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Sample records for a549 cells proteomic

  1. [Comparative Proteomic Analysis of Human Lung Adenocarcinoma Cisplatin-resistant Cell Strain A549/CDDP.].

    PubMed

    Shi, Sien; Wang, Jianjun; Zhai, Wei; Gao, Guizhou; Tang, Jian; Fan, Kai; Tang, Zheng; Wei, Liang

    2009-11-20

    Chemotherapy plays an important role in the comprehensive therapy of lung cancer. However, the drug-resistance often causes the failure of the chemotherapy. The aim of this study is to identify differently expressed protein before and after cisplatin resistance of human lung adenocarcinoma cell A549 by proteomic analysis. Cisplatin-resistant cell strain A549/CDDP was established by combining gradually increasing concentration of cisplatin with large dosage impact. Comparative proteomic analysis of A549 and A549/CDDP were carried out by means of two-dimensional gel electrophoresis. The differentially expressed proteins were detected and identified by MALDI-TOF mass spectrometry. Eighty-two differentially expressed proteins were screened by analysis the electrophoretic maps of A549 and A549/CDDP. Six differential proteins were analyzed by peptide mass fingerprinting. Glucose regulating protein 75, ribosomal protein S4, mitochondrial ATP synthase F1 complex beta subunit and immunoglobulin heavy chain variable region were identified. All four differentially expressed proteins were over-expressed in A549/CDDP, whereas low-expressed or no-expressed in A549. These differentially expressed proteins give some clues to elucidate the mechanism of lung cancer cell resistant of cisplatin, providing the basis of searching for potential target of chemotherapy of lung cancer.

  2. SAHA treatment reveals the link between histone lysine acetylation and proteome in nonsmall cell lung cancer A549 Cells.

    PubMed

    Wu, Quan; Xu, Weiqing; Cao, Lejie; Li, Xin; He, Tieming; Wu, Zhiwei; Li, Wenting

    2013-09-06

    Suberoylanilide hydroxamic acid (SAHA) is a well-known pan HDAC inhibitor, and its clinical application (Vorinostat) has been demonstrated to treat nonsmall cell lung cancer (NSCLS). Nevertheless, the impact of SAHA treatment on histone lysine acetylation and proteome in NSCLS cells still need further elucidate. In NSCLS A549 cells, by using stable isotope labeling for cell culture (SILAC)-based quantitative proteomics, biochemistry assay, and bioinformatic analysis, here we for the first time comprehensively identified and quantified histone lysine acetylation in A549 cells toward SAHA treatment. Despite the fact that SAHA treatment significantly increased histone lysine acetylation in specific sites, unexpectedly, some important "histone markers" showed markedly decreased acetylation level. Further quantitative proteome studies showed that among totally quantifiable 2818 nonredundant proteins, 1355 proteins were with increased level and 1463 with decreased level in response to SAHA treatment. Bioinformatic analysis further revealed that those quantifiable proteins were mainly involved in multiple biological functions and metabolic and enzyme-regulated pathways as well as protein complexes. By establishing the link between histone modification and whole proteome in response to SAHA treatment in NSCLS cells, this study therefore may deepen our understanding of HDAC inhibitor-mediated cancer therapeutics.

  3. 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

  4. 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

  5. Effects of Green Tea Extract on Lung Cancer A549 Cells: Proteomic Identification of Proteins Associated with Cell Migration

    PubMed Central

    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

    2009-01-01

    Green tea polyphenols exhibit multiple anti-tumor 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 two-dimensional gel electrophoresis LC-tandem mass spectrometry of A549 cells before and after GTE exposure. We have identified 14 protein spots that changed in expression (≥2 fold) after GTE treatment. These proteins are involved in calcium-binding, cytoskeleton and motility, metabolism, detoxification or gene regulation. In particular we found up-regulation of several genes that modulate actin remodeling and cell migration, including lamin A/C. Our data indicated that GTE-induced lamin A/C up-regulation appears to be at the transcriptional level and the increased expression results in the decrease in the 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 anti-tumor activities of GTE. PMID:19137550

  6. Proteomic analysis of selective cytotoxic anticancer properties of flavonoids isolated from Citrus platymamma on A549 human lung cancer cells.

    PubMed

    Nagappan, Arulkumar; Venkatarame Gowda Saralamma, Venu; Hong, Gyeong Eun; Lee, Ho Jeong; Shin, Sung Chul; Kim, Eun Hee; Lee, Won Sup; Kim, Gon Sup

    2016-10-01

    Citrus platymamma Hort. ex Tanaka (Byungkyul in Korean) has been used in Korean folk medicine for the treatment of inflammatory disorders and cancer. However, the molecular mechanism underlying the anticancer properties of flavonoids isolated from C. platymamma (FCP) remains to be elucidated. Therefore, the present study attempted to identify the key proteins, which may be important in the anticancer effects of FCP on A549 cells using a proteomic approach. FCP showed a potent cytotoxic effect on the A549 human lung cancer cells, however, it had no effect on WI‑38 human fetal lung fibroblasts at the same concentrations. Furthermore, 15 differentially expressed protein spots (spot intensities ≥2‑fold change; P<0.05) were obtained from comparative proteome analysis of two‑dimensional gel electrophoresis maps of the control (untreated) and FCP‑treated A549 cells. Finally, eight differentially expressed proteins, one of which was upregulated and seven of which were downregulated, were successfully identified using matrix‑assisted laser desorption/ionization time‑of‑flight/time‑of‑flight tandem mass spectrometry and peptide mass fingerprinting analysis. Specifically, proteins involved in signal transduction were significantly downregulated, including annexin A1 (ANXA1) and ANXA4, whereas 14‑3‑3ε was upregulated. Cytoskeletal proteins, including cofilin‑1 (CFL1), cytokeratin 8 (KRT8) and KRT79, and molecular chaperones/heat shock proteins, including endoplasmin, were downregulated. Proteins involved in protein metabolism, namely elongation factor Ts were also downregulated. Consistent with results of the proteome analysis, the immunoblotting results showed that 14‑3‑3ε was upregulated, whereas CFL1, ANXA4 and KRT8 were downregulated in the FCP‑treated A549 cells. The majority of the proteins were involved in tumor growth, cell cycle, apoptosis, migration and signal transduction. These findings provide novel insights into the molecular

  7. 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

  8. Combination therapy in A549 cells.

    PubMed

    Yuan, Menghui; Wang, Jing; Deng, Jinglan; Wang, Zhe; Yang, Weidong; Li, Guoquan; Ren, Bingxiu

    2010-04-01

    We investigated the anti-tumor effect induced by the combination of the radiotherapeutic agent (131)I-RC-160 and the prodrug 5-FC in human non-small cell lung cancer (NSCLC) A549 cells that were co-expressing the human somatostatin receptor 2 gene (hSSTR2) and E. coli cytosine deaminase gene (CD). We cloned both hSSTR2 and CD into a bicistronic mammalian expression plasmid and stably transfected it into A549 cells (pCIS-A549 cells). After antibiotic selection, SSTR expression in stable clones was determined by reverse transcription and polymerase chain reaction (RT-PCR), Western blot, flow cytometry and immunofluorescence analyses. To assess the in vivo targeting efficiency of the "engineered" A549 cells, the cells were subcutaneously injected into nude mice and the biodistribution of (99m)Tc-RC-160 was assessed at different time points. The tumor inhibitory effects of (131)I-RC-160 and/or 5-FC were evaluated by measurement of tumor growth and immunohistochemical analysis. Multiple analyses demonstrated the successful expression of hSSTR2 in A549 cells. In vivo radioimaging revealed specific targeting of RC-160 to the tumors derived from pCIS-A549 cells when compared to those from control A549 cells. The tumor inhibitory rate of pCIS-A549 tumors in the (131)I-RC-160 plus 5-FC-treated group was significantly higher than that in the single agent-treated group, control group and control tumors. Co-expression of the hSSTR2 and CD genes in tumor cells can selectively sensitize these cells to the infra-additive effects of radioisotope-labeled RC-160 and 5-FC in vivo. This approach offers a potential therapeutic strategy for the treatment of lung cancer. Crown Copyright 2010. Published by Elsevier Inc. All rights reserved.

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

    PubMed

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

    2014-05-01

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

  10. 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.

  11. 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.

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

    PubMed

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

    2017-07-01

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

  13. [Radiosensitizing effect of erlotinib on human lung adenocarcinoma cell line A549].

    PubMed

    Liu, Xiao-qun; Qiao, Tian-kui

    2013-11-01

    To explore the radiosensitizing effect of erlotinib on human lung adenocarcinoma cell line A549 cells and the related mechanisms. The inhibitory effect of erlotinib on A549 cells was assessed by MTT assay, and its IC50 concentration was calculated. The radiosensitization was evaluated by the method of clone forming assay. Flow cytometry was used to analyze the effect of erlotinib on cell cycle and apoptosis. The growth of A549 cells was inhibited after the cells were exposed to erlotinib for 48 hours. Moreover, the inhibitory rates increased with the increase of erlotinib concentrations, and IC50 was 19.26 µmol/L. In contrast to the irradiation alone group, the survival rates of the cells in erlotinib plus irradiation groups decreased, and erlotinib enhanced the radiosensitivity of the A549 cells. This effect was further increased as cells were exposed to erlotinib for a longer time. In the irradiation alone group and the two groups exposed to erlotinib for 24 hours and 48 hours before irradiation, D0 values were 3.01 Gy, 2.58 Gy and 2.45 Gy respectively, and Dq values were 2.16 Gy, 1.94 Gy and 1.61 Gy, respectively. In the last two groups, SERD0 values were 1.17 and 1.23, respectively. The flow cytometry analysis showed that erlotinib induced G2/M phase arrest and increased the apoptosis rate in A549 cells. With the increase of exposure time, the effects were more significant. Erlotinib inhibits the A549 cell growth and enhances the radiosensitivity of A549 cells in vitro. The radiosensitizing mechanisms might be related to inhibiting repair of sublethal injury and inducing G2/M phase arrest and apoptosis.

  14. Endotoxin potency in the A549 lung epithelial cell bioassay and the limulus amebocyte lysate assay.

    PubMed

    Hansen, L A; Poulsen, O M; Würtz, H

    1999-06-24

    The purpose of this study was to elucidate to what extent the potency of endotoxins measured by the limulus amebocyte lysate (LAL) assay is reflected in the potency in an in vitro assay based on release of interleukin-8 (IL-8) from a lung epithelial cell line, A549. Lipopolysaccharides (LPS) from Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella enteritidis and detoxified LPS from E. coli were applied in serial dilutions in the LAL assay and in the A549 bioassay. Also 19 organic dust samples from waste recycling plants were tested. The A549 cells were incubated for 24 h with LPS or dust, and the IL-8 secretion was determined by ELISA. The method for evaluation of the LAL assay showed linearity for the four endotoxins. Using the slope as a measure of the potency factor (PF), LPS from E. coli and S. enteritidis was about four times more potent than that for LPS from K. pneumoniae and P. aeruginosa. In the A549 bioassay each of the different types of endotoxin had characteristic and very different dose-response curves. The potency of the LPS, in the A549 bioassay, ranked as follows K. pneumoniae > P. aeruginosa > E. coli > or = S. enteritidis. The content of endotoxin in the dust samples did not correlate with their potency in the A549 bioassay. The present study indicates a poor correlation between the potency of endotoxin in the LAL assay compared with the A549 bioassay. The lack of correlation when organic dust samples are tested may reflect the fact that these samples contain biological active compounds, which are non-reactive in the LAL-assay but stimulate IL-8 secretion from epithelial cells.

  15. 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.

  16. PARTICULATE MATTER (PM) INHIBITS NEUROTROPHIN RELEASE FROM A549 CELLS

    EPA Science Inventory

    Several investigations have linked PM exposure to the exacerbation of allergic lung diseases. Many PM effects are mediated by cells within the lung including the airway epithelium, eosinophils, and lymphocytes. These cells also produce neurotophins such as NGF and/or express neur...

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

    PubMed

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

    2018-02-01

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

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

    SciTech Connect

    Huang, Chunrong; Zheng, Haichong; He, Wanmei

    2016-05-20

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

  19. 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.

  20. 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. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

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

    PubMed

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

    2015-01-01

    To investigate the influence of exogenous p53 upregulated modulator of apoptosis (PUMA) expression on cell proliferation and apoptosis in human non-small cell lung cancer A549 cells and transplanted tumor cell growth in nude mice. A549 cells were divided into the following groups: control, non- carrier (NC), PUMA (transfected with pCEP4- (HA) 2-PUMA plasmid), DDP (10 μg/mL cisplatin treatment) and PUMA+DDP (transfected with pCEP4-(HA)2-PUMA plasmid and 10 μg/mL cisplatin treatment). The MTT method was used to detect the cell survival rate. Cell apoptosis rates were measured by flow cytometry, and PUMA, Bax and Bcl-2 protein expression levels were measured by Western blotting. Compared to the control group, the PUMA, DDP and PUMA+DDP groups all had significantly decreased A549 cell proliferation (p<0.01), with the largest reduction in the PUMA+DDP group. Conversely, the apoptosis rates of the three groups were significantly increased (P<0.01), and the PUMA and DDP treatments were synergistic. Moreover, Bax protein levels significantly increased (p<0.01), while Bcl-2 protein levels significantly decreased (p<0.01). Finally, both the volume and the weights of transplanted tumors were significantly reduced (p<0.01), and the inhibition ratio of the PUMA+DDP group was significantly higher than in the single DDP or PUMA groups. Exogenous PUMA effectively inhibited lung cancer A549 cell proliferation and transplanted tumor growth by increasing Bax protein levels and reducing Bcl-2 protein levels.

  2. Selective killing effect of oxytetracycline, propafenone and metamizole on A549 or Hela cells

    PubMed Central

    Feng, Guihua

    2013-01-01

    Objective To determine the selective killing effect of oxytetracycline, propafenone and metamizole on A549 or Hela cells. Methods Proliferation assay, lactate dehydrogenase (LDH) assay, apoptosis detecting, flow cytometry and western blot were performed. Results It was found that treatment with propafenone at the concentration of 0.014 g/L or higher for 48 h could induce apoptosis in Hela cells greatly, while it was not observed in oxytetracycline and metamizole at the concentration of 0.20 g/L for 48 h. Oxytetracycline, propafenone and metamizole all displayed evident inhibitory effects on the proliferation of A549 cells. The results of LDH assay demonstrated that the drugs at the test range of concentration did not cause necrosis in the cells. Propafenone could elevate the protein level of P53 effectively (P<0.01). Conclusions Oxytetracycline, propafenone and metamizol (dipyrone) all displayed evident inhibitory effects on the proliferation of A549 cells. Propafenone also displayed evident inhibitory effects on the proliferation of Hela cells. PMID:24385693

  3. [Expression of MADD in lung adenocarcinoma tissues and its effects on proliferation and apoptosis of lung adenocarcinoma A549 cells].

    PubMed

    Wei, Yu-Ping; Wu, Jin-Xiang; Guo, Yuan-Fang; Sun, Gao-Ying; Zhang, Qiang; Bi, Wen-Xiang; Dong, Liang

    2012-04-01

    To investigate the expression of MAPK-activating death domain protein (MADD) in lung adenocarcinoma tissues and its effects on proliferation and apoptosis of lung adenocarcinoma A549 cells. Immunohistochemistry was used to detect the expression of MADD in lung normal and tumor tissues. The expression of IG20 gene in A549 cells was measured by reverse transcription polymerase chain reaction. A549 cells were transfected with pEYFP-MADD plasmids carrying MADD gene or pNL-SIN-GFP-MID lentiviral vectors used for RNA interference. MADD expression and cell proliferation and apoptosis were determined by Western blot, MTT assay, and flow cytometry. The expression levels of MADD were higher in lung adenocarcinoma and squamous cell carcinoma tissues than that in lung normal tissues, and lung adenocarcinoma tissues expressed more MADD than lung squamous cell carcinoma tissues. The transcript encoding MADD was expressed in A549 cells. The transfection of pEYFP-MADD plasmids could increase MADD expression and cell proliferation of A549 cells, while the A549 cells transfected with pNL-SIN-GFP-MID lentiviral vectors showed significantly decreases in the MADD level and proliferation. It is shown that MADD overexpression could inhibit A549 cell apoptosis, and knock down of MADD could promote apoptosis of them. The expression of MADD increases obviously in lung adenocarcinoma, and MADD can promote survival of lung adenocarcinoma cells by inhibiting apoptosis.

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

    PubMed

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

    2012-08-01

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

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

    PubMed

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

    2017-03-01

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

  6. 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. Copyright © 2011 John Wiley & Sons, Ltd.

  7. 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. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

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

    PubMed

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

    2017-06-01

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

  9. 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.

  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. Inhibition of Raf-MEK-ERK and Hypoxia pathways by Phyllanthus prevents metastasis in human lung (A549) cancer cell line

    PubMed Central

    2013-01-01

    Background Lung cancer constitutes one of the malignancies with the greatest incidence and mortality rates with 1.6 million new cases and 1.4 million deaths each year. Prognosis remains poor due to deleterious development of multidrug resistance resulting in less than 15% lung cancer patients reaching five years survival. We have previously shown that Phyllanthus induced apoptosis in conjunction with its antimetastastic action. In the current study, we aimed to determine the signaling pathways utilized by Phyllanthus to exert its antimetastatic activities. Methods Cancer 10-pathway reporter array was performed to screen the pathways affected by Phyllanthus in lung carcinoma cell line (A549) to exert its antimetastatic effects. Results from this array were then confirmed with western blotting, cell cycle analysis, zymography technique, and cell based ELISA assay for human total iNOS. Two-dimensional gel electrophoresis was subsequently carried out to study the differential protein expressions in A549 after treatment with Phyllanthus. Results Phyllanthus was observed to cause antimetastatic activities by inhibiting ERK1/2 pathway via suppression of Raf protein. Inhibition of this pathway resulted in the suppression of MMP2, MMP7, and MMP9 expression to stop A549 metastasis. Phyllanthus also inhibits hypoxia pathway via inhibition of HIF-1α that led to reduced VEGF and iNOS expressions. Proteomic analysis revealed a number of proteins downregulated by Phyllanthus that were involved in metastatic processes, including invasion and mobility proteins (cytoskeletal proteins), transcriptional proteins (proliferating cell nuclear antigen; zinc finger protein), antiapoptotic protein (Bcl2) and various glycolytic enzymes. Among the four Phyllanthus species tested, P. urinaria showed the greatest antimetastatic activity. Conclusions Phyllanthus inhibits A549 metastasis by suppressing ERK1/2 and hypoxia pathways that led to suppression of various critical proteins for A549

  12. Inhibition of Raf-MEK-ERK and hypoxia pathways by Phyllanthus prevents metastasis in human lung (A549) cancer cell line.

    PubMed

    Lee, Sau Har; Jaganath, Indu Bala; Manikam, Rishya; Sekaran, Shamala Devi

    2013-10-20

    Lung cancer constitutes one of the malignancies with the greatest incidence and mortality rates with 1.6 million new cases and 1.4 million deaths each year. Prognosis remains poor due to deleterious development of multidrug resistance resulting in less than 15% lung cancer patients reaching five years survival. We have previously shown that Phyllanthus induced apoptosis in conjunction with its antimetastastic action. In the current study, we aimed to determine the signaling pathways utilized by Phyllanthus to exert its antimetastatic activities. Cancer 10-pathway reporter array was performed to screen the pathways affected by Phyllanthus in lung carcinoma cell line (A549) to exert its antimetastatic effects. Results from this array were then confirmed with western blotting, cell cycle analysis, zymography technique, and cell based ELISA assay for human total iNOS. Two-dimensional gel electrophoresis was subsequently carried out to study the differential protein expressions in A549 after treatment with Phyllanthus. Phyllanthus was observed to cause antimetastatic activities by inhibiting ERK1/2 pathway via suppression of Raf protein. Inhibition of this pathway resulted in the suppression of MMP2, MMP7, and MMP9 expression to stop A549 metastasis. Phyllanthus also inhibits hypoxia pathway via inhibition of HIF-1α that led to reduced VEGF and iNOS expressions. Proteomic analysis revealed a number of proteins downregulated by Phyllanthus that were involved in metastatic processes, including invasion and mobility proteins (cytoskeletal proteins), transcriptional proteins (proliferating cell nuclear antigen; zinc finger protein), antiapoptotic protein (Bcl2) and various glycolytic enzymes. Among the four Phyllanthus species tested, P. urinaria showed the greatest antimetastatic activity. Phyllanthus inhibits A549 metastasis by suppressing ERK1/2 and hypoxia pathways that led to suppression of various critical proteins for A549 invasion and migration.

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

    PubMed

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

    2015-05-01

    Studying different concentrations of nickel smelting smoke subjects of human lung adenocarcinoma cells (A549) carcinogenic effects, discusses the influence of L-ascorbic acid protection. The A549 cells were divided into experimental and L-ascorbic acid in the intervention group. Plus exposure group concentration of nickel refining dusts were formulated 0.00, 6.25, 12.50, 25.00, 50.00, 100.00 µg/ml suspension, the intervention group on the basis of the added exposure group containing L-ascorbic acid (100 mmol/L), contact 24 h. Detection of cell viability by MTT assay. When the test substance concentration select 0.00, 25.00, 50.00, 100.00 µg/ml experiment for internal Flou-3 fluorescent probe to detect cell Ca²⁺ concentration, within DCFH-DA detect intracellular reactive oxygen (ROS) content, real-time quantitative PCR (real time, in the RT-PCR) was used to detect cell HIF-1α gene expression. With the increase of concentration, subjects increased cell growth inhibition rate, intracellular Ca²⁺ concentration increases, ROS content increased, HIF-1α gene expression increased, differences were statistically significant (P < 0.05). After L-ascorbic acid intervention treatment, the results of the intervention group were lower than that of the experimental group, and the difference was statistically significant (P < 0.05), so L-ascorbic acid can effectively protect the nickel exposure damage to cells. With subjects following exposure to nickel concentration increased, its effect on A549 cell damage increases, L-ascorbic acid cell damage caused by nickel has certain protective effect.

  14. Adiponectin down-regulates CREB and inhibits proliferation of A549 lung cancer cells.

    PubMed

    Illiano, Michela; Nigro, Ersilia; Sapio, Luigi; Caiafa, Ilaria; Spina, Annamaria; Scudiero, Olga; Bianco, Andrea; Esposito, Sabrina; Mazzeo, Filomena; Pedone, Paolo Vincenzo; Daniele, Aurora; Naviglio, Silvio

    2017-08-01

    Adipokines are known to play a relevant role in a number of cancer related molecular pathways. Adiponectin is a major adipokine with anti-inflammatory and beneficial metabolic actions. Furthermore, it has been shown to exert anti-carcinogenic effects in various tumor models and some clinical studies suggested an inverse relationship between circulating levels of adiponectin and an increased risk for development of malignancies. On the other hand, the cyclic AMP response element binding (CREB) transcription factor has been clearly linked to lung cancer. we analyzed cell proliferation, cell cycle of A549 cells treated with adiponectin as well as CREB activation status in human lung adenocarcinoma A549 cells and in non-small cell lung cancer (NSCLC) samples. adiponectin treatment, at concentrations ranging between 5 and 50 μg/ml mimicking human serum levels, has a significant effect on reducing tumor cell proliferation of A549 cells, mainly by altering cell cycle progression. Importantly, we provide evidence that adiponectin clearly inhibits in a dose- and time-dependent manner CREB phosphorylation (activation) and, at least in part, also the level of CREB protein itself, preceding and accompanying the anti-proliferative effects in response to adiponectin. Moreover, in agreement with previous studies demonstrating that CREB over-expression occurs in many tumors, we also show by western-blotting from lung specimen that CREB is significantly up-regulated in NSCLC samples compared to adjacent normal tissues from six patients. Overall, our results represent the first evidence of CREB inhibition by adiponectin and may provide new insight into therapeutic strategies for lung cancer. Copyright © 2017. Published by Elsevier Ltd.

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

    PubMed

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

    2015-01-01

    A series of structurally unique second mitochondria-derived activators of caspase (Smac) that act as antagonists of inhibitor of apoptosis proteins (IAPs) directly have been discovered and have been shown to promote chemotherapy-induced apoptosis. In this study, we investigate the role of Smac in Taxol-induced apoptosis of lung cancer cell in vitro. PcDNA3.1/Smac recombinants were transfected into the non-small cell lung cancer cell line A549. Smac expression was detected by RT-PCR and Western blot. The invasive ability of cells was examined. Flow cytometry was used to analyze apoptosis of cells induced by Taxol with Annexin V/PI double staining technique. Smac expression was significantly higher in the PcDNA3.1/Smac recombinant group than in the untransfected group at mRNA and protein level (p < 0.05) and lower invasion through a basal membrane was apparent after transfection (p < 0.05). A small number of cells were promoted to apoptosis in the PcDNA3.1/Smac group. There were significant differences compared to the empty vector group and control group. The apoptosis rate was significantly higher in PcDNA3.1/Smac + Taxol group than in other groups (p < 0.05). Transfected Smac can enhance the chemosensitivity of the non-small cell lung cancer cell line A549 to Taxol.

  16. [Astaxanthin inhibits proliferation and promotes apoptosis of A549 lung cancer cells via blocking JAK1/STAT3 pathway].

    PubMed

    Wu, Chuntao; Zhang, Jinji; Liu, Tienan; Jiao, Guimei; Li, Changzai; Hu, Baoshan

    2016-06-01

    Objective To investigate the anti-tumor effects of astaxanthin on A549 lung cancer cells and the related mechanisms. Methods A549 cells were cultured with various concentrations of astaxanthin (20, 40, 60, 80, 100 μmol/L), and DMSO at the same concentrations served as vehicle controls. The viability of A549 cells was detected by CCK-8 assay; cell cycle and apoptosis were observed by flow cytometry; and the expressions of B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), signal transducers and activators of transcription 3 (STAT3), and Janus kinase 1 (JAK1) were evaluated by Western blotting. Results CCK-8 assay showed that astaxanthin decreased the proliferation of A549 cells in a dose-dependent manner. Flow cytometry showed that astaxanthin increased the number of cells in the G0/G1 phase and induced apoptosis in A549 cells. Western blotting showed that astaxanthin up-regulated the expression of Bax and down-regulated the expressions of Bcl-2, STAT3 and JAK1. Conclusion Astaxanthin functions as a potent inhibitor of A549 lung cancer cell growth by targeting JAK1/STAT3 signaling pathway.

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

    PubMed

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

    2011-11-01

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

  18. Ciclesonide uptake and metabolism in human alveolar type II epithelial cells (A549)

    PubMed Central

    Nonaka, Takashi; Nave, Rüdiger; McCracken, Nigel; Kawashimo, Atsuko; Katsuura, Yasuhiro

    2007-01-01

    Background Ciclesonide is a novel inhaled corticosteroid for the treatment of airway inflammation. In this study we investigated uptake and in vitro metabolism of ciclesonide in human alveolar type II epithelial cells (A549). Ciclesonide uptake was compared with fluticasone propionate, an inhaled corticosteroid that is not metabolized in lung tissue. A549 cells were incubated with 2 × 10-8 M ciclesonide or fluticasone propionate for 3 to 30 min to determine uptake; or with 2 × 10-8 M ciclesonide for 1 h, followed by incubation with drug-free buffer for 3, 6, and 24 h to analyze in vitro metabolism. High performance liquid chromatography with tandem mass spectrometry was used to measure the concentrations of both corticosteroids and metabolites. Results At all time points the mean intracellular concentration was higher for ciclesonide when compared with fluticasone propionate. Activation of ciclesonide to desisobutyryl-ciclesonide (des-CIC) was confirmed and conjugates of des-CIC with fatty acids were detected. The intracellular concentration of ciclesonide decreased over time, whereas the concentration of des-CIC remained relatively stable: 2.27 to 3.19 pmol/dish between 3 and 24 h. The concentration of des-CIC fatty acid conjugates increased over time, with des-CIC-oleate being the main metabolite. Conclusion Uptake of ciclesonide into A549 cells was more efficient than that of the less lipophilic fluticasone propionate. Intracellular concentrations of the pharmacologically active metabolite des-CIC were maintained for up to 24 h. The local anti-inflammatory activity of ciclesonide in the lung may be prolonged by the slow release of active drug from the depot of fatty acid esters. PMID:17900334

  19. Deletion of Adipose Triglyceride Lipase Links Triacylglycerol Accumulation to a More-Aggressive Phenotype in A549 Lung Carcinoma Cells.

    PubMed

    Tomin, Tamara; Fritz, Katarina; Gindlhuber, Juergen; Waldherr, Linda; Pucher, Bettina; Thallinger, Gerhard G; Nomura, Daniel K; Schittmayer, Matthias; Birner-Gruenberger, Ruth

    2018-04-06

    Adipose triglyceride lipase (ATGL) catalyzes the rate limiting step in triacylglycerol breakdown in adipocytes but is expressed in most tissues. The enzyme was shown to be lost in many human tumors, and its loss may play a role in early stages of cancer development. Here, we report that loss of ATGL supports a more-aggressive cancer phenotype in a model system in which ATGL was deleted in A549 lung cancer cells by CRISPR/Cas9. We observed that loss of ATGL led to triacylglycerol accumulation in lipid droplets and higher levels of cellular phospholipid and bioactive lipid species (lyso- and ether-phospholipids). Label-free quantitative proteomics revealed elevated expression of the pro-oncogene SRC kinase in ATGL depleted cells, which was also found on mRNA level and confirmed on protein level by Western blot. Consistently, higher expression of phosphorylated (active) SRC (Y416 phospho-SRC) was observed in ATGL-KO cells. Cells depleted of ATGL migrated faster, which was dependent on SRC kinase activity. We propose that loss of ATGL may thus increase cancer aggressiveness by activation of pro-oncogenic signaling via SRC kinase and increased levels of bioactive lipids.

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

    PubMed

    Niu, Ben; Lin, Jingshuang; Feng, Tao

    2015-04-01

    To observe the effect of 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) on the apoptosis of human lung cancer cell lines A549 and H446, and to investigate the potential mechanisms. Proliferation inhibition and apoptosis assays, and the cell cycles were detected by MTT and flow cytometry respectively. Western blot was used to determine the expression level of proteins such as Hsp90, Hsp70, AKt, Her-2, Bcl-2 and Bax. After treated with 17-AAG, the proliferation of both A549 and H446 cells was inhibited significantly in a dose-dependent manner; as the concentration of 17-AAG was from 50 to 500 nmol/L, the IC₅₀ values to A549 and H446 cell lines were (222 ± 13) nmol/L and (189 ± 7) nmol/L respectively at 48 h. Cell cycle assays showed that 17-AAG was able to arrest cell cycles of A549 and H446 cell lines at the G₂/M phase. Apoptosis assay showed that 17-AAG was capable of inducing apoptosis in A549 and H446 cell lines. After treated with 17-AAG for 48 h, there were significant differences between the 400 nmol/L groups(46.3% for A549 cell line and 56.9% for H446 cell line) and the control group (11.9% for A549 cell line and 6.9% for H446 cell line, P < 0.01). Western blot results showed that the relative proteins of the Hsp90 signal pathway in both A549 and H446 cell lines underwent similar changes after 17-AAG treatment: Akt and Her-2 decreased significantly while the expression of Hsp70 increased. Meanwhile, the expression of Bcl-2 decreased but that of Bax increased, indicating that 17-AAG was able to promote apoptosis mode in A549 and H446 cells. 17-AAG can regulate the expression level of apoptosis-related proteins such as Bax and Bcl-2 by Hsp90 signaling pathway in A549 and H446 cells, and ultimately inhibit cell proliferation and induce apoptosis.

  1. Evaluation of whole cigarette smoke induced oxidative stress in A549 and BEAS-2B cells.

    PubMed

    Zhang, Shimin; Li, Xiang; Xie, Fuwei; Liu, Kejian; Liu, Huimin; Xie, Jianping

    2017-09-01

    Cigarette smoke is a complex and oxidative aerosol. Previous researches on the hazards of cigarette smoke mainly focused on the adverse bioeffects induced by its condensates or gas vapor phase, which ignored the dynamic processes of smoking and the cigarette smoke aging. To overcome these disadvantages, we performed air-liquid interface exposure of whole smoke, which used native and unmodified smoke and ensured the exposure similar to physiological inhalation. Our results indicated that whole cigarette smoke induced lung epithelial cells (A549) and bronchial epithelial cells (BEAS-2B) damages in cytotoxicity assays (methyl thiazoly tetrazolium and neutral red uptake assays). In addition, A549 and BEAS-2B cells showed oxidative damages in whole smoke exposure, with concentration change of several biomarkers (reduced and oxidized glutathione, malondialdehyde, 4-hydroxyhydroxy-2-nonenal, extracellular superoxide dismutase, and 8-hydroxyl deoxyguanosine). These results indicate that whole smoke-induced oxidative stress occurs in two different kinds of cells at air-liquid interface. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. [Effect of Inhibiting NGAL Gene Expression on A549 Lung Cancer Cell Migration and Invasion].

    PubMed

    Tang, Jian; Li, Jie; Li, Shaojun; Li, Jingbo; Yu, Changhai; Wei, Chengze

    2015-04-01

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

  3. 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.

  4. Antimetastatic effects of Phyllanthus on human lung (A549) and breast (MCF-7) cancer cell lines.

    PubMed

    Lee, Sau Har; Jaganath, Indu Bala; Wang, Seok Mui; Sekaran, Shamala Devi

    2011-01-01

    Current chemotherapeutic drugs kill cancer cells mainly by inducing apoptosis. However, they become ineffective once cancer cell has the ability to metastasize, hence the poor prognosis and high mortality rate. Therefore, the purpose of this study was to evaluate the antimetastatic potential of Phyllanthus (P. niruri, P. urinaria, P. watsonii, and P. amarus) on lung and breast carcinoma cells. Cytotoxicity of Phyllanthus plant extracts were first screened using the MTS reduction assay. They were shown to inhibit MCF-7 (breast carcinoma) and A549 (lung carcinoma) cells growth with IC(50) values ranging from 50-180 µg/ml and 65-470 µg/ml for methanolic and aqueous extracts respectively. In comparison, they have lower toxicity on normal cells with the cell viability percentage remaining above 50% when treated up to 1000 µg/ml for both extracts. After determining the non-toxic effective dose, several antimetastasis assays were carried out and Phyllanthus extracts were shown to effectively reduce invasion, migration, and adhesion of both MCF-7 and A549 cells in a dose-dependent manner, at concentrations ranging from 20-200 µg/ml for methanolic extracts and 50-500 µg/ml for aqueous extracts. This was followed by an evaluation of the possible modes of cell death that occurred along with the antimetastatic activity. Phyllanthus was shown to be capable of inducing apoptosis in conjunction with its antimetastastic action, with more than three fold increase of caspases-3 and -7, the presence of DNA-fragmentation and TUNEL-positive cells. The ability of Phyllanthus to exert antimetastatic activities is mostly associated to the presence of polyphenol compounds in its extracts. The presence of polyphenol compounds in the Phyllanthus plant is critically important in the inhibition of the invasion, migration, and adhesion of cancer cells, along with the involvement of apoptosis induction. Hence, Phyllanthus could be a valuable candidate in the treatment of metastatic cancers.

  5. Alterations of A549 lung cell gene expression in response to biochemical toxins.

    PubMed

    Boesewetter, D E; Collier, J L; Kim, A M; Riley, M R

    2006-03-01

    Health risks associated with the inhalation of potentially toxic materials have been a topic of great public concern. In vitro cellular analyses can provide mechanistic information on the molecular-level responses of lung-derived cell lines to a variety of these hazards. This understanding may be used to develop methods to reduce the damage from such toxins or to detect early stages of their effects. Here we describe an evaluation of the alterations in gene expression of an immortalized lung cell line (A549, human type II epithelia) to a variety of inhalation health hazards including etoposide, gliotoxin, streptolysin O, methyl methansesulfonate (MMS), and Triton X-100. The A549 cells display a dose-response relationship to each toxin with initial responses including alterations in metabolic activity, increases in membrane permeability, and initiation of response genes. In general, membrane-damaging agents (streptolysin O and Triton X-100) induce production of new ion channel proteins, structural proteins, and metabolic enzymes. Gliotoxin impacted the metabolic machinery, but also altered ion channels. Etoposide and MMS caused alterations in the cell cycle, induced DNA repair enzymes, and initiated apoptotic pathways, but MMS also induced immune response cascades. The mechanism of cell response to each toxin is supported by physiological analyses that indicated a fairly slow initiation of cell response to all compounds tested, except for Triton, which caused rapid decline in cell function due to solubilization of the cell membrane. However, Triton does induce production of a number of cell membrane-associated proteins and so its effects at low concentrations are likely translated throughout the cell. Together these results indicate a broader array of cellular responses to each of the test toxins than have previously been reported.

  6. High pemetrexed sensitivity of docetaxel-resistant A549 cells is mediated by TP53 status and downregulated thymidylate synthase

    PubMed Central

    Kuo, Wei-Ting; Tu, Dom-Gene; Chiu, Ling-Yen; Sheu, Gwo-Tarng; Wu, Ming-Fang

    2017-01-01

    The chemoresistance of non-small cell lung cancer (NSCLC) that occurs in docetaxel (DOC) chemotherapy substantially decreases the survival of patients. To overcome DOC-induced chemoresistance, we established DOC-selected A549 lung cancer sublines (A549/D16 and A549/D32) and revealed that both sublines were cross-resistant to vincristine (VCR) and doxorubicin (DXR). Notably, both sublines were more sensitive to pemetrexed (PEM) than parental cells according to MTT and clonogenic assays. The expression levels of thymidylate synthase (TS) and γ-glutamyl hydrolase (GGH) were downregulated in DOC-resistant sublines. When exogenous TS was overexpressed in A549/D16 cells, PEM sensitivity was significantly decreased, however it was not decreased by overexpression of exogenous GGH. PEM treatment induced more apoptotic sub-G1 cells in both DOC-resistant sublines and in the in vivo PEM sensitivities of A549/D16 cells. These findings were further confirmed by a xenografted tumor model. To unmask the mediator of TS downregulation, we investigated human lung cancer cell lines that have various TP53 statuses using DOC treatment. The level of TS protein was significantly decreased in wild-type TP53-containing cells with DOC treatment; TS expression levels were not affected in mutant-TP53 and TP53-null cells under the same conditions. Furthermore, when the expression of TP53 was inhibited in A549 cells, the expression level of TS was increased. Our data indicated that DOC activated wild-type TP53 and suppressed TS expression under continuous DOC exposure. Therefore, the expression of TS remained at low levels in DOC-resistant A549 cancer cells. Our data revealed that for lung cancer with DOC resistance and wild-type TP53 status, the administration of PEM as a second-line agent to overcome DOC-resistance may benefit patients. PMID:28901493

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

    PubMed

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

    2015-06-01

    To establish a preprocessing method for cell morphometry in microscopic images of A549 cells in epithelial-mesenchymal transition (EMT). Adobe Photoshop CS2 (Adobe Systems, Inc.) was used for preprocessing the images. First, all images were processed for size uniformity and high distinguishability between the cell and background area. Then, a blank image with the same size and grids was established and cross points of the grids were added into a distinct color. The blank image was merged into a processed image. In the merged images, the cells with 1 or more cross points were chosen, and then the cell areas were enclosed and were replaced in a distinct color. Except for chosen cellular areas, all areas were changed into a unique hue. Three observers quantified roundness of cells in images with the image preprocess (IPP) or without the method (Controls), respectively. Furthermore, 1 observer measured the roundness 3 times with the 2 methods, respectively. The results between IPPs and Controls were compared for repeatability and reproducibility. As compared with the Control method, among 3 observers, use of the IPP method resulted in a higher number and a higher percentage of same-chosen cells in an image. The relative average deviation values of roundness, either for 3 observers or 1 observer, were significantly higher in Controls than in IPPs (p < 0.01 or 0.001). The values of intraclass correlation coefficient, both in Single Type or Average, were higher in IPPs than in Controls both for 3 observers and 1 observer. Processed with Adobe Photoshop, a chosen cell from an image was more objective, regular, and accurate, creating an increase of reproducibility and repeatability on morphometry of A549 cells in epithelial to mesenchymal transition.

  8. 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.

  9. 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.

  10. A549 Lung Epithelial Cells Grown as Three-Dimensional Aggregates: Alternative Tissue Culture Model for Pseudomonas aeruginosa Pathogenesis

    PubMed Central

    Carterson, A. J.; Höner zu Bentrup, K.; Ott, C. M.; Clarke, M. S.; Pierson, D. L.; Vanderburg, C. R.; Buchanan, K. L.; Nickerson, C. A.; Schurr, M. J.

    2005-01-01

    A three-dimensional (3-D) lung aggregate model was developed from A549 human lung epithelial cells by using a rotating-wall vessel bioreactor to study the interactions between Pseudomonas aeruginosa and lung epithelial cells. The suitability of the 3-D aggregates as an infection model was examined by immunohistochemistry, adherence and invasion assays, scanning electron microscopy, and cytokine and mucoglycoprotein production. Immunohistochemical characterization of the 3-D A549 aggregates showed increased expression of epithelial cell-specific markers and decreased expression of cancer-specific markers compared to their monolayer counterparts. Immunohistochemistry of junctional markers on A549 3-D cells revealed that these cells formed tight junctions and polarity, in contrast to the cells grown as monolayers. Additionally, the 3-D aggregates stained positively for the production of mucoglycoprotein while the monolayers showed no indication of staining. Moreover, mucin-specific antibodies to MUC1 and MUC5A bound with greater affinity to 3-D aggregates than to the monolayers. P. aeruginosa attached to and penetrated A549 monolayers significantly more than the same cells grown as 3-D aggregates. Scanning electron microscopy of A549 cells grown as monolayers and 3-D aggregates infected with P. aeruginosa showed that monolayers detached from the surface of the culture plate postinfection, in contrast to the 3-D aggregates, which remained attached to the microcarrier beads. In response to infection, proinflammatory cytokine levels were elevated for the 3-D A549 aggregates compared to monolayer controls. These findings suggest that A549 lung cells grown as 3-D aggregates may represent a more physiologically relevant model to examine the interactions between P. aeruginosa and the lung epithelium during infection. PMID:15664956

  11. Adhesion of MRC-5 and A549 cells on poly(dimethylsiloxane) surface modified by proteins.

    PubMed

    Zuchowska, Agnieszka; Kwiatkowski, Piotr; Jastrzebska, Elzbieta; Chudy, Michal; Dybko, Artur; Brzozka, Zbigniew

    2016-02-01

    PDMS is a very popular material used for fabrication of Lab-on-a-Chip systems for biological applications. Although PDMS has numerous advantages, it is a highly hydrophobic material, which inhibits adhesion and proliferation of the cells. PDMS surface modifications are used to enrich growth of the cells. However, due to the fact that each cell type has specific adhesion, it is necessary to optimize the parameters of these modifications. In this paper, we present an investigation of normal (MRC-5) and carcinoma (A549) human lung cell adhesion and proliferation on modified PDMS surfaces. We have chosen these cell types because often they are used as models for basic cancer research. To the best of our knowledge, this is the first presentation of this type of investigation. The combination of a gas-phase processing (oxygen plasma or ultraviolet irradiation) and wet chemical methods based on proteins' adsorption was used in our experiments. Different proteins such as poly-l-lysine, fibronectin, laminin, gelatin, and collagen were incubated with the activated PDMS samples. To compare with other works, here, we also examined how ratio of prepolymer to curing agent (5:1, 10:1, and 20:1) influences PDMS hydrophilicity during further modifications. The highest adhesion of the tested cells was observed for the usage of collagen, regardless of PDMS ratio. However, the MRC-5 cell line demonstrated better adhesion than A549 cells. This is probably due to the difference in their morphology and type (normal/cancer). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Cell uptake of paclitaxel solid lipid nanoparticles modified by cell-penetrating peptides in A549 cells.

    PubMed

    Zhang, Yin-Long; Zhang, Zhen-Hai; Jiang, Tian-Yue; Ayman-Waddad; Jing-Li; Lv, Hui-Xia; Zhou, Jian-Ping

    2013-01-01

    The aim of this study was to investigate the cytotoxicity of paclitaxel solid lipid nanoparticles (SLN) modified with stearic acid octaarginine (SA-R8-PTX-SLN) as well as the cellular uptake of coumarin-6-loaded SLN modified with SA-R8 (SA-R8-C6-SLN) in human lung cancer cells, A549. SLN were prepared using a film dispersion method; and then their particle size, zeta potential, morphology, bound efficiency of SAR8, drug loading efficiency, and in vitro release were characterized. SA-R8-PTX-SLN and SA-R8-C6-SLN were incubated with A549 cells to measure their cytotoxicity and cellular uptake, respectively. The results indicated that the cytotoxicity of SA-R8-PTX-SLN was enhanced significantly with the increasing amount of SA-R8 and the cellular uptakes of SLN increased with the incubated concentrations and the incubated time of SLN. In contrast, SA-R8-SLN could significantly enhance the cellular uptake of SLN and the cytotoxicity of PTX in A549 cells. These in vitro results suggest that SA-R8-SLN could be proposed as alternative drug delivery system.

  13. Interleukin-15 increases neutrophil adhesion onto human respiratory epithelial A549 cells and attracts neutrophils in vivo

    PubMed Central

    Pelletier, M; Girard, D

    2005-01-01

    Interleukin-15 (IL-15) is a neutrophil agonist that plays a role in inflammatory disorders, including a variety of pulmonary diseases. Adhesion of neutrophils onto pulmonary cells is a major event leading to development of inflammation. Recently, elevated levels of IL-15 have been associated with different pulmonary diseases. There is no clear evidence that IL-15 modulates cell surface expression of adhesion molecules in neutrophils, or that IL-15 is involved in neutrophil adhesion onto pulmonary cells. Also, it is not clear if IL-15 induces a neutrophilic inflammation in vivo. This study was aimed at elucidation of these issues. Neutrophils were treated with IL-15 and cell surface expression of CD11a, CD11b, CD11c and CD18 was monitored by flow cytometry. The human respiratory epithelial A549 cell line was used as a substrate for the neutrophil adhesion assay and cell surface expression of CD50, CD54 and CD106 was monitored in IL-15-induced A549 cells. The murine air pouch model was used for investigating potential neutrophilic inflammation induced by IL-15 in vivo. IL-15 significantly increased neutrophil cell surface expression of CD11b and CD18 and up-regulated A549 cell surface expression of CD54. Moreover, A549 cells were found to express IL-15R components and adhesion of neutrophils onto A549 cells was increased when neutrophils or A549 cells were treated with IL-15. Finally, IL-15 induced neutrophilic inflammation in vivo and concentrations of IL-6 and CXCL2/MIP-2 were increased in IL-15-induced pouches. IL-15 might participate in inflammatory pulmonary diseases by attracting neutrophils, modulating cell surface expression molecules and increasing neutrophil adhesion onto pulmonary cells. PMID:15996196

  14. [Effect of ginsenoside on the cellular proliferation, apoptosis and cell cycles in LC A549 and HUVEC 304 cell lines].

    PubMed

    Chen, Ming-wei; Ma, Ai-qun; Ni, Lei; Huang, Chen; Zhang, Dian-zeng; Niu, Xiao-ying

    2005-04-01

    To determine the effect of ginsenoside on the cellular proliferation, apoptosis and cell cycles in LC A549 and HUVEC 304 cell lines. A549 and HUVEC 304 cell lines were cultured with different concentrations of ginsenoside. Cellular proliferation was detected with MTT, apoptosis and cell cycles were checked with Flow Cytometer, and change of microstructure was observed by transmission electron microscope. The apoptosis rate was 29.8% in A549 cell lines after being interfered with ginsenoside at 3 x 10(-6) mol/L, significantly higher than that in the control group ( P < 0.05). No change was observed in the cell cycles after being interfered with ginsenoside. The inhibitive rate of ginsenoside was 12.53% for HUVEC 304 cell line at 1 x 10(-4) mol/L (P < 0.05 ). The cells induced by conditioned medium could be inhibited by ginsenoside, and apoptotic body could be found in cells induced by conditioned medium at 10(-6) mol/L. The proliferation of vascular endothelial cell could be inhibited by ginsenoside, and apoptosis could also be found in both tumor cells and cells induced by conditioned medium after being interfered with ginsenoside.

  15. 4-Nitroquinoline-1-oxide effects human lung adenocarcinoma A549 cells by regulating the expression of POLD4

    PubMed Central

    HUANG, QIN-MIAO; ZENG, YI-MING; ZHANG, HUA-PING; LV, LIANG-CHAO; YANG, DONG-YONG; LIN, HUI-HUANG

    2016-01-01

    The aim of the present study was to explore the expression of POLD4 in human lung adenocarcinoma A549 cells under 4-nitroquinoline-1-oxide (4NQO) stimulation to investigate the role of POLD4 in smoking-induced lung cancer. The lung cancer A549 cell line was treated with 4NQO, with or without MG132 (an inhibitor of proteasome activity), and subsequently the POLD4 level was determined by western blot analysis. Secondly, the cell sensitivity to 4NQO and Taxol was determined when the POLD4 expression level was downregulated by siRNA. The POLD4 protein levels in the A549 cells decreased following treatment with 4NQO; however, MG132 could reverse this phenotype. Downregulation of the POLD4 expression by siRNA enhanced A549 cell sensitivity to 4NQO, but not to Taxol. In conclusion, 4NQO affects human lung adenocarcinoma A549 cells by regulating the expression of POLD4. PMID:26998273

  16. 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.

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

    PubMed

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

    2016-12-01

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

  18. Antimetastatic Effects of Phyllanthus on Human Lung (A549) and Breast (MCF-7) Cancer Cell Lines

    PubMed Central

    Lee, Sau Har; Jaganath, Indu Bala; Wang, Seok Mui; Sekaran, Shamala Devi

    2011-01-01

    Background Current chemotherapeutic drugs kill cancer cells mainly by inducing apoptosis. However, they become ineffective once cancer cell has the ability to metastasize, hence the poor prognosis and high mortality rate. Therefore, the purpose of this study was to evaluate the antimetastatic potential of Phyllanthus (P. niruri, P. urinaria, P. watsonii, and P. amarus) on lung and breast carcinoma cells. Methodology/Principal Findings Cytotoxicity of Phyllanthus plant extracts were first screened using the MTS reduction assay. They were shown to inhibit MCF-7 (breast carcinoma) and A549 (lung carcinoma) cells growth with IC50 values ranging from 50–180 µg/ml and 65–470 µg/ml for methanolic and aqueous extracts respectively. In comparison, they have lower toxicity on normal cells with the cell viability percentage remaining above 50% when treated up to 1000 µg/ml for both extracts. After determining the non-toxic effective dose, several antimetastasis assays were carried out and Phyllanthus extracts were shown to effectively reduce invasion, migration, and adhesion of both MCF-7 and A549 cells in a dose-dependent manner, at concentrations ranging from 20–200 µg/ml for methanolic extracts and 50–500 µg/ml for aqueous extracts. This was followed by an evaluation of the possible modes of cell death that occurred along with the antimetastatic activity. Phyllanthus was shown to be capable of inducing apoptosis in conjunction with its antimetastastic action, with more than three fold increase of caspases-3 and -7, the presence of DNA-fragmentation and TUNEL-positive cells. The ability of Phyllanthus to exert antimetastatic activities is mostly associated to the presence of polyphenol compounds in its extracts. Conclusions/Significance The presence of polyphenol compounds in the Phyllanthus plant is critically important in the inhibition of the invasion, migration, and adhesion of cancer cells, along with the involvement of apoptosis induction. Hence

  19. α5-nAChR modulates nicotine-induced cell migration and invasion in A549 lung cancer cells.

    PubMed

    Sun, Haiji; Ma, Xiaoli

    2015-09-01

    Cigarette smoking is the most important risk factor in the development of human lung cancer. Nicotine, the major component in tobacco, not only contributes to carcinogenesis but also promotes tumor metastasis. By binding to nicotinic acetylcholine receptors (nAChRs), nicotine induces the proliferation and migration of non-small cell lung cancer. Recently studies have indicated that α5-nAChR is highly associated with lung cancer risk and nicotine dependence. Nevertheless, it is unclear whether nicotine promotes the migration and invasion through activation of α5-nAChR in lung cancer. In the present study, A549 cell was exposed to 1μN nicotine for 8, 24 or 48h. Wound-healing assay and transwell assay were used to evaluate the capability of A549 cell migration and cell invasion, respectively. Silencing of α5-nAChR was done by siRNA. Western blotting and PCR were used to detect α5-nAChR expression. Nicotine can induce activation of α5-nAChR in association with increased migration and invasion of human lung cancer A549 cell. Treatment of cells with α5-nAChR specific siRNA blocks nicotine-stimulated activation of α5-nAChR and suppresses A549 cell migration and invasion. Reduction of α5-nAChR resulted in upregulation of E-cadherin, consistent with E-cadherin being inhibitive of cancer cell invasion. These findings suggest that nicotine-induced migration and invasion may occur in a mechanism through activation of α5-nAChR, which can contribute to metastasis or development of human lung cancer. Copyright © 2015 Elsevier GmbH. All rights reserved.

  20. 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.

  1. 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. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Apoptosis of human lung adenocarcinoma A549 cells induced by prodigiosin analogue obtained from an entomopathogenic bacterium Serratia marcescens.

    PubMed

    Zhou, Wei; Jin, Zhi-Xiong; Wan, Yong-Ji

    2010-12-01

    An entomopathogenic bacterial strain SCQ1 was isolated from silkworm (Bombyx mori) and identified as Serratia marcescens via 16S rRNA gene analysis. This strain produces a red pigment that causes acute septicemia of silkworm. The red pigment of strain SCQ1 was identified as prodigiosin analogue (PGA) with various reported biological activities. In this study, we found that low concentration of PGA showed significant anticancer activity in human lung adenocarcinoma A549 cells, but has little effect in human bone marrow stem cells, in vitro. By exposure to different concentrations of PGA for 24 h, morphological changes and the MTT assay showed that A549 cell line was very sensitive to PGA, with IC(50) value about 2.2 mg/L. Early stage of apoptosis was detected by flow cytometry while A549 cells were treated with PGA for 4 and 12 h, respectively. The proportion of dead cells was increased with treatment time or the concentrations of PGA, but it was inversely proportional to that of apoptotic cells. These results indicate that PGA obtained from strain SCQ1 induces apoptosis in A549 cells, but the molecular mechanisms of cell death are complicated, and the S. marcescens strain SCQ1 may serve as a source of the anticancer compound, PGA.

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

    PubMed

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

    2018-02-01

    Many studies have demonstrated that heavy metals existing as a mixture in the atmospheric environment cause adverse effects on human health and are important key factors of cytotoxicity; however, little investigation has been conducted on a toxicological study of a metal mixture from atmospheric fine particulate matter. The objective of this study was to predict the combined effects of heavy metals in aerosol by using in vitro human cells and obtain a suitable mixture toxicity model. Arsenic, nickel, and lead were selected for mixtures exposed to A549 human lung cancer cells. Cell proliferation (WST-1), glutathione (GSH), and interleukin (IL)-8 inhibition were observed and applied to the prediction models of mixture toxicity, concentration addition (CA) and independent action (IA). The total mixture concentrations were set by an IC 10 -fixed ratio of individual toxicity to be more realistic for mortality and enzyme inhibition tests. The results showed that the IA model was statistically closer to the observed results than the CA model in mortality, indicating dissimilar modes of action. For the GSH inhibition, the results predicted by the IA and CA models were highly overestimated relative to mortality. Meanwhile, the IL-8 results were stable with no significant change in immune reaction related to inflammation. In conclusion, the IA model is a rapid prediction model in heavy metals mixtures; mortality, as a total outcome of cell response, is a good tool for demonstrating the combined toxicity rather than other biochemical responses.

  4. Chloroquine inhibits cell growth in human A549 lung cancer cells by blocking autophagy and inducing mitochondrial‑mediated apoptosis.

    PubMed

    Liu, Likun; Han, Cuicui; Yu, Haitao; Zhu, Wenbin; Cui, Hongxia; Zheng, Lihong; Zhang, Chunjing; Yue, Liling

    2018-04-12

    Chloroquine (CQ) has been revealed to exhibit antitumor activity in several human tumors including lung cancer as mono‑ or add‑on therapy. The antitumor effect of CQ appears to depend on the tumor type, stage and genetic context. Few studies have focused on the mechanism concerning the antitumor effect of CQ monotherapy and the cause and effect relationship among autophagy, apoptosis and CQ in human lung A549 cells. Therefore, the present study aimed to identify the antitumor effect of CQ monotherapy and analyze the possible mechanism. In the present study, we demonstrated that CQ suppressed human A549 cell growth in a dose‑ and time‑dependent manner. CQ‑mediated growth inhibition in A549 cells was characterized by the targeting of the PI3K/AKT pathway, thus, inducing mitochondria‑mediated apoptosis at relatively higher concentrations by downregulating Bcl‑2 expression, increasing the expression level of Bax, decreasing mitochondrial membrane potential, releasing cytochrome c from the mitochondria into the cytosol, activating caspase‑3 and cleaving PARP. Collectively, these findings may offer a new rationale for using CQ as a lung cancer therapy drug in clinical practice.

  5. 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

  6. 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.

  7. 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

  8. The influence of incubation time on adenovirus quantitation in A549 cells by most probable number.

    PubMed

    Cashdollar, Jennifer L; Huff, Emma; Ryu, Hodon; Grimm, Ann C

    2016-11-01

    Cell culture based assays used to detect waterborne viruses typically call for incubating the sample for at least two weeks in order to ensure that all the culturable virus present is detected. Historically, this estimate was based, at least in part, on the length of time used for detecting poliovirus. In this study, we have examined A549 cells infected with human adenovirus type 2, and have found that a three week incubation of virus infected cells results in a higher number of detected viruses by quantal assay than what is seen after two weeks of incubation, with an average 955% increase in Most Probable Number (MPN) from 2 weeks to 3 weeks. This increase suggests that the extended incubation time is essential for accurately estimating viral titer, particularly for slow-growing viruses, UV treated samples, or samples with low titers of virus. In addition, we found that for some UV-treated samples, there was no detectable MPN at 2 weeks, but after 3 weeks, MPN values were obtained. For UV-treated samples, the average increase in MPN from 2 weeks to 3 weeks was 1401%, while untreated samples averaged a change in MPN of 674%, leading us to believe that the UV-damaged viral DNA may be able to be repaired such that viral replication then occurs. Published by Elsevier B.V.

  9. Overview on biological implications of metal oxide nanoparticle exposure to human alveolar A549 cell line.

    PubMed

    Martin, Ansie; Sarkar, Angshuman

    2017-08-01

    Metal oxides (MeOx) are exponentially being used in a wide range of applications and are the largest class of commercially produced nanomaterials. This presents unprecedented human exposure. Thus, understanding nanoparticle induced cellular stress can greatly help design strategies to combat them. Scores of studies have been carried out to understand the effects of MeOx nanoparticle exposure on human alveolar cells, which are highly susceptible to aerosolized matter. There is a huge redundancy of information generated, also, a lack of a comprehensive conglomeration of this information. We have built here in a sincere summary of the cellular responses reported till date as a direct consequence of MeOx nanoparticle exposure on human alveolar (A549) cells. Detailed accounts of cellular morphology modulation, generation of reactive oxygen species (ROS) and oxidative stress, inflammation and cytokine release, genotoxic and epi-genotoxic insults, toxicological trend, nanoparticle internalization, modes of cell death, protein synthesis, and membrane damage among others are discussed. Finally, to aid predictability of the highly dynamic and multifactorial nature of this toxicity, we have hypothesized models that describe the ensuing mechanisms based on common patterns discovered throughout our literature survey.

  10. Involvement of lysosomal dysfunction in silver nanoparticle-induced cellular damage in A549 human lung alveolar epithelial cells.

    PubMed

    Miyayama, Takamitsu; Matsuoka, Masato

    2016-01-01

    While silver nanoparticles (AgNPs) are widely used in consumer and medical products, the mechanism by which AgNPs cause pulmonary cytotoxicity is not clear. AgNP agglomerates are found in endo-lysosomal structures within the cytoplasm of treated cells. In this study, the functional role of lysosomes in AgNP-induced cellular damage was examined in A549 human lung alveolar epithelial cells. We evaluated the intracellular distribution of AgNPs, lysosomal pH, cellular viability, Ag dissolution, and metallothionein (MT) mRNA levels in AgNP-exposed A549 cells that were treated with bafilomycin A1, the lysosomal acidification inhibitor. Exposure of A549 cells to citrate-coated AgNPs (20 nm diameter) for 24 h induced cellular damage and cell death at 100 and 200 μg Ag/ml, respectively. Confocal laser microscopic examination of LysoTracker-stained cells showed that AgNPs colocalized with lysosomes and their agglomeration increased in a dose-dependent manner (50-200 μg Ag/ml). In addition, the fluorescence signals of LysoTracker were reduced following exposure to AgNPs, suggesting the elevation of lysosomal pH. Treatment of A549 cells with 200 nM bafilomycin A1 and AgNPs (50 μg Ag/ml) further reduced the fluorescence signals of LysoTracker. AgNP-induced cell death was also increased by bafilomycin A1 treatment. Finally, treatment with bafilomycin A1 suppressed the dissolution of Ag and decreased the mRNA expression levels of MT-I and MT-II following exposure to AgNPs. The perturbation of lysosomal pH by AgNP exposure may play a role in AgNP agglomeration and subsequent cellular damage in A549 cells.

  11. 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. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Cinchonine induces apoptosis of HeLa and A549 cells through targeting TRAF6.

    PubMed

    Qi, Yonghao; Pradipta, Ambara R; Li, Miao; Zhao, Xuan; Lu, Lulu; Fu, Xuegang; Wei, Jing; Hsung, Richard P; Tanaka, Katsunori; Zhou, Lijun

    2017-02-23

    Cancer cells are known to over-express TRAF6 that is critical for both AKT and TAK1 activations. The Really Interesting New Gene (RING) domain of TRAF6 is believed to be responsible for the E3 ligase activity, ZINC fingers of TRAF6 provide critical support for the activity of the RING domain which is critical for both AKT and TAK1 activations. We employed computational docking program to identify small molecules that could effectively and competitively bind with the RING domain of TRAF6, which is believed to be responsible for its E3 ligase activity. MTT assay and flow cytometry were employed to analyze apoptosis of cancer cells. Signaling pathways were detected using immunoprecipitation and western blotting, and immunofluorescence was pursued to assess the nature of binding of cinchonine to TRAF6. We also performed animal experiments to test effect of cinchonine in vivo. Cinchonine, a naturally occurring Cinchona alkaloid identified from the docking study, could bind to TRAF6 in HeLa and A549 cells and induce apoptosis of these cancer cells. We found that AKT ubiquitination and phosphorylation as well as phosphorylation of TAK1 were decreased. These activities would lead to subsequent suppression anti-apoptotic protein Bcl-2, while elevating pro-apoptotic protein Bax. Immunofluorescence staining unambiguously demonstrated the binding of cinchonine specifically at the RING domain of TRAF6 in cells, thereby validating the computational modeling. Animal experiments showed that cinchonine could suppress tumor growth in mice without showing significant acute toxicity. These investigations suggest that through competitive binding with the RING domain of TRAF6, cinchonine could induce apoptosis via inhibiting AKT and TAK1 signaling pathways.

  13. Induction of cell death by pyropheophorbide-α methyl ester-mediated photodynamic therapy in lung cancer A549 cells.

    PubMed

    Tu, Ping-Hua; Huang, Wen-Jun; Wu, Zhan-Ling; Peng, Qing-Zhen; Xie, Zhi-Bin; Bao, Ji; Zhong, Ming-Hua

    2017-03-01

    Pyropheophorbide-α methyl ester (MPPa) was a promising photosensitizer with stable chemical structure, strong absorption, higher tissue selectivity and longer activation wavelengths. The present study investigated the effect of MPPa-mediated photodynamic treatment on lung cancer A549 cells as well as the underlying mechanisms. Cell Counting Kit-8 was employed for cell viability assessment. Reactive oxygen species levels were determined by fluorescence microscopy and flow cytometry. Cell morphology was evaluated by Hoechst staining and transmission electron microscopy. Mitochondrial membrane potential, cellular apoptosis and cell cycle distribution were evaluated flow-cytometrically. The protein levels of apoptotic effectors were examined by Western blot. We found that the photocytotoxicity of MPPa showed both drug- and light- dose dependent characteristics in A549 cells. Additionally, MPPa-PDT caused cell apoptosis by reducing mitochondrial membrane potential, increasing reactive oxygen species (ROS) production, inducing caspase-9/caspase-3 signaling activation as well as cell cycle arrest at G 0 /G 1 phase. These results suggested that MPPa-PDT mainly kills cells by apoptotic mechanisms, with overt curative effects, indicating that MPPa should be considered a potent photosensitizer for lung carcinoma treatment. © 2017 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

  14. The effects of dietary phenolic compounds on cytokine and antioxidant production by A549 cells.

    PubMed

    Gauliard, Benoit; Grieve, Douglas; Wilson, Rhoda; Crozier, Alan; Jenkins, Carol; Mullen, William D; Lean, Michael

    2008-06-01

    Levels of inflammatory cytokines are raised in chronic obstructive pulmonary disease (COPD). A diet rich in antioxidant vitamins may protect against the development of COPD. This study examined the effects of phenolic compounds and food sources on cytokine and antioxidant production by A549 cells. The effects of the following phenolic compounds on basal and interleukin (IL)-1-stimulated release of IL-8, IL-6, and reduced glutathione (GSH) were examined: resveratrol; Bouvrage, a commercially available raspberry juice (Ella Drinks Ltd., Alloa, Clacksmannanshire, UK); and quercetin 3'-sulfate. Purification of the raspberry juice by high-performance liquid chromatography gave three fractions: Fraction 1 contained phenolic acid and vitamin C, Fraction 2 contained flavonoids and ellagic acid, and Fraction 3 contained anthocyanins and ellagitannins. IL-8 production was increased in the presence of IL-1 (165 vs. 6,011 pg/mL, P < .0001). None of the compounds tested had any significant effect on GSH. Resveratrol at concentrations > or =50 micromol/mL significantly inhibited IL-8 and IL-6 production. Similar findings were made with raspberry juice at concentrations > or =25 microL/mL, and Fractions 1 and 3 were best able to inhibit IL-8 production. Quercetin 3'-sulfate, at 25 micromol/mL, inhibited IL-8 and IL-6 production. The changes observed in IL-8 were paralleled by changes in tumor necrosis factor-alpha. Thus, phenolic compounds can significantly alter cytokine and antioxidant production.

  15. Bostrycin inhibits proliferation of human lung carcinoma A549 cells via downregulation of the PI3K/Akt pathway

    PubMed Central

    2011-01-01

    Background Bostrycin is a novel compound isolated from marine fungi that inhibits proliferation of many cancer cells. However, the inhibitory effect of bostrycin on lung cancers has not been reported. This study is to investigate the inhibitory effects and mechanism of bostrycin on human lung cancer cells in vitro. Methods We used MTT assay, flow cytometry, microarray, real time PCR, and Western blotting to detect the effect of bostrycin on A549 human pulmonary adenocarcinoma cells. Results We showed a significant inhibition of cell proliferation and induction of apoptosis in bostrycin-treated lung adenocarcinoma cells. Bostrycin treatment caused cell cycle arrest in the G0/G1 phase. We also found the upregulation of microRNA-638 and microRNA-923 in bostrycin-treated cells. further, we found the downregulation of p110α and p-Akt/PKB proteins and increased activity of p27 protein after bostrycin treatment in A549 cells. Conclusions Our study indicated that bostrycin had a significant inhibitory effect on proliferation of A549 cells. It is possible that upregulation of microRNA-638 and microRNA-923 and downregulaton of the PI3K/AKT pathway proteins played a role in induction of cell cycle arrest and apoptosis in bostrycin-treated cells. PMID:21303527

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

    PubMed

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

    2014-04-01

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

  17. 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; Jung, Hae Hyun

    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 A549more » cells.« less

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2018-03-01

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

  20. Indole-3-carbinol induces apoptosis through p53 and activation of caspase-8 pathway in lung cancer A549 cells.

    PubMed

    Choi, Hee-Sook; Cho, Min-Chul; Lee, Hee Gu; Yoon, Do-Young

    2010-03-01

    Indole-3-carbinol (I3C) has anti-tumor effects in various cancer cell lines. However, the anti-tumor effect of I3C on human lung cancers has been rarely reported. We investigated the anti-tumor effects and its mechanism of I3C on human lung carcinoma A549 cell line. Treatment of the A549 cells with I3C significantly reduced cell proliferation, increased formations of fragmented DNA and apoptotic body, and induced cell cycle arrest at G0/G1 phase. I3C increased not only the protein levels of cyclin D1, phosphorylated p53, and p21 but also the expression of Fas mRNA. Cleavage of caspase-9, -8, -3 and PARP also was increased by I3C. Treatment with wortmannin significantly suppressed both I3C-induced Ser15 phosphorylation and accumulation of p53 protein. The inhibition of caspase-8 by z-IETD-FMK significantly decreased cleavage of procaspase-8,-3 and PARP in I3C-treated A549 cells. Taken together, these results demonstrate that I3C induces cell cycle arrest at G0/G1 through the activation of p-p53 at Ser 15 and induces caspase-8 mediated apoptosis via the Fas death receptor. This molecular mechanism for apoptotic effect of I3C on A549 lung carcinoma cells may be a first report and suggest that I3C may be a preventive and therapeutic agent against lung cancer. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  1. Inhibition of growth and induction of apoptosis in A549 cells by compounds from oxheart cabbage extract.

    PubMed

    Wang, Nan; Wang, Wei; Liu, Caiqin; Jin, Jianchang; Shao, Bo; Shen, Lianqing

    2016-08-01

    Oxheart cabbage (Brassica oleracea var. capitata) is a member of the Brassica genus. Although some studies on the anticancer effects of extracts from oxheart cabbage have been reported, comprehensive information on the bioactive fractions and components from oxheart cabbage extracts is still lacking. The aim of this study was to isolate and identify the bioactive fractions and components from oxheart cabbage seeds using activity-guided isolation methods. The cytotoxicity and apoptotic effects of fraction II, fraction III, iberverin, sulforaphane and iberin from oxheart cabbage seed extract were investigated. The results showed that all five components had inhibitory effects on the in vitro growth of A549 cells which were dose-dependent. These compounds also changed the morphology of A549 cells, and their inhibitory activity on A549 cells was as follows: sulforaphane > iberin > iberverin > fraction III > fraction II. The IC50 values were 3.53 ± 0.63, 4.93 ± 1.02, 7.07 ± 0.51, 15.56 ± 0.24 and 27.32 ± 0.63 µg mL(-1) respectively. Fraction II, fraction III, iberverin, sulforaphane and iberin induced cell apoptosis by increasing early apoptosis and late apoptosis/necrosis, and activation of caspase-3, -8 and -9. These results indicated that the decrease in A549 cell viability by active compounds from oxheart cabbage seed extract was due to the induction of apoptosis. © 2015 Society of Chemical Industry. © 2015 Society of Chemical Industry.

  2. Oligomeric proanthocyanidins protects A549 cells against H2O2-induced oxidative stress via the Nrf2-ARE pathway.

    PubMed

    Sun, Chao; Jin, Weiguo; Shi, Hongcan

    2017-06-01

    Oxidative signaling and oxidative stress contribute to aging, cancer and diseases resulting from lung fibrosis. In this study, we explored the anti-oxidative potential of oligomeric proanthocyanidins (OPCs), natural flavonoid compounds. We examined the protective effects of OPCs against hydrogen peroxide (H2O2)-induced oxidative stress in non-small cell lung cancer cells (A549). We demonstrated that OPC markedly attenuated H2O2-induced A549 cell viability, as shown by by 3-[4,5-dimethylthiazol-2-yl)]-2,5-diphenyl-tetrazolium bromide (MTT) assay. At the same time, OPC inhibited H2O2-induced oxidative stress by significantly increasing the activities of superoxide dismutase, catalase and glutathione, and reducing the levels of reactive oxygen species (ROS) and malondialdehyde (MDA). Treatment of the A549 cells with OPC significantly promoted the nuclear translocation of NF-E2-related factor 2 (Nrf2) and significantly enhanced the expression of its target genes [heme oxygenase-1 (HO-1), NAD(P)H quinone dehydrogenase 1 (NQO1) and thioredoxin reductase 1 (TXNRD1)] with different fold change values at both the mRNA and protein level. The knockout of Nrf2 using CRISPR/Cas9 technology attenuated OPC-mediated ARE gene transcription, and almost abolished the OPC-mediated protective effects against H2O2-induced oxidative stress. On the whole, our study suggests that OPC plays an important role in controlling the antioxidant response of A549 cells via the Nrf2-ARE pathway.

  3. Discovery of novel HSP90 inhibitors that induced apoptosis and impaired autophagic flux in A549 lung cancer cells.

    PubMed

    Wei, Qun; Ning, Jun-Ya; Dai, Xi; Gao, Yuan-Di; Su, Le; Zhao, Bao-Xiang; Miao, Jun-Ying

    2018-02-10

    Heat shock protein 90 (HSP90) inhibition has aroused increasing enthusiasm in antitumor strategies in recent years. According to our previous studies, we synthesized a series of coumarin pyrazoline compounds HCP1-HCP6 that might be HSP90 inhibitors. Interactions between HCP1-HCP6 and HSP90 were examined and antitumor activities of them were investigated in A549 lung cancer cells. Results showed that all the six derivatives could interact with HSP90, in which HCP1 exhibited the best binding ability and inhibited the activity of HSP90. Meanwhile, HCP1-HCP6 reduced the cell viability of A549cells and HCP1 possessed the lowest IC 50 value. Above all HCP1 exerted better HSP90 inhibitory and anticancer effects than our initially identified HSP90 inhibitor DPB. As to the underlying mechanism, HCP1-HCP6 not only induced apoptosis as DPB but also blocked autophagic flux in A549cells. Therefore, we discovered a novel HSP90 inhibitor HCP1 that had better biological activity and provided us a useful tool to explore the underlying mechanism of lung cancer therapy. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  4. 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. © 2016 Wiley Periodicals, Inc.

  5. BZML, a novel colchicine binding site inhibitor, overcomes multidrug resistance in A549/Taxol cells by inhibiting P-gp function and inducing mitotic catastrophe.

    PubMed

    Bai, Zhaoshi; Gao, Meiqi; Zhang, Huijuan; Guan, Qi; Xu, Jingwen; Li, Yao; Qi, Huan; Li, Zhengqiang; Zuo, Daiying; Zhang, Weige; Wu, Yingliang

    2017-08-28

    Multidrug resistance (MDR) interferes with the efficiency of chemotherapy. Therefore, developing novel anti-cancer agents that can overcome MDR is necessary. Here, we screened a series of colchicine binding site inhibitors (CBSIs) and found that 5-(3, 4, 5-trimethoxybenzoyl)-4-methyl-2-(p-tolyl) imidazol (BZML) displayed potent cytotoxic activity against both A549 and A549/Taxol cells. We further explored the underlying mechanisms and found that BZML caused mitosis phase arrest by inhibiting tubulin polymerization in A549 and A549/Taxol cells. Importantly, BZML was a poor substrate for P-glycoprotein (P-gp) and inhibited P-gp function by decreasing P-gp expression at the protein and mRNA levels. Cell morphology changes and the expression of cycle- or apoptosis-related proteins indicated that BZML mainly drove A549/Taxol cells to die by mitotic catastrophe (MC), a p53-independent apoptotic-like cell death, whereas induced A549 cells to die by apoptosis. Taken together, our data suggest that BZML is a novel colchicine binding site inhibitor and overcomes MDR in A549/Taxol cells by inhibiting P-gp function and inducing MC. Our study also offers a new strategy to solve the problem of apoptosis-resistance. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    PubMed

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

    2017-10-01

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

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

    SciTech Connect

    Zhang, Jian, E-mail: zhangjian197011@yahoo.com; Zhang, Tao; Ti, Xinyu

    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 ofmore » 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.« less

  8. Side population cells separated from A549 lung cancer cell line possess cancer stem cell-like properties and inhibition of autophagy potentiates the cytotoxic effect of cisplatin.

    PubMed

    Yang, Yang; Fan, Yuxia; Qi, Yu; Liu, Donglei; Wu, Kai; Wen, Fengbiao; Zhao, Song

    2015-08-01

    Recent studies have suggested that cancer stem cells (CSCs) may be responsible for tumorigenesis and contribute to resistance to chemotherapy. Side population (SP) cells are thought to be enriched for CSCs in most types of human tumors. Therefore, the aim of the present study was to sort SP cells using an A549 lung cancer cell line, identify the cancer stem cell-like properties of SP and determine the role of autophagy in the survival of SP cells of lung cancer. SP cells were isolated by fluorescence-activated cell sorter (FACS) from A549 lung cancer cells, and the CSC-like properties were verified through confocal fluorescence imaging, sphere formation assays, cell proliferation and colony formation assay, gene expression in vitro and tumor formation in vivo. The role of autophagy in the survival of SP cells was assessed by western blotting and flow cytometric analysis. A549 lung cancer cells contained 1.10% SP cells. SP cells showed higher abilities of sphere and colony formation, cell proliferation and self-renewal. Moreover, compared to non-SP, SP cells demonstrated a higher mRNA expression of stem cell markers (MDR1, ABCG2 and OCT-4). The clone formation efficiency of SP cells was significantly higher than that non-SP cells under the same conditions. Expression of autophagosomes in SP cells was markedly lower than that in non-SP cells. However, the level of autophagy in SP cells was found to be markedly increased in the presence of cisplatin. In addition, inhibition of autophagy enhanced the effects of apoptosis induced by cisplatin. SP cells from the A549 lung cancer cell line possessed the properties of CSCs and were used to investigate the further characteristics of lung CSCs. SP cells were more resistant to chemotherapy and inhibition of autophagy enhanced the effects of apoptosis induced by the chemotherapeutic agent, cisplatin. These results may provide insight into novel therapeutic targets.

  9. Formoxanthone C, isolated from Cratoxylum formosum ssp. pruniflorum, reverses anticancer drug resistance by inducing both apoptosis and autophagy in human A549 lung cancer cells.

    PubMed

    Kaewpiboon, Chutima; Boonnak, Nawong; Kaowinn, Sirichat; Chung, Young-Hwa

    2018-02-15

    Multidrug resistance (MDR) cancer toward cancer chemotherapy is one of the obstacles in cancer therapy. Therefore, it is of interested to use formoxanthone C (1,3,5,6-tetraoxygenated xanthone; XanX), a natural compound, which showed cytotoxicity against MDR human A549 lung cancer (A549RT-eto). The treatment with XanX induced not only apoptosis- in A549RT-eto cells, but also autophagy-cell death. Inhibition of apoptosis did not block XanX-induced autophagy in A549RT-eto cells. Furthermore, suppression of autophagy by beclin-1 small interfering RNAs (siRNAs) did not interrupt XanX-induced apoptosis, indicating that XanX can separately induce apoptosis and autophagy. Of interest, XanX treatment reduced levels of histone deacetylase 4 (HDAC4) protein overexpressed in A549RT-etocells. The co-treatment with XanX and HDAC4 siRNA accelerated both autophagy and apoptosis more than that by XanX treatment alone, suggesting survival of HDAC4 in A549RT-eto cells. XanX reverses etoposide resistance in A549RT-eto cells by induction of both autophagy and apoptosis, and confers cytotoxicity through down-regulation of HDAC4. Copyright © 2017. Published by Elsevier Ltd.

  10. Cytotoxicity of withasteroids: withametelin induces cell cycle arrest at G2/M phase and mitochondria-mediated apoptosis in non-small cell lung cancer A549 cells.

    PubMed

    Rao, Poorna Chandra; Begum, Sajeli; Jahromi, Mohammad Ali Farboodniay; Jahromi, Zahra Hosseini; Sriram, Saketh; Sahai, Mahendra

    2016-09-01

    Considerable interest has been gained by withasteroids because of their structural uniqueness and wide spectrum of biological activities. However, limited systematic studies for proving their cytotoxic potential have so far been reported. Hence, an attempt was made to test the cytotoxicity of six withasteroids viz., withametelin (WM), withaphysalin D, withaphysalin E, 12-deoxywithastramonolide, Withaperuvin B, and physalolactone against A549, HT-29, and MDA-MB-231 cancer cell lines. Significant cytotoxic effect of WM against A549 cells (IC 50 value of 6.0 μM), MDA-MB-231 cells (IC 50 value of 7.6 μM), and HT-29 cells (IC 50 value of 8.2 μM) was observed. Withaperuvin B and physalolactone were found to be effective against MDA-MB-231 cells. The significantly active WM arrested the A549 cells at G2/M phase and downregulated the expression of G2/M regulatory proteins such as cdc2, cyclin B1, and cdc25C. Apoptosis induced by WM in A549 cells was associated with the generation of ROS and depletion of MMP. Furthermore, WM treatment resulted in Bax upregulation, Bcl-2 downregulation, translocation of cytochrome c to mitochondria, activation of caspase-9 and -3, and PARP cleavage corroborating the apoptosis induction through intrinsic apoptotic pathway. Thus, WM possessing broader cytotoxic effect is a promising lead molecule which has the potential to be developed as a new therapeutic agent for NSCLC.

  11. The effects of combined treatment with sevoflurane and cisplatin on growth and invasion of human adenocarcinoma cell line A549.

    PubMed

    Liang, Hua; Wang, Han Bing; Liu, Hong Zhen; Wen, Xian Jie; Zhou, Qiao Ling; Yang, Cheng Xiang

    2013-07-01

    Sevoflurane, an inhalational anesthetic, and cisplatin (DDP)-based chemotherapy have been widely used during lung cancer surgery. However, the effect of sevoflurane on the sensitivity of lung cancer cells to DDP chemotherapy remains unclear. In this study, the effects of combined treatment with sevoflurane and cisplatin on the growth and invasion of human lung adenocarcinoma A549 cell line have been investigated. The underlying mechanism has also been explored. In our experiment, A549 cells were treated with 2.5% sevoflurane, 10μmol/L DDP, or the co-treatment of sevoflurane and DDP for 4h, respectively. Cell proliferation was evaluated by the MTT assay and colony formation assay. Apoptosis was assessed by flow cytometry. Cell invasion was detected by Transwell assay. The expressions of X-linked inhibitor of apoptosis protein (XIAP), Survivin, matrix metalloproteinase (MMP)-2 and MMP-9 were determined by western blotting. Our results showed that sevoflurane combined with DDP resulted in a more pronounced inhibition of tumor cells growth and invasion as compared with either drug alone. Besides, XIAP, Survivin, MMP-2, and MMP-9 were downregulated more significantly by the co-treatment of the two drugs as compared to sevoflurane treatment or DDP treatment alone. Taken together, the growth-inhibitory and invasion-inhibitory synergy between sevoflurane and DDP in human adenocarcinoma A549 cell line was found in this study. Furthermore, we showed that the growth-inhibitory synergy between sevoflurane and DDP might be associated with the downregulation of XIAP and Survivin, and the invasion-inhibitory synergy between sevoflurane and DDP might be involved in the downregulation of MMP-2 and MMP-9. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  12. 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

  13. 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.

  14. Theranostic-PDT with the antibody anti isoform 4 SOD mitocondrial labeled with PpIX in the lung cancer cell line A-549.

    PubMed

    Martínez-García, Claudia; Medina-Flores, Yolanda; de la Rosa-Vázquez, José Manuel; Soriano-Pérez, Eva Elda; Villalobos-Hernández, Juan Ramón; Ramón-Gallegos, Eva

    2018-03-27

    In this work, a drug product composed of an IgM antibody derived from a hybridoma subclone 4C1F6D5G7B8 was prepared and further labeled with PpIX to be used in cell lines A-549 and MRC-5. The aim of this work was to evaluate the potential theranostic activity of the obtained product together with photodynamic therapy (PDT). The IgM antibody labeled with PpIX was used in different concentrations to perform theranostics with PDT in cell lines A-549 and MRC-5 in order to identify the specificity of IgM antibody in lung cancer cells by means of a LED-irradiation system set at 630 nm. The location of the conjugate was further determined by confocal microscopy. The theranostic with conjugate Ab-PpIX in the A-549 cell lines showed fluorescence by confocal microscopy, whereas the MRC-5 cell line showed no reactivity. The PDT with the conjugate in the cell line A-549 decreased its viability 70 % compared to the control. On the contrary, with the MRC-5 cell line no viability diference was shown. The confocal microscopy applied to the cell line A-549 showed that the Ab-PpIX was majorly located at the cytoplasm. Ab-PpIX showed therapeutical potential in lung cancer cells A-549 and had no activity in non-cancerous lung cells (MCR-5). Copyright © 2018. Published by Elsevier B.V.

  15. Copper doping enhanced the oxidative stress-mediated cytotoxicity of TiO2nanoparticles in A549 cells.

    PubMed

    Ahmad, J; Siddiqui, M A; Akhtar, M J; Alhadlaq, H A; Alshamsan, A; Khan, S T; Wahab, R; Al-Khedhairy, A A; Al-Salim, A; Musarrat, J; Saquib, Q; Fareed, M; Ahamed, M

    2018-05-01

    Physicochemical properties of titanium dioxide nanoparticles (TiO 2 NPs) can be tuned by doping with metals or nonmetals. Copper (Cu) doping improved the photocatalytic behavior of TiO 2 NPs that can be applied in various fields such as environmental remediation and nanomedicine. However, interaction of Cu-doped TiO 2 NPs with human cells is scarce. This study was designed to explore the role of Cu doping in cytotoxic response of TiO 2 NPs in human lung epithelial (A549) cells. Characterization data demonstrated the presence of both TiO 2 and Cu in Cu-doped TiO 2 NPs with high-quality lattice fringes without any distortion. The size of Cu-doped TiO 2 NPs (24 nm) was lower than pure TiO 2 NPs (30 nm). Biological results showed that both pure and Cu-doped TiO 2 NPs induced cytotoxicity and oxidative stress in a dose-dependent manner. Low mitochondrial membrane potential and higher caspase-3 enzyme (apoptotic markers) activity were also observed in A549 cells exposed to pure and Cu-doped TiO 2 NPs. We further observed that cytotoxicity caused by Cu-doped TiO 2 NPs was higher than pure TiO 2 NPs. Moreover, antioxidant N-acetyl cysteine effectively prevented the reactive oxygen species generation, glutathione depletion, and cell viability reduction caused by Cu-doped TiO 2 NPs. This is the first report showing that Cu-doped TiO 2 NPs induced cytotoxicity and oxidative stress in A549 cells. This study warranted further research to explore the role of Cu doping in toxicity mechanisms of TiO 2 NPs.

  16. Flavonoids isolated from Citrus platymamma induced G2/M cell cycle arrest and apoptosis in A549 human lung cancer cells

    PubMed Central

    Nagappan, Arulkumar; Lee, Ho Jeong; Saralamma, Venu Venkatarame Gowda; Park, Hyeon Soo; Hong, Gyeong Eun; Yumnam, Silvia; Raha, Suchismita; Charles, Shobana Nancy; Shin, Sung Chul; Kim, Eun Hee; Lee, Won Sup; Kim, Gon Sup

    2016-01-01

    Citrus platymamma hort. ex Tanaka belongs to the Rutaceae family and is widely used in folk medicines in Korea due to its anti-proliferative, anti-cancer, anti-oxidant, anti-inflammatory and anti-diabetic activities. However, the molecular mechanism of its anti-cancer effect is not well understood. The present study was conducted to elucidate the anti-cancer effect and molecular mechanism of flavonoids from Citrus platymamma (FCP) on A549 cells. FCP displayed concentration-dependent inhibition on A549 cells proliferation. Further, flow cytometry revealed that FCP significantly increased the sub-G1 (apoptotic cell population) and G2/M phase population, and the total number of apoptotic cells, in a dose-dependent manner. Nuclear condensation and fragmentation were also observed upon staining with Hoechst 33342 in FCP-treated A549 cells. Immunoblotting demonstrated a dose-dependent downregulation of cyclin B1, cyclin-dependent kinase 1, cell division cycle 25c, pro-caspases −3, −6, −8 and −9, and poly (adenosine diphosphate-ribose) polymerase (PARP) in FCP-treated A549 cells. In addition, FCP induced caspase-3 activation and subsequent PARP cleavage, and increased the B-cell lymphoma (Bcl)-2-associated X protein/Bcl-extra large ratio in A549 cells. These findings suggest that FCP induced G2/M arrest and apoptosis of A549 cells. The present study provides evidence that FCP may be useful in the treatment of human lung cancer. PMID:27446443

  17. 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

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

    PubMed

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

    2004-07-01

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

  19. Telmisartan inhibits NSCLC A549 cell proliferation and migration by regulating the PI3K/AKT signaling pathway.

    PubMed

    Zhang, Suolin; Wang, Yayan

    2018-04-01

    Expression of angiotensin II (Ang II), a key biological peptide in the renin-angiotensin system, is closely associated with the occurrence and development of cancer. Ang II binds two receptor subtypes, the Ang II type 1 receptor (AT1R) and the AT2R, to mediate a series of biological effects. Telmisartan, a specific AT1R blocker, has been reported to have potential as an anticancer drug for treating renal cancer. In the present study, whether telmisartan had an effect on non-small cell lung cancer (NSCLC) cell proliferation and migration was investigated. The Cell Counting kit-8 assay revealed that telmisartan significantly inhibited the growth of the NSCLC A549 cell line in a time- and dose-dependent manner. In a transwell assay, telmisartan significantly inhibited cellular invasion and migration. Furthermore, it was determined that the expression of the anti-apoptotic protein B-cell lymphoma was decreased, and that of the pro-apoptotic proteins caspase-3 and Bcl-associated X increased in the A549 cells treated with telmisartan. Additionally, levels of phosphorylated RAC serine/threonine-protein kinase (p-AKT), p-mechanistic target of rapamycin, p70-S6 kinase and cyclin D1 was decreased in the telmisartan-treated group. Therefore, the current study reveals that telmisartan-induced NSCLC apoptosis may be regulated via the phosphoinositide 3-kinase/AKT signaling pathway, which indicates that it may be a potential novel drug for clinical NSCLC treatment.

  20. Polyurethane dispersion containing quaternized ammonium groups: An efficient nanosize gene delivery carrier for A549 cancer cell line transfection.

    PubMed

    Yousefpour Marzbali, Mahsa; Yari Khosroushahi, Ahmad; Movassaghpour, AliAkbar; Yeganeh, Hamid

    2016-01-25

    A novel polyurethane containing cationic ammonium groups (QPU) was synthesized and used as vector for gene therapy and cancer gene targeting. The synthesized QPU was characterized by Fourier transform infrared and nuclear magnetic resonance spectroscopy methods. An agarose gel retardation electrophoresis assay was conducted to verify the complete complex formation between QPU and pDNA. The particles size and zeta potential of neat polymers, plasmid DNA, polymers/DNA polyplexes were determined by the dynamic light scattering technique. The polyplexes cytotoxicity was determined using [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and its transfection efficiency was examined qualitatively by fluorescent microscopy and quantitatively by flow cytometery methods. The gel retardation assay, particle size and zeta potential measurements were confirmed that the synthesized cationic polymer could condense DNA efficiently in the physiologic condition. QPU polyplexes showed a significantly lower cytotoxicity compared to Polyfect polyplexes in the examined human cancerous (A549) or normal cells (KDR). Based on our findings, the transfection efficiency by QPU was 2.2 fold higher than Polyfect in the A549 cells whereas in the KDR cells, the cell transfection by Polyfect was 18.1 fold higher than QPU. Due to low cytotoxicity for normal cells and high transfection efficiency in cancer cells, the potential applicability of designed QPU as a non-viral gene carrier for targeting of cancer gene therapy was confirmed. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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

    PubMed

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

    2013-02-01

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

  2. Chlorogenic acid regulates apoptosis and stem cell marker-related gene expression in A549 human lung cancer cells.

    PubMed

    Yamagata, Kazuo; Izawa, Yuri; Onodera, Daiki; Tagami, Motoki

    2018-04-01

    Previous studies indicated that chlorogenic acid, a compound present in many fruits and vegetables, has anti-cancer activities. We report that chlorogenic acid regulates the expression of apoptosis-related genes and self-renewal-related stem cell markers in cancer cells. The lung cancer cell line A549 was cultured with or without chlorogenic acid. The presence of chlorogenic acid decreased cell proliferation as measured by MTT activity. Polymerase chain reaction (PCR) showed that treatment of cells with chlorogenic acid reduced the expression of BCL2 but increased that of both BAX and CASP3. Chlorogenic acid enhanced annexin V expression as measured using fluorescently labeled annexin V. Chlorogenic acid also induced p38 MAPK and JNK gene expression. Meanwhile, several agents, including SB203580 (p38 MAP kinase inhibitor), N-acetylcysteine (antioxidant inhibitor), dipyridamole (phosphodiesterase inhibitor), and apocynin (NADPH-oxidase inhibitor) blocked chlorogenic acid-induced BAX gene expression. Chlorogenic acid reduced gene expression levels of stem cell-associated markers NANOG, POU5F1, and SOX2. Together these results indicate that chlorogenic acid affects the expression of apoptosis-related genes that are part of oxidative stress and p38 MAP-dependent pathways, as well as genes encoding stem cell markers. In conclusion, chlorogenic acid may contribute to the polyphenolic anti-cancer effect associated with consumption of vegetables and fruits.

  3. 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.

  4. Part II-mechanism of adaptation: A549 cells adapt to high concentration of nitric oxide through bypass of cell cycle checkpoints.

    PubMed

    Aqil, Madeeha; Deliu, Zane; Elseth, Kim M; Shen, Grace; Xue, Jiaping; Radosevich, James A

    2014-03-01

    Previous work has shown enhanced survival capacity in high nitric oxide (HNO)-adapted tumor cells. In Part I of this series of manuscripts, we have shown that A549-HNO cells demonstrate an improved growth profile under UV and X-ray radiation treatment. These cells exhibit increased expression of proteins involved in DNA damage recognition and repair pathway, both the non-homologous end joining pathway and homologous recombination. These include Ku80, DNA-PK, XLF ligase and MRN complex proteins. Further, the A549-HNO cells show high levels of ATM, ATR, Chk1 and Chk2, and phospho-p53. Activation of these molecules may lead to cell cycle arrest and apoptosis due to DNA damage. This is observed in parent A549 cells in response to NO donor treatment; however, the A549-HNO cells proliferate and inhibit apoptosis. Cell cycle analysis showed slowed progression through S phase which will allow time for DNA repair. Thus, to better understand the increased growth rate in A549-HNO when compared to the parent cell line A549, we studied molecular mechanisms involved in cell cycle regulation in A549-HNO cells. During the initial time period of NO donor treatment, we observe high levels of cyclin/Cdk complexes involved in regulating various stages of the cell cycle. This would lead to bypass of G1-S and G2-M checkpoints. The HNO cells also show much higher expression of Cdc25A. Cdc25A activates Cdk molecules involved in different phases of the cell cycle. In addition, there is enhanced phosphorylation of the Rb protein in HNO cells. This leads to inactivation of Rb/E2F checkpoint regulating G1-S transition. This may lead to faster progression in S phase. Thus, all of these perturbations in HNO cells lead to accelerated cell cycle progression and a higher growth rate. We also assessed expression of cell cycle inhibitors in HNO cells. Interestingly, the HNO cells show a significant decline in p21CIP1 at initial time points, but with prolonged exposure, the levels were much higher

  5. Salvianolic acid A reverses cisplatin resistance in lung cancer A549 cells by targeting c-met and attenuating Akt/mTOR pathway.

    PubMed

    Tang, Xia-Li; Yan, Li; Zhu, Ling; Jiao, De-Min; Chen, Jun; Chen, Qing-Yong

    2017-09-01

    Drug resistance is one of the leading causes of chemotherapy failure in non-small cell lung cancer (NSCLC) treatment. The purpose of this study was to investigate the role of c-met in human lung cancer cisplatin resistance cell line (A549/DDP) and the reversal mechanism of salvianolic acid A (SAA), a phenolic active compound extracted from Salvia miltiorrhiza. In this study, we found that A549/DDP cells exert up-regulation of c-met by activating the Akt/mTOR signaling pathway. We also show that SAA could increase the chemotherapeutic efficacy of cisplatin, suggesting a synergistic effect of SAA and cisplatin. Moreover, we revealed that SAA enhanced sensitivity to cisplatin in A549/DDP cells mainly through suppression of the c-met/AKT/mTOR signaling pathway. Knockdown of c-met revealed similar effects as that of SAA in A549/DDP cells. In addition, SAA effectively prevented multidrug resistance associated protein1 (MDR1) up-regulation in A549/DDP cells. Taken together, our results indicated that SAA suppressed c-met expression and enhanced the sensitivity of lung adenocarcinoma A549 cells to cisplatin through AKT/mTOR signaling pathway. Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  6. Aqueous extract of Sapindus mukorossi induced cell death of A549 cells and exhibited antitumor property in vivo.

    PubMed

    Liu, Min; Chen, Yen-Lin; Kuo, Yao-Haur; Lu, Mei-Kuang; Liao, Chia-Ching

    2018-03-19

    Sapindus mukorossi is a deciduous plant and has recently been recognized to have anticancer property. In the present study, we discovered that S. mukorossi leaf and stem aqueous extract (SaM) contained two polysaccharides mainly made of myo-inositol, galactose, glucose, and fructose and the aim of this study was to investigate the antitumor property the aqueous extract SaM. In vitro treatment of SaM diminished proliferative potential of lung adenocarcinomic cells and induced intracellular oxidative stress, as well as necrotic cell death. Moreover, exposure to SaM attenuated cell migration, demonstrating the effectiveness at reducing invasive property of malignant lung cells. Gene and protein expression studies indicated that SaM treatment altered the expression of proliferation/survival modulator NF-κB, tumor growth modulator ERK2, metastasis-associated molecules MMP9/12, and tumor suppressor p53 in A549 cells. Using model animals bearing Lewis lung cancer cell LL/2, we demonstrated that SaM was antitumoral and did not induce any undesired organ damage, immunotoxicity, and off-target inflammation. This work, to our knowledge, is the first study documents the antitumor bioactivity of aqueous extract riched in polysaccharides from S. mukorossi and provides insights into the potential pharmacological application of SaM as antitumor agent against lung cancer.

  7. Screening of Stat3 inhibitory effects of Korean herbal medicines in the A549 human lung cancer cell line.

    PubMed

    Park, Jong-Shik; Bang, Ok-Sun; Kim, Jinhee

    2014-06-01

    The transcription factor signal transducer and activator of transcription 3 (Stat3) is constitutively activated in many human cancers. It promotes tumor cell proliferation, inhibits apoptosis, induces angiogenesis and metastasis, and suppresses antitumor host immune responses. Therefore, Stat3 has emerged as a promising molecular target for cancer therapies. In this study, we evaluated the Stat3-suppressive activity of 38 herbal medicines traditionally used in Korea. Medicinal herb extracts in 70% ethanol were screened for their ability to suppress Stat3 in the A549 human lung cancer cell line. A Stat3-responsive reporter assay system was used to detect intracellular Stat3 activity in extract-treated cells, and Western blot analyses were performed to measure the expression profiles of Stat3-regulated proteins. Fifty percent of the 38 extracts possessed at least mild Stat3-suppressive activities (i.e., activity less than 75% of the vehicle control). Ethanol extracts of Bupleurum falcatum L., Taraxacum officinale Weber, Solanum nigrum L., Ulmus macrocarpa Hance, Euonymus alatus Sieb., Artemisia capillaris Thunb., and Saururus chinensis (Lour.) Baill inhibited up to 75% of the vehicle control Stat3 activity level. A549 cells treated with these extracts also had reduced Bcl-xL, Survivin, c-Myc, and Mcl-1 expression. Many medicinal herbs traditionally used in Korea contain Stat3 activity-suppressing substances. Because of the therapeutic impact of Stat3 inhibition, these results could be useful when developing novel cancer therapeutics from medicinal herbs.

  8. Acrolein activates cell survival and apoptotic death responses involving the endoplasmic reticulum in A549 lung cells.

    PubMed

    Tanel, André; Pallepati, Pragathi; Bettaieb, Ahmed; Morin, Patrick; Averill-Bates, Diana A

    2014-05-01

    Acrolein, a highly reactive α,β-unsaturated aldehyde, is a product of endogenous lipid peroxidation. It is a ubiquitous environmental pollutant that is generated mainly by smoke, overheated cooking oil and vehicle exhaust. Acrolein damages cellular proteins, which could lead to accumulation of aberrantly-folded proteins in the endoplasmic reticulum (ER). This study determines the mechanisms involved in acrolein-induced apoptosis mediated by the ER and possible links with the ER stress response in human A549 lung cells. The exposure of cells to acrolein (15-50μM) for shorter times of 15 to 30min activated several ER stress markers. These included the ER chaperone protein BiP and the three ER sensors: (i) the survival/rescue molecules protein kinase RNA (PKR)-like ER kinase (PERK) and eukaryotic initiation factor 2 alpha (eIF2α) were phosphorylated; (ii) cleavage of activating transcription factor 6 (ATF6) occurred, and (iii) inositol-requiring protein-1 alpha (IRE1α) was phosphorylated. Acrolein (25-50μM) caused apoptotic cell death mediated by the ER after 2h, which was characterised by the induction of CHOP and activation of ER proteases calpain and caspase-4. Calpain and caspase-7 were the initiating factors for caspase-4 activation in acrolein-induced apoptosis. These results increase our knowledge about cellular responses to acrolein in lung cells, which have implications for human health. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Development of drug-loaded chitosan hollow nanoparticles for delivery of paclitaxel to human lung cancer A549 cells.

    PubMed

    Jiang, Jie; Liu, Ying; Wu, Chao; Qiu, Yang; Xu, Xiaoyan; Lv, Huiling; Bai, Andi; Liu, Xuan

    2017-08-01

    In this study, biodegradable chitosan hollow nanospheres (CHN) were fabricated using polystyrene nanospheres (PS) as templates. CHN were applied to increase the solubility of poorly water-soluble drugs. The lung cancer drug paclitaxel (PTX), which is used as a model drug, was loaded into CHN by the adsorption equilibrium method. The drug-loaded sample (PTX-CHN) offered sustained PTX release and good bioavailability. The state characterization of PTX by differential scanning calorimetry (DSC), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) showed that the PTX absorbed into CHN existed in an amorphous state. An in vitro toxicity experiment indicated that CHN were nontoxic as carriers of poorly water-soluble drugs. The PTX-CHN produced a marked inhibition of lung cancer A549 cells proliferation and encouraged apoptosis. A cell uptake experiment indicated that PTX-CHN was successfully taken up by lung cancer A549 cells. Furthermore, a degradation experiment revealed that CHN were readily biodegradable. These findings state clearly that CHN can be regarded as promising biomaterials for lung cancer treatment.

  10. Oxidative stress and cell cycle arrest induced by short-term exposure to dustfall PM2.5 in A549 cells.

    PubMed

    Yang, Jie; Huo, Tingting; Zhang, Xu; Ma, Jie; Wang, Yulin; Dong, Faqin; Deng, Jianjun

    2017-11-02

    It was reported that in vitro short-term exposure to PM 2.5 caused different lung diseases through inflammatory response, immune toxicity, oxidative stress, and genetic mutations. However, the complex molecular biological mechanism for its toxicity had not been fully elucidated. Therefore, the present study investigated the cytotoxicity, oxidative damage, mitochondria damage, apoptosis, and cell cycle arrest of NX and QH PM 2.5 in A549 cells. Further, cell cycle arrest-related gene levels in PM 2.5 -induced A549 cells were also detected. Our results suggested that PM 2.5 reduced the cell viability in A549 cells. Simultaneously, excessive ROS decreased MMP levels and damaged mitochondrial membrane integrity and induced mitochondrial oxidative damage through the oxygen-dependent killer route, resulting in mitochondrial damage and cell apoptosis. Besides, the results also showed that PM 2.5 induced A549 cell cycle alteration in G2/M phase after co-culture for 24 h. G2/M phase arrest was induced by upregulation of p53 and p21 and downregulation of CDK1 mRNA expression. In addition, lncRNA Sox2ot might play an important role as the specific oncogenes and it participated in G2/M phase arrest by regulating the expression of EZH 2 .

  11. Garcinol from Garcinia indica Downregulates Cancer Stem-like Cell Biomarker ALDH1A1 in Nonsmall Cell Lung Cancer A549 Cells through DDIT3 Activation.

    PubMed

    Wang, Jinhan; Wang, Liwen; Ho, Chi-Tang; Zhang, Kunsheng; Liu, Qiang; Zhao, Hui

    2017-05-10

    Nonsmall cell lung cancer (NSCLC) is the predominant type of lung cancer. Patients with NSCLC show high mortality rates because of failure to clean up cancer stem cells (CSCs). The anticancer activity of phytochemical garcinol has been identified in various cancer cell models. However, the effect of garcinol on NSCLC cell lines is still lacking. Of the NSCLC cell lines we tested, A549 cells were the most sensitive to garcinol. Interestingly, Aldehyde Dehydrogenase 1 Family Member A1 (ALDH1A1) was preferentially expressed in A549 cells and downregulated by the addition of garcinol. We also found that garcinol enriched DNA damage-inducible transcript 3 (DDIT3) and then altered DDIT3-CCAAT-enhancer-binding proteins beta (C/EBPβ) interaction resulting in a decreased binding of C/EBPβ to the endogenous ALDH1A1 promoter. Furthermore, garcinol's inhibition of ALDH1A1 was identified in a xenograft mice model. Garcinol repressed ALDH1A1 transcription in A549 cells through alterations in the interaction between DDIT3 and C/EBPβ. Garcinol could be a potential dietary phytochemical candidate for NSCLCs patients whose tumors harbored high ALDH1A1 expression.

  12. Radiosensitizing effect of schinifoline from Zanthoxylum schinifolium Sieb et Zucc on human non-small cell lung cancer A549 cells: a preliminary in vitro investigation.

    PubMed

    Wang, Cheng-Fang; Fan, Li; Tian, Mei; Qi, Xue-Song; Liu, Jian-Xiang; Feng, Jiang-Bin; Du, Shu-Shan; Su, Xu; Wang, Yong-Yan

    2014-12-01

    Schinifoline (SF), a 4-quinolinone derivative, was found in Zanthoxylum schinifolium for the first time. 4-Quinolinone moieties are thought to have cytotoxic activity and are often used as a tubulin polymerization inhibitors, heterogeneous enzyme inhibitors and antiplatelet agents. However, very little information respect to radiosensitization has focused on SF. This work aimed to investigate the radiosensitizing effect of SF on A549 cells. The cell viability results indicated cytotoxicity of SF on A549 cells, with IC50 values of 33.7 ± 2.4, 21.9 ± 1.9 and 16.8 ± 2.2 μg/mL, respectively, after 6, 12, 24 h treatment with different concentrations, and the 10% or 20% IC50 concentration during 12 h was applied in later experiments. The results of cell proliferative inhibition and clonogenic assay showed that SF enhanced the radiosensitivity of A549 cells when applied before 60Co γ-irradiation and this effect was mainly time and concentration dependent. The flow cytometric data indicated that SF treatment before the irradiation increased the G2/M phase, thus improving the radiosensitivity of A549, leading to cell apoptosis. This paper is the first study that describes the in vitro radiosensitising, cell cycle and apoptotic-inducing effects of schinifoline.

  13. Glutamine drives glutathione synthesis and contributes to radiation sensitivity of A549 and H460 lung cancer cell lines

    PubMed Central

    Sappington, Daniel R.; Siegel, Eric R.; Hiatt, Gloria; Desai, Abhishek; Penney, Rosalind B.; Jamshidi-Parsian, Azemat; Griffin, Robert J.; Boysen, Gunnar

    2016-01-01

    Background Increased glutamine uptake is known to drive cancer cell proliferation, making tumor cells glutamine-dependent. Glutamine provides additional carbon and nitrogen sources for cell growth. The first step in glutamine utilization is its conversion to glutamate by glutaminase (GLS). Glutamate is a precursor for glutathione synthesis, and we investigated the hypothesis that glutamine drives glutathione synthesis and thereby contributes to cellular defense systems. Methods The importance of glutamine for glutathione synthesis was studied in H460 and A549 lung cancer cell lines using glutamine-free medium and Bis-2-(5-phenyl-acetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) a GLS inhibitor. Metabolic activities were determined by targeted mass spectrometry. Results A significant correlation between glutamine consumption and glutathione excretion was demonstrated in H460 and A549 tumor cells. Culturing in the presence of [13C5]glutamine demonstrated that by 12 hrs >50% of excreted glutathione is derived from glutamine. Culturing in glutamine-free medium or treatment with BPTES, a glutaminase (GLS)-specific inhibitor, reduced cell proliferation and viability, and abolished glutathione excretion. Treatment with glutathione-ester prevented BPTES induced cytotoxicity. Inhibition of GLS markedly radiosensitized the lung tumor cell lines, suggesting an important role of glutamine-derived glutathione in determining radiation sensitivity. Conclusions We demonstrate here for the first time that a significant amount of extracellular glutathione is directly derived from glutamine. This finding adds yet another important function to the already known glutamine dependence of tumor cells and probably tumors as well. General significance Glutamine is essential for synthesis and excretion of glutathione to promote cell growth and viability. PMID:26825773

  14. Repeated Aurora-A siRNA Transfection Results in Effective Apoptosis of A549 Cells Compared to Single Transfection.

    PubMed

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

    2016-01-01

    Suppression of Aurora kinase A (Aurora-A, AURKA) by Aurora-A siRNA has been proposed for lung tumor treatment. However, protocols using single administration have shown little benefit in some types of lung tumor. Given that transfection efficiency of Aurora-A siRNA is low due to tightly packed cells in the tumor, we hypothesized that repeated administration would result in efficient cell apoptosis. We compared single vs. repeated transfection (thrice) in A549 cells by transfecting Aurora-A siRNA (siA) on the 1st or 1st, 2nd and 3rd day after cell seeding. A random sequence was used as the negative siRNA control (siC). Cells in the single transfection group received only transfection reagent without siRNAs on the 2nd and 3rd day. Two days after the third transfection, both single and repeated siA administration decreased mRNA expression of Aurora-A and cell viability compared to no administration and siC single administration. However, the decrease in these two indices with repeated transfection was more obvious than that following single administration: cell viability decreased to 72.8 ± 3.05% (p < 0.05) following siA single transfection and to 64.2 ± 1.99% (p < 0.05) following siA repeated transfection, compared with normal control cells, respectively. Gene expression decreased to 17 ± 16.6% (p < 0.05 vs. normal control) following siA repeated transfection and to 43.2 ± 13.0% (p < 0.05 vs. normal control) following siA single transfection. Compared to single transfection, repeated Aurora-A siRNA transfection decreased Aurora-A, which, in turn, resulted in effective apoptosis of A549 cells.

  15. The Pseudomonas aeruginosa Exopolysaccharide Psl Facilitates Surface Adherence and NF-κB Activation in A549 Cells

    PubMed Central

    Byrd, Matthew S.; Pang, Bing; Mishra, Meenu; Swords, W. Edward; Wozniak, Daniel J.

    2010-01-01

    In order for the opportunistic Gram-negative pathogen Pseudomonas aeruginosa to cause an airway infection, the pathogen interacts with epithelial cells and the overlying mucous layer. We examined the contribution of the biofilm polysaccharide Psl to epithelial cell adherence and the impact of Psl on proinflammatory signaling by flagellin. Psl has been implicated in the initial attachment of P. aeruginosa to biotic and abiotic surfaces, but its direct role in pathogenesis has not been evaluated (L. Ma, K. D. Jackson, R. M. Landry, M. R. Parsek, and D. J. Wozniak, J. Bacteriol. 188:8213–8221, 2006). Using an NF-κB luciferase reporter system in the human epithelial cell line A549, we show that both Psl and flagellin are necessary for full activation of NF-κB and production of the interleukin 8 (IL-8) chemokine. We demonstrate that Psl does not directly stimulate NF-κB activity, but indirectly as a result of increasing contact between bacterial cells and epithelial cells, it facilitates flagellin-mediated proinflammatory signaling. We confirm differential adherence of Psl and/or flagellin mutants by scanning electron microscopy and identify Psl-dependent membrane structures that may participate in adherence. Although we hypothesized that Psl would protect P. aeruginosa from recognition by the epithelial cell line A549, we instead observed a positive role for Psl in flagellin-mediated NF-κB activation, likely as a result of increasing contact between bacterial cells and epithelial cells. PMID:20802825

  16. Epigallocatechin-3-Gallate Enhances the Therapeutic Effects of Leptomycin B on Human Lung Cancer A549 Cells

    PubMed Central

    Cromie, Meghan M.; Gao, Weimin

    2015-01-01

    Our previous studies have shown Leptomycin B (LMB) is a promising antilung cancer drug. Epigallocatechin-3-gallate (EGCG) has antitumor properties but a debatable clinical application. The objective of this study is to evaluate the combination therapeutic effect of LMB and EGCG and its molecular mechanisms in human lung cancer A549 cells. Increased cytotoxicity was observed in LMB+EGCG-treated cells compared to LMB-treated cells. Elevated ROS was maximized 2 h after treatment, and LMB+EGCG-treated cells had higher ROS levels compared to LMB. N-Acetyl-L-cysteine (NAC) studies confirmed the oxidative role of LMB and/or EGCG treatment. In comparison to the control, CYP3A4, SOD, GPX1, and p21 mRNA expression levels were increased 7.1-, 2.0-, 4.6-, and 13.1-fold in LMB-treated cells, respectively, while survivin was decreased 42.6-fold. Additionally, these increases of CYP3A4, SOD, and GPX1 were significantly reduced, while p21 was significantly increased in LMB+EGCG-treated cells compared to LMB-treated cells. The qRT-PCR results for p21 and survivin were further confirmed by Western blot. Our study first shows that LMB produces ROS and is possibly metabolized by CYP3A4, GPX1, and SOD in A549 cells, and combination treatment of LMB and EGCG augments LMB-induced cytotoxicity through enhanced ROS production and the modulation of drug metabolism and p21/survivin pathways. PMID:25922640

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

    PubMed

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

    2013-11-01

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

  18. LW6, a hypoxia-inducible factor 1 inhibitor, selectively induces apoptosis in hypoxic cells through depolarization of mitochondria in A549 human lung cancer cells.

    PubMed

    Sato, Mariko; Hirose, Katsumi; Kashiwakura, Ikuo; Aoki, Masahiko; Kawaguchi, Hideo; Hatayama, Yoshiomi; Akimoto, Hiroyoshi; Narita, Yuichiro; Takai, Yoshihiro

    2015-09-01

    Hypoxia‑inducible factor 1 (HIF‑1) activates the transcription of genes that act upon the adaptation of cancer cells to hypoxia. LW6, an HIF‑1 inhibitor, was hypothesized to improve resistance to cancer therapy in hypoxic tumors by inhibiting the accumulation of HIF‑1α. A clear anti‑tumor effect under low oxygen conditions would indicate that LW6 may be an improved treatment strategy for cancer in hypoxia. In the present study, the HIF‑1 inhibition potential of LW6 on the growth and apoptosis of A549 lung cancer cells in association with oxygen availability was evaluated. LW6 was observed to inhibit the expression of HIF‑1α induced by hypoxia in A549 cells at 20 mM, independently of the von Hippel‑Lindau protein. In addition, at this concentration, LW6 induced hypoxia‑selective apoptosis together with a reduction in the mitochondrial membrane potential. The intracellular reactive oxygen species levels increased in LW6‑treated hypoxic A549 cells and LW6 induced a hypoxia‑selective increase of mitochondrial O2•‑. In conclusion, LW6 inhibited the growth of hypoxic A549 cells by affecting the mitochondria. The inhibition of the mitochondrial respiratory chain is suggested as a potentially effective strategy to target apoptosis in cancer cells.

  19. Identification of side population cells in human lung adenocarcinoma A549 cell line and elucidation of the underlying roles in lung cancer.

    PubMed

    Xie, Tong; Mo, Lingzhao; Li, Li; Mao, Naiquan; Li, Danrong; Liu, Deseng; Zuo, Chuantian; Huang, Dingming; Pan, Qi; Yang, Li; Wang, Shoufeng

    2018-04-01

    The present study aimed to isolate and characterize side population (SP) cells in the human lung cancer A549 cell line, and elucidate the molecular mechanism of SP cells underlying lung cancer. The SP and non-SP (NSP) cells in A549 cells were isolated and their differentiation was analyzed by fluorescence-activated cell sorting. An in vitro plate clone assay, Matrigel ® Transwell assay and chemoresistance analysis of the sorted SP and NSP cells were performed. In addition, the sorted SP and NSP cells were injected into BALB/c nude mice to detect their tumorigenic potential in vivo . The expression of ATP-binding cassette sub-family G member 2 (ABCG2) in transplanted tumors was detected by immunohistochemistry. The SP and NSP cells were successfully isolated. The results demonstrated that SP cells accounted for 1.09% of live A549 cells. SP cells produced SP and NSP cells, while NSP cells only produced NSP cells. In addition, SP cells formed more colonies, exhibited improved invasive ability and increased levels of chemoresistance compared with NSP cells in vitro . SP cells demonstrated a higher tumorigenic potential in BALB/c nude mice, and the number of ABCG2-positive cells in the SP xenograft tumors were significantly increased compared with that in the NSP xenograft tumors. The present study indicated that SP cells isolated from the human lung cancer A549 cell line demonstrated increased tumorigenicity, and improved invasive ability and chemoresistance compared with NSP cells. In addition, detection of ABCG2 expression may assist in predicting the chemotherapeutic outcome of patients, and serve as a target for treating lung cancer.

  20. 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

  1. TRIM25 is associated with cisplatin resistance in non-small-cell lung carcinoma A549 cell line via downregulation of 14-3-3σ.

    PubMed

    Qin, Xia; Qiu, Feng; Zou, Zhen

    2017-11-04

    Lung cancer, in particular, non-small cell lung cancer (NSCLC), is the leading cause of cancer-related mortality. Cis-Diamminedichloroplatinum (cisplatin, CDDP) as first-line chemotherapy for NSCLC, but resistance occurs frequently. We previously reported that Tripartite motif protein 25 (TRIM25) was highly expressed in cisplatin-resistant human lung adenocarcinoma A549 cells (A549/CDDP) in comparison with its parental A549 cells. Herein, we take a further step to demonstrate the association of TRIM25 and cisplatin resistance and also the underlying mechanisms. Knockdown of TRIM25 by RNA interference in A549/CDDP cells decreased half maximal inhibitory concentration (IC 50 ) values and promoted apoptosis in response to cisplatin, whereas overexpression of TRIM25 had opposite effects. More importantly, we found that concomitant knockdown of 14-3-3σ and TRIM25 absolutely reversed the decreased MDM2, increased p53, increased cleaved-Capsese3 and decreased IC 50 value induced by knockdown of TRIM25 individually, suggesting that TRIM25 mediated cisplatin resistance primarily through downregulation of 14-3-3σ. Our results indicate that TRIM25 is associated with cisplatin resistance and 14-3-3σ-MDM2-p53 signaling pathway is involved in this process, suggesting targeting TRIM25 may be a potential strategy for the reversal of cisplatin resistance. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. 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.

  3. 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. Copyright © 2011 Elsevier GmbH. All rights reserved.

  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. 4-methoxychalcone enhances cisplatin-induced oxidative stress and cytotoxicity by inhibiting the Nrf2/ARE-mediated defense mechanism in A549 lung cancer cells.

    PubMed

    Lim, Juhee; Lee, Sung Ho; Cho, Sera; Lee, Ik-Soo; Kang, Bok Yun; Choi, Hyun Jin

    2013-10-01

    Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcriptional regulator for the protection of cells against oxidative and xenobiotic stresses. Recent studies have demonstrated that high constitutive expression of Nrf2 is observed in many types of cancer cells showing resistance to anti-cancer drugs, suggesting that the suppression of overexpressed Nrf2 could be an attractive therapeutic strategy to overcome cancer drug resistance. In the present study, we aimed to find small molecule compounds that enhance the sensitivity of tumor cells to cisplatin induced cytotoxicity by suppressing Nrf2-mediated defense mechanism. A549 lung cancer cells were shown to be more resistant to the anti-cancer drug cisplatin than HEK293 cells, with higher Nrf2 signaling activity; constitutively high amounts of Nrf2-downstream target proteins were observed in A549 cells. Among the three chalcone derivatives 4-methoxy-chalcone (4-MC), hesperidin methylchalcone, and neohesperidin dihydrochalcone, 4-MC was found to suppress transcriptional activity of Nrf2 in A549 cells but to activate it in HEK293 cells. 4-MC was also shown to down-regulate expression of Nrf2 and the downstream phase II detoxifying enzyme NQO1 in A549 cells. The PI3K/Akt pathway was found to be involved in the 4-MC-induced inhibition of Nrf2/ARE activity in A549 cells. This inhibition of Nrf2 signaling results in the accelerated generation of reactive oxygen species and exacerbation of cytotoxicity in cisplatin-treated A549 cells. Taken together, these results suggest that the small molecule compound 4-MC could be used to enhance the sensitivity of tumor cells to the therapeutic effect of cisplatin through the regulation of Nrf2/ARE signaling.

  6. 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.

  7. Inhibitory effect of liquiritigenin on migration via downregulation proMMP-2 and PI3K/Akt signaling pathway in human lung adenocarcinoma A549 cells.

    PubMed

    Wang, Yu; Xie, Sirou; Liu, Changwei; Wu, Yuchen; Liu, Yuxin; Cai, Yunqing

    2012-01-01

    Liquiritigenin (LQ) is a flavanone extracted from Glycyrrhizae, which has multiple biological effects, such as antiinflammation and anticancer. This study is the first to investigate the effect of LQ on the migration of human lung adenocarcinoma A549 cells in vitro. First, LQ exhibited inhibitory effects on the adhesion and migration of A549 cells in the absence of cytotoxicity. Gelatin zymography and Western blot analysis showed that LQ significantly reduced the expression of promatrix metalloproteinase-2 (proMMP-2) in A549 cells in terms of both activity and protein level. Second, LQ inhibited the phosphorylation of Akt and activated the phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Furthermore, the treatment of inhibitors specific for Akt (LY294002) and ERK1/2 (U0126) to A549 cells resulted in reduced activity of proMMP-2. These results suggested that the inhibition on proMMP-2 expression by LQ may be through suppression on PI3K/Akt signaling pathway, which in turn led to the inhibition of lung adenocarcinoma A549 cells migration. However, activation of ERK might not be involved in the regulation of proMMP-2. Taken together, LQ may be considered as a potential interfering agent of cancer progression.

  8. Depletion of hepatoma-derived growth factor-related protein-3 induces apoptotic sensitization of radioresistant A549 cells via reactive oxygen species-dependent p53 activation

    SciTech Connect

    Yun, Hong Shik; Hong, Eun-Hee; Department of Chemistry, College of Natural Sciences, Hanyang University, Seoul 133-791

    2013-09-27

    Highlights: •HRP-3 is a radiation- and anticancer drug-responsive protein in A549 cells. •Depletion of HRP-3 induces apoptosis of radio- and chemoresistant A549 cells. •Depletion of HRP-3 promotes ROS generation via inhibition of the Nrf2/HO-1 pathway. •Depletion of HRP-3 enhances ROS-dependent p53 activation and PUMA expression. -- Abstract: Biomarkers based on functional signaling have the potential to provide greater insight into the pathogenesis of cancer and may offer additional targets for anticancer therapeutics. Here, we identified hepatoma-derived growth factor-related protein-3 (HRP-3) as a radioresistance-related gene and characterized the molecular mechanism by which its encoded protein regulates the radio- and chemoresistant phenotypemore » of lung cancer-derived A549 cells. Knockdown of HRP-3 promoted apoptosis of A549 cells and potentiated the apoptosis-inducing action of radio- and chemotherapy. This increase in apoptosis was associated with a substantial generation of reactive oxygen species (ROS) that was attributable to inhibition of the Nrf2/HO-1 antioxidant pathway and resulted in enhanced ROS-dependent p53 activation and p53-dependent expression of PUMA (p53 upregulated modulator of apoptosis). Therefore, the HRP-3/Nrf2/HO-1/ROS/p53/PUMA cascade is an essential feature of the A549 cell phenotype and a potential radiotherapy target, extending the range of targets in multimodal therapies against lung cancer.« less

  9. APE1 modulates cellular responses to organophosphate pesticide-induced oxidative damage in non-small cell lung carcinoma A549 cells.

    PubMed

    Thakur, Shweta; Dhiman, Monisha; Mantha, Anil K

    2018-04-01

    Monocrotophos (MCP) and chlorpyrifos (CP) are widely used organophosphate pesticides (OPPs), speculated to be linked with human pathologies including cancer. Owing to the fact that lung cells are most vulnerable to the environmental toxins, the development and progression of lung cancer can be caused by the exposure of OPPs. The present study investigates the oxidative DNA damage response evoked by MCP and CP in human non-small cell lung carcinoma A549 cells. A549 cells were exposed to MCP and CP; cytotoxicity and reactive oxygen species (ROS) generation were measured to select the non-toxic dose. In order to establish whether MCP and CP can initiate the DNA repair and cell survival signalling pathways in A549 cells, qRT-PCR and Western blotting techniques were used to investigate the mRNA and protein expression levels of DNA base excision repair (BER)-pathway enzymes and transcription factors (TFs) involved in cell survival mechanisms. A significant increase in cell viability and ROS generation was observed when exposed to low and moderate doses of MCP and CP at different time points (24, 48 and 72 h) studied. A549 cells displayed a dose-dependent accumulation of apurinic/apyrimidinic (AP) sites after 24 h exposure to MCP advocating for the activation of AP endonuclease-mediated DNA BER-pathway. Cellular responses to MCP- and CP-induced oxidative stress resulted in an imbalance in the mRNA and protein expression of BER-pathway enzymes, viz. PARP1, OGG1, APE1, XRCC1, DNA pol β and DNA ligase III α at different time points. The treatment of OPPs resulted in the upregulation of TFs, viz. Nrf2, c-jun, phospho-c-jun and inducible nitric oxide synthase. Immunofluorescent confocal imaging of A549 cells indicated that MCP and CP induces the translocation of APE1 within the cytoplasm at an early 6 h time point, whereas it promotes nuclear localization after 24 h of treatment, which suggests that APE1 subcellular distribution is dynamically regulated in response to

  10. Heat-Modified Citrus Pectin Induces Apoptosis-Like Cell Death and Autophagy in HepG2 and A549 Cancer Cells

    PubMed Central

    Leclere, Lionel; Fransolet, Maude; Cote, Francois; Cambier, Pierre; Arnould, Thierry; Van Cutsem, Pierre; Michiels, Carine

    2015-01-01

    Cancer is still one of the leading causes of death worldwide, and finding new treatments remains a major challenge. Previous studies showed that modified forms of pectin, a complex polysaccharide present in the primary plant cell wall, possess anticancer properties. Nevertheless, the mechanism of action of modified pectin and the pathways involved are unclear. Here, we show that citrus pectin modified by heat treatment induced cell death in HepG2 and A549 cells. The induced cell death differs from classical apoptosis because no DNA cleavage was observed. In addition, Z-VAD-fmk, a pan-caspase inhibitor, did not influence the observed cell death in HepG2 cells but appeared to be partly protective in A549 cells, indicating that heat-modified citrus pectin might induce caspase-independent cell death. An increase in the abundance of the phosphatidylethanolamine-conjugated Light Chain 3 (LC3) protein and a decrease in p62 protein abundance were observed in both cell types when incubated in the presence of heat-modified citrus pectin. These results indicate the activation of autophagy. To our knowledge, this is the first time that autophagy has been revealed in cells incubated in the presence of a modified form of pectin. This autophagy activation appears to be protective, at least for A549 cells, because its inhibition with 3-methyladenine increased the observed modified pectin-induced cytotoxicity. This study confirms the potential of modified pectin to improve chemotherapeutic cancer treatments. PMID:25794149

  11. Heat-modified citrus pectin induces apoptosis-like cell death and autophagy in HepG2 and A549 cancer cells.

    PubMed

    Leclere, Lionel; Fransolet, Maude; Cote, Francois; Cambier, Pierre; Arnould, Thierry; Van Cutsem, Pierre; Michiels, Carine

    2015-01-01

    Cancer is still one of the leading causes of death worldwide, and finding new treatments remains a major challenge. Previous studies showed that modified forms of pectin, a complex polysaccharide present in the primary plant cell wall, possess anticancer properties. Nevertheless, the mechanism of action of modified pectin and the pathways involved are unclear. Here, we show that citrus pectin modified by heat treatment induced cell death in HepG2 and A549 cells. The induced cell death differs from classical apoptosis because no DNA cleavage was observed. In addition, Z-VAD-fmk, a pan-caspase inhibitor, did not influence the observed cell death in HepG2 cells but appeared to be partly protective in A549 cells, indicating that heat-modified citrus pectin might induce caspase-independent cell death. An increase in the abundance of the phosphatidylethanolamine-conjugated Light Chain 3 (LC3) protein and a decrease in p62 protein abundance were observed in both cell types when incubated in the presence of heat-modified citrus pectin. These results indicate the activation of autophagy. To our knowledge, this is the first time that autophagy has been revealed in cells incubated in the presence of a modified form of pectin. This autophagy activation appears to be protective, at least for A549 cells, because its inhibition with 3-methyladenine increased the observed modified pectin-induced cytotoxicity. This study confirms the potential of modified pectin to improve chemotherapeutic cancer treatments.

  12. Cell division cycle 25 homolog c effects on low-dose hyper-radiosensitivity and induced radioresistance at elevated dosage in A549 cells.

    PubMed

    Zhao, Yanxia; Cui, Yingshan; Han, Jun; Ren, Jinghua; Wu, Gang; Cheng, Jing

    2012-09-01

    The underlying mechanisms behind both low-dose hyper-radiosensitivity (HRS) and induced radioresistance (IRR), generally occurring at elevated radiation levels, remain unclear; however, elucidation of the relationship between cell cycle division 25 homolog c (Cdc25c) phosphatase and HRS/IRR may provide important insights into this process. Two cell lines with disparate HRS status, A549 and SiHa cells, were selected as cell models for comparison of dose-dependent Cdc25c phosphatase expression subsequent to low-dose irradiation. Knockdown of Cdc25c in A549 cells was mediated by transfection with a pGCsi-RAN-U6neo vector containing hairpin siRNA sequences. S216-phosphorylated Cdc25c protein [p-Cdc25c (Ser216)], cell survival and mitotic ratio were measured by western blot, colony-forming assay and histone H3 phosphorylation analysis. Variant p-Cdc25c (Ser216) expression was observed in the two cell lines after irradiation. The p-Cdc25c (Ser216) expression noted in SiHa cells after administration of 0-1 Gy radiation was similar to the radioresistance model; however, in A549 cells, the dose response for the phosphorylation of the Cdc25c Ser216 residue overlapped the level required to overcome the HRS response. Furthermore, Cdc25c repression prior to low-dose radiation induced more distinct HRS and prevented the development of IRR. The dose required to overcome the HRS response coincided with the effect of early G2-phase checkpoint arrest in A549 cells (approximately 0.3 Gy), and Cdc25c knockdown in A549 cells (approximately 0.5 Gy) corresponded to the phosphorylation of the Cdc25c Ser216 residue. Resultant data confirmed that dose-dependent Cdc25c phosphatase does effectively act as an early G2-phase checkpoint, thus indicating mechanistic importance in the HRS to IRR transition in A549 cells.

  13. Deguelin inhibits the migration and invasion of lung cancer A549 and H460 cells via regulating actin cytoskeleton rearrangement.

    PubMed

    Zhao, Honggang; Jiao, Yan; Zhang, Zuncheng

    2015-01-01

    Deguelin, the main components from Mundulea sericea, was reported to suppress the growth of various cancer cells. However, the effect of Deguelin on tumor cell invasion and metastasis and its mechanism still unclear so far. In this study, we investigated the effects of Deguelin on the cell invasion in human lung cancer A549 and H460 cells. Our results demonstrate that Deguelin can significantly inhibited cell proliferation, cell migration and cell invasion. Moreover, Deguelin could also affected reorganization of the actin cytoskeleton and decreased filopodia and lamellipodia formation. Furthermore, deguelin-treated tumors showed decreased the tumor metastasis related genes such as CD44, MMP2 and MMP9 at protein and mRNA levels and the content of CEA, SCC, NSE, CYFAR21-1. In addition, Deguelin down-regulated protein expression of Rac1 and Rock1, which are impotent in actin cytoskeleton rearrangements and cell motility. Together, our results suggest that Deguelin inhibit tumor growth and metastasis of lung cancer cells and might be a candidate compound for curing lung cancer.

  14. Targeted interfering DEP domain containing 1 protein induces apoptosis in A549 lung adenocarcinoma cells through the NF-κB signaling pathway.

    PubMed

    Wang, Qingqing; Li, Aili; Jin, Junfei; Huang, Guojin

    2017-01-01

    Ectopic expression of DEP domain containing 1 (DEPDC1) in lung adenocarcinomas is associated with poor prognosis, but its role and the underlying mechanism remain unknown. In this study, DEPDC1 expression in lung cancer cell lines was examined with Western blot assay, and DEPDC1-positive cell A549 was selected for further experiments. DEPDC1 inhibitor miR-130a was overexpressed in A549 cells, and the proliferation and apoptosis of these cells were analyzed with cell counting and flow cytometry assay. Interfering peptide 11R-DEP:611-628 and JNK inhibitor SP600125 were used alone or in combination to treat A549 cells, and the cell proliferation and apoptosis were assessed by flow cytometry assay; caspase 3 and cleaved caspase 3, phosphor-JNK, and total JNK were detected by Western blotting; and nuclear factor kappa B (NF-κB) localization was determined by immunofluorescence staining. We found that miR-130a and 11R-DEP:611-628 peptides (5 μM) both inhibited A549 proliferation and induced apoptosis. We observed that 11R-DEP:611-628 peptide treatment resulted in elevated A20 expression, dramatically reduced nuclear NF-κB, and increased phosphor-JNK. These findings indicate that DEPDC1 inhibits apoptosis of A549 cell by suppressing A20 expression to regulate NF-κB activity, and that JNK plays a protective role upon 11R-DEP:611-628 peptide treatment. In conclusion, DEPDC1 might be a novel therapeutic target for lung cancer, and the 11R-DEP:611-628 peptide is a potent apoptosis inducer in A549 cells.

  15. Targeted interfering DEP domain containing 1 protein induces apoptosis in A549 lung adenocarcinoma cells through the NF-κB signaling pathway

    PubMed Central

    Wang, Qingqing; Li, Aili; Jin, Junfei; Huang, Guojin

    2017-01-01

    Ectopic expression of DEP domain containing 1 (DEPDC1) in lung adenocarcinomas is associated with poor prognosis, but its role and the underlying mechanism remain unknown. In this study, DEPDC1 expression in lung cancer cell lines was examined with Western blot assay, and DEPDC1-positive cell A549 was selected for further experiments. DEPDC1 inhibitor miR-130a was overexpressed in A549 cells, and the proliferation and apoptosis of these cells were analyzed with cell counting and flow cytometry assay. Interfering peptide 11R-DEP:611–628 and JNK inhibitor SP600125 were used alone or in combination to treat A549 cells, and the cell proliferation and apoptosis were assessed by flow cytometry assay; caspase 3 and cleaved caspase 3, phosphor-JNK, and total JNK were detected by Western blotting; and nuclear factor kappa B (NF-κB) localization was determined by immunofluorescence staining. We found that miR-130a and 11R-DEP:611–628 peptides (5 μM) both inhibited A549 proliferation and induced apoptosis. We observed that 11R-DEP:611–628 peptide treatment resulted in elevated A20 expression, dramatically reduced nuclear NF-κB, and increased phosphor-JNK. These findings indicate that DEPDC1 inhibits apoptosis of A549 cell by suppressing A20 expression to regulate NF-κB activity, and that JNK plays a protective role upon 11R-DEP:611–628 peptide treatment. In conclusion, DEPDC1 might be a novel therapeutic target for lung cancer, and the 11R-DEP:611–628 peptide is a potent apoptosis inducer in A549 cells. PMID:28979136

  16. On the cytotoxicity of some microbial volatile organic compounds as studied in the human lung cell line A549.

    PubMed

    Kreja, Ludwika; Seidel, Hans-Joachim

    2002-10-01

    The cytotoxicity of 13 microbial volatile organic compounds (MVOC) was studied using a human lung carcinoma epithelial cell line A549 in a colony formation assay and two colorimetric assays: the microculture tetrazolium assay (MTT assay) and the cellular protein assay (methylene blue-MB assay). For comparison, two known cytotoxic substances: the non-volatile mycotoxin gliotoxin and the mono-functional alkylating agent methyl methanesulfonate (MMS) were studied. Concentration-response curves for each agent were established and the IC50 value (concentration resulting in 50% inhibition of colony growth or absorbance) was estimated. There are differences in toxicity levels between the MVOC tested and gliotoxin and MMS. The most toxic MVOC was 1-decanol which was as effective as MMS in all test systems. 1-decanol was about 10-fold more toxic than the other MVOC. All MVOC tested were more than 1000-fold less toxic than gliotoxin.

  17. Effects of quercetin on CDK4 mRNA and protein expression in A549 cells infected by H1N1

    PubMed Central

    WAN, QIAOFENG; WANG, HAO; LIN, YUAN; GU, LIGANG; HAN, MEI; YANG, ZHIWEI; ZHANG, YANLI; MA, RUI; WANG, LI; WANG, ZHISHENG

    2013-01-01

    This study was conducted to investigate the effects of quercetin on the expression of cyclin-dependent kinase (CDK4) mRNA and protein in A549 lung epithelial tumor cells infected by H1N1. First, the Thiazolyl Blue Tetrazolium Bromide (MTT) method was used to determine H1N1 virulence, quercetin cytotoxicity and inhibition of the cytopathic effect of H1N1 on A549 cells by quercetin. Subsequently, 100 TCID50 H1N1 was used to infect A549 cells for 2 h prior to culture in maintenance media containing 10 mg/l quercetin. After 4, 12, 24 and 48 h of culture, the cells were collected and total RNA and protein were extracted. Fluorescent quantitative polymerase chain reaction and western blot analysis were then performed to assess the expression of CDK4 mRNA and protein. The experiment demonstrated that the TCID50 of H1N1 in A549 cells was 10−4.75, the maximum non-toxic concentration of quercetin in A549 cells was 30–60 mg/l and the minimum effective concentration of quercetin for the inhibition of the H1N1 cytopathic effect on A549 cells was 10 mg/l. The results indicated that quercetin may significantly inhibit CDK4 mRNA and protein overexpression caused by H1N1 within 4–48 h. In conclusion, quercetin may protect against H1N1 infection by effectively reducing the mRNA and protein expression of CDK4 caused by H1N1 infection. PMID:24649026

  18. 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

    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 treatmentsmore » 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

  19. 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.

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

    PubMed

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

    2018-02-01

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

  1. SGK1 inhibits PM2.5-induced apoptosis and oxidative stress in human lung alveolar epithelial A549cells.

    PubMed

    Li, Jin; Zhou, Qiulian; Yang, Tingting; Li, Yongqin; Zhang, Yuhui; Wang, Jinhua; Jiao, Zheng

    2018-02-19

    Emerging evidence demonstrated that particulate matter 2.5 (PM2.5) is an important environmental risk factor for lung diseases. Serum- and glucocorticoid-inducible kinase 1(SGK1) was reported to be a crucial factor for cell survival. However, the role of SGK1 in PM2.5-induced cell injury is still unclear. In this work, we firstly found that the expression of SGK1 was decreased in PM2.5-treated human lung alveolar epithelial (A549) cells by western blot. In addition, overexpression of SGK1 significantly attenuated A549cell apoptosis and reduced the reactive oxygen species (ROS) generation induced by PM2.5. Moreover, we found that PM2.5 exposure significantly promoted the ERK1/2 activation and inhibited the AKT activation, whereas overexpression of SGK1 could reverse that. Finally, the results of the rescue experiment showed that MK2206 (AKT inhibitor) could rescue the impact of SGK1 on A549cell apoptosis, while PD98059 (ERK1/2 inhibitor) could not further aggravate the impact. Taken together, our results suggest that SGK1 inhibits PM2.5-induced cell apoptosis and ROS generation via ERK1/2 and AKT signaling pathway in human lung alveolar epithelial A549cells. Copyright © 2018 Elsevier Inc. All rights reserved.

  2. 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.

  3. 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.

  4. 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.

  5. Hypoxia-inducible factor 1 promoter-induced JAB1 overexpression enhances chemotherapeutic sensitivity of lung cancer cell line A549 in an anoxic environment.

    PubMed

    Hu, Ming-Dong; Xu, Jian-Cheng; Fan, Ye; Xie, Qi-Chao; Li, Qi; Zhou, Chang-Xi; Mao, Mei; Yang, Yu

    2012-01-01

    The presence of lung cancer cells in anoxic zones is a key cause od chemotherapeutic resistance. Thus, it is necessary to enhance the sensitivity of such lung cancer cells. However, loss of efficient gene therapeutic targeting and inefficient objective gene expression in the anoxic zone in lung cancer are dilemmas. In the present study, a eukaryotic expression plasmid pUC57-HRE-JAB1 driven by a hypoxia response elements promoter was constructed and introduced into lung cancer cell line A549. The cells were then exposed to a chemotherapeutic drug cis-diamminedichloroplatinum (C-DDP). qRT-PCR and western blotting were used to determine the mRNA and protein level and flow cytometry to examine the cell cycle and apoptosis of A549 transfected pUC57-HRE-JAB1. The results showed that JAB1 gene in the A549 was overexpressed after the transfection, cell proliferation being arrested in G1 phase and the apoptosis ratio significantly increased. Importantly, introduction of pUC57-HRE-JAB1 significantly increased the chemotherapeutic sensitivity of A549 in an anoxic environment. In conclusion, JAB1 overexpression might provide a novel strategy to overcome chemotherapeutic resistance in lung cancer.

  6. 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.

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

    PubMed

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

    2016-09-01

    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-7h 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. Copyright © 2016. Published by Elsevier Inc.

  8. Bio-fabrication of catalytic platinum nanoparticles and their in vitro efficacy against lungs cancer cells line (A549).

    PubMed

    Ullah, Sadeeq; Ahmad, Aftab; Wang, Aoke; Raza, Muslim; Jan, Amin Ullah; Tahir, Kamran; Rahman, Aziz Ur; Qipeng, Yuan

    2017-08-01

    Platinum based drugs are considered as effective agents against various types of carcinoma; however, the severe toxicity associated with the chemically prepared platinum complexes limit their practical applications. Similarly, water pollution caused by various organic moieties is another serious health problem worldwide. Hence, an intense need exists to develop new, effective and biocompatible materials with catalytic and biomedical applications. In the present contribution, we prepared platinum nanoparticles (PtNPs) by a green route using phytochemicals as a source of reducing and stabilizing agents. Well dispersed and crystalline PtNPs of spherical shapes were prepared and characterized. The bio-fabricated PtNPs were used as catalyst and anticancer agents. Catalytic performance of the PtNPs showed that 84% of the methylene blue can be reduced in 32min under visible light irradiation (K=0.078min -1 ). Similarly the catalytic conversion of 4-nitrophenol to 4-aminophenol was achieved in <20min (K=0.124min -1 ). The in vitro anticancer study revealed that biogenic PtNPs are the efficient nano-agents possessing strong anticancer activity against the lungs cancer cells line (A549). Interestingly, the as prepared PtNPs were well tolerated by normal human cells, and therefore, could be effective and biocompatible agents in the treatment of different cancer cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. 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.

  10. In vitro inflammatory effects of polyhexamethylene biguanide through NF-κB activation in A549 cells.

    PubMed

    Kim, Ha Ryong; Shin, Da Young; Chung, Kyu Hyuck

    2017-02-01

    Polyhexamethylene biguanide (PHMB) is a member of the polymeric guanidine family, which is used as a biocide and preservative in industrial, medicinal, and consumer products. Some studies reported that polyhexamethylene guanidine phosphate, which is also a member of the guanidine family, induced severe inflammation and fibrosis in the lungs. However, limited studies have evaluated the pulmonary toxicity of PHMB associated with inflammatory responses. The aim of this study was to elucidate the inflammatory responses and its mechanisms induced by PHMB in lung cells. A549 cells exposed to PHMB showed decreased viability, reactive oxygen species (ROS) generation, inflammatory cytokine secretion, and nuclear factor kappa B (NF-κB) activation. The cells showed dose-dependent cytotoxicity and slight generation of ROS. PHMB triggered inflammatory cytokine secretion and NF-κB activation by modulating the degradation of IκB-α and the accumulation of nuclear p65. TNF-α plays important roles in IL-8 expression as well as NF-κB activation. Moreover, IL-8 production induced by PHMB was completely suppressed by a NF-κB inhibitor, but not by a ROS scavenger. In conclusion, we suggest that PHMB induces the inflammatory responses via the NF-κB signaling pathway. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Effective deactivation of A549 tumor cells in vitro and in vivo by RGD-decorated chitosan-functionalized single-walled carbon nanotube loading docetaxel.

    PubMed

    Li, Bin; Zhang, Xiao-Xue; Huang, Hao-Yan; Chen, Li-Qing; Cui, Jing-Hao; Liu, Yanli; Jin, Hehua; Lee, Beom-Jin; Cao, Qing-Ri

    2018-03-10

    This study aims to construct and evaluate RGD-decorated chitosan (CS)-functionalized pH-responsive single-walled carbon nanotube (SWCNT) carriers using docetaxel (DTX) as a model anticancer drug. DTX was loaded onto SWCNT via π-π stacking interaction (SWCNT-DTX), followed by the non-covalent conjugation of RGD-decorated CS to SWCNT-DTX to prepare RGD-CS-SWCNT-DTX. The RGD-CS-SWCNT-DTX showed significantly higher drug release than the pure drug, giving higher release rate at pH 5.0 (68%) than pH 7.4 (49%). The RGD-CS-SWCNT-DTX could significantly inhibit the growth of A549 tumor cells in vitro, and the uptake amount of A549 cells was obviously higher than that of MCF-7 cells. Meanwhile, the cellular uptake of RGD-CS-SWCNT-DTX was higher than that of CS-SWCNT-DTX in A549 cells, mainly through clathrin and caveolae-mediated endocytosis. The RGD-CS-SWCNT-DTX significantly inhibited tumor growth of A549 cell-bearing nude mice through active tumor-targeting ability. Furthermore, no pathological changes were found in tissues and organs. The result demonstrated that RGD-CS-SWCNT-DTX displayed high drug loading, pH-responsive drug release, remarkable antitumor effect in vitro and in vivo, and also good safety to animal body. Copyright © 2018 Elsevier B.V. All rights reserved.

  12. AMP-activated protein kinase (AMPK) activation is involved in chrysin-induced growth inhibition and apoptosis in cultured A549 lung cancer cells.

    PubMed

    Shao, Jun-jie; Zhang, Ai-ping; Qin, Wei; Zheng, Lin; Zhu, Yi-fan; Chen, Xin

    2012-07-06

    Here we show that chrysin induces growth inhibition and apoptosis in cultured lung cancer A549 cells, and activation of AMP-activated protein kinase (AMPK) may contribute to this process. Our Western-blots results demonstrated a significant AMPK activation after chrysin treatment in A549 cells. Inhibition of AMPK by shRNA-mediated gene silencing, or by its inhibitor, diminished chrysin-induced A549 cell growth inhibition and apoptosis. Forced activation of AMPK by introducing a constitutively active form of AMPKα (CA-AMPKα), or by its activators, mimicked chrysin's effect. For mechanism analysis, we found chrysin inhibited Akt/mammalian target of rapamycin (mTOR) activation, and knocking-down of AMPK by shRNA almost reversed this effect. Finally, we observed that a relative low dose of chrysin enhanced doxorubicin-induced AMPK activation to promote A549 cell apoptosis. Our study suggests that activation of AMPK by chrysin contributes to Akt suppression, growth inhibition and apoptosis in human lung cancer cells, and agents that could activate AMPK may serve as useful adjuvants for traditional chemotherapy against lung cancer. Copyright © 2012 Elsevier Inc. All rights reserved.

  13. The influence of incubation time on adenovirus quantitation in A549 cells by most probable number

    EPA Science Inventory

    Cell culture based assays used to detect waterborne viruses typically call for incubating the sample for at least two weeks in order to ensure that all the culturable virus present is detected. Historically, this estimate was based, at least in part, on the length of time used fo...

  14. 2,3,5,4-tetrahydroxy diphenylethylene-2-O-glucoside inhibits the adhesion and invasion of A549 human lung cancer cells

    PubMed Central

    Xu, Ming; Wang, Cong; Zhu, Minglin; Wang, Xianguo; Zhang, Li; Zhao, Jinping

    2017-01-01

    Lung cancer is considered to be a serious disease that poses a significant threat to human health. 2,3,5,4-tetrahydroxy diphenylethylene-2-O-glucoside (THSG) is a bioactive compound derived from Polygonum multiflorum Thunb. That has been demonstrated to possess antioxidative, anti-inflammatory and antitumor activities. However, little is currently known regarding the potential anticancer effects of this compound in lung cancer. Therefore, the present study aimed to investigate the effects of THSG on the adhesion and invasion of A549 human lung cancer cells in vitro, and to identify the putative mechanisms involved. Cell Counting kit-8 assay was performed to determine A549 cell viability following treatment with various doses (0, 5, 10, 25, 50, 100, 150 and 200 µM) of THSG for 12, 24 and 48 h. In addition, cell adhesion and invasion were determined following treatment of A549 cells with 0, 10, 25 or 50 µM THSG for 1, 2 or 3 h, respectively. Reverse transcription-quantitative polymerase chain reaction analysis was performed to examine the mRNA expression levels of Snail, E-cadherin, vimentin, matrix metalloproteinase (MMP) 2 and MMP9 following THSG treatment for 12 h. Western blot analysis was conducted to detect the protein expression levels of Snail, E-cadherin, vimentin, MMP2 and MMP9 following THSG treatment for 24 h. Treatment with THSG (10, 25 and 50 µM) significantly suppressed the adhesion and invasion of A549 human lung cancer cells in a dose-dependent manner. In addition, the mRNA and protein expression levels of adhesion and invasion-associated factors were decreased significantly in A549 cells treated with THSG. In conclusion, THSG effectively suppressed the adhesion and invasion of human lung cancer cells potentially by inhibiting the expression of adhesion and invasion-related genes. PMID:28990072

  15. 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.

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

    PubMed

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

    2016-01-15

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

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

    PubMed

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

    2013-12-01

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

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

    PubMed

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

    2017-04-21

    Tuberculosis (TB) is a major global health problem, and there is an association between tobacco smoke and TB. Water pipe smoking has become an increasing problem not only in Middle Eastern countries but also globally because users consider it as safer than cigarettes. The presence of high levels of toxic substances in water-pipe smoke may be a predisposing factor that enhances the incidence of pulmonary disorders. For example, uncontrolled macropinocytosis in alveolar epithelial cells following exposure to water-pipe smoke may predispose subjects to pulmonary infection. Here, we studied the effects of water-pipe condense (WPC) on the internalization of Mycobacterium Bovis BCG by macropinocytosis in the alveolar epithelial cell line A549. A549 cells were exposed to WPC (4 mg/ml) for 24, 48, 72 and 96 h. Cell viability was studied using the methyl thiazolyldipenyl-tetrazolium bromide (MTT) reduction assay and proliferation by bromodeoxyUridine (BrdU) incorporation. Cells were exposed to FITC-Dextran (1 mg/ml) (as a control) and FITC-BCG (MOI = 10) for 20 min at 37 °C before cells were collected and the uptake of BCG-FITC determined by flow cytometry. Similar experiments were performed at 4 °C as a control. The Rho-associated protein kinase (ROCK) inhibitor Y-27632 (1 μM) was used to assess the mechanism by which WPC enhanced BCG uptake. WPC (4 mg/ml) increased the uptake of BCG-FITC after 72 (1.3 ± 0.1 fold, p < 0.05) and 96 (1.4 ± 0.05 fold, p < 0.05) hours. No effect on BCG-FITC uptake was observed at 24 or 48 h. WPC also significantly increased the uptake of FITC-Dextran (2.9 ± 0.3 fold, p < 0.05) after 24 h. WPC significantly decreased cell viability after 24 (84 ± 2%, p < 0.05), 48 (78±, 3%, p < 0.05), 72 (64 ± 2%, p < 0.05) and 96 h (45 ± 2%, p < 0.05). Y-27632 completely attenuated the increased uptake of BCG by WPC. Cell proliferation showed a decreasing trend in a time

  19. Enhanced cytotoxic activity of endophytic bacterial extracts fromAdhatoda beddomeileaves in A549 lung cancer cell lines.

    PubMed

    Swarnalatha, Y; Saha, Bhaswti

    2016-01-01

    The current study is aimed to isolate and study the efficacy of the anticancer activity of endophytic bacteria from adhatoda beddomei leaves. Endophytic bacteria, microorganisms can found in the plant tissues, like leaves, branches and roots and able to produce various novel secondary metabolites for the medicinal applications. Endophytic bacteria were isolated from the leaves of the adhatoda beddomei leaves. The extract from the culture was tested for the cytotoxicity in A549 cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)assay, dual staining and nuclear staining. The expression of the apoptotic and proliferative genes were assessed with the reverse transcriptase polymerase chain reaction (RT-PCR) comparing with the control gene. The inhibitory concentration (IC50) of the bacterial extract was found to be 43.97 μg/ml. With the dual staining the apoptotic cell percentage was significantly increased (P < 0.001) and with the 40μg/ml and 80μg/ml concentration the apoptotic percentages observed as 67% and 89% respectively. Similar concentrations were used for the nuclear fragmentation (PI) and the cell cycle analysis (FACS) using WinMDI 2.9 software. During cell cycle analysis the accumulation of the cells at G0-G1 stage was observed with increasing concentrations of the chi-alg encophytic bacterial extract nanoparticles. Finally the proapoptotic and proliferative gene expression for the bax, Bcl-2 and caspase was significantly regulated (P < 0.01; P < 0.05). The Bax and Caspase were up-regulated and Bcl-2 was down regulated. The results conclude that enophytic bacterial extract possess good cytotoxic activity.

  20. Identification of curcumin-inhibited extracellular matrix receptors in non-small cell lung cancer A549 cells by RNA sequencing.

    PubMed

    Li, Huiping; Wu, Hongjin; Zhang, Hongfang; Li, Ying; Li, Shuang; Hou, Qiang; Wu, Shixiu; Yang, Shuan-Ying

    2017-06-01

    Curcumin is a potent anti-cancer drug in several types of human cancers. Despite of several preclinical and clinical studies of curcumin, the precise mechanism of curcumin in cancer prevention has remained unclear. In our study, we for the first time investigated whole transcriptome alteration in A549 non-small cell lung cancer (NSCLC) cell lines after treatment with curcumin using RNA sequencing. We found that lots of genes and signaling pathways were significantly altered after curcumin treatment in A549 cells. With bioinformatics approaches (gene ontology, Kyoto Encyclopedia of Genes and Genomes, and STRING), we found that those curcumin altered genes were not only the genes that induce cell death but also those extracellular matrix receptors and mitogen-activated protein kinase signaling pathway genes which regulate cell migration and proliferation. Among those significantly altered genes, eight genes ( COL1A1, COL4A1, COL5A1, LAMA5, ITGA3, ITGA2B, DDIT3, and DUSP1) were further examined by quantitative reverse transcription polymerase chain reaction and western blot analysis in four non-small cell lung cancer cell lines. Both in cell lines and in mouse model, the extracellular matrix receptors including the integrin ( ITGA3 and ITGA2B), collagen ( COL5A1), and laminin ( LAMA5) were significantly inhibited by curcumin at messenger RNA and protein levels. Functional studies confirmed that curcumin not only induced A549 cell death but also repressed cell proliferation and migration by regulating extracellular matrix receptors. Collectively, our study suggests that curcumin may be used as a promising drug candidate for intervening lung cancer in future studies.

  1. Nur77 attenuates endothelin-1 expression via downregulation of NF-κB and p38 MAPK in A549 cells and in an ARDS rat model.

    PubMed

    Jiang, Yujie; Zeng, Yi; Huang, Xia; Qin, Yueqiu; Luo, Weigui; Xiang, Shulin; Sooranna, Suren R; Pinhu, Liao

    2016-12-01

    Acute respiratory distress syndrome (ARDS) is characterized by inflammatory injury to the alveolar and capillary barriers that results in impaired gas exchange and severe acute respiratory failure. Nuclear orphan receptor Nur77 has emerged as a regulator of gene expression in inflammation, and its role in the pathogenesis of ARDS is not clear. The objective of this study is to investigate the potential role of Nur77 and its underlying mechanism in the regulation of endothelin-1 (ET-1) expression in lipopolysaccharide (LPS)-induced A549 cells and an ARDS rat model. We demonstrate that LPS induced Nur77 expression and nuclear export in A549 cells. Overexpression of Nur77 markedly decreased basal and LPS-induced ET-1 expression in A549 cells, whereas knockdown of Nur77 increased the ET-1 expression. LPS-induced phosphorylation and nuclear translocation of NF-κB and p38 MAPK were blocked by Nur77 overexpression and augmented by Nur77 knockdown in A549 cells. In vivo, LPS induced Nur77 expression in lung in ARDS rats. Pharmacological activation of Nur77 by cytosporone B (CsnB) inhibited ET-1 expression in ARDS rats, decreased LPS-induced phosphorylation of NF-κB and p38 MAPK, and relieved lung, liver, and kidney injury. Pharmacological deactivation of Nur77 by 1,1-bis-(3'-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH, C-DIM8) had no effect on ET-1 expression and lung injury. These results indicated that Nur77 decreases ET-1 expression by suppressing NF-κB and p38 MAPK in LPS-stimulated A549 cells in vitro, and, in an LPS-induced ARDS rat model, CsnB reduced ET-1 expression and lung injury in ARDS rats. Copyright © 2016 the American Physiological Society.

  2. Delphinidin inhibits angiogenesis through the suppression of HIF-1α and VEGF expression in A549 lung cancer cells.

    PubMed

    Kim, Mun-Hyeon; Jeong, Yun-Jeong; Cho, Hyun-Ji; Hoe, Hyang-Sook; Park, Kwan-Kyu; Park, Yoon-Yub; Choi, Yung Hyun; Kim, Cheorl-Ho; Chang, Hyeun-Wook; Park, Young-Ja; Chung, Il-Kyung; Chang, Young-Chae

    2017-02-01

    Delphinidin, a polyphenol that belongs to the group of anthocyanidins and is abundant in many pigmented fruits and vegetables, possesses important antioxidant, anti‑inflammatory, anti-mutagenic and anticancer properties. In the present study, we investigated the inhibitory effects of delphinidin on vascular endothelial growth factor (VEGF) expression, an important factor involved in angiogenesis and tumor progression, in A549 human lung cancer cells. Delphinidin inhibited CoCl2- and epidermal growth factor (EGF)-induced VEGF mRNA expression and VEGF protein production. Delphinidin also decreased CoCl2- and EGF-stimulated expression of hypoxia‑inducible factor (HIF)‑1α, which is a transcription factor of VEGF. Delphinidin suppressed CoCl2- and EGF-induced hypoxia‑response element (HRE) promoter activity, suggesting that the inhibitory effects of delphinidin on VEGF expression are caused by the suppression of the binding of HIF-1 to the HRE promoter. We also found that delphinidin specifically decreased the CoCl2- and EGF-induced HIF-1α protein expression by blocking the ERK and PI3K/Akt/mTOR/p70S6K signaling pathways, whereas the p38-mediated pathways were not involved. In animal models, EGF-induced new blood vessel formation was significantly inhibited by delphinidin. Therefore, our results indicate that delphinidin has a potentially new role in anti‑angiogenic action by inhibiting HIF-1α and VEGF expression.

  3. Inhibition of acrolein-stimulated MUC5AC expression by Platycodon grandiflorum root-derived saponin in A549 cells.

    PubMed

    Choi, Jae Ho; Hwang, Yong Pil; Han, Eun Hee; Kim, Hyung Gyun; Park, Bong Hwan; Lee, Hyun Sun; Park, Byung Keun; Lee, Young Chun; Chung, Young Chul; Jeong, Hye Gwang

    2011-09-01

    Mucin overproduction is a hallmark of chronic airway diseases such as chronic obstructive pulmonary disease. In this study, we investigated the inhibition of acrolein-induced expression of mucin 5, subtypes A and C (MUC5AC) by Changkil saponin (CKS) in A549 cells. Acrolein, a known toxin in tobacco smoke and an endogenous mediator of oxidative stress, increases the expression of airway MUC5AC, a major component of airway mucus. CKS, a Platycodon grandiflorum root-derived saponin, inhibited acrolein-induced MUC5AC expression and activity, through the suppression of NF-κB activation. CKS also repressed acrolein-induced phosphorylation of ERK1/2, JNK1/2, and p38MAPK, which are upstream signaling molecules that control MUC5AC expression. In addition, the MAPK inhibitors PD98059 (ERK1/2), SP600125 (JNK1/2), and SB203580 (p38 MAPK), and a PKC delta inhibitor (rottlerin; PKCδ) inhibited acrolein-induced MUC5AC expression and activity. CKS repressed acrolein-induced phosphorylation of PKCδ. Moreover, a reactive oxygen species (ROS) inhibitor, N-acetylcysteine, inhibited acrolein-induced MUC5AC expression and activity through the suppression of PKCδ and MAPK activation, and CKS repressed acrolein-induced ROS production. These results suggest that CKS suppresses acrolein-induced MUC5AC expression by inhibiting the activation of NF-κB via ROS-PKCδ-MAPK signaling. Copyright © 2011 Elsevier Ltd. All rights reserved.

  4. 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.

  5. 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

  6. SU-F-T-677: Synergistic Effect(s) of Clotrimazole On Radiation Cell Survival of A549 Lung Cancer Cells in Glucose Vs. Galactose Media

    SciTech Connect

    Boss, G; Tambasco, M; Garakani, M

    2016-06-15

    Purpose: In order to determine the synergistic effect of clotrimazole on radiosensitivity of A549 lung cancer cells, and the effect of oxidative pathways on modulating radiosensitivity, we studied how these cells survived under varying amounts of radiation and clotrimazole as well ass when glucose was switched for galactose media. Methods: The glucose media was used to determine the presence of any synergistic effect of clotrimazole on radiation using values of radiation and clotrimazole concentrations, varying from 0 – 8 Gy and 0 – 20 µM, respectively. As a galactose diet is known to activate oxidative pathways, which do not relymore » on hexokinase II (HK2), all trials were repeated using galactose media to determine the extent that HK2 unbinding from the mitochondrial membrane plays a role in modulating the observed radiosensitivity. An apoptosis vs. necrosis assay was implemented to find out the modality by which cell death occurred. An intracellular lactate assay was performed to exhibit the extent of anaerobic glycolysis. Results: After running the primary experiments, it was found that in glucose media, the cancer cells showed higher cell kill when clotrimazole was added to the media, followed by the cells being irradiated. Conclusion: Given the preliminary results it is validated that under higher concentrations of clotrimazole, in glucose media, A549 lung cancer cells exhibit a lower amount of survival. While all results have not yet been gathered. We anticipate that in galactose media the A549 cells will exhibit this effect to a much smaller degree, if at all.« less

  7. 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.

  8. A flavonoid isolated from Streptomyces sp. (ERINLG-4) induces apoptosis in human lung cancer A549 cells through p53 and cytochrome c release caspase dependant pathway.

    PubMed

    Balachandran, C; Sangeetha, B; Duraipandiyan, V; Raj, M Karunai; Ignacimuthu, S; Al-Dhabi, N A; Balakrishna, K; Parthasarathy, K; Arulmozhi, N M; Arasu, M Valan

    2014-12-05

    The aim of this study was to investigate the anticancer activity of a flavonoid type of compound isolated from soil derived filamentous bacterium Streptomyces sp. (ERINLG-4) and to explore the molecular mechanisms of action. Cytotoxic properties of ethyl acetate extract was carried out against A549 lung cancer cell line using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cytotoxic properties of isolated compound were investigated in A549 lung cancer cell line, COLO320DM cancer cell line and Vero cells. The compound showed potent cytotoxic properties against A549 lung cancer cell line and moderate cytotoxic properties against COLO320DM cancer cell line. Isolated compound showed no toxicity up to 2000 μg/mL in Vero cells. So we have chosen the A549 lung cancer cell line for further anticancer studies. Intracellular visualization was done by using a laser scanning confocal microscope. Apoptosis was measured using DNA fragmentation technique. Treatment of the A549 cancer cells with isolated compound significantly reduced cell proliferation, increased formation of fragmented DNA and apoptotic body. Activation of caspase-9 and caspase-3 indicated that compound may be inducing intrinsic and extrinsic apoptosis pathways. Bcl-2, p53, pro-caspases, caspase-3, caspase-9 and cytochrome c release were detected by western blotting analysis after compound treatment (123 and 164 μM). The activities of pro-caspases-3, caspase-9 cleaved to caspase-3 and caspase-9 gradually increased after the addition of isolated compound. But Bcl-2 protein was down regulated after treatment with isolated compound. Molecular docking studies showed that the compound bound stably to the active sites of caspase-3 and caspase-9. These results strongly suggest that the isolated compound induces apoptosis in A549 cancer cells via caspase activation through cytochrome c release from mitochondria. The present results might provide helpful suggestions for the design of

  9. Effects of ultrafine petrol exhaust particles on cytotoxicity, oxidative stress generation, DNA damage and inflammation in human A549 lung cells and murine RAW 264.7 macrophages.

    PubMed

    Durga, Mohan; Nathiya, Soundararajan; Rajasekar, Abbu; Devasena, Thiyagarajan

    2014-09-01

    Air pollution has persistently been the major cause of respiratory-related illness and death. Environmental pollutants such as diesel and petrol exhaust particles (PEPs) are the major contributors to urban air pollution. The aim of the present study was to characterize and investigate the in vitro cytotoxicity, oxidative stress, DNA damage and inflammation induced by PEPs. Cultured type II epithelium cells (human A549 lung cells) and alveolar macrophages (murine RAW 264.7 cells) were exposed to control, vehicle control and to different concentrations of PEPs for up to 24h. Each treatment was evaluated by cell viability, cytotoxicity, oxidative stress, DNA damage and inflammatory parameters. Overall in vitro studies demonstrated that both cell lines showed similar patterns in response to the above studies induced by petrol exhaust nanoparticles (PENPs). Vehicle control showed no changes compared with the control. In both cell lines, significant changes at the dose of 20 and 50μg/mL (A549 cell lines) and 10and 20μg/mL (macrophages) for PENPs were found. The reactive oxygen species production in both cell lines shot up in minutes, reached the maximum within an hour and came down after 4h. Hence, exposure to PENPs resulted in dose-dependent toxicity in cultured A549 cells and RAW 264.7 cells and was closely correlated to increased oxidative stress, DNA damage and inflammation. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Cytotoxic, Antiproliferative and Apoptotic Effects of Perillyl Alcohol and Its Biotransformation Metabolite on A549 and HepG2 Cancer Cell Lines.

    PubMed

    Oturanel, Ceren E; Kıran, İsmail; Özşen, Özge; Çiftçi, Gülşen A; Atlı, Özlem

    2017-01-01

    A monoterpene, perillyl alcohol, has attracted attention in medicinal chemistry since it exhibited chemo-preventive and therapeutic properties against a variety of cancers. In the present work, it was aimed to obtain derivatives of perillyl alcohol through microbial biotransformation and investigate their anticancer activities against A549 and HepG2 cancer cell lines. Biotransformation studies were carried out in a α-medium for 7 days at 25oC. XTT assay was performed to investigate the anticancer activities of perillyl alcohol and its biotransformation metabolite, dehydroperillic acid, against A549 and HepG2 cell lines and their selectivity using healthy cell line, NIH/3T3. Cell proliferation ELISA, BRDU (colorimetric) assay was used for measurement of proliferation in replicative cells in which DNA synthesis occurs. Flow cytometric analyses were also carried out for measuring apoptotic cell percentages, caspase 3 activation and mitochondrial membrane potential. Biotransformation of perillyl alcohol with Fusarium culmorum yielded dehydroperillic acid in a yield of 20.4 %. In in vitro anticancer studies, perillyl alcohol was found to exert cytotoxicity against HepG2 cell line with an IC50 value of 409.2 μg/mL. However, this effect was not found to be selective because of its higher IC50 (250 μg/mL) value against NIH/3T3 cell line. On the other hand, dehydroperillic acid was found to be effective and also selective against A549 cell line with an IC50 value of 125 μg/mL and a selectivity index (SI) value of 400. Apoptosis inducing effects of dehydroperillic acid was better in A549 cell line. Dehydroperillic acid may be a good candidate for therapy of lung adenocarcinoma and may show this anticancer activity by inducing apoptosis. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  11. 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.

  12. 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

  13. 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.

  14. Cooking oil fume-derived PM2.5 induces apoptosis in A549 cells and MAPK/NF-кB/STAT1 pathway activation.

    PubMed

    Dou, Changming; Zhang, Jie; Qi, Cuicui

    2018-04-01

    Cooking oil fumes (COFs) are the major sources of indoor air pollution in Asia. It is well known that alveolar cells are key participants in the development of respiratory system; however, it still remains unknown whether alveolar cells are affected by COFs. Therefore, the present study investigated the effects of COFs on alveolar cells (A549 cells) and illuminated its apoptotic mechanism in response to COF-PM 2.5 exposure. When A549 cells were exposed to COF-PM 2.5 , cell viability was substantially decreased, while the generation of ROS increased, and LDH levels and CCK-8 levels gradually changed within a dose-dependent manner. The nitrite concentration in the supernatants was augmented, while the SOD activity and GSH recycling were decreased upon COF-PM 2.5 . Moreover, COF-PM 2.5 treatment increased mRNA levels of COX-2, inducible NO synthase, and TNF-α, and Elisa assay suggested that secretory proteins IL-6 and TNF-α were also increased. Furthermore, the Bax/Bcl-2 mRNA ratio was increased, and cleaved caspase-3 protein was activated in the A549 cells. Strikingly, COF-PM 2.5 induced the phosphorylation of STAT1 at Tyr701/Ser727 and activation of NF-кB and ERK1/2, p38, and JNK of the MAPK pathway. In short, our study suggested that COF-PM 2.5 resulted in inflammation, apoptosis, and cell damage in A549 cells, which might be modulated via the activation of MAPK/NF-кB/STAT1 pathway.

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

    SciTech Connect

    Lin, Hui-Hsuan; Department of Medical Research, Chung Shan Medical University Hospital, Taichung, Taiwan; Tsai, Chia-Wen

    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 bymore » 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.« less

  16. Low-dose carbon-based nanoparticle-induced effects in A549 lung cells determined by biospectroscopy are associated with increases in genomic methylation.

    PubMed

    Li, Junyi; Tian, Meiping; Cui, Li; Dwyer, John; Fullwood, Nigel J; Shen, Heqing; Martin, Francis L

    2016-02-02

    Nanotechnology has introduced many manufactured carbon-based nanoparticles (CNPs) into our environment, generating a debate into their risks and benefits. Numerous nanotoxicology investigations have been carried, and nanoparticle-induced toxic effects have been reported. However, there remain gaps in our knowledge, primarily regarding mechanism. Herein, we assessed the global alterations induced by CNPs in A549 lung cells using biospectroscopy techniques, including attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy and surface-enhanced Raman spectroscopy (SERS). A549 cells were treated with fullerene (C60), long or short multi-walled carbon nanotubes, or single-walled carbon nanotubes at concentrations of 0.1 mg/L, 0.01 mg/L and 0.001 mg/L. Exposed cells were then analysed by ATR-FTIR spectroscopy and SERS. Spectra were pre-processed via computational analysis, and information on biochemical alterations in exposed cells were identified. Additionally, global DNA methylation levels in cells exposed to CNPs at 0.1 mg/L were determined using HPLC-MS and genetic regulators (for DNA methylation) were checked by quantitative real-time RT-PCR. It was found that CNPs exert marked effects in A549 cells and also contribute to increases in global DNA methylation. For the first time, this study highlights that real-world levels of nanoparticles can alter the methylome of exposed cells; this could have enormous implications for their regulatory assessment.

  17. Low-dose carbon-based nanoparticle-induced effects in A549 lung cells determined by biospectroscopy are associated with increases in genomic methylation

    NASA Astrophysics Data System (ADS)

    Li, Junyi; Tian, Meiping; Cui, Li; Dwyer, John; Fullwood, Nigel J.; Shen, Heqing; Martin, Francis L.

    2016-02-01

    Nanotechnology has introduced many manufactured carbon-based nanoparticles (CNPs) into our environment, generating a debate into their risks and benefits. Numerous nanotoxicology investigations have been carried, and nanoparticle-induced toxic effects have been reported. However, there remain gaps in our knowledge, primarily regarding mechanism. Herein, we assessed the global alterations induced by CNPs in A549 lung cells using biospectroscopy techniques, including attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy and surface-enhanced Raman spectroscopy (SERS). A549 cells were treated with fullerene (C60), long or short multi-walled carbon nanotubes, or single-walled carbon nanotubes at concentrations of 0.1 mg/L, 0.01 mg/L and 0.001 mg/L. Exposed cells were then analysed by ATR-FTIR spectroscopy and SERS. Spectra were pre-processed via computational analysis, and information on biochemical alterations in exposed cells were identified. Additionally, global DNA methylation levels in cells exposed to CNPs at 0.1 mg/L were determined using HPLC-MS and genetic regulators (for DNA methylation) were checked by quantitative real-time RT-PCR. It was found that CNPs exert marked effects in A549 cells and also contribute to increases in global DNA methylation. For the first time, this study highlights that real-world levels of nanoparticles can alter the methylome of exposed cells; this could have enormous implications for their regulatory assessment.

  18. Annona muricata leaves induced apoptosis in A549 cells through mitochondrial-mediated pathway and involvement of NF-κB.

    PubMed

    Moghadamtousi, Soheil Zorofchian; Kadir, Habsah Abdul; Paydar, Mohammadjavad; Rouhollahi, Elham; Karimian, Hamed

    2014-08-15

    Annona muricata leaves have been reported to have antiproliferative effects against various cancer cell lines. However, the detailed mechanism has yet to be defined. The current study was designed to evaluate the molecular mechanisms of A. muricata leaves ethyl acetate extract (AMEAE) against lung cancer A549 cells. The effect of AMEAE on cell proliferation of different cell lines was analyzed by MTT assay. High content screening (HCS) was applied to investigate the suppression of NF-κB translocation, cell membrane permeability, mitochondrial membrane potential (MMP) and cytochrome c translocation from mitochondria to cytosol. Reactive oxygen species (ROS) formation, lactate dehydrogenase (LDH) release and activation of caspase-3/7, -8 and -9 were measured while treatment. The western blot analysis also carried out to determine the protein expression of cleaved caspase-3 and -9. Flow cytometry analysis was used to determine the cell cycle distribution and phosphatidylserine externalization. Quantitative PCR analysis was performed to measure the gene expression of Bax and Bcl-2 proteins. Cell viability analysis revealed the selective cytotoxic effect of AMEAE towards lung cancer cells, A549, with an IC50 value of 5.09 ± 0.41 μg/mL after 72 h of treatment. Significant LDH leakage and phosphatidylserine externalization were observed in AMEAE treated cells by fluorescence analysis. Treatment of A549 cells with AMEAE significantly elevated ROS formation, followed by attenuation of MMP via upregulation of Bax and downregulation of Bcl-2, accompanied by cytochrome c release to the cytosol. The incubation of A549 cells with superoxide dismutase and catalase significantly attenuated the cytotoxicity caused by AMEAE, indicating that intracellular ROS plays a pivotal role in cell death. The released cytochrome c triggered the activation of caspase-9 followed by caspase-3. In addition, AMEAE-induced apoptosis was accompanied by cell cycle arrest at G0/G1 phase. Moreover

  19. 7-Epiclusianone, a Benzophenone Extracted from Garcinia brasiliensis (Clusiaceae), Induces Cell Cycle Arrest in G1/S Transition in A549 Cells.

    PubMed

    Ionta, Marisa; Ferreira-Silva, Guilherme A; Niero, Evandro L; Costa, Éderson D'Martin; Martens, Adam A; Rosa, Welton; Soares, Marisi G; Machado-Santelli, Gláucia M; Lago, João Henrique G; Santos, Marcelo H

    2015-07-15

    Lung cancer is the leading cause of cancer deaths in the world. Disease stage is the most relevant factor influencing mortality. Unfortunately, most patients are still diagnosed at an advanced stage and their five-year survival rate is only 4%. Thus, it is relevant to identify novel drugs that can improve the treatment options for lung cancer. Natural products have been an important source for the discovery of new compounds with pharmacological potential including antineoplastic agents. We have previously isolated a prenylated benzophenone (7-epiclusianone) from Garcinia brasiliensis (Clusiaceae) that has several biological properties including antiproliferative activity against cancer cell lines. In continuation with our studies, the present work aimed to investigate the mechanisms involved with antiproliferative activity of 7-epiclusianone in A549 cells. Our data showed that 7-epiclusianone reduced the viability of A549 cells in a concentration-dependent manner (IC50 of 16.13 ± 1.12 μM). Cells were arrested in G1/S transition and apoptosis was induced. In addition, we observed morphological changes with cytoskeleton disorganization in consequence of the treatment. Taken together, the results showed that cell cycle arrest in G1/S transition is the main mechanism involved with antiproliferative activity of 7-epiclusianone. Our results are promising and open up the prospect of using this compound in further anticancer in vivo studies.

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

    PubMed

    Holownia, A; Wielgat, P; Eljaszewicz, A

    2018-03-01

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

  1. Inhibition of cellular proliferation and induction of apoptosis in human lung adenocarcinoma A549 cells by T-type calcium channel antagonist.

    PubMed

    Choi, Doo Li; Jang, Sun Jeong; Cho, Sehyeon; Choi, Hye-Eun; Rim, Hong-Kun; Lee, Kyung-Tae; Lee, Jae Yeol

    2014-03-15

    The anti-proliferative and apoptotic activities of new T-type calcium channel antagonist, 6e (BK10040) on human lung adenocarcinoma A549 cells were investigated. The MTT assay results indicated that BK10040 was cytotoxic against human lung adenocarcinoma (A549) and pancreatic cancer (MiaPaCa2) cells in a dose-dependent manner with IC50 of 2.25 and 0.93μM, respectively, which is ca. 2-fold more potent than lead compound KYS05090 despite of its decreased T-type calcium channel blockade. As a mode of action for cytotoxic effect of BK10040 on lung cancer (A549) cells, this cancer cell death was found to have the typical features of apoptosis, as evidenced by the accumulation of positive cells for annexin V. In addition, BK10040 triggered the activations of caspases 3 and 9, and the cleavages of poly (ADP-ribose) polymerase (PARP). Moreover, the treatment with z-VAD-fmk (a broad spectrum caspase inhibitor) significantly prevented BK10040-induced apoptosis. Based on these results, BK10040 may be used as a potential therapeutic agent for human lung cancer via the potent apoptotic activity. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Neferine augments therapeutic efficacy of cisplatin through ROS- mediated non-canonical autophagy in human lung adenocarcinoma (A549 cells).

    PubMed

    Kalai Selvi, Sivalingam; Vinoth, Amirthalingam; Varadharajan, Thiyagarajan; Weng, Ching Feng; Vijaya Padma, Viswanadha

    2017-05-01

    Combination of dietary components with chemotherapy drugs is an emerging new strategy for cancer therapy to increase antitumor responses. Neferine, major bisbenzylisoquinoline alkaloid isolated from the seed embryo of Nelumbo nucifera (Lotus). In the present study, we investigated the efficacy of the combinatorial regimen of neferine and cisplatin compared to cisplatin high dose in human lung adenocarcinoma (A549) cells. Co-treatment with neferine enhanced cisplatin-induced autophagy in A549 cells was accompanied by Acidic vesicular accumulation (AVO), enhanced generation of reactive oxygen species (ROS) and depletion of intracellular glutathione (GSH), down regulation of PI3K/AKT/mTOR pathway, conversion of LC3B-I to LC3B-II. This enhanced autophagy developed via a non-canonical mechanism that did not require Beclin-1, PI3KCIII. In conclusion, these results suggest that neferine enhances cisplatin -induced autophagic cancer cell death through downregulation of PI3K/Akt/mTOR signaling pro-survival pathway and ROS- mediated Beclin-1 and PI3K CIII independent autophagy in human lung adenocarcinoma (A549 cells). Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Baicalin, a Chinese Herbal Medicine, Inhibits the Proliferation and Migration of Human Non-Small Cell Lung Carcinoma (NSCLC) Cells, A549 and H1299, by Activating the SIRT1/AMPK Signaling Pathway.

    PubMed

    You, Jiawen; Cheng, Jun; Yu, Bing; Duan, Changhua; Peng, Jinghua

    2018-04-10

    BACKGROUND Baicalin is a flavonoid derived from Scutellaria baicalensis, used in Chinese herbal medicine. Activation of the sirtuin 1 gene (SIRT1) and adenosine monophosphate (AMP)-activated protein kinase gene (AMPK), the SIRT1/AMPK signaling pathway, is associated with human malignant tumors. The aim of this study was to investigate the effects of baicalin on the cell viability, apoptosis, proliferation, and migration of human non-small cell lung cancer (NSCLC) cells, A549 and H1299, in vitro. MATERIAL AND METHODS Human NSCLC cells, A549 and H1299, were treated with serial doses of baicalin. Small interfering RNA (siRNA) silencing of the SIRT1 and AMPK genes was performed using cell transfection. The MTT assay was used to determine cell viability, flow cytometry was used to measure cell apoptosis, wound healing and transwell assays were used to assess cell migration of A549 and H1299 cells. Western blotting was used to measure protein expression and phosphorylation levels in untreated A549 and H1299 cells, and cells treated with increasing doses of baicalin. RESULTS Baicalin inhibited the viability, migration, and invasion of A549 and H1299 cells, and increased cell apoptosis in a dose-dependent manner. Baicalin activated the SIRT1/AMPK and mechanistic target of rapamycin (mTOR), and SIRT1/AMPK and matrix metalloproteinase (MMP) signaling in A549 and H1299 cells in a dose-dependent manner. siRNA silencing of SIRT1 and AMPK reduced the effects of baicalin on cell proliferation and migration. CONCLUSIONS Baicalin, a flavonoid used in Chinese herbal medicine, inhibited the proliferation and migration of human NSCLC cells, A549 and H1299, by activating the SIRT1/AMPK signaling pathway.

  4. Increase in cell motility by carbon ion irradiation via the Rho signaling pathway and its inhibition by the ROCK inhibitor Y-27632 in lung adenocarcinoma A549 cells.

    PubMed

    Murata, Kazutoshi; Noda, Shin-ei; Oike, Takahiro; Takahashi, Akihisa; Yoshida, Yukari; Suzuki, Yoshiyuki; Ohno, Tatsuya; Funayama, Tomoo; Kobayashi, Yasuhiko; Takahashi, Takeo; Nakano, Takashi

    2014-07-01

    This study aimed to investigate the effect of carbon ion (C-ion) irradiation on cell motility through the ras homolog gene family member (Rho) signaling pathway in the human lung adenocarcinoma cell line A549. Cell motility was assessed by a wound-healing assay, and the formation of cell protrusions was evaluated by F-actin staining. Cell viability was examined by the WST-1 assay. The expression of myosin light chain 2 (MLC2) and the phosphorylation of MLC2 at Ser19 (P-MLC2-S19) were analyzed by Western blot. At 48 h after irradiation, the wound-healing assay demonstrated that migration was significantly greater in cells irradiated with C-ion (2 or 8 Gy) than in unirradiated cells. Similarly, F-actin staining showed that the formation of protrusions was significantly increased in cells irradiated with C-ion (2 or 8 Gy) compared with unirradiated cells. The observed increase in cell motility due to C-ion irradiation was similar to that observed due to X-ray irradiation. Western-blot analysis showed that C-ion irradiation (8 Gy) increased P-MLC2-S19 expression compared with in unirradiated controls, while total MLC2 expression was unchanged. Exposure to a non-toxic concentration of Y-27632, a specific inhibitor of Rho-associated coiled-coil-forming protein kinase (ROCK), reduced the expression of P-MLC2-S19 after C-ion irradiation (8 Gy), resulting in a significant reduction in migration. These data suggest that C-ion irradiation increases cell motility in A549 cells via the Rho signaling pathway and that ROCK inhibition reduces that effect. © The Author 2014. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

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

    PubMed

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

    2016-11-16

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

  6. Silica nanoparticles and lead acetate co-exposure triggered synergistic cytotoxicity in A549 cells through potentiation of mitochondria-dependent apoptosis induction.

    PubMed

    Lu, Chun-Feng; Li, Li-Zhong; Zhou, Wei; Zhao, Jun; Wang, Yi-Mei; Peng, Shuang-Qing

    2017-06-01

    The adverse effects of PM2.5 are the results of combined toxicities of finer particles and their adsorbed toxic pollutants. Nevertheless, the combined toxicity of finer particles and air pollutants still remains unclear. The present study was therefore undertaken to investigate the combined cytotoxicity of silica nanoparticles (nano-SiO 2 , a typical atmospheric ultrafine particle) and lead acetate (Pb, a representative air pollutant) in A549 cells focusing on mitochondria-dependent apoptosis induction. The results showed that Pb exposure alone induced mitochondria-dependent apoptosis in A549 cells, as evidenced by increased apoptotic rate and Bax/Bcl-2 ratio, up-regulated caspases 3 and 9 expressions as well as decreased mitochondrial membrane potential. Non-cytotoxic concentration of nano-SiO 2 exposure alone did not trigger apoptosis in A549 cells, but potentialized the apoptotic changes when co-exposure with Pb. Factorial analyses revealed synergistic interactions were responsible for the potentiation of joint apoptotic responses. Copyright © 2017. Published by Elsevier B.V.

  7. Genistein enhances the effect of trichostatin A on inhibition of A549 cell growth by increasing expression of TNF receptor-1

    SciTech Connect

    Wu, Tzu-Chin; Yang, Ying-Chihi; Huang, Pei-Ru

    2012-08-01

    Our previous study has shown that genistein enhances apoptosis in A549 lung cancer cells induced by trichostatin A (TSA). The precise molecular mechanism underlying the effect of genistein, however, remains unclear. In the present study, we investigated whether genistein enhances the anti-cancer effect of TSA through up-regulation of TNF receptor-1 (TNFR-1) death receptor signaling. We incubated A549 cells with TSA (50 ng/mL) alone or in combination with genistein and then determined the mRNA and protein expression of TNFR-1 as well as the activation of downstream caspases. Genistein at 5 and 10 μM significantly enhanced the TSA-induced decrease in cell numbermore » and apoptosis in a dose-dependent manner. The combined treatment significantly increased mRNA and protein expression of TNFR-1 at 6 and 12 h, respectively, compared with that of the control group; while TSA alone had no effect. TSA in combination with 10 μM of genistein increased TNFR-1 mRNA and protein expression by about 70% and 40%, respectively. The underlying mechanism for this effect of genistein may be partly associated with the estrogen receptor pathway. The combined treatment also increased the activation of caspase-3 and ‐10 as well as p53 protein expression in A549 cells. The enhancing effects of genistein on the TSA-induced decrease in cell number and on the expression of caspase-3 in A549 cells were suppressed by silencing TNFR-1 expression. These data demonstrated that the upregulation of TNFR-1 death receptor signaling plays an important role, at least in part, in the enhancing effect of genistein on TSA-induced apoptosis in A549 cells. -- Highlights: ► TSA combined with genistein rather than TSA alone increases the expression of TNFR-1. ► Genistein may exert such an effect partly through estrogen receptor pathway. ► The combined treatment increases the activation of caspase-10 and caspase-3. ► The combined treatment also increases the expression of p53 protein. ► TNFR-1

  8. Inhibition of DNA‑PK activity sensitizes A549 cells to X‑ray irradiation by inducing the ATM‑dependent DNA damage response.

    PubMed

    Yang, Lina; Yang, Xinrui; Tang, Yiwei; Zhang, Defu; Zhu, Lijie; Wang, Shengnan; Wang, Bo; Ma, Tao

    2018-03-29

    Non‑small cell lung cancer (NSCLC) is radioresistant to X‑rays due to powerful cellular DNA damage repair mechanisms. DNA‑dependent protein kinase (DNA‑PK) is a key enzyme involved in DNA damage repair and the phenomenon and molecular mechanism of NSCLC radionsensitivity were investigated following inhibition of DNA‑PK activity. In the present study A549 cells were treated with the DNA‑PK inhibitor NU7026 and/or siRNA directed against ataxia telangiectasia mutated (ATM), followed by exposure to 4 Gy X‑ray irradiation. Radiosensitivity, DNA damage, apoptosis and protein expression were measured by colony formation assay, γH2AX foci immunofluorescence, Annexin V/PI staining and western blotting, respectively. A Balb/c‑nu/nu xenograft mouse model was established by subcutaneous injection of A549 cells and was used to examine the effect of administering NU7026 via intraperitoneal injection prior to 4 Gy X‑ray exposure. The xenograft tumors were weighed and observed by hematoxylin and eosin staining after irradiation. NU7026 treatment followed by X‑ray irradiation significantly decreased the colony formation ratio of A549 cells, and increased γH2AX foci and cell apoptosis. Furthermore, the combined treatment of NU7026 and X‑rays resulted in growth inhibition and cell apoptosis in A549 xenograft tumors. Consequently, apoptosis regulators full‑length transactivating (TA) p73 and an N‑terminally truncated (DN) p73 were upregulated and downregulated respectively, leading to activation of glucosyltransferases and Rab‑like GTPase activators and myotubularins domain‑containing 4 (GRAMD4) protein to reduce the Bcl‑2/Bax protein ratio. In addition, ATM siRNA efficiently prevented γH2AX foci formation, and enhanced NU7026‑induced inhibition of survival and promoted apoptosis. In conclusion, inhibition of DNA‑PK activity increased the radiosensitivity of A549 cells to X‑ray irradiation. NU7026 treatment activated the ATM‑dependent DNA

  9. RNA silencing of integrin-linked kinase increases the sensitivity of the A549 lung cancer cell line to cisplatin and promotes its apoptosis.

    PubMed

    Zhao, Xiaozhen; Xu, Zhenye; Wang, Zhongqi; Wu, Zhonghua; Gong, Yabin; Zhou, Lijuan; Xiang, Yi

    2015-07-01

    The expression of integrin-linked kinase (ILK) has been reported to be involved in the regulation of integrin-mediated processes, including cancer cell proliferation, migration and invasion. Previous studies have demonstrated that inhibition of ILK may be an underlying approach for treating cancer. However, whether the knock down of ILK affects growth and apoptosis of lung cancer cells remains to be elucidated. Importantly, whether downregulation of ILK increases the sensitivity of lung cancer cells to cisplatin and amplifies cell apoptosis also remains to be elucidated. In the present study, ILK downregulation was mediated by lentivirus-mediated RNA interference. The expression levels of associated genes were determined by reverse-transcription quantitative polymerase chain reaction and western blotting. Cell proliferation was evaluated using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and clone formation assay. The cell cycle and apoptosis were analyzed using flow cytometry. The current data revealed that lentivirus-mediated ILK gene silencing alone inhibited A549 cell proliferation and promotes cell cycle arrest, however, had no detectable effect on cell apoptosis. However, combined treatment with lentivirus-mediated ILK interference and cisplatin chemotherapy induced significantly more cell apoptosis than mono-chemotherapy or knockdown. The increased cell apoptosis and proliferation inhibition were attributed to abnormal downstream protein expression of ILK, including phospho-glycogen synthase kinase 3β, p-AKT, activator protein-1, β-catenin, cyclin D1 and matrix metalloproteinase-9. ILK inhibition may suppress the proliferation of A549 and increase A549 sensitivity to cisplatin. The combined treatment of ILK gene knockdown and chemotherapy has the potential to improve anticancer efficacy.

  10. Meloxicam increases intracellular accumulation of doxorubicin via downregulation of multidrug resistance-associated protein 1 (MRP1) in A549 cells.

    PubMed

    Chen, S F; Zhang, Z Y; Zhang, J L

    2015-11-19

    It has been suggested that selected COX inhibitors can overcome multidrug resistance through the inhibition of ATP‑binding cassette-transporter proteins thereby enhancing the inhibitory effect of doxorubicin on human tumor growth and promoting the actions of cytostatics. However, their effect on lung cancer and the molecular mechanisms involved in the overcoming of multidrug resistance are unclear. In the present study, the ability of meloxicam, a COX-2-specific inhibitor to enhance doxorubicin‑mediated inhibition was investigated in human A549 lung cancer in vivo and in vitro. In order to unravel the molecular mechanisms involved in doxorubicin accumulation, we measured the levels of multidrug resistance-associated protein (MRP)-transporter protein activity and expression by western blotting, since this has been implicated in meloxicam action as well as in chemoresistance. We found that, in A549 cells, meloxicam could increase intracellular accumulation of doxorubicin, a substrate for MRP, through inhibition of cellular export. Western blot analysis indicated that meloxicam reduced the expression of MRP1 and MRP4. The results reported in the present study demonstrate for the first time that the specific COX-2 inhibitor meloxicam can increase the intracellular accumulation of doxorubicin and enhance doxorubicin-induced cytotoxicity in A549 cancer cells by reducing the expression of MRP1 and MRP4.

  11. The synergistic antitumor effects of all-trans retinoic acid and C-phycocyanin on the lung cancer A549 cells in vitro and in vivo.

    PubMed

    Li, Bing; Gao, Mei-Hua; Chu, Xian-Ming; Teng, Lei; Lv, Cong-Yi; Yang, Peng; Yin, Qi-Feng

    2015-02-15

    The anticancer effects and mechanism of all-trans retinoic acid (ATRA), C-phycocyanin (C-PC) or ATRA+C-PC on the growth of A549 cells were studied in in vitro and in vivo experiments. The effects of C-PC and ATRA on the growth of A549 cells were determined. The expression of CDK-4 and caspase-3, and the cellular apoptosis levels were detected. The tumor model was established by subcutaneous injection of A549 cells to the left axilla of the NU/NU mice. The weights of tumor and the spleen were tested. The viabilities of T-cells and spleen cells, TNF levels, the expression of Bcl-2 protein and Cyclin D1 gene were examined. Results showed both C-PC and ATRA could inhibit the growth of tumor cells in vivo and in vitro. ATRA+C-PC cooperatively showed a higher antitumor activity. The dosage of ATRA was reduced when it was administered with C-PC together, and the toxicity was reduced as well. ATRA+C-PC could decrease CDK-4 but increase caspase-3 protein expression level and induce cell apoptosis. ATRA alone could lower the activities of T lymphocytes and spleen weights, but the combination with C-PC could effectively promote viability of T cells and spleen. C-PC+ATRA could up-regulate TNF, and down-regulate Bcl-2 and Cyclin D1 gene. The combination might inhibit tumor growth by inhibiting the progress of cell cycle, inducing cell apoptosis and enhancing the body immunity. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. MiR-509-3-5p causes aberrant mitosis and anti-proliferative effect by suppression of PLK1 in human lung cancer A549 cells.

    PubMed

    Wang, Xian-Hui; Lu, Yao; Liang, Jing-Jing; Cao, Ji-Xiang; Jin, Ya-Qiong; An, Guo-Shun; Ni, Ju-Hua; Jia, Hong-Ti; Li, Shu-Yan

    2016-09-16

    MicroRNAs (miRNAs) are potent post-transcriptional regulators of gene expression and play roles in DNA damage response (DDR). PLK1 is identified as a modulator of DNA damage checkpoint. Although down-regulation of PLK1 by certain microRNAs has been reported, little is known about the interplay between PLK1 and miR-509-3-5p in DDR. Here we have demonstrated that miR-509-3-5p repressed PLK1 expression by targeting PLK1 3'-UTR, thereby causing mitotic aberration and growth arrest of human lung cancer A549 cells. Repression of PLK1 by miR-509-3-5p was further evidenced by over-expression of miR-509-3-5p in A549, HepG2 and HCT116p53(-/-) cancer cells, in which PLK1 protein was suppressed. Consistently, miR-509-3-5p was stimulated, while PLK1 protein was down-regulated in A549 cells exposed to CIS and ADR, suggesting that suppression of PLK1 by miR-509-3-5p is a component of CIS/ADR-induced DDR pathway. Flow cytometry and immunofluorescence labeling showed that over-expression of miR-509-3-5p in A549 induced G2/M arrest and aberrant mitosis characterized by abnormal bipolar mitotic spindles, condensed chromosomes, lagging DNA and chromosome bridges. In addition, over-expression of miR-509-3-5p markedly blocked A549 cell proliferation and sensitized the cells to CIS and ADR treatment. Taken together, miR-509-3-5p is a feasible suppressor for cancer by targeting PLK1. Our data may provide aid in potential design of combined chemotherapy and in our better understanding of the roles of microRNAs in response to DNA damage. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Revelation of Different Nanoparticle-Uptake Behavior in Two Standard Cell Lines NIH/3T3 and A549 by Flow Cytometry and Time-Lapse Imaging.

    PubMed

    Jochums, André; Friehs, Elsa; Sambale, Franziska; Lavrentieva, Antonina; Bahnemann, Detlef; Scheper, Thomas

    2017-07-19

    The uptake of nanomaterials into different cell types is a central pharmacological issue for the determination of nanotoxicity as well as for the development of drug delivery strategies. Most responses of the cells depend on their intracellular interactions with nanoparticles (NPs). Uptake behavior can be precisely investigated in vitro, with sensitive high throughput methods such as flow cytometry. In this study, we investigated two different standard cell lines, human lung carcinoma (A549) and mouse fibroblast (NIH/3T3) cells, regarding their uptake behavior of titanium dioxide NPs. Cells were incubated with different concentrations of TiO₂ NPs and samples were taken at certain time points to compare the uptake kinetics of both cell lines. Samples were analyzed with the help of flow cytometry by studying changes in the side and forward scattering signal. To additionally enable a detection via fluorescence, NPs were labeled with the fluorescent dye fluorescein isothiocyanate (FITC) and propidium iodide (PI). We found that NIH/3T3 cells take up the studied NPs more efficiently than A549 cells. These findings were supported by time-lapse microscopic imaging of the cells incubated with TiO₂ NPs. Our results confirm that the uptake behavior of individual cell types has to be considered before interpreting any results of nanomaterial studies.

  14. Revelation of Different Nanoparticle-Uptake Behavior in Two Standard Cell Lines NIH/3T3 and A549 by Flow Cytometry and Time-Lapse Imaging

    PubMed Central

    Jochums, André; Friehs, Elsa; Sambale, Franziska; Lavrentieva, Antonina; Bahnemann, Detlef; Scheper, Thomas

    2017-01-01

    The uptake of nanomaterials into different cell types is a central pharmacological issue for the determination of nanotoxicity as well as for the development of drug delivery strategies. Most responses of the cells depend on their intracellular interactions with nanoparticles (NPs). Uptake behavior can be precisely investigated in vitro, with sensitive high throughput methods such as flow cytometry. In this study, we investigated two different standard cell lines, human lung carcinoma (A549) and mouse fibroblast (NIH/3T3) cells, regarding their uptake behavior of titanium dioxide NPs. Cells were incubated with different concentrations of TiO2 NPs and samples were taken at certain time points to compare the uptake kinetics of both cell lines. Samples were analyzed with the help of flow cytometry by studying changes in the side and forward scattering signal. To additionally enable a detection via fluorescence, NPs were labeled with the fluorescent dye fluorescein isothiocyanate (FITC) and propidium iodide (PI). We found that NIH/3T3 cells take up the studied NPs more efficiently than A549 cells. These findings were supported by time-lapse microscopic imaging of the cells incubated with TiO2 NPs. Our results confirm that the uptake behavior of individual cell types has to be considered before interpreting any results of nanomaterial studies. PMID:29051447

  15. Effect of etoposide-induced alteration of the Mdm2-Rb signaling pathway on cellular senescence in A549 lung adenocarcinoma cells.

    PubMed

    Dai, Wenjing; Jiang, Yi; Chen, Kairong; Qiu, Jing; Sun, Jian; Zhang, Wei; Zhou, Xiafei; Huang, Na; Li, Yunhui; Li, Wancheng

    2017-10-01

    The present study aimed to investigate the effect of various concentrations of etoposide (VP-16) on the E3 ubiquitin-protein ligase Mdm2 (Mdm2)-retinoblastoma (Rb) signaling pathway in the cellular senescence of A549 lung adenocarcinoma cells. A549 cells were randomly divided into the following four groups: Control group (no treatment), group 1 (1 µmol/l VP-16), group 2 (5 µmol/l VP-16) and group 3 (25 µmol/l VP-16). Each group was cultured for 48 h after treatment prior to observation of the alterations to cellular morphology. The cell cycle distribution of each group was also detected by flow cytometry. In addition, the activity of cellular senescence-associated β-galactosidase, and the expression of Mdm2 and phosphorylated (p-) Rb protein, was measured. The percentage of senescent cells was significantly higher following VP-16 treatment compared with the control group. The percentage of G 1 phase cells, and p-Rb protein and Mdm2 protein expression were also significantly different following VP-16 treatment compared with the control group. VP-16 increased the activity of β-galactosidase in the A459 cells. VP-16 also decreased the expression level of Mdm2 and p-Rb protein and inhibited cell cycle progression in G 1 . These results indicate that VP-16 induces the cellular senescence of A549 cells via the Mdm2-Rb signaling pathway. However, further investigations are required to validate the mechanisms underlying these effects of VP-16.

  16. α9- and α7-containing receptors mediate the pro-proliferative effects of nicotine in the A549 adenocarcinoma cell line.

    PubMed

    Mucchietto, Vanessa; Fasoli, Francesca; Pucci, Susanna; Moretti, Milena; Benfante, Roberta; Maroli, Annalisa; Di Lascio, Simona; Bolchi, Cristiano; Pallavicini, Marco; Dowell, Cheryl; McIntosh, Michael; Clementi, Francesco; Gotti, Cecilia

    2017-07-20

    Tobacco smoke contains many classes of carcinogens and although nicotine is unable to initiate tumourigenesis in humans and rodents, it promotes tumour growth and metastasis in lung tumours by acting on neuronal nicotinic ACh receptors (nAChRs). The aim of this study was to identify molecularly, biochemically and pharmacologically which nAChR subtypes are expressed and functionally activated by nicotine in lung cancer cell lines. We used A549 and H1975 adenocarcinoma cell lines derived from lung tumours to test the in vitro effects of nicotine, and nAChR subtype-specific peptides and compounds. The two adenocarcinoma cell lines express distinctive nAChR subtypes, and this affects their nicotine-induced proliferation. In A549 cells, nAChRs containing the α7 or α9 subunits not only regulate nicotine-induced cell proliferation but also the activation of the Akt and ERK pathways. Blocking these nAChRs by means of subtype-specific peptides, or silencing their expression by means of subunit-specific siRNAs, abolishes nicotine-induced proliferation and signalling. Moreover, we found that the α7 antagonist MG624 also acts on α9-α10 nAChRs, blocks the effects of nicotine on A549 cells and has dose-dependent cytotoxic activity. These results highlight the pathophysiological role of α7- and α9-containing receptors in promoting non-small cell lung carcinoma cell growth and intracellular signalling and provide a framework for the development of new drugs that specifically target the receptors expressed in lung tumours. © 2017 The British Pharmacological Society.

  17. Inhibition of mitogen activated protein kinases increases the sensitivity of A549 lung cancer cells to the cytotoxicity induced by a kava chalcone analog.

    PubMed

    Warmka, Janel K; Solberg, Eric L; Zeliadt, Nicholette A; Srinivasan, Balasubramanian; Charlson, Aaron T; Xing, Chengguo; Wattenberg, Elizabeth V

    2012-08-03

    We are interested in investigating the biological activity of chalcones, a major class of compounds found in the beverage kava, in order to develop potent and selective chemopreventive candidates. Consumption of kava in the South Pacific Islands is inversely correlated with cancer incidence, even among smokers. Accordingly, chalcones have anti-cancer activities in animal and cell culture models. To investigate signaling pathways that affect chalcone action we studied a potent analog, (E)-3-(3-hydroxy-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (chalcone-24). Chalcone-24 was selected from a series of chalcone analogs that were synthesized based on the structures derived from flavokawain compounds found in kava, and screened in A549 lung cancer cells for induction of cytotoxicity and inhibition of NF-κB, a transcription factor associated with cell survival. Incubation of A549 cells with chalcone-24 resulted in a dose-dependent inhibition of cell viability, inhibition of NF-κB, activation of caspases, and activation of extracellular signal regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK); ERK1/2 and JNK are mitogen activated protein kinases that play central roles in regulating cell fate. Pharmacological inhibitors of ERK1/2 or JNK increased the sensitivity of A549 cells to chalcone-24-induced cytotoxicity, without affecting NF-κB or caspase activity. These results will help refine the synthesis of chalcone analogs to maximize the combination of actions required to prevent and treat cancer. Copyright © 2012 Elsevier Inc. All rights reserved.

  18. Inhibition of mitogen activated protein kinases increases the sensitivity of A549 lung cancer cells to the cytotoxicity induced by a kava chalcone analog

    PubMed Central

    Warmka, Janel K.; Solberg, Eric L.; Zeliadt, Nicholette A.; Srinivasan, Balasubramanian; Charlson, Aaron T.; Xing, Chengguo; Wattenberg, Elizabeth V.

    2012-01-01

    We are interested in investigating the biological activity of chalcones, a major class of compounds found in the beverage kava, in order to develop potent and selective chemopreventive candidates. Consumption of kava in the South Pacific Islands is inversely correlated with cancer incidence, even among smokers. Accordingly, chalcones have anti-cancer activities in animal and cell culture models. To investigate signaling pathways that affect chalcone action we studied a potent analog, (E)-3-(3-hydroxy-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (chalcone-24). Chalcone-24 was selected from a series of chalcone analogs that were synthesized based on the structures derived from flavokawain compounds found in kava, and screened in A549 lung cancer cells for induction of cytotoxicity and inhibition of NF-κB, a transcription factor associated with cell survival. Incubation of A549 cells with chalcone-24 resulted in a dose-dependent inhibition of cell viability, inhibition of NF-κB, activation of caspases, and activation of extracellular signal regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK); ERK1/2 and JNK are mitogen activated protein kinases that play central roles in regulating cell fate. Pharmacological inhibitors of ERK1/2 or JNK increased the sensitivity of A549 cells to chalcone-24-induced cytotoxicity, without affecting NF-κB or caspase activity. These results will help refine the synthesis of chalcone analogs to maximize the combination of actions required to prevent and treat cancer. PMID:22771807

  19. In vitro effects induced by diesel exhaust at an air-liquid interface in a human lung alveolar carcinoma cell line A549.

    PubMed

    Okubo, Tomoko; Hosaka, Mitsugu; Nakae, Dai

    2015-01-01

    The present study examined the effects induced in vitro in human adenocarcinoma-derived alveolar basal epithelial A549 cells by diesel particulate matter (DPM) administered into the culture medium or by diesel exhaust administered at an air-liquid interface. When A549 cells were exposed to DPM in the culture medium, cell proliferation was inhibited at doses of 10-100 μg/mL; generation of interleukin (IL)-8 and the antioxidant enzyme, heme oxygenase-1 (HO-1), were inhibited at a dose of 100 μg/mL, and hydroxyl radicals were produced, but could be inhibited by catalase or superoxide dismutase. In contrast, when A549 cells were exposed to diesel exhaust, cell proliferation was inhibited in the absence, but not in the presence, of a diesel particulate filter (DPF); in the absence of a DPF IL-8 was produced in the same amount as in the control cells but was suppressed in the presence of a DPF; HO-1 mRNA was transiently over-expressed in the presence of a DPF, and it was also increased slightly produced in the absence of a DPF but statistically not significant in the presence of a DPF, and it was also increased slightly produced in the absence of a DPF but statistically not significant; HO-1 was transiently produced independent of the absence or the presence of a DPF; and hydroxyl radicals were weakly produced, even in the presence of a DPF but could be inhibited by catalase or superoxide dismutase. It is thus suggested that oxidative stress may be induced by exposure to DPM or diesel exhaust and thereby exerts cytotoxic effect. The introduction of a DPF is effective to protect cells from the toxicity of diesel exhaust presumably by suppression of an oxidative stress. Copyright © 2015 Elsevier GmbH. All rights reserved.

  20. Titanium dioxide nanoparticles-mediated in vitro cytotoxicity does not induce Hsp70 and Grp78 expression in human bronchial epithelial A549 cells.

    PubMed

    Aueviriyavit, Sasitorn; Phummiratch, Duangkamol; Kulthong, Kornphimol; Maniratanachote, Rawiwan

    2012-10-01

    Titanium dioxide nanoparticles (TiO(2)NPs) are increasingly being used in various industrial applications including the production of paper, plastics, cosmetics and paints. With the increasing number of nano-related products, the concern of governments and the general public about the health and environmental risks, especially with regard to occupational and other environmental exposure, are gradually increasing. However, there is insufficient knowledge about the actual affects upon human health and the environment, as well as a lack of suitable biomarkers for assessing TiO(2)NP-induced cytotoxicity. Since the respiratory tract is likely to be the main exposure route of industrial workers to TiO(2)NPs, we investigated the cytotoxicity of the anatase and rutile crystalline forms of TiO(2)NPs in A549 cells, a human alveolar type II-like epithelial cell line. In addition, we evaluated the transcript and protein expression levels of two heat shock protein (HSP) members, Grp78 and Hsp70, to ascertain their suitability as biomarkers of TiO(2)NP-induced toxicity in the respiratory system. Ultrastructural observations confirmed the presence of TiO(2)NPs inside cells. In vitro exposure of A549 cells to the anatase or rutile forms of TiO(2)NPs led to cell death and induced intracellular ROS generation in a dose-dependent manner, as determined by the MTS and dichlorofluorescein (DCF) assays, respectively. In contrast, the transcript and protein expression levels of Hsp70 and Grp78 did not change within the same TiO(2)NPs dose range (25-500 μg/ml). Thus, whilst TiO(2)NPs can cause cytotoxicity in A549 cells, and thus potentially in respiratory cells, Hsp70 and Grp78 are not suitable biomarkers for evaluating the acute toxicological effects of TiO(2)NPs in the respiratory system.

  1. Overexpression of Smac promotes Cisplatin-induced apoptosis by activating caspase-3 and caspase-9 in lung cancer A549 cells.

    PubMed

    Qin, Sida; Yang, Chengcheng; Wang, Xifang; Xu, Chongwen; Li, Shuo; Zhang, Boxiang; Ren, Hong

    2013-03-01

    Second mitochondrial-derived activator of caspase (Smac) plays crucial roles in mitochondrial apoptosis pathways and promotes chemotherapy-induced apoptosis. In this study, Smac levels were examined in various lung cancer cell lines, and the effects of overexpressed Smac in the nonsmall-cell lung cancer cell line A549 were assayed by stable transfection of Smac. Subsequently, MTT assays, cell counting, and flow cytometry were applied to show that overexpression of Smac inhibits cell viability and cell growth and enhances apoptosis after cisplatin treatment. Western blotting was performed before and after cisplatin treatment to demonstrate that drug treatment could release Smac from mitochondria into the cytosol and promote apoptosis by activating caspase-3 and caspase-9. Promotion of apoptosis by cytosolic Smac could be blocked by pretreating cells with the caspase-9 inhibitor z-LEHD-FMK. Our findings indicate that overexpressed Smac significantly inhibited A549 cell growth and promoted apoptosis following cisplatin treatment due to the release of Smac from mitochondria into the cytosol, which increased the activities of caspase-3 and caspase-9.

  2. Study of Bioreductive Anticancer Agent RH-1-Induced Signals Leading the Wild-Type p53-Bearing Lung Cancer A549 Cells to Apoptosis.

    PubMed

    Stulpinas, Aurimas; Imbrasaitė, Aušra; Krestnikova, Natalija; Šarlauskas, Jonas; Čėnas, Narimantas; Kalvelytė, Audronė Valerija

    2016-01-19

    Aziridinylquinone RH-1 (2,5-diaziridinyl-3-hydroxymethyl-6-methyl-cyclohexa-2,5-diene-1,4-dione) is a potential anticancer agent. RH-1 action is associated with quinone oxidoreductase (NQO1) which reduces this diaziridinylbenzoquinone into DNA-alkylating hydroquinone and is overexpressed in many tumors. Another suggested mechanism of RH-1 toxicity is the formation of reactive oxygen species (ROS) arising from its redox cycling. In order to improve anticancer action of this and similar antitumor quinones, we investigated the involvement of different signaling molecules in cytotoxicity induced by RH-1 by using wild-type tumor suppressor p53 bearing nonsmall cell lung carcinoma A549 cells as a model. Gradual and prolonged increase of mitogen-activated protein kinases (MAPK) ERK, P38, and JNK phosphorylation was observed during 24-h RH-1 treatment. In parallel, activation of DNA damage-sensing ATM kinase, upregulation, and phosphorylation of TP53 (human p53) took place. Inhibition studies revealed that RH-1-induced A549 apoptosis involved the NQO1-ATM-p53 signaling pathway and ROS generation. TP53 participated in ROS- and DNA damage-induced cell death differently. Moreover, MAP kinase JNK was another TP53 activator and death inducer in A549 cells. At the same time, rapid and prolonged activation of AKT kinase during RH-1 treatment was found, and it proved to be antiapoptotic kinase in our model system. Therefore, we identified that different and opposite cell death regulating signaling pathways, which may counteract one another, are induced in cancer cells during chemotherapeutic RH-1 treatment.

  3. Anti-metastatic effects of isolinderalactone via the inhibition of MMP-2 and up regulation of NM23-H1 expression in human lung cancer A549 cells

    PubMed Central

    Chuang, Cheng-Hung; Wang, Li-Yu; Wong, Yuen Man; Lin, En-Shyh

    2018-01-01

    Metastatic lung cancer is a leading cause of mortality and has a mortality rate of ≥90%. Isolinderalactone (ILL) is a sesquiterpene lactone compound that has been used in traditional Chinese medicine. Research has demonstrated that ILL has anti-inflammatory and anti-proliferative properties; however, to the best of our knowledge, studies investigating whether ILL can inhibit lung cancer cell metastasis have not been conducted. In the present study, 1–10 µM ILL was applied in the culturing of the A549 lung cancer cell line to investigate the effects of ILL on the invasion and migration of lung cancer cells, including whether the possible mechanisms of ILL are associated with the expression of matrix metalloproteinase (MMP)-2 and NME/NM23 nucleoside diphosphate kinase 1 (NM23-H1) genes. The results of the present study indicated that ILL inhibited the invasion and migration of the A549 cancer cells and exhibited a dose-response association. ILL also significantly inhibited the protein expression and activity of MMP-2 (P<0.05), exhibiting a trend similar to that of its invasion- and migration-associated properties. Further research revealed that ILL significantly increased the expression of NM23-H1 protein and inhibited the expression of β-catenin protein (P<0.05). The results of the present study is, to the best of our knowledge, the first to confirm that ILL can inhibit the invasion and migration of A549 cancer cells, with the possible mechanisms potentially involving the inhibition of MMP-2 and β-catenin protein expression resulting from the up regulation of NM23-H1 expression. PMID:29541242

  4. Synergistic induction of apoptosis by sulindac and simvastatin in A549 human lung cancer cells via reactive oxygen species-dependent mitochondrial dysfunction.

    PubMed

    Hwang, Ki-Eun; Park, Chul; Kwon, Su-Jin; Kim, Young-Suk; Park, Do-Sim; Lee, Mi-Kyung; Kim, Byoung-Ryun; Park, Seong-Hoon; Yoon, Kwon-Ha; Jeong, Eun-Taik; Kim, Hak-Ryul

    2013-07-01

    Prevention of lung cancer is more feasible and holds greater promise when different agents are used in combination to target multiple processes during carcinogenesis. The mechanisms by which non-steroidal anti-inflammatory drugs and statins inhibit cancer cell growth and induce apoptosis are not fully understood. This study was designed to investigate lung cancer chemoprevention through a mechanism-based approach using sulindac at low doses in combination with simvastatin. We found that sulindac-induced cytotoxicity was significantly enhanced in the presence of simvastatin. The combination of sulindac and simvastatin induced more extensive caspase-dependent apoptosis in A549 cells compared to that induced with either drug alone. The combination of sulindac and simvastatin also increased the loss of mitochondrial transmembrane potential (∆Ψm) and the cytosolic release of cytochrome c. In addition, ROS generation in cells treated with both sulindac and simvastatin was markedly increased compared to cells treated with either sulindac or simvastatin alone. The enhancement of ROS generation by sulindac and simvastatin was abrogated by pretreatment with NAC, which also prevented apoptosis and mitochondrial dysfunction induced by sulindac and simvastatin. These results suggest that sulindac and simvastatin-induced ROS generation in A549 lung cancer cells causes their accumulation in mitochondria, triggering the release of apoptogenic molecules from the mitochondria to the cytosol, and thus leading to caspase activation and cell death.

  5. MicroRNA-9 functions as a tumor suppressor and enhances radio-sensitivity in radio-resistant A549 cells by targeting neuropilin 1

    PubMed Central

    Xiong, Kai; Shao, Li Hong; Zhang, Hai Qin; Jin, Linlin; Wei, Wei; Dong, Zhuo; Zhu, Yue Quan; Wu, Ning; Jin, Shun Zi; Xue, Li Xiang

    2018-01-01

    Radiotherapy is commonly used to treat lung cancer but may not kill all cancer cells, which may be attributed to the radiotherapy resistance that often occurs in non-small cell lung cancer (NSCLC). At present, the molecular mechanism of radio-resistance remains unclear. Neuropilin 1 (NRP1), a co-receptor for vascular endothelial growth factor (VEGF), was demonstrated to be associated with radio-resistance of NSCLC cells via the VEGF-phosphoinositide 3-kinase-nuclear factor-κB pathway in our previous study. It was hypothesized that certain microRNAs (miRs) may serve crucial functions in radio-sensitivity by regulating NRP1. Bioinformatics predicted that NRP1 was a potential target of miR-9, and this was validated by luciferase reporter assays. Functionally, miR-9-transfected A549 cells exhibited a decreased proliferation rate, increased apoptosis rate and attenuated migratory and invasive abilities. Additionally, a high expression of miR-9 also significantly enhanced the radio-sensitivity of A549 cells in vitro and in vivo. These data improve understanding of the mechanisms of cell radio-resistance, and suggest that miR-9 may be a molecular target for the prediction of radio-sensitivity in NSCLC. PMID:29435012

  6. MicroRNA-9 functions as a tumor suppressor and enhances radio-sensitivity in radio-resistant A549 cells by targeting neuropilin 1.

    PubMed

    Xiong, Kai; Shao, Li Hong; Zhang, Hai Qin; Jin, Linlin; Wei, Wei; Dong, Zhuo; Zhu, Yue Quan; Wu, Ning; Jin, Shun Zi; Xue, Li Xiang

    2018-03-01

    Radiotherapy is commonly used to treat lung cancer but may not kill all cancer cells, which may be attributed to the radiotherapy resistance that often occurs in non-small cell lung cancer (NSCLC). At present, the molecular mechanism of radio-resistance remains unclear. Neuropilin 1 (NRP1), a co-receptor for vascular endothelial growth factor (VEGF), was demonstrated to be associated with radio-resistance of NSCLC cells via the VEGF-phosphoinositide 3-kinase-nuclear factor-κB pathway in our previous study. It was hypothesized that certain microRNAs (miRs) may serve crucial functions in radio-sensitivity by regulating NRP1. Bioinformatics predicted that NRP1 was a potential target of miR-9, and this was validated by luciferase reporter assays. Functionally, miR-9-transfected A549 cells exhibited a decreased proliferation rate, increased apoptosis rate and attenuated migratory and invasive abilities. Additionally, a high expression of miR-9 also significantly enhanced the radio-sensitivity of A549 cells in vitro and in vivo . These data improve understanding of the mechanisms of cell radio-resistance, and suggest that miR-9 may be a molecular target for the prediction of radio-sensitivity in NSCLC.

  7. Dual effects of human adipose tissue-derived mesenchymal stem cells in human lung adenocarcinoma A549 xenografts and colorectal adenocarcinoma HT-29 xenografts in mice.

    PubMed

    Rhyu, Jung Joo; Yun, Jun-Won; Kwon, Euna; Che, Jeong-Hwan; Kang, Byeong-Cheol

    2015-10-01

    Human adipose tissue-derived mesenchymal stem cells (hATMSCs) have great potential as a therapy for various diseases. However, emerging evidence shows that there are conflicting results concerning effects of hATMSCs on tumor progression. Our objective was to determine whether and how hATMSCs modulate tumor growth. After cancer cell lines were subcutaneously inoculated into BALB/c-nude and hairless severe combined immunodeficient mice, hATMSCs were intratumorally injected into the mice. The growth of the A549 tumors was inhibited by hATMSCs, yet that of the HT-29 tumors was significantly promoted by hATMSCs in the in vivo xenograft models. In vitro study using a co-culture system of cancer cells and hATMSCs was consistent with the in vivo experiments. To reveal the molecular events induced by hATMSCs in the xenograft models, global gene expression profiles of the A549 and HT-29 tumors in the absence or presence of hATMSCs were determined. Significant numbers of genes involved in biological processes were altered in the hATMSC-treated A549 tumors, whereas no biological process was regulated by treatment with hATMSCs in the HT-29 tumors, reflecting the different effects of hATMSCs in the different types of cancer. Notably, histone cluster 1, H2aj and neuropeptide Y receptor Y4 were found to be expressed in direct or inverse proportion to tumor size in both xenograft models. In addition, nuclear factor κB (NF-κB) p65 was differentially phosphorylated by the hATMSCs dependent on the source of the cancer cells. In conclusion, the identified gene profiling and NF-κB signaling provide molecular evidence to explain the conflicting findings in tumor‑MSC studies, although further study is needed to confirm these findings using various types of cancer.

  8. MicroRNA-122-3p inhibits tumor cell proliferation and induces apoptosis by targeting Forkhead box O in A549 cells.

    PubMed

    Wang, Wen; Yang, Jinsong; Yu, Fenglei; Li, Wenjie; Wang, Li; Zou, Haoyu; Long, Xia

    2018-02-01

    The imbalance between cell proliferation and apoptosis was implicated to serve key roles in cancer pathogenesis. The characteristics of microRNAs (miRNAs/miRs) have attracted much attention in research focusing on cancer pathogenesis in recent years. miR-122-3p has been reported to be associated with a number of disease processes and pathogenesis, including lung cancer. The present study aimed to investigate the association of miR-122-3p expression level with cell proliferation and apoptosis in a lung cancer cell line. A549 cells were transfected with miR-122-3p to interrupt the expression of miR-122-3p. Subsequently, MTT and BrdU assay, and western blot were used to analyze the influence of miR-122-3p on lung cancer cell proliferation, cell viability and its underlying mechanism. The present study revealed that, by targeting p27, overexpression of miR-122-3p inhibited cell proliferation in lung cancer. Furthermore, the cell apoptosis analysis suggested that overexpression of miR-122-3p was able to inhibit cell apoptosis by targeting Forkhead box O. These findings suggest that miR-122-3p may be associated with the pathology and progression of lung cancer and be a new therapeutic target for this disease.

  9. Coptisine-induced cell cycle arrest at G2/M phase and reactive oxygen species-dependent mitochondria-mediated apoptosis in non-small-cell lung cancer A549 cells.

    PubMed

    Rao, Poorna Chandra; Begum, Sajeli; Sahai, Mahendra; Sriram, D Saketh

    2017-03-01

    This study aimed to explore the effect of coptisine on non-small-cell lung cancer and its mechanism through various in vitro cellular models (A549). Results claimed significant inhibition of proliferation by coptisine against A549, H460, and H2170 cells with IC 50 values of 18.09, 29.50, and 21.60 µM, respectively. Also, coptisine exhibited upregulation of pH2AX, cell cycle arrest at G2/M phase, and downregulation of the expression of cyclin B1, cdc2, and cdc25C and upregulation of p21 dose dependently. Furthermore, induction of apoptosis in A549 cells by coptisine was characterized by the activation of caspase 9, caspase 8, and caspase 3, and cleavage of poly adenosine diphosphate ribose polymerase. In addition, coptisine was found to increase reactive oxygen species generation, upregulate Bax/Bcl-2 ratio, disrupt mitochondrial membrane potential, and cause cytochrome c release into the cytosol. Besides, treatment with a reactive oxygen species inhibitor (N-acetyl cysteine) abrogated coptisine-induced growth inhibition, apoptosis, reactive oxygen species generation, and mitochondrial dysfunction. Thus, the mediation of reactive oxygen species in the apoptosis-induced effect of coptisine in A549 cells was corroborated. These findings have offered new insights into the effect and mechanisms of action of coptisine against non-small-cell lung cancer.

  10. 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

    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 amore » 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.« less

  11. 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. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Artemisinin induces A549 cell apoptosis dominantly via a reactive oxygen species-mediated amplification activation loop among caspase-9, -8 and -3.

    PubMed

    Gao, Weijie; Xiao, Fenglian; Wang, Xiaoping; Chen, Tongsheng

    2013-10-01

    This report is designed to explore the roles of caspase-8, -9 and -3 in artemisinin (ARTE)-induced apoptosis in non-small cell lung cancer cells (A549 cells). ARTE induced reactive oxygen species (ROS)-mediated apoptosis in dose- and time-dependent fashion. Although ARTE treatment did not induce Bid cleavage and significant loss of mitochondrial membrane potential, it induced release of Smac and AIF but not cytochrome c from mitochondria, and silencing of Bak but not Bax significantly prevented ARTE-induced cytotoxicity. Moreover, ARTE treatment induced ROS-dependent activation of caspase-9, -8 and -3. Of the utmost importance, silencing or inhibiting any one of caspase-8, -9 and -3 almost completely prevented ARTE-induced activation of all the three caspases and remarkably abrogated the cytotoxicity of ARTE, suggesting that ARTE triggered an amplification activation loop among caspase-9, -8 and -3. Collectively, our data demonstrate that ARTE induces a ROS-mediated amplification activation loop among caspase-9, -8 and -3 to dominantly mediate the apoptosis of A549 cells.

  13. 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.

  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 Viqi Chutan Recipe on Caspase-4 and DNA-PK of Cell Apoptosis Approach in Transplanted Lung Cancer A549 Cells in Nude Mice].

    PubMed

    Li, Shu; Wang, Shu-mei; Yang, Yang-bo-jun; Liu, Qi-ou

    2015-06-01

    To study the inhibitory effect of Yiqi Chutan Recipe on the transplanted tumor through endoplasmic reticulum UPR-mediated approach. 40 lung cancer A549 cells models transplanted in nude mice were established. On the 7th day of inoculation, mice were randomly divided into model group( saline group) , Cisplatin group (0.002 g/kg), Yiqi Chutan Recipe low dose group (3.0 g/kg), Yiqi Chutan Recipe high dose group(6. 0 g/kg)and Yiqi Chutan Recipe (3.0 g/kg)with Cisplatin group (0.002 g/kg). Each aforementioned group had eight mice. Mice were treated by Yiqi Chutan Recipe to gavage one time a day, for 21 days, and by Cisplatin Injection to intraperitoneal injection one time a day, for 7 days. On the 22th day, all mice were executed to death. Then each tumor's weight and volume were measured, and the expression of Caspase-4 and DNA-PK protein were detected through immunohistochemical method and Western blot method. Compared with model group, the tumors' volume and weight of Yiqi Chutan Recipe high dose group and Yiqi Chutan Recipe with Cisplatin group were decreased, but the expressions of Caspase-4 and DNA-PK protein in tumors were increased (P < 0.01). Yiqi Chutan Recipe with Cisplatin Group had the better effect (P < 0.05). Yiqi Chutan Recipe has a certain inhibitory effect on A549 lung cancer in mice and its possible mechanism is relevant to the increase of expression of Caspase-4 and DNA-PK protein.

  16. 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. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  17. 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.

  18. BRM270 inhibits cancer stem cell maintenance via microRNA regulation in chemoresistant A549 lung adenocarcinoma cells.

    PubMed

    Kwon, Taeho; Chandimali, Nisansala; Huynh, Do Luong; Zhang, Jiao Jiao; Kim, Nameun; Bak, Yesol; Yoon, Do-Young; Yu, Dae-Yeul; Lee, Jae Cheol; Gera, Meeta; Ghosh, Mrinmoy; Park, Yang Ho; Jeong, Dong Kee

    2018-02-14

    Chemotherapy is a standard treatment for non-small-cell lung cancer (NSCLC). However, the dose-limiting toxicity of drugs and the development of chemoresistance are major clinical challenges to successful management of NSCLC. Asian traditional medicine is gaining global attention as a non-toxic alternative to chemotherapy. BRM270 is an extract formulated from seven Asian medicinal plants that has been shown to inhibit tumor cell proliferation in diverse cancer types. We previously demonstrated that BRM270 suppresses tumorigenesis by negatively regulating nuclear factor-κB signaling in multidrug-resistant cancer stem cells (CSCs). In this study we report that the growth, migration, and invasion of normal human lung adenocarcinoma cells and their chemoresistant derivatives was inhibited by BRM270 treatment. Notably, BRM270 was found to modulate CSC self-renewal and tumor-initiating capacity via positive regulation of the miRNA-128. Thus, combination therapy with miRNA-128 and BRM270 may be an effective treatment strategy for chemoresistant NSCLC.

  19. New geranylated flavanones from the fruits of Paulownia catalpifolia Gong Tong with their anti-proliferative activity on lung cancer cells A549.

    PubMed

    Gao, Tian-yang; Jin, Xing; Tang, Wen-zhao; Wang, Xiao-jing; Zhao, Yun-xue

    2015-09-01

    Three new geranylated flavanones, named as paucatalinone A (1), B (2), and isopaucatalinone B (3), were isolated from the fruits of Paulownia catalpifolia Gong Tong (Scrophulariaceae). Their structures were well determined by means of IR, MS, 1D and 2D NMR, and CD techniques. Paucatalinone A (1) is the first sample as a dimeric geranylated flavanone derivative isolated from natural products. Paucatalinone A (1) displayed good antiproliferative effects on human lung cancer cells A549 and resulted in a clear increase of the percentage of cells in G1 phase and a decrease in the percentage of cells in S and G2/M phases in comparison with control cells. Copyright © 2015. Published by Elsevier Ltd.

  20. Evaluation of anticancer activity of water and juice extracts of young Hordeum vulgare in human cancer cell lines HT-29 and A549.

    PubMed

    Czerwonka, Arkadiusz; Kawka, Katarzyna; Cykier, Klaudia; Lemieszek, Marta Kinga; Rzeski, Wojciech

    2017-06-12

    Introduction and objective. Barley (Hordeum vulgare L.) is known as a rich source of different bioactive compounds. At present, considerable attention of researchers is focused on young barley grass. It can be a good source of dietary minerals, vitamins, carbohydrates, amino acids, phenolic compounds and proteins. It is possible that the composition of chemical ingredients beneficial for health may induce an anticancer potential of young barley in human colon adenocarcinoma (HT-29) and human lung adenocarcinoma (A549) cell lines. Materials and method. Hordeum vulgare water extract (HWE) and Hordeum vulgare juice extract (HJE) were prepared. Cell proliferation and viability were examined with the use of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and NR (neutral red) methods. Induction of necrosis was assessed by propidium iodide/Hoechst staining. Progress of the cell cycle involved in cell proliferation, apoptosis, and regulation of transcription was estimated using flow cytometry analysis. Additionally, the capability of free radical scavenging was evaluated with the DPPH assay. Results. The study revealed that extracts inhibited the proliferation of cancer cells. The NR study confirmed the low cytotoxic activity of the tested extracts to normal human colon epithelial cells (CCD 841 CoTr) and human skin fibroblasts (HSF). Furthermore, a dose-dependent cytotoxicity against HT-29 cells, but not A549 cells, has been reported. The free radical scavenging activity was observed in the case of the HWE but not the HJE. Conclusions. The obtained results indicate a cancer chemopreventive potential of young barley as a safe dietary agent in colon carcinoma.

  1. 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.

  2. Effect of three fatty acids from the leaf extract of Tiliacora triandra on P-glycoprotein function in multidrug-resistant A549RT-eto cell line

    PubMed Central

    Kaewpiboon, Chutima; Winayanuwattikun, Pakorn; Yongvanich, Tikamporn; Phuwapraisirisan, Preecha; Assavalapsakul, Wanchai

    2014-01-01

    Background: Cancer cells have the ability to develop resistance to chemotherapy drugs, which then leads to a reduced effectiveness and success of the treatment. Multidrug resistance (MDR) involves the resistance in the same cell/tissue to a diverse range of drugs of different structures. One of the characteristics of MDR is an overexpression of P-glycoprotein (P-gp), which causes the efflux of the accumulated drug out of the cell. The MDR human non-small cell lung carcinoma cell line with a high P-gp expression level (A549RT-eto) was used to investigate the bioactive compounds capable of reversing the etoposide resistance in this cell line. Materials and Methods: The leaves of Tiliacora triandra were sequentially extracted with hexane, dichloromethane, methanol and water. Only the hexane extract reduced the etoposide resistance of the A549RT-eto cell line, and was further fractionated by column chromatography using the TLC-pattern and the restoration of etoposide sensitivity as the selection criteria. Results: The obtained active fraction (F22) was found by nuclear magnetic resonance and gas chromatography-mass spectroscopy analyses to be comprised of a 49.5:19.6:30.9 (w/w/w) mixture of hexadecanoic: octadecanoic acid: (Z)-6-octadecenoic acids. This stoichiometric mixture was recreated using pure fatty acids (MSFA) and gave a similar sensitization to etoposide and enhanced the relative rate of rhodamine-123 accumulation to a similar extent as F22, supporting the action via reducing P-gp activity. In contrast, the fatty acids alone did not show this effect. Conclusion: This is the first report of the biological activity from the leaves of T. triandra as a potential source of a novel chemosensitizer. PMID:25298673

  3. Effect of three fatty acids from the leaf extract of Tiliacora triandra on P-glycoprotein function in multidrug-resistant A549RT-eto cell line.

    PubMed

    Kaewpiboon, Chutima; Winayanuwattikun, Pakorn; Yongvanich, Tikamporn; Phuwapraisirisan, Preecha; Assavalapsakul, Wanchai

    2014-08-01

    Cancer cells have the ability to develop resistance to chemotherapy drugs, which then leads to a reduced effectiveness and success of the treatment. Multidrug resistance (MDR) involves the resistance in the same cell/tissue to a diverse range of drugs of different structures. One of the characteristics of MDR is an overexpression of P-glycoprotein (P-gp), which causes the efflux of the accumulated drug out of the cell. The MDR human non-small cell lung carcinoma cell line with a high P-gp expression level (A549RT-eto) was used to investigate the bioactive compounds capable of reversing the etoposide resistance in this cell line. The leaves of Tiliacora triandra were sequentially extracted with hexane, dichloromethane, methanol and water. Only the hexane extract reduced the etoposide resistance of the A549RT-eto cell line, and was further fractionated by column chromatography using the TLC-pattern and the restoration of etoposide sensitivity as the selection criteria. The obtained active fraction (F22) was found by nuclear magnetic resonance and gas chromatography-mass spectroscopy analyses to be comprised of a 49.5:19.6:30.9 (w/w/w) mixture of hexadecanoic: octadecanoic acid: (Z)-6-octadecenoic acids. This stoichiometric mixture was recreated using pure fatty acids (MSFA) and gave a similar sensitization to etoposide and enhanced the relative rate of rhodamine-123 accumulation to a similar extent as F22, supporting the action via reducing P-gp activity. In contrast, the fatty acids alone did not show this effect. This is the first report of the biological activity from the leaves of T. triandra as a potential source of a novel chemosensitizer.

  4. PKM2 Thr454 phosphorylation increases its nuclear translocation and promotes xenograft tumor growth in A549 human lung cancer cells

    SciTech Connect

    Yu, Zhenhai, E-mail: tomsyu@163.com; Huang, Liangqian; Qiao, Pengyun

    2016-05-13

    Pyruvate kinase M2 (PKM2) is a key enzyme of glycolysis which is highly expressed in many tumor cells, and plays an important role in the Warburg effect. In previous study, we found PIM2 phosphorylates PKM2 at Thr454 residue (Yu, etl 2013). However, the functions of PKM2 Thr454 modification in cancer cells still remain unclear. Here we find PKM2 translocates into the nucleus after Thr454 phosphorylation. Replacement of wild type PKM2 with a mutant (T454A) enhances mitochondrial respiration, decreases pentose phosphate pathway, and enhances chemosensitivity in A549 cells. In addition, the mutant (T454A) PKM2 reduces xenograft tumor growth in nude mice. Thesemore » findings demonstrate that PKM2 T454 phosphorylation is a potential therapeutic target in lung cancer.« less

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

    PubMed

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

    2006-05-01

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

  6. Cytotoxicity study of Piper nigrum seed mediated synthesized SnO2nanoparticles towards colorectal (HCT116) and lung cancer (A549) cell lines.

    PubMed

    Tammina, Sai Kumar; Mandal, Badal Kumar; Ranjan, Shivendu; Dasgupta, Nandita

    2017-01-01

    Different sized tetragonal tin oxide nanoparticles (SnO 2 NPs) were synthesized using Piper nigrum seed extract at three different calcination temperatures (300, 500, 900°C) and these nanoparticles (NPs) were characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD), dynamic light scattering (DLS) and Fourier transform infrared spectrophotometry (FT-IR). The optical properties were studied using UV-Vis and photoluminescence (PL) spectrophotometers. The generation of reactive oxygen species (ROS) was monitored by using a fluorescence spectrophotometer and fluorescence microscope. The cytotoxicity of the synthesized SnO 2 NPs was checked against the colorectal (HCT116) and lung (A549) cancer cell lines and the study results show that SnO 2 NPs were toxic against cancer cell lines depending on their size and dose. IC 50 values of SnO 2 NPs having average particle sizes of 8.85±3.5, 12.76±3.9 and 29.29±10.9nm are 165, 174 and 208μgL -1 against HCT116, while these values are 135, 157 and 187μgL -1 against A549 carcinoma cell lines, respectively. The generated ROS were responsible for the cytotoxicity of SnO 2 NPs to the studied cancer cells and smaller size NPs generated more ROS and hence showed higher cytotoxicity over larger size NPs. The results of this study suggest that the synthesized stable nanoparticles could be a potent therapeutic agent towards cancerous cell lines. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Induction of apoptosis and autophagy by sodium selenite in A549 human lung carcinoma cells through generation of reactive oxygen species.

    PubMed

    Park, Shin-Hyung; Kim, Jeong-Hwan; Chi, Gyoo Yong; Kim, Gi-Young; Chang, Young-Chae; Moon, Sung-Kwon; Nam, Soo-Wan; Kim, Wun-Jae; Yoo, Young Hyun; Choi, Yung Hyun

    2012-08-03

    Selenium in the form of sodium selenite has been reported to exert anti-tumor effects in several cancer cell types by inducing autophagic cell death and apoptosis mediated by reactive oxygen species (ROS). However, the exact molecular pathways underlying these effects have not been fully established. The present study used A549 human lung carcinoma cells for further investigation of the anti-cancer mechanism of sodium selenite. We showed that sodium selenite modulated both the extrinsic and intrinsic apoptotic pathways, which were interconnected by Bid truncation. We used z-VAD-fmk, a pan-caspase inhibitor, to demonstrate that sodium selenite-induced apoptosis was dependent on the activation of caspases. Sodium selenite also increased autophagy, as indicated by an increase in microtubule-associated protein light chain-3 (LC3) puncta, accumulation of LC3II, and elevation of autophagic flux. Pretreatment with bafilomycin A1 enhanced sodium selenite-induced apoptosis, indicating that sodium selenite-induced autophagy functioned as a survival mechanism. Sodium selenite treatment also resulted in generation of ROS, which abrogated mitochondrial membrane potential (MMP) and regulated both apoptosis and autophagy. Phospho-nuclear factor erythroid 2-related factor 2 (p-Nrf2) showed a ROS-dependent translocation to the nucleus, which suggested that Nrf2 might increase cell survival by suppressing ROS accumulation and apoptosis mediated by oxidative stress. Sodium selenite treatment of A549 cells therefore appeared to trigger both apoptosis and cytoprotective autophagy, which were both mediated by ROS. The data suggest that regulation of ROS generation and autophagy can be a potential strategy for treating lung cancer that is resistant to pro-apoptotic therapeutics. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  8. Tumor necrosis factor-{alpha} induces MMP-9 expression via p42/p44 MAPK, JNK, and nuclear factor-{kappa}B in A549 cells

    SciTech Connect

    Lin, C.-C.; Tseng, Hsiao-Wei; Hsieh, Hsi-Lung

    2008-06-15

    Matrix metalloproteinases (MMPs), in particular MMP-9, have been shown to be induced by cytokines including tumor necrosis factor-{alpha} (TNF-{alpha}) and contributes to airway inflammation. However, the mechanisms underlying MMP-9 expression induced by TNF-{alpha} in human A549 cells remain unclear. Here, we showed that TNF-{alpha} induced production of MMP-9 protein and mRNA is determined by zymographic, Western blotting, RT-PCR and ELISA assay, which were attenuated by inhibitors of MEK1/2 (U0126), JNK (SP600125), and NF-{kappa}B (helenalin), and transfection with dominant negative mutants of ERK2 ({delta}ERK) and JNK ({delta}JNK), and siRNAs for MEK1, p42 and JNK2. TNF-{alpha}-stimulated phosphorylation of p42/p44 MAPK and JNKmore » were attenuated by pretreatment with the inhibitors U0126 and SP600125 or transfection with dominant negative mutants of {delta}ERK and {delta}JNK. Furthermore, the involvement of NF-{kappa}B in TNF-{alpha}-induced MMP-9 production was consistent with that TNF-{alpha}-stimulated degradation of I{kappa}B-{alpha} and translocation of NF-{kappa}B into the nucleus which were blocked by helenalin, but not by U0126 and SP600125, revealed by immunofluorescence staining. The regulation of MMP-9 gene transcription by MAPKs and NF-{kappa}B was further confirmed by gene luciferase activity assay. MMP-9 promoter activity was enhanced by TNF-{alpha} in A549 cells transfected with wild-type MMP-9-Luc, which was inhibited by helenalin, U0126, or SP600125. In contrast, TNF-{alpha}-stimulated MMP-9 luciferase activity was totally lost in cells transfected with mutant-NF-{kappa}B MMP-9-luc. Moreover, pretreatment with actinomycin D and cycloheximide attenuated TNF-{alpha}-induced MMP-9 expression. These results suggest that in A549 cells, phosphorylation of p42/p44 MAPK, JNK, and transactivation of NF-{kappa}B are essential for TNF-{alpha}-induced MMP-9 gene expression.« less

  9. Influenza virus infection induces translocation of apoptosis-inducing factor (AIF) in A549 cells: role of AIF in apoptosis and viral propagation.

    PubMed

    Qu, Xinyan; Ding, Xiaoran; Duan, Ming; Yang, Jing; Lin, Ruxian; Zhou, Zhe; Wang, Shengqi

    2017-03-01

    It is recognized that influenza virus induces caspase-dependent apoptosis by activating caspase-3. Apoptosis-inducing factor (AIF) is a caspase-independent cell death effector, and its mitochondrial-nuclear translocation plays an important role in apoptosis. It is demonstrated in this study how influenza virus infection can induce caspase-independent apoptosis in the human alveolar epithelial cell line A549. AIF is translocated from the mitochondria to the nucleus in a caspase-independent manner in response to infection with influenza virus. Knockdown of AIF expression by small interfering RNA (siRNA) led to a reduction in virus-infection-induced apoptosis and virus yield. These results indicate that AIF translocation has a role in influenza-virus-induced apoptosis.

  10. Cytoprotective effect of bioactive sea buckthorn extract on paraquat-exposed A549 cells via induction of Nrf2 and its downstream genes.

    PubMed

    Podder, Biswajit; Kim, Yong-Sik; Song, Ho-Yeon

    2013-12-01

    The extract of sea buckthorn (SBT) [Hippophae rhamnoides L. (Elaeagnaceae)], is used as a food supplement and traditional medicine in numerous countries. This study investigated the protective effects of the functional extract of SBT against paraquat (PQ)-induced toxicity via antioxidant mechanisms in A549 cells. The methanol extract of SBT (25-200 µg/ml) was used to protect cells against PQ (200 µM)-induced cell death. A viability assay was conducted using 3-(4,5-dimethylthioazol-2-ly)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase (LDH). Total intracellular reactive oxygen species (ROS) were measured and plotted. For validation of the SBT-induced expression of nuclear factor-E2-related factor 2 (Nrf2) and its target genes, western blot analysis and qPCR were performed. The present study showed that pretreatment of A549 cells with SBT extract significantly attenuated PQ (200 µM)-induced cellular toxicity. The maximum cytoprotective effect was identified using 200 µg/ml SBT extract; it began 24 h following exposure and was sustained up to 120 h (P<0.05). SBT extract significantly reduced LDH activity by 35.63% and ROS levels by 30.90% (P<0.05). Pretreatment with SBT extract activated Nrf2 mRNA and protein expression and its nuclear translocation. The SBT extract effectively induced Nrf2 target genes, such as NAD(P)H dehydrogenase quinone 1, glutathione peroxidase 1, glutathione reductase and catalase following treatment with PQ. Based on these results, it was hypothesized that SBT extract may be used as a potential therapeutic agent for the treatment of various oxidative stress-related diseases.

  11. 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. Published by Elsevier Ltd.

  12. ANTITUMOR AND APOPTOTIC EFFECTS OF CUCURBITACIN A IN A-549 LUNG CARCINOMA CELLS IS MEDIATED VIA G2/M CELL CYCLE ARREST AND M-TOR/PI3K/AKT SIGNALLING PATHWAY.

    PubMed

    Wang, Wen-Dong; Liu, Yan; Su, Yuan; Xiong, Xian-Zhi; Shang, Dan; Xu, Juan-Juan; Liu, Hong-Ju

    2017-01-01

    The main aim of this study was to demonstrate the antitumor potential of cucurbitacin A on A-549 NSCLC (non-small cell lung cancer cells). The effects of Cucurbitacin A on apoptotic induction, cell physic, cell cycle failure and m-TOR/PI3K/Akt signalling pathway were also investigated in the present study. MTT assay and clonogenic assay were carried out to study effects of this compound on cell cytotoxicity and colony forming tendency in A-549 cells. Moreover, phase and fluorescence microscopic techniques were used to examine the effects on cell morphology and induction of apoptosis. The effects on cell cycle phase distribution were investigated by flow cytometry and effects on m-TOR/PI3K/Akt signalling proteins were assessed by western blot analysis. Results showed that cucurbitacin A induced dose-dependent cytotoxic effects along with suppressing the colony forming tendency in these cells. Cucurbitacin A also induced morphological changes in these cells featuring chromatin condensation, cell shrinkage and apoptotic body formation. G2/M phase cell cycle collapse was also induced by Cucurbitacin A along with inhibition of expression levels of m-TOR/PI3K/Akt proteins. In conclusion, cucurbitacin A inhibits cancer growth in A-549 NSCLC cells by inducing apoptosis, targeting m-TOR/PI3K/Akt signalling pathway and G2/M cell cycle.

  13. 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. © The Author(s) 2015.

  14. 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.

  15. Combined metformin and resveratrol confers protection against UVC-induced DNA damage in A549 lung cancer cells via modulation of cell cycle checkpoints and DNA repair.

    PubMed

    Lee, Yong-Syu; Doonan, Barbara B; Wu, Joseph M; Hsieh, Tze-Chen

    2016-06-01

    Aging in humans is a multi-factorial cellular process that is associated with an increase in the risk of numerous diseases including diabetes, coronary heart disease and cancer. Aging is linked to DNA damage, and a persistent source of DNA damage is exposure to ultraviolet (UV) radiation. As such, identifying agents that confer protection against DNA damage is an approach that could reduce the public health burden of age-related disorders. Metformin and resveratrol have both shown effectiveness in preventing several age-related diseases; using human A549 cells, we investigated whether metformin or resveratrol, alone or combined, prevent UVC-induced DNA damage. We found that metformin inhibited UVC-induced upregulation of p53, as well as downregulated the expression of two DNA damage markers: γH2AX and p-chk2. Metformin also upregulated DNA repair as evidenced by the increase in expression of p53R2. Treatment with metformin also induced cell cycle arrest in UVC-induced cells, in correlation with a reduction in the levels of cyclin E/cdk2/Rb and cyclin B1/cdk1. Compared to metformin, resveratrol as a single agent showed less effectiveness in counteracting UVC-elicited cellular responses. However, resveratrol displayed synergism when combined with metformin as shown by the downregulation of p53/γH2AX/p-chk2. In conclusion, the results of the present study validate the effectiveness of metformin, alone or with the addition of resveratrol, in reducing the risk of aging by conferring protection against UV-induced DNA damage.

  16. 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. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

  17. 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. 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.

    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 changesmore » 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.« less

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

    PubMed

    Adcock, Ian M; Mortaz, Esmaeil; Alipoor, Shamila D; Garssen, Johan; Akbar Velayati, Ali

    2016-12-01

    Tuberculosis (TB) is a major global health problem and poses immense threats to many populations. The association between tobacco smoke and TB has already been studied. Water-pipe smoking has become an increasing problem not only in Middle Eastern countries but also globally as it is considered by users as being safer than cigarettes. The presence of high levels of toxic substances in water-pipe smoke may be predisposing factors that enhance the incidence of pulmonary disorders in water-pipe smokers. For example, uncontrolled macropinocytosis occurs in alveolar epithelial cells following exposure to water-pipe smoke, which may predispose individuals to pulmonary infection. In this work, we studied the effects of water-pipe condense (WPC) on the internalization of Mycobacterium bovis (bacillus Calmette-Guérin [BCG]) by macropinocytosis in Type II alveolar epithelial cells (A549). A549 cells were treated by WPC (4mg/mL) for 24 h, 48 h, 72 h, and 96 h, respectively. The effect on cell proliferation was studied using a methylthiazolyldiphenyl-tetrazolium bromide (MTT) reduction assay. Cells were exposed to fluorescein isothiocyanate (FITC)-dextran (1mg/mL; control) and FITC-BCG (multiplicity of infection, 10) for 20min at 37°C before their collection and the uptake of BCG-FITC was determined by flow cytometry. Similar experiments were performed at 4°C as a control. WPC (4mg/mL) after 72h (1.4±0.2-fold, p<0.05) and 96h (1.6±0.2-fold, p<0.05) hours increased the uptake of BCG-FITC. No effect on BCG-FITC uptake was observed at 24h or 48h. WPC also significantly increased the uptake of FITC-dextran (2.9±0.3-fold, p<0.05) after 96h. WPC also significantly decreased cell proliferation after 24h (84±2%), 48h (78±3%), 72h (64±2%, p<0.05), and 96h (45±2%, p<0.05). WPC exposure increased epithelial cells' permeability and death and enhanced their capacity for macropinocytosis. Our in vitro data suggest possible harmful effects of WPC on the ability of lung epithelial

  20. Proteomics Applications in Dental Derived Stem Cells.

    PubMed

    Li, Jie; Tian, Weidong; Song, Jinlin

    2017-07-01

    At present, the existence of a variety of dental derived stem cells has been documented. These cells displayed promising clinical application potential not only for teeth and its surrounding tissue regeneration, but also for other tissues, such as nerve and bone regeneration. Proteomics is an unbiased, global informatics tool that provides information on all protein expression levels as well as post-translational modification in cells or tissues and is applicable to dental derived stem cells research. Over the last decade, considerable progress has been made to study the global proteome, secrotome, and membrane proteome of dental derived stem cells. Here, we present an overview of the proteomics studies in the context of stem cell research. Particular attention is given to dental derived stem cell types as well as current challenges and opportunities. J. Cell. Physiol. 232: 1602-1610, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  1. Inhibition of Matrix Metalloproteinase-2 Enhances Radiosensitivity by Abrogating Radiation-induced FoxM1-mediated G2/M Arrest in A549 Lung Cancer Cells

    PubMed Central

    Chetty, Chandramu; Bhoopathi, Praveen; Rao, Jasti S.; Lakka, Sajani S.

    2009-01-01

    Matrix metalloproteinase-2 (MMP-2) is known to degrade the collagen IV, play a role in radiation-induced lung injury. We therefore investigated the anti-tumor effects of combining MMP-2 inhibition using an adenovirus expressing siRNA against MMP-2 (Ad-MMP-2-Si) with radiation therapy (IR) on A549 lung cancer cells in vitro and in vivo. IR increased MMP-2 mRNA, protein and activity in lung cancer cells. MMP-2 inhibition along with IR enhanced radiosensitivity as determined by clonogenic assay, flow cytometry, and TUNEL assay. We show that MMP-2 inhibition prior to irradiation reduced p53 phosphorylation, with a corresponding reduction in the expression of the p53 downstream target gene p21Cip1/Waf1. Irradiated tumor cells induced the FoxM1-mediated DNA repair gene, XRCC1 and Checkpoint-2/1, which were abrogated with combined treatment of Ad-MMP-2-Si and IR. Further, the combination of Ad-MMP-2-Si with radiotherapy significantly increased anti-tumor efficacy in vivo compared to either agent alone. Indeed, histological analysis of tumor sections collected from the combination group revealed more apoptotic cells. These studies suggest that MMP-2 inhibition in combination with radiotherapy abrogates G2 cell cycle arrest leading to apoptosis and provide evidence of the anti-tumor efficacy of combining MMP-2 inhibition with irradiation as a new therapeutic strategy for the effective treatment of NSCLC patients. PMID:19165865

  2. TU-H-CAMPUS-TeP3-01: Gold Nanoparticle-Enhanced Radiation Therapy in In Vitro A549 Lung Carcinoma: Studies in Both Traditional Monolayer and Three Dimensional Cell Culture Models

    SciTech Connect

    Oumano, M; University of Massachusetts Lowell, Lowell, MA; Ngwa, W

    2016-06-15

    Purpose: To measure the increase in in vitro radiosensitivity for A549 lung carcinoma cells due to gold nanoparticle (GNP) radiation dose enhancement in both traditional monolayer and three dimensional (3D) cell culture models. Methods: A γH2AX immunofluorescence assay is performed on monolayer A549 cell culture and quantitatively analyzed to measure the increase in double strand breaks (DSBs) resulting from GNP dose enhancement. A clonogenic survival assay (CSA) is then performed on monolayer A549 cell culture to assess true viability after treatment. And lastly, another γH2AX assay is performed on 3D A549 multicellular nodules overlaid on a bed of growth factormore » reduced matrigel to measure dose response in a model that better recapitulates treatment response to actual tumors in vivo. Results: The first γH2AX assay performed on the monolayer cell culture shows a significant increase in DSBs due to GNP dose enhancement. The maximum average observed increase in normalized fluorescent intensity for monolayer cell culture is 171% for the 6Gy-treatment groups incubated in 0.556 mg Au/ml solution. The CSA performed on monolayer cell culture also shows considerable GNP dose enhancement. The maximum decrease in the normalized surviving fraction is 12% for the 4Gy-treatment group incubated in 0.556 mg Au/ml. And lastly, the GNP dose enhancement is confirmed to be mitigated in three dimensional cell culture models as compared to the traditional monolayer model. The maximum average observed dose enhancement for 3D cell culture is 19% for the 6Gy-treatment groups and incubated in 0.556 mg Au/ml. Conclusion: A marked increase in radiosensitivity is observed for A549 lung carcinoma cells when treated with GNPs plus radiation as opposed to radiation alone. Traditional monolayer cell culture also shows a much more pronounced radiation dose enhancement than 3D cell culture.« less

  3. Simple 2(5H)-furanone derivatives with selective cytotoxicity towards non-small cell lung cancer cell line A549 - Synthesis, structure-activity relationship and biological evaluation.

    PubMed

    Byczek-Wyrostek, Anna; Kitel, Radoslaw; Rumak, Klaudia; Skonieczna, Magdalena; Kasprzycka, Anna; Walczak, Krzysztof

    2018-04-25

    A series of 5-alkoxy derivatives of 3,4-dichloro-5-hydroxyfuran-2-(5H)-one (mucochloric acid, MCA) were obtained and subsequently subjected to modification in the C-4 position of 2(5H)-furanone ring. The cytotoxicity of newly synthesized compounds was evaluated in MTT assay against non-small cell lung cancer (A549) and healthy lung epithelial cell line (BEAS-2B). The derivatives containing a branched alkoxy substituent in the C-5 position demonstrated the highest anticancer properties, whereas modification of compounds in the C-4 position of 2(5H)-furanone ring only slightly improve their antiproliferative properties. Compounds 12 and 15 exhibited the best selectivity towards A549cells and were also evaluated in a panel of cancer cell lines of different origin. Further investigation revealed that treatment of A549cell line with compounds 12 and 15 led to G2 phase cell cycle arrest and induction of caspase-independent cell death. Moreover, compound 12 was found to act synergistically with erlotinib. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  4. Suppression of Cell Growth, Migration and Drug Resistance by Ethanolic Extract of Antrodia cinnamomea in Human Lung Cancer A549 Cells and C57BL/6J Allograft Tumor Model

    PubMed Central

    Wu, Chi-Han; Liu, Fon-Chang; Lai, Ming-Tsung; Lan, Shou-Jen; Wu, Chieh-Hsi; Sheu, Ming-Jyh

    2018-01-01

    The purpose of this study was to investigate the inhibitory activities of ethanolic extracts from Antrodia cinnamomea (EEAC) on lung cancer. Cell proliferation and cell cycle distribution were analyzed using (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay and flow cytometry, respectively. Wound-healing assay, Western blotting, and a murine tumor model were separately used to examine cell migration, protein expression, and tumor repression. Our results showed that EEAC induced cell cycle arrest at the G0/G1 phase resulting decreased cell viability in A549 cells. Moreover, EEAC up-regulated the growth-suppressing proteins, adenosine 5′-monophosphate-activated protein kinase (AMPK), p21 and p27, but down-regulated the growth-promoting proteins, protein kinase B (Akt), mammalian tarfet of rapamycin (mTOR), extracellular signal-regulating kinase 1/2 (ERK1/2), retinoblastoma protein (Rb), cyclin E, and cyclin D1. EEAC also inhibited A549 cell migration and reduced expression of gelatinases. In addition, our data showed that tumor growth was suppressed after treatment with EEAC in a murine allograft tumor model. Some bioactive compounds from EEAC, such as cordycepin and zhankuic acid A, were demonstrated to reduce the protein expressions of matrix metalloproteinase (MMP)-9 and cyclin D1 in A549 cells. Furthermore, EEAC enhanced chemosensitivity of A549 to paclitaxel by reducing the protein levels of caveolin-1. Our data suggests that EEAC has the potential to be an adjuvant medicine for the treatment of lung cancer. PMID:29522490

  5. A methoxyflavanone derivative from the Asian medicinal herb (Perilla frutescens) induces p53-mediated G2/M cell cycle arrest and apoptosis in A549 human lung adenocarcinoma.

    PubMed

    Abd El-Hafeez, Amer Ali; Fujimura, Takashi; Kamei, Rikiya; Hirakawa, Noriko; Baba, Kenji; Ono, Kazuhisa; Kawamoto, Seiji

    2017-07-14

    Perilla frutescens is an Asian dietary herb consumed as an essential seasoning in Japanese cuisine as well as used for a Chinese medicine. Here, we report that a newly found methoxyflavanone derivative from P. frutescens (Perilla-derived methoxyflavanone, PDMF; 8-hydroxy-5,7-dimethoxyflavanone) shows carcinostatic activity on human lung adenocarcinoma, A549. We found that treatment with PDMF significantly inhibited cell proliferation and decreased viability through induction of G 2 /M cell cycle arrest and apoptosis. The PDMF stimulation induces phosphorylation of tumor suppressor p53 on Ser15, and increases its protein amount in conjunction with up-regulation of downstream cyclin-dependent kinase inhibitor p21 Cip1/Waf1 and proapoptotic caspases, caspase-9 and caspase-3. We also found that small interfering RNA knockdown of p53 completely abolished the PDMF-induced G 2 /M cell cycle arrest, and substantially abrogated its proapoptotic potency. These results suggest that PDMF represents a useful tumor-preventive phytochemical that triggers p53-driven G 2 /M cell cycle arrest and apoptosis.

  6. Oxygen Enhancement Ratio in Radiation-Induced Initial DSBs by an Optimized Flow Cytometry-based Gamma-H2AX Analysis in A549 Human Cancer Cells.

    PubMed

    Sunada, Shigeaki; Hirakawa, Hirokazu; Fujimori, Akira; Uesaka, Mitsuru; Okayasu, Ryuichi

    2017-11-01

    High-linear energy transfer (LET) heavy ions cause higher therapeutic effects than low-LET radiation due to lower dependency on oxygen concentration in tumor cell killing. The lethality after irradiation largely depends on DNA double-strand breaks (DSBs), however the detailed LET dependency for DSB induction under oxic and hypoxic conditions has not been reported. Therefore, we evaluated the oxygen enhancement ratio (OER) of heavy ion-induced DSB induction using a highly-optimized flow cytometry-based method of γ-H2AX detection. Non-small cell lung cancer (NSCLC) A549 cells were exposed to X-ray, carbon-ion and iron-ion radiations under oxic or hypoxic condition. As a DSB marker, the γ-H2AX signal was measured 1 h postirradiation and analyzed by flow cytometry. DSB slope values were calculated as DSB induction per Gy. Our method was able to detect high-LET radiation-induced DSBs even from clustered DNA damage sites. We also showed a decrease in OER value in an LET-dependent manner regardless of radiation type. In summary, we demonstrated a simple, quick and highly-optimized flow cytometry-based method of DSB analysis that detects DSBs induced by heavy-ion radiation for hypoxic and nonhypoxic cancer cells. Our study may provide a useful biological basis for heavy-ion radiotherapy.

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

    PubMed

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

    2017-04-15

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

  8. A novel system enhancing the endosomal escapes of peptides promotes Bak BH3 peptide inducing apoptosis in lung cancer A549 cells.

    PubMed

    Lin, Nanjing; Zheng, Wenyun; Li, Linfeng; Liu, Hui; Wang, Tianwen; Wang, Ping; Ma, Xingyuan

    2014-06-01

    Therapeutic peptides have been proven useful for treating various diseases. However, it is difficult for therapeutic peptides to reach their target sites with an effective concentration due to inefficient intracellular delivery. Although Tat transduction peptide is a promising tool to deliver therapeutic peptides into cells, the entrapment within endosomes and the nuclear localization of Tat transduction peptide severely limited the biological effects of Tat-linked cargos. In this study, we designed a novel peptide delivering system, Tat-INF7-ubiquitin (TIU), which consisted of Tat transduction peptide, endosomal escape enhancer peptide INF7, and ubiquitin protein. We found that the TIU system was able to efficiently deliver the mCherry fluorescent proteins and the apoptosis-inducing Bak BH3 peptide into the cytosol. The Bak BH3 peptide transported into the cells by the TIU system increased the apoptotic rate to 46.15 ± 4.86% (p < 0.001) in A549 cells, while Tat-BH3 could result in only 20.45 ± 2.89%. These results demonstrated that the TIU system could enhance the effects of therapeutic peptides by facilitating the transmembrane delivery of peptides into the cells and the escape of target proteins from the endosome efficiently.

  9. DNA damage and endoplasmic reticulum stress mediated curcumin-induced cell cycle arrest and apoptosis in human lung carcinoma A-549 cells through the activation caspases cascade- and mitochondrial-dependent pathway.

    PubMed

    Lin, Song-Shei; Huang, Hsuan-Pang; Yang, Jai-Sing; Wu, Jeng-Yuan; Hsia, Te-Chun; Hsai, Te-Chun; Lin, Chin-Chung; Lin, Cheng-Wen; Kuo, Chao-Lin; Gibson Wood, W; Chung, Jing-Gung

    2008-12-08

    Curcumin, a major component of the Curcuma species, is known to have antioxidant, anti-inflammatory properties and induce apoptosis of cancer cells, however, the precise molecular mechanisms of apoptosis in vitro are unclear. In this study, we showed that curcumin, a plant product containing the phenolic phytochemical, caused DNA damage and endoplasmic reticulum (ER) stress and mitochondrial-dependent-induced apoptosis through the activation of caspase-3 at a treatment concentration of 30 microM in human lung cancer A-549 cells. In contrast, treatment with 5-10 microM of curcumin did not induce significant apoptosis, but rather induced G2/M-phase arrest in A-549 cells. Flow cytometric analysis indicated that curcumin directly increased intracellular oxidative stress based on the cell permeable dye, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) acting as an indicator of reactive oxygen species (ROS) generation. GADD153 and GRP78 were increased by curcumin which was indicative of ER stress. Curcumin increased Ca(2+) levels and the mitochondrial membrane potential (DeltaPsi(m)), was decreased in A-549 cells. Overall, our results demonstrated that curcumin treatment causes cell death by activating pathways inducing G2/M-phase arrest and apoptosis.

  10. 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.

  11. Pleuropterus multiflorus (Hasuo) mediated straightforward eco-friendly synthesis of silver, gold nanoparticles and evaluation of their anti-cancer activity on A549 lung cancer cell line.

    PubMed

    Castro-Aceituno, Verónica; Abbai, Ragavendran; Moon, Seong Soo; Ahn, Sungeun; Mathiyalagan, Ramya; Kim, Yu-Jin; Kim, Yeon-Ju; Yang, Deok Chun

    2017-09-01

    Pleuropterus multiflorus (Hasuo) is a widely used medicinal plant in Korea and China for treating amnesia, isnomia, heart throbbing etc. With the constructive idea of promoting the wide-spread usage of P. multiflorus, we propose its indirect usage in the form of biologically active silver (Pm-AgNPs) and gold nanoparticles (Pm-AuNPs). The synthesized nanoparticles were predominantly spherical, crystalline with the Z-average hydrodynamic diameter of 274.8nm and 104.8nm respectively. Also, proteins and phenols were identified as the major players involved in their synthesis and stability. Further, Pm-AgNPs at 25μg/mL were significantly cytotoxic to lung cancer cells, whereas, Pm-AuNPs were not cytotoxic to both normal keratinocyte and lung cancer cells even at 100μg/mL. In addition, further evaluation of the anti-cancer activity of these new nanoparticles, such as migration and apoptosis, shown that Pm-AgNPs have a potential therapeutic effect on A549 lung cancer cell treatment. To the best of our knowledge, this is the first report dissecting out the ability of the endemic P. multiflorus for the synthesis of bioactive silver and gold nanoparticle which would open up doors for its extensive usage in medicinal field. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  12. 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-sensitive protein synthesis and cell proliferation signaling. Our results show that JP-1 activates p53/miR-34a tumor suppressor axis and decreases proteins related to proliferation, apoptosis resistance, and metastasis, suggesting its potential as a complementary medicine for LADC treatment. PMID:28074102

  13. Picroside II Protects Rat Lung and A549 Cell Against LPS-Induced Inflammation by the NF-κB Pathway.

    PubMed

    Shen, Bingyu; Zhao, Chenxu; Chen, Chengzhen; Li, Zheng; Li, Yang; Tian, Ye; Feng, Haihua

    2017-06-01

    Picroside II is the main active ingredient in the root department of Chinese medicine Picrorhiza scrophulariiflora which has been proved to have beneficial effects on health, such as ameliorating the cerebral ischemia and protecting the liver. However, its effects on acute lung injury remain unclear. The purpose of the study was to evaluate the effects of picroside II on acute lung injury in mice and the inflammation in A549 cells which are lipopolysaccharide (LPS) induced. We evaluated the levels of tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1β), and interleukin-6 (IL-6) in vivo and vitro by enzyme-linked immunosorbent assay (ELISA) and quantitative polymerase chain reaction (qRT-PCR). Results showed picroside II significantly decreased the concentrations of TNF-α, IL-1β, and IL-6 in cells and mice. In addition, Western blot and immunofluorescence analysis indicated that picroside II suppressed the activation of p65 NF-κB signaling pathway compared with LPS stimulation. In the acute lung injury model of mice, after picroside II treatment, the pathologic changes of lung tissues had been alleviated and lung wet/dry weight ratio was decreased. In summary, picroside II showed the promising effects of anti-inflammation in cells and animals.

  14. Molecular analysis of hepatitis E virus from farm rabbits in Inner Mongolia, China and its successful propagation in A549 and PLC/PRF/5 cells.

    PubMed

    Jirintai, Suljid; Jinshan; Tanggis; Manglai, Dugarjavin; Mulyanto; Takahashi, Masaharu; Nagashima, Shigeo; Kobayashi, Tominari; Nishizawa, Tsutomu; Okamoto, Hiroaki

    2012-12-01

    Rabbit hepatitis E virus (HEV) strains have recently been isolated in several areas of China and in the US and France. However, the host range, distribution and zoonotic potential of these HEV strains remain unknown and their propagation in cultured cells has not yet been reported. A total of 211 4-month-old rabbits raised on a farm in Inner Mongolia were tested for the presence of anti-HEV antibodies and HEV RNA. Overall, 121 rabbits (57.3%) tested positive for anti-HEV antibodies, and 151 (71.6%) had detectable HEV RNA. The 174 HEV strains recovered from these viremic rabbits, including two distinct strains each from 23 rabbits, differed from each other by up to 13.6% in a 412-nucleotide (nt) sequence within ORF2, and were 89.3-95.9% identical to the reported rabbit HEV strains in other provinces of China. Three representative Inner Mongolian strains, one each from three phylogenetic clusters, whose entire genomic sequences were determined, shared 79.6-96.7% identities with reported rabbit HEV strains within the entire or 242- to 1349-nt partial genomic sequence. Rabbit HEV strains recovered from liver tissues of rabbits with a high HEV load propagated efficiently in human cell lines (A549 and PLC/PRF/5 cells), suggesting the potential zoonotic risk of rabbit HEV. Copyright © 2012 Elsevier B.V. All rights reserved.

  15. Integrating cell biology and proteomic approaches in plants.

    PubMed

    Takáč, Tomáš; Šamajová, Olga; Šamaj, Jozef

    2017-10-03

    Significant improvements of protein extraction, separation, mass spectrometry and bioinformatics nurtured advancements of proteomics during the past years. The usefulness of proteomics in the investigation of biological problems can be enhanced by integration with other experimental methods from cell biology, genetics, biochemistry, pharmacology, molecular biology and other omics approaches including transcriptomics and metabolomics. This review aims to summarize current trends integrating cell biology and proteomics in plant science. Cell biology approaches are most frequently used in proteomic studies investigating subcellular and developmental proteomes, however, they were also employed in proteomic studies exploring abiotic and biotic stress responses, vesicular transport, cytoskeleton and protein posttranslational modifications. They are used either for detailed cellular or ultrastructural characterization of the object subjected to proteomic study, validation of proteomic results or to expand proteomic data. In this respect, a broad spectrum of methods is employed to support proteomic studies including ultrastructural electron microscopy studies, histochemical staining, immunochemical localization, in vivo imaging of fluorescently tagged proteins and visualization of protein-protein interactions. Thus, cell biological observations on fixed or living cell compartments, cells, tissues and organs are feasible, and in some cases fundamental for the validation and complementation of proteomic data. Validation of proteomic data by independent experimental methods requires development of new complementary approaches. Benefits of cell biology methods and techniques are not sufficiently highlighted in current proteomic studies. This encouraged us to review most popular cell biology methods used in proteomic studies and to evaluate their relevance and potential for proteomic data validation and enrichment of purely proteomic analyses. We also provide examples of

  16. Effect of gemcitabine on the uptake of (18)F-fluorodeoxyglucose and (18)F-fluorothymidine in lung adenocarcinoma A549 cells and the animal tumor model.

    PubMed

    Zhang, Bin; Deng, Sheng-Ming; Guo, Ling-Chuan; Dong, Jia-Jia; Zhu, Yan-Bo; Gao, Yuan; Wang, Zhen-Xin; Cho, William C

    2016-01-01

    Gemcitabine is the first-line drug for nonsmall cell lung cancer, and 18F-fluorodeoxyglucose. (18F-FDG) and 18F-fluorothymidine. (18F-FLT) are positron emission tomography. (PET) imaging agents. The aim of this study was to explore the effect of gemcitabine on the uptake of 18F-FDG and 18F-FLT in A549 cells and the animal tumor model. The inhibitory effects of gemcitabine on cell growth were determined by tetrazolium blue method, and uptake rates of 18F-FDG and 18F-FLT were determined under the same conditions. The adenocarcinoma-bearing nude mice before and after gemcitabine treatments were performed microPET imaging with 18F-FDG and 18F-FLT. Hematoxylin and eosin staining and immunohistochemical analysis of tumor specimens were conducted. After the administration of gemcitabine, positive correlations were observed between inhibition of 18F-FDG or 18F.FLT uptake and cell growth. (r = 0.957 or 0.981, P < 0.01). SUVmax values by 18F-FDG in the tumor, before and after administration of gemcitabine at the dose of 60 mmol/L, revealed an increase by. (35.83 ± 10.58) %. After administration of 120 mmol/L gemcitabine, the SUVmax values decreased by (12.37 ± 7.33) %. The SUVmax values by 18F-FLT at the dose of 60 mmol/L gemcitabine revealed a decrease by (56.47 ± 10.83) %. Pathological staining showed obvious vasodilation and invasion of lymphocytes and plasma cells at the dose of 60 mmol/L, and the expression of glucose transporter protein-1, Ki-67 and proliferating cell nuclear antigen in tumor cells were inhibited. 18F-FLT imaging can assess the proliferation of tumor cells and 18F-FDG imaging can reflect the changes of the tumor microenvironment after administration of gemcitabine.

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

    PubMed

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

    2014-08-01

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

  18. 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; 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 bymore » 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.« less

  19. 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.

  20. Microcystin-LR induces a wide variety of biochemical changes in the A549 human non-small cell lung cancer cell line: Roles for protein phosphatase 2A and its substrates.

    PubMed

    Wang, Hanying; Xu, Kailun; Wang, Beilei; Liu, Jinghui; Wang, Xiaofeng; Xing, Mingluan; Huang, Pu; Guo, Zonglou; Xu, Lihong

    2017-03-01

    Our previous studies have described the toxic effects of microcystin-LR (MC-LR) in various normal cell lines and human hepatoma SMMC-7721 cells, but the specific effects of MC-LR in other types of cancer cells with respect to protein phosphatase 2A (PP2A) have not been fully elaborated. A549 human lung adenocarcinoma cells have been identified to express organic anion-transporting polypeptides (OATP) involved in cellular uptake of MC-LR, and thus probably make an appropriate in vitro model to assess MC-LR's cytotoxicity. Hence, in our present study, A549 cells were treated with various concentrations of MC-LR for 24 h. The presence of MC-LR in A549 cells was confirmed, and PP2A activity, PP2A substrates, cytoskeleton, apoptosis, and proliferation were subsequently explored. The results showed that 5-10 μM MC-LR inhibited PP2A activity significantly but 0.5-1 μM MC-LR did not change PP2A activity dramatically. The inhibition could result from the hyperphosphorylation of PP2A/C at Tyr307, an elevation in the total PP2A/C expression and the dissociation of α4/PP2A/C complexes. Moreover, MC-LR led to rearrangements of filamentous actin and microtubules, which might be correlated with the hyperphosphorylation of Ezrin, VASP and HSP27 due to PP2A inhibition and mitogen-activated protein kinase (MAPK) activation. However, exposure to MC-LR for 24 h failed to trigger either apoptosis or proliferation, which might be related to PP2A-inhibition-induced hyperphosphorylation of Bcl-2 and Bad and the activation status of Akt. In conclusion, our data indicated that MC-LR induced extensive molecular and cellular alterations in A549 cells through a PP2A-centered pathway, which differed in some respects from our previous study in SMMC-7721 cells. To our knowledge, this is the first report comprehensively demonstrating the effects of MC-LR in A549 cells, and our findings provide insights into the mechanism of MC-LR toxicity in cancer cells. © 2016 Wiley Periodicals, Inc. Environ

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

    PubMed

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

    2017-09-01

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

  2. Terbutaline causes immobilization of single β2-adrenergic receptor-ligand complexes in the plasma membrane of living A549 cells as revealed by single-molecule microscopy

    NASA Astrophysics Data System (ADS)

    Sieben, Anne; Kaminski, Tim; Kubitscheck, Ulrich; Häberlein, Hanns

    2011-02-01

    G-protein-coupled receptors are important targets for various drugs. After signal transduction, regulatory processes, such as receptor desensitization and internalization, change the lateral receptor mobility. In order to study the lateral diffusion of β2-adrenergic receptors (β2AR) complexed with fluorescently labeled noradrenaline (Alexa-NA) in plasma membranes of A549 cells, trajectories of single receptor-ligand complexes were monitored using single-particle tracking. We found that a fraction of 18% of all β2ARs are constitutively immobile. About 2/3 of the β2ARs moved with a diffusion constant of D2 = 0.03+/-0.001 μm2/s and about 17% were diffusing five-fold faster (D3 = 0.15+/-0.02 μm2/s). The mobile receptors moved within restricted domains and also showed a discontinuous diffusion behavior. Analysis of the trajectory lengths revealed two different binding durations with τ1 = 77+/-1 ms and τ2 = 388+/-11 ms. Agonistic stimulation of the β2AR-Alexa-NA complexes with 1 μM terbutaline caused immobilization of almost 50% of the receptors within 35 min. Simultaneously, the mean area covered by the mobile receptors decreased significantly. Thus, we demonstrated that agonistic stimulation followed by cell regulatory processes results in a change in β2AR mobility suggesting that different receptor dynamics characterize different receptor states.

  3. Neural Stem Cells (NSCs) and Proteomics.

    PubMed

    Shoemaker, Lorelei D; Kornblum, Harley I

    2016-02-01

    Neural stem cells (NSCs) can self-renew and give rise to the major cell types of the CNS. Studies of NSCs include the investigation of primary, CNS-derived cells as well as animal and human embryonic stem cell (ESC)-derived and induced pluripotent stem cell (iPSC)-derived sources. NSCs provide a means with which to study normal neural development, neurodegeneration, and neurological disease and are clinically relevant sources for cellular repair to the damaged and diseased CNS. Proteomics studies of NSCs have the potential to delineate molecules and pathways critical for NSC biology and the means by which NSCs can participate in neural repair. In this review, we provide a background to NSC biology, including the means to obtain them and the caveats to these processes. We then focus on advances in the proteomic interrogation of NSCs. This includes the analysis of posttranslational modifications (PTMs); approaches to analyzing different proteomic compartments, such the secretome; as well as approaches to analyzing temporal differences in the proteome to elucidate mechanisms of differentiation. We also discuss some of the methods that will undoubtedly be useful in the investigation of NSCs but which have not yet been applied to the field. While many proteomics studies of NSCs have largely catalogued the proteome or posttranslational modifications of specific cellular states, without delving into specific functions, some have led to understandings of functional processes or identified markers that could not have been identified via other means. Many challenges remain in the field, including the precise identification and standardization of NSCs used for proteomic analyses, as well as how to translate fundamental proteomics studies to functional biology. The next level of investigation will require interdisciplinary approaches, combining the skills of those interested in the biochemistry of proteomics with those interested in modulating NSC function. © 2016 by The

  4. Neural Stem Cells (NSCs) and Proteomics*

    PubMed Central

    Shoemaker, Lorelei D.; Kornblum, Harley I.

    2016-01-01

    Neural stem cells (NSCs) can self-renew and give rise to the major cell types of the CNS. Studies of NSCs include the investigation of primary, CNS-derived cells as well as animal and human embryonic stem cell (ESC)-derived and induced pluripotent stem cell (iPSC)-derived sources. NSCs provide a means with which to study normal neural development, neurodegeneration, and neurological disease and are clinically relevant sources for cellular repair to the damaged and diseased CNS. Proteomics studies of NSCs have the potential to delineate molecules and pathways critical for NSC biology and the means by which NSCs can participate in neural repair. In this review, we provide a background to NSC biology, including the means to obtain them and the caveats to these processes. We then focus on advances in the proteomic interrogation of NSCs. This includes the analysis of posttranslational modifications (PTMs); approaches to analyzing different proteomic compartments, such the secretome; as well as approaches to analyzing temporal differences in the proteome to elucidate mechanisms of differentiation. We also discuss some of the methods that will undoubtedly be useful in the investigation of NSCs but which have not yet been applied to the field. While many proteomics studies of NSCs have largely catalogued the proteome or posttranslational modifications of specific cellular states, without delving into specific functions, some have led to understandings of functional processes or identified markers that could not have been identified via other means. Many challenges remain in the field, including the precise identification and standardization of NSCs used for proteomic analyses, as well as how to translate fundamental proteomics studies to functional biology. The next level of investigation will require interdisciplinary approaches, combining the skills of those interested in the biochemistry of proteomics with those interested in modulating NSC function. PMID:26494823

  5. Induction and repair of DNA cross-links induced by sulfur mustard in the A-549 cell line followed by a comet assay.

    PubMed

    Jost, Petr; Svobodova, Hana; Stetina, Rudolf

    2015-07-25

    Sulfur mustard is a highly toxic chemical warfare agent with devastating impact on intoxicated tissues. DNA cross-links are probably the most toxic DNA lesions induced in the cell by sulfur mustard. The comet assay is a very sensitive method for measuring DNA damage. In the present study using the A-549 lung cell line, the comet assay protocol was optimized for indirect detection of DNA cross-links induced by sulfur mustard. The method is based on the additional treatment of the assayed cells containing cross-links with the chemical mutagen, styrene oxide. Alkali-labile adducts of styrene oxide cause DNA breaks leading to the formation of comets. A significant dose-dependent reduction of DNA migration of the comet's tail was found after exposing cells to sulfur mustard, indicative of the amount of sulfur mustard induced cross-links. The remarkable decrease of % tail DNA could be observed as early as 5min following exposure to sulfur mustard and the maximal effect was found after 30min, when DNA migration was reduced to the minimum. Sulfur mustard preincubated in culture medium without cells lost its ability to induce cross-links and had a half-life of about 15min. Pre-incubation longer than 30min does not lead to a significant increase in cross-links when applied to cells. However, the amount of cross-links is decreased during further incubation due to repair. The current modification of the comet assay provides a useful tool for detecting DNA cross-links induced by sulfur mustard and could be used for detection of other DNA cross-linking agents such as chemotherapeutic drugs. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  6. Particulate emissions from the combustion of birch, beech, and spruce logs cause different cytotoxic responses in A549 cells.

    PubMed

    Kasurinen, Stefanie; Jalava, Pasi I; Happo, Mikko S; Sippula, Olli; Uski, Oskari; Koponen, Hanna; Orasche, Jürgen; Zimmermann, Ralf; Jokiniemi, Jorma; Hirvonen, Maija-Riitta

    2017-05-01

    According to the World Health Organization particulate emissions from the combustion of solid fuels caused more than 110,000 premature deaths worldwide in 2010. Log wood combustion is the most prevalent form of residential biomass heating in developed countries, but it is unknown how the type of wood logs used in furnaces influences the chemical composition of the particulate emissions and their toxicological potential. We burned logs of birch, beech and spruce, which are used commonly as firewood in Central and Northern Europe in a modern masonry heater, and compared them to the particulate emissions from an automated pellet boiler fired with softwood pellets. We determined the chemical composition (elements, ions, and carbonaceous compounds) of the particulate emissions with a diameter of less than 1 µm and tested their cytotoxicity, genotoxicity, inflammatory potential, and ability to induce oxidative stress in a human lung epithelial cell line. The chemical composition of the samples differed significantly, especially with regard to the carbonaceous and metal contents. Also the toxic effects in our tested endpoints varied considerably between each of the three log wood combustion samples, as well as between the log wood combustion samples and the pellet combustion sample. The difference in the toxicological potential of the samples in the various endpoints indicates the involvement of different pathways of toxicity depending on the chemical composition. All three emission samples from the log wood combustions were considerably more toxic in all endpoints than the emissions from the pellet combustion. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1487-1499, 2017. © 2016 Wiley Periodicals, Inc.

  7. 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.

  8. Impact of after-treatment devices and biofuels on diesel exhausts genotoxicity in A549 cells exposed at air-liquid interface.

    PubMed

    Barraud, C; Corbière, C; Pottier, I; Estace, E; Blanchard, K; Logie, C; Lagadu, S; Kéravec, V; Pottier, D; Dionnet, F; Morin, J P; Préterre, D; André, V; Monteil, C; Sichel, F

    2017-12-01

    Using an air-liquid interface (ALI) device in dynamic conditions, we evaluated the efficiency of fuel after-treatment strategies (diesel oxidation catalysis, DOC, and diesel particulate filter, DPF, devices) and the impact of 7% and 30% rapeseed methyl esters (RME) blending on oxidative stress and genotoxicity induced in A549 lung cells after 3h exposure to whole Diesel exhausts. Oxidative stress was studied using assays of ROS production, glutathione level, catalase and superoxide-dismutase (SOD) activities. No oxidative stress and no clear differences on cytotoxicity patterns between biodiesel and standard Diesel exhausts were found. A weak but significant genotoxicity (8-oxodGuo adducts) and, for standard Diesel only, a DNA damage response (DDR) as evidenced by ƔH2AX foci, remained after DOC+DPF flowing. All together, these data could contribute to the improvement of the after treatment strategies and to health risk assessment of current diesel exhausts. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. A Cell-type-resolved Liver Proteome*

    PubMed Central

    Ding, Chen; Li, Yanyan; Guo, Feifei; Jiang, Ying; Ying, Wantao; Li, Dong; Yang, Dong; Xia, Xia; Liu, Wanlin; Zhao, Yan; He, Yangzhige; Li, Xianyu; Sun, Wei; Liu, Qiongming; Song, Lei; Zhen, Bei; Zhang, Pumin; Qian, Xiaohong; Qin, Jun; He, Fuchu

    2016-01-01

    Parenchymatous organs consist of multiple cell types, primarily defined as parenchymal cells (PCs) and nonparenchymal cells (NPCs). The cellular characteristics of these organs are not well understood. Proteomic studies facilitate the resolution of the molecular details of different cell types in organs. These studies have significantly extended our knowledge about organogenesis and organ cellular composition. Here, we present an atlas of the cell-type-resolved liver proteome. In-depth proteomics identified 6000 to 8000 gene products (GPs) for each cell type and a total of 10,075 GPs for four cell types. This data set revealed features of the cellular composition of the liver: (1) hepatocytes (PCs) express the least GPs, have a unique but highly homogenous proteome pattern, and execute fundamental liver functions; (2) the division of labor among PCs and NPCs follows a model in which PCs make the main components of pathways, but NPCs trigger the pathways; and (3) crosstalk among NPCs and PCs maintains the PC phenotype. This study presents the liver proteome at cell resolution, serving as a research model for dissecting the cell type constitution and organ features at the molecular level. PMID:27562671

  10. 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

  11. Heteroleptic monometallic and trimetallic ruthenium(II) complexes incorporating a π-extended dipyrrin ligand: Light-activated reactions with the A549 lung cancer cell line.

    PubMed

    Swavey, Shawn; Morford, Krista; Tsao, Max; Comfort, Kristen; Kilroy, Mary Kate

    2017-10-01

    A heteroleptic monometallic ruthenium(II) and a heteroleptic trimetallic ruthenium(II) complex have been synthesized and characterized. Both complexes have an overall 3+ charge, with the charge density greater for the monometallic complex. The electronic spectra of the monometallic ruthenium(II) complex exhibits intense π-π* transitions associated with the bipyridyl groups along with overlapping metal to ligand charge transfer (MLCT) and ligand centered π-π* transitions ranging from 520nm to approximately 600nm. The trimetallic ruthenium(II) complex, on the other hand, displays more well defined transitions with the expected π-π* transition of the bipyridyl groups at 294nm and Ru(dπ) to bpy(π*) MLCT transitions at 355nm and 502nm. In addition to these absorption bands an intense transition, 578nm, resulting from overlapping dipyrrin (π-π*) and Ru(dπ) to dipyrrin(π*) transitions is observed. Electrochemical and spectroelectrochemical experiments were used to help in assigning these transitions. Irradiation of the complexes in the presence of plasmid DNA within the photodynamic therapy window (600nm to 850nm) reveal, using electrophoresis, that both complexes are capable of causing photo-damage to the DNA backbone. The trimetallic ruthenium(II) complex; however, also shows the ability to generate photoinduced DNA damage in the absence of oxygen, suggesting a photo-oxidative process. Studies of the complexes toward lung cancer cells (A549 cell line) in the absence of light indicate little cytotoxicity up to 50μM. Upon irradiation of the cells with a low power 420nm light source the trimetallic complex showed considerably greater photo-cytotoxicity compared to the monometallic analog. A dose-dependent response curve gives an IC50 of 92μM for complex B. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Molecular Mode of Action and Role of TP53 in the Sensitivity to the Novel Epothilone Sagopilone (ZK-EPO) in A549 Non-Small Cell Lung Cancer Cells

    PubMed Central

    Winsel, Sebastian; Sommer, Anette; Eschenbrenner, Julia; Mittelstaedt, Kevin; Klar, Ulrich; Hammer, Stefanie; Hoffmann, Jens

    2011-01-01

    Sagopilone, an optimized fully synthetic epothilone, is a microtubule-stabilizing compound that has shown high in vitro and in vivo activity against a broad range of human tumor models. We analyzed the differential mechanism of action of sagopilone in non-small cell lung cancer cell lines in vitro. Sagopilone inhibited proliferation of non-small cell lung cancer cell lines at lower nanomolar concentration. The treatment with sagopilone caused strong disturbances of cellular cytoskeletal organization. Two concentration-dependent phenotypes were observed. At 2.5 nM sagopilone or 4 nM paclitaxel an aneuploid phenotype occur whereas a mitotic arrest phenotype was induced by 40 nM sagopilone or paclitaxel. Interestingly, treatment with 2.5 nM of sagopilone effectively inhibited cell proliferation, but - compared to high concentrations (40 nM) - only marginally induced apoptosis. Treatment with a high versus a low concentration of sagopilone or paclitaxel regulates a non-overlapping set of genes, indicating that both phenotypes substantially differ from each other. Genes involved in G2/M phase transition and the spindle assembly checkpoint, like Cyclin B1 and BUBR1 were upregulated by treatment with 40 nM sagopilone. Unexpectedly, also genes involved in DNA damage response were upregulated under that treatment. In contrast, treatment of A549 cells with a low concentration of sagopilone revealed an upregulation of direct transcriptional target genes of TP53, like CDKN1A, MDM2, GADD45A, FAS. Knockdown of TP53, which inhibited the transcriptional induction of TP53 target genes, led to a significant increase in apoptosis induction in A549 cells when treated with a low concentration of sagopilone. The results indicate that activation of TP53 and its downstream effectors like CDKN1A by low concentrations of sagopilone is responsible for the relative apoptosis resistance of A549 cells and might represent a mechanism of resistance to sagopilone. PMID:21559393

  13. 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. Copyright © 2010 Wiley-Liss, Inc.

  14. MMP-2 siRNA Induced Fas/CD95 Mediated Extrinsic II Apoptotic Pathway in the A549 Lung Adenocarcinoma Cell Line

    PubMed Central

    Chetty, Chandramu; Bhoopathi, Praveen; Lakka, Sajani S.; Rao, Jasti S.

    2007-01-01

    We have previously reported that the downregulation of MMP-2 by adenovirus-mediated delivery of MMP-2 siRNA (Ad-MMP-2) reduced spheroid invasion and angiogenesis in vitro, and, metastasis and tumor growth in vivo. In this study, we investigated the mechanism of Ad-MMP-2-mediated growth inhibition in vitro and in vivo. Ad-MMP-2 infection led to the induction of apoptosis as determined by TUNEL assay, Annexin-V staining and PARP-1 cleavage in a dose-dependent manner in A549 cells. Ad-MMP-2 decreased the content of the anti-apoptotic members of the Bcl-2 family proteins (Bcl-2 and Bcl-xL) and increased the content of the pro-apoptotic members of the Bcl-2 family (Bax and Bcl-xS) as determined by immunoblotting analysis. Furthermore, Ad-MMP-2-mediated apoptosis was accompanied by increase in truncated Bid, release of cytochrome-c, and the activation of caspases-8, -9 and -3. Immunoblot analysis showed that Ad-MMP-2 infection caused upregulation of Fas/Fas-L and FADD. And Anti-Fas-L antibody reversed Ad-MMP-2-induced apoptosis. TIMP-3, an endogenous inhibitor of MMP-2, which cleaves Fas-L and activates the Fas/Fas-L inducing apoptotic pathway, was increased in Ad-MMP-2-treated cells. Adenovirus-mediated expression of MMP-2 siRNA in human lung xenografts in vivo resulted in increased immunostaining of Fas, Fas-L, cleaved Bid and TIMP-3. This is the first report, to our knowledge, showing that MMP-2 inhibition upregulates TIMP-3 levels, which in turn, promotes apoptosis in lung cancer. PMID:17599056

  15. MMP-2 siRNA induced Fas/CD95-mediated extrinsic II apoptotic pathway in the A549 lung adenocarcinoma cell line.

    PubMed

    Chetty, C; Bhoopathi, P; Lakka, S S; Rao, J S

    2007-12-06

    We have previously reported that the downregulation of MMP-2 by adenovirus-mediated delivery of MMP-2 siRNA (Ad-MMP-2) reduced spheroid invasion and angiogenesis in vitro, and, metastasis and tumor growth in vivo. In this study, we investigated the mechanism of Ad-MMP-2-mediated growth inhibition in vitro and in vivo. Ad-MMP-2 infection led to the induction of apoptosis as determined by TUNEL assay, Annexin-V staining and PARP-1 cleavage in a dose-dependent manner in A549 cells. Ad-MMP-2 decreased the content of the antiapoptotic members of the Bcl-2 family proteins (Bcl-2 and Bcl-xL) and increased the content of the pro-apoptotic members of the Bcl-2 family (Bax and Bcl-xS) as determined by immunoblotting analysis. Furthermore, Ad-MMP-2-mediated apoptosis was accompanied by increase in truncated Bid, release of cytochrome c and the activation of caspase-8, -9 and -3. Immunoblot analysis showed that Ad-MMP-2 infection caused upregulation of Fas/Fas-L and FADD, and Anti-Fas-L antibody reversed Ad-MMP-2-induced apoptosis. Tissue inhibitor of metalloproteinases (TIMP)-3, an endogenous inhibitor of MMP-2, which cleaves Fas-L and activates the Fas/Fas-L inducing apoptotic pathway, was increased in Ad-MMP-2-treated cells. Adenovirus-mediated expression of MMP-2 siRNA in human lung xenografts in vivo resulted in increased immunostaining of Fas, Fas-L, cleaved Bid and TIMP-3. This is the first report, to our knowledge, showing that MMP-2 inhibition upregulates TIMP-3 levels, which in turn, promotes apoptosis in lung cancer.

  16. Effects of EP4 solution and LPD solution vs Euro-Collins solution on Na(+)/K(+)-ATPase activity in rat alveolar type II cells and human alveolar epithelial cell line A549 cells.

    PubMed

    Suzuki, S; Inoue, K; Sugita, M; Tsubochi, H; Kondo, T; Fujimura, S

    2000-09-01

    Intact alveolar epithelial Na(+)/K(+)- adenosinetriphosphatase (ATPase) function is important in preventing alveolar fluid accumulation after lung transplantation. We examined whether the type of preservation solution used influences Na(+)/K(+)-ATPase activity in alveolar epithelial cells. Rat alveolar type II cells were preserved with EP4, low-potassium dextran (LPD), or Euro-Collins solution at 7 degrees C for 5 and 20 hours. To assess cell toxicity, we measured cell viability and lactate dehydrogenase release. Na(+)/K(+)-ATPase activity was measured as ouabain-sensitive ATPase hydrolysis. We also examined the effect of terbutaline (10(-3) mol/liter) and dibutyryl cyclic adenosine monophosphate (dbcAMP) (10(-3) mol/liter) on Na(+)/K(+)-ATPase activity in A549 cells preserved for 5 hours. All solutions caused significant damage of rat alveolar type II cells at 20 hours. However, Na(+)/K(+)-ATPase activity was preserved at normal levels with EP4 and LPD over 20 hours. Terbutaline and dbcAMP significantly increased Na(+)/K(+)-ATPase activity in A549 cells preserved with EP4 and LPD solutions for 5 hours. However, we observed no activation in the cells preserved with Euro-Collins solution. We found no significant difference in intracellular cAMP levels after terbutaline challenge among the types of preservation solution. We conclude that extracellular-type solutions such as EP4 and LPD may be preferable for maintaining not only the basal activity but also the ability to activate Na(+)/K(+)-ATPase in response to beta-adrenergic agonists, in alveolar epithelial cells.

  17. Biochemical synthesis of silver nanoprticles using filamentous fungi Penicillium decumbens (MTCC-2494) and its efficacy against A-549 lung cancer cell line.

    PubMed

    Majeed, Shahnaz; Abdullah, Mohd Syafiq Bin; Dash, Gouri Kumar; Ansari, Mohammed Tahir; Nanda, Anima

    2016-08-01

    Biosynthesis of silver and other metallic nanoparticles is one of the emerging research area in the field of science and technology due to their potentiality, especially in the field of nano-biotechnology and biomedical sciences in order to develop nanomedicine. In our present study, Penicillium decumbens (MTCC-2494) was brought from Institute of Microbial Technology (IMTECH) Chandigarh and employed for extracellular biological synthesis of silver nanoparticles. Ag-NPs formation was appeared with a dark brown color inside the conical flask. Characterization of Ag-NPs were done by UV-Spectrophotometric analysis which showed absorption peak at 430 nm determines the presence of nanoparticles, Fourier transform infrared (FT-IR) spectroscopic analysis, showed amines and amides are the possible proteins involved in the stabilization of nanoparticles as capping agent. Atomic force Microscopy (AFM) confirmed the particle are spherical, size was around 30 to 60 nm and also the roughness of nanoparticles. Field emission scanning electron microscopy (FE-SEM) showed the topology of the nanoparticles and were spherical in shape. The biosynthesis process was found fast, ecofriendly and cost effective. Nano-silver particle was found to have a broad antimicrobial activity and also it showed good enhancement of antimicrobial activity of Carbenicillin, Piperacillin, Cefixime, Amoxicillin, Ofloxacin and Sparfloxacin in a synergistic mode. These Ag-NPs showed good anti-cancer activity at 80 μg·mL(-1)upon 24 hours of incubation and toxicity increases upon 48 hours of incubation against A-549 human lung cancer cell line and the synergistic formulation of the antibiotic with the synthesized nanoparticles was found more effective against the pathogenic bacteria studied. Copyright © 2016 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

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

    PubMed

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

    2012-03-01

    Exposure to emissions from laser printers during the printing process is commonplace worldwide, both in the home and workplace environment. In the present study, cytotoxic and genotoxic effects of the emission from five low to medium-throughput laser printers were investigated with respect to the release of ozone (O(3) ), volatile organic compounds (VOC), particulate matter (PM), and submicrometer particles (SMP) during standby and operation. Experiments were conducted in a 1 m(3) emission chamber connected to a Vitrocell® exposure system. Cytotoxicity was determined by the WST-1 assay and genotoxicity by the micronucleus test in human A549 lung cells. The five laser printers emitted varying but generally small amounts of O(3) , VOC, and PM. VOC emissions included 13 compounds with total VOC concentrations ranging from 95 to 280 μg/m(3) (e.g., 2-butanone, hexanal, m,p-xylene, and o-xylene). Mean PM concentrations were below 2.4 μg/m(3). SMP number concentration levels during standby ranged from 9 to 26 particles/cm(3). However, three of the printers generated a 90 to 16 × 10(3) -fold increase of SMP during the printing process (maximum 294,460 particles/cm(3)). Whereas none of the printer emissions were found to cause cytotoxicity, emissions from two printers induced formation of micronuclei (P < 0.001), thus providing evidence for genotoxicity. As yet, differences in biological activity cannot be explained on the basis of the specific emission characteristics of the different printers. Because laser printing technology is widely used, studies with additional cytogenetic endpoints are necessary to confirm the DNA-damaging potency and to identify emission components responsible for genotoxicity. Copyright © 2011 Wiley-Liss, Inc.

  19. K9(C4H4FN2O2)2Nd(PW11O39)2·25H2O induces apoptosis in human lung cancer A549 cells

    PubMed Central

    Xia, Rong-Yao; Zhang, Ran-Ran; Jiang, Zhe; Sun, Ya-Jiao; Liu, Jing; Chen, Fu-Hui

    2017-01-01

    Lung cancer is the leading cause of cancer-associated mortality worldwide. The present study investigated the effects of K9(C4H4FN2O2)2Nd(PW11O39)2·25H2O (FNdPW), a chemically synthesized polyoxometalate that contains rare earth elements, on lung cancer growth, and explored the mechanism underlying its actions. The effects of FNdPW on the cell viability and apoptosis of human lung cancer A549 cells were measured using MTT assay, acridine orange/ethidium bromide staining and electron microscopy. The expression of apoptosis-related proteins, including B-cell lymphoma (Bcl)-2-associated death promoter (Bad), phosphorylated (p)-Bad, X-linked inhibitor of apoptosis (XIAP), apoptosis-inducing factor (AIF), Bcl-2-associated X protein (Bax) and Bcl-2, was determined by western blotting. Caspase-3 activity was measured using a caspase-3 activity kit. After 72 h of incubation, FNdPW reduced cell viability and induced apoptosis in A549 cells in a concentration- and time-dependent manner. FNdPW upregulated the pro-apoptotic Bad and Bax proteins, and downregulated the anti-apoptotic p-Bad, Bcl-2 and XIAP proteins. Furthermore, FNdPW also enhanced caspase-3 activity and increased the protein level of AIF in A549 cells, which was independent of the caspase-3 pathway. These events were associated with the regulation exerted by FNdPW on multiple targets involved in A549 cell proliferation. Therefore, FNdPW may be a novel drug for the treatment of lung cancer. PMID:28454260

  20. K9(C4H4FN2O2)2Nd(PW11O39)2·25H2O induces apoptosis in human lung cancer A549 cells.

    PubMed

    Xia, Rong-Yao; Zhang, Ran-Ran; Jiang, Zhe; Sun, Ya-Jiao; Liu, Jing; Chen, Fu-Hui

    2017-03-01

    Lung cancer is the leading cause of cancer-associated mortality worldwide. The present study investigated the effects of K 9 (C 4 H 4 FN 2 O 2 ) 2 Nd(PW 11 O 39 ) 2 ·25H 2 O (FNdPW), a chemically synthesized polyoxometalate that contains rare earth elements, on lung cancer growth, and explored the mechanism underlying its actions. The effects of FNdPW on the cell viability and apoptosis of human lung cancer A549 cells were measured using MTT assay, acridine orange/ethidium bromide staining and electron microscopy. The expression of apoptosis-related proteins, including B-cell lymphoma (Bcl)-2-associated death promoter (Bad), phosphorylated (p)-Bad, X-linked inhibitor of apoptosis (XIAP), apoptosis-inducing factor (AIF), Bcl-2-associated X protein (Bax) and Bcl-2, was determined by western blotting. Caspase-3 activity was measured using a caspase-3 activity kit. After 72 h of incubation, FNdPW reduced cell viability and induced apoptosis in A549 cells in a concentration- and time-dependent manner. FNdPW upregulated the pro-apoptotic Bad and Bax proteins, and downregulated the anti-apoptotic p-Bad, Bcl-2 and XIAP proteins. Furthermore, FNdPW also enhanced caspase-3 activity and increased the protein level of AIF in A549 cells, which was independent of the caspase-3 pathway. These events were associated with the regulation exerted by FNdPW on multiple targets involved in A549 cell proliferation. Therefore, FNdPW may be a novel drug for the treatment of lung cancer.

  1. Application of a lipid-coated hollow calcium phosphate nanoparticle in synergistic co-delivery of doxorubicin and paclitaxel for the treatment of human lung cancer A549 cells.

    PubMed

    Wu, Chao; Xu, Jie; Hao, Yanna; Zhao, Ying; Qiu, Yang; Jiang, Jie; Yu, Tong; Ji, Peng; Liu, Ying

    2017-01-01

    In this study, we developed a lipid-coated hollow calcium phosphate (LCP) nanoparticle for the combined application of two chemotherapeutic drugs to human lung cancer A549 cells. Hydrophilic doxorubicin (DOX) was incorporated into the hollow structure of hollow calcium phosphate (HCP), and a lipid bilayer containing hydrophobic paclitaxel (PTX) was subsequently coated on the surface of HCP. The study on combinational effects demonstrated that the combination of DOX and PTX at a mass ratio of 12:1 showed a synergistic effect against A549 cells. The particle size, zeta potential, and encapsulation efficiency were measured to obtain optimal values: particle size was 335.0 3.2 nm, zeta potential -41.1 mV, and encapsulation efficiency 80.40%±2.24%. An in vitro release study indicated that LCP produced a sustained drug release. A549 cells had a better uptake of LCP with good biocompatibility. Furthermore, in vitro cytotoxicity experiment, apoptosis analysis, in vivo anti-tumor efficacy and protein expression analysis of Bax, Bcl-2, and Caspase-3 demonstrated that the co-delivery system based on LCP had significant synergistic anti-tumor activity. All conclusions suggested that LCP is a promising platform for co-delivery of multiple anti-tumor drugs.

  2. Application of a lipid-coated hollow calcium phosphate nanoparticle in synergistic co-delivery of doxorubicin and paclitaxel for the treatment of human lung cancer A549 cells

    PubMed Central

    Wu, Chao; Xu, Jie; Hao, Yanna; Zhao, Ying; Qiu, Yang; Jiang, Jie; Yu, Tong; Ji, Peng; Liu, Ying

    2017-01-01

    In this study, we developed a lipid-coated hollow calcium phosphate (LCP) nanoparticle for the combined application of two chemotherapeutic drugs to human lung cancer A549 cells. Hydrophilic doxorubicin (DOX) was incorporated into the hollow structure of hollow calcium phosphate (HCP), and a lipid bilayer containing hydrophobic paclitaxel (PTX) was subsequently coated on the surface of HCP. The study on combinational effects demonstrated that the combination of DOX and PTX at a mass ratio of 12:1 showed a synergistic effect against A549 cells. The particle size, zeta potential, and encapsulation efficiency were measured to obtain optimal values: particle size was 335.0 3.2 nm, zeta potential −41.1 mV, and encapsulation efficiency 80.40%±2.24%. An in vitro release study indicated that LCP produced a sustained drug release. A549 cells had a better uptake of LCP with good biocompatibility. Furthermore, in vitro cytotoxicity experiment, apoptosis analysis, in vivo anti-tumor efficacy and protein expression analysis of Bax, Bcl-2, and Caspase-3 demonstrated that the co-delivery system based on LCP had significant synergistic anti-tumor activity. All conclusions suggested that LCP is a promising platform for co-delivery of multiple anti-tumor drugs. PMID:29184399

  3. Low-dose etoposide-treatment induces endoreplication and cell death accompanied by cytoskeletal alterations in A549 cells: Does the response involve senescence? The possible role of vimentin

    PubMed Central

    2013-01-01

    Background Senescence in the population of cells is often described as a program of restricted proliferative capacity, which is manifested by broad morphological and biochemical changes including a metabolic shift towards an autophagic-like response and a genotoxic-stress related induction of polyploidy. Concomitantly, the cell cycle progression of a senescent cell is believed to be irreversibly arrested. Recent reports suggest that this phenomenon may have an influence on the therapeutic outcome of anticancer treatment. The aim of this study was to verify the possible involvement of this program in the response to the treatment of the A549 cell population with low doses of etoposide, as well as to describe accompanying cytoskeletal alterations. Methods After treatment with etoposide, selected biochemical and morphological parameters were examined, including: the activity of senescence-associated ß-galactosidase, SAHF formation, cell cycle progression, the induction of p21Cip1/Waf1/Sdi1 and cyclin D1, DNA strand breaks, the disruption of cell membrane asymmetry/integrity and ultrastructural alterations. Vimentin and G-actin cytoskeleton was evaluated both cytometrically and microscopically. Results and conclusions Etoposide induced a senescence-like phenotype in the population of A549 cells. Morphological alterations were nevertheless not directly coupled with other senescence markers including a stable cell cycle arrest, SAHF formation or p21Cip1/Waf1/Sdi1 induction. Instead, a polyploid, TUNEL-positive fraction of cells visibly grew in number. Also upregulation of cyclin D1 was observed. Here we present preliminary evidence, based on microscopic analyses, that suggest a possible role of vimentin in nuclear alterations accompanying polyploidization-depolyploidization events following genotoxic insults. PMID:23383739

  4. In vitro chemoresistance profile and expression/function of MDR associated proteins in resistant cell lines derived from CCRF-CEM, K562, A549 and MDA MB 231 parental cells.

    PubMed

    Noskova, V; Dzubak, P; Kuzmina, G; Ludkova, A; Stehlik, D; Trojanec, R; Janostakova, A; Korinkova, G; Mihal, V; Hajduch, M

    2002-01-01

    Although cellular experiments have elucidated a number of active principles in the study of the multidrug resistance (MDR) phenomena, most of the drug resistant tumor cells were derived from different parental cell lines. This fact limits generalization of some experimental data and conclusions, and therefore we selected and characterized cell lines resistant to various anti-cancer agents derived from four parental cell lines: CEM (human T-lymphoblastic leukemia), K562 (human myeloid leukemia), A549 (human lung adenocarcinoma) and MDAMB 231 (human breast adenocarcinoma). In total we obtained a set of 42 resistant sublines, which is an excellent tool for the future studies of different aspects of MDR. In this study we report on some basic characteristics of these sublines, namely, cross-resistance to other anti-cancer drugs investigated by in vitro MTT assay, expression of MDR associated proteins (Pgp, MRP1, LRP, GST-pi and Topo IIalpha) as well as the functional activity of Pgp and MRP.

  5. A platycoside-rich fraction from the root of Platycodon grandiflorum enhances cell death in A549 human lung carcinoma cells via mainly AMPK/mTOR/AKT signal-mediated autophagy induction.

    PubMed

    Yim, Nam-Hui; Hwang, Youn-Hwan; Liang, Chun; Ma, Jin Yeul

    2016-12-24

    The root of Platycodon grandiflorum (PG), commonly known as Kilkyong in Korea, Jiegeng in China, and Kikyo in Japan, has been extensively used as a traditional anti-inflammatory medicine in Asia for the treatment of respiratory conditions, such as bronchitis, asthma, and tonsillitis. Platycosides isolated from PG are especially well-known for their anti-cancer effects. We investigated the involvement of autophagic cell death and other potential molecular mechanisms induced by the platycoside-containing butanol fraction of PG (PGB) in human lung carcinoma cells. PGB-induced growth inhibition and cell death were measured using a 5-diphenyl-tetrazolium bromide (MTT) assay. The effects of PGB on autophagy were determined by observing microtubule-associated protein 1 light chain 3 (LC3) redistribution with confocal microscopy. The PGB-mediated regulation of autophagy-associated proteins was investigated using Western blotting analysis. Furthermore, the anti-cancer mechanism of PGB was confirmed using chemical inhibitors. A high-performance liquid chromatography (HPLC)-DAD system was used to analyze the platycosides in PGB. In A549 cells, PGB induced significant autophagic cell death. Specifically, PGB upregulated LC3-II in a time- and dose-dependent manner, and it redistributed LC3 via autophagosome formation in the cytoplasm. PGB treatment increased the phosphorylation of AMP-activated protein kinase (AMPK) and subsequently suppressed the AKT/mammalian target of the rapamycin (mTOR) pathway. Furthermore, PGB inhibited cell proliferation by regulating the mitogen-activated protein kinase (MAPK) pathways. In this study, six types of platycosides were identified in the PGB using HPLC. PGB efficiently induced cancer cell death via autophagy and the modulation of the AMPK/mTOR/AKT and MAPK signaling pathways in A549 cells. Therefore, PGB may be an efficacious herbal anti-cancer therapy. Copyright © 2016. Published by Elsevier Ireland Ltd.

  6. Proteomics in studying cancer stem cell biology.

    PubMed

    Kranenburg, Onno; Emmink, Benjamin L; Knol, Jaco; van Houdt, Winan J; Rinkes, Inne H M Borel; Jimenez, Connie R

    2012-06-01

    Normal multipotent tissue stem cells (SCs) are the driving force behind tissue turnover and repair. The cancer stem cell theory holds that tumors also contain stem-like cells that drive tumor growth and metastasis formation. However, very little is known about the regulation of SC maintenance pathways in cancer and how these are affected by cancer-specific genetic alterations and by treatment. Proteomics is emerging as a powerful tool to identify the signaling complexes and pathways that control multi- and pluri-potency and SC differentiation. Here, the authors review the novel insights that these studies have provided and present a comprehensive strategy for the use of proteomics in studying cancer SC biology.

  7. Synthesis of 6-cinnamoyl-2H-benzo[b][1,4]oxazin-3(4H)-ones and their effects on A549 lung cancer cell growth.

    PubMed

    Zhou, Xue-Wen; Ma, Han-Lin; Zhang, Xuan; Jing, Shi-Yao; Miao, Jun-Ying; Zhao, Bao-Xiang

    2014-05-22

    A series of novel 6-cinnamoyl-2H-benzo[b][1,4]oxazin-3(4H)-one derivatives was synthesized. The structures of compounds were characterized by (1)H NMR, IR, and MS. Moreover, representative crystal structure was determined by X-ray diffraction analysis. The preliminary biological evaluation of all these compounds showed that compounds 3a-3d would suppress the growth of A549 lung cells effectively by inducing autophagy and cell cycle arrest. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  8. Evaluation of 5-HT7 Receptor Trafficking on In Vivo and In Vitro Model of Lipopolysaccharide (LPS)-Induced Inflammatory Cell Injury in Rats and LPS-Treated A549 Cells.

    PubMed

    Ayaz, Gulsen; Halici, Zekai; Albayrak, Abdulmecit; Karakus, Emre; Cadirci, Elif

    2017-02-01

    This study aimed to investigate the effects of the 5-HT7 receptor agonist (LP44) and antagonist (SB269970) on LPS-induced in vivo tissue damage and cell culture by molecular methods. This study was conducted in two steps. For in vivo studies, 24 female rats were divided into four groups. Group I: healthy; II (2nd h): LPS 5 mg/kg administered intraperitoneally (i.p.); III (4th h): LPS 5 mg/kg administered i.p.; IV (8th h): LPS 5 mg/kg administered i.p. For in vitro studies, we used the A549 cell line. Groups: I control (healthy) (2-4 h); II LPS: 1 µg/ml E. Coli O55:B5 strain (2-4 h); III agonist (LP44) 10 -9 M (2-4 h); IV antagonist (SB269970) 10 -9 M (2-4 h); V LPS+agonist 10 -9 M (LP44 1 µg/ml) (2-4 h); VI LPS+antagonist 10 -9 M (2-4 h). In molecular analyses, we determined increased TNF-α, IL-1β, NF-κB, and 5-HT7 mRNA expressions in rat lung tissues and increased TNF-α, iNOS, and 5-HT7 mRNA expressions in the A549 cell line. In in vitro parameters, LP44 agonist administration-related decrease was observed. Our study showed that lung 5-HT7 receptor expression is increased in LPS-induced endotoxemia. All this data suggest that 5-HT7 receptor overexpression is an important protective mechanism during LPS-induced sepsis-related cell damage.

  9. 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

  10. Design, synthesis, and characterization of α, β-unsaturated carboxylic acid, and its urea based derivatives that explores novel epigenetic modulators in human non-small cell lung cancer A549 cell line.

    PubMed

    Chidambaram, Anusha; Sundararaju, Kavya; Chidambaram, Ramesh K; Subbiah, Rajasekaran; Jayaraj, John M; Muthusamy, Karthikeyan; Vilwanathan, Ravikumar

    2018-07-01

    Histone deacetylase inhibitors (HDACi) are a small molecule chemotherapeutics that target the chromatin remodeling through the regulation of histone and non-histone proteins. These inhibitors directed against histone deacetylase (HDAC) enzymes have become an important therapeutic tool in oncology; consequently, scientific efforts have fortified the quest for newer and novel HDACi, which forces the design of structurally innovative HDACi. Various urea containing compounds exhibited admirable anticancer activity. On the basis of these observations, we design and synthesize HDAC specific blocker molecules which are specifically besieged towards class I, class II, and class IV HDAC isoforms to enhance the structural assortment for HDACi. Through docking experiments, we identified that the compounds were tightly bound to the isoforms of the HDAC enzymes at their receptor regions. These derivatives potently inhibited the different isoforms, namely, class I, II, and IV of HDACs, by hyperacetylation of lysine residues in A549 cells. The mechanism of apoptosis is evident, regulating tumor suppressor genes and proteins, thereby facilitating the activation of the death receptor pathway by the tumor necrosis factor (TNF) receptor. These derivative facilitated the induction of reactive oxygen species (ROS) generation leading to downregulation of Bcl 2 , and upregulation of Bax expression, thereby dysregulating mitochondrial membrane potential (ΔΨ m ) to release cytochrome c, and activation of intrinsic pathway. These compounds downregulate the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway to inhibit cell growth, proliferation, and metastasis through the matrix metalloproteinases (MMPs) MMP2 and MMP9 in A549 cells. These results suggest that our designed urea based derivatives act as epigenetic targeting agents through HDAC inhibition. © 2017 Wiley Periodicals, Inc.

  11. A549 and PLC/PRF/5 cells can support the efficient propagation of swine and wild boar hepatitis E virus (HEV) strains: demonstration of HEV infectivity of porcine liver sold as food.

    PubMed

    Takahashi, Hideyuki; Tanaka, Toshinori; Jirintai, Suljid; Nagashima, Shigeo; Takahashi, Masaharu; Nishizawa, Tsutomu; Mizuo, Hitoshi; Yazaki, Yasuyuki; Okamoto, Hiroaki

    2012-02-01

    Recent evidence has indicated the cross-species transmission of hepatitis E virus (HEV) from pigs and wild boars to humans, causing zoonosis, mostly via consumption of uncooked or undercooked animal meat/viscera. However, no efficient cell culture system for swine and boar HEV strains has been established. We inoculated A549 cells with 12 swine and boar HEV strains of liver, feces, or serum origin at an HEV load of ≥2.0 × 10(4) copies per well and found that the HEV progeny replicated as efficiently as human HEV strains, with a maximum load of ~10(8) copies/ml. However, the HEV load in the culture medium at 30 days post-inoculation differed markedly by inoculum, ranging from 1.0 × 10(2) to 1.1 × 10(7) copies/ml upon inoculation at a lower load of approximately 10(5) copies per well. All progeny were passaged successfully onto A549 and PLC/PRF/5 cells. In sharp contrast, no progeny viruses were detectable in the culture supernatant upon inoculation with 13 swine and boar HEV strains at an HEV load of <1.8 × 10(4) copies per well. The present study also demonstrates that swine liver sold as food can be infectious, supporting the risk of zoonotic food-borne HEV infection.

  12. Spatial-Resolution Cell Type Proteome Profiling of Cancer Tissue by Fully Integrated Proteomics Technology.

    PubMed

    Xu, Ruilian; Tang, Jun; Deng, Quantong; He, Wan; Sun, Xiujie; Xia, Ligang; Cheng, Zhiqiang; He, Lisheng; You, Shuyuan; Hu, Jintao; Fu, Yuxiang; Zhu, Jian; Chen, Yixin; Gao, Weina; He, An; Guo, Zhengyu; Lin, Lin; Li, Hua; Hu, Chaofeng; Tian, Ruijun

    2018-05-01

    Increasing attention has been focused on cell type proteome profiling for understanding the heterogeneous multicellular microenvironment in tissue samples. However, current cell type proteome profiling methods need large amounts of starting materials which preclude their application to clinical tumor specimens with limited access. Here, by seamlessly combining laser capture microdissection and integrated proteomics sample preparation technology SISPROT, specific cell types in tumor samples could be precisely dissected with single cell resolution and processed for high-sensitivity proteome profiling. Sample loss and contamination due to the multiple transfer steps are significantly reduced by the full integration and noncontact design. H&E staining dyes which are necessary for cell type investigation could be selectively removed by the unique two-stage design of the spintip device. This easy-to-use proteome profiling technology achieved high sensitivity with the identification of more than 500 proteins from only 0.1 mm 2 and 10 μm thickness colon cancer tissue section. The first cell type proteome profiling of four cell types from one colon tumor and surrounding normal tissue, including cancer cells, enterocytes, lymphocytes, and smooth muscle cells, was obtained. 5271, 4691, 4876, and 2140 protein groups were identified, respectively, from tissue section of only 5 mm 2 and 10 μm thickness. Furthermore, spatially resolved proteome distribution profiles of enterocytes, lymphocytes, and smooth muscle cells on the same tissue slices and across four consecutive sections with micrometer distance were successfully achieved. This fully integrated proteomics technology, termed LCM-SISPROT, is therefore promising for spatial-resolution cell type proteome profiling of tumor microenvironment with a minute amount of clinical starting materials.

  13. Comparative analysis of microRNA expression profiles between A549, A549/DDP and their respective exosomes.

    PubMed

    Qin, Xiaobing; Yu, Shaorong; Xu, Xiaoyue; Shen, Bo; Feng, Jifeng

    2017-06-27

    Exosomes were reported to transport bioactive molecules and influence the biology behavior of recipient cells. In order to study the role of exosomal microRNAs in the mechanism of cisplatin resistance to lung cancer cells, we analyzed the expression profiles of microRNAs in A549, A549/DDP cells and their exosomes by microarray. The results showed that a certain proportion of microRNAs were co-expressed in the cells and exosomes. Linear regression analysis showed that the expression of microRNAs in A549 and A549/DDP cells were strongly correlated with those in their respective exosomes. The expression level of 5 microRNAs (miR-197-5p, miR-4443, miR-642a-3p, miR-27b-3p and miR-100-5p) with the most differential expression were verified by qRT-PCR. The results were consistent with those of the microarray. Target gene prediction and pathway analysis discovered that the microRNAs in the intersections may participate in drug resistance. And the prediction of their association with diseases found that most of these microRNAs was associated with lung cancer. We could draw a preliminary conclusion that microRNAs in exosomes may be involved in the drug resistance of lung cancer cells to cisplatin.

  14. Nanosecond-Pulsed DBD Plasma-Generated Reactive Oxygen Species Trigger Immunogenic Cell Death in A549 Lung Carcinoma Cells through Intracellular Oxidative Stress

    PubMed Central

    Lin, Abraham; Truong, Billy; Patel, Sohil; Kaushik, Nagendra; Choi, Eun Ha; Fridman, Gregory; Fridman, Alexander; Miller, Vandana

    2017-01-01

    A novel application for non-thermal plasma is the induction of immunogenic cancer cell death for cancer immunotherapy. Cells undergoing immunogenic death emit danger signals which facilitate anti-tumor immune responses. Although pathways leading to immunogenic cell death are not fully understood; oxidative stress is considered to be part of the underlying mechanism. Here; we studied the interaction between dielectric barrier discharge plasma and cancer cells for oxidative stress-mediated immunogenic cell death. We assessed changes to the intracellular oxidative environment after plasma treatment and correlated it to emission of two danger signals: surface-exposed calreticulin and secreted adenosine triphosphate. Plasma-generated reactive oxygen and charged species were recognized as the major effectors of immunogenic cell death. Chemical attenuators of intracellular reactive oxygen species successfully abrogated oxidative stress following plasma treatment and modulated the emission of surface-exposed calreticulin. Secreted danger signals from cells undergoing immunogenic death enhanced the anti-tumor activity of macrophages. This study demonstrated that plasma triggers immunogenic cell death through oxidative stress pathways and highlights its potential development for cancer immunotherapy. PMID:28467380

  15. Nanosecond-Pulsed DBD Plasma-Generated Reactive Oxygen Species Trigger Immunogenic Cell Death in A549 Lung Carcinoma Cells through Intracellular Oxidative Stress.

    PubMed

    Lin, Abraham; Truong, Billy; Patel, Sohil; Kaushik, Nagendra; Choi, Eun Ha; Fridman, Gregory; Fridman, Alexander; Miller, Vandana

    2017-05-03

    A novel application for non-thermal plasma is the induction of immunogenic cancer cell death for cancer immunotherapy. Cells undergoing immunogenic death emit danger signals which facilitate anti-tumor immune responses. Although pathways leading to immunogenic cell death are not fully understood; oxidative stress is considered to be part of the underlying mechanism. Here; we studied the interaction between dielectric barrier discharge plasma and cancer cells for oxidative stress-mediated immunogenic cell death. We assessed changes to the intracellular oxidative environment after plasma treatment and correlated it to emission of two danger signals: surface-exposed calreticulin and secreted adenosine triphosphate. Plasma-generated reactive oxygen and charged species were recognized as the major effectors of immunogenic cell death. Chemical attenuators of intracellular reactive oxygen species successfully abrogated oxidative stress following plasma treatment and modulated the emission of surface-exposed calreticulin. Secreted danger signals from cells undergoing immunogenic death enhanced the anti-tumor activity of macrophages. This study demonstrated that plasma triggers immunogenic cell death through oxidative stress pathways and highlights its potential development for cancer immunotherapy.

  16. Cell cycle synchronization and BrdU incorporation as a tool to study the possible selective elimination of ErbB1 gene in the micronuclei in A549 cells

    PubMed Central

    Lauand, C.; Niero, E.L.; Dias, V.M.; Machado-Santelli, G.M.

    2015-01-01

    Lung cancer often exhibits molecular changes, such as the overexpression of the ErbB1 gene that encodes epidermal growth factor receptor (EGFR). ErbB1 amplification and mutation are associated with tumor aggressiveness and low response to therapy. The aim of the present study was to design a schedule to synchronize the cell cycle of A549 cell line (a non-small cell lung cancer) and to analyze the possible association between the micronuclei (MNs) and the extrusion of ErbB1 gene extra-copies. After double blocking, by the process of fetal bovine serum deprivation and vincristine treatment, MNs formation was monitored with 5-bromo-2-deoxyuridine (BrdU) incorporation, which is an S-phase marker. Statistical analyses allowed us to infer that MNs may arise both in mitosis as well as in interphase. The MNs were able to replicate their DNA and this process seemed to be non-synchronous with the main cell nuclei. The presence of ErbB1 gene in the MNs was evaluated by fluorescent in situ hybridization (FISH). ErbB1 sequences were detected in the MNs, but a relation between the MNs formation and extrusion of amplified ErbB1could not be established. The present study sought to elucidate the meaning of MNs formation and its association with the elimination of oncogenes or other amplified sequences from the tumor cells. PMID:25760027

  17. Hyaluronic acid-fabricated nanogold delivery of the inhibitor of apoptosis protein-2 siRNAs inhibits benzo[a]pyrene-induced oncogenic properties of lung cancer A549 cells

    NASA Astrophysics Data System (ADS)

    Lin, Chung-Ming; Kao, Wei-Chien; Yeh, Chun-An; Chen, Hui-Jye; Lin, Shinn-Zong; Hsieh, Hsien-Hsu; Sun, Wei-Shen; Chang, Chih-Hsuan; Hung, Huey-Shan

    2015-03-01

    Benzo[a]pyrene (BaP), a component of cooking oil fumes (COF), promotes lung cancer cell proliferation and survival via the induction of inhibitor of apoptosis protein-2 (IAP-2) proteins. Thus knockdown of IAP-2 would be a promising way to battle against lung cancer caused by COF. Functionalized gold nanoparticle (AuNP) is an effective delivery system for bio-active materials. Here, biocompatible hyaluronic acid (HA) was fabricated into nanoparticles to increase the target specificity by binding to CD44-over-expressed cancer cells. IAP-2-specific small-interfering RNA (siRNAs) or fluorescein isothiocyanate (FITC) were then incorporated into AuNP-HA. Conjugation of IAP-2 siRNA into AuNPs-HA was verified by the UV-vis spectrometer and Fourier transform infrared spectrometer. Further studies showed that AuNP-HA/FITC were effectively taken up by A549 cells through CD44-mediated endocytosis. Incubation of BaP-challenged cells with AuNP-HA-IAP-2 siRNAs silenced the expression of IAP-2, decreased cell proliferation and triggered pronounced cell apoptosis by the decrease in Bcl-2 protein and the increase in Bax protein as well as the active form of caspases-3. The BaP-elicited cell migration and enzymatic activity of the secreted matrix metalloproteinase-2 were also substantially suppressed by treatment with AuNP-HA-IAP-2 siRNAs. These results indicated that IAP-2 siRNAs can be efficiently delivered into A549 cells by functionalized AuNP-HA to repress the IAP-2 expression and BaP-induced oncogenic events, suggesting the potential therapeutic application of IAP-2 siRNA or other siRNA-conjugated AuNP-HA composites to COF-induced lung cancer and other gene-caused diseases in the future.

  18. Extracellular HSP70 Activates ERK1/2, NF-kB and Pro-Inflammatory Gene Transcription Through Binding with RAGE in A549 Human Lung Cancer Cells.

    PubMed

    Somensi, Nauana; Brum, Pedro Ozorio; de Miranda Ramos, Vitor; Gasparotto, Juciano; Zanotto-Filho, Alfeu; Rostirolla, Diana Carolina; da Silva Morrone, Maurilio; Moreira, José Claudio Fonseca; Pens Gelain, Daniel

    2017-01-01

    Heat shock protein 70 (HSP70) has been recently described with extracellular actions, where it is actively released in inflammatory conditions. Acting as DAMPs (damage associated molecular pattern), extracellular HSP70 (eHSP70) interacts with membrane receptors and activates inflammatory pathways. At this context, the receptor for advanced glycation endproducts (RAGE) emerges as a possible candidate for interaction with eHSP70. RAGE is a pattern-recognition receptor and its expression is increased in several diseases related to a chronic pro-inflammatory state. One of the main consequences of RAGE ligand-binding is the ERK1/2 (extracellular signal-regulated kinases)-dependent activation of NF-kB (nuclear factor kappa B), which leads to expression of TNF-α (tumor necrosis factor alpha) and other cytokines. The purpose of this work is to elucidate if eHSP70 is able to evoke RAGE-dependent signaling using A549 human lung cancer cells, which constitutively express RAGE. Immunoprecipitation and protein proximity assay were utilized to demonstrate the linkage between RAGE and eHSP70. To investigate RAGE relevance on cell response to eHSP70, siRNA was used to knockdown the receptor expression. Signaling pathways activation were evaluated by western blotting, gene reporter luciferase and real time quantitative PCR. Protein eHSP70 shown to be interacting physically with the receptor RAGE in our cell model. Treatment with eHSP70 caused ERK1/2 activation and NF-κB transactivation impaired by RAGE knockdown. Moreover, the stimulation of pro-inflammatory cytokines expression by eHSP70 was inhibited in RAGE-silenced cells. Finally, conditioned medium of eHSP70-treated A549 cells caused differential effects in monocytes cytokine expression when A549 RAGE expression is inhibited. Our results evidence eHSP70 as a novel RAGE agonist capable of influence the cross-talk between cancer and immune system cells. © 2017 The Author(s). Published by S. Karger AG, Basel.

  19. The influence of Hurricanes Katrina and Rita on the inflammatory cytokine response and protein expression in A549 cells exposed to PM2.5 collected in the Baton Rouge-Port Allen industrial corridor of Southeastern Louisiana in 2005.

    PubMed

    Bourgeois, Brian; Owens, John Wesley

    2014-03-01

    Hurricanes Katrina and Rita hit the coast of Louisiana in 2005 and killed more than 2000 people. The two storms resulted in a significant spike in particulate matter (PM2.5) levels across the state of Louisiana. This report focuses on PM2.5 samples collected in 2005 from two monitoring sites in the neighboring cities of Baton Rouge and Port Allen, Louisiana. Inductively coupled plasma (ICP) revealed the presence of PM2.5-adsorbed representative and Fenton-active transition metals. Gas chromatography/mass spectrometry (GC-MS) analyses revealed the presence of 23 PAH compounds. Endotoxins were also detected. Metals and endotoxins were extracted with water. PAH were extracted with dichloromethane. In order to assess cytotoxicity, aqueous PM2.5 extracts were introduced to A549 Human Epithelial Lung Carcinoma Cells. Results indicated decreased cell viability in a dose-dependent manner, with an LC50 of 235 µg/ml and 250 µg/ml, respectively, for the two sites featured here. Endotoxins alone were not cytotoxic. The concentration of reactive oxygen species (ROS) and released LDH activity increased following exposure of A549 cells to aqueous PM2.5 extracts. Fluorescence microscopy revealed apoptotic and necrotic cell death mechanisms. ELISA revealed increased secretion of primary pro-inflammatory cytokines, IL-6, IL-8, and TNF-α. Global PCR gene expression revealed up-regulation of proteins associated with the cytokine storm; e.g. interleukins, chemokines, and TNF-α. Global antibody microarray was consistent with an inflammatory response, with up-regulation of cytokines involved in the down-field activation of the caspase cascade and kinase pathways. The up-regulation of metal-redox sensitive transcription factors, NF-κβ and AP-1, is consistent with a cell death mechanism initiated by Fenton-active transition metal redox catalysis.

  20. Grape Seed Proanthocyanidin Inhibits Mucin Synthesis and Viral Replication by Suppression of AP-1 and NF-κB via p38 MAPKs/JNK Signaling Pathways in Respiratory Syncytial Virus-Infected A549 Cells.

    PubMed

    Lee, Jin-Woo; Kim, Young Il; Im, Chang-Nim; Kim, Sung Wan; Kim, Su Jin; Min, Seoyeon; Joo, Yong Hoon; Yim, Sung-Vin; Chung, Namhyun

    2017-06-07

    Airway epithelial cells are often infected by respiratory syncytial virus (RSV), one of the most common causes of asthma, bronchiolitis, chronic obstructive pulmonary disease, and pneumonia. During the infection process, excessive mucins instigate airway inflammation. However, the mechanism underlying RSV-induced airway hyper-responsiveness and inflammation is poorly understood. Furthermore, no reliable vaccines or drugs for antiviral therapy are available. In this study, the effect of the natural compound grape seed proanthocyanidin (GSP) on RSV-infected human airway epithelial cells A549 was evaluated. After pretreatment of the cells with or without exposure to RSV with 5-10 μg GSP/mL, the expression of various mucins (MUC1, MUC2, MUC5AC, MUC5B, and MUC8) was evaluated by real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blotting, as well as confocal microscopy. We found that GSP significantly decreased RSV-induced mucin synthesis at the mRNA and protein levels. In addition, GSP suppressed the RSV-induced signaling pathways, including extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38, together with nuclear factor kappa B (NF-κB) and activating protein-1 family members (c-Jun and c-Fos). Concomitantly, GSP inhibited the replication of RSV within A549 cells. Taken together, all our results suggest that GSP could be a potent therapeutic agent to suppress excessive mucus production and viral replication in RSV-induced airway inflammatory disorders.

  1. Naringin attenuates EGF-induced MUC5AC secretion in A549 cells by suppressing the cooperative activities of MAPKs-AP-1 and IKKs-IκB-NF-κB signaling pathways.

    PubMed

    Nie, Yi-chu; Wu, Hao; Li, Pei-bo; Xie, Li-ming; Luo, Yu-long; Shen, Jian-gang; Su, Wei-wei

    2012-09-05

    Naringenin, the aglycone of naringin, has been reported to attenuate MUC5AC secretion by inhibiting activity of nuclear factor kappa B (NF-κB) via EGFR-PI3K-Akt/ERK MAPKinase signaling pathways. However, previous studies demonstrated that the MUC5AC promoter was located in two different regions: an activator protein-1 (AP-1) binding site and a NF-κB binding site. The current study comprehensively determined the involvement of MAPKs/AP-1 and IKKs/IκB/NF-κB in epidermal growth factor (EGF)-induced A549 cells, and sought to ascertain the signaling pathways of naringin imparted in suppression of EGF-induced MUC5AC secretion. The results showed that naringin of 100 μM not only significantly decreased EGF-induced overexpressions of both MUC5AC mucin and mRNA in A549 cells, but also suppressed the phosphorylation of EGF receptor, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK1/2), and c-Jun N-terminal kinase (JNK), as well as nucleus NF-κB p65 and AP-1. Moreover, any of three MAPKs inhibitors (PD98059, SB203580, and SP600125) significantly inhibited EGF-induced MUC5AC secretion. And as compared to MG132, the inhibitor κB (IκB) phosphorylation inhibitor of SN50 was more effective in reducing EGF-induced MUC5AC secretion because of suppression of nucleus AP-1. Meanwhile, as compared to naringin, both SP600125 and azithromycin were less effective in suppressing EGF-induced secretion of MUC5AC because of the unchanged nucleus NF-κB p65. These results indicated that naringin attenuates EGF-induced MUC5AC secretion in A549 cells by suppressing the cooperative activities of MAPKs/AP-1 and IKKs/IκB/NF-κB signaling pathways. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. Proteomic and Bioinformatic Profile of Primary Human Oral Epithelial Cells

    PubMed Central

    Ghosh, Santosh K.; Yohannes, Elizabeth; Bebek, Gurkan; Weinberg, Aaron; Jiang, Bin; Willard, Belinda; Chance, Mark R.; Kinter, Michael T.; McCormick, Thomas S.

    2012-01-01

    Wounding of the oral mucosa occurs frequently in a highly septic environment. Remarkably, these wounds heal quickly and the oral cavity, for the most part, remains healthy. Deciphering the normal human oral epithelial cell (NHOEC) proteome is critical for understanding the mechanism(s) of protection elicited when the mucosal barrier is intact, as well as when it is breached. Combining 2D gel electrophoresis with shotgun proteomics resulted in identification of 1662 NHOEC proteins. Proteome annotations were performed based on protein classes, molecular functions, disease association and membership in canonical and metabolic signaling pathways. Comparing the NHOEC proteome with a database of innate immunity-relevant interactions (InnateDB) identified 64 common proteins associated with innate immunity. Comparison with published salivary proteomes revealed that 738/1662 NHOEC proteins were common, suggesting that significant numbers of salivary proteins are of epithelial origin. Gene ontology analysis showed similarities in the distributions of NHOEC and saliva proteomes with regard to biological processes, and molecular functions. We also assessed the inter-individual variability of the NHOEC proteome and observed it to be comparable with other primary cells. The baseline proteome described in this study should serve as a resource for proteome studies of the oral mucosa, especially in relation to disease processes. PMID:23035736

  3. Regulation of deprotonation of 3,3-di(1H-tetrazol-5-yl)pentanedioic acid: Solvothermal synthesis of La(III) and heterometallic La(III)/Cu(II) compounds for ablation of A549 cells

    NASA Astrophysics Data System (ADS)

    Guo, Meng Yue; Zhang, Xin; Zhao, Liang; Li, Yue Kang; Chen, Dian Yu; Yang, Gao Wen; Li, Qiao Yun

    2018-03-01

    3,3-di(1H-tetrazol-5-yl)pentanedioic acid (H4dtzpda) can display tunable valence when reacted with different metal ions because it has four acidic hydrogen atoms of both tetrazole rings and carboxylate groups. Solvothermal reactions of H4dtzpda with La(NO3)2·6H2O or La(NO3)3·6H2O/Cu(NO3)2·6H2O afforded a one dimensional [La(Hdtzpda)(H2O)4]·4H2O (1) and a three dimensional [La2Cu(dtzpda)2(H2O)10]·4H2O (2), respectively, where only three acidic hydrogen atoms of H4dtzpda are deprotonated in compound 1 while all the four acidic ones are deprotonated in compound 2. In compound 1, Hdtzpda3- is a penta-dentate ligand to bridge La(III) centers via only oxygen atoms of the carboxylate group while in compound 2, dtzpda4- is a hepta-dentate one via not only the oxygen atoms of the carboxylate group but also the nitrogen atoms of the tetrazole rings. PEG-5000 (poly(ethyleneglycol-5000)) coated compound 1 or 2 nanoparticles (NPs) have good dispersity in water. Cytotoxicity study on A549 cells (human caucasian lung carcinoma) shows that compound 2 (half-maximal inhibitory concentration, IC50~18 μg/mL) is superior to compound 1 (IC50~38 μg/mL). Furthermore, both NPs of the two compounds are able to inhibit the migration of A549 cells, indicating their potential to inhibit the transfer of tumors in vivo.

  4. The impact of anticancer activity upon Beta vulgaris extract mediated biosynthesized silver nanoparticles (ag-NPs) against human breast (MCF-7), lung (A549) and pharynx (Hep-2) cancer cell lines.

    PubMed

    Venugopal, K; Ahmad, H; Manikandan, E; Thanigai Arul, K; Kavitha, K; Moodley, M K; Rajagopal, K; Balabhaskar, R; Bhaskar, M

    2017-08-01

    The present study tried for a phyto-synthetic method of producing silver nanoparticles (Ag-NPs) with size controlled as and eco-friendly route that can lead to their advanced production with decorative tranquil morphology. By inducing temperature fluctuation of the reaction mixture from 25 to 80°C the plasmon resonance band raised slowly which had an ultimate effect on size and shape of Ag-NPs as shown by UV-visible spectroscopy and TEM results. The biosynthesized nanoparticles showed good cytotoxic impact against MCF-7, A549 and Hep2 cells compared to normal cell lines. Compared to control plates, the percentage of cell growth inhibition was found to be high with as concentrations of Ag-NPs becomes more as determined by MTT assay. The AO/EtBr staining observations demonstrated that the mechanism of cell death induced by Ag-NPs was due to apoptosis in cancer cells. These present results propose that the silver nanoparticles (Ag-NPs) may be utilized as anticancer agents for the treatment of various cancer types. However, there is a need for study of in vivo examination of these nanoparticles to find their role and mechanism inside human body. Further, studies we plan to do biomarker fabrication from the green synthesized plant extract nanoparticles like silver, gold and copper nanoparticles with optimized shape and sizes and their enhancement of these noble nanoparticles. Copyright © 2017. Published by Elsevier B.V.

  5. 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

  6. 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.

  7. All-Trans Retinoic Acid Induces Proliferation, Survival, and Migration in A549 Lung Cancer Cells by Activating the ERK Signaling Pathway through a Transcription-Independent Mechanism.

    PubMed

    Quintero Barceinas, Reyna Sara; García-Regalado, Alejandro; Aréchaga-Ocampo, Elena; Villegas-Sepúlveda, Nicolás; González-De la Rosa, Claudia Haydée

    2015-01-01

    All-trans retinoic acid (ATRA) has been used as an antineoplastic because of its ability to promote proliferation, inhibition, and differentiation, primarily in leukemia; however, in other types of cancer, such as lung cancer, treatment with ATRA is restricted because not all the patients experience the same results. The ERK signaling pathway is dysregulated in cancer cells, including lung cancer, and this dysregulation promotes proliferation and cell invasion. In this study, we demonstrate that treatment with ATRA can activate the ERK signaling pathway by a transcription-independent mechanism through a signaling cascade that involves RARα and PI3K, promoting growth, survival, and migration in lung cancer cells. Until now, this mechanism was unknown in lung cancer cells. The inhibition of the ERK signaling pathway restores the beneficial effects of ATRA, reduces proliferation, increases apoptosis, and blocks the cell migration process in lung cancer cells. In conclusion, our results suggest that the combination of ATRA with ERK inhibitor in clinical trials for lung cancer is warranted.

  8. A Cell-Based Approach to the Human Proteome Project

    NASA Astrophysics Data System (ADS)

    Kelleher, Neil L.

    2012-10-01

    The general scope of a project to determine the protein molecules that comprise the cells within the human body is framed. By focusing on protein primary structure as expressed in specific cell types, this concept for a cell-based version of the Human Proteome Project (CB-HPP) is crafted in a manner analogous to the Human Genome Project while recognizing that cells provide a primary context in which to define a proteome. Several activities flow from this articulation of the HPP, which enables the definition of clear milestones and deliverables. The CB-HPP highlights major gaps in our knowledge regarding cell heterogeneity and protein isoforms, and calls for development of technology that is capable of defining all human cell types and their proteomes. The main activities will involve mapping and sorting cell types combined with the application of beyond the state-of-the art in protein mass spectrometry.

  9. Knockdown of end-binding protein 1 induces apoptosis in radioresistant A549 lung cancer cells via p38 kinase-dependent COX-2 upregulation.

    PubMed

    Baek, Jeong-Hwa; Yim, Ji-Hye; Song, Jie-Young; Um, Hong-Duck; Park, Jong Kuk; Park, In-Chul; Kim, Jae-Sung; Lee, Chang-Woo; Hong, Eun-Hee; Kim, Eun Ho; Hwang, Sang-Gu

    2018-04-01

    The role of end-binding protein 1 (EB1) in lung cancer tumorigenesis and radiotherapy remains poorly understood. In the present study, we observed that EB1 was highly expressed in lung tumor tissues compared with normal non-tumor tissues based on immunohistochemical analysis of lung cancer tissue samples obtained from human tissue microarrays. EB1 was also highly overexpressed in radioresistant lung and cervical cancer cells, which exhibited increased cell death after EB1 silencing. The cytotoxicity induced by EB1 gene knockdown was due to the activation and generation of reactive oxygen species by p38 mitogen-activated protein kinase. Notably, this signaling cascade, however not nuclear factor-κB-mediated signaling, induced the expression of cyclooxygenase-2, a key effector of apoptotic death. Our results provided new molecular evidence supporting the use of EB1 as a novel target in lung cancer therapy, especially in the case of radioresistance.

  10. Mir-513a-3p contributes to the controlling of cellular migration processes in the A549 lung tumor cells by modulating integrin β-8 expression.

    PubMed

    da Silveira, Marina Bonfogo; Lima, Kelvin Furtado; da Silva, Andrea Renata; Dos Santos, Robson Augusto Souza; Moraes, Karen C M

    2017-12-04

    Lung tumors are a frequent type of cancer in humans and a leading cause of death, and the late diagnostic contributes to high mortality rates. New therapeutic strategies are needed, and the heptapeptide angiotensin-(1-7) [ang-(1-7)] demonstrated the ability to control cancer growth rates and migration in vitro and in vivo. However, the possible use of the heptapeptide in clinical trials demands deeper analyses to elucidate molecular mechanisms of its effect in the target cells. In this study, we investigated relevant elements that control pro-inflammatory environment and cellular migration, focusing in the post-transcription mechanism using lung tumor cell line. In our cellular model, the microRNA-513a-3p was identified as a novel element targeting ITG-β8, thereby controlling the protein level and its molecular function in the controlling of migration and pro-inflammatory environment. These findings provide useful information for future studies, using miR-513a-3p as an innovative molecular tool to control lung tumor cell migration, which will support more effective clinical treatment of the patients with the widely used chemotherapeutic agents, increasing survival rates.

  11. Curcumin Inhibits LIN-28A through the Activation of miRNA-98 in the Lung Cancer Cell Line A549.

    PubMed

    Liu, Wei-Lun; Chang, Jia-Ming; Chong, Inn-Wen; Hung, Yi-Li; Chen, Yung-Hsiang; Huang, Wen-Tsung; Kuo, Hsuan-Fu; Hsieh, Chong-Chao; Liu, Po-Len

    2017-06-03

    Metastasis is common in lung cancer and is associated with poor clinical outcomes and increased mortality. Curcumin is a natural anti-cancer agent that inhibits the metastasis of various cancers by modulating the expression of micro (mi) RNAs such as miR-98, which acts as a tumor suppressor. This study investigated the effect of curcumin on miR-98 expression and in vitro cell line growth and invasiveness in lung cancer. Curcumin treatment enhanced the expression of miR-98 and reduced that of the miR-98 target gene LIN28A as well as matrix metalloproteinase ( MMP ) 2 and MMP9 in vitro and in vivo. MiR-98 overexpression suppressed lung cancer cell migration and invasion by inhibiting LIN28A-induced MMP2 and MMP9 expression. Meanwhile, LIN28A level was downregulated by overexpression of miR-98 mimic. Induction of miR-98 by curcumin treatment suppressed MMP2 and MMP9 by targeting LIN28A. These findings provide insight into the mechanisms by which curcumin suppresses lung cancer cell line growth in vitro and in vivo and invasiveness in vitro.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-07-01

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

  14. 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. Copyright © 2015 John Wiley & Sons, Ltd.

  15. Synthesis of silver nanoparticles (Ag NPs) for anticancer activities (MCF 7 breast and A549 lung cell lines) of the crude extract of Syzygium aromaticum.

    PubMed

    Venugopal, K; Rather, H A; Rajagopal, K; Shanthi, M P; Sheriff, K; Illiyas, M; Rather, R A; Manikandan, E; Uvarajan, S; Bhaskar, M; Maaza, M

    2017-02-01

    In the present report, silver nanoparticles were synthesized using Piper nigrum extract for in vitro cytotoxicity efficacy against MCF-7 and HEP-2 cells. The silver nanoparticles (AgNPs) were formed within 20min and after preliminarily confirmation by UV-Visible spectroscopy (strong peak observed at ~441nm), they were characterized by using FT-IR and HR-TEM. The TEM images show spherical shape of biosynthesized AgNPs with particle size in the range 5-40nm while as compositional analysis were observed by EDAX. MTT assays were carried out for cytotoxicity of various concentrations of biosynthesized silver nanoparticles and Piper nigrum extract ranging from 10 to 100μg. The biosynthesized silver nanoparticles showed a significant anticancer activity against both MCF-7 and Hep-2 cells compared to Piper nigrum extract which was dose dependent. Our study thus revealed an excellent application of greenly synthesized silver nanoparticles using Piper nigrum. The study further suggested the potential therapeutic use of these nanoparticles in cancer study. Copyright © 2016. Published by Elsevier B.V.

  16. In vitro and in vivo characteristics of biogenic high surface silica nanoparticles in A549 lung cancer cell lines and Danio rerio model systems for inorganic biomaterials development.

    PubMed

    Rangaraj, Suriyaprabha; Venkatachalam, Rajendran

    2017-08-24

    Silica based organic-inorganic hybrids are turned over the most necessitate biomaterial due to their exotic biomedical properties. Colloidal silica nanoparticles (SNPs) of high surface area are synthesized from the bamboo wastes (leave biomass) as a viable and promising alternative to synthetic silica sol through alkaline extraction process. Physico-chemical properties of the prepared silica powders are examined employing extensive characterization tools. The characteristic results of the silica sol demonstrate amorphous particles (average size: 25 nm) with relatively high surface area (428 m 2  g -1 ) and spherical morphology. The teratogenicity of the surface and size dependant SNPs is evaluated using an alternative animal model, zebrafish (Danio rerio) in comparison with micron sized particles. LDH based cytotoxicity assay reveals non-significant cell damage in all the tested silica particles. Less mortality, uptake and dysfunctionalities of the organs during the developmental stages of zebrafish treated with bulk and nanoparticles confirm their biocompatibility. The least DNA strand breakage during genotoxic assay and teratogenic parameters are found to exhibit desirable bioactivity of SNPs for clinical applications even at higher concentrations. For the first time, bamboo derived silica sol induced genotoxicity is assessed at molecular level to understand the interaction mechanism with the fish genome.

  17. 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. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

  18. Polymeric Nano-Encapsulation of Curcumin Enhances its Anti-Cancer Activity in Breast (MDA-MB231) and Lung (A549) Cancer Cells Through Reduction in Expression of HIF-1α and Nuclear p65 (Rel A).

    PubMed

    Khan, Mohammed N; Haggag, Yusuf A; Lane, Majella E; McCarron, Paul A; Tambuwala, Murtaza M

    2018-02-14

    The anti-cancer potential of curcumin, a natural NFκβ inhibitor, has been reported extensively in breast, lung and other cancers. In vitro and in vivo studies indicate that the therapeutic efficacy of curcumin is enhanced when formulated in a nanoparticulate carrier. However, the mechanism of action of curcumin at the molecular level in the hypoxic tumour micro-environment is not fully understood. Hence, the aim of our study was to investigate the mechanism of action of curcumin formulated as nanoparticles in in vitro models of breast and lung cancer under an hypoxic microenvironment. Biodegradable poly(lactic-co-glycolic acid) PLGA nanoparticles (NP), loaded with curcumin (cur-PLGA-NP), were fabricated using a solvent evaporation technique to overcome solubility issues and to facilitate intracellular curcumin delivery. Cytotoxicity of free curcumin and cur-PLGA-NP was evaluated in MDA-MB-231 and A549 cell lines using migration, invasion and colony formation assays. All treatments were performed under an hypoxic micro-environment and whole cell lysates from controls and test groups were used to determine the expression of HIF-1α and p65 levels using ELISA assays. A ten-fold increase in solubility, three-fold increase in anti-cancer activity and a significant reduction in the levels of cellular HIF-1α and nuclear p65 (Rel A) were observed for cur-PLGA-NP, when compared to free curcumin. Our findings indicate that curcumin can effectively lower the elevated levels of HIF-1α and nuclear p65 (Rel A) in breast and lung cancer cells under an hypoxic tumour micro-environment when delivered in nanoparticulate form. This applied means of colloidal delivery could explain the improved anti-cancer efficacy of curcumin and has further potential applications in enhancing the activity of anti-cancer agents of low solubility. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  19. Advancing Cell Biology Through Proteomics in Space and Time (PROSPECTS)*

    PubMed Central

    Lamond, Angus I.; Uhlen, Mathias; Horning, Stevan; Makarov, Alexander; Robinson, Carol V.; Serrano, Luis; Hartl, F. Ulrich; Baumeister, Wolfgang; Werenskiold, Anne Katrin; Andersen, Jens S.; Vorm, Ole; Linial, Michal; Aebersold, Ruedi; Mann, Matthias

    2012-01-01

    The term “proteomics” encompasses the large-scale detection and analysis of proteins and their post-translational modifications. Driven by major improvements in mass spectrometric instrumentation, methodology, and data analysis, the proteomics field has burgeoned in recent years. It now provides a range of sensitive and quantitative approaches for measuring protein structures and dynamics that promise to revolutionize our understanding of cell biology and molecular mechanisms in both human cells and model organisms. The Proteomics Specification in Time and Space (PROSPECTS) Network is a unique EU-funded project that brings together leading European research groups, spanning from instrumentation to biomedicine, in a collaborative five year initiative to develop new methods and applications for the functional analysis of cellular proteins. This special issue of Molecular and Cellular Proteomics presents 16 research papers reporting major recent progress by the PROSPECTS groups, including improvements to the resolution and sensitivity of the Orbitrap family of mass spectrometers, systematic detection of proteins using highly characterized antibody collections, and new methods for absolute as well as relative quantification of protein levels. Manuscripts in this issue exemplify approaches for performing quantitative measurements of cell proteomes and for studying their dynamic responses to perturbation, both during normal cellular responses and in disease mechanisms. Here we present a perspective on how the proteomics field is moving beyond simply identifying proteins with high sensitivity toward providing a powerful and versatile set of assay systems for characterizing proteome dynamics and thereby creating a new “third generation” proteomics strategy that offers an indispensible tool for cell biology and molecular medicine. PMID:22311636

  20. Sulforaphane epigenetically demethylates the CpG sites of the miR-9-3 promoter and reactivates miR-9-3 expression in human lung cancer A549 cells.

    PubMed

    Gao, Linbo; Cheng, David; Yang, Jie; Wu, Renyi; Li, Wenji; Kong, Ah-Ng

    2018-02-09

    Increasing evidence suggests that epigenetic aberrations contribute to the development and progression of cancers such as lung cancer. The promoter region of miR-9-3 was recently found to be hypermethylated in lung cancer, resulting in down-regulation of miR-9-3 and poor patient prognosis. Sulforaphane (SFN), a natural compound that is obtained from cruciferous vegetables, has potent anticancer activities. In this study, we aimed to investigate the effect of SFN on restoring the miR-9-3 level in lung cancer A549 cells through epigenetic regulation. DNA methylation of the miR-9-3 promoter was examined using bisulfite genomic sequencing and methylated DNA immunoprecipitation analysis. The expression levels of miR-9-3 and several epigenetic modifying enzymes were measured using quantitative real-time polymerase chain reaction and Western blotting, respectively. The transcriptional activity of the miR-9-3 promoter was evaluated by patch methylation, and histone modifications were analyzed using chromatin immunoprecipitation (ChIP) assays. We found that CpG methylation was reduced in the miR-9-3 promoter and that miR-9-3 expression was increased after 5 days of treatment with SFN. In vitro methylation analysis showed that the methylated recombinant construct exhibited lower luciferase reporter activity than the unmethylated counterpart. ChIP assays revealed that SFN treatment increased H3K4me1 enrichment at the miR-9-3 promoter. Furthermore, SFN treatment attenuated enzymatic DNMT activity and DNMT3a, HDAC1, HDAC3, HDAC6 and CDH1 protein expression. Taken together, these findings indicate that SFN may exert its chemopreventive effects partly through epigenetic demethylation and restoration of miR-9-3. Copyright © 2017. Published by Elsevier Inc.

  1. A proteomic view of cell physiology of Bacillus licheniformis.

    PubMed

    Voigt, Birgit; Schweder, Thomas; Becher, Dörte; Ehrenreich, Armin; Gottschalk, Gerhard; Feesche, Jörg; Maurer, Karl-Heinz; Hecker, Michael

    2004-05-01

    The still ongoing sequencing of Bacillus licheniformis at the Göttingen Sequencing Laboratory provides the basis for proteome studies of the bacterium. By using two-dimensional (2-D) electrophoresis and protein identification by mass spectrometry, we were able to create master gels for B. licheniformis cells grown either in minimal medium or in complex medium containing about 300 and 180 entries, respectively. With the DECODON Delta 2D software we identified the most abundant protein spots on the gels, which were shown to perform mainly basic metabolic functions in the cell such as translation, amino acid metabolism, glycolysis, and tricarboxylic acid (TCA) cycle. Based on the master gels, we were able to study the regulation of metabolic pathways such as glycolysis and TCA cycle. In cells grown in the presence of glucose a significant increase of the amount of some glycolytic enzymes (TpiA, GapA, Pgk, Pgm, Eno, Pyk) and of the pyruvate dehydrogenase (PdhA-D) was found. At the same time, there is a strong repression of almost all TCA cycle enzymes and of the ATP synthase. Glucose also stimulates the acetate kinase (AckA) and the phosphotransacetylase (Pta) which are known to be involved in the overflow metabolism in B. subtilis. Furthermore, we began developing proteomic signatures for growth of B. licheniformis in complex medium. For this purpose, we compared the proteome pattern of exponentially growing cells with that of cells in different stages during stationary phase. The most obvious proteomic signature indicates that cells during stationary phase are subjected to a severe oxidative stress and a resulting protein stress. Furthermore, the level of many vegetative proteins is strongly reduced when the growth is arrested after entry into stationary phase. The data indicate that proteomics can be a valuable tool to describe the physiological state of B. licheniformis cell populations, e.g., of cells growing in a bioreactor.

  2. Biomaterial surface proteomic signature determines interaction with epithelial cells.

    PubMed

    Abdallah, Mohamed-Nur; Tran, Simon D; Abughanam, Ghada; Laurenti, Marco; Zuanazzi, David; Mezour, Mohamed A; Xiao, Yizhi; Cerruti, Marta; Siqueira, Walter L; Tamimi, Faleh

    2017-05-01

    Cells interact with biomaterials indirectly through extracellular matrix (ECM) proteins adsorbed onto their surface. Accordingly, it could be hypothesized that the surface proteomic signature of a biomaterial might determine its interaction with cells. Here, we present a surface proteomic approach to test this hypothesis in the specific case of biomaterial-epithelial cell interactions. In particular, we determined the surface proteomic signature of different biomaterials exposed to the ECM of epithelial cells (basal lamina). We revealed that the biomaterial surface chemistry determines the surface proteomic profile, and subsequently the interaction with epithelial cells. In addition, we found that biomaterials with surface chemistries closer to that of percutaneous tissues, such as aminated PMMA and aminated PDLLA, promoted higher selective adsorption of key basal lamina proteins (laminins, nidogen-1) and subsequently improved their interactions with epithelial cells. These findings suggest that mimicking the surface chemistry of natural percutaneous tissues can improve biomaterial-epithelial integration, and thus provide a rationale for the design of improved biomaterial surfaces for skin regeneration and percutaneous medical devices. Failure of most biomaterials originates from the inability to predict and control the influence of their surface properties on biological phenomena, particularly protein adsorption, and cellular behaviour, which subsequently results in unfavourable host response. Here, we introduce a surface-proteomic screening approach using a label-free mass spectrometry technique to decipher the adsorption profile of extracellular matrix (ECM) proteins on different biomaterials, and correlate it with cellular behaviour. We demonstrated that the way a biomaterial selectively interacts with specific ECM proteins of a given tissue seems to determine the interactions between the cells of that tissue and biomaterials. Accordingly, this approach can

  3. Plant organelle proteomics: collaborating for optimal cell function.

    PubMed

    Agrawal, Ganesh Kumar; Bourguignon, Jacques; Rolland, Norbert; Ephritikhine, Geneviève; Ferro, Myriam; Jaquinod, Michel; Alexiou, Konstantinos G; Chardot, Thierry; Chakraborty, Niranjan; Jolivet, Pascale; Doonan, John H; Rakwal, Randeep

    2011-01-01

    Organelle proteomics describes the study of proteins present in organelle at a particular instance during the whole period of their life cycle in a cell. Organelles are specialized membrane bound structures within a cell that function by interacting with cytosolic and luminal soluble proteins making the protein composition of each organelle dynamic. Depending on organism, the total number of organelles within a cell varies, indicating their evolution with respect to protein number and function. For example, one of the striking differences between plant and animal cells is the plastids in plants. Organelles have their own proteins, and few organelles like mitochondria and chloroplast have their own genome to synthesize proteins for specific function and also require nuclear-encoded proteins. Enormous work has been performed on animal organelle proteomics. However, plant organelle proteomics has seen limited work mainly due to: (i) inter-plant and inter-tissue complexity, (ii) difficulties in isolation of subcellular compartments, and (iii) their enrichment and purity. Despite these concerns, the field of organelle proteomics is growing in plants, such as Arabidopsis, rice and maize. The available data are beginning to help better understand organelles and their distinct and/or overlapping functions in different plant tissues, organs or cell types, and more importantly, how protein components of organelles behave during development and with surrounding environments. Studies on organelles have provided a few good reviews, but none of them are comprehensive. Here, we present a comprehensive review on plant organelle proteomics starting from the significance of organelle in cells, to organelle isolation, to protein identification and to biology and beyond. To put together such a systematic, in-depth review and to translate acquired knowledge in a proper and adequate form, we join minds to provide discussion and viewpoints on the collaborative nature of organelles in

  4. Analysis of the soluble cell wall proteome of gymnosperms.

    PubMed

    Uzal, Esther Novo; Gómez-Ros, Laura V; Hernández, Jose A; Pedreño, María A; Cuello, Juan; Ros Barceló, Alfonso

    2009-05-15

    We analyzed the cell wall proteome of lignifying suspension cell cultures (SCCs) from four gymnosperms that differ in evolution degree. This analysis showed the presence of "peptide sequence tags" (PSTs) corresponding to glucan endo-1,3-beta-D-glucosidase, xyloglucan-endotrans-glucosylase/hydrolase, chitinases, thaumatin-like proteins and proteins involved in lignin/lignan biosynthesis, such as dirigent-like proteins and peroxidases. Surprisingly, and given the abundance of peroxidases in the cell wall proteome of these gymnosperms, PSTs corresponding to peroxidases were only detected in tryptic fragments of the cell wall proteome of Cycas revoluta. The current lack of knowledge regarding C. revoluta peroxidases led us to purify, characterize and partially sequence the peroxidases responsible for lignin biosynthesis in this species. This yielded three peroxidase-enriched fractions: CrPrx 1, CrPrx 2 and CrPrx 3. Analyses of tryptic peptides of CrPrx 2 (32kDa) and CrPrx 3 (26kDa) suggest that CrPrx 3 arises from CrPrx 2 by protein truncation, and that CrPrx 3 apparently constitutes a post-translational modification of CrPrx 2. That CrPrx 2 and CrPrx 3 are apparently the same enzyme was also deduced from the similarity between the k(cat) shown by both peroxidases for the three monolignols. These results emphasize the analogies between the cell wall proteome of gymnosperms and angiosperms, the complexity of the peroxidase proteome, and the difficulties involved in establishing fine structure-function relationships.

  5. Plant cell organelle proteomics in response to abiotic stress.

    PubMed

    Hossain, Zahed; Nouri, Mohammad-Zaman; Komatsu, Setsuko

    2012-01-01

    Proteomics is one of the finest molecular techniques extensively being used for the study of protein profiling of a given plant species experiencing stressed conditions. Plants respond to a stress by alteration in the pattern of protein expression, either by up-regulating of the existing protein pool or by the synthesizing novel proteins primarily associated with plants antioxidative defense mechanism. Improved protein extraction protocols and advance techniques for identification of novel proteins have been standardized in different plant species at both cellular and whole plant level for better understanding of abiotic stress sensing and intracellular stress signal transduction mechanisms. In contrast, an in-depth proteome study of subcellular organelles could generate much detail information about the intrinsic mechanism of stress response as it correlates the possible relationship between the protein abundance and plant stress tolerance. Although a wealth of reviews devoted to plant proteomics are available, review articles dedicated to plant cell organelle proteins response under abiotic stress are very scanty. In the present review, an attempt has been made to summarize all significant contributions related to abiotic stresses and their impacts on organelle proteomes for better understanding of plants abiotic stress tolerance mechanism at protein level. This review will not only provide new insights into the plants stress response mechanisms, which are necessary for future development of genetically engineered stress tolerant crop plants for the benefit of humankind, but will also highlight the importance of studying changes in protein abundance within the cell organelles in response to abiotic stress.

  6. Proteomic techniques for characterisation of mesenchymal stem cell secretome.

    PubMed

    Kupcova Skalnikova, Helena

    2013-12-01

    Mesenchymal stem cells (MSCs) are multipotent cells with a substantial potential in human regenerative medicine due to their ability to migrate to sites of injury, capability to suppress immune response and accessibility in large amount from patient's own bone marrow or fat tissue. It has been increasingly observed that the transplanted MSCs did not necessarily engraft and differentiate at the site of injury but might exert their therapeutic effects through secreted trophic signals. The MSCs secrete a variety of autocrine/paracrine factors, called secretome, that support regenerative processes in the damaged tissue, induce angiogenesis, protect cells from apoptotic cell death and modulate immune system. The cell culture medium conditioned by MSCs or osteogenic, chondrogenic as well as adipogenic precursors derived from MSCs has become a subject of intensive proteomic profiling in the search for and identification of released factors and microvesicles that might be applicable in regenerative medicine. Jointly with the methods for MSC isolation, expansion and differentiation, proteomic analysis of MSC secretome was enabled recently mainly due to the extensive development in protein separation techniques, mass spectrometry, immunological methods and bioinformatics. This review describes proteomic techniques currently applied or prospectively applicable in MSC secretomics, with a particular focus on preparation of the secretome sample, protein/peptide separation, mass spectrometry and protein quantification techniques, analysis of posttranslational modifications, immunological techniques, isolation and characterisation of secreted vesicles and exosomes, analysis of cytokine-encoding mRNAs and bioinformatics. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  7. Proteome and phosphoproteome of primary cultured pig urothelial cells.

    PubMed

    Verma, Nisha; Bäuerlein, Carolin; Pink, Mario; Rettenmeier, Albert W; Schmitz-Spanke, Simone

    2011-12-01

    Epithelial tissue lining the inner side of the urinary bladder is the most common target for bladder cancer-related diseases. Bladders of freshly slaughtered pigs were utilised for a comprehensive analysis of the proteome and phosphoproteome of bladder epithelial cells. Following protein separation by 2-D gel electrophoresis and identification by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) the first proteome and phosphoproteome maps of pig urinary bladder epithelial cells (PUBEC) were established. A total of 120 selected protein spots were identified. By using the La(3+) enrichment method further developed in our laboratory we identified 31 phosphoproteins with minimal contamination by non-phosphopeptides. The 2-DE map of pig urothelial cells may prove as a useful tool for studies on uroepithelial biology, and the analysed phosphoproteins expression pattern, together with the whole cell proteome, will be helpful for identifying the proteins involved in bladder-related diseases. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Quantitative Analysis of Human Red Blood Cell Proteome.

    PubMed

    Bryk, Agata H; Wiśniewski, Jacek R

    2017-08-04

    Red blood cells (RBCs) are the most abundant cell type in the human body. RBCs and, in particular, their plasma membrane composition have been extensively studied for many years. During the past decade proteomics studies have extended our knowledge on RBC composition; however, these studies did not provide quantitative insights. Here we report a large-scale proteomics investigation of RBCs and their "white ghost" membrane fraction. Samples were processed using the multienzyme digestion filter-aided sample preparation (MED-FASP) and analyzed using Q-Exactive mass spectrometer. Protein abundances were computed using the total protein approach (TPA). The validation of the data with stable isotope-labeled peptide-based protein quantification followed. Our in-depth analysis resulted in the identification of 2650 proteins, of which 1890 occurred at more than 100 copies per cell. We quantified 41 membrane transporter proteins spanning an abundance range of five orders of magnitude. Some of these, including the drug transporter ABCA7 and choline transporters SLC44A1 and SLC44A2, have not previously been identified in RBC membranes. Comparison of protein copy numbers assessed by proteomics showed a good correlation with literature data; however, abundances of several proteins were not consistent with the classical references. Because we validated our findings by a targeted analysis using labeled standards, our data suggest that some older reference data from a variety of biochemical approaches are inaccurate. Our study provides the first "in-depth" quantitative analysis of the RBC proteome and will promote future studies of erythrocyte structure, functions, and disease.

  9. Proteome analysis of human embryonic stem cells organelles.

    PubMed

    Shekari, Faezeh; Nezari, Hossein; Larijani, Mehran Rezaei; Han, Chia-Li; Baharvand, Hossein; Chen, Yu-Ju; Salekdeh, Ghasem Hosseini

    2017-06-06

    As the functions of proteins are associated with their cellular localization, the comprehensive sub-cellular proteome knowledge of human embryonic stem cells (hESCs) is indispensable for ensuring a therapeutic effect. Here, we have utilized a sub-cellular proteomics approach to analyze the localization of proteins in the nucleus, mitochondria, crude membrane, cytoplasm, heavy and light microsomes. Out of 2002 reproducibly identified proteins, we detected 762 proteins in a single organelle whereas 160 proteins were found in all sub-cellular fractions. We verified the localization of identified proteins through databases and discussed the consistency of the obtained results. With regards to the ambiguity in the definition of a membrane protein, we tried to clearly define the plasma membrane, peripheral membrane and membrane proteins by annotation of these proteins in databases, along with predictions of transmembrane helices. Among ten enriched signaling pathways highlighted in our results, non-canonical Wnt signaling were analyzed in greater detail. The functions of three novel hESC membrane proteins (ERBB4, GGT1 and ZDHHC13) have been assessed in terms of pluripotency. Our report is the most comprehensive for organellar proteomics of hESCs. Mass spectrometric identification of proteins using a TripleTOF 5600 from nucleus, mitochondria, crude membrane, cytoplasm, heavy and light microsomal fractions highlighted the significance of the non-canonical Wnt signaling in human embryonic stem cells. Copyright © 2017. Published by Elsevier B.V.

  10. Mapping the Spatial Proteome of Metastatic Cells in Colorectal Cancer.

    PubMed

    Mendes, Marta; Peláez-García, Alberto; López-Lucendo, María; Bartolomé, Rubén A; Calviño, Eva; Barderas, Rodrigo; Casal, J Ignacio

    2017-10-01

    Colorectal cancer (CRC) is the second deadliest cancer worldwide. Here, we aimed to study metastasis mechanisms using spatial proteomics in the KM12 cell model. Cells were SILAC-labeled and fractionated into five subcellular fractions corresponding to: cytoplasm, plasma, mitochondria and ER/golgi membranes, nuclear, chromatin-bound and cytoskeletal proteins and analyzed with high resolution mass spectrometry. We provide localization data of 4863 quantified proteins in the different subcellular fractions. A total of 1318 proteins with at least 1.5-fold change were deregulated in highly metastatic KM12SM cells respect to KM12C cells. The protein network organization, protein complexes and functional pathways associated to CRC metastasis was revealed with spatial resolution. Although 92% of the differentially expressed proteins showed the same deregulation in all subcellular compartments, a subset of 117 proteins (8%) showed opposite changes in different subcellular localizations. The chaperonin CCT, the Eif2 and Eif3 initiation of translation and the oxidative phosphorylation complexes together with an important number of guanine nucleotide-binding proteins, were deregulated in abundance and localization within the metastatic cells. Particularly relevant was the relationship of deregulated protein complexes with exosome secretion. The knowledge of the spatial proteome alterations at subcellular level contributes to clarify the molecular mechanisms underlying colorectal cancer metastasis and to identify potential targets of therapeutic intervention. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Cell wall proteome analysis of Arabidopsis thaliana mature stems.

    PubMed

    Duruflé, Harold; Clemente, Hélène San; Balliau, Thierry; Zivy, Michel; Dunand, Christophe; Jamet, Elisabeth

    2017-04-01

    Plant stems carry flowers necessary for species propagation and need to be adapted to mechanical disturbance and environmental factors. The stem cell walls are different from other organs and can modify their rigidity or viscoelastic properties for the integrity and the robustness required to withstand mechanical impacts and environmental stresses. Plant cell wall is composed of complex polysaccharide networks also containing cell wall proteins (CWPs) crucial to perceive and limit the environmental effects. The CWPs are fundamental players in cell wall remodeling processes, and today, only 86 have been identified from the mature stems of the model plant Arabidopsis thaliana. With a destructive method, this study has enlarged its coverage to 302 CWPs. This new proteome is mainly composed of 27.5% proteins acting on polysaccharides, 16% proteases, 11.6% oxido-reductases, 11% possibly related to lipid metabolism and 11% of proteins with interacting domains with proteins or polysaccharides. Compared to stem cell wall proteomes already available (Brachypodium distachyon, Sacharum officinarum, Linum usitatissimum, Medicago sativa), that of A. thaliana stems has a higher proportion of proteins acting on polysaccharides and of proteases, but a lower proportion of oxido-reductases. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Potential Salivary Proteomic Markers of Oral Squamous Cell Carcinoma.

    PubMed

    Gallo, Crescenzio; Ciavarella, Domenico; Santarelli, Andrea; Ranieri, Elena; Colella, Giuseppe; Lo Muzio, Lorenzo; Lo Russo, Lucio

    2016-01-01

    The aim of the present study was to investigate the diagnostic and prognostic potential of proteomic signatures in saliva of patients with oral squamous cell carcinoma (OSCC). Data from SELDI-TOF mass spectrometry of saliva from 45 OSCC patients and 30 healthy controls were analyzed by means of univariate and multivariate statistical approaches, in order to identify proteomic OSCC signatures, reduce dimensionality and build models for discriminating between OSCC and controls, as well as predict nodal status. The saliva proteome presents significant modifications in OSCC patients; some of them seem to be related to nodal involvement, and may be useful for knowledge advancement regarding oral carcinogenesis and definition of diagnostic and prognostic biomarkers. Our attempt to create a predictive model using different artificial neural networks (i.e. feed-forward (FF), radial basis function (RBF), vector quantization (VQ)) demonstrated that such biostatistical tools are powerful but not all network architectures have similar performance. RBF architecture showed the best diagnostic performance (91.89%), whereas FF had the best (77.27%) prognostic accuracy (distinguishing between N(-) and N(+)). Searching for potential biomarkers among differently expressed peptides is a challenge requiring for appropriate strategies that still remain to be defined. A number of factors may potentially impair results, e.g.: (i) a group's definition for adequate comparison; (ii) reduction of data dimensionality and selection of variables to be tested in predictive models; (iii) selection of the biostatistical tool for predictive models. Copyright© 2016, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.

  13. Role of Proteome Physical Chemistry in Cell Behavior.

    PubMed

    Ghosh, Kingshuk; de Graff, Adam M R; Sawle, Lucas; Dill, Ken A

    2016-09-15

    We review how major cell behaviors, such as bacterial growth laws, are derived from the physical chemistry of the cell's proteins. On one hand, cell actions depend on the individual biological functionalities of their many genes and proteins. On the other hand, the common physics among proteins can be as important as the unique biology that distinguishes them. For example, bacterial growth rates depend strongly on temperature. This dependence can be explained by the folding stabilities across a cell's proteome. Such modeling explains how thermophilic and mesophilic organisms differ, and how oxidative damage of highly charged proteins can lead to unfolding and aggregation in aging cells. Cells have characteristic time scales. For example, E. coli can duplicate as fast as 2-3 times per hour. These time scales can be explained by protein dynamics (the rates of synthesis and degradation, folding, and diffusional transport). It rationalizes how bacterial growth is slowed down by added salt. In the same way that the behaviors of inanimate materials can be expressed in terms of the statistical distributions of atoms and molecules, some cell behaviors can be expressed in terms of distributions of protein properties, giving insights into the microscopic basis of growth laws in simple cells.

  14. Advances in the proteomic investigation of the cell secretome

    PubMed Central

    Brown, Kristy J; Formolo, Catherine A; Seol, Haeri; Marathi, Ramya L; Duguez, Stephanie; An, Eunkyung; Pillai, Dinesh; Nazarian, Javad; Rood, Brian R; Hathout, Yetrib

    2012-01-01

    Studies of the cell secretome have greatly increased in recent years owing to improvements in proteomic platforms, mass spectrometry instrumentation and to the increased interaction between analytical chemists, biologists and clinicians. Several secretome studies have been implemented in different areas of research, leading to the generation of a valuable secretome catalogs. Secreted proteins continue to be an important source of biomarkers and therapeutic target discovery and are equally valuable in the field of microbiology. Several discoveries have been achieved in vitro using cell culture systems, ex vivo using human tissue specimens and in vivo using animal models. In this review, some of the most recent advances in secretome studies and the fields that have benefited the most from this evolving technology are highlighted. PMID:22809211

  15. Optimization and Modeling of Quadrupole Orbitrap Parameters for Sensitive Analysis toward Single-Cell Proteomics.

    PubMed

    Sun, Bingyun; Kovatch, Jessica Rae; Badiong, Albert; Merbouh, Nabyl

    2017-10-06

    Single-cell proteomics represents a field of extremely sensitive proteomic analysis, owing to the minute amount of yet complex proteins in a single cell. Without amplification potential as of nucleic acids, single-cell mass spectrometry (MS) analysis demands special instrumentation running with optimized parameters to maximize the sensitivity and throughput for comprehensive proteomic discovery. To facilitate such analysis, we here investigated two factors critical to peptide sequencing and protein detection in shotgun proteomics, i.e. precursor ion isolation window (IW) and maximum precursor ion injection time (ITmax), on an ultrahigh-field quadrupole Orbitrap (Q-Exactive HF). Counterintuitive to the frequently used proteomic parameters for bulk samples (>100 ng), our experimental data and subsequent modeling suggested a universally optimal IW of 4.0 Th for sample quantity ranging from 100 ng to 1 ng, and a sample-quantity dependent ITmax of more than 250 ms for 1-ng samples. Compared with the benchmark condition of IW = 2.0 Th and ITmax = 50 ms, our optimization generated up to 300% increase to the detected protein groups for 1-ng samples. The additionally identified proteins allowed deeper penetration of proteome for better revealing crucial cellular functions such as signaling and cell adhesion. We hope this effort can prompt single-cell and trace proteomic analysis and enable a rational selection of MS parameters.

  16. Changes in morphology, cell wall composition and soluble proteome in Rhodobacter sphaeroides cells exposed to chromate.

    PubMed

    Italiano, Francesca; Rinalducci, Sara; Agostiano, Angela; Zolla, Lello; De Leo, Francesca; Ceci, Luigi R; Trotta, Massimo

    2012-10-01

    The response of the carotenoidless Rhodobacter sphaeroides mutant R26 to chromate stress under photosynthetic conditions is investigated by biochemical and spectroscopic measurements, proteomic analysis and cell imaging. Cell cultures were found able to reduce chromate within 3-4 days. Chromate induces marked changes in the cellular dimension and morphology, as revealed by atomic force microscopy, along with compositional changes in the cell wall revealed by infrared spectroscopy. These effects are accompanied by significant changes in the level of several proteins: 15 proteins were found up-regulated and 15 down-regulated. The protein content found in chromate exposed cells is in good agreement with the biochemical, spectroscopic and microscopic results. Moreover at the present stage no specific chromate-reductase could be found in the soluble proteome, indicating that detoxification of the pollutant proceeds via aspecific reductants.

  17. Proteomic analysis of serum deprivation in tongue squamous cell carcinoma

    PubMed Central

    Zhang, Junfeng; Dong, Wei; Meng, Yufen; Jiang, Miao; Zhan, Zhen

    2017-01-01

    The occurrence of tongue squamous cell carcinoma (TSCC) is closely correlated with serum components; however, the detailed mechanism remains to be fully elucidated. Proteomic analysis contributed to the discovery of potential biomarkers and provided an insight into TSCC at a molecular level. The present study investigated the effect of serum deprivation on the Tca-8113 TSCC cell line through protein profiling using two-dimensional gel electrophoresis and mass spectrometry, with the aim of improving TSCC diagnosis. The results showed that the Tca-8113 cells maintained proliferative capacity and resisted apoptosis following serum deprivation. A total of 43 proteins were upregulated and 45 were downregulated following serum deprivation for 24 h, compared with untreated controls (0 h). The upregulated caspase-7, heat shock protein 27 and Annexin A1, and the downregulated peroxiredoxin-6 and heat shock protein 70, were selected for verification using reverse transcription-polymerase chain reaction analysis following serum deprivation for 16 h. The results indicated that reactive oxygen species may be important in serum deprivation-induced oxidative stress. PMID:29039553

  18. Human borna disease virus infection impacts host proteome and histone lysine acetylation in human oligodendroglia cells

    SciTech Connect

    Liu, Xia; Department of Neurology, The Fifth People's Hospital of Shanghai, School of Medicine, Fudan University, Shanghai, 200240; Zhao, Libo

    2014-09-15

    Background: Borna disease virus (BDV) replicates in the nucleus and establishes persistent infections in mammalian hosts. A human BDV strain was used to address the first time, how BDV infection impacts the proteome and histone lysine acetylation (Kac) of human oligodendroglial (OL) cells, thus allowing a better understanding of infection-driven pathophysiology in vitro. Methods: Proteome and histone lysine acetylation were profiled through stable isotope labeling for cell culture (SILAC)-based quantitative proteomics. The quantifiable proteome was annotated using bioinformatics. Histone acetylation changes were validated by biochemistry assays. Results: Post BDV infection, 4383 quantifiable differential proteins were identified and functionally annotated tomore » metabolism pathways, immune response, DNA replication, DNA repair, and transcriptional regulation. Sixteen of the thirty identified Kac sites in core histones presented altered acetylation levels post infection. Conclusions: BDV infection using a human strain impacted the whole proteome and histone lysine acetylation in OL cells. - Highlights: • A human strain of BDV (BDV Hu-H1) was used to infect human oligodendroglial cells (OL cells). • This study is the first to reveal the host proteomic and histone Kac profiles in BDV-infected OL cells. • BDV infection affected the expression of many transcription factors and several HATs and HDACs.« less

  19. Proteomic analysis of osteogenic differentiation of dental follicle precursor cells.

    PubMed

    Morsczeck, Christian; Petersen, Jørgen; Völlner, Florian; Driemel, Oliver; Reichert, Torsten; Beck, Hans Christian

    2009-04-01

    Recently, there has been an increased interest in unravelling the molecular mechanisms and cellular pathways controlling the differentiation and proliferation of human stem cell lines. Proteome analysis has proven to be an effective approach to comprehensive analysis of the regulatory network of differentiation. In the present study we applied 2-DE combined with capillary-LC-MS/MS analysis to profile differentially regulated proteins upon differentiation of dental follicle precursor cells (DFPCs). Out of 115 differentially regulated proteins, glutamine synthetase, lysosomal proteinase cathepsin B proteins, plastin 3 T-isoform, beta-actin, superoxide dismutases, and transgelin were found to be highly up-regulated, whereas cofilin-1, pro-alpha 1 collagen, destrin, prolyl 4-hydrolase and dihydrolipoamide dehydrogenase were found to be highly down-regulated. The group of up-regulated proteins is associated with actin-bundling and defence against oxidative cellular stress, whereas down-regulated proteins were associated with collagen biosynthesis. Bioinformatic analyses of the entire data set confirmed these findings that represent significant steps towards the understanding of DFPC differentiation. The bioinformatic analyses suggest that proteins associated with cell cycle progression and protein metabolism were down-regulated and proteins involved in catabolism, cell motility and biological quality were up-regulated. These results display the general physiological state of DFPCs before and after osteogenic differentiation. We also identified regulatory proteins, such as the transcription factors TP53 and Sp-1, associated with the differentiation process. Further studies will investigate the impact of identified regulatory proteins for cell proliferation and osteogenic differentiation in DFPCs.

  20. Proteomic profiling of acrolein adducts in human lung epithelial cells

    PubMed Central

    Spiess, Page C.; Deng, Bin; Hondal, Robert J.; Matthews, Dwight E.; van der Vliet, Albert

    2011-01-01

    Acrolein (2,3-propenal) is a major indoor and outdoor air pollutant originating largely from tobacco smoke or organic combustion. Given its high reactivity, the adverse effects of inhaled acrolein are likely due to direct interactions with the airway epithelium, resulting in altered epithelial function, but only limited information exists to date regarding the primary direct cellular targets for acrolein. Here, we describe a global proteomics approach to characterize the spectrum of airway epithelial protein targets for Michael adduction in acrolein-exposed bronchial epithelial (HBE1) cells, based on biotin hydrazide labeling and avidin purification of biotinylated proteins or peptides for analysis by LC-MS/MS. Identified protein targets included a number of stress proteins, cytoskeletal proteins, and several key proteins involved in redox signaling, including thioredoxin reductase, thioredoxin, peroxiredoxins, and glutathione S-transferase π. Because of the central role of thioredoxin reductase in cellular redox regulation, additional LC-MS/MS characterization was performed on purified mitochondrial thioredoxin reductase to identify the specific site of acrolein adduction, revealing the catalytic selenocysteine residue as the target responsible for enzyme inactivation. Our findings indicate that these approaches are useful in characterizing major protein targets for acrolein, and will enhance mechanistic understanding of the impact of acrolein on cell biology. PMID:21704744

  1. Investigation of the Cell Surface Proteome of Human Periodontal Ligament Stem Cells.

    PubMed

    Xiong, Jimin; Menicanin, Danijela; Zilm, Peter S; Marino, Victor; Bartold, P Mark; Gronthos, Stan

    2016-01-01

    The present study examined the cell surface proteome of human periodontal ligament stem cells (PDLSC) compared to human fibroblasts. Cell surface proteins were prelabelled with CyDye before processing to extract the membrane lysates, which were separated using 2D electrophoresis. Selected differentially expressed protein "spots" were identified using Mass spectrometry. Four proteins were selected for validation: CD73, CD90, Annexin A2, and sphingosine kinase 1 previously associated with mesenchymal stem cells. Flow cytometric analysis found that CD73 and CD90 were highly expressed by human PDLSC and gingival fibroblasts but not by keratinocytes, indicating that these antigens could be used as potential markers for distinguishing between mesenchymal cells and epithelial cell populations. Annexin A2 was also found to be expressed at low copy number on the cell surface of human PDLSC and gingival fibroblasts, while human keratinocytes lacked any cell surface expression of Annexin A2. In contrast, sphingosine kinase 1 expression was detected in all the cell types examined using immunocytochemical analysis. These proteomic studies form the foundation to further define the cell surface protein expression profile of PDLSC in order to better characterise this cell population and help develop novel strategies for the purification of this stem cell population.

  2. The cell wall and secretory proteome of a tobacco cell line synthesising secondary wall.

    PubMed

    Millar, David J; Whitelegge, Julian P; Bindschedler, Laurence V; Rayon, Catherine; Boudet, Alain-Michel; Rossignol, Michel; Borderies, Gisèle; Bolwell, G Paul

    2009-05-01

    The utility of plant secondary cell wall biomass for industrial and biofuel purposes depends upon improving cellulose amount, availability and extractability. The possibility of engineering such biomass requires much more knowledge of the genes and proteins involved in the synthesis, modification and assembly of cellulose, lignin and xylans. Proteomic data are essential to aid gene annotation and understanding of polymer biosynthesis. Comparative proteomes were determined for secondary walls of stem xylem and transgenic xylogenic cells of tobacco and detected peroxidase, cellulase, chitinase, pectinesterase and a number of defence/cell death related proteins, but not marker proteins of primary walls such as xyloglucan endotransglycosidase and expansins. Only the corresponding detergent soluble proteome of secretory microsomes from the xylogenic cultured cells, subjected to ion-exchange chromatography, could be determined accurately since, xylem-specific membrane yields were of poor quality from stem tissue. Among the 109 proteins analysed, many of the protein markers of the ER such as BiP, HSP70, calreticulin and calnexin were identified, together with some of the biosynthetic enzymes and associated polypeptides involved in polymer synthesis. However 53% of these endomembrane proteins failed identification despite the use of two different MS methods, leaving considerable possibilities for future identification of novel proteins involved in secondary wall polymer synthesis once full genomic data are available.

  3. Trypsin-induced proteome alteration during cell subculture in mammalian cells.

    PubMed

    Huang, Hsiang-Ling; Hsing, Hsiang-Wei; Lai, Tzu-Chia; Chen, Yi-Wen; Lee, Tian-Ren; Chan, Hsin-Tsu; Lyu, Ping-Chiang; Wu, Chieh-Lin; Lu, Ying-Chieh; Lin, Szu-Ting; Lin, Cheng-Wen; Lai, Chih-Ho; Chang, Hao-Teng; Chou, Hsiu-Chuan; Chan, Hong-Lin

    2010-05-11

    It is essential to subculture the cells once cultured cells reach confluence. For this, trypsin is frequently applied to dissociate adhesive cells from the substratum. However, due to the proteolytic activity of trypsin, cell surface proteins are often cleaved, which leads to dysregulation of the cell functions. In this study, a triplicate 2D-DIGE strategy has been performed to monitor trypsin-induced proteome alterations. The differentially expressed spots were identified by MALDI-TOF MS and validated by immunoblotting. 36 proteins are found to be differentially expressed in cells treated with trypsin, and proteins that are known to regulate cell metabolism, growth regulation, mitochondrial electron transportation and cell adhesion are down-regulated and proteins that regulate cell apoptosis are up-regulated after trypsin treatment. Further study shows that bcl-2 is down-regulated, p53 and p21 are both up-regulated after trypsinization. In summary, this is the first report that uses the proteomic approach to thoroughly study trypsin-induced cell physiological changes and provides researchers in carrying out their experimental design.

  4. Developing Top Down Proteomics to Maximize Proteome and Sequence Coverage from Cells and Tissues

    PubMed Central

    Ahlf, Dorothy R.; Thomas, Paul M.; Kelleher, Neil L.

    2013-01-01

    Mass spectrometry based proteomics generally seeks to identify and characterize protein molecules with high accuracy and throughput. Recent speed and quality improvements to the independent steps of integrated platforms have removed many limitations to the robust implementation of top down proteomics (TDP) for proteins below 70 kDa. Improved intact protein separations coupled to high-performance instruments have increased the quality and number of protein and proteoform identifications. To date, TDP applications have shown >1,000 protein identifications, expanding to an average of ~3–4 more proteoforms for each protein detected. In the near future, increased fractionation power, new mass spectrometers and improvements in proteoform scoring will combine to accelerate the application and impact of TDP to this century’s biomedical problems. PMID:23988518

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

    PubMed Central

    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. PMID:27022256

  6. Microchip platforms for multiplex single-cell functional proteomics with applications to immunology and cancer research

    PubMed Central

    2013-01-01

    Single-cell functional proteomics assays can connect genomic information to biological function through quantitative and multiplex protein measurements. Tools for single-cell proteomics have developed rapidly over the past 5 years and are providing approaches for directly elucidating phosphoprotein signaling networks in cancer cells or for capturing high-resolution snapshots of immune system function in patients with various disease conditions. We discuss advances in single-cell proteomics platforms, with an emphasis on microchip methods. These methods can provide a direct correlation of morphological, functional and molecular signatures at the single-cell level. We also provide examples of how those platforms are being applied to both fundamental biology and clinical studies, focusing on immune-system monitoring and phosphoprotein signaling networks in cancer. PMID:23998271

  7. Social network architecture of human immune cells unveiled by quantitative proteomics.

    PubMed

    Rieckmann, Jan C; Geiger, Roger; Hornburg, Daniel; Wolf, Tobias; Kveler, Ksenya; Jarrossay, David; Sallusto, Federica; Shen-Orr, Shai S; Lanzavecchia, Antonio; Mann, Matthias; Meissner, Felix

    2017-05-01

    The immune system is unique in its dynamic interplay between numerous cell types. However, a system-wide view of how immune cells communicate to protect against disease has not yet been established. We applied high-resolution mass-spectrometry-based proteomics to characterize 28 primary human hematopoietic cell populations in steady and activated states at a depth of >10,000 proteins in total. Protein copy numbers revealed a specialization of immune cells for ligand and receptor expression, thereby connecting distinct immune functions. By integrating total and secreted proteomes, we discovered fundamental intercellular communication structures and previously unknown connections between cell types. Our publicly accessible (http://www.immprot.org/) proteomic resource provides a framework for the orchestration of cellular interplay and a reference for altered communication associated with pathology.

  8. AMP-Activated Protein Kinase Regulates the Cell Surface Proteome and Integrin Membrane Traffic

    PubMed Central

    Thavarajah, Thanusi; Medvedev, Sergei; Bowden, Peter; Marshall, John G.; Antonescu, Costin N.

    2015-01-01

    The cell surface proteome controls numerous cellular functions including cell migration and adhesion, intercellular communication and nutrient uptake. Cell surface proteins are controlled by acute changes in protein abundance at the plasma membrane through regulation of endocytosis and recycling (endomembrane traffic). Many cellular signals regulate endomembrane traffic, including metabolic signaling; however, the extent to which the cell surface proteome is controlled by acute regulation of endomembrane traffic under various conditions remains incompletely understood. AMP-activated protein kinase (AMPK) is a key metabolic sensor that is activated upon reduced cellular energy availability. AMPK activation alters the endomembrane traffic of a few specific proteins, as part of an adaptive response to increase energy intake and reduce energy expenditure. How increased AMPK activity during energy stress may globally regulate the cell surface proteome is not well understood. To study how AMPK may regulate the cell surface proteome, we used cell-impermeable biotinylation to selectively purify cell surface proteins under various conditions. Using ESI-MS/MS, we found that acute (90 min) treatment with the AMPK activator A-769662 elicits broad control of the cell surface abundance of diverse proteins. In particular, A-769662 treatment depleted from the cell surface proteins with functions in cell migration and adhesion. To complement our mass spectrometry results, we used other methods to show that A-769662 treatment results in impaired cell migration. Further, A-769662 treatment reduced the cell surface abundance of β1-integrin, a key cell migration protein, and AMPK gene silencing prevented this effect. While the control of the cell surface abundance of various proteins by A-769662 treatment was broad, it was also selective, as this treatment did not change the cell surface abundance of the transferrin receptor. Hence, the cell surface proteome is subject to acute

  9. ATP-Sensitive K+ Channel-Deficient Dilated Cardiomyopathy Proteome Remodeled by Embryonic Stem Cell Therapy

    PubMed Central

    Zlatkovic-Lindor, Jelena; Arrell, D. Kent; Yamada, Satsuki; Nelson, Timothy J.; Terzic, Andre

    2011-01-01

    Transplantation of pluripotent stem cells has proven beneficial in heart failure, yet the proteomic landscape underlying repair remains largely uncharacterized. In a genetic model of dilated cardiomyopathy elicited by pressure overload in the KCNJ11 (potassium inwardly rectifying channel, subfamily J, member 11) null mutant, proteome-wide profiles were here resolved by means of a systems approach prior to and following disease manifestation in the absence or presence of embryonic stem cell treatment. Comparative two-dimensional gel electrophoresis revealed a unique cardiomyopathic proteome in the absence of therapy, remodeled in response to stem cell treatment. Specifically, linear ion trap quadrupole-Orbitrap mass spectrometry determined the identities of 93 and 109 differentially expressed proteins from treated and untreated cardiomyopathic hearts, respectively. Mapped protein– protein relationships and corresponding neighborhoods incorporated the stem cell-dependent subproteome into a nonstochastic network with divergent composition from the stem cell-independent counterpart. Stem cell intervention produced a distinct proteome signature across a spectrum of biological processes ranging from energetic metabolism, oxidoreductases, and stress-related chaper-ones to processes supporting protein synthesis/degradation, signaling, and transport regulation, cell structure and scaffolding. In the absence of treatment, bioinformatic interrogation of the disease-only proteome network prioritized adverse cardiac outcomes, ablated or ameliorated following stem cell transplantation. Functional and structural measurements validated improved myocardial contractile performance, reduced ventricular size and decreased cardiac damage in the treated cohort. Unbiased systems assessment unmasked “cardiovascular development” as a prioritized biological function in stem cell-reconstructed cardiomyopathic hearts. Thus, embryonic stem cell treatment transformed the cardiomyopathic

  10. MicroRNA Profiling of the Effect of the Heptapeptide Angiotensin-(1-7) in A549 Lung Tumor Cells Reveals a Role for miRNA149-3p in Cellular Migration Processes.

    PubMed

    Silva, Brenda de Oliveira da; Lima, Kelvin Furtado; Gonçalves, Letícia Rocha; Silveira, Marina Bonfogo da; Moraes, Karen C M

    2016-01-01

    Lung cancer is one of the most frequent types of cancer in humans and a leading cause of death worldwide. The high mortality rates are correlated with late diagnosis, which leads to high rates of metastasis found in patients. Thus, despite all the improvement in therapeutic approaches, the development of new drugs that control cancer cell migration and metastasis are required. The heptapeptide angiotensin-(1-7) [ang-(1-7)] has demonstrated the ability to control the growth rates of human lung cancer cells in vitro and in vivo, and the elucidation of central elements that control the fine-tuning of cancer cells migration in the presence of the ang-(1-7), will support the development of new therapeutic approaches. Ang-(1-7) is a peptide hormone of the renin-angiotensin system (RAS) and this study investigates the modulatory effect of the heptapeptide on the expression pattern of microRNAs (miRNAs) in lung tumor cells, to elucidate mechanistic concerns about the effect of the peptide in the control of tumor migratory processes. Our primary aim was to compare the miRNA profiling between treated and untreated-heptapeptide cells to characterize the relevant molecule that modulates cellular migration rates. The analyses selected twenty one miRNAs, which are differentially expressed between the groups; however, statistical analyses indicated miRNA-149-3p as a relevant molecule. Once functional analyses were performed, we demonstrated that miRNA-149-3p plays a role in the cellular migration processes. This information could be useful for future investigations on drug development.

  11. Green fluorescent protein expression triggers proteome changes in breast cancer cells.

    PubMed

    Coumans, J V F; Gau, D; Poljak, A; Wasinger, V; Roy, P; Moens, P

    2014-01-01

    Green fluorescent protein (GFP) is the most commonly used reporter of expression in cell biology despite evidence that it affects the cell physiology. The molecular mechanism of GFP-associated modifications has been largely unexplored. In this paper we investigated the proteome modifications following stable expression of GFP in breast cancer cells (MDA-MB-231). A combination of three different proteome analysis methods (2-DE, iTRAQ, label-free) was used to maximise proteome coverage. We found that GFP expression induces changes in expression of proteins that are associated with protein folding, cytoskeletal organisation and cellular immune response. In view of these findings, the use of GFP as a cell reporter should be carefully monitored. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. Plant subcellular proteomics: Application for exploring optimal cell function in soybean.

    PubMed

    Wang, Xin; Komatsu, Setsuko

    2016-06-30

    Plants have evolved complicated responses to developmental changes and stressful environmental conditions. Subcellular proteomics has the potential to elucidate localized cellular responses and investigate communications among subcellular compartments during plant development and in response to biotic and abiotic stresses. Soybean, which is a valuable legume crop rich in protein and vegetable oil, can grow in several climatic zones; however, the growth and yield of soybean are markedly decreased under stresses. To date, numerous proteomic studies have been performed in soybean to examine the specific protein profiles of cell wall, plasma membrane, nucleus, mitochondrion, chloroplast, and endoplasmic reticulum. In this review, methods for the purification and purity assessment of subcellular organelles from soybean are summarized. In addition, the findings from subcellular proteomic analyses of soybean during development and under stresses, particularly flooding stress, are presented and the proteins regulated among subcellular compartments are discussed. Continued advances in subcellular proteomics are expected to greatly contribute to the understanding of the responses and interactions that occur within and among subcellular compartments during development and under stressful environmental conditions. Subcellular proteomics has the potential to investigate the cellular events and interactions among subcellular compartments in response to development and stresses in plants. Soybean could grow in several climatic zones; however, the growth and yield of soybean are markedly decreased under stresses. Numerous proteomics of cell wall, plasma membrane, nucleus, mitochondrion, chloroplast, and endoplasmic reticulum was carried out to investigate the respecting proteins and their functions in soybean during development or under stresses. In this review, methods of subcellular-organelle enrichment and purity assessment are summarized. In addition, previous findings of

  13. High Resolution Proteomic Analysis of the Cervical Cancer Cell Lines Secretome Documents Deregulation of Multiple Proteases.

    PubMed

    Pappa, Kalliopi I; Kontostathi, Georgia; Makridakis, Manousos; Lygirou, Vasiliki; Zoidakis, Jerome; Daskalakis, George; Anagnou, Nicholas P

    2017-01-01

    Oncogenic infection by HPV, eventually leads to cervical carcinogenesis, associated by deregulation of specific pathways and protein expression at the intracellular and secretome level. Thus, secretome analysis can elucidate the biological mechanisms contributing to cervical cancer. In the present study we systematically analyzed its constitution in four cervical cell lines employing a highly sensitive proteomic technology coupled with bioinformatics analysis. LC/MS-MS proteomics and bioinformatics analysis were performed in the secretome of four informative cervical cell lines SiHa (HPV16 + ), HeLa (HPV18 + ), C33A (HPV - ) and HCK1T (normal). The proteomic pattern of each cancer cell line compared to HCK1T was identified and a detailed bioinformatics analysis disclosed inhibition of matrix metalloproteases in cancer cell lines. This prediction was further confirmed via zymography for MMP-2 and MMP-9, western blot analysis for ADAM10 and by MRM for TIMP1. The differential expression of important secreted proteins such as CATD, FUCA1 and SOD2 was also confirmed by western blot analysis. MRM-targeted proteomics analysis confirmed the differential expression of CATD, CATB, SOD2, QPCT and NEU1. High resolution proteomics analysis of cervical cancer secretome revealed significantly deregulated biological processes and proteins implicated in cervical carcinogenesis. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  14. A proteomic chronology of gene expression through the cell cycle in human myeloid leukemia cells

    PubMed Central

    Ly, Tony; Ahmad, Yasmeen; Shlien, Adam; Soroka, Dominique; Mills, Allie; Emanuele, Michael J; Stratton, Michael R; Lamond, Angus I

    2014-01-01

    Technological advances have enabled the analysis of cellular protein and RNA levels with unprecedented depth and sensitivity, allowing for an unbiased re-evaluation of gene regulation during fundamental biological processes. Here, we have chronicled the dynamics of protein and mRNA expression levels across a minimally perturbed cell cycle in human myeloid leukemia cells using centrifugal elutriation combined with mass spectrometry-based proteomics and RNA-Seq, avoiding artificial synchronization procedures. We identify myeloid-specific gene expression and variations in protein abundance, isoform expression and phosphorylation at different cell cycle stages. We dissect the relationship between protein and mRNA levels for both bulk gene expression and for over ∼6000 genes individually across the cell cycle, revealing complex, gene-specific patterns. This data set, one of the deepest surveys to date of gene expression in human cells, is presented in an online, searchable database, the Encyclopedia of Proteome Dynamics (http://www.peptracker.com/epd/). DOI: http://dx.doi.org/10.7554/eLife.01630.001 PMID:24596151

  15. Mitochondrial biogenesis and proteome remodeling promotes one carbon metabolism for T cell activation

    PubMed Central

    Ron-Harel, Noga; Santos, Daniel; Ghergurovich, Jonathan M.; Sage, Peter T.; Reddy, Anita; Lovitch, Scott B.; Dephoure, Noah; Satterstrom, F. Kyle; Sheffer, Michal; Spinelli, Jessica B.; Gygi, Steven; Rabinowitz, Joshua D.; Sharpe, Arlene H.; Haigis, Marcia C.

    2017-01-01

    Summary Naïve T cell stimulation activates anabolic metabolism to fuel the transition from quiescence to growth and proliferation. Here we show that naïve CD4+ T cell activation induces a unique program of mitochondrial biogenesis and remodeling. Using mass spectrometry, we quantified protein dynamics during T cell activation. We identified substantial remodeling of the mitochondrial proteome over the first 24 hr of T cell activation to generate mitochondria with a distinct metabolic signature, with one carbon metabolism as the most induced pathway. Salvage pathways and mitochondrial one carbon metabolism, fed by serine, contribute to purine and thymidine synthesis to enable T cell proliferation and survival. Genetic inhibition of the mitochondrial serine catabolic enzyme SHMT2 impaired T cell survival in culture, and antigen-specific T cell abundance in vivo. Thus, during T cell activation, mitochondrial proteome remodeling generates specialized mitochondria with enhanced one carbon metabolism that is critical for T cell activation and survival. PMID:27411012

  16. How nitrogen sources influence Mortierella alpina aging: From the lipid droplet proteome to the whole-cell proteome and metabolome.

    PubMed

    Yu, Yadong; Zhang, Lei; Li, Tao; Wu, Na; Jiang, Ling; Ji, Xiaojun; Huang, He

    2018-05-15

    Arachidonic acid (ARA) is a valuable polyunsaturated fatty acid produced by Mortierella alpina. Although some strategies such as nitrogen supplementation have shown the potential to affect the aging of M. alpina in ways which enable it to produce more ARA, the underlying mechanism remains elusive. Herein, we conducted a systematical analysis of the lipid droplet proteome, as well as the whole-cell proteome and metabolome, in order to elucidate how and why two different nitrogen sources (KNO 3 and urea) affect the aging of M. alpina and the corresponding ARA concentration. We found that KNO 3 promoted the ARA concentration, while urea accelerated lipid consumption and stimulated the decomposition of mycelia. Although both KNO 3 and urea activated carbohydrate metabolic pathways, KNO 3 exerted a stronger promoting effect on the pentose phosphate pathway and induced the lipid droplets to participate in the citrate-pyruvate cycle. The activities of malic enzyme and isocitrate dehydrogenase were also promoted more by KNO 3 . These pathways provided additional substrates and reducing power for ARA synthesis and ROS elimination. Accordingly, since urea showed a weaker promotion of the related pathways, it caused a depression of the antioxidant system and a consequent increase of ROS. These findings facilitate the design of nitrogen supplementation strategies to achieve higher ARA concentrations, and provide guidance for deciphering the mechanisms of similar aging phenomena in other oleaginous microorganisms. Polyunsaturated fatty acids such as arachidonic acid (ARA) are valuable nutrients, which play important roles in preventing numerous diseases and facilitating development. Although it has been found for years that ARA production will be increased in the aging process of Mortierella alpina (M. alpina) and nitrogen sources are involved in this process, the underlying mechanism for this phenomenon remains unknown. In this work, we used the subcellular proteomics, whole-cell

  17. Current clinical application of genomic and proteomic profiling in non-small-cell lung cancer.

    PubMed

    Tanvetyanon, Tawee; Creelan, Benjamin C; Chiappori, Alberto A

    2014-01-01

    Genomic or proteomic profiling of cancer can be broadly defined as a systematic grouping of cancer based on its genetic or protein makeup. In the management of non-small-cell lung cancer (NSCLC), genomic and proteomic profiling applications have become useful in early disease detection, diagnosis, treatment, and prognostication. We reviewed the recent literature on the applications of genomic and proteomic profiling in NSCLC. Important applications were summarized into those already adopted as standard care and those still under investigation. For genomic profiling, testing for EGFR mutation and ALK rearrangement has become routine for adenocarcinoma. Multiplex assay and malignancy-risk gene signature are both important applications in development. A test to predict outcome after treatment with an epidermal growth factor rector/tyrosine kinase inhibitor and a screening blood test for lung cancer are being investigated for use in proteomic profiling. Genomic profiling is routine in patients with NSCLC, and proteomic profiling shows promise. Additional genomic and proteomic profiling applications may also prove to be useful contributions in the care of these patients.

  18. Single-Cell Expression Profiling and Proteomics of Primordial Germ Cells, Spermatogonial Stem Cells, Adult Germ Stem Cells, and Oocytes.

    PubMed

    Conrad, Sabine; Azizi, Hossein; Skutella, Thomas

    2018-01-04

    The mammalian germ cells, cell assemblies, tissues, and organs during development and maturation have been extensively studied at the tissue level. However, to investigate and understand the fundamental insights at the molecular basis of germ and stem cells, their cell fate plasticity, and determination, it is of most importance to analyze at the large scale on the single-cell level through different biological windows. Here, modern molecular techniques optimized for single-cell analysis, including single fluorescence-activated cell sorting (FACS) and single-cell RNA sequencing (scRNA-seq) or microfluidic high-throughput quantitative real-time polymerase chain reaction (qRT-PCR) for single-cell gene expression and liquid chromatography coupled to tandem mass spectrometry (LC-MSMS) for protein profiling, have been established and are still getting optimized.This review aims on describing and discussing recent single-cell expression profiling and proteomics of different types of human germ cells, including primordial germ cells (PGCs), spermatogonial stem cells (SSCs), human adult germ stem cells (haGSCs), and oocytes.

  19. Proteome-wide analysis of neural stem cell differentiation to facilitate transition to cell replacement therapies.

    PubMed

    Zizkova, Martina; Sucha, Rita; Tyleckova, Jirina; Jarkovska, Karla; Mairychova, Katerina; Kotrcova, Eva; Marsala, Martin; Gadher, Suresh Jivan; Kovarova, Hana

    2015-02-01

    Neurodegenerative diseases are devastating disorders and the demands on their treatment are set to rise in connection with higher disease incidence. Knowledge of the spatiotemporal profile of cellular protein expression during neural differentiation and definition of a set of markers highly specific for targeted neural populations is a key challenge. Intracellular proteins may be utilized as a readout for follow-up transplantation and cell surface proteins may facilitate isolation of the cell subpopulations, while secreted proteins could help unravel intercellular communication and immunomodulation. This review summarizes the potential of proteomics in revealing molecular mechanisms underlying neural differentiation of stem cells and presents novel candidate proteins of neural subpopulations, where understanding of their functionality may accelerate transition to cell replacement therapies.

  20. The E. coli S30 lysate proteome: A prototype for cell-free protein production.

    PubMed

    Foshag, Daniel; Henrich, Erik; Hiller, Ekkehard; Schäfer, Miriam; Kerger, Christian; Burger-Kentischer, Anke; Diaz-Moreno, Irene; García-Mauriño, Sofía M; Dötsch, Volker; Rupp, Steffen; Bernhard, Frank

    2018-01-25

    Protein production using processed cell lysates is a core technology in synthetic biology and these systems are excellent to produce difficult toxins or membrane proteins. However, the composition of the central lysate of cell-free systems is still a "black box". Escherichia coli lysates are most productive for cell-free expression, yielding several mgs of protein per ml of reaction. Their preparation implies proteome fractionation, resulting in strongly biased and yet unknown lysate compositions. Many metabolic pathways are expected to be truncated or completely removed. The lack of knowledge of basic cell-free lysate proteomes is a major bottleneck for directed lysate engineering approaches as well as for assay design using non-purified reaction mixtures. This study is starting to close this gap by providing a blueprint of the S30 lysate proteome derived from the commonly used E. coli strain A19. S30 lysates are frequently used for cell-free protein production and represent the basis of most commercial E. coli cell-free expression systems. A fraction of 821 proteins was identified as the core proteome in S30 lysates, representing approximately a quarter of the known E. coli proteome. Its classification into functional groups relevant for transcription/translation, folding, stability and metabolic processes will build the framework for tailored cell-free reactions. As an example, we show that SOS response induction during cultivation results in tuned S30 lysate with better folding capacity, and improved solubility and activity of synthesized proteins. The presented data and protocols can serve as a platform for the generation of customized cell-free systems and product analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Identification of factors that function in Drosophila salivary gland cell death during development using proteomics

    PubMed Central

    McPhee, C K; Balgley, B M; Nelson, C; Hill, J H; Batlevi, Y; Fang, X; Lee, C S; Baehrecke, E H

    2013-01-01

    Proteasome inhibitors induce cell death and are used in cancer therapy, but little is known about the relationship between proteasome impairment and cell death under normal physiological conditions. Here, we investigate the relationship between proteasome function and larval salivary gland cell death during development in Drosophila. Drosophila larval salivary gland cells undergo synchronized programmed cell death requiring both caspases and autophagy (Atg) genes during development. Here, we show that ubiquitin proteasome system (UPS) function is reduced during normal salivary gland cell death, and that ectopic proteasome impairment in salivary gland cells leads to early DNA fragmentation and salivary gland condensation in vivo. Shotgun proteomic analyses of purified dying salivary glands identified the UPS as the top category of proteins enriched, suggesting a possible compensatory induction of these factors to maintain proteolysis during cell death. We compared the proteome following ectopic proteasome impairment to the proteome during developmental cell death in salivary gland cells. Proteins that were enriched in both populations of cells were screened for their function in salivary gland degradation using RNAi knockdown. We identified several factors, including trol, a novel gene CG11880, and the cop9 signalsome component cop9 signalsome 6, as required for Drosophila larval salivary gland degradation. PMID:22935612

  2. Proteomic profiling reveals dopaminergic regulation of progenitor cell functions of goldfish radial glial cells in vitro.

    PubMed

    Xing, Lei; Martyniuk, Christopher J; Esau, Crystal; Da Fonte, Dillon F; Trudeau, Vance L

    2016-07-20

    Radial glial cells (RGCs) are stem-like cells found in the developing and adult central nervous system. They function as both a scaffold to guide neuron migration and as progenitor cells that support neurogenesis. Our previous study revealed a close anatomical relationship between dopamine neurons and RGCs in the telencephalon of female goldfish. In this study, label-free proteomics was used to identify the proteins in a primary RGC culture and to determine the proteome response to the selective dopamine D1 receptor agonist SKF 38393 (10μM), in order to better understand dopaminergic regulation of RGCs. A total of 689 unique proteins were identified in the RGCs and these were classified into biological and pathological pathways. Proteins such as nucleolin (6.9-fold) and ependymin related protein 1 (4.9-fold) were increased in abundance while proteins triosephosphate isomerase (10-fold) and phosphoglycerate dehydrogenase (5-fold) were decreased in abundance. Pathway analysis revealed that proteins that consistently changed in abundance across biological replicates were related to small molecules such as ATP, lipids and steroids, hormones, glucose, cyclic AMP and Ca(2+). Sub-network enrichment analysis suggested that estrogen receptor signaling, among other transcription factors, is regulated by D1 receptor activation. This suggests that these signaling pathways are correlated to dopaminergic regulation of radial glial cell functions. Most proteins down-regulated by SKF 38393 were involved in cell cycle/proliferation, growth, death, and survival, which suggests that dopamine inhibits the progenitor-related processes of radial glial cells. Examples of differently expressed proteins including triosephosphate isomerase, nucleolin, phosphoglycerate dehydrogenase and capping protein (actin filament) muscle Z-line beta were validated by qPCR and western blot, which were consistent with MS/MS data in the direction of change. This is the first study to characterize the RGC

  3. Proteomics of Dense Core Secretory Vesicles Reveal Distinct Protein Categories for Secretion of Neuroeffectors for Cell-Cell Communication

    PubMed Central

    Wegrzyn, Jill L.; Bark, Steven J.; Funkelstein, Lydiane; Mosier, Charles; Yap, Angel; Kazemi-Esfarjani, Parasa; La Spada, Albert; Sigurdson, Christina; O’Connor, Daniel T.; Hook, Vivian

    2010-01-01

    Regulated secretion of neurotransmitters and neurohumoural factors from dense core secretory vesicles provides essential neuroeffectors for cell-cell communication in the nervous and endocrine systems. This study provides comprehensive proteomic characterization of the categories of proteins in chromaffin dense core secretory vesicles that participate in cell-cell communication from the adrenal medulla. Proteomic studies were conducted by nano-HPLC Chip MS/MS tandem mass spectrometry. Results demonstrate that these secretory vesicles contain proteins of distinct functional categories consisting of neuropeptides and neurohumoural factors, protease systems, neurotransmitter enzymes and transporters, receptors, enzymes for biochemical processes, reduction/oxidation regulation, ATPases, protein folding, lipid biochemistry, signal transduction, exocytosis, calcium regulation, as well as structural and cell adhesion proteins. The secretory vesicle proteomic data identified 371 distinct proteins in the soluble fraction and 384 distinct membrane proteins, for a total of 686 distinct secretory vesicle proteins. Notably, these proteomic analyses illustrate the presence of several neurological disease-related proteins in these secretory vesicles, including huntingtin interacting protein, cystatin C, ataxin 7, and prion protein. Overall, these findings demonstrate that multiple protein categories participate in dense core secretory vesicles for production, storage, and secretion of bioactive neuroeffectors for cell-cell communication in health and disease. PMID:20695487

  4. Nuclear proteome analysis of undifferentiated mouse embryonic stem and germ cells.

    PubMed

    Buhr, Nicolas; Carapito, Christine; Schaeffer, Christine; Kieffer, Emmanuelle; Van Dorsselaer, Alain; Viville, Stéphane

    2008-06-01

    Embryonic stem cells (ESCs) and embryonic germ cells (EGCs) provide exciting models for understanding the underlying mechanisms that make a cell pluripotent. Indeed, such understanding would enable dedifferentiation and reprogrammation of any cell type from a patient needing a cell therapy treatment. Proteome analysis has emerged as an important technology for deciphering these biological processes and thereby ESC and EGC proteomes are increasingly studied. Nevertheless, their nuclear proteomes have only been poorly investigated up to now. In order to investigate signaling pathways potentially involved in pluripotency, proteomic analyses have been performed on mouse ESC and EGC nuclear proteins. Nuclei from ESCs and EGCs at undifferentiated stage were purified by subcellular fractionation. After 2-D separation, a subtractive strategy (subtracting culture environment contaminating spots) was applied and a comparison of ESC, (8.5 day post coïtum (dpc))-EGC and (11.5 dpc)-EGC specific nuclear proteomes was performed. A total of 33 ESC, 53 (8.5 dpc)-EGC, and 36 (11.5 dpc)-EGC spots were identified by MALDI-TOF-MS and/or nano-LC-MS/MS. This approach led to the identification of two isoforms (with and without N-terminal acetylation) of a known pluripotency marker, namely developmental pluripotency associated 5 (DPPA5), which has never been identified before in 2-D gel-MS studies of ESCs and EGCs. Furthermore, we demonstrated the efficiency of our subtracting strategy, in association with a nuclear subfractionation by the identification of a new protein (protein arginine N-methyltransferase 7; PRMT7) behaving as proteins involved in pluripotency.

  5. Isolation and characterization of a canine mammary cell line prepared for proteomics analysis.

    PubMed

    Zamani-Ahmadmahmudi, Mohamad; Nassiri, Seyed Mahdi; Jahanzad, Issa; Shirani, Dariush; Rahbarghazi, Reza; Yazdani, Babak

    2013-06-01

    Mammary cancer is the most common tumor in female dogs. Canine mammary tumor serves as an excellent model for human breast cancer biology. Cancer cell lines are routinely used as the source of protein for proteomics studies because antigen homogeneity is essential for protein profiling of tumors. In this study, we sought to isolate and characterize a canine mammary cell line that was subject to protein profiling analysis through 2-dimensional electrophoresis (2-DE) method. Mammary tumor was collected from a 6-year-old terrier dog. Tumor fragments were treated with collagenase, and dissociated cells were cultured. The cell line was subcultured over 50 times. Characterization profile included population doubling time, colony forming assay, spheroid formation/migration potency, immunocytochemistry for steroid receptors and intermediate filaments, karyotyping, RT-PCR for cytokeratins 8, 14, and 18, and 2-DE pattern. The cell line revealed three growth phases including normal, dormant, and immortal phase. Immunocytochemistry showed that the cell line was positive for estrogen receptor, pancytokeratin, cytokeratin-low and vimentin, and negative for progesterone receptor, cytokeratin-high. RT-PCR supported the immunocytochemistry results. 2-DE pattern and proteome analysis of the cell line revealed that protein composition was stable, indicating the cell line as an appropriate source of protein for canine mammary proteomics studies. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. Chalepin: A Compound from Ruta angustifolia L. Pers Exhibits Cell Cycle Arrest at S phase, Suppresses Nuclear Factor-Kappa B (NF-κB) Pathway, Signal Transducer and Activation of Transcription 3 (STAT3) Phosphorylation and Extrinsic Apoptotic Pathway in Non-small Cell Lung Cancer Carcinoma (A549).

    PubMed

    Richardson, Jaime Stella Moses; Aminudin, Norhaniza; Abd Malek, Sri Nurestri

    2017-10-01

    Plants have been a major source of inspiration in developing novel drug compounds in the treatment of various diseases that afflict human beings worldwide. Ruta angustifolia L. Pers known locally as Garuda has been conventionally used for various medicinal purposes such as in the treatment of cancer. A dihydrofuranocoumarin named chalepin, which was isolated from the chloroform extract of the plant, was tested on its ability to inhibit molecular pathways of human lung carcinoma (A549) cells. Cell cycle analysis and caspase 8 activation were conducted using a flow cytometer, and protein expressions in molecular pathways were determined using Western blot technique. Cell cycle analysis showed that cell cycle was arrested at the S phase. Further studies using Western blotting technique showed that cell cycle-related proteins such as cyclins, cyclin-dependent kinases (CDKs), and inhibitors of CDKs correspond to a cell cycle arrest at the S phase. Chalepin also showed inhibition in the expression of inhibitors of apoptosis proteins. Nuclear factor-kappa B (NF-κB) pathway, signal transducer and activation of transcription 3 (STAT-3), cyclooxygenase-2, and c-myc were also downregulated upon treatment with chalepin. Chalepin was found to induce extrinsic apoptotic pathway. Death receptors 4 and 5 showed a dramatic upregulation at 24 h. Analysis of activation of caspase 8 with the flow cytometer showed an increase in activity in a dose- and time-dependent manner. Activation of caspase 8 induced cleavage of BH3-interacting domain death agonist, which initiated a mitochondrial-dependent or -independent apoptosis. Chalepin causes S phase cell cycle arrest, NF-κB pathway inhibition, and STAT-3 inhibition, induces extrinsic apoptotic pathway, and could be an excellent chemotherapeutic agent. This study reports the capacity of an isolated bioactive compound known as chalepin to suppress the nuclear factor kappa-light-chain-enhancer of activated B cells pathway, signal

  7. High-resolution proteome maps of Bacillus licheniformis cells growing in minimal medium.

    PubMed

    Voigt, Birgit; Albrecht, Dirk; Sievers, Susanne; Becher, Dörte; Bongaerts, Johannes; Evers, Stefan; Schweder, Thomas; Maurer, Karl-Heinz; Hecker, Michael

    2015-08-01

    Bacillus licheniformis is an important host for the industrial production of enzymes mainly because of its ability to secrete large amounts of protein. We analyzed the proteome of B. licheniformis cells growing in a minimal medium. Beside the cytosolic proteome, the membrane and the extracellular proteome were studied. We could identify 1470 proteins; 1168 proteins were classified as cytosolic proteins, 195 proteins with membrane-spanning domains were classified as membrane proteins, and 107 proteins, with either putative signals peptides or flagellin-like sequences, were classified as secreted proteins. The identified proteins were grouped into functional categories and used to reconstruct cellular functions and metabolic pathways of growing B. licheniformis cells. The largest group was proteins with functions in basic metabolic pathways such as carbon metabolism, amino acid and nucleotide synthesis and synthesis of fatty acids and cofactors. Many proteins detected were involved in DNA replication, transcription, and translation. Furthermore, a high number of proteins employed in the transport of a wide variety of compounds were found to be expressed in the cells. All MS data have been deposited in the ProteomeXchange with identifier PXD000791 (http://proteomecentral.proteomexchange.org/dataset/PXD000791). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Proteomic View of the Crosstalk betweenLactobacillus mucosaeand Intestinal Epithelial Cells in Co-culture Revealed by Q Exactive-Based Quantitative Proteomics.

    PubMed

    Pajarillo, Edward Alain B; Kim, Sang Hoon; Valeriano, Valerie Diane; Lee, Ji Yoon; Kang, Dae-Kyung

    2017-01-01

    Lactobacilli are bacteria that are beneficial to host health, but information on communication between Lactobacilli and host cells in the intestine is lacking. In this study, we examined the proteomes of the Lactobacillus mucosae strain LM1, as a model of beneficial bacteria, and the intestinal porcine epithelial cell line (IPEC-J2) after co-culture. Label-free proteomics demonstrated the high-throughput capability of the technique, and robust characterization of the functional profiles and changes in the bacteria and intestinal cells was achieved in pure and mixed cultures. After co-culture, we identified totals of 376 and 653 differentially expressed proteins in the LM1 and IPEC-J2 proteomes, respectively. The major proteomic changes in the LM1 strain occurred in the functional categories of transcription, general function, and translation, whereas those in IPEC-J2 cells involved metabolic and cellular processes, and cellular component organization/biogenesis. Among them, elongation factor Tu, glyceraldehyde 3-phosphate dehydrogenase, and phosphocarrier protein HPr, which are known to be involved in bacterial adhesion, were upregulated in LM1. In contrast, proteins involved in tight junction assembly, actin organization, and genetic information processing (i.e., histones and signaling pathways) were significantly upregulated in IPEC-J2 cells. Furthermore, we identified functional pathways that are possibly involved in host-microbe crosstalk and response. These findings will provide novel insights into host-bacteria communication and the molecular mechanism of probiotic establishment in the intestine.

  9. Identification of Thalidomide-Specific Transcriptomics and Proteomics Signatures during Differentiation of Human Embryonic Stem Cells

    PubMed Central

    Meganathan, Kesavan; Jagtap, Smita; Wagh, Vilas; Winkler, Johannes; Gaspar, John Antonydas; Hildebrand, Diana; Trusch, Maria; Lehmann, Karola; Hescheler, Jürgen; Schlüter, Hartmut; Sachinidis, Agapios

    2012-01-01

    Embryonic development can be partially recapitulated in vitro by differentiating human embryonic stem cells (hESCs). Thalidomide is a developmental toxicant in vivo and acts in a species-dependent manner. Besides its therapeutic value, thalidomide also serves as a prototypical model to study teratogenecity. Although many in vivo and in vitro platforms have demonstrated its toxicity, only a few test systems accurately reflect human physiology. We used global gene expression and proteomics profiling (two dimensional electrophoresis (2DE) coupled with Tandem Mass spectrometry) to demonstrate hESC differentiation and thalidomide embryotoxicity/teratogenecity with clinically relevant dose(s). Proteome analysis showed loss of POU5F1 regulatory proteins PKM2 and RBM14 and an over expression of proteins involved in neuronal development (such as PAK2, PAFAH1B2 and PAFAH1B3) after 14 days of differentiation. The genomic and proteomic expression pattern demonstrated differential expression of limb, heart and embryonic development related transcription factors and biological processes. Moreover, this study uncovered novel possible mechanisms, such as the inhibition of RANBP1, that participate in the nucleocytoplasmic trafficking of proteins and inhibition of glutathione transferases (GSTA1, GSTA2), that protect the cell from secondary oxidative stress. As a proof of principle, we demonstrated that a combination of transcriptomics and proteomics, along with consistent differentiation of hESCs, enabled the detection of canonical and novel teratogenic intracellular mechanisms of thalidomide. PMID:22952932

  10. Proteomics analysis of kojic acid treated A375 human malignant melanoma cells.

    PubMed

    Nawarak, Jiraporn; Huang-Liu, Rosa; Kao, Shao-Hsuan; Liao, Hsien-Hua; Sinchaikul, Supachok; Chen, Shui-Tein; Cheng, Sun-Long

    2008-09-01

    Although the toxicogenomics of kojic acid treated A375 human malignant melanoma cells has been elucidated, the proteomics of cellular response is still poorly understood. We performed proteomic analysis to investigate the anticancer effect of kojic acid on protein expression profile in A375 cells. A375 cells were treated with kojic acid at 8 microg/mL for 24, 48, and 72 h. With the use of 2-D PAGE and MALDI-Q-TOF MS and MS/MS analyses, proteomic profiles of A375 cells between control and kojic acid treatment were compared, and 30 differentially expressed proteins, containing 2 up-regulated proteins and 28 down-regulated proteins, were identified. Among these proteins, 17 isoforms of 5 identical proteins were observed and 11 chaperone proteins showed the high proportion of protein spots with 36.7% of total proteins. Bioinformatic tools were used to search for protein function and prediction of protein interaction. Sixteen differentially expressed proteins exhibited interaction network linked to the downstream regulations of p53 tumor suppressor and cell apoptosis, which may lead to suppress the melanogenesis and tumorigenesis of kojic acid treated A375 cells. In addition, GRP75, VIME and 2AAA were validated by Western blot analysis, whereas GRP75, 2AAA, HS90B, ENPL and KPYM were validated by RT-PCR. Therefore, these proteins play the important roles in cancer progression and may be potential biomarkers that are useful for diagnostic and therapeutic applications of malignant melanoma cancer.

  11. A proteomic view of cell physiology of Bacillus subtilis--bringing the genome sequence to life.

    PubMed

    Hecker, Michael

    2003-01-01

    The genome sequence is the "blue-print of life", and the proteomic approach brings this genome sequence to life. Simple model systems are urgently required to "train" this transformation of the genome sequence into life: why not Bacillus subtilis, the model organism for gram-positive bacteria and of functional genomics? By combination of the highly sensitive 2D protein gel electrophoresis with the identification of the protein spots by microsequencing or mass spectrometry we established a 2D protein index of Bacillus subtilis. In order to depict the entire proteome of a B. subtilis cell, alkaline, cell-wall associated, or extracellular proteins were also included. The proteins of this database (see http://microbio2.biologie.uni-greifswald.de:8880/sub2d.htm) were allocated to proteins with house-keeping functions typical of growing cells and to proteins synthesized particularly in non-growing cells. A computer-aided evaluation of the 2D gels loaded with radioactively-labeled proteins from growing or stressed/starved cells proved to be a powerful tool for the analysis of global regulation of the expression of the entire genome. This is shown for the analysis of glycolysis/TCA cycle (house keeping proteins) and for the analysis of the heat stress stimulon. For the heat stress stimulon it is demonstrated how the proteomic approach can be used: (i) to define the structure of a stimulon, (ii) to dissect stimulons into regulons, (iii) to analyze the regulation, structure, and function of unknown regulons, (iv) to define overlapping reguIons or modulons, and finally (v) to explore complex adaptational networks. Furthermore, it will be demonstrated how the "dual channel pattern comparison" or "proteomics signature" (R. VanBogelen) can be used for a comprehensive understanding or prediction of the physiological state of growing or starving cell populations. This is shown for glucose-starved cells. In order to describe the structure and function of gene regulation groups it

  12. Bone Proteomics experiment (BOP): the first proteomics analysis of mammalian cells cultivated in weightlessness conditions

    NASA Astrophysics Data System (ADS)

    Costessi, A.; Vascotto, C.; Pines, A.; Romanello, M.; Schonenborg, R.; Schiller, P.; Moro, L.; Tell, G.

    Bone mass loss is a major consequence of extended periods of weightlessness Many studies have been performed on astronauts and animal models establishing that a decrease of the maturation process and of the bone synthesising activity of osteoblast cells play a key role in microgravity-dependent bone mass loss Several experiments on single cells and tissues showed that weightlessness can also influence cells cultivated in vitro Many molecular mechanisms are affected among which the cytoskeleton and intracellular signal transduction cascades However the underlying mechanisms of these changes and their molecular consequences are far from being fully understood and the cellular gravisensing machinery is still unknown In contrast to weightlessness dynamic mechanical loading increases bone density and strength and promotes osteoblast proliferation differentiation and matrix production by acting at the gene expression level However the molecular mechanisms by which mechanical forces are converted into biochemical signalling in bone are also poorly understood A growing body of evidence points to extracellular nucleotides i e ATP and UTP as soluble factors that are released by several cell types in response to mechanical stimulation and that eventually trigger an intracellular signal We have recently demonstrated in the HOBIT osteoblast cell line that ATP and UTP treatments can activate two fundamental transcription factors that promote osteoblast differentiation and physiology Runx2 and Egr-1 as well as their target genes galectin-3 and

  13. Nanodroplet processing platform for deep and quantitative proteome profiling of 10–100 mammalian cells

    SciTech Connect

    Zhu, Ying; Piehowski, Paul D.; Zhao, Rui

    2018-02-28

    Nanoscale or single-cell technologies are critical for biomedical applications. However, current mass spectrometry (MS)-based proteomic approaches require samples comprising a minimum of thousands of cells to provide in-depth profiling. Here in this paper, we report the development of a nanoPOTS (nanodroplet processing in one pot for trace samples) platform for small cell population proteomics analysis. NanoPOTS enhances the efficiency and recovery of sample processing by downscaling processing volumes to <200 nL to minimize surface losses. When combined with ultrasensitive liquid chromatography-MS, nanoPOTS allows identification of ~1500 to ~3000 proteins from ~10 to ~140 cells, respectively. By incorporating the Match Betweenmore » Runs algorithm of MaxQuant, >3000 proteins are consistently identified from as few as 10 cells. Furthermore, we demonstrate quantification of ~2400 proteins from single human pancreatic islet thin sections from type 1 diabetic and control donors, illustrating the application of nanoPOTS for spatially resolved proteome measurements from clinical tissues.« less

  14. Proteomics analysis of A375 human malignant melanoma cells in response to arbutin treatment.

    PubMed

    Nawarak, Jiraporn; Huang-Liu, Rosa; Kao, Shao-Hsuan; Liao, Hsien-Hua; Sinchaikul, Supachok; Chen, Shui-Tein; Cheng, Sun-Long

    2009-02-01

    Although the toxicogenomics of A375 human malignant melanoma cells treated with arbutin have been elucidated using DNA microarray, the proteomics of the cellular response to this compound are still poorly understood. In this study, we performed proteomic analyses to investigate the anticancer effect of arbutin on the protein expression profile in A375 cells. After treatment with arbutin (8 microg/ml) for 24, 48 and 72 h, the proteomic profiles of control and arbutin-treated A375 cells were compared, and 26 differentially expressed proteins (7 upregulated and 19 downregulated proteins) were identified by MALDI-Q-TOF MS and MS/MS. Among these proteins, 13 isoforms of six identical proteins were observed. Bioinformatic tools were used to search for protein function and to predict protein interactions. The interaction network of 14 differentially expressed proteins was found to be correlated with the downstream regulation of p53 tumor suppressor and cell apoptosis. In addition, three upregulated proteins (14-3-3G, VDAC-1 and p53) and five downregulated proteins (ENPL, ENOA, IMDH2, PRDX1 and VIME) in arbutin-treated A375 cells were validated by RT-PCR analysis. These proteins were found to play important roles in the suppression of cancer development.

  15. Systems analysis of endothelial cell plasma membrane proteome of rat lung microvasculature.

    PubMed

    Li, Yan; Massey, Kerri; Witkiewicz, Halina; Schnitzer, Jan E

    2011-03-29

    Endothelial cells line all blood vessels to form the blood-tissue interface which is critical for maintaining organ homeostasis and facilitates molecular exchange. We recently used tissue subcellular fractionation combined with several multi-dimensional mass spectrometry-based techniques to enhance identification of lipid-embedded proteins for large-scale proteomic mapping of luminal endothelial cell plasma membranes isolated directly from rat lungs in vivo. The biological processes and functions of the proteins expressed at this important blood-tissue interface remain unexplored at a large scale. We performed an unbiased systems analysis of the endothelial cell surface proteome containing over 1800 proteins to unravel the major functions and pathways apparent at this interface. As expected, many key functions of plasma membranes in general (i.e., cell surface signaling pathways, cytoskeletal organization, adhesion, membrane trafficking, metabolism, mechanotransduction, membrane fusion, and vesicle-mediated transport) and endothelial cells in particular (i.e., blood vessel development and maturation, angiogenesis, regulation of endothelial cell proliferation, protease activity, and endocytosis) were significantly overrepresented in this proteome. We found that endothelial cells express multiple proteins that mediate processes previously reported to be restricted to neuronal cells, such as neuronal survival and plasticity, axon growth and regeneration, synaptic vesicle trafficking and neurotransmitter metabolic process. Surprisingly, molecular machinery for protein synthesis was also detected as overrepresented, suggesting that endothelial cells, like neurons, can synthesize proteins locally at the cell surface. Our unbiased systems analysis has led to the potential discovery of unexpected functions in normal endothelium. The discovery of the existence of protein synthesis at the plasma membrane in endothelial cells provides new insight into the blood

  16. Integrated Metabolomics, Transcriptomics and Proteomics Identifies Metabolic Pathways Affected by Anaplasma phagocytophilum Infection in Tick Cells*

    PubMed Central

    Villar, Margarita; Ayllón, Nieves; Alberdi, Pilar; Moreno, Andrés; Moreno, María; Tobes, Raquel; Mateos-Hernández, Lourdes; Weisheit, Sabine; Bell-Sakyi, Lesley; de la Fuente, José

    2015-01-01

    Anaplasma phagocytophilum is an emerging zoonotic pathogen that causes human granulocytic anaplasmosis. These intracellular bacteria establish infection by affecting cell function in both the vertebrate host and the tick vector, Ixodes scapularis. Previous studies have characterized the tick transcriptome and proteome in response to A. phagocytophilum infection. However, in the postgenomic era, the integration of omics datasets through a systems biology approach allows network-based analyses to describe the complexity and functionality of biological systems such as host–pathogen interactions and the discovery of new targets for prevention and control of infectious diseases. This study reports the first systems biology integration of metabolomics, transcriptomics, and proteomics data to characterize essential metabolic pathways involved in the tick response to A. phagocytophilum infection. The ISE6 tick cells used in this study constitute a model for hemocytes involved in pathogen infection and immune response. The results showed that infection affected protein processing in endoplasmic reticulum and glucose metabolic pathways in tick cells. These results supported tick–Anaplasma co-evolution by providing new evidence of how tick cells limit pathogen infection, while the pathogen benefits from the tick cell response to establish infection. Additionally, ticks benefit from A. phagocytophilum infection by increasing survival while pathogens guarantee transmission. The results suggested that A. phagocytophilum induces protein misfolding to limit the tick cell response and facilitate infection but requires protein degradation to prevent ER stress and cell apoptosis to survive in infected cells. Additionally, A. phagocytophilum may benefit from the tick cell's ability to limit bacterial infection through PEPCK inhibition leading to decreased glucose metabolism, which also results in the inhibition of cell apoptosis that increases infection of tick cells. These

  17. Integrated Metabolomics, Transcriptomics and Proteomics Identifies Metabolic Pathways Affected by Anaplasma phagocytophilum Infection in Tick Cells.

    PubMed

    Villar, Margarita; Ayllón, Nieves; Alberdi, Pilar; Moreno, Andrés; Moreno, María; Tobes, Raquel; Mateos-Hernández, Lourdes; Weisheit, Sabine; Bell-Sakyi, Lesley; de la Fuente, José

    2015-12-01

    Anaplasma phagocytophilum is an emerging zoonotic pathogen that causes human granulocytic anaplasmosis. These intracellular bacteria establish infection by affecting cell function in both the vertebrate host and the tick vector, Ixodes scapularis. Previous studies have characterized the tick transcriptome and proteome in response to A. phagocytophilum infection. However, in the postgenomic era, the integration of omics datasets through a systems biology approach allows network-based analyses to describe the complexity and functionality of biological systems such as host-pathogen interactions and the discovery of new targets for prevention and control of infectious diseases. This study reports the first systems biology integration of metabolomics, transcriptomics, and proteomics data to characterize essential metabolic pathways involved in the tick response to A. phagocytophilum infection. The ISE6 tick cells used in this study constitute a model for hemocytes involved in pathogen infection and immune response. The results showed that infection affected protein processing in endoplasmic reticulum and glucose metabolic pathways in tick cells. These results supported tick-Anaplasma co-evolution by providing new evidence of how tick cells limit pathogen infection, while the pathogen benefits from the tick cell response to establish infection. Additionally, ticks benefit from A. phagocytophilum infection by increasing survival while pathogens guarantee transmission. The results suggested that A. phagocytophilum induces protein misfolding to limit the tick cell response and facilitate infection but requires protein degradation to prevent ER stress and cell apoptosis to survive in infected cells. Additionally, A. phagocytophilum may benefit from the tick cell's ability to limit bacterial infection through PEPCK inhibition leading to decreased glucose metabolism, which also results in the inhibition of cell apoptosis that increases infection of tick cells. These results

  18. Label free quantitative proteomics analysis on the cisplatin resistance in ovarian cancer cells.

    PubMed

    Wang, F; Zhu, Y; Fang, S; Li, S; Liu, S

    2017-05-20

    Quantitative proteomics has been made great progress in recent years. Label free quantitative proteomics analysis based on the mass spectrometry is widely used. Using this technique, we determined the differentially expressed proteins in the cisplatin-sensitive ovarian cancer cells COC1 and cisplatin-resistant cells COC1/DDP before and after the application of cisplatin. Using the GO analysis, we classified those proteins into different subgroups bases on their cellular component, biological process, and molecular function. We also used KEGG pathway analysis to determine the key signal pathways that those proteins were involved in. There are 710 differential proteins between COC1 and COC1/DDP cells, 783 between COC1 and COC1/DDP cells treated with cisplatin, 917 between the COC1/DDP cells and COC1/DDP cells treated with LaCl3, 775 between COC1/DDP cells treated with cisplatin and COC1/DDP cells treated with cisplatin and LaCl3. Among the same 411 differentially expressed proteins in cisplatin-sensitive COC1 cells and cisplain-resistant COC1/DDP cells before and after cisplatin treatment, 14% of them were localized on the cell membrane. According to the KEGG results, differentially expressed proteins were classified into 21 groups. The most abundant proteins were involved in spliceosome. This study lays a foundation for deciphering the mechanism for drug resistance in ovarian tumor.

  19. Proteomic Profiling Identified Multiple Short-lived Members of the Central Proteome as the Direct Targets of the Addicted Oncogenes in Cancer Cells

    PubMed Central

    Qi, Tonggang; Zhang, Wei; Luan, Yun; Kong, Feng; Xu, Dawei; Cheng, Guanghui; Wang, Yunshan

    2014-01-01

    “Oncogene addiction” is an unexplained phenomenon in the area of cancer targeted therapy. In this study, we have tested a hypothesis that rapid apoptotic response of cancer cells following acute inhibition of the addicted oncogenes is because of loss of multiple short-lived proteins whose activity normally maintain cell survival by blocking caspase activation directly or indirectly. It was shown that rapid apoptotic response or acute apoptosis could be induced in both A431 and MiaPaCa-2 cells, and quick down-regulation of 17 proteins, which were all members of the central proteome of human cells, was found to be associated with the onset of acute apoptosis. Knockdown of PSMD11 could partially promote the occurrence of acute apoptosis in both MiaPaCa-2 and PANC-1 pancreatic cancer cells. These findings indicate that maintaining the stability of central proteome may be a primary mechanism for addicted oncogenes to maintain the survival of cancer cells through various signaling pathways, and quick loss of some of the short-lived members of the central proteome may be the direct reason for the rapid apoptotic response or acute apoptosis following acute inhibition of the addicted oncogenes in cancer cells. These findings we have presented can help us better understand the phenomenon of oncogene-addiction and may have important implications for the targeted therapy of cancer. PMID:24105791

  20. Impact of the antiproliferative agent ciclopirox olamine treatment on stem cells proteome

    PubMed Central

    Dihazi, Gry H; Bibi, Asima; Jahn, Olaf; Nolte, Jessica; Mueller, Gerhard A; Engel, Wolfgang; Dihazi, Hassan

    2013-01-01

    AIM: To investigate the proteome changes of stem cells due to ciclopirox olamine (CPX) treatment compared to control and retinoic acid treated cells. METHODS: Stem cells (SCs) are cells, which have the ability to continuously divide and differentiate into various other kinds of cells. Murine embryonic stem cells (ESCs) and multipotent adult germline stem cells (maGSCs) were treated with CPX, which has been shown to have an antiproliferative effect on stem cells, and compared to stem cells treated with retinoic acid (RA), which is known to have a differentiating effect on stem cells. Classical proteomic techniques like 2-D gel electrophoresis and differential in-gel electrophoresis (DIGE) were used to generate 2D protein maps from stem cells treated with RA or CPX as well as from non-treated stem cells. The resulting 2D gels were scanned and the digitalized images were collated with the help of Delta 2D software. The differentially expressed proteins were analyzed by a MALDI-TOF-TOF mass spectrometer, and the identified proteins were investigated and categorized using bioinformatics. RESULTS: Treatment of stem cells with CPX, a synthetic antifungal clinically used to treat superficial mycoses, resulted in an antiproliferative effect in vitro, without impairment of pluripotency. To understand the mechanisms induced by CPX treatments which results in arrest of cell cycle without any marked effect on pluripotency, a comparative proteomics study was conducted. The obtained data revealed that the CPX impact on cell proliferation was accompanied with a significant alteration in stem cell proteome. By peptide mass fingerprinting and tandem mass spectrometry combined with searches of protein sequence databases, a set of 316 proteins was identified, corresponding to a library of 125 non-redundant proteins. With proteomic analysis of ESCs and maGSCs treated with CPX and RA, we could identify more than 90 single proteins, which were differently expressed in both cell lines. We

  1. Impact of the antiproliferative agent ciclopirox olamine treatment on stem cells proteome.

    PubMed

    Dihazi, Gry H; Bibi, Asima; Jahn, Olaf; Nolte, Jessica; Mueller, Gerhard A; Engel, Wolfgang; Dihazi, Hassan

    2013-01-26

    To investigate the proteome changes of stem cells due to ciclopirox olamine (CPX) treatment compared to control and retinoic acid treated cells. Stem cells (SCs) are cells, which have the ability to continuously divide and differentiate into various other kinds of cells. Murine embryonic stem cells (ESCs) and multipotent adult germline stem cells (maGSCs) were treated with CPX, which has been shown to have an antiproliferative effect on stem cells, and compared to stem cells treated with retinoic acid (RA), which is known to have a differentiating effect on stem cells. Classical proteomic techniques like 2-D gel electrophoresis and differential in-gel electrophoresis (DIGE) were used to generate 2D protein maps from stem cells treated with RA or CPX as well as from non-treated stem cells. The resulting 2D gels were scanned and the digitalized images were collated with the help of Delta 2D software. The differentially expressed proteins were analyzed by a MALDI-TOF-TOF mass spectrometer, and the identified proteins were investigated and categorized using bioinformatics. Treatment of stem cells with CPX, a synthetic antifungal clinically used to treat superficial mycoses, resulted in an antiproliferative effect in vitro, without impairment of pluripotency. To understand the mechanisms induced by CPX treatments which results in arrest of cell cycle without any marked effect on pluripotency, a comparative proteomics study was conducted. The obtained data revealed that the CPX impact on cell proliferation was accompanied with a significant alteration in stem cell proteome. By peptide mass fingerprinting and tandem mass spectrometry combined with searches of protein sequence databases, a set of 316 proteins was identified, corresponding to a library of 125 non-redundant proteins. With proteomic analysis of ESCs and maGSCs treated with CPX and RA, we could identify more than 90 single proteins, which were differently expressed in both cell lines. We could highlight, that

  2. Study on the effect of BMSCs-EGFP-tk as mediator of HSV1-tk/GCV suicide gene therapy directed against A549 in vitro.

    PubMed

    Yang, Kun; Xu, Wen-Gui; Liu, Yong-Zhe; Meng, Xiang-Rui; Chen, Peng; Wu, Li-Chuan

    2014-01-01

    This study aims to observe the expression of HSV1-tk in mouse bone marrow mesenchymal stem cells (BMSCs-EGFP-tk) and detect the inhibition and killing effects of BMSCs as mediator of HSV1-tk/GCV on A549 cells in vitro, which can provide the experimental basis for gene therapy of lung cancer. We constructed the recombinant plasmid Vector pDON-AI-2 Neo-HSV1-tk-IRES2-EGFP with genetic engineering methods. Then we obtained the virus-like particles with infection ability after packaging the virus. The recombinant plasmid was transfected into mouse bone marrow mesenchymal stem cells in vitro. The expressions of EGFP in cells were observed by fluorescence microscopy and HSV1-tk gene was detected with RT-PCR. At last, the A549 cells and BMSCs-EGFP-tk cells were co-cultured with in vitro contact method, and the effect of BMSCs-EGFP-tk/GCV system was determined by MTT. Results indicated that the biological characteristics of BMSCs-EGFP-tk were consistent with those of BMSCs and fluorescent light expression and HSV1-tk gene expression can persist at least 15 days. The A549 cells and BMSCs-EGFP-tk cells were co-cultured and BMSCs-EGFP-tk:A549 = 2:1, adding 1 μg/mL GCV, the theory mortality is 58.44%, but actually the mortality is 90%. There is almost no difference between BMSCs-EGFP-tk and BMSCs cells in biological characteristics. The growth of A549 cells have an obviously inhibition and the bystander effect is outstanding in vitro after co-culture and this experiment lays solid foundation for the future research.

  3. Proteomic tools for the characterization of cell death mechanisms in drug discovery.

    PubMed

    Anania, Veronica G; Lill, Jennie R

    2015-08-01

    This review focuses on the use of proteomic tools for the characterization of cell death mechanisms that have contributed to drug discovery efforts. Resistance to cell death plays a major role in the development of many diseases, including numerous types of malignancies. Using a multitude of proteomic approaches, including protein-protein interaction studies, phosphorylation site mapping, ubiquitination site identification, and differential quantitative approaches, various cellular death pathways such as apoptosis and necroptosis have been investigated. These studies have aided in the development of therapeutic strategies or allowed dissection of clinical results to evaluate the success of clinical trials in addition to contributing to our understanding of these biological pathways. Here, we address the new wave of discoveries enabled by advancements in mass spectrometric technology and bioinformatic infrastructure that will hopefully lead to clinically efficacious strategies to overcome resistance to apoptosis and therefore offer improved treatment options for patients. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Proteome Changes during Transition from Human Embryonic to Vascular Progenitor Cells.

    PubMed

    Tsolis, Konstantinos C; Bagli, Eleni; Kanaki, Katerina; Zografou, Sofia; Carpentier, Sebastien; Bei, Ekaterini S; Christoforidis, Savvas; Zervakis, Michalis; Murphy, Carol; Fotsis, Theodore; Economou, Anastassios

    2016-06-03

    Human embryonic stem cells (hESCs) are promising in regenerative medicine (RM) due to their differentiation plasticity and proliferation potential. However, a major challenge in RM is the generation of a vascular system to support nutrient flow to newly synthesized tissues. Here we refined an existing method to generate tight vessels by differentiating hESCs in CD34(+) vascular progenitor cells using chemically defined media and growth conditions. We selectively purified these cells from CD34(-) outgrowth populations also formed. To analyze these differentiation processes, we compared the proteomes of the hESCs with those of the CD34(+) and CD34(-) populations using high resolution mass spectrometry, label-free quantification, and multivariate analysis. Eighteen protein markers validate the differentiated phenotypes in immunological assays; nine of these were also detected by proteomics and show statistically significant differential abundance. Another 225 proteins show differential abundance between the three cell types. Sixty-three of these have known functions in CD34(+) and CD34(-) cells. CD34(+) cells synthesize proteins implicated in endothelial cell differentiation and smooth muscle formation, which support the bipotent phenotype of these progenitor cells. CD34(-) cells are more heterogeneous synthesizing muscular/osteogenic/chondrogenic/adipogenic lineage markers. The remaining >150 differentially abundant proteins in CD34(+) or CD34(-) cells raise testable hypotheses for future studies to probe vascular morphogenesis.

  5. Quantitative Analysis of Global Proteome and Lysine Acetylome Reveal the Differential Impacts of VPA and SAHA on HL60 Cells.

    PubMed

    Zhu, Xiaoyu; Liu, Xin; Cheng, Zhongyi; Zhu, Jun; Xu, Lei; Wang, Fengsong; Qi, Wulin; Yan, Jiawei; Liu, Ning; Sun, Zimin; Liu, Huilan; Peng, Xiaojun; Hao, Yingchan; Zheng, Nan; Wu, Quan

    2016-01-29

    Valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA) are both HDAC inhibitors (HDACi). Previous studies indicated that both inhibitors show therapeutic effects on acute myeloid leukaemia (AML), while the differential impacts of the two different HDACi on AML treatment still remains elusive. In this study, using 3-plex SILAC based quantitative proteomics technique, anti-acetyllysine antibody based affinity enrichment, high resolution LC-MS/MS and intensive bioinformatic analysis, the quantitative proteome and acetylome in SAHA and VPA treated AML HL60 cells were extensively studied. In total, 5,775 proteins and 1,124 lysine acetylation sites were successfully obtained in response to VAP and SAHA treatment. It is found that VPA and SAHA treatment differently induced proteome and acetylome profiling in AML HL60 cells. This study revealed the differential impacts of VPA and SAHA on proteome/acetylome in AML cells, deepening our understanding of HDAC inhibitor mediated AML therapeutics.

  6. Traumatically injured astrocytes release a proteomic signature modulated by STAT3 dependent cell survival

    PubMed Central

    Levine, Jaclynn; Kwon, Eunice; Paez, Pablo; Yan, Weihong; Czerwieniec, Gregg; Loo, Joseph A.; Sofroniew, Michael V.; Wanner, Ina-Beate

    2015-01-01

    Molecular markers associated with CNS injury are of diagnostic interest. Mechanical trauma generates cellular deformation associated with membrane permeability with unknown molecular consequences. We used an in vitro model of stretch-injury and proteomic analyses to determine protein changes in murine astrocytes and their surrounding fluids. Abrupt pressure-pulse stretching resulted in the rapid release of 59 astrocytic proteins with profiles reflecting cell injury and cell death, i.e. mechanoporation and cell lysis. This acute trauma-release proteome was overrepresented with metabolic proteins compared to the uninjured cellular proteome, bearing relevance for post-traumatic metabolic depression. Astrocyte-specific deletion of signal transducer and activator of transcription 3 (STAT3-CKO) resulted in reduced stretch-injury tolerance, elevated necrosis and increased protein release. Consistent with more lysed cells, more protein complexes, nuclear and transport proteins were released from STAT3-CKO versus non-transgenic astrocytes. STAT3-CKO astrocytes had reduced basal expression of GFAP, lactate dehydrogenase B (LDHB), aldolase C (ALDOC) and astrocytic phosphoprotein 15 (PEA15), and elevated levels of tropomyosin (TPM4) and α actinin 4 (ACTN4). Stretching caused STAT3 dependent cellular depletion of PEA15 and GFAP, and its filament disassembly in subpopulations of injured astrocytes. PEA15 and ALDOC signals were low in injured astrocytes acutely after mouse spinal cord crush injury and robustly expressed in reactive astrocytes one day post-injury. In contrast, α crystallin (CRYAB) was present in acutely injured astrocytes, and absent from uninjured and reactive astrocytes, demonstrating novel marker differences among post-injury astrocytes. These findings reveal a proteomic signature of traumatically-injured astrocytes reflecting STAT3-dependent cellular survival with potential diagnostic value. PMID:26683444

  7. A Dual SILAC Proteomic Labeling Strategy for Quantifying Constitutive and Cell-Cell Induced Protein Secretion.

    PubMed

    Stiess, Michael; Wegehingel, Sabine; Nguyen, Chuong; Nickel, Walter; Bradke, Frank; Cambridge, Sidney B

    2015-08-07

    Recent evidence suggests that the extracellular protein milieu is much more complex than previously assumed as various secretome analyses from different cell types described the release of hundreds to thousands of proteins. The extracellular function of many of these proteins has yet to be determined particularly in the context of three-dimensional tissues with abundant cell-cell contacts. Toward this goal, we developed a strategy of dual SILAC labeling astrocytic cultures for in silico exclusion of unlabeled proteins from serum or neurons used for stimulation. For constitutive secretion, this strategy allowed the precise quantification of the extra-to-intracellular protein ratio of more than 2000 identified proteins. Ratios covered 4 orders of magnitude indicating that the intracellular vs extracellular contributions of different proteins can be variable. Functionally, the secretome of labeled forebrain astrocytic cultures specifically changed within hours after adding unlabeled, "physiological" forebrain neurons. "Nonphysiological" cerebellar hindbrain neurons, however, elicited a different, highly repulsive secretory response. Our data also suggest a significant association of constitutive secretion with the classical secretion pathway and regulated secretion with unconventional pathways. We conclude that quantitative proteomics can help to elucidate general principles of cellular secretion and provide functional insight into the abundant extracellular presence of proteins.

  8. Proteome Profile of Swine Testicular Cells Infected with Porcine Transmissible Gastroenteritis Coronavirus

    PubMed Central

    Ma, Ruili; Zhang, Yanming; Liu, Haiquan; Ning, Pengbo

    2014-01-01

    The interactions occurring between a virus and a host cell during a viral infection are complex. The purpose of this paper was to analyze altered cellular protein levels in porcine transmissible gastroenteritis coronavirus (TGEV)-infected swine testicular (ST) cells in order to determine potential virus-host interactions. A proteomic approach using isobaric tags for relative and absolute quantitation (iTRAQ)-coupled two-dimensional liquid chromatography-tandem mass spectrometry identification was conducted on the TGEV-infected ST cells. The results showed that the 4-plex iTRAQ-based quantitative approach identified 4,112 proteins, 146 of which showed significant changes in expression 48 h after infection. At 64 h post infection, 219 of these proteins showed significant change, further indicating that a larger number of proteomic changes appear to occur during the later stages of infection. Gene ontology analysis of the altered proteins showed enrichment in multiple biological processes, including cell adhesion, response to stress, generation of precursor metabolites and energy, cell motility, protein complex assembly, growth, developmental maturation, immune system process, extracellular matrix organization, locomotion, cell-cell signaling, neurological system process, and cell junction organization. Changes in the expression levels of transforming growth factor beta 1 (TGF-β1), caspase-8, and heat shock protein 90 alpha (HSP90α) were also verified by western blot analysis. To our knowledge, this study is the first time the response profile of ST host cells following TGEV infection has been analyzed using iTRAQ technology, and our description of the late proteomic changes that are occurring after the time of vigorous viral production are novel. Therefore, this study provides a solid foundation for further investigation, and will likely help us to better understand the mechanisms of TGEV infection and pathogenesis. PMID:25333634

  9. Proteome of human stem cells from periodontal ligament and dental pulp.

    PubMed

    Eleuterio, Enrica; Trubiani, Oriana; Sulpizio, Marilisa; Di Giuseppe, Fabrizio; Pierdomenico, Laura; Marchisio, Marco; Giancola, Raffaella; Giammaria, Gianluigi; Miscia, Sebastiano; Caputi, Sergio; Di Ilio, Carmine; Angelucci, Stefania

    2013-01-01

    Many adult tissues contain a population of stem cells with the ability to regenerate structures similar to the microenvironments from which they are derived in vivo and represent a promising therapy for the regeneration of complex tissues in the clinical disorder. Human adult stem cells (SCs) including bone marrow stem cells (BMSCs), dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) have been characterized for their high proliferative potential, expression of characteristic SC-associated markers and for the plasticity to differentiate in different lineage in vitro. The aim of this study is to define the molecular features of stem cells from oral tissue by comparing the proteomic profiles obtained with 2-DE followed by MALDI-TOF/TOF of ex-vivo cultured human PDLSCs, DPSCs and BMSCs. Our results showed qualitative similarities in the proteome profiles among the SCs examined including some significant quantitative differences. To enrich the knowledge of oral SCs proteome we performed an analysis in narrow range pH 4-7 and 6-9, and we found that DPSCs vs PDLSCs express differentially regulated proteins that are potentially related to growth, regulation and genesis of neuronal cells, suggesting that SCs derived from oral tissue source populations may possess the potential ability of neuronal differentiation which is very consistent with their neural crest origin. This study identifies some differentially expressed proteins by using comparative analysis between DPSCs and PDLSCs and BMSCs and suggests that stem cells from oral tissue could have a different cell lineage potency compared to BMSCs.

  10. Profound Re-Organization of Cell Surface Proteome in Equine Retinal Pigment Epithelial Cells in Response to In Vitro Culturing

    PubMed Central

    Szober, Christoph M.; Hauck, Stefanie M.; Euler, Kerstin N.; Fröhlich,