Human Epithelial Cell Line A549 Research Articles

Class A Scavenger Receptor-Mediated Double-Stranded RNA Internalization Is Independent of Innate Antiviral Signaling and Does Not Require Phosphatidylinositol 3-Kinase Activity.

dsRNA is a potent trigger of innate immune signaling, eliciting effects within virally infected cells and after release from dying cells. Given its inherent stability, extracellular dsRNA induces both local and systemic effects. Although the class A scavenger receptors (SR-As) mediate dsRNA entry, it is unknown whether they contribute to signaling beyond ligand internalization. In this study, we investigated whether SR-As contribute to innate immune signaling independent of the classic TLR and retinoic acid-inducible gene-I-like receptor (RLR) pathways. We generated a stable A549 human epithelial cell line with inducible expression of the hepatitis C virus protease NS3/4A, which efficiently cleaves TRIF and IFN-β promoter stimulator 1, adaptors for TLR3 and the RLRs, respectively. Cells expressing NS3/4A and TLR3/MyD88/IFN-β promoter stimulator 1(-/-) mouse embryonic fibroblasts completely lacked antiviral activity to extracellular dsRNA relative to control cells, suggesting that SR-As do not possess signaling capacity independent of TLR3 or the RLRs. Previous studies implicated PI3K signaling in SR-A-mediated activities and in downstream production of type I IFN. We found that SR-A-mediated dsRNA internalization occurs independent of PI3K activation, whereas downstream signaling leading to IFN production was partially dependent on PI3K activity. Overall, these findings suggest that SR-A-mediated dsRNA internalization is independent of innate antiviral signaling.

ID: 31: The diverse roles of type I interferon in response to EMCV and SeV infections

Type I interferon (IFN) is one of the body’s first line of defense against invading pathogens. The IFN- α subgroup in the type I IFN family consists of 12 different proteins that are known to have antiviral properties. However, the specific functions, efficacies, and induction patterns of each IFN- α subtype in response to viral pathogens have not been fully assessed. This study examines these properties using two RNA viruses, Sendai virus (SeV) and encephalomyocarditis virus (EMCV) and two human cell lines. Using the human lung epithelial cell line A549 and monocytoid cell line U937 we show that IFN- α subtypes are unique in their antiviral properties. IFN- α subtype pretreatment of both cell lines challenged with EMCV results in a range of antiviral activities. Subtype pretreatment followed by SeV challenge, however, did not protect either cell type. A549 cells were shown to induce very little type I IFN RNA upon SeV infection, but this induction results in the upregulation of IFN stimulated genes and biologically active IFN protein. Together, our study shows that IFN- α s have cell type and virus specific functions. The inability of type I IFN to protect against SeV infection must be further explored mechanistically to uncover why biologically active type I IFN is unable to induce an antiviral state.

Paracoccidioides brasiliensis induces recruitment of α3 and α5 integrins into epithelial cell membrane rafts, leading to cytokine secretion

Paracoccidioides brasiliensis is one of the etiological agents of paracoccidioidomycosis, a human systemic mycosis, highly prevalent in Latin America. In the present work, we demonstrate that P. brasiliensis yeasts promote IL-6 and IL-8 secretion by the human lung epithelial cell line A549 in an integrin-dependent manner. In fact, small interfering RNA directed to α3 and α5 integrins decreased IL-6 and IL-8 levels in P. brasiliensis-infected A549 cell cultures. This fungus also led to an increase in the expression of α3 and α5 integrins in this epithelial cell line. In addition, P. brasiliensis yeasts promoted α3 and α5 integrins clustering into A549 cell membrane rafts. Furthermore, epithelial cell membrane raft disruption with nystatin decreased IL-6 and IL-8 levels in P. brasiliensis-A549 cell cultures. Therefore, by increasing host α3 and α5 integrins levels and clustering these receptors into membrane rafts, P. brasiliensis yeasts may modulate host inflammation.

Validation of the 2nd Generation Proteasome Inhibitor Oprozomib for Local Therapy of Pulmonary Fibrosis.

Proteasome inhibition has been shown to prevent development of fibrosis in several organs including the lung. However, effects of proteasome inhibitors on lung fibrosis are controversial and cytotoxic side effects of the overall inhibition of proteasomal protein degradation cannot be excluded. Therefore, we hypothesized that local lung-specific application of a novel, selective proteasome inhibitor, oprozomib (OZ), provides antifibrotic effects without systemic toxicity in a mouse model of lung fibrosis. Oprozomib was first tested on the human alveolar epithelial cancer cell line A549 and in primary mouse alveolar epithelial type II cells regarding its cytotoxic effects on alveolar epithelial cells and compared to the FDA approved proteasome inhibitor bortezomib (BZ). OZ was less toxic than BZ and provided high selectivity for the chymotrypsin-like active site of the proteasome. In primary mouse lung fibroblasts, OZ showed significant anti-fibrotic effects, i.e. reduction of collagen I and α smooth muscle actin expression, in the absence of cytotoxicity. When applied locally into the lungs of healthy mice via instillation, OZ was well tolerated and effectively reduced proteasome activity in the lungs. In bleomycin challenged mice, however, locally applied OZ resulted in accelerated weight loss and increased mortality of treated mice. Further, OZ failed to reduce fibrosis in these mice. While upon systemic application OZ was well tolerated in healthy mice, it rather augmented instead of attenuated fibrotic remodelling of the lung in bleomycin challenged mice. To conclude, low toxicity and antifibrotic effects of OZ in pulmonary fibroblasts could not be confirmed for pulmonary fibrosis of bleomycin-treated mice. In light of these data, the use of proteasome inhibitors as therapeutic agents for the treatment of fibrotic lung diseases should thus be considered with caution.

Lariciresinol-4-O-β-D-glucopyranoside from the root of Isatis indigotica inhibits influenza A virus-induced pro-inflammatory response

Ethnopharmacological relevanceIsatis indigotica is a traditional Chinese medicine. Its dried roots named “ban lan gen” in Chinese, are used for clinical treatment of virus infection, tumor, inflammation with a long history. However, its anti-influenza active ingredient and the underlying mechanism remain unclear. In this study, the anti-influenza and anti-inflammatory effects of a lignan glycoside: lariciresinol-4-O-β-D-glucopyranoside isolated from the root of I. indigotica on human alveolar epithelial cell line A549 infected with influenza A virus were investigated. Materials and methodsChemical and spectroscopic methods were employed to identify the structure of the lignan glycoside. Cytotoxicity of the lignan glycoside was analyzed using methylthiazolyltetrazolium (MTT) assay. The inhibitory activity against influenza virus of the lignan was determined by CPE inhibition assay. HEK-293 cells stably co-transfected with NF-κB responsive firefly luciferase and constitutively expressing GFP were employed for monitoring the effect of the lignan on NF-κB signal pathway activation. Nuclear export of viral ribonucleoprotein (RNP) complexes was monitored by indirect immunofluorescence. Quantitative real-time PCR was used to quantify the expression profiling of cytokines and chemokines after infection with influenza virus. ResultsWe showed that the lignan glycoside treatment was effective against the influenza A virus-induced cytopathic effect (CPE) in MDCK cells. Further study demonstrated the lignan glycoside attenuated virus-induced NF-κB activation, but did not affect export of viral ribonucleoprotein (RNP) complexes from the nucleus in late stages of infection. We revealed that the lignan glycoside suppressed influenza A virus (H1N1)-induced expression of the pro-inflammatory molecules IL-6, TNF-α, IL-8, MCP-1, IP-10 and IFN-α. Moreover, the cytokines and chemokines profiles induced by H9N2 virus resembled those of influenza virus H1N1, but the lignan glycoside reduced the expression of IP-10 and TNF-α. ConclusionsOur results suggest that the lignan glycoside is a bioactive component of I. indigotica which may contribute an adjunct to pharmacotherapy for influenza virus infection.

Abstract 3872: Genotoxicity of zinc oxid nanoparticles and the activation of ATM-Chk2 DNA-damage-response pathway are caused by zinc-ions

Abstract Zinc oxide nanoparticles (ZnO-NP) are used in a variety of applications including cosmetics and pharmaceuticals. The common utilization of ZnO-NPs results in an increasing environmental release with unknown health effects. Recent studies have shown that ZnO-NP exposure cause cellular toxicity including DNA-damages. Discussed mechanisms are interactions of the ZnO-NP with intracellular structures and molecules after internalization, extracellular or intracellular dissociation of the particles, and the generation of ROS. In the present study we evaluated the genotoxic properties of ZnO-NP as well as the cellular DNA damage response in dependence of oxidative distress and ZnO-NP dissociation into Zn2+. For this purpose, we exposed the human epithelial cell line A549 to defined ZnO-NP (100μg/ml). Genotoxic effects of the particles were evaluated by Comet assay and γH2AX-foci, measuring DNA double-strand breaks (DNA-DSBs). Dissociation of ZnO-NP and the intracellular increase of Zn2+ were performed with spectrofluorimetric and cLSM measurements by the use of FluoZin3. Oxidative distress was determined by the compound dihydrodichlorofluorescein diacetate (H2DCF-DA). The Activation of the DNA damage response pathway was proven by analyses of H2AX-, ATM-, Chk1- and Chk2 phosphorylation, and p21, p53 up-regulation after ZnO-NP exposure by western blot or ELISA. To figure out, if the genotoxic potential of ZnO-NP is dependent of released Zn2+ or the generation of ROS we inhibited oxidative distress by the antioxidant N-acetyl-L-cysteine (NAC) and Zn2+ were extracellular chelated by diethylene triamine pentaacetic acid (DTPA). We found a fast extracellular dissociation of ZnO-NP and a quick intracellular increase of Zn2+. Furthermore, we ascertained an activation of H2AX induced by ZnO-NP; this phosphorylation is totally inhibited by DTPA. ZnO-NP activated the DSB-damage response indicated by ATM-, Chk2-phosphorylation as well as an up-regulation of p53 and p21. Pretreatment with NAC scavenging ROS leads to a reduced and Zn2+ complexation to no activation of ATM-Chk2-DNA damage pathway. The ATR/Chk1 pathway was not activated by ZnO-NP. We demonstrated ZnO-NP induced DNA-damages and the activation of the ATM/Chk2 DNA damage response pathway but not the ATR/Chk1 pathway, indicating that ZnO-NP exposure induces DNA-DSBs. Scavanging of ROS leads to a slight reduction of DNA-DSBs but extracellular Zn2+ compelxation to a complete impairment suggesting that the mechanism of ZnO-NP genotoxicity is not mediated via ROS-generation but rather a direct interaction of dissociated Zn-ions with the DNA. Citation Format: Julia Heim, Nawaz Muhammad Tahir, Anke Kaltbeitzel, Volker Mailänder, Hartmut Lüddens, Wolfgang Tremel, Jürgen Brieger. Genotoxicity of zinc oxid nanoparticles and the activation of ATM-Chk2 DNA-damage-response pathway are caused by zinc-ions. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3872. doi:10.1158/1538-7445.AM2015-3872

Cigarette smoke affects IL-17A, IL-17F and IL-17 receptor expression in the lung tissue: Ex vivo and in vitro studies

Cigarette smoke is a risk factor for Chronic Obstructive Pulmonary Disease (COPD). Th-17 cytokines are involved in the pathogenesis of COPD. We aimed to evaluate the role of cigarette smoke on the expression of IL-17A, IL-17F and IL-17R in airways of COPD patients. Epithelial and subepithelial immunoreactivity for IL-17A, IL-17F and IL-17R was assessed in surgical specimens from COPD patients (n=15) and from healthy subjects (HC) (n=10) by immunohistochemistry. In vitro, human epithelial cell line 16HBE and A549 as well as PBMC from normal donors were stimulated with cigarette smoke extract (CSE) (0%, 2.5%, 5%, 10%) to evaluate the IL-17A, IL-17F and IL-17R expression by flow cytometry. Furthermore, rhIL-17A and CSE stimulation was evaluated on proliferation and apoptosis in 16HBE and in A549. In central and distal airways immunoreactivity for IL-17A, IL-17F and IL-17R significantly increased in the epithelium and IL-17A in the subepithelium from COPD than in HC. In distal airway, immunoreactivity for IL-17F increased in the subepithelium of COPD than in HC. IL-17A immunoreactivity positively correlate with IL-17R and total pack years in the epithelium from central and distal airways of COPD patients. In vitro, CSE stimulation significantly increased IL-17F and IL-17R in 16HBE (2.5%) and A549 (5%) while IL-17A and IL-17F in PBMC (10%). IL-17A and CSE stimulation, rather than CSE or rhIL-17A alone, significantly increased proliferation in 16HBE and apoptosis in A549. Cigarette smoke increases Th17 immunity in lung tissue of COPD patients, promoting the mechanism of proliferation and apoptosis in airway epithelial cells.

Type II Secretion-Dependent Degradative and Cytotoxic Activities Mediated by Stenotrophomonas maltophilia Serine Proteases StmPr1 and StmPr2.

Stenotrophomonas maltophilia is an emerging opportunistic pathogen that primarily causes pneumonia and bacteremia in immunocompromised individuals. We recently reported that S. maltophilia strain K279a encodes the Xps type II secretion system and that Xps promotes rounding, actin rearrangement, detachment, and death in the human lung epithelial cell line A549. Here, we show that Xps-dependent cell rounding and detachment occur with multiple human and murine cell lines and that serine protease inhibitors block Xps-mediated rounding and detachment of A549 cells. Using genetic analysis, we determined that the serine proteases StmPr1 and StmPr2, which were confirmed to be Xps substrates, are predominantly responsible for secreted proteolytic activities exhibited by strain K279a, as well as the morphological and cytotoxic effects on A549 cells. Supernatants from strain K279a also promoted the degradation of type I collagen, fibrinogen, and fibronectin in a predominantly Xps- and protease-dependent manner, although some Xps-independent degradation of fibrinogen was observed. Finally, Xps, and predominantly StmPr1, degraded interleukin 8 (IL-8) secreted by A549 cells during coculture with strain K279a. Our findings indicate that while StmPr1 and StmPr2 are predominantly responsible for A549 cell rounding, extracellular matrix protein degradation, and IL-8 degradation, additional Xps substrates also contribute to these activities. Altogether, our data provide new insight into the virulence potential of the S. maltophilia Xps type II secretion system and its StmPr1 and StmPr2 substrates.

Methotrexate influx via folate transporters into alveolar epithelial cell line A549

Methotrexate (MTX), a drug used for the treatment of certain cancers as well as rheumatoid arthritis, sometimes induces serious interstitial lung injury. Although lung toxicity of MTX is related to its accumulation, the information concerning MTX transport in the lungs is lacking. In this study, we investigated the mechanisms underlying MTX influx into human alveolar epithelial cell line A549. MTX influx into A549 cells was time-, pH-, and temperature-dependent and showed saturation kinetics. The influx was inhibited by folic acid with IC50 values of 256.1 μM at pH 7.4 and 1.6 μM at pH 5.5, indicating that the mechanisms underlying MTX influx would be different at these pHs. We then examined the role of two folate transporters in MTX influx, reduced folate carrier (RFC) and proton-coupled folate transporter (PCFT). The expression of RFC and PCFT mRNAs in A549 cells was confirmed by reverse transcription polymerase chain reaction. In addition, MTX influx was inhibited by thiamine monophosphate, an RFC inhibitor, at pH 7.4, and by sulfasalazine, a PCFT inhibitor, at pH 5.5. These results indicated that RFC and PCFT are predominantly involved in MTX influx into A549 cells at pH 7.4 and pH 5.5, respectively.

The involvement of Mycobacterium leprae Hlp-DNA complex in bacterial recognition and airways epithelial activation (INM3P.404)

Abstract Airways are considered the main site of entry of Mycobacterium leprae (ML), the causative agent of leprosy. ML expresses a histone-like protein (Hlp), present in the cell envelope, which has been implicated in the binding of this mycobacterium to respiratory epithelial cells (ECs). So we investigated whether mycobacterial DNA-Hlp complexes are able to increase the human alveolar epithelial cell line A549 responses to ML through recognition by TLR9. ELISA results showed that ML and soluble CpG-Hlp complexes increase IL-8 secretion in ECs. Moreover, immunoblotting and ELISA assays revealed that ML induces the NF-κB nuclear translocation in A549 cells and the IL-8 production was dependent of that translocation. The antagonist of the nucleic acid PRRs also affected the ML-induced IL-8 production by ECs. In culture medium, the M. smegmatis wild-type (wt), but not the mutant strain for the gene hlp, was able to form clumps in the presence of exogenous mycobacterial DNA. Furthermore, the addition of CpG to M. smegmatis wt increased the production of IL-8 by A549 cells. Finally, conditioned medium from ECs pre-treated with ML was able to increase the CD40 and CD86 co-stimulatory molecules expression on plasmacytoid dendritic cells (DCs). Altogether, these observations suggest a potential role for the M. leprae DNA-Hlp complex in the activation of the airways epithelium, and an involvement of DCs in the protection of the host at the initiation of infection.

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

Man-made vitreous fibers have been widely used as insulation material as asbestos substitutes; however their morphology and composition raises concerns. In 1988 the International Agency for Research on Cancer classified fiberglass, rock wool, slag wool, and ceramic fibers as Group 2B, i.e. possibly carcinogenic to humans. In 2002 it reassigned fiberglass, rock and slag wool, and continuous glass filaments to Group 3, not classifiable as carcinogenic to humans. The aim of this study was to verify the cytotoxic and genotoxic effects and oxidative stress production induced by in vitro exposure of human alveolar epithelial cells A549 to glass fibers with a predominant diameter <3μm (97%) and length >5μm (93%). A549 cells were incubated with 5, 50, or 100μg/ml (2.1, 21, and 42μg/cm2, respectively) of glass fibers for 72h. Cytotoxicity and DNA damage were tested by the MTT and the Comet assay, respectively. Oxidative stress was determined by measuring inducible nitric oxide synthase (iNOS) expression by Western blotting, production of nitric oxide (NO) with Griess reagent, and concentration of reactive oxygen species by fluorescent quantitative analysis with 2′,7′-dichlorofluorescein-diacetate (DCFH-DA). The results showed that glass fiber exposure significantly reduced cell viability and increased DNA damage and oxidative stress production in a concentration-dependent manner, demonstrating that glass fibers exert cytotoxic and genotoxic effects related to increased oxidative stress on the human alveolar cell line A549.

Desialylation of airway epithelial cells during influenza virus infection enhances pneumococcal adhesion via galectin binding

The continued threat of worldwide influenza pandemics, together with the yearly emergence of antigenically drifted influenza A virus (IAV) strains, underscore the urgent need to elucidate not only the mechanisms of influenza virulence, but also those mechanisms that predispose influenza patients to increased susceptibility to subsequent infection with Streptococcus pneumoniae. Glycans displayed on the surface of epithelia that are exposed to the external environment play important roles in microbial recognition, adhesion, and invasion. It is well established that the IAV hemagglutinin and pneumococcal adhesins enable their attachment to the host epithelia. Reciprocally, the recognition of microbial glycans by host carbohydrate-binding proteins (lectins) can initiate innate immune responses, but their relevance in influenza or pneumococcal infections is poorly understood. Galectins are evolutionarily conserved lectins characterized by affinity for β-galactosides and a unique sequence motif, with critical regulatory roles in development and immune homeostasis. In this study, we examined the possibility that galectins expressed in the airway epithelial cells might play a significant role in viral or pneumococcal adhesion to airway epithelial cells. Our results in a mouse model for influenza and pneumococcal infection revealed that the murine lung expresses a diverse galectin repertoire, from which selected galectins, including galectin 1 (Gal1) and galectin 3 (Gal3), are released to the bronchoalveolar space. Further, the results showed that influenza and subsequent S. pneumoniae infections significantly alter the glycosylation patterns of the airway epithelial surface and modulate galectin expression. In vitro studies on the human airway epithelial cell line A549 were consistent with the observations made in the mouse model, and further revealed that both Gal1 and Gal3 bind strongly to IAV and S. pneumoniae, and that exposure of the cells to viral neuraminidase or influenza infection increased galectin-mediated S. pneumoniae adhesion to the cell surface. Our results suggest that upon influenza infection, pneumococcal adhesion to the airway epithelial surface is enhanced by an interplay among the host galectins and viral and pneumococcal neuraminidases. The observed enhancement of pneumococcal adhesion may be a contributing factor to the observed hypersusceptibility to pneumonia of influenza patients.

Efficient intratracheal delivery of airway epithelial cells in mice and pigs.

Cellular therapy via direct intratracheal delivery has gained interest as a novel therapeutic strategy for treating various pulmonary diseases including cystic fibrosis lung disease. However, concerns such as insufficient cell engraftment in lungs and lack of large animal model data remain to be resolved. This study aimed to establish a simple method for evaluating cell retention in lungs and to develop reproducible approaches for efficient cell delivery into mouse and pig lungs. Human lung epithelial cells including normal human bronchial/tracheal epithelial (NHBE) cells and human lung epithelial cell line A549 were infected with pSicoR-green fluorescent protein (GFP) lentivirus. GFP-labeled NHBE cells were delivered via a modified intratracheal cell instillation method into the lungs of C57BL/6J mice. Two days following cell delivery, GFP ELISA-based assay revealed a substantial cell-retention efficiency (10.48 ± 2.86%, n = 7) in mouse lungs preinjured with 2% polidocanol. When GFP-labeled A549 cells were transplanted into Yorkshire pig lungs with a tracheal intubation fiberscope, a robust initial cell attachment (22.32% efficiency) was observed at 24 h. In addition, a lentiviral vector was developed to induce the overexpression and apical localization of cystic fibrosis transmembrane conductance regulator (CFTR)-GFP fusion proteins in NHBE cells as a means of ex vivo CFTR gene transfer in nonprogenitor (relatively differentiated) lung epithelial cells. These results have demonstrated the convenience and efficiency of direct delivery of exogenous epithelial cells to lungs in mouse and pig models and provided important background for future preclinical evaluation of intratracheal cell transplantation to treat lung diseases.

Liposomal β-Glucan: Preparation, Characterization and Anticancer Activities

Context: An assortment of 1,3-β-glucans have been explored for their structural and pharmacological capabilities. The immunomodulatory effects of 1,3-β-glucans have served to fight cancer. Objectives: The aim of this work was the investigation of the physiocochemical characterizations of 1,3-β-D-glucan encapsulation of liposomes and their effects on human lung carcinoma epithelial cell line A549. aterials and Methods: β-glucan was encapsulated into liposomes composed of different lipids and cholesterol [PC, PC:Chol (6:1), DDAB:DPPC:Chol (4:2:1), DCP:DPPC:Chol (4:2:1), DPPC:Chol (6:1), DSPC:Chol (6:1), DSPC]. The liposomal formulations were prepared by the hydration-dehydration method. The mean diameter of liposomes and the polydispersity index were determined by photon correlation spectroscopy (PCS) with the use of Submicron Particle Sizer. The hemocompatibility of liposome on human erythrocytes was evaluated. The MTT assay was also performed to evaluate the A549 cells viability. Results: The mean particle size of vesicles loaded with β-glucan varied from 130.20 ± 10.5 to 180.10 ± 11.3 nm. The polydispersity index ranged from 0.66 ± 0.0004 to 0.90 ± 0.08. The encapsulation efficiencies in DDAB:DPPC:Chol, DCP:DPPC:Chol and DPPC:Chol formulations were 17.20 ± 0.08%, 23.60 ± 0.20% and 18.23 ± 0.06%, respectively. The hemolysis rates did not exceed the negative control value by more than 2% and 8% at 10 and 24 h, respectively. These three formulations showed higher growth suppression of A549 cell compared to free β-glucan. This growth suppression of A549 cell by 1 μM doxorubicin was further promoted by liposomal β-glucan. Conclusion: Liposomal 1,3-β-D-glucan might potentially enhance antitumor activity.

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

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

Abstract 3369: Differential biotherapeutic advantages of honey in targeting the Warburg effect and survival of MRC-5 and A549 cell lines

Abstract Lung cancer is a one of the most prevalent and deadly cancers in United States. Experimental evidence support that cancer cells do exhibit higher glycolytic rates than normal cells (Warburg effect). To exploit this unique cancer-dependent ATP generation phenomenon, we hypothesize that exposure of cancer cells to organic inhibitors of glycolysis would negatively impact their survival and alter their growth and viability resulting from the vast decrease in their essential glycolytic ATP production; no negative consequences will be seen on normal lung cells. The human lung fibroblast cell line MRC-5 and the human alveolar epithelial cell line A549 were used as models for normal lung and lung cancer in vitro in this study. Using standard methods, both cell lines were maintained and exposed to honey and D-glucose 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. Our results indicate that exposure of both cell lines to these natural nutraceutic organics resulted in concentration dependent cell destruction/cell survival depending on the cell line exposed. Honey and D-glucose showed statistically significant (p&amp;lt;0.05) differential negative effects on the A549 line in comparison to its unexposed control as well as to their effects on the MRC-5 cell line. Results show a promising role of honey and D-glucose as metabolites of interest for selective management of cancerous cells. Citation Format: Ibrahim O. Farah, Veshell L. Lewis, Wellington K. Ayensu, Joseph A. Cameron. Differential biotherapeutic advantages of honey in targeting the Warburg effect and survival of MRC-5 and A549 cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3369. doi:10.1158/1538-7445.AM2014-3369

Xanthones with anti-tumor activity isolated from Securidaca inappendiculata

A new xanthone, 1,2-dihydroxy-6,7,8-trimethoxyxanthone, together with ten known xanthones and nine other compounds was isolated from the stem of Securidaca inappendiculata. The structure of the new compound was elucidated based on spectroscopic data analyses, and other compounds were identified by comparison with data from the literature. The anti-tumor activities of the xanthones were evaluated by MTT assay on human lung adenocarcinoma epithelial cell line A549, and a primary structure–activity relationship analysis was carried out based on results from MTT assay. It is revealed that all the xanthones possessed anti-tumor activity in vitro in a concentration-dependent manner. The IC50 values varied a lot among xanthones. 1,7-Dihydroxy-3,4-dimethoxyxanthone was proved to be the most efficient anti-tumor agent with IC50 of 50.66 μM, while 1,3,7-trihydroxy-2,8-dimethoxyxanthone and 1,7-dihydroxyxanthone possessed the weakest activities. The structure–activity relationship analysis revealed that the anti-tumor activity of xanthones was affected mostly by the position and number of hydroxyl and methoxyl groups. Free hydroxyl and ortho-methoxyl groups were believed to possess important potentials to the activity.

Characteristics and cellular effects of ambient particulate matter from Beijing

In vitro tests using human adenocarcinomic alveolar epithelial cell line A549 and small mouse monocyte-macrophage cell line J774A.1 were conducted to test toxicity of six PM (particulate matter) samples from Beijing. The properties of the samples differ significantly. The production of inflammatory cytokine (TNF-α for J774A.1) and chemokine (IL-8 for A549) and the level of intracellular reactive oxygen species (ROS) were used as endpoints. There was a positive correlation between water soluble organic carbon and DTT-based redox activity. Both cell types produced increased levels of inflammatory mediators and had higher level of intracelllar ROS, indicating the presence of PM-induced inflammatory response and oxidative stress, which were dose-dependent and significantly different among the samples. The releases of IL-8 from A549 and TNF-α from J774A.1 were significantly correlated to PM size, Zeta potential, endotoxin, major metals, and polycyclic aromatic hydrocarbons. No correlation between ROS and these properties was identified.

TMEM16A protein attenuates lipopolysaccharide-mediated inflammatory response of human lung epithelial cell line A549

ABSTRACTObjective: To observe the expression of endogenous TMEM16A in rat alveolar type II epithelial cells (AT-II) and A549, and study the effect of TMEM16A on lipopolysaccharide (LPS)-induced proinflammatory cytokine secretion. Methods: Rat AT-II cells were isolated and TMEM16A protein expression in rat AT-II cells was measured by Western blot. TMEM16A mRNA and protein expressions in A549 were measured by real-time quantitative polymerase chain reaction (PCR) and Western blot, respectively. TMEM16A gene was transfected into A549 using Lipofectamine 2000. Transfected cells were selected in the presence of G418 to create a stable TMEM16A overexpression A549 cell line. The expression of TMEM16A in A549 was knocked down by lentiviral vector-mediated RNA interference. TNF-α and IL-8 levels were determined by enzyme-linked immunosorbent assay (ELISA). A dual-luciferase reporter assay system was used to measure the transcriptional activity of NF-κB. Results: (1) Endogenous TMEM16A was expressed in rat AT-II and A549. (2) TMEM16A expression in A549 significantly increased at 24 hours and 36 hours, and then decreased at 48 hours after LPS treatment. (3) TMEM16A mRNA and protein expressions were increased in the stable TMEM16A overexpression A549 cell line. (4) TMEM16A overexpression decreased the LPS-induced TNF-α and IL-8 secretions. (5) TMEM16A mRNA and protein expressions were knocked down in TMEM16A-siRNA lentivirus transfected A549. (6) TMEM16A knockdown increased the LPS-induced TNF-α and IL-8 secretions. (7) TMEM16A overexpression inhibited LPS-induced NF-κB activation. Conclusions: TMEM16A is expressed in AT-II. TMEM16A in A549 inhibits LPS-induced NF-κB activation and decreases proinflammatory cytokines release, protecting A549 from acute LPS-mediated damage.

Highly Stable Positively Charged Dendron-Encapsulated Gold Nanoparticles

We report the development of a novel cationic dendron (TAG1-PCD) and a positively charged gold nanoparticle-dendron conjugate (PCD-AuNP). TAG1-PCD was designed by considering the reactivity, hydrophilicity, and cationic nature that is required to yield a stable gold conjugate in aqueous media. The PCD-AuNPs, nominally 10 nm in size, were synthesized by reduction of chloroauric acid in the presence of TAG1-PCD. The physicochemical properties of PCD-AuNPs were characterized by dynamic light scattering, transmission electron microscopy, UV-vis absorbance, and X-ray photoelectron spectroscopy for investigation of size distribution, shape uniformity, surface plasmon resonance bands, and Au-dendron bonding. Asymmetric-flow field flow fractionation was employed to confirm the in situ size, purity, and surface properties of the PCD-AuNPs. Additionally, the stability of PCD-AuNPs was systematically evaluated with respect to shelf life determination, stability in biological media and a wide range of pH values, chemical resistance against cyanide, redispersibility from lyophilized state, and stability at temperatures relevant to biological systems. Dose dependent cell viability was evaluated in vitro using the human lung epithelial cell line A549 and a monkey kidney Vero cell line. Observations from in vitro studies are discussed. Overall, the investigation confirmed the successful development of stable PCD-AuNPs with excellent stability in biologically relevant test media containing proteins and electrolytes, and with a shelf life exceeding 6 months. The excellent aqueous stability and apparent lack of toxicity for this conjugate enhances its potential use as a test material for investigating interactions between positively charged NPs and biocellular and biomolecular systems, or as a vehicle for drug delivery.