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Sample records for a549 human pulmonary

  1. Extract from Nandina domestica inhibits lipopolysaccharide-induced cyclooxygenase-2 expression in human pulmonary epithelial A549 cells.

    PubMed

    Ueki, Takuro; Akaishi, Tatsuhiro; Okumura, Hidenobu; Abe, Kazuho

    2012-01-01

    Extract from fruits of Nandina domestica THUNBERG (NDE) has been used to improve cough and breathing difficulty in Japan for many years. To explore whether NDE may alleviate respiratory inflammation, we investigated its effect on expression of cyclooxygenase-2 (COX-2) and production of prostaglandin E₂ (PGE₂) in human pulmonary epithelial A549 cells in culture. Treatment with lipopolysaccharide (LPS; 6 µg/mL) resulted in an increase of COX-2 expression and PGE₂ production in A549 cells. Both the LPS-induced COX-2 expression and PGE₂ production were significantly inhibited by NDE (1-10 µg/mL) in a concentration-dependent manner. NDE did not affect COX-1 expression nor COX activity. These results suggest that NDE downregulates LPS-induced COX-2 expression and inhibits PGE₂ production in pulmonary epithelial cells. Furthermore, higenamine and nantenine, two major constituents responsible for tracheal relaxing effect of NDE, did not mimic the inhibitory effect of NDE on LPS-induced COX-2 expression in A549 cells. To identify active constituent(s) of NDE responsible for the anti-inflammatory effect, NDE was introduced in a polyaromatic absorbent resin column and stepwise eluted to yield water fraction, 20% methanol fraction, 40% methanol fraction, 99.8% methanol fraction, and 99.5% acetone fraction. However, none of these five fractions alone inhibited LPS-induced COX-2 expression. On the other hand, exclusion of water fraction from NDE abolished the inhibitory effect of NDE on LPS-induced COX-2 expression. These results suggest that constituent(s) present in water fraction is required but not sufficient for the anti-inflammatory activity of NDE, which may result from interactions among multiple constituents.

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

    PubMed

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

    2016-02-01

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

  3. Inhibitory effect of radiotherapy combined with weekly recombinant human endostatin on the human pulmonary adenocarcinoma A549 xenografts in nude mice.

    PubMed

    Jiang, Xiao-dong; Dai, Peng; Wu, Jin; Song, Da-an; Yu, Jin-ming

    2011-05-01

    The aim of this study was to investigate the inhibitory effect of radiotherapy combined with weekly recombinant human endostatin (RHES) on the human pulmonary adenocarcinoma A549 xenografts in nude mice. The 40 A549 xenograft nude mice models were randomly divided into 4 groups (each group with 10 nude mice). Single radiotherapy group (group 1) was given a single external irradiation (6MV-X ray, 10 Gy) and peritumoral subcutaneous injection of 0.2 ml normal saline every day for 7 days. Single RHES group (group 2) was given peritumoral subcutaneous injection of 0.2 ml RHES (0.75 mg/ml) for 7 days. Combination therapy group (group 3) was given radiotherapy as the same as group 1 and RHES as the same as group 2. Control group was given normal saline as the same as group 1. The tumor volume was smaller in group 3 than in control group from the 8th day after treatment (P<0.05) and tumor regression occurred from the second week after treatment in group 3. On the 15th day after treatment, the inhibitory rates of tumor volume were 69.65%, 92.64% and 116.4% in groups 2, 1 and 3, respectively; MVD number was lower in group 3 than in group 1 (P<0.05); there was no statistical significance in VEGF expression between group 2 and control group as well as between group 3 and group 1 (P>0.05). Apoptosis was marked in group 3. Radiotherapy combined with weekly RHES can significantly inhibit tumor growth and earlier induce tumor regression, which may be related to the improvement of tumor hypoxia and the inhibition of radiation-induced tumor angiogenesis. Short-term application (1 week) of RHES is beneficial to clinical practice.

  4. Winter fine particulate matter from Milan induces morphological and functional alterations in human pulmonary epithelial cells (A549).

    PubMed

    Gualtieri, Maurizio; Mantecca, Paride; Corvaja, Viviana; Longhin, Eleonora; Perrone, Maria Grazia; Bolzacchini, Ezio; Camatini, Marina

    2009-07-10

    Samples of PM(2.5) were gravimetrically collected during the winter 2005/2006 in the urban area of Milan (North Italy). Samples were chemically characterized and the particles were detached from filters to determine their cytotoxic effects on the A549 cell line. Based on the potential toxicological relevance of its components, Milan winter PM(2.5) contained high concentrations of pro-oxidant transition metals and PAHs, while re-suspended particles showed a relatively high frequency of dimensional classes ranging from 40 nm to 300 nm. A549 cells exposed to particle suspensions showed a concentration-dependent decrease in viability, starting from 10 microg/cm(2). Phagocytosis of particles by A549 cells and particle aggregates were morphologically characterized and seemed to depend on both particle concentration and exposure time, with the majority of particles being engulfed in membrane-bound vacuoles after 24h of exposure. The ability of ultrafine particles to penetrate and spread throughout the cells was also verified. Cell membrane lysis and mitochondrial ultrastructural disruption appeared to be the main modifications induced by PM(2.5) on A549 cells. Concomitantly to the adverse effects observed in terms of cell mortality and ultrastructural lesions, a significant intracellular production of reactive oxygen species (ROS) was observed, suggesting that the cytotoxicity, exerted by the winter PM(2.5) in Milan, derived also from its oxidative potential, probably associated with particle-adsorbed metals and PAHs.

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

    PubMed

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

    1991-10-01

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

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

    PubMed Central

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

    1991-01-01

    Synthesis of pulmonary surfactant (PS) is necessary for normal functioning of the lungs and its production is indicative of normal differentiated lung. The human alveolar carcinoma, A549, has been found to synthesis and secrete PS in vitro. The purpose of this study was to optimise the culture conditions for PS synthesis by A549 as well as to determine the potential role of foetal lung fibroblasts in the induction of PS by glucocorticoids. A549 cells growing in filter wells produced higher levels of PS in response to steroid, a 5-fold increase on the filter well compared to only a 1.5-fold increase when the cells were cultured on a conventional plastic substrate. A549 cells grown in filter wells responded to coculture with fibroblasts whether in direct contact or separated co-culture. A 20-fold increase in PS over control values was observed in separated steroid-treated co-cultures, suggesting the presence of a diffusible factor. A partially purified factor was isolated from fibroblast conditioned medium which was capable of inducing differentiation and other phenotypic changes in A549, namely induction of PS, reduction of plasminogen activator activity and reduction in the in vivo growth of A549 xenografts in nude mice. These results suggest that, under the correct conditions, A549 cells, although transformed, still retain the capacity to respond to differentiation-inducing signals from normal fibroblasts. Images Figure 5 PMID:1654985

  7. Pulmonary metastases of the A549-derived lung adenocarcinoma tumors growing in nude mice. A multiple case study.

    PubMed

    Jakubowska, Monika; Sniegocka, Martyna; Podgórska, Ewa; Michalczyk-Wetula, Dominika; Urbanska, Krystyna; Susz, Anna; Fiedor, Leszek; Pyka, Janusz; Płonka, Przemysław M

    2013-01-01

    Lung adenocarcinoma is a leading human malignancy with fatal prognosis. Ninety percent of the deaths, however, are caused by metastases. The model of subcutaneous tumor xenograft in nude mice was adopted to study the growth of control and photodynamically treated tumors derived from the human A549 lung adenocarcinoma cell line. As a side-result of the primary studies, observations on the metastasis of these tumors to the murine lungs were collected, and reported in the present paper. The metastasizing primary tumors were drained by a prominent number of lymphatic vessels. The metastatic tissue revealed the morphology of well-differentiated or trans-differentiated adenocarcinoma. Further histological and histochemical analyses demonstrated the presence of golden-brown granules in the metastatic tissue, similar to these found in the tumor tissue. In contrast to the primary tumors, the electron paramagnetic resonance spectroscopy revealed no nitric oxide - hemoglobin complexes (a source of intense paramagnetic signals), in the metastases. No metastases were found in other murine organs; however, white infarctions were identified in a single liver. Taken together, the A549-derived tumors growing subcutaneously in nude mice can metastasize and grow on site in the pulmonary tissue. Thus, they can represent an alternative for the model of induced metastatic nodule formation, following intravenous administration of the cancerous cells.

  8. Phosphorylation of p53 at serine 15 in A549 pulmonary epithelial cells exposed to vanadate: Involvement of ATM pathway

    SciTech Connect

    Suzuki, Katsura; Inageda, Kiyoshi; Nishitai, Gen; Matsuoka, Masato . E-mail: matsuoka@research.twmu.ac.jp

    2007-04-01

    When A549 cells were exposed to sodium metavanadate (NaVO{sub 3}), the pentavalent species of vanadium (vanadate), phosphorylation of p53 protein at Ser15 was found in a time (8-48 h)- and dose (10-200 {mu}M)-dependent manner. After the incubation with 50 or 100 {mu}M NaVO{sub 3} for 48 h, accumulation of p53 protein was accompanied with Ser15 phosphorylation. Among serines in p53 protein immunoprecipitated from A549 cells treated with 100 {mu}M NaVO{sub 3} for 48 h, only Ser15 was markedly phosphorylated. Treatment with other vanadate compounds, sodium orthovanadate (Na{sub 3}VO{sub 4}) and ammonium metavanadate (NH{sub 4}VO{sub 3}), also induced Ser15 phosphorylation and accumulation of p53 protein. While phosphorylation of extracellular signal-regulated protein kinase (ERK) was found in cells treated with NaVO{sub 3}, treatment with U0126 did not suppress Ser15 phosphorylation. On the other hand, treatment with wortmannin or caffeine, the inhibitors to phosphatidylinositol 3-kinase related kinases (PIKKs), suppressed both NaVO{sub 3}-induced Ser15 phosphorylation and accumulation of p53 protein. The silencing of ataxia telangiectasia mutated (ATM) expression using short-interference RNA resulted in the marked suppression of Ser15 phosphorylation in A549 cells exposed to NaVO{sub 3}. However, treatment with antioxidants such as catalase and N-acetylcysteine did not suppress NaVO{sub 3}-induced Ser15 phosphorylation. Transcriptional activation of p53 and DNA fragmentation in A549 cells treated with NaVO{sub 3} were suppressed only slightly by S15A mutation, suggesting that Ser15 phosphorylation is not essential for these responses. The present results showed that vanadate induces the phosphorylation of p53 at Ser15 depending on ATM, one of the members of PIKK family, in this human pulmonary epithelial cell line.

  9. Effect of copper overload on the survival of HepG2 and A-549 human-derived cells.

    PubMed

    Arnal, N; de Alaniz, M J T; Marra, C A

    2013-03-01

    We investigated the effect of copper (Cu) overload (20-160 µM/24 h) in two cell lines of human hepatic (HepG2) and pulmonary (A-549) origin by determining lipid and protein damage and the response of the antioxidant defence system. A-549 cells were more sensitive to Cu overload than HepG2 cells. A marked increase was observed in both the cell lines in the nitrate plus nitrite concentration, protein carbonyls and thiobarbituric acid reactive substances (TBARS). The TBARS increase was consistent with an increment in saturated fatty acids at the expense of polyunsaturated acids in a Cu concentration-dependent fashion. Antioxidant enzymes were stimulated by Cu overload. Superoxide dismutase activity increased significantly in both the cell lines, with greater increases in HepG2 than in A-549 cells. A marked increase in ceruloplasmin and metallothionein content in both the cell types was also observed. Dose-dependent decreases in α-tocopherol and ferric reducing ability were observed. Total glutathione content was lower in A-549 cells and higher in HepG2. Calpain and caspase-3 were differentially activated in a dose-dependent manner under copper-induced reactive oxygen species production. We conclude that Cu exposure of human lung- and liver-derived cells should be considered a reliable experimental system for detailed study of mechanism/mechanisms by which Cu overload exerts its deleterious effects.

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

    PubMed

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

    2014-12-01

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

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

    PubMed Central

    Zhong, Liangrui; Zheng, Junxian; Sun, Qianqian; Wei, Kemin; Hu, Yijuan

    2016-01-01

    Radix Tetrastigma hemsleyani flavone (RTHF) is widely used as a traditional herb and has detoxification and anti-inflammatory effects. In this study, we investigated the potential effects of RTHF on the growth and metastasis of human lung adenocarcinoma A549 cells and evaluated its mechanisms. A549 cells were treated with RTHF at various concentrations for different periods. In vitro Cell Counting Kit-8 assay and colony formation methods showed that RTHF had dose- and time-dependent antiproliferation effects on A549 cells. A cell adhesion assay showed that RTHF decreased A549 cell adhesion in a dose-dependent manner. Cell invasion and migration were investigated using the Transwell assay and observed using an inverted microscope; the results showed that cell metastasis was significantly lower in the treatment group than that in the control group (P<0.01). Expression of metastasis-related matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) was detected by real-time polymerase chain reaction and Western blotting. The results showed that the expression of MMP-2, MMP-9, and TIMP-1 decreased, while that of TIMP-2 increased significantly in the RTHF group when compared with the results of the control group. These results show that RTHF exhibits antigrowth and antimetastasis activity in lung cancer A549 cells by decreasing the expression of MMP-2/-9 and TIMP-1 and increasing that of TIMP-2. PMID:26893573

  12. Functional expression of nicotine influx transporter in A549 human alveolar epithelial cells.

    PubMed

    Tega, Yuma; Yuzurihara, Chihiro; Kubo, Yoshiyuki; Akanuma, Shin-ichi; Ehrhardt, Carsten; Hosoya, Ken-ichi

    2016-02-01

    Nicotine is a potent addictive alkaloid, and is rapidly absorbed through the alveoli of the lung. However, the transport mechanism of nicotine at the human alveolar epithelial barrier has not been investigated in great detail. In the present study, the transport mechanism of nicotine across alveolar epithelium was investigated in vitro using A549 cells, a human adenocarcinoma-derived cell line with an alveolar epithelial cell like phenotype. Nicotine uptake by A549 cells exhibited time-, temperature-, and concentration-dependence with a Km of 50.4 μM. These results suggest that a carrier-mediated transport process is involved in nicotine transport in human alveolar epithelial cells. Nicotine uptake by A549 cells was insensitive to change in extracellular pH. Moreover, nicotine uptake by A549 cells could be inhibited by organic cations such as verapamil and pyrilamine, but not typical substrates of organic cation transporters and β2-agonist. These results suggest that a novel, not yet molecularly identified, organic cation transporter plays a role in nicotine transport which is unlikely to interact with β2-agonist transport. This nicotine influx transporter in human alveolar epithelium might have implications for the rapid absorption of nicotine into the systemic circulation.

  13. The verapamil transporter expressed in human alveolar epithelial cells (A549) does not interact with β2-receptor agonists.

    PubMed

    Salomon, Johanna J; Ehrhardt, Carsten; Hosoya, Ken-Ichi

    2014-01-01

      Affinity of different organs for verapamil is highly variable and organ-specific. For example, the drug exhibits high levels of accumulation in lung tissues. A transporter recognising verapamil as a substrate has previously been identified in human retinal pigment epithelial (RPE) and in rat retinal capillary endothelial (TR-iBRB2) cells. This transporter is distinct from any of the cloned organic cation transporters. Therefore, we hypothesised that the verapamil transporter is also functionally expressed in the human respiratory mucosa. Moreover, we tested the hypothesis that this transporter interacts with pulmonary administered cationic drugs such as β2-agonists. The uptake of [(3)H]verapamil was studied in A549 human alveolar epithelial cell monolayers at different times and concentrations. The influence of extracellular proton concentration and various organic cations on verapamil uptake was determined. Verapamil uptake into A549 cells was time- and concentration-dependent, sensitive to pH and had a Km value of 39.8 ± 8.2 µM. Verapamil uptake was also sensitive to inhibition by amantadine, quinidine and pyrilamine, but insensitive to other typical modulators of organic cation and choline transporters. Whilst we demonstrated functional activity of the elusive verapamil transporter at the lung epithelium, our data suggest that this transporter does not interact with β2-agonists at therapeutic concentrations.

  14. Sinomenine inhibits A549 human lung cancer cell invasion by mediating the STAT3 signaling pathway

    PubMed Central

    Jiang, Shulong; Gao, Yebo; Hou, Wei; Liu, Rui; Qi, Xin; Xu, Xia; Li, Jie; Bao, Yanju; Zheng, Honggang; Hua, Baojin

    2016-01-01

    Increasing evidence suggests that the failure of lung cancer treatment may occur as a result of tumor invasion and metastasis. Signal transducer and activator of transcription 3 (STAT3), an epithelial-mesenchymal transition-inducing transcription factor, is a key signaling molecule involved in the proliferation, apoptosis, invasion and metastasis of tumor cells. Sinomenine is an alkaloid compound with an antineoplastic potential against a variety of cancer cells. The aim of the present study was to assess the antitumor mechanisms of sinomenine in the A549 human lung cancer cell line. The results demonstrated that sinomenine manifested dose-dependent cytotoxicity and induced apoptosis in A549 cells. The protein expression of Janus kinase 2, STAT3, phosphorylated-STAT3, Snail, N-cadherin and vimentin decreased in sinomenine-treated cells, while E-cadherin protein expression increased. The regulation of STAT3, N-cadherin and E-cadherin by sinomenine was further confirmed by reverse transcription-quantitative polymerase chain reaction and immunofluorescent staining. It was demonstrated that sinomenine exerts inhibitory effects on A549 human lung cancer cell invasion, possibly through the inhibition of STAT3 signaling. These results provide a novel insight into the role of sinomenine in the treatment of non-small cell lung cancer. PMID:27446441

  15. Fucoidan from Undaria pinnatifida induces apoptosis in A549 human lung carcinoma cells.

    PubMed

    Boo, Hye-Jin; Hyun, Jae-Hee; Kim, Sang-Cheol; Kang, Jung-Il; Kim, Min-Kyoung; Kim, Sun-Yeou; Cho, Heeyeong; Yoo, Eun-Sook; Kang, Hee-Kyoung

    2011-07-01

    Fucoidan, a sulfated polysaccharide, has various biological activities, such as anticancer, antiangiogenic and antiinflammatory effects; however, the mechanisms of action of fucoidan on anticancer activity have not been fully elucidated. The anticancer effects of fucoidan from Undaria pinnatifida on A549 human lung carcinoma cells were examined. Treatment of A549 cells with fucoidan resulted in potent antiproliferative activity. Also, some typical apoptotic characteristics, such as chromatin condensation and an increase in the population of sub-G1 hypodiploid cells, were observed. With respect to the mechanism underlying the induction of apoptosis, fucoidan reduced Bcl-2 expression, but the expression of Bax was increased in a dose-dependent manner compared with the controls. Furthermore, fucoidan induced caspase-9 activation, but decreased the level of procaspase-3. Cleavage of poly-ADP-ribose polymerase (PARP), a vital substrate of effector caspase, was found. The study further investigated the role of the MAPK and PI3K/Akt pathways with respect to the apoptotic effect of fucoidan, and showed that fucoidan activates ERK1/2 in A549 cells. Unlike ERK1/2, however, treatment with fucoidan resulted in the down-regulation of phospho-p38 expression. In addition, fucoidan resulted in the down-regulation of phospho-PI3K/Akt. Together, these results indicate that fucoidan induces apoptosis of A549 human lung cancer cells through down-regulation of p38, PI3K/Akt, and the activation of the ERK1/2 MAPK pathway.

  16. Tomatidine inhibits invasion of human lung adenocarcinoma cell A549 by reducing matrix metalloproteinases expression.

    PubMed

    Yan, Kun-Huang; Lee, Liang-Ming; Yan, Shao-Han; Huang, Hsiang-Ching; Li, Chia-Chen; Lin, Hui-Ting; Chen, Pin-Shern

    2013-05-25

    Tomatidine is an aglycone of glycoalkaloid tomatine in tomato. Tomatidine is found to possess anti-inflammatory properties and may serve as a chemosensitizer in multidrug-resistant tumor cells. However, the effect of tomatidine on cancer cell metastasis remains unclear. This study examines the effect of tomatidine on the migration and invasion of human lung adenocarcinoma A549 cell in vitro. The data demonstrates that tomatidine does not effectively inhibit the viability of A549 cells. When treated with non-toxic doses of tomatidine, cell invasion is markedly suppressed by Boyden chamber invasion assay, while cell migration is not affected. Tomatidine reduces the mRNA level of matrix metalloproteinase-2 (MMP-2), MMP-9 and increases the expression of reversion-inducing cysteine-rich protein with kazal motifs (RECK), as well as tissue inhibitor of metalloproteinase-1 (TIMP-1). The immunoblotting assays indicate that tomatidine is very effective in suppressing the phosphorylation of Akt and extracellular signal regulating kinase (ERK). In addition, tomatidine significantly decreases the nuclear level of nuclear factor kappa B (NF-κB), which suggests that tomatidine inhibits NF-κB activity. Furthermore, the treatment of inhibitors specific for PI3K/Akt (LY294002), ERK (U0126), or NF-κB (pyrrolidine dithiocarbamate) to A549 cells reduced cell invasion and MMP-2/9 expression. The results suggest that tomatidine inhibits the invasion of A549 cells by reducing the expression of MMPs. It also inhibits ERK and Akt signaling pathways and NF-κB activity. These findings demonstrate a new therapeutic potential for tomatidine in anti-metastatic therapy.

  17. Sodium orthovanadate affects growth of some human epithelial cancer cells (A549, HTB44, DU145).

    PubMed

    Klein, Andrzej; Holko, Przemyslaw; Ligeza, Janusz; Kordowiak, Anna M

    2008-01-01

    Within the concentration range of 1-20 microM, orthovanadate (Na3VO4) demonstrated a time and dose-dependent inhibition of autocrine growth of the human carcinoma cell lines A549 (lung), HTB44 (kidney) and DU145 (prostate), as compared to appropriate controls (without Na3VO4). The investigation was conducted by two methods: staining with N-hexa-methylpararosaniline (crystal violet=CV) or bromide3-(4,5-dimethyltio-azo-2)-2,5-diphenyl-tetrazole (MTT). In 5, 10 and 20 microM of Na3VO4 in serum-free medium, the mean values of these two tests for A549 were approximately 40%, 45% or 65% as compared to the appropriate controls. HTB44 had the greatest opportunity (statistically insignificant) at lower vanadium concentrations (up to 10 microM), whereas at 20 microM growth inhibition of these cells was approximately 50% of the controls. DU145 showed approximately 33%, 65% and 98% growth inhibition for 5, 10 and 20 microM of Na3VO4, respectively Additionally, hypothetical curves obtained by a MANOVA test based on the CV results after 72 h incubation with Na3VO4 in serum-free medium, and an example of a time-dependent effect of Na3VO4 on A549 cells, were also presented. Sodium orthovanadate was also examined for its cytotoxic capabilities, especially its ability to induce tumor cell apoptosis; the results were compared with the effect of paclitaxel. The target cells were dyed by differential staining (HOECHST33258 and propidium iodide) after 3 h and 24 h (DU145) or 3 h and 72 h (A549) of incubation with the vanadium compound. Contrary to the two cancer cell lines (viable, apoptotic or necrotic in experimental conditions), the renal HTB44 cells were insensitive up to 15 microM Na3VO4 concentrations. After 3 h incubation with Na3VO4, both lung (A549) and prostate (DU145) cancer cells showed a slight but significant reduction in the percentage of viable cells, and an increased amount of apoptotic cells. In contrast to the lung cells, DU145 prostate cells after 24 h were more

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

    PubMed

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

    2012-01-01

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

  19. Previous heat shock treatment inhibits Mayaro virus replication in human lung adenocarcinoma (A549) cells.

    PubMed

    Virgilio, P L; Godinho-Netto, M C; Carvalho Mda, G

    1997-01-01

    Human lung adenocarcinoma cells (A549) were submitted to mild or severe heat shock (42 degrees C or 44 degrees C) for 1 h, while another group of cells was double-heat-shocked (submitted to 42 degrees C for 1 h, returned to 37 degrees C for 3 h, then exposed to 44 degrees C for 1 h). After each heat treatment, the cells were infected with Mayaro virus for 24 h and incubated at 37 degrees C. The results showed that the double-heat-shocked thermotolerant cells exhibited a 10(4)-fold virus titre inhibition, despite the recovery of protein synthesis and original morphology 24 h post-infection. In contrast, cells submitted to mild or severe heat shock exhibited weaker inhibition of Mayaro virus titre (10(2)-fold). The mildly heat-shocked cells also presented a full recovery in protein synthesis, which was not observed in severely heat-shocked cells. These results indicate that exposure of A549 cells to a mild or to a double heat shock treatment before Mayaro virus infection induces an antiviral state.

  20. Genistein inhibits A549 human lung cancer cell proliferation via miR-27a and MET signaling

    PubMed Central

    Yang, Yang; Zang, Aimin; Jia, Youchao; Shang, Yanhong; Zhang, Zhuoqi; Ge, Kun; Zhang, Jinchao; Fan, Wufang; Wang, Bei

    2016-01-01

    Genistein is a soybean isoflavone; in its aglycone it has various biological activities. Animal experiments, clinical studies and epidemiological investigations suggest that genistein has preventative and curative functions for a number of diseases, particularly in cancer. The present study explored the potential anti-cancer effect of genistein by observing its role in inhibiting A549 human lung cancer cell proliferation and investigating the possible mechanism. A549 cells were exposed to various concentrations of genistein (0, 10, 25, 50, 100 and 200 µM; dissolved in physiological saline) for 1, 2 and 3 days. Subsequently, the viability of A549 cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, cell apoptosis was examined using a flow cytometer, caspase 3/9 activity was measured using commercial kits, reverse transcription quantitative polymerase chain reaction was used to analyze the miR-27a expression and western blotting was used to investigate MET protein expression. The results suggested a significant inhibition of A549 cell growth following treatment with genistein in a time- and dose-dependent manner. The current study also indicated that treatment with genistein significantly induces cell apoptosis and promotes caspase-3/9 activation of A549 cells in a dose-dependent manner. Further functional assays revealed that the anti-cancer effect of genistein activated microRNA-27a (miR-27a) expression levels and reduced MET protein expression in A549 cells. In conclusion, the present study demonstrates that genistein inhibits A549 human lung cancer cell proliferation. Furthermore, this study reports, for the first time, a correlation between the anti-cancer effect of genistein and miR-27a-mediated MET signaling. PMID:27602162

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

    PubMed Central

    Shen, Lei; Zhang, Shan-Qiang; Liu, Lei; Sun, Yu; Wu, Yu-Xuan; Xie, Li-Ping; Liu, Ji-Cheng

    2017-01-01

    Background Jolkinolide A (JA) and Jolkinolide B (JB) are diterpenoids extracted from the roots of Euphorbia fischeriana Steud and have been shown to have anti-tumor activity. However, their effects on the ability of tumor cells to invade blood vessels and metastasize remain largely unknown. Investigations into the effects of JA and JB on the angiogenesis of tumor tissues may facilitate the identification of new natural drugs with anti-tumor growth and metastasis activities. Material/Methods We used different concentrations of JA and JB (20 μg/ml, 40 μg/ml, 60 μg/ml, 80 μg/ml, and 100 μg/ml) to stimulate A549 cells and then studied the effects on the growth and metastasis of lung cancers. In addition, we used conditional media from A549 cells (A549-CM) stimulated by either JA or JB in different concentrations to culture human umbilical vein endothelial cells (HUVECs). Results We found that both JA and JB significantly inhibited the Akt-STAT3-mTOR signaling pathway and reduced the expression of VEGF in A549 cells, but JB exhibited more significant inhibitory effects than JA. The JB-stimulated A549 cell conditional media had a greater inhibitory effect on the proliferation and migration of HUVECs than did the conditional media of JA-stimulated A549 cells. This effect gradually increased with increasing concentrations of either type of Jolkinolide. Conclusions Our results suggest that JA and JB inhibited VEGF expression in A549 cells through the inhibition of the Akt-STAT3-mTOR signaling pathway, and directly inhibited the proliferation and migration of HUVECs. These findings are of great significance for the development of new plant-derived chemotherapy agents for the treatment of cancer. PMID:28087861

  2. Schisandrin B inhibits the proliferation of human lung adenocarcinoma A549 cells by inducing cycle arrest and apoptosis

    PubMed Central

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

    2015-01-01

    Lung cancer is the leading cause of cancer death in the world. Schizandrin B (Sch B) is one of the main dibenzocyclooctadiene lignans present in the fruit of Schisandra chinensis (Schisandraceae). Sch B has multiple functions against cancer. The aim of this study was to determine the effect of Sch B on the proliferation, cell cycling, apoptosis and invasion of lung adenocarcinoma A549 cells by MTT, flow cytometry, wound healing and transwell invasion assays. Treatment with Sch B inhibited the proliferation of A549 cells in a dose-dependent manner. Sch B induced cell cycle arrest at G0/G1 phase by down-regulating the expression of cyclin D1, cyclin-dependent kinase (CDK)4, and CDK6, but up-regulating p53 and p21 expression in A549 cells. Furthermore, Sch B triggered A549 cell apoptosis by increasing Bax, cleaved caspase-3, 9, Cyto C, but decreasing Bcl-2 and PCNA expression. In addition, Sch B inhibited the invasion and migration of A549 cells by down-regulating the expressions of HIF-1, VEGF, MMP-9 and MMP-2. Therefore, Sch B has potent anti-tumor activity and may be a promising traditional Chinese medicine for human lung carcinoma. PMID:26221229

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

    NASA Astrophysics Data System (ADS)

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

    2007-04-01

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

  4. Combined toxic effect of airborne heavy metals on human lung cell line A549.

    PubMed

    Choi, Yeowool; Park, Kihong; Kim, Injeong; Kim, Sang D

    2016-11-25

    Many studies have demonstrated that heavy metals existing as a mixture in the atmospheric environment cause adverse effects on human health and are important key factors of cytotoxicity; however, little investigation has been conducted on a toxicological study of a metal mixture from atmospheric fine particulate matter. The objective of this study was to predict the combined effects of heavy metals in aerosol by using in vitro human cells and obtain a suitable mixture toxicity model. Arsenic, nickel, and lead were selected for mixtures exposed to A549 human lung cancer cells. Cell proliferation (WST-1), glutathione (GSH), and interleukin (IL)-8 inhibition were observed and applied to the prediction models of mixture toxicity, concentration addition (CA) and independent action (IA). The total mixture concentrations were set by an IC10-fixed ratio of individual toxicity to be more realistic for mortality and enzyme inhibition tests. The results showed that the IA model was statistically closer to the observed results than the CA model in mortality, indicating dissimilar modes of action. For the GSH inhibition, the results predicted by the IA and CA models were highly overestimated relative to mortality. Meanwhile, the IL-8 results were stable with no significant change in immune reaction related to inflammation. In conclusion, the IA model is a rapid prediction model in heavy metals mixtures; mortality, as a total outcome of cell response, is a good tool for demonstrating the combined toxicity rather than other biochemical responses.

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

    PubMed Central

    2011-01-01

    Background 5-allyl-7-gen-difluoromethoxychrysin (AFMC) is a novel synthetic analogue of chrysin that has been reported to inhibit proliferation in various cancer cell lines. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-cancer agent. Methods The cytotoxicity of A549 and WI-38 cells were determined using colorimetry. Apoptosis was detected by flow cytometry (FCM) after propidium iodide (PI) fluorescence staining and agarose gel electrophoresis. Caspase activities were evaluated using enzyme-linked immunosorbent assay (ELISA).The expressions of DR4 and DR5 were analyzed using FCM and western blot. Results Subtoxic concentrations of AFMC sensitize human non-small cell lung cancer (NSCLC) A549 cells to TRAIL-mediated apoptosis. Combined treatment of A549 cells with AFMC and TRAIL significantly activated caspase-3, -8 and -9. The caspase-3 inhibitor zDEVD-fmk and the caspase-8 inhibitor zIETD-fmk blocked the apoptosis of A549 cells induced by co-treatment with AFMC and TRAIL. In addition, we found that treatment of A549 cells with AFMC significantly induced the expression of death receptor 5 (DR5). AFMC-mediated sensitization of A549 cells to TRAIL was efficiently reduced by administration of a blocking antibody or small interfering RNAs against DR5. AFMC also caused increase of the Sub-G1 cells by TRAIL treatment and increased the expression levels of DR5 in other NSCLC H460 and H157 cell lines. In contrast, AFMC-mediated induction of DR5 expression was not observed in human embryo lung WI-38 cells, and AFMC did not sensitize WI-38 cells to TRAIL-induced apoptosis. Conclusions AFMC synergistically enhances TRAIL-mediated apoptosis in NSCLC cells through up-regulating DR5 expression. PMID:21801359

  6. Transcriptome Profiles of Human Lung Epithelial Cells A549 Interacting with Aspergillus fumigatus by RNA-Seq

    PubMed Central

    Jia, Xiaodong; Wang, Shuo; Wang, Jing; Chen, Yong; Zhao, Jingya; Tian, Shuguang; Han, Xuelin; Han, Li

    2015-01-01

    Lung epithelial cells constitute the first defense line of host against the inhaled Aspergillus fumigatus; however, the transcriptional response of human alveolar type II epithelial cells was still unclear. Here we used RNA-Seq technology to assess the transcriptome profiles of A549 cells following direct interaction with conidia of A. fumigatus. The total number of identified genes was 19118. Compared with uninfected A549 cells, 459 genes were differentially expressed in cells co-incubated with conidia for 8 h, including 302 up-regulated genes and 157 down-regulated genes. GO and KEGG pathway enrichment analysis showed that most of the up-regulated genes were related to immune response, chemotaxis and inflammatory response and enriched in cytokine-cytokine receptor interaction, JAK-STAT and MAPK signaling pathways. The down-regulated genes were mainly enriched for terms associated with development, hemopoiesis and ion transport. Among them, EGR4 and HIST1H4J gene had the maximum of fold change in up-regulated and down-regulated genes, respectively. Fourteen up-regulated genes and three down-regulated genes were further validated and significant increase on expression of IL-6, IL-8 and TNF-α in A549 cells were confirmed by qRT-PCR during the interaction of A549 cells with A. fumigatus. Besides, western blot showed that expression of two proteins (ARC, EGR1) significantly increased in A549 cells during interaction with A. fumigatus conidia for 8h. Interference of endogenous expression of ARC or EGR1 protein in A549 cells reduced the internalization of A. fumigatus. These results provided important insights into dynamic changes of gene expression in lung epithelial cells, especially its strong immunological response against A. fumigatus infection. PMID:26273834

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

    PubMed

    Khan, Naghma; Hadi, Naghma; Afaq, Farrukh; Syed, Deeba N; Kweon, Mee-Hyang; Mukhtar, Hasan

    2007-01-01

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

  8. Enhancement of radiosensitivity by CpG-oligodeoxyribonucleotide-7909 in human non-small cell lung cancer A549 cells.

    PubMed

    Zha, Lin; Qiao, Tiankui; Yuan, Sujuan; Lei, Linjie

    2010-04-01

    CpG-oligodeoxyribonucleotides (CpG-ODNs), which induce signaling through the toll-like receptor 9, are currently under investigation as immunity stimulators against cancer. It has recently been suggested that CpG-ODNs may also enhance sensitivity to traditional therapies including chemotherapy in certain cancer-cell lines. The purpose of this study was to define the activity of CpG-ODN7909 in increasing radiosensitivity of the human non-small cell lung cancer cell line A549 in vitro. First, a dose- and time-dependent inhibitory effect on cell viability was observed after A549 cells were treated with different concentrations of CpG-ODN7909 (5, 10, 30, and 60 microg/mL). Second, decreased cell clonogenic survival, enhanced cell apoptotic index, accumulated percentage of cells in the G2/M phase, and increased tumor necrosis factor (TNF)-alpha secretion were found after combined treatments with 10 microg/mL of CpG-ODN7909 and radiation compared to either treatment alone (p < 0.05). Furthermore, the toll-like receptor 9 mRNA was found to express in A549. The results suggest that CpG-ODN7909 can increase the radiosensitivity of human non-small cell lung cancer A549 cells, which may be associated with reduced cell clonogenic survival, enhanced apoptosis, prolonged cell-cycle arrest in G2/M, and stimulation of TNF-alpha secretion.

  9. Molecular mechanisms underlying mangiferin-induced apoptosis and cell cycle arrest in A549 human lung carcinoma cells

    PubMed Central

    SHI, WEI; DENG, JIAGANG; TONG, RONGSHENG; YANG, YONG; HE, XIA; LV, JIANZHEN; WANG, HAILIAN; DENG, SHAOPING; QI, PING; ZHANG, DINGDING; WANG, YI

    2016-01-01

    Mangiferin, which is a C-glucosylxanthone (1,3,6,7-tetrahydroxyxanthone-C2-β-D-glucoside) purified from plant sources, has recently gained attention due to its various biological activities. The present study aimed to determine the apoptotic effects of mangiferin on A549 human lung adenocarcinoma cells. In vitro studies demonstrated that mangiferin exerted growth-inhibitory and apoptosis-inducing effects against A549 cells. In addition, mangiferin exhibited anti-tumor properties in A549 xenograft mice in vivo. Mangiferin triggered G2/M phase cell cycle arrest via down-regulating the cyclin-dependent kinase 1-cyclin B1 signaling pathway, and induced apoptotic cell death by inhibiting the protein kinase C-nuclear factor-κB pathway. In addition, mangiferin was able to enhance the antiproliferative effects of cisplatin on A549 cells, thus indicating the potential for a combined therapy. Notably, mangiferin exerted anticancer effects in vivo, where it was able to markedly decrease the volume and weight of subcutaneous tumor mass, and expand the lifespan of xenograft mice. The present study clarified the molecular mechanisms underlying mangiferin-induced antitumor activities, and suggested that mangiferin may be considered a potential antineoplastic drug for the future treatment of cancer. PMID:26935347

  10. Capsaicin-induced genotoxic stress does not promote apoptosis in A549 human lung and DU145 prostate cancer cells.

    PubMed

    Lewinska, Anna; Jarosz, Paulina; Czech, Joanna; Rzeszutek, Iwona; Bielak-Zmijewska, Anna; Grabowska, Wioleta; Wnuk, Maciej

    2015-02-01

    Capsaicin is the major pungent component of the hot chili peppers of the genus Capsicum, which are consumed worldwide as a food additive. More recently, the selective action of capsaicin against cancer cells has been reported. Capsaicin was found to induce apoptosis and inhibit proliferation of a wide range of cancer cells in vitro, whereas being inactive against normal cells. As data on capsaicin-induced genotoxicity are limited and the effects of capsaicin against human lung A549 and DU145 prostate cancer cells were not explored in detail, we were interested in determining whether capsaicin-associated genotoxicity may also provoke A549 and DU145 cell death. Capsaicin-induced decrease in metabolic activity and cell proliferation, and changes in the cell cycle were limited to high concentrations used (≥ 100 μM), whereas, at lower concentrations, capsaicin stimulated both DNA double strand breaks and micronuclei production. Capsaicin was unable to provoke apoptotic cell death when used up to 250 μM concentrations. Capsaicin induced oxidative stress, but was ineffective in provoking the dissipation of the mitochondrial inner transmembrane potential. A different magnitude of p53 binding protein 1 (53BP1) recruitment contributed to diverse capsaicin-induced genotoxic effects in DU145 and A549 cells. Capsaicin was also found to be a DNA hypermethylating agent in A549 cells. In summary, we have shown that genotoxic effects of capsaicin may contribute to limited susceptibility of DU145 and A549 cancer cells to apoptosis in vitro, which may question the usefulness of capsaicin-based anticancer therapy, at least in a case of lung and prostate cancer.

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

    PubMed

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

    2013-02-04

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

  12. Induction of apoptosis in human lung carcinoma A549 epithelial cells with an ethanol extract of Tremella mesenterica.

    PubMed

    Chen, Nan-Yin; Lai, Hsi-Huai; Hsu, Tai-Hao; Lin, Fang-Yi; Chen, Jian-Zhi; Lo, Hui-Chen

    2008-05-01

    Tremella mesenterica (TM) is a common food and folk medicine widely used in several Asian countries as a tonic for the lungs. In the present study, we compared the effects of extracellular polysaccharides (EPS), intracellular polysaccharides (IPS), and ethanol extract (EE) of Tremella mesenterica on the induction of apoptosis into human lung carcinoma A549 epithelial cells. The EE, but not the EPS or the IPS, almost completely inhibited the growth of A549 cells. The results of Annexin V-FITC/PI staining and flow cytometric analysis indicated that the percentage of Annexin V(+)/PI(-) cells in EE-treated cells increased to 32.8%. The results of further investigation showed a disruption of mitochondrial transmembrane potential (DeltaPsi(m)), the production of reactive oxygen species (ROS), and the activation of caspase-3 protein in EE-treated cells. These findings suggest that EE can decrease cell viability and induce apoptosis in A549 cell lines by activating a mitochondrial pathway.

  13. Dexamethasone-(C21-phosphoramide)-[anti-EGFR]: molecular design, synthetic organic chemistry reactions, and antineoplastic cytotoxic potency against pulmonary adenocarcinoma (A549)

    PubMed Central

    Coyne, Cody P; Narayanan, Lakshmi

    2016-01-01

    Purpose Corticosteroids are effective in the management of a variety of disease states, such as several forms of neoplasia (leukemia and lymphoma), autoimmune conditions, and severe inflammatory responses. Molecular strategies that selectively “target” delivery of corticosteroids minimize or prevents large amounts of the pharmaceutical moiety from passively diffusing into normal healthy cell populations residing within tissues and organ systems. Materials and methods The covalent immunopharmaceutical, dexamethasone-(C21-phosphoramide)-[anti-EGFR] was synthesized by reacting dexamethasone-21-monophosphate with a carbodiimide reagent to form a dexamethasone phosphate carbodiimide ester that was subsequently reacted with imidazole to create an amine-reactive dexamethasone-(C21-phosphorylimidazolide) intermediate. Monoclonal anti-EGFR immunoglobulin was combined with the amine-reactive dexamethasone-(C21-phosphorylimidazolide) intermediate, resulting in the synthesis of the covalent immunopharmaceutical, dexamethasone-(C21-phosphoramide)-[anti-EGFR]. Following spectrophotometric analysis and validation of retained epidermal growth factor receptor type 1 (EGFR)-binding avidity by cell-ELISA, the selective anti-neoplasic cytotoxic potency of dexamethasone-(C21-phosphoramide)-[anti-EGFR] was established by MTT-based vitality stain methodology using adherent monolayer populations of human pulmonary adenocarcinoma (A549) known to overexpress the tropic membrane receptors EGFR and insulin-like growth factor receptor type 1. Results The dexamethasone:IgG molar-incorporation-index for dexamethasone-(C21-phosphoramide)-[anti-EGFR] was 6.95:1 following exhaustive serial microfiltration. Cytotoxicity analysis: covalent bonding of dexamethasone to monoclonal anti-EGFR immunoglobulin did not significantly modify the ex vivo antineoplastic cytotoxicity of dexamethasone against pulmonary adenocarcinoma at and between the standardized dexamethasone equivalent concentrations of 10

  14. Therapeutic effects of sorafenib on the A549/DDP human lung adenocarcinoma cell line in vitro.

    PubMed

    Chen, Xiang-Qi; Wang, Yu-Lan; Li, Zhi-Ying; Lin, Ting-Yan

    2014-07-01

    The aim of the present study was to observe the effects of sorafenib on the proliferation, apoptosis and invasion of A549/DDP cisplatin-resistant lung adenocarcinoma cells cultured in vitro. The A549/DDP cisplatin-resistant lung adenocarcinoma cell strain was cultured in vitro, the cell culture group incubated in culture medium only was set as the control group (Group S0) and the four concentration gradients of sorafenib were added to the culture groups as the experimental groups: S1, 2 µmol/l; S2, 4 µmol/l; S3, 8 µmol/l; and S4, 16 µmol/l. The MTT assay was used to determine the growth inhibition rate of the cells, which were respectively subjected to sorafenib treatment for 24, 48 and 72 h. Flow cytometry was used to determine the rate of apoptosis of cells in each group following sorafenib treatment for 72 h. Furthermore, the Transwell invasion experiment was used to determine the effect on A549/DDP cell invasion following sorafenib treatment for 24 h. Based on the MTT assay, it was found that the inhibition rates of A549/DDP cisplatin-resistant lung adenocarcinoma cells in groups S1-4 following sorafenib treatment for 24 h were 4.58±2.82, 14.93±2.62, 37.58±7.13 and 58.39±8.15%, respectively. For 48 h, inhibition rates in S1-4 were 14.98±2.93, 26.28±7.31, 63.00±3.05 and 78.84±3.96%, respectively, and for 72 h, inhibition rates were 18.80±2.82, 32.71±2.55, 75.51±4.73 and 87.50±3.36%, respectively. The difference in the inhibition rates of cells among the experimental groups for the same incubation time showed statistical significance (P<0.05). Flow cytometric analysis indicated that the rate of apoptosis in the control group was 8.88±0.81% following sorafenib treatment for 72 h, and the rates of apoptosis in groups S1-4 were, 12.84±0.24, 17.27±0.78, 21.98±0.75 and 49.67±1.38%, respectively. The rate of apoptosis in each experimental group was higher compared with that in the control group (P<0.05). The difference in the rate of apoptosis

  15. Cytotoxicity, oxidative stress and genotoxicity induced by glass fibers on human alveolar epithelial cell line A549.

    PubMed

    Rapisarda, Venerando; Loreto, Carla; Ledda, Caterina; Musumeci, Giuseppe; Bracci, Massimo; Santarelli, Lory; Renis, Marcella; Ferrante, Margherita; Cardile, Venera

    2015-04-01

    Man-made vitreous fibers have been widely used as insulation material as asbestos substitutes; however their morphology and composition raises concerns. In 1988 the International Agency for Research on Cancer classified fiberglass, rock wool, slag wool, and ceramic fibers as Group 2B, i.e. possibly carcinogenic to humans. In 2002 it reassigned fiberglass, rock and slag wool, and continuous glass filaments to Group 3, not classifiable as carcinogenic to humans. The aim of this study was to verify the cytotoxic and genotoxic effects and oxidative stress production induced by in vitro exposure of human alveolar epithelial cells A549 to glass fibers with a predominant diameter <3 μm (97%) and length >5 μm (93%). A549 cells were incubated with 5, 50, or 100 μg/ml (2.1, 21, and 42 μg/cm(2), respectively) of glass fibers for 72 h. Cytotoxicity and DNA damage were tested by the MTT and the Comet assay, respectively. Oxidative stress was determined by measuring inducible nitric oxide synthase (iNOS) expression by Western blotting, production of nitric oxide (NO) with Griess reagent, and concentration of reactive oxygen species by fluorescent quantitative analysis with 2',7'-dichlorofluorescein-diacetate (DCFH-DA). The results showed that glass fiber exposure significantly reduced cell viability and increased DNA damage and oxidative stress production in a concentration-dependent manner, demonstrating that glass fibers exert cytotoxic and genotoxic effects related to increased oxidative stress on the human alveolar cell line A549.

  16. Curcumin promotes apoptosis in A549/DDP multidrug-resistant human lung adenocarcinoma cells through an miRNA signaling pathway

    SciTech Connect

    Zhang, Jian; Zhang, Tao; Ti, Xinyu; Shi, Jieran; Wu, Changgui; Ren, Xinling; Yin, Hong

    2010-08-13

    Research highlights: {yields} Curcumin had anti-cancer effects on A549/DDP multidrug-resistant human lung adenocarcinoma cells {yields} Curcumin promotes apoptosis in A549/DDP cells through a miRNA signaling pathway {yields} Curcumin induces A549/DDP cell apoptosis by downregulating miR-186* {yields} miR-186* may serve as a potential gene therapy target for refractory lung cancer that is sensitive to curcumin -- Abstract: Curcumin extracted from the rhizomes of Curcuma longa L. has been shown to have inhibitory effects on cancers through its anti-proliferative and pro-apoptotic activities. Emerging evidence demonstrates that curcumin can overcome drug resistance to classical chemotherapies. Thus, the mechanisms underlying the anti-tumor activities of curcumin require further study. In our study, we first demonstrated that curcumin had anti-cancer effects on A549/DDP multidrug-resistant human lung adenocarcinoma cells. Further studies showed that curcumin altered miRNA expression; in particular, significantly downregulated the expression of miR-186* in A549/DDP. In addition, transfection of cells with a miR-186* inhibitor promoted A549/DDP apoptosis, and overexpression of miR-186* significantly inhibited curcumin-induced apoptosis in A549/DDP cells. These observations suggest that miR-186* may serve as a potential gene therapy target for refractory lung cancer that is sensitive to curcumin.

  17. A Comprehensive Proteomic View of Responses of A549 Type II Alveolar Epithelial Cells to Human Respiratory Syncytial Virus Infection*

    PubMed Central

    Dave, Keyur A.; Norris, Emma L.; Bukreyev, Alexander A.; Headlam, Madeleine J.; Buchholz, Ursula J.; Singh, Toshna; Collins, Peter L.; Gorman, Jeffrey J.

    2014-01-01

    Human respiratory syncytial virus is a major respiratory pathogen for which there are no suitable antivirals or vaccines. A better understanding of the host cell response to this virus may redress this problem. The present report concerns analysis of multiple independent biological replicates of control and 24 h infected lysates of A549 cells by two different proteomic workflows. One workflow involved fractionation of lysates by in-solution protein IEF and individual fractions were digested using trypsin prior to capillary HPLC-LTQ-OrbitrapXL-MS/MS. A second workflow involved digestion of whole cell lysates and analysis by nanoUltraHPLC-LTQ-OrbitrapElite-MS/MS. Both workflows resulted in the quantification of viral proteins exclusively in lysates of infected cells in the relative abundances anticipated from previous studies. Unprecedented numbers (3247 - 5010) of host cell protein groups were also quantified and the infection-specific regulation of a large number (191) of these protein groups was evident based on a stringent false discovery rate cut-off (<1%). Bioinformatic analyses revealed that most of the regulated proteins were potentially regulated by type I, II, and III interferon, TNF-α and noncanonical NF-κB2 mediated antiviral response pathways. Regulation of specific protein groups by infection was validated by quantitative Western blotting and the cytokine-/key regulator-specific nature of their regulation was confirmed by comparable analyses of cytokine treated A549 cells. Overall, it is evident that the workflows described herein have produced the most comprehensive proteomic characterization of host cell responses to human respiratory syncytial virus published to date. These workflows will form the basis for analysis of the impacts of specific genes of human respiratory syncytial virus responses of A549 and other cell lines using a gene-deleted version of the virus. They should also prove valuable for the analysis of the impact of other infectious

  18. A comprehensive proteomic view of responses of A549 type II alveolar epithelial cells to human respiratory syncytial virus infection.

    PubMed

    Dave, Keyur A; Norris, Emma L; Bukreyev, Alexander A; Headlam, Madeleine J; Buchholz, Ursula J; Singh, Toshna; Collins, Peter L; Gorman, Jeffrey J

    2014-12-01

    Human respiratory syncytial virus is a major respiratory pathogen for which there are no suitable antivirals or vaccines. A better understanding of the host cell response to this virus may redress this problem. The present report concerns analysis of multiple independent biological replicates of control and 24 h infected lysates of A549 cells by two different proteomic workflows. One workflow involved fractionation of lysates by in-solution protein IEF and individual fractions were digested using trypsin prior to capillary HPLC-LTQ-OrbitrapXL-MS/MS. A second workflow involved digestion of whole cell lysates and analysis by nanoUltraHPLC-LTQ-OrbitrapElite-MS/MS. Both workflows resulted in the quantification of viral proteins exclusively in lysates of infected cells in the relative abundances anticipated from previous studies. Unprecedented numbers (3247 - 5010) of host cell protein groups were also quantified and the infection-specific regulation of a large number (191) of these protein groups was evident based on a stringent false discovery rate cut-off (<1%). Bioinformatic analyses revealed that most of the regulated proteins were potentially regulated by type I, II, and III interferon, TNF-α and noncanonical NF-κB2 mediated antiviral response pathways. Regulation of specific protein groups by infection was validated by quantitative Western blotting and the cytokine-/key regulator-specific nature of their regulation was confirmed by comparable analyses of cytokine treated A549 cells. Overall, it is evident that the workflows described herein have produced the most comprehensive proteomic characterization of host cell responses to human respiratory syncytial virus published to date. These workflows will form the basis for analysis of the impacts of specific genes of human respiratory syncytial virus responses of A549 and other cell lines using a gene-deleted version of the virus. They should also prove valuable for the analysis of the impact of other infectious

  19. CDK-associated Cullin 1 promotes cell proliferation with activation of ERK1/2 in human lung cancer A549 cells

    SciTech Connect

    Chen, Tian Jun; Gao, Fei; Yang, Tian; Thakur, Asmitanand; Ren, Hui; Li, Yang; Zhang, Shuo; Wang, Ting; Chen, Ming Wei

    2013-07-19

    Highlights: •CDK-associated Cullin 1 (CAC1) expression increases in human lung carcinoma. •CAC1 promotes the proliferation of lung cancer A549 cells. •CAC1 promotes human lung cancer A549 cell proliferation with activation of ERK1/2. -- Abstract: Lung cancer is one of the most common causes of cancer-related death in the world, but the mechanisms remain unknown. In this study, we investigated the expression of CDK-associated Cullin 1 (CAC1) in lung cancer, the effect of CAC1 on the proliferation of human lung cancer A549 cells, and the activation of signaling pathways of mitogen-activated protein kinases (MAPKs). Results showed that CAC1 expression was higher levels in human lung carcinoma than normal lung tissue, and CAC1 siRNA reduced the proliferation of lung cancer A549 cells by decreasing cell activity and cell division in vitro. The proportion of cells treated with CAC1 siRNA increased in the G1 phase and decreased in the S and G2/M phase, indicative of G1 cell cycle arrest. Furthermore, the proportions of early/late apoptosis in lung cancer A549 cells were enhanced with CAC1 siRNA treatment. It was also found that activation of extracellular signal-regulated protein kinase (ERK) and p38 signaling pathways were involved in the proliferation of A549 cells. After CAC1 siRNA treatment, p-ERK1/2 levels decreased, and meanwhile p-p38 level increased, A549 cell proliferation increased when ERK1/2 signaling is activated by PMA. Our findings demonstrated that CAC1 promoted the proliferation of human lung cancer A549 cells with activation of ERK1/2 signaling pathways, suggesting a potential cure target for treatment of human lung cancer.

  20. Coenzyme Q0 from Antrodia cinnamomea in Submerged Cultures Induces Reactive Oxygen Species-Mediated Apoptosis in A549 Human Lung Cancer Cells

    PubMed Central

    Chung, Cheng-Han; Lee, Kung-Ta

    2014-01-01

    We investigated the anticancer effects of Antrodia cinnamomea, a medicinal mushroom from Taiwan, on A549 human lung cancer cells using the ethyl acetate extract from submerged culture filtrates. Our results showed that 2,3-dimethoxy-5-methyl-1,4-benzoquinone (coenzyme Q0; CoQ0) derived from A. cinnamomea submerged culture filtrates has anticancer activity. CoQ0 treatment reduced the viability of A549, HepG2, and SW480 cancer cell lines. Furthermore, CoQ0 induced reactive oxygen species (ROS) generation and apoptosis in A549 cells, which was inhibited by the antioxidant ascorbic acid. To our knowledge, these data demonstrate for the first time that CoQ0 derived from A. cinnamomea submerged culture filtrates exerts its anticancer effect through the induction of ROS-mediated apoptosis in A549 human lung cancer cells. PMID:25431605

  1. Nintedanib modulates surfactant protein-D expression in A549 human lung epithelial cells via the c-Jun N-terminal kinase-activator protein-1 pathway.

    PubMed

    Kamio, Koichiro; Usuki, Jiro; Azuma, Arata; Matsuda, Kuniko; Ishii, Takeo; Inomata, Minoru; Hayashi, Hiroki; Kokuho, Nariaki; Fujita, Kazue; Saito, Yoshinobu; Miya, Toshimichi; Gemma, Akihiko

    2015-06-01

    Idiopathic pulmonary fibrosis (IPF) is a progressive disease with a high mortality rate. Signalling pathways activated by several tyrosine kinase receptors are known to be involved in lung fibrosis, and this knowledge has led to the development of the triple tyrosine kinase inhibitor nintedanib, an inhibitor of vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and fibroblast growth factor receptor (FGFR), for the treatment of IPF. Pulmonary surfactant protein D (SP-D), an important biomarker of IPF, reportedly attenuates bleomycin-induced pulmonary fibrosis in mice. In this study, we investigated whether nintedanib modulates SP-D expression in human lung epithelial (A549) cells using quantitative real-time reverse transcriptase polymerase chain reaction and western blotting. To investigate the mechanisms underlying the effects of nintedanib, we evaluated the phosphorylation of c-Jun N-terminal kinase (JNK) and its downstream target c-Jun. The effect of the JNK inhibitor SP600125 on c-Jun phosphorylation was also tested. Activation of activator protein-1 (AP-1) was examined using an enzyme-linked immunosorbent assay-based test, and cell proliferation assays were performed to estimate the effect of nintedanib on cell proliferation. Furthermore, we treated mice with nintedanib to examine its in vivo effect on SP-D levels in lungs. These experiments showed that nintedanib up-regulated SP-D messenger RNA expression in a dose-dependent manner at concentrations up to 5 μM, with significant SP-D induction observed at concentrations of 3 μM and 5 μM, in comparison with that observed in vehicle controls. Nintedanib stimulated a rapid increase in phosphorylated JNK in A549 cells within 30 min of treatment and stimulated c-Jun phosphorylation, which was inhibited by the JNK inhibitor SP600125. Additionally, nintedanib was found to activate AP-1. A549 cell proliferation was not affected by nintedanib at any of the tested

  2. Cellular uptake and toxic effects of fine and ultrafine metal-sulfate particles in human A549 lung epithelial cells.

    PubMed

    Könczöl, Mathias; Goldenberg, Ella; Ebeling, Sandra; Schäfer, Bianca; Garcia-Käufer, Manuel; Gminski, Richard; Grobéty, Bernard; Rothen-Rutishauser, Barbara; Merfort, Irmgard; Gieré, Reto; Mersch-Sundermann, Volker

    2012-12-17

    Ambient airborne particulate matter is known to cause various adverse health effects in humans. In a recent study on the environmental impacts of coal and tire combustion in a thermal power station, fine crystals of PbSO(4) (anglesite), ZnSO(4)·H(2)O (gunningite), and CaSO(4) (anhydrite) were identified in the stack emissions. Here, we have studied the toxic potential of these sulfate phases as particulates and their uptake in human alveolar epithelial cells (A549). Both PbSO(4) and CaSO(4) yielded no loss of cell viability, as determined by the WST-1 and NR assays. In contrast, a concentration-dependent increase in cytotoxicity was observed for Zn sulfate. For all analyzed sulfates, an increase in the production of reactive oxygen species (ROS), assessed by the DCFH-DA assay and EPR, was observed, although to a varying extent. Again, Zn sulfate was the most active compound. Genotoxicity assays revealed concentration-dependent DNA damage and induction of micronuclei for Zn sulfate and, to a lower extent, for CaSO(4), whereas only slight effects could be found for PbSO(4). Moreover, changes of the cell cycle were observed for Zn sulfate and PbSO(4). It could be shown further that Zn sulfate increased the nuclear factor kappa-B (NF-κB) DNA binding activity and activated JNK. During our TEM investigations, no effect on the appearance of the A549 cells exposed to CaSO(4) compared to the nonexposed cells was observed, and in our experiments, only one CaSO(4) particle was detected in the cytoplasm. In the case of exposure to Zn sulfate, no particles were found in the cytoplasm of A549 cells, but we observed a concentration-dependent increase in the number and size of dark vesicles (presumably zincosomes). After exposure to PbSO(4), the A549 cells contained isolated particles as well as agglomerates both in vesicles and in the cytoplasm. Since these metal-sulfate particles are emitted into the atmosphere via the flue gas of coal-fired power stations, they may be

  3. Curcumin inhibits interferon-{alpha} induced NF-{kappa}B and COX-2 in human A549 non-small cell lung cancer cells

    SciTech Connect

    Lee, Jeeyun |; Im, Young-Hyuck | E-mail: imyh@smc.samsung.co.kr; Jung, Hae Hyun; Kim, Joo Hyun; Park, Joon Oh |; Kim, Kihyun |; Kim, Won Seog |; Ahn, Jin Seok

    2005-08-26

    The A549 cells, non-small cell lung cancer cell line from human, were resistant to interferon (IFN)-{alpha} treatment. The IFN-{alpha}-treated A549 cells showed increase in protein expression levels of NF-{kappa}B and COX-2. IFN-{alpha} induced NF-{kappa}B binding activity within 30 min and this increased binding activity was markedly suppressed with inclusion of curcumin. Curcumin also inhibited IFN-{alpha}-induced COX-2 expression in A549 cells. Within 10 min, IFN-{alpha} rapidly induced the binding activity of a {gamma}-{sup 32}P-labeled consensus GAS oligonucleotide probe, which was profoundly reversed by curcumin. Taken together, IFN-{alpha}-induced activations of NF-{kappa}B and COX-2 were inhibited by the addition of curcumin in A549 cells.

  4. Nanoparticles of Selaginella doederleinii leaf extract inhibit human lung cancer cells A549

    NASA Astrophysics Data System (ADS)

    Syaefudin; Juniarti, A.; Rosiyana, L.; Setyani, A.; Khodijah, S.

    2016-01-01

    The aim of the present study is to evaluate cytotoxicity effect of nanoparticles of Selaginella doederleinii (S. doederleinii) leaves extract. S. doederleinii was extracted by maceration method using 70%(v/v) ethanol as solvent. Phytochemical content was analyzed qualitatively by using Harborne and Thin Layer Chromatography (TLC) methods. Nanoparticle extract was prepared by ionic gelation using chitosan as encapsulant agent. Anticancer activity was performed by using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The results showed that S. doederleinii contains of flavonoids. Nanoparticle of S. doederleinii leaves extract greatly inhibited A549 cells growth (cancer cells), with IC50 of 3% or 1020 μg/ml. These nanoparticles extract also inhibited the growth of Chang cells (normal cells), with IC50 of 4% or 1442 μg/ml. The effective concentration of nanoparticles extract which inhibits cancer cells without harming the normal cells is 0.5% or 167 μg/ml. Further studies are needed to obtain the concentration of nanoparticles extract which can selectively suppress cancer cells.

  5. Oleifolioside B-mediated autophagy promotes apoptosis in A549 human non-small cell lung cancer cells.

    PubMed

    Jin, Cheng-Yun; Yu, Hai Yang; Park, Cheol; Han, Min Ho; Hong, Su Hyun; Kim, Kyoung-Sook; Lee, Young-Choon; Chang, Young-Chae; Cheong, Jaehun; Moon, Sung-Kwon; Kim, Gi-Young; Moon, Hyung-In; Kim, Wun-Jae; Lee, Jai-Heon; Choi, Yung Hyun

    2013-12-01

    The biochemical mechanisms of cell death by oleifolioside B (OB), a cycloartane-type triterpene glycoside isolated from Dendropanax morbifera Leveille, were investigated in A549 human lung carcinoma cells. Our data indicated that exposure to OB led to caspase activation and typical features of apoptosis; however, apoptotic cell death was not prevented by z-VAD-fmk, a pan-caspase inhibitor, demonstrating that OB-induced apoptosis was independent of caspase activation. Subsequently, we found that OB increased autophagy, as indicated by an increase in monodansylcadaverine fluorescent dye-labeled autophagosome formation and in the levels of the autophagic form of microtubule-associated protein 1 light chain 3 and Atg3, an autophagy-specific gene, which is associated with inhibiting phospho-nuclear factor erythroid 2-related factor 2 (Nrf2) expression. However, pretreatment with bafilomycin A1, an autophagy inhibitor, attenuated OB-induced apoptosis and dephosphorylation of Nrf2. The data suggest that OB-induced autophagy functions as a death mechanism in A549 cells and OB has potential as a novel anticancer agent capable of targeting apoptotic and autophagic cell death and the Nrf2 signaling pathway.

  6. In vitro cytotoxicity and morphological assessment of smoke from polymer combustion in human lung derived cells (A549).

    PubMed

    Lestari, F; Hayes, A J; Green, A R; Chattopadhyay, G

    2012-04-01

    The application of polymer and composites in building and modern transport interiors raises concerns of potential health hazards during combustion. Cytotoxicity and morphological assessment of smoke from polymer combustion in human lung derived cells (A549) has been investigated. A laboratory scale vertical tube furnace was used for the generation of combustion products. A range of materials used in the building and transport industry including high density-polyethylene (HDPE), polypropylene (PP), polycarbonate (PC), and polyvinyl chloride (PVC), fiberglass reinforced polymers (FRPs), and melamine faced plywood (MFP) were studied. The exposure of combustion toxicants to human lung cells (A549) at the air/liquid interface was acquired using a Harvard Navicyte Chamber. Cytotoxic effects on human cells were assessed based on cell viability using a selected in vitro cytotoxicity assays, including NRU (neutral red uptake) and ATP (adenosine triphosphate). Morphological assessment on the effects of combustion products in human lung cells from selected materials including PVC, FRP and MFP was assessed using scanning electron microscopy (SEM). The volatile organic compounds from thermal decomposition products were identified using ATD-GCMS (Automatic Thermal Desorption Gas Chromatography Mass Spectrometry). NOAEC (No Observable Adverse Effect Concentration), IC(10) (10% inhibitory concentration), IC(50) (50% inhibitory concentration), and TLC (Total Lethal Concentration) values (mg/l) were generated. The following toxicity ranking was observed from the most toxic material to the least toxic using the NRU assay: PVC>PP>HDPE>PC >FRP-10>MFP>FRP-16; and the ATP assay: PVC>HDPE>PP>FRP-10>FRP-16>MFP>PC. The method described here could potentially be an alternative to current fire toxicity standards.

  7. Portulaca oleracea Seed Oil Exerts Cytotoxic Effects on Human Liver Cancer (HepG2) and Human Lung Cancer (A-549) Cell Lines.

    PubMed

    Al-Sheddi, Ebtesam Saad; Farshori, Nida Nayyar; Al-Oqail, Mai Mohammad; Musarrat, Javed; Al-Khedhairy, Abdulaziz Ali; Siddiqui, Maqsood Ahmed

    2015-01-01

    Portulaca oleracea (Family: Portulacaceae), is well known for its anti-inflammatory, antioxidative, anti- bacterial, and anti-tumor activities. However, cytotoxic effects of seed oil of Portulaca oleracea against human liver cancer (HepG2) and human lung cancer (A-549) cell lines have not been studied previously. Therefore, the present study was designed to investigate the cytotoxic effects of Portulaca oleracea seed oil on HepG2 and A-549 cell lines. Both cell lines were exposed to various concentrations of Portulaca oleracea seed oil for 24h. After the exposure, percentage cell viability was studied by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT), neutral red uptake (NRU) assays, and cellular morphology by phase contrast inverted microscopy. The results showed a concentration-dependent significant reduction in the percentage cell viability and an alteration in the cellular morphology of HepG2 and A-549 cells. The percentage cell viability was recorded as 73%, 63%, and 54% by MTT assay and 76%, 61%, and 50% by NRU assay at 250, 500, and 1000 μg/ml, respectively in HepG2 cells. Percentage cell viability was recorded as 82%, 72%, and 64% by MTT assay and 83%, 68%, and 56% by NRU assay at 250, 500, and 1000 μg/ml, respectively in A-549 cells. The 100 μg/ml and lower concentrations were found to be non cytotoxic to A-549 cells, whereas decrease of 14% and 12% were recorded by MTT and NRU assay, respectively in HepG2 cells. Both HepG2 and A-549 cell lines exposed to 250, 500, and 1000 μg/ ml of Portulaca oleracea seed oil lost their normal morphology, cell adhesion capacity, become rounded, and appeared smaller in size. The data from this study showed that exposure to seed oil of Portulaca oleracea resulted in significant cytotoxicity and inhibition of growth of the human liver cancer (HepG2) and human lung cancer (A-549) cell lines.

  8. Gene expression modulation in A549 human lung cells in response to combustion-generated nano-sized particles.

    PubMed

    Arenz, Andrea; Hellweg, Christine E; Stojicic, Nevena; Baumstark-Khan, Christa; Grotheer, Horst-Henning

    2006-12-01

    High levels of ambient air pollution are associated in humans with aggravation of asthma and of respiratory and cardiopulmonary morbidity; long-term exposures to particulate matter (PM) have been linked to possible increases in lung cancer risk, chronic respiratory disease, and increased death rates. The Biodiagnostics Group of the DLR Institute of Aerospace Medicine develops cellular test systems capable of monitoring the biological consequences of environmental conditions on humans already on cellular and molecular level. Such bioassays rely on the receptor-reporter principle, where cell lines are transfected with plasmids carrying a reporter gene under control of environment-dependent promoters (receptor), which play a key role in regulating gene expressions in response to extracellular signals. We developed the recombinant human lung epithelial cell line A549-NF-kappaB-EGFP/Neo carrying a genetically encoded fluorescent indicator for monitoring activation of the NF-kappaB signaling pathway in living cells in response to genotoxic and cytotoxic environmental influences. With this cell line we screened several candidate human radiation-responsive genes (GADD45beta, CDKN1A) and NF-kappaB-dependent genes (IL-6, NFkappaBIA, and pNF-kappaB-EGFP) for gene expression changes by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assay, using cDNA obtained from total RNA isolated at various time points after exposure to combustion generated nano-sized particle samples.

  9. In vitro and in vivo antitumor activity of scutebarbatine A on human lung carcinoma A549 cell lines.

    PubMed

    Yang, Xiao-Kun; Xu, Ming-Yuan; Xu, Gui-Sen; Zhang, Yu-Lan; Xu, Zhao-Xia

    2014-06-25

    During our systematic study on the anticancer activities of Scutellaria barbata, scutebarbatine A (SBT-A), one of the major alkaloids in S. barbata, was found to have antitumor effects on A549 cells. Thus, we designed the present study to investigate in detail the antitumor effects of SBT-A. The cytotoxic effect of SBT-A on A549 in vitro were determined by an MTT assay and evaluated by IC50 values. Furthermore, results of Hoechst 33258 and Annexin V/PI staining assays demonstrated that SBT-A had significant antitumor effects on A549 cells via apoptosis, in a concentration-dependent manner. What's more, the mechanism was explored by western blotting, and our study revealed that SBT-A can up-regulate the expressions of cytochrome c, caspase-3 and 9, and down-regulate the levels of Bcl-2 in A549 cells. Finally, the antitumor effects of SBT-A were evaluated in vivo by using transplanted tumor nude mice, and the results confirmed that SBT-A has a notable antitumor effect on A549 cancer via mitochondria-mediated apoptosis. Collectively, our results demonstrated that SBT-A showed significant antitumor effects on A549 cells in vivo and in vitro via mitochondria-mediated apoptosis by up-regulating expressions of caspase-3 and 9, and down-regulating Bcl-2.

  10. Effects of sodium lactate Ringer's injection on transfection of human protein kinase C-α antisense oligonucleotide in A549 lung cancer cells.

    PubMed

    Wang, Z H; Sun, W W; Han, Y L; Ma, Z

    2016-08-26

    In the present study, we evaluated the effects of four solutions [Dulbecco's modified Eagle's medium (DMEM), sodium lactate Ringer's injection (SLRI), phosphate-buffered saline (PBS), and NaCl] on the transfection of the human protein kinase C-a antisense oligonucleotide (PKC-a ASO) aprinocarsen in human lung carcinoma A549 cells. Specifically, SLRI, DMEM, PBS, or NaCl were used as the growth solutions for A549 cells, and OPTI-MEM was used as the PKC-a ASO diluent for transfection. Additionally, SLRI, DMEM, PBS, or NaCl were used as both the growth solutions and diluents for transfection. The cell viability and transfection efficiency were determined. The results demonstrated that when SLRI was used as either the growth solution or both the growth solution and diluent for aprinocarsen transfection in A549 cells, the effects were close to the best effects observed with DMEM as the growth solution and OPTI-MEM as the diluent, which supported the transfection of aprinocarsen into the cells. Moreover, SLRI resulted in higher transfection efficiency than those of PBS and NaCl. In in vitro experiments, aprinocarsen effectively induced apoptosis in A549 cells. In conclusion, SLRI may replace PBS or NaCl in clinical trials as a transfection solution readily accepted by the human body. To our knowledge, this is the first report demonstrating the use of SLRI as a transfection solution in lung-cancer cell lines.

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

    PubMed

    Farzin, Hamidreza; Toroghi, Reza; Haghparast, Alireza

    2016-01-01

    Influenza H9N2 virus mostly infects avian species but poses a potential health risk to humans. Little is known about the mammalian host immune responses to H9N2 virus. To obtain insight into the innate immune responses of human lung epithelial cells to the avian H9N2 virus, the expressions of pro-inflammatory cytokines and chemokine in the human airway epithelial cells infected with avian H9N2 virus were examined by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). H9N2 virus was able to cultivate in the human lung epithelial cell line (A549) and stimulate production of pro-inflammatory cytokines (IL-1β, IL-6) and chemokine (IL-8). Expressions of cytokine genes were up-regulated to a significantly higher level for IL-1β (p < 0.01), IL-6 (p < 0.01 after 12 hours and p < 0.05 after 24 hours) and IL-8 (p < 0.01 after 12 hours and p < 0.001 after 24 hours) in virus-cultured A549 cells as compared with non-virus-cultured cells. The amount of IL-6 and IL-1β proteins secreted into the culture medium was also increased after virus culture infection of A549 cell line compared to non-virus-cultured A549 cells and were significant in both IL-1β (p < 0.05 in 18 hours and p < 0.001 in 24-48 hours harvested supernatant) and IL-6 (p < 0.001). Silencing the p65 component of NF-κB in A549 cells suppressed the stimulatory effects of influenza virus on secretion of pro-inflammatory cytokines and chemokine. The findings in this study will broaden our understanding of host innate immune mechanisms and the pathogenesis of H9N2 influenza viruses in human respiratory epithelium.

  12. Psoralen reverses docetaxel-induced multidrug resistance in A549/D16 human lung cancer cells lines.

    PubMed

    Hsieh, Ming-Ju; Chen, Mu-Kuan; Yu, Ya-Yen; Sheu, Gwo-Tarng; Chiou, Hui-Ling

    2014-06-15

    Chemotherapy is the recommended treatment for advanced-stage cancers. However, the emergence of multidrug resistance (MDR), the ability of cancer cells to become simultaneously resistant to different drugs, limits the efficacy of chemotherapy. Previous studies have shown that herbal medicine or natural food may be feasible for various cancers as potent chemopreventive drug. This study aims to explore the capablility of reversing the multidrug resistance of docetaxel (DOC)-resistant A549 cells (A549/D16) of psoralen and the underlying mechanisms. In this study, results showed that the cell viability of A549/D16 subline is decreased when treated with psoralen plus DOC, while psoralen has no effect on the cell proliferation on A549 and A549/D16 cells. Furthermore, mRNA and proteins levels of ABCB1 were decreased in the presence of psoralen, while decreased ABCB1 activity was also revealed by flow cytometry. Based on these results, we believe that psoralen may be feasible for reversing the multidrug resistance by inhibiting ABCB1 gene and protein expression. Such inhibition will lead to a decrease in ABCB1 activity and anti-cancer drug efflux, which eventually result in drug resistance reversal and therefore, sensitizing drug-resistant cells to death in combination with chemotherapeutic drugs.

  13. Gliotoxin promotes Aspergillus fumigatus internalization into type II human pneumocyte A549 cells by inducing host phospholipase D activation.

    PubMed

    Jia, Xiaodong; Chen, Fangyan; Pan, Weihua; Yu, Rentao; Tian, Shuguang; Han, Gaige; Fang, Haiqin; Wang, Shuo; Zhao, Jingya; Li, Xianping; Zheng, Dongyu; Tao, Sha; Liao, Wanqing; Han, Xuelin; Han, Li

    2014-06-01

    The internalization of Aspergillus fumigatus into lung epithelial cells is critical for the infection process in the host. Gliotoxin is the most potent toxin produced by A. fumigatus. However, its role in A. fumigatus internalization into the lung epithelial cells is still largely unknown. In the present study, the deletion of the gliP gene regulating the production of gliotoxin in A. fumigatus suppressed the internalization of conidia into the A549 lung epithelial cells, and this suppression could be rescued by the exogenous addition of gliotoxin. At lower concentrations, gliotoxin enhanced the internalization of the conidia of A. fumigatus into A549 cells; in contrast, it inhibited the phagocytosis of J774 macrophages in a dose-dependent manner. Under a concentration of 100 ng/ml, gliotoxin had no effect on A549 cell viability but attenuated ROS production in a dose-dependent manner. Gliotoxin significantly stimulated the phospholipase D activity in the A549 cells at a concentration of 50 ng/ml. This stimulation was blocked by the pretreatment of host cells with PLD1- but not PLD2-specific inhibitor. Morphological cell changes induced by gliotoxin were observed in the A549 cells accompanying with obvious actin cytoskeleton rearrangement and a moderate alteration of phospholipase D distribution. Our data indicated that gliotoxin might be responsible for modulating the A. fumigatus internalization into epithelial cells through phospholipase D1 activation and actin cytoskeleton rearrangement.

  14. EGCG reverses human neutrophil elastase-induced migration in A549 cells by directly binding to HNE and by regulating α1-AT

    NASA Astrophysics Data System (ADS)

    Xiaokaiti, Yilixiati; Wu, Haoming; Chen, Ya; Yang, Haopeng; Duan, Jianhui; Li, Xin; Pan, Yan; Tie, Lu; Zhang, Liangren; Li, Xuejun

    2015-07-01

    Lung carcinogenesis is a complex process that occurs in unregulated inflammatory environment. EGCG has been extensively investigated as a multi-targeting anti-tumor and anti-inflammatory compound. In this study, we demonstrated a novel mechanism by which EGCG reverses the neutrophil elastase-induced migration of A549 cells. We found that neutrophil elastase directly triggered human adenocarcinoma A549 cell migration and that EGCG suppressed the elevation of tumor cell migration induced by neutrophil elastase. We observed that EGCG directly binds to neutrophil elastase and inhibits its enzymatic activity based on the CDOCKER algorithm, MD stimulation by GROMACS, SPR assay and elastase enzymatic activity assay. As the natural inhibitor of neutrophil elastase, α1-antitrypsin is synthesized in tumor cells. We further demonstrated that the expression of α1-antitrypsin was up-regulated after EGCG treatment in neutrophil elastase-treated A549 cells. We preliminarily discovered that the EGCG-mediated induction of α1-antitrypsin expression might be correlated with the regulatory effect of EGCG on the PI3K/Akt pathway. Overall, our results suggest that EGCG ameliorates the neutrophil elastase-induced migration of A549 cells. The mechanism underlying this effect may include two processes: EGCG directly binds to neutrophil elastase and inhibits its enzymatic activity; EGCG enhances the expression of α1-antitrypsin by regulating the PI3K/AKT pathway.

  15. Bufalin inhibits TGF-β-induced epithelial-to-mesenchymal transition and migration in human lung cancer A549 cells by downregulating TGF-β receptors

    PubMed Central

    ZHAO, LEI; LIU, SHIZHOU; CHE, XIAOFANG; HOU, KEZUO; MA, YANJU; LI, CE; WEN, TI; FAN, YIBO; HU, XUEJUN; LIU, YUNPENG; QU, XIUJUAN

    2015-01-01

    The epithelial-to-mesenchymal transition (EMT) is a well-known prerequisite for cancer cells to acquire the migratory and invasive capacity, and to subsequently metastasize. Bufalin is one of the major active components of the traditional Chinese medicine Chan Su, and accumulating evidence has shown its anticancer effect in multipe types of cancer. However, the role of bufalin in transforming growth factor-β (TGF-β)-induced EMT and migration remains unclear. In the present study, the effect of bufalin on TGF-β-induced EMT and migration was investigated in human lung cancer A549 cells. TGF-β induced EMT in A549 cells and increased their migratory ability, which were markedly suppressed by bufalin. Additionally, TGF-β-induced upregulation of Twist2 and zinc finger E-box binding homeobox 2 (ZEB2), as well as the phosphorylation of Smad2 and Smad3 were also inhibited by bufalin. However, the Smad-independent signaling pathways were not affected. Further analysis showed that the TGF-β receptor I (TβRI) and TGF-β receptor II (TβRII) were downregulated in the presence of bufalin. Pretreatment with SB431542, a potent inhibitor of the phosphorylation of TβRI, significantly attenuated TGF-β-induced EMT, mimicking the effect of bufalin on A549 cells. Taken together, these results suggest that bufalin suppresses TGF-β-induced EMT and migration by downregulating TβRI and TβRII in A549 cells. PMID:26133118

  16. Dichloroacetate alters Warburg metabolism, inhibits cell growth, and increases the X-ray sensitivity of human A549 and H1299 NSC lung cancer cells.

    PubMed

    Allen, Kah Tan; Chin-Sinex, Helen; DeLuca, Thomas; Pomerening, Joseph R; Sherer, Jeremy; Watkins, John B; Foley, John; Jesseph, Jerry M; Mendonca, Marc S

    2015-12-01

    We investigated whether altering Warburg metabolism (aerobic glycolysis) by treatment with the metabolic agent dichloroacetate (DCA) could increase the X-ray-induced cell killing of the radiation-resistant human non-small-cell lung cancer (NSCLC) cell lines A549 and H1299. Treatment with 50mM DCA decreased lactate production and glucose consumption in both A549 and H1299, clear indications of attenuated aerobic glycolysis. In addition, we found that DCA treatment also slowed cell growth, increased population-doubling time, and altered cell cycle distribution. Furthermore, we report that treatment with 50mM DCA significantly increased single and fractionated X-ray-induced cell killing of A549 and H1299 cells. Assay of DNA double-strand break repair by neutral comet assays demonstrated that DCA inhibited both the fast and the slow kinetics of X-ray-induced DSB repair in both A549 and H1299 NSCL cancer cells. Taken together the data suggest a correlation between an attenuated aerobic glycolysis and enhanced cytotoxicity and radiation-induced cell killing in radiation-resistant NSCLC cells.

  17. Alpha-tomatine inactivates PI3K/Akt and ERK signaling pathways in human lung adenocarcinoma A549 cells: effect on metastasis.

    PubMed

    Shih, Yuan-Wei; Shieh, Jiunn-Min; Wu, Pei-Fen; Lee, Yi-Chieh; Chen, Yi-Zhi; Chiang, Tai-An

    2009-08-01

    This study first investigates the anti-metastatic effect of alpha-tomatine in the human lung adenocarcinoma cell line: A549. In this study, we first noted alpha-tomatine inhibited A549 cells invasion and migration by wound-healing assay and Boyden chamber assay. The data also showed alpha-tomatine could inhibit phosphorylation of Akt and extracellular signal-regulated kinase 1 and 2 (ERK1/2), which is involved in the up-regulating matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) or urokinase-type plasminogen activator (u-PA), whereas it did not affect phosphorylation of c-Jun N-terminal kinase (JNK) and p38. Next, alpha-tomatine significantly decreased the nuclear levels of nuclear factor kappa B (NF-kappaB), c-Fos, and c-Jun. Also, treating A549 cells with alpha-tomatine also leads to a dose-dependent inhibition on the binding abilities of NF-kappaB and activator protein-1 (AP-1). Further, the treatment of inhibitors specific for PI3K (Wortmannin) or ERK (U0126) to A549 cells could cause reduced activities of MMP-2, MMP-9, and u-PA. These results showed alpha-tomatine could inhibit the metastatic ability of A549 cells by reducing MMP-2, MMP-9, and u-PA activities through suppressing phosphoinositide 3-kinase/Akt (PI3K/Akt) or ERK1/2 signaling pathway and inhibition NF-kappaB or AP-1 binding activities. These findings proved alpha-tomatine might be an anti-metastatic agent against human lung adenocarcinoma.

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

    PubMed Central

    Lee, In-Seung; Uh, InJoon; Kim, Ki-Suk; Kim, Kang-Hoon; Park, Jiyoung; Kim, Yumi; Jung, Ji-Hoon; Jung, Hee-Jae

    2016-01-01

    Objective. There is limited information of the anti-inflammatory effects of Rg3 on inflamed lung cells and tissues. Therefore, we confirmed the anti-inflammatory mechanism of ginsenoside Rg3 in inflamed human airway epithelial cells (A549) and tissues whether Rg3 regulates nuclear factor kappa B (NF-κB) activity. Methods. To induce the inflammation, IL-1β (10 ng/ml) was treated to A549 cells for 4 h. The effects of Rg3 on NF-κB activity and COX-2 expression were evaluated by western blotting analysis in both IL-1β-induced inflamed A549 cell and human asthmatic airway epithelial tissues. Using multiplex cytokines assay, the secretion levels of NF-κB-mediated cytokines/chemokines were measured. Result. Rg3 showed the significant inhibition of NF-κB activity thereby reduced COX-2 expression was determined in both IL-1β-induced inflamed A549 cell and human asthmatic airway epithelial tissues. In addition, among NF-κB-mediated cytokines, the secretion levels of IL-4, TNF-α, and eotaxin were significantly decreased by Rg3 in asthma tissues. Even though there was no significant difference, IL-6, IL-9, and IL-13 secretion showed a lower tendency compared to saline-treated human asthmatic airway epithelial tissues. Conclusion. The results from this study demonstrate the potential of Rg3 as an anti-inflammatory agent through regulating NF-κB activity and reducing the secretion of NF-κB-mediated cytokines/chemokines. PMID:28116321

  19. Human microRNA-24 modulates highly pathogenic avian-origin H5N1 influenza A virus infection in A549 cells by targeting secretory pathway furin.

    PubMed

    Loveday, Emma-Kate; Diederich, Sandra; Pasick, John; Jean, François

    2015-01-01

    A common critical cellular event that many human enveloped viruses share is the requirement for proteolytic cleavage of the viral glycoprotein by furin in the host secretory pathway. For example, the furin-dependent proteolytic activation of highly pathogenic (HP) influenza A (infA) H5 and H7 haemagglutinin precursor (HA0) subtypes is critical for yielding fusion-competent infectious virions. In this study, we hypothesized that viral hijacking of the furin pathway by HP infA viruses to permit cleavage of HA0 could represent a novel molecular mechanism controlling the dynamic production of fusion-competent infectious virus particles during the viral life cycle. We explored the biological role of a newly identified furin-directed human microRNA, miR-24, in this process as a potential post-transcriptional regulator of the furin-mediated activation of HA0 and production of fusion-competent virions in the host secretory pathway. We report that miR-24 and furin are differentially expressed in human A549 cells infected with HP avian-origin infA H5N1. Using miR-24 mimics, we demonstrated a robust decrease in both furin mRNA levels and intracellular furin activity in A549 cells. Importantly, pretreatment of A549 cells with miR-24 mimicked these results: a robust decrease of H5N1 infectious virions and a complete block of H5N1 virus spread that was not observed in A549 cells infected with low-pathogenicity swine-origin infA H1N1 virus. Our results suggest that viral-specific downregulation of furin-directed microRNAs such as miR-24 during the life cycle of HP infA viruses may represent a novel regulatory mechanism that governs furin-mediated proteolytic activation of HA0 glycoproteins and production of infectious virions.

  20. Monitoring of TGF-β 1-Induced Human Lung Adenocarcinoma A549 Cells Epithelial-Mesenchymal Transformation Process by Measuring Cell Adhesion Force with a Microfluidic Device.

    PubMed

    Li, Yuan; Gao, AnXiu; Yu, Ling

    2016-01-01

    The epithelial-mesenchymal transition (EMT) is a process in which epithelial cells lose their cell polarity and cell-cell adhesion, and gain migratory and invasive properties. It is believed that EMT is associated with initiation and completion of the invasion-metastasis cascade. In this study, an economic approach was developed to fabricate a microfluidic device with less instrumentation requirement for the investigation of EMT by quantifying cell adhesion force. Fluid shear force was precisely controlled by a homemade microfluidic perfusion apparatus and interface. The adhesion capability of the human lung adenocarcinoma cell line A549 on different types of extracellular matrix protein was studied. In addition, effects of transforming growth factor-β (TGF-β) on EMT in A549 cells were investigated by characterizing the adhesion force changes and on-chip fluorescent staining. The results demonstrate that the microfluidic device is a potential tool to characterize the epithelial-mesenchymal transition process by measuring cell adhesion force.

  1. Erucin, a new promising cancer chemopreventive agent from rocket salads, shows anti-proliferative activity on human lung carcinoma A549 cells.

    PubMed

    Melchini, A; Costa, C; Traka, M; Miceli, N; Mithen, R; De Pasquale, R; Trovato, A

    2009-07-01

    Erucin (ER) is a dietary isothiocyanate present in cruciferous vegetables, such as rocket salads (Erucasativa Mill., Diplotaxis sp.), that has been recently considered a promising cancer chemopreventive phytochemical. Biological activity of ER was investigated on human lung adenocarcinoma A549 cells, analyzing its effects on molecular pathways involved in apoptosis and cell cycle arrest, such as PARP-1 cleavage, p53 and p21 protein expression. Our results show that ER affects the A549 cell proliferation, enhancing significantly p53 and p21 protein expression in a dose-dependent manner (p<0.001). PARP-1 cleavage occurs only after exposure to high concentrations of ER (50 microM), in accordance to previous studies showing similar bioactivity of other isothiocyanates (ITCs). Our study reports for the first time that the induction of p53, p21 and PARP-1 cleavage may participate in the anti-proliferative activity of ER in human lung adenocarcinoma A549 cells. Comparison of data with those obtained with the isothiocyanate sulforaphane (SF), structurally related to ER, underlines the strong relationship between structural analogy of ITCs and their biological activity. The ability of dietary compounds to modulate molecular mechanisms that affect cancer cell proliferation is certainly a key point of the cancer prevention potential by functional foods.

  2. Effect of silencing SATB1 on proliferation, invasion and apoptosis of A549 human lung adenocarcinoma cells

    PubMed Central

    Huang, Bo; Zhou, Hongli; Wang, Siwang; Lang, Xian Ping; Wang, Xiaodong

    2016-01-01

    The present study aimed to explore the clinical characteristics of special adenine-thymine-rich sequence-binding protein 1 (SATB1) in lung adenocarcinoma and its role in the proliferation, invasion, migration and apoptosis of the lung adenocarcinoma cell line A549. The expression of SATB1 was first studied in tumor tissues of lung adenocarcinoma and adjacent non-tumor tissues. The siRNA green fluorescent protein expression vector of SATB1 was constructed and transfected into the lung adenocarcinoma cell line A549, then a fluorescence microscope was used to study the transfection efficiency. Western blot analysis was adopted to measure the silencing efficiency. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Transwell and scratch assays were used to study cell proliferation, invasion and migration activity, and the apoptosis rate was tested by flow cytometry. SATB1 expression was low in the adjacent non-tumor tissues but high in lung adenocarcinoma tissues, and it was reversely proportional to the differentiation degree. Following transfection with SATB1-siRNA, the expression of SATB1 in A549 cells was blocked (P<0.01). In addition, the proliferation, invasion and migration abilities of cells decreased significantly while the apoptosis rate increased significantly (P<0.01). In conclusion SATB1 is closely associated with the pathogenesis and development of lung adenocarcinoma. PMID:27895736

  3. Identification of epigallocatechin-3-gallate in green tea polyphenols as a potent inducer of p53-dependent apoptosis in the human lung cancer cell line A549.

    PubMed

    Yamauchi, Rieko; Sasaki, Kaori; Yoshida, Kenichi

    2009-08-01

    The effects of green tea polyphenols on cultured cancer cells have been well characterized, especially the effects of epigallocatechin-3-gallate (EGCg), since EGCg suppresses oncogenic signaling pathways and induces cell cycle arrest or apoptosis by regulating cell cycle-associated proteins. In the present study, we attempted to identify signaling pathways or target molecules regulated by each of or a mixture of green tea polyphenols, including epicatechin (EC), epicatechin-3-gallate (ECg), epigallocatechin (EGC), and EGCg, in the human lung cancer cell line A549. ECg, EGC, and a catechin mixture, in addition to EGCg, significantly decreased cell viability. In contrast, caspase 3/7 activity, an apoptosis indicator, was specifically induced by EGCg. By conducting a series of luciferase-based reporter assays, we revealed that the catechin mixture only up-regulates the p53 reporter. EGCg was a more potent inducer of p53-dependent transcription, and this induction was further supported by the induced level of p53 protein. RNA interference (RNAi)-mediated p53 knockdown completely abolished EGCg-induced apoptosis. Finally, a proteome and western blot analysis using approximately 70 different antibodies failed to detect up-regulated proteins in catechin mixture-treated A549 cells. Taken together, these results indicate that EGCg, among several green tea polyphenols, is a potent apoptosis inducer that functions exclusively through a p53-dependent pathway in A549 cells.

  4. Green tea catechin, epigallocatechin-3-gallate, attenuates the cell viability of human non-small-cell lung cancer A549 cells via reducing Bcl-xL expression.

    PubMed

    Sonoda, Jun-Ichiro; Ikeda, Ryuji; Baba, Yasutaka; Narumi, Keiko; Kawachi, Akio; Tomishige, Erisa; Nishihara, Kazuya; Takeda, Yasuo; Yamada, Katsushi; Sato, Keizo; Motoya, Toshiro

    2014-07-01

    Clinical and epidemiological studies have indicated that the consumption of green tea has a number of beneficial effects on health. Epigallocatechin-3-gallate (EGCg), the major polyphenolic compound present in green tea, has received much attention as an active ingredient. Among the numerous promising profiles of EGCg, the present study focused on the anticancer effects. Apoptosis induced by EGCg and subsequent cell growth suppression have been demonstrated in a number of cell culture studies. However, the underlying mechanism of apoptotic cell death remains unclear. Thus, the aim of the present study was to identify the major molecule that mediates proapoptotic cell death by EGCg. The effect of EGCg on cell proliferation and the induction of mRNA that modulates apoptotic cell death was evaluated in the A549 human non-small-cell lung cancer cell line. In addition, morphological changes were assessed by microscopy in A549 cells that had been treated with 100 μM EGCg for 24 h. The MTT assay revealed that cell proliferation was significantly reduced by EGCg in a dose-dependent manner (3-100 μM). The mRNA expression level of B-cell lymphoma-extra large (Bcl-xL) was decreased in A549 cells following 24 h incubation with 100 μM EGCg. Therefore, the results indicated that the inhibition of cell proliferation by EGCg may be achieved via suppressing the expression of the cell death-inhibiting gene, Bcl-xL.

  5. Green tea catechin, epigallocatechin-3-gallate, attenuates the cell viability of human non-small-cell lung cancer A549 cells via reducing Bcl-xL expression

    PubMed Central

    SONODA, JUN-ICHIRO; IKEDA, RYUJI; BABA, YASUTAKA; NARUMI, KEIKO; KAWACHI, AKIO; TOMISHIGE, ERISA; NISHIHARA, KAZUYA; TAKEDA, YASUO; YAMADA, KATSUSHI; SATO, KEIZO; MOTOYA, TOSHIRO

    2014-01-01

    Clinical and epidemiological studies have indicated that the consumption of green tea has a number of beneficial effects on health. Epigallocatechin-3-gallate (EGCg), the major polyphenolic compound present in green tea, has received much attention as an active ingredient. Among the numerous promising profiles of EGCg, the present study focused on the anticancer effects. Apoptosis induced by EGCg and subsequent cell growth suppression have been demonstrated in a number of cell culture studies. However, the underlying mechanism of apoptotic cell death remains unclear. Thus, the aim of the present study was to identify the major molecule that mediates proapoptotic cell death by EGCg. The effect of EGCg on cell proliferation and the induction of mRNA that modulates apoptotic cell death was evaluated in the A549 human non-small-cell lung cancer cell line. In addition, morphological changes were assessed by microscopy in A549 cells that had been treated with 100 μM EGCg for 24 h. The MTT assay revealed that cell proliferation was significantly reduced by EGCg in a dose-dependent manner (3–100 μM). The mRNA expression level of B-cell lymphoma-extra large (Bcl-xL) was decreased in A549 cells following 24 h incubation with 100 μM EGCg. Therefore, the results indicated that the inhibition of cell proliferation by EGCg may be achieved via suppressing the expression of the cell death-inhibiting gene, Bcl-xL. PMID:24944597

  6. Comparison of oxycodone and morphine on the proliferation, apoptosis and expression of related molecules in the A549 human lung adenocarcinoma cell line

    PubMed Central

    Tian, Mi; Jin, Li; Li, Renqi; Zhu, Sihai; Ji, Muhuo; Li, Weiyan

    2016-01-01

    The present study aimed to compare the effects of oxycodone and morphine hydrochloride on the proliferation, apoptosis and migration of A549 lung cancer cells. A549 human lung cancer cells were cultured in vitro and treated with oxycodone or morphine at various concentrations (10, 20 and 40 µg/ml). Cell migration was determined using a wound healing assay, whereas apoptosis was detected using flow cytometry. Reverse transcription quantitative-polymerase chain reaction was performed in order to assess the apoptosis-related gene expression levels, including p53, B-cell lymphoma (Bcl)-2 and Bcl-2-associated X protein (Bax). The levels of vascular endothelial growth factor (VEGF) and urokinase-type plasminogen activator (uPA) were detected using enzyme-linked immunosorbent assays. The expression levels of intercellular cell adhesion molecule (ICAM)-1 were determined by immunofluorescence. In the present study, oxycodone and morphine induced apoptosis in A549 lung cancer cells with similar potency; however, >20 µg/ml oxycodone was more effective at inhibiting cell proliferation (P<0.05) and migration (P<0.05), as compared with morphine at the same concentration. Oxycodone induced a dose-dependent increase in the expression levels of p53 and Bax apoptosis-related genes, whereas it decreased the gene expression levels of Bcl-2. Furthermore, oxycodone decreased, whereas morphine increased, the expression levels of ICAM-1 in a concentration-dependent manner. In addition, at 40 µg/ml, the expression levels of VEGF and uPA in the morphine group were significantly higher than those demonstrated in the oxycodone group (P<0.05). In conclusion, oxycodone was more effective in inhibiting the proliferation and migration of A549 lung cancer cells, as compared with morphine. PMID:27446244

  7. Aspergillus fumigatus germ tube growth and not conidia ingestion induces expression of inflammatory mediator genes in the human lung epithelial cell line A549.

    PubMed

    Bellanger, Anne-Pauline; Millon, Laurence; Khoufache, Khaled; Rivollet, Danièle; Bièche, Ivan; Laurendeau, Ingrid; Vidaud, Michel; Botterel, Françoise; Bretagne, Stéphane

    2009-02-01

    Inhalation of conidia is the main cause of invasive pulmonary aspergillosis (IPA) and the respiratory epithelium is the first line of defence. To explore the triggering factor for the inflammatory response to Aspergillus fumigatus, the species mainly responsible for IPA, this study analysed the differential expression of three inflammatory genes in A549 cells after challenge with live and killed conidia. The influence of steroids, one of the main risk factors for developing IPA, was also investigated. Quantification of mRNAs of the inflammatory mediator genes encoding interleukin (IL)-8, tumour necrosis factor (TNF)-alpha and granulocyte-monocyte colony-stimulating factor (GM-CSF) was carried out using real-time PCR. Ingestion rates were studied for the conidia of A. fumigatus and Penicillium chrysogenum using a fluorescence brightener. Similar results were obtained for both species, with ingestion rates ranging from 35 to 40 %. Exposure of A549 cells to live A. fumigatus conidia only induced a four- to fivefold increase in the mRNA levels of the three genes, starting 8 h after the initial contact. Both inactivation of live A. fumigatus conidia and treatment by dexamethasone (10(-7) M) prevented the overexpression of TNF-alpha, IL-8 and GM-CSF. Fungal growth, rather than conidia ingestion, appears to be the main stimulus for the production of inflammatory mediators by epithelial cells, and this production is inhibited by steroid therapy. These results underline the role that the epithelium plays in the innate response against IPA.

  8. Streptococcus pneumoniae ClpL Modulates Adherence to A549 Human Lung Cells through Rap1/Rac1 Activation

    PubMed Central

    Nguyen, Cuong Thach; Le, Nhat-Tu; Tran, Thao Dang-Hien; Kim, Eun-Hye; Park, Sang-Sang; Luong, Truc Thanh; Chung, Kyung-Tae; Pyo, Suhkneung

    2014-01-01

    Caseinolytic protease L (ClpL) is a member of the HSP100/Clp chaperone family, which is found mainly in Gram-positive bacteria. ClpL is highly expressed during infection for refolding of stress-induced denatured proteins, some of which are important for adherence. However, the role of ClpL in modulating pneumococcal virulence is poorly understood. Here, we show that ClpL impairs pneumococcal adherence to A549 lung cells by inducing and activating Rap1 and Rac1, thus increasing phosphorylation of cofilin (inactive form). Moreover, infection with a clpL mutant (ΔclpL) causes a greater degree of filopodium formation than D39 wild-type (WT) infection. Inhibition of Rap1 and Rac1 impairs filopodium formation and pneumococcal adherence. Therefore, ClpL can reduce pneumococcal adherence to A549 cells, likely via modulation of Rap1- and Rac1-mediated filopodium formation. These results demonstrate a potential role for ClpL in pneumococcal resistance to host cell adherence during infection. This study provides insight into further understanding the interactions between hosts and pathogens. PMID:24980975

  9. Determination of in vitro free radical scavenging and antiproliferative effect of Pennisetum alopecuroides on cultured A549 human lung cancer cells

    PubMed Central

    Mathew, Githa Elizabeth; Mathew, Bijo; Gokul, S.; Krishna, Rahul; Farisa, M. P.

    2015-01-01

    Context: Pennisetum alopecuroides (Poaceae) is a grass predominantly distributed in tropics and sub tropics. It is used as a cattle feed in many regions. Aim: The objective of the present study was to investigate the in vitro free radical scavenging and antiproliferative activity of ethanol extract of P. alopecuroides (EEPA) on cultured A549 human lung cancer cell lines. Settings and Design: The anti-oxidant activity of ethanol extract was evaluated at dose level 12.5, 25, 50, 100, and 200 μg/ml. The in vitro antiproliferative activity was measured at doses of 10, 50, and 100 μg/ml. Materials and Methods: The free radical scavenging activity of the EEPA was determined by 2,2-Diphenyl-1-picrylhydrazyl (DPPH) method and in vitro antiproliferative activity on A549 human lung cancer cells was conducted by using MTT assay method. Results: The phytochemical screening revealed that the P. alopecuroides contained alkaloids, tannins, saponins, and flavonoids as the major secondary metabolites. The IC50 value of DPPH scavenging activity was found to be 44.41 μg/ml and 31.02 μg/ml  for a mixture of EEPA and standard ascorbic acid, respectively. In vitro MTT assay showed that EEPA had anti-proliferation effects on A549 cells in a dose dependent manner. Conclusions: This is the 1st time a pharmacological exploration of P. alopecuroides grasses has been conducted. We have shown that P. alopecuroides exhibits good free radical scavenging and strong in vitro cytotoxic activities against human lung cancer cell lines. PMID:26120234

  10. In vivo-in vitro comparison of acute respiratory tract toxicity using human 3D airway epithelial models and human A549 and murine 3T3 monolayer cell systems.

    PubMed

    Sauer, Ursula G; Vogel, Sandra; Hess, Annemarie; Kolle, Susanne N; Ma-Hock, Lan; van Ravenzwaay, Bennard; Landsiedel, Robert

    2013-02-01

    The usefulness of in vitro systems to predict acute inhalation toxicity was investigated. Nineteen substances were tested in three-dimensional human airway epithelial models, EpiAirway™ and MucilAir™, and in A549 and 3T3 monolayer cell cultures. IC(50) values were compared to rat four-hour LC(50) values classified according to EPA and GHS hazard categories. Best results were achieved with a prediction model distinguishing toxic from non-toxic substances, with satisfactory specificities and sensitivities. Using a self-made four-level prediction model to classify substances into four in vitro hazard categories, in vivo-in vitro concordance was mediocre, but could be improved by excluding substances causing pulmonary edema and emphysema in vivo. None of the test systems was outstanding, and there was no evidence that tissue or monolayer systems using respiratory tract cells provide an added value. However, the test systems only reflected bronchiole epithelia and alveolar cells and investigated cytotoxicity. Effects occurring in other cells by other mechanisms could not be recognised. Further work should optimise test protocols and expand the set of substances tested to define applicability domains. In vivo respiratory toxicity data for in vitro comparisons should distinguish different modes of action, and their relevance for human health effects should be ensured.

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

    PubMed

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

    2015-05-01

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

  12. Rosemary extract reduces Akt/mTOR/p70S6K activation and inhibits proliferation and survival of A549 human lung cancer cells.

    PubMed

    Moore, Jessy; Megaly, Mark; MacNeil, Adam J; Klentrou, Panagiota; Tsiani, Evangelia

    2016-10-01

    Compounds of plant origin and food components have attracted scientific attention for use as agents for cancer prevention and treatment. Rosemary extract contains polyphenols that were shown to have anti-cancer and other health benefits. The survival pathways of Akt, mammalian target of rapamycin (mTOR) and p70S6K, and the apoptotic protein poly ADP ribose polymerase (PARP) are key modulators of cancer cell growth and survival. In this study, we examined the effects of rosemary extract on proliferation, survival and apoptosis of human non-small cell lung cancer (NSCLC) cells and its influence on signaling events. Human NSCLC adenocarcinoma A549 cells were used. Cell proliferation and clonogenic survival were assessed using specific assays. Immunoblotting was used to examine total and phosphorylated levels of Akt, mTOR and p70S6K, and cleavage of PARP. Rosemary extract dose-dependently inhibited cell proliferation and reduced clonogenic survival of A549 cells, while PARP cleavage, an indicator of apoptosis, was enhanced. Rosemary extract significantly reduced total and phosphorylated/activated Akt, mTOR and p70S6K levels. In conclusion, rosemary extract inhibited proliferation, blocked clonogenic survival, and enhanced apoptosis of A549 lung cancer cells. These effects were associated with inhibition of Akt and downstream mTOR and p70S6K activity. Our data suggest that rosemary extract may have considerable anti-tumor and chemoprevention properties in lung cancer and deserves further systematic investigation in animal models of lung cancer.

  13. ROS/Autophagy/Nrf2 Pathway Mediated Low-Dose Radiation Induced Radio-Resistance in Human Lung Adenocarcinoma A549 Cell.

    PubMed

    Chen, Ni; Wu, Lijun; Yuan, Hang; Wang, Jun

    2015-01-01

    Low-dose ionizing radiation (LDIR) can induce radio-resistance to following high dose radiation in various mammalian cells. The protective role of LDIR has been thought to be associated with the overall outcomes of cancer radiotherapy. NF-E2 related factor 2 (Nrf2) is a transcription factor that plays pivotal roles in maintaining cellular oxidative equilibrium. Since oxidative stress has been indicated to be a mediator of LDIR induced radio-resistance, the role of Nrf2 in this process was investigated in this research. Our results showed that in human lung adenocarcinoma A549 cell, 5cGy alpha particle induced radio-resistance to following 75cGy alpha particle radiation. The expression level of Nrf2 and its target Heme Oxygenase-1(HO-1) increased after 5cGy radiation. Both the shRNA of Nrf2 and the chemical inhibitor of HO-1 suppressed the induced radio-resistance, indicating the involvement of Nrf2 antioxidant pathway in this process. Further, we found 5cGy radiation stimulated autophagy process in A549. Inhibition of the autophagy process resulted in suppression of the radio-resistance and the induced expression of Nrf2 and HO-1. ROS scavenger N-acetyl-L-cysteine (NAC) blocked the autophagy process induced by 5cGy alpha particle, the upregulation of Nrf2 and HO-1, as well as the induced radio-resistance. In conclusion, ROS elevation caused by LDIR promoted Autophagy/Nrf2-HO-1 and conferred radio-resistance in A549.

  14. ROS/Autophagy/Nrf2 Pathway Mediated Low-Dose Radiation Induced Radio-Resistance in Human Lung Adenocarcinoma A549 Cell

    PubMed Central

    Chen, Ni; Wu, Lijun; Yuan, Hang; Wang, Jun

    2015-01-01

    Low-dose ionizing radiation (LDIR) can induce radio-resistance to following high dose radiation in various mammalian cells. The protective role of LDIR has been thought to be associated with the overall outcomes of cancer radiotherapy. NF-E2 related factor 2 (Nrf2) is a transcription factor that plays pivotal roles in maintaining cellular oxidative equilibrium. Since oxidative stress has been indicated to be a mediator of LDIR induced radio-resistance, the role of Nrf2 in this process was investigated in this research. Our results showed that in human lung adenocarcinoma A549 cell, 5cGy alpha particle induced radio-resistance to following 75cGy alpha particle radiation. The expression level of Nrf2 and its target Heme Oxygenase-1(HO-1) increased after 5cGy radiation. Both the shRNA of Nrf2 and the chemical inhibitor of HO-1 suppressed the induced radio-resistance, indicating the involvement of Nrf2 antioxidant pathway in this process. Further, we found 5cGy radiation stimulated autophagy process in A549. Inhibition of the autophagy process resulted in suppression of the radio-resistance and the induced expression of Nrf2 and HO-1. ROS scavenger N-acetyl-L-cysteine (NAC) blocked the autophagy process induced by 5cGy alpha particle, the upregulation of Nrf2 and HO-1, as well as the induced radio-resistance. In conclusion, ROS elevation caused by LDIR promoted Autophagy/Nrf2-HO-1 and conferred radio-resistance in A549. PMID:26078725

  15. Effect of Paclitaxel-Mesoporous Silica Nanoparticles with a Core-Shell Structure on the Human Lung Cancer Cell Line A549

    NASA Astrophysics Data System (ADS)

    Wang, Tieliang; Liu, Ying; Wu, Chao

    2017-01-01

    A nanodrug delivery system of paclitaxel-mesoporous silica nanoparticles with a core-shell structure (PAC-csMSN) was used to increase the dissolution of paclitaxel (PAC) and improve its treatment of lung cancer. PAC was loaded into the core-shell mesoporous silica nanoparticles (csMSN) by the adsorption equilibrium method and was in an amorphous state in terms of its mesoporous structure. In vitro and in vivo studies showed that csMSN increased the dissolution rate of PAC and improved its lung absorption. The area under concentration-time curve (AUC) value of PAC-csMSN used for pulmonary delivery in rabbits was 2.678-fold higher than that obtained with the PAC. After continuous administration for 3 days, a lung biopsy showed no signs of inflammation. Cell apoptosis results obtained by flow cytometry indicated that PAC-csMSN was more potent than pure PAC in promoting cell apoptosis. An absorption investigation of PAC-csMSN in A549 cells was carried out by transmission electron microscopy (TEM) and laser scanning confocal microscopy (LSCM). The obtained results indicated that the cellular uptake was time-dependent and csMSN was uptaken into the cytoplasm. All these results demonstrate that csMSN have the potential to achieve pulmonary inhalation administration of poorly water-soluble drugs for the treatment of lung cancer.

  16. miR-129b suppresses cell proliferation in the human lung cancer cell lines A549 and H1299.

    PubMed

    Zheng, L; Qi, Y X; Liu, S; Shi, M L; Yang, W P

    2016-10-17

    Lung cancer is one of the most prevalent malignant tumors, and is one of the primary causes of cancer-associated deaths. In 2002, an estimated 1.18 million lung cancer-associated deaths were recorded, accounting for 18% of cancer-related deaths and 2% of total mortality. Despite the great progress that has been made in lung cancer therapies, the mechanisms underlying lung cancer formation and development remain largely unknown. Meanwhile, the microRNA miR-129 has been shown to be involved in the formation of many types of cancer. Therefore, this study aims to investigate whether miR129b could suppress proliferation of lung cancer cell lines. NSCLC tissue samples were collected from the Department of Respiratory Medicine between April 2013 and December 2015. Ten normal health individuals were recruited as controls. Lung cancer cell lines A549 and H1299 were used to examine the suppressive effects of miR129b. Quantitative real-time PCR was used to detect miR129b expression. The MTT assay was used to analyze cell proliferation. Results indicated that miR-129b is down-regulated in lung cancer cell lines and NSCLC tissues. Furthermore, overexpression of miR-129b inhibited proliferation of lung cancer cells. In conclusion, miR-129b suppresses lung cancer cell proliferation, and can be a potential therapeutic target for treatment of lung cancers.

  17. Human primary bronchial epithelial cells respond differently to titanium dioxide nanoparticles than the lung epithelial cell lines A549 and BEAS-2B.

    PubMed

    Ekstrand-Hammarström, Barbro; Akfur, Christine M; Andersson, Per Ola; Lejon, Christian; Osterlund, Lars; Bucht, Anders

    2012-09-01

    We have compared the cellular uptake and responses of five preparations of nanocrystalline titanium dioxide (TiO(2)) between normal human bronchial epithelial (NHBE) cells and epithelial cell lines (A549 and BEAS-2B). The P25 nanoparticles, containing both anatase and rutile modifications, induced reactive oxygen species (ROS) and secretion of the neutrophil chemoattractant IL-8 in all three cell types used. Pure anatase and rutile particles provoked differential IL-8 response in A549 and no response in BEAS-2B cells despite similar formation of ROS. The pure TiO(2) modifications also provoked release of the inflammatory mediators: IL-6, G-CSF and VEGF, in NHBE cells but not in the two cell lines. We conclude that the responsiveness of lung epithelial cells is strongly dependent on both the physicochemical properties of TiO(2) nanoparticles and the type of responder cells. The differential pro-inflammatory responsiveness of primary lung epithelial cells compared with immortalized cell lines should be considered in the assessment of adverse reactions to inhaled nanoparticles.

  18. Biological impacts of TiO2 on human lung cell lines A549 and H1299: particle size distribution effects

    NASA Astrophysics Data System (ADS)

    Tedja, Roslyn; Marquis, Christopher; Lim, May; Amal, Rose

    2011-09-01

    Increasing use of titanium dioxide (TiO2) nanoparticles in many commercial applications has led to emerging concerns regarding the safety and environmental impact of these materials. In this study, we have investigated the biological impact of nano-TiO2 (with particle primary size of 20 nm Aeroxide P25) on human lung cell lines in vitro and also the effect of particle size distribution on the particle uptake and apparent toxicity. The biological impact of nano-TiO2 is shown to be influenced by the concentration and particle size distribution of the TiO2 and the impact was shown to differ between the two cell lines (A549 and H1299) investigated herein. A549 cell line was shown to be relatively resistant to the total amount of TiO2 particles uptaken, as measured by cell viability and metabolic assays, while H1299 had a much higher capacity to ingest TiO2 particles and aggregates, with consequent evidence of impact at concentrations as low as 30-150 μg/mL TiO2. Evidence gathered from this study suggests that both viability and metabolic assays (measuring metabolic and mitochondrial activities and also cellular ATP level) should be carried out collectively to gain a true assessment of the impact of exposure to TiO2 particles.

  19. Three-dimensional quantitative structure-activity relationship study on anti-cancer activity of 3,4-dihydroquinazoline derivatives against human lung cancer A549 cells

    NASA Astrophysics Data System (ADS)

    Cho, Sehyeon; Choi, Min Ji; Kim, Minju; Lee, Sunhoe; Lee, Jinsung; Lee, Seok Joon; Cho, Haelim; Lee, Kyung-Tae; Lee, Jae Yeol

    2015-03-01

    A series of 3,4-dihydroquinazoline derivatives with anti-cancer activities against human lung cancer A549 cells were subjected to three-dimensional quantitative structure-activity relationship (3D-QSAR) studies using the comparative molecular similarity indices analysis (CoMSIA) approaches. The most potent compound, 1 was used to align the molecules. As a result, the best prediction was obtained with CoMSIA combined the steric, electrostatic, hydrophobic, hydrogen bond donor, and hydrogen bond acceptor fields (q2 = 0.720, r2 = 0.897). This model was validated by an external test set of 6 compounds giving satisfactory predictive r2 value of 0.923 as well as the scrambling stability test. This model would guide the design of potent 3,4-dihydroquinazoline derivatives as anti-cancer agent for the treatment of human lung cancer.

  20. Role of {alpha}{sub v}{beta}{sub 5} integrin receptor in endocytosis of crocidolite and its effect on intracellular glutathione levels in human lung epithelial (A549) cells

    SciTech Connect

    Pande, Priyadarshini; Mosleh, Tariq A.; Aust, Ann E. . E-mail: aaust@cc.usu.edu

    2006-01-15

    Crocidolite, containing 27% iron by weight, is the most carcinogenic form of asbestos. Crocidolite fibers are endocytized by {alpha}{sub v}{beta}{sub 5} integrin receptors in rabbit pleural mesothelial cells. We show here that crocidolite fibers are endocytized in human lung epithelial (A549) cells and in primary small airway epithelial (SAEC) cells. Presence of the integrin {alpha}{sub v}{beta}{sub 5} blocking antibody, P1F6, significantly reduced the uptake of crocidolite fibers in A549 cells. Thus, the integrin {alpha}{sub v}{beta}{sub 5} receptor is involved in endocytosis of crocidolite fibers in A549 cells as well. Previously, it has been observed that asbestos fibers lead to changes in the intracellular redox environment, i.e. a marked decrease in intracellular glutathione concentrations and an increase in the extracellular glutathione in A549 cells. In addition, the decrease in intracellular glutathione was found to be largely independent of iron present on the surface of the fiber. A549 cells were treated with crocidolite in the presence of endocytosis inhibitor cytochalasin D. Our data indicate that, upon preventing endocytosis, we were able to reverse the decrease in total intracellular glutathione. The decrease in total intracellular glutathione could also be prevented in the presence of the monoclonal antibody P1F6. Thus, we observed that endocytosis of crocidolite fibers via integrin {alpha}{sub v}{beta}{sub 5} receptor is linked to the marked decrease in total intracellular glutathione in A549 cells.

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

    PubMed

    Rossner, Pavel; Strapacova, Simona; Stolcpartova, Jitka; Schmuczerova, Jana; Milcova, Alena; Neca, Jiri; Vlkova, Veronika; Brzicova, Tana; Machala, Miroslav; Topinka, Jan

    2016-08-26

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

  2. Toxicity of wood smoke particles in human A549 lung epithelial cells: the role of PAHs, soot and zinc.

    PubMed

    Dilger, Marco; Orasche, Jürgen; Zimmermann, Ralf; Paur, Hanns-Rudolf; Diabaté, Silvia; Weiss, Carsten

    2016-12-01

    Indoor air pollution is associated with increased morbidity and mortality. Specifically, the health impact of emissions from domestic burning of biomass and coal is most relevant and is estimated to contribute to over 4 million premature deaths per year worldwide. Wood is the main fuel source for biomass combustion and the shift towards renewable energy sources will further increase emissions from wood combustion even in developed countries. However, little is known about the constituents of wood smoke and biological mechanisms that are responsible for adverse health effects. We exposed A549 lung epithelial cells to collected wood smoke particles and found an increase in cellular reactive oxygen species as well as a response to bioavailable polycyclic aromatic hydrocarbons. In contrast, cell vitality and regulation of the pro-inflammatory cytokine interleukin-8 were not affected. Using a candidate approach, we could recapitulate WSP toxicity by the combined actions of its constituents soot, metals and PAHs. The soot fraction and metals were found to be the most important factors for ROS formation, whereas the PAH response can be mimicked by the model PAH benzo[a]pyrene. Strikingly, PAHs adsorbed to WSPs were even more potent in activating target gene expression than B[a]P individually applied in suspension. As PAHs initiate multiple adverse outcome pathways and are prominent carcinogens, their role as key pollutants in wood smoke and its health effects warrants further investigation. The presented results suggest that each of the investigated constituents soot, metals and PAHs are major contributors to WSP toxicity. Mitigation strategies to prevent adverse health effects of wood combustion should therefore not only aim at reducing the emitted soot and PAHs but also the metal content, through the use of more efficient combustion appliances, and particle precipitation techniques, respectively.

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

    PubMed Central

    Rossner, Pavel; Strapacova, Simona; Stolcpartova, Jitka; Schmuczerova, Jana; Milcova, Alena; Neca, Jiri; Vlkova, Veronika; Brzicova, Tana; Machala, Miroslav; Topinka, Jan

    2016-01-01

    We investigated the toxicity of benzo[a]pyrene (B[a]P), 1-nitropyrene (1-NP) and 3-nitrobenzanthrone (3-NBA) in A549 cells. Cells were treated for 4 h and 24 h with: B[a]P (0.1 and 1 μM), 1-NP (1 and 10 μM) and 3-NBA (0.5 and 5 μM). Bulky DNA adducts, lipid peroxidation, DNA and protein oxidation and mRNA expression of CYP1A1, CYP1B1, NQO1, POR, AKR1C2 and COX2 were analyzed. Bulky DNA adducts were induced after both treatment periods; the effect of 1-NP was weak. 3-NBA induced high levels of bulky DNA adducts even after 4-h treatment, suggesting rapid metabolic activation. Oxidative DNA damage was not affected. 1-NP caused protein oxidation and weak induction of lipid peroxidation after 4-h incubation. 3-NBA induced lipid peroxidation after 24-h treatment. Unlike B[a]P, induction of the aryl hydrocarbon receptor, measured as mRNA expression levels of CYP1A1 and CYP1B1, was low after treatment with polycyclic aromatic hydrocarbon (PAH) nitro-derivatives. All test compounds induced mRNA expression of NQO1, POR, and AKR1C2 after 24-h treatment. AKR1C2 expression indicates involvement of processes associated with reactive oxygen species generation. This was supported further by COX2 expression induced by 24-h treatment with 1-NP. In summary, 3-NBA was the most potent genotoxicant, whereas 1-NP exhibited the strongest oxidative properties. PMID:27571070

  4. Fyn mediates transforming growth factor-beta1-induced down-regulation of E-cadherin in human A549 lung cancer cells.

    PubMed

    Kim, An Na; Jeon, Woo-Kwang; Lim, Kyu-Hyoung; Lee, Hui-Young; Kim, Woo Jin; Kim, Byung-Chul

    2011-04-01

    Transforming growth factor-beta (TGF-β) signaling positively contributes to the regulation of tumor metastasis. However, the underlying molecular mechanisms are less well defined. We here show that Fyn, a member of Src family tyrosine kinases, plays a critical role in mediating TGF-β1-induced down-regulation of E-cadherin in human A549 lung cancer cells. Blockade of Fyn with siRNA knockdown or ligand-binding defective mutant significantly lowered the ability of TGF-β1 to repress E-cadherin expression. Furthermore, our results demonstrated that Fyn facilitates TGF-β1-mediated suppression of E-cadherin through p38 kinase-dependent induction of Snail. Collectively, our findings identify a Fyn-p38-Snail cascade as a new signaling pathway mediating oncogenic TGF-β function.

  5. Separation of an aqueous extract Inonotus obliquus (Chaga). A novel look at the efficiency of its influence on proliferation of A549 human lung carcinoma cells.

    PubMed

    Mazurkiewicz, Witold; Rydel, Katarzyna; Pogocki, Dariusz; Lemieszek, Marta Kinga; Langner, Ewa; Rzeski, Wojciech

    2010-01-01

    Aqueous extract of Inonotus obliquus was hydrolyzed in dilute hydrochloric acid. The products were extracted applying organic solvents, and separated chromatographically on a silica gel-packed column. Eluted fractions were analyzed by means of GC-MS. The presence of hydrocarbons, alcohols, phenols and various carbonyl compounds in analyzed fractions has been detected and quantified. Preliminarily experiments on the influence of certain separated samples on the proliferation of A549 human lung carcinoma cells were performed. Therefore, we hypothesize that the major antiproliferative effects are related to the presence of benzaldehyde, which is a benzyl alcohol metabolite formed in situ in the cells culture with the yield moderated by the presence of trace amounts of "high molecular mass compounds".

  6. Multidimensional effects of biologically synthesized silver nanoparticles in Helicobacter pylori, Helicobacter felis, and human lung (L132) and lung carcinoma A549 cells

    NASA Astrophysics Data System (ADS)

    Gurunathan, Sangiliyandi; Jeong, Jae-Kyo; Han, Jae Woong; Zhang, Xi-Feng; Park, Jung Hyun; Kim, Jin-Hoi

    2015-02-01

    Silver nanoparticles (AgNPs) are prominent group of nanomaterials and are recognized for their diverse applications in various health sectors. This study aimed to synthesize the AgNPs using the leaf extract of Artemisia princeps as a bio-reductant. Furthermore, we evaluated the multidimensional effect of the biologically synthesized AgNPs in Helicobacter pylori, Helicobacter felis, and human lung (L132) and lung carcinoma (A549) cells. UV-visible (UV-vis) spectroscopy confirmed the synthesis of AgNPs. X-ray diffraction (XRD) indicated that the AgNPs are specifically indexed to a crystal structure. The results from Fourier transform infrared spectroscopy (FTIR) indicate that biomolecules are involved in the synthesis and stabilization of AgNPs. Dynamic light scattering (DLS) studies showed the average size distribution of the particle between 10 and 40 nm, and transmission electron microscopy (TEM) confirmed that the AgNPs were significantly well separated and spherical with an average size of 20 nm. AgNPs caused dose-dependent decrease in cell viability and biofilm formation and increase in reactive oxygen species (ROS) generation and DNA fragmentation in H. pylori and H. felis. Furthermore, AgNPs induced mitochondrial-mediated apoptosis in A549 cells; conversely, AgNPs had no significant effects on L132 cells. The results from this study suggest that AgNPs could cause cell-specific apoptosis in mammalian cells. Our findings demonstrate that this environmentally friendly method for the synthesis of AgNPs and that the prepared AgNPs have multidimensional effects such as anti-bacterial and anti-biofilm activity against H. pylori and H. felis and also cytotoxic effects against human cancer cells. This report describes comprehensively the effects of AgNPs on bacteria and mammalian cells. We believe that biologically synthesized AgNPs will open a new avenue towards various biotechnological and biomedical applications in the near future.

  7. Cytotoxicity and genotoxicity in human lung epithelial A549 cells caused by airborne volatile organic compounds emitted from pine wood and oriented strand boards.

    PubMed

    Gminski, Richard; Tang, Tao; Mersch-Sundermann, Volker

    2010-06-16

    Due to the massive reduction of air-change rates in modern, energy-saving houses and dwellings, the contribution of volatile organic compound (VOCs) emissions from wood-based materials to indoor air quality has become increasingly important. To evaluate toxicity of VOC mixtures typically emitted from pine wood and oriented strand boards (OSB) and their main constituents (selected terpenes and aldehydes), cytotoxicity and genotoxicity were investigated in human A549 lung cells. To facilitate exposure directly via gas phase, a 250 L emission chamber was combined with a Vitrocell exposure system. VOC exposure concentrations were measured by GC/MSD. Biological effects were determined after an exposure time of 1h by measuring cytotoxicity (erythrosine B staining) and genotoxicity (comet assay). Neither cytotoxic nor genotoxic effects were observed for VOC mixtures emitted from pine wood or OSB at loading factors of approximately 13 m(2)/m(3) (worst case conditions) of the panels (with maximum VOC levels of about 80 mg/m(3)) in comparison to clean air. While alpha-pinene and Delta(3)-carene did not induce toxic effects even at exposure concentrations of up to 1800 mg/m(3) and 600 mg/m(3), respectively, hexanal showed a cytotoxic effect at 2000 mg/m(3). The alpha,beta-unsaturated aldehydes 2-heptenal and 2-octenal caused genotoxic effects in concentrations exceeding 100mg/m(3) and 40 mg/m(3), respectively. In conclusion, high concentrations of VOCs and VOC mixtures emitted from pine wood and OSB did not lead to adverse effects in A549 human lung cells even at concentrations 10(2) to 10(5)-fold higher than those found in normal indoor air. Attention must be paid to mutagenic and possibly carcinogenic alpha,beta-unsaturated aldehydes.

  8. Therapeutic effects of gold nanoparticles synthesized using Musa paradisiaca peel extract against multiple antibiotic resistant Enterococcus faecalis biofilms and human lung cancer cells (A549).

    PubMed

    Vijayakumar, S; Vaseeharan, B; Malaikozhundan, B; Gopi, N; Ekambaram, P; Pachaiappan, R; Velusamy, P; Murugan, K; Benelli, G; Suresh Kumar, R; Suriyanarayanamoorthy, M

    2017-01-01

    Botanical-mediated synthesis of nanomaterials is currently emerging as a cheap and eco-friendly nanotechnology, since it does not involve the use of toxic chemicals. In the present study, we focused on the synthesis of gold nanoparticles using the aqueous peel extract of Musa paradisiaca (MPPE-AuNPs) following a facile and cheap fabrication process. The green synthesized MPPE-AuNPs were bio-physically characterized by UV-Vis spectroscopy, FTIR, XRD, TEM, Zeta potential analysis and EDX. MPPE-AuNPs were crystalline in nature, spherical to triangular in shape, with particle size ranging within 50 nm. The biofilm inhibition activity of MPPE-AuNPs was higher against multiple antibiotic resistant (MARS) Gram-positive Enterococcus faecalis. Light and confocal laser scanning microscopic observations evidenced that the MPPE-AuNPs effectively inhibited the biofilm of E. faecalis when tested at 100 μg mL(-1). Cytotoxicity studies demonstrated that MPPE-AuNPs were effective in inhibiting the viability of human A549 lung cancer cells at higher concentrations of 100 μg mL(-1). The morphological changes in the MPPE-AuNPs treated A549 lung cancer cells were visualized under phase-contrast microscopy. Furthermore, the ecotoxicity of MPPE-AuNPs on the freshwater micro crustacean Ceriodaphnia cornuta were evaluated. Notably, no mortality was recorded in MPPE-AuNPs treated C. cornuta at 250 μg mL(-1). This study concludes that MPPE-AuNPs are non-toxic, eco-friendly and act as a multipurpose potential biomaterial for biomedical applications.

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

    PubMed

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

    2013-12-01

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

  10. Avastin® in combination with gemcitabine and cisplatin significantly inhibits tumor angiogenesis and increases the survival rate of human A549 tumor-bearing mice

    PubMed Central

    LIU, YING; XIA, XIZHENG; ZHOU, MINGKAI; LIU, XIAOJUN

    2015-01-01

    The aim of this study was to investigate the effect of Avastin® in combination with gemcitabine and cisplatin (GP) on the tumor growth of A549 tumor-bearing mice and the potential anti-tumor mechanism. A total of 30 human A549 tumor-bearing nude mice were randomly divided into the Avastin, chemotherapy and combined treatment groups for treatment with an intraperitoneal injection of Avastin (5 mg/kg) (Avastin group); an intraperitoneal injection of gemcitabine (4 mg/kg) and cisplatin (4 mg/kg) (chemotherapy group); or intraperitoneal injections of Avastin and GP (combined treatment group). The mice were observed for 30 days and the tumor growth, survival and body weight of the mice in the three groups were analyzed. The protein level of vascular endothelial growth factor (VEGF) in the tumor tissues was analyzed by ELISA. The vascular density and structural changes of the tumor were analyzed using immunohistochemistry. Compared with the Avastin and chemotherapy groups, the tumor growth of mice in the combined treatment group was significantly inhibited, and the survival rate of the mice was increased significantly. No difference in body weight was observed among the three groups of mice (P>0.05). The levels of VEGF in the combined treatment group tumor tissues were significantly reduced compared with those in the chemotherapy group tumor tissues (P<0.05). Furthermore, the vessel density of the tumor tissue in the combined treatment group was significantly reduced compared with that in the chemotherapy group (P<0.05), and the number of normal vessels in the combined treatment group tumors was significantly higher than that in the chemotherapy group tumors after 7 days of treatment (P<0.05). In conclusion, Avastin can significantly decrease the level of VEGF in tumor tissue, inhibit tumor angiogenesis and promote the normalization of tumor vascular structure, which may explain the enhanced efficacy of Avastin in combination with chemotherapy. PMID:26136956

  11. Isolinderalactone inhibits proliferation of A549 human non‑small cell lung cancer cells by arresting the cell cycle at the G0/G1 phase and inducing a Fas receptor and soluble Fas ligand-mediated apoptotic pathway.

    PubMed

    Chang, Wei-An; Lin, En-Shyh; Tsai, Ming-Ju; Huang, Ming-Shyan; Kuo, Po-Lin

    2014-05-01

    Lung cancer is currently the leading cause of cancer-related mortality worldwide. In Taiwan, lung cancer is also the type of malignancy that is the major cause of cancer-mortality. Investigating the mechanism of apoptosis of lung cancer cells is important in the treatment of lung cancer. In the present study, isolinderalactone was demonstrated to exhibit anticancer effects in A549 human non-small cell lung cancer cells. The effect of isolinderalactone on apoptosis, cell cycle distribution p21 levels and the Fas receptor and soluble Fas ligand (sFasL) were assayed in order to determine the mechanism underlying the anticancer effect of isolinderalactone. It was demonstrated that isolinderalactone may induce p21 expression and then cause the cell cycle arrest of A549 cells. The data of the present study also revealed that the Fas/sFasL apoptotic system is significant in the mechanism of isolinderalactone‑induced apoptosis of A549 cells. These novel findings demonstrated that isolinderalactone may cause the cell cycle arrest of A549 cells by induction of p21, and induce apoptosis of A549 human non-small-cell lung carcinoma cells through the Fas/sFasL apoptotic system.

  12. [Effect of two different acellular lung matrices on α-SMA expression in A549 cells].

    PubMed

    Chen, C; Wang, Z Y; Weng, J; Wang, Z B; Mei, J; Du, X H; Wang, L

    2017-01-24

    Objective: To explore the effect of acellular normal and fibrotic lung matrices on alpha smooth muscle actin (α-SMA) expression in human lung adenocarcinoma cell line A549. Methods: Twenty adult SD rats were randomly divided into normal group and idiopathic pulmonary fibrosis(IPF)group (n=10 each). The pulmonary fibrosis was induced by Bleomycin. Normal and fibrotic decellularized lungs were made, then sections with 500 μm thick were cut by a standard Vibratome. None scaffold was set as control group. A549 cells were seeded dropwise into different slices (normal and fibrotic scaffolds), and cultured for one week in vitro. The expression of α-SMA was measured by immunofluorescence staining and quantitative real time polymerase chain reaction (qRT-PCR). Results: In control group, the expression of α-SMA protein was positive in A549 cells by immunofluorescence staining. However, it expressed weakly both in normal and fibrotic scaffold group, and the fluorescence intensity in fibrotic scaffold group was significant lower than that in normal group (P<0.05). The relative expression amount of α-SMA mRNA in normal and fibrotic scaffold group were (0.70±0.11) and (0.55±0.12), which were significant lower than that of control group (1.28±0.21) (P<0.05). Moreover, the relative expression of α-SMA mRNA in fibrotic scaffold group was decreased compared to that in normal scaffold group (P<0.05). Conclusions: Acellular normal and fibrotic lung scaffold can downregulate the expression of α-SMA in human lung adenocarcinoma cell line A549. It may inhibit the movement of A549 cells in acellular normal and fibrotic lung matrices, especially in acellular fibrotic lung scaffold.

  13. Expression of human eukaryotic initiation factor 3f oscillates with cell cycle in A549 cells and is essential for cell viability

    PubMed Central

    2010-01-01

    Background Transcriptional and postranslational regulation of the cell cycle has been widely studied. However, there is scarce knowledge concerning translational control of this process. Several mammalian eukaryotic initiation factors (eIFs) seem to be implicated in controlling cell proliferation. In this work, we investigated if the human eIF3f expression and function is cell cycle related. Results The human eIF3f expression has been found to be upregulated in growth-stimulated A549 cells and downregulated in G0. Western blot analysis and eIF3f promotor-luciferase fusions revealed that eIF3f expression peaks twice in the cell cycle: in the S and the M phases. Deregulation of eIF3f expression negatively affects cell viability and induces apoptosis. Conclusions The expression pattern of human eIF3f during the cell cycle confirms that this gene is cell division related. The fact that eIF3f expression peaks in two cell cycle phases raises the possibility that this gene may exert a differential function in the S and M phases. Our results strongly suggest that eIF3f is essential for cell proliferation. PMID:20462454

  14. Down-regulation of the DNA-repair endonuclease 8-oxo-guanine DNA glycosylase 1 (hOGG1) by sodium dichromate in cultured human A549 lung carcinoma cells.

    PubMed

    Hodges, N J; Chipman, J K

    2002-01-01

    Hexavalent chromium is a genotoxic human pulmonary carcinogen that elevates DNA oxidation, apparently through the generation of reactive DNA-damaging intermediates including Cr(V), Cr(IV) and reactive oxygen species. We tested the hypothesis that elevation of DNA oxidation may also be through inhibition of the expression of the repair glycosylase for 8-oxo deoxyguanine (hOGG1) in cultured A549 human lung epithelial cells. Treatment with sodium dichromate (0-100 microM, 16 h) resulted in a concentration-dependent decrease in the levels of OGG1 mRNA as measured by both RT-PCR and RNase protection assay. Sodium dichromate at 25 microM and above gave a marked reduction of OGG1 mRNA expression which was not seen at 1 microM and below. No effect on the expression of the apurinic endonuclease hAPE or the house-keeping gene GAPDH was observed at any of the concentrations of sodium dichromate investigated. Treatment of cells with the pro-oxidant H(2)O(2) (0-200 microM) for 16 h had no detectable effect on the levels of OGG1 mRNA or protein expression suggesting that the effect of sodium dichromate is not mediated by H(2)O(2). Western blotting demonstrated that sodium dichromate (100 microM; 16 h and >25 microM; 28 h) markedly reduced levels of OGG1 protein in nuclear cell extracts. Additionally, treatment of cells with sodium dichromate (>25 microM, 28 h) resulted in a concentration-dependent decrease in the ability of nuclear extracts to nick a synthetic oligonucleotide containing 8-oxo deoxyguanine (8-oxo dG). We conclude that the elevation of 8-oxo dG levels observed in A549 cells treated with sodium dichromate may be, at least in part, due to a reduced capacity to repair endogenous and hexavalent chromium-induced 8-oxo dG.

  15. The fruit juice of Morinda citrifolia (noni) downregulates HIF-1α protein expression through inhibition of PKB, ERK-1/2, JNK-1 and S6 in manganese-stimulated A549 human lung cancer cells.

    PubMed

    Jang, Byeong-Churl

    2012-03-01

    High exposure of manganese is suggested to be a risk factor for many lung diseases. Evidence suggests anticancerous and antiangiogenic effects by products derived from Morinda citrifolia (noni) fruit. In this study, we investigated the effect of noni fruit juice (NFJ) on the expression of HIF-1α, a tumor angiogenic transcription factor in manganese-chloride (manganese)-stimulated A549 human lung carcinoma cells. Treatment with manganese largely induced expression of HIF-1α protein but did not affect HIF-1α mRNA expression in A549 cells, suggesting the metal-mediated co- and/or post-translational HIF-1α upregulation. Manganese treatment also led to increased phosphorylation of extracellular-regulated protein kinase-1/2 (ERK-1/2), c-Jun N-terminal kinase-1 (JNK-1), protein kinase B (PKB), S6 and eukaryotic translation initiation factor-2α (eIF-2α) in A549 cells. Of note, the exposure of NFJ inhibited the manganese-induced HIF-1α protein upregulation in a concentration-dependent manner. Importantly, as assessed by results of pharmacological inhibition and siRNA transfection studies, the effect of NFJ on HIF-1α protein downregulation seemed to be largely associated with the ability of NFJ to interfere with the metal's signaling to activate PKB, ERK-1/2, JNK-1 and S6 in A549 cells. It was further shown that NFJ could repress the induction of HIF-1α protein by desferoxamine or interleukin-1β (IL-1β), another HIF-1α inducer in A549 cells. Thus, the present study provides the first evidence that NFJ has the ability to strongly downregulate manganese-induced HIF-1α protein expression in A549 human lung cancer cells, which may suggest the NFJ-mediated beneficial effects on lung pathologies in which manganese and HIF-1α overexpression play pathogenic roles.

  16. Organic compounds in tire particle induce reactive oxygen species and heat-shock proteins in the human alveolar cell line A549.

    PubMed

    Gualtieri, Maurizio; Mantecca, Paride; Cetta, Francesco; Camatini, Marina

    2008-05-01

    Debris produced from the attrition of tires of motor vehicles constitutes 5-7% of the atmospheric particulate matter (PM10). Debris particles are indeed small enough to enter human lung and thus morphological and chemical characterization has been performed. We demonstrated that the organic fraction of tire debris induces a dose-dependent increase in cell mortality, DNA damage, as well as a significant modification of cell morphology at the dose of 60 microg/ml, which may correspond to the quantity present in the air humans inhale daily. The present research aims at investigating if reactive oxygen species (ROS) production and Hsp70 expression are involved in the cascade of toxic effects produced on the A549 cell line, as it has been suggested for the ultrafine atmospheric particles and diesel exhaust. To this end, cells were exposed at the doses of 10, 50, 60, 75 microg/ml of TD organic extract (TDOE) and analyzed at different exposure time. ROS were detected by the oxidation of 2'7'-dichlorodihydrofluorescein diacetate to dichlorofluorescein, and fluorescence was measured by flow cytometry. Hsp70 protein expression was determined by immunochemical analysis, and protein expression quantification performed by optical densitometry. ROS production was analysed after 2 h of treatment. A statistically significant increase in fluorescence was observed and the intensity of the stress response was parallel to the increasing concentrations used. An evident increase of Hsp70 expression at lower doses (10, 50 microg/ml) and at longer exposure times (72 h) was observed, during the time that our previous studies showed that cell viability, plasma membrane integrity, and DNA molecules were not affected. Thus it can be deduced that the increase in Hsp70 expression protected the cells from those damages, which became evident at the higher doses, and that this parameter might be used as a sensitive indicator of exposure. These data suggest that ROS production may be the first

  17. Protein C inhibits endocytosis of thrombin-thrombomodulin complexes in A549 lung cancer cells and human umbilical vein endothelial cells

    SciTech Connect

    Maruyama, I.; Majerus, P.W.

    1987-05-01

    We investigated the effect of protein C on the endocytosis of thrombin-thrombomodulin complexes. We previously showed that exposure of umbilical vein endothelial cells to thrombin stimulated the internalization and degradation of thrombin. A similar internalization was stimulated by a monoclonal antithrombomodulin antibody. We have repeated these studies in the presence of protein C and found that endocytosis of /sup 125/I-thrombin-thrombomodulin complexes, but not /sup 125/I-antithrombomodulin-thrombomodulin complexes, is inhibited. Activated protein C did not inhibit endocytosis of thrombin-thrombomodulin complexes. Protein C inhibited both internalization and degradation of /sup 125/I-thrombin and diisopropylphosphoryl (DIP) /sup 125/I-thrombin in human lung cancer cells (A549). These effects were observed at protein C concentrations found in human plasma. Protein S had no effect on the inhibition of endocytosis of thrombin-thrombomodulin complexes by protein C. We propose that protein C may regulate the rate of endocytosis of thrombin-thrombomodulin complexes in vivo and thereby control the capacity for endothelium to activate protein C.

  18. Claudin-18 inhibits cell proliferation and motility mediated by inhibition of phosphorylation of PDK1 and Akt in human lung adenocarcinoma A549 cells.

    PubMed

    Shimobaba, Shun; Taga, Saeko; Akizuki, Risa; Hichino, Asami; Endo, Satoshi; Matsunaga, Toshiyuki; Watanabe, Ryo; Yamaguchi, Masahiko; Yamazaki, Yasuhiro; Sugatani, Junko; Ikari, Akira

    2016-06-01

    Abnormal expression of claudin subtypes has been reported in various cancers. However, the pathological role of each claudin has not been clarified in detail. Claudin-18 was absent in human non-small cell and small cell lung cancers, although it is expressed in normal lung tissues. Here, we examined the effect of claudin-18 expression on the expression of junctional proteins, cell proliferation, and cell motility using human lung adenocarcinoma A549 cells. Real-time PCR and western blotting showed that exogenous expression of claudin-18 had no effect on the expression of junctional proteins including claudin-1, zonula occludens-1 (ZO-1), occludin, and E-cadherin. Claudin-18 was mainly distributed in cell-cell contact areas concomitant with ZO-1. Cell proliferation was significantly decreased at 48 and 72h after seeding of claudin 18-expressing cells. Claudin-18 suppressed cell motility, whereas it increased cell death in anoikis. Claudin-18 decreased phosphorylated (p)-3-phosphoinositide-dependent protein kinase-1 (PDK1) and p-Akt levels without affecting p-epidermal growth factor receptor and p-phosphatidylinositol-3 kinase (PI3K) levels. Furthermore, claudin-18 was bound with PDK1 and suppressed the nuclear localization of PDK1. We suggest that claudin-18 suppresses the abnormal proliferation and motility of lung epithelial cells mediated by inhibition of the PI3K/PDK1/Akt signaling pathway.

  19. Combined treatment of curcumin and small molecule inhibitors suppresses proliferation of A549 and H1299 human non-small-cell lung cancer cells.

    PubMed

    Lin, Hui-Ping; Kuo, Li-Kuo; Chuu, Chih-Pin

    2012-01-01

    Curcumin (diferuloylmethane) is a phenolic compound present in turmeric and is ingested daily in many parts of the world. Curcumin has been reported to cause inhibition on proliferation and induction of apoptosis in many human cancer cell lines, including non-small cell lung cancer cells (NSCLC). However, the clinical application of curcumin is restricted by its low bioavailability. In this report, it was observed that combined treatment of a low dosage of curcumin (5-10 µM) with a low concentration (0.1-2.5 µM) of small molecule inhibitors, including AG1478, AG1024, PD173074, LY294002 and caffeic acid phenethyl ester (CAPE) increased the growth inhibition in two human NSCLC cell lines: A549 and H1299 cells. The observation suggested that combined treatment of a low dosage of curcumin with inhibitors against epidermal growth factor receptor (EGFR), insulin-like growth factor 1 (IGF-1R), fibroblast growth factors receptor (FGFR), phosphatidylinositol 3-kinases (PI3K) or NF-κB signaling pathway may be a potential adjuvant therapy beneficial to NSCLC patients.

  20. Human Lung Cancer Cell Line A-549 ATCC Is Differentially Affected by Supranutritional Organic and Inorganic Selenium

    PubMed Central

    Flores Villavicencio, Lérida Liss; Cruz-Jiménez, Gustavo; Barbosa-Sabanero, Gloria; Kornhauser-Araujo, Carlos; Mendoza-Garrido, M. Eugenia; de la Rosa, Guadalupe; Sabanero-López, Myrna

    2014-01-01

    The effects of organic and inorganic forms of selenium (Se) on human cells have been extensively studied for nutritional concentrations; however, to date, little is known about the potential toxicity at supranutritional levels. In the present study we determined the effects of sodium selenite (SSe) and selenomethionine (SeMet) on cell growth and intracellular structures in lung cancer cells exposed at Se concentrations between 0 and 3 mM. Our results showed that SSe affected cell growth more rapidly than SeMet (24 h and 48 h, resp.). After 24 h of cells exposure to 0.5, 1.5, and 3 mM SSe, cell growth was reduced by 10, 50, and 60%, as compared to controls. After 48 h, nuclear fragmentation was evident in cells exposed to SSe, suggesting an induction to cell death. In contrast, SeMet did not affect cell proliferation, and the cells were phenotypically similar to controls. Microtubules and microfilaments structures were also affected by both Se compounds, again SSe being more toxic than SeMet. To our knowledge, this is the first report on the differential effects of organic and inorganic Se in supranutritional levels in lung cancer cells. PMID:25477771

  1. Identification of a Short Cell-Penetrating Peptide from Bovine Lactoferricin for Intracellular Delivery of DNA in Human A549 Cells.

    PubMed

    Liu, Betty R; Huang, Yue-Wern; Aronstam, Robert S; Lee, Han-Jung

    2016-01-01

    Cell-penetrating peptides (CPPs) have been shown to deliver cargos, including protein, DNA, RNA, and nanomaterials, in fully active forms into live cells. Most of the CPP sequences in use today are based on non-native proteins that may be immunogenic. Here we demonstrate that the L5a CPP (RRWQW) from bovine lactoferricin (LFcin), stably and noncovalently complexed with plasmid DNA and prepared at an optimal nitrogen/phosphate ratio of 12, is able to efficiently enter into human lung cancer A549 cells. The L5a CPP delivered a plasmid containing the enhanced green fluorescent protein (EGFP) coding sequence that was subsequently expressed in cells, as revealed by real-time PCR and fluorescent microscopy at the mRNA and protein levels, respectively. Treatment with calcium chloride increased the level of gene expression, without affecting CPP-mediated transfection efficiency. Zeta-potential analysis revealed that positively electrostatic interactions of CPP/DNA complexes correlated with CPP-mediated transport. The L5a and L5a/DNA complexes were not cytotoxic. This biomimetic LFcin L5a represents one of the shortest effective CPPs and could be a promising lead peptide with less immunogenic for DNA delivery in gene therapy.

  2. Fabrication of nano-silver particles using Cymodocea serrulata and its cytotoxicity effect against human lung cancer A549 cells line

    NASA Astrophysics Data System (ADS)

    Palaniappan, P.; Sathishkumar, G.; Sankar, R.

    2015-03-01

    The present study reports, green synthesis of bioactive silver nanoparticles (AgNPs) under different temperature (60 °C, room temperature and 4° refrigerator) using the aqueous extract of sea grass Cymodocea serrulata as a potential bioreductant. Increased temperature fabricates more AgNPs compare to room temperature and refrigerator condition. At first the reduction of Ag+ ions were confirmed through color change which produces an absorbance spectra at 420 nm in UV-Visible spectrophotometer. Additionally various exclusive instrumentations such as X-ray diffraction (XRD), Dynamic light scattering (DLS), scanning electron microscope (SEM) analysis and Transmission electron microscope (TEM) were authorizes the biosynthesis and physio-chemical characterization of AgNPs. From Fourier transform infrared spectroscopy (FTIR) analysis, it was identified that the water soluble fractions of the sea grass mainly responsible for reduction of ionic silver (Ag+) into (Ag0) nano-ranged particles and also they act as stabilizing agent to sustain the durability of NPs for long period of time. Further, synthesized AgNPs shows potential cytotoxicity against human lung cancer A549 cells (LD50-100 μg/ml). The overall results suggest that C. serrulata is a valuable bioresource to generate rapid and eco-friendly bioactive AgNPs towards cancer therapy.

  3. Fabrication of nano-silver particles using Cymodocea serrulata and its cytotoxicity effect against human lung cancer A549 cells line.

    PubMed

    Palaniappan, P; Sathishkumar, G; Sankar, R

    2015-03-05

    The present study reports, green synthesis of bioactive silver nanoparticles (AgNPs) under different temperature (60°C, room temperature and 4° refrigerator) using the aqueous extract of sea grass Cymodocea serrulata as a potential bioreductant. Increased temperature fabricates more AgNPs compare to room temperature and refrigerator condition. At first the reduction of Ag(+) ions were confirmed through color change which produces an absorbance spectra at 420nm in UV-Visible spectrophotometer. Additionally various exclusive instrumentations such as X-ray diffraction (XRD), Dynamic light scattering (DLS), scanning electron microscope (SEM) analysis and Transmission electron microscope (TEM) were authorizes the biosynthesis and physio-chemical characterization of AgNPs. From Fourier transform infrared spectroscopy (FTIR) analysis, it was identified that the water soluble fractions of the sea grass mainly responsible for reduction of ionic silver (Ag(+)) into (Ag(0)) nano-ranged particles and also they act as stabilizing agent to sustain the durability of NPs for long period of time. Further, synthesized AgNPs shows potential cytotoxicity against human lung cancer A549 cells (LD50-100μg/ml). The overall results suggest that C. serrulata is a valuable bioresource to generate rapid and eco-friendly bioactive AgNPs towards cancer therapy.

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

    PubMed

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

    2011-05-01

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

  5. Evaluation of cytotoxic, oxidative stress, proinflammatory and genotoxic responses of micro- and nano-particles of dolomite on human lung epithelial cells A(549).

    PubMed

    Patil, Govil; Khan, Mohd Imran; Patel, Devendra Kumar; Sultana, Sarwat; Prasad, Rajendra; Ahmad, Iqbal

    2012-09-01

    Dolomite is a natural mineral of great industrial importance and used worldwide, thus millions of workers are at risk of occupational exposure. Its toxicity is however, meagerly documented. In the present investigation, a dolomite powder obtained from its milling unit was analyzed by some standard methods namely, optical microscopy, transmission electron microscopy and dynamic light scattering. Results showed that dolomite powder contained particles of different shapes and size both microparticles (MPs) and nanoparticles (NPs), suggesting potential occupational exposure of these particles. An attempt was therefore, made to investigate dolomite toxicity in a particle size-dependent manner in human lung epithelial cells A(549). The comparative toxicity evaluation of MPs and NPs was carried out by assessing their effects on cell viability, membrane damage, glutathione, reactive oxygen species (ROS), lipid peroxidation (LPO), micronucleus (MN) and proinflammatory cytokines, namely tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6). These markers of cytotoxicity, genotoxicity and inflammation were assayed in cells exposed to MPs and NPs in a dose-and time-dependent manner. Invariably, their toxic effects were dose-and time-dependent while NPs in general were significantly more toxic. Notably, NPs caused oxidative stress, genotoxicity and inflammatory responses, as seen by significant induction of ROS, LPO, MN, TNF-α, IL-1β and IL-6. Thus, the study tends to suggest that separate health safety standards would be required for micrometer and nanometer scale particles of dolomite.

  6. The environmental carcinogen 3-nitrobenzanthrone and its main metabolite 3-aminobenzanthrone enhance formation of reactive oxygen intermediates in human A549 lung epithelial cells.

    PubMed

    Hansen, Tanja; Seidel, Albrecht; Borlak, Jürgen

    2007-06-01

    The environmental contaminant 3-nitrobenzanthrone (3-NBA) is highly mutagenic and a suspected human carcinogen. We aimed to evaluate whether 3-NBA is able to deregulate critical steps in cell cycle control and apoptosis in human lung epithelial A549 cells. Increased intracellular Ca(2+) and caspase activities were detected upon 3-NBA exposure. As shown by cell cycle analysis, an increased number of S-phase cells was observed after 24 h of treatment with 3-NBA. Furthermore, 3-NBA was shown to inhibit cell proliferation when added to subconfluent cell cultures. The main metabolite of 3-NBA, 3-ABA, induced statistically significant increases in tail moment as judged by alkaline comet assay. The potential of 3-NBA and 3-ABA to enhance the production of reactive oxygen species (ROS) was demonstrated by flow cytometry using 2',7'-dichlorofluorescein-diacetate (DCFH-DA). The enzyme inhibitors allopurinol, dicumarol, resveratrol and SKF525A were used to assess the impact of metabolic conversion on 3-NBA-mediated ROS production. Resveratrol decreased dichlorofluorescein (DCF) fluorescence by 50%, suggesting a role for CYP1A1 in 3-NBA-mediated ROS production. Mitochondrial ROS production was significantly attenuated (20% reduction) by addition of rotenone (complex I inhibition) and thenoyltrifluoroacetone (TTFA, complex II inhibition). Taken together, the results of the present study provide evidence for a genotoxic potential of 3-ABA in human epithelial lung cells. Moreover, both compounds lead to increased intracellular ROS and create an environment favorable to DNA damage and the promotion of cancer.

  7. The environmental carcinogen 3-nitrobenzanthrone and its main metabolite 3-aminobenzanthrone enhance formation of reactive oxygen intermediates in human A549 lung epithelial cells

    SciTech Connect

    Hansen, Tanja . E-mail: tanja.hansen@item.fraunhofer.de; Seidel, Albrecht; Borlak, Juergen

    2007-06-01

    The environmental contaminant 3-nitrobenzanthrone (3-NBA) is highly mutagenic and a suspected human carcinogen. We aimed to evaluate whether 3-NBA is able to deregulate critical steps in cell cycle control and apoptosis in human lung epithelial A549 cells. Increased intracellular Ca{sup 2+} and caspase activities were detected upon 3-NBA exposure. As shown by cell cycle analysis, an increased number of S-phase cells was observed after 24 h of treatment with 3-NBA. Furthermore, 3-NBA was shown to inhibit cell proliferation when added to subconfluent cell cultures. The main metabolite of 3-NBA, 3-ABA, induced statistically significant increases in tail moment as judged by alkaline comet assay. The potential of 3-NBA and 3-ABA to enhance the production of reactive oxygen species (ROS) was demonstrated by flow cytometry using 2',7'-dichlorofluorescein-diacetate (DCFH-DA). The enzyme inhibitors allopurinol, dicumarol, resveratrol and SKF525A were used to assess the impact of metabolic conversion on 3-NBA-mediated ROS production. Resveratrol decreased dichlorofluorescein (DCF) fluorescence by 50%, suggesting a role for CYP1A1 in 3-NBA-mediated ROS production. Mitochondrial ROS production was significantly attenuated (20% reduction) by addition of rotenone (complex I inhibition) and thenoyltrifluoroacetone (TTFA, complex II inhibition). Taken together, the results of the present study provide evidence for a genotoxic potential of 3-ABA in human epithelial lung cells. Moreover, both compounds lead to increased intracellular ROS and create an environment favorable to DNA damage and the promotion of cancer.

  8. Mimulone-induced autophagy through p53-mediated AMPK/mTOR pathway increases caspase-mediated apoptotic cell death in A549 human lung cancer cells.

    PubMed

    An, Hyun-Kyu; Kim, Kyoung-Sook; Lee, Ji-Won; Park, Mi-Hyun; Moon, Hyung-In; Park, Shin-Ji; Baik, Ji-Sue; Kim, Cheorl-Ho; Lee, Young-Choon

    2014-01-01

    Anticancer properties and mechanisms of mimulone (MML), C-geranylflavonoid isolated from the Paulownia tomentosa fruits, were firstly elucidated in this study. MML prevented cell proliferation in a dose- and time-dependent way and triggered apoptosis through the extrinsic pathway in A549 human lung adenocarcinoma cells. Furthermore, MML-treated cells displayed autophagic features, such as the formation of autophagic vacuoles, a primary morphological feature of autophagy, and the accumulation of microtubule-associated protein 1 light chain 3 (LC3) puncta, another typical maker of autophagy, as determined by FITC-conjugated immunostaining and monodansylcadaverine (MDC) staining, respectively. The expression levels of LC3-I and LC3-II, specific markers of autophagy, were also augmented by MML treatment. Autophagy inhibition by 3-methyladenine (3-MA), pharmacological autophagy inhibitor, and shRNA knockdown of Beclin-1 reduced apoptotic cell death induced by MML. Autophagic flux was not significantly affected by MML treatment and lysosomal inhibitor, chloroquine (CQ) suppressed MML-induced autophagy and apoptosis. MML-induced autophagy was promoted by decreases in p53 and p-mTOR levels and increase of p-AMPK. Moreover, inhibition of p53 transactivation by pifithrin-α (PFT-α) and knockdown of p53 enhanced induction of autophagy and finally promoted apoptotic cell death. Overall, the results demonstrate that autophagy contributes to the cytotoxicity of MML in cancer cells harboring wild-type p53. This study strongly suggests that MML is a potential candidate for an anticancer agent targeting both autophagy and apoptotic cell death in human lung cancer. Moreover, co-treatment of MML and p53 inhibitor would be more effective in human lung cancer therapy.

  9. Mimulone-Induced Autophagy through p53-Mediated AMPK/mTOR Pathway Increases Caspase-Mediated Apoptotic Cell Death in A549 Human Lung Cancer Cells

    PubMed Central

    Lee, Ji-Won; Park, Mi-Hyun; Moon, Hyung-In; Park, Shin-Ji; Baik, Ji-Sue; Kim, Cheorl-Ho; Lee, Young-Choon

    2014-01-01

    Anticancer properties and mechanisms of mimulone (MML), C-geranylflavonoid isolated from the Paulownia tomentosa fruits, were firstly elucidated in this study. MML prevented cell proliferation in a dose- and time-dependent way and triggered apoptosis through the extrinsic pathway in A549 human lung adenocarcinoma cells. Furthermore, MML-treated cells displayed autophagic features, such as the formation of autophagic vacuoles, a primary morphological feature of autophagy, and the accumulation of microtubule-associated protein 1 light chain 3 (LC3) puncta, another typical maker of autophagy, as determined by FITC-conjugated immunostaining and monodansylcadaverine (MDC) staining, respectively. The expression levels of LC3-I and LC3-II, specific markers of autophagy, were also augmented by MML treatment. Autophagy inhibition by 3-methyladenine (3-MA), pharmacological autophagy inhibitor, and shRNA knockdown of Beclin-1 reduced apoptotic cell death induced by MML. Autophagic flux was not significantly affected by MML treatment and lysosomal inhibitor, chloroquine (CQ) suppressed MML-induced autophagy and apoptosis. MML-induced autophagy was promoted by decreases in p53 and p-mTOR levels and increase of p-AMPK. Moreover, inhibition of p53 transactivation by pifithrin-α (PFT-α) and knockdown of p53 enhanced induction of autophagy and finally promoted apoptotic cell death. Overall, the results demonstrate that autophagy contributes to the cytotoxicity of MML in cancer cells harboring wild-type p53. This study strongly suggests that MML is a potential candidate for an anticancer agent targeting both autophagy and apoptotic cell death in human lung cancer. Moreover, co-treatment of MML and p53 inhibitor would be more effective in human lung cancer therapy. PMID:25490748

  10. Oxidative damage to DNA and repair induced by Norwegian wood smoke particles in human A549 and THP-1 cell lines.

    PubMed

    Danielsen, Pernille Høgh; Loft, Steffen; Kocbach, Anette; Schwarze, Per E; Møller, Peter

    2009-03-31

    Genotoxic effects of traffic-generated particulate matter (PM) are well described, whereas little data are available on PM from combustion of biomass and wood, which contributes substantially to air pollution world wide. The aim of this study was to compare the genotoxicity of wood smoke particulate matter (WSPM), authentic traffic-generated particles, mineral PM and standard reference material (SRM2975) of diesel exhaust particles in human A549 lung epithelial and THP-1 monocytic cell lines. DNA damage was measured as strand breaks (SB) and formamidopyrimidine DNA glycosylase (FPG) sites by the comet assay, whereas cell cytotoxicity was determined as lactate dehydrogenase release. The exposure to WSPM generated SB and FPG sites in both cell lines at concentrations from 2.5 or 25 microg/ml, which were not cytotoxic. Compared to all other studied particles, WSPM generated greater responses in terms of both SB and FPG sites. Organic extracts of WSPM and SRM2975 elicited higher levels of SB than native and washed PM at 25 and 100 microg/ml, whereas assay saturation precluded reliable assessment of FPG sites. During a 6h post-exposure period, in which the medium with PM had been replaced by fresh medium, 60% of the DNA lesions generated by WSPM were removed. In conclusion, WSPM generated more DNA damage than traffic-generated PM per unit mass in human cell lines, possibly due to the high level of polycyclic aromatic hydrocarbons in WSPM. This suggests that exposure to WSPM might be more hazardous than PM collected from vehicle exhaust with respect to development of lung cancer.

  11. Upregulation of AQP3 and AQP5 induced by dexamethasone and ambroxol in A549 cells.

    PubMed

    Ben, Yong; Chen, Jie; Zhu, Rong; Gao, Lei; Bai, Chunxue

    2008-04-30

    Aquaporins (AQPs) are membrane channel proteins that play roles in the regulation of water permeability in many tissues. AQP1 and AQP5 expressed in lung provide the principal route for osmotically driven water transport. In the airways, AQP3 and AQP4 facilitate water transport. Dexamethasone and ambroxol are often used to treat patients with pulmonary diseases accompanied by airway hypersecretion. The role of AQPs in these effective treatments has not been addressed. In this study, we analyzed the expression of AQPs in a human airway epithelial cell line (A549 cells) and showed that AQP3 and 5, but not AQP1 and 4, were expressed in A549 cells. Both dexamethasone and ambroxol stimulated the expression of AQP3 and 5 at the mRNA and protein levels. The data suggest potential roles of AQP3 and 5 in the regulation of airway hypersecretion, perhaps ultimately providing a target for treating such diseases.

  12. Nimesulide, a selective COX-2 inhibitor, acts synergistically with ionizing radiation against A549 human lung cancer cells through the activation of caspase-8 and caspase-3.

    PubMed

    Kim, Byeong Mo; Won, Juyoon; Maeng, Kyung Ah; Han, Young Soo; Yun, Yeon-Sook; Hong, Sung Hee

    2009-05-01

    Several lines of evidence suggest that non-steroidal anti-inflammatory drugs (NSAIDs) have a radiosensitizing effect on cancer cells in vitro and in vivo, but little is known about the underlying cellular mechanism. In this study, we found that the treatment with the NSAID nimesulide significantly increased the sensitivity of A549 human non-small cell lung cancer cells to radiotherapy. The combined nimesulide-radiation treatment increased apoptosis, induced the cleavage of caspase-3, caspase-9, and poly(ADP-ribose) polymerase (PARP), activated caspase-8, and induced cleavage of Bid. A pan-caspase inhibitor, z-VAD-fmk, suppressed this increase in apoptosis and also suppressed the cleavage of caspase-8, caspase-3, and PARP, suggesting a caspase-dependent mechanism. In addition, z-IETD-fmk, a selective caspase-8 inhibitor, suppressed the nimesulide- and radiation-induced cleavage activation of caspase-9, caspase-3, caspase-8, and Bid, and suppressed the concomitant apoptosis, indicating that the nimesulide-induced increase in radiosensitivity was initiated by caspase-8. However, the caspase-3 inhibitor z-DEVD-fmk failed to suppress activation of the caspase-8/Bid pathway, indicating that caspase-3 activation occurred downstream of caspase-8 activation in our experiments. Marked antitumor effects, which were evaluated by measuring protracted tumor regression, were observed when nude mice were treated with a combination of nimesulide at a clinically achievable dose (0.5 mg/kg) and radiation therapy. Our results, demonstrating the radiosensitivity-increasing and tumor growth-inhibiting effects of nimesulide, suggest that nimesulide may be suitable as an adjuvant to enhance the efficacy and selectivity of radiotherapy.

  13. Alpha-chaconine-reduced metastasis involves a PI3K/Akt signaling pathway with downregulation of NF-kappaB in human lung adenocarcinoma A549 cells.

    PubMed

    Shih, Yuan-Wei; Chen, Pin-Shern; Wu, Cheng-Hsun; Jeng, Ya-Fang; Wang, Chau-Jong

    2007-12-26

    Alpha-chaconine, isolated from Solanum tuberosum Linn., is a naturally occurring steroidal glycoalkaloid in potato sprouts. Some reports demonstrated that alpha-chaconine had various anticarcinogenic properties. The aim of this study is to investigate the inhibitory effect of alpha-chaconine on lung adenocarcinoma cell metastasis in vitro. We chose the highly metastatic A549 cells, which were treated with various concentrations of alpha-chaconine to clarify the potential of inhibiting A549 cells invasion and migration. Data showed that alpha-chaconine inhibited A549 cell invasion/migration according to wound healing assay and Boyden chamber assay. Our results also showed that alpha-chaconine could inhibit phosphorylation of c-Jun N-terminal kinase (JNK) and Akt, whereas it did not affected phosphorylation of extracellular signal regulating kinase (ERK) and p38. In addition, alpha-chaconine significantly decreased the nuclear level of nuclear factor kappa B (NF-kappaB) and the binding ability of NF-kappaB. These results suggested that alpha-chaconine inhibited A549 cell metastasis by a reduction of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) activities involving suppression of phosphoinositide 3-kinase/Akt/NF-kappaB (PI3K/Akt/NF-kappaB) signaling pathway. Inhibiting metastasis by alpha-chaconine might offer a pivotal mechanism for its effective chemotherapeutic action.

  14. Comparison of wood smoke PM2.5 obtained from the combustion of FIR and beech pellets on inflammation and DNA damage in A549 and THP-1 human cell lines.

    PubMed

    Corsini, Emanuela; Budello, Silvia; Marabini, Laura; Galbiati, Valentina; Piazzalunga, Andrea; Barbieri, Pierluigi; Cozzutto, Sergio; Marinovich, Marina; Pitea, Demetrio; Galli, Corrado L

    2013-12-01

    The aim of this study was to investigate the effect on the induction of interleukin-8 of particulate matter (PM) from fir and beech pellets burnt in domestic appliances on two human cells lines, namely the lung epithelial cell line A549 and the promyelocytic cell line THP-1. The effects of PM2.5 obtained from combustion of beech and fir pellets were compared to reference diesel exhaust particulates (DEP). In parallel, wood smoke PM-induced genotoxicity and oxidative stress were also investigated in A549 cells. Cells were treated for different times (3-72 h) with increasing concentrations of PM2.5 obtained from sequential combustions of fir and beech pellets or reference DEP. Cell viability was assessed by lactate dehydrogenase leakage, and the release of interleukin-8 or CXCL8 (IL-8) was measured to evaluate the pro-inflammatory effect. Oxidative stress was evaluated by the 5(6)-carboxy-2',7'dichlorofluorescein diacetate (DCFH-DA) assay and DNA damage by the alkaline comet assay and micronucleus frequency by flow cytometry. Both A549 and THP-1 cells responded in a dose- and time-related manner to wood smoke PM2.5 with IL-8 release, particles obtained from late combustions being the most active. THP-1 cells were more sensitive than A549 cells. On a mass base, similar effects were observed for both fir and beech PM2.5. However, the combustion of beech pellets generated approximately three times more PM2.5 than fir pellets. Regarding the mechanism of PM2.5 uptake, in both THP-1 and A549 cells, cytochalasin D prevented PM2.5-induced IL-8 mRNA expression and cytokine release, indicating a key role for actin polymerization in particles uptake and that the production of IL-8 correlated with particle phagocytosis. As signal transduction pathway involvement, in both THP-1 and A549 cells, PM2.5-induced IL-8 release could be completely blocked by the selective inhibitor SB203580, indicating a role of p38 MAPK activation. PM2.5 from both fir and beech pellets also induced

  15. Digoxin downregulates NDRG1 and VEGF through the inhibition of HIF-1α under hypoxic conditions in human lung adenocarcinoma A549 cells.

    PubMed

    Wei, Dong; Peng, Jing-Jing; Gao, Hui; Li, Hua; Li, Dong; Tan, Yong; Zhang, Tao

    2013-04-02

    Digoxin, an inhibitor of Na+/K+ ATPase, has been used in the treatment of heart-related diseases (such as congestive heart failure and atrial arrhythmia) for decades. Recently, it was reported that digoxin is also an effective HIF-1α inhibitor. We investigated whether digoxin could suppress tumor cell growth through HIF-1α in non-small cell lung cancer cells (A549 cells) under hypoxic conditions. An MTT assay was used to measure cell viability. RT-PCR and western blotting were performed to analyze the mRNA and protein expression of VEGF, NDRG1, and HIF-1α. HIF-1α nuclear translocation was then determined by EMSA. Digoxin was found to inhibit the proliferation of A549 cells under hypoxic conditions. Our results showed that hypoxia led to the upregulation of VEGF, NDRG1, and HIF-1α both at the mRNA and protein levels. We also found that the hypoxia-induced overexpression of VEGF, NDRG1, and HIF-1α was suppressed by digoxin in a concentration-dependent manner. As expected, our EMSA results demonstrated that under hypoxic conditions HIF-1α nuclear translocation was also markedly reduced by digoxin in a concentration-dependent manner. Our results suggest that digoxin downregulated hypoxia-induced overexpression of VEGF and NDRG1 at the transcriptional level probably through the inhibition of HIF-1α synthesis in A549 cells.

  16. Impairment of cell cycle progression by sterigmatocystin in human pulmonary cells in vitro.

    PubMed

    Huang, Shujuan; Wang, Juan; Xing, Lingxiao; Shen, Haitao; Yan, Xia; Wang, Junling; Zhang, Xianghong

    2014-04-01

    Sterigmatocystin (ST) is a carcinogenic mycotoxin that is commonly found in human food, animal feed and in the indoor environment. Although the correlation between ST exposure and lung cancer has been widely reported in many studies, the cytotoxicity of ST on human pulmonary cells is not yet fully understood. In the current study, we found that ST could induce DNA double-strand breaks in a human immortalized bronchial epithelial cell line (BEAS-2B cells) and a human lung cancer cell line (A549 cells). In addition, the effects of ST on cell cycle arrest were complex and dependent on the tested ST concentration and cell type. Low concentrations of ST arrested cells in the G2/M phase in BEAS-2B cells and in the S phase in A549 cells, while at high concentration both cells lines were arrested in S and G2/M phases. Furthermore, we observed that the modulation of cyclins and CDK expression showed concomitant changes with cell cycle arrest upon ST exposure in BEAS-2B and A549 cells. In conclusion, ST induced DNA damage and affected key proteins involved in cell cycle regulation to trigger genomic instability, which may be a potential mechanism underlying the developmental basis of lung carcinogenesis.

  17. Regulation of different components from Ophiopogon japonicus on autophagy in human lung adenocarcinoma A549Cells through PI3K/Akt/mTOR signaling pathway.

    PubMed

    Chen, Juan; Yuan, Jiarui; Zhou, Liqiang; Zhu, Maomao; Shi, Ziqi; Song, Jie; Xu, Qingyu; Yin, Guowen; Lv, You; Luo, Yi; Jia, Xiaobin; Feng, Liang

    2017-03-01

    Autophagy plays a dual role in the development of cancer, acting as both a tumor suppressor and a cell survival inducer. Ophiopogon japonicus (L.f) Ker-Gawl (OJ), as a traditional Chinese medicine, specially possesses remarkable anti-cancer activity in the clinical. Previously, studies have indicated that flavonoids (FOJ) and steroidal saponins (SSOJ) are the main active substances of OJ. However, the effects of FOJ and SSOJ on autophagy of A549 cells have not been fully elucidated. In this study, we found that the expressions of autophagy-related mediators (LC3-II/LC3-I ratio, Atg-3, Atg-7 and Beclin-1) were increased in A549 cells by the treatment with FOJ (7.9mg crude drug/mL) and SSOJ (12.2mg crude drug/mL). Meanwhile, FOJ or SSOJ could induce the up-regulation of LC3-II at both protein and mRNA levels. Moreover, we observed the cytoplasmic vaculoes which formed double-layered membranes and only some cytoplasmic organelles or myelin figures remained in FOJ or SSOJ-treated A549 cells for 24h by Transmission Electron Microscopy (TEM). Further detection about the PI3K/Akt/mTOR signaling pathway showed that the levels of PI3K, Akt and mTOR were significantly suppressed with the FOJ or SSOJ treatment. The 3-MA (an autophagy inhibitor) and LY294002 (a PI3K inhibitor) further confirmed the underlying mechanism in the FOJ or SSOJ-induced autophagy of A549 cells. Additionally, the pretreatment with FOJ and SSOJ increased the level of p53, whereas decreased the expression of Ki67. These findings suggested that FOJ or SSOJ could activate the autophagy of A549 cells, wherein the mechanism might be associated with their inhibition of PI3K/Akt/mTOR signaling pathway. Thus, FOJ or SSOJ could be a potential autophagy inducer to prevent the process of lung cancer.

  18. Drug Transporter Protein Quantification of Immortalized Human Lung Cell Lines Derived from Tracheobronchial Epithelial Cells (Calu-3 and BEAS2-B), Bronchiolar-Alveolar Cells (NCI-H292 and NCI-H441), and Alveolar Type II-like Cells (A549) by Liquid Chromatography-Tandem Mass Spectrometry.

    PubMed

    Sakamoto, Atsushi; Matsumaru, Takehisa; Yamamura, Norio; Suzuki, Shinobu; Uchida, Yasuo; Tachikawa, Masanori; Terasaki, Tetsuya

    2015-09-01

    Understanding the mechanisms of drug transport in the human lung is an important issue in pulmonary drug discovery and development. For this purpose, there is an increasing interest in immortalized lung cell lines as alternatives to primary cultured lung cells. We recently reported the protein expression in human lung tissues and pulmonary epithelial cells in primary culture, (Sakamoto A, Matsumaru T, Yamamura N, Uchida Y, Tachikawa M, Ohtsuki S, Terasaki T. 2013. J Pharm Sci 102(9):3395-3406) whereas comprehensive quantification of protein expressions in immortalized lung cell lines is sparse. Therefore, the aim of the present study was to clarify the drug transporter protein expression of five commercially available immortalized lung cell lines derived from tracheobronchial cells (Calu-3 and BEAS2-B), bronchiolar-alveolar cells (NCI-H292 and NCI-H441), and alveolar type II cells (A549), by liquid chromatography-tandem mass spectrometry-based approaches. Among transporters detected, breast cancer-resistance protein in Calu-3, NCI-H292, NCI-H441, and A549 and OCTN2 in BEAS2-B showed the highest protein expression. Compared with data from our previous study,(Sakamoto A, Matsumaru T, Yamamura N, Uchida Y, Tachikawa M, Ohtsuki S, Terasaki T. 2013. J Pharm Sci 102(9):3395-3406) NCI-H441 was the most similar with primary lung cells from all regions in terms of protein expression of organic cation/carnitine transporter 1 (OCTN1). In conclusion, the protein expression profiles of transporters in five immortalized lung cell lines were determined, and these findings may contribute to a better understanding of drug transport in immortalized lung cell lines.

  19. Inhibitory Effects of Salinomycin on Cell Survival, Colony Growth, Migration, and Invasion of Human Non-Small Cell Lung Cancer A549 and LNM35: Involvement of NAG-1.

    PubMed

    Arafat, Kholoud; Iratni, Rabah; Takahashi, Takashi; Parekh, Khatija; Al Dhaheri, Yusra; Adrian, Thomas E; Attoub, Samir

    2013-01-01

    A major challenge for oncologists and pharmacologists is to develop more potent and less toxic drugs that will decrease the tumor growth and improve the survival of lung cancer patients. Salinomycin is a polyether antibiotic used to kill gram-positive bacteria including mycobacteria, protozoans such as plasmodium falciparum, and the parasites responsible for the poultry disease coccidiosis. This old agent is now a serious anti-cancer drug candidate that selectively inhibits the growth of cancer stem cells. We investigated the impact of salinomycin on survival, colony growth, migration and invasion of the differentiated human non-small cell lung cancer lines LNM35 and A549. Salinomycin caused concentration- and time-dependent reduction in viability of LNM35 and A549 cells through a caspase 3/7-associated cell death pathway. Similarly, salinomycin (2.5-5 µM for 7 days) significantly decreased the growth of LNM35 and A549 colonies in soft agar. Metastasis is the main cause of death related to lung cancer. In this context, salinomycin induced a time- and concentration-dependent inhibition of cell migration and invasion. We also demonstrated for the first time that salinomycin induced a marked increase in the expression of the pro-apoptotic protein NAG-1 leading to the inhibition of lung cancer cell invasion but not cell survival. These findings identify salinomycin as a promising novel therapeutic agent for lung cancer.

  20. Andrographolide down-regulates hypoxia-inducible factor-1α in human non-small cell lung cancer A549 cells.

    PubMed

    Lin, Hui-Hsuan; Tsai, Chia-Wen; Chou, Fen-Pi; Wang, Chau-Jong; Hsuan, Shu-Wen; Wang, Cheng-Kun; Chen, Jing-Hsien

    2011-02-01

    Andrographolide (Andro), a diterpenoid lactone isolated from a traditional herbal medicine Andrographis paniculata, is known to possess multiple pharmacological activities. In our previous study, Andro had been shown to inhibit non-small cell lung cancer (NSCLC) A549 cell migration and invasion via down-regulation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Here we demonstrated that Andro inhibited the expression of hypoxia-inducible factor-1α (HIF-1α) in A549 cells. HIF-1α plays an important role in tumor growth, angiogenesis and lymph node metastasis of NSCLC. The Andro-induced decrease of cellular protein level of HIF-1α was correlated with a rapid ubiquitin-dependent degradation of HIF-1α, and was accompanied by increased expressions of hydroxyl-HIF-1α and prolyl hydroxylase (PHD2), and a later decrease of vascular endothelial growth factor (VEGF) upon the treatment of Andro. The Andro-inhibited VEGF expression appeared to be a consequence of HIF-1α inactivation, because its DNA binding activity was suppressed by Andro. Molecular data showed that all these effects of Andro might be mediated via TGFβ1/PHD2/HIF-1α pathway, as demonstrated by the transfection of TGFβ1 overexpression vector and PHD2 siRNA, and the usage of a pharmacological MG132 inhibitor. Furthermore, we elucidated the involvement of Andro in HIF-1α transduced VEGF expression in A549 cells and other NSCLC cell lines. In conclusion, these results highlighted the potential effects of Andro, which may be developed as a chemotherapeutic or an anti-angiogenesis agent for NSCLC in the future.

  1. Andrographolide down-regulates hypoxia-inducible factor-1{alpha} in human non-small cell lung cancer A549 cells

    SciTech Connect

    Lin, Hui-Hsuan; Tsai, Chia-Wen; Chou, Fen-Pi; Wang, Chau-Jong; Hsuan, Shu-Wen; Wang, Cheng-Kun; Chen, Jing-Hsien

    2011-02-01

    Andrographolide (Andro), a diterpenoid lactone isolated from a traditional herbal medicine Andrographis paniculata, is known to possess multiple pharmacological activities. In our previous study, Andro had been shown to inhibit non-small cell lung cancer (NSCLC) A549 cell migration and invasion via down-regulation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Here we demonstrated that Andro inhibited the expression of hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) in A549 cells. HIF-1{alpha} plays an important role in tumor growth, angiogenesis and lymph node metastasis of NSCLC. The Andro-induced decrease of cellular protein level of HIF-1{alpha} was correlated with a rapid ubiquitin-dependent degradation of HIF-1{alpha}, and was accompanied by increased expressions of hydroxyl-HIF-1{alpha} and prolyl hydroxylase (PHD2), and a later decrease of vascular endothelial growth factor (VEGF) upon the treatment of Andro. The Andro-inhibited VEGF expression appeared to be a consequence of HIF-1{alpha} inactivation, because its DNA binding activity was suppressed by Andro. Molecular data showed that all these effects of Andro might be mediated via TGF{beta}1/PHD2/HIF-1{alpha} pathway, as demonstrated by the transfection of TGF{beta}1 overexpression vector and PHD2 siRNA, and the usage of a pharmacological MG132 inhibitor. Furthermore, we elucidated the involvement of Andro in HIF-1{alpha} transduced VEGF expression in A549 cells and other NSCLC cell lines. In conclusion, these results highlighted the potential effects of Andro, which may be developed as a chemotherapeutic or an anti-angiogenesis agent for NSCLC in the future.

  2. A polysaccharide fraction of adlay seed (Coixlachryma-jobi L.) induces apoptosis in human non-small cell lung cancer A549 cells

    SciTech Connect

    Lu, Xiangyi; Liu, Wei; Wu, Junhua; Li, Mengxian; Wang, Juncheng; Wu, Jihui; Luo, Cheng

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer A polysaccharide from adlay seed, its molecular mass, optical rotation and sugars was determined. Black-Right-Pointing-Pointer We demonstrated that a polysaccharide from adlay can induce apoptosis in cancer cells. Black-Right-Pointing-Pointer The polysaccharide inhibited the metabolism and proliferation of NSCLC A549 cells. Black-Right-Pointing-Pointer The polysaccharide may trigger apoptosis via the mitochondria-dependent pathway. -- Abstract: Different seed extracts from Coix lachryma-jobi (adlay seed) have been used for the treatment of various cancers in China, and clinical data support the use of these extracts for cancer therapy; however, their underlying molecular mechanisms have not been well defined. A polysaccharide fraction, designated as CP-1, was extracted from the C.lachryma-jobi L. var. using the ethanol subsiding method. CP-1 induced apoptosis in A549 cells in a dose-dependent manner, as determined by MTT assay. Apoptotic bodies were observed in the cells by scanning electronic microscopy. Apoptosis and DNA accumulation during S-phase of the cell cycle were determined by annexin V-FITC and PI staining, respectively, and measured by flow cytometry. CP-1 also extended the comet tail length on single cell gel electrophoresis, and disrupted the mitochondrial membrane potential. Further analysis by western blotting showed that the expression of caspase-3 and caspase-9 proteins was increased. Taken together, our results demonstrate that CP-1 is capable of inhibiting A549 cell proliferation and inducing apoptosis via a mechanism primarily involving the activation of the intrinsic mitochondrial pathway. The assay data suggest that in addition to its nutritional properties, CP-1 is a very promising candidate polysaccharide for the development of anti-cancer medicines.

  3. A polysaccharide fraction of adlay seed (Coixlachryma-jobi L.) induces apoptosis in human non-small cell lung cancer A549 cells.

    PubMed

    Lu, Xiangyi; Liu, Wei; Wu, Junhua; Li, Mengxian; Wang, Juncheng; Wu, Jihui; Luo, Cheng

    2013-01-11

    Different seed extracts from Coix lachryma-jobi (adlay seed) have been used for the treatment of various cancers in China, and clinical data support the use of these extracts for cancer therapy; however, their underlying molecular mechanisms have not been well defined. A polysaccharide fraction, designated as CP-1, was extracted from the C.lachryma-jobi L. var. using the ethanol subsiding method. CP-1 induced apoptosis in A549 cells in a dose-dependent manner, as determined by MTT assay. Apoptotic bodies were observed in the cells by scanning electronic microscopy. Apoptosis and DNA accumulation during S-phase of the cell cycle were determined by annexin V-FITC and PI staining, respectively, and measured by flow cytometry. CP-1 also extended the comet tail length on single cell gel electrophoresis, and disrupted the mitochondrial membrane potential. Further analysis by western blotting showed that the expression of caspase-3 and caspase-9 proteins was increased. Taken together, our results demonstrate that CP-1 is capable of inhibiting A549 cell proliferation and inducing apoptosis via a mechanism primarily involving the activation of the intrinsic mitochondrial pathway. The assay data suggest that in addition to its nutritional properties, CP-1 is a very promising candidate polysaccharide for the development of anti-cancer medicines.

  4. Rubus idaeus L Inhibits Invasion Potential of Human A549 Lung Cancer Cells by Suppression Epithelial-to-Mesenchymal Transition and Akt Pathway In Vitro and Reduces Tumor Growth In Vivo.

    PubMed

    Chu, Shu-Chen; Hsieh, Yih-Shou; Hsu, Li-Sung; Chen, Kuo-Shuen; Chiang, Chien-Cheng; Chen, Pei-Ni

    2014-05-01

    The metastasis of lung cancer is the most prevalent cause of patient death. Various treatment strategies have targeted the prevention of the occurrence of metastasis. The epithelial-mesenchymal transition (EMT) in lung cancer cells is considered a prerequisite to acquire the invasive/migratory phenotype and to subsequently achieve metastasis. However, the effects ofRubus idaeuson cancer invasion and the EMT of the human lung carcinoma remain unclear. In this article, we test the hypothesis thatR idaeusethyl acetate (RIAE) possesses an antimetastatic effect and reverses the EMT potential of human lung A549 cells. We extract the raspberryR idaeuswith methanol (RIME), chloroform (RICE), ethyl acetate (RIAE),n-butanol (RIBE), and water (RIWE). The RIAE treatment obviously inhibits the invasive (P< .001), motility (P< .001), spreading, and migratory potential (P< .001) of highly metastatic human lung cancer A549 cells. The zymography and promoter luciferase analysis reveals that RIAE decreases the proteinase and transcription activities of MMP-2 and u-PA. Molecular analyses show that RIAE increases the E-cadherin level that is mainly localized at the cellular membrane. This result was also verified through confocal analyses. RIAE also induces the upregulation of an epithelial marker, such as α-catenin, and decreases mesenchymal markers, such as snail-1 and N-cadherin, that promote cell invasion and metastasis. RIAE inhibits MMP-2 and u-PA by attenuating the NF-κB and p-Akt expression. The inhibition of RIAE on the growth of A549 cells in vivo was also verified using a cancer cell xenograft nude mice model. Our results show the anti-invasive/antitumor effects of RIAE and associated mechanisms, which suggest that RIAE should be further tested in clinically relevant models to exploit its potential benefits against metastatic lung cancer cells.

  5. Adverse effects of industrial multiwalled carbon nanotubes on human pulmonary cells

    PubMed Central

    Tabet, Lyes; Bussy, Cyrill; Amara, Nadia; Setyan, Ari; Grodet, Alain; Rossi, Michel J.; Pairon, Jean-Claude; Boczkowski, Jorge; Lanone, Sophie

    2009-01-01

    The aim of this study was to evaluate adverse effects of multi-walled carbon nanotubes (MWCNT) produced for industrial purposes, on the human epithelial cell line A549. MWCNT were dispersed in dipalmitoyl lecithin (DPL), a component of pulmonary surfactant, and the effects of dispersion in DPL were compared to those in 2 other media: ethanol (EtOH) and phosphate buffer saline (PBS). Effects of MWCNT were also compared to those of 2 asbestos fibers (chrysotile and crocidolite) and carbon black (CB) nanoparticles, not only in A549 cells, but also on mesothelial cells (MeT5A human cell line), used as an asbestos-sensitive cell type. MWCNT formed agglomerates on top of both cell lines (surface area 15–35 μm2), that were significantly larger and more numerous in PBS than in EtOH and DPL. Whatever the dispersion media, incubation with 100 μg/ml MWCNT induced a similar decrease in metabolic activity without changing cell membrane permeability or apoptosis. Neither MWCNT cellular internalization nor oxidative stress were observed. In contrast, asbestos fibers penetrated into the cells, decreased metabolic activity but not cell membrane permeability and increased apoptosis, without decreasing cell number. CB was internalized without any adverse effects. In conclusion, this study demonstrates that MWCNT produced for industrial purposes exert adverse effects without being internalized by human epithelial and mesothelial pulmonary cell lines. PMID:19034795

  6. Vitamin A (retinol) downregulates the receptor for advanced glycation endproducts (RAGE) by oxidant-dependent activation of p38 MAPK and NF-kB in human lung cancer A549 cells.

    PubMed

    de Bittencourt Pasquali, Matheus Augusto; Gelain, Daniel Pens; Zeidán-Chuliá, Fares; Pires, André Simões; Gasparotto, Juciano; Terra, Silvia Resende; Moreira, José Cláudio Fonseca

    2013-04-01

    As an essential component of the diet, retinol supplementation is often considered harmless and its application is poorly controlled. However, recent works demonstrated that retinol may induce a wide array of deleterious effects, especially when doses used are elevated. Controlled clinical trials have demonstrated that retinol supplementation increased the incidence of lung cancer and mortality in smokers. Experimental works in cell cultures and animal models showed that retinol may induce free radical production, oxidative stress and extensive biomolecular damage. Here, we evaluated the effect of retinol on the regulation of the receptor for advanced glycation end-products (RAGE) in the human lung cancer cell line A549. RAGE is constitutively expressed in lungs and was observed to be down-regulated in lung cancer patients. A549 cells were treated with retinol doses reported as physiologic (2 μM) or therapeutic (5, 10 or 20 μM). Retinol at 10 and 20 μM increased free radical production, oxidative damage and antioxidant enzyme activity in A549 cells. These doses also downregulated RAGE expression. Antioxidant co-treatment with Trolox®, a hydrophilic analog of α-tocopherol, reversed the effects of retinol on oxidative parameters and RAGE downregulation. The effect of retinol on RAGE was mediated by p38 MAPK activation, as blockade of p38 with PD169316 (10 μM), SB203580 (10 μM) or siRNA to either p38α (MAPK14) or p38β (MAPK11) reversed the effect of retinol on RAGE. Trolox also inhibited p38 phosphorylation, indicating that retinol induced a redox-dependent activation of this MAPK. Besides, we observed that NF-kB acted as a downstream effector of p38 in RAGE downregulation by retinol, as NF-kB inhibition by SN50 (100 μg/mL) and siRNA to p65 blocked the effect of retinol on RAGE, and p38 inhibitors reversed NF-kB activation. Taken together, our results indicate a pro-oxidant effect of retinol on A549 cells, and suggest that modulation of RAGE expression by

  7. Green tea induces annexin-I expression in human lung adenocarcinoma A549 cells: involvement of annexin-I in actin remodeling.

    PubMed

    Lu, Qing-Yi; Jin, Yu Sheng; Zhang, Zuo-Feng; Le, Anh D; Heber, David; Li, Frederick P; Dubinett, Steven M; Rao, Jian Yu

    2007-05-01

    Green tea polyphenols exhibit multiple antitumor activities in various in vitro and in vivo tumor models, and the mechanisms of action are not clear. Previously, we found that green tea extract (GTE) regulates actin remodeling in different cell culture systems. Actin remodeling plays an important role in cancer cell morphology, cell adhesion, motility, and invasion. Using proteomic approaches, we found GTE-induced expression of annexin-I, a multifunctional actin binding protein, in these cell lines. In this study, we aimed to further define the functional role of GTE-induced annexin-I expression in actin remodeling, cell adhesion, and motility in lung adenocarcinoma A549 cells. We found that GTE stimulates the expression of annexin-I in a dose-dependent fashion. The GTE-induced annexin-I expression appears to be at the transcription level, and the increased annexin-I expression mediates actin polymerization, resulting in enhanced cell adhesion and decreased motility. Annexin-I specific interference resulted in loss of GTE-induced actin polymerization and cell adhesion, but not motility. In fact, annexin-I specific interference itself inhibited motility even without GTE. Together, annexin-I plays an important role in GTE-induced actin remodeling, and it may serve as a potential molecular target associated with the anticancer activities of green tea.

  8. Gracilaria edulis exhibit antiproliferative activity against human lung adenocarcinoma cell line A549 without causing adverse toxic effect in vitro and in vivo.

    PubMed

    Sakthivel, Ravi; Muniasamy, Samuthirapandi; Archunan, Govindaraju; Devi, Kasi Pandima

    2016-02-01

    In the present study, the antiproliferative potential of various solvent extracts of Gracilaria edulis (GE) was tested against various cancer cell lines. In the A549 lung cancer cell line model, GE ethyl acetate extract (GEEA) (100 μg mL(-1)) treated group showed the maximum and significant (P < 0.05) growth inhibition at 48 h. The IC50 value was found to be 24.5 ± 19.1 μg mL(-1) at 48 h. Moreover, a low level of LDH release was observed at 48 h at various concentrations of (40, 60, 80 and 100 μg mL(-1)) GEEA extract-treated group compared to a control group. Changes in the cell morphology and echinoid spikes formation were observed at 48 h. Safety evaluation of GEEA in a non-cancerous liver cell line, PBMC and in Wistar rats positively revealed that the extract did not show any adverse toxic effects. The GEEA extract was partially purified by column chromatography and the active fraction was characterized through LC-MS analysis. Furthermore, HPLC and FT-IR analysis of the active fractions confirmed the presence of phytol, a diterpene compound with potent antiproliferative activity, which positively suggests that the red alga G. edulis contains a potent anticancer active principle.

  9. Irciniastatin A induces potent and sustained activation of extracellular signal-regulated kinase and thereby promotes ectodomain shedding of tumor necrosis factor receptor 1 in human lung carcinoma A549 cells.

    PubMed

    Quach, Hue Tu; Hirano, Seiya; Fukuhara, Sayuri; Watanabe, Tsubasa; Kanoh, Naoki; Iwabuchi, Yoshiharu; Usui, Takeo; Kataoka, Takao

    2015-01-01

    Irciniastatin A is a pederin-type marine product that potently inhibits translation. We have recently shown that irciniastatin A induces ectodomain shedding of tumor necrosis factor (TNF) receptor 1 with slower kinetics than other translation inhibitors. In human lung carcinoma A549 cells, irciniastatin A induced a marked and sustained activation of extracellular signal-regulated kinase (ERK) and induced little activation of p38 mitogen-activated protein (MAP) kinase and c-Jun N-terminal kinase (JNK). Moreover, the TNF receptor 1 shedding induced by irciniastatin A was blocked by the MAP kinase/ERK kinase inhibitor U0126, but not by the p38 MAP kinase inhibitor SB203580 or the JNK inhibitor SP600125. Thus unlike other translation inhibitors that trigger ribotoxic stress response, our results show that irciniastatin A is a unique translation inhibitor that induces a potent and sustained activation of the ERK pathway, and thereby promotes the ectodomain shedding of TNF receptor 1 in A549 cells.

  10. Modulation of intrinsic in vitro resistance to carboplatin by edatrexate in the A549 human nonsmall cell lung cancer cell line.

    PubMed

    Perez, E A; Hack, F M; Fletcher, T S; Chou, T C

    1994-01-01

    Edatrexate (10-ethyl-deazaaminopterin) is a methotrexate analog that has been shown to have greater antitumor activity and improved therapeutic index compared to its parent compound in preclinical systems. We have evaluated the ability of edatrexate to modulate the intrinsic resistance of the lung adenocarcinoma A549 cell line to carboplatin. Concentration effects, exposure time and schedule dependence were assessed. Modulation of resistance was observed with edatrexate treatment (0.2 microM for 1 h) prior to carboplatin. The concentrations of carboplatin to achieve IC50 at the 1-, 3-, and 24-h IC50 were decreased by a mean of 16.8 times (12.2-22.2) with edatrexate preexposure. In contrast, there was little modulation observed of carboplatin resistance when carboplatin was administered prior to edatrexate. In addition, schedule dependency experiments were performed using the method described by Chou and Talalay, in which the ratio of carboplatin to edatrexate was constant or nonconstant, and both the potency of effects and the shapes of the concentration-effect curves were taken into account in a computerized analysis. These experiments also demonstrated schedule dependency. Although both treatments resulted in a reduced IC50 vs. carboplatin alone, the reduction was much greater when edatrexate was added first (12.59 vs. 2.59 times). We conclude that the combination of edatrexate and carboplatin demonstrates schedule-dependent modulation of intrinsic carboplatin resistance in this in vitro model at clinically achievable edatrexate plasma levels (0.01 to 10 microM). The greatest modulatory synergism was observed in the setting of edatrexate treatment before carboplatin. Our findings suggest a potentially useful schedule when combining edatrexate and carboplatin for the treatment of malignant disease.

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

    SciTech Connect

    Choi, Hye Jin; Lee, Dong-Hyung; Park, Seong-Hwan; Kim, Juil; Do, Kee Hun; An, Tae Jin; Ahn, Young Sup; Park, Chung Berm; Moon, Yuseok

    2011-09-30

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

  12. Toward therapeutic pulmonary alveolar regeneration in humans.

    PubMed

    Massaro, Donald; Massaro, Gloria Decarlo

    2006-11-01

    In humans, age results in loss of pulmonary alveoli; menopause accelerates loss of diffusing capacity, an index of alveolar surface area; and disease (e.g., chronic obstructive pulmonary disease) results in loss of alveoli. Thus, an important goal for investigators is to generate knowledge that allows induction of pulmonary alveolar regeneration in humans. Our enthusiasm for this goal and our assessment of its feasibility are based on work in several laboratories over the last decade that has disproved the notion that pulmonary alveoli are incapable of regeneration, and on the growing evidence that signals that regulate programs of alveolar turnover (loss and regeneration) are conserved from rodents to humans. We review animal models of alveolar loss and regeneration and their conservation during evolution, and hence their relevance to humans.

  13. Infection of A549 human type II epithelial cells with Mycobacterium tuberculosis induces changes in mitochondrial morphology, distribution and mass that are dependent on the early secreted antigen, ESAT-6.

    PubMed

    Fine-Coulson, Kari; Giguère, Steeve; Quinn, Frederick D; Reaves, Barbara J

    2015-10-01

    Pulmonary infection by Mycobacterium tuberculosis (Mtb) involves the invasion of alveolar epithelial cells (AECs). We used Mitotracker Red(®) to assess changes in mitochondrial morphology/distribution and mass from 6 to 48 h post infection (hpi) by confocal microscopy and flow cytometry in Mtb-infected A549 type II AECs. During early infection there was no effect on mitochondrial morphology, however, by 48 hpi mitochondria appeared fragmented and concentrated around the nucleus. In flow cytometry experiments, the median fluorescence intensity (MFI) decreased by 44% at 48 hpi; double-labelling using antibodies to the integral membrane protein COXIV revealed that these changes were due to a decrease in mitochondrial mass. These changes did not occur with the apathogenic strain, Mycobacterium bovis BCG. ESAT-6 is a virulence factor present in Mtb Erdman but lacking in M. bovis BCG. We performed similar experiments using Mtb Erdman, an ESAT-6 deletion mutant and its complement. MFI decreased at 48 hpi in the parent and complemented strains versus uninfected controls by 52% and 36% respectively; no decrease was detected in the deletion mutant. These results indicate an involvement of ESAT-6 in the perturbation of mitochondria induced by virulent Mtb in AECs and suggest mitophagy may play a role in the infection process.

  14. Pulmonary oedema following exercise in humans.

    PubMed

    Hodges, Alastair N H; Mayo, John R; McKenzie, Donald C

    2006-01-01

    Pulmonary physiologists have documented many transient changes in the lung and the respiratory system during and following exercise, including the incomplete oxygen saturation of arterial blood in some subjects, possibly due to transient pulmonary oedema. The large increase in pulmonary arterial pressure during exercise, leading to either increased pulmonary capillary leakage and/or pulmonary capillary stress failure, is likely to be responsible for any increase in extravascular lung water during exercise. The purpose of this article is to summarise the studies to date that have specifically examined lung water following exercise. A limited number of studies have been completed with the specific purpose of identifying pulmonary oedema following exercise or a similar intervention. Of these, approximately 50% have observed a positive change and the remaining have provided results that are either inconclusive or show no change in extravascular lung water. While it is difficult to draw a firm conclusion from these studies, we believe that pulmonary oedema does occur in some humans following exercise. As such, this is a phenomenon of significance to pulmonary and exercise physiologists. This possibility warrants further study in the area with more precise measurement tools than has previously been undertaken.

  15. High Throughput Determination of TGFβ1/SMAD3 Targets in A549 Lung Epithelial Cells

    PubMed Central

    Kaplan, Tommy; Yu, Haiying; Bais, Abha S.; Richards, Thomas; Pandit, Kusum V.; Zeng, Qilu; Benos, Panayiotis V.; Friedman, Nir; Eickelberg, Oliver; Kaminski, Naftali

    2011-01-01

    Background Transforming growth factor beta 1 (TGFβ1) plays a major role in many lung diseases including lung cancer, pulmonary hypertension, and pulmonary fibrosis. TGFβ1 activates a signal transduction cascade that results in the transcriptional regulation of genes in the nucleus, primarily through the DNA-binding transcription factor SMAD3. The objective of this study is to identify genome-wide scale map of SMAD3 binding targets and the molecular pathways and networks affected by the TGFβ1/SMAD3 signaling in lung epithelial cells. Methodology We combined chromatin immunoprecipitation with human promoter region microarrays (ChIP-on-chip) along with gene expression microarrays to study global transcriptional regulation of the TGFβ1/SMAD3 pathway in human A549 alveolar epithelial cells. The molecular pathways and networks associated with TGFβ1/SMAD3 signaling were identified using computational approaches. Validation of selected target gene expression and direct binding of SMAD3 to promoters were performed by quantitative real time RT-PCR and electrophoretic mobility shift assay on A549 and human primary lung epithelial cells. Results and Conclusions Known TGFβ1 target genes such as SERPINE1, SMAD6, SMAD7, TGFB1 and LTBP3, were found in both ChIP-on-chip and gene expression analyses as well as some previously unrecognized targets such as FOXA2. SMAD3 binding of FOXA2 promoter and changed expression were confirmed. Computational approaches combining ChIP-on-chip and gene expression microarray revealed multiple target molecular pathways affected by the TGFβ1/SMAD3 signaling. Identification of global targets and molecular pathways and networks associated with TGFβ1/SMAD3 signaling allow for a better understanding of the mechanisms that determine epithelial cell phenotypes in fibrogenesis and carcinogenesis as does the discovery of the direct effect of TGFβ1 on FOXA2. PMID:21625455

  16. Hyperthermia in the febrile range induces HSP72 expression proportional to exposure temperature but not to HSF-1 DNA-binding activity in human lung epithelial A549 cells.

    PubMed

    Tulapurkar, Mohan E; Asiegbu, Benedict E; Singh, Ishwar S; Hasday, Jeffrey D

    2009-09-01

    Expression of heat shock proteins (HSPs) is classically activated at temperatures above the physiologic range (>or=42 degrees C) via activation of the stress-activated transcription factor, heat shock factor-1 (HSF-1). Several studies suggest that less extreme hyperthermia, especially within the febrile range, as occurs during fever and exertional/environmental hyperthemia, can also activate HSF-1 and enhance HSP expression. We compared HSP72 protein and mRNA expression in human A549 lung epithelial cells continuously exposed to 38.5 degrees C, 39.5 degrees C, or 41 degrees C or exposed to a classic heat shock (42 degrees C for 2 h). We found that expression of HSP72 protein and mRNA increased linearly as incubation temperature was increased from 37 degrees C to 41 degrees C, but increased abruptly when the incubation temperature was raised to 42 degrees C. A similar response in luciferase activity was observed using A549 cells stably transfected with an HSF-1-responsive luciferase reporter plasmid. However, activation of intranuclear HSF-1 DNA-binding activity was comparable at 38.5 degrees C, 39.5 degrees C, and 41 degrees C and only modestly greater at 42 degrees C but the mobility of HSF1 protein on a denaturing gel was altered with increasing exposure temperature and was distinctly different at 42 degrees C. These findings indicate that the proportional changes in HSF-1-dependent HSP72 expression at febrile-range temperatures are dependent upon exposure time and temperature but not on the degree of HSF-1 DNA-binding activity. Instead, HSF-1-mediated HSP expression following hyperthermia and heat shock appears to be mediated, in addition to HSF-1 activation, by posttranslational modifications of HSF-1 protein.

  17. The effect of ataxia-telangiectasia mutated kinase-dependent hyperphosphorylation of checkpoint kinase-2 on oligodeoxynucleotide 7909 containing CpG motifs-enhanced sensitivity to X-rays in human lung adenocarcinoma A549 cells

    PubMed Central

    Liu, Xiaoqun; Liu, Xiangdong; Qiao, Tiankui; Chen, Wei; Yuan, Sujuan

    2015-01-01

    Objective The aim of the study reported here was to further investigate the potential effect of ataxia-telangiectasia mutated (ATM) kinase-dependent hyperphosphorylation of checkpoint kinase-2 (Chk2) on radiosensitivity enhanced by oligodeoxynucleotide 7909 containing CpG motifs (CpG ODN7909) in human lung adenocarcinoma A549 cells. Methods In vitro A549 cells were randomly separated into control, CpG, X-ray, CpG+ X-ray, ATM kinase-small interfering RNA (siRNA)+CpG+X-ray (ATM-siRNA), and Chk2-siRNA+CpG+X-ray (Chk2-siRNA) groups. siRNAs were adopted to silence the ATM and Chk2 genes. Expression and phosphorylation of ATM kinase and Chk2 were detected by Western blot assay. Cell colonies were observed under inverted phase-contrast microscopy. Cellular survival curves were fitted using a multi-target single-hitting model. Cell cycle and apoptosis were analyzed by flow cytometry. Results Expression of ATM kinase and Chk2 was similar among the control, CpG, X-ray, and CpG+X-ray groups. Phosphorylated ATM kinase and Chk2 were significantly increased in the CpG+X-ray group compared with in the X-ray group (t=6.00, P<0.01 and t=3.13, P<0.05, respectively), though these were hardly detected in the control and CpG groups. However, expression of ATM kinase and Chk2 was clearly downregulated in the ATM-siRNA and Chk2-siRNA groups, respectively. Similarly, their phosphorylation levels were also significantly decreased in the ATM-siRNA group (t=14.35, P<0.01 and t=8.46, P<0.01, respectively) and a significant decrease in phosphorylated Chk2 was observed in the Chk2-siRNA group (t=7.28, P<0.01) when compared with the CpG+X-ray group. Further, the number of A549 cells at Gap 2/mitotic phase and the apoptosis rate were clearly increased in the CpG+X-ray group compared with in the other groups (all P<0.05). The multi-target single-hitting model showed that the sensitization enhancement ratio calculated by mean death dose was 1.39 in CpG+X-ray group (vs 1.04 and 1.03 in the ATM

  18. Artesunate induces AIF-dependent apoptosis in A549 cells

    NASA Astrophysics Data System (ADS)

    Zhou, Chen-juan; Chen, Tong-Sheng

    2012-03-01

    Artesunate (ART), a semi-synthetic derivative of the sesquiterpene artemisinin extracted from the Chinese herb Artemisia annua, exerts a broad spectrum of clinical activity against human cancers. It has been shown that ART induces cancer cells death through apoptosis pathway. This study investigated whether ART treatment induced reactive oxygen species (ROS)-dependent cell death in the apoptosis fashion in human lung adenocarconoma A549 cell line and the proapoptotic protein apoptosis inducing factor (AIF) is involved in ART-induced apoptosis. Cells treated with ART exhibited typical apoptotic morphology as chromatin condensation, margination and shrunken nucleus. ART treatment also induced a loss of mitochondrial membrane potential and AIF release from mitochondria. Silencing AIF can remarkable attenuated ART-induced apoptosis. Collectively, ART induces apoptosis by caspase-independent intrinsic pathway in A549 cells.

  19. Effect of Phospholipid Transfer Protein on Cigarette Smoke Extract-Induced IL-8 Production in Human Pulmonary Epithelial Cells.

    PubMed

    Li, Youlun; Yu, Xiuying; Fu, Xiaofeng; Wu, Fengping; Zou, Linlin; Chen, Yuhan; Chen, Yajuan

    2016-12-01

    To investigate the effect of phospholipid transfer protein (PLTP) on the cigarette smoke extract (CSE)-induced production of interleukin-8 (IL-8) in human pulmonary epithelial cells, male Wistar rats were exposed to air and cigarette smoke (n = 10/exposure) for 6 h/day on three consecutive days. Their lungs were sectioned and bronchoalveolar lavage fluid (BALF) examined. The expression of PLTP and IL-8 in the lung was detected immunohistochemically. Lung injury was accompanied by the upregulation of PLTP and IL-8 in the CSE-exposed rat model, and the number of white blood cells in the BALF was significantly increased compared with those of the controls. Both neutrophils and macrophages were clearly increased. Human alveolar epithelial cells (A549) and human bronchial epithelial cells (HBECs) were treated with different concentrations of CSE for various times. The cells were also transfected with small interfering RNA directed against PLTP, and U0126, an inhibitor of the ERK1/2 pathway, was administered before CSE exposure. The expression of PLTP and IL-8 mRNAs and PLTP, IL-8, total ERK, and phosphorylated ERK proteins was analyzed. The expression of IL-8 and phosphorylated ERK was significantly increased in A549 cells and HBECs after CSE stimulation, and CSE upregulated the expression of PLTP in A549 cells. In contrast, CSE inhibited the expression of PLTP in HBECs. The CSE-induced expression of IL-8 and p-ERK was significantly increased by the knockdown of PLTP. Therefore, PLTP may regulate CSE-induced IL-8 expression via the ERK1/2 signaling pathway in human pulmonary epithelial cells.

  20. Comparative Cytotoxicity of Glycyrrhiza glabra Roots from Different Geographical Origins Against Immortal Human Keratinocyte (HaCaT), Lung Adenocarcinoma (A549) and Liver Carcinoma (HepG2) Cells.

    PubMed

    Basar, Norazah; Oridupa, Olayinka Ayotunde; Ritchie, Kenneth J; Nahar, Lutfun; Osman, Nashwa Mostafa M; Stafford, Angela; Kushiev, Habibjon; Kan, Asuman; Sarker, Satyajit D

    2015-06-01

    Glycyrrhiza glabra L. (Fabaceae), commonly known as 'liquorice', is a well-known medicinal plant. Roots of this plant have long been used as a sweetening and flavouring agent in food and pharmaceutical products, and also as a traditional remedy for cough, upper and lower respiratory ailments, kidney stones, hepatitis C, skin disorder, cardiovascular diseases, diabetes, gastrointestinal ulcers and stomach ache. Previous pharmacological and clinical studies have revealed its antitussive, antiinflammatory, antiviral, antimicrobial, antioxidant, immunomodulatory, hepatoprotective and cardioprotective properties. While glycyrrhizin, a sweet-tasting triterpene saponin, is the principal bioactive compound, several bioactive flavonoids and isoflavonoids are also present in the roots of this plant. In the present study, the cytotoxicity of the methanol extracts of nine samples of the roots of G. glabra, collected from various geographical origins, was assessed against immortal human keratinocyte (HaCaT), lung adenocarcinoma (A549) and liver carcinoma (HepG2) cell lines using the in vitro 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide cell toxicity/viability assay. Considerable variations in levels of cytotoxicity were observed among various samples of G. glabra.

  1. Lycium europaeum fruit extract: antiproliferative activity on A549 human lung carcinoma cells and PC12 rat adrenal medulla cancer cells and assessment of its cytotoxicity on cerebellum granule cells.

    PubMed

    Ghali, Wafa; Vaudry, David; Jouenne, Thierry; Marzouki, Mohamed Nejib

    2015-01-01

    Cancer is a major worldwide health problem and one of the leading causes of death either in developed or developing countries. Plant extracts and derivatives have always been used for various disease treatments and many anticancer agents issued from plants and vegetables are clinically recognized and used all over the world. Lycium europaeum (Solanaceae) also called "wolfberry" was known since ancient times in the Mediterranean area as a medicinal plant and used in several traditional remedies. The Lycium species capacity of reducing the incidence of cancer and also of halting or reserving the growth of cancer was reported by traditional healers. In this study, the antiproliferative capacity, protective properties, and antioxidant activity of the hydro-alcoholic fruit extract of Lycium europaeum were investigated. Results showed that Lycium extract exhibits the ability to reduce cancer cell viability, inhibits proliferation, and induces apoptosis in A549 human lung cancer cells and PC12 rat adrenal medulla cancer cells, in a concentration- and time-dependent manner. Cytotoxic effect on normal rat cerebellum granule cells was assessed to be nonsignificant. Results also showed that Lycium fruit extract protected lipids, proteins, and DNA against oxidative stress damages induced by H2O2 via scavenging reactive oxygen species.

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

    PubMed Central

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

    2016-01-01

    Pulmonary research requires models that represent the physiology of alveolar epithelium but concerns with reproducibility, consistency and the technical and ethical challenges of using primary or stem cells has resulted in widespread use of continuous cancer or other immortalized cell lines. The A549 ‘alveolar’ cell line has been available for over four decades but there is an inconsistent view as to its suitability as an appropriate model for primary alveolar type II (ATII) cells. Since most work with A549 cells involves short term culture of proliferating cells, we postulated that culture conditions that reduced proliferation of the cancer cells would promote a more differentiated ATII cell phenotype. We examined A549 cell growth in different media over long term culture and then used microarray analysis to investigate temporal regulation of pathways involved in cell cycle and ATII differentiation; we also made comparisons with gene expression in freshly isolated human ATII cells. Analyses indicated that long term culture in Ham’s F12 resulted in substantial modulation of cell cycle genes to result in a quiescent population of cells with significant up-regulation of autophagic, differentiation and lipidogenic pathways. There were also increased numbers of up- and down-regulated genes shared with primary cells suggesting adoption of ATII characteristics and multilamellar body (MLB) development. Subsequent Oil Red-O staining and Transmission Electron Microscopy confirmed MLB expression in the differentiated A549 cells. This work defines a set of conditions for promoting ATII differentiation characteristics in A549 cells that may be advantageous for studies with this cell line. PMID:27792742

  3. Atrial natriuretic peptide: A novel mediator for TGF-β1-induced epithelial-mesenchymal transition in 16HBE-14o and A549 cells.

    PubMed

    Chu, Shuyuan; Zhang, Xiufeng; Sun, Yabing; Yu, Yuanyuan; Liang, Yaxi; Jiang, Ming; Huang, Jianwei; Ma, Libing

    2017-02-13

    Atrial natriuretic peptide (ANP) is increasingly expressed on airway and inhibits pulmonary arterial remodeling. However, the role of ANP in remodeling of respiratory system is still unclear. The role of ANP on airway remodeling and the possible mechanism was explored in this study. Both human bronchial epithelial 16HBE-14o cells and alveolar epithelial A549 cells were stimulated by TGF-β1, ANP, cGMP inhibitor, PKG inhibitor, and cGMP analogue. The expressions of epithelial markers, mesenchymal markers, and Smad3 were assessed by quantitative real-time PCR and western blotting. Immunohistochemical staining was employed to assess Smad3 expression once it was silenced by siRNA in 16HBE-14o or A549 cells. Our results showed that the mRNA and protein expressions of E-Cadherin were decreased, whereas α-SMA expressions were increased after induction by TGF-β1 in 16HBE-14o and A549 cells. The E-Cadherin expressions were increased and α-SMA expressions were decreased after ANP stimulation. Inhibition of cGMP or PKG decreased E-Cadherin expression but increased α-SMA expression, which could be reversed by cGMP analogue. Moreover, the phosphorylated Smad3 expression was consistent with α-SMA expression. After smad3 was silenced, Smad3 was mostly expressed in cytoplasm instead of nucleus as non-silenced cells during epithelial-mesenchymal transition (EMT). In conclusion, ANP inhibits TGF-β1-induced EMT in 16HBE-14o and A549 cells through cGMP/PKG signaling, by which it targets TGF-β1/Smad3 via attenuating phosphorylation of Smad3. These findings suggest the potential of ANP in the treatment on pulmonary diseases with airway remodeling.

  4. Leukotriene receptors on human pulmonary vascular endothelium.

    PubMed

    Ortiz, J L; Gorenne, I; Cortijo, J; Seller, A; Labat, C; Sarria, B; Abram, T S; Gardiner, P J; Morcillo, E; Brink, C

    1995-08-01

    1. Cysteinyl-leukotrienes cause contractions and/or relaxations of human isolated pulmonary vascular preparations. Although, the localization and nature of the receptors through which these effects are mediated have not been fully characterized, some effects are indirect and not mediated via the well-described LT1 receptor. 2. In human pulmonary veins (HPV) with an intact endothelium, leukotriene D4 (LTD4) induced contraction above basal tone. This response was observed at lower concentrations of LTD4 in the presence of nitric oxide synthase inhibitor N omega-nitro-L-arginine (L-NOARG). Contractions (in the absence and presence of L-NOARG) were partially blocked by the LT1 antagonists (MK 571 and ICI 198615). 3. LTD4 relaxed HPV previously contracted with noradrenaline. This relaxation was potentiated by LT1 antagonists, but was abolished by removal of the endothelium. LTD4 also relaxed human pulmonary arteries (HPA) precontracted with noradrenaline but this effect was not modified by LT1 antagonists. 4. The results suggest that contraction of endothelium-intact HPV by LTD4 is partially mediated via LT1 receptors. Further, in endothelium-intact HPV, this contraction was opposed by a relaxation induced by LTD4, dependent on the release of nitric oxide, which was mediated, at least in part, via a non-LT1 receptor. In addition, LTD4 relaxation on contracted HPA was not mediated by LT1 receptors. 5. The mechanical effects of LTD4 on human pulmonary vasculature are complex and involve both direct and indirect mechanisms mediated via at least two types of cysteinyl-leukotriene receptors.

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

    PubMed Central

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

    2016-01-01

    Hepatitis E virus (HEV) is a human pathogen with increasing importance. The lack of efficient cell culture systems hampers systematic studies on its replication cycle, virus neutralization and inactivation. Here, several cell lines were inoculated with the HEV genotype 3c strain 47832c, previously isolated from a chronically infected transplant patient. At 14 days after inoculation the highest HEV genome copy numbers were found in A549 cells, followed by PLC/PRF/5 cells, whereas HepG2/C3A, Huh-7 Lunet BLR and MRC-5 cells only weakly supported virus replication. Inoculation of A549-derived subclone cell lines resulted in most cases in reduced HEV replication. However, the subclone A549/D3 was susceptible to lower virus concentrations and resulted in higher virus yields as compared to parental A549 cells. Transcriptome analysis indicated a downregulation of genes for carcinoembryonic antigen-related cell adhesion molecules (CEACAM) 5 and 6, and an upregulation of the syndecan 2 (SDC2) gene in A549/D3 cells compared to A549 cells. However, treatment of A549/D3 cells or A549 cells with CEACAM- or syndecan 2-specific antisera did not influence HEV replication. The results show that cells supporting more efficient HEV replication can be selected from the A549 cell line. The specific mechanisms responsible for the enhanced replication remain unknown. PMID:27690085

  6. Rab27A regulates exosome secretion from lung adenocarcinoma cells A549: involvement of EPI64.

    PubMed

    Li, Wenhai; Hu, Yunsheng; Jiang, Tao; Han, Yong; Han, Guoliang; Chen, Jiakuan; Li, Xiaofei

    2014-11-01

    Exosomes are small membrane vesicles secreted into the extracellular compartment by exocytosis. The unique composition of exosomes can be transported to other cells which allow cells to exert biological functions at distant sites. However, in lung cancer, the regulation of exosome secretion was poorly understood. In this study, we employed human lung adenocarcinoma A549 cells to determine the exosome secretion and involved regulation mechanism. We found that Rab27A was expressed in A549 cells and the reduction of Rab27A by Rab27A-specific shRNA could significantly decrease the secretion of exosome by A549 cells. EPI64, a candidate GAP that is specific for Rab27, was also detected in A549 cells. By pull-down assay, we found that EPI64 participated in the exosome secretion of A549 cells by acting as a specific GAP for Rab27A, not Rab27B. Overexpression of EPI64 enhanced exosome secretion. Taken together, in A549 cells, EPI64 could regulate the exosome secretion by functioning as a GAP specific for Rab27A.

  7. Proteomic response to 5,6-dimethylxanthenone 4-acetic acid (DMXAA, vadimezan) in human non-small cell lung cancer A549 cells determined by the stable-isotope labeling by amino acids in cell culture (SILAC) approach.

    PubMed

    Pan, Shu-Ting; Zhou, Zhi-Wei; He, Zhi-Xu; Zhang, Xueji; Yang, Tianxin; Yang, Yin-Xue; Wang, Dong; Qiu, Jia-Xuan; Zhou, Shu-Feng

    2015-01-01

    5,6-Dimethylxanthenone 4-acetic acid (DMXAA), also known as ASA404 and vadimezan, is a potent tumor blood vessel-disrupting agent and cytokine inducer used alone or in combination with other cytotoxic agents for the treatment of non-small cell lung cancer (NSCLC) and other cancers. However, the latest Phase III clinical trial has shown frustrating outcomes in the treatment of NSCLC, since the therapeutic targets and underlying mechanism for the anticancer effect of DMXAA are not yet fully understood. This study aimed to examine the proteomic response to DMXAA and unveil the global molecular targets and possible mechanisms for the anticancer effect of DMXAA in NSCLC A549 cells using a stable-isotope labeling by amino acids in cell culture (SILAC) approach. The proteomic data showed that treatment with DMXAA modulated the expression of 588 protein molecules in A549 cells, with 281 protein molecules being up regulated and 306 protein molecules being downregulated. Ingenuity pathway analysis (IPA) identified 256 signaling pathways and 184 cellular functional proteins that were regulated by DMXAA in A549 cells. These targeted molecules and signaling pathways were mostly involved in cell proliferation and survival, redox homeostasis, sugar, amino acid and nucleic acid metabolism, cell migration, and invasion and programed cell death. Subsequently, the effects of DMXAA on cell cycle distribution, apoptosis, autophagy, and reactive oxygen species (ROS) generation were experimentally verified. Flow cytometric analysis showed that DMXAA significantly induced G1 phase arrest in A549 cells. Western blotting assays demonstrated that DMXAA induced apoptosis via a mitochondria-dependent pathway and promoted autophagy, as indicated by the increased level of cytosolic cytochrome c, activation of caspase 3, and enhanced expression of beclin 1 and microtubule-associated protein 1A/1B-light chain 3 (LC3-II) in A549 cells. Moreover, DMXAA significantly promoted intracellular ROS

  8. Up-Regulation and Profibrotic Role of Osteopontin in Human Idiopathic Pulmonary Fibrosis

    PubMed Central

    2005-01-01

    Background Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal disorder characterized by fibroproliferation and excessive accumulation of extracellular matrix in the lung. Methods and Findings Using oligonucleotide arrays, we identified osteopontin as one of the genes that significantly distinguishes IPF from normal lungs. Osteopontin was localized to alveolar epithelial cells in IPF lungs and was also significantly elevated in bronchoalveolar lavage from IPF patients. To study the fibrosis-relevant effects of osteopontin we stimulated primary human lung fibroblasts and alveolar epithelial cells (A549) with recombinant osteopontin. Osteopontin induced a significant increase of migration and proliferation in both fibroblasts and epithelial cells. Epithelial growth was inhibited by the pentapeptide Gly-Arg-Gly-Asp-Ser (GRGDS) and antibody to CD44, while fibroproliferation was inhibited by GRGDS and antibody to αvβ3 integrin. Fibroblast and epithelial cell migration were inhibited by GRGDS, anti-CD44, and anti-αvβ3. In fibroblasts, osteopontin up-regulated tissue inhibitor of metalloprotease-1 and type I collagen, and down-regulated matrix metalloprotease-1 (MMP-1) expression, while in A549 cells it caused up-regulation of MMP-7. In human IPF lungs, osteopontin colocalized with MMP-7 in alveolar epithelial cells, and application of weakest link statistical models to microarray data suggested a significant interaction between osteopontin and MMP-7. Conclusions Our results provide a potential mechanism by which osteopontin secreted from the alveolar epithelium may exert a profibrotic effect in IPF lungs and highlight osteopontin as a potential target for therapeutic intervention in this incurable disease. PMID:16128620

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

    PubMed

    Xu, Jian; Wu, Fan; Tian, Deying; Wang, Jingjing; Zheng, Zizheng; Xia, Ningshao

    2014-01-01

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

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

    PubMed Central

    Han, Mei-ling; Zhao, Yi-fan; Tan, Cai-hong; Xiong, Ya-jie; Wang, Wen-juan; Wu, Feng; Fei, Yao; Wang, Long; Liang, Zhong-qin

    2016-01-01

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

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

    PubMed

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

    2015-05-01

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

  12. In vitro cytotoxicity of gold nanorods in A549 cells.

    PubMed

    Tang, Ying; Shen, Yafeng; Huang, Libin; Lv, Gaojian; Lei, Changhai; Fan, Xiaoyan; Lin, Fangxing; Zhang, Yuxia; Wu, Lihui; Yang, Yongji

    2015-03-01

    Gold nanoparticles, which have unique physicochemical characteristics, are being used for an increasingly wide range of applications in biomedical research. In this study, gold nanorods (width of 25 nm, length of 52 nm) were found to be internalized by A549 cells and were primarily localized in the lysosomes and membranous vesicles. The integrity of the membranes of A549 cells exposed to gold nanorods for 4h was damaged, as indicated by laser scanning confocal microscopy (LSCM). Increased lactate dehydrogenase (LDH) leakage and decreased cell viability further indicated the concentration-dependent cytotoxicity of the gold nanorods to the A549 cells. Reactive oxygen species (ROS) production was induced in the A549 cells by the gold nanorods, and this effect was positively correlated with the concentration of the gold nanorods. The results of this study indicated that exposure to gold nanorods caused dose-dependent cytotoxicity in A549 cells and that oxidative stress may be the main factor causing cytotoxicity.

  13. The influence of ciprofloxacin on viability of A549, HepG2, A375.S2, B16 and C6 cell lines in vitro.

    PubMed

    Kloskowski, Tomasz; Gurtowska, Natalia; Nowak, Monika; Joachimiak, Romana; Bajek, Anna; Olkowska, Joanna; Drewa, Tomasz

    2011-01-01

    Ciprofloxacin is a chemotherapeutic agent mainly used in the treatment of the pulmonary and urinary tract infections but is also known for its anticancer properties. The aim of these study was to check the anticancer effect of ciprofloxacin on selected five cell lines. Human non-small cell lung cancer line A549, human hepatocellular carcinoma line HepG2, human and mouse melanoma lines (A375.S2 and B16) and rat glioblastoma line C6 were used for evaluation of cytotoxic properties of ciprofloxacin (in concentration range: 10-1000 microg/mL). Viability was established using trypan blue assay and MTT. Ciprofloxacin induced morphological changes and decreased viability of A549 cells in a concentration and time dependent manner. In case of A375.S2 and B16 cell lines, cytotoxicyty of ciprofloxacin was observed but we were not able to eradicate all cells from A375.S2 and B16 cultures. HepG2 line was sensitive to ciprofloxacin, but this effect was independent from concentration and incubation time. The C6 cells were insensitive to ciprofloxacin. Our results showed that ciprofloxacin can be potentially used for the experimental adjunctive therapy of lung cancer.

  14. Xanthohumol induces apoptosis and S phase cell cycle arrest in A549 non-small cell lung cancer cells

    PubMed Central

    Yong, Wai Kuan; Ho, Yen Fong; Malek, Sri Nurestri Abd

    2015-01-01

    Background: Xanthohumol, a major prenylated chalcone found in female hop plant, Humulus lupulus, was reported to have various chemopreventive and anti-cancer properties. However, its apoptotic effect on human alveolar adenocarcinoma cell line (A549) of non-small cell lung cancer (NSCLC) was unknown. Objective: This study aimed to investigate the effects of xanthohumol on apoptosis in A549 human NSCLC cells. Materials and Methods: A549 cell proliferation was determined by sulforhodamine B assay. Morphological changes of the cells were studied via phase contrast and fluorescent microscopy. Induction of apoptosis was assessed by Annexin-V fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) staining, DNA fragmentation (TUNEL) assay mitochondrial membrane potential assay, cell cycle analysis, and caspase activity studies. Results: Xanthohumol was found to decrease cell proliferation in A549 cells but had relatively low cytotoxicity on normal human lung fibroblast cell line (MRC-5). Typical cellular and nuclear apoptotic features were also observed in A549 cells treated with xanthohumol. Onset of apoptosis in A549 cells was further confirmed by externalization of phosphatidylserine, changes in mitochondrial membrane potential, and DNA fragmentation in the cells after treatment. Xanthohumol induced accumulation of cells in sub G1 and S phase based on cell cycle analysis and also increased the activities of caspase-3, -8, and -9. Conclusion: This work suggests that xanthohumol as an apoptosis inducer, may be a potent therapeutic compound for NSCLC. PMID:26664015

  15. [Pulmonary complications in children with human immunodeficiency virus infection].

    PubMed

    Brockmann V, Pablo; Viviani S, Támara; Peña D, Anamaría

    2007-08-01

    Pulmonary complications in children infected by human immunodeficiency virus (HIV) are common and may be the first manifestation of acquired immunodeficiency syndrome (AIDS). The aim of our study was to review pulmonary diseases and complications in pediatric patients with HIV infection in a large tertiary hospital in Santiago, Chile. We performed a retrospective, descriptive analysis of 17 patients with HIV infection controlled at the Hospital Dr. Sótero del Rio. Respiratory complications/diseases were: overall pneumonia (n: 14), recurrent pneumonia (n: 10), citomegalovirus associated pneumonia (n: 4), Pneumocystis jiroveci associated pneumonia (n: 1) pulmonary tuberculosis (n: 1), lymphoid interstitial pneumonia (n: 3) and chronic pulmonary disease (n: 7). Microorganisms isolated were mostly atypical and frequently associated with severe and chronic pulmonary damage. A high degree of suspicion is required to detect atypical microorganisms promptly, in order to rapidly implement pathogen targeted therapy that could potentially decrease the possibility of sequelae.

  16. PVM/MA-shelled selol nanocapsules promote cell cycle arrest in A549 lung adenocarcinoma cells

    PubMed Central

    2014-01-01

    Background Selol is an oily mixture of selenitetriacylglycerides that was obtained as a semi-synthetic compound containing selenite. Selol is effective against cancerous cells and less toxic to normal cells compared with inorganic forms of selenite. However, Selol’s hydrophobicity hinders its administration in vivo. Therefore, the present study aimed to produce a formulation of Selol nanocapsules (SPN) and to test its effectiveness against pulmonary adenocarcinoma cells (A549). Results Nanocapsules were produced through an interfacial nanoprecipitation method. The polymer shell was composed of poly(methyl vinyl ether-co-maleic anhydride) (PVM/MA) copolymer. The obtained nanocapsules were monodisperse and stable. Both free Selol (S) and SPN reduced the viability of A549 cells, whereas S induced a greater reduction in non-tumor cell viability than SPN. The suppressor effect of SPN was primarily associated to the G2/M arrest of the cell cycle, as was corroborated by the down-regulations of the CCNB1 and CDC25C genes. Apoptosis and necrosis were induced by Selol in a discrete percentage of A549 cells. SPN also increased the production of reactive oxygen species, leading to oxidative cellular damage and to the overexpression of the GPX1, CYP1A1, BAX and BCL2 genes. Conclusions This study presents a stable formulation of PVM/MA-shelled Selol nanocapsules and provides the first demonstration that Selol promotes G2/M arrest in cancerous cells. PMID:25149827

  17. Enhanced expression levels of aquaporin-1 and aquaporin-4 in A549 cells exposed to silicon dioxide.

    PubMed

    Hao, Xiaohui; Wang, Hongli; Liu, Wei; Liu, Shupeng; Peng, Zihe; Sun, Yue; Zhao, Jinyuan; Jiang, Qiujie; Liu, Heliang

    2016-09-01

    Aquaporins (AQPs), water channel proteins in the cell membranes of mammals, have been reported to be important in maintaining the water balance of the respiratory system. However, little is known regarding the role of AQP in occupational pulmonary diseases such as silicosis. The present study investigated the expression of AQP1 and AQP4 in the human A549 alveolar epithelial cell line stimulated by silica (SiO2). A549 cells were cultured and divided into four groups: Control, SiO2‑stimulated, AQP1 inhibitor and AQP4 inhibitor. The cells of the SiO2‑stimulated group were stimulated with SiO2 dispersed suspension (50 mg/ml). The cells of the inhibitor group were pretreated with mercury (II) chloride (HgCl2; a specific channel inhibitor of AQP1) and 2‑(nicotinamide)‑1,3,4‑thiadiazole (TGN‑020; a specific channel inhibitor of AQP4) and stimulated with SiO2. The mRNA expression levels of AQP1 and AQP4 were detected by reverse transcription‑quantitative polymerase chain reaction, and the protein expression levels of AQP1 and AQP4 were detected by western blotting and immunocytochemistry. Compared with the control group, the expression levels of AQP1 and AQP4 mRNA and protein in SiO2‑stimulated groups increased and subsequently decreased (AQP1 peaked at 2 h and AQP4 at 1h; both P<0.001 compared with control group). In the inhibitor group, expression levels were increased compared with controls; however, they were significantly decreased compared with the SiO2‑stimulated group at 2 h (AQP1; P<0.001) and 1 h (AQP4; P<0.001). The expression of AQP1 and AQP4 increased when exposed to SiO2, and this was inhibited by HgCl2 and TGN‑020, suggesting that AQP1 and AQP4 may contribute to A549 cell damage induced by SiO2. AQP1 and AQP4 may thus be involved in the initiation and development of silicosis.

  18. Lymphatic Stomata in the Adult Human Pulmonary Ligament

    PubMed Central

    Miura, Masahiro; Iobe, Hiroaki; Kudo, Tomoo; Shimazu, Yoshihito; Aoba, Takaaki; Okudela, Koji; Nagahama, Kiyotaka; Sakamaki, Kentaro; Yoshida, Maki; Nagao, Toshitaka; Nakaya, Takeo; Kurata, Atsushi; Ohtani, Osamu

    2015-01-01

    Abstract Background: Lymphatic stomata are small lymphatic openings in the serosal membrane that communicate with the serosal cavity. Although these stomata have primarily been studied in experimental mammals, little is known concerning the presence and properties of lymphatic stomata in the adult human pleura. Thus, adult human pleurae were examined for the presence or absence of lymphatic stomata. Methods and Results: A total of 26 pulmonary ligaments (13 left and 13 right) were obtained from 15 adult human autopsy cases and examined using electron and light microscopy. The microscopic studies revealed the presence of apertures fringed with D2-40-positive, CD31-positive, and cytokeratin-negative endothelial cells directly communicating with submesothelial lymphatics in all of the pulmonary ligaments. The apertures' sizes and densities varied from case to case according to the serial tissue section. The medians of these aperture sizes ranged from 2.25 to 8.75 μm in the left pulmonary ligaments and from 2.50 to 12.50 μm in the right pulmonary ligaments. The densities of the apertures ranged from 2 to 9 per mm2 in the left pulmonary ligaments and from 2 to 18 per mm2 in the right pulmonary ligaments. However, no significant differences were found regarding the aperture size (p=0.359) and density (p=0.438) between the left and the right pulmonary ligaments. Conclusions: Our study revealed that apertures exhibit structural adequacy as lymphatic stomata on the surface of the pulmonary ligament, thereby providing evidence that lymphatic stomata are present in the adult human pleura. PMID:25526320

  19. Surfactant lipids regulate LPS-induced interleukin-8 production in A549 lung epithelial cells by inhibiting translocation of TLR4 into lipid raft domains

    PubMed Central

    Abate, Wondwossen; Alghaithy, Abdulaziz A.; Parton, Joan; Jones, Kenneth P.; Jackson, Simon K.

    2010-01-01

    In addition to providing mechanical stability, growing evidence suggests that surfactant lipid components can modulate inflammatory responses in the lung. However, little is known of the molecular mechanisms involved in the immunomodulatory action of surfactant lipids. This study investigates the effect of the lipid-rich surfactant preparations Survanta®, Curosurf®, and the major surfactant phospholipid dipalmitoylphosphatidylcholine (DPPC) on interleukin-8 (IL-8) gene and protein expression in human A549 lung epithelial cells using immunoassay and PCR techniques. To examine potential mechanisms of the surfactant lipid effects, Toll-like receptor 4 (TLR4) expression was analyzed by flow cytometry, and membrane lipid raft domains were separated by density gradient ultracentrifugation and analyzed by immunoblotting with anti-TLR4 antibody. The lipid-rich surfactant preparations Survanta®, Curosurf®, and DPPC, at physiological concentrations, significantly downregulated lipopolysaccharide (LPS)-induced IL-8 expression in A549 cells both at the mRNA and protein levels. The surfactant preparations did not affect the cell surface expression of TLR4 or the binding of LPS to the cells. However, LPS treatment induced translocation of TLR4 into membrane lipid raft microdomains, and this translocation was inhibited by incubation of the cells with the surfactant lipid. This study provides important mechanistic details of the immune-modulating action of pulmonary surfactant lipids. PMID:19648651

  20. Rapamycin‐induced autophagy sensitizes A549 cells to radiation associated with DNA damage repair inhibition

    PubMed Central

    Li, Yong; Liu, Fen; Wang, Yong; Li, Donghai; Guo, Fei; Xu, Liyao; Zeng, Zhengguo; Zhong, Xiaojun

    2016-01-01

    Abstract Background Autophagy has been reported to increase in cancer cells after radiation. However, it remains unknown whether increased autophagy as a result of radiation affects DNA damage repair and sensitizes cancer cells. In this study, the radiosensitization effect of rapamycin, a mammalian target of rapamycin inhibitor that induces autophagy, on human lung adenocarcinoma A549 cells was investigated. Methods A549 cells were treated with different concentrations of rapamycin. Cell viability was evaluated by methyl‐thiazolyl‐tetrazolium assay. Survival fraction values of A549 cells after radiotherapy were detected by colony formation assay. Autophagosome was observed by a transmission electron microscope. Furthermore, Western blot was employed to examine alterations in autophagy protein LC3 and p62, DNA damage protein γ–H2AX, and DNA damage repair proteins Rad51, Ku70, and Ku80. Rad51, Ku70, and Ku80 messenger ribonucleic acid (mRNA) expression levels were examined by real‐time polymerase chain reaction. Results Rapamycin suppressed A549 cell proliferation in dose and time‐dependent manners. An inhibitory concentration (IC) 10 dose of rapamycin could induce autophagy in A549 cells. Rapamycin combined with radiation significantly decreased the colony forming ability of cells, compared with rapamycin or radiation alone. Rapamycin and radiation combined increased γ–H2AX expression levels and decreased Rad51 and Ku80 expression levels, compared with single regimens. However, rapamycin treatment did not induce any change in Rad51, Ku70, and Ku80 mRNA levels, regardless of radiation. Conclusions These findings indicate that increasing autophagy sensitizes lung cancer cells to radiation. PMID:27385978

  1. Primary pulmonary hypertension associated with human immunodeficiency virus infection.

    PubMed Central

    Golpe, R.; Fernandez-Infante, B.; Fernandez-Rozas, S.

    1998-01-01

    Several cardiorespiratory diseases can complicate human immunodeficiency virus infection. Primary pulmonary hypertension is a rare clinical disorder which carries a bad prognosis. More than 90 cases of HIV-associated primary pulmonary hypertension have been reported to date. Although its pathogenesis remains unknown, some evidence suggests a possible role for the virus itself in its development. Genetic susceptibility may also be implicated. The clinical and histopathologic features of this entity do not differ from those of classic primary pulmonary hypertension. The diagnosis requires a high degree of clinical suspicion and a careful evaluation to rule out causes of secondary pulmonary hypertension. In addition to supportive measures, anticoagulation and vasodilators have been used to treat this disorder, although sufficient data regarding long-term results with these therapies are lacking. PMID:9799910

  2. Primary pulmonary hypertension associated with human immunodeficiency virus infection.

    PubMed

    Golpe, R; Fernandez-Infante, B; Fernandez-Rozas, S

    1998-07-01

    Several cardiorespiratory diseases can complicate human immunodeficiency virus infection. Primary pulmonary hypertension is a rare clinical disorder which carries a bad prognosis. More than 90 cases of HIV-associated primary pulmonary hypertension have been reported to date. Although its pathogenesis remains unknown, some evidence suggests a possible role for the virus itself in its development. Genetic susceptibility may also be implicated. The clinical and histopathologic features of this entity do not differ from those of classic primary pulmonary hypertension. The diagnosis requires a high degree of clinical suspicion and a careful evaluation to rule out causes of secondary pulmonary hypertension. In addition to supportive measures, anticoagulation and vasodilators have been used to treat this disorder, although sufficient data regarding long-term results with these therapies are lacking.

  3. FGFR3 silencing by siRNA inhibits invasion of A549 cells

    PubMed Central

    Li, Yuhua; Liu, Xiguang; Zhang, Hongjun; Jiang, Tao; Xiao, Wenjing; Zhao, Shufen; Yu, Xiaoyun; Han, Fanjie

    2016-01-01

    The present study identified that fibroblast growth factor receptor 3 (FGFR3) was significantly upregulated in bone metastasis of lung adenocarcinoma. RNA interference (RNAi) is a powerful approach for treating a wide range of human diseases, including cancer, through downregulating the expression of selected genes. In the present study, the invasiveness of A549 cells cultured in vitro was altered by small interfering (si)RNA targeting FGFR3, and the regulatory effect of silencing FGFR3 on the expression levels of E-cadherin and matrix metalloproteinase (MMP)9 was investigated. Human lung adenocarcinoma A549 cells were transfected with synthetic specific siRNAs targeting a fragment of the FGFR3 gene (namely, siRNA-855, siRNA-1447 and siRNA-2076) or with negative control (NC) siRNA. Cells were divided into five groups (A, siRNA-855 group; B, siRNA-1447 group; C, siRNA-2076 group; D, NC-siRNA group; and E, blank control group). The effect of the above siRNAs targeting FGFR3 on the invasion capacity of A549 cells was detected by Transwell assay. siRNAs against FGFR3 were transfected into A549 cells with by Lipofectamine® 2000, and the expression levels of FGFR3, E-cadherin and MMP9 were measured by reverse transcription-quantitative polymerase chain reaction and western blot assay. The experimental findings indicated that the expression levels of FGFR3 and MMP9 were significantly reduced in the siRNA-FGFR3-transfected groups (A-C groups), compared with those in the D and E groups (P<0.01). In addition, the expression levels of E-cadherin were markedly elevated in the A-C groups, compared with those in the D and E groups (P<0.01). There was no significant difference in E-cadherin expression between the A-C groups, or between the D and E groups (P>0.05). These results indicated that siRNA-FGFR3 was able to decrease the invasiveness of A549 cells, inhibit the expression of MMP9 and increase the expression of E-cadherin by downregulating the expression of FGFR3. Taken

  4. [Pulmonary arterial hypertension associated to human immunodeficiency virus].

    PubMed

    Sandoval-Gutiérrez, José Luis; Santos-Martínez, Luis Efren; Rodríguez-Silverio, Juan; Baranda-Tovar, Francisco Martín; Rivera-Rosales, Rosa María; Flores-Murrieta, Francisco Javier

    2015-01-01

    From the advent of the highly effective antiretroviral treatment, the life expectancy of patients with human immunodeficiency virus has increased significantly. At present, the causes of death are non-infectious complications. Between them, the pulmonary arterial hypertension has a special importance. It is important early detection to establish the therapeutic, with the objective of preventing a fatal outcome to future.

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

    NASA Astrophysics Data System (ADS)

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

    2016-10-01

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

  6. Identification of human pulmonary alkaline phosphatase isoenzymes.

    PubMed

    Capelli, A; Cerutti, C G; Lusuardi, M; Donner, C F

    1997-04-01

    An increase of alkaline phosphatase (ALP) activity has been observed in the bronchoalveolar lavage fluid (BALF) of patients affected by pulmonary fibrosis in chronic interstitial lung disorders. To characterize the ALP isoenzymes in such cases, we used gel filtration, agarose gel electrophoresis, heat and amino acid inhibition assays, wheat-germ agglutinin (WGA) precipitation, and an immunoassay specific for the bone-isoform of ALP. Only one anodic band representing a high-molecular-weight isoform of ALP (Mr approximately 2,000 kDa) was observed on electrophoresis of BALF. The inhibition assay results were consistent for a tissue-nonspecific isoenzyme sensitive to a temperature of 56 degrees C (71.9 +/- 2.5% inhibition) and to homoarginine (65.7 +/- 1.9%), and resistant to L-phenylalanine and L-leucine. Less than 13% of ALP activity was heat-stable. After incubation of BALF specimens with glycosyl-phosphatidylinositol-phospholipase D plus Nonidet P-40, or with phosphatidylinositol-phospholipase C alone, an electrophoretic cathodic band (Mr approximately 220 kDa) appeared near the bone band of a standard serum. With the WGA assay, 84.4 +/- 3.3% of ALP precipitated and the band disappeared. After immunoassay for the bone isoform, a mean of less than 5% enzyme activity was measured. We conclude that the ALP found in BALF is a pulmonary isoform of a tissue nonspecific isoenzyme.

  7. Hypoxic Pulmonary Vasoconstriction in Humans: Tale or Myth

    PubMed Central

    Hussain, A.; Suleiman, M.S.; George, S.J.; Loubani, M.; Morice, A.

    2017-01-01

    Hypoxic Pulmonary vasoconstriction (HPV) describes the physiological adaptive process of lungs to preserves systemic oxygenation. It has clinical implications in the development of pulmonary hypertension which impacts on outcomes of patients undergoing cardiothoracic surgery. This review examines both acute and chronic hypoxic vasoconstriction focusing on the distinct clinical implications and highlights the role of calcium and mitochondria in acute versus the role of reactive oxygen species and Rho GTPases in chronic HPV. Furthermore it identifies gaps of knowledge and need for further research in humans to clearly define this phenomenon and the underlying mechanism. PMID:28217180

  8. Hypoxia Does neither Stimulate Pulmonary Artery Endothelial Cell Proliferation in Mice and Rats with Pulmonary Hypertension and Vascular Remodeling nor in Human Pulmonary Artery Endothelial Cells

    PubMed Central

    Yu, Lunyin; Hales, Charles A.

    2011-01-01

    Background Hypoxia results in pulmonary hypertension and vascular remodeling due to induction of pulmonary artery cell proliferation. Besides pulmonary artery smooth muscle cells, pulmonary artery endothelial cells (PAECs) are also involved in the development of pulmonary hypertension, but the effect of hypoxia on PAEC proliferation has not been completely understood. Methods We investigated PAEC proliferation in mice and rats with hypoxia-induced pulmonary hypertension and vascular remodeling as well as in human PAECs under hypoxia. Results and Conclusion We did not find significant PAEC proliferation in chronically hypoxic rats or mice. There was a slight decrease in proliferation in mice and rats with pulmonary hypertension and vascular remodeling. We also did not find significant human PAEC proliferation and cell cycle progression under different levels of oxygen (1, 2, 3, 5 and 10%) for one day, although the same conditions of hypoxia induced significant proliferation and cell cycle progression in pulmonary artery smooth muscle cells and pulmonary artery fibroblasts. Exposure to hypoxia for 7 days also did not increase PAEC proliferation. These results demonstrated that hypoxia alone is not a stimulus to PAEC proliferation in vivo and in vitro. The present study provides a novel role for PAECs in hypoxia-induced pulmonary hypertension and vascular remodeling. PMID:21691120

  9. Human insulin microcrystals with lactose carriers for pulmonary delivery.

    PubMed

    Lim, Se-Hwan; Park, Hye Won; Shin, Chang-Hoon; Kwon, Jai-Hyun; Kim, Chan-Wha

    2009-12-01

    Dry powder formulations for pulmonary delivery are attractive because many issues of solubility and stability can be minimized. Human insulin microcrystals with lactose carriers were produced for pulmonary delivery. The average particle diameter was 2.3 microm, with a narrow, monodispersed size distribution. The percentages of high molecular weight proteins (%HMWPs), other insulin-related compounds (%OIRCs), and A-21 desamido insulin (%D(es)) were very low throughout the microcrystal preparation process. Administration of the microcrystal powder by intratracheal insufflation significantly reduced the blood glucose levels of Sprague-Dawley rats. The percent minimum reductions of the blood glucose concentration (%MRBG) produced by the insulin microcrystal powder and by an insulin solution reached 40.4% and 33.4% of the initial glucose levels respectively, and their bioavailability relative to subcutaneous injection (F) was 15% and 10% respectively. These results confirm that the insulin microcrystal powder prepared is suitable for pulmonary delivery in an effective dosage form.

  10. TLE1 promotes EMT in A549 lung cancer cells through suppression of E-cadherin.

    PubMed

    Yao, Xin; Ireland, Shubha Kale; Pham, Tri; Temple, Brandi; Chen, Renwei; Raj, Madhwa H G; Biliran, Hector

    2014-12-12

    The Groucho transcriptional corepressor TLE1 protein has recently been shown to be a putative lung specific oncogene, but its underlying oncogenic activity in lung cancer has not been fully elucidated. In this report, we investigated whether TLE1 regulates lung cancer aggressiveness using the human lung adenocarcinoma cell line A549 as a model system. Through a combination of genetic approaches, we found that TLE1 potentiates epithelial-to-mesenchymal transition (EMT) in A549 cells in part through suppression of the tumor suppressor gene E-cadherin. Exogenous expression of TLE1 in A549 cells resulted in heightened EMT phenotypes (enhanced fibroblastoid morphology and increased cell migratory potential) and in molecular alterations characteristic of EMT (downregulation of the epithelial marker E-cadherin and upregulation of the mesenchymal marker Vimentin). Conversely, downregulation of endogenous TLE1 expression in these cells resulted in reversal of basal EMT characterized by a cuboidal-like epithelial cell phenotype, reduced cell motility, and upregulated E-cadherin expression. Mechanistic studies showed that TLE1 suppresses E-cadherin expression at the transcriptional level in part by recruiting histone deacetylase (HDAC) activity to the E-cadherin promoter. Consistently, the HDAC inhibitor TSA partially reversed the TLE1-induced E-cadherin downregulation and cell migration, suggesting a role for HDACs in TLE1-mediated transcriptional repression of E-cadherin and EMT function. These findings uncover a novel role of TLE1 in regulating EMT in A549 cells through its repressive effect on E-cadherin and provide a mechanism for TLE1 oncogenic activity in lung cancer.

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

    PubMed

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

    2014-01-01

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

  12. Oxidative stress mediated apoptosis induced by nickel ferrite nanoparticles in cultured A549 cells.

    PubMed

    Ahamed, Maqusood; Akhtar, Mohd Javed; Siddiqui, Maqsood A; Ahmad, Javed; Musarrat, Javed; Al-Khedhairy, Abdulaziz A; AlSalhi, Mohamad S; Alrokayan, Salman A

    2011-05-10

    Due to the interesting magnetic and electrical properties with good chemical and thermal stabilities, nickel ferrite nanoparticles are being utilized in many applications including magnetic resonance imaging, drug delivery and hyperthermia. Recent studies have shown that nickel ferrite nanoparticles produce cytotoxicity in mammalian cells. However, there is very limited information concerning the toxicity of nickel ferrite nanoparticles at the cellular and molecular level. The aim of this study was to investigate the cytotoxicity, oxidative stress and apoptosis induction by well-characterized nickel ferrite nanoparticles (size 26 nm) in human lung epithelial (A549) cells. Nickel ferrite nanoparticles induced dose-dependent cytotoxicity in A549 cells demonstrated by MTT, NRU and LDH assays. Nickel ferrite nanoparticles were also found to induce oxidative stress evidenced by generation of reactive oxygen species (ROS) and depletion of antioxidant glutathione (GSH). Further, co-treatment with the antioxidant L-ascorbic acid mitigated the ROS generation and GSH depletion due to nickel ferrite nanoparticles suggesting the potential mechanism of oxidative stress. Quantitative real-time PCR analysis demonstrated that following the exposure of A549 cells to nickel ferrite nanoparticles, the level of mRNA expressions of cell cycle checkpoint protein p53 and apoptotic proteins (bax, caspase-3 and caspase-9) were significantly up-regulated, whereas the expression of anti-apoptotic proteins (survivin and bcl-2) were down-regulated. Moreover, activities of caspase-3 and caspase-9 enzymes were also significantly higher in nickel ferrite nanoparticles exposed cells. To the best of our knowledge this is the first report showing that nickel ferrite nanoparticles induced apoptosis in A549 cells through ROS generation and oxidative stress via p53, survivin, bax/bcl-2 and caspase pathways.

  13. Pulmonary ultrastructure of the late aspects of human paraquat poisoning.

    PubMed Central

    Dearden, L. C.; Fairshter, R. D.; McRae, D. M.; Smith, W. R.; Glauser, F. L.; Wilson, A. F.

    1978-01-01

    The pulmonary ultrastructure of the late aspects of a case of human paraquat poisoning is investigated and compared with normal human pulmonary ultrastructure. Alveoli in the paraquat patient are numerically reduced in comparison to the control. They are filled with edematous proteinaceous plasma-like fluid containing erythrocytes, macrophages, leukocytes, fibroblast-like cells, platelets, and fibrin. These alveoli are lined by granular pneumocytes. Interstitial areas in the paraquat patient are greatly expanded and there are no alveolar septums. Interstitial areas contain proteinaceous plasma-like material, collagen, fibrin, platelets, mature fibroblasts, plasma cells, many leukocytes, numerous erythrocytes, and capillaries. Capillary permeability seems to be enhanced in the paraquat patient either by vesicles forming transendothelial channels or pores or by disruption of endothelial cells. Images Figure 1 Figure 2 Figures 3-7 Figure 8 Figure 9 Figure 10 Figure 11 PMID:213978

  14. Halothane-induced alterations in cellular structure and proliferation of A549 cells.

    PubMed

    Stephanova, E; Topouzova-Hristova, T; Hazarosova, R; Moskova, V

    2008-12-01

    Genotoxicity, cytotoxicity or teratogenicity are among the well-known detrimental effects of the volatile anaesthetics. The aim of the present work was to study the structural changes, proliferative activity and the possibility of alveolar A549 cells to recover after in vitro exposure to halothane at 1.5 and 2.1mM concentrations. Our data indicated significant reduction of viability, suppression of mitotic activity more than 60%, and that these alterations were accompanied by disturbances of nuclear and nucleolar structures. The most prominent negative effect was the destruction of the lamellar bodies, the main storage organelles of pulmonary surfactant, substantial for the lung physiology. In conclusion, halothane applied at clinically relevant concentrations exerts genotoxic and cytotoxic effect on the alveolar cells in vitro, most likely as a consequence of stress-induced apoptosis, thus modulating the respiratory function.

  15. Transcriptome Sequencing Reveals Key Pathways and Genes Associated with Cisplatin Resistance in Lung Adenocarcinoma A549 Cells

    PubMed Central

    Fang, Yani; Zhang, Cheng; Wu, Tong; Wang, Qi; Liu, Jinhui; Dai, Penggao

    2017-01-01

    Acquired resistance to cisplatin-based chemotherapy frequently occurs in patients with non-small cell lung cancer, and the underlying molecular mechanisms are not well understood. The aim of this study was to investigate whether a distinct gene expression pattern is associated with acquired resistance to cisplatin in human lung adenocarcinoma. Whole-transcriptome sequencing was performed to compare the genome-wide gene expression patterns of the human lung adenocarcinoma A549 cisplatin-resistant cell line A549/DDP with those of its progenitor cell line A549. A total of 1214 differentially expressed genes (DEGs) were identified, 656 of which were upregulated and 558 were downregulated. Functional annotation of the DEGs in the Kyoto Encyclopedia of Genes and Genomes database revealed that most of the identified genes were enriched in the PI3K/AKT, mitogen-activated protein kinase, actin cytoskeleton regulation, and focal adhesion pathways in A549/DDP cells. These results support previous studies demonstrating that the pathways regulating cell proliferation and invasion confer resistance to chemotherapy. Furthermore, the results proved that cell adhesion and cytoskeleton regulation is associated with cisplatin resistance in human lung cancer. Our study provides new promising biomarkers for lung cancer prognosis and potential therapeutic targets for lung cancer treatment. PMID:28114404

  16. Primary pulmonary hypertension, Castleman's disease and human herpesvirus-8.

    PubMed

    Bull, T M; Cool, C D; Serls, A E; Rai, P R; Parr, J; Neid, J M; Geraci, M W; Campbell, T B; Voelkel, N F; Badesch, D B

    2003-09-01

    Primary pulmonary hypertension (PPH) and Castleman's disease (CD) are rare conditions infrequently encountered in clinical practice. In this paper, two patients diagnosed with both of these diseases are reported. The authors speculate that rather than being a chance occurrence, these conditions are linked by a common angio-proliferative mechanism. Therefore, an association between infection with the human herpesvirus-8 and the diseases of PPH and CD was sought. Evidence of human herpesvirus-8 infection was found in the lung tissue and, specifically, in the plexiform lesions from one of the patients.

  17. Toll-like receptor 5 agonist inhibition of growth of A549 lung cancer cells in vivo in a Myd88 dependent manner.

    PubMed

    Zhou, Shi-Xiang; Li, Feng-Sheng; Qiao, Yu-Lei; Zhang, Xue-Qing; Wang, Zhi-Dong

    2012-01-01

    The purpose of this study was to examine the effect of a Toll-like receptor 5 (TLR5) agonist, CBLB502, on the growth and radiosensitivity of A549 lung cancer cells in vivo. Expression of myeloid differentiation factor 88 (MyD88) or TLR5 was stably knocked down in human lung cancer cells (A549) using lentivirus expressing short hairpin RNA targeting human MyD88 or TLR5. Lack of MyD88 or TLR5 expression enhanced tumor growth in mouse xenografts of A549 lung cancer cells. CBLB502 inhibited the growth of A549 lung cancer cells, not A549-MyD88-KD cells in vivo in the murine xenograft model. Our results showed that the inhibition of A549 by CBLB502 in vivo was realized through regulating the expression of neutrophil recruiting cytokines and neutrophil infiltration. Finally, we found that activation of TLR5 signaling did not affect the radiosensitivity of tumors in vivo.

  18. Discovery of 2'-hydroxychalcones as autophagy inducer in A549 lung cancer cells.

    PubMed

    Wang, Fang-Wu; Wang, Sheng-Qing; Zhao, Bao-Xiang; Miao, Jun-Ying

    2014-05-21

    A series of 2'-hydroxychalcone derivatives was synthesized and the effects of all the compounds on growth of A549 lung cancer cell were investigated. The results showed that all compounds had inhibitory effects on the growth of A549 lung cancer cells and compound possessed the highest growth inhibitory effect and induced autophagy of A549 lung cancer cells.

  19. Characterization of optimal resting tension in human pulmonary arteries

    PubMed Central

    Hussain, Azar; Bennett, Robert T; Chaudhry, Mubarak A; Qadri, Syed S; Cowen, Mike; Morice, Alyn H; Loubani, Mahmoud

    2016-01-01

    AIM To determine the optimum resting tension (ORT) for in vitro human pulmonary artery (PA) ring preparations. METHODS Pulmonary arteries were dissected from disease free sections of the resected lung in the operating theatre and tissue samples were directly sent to the laboratory in Krebs-Henseleit solution (Krebs). The pulmonary arteries were then cut into 2 mm long rings. PA rings were mounted in 25 mL organ baths or 8 mL myograph chambers containing Krebs compound (37 °C, bubbled with 21% O2: 5% CO2) to measure changes in isometric tension. The resting tension was set at 1-gram force (gf) with vessels being left static to equilibrate for duration of one hour. Baseline contractile reactions to 40 mmol/L KCl were obtained from a resting tension of 1 gf. Contractile reactions to 40 mmol/L KCl were then obtained from stepwise increases in resting tension (1.2, 1.4, 1.6, 1.8 and 2.0 gf). RESULTS Twenty PA rings of internal diameter between 2-4 mm were prepared from 4 patients. In human PA rings incrementing the tension during rest stance by 0.6 gf, up to 1.6 gf significantly augmented the 40 mmol/L KCl stimulated tension. Further enhancement of active tension by 0.4 gf, up to 2.0 gf mitigate the 40 mmol/L KCl stimulated reaction. Both Myograph and the organ bath demonstrated identical conclusions, supporting that the radial optimal resting tension for human PA ring was 1.61 g. CONCLUSION The radial optimal resting tension in our experiment is 1.61 gf (15.78 mN) for human PA rings. PMID:27721938

  20. Apoptosis inducing ability of silver decorated highly reduced graphene oxide nanocomposites in A549 lung cancer.

    PubMed

    Khan, Merajuddin; Khan, Mujeeb; Al-Marri, Abdulhadi H; Al-Warthan, Abdulrahman; Alkhathlan, Hamad Z; Siddiqui, Mohammed Rafiq H; Nayak, Vadithe Lakshma; Kamal, Ahmed; Adil, Syed F

    2016-01-01

    Recently, graphene and graphene-based materials have been increasingly used for various biological applications due to their extraordinary physicochemical properties. Here, we demonstrate the anticancer properties and apoptosis-inducing ability of silver doped highly reduced graphene oxide nanocomposites synthesized by employing green approach. These nano composites (PGE-HRG-Ag) were synthesized by using Pulicaria glutinosa extract (PGE) as a reducing agent and were evaluated for their anticancer properties against various human cancer cell lines with tamoxifen as the reference drug. A correlation between the amount of Ag nanoparticles on the surface of highly reduced graphene oxide (HRG) and the anticancer activity of nanocomposite was observed, wherein an increase in the concentration of Ag nanoparticles on the surface of HRG led to the enhanced anticancer activity of the nanocomposite. The nanocomposite PGE-HRG-Ag-2 exhibited more potent cytotoxicity than standard drug in A549 cells, a human lung cancer cell line. A detailed investigation was undertaken and Fluorescence activated cell sorting (FACS) analysis demonstrated that the nanocomposite PGE-HRG-Ag-2 showed G0/G1 phase cell cycle arrest and induced apoptosis in A549 cells. Studies such as, measurement of mitochondrial membrane potential, generation of reactive oxygen species (ROS) and Annexin V-FITC staining assay suggested that this compound induced apoptosis in human lung cancer cells.

  1. Pulmonary epithelial CCR3 promotes LPS-induced lung inflammation by mediating release of IL-8.

    PubMed

    Li, Bo; Dong, Chunling; Wang, Guifang; Zheng, Huiru; Wang, Xiangdong; Bai, Chunxue

    2011-09-01

    Interleukin (IL)-8 from pulmonary epithelial cells has been suggested to play an important role in the airway inflammation, although the mechanism remains unclear. We envisioned a possibility that pulmonary epithelial CCR3 could be involved in secretion and regulation of IL-8 and promote lipopolysaccharide (LPS)-induced lung inflammation. Human bronchial epithelial cell line NCI-H292 and alveolar type II epithelial cell line A549 were used to test role of CCR3 in production of IL-8 at cellular level. In vivo studies were performed on C57/BL6 mice instilled intratracheally with LPS in a model of acute lung injury (ALI). The activity of a CCR3-specific inhibitor (SB-328437) was measured in both in vitro and in vivo systems. We found that expression of CCR3 in NCI-H292 and A549 cells were increased by 23% and 16%, respectively, 24 h after the challenge with LPS. LPS increased the expression of CCR3 in NCI-H292 and A549 cells in a time-dependent manner, which was inhibited significantly by SB-328437. SB-328437 also diminished neutrophil recruitment in alveolar airspaces and improved LPS-induced ALI and production of IL-8 in bronchoalveolar lavage fluid. These results suggest that pulmonary epithelial CCR3 be involved in progression of LPS-induced lung inflammation by mediating release of IL-8. CCR3 in pulmonary epithelia may be an attractive target for development of therapies for ALI.

  2. Pulmonary Tuberculosis in Humanized Mice Infected with HIV-1

    PubMed Central

    Nusbaum, Rebecca J.; Calderon, Veronica E.; Huante, Matthew B.; Sutjita, Putri; Vijayakumar, Sudhamathi; Lancaster, Katrina L.; Hunter, Robert L.; Actor, Jeffrey K.; Cirillo, Jeffrey D.; Aronson, Judith; Gelman, Benjamin B.; Lisinicchia, Joshua G.; Valbuena, Gustavo; Endsley, Janice J.

    2016-01-01

    Co-infection with HIV increases the morbidity and mortality associated with tuberculosis due to multiple factors including a poorly understood microbial synergy. We developed a novel small animal model of co-infection in the humanized mouse to investigate how HIV infection disrupts pulmonary containment of Mtb. Following dual infection, HIV-infected cells were localized to sites of Mtb-driven inflammation and mycobacterial replication in the lung. Consistent with disease in human subjects, we observed increased mycobacterial burden, loss of granuloma structure, and increased progression of TB disease, due to HIV co-infection. Importantly, we observed an HIV-dependent pro-inflammatory cytokine signature (IL-1β, IL-6, TNFα, and IL-8), neutrophil accumulation, and greater lung pathology in the Mtb-co-infected lung. These results suggest that in the early stages of acute co-infection in the humanized mouse, infection with HIV exacerbates the pro-inflammatory response to pulmonary Mtb, leading to poorly formed granulomas, more severe lung pathology, and increased mycobacterial burden and dissemination. PMID:26908312

  3. Pulmonary Tuberculosis in Humanized Mice Infected with HIV-1.

    PubMed

    Nusbaum, Rebecca J; Calderon, Veronica E; Huante, Matthew B; Sutjita, Putri; Vijayakumar, Sudhamathi; Lancaster, Katrina L; Hunter, Robert L; Actor, Jeffrey K; Cirillo, Jeffrey D; Aronson, Judith; Gelman, Benjamin B; Lisinicchia, Joshua G; Valbuena, Gustavo; Endsley, Janice J

    2016-02-24

    Co-infection with HIV increases the morbidity and mortality associated with tuberculosis due to multiple factors including a poorly understood microbial synergy. We developed a novel small animal model of co-infection in the humanized mouse to investigate how HIV infection disrupts pulmonary containment of Mtb. Following dual infection, HIV-infected cells were localized to sites of Mtb-driven inflammation and mycobacterial replication in the lung. Consistent with disease in human subjects, we observed increased mycobacterial burden, loss of granuloma structure, and increased progression of TB disease, due to HIV co-infection. Importantly, we observed an HIV-dependent pro-inflammatory cytokine signature (IL-1β, IL-6, TNFα, and IL-8), neutrophil accumulation, and greater lung pathology in the Mtb-co-infected lung. These results suggest that in the early stages of acute co-infection in the humanized mouse, infection with HIV exacerbates the pro-inflammatory response to pulmonary Mtb, leading to poorly formed granulomas, more severe lung pathology, and increased mycobacterial burden and dissemination.

  4. 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

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

    PubMed

    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.

  6. Human immunodeficiency virus, herpes virus infections, and pulmonary vascular disease

    PubMed Central

    Flores, Sonia C.; Almodovar, Sharilyn

    2013-01-01

    The following state-of-the-art seminar was delivered as part of the Aspen Lung Conference on Pulmonary Hypertension and Vascular Diseases held in Aspen, Colorado in June 2012. This paper will summarize the lecture and present results from a nonhuman primate model of infection with Simian (Human) Immunodeficiency Virus - nef chimeric virions as well as the idea that polymorphisms in the HIV-1 nef gene may be driving the immune response that results in exuberant inflammation and aberrant endothelial cell (EC) function. We will present data gathered from primary HIV nef isolates where we tested the biological consequences of these polymorphisms and how their presence in human populations may predict patients at risk for developing this disease. In this article, we also discuss how a dysregulated immune system, in conjunction with a viral infection, could contribute to pulmonary arterial hypertension (PAH). Both autoimmune diseases and some viruses are associated with defects in the immune system, primarily in the function of regulatory T cells. These T-cell defects may be a common pathway in the formation of plexiform lesions. Regardless of the route by which viruses may lead to PAH, it is important to recognize their role in this rare disease. PMID:23662195

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

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

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

  8. [High altitude pulmonary edema. An experiment of nature to study the underlying mechanisms of hypoxic pulmonary hypertension and pulmonary edema in humans].

    PubMed

    Schwab, Marcos; Jayet, Pierre-Yves; Allemann, Yves; Sartori, Claudio; Scherrer, Urs

    2007-01-01

    High altitude constitutes an exciting natural laboratory for medical research. Over the past decade, it has become clear that the results of high-altitude research may have important implications not only for the understanding of diseases in the millions of people living permanently at high altitude, but also for the treatment of hypoxemia-related disease states in patients living at low altitude. High-altitude pulmonary edema (HAPE) is a life-threatening condition occurring in predisposed, but otherwise healthy subjects, and, therefore, allows to study underlying mechanisms of pulmonary edema in humans, in the absence of confounding factors. Over the past decade, evidence has accumulated that HAPE results from the conjunction of two major defects, augmented alveolar fluid flooding resulting from exaggerated hypoxic pulmonary hypertension, and impaired alveolar fluid clearance related to defective respiratory transepithelial sodium transport. Here, after a brief presentation of the clinical features of HAPE, we review this novel concept. We provide experimental evidence for the novel concept that impaired pulmonary endothelial and epithelial nitric oxide synthesis and/or bioavailability may represent the central underlying defect predisposing to exaggerated hypoxic pulmonary vasoconstriction and alveolar fluid flooding. We demonstrate that exaggerated pulmonary hypertension, while possibly a condition sine qua non, may not be sufficient to cause HAPE, and how defective alveolar fluid clearance may represent a second important pathogenic mechanism. Finally, we outline how this insight gained from studies in HAPE may be translated into the management of hypoxemia related disease states in general.

  9. Duration of pulmonary function adaptation to ozone in humans

    SciTech Connect

    Kulle, T.J.; Sauder, L.R.; Kerr, H.D.; Farrell, B.P.; Bermel, M.S.; Smith, D.M.

    1982-11-01

    The duration of pulmonary function adaptation subsequent to cessation of a 5-day repeated ozone (O/sub 3/) exposure was studied in 24 nonsmoking human subjects. A three-week, 3 hr/day study ws conducted. The subjects received filtered air on Week 1 and 0.4 ppm O/sub 3/ on Week 2. During Week 3, 13 subjects were re-exposed to O/sub 3/ on Friday and 11 were re-exposed to O/sub 3/ on Tuesday. Spirometric measurements (FVC and FEV/sub 1/) and bronchial reactivity to methacholine showed adapation within 2-3 days of the repeated daily exposures (Week 2). Although the duration of adaptation seen with bronchial reactivity appears longer than 7-days, the FVC and FEV/sub 1/ clearly demonstrated complete loss of adaptation by 7 days, with a trend toward significance by 4 days. We concluded, therefore, the loss of ozone adaptation in pulmonary function is a gradual phenomenon lasting less than 7 days following cessation of repeated daily exposures.

  10. Gold nanoparticles induce cytotoxicity in the alveolar type-II cell lines A549 and NCIH441

    PubMed Central

    Uboldi, Chiara; Bonacchi, Daniele; Lorenzi, Giada; Hermanns, M Iris; Pohl, Christine; Baldi, Giovanni; Unger, Ronald E; Kirkpatrick, C James

    2009-01-01

    Background During the last years engineered nanoparticles (NPs) have been extensively used in different technologies and consequently many questions have arisen about the risk and the impact on human health following exposure to nanoparticles. Nevertheless, at present knowledge about the cytotoxicity induced by NPs is still largely incomplete. In this context, we have investigated the cytotoxicity induced by gold nanoparticles (AuNPs), which differed in size and purification grade (presence or absence of sodium citrate residues on the particle surface) in vitro, in the human alveolar type-II (ATII)-like cell lines A549 and NCIH441. Results We found that the presence of sodium citrate residues on AuNPs impaired the viability of the ATII-like cell lines A549 and NCIH441. Interestingly, the presence of an excess of sodium citrate on the surface of NPs not only reduced the in vitro viability of the cell lines A549 and NCIH441, as shown by MTT assay, but also affected cellular proliferation and increased the release of lactate dehydrogenase (LDH), as demonstrated by Ki-67 and LDH-release assays respectively. Furthermore, we investigated the internalization of AuNPs by transmission electron microscopy (TEM) and we observed that particles were internalized by active endocytosis in the cell lines A549 and NCIH441 within 3 hr. In addition, gold particles accumulated in membrane-bound vesicles and were not found freely dispersed in the cytoplasm. Conclusion Our data suggest that the presence of contaminants, such as sodium citrate, on the surface of gold nanoparticles might play a pivotal role in inducing cytotoxicity in vitro, but does not influence the uptake of the particles in human ATII-like cell lines. PMID:19545423

  11. Inflammatory stimuli inhibit glucocorticoid-dependent transactivation in human pulmonary epithelial cells: rescue by long-acting beta2-adrenoceptor agonists.

    PubMed

    Rider, Christopher F; King, Elizabeth M; Holden, Neil S; Giembycz, Mark A; Newton, Robert

    2011-09-01

    By repressing inflammatory gene expression, glucocorticoids are the most effective treatment for chronic inflammatory diseases such as asthma. However, in some patients with severe disease, or who smoke or suffer from chronic obstructive pulmonary disease, glucocorticoids are poorly effective. Although many investigators focus on defects in the repression of inflammatory gene expression, glucocorticoids also induce (transactivate) the expression of numerous genes to elicit anti-inflammatory effects. Using human bronchial epithelial (BEAS-2B) and pulmonary (A549) cells, we show that cytokines [tumor necrosis factor α (TNFα) and interleukin 1β], mitogens [fetal calf serum (FCS) and phorbol ester], cigarette smoke, and a G(q)-linked G protein-coupled receptor agonist attenuate simple glucocorticoid response element (GRE)-dependent transcription. With TNFα and FCS, this effect was not overcome by increasing concentrations of dexamethasone, budesonide, or fluticasone propionate. Thus, the maximal ability of the glucocorticoid to promote GRE-dependent transcription was reduced, and this was shown additionally for the glucocorticoid-induced gene p57(KIP2). The long-acting β(2)-adrenoceptor agonists (LABAs) formoterol fumarate and salmeterol xinafoate enhanced simple GRE-dependent transcription to a level that could not be achieved by glucocorticoid alone. In the presence of TNFα or FCS, which repressed glucocorticoid responsiveness, these LABAs restored glucocorticoid-dependent transcription to levels that were achieved by glucocorticoid alone. Given the existence of genes, such as p57(KIP2), which may mediate anti-inflammatory actions of glucocorticoids, we propose that repression of transactivation represents a mechanism for glucocorticoid resistance and for understanding the clinical benefit of LABAs as an add-on therapy in asthma and chronic obstructive pulmonary disease.

  12. A novel polysaccharide from Sargassum integerrimum induces apoptosis in A549 cells and prevents angiogensis in vitro and in vivo

    PubMed Central

    Liu, Ge; Kuang, Shan; Wu, Shimei; Jin, Weihua; Sun, Chaomin

    2016-01-01

    Many polysaccharides isolated from plants have exhibited promising antitumor activities. The aim of this study is to investigate the antitumor activity of the novel polysaccharide named SPS from Sargassum integerrimum, elucidate the underlying anticancer mechanism in a human lung cancer cell line A549, and evaluate its anti-angiogenic activity both in vitro and in vivo. The results show that SPS significantly reduces A549 cells viability in a dose- and time-dependent manner via MTT method. Flow cytometry analysis indicates that SPS could induce cell apoptosis, the loss of mitochondrial membrane potential (MMP), generation of reactive oxygen species (ROS) and G2/M phase cell cycle arrest of A549 cells. Up-regulation of the expressions of P53 and Bax, down-regulation of the expression of Bcl-2, and activation of cleaved caspase-3, caspase-9 and PARP are also detected by western blotting after the treatment of SPS. In addition, SPS inhibits the proliferation, migration and cord formation of human umbilical vein endothelial cells (HUVECs) in vitro, and prevents the vascular development of zebrafish embryos in vivo. Altogether, our data prove the anticancer and anti-angiogenesis properties of SPS, and provide further insights into the potential pharmacological application of SPS as antitumor and anti-angiogenic agent against lung cancer. PMID:27216943

  13. Applying cybernetic technology to diagnose human pulmonary sounds.

    PubMed

    Chen, Mei-Yung; Chou, Cheng-Han

    2014-06-01

    Chest auscultation is a crucial and efficient method for diagnosing lung disease; however, it is a subjective process that relies on physician experience and the ability to differentiate between various sound patterns. Because the physiological signals composed of heart sounds and pulmonary sounds (PSs) are greater than 120 Hz and the human ear is not sensitive to low frequencies, successfully making diagnostic classifications is difficult. To solve this problem, we constructed various PS recognition systems for classifying six PS classes: vesicular breath sounds, bronchial breath sounds, tracheal breath sounds, crackles, wheezes, and stridor sounds. First, we used a piezoelectric microphone and data acquisition card to acquire PS signals and perform signal preprocessing. A wavelet transform was used for feature extraction, and the PS signals were decomposed into frequency subbands. Using a statistical method, we extracted 17 features that were used as the input vectors of a neural network. We proposed a 2-stage classifier combined with a back-propagation (BP) neural network and learning vector quantization (LVQ) neural network, which improves classification accuracy by using a haploid neural network. The receiver operating characteristic (ROC) curve verifies the high performance level of the neural network. To expand traditional auscultation methods, we constructed various PS diagnostic systems that can correctly classify the six common PSs. The proposed device overcomes the lack of human sensitivity to low-frequency sounds and various PS waves, characteristic values, and a spectral analysis charts are provided to elucidate the design of the human-machine interface.

  14. Wheatgrass Extract Ameliorates Hypoxia-induced Mucin Gene Expression in A549 cells

    PubMed Central

    Sim, Ju hwan; Choi, Moon-Hee; Shin, Hyun-Jae; Lee, Ji-Eun

    2017-01-01

    Background: Wheatgrass is known to have antioxidant, antiaging, and anti-inflammatory effect. However, its protective effect against hypoxia is not yet evaluated. Objective: In this study, we evaluated the protective and anti-inflammatory effect of wheatgrass against the hypoxia in airway epithelial cells. Materials and Methods: A549 human lung adenocarcinoma cells were incubated in a hypoxic condition (CO2 5%/O2 1%) for 24 hr in the presence of different concentration of wheatgrass 50, 75, 100, and 150 μg/mL, and the magnitude of each immunologic response produced by the A549 cells was compared. The mRNA expression level of mucin gene (MUC), 5A, 5B, 8, GM-CSF, TNF-α, and VEGF were evaluated by using real-time polymerase chain reaction. The MUC proteins level before and after knocking out the hypoxia-inducible factor (hif)-1α via short interfering (si) RNA transfection were assessed by immunoblot analysis. Accordingly, the involved cell signaling pathway was evaluated by immunoblot analysis. Results: The inflammatory cytokines (GM-CSF, TNF- α) and the expressions of MUC 5A, 5B, and 8 were augmented by hypoxia. The augmented MUC expression was decreased by the wheatgrass extract administration. Hif-1α gene expression after hypoxia exposure was decreased by wheatgrass. Knockdown of hif-1α by siRNA reduced the mucin gene expression and which was more enhanced by wheatgrass extract. Conclusion: Theses results suggest that wheatgrass may be useful in the treatment of sinonasal disease by inhibiting mucus hypersecretion in airway epithelium. SUMMARY Wheatgrass extract decreases the hypoxia-induced MUC 5A, 5B and 8 expression.Hif-1α gene expression after hypoxia exposure was decreased by wheatgrass.Wheatgrass inhibits p44/42 phosphorylation in hypoxia-exposed airway epithelial cells. Abbreviations used: A549: human lung adenocarcinoma cells, GM-CSF: granulocyte-macrophage colony stimulating factor, HIF: hypoxia inducible factor, IL: interleukin, MUC: mucin, MTT: 3

  15. Copper dependence of angioproliferation in pulmonary arterial hypertension in rats and humans.

    PubMed

    Bogaard, Harm J; Mizuno, Shiro; Guignabert, Christophe; Al Hussaini, Aysar A; Farkas, Daniela; Ruiter, Gerrina; Kraskauskas, Donatas; Fadel, Elie; Allegood, Jeremy C; Humbert, Marc; Vonk Noordegraaf, Anton; Spiegel, Sarah; Farkas, Laszlo; Voelkel, Norbert F

    2012-05-01

    Obliteration of the vascular lumen by endothelial cell growth is a hallmark of many forms of severe pulmonary arterial hypertension. Copper plays a significant role in the control of endothelial cell proliferation in cancer and wound-healing. We sought to determine whether angioproliferation in rats with experimental pulmonary arterial hypertension and pulmonary microvascular endothelial cell proliferation in humans depend on the proangiogenic action of copper. A copper-depleted diet prevented, and copper chelation with tetrathiomolybdate reversed, the development of severe experimental pulmonary arterial hypertension. The copper chelation-induced reopening of obliterated vessels was caused by caspase-independent apoptosis, reduced vessel wall cell proliferation, and a normalization of vessel wall structure. No evidence was found for a role of super oxide-1 inhibition or lysyl-oxidase-1 inhibition in the reversal of angioproliferation. Tetrathiomolybdate inhibited the proliferation of human pulmonary microvascular endothelial cells, isolated from explanted lungs from control subjects and patients with pulmonary arterial hypertension. These data suggest that the inhibition of endothelial cell proliferation by a copper-restricting strategy could be explored as a new therapeutic approach in pulmonary arterial hypertension. It remains to be determined, however, whether potential toxicity to the right ventricle is offset by the beneficial pulmonary vascular effects of antiangiogenic treatment in patients with pulmonary arterial hypertension.

  16. 13-Methyl-palmatrubine induces apoptosis and cell cycle arrest in A549 cells in vitro and in vivo

    PubMed Central

    Chen, Jingxian; Lu, Xingang; Lu, Chenghua; Wang, Chunying; Xu, Haizhu; Xu, Xiaoli; Gou, Haixin; Zhu, Bing; Du, Wangchun

    2016-01-01

    Corydalis yanhusuo, a well-known herbaceous plant, is commonly used in the treatment of inflammation, injury and pain. One natural agent isolated from Corydalis yanhusuo, 13-methyl-palmatrubine, was found to have a cytotoxic effect on cancer cells as reported in published studies. In the present study, we synthesized a potential anti-lung tumor agent, 13-methyl-palmatrubine and analyzed its activity. 13-Methyl-palmatrubine exhibited a cytotoxic effect on a panel of cancer cell lines in a time- and concentration-dependent manner. Among all the tested cancer cell lines, lung cancer A549 cells were most sensitive to 13-methyl-palmatrubine treatment. Meanwhile 13-methyl-palmatrubine showed less cytotoxicity in human normal cells. Our investigation revealed that 13-methyl-palmatrubine induced apoptosis and cell cycle arrest in A549 cells in a dose-dependent manner. Furthermore, 13-methyl-palmatrubine treatment caused activation of P38 and JNK pathways and blocked the EGFR pathway. In conclusion, our findings demonstrated that 13-methyl-palmatrubine inhibited the growth of A549 cells mediated by blocking of the EGFR signaling pathway and activation of the MAPK signaling pathway and provides a better understanding of the molecular mechanisms of 13-methyl-palmatrubine. PMID:27633656

  17. Suppression of SCARA5 by Snail1 is essential for EMT-associated cell migration of A549 cells

    PubMed Central

    Liu, J; Hu, G; Chen, D; Gong, A-Y; Soori, G S; Dobleman, T J; Chen, X-M

    2013-01-01

    Accumulating evidence indicates that epithelial-to-mesenchymal transition (EMT) might be a key event for cancer progression. The upregulation of Snail1, one of the most extensively studied EMT regulators, has been implicated in cancer metastasis, but the underlying mechanisms remain unclear. This study aims to identify that Snail1 targets regulating EMT-associated cancer cell migration. Human lung carcinoma A549 cells were treated with transforming growth factor beta 1 (TGF-β1), and EMT-associated phenotypic and functional alterations were monitored. TGF-β1 induced typical EMT-like morphological changes, ‘cadherin switching' and cell migration in A549 cells. TGF-β1 stimulation induced rapid and persistent upregulation of Snail1. Moreover, Snail1 upregulation was required for EMT-associated cell migration. Several metastasis suppressors with putative Snail1-binding sites in their promoters were dramatically repressed in A549 cells during TGF-β1-induced EMT. Gain- and loss-of Snail1 function experiments demonstrated that scavenger receptor class A member 5 (SCARA5) was negatively regulated by Snail1. Importantly, SCARA5 downregulation was essential for EMT-induced migration in A549 cells. The chromatin immunoprecipitation assay revealed that Snail1 could bind to the E-box elements in SCARA5 promoter, implying that SCARA5 is a direct Snail1 target modulating cancer cell mobility during EMT. In addition, we showed that DNA methyltransferase 1 was physically associated with Snail1 to silence SCARA5 expression with an unidentified DNA methylation-independent mechanism, suggesting the complexity of Snail1-mediated epigenetic regulation. Collectively, our data demonstrated that EMT-regulator Snail1 suppresses the expression of SCARA5 to promote cancer progression, highlighting the possibility to target Snail1 and SCARA5 for cancer treatment. PMID:24061576

  18. Suppression of SCARA5 by Snail1 is essential for EMT-associated cell migration of A549 cells.

    PubMed

    Liu, J; Hu, G; Chen, D; Gong, A-Y; Soori, G S; Dobleman, T J; Chen, X-M

    2013-09-23

    Accumulating evidence indicates that epithelial-to-mesenchymal transition (EMT) might be a key event for cancer progression. The upregulation of Snail1, one of the most extensively studied EMT regulators, has been implicated in cancer metastasis, but the underlying mechanisms remain unclear. This study aims to identify that Snail1 targets regulating EMT-associated cancer cell migration. Human lung carcinoma A549 cells were treated with transforming growth factor beta 1 (TGF-β1), and EMT-associated phenotypic and functional alterations were monitored. TGF-β1 induced typical EMT-like morphological changes, 'cadherin switching' and cell migration in A549 cells. TGF-β1 stimulation induced rapid and persistent upregulation of Snail1. Moreover, Snail1 upregulation was required for EMT-associated cell migration. Several metastasis suppressors with putative Snail1-binding sites in their promoters were dramatically repressed in A549 cells during TGF-β1-induced EMT. Gain- and loss-of Snail1 function experiments demonstrated that scavenger receptor class A member 5 (SCARA5) was negatively regulated by Snail1. Importantly, SCARA5 downregulation was essential for EMT-induced migration in A549 cells. The chromatin immunoprecipitation assay revealed that Snail1 could bind to the E-box elements in SCARA5 promoter, implying that SCARA5 is a direct Snail1 target modulating cancer cell mobility during EMT. In addition, we showed that DNA methyltransferase 1 was physically associated with Snail1 to silence SCARA5 expression with an unidentified DNA methylation-independent mechanism, suggesting the complexity of Snail1-mediated epigenetic regulation. Collectively, our data demonstrated that EMT-regulator Snail1 suppresses the expression of SCARA5 to promote cancer progression, highlighting the possibility to target Snail1 and SCARA5 for cancer treatment.

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

    PubMed

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

    2017-01-01

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

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

    PubMed Central

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

    2017-01-01

    Period2 (Per2) is a key mammalian circadian clock protein, and additionally has a tumor suppressive function. The present study aimed to investigate its role in drug resistance in A549/cisplatin (DDP) lung adenocarcinoma cells. Per2 knockdown and overexpression in A549/DDP cells were used to compare cell proliferation (by MTT assay), apoptosis (active-caspase 3 western blot) and clone forming assay. The activation of AKT/mechanistic target of rapamycin (mTOR) was investigated by a western blot assay. The Per2 expression level was decreased in A549/DDP cells compared with A549 cells. Per2 knockdown by short hairpin RNA protects A549/DDP cells from apoptosis, and promotes proliferation and migration. Per2 knockdown results in increased activation of the phosphoinositide 3-kinase (PI3K)/AKT/mTOR signaling pathway. Overexpression of Per2 in A549/DDP cells may reduce the activity of the PI3K/AKT/mTOR signaling pathway, and promote apoptosis of A549 cells. The results of the present study suggest that Per2 participates in AKT-mediated drug resistance in A549/DDP lung adenocarcinoma cells. PMID:28123577

  1. Calcium is not required for triggering volume restoration in hypotonically challenged A549 epithelial cells.

    PubMed

    Ponomarchuk, Olga; Boudreault, Francis; Orlov, Sergei N; Grygorczyk, Ryszard

    2016-11-01

    Maintenance of cell volume is a fundamental housekeeping function in eukaryotic cells. Acute cell swelling activates a regulatory volume decrease (RVD) process with poorly defined volume sensing and intermediate signaling mechanisms. Here, we analyzed the putative role of Ca(2+) signaling in RVD in single substrate-adherent human lung epithelial A549 cells. Acute cell swelling was induced by perfusion of the flow-through imaging chamber with 50 % hypotonic solution at a defined fluid turnover rate. Changes in cytosolic Ca(2+) concentration ([Ca(2+)]i) and cell volume were monitored simultaneously with ratiometric Fura-2 fluorescence and 3D reconstruction of stereoscopic single-cell images, respectively. Hypotonic challenge caused a progressive swelling peaking at ∼20 min and followed, during the next 20 min, by RVD of 60 ± 7 % of the peak volume increase. However, at the rate of swelling used in our experiments, these processes were not accompanied by a measurable increment of [Ca(2+)]i. Loading with intracellular Ca(2+) chelator BAPTA slightly delayed peak of swelling but did not prevent RVD in 82 % of cells. Further, electrophysiology whole-cell patch-clamp experiments showed that BAPTA did not block activation of volume-regulated anion channel (VRAC) measured as swelling-induced outwardly rectifying 5-nitro-2-(3-phenylpropyl-amino) benzoic acid sensitive current. Together, our data suggest that intracellular Ca(2+)-mediated signaling is not essential for VRAC activation and subsequent volume restoration in A549 cells.

  2. Human Regional Pulmonary Gas Exchange with Xenon Polarization Transfer (XTC)

    NASA Astrophysics Data System (ADS)

    Muradian, Iga; Butler, James; Hrovat, Mirko; Topulos, George; Hersman, Elizabeth; Ruset, Iulian; Covrig, Silviu; Frederick, Eric; Ketel, Stephen; Hersman, F. W.; Patz, Samuel

    2007-03-01

    Xenon Transfer Contrast (XTC) is an existing imaging method (Ruppert et al, Magn Reson Med, 51:676-687, 2004) that measures the fraction F of ^129Xe magnetization that diffuses from alveolar gas spaces to septal parenchymal tissue in lungs in a specified exchange time. As previously implemented, XTC is a 2-breath method and has been demonstrated in anesthetized animals. To use XTC in humans and to avoid issues associated with obtaining identical gas volumes on subsequent breath-hold experiments as well as precise image registration in post-processing, a single breath XTC method was developed that acquires three consecutive gradient echo images in an 8s acquisition. We report here initial measurements of the mean and variance of F for 5 normal healthy subjects as well as 7 asymptomatic smokers. The experiments were performed at two lung volumes (˜45 and 65% of TLC). We found that both the mean and variance of F increased with smoking history. In comparison, standard pulmonary function tests such as DLCO FEV1 showed no correlation with smoking history.

  3. Long-chain carboxychromanols are the major metabolites of tocopherols and tocotrienols in A549 lung epithelial cells but not HepG2 cells.

    PubMed

    You, Cha-Sook; Sontag, Timothy J; Swanson, Joy E; Parker, Robert S

    2005-02-01

    Human lung type II cell derived A549 epithelial cancer cells and HepG2 hepatocytes constitutively express cytochrome P4504F2, a P450 we previously identified as a tocopherol-omega-hydroxylase. To determine if A549 cells would metabolize tocochromanols via the omega-hydroxylase pathway, we compared the metabolism of tocopherols (alpha-, gamma-, delta-TOH) and tocotrienols (alpha-, gamma-, delta-T3) in these 2 cell lines. Cultures were incubated with alpha-, gamma-, or delta-TOH, or the analogous T3s, and synthesis of their metabolites quantitated by GC-MS. A549 cells metabolized all tocochromanols 2-3 times more extensively than HepG2 cells (P < 0.001) except alpha-TOH, a difference not related to cell uptake of substrate but rather was reflective of greater microsomal TOH-omega-hydroxylase enzyme activity. Notably, 9'-carboxychromanols were the major metabolites of all gamma- and delta-TOHs and T3s in A549 cultures, whereas 3'- and 5'-carboxychromanols predominated in HepG2 cultures. Accumulation of 9'-carboxychromanols in A549 cultures was due to their inefficient conversion to 7'-carboxychromanols relative to HepG2 cells. Sesamin inhibited tocochromanol metabolism in both cells types, and neither cell type exhibited evidence of alternative (sesamin-insensitive) pathways of metabolism. TOH-omega-hydroxylase activity was undetectable in rat primary lung type II cells, suggesting that expression of activity was associated with transformation of normal type II cells to cancer cells. Long-chain carboxychromanol metabolites of gamma-TOH and other forms of vitamin E can be biosynthesized in A549 cultures for assessment of their biological activity, including their potential inhibition of synthesis of inflammatory mediators.

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

    PubMed

    Yamada, Atsushi; Aki, Toshihiko; Unuma, Kana; Funakoshi, Takeshi; Uemura, Koichi

    2015-01-01

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

  5. Lipopolysaccharide and Interleukin 1 Augment the Effects of Hypoxia and Inflammation in Human Pulmonary Arterial Tissue

    NASA Astrophysics Data System (ADS)

    Ziesche, Rolf; Petkov, Venzeslav; Williams, John; Zakeri, Schaker M.; Mosgoller, Wilhelm; Knofler, Martin; Block, Lutz H.

    1996-10-01

    The combined effects of hypoxia and interleukin 1, lipopolysaccharide, or tumor necrosis factor α on the expression of genes encoding endothelial constitutive and inducible nitric oxide synthases, endothelin 1, interleukin 6, and interleukin 8 were investigated in human primary pulmonary endothelial cells and whole pulmonary artery organoid cultures. Hypoxia decreased the expression of constitutive endothelial nitric oxide synthase (NOS-3) mRNA and NOS-3 protein as compared with normoxic conditions. The inhibition of expression of NOS-3 corresponded with a reduced production of NO. A combination of hypoxia with bacterial lipopolysaccharide, interleukin 1β , or tumor necrosis factor α augmented both effects. In contrast, the combination of hypoxia and the inflammatory mediators superinduced the expression of endothelin 1, interleukin 6, and interleukin 8. Here, we have shown that inflammatory mediators aggravate the effect of hypoxia on the down-regulation of NOS-3 and increase the expression of proinflammatory cytokines in human pulmonary endothelial cells and whole pulmonary artery organoid cultures.

  6. Sp1 inhibition-mediated upregulation of VEGF 165 b induced by rh-endostatin enhances antiangiogenic and anticancer effect of rh-endostatin in A549.

    PubMed

    Li, Zhen-yu; Zhu, Fang; Hu, Jian-li; Peng, Gang; Chen, Jing; Zhang, Sheng; Chen, Xu; Zhang, Rui-guang; Chen, Ling-juan; Liu, Pian; Luo, Ming; Sun, Zhi-hua; Ren, Jing-hua; Huang, Li-li; Wu, Gang

    2011-08-01

    Recombinant human endostatin (rh-endostatin), a potential antiangiogenic agent, is used in non-small cell lung carcinoma treatment and represses vascular endothelial cell growth factor (VEGF) levels in tumor cell. However, precise affection of rh-endostatin on the proangiogenic VEGF isoforms (VEGF(165)) or antiangiogenic VEGF isoforms (VEGF(165)b) is not clear. We therefore tested the hypothesis that rh-endostatin could alter expression of these isoforms to regulate tumor growth. A549 cells were exposed to rh-endostatin, and the expression of VEGF(165) and VEGF(165)b was detected. The role of SP1 as a regulator of isoform expression was investigated. We then examined the anticancer and antiangiogenic efficacy of rh-endostatin in combination with exogenous VEGF(165)b against A549 cells, EA.HY 926 cells and xenograft model of human lung cancer. rh-Endostatin reduced VEGF(165) and induced VEGF(165)b as well as inhibited SP1 in A549 cells. SP1 inhibitor (betulinic acid) also developed those changes. VEGF(165)b-rh-endostatin combination was highly synergistic and inhibited growth, survival, and migration of A549 cells, VEGF-mediated VEGFR2 phosphorylation in EA.HY 926 cells, and tumor growth in xenograft model of human lung cancer. rh-Endostatin downregulates proangiogenic vascular endothelial growth factor A (VEGFA) isoform and upregulates antiangiogenic VEGFA isoform, possibly through inhibition of SP1. Furthermore, VEGF(165)b sensitizes A549 to rh-endostatin treatment and enhances the anticancer effect of rh-endostatin.

  7. Sildenafil as treatment for Human Immunodeficiency Virus-related pulmonary hypertension in a child.

    PubMed

    Wong, Abdul Rahim; Rasool, Aida Hanum G; Abidin, Nik Zainal; Noor, Abdul Rahmand; Quah, Ban Seng

    2006-03-01

    Human Immunodeficiency Virus (HIV)-related pulmonary hypertension is a relatively rare disease that can affect HIV sufferers. This is almost always associated with a poor outcome and death. An 18 month-old girl, probably the youngest on record, was diagnosed to have pulmonary hypertension (PHT) and retrospectively found to have HIV infection. Sildenafil was used to control her PHT and she remains alive even after 2 years.

  8. Contrasting effects of ascorbate and iron on the pulmonary vascular response to hypoxia in humans.

    PubMed

    Talbot, Nick P; Croft, Quentin P; Curtis, M Kate; Turner, Brandon E; Dorrington, Keith L; Robbins, Peter A; Smith, Thomas G

    2014-12-01

    Hypoxia causes an increase in pulmonary artery pressure. Gene expression controlled by the hypoxia-inducible factor (HIF) family of transcription factors plays an important role in the underlying pulmonary vascular responses. The hydroxylase enzymes that regulate HIF are highly sensitive to varying iron availability, and iron status modifies the pulmonary vascular response to hypoxia, possibly through its effects on HIF. Ascorbate (vitamin C) affects HIF hydroxylation in a similar manner to iron and may therefore have similar pulmonary effects. This study investigated the possible contribution of ascorbate availability to hypoxic pulmonary vasoconstriction in humans. Seven healthy volunteers undertook a randomized, controlled, double-blind, crossover protocol which studied the effects of high-dose intravenous ascorbic acid (total 6 g) on the pulmonary vascular response to 5 h of sustained hypoxia. Systolic pulmonary artery pressure (SPAP) was assessed during hypoxia by Doppler echocardiography. Results were compared with corresponding data from a similar study investigating the effect of intravenous iron, in which SPAP was measured in seven healthy volunteers during 8 h of sustained hypoxia. Consistent with other studies, iron supplementation profoundly inhibited hypoxic pulmonary vasoconstriction (P < 0.001). In contrast, supraphysiological supplementation of ascorbate did not affect the increase in pulmonary artery pressure induced by several hours of hypoxia (P = 0.61). We conclude that ascorbate does not interact with hypoxia and the pulmonary circulation in the same manner as iron. Whether the effects of iron are HIF-mediated remains unknown, and the extent to which ascorbate contributes to HIF hydroxylation in vivo is also unclear.

  9. A rare case of human pulmonary dirofilariasis with a growing pulmonary nodule after migrating infiltration shadows, mimicking primary lung carcinoma

    PubMed Central

    Haro, Akira; Tamiya, Sadafumi; Nagashima, Akira

    2016-01-01

    Introduction Pulmonary dirofilariasis is a rare pulmonary parasitic infection by the nematode Dirofilaria immitis. It is characterized by an asymptomatic pulmonary nodule usually seen on chest X-ray. The differential diagnosis of pulmonary dirofilariasis includes other pulmonary diseases, primary lung carcinoma and metastatic lung tumor. Case presentation Pulmonary dirofilariasis was diagnosed in a woman who presented with interstitial pneumonia. Growth of the pulmonary nodule was detected subsequent to hemoptysis. The histological diagnosis was made based on a wedge resection performed under video-associated thoracic surgery (VATS). Conclusion Pulmonary dirofilariasis often varies in its clinical course. The diagnosis is best made using wedge resection under VATS. PMID:27015012

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

    PubMed

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

    2015-07-01

    The aim of the study is to explore the bystander effects in A549 cells that have been exposed to 6MV X-ray. Control group, irradiated group, irradiated conditioned medium (ICM)-received group, and fresh medium group were designed in this study. A549 cells in the logarithmic growth phase were irradiated with 6MV X-ray at 0, 0.5, 1, 1.5, and 2. In ICM-received group, post-irradiation A549 cells were cultured for 3 h and were transferred into non-irradiated A549 cells for further cultivation. Clone forming test was applied to detect the survival fraction of cells. Annexin V-FITC/PI double-staining assay was used to detect the apoptosis of A549 cells 24, 48, 72, and 96 h after 2-Gy 6MV X-ray irradiation, and the curves of apoptosis were drawn. The changes in the cell cycles 4, 48, 72, and 96 h after 2-Gy 6MV X-ray irradiation were detected using PI staining flow cytometry. With the increase of irradiation dose, the survival fraction of A549 cells after the application of 0.5 Gy irradiation was decreasing continuously. In comparison to the control group, the apoptosis rate of the ICM-received group was increased in a time-dependent pattern, with the highest apoptosis rate observed at 72 h (p < 0.05). Cell count in G2/M stages was obviously increased compared with that of the control group (p < 0.05), with the highest count observed at 72 h, after which G2/M stage arrest was diminished. ICM can cause apparent A549 cell damage, indicating that 6MV X-ray irradiation can induce bystander effect on A549 cells, which reaches a peak at 72 h.

  11. Regional pulmonary perfusion following human heart-lung transplantation

    SciTech Connect

    Lisbona, R.; Hakim, T.S.; Dean, G.W.; Langleben, D.; Guerraty, A.; Levy, R.D. )

    1989-08-01

    Ventilation and perfusion scans were obtained in six subjects who had undergone heart-lung transplantation with consequent denervation of the cardiopulmonary axis. Two of the subjects had developed obliterative bronchiolitis, which is believed to be a form of chronic rejection. Their pulmonary function tests demonstrated airflow obstruction and their scintigraphic studies were abnormal. In the remaining four subjects without obstructive airways disease, ventilation and planar perfusion scans were normal. Single photon emission computed tomography imaging of pulmonary perfusion in these patients revealed a layered distribution of blood flow indistinguishable from that of normal individuals. It is concluded that neurogenic mechanisms have little influence on the pattern of local pulmonary blood flow at rest.

  12. 3′,4′,5′,5,7-Pentamethoxyflavone Sensitizes Cisplatin-Resistant A549 Cells to Cisplatin by Inhibition of Nrf2 Pathway

    PubMed Central

    Hou, Xiangyu; Bai, Xupeng; Gou, Xiaoli; Zeng, Hang; Xia, Chen; Zhuang, Wei; Chen, Xinmeng; Zhao, Zhongxiang; Huang, Min; Jin, Jing

    2015-01-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is an important redox-sensitive transcription factor that regulates the expression of several cytoprotective genes. More recently, genetic analyses of human tumors have indicated that Nrf2 may cause resistance to chemotherapy. In this study, we found that the expression levels of Nrf2 and its target genes GCLC, HO-1, NQO1 were significantly higher in cisplatin-resistant A549 (A549/CDDP) cells than those in A549 cells, and this resistance was partially reversed by Nrf2 siRNA. 3′,4′,5′,5,7-Pentamethoxyflavone (PMF), a natural flavonoid extracted from Rutaceae plants, sensitized A549/CDDP to CDDP and substantially induced apoptosis compared with that of CDDP alone treated group, and this reversal effect decreased when Nrf2 was downregulated by siRNA. Mechanistically, PMF reduced Nrf2 expression leading to a reduction of Nrf2 downstream genes, and in contrast, this effect was decreased by blocking Nrf2 with siRNA. Taken together, these results demonstrated that PMF could be used as an effective adjuvant sensitizer to increase the efficacy of chemotherapeutic drugs by downregulating Nrf2 signaling pathway. PMID:25843086

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

    PubMed

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

    2016-09-01

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

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

    PubMed

    Yevdokimova, N; Freshney, R I

    1997-01-01

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

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

    PubMed Central

    Yevdokimova, N.; Freshney, R. I.

    1997-01-01

    Alkaline phosphatase, a marker of differentiation in the human alveolar adenocarcinoma cell line A549, is inducible by conditioned medium from lung fibroblasts and by cytokines including oncostatin M and interleukin 6, but only in the presence of a glucocorticoid, dexamethasone. Dexamethasone was shown to induce incorporation of [3H]glucosamine into three fractions of medium and cell trypsinate from subconfluent A549 cells, eluting from DEAE ion-exchange chromatography. The first peak did not correspond to any of the unlabelled glycosaminoglycans and was not characterized further. Induction was seen in two other peaks, corresponding to hyaluronic acid and heparan sulphate. Of these, heparan sulphate, eluting as one well-defined peak (referred to as HS1) and another of lower activity and less well defined (HS2), was selected as the most likely to interact with growth factors and cytokines and was isolated from the eluate, concentrated and desalted, and used in alkaline phosphatase induction experiments in place of dexamethasone. HS1 isolated from the medium (HS1m) of subconfluent A549 cells was shown to replace dexamethasone in induction experiments with fibroblast-conditioned medium, oncostatin M and interleukin 6. HS1 from the cell trypsinate and HS2 from the medium and trypsinate were inactive. As the activity of HS1m could be abolished by heparinase and heparitinase but not by chondroitinase ABC, it was concluded that HS1m was a fraction of heparan sulphate involved in the regulation of paracrine growth factor activity in lung fibroblast-conditioned medium, and in the regulation of other growth factors with potential roles in the paracrine control of cell differentiation. PMID:9252193

  16. Therapeutic Targeting of CC Ligand 21 or CC Chemokine Receptor 7 Abrogates Pulmonary Fibrosis Induced by the Adoptive Transfer of Human Pulmonary Fibroblasts to Immunodeficient Mice

    PubMed Central

    Pierce, Elizabeth M.; Carpenter, Kristin; Jakubzick, Claudia; Kunkel, Steven L.; Flaherty, Kevin R.; Martinez, Fernando J.; Hogaboam, Cory M.

    2007-01-01

    Idiopathic interstitial pneumonias (IIPs) are a collection of pulmonary fibrotic diseases of unknown etiopathogenesis. CC chemokine receptor 7 (CCR7) is expressed in IIP biopsies and primary fibroblast lines, but its role in pulmonary fibrosis was not previously examined. To study the in vivo role of CCR7 in a novel model of pulmonary fibrosis, 1.0 × 106 primary fibroblasts grown from idiopathic pulmonary fibrosis/usual interstitial pneumonia, nonspecific interstitial pneumonia, or histologically normal biopsies were injected intravenously into C.B-17 severe combined immunodeficiency (SCID)/beige (bg) mice. At days 35 and 63 after idiopathic pulmonary fibrosis/usual interstitial pneumonia fibroblast injection, patchy interstitial fibrosis and increased hydroxyproline were present in the lungs of immunodeficient mice. Adoptively transferred nonspecific interstitial pneumonia fibroblasts caused a more diffuse interstitial fibrosis and increased hydroxyproline levels at both times, but injected normal human fibroblasts did not induce interstitial remodeling changes in C.B-17SCID/bg mice. Systemic therapeutic immunoneutralization of either human CCR7 or CC ligand 21, its ligand, significantly attenuated the pulmonary fibrosis in groups of C.B-17SCID/bg mice that received either type of IIP fibroblasts. Thus, the present study demonstrates that pulmonary fibrosis is initiated by the intravenous introduction of primary human fibroblast lines into immunodeficient mice, and this fibrotic response is dependent on the interaction between CC ligand 21 and CCR7. PMID:17392156

  17. Proteomic Analysis of Cellular Response Induced by Multi-Walled Carbon Nanotubes Exposure in A549 Cells

    PubMed Central

    Zhang, Xing; Jia, Zhenyu; Gao, Xiangjing; Jiang, Ying; Yan, Chunlan; Duerksen-Hughes, Penelope J.; Chen, Fanqing Frank; Li, Hongjuan; Zhu, Xinqiang; Yang, Jun

    2014-01-01

    The wide application of multi-walled carbon nanotubes (MWCNT) has raised serious concerns about their safety on human health and the environment. However, the potential harmful effects of MWCNT remain unclear and contradictory. To clarify the potentially toxic effects of MWCNT and to elucidate the associated underlying mechanisms, the effects of MWCNT on human lung adenocarcinoma A549 cells were examined at both the cellular and the protein level. Cytotoxicity and genotoxicity were examined, followed by a proteomic analysis (2-DE coupled with LC-MS/MS) of the cellular response to MWCNT. Our results demonstrate that MWCNT induces cytotoxicity in A549 cells only at relatively high concentrations and longer exposure time. Within a relatively low dosage range (30 µg/ml) and short time period (24 h), MWCNT treatment does not induce significant cytotoxicity, cell cycle changes, apoptosis, or DNA damage. However, at these low doses and times, MWCNT treatment causes significant changes in protein expression. A total of 106 proteins show altered expression at various time points and dosages, and of these, 52 proteins were further identified by MS. Identified proteins are involved in several cellular processes including proliferation, stress, and cellular skeleton organization. In particular, MWCNT treatment causes increases in actin expression. This increase has the potential to contribute to increased migration capacity and may be mediated by reactive oxygen species (ROS). PMID:24454774

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

    PubMed

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

    2015-04-01

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

  19. Taxol-induced paraptosis-like A549 cell death is not senescence

    NASA Astrophysics Data System (ADS)

    Wang, Chao-yang; Chen, Tong-Sheng

    2011-03-01

    Our previous studies have shown that taxol, a potent anticancer agent, induces caspase-independent cell death and cytoplasmic vacuolization in human lung cancer cells. However, the mechanisms of taxol-induced cytoplasmic vacuolization are poorly understood. Cytoplasmic vacuolization have been reported to be a characteristic of cell senescence. Here, we employed confocal fluorescence microscopy imaging to study the reversibility of taxol-induced cytoplasmic vacuolization and whether taxol triggers senescence in A549 cells. We found that taxol-induced cytoplasmic vacuolization at 6 or 9 h after treatment with taxol did not decrease but increase at 24 h or 72 h after refreshing the culture medium without taxol, indicating taxol-induced cytoplasmic vacuolization is irreversible. We used SA-β-Gal (senescence-associated β-galactosidase) to assess whether taxol-induced cell death in cytoplasmic vacuolization fashion is senescence, and found that hydrogen peroxide (H2O2)-treated, but not taxol-treated cells is significantly stained by the SA-β-Gal, a senescence testing kit, indicating that the form of taxol-induced cell death is not senescence.

  20. In vivo evaluation of curcumin-loaded nanoparticles in a A549 xenograft mice model.

    PubMed

    Yin, Hai-Tao; Zhang, De-Geng; Wu, Xiao-Li; Huang, Xin-En; Chen, Gang

    2013-01-01

    Curcumin (Cum) has been reported to have potential chemo-preventive and chemotherapeutic activity through influencing various processes, inducing cell cycle arrest, differentiation and apoptosis in a series of cancers. However, the poor solubility of Cum limits its further applications in the treatment of cancer. We have previously reported Cum-loaded nanoparticles (Cum-NPs) prepared with amphilic methoxy poly(ethylene glycol)-polycaprolactone (mPEG-PCL) block copolymers. The current study demonstrated superior antitumor efficacy of Cum-NPs over free Cum in the treatment of lung cancer. In vivo evaluation further demonstrated superior anticancer effects of Cum-NPs by delaying tumor growth compared to free Cum in an established A549 transplanted mice model. Moreover, Cum-NPs showed little toxicity to normal tissues including bone marrow, liver and kidney at a therapeutic dose. These results suggest that Cum-NPs are effective to inhibit the growth of human lung cancer with little toxicity to normal tissues, and could provide a clinically useful therapeutic regimen. They thus merit more research to evaluate the feasibility of clinical application.

  1. Pinus massoniana bark extract inhibits migration of the lung cancer A549 cell line

    PubMed Central

    Mao, Ping; Zhang, Ershao; Chen, Yang; Liu, Likun; Rong, Daqing; Liu, Qingfeng; Li, Weiling

    2017-01-01

    The bark of Pinus massoniana is a traditional Chinese medicine for the treatment of various health disorders. Previous studies have demonstrated that P. massoniana bark extract (PMBE) may induce the apoptosis of hepatoma and cervical cancer cells. However, whether PMBE is able to inhibit the migration of lung cancer cells requires further investigation. In the current study, the effects of PMBE on the viability of human lung cancer A549 cells were detected using an MTT assay. The migration of lung cancer cells following exposure to PMBE were quantified using wound healing and Transwell assays, respectively. The expression levels of matrix metalloproteinase (MMP)-9 were determined using western blotting. The results revealed that PMBE significantly inhibited the growth of the lung cancer cells. In addition, the wound closure rate and the migration of the lung cancer cells were suppressed by PMBE. Furthermore, the expression levels of MMP-9 were reduced. These findings indicated that PMBE is able to restrict the migration and invasion of lung cancer cells, and that PMBE may serve as a novel therapeutic agent for patients with metastatic lung cancer in the future. PMID:28356994

  2. Anti-Proliferative and Apoptosis-Inducing Effect of Theabrownin against Non-small Cell Lung Adenocarcinoma A549 Cells

    PubMed Central

    Wu, Feifei; Zhou, Li; Jin, Wangdong; Yang, Weiji; Wang, Ying; Yan, Bo; Du, Wenlin; Zhang, Qiang; Zhang, Lei; Guo, Yonghua; Zhang, Jin; Shan, Letian; Efferth, Thomas

    2016-01-01

    With the highest cancer incidence rate, lung cancer, especially non-small cell lung cancer (NSCLC), is the leading cause of cancer death in the world. Tea (leaves of Camellia sinensis) has been widely used as a traditional beverage beneficial to human health, including anti-NSCLC activity. Theabrownin (TB) is one major kind of tea pigment responsible for the beneficial effects of tea liquor. However, its effect on NSCLC is unknown. The aim of the present study was to evaluate anti-proliferative and apoptosis-inducing effect of TB on NSCLC (A549) cells, using MTT assay, morphological observation (DAPI staining), in situ terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and annexin-V/PI flow cytometry. Subsequently, the expression of several genes associated with cell proliferation and apoptosis were detected by real time PCR assay to explore its potential underlying mechanism. TB was revealed to inhibit cell proliferation of A549 cells in a concentration-dependent and time-dependent manner. Morphological observation, TUNEL assay and flow cytometric analysis evidenced an apoptosis-inducing effect of TB on A549 cells in a concentration-dependent manner. The real time PCR assay demonstrated that TB down-regulated the expression of TOPO I, TOPO II, and BCL-2, and up-regulated the expression of E2F1, P53, GADD45, BAX, BIM, and CASP 3,7,8,9, which suggests an activation of P53-mediated apoptotic (caspase-dependent) pathway in response to TB treatment. The western blot analysis showed a similar trend for the corresponding protein expression (P53, Bax, Bcl-2, caspase 3,9, and PARP) and further revealed DNA damage as a trigger of the apoptosis (phosphorylation of histone H2A.X). Accordingly, TB can be speculated as a DNA damage inducer and topoisomerase (Topo I and Topo II) inhibitor that can up-regulate P53 expression and subsequently modulate the expression of the downstream genes to induce cell proliferation inhibition and apoptosis of A549 cells

  3. Venom present in sea anemone (Heteractis magnifica) induces apoptosis in non-small-cell lung cancer A549 cells through activation of mitochondria-mediated pathway.

    PubMed

    Ramezanpour, Mahnaz; da Silva, Karen Burke; Sanderson, Barbara J S

    2014-03-01

    Lung cancer is a major cause of cancer deaths throughout the world and the complexity of apoptosis resistance in lung cancer is apparent. Venom from Heteractis magnifica caused dose-dependent decreases in survival of the human non-small-cell lung cancer cell line, as determined by the MTT and Crystal Violet assays. The H. magnifica venom induced cell cycle arrest and induced apoptosis of A549 cells, as confirmed by annexin V/propidium iodide staining. The venom-induced apoptosis in A549 cells was characterized by cleavage of caspase-3 and a reduction in the mitochondrial membrane potential. Interestingly, crude extracts from H. magnifica had less effect on the survival of non-cancer cell lines. In the non-cancer cells, the mechanism via which cell death occurred was through necrosis not apoptosis. These findings are important for future work using H. magnifica venom for pharmaceutical development to treat human lung cancer.

  4. Activities of ten essential oils towards Propionibacterium acnes and PC-3, A-549 and MCF-7 cancer cells.

    PubMed

    Zu, Yuangang; Yu, Huimin; Liang, Lu; Fu, Yujie; Efferth, Thomas; Liu, Xia; Wu, Nan

    2010-04-30

    Ten essential oils, namely, mint (Mentha spicata L., Lamiaceae), ginger (Zingiber officinale Rosc., Zingiberaceae), lemon (Citrus limon Burm.f., Rutaceae), grapefruit (Citrus paradisi Macf., Rutaceae), jasmine (Jasminum grandiflora L., Oleaceae), lavender (Mill., Lamiaceae), chamomile (Matricaria chamomilla L., Compositae), thyme (Thymus vulgaris L., Lamiaceae), rose (Rosa damascena Mill., Rosaceae) and cinnamon (Cinnamomum zeylanicum N. Lauraceae) were tested for their antibacterial activities towards Propionibacterium acnes and in vitro toxicology against three human cancer cell lines. Thyme, cinnamon and rose essential oils exhibited the best antibacterial activities towards P. acnes, with inhibition diameters of 40 +/- 1.2 mm, 33.5 +/- 1.5 mm and 16.5 +/- 0.7 mm, and minimal inhibitory concentrations of 0.016% (v/v), 0.016% (v/v) and 0.031% (v/v), respectively. Time-kill dynamic procedures showed that thyme, cinnamon, rose, and lavender essential oils exhibited the strongest bactericidal activities at a concentration of 0.25% (v/v), and P. acnes was completely killed after 5 min. The thyme essential oil exhibited the strongest cytotoxicity towards three human cancer cells. Its inhibition concentration 50% (IC(50)) values on PC-3, A549 and MCF-7 tumor cell lines were 0.010% (v/v), 0.011% (v/v) and 0.030% (v/v), respectively. The cytotoxicity of 10 essential oils on human prostate carcinoma cell (PC-3) was significantly stronger than on human lung carcinoma (A549) and human breast cancer (MCF-7) cell lines.

  5. Protocatechuic aldehyde ameliorates experimental pulmonary fibrosis by modulating HMGB1/RAGE pathway

    SciTech Connect

    Zhang, Liang Ji, Yunxia Kang, Zechun Lv, Changjun Jiang, Wanglin

    2015-02-15

    An abnormal high mobility group box 1 (HMGB1) activation and a decrease in receptor for advanced glycation end-product (RAGE) play a key role in the pathogenesis of pulmonary fibrosis. Protocatechuic aldehyde (PA) is a naturally occurring compound, which is extracted from the degradation of phenolic acids. However, whether PA has anti-fibrotic functions is unknown. In this study, the effects of PA on the transforming growth factor-β1 (TGF-β1)-mediated epithelial–mesenchymal transition (EMT) in A549 cells, on the apoptosis of human type I alveolar epithelial cells (AT I), on the proliferation of human lung fibroblasts (HLF-1) in vitro, and on bleomycin (BLM)-induced pulmonary fibrosis in vivo were investigated. PA treatment resulted in a reduction of EMT in A549 cells with a decrease in vimentin and HMGB, an increase of E-cadherin and RAGE, a reduction of HLF-1 proliferation with a decrease of fibroblast growth factor 2 (FGF-2) and platelet-derived growth factor (PDGF). Apoptosis of AT I was attenuated with an increase of RAGE. PA ameliorated BLM-induced pulmonary fibrosis in rats with a reduction of histopathological scores and collagen deposition, and a lower FGF-2, PDGF, α-smooth muscle actin (α-SMA) and HMGB1 expression, whereas higher RAGE was found in BLM-instilled lungs. Through the decrease of HGMB1 and the regulation of RAGE, PA reversed the EMT, inhibited HLF-1 proliferation as well as reduced apoptosis in AT I, and prevented pulmonary fibrosis in vivo. Collectively, our results demonstrate that PA prevents experimental pulmonary fibrosis by modulating HMGB1/RAGE pathway. - Highlights: • PA prevents EMT, reduces the apoptosis of AT1 in vitro. • PA decreases proliferation of HLF-1, reduces PDGF and FGF expression in vitro. • PA prevents experimental pulmonary fibrosis by modulating the HMGB1/RAGE pathway.

  6. Induction of p53-independent growth inhibition in lung carcinoma cell A549 by gypenosides

    PubMed Central

    Liu, Jung-Sen; Chiang, Tzu-Hsuan; Wang, Jinn-Shyan; Lin, Li-Ju; Chao, Wei-Chih; Inbaraj, Baskaran Stephen; Lu, Jyh-Feng; Chen, Bing-Huei

    2015-01-01

    The objectives of this study are to investigate antiproliferative effect and mechanisms of bioactive compounds from Gynostemma pentaphyllum (G. pentaphyllum) on lung carcinoma cell A549. Saponins, carotenoids and chlorophylls were extracted and fractionated by column chromatography, and were subjected to high-performance liquid chromatography-mass spectrometry analyses. The saponin fraction, which consisted mainly of gypenoside (Gyp) XXII and XXIII, rather than the carotenoid and chlorophyll ones, was effective in inhibiting A549 cell growth in a concentration- and a time-dependent manner as evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The estimated half maximal inhibitory concentration (IC50) of Gyp on A549 cells was 30.6 μg/ml. Gyp was further demonstrated to induce an apparent arrest of the A549 cell cycle at both the S phase and the G2/M phase, accompanied by a concentration- and a time-dependent increase in the proportions of both the early and late apoptotic cells. Furthermore, Gyp down-regulated cellular expression of cyclin A and B as well as BCL-2, while up-regulated the expression of BAX, DNA degradation factor 35 KD, poly [ADP-ribose] polymerase 1, p53, p21 and caspase-3. Nevertheless, both the treatment of a p53 inhibitor, pifithrin-α, and the small hairpin RNA-mediated p53 knockdown in the A549 cells did not alter the growth inhibition effect induced by Gyp. As a result, the cell cycle arrest and apoptosis of A549 cells induced by Gyp would most likely proceed through p53-independent pathway(s). PMID:25781909

  7. Cytoprotective Effect of Makgeolli Lees on Paraquat Induced Oxidative Stress in A549 Cells via Activation of NRF2 and Antioxidant Genes.

    PubMed

    Jeon, Miso; Rahman, Naimur; Kim, Yong-Sik

    2016-02-01

    Makgeolli lees (ML) has several physiological effects such as antioxidant, antidiabetic, and anticancer properties, but its biological functions have not been determined definitively. Here, we tested whether ML has a cytoprotective effect on paraquat (PQ)-induced oxidative stress in the human lung carcinoma cell line A549. At 0.1 mg/ml ML, viability of PQ-exposed A549 cells was restored by 12.4%, 18.5%, and 48.6% after 24, 48, and 72 h, respectively. ML also reduced production of the intracellular reactive oxygen species (ROS) that were generated by PQ treatment. Further experiments revealed that ML treatment enhanced the expression and nuclear translocation of nuclear factor erythroid 2-related factor 2 (NRF2) as well as ARE-GFP reporter activity. ML treatment also effectively increased the expression of NRF2's target genes NAD(P)H dehydrogenase quinone 1 (NQO1) and heme oxygenase 1 (HO-1). Moreover, we found that expression of cytoprotective genes, including glutathione peroxidases (GPXs), superoxide dismutase (SOD1), catalase (CAT), peroxiredoxin 3 (PRDX3), and peroxiredoxin 4 (PRDX4), was greatly enhanced by treatment with ML during PQ exposure. Taken together, the data suggest that treatment of PQ-exposed A549 cells with ML ameliorates cytotoxicity through induction of NRF2 expression and its target genes HO-1, NQO1, and other antioxidant genes. Thus, ML may serve as a functional food applicable to ROS-mediated human diseases.

  8. Evidence for a M1 muscarinic receptor on the endothelium of human pulmonary veins

    PubMed Central

    Walch, Laurence; Gascard, Jean-Pierre; Dulmet, Elisabeth; Brink, Charles; Norel, Xavier

    2000-01-01

    To characterize the muscarinic receptors on human pulmonary veins associated with the acetylcholine (ACh)-induced relaxation, isolated venous and arterial preparations were pre-contracted with noradrenaline (10 μM) and were subsequently challenged with ACh in the absence or presence of selective muscarinic antagonists.ACh relaxed venous preparations derived from human lung with a pD2 value of 5.82±0.09 (n=16). In venous preparations where the endothelium had been removed, the ACh relaxations were abolished (n=4). ACh relaxed arterial preparations with a pD2 value of 7.06±0.14 (n=5).Atropine (1 μM), the non selective antagonist for muscarinic receptors, inhibited ACh-induced relaxations in human pulmonary veins. The affinity value (pKB value) for atropine was: 8.64±0.10 (n=5). The selective muscarinic antagonists (darifenacin (M3), himbacine (M2,M4), methoctramine (M2) and pFHHSiD (M1,M3)) also inhibited ACh-induced relaxations in venous preparations. The pKB values obtained for these antagonists were not those predicted for the involvement of M2–5 receptors in the ACh-induced relaxation in human pulmonary veins.The pKB value for darifenacin (1 μM) was significantly greater in human pulmonary arterial (8.63±0.14) than in venous (7.41±0.20) preparations derived from three lung samples.In human pulmonary veins, the pKB values for pirenzepine (0.5 and 1 μM), a selective antagonist for M1 receptors, were: 7.89±0.24 (n=7) and 8.18±0.22 (n=5), respectively. In the venous preparations, the pKB values derived from the functional studies with all the different muscarinic antagonists used were correlated (r=0.89; P=0.04; slope=0.78) with the affinity values (pKi values) previously published for human cloned m1 receptors in CHO cells.These results suggest that the relaxations induced by ACh are due to the activation of M1 receptors on endothelial cells in isolated human pulmonary veins. PMID:10781000

  9. Imaging and characterization of stretch-induced ATP release from alveolar A549 cells.

    PubMed

    Grygorczyk, Ryszard; Furuya, Kishio; Sokabe, Masahiro

    2013-03-01

    Abstract  Mechano-transduction at cellular and tissue levels often involves ATP release and activation of the purinergic signalling cascade. In the lungs, stretch is an important physical stimulus but its impact on ATP release, the underlying release mechanisms and transduction pathways are poorly understood. Here, we investigated the effect of unidirectional stretch on ATP release from human alveolar A549 cells by real-time luciferin-luciferase bioluminescence imaging coupled with simultaneous infrared imaging, to monitor the extent of cell stretch and to identify ATP releasing cells. In subconfluent (<90%) cell cultures, single 1 s stretch (10-40%)-induced transient ATP release from a small fraction (1.5%) of cells that grew in number dose-dependently with increasing extent of stretch. ATP concentration in the proximity (150 μm) of releasing cells often exceeded 10 μm, sufficient for autocrine/paracrine purinoreceptor stimulation of neighbouring cells. ATP release responses were insensitive to the putative ATP channel blockers carbenoxolone and 5-nitro-2-(3-phenylpropyl-amino) benzoic acid, but were inhibited by N-ethylmaleimide and bafilomycin. In confluent cell cultures, the maximal fraction of responding cells dropped to <0.2%, but was enhanced several-fold in the wound/scratch area after it was repopulated by new cells during the healing process. Fluo8 fluorescence experiments revealed two types of stretch-induced intracellular Ca(2+) responses, rapid sustained Ca(2+) elevations in a limited number of cells and delayed secondary responses in neighbouring cells, seen as Ca(2+) waves whose propagation was consistent with extracellular diffusion of released ATP. Our experiments revealed that a single >10% stretch was sufficient to initiate intercellular purinergic signalling in alveolar cells, which may contribute to the regulation of surfactant secretion and wound healing.

  10. PM10-biogenic fraction drives the seasonal variation of proinflammatory response in A549 cells.

    PubMed

    Camatini, Marina; Corvaja, Viviana; Pezzolato, Eleonora; Mantecca, Paride; Gualtieri, Maurizio

    2012-02-01

    PM10 was collected in a Milan urban site, representative of the city air quality, during winter and summer 2006. Mean daily PM10 concentration was 48 μg m(-3) during summer and 148 μg m(-3) during winter. Particles collected on Teflon filters were chemically characterized and the endotoxin content determined by the LAL test. PM10-induced cell toxicity, assessed with MTT and LDH methods, and proinflammatory potential, monitored by IL-6 and IL-8 cytokines release, were investigated on the human alveolar epithelial cell line A549 exposed to increasing doses of PM. Besides untreated cells, exposure to inert carbon particles (2-12 μm) was also used as additional control. Both cell toxicity and proinflammatory potency resulted to be higher for summer PM10 with respect of winter PM10, with IL-6 showing the highest dose-dependent release. The relevance of biogenic components adsorbed onto PM10 in eliciting the proinflammatory mediators release was investigated by inhibition experiments. Polymixin B (Poly) was used to inhibit particle-bind LPS while Toll-like receptor-2 antibody (a-TLR2) to specifically block the activation of this receptor. While cell viability was not modulated in cells coexposed to PM10 and Poly or a-TLR2 or both, inflammatory response did it, with IL-6 release being the most inhibited. In conclusion, Milan PM10-induced seasonal-dependent biological effects, with summer particles showing higher cytotoxic and proinflammatory potential. Cytotoxicity seemed to be unaffected by the PM biogenic components, while inflammation was significantly reduced after the inhibition of some biogenic activated pathways. Besides, the PM-associated biogenic activity does not entirely justify the PM-induced inflammatory effects. © 2010 Wiley Periodicals, Inc. Environ Toxicol 2012.

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

    PubMed Central

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

    2015-01-01

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

  12. DNA double-strand breaks activate ATM independent of mitochondrial dysfunction in A549 cells.

    PubMed

    Kalifa, Lidza; Gewandter, Jennifer S; Staversky, Rhonda J; Sia, Elaine A; Brookes, Paul S; O'Reilly, Michael A

    2014-10-01

    Excessive nuclear or mitochondrial DNA damage can lead to mitochondrial dysfunction, decreased energy production, and increased generation of reactive oxygen species (ROS). Although numerous cell signaling pathways are activated when cells are injured, the ataxia telangiectasia mutant (ATM) protein has emerged as a major regulator of the response to both mitochondrial dysfunction and nuclear DNA double-strand breaks (DSBs). Because mitochondrial dysfunction is often a response to excessive DNA damage, it has been difficult to determine whether nuclear and/or mitochondrial DNA DSBs activate ATM independent of mitochondrial dysfunction. In this study, mitochondrial and nuclear DNA DSBs were generated in the A549 human lung adenocarcinoma cell line by infecting with retroviruses expressing the restriction endonuclease PstI fused to a mitochondrial targeting sequence (MTS) or nuclear localization sequence (NLS) and a hemagglutinin antigen epitope tag (HA). Expression of MTS-PstI-HA or NLS-PstI-HA activated the DNA damage response defined by phosphorylation of ATM, the tumor suppressor protein p53 (TP53), KRAB-associated protein (KAP)-1, and structural maintenance of chromosomes (SMC)-1. Phosphorylated ATM and SMC1 were detected in nuclear fractions, whereas phosphorylated TP53 and KAP1 were detected in both mitochondrial and nuclear fractions. PstI also enhanced expression of the cyclin-dependent kinase inhibitor p21 and inhibited cell growth. This response to DNA damage occurred in the absence of detectable mitochondrial dysfunction and excess production of ROS. These findings reveal that DNA DSBs are sufficient to activate ATM independent of mitochondrial dysfunction and suggest that the activated form of ATM and some of its substrates are restricted to the nuclear compartment, regardless of the site of DNA damage.

  13. Gene expression profile of A549 cells from tissue of 4D model predicts poor prognosis in lung cancer patients.

    PubMed

    Mishra, Dhruva K; Creighton, Chad J; Zhang, Yiqun; Gibbons, Don L; Kurie, Jonathan M; Kim, Min P

    2014-02-15

    The tumor microenvironment plays an important role in regulating cell growth and metastasis. Recently, we developed an ex vivo lung cancer model (four dimensional, 4D) that forms perfusable tumor nodules on a lung matrix that mimics human lung cancer histopathology and protease secretion pattern. We compared the gene expression profile (Human OneArray v5 chip) of A549 cells, a human lung cancer cell line, grown in a petri dish (two-dimensional, 2D), and of the same cells grown in the matrix of our ex vivo model (4D). Furthermore, we obtained gene expression data of A549 cells grown in a petri dish (2D) and matrigel (three-dimensional, 3D) from a previous study and compared the 3D expression profile with that of 4D. Expression array analysis showed 2,954 genes differentially expressed between 2D and 4D. Gene ontology (GO) analysis showed upregulation of several genes associated with extracellular matrix, polarity and cell fate and development. Moreover, expression array analysis of 2D vs. 3D showed 1,006 genes that were most differentially expressed, with only 36 genes (4%) having similar expression patterns as observed between 2D and 4D. Finally, the differential gene expression signature of 4D cells (vs. 2D) correlated significantly with poor survival in patients with lung cancer (n = 1,492), while the expression signature of 3D vs. 2D correlated with better survival in lung cancer patients with lung cancer. As patients with larger tumors have a worse rate of survival, the ex vivo 4D model may be a good mimic of natural progression of tumor growth in lung cancer patients.

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

    PubMed

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

    2016-03-01

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

  15. The role of PRRX1 in the apoptosis of A549 cells induced by cisplatin

    PubMed Central

    Zhu, Hongbin; Sun, Gengyun; Dong, Jiahui; Fei, Liming

    2017-01-01

    Paired related homeobox1 (PRRX1) was a newly identified Epithelial mesenchymal transition (EMT) inducer. It was found that the decreased expression of PRRX1 in breast cancer and liver cancer could enable tumor cells to obtain tumor stem cell characteristics in vitro studies. However, the role of PRRX1 in lung cancer was still unknown. The down-regulated PRRX1 gene in A549 cells was established by slow virus infection in this study. The apoptosis of A549 cells was observed after the treatment of different concentrations of cisplatin and the role of PRRX1 in the apoptosis of A549 cells was explored. MTT results showed that down-regulated PRRX1 gene could resist the inhibitory effect of cisplatin on cell proliferation. The results of flow cytometry assay showed that down-regulated PRRX1 gene could reduce the apoptosis and promote A549 cells to enter G2 phase. Mitochondrial membrane potential detection showed that PRRX1 gene could inhibit the decrease of mitochondrial membrane potential. Western blotting results showed that down-regulated PRRX1 gene could reduce the expression levels of Caspase3, caspase9, Apaf-1 and cytochrome C. In a word, down-regulation of PRRX1 could cause lung cancer cells to produce anti apoptotic ability and resistance to cisplatin, which maybe through caspase3 pathway. PMID:28337269

  16. Edaravone Decreases Paraquat Toxicity in A549 Cells and Lung Isolated Mitochondria

    PubMed Central

    Shokrzadeh, Mohammad; Shaki, Fatemeh; Mohammadi, Ebrahim; Rezagholizadeh, Neda; Ebrahimi, Fatemeh

    2014-01-01

    Edaravone, an antioxidant and radical scavenger, showed protective effects against oxidative stress-like condition. Paraquat (PQ) is toxic herbicide considerable evidence suggests that oxidative stress and mitochondrial dysfunction contribute to PQ toxicity. In this study, protective effect of edaravone against PQ induced toxicity and reactive oxygen species (ROS) generation in A549 cells and lung isolated mitochondria were evaluated. A549 cells and lung isolated mitochondria were divided into control group, PQ group, edaravone group and PQ plus edaravone-pretreated group. Cellular and mitochondrial viability assayed using MTT test and ROS generations in both cellular and mitochondrial fraction were determined by fluorometry using DCFH-DA as indicator. Our results showed that edaravone (5–100 µM) prevented PQ (500 µM) induced cytotoxicity in A549 cells that the best protective effect was observed at concentration of 50 µM of edaravone. In addition, PQ-induced ROS generation in A549 cells significantly inhibited by edaravone. Moreover, PQ decreased mitochondria viability and also increased ROS generation in lung isolated mitochondria that edaravone (25–400 µM) markedly inhibited these toxic effects. In overall, the results of this study suggest that lung mitochondria maintenance is essential for maintaining PQt cytotoxicity and Edaravone is a protective drug against PQ toxicity in-vitro. PMID:25237364

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

    PubMed

    Shabram, Paul; Kolman, John L

    2014-01-01

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

  18. Changes in the cellular proteins of A549 infected with Hepatitis E virus by proteomics analysis

    PubMed Central

    2014-01-01

    Background Our understanding of Hepatitis E virus (HEV) has changed enormously over the past 30 years, from a waterborne infection causing outbreaks of acute hepatitis in developing countries to an infection of global distribution causing a range of hepatic and extra-hepatic illness. However, the key proteins playing important parts in the virus infection were still unknown. Understanding the changes of cellular proteins in these cells exposed to HEV is helpful for elucidating molecular mechanisms associated with function alterations of HEV-infected susceptible cells. In the present study, a comparative gel-based proteomic analysis was employed to study the changes in cellular proteins of A549 exposed to HEV in vitro to provide novel information for understanding the functional alterations of A549 induced by HEV infection. Result Of 2 585-3 152 protein spots visualized on each gel using silver staining, a total of 31 protein spots were found to be differentially expressed in HEV-infected A549 cells compared with mock-infected A549, including 10 significantly up-regulated protein spots and 21 significantly down-regulated protein spots. Conclusion Our work is the first time regarding the proteomic analysis on the cellular responses to HEV infection. This work is helpful for investigating the molecular basis associated with the interaction between HEV and the host cells although more efforts should be required to discover the mechanisms. PMID:25175408

  19. TSPYL5 is involved in cell growth and the resistance to radiation in A549 cells via the regulation of p21{sup WAF1/Cip1} and PTEN/AKT pathway

    SciTech Connect

    Kim, Eun Jin; Lee, So Yong; Kim, Tae Rim; Choi, Soo Im; Cho, Eun Wie; Kim, Kug Chan; Kim, In Gyu

    2010-02-12

    TSPYL5, encoding testis-specific Y-like protein, has been postulated to be a tumor suppressor gene, and its hypermethylation is often associated with human disease, especially cancer. In this study, we report that the TSPYL5 gene was less methylated (30%) in A549 lung adenocarcinoma cells, which are relatively resistant to {gamma}-radiation, than in H460 lung cancer cells, in which the TSPYL5 gene was hypermethylated (95%); thus, the expression level of TSPYL5 is much higher in A549 cells than in H460 cells. We showed that TSPYL5 suppression with silencing RNA in A549 cells up-regulated cellular PTEN, followed by down-regulation of AKT activation. Therefore, blockage of TSPYL5 sensitized A549 cells to cytotoxic agents such as {gamma}-radiation. In addition, TSPYL5 suppression also showed an increased level of p21{sup WAF1/Cip1} and subsequently induced inhibition of cell growth in A549 cells. The overexpression of TSPYL5 in H460 cells showed the opposite effects. This study provides the first demonstration that TSPYL5 modulates cell growth and sensitization of cells to the detrimental effects of damaging agents via regulation of p21{sup WAF1/Cip1} and PTEN/AKT pathway.

  20. Role of gambogic acid and NaI131 in A549/DDP cells

    PubMed Central

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

    2017-01-01

    Resistance to platinum in tumor tissue is a considerable barrier against effective lung cancer treatment. Radionuclide therapy is the primary adjuvant treatment, however, the toxic side effects limit its dosage in the clinical setting. Therefore, the present study aimed to determine whether an NaI131 radiosensitizer could help reduce the toxic side effects of radionuclide therapy. In vitro experiments were conducted to determine whether NaI131 can inhibit platinum resistance in A549/DDP cells, which are cisplatin-resistant non-small cell lung cancer cells, and whether gambogic acid (GA) is an effective NaI131 radiosensitizer. Cell proliferation following drug intervention was analyzed using MTT and isobolographic analysis. Apoptosis was assessed by flow cytometry. In addition, the mechanisms of drug intervention were analyzed by measuring the expression of P-glycoprotein (P-gP), B cell lymphoma 2 (Bcl-2), Bcl2-associated X protein (Bax) and P53 using western blot analysis and immunocytochemistry. According to isobolographic analysis, a low concentration of NaI131 combined with GA had a synergistic effect on the inhibition of A549/DDP cell proliferation, which was consistent with an increased rate of apoptosis. Furthermore, the overexpression of Bax, and the downregulation of P-gP, P53 and Bcl-2 observed demonstrated the potential mechanism(s) of NaI131 and GA intervention. NaI131 may induce apoptosis in A549/DDP cells by regulating apoptosis-related proteins. A low concentration combination of NaI131 and GA was able to significantly inhibit A549/DDP cell proliferation and induce cell apoptosis. Thus, the two drugs appear to have a synergistic effect on apoptosis of A549/DDP cells. PMID:28123519

  1. Scanning electron microscopic examination of human pulmonary capillaries using a latex replication method.

    PubMed

    Kendall, M W; Eissmann, E

    1980-03-01

    The human pulmonary microvasculature from the apical bronchopulmonary segment was studied by scanning electron microscopy using latex replicas. The latex replica was composed of a blend of vinyl chloride latexes using a plasticized vinyl chloride copolymer with a vinyl chloride copolymer. The polymerized latex produced a cast of the pulmonary arterial vascular tree, including the capillary patterns, which freely anastomose, thereby draining blood into pulmonary veinules and veins. The latex was injected via a gravity flow system modified from its earlier application in Guinea pig lungs. The apparently normal lungs from two recently deceased humans (dead for 5-6 hours and held in refrigeration) were perfused with heparinized Ringer's solution and subsequently injected with latex. The resulting latex casts of the capillaries revealed a three-dimensional network arranged in irregular vascular rings or ovals. This pattern was most conspicuous in deep and intermediate bronchopulmonary segmental areas. However, the subpleural capillaries produced casts that often terminated blindly, as observed with stereo SEm, suggesting that these vessels may tend to form thrombi more easily as compared with capillaries from other regions of the lung alveoli. The pulmonary arteriolar replicas contained indentations representing endothelial cell nuclei, and the capillary replicas projected oval evaginations that may represent discrete loci or capillary mural attenuations.

  2. Down-regulation of protein kinase Ceta by antisense oligonucleotides sensitises A549 lung cancer cells to vincristine and paclitaxel.

    PubMed

    Sonnemann, Jürgen; Gekeler, Volker; Ahlbrecht, Katrin; Brischwein, Klaus; Liu, Chao; Bader, Peter; Müller, Cornelia; Niethammer, Dietrich; Beck, James F

    2004-06-25

    Previous studies point to protein kinase C (PKC) isozyme eta as a resistance factor in cancer cells. Therefore, we investigated whether down-regulation of PKCeta with second generation antisense oligonucleotides (ODNs) would sensitise A549 human lung carcinoma cells to cytostatics. The effects were compared to the outcome of Bcl-xL down-regulation. Upon treatment with antisense ODNs, PKCeta and Bcl-xL were both significantly reduced on mRNA and protein level. Down-regulation of either PKCeta or Bcl-xL in combination with vincristine or paclitaxel resulted in a significant increase in caspase-3 activity compared to that in the control oligonucleotide treated cells. In addition, PKCeta down-regulation augmented vincristine-induced dissipation of mitochondrial transmembrane potential. In conclusion, these results confirm that PKCeta might represent a considerable resistance factor and an interesting target to improve anticancer chemotherapy.

  3. Rapid induction and persistence of paracrine-induced cellular antiviral states arrest viral infection spread in A549 cells

    PubMed Central

    Voigt, Emily A; Swick, Adam; Yin, John

    2016-01-01

    The virus/host interaction is a complex interplay between pro- and anti-viral factors that ultimately determines the spread or halt of virus infections in tissues. This interplay develops over multiple rounds of infection. The purpose of this study was to determine how cellular-level processes combine to impact the spatial spread of infection. We measured the kinetics of virus replication (VSV), antiviral paracrine signal upregulation and secretion, spatial spread of virus and paracrine antiviral signaling, and inhibition of virus production in antiviral-exposed A549 human lung epithelial cells. We found that initially infected cells released antiviral signals 4-to-7 hours following production of virus. However, the subsequent rapid dissemination of signal and fast induction of a robust and persistent antiviral state ultimately led to a suppression of infection spread. This work shows how cellular responses to infection and activation of antiviral responses can integrate to ultimately control infection spread across host cell populations. PMID:27254596

  4. Determinants of ventilation and pulmonary artery pressure during early acclimatization to hypoxia in humans.

    PubMed

    Fatemian, Marzieh; Herigstad, Mari; Croft, Quentin P P; Formenti, Federico; Cardenas, Rosa; Wheeler, Carly; Smith, Thomas G; Friedmannova, Maria; Dorrington, Keith L; Robbins, Peter A

    2016-03-01

    Pulmonary ventilation and pulmonary arterial pressure both rise progressively during the first few hours of human acclimatization to hypoxia. These responses are highly variable between individuals, but the origin of this variability is unknown. Here, we sought to determine whether the variabilities between different measures of response to sustained hypoxia were related, which would suggest a common source of variability. Eighty volunteers individually underwent an 8-h isocapnic exposure to hypoxia (end-tidal P(O2)=55 Torr) in a purpose-built chamber. Measurements of ventilation and pulmonary artery systolic pressure (PASP) assessed by Doppler echocardiography were made during the exposure. Before and after the exposure, measurements were made of the ventilatory sensitivities to acute isocapnic hypoxia (G(pO2)) and hyperoxic hypercapnia, the latter divided into peripheral (G(pCO2)) and central (G(cCO2)) components. Substantial acclimatization was observed in both ventilation and PASP, the latter being 40% greater in women than men. No correlation was found between the magnitudes of pulmonary ventilatory and pulmonary vascular responses. For G(pO2), G(pCO2) and G(cC O2), but not the sensitivity of PASP to acute hypoxia, the magnitude of the increase during acclimatization was proportional to the pre-acclimatization value. Additionally, the change in G(pO2) during acclimatization to hypoxia correlated well with most other measures of ventilatory acclimatization. Of the initial measurements prior to sustained hypoxia, only G(pCO2) predicted the subsequent rise in ventilation and change in G(pO2) during acclimatization. We conclude that the magnitudes of the ventilatory and pulmonary vascular responses to sustained hypoxia are predominantly determined by different factors and that the initial G(pCO2) is a modest predictor of ventilatory acclimatization.

  5. Telomerase Cajal body protein 1 depletion inhibits telomerase trafficking to telomeres and induces G1 cell cycle arrest in A549 cells.

    PubMed

    Yuan, Ping; Wang, Zhitian; Lv, Wang; Pan, Hui; Yang, Yunhai; Yuan, Xiaoshuai; Hu, Jian

    2014-09-01

    Telomerase Cajal body protein 1 (TCAB1) is a telomerase holoenzyme, which is markedly enriched in Cajal bodies (CBs) and facilitates the recruitment of telomerase to CBs in the S phase of the cell cycle. This recruitment is dependent on TCAB1 binding to a telomerase RNA component. The majority of cancer cells are able to grow indefinitely due to telomerase and its mechanism of trafficking to telomeres. In the present study, a certain level of TCAB1 expression in A549 human lung cells was identified and TCAB1 knockdown exhibited a potent antiproliferative effect on these cells, which was coupled with a decrease in the cell density and activity of the cellular enzymes. In addition, TCAB1-depletion was demonstrated to inhibit telomerase trafficking to telomeres in the A549 cells, leading to subsequent G1 cell cycle arrest without inducing apoptotic cell death. Overall, these observations indicated that TCAB1 may be essential for A549 cell proliferation and cell cycle regulation, and may be a potential candidate for the development of a therapeutic target for lung adenocarcinomas.

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

    PubMed Central

    2011-01-01

    Background In mechanically ventilated preterm infants with respiratory distress syndrome (RDS), exogenous surfactant application has been demonstrated both to decrease DNA-synthesis but also and paradoxically to increase epithelial cell proliferation. However, the effect of exogenous surfactant has not been studied directly on alveolar type II cells (ATII cells), a key cell type responsible for alveolar function and repair. Objective The aim of this study was to investigate the effects of two commercially available surfactant preparations on ATII cell viability and DNA synthesis. Methods Curosurf® and Alveofact® were applied to two ATII cell lines (human A549 and mouse iMATII cells) and to primary rat ATII cells for periods of up to 24 h. Cell viability was measured using the redox indicator resazurin and DNA synthesis was measured using BrdU incorporation. Results Curosurf® resulted in slightly decreased cell viability in all cell culture models. However, DNA synthesis was increased in A549 and rat ATII cells but decreased in iMATII cells. Alveofact® exhibited the opposite effects on A549 cells and had very mild effects on the other two cell models. Conclusion This study showed that commercially available exogenous surfactants used to treat preterm infants with RDS can have profound effects on cell viability and DNA synthesis. PMID:21324208

  7. Relative sensitivity of immunohistochemistry, multiple reaction monitoring mass spectrometry, in situ hybridization and PCR to detect Coxsackievirus B1 in A549 cells

    PubMed Central

    Laiho, Jutta E.; Oikarinen, Maarit; Richardson, Sarah J.; Frisk, Gun; Nyalwidhe, Julius; Burch, Tanya C.; Morris, Margaret A.; Oikarinen, Sami; Pugliese, Alberto; Dotta, Francesco; Campbell-Thompson, Martha; Nadler, Jerry; Morgan, Noel G.; Hyöty, Heikki

    2017-01-01

    Background Enteroviruses (EVs) have been linked to the pathogenesis of several diseases and there is a collective need to develop improved methods for the detection of these viruses in tissue samples. Objectives This study evaluates the relative sensitivity of immunohistochemistry (IHC), proteomics, in situ hybridization (ISH) and RT-PCR to detect one common EV, Coxsackievirus B1 (CVB1), in acutely infected human A549 cells in vitro. Study design A549 cells were infected with CVB1 and diluted with uninfected A549 cells to produce a limited dilution series in which the proportion of infected cells ranged from 10−1 to 10−8. Analyses were carried out by several laboratories using IHC with different anti-EV antibodies, ISH with both ViewRNA and RNAScope systems, liquid chromatography multiple reaction monitoring mass spectrometry (LC/MRM/MS/MS), and two modifications of RT-PCR. Results RT-PCR was the most sensitive method for EV detection yielding positive signals in the most diluted sample (10−8). LC/MRM/MS/MS detected viral peptides at dilutions as high as 10−7. The sensitivity of IHC depended on the antibody used, and the most sensitive antibody (Dako clone 5D8/1) detected virus proteins at a dilution of 10−6, while ISH detected the virus at dilutions of 10−4. Conclusions All methods were able to detect CVB1 in infected A549 cells. RT-PCR was most sensitive followed by LC/MRM/MS/MS and then IHC. The results from this in vitro survey suggest that all methods are suitable tools for EV detection but that their differential sensitivities need to be considered when interpreting the results from such studies. PMID:26875099

  8. Cell cycle inhibitory activity of Piper longum against A549 cell line and its protective effect against metal-induced toxicity in rats.

    PubMed

    Sharma, Amit Kumar; Kumar, Shashank; Chashoo, Gousia; Saxena, Ajit K; Pandey, Abhay K

    2014-10-01

    Anticancer potential of Piper longum fruit against human cancer cell lines (DU-145 prostate, A549 lung, THP-1 leukemia, IGR-OVI-1 ovary and MCF-7 breast) as well as its in vitro and in vivo biochemical efficacy in A1Cl3-induced hepatotoxicity were evaluated in the rats. Dried samples were extracted with several solvents using soxhlet apparatus. Flavonoid content in chloroform, benzene, ethyl alcohol and aqueous extracts of fruit was 19, 14, 12 and 11 μg quercetin equivalent/mg of sample, respectively. Hexane extracts exhibited 90-92% cytotoxicity against most of the test cell lines (A549, THP-1, IGR-OVI-1 and MCF-7), while benzene extract displayed 84-87% cytotoxicity against MCF-7, IGR-OV-1 and THP-1 cell lines. Among extracts, hexane, benzene and acetone extracts demonstrated considerable cytotoxicity (91-95%) against A549 (lung cancer) cell line in Sulforhodamine B dye (SRB) assay. Cell cycle analysis revealed that hexane, benzene and acetone extracts produced 41, 63 and 43% sub-G1 DNA fraction, demonstrating cell cycle inhibitory potential of these extracts against A549 cell line. Chloroform, ethyl alcohol and aqueous extracts displayed 71, 64 and 65% membrane protective activity, respectively in lipid peroxidation inhibition assay. P. longum fruit extracts also ameliorated A1Cl3-induced hepatotoxicity, as indicated by alterations observed in serum enzymes ALP, SGOT and SGPT activity, as well as creatinine and bilirubin contents. In conclusion, study established the cytotoxic and hepatoprotective activity in P. longum extracts.

  9. miR-107 regulates cisplatin chemosensitivity of A549 non small cell lung cancer cell line by targeting cyclin dependent kinase 8

    PubMed Central

    Zhang, Zhe; Zhang, Lu; Yin, Zhi-Yi; Fan, Xing-Long; Hu, Bo; Wang, Lun-Qing; Zhang, Di

    2014-01-01

    Previous studies demonstrated that the acquired drug resistance of non-small cell lung cancer (NSCLC) was related to deregulation of miRNAs. However, the effects of miR-107 and the mechanism through which miR-107 affects the cisplatin chemoresistance in NSCLC have not been reported. TaqMan RT-PCR or Western blot assay was performed to detect the expression of mature miR-107 and cyclin dependent kinase 8 (CDK8) protein. The viabilities of treated cells were analyzed using MTT assay. We found that the expression level of miR-107 in A549 cells was significantly lower than that in normal human bronchial epithelial cells (0.45 ± 0.26 vs. 1.00 ± 0.29, P = 0.032). The MTT assay showed that the A549 cells transfected with miR-107 mimics were significantly more sensitive to the therapy of cisplatin than control cells. A549 cells transfected with miR-107 mimics showed a decreased CDK8 protein expression. Downregulation of CDK8 expression by siRNAs, A549 cells became more sensitive to the therapy of cisplatin. In addition, the enhanced growth-inhibitory effect by the miR-107 mimic transfection was enhanced after the addition of CDK8 siRNA. In conclusion, the present study provides the first evidence that miR-107 plays a key role in cisplatin resistance by targeting the CDK8 protein in NSCLC cell lines, suggesting that miR-107 can be used to predict a patient’s response to chemotherapy as well as serve as a novel potential maker for NSCLC therapy. PMID:25400821

  10. TIMP-2 modulates cancer cell transcriptional profile and enhances E-cadherin/beta-catenin complex expression in A549 lung cancer cells

    PubMed Central

    Bourboulia, Dimitra; Han, HuiYing; Isaac, Biju; Wei, Beiyang; Neckers, Len; Stetler-Stevenson, William G.

    2013-01-01

    Tissue Inhibitor of Metalloproteinase 2 (TIMP-2) plays an essential role in regulating matrix remodeling, cell growth, differentiation, angiogenesis and apoptosis in vitro and in vivo. We have recently shown that TIMP-2-mediated inhibition of tumor growth is independent of matrix metalloproteinase-mediated mechanisms, and is a consequence of modulating both the tumor cells and the tumor microenvironment. In the current study we aim to identify the molecular pathways associated with these effects. We analyzed the transcriptional profile of the human lung cancer cell line A549 upon overexpression of TIMP-2 and Ala+TIMP-2 (mutant that does not inhibit MMP activity), and we found changes in gene expression predominantly related to decreased tumor development and metastasis. Increased E-cadherin expression in response to both TIMP-2 and Ala+TIMP-2 expression was confirmed by real time quantitative RT-PCR and immunoblotting. A549 cells treated with epidermal growth factor (EGF) displayed loss of cobblestone morphology and cell-cell contact, while cells overexpressing TIMP-2 or Ala+TIMP-2 were resistant to EGF-induced morphological changes. Moreover, exogenous treatment with recombinant Ala+TIMP-2 blocked EGF induced down-regulation of E-cadherin. In vivo, immunohistochemistry of A549 xenografts expressing either TIMP-2 or Ala+TIMP-2 demonstrated increased E-cadherin protein levels. More importantly, transcriptional profile analysis of tumor tissue revealed critical pathways associated with effects on tumor-host interaction and inhibition of tumor growth. In conclusion, we show that TIMP-2 promotes an anti-tumoral transcriptional profile in vitro and in vivo, including upregulation of E-cadherin, in A549 lung cancer cells. PMID:23371049

  11. Impact of CHK2-small interfering RNA on CpG ODN7909-enhanced radiosensitivity in lung cancer A549 cells

    PubMed Central

    Chen, Wei; Liu, Xiaoqun; Qiao, Tiankui; Yuan, Sujuan

    2012-01-01

    Objective To investigate the impact of checkpoint kinase 2 (CHK2)-small interfering RNA (CHK2-siRNA) on the enhancement of radiosensitivity by CpG oligodeoxynucleotide (ODN) 7909 in lung cancer A549 cells. Methods The A549 cells were randomly divided into five groups: control, CpG, X-ray, CpG + X-ray, and CHK2-siRNA + CpG + X-ray. Cell colonization was observed using inverted microscopy. Cell cycle and apoptosis were analyzed by flow cytometry. CHK2 expression was detected by Western blot. CHK2-siRNA was adopted to silence the expression of CHK2. Results The level of CHK2 phosphorylation was higher in the CpG + X-ray group than in the X-ray group. Increases in G2/mitotic (M) phase arrest and apoptosis and a decrease of cell survival rate in the CpG + X-ray group were statistically significant (P < 0.05) when compared with the CHK2-siRNA + CpG + X-ray group in which the expression of CHK2 was obviously inhibited. The combination of CpG ODN7909 and X-ray irradiation was found to enhance the mitotic death of A549 cells. The sensitization enhancement ratio of mean death dose (D0) was 1.42 in the CpG + X-ray group, which was higher than that of the CHK2-siRNA + CpG + X-ray group, in which D0 was 1.05. Conclusion To a certain extent, the impact of a combination of CpG ODN7909 and X-ray on G2/M phase arrest, apoptosis, and rate of cell survival was attenuated by CHK2-siRNA in human lung adenocarcinoma A549 cells, indicating that increased phosphorylation of CHK2 might be a radiosensitive pathway. PMID:23233807

  12. Telomerase downregulation induced by the G-quadruplex ligand 12459 in A549 cells is mediated by hTERT RNA alternative splicing

    PubMed Central

    Gomez, Dennis; Lemarteleur, Thibault; Lacroix, Laurent; Mailliet, Patrick; Mergny, Jean-Louis; Riou, Jean-François

    2004-01-01

    Ligand 12459, a potent G-quadruplex-interacting agent that belongs to the triazine series, was previously shown to downregulate telomerase activity in the human A549 lung carcinoma cell line. We show here that the downregulation of telomerase activity is caused by an alteration of the hTERT splicing pattern induced by 12459, i.e. an almost complete disappearance of the active (+α,+β) transcript and an over-expression of the inactive –β transcript. Spliced intron 6 forming the –β hTERT transcript contained several tracks of G-rich sequences able to form G-quadruplexes. By using a specific PCR-stop assay, we show that 12459 is able to stabilize the formation of these G-quadruplex structures. A549 cell line clones selected for resistance to 12459 have been analyzed for their hTERT splicing pattern. Resistant clones are able to maintain the active hTERT transcript under 12459 treatment, suggesting the appearance of mechanisms able to bypass the 12459-induced splicing alterations. In contrast to 12459, telomestatin and BRACO19, two other G-quadruplex-interacting agents, have no effect on the hTERT splicing pattern in A549 cells, are cytotoxic against the A549-resistant clones and display a lower efficiency to stabilize hTERT G-quadruplexes. These results lead us to propose that 12459 impairs the splicing machinery of hTERT through stabilization of quadruplexes located in the hTERT intron 6. Differences of selectivity between 12459, BRACO19 and telomestatin for these hTERT quadruplexes may be important to explain their respective activity and inactivity against hTERT splicing. PMID:14729921

  13. Pulmonary hypertension

    MedlinePlus

    Pulmonary arterial hypertension; Sporadic primary pulmonary hypertension; Familial primary pulmonary hypertension; Idiopathic pulmonary arterial hypertension; Primary pulmonary hypertension; PPH; Secondary pulmonary ...

  14. Plasma Metabolomics in Human Pulmonary Tuberculosis Disease: A Pilot Study

    PubMed Central

    Frediani, Jennifer K.; Jones, Dean P.; Tukvadze, Nestan; Uppal, Karan; Sanikidze, Eka; Kipiani, Maia; Tran, ViLinh T.; Hebbar, Gautam; Walker, Douglas I.; Kempker, Russell R.; Kurani, Shaheen S.; Colas, Romain A.; Dalli, Jesmond; Tangpricha, Vin; Serhan, Charles N.; Blumberg, Henry M.; Ziegler, Thomas R.

    2014-01-01

    We aimed to characterize metabolites during tuberculosis (TB) disease and identify new pathophysiologic pathways involved in infection as well as biomarkers of TB onset, progression and resolution. Such data may inform development of new anti-tuberculosis drugs. Plasma samples from adults with newly diagnosed pulmonary TB disease and their matched, asymptomatic, sputum culture-negative household contacts were analyzed using liquid chromatography high-resolution mass spectrometry (LC-MS) to identify metabolites. Statistical and bioinformatics methods were used to select accurate mass/charge (m/z) ions that were significantly different between the two groups at a false discovery rate (FDR) of q<0.05. Two-way hierarchical cluster analysis (HCA) was used to identify clusters of ions contributing to separation of cases and controls, and metabolomics databases were used to match these ions to known metabolites. Identity of specific D-series resolvins, glutamate and Mycobacterium tuberculosis (Mtb)-derived trehalose-6-mycolate was confirmed using LC-MS/MS analysis. Over 23,000 metabolites were detected in untargeted metabolomic analysis and 61 metabolites were significantly different between the two groups. HCA revealed 8 metabolite clusters containing metabolites largely upregulated in patients with TB disease, including anti-TB drugs, glutamate, choline derivatives, Mycobacterium tuberculosis-derived cell wall glycolipids (trehalose-6-mycolate and phosphatidylinositol) and pro-resolving lipid mediators of inflammation, known to stimulate resolution, efferocytosis and microbial killing. The resolvins were confirmed to be RvD1, aspirin-triggered RvD1, and RvD2. This study shows that high-resolution metabolomic analysis can differentiate patients with active TB disease from their asymptomatic household contacts. Specific metabolites upregulated in the plasma of patients with active TB disease, including Mtb-derived glycolipids and resolvins, have potential as biomarkers

  15. Carbocisteine attenuates hydrogen peroxide-induced inflammatory injury in A549 cells via NF-κB and ERK1/2 MAPK pathways.

    PubMed

    Wang, Wei; Zheng, Jin-Ping; Zhu, Shao-Xuan; Guan, Wei-Jie; Chen, Mao; Zhong, Nan-Shan

    2015-02-01

    Carbocisteine is a mucolytic drug with anti-oxidative effect, we had previously proved that carbocisteine remarkably reduced the rate of acute exacerbations and improved the quality of life in patients with chronic obstructive pulmonary disease (COPD), however, very little is known about its mechanisms. In this study, we aimed to investigate the anti-inflammatory effects of carbocisteine against hydrogen peroxide (H2O2). A549 cells were cultured in vitro and treated with H2O2 as damaged cell models, carbocisteine was administered 24h prior to or after H2O2 exposure, and the protective effects of carbocisteine were determined by MTT, qRT-PCR, ELISA, western blot and immunofluorescence assays. The results showed that carbocisteine could increase cell viability and decrease LDH, IL-6 and IL-8 levels in the supernatant. Additionally, carbocisteine decreased IL-6, IL-8, TNF-α, IP-10 and MIP-1β mRNA in a dose-dependent manner. Moreover, carbocisteine could attenuate phosphorylation of NF-κB p65 and ERK1/2 and inhibit the nuclear translocation of pNF-κB p65 induced by H2O2. In conclusion, carbocisteine inhibited H2O2-induced inflammatory injury in A549 cells, NF-κB and ERK1/2 MAPK were the target pathways.

  16. Theophylline prevents NAD{sup +} depletion via PARP-1 inhibition in human pulmonary epithelial cells

    SciTech Connect

    Moonen, Harald J.J. . E-mail: h.moonen@grat.unimaas.nl; Geraets, Liesbeth; Vaarhorst, Anika; Bast, Aalt; Wouters, Emiel F.M.; Hageman, Geja J.

    2005-12-30

    Oxidative DNA damage, as occurs during exacerbations in chronic obstructive pulmonary disease (COPD), highly activates the nuclear enzyme poly(ADP-ribose)polymerase-1 (PARP-1). This can lead to cellular depletion of its substrate NAD{sup +}, resulting in an energy crisis and ultimately in cell death. Inhibition of PARP-1 results in preservation of the intracellular NAD{sup +} pool, and of NAD{sup +}-dependent cellular processes. In this study, PARP-1 activation by hydrogen peroxide decreased intracellular NAD{sup +} levels in human pulmonary epithelial cells, which was found to be prevented in a dose-dependent manner by theophylline, a widely used compound in the treatment of COPD. This enzyme inhibition by theophylline was confirmed in an ELISA using purified human PARP-1 and was found to be competitive by nature. These findings provide new mechanistic insights into the therapeutic effect of theophylline in oxidative stress-induced lung pathologies.

  17. Protection of A549 cells against the toxic effects of sulphur mustard by hexamethylenetetramine.

    PubMed

    Lindsay, C D; Hambrook, J L

    1997-02-01

    The A549 cell line was used as a model of the deep lung to study the toxicity and mechanism of action of sulphur mustard (HD), using the neutral red (NR) dye retention and gentian violet (GV) assays as indices of cell viability. It was found that exposure to concentrations in excess of 40 microM HD resulted in a rapid onset of toxicity. Exposure to 1000 microM HD reduced viability in A549 cell cultures to 61% after 2 h (control cultures = 100%), whereas exposure to 40 microM HD did not result in deleterious effects until 26 h at which point viability fell to only 84% (NR assay). Agarose gel electrophoresis of cell cultures exposed to 40 and 1000 microM HD and harvested at 4.5, 19 and 43 h after exposure to HD, indicated that cell death was due to necrosis, despite the observation that at the higher concentration of HD cells displayed many of the features common to cells undergoing apoptotic death. The ability of hexamethylenetetramine (HMT) to protect A549 cells against the effects of an LC50 challenge dose of HD was assessed using the GV and NR assays. It was found that HMT (15 mM) could protect cells against the effects of HD though HMT had to be present at the time of HD challenge. Cultures treated with HD only were 49% viable at 48 h after HD challenge, compared to 101% for protected cultures (NR assay) and 58% and 91% for unprotected and protected cultures respectively using the GV assay. Morphological observations of GV and NR stained cultures confirmed these findings. HMT concentrations of 2.5 to 25 mM were used. Maximal protection against the toxic effects of HD (LC50) was found at 10 to 25 mM HMT. Over this concentration range, HMT did not exert any toxic effects on A549 cells. Pretreatment of A549 cultures with HMT followed by its removal prior to HD challenge had no protective effect. Similarly, treating cultures with HD followed by addition of HMT did not increase the viability of the cultures, even if the HMT was added immediately after HD exposure

  18. Interactions between heart rate variability and pulmonary gas exchange efficiency in humans.

    PubMed

    Sin, Peter Y W; Webber, Matthew R; Galletly, Duncan C; Ainslie, Philip N; Brown, Stephen J; Willie, Chris K; Sasse, Alexander; Larsen, Peter D; Tzeng, Yu-Chieh

    2010-07-01

    The respiratory component of heart rate variability (respiratory sinus arrhythmia, RSA) has been associated with improved pulmonary gas exchange efficiency in humans via the apparent clustering and scattering of heart beats in time with the inspiratory and expiratory phases of alveolar ventilation, respectively. However, since human RSA causes only marginal redistribution of heart beats to inspiration, we tested the hypothesis that any association between RSA amplitude and pulmonary gas exchange efficiency may be indirect. In 11 patients with fixed-rate cardiac pacemakers and 10 healthy control subjects, we recorded R-R intervals, respiratory flow, end-tidal gas tension and the ventilatory equivalents for carbon dioxide and oxygen during 'fast' (0.25 Hz) and 'slow' paced breathing (0.10 Hz). Mean heart rate, mean arterial blood pressure, mean arterial pressure fluctuations, tidal volume, end-tidal CO(2), and were similar between pacemaker and control groups in both the fast and slow breathing conditions. Although pacemaker patients had no RSA and slow breathing was associated with a 2.5-fold RSA amplitude increase in control subjects (39 +/- 21 versus 97 +/- 45 ms, P < 0.001), comparable (main effect for breathing frequency, F(1,19) = 76.54, P < 0.001) and reductions (main effect for breathing frequency, F(1,19) = 23.90, P < 0.001) were observed for both cohorts during slow breathing. In addition, the degree of (r = 0.36, P = 0.32) and reductions (r = 0.29, P = 0.43) from fast to slow breathing were not correlated to the degree of associated RSA amplitude enhancements in control subjects. These findings suggest that the association between RSA amplitude and pulmonary gas exchange efficiency during variable-frequency paced breathing observed in prior human work is not contingent on RSA being present. Therefore, whether RSA serves an intrinsic physiological function in optimizing pulmonary gas exchange efficiency in humans requires further experimental validation.

  19. Vagal nerve activity contributes to improve the efficiency of pulmonary gas exchange in hypoxic humans.

    PubMed

    Ito, Shoji; Sasano, Hiroshi; Sasano, Nobuko; Hayano, Junichiro; Fisher, Joseph A; Katsuya, Hirotada

    2006-09-01

    The aim of this study was to test our hypothesis that both phasic cardiac vagal activity and tonic pulmonary vagal activity, estimated as respiratory sinus arrhythmia (RSA) and anatomical dead space volume, respectively, contribute to improve the efficiency of pulmonary gas exchange in humans. We examined the effect of blocking vagal nerve activity with atropine on pulmonary gas exchange. Ten healthy volunteers inhaled hypoxic gas with constant tidal volume and respiratory frequency through a respiratory circuit with a respiratory analyser. Arterial partial pressure of O(2) (P(aO(2))) and arterial oxygen saturation (S(pO(2))) were measured, and alveolar-to-arterial P(O(2)) difference (D(A-aO(2))) was calculated. Anatomical dead space (V(D,an)), alveolar dead space (V(D,alv)) and the ratio of physiological dead space to tidal volume (V(D,phys)/V(T)) were measured. Electrocardiogram was recorded, and the amplitude of R-R interval variability in the high-frequency component (RRIHF) was utilized as an index of RSA magnitude. These parameters of pulmonary function were measured before and after administration of atropine (0.02 mg kg(-1)). Decreased RRIHF (P < 0.01) was accompanied by decreases in P(aO(2)) and S(pO(2)) (P < 0.05 and P < 0.01, respectively) and an increase in D(A-aO(2)) (P < 0.05). Anatomical dead space, V(D,alv) and V(D,phys)/V(T) increased (P < 0.01, P < 0.05 and P < 0.01, respectively) after atropine administration. The blockade of the vagal nerve with atropine resulted in an increase in V(D,an) and V(D,alv) and a deterioration of pulmonary oxygenation, accompanied by attenuation of RSA. Our findings suggest that both phasic cardiac and tonic pulmonary vagal nerve activity contribute to improve the efficiency of pulmonary gas exchange in hypoxic conscious humans.

  20. Doppler echo evaluation of pulmonary venous-left atrial pressure gradients: human and numerical model studies

    NASA Technical Reports Server (NTRS)

    Firstenberg, M. S.; Greenberg, N. L.; Smedira, N. G.; Prior, D. L.; Scalia, G. M.; Thomas, J. D.; Garcia, M. J.

    2000-01-01

    The simplified Bernoulli equation relates fluid convective energy derived from flow velocities to a pressure gradient and is commonly used in clinical echocardiography to determine pressure differences across stenotic orifices. Its application to pulmonary venous flow has not been described in humans. Twelve patients undergoing cardiac surgery had simultaneous high-fidelity pulmonary venous and left atrial pressure measurements and pulmonary venous pulsed Doppler echocardiography performed. Convective gradients for the systolic (S), diastolic (D), and atrial reversal (AR) phases of pulmonary venous flow were determined using the simplified Bernoulli equation and correlated with measured actual pressure differences. A linear relationship was observed between the convective (y) and actual (x) pressure differences for the S (y = 0.23x + 0.0074, r = 0.82) and D (y = 0.22x + 0.092, r = 0.81) waves, but not for the AR wave (y = 0. 030x + 0.13, r = 0.10). Numerical modeling resulted in similar slopes for the S (y = 0.200x - 0.127, r = 0.97), D (y = 0.247x - 0. 354, r = 0.99), and AR (y = 0.087x - 0.083, r = 0.96) waves. Consistent with numerical modeling, the convective term strongly correlates with but significantly underestimates actual gradient because of large inertial forces.

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

    PubMed

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

    2015-03-01

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

  2. Isolation of eastern equine encephalitis virus in A549 and MRC-5 cell cultures.

    PubMed

    Sotomayor, E A; Josephson, S L

    1999-07-01

    Eastern equine encephalitis (EEE) has been diagnosed either serologically or by virus isolation. Until now, the recovery of EEE virus has been delegated to reference laboratories with the expertise and resources needed to amplify the virus in a susceptible vertebrate host and/or to isolate and identify the virus in cell culture. We report a case in which EEE virus was recovered directly from a patient's cerebrospinal fluid in A549 and MRC-5 cell cultures. Many clinical virology laboratories routinely use these cells to recover adenovirus, herpes simplex virus, and enterovirus. To the best of our knowledge, this is the first report of isolation of EEE virus in A549 cell culture. This report demonstrates the possibility of recovery of EEE virus in cell culture without the necessity of bioamplification or maintaining unusual cell lines.

  3. Deactivation of A549 cancer cells in vitro by a dielectric barrier discharge plasma needle

    NASA Astrophysics Data System (ADS)

    Huang, Jun; Chen, Wei; Li, Hui; Wang, Xing-Quan; Lv, Guo-Hua; Khohsa, M. Latif; Guo, Ming; Feng, Ke-Cheng; Wang, Peng-Ye; Yang, Si-Ze

    2011-03-01

    An inactivation mechanism study on A549 cancer cells by means of a dielectric barrier discharge plasma needle is presented. The neutral red uptake assay provides a quantitative estimation of cell viability after plasma treatment. Experimental results show that the efficiency of argon plasma for the inactivation process is very dependent on power and treatment time. A 27 W power and 120 s treatment time along with 900 standard cubic centimeter per minute Ar flow and a nozzle-to-sample separation of 3 mm are the best parameters of the process. According to the argon emission spectra of the plasma jet and the optical microscope images of the A549 cells after plasma treatment, it is concluded that the reactive species (for example, OH and O) in the argon plasma play a major role in the cell deactivation.

  4. Rodent models of pulmonary hypertension: harmonisation with the world health organisation's categorisation of human PH.

    PubMed

    Ryan, J; Bloch, K; Archer, S L

    2011-08-01

    The WHO classification of pulmonary hypertension (PH) recognises five distinct groups, all sharing a mean, resting, pulmonary artery pressure (PAP) > 25 mmHg. The aetiology of PH varies by group (1-pulmonary vascular disease, 2-high left heart filling pressures, 3-hypoxia, 4-unresolved pulmonary embolism and 5-miscellaneous). Inclusion in a group reflects shared histological, haemodynamic and pathophysiological features and has therapeutic implications. Advantages of using rodent models to understand the pathophysiology of human PH and to test experimental therapies include the economy, safety and mechanistic certainty they provide. As rodent models are meant to reflect human PH, they should be categorised by a parallel PH classification and limitations in achieving this ideal recognised. Challenges with rodent models include: accurate phenotypic characterisation (haemodynamics, histology and imaging), species and strain variations in the natural history of PH, and poor fidelity to the relevant human PH group. Rat models of group 1 PH include: monocrotaline (± pneumonectomy), chronic hypoxia + SU-5416 (a VEGF receptor inhibitor) and the fawn-hooded rat (FHR). Mouse models of group 1 PH include: transgenic mice overexpressing the serotonin transporter or dominant-negative mutants of bone morphogenetic protein receptor-2. Group 1 PH is also created by infecting S100A4/Mts1 mice with γ-herpesvirus. The histological features of group 1 PH, but not PH itself, are induced by exposure to Schistosoma mansoni or Stachybotrys chartarum. Group 3 PH is modelled by exposure of rats or mice to chronic hypoxia. Rodent models of groups 2, 4 and 5 PH are needed. Comprehensive haemodynamic, histological and molecular phenotyping, coupled with categorisation into WHO PH groups, enhances the utility of rodent models.

  5. NADPH:cytochrome c (P450) reductase activates tirapazamine (SR4233) to restore hypoxic and oxic cytotoxicity in an aerobic resistant derivative of the A549 lung cancer cell line

    PubMed Central

    Saunders, M P; Patterson, A V; Chinje, E C; Harris, A L; Stratford, I J

    2000-01-01

    Tirapazamine (TPZ, SR4233, WIN 59075) is a bioreductive drug that is activated in regions of low oxygen tension to a cytotoxic radical intermediate. This labile metabolite shows high selective toxicity towards hypoxic cells, such as those found in solid tumours. Under aerobic conditions, redox cycling occurs with subsequent generation of superoxide radicals, which are also cytotoxic. NADPH:cytochrome c (P450) reductase (P450R) is a one-electron reducing enzyme that efficiently activates TPZ. Recently a derivative of the A549 non-small cell lung cancer cell line (A549c50) was generated that showed substantially reduced P450R activity compared to its parental line (Elwell et al (1997) Biochem Pharmacol54: 249–257). Here, it is demonstrated that the A549c50 cells are markedly more resistant to TPZ under both aerobic and hypoxic conditions. In addition, these cells have a dramatically impaired ability to metabolize TPZ to its two-electron reduction product, SR4317, under hypoxic conditions when compared to wild-type cells. P450R activity in the A549c50 cells was reintroduced to similar levels as that seen in the parental A549 cells by transfection of the full-length cDNA for human P450R. These P450R over-expressing cells exhibit restored sensitivity to TPZ under both aerobic and hypoxic conditions, comparable to that found in the original parental A549 cells. Further, the ability of the transfected cells to metabolize TPZ to SR4317 under hypoxic conditions is also shown to be restored. This provides further evidence that P450R can play an important role in the activation, metabolism and toxicity of this lead bioreductive drug. © 2000 Cancer Research Campaign PMID:10682679

  6. The common anesthetic, sevoflurane, induces apoptosis in A549 lung alveolar epithelial cells.

    PubMed

    Wei, Gui-Hua; Zhang, Juan; Liao, Da-Qing; Li, Zhuo; Yang, Jing; Luo, Nan-Fu; Gu, Yan

    2014-01-01

    Lung alveolar epithelial cells are the first barrier exposed to volatile anesthetics, such as sevoflurane, prior to reaching the targeted neuronal cells. Previously, the effects of volatile anesthetics on lung surfactant were studied primarily with physicochemical models and there has been little experimental data from cell cultures. Therefore it was investigated whether sevoflurane induces apoptosis of A549 lung epithelial cells. A549 cells were exposed to sevoflurane via a calibrated vaporizer with a 2 l/min flow in a gas‑tight chamber at 37˚C. The concentration of sevoflurane in Dulbecco's modified Eagle's medium was detected with gas chromatography. Untreated cells and cells treated with 2 µM daunorubicin hydrochloride (DRB) were used as negative and positive controls, respectively. Apoptosis factors, including the level of ATP, apoptotic‑bodies by terminal deoxynucleotidyl transferase‑mediated dUTP nick end labeling (TUNEL) assay, DNA damage and the level of caspase 3/7 were analyzed. Cells treated with sevoflurane showed a significant reduction in ATP compared with untreated cells. Effects in the DRB group were greater than in the sevoflurane group. The difference of TUNEL staining between the sevoflurane and untreated groups was statistically significant. DNA degradation was observed in the sevoflurane and DRB groups, however this was not observed in the untreated group. The sevoflurane and DRB groups induced increased caspase 3/7 activation compared with untreated cells. These results suggest that sevoflurane induces apoptosis in A549 cells. In conclusion, 5% sevoflurane induced apoptosis of A549 lung alveolar epithelial cells, which resulted in decreased cell viability, increased apoptotic bodies, impaired DNA integrality and increased levels of caspase 3/7.

  7. Defect of hepatocyte growth factor production by fibroblasts in human pulmonary emphysema.

    PubMed

    Plantier, Laurent; Marchand-Adam, Sylvain; Marchal-Sommé, Joëlle; Lesèche, Guy; Fournier, Michel; Dehoux, Monique; Aubier, Michel; Crestani, Bruno

    2005-04-01

    Pulmonary emphysema results from an excessive degradation of lung parenchyma associated with a failure of alveolar repair. Secretion by pulmonary fibroblasts of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) is crucial to an effective epithelial repair after lung injury. We hypothesized that abnormal HGF or KGF secretion by pulmonary fibroblasts could play a role in the development of emphysema. We measured in vitro production of HGF and KGF by human fibroblasts cultured from emphysematous and normal lung samples. HGF and KGF production was quantified at basal state and after stimulation. Intracellular content of HGF was lower in emphysema (1.52 pg/mug, range of 0.15-7.40 pg/mug) than in control fibroblasts (14.16 pg/mug, range of 2.50-47.62 pg/mug; P = 0.047). HGF production by emphysema fibroblasts (19.3 pg/mug protein, range of 10.4-39.2 pg/mug) was lower than that of controls at baseline (57.5 pg/mug, range of 20.4-116 pg/mug; P = 0.019) and after stimulation with interleukin-1beta or prostaglandin E(2). Neither retinoic acids (all-trans and 9-cis) nor N-acetylcysteine could reverse this abnormality. KGF production by emphysema fibroblasts (5.3 pg/mug, range of 2.2-9.3 pg/mug) was similar to that of controls at baseline (2.6 pg/mug, range of 1-6.1 pg/mug; P = 0.14) but could not be stimulated with interleukin-1beta. A decreased secretion of HGF by pulmonary fibroblasts could contribute to the insufficient alveolar repair in pulmonary emphysema.

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

    PubMed

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

    2016-12-09

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

  9. Wnt/{beta}-catenin signaling regulates cancer stem cells in lung cancer A549 cells

    SciTech Connect

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

    2010-02-12

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-12-01

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

  11. Establishment of a first-line second-line treatment model for human pulmonary adenocarcinoma

    PubMed Central

    Wang, Lining; Wang, Yu; Guan, Qi; Liu, Yong; He, Tianyi; Wang, Jiaru

    2016-01-01

    Lung cancer is one of the most prevalent types of cancer in the world. Surgery, chemotherapy and radiotherapy are used clinically as treatments for numerous cancers. Due to the appearance of drug resistance, the remission rate is limited to 40–50%. Docetaxel and pemetrexed are two drugs commonly used, and their effects in single-phase cell culture are well known. From the pharmacological point of view, it appears rational to hypothesize that sequential therapy effects can show better outcomes compared with traditional single-phase experiments. Considering this, the present study aimed to establish a first-line second-line adenocarcinoma treatment model, using the combination of cisplatin with docetaxel or pemetrexed in vitro in different sequential therapy timings. To test this, the human lung cancer A549 cell line was used. The inhibitory effect was determined by adding docetaxel following treatment with cisplatin and pemetrexed (Pem-Doc group) and comparing this with a group in which pemetrexed was added subsequent to treatment with cisplatin and docetaxel (Doc-Pem group). Additionally, the differences in the gene and protein expression levels of excision repair cross-completion gene 1 (ERCC1), a gene that promotes drug resistance to cisplatin, were compared between the two groups. The present results showed that the inhibitory effect of cell proliferation in the Pem-Doc group was increased compared with that of Doc-Pem group, while the gene expression and protein levels of ERCC1 in the Pem-Doc group were decreased compared with those of Doc-Pem group. The Pem-Doc treatment plan is more effective in inhibiting cell proliferation and in lowering the expression of the ERCC1 gene. Therefore, Pem-Doc may be a more effective adenocarcinoma treatment. PMID:28105156

  12. Fused pulmonary lobes is a rat model of human Fraser syndrome

    SciTech Connect

    Kiyozumi, Daiji; Nakano, Itsuko; Takahashi, Ken L.; Hojo, Hitoshi; Aoyama, Hiroaki; Sekiguchi, Kiyotoshi

    2011-07-29

    Highlights: {yields} Fused pulmonary lobes (fpl) mutant rats exhibit similar phenotypes to Fraser syndrome. {yields} The fpl gene harbors a nonsense mutation in Fraser syndrome-associated gene Frem2. {yields} Fpl mutant is defined as a first model of human Fraser syndrome in rats. -- Abstract: Fused pulmonary lobes (fpl) is a mutant gene that is inherited in an autosomal recessive manner and causes various developmental defects, including fusion of pulmonary lobes, and eyelid and digit anomalies in rats. Since these developmental defects closely resemble those observed in patients with Fraser syndrome, a recessive multiorgan disorder, and its model animals, we investigated whether the abnormal phenotypes observed in fpl/fpl mutant rats are attributable to a genetic disorder similar to Fraser syndrome. At the epidermal basement membrane in fpl/fpl mutant neonates, the expression of QBRICK, a basement membrane protein whose expression is attenuated in Fraser syndrome model mice, was greatly diminished compared with control littermates. Quantitative RT-PCR analyses of Fraser syndrome-related genes revealed that Frem2 transcripts were markedly diminished in QBRICK-negative embryos. Genomic DNA sequencing of the fpl/fpl mutant identified a nonsense mutation that introduced a stop codon at serine 2005 in Frem2. These findings indicate that the fpl mutant is a rat model of human Fraser syndrome.

  13. Flavonoids from Gynostemma pentaphyllum exhibit differential induction of cell cycle arrest in H460 and A549 cancer cells.

    PubMed

    Tsui, Ko-Chung; Chiang, Tzu-Hsuan; Wang, Jinn-Shyan; Lin, Li-Ju; Chao, Wei-Chih; Chen, Bing-Huei; Lu, Jyh-Feng

    2014-10-31

    Flavonoids, containing mainly kaempferol rhamnohexoside derivatives, were extracted from Gynostemma pentaphyllum (G. pentaphyllum) and their potential growth inhibition effects against H460 non-small cell lung cancer cells was explored and compared to that on A549 cells. The extracted flavonoids were found to exhibit antiproliferation effects against H460 cells (IC50 = 50.2 μg/mL), although the IC50 of H460 is 2.5-fold that of A549 cells (IC50 = 19.8 μg/mL). Further investigation revealed that H460 cells are more susceptible to kaempferol than A549, whereas A549 cell growth is better inhibited by kaempferol rhamnohexoside derivatives as compared with H460. In addition, flavonoids from G. pentaphyllum induced cell cycle arrest at both S and G2/M phases with concurrent modulated expression of the cellular proteins cyclin A, B, p53 and p21 in A549 cells, but not H460. On the contrary, apoptosis and concomitant alteration in balance of BCL-2 and BAX expression as well as activation of caspase-3 were equally affected between both cells by flavonoid treatment. These observations strongly suggest the growth inhibition discrepancy between H460 and A549 following flavonoid treatment can be attributed to the lack of cell cycle arrest in H460 cells and the differences between H460 and A549 cells may serve as contrasting models for further mechanistic investigations.

  14. Hantavirus antibodies in rodents and human cases with pulmonary syndrome, Rio Negro, Argentina.

    PubMed

    Larrieu, Edmundo; Cantoni, Gustavo; Herrero, Eduardo; Pérez, Alicia; Talmon, Gabriel; Vázquez, Gabriela; Arellano, Odila; Padula, Paula

    2008-01-01

    In Río Negro Province, Argentina, human cases of hantavirus pulmonary syndrome (HPS) appeared in the region of subantarctic forests. The Andes virus (ANDV) has been identified in the region both in Oligoryzomys longicaudatus rodents and in humans, with the main transmission being from rodents to humans but also showing the possibility of human to human transmission. Between 1996 and 2004, in 40 campaigns, 29.960 night-traps for capturing live rodents were set up. Blood samples were obtained from the rodents and processed using enzyme immunoassay with recombinant antigens made from ANDV. A total of 1767 rodents were captured, with a capture success of 5.9% and an antibody prevalence of 2.1%. Important differences were observed among the species captured from Andes and Steppe regions. Seropositive Oligoryzomys longicaudatus, Abrotrix olivaceus, Abrotrix xanhtothinus and Loxodontomus microtus were captured. During the 1993-2004 period, 40 HPS cases were registered.

  15. Determinants of ventilation and pulmonary artery pressure during early acclimatization to hypoxia in humans

    PubMed Central

    Fatemian, Marzieh; Herigstad, Mari; Croft, Quentin P. P.; Formenti, Federico; Cardenas, Rosa; Wheeler, Carly; Smith, Thomas G.; Friedmannova, Maria; Dorrington, Keith L.

    2015-01-01

    Key points Lung ventilation and pulmonary artery pressure rise progressively in response to 8 h of hypoxia, changes described as ‘acclimatization to hypoxia’. Acclimatization responses differ markedly between humans for unknown reasons.We explored whether the magnitudes of the ventilatory and vascular responses were related, and whether the degree of acclimatization could be predicted by acute measurements of ventilatory and vascular sensitivities.In 80 healthy human volunteers measurements of acclimatization were made before, during, and after a sustained exposure to 8 h of isocapnic hypoxia.No correlation was found between measures of ventilatory and pulmonary vascular acclimatization.The ventilatory chemoreflex sensitivities to acute hypoxia and hypercapnia all increased in proportion to their pre‐acclimatization values following 8 h of hypoxia. The peripheral (rapid) chemoreflex sensitivity to CO2, measured before sustained hypoxia against a background of hyperoxia, was a modest predictor of ventilatory acclimatization to hypoxia. This finding has relevance to predicting human acclimatization to the hypoxia of altitude. Abstract Pulmonary ventilation and pulmonary arterial pressure both rise progressively during the first few hours of human acclimatization to hypoxia. These responses are highly variable between individuals, but the origin of this variability is unknown. Here, we sought to determine whether the variabilities between different measures of response to sustained hypoxia were related, which would suggest a common source of variability. Eighty volunteers individually underwent an 8‐h isocapnic exposure to hypoxia (end‐tidal P O2=55 Torr) in a purpose‐built chamber. Measurements of ventilation and pulmonary artery systolic pressure (PASP) assessed by Doppler echocardiography were made during the exposure. Before and after the exposure, measurements were made of the ventilatory sensitivities to acute isocapnic hypoxia (GpO2) and

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

    PubMed

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

    2017-03-01

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

  17. Human Recombinant Apyrase Therapy Protects Against Canine Pulmonary Ischemia-Reperfusion Injury

    PubMed Central

    Ibrahim, Mohsen; Wang, Xingan; Puyo, Carlos A.; Montecalvo, Alessandro; Huang, Howard J.; Hachem, Ramsey R.; Andreetti, Claudio; Menna, Cecilia; Chen, Ridong; Krupnick, Alexander S.; Kreisel, Daniel; Rendina, Erino A.; Gelman, Andrew E.

    2014-01-01

    INTRODUCTION There is accumulating evidence that extracellular adenosine triphosphate (eATP) promotes many of the underlying mechanisms that exacerbate acute lung injury. However, much of this data is from inbred rodent models indicating the need for further investigation in higher vertebrates to better establish clinical relevance. To this end we evaluated a human recombinant apyrase therapy in a canine warm pulmonary ischemia-reperfusion injury (IRI) model and measured eATP levels in human lung recipients with or without primary lung allograft dysfunction (PGD). METHODS Warm ischemia was induced for 90 minutes in the left lung of 14 mongrel dogs. Seven minutes after reperfusion, the apyrase APT102 (1 mg/kg, N=7) or saline vehicle (N=7) was injected into the pulmonary artery. Arterial blood gases were obtained every 30 minutes up to 180 minutes after reperfusion. Bronchioalveolar lavage fluid (BALF) was analyzed for eATP concentration, cellularity and inflammatory mediator accumulation. Thirty bilateral human lung transplant recipients were graded for immediate early PGD and assessed for BALF eATP levels. RESULTS APT102-treated dogs had progressively better lung function and less pulmonary edema over the 3-hour reperfusion period when compared to vehicle-treated controls. Protection from IRI was observed with lower BALF eATP levels, fewer airway leukocytes and blunted inflammatory mediator expression. Additionally, human lung recipients with moderate to severe PGD had significantly higher eATP levels when compared to recipients without this injury. CONCLUSIONS Extracellular ATP accumulates in acutely injured canine and human lungs. Strategies that target eATP reduction may help protect lung recipients from IRI. PMID:25455749

  18. Deduced amino acid sequence of human pulmonary surfactant proteolipid: SPL(pVal)

    SciTech Connect

    Whitsett, J.A.; Glasser, S.W.; Korfhagen, T.R.; Weaver, T.E.; Clark, J.; Pilot-Matias, T.; Meuth, J.; Fox, J.L.

    1987-05-01

    Hydrophobic, proteolipid-like protein of Mr 6500 was isolated from ether/ethanol extracts of human, canine and bovine pulmonary surfactant. Amino acid composition of the protein demonstrated a remarkable abundance of hydrophobic residues, particularly valine and leucine. The N-terminal amino acid sequence of the human protein was determined: N-Leu-Ile-Pro-Cys-Cys-Pro-Val-Asn-Leu-Lys-Arg-Leu-Leu-Ile-Val4... An oligonucleotide probe was used to screen an adult human lung cDNA library and resulted in detection of cDNA clones with predicted amino acid sequence with close identity to the N-terminal amino acid sequence of the human peptide. SPL(pVal) was found within the reading frame of a larger peptide. SPL(pVal) results from proteolytic processing of a larger preprotein. Northern blot analysis detected in a single 1.0 kilobase SPL(pVal) RNA which was less abundant in fetal than in adult lung. Mixtures of purified canine and bovine SPL(pVal) and synthetic phospholipids display properties of rapid adsorption and surface tension lowering activity characteristic of surfactant. Human SPL(pVal) is a pulmonary surfactant proteolipid which may therefore be useful in combination with phospholipids and/or other surfactant proteins for the treatment of surfactant deficiency such as hyaline membrane disease in newborn infants.

  19. Dielectric barrier discharge plasma in Ar/O{sub 2} promoting apoptosis behavior in A549 cancer cells

    SciTech Connect

    Huang Jun; Li Hui; Chen Wei; Lv Guohua; Wang Xingquan; Zhang Guoping; Wang Pengye; Ostrikov, Kostya; Yang Size

    2011-12-19

    The Ar/O{sub 2} plasma needle in the induction of A549 cancer cells apoptosis process is studied by means of real-time observation. The entire process of programmed cell death is observed. The typical morphological changes of A549 apoptosis are detected by 4', 6-diamidino-2-phenylindole staining, for example, chromatin condensation and nuclear fragmentation. Cell viability is determined and quantified by neutral red uptake assay, and the survival rate of A549 from Ar/O{sub 2} plasmas is presented. Further spectral analysis indicates the reactive species, including O and OH play crucial roles in the cell inactivation.

  20. Dielectric barrier discharge plasma in Ar/O2 promoting apoptosis behavior in A549 cancer cells

    NASA Astrophysics Data System (ADS)

    Huang, Jun; Li, Hui; Chen, Wei; Lv, Guo-Hua; Wang, Xing-Quan; Zhang, Guo-Ping; Ostrikov, Kostya; Wang, Peng-Ye; Yang, Si-Ze

    2011-12-01

    The Ar/O2 plasma needle in the induction of A549 cancer cells apoptosis process is studied by means of real-time observation. The entire process of programmed cell death is observed. The typical morphological changes of A549 apoptosis are detected by 4', 6-diamidino-2-phenylindole staining, for example, chromatin condensation and nuclear fragmentation. Cell viability is determined and quantified by neutral red uptake assay, and the survival rate of A549 from Ar/O2 plasmas is presented. Further spectral analysis indicates the reactive species, including O and OH play crucial roles in the cell inactivation.

  1. Low level CO2 effects on pulmonary function in humans

    NASA Technical Reports Server (NTRS)

    Sexton, J.; Mueller, K.; Elliott, A.; Gerzer, D.; Strohl, K. P.; West, J. B. (Principal Investigator)

    1998-01-01

    The purpose of the study was to determine whether chamber exposure to low levels of CO2 results in functional alterations in gas mixing and closing volume in humans. Four healthy volunteer subjects were exposed to 0.7% CO2 and to 1.2% CO2. Spirometry, lung volumes, single breath nitrogen washout, diffusing capacity for carbon monoxide (DLCO) by two methods, and cardiac output were measured in triplicate. Values were obtained over two non-consecutive days during the training period (control) and on days 2 or 3, 4, 6, 10, 13, and 23 of exposure to each CO2 level. Measurements were made during the same time of day. There was one day of testing after exposure, while still in the chamber but off carbon dioxide. The order of testing, up until measurements of DLCO and cardiac output, were randomized to avoid presentation effects. The consistent findings were a reduction in diffusing capacity for carbon monoxide and a fall in cardiac output, occurring to a similar degree with both exposures. For the group as a whole, there was no indication of major effects on spirometry, lung volumes, gas mixing or dead space. We conclude that small changes may occur in the function of distal gas exchanging units; however, these effects were not associated with any adverse health effects. The likelihood of pathophysiologic changes in lung function or structure with 0.7 or 1.2% CO2 exposure for this period of time, is therefore, low.

  2. Air Pollution by Hydrothermal Volcanism and Human Pulmonary Function.

    PubMed

    Linhares, Diana; Ventura Garcia, Patrícia; Viveiros, Fátima; Ferreira, Teresa; dos Santos Rodrigues, Armindo

    2015-01-01

    The aim of this study was to assess whether chronic exposure to volcanogenic air pollution by hydrothermal soil diffuse degassing is associated with respiratory defects in humans. This study was carried in the archipelago of the Azores, an area with active volcanism located in the Atlantic Ocean where Eurasian, African, and American lithospheric plates meet. A cross-sectional study was performed on a study group of 146 individuals inhabiting an area where volcanic activity is marked by active fumarolic fields and soil degassing (hydrothermal area) and a reference group of 359 individuals inhabiting an area without these secondary manifestations of volcanism (nonhydrothermal area). Odds ratio (OR) and 95% confidence intervals (CIs) were adjusted for age, gender, fatigue, asthma, and smoking. The OR for restrictive defects and for exacerbation of obstructive defects (COPD) in the hydrothermal area was 4.4 (95% CI 1.78-10.69) and 3.2 (95% CI 1.82-5.58), respectively. Increased prevalence of restrictions and all COPD severity ranks (mild, moderate, and severe) was observed in the population from the hydrothermal area. These findings may assist health officials in advising and keeping up with these populations to prevent and minimize the risk of respiratory diseases.

  3. Air Pollution by Hydrothermal Volcanism and Human Pulmonary Function

    PubMed Central

    Linhares, Diana; Garcia, Patrícia Ventura; Viveiros, Fátima; Ferreira, Teresa; Rodrigues, Armindo dos Santos

    2015-01-01

    The aim of this study was to assess whether chronic exposure to volcanogenic air pollution by hydrothermal soil diffuse degassing is associated with respiratory defects in humans. This study was carried in the archipelago of the Azores, an area with active volcanism located in the Atlantic Ocean where Eurasian, African, and American lithospheric plates meet. A cross-sectional study was performed on a study group of 146 individuals inhabiting an area where volcanic activity is marked by active fumarolic fields and soil degassing (hydrothermal area) and a reference group of 359 individuals inhabiting an area without these secondary manifestations of volcanism (nonhydrothermal area). Odds ratio (OR) and 95% confidence intervals (CIs) were adjusted for age, gender, fatigue, asthma, and smoking. The OR for restrictive defects and for exacerbation of obstructive defects (COPD) in the hydrothermal area was 4.4 (95% CI 1.78–10.69) and 3.2 (95% CI 1.82–5.58), respectively. Increased prevalence of restrictions and all COPD severity ranks (mild, moderate, and severe) was observed in the population from the hydrothermal area. These findings may assist health officials in advising and keeping up with these populations to prevent and minimize the risk of respiratory diseases. PMID:26301247

  4. Prostanoid EP1- and TP-receptors involved in the contraction of human pulmonary veins

    PubMed Central

    Walch, Laurence; de Montpreville, Vincent; Brink, Charles; Norel, Xavier

    2001-01-01

    To characterize the prostanoid receptors (TP, FP, EP1 and/or EP3) involved in the vasoconstriction of human pulmonary veins, isolated venous preparations were challenged with different prostanoid-receptor agonists in the absence or presence of selective antagonists. The stable thromboxane A2 mimetic, U46619, was a potent constrictor agonist on human pulmonary veins (pEC50=8.60±0.11 and Emax=4.61±0.46 g; n=15). The affinity values for two selective TP-antagonists (BAY u3405 and GR32191B) versus U46619 were BAY u3405: pA2=8.94±0.23 (n=3) and GR32191B: apparent pKB=8.25±0.34 (n=3), respectively. These results are consistent with the involvement of TP-receptor in the U46619 induced contractions. The two EP1-/EP3- agonists (17-phenyl-PGE2 and sulprostone) induced contraction of human pumonary veins (pEC50=8.56±0.18; Emax=0.56±0.24 g; n=5 and pEC50=7.65±0.13; Emax=1.10±0.12 g; n=14, respectively). The potency ranking for these agonists: 17-phenyl-PGE2>sulprostone suggests the involvement of an EP1-receptor rather than EP3. In addition, the contractions induced by sulprostone, 17-phenyl-PGE2 and the IP-/EP1- agonist (iloprost) were blocked by the DP-/EP1-/EP2-receptor antagonist (AH6809) as well as by the EP1 antagonist (SC19220). PGF2α induced small contractions which were blocked by AH6809 while fluprostenol was ineffective. These results indicate that FP-receptors are not implicated in the contraction of human pulmonary veins. These data suggest that the contractions induced by prostanoids involved TP- and EP1-receptors in human pulmonary venous smooth muscle. PMID:11739243

  5. Developmental Origins of Hypoxic Pulmonary Hypertension and Systemic Vascular Dysfunction: Evidence from Humans.

    PubMed

    Sartori, Claudio; Rimoldi, Stefano F; Duplain, Hervé; Stuber, Thomas; Garcin, Sophie; Rexhaj, Emrush; Allemann, Yves; Scherrer, Urs

    2016-01-01

    Epidemiological studies have shown an association between pathologic events occurring during fetal/perinatal life and the development of cardiovascular and metabolic disease in adulthood. These observations have led to the so-called developmental origin of adult disease hypothesis. More recently, evidence has been provided that the pulmonary circulation is also an important target for the developmental programming of adult disease in both experimental animal models and in humans. Here we will review this evidence and provide insight into mechanisms that may play a pathogenic role.

  6. Histopathological Analogies in Chronic Pulmonary Lesions between Cattle and Humans: Basis for an Alternative Animal Model

    PubMed Central

    Ramírez-Romero, Rafael; Nevárez-Garza, Alicia M.; Rodríguez-Tovar, Luis E.; Wong-González, Alfredo; Ledezma-Torres, Rogelio A.; Hernández-Vidal, Gustavo

    2012-01-01

    Most of the natural cases of pneumonia in feedlot cattle are characterized by a longer clinical course due to chronic lung lesions. Microscopically, these lesions include interstitial fibroplasia, bronchitis, bronchiectasis, bronchiolitis obliterans, and epithelial metaplasia of the airways. Herein, the aim was to review, under a medical perspective, the pathologic mechanisms operating in these chronic pneumonic lesions in calves. Based on the similarities of these changes to those reported in bronchiolitis obliterans/organising pneumonia (BO/OP) and chronic obstructive pulmonary disease (COPD) in human beings, calves are proposed as an alternative animal model. PMID:22629176

  7. SB203580 enhances the RV-induced loss of mitochondrial membrane potential and apoptosis in A549 cells

    NASA Astrophysics Data System (ADS)

    Li, Hai-yang; Zhuang, Cai-ping; Wang, Xiao-ping; Chen, Tong-sheng

    2012-03-01

    Resveratrol (RV), a naturally occurring phytoalexin, is known to possess a wide spectrum of chemopreventive and chemotherapeutic effects in various stages of human tumors. p38, a member of the mitogen-activated protein kinase (MAPK) superfamily, is always activated by some extracellular stimulus to regulate many cellular signal transduction pathways, such as apoptosis, proliferation, and inflammation and so on. In this report, we assessed the effect of SB203580, a specific inhibitor of p38 MAPK signaling pathway, on the RV-induced apoptosis in human lung adenocarcinoma (A549) cells. CCK-8 assay showed that pretreatment with SB203580 significantly enhanced the cytotoxicity of RV, which was further verified by analyzing the phosphatidylserine externalization using flow cytometry. In order to further confirm whether SB203580 accelerated apoptosis via the intrinsic apoptosis pathway, we analyzed the dysfunction of mitochondrial membrane potential (Δψm) of cells stained with rhodamine 123 by using flow cytometry after treatment with RV in the absence and presence of SB203580. Our data for the first time reported that p38 inhibitor SB203580 enhanced the RV-induced apoptosis via a mitochondrial pathway.

  8. Animal Models of Pulmonary Hypertension: Matching Disease Mechanisms to Etiology of the Human Disease

    PubMed Central

    Colvin, Kelley L.; Yeager, Michael E.

    2015-01-01

    Recently a great deal of progress has been made in our understanding of pulmonary hypertension (PH). Research from the past 30 years has resulted in newer treatments that provide symptomatic improvements and delayed disease progression. Unfortunately, the cure for patients with this lethal syndrome remains stubbornly elusive. With the relative explosion of scientific literature regarding PH, confusion has arisen regarding animal models of the disease and their correlation to the human condition. This short review uniquely focuses on the clear and present need to better correlate mechanistic insights from existing and emerging animal models of PH to specific etiologies and histopathologies of human PH. A better understanding of the pathologic processes in various animal models and how they relate to the human disease should accelerate the development of newer and more efficacious therapies. PMID:25705569

  9. Induction of apoptosis by FFJ-5, a novel naphthoquinone compound, occurs via downregulation of PKM2 in A549 and HepG2 cells

    PubMed Central

    Wei, Xiaoli; Li, Ming; Ma, Mingming; Jia, Huina; Zhang, Yu; Kang, Wenyi; Wang, Tianxiao; Shi, Xiaoyan

    2017-01-01

    Pyruvate kinase isoenzyme M2 (PKM2) has previously been identified as a tumor biomarker and as a potential target for cancer therapy. In this study, F§FJ-5, a characterized naphthoquinone modifier of mollugin, was synthesized in order to investigate its anticancer activity and the potential mechanisms. It was observed that FFJ-5 inhibited the cell growth of human lung adenocarcinoma cells A549 and human hepatoma cells HepG2 by MTT assays. FFJ-5 arrested cell cycle at the G2/M phase. Further analyses demonstrated that FFJ-5 attenuated the expression of PKM2 and reduced the production of adenosine triphosphate (ATP). Reduced expression and activity of epidermal growth factor receptor (EGFR) and Akt were observed in A549 and HepG2 cells exposed to FFJ-5. FFJ-5 exposure also resulted in cell apoptosis, in association with decreased intracellular pH level and mitochondrial membrane potential. In addition, FFJ-5 activated the caspase-3 cascade. In conclusion, FFJ-5 inhibited cancer cell growth via the blocking the EGFR-Akt-PKM2 pathway or through the synergistic action of EGFR, Akt and PKM2 proteins, alongside a decrease in ATP production. In addition, FFJ-5 induced cancer cell apoptosis by decreasing the intracellular pH level and the mitochondrial apoptosis pathway. The present results suggest a potential role of FFJ-5 on the therapy of human cancer. PMID:28356960

  10. SIRT1 is required for mitochondrial biogenesis reprogramming in hypoxic human pulmonary arteriolar smooth muscle cells.

    PubMed

    Li, Pengyun; Liu, Yan; Burns, Nana; Zhao, Ke-Seng; Song, Rui

    2017-03-22

    Although recent studies have reported that mitochondria are putative oxygen sensors underlying hypoxic pulmonary vasoconstriction, little is known concerning the sirtuin 1 (SIRT1)-mediated mitochondrial biogenesis regulatory program in pulmonary arteriolar smooth muscle cells (PASMCs) during hypoxia/reoxygenation (H/R). We investigated the epigenetic regulatory mechanism of mitochondrial biogenesis and function in human PASMCs during H/R. Human PASMCs were exposed to hypoxia of 24-48 h and reoxygenation of 24-48 h. The expression of SIRT1 was reduced in a time-dependent manner. Mitochondrial transcription factor A (TFAM) expression was increased during hypoxia and decreased during reoxygenation, while the release of TFAM was increased in a time-dependent manner. Lentiviral overexpression of SIRT1 preserved SIRT3 deacetylase activity in human PASMCs exposed to H/R. Knockdown of PGC-1α suppressed the effect of SIRT1 on SIRT3 activity. Knockdown of SIRT3 abrogated SIRT1-mediated deacetylation of cyclophilin D (CyPD). Notably, knockdown of SIRT3 or PGC-1α suppressed the incremental effect of SIRT1 on mitochondrial TFAM, mitochondrial DNA (mtDNA) content and cellular ATP levels. Importantly, polydatin restored SIRT1 levels in human PASMCs exposed to H/R. Knockdown of SIRT1 suppressed the effect of polydatin on mitochondrial TFAM, mtDNA content and cellular ATP levels. In conclusion, SIRT1 expression is decreased in human PASMCs during H/R. TFAM expression in mitochondria is reduced and the release of TFAM is increased by H/R. PGC-1α/SIRT3/CyPD mediates the protective effect of SIRT1 on expression and release of TFAM and mitochondrial biogenesis and function. Polydatin improves mitochondrial biogenesis and function by enhancing SIRT1 expression in hypoxic human PASMCs.

  11. microRNA-99a is downregulated and promotes proliferation, migration and invasion in non-small cell lung cancer A549 and H1299 cells.

    PubMed

    Chen, Changjin; Zhao, Ziyi; Liu, Yu; Mu, Dezhi

    2015-03-01

    There is increasing evidence that microRNAs (miRNAs) are able to play a key role in the diagnosis and therapy of cancer. miRNA-99a (miR-99a), which is downregulated in several human malignancies, has been reported as a potential tumor suppressor. However, to the best of our knowledge, the expression and function of miR-99a has not been investigated in human non-small cell lung cancer (NSCLC) at present. The aim of the current study was to evaluate the association between NSCLC and miR-99a. miR-99a expression was analyzed in 15 pairs of NSCLC and non-cancerous tissue samples by reverse transcription-quantitative polymerase chain reaction. In addition, the NSCLC A549 and H1299 cell lines were transfected with miR-99a mimics, and the effect of miR-99a on the cell cycle, cell proliferation, migration and colony formation of A549 and H1299 cells was investigated. It was found that the level of miR-99a expression was significantly downregulated in NSCLC tissues and that ectopic overexpression of miR-99a significantly inhibited the growth of A549 and H1299 cells. Additionally, ectopic overexpression of miR-99a inhibited A549 and H1299 cell migration and invasion by inhibiting epithelial to mesenchymal transition. The downregulation of insulin-like growth factor 1 receptor (IGF-1R) by miR-99a and knockdown of IGF-1R mediated by siRNA were each found to phenocopy the effect of miR-99a overexpression in NSCLC. To the best of our knowledge, the present study demonstrated for the first time that, in NSCLC, miR-99a is downregulated and thus regulates proliferation, colony formation and migration through the IGF-1R pathway, which indicates that miR-99a is a diagnostic biomarker for NSCLC.

  12. TGF-β and Hypoxia/Reoxygenation Promote Radioresistance of A549 Lung Cancer Cells through Activation of Nrf2 and EGFR

    PubMed Central

    Lee, Sae-lo-oom; Ryu, Hwani; Son, A-rang; Seo, Bitna; Kim, Jooyoung; Jung, Seung-Youn; Song, Jie-Young; Hwang, Sang-Gu; Ahn, Jiyeon

    2016-01-01

    Although many studies have examined the roles of hypoxia and transforming growth factor- (TGF-) β separately in the tumor microenvironment, the effects of simultaneous treatment with hypoxia/reoxygenation and TGF-β on tumor malignancy are unclear. Here, we investigated the effects of redox signaling and oncogenes on cell proliferation and radioresistance in A549 human lung cancer cells in the presence of TGF-β under hypoxia/reoxygenation conditions. Combined treatment with TGF-β and hypoxia activated epidermal growth factor receptor (EGFR) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a redox-sensitive transcription factor. Interestingly, Nrf2 knockdown suppressed the effects of combined treatment on EGFR phosphorylation. In addition, blockade of EGFR signaling also suppressed induction of Nrf2 following combined treatment with hypoxia and TGF-β, indicating that the combined treatment induced positive crosstalk between Nrf2 and EGFR. TGF-β and hypoxia/reoxygenation increased the accumulation of reactive oxygen species (ROS), while treatment with N-acetyl-l-cysteine abolished the activation of Nrf2 and EGFR. Treatment with TGF-β under hypoxic conditions increased the proliferation of A549 cells compared with that after vehicle treatment. Moreover, cells treated with the combined treatment exhibited resistance to ionizing radiation (IR), and knockdown of Nrf2 increased IR-induced cell death under these conditions. Thus, taken together, our findings suggested that TGF-β and hypoxia/reoxygenation promoted tumor progression and radioresistance of A549 cells through ROS-mediated activation of Nrf2 and EGFR. PMID:26904167

  13. ROS-mediated endoplasmic reticulum stress and mitochondrial dysfunction underlie apoptosis induced by resveratrol and arsenic trioxide in A549 cells.

    PubMed

    Gu, Shiyan; Chen, Chengzhi; Jiang, Xuejun; Zhang, Zunzhen

    2016-02-05

    Although it is well documented that endoplasmic reticulum (ER) stress and mitochondrial dysfunction are associated with apoptosis, little is known about whether they are involved in the apoptotic cell death induced by resveratrol and arsenic trioxide (ATO) combination. In this study, we identified a series of sensitization effects of resveratrol on human lung adenocarcinoma A549 cells to ATO treatment, with the combination index (CI) of resveratrol and ATO less than 1. Then, we demonstrated that ER stress was contributed to this synergistic effect, which was manifested by increased the expression levels of ER stress hallmarks, including 78-kDa glucose-regulated protein (GRP 78), caspase 12 and C/EBP-homologous protein (CHOP), In addition, mitochondrial dysfunction was observed after exposure of A549 cells to resveratrol or/and ATO, which was displayed by some alterations of mitochondria-related events, such as loss of mitochondrial membrane potential, cytochrome c release and changes of Bax and Bcl-2 expressions. Our results further demonstrated that resveratrol and ATO-induced ER stress and mitochondrial dysfunction were mediated by reactive oxygen species (ROS), showing that pre-treatment of N-acetyl-l-cysteine, a potent ROS scavenger, restored the ER stress and mitochondrial dysfunction in cells co-treated with resveratrol and ATO, thereby leading to the reduction of the apoptosis. Collectively, these results clearly suggest that ROS-mediated ER stress and mitochondrial dysfunction were involved in the apoptosis induced by resveratrol and ATO in A549 cells, which provides a novel insight into the molecular mechanisms of resveratrol-mediated ATO-sensitization.

  14. Neutrophil-Derived MMP-8 Drives AMPK-Dependent Matrix Destruction in Human Pulmonary Tuberculosis.

    PubMed

    Ong, Catherine W M; Elkington, Paul T; Brilha, Sara; Ugarte-Gil, Cesar; Tome-Esteban, Maite T; Tezera, Liku B; Pabisiak, Przemyslaw J; Moores, Rachel C; Sathyamoorthy, Tarangini; Patel, Vimal; Gilman, Robert H; Porter, Joanna C; Friedland, Jon S

    2015-05-01

    Pulmonary cavities, the hallmark of tuberculosis (TB), are characterized by high mycobacterial load and perpetuate the spread of M. tuberculosis. The mechanism of matrix destruction resulting in cavitation is not well defined. Neutrophils are emerging as key mediators of TB immunopathology and their influx are associated with poor outcomes. We investigated neutrophil-dependent mechanisms involved in TB-associated matrix destruction using a cellular model, a cohort of 108 patients, and in separate patient lung biopsies. Neutrophil-derived NF-kB-dependent matrix metalloproteinase-8 (MMP-8) secretion was up-regulated in TB and caused matrix destruction both in vitro and in respiratory samples of TB patients. Collagen destruction induced by TB infection was abolished by doxycycline, a licensed MMP inhibitor. Neutrophil extracellular traps (NETs) contain MMP-8 and are increased in samples from TB patients. Neutrophils lined the circumference of human pulmonary TB cavities and sputum MMP-8 concentrations reflected TB radiological and clinical disease severity. AMPK, a central regulator of catabolism, drove neutrophil MMP-8 secretion and neutrophils from AMPK-deficient patients secrete lower MMP-8 concentrations. AMPK-expressing neutrophils are present in human TB lung biopsies with phospho-AMPK detected in nuclei. These data demonstrate that neutrophil-derived MMP-8 has a key role in the immunopathology of TB and is a potential target for host-directed therapy in this infectious disease.

  15. Human pulmonary dirofilariasis coexisting with intercostal neurilemmoma: a case report and literature review.

    PubMed

    Li, Chia-Ying; Chang, Yih-Leong; Lee, Yung-Chie

    2013-10-01

    Human pulmonary dirofilariasis (HPD) is a rare zoonotic infection caused by Dirofilaria immitis. Dogs are the definite hosts and humans are infected occasionally via a vector, generally a mosquito. Most thoracic neurilemmoma arise in the mediastinum and fewer tumors originate peripherally from the intercostal nerves. Most patients with HPD or thoracic neurilemmoma are asymptomatic and these diseases are often discovered incidentally. We present a 53-year-old female who was found to have a pulmonary nodule and a chest wall nodule during a routine health examination. She underwent a video-assisted thoracoscopic surgery (VATS) with partial lung resection and local excision of the chest wall. The pathological examination revealed a coiled, degenerating Dirofilariasis immitis worm surrounded by granulomatous inflammation with caseous necrosis and a neurilemmoma composed of S-100 protein immunoreactive but smooth muscle actin negative spindle cells. Because these diseases are self-limiting and make further treatment unnecessary, video-assisted thoracoscopic surgery (VATS) is considered preferable and less invasive for definitive diagnosis and management.

  16. TGFβ upregulates PAR-1 expression and signalling responses in A549 lung adenocarcinoma cells

    PubMed Central

    Smoktunowicz, Natalia; Platé, Manuela; Stern, Alejandro Ortiz; D'Antongiovanni, Vanessa; Robinson, Eifion; Chudasama, Vijay; Caddick, Stephen; Scotton, Chris J.; Jarai, Gabor; Chambers, Rachel C.

    2016-01-01

    The major high-affinity thrombin receptor, proteinase activated receptor-1 (PAR-1) is expressed at low levels by the normal epithelium but is upregulated in many types of cancer, including lung cancer. The thrombin-PAR-1 signalling axis contributes to the activation of latent TGFβ in response to tissue injury via an αvβ6 integrin-mediated mechanism. TGFβ is a pleiotropic cytokine that acts as a tumour suppressor in normal and dysplastic cells but switches into a tumour promoter in advanced tumours. In this study we demonstrate that TGFβ is a positive regulator of PAR-1 expression in A549 lung adenocarcinoma cells, which in turn increases the sensitivity of these cells to thrombin signalling. We further demonstrate that this effect is Smad3-, ERK1/2- and Sp1-dependent. We also show that TGFβ-mediated PAR-1 upregulation is accompanied by increased expression of integrin αv and β6 subunits. Finally, TGFβ pre-stimulation promotes increased migratory potential of A549 to thrombin. These data have important implications for our understanding of the interplay between coagulation and TGFβ signalling responses in lung cancer. PMID:27566553

  17. Middle infrared radiation induces G2/M cell cycle arrest in A549 lung cancer cells.

    PubMed

    Chang, Hsin-Yi; Shih, Meng-Her; Huang, Hsuan-Cheng; Tsai, Shang-Ru; Juan, Hsueh-Fen; Lee, Si-Chen

    2013-01-01

    There were studies investigating the effects of broadband infrared radiation (IR) on cancer cell, while the influences of middle-infrared radiation (MIR) are still unknown. In this study, a MIR emitter with emission wavelength band in the 3-5 µm region was developed to irradiate A549 lung adenocarcinoma cells. It was found that MIR exposure inhibited cell proliferation and induced morphological changes by altering the cellular distribution of cytoskeletal components. Using quantitative PCR, we found that MIR promoted the expression levels of ATM (ataxia telangiectasia mutated), ATR (ataxia-telangiectasia and Rad3-related and Rad3-related), TP53 (tumor protein p53), p21 (CDKN1A, cyclin-dependent kinase inhibitor 1A) and GADD45 (growth arrest and DNA-damage inducible), but decreased the expression levels of cyclin B coding genes, CCNB1 and CCNB2, as well as CDK1 (Cyclin-dependent kinase 1). The reduction of protein expression levels of CDC25C, cyclin B1 and the phosphorylation of CDK1 at Thr-161 altogether suggest G(2)/M arrest occurred in A549 cells by MIR. DNA repair foci formation of DNA double-strand breaks (DSB) marker γ-H2AX and sensor 53BP1 was induced by MIR treatment, it implies the MIR induced G(2)/M cell cycle arrest resulted from DSB. This study illustrates a potential role for the use of MIR in lung cancer therapy by initiating DSB and blocking cell cycle progression.

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

    NASA Astrophysics Data System (ADS)

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

    2015-10-01

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

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

    PubMed

    Kuźnar-Kamińska, Barbara; Mikuła-Pietrasik, Justyna; Sosińska, Patrycja; Książek, Krzysztof; Batura-Gabryel, Halina

    2016-01-01

    Patients with COPD develop lung cancer more frequently than healthy smokers. At the same time, molecular mediators promoting various aspects of cancer cell progression are still elusive. In this report, we examined whether COPD can be coupled with increased migration of non-small-cell lung cancer cells A549 and, if so, whether this effect may be related to altered production and activity of chemokines CCL21, CXCL5, and CXCL12. The study showed that the migration of A549 cells through the polycarbonate membrane and basement membrane extract toward a chemotactic gradient elicited by serum from patients with COPD was markedly higher as compared with serum from healthy donors. The concentration of CCL21 and CXCL12, but not CXCL5, in serum from patients with COPD was also increased. Experiments in which CCL21- and CXCL12-dependent signaling was blocked revealed that increased migration of the cancer cells upon treatment with serum from patients with COPD was mediated exclusively by CCL21. Collectively, our results indicate that COPD may contribute to the progression of lung cancer via CCL21-dependent intensification of cancer cell migration.

  20. Oxidative damage induced in A549 cells by physically and chemically characterized air particulate matter (PM2.5) collected in Abidjan, Côte d'Ivoire.

    PubMed

    Kouassi, Kouakou S; Billet, Sylvain; Garçon, Guillaume; Verdin, Anthony; Diouf, Amadou; Cazier, Fabrice; Djaman, Joseph; Courcot, Dominique; Shirali, Pirouz

    2010-05-01

    Exposure to high levels of air pollution particulate matter (PM) is strongly associated with increased pulmonary morbidity and mortality. However, the underlying mechanisms of action whereby PM cause adverse health effects are still unclear. In developing countries, like in the sub-Saharian region of Africa, people are often exposed to high PM levels. Hence, three PM(2.5) samples were collected in the District of Abidjan (Côte d'Ivoire), under rural, urban or industrial influences. Their most toxicologically relevant physical and chemical characteristics were determined--thereby showing that most of them were equal or smaller than 2.5 microm--and the influence of both natural (Ca, Na, Mg, Ti, etc.) and anthropic (Al, Fe, Mn, Cr, Pb, Zn, Cu, Ni, benzene and its derivatives, paraffins, etc.) emission sources. The toxicity induced by the three PM samples was studied through 5-bromodeoxyuridine incorporation to DNA, mitochondrial dehydrogenase activity and extracellular lactate dehydrogenase activity. Hence, effect concentrations at 10 and 50% (EC(10) and EC(50), respectively) were as follows: (i) rural PM--EC(10) = 5.91 microg cm(-2) and EC(50) = 29.55 microg cm(-2); (ii) urban PM--EC(10) = 5.45 microg cm(-2) and EC(50) = 27.23 microg cm(-2); and (iii) industrial PM--EC(10) = 6.86 microg cm(-2) and EC(50) = 34.29 microg cm(-2). Moreover, PM-induced oxidative damage in A549 cells was observed through the induction of lipid peroxidation, the alteration of superoxide dismutase activity, and the disruption of glutathione status. Both the transition metals and the organic chemicals within the three collected PM samples under study might be involved in the oxidative damage and, therefore, the toxicity they induced in A549 cells.

  1. The pathogenesis of bleomycin-induced lung injury in animals and its applicability to human idiopathic pulmonary fibrosis.

    PubMed

    Williamson, James D; Sadofsky, Laura R; Hart, Simon P

    2015-03-01

    Idiopathic pulmonary fibrosis (IPF) is a devastating disease of unknown etiology, for which there is no curative pharmacological therapy. Bleomycin, an anti-neoplastic agent that causes lung fibrosis in human patients has been used extensively in rodent models to mimic IPF. In this review, we compare the pathogenesis and histological features of human IPF and bleomycin-induced pulmonary fibrosis (BPF) induced in rodents by intratracheal delivery. We discuss the current understanding of IPF and BPF disease development, from the contribution of alveolar epithelial cells and inflammation to the role of fibroblasts and cytokines, and draw conclusions about what we have learned from the intratracheal bleomycin model of lung fibrosis.

  2. Wheatgrass extract inhibits hypoxia-inducible factor-1-mediated epithelial-mesenchymal transition in A549 cells

    PubMed Central

    Do, Nam Yong; Shin, Hyun-Jae

    2017-01-01

    BACKGROUND/OBJECTIVES Epithelial-mesenchymal transition (EMT) is involved in not only cancer development and metastasis but also non-cancerous conditions. Hypoxia is one of the proposed critical factors contributing to formation of chronic rhinosinusitis or nasal polyposis. Wheatgrass (Triticum aestivum) has antioxidant, anti-aging, and anti-inflammatory effects. In this study, we analyzed whether wheatgrass has an inhibitory effect on the EMT process in airway epithelial cells. MATERIALS/METHODS A549 human lung adenocarcinoma cells were incubated in hypoxic conditions (CO2 5%/O2 1%) for 24 h in the presence of different concentrations of wheatgrass extract (50, 75, 100, and 150 µg/mL) and changes in expression of epithelial or mesenchymal markers were evaluated by immunoblotting and immunofluorescence. Accordingly, associated EMT-related transcriptional factors, Snail and Smad, were also evaluated. RESULTS Hypoxia increased expression of N-cadherin and reduced expression of E-cadherin. Mechanistically, E-cadherin levels were recovered during hypoxia by silencing hypoxia inducible factor (HIF)-1α or administering wheatgrass extract. Wheatgrass inhibited the hypoxia-mediated EMT by reducing the expression of phosphorylated Smad3 (pSmad3) and Snail. It suppressed the hypoxia-mediated EMT processes of airway epithelial cells via HIF-1α and the pSmad3 signaling pathway. CONCLUSION These results suggest that wheatgrass has potential as a therapeutic or supplementary agent for HIF-1-related diseases. PMID:28386380

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

    PubMed

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

    1995-02-01

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

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

    PubMed Central

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

    1995-01-01

    The differentiation of A549, a human tumour cell line from type II pneumocytes, can be induced by a crude fibroblast-derived factor (FDF) isolated from the conditioned medium of glucocorticoid-treated lung fibroblasts. In the present report, we have used alkaline phosphatase as a differentiation marker to investigate the activity of a number of growth factors as potential candidates for this paracrine activity. This showed that insulin, interleukin 6 (IL-6), and interferon alpha (IFN-alpha) could simulate the activity of conditioned medium. Their effects were dexamethasone (DX) dependent, additive and reversible with a half-life of 1 week. Transforming growth factor alpha and beta, IL-1 alpha and epidermal growth factor, were all inhibitory, and inhibition was opposed, partially or completely, by DX. The most potent inducer was IL-6, but as DX was shown to decrease the concentration of IL-6 in lung fibroblast-conditioned medium it seems an unlikely candidate for FDF. Unlike FDF, all of the positive-acting factors were shown to induce plasminogen activator. FDF has also been shown to be active in the absence of DX. This suggests that differentiation-inducing activity may be present in several paracrine factors, but that so far a candidate for FDF has not been identified. PMID:7841035

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

    NASA Astrophysics Data System (ADS)

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

    2015-05-01

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

  6. Fenugreek extract diosgenin and pure diosgenin inhibit the hTERT gene expression in A549 lung cancer cell line.

    PubMed

    Rahmati-Yamchi, Mohammad; Ghareghomi, Somayyeh; Haddadchi, Gholamreza; Milani, Morteza; Aghazadeh, Mohammad; Daroushnejad, Hasan

    2014-09-01

    Trigonella foenum-graecum generally known as fenugreek, has been normally cultivated in Asia and Africa for the edible and medicinal values of its seeds. Fenugreek leaves and seeds have been used widely for therapeutic purposes. Fenugreek seed is recognized to show anti-diabetic and anti-nociceptive properties and other things such as hypocholesterolaemic, and anti-cancer. Diosgenin is a steroidal saponin from therapeutic herbs, fenugreek (T. foenum-graceum L.), has been well-known to have anticancer properties. Telomerase activity is not identified in usual healthy cells, while in carcinogenic cell telomerase expression is reactivated. Therefore telomerase illustrates a promising cancer therapeutic target. We deliberate the inhibitory effect of pure diosgenin and fenugreek extract diosgenin on human telomerase reverse transcriptase gene (hTERT) expression which is critical for telomerase activity. MTT-assay and qRT-PCR analysis were achieved to discover cytotoxicity effects and hTERT gene expression inhibition properties, separately. MTT results exhibited that IC50 for pure diosgenin were 47, 44 and 43 µM and for fenugreek extract diosgenin were 49, 48 and 47 µM for 24, 48 and 72 h after treatment. Culturing cells with pure diosgenin and fenugreek extract diosgenin treatment caused in down regulation of hTERT expression. These results indication that pure and impure diosgenin prevents telomerase activity by down regulation of the hTERT gene expression in A549 lung cancer cell line, with the difference that pure compound is more effective than another.

  7. Massive Pulmonary Embolism: Treatment with Thrombus Fragmentation and Local Fibrinolysis with Recombinant Human-Tissue Plasminogen Activator

    SciTech Connect

    Stock, Klaus Wilhelm; Jacob, Augustinus Ludwig; Schnabel, Karl Jakob; Bongartz, Georg; Steinbrich, Wolfgang

    1997-09-15

    Purpose: To report the results of thrombus fragmentation in combination with local fibrinolysis using recombinant human-tissue plasminogen activator (rtPA) in patients with massive pulmonary embolism. Methods: Five patients with massive pulmonary embolism were treated with thrombus fragmentation followed by intrapulmonary injection of rtPA. Clot fragmentation was performed with a guidewire, angiographic catheter, and balloon catheter. Three patients had undergone recent surgery; one of them received a reduced dosage of rtPA. Results: All patients survived and showed clinical improvement with a resultant significant (p < 0.05) decrease in the pulmonary blood pressure (mean systolic pulmonary blood pressure before treatment, 49 mmHg; 4 hr after treatment, 28 mmHg). Angiographic follow-up in three patients revealed a decrease in thrombus material and an increase in pulmonary perfusion. Two patients developed retroperitoneal hematomas requiring transfusion. Conclusion: Clot fragmentation and local fibrinolysis with rtPA was an effective therapy for massive pulmonary embolism. Bleeding at the puncture site was a frequent complication.

  8. Lipocalin-2 Promotes Endoplasmic Reticulum Stress and Proliferation by Augmenting Intracellular Iron in Human Pulmonary Arterial Smooth Muscle Cells

    PubMed Central

    Wang, Guoliang; Liu, Shenghua; Wang, Li; Meng, Liukun; Cui, Chuanjue; Zhang, Hao; Hu, Shengshou; Ma, Ning; Wei, Yingjie

    2017-01-01

    Endoplasmic reticulum (ER) stress, a feature of many conditions associated with pulmonary hypertension (PH), is increasingly recognized as a common response to promote proliferation in the walls of pulmonary arteries. Increased expression of Lipocalin-2 in PH led us to test the hypothesis that Lipocalin-2, a protein known to sequester iron and regulate it intracellularly, might facilitate the ER stress and proliferation in pulmonary arterial smooth muscle cells (PASMCs). In this study, we observed greatly increased Lcn2 expression accompanied with increased ATF6 cleavage in a standard rat model of pulmonary hypertension induced by monocrotaline. In cultured human PASMCs, Lcn2 significantly promoted ER stress (determined by augmented cleavage and nuclear localization of ATF6, up-regulated transcription of GRP78 and NOGO, increased expression of SOD2, and mild augmented mitochondrial membrane potential) and proliferation (assessed by Ki67 staining and BrdU incorporation). Lcn2 promoted ER stress accompanied with augmented intracellular iron levels in human PASMCs. Treatment human PASMCs with FeSO4 induced the similar ER stress and proliferation response and iron chelator (deferoxamine) abrogated the ER stress and proliferation induced by Lcn2 in cultured human PASMCs. In conclusion, Lcn2 significantly promoted human PASMC ER stress and proliferation by augmenting intracellular iron. The up-regulation of Lcn2 probably involved in the pathogenesis and progression of PH. PMID:28255266

  9. A role for the CXCL12 receptor, CXCR7, in the pathogenesis of human pulmonary vascular disease.

    PubMed

    Costello, Christine M; McCullagh, Brian; Howell, Katherine; Sands, Michelle; Belperio, John A; Keane, Michael P; Gaine, Sean; McLoughlin, Paul

    2012-06-01

    Given the critical role that endothelial cell dysfunction plays in the pathogenesis of pulmonary hypertensive diseases, we set out to establish if CXCR7, a receptor for the pro-angiogenic ligand CXCL12, is expressed in the vasculature of human lung diseases and examine its role in mediating CXCL12-induced responses in primary pulmonary human microvascular endothelial cells. Receptor and ligand expression was examined in control and explanted human hypertensive lungs, in human plasma and in hypoxic rodent lungs, by ELISA and immunohistochemical studies. Functional in vitro experiments examined the role of CXCR7 in CXCL12-induced lung microvascular endothelial cell proliferation, migration, and wound regeneration and repair. CXCR7 is elevated in the endothelium of explanted human hypertensive lungs and circulating CXCL12 concentrations are significantly elevated in disease. We demonstrate that alveolar hypoxia similar to that found in lung disease increases CXCR7 expression in the pulmonary endothelium. Furthermore, CXCR7 is the receptor through which endothelial cell regeneration and repair, and proliferation, is mediated, whereas signalling via CXCR4 is essential for chemotactic cell migration. Our findings demonstrate that CXCR7 has a critical but previously unrecognised role to play in endothelial cell proliferation, suggesting that CXCR7-mediated signalling may be functionally important in pulmonary vascular diseases.

  10. [Influence of Berberine on Cisplatin Antineoplastic Effect in A549 Cells].

    PubMed

    Jiang, Guojun; Li, Li; Wu, Xiaoxiang; Dong, Shuying; Tong, Xuhui

    2015-08-01

    背景与目的 以顺铂为基础的化疗方案是晚期非小细胞肺癌的一线化疗方案,但是由于顺铂的不良反应严重及耐药性的产生均限制了它的临床应用,本研究采用联合用药的方式观察黄连素对顺铂抗肿瘤作用的影响,并探讨其可能机制。方法 分别观察黄连素对肺腺癌细胞A549细胞中总Cx43蛋白、细胞膜Cx43蛋白的表达以及细胞缝隙连接功能的改变,通过标准细胞集落克隆实验观察黄连素对顺铂细胞毒性的影响;并观察PKC激酶的表达。结果 黄连素在0 μM-10 μM浓度范围内对细胞无毒性,通过增加细胞内总Cx43蛋白和胞膜Cx43蛋白的表达而增强细胞缝隙连接功能,0.1 μM、1 μM、10 μM黄连素可以显著增强细胞间的荧光传递,与空白对照组相比,黄连素预处理后的细胞间荧光传递功能分别增加了33.3% (P=0.002,3)、67.0% (P<0.001)、160.0% (P<0.001),这种作用与PKC的活性被抑制相关,抑制PKC活性可以进一步增加顺铂对A549细胞的毒性作用。结论 黄连素可通过增加A549细胞的缝隙连接功能而明显增强顺铂的细胞毒性。.

  11. Fused pulmonary lobes is a rat model of human Fraser syndrome.

    PubMed

    Kiyozumi, Daiji; Nakano, Itsuko; Takahashi, Ken L; Hojo, Hitoshi; Aoyama, Hiroaki; Sekiguchi, Kiyotoshi

    2011-07-29

    Fused pulmonary lobes (fpl) is a mutant gene that is inherited in an autosomal recessive manner and causes various developmental defects, including fusion of pulmonary lobes, and eyelid and digit anomalies in rats. Since these developmental defects closely resemble those observed in patients with Fraser syndrome, a recessive multiorgan disorder, and its model animals, we investigated whether the abnormal phenotypes observed in fpl/fpl mutant rats are attributable to a genetic disorder similar to Fraser syndrome. At the epidermal basement membrane in fpl/fpl mutant neonates, the expression of QBRICK, a basement membrane protein whose expression is attenuated in Fraser syndrome model mice, was greatly diminished compared with control littermates. Quantitative RT-PCR analyses of Fraser syndrome-related genes revealed that Frem2 transcripts were markedly diminished in QBRICK-negative embryos. Genomic DNA sequencing of the fpl/fpl mutant identified a nonsense mutation that introduced a stop codon at serine 2005 in Frem2. These findings indicate that the fpl mutant is a rat model of human Fraser syndrome.

  12. Human pulmonary artery endothelial cells in the model of mucopolysaccharidosis VI present a prohypertensive phenotype

    PubMed Central

    Golda, Adam; Jurecka, Agnieszka; Gajda, Karolina; Tylki-Szymańska, Anna; Lalik, Anna

    2015-01-01

    Background Mucopolysaccharidosis type VI (MPS VI) is an autosomal recessive lysosomal disorder caused by a deficient activity of N-acetylgalactosamine-4-sulfatase (ARSB). Pulmonary hypertension (PH) occurs in MPS VI patients and is a marker of bad prognosis. Malfunction of endothelium, which regulates vascular tonus and stimulates angiogenesis, can contribute to the occurrence of PH in MPS VI. Aim The aim of the study was to establish a human MPS VI cellular model of pulmonary artery endothelial cells (HPAECs) and evaluate how it affects factors that may trigger PH such as proliferation, apoptosis, expression of endothelial nitric oxide synthase (eNOS), natriuretic peptide type C (NPPC), and vascular endothelial growth factor A (VEGFA). Results Increasing concentrations of dermatan sulfate (DS) reduce the viability of the cells in both ARSB deficiency and controls, but hardly influence apoptosis. The expression of eNOS in HPAECs is reduced up to two thirds in the presence of DS. NPPC shows a biphasic expression reaction with an increase at 50 μg/mL DS and reduction at 0 and 100 μg/mL DS. The expression of VEGFA decreases with increasing DS concentrations and absence of elastin, and increases with increasing DS in the presence of elastin. Conclusion Our data suggest that MPS VI endothelium presents a prohypertensive phenotype due to the reduction of endothelium's proliferation ability and expression of vasorelaxing factors. PMID:26937388

  13. p53/PUMA expression in human pulmonary fibroblasts mediates cell activation and migration in silicosis

    PubMed Central

    Wang, Wei; Liu, Haijun; Dai, Xiaoniu; Fang, Shencun; Wang, Xingang; Zhang, Yingming; Yao, Honghong; Zhang, Xilong; Chao, Jie

    2015-01-01

    Phagocytosis of SiO2 into the lung causes an inflammatory cascade that results in fibroblast proliferation and migration, followed by fibrosis. Clinical evidence has indicated that the activation of alveolar macrophages by SiO2 produces rapid and sustained inflammation characterized by the generation of monocyte chemotactic protein 1, which, in turn, induces fibrosis. However, the details of events downstream of monocyte chemotactic protein 1 activity in pulmonary fibroblasts remain unclear. Here, to elucidate the role of p53 in fibrosis induced by silica, both the upstream molecular mechanisms and the functional effects on cell proliferation and migration were investigated. Experiments using primary cultured adult human pulmonary fibroblasts led to the following results: 1) SiO2 treatment resulted in a rapid and sustained increase in p53 and PUMA protein levels; 2) the MAPK and PI3K pathways were involved in the SiO2-induced alteration of p53 and PUMA expression; and 3) RNA interference targeting p53 and PUMA prevented the SiO2-induced increases in fibroblast activation and migration. Our study elucidated a link between SiO2-induced p53/PUMA expression in fibroblasts and cell migration, thereby providing novel insight into the potential use of p53/PUMA in the development of novel therapeutic strategies for silicosis treatment. PMID:26576741

  14. Predicting pulmonary fibrosis in humans after exposure to multi-walled carbon nanotubes (MWCNTs).

    PubMed

    Sharma, Monita; Nikota, Jake; Halappanavar, Sabina; Castranova, Vincent; Rothen-Rutishauser, Barbara; Clippinger, Amy J

    2016-07-01

    The increased production and use of multi-walled carbon nanotubes (MWCNTs) in a diverse array of consumer, medical, and industrial applications have raised concerns about potential human exposure to these materials in the workplace and ambient environments. Inhalation is a primary route of exposure to MWCNTs, and the existing data indicate that they are potentially hazardous to human health. While a 90-day rodent inhalation test (e.g., OECD Test No. 413: subchronic inhalation toxicity: 90-day study or EPA Health Effects Test Guidelines OPPTS 870.3465 90-day inhalation toxicity) is recommended by the U.S. Environmental Protection Agency Office of Pollution Prevention and Toxics for MWCNTs (and other CNTs) if they are to be commercially produced (Godwin et al. in ACS Nano 9:3409-3417, 2015), this test is time and cost-intensive and subject to scientific and ethical concerns. As a result, there has been much interest in transitioning away from studies on animals and moving toward human-based in vitro and in silico models. However, given the multiple mechanisms of toxicity associated with subchronic exposure to inhaled MWCNTs, a battery of non-animal tests will likely be needed to evaluate the key endpoints assessed by the 90-day rodent study. Pulmonary fibrosis is an important adverse outcome related to inhalation exposure to MWCNTs and one that the non-animal approach should be able to assess. This review summarizes the state-of-the-science regarding in vivo and in vitro toxicological methods for predicting MWCNT-induced pulmonary fibrosis.

  15. Pharmacological evidence for a novel cysteinyl-leukotriene receptor subtype in human pulmonary artery smooth muscle

    PubMed Central

    Walch, Laurence; Norel, Xavier; Bäck, Magnus; Gascard, Jean-Pierre; Dahlén, Sven-Erik; Brink, Charles

    2002-01-01

    To characterize the cysteinyl-leukotriene receptors (CysLT receptors) in isolated human pulmonary arteries, ring preparations were contracted with leukotriene C4 (LTC4) and leukotriene D4 (LTD4) in either the absence or presence of the selective CysLT1 receptor antagonists, ICI 198615, MK 571 or the dual CysLT1/CysLT2 receptor antagonist, BAY u9773. Since the contractions induced by the cysteinyl-leukotrienes (cysLTs) in intact preparations failed to attain a plateau response over the concentration range studied, the endothelium was removed and the tissue treated continuously with indomethacin (Rubbed+INDO). In these latter preparations, the pEC50 for LTC4 and LTD4 were not significantly different (7.61±0.07, n=20 and 7.96±0.09, n=22, respectively). However, the LTC4 and LTD4 contractions were markedly potentiated when compared with data from intact tissues. Leukotriene E4 (LTE4) did not contract human isolated pulmonary arterial preparations. In addition, treatment of preparations with LTE4 (1 μM; 30 min) did not modify either the LTC4 or LTD4 contractions. Treatment of preparations with the S-conjugated glutathione (S-hexyl-GSH; 100 μM, 30 min), an inhibitor of the metabolism of LTC4 to LTD4, did not modify LTC4 contractions. The pEC50 values for LTC4 were significantly reduced by treatment of the preparations with either ICI 198615, MK 571 or BAY u9773 and the pKB values were: 7.20, 7.02 and 6.26, respectively. In contrast, these antagonists did not modify the LTD4 pEC50 values. These findings suggest the presence of two CysLT receptors on human pulmonary arterial vascular smooth muscle. A CysLT1 receptor with a low affinity for CysLT1 antagonists and a novel CysLT receptor subtype, both responsible for vasoconstriction. Activation of this latter receptor by LTC4 and LTD4 induced a contractile response which was resistant to the selective CysLT1 antagonists (ICI 198615 and MK 571) as well as the non-selective (CysLT1/CysLT2) antagonist, BAY u9773. PMID

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

    PubMed

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

    2015-10-01

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

  17. Enhanced sensitivity of A549 cells to the cytotoxic action of anticancer drugs via suppression of Nrf2 by procyanidins from Cinnamomi Cortex extract

    SciTech Connect

    Ohnuma, Tomokazu; Matsumoto, Takashi; Itoi, Ayano; Kawana, Ayako; Nishiyama, Takahito; Ogura, Kenichiro; Hiratsuka, Akira

    2011-10-07

    Highlights: {yields} We found a novel inhibitor of Nrf2 known as a chemoresistance factor. {yields} Overexpressed Nrf2 in lung cancer cells was suppressed by Cinnamomi Cortex extract. {yields} Cytotoxic action of anticancer drugs in cells treated with the extract was enhanced. {yields} Procyanidin tetramers and pentamers were active components in suppressing Nrf2. -- Abstract: Nuclear factor-E2-related factor 2 (Nrf2) is an important cytoprotective transcription factor because Nrf2-regulated enzymes play a key role in antioxidant and detoxification processes. Recent studies have reported that lung cancer cells overexpressing Nrf2 exhibit increased resistance to chemotherapy. Suppression of overexpressed Nrf2 is needed for a new therapeutic approach against lung cancers. In the present study, we found that Cinnamomi Cortex extract (CCE) has an ability to suppress Nrf2-regulated enzyme activity and Nrf2 expression in human lung cancer A549 cells with high Nrf2 activity. Moreover, we demonstrated that CCE significantly enhances sensitivity of A549 cells to the cytotoxic action of doxorubicin and etoposide as well as increasing the intracellular accumulation of both drugs. These results suggest that CCE might be an effective concomitant agent to reduce anticancer drug resistance derived from Nrf2 overexpression. Bioactivity-guided fractionation revealed that procyanidin tetramers and pentamers contained in CCE were active components in suppressing Nrf2.

  18. Anticancer effects of novel thalidomide analogs in A549 cells through inhibition of vascular endothelial growth factor and matrix metalloproteinase-2.

    PubMed

    El-Aarag, Bishoy; Kasai, Tomonari; Masuda, Junko; Agwa, Hussein; Zahran, Magdy; Seno, Masaharu

    2017-01-01

    Lung cancer is one of the major causes of cancer-related mortality worldwide, and non-small-cell lung cancer is the most common form of lung cancer. Several studies had shown that thalidomide has potential for prevention and therapy of cancer. Therefore, the current study aimed to investigate the antitumor effects of two novel thalidomide analogs in human lung cancer A549 cells. The antiproliferative, antimigratory, and apoptotic effects in A549 cells induced by thalidomide analogs were examined. In addition, their effects on the expression of mRNAs encoding vascular endothelial growth factor165 (VEGF165) and matrix metalloproteinase-2 (MMP-2) were evaluated. Their influence on the tumor volume in nude mice was also determined. Results revealed that thalidomide analogs exhibited antiproliferative, antimigratory, and apoptotic activities with more pronounced effect than thalidomide drug. Furthermore, analogs 1 and 2 suppressed the expression levels of VEGF165 by 42% and 53.2% and those of MMP-2 by 45% and 52%, respectively. Thalidomide analogs 1 and 2 also reduced the tumor volume by 30.11% and 53.52%, respectively. Therefore, this study provides evidence that thalidomide analogs may serve as a new therapeutic option for treating lung cancer.

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

    PubMed

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

    2015-01-01

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

  20. Cytokines from the tumor microenvironment modulate sirtinol cytotoxicity in A549 lung carcinoma cells.

    PubMed

    Pal, Shyama; Shankar, Bhavani S; Sainis, Krishna B

    2013-10-01

    Cytokines in tumor microenvironment play an important role in the success or failure of molecular targeted therapies. We have chosen tumor necrosis factor α (TNF-α), TNF related apoptosis inducing ligand (TRAIL), insulin-like growth factor 1 (IGF-1) and transforming growth factor β (TGF-β) as representative pro-inflammatory, pro-apoptotic, anti-apoptotic and anti-inflammatory tumor derived cytokines. Analysis of Oncomine database revealed the differential expression of these cytokines in a subset of cancer patients. The effects of these cytokines on cytotoxicity of FDA approved drugs - cisplatin and taxol and inhibitors of epidermal growth factor receptor - AG658, Janus kinase - AG490 and SIRT1 - sirtinol were assessed in A549 lung cancer cells. TRAIL augmented cytotoxicity of sirtinol and IGF-1 had a sparing effect. Since TRAIL and IGF-1 differentially modulated sirtinol cytotoxicity, further studies were carried out to identify the mechanisms. Sirtinol or knockdown of SIRT1 increased the expression of death receptors DR4 and DR5 and sensitized A549 cells to TRAIL. Increased cell death in presence of TRAIL and sirtinol was caspase independent and demonstrated classical features of necroptosis. Inhibition of iNOS increased caspase activity and switched the mode of cell death to caspase mediated apoptosis. Interestingly, sirtinol or SIRT1 knockdown did not increase IGF-1R expression. Instead, it abrogated ligand induced downregulation of IGF-1R and increased cell survival through PI3K-AKT pathway. In conclusion, these findings reveal that the tumor microenvironment contributes to modulation of cytotoxicity of drugs and that combination therapy, with agents that increase TRAIL signaling and suppress IGF-1 pathway may potentiate anticancer effect.

  1. Salvianolic acid A positively regulates PTEN protein level and inhibits growth of A549 lung cancer cells

    PubMed Central

    BI, LEI; CHEN, JIANPING; YUAN, XIAOJING; JIANG, ZEQUN; CHEN, WEIPING

    2013-01-01

    Salvianolic acid A (Sal A) is an effective compound extracted from Salvia miltiorrhiza which has been used in the treatment of various diseases. Preliminary data indicate that Sal A treatment has a specific anti-lung cancer effect. However, the manner in which Sal A regulates cancer growth remains unknown. In this study, the A549 lung cancer cell line and its response to Sal A treatment was examined. Results showed that Sal A treatment significantly decreased A549 cell growth, promoted partial apoptosis and increased mitochondrial membrane permeability. Western blot analysis showed that Sal A upregulated the phosphatase and tensin homolog (PTEN) protein level, while consistently downregulating Akt phosphorylation. These results indicate that Sal A negatively mediates A549 lung cancer cell line growth or apoptosis, most likely by positively regulating PTEN protein level. PMID:24648921

  2. Proliferative and Inhibitory Activity of Siberian ginseng (Eleutherococcus senticosus) Extract on Cancer Cell Lines; A-549, XWLC-05, HCT-116, CNE and Beas-2b.

    PubMed

    Cichello, Simon Angelo; Yao, Qian; Dowell, Ashley; Leury, Brian; He, Xiao-Qiong

    2015-01-01

    Siberian ginseng (Eleutherococcus senticosus) is used primarily as an adaptogen herb and also for its immune stimulant properties in Western herbal medicine. Another closely related species used in East Asian medicine systems i.e. Kampo, TCM (Manchuria, Korea, Japan and Ainu of Hokkaido) and also called Siberian ginseng (Acanthopanax senticosus) also displays immune-stimulant and anti-cancer properties. These may affect tumour growth and also provide an anti-fatigue effect for cancer patients, in particular for those suffering from lung cancer. There is some evidence that a carbohydrate in Siberian ginseng may possess not only immune stimulatory but also anti-tumour effects and also display other various anti-cancer properties. Our study aimed to determine the inhibitory and also proliferative effects of a methanol plant extract of Siberan ginseng (E. senticosus) on various cancer and normal cell lines including: A-549 (small cell lung cancer), XWLC-05 (Yunnan lung cancer cell line), CNE (human nasopharyngeal carcinoma cell line), HCT-116 (human colon cancer) and Beas-2b (human lung epithelial). These cell lines were treated with an extract from E. senticosus that was evaporated and re- constituted in DMSO. Treatment of A-549 (small cell lung cancer) cells with E. senticosus methanolic extract showed a concentration-dependent inhibitory trend from 12.5 - 50μg/mL, and then a plateau, whereas at 12.5 and 25 μg/mL, there is a slight growth suppression in QBC-939 cells, but then a steady suppression from 50, 100 and 200μg/mL. Further, in XWLC-05 (Yunnan lung cancer cell line), E. senticosus methanolic extract displayed an inhibitory effect which plateaued with increasing dosage. Next, in CNE (human nasopharyngeal carcinoma cell line) there was a dose dependent proliferative response, whereas in Beas-2 (human lung epithelial cell line), an inhibitory effect. Finally in colon cancer cell line (HCT-116) we observed an initially weak inhibitory effect and then plateau.

  3. Pulmonary inflation reflex: its lack of physiological significance in coronary circulation of humans.

    PubMed

    Wilson, R F; Marcus, M L; White, C W

    1988-10-01

    In awake dogs, voluntary deep inspiration results in a marked, reflex-mediated increase in coronary blood flow. To study this reflex in humans, we subselectively measured coronary blood flow velocity (CBFV) with a 3-F coronary Doppler catheter in 12 subjects with angiographically normal coronary arteries. In each subject, intracoronary papaverine increased CBFV to 4.8 +/- 0.2 X resting CBFV and reduced coronary resistance to 0.20 +/- 0.01 X resting coronary resistance, demonstrating normal vasodilator reserve in the vessel under study. Valsalva maneuver reduced CBFV to 0.67 +/- 0.09 X resting CBFV and increased coronary resistance to 1.37 +/- 0.20 X resting coronary resistance (P less than 0.05 vs. control). Maximum voluntary inspiration, however, did not significantly change CBFV (peak response 1.03 +/- 0.05 X resting CBFV) or coronary resistance (0.96 +/- 0.04 X resting). To determine whether augmentation of resting alpha-adrenergic tone would potentiate the reflex, eight patients performed a sustained 33% maximal handgrip for 2 min. Maximal deep inspiration during handgrip failed to result in any significant change in CBFV or coronary resistance. These studies demonstrate that the canine pulmonary inflation reflex has little or no physiological significance in the coronary circulation of conscious humans. Additionally, these data suggest that the magnitude of reflex control of the coronary circulation may vary considerably between dogs and humans.

  4. A critical role for p130Cas in the progression of pulmonary hypertension in humans and rodents

    PubMed Central

    Tu, Ly; De Man, Frances; Girerd, Barbara; Huertas, Alice; Chaumais, Marie-Camille; Lecerf, Florence; François, Charlène; Perros, Frédéric; Dorfmüller, Peter; Fadel, Elie; Montani, David; Eddahibi, Saadia; Humbert, Marc; Guignabert, Christophe

    2012-01-01

    Rationale Pulmonary arterial hypertension (PAH) is a progressive and fatal disease characterized by pulmonary arterial muscularization due to excessive pulmonary vascular cell proliferation and migration, a phenotype dependent upon growth factors and activation of receptor tyrosine kinases (RTKs). p130Cas is an adaptor protein involved in several cellular signaling pathways that control cell migration, proliferation and survival. Objectives We hypothesized that in experimental and human PAH p130Cas signaling is over-activated, thereby facilitating the intracellular transmission of signal induced by fibroblast growth factor (FGF)-2, epidermal growth factor (EGF), and platelet-derived growth factor (PDGF). Measurements and Main Results In PAH patients, levels of p130Cas protein and/or activity are higher in the serum, in walls of distal pulmonary arteries, in cultured smooth muscle (PA-SMCs) and pulmonary endothelial cells (P-ECs) than controls. These abnormalities in the p130Cas signaling were also found to be in the chronically hypoxic mice and monocrotaline-injected rats as models of human PAH. We next obtained evidence for convergence and amplification of the growth-stimulating effect of EGF, FGF2 and PDGF signaling pathways via p130Cas signaling pathway. Finally, we found that daily treatment with each of the EGF-R inhibitor gefitinib, the FGF-R inhibitor dovitinib and the PDGF-R inhibitor imatinib started 2 weeks after a subcutaneous monocrotaline injection substantially attenuate the abnormal increase in p130cas and ERK1/2 activation and regress established PH. Conclusions Our findings demonstrate that p130Cas signaling plays a critical role in experimental and iPAH by modulating pulmonary vascular cell migration, proliferation and by acting as an amplifier of RTKs downstream signals. PMID:22798315

  5. Hypertonic saline attenuates TNF-alpha-induced NF-kappaB activation in pulmonary epithelial cells.

    PubMed

    Nydam, Trevor L; Moore, Ernest E; McIntyre, Robert C; Wright, Franklin L; Gamboni-Robertson, Fabia; Eckels, Phillip C; Banerjee, Anirban

    2009-05-01

    Resuscitation with hypertonic saline (HTS) attenuates acute lung injury (ALI) and modulates postinjury hyperinflammation. TNF-alpha-stimulated pulmonary epithelium is a major contributor to hemorrhage-induced ALI. We hypothesized that HTS would inhibit TNF-alpha-induced nuclear factor (NF)-kappaB proinflammatory signaling in pulmonary epithelial cells. Therefore, we pretreated human pulmonary epithelial cells (A549) with hypertonic medium (180 mM NaCl) for 30 min, followed by TNF-alpha stimulation (10 ng/mL). Key regulatory steps and protein concentrations in this pathway were assessed for significant alterations. Hypertonic saline significantly reduced TNF-alpha-induced intercellular adhesion molecule 1 levels and NF-kappaB nuclear localization. The mechanism is attenuated phosphorylation and delayed degradation of IkappaB alpha. Hypertonic saline did not alter TNF-alpha-induced p38 mitogen-activated protein kinase phosphorylation or constitutive vascular endothelial growth factor expression, suggesting that the observed inhibition is not a generalized suppression of protein phosphorylation or cellular function. These results show that HTS inhibits TNF-alpha-induced NF-kappaB activation in the pulmonary epithelium and, further, our understanding of its beneficial effects in hemorrhage-induced ALI.

  6. Inflammatory Responses in Blood Samples of Human Immunodeficiency Virus-Infected Patients with Pulmonary Infections

    PubMed Central

    Benito, Natividad; Moreno, Asunción; Filella, Xavier; Miró, José M.; González, Julià; Pumarola, Tomás; Valls, María Eugenia; Luna, Montserrat; García, Felipe; Rañó, Ana; Torres, Antoni; Gatell, José M.

    2004-01-01

    We analyzed the characteristics of the inflammatory response occurring in blood during pulmonary infections in human immunodeficiency virus (HIV)-infected patients. A prospective study of consecutive hospital admissions of HIV-infected patients with new-onset radiologic pulmonary infiltrates was carried out in a tertiary university hospital from April 1998 to May 2001. Plasma cyclic AMP receptor protein (CRP), interleukin 1β (IL-1β), IL-6, IL-8, IL-10, and tumor necrosis factor alpha (TNF-α) levels were determined at the time of admission and 4, 5, and 6 days later. Patients were included in a protocol addressed to study etiology and outcome of disease. A total of 249 episodes of infection were included, with the main diagnoses being bacterial pneumonia (BP) (118 episodes), Pneumocystis carinii pneumonia (PCP) (41 episodes), and mycobacteriosis (36 episodes). For these three patient groups, at the time of admission the median CRP and cytokine levels were as follows: CRP, 10.2, 3.8 and 5 mg/dl, respectively (P = 0.0001); IL-8, 19, 3, and 2.9 pg/ml (P = 0.045); and TNF-α, 46.4, 44, and 75 pg/ml, respectively (P = 0.029). There were no significant differences in levels of IL-1β, IL-6, or IL-10 among the patient groups. A total of 23 patients died. At the time of admission, HIV-infected patients with BP had higher plasma CRP and IL-8 levels than did PCP and mycobacteriosis patients. TNF-α levels were higher in patients with mycobacteriosis. An elevated IL-8 level (>61 pg/ml) at the time of admission was an independent factor associated with higher mortality (odds ratio, 12; 95% confidence interval, 1.2 to 235.5). PMID:15138189

  7. MCPIP1 mediates silica-induced cell migration in human pulmonary fibroblasts.

    PubMed

    Liu, Haijun; Dai, Xiaoniu; Cheng, Yusi; Fang, Shencun; Zhang, Yingming; Wang, Xingang; Zhang, Wei; Liao, Hong; Yao, Honghong; Chao, Jie

    2016-01-15

    Silicosis is a systemic disease caused by inhaling silicon dioxide (SiO2). Phagocytosis of SiO2 in the lungs initiates an inflammatory cascade that results in fibroblast proliferation and migration followed by fibrosis. According to previous data from our laboratory, monocyte chemotactic protein-1 (MCP-1) plays a critical role in fibroblast proliferation and migration in conventional two-dimensional (2D) monolayer cultures. The present study aimed to explore the downstream cascade of MCP-1 in both 2D and three-dimensional (3D) cell culture models of silicosis. Experiments using primary cultured adult human pulmonary fibroblasts (HPF-a) demonstrated the following: 1) SiO2 treatment induces expression of MCP-1-induced protein (MCPIP1) in a time- and dose-dependent manner in both 2D and 3D cultures; 2) the MAPK and phosphatidylinositol-3-kinase (PI3K)/Akt pathways are involved in SiO2-induced MCPIP1 expression; and 3) MCPIP1 induction mediates the SiO2-induced increase in cell migration in both 2D and 3D cultures. The effect of MCP-1 in silicosis occurs mainly through MCPIP1, which, in turn, mediates the observed SiO2-induced increase in pulmonary fibroblast migration. However, the time frame for MCPIP1 induction differed between 2D and 3D cultures, indicating that, compared with conventional 2D cell culture systems, 3D culture may be useful for analyses of fibroblast physiology under conditions that more closely resemble in vivo environments. Our study determined the link between fibroblast-derived MCPIP1 and SiO2-induced cell migration, and this finding provides novel evidence of the potential of MCPIP1 in the development of novel therapeutic strategies for silicosis.

  8. Blocking the NOTCH pathway can inhibit the growth of CD133-positive A549 cells and sensitize to chemotherapy.

    PubMed

    Liu, Juntao; Mao, Zhangfan; Huang, Jie; Xie, Songping; Liu, Tianshu; Mao, Zhifu

    2014-02-21

    Cancer stem cells (CSCs) are believed to play an important role in tumor growth and recurrence. These cells exhibit self-renewal and proliferation properties. CSCs also exhibit significant drug resistance compared with normal tumor cells. Finding new treatments that target CSCs could significantly enhance the effect of chemotherapy and improve patient survival. Notch signaling is known to regulate the development of the lungs by controlling the cell-fate determination of normal stem cells. In this study, we isolated CSCs from the human lung adenocarcinoma cell line A549. CD133 was used as a stem cell marker for fluorescence-activated cell sorting (FACS). We compared the expression of Notch signaling in both CD133+ and CD133- cells and blocked Notch signaling using the γ-secretase inhibitor DAPT (GSI-IX). The effect of combining GSI and cisplatin (CDDP) was also examined in these two types of cells. We observed that both CD133+ and CD133- cells proliferated at similar rates, but the cells exhibited distinctive differences in cell cycle progression. Few CD133+ cells were observed in the G2/M phase, and there were half as many cells in S phase compared with the CD133- cells. Furthermore, CD133+ cells exhibited significant resistance to chemotherapy when treated with CDDP. The expression of Notch signaling pathway members, such as Notch1, Notch2 and Hes1, was lower in CD133+ cells. GSI slightly inhibited the proliferation of both cell types and exhibited little effect on the cell cycle. The inhibitory effects of DPP on these two types of cells were enhanced when combined with GSI. Interestingly, this effect was especially significant in CD133+ cells, suggesting that Notch pathway blockade may be a useful CSC-targeted therapy in lung cancer.

  9. Irradiation-Dependent Effects on Tumor Perfusion and Endogenous and Exogenous Hypoxia Markers in an A549 Xenograft Model

    SciTech Connect

    Fokas, Emmanouil; Haenze, Joerg; Kamlah, Florentine; Eul, Bastian G.; Lang, Nico; Keil, Boris; Heverhagen, Johannes T.; Engenhart-Cabillic, Rita; An Hanxiang; Rose, Frank

    2010-08-01

    Purpose: Hypoxia is a major determinant of tumor radiosensitivity, and microenvironmental changes in response to ionizing radiation (IR) are often heterogenous. We analyzed IR-dependent changes in hypoxia and perfusion in A549 human lung adenocarcinoma xenografts. Materials and Methods: Immunohistological analysis of two exogenously added chemical hypoxic markers, pimonidazole and CCI-103F, and of the endogenous marker Glut-1 was performed time dependently after IR. Tumor vessels and apoptosis were analyzed using CD31 and caspase-3 antibodies. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and fluorescent beads (Hoechst 33342) were used to monitor vascular perfusion. Results: CCI-103F signals measuring the fraction of hypoxic areas after IR were significantly decreased by approximately 50% when compared with pimonidazole signals, representing the fraction of hypoxic areas from the same tumors before IR. Interestingly, Glut-1 signals were significantly decreased at early time point (6.5 h) after IR returning to the initial levels at 30.5 h. Vascular density showed no difference between irradiated and control groups, whereas apoptosis was significantly induced at 10.5 h post-IR. DCE-MRI indicated increased perfusion 1 h post-IR. Conclusions: The discrepancy between the hypoxic fractions of CCI-103F and Glut-1 forces us to consider the possibility that both markers reflect different metabolic alterations of tumor microenvironment. The reliability of endogenous markers such as Glut-1 to measure reoxygenation in irradiated tumors needs further consideration. Monitoring tumor microvascular response to IR by DCE-MRI and measuring tumor volume alterations should be encouraged.

  10. Triptolide inhibits COX-2 expression by regulating mRNA stability in TNF-{alpha}-treated A549 cells

    SciTech Connect

    Sun, Lixin; Zhang, Shuang; Jiang, Zhenzhou; Huang, Xin; Wang, Tao; Huang, Xiao; Li, Han; Zhang, Luyong

    2011-12-09

    Highlights: Black-Right-Pointing-Pointer Triptolide inhibited COX-2 expression and the half-life of COX-2 mRNA is decreased. Black-Right-Pointing-Pointer The HuR protein shuttling from nucleus to cytoplasm is inhibited by triptolide. Black-Right-Pointing-Pointer Triptolide inhibited 3 Prime -UTR fluorescence reporter gene activity. Black-Right-Pointing-Pointer COX-2 mRNA binding to HuR is decreased by triptolide in pull-down experiments. -- Abstract: Cyclooxygenase-2 (COX-2) over-expression is frequently associated with human non-small-cell lung cancer (NSCLC) and involved in tumor proliferation, invasion, angiogenesis and resistance to apoptosis. In the present study, the effects of triptolide on COX-2 expression in A549 cells were investigated and triptolide was found to inhibit TNF-{alpha}-induced COX-2 expression. In our further studies, it was found that triptolide decreased the half-life of COX-2 mRNA dramatically and that it inhibited 3 Prime -untranslated region (3 Prime -UTR) fluorescence reporter gene activity. Meanwhile, triptolide inhibited the HuR shuttling from nucleus to cytoplasm. After triptolide treatment, decreased COX-2 mRNA in pull-down experiments with anti-HuR antibodies was observed, indicating that the decreased cytoplasmic HuR is responsible for the decreased COX-2 mRNA. Taken together, our results provided evidence for the first time that triptolide inhibited COX-2 expression by COX-2 mRNA stability modulation and post-transcriptional regulation. These results provide a novel mechanism of action for triptolide which may be important in the treatment of lung cancer.

  11. Overexpression of Bcl-2–Associated Death Inhibits A549 Cell Growth In Vitro and In Vivo

    PubMed Central

    Huang, Na; Zhu, Jing; Liu, Dan; Li, Ya-Lun; Chen, Bo-Jiang; He, Yan-Qi; Liu, Kun; Mo, Xian-Ming

    2012-01-01

    Abstract The importance of apoptosis during the process of inhibiting tumorigenesis has been recognized. The role of BH3-only proapoptotic protein Bcl-2–associated death (BAD) in tumor growth remains controversial. The aim of this study was to explore the role of BAD in lung cancer cells. Our study showed that expression of BAD was upregulated in A549 cells by a recombinant lentivirus overexpressing BAD. In vitro, BAD overexpression significantly inhibited A549 cell proliferation and induced apoptosis in cell proliferation and apoptosis assays, respectively. The effect of BAD on A549 cells was studied in tumor xenograft of nude mice and the results showed that the tumor volume in the experimental group was smaller than the control groups. Further, immunohistochemical technique was used to determine the cell proliferation and apoptosis status of the lung tumor xenograft cells. This demonstrated that the in vivo and in vitro results were consistent. Taken together, our results indicate that overexpression of BAD inhibits the growth of A549 cells in vitro and in vivo, through inhibiting cell proliferation and inducing apoptosis. Thus, BAD could be a potential therapeutic target. PMID:22011203

  12. Enhanced production of nitric oxide in A549 cells through activation of TRPA1 ion channel by cold stress.

    PubMed

    Sun, Wenwu; Wang, Zhonghua; Cao, Jianping; Wang, Xu; Han, Yaling; Ma, Zhuang

    2014-08-31

    The respiratory epithelium is exposed to the external environment, and inhalation of cold air is common during the season of winter. In addition, the lung is a major source of nitric oxide (NO). However, the effect of cold stress on the production of NO is still unclear. In the present work, We measured the change of NO in single cell with DACF-DA and the change in cytosolic Ca(2+) concentration ([Ca(2+)]c) in A549 cell. We observed that cold stress (from 20 °C to 5 °C) induced an increase of NO in A549 cell, which was completely abolished by applying an extracellular Ca(2+) free medium. Further experiments showed that cold-sensing transient receptor potential subfamily member 1 (TRPA1) channel agonist (allyl isothiocyanate, AITC) increased the production of NO and the level of [Ca(2+)]c in A549 cell. Additionally, TRPA1 inhibitor, Ruthenium red (RR) and camphor, significantly blocked the enhanced production of NO and the rise of [Ca(2+)]c induced by AITC or cold stimulation, respectively. Taken together, these data indicated that cold-induced TRPA1 activation was responsible for the enhanced production of NO in A549 cell.

  13. Green tea polyphenol EGCG reverse cisplatin resistance of A549/DDP cell line through candidate genes demethylation.

    PubMed

    Zhang, Youwei; Wang, Xiang; Han, Liang; Zhou, Yizhou; Sun, Sanyuan

    2015-02-01

    Epigallocatechin-3-gallate (EGCG), the major polyphenol in green tea, has been extensively studied as a potential demethylating agent. Our hypothesis is that EGCG could resensitize non-small-cell lung cancer (NSCLC) cells to cisplatin (DDP) through candidate genes demethylation. The A549/DDP cell line was established by continuous exposure of A549 cells to increasing concentrations of DDP. MTT, colony formation assay, flow cytometric analysis, Hoechst staining, real time-PCR, quantitative methylation-specific PCR and in vivo experiments were performed in this study. EGCG+DDP treatment significantly caused proliferation inhibition, cell cycle arrest in G1 phase, increase of apoptosis in A549/DDP cells, along with inhibition of DNA methyltransferase (DNMT) activity and histone deacetylase (HDAC) activity, reversal of hypermethylated status and downregulated expression of GAS1, TIMP4, ICAM1 and WISP2 gene in A549/DDP cells. Furthermore, pre-treatment with EGCG followed by DDP caused significant tumor inhibition in vivo. Methylation levels of GAS1, TIMP4, ICAM1 and WISP2 were decreased and their expression levels were increased in EGCG-treatment groups, but only combinatorial treatment group caused growth inhibition. In conclusion, we identified EGCG pretreatment resensitized cells to DDP, along with the demethylation and restoration of expression of candidate genes.

  14. The synergistic effect of resveratrol in combination with cisplatin on apoptosis via modulating autophagy in A549 cells.

    PubMed

    Hu, Song; Li, Xiaolin; Xu, Rongrong; Ye, Lingyun; Kong, Hui; Zeng, Xiaoning; Wang, Hong; Xie, Weiping

    2016-06-01

    Several studies have shown that combination treatment with natural products and chemotherapy agents can improve the sensitivity and cytotoxicity of chemotherapy agents. Resveratrol, a natural product, has many biological effects including antitumor and antiviral activities, as well as vascular protective effect. The aim of this study is to investigate the synergistic anticancer effect of resveratrol in combination with cisplatin and the potential anticancer mechanisms involved in A549 cells. The results obtained from Cell Counting Kit-8 and isobolographic analysis demonstrated that combination of resveratrol and cisplatin resulted in synergistic cytotoxic effects in A549 cells. Results from Hoechst staining, flow cytometry and western blot analysis suggested that resveratrol enhanced cisplatin-mediated apoptosis. Meanwhile, the changes of LC3-II and P62 levels and formation of autophagosome suggested that resveratrol in combination with cisplatin triggered autophagy. More importantly, inhibiting autophagy by 3-methyladenine markedly attenuated the apoptosis caused by combination of resveratrol and cisplatin in A549 cells. Taken together, our study provides the first evidence that resveratrol combined with cisplatin synergistically induce apoptosis via modulating autophagic cell death in A549 cells. These findings also help us to understand the role of natural products in combination with chemotherapy agents in lung cancer.

  15. TLR9 Differentiates Rapid from Slowly Progressive Forms of Idiopathic Pulmonary Fibrosis

    PubMed Central

    Trujillo, Glenda; Meneghin, Alessia; Flaherty, Kevin R.; Sholl, Lynette M.; Myers, Jeffrey L.; Kazerooni, Ella A.; Gross, Barry H.; Oak, Sameer R.; Coelho, Ana Lucia; Evanoff, Holly; Day, Elizabeth; Toews, Galen B.; Joshi, Amrita D.; Schaller, Matthew A.; Waters, Beatrice; Jarai, Gabor; Westwick, John; Kunkel, Steve L.; Martinez, Fernando J.; Hogaboam, Cory M.

    2011-01-01

    Idiopathic pulmonary fibrosis is a generally progressive disorder with highly heterogeneous disease progression. The most common of the idiopathic interstitial pneumonias, idiopathic pulmonary fibrosis is characterized by a steady worsening of lung function and gas exchange cause by diffuse alveolar damage and severe fibrosis. We examined clinical features of patients with idiopathic pulmonary fibrosis to classify them as exhibiting rapid or slowly progressive over the first year of follow-up. We identified differences between the two groups in order to investigate the mechanism of rapid progression. Previous work from our laboratory has demonstrated that Toll-like receptor 9, a pathogen recognition receptor, promotes myofibroblast differentiation in lung fibroblasts cultured from biopsies of patients with idiopathic pulmonary fibrosis. Therefore, we hypothesized that TLR9 functions as both a sensor of pathogenic molecules and a profibrotic signal in rapidly progressive idiopathic pulmonary fibrosis. TLR9 was present at higher concentrations in surgical lung biopsies from rapidly progressive patients than in tissue from normal controls. Fibroblasts from rapid progressors were more responsive to the TLR9 agonist, CpG, than were fibroblasts from control patients. We used a humanized SCID mouse and demonstrated that there was increased fibrosis in murine lungs receiving human lung fibroblasts from rapid progressors than in mice receiving normal fibroblasts. This fibrosis was exacerbated by intranasal CpG challenges. Furthermore, CpG induced the differentiation of blood monocytes into fibrocytes and the epithelial-to-mesenchymal transition of A549 lung epithelial cells. These data suggest that TLR9 may drive the pathogenesis of rapidly progressive idiopathic pulmonary fibrosis and is a potential indicator of this subset of the disease. PMID:21068441

  16. Variations in Alveolar Partial Pressure for Carbon Dioxide and Oxygen Have Additive Not Synergistic Acute Effects on Human Pulmonary Vasoconstriction

    PubMed Central

    Croft, Quentin P. P.; Formenti, Federico; Talbot, Nick P.; Lunn, Daniel; Robbins, Peter A.; Dorrington, Keith L.

    2013-01-01

    The human pulmonary vasculature constricts in response to hypercapnia and hypoxia, with important consequences for homeostasis and adaptation. One function of these responses is to direct blood flow away from poorly-ventilated regions of the lung. In humans it is not known whether the stimuli of hypercapnia and hypoxia constrict the pulmonary blood vessels independently of each other or whether they act synergistically, such that the combination of hypercapnia and hypoxia is more effective than the sum of the responses to each stimulus on its own. We independently controlled the alveolar partial pressures of carbon dioxide (Paco2) and oxygen (Pao2) to examine their possible interaction on human pulmonary vasoconstriction. Nine volunteers each experienced sixteen possible combinations of four levels of Paco2 (+6, +1, −4 and −9 mmHg, relative to baseline) with four levels of Pao2 (175, 100, 75 and 50 mmHg). During each of these sixteen protocols Doppler echocardiography was used to evaluate cardiac output and systolic tricuspid pressure gradient, an index of pulmonary vasoconstriction. The degree of constriction varied linearly with both Paco2 and the calculated haemoglobin oxygen desaturation (1-So2). Mixed effects modelling delivered coefficients defining the interdependence of cardiac output, systolic tricuspid pressure gradient, ventilation, Paco2 and So2. No interaction was observed in the effects on pulmonary vasoconstriction of carbon dioxide and oxygen (p>0.64). Direct effects of the alveolar gases on systolic tricuspid pressure gradient greatly exceeded indirect effects arising from concurrent changes in cardiac output. PMID:23935847

  17. Developmental Reprogramming in Mesenchymal Stromal Cells of Human Subjects with Idiopathic Pulmonary Fibrosis

    PubMed Central

    Chanda, Diptiman; Kurundkar, Ashish; Rangarajan, Sunad; Locy, Morgan; Bernard, Karen; Sharma, Nirmal S.; Logsdon, Naomi J.; Liu, Hui; Crossman, David K.; Horowitz, Jeffrey C.; De Langhe, Stijn; Thannickal, Victor J.

    2016-01-01

    Cellular plasticity and de-differentiation are hallmarks of tissue/organ regenerative capacity in diverse species. Despite a more restricted capacity for regeneration, humans with age-related chronic diseases, such as cancer and fibrosis, show evidence of a recapitulation of developmental gene programs. We have previously identified a resident population of mesenchymal stromal cells (MSCs) in the terminal airways-alveoli by bronchoalveolar lavage (BAL) of human adult lungs. In this study, we characterized MSCs from BAL of patients with stable and progressive idiopathic pulmonary fibrosis (IPF), defined as <5% and ≥10% decline, respectively, in forced vital capacity over the preceding 6-month period. Gene expression profiles of MSCs from IPF subjects with progressive disease were enriched for genes regulating lung development. Most notably, genes regulating early tissue patterning and branching morphogenesis were differentially regulated. Network interactive modeling of a set of these genes indicated central roles for TGF-β and SHH signaling. Importantly, fibroblast growth factor-10 (FGF-10) was markedly suppressed in IPF subjects with progressive disease, and both TGF-β1 and SHH signaling were identified as critical mediators of this effect in MSCs. These findings support the concept of developmental gene re-activation in IPF, and FGF-10 deficiency as a potentially critical factor in disease progression. PMID:27869174

  18. Pulmonary Surfactant Phosphatidylglycerol Inhibits Mycoplasma pneumoniae-stimulated Eicosanoid Production from Human and Mouse Macrophages*

    PubMed Central

    Kandasamy, Pitchaimani; Zarini, Simona; Chan, Edward D.; Leslie, Christina C.; Murphy, Robert C.; Voelker, Dennis R.

    2011-01-01

    Mycoplasma pneumoniae is a human pathogen causing respiratory infections that are also associated with serious exacerbations of chronic lung diseases. Membranes and lipoproteins from M. pneumoniae induced a 4-fold increase in arachidonic acid (AA) release from RAW264.7 and a 2-fold increase in AA release from primary human alveolar macrophages. The bacterial lipoprotein mimic and TLR2/1 agonist Pam3Cys and the TLR2/6 agonist MALP-2 produced effects similar to those elicited by M. pneumoniae in macrophages by inducing the phosphorylation of p38MAPK and p44/42ERK1/2 MAP kinases and cyclooxygenase-2 (COX-2) expression. M. pneumoniae induced the generation of prostaglandins PGD2 and PGE2 from RAW264.7 cells and thromboxane B2 (TXB2) from human alveolar macrophages. Anti-TLR2 antibody completely abolished M. pneumoniae-induced AA release and TNFα secretion from RAW264.7 cells and human alveolar macrophages. Disruption of the phosphorylation of p44/42ERK1/2 or inactivation of cytosolic phospholipase A2α (cPLA2α) completely inhibited M. pneumoniae-induced AA release from macrophages. The minor pulmonary surfactant phospholipid, palmitoyl-oleoyl-phosphatidylglycerol (POPG), antagonized the proinflammatory actions of M. pneumoniae, Pam3Cys, and MALP-2 by reducing the production of AA metabolites from macrophages. The effect of POPG was specific, insofar as saturated PG, and saturated and unsaturated phosphatidylcholines did not have significant effect on M. pneumoniae-induced AA release. Collectively, these data demonstrate that M. pneumoniae stimulates the production of eicosanoids from macrophages through TLR2, and POPG suppresses this pathogen-induced response. PMID:21205826

  19. Novel curcumin analogue IHCH exhibits potent anti‑proliferative effects by inducing autophagy in A549 lung cancer cells.

    PubMed

    Zhou, Guang-Zhou; Xu, Su-Li; Sun, Gang-Chun; Chen, Xiao-Bing

    2014-07-01

    Curcumin is a natural polyphenolic compound that exhibits strong antioxidant and anticancer activities; however, low bioavailability has restricted its application in chemotherapeutic trials. The present study aimed to investigate the anticancer effect of the novel curcumin derivative 2E,6E‑2‑(1H‑indol‑3‑yl) methylene)‑6‑(4‑hydroxy‑3‑methoxy benzylidene)‑cyclohexanone (IHCH) on A549 lung cancer cells. Cells were treated with IHCH at different concentrations (1‑40 µM) for different time periods (1‑36 h). Microscopic analysis revealed that IHCH inhibited A549 cell growth and induced the formation of characteristic autophagolysosomes in a dose‑ and time‑dependent manner. Furthermore, the inhibitory rate of IHCH (40 µM) on A549 cell viability was 77.34% after 36 h of treatment. Acridine orange staining revealed an increase in autophagic vacuoles in the IHCH‑treated A549 cells. Monodansylcadaverine staining was used to analyze autophagy rate. Immunocytochemistry revealed an increase in light chain (LC) 3 protein expression in the IHCH‑treated cells and western blot analysis detected the conversion of LC3‑I to LC3‑II, as well as the recruitment of LC3 to autophagosomes in the cytoplasmatic compartment, suggesting the occurrence of autophagy. These findings show that IHCH induced autophagy in A549 cells, which is a novel cell death mechanism induced by curcumin derivatives.

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

    SciTech Connect

    Gu, Haihua; Yang, Tao; Fu, Shaozi; Chen, Xiaofan; Guo, Lei; Ni, Yiming

    2014-01-31

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

  1. Proteomic Analysis of Differential Expression of Cellular Proteins in Response to Avian H9N2 Virus Infection of A549 Cells

    PubMed Central

    Yu, Guanliu; Liang, Wei; Liu, Jiyuan; Meng, Dan; Wei, Liangmeng; Chai, Tongjie; Cai, Yumei

    2016-01-01

    In this study, differentially expressed proteins in A549 cells (human lung adenocarcinoma epithelial cell line) infected with H9N2 avian influenza virus (AIV) were investigated by two-dimensional electrophoresis (2-DE). Sixteen different spots between the groups (ratio > 2, p < 0.05) were identified with mass spectrometry identification. Proteins located in the downstream of the NF-κB and IFN transcription factor pathways were identified, e.g., ISG15. Actin and keratin were also identified, suggesting that the cytoskeleton may plays an important role in the AIV infection of mammalian cells. These findings could provide insights into the interaction between host and influenza viruses and might provide valuable information for clarifying the pathogenesis of viral infections as well. PMID:28018302

  2. Molecular Role of EGFR-MAPK Pathway in Patchouli Alcohol-Induced Apoptosis and Cell Cycle Arrest on A549 Cells In Vitro and In Vivo

    PubMed Central

    Yang, Liu; Lu, ChengHua; Xu, ZhenYu; Qiu, HongFu; Wang, JingWen; Zhu, Yin

    2016-01-01

    Nowadays, chemotherapy is still the main effective treatment for cancer. Herb prescriptions containing Pogostemon cablin Benth (also known as “Guang-Huo-Xiang”) have been widely used in Chinese medicine today. In our research, we found that patchouli alcohol, a compound isolated from the oil of Pogostemon cablin Benth, exerted antitumor ability against human lung cancer A549 cells ability both in vitro and in vivo. MTT assay was used to assess cell viability. Hoechst 33342 staining and TUNEL cover glass staining provided the visual evidence of apoptosis. Caspase activity measurement showed that patchouli alcohol activated caspase 9 and caspase 3 of mitochondria-mediated apoptosis. Consistently, patchouli alcohol inhibited the xenograft tumor in vivo. Further investigation of the underlying molecular mechanism showed that MAPK and EGFR pathway might contribute to the antitumor effect of patchouli alcohol. Our study proved that patchouli alcohol might be able to serve as a novel antitumor compound in the clinical treatment of lung cancer. PMID:27830146

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

    PubMed

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

    2013-10-01

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

  4. Raw and thermally treated cement asbestos exerts different cytotoxicity effects on A549 cells in vitro.

    PubMed

    Pugnaloni, Armanda; Lucarini, Guendalina; Rubini, Corrado; Smorlesi, Arianna; Tomasetti, Marco; Strafella, Elisabetta; Armeni, Tatiana; Gualtieri, Alessandro F

    2015-01-01

    Raw cement asbestos (RCA) undergoes a complete solid state transformation when heated at high temperatures. The secondary raw material produced, high temperatures-cement asbestos (HT-CA) is composed of newly-formed crystals in place of the asbestos fibers present in RCA. Our previous study showed that HT-CA exerts lower cytotoxic cell damage compared to RCA. Nevertheless further investigations are needed to deepen our understanding of pathogenic pathways involving oxidative and nitrative damage. Our aim is to deepen the understanding of the biological effects on A549 cells of these materials regarding DNA damage related proteins (p53, its isoform p73 and TRAIL) and nitric oxide (NO) production during inducible nitric oxide synthase (iNOS)-mediated inflammation. Increments of p53/p73 expression, iNOS positive cells and NO concentrations were found with RCA, compared to HT-CA and controls mainly at 48 h. Interestingly, ferrous iron causing reactive oxygen species (ROS)-mediated DNA damage was found in RCA as a contaminant. HT-CA thermal treatment induces a global recrystallization with iron in a crystal form poorly released in media. HT-CA slightly interferes with genome expression and exerts lower inflammatory potential compared to RCA on biological systems. It could represent a safe approach for storing or recycling asbestos and an environmentally friendly alternative to asbestos waste.

  5. Study of gaseous benzene effects upon A549 lung epithelial cells using a novel exposure system.

    PubMed

    Mascelloni, Massimiliano; Delgado-Saborit, Juana Maria; Hodges, Nikolas J; Harrison, Roy M

    2015-08-19

    Volatile organic compounds (VOCs) are ubiquitous pollutants known to be present in both indoor and outdoor air arising from various sources. Indoor exposure has increasingly become a major cause of concern due to the effects that such pollutants can have on health. Benzene, along with toluene, is one of the main components of the VOC mixture and is a known carcinogen due to its genotoxic effects. The aim of this study was to test the feasibility of an in vitro model to study the short-term effects of exposure of lung cells to airborne benzene. We studied the effects of exposure on DNA and the production of reactive oxygen species (ROS) in A549 cells, exposed to various concentrations of benzene (0.03; 0.1; 0.3 ppm) in gaseous form using a custom designed cell exposure chamber. Results showed a concentration-dependent increase of DNA breaks and an increase of ROS production, confirming the feasibility of the experimental procedure and validating the model for further in vitro studies of exposure to other VOCs.

  6. Superinfection exclusion is absent during acute Junin virus infection of Vero and A549 cells

    PubMed Central

    Gaudin, Raphaël; Kirchhausen, Tomas

    2015-01-01

    Many viruses have evolved strategies of so-called “superinfection exclusion” to prevent re-infection of a cell that the same virus has already infected. Although Old World arenavirus infection results in down-regulation of its viral receptor and thus superinfection exclusion, whether New World arenaviruses have evolved such a mechanism remains unclear. Here we show that acute infection by the New World Junin virus (JUNV) failed to down-regulate the transferrin receptor and did not induce superinfection exclusion. We observed that Vero cells infected by a first round of JUNV (Candid1 strain) preserve an ability to internalize new incoming JUNV particles that is comparable to that of non-infected cells. Moreover, we developed a dual infection assay with the wild-type Candid1 JUNV and a recombinant JUNV-GFP virus to discriminate between first and second infections at the transcriptional and translational levels. We found that Vero and A549 cells already infected by JUNV were fully competent to transcribe viral RNA from a second round of infection. Furthermore, flow cytometry analysis of viral protein expression indicated that viral translation was normal, regardless of whether cells were previously infected or not. We conclude that in acutely infected cells, Junin virus lacks a superinfection exclusion mechanism. PMID:26549784

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

    PubMed

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

    2015-10-01

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

  8. Superinfection exclusion is absent during acute Junin virus infection of Vero and A549 cells.

    PubMed

    Gaudin, Raphaël; Kirchhausen, Tomas

    2015-11-09

    Many viruses have evolved strategies of so-called "superinfection exclusion" to prevent re-infection of a cell that the same virus has already infected. Although Old World arenavirus infection results in down-regulation of its viral receptor and thus superinfection exclusion, whether New World arenaviruses have evolved such a mechanism remains unclear. Here we show that acute infection by the New World Junin virus (JUNV) failed to down-regulate the transferrin receptor and did not induce superinfection exclusion. We observed that Vero cells infected by a first round of JUNV (Candid1 strain) preserve an ability to internalize new incoming JUNV particles that is comparable to that of non-infected cells. Moreover, we developed a dual infection assay with the wild-type Candid1 JUNV and a recombinant JUNV-GFP virus to discriminate between first and second infections at the transcriptional and translational levels. We found that Vero and A549 cells already infected by JUNV were fully competent to transcribe viral RNA from a second round of infection. Furthermore, flow cytometry analysis of viral protein expression indicated that viral translation was normal, regardless of whether cells were previously infected or not. We conclude that in acutely infected cells, Junin virus lacks a superinfection exclusion mechanism.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2013-01-01

    Lung cancer is among the most prevalent and deadly cancers in United States. In general, cancer cells are known to exhibit higher rates of glycolysis in comparison to normal cells. In attempting to exploit this unique cancer-dependent ATP generation phenomenon, it was our hypothesis that upon exposure to organic inhibitors of glycolysis, cancer cells would not survive normally and that their growth and viability would be vastly decreased; essential glycolytic ATP production will be exhausted to the point of collapsing energy utilization. Furthermore, we hypothesize that no negative effect would be seen with exposures to organic inhibitors for normal lung cells. The human lung fibroblast MRC-5 and the human A549 alveolar epithelial cell lines were used as in vitro models of normal lung and lung cancers respectively. Using standard methods, both cell lines were maintained and exposed to pyruvic acid, sodium citrate and sodium bicarbonate reagents at concentration levels ranging from 31.3-2,000 µg/ml in 96 well plates in quadruplets and experiments repeated at least three times using MTT, and cell counting (T4 Cellometer) assays as well as phase-contrast photo-imaging for parallel morphological displays of any changes in the course of their vitality and metabolic activities. Our results indicate that exposure of both cell lines to these organics resulted in concentration dependent cell destruction/cell survival depending on the cell line exposed. Pyruvic acid, sodium citrate and sodium bicarbonate showed statistically significant (p<0.05) differential negative effects on the A549 cell line in comparison to its unexposed control as well as to their effects on the MRC-5 cell line, presenting a potential promise for their use as cancer biotherapeutics.

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

    PubMed

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

    2016-05-01

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

  12. Prostacyclin release and receptor activation: differential control of human pulmonary venous and arterial tone

    PubMed Central

    Norel, Xavier; Walch, Laurence; Gascard, Jean-Pierre; Montpreville, Vincent de; Brink, Charles

    2004-01-01

    In human pulmonary vascular preparations, precontracted arteries were more sensitive to the relaxant effect of acetylcholine (ACh) than veins (pD2 values: 7.25±0.08 (n=23) and 5.92±0.09 (n=25), respectively). Therefore, the role of prostacyclin (PGI2) was explored to examine whether this mediator may be responsible for the difference in relaxation. In the presence of the cyclooxygenase (COX) inhibitor, indomethacin (INDO), the ACh relaxations were reduced in arteries but not in veins. On the contrary, an inhibitor (L-NOARG) of the nitric oxide synthase blocked preferentially the relaxation in veins. A greater release of 6-keto-PGF1α, the stable metabolite of PGI2, was observed in arterial preparations than in venous preparations when stimulated with either ACh or arachidonic acid (AA). Exogenous PGI2 produced a reduced relaxant effect in the precontracted vein when compared with the artery. In the presence of the EP1-receptor antagonist AH6809, the PGI2 relaxation of veins was similar to arteries. In veins, AA (0.1 mM) produced a biphasic response, namely, a contraction peak (0.4–0.5 g) followed by a relaxation. These contractions in venous preparations were abolished either in the absence of endothelium or in the presence of INDO or an EP1-receptor antagonist (AH6809, SC19220). In the arterial preparations AA induced only relaxations. In both vascular preparations, COX-1 but not the COX-2 protein was detected in microsomal preparations derived from homogenized tissues or freshly isolated endothelial cells. The differential vasorelaxations induced by ACh may be explained, in part, by a more pronounced production and release of PGI2 in human pulmonary arteries than in the veins. In addition, while PGI2 induced relaxation by activation of IP-receptors in both types of vessels, a PGI2 constrictor effect was responsible for masking the relaxation in the veins by activation of the EP1-receptor. PMID:15172959

  13. Induction of H2AX phosphorylation in pulmonary cells by tobacco smoke: a new assay for carcinogens.

    PubMed

    Albino, A P; Huang, X; Jorgensen, E; Yang, J; Gietl, D; Traganos, F; Darzynkiewicz, Z

    2004-08-01

    DNA double strand breaks (DSBs) are potentially carcinogenic lesions. The induction of DSBs triggers phosphorylation of histone H2AX. Phosphorylated H2AX, denoted p-H2AX, may be detected immunocytochemically and the intensity of p-H2AX immunofluorescence (IF) reveals the frequency of DSBs. Using this assay we tested whether the exposure of A549 human pulmonary adenocarcinoma cells to tobacco smoke, and normal human bronchial epithelial cells (NHBE) to tobacco smoke condensate, induces DSBs. Cellular p-H2AX IF and DAPI fluorescence of individual cells were measured by laser scanning cytometry (LSC). Exposure of A549 cells to tobacco smoke and NHBE cells to smoke condensate led to H2AX phosphorylation in both a time and dose dependent manner. The maximal rate of H2AX phosphorylation was seen during the initial 4h of cell treatment. At high doses (50 microg/ml of smoke condensate), H2AX phosphorylation continued to increase for up to 24h. No differences in the level of H2AX phosphorylation were apparent between cells in G(1) vs S vs G(2)/M phase of the cell cycle in response to treatment with smoke condensate. The data provide strong evidence that exposure of A549 cells to tobacco smoke or NHBE cells to smoke condensate rapidly induces DSBs in these cells. The present assay to detect and measure DSBs induced by tobacco products complements other mutagenicity assays and may be applied to test potential carcinogens in other products.

  14. Human papillomavirus type 16 DNA detected in pulmonary metastases from a penile squamous cell carcinoma: a case study.

    PubMed

    Lorenzon, Laura; Benevolo, Maria; Visca, Paolo; Venturo, Irene; Filippetti, Massimo; Piro, Francesca Romana; Rollo, Francesca; Vocaturo, Amina

    2013-02-01

    This report describe the case of a patient presenting with pulmonary metastases from a penile cancer, where the presence of the human papillomavirus (HPV) type 16 DNA both in the primary tumor and in the distant metastases confirmed the spreading of the disease, ruling out a possible primary lung squamous cell carcinoma. Indeed, according to the findings, the HPV genotyping test might help in the identification of metastatic disease from anogenital malignancies or other HPV-related cancers.

  15. The primary role of iron-mediated lipid peroxidation in the differential cytotoxicity caused by two varieties of talc nanoparticles on A549 cells and lipid peroxidation inhibitory effect exerted by ascorbic acid.

    PubMed

    Akhtar, Mohd Javed; Kumar, Sudhir; Murthy, Ramesh Chandra; Ashquin, Mohd; Khan, Mohd Imran; Patil, Govil; Ahmad, Iqbal

    2010-06-01

    Talc particles, the basic ingredient in different kinds of talc-based cosmetic and pharmaceutical products, pose a health risk to pulmonary and ovarian systems due to domestic and occupational exposures. Two types of talc nanoparticles depending on the source of geographical origin - indigenous- and commercial talc nanoparticles were assessed for their potential in vitro toxicity on A(549) cells; along with indigenous conventionally used microtalc particles. Cell viability, determined through live/dead staining and 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, decreased as a function of concentration, origin and size of particles. Both varieties of talc nanoparticles differentially induced lipid peroxidation (LPO), which was correlated with the pattern of lactate dehydrogenase (LDH) leakage, reactive oxygen species (ROS) generation, and glutathione (GSH) depletion. Relatively higher cytotoxicity of indigenous nanotalc could be attributed to its higher content of iron as compared to commercial nanotalc. The known scavenger of ROS, l-ascorbic acid significantly inhibited LPO induction due to talc particles. Data suggest that nanotalc toxicity on A(549) cells was mediated through oxidative stress, wherein role of iron-mediated LPO was much pronounced in differential cytotoxicity.

  16. Pulmonary edema

    MedlinePlus

    ... congestion; Lung water; Pulmonary congestion; Heart failure - pulmonary edema ... Pulmonary edema is often caused by congestive heart failure . When the heart is not able to pump efficiently, blood ...

  17. Pulmonary surfactant: hydrophobic nature of the mucosal surface of the human amnion.

    PubMed

    Cotton, D B; Hills, B A

    1984-04-01

    The contact angle has been measured for a drop of saline placed upon the rinsed mucosal surface of the amnion in eleven human placental membranes obtained from normal births at full term. The contact angle averaged 70 degrees, indicating a hydrophobic surface comparable with graphite (86 degrees), polyethylene (94 degrees) or oxyntic tissue (85 degrees) which is also exposed to endogenous surface-active phospholipids in vivo. By comparison, four pre-term placentas with an average gestation period of 29.5 weeks gave a mean contact angle of 32 degrees, indicating that hydrophobicity of the placenta increases with maturity (41 weeks) and might well be imparted by adsorbed surfactants present in amniotic fluid and known to render other surfaces hydrophobic. Since the mucosal epithelium of the amnion is exposed to the same surfactants in the same physical state as the fetal alveolar wall, the above results imply that this surface may also be hydrophobic, as indicated in the adult lung by other studies. The concept of surfactant directly adsorbed to the pulmonary tissue surfaces is discussed in connexion with its possible functional advantages in 'de-watering' the lung at birth, maintaining homeostasis by water repellency , releasing airway surfaces and lymph ducts glued by protein and lubricating tissue respiratory movement.

  18. Lipopolysaccharide Induces Human Pulmonary Micro-Vascular Endothelial Apoptosis via the YAP Signaling Pathway

    PubMed Central

    Yi, Lei; Huang, Xiaoqin; Guo, Feng; Zhou, Zengding; Chang, Mengling; Tang, Jiajun; Huan, Jingning

    2016-01-01

    Gram-negative bacterial lipopolysaccharide (LPS) induces a pathologic increase in lung vascular leakage under septic conditions. LPS-induced human pulmonary micro-vascular endothelial cell (HPMEC) apoptosis launches and aggravates micro-vascular hyper-permeability and acute lung injury (ALI). Previous studies show that the activation of intrinsic apoptotic pathway is vital for LPS-induced EC apoptosis. Yes-associated protein (YAP) has been reported to positively regulate intrinsic apoptotic pathway in tumor cells apoptosis. However, the potential role of YAP protein in LPS-induced HPMEC apoptosis has not been determined. In this study, we found that LPS-induced activation and nuclear accumulation of YAP accelerated HPMECs apoptosis. LPS-induced YAP translocation from cytoplasm to nucleus by the increased phosphorylation on Y357 resulted in the interaction between YAP and transcription factor P73. Furthermore, inhibition of YAP by small interfering RNA (siRNA) not only suppressed the LPS-induced HPMEC apoptosis but also regulated P73-mediated up-regulation of BAX and down-regulation of BCL-2. Taken together, our results demonstrated that activation of the YAP/P73/(BAX and BCL-2)/caspase-3 signaling pathway played a critical role in LPS-induced HPMEC apoptosis. Inhibition of the YAP might be a potential therapeutic strategy for lung injury under sepsis. PMID:27807512

  19. Differences in pulmonary responses of rats, other animals, and humans to chronic inhalation of silica and other particles

    SciTech Connect

    Mauderly, J.L.

    1993-12-31

    The pulmonary carcinogenicity of quartz in rats supports the plausibility of silica-induced lung cancer in humans. However, pulmonary responses of rats to dusts differ from those of other rodents, and may differ from those of humans. Dust-exposed rats have a greater propensity than mice or hamsters for epithelial hyperplasia, metaplasia, and fibrosis. Lung tumors occur in rats, but not mice or hamsters, treated with quartz, or exposed chronically to several other dusts. There are few opportunities for directly comparing the susceptibilities of rats and humans to dust-induced lung tumors. Because of the uncertain human responses to silica and many other particles, the negative human lung cancer response to coal dust may provide the best opportunity to calibrate responses of rats against those of humans. Historical dust lung burdens in coal miners were in the range of those associated with carcinogenicity in rats exposed to several dusts, but the carcinogenicity of coal dust in rats is unknown. The usefulness of tumor data from rats for predicting human lung cancer risk from inhaled silica and other dusts remains uncertain.

  20. Iptakalim influences the proliferation and apoptosis of human pulmonary artery smooth muscle cells

    PubMed Central

    LI, QINGLIN; YAN, XIAOPEI; KONG, HUI; XIE, WEIPING; WANG, HONG

    2016-01-01

    The aim of the present study was to determine the effect of an ATP-sensitive K+ (KATP) channel opener iptakalim (IPT) on the proliferation and apoptosis of human pulmonary artery smooth muscle cells (HPASMCs), and examine the potential value of IPT to hypoxic pulmonary hypertension (HPH) at a cellular level. HPASMCs were divided into the control, ET-1, ET-1+IPT and ET-1+IPT+glibenclamide (GLI) groups. GLI was administered 30 min prior to ET-1 and IPT. The 4 groups were incubated with corresponding reagents for 24 h. Cell viability was evaluated using a CCK-8 assay, cell proliferation by 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay, and cell apoptosis via the expression of apoptosis-related proteins, i.e., Bcl-2-associated X protein (Bax) and B-cell lymphoma 2 (Bcl-2) using western blotting. We incubated HPASMCs with varying concentrations of ET-1 for 24, 48 and 72 h, and found that cell survival rate was increased in a dose-dependent manner (P<0.05) rather than in a time-dependent manner (P>0.05). After co-incubation of HPASMCs with varying concentrations of IPT and ET-1 for 24 h, the cell survival rate was decreased in a dose-dependent manner. The cell survival rate in the IPT+ET-1 group was significantly lower than that in the ET-1 group (P<0.05). The cell viability (P<0.05) and proliferation (P<0.05) in the ET-1 group were higher than those in the control group, and the expression of Bax/Bcl-2 was lower than the control group (P<0.05). The cell viability (P<0.05) and proliferation (P<0.05) in the ET-1+IPT group were lower than those in the ET-1 group, and the expression of Bax/Bcl-2 was higher than that in the ET-1 group (P<0.05). The cell viability (P<0.05) and proliferation (P<0.05) in the ET-1+IPT+GLI group were higher than those in the ET-1+IPT group, and the expression of Bax/Bcl-2 was lower than that in the ET-1+IPT group (P<0.05). In conclusion, IPT inhibited ET-1-induced HPASMC proliferation and promoted cell apoptosis. Thus, it may play an

  1. Acute high-dose X-radiation-induced genomic changes in A549 cells.

    PubMed

    Muradyan, A; Gilbertz, K; Stabentheiner, S; Klause, S; Madle, H; Meineke, V; Ullmann, R; Scherthan, H

    2011-06-01

    Accidents with ionizing radiation often involve single, acute high-dose exposures that can lead to acute radiation syndrome and late effects such as carcinogenesis. To study such effects at the cellular level, we investigated acute ionizing radiation-induced chromosomal aberrations in A549 adenocarcinoma cells at the genome-wide level by exposing the cells to an acute dose of 6 Gy 240 kV X rays. One sham-irradiated clone and four surviving irradiated clones were recovered by minimal dilution and further expanded and analyzed by chromosome painting and tiling-path array CGH, with the nonirradiated clone 0 serving as the control. Acute X-ray exposure induced specific translocations and changes in modal chromosome number in the four irradiated clones. Array CGH disclosed unique and recurrent genomic changes, predominantly losses, and revealed that the fragile sites FRA3B and FRA16D were preferential regions of genomic alterations in all irradiated clones, which is likely related to radioresistant S-phase progression and genomic stress. Furthermore, clone 4 displayed an increased radiosensitivity at doses >5 Gy. Pairwise comparisons of the gene expression patterns of all irradiated clones to the sham-irradiated clone 0 revealed an enrichment of the Gene Ontology term "M Phase" (P = 6.2 × 10(-7)) in the set of differentially expressed genes of clone 4 but not in those of clones 1-3. Ionizing radiation-induced genomic changes and fragile site expression highlight the capacity of a single acute radiation exposure to affect the genome of exposed cells by inflicting genomic stress.

  2. Hyperoxia enhances VEGF release from A549 cells via post-transcriptional processes

    PubMed Central

    Shenberger, Jeffrey S.; Zhang, Lianqin; Powell, Richard J.; Barchowsky, Aaron

    2007-01-01

    Exposure of animals to hyperoxia decreases lung VEGF mRNA expression concomitant with an acute increase in VEGF protein within the epithelial lining fluid (ELF). The VEGF concentration in ELF is in excess of that found in the plasma, leading to the hypothesis that hyperoxia stimulates the release of VEGF protein from stores within the extracellular matrix. To test this hypothesis in a cell culture system, we exposed A549 cells to 95% O2 for 48 hrs followed by recovery in room air (RA) for 24 hrs. We found that Ox increased VEGF protein 2- to 3-fold within the medium at 48 hrs of exposure and during recovery. Heparin clearing revealed the medium to contain a 50:50 mixture of the heparin-binding (VEGF165) and heparin-non-binding (VEGF121) proteins and Ox to increase both proteins equally. Transcriptional activation of VEGF appears unlikely to explain the increase in VEGF protein as full-length and splice variant VEGF mRNA expression were unchanged by hyperoxia. Analysis of cell-associated VEGF proteins found that Ox increased the expression of VEGF121 and VEGF165 proteins. Blocking binding sites with exogenous heparin enhanced VEGF protein in the medium from RA grown cells while heparinase digestion of bound VEGF revealed a greater reserve of VEGF protein in RA cells. Collectively these findings indicate that hyperoxia enhances the expression of VEGF121/165 proteins and facilitates the release of VEGF165 from cell-associated stores. Increases in VEGF in ELF may represent an adaptive response fostering cell survival and type II cell proliferation in O2-induced lung injury. PMID:17664148

  3. Study of the synergistic effects of all-transretinoic acid and C-phycocyanin on the growth and apoptosis of A549 cells.

    PubMed

    Li, Bing; Gao, Mei-Hua; Lv, Cong-Yi; Yang, Peng; Yin, Qi-Feng

    2016-03-01

    In the present study, we investigated the effects of the combination of all-transretinoic acid (ATRA) and natural nontoxic C-phycocyanin (C-PC) on the growth of A549 lung cancer cells in vitro and in vivo. Furthermore, the anticancer mechanism of the drug combination was revealed. Results showed both C-PC and ATRA could inhibit the growth of A549 cells in vivo. The combination of ATRA+C-PC could yield a higher inhibition rate. C-PC exerted a major effect on the proliferation of human embryo lung cells, but ATRA at a high concentration exerted an inhibitory effect. In addition, ATRA+C-PC could decrease the CDK4 mRNA level, but upregulated caspase-3 protein expression and induced cell apoptosis. A mouse model with tumor was constructed by a subcutaneous injection to the left axilla of nu nude (NU/NU) mice. Compared with the control group, the tumor weight was decreased in the single-drug treatment group and was the lowest in the combination group. C-PC+ATRA could upregulate tumor necrosis factor levels and downregulate Bcl-2 expression and the cyclin D1 gene in the tumor. C-PC could promote T cells' activities and spleen weight, but a single use of ATRA exerted an opposite effect. The dosage of ATRA could be reduced when combined with C-PC to reduce the toxic side-effects. In summary, the antitumor effects of the C-PC+ATRA combination were more significant than a single drug in vivo and in vitro.

  4. Multifunctional polyamidoamine-modified selenium nanoparticles dual-delivering siRNA and cisplatin to A549/DDP cells for reversal multidrug resistance.

    PubMed

    Zheng, Wenjing; Cao, Chengwen; Liu, Yanan; Yu, Qianqian; Zheng, Chuping; Sun, Dongdong; Ren, Xiaofan; Liu, Jie

    2015-01-01

    Multidrug resistance (MDR) is a major barrier against effective cancer treatment. Dual-delivering a therapeutic small interfering RNA (siRNA) and chemotherapeutic agents has been developed to reverse drug resistance in tumor cells. In this study, amine-terminated generation 5 polyamidoamine (PAMAM) dendrimers (G5.NH2)-modified selenium nanoparticles (G5@Se NP) were synthesized for the systemic dual-delivery of mdr1 siRNA and cisplatin (cis-diamminedichloroplatinum-(II), DDP), which was demonstrated to enhance siRNA loading, releasing efficiency and gene-silencing efficacy. When the mdr1 siRNA was conjugated with G5@Se NP via electrostatic interaction, a significant down-regulation of P-glycoprotein and multidrug resistance-associated protein expression was observed; G5@Se-DDP-siRNA arrested A549/DDP cells at G1 phase and led to enhanced cytotoxicity in A549/DDP cells through induction of apoptosis involving the AKT and ERK signaling pathways. Interestingly, G5@Se-DDP NP were much less reactive than DDP in the reactions with both MT and GSH, indicating that loading of DDP in a nano-delivery system could effectively prevent cell detoxification. Furthermore, animal studies demonstrated that the new delivery system of G5@Se-DDP-siRNA significantly enhanced the anti-tumor effect on tumor-bearing nude mice, with no appreciable abnormality in the major organs. These results suggest that G5@Se NP could be a potential platform to combine chemotherapy and gene therapy technology in the treatment of human disease.

  5. Radio-sensitization effect of an mTOR inhibitor, temsirolimus, on lung adenocarcinoma A549 cells under normoxic and hypoxic conditions

    PubMed Central

    Ushijima, Hiroki; Suzuki, Yoshiyuki; Oike, Takahiro; Komachi, Mayumi; Yoshimoto, Yuya; Ando, Ken; Okonogi, Noriyuki; Sato, Hiro; Noda, Shin-ei; Saito, Jun-ichi; Nakano, Takashi

    2015-01-01

    The mammalian target of rapamycin (mTOR) correlates with cell survival under hypoxia and regulates hypoxia-inducible factor-1α (HIF-1α), a key protein in hypoxia-related events. However, the role of mTOR in radio-resistance has not been fully investigated. Therefore, the effect of mTOR on the radio-resistance of cancer cells under hypoxia was evaluated using the mTOR inhibitor temsirolimus. Clonogenic survival was examined in the A549 human lung adenocarcinoma cell line under normoxia or hypoxia, with or without temsirolimus. An oxygen enhancement ratio (OER) was calculated using the D10 values, the doses giving 10% survival. Western blotting was performed to investigate the effect of temsirolimus on mTOR and the HIF-1α pathway under normoxia and hypoxia. A549 cells showed a radio-resistance of 5.1 and 14.2 Gy, as indicated by D10 values under normoxia and hypoxia, respectively; the OER was 2.8. The cell survival rates under hypoxia and with temsirolimus remarkably decreased compared with those under normoxia. The D10 values of the cells under normoxia and hypoxia were 4.8 and 5.4 Gy, respectively (OER = 1.1). mTOR expression was suppressed by temsirolimus under both normoxia and hypoxia. HIF-1α expression decreased under hypoxia in the presence of temsirolimus. These results suggest that temsirolimus can overcome the radio-resistance induced by hypoxia. When the fact that mTOR acts upstream of HIF-1α is considered, our data suggest that the restoration of radiation sensitivity by temsirolimus under hypoxia may be associated with the suppression of the HIF-1α pathway. Temsirolimus could therefore be used as a hypoxic cell radio-sensitizer. PMID:25887043

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

    PubMed

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

    2013-09-17

    Particulate matter (PM) is a major ambient air pollutant causing millions of premature deaths each year in China. The toxicity of PM is property and size dependent. In this study, ambient PM samples collected in Beijing were divided into five size fractions with nominal aerodynamic ranges of <0.40, 0.40-1.1, 1.1-3.3, 3.3-5.8, and 5.8-10 μm. Individual size fractions were characterized for a number of properties including particle size distribution, specific surface area, zeta potential, dithiothreitol (DTT)-based redox ability, and contents of water-soluble organic carbon (WSOC), polycyclic aromatic hydrocarbons (PAHs), selected metals, and endotoxin. Human adenocarcinomic alveolar epithelial cell line A549 and small mouse monocyte-macrophage cell line J774A.1 were tested for their relative viabilities and inflammatory effects (interleukine-8 for A549 and tumor necrosis factor-α for J774A.1) after exposure to PM of various sizes. It was found that PM specific area was positively correlated with WSOC, high molecular weight PAHs, DTT-based redox ability, negatively correlated with surface zeta potential and lithophile metals. Several trace metals from combustion sources were enriched in intermediate size fractions. For both endotoxin concentrations of the PM and PM induced inflammatory cytokine expressions by the two cell lines, there were general increasing trends as PM size increased with an exception of the finest fraction, which induced the highest inflammatory effects. It seems that the size dependence of cytokine expression was associated with a number of properties including endotoxin content, zeta potential, settling velocity, metal content, and DTT-based redox ability.

  7. Radiosensitization of TPGS-emulsified docetaxel-loaded poly(lactic-co-glycolic acid) nanoparticles in CNE-1 and A549 cells.

    PubMed

    Shi, Wei; Yuan, Yin; Chu, Min; Zhao, Shuang; Song, Qingle; Mu, Xiaoqian; Xu, Shuangbing; Zhang, Zhiping; Yang, Kunyu

    2016-03-01

    Docetaxel is among the most effective radiosensitizers. It is widely used as radiosensitizer in many tumors, including head and neck carcinoma. Nevertheless, poor solubility and severe hypersensitivity limit its clinical use and its therapeutic effect remains to be improved. In this study, docetaxel-loaded polymeric nanoparticles were prepared by nanoprecipitation method to be new radiosensitizer with lower side effects and higher efficacy. The physiochemical characteristics of the nanoparticles were studied. Two human tumor cell lines which are resistant to radiotherapy were used in this research. We have compared the radioenhancement efficacy of docetaxel-loaded nanoparticles with docetaxel in A549 and CNE-1 cells. Compared with docetaxel, radiosensitization of docetaxel-loaded nanoparticles was improved significantly (sensitization enhancement ratio in A549 increased 1.24-fold to 1.68-fold when the radiation was applied 2 h after the drug, p < 0.01, sensitization enhancement ratio in CNE-1 increased 1.32-fold to 1.61-fold, p < 0.05). We explored the mechanisms for the radiosensitization efficiency and the difference between docetaxel and docetaxel-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles. The improved radiosensitization efficacy was associated with enhanced G2/M arrest, promoted apoptosis and the role of D-alpha-tocopheryl polyethylene glycol 1000 succinate which will enhance the cell uptake and inhibit the multiple drug resistance. Moreover, the radiosensitization efficacy of docetaxel-loaded nanoparticles was more prominent than docetaxel. In conclusion, tocopheryl polyethylene glycol 1000 succinate-emulsified docetaxel-loaded PLGA nanoparticles were more efficacious and fewer adverse effects were observed than with the commercial docetaxel formulation. Thus, PLGA nanoparticles hold promise as a radiosensitizing agent.

  8. E2F1 enhances 8-chloro-adenosine-induced G2/M arrest and apoptosis in A549 and H1299 lung cancer cells.

    PubMed

    Duan, Hong-Ying; Cao, Ji-Xiang; Qi, Jun-Juan; Wu, Guo-Sheng; Li, Shu-Yan; An, Guo-Shun; Jia, Hong-Ti; Cai, Wang-Wei; Ni, Ju-Hua

    2012-03-01

    The E2F1 transcription factor is a well known regulator of cell proliferation and apoptosis, but its role in response to DNA damage is less clear. 8-Chloro-adenosine (8-Cl-Ado), a nucleoside analog, can inhibit proliferation in a variety of human tumor cells. However, it is still elusive how the agent acts on tumors. Here we show that A549 and H1299 cells formed DNA double-strand breaks after 8-Cl-Ado exposure, accompanied by E2F1 upregulation at protein level. Overexpressed wild-type (E2F1-wt) colocalized with double-strand break marker γ-H2AX and promoted G2/M arrest in 8-Cl-Ado-exposed A549 and H1299, while expressed S31A mutant of E2F1 (E2F1-mu) significantly reduced ability to accumulate at sites of DNA damage and G2/M arrest, suggesting that E2F1 is required for activating G2/M checkpoint pathway upon DNA damage. Transfection of either E2F1-wt or E2F1-mu plasmid promoted apoptosis in 8-Cl-Ado-exposed cells, indicating that 8-Cl-Ado may induce apoptosis in E2F1-dependent and E2F1-independent ways. These findings demonstrate that E2F1 plays a crucial role in 8-Cl-Ado-induced G2/M arrest but is dispensable for 8-Cl-Ado-induced apoptosis. These data also suggest that the mechanism of 8-Cl-Ado action is complicated.

  9. [Hantavirus in human and rodent population in an endemic area for hantavirus pulmonary syndrome in Argentina].

    PubMed

    Sosa-Estani, Sergio; Martínez, Valeria P; González Della Valle, Marcelo; Edelstein, Alexis; Miguel, Sergio; Padula, Paula J; Cacase, María L; Segura, Elsa L

    2002-01-01

    This paper analyzed the prevalence and distribution of serological reactivity to hantavirus (antibody against ANDES virus) of human population exposed to hantavirus and rodents trapped in the studied area. This study was developed in Salta (Orán and San Martín Departments), area with the highest incidence for Hantavirus Pulmonary Syndrome (HPS) in Argentina. In December 1997, 453 healthy people were studied by serology and 39 rodents by serology and PCR. The studied individuals were distributed as: 145 farm inhabitants (FI), 212 people living in the same dwelling with healthy individuals (controls) (Cco), 87 people living in the same dwelling with persons undergoing SPH in 1997 (cases) (Cca). Moreover, 19 physicians and nurses who cared for patients with SPH in 1997 were also studied. The prevalence of hantavirus infection among the studied population was 6.3%. The prevalence was 10.3% among FI, 6.9% among Cca and 3.3% among Cco (p < 0.02). There was no serological reactivity among PS. The prevalence in 39 trapped rodents was 10.2%, with infection only for Oligoryzomys chacoensis, O. flavescens and Akodon varius species. The prevalence of human cases with asymptomatic infection in Salta is higher than in other regions of the country, and we are presenting a hypothesis to explain these differences. The analyzed data suggest that in this region up to the time this study was performed, there would not have been person to person transmission of hantavirus. The transmission would be from rodent contact exclusively and mainly in ongoing deforestation areas and domestic habitat surrounding rural dwellings.

  10. Combined exposure to nano-silica and lead induced potentiation of oxidative stress and DNA damage in human lung epithelial cells.

    PubMed

    Lu, Chun-Feng; Yuan, Xiao-Yan; Li, Li-Zhong; Zhou, Wei; Zhao, Jun; Wang, Yi-Mei; Peng, Shuang-Qing

    2015-12-01

    Growing evidence has confirmed that exposure to ambient particulate matters (PM) is associated with increased morbidity and mortality of cardiovascular and pulmonary diseases. Ambient PM is a complex mixture of particles and air pollutants. Harmful effects of PM are specifically associated with ultrafine particles (UFPs) that can adsorb high concentrations of toxic air pollutants and are easily inhaled into the lungs. However, combined effects of UFPs and air pollutants on human health remain unclear. In the present study, we elucidated the combined toxicity of silica nanoparticles (nano-SiO2), a typical UFP, and lead acetate (Pb), a typical air pollutant. Lung adenocarcinoma A549 cells were exposed to nano-SiO2 and Pb alone or their combination, and their combined toxicity was investigated by focusing on cellular oxidative stress and DNA damage. Factorial analyses were performed to determine the potential interactions between nano-SiO2 and Pb. Our results showed that exposure of A549 cells to a modest cytotoxic concentration of Pb alone induced oxidative stress, as evidenced by elevated reactive oxygen species generation and lipid peroxidation, and reduced glutathione content and superoxide dismutase and glutathione peroxidase activities. In addition, exposure of A549 cells to Pb alone induced DNA damage, as evaluated by alkaline comet assay. Exposure of A549 cells to non-cytotoxic concentration of nano-SiO2 did not induce cellular oxidative stress and DNA damage. However, exposure to the combination of nano-SiO2 and Pb potentiated oxidative stress and DNA damage in A549 cells. Factorial analyses indicated that the potentiation of combined toxicity of nano-SiO2 and Pb was induced by additive or synergistic interactions.

  11. The PDZ protein TIP-1 facilitates cell migration and pulmonary metastasis of human invasive breast cancer cells in athymic mice

    SciTech Connect

    Han, Miaojun; Wang, Hailun; Zhang, Hua-Tang; Han, Zhaozhong

    2012-05-25

    Highlights: Black-Right-Pointing-Pointer This study has revealed novel oncogenic functions of TIP-1 in human invasive breast cancer. Black-Right-Pointing-Pointer Elevated TIP-1 expression levels in human breast cancers correlate to the disease prognosis. Black-Right-Pointing-Pointer TIP-1 knockdown suppressed the cell migration and pulmonary metastasis of human breast cancer cells. Black-Right-Pointing-Pointer TIP-1 knockdown suppressed the expression and functionality of motility-related genes. -- Abstract: Tax-interacting protein 1 (TIP-1, also known as Tax1bp3) inhibited proliferation of colon cancer cells through antagonizing the transcriptional activity of beta-catenin. However, in this study, elevated TIP-1 expression levels were detected in human invasive breast cancers. Studies with two human invasive breast cancer cell lines indicated that RNAi-mediated TIP-1 knockdown suppressed the cell adhesion, proliferation, migration and invasion in vitro, and inhibited tumor growth in mammary fat pads and pulmonary metastasis in athymic mice. Biochemical studies showed that TIP-1 knockdown had moderate and differential effects on the beta-catenin-regulated gene expression, but remarkably down regulated the genes for cell adhesion and motility in breast cancer cells. The decreased expression of integrins and paxillin was accompanied with reduced cell adhesion and focal adhesion formation on fibronectin-coated surface. In conclusion, this study revealed a novel oncogenic function of TIP-1 suggesting that TIP-1 holds potential as a prognostic biomarker and a therapeutic target in the treatment of human invasive breast cancers.

  12. Pulmonary Inflammatory Responses To Acute Meteorite Dust Exposures - Implications For Human Space Exploration

    NASA Technical Reports Server (NTRS)

    Harrington, A. D.; McCubbin, F. M.; Kaur, J.; Smirnov, A.; Galdanes, K.; Schoonen, M. A. A.; Chen, L. C.; Tsirka, S. E.; Gordon, T.

    2017-01-01

    The previous manned missions to the Moon represent milestones of human ingenuity, perseverance, and intellectual curiosity. However, one of the major ongoing concerns is the array of hazards associated with lunar surface dust. Not only did the dust cause mechanical and structural integrity issues with the suits, the dust 'storm' generated upon reentrance into the crew cabin caused "lunar hay fever" and "almost blindness" (Figure 1). It was further reported that the allergic response to the dust worsened with each exposure. The lack of gravity exacerbated the exposure, requiring the astronauts to wear their helmet within the module in order to avoid breathing the irritating particles. Due to the prevalence of these high exposures, the Human Research Roadmap developed by NASA identifies the Risk of Adverse Health and Performance Effects of Celestial Dust Exposure as an area of concern. Extended human exploration will further increase the probability of inadvertent and repeated exposures to celestial dusts. Going forward, hazard assessments of celestial dusts will be determined through sample return efforts prior to astronaut deployment. Studies on the lunar highland regolith indicate that the dust is not only respirable but also reactive, and previous studies concluded that it is moderately toxic; generating a greater response than titanium oxide but a lower response than quartz. The presence of reactive oxygen species (ROS) on the surface of the dust has been implicated. However, there is actually little data related to physicochemical characteristics of particulates and pulmonary toxicity, especially as it relates to celestial dust exposure. As a direct response to this deficit, the present study evaluates the role of a particulate's innate geochemical features (e.g., bulk chemistry, internal composition, morphology, size, and reactivity) in generating adverse toxicological responses in vitro and in vivo. This highly interdisciplinary study evaluates the relative

  13. Knockdown of Aurora-B inhibits the growth of non-small cell lung cancer A549 cells.

    PubMed

    Yu, Jing Jing; Zhou, Long Dian; Zhao, Tian Tian; Bai, Wei; Zhou, Jing; Zhang, Wei

    2015-09-01

    Elevated expression of Aurora-B affects cell apoptosis and proliferation in a variety of solid tumors. However, the role of Aurora-B has been poorly evaluated in non-small cell lung cancer (NSCLC). In the present study, it was found that Aurora-B was overexpressed in tissue specimens obtained from 174 patients with lung cancer. It was also demonstrated that knockdown of Aurora-B induces apoptosis and inhibits the growth of lung cancer A549 cells in vitro and in vivo. Furthermore, it was found that silencing Aurora-B decreased the activity of the phosphoinositide 3-kinase (PI3K)/AKT pathway. Therefore, it was concluded that knockdown of Aurora-B induces apoptosis and inhibits growth in NSCLC A549 cells, in addition to inhibiting the activity of the PI3K/AKT signaling pathway. Targeting Aurora-B may provide a novel target for lung cancer therapy.

  14. The effect of urapidil, an alpha-1 adrenoceptor antagonist and a 5-HT1A agonist, on the vascular tone of the porcine coronary and pulmonary arteries, the rat aorta and the human pulmonary artery.

    PubMed

    Bopp, Claire; Auger, Cyril; Diemunsch, Pierre; Schini-Kerth, Valérie

    2016-05-15

    Urapidil (Eupressyl(®)) an antihypertensive drug acting as an α1 antagonist and a 5-HT1A agonist, may be of special interest in the treatment of hypertension associated with preeclamptic toxaemia and hypoxia-induced pulmonary arterial vasoconstriction. However, the effect of urapidil on vascular tone has been poorly investigated. Vascular reactivity was evaluated using pulmonary and coronary arteries from 36 pigs, aortae from 22 rats and 9 human pulmonary artery samples suspended in organ chambers. Concentration-relaxation curves either to urapidil, 5-HT, or the 5-HT1A receptor agonist 8-OH-DPAT were constructed after pre-contraction of rings. Pig pulmonary and coronary artery rings were contracted with U46619, a thromboxane mimetic, rat aortic rings with either endothelin-1 or phenylephrine, and human pulmonary artery rings with U46619 or phenylephrine. Urapidil markedly inhibited phenylephrine-induced contractions in rat aortic rings with and without endothelium with a more pronounced effect observed in rings without endothelium. Both 5-HT and 8-OH-DPAT failed to induce relaxation in rat aortic rings with an intact endothelium. 5-HT, but not urapidil and 8-OH-DPAT, induced a concentration-dependent relaxation in the porcine coronary and pulmonary artery rings with an intact endothelium (P<0.05). 5-HT and phenylephrine but not urapidil caused concentration-dependent contractions in human pulmonary artery rings. The present findings, while confirming that urapidil is a potent inhibitor of α1-adrenoceptor-induced contraction, do not support the role of 5-HT1A receptor activation in the control of the vascular tone of the different types of arteries tested in response to urapidil. In addition, they indicate that urapidil seems to preferentially target arteries with endothelial dysfunction.

  15. Human Pulmonary Microvascular Endothelial Cells Support Productive Replication of Highly Pathogenic Avian Influenza Viruses: Possible Involvement in the Pathogenesis of Human H5N1 Virus Infection

    PubMed Central

    Zeng, Hui; Pappas, Claudia; Belser, Jessica A.; Houser, Katherine V.; Zhong, Weiming; Wadford, Debra A.; Stevens, Troy; Balczon, Ron; Katz, Jacqueline M.

    2012-01-01

    Highly pathogenic avian influenza (HPAI) H5N1 viruses continue to cause sporadic human infections with a high fatality rate. Respiratory failure due to acute respiratory distress syndrome (ARDS) is a complication among hospitalized patients. Since progressive pulmonary endothelial damage is the hallmark of ARDS, we investigated host responses following HPAI virus infection of human pulmonary microvascular endothelial cells. Evaluation of these cells for the presence of receptors preferred by influenza virus demonstrated that avian-like (α2-3-linked) receptors were more abundant than human-like (α2-6-linked) receptors. To test the permissiveness of pulmonary endothelial cells to virus infection, we compared the replication of selected seasonal, pandemic (2009 H1N1 and 1918), and potentially pandemic (H5N1) influenza virus strains. We observed that these cells support productive replication only of HPAI H5N1 viruses, which preferentially enter through and are released from the apical surface of polarized human endothelial monolayers. Furthermore, A/Thailand/16/2004 and A/Vietnam/1203/2004 (VN/1203) H5N1 viruses, which exhibit heightened virulence in mammalian models, replicated to higher titers than less virulent H5N1 strains. VN/1203 infection caused a significant decrease in endothelial cell proliferation compared to other subtype viruses. VN/1203 virus was also found to be a potent inducer of cytokines and adhesion molecules known to regulate inflammation during acute lung injury. Deletion of the H5 hemagglutinin (HA) multibasic cleavage site did not affect virus infectivity but resulted in decreased virus replication in endothelial cells. Our results highlight remarkable tropism and infectivity of the H5N1 viruses for human pulmonary endothelial cells, resulting in the potent induction of host inflammatory responses. PMID:22072765

  16. Pulmonary embolus

    MedlinePlus

    ... clot - lung; Embolus; Tumor embolus; Embolism - pulmonary; DVT-pulmonary embolism; Thrombosis - pulmonary embolism ... Main symptoms of a pulmonary embolism include chest pain that may be any of the following: Under the breastbone or on one side Sharp or stabbing ...

  17. Blocking the NOTCH pathway can inhibit the growth of CD133-positive A549 cells and sensitize to chemotherapy

    SciTech Connect

    Liu, Juntao; Mao, Zhangfan; Huang, Jie; Xie, Songping; Liu, Tianshu; Mao, Zhifu

    2014-02-21

    Highlights: • Notch signaling pathway members are expressed lower levels in CD133+ cells. • CD133+ cells are not as sensitive as CD133− cells to chemotherapy. • GSI could inhibit the growth of both CD133+ and CD133− cells. • Blockade of Notch signaling pathway enhanced the effect of chemotherapy with CDDP. • DAPT/CDDP co-therapy caused G2/M arrest and elimination in CD133+ cells. - Abstract: Cancer stem cells (CSCs) are believed to play an important role in tumor growth and recurrence. These cells exhibit self-renewal and proliferation properties. CSCs also exhibit significant drug resistance compared with normal tumor cells. Finding new treatments that target CSCs could significantly enhance the effect of chemotherapy and improve patient survival. Notch signaling is known to regulate the development of the lungs by controlling the cell-fate determination of normal stem cells. In this study, we isolated CSCs from the human lung adenocarcinoma cell line A549. CD133 was used as a stem cell marker for fluorescence-activated cell sorting (FACS). We compared the expression of Notch signaling in both CD133+ and CD133− cells and blocked Notch signaling using the γ-secretase inhibitor DAPT (GSI-IX). The effect of combining GSI and cisplatin (CDDP) was also examined in these two types of cells. We observed that both CD133+ and CD133− cells proliferated at similar rates, but the cells exhibited distinctive differences in cell cycle progression. Few CD133+ cells were observed in the G{sub 2}/M phase, and there were half as many cells in S phase compared with the CD133− cells. Furthermore, CD133+ cells exhibited significant resistance to chemotherapy when treated with CDDP. The expression of Notch signaling pathway members, such as Notch1, Notch2 and Hes1, was lower in CD133+ cells. GSI slightly inhibited the proliferation of both cell types and exhibited little effect on the cell cycle. The inhibitory effects of DPP on these two types of cells were

  18. Lysyl oxidase mediates hypoxia-induced radioresistance in non-small cell lung cancer A549 cells

    PubMed Central

    Gong, Chongwen; Gu, Runxia; Jin, Honglin; Sun, Yao; Li, Zhenyu; Wu, Gang

    2016-01-01

    Hypoxia-induced radioresistance has been well known as the main obstacle in cancer radiotherapy. Lysyl oxidase (LOX) was previously demonstrated to play an important role in hypoxia-induced biological behaviors, such as metastasis and angiogenesis, through hypoxia-inducible factor-1α (HIF-1α), which is an important contributing factor to radioresistance in tumor cells. However, how LOX plays a role in hypoxia-induced radioresistance has yet to be determined. Here, we found that LOX expression was in accordance with HIF-1α expression, and LOX expression at the mRNA and protein level, and enzymatic activity were remarkably upregulated in the hypoxic A549 cells, compared with normoxic A549 cells. Inhibition of LOX resulted in the reduction of the ability to repair double-stranded breaks (DSBs), promotion of apoptosis, relief of G2/M cycle arrest, and eventually reduction of hypoxia-induced radioresistance in the hypoxic A549 cells. This suggests that LOX may play an important role in hypoxia-induced radioresistance. Together, our results might suggest a novel potential therapeutic target in the management of non-small cell lung cancer (NSCLC). PMID:26515140

  19. Over expression of miR-200c suppresses invasion and restores methotrexate sensitivity in lung cancer A549 cells.

    PubMed

    Shan, Wulin; Zhang, Xiaolei; Li, Ming; Deng, Fang; Zhang, Jing

    2016-11-30

    MicroRNAs have become recognized as key players in the development of malignancy. MiR-200c can function as a tumor suppressor gene. However, the effect of miR-200c on methotrexate resistance remains unclear to date. This study aims to evaluate the function of miR-200c in lung cancer A549 cells. The data presented in our study demonstrated that the expression of miR-200c was down-regulated in methotrexate-resistant A549 cells. Over expression of miR-200c could significantly inhibit cell proliferation, induce G0/G1 cell cycle arrest and induce cell apoptosis. RT-PCR and Western blot assays showed that the expression of P53 and P21 were significantly increased with miR-200c overexpression. These results indicated that over expression of miR-200c might enhance the sensitivity of A549 cells to methotrexate through the P53/P21 pathway. Furthermore, miR-200c overexpression significantly inhibited cell migration and invasion with increasing the expression of E-cad and decreasing the expression of EZH2. In consequence, we provide a mechanism of acquired resistance to methotrexate that is caused by the loss of miR-200c in lung cancer cells. Along with this, our study demonstrates the complex network of microRNA mediated chemoresistance.

  20. Effects of green tea extract on lung cancer A549 cells: proteomic identification of proteins associated with cell migration.

    PubMed

    Lu, Qing-Yi; Yang, Yanan; Jin, Yu Sheng; Zhang, Zuo-Feng; Heber, David; Li, Frederick P; Dubinett, Steven M; Sondej, Melissa A; Loo, Joseph A; Rao, Jian Yu

    2009-02-01

    Green tea polyphenols exhibit multiple antitumor activities, and the mechanisms of action are not completely understood. Previously, we reported that green tea extract (GTE)-induced actin remolding is associated with increased cell adhesion and decreased motility in A549 lung cancer cells. To identify the cellular targets responsible for green tea-induced actin remodeling, we performed 2-DE LC-MS/MS of A549 cells before and after GTE exposure. We have identified 14 protein spots that changed in expression (> or =2-fold) after GTE treatment. These proteins are involved in calcium-binding, cytoskeleton and motility, metabolism, detoxification, or gene regulation. In particular we found upregulation of several genes that modulate actin remodeling and cell migration, including lamin A/C. Our data indicated that GTE-induced lamin A/C upregulation appears to be at the transcriptional level and the increased expression results in the decrease in cell motility, as confirmed by siRNA. The result of the study demonstrates that GTE alters the levels of many proteins involved in growth, motility and apoptosis of A549 cells and their identification may explain the multiple antitumor activities of GTE.

  1. The effects of Davallic acid from Davallia divaricata Blume on apoptosis induction in A549 lung cancer cells.

    PubMed

    Cheng, An-Sheng; Chang, We-Chang; Cheng, Yu-Hsiang; Chen, Kai-Yu; Chen, Kai-Hsien; Chang, Tsu-Liang

    2012-11-01

    Traditional or folk medicinal herbs continue to be prescribed in the treatment of various diseases and conditions in many cultures. Recent scientific efforts have focused on the potential roles of extracts of traditional herbs as alternative and complementary medications for cancer treatment. In Taiwan, Davallia divaricata Blume has been traditionally employed in folk medicine for therapy of lung cancer, davallic acid being the major active compound of D. divaricata Blume. In this study, we investigated the inhibitory activity of davallic acid on the proliferation of A549 lung cancer cells. Davallic acid was extracted from D. divaricata Blume, and its effects on cell viability, cell cycle distribution, ROS level, and apoptotic protein expression in A549 cells were determined. Davallic acid significantly induced reactive oxygen species (ROS) generation as well as caspase-3, -8, and -9 activation, thereby repressing A549 cell growth and elevating apoptotic activity. Since lung cancer has a high incidence of recurrence, these results indicate that davallic acid may have the potential to be a natural anti-lung cancer compound, and may provide a basis for further study of its use in combating cancer.

  2. Silver nanoparticles induce apoptosis and G2/M arrest via PKCζ-dependent signaling in A549 lung cells.

    PubMed

    Lee, Young Sook; Kim, Dong Woon; Lee, Young Ho; Oh, Jung Hwa; Yoon, Seokjoo; Choi, Mi Sun; Lee, Sung Kyu; Kim, Ji Won; Lee, Kyuhong; Song, Chang-Woo

    2011-12-01

    The use of silver nanoparticles is one of the fastest growing product categories in the nanotechnology industry, with a focus on antimicrobial activity. However, thus far, toxicity data for silver nanoparticles are limited. In this study, we investigated the cytotoxic effects of silver nanoparticles (Ag NPs) and the pathway by which they affect A549 lung epithelial cells. The effects of Ag NPs on cell survival, cell cycle progression, and mRNA and protein alterations of selected cell cycle- and apoptosis-related genes were studied using formazan dye and LDH release assays, flow cytometric analysis, semi-quantitative RT-PCR, and Western blot analysis. Ag NPs reduced cell viability, increased LDH release, and modulated cell cycle distribution through the accumulation of cells at G2/M and sub-G1 phases (cell death), with a concurrent decrease in cells at G1. Ag NP treatment increased Bax and Bid mRNA levels and downregulated Bcl-2 and Bcl-w mRNAs in a dose-dependent manner. Furthermore, Ag NPs altered the mRNA levels of protein kinase C (PKC) family members. In particular, ectopic overexpression of PKCζ led to the enhancement of cellular proliferation and reduced sensitivity to Ag NPs in A549 cells. Together, these results suggest that Ag NPs induce strong toxicity and G2/M cell cycle arrest by a mechanism involving PKCζ downregulation in A549 cells.

  3. Targeting Interleukin-13 with Tralokinumab Attenuates Lung Fibrosis and Epithelial Damage in a Humanized SCID Idiopathic Pulmonary Fibrosis Model

    PubMed Central

    Zhang, Huilan; Oak, Sameer R.; Coelho, Ana Lucia; Herath, Athula; Flaherty, Kevin R.; Lee, Joyce; Bell, Matt; Knight, Darryl A.; Martinez, Fernando J.; Sleeman, Matthew A.; Herzog, Erica L.; Hogaboam, Cory M.

    2014-01-01

    The aberrant fibrotic and repair responses in the lung are major hallmarks of idiopathic pulmonary fibrosis (IPF). Numerous antifibrotic strategies have been used in the clinic with limited success, raising the possibility that an effective therapeutic strategy in this disease must inhibit fibrosis and promote appropriate lung repair mechanisms. IL-13 represents an attractive target in IPF, but its disease association and mechanism of action remains unknown. In the present study, an overexpression of IL-13 and IL-13 pathway markers was associated with IPF, particularly a rapidly progressive form of this disease. Targeting IL-13 in a humanized experimental model of pulmonary fibrosis using tralokinumab (CAT354) was found to therapeutically block aberrant lung remodeling in this model. However, targeting IL-13 was also found to promote lung repair and to restore epithelial integrity. Thus, targeting IL-13 inhibits fibrotic processes and enhances repair processes in the lung. PMID:24325475

  4. Resveratrol prevents hypoxia-induced arginase II expression and proliferation of human pulmonary artery smooth muscle cells via Akt-dependent signaling.

    PubMed

    Chen, Bernadette; Xue, Jianjing; Meng, Xiaomei; Slutzky, Jessica L; Calvert, Andrea E; Chicoine, Louis G

    2014-08-15

    Pulmonary artery smooth muscle cell (PASMC) proliferation plays a fundamental role in the vascular remodeling seen in pulmonary hypertensive diseases associated with hypoxia. Arginase II, an enzyme regulating the first step in polyamine and proline synthesis, has been shown to play a critical role in hypoxia-induced proliferation of human PASMC (hPASMC). In addition, there is evidence that patients with pulmonary hypertension have elevated levels of arginase in the vascular wall. Resveratrol, a natural polyphenol found in red wine and grape skins, has diverse biochemical and physiological actions including antiproliferative properties. Furthermore, resveratrol has been shown to attenuate right ventricular and pulmonary artery remodeling, both pathological components of pulmonary hypertension. The present studies tested the hypothesis that resveratrol would prevent hypoxia-induced pulmonary artery smooth muscle cell proliferation by inhibiting hypoxia-induced arginase II expression. Our data indicate that hypoxia-induced hPASMC proliferation is abrogated following treatment with resveratrol. In addition, the hypoxic induction of arginase II was directly attenuated by resveratrol treatment. Furthermore, we found that the inhibitory effect of resveratrol on arginase II in hPASMC was mediated through the PI3K-Akt signaling pathway. Supporting these in vitro findings, resveratrol normalized right ventricular hypertrophy in an in vivo neonatal rat model of chronic hypoxia-induced pulmonary hypertension. These novel data support the notion that resveratrol may be a potential therapeutic agent in pulmonary hypertension by preventing PASMC arginase II induction and proliferation.

  5. Surfactant protein A2 mutations associated with pulmonary fibrosis lead to protein instability and endoplasmic reticulum stress.

    PubMed

    Maitra, Meenakshi; Wang, Yongyu; Gerard, Robert D; Mendelson, Carole R; Garcia, Christine Kim

    2010-07-16

    Rare heterozygous mutations in the gene encoding surfactant protein A2 (SP-A2, SFTPA2) are associated with adult-onset pulmonary fibrosis and adenocarcinoma of the lung. We have previously shown that two recombinant SP-A2 mutant proteins (G231V and F198S) remain within the endoplasmic reticulum (ER) of A549 cells and are not secreted into the culture medium. The pathogenic mechanism of the mutant proteins is unknown. Here we analyze all common and rare variants of the surfactant protein A2, SP-A2, in both A549 cells and in primary type II alveolar epithelial cells. We show that, in contrast with all other SP-A2 variants, the mutant proteins are not secreted into the medium with wild-type SP-A isoforms, form fewer intracellular dimer and trimer oligomers, are partially insoluble in 0.5% Nonidet P-40 lysates of transfected A549 cells, and demonstrate greater protein instability in chymotrypsin proteolytic digestions. Both the G231V and F198S mutant SP-A2 proteins are destroyed via the ER-association degradation pathway. Expression of the mutant proteins increases the transcription of a BiP-reporter construct, expression of BiP protein, and production of an ER stress-induced XBP-1 spliced product. Human bronchoalveolar wash samples from individuals who are heterozygous for the G231V mutation have similar levels of total SP-A as normal family members, which suggests that the mechanism of disease does not involve an overt lack of secreted SP-A but instead involves an increase in ER stress of resident type II alveolar epithelial cells.

  6. Genome sequencing of idiopathic pulmonary fibrosis in conjunction with a medical school human anatomy course.

    PubMed

    Kumar, Akash; Dougherty, Max; Findlay, Gregory M; Geisheker, Madeleine; Klein, Jason; Lazar, John; Machkovech, Heather; Resnick, Jesse; Resnick, Rebecca; Salter, Alexander I; Talebi-Liasi, Faezeh; Arakawa, Christopher; Baudin, Jacob; Bogaard, Andrew; Salesky, Rebecca; Zhou, Qian; Smith, Kelly; Clark, John I; Shendure, Jay; Horwitz, Marshall S

    2014-01-01

    Even in cases where there is no obvious family history of disease, genome sequencing may contribute to clinical diagnosis and management. Clinical application of the genome has not yet become routine, however, in part because physicians are still learning how best to utilize such information. As an educational research exercise performed in conjunction with our medical school human anatomy course, we explored the potential utility of determining the whole genome sequence of a patient who had died following a clinical diagnosis of idiopathic pulmonary fibrosis (IPF). Medical students performed dissection and whole genome sequencing of the cadaver. Gross and microscopic findings were more consistent with the fibrosing variant of nonspecific interstitial pneumonia (NSIP), as opposed to IPF per se. Variants in genes causing Mendelian disorders predisposing to IPF were not detected. However, whole genome sequencing identified several common variants associated with IPF, including a single nucleotide polymorphism (SNP), rs35705950, located in the promoter region of the gene encoding mucin glycoprotein MUC5B. The MUC5B promoter polymorphism was recently found to markedly elevate risk for IPF, though a particular association with NSIP has not been previously reported, nor has its contribution to disease risk previously been evaluated in the genome-wide context of all genetic variants. We did not identify additional predicted functional variants in a region of linkage disequilibrium (LD) adjacent to MUC5B, nor did we discover other likely risk-contributing variants elsewhere in the genome. Whole genome sequencing thus corroborates the association of rs35705950 with MUC5B dysregulation and interstitial lung disease. This novel exercise additionally served a unique mission in bridging clinical and basic science education.

  7. Hyperosmolarity attenuates TNFα–mediated pro-inflammatory activation of human pulmonary microvascular endothelial cells

    PubMed Central

    Banerjee, Anirban; Moore, Ernest E.; McLaughlin, Nathan J.; Lee, Luis; Jones, Wilbert L.; Johnson, Jeffrey L.; Nydam, Trevor L.; Silliman, Christopher C.

    2013-01-01

    Firm neutrophil (PMN)-endothelial (EC) adhesion is crucial to the PMN-mediated hyperinflammation observed in acute lung injury. Hypertonic saline (HTS) used for resuscitation of hemorrhagic shock has been associated with a decreased incidence of PMN-mediated lung injury/acute respiratory distress syndrome. We hypothesize that physiologically accessible hypertonic incubation (170mM vs. 140mM, osmolarity ranging from 360-300 mOsm/L) inhibits pro-inflammatory activation of human pulmonary microvascular endothelial cells (HMVECs). Pro-inflammatory activation of HMVECs was investigated in response to TNFα including IL-8 release, ICAM-1 surface expression, PMN adhesion, and signaling mechanisms under both isotonic (control) and hypertonic conditions. Hyperosmolarity alone had no effect on either basal IL-8 release or ICAM-1 surface expression, but did lead to concentration-dependent decreases in TNFα–induced IL-8 release, ICAM-1 surface expression, and PMN:HMVEC adhesion. Conversely, HTS activated p38 mitogen-activated protein kinase (MAPK) and enhanced TNFα activation of p38 MAPK. Despite this basal activation, hyperosmolar incubation attenuated TNFα stimulated IL-8 release and ICAM-1 surface expression and subsequent PMN adherence, while p38 MAPK inhibition did not further influence the effects of hyperosmolar conditions on ICAM-1 surface expression. In addition, TNFα induced NF-kB DNA binding, but HTS conditions attenuated this by 31% (p<0.01). In conclusion, HTS reduces PMN:HMVEC adhesion as well as TNFα-induced pro-inflammatory activation of primary HMVECs via attenuation of NF-kB signaling. PMID:23364439

  8. Moringa oleifera Gold Nanoparticles Modulate Oncogenes, Tumor Suppressor Genes, and Caspase-9 Splice Variants in A549 Cells.

    PubMed

    Tiloke, Charlette; Phulukdaree, Alisa; Anand, Krishnan; Gengan, Robert M; Chuturgoon, Anil A

    2016-10-01

    Gold nanoparticles (AuNP's) facilitate cancer cell recognition and can be manufactured by green synthesis using nutrient rich medicinal plants such as Moringa oleifera (MO). Targeting dysregulated oncogenes and tumor suppressor genes is crucial for cancer therapeutics. We investigated the antiproliferative effects of AuNP synthesized from MO aqueous leaf extracts (MLAuNP ) in A549 lung and SNO oesophageal cancer cells. A one-pot green synthesis technique was used to synthesise MLAuNP . A549, SNO cancer cells and normal peripheral blood mononuclear cells (PBMCs) were exposed to MLAuNP and CAuNP to evaluate cytotoxicity (MTT assay); apoptosis was measured by phosphatidylserine (PS) externalization, mitochondrial depolarization (ΔΨm) (flow cytometry), caspase-3/7, -9 activity, and ATP levels (luminometry). The mRNA expression of c-myc, p53, Skp2, Fbw7α, and caspase-9 splice variants was determined using qPCR, while relative protein expression of c-myc, p53, SRp30a, Bax, Bcl-2, Smac/DIABLO, Hsp70, and PARP-1 were determined by Western blotting. MLAuNP and CAuNP were not cytotoxic to PBMCs, whilst its pro-apoptotic properties were confirmed in A549 and SNO cells. MLAuNP significantly increased caspase activity in SNO cells while MLAuNP significantly increased PS externalization, ΔΨm, caspase-9, caspase-3/7 activities, and decreased ATP levels in A549 cells. Also, p53 mRNA and protein levels, SRp30a (P = 0.428), Bax, Smac/DIABLO and PARP-1 24 kDa fragment levels were significantly increased. Conversely, MLAuNP significantly decreased Bcl-2, Hsp70, Skp2, Fbw7α, c-myc mRNA, and protein levels and activated alternate splicing with caspase-9a splice variant being significantly increased. MLAuNP possesses antiproliferative properties and induced apoptosis in A549 cells by activating alternate splicing of caspase-9. J. Cell. Biochem. 117: 2302-2314, 2016. © 2016 Wiley Periodicals, Inc.

  9. Preferential metabolism of N-nitrosodiethylamine by two cell lines derived from human pulmonary adenocarcinomas

    SciTech Connect

    Falzon, M.; McMahon, J.B.; Gazdar, A.F.; Schuller, H.M.

    1986-01-01

    Diethylnitrosamine (DEN), in common with other nitrosamines, is a carcinogenic agent which produces tumors in a wide variety of tissues in experimental animals. The pulmonary Clara cell is a major target of N-nitrosamine-induced carcinogenesis in hamsters and rats. DEN is believed to require metabolic activation to elicit its carcinogenic effects. The metabolism of (/sup 14/C)DEN was studied in two cell lines derived from human lung adenocarcinomas and two cell lines derived from human small cell lung cancers by monitoring /sup 14/CO/sub 2/ production and covalent binding of radiolabel from (/sup 14/C)DEN to the cell protein and DNA fractions. (/sup 14/C)DEN was metabolized by adenocarcinoma-derived NCI-H322 (with Clara cell features) and NCI-H358 (with features of alveolar type II cells) but not by NCI-H69 and NCI-H128 (derived from small cell carcinoma). Metabolism was markedly inhibited by heat denaturation of the cell protein. (/sup 14/C)DEN metabolism by NCI-H322 was greatly decreased when the incubation was carried out under anaerobic conditions and in the presence of a carbon monoxide enriched atmosphere. These results suggested the involvement of the cytochrome P-450-dependent monooxygenase enzyme system. Metabolism by NCI-H358 was also decreased in the absence of oxygen or presence of carbon monoxide although the effects were relatively small compared with the results with NCI-H322. On the other hand, aspirin or indomethacin, which are inhibitors of the fatty acid cyclooxygenase component of prostaglandin endoperoxide synthetase, preferentially inhibited (/sup 14/C)DEN metabolism by NIC-H358. There were little or no effects of these inhibitors on the metabolism of DEN in NCI-H322. The data suggest that DEN metabolism in different lung cell types may be carried out by different enzyme systems which in turn may contribute to the selective effect of DEN in the lung.

  10. Strains of Mycobacterium tuberculosis differ in affinity for human osteoblasts and alveolar cells in vitro.

    PubMed

    Sarkar, Shrabanti; Dlamini, Muyalo G; Bhattacharya, Debapriya; Ashiru, Olubisi T; Sturm, A Willem; Moodley, Prashini

    2016-01-01

    Although the lung is the primary site of infection of tuberculosis, Mycobacterium tuberculosis is capable of causing infection at other sites. In 5-10 % such extra-pulmonary tuberculosis is located in bone tissue of the spine. It is unknown whether host or microbial factors are responsible for the site where extra-pulmonary tuberculosis manifests itself. One MDR isolate belonging to strain F28, one susceptible F11 and one isolate each of susceptible, MDR and XDR F15/LAM4/KZN were cultured in Middlebrook 7H9 media. Human osteoblasts (SaOS-2) and human alveolar epithelial cells (A549) were exposed to these different isolates of M. tuberculosis and invasion capacity and intra-cellular multiplication rates were established. Mouse macrophage (MHS) cells exposed to M. tuberculosis H37Rv served as control. The invasion capacity of F15/LAM4/KZN representatives increased with the level of resistance. The F28 MDR strain showed similar invasion capacity as the XDR F15/LAM4/KZN for pulmonary epthelial cells, whilst the fully susceptible F11 strain displayed a propensity for osteoblasts. The differences observed may in part explain why certain strains are able to cause infection at specific extra-pulmonary sites. We postulated that the development of extra-pulmonary tuberculosis depends on the ability of the microbe to pass effectively through the alveolar epithelial lining and its affinity for cells other than those in pulmonary tissue.

  11. Time-course of ventilation, arterial and pulmonary CO(2) tension during CO (2) increase in humans.

    PubMed

    Satoh, Toru; Okada, Yasumasa; Hara, Yasushi; Sakamaki, Fumio; Kyotani, Shingo; Tomita, Takeshi; Nagaya, Noritoshi; Nakanishi, Norifumi

    2012-01-01

    A change of ventilation (VE), PaCO( 2 ) (arterial CO( 2 ) tension) and PvCO( 2 ) (pulmonary arterial CO( 2 ) tension) with time was not evaluated precisely during exercise or CO( 2 ) rebreathing in humans. In this study, changes of these variables with time were fitted to exponential curves {y = Exp ( x/ T + A ) + k} and compared. When exercise pulmonary hemodynamics was examined in 15 cardiac patients to decide therapies, we asked the patients to undergo CO( 2 ) rebreathing using air with supplementation of consumed O( 2 ). Arterial and pulmonary blood was drawn every minute. During exercise, T was 28.2 ± 8.4 and 26.8 ± 12.4, and A was 0.80 ± 0.50 and 0.50 ± 0.90 in VE and PvCO( 2 ), respectively, with no statistical differences. During CO( 2 ) rebreathing, T was 18.6 ± 5.8, 41.8 ± 38.0 and 21.6 ± 9.7 and A was 0.39 ± 0.67, 1.64 ± 1.35 and 0.17 ± 0.83 in VE, PaCO( 2 ) and PvCO( 2 ), respectively, with statistical difference of PaCO( 2 ) from other variables, suggesting that VE and PvCO( 2 ) showed same mode of change according to time but PaCO( 2 ) did not.

  12. Grainyhead-like 2 (GRHL2) distribution reveals novel pathophysiological differences between human idiopathic pulmonary fibrosis and mouse models of pulmonary fibrosis

    PubMed Central

    Mahavadi, Poornima; Sasikumar, Satish; Cushing, Leah; Hyland, Tessa; Rosser, Ann E.; Riccardi, Daniela; Lu, Jining; Kalin, Tanya V.; Kalinichenko, Vladimir V.; Guenther, Andreas; Ramirez, Maria I.; Pardo, Annie; Selman, Moisés; Warburton, David

    2013-01-01

    Chronic injury of alveolar lung epithelium leads to epithelial disintegrity in idiopathic pulmonary fibrosis (IPF). We had reported earlier that Grhl2, a transcriptional factor, maintains alveolar epithelial cell integrity by directly regulating components of adherens and tight junctions and thus hypothesized an important role of GRHL2 in pathogenesis of IPF. Comparison of GRHL2 distribution at different stages of human lung development showed its abundance in developing lung epithelium and in adult lung epithelium. However, GRHL2 is detected in normal human lung mesenchyme only at early fetal stage (week 9). Similar mesenchymal reexpression of GRHL2 was also observed in IPF. Immunofluorescence analysis in serial sections from three IPF patients revealed at least two subsets of alveolar epithelial cells (AEC), based on differential GRHL2 expression and the converse fluorescence intensities for epithelial vs. mesenchymal markers. Grhl2 was not detected in mesenchyme in intraperitoneal bleomycin-induced injury as well as in spontaneously occurring fibrosis in double-mutant HPS1 and HPS2 mice, whereas in contrast in a radiation-induced fibrosis model, with forced Forkhead box M1 (Foxm1) expression, an overlap of Grhl2 with a mesenchymal marker was observed in fibrotic regions. Grhl2's role in alveolar epithelial cell plasticity was confirmed by altered Grhl2 gene expression analysis in IPF and further validated by in vitro manipulation of its expression in alveolar epithelial cell lines. Our findings reveal important pathophysiological differences between human IPF and specific mouse models of fibrosis and support a crucial role of GRHL2 in epithelial activation in lung fibrosis and perhaps also in epithelial plasticity. PMID:24375798

  13. Transforming growth factor-β evokes Ca2+ waves and enhances gene expression in human pulmonary fibroblasts.

    PubMed

    Mukherjee, Subhendu; Kolb, Martin R J; Duan, Fuqin; Janssen, Luke J

    2012-06-01

    Fibroblasts maintain the structural framework of animal tissue by synthesizing extracellular matrix molecules. Chronic lung diseases are characterized in part by changes in fibroblast numbers, properties, and more. Fibroblasts respond to a variety of growth factors, cytokines, and proinflammatory mediators. However, the signaling mechanisms behind these responses have not been fully explored. We sought to determine the role of Ca(2+) waves in transforming growth factor-β (TGF-β)-mediated gene expression in human pulmonary fibroblasts. Primary human pulmonary fibroblasts were cultured and treated with TGF-β and different blockers under various conditions. Cells were then loaded with the Ca(2+) indicator dye Oregon green, and Ca(2+) waves were monitored by confocal [Ca(2+)](i) fluorimetry. Real-time PCR was used to probe gene expression. TGF-β (1 nM) evoked recurring Ca(2+) waves. A 30-minute pretreatment of SD 208, a TGF-β receptor-1 kinase inhibitor, prevented Ca(2+) waves from being evoked by TGF-β. The removal of external Ca(2+) completely occluded TGF-β-evoked Ca(2+) waves. Cyclopiazonic acid, an inhibitor of the internal Ca(2+) pump, evoked a relatively slowly developing rise in Ca(2+) waves compared with the rapid changes evoked by TGF-β, but the baseline fluorescence was increased. Ryanodine (10(-5) M) also blocked TGF-β-mediated Ca(2+) wave activity. Real-time PCR showed that TGF-β rapidly and dramatically increased the gene expression of collagen A1 and fibronectin. This increase was blocked by ryanodine treatment and cyclopiazonic acid. We conclude that, in human pulmonary fibroblasts, TGF-β acts on ryanodine-sensitive channels, leading to Ca(2+) wave activity, which in turn amplifies extracellular matrix gene expression.

  14. D-4F, an apolipoprotein A-I mimetic, inhibits TGF-β1 induced epithelial-mesenchymal transition in human alveolar epithelial cell.

    PubMed

    You, Jia; Wang, Jintao; Xie, Linshen; Zhu, Chengwen; Xiong, Jingyuan

    2016-10-01

    Emerging evidences support that transforming growth factor β1 (TGF-β1) induced epithelial-mesenchymal transition (EMT) participates in the pathogenesis of pulmonary fibrosis and asthmatic airway remodeling. Recent studies demonstrated that apolipoprotein A-I (Apo A-I) is the only known substance that can resolve established pulmonary fibrotic nodules, and Apo A-I mimetic D-4F (a synthetic polypeptide consisting of 18 amino acids) plays an inhibitory role in murine asthmatic model. However, cellular mechanisms for such therapeutic effects of Apo A-I and D-4F remain to be elucidated. This study evaluated the effects of D-4F on TGF-β1 induced EMT in human type II alveolar epithelial cell line A549. A549 cells treated with 10ng/ml of TGF-β1 manifested distinct EMT, including fibroblastic morphological changes, down-regulation of epithelial marker E-cadherin and up-regulation of mesenchymal marker vimentin. These EMT related changes were all inhibited by D-4F in a concentration dependent manner. Transcriptional investigation demonstrated clearly that D-4F dose-dependently compensated for the reduced E-cadherin mRNA level and the increased vimentin mRNA level in TGF-β1 treated A549 cells. Translational analysis revealed that D-4F significantly reversed the TGF-β1 induced changes of E-cadherin and vimentin levels. These results suggested that D-4F inhibits TGF-β1 induced EMT in human alveolar epithelial cell. Given the functional similarities between D-4F and Apo A-I, it is speculated that D-4F and Apo A-I are able to exert possible anti-fibrotic and anti-asthmatic effects via inhibiting alveolar EMT, and D-4F may possess beneficial clinical potential for patients suffering from pulmonary fibrosis and asthma.

  15. Apigenin inhibits PMA-induced expression of pro-inflammatory cytokines and AP-1 factors in A549 cells.

    PubMed

    Patil, Rajeshwari H; Babu, R L; Naveen Kumar, M; Kiran Kumar, K M; Hegde, Shubha M; Ramesh, Govindarajan T; Chidananda Sharma, S

    2015-05-01

    Acute and chronic alveolar or bronchial inflammation is thought to be central to the pathogenesis of many respiratory disorders. Cytokines and granulocyte macrophage colony-stimulating factors (GM-CSF) play an important role in chronic inflammation. Activator protein-1 (AP-1) the superfamily of transcription factors is involved in proliferation, differentiation, apoptosis, and transformation including inflammation. Understanding the function and regulation of proinflammatory factors involved in inflammation may provide the novel therapeutic strategies in the treatment of inflammatory diseases. Our aim of the present study is to investigate the pro-inflammatory cytokines and pattern of AP-1 factors expressed during activation of lung adenocarcinoma A549 cells by Phorbol-12-myristate-13-acetate (PMA) and to understand the anti-inflammatory effect of apigenin. A549 cells were treated with and without PMA or apigenin, and the cell viability was assessed by MTT assay. Expressions of inflammatory mediators and different AP-1 factors were analyzed by semi-quantitative RT-PCR. IL-6 protein secreted was analyzed by ELISA, and expressions of IL-1β, c-Jun, and c-Fos proteins were analyzed by Western blotting. Activation of A549 cells by PMA, induced the expression of pro-inflammatory cytokine (IL-1β, IL-2, IL-6, IL-8, and TNF-α) mRNAs and secretion of IL-6 and the expression of specific AP-1 factors (c-Jun, c-Fos, and Fra-1). Treatment of cells with apigenin, significantly inhibited PMA-stimulated mRNA expression of above pro-inflammatory cytokines, AP-1 factors, cyclooxygenase-2, and secretion of IL-6 protein. Results suggested that the AP-1 factors may be involved in inflammation and apigenin has anti-inflammatory effect, which may be useful for therapeutic management of lung inflammatory diseases.

  16. Interleukin-4-Mediated 15-Lipoxygenase-1 Trans-Activation Requires UTX Recruitment and H3K27me3 Demethylation at the Promoter in A549 Cells

    PubMed Central

    Han, Hongya; Xu, Dawei; Liu, Cheng; Claesson, Hans-Erik; Björkholm, Magnus; Sjöberg, Jan

    2014-01-01

    Arachidonate 15-lipoxygenase-1 (ALOX15) oxygenates polyunsaturated fatty acids and bio-membranes, generating multiple lipid signalling mediators involved in inflammation. Several lines of evidence indicate that ALOX15 activation in the respiratory tract contributes to asthma progression. Recent experimental data reveals that histone modification at the promoter plays a critical role in ALOX15 gene transcription. In the present study, we examined the status of histone H3 trimethyl-lysine 27 (H3K27me3) at the ALOX15 promoter by chromatin immunoprecipitation assay in human lung epithelial carcinoma A549 cells incubated with or without interleukin (IL)-4. We identified demethylation of H3K27me3 at the ALOX15 promoter after IL-4 treatment. Furthermore, we found that the H3K27me2/3-specific demethylase, ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX), mediates the H3K27me3 demethylation during ALOX15 transcriptional activation. When UTX expression was knocked down using siRNA, IL-4-mediated H3K27me3 demethylation and ALOX15 induction were significantly attenuated. The critical role of UTX in ALOX15 expression was confirmed in human monocytes and the Hodgkin lymphoma (HL) cell line L1236, but was in these cells not related to H3K27me3-demethylase activity. These results demonstrate that UTX is implicated in IL-4 mediated transcriptional activation of the ALOX15 gene. PMID:24465480

  17. Cis-acting sequences from a human surfactant protein gene confer pulmonary-specific gene expression in transgenic mice

    SciTech Connect

    Korfhagen, T.R.; Glasser, S.W.; Wert, S.E.; Bruno, M.D.; Daugherty, C.C.; McNeish, J.D.; Stock, J.L.; Potter, S.S.; Whitsett, J.A. )

    1990-08-01

    Pulmonary surfactant is produced in late gestation by developing type II epithelial cells lining the alveolar epithelium of the lung. Lack of surfactant at birth is associated with respiratory distress syndrome in premature infants. Surfactant protein C (SP-C) is a highly hydrophobic peptide isolated from pulmonary tissue that enhances the biophysical activity of surfactant phospholipids. Like surfactant phospholipid, SP-C is produced by epithelial cells in the distal respiratory epithelium, and its expression increases during the latter part of gestation. A chimeric gene containing 3.6 kilobases of the promoter and 5{prime}-flanking sequences of the human SP-C gene was used to express diphtheria toxin A. The SP-C-diphtheria toxin A fusion gene was injected into fertilized mouse eggs to produce transgenic mice. Affected mice developed respiratory failure in the immediate postnatal period. Morphologic analysis of lungs from affected pups showed variable but severe cellular injury confined to pulmonary tissues. Ultrastructural changes consistent with cell death and injury were prominent in the distal respiratory epithelium. Proximal components of the tracheobronchial tree were not severely affected. Transgenic animals were of normal size at birth, and structural abnormalities were not detected in nonpulmonary tissues. Lung-specific diphtheria toxin A expression controlled by the human SP-C gene injured type II epithelial cells and caused extensive necrosis of the distal respiratory epithelium. The absence of type I epithelial cells in the most severely affected transgenic animals supports the concept that developing type II cells serve as precursors to type I epithelial cells.

  18. Human Adipose-derived Mesenchymal Stem Cells Attenuate Early Stage of Bleomycin Induced Pulmonary Fibrosis: Comparison with Pirfenidone

    PubMed Central

    Reddy, Manoj; Fonseca, Lyle; Gowda, Shashank; Chougule, Basavraj; Hari, Aarya; Totey, Satish

    2016-01-01

    Background and Objectives Idiopathic pulmonary fibrosis (IPF) is a progressive, irreversible, invariably fatal fibrotic lung disease with no lasting option for therapy. Mesenchymal stem cells (MSCs) could be a promising modality for the treatment of IPF. Aim of the study was to investigate improvement in survivability and anti-fibrotic efficacy of human adipose-derived mesenchymal stem cells (AD-MSCs) in comparison with pirfenidone in the bleomycin-induced pulmonary fibrosis model. Methods Human AD-MSCs were administered intravenously on day 3, 6 and 9 after an intra-tracheal challenge with bleomycin, whereas, pirfenidone was given orally in drinking water at the rate of 100 mg/kg body weight three times a day daily from day 3 onward. AD-MSCs were labelled with PKH-67 before administration to detect engraftment. Disease severity and improvement was assessed and compared between sham control and vehicle control groups using Kaplan-Meier survival analysis, biochemical and molecular analysis, histopathology and high resolution computed tomography (HRCT) parameters at the end of study. Results Results demonstrated that AD-MSCs significantly increase survivability; reduce organ weight and collagen deposition better than pirfenidone group. Histological analyses and HRCT of the lung revealed that AD-MSCs afforded protection against bleomycin induced fibrosis and protect architecture of the lung. Gene expression analysis revealed that AD-MSCs potently suppressed pro-fibrotic genes induced by bleomycin. More importantly, AD-MSCs were found to inhibit pro-inflammatory related transcripts. Conclusions Our results provided direct evidence that AD-MSC-mediated immunomodulation and anti-fibrotic effect in the lungs resulted in marked protection in pulmonary fibrosis, but at an early stage of disease. PMID:27871152

  19. Bioengineered Human and Allogeneic Pulmonary Valve Conduits Chronically Implanted Orthotopically in Baboons: Hemodynamic Performance and Immunological Consequences

    PubMed Central

    Hopkins, Richard A.; Bert, Arthur A.; Hilbert, Stephen L.; Quinn, Rachael W.; Brasky, Kathleen M.; Drake, William B.; Lofland, Gary K.

    2012-01-01

    Objective This study assesses in a baboon model the hemodynamics and HLA immunogenicity of chronically implanted bioengineered (decellularized with collagen conditioning treatments) human and baboon heart valve scaffolds. Methods Fourteen baboons underwent pulmonary valve replacement, eight with decellularized and conditioned (bioengineered) pulmonary valves derived from either allogeneic (N=3) or xenogeneic (human) (N=5) hearts; for comparison, six baboons received clinically relevant reference cryopreserved or porcine valved conduits. Panel reactive serum antibodies (HLA Class I&II), complement fixing antibodies (C1q binding), and C-reactive protein titers were measured serially until elective sacrifice at 10 or 26 weeks. Serial transesophageal echocardiograms (TEE) measured valve function and geometry. Differences were analyzed with Kruskal-Wallis and Wilcoxon Rank Sum. P≤ 0.05 significant. Results All animals survived and thrived, exhibiting excellent immediate implanted valve function by TEE. Over time, reference valves developed smaller indexed effective orifice areas, EOAI=0.84(1.22) cm2/m2 median (range) while all bioengineered valves remained normal, EOAI=2.45 (1.35) cm2/m2; P=0.005. None of the bioengineered valves developed elevated peak transvalvular gradients, 5.5(6.0) versus 12.5(23.0) mmHg, P=0.003. Cryopreserved valves provoked the most intense antibody responses. Two of five human bioengineered and two of three baboon bioengineered valves did not provoke any Class I antibodies. Bioengineered human (but not baboon) scaffolds provoked Class II antibodies. C1q+ antibodies developed in four recipients. Conclusions Valve dysfunction correlated with markers for more intense inflammatory provocation. The tested bioengineering methods reduced antigenicity of both human and baboon valves. Bioengineered replacement valves from both species were hemodynamically equivalent to native valves. PMID:22841171

  20. Human metapneumovirus infection activates the TSLP pathway that drives excessive pulmonary inflammation and viral replication in mice.

    PubMed

    Lay, Margarita K; Céspedes, Pablo F; Palavecino, Christian E; León, Miguel A; Díaz, Rodrigo A; Salazar, Francisco J; Méndez, Gonzalo P; Bueno, Susan M; Kalergis, Alexis M

    2015-06-01

    Human metapneumovirus (hMPV) is a leading cause of acute respiratory tract infections in children and the elderly. The mechanism by which this virus triggers an inflammatory response still remains unknown. Here, we evaluated whether the thymic stromal lymphopoietin (TSLP) pathway contributes to lung inflammation upon hMPV infection. We found that hMPV infection promotes TSLP expression both in human airway epithelial cells and in the mouse lung. hMPV infection induced lung infiltration of OX40L(+) CD11b(+) DCs. Mice lacking the TSLP receptor deficient mice (tslpr(-/-) ) showed reduced lung inflammation and hMPV replication. These mice displayed a decreased number of neutrophils as well a reduction in levels of thymus and activation-regulated chemokine/CCL17, IL-5, IL-13, and TNF-α in the airways upon hMPV infection. Furthermore, a higher frequency of CD4(+) and CD8(+) T cells was found in tslpr(-/-) mice compared to WT mice, which could contribute to controlling viral spread. Depletion of neutrophils in WT and tslpr(-/-) mice decreased inflammation and hMPV replication. Remarkably, blockage of TSLP or OX40L with specific Abs reduced lung inflammation and viral replication following hMPV challenge in mice. Altogether, these results suggest that activation of the TSLP pathway is pivotal in the development of pulmonary pathology and pulmonary hMPV replication.

  1. Pulmonary angiography

    MedlinePlus

    ... Pulmonary arteriography; Pulmonary angiogram; Angiogram of the lungs Images Pulmonary arteries References Jackson JE, Meaney JFM. Angiography. ... urac.org). URAC's accreditation program is an independent audit to verify that A.D.A.M. follows ...

  2. Pulmonary Rehabilitation

    MedlinePlus

    ... Topics Bronchitis COPD Cystic Fibrosis Idiopathic Pulmonary Fibrosis Sarcoidosis Send a link to NHLBI to someone by ... people who have COPD (chronic obstructive pulmonary disease), sarcoidosis (sar-koy-DOE-sis), idiopathic pulmonary fibrosis , or ...

  3. Over-expression of human endosulfatase-1 exacerbates cadmium-induced injury to transformed human lung cells in vitro

    SciTech Connect

    Zhang, Huiying; Newman, Donna R.; Bonner, James C.; Sannes, Philip L.

    2012-11-15

    Environmental exposure to cadmium is known to cause damage to alveolar epithelial cells of the lung, impair their capacity to repair, and result in permanent structural alterations. Cell surface heparan sulfate proteoglycans (HSPGs) can modulate cell responses to injury through their interactions with soluble effector molecules. These interactions are often sulfate specific, and the removal of sulfate groups from HS side chains could be expected to influence cellular injury, such as that caused by exposure to cadmium. The goal of this study was to define the role 6-O-sulfate plays in cellular responses to cadmium exposure in two pulmonary epithelial cancer cell lines (H292 and A549) and in normal human primary alveolar type II (hAT2) cells. Sulfate levels were modified by transduced transient over-expression of 6-O-endosulfatase (HSulf-1), a membrane-bound enzyme which specifically removes 6-O-sulfate groups from HSPG side chains. Results showed that cadmium decreased cell viability and activated apoptosis pathways at low concentrations in hAT2 cells but not in the cancer cells. HSulf-1 over-expression, on the contrary, decreased cell viability and activated apoptosis pathways in H292 and A549 cells but not in hAT2 cells. When combined with cadmium, HSulf-1 over-expression further decreased cell viability and exacerbated the activation of apoptosis pathways in the transformed cells but did not add to the toxicity in hAT2 cells. The finding that HSulf-1 sensitizes these cancer cells and intensifies the injury induced by cadmium suggests that 6-O-sulfate groups on HSPGs may play important roles in protection against certain environmental toxicants, such as heavy metals. -- Highlights: ► Primary human lung alveolar type 2 (hAT2) cells and H292 and A549 cells were used. ► Cadmium induced apoptosis in hAT2 cells but not in H292 or A549 cells. ► HSulf-1exacerbates apoptosis induced by cadmium in H292 and A549 but not hAT2 cells.

  4. Pulmonary Kaposi's sarcoma as the initial presentation of human immunodeficiency virus infection

    PubMed Central

    Imran, Tasnim F.; Al-Khateeb, Ziyaad; Jung, Jin; Peters, Stephen; Dever, Lisa L.

    2014-01-01

    Kaposi's sarcoma (KS) usually presents in HIV-infected patients with cutaneous lesions that may advance to extensive visceral disease. There have been only a few documented cases in which the initial presentation of Kaposi's sarcoma involved the bronchopulmonary system. We describe a newly diagnosed patient who presented with pulmonary KS as his initial presentation of the disease. Our report is intended to increase clinicians’ awareness that pulmonary Kaposi's sarcoma should be considered in HIV-infected patients who present with respiratory symptoms, even if they do not manifest the typical mucocutaneous manifestations of KS or have low CD4 counts. Early diagnosis and therapy are essential in improving outcomes as this condition carries a high mortality. PMID:26839780

  5. Pulmonary Kaposi's sarcoma as the initial presentation of human immunodeficiency virus infection.

    PubMed

    Imran, Tasnim F; Al-Khateeb, Ziyaad; Jung, Jin; Peters, Stephen; Dever, Lisa L

    2014-01-01

    Kaposi's sarcoma (KS) usually presents in HIV-infected patients with cutaneous lesions that may advance to extensive visceral disease. There have been only a few documented cases in which the initial presentation of Kaposi's sarcoma involved the bronchopulmonary system. We describe a newly diagnosed patient who presented with pulmonary KS as his initial presentation of the disease. Our report is intended to increase clinicians' awareness that pulmonary Kaposi's sarcoma should be considered in HIV-infected patients who present with respiratory symptoms, even if they do not manifest the typical mucocutaneous manifestations of KS or have low CD4 counts. Early diagnosis and therapy are essential in improving outcomes as this condition carries a high mortality.

  6. Elevation of iron storage in humans attenuates the pulmonary vascular response to hypoxia.

    PubMed

    Bart, Nicole K; Curtis, M Kate; Cheng, Hung-Yuan; Hungerford, Sara L; McLaren, Ross; Petousi, Nayia; Dorrington, Keith L; Robbins, Peter A

    2016-08-01

    Sustained hypoxia over several hours induces a progressive rise in pulmonary artery systolic pressure (PASP). Administration of intravenous iron immediately prior to the hypoxia exposure abrogates this effect, suggesting that manipulation of iron stores may modify hypoxia-induced pulmonary hypertension. Iron (ferric carboxymaltose) administered intravenously has a plasma half-life of 7-12 h. Thus any therapeutic use of intravenous iron would require its effect on PASP to persist long after the iron-sugar complex has been cleared from the blood. To examine this, we studied PASP during sustained (6 h) hypoxia on 4 separate days (days 0, 1, 8, and 43) in 22 participants. On day 0, the rise in PASP with hypoxia was well matched between the iron and saline groups. On day 1, each participant received either 1 g of ferric carboxymaltose or saline in a double-blind manner. After administration of intravenous iron, the rise in PASP with hypoxia was attenuated by ∼50%, and this response remained suppressed on both days 8 and 43 (P < 0.001). Following administration of intravenous iron, values for ferritin concentration, transferrin saturation, and hepcidin concentration rose significantly (P < 0.001, P < 0.005, and P < 0.001, respectively), and values for transferrin concentration fell significantly (P < 0.001). These changes remained significant at day 43 We conclude that the attenuation of the pulmonary vascular response to hypoxia by elevation of iron stores persists long after the artificial iron-sugar complex has been eliminated from the blood. The persistence of this effect suggests that intravenous iron may be of benefit in some forms of pulmonary hypertension.

  7. Effect of miR-335 upregulation on the apoptosis and invasion of lung cancer cell A549 and H1299.

    PubMed

    Wang, Huaqi; Li, Min; Zhang, Ren; Wang, Yuanyuan; Zang, Wenqiao; Ma, Yunyun; Zhao, Guoqiang; Zhang, Guojun

    2013-10-01

    MicroRNAs are small non-coding RNAs that may also function as oncogenes and tumor-suppressor genes, as the abnormal expression of microRNAs is associated with various human tumors. However, the effect of miR-335 on the lung cancer cells remains unclear. The aim of the paper was to study the expression of miR335 in non-small cell lung cancer (NSCLC) and miR335's relation to the metastasis, invasion, and apoptosis in lung cancer cells A549 and H1299. qRT-PCR was used to identify the miR-335 expression. The effects of miR-335 on cell proliferation, apoptosis, and invasion were further analyzed. Luciferase reporter assay and Western blot were to verify Bcl-w and SP1 as potential major target genes of miR-335. Finally, the effect of Bcl-w on miR-335-induced cell survival was determined. Our results showed that miR-335 expression was significantly lower in NSCLC tissue, which was significantly associated with lymph node metastasis. In contrast to cells in blank and negative control groups, incidence of apoptosis was significantly higher (P < 0.05) and the number of cells migrating through matrigel was significantly lower (P < 0.05) in miR-335 mimics transfected group. Western blot and luciferase reporter assay demonstrated that miR-335 could bind to the putative binding sites in Bcl-w (or SP1) mRNA 3'-untranslated region to visibly lower the expression of Bcl-w (or SP1). The introduction of Bcl-w cDNA without 3'-untranslated region abrogated miR-335-induced cell survival. These results indicated that upregulation of miR-335 can simultaneously suppress the invasiveness and promote apoptosis of lung cancer cell A549 and H1299 by targeting Bcl-w and SP1. Therefore, miR-335 may be a potential therapeutic target in NSCLC treatment.

  8. Exercise-induced interstitial pulmonary edema at sea-level in young and old healthy humans

    PubMed Central

    Taylor, Bryan J.; Carlson, Alex R.; Miller, Andrew D.; Johnson, Bruce D.

    2014-01-01

    We asked whether aged adults are more susceptible to exercise-induced pulmonary edema relative to younger individuals. Lung diffusing capacity for carbon monoxide (DLCO), alveolar-capillary membrane conductance (Dm) and pulmonary-capillary blood volume (Vc) were measured before and after exhaustive discontinuous incremental exercise in 10 young (YNG; 27±3 yr) and 10 old (OLD; 69±5 yr) males. In YNG subjects, Dm increased (11±7%, P=0.031), Vc decreased (−10±9%, P=0.01) and DLCO was unchanged (30.5±4.1 vs. 29.7±2.9 ml/min/mmHg, P=0.44) pre- to post-exercise. In OLD subjects, DLCO and Dm increased (11±14%, P=0.042; 16±14%, P=0.025) but Vc was unchanged (58±23 vs. 56±23 ml, P=0.570) pre- to post-exercise. Group-mean Dm/Vc was greater after vs. before exercise in the YNG and OLD subjects. However, Dm/Vc was lower post-exercise in 2 of the 10 YNG (−7±4%) and 2 of the 10 OLD subjects (−10±5%). These data suggest that exercise decreases interstitial lung fluid in most YNG and OLD subjects, with a small number exhibiting evidence for exercise-induced pulmonary edema. PMID:24200644

  9. Release behavior and toxicity profiles towards A549 cell lines of ciprofloxacin from its layered zinc hydroxide intercalation compound

    PubMed Central

    2013-01-01

    Background Layered hydroxides salts (LHS), a layered inorganic compound is gaining attention in a wide range of applications, particularly due to its unique anion exchange properties. In this work, layered zinc hydroxide nitrate (LZH), a family member of LHS was intercalated with anionic ciprofloxacin (CFX), a broad spectrum antibiotic via ion exchange in a mixture solution of water:ethanol. Results Powder x-ray diffraction (XRD), Fourier transform infrared (FTIR) and thermogravimetric analysis (TGA) confirmed the drug anions were successfully intercalated in the interlayer space of LZH. Specific surface area of the obtained compound was increased compared to that of the host due to the different pore textures between the two materials. CFX anions were slowly released over 80 hours in phosphate-buffered saline (PBS) solution due to strong interactions that occurred between the intercalated anions and the host lattices. The intercalation compound demonstrated enhanced antiproliferative effects towards A549 cancer cells compared to the toxicity of CFX alone. Conclusions Strong host-guest interactions between the LZH lattice and the CFX anion give rise to a new intercalation compound that demonstrates sustained release mode and enhanced toxicity effects towards A549 cell lines. These findings should serve as foundations towards further developments of the brucite-like host material in drug delivery systems. PMID:23849189

  10. Antitumor activity of EGFR targeted pH-sensitive immunoliposomes encapsulating gemcitabine in A549 xenograft nude mice.

    PubMed

    Kim, In-Young; Kang, Young-Sook; Lee, Doo Sung; Park, Heon-Joo; Choi, Eun-Kyung; Oh, Yu-Kyoung; Son, Hye-Jung; Kim, Jin-Seok

    2009-11-16

    Immunoliposomes directed by monoclonal antibodies are promising vehicles for tumor targeted drug delivery. Development of a long-circulating formulation of pH-sensitive liposomes (PSLs) with epidermal growth factor receptor (EGFR) antibody attached was designed and tested using A549 cells and BALB/c-nu/nu mouse tumor model. PSL formulation was prepared using small unilamellar vesicles of DOPE and CHEMS (6:4 molar ratio) by REV method. The average size and zeta-potential of the formulation measured by dynamic laser-light scattering were approximately 146+/-43.9 nm (PDI=0.09+/-0.02) and -1.77+/-0.03 mV, respectively. A549 cells were xenotransplanted into BALB/c-nu/nu mice and various formulations of gemcitabine (gem), such as in its free form, PSLs or Ab-PSLs, were injected intravenously via a tail vein. The rate of tumor volume increment in Ab-PSLs with gem-treated group was remarkably slower than that of other drug-treated group. The tumor from Ab-PSLs with gem 160 mg/kg-injected group exhibited a markedly lowest account of PCNA labeled cells and had highest TUNEL-positive cells among tested. This suggests that treatment of Ab-PSLs with gem resulted in an increased apoptosis of tumor cells, leading to tumor growth inhibition. These results demonstrate that PSLs provide an efficient and targeted delivery of gemcitabine and may represent a useful new treatment approach for tumors which overexpress the EGFR.

  11. Activation of the phosphatidylinositol 3-kinase/Akt pathway is involved in lipocalin-2-promoted human pulmonary artery smooth muscle cell proliferation.

    PubMed

    Wang, Guoliang; Ma, Ning; Meng, Liukun; Wei, Yingjie; Gui, Jingang

    2015-12-01

    Over-activated PI3K/Akt signaling, a pathway strongly related to cancer survival and proliferation, has been reported recently to be involved in pulmonary artery smooth muscle cell apoptosis and proliferation in pulmonary hypertension (PH). In this study, we observed greatly increased lipocalin-2 (Lcn2) expression accompanied with over-activated PI3K/Akt signaling in a standard rat model of PH induced by monocrotaline. In view of the close relationship between Lcn2 and PI3K/Akt pathway, we hypothesized that the up-regulated Lcn2 might be a trigger of over-activated PI3K/Akt signaling in PH. Our results showed that Lcn2 significantly activated the PI3K/Akt pathway (determined by augmented Akt phosphorylation and up-regulated Mdm2) and significantly promoted proliferation (assessed by Ki67 staining) in cultured human pulmonary artery smooth muscle cells. Furthermore, we demonstrated that inhibition of Akt phosphorylation (LY294002) abrogated the Lcn2-promoted proliferation in cultured human pulmonary artery smooth muscle cells. In conclusion, Lcn2 significantly promoted human pulmonary artery smooth muscle cell proliferation by activating PI3K/Akt pathway. Further study on the role and mechanism of Lcn2 will help explore novel therapeutic strategies based on attenuating over-activated PI3K/Akt signaling in PH.

  12. Multi-walled carbon nanotube length as a critical determinant of bioreactivity with primary human pulmonary alveolar cells

    PubMed Central

    Sweeney, Sinbad; Berhanu, Deborah; Misra, Superb K.; Thorley, Andrew J.; Valsami-Jones, Eugenia; Tetley, Teresa D.

    2015-01-01

    Multiwalled carbon nanotube (MWCNT) length is suggested to critically determine their pulmonary toxicity. This stems from in vitro and in vivo rodent studies and in vitro human studies using cell lines (typically cancerous). There is little data using primary human lung cells. We addressed this knowledge gap, using highly relevant, primary human alveolar cell models exposed to precisely synthesized and thoroughly characterized MWCNTs. In this work, transformed human alveolar type-I-like epithelial cells (TT1), primary human alveolar type-II epithelial cells (ATII) and alveolar macrophages (AM) were treated with increasing concentrations of MWCNTs before measuring cytotoxicity, inflammatory mediator release and MAP kinase signalling. Strikingly, we observed that short MWCNTs (~0.6 µm in length) induced significantly greater responses from the epithelial cells, whilst AM were particularly susceptible to long MWCNTs (~20 µm). These differences in the pattern of mediator release were associated with alternative profiles of JNK, p38 and ERK1/2 MAP kinase signal transduction within each cell type. This study, using highly relevant target human alveolar cells and well defined and characterized MWCNTs, shows marked cellular responses to the MWCNTs that vary according to the target cell type, as well as the aspect ratio of the MWCNT. PMID:25780270

  13. Cyto-genotoxic effects of smoke from commercial filter and non-filter cigarettes on human bronchial and pulmonary cells.

    PubMed

    Cavallo, Delia; Ursini, Cinzia L; Fresegna, Anna M; Maiello, Raffaele; Ciervo, Aureliano; Ferrante, Riccardo; Buresti, Giuliana; Iavicoli, Sergio

    2013-01-20

    Cigarette smoke is a complex mixture of chemicals, some of which are known as carcinogens. The cyto-genotoxic effects of cigarette-smoke extract (CSE) from commercial cigarettes without (A and B) and with filter (C and D) were evaluated at different CSE concentrations on A549 and BEAS-2B cells. The particle content of the cigarette smoke and the metal composition of the CSE were also analyzed. The cells were exposed to 1-10% of the CSE from one cigarette per experiment. Cytotoxicity was evaluated by use of the MTT assay after 24h, and the lactate dehydrogenase (LDH) assay after 30min and 24h. The Fpg-modified comet assay was used to evaluate direct-oxidative DNA damage on cells exposed for 30min. As expected, unfiltered cigarette smoke (particularly from the B cigarette) contained a higher number of particles than filtered smoke. With smoke extract from the B cigarette we found a decrease in cell viability only in BEAS-2B cells. The results of the LDH test showed membrane damage for B-cigarette smoke extract, particularly in BEAS-2B cells. Extracts from unfiltered cigarette smoke induced significant direct DNA damage, to a larger extent in A549 cells. Filtered cigarette-smoke extract induced a significant direct DNA damage at 5-10%. A significant induction of oxidative DNA damage was found at the highest CSE concentration in both cell types (by smoke extracts from B and C cigarettes in A549 cells, and from A and D cigarettes in BEAS-2B cells). Smoke extracts from filter cigarettes induced less direct DNA damage than those from unfiltered cigarettes in A549 cells, probably due to a protective effect of filter. In BEAS-2B cells the smoke extract from the B-cigarette showed the highest genotoxic effect, with a concentration-dependent trend. These findings show a higher cyto-genotoxicity for smoke extracts from the B-cigarette and oxidative effects for those from the A and D cigarettes, particularly in BEAS-2B cells. Moreover, there was a higher responsiveness of A549

  14. Eradication of Human Hepatic and Pulmonary Melanoma Metastases in SCID Mice by Antibody--Interleukin 2 Fusion Proteins

    NASA Astrophysics Data System (ADS)

    Becker, Jurgen C.; Pancook, James D.; Gillies, Stephen D.; Mendelsohn, John; Reisfeld, Ralph A.

    1996-04-01

    Antibody--cytokine fusion proteins combine the unique targeting ability of antibodies with the multifunctional activity of cytokines. Here, we demonstrate the therapeutic efficacy of such constructs for the treatment of hepatic and pulmonary metastases of different melanoma cell lines. Two antibody--interleukin 2 (IL-2) fusion proteins, ch225-IL2 and ch14.18-IL2, constructed by fusion of a synthetic sequence coding for human IL-2 to the carboxyl end of the Cγ 1 gene of the corresponding antibodies, were tested for their therapeutic efficacy against xenografted human melanoma in vivo. Tumorspecific fusion proteins completely inhibited the growth of hepatic and pulmonary metastases in C.B-17 scid/scid mice previously reconstituted with human lymphokine-activated killer cells, whereas treatment with combinations of the corresponding antibodies plus recombinant IL-2 only reduced the tumor load. Even when treatment with fusion proteins was delayed up to 8 days after inoculation of tumor cells, it still resulted in complete eradication of micrometastases that were established at that time point. Selection of tumor cell lines expressing or lacking the targeted antigen of the administered fusion protein proved the specificity of the observed antitumor effect. Biodistribution analysis demonstrated that the tumorspecific fusion protein accumulated not only in subcutaneous tumors but also in lungs and livers affected with micrometastases. Survival times of animals treated with the fusion protein were more than doubled as compared to those treated with the combination of the corresponding antibody plus IL-2. Our data demonstrate that an immunotherapeutic approach using cytokines targeted by antibodies to tumor sites has potent effects against disseminated human melanoma.

  15. cDNA and deduced amino acid sequence of human pulmonary surfactant-associated proteolipid SPL(Phe)

    SciTech Connect

    Glasser, S.W.; Korfhagen, T.R.; Weaver, T.; Pilot-Matias, T.; Fox, J.L.; Whitsett, J.A.

    1987-06-01

    Hydrophobic surfactant-associated protein of M/sub r/ 6000-14,000 was isolated from either/ethanol or chloroform/methanol extracts of mammalian pulmonary surfactant. Automated Edman degradation in a gas-phase sequencer showed the major N-terminus of the human low molecular weight protein to be Phe-Pro-Ile-Pro-Leu-Pro-Try-Cys-Trp-Leu-Cys-Arg-Ala-Leu-. Because of the N-terminal phenylalanine, the surfactant protein was designated SPL(Phe). Antiserum generated against hydrophobic surfactant protein(s) from bovine pulmonary surfactant recognized protein of M/sub r/ 6000-14,000 in immunoblot analysis and was used to screen a lambdagt11 expression library constructed from adult human lung poly(A)/sup +/ RNA. This resulted in identification of a 1.4-kilobase cDNA clone that was shown to encode the N-terminus of the surfactant polypeptide SPL(Phe) (Phe-Pro-Ile-Pro-Leu-Pro-) within an open reading frame for a larger protein. Expression of a fused ..beta..-galactosidase-SPL (Phe) gene in Escherichia coli yielded an immunoreactive M/sub r/ 34,000 fusion peptide. Hybrid-arrested translation with the cDNA and immunoprecipitation of (/sup 35/S)methionine-labeled in vitro translation products of human poly(A)/sup +/ RNA with a surfactant polyclonal antibody resulted in identification of a M/sub r/ 40,000 precursor protein. Blot hybridization analysis of electrophoretically fractionated RNA from human lung detected a 2.0-kilobase RNA that was more abundant in adult lung than in fetal lung. These proteins, and specifically SPL(Phe), may therefore be useful for synthesis of replacement surfactants for treatment of hyaline membrane disease in newborn infants or of other surfactant-deficient states.

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

    PubMed

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

    2016-04-01

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

  17. The production of reactive oxygen species and the mitochondrial membrane potential are modulated during onion oil-induced cell cycle arrest and apoptosis in A549 cells.

    PubMed

    Wu, Xin-jiang; Stahl, Thorsten; Hu, Ying; Kassie, Fekadu; Mersch-Sundermann, Volker

    2006-03-01

    Protective effects of Allium vegetables against cancers have been shown extensively in experimental animals and epidemiologic studies. We investigated cell proliferation and the induction of apoptosis by onion oil extracted from Allium cepa, a widely consumed Allium vegetable, in human lung cancer A549 cells. GC/MS analysis suggested that propyl sulfides but not allyl sulfides are major sulfur-containing constituents of onion oil. Onion oil at 12.5 mg/L significantly induced apoptosis (13% increase of apoptotic cells) as indicated by sub-G1 DNA content. It also caused cell cycle arrest at the G2/M phase; 25 mg/L onion oil increased the percentage of G2/M cells almost 6-fold compared with the dimethyl sulfoxide control. The action of onion oil may occur via a reactive oxygen species-dependent pathway because cell cycle arrest and apoptosis were blocked by the antioxidants N-acetylcysteine and exogenous glutathione. Marked collapse of the mitochondrial membrane potential suggested that dysfunction of the mitochondria may be involved in the oxidative burst and apoptosis induced by onion oil. Expression of phospho-cdc2 and phospho-cyclin B1 were downregulated by onion oil, perhaps accounting for the G2/M arrest. Overall, these results suggest that onion oil may exert chemopreventive action by inducing cell cycle arrest and apoptosis in tumor cells.

  18. MicroRNA profiling of Sendai virus-infected A549 cells identifies miR-203 as an interferon-inducible regulator of IFIT1/ISG56.

    PubMed

    Buggele, William A; Horvath, Curt M

    2013-08-01

    The mammalian type I interferon (IFN) response is a primary barrier for virus infection and is essential for complete innate and adaptive immunity. Both IFN production and IFN-mediated antiviral signaling are the result of differential cellular gene expression, a process that is tightly controlled at transcriptional and translational levels. To determine the potential for microRNA (miRNA)-mediated regulation of the antiviral response, small-RNA profiling was used to analyze the miRNA content of human A549 cells at steady state and following infection with the Cantell strain of Sendai virus, a potent inducer of IFN and cellular antiviral responses. While the miRNA content of the cells was largely unaltered by infection, specific changes in miRNA abundance were identified during Sendai virus infection. One miRNA, miR-203, was found to accumulate in infected cells and in response to IFN treatment. Results indicate that miR-203 is an IFN-inducible miRNA that can negatively regulate a number of cellular mRNAs, including an IFN-stimulated gene target, IFIT1/ISG56, by destabilizing its mRNA transcript.

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

    PubMed

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

    2014-10-05

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

  20. Activation of p53/miR-34a Tumor Suppressor Axis by Chinese Herbal Formula JP-1 in A549 Lung Adenocarcinoma Cells

    PubMed Central

    Chow, Jyh-Ming; Lin, Pei-Chun; Hu, Tsai-Shu; Kuo, Hui-Ching; Huang, Jhy-Shrian

    2016-01-01

    Lung cancer is the leading cause of cancer death worldwide; the most common pathologic type is lung adenocarcinoma (LADC). In spite of the recent progress in targeted therapy, most LADC patients eventually expired due to the inevitable recurrence and drug resistance. New complementary agent with evidence-based molecular mechanism is urgently needed. MiR-34a is an important p53 downstream tumor suppressor, which regulates apoptosis, cell-cycle, EMT (epithelial mesenchymal transition), and so forth. Its expression is deficient in many types of cancers including LADC. Here, we show that a Chinese herbal formula JP-1 activates p53/miR-34a axis in A549 human LADC cells (p53 wild-type). Treatment with JP-1 induces p53 and its downstream p21 and BAX proteins as well as the miR-34a, resulting in growth inhibition, colony formation reduction, migration repression, and apoptosis induction. Accordingly, the decreases of miR-34a downstream targets such as CDK6, SIRT1, c-Myc, survivin, Snail, and AXL were observed. Moreover, JP-1 activates AMPKα and reduces mTOR activity, implying its inhibitory effect on the energy-sensiti