE-stimulated tumor necrosis factor (TNF)-α-converting enzyme (TACE; ADAM-17) activity and subsequent TNF-α release (P < 0.001). 8-Br-cAMP also blocked HDE-stimulated IL-6 and keratinocyte-derived chemokine release in precision-cut mouse lung slices (P < 0.05). These data show bidirectional regulation of PKC-ε via a PKA-mediated inhibition of TACE activity resulting in reduced PKC-ε-mediated release of IL-6 and IL-8. PMID:25150062

  11. Analysis of global gene expression changes in human bronchial epithelial cells exposed to spores of the allergenic fungus, Alternaria alternata

    PubMed Central

    Babiceanu, M. C.; Howard, B. A.; Rumore, A. C.; Kita, H.; Lawrence, C. B.

    2013-01-01

    Exposure and sensitivity to ubiquitous airborne fungi such as Alternaria alternata have long been implicated in the development, onset, and exacerbation of chronic allergic airway disorders. This present study is the first to investigate global changes in host gene expression during the interaction of cultured human bronchial epithelial cells and live Alternaria spores. In in vitro experiments human bronchial epithelial cells (BEAS-2B) were exposed to spores or media alone for 24 h. RNA was collected from three biological replicates per treatment and was used to assess changes in gene expression patterns using Affymetrix Human Genome U133 Plus 2.0 Arrays. In cells treated with Alternaria spores compared to controls, 613 probe sets representing 460 individual genes were found differentially expressed (p ≤ 0.05). In this set of 460 statistically significant, differentially expressed genes, 397 genes were found to be up-regulated and 63 were down-regulated. Of these 397 up-regulated genes, 156 genes were found to be up-regulated ≥2 fold. Interestingly, none of the 63 down-regulated genes were found differentially expressed at ≤−2 fold. Differentially expressed genes were identified following statistical analysis and subsequently used for pathway and network evaluation. Interestingly, many cytokine and chemokine immune response genes were up-regulated with a particular emphasis on interferon-inducible genes. Genes involved in cell death, retinoic acid signaling, and TLR3 response pathways were also significantly up-regulated. Many of the differentially up-regulated genes have been shown in other systems to be associated with innate immunity, inflammation and/or allergic airway diseases. This study now provides substantial information for further investigating specific genes and innate immune system pathways activated by Alternaria in the context of allergic airway diseases. PMID:23882263

  12. Toxicological effects of polycyclic aromatic hydrocarbons and their derivatives on respiratory cells

    NASA Astrophysics Data System (ADS)

    Koike, Eiko; Yanagisawa, Rie; Takano, Hirohisa

    2014-11-01

    Polycyclic aromatic hydrocarbons (PAHs) are found in ambient aerosols and particulate matter. Experimental studies have shown that PAHs and related chemicals can induce toxicological effects. The present study aimed to investigate the effects of PAHs and their derivatives on the respiratory and immune systems and the underlying mechanisms. The human bronchial epithelial cell line BEAS-2B was exposed to PAHs and their derivatives, and the cytotoxicity and proinflammatory protein expression were then investigated. A cytotoxic effect was observed in BEAS-2B exposed to PAH derivatives such as naphthoquinone (NQ), phenanthrenequinone (PQ), 1-nitropyrene (1-NP), and 1-aminopyrene (1-AP). In addition, 1,2-NQ and 9,10-PQ showed more effective cytotoxicity than 1,4-NQ and 1,4-PQ, respectively. Pyrene showed a weak cytotoxic effect. On the other hand, naphthalene and phenanthrene showed no significant effects. Pyrene, 1-NP, and 1-AP also increased intercellular adhesion molecule-1 expression and interleukin-6 production in BEAS-2B. The increase was partly suppressed by protein kinase inhibitors such as the epidermal growth factor receptor-selective tyrosine kinase inhibitor and nuclear receptor antagonists such as the thyroid hormone receptor antagonist. The present study suggests that the toxicological effects of chemicals may be related to the different activities resulting from their structures, such as numbers of benzene rings and functional groups. Furthermore, the chemical-induced increase in proinflammatory protein expression in bronchial epithelial cells was possibly a result of the activation of protein kinase pathways and nuclear receptors. The increase may partly contribute to the adverse health effects of atmospheric PAHs.

  13. Curcumin and Vitamin E Protect against Adverse Effects of Benzo[a]pyrene in Lung Epithelial Cells

    PubMed Central

    Cai, Qingsong; Lv, Tangfeng; Singh, Kamaleshwar; Gao, Weimin

    2014-01-01

    Benzo[a]pyrene (BaP), a well-known environmental carcinogen, promotes oxidative stress and DNA damage. Curcumin and vitamin E (VE) have potent antioxidative activity that protects cells from oxidative stress and cellular damage. The objectives of the present study were to investigate the adverse effects of BaP on normal human lung epithelial cells (BEAS-2B), the potential protective effects of curcumin and VE against BaP-induced cellular damage, and the molecular mechanisms of action. MTT assay, flow cytometry, fluorescence microplate assay, HPLC, qRT-PCR, and western blot were performed to analyze cytotoxicity, cell cycle, reactive oxygen species (ROS), BaP diol-epoxidation (BPDE)-DNA adducts, gene expression, and protein expression, respectively. Curcumin or VE prevented cells from BaP-induced cell cycle arrest and growth inhibition, significantly suppressed BaP-induced ROS levels, and decreased BPDE-DNA adducts. While CYP1A1 and 1B1 were induced by BaP, these inductions were not significantly reduced by curcumin or VE. Moreover, the level of activated p53 and PARP-1 were significantly induced by BaP, whereas this induction was markedly reduced after curcumin and VE co-treatment. Survivin was significantly down-regulated by BaP, and curcumin significantly restored survivin expression in BaP-exposed cells. The ratio of Bax/Bcl-2 was also significantly increased in cells exposed to BaP and this increase was reversed by VE co-treatment. Taken together, BaP-induced cytotoxicity occurs through DNA damage, cell cycle arrest, ROS production, modulation of metabolizing enzymes, and the expression/activation of p53, PARP-1, survivin, and Bax/Bcl-2. Curcumin and VE could reverse some of these BaP-mediated alterations and therefore be effective natural compounds against the adverse effects of BaP in lung cells. PMID:24664296

  14. Curcumin and vitamin E protect against adverse effects of benzo[a]pyrene in lung epithelial cells.

    PubMed

    Zhu, Wenbin; Cromie, Meghan M; Cai, Qingsong; Lv, Tangfeng; Singh, Kamaleshwar; Gao, Weimin

    2014-01-01

    Benzo[a]pyrene (BaP), a well-known environmental carcinogen, promotes oxidative stress and DNA damage. Curcumin and vitamin E (VE) have potent antioxidative activity that protects cells from oxidative stress and cellular damage. The objectives of the present study were to investigate the adverse effects of BaP on normal human lung epithelial cells (BEAS-2B), the potential protective effects of curcumin and VE against BaP-induced cellular damage, and the molecular mechanisms of action. MTT assay, flow cytometry, fluorescence microplate assay, HPLC, qRT-PCR, and western blot were performed to analyze cytotoxicity, cell cycle, reactive oxygen species (ROS), BaP diol-epoxidation (BPDE)-DNA adducts, gene expression, and protein expression, respectively. Curcumin or VE prevented cells from BaP-induced cell cycle arrest and growth inhibition, significantly suppressed BaP-induced ROS levels, and decreased BPDE-DNA adducts. While CYP1A1 and 1B1 were induced by BaP, these inductions were not significantly reduced by curcumin or VE. Moreover, the level of activated p53 and PARP-1 were significantly induced by BaP, whereas this induction was markedly reduced after curcumin and VE co-treatment. Survivin was significantly down-regulated by BaP, and curcumin significantly restored survivin expression in BaP-exposed cells. The ratio of Bax/Bcl-2 was also significantly increased in cells exposed to BaP and this increase was reversed by VE co-treatment. Taken together, BaP-induced cytotoxicity occurs through DNA damage, cell cycle arrest, ROS production, modulation of metabolizing enzymes, and the expression/activation of p53, PARP-1, survivin, and Bax/Bcl-2. Curcumin and VE could reverse some of these BaP-mediated alterations and therefore be effective natural compounds against the adverse effects of BaP in lung cells. PMID:24664296

  15. Constitutive activation of epidermal growth factor receptor promotes tumorigenesis of Cr(VI)-transformed cells through decreased reactive oxygen species and apoptosis resistance development.

    PubMed

    Kim, Donghern; Dai, Jin; Fai, Leonard Yenwong; Yao, Hua; Son, Young-Ok; Wang, Lei; Pratheeshkumar, Poyil; Kondo, Kazuya; Shi, Xianglin; Zhang, Zhuo

    2015-01-23

    Hexavalent chromium (Cr(VI)) compounds are well-established lung carcinogens. Epidermal growth factor receptor (EGFR) is a tyrosine kinase transmembrane receptor that regulates cell survival, tumor invasion, and angiogenesis. Our results show that chronic exposure of human bronchial epithelial (BEAS-2B) cells to Cr(VI) is able to cause malignant cell transformation. These transformed cells exhibit apoptosis resistance with reduced poly ADP-ribose polymerase cleavage (C-PARP) and Bax expression and enhanced expressions of Bcl-2 and Bcl-xL. These transformed cells also exhibit reduced capacity of reactive oxygen species (ROS) generation along with elevated expression of antioxidant manganese superoxide dismutase 2 (SOD2). The expression of this antioxidant was also elevated in lung tumor tissue from a worker exposed to Cr(VI) for 19 years. EGFR was activated in Cr(VI)-transformed BEAS-2B cells, lung tissue from animals exposed to Cr(VI) particles, and human lung tumor tissue. Further study indicates that constitutive activation of EGFR in Cr(VI)-transformed cells was due to increased binding to its ligand amphiregulin (AREG). Inhibition of EGFR or AREG increased Bax expression and reduced Bcl-2 expression, resulting in reduced apoptosis resistance. Furthermore, inhibition of AREG or EGFR restored capacity of ROS generation and decreased SOD2 expression. PI3K/AKT was activated, which depended on EGFR in Cr(VI)-transformed BEAS-2B cells. Inhibition of PI3K/AKT increased ROS generation and reduced SOD2 expression, resulting in reduced apoptosis resistance with commitment increase in Bax expression and reduction of Bcl-2 expression. Xenograft mouse tumor study further demonstrates the essential role of EGFR in tumorigenesis of Cr(VI)-transformed cells. In summary, the present study suggests that ligand-dependent constitutive activation of EGFR causes reduced ROS generation and increased antioxidant expression, leading to development of apoptosis resistance, contributing

  16. Expression of Aquaporin 5 (AQP5) Promotes Tumor Invasion in Human Non Small Cell Lung Cancer

    PubMed Central

    Kang, Sung Koo; Kim, Myoung Sook; Lee, Juna; Lee, Seung Koo; Gong, Gyungyub; Kim, Yong Hee; Soria, Jean Charles; Jang, Se Jin; Sidransky, David; Moon, Chulso

    2008-01-01

    The aquaporins (AQP) are water channel proteins playing a major role in transcellular and transepithelial water movement. Recently, the role of AQPs in human carcinogenesis has become an area of great interest. Here, by immunohistochemistry (IHC), we have found an expression of AQP5 protein in 35.3% (IHC-score: ≥1, 144/408) of the resected NSCLC tissue samples. Cases with AQP5-positive status (IHC-score: ≥2) displayed a higher rate of tumor recurrence than negative ones in NSCLC (54.7% vs. 35.1%, p = 0.005) and worse disease-free survival (p = 0.033; OR = 1.52; 95%CI:1.04−2.23). Further in vitro invasion assay using BEAS-2B and NIH3T3 cells stably transfected with overexpression constructs for full length wild-type AQP5 (AQP5) and its two mutants, N185D which blocks membrane trafficking and S156A which blocks phosphorylation on Ser156, showed that AQP5 induced cell invasions while both mutants did not. In BEAS-2B cells, the expression of AQP5 caused a spindle-like and fibroblastic morphologic change and losses of cell-cell contacts and cell polarity. Only cells with AQP5, not either of two mutants, exhibited a loss of epithelial cell markers and a gain of mesenchymal cell markers. In a human SH3-domains protein array, cellular extracts from BEAS-2B with AQP5 showed a robust binding activity to SH3-domains of the c-Src, Lyn, and Grap2 C-terminal. Furthermore, in immunoprecipitation assay, activated c-Src, phosphorylated on Tyr416, showed a stronger binding activity to cellular extracts from BEAS-2B with AQP5 compared with N185D or S156A mutant. Fluorescence in situ hybridization (FISH) analysis failed to show evidence of genomic amplification, suggesting AQP5 expression as a secondary event. Based on these clinical and molecular observations, we conclude that AQP5, through its phosphorylation on Ser156 and subsequent interaction with c-Src, plays an important role in NSCLC invasion and, therefore, may provide a unique opportunity for developing a

  17. Long-term low-dose α-particle enhanced the potential of malignant transformation in human bronchial epithelial cells through MAPK/Akt pathway

    SciTech Connect

    Liu, Weili; Xiao, Linlin; Dong, Chen; He, Mingyuan; Pan, Yan; Xie, Yuexia; Tu, Wenzhi; Fu, Jiamei; Shao, Chunlin

    2014-05-09

    Highlights: • Multi-exposures of 25 mGy α-ray enhanced cell proliferation, adhesion, and invasion. • MAPK/Akt but not JNK/P66 was positively correlated with cell invasive phenotypes. • LDR of α-irradiation triggers cell malignant transformation through MAPK/Akt. - Abstract: Since the wide usage of ionizing radiation, the cancer risk of low dose radiation (LDR) (<0.1 Gy) has become attractive for a long time. However, most results are derived from epidemiologic studies on atomic-bomb survivors and nuclear accidents surrounding population, and the molecular mechanism of this risk is elusive. To explore the potential of a long-term LDR-induced malignant transformation, human bronchial epithelial cells Beas-2B were fractionally irradiated with 0.025 Gy α-particles for 8 times in total and then further cultured for 1–2 months. It was found that the cell proliferation, the abilities of adhesion and invasion, and the protein expressions of p-ERK, p-Akt, especially p-P38 were not only increased in the multiply-irradiated cells but also in their offspring 1–2 months after the final exposure, indicating high potentiality of cell malignant transformation. On opposite, the expressions of p-JNK and p-P66 were diminished in the subcultures of irradiated cells and thus may play a role of negative regulation in canceration. When the cells were transferred with p38 siRNA, the LDR-induced enhancements of cell adhesion and invasion were significantly reduced. These findings suggest that long-term LDR of α-particles could enhance the potential of malignant transformation incidence in human bronchial epithelial cells through MAPK/Akt pathway.

  18. Gene expression profiling and pathway analysis of human bronchial epithelial cells exposed to airborne particulate matter collected from Saudi Arabia

    SciTech Connect

    Sun, Hong; Shamy, Magdy; Kluz, Thomas; Muñoz, Alexandra B.; Zhong, Mianhua; Laulicht, Freda; Alghamdi, Mansour A.; Khoder, Mamdouh I.; Chen, Lung-Chi; Costa, Max

    2012-12-01

    Epidemiological studies have established a positive correlation between human mortality and increased concentration of airborne particulate matters (PM). However, the mechanisms underlying PM related human diseases, as well as the molecules and pathways mediating the cellular response to PM, are not fully understood. This study aims to investigate the global gene expression changes in human cells exposed to PM{sub 10} and to identify genes and pathways that may contribute to PM related adverse health effects. Human bronchial epithelial cells were exposed to PM{sub 10} collected from Saudi Arabia for 1 or 4 days, and whole transcript expression was profiled using the GeneChip human gene 1.0 ST array. A total of 140 and 230 genes were identified that significantly changed more than 1.5 fold after PM{sub 10} exposure for 1 or 4 days, respectively. Ingenuity Pathway Analysis revealed that different exposure durations triggered distinct pathways. Genes involved in NRF2-mediated response to oxidative stress were up-regulated after 1 day exposure. In contrast, cells exposed for 4 days exhibited significant changes in genes related to cholesterol and lipid synthesis pathways. These observed changes in cellular oxidative stress and lipid synthesis might contribute to PM related respiratory and cardiovascular disease. -- Highlights: ► PM exposure modulated gene expression and associated pathways in BEAS-2B cells. ► One-day exposure to PM induced genes involved in responding to oxidative stress. ► 4-day exposure to PM changed genes associated to cholesterol and lipid synthesis.

  19. Let-7a modulates particulate matter (≤ 2.5 μm)-induced oxidative stress and injury in human airway epithelial cells by targeting arginase 2.

    PubMed

    Song, Lei; Li, Dan; Gu, Yue; Li, Xiaoping; Peng, Liping

    2016-10-01

    Epidemiological studies show that particulate matter (PM) with an aerodynamic diameter ≤ 2.5 μm (PM2.5) is associated with cardiorespiratory diseases via the induction of excessive oxidative stress. However, the precise mechanism underlying PM2.5-mediated oxidative stress injury has not been fully elucidated. Accumulating evidence has indicated the microRNA let-7 family might play a role in PM-mediated pathological processes. In this study, we investigated the role of let-7a in oxidative stress and cell injury in human bronchial epithelial BEAS2B (B2B) cells after PM2.5 exposure. The let-7a level was the most significantly decreased in B2B cells after PM2.5 exposure. The overexpression of let-7a suppressed intracellular reactive oxygen species levels and the percentage of apoptotic cells after PM2.5 exposure, while the let-7a level decreased arginase 2 (ARG2) mRNA and protein levels in B2B cells by directly targeting the ARG2 3'-untranslated region. ARG2 expression was upregulated in B2B cells during PM2.5 treatment, and ARG2 knockdown could remarkably reduce oxidative stress and cellular injury. Moreover, its restoration could abrogate the protective effects of let-7a against PM2.5-induced injury. In conclusion, let-7a decreases and ARG2 increases resulting from PM2.5 exposure may exacerbate oxidative stress, cell injury and apoptosis of B2B cells. The let-7a/ARG2 axis is a likely therapeutic target for PM2.5-induced airway epithelial injury. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26989813

  20. Gene Expression Profile and Toxic Effects in Human Bronchial Epithelial Cells Exposed to Zearalenone

    PubMed Central

    So, Mei Yu; Tian, ZhiPeng; Phoon, Yong Shian; Sha, Sha; Antoniou, Michael N.; Zhang, JiangWen; Wu, Rudolf S. S.; Tan-Un, Kian C

    2014-01-01

    Zearalenone (ZEA), a mycoestrogen produced by Fusarium fungal species, is mainly found in cereal crops such as maize, wheat and barley. Although ZEA has been reported to be present in air, little is known about the health risk or the molecular basis of action when lung cells are exposed to ZEA. As ZEA has a similar structure to estrogen, its potential risk as an endocrine disrupting chemical (EDC) has thus aroused both environmental and public health concerns. The purpose of this study is to identify the responses and underlying molecular changes that occur when human bronchial epithelial BEAS-2B cells are exposed to ZEA. Differential gene expression profiles were identified in cells that were treated with 40 µM ZEA for 6 h and 24 h by high-throughput microarray analysis using Affymetrix Human Gene 2.0 GeneChip. The array results showed that after ZEA treatment, 262 genes at 6 h and 1073 genes at 24 h were invovled in the differential regulation. Pathway analysis revealed that diverse cellular processes were affected when lung cells were exposed to ZEA resulting in impaired response to DNA damage, cell cycle arrest, down-regulation of inflammatory responses and alterations of epigenetic marks. Results of further experiments indicated that 40 µM ZEA decreased cell viability, induced apoptosis and promoted reactive oxygen species (ROS) generation in a time-dependent manner. Immuno-suppressive effects of ZEA were further revealed through the suppression of lipopolysaccharide (LPS)-induced expression of pro-inflammatory cytokines (IL-6, IL-8 and IL-1β). Interestingly, the level of global DNA methylation was markedly decreased after 24 h exposure to ZEA. Collectively, these observations suggested that a broad range of toxic effects are elicited by ZEA. Particularly, ROS may play a pivotal role in ZEA-induced cell death. These adverse effects observed in lung cells suggest that exposure to ZEA may increase susceptibility of lung cells to diseases and required further

  1. Quantitative evaluation of berberine subcellular distribution and cellular accumulation in non-small cell lung cancer cells by UPLC-MS/MS.

    PubMed

    Yuan, Zhong-Wen; Leung, Elaine Lai-Han; Fan, Xing-Xing; Zhou, Hua; Ma, Wen-Zhe; Liu, Liang; Xie, Ying

    2015-11-01

    Berberine, an isoquinoline alkaloid, has been demonstrated to be a safe anti-cancer agent with multiple effects on mitochondria. Intracellular concentration and distribution around the targeting sites are determinants of efficacy, but subcellular distribution of berberine has not been fully elucidated yet, which relies on the sensitive and robustness assay. In this study, a sensitive and robust UPLC-MS/MS method has been developed and validated with optimized extraction solvents and detection conditions. Key factors such as the purity and integrity of isolated organelle fractions, and the effects of isolation procedures on the subcellular concentration of berberine were systemically evaluated. With the developed assay, we found that the intracellular accumulations of berberine in two gefitinib resistant NSCLC cell lines H1650 and H1975 were 2-3 folds higher than that of normal epithelial cells BEAS-2B. Moreover, significantly different subcellular distribution profiles in NSCLC cancer cells from that of BEAS-2B cells with a striking increase in content in most organelles may contribute to its selective cytotoxicity to cancer cells. Furthermore, a predominant accumulation of berberine was observed for the first time in microsomal fraction for all three cell lines. Therefore, this method could be used for quantitative evaluation of subcellular distribution and cellular accumulation of berberine and for further evaluation of the concentration-effects relationship. PMID:26452787

  2. Effect of particle size and dispersion status on cytotoxicity and genotoxicity of zinc oxide in human bronchial epithelial cells.

    PubMed

    Roszak, Joanna; Catalán, Julia; Järventaus, Hilkka; Lindberg, Hanna K; Suhonen, Satu; Vippola, Minnamari; Stępnik, Maciej; Norppa, Hannu

    2016-07-01

    Data available on the genotoxicity of zinc oxide (ZnO) nanoparticles (NPs) are controversial. Here, we examined the effects of particle size and dispersion status on the cytotoxicity and genotoxicity of nanosized and fine ZnO, in the presence and absence of bovine serum albumin (BSA; 0.06%) in human bronchial epithelial BEAS-2B cells. Dynamic light scattering analysis showed the most homogenous dispersions in water alone for nanosized ZnO and in water with BSA for fine ZnO. After a 48-h treatment, both types of ZnO were cytotoxic within a similar, narrow dose range (1.5-3.0μg/cm(2)) and induced micronuclei at a near toxic dose range (1.25-1.75μg/cm(2)), both with and without BSA. In the comet assay, nanosized ZnO (1.25-1.5μg/cm(2)), in the absence of BSA, caused a statistically significant increase in DNA damage after 3-h and 6-h treatments, while fine ZnO did not. Our findings may be explained by better uptake or faster intracellular dissolution of nanosized ZnO without BSA during short treatments (3-6h; the comet assay), with less differences between the two ZnO forms after longer treatments (>48h; the in vitro micronucleus test). As ZnO is genotoxic within a narrow dose range partly overlapping with cytotoxic doses, small experimental differences e.g. in the dispersion of ZnO particles may have a substantial effect on the genotoxicity of the nominal doses added to the cell culture. PMID:27402478

  3. Anti-oxidative and inflammatory responses induced by fly ash particles and carbon black in lung epithelial cells.

    PubMed

    Diabaté, Silvia; Bergfeldt, Britta; Plaumann, Diana; Ubel, Caroline; Weiss, Carsten

    2011-12-01

    Combustion-derived nanoparticles as constituents of ambient particulate matter have been shown to induce adverse health effects due to inhalation. However, the components inducing these effects as well as the biological mechanisms are still not fully understood. The fine fraction of fly ash particles collected from the electrostatic precipitator of a municipal solid waste incinerator was taken as an example for real particles with complex composition released into the atmosphere to study the mechanism of early biological responses of BEAS-2B human lung epithelial cells. The studies include the effects of the water-soluble and -insoluble fractions of the fly ash and the well-studied carbon black nanoparticles were used as a reference. Fly ash induced reactive oxygen species (ROS) and increased the total cellular glutathione (tGSH) content. Carbon black also induced ROS generation; however, in contrast to the fly ash, it decreased the intracellular tGSH. The fly ash-induced oxidative stress was correlated with induction of the anti-oxidant enzyme heme oxygenase-1 and increase of the redox-sensitive transcription factor Nrf2. Carbon black was not able to induce HO-1. ROS generation, tGSH increase and HO-1 induction were only induced by the insoluble fraction of the fly ash, not by the water-soluble fraction. ROS generation and HO-1 induction were markedly inhibited by pre-incubation of the cells with the anti-oxidant N-acetyl cysteine which confirmed the involvement of oxidative stress. Both effects were also reduced by the metal chelator deferoxamine indicating a contribution of bioavailable transition metals. In summary, both fly ash and carbon black induce ROS but only fly ash induced an increase of intracellular tGSH and HO-1 production. Bioavailable transition metals in the solid water-insoluble matrix of the fly ash mostly contribute to the effects. PMID:21626191

  4. Role of H2O2 in the oxidative effects of zinc exposure in human airway epithelial cells.

    PubMed

    Wages, Phillip A; Silbajoris, Robert; Speen, Adam; Brighton, Luisa; Henriquez, Andres; Tong, Haiyan; Bromberg, Philip A; Simmons, Steven O; Samet, James M

    2014-01-01

    Human exposure to particulate matter (PM) is a global environmental health concern. Zinc (Zn(2+)) is a ubiquitous respiratory toxicant that has been associated with PM health effects. However, the molecular mechanism of Zn(2+) toxicity is not fully understood. H2O2 and Zn(2+) have been shown to mediate signaling leading to adverse cellular responses in the lung and we have previously demonstrated Zn(2+) to cause cellular H2O2 production. To determine the role of Zn(2+)-induced H2O2 production in the human airway epithelial cell response to Zn(2+) exposure. BEAS-2B cells expressing the redox-sensitive fluorogenic sensors HyPer (H2O2) or roGFP2 (EGSH) in the cytosol or mitochondria were exposed to 50µM Zn(2+) for 5min in the presence of 1µM of the zinc ionophore pyrithione. Intracellular H2O2 levels were modulated using catalase expression either targeted to the cytosol or ectopically to the mitochondria. HO-1 mRNA expression was measured as a downstream marker of response to oxidative stress induced by Zn(2+) exposure. Both cytosolic catalase overexpression and ectopic catalase expression in mitochondria were effective in ablating Zn(2+)-induced elevations in H2O2. Compartment-directed catalase expression blunted Zn(2+)-induced elevations in cytosolic EGSH and the increased expression of HO-1 mRNA levels. Zn(2+) leads to multiple oxidative effects that are exerted through H2O2-dependent and independent mechanisms. PMID:25462065

  5. Cigarette smoke-induced epithelial expression of WNT-5B: implications for COPD.

    PubMed

    Heijink, Irene H; de Bruin, Harold G; Dennebos, Robin; Jonker, Marnix R; Noordhoek, Jacobien A; Brandsma, Corry-Anke; van den Berge, Maarten; Postma, Dirkje S

    2016-08-01

    Wingless/integrase-1 (WNT) signalling is associated with lung inflammation and repair, but its role in chronic obstructive pulmonary disease (COPD) pathogenesis is unclear. We investigated whether cigarette smoke-induced dysregulation of WNT-5B contributes to airway remodelling in COPD.We analysed WNT-5B protein expression in the lung tissue of COPD patients and (non)smoking controls, and investigated the effects of cigarette smoke exposure on WNT-5B expression in COPD and control-derived primary bronchial epithelial cells (PBECs). Additionally, we studied downstream effects of WNT-5B on remodelling related genes fibronectin, matrix metalloproteinase (MMP)-2, MMP-9 and SnaiI in BEAS-2B and air-liquid interface (ALI)-cultured PBECs.We observed that airway epithelial WNT-5B expression is significantly higher in lung tissue from COPD patients than controls. Cigarette smoke extract significantly increased mRNA expression of WNT-5B in COPD, but not control-derived PBECs. Exogenously added WNT-5B augmented the expression of remodelling related genes in BEAS-2B cells, which was mediated by transforming growth factor (TGF)-β/Smad3 signalling. In addition, WNT-5B upregulated the expression of these genes in ALI-cultured PBECs, particularly PBECs from COPD patients.Together, our results provide evidence that exaggerated WNT-5B expression upon cigarette smoke exposure in the bronchial epithelium of COPD patients leads to TGF-β/Smad3-dependent expression of genes related to airway remodelling. PMID:27126693

  6. High-throughput, Quantitative Analysis of Acrolein-derived DNA Adducts in Human Oral Cells by Immunohistochemistry

    PubMed Central

    Greenspan, Emily J.; Lee, Hanjoo; Dyba, Marcin; Pan, Jishen; Mekambi, Kepher; Johnson, Tierra; Blancato, Jan; Mueller, Susette; Berry, Deborah L.

    2012-01-01

    Acrolein (Acr) is a ubiquitous environmental pollutant as well as an endogenous compound. Acrolein-derived 1,N2-propanodeoxyguanosines (Acr-dG) are exocyclic DNA adducts formed following exposure to cigarette smoke or from lipid peroxidation. Acr-dG is mutagenic and potentially carcinogenic and may represent a useful biomarker for the early detection of cancers related to smoking or other oxidative conditions, such as chronic inflammation. In this study, we have developed a high-throughput, automated method using a HistoRx PM-2000 imaging system combined with MetaMorph software for quantifying Acr-dG adducts in human oral cells by immunohistochemical detection using a monoclonal antibody recently developed by our laboratory. This method was validated in a cell culture system using BEAS-2B human bronchial epithelial cells treated with known concentrations of Acr. The results were further verified by quantitative analysis of Acr-dG in DNA of BEAS-2B cells using a liquid chromatography/tandem mass spectrometry/multiple-reaction monitoring method. The automated method is a quicker, more accurate method than manual evaluation of counting cells expressing Acr-dG and quantifying fluorescence intensity. It may be applied to other antibodies that are used for immunohistochemical detection in tissues as well as cell lines, primary cultures, and other cell types. PMID:22899861

  7. Integrins and epithelial cell polarity

    PubMed Central

    Lee, Jessica L.; Streuli, Charles H.

    2014-01-01

    ABSTRACT Cell polarity is characterised by differences in structure, composition and function between at least two poles of a cell. In epithelial cells, these spatial differences allow for the formation of defined apical and basal membranes. It has been increasingly recognised that cell–matrix interactions and integrins play an essential role in creating epithelial cell polarity, although key gaps in our knowledge remain. This Commentary will discuss the mounting evidence for the role of integrins in polarising epithelial cells. We build a model in which both inside-out signals to polarise basement membrane assembly at the basal surface, and outside-in signals to control microtubule apical–basal orientation and vesicular trafficking are required for establishing and maintaining the orientation of epithelial cell polarity. Finally, we discuss the relevance of the basal integrin polarity axis to cancer. This article is part of a Minifocus on Establishing polarity. For further reading, please see related articles: ‘ERM proteins at a glance’ by Andrea McClatchey (J. Cell Sci. 127, 3199–3204). ‘Establishment of epithelial polarity – GEF who's minding the GAP?’ by Siu Ngok et al. (J. Cell Sci. 127, 3205–3215). PMID:24994933

  8. JNK and STAT3 signaling pathways converge on Akt-mediated phosphorylation of EZH2 in bronchial epithelial cells induced by arsenic

    PubMed Central

    Chen, Bailing; Liu, Jia; Chang, Qingshan; Beezhold, Kevin; Lu, Yongju; Chen, Fei

    2013-01-01

    The molecular mechanisms by which arsenic (As3+) causes human cancers remain to be fully elucidated. Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of polycomb-repressive complexes 2 (PRC2) that promotes trimethylation of lysine 27 of histone H3, leading to altered expression of tumor suppressors or oncogenes. In the present study, we determined the effect of As3+ on EZH2 phosphorylation and the signaling pathways important for As3+-induced EZH2 phosphorylation in human bronchial epithelial cell line BEAS-2B. The involvement of kinases in As3+-induced EZH2 phosphorylation was validated by siRNA-based gene silencing. The data showed that As3+ can induce phosphorylation of EZH2 at serine 21 in human bronchial epithelial cells and that the phosphorylation of EZH2 requires an As3+-activated signaling cascade from JNK and STAT3 to Akt. Transfection of the cells with siRNA specific for JNK1 revealed that JNK silencing reduced serine727 phosphorylation of STAT3, Akt activation and EZH2 phosphorylation, suggesting that JNK is the upstream kinase involved in As3+-induced EZH2 phosphorylation. Because As3+ is capable of inducing miRNA-21 (miR-21), a STAT3-regulated miRNA that represses protein translation of PTEN or Spry2, we also tested the role of STAT3 and miR-21 in As3+-induced EZH2 phosphorylation. Ectopic overexpression of miR-21 promoted Akt activation and phosphorylation of EZH2, whereas inhibiting miR-21 by transfecting the cells with anti-miR-21 inhibited Akt activation and EZH2 phosphorylation. Taken together, these results demonstrate a contribution of the JNK, STAT3 and Akt signaling axis to As3+-induced EZH2 phosphorylation. Importantly, these findings may reveal new molecular mechanisms underlying As3+-induced carcinogenesis. PMID:23255093

  9. Mammalian Cells Exhibit a Range of Sensitivities to Silver Nanoparticles that are Partially Explicable by Variations in Antioxidant Defense and Metallothionein Expression.

    PubMed

    Zhang, Haiyuan; Wang, Xiang; Wang, Meiying; Li, Linjiang; Chang, Chong Hyun; Ji, Zhaoxia; Xia, Tian; Nel, Andre E

    2015-08-01

    While it is well known that there are interspecies differences in Ag sensitivity, differences in the cytotoxic responses of mammalian cells to silver nanoparticles (Ag NPs) are also observed. In order to explore these response outcomes, six cell lines, including epithelial cells (Caco-2, NHBE, RLE-6TN, and BEAS-2B) and macrophages (RAW 264.7 and THP-1) of human and rodent origin, are exposed to 20 nm citrate- and PVP-coated Ag NPs with Au cores, as well as 20 nm citrate-coated particles without cores. An MTS assay shows that while Caco-2 and NHBE cells are resistant to particles over a 0.1-50 μg mL(-1) dose range, RAW 264.7, THP-1, RLE-6TN, and BEAS-2B cells are more susceptible. While there are small differences in dissolution rates, there are no major differences in the cytotoxic potential of the different particles. However, differences in anti-oxidant defense and metallothionein expression among different cell types are observed, which can partially explain differential Ag NP sensitivity. So, it is important to consider these differences in understanding the potential heterogeneous effects of nano Ag on mammalian biological systems. PMID:25930061

  10. Ion Channels in Epithelial Cells

    NASA Astrophysics Data System (ADS)

    Palmer, Lawrence G.

    Ion channels in epithelial cells serve to move ions, and in some cases fluid, between compartments of the body. This function of the transfer of material is fundamentally different from that of the transfer of information, which is the main job of most channels in excitable cells. Nevertheless the basic construction of the channels is similar in many respects in the two tissue types. This chapter reviews the nature of channels in epithelia and discusses how their functions have evolved to accomplish the basic tasks for which they are responsible. I will focus on three channel types: epithelial Na+ channels, inward-rectifier K+ channels, and CFTR Cl- channels.

  11. Epithelial Sodium Channels in Pulmonary Epithelial Progenitor and Stem Cells

    PubMed Central

    Liu, Yang; Jiang, Bi-Jie; Zhao, Run-Zhen; Ji, Hong-Long

    2016-01-01

    Regeneration of the epithelium of mammalian lungs is essential for restoring normal function following injury, and various cells and mechanisms contribute to this regeneration and repair. Club cells, bronchioalveolar stem cells (BASCs), and alveolar type II epithelial cells (ATII) are dominant stem/progenitor cells for maintaining epithelial turnover and repair. Epithelial Na+ channels (ENaC), a critical pathway for transapical salt and fluid transport, are expressed in lung epithelial progenitors, including club and ATII cells. Since ENaC activity and expression are development- and differentiation-dependent, apically located ENaC activity has therefore been used as a functional biomarker of lung injury repair. ENaC activity may be involved in the migration and differentiation of local and circulating stem/progenitor cells with diverse functions, eventually benefiting stem cells spreading to re-epithelialize injured lungs. This review summarizes the potential roles of ENaC expressed in native progenitor and stem cells in the development and regeneration of the respiratory epithelium. PMID:27570489

  12. Hyperoside induces both autophagy and apoptosis in non-small cell lung cancer cells in vitro

    PubMed Central

    Fu, Ting; Wang, Ling; Jin, Xiang-nan; Sui, Hai-juan; Liu, Zhou; Jin, Ying

    2016-01-01

    Aim: Hyperoside (quercetin-3-O-β-D-galactopyranoside) is a flavonol glycoside found in plants of the genera Hypericum and Crataegus, which exhibits anticancer, anti-oxidant, and anti-inflammatory activities. In this study we investigated whether autophagy was involved in the anticancer mechanisms of hyperoside in human non-small cell lung cancer cells in vitro. Methods: Human non-small cell lung cancer cell line A549 was tested, and human bronchial epithelial cell line BEAS-2B was used for comparison. The expression of LC3-II, apoptotic and signaling proteins was measured using Western blotting. Autophagosomes were observed with MDC staining, LC3 immunocytochemistry, and GFP-LC3 fusion protein techniques. Cell viability was assessed using MTT assay. Results: Hyperoside (0.5, 1, 2 mmol/L) dose-dependently increased the expression of LC3-II and autophagosome numbers in A549 cells, but had no such effects in BEAS-2B cells. Moreover, hyperoside dose-dependently inhibited the phosphorylation of Akt, mTOR, p70S6K and 4E-BP1, but increased the phosphorylation of ERK1/2 in A549 cells. Insulin (200 nmol/L) markedly enhanced the phosphorylation of Akt and decreased LC3-II expression in A549 cells, which were reversed by pretreatment with hyperoside, whereas the MEK1/2 inhibitor U0126 (20 μmol/L) did not blocked hyperoside-induced LC3-II expression. Finally, hyperoside dose-dependently suppressed the cell viability and induced apoptosis in A549 cells, which were significantly attenuated by pretreatment with the autophagy inhibitor 3-methyladenine (2.5 mmol/L). Conclusion: Hyperoside induces both autophagy and apoptosis in human non-small cell lung cancer cells in vitro. The autophagy is induced through inhibiting the Akt/mTOR/p70S6K signal pathways, which contributes to anticancer actions of hyperoside. PMID:26948085

  13. Cell cycle alterations induced by urban PM2.5 in bronchial epithelial cells: characterization of the process and possible mechanisms involved

    PubMed Central

    2013-01-01

    Background This study explores and characterizes cell cycle alterations induced by urban PM2.5 in the human epithelial cell line BEAS-2B, and elucidates possible mechanisms involved. Methods The cells were exposed to a low dose (7.5 μg/cm2) of Milan winter PM2.5 for different time points, and the cell cycle progression was analyzed by fluorescent microscopy and flow cytometry. Activation of proteins involved in cell cycle control was investigated by Western blotting and DNA damage by 32P-postlabelling, immunostaining and comet assay. The formation of reactive oxygen species (ROS) was quantified by flow cytometry. The role of PM organic fraction versus washed PM on the cell cycle alterations was also examined. Finally, the molecular pathways activated were further examined using specific inhibitors. Results Winter PM2.5 induced marked cell cycle alteration already after 3 h of exposure, represented by an increased number of cells (transient arrest) in G2. This effect was associated with an increased phosphorylation of Chk2, while no changes in p53 phosphorylation were observed at this time point. The increase in G2 was followed by a transient arrest in the metaphase/anaphase transition point (10 h), which was associated with the presence of severe mitotic spindle aberrations. The metaphase/anaphase delay was apparently followed by mitotic slippage at 24 h, resulting in an increased number of tetraploid G1 cells and cells with micronuclei (MN), and by apoptosis at 40 h. Winter PM2.5 increased the level of ROS at 2 h and DNA damage (8-oxodG, single- and double stand breaks) was detected after 3 h of exposure. The PM organic fraction caused a similar G2/M arrest and augmented ROS formation, while washed PM had no such effects. DNA adducts were detected after 24 h. Both PM-induced DNA damage and G2 arrest were inhibited by the addition of antioxidants and α-naphthoflavone, suggesting the involvement of ROS and reactive electrophilic metabolites formed via a P

  14. The long-acting β2-adrenoceptor agonist, indacaterol, enhances glucocorticoid receptor-mediated transcription in human airway epithelial cells in a gene- and agonist-dependent manner

    PubMed Central

    Joshi, T; Johnson, M; Newton, R; Giembycz, M A

    2015-01-01

    Background and Purpose Inhaled glucocorticoid (ICS)/long-acting β2-adrenoceptor agonist (LABA) combination therapy is a recommended treatment option for patients with moderate/severe asthma in whom adequate control cannot be achieved by an ICS alone. Previously, we discovered that LABAs can augment dexamethasone-inducible gene expression and proposed that this effect may explain how these two drugs interact to deliver superior clinical benefit. Herein, we extended that observation by analysing, pharmacodynamically, the effect of the LABA, indacaterol, on glucocorticoid receptor (GR)-mediated gene transcription induced by seven ligands with intrinsic activity values that span the spectrum of full agonism to antagonism. Experimental Approach BEAS-2B human airway epithelial cells stably transfected with a 2× glucocorticoid response element luciferase reporter were used to model gene transcription together with an analysis of several glucocorticoid-inducible genes. Key Results Indacaterol augmented glucocorticoid-induced reporter activation in a manner that was positively related to the intrinsic activity of the GR agonist. This effect was demonstrated by an increase in response maxima without a change in GR agonist affinity or efficacy. Indacaterol also enhanced glucocorticoid-inducible gene expression. However, the magnitude of this effect was dependent on both the GR agonist and the gene of interest. Conclusions and Implications These data suggest that indacaterol activates a molecular rheostat, which increases the transcriptional competency of GR in an agonist- and gene-dependent manner without apparently changing the relationship between fractional GR occupancy and response. These findings provide a platform to rationally design ICS/LABA combination therapy that is based on the generation of agonist-dependent gene expression profiles in target and off-target tissues. PMID:25598440

  15. Temporal-spatial analysis of U.S.-Mexico border environmental fine and coarse PM air sample extract activity in human bronchial epithelial cells

    SciTech Connect

    Lauer, Fredine T.; Mitchell, Leah A.; Bedrick, Edward; McDonald, Jacob D.; Lee, Wen-Yee; Li, Wen-Whai; Olvera, Hector; Amaya, Maria A.; Berwick, Marianne; Gonzales, Melissa; Currey, Robert; Pingitore, Nicholas E.

    2009-07-01

    Particulate matter less than 10 {mu}m (PM10) has been shown to be associated with aggravation of asthma and respiratory and cardiopulmonary morbidity. There is also great interest in the potential health effects of PM2.5. Particulate matter (PM) varies in composition both spatially and temporally depending on the source, location and seasonal condition. El Paso County which lies in the Paso del Norte airshed is a unique location to study ambient air pollution due to three major points: the geological land formation, the relatively large population and the various sources of PM. In this study, dichotomous filters were collected from various sites in El Paso County every 7 days for a period of 1 year. The sampling sites were both distant and near border crossings, which are near heavily populated areas with high traffic volume. Fine (PM2.5) and Coarse (PM10-2.5) PM filter samples were extracted using dichloromethane and were assessed for biologic activity and polycyclic aromatic (PAH) content. Three sets of marker genes human BEAS2B bronchial epithelial cells were utilized to assess the effects of airborne PAHs on biologic activities associated with specific biological pathways associated with airway diseases. These pathways included in inflammatory cytokine production (IL-6, IL-8), oxidative stress (HMOX-1, NQO-1, ALDH3A1, AKR1C1), and aryl hydrocarbon receptor (AhR)-dependent signaling (CYP1A1). Results demonstrated interesting temporal and spatial patterns of gene induction for all pathways, particularly those associated with oxidative stress, and significant differences in the PAHs detected in the PM10-2.5 and PM2.5 fractions. Temporally, the greatest effects on gene induction were observed in winter months, which appeared to correlate with inversions that are common in the air basin. Spatially, the greatest gene expression increases were seen in extracts collected from the central most areas of El Paso which are also closest to highways and border crossings.

  16. Astragalin inhibits airway eotaxin-1 induction and epithelial apoptosis through modulating oxidative stress-responsive MAPK signaling

    PubMed Central

    2014-01-01

    Background Eotaxin proteins are a potential therapeutic target in treating the peribronchial eosinophilia associated with allergic airway diseases. Since inflammation is often associated with an increased generation of reactive oxygen species (ROS), oxidative stress is a mechanistically imperative factor in asthma. Astragalin (kaempferol-3-O-glucoside) is a flavonoid with anti-inflammatory activity and newly found in persimmon leaves and green tea seeds. This study elucidated that astragalin inhibited endotoxin-induced oxidative stress leading to eosinophilia and epithelial apoptosis in airways. Methods Airway epithelial BEAS-2B cells were exposed to lipopolysaccharide (LPS) in the absence and presence of 1–20 μM astragalin. Western blot and immunocytochemical analyses were conducted to determine induction of target proteins. Cell and nuclear staining was also performed for ROS production and epithelial apoptosis. Results When airway epithelial cells were exposed to 2 μg/ml LPS, astragalin nontoxic at ≤20 μM suppressed cellular induction of Toll-like receptor 4 (TLR4) and ROS production enhanced by LPS. Both LPS and H2O2 induced epithelial eotaxin-1 expression, which was blocked by astragalin. LPS activated and induced PLCγ1, PKCβ2, and NADPH oxidase subunits of p22phox and p47phox in epithelial cells and such activation and induction were demoted by astragalin or TLR4 inhibition antagonizing eotaxin-1 induction. H2O2-upregulated phosphorylation of JNK and p38 MAPK was dampened by adding astragalin to epithelial cells, while this compound enhanced epithelial activation of Akt and ERK. H2O2 and LPS promoted epithelial apoptosis concomitant with nuclear condensation or caspase-3 activation, which was blunted by astragalin. Conclusions Astragalin ameliorated oxidative stress-associated epithelial eosinophilia and apoptosis through disturbing TLR4-PKCβ2-NADPH oxidase-responsive signaling. Therefore, astragalin may be a potent agent antagonizing endotoxin

  17. Epithelial cells and Von Gierke's disease.

    PubMed

    Negishi, H; Benke, P J

    1977-08-01

    Epithelial cells and not fibroblasts from human liver and amniotic fluid contain inducible glucose-6-phosphatase (G-6-Pase) activity. The diagnosis of Von Gierke's disease has been made in a patient with hepatomegaly utilizing cultured epithelial cells grown from a liver biopsy. G-6-Pase activity in epithelial cells from this patient could not be induced by dibutyryl cyclic AMP and theophylline. This is the first use of epithelial cells for diagnosis of a metabolic disease. G-6-Pase activity in cloned epithelial cells from amniotic fluid increases 2- to 3-fold after 24-hr exposure to dibutyryl cyclic AMP and theophylline. The prenatal diagnosis of Von Gierke's disease may be possible in a laboratory experienced with these techniques if epithelial cell growth is obtained from amniotic fluid. PMID:196249

  18. Diatom-derived polyunsaturated aldehydes activate cell death in human cancer cell lines but not normal cells.

    PubMed

    Sansone, Clementina; Braca, Alessandra; Ercolesi, Elena; Romano, Giovanna; Palumbo, Anna; Casotti, Raffaella; Francone, Maria; Ianora, Adrianna

    2014-01-01

    Diatoms are an important class of unicellular algae that produce bioactive polyunsaturated aldehydes (PUAs) that induce abortions or malformations in the offspring of invertebrates exposed to them during gestation. Here we compare the effects of the PUAs 2-trans,4-trans-decadienal (DD), 2-trans,4-trans-octadienal (OD) and 2-trans,4-trans-heptadienal (HD) on the adenocarcinoma cell lines lung A549 and colon COLO 205, and the normal lung/brunch epithelial BEAS-2B cell line. Using the viability MTT/Trypan blue assays, we show that PUAs have a toxic effect on both A549 and COLO 205 tumor cells but not BEAS-2B normal cells. DD was the strongest of the three PUAs tested, at all time-intervals considered, but HD was as strong as DD after 48 h. OD was the least active of the three PUAs. The effect of the three PUAs was somewhat stronger for A549 cells. We therefore studied the death signaling pathway activated in A549 showing that cells treated with DD activated Tumor Necrosis Factor Receptor 1 (TNFR1) and Fas Associated Death Domain (FADD) leading to necroptosis via caspase-3 without activating the survival pathway Receptor-Interacting Protein (RIP). The TNFR1/FADD/caspase pathway was also observed with OD, but only after 48 h. This was the only PUA that activated RIP, consistent with the finding that OD causes less damage to the cell compared to DD and HD. In contrast, cells treated with HD activated the Fas/FADD/caspase pathway. This is the first report that PUAs activate an extrinsic apoptotic machinery in contrast to other anticancer drugs that promote an intrinsic death pathway, without affecting the viability of normal cells from the same tissue type. These findings have interesting implications also from the ecological viewpoint considering that HD is one of the most common PUAs produced by diatoms. PMID:24992192

  19. Diatom-Derived Polyunsaturated Aldehydes Activate Cell Death in Human Cancer Cell Lines but Not Normal Cells

    PubMed Central

    Sansone, Clementina; Braca, Alessandra; Ercolesi, Elena; Romano, Giovanna; Palumbo, Anna; Casotti, Raffaella; Francone, Maria; Ianora, Adrianna

    2014-01-01

    Diatoms are an important class of unicellular algae that produce bioactive polyunsaturated aldehydes (PUAs) that induce abortions or malformations in the offspring of invertebrates exposed to them during gestation. Here we compare the effects of the PUAs 2-trans,4-trans-decadienal (DD), 2-trans,4-trans-octadienal (OD) and 2-trans,4-trans-heptadienal (HD) on the adenocarcinoma cell lines lung A549 and colon COLO 205, and the normal lung/brunch epithelial BEAS-2B cell line. Using the viability MTT/Trypan blue assays, we show that PUAs have a toxic effect on both A549 and COLO 205 tumor cells but not BEAS-2B normal cells. DD was the strongest of the three PUAs tested, at all time-intervals considered, but HD was as strong as DD after 48 h. OD was the least active of the three PUAs. The effect of the three PUAs was somewhat stronger for A549 cells. We therefore studied the death signaling pathway activated in A549 showing that cells treated with DD activated Tumor Necrosis Factor Receptor 1 (TNFR1) and Fas Associated Death Domain (FADD) leading to necroptosis via caspase-3 without activating the survival pathway Receptor-Interacting Protein (RIP). The TNFR1/FADD/caspase pathway was also observed with OD, but only after 48 h. This was the only PUA that activated RIP, consistent with the finding that OD causes less damage to the cell compared to DD and HD. In contrast, cells treated with HD activated the Fas/FADD/caspase pathway. This is the first report that PUAs activate an extrinsic apoptotic machinery in contrast to other anticancer drugs that promote an intrinsic death pathway, without affecting the viability of normal cells from the same tissue type. These findings have interesting implications also from the ecological viewpoint considering that HD is one of the most common PUAs produced by diatoms. PMID:24992192

  20. A Novel Natural Product, KL-21, Inhibits Proliferation and Induces Apoptosis in Chronic Lymphocytic Leukemia Cells

    PubMed Central

    Adan Gökbulut, Aysun; Yaşar, Mustafa; Baran, Yusuf

    2015-01-01

    Objective: The aims of this study were to examine the cytotoxic and apoptotic effects of KL-21, a novel plant product (produced by Naturin Natural Products, İzmir, Turkey), on 232B4 chronic lymphocytic leukemia (CLL) cells and to determine the cytotoxic effects on healthy BEAS-2B human bronchial epithelial cells. Materials and Methods: The cytotoxic effect of KL-21 was determined by MTT cell proliferation assay. Changes in caspase-3 enzyme activity were measured using the caspase-3 colorimetric assay. Changes in mitochondrial membrane potential were determined using the JC-1 dye-based method. Annexin V-FITC/PI double staining was performed to measure the apoptotic cell population. Effects of KL-21 on cell cycle profiles of CLL cells were investigated by flow cytometry. Results: We detected time- and concentration-dependent increases in the cytotoxic effect of KL-21 on 232B4 CLL cells. However, we also showed that, especially at higher concentrations, KL-21 was less cytotoxic towards BEAS-2B healthy cells than towards CLL cells. Annexin-V/PI double staining results showed that the apoptotic cell population increased in 232B4 cells. Increasing concentrations of KL-21 increased caspase-3 enzyme activity and induced loss of mitochondrial membrane potential. KL-21 administration resulted in small increases in the percentage of the cells in the G0/G1 phase while it decreased the S phase cell population up to 1 mg/mL. At the highest concentration, most of the cells accumulated in the G0/G1 phase. Conclusion: KL-21 has a growth-inhibitory effect on 232B4 CLL cells. KL-21 causes apoptosis and cell cycle arrest at G0/G1. PMID:26316479

  1. Effect of Elevated Carbon Dioxide on Bronchial Epithelial Innate Immune Receptor Response to Organic Dust from Swine Confinement Barns

    PubMed Central

    Schneberger, D.; Cloonan, D.; DeVasure, J. M.; Bailey, K. L.; Romberger, D. J.; Wyatt, T. A.

    2015-01-01

    Hypercapnia is known to have immunoregulatory effects within the lung. Cell culture systems demonstrate this in both macrophages and alveolar cell lines, suggesting that alveoli are affected by changes in CO2 levels. We hypothesized that hypercapnia would also modulate human bronchial epithelial cell immune responses. Innate immune responses to Pam3CSK4 (TLR2 ligand), LPS (TLR4 ligand) and a complex innate immune stimulus, an extract from the organic dust of swine confinement barns (barn dust extract or BDE), were tested in a human bronchial epithelial cell line, BEAS-2B. Both TLR ligands showed a decrease in IL-6 and IL-8 production, and an increase in MCP-1 in response to elevated CO2 indicating an enhancement in cytokine production to hypercapnia. This change was not reflected in expression levels of TLR receptor RNA which remained unchanged in response to elevated CO2. Interestingly, barn dust showed an increase in IL-6, IL-8 and MCP-1 response at 9% CO2, suggesting that elevated CO2 exerts different effects on different stimuli. Our results show that airway epithelial cell immune responses to barn dust respond differently to hypercapnic conditions than individual TLR ligands. PMID:25921030

  2. Epithelial organization, cell polarity and tumorigenesis.

    PubMed

    McCaffrey, Luke Martin; Macara, Ian G

    2011-12-01

    Epithelial cells comprise the foundation for the majority of organs in the mammalian body, and are the source of approximately 90% of all human cancers. Characteristically, epithelial cells form intercellular adhesions, exhibit apical/basal polarity, and orient their mitotic spindles in the plane of the epithelial sheet. Defects in these attributes result in the tissue disorganization associated with cancer. Epithelia undergo self-renewal from stem cells, which might in some cases be the cell of origin for cancers. The PAR polarity proteins are master regulators of epithelial organization, and are closely linked to signaling pathways such as Hippo, which orchestrate proliferation and apoptosis to control organ size. 3D ex vivo culture systems can now faithfully recapitulate epithelial organ morphogenesis, providing a powerful approach to study both normal development and the initiating events in carcinogenesis. PMID:21782440

  3. A Novel Cell-Penetrating Peptide Derived from Human Eosinophil Cationic Protein

    PubMed Central

    Fang, Shun-lung; Fan, Tan-chi; Fu, Hua-Wen; Chen, Chien-Jung; Hwang, Chi-Shin; Hung, Ta-Jen; Lin, Lih-Yuan; Chang, Margaret Dah-Tsyr

    2013-01-01

    Cell-penetrating peptides (CPPs) are short peptides which can carry various types of molecules into cells; however, although most CPPs rapidly penetrate cells in vitro, their in vivo tissue-targeting specificities are low. Herein, we describe cell-binding, internalization, and targeting characteristics of a newly identified 10-residue CPP, denoted ECP32–41, derived from the core heparin-binding motif of human eosinophil cationic protein (ECP). Besides traditional emphasis on positively charged residues, the presence of cysteine and tryptophan residues was demonstrated to be essential for internalization. ECP32–41 entered Beas-2B and wild-type CHO-K1 cells, but not CHO cells lacking of cell-surface glycosaminoglycans (GAGs), indicating that binding of ECP32–41 to cell-surface GAGs was required for internalization. When cells were cultured with GAGs or pre-treated with GAG-digesting enzymes, significant decreases in ECP32–41 internalization were observed, suggesting that cell-surface GAGs, especially heparan sulfate proteoglycans were necessary for ECP32–41 attachment and penetration. Furthermore, treatment with pharmacological agents identified two forms of energy-dependent endocytosis, lipid-raft endocytosis and macropinocytosis, as the major ECP32–41 internalization routes. ECP32–41 was demonstrated to transport various cargoes including fluorescent chemical, fluorescent protein, and peptidomimetic drug into cultured Beas-2B cells in vitro, and targeted broncho-epithelial and intestinal villi tissues in vivo. Hence this CPP has the potential to serve as a novel vehicle for intracellular delivery of biomolecules or medicines, especially for the treatment of pulmonary or gastrointestinal diseases. PMID:23469189

  4. Epithelial TRPV1 signaling accelerates gingival epithelial cell proliferation.

    PubMed

    Takahashi, N; Matsuda, Y; Yamada, H; Tabeta, K; Nakajima, T; Murakami, S; Yamazaki, K

    2014-11-01

    Transient receptor potential cation channel subfamily V member 1 (TRPV1), a member of the calcium-permeable thermosensitive transient receptor potential superfamily, is a sensor of thermal and chemical stimuli. TRPV1 is activated by noxious heat (> 43°C), acidic conditions (pH < 6.6), capsaicin, and endovanilloids. This pain receptor was discovered on nociceptive fibers in the peripheral nervous system. TRPV1 was recently found to be expressed by non-neuronal cells, such as epithelial cells. The oral gingival epithelium is exposed to multiple noxious stimuli, including heat and acids derived from endogenous and exogenous substances; however, whether gingival epithelial cells (GECs) express TRPV1 is unknown. We show that both TRPV1 mRNA and protein are expressed by GECs. Capsaicin, a TRPV1 agonist, elevated intracellular Ca(2+) levels in the gingival epithelial cell line, epi 4. Moreover, TRPV1 activation in epi 4 cells accelerated proliferation. These responses to capsaicin were inhibited by a specific TRPV1 antagonist, SB-366791. We also observed GEC proliferation in capsaicin-treated mice in vivo. No effects were observed on GEC apoptosis by epithelial TRPV1 signaling. To examine the molecular mechanisms underlying this proliferative effect, we performed complementary (c)DNA microarray analysis of capsaicin-stimulated epi 4 cells. Compared with control conditions, 227 genes were up-regulated and 232 genes were down-regulated following capsaicin stimulation. Several proliferation-related genes were validated by independent experiments. Among them, fibroblast growth factor-17 and neuregulin 2 were significantly up-regulated in capsaicin-treated epi 4 cells. Our results suggest that functional TRPV1 is expressed by GECs and contributes to the regulation of cell proliferation. PMID:25266715

  5. Symmetry breaking mechanism for epithelial cell polarization

    NASA Astrophysics Data System (ADS)

    Veglio, A.; Gamba, A.; Nicodemi, M.; Bussolino, F.; Serini, G.

    2009-09-01

    In multicellular organisms, epithelial cells form layers separating compartments responsible for different physiological functions. At the early stage of epithelial layer formation, each cell of an aggregate defines an inner and an outer side by breaking the symmetry of its initial state, in a process known as epithelial polarization. By integrating recent biochemical and biophysical data with stochastic simulations of the relevant reaction-diffusion system, we provide evidence that epithelial cell polarization is a chemical phase-separation process induced by a local bistability in the signaling network at the level of the cell membrane. The early symmetry breaking event triggering phase separation is induced by adhesion-dependent mechanical forces localized in the point of convergence of cell surfaces when a threshold number of confluent cells is reached. The generality of the emerging phase-separation scenario is likely common to many processes of cell polarity formation.

  6. Wood Smoke Particle Sequesters Cell Iron to Impact a Biological Effect.

    PubMed

    Ghio, Andrew J; Soukup, Joleen M; Dailey, Lisa A; Tong, Haiyan; Kesic, Matthew J; Budinger, G R Scott; Mutlu, Gökhan M

    2015-11-16

    The biological effect of an inorganic particle (i.e., silica) can be associated with a disruption in cell iron homeostasis. Organic compounds included in particles originating from combustion processes can also complex sources of host cell iron to disrupt metal homeostasis. We tested the postulate that (1) wood smoke particle (WSP) sequesters host cell iron resulting in a disruption of metal homeostasis, (2) this loss of essential metal results in both an oxidative stress and biological effect in respiratory epithelial cells, and (3) humic-like substances (HULIS), a component of WSP, have a capacity to appropriate cell iron and initiate a biological effect. BEAS-2B cells exposed to WSP resulted in diminished concentrations of mitochondrial (57)Fe, whereas preincubation with ferric ammonium citrate (FAC) prevented significant mitochondrial iron loss after such exposure. Cellular oxidant generation was increased after WSP exposure, but this signal was diminished by coincubation with FAC. Similarly, exposure of BEAS-2B cells to 100 μg/mL WSP activated mitogen-activated protein (MAP) kinases, elevated NF-E2-related factor 2/antioxidant responsive element (Nrf2 ARE) expression, and provoked interleukin (IL)-6 and IL-8 release, but all these changes were diminished by coincubation with FAC. The biological response to WSP was reproduced by exposure to 100 μg/mL humic acid, a polyphenol comparable to HULIS included in the WSP that complexes iron. We conclude that (1) the biological response following exposure to WSP is associated with sequestration of cell iron by the particle, (2) increasing available iron in the cell diminished the biological effects after particle exposure, and (3) HULIS included in WSP can sequester the metal initiating the cell response. PMID:26462088

  7. Modulation of airway epithelial cell functions by Pidotimod: NF-kB cytoplasmatic expression and its nuclear translocation are associated with an increased TLR-2 expression

    PubMed Central

    2013-01-01

    Background Recurrent respiratory infections are one of the most important causes of morbidity in childhood. When immune functions are still largely immature, the airway epithelium plays a primary defensive role since, besides providing a physical barrier, it is also involved in the innate and the adaptive immune responses. A study was therefore designed to evaluate in vitro whether pidotimod, a synthetic dipeptide able to stimulate the inflammatory and immune effector cells, could activate bronchial epithelial cell functions involved in response to infections. Methods BEAS-2B cell line (human bronchial epithelial cells infected with a replication-defective Adenovirus 12-SV40 virus hybrid) were cultured in the presence of pidotimod, with or without tumor necrosis factor (TNF)-α or zymosan to assess: a) intercellular adhesion molecule (ICAM)-1 expression, by flow cytometry; b) toll-like receptor (TLR)-2 expression and production, by immunofluorescence flow cytometry and western blotting; d) interleukin (IL)-8 release, by enzyme-linked immunosorbent assay (ELISA); e) activated extracellular-signal-regulated kinase (ERK1/2) phosphorylation and nuclear factor-kappa B (NF-kB) activation, by western blotting. Results The constitutive expression of ICAM-1 and IL-8 release were significant up-regulated by TNF-α (ICAM-1) and by TNF-α and zymosan (IL-8), but not by pidotimod. In contrast, an increased TLR-2 expression was found after exposure to pidotimod 10 and 100 μg/ml (p < 0.05) and to the association pidotimod 100 μg/ml + TNF-α (p < 0.05). Western blot analysis substantiated that the constitutive TLR-2 expression was significantly increased after exposure to all the stimuli. Finally, while a remarkable inhibition of TNF-α -induced ERK1/2 phosphorylation was observed in the presence of pidotimod, both TNF-α and pidotimod were effective in inducing NF-kB protein expression in the cytoplasm and its nuclear translocation. Conclusion Through different

  8. Airway epithelial cell responses to ozone injury

    SciTech Connect

    Leikauf, G.D.; Simpson, L.G.; Zhao, Qiyu

    1995-03-01

    The airway epithelial cell is an important target in ozone injury. Once activated, the airway epithelium responds in three phases. The initial, or immediate phase, involves activation of constitutive cells, often through direct covalent interactions including the formation of secondary ozonolysis products-hydroxyhydroperoxides, aldehydes, and hydrogen peroxide. Recently, we found hydroxyhydroperoxides to be potent agonists; of bioactive eicosanoid formation by human airway epithelial cells in culture. Other probable immediate events include activation and inactivation of enzymes present on the epithelial surface (e.g., neutral endopeptidase). During the next 2 to 24 hr, or early phase, epithelial cells respond by synthesis and release of chemotactic factors, including chemokines-macrophage inflammatory protein-2, RANTES, and interleukin-8. Infiltrating leukocytes during this period also release elastase, an important agonist of epithelial cell mucus secretion and additional chemokine formation. The third (late) phase of ozone injury is characterized by eosinophil or monocyte infiltration. Cytokine expression leads to alteration of structural protein synthesis, with increases in fibronectin evident by in situ hybridization. Synthesis of epithelial antiproteases, e.g., secretary leukocyte protease inhibitor, may also increase locally 24 to 48 hr after elastase concentrations become excessive. Thus, the epithelium is not merely a passive barrier to ozone injury but has a dynamic role in directing the migration, activating, and then counteracting inflammatory cells. Through these complex interactions, epithelial cells can be viewed as the initiators (alpha) and the receptors (omega) of ozone-induced airway disease. 51 refs., 2 figs., 3 tabs.

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

    PubMed Central

    Chang, Hong-Bin; Chen, Bing-Huei

    2015-01-01

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

  10. Human bronchial epithelial cells exposed in vitro to diesel exhaust particles exhibit alterations in cell rheology and cytotoxicity associated with decrease in antioxidant defenses and imbalance in pro- and anti-apoptotic gene expression.

    PubMed

    Seriani, Robson; de Souza, Claudia Emanuele Carvalho; Krempel, Paloma Gava; Frias, Daniela Perroni; Matsuda, Monique; Correia, Aristides Tadeu; Ferreira, Márcia Zotti Justo; Alencar, Adriano Mesquita; Negri, Elnara Marcia; Saldiva, Paulo Hilário Nascimento; Mauad, Thais; Macchione, Mariangela

    2016-05-01

    Diesel exhaust particles (DEPs) from diesel engines produce adverse alterations in cells of the airways by activating intracellular signaling pathways and apoptotic gene overexpression, and also by influencing metabolism and cytoskeleton changes. This study used human bronchial epithelium cells (BEAS-2B) in culture and evaluates their exposure to DEPs (15ug/mL for 1 and 2 h) in order to determine changes to cell rheology (viscoelasticity) and gene expression of the enzymes involved in oxidative stress, apoptosis, and cytotoxicity. BEAS-2B cells exposed to DEPs were found to have a significant loss in stiffness, membrane stability, and mitochondrial activity. The genes involved in apoptosis [B cell lymphoma 2 (BCL-2 and caspase-3)] presented inversely proportional expressions (p = 0.05, p = 0.01, respectively), low expression of the genes involved in antioxidant responses [SOD1 (superoxide dismutase 1); SOD2 (superoxide dismutase 2), and GPx (glutathione peroxidase) (p = 0.01)], along with an increase in cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1) (p = 0.01). These results suggest that alterations in cell rheology and cytotoxicity could be associated with oxidative stress and imbalance between pro- and anti-apoptotic genes. PMID:26856867

  11. Cell Type-Dependent Changes in CdSe/ZnS Quantum Dot Uptake and Toxic Endpoints

    PubMed Central

    Soenen, Stefaan J.; Al-Ali, Abdullah; Brown, Andy; Hondow, Nicole; Wills, John; Jenkins, Gareth J. S.; Doak, Shareen H.

    2015-01-01

    Toxicity of nanoparticles (NPs) is often correlated with the physicochemical characteristics of the materials. However, some discrepancies are noted in in-vitro studies on quantum dots (QDs) with similar physicochemical properties. This is partly related to variations in cell type. In this study, we show that epithelial (BEAS-2B), fibroblast (HFF-1), and lymphoblastoid (TK6) cells show different biological responses following exposure to QDs. These cells represented the 3 main portals of NP exposure: bronchial, skin, and circulatory. The uptake and toxicity of negatively and positively charged CdSe:ZnS QDs of the same core size but with different surface chemistries (carboxyl or amine polymer coatings) were investigated in full and reduced serum containing media following 1 and 3 cell cycles. Following thorough physicochemical characterization, cellular uptake, cytotoxicity, and gross chromosomal damage were measured. Cellular damage mechanisms in the form of reactive oxygen species and the expression of inflammatory cytokines IL-8 and TNF-α were assessed. QDs uptake and toxicity significantly varied in the different cell lines. BEAS-2B cells demonstrated the highest level of QDs uptake yet displayed a strong resilience with minimal genotoxicity following exposure to these NPs. In contrast, HFF-1 and TK6 cells were more susceptible to toxicity and genotoxicity, respectively, as a result of exposure to QDs. Thus, this study demonstrates that in addition to nanomaterial physicochemical characterization, a clear understanding of cell type-dependent variation in uptake coupled to the inherently different capacities of the cell types to cope with exposure to these exogenous materials are all required to predict genotoxicity. PMID:25601991

  12. Cell type-dependent changes in CdSe/ZnS quantum dot uptake and toxic endpoints.

    PubMed

    Manshian, Bella B; Soenen, Stefaan J; Al-Ali, Abdullah; Brown, Andy; Hondow, Nicole; Wills, John; Jenkins, Gareth J S; Doak, Shareen H

    2015-04-01

    Toxicity of nanoparticles (NPs) is often correlated with the physicochemical characteristics of the materials. However, some discrepancies are noted in in-vitro studies on quantum dots (QDs) with similar physicochemical properties. This is partly related to variations in cell type. In this study, we show that epithelial (BEAS-2B), fibroblast (HFF-1), and lymphoblastoid (TK6) cells show different biological responses following exposure to QDs. These cells represented the 3 main portals of NP exposure: bronchial, skin, and circulatory. The uptake and toxicity of negatively and positively charged CdSe:ZnS QDs of the same core size but with different surface chemistries (carboxyl or amine polymer coatings) were investigated in full and reduced serum containing media following 1 and 3 cell cycles. Following thorough physicochemical characterization, cellular uptake, cytotoxicity, and gross chromosomal damage were measured. Cellular damage mechanisms in the form of reactive oxygen species and the expression of inflammatory cytokines IL-8 and TNF-α were assessed. QDs uptake and toxicity significantly varied in the different cell lines. BEAS-2B cells demonstrated the highest level of QDs uptake yet displayed a strong resilience with minimal genotoxicity following exposure to these NPs. In contrast, HFF-1 and TK6 cells were more susceptible to toxicity and genotoxicity, respectively, as a result of exposure to QDs. Thus, this study demonstrates that in addition to nanomaterial physicochemical characterization, a clear understanding of cell type-dependent variation in uptake coupled to the inherently different capacities of the cell types to cope with exposure to these exogenous materials are all required to predict genotoxicity. PMID:25601991

  13. Cell Division Drives Epithelial Cell Rearrangements during Gastrulation in Chick.

    PubMed

    Firmino, Joao; Rocancourt, Didier; Saadaoui, Mehdi; Moreau, Chloe; Gros, Jerome

    2016-02-01

    During early embryonic development, cells are organized as cohesive epithelial sheets that are continuously growing and remodeled without losing their integrity, giving rise to a wide array of tissue shapes. Here, using live imaging in chick embryo, we investigate how epithelial cells rearrange during gastrulation. We find that cell division is a major rearrangement driver that powers dramatic epithelial cell intercalation events. We show that these cell division-mediated intercalations, which represent the majority of epithelial rearrangements within the early embryo, are absolutely necessary for the spatial patterning of gastrulation movements. Furthermore, we demonstrate that these intercalation events result from overall low cortical actomyosin accumulation within the epithelial cells of the embryo, which enables dividing cells to remodel junctions in their vicinity. These findings uncover a role for cell division as coordinator of epithelial growth and remodeling that might underlie various developmental, homeostatic, or pathological processes in amniotes. PMID:26859350

  14. Odontogenic epithelial stem cells: hidden sources.

    PubMed

    Padma Priya, Sivan; Higuchi, Akon; Abu Fanas, Salem; Pooi Ling, Mok; Kumari Neela, Vasantha; Sunil, P M; Saraswathi, T R; Murugan, Kadarkarai; Alarfaj, Abdullah A; Munusamy, Murugan A; Kumar, Suresh

    2015-12-01

    The ultimate goal of dental stem cell research is to construct a bioengineered tooth. Tooth formation occurs based on the well-organized reciprocal interaction of epithelial and mesenchymal cells. The dental mesenchymal stem cells are the best explored, but because the human odontogenic epithelium is lost after the completion of enamel formation, studies on these cells are scarce. The successful creation of a bioengineered tooth is achievable only when the odontogenic epithelium is reconstructed to produce a replica of natural enamel. This article discusses the untapped sources of odontogenic epithelial stem cells in humans, such as those present in the active dental lamina in postnatal life, in remnants of dental lamina (the gubernaculum cord), in the epithelial cell rests of Malassez, and in reduced enamel epithelium. The possible uses of these stem cells in regenerative medicine, not just for enamel formation, are discussed. PMID:26367485

  15. Anti-Inflammatory Activity of a Novel Family of Aryl Ureas Compounds in an Endotoxin-Induced Airway Epithelial Cell Injury Model

    PubMed Central

    Cabrera-Benitez, Nuria E.; Pérez-Roth, Eduardo; Casula, Milena; Ramos-Nuez, Ángela; Ríos-Luci, Carla; Rodríguez-Gallego, Carlos; Sologuren, Ithaisa; Jakubkiene, Virginija; Slutsky, Arthur S.; Padrón, José M.; Villar, Jesús

    2012-01-01

    Background Despite our increased understanding of the mechanisms involved in acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS), there is no specific pharmacological treatment of proven benefit. We used a novel screening methodology to examine potential anti-inflammatory effects of a small structure-focused library of synthetic carbamate and urea derivatives in a well established cell model of lipopolysaccharide (LPS)-induced ALI/ARDS. Methodology/Principal Findings After a pilot study to develop an in vitro LPS-induced airway epithelial cell injury model, a library of synthetic carbamate and urea derivates was screened against representative panels of human solid tumor cell lines and bacterial and fungal strains. Molecules that were non-cytotoxic and were inactive in terms of antiproliferative and antimicrobial activities were selected to study the effects on LPS-induced inflammatory response in an in vitro cell culture model using A549 human alveolar and BEAS-2B human bronchial cells. These cells were exposed for 18 h to LPS obtained from Escherichia coli, either alone or in combination with the test compounds. The LPS antagonists rhein and emodin were used as reference compounds. The most active compound (CKT0103) was selected as the lead compound and the impact of CKT0103 on pro-inflammatory IL-6 and IL-8 cytokine levels, expression of toll-like receptor-4 (TLR4) and nuclear factor kappa B inhibitor alpha (IκBα) was measured. CKT0103 significantly inhibited the synthesis and release of IL-6 and IL-8 induced by LPS. This suppression was associated with inhibition of TLR4 up-regulation and IκBα down-regulation. Immunocytochemical staining for TLR4 and IκBα supported these findings. Conclusions/Significance Using a novel screening methodology, we identified a compound – CKT0103 – with potent anti-inflammatory effects. These findings suggest that CKT0103 is a potential target for the treatment of the acute phase of sepsis and

  16. Superiority of combined phosphodiesterase PDE3/PDE4 inhibition over PDE4 inhibition alone on glucocorticoid- and long-acting β2-adrenoceptor agonist-induced gene expression in human airway epithelial cells.

    PubMed

    BinMahfouz, Hawazen; Borthakur, Bibhusana; Yan, Dong; George, Tresa; Giembycz, Mark A; Newton, Robert

    2015-01-01

    Glucocorticoids, also known as corticosteroids, induce effector gene transcription as a part of their anti-inflammatory mechanisms of action. Such genomic effects can be significantly enhanced by long-acting β2-adrenoceptor agonists (LABAs) and may contribute to the clinical superiority of inhaled corticosteroid (ICS)/LABA combinations in asthma and chronic obstructive pulmonary disease (COPD) over ICSs alone. Using models of cAMP- and glucocorticoid-induced transcription in human bronchial epithelial BEAS-2B cells, we show that combining inhibitors of phosphodiesterase (PDE) 3 and PDE4 provides greater benefits compared with inhibiting either PDE alone. In respect to cAMP-dependent transcription, inhibitors of PDE3 (siguazodan, cilostazol) and PDE4 (rolipram, GSK256066, roflumilast N-oxide) each sensitized to the LABA, formoterol. This effect was magnified by dual PDE3 and PDE4 inhibition. Siguazodan plus rolipram was also more effective at inducing cAMP-dependent transcription than either inhibitor alone. Conversely, the concentration-response curve describing the enhancement of dexamethasone-induced, glucocorticoid response element-dependent transcription by formoterol was displaced to the left by PDE4, but not PDE3, inhibition. Overall, similar effects were described for bona fide genes, including RGS2, CD200, and CRISPLD2. Importantly, the combination of siguazodan plus rolipram prolonged the duration of gene expression induced by formoterol, dexamethasone, or dexamethasone plus formoterol. This was most apparent for RGS2, a bronchoprotective gene that may also reduce the proinflammatory effects of constrictor mediators. Collectively, these data provide a rationale for the use of PDE3 and PDE4 inhibitors in the treatment of COPD and asthma where they may enhance, sensitize, and prolong the effects of LABA/ICS combination therapies. PMID:25324049

  17. Different roles of ROS and Nrf2 in Cr(VI)-induced inflammatory responses in normal and Cr(VI)-transformed cells.

    PubMed

    Roy, Ram Vinod; Pratheeshkumar, Poyil; Son, Yong-Ok; Wang, Lei; Hitron, John Andrew; Divya, Sasidharan Padmaja; Zhang, Zhuo; Shi, Xianglin

    2016-09-15

    Hexavalent chromium (Cr(VI)) is classified as a human carcinogen. Cr(VI) has been associated with adenocarcinomas and squamous cell carcinoma of the lung. The present study shows that acute Cr(VI) treatment in human bronchial epithelial cells (BEAS-2B) increased inflammatory responses (TNF-α, COX-2, and NF-кB/p65) and expression of Nrf2. Cr(VI)-induced generation of reactive oxygen species (ROS) are responsible for increased inflammation. Despite the fact that Nrf2 is a master regulator of response to oxidative stress, silencing of Nrf2 in the acute Cr(VI) treatment had no effect on Cr(VI)-induced inflammation. In contrast, in Cr(VI)-transformed (CrT) cells, Nrf2 is constitutively activated. Knock-down of this protein resulted in decreased inflammation, while silencing of SOD2 and CAT had no effect in the expression of these inflammatory proteins. Results obtained from the knock-down of Nrf2 in CrT cells are very different from the results obtained in the acute Cr(VI) treatment. In BEAS-2B cells, knock-down of Nrf2 had no effect in the inflammation levels, while in CrT cells a decrease in the expression of inflammation markers was observed. These results indicate that before transformation, ROS plays a critical role while Nrf2 not in Cr(VI)-induced inflammation, whereas after transformation (CrT cells), Nrf2 is constitutively activated and this protein maintains inflammation while ROS not. Constitutively high levels of Nrf2 in CrT binds to the promoter regions of COX-2 and TNF-α, leading to increased inflammation. Collectively, our results demonstrate that before cell transformation ROS are important in Cr(VI)-induced inflammation and after transformation a constitutively high level of Nrf2 is important. PMID:27470422

  18. Three-Dimensionally Engineered Normal Human Broncho-epithelial Tissue-Like Assemblies: Target Tissues for Human Respiratory Viral Infections

    NASA Technical Reports Server (NTRS)

    Goodwin, T. J.; McCarthy, M.; Lin, Y-H

    2006-01-01

    In vitro three-dimensional (3D) human broncho-epithelial (HBE) tissue-like assemblies (3D HBE TLAs) from this point forward referred to as TLAs were engineered in Rotating Wall Vessel (RWV) technology to mimic the characteristics of in vivo tissues thus providing a tool to study human respiratory viruses and host cell interactions. The TLAs were bioengineered onto collagen-coated cyclodextran microcarriers using primary human mesenchymal bronchial-tracheal cells (HBTC) as the foundation matrix and an adult human bronchial epithelial immortalized cell line (BEAS-2B) as the overlying component. The resulting TLAs share significant characteristics with in vivo human respiratory epithelium including polarization, tight junctions, desmosomes, and microvilli. The presence of tissue-like differentiation markers including villin, keratins, and specific lung epithelium markers, as well as the production of tissue mucin, further confirm these TLAs differentiated into tissues functionally similar to in vivo tissues. Increasing virus titers for human respiratory syncytial virus (wtRSVA2) and parainfluenza virus type 3 (wtPIV3 JS) and the detection of membrane bound glycoproteins over time confirm productive infections with both viruses. Therefore, TLAs mimic aspects of the human respiratory epithelium and provide a unique capability to study the interactions of respiratory viruses and their primary target tissue independent of the host's immune system.

  19. Induced pluripotency of human prostatic epithelial cells.

    PubMed

    Zhao, Hongjuan; Sun, Ning; Young, Sarah R; Nolley, Rosalie; Santos, Jennifer; Wu, Joseph C; Peehl, Donna M

    2013-01-01

    Induced pluripotent stem (iPS) cells are a valuable resource for discovery of epigenetic changes critical to cell type-specific differentiation. Although iPS cells have been generated from other terminally differentiated cells, the reprogramming of normal adult human basal prostatic epithelial (E-PZ) cells to a pluripotent state has not been reported. Here, we attempted to reprogram E-PZ cells by forced expression of Oct4, Sox2, c-Myc, and Klf4 using lentiviral vectors and obtained embryonic stem cell (ESC)-like colonies at a frequency of 0.01%. These E-PZ-iPS-like cells with normal karyotype gained expression of pluripotent genes typical of iPS cells (Tra-1-81, SSEA-3, Nanog, Sox2, and Oct4) and lost gene expression characteristic of basal prostatic epithelial cells (CK5, CK14, and p63). E-PZ-iPS-like cells demonstrated pluripotency by differentiating into ectodermal, mesodermal, and endodermal cells in vitro, although lack of teratoma formation in vivo and incomplete demethylation of pluripotency genes suggested only partial reprogramming. Importantly, E-PZ-iPS-like cells re-expressed basal epithelial cell markers (CD44, p63, MAO-A) in response to prostate-specific medium in spheroid culture. Androgen induced expression of androgen receptor (AR), and co-culture with rat urogenital sinus further induced expression of prostate-specific antigen (PSA), a hallmark of secretory cells, suggesting that E-PZ-iPS-like cells have the capacity to differentiate into prostatic basal and secretory epithelial cells. Finally, when injected into mice, E-PZ-iPS-like cells expressed basal epithelial cell markers including CD44 and p63. When co-injected with rat urogenital mesenchyme, E-PZ-iPS-like cells expressed AR and expression of p63 and CD44 was repressed. DNA methylation profiling identified epigenetic changes in key pathways and genes involved in prostatic differentiation as E-PZ-iPS-like cells converted to differentiated AR- and PSA-expressing cells. Our results suggest that

  20. Epithelial Cell Shedding and Barrier Function

    PubMed Central

    Williams, J. M.; Duckworth, C. A.; Burkitt, M. D.; Watson, A. J. M.; Campbell, B. J.

    2015-01-01

    The intestinal epithelium is a critical component of the gut barrier. Composed of a single layer of intestinal epithelial cells (IECs) held together by tight junctions, this delicate structure prevents the transfer of harmful microorganisms, antigens, and toxins from the gut lumen into the circulation. The equilibrium between the rate of apoptosis and shedding of senescent epithelial cells at the villus tip, and the generation of new cells in the crypt, is key to maintaining tissue homeostasis. However, in both localized and systemic inflammation, this balance may be disturbed as a result of pathological IEC shedding. Shedding of IECs from the epithelial monolayer may cause transient gaps or microerosions in the epithelial barrier, resulting in increased intestinal permeability. Although pathological IEC shedding has been observed in mouse models of inflammation and human intestinal conditions such as inflammatory bowel disease, understanding of the underlying mechanisms remains limited. This process may also be an important contributor to systemic and intestinal inflammatory diseases and gut barrier dysfunction in domestic animal species. This review aims to summarize current knowledge about intestinal epithelial cell shedding, its significance in gut barrier dysfunction and host-microbial interactions, and where research in this field is directed. PMID:25428410

  1. Interaction with Epithelial Cells Modifies Airway Macrophage Response to Ozone

    EPA Science Inventory

    The initial innate immune response to ozone (03) in the lung is orchestrated by structural cells, such as epithelial cells, and resident immune cells, such as airway macrophages (Macs). We developed an epithelial cell-Mac coculture model to investigate how epithelial cell-derived...

  2. Epithelial Cell Regulation of Allergic Diseases.

    PubMed

    Gour, Naina; Lajoie, Stephane

    2016-09-01

    Allergic diseases, which have escalated in prevalence in recent years, arise as a result of maladaptive immune responses to ubiquitous environmental stimuli. Why only certain individuals mount inappropriate type 2 immune responses to these otherwise harmless allergens has remained an unanswered question. Mounting evidence suggests that the epithelium, by sensing its environment, is the central regulator of allergic diseases. Once considered to be a passive barrier to allergens, epithelial cells at mucosal surfaces are now considered to be the cornerstone of the allergic diathesis. Beyond their function as maintaining barrier at mucosal surfaces, mucosal epithelial cells through the secretion of mediators like IL-25, IL-33, and TSLP control the fate of downstream allergic immune responses. In this review, we will discuss the advances in recent years regarding the process of allergen recognition and secretion of soluble mediators by epithelial cells that shape the development of the allergic response. PMID:27534656

  3. Rosemary components inhibit benzo[a]pyrene-induced genotoxicity in human bronchial cells.

    PubMed

    Offord, E A; Macé, K; Ruffieux, C; Malnoë, A; Pfeifer, A M

    1995-09-01

    The commonly used spice and flavouring agent, rosemary, derived from the leaves of the plant Rosmarinus officinalis L., displays antioxidant properties in foods and in biological systems. Moreover, in animal models rosemary components were found to inhibit the initiation and tumour promotion phases of carcinogenesis. In this work, we studied the mechanisms by which rosemary components block initiation of carcinogenesis by the procarcinogen benzo[a]pyrene (B[a]P) in human bronchial epithelial cells (BEAS-2B). Whole rosemary extract (6 micrograms/ml) or an equivalent concentration of its most potent antioxidant constituents, carnosol or carnosic acid, inhibited DNA adduct formation by 80% after 6 h co-incubation with 1.5 muM B[a]P. Under similar conditions, cytochrome P450 (CYP) 1A1 mRNA expression was 50% lower in the presence of rosemary components, and CYP1A1 activity was inhibited 70-90%. The observed reduction of DNA adduct formation by rosemary components may mostly result from the inhibition of the activation of benzo[a]pyrene to its ultimate metabolites. Carnosol also affected expression of the phase II enzyme glutathione-S-transferase which is known to detoxify the proximate carcinogenic metabolite of B[a]P. Treatment of BEAS-2B cells with carnosol (1 microgram/ml) for 24 h resulted in a 3- to 4-fold induction of GST pi mRNA. Moreover, expression of a second important phase II enzyme, NAD(P)H: quinone reductase, was induced by carnosol in parallel with GST pi. Therefore, rosemary components have the potential to decrease activation and increase detoxification of an important human carcinogen, identifying them as promising candidates for chemopreventive programs. PMID:7554054

  4. Expression of secretory phospholipase A2 enzymes in lungs of humans with pneumonia and their potential prostaglandin-synthetic function in human lung-derived cells.

    PubMed

    Masuda, Seiko; Murakami, Makoto; Mitsuishi, Michiko; Komiyama, Kazuo; Ishikawa, Yukio; Ishii, Toshiharu; Kudo, Ichiro

    2005-04-01

    Although a number of sPLA2 (secretory phospholipase A2) enzymes have been identified in mammals, the localization and functions of individual enzymes in human pathologic tissues still remain obscure. In the present study, we have examined the expression and function of sPLA2s in human lung-derived cells and in human lungs with pneumonia. Group IID, V and X sPLA2s were expressed in cultured human bronchial epithelial cells (BEAS-2B) and normal human pulmonary fibroblasts with distinct requirement for cytokines (interleukin-1b, tumour necrosis factor a and interferon-g). Lentivirus- or adenovirus-mediated transfection of various sPLA2s into BEAS-2B or normal human pulmonary fibroblast cells revealed that group V and X sPLA2s increased arachidonate release and prostaglandin production in both cell types, whereas group IIA and IID sPLA2s failed to do so. Immunohistochemistry of human lungs with pneumonia demonstrated that group V and X sPLA2s were widely expressed in the airway epithelium, interstitium and alveolar macrophages, in which group IID sPLA2 was also positive, whereas group IIA sPLA2 was restricted to the pulmonary arterial smooth muscle layers and bronchial chondrocytes, and group IIE and IIF sPLA2s were minimally detected. These results suggest that group V and X sPLA2s affect lung pathogenesis by facilitating arachidonate metabolism or possibly through other functions. PMID:15509193

  5. Innate lymphoid cells regulate intestinal epithelial cell glycosylation.

    PubMed

    Goto, Yoshiyuki; Obata, Takashi; Kunisawa, Jun; Sato, Shintaro; Ivanov, Ivaylo I; Lamichhane, Aayam; Takeyama, Natsumi; Kamioka, Mariko; Sakamoto, Mitsuo; Matsuki, Takahiro; Setoyama, Hiromi; Imaoka, Akemi; Uematsu, Satoshi; Akira, Shizuo; Domino, Steven E; Kulig, Paulina; Becher, Burkhard; Renauld, Jean-Christophe; Sasakawa, Chihiro; Umesaki, Yoshinori; Benno, Yoshimi; Kiyono, Hiroshi

    2014-09-12

    Fucosylation of intestinal epithelial cells, catalyzed by fucosyltransferase 2 (Fut2), is a major glycosylation mechanism of host-microbiota symbiosis. Commensal bacteria induce epithelial fucosylation, and epithelial fucose is used as a dietary carbohydrate by many of these bacteria. However, the molecular and cellular mechanisms that regulate the induction of epithelial fucosylation are unknown. Here, we show that type 3 innate lymphoid cells (ILC3) induced intestinal epithelial Fut2 expression and fucosylation in mice. This induction required the cytokines interleukin-22 and lymphotoxin in a commensal bacteria-dependent and -independent manner, respectively. Disruption of intestinal fucosylation led to increased susceptibility to infection by Salmonella typhimurium. Our data reveal a role for ILC3 in shaping the gut microenvironment through the regulation of epithelial glycosylation. PMID:25214634

  6. Protons Sensitize Epithelial Cells to Mesenchymal Transition

    PubMed Central

    Wang, Minli; Hada, Megumi; Saha, Janapriya; Sridharan, Deepa M.; Pluth, Janice M.; Cucinotta, Francis A.

    2012-01-01

    Proton radiotherapy has gained more favor among oncologists as a treatment option for localized and deep-seated tumors. In addition, protons are a major constituent of the space radiation astronauts receive during space flights. The potential for these exposures to lead to, or enhance cancer risk has not been well studied. Our objective is to study the biological effects of low energy protons on epithelial cells and its propensity to enhance transforming growth factor beta 1 (TGFβ1)-mediated epithelial-mesenchymal transition (EMT), a process occurring during tumor progression and critical for invasion and metastasis. Non-transformed mink lung epithelial cells (Mv1Lu) and hTERT- immortalized human esophageal epithelial cells (EPC) were used in this study. EMT was identified by alterations in cell morphology, EMT-related gene expression changes determined using real-time PCR, and EMT changes in specific cellular markers detected by immunostaining and western blotting. Although TGFβ1 treatment alone is able to induce EMT in both Mv1Lu and EPC cells, low energy protons (5 MeV) at doses as low as 0.1 Gy can enhance TGFβ1 induced EMT. Protons alone can also induce a mild induction of EMT. SD208, a potent TGFβ Receptor 1 (TGFβR1) kinase inhibitor, can efficiently block TGFβ1/Smad signaling and attenuate EMT induction. We suggest a model for EMT after proton irradiation in normal and cancerous tissue based on our results that showed that low and high doses of protons can sensitize normal human epithelial cells to mesenchymal transition, more prominently in the presence of TGFβ1, but also in the absence of TGFβ1. PMID:22844446

  7. Respiratory epithelial cells orchestrate pulmonary innate immunity.

    PubMed

    Whitsett, Jeffrey A; Alenghat, Theresa

    2015-01-01

    The epithelial surfaces of the lungs are in direct contact with the environment and are subjected to dynamic physical forces as airway tubes and alveoli are stretched and compressed during ventilation. Mucociliary clearance in conducting airways, reduction of surface tension in the alveoli, and maintenance of near sterility have been accommodated by the evolution of a multi-tiered innate host-defense system. The biophysical nature of pulmonary host defenses are integrated with the ability of respiratory epithelial cells to respond to and 'instruct' the professional immune system to protect the lungs from infection and injury. PMID:25521682

  8. Esophageal epithelial cells acquire functional characteristics of activated myofibroblasts after undergoing an epithelial to mesenchymal transition

    PubMed Central

    Muir, Amanda B.; Dods, Kara; Noah, Yuli; Toltzis, Sarit; Chandramouleeswaran, Prasanna Modayur; Lee, Anna; Benitez, Alain; Bedenbaugh, Adam; Falk, Gary W.; Wells, Rebecca G.; Nakagawa, Hiroshi; Wang, Mei-Lun

    2015-01-01

    Background and Aims Eosinophilic esophagitis (EoE) is an allergic inflammatory disease that leads to esophageal fibrosis and stricture. We have recently shown that in EoE, esophageal epithelial cells undergo an epithelial to mesenchymal transition (EMT), characterized by gain of mesenchymal markers and loss of epithelial gene expression. Whether epithelial cells exposed to profibrotic cytokines can also acquire the functional characteristics of activated myofibroblasts, including migration, contraction, and extracellular matrix deposition, is relevant to our understanding and treatment of EoE-associated fibrogenesis. In the current study, we characterize cell migration, contraction, and collagen production by esophageal epithelial cells that have undergone cytokine-induced EMT in vitro. Methods and Results Stimulation of human non-transformed immortalized esophageal epithelial cells (EPC2-hTERT) with profibrotic cytokines TNFα, TGFβ, and IL1β for three weeks led to acquisition of mesenchymal αSMA and vimentin, and loss of epithelial E-cadherin expression. Upon removal of the profibrotic stimulus, epithelial characteristics were partially rescued. TGFβ stimulation had a robust effect upon epithelial collagen production. Surprisingly, TNFα stimulation had the most potent effect upon cell migration and contraction, exceeding the effects of the prototypical profibrotic cytokine TGFβ. IL1β stimulation alone had minimal effect upon esophageal epithelial migration, contraction, and collagen production. Conclusions Esophageal epithelial cells that have undergone EMT acquire functional characteristics of activated myofibroblasts in vitro. Profibrotic cytokines exert differential effects upon esophageal epithelial cells, underscoring complexities of fibrogenesis in EoE, and implicating esophageal epithelial cells as effector cells in EoE-associated fibrogenesis. PMID:25183431

  9. EDAC: Epithelial defence against cancer-cell competition between normal and transformed epithelial cells in mammals.

    PubMed

    Kajita, Mihoko; Fujita, Yasuyuki

    2015-07-01

    During embryonic development or under certain pathological conditions, viable but suboptimal cells are often eliminated from the cellular society through a process termed cell competition. Cell competition was originally identified in Drosophila where cells with different properties compete for survival; 'loser' cells are eliminated from tissues and consequently 'winner' cells become dominant. Recent studies have shown that cell competition also occurs in mammals. While apoptotic cell death is the major fate for losers in Drosophila, outcompeted cells show more variable phenotypes in mammals, such as cell death-independent apical extrusion and cellular senescence. Molecular mechanisms underlying these processes have been recently revealed. Especially, in epithelial tissues, normal cells sense and actively eliminate the neighbouring transformed cells via cytoskeletal proteins by the process named epithelial defence against cancer (EDAC). Here, we introduce this newly emerging research field: cell competition in mammals. PMID:25991731

  10. Comparative DNA damage and transcriptomic effects of engineered nanoparticles in human lung cells in vitro

    EPA Science Inventory

    A series of six titanium dioxide and two cerium oxide engineered nanomaterials were assessed for their ability to induce cytotoxicity, reactive oxygen species (ROS), various types of DNA damage, and transcriptional changes in human respiratory BEAS-2B cells exposed in vitro at se...

  11. Human Mammary Luminal Epithelial Cells Contain Progenitors to Myoepithelial Cells

    SciTech Connect

    Pechoux, Christine; Gudjonsson, Thorarinn; Ronnov-Jessen, Lone; Bissell, Mina J; Petersen, Ole

    1999-02-01

    The origin of the epithelial and myoepithelial cells in the human breast has not been delineated. In this study we have addressed whether luminal epithelial cells and myoepithelial cells are vertically connected, i.e., whether one is the precursor for the other. We used a primary culture assay allowing preservation of basic phenotypic traits of luminal epithelial and myoepithelial cells in culture. The two cell types were then separated immunomagnetically using antibodies directed against lineage-specific cell surface antigens into at best 100% purity. The cellular identity was ascertained by cytochemistry, immunoblotting, and 2-D gel electrophoresis. Luminal epithelial cells were identified by strong expression of cytokeratins 18 and 19 while myoepithelial cells were recognized by expression of vimentin and {alpha}-smooth muscle actin. We used a previously devised culture medium (CDM4) that allows vigorous expansion of proliferative myoepithelial cells and also devised a medium (CDM6) that allowed sufficient expansion of differentiated luminal epithelial cells based on addition of hepatocyte growth factor/scatter factor. The two different culture media supported each lineage for at least five passages without signs of interconversion. We used parallel cultures where we switched culture media, thus testing the ability of each lineage to convert to the other. Whereas the myoepithelial lineage showed no signs of interconversion, a subset of luminal epithelial cells, gradually, but distinctly, converted to myoepithelial cells. We propose that in the mature human breast, it is the luminal epithelial cell compartment that gives rise to myoepithelial cells rather than the other way around.

  12. DNA typing of epithelial cells after strangulation.

    PubMed

    Wiegand, P; Kleiber, M

    1997-01-01

    DNA typing was carried out on epithelial cells which were transferred from the hands of the suspect onto the neck of the victim. In an experimental study 16 suspect-victim combinations were investigated for estimating the typing success. Alternatively to an attack against the neck, the upper arm was used for "strangulation". PCR typing was carried out using the short tandem repeat systems (STRs) HumCD4, HumVWF31A (VWA) and Hum-FIBRA (FGA) and the success rate was > 70% for all 3 systems. In most of the cases mixed patterns containing the phenotype of the suspect and the victim were obtained. In a case where strangulation was the cause of death, epithelial cells could be removed from the neck of the victim. The DNA pattern of the suspect could be successfully amplified using four STRs, demonstrating the applicability of this approach for practical casework. PMID:9274940

  13. Control of local immunity by airway epithelial cells.

    PubMed

    Weitnauer, M; Mijošek, V; Dalpke, A H

    2016-03-01

    The lung is ventilated by thousand liters of air per day. Inevitably, the respiratory system comes into contact with airborne microbial compounds, most of them harmless contaminants. Airway epithelial cells are known to have innate sensor functions, thus being able to detect microbial danger. To avoid chronic inflammation, the pulmonary system has developed specific means to control local immune responses. Even though airway epithelial cells can act as proinflammatory promoters, we propose that under homeostatic conditions airway epithelial cells are important modulators of immune responses in the lung. In this review, we discuss epithelial cell regulatory functions that control reactivity of professional immune cells within the microenvironment of the airways and how these mechanisms are altered in pulmonary diseases. Regulation by epithelial cells can be divided into two mechanisms: (1) mediators regulate epithelial cells' innate sensitivity in cis and (2) factors are produced that limit reactivity of immune cells in trans. PMID:26627458

  14. Generation of Mouse Lung Epithelial Cells

    PubMed Central

    Kasinski, Andrea L.; Slack, Frank J.

    2016-01-01

    Although in vivo models are excellent for assessing various facets of whole organism physiology, pathology, and overall response to treatments, evaluating basic cellular functions, and molecular events in mammalian model systems is challenging. It is therefore advantageous to perform these studies in a refined and less costly setting. One approach involves utilizing cells derived from the model under evaluation. The approach to generate such cells varies based on the cell of origin and often the genetics of the cell. Here we describe the steps involved in generating epithelial cells from the lungs of KrasLSL-G12D/+; p53LSL-R172/+ mice (Kasinski and Slack, 2012). These mice develop aggressive lung adenocarcinoma following cre-recombinase dependent removal of a stop cassette in the transgenes and subsequent expression of Kra-G12D and p53R172. While this protocol may be useful for the generation of epithelial lines from other genetic backgrounds, it should be noted that the Kras; p53 cell line generated here is capable of proliferating in culture without any additional genetic manipulation that is often needed for less aggressive backgrounds.

  15. Transcriptional Landscape of Glomerular Parietal Epithelial Cells

    PubMed Central

    Gharib, Sina A.; Pippin, Jeffrey W.; Ohse, Takamoto; Pickering, Scott G.; Krofft, Ronald D.; Shankland, Stuart J.

    2014-01-01

    Very little is known about the function of glomerular parietal epithelial cells (PECs). In this study, we performed genome-wide expression analysis on PEC-enriched capsulated vs. PEC-deprived decapsulated rat glomeruli to determine the transcriptional state of PECs under normal conditions. We identified hundreds of differentially expressed genes that mapped to distinct biologic modules including development, tight junction, ion transport, and metabolic processes. Since developmental programs were highly enriched in PECs, we characterized several of their candidate members at the protein level. Collectively, our findings confirm that PECs are multifaceted cells and help define their diverse functional repertoire. PMID:25127402

  16. Celecoxib increases lung cancer cell lysis by lymphokine-activated killer cells via upregulation of ICAM-1

    PubMed Central

    Frank, Marcus; Linnebacher, Michael; Hinz, Burkhard

    2015-01-01

    The antitumorigenic mechanism of the selective cyclooxygenase-2 (COX-2) inhibitor celecoxib is still a matter of debate. Using lung cancer cell lines (A549, H460) and metastatic cells derived from a lung cancer patient, the present study investigates the impact of celecoxib on the expression of intercellular adhesion molecule 1 (ICAM-1) and cancer cell lysis by lymphokine-activated killer (LAK) cells. Celecoxib, but not other structurally related selective COX-2 inhibitors (i.e., etoricoxib, rofecoxib, valdecoxib), was found to cause a substantial upregulation of ICAM-1 protein levels. Likewise, ICAM-1 mRNA expression was increased by celecoxib. Celecoxib enhanced the susceptibility of cancer cells to be lysed by LAK cells with the respective effect being reversed by a neutralizing ICAM-1 antibody. In addition, enhanced killing of celecoxib-treated cancer cells was reversed by preincubation of LAK cells with an antibody to lymphocyte function associated antigen 1 (LFA-1), suggesting intercellular ICAM-1/LFA-1 crosslink as crucial event within this process. Finally, celecoxib elicited no significant increase of LAK cell-mediated lysis of non-tumor bronchial epithelial cells, BEAS-2B, associated with a far less ICAM-1 induction as compared to cancer cells. Altogether, our data demonstrate celecoxib-induced upregulation of ICAM-1 on lung cancer cells to be responsible for intercellular ICAM-1/LFA-1 crosslink that confers increased cancer cell lysis by LAK cells. These findings provide proof for a novel antitumorigenic mechanism of celecoxib. PMID:26513172

  17. Cationic polystyrene nanospheres induce autophagic cell death through the induction of endoplasmic reticulum stress

    NASA Astrophysics Data System (ADS)

    Chiu, Hui-Wen; Xia, Tian; Lee, Yu-Hsuan; Chen, Chun-Wan; Tsai, Jui-Chen; Wang, Ying-Jan

    2014-12-01

    Nanoparticles (NPs) have been used to produce a wide range of products that have applications in imaging and drug delivery in medicine. Due to their chemical stability, well-controlled sizes and surface charges, polystyrene (PS) NPs have been developed as biosensors and drug delivery carriers. However, the possible adverse biological effects and underlying mechanisms are still unclear. Recently, autophagy has been implicated in the regulation of cell death. In this study, we evaluated a library of PS NPs with different surface charges. We found that NH2-labeled polystyrene (NH2-PS) nanospheres were highly toxic with enhanced uptake in macrophage (RAW 264.7) and lung epithelial (BEAS-2B) cells. Furthermore, NH2-PS could induce autophagic cell death. NH2-PS increased autophagic flux due to reactive oxygen species (ROS) generation and endoplasmic reticulum (ER) stress caused by misfolded protein aggregation. The inhibition of ER stress decreased cytotoxicity and autophagy in the NH2-PS-treated cells. In addition, the Akt/mTOR and AMPK signaling pathways were involved in the regulation of NH2-PS-triggered autophagic cell death. These results suggest an important role of autophagy in cationic NP-induced cell death and provide mechanistic insights into the inhibition of the toxicity and safe material design.Nanoparticles (NPs) have been used to produce a wide range of products that have applications in imaging and drug delivery in medicine. Due to their chemical stability, well-controlled sizes and surface charges, polystyrene (PS) NPs have been developed as biosensors and drug delivery carriers. However, the possible adverse biological effects and underlying mechanisms are still unclear. Recently, autophagy has been implicated in the regulation of cell death. In this study, we evaluated a library of PS NPs with different surface charges. We found that NH2-labeled polystyrene (NH2-PS) nanospheres were highly toxic with enhanced uptake in macrophage (RAW 264.7) and lung

  18. Formation of a Neurosensory Organ by Epithelial Cell Slithering.

    PubMed

    Kuo, Christin S; Krasnow, Mark A

    2015-10-01

    Epithelial cells are normally stably anchored, maintaining their relative positions and association with the basement membrane. Developmental rearrangements occur through cell intercalation, and cells can delaminate during epithelial-mesenchymal transitions and metastasis. We mapped the formation of lung neuroepithelial bodies (NEBs), innervated clusters of neuroendocrine/neurosensory cells within the bronchial epithelium, revealing a targeted mode of cell migration that we named "slithering," in which cells transiently lose epithelial character but remain associated with the membrane while traversing neighboring epithelial cells to reach cluster sites. Immunostaining, lineage tracing, clonal analysis, and live imaging showed that NEB progenitors, initially distributed randomly, downregulate adhesion and polarity proteins, crawling over and between neighboring cells to converge at diametrically opposed positions at bronchial branchpoints, where they reestablish epithelial structure and express neuroendocrine genes. There is little accompanying progenitor proliferation or apoptosis. Activation of the slithering program may explain why lung cancers arising from neuroendocrine cells are highly metastatic. PMID:26435104

  19. Ouabain modulates ciliogenesis in epithelial cells

    PubMed Central

    Larre, Isabel; Castillo, Aida; Flores-Maldonado, Catalina; Contreras, Ruben G.; Galvan, Ivan; Muñoz-Estrada, Jesus; Cereijido, Marcelino

    2011-01-01

    The exchange of substances between higher organisms and the environment occurs across transporting epithelia whose basic features are tight junctions (TJs) that seal the intercellular space, and polarity, which enables cells to transport substances vectorially. In a previous study, we demonstrated that 10 nM ouabain modulates TJs, and we now show that it controls polarity as well. We gauge polarity through the development of a cilium at the apical domain of Madin-Darby canine kidney cells (MDCK, epithelial dog kidney). Ouabain accelerates ciliogenesis in an ERK1/2-dependent manner. Claudin-2, a molecule responsible for the Na+ and H2O permeability of the TJs, is also present at the cilium, as it colocalizes and coprecipitates with acetylated α-tubulin. Ouabain modulates claudin-2 localization at the cilium through ERK1/2. Comparing wild-type and ouabain-resistant MDCK cells, we show that ouabain acts through Na+,K+-ATPase. Taken together, our previous and present results support the possibility that ouabain constitutes a hormone that modulates the transporting epithelial phenotype, thereby playing a crucial role in metazoan life. PMID:22143774

  20. Is the inflammasome relevant for epithelial cell function?

    PubMed

    Santana, Patricia T; Martel, Jan; Lai, Hsin-Chih; Perfettini, Jean-Luc; Kanellopoulos, Jean M; Young, John D; Coutinho-Silva, Robson; Ojcius, David M

    2016-02-01

    Inflammasomes are intracellular protein complexes that sense microbial components and damage of infected cells. Following activation by molecules released by pathogens or injured cells, inflammasomes activate caspase-1, allowing secretion of the pro-inflammatory cytokines IL-1β and IL-18 from innate immune cells. Inflammasomes are also expressed in epithelial cells, where their function has attracted less attention. Nonetheless, depending on the tissue, epithelial inflammasomes can mediate inflammation, wound healing, and pain sensitivity. We review here recent findings on inflammasomes found in epithelial tissues, highlighting the importance of these protein complexes in the response of epithelial tissues to microbial infections. PMID:26546965

  1. Coevolution of neoplastic epithelial cells and multilineage stroma via polyploid giant cells during immortalization and transformation of mullerian epithelial cells

    PubMed Central

    Zhang, Shiwu; Mercado-Uribe, Imelda; Sood, Anil; Bast, Robert C.; Liu, Jinsong

    2016-01-01

    Stromal cells are generally considered to be derived primarily from the host's normal mesenchymal stromal cells or bone marrow. However, the origins of stromal cells have been quite controversial. To determine the role of polyploidy in tumor development, we examined the fate of normal mullerian epithelial cells during the immortalization and transformation process by tracing the expression of SV40 large T antigen. Here we show that immortalized or HRAS-transformed mullerian epithelial cells contain a subpopulation of polyploid giant cells that grow as multicellular spheroids expressing hematopoietic markers in response to treatment with CoCl2. The immortalized or transformed epithelial cells can transdifferentiate into stromal cells when transplanted into nude mice. Immunofluorescent staining revealed expression of stem cell factors OCT4, Nanog, and SOX-2 in spheroid, whereas expression of embryonic stem cell marker SSEA1 was increased in HRAS-transformed cells compared with their immortalized isogenic counterparts. These results suggest that normal mullerian epithelial cells are intrinsically highly plastic, via the formation of polyploid giant cells and activation of embryonic stem-like program, which work together to promote the coevolution of neoplastic epithelial cells and multiple lineage stromal cells. PMID:27382431

  2. Establishment of Hertwig's epithelial root sheath/epithelial rests of Malassez cell line from human periodontium.

    PubMed

    Nam, Hyun; Kim, Ji-Hye; Kim, Jae-Won; Seo, Byoung-Moo; Park, Joo-Cheol; Kim, Jung-Wook; Lee, Gene

    2014-07-01

    Human Hertwig's epithelial root sheath/epithelial rests of Malassez (HERS/ERM) cells are epithelial remnants of teeth residing in the periodontium. Although the functional roles of HERS/ERM cells have yet to be elucidated, they are a unique epithelial cell population in adult teeth and are reported to have stem cell characteristics. Therefore, HERS/ERM cells might play a role as an epithelial component for the repair or regeneration of dental hard tissues; however, they are very rare population in periodontium and the primary isolation of them is considered to be difficult. To overcome these problems, we immortalized primary HERS/ERM cells isolated from human periodontium using SV40 large T antigen (SV40 LT) and performed a characterization of the immortalized cell line. Primary HERS/ERM cells could not be maintained for more than 6 passages; however, immortalized HERS/ERM cells were maintained for more than 20 passages. There were no differences in the morphological and immunophenotypic characteristics of HERS/ERM cells and immortalized HERS/ERM cells. The expression of epithelial stem cell and embryonic stem cell markers was maintained in immortalized HERS/ERM cells. Moreover, immortalized HERS/ERM cells could acquire mesenchymal phenotypes through the epithelial-mesenchymal transition via TGF-β1. In conclusion, we established an immortalized human HERS/ERM cell line with SV40 LT and expect this cell line to contribute to the understanding of the functional roles of HERS/ERM cells and the tissue engineering of teeth. PMID:25081036

  3. Stromal-epithelial interactions in aging and cancer: Senescent fibroblasts alter epithelial cell differentiation

    SciTech Connect

    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.

  4. Stromal-epithelial interactions in aging and cancer: senescent fibroblasts alter epithelial cell differentiation

    PubMed Central

    Parrinello, Simona; Coppe, Jean-Philippe; Krtolica, Ana; Campisi, Judith

    2016-01-01

    Summary Cellular senescence suppresses cancer by arresting cells at risk of 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. Furthermore, 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. PMID:15657080

  5. Henipavirus pathogenesis in human respiratory epithelial cells.

    PubMed

    Escaffre, Olivier; Borisevich, Viktoriya; Carmical, J Russ; Prusak, Deborah; Prescott, Joseph; Feldmann, Heinz; Rockx, Barry

    2013-03-01

    Hendra virus (HeV) and Nipah virus (NiV) are deadly zoonotic viruses for which no vaccines or therapeutics are licensed for human use. Henipavirus infection causes severe respiratory illness and encephalitis. Although the exact route of transmission in human is unknown, epidemiological studies and in vivo studies suggest that the respiratory tract is important for virus replication. However, the target cells in the respiratory tract are unknown, as are the mechanisms by which henipaviruses can cause disease. In this study, we characterized henipavirus pathogenesis using primary cells derived from the human respiratory tract. The growth kinetics of NiV-Malaysia, NiV-Bangladesh, and HeV were determined in bronchial/tracheal epithelial cells (NHBE) and small airway epithelial cells (SAEC). In addition, host responses to infection were assessed by gene expression analysis and immunoassays. Viruses replicated efficiently in both cell types and induced large syncytia. The host response to henipavirus infection in NHBE and SAEC highlighted a difference in the inflammatory response between HeV and NiV strains as well as intrinsic differences in the ability to mount an inflammatory response between NHBE and SAEC. These responses were highest during HeV infection in SAEC, as characterized by the levels of key cytokines (interleukin 6 [IL-6], IL-8, IL-1α, monocyte chemoattractant protein 1 [MCP-1], and colony-stimulating factors) responsible for immune cell recruitment. Finally, we identified virus strain-dependent variability in type I interferon antagonism in NHBE and SAEC: NiV-Malaysia counteracted this pathway more efficiently than NiV-Bangladesh and HeV. These results provide crucial new information in the understanding of henipavirus pathogenesis in the human respiratory tract at an early stage of infection. PMID:23302882

  6. Henipavirus Pathogenesis in Human Respiratory Epithelial Cells

    PubMed Central

    Escaffre, Olivier; Borisevich, Viktoriya; Carmical, J. Russ; Prusak, Deborah; Prescott, Joseph; Feldmann, Heinz

    2013-01-01

    Hendra virus (HeV) and Nipah virus (NiV) are deadly zoonotic viruses for which no vaccines or therapeutics are licensed for human use. Henipavirus infection causes severe respiratory illness and encephalitis. Although the exact route of transmission in human is unknown, epidemiological studies and in vivo studies suggest that the respiratory tract is important for virus replication. However, the target cells in the respiratory tract are unknown, as are the mechanisms by which henipaviruses can cause disease. In this study, we characterized henipavirus pathogenesis using primary cells derived from the human respiratory tract. The growth kinetics of NiV-Malaysia, NiV-Bangladesh, and HeV were determined in bronchial/tracheal epithelial cells (NHBE) and small airway epithelial cells (SAEC). In addition, host responses to infection were assessed by gene expression analysis and immunoassays. Viruses replicated efficiently in both cell types and induced large syncytia. The host response to henipavirus infection in NHBE and SAEC highlighted a difference in the inflammatory response between HeV and NiV strains as well as intrinsic differences in the ability to mount an inflammatory response between NHBE and SAEC. These responses were highest during HeV infection in SAEC, as characterized by the levels of key cytokines (interleukin 6 [IL-6], IL-8, IL-1α, monocyte chemoattractant protein 1 [MCP-1], and colony-stimulating factors) responsible for immune cell recruitment. Finally, we identified virus strain-dependent variability in type I interferon antagonism in NHBE and SAEC: NiV-Malaysia counteracted this pathway more efficiently than NiV-Bangladesh and HeV. These results provide crucial new information in the understanding of henipavirus pathogenesis in the human respiratory tract at an early stage of infection. PMID:23302882

  7. Nuclear microscopy of rat colon epithelial cells

    NASA Astrophysics Data System (ADS)

    Ren, M.; Rajendran, Reshmi; Ng, Mary; Udalagama, Chammika; Rodrigues, Anna E.; Watt, Frank; Jenner, Andrew Michael

    2011-10-01

    Using Nuclear microscopy, we have investigated iron distributions in the colons of Sprague Dawley rats, in order to elucidate heme uptake. Four groups of five Sprague Dawley rats (mean weight 180 g) were fed different purified diets containing either heme diet (2.5% w/w hemoglobin), high fat diet (HFD) (18% w/w fat, 1% w/w cholesterol), 'western' diet (combination of hemoglobin 2.5% and 18% fat, 1% cholesterol) or control diet (7% w/w fat). After 4 weeks, animals were sacrificed by exsanguination after anaesthesia. Thin sections of frozen colon tissue were taken, freeze dried and scanned using nuclear microscopy utilising the techniques PIXE, RBS and STIM. The new data acquisition system (IonDaq) developed in CIBA was used to obtain high resolution images and line scans were used to map the iron distributions across the colon boundaries. The nuclear microscope results indicate that when HFD is given in addition to heme, the iron content of the epithelial cells that line the colon decreases, and the zinc in the smooth muscle wall increases. This implies that the level of heme and fat in diet has an important role in colon health, possibly by influencing epithelial cells directly or changing luminal composition such as bacterial flora or levels of metabolites and cytotoxins.

  8. Regulation of intestinal epithelial cells transcriptome by enteric glial cells: impact on intestinal epithelial barrier functions

    PubMed Central

    2009-01-01

    Background Emerging evidences suggest that enteric glial cells (EGC), a major constituent of the enteric nervous system (ENS), are key regulators of intestinal epithelial barrier (IEB) functions. Indeed EGC inhibit intestinal epithelial cells (IEC) proliferation and increase IEB paracellular permeability. However, the role of EGC on other important barrier functions and the signalling pathways involved in their effects are currently unknown. To achieve this goal, we aimed at identifying the impact of EGC upon IEC transcriptome by performing microarray studies. Results EGC induced significant changes in gene expression profiling of proliferating IEC after 24 hours of co-culture. 116 genes were identified as differentially expressed (70 up-regulated and 46 down-regulated) in IEC cultured with EGC compared to IEC cultured alone. By performing functional analysis of the 116 identified genes using Ingenuity Pathway Analysis, we showed that EGC induced a significant regulation of genes favoring both cell-to-cell and cell-to-matrix adhesion as well as cell differentiation. Consistently, functional studies showed that EGC induced a significant increase in cell adhesion. EGC also regulated genes involved in cell motility towards an enhancement of cell motility. In addition, EGC profoundly modulated expression of genes involved in cell proliferation and cell survival, although no clear functional trend could be identified. Finally, important genes involved in lipid and protein metabolism of epithelial cells were shown to be differentially regulated by EGC. Conclusion This study reinforces the emerging concept that EGC have major protective effects upon the IEB. EGC have a profound impact upon IEC transcriptome and induce a shift in IEC phenotype towards increased cell adhesion and cell differentiation. This concept needs to be further validated under both physiological and pathophysiological conditions. PMID:19883504

  9. Epithelial-mesenchymal transition can suppress major attributes of human epithelial tumor-initiating cells

    PubMed Central

    Celià-Terrassa, Toni; Meca-Cortés, Óscar; Mateo, Francesca; Martínez de Paz, Alexia; Rubio, Nuria; Arnal-Estapé, Anna; Ell, Brian J.; Bermudo, Raquel; Díaz, Alba; Guerra-Rebollo, Marta; Lozano, Juan José; Estarás, Conchi; Ulloa, Catalina; ρlvarez-Simón, Daniel; Milà, Jordi; Vilella, Ramón; Paciucci, Rosanna; Martínez-Balbás, Marian; García de Herreros, Antonio; Gomis, Roger R.; Kang, Yibin; Blanco, Jerónimo; Fernández, Pedro L.; Thomson, Timothy M.

    2012-01-01

    Malignant progression in cancer requires populations of tumor-initiating cells (TICs) endowed with unlimited self renewal, survival under stress, and establishment of distant metastases. Additionally, the acquisition of invasive properties driven by epithelial-mesenchymal transition (EMT) is critical for the evolution of neoplastic cells into fully metastatic populations. Here, we characterize 2 human cellular models derived from prostate and bladder cancer cell lines to better understand the relationship between TIC and EMT programs in local invasiveness and distant metastasis. The model tumor subpopulations that expressed a strong epithelial gene program were enriched in highly metastatic TICs, while a second subpopulation with stable mesenchymal traits was impoverished in TICs. Constitutive overexpression of the transcription factor Snai1 in the epithelial/TIC-enriched populations engaged a mesenchymal gene program and suppressed their self renewal and metastatic phenotypes. Conversely, knockdown of EMT factors in the mesenchymal-like prostate cancer cell subpopulation caused a gain in epithelial features and properties of TICs. Both tumor cell subpopulations cooperated so that the nonmetastatic mesenchymal-like prostate cancer subpopulation enhanced the in vitro invasiveness of the metastatic epithelial subpopulation and, in vivo, promoted the escape of the latter from primary implantation sites and accelerated their metastatic colonization. Our models provide new insights into how dynamic interactions among epithelial, self-renewal, and mesenchymal gene programs determine the plasticity of epithelial TICs. PMID:22505459

  10. Documentation of angiotensin II receptors in glomerular epithelial cells

    NASA Technical Reports Server (NTRS)

    Sharma, M.; Sharma, R.; Greene, A. S.; McCarthy, E. T.; Savin, V. J.; Cowley, A. W. (Principal Investigator)

    1998-01-01

    Angiotensin II decreases glomerular filtration rate, renal plasma flow, and glomerular capillary hydraulic conductivity. Although angiotensin II receptors have been demonstrated in mesangial cells and proximal tubule cells, the presence of angiotensin II receptors in glomerular epithelial cells has not previously been shown. Previously, we have reported that angiotensin II caused an accumulation of cAMP and a reorganization of the actin cytoskeleton in cultured glomerular epithelial cells. Current studies were conducted to verify the presence of angiotensin II receptors by immunological and non-peptide receptor ligand binding techniques and to ascertain the activation of intracellular signal transduction in glomerular epithelial cells in response to angiotensin II. Confluent monolayer cultures of glomerular epithelial cells were incubated with angiotensin II, with or without losartan and/or PD-123,319 in the medium. Membrane vesicle preparations were obtained by homogenization of washed cells followed by centrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane proteins followed by multiscreen immunoblotting was used to determine the presence of angiotensin II receptor type 1 (AT1) or type 2 (AT2). Angiotensin II-mediated signal transduction in glomerular epithelial cells was studied by measuring the levels of cAMP, using radioimmunoassay. Results obtained in these experiments showed the presence of both AT1 and AT2 receptor types in glomerular epithelial cells. Angiotensin II was found to cause an accumulation of cAMP in glomerular epithelial cells, which could be prevented only by simultaneous use of losartan and PD-123,319, antagonists for AT1 and AT2, respectively. The presence of both AT1 and AT2 receptors and an increase in cAMP indicate that glomerular epithelial cells respond to angiotensin II in a manner distinct from that of mesangial cells or proximal tubular epithelial cells. Our results suggest that glomerular epithelial

  11. Applications of mouse airway epithelial cell culture for asthma research.

    PubMed

    Horani, Amjad; Dickinson, John D; Brody, Steven L

    2013-01-01

    Primary airway epithelial cell culture provides a valuable tool for studying cell differentiation, cell-cell interactions, and the role of immune system factors in asthma pathogenesis. In this chapter, we discuss the application of mouse tracheal epithelial cell cultures for the study of asthma biology. A major advantage of this system is the ability to use airway epithelial cells from mice with defined genetic backgrounds. The in vitro proliferation and differentiation of mouse airway epithelial cells uses the air-liquid interface condition to generate well-differentiated epithelia with characteristics of native airways. Protocols are provided for manipulation of differentiation, induction of mucous cell metaplasia, genetic modification, and cell and pathogen coculture. Assays for the assessment of gene expression, responses of cells, and analysis of specific cell subpopulations within the airway epithelium are included. PMID:23943446

  12. Klebsiella pneumoniae Is Able to Trigger Epithelial-Mesenchymal Transition Process in Cultured Airway Epithelial Cells

    PubMed Central

    Leone, Laura; Mazzetta, Francesca; Martinelli, Daniela; Valente, Sabatino; Alimandi, Maurizio; Raffa, Salvatore; Santino, Iolanda

    2016-01-01

    The ability of some bacterial pathogens to activate Epithelial-Mesenchymal Transition normally is a consequence of the persistence of a local chronic inflammatory response or depends on a direct interaction of the pathogens with the host epithelial cells. In this study we monitored the abilities of the K. pneumoniae to activate the expression of genes related to EMT-like processes and the occurrence of phenotypic changes in airway epithelial cells during the early steps of cell infection. We describe changes in the production of intracellular reactive oxygen species and increased HIF-1α mRNA expression in cells exposed to K. pneumoniae infection. We also describe the upregulation of a set of transcription factors implicated in the EMT processes, such as Twist, Snail and ZEB, indicating that the morphological changes of epithelial cells already appreciable after few hours from the K. pneumoniae infection are tightly regulated by the activation of transcriptional pathways, driving epithelial cells to EMT. These effects appear to be effectively counteracted by resveratrol, an antioxidant that is able to exert a sustained scavenging of the intracellular ROS. This is the first report indicating that strains of K. pneumoniae may promote EMT-like programs through direct interaction with epithelial cells without the involvement of inflammatory cells. PMID:26812644

  13. Epithelial in vitro cell systems in carcinogenesis studies

    SciTech Connect

    Borek, C.

    1983-01-01

    The development of epithelial cells systems to study oncogenic transformation has presented a major challenge in the field of carcinogenesis. Because there exists in man a preponderance of carcinomas over sarcomas, the importance of studying oncogenic transformation in epithelial cells is of great relevance to human disease. The difficulty lies in the fact that different tissues contain epithelial cells with singular differentiated characteristics, which must be defined to assert the different nature of the cells being used. Liver cells in culture are a case in point. By careful maintenance and optimal culture conditions, one can maintain many of the differentiated characteristics of the cells for prolonged periods of time.

  14. Heterogeneity and stochastic growth regulation of biliary epithelial cells dictate dynamic epithelial tissue remodeling.

    PubMed

    Kamimoto, Kenji; Kaneko, Kota; Kok, Cindy Yuet-Yin; Okada, Hajime; Miyajima, Atsushi; Itoh, Tohru

    2016-01-01

    Dynamic remodeling of the intrahepatic biliary epithelial tissue plays key roles in liver regeneration, yet the cellular basis for this process remains unclear. We took an unbiased approach based on in vivo clonal labeling and tracking of biliary epithelial cells in the three-dimensional landscape, in combination with mathematical simulation, to understand their mode of proliferation in a mouse liver injury model where the nascent biliary structure formed in a tissue-intrinsic manner. An apparent heterogeneity among biliary epithelial cells was observed: whereas most of the responders that entered the cell cycle upon injury exhibited a limited and tapering growth potential, a select population continued to proliferate, making a major contribution in sustaining the biliary expansion. Our study has highlighted a unique mode of epithelial tissue dynamics, which depends not on a hierarchical system driven by fixated stem cells, but rather, on a stochastically maintained progenitor population with persistent proliferative activity. PMID:27431614

  15. Isolation of Cancer Epithelial Cells from Mouse Mammary Tumors

    PubMed Central

    Johnson, Sara; Chen, Hexin; Lo, Pang-Kuo

    2016-01-01

    The isolation of cancer epithelial cells from mouse mammary tumor is accomplished by digestion of the solid tumor. Red blood cells and other contaminates are removed using several washing techniques such that primary epithelial cells can further enriched. This procedure yields primary tumor cells that can be used for in vitro tissue culture, fluorescence-activated cell sorting (FACS) and a wide variety of other experiments (Lo et al., 2012).

  16. Multi-functionality and plasticity characterize epithelial cells in Hydra

    PubMed Central

    Buzgariu, W; Al Haddad, S; Tomczyk, S; Wenger, Y; Galliot, B

    2015-01-01

    Epithelial sheets, a synapomorphy of all metazoans but porifers, are present as 2 layers in cnidarians, ectoderm and endoderm, joined at their basal side by an extra-cellular matrix named mesoglea. In the Hydra polyp, epithelial cells of the body column are unipotent stem cells that continuously self-renew and concomitantly express their epitheliomuscular features. These multifunctional contractile cells maintain homeostasis by providing a protective physical barrier, by digesting nutrients, by selecting a stable microbiota, and by rapidly closing wounds. In addition, epithelial cells are highly plastic, supporting the adaptation of Hydra to physiological and environmental changes, such as long starvation periods where survival relies on a highly dynamic autophagy flux. Epithelial cells also play key roles in developmental processes as evidenced by the organizer activity they develop to promote budding and regeneration. We propose here an integrative view of the homeostatic and developmental aspects of epithelial plasticity in Hydra. PMID:26716072

  17. Multi-functionality and plasticity characterize epithelial cells in Hydra.

    PubMed

    Buzgariu, W; Al Haddad, S; Tomczyk, S; Wenger, Y; Galliot, B

    2015-01-01

    Epithelial sheets, a synapomorphy of all metazoans but porifers, are present as 2 layers in cnidarians, ectoderm and endoderm, joined at their basal side by an extra-cellular matrix named mesoglea. In the Hydra polyp, epithelial cells of the body column are unipotent stem cells that continuously self-renew and concomitantly express their epitheliomuscular features. These multifunctional contractile cells maintain homeostasis by providing a protective physical barrier, by digesting nutrients, by selecting a stable microbiota, and by rapidly closing wounds. In addition, epithelial cells are highly plastic, supporting the adaptation of Hydra to physiological and environmental changes, such as long starvation periods where survival relies on a highly dynamic autophagy flux. Epithelial cells also play key roles in developmental processes as evidenced by the organizer activity they develop to promote budding and regeneration. We propose here an integrative view of the homeostatic and developmental aspects of epithelial plasticity in Hydra. PMID:26716072

  18. Sonic Hedgehog regulates thymic epithelial cell differentiation.

    PubMed

    Saldaña, José Ignacio; Solanki, Anisha; Lau, Ching-In; Sahni, Hemant; Ross, Susan; Furmanski, Anna L; Ono, Masahiro; Holländer, Georg; Crompton, Tessa

    2016-04-01

    Sonic Hedgehog (Shh) is expressed in the thymus, where it regulates T cell development. Here we investigated the influence of Shh on thymic epithelial cell (TEC) development. Components of the Hedgehog (Hh) signalling pathway were expressed by TEC, and use of a Gli Binding Site-green fluorescence protein (GFP) transgenic reporter mouse demonstrated active Hh-dependent transcription in TEC in the foetal and adult thymus. Analysis of Shh-deficient foetal thymus organ cultures (FTOC) showed that Shh is required for normal TEC differentiation. Shh-deficient foetal thymus contained fewer TEC than wild type (WT), the proportion of medullary TEC was reduced relative to cortical TEC, and cell surface expression of MHC Class II molecules was increased on both cortical and medullary TEC populations. In contrast, the Gli3-deficient thymus, which shows increased Hh-dependent transcription in thymic stroma, had increased numbers of TEC, but decreased cell surface expression of MHC Class II molecules on both cortical and medullary TEC. Neutralisation of endogenous Hh proteins in WT FTOC led to a reduction in TEC numbers, and in the proportion of mature Aire-expressing medullary TEC, but an increase in cell surface expression of MHC Class II molecules on medullary TEC. Likewise, conditional deletion of Shh from TEC in the adult thymus resulted in alterations in TEC differentiation and consequent changes in T cell development. TEC numbers, and the proportion of mature Aire-expressing medullary TEC were reduced, and cell surface expression of MHC Class II molecules on medullary TEC was increased. Differentiation of mature CD4 and CD8 single positive thymocytes was increased, demonstrating the regulatory role of Shh production by TEC on T cell development. Treatment of human thymus explants with recombinant Shh or neutralising anti-Shh antibody indicated that the Hedgehog pathway is also involved in regulation of differentiation from DP to mature SP T cells in the human thymus. PMID

  19. Sonic Hedgehog regulates thymic epithelial cell differentiation

    PubMed Central

    Saldaña, José Ignacio; Solanki, Anisha; Lau, Ching-In; Sahni, Hemant; Ross, Susan; Furmanski, Anna L.; Ono, Masahiro; Holländer, Georg; Crompton, Tessa

    2016-01-01

    Sonic Hedgehog (Shh) is expressed in the thymus, where it regulates T cell development. Here we investigated the influence of Shh on thymic epithelial cell (TEC) development. Components of the Hedgehog (Hh) signalling pathway were expressed by TEC, and use of a Gli Binding Site-green fluorescence protein (GFP) transgenic reporter mouse demonstrated active Hh-dependent transcription in TEC in the foetal and adult thymus. Analysis of Shh-deficient foetal thymus organ cultures (FTOC) showed that Shh is required for normal TEC differentiation. Shh-deficient foetal thymus contained fewer TEC than wild type (WT), the proportion of medullary TEC was reduced relative to cortical TEC, and cell surface expression of MHC Class II molecules was increased on both cortical and medullary TEC populations. In contrast, the Gli3-deficient thymus, which shows increased Hh-dependent transcription in thymic stroma, had increased numbers of TEC, but decreased cell surface expression of MHC Class II molecules on both cortical and medullary TEC. Neutralisation of endogenous Hh proteins in WT FTOC led to a reduction in TEC numbers, and in the proportion of mature Aire-expressing medullary TEC, but an increase in cell surface expression of MHC Class II molecules on medullary TEC. Likewise, conditional deletion of Shh from TEC in the adult thymus resulted in alterations in TEC differentiation and consequent changes in T cell development. TEC numbers, and the proportion of mature Aire-expressing medullary TEC were reduced, and cell surface expression of MHC Class II molecules on medullary TEC was increased. Differentiation of mature CD4 and CD8 single positive thymocytes was increased, demonstrating the regulatory role of Shh production by TEC on T cell development. Treatment of human thymus explants with recombinant Shh or neutralising anti-Shh antibody indicated that the Hedgehog pathway is also involved in regulation of differentiation from DP to mature SP T cells in the human thymus. PMID

  20. Collective Epithelial Migration and Cell Rearrangements Drive Mammary Branching Morphogenesis

    PubMed Central

    Ewald, Andrew J.; Brenot, Audrey; Duong, Myhanh; Chan, Bianca S.; Werb, Zena

    2009-01-01

    Summary Epithelial organs are built through the movement of groups of interconnected cells. We observed cells in elongating mammary ducts reorganize into a multilayered epithelium, migrate collectively, and rearrange dynamically, all without forming leading cellular extensions. Duct initiation required proliferation, Rac, and myosin light-chain kinase, whereas repolarization to a bilayer depended on Rho kinase. We observed that branching morphogenesis results from the active motility of both luminal and myoepithelial cells. Luminal epithelial cells advanced collectively, whereas myoepithelial cells appeared to restrain elongating ducts. Significantly, we observed that normal epithelium and neoplastic hyperplasias are organized similarly during morphogenesis, suggesting common mechanisms of epithelial growth. PMID:18410732

  1. Observing planar cell polarity in multiciliated mouse airway epithelial cells

    PubMed Central

    Vladar, Eszter K.; Lee, Yin Loon; Stearns, Tim; Axelrod, Jeffrey D.

    2015-01-01

    The concerted movement of cilia propels inhaled contaminants out of the lungs, safeguarding the respiratory system from toxins, pathogens, pollutants, and allergens. Motile cilia on the multiciliated cells (MCCs) of the airway epithelium are physically oriented along the tissue axis for directional motility, which depends on the planar cell polarity (PCP) signaling pathway. The MCCs of the mouse respiratory epithelium have emerged as an important model for the study of motile ciliogenesis and the PCP signaling mechanism. Unlike other motile ciliated or planar polarized tissues, airway epithelial cells are relatively easily accessible and primary cultures faithfully model many of the essential features of the in vivo tissue. There is growing interest in understanding how cells acquire and polarize motile cilia due to the impact of mucociliary clearance on respiratory health. Here, we present methods for observing and quantifying the planar polarized orientation of motile cilia both in vivo and in primary culture airway epithelial cells. We describe how to acquire and evaluate electron and light microscopy images of ciliary ultrastructural features that reveal planar polarized orientation. Furthermore, we describe the immunofluorescence localization of PCP pathway components as a simple readout for airway epithelial planar polarization and ciliary orientation. These methods can be adapted to observe ciliary orientation in other multi- and monociliated cells and to detect PCP pathway activity in any tissue or cell type. PMID:25837385

  2. Arsenic exposure induces the Warburg effect in cultured human cells

    SciTech Connect

    Zhao, Fei; Severson, Paul; Pacheco, Samantha; Futscher, Bernard W.; Klimecki, Walter T.

    2013-08-15

    Understanding how arsenic exacts its diverse, global disease burden is hampered by a limited understanding of the particular biological pathways that are disrupted by arsenic and underlie pathogenesis. A reductionist view would predict that a small number of basic pathways are generally perturbed by arsenic, and manifest as diverse diseases. Following an initial observation that arsenite-exposed cells in culture acidify their media more rapidly than control cells, the report here shows that low level exposure to arsenite (75 ppb) is sufficient to induce aerobic glycolysis (the Warburg effect) as a generalized phenomenon in cultured human primary cells and cell lines. Expanded studies in one such cell line, the non-malignant pulmonary epithelial line, BEAS-2B, established that the arsenite-induced Warburg effect was associated with increased accumulation of intracellular and extracellular lactate, an increased rate of extracellular acidification, and inhibition by the non-metabolized glucose analog, 2-deoxy-D-glucose. Associated with the induction of aerobic glycolysis was a pathway-wide induction of glycolysis gene expression, as well as protein accumulation of an established glycolysis master-regulator, hypoxia-inducible factor 1A. Arsenite-induced alteration of energy production in human cells represents the type of fundamental perturbation that could extend to many tissue targets and diseases. - Highlights: • Chronic arsenite exposure induces aerobic glycolysis, dubbed the “Warburg effect”. • Arsenite-induced Warburg effect is a general phenomenon in cultured human cells. • HIF-1A may mediate arsenite induced Warburg effect.

  3. Human Metapneumovirus Is Capable of Entering Cells by Fusion with Endosomal Membranes

    PubMed Central

    Cox, Reagan G.; Mainou, Bernardo A.; Johnson, Monika; Hastings, Andrew K.; Schuster, Jennifer E.; Dermody, Terence S.; Williams, John V.

    2015-01-01

    Human metapneumovirus (HMPV), a member of the Paramyxoviridae family, is a leading cause of lower respiratory illness. Although receptor binding is thought to initiate fusion at the plasma membrane for paramyxoviruses, the entry mechanism for HMPV is largely uncharacterized. Here we sought to determine whether HMPV initiates fusion at the plasma membrane or following internalization. To study the HMPV entry process in human bronchial epithelial (BEAS-2B) cells, we used fluorescence microscopy, an R18-dequenching fusion assay, and developed a quantitative, fluorescence microscopy assay to follow virus binding, internalization, membrane fusion, and visualize the cellular site of HMPV fusion. We found that HMPV particles are internalized into human bronchial epithelial cells before fusing with endosomes. Using chemical inhibitors and RNA interference, we determined that HMPV particles are internalized via clathrin-mediated endocytosis in a dynamin-dependent manner. HMPV fusion and productive infection are promoted by RGD-binding integrin engagement, internalization, actin polymerization, and dynamin. Further, HMPV fusion is pH-independent, although infection with rare strains is modestly inhibited by RNA interference or chemical inhibition of endosomal acidification. Thus, HMPV can enter via endocytosis, but the viral fusion machinery is not triggered by low pH. Together, our results indicate that HMPV is capable of entering host cells by multiple pathways, including membrane fusion from endosomal compartments. PMID:26629703

  4. Technical note: Isolation and characterization of porcine mammary epithelial cells.

    PubMed

    Dahanayaka, S; Rezaei, R; Porter, W W; Johnson, G A; Burghardt, R C; Bazer, F W; Hou, Y Q; Wu, Z L; Wu, G

    2015-11-01

    Within the mammary gland, functional synthesis of milk is performed by its epithelial (alveolar) cells. The availability of a stable mammary epithelial cell line is essential for biochemical studies to elucidate cellular and molecular mechanisms responsible for nutritional regulation of lactation. Therefore, porcine mammary epithelial cells (PMEC) were isolated from mammary glands of a 9-mo-old nonpregnant and nonlactating gilt and cultured to establish a nonimmortalized cell line. These cells were characterized by expression of cytokeratin-18 (an intermediate filament specific for epithelial cells), β-casein (a specific marker for mammary epithelial cells), and α-lactalbumin. In culture, the PMEC doubled in number every 24 h and maintained a cobblestone morphology, typical for cultured epithelial cells, for at least 15 passages. Addition of 0.2 to 2 μg/mL prolactin to culture medium for 3 d induced the production of β-casein and α-lactalbumin by PMEC in a dose-dependent manner. Thus, we have successfully developed a useful PMEC line for future studies of cellular and molecular regulation of milk synthesis by mammary epithelial cells of the sow. PMID:26641038

  5. Sodium metavanadate exhibits carcinogenic tendencies in vitro in immortalized human bronchial epithelial cells†

    PubMed Central

    Passantino, Lisa; Muñoz, Alexandra B.

    2014-01-01

    Pentavalent vanadium compounds induce intracellular changes in vitro that are consistent with those of other carcinogenic substances. While there is no clear evidence that vanadium compounds cause cancer in humans, vanadium pentoxide causes lung cancer in rodents after long-term inhalation exposures and in turn IARC has categorized it as a group 2B possible human carcinogen. The goal of this study was to investigate the carcinogenicity of NaVO3 in the human immortalized bronchial epithelial cell line, Beas-2B. Cells were treated with 10 μM NaVO3 for 5 weeks, with or without recovery time, followed by gene expression microarray analysis. In a separate experiment, cells were exposed to 1–10 μM NaVO3 for 4 weeks and then grown in soft agar to test for anchorage-independent growth. A dose-dependent increase in the number of colonies was observed. In scratch tests, NaVO3-transformed clones could repair a wound faster than controls. In a gene expression microarray analysis of soft agar clones there were 2010 differentially expressed genes (DEG) (adjusted p-value ≤ 0.05) in NaVO3-transformed clones relative to control clones. DEG from this experiment were compared with the DEG of 5 week NaVO3 exposure with or without recovery, all with adjusted p-values < 0.05, and 469 genes were altered in the same direction for transformed clones, 5 week NaVO3-treated cells, and the recovered cells. The data from this study imply that chronic exposure to NaVO3 causes changes that are consistent with cellular transformation including anchorage-independent growth, enhanced migration ability, and gene expression changes that were likely epigenetically inherited. PMID:23963610

  6. Mesenchymal-epithelial interactions during digestive tract development and epithelial stem cell regeneration.

    PubMed

    Le Guen, Ludovic; Marchal, Stéphane; Faure, Sandrine; de Santa Barbara, Pascal

    2015-10-01

    The gastrointestinal tract develops from a simple and uniform tube into a complex organ with specific differentiation patterns along the anterior-posterior and dorso-ventral axes of asymmetry. It is derived from all three germ layers and their cross-talk is important for the regulated development of fetal and adult gastrointestinal structures and organs. Signals from the adjacent mesoderm are essential for the morphogenesis of the overlying epithelium. These mesenchymal-epithelial interactions govern the development and regionalization of the different gastrointestinal epithelia and involve most of the key morphogens and signaling pathways, such as the Hedgehog, BMPs, Notch, WNT, HOX, SOX and FOXF cascades. Moreover, the mechanisms underlying mesenchyme differentiation into smooth muscle cells influence the regionalization of the gastrointestinal epithelium through interactions with the enteric nervous system. In the neonatal and adult gastrointestinal tract, mesenchymal-epithelial interactions are essential for the maintenance of the epithelial regionalization and digestive epithelial homeostasis. Disruption of these interactions is also associated with bowel dysfunction potentially leading to epithelial tumor development. In this review, we will discuss various aspects of the mesenchymal-epithelial interactions observed during digestive epithelium development and differentiation and also during epithelial stem cell regeneration. PMID:26126787

  7. Cell Chirality Induces Collective Cell Migration in Epithelial Sheets

    NASA Astrophysics Data System (ADS)

    Sato, Katsuhiko; Hiraiwa, Tetsuya; Shibata, Tatsuo

    2015-10-01

    During early development, epithelial cells form a monolayer sheet and migrate in a uniform direction. Here, we address how this collective migration can occur without breaking the cell-to-cell attachments. Repeated contraction and expansion of the cell-to-cell interfaces enables the cells to rearrange their positions autonomously within the sheet. We show that when the interface tension is strengthened in a direction that is tilted from the body axis, cell rearrangements occur in such a way that unidirectional movement is induced. We use a vertex model to demonstrate that such anisotropic tension can generate the unidirectional motion of cell sheets. Our results suggest that cell chirality facilitates collective cell migration during tissue morphogenesis.

  8. Fungal glycan interactions with epithelial cells in allergic airway disease

    PubMed Central

    Roy, René M.; Klein, Bruce S.

    2014-01-01

    Human exposure to fungi results in a wide range of health outcomes, from invasive disease or allergy to immune tolerance. Inhaled fungi contact airway epithelial cells as an early event, and this host:fungal interaction can shape the eventual immunological outcome. Emerging evidence points to exposure to fungal cell wall carbohydrates in the development of allergic airway disease. Herein, we describe determinants of fungal allergenicity, and review the responses of airway epithelial cells to fungal carbohydrates. A greater understanding of the recognition of and response to fungal carbohydrates by airway epithelial cells may lead to the development of targeted therapies that ameliorate allergic airway disease. PMID:23602359

  9. Quantitative Assessment of Cytosolic Salmonella in Epithelial Cells

    PubMed Central

    Knodler, Leigh A.; Nair, Vinod; Steele-Mortimer, Olivia

    2014-01-01

    Within mammalian cells, Salmonella enterica serovar Typhimurium (S. Typhimurium) inhabits a membrane-bound vacuole known as the Salmonella-containing vacuole (SCV). We have recently shown that wild type S. Typhimurium also colonizes the cytosol of epithelial cells. Here we sought to quantify the contribution of cytosolic Salmonella to the total population over a time course of infection in different epithelial cell lines and under conditions of altered vacuolar escape. We found that the lysosomotropic agent, chloroquine, acts on vacuolar, but not cytosolic, Salmonella. After chloroquine treatment, vacuolar bacteria are not transcriptionally active or replicative and appear degraded. Using a chloroquine resistance assay, in addition to digitonin permeabilization, we found that S. Typhimurium lyses its nascent vacuole in numerous epithelial cell lines, albeit with different frequencies, and hyper-replication in the cytosol is also widespread. At later times post-infection, cytosolic bacteria account for half of the total population in some epithelial cell lines, namely HeLa and Caco-2 C2Bbe1. Both techniques accurately measured increased vacuole lysis in epithelial cells upon treatment with wortmannin. By chloroquine resistance assay, we also determined that Salmonella pathogenicity island-1 (SPI-1), but not SPI-2, the virulence plasmid nor the flagellar apparatus, was required for vacuolar escape and cytosolic replication in epithelial cells. Together, digitonin permeabilization and the chloroquine resistance assay will be useful, complementary tools for deciphering the mechanisms of SCV lysis and Salmonella replication in the epithelial cell cytosol. PMID:24400108

  10. Lung epithelial cells modulate the inflammatory response of alveolar macrophages.

    PubMed

    Rubovitch, Vardit; Gershnabel, Shoham; Kalina, Moshe

    2007-12-01

    The goal of this study was to examine the effect of alveolar epithelial cells on inflammatory responses in macrophages. Lung epithelial cells (either rat RLE-6TN or human A549 cells) reduced LPS-induced NO production in alveolar macrophages (AM) in a contact-independent mechanism. The inhibitory effect of the epithelial cells was present already at the transcriptional level: LPS-induced inducible NO synthase (iNOS) expression was significantly smaller. Surfactant protein A (SP-A)-induced NO production by alveolar macrophages was also reduced in the presence of A549 cells, though, by a different kinetics. LPS-induced interleukin-6 (IL-6) production (another inflammatory pathway) by alveolar macrophages was also reduced in the presence of RLE-6TN cells. These data suggest a role for lung epithelial cells in the complicated modulation of inflammatory processes, and provide an insight into the mechanism underlying. PMID:17851743