A549 Pulmonary Epithelial Cells Research Articles

Synthesis and In Vitro Anticancer Activity of Pyrrolidone Derivatives Bearing 3,4,5-Trimethoxyphenyl Moiety as a Promising Anticancer Scaffold

A series of 5-oxo-1-(3,4,5-trimethoxyphenyl)pyrrolidine-3-carboxylic acid derivatives–hydrazones, N-ethylhydrazones, pyrrole, pyrazole, oxadiazole, and triazole were synthesized and evaluated for their anticancer activity using human A549 pulmonary epithelial cells (ATCC CCl-185). The in vitro viability inhibitory effects of the compounds were assessed using the MTT assay. The characterization of the anticancer activity of the synthesized compounds showed that the incorporation of 1,3,4-oxadiazolethione and 4-aminotriazolethione rings into the molecular structures obviously enhances the anticancer activity against human A549 lung epithelial cells, reducing their viability to 28.0% and 29.6%, respectively. The anticancer activity of these azole derivatives was significantly higher than that of cytarabine. Further studies are needed to better optimize 5-oxo-1-(3,4,5-trimethoxyphenyl)pyrrolidine-3-carboxylic acid derivatives and enhance their in vitro anticancer activity.

MiR-181a-3p from Exosome Excreted by BMSCs Promotes Apoptosis of Lung Cancer Cells Through Activating PR-ERAD Signal Pathway

Our study aimed to assess the effect of miR-181a-3p from exosome excreted by BMSCs on lung cancer cell apoptosis. Lung cancer cells A549 and normal pulmonary epithelial cells were cultivated in vitro to measure ERAD and PR mRNA level by qRT-PCR or Western blot assay along with analysis of cell proliferative activity by CCK-8, apoptosis by flow cytometry and level of ERAD, PR and p-AKT. ERAD in A549 cells was significantly elevated compared with BEAS-2B cells and PR was reduced. A549 cell proliferation was restrained after treated with miR-181a-3p from exosome excreted by BMSCs and cell apoptosis was promoted in a dose-dependent manner. ERAD was down-regulated and PR was up-regulated by miR-181a-3p from exosome excreted by BMSCs in varied concentrations. The proliferation and cell growth of lung cancer could be restrained by exosome derived from BMSCs through restraining the proliferative signal pathway. The activity of PR-ERAD was affected by the miR-181a-3p from exosome excreted by BMSCs, leading to inhibited proliferation and promoted apoptosis of lung cancer cells.

Inhibition of AHNAK nucleoprotein 2 alleviates pulmonary fibrosis by downregulating the TGF-β1/Smad3 signaling pathway.

Idiopathic pulmonary fibrosis (IPF) is a chronic and advanced interstitial lung disease with poor prognosis. AHNAK nucleoprotein 2 (AHNAK2) is a macromolecular protein that is important for cell migration and muscle membrane repair. The protein acts via epithelial-mesenchymal transition (EMT), which is a key mechanism in the pathogenesis of IPF. However, very few studies have elucidated the effect of AHNAK2 in the development of IPF. Therefore, we aimed to determine the role of AHNAK2 in IPF development. C57BL/6 mice were induced with bleomycin, while A549 and Beas-2b pulmonary epithelial cell lines were treated with TGF-β1 to induce IPF model. The expression of AHNAK2 was detected using immunohistochemistry staining in vivo, and real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting (WB) in vitro. C57BL/6 mice were injected with adeno-associated virus (AAV)-sh NC or AAV-sh AHNAK2 and the pulmonary function and EMT marker expression were measured. The migratory abilities of the two transforming growth factor beta 1 (TGF-β1)-induced cell lines were examined using wound-healing and Transwell assays after transfection with si-NC, si-AHNAK2-1 and -2. EMT marker expression was detected using RT-qPCR and WB. Smad3 and phosphorylated-Smad3 of the two cells were examined using WB. Following Smad3 inhibition by Smad3 phosphorylation inhibitor (SIS3), TGF-β1-induced cell migration and EMT marker expression were evaluated again after different transfections. AHNAK2 expression was higher in the IPF model than in the normal model in vivo and in vitro. Partial inhibition of AHNAK2 suppressed the EMT process and improved pulmonary ventilation and compliance in the mouse model of IPF. Similarly, knockdown of AHNAK2 suppressed the migration of pulmonary epithelial cells and reversed EMT. Furthermore, Smad3 of the two TGF-β1-induced cell lines was not activated when AHNAK2 was inhibited. When SIS3 inhibited the activation of Smad3, the suppression of AHNAK2 had no effect on A549 and Beas-2b, regardless of TGF-β1 induction. Inhibition of AHNAK2 alleviates pulmonary fibrosis and partially reverses EMT by inhibiting the TGF-β1/Smad3 signaling pathway. Therefore, AHNAK2 is a potential therapeutic target for IPF.

Effects of Taurine against benzo[α]pyrene-Induced Cell Cycle Arrest and Reactive Oxygen Species-Mediated Nuclear Factor-Kappa B Apoptosis via Reduction of Mitochondrial Stress in A549 Cells

Taurine is a free amino acid that prevents reactive oxygen species (ROS) formation. ROS production is associated with oxidative stress, cell proliferation, apoptosis, inflammation, and DNA alterations in benzo[α]pyrene (BaP)-induced lung cells. Here, we assessed the effect of adding of 25 mM taurine on human pulmonary alveolar epithelial A549 cells treated with different concentrations of BaP. After culturing for 24 h, the cells were tested for biomarkers including cell viability, cellular morphology, Annexin V-FITC/propidium iodide, cell cycle regulation, ROS accumulation, mitochondrial membrane potential (MMP), and expression of related signaling genes and proteins. BaP induced cell cycle arrest and decreased cell viability in a dose-dependent manner. In addition, 50 μM BaP induced a 52.2% increase in ROS levels and inhibited MMP by up to 80%; however, taurine decreased BaP-induced ROS production by 19.5% and restored MMP. The expression of nuclear factor-kappa B (NF-κB), B-cell lymphoma-2 (BCL-2) homologous antagonist killer (Bak), BCL-2-associated X protein (Bax), and cytochrome c at both the mRNA and protein levels were increased, and the expression of BCL-2 and BCL-x1 was decreased by BaP treatment. Furthermore, BaP activated caspase-3/7 expression by up to 25%. However, taurine decreased the expression of NF-κB, Bak, Bax and cytochrome c levels, reduced caspase-3/7 activities, and increased the expression of BCL-2 and BCL-x1. Hence, taurine attenuates BaP-induced oxidative stress and mitochondrial dysfunction by inhibiting the NF-κB-mediated intrinsic apoptosis pathway in A549 cells. Taurine can be considered as a preventive molecule to prevent lung damage.

Anti-Inflammatory and Anti-Fibrotic Effect of Immortalized Mesenchymal-Stem-Cell-Derived Conditioned Medium on Human Lung Myofibroblasts and Epithelial Cells.

Idiopathic pulmonary fibrosis (IPF) is caused by progressive lung tissue impairment due to extended chronic fibrosis, and it has no known effective treatment. The use of conditioned media (CM) from an immortalized human adipose mesenchymal stem cell line could be a promising therapeutic strategy, as it can reduce both fibrotic and inflammatory responses. We aimed to investigate the anti-inflammatory and anti-fibrotic effect of CM on human pulmonary subepithelial myofibroblasts (hPSM) and on A549 pulmonary epithelial cells, treated with pro-inflammatory or pro-fibrotic mediators. CM inhibited the proinflammatory cytokine-induced mRNA and protein production of various chemokines in both hPSMs and A549 cells. It also downregulated the mRNA expression of IL-1α, but upregulated IL-1β and IL-6 mRNA production in both cell types. CM downregulated the pro-fibrotic-induced mRNA expression of collagen Type III and the migration rate of hPSMs, but upregulated fibronectin mRNA production and the total protein collagen secretion. CM’s direct effect on the chemotaxis and cell recruitment of immune-associated cells, and its indirect effect on fibrosis through the significant decrease in the migration capacity of hPSMs, makes it a plausible candidate for further development towards a therapeutic treatment for IPF.

SARS-CoV-2 Ion Channel ORF3a Enables TMEM16F-Dependent Phosphatidylserine Externalization to Augment Procoagulant Activity of the Tenase and Prothrombinase Complexes

Severe SARS-CoV-2 infection is complicated by dysregulation of the blood coagulation system and high rates of thrombosis, but virus-intrinsic mechanisms underlying this phenomenon are poorly understood. Increased intracellular calcium concentrations promote externalization of phosphatidylserine (PS), the membrane anionic phospholipid required for assembly and activation of the tenase and prothrombinase complexes to drive blood coagulation. TMEM16F is a ubiquitous phospholipid scramblase that mediates externalization of PS in a calcium-dependent manner. As SARS-CoV-2 ORF3a encodes a presumed cation channel with the ability to transport calcium, we hypothesized that ORF3a expression by infected host cells perturbs the cellular calcium rheostat, driving TMEM16F-dependent externalization of PS and enhancing procoagulant activity.Using a doxycycline-inducible system, synchronized expression of ORF3a in A549 pulmonary epithelial cells resulted in a time-dependent augmentation of tissue factor (TF) procoagulant activity exceeding 9-fold by 48 hours (p < 0.0001), with no change in TF cell-surface expression. This enhancement was dependent upon PS as determined by inhibition with the PS-binding protein lactadherin. Over 2-fold enhancement of prothrombinase activity (p < 0.0001) was also observed by 48 hours. ORF3a increased intracellular calcium levels by 18-fold at 48 hours (p < 0.0001), as determined by the intracellular calcium indicator fluo-4. After 16 hours of ORF3a expression, more than 60% of cells had externalized PS (p < 0.001) without increased cell death, as quantified by flow cytometry following annexin V binding. Immunofluorescence microscopy staining for ORF3a, annexin V, and nuclei confirmed ORF3a expression within internal and cell surface membranes and increased PS externalization. PS externalization was insensitive to the pan-caspase inhibitor z-VAD-FMK, and there was no evidence of apoptotic activation as determined by caspase-3 cleavage. By contrast, ORF3a expression did not augment coagulation in cells deficient in the calcium-dependent phospholipid scramblase TMEM16F. Similarly, ORF3a-enhanced TF procoagulant activity (p < 0.01) and prothrombinase activity (p<0.05) was completely abrogated using TMEM16 inhibitors, including the uricosuric agent benzbromarone that has been registered for human use in over 20 countries. Live SARS-CoV-2 infection of A549-ACE2 cells increased cell surface factor Xa generation at MOI 0.1 (p < 0.01) but not MOI 0.01 or following heat inactivation of the virus, and RNA sequencing confirmed ORF3a induction without increased F3 expression. RNA sequencing of human SARS-CoV-2 infected lung autopsy and control tissue (n= 53) confirmed these findings in vivo. Immunofluorescence staining for ORF3a and KRT8/18 and CD31 in SARS-CoV-2 infected human lung autopsy specimens demonstrated ORF3a expression in pulmonary epithelium and endothelial cells, highlighting the potential pathologic relevance of this mechanism.Here we demonstrate that expression of the SARS-CoV-2 accessory protein ORF3a increases the intracellular calcium concentration and TMEM16F-dependent PS scrambling to augment procoagulant activity of the tenase and prothrombinase complexes. Our studies of human cells and tissues infected with SARS-CoV-2 support the pathologic relevance of this mechanism. We highlight the therapeutic potential to target the ORF3a-TMEM16F axis as with benzbromarone to mitigate dysregulation of coagulation and thrombosis during severe SARS-CoV-2 infection. DisclosuresSchwartz: Miromatrix Inc: Membership on an entity's Board of Directors or advisory committees; Alnylam Inc.: Consultancy, Speakers Bureau. Schulman: CSL Behring: Consultancy, Research Funding.

Role of nuclear factor‐κB (NF‐κB) in the regulation of pro‐inflammatory IκB‐kinase‐ε (IKKε) gene expression in human pulmonary epithelial cells

Rationale Clinically, mild-to-moderate asthma is well-controlled by inhaled glucocorticoids. However, severe asthma is poorly controlled by this key pharmacological intervention. Understanding which pro-inflammatory genes and signalling pathways avoid repression by glucocorticoids represents an important step towards improving current pharmacological interventions. IκB-kinase-ε (IKKε) was shown to escape full repression by glucocorticoids in airway epithelial cells, the primary site of action for glucocorticoid therapies in asthma. IKKε is also highly homologous to the canonical IκB kinases known to activate a family of pro-inflammatory transcription factors: nuclear factor-κB (NF-κB). However, the role and regulation of IKKε is largely uncharacterized. This study aimed to elucidate the regulation of IKKε gene expression by NF-κB in airway epithelial cells. Methods Human pulmonary epithelial A549 or bronchial airway epithelial BEAS-2B cells were used to model gene expression in airway epithelial cells. Cells were treated with cytokines interleukin-1β (IL1B, 1 ng/ml), tumour necrosis factor-α (TNF, 10 ng/ml), and/or glucocorticoid (dexamethasone, 1µM). Western blotting, qPCR, and RNA-sequencing were performed to characterize protein and mRNA expression of IKKε. Cells harbouring an NF-κB driven luciferase reporter construct were used to assess overall NF-κB activity. Chromatin immunoprecipitation (ChIP) sequencing data from BEAS-2B cells was used to explore binding of NF-κB subunit RelA/p65 to DNA using the UCSC genome browser. Results In A549 cells, IKKε protein was upregulated by both and IL1B and TNF with highest expression observed at 24h post stimulus. This peak expression was significantly, albeit partially, repressed by the glucocorticoid, dexamethasone. IKKε mRNA expression peaked at 6h, with partial repression by dexamethasone observed at 24h. ChIP-seq data from BEAS-2B cells treated with TNF showed p65 binding to the promoter region of IKKε. This suggests regulation of IKKε expression by NF-κB. In A549 cells, silencing of p65 decreased the ability of IL1B to induce NF-κB reporter activity and also reduced IL1B-induced expression of IKKε. Likewise, adenoviral-mediated overexpression of IκBαΔN, a dominant inhibitor of NF-κB, markedly reduced IKKε mRNA expression that was induced by IL1B. Conclusions These data suggest that induction of IKKε expression by IL1B or TNF is dependent on NF-κB activity in pulmonary epithelial cells. This supports the conclusion that early onset NF-κB activity leads to the expression of inflammatory genes, such as IKKε, to activate later onset signal transduction cascades to prolong inflammatory gene expression in response to a given stimuli. As IKKε induction was only partially repressed by glucocorticoid, the induction of downstream effector processes may also be largely resistant to the repressive effects of glucocorticoid. These findings represent an important step in characterizing the role of IKKε in inflammatory signalling cascades and paves the way for interventions in severe asthma therapy that may be more able to repress the activity of this pro-inflammatory pathway.

Effects of Acrolein on the Proliferation of and Per1 Gene Expression in Pulmonary Epithelial Cells

To investigate the effect of acrolein on the proliferation of pulmonary epithelial cells and its possible mechanism. Two strains of pulmonary epithelial cells, A549 cells and MLE15 cells, were used as in vitro models of pulmonary epithelial cell, and were treated with 80 μmol/L acrolein or phosphate buffer saline (PBS) as the control. The proliferation of pulmonary epithelial cells were determined with CCK-8 kit after cell culturing resumed for 12 h, 24 h, 36 h and 48 h post acrolein treatment, and the expression of period circadian regulator gene 1 ( Per1) was examined using Western blot test 24 h after acrolein treatment. In addition, after acrolein treatment, the cells were restored with transforming growth factor-β (TGF-β) added in the medium, and the cell proliferation and the expression of Per1 protein were also examined. The proliferation of A549 cells and MLE15 cells decreased significantly after being treated with 80 μmol/L acrolein for 30 min, and the expression of Per1 protein was also downregulated significantly ( P<0.05). The addition of TGF-β after acrolein treatment did not significantly change the reduction in cell proliferation caused by acrolein, but the expression of Per1 protein in pulmonary epithelial cells was significantly higher than that in cells restored without TGF-β ( P<0.05). Acrolein treatment resulted in the decreased proliferation of pulmonary epithelial cells and the Per1 expression in pulmonary epithelial cells. Although TGF-β addition did not reverse the reduction of cell proliferation after acrolein treatment, the Per1 expression levels were recovered to a certain extent compared to that in cells restored in medium without TGF-β after acrolein treatment.

Genomic determinants implicated in the glucocorticoid-mediated induction of KLF9 in pulmonary epithelial cells

Ligand-activated glucocorticoid receptor (GR) elicits variable glucocorticoid-modulated transcriptomes in different cell types. However, some genes, including Krüppel-like factor 9 (KLF9), a putative transcriptional repressor, demonstrate conserved responses. We show that glucocorticoids induce KLF9 expression in the human airways in vivo and in differentiated human bronchial epithelial (HBE) cells grown at air–liquid interface (ALI). In A549 and BEAS-2B pulmonary epithelial cells, glucocorticoids induce KLF9 expression with similar kinetics to primary HBE cells in submersion culture. A549 and BEAS-2B ChIP-seq data reveal four common glucocorticoid-induced GR binding sites (GBSs). Two GBSs mapped to the 5ʹ-proximal region relative to KLF9 transcription start site (TSS) and two occurred at distal sites. These were all confirmed in primary HBE cells. Global run-on (GRO) sequencing indicated robust enhancer RNA (eRNA) production from three of these GBSs in BEAS-2B cells. This was confirmed in A549 cells, plus submersion, and ALI culture of HBE cells. Cloning each GBS into luciferase reporters revealed glucocorticoid-induced activity requiring a glucocorticoid response element (GRE) within each distal GBS. While the proximal GBSs drove modest reporter induction by glucocorticoids, this region exhibited basal eRNA production, RNA polymerase II enrichment, and looping to the TSS, plausibly underlying constitutive KLF9 expression. Post glucocorticoid treatment, interactions between distal and proximal GBSs and the TSS correlated with KLF9 induction. CBP/P300 silencing reduced proximal GBS activity, but negligibly affected KLF9 expression. Overall, a model for glucocorticoid-mediated regulation of KLF9 involving multiple GBSs is depicted. This work unequivocally demonstrates that mechanistic insights gained from cell lines can translate to physiologically relevant systems.

High mobility group nucleosomal binding 2 reduces integrin α5/β1‐mediated adhesion of Klebsiella pneumoniae on human pulmonary epithelial cells via nuclear factor I

It has been reported that high mobility group nucleosomal binding domain 2 (HMGN2) is a nucleus-related protein that regulates gene transcription and plays a critical role in bacterial clearance. An elevated level of HMGN2 reduced integrin α5/β1 expression of human pulmonary epithelial A549 cells was demonstrated during Klebsiella pneumoniae infection, thus weakening bacterial adhesion and invasion. However, the mechanism by which HMGN2 regulates integrin expression remains unclear. This study found that a transcription factor-nuclear factor I (NFI), which serves as the potential target of HMGN2 regulated integrin expression. The results showed that HMGN2 was able to promote NFIA and NFIB expression by increasing H3K27 acetylation of NFIA/B promoter regions. The integrin α5/β1 expression was significantly enhanced by knockdown of NFIA/B via a siRNA approach. Meanwhile, NFIA/B silence could also compromise the inhibition effect of HMGN2 on the integrin α5/β1 expression. Mechanistically, it was demonstrated that HMGN2 facilitated the recruitment of NFI on the promoter regions of integrin α5/β1 according to the chromatin immunoprecipitation assay. In addition, it was further demonstrated that the knockdown of NFIA/B induced more adhesion of Klebsiella pneumoniae on pulmonary epithelial A549 cells, which could be reversed by the application of an integrin inhibitor RGD. The results revealed a regulatory role of HMGN2 on the transcription level of integrin α5/β1, indicating a potential treatment strategy against Klebsiella pneumoniae-induced infectious lung diseases.

MicroRNA-92a serves as a risk factor in sepsis-induced ARDS and regulates apoptosis and cell migration in lipopolysaccharide-induced HPMEC and A549 cell injury

AimsSepsis-induced acute respiratory distress syndrome (ARDS) is a common, high mortality complication in intensive care unit (ICU) patients. MicroRNA-92a (miR-92a) plays a role in many diseases, but its association with sepsis-induced ARDS is unclear. Materials and methodsWe enrolled 53 patients, including 17 with sepsis only, and 36 with sepsis-induced ARDS. Lipopolysaccharide (LPS) was used to stimulate pulmonary microvascular endothelial cells (HPMEC) and alveolar epithelial A549 cells, which were used to investigate the miR-92a roles in ARDS. MiR-92a expression levels in patient serum and cells were quantified using quantitative reverse transcription-polymerase chain reaction (RT-PCR), and protein expression was examined using Western blotting. The effect of miR-92a on apoptosis was examined using flow cytometry. Wound healing and transwell migration assays were used to evaluate cell migration. Key findingsSerum miR-92a expression was higher in patients with sepsis-induced ARDS, when compared to patients with sepsis only. After LPS treatment in cells, miR-92a expression was higher when compared with control group, cell apoptosis and inflammatory responses were increased and cell migration was inhibited. However, cell apoptosis and inflammatory responses were decreased and cell migration was enhanced after miR-92a downregulation, when compared with inhibitor negative control (NC) group. Moreover, phosphorylated-Akt and phosphorylated-mTOR expression were increased after miR-92a inhibition. SignificanceOur study provides evidence that circulating serum miR-92a could act as a risk factor for sepsis-induced ARDS. MiR-92a inhibition attenuated the adverse effects of LPS on ARDS through the Akt/mTOR signaling pathway.

Ethanol Extract of Caesalpinia decapetala Inhibits Influenza Virus Infection In Vitro and In Vivo.

Influenza virus infections can lead to viral pneumonia and acute respiratory distress syndrome in severe cases, causing significant morbidity and mortality and posing a great threat to human health. Because of the diversity of influenza virus strains and drug resistance to the current direct antiviral agents, there have been no effective drugs as yet to cure all patients infected by influenza viruses. Natural products from plants contain compounds with diverse structures that have the potential to interact with multiple host and virus factors. In this study, we identified the ethanol extract of Caesalpinia decapetala (Roth) Alston (EEC) as an inhibitor against the replication of a panel of influenza A and B viruses both on human pulmonary epithelial A549 and human monocytic U937 cells. The animal study revealed that EEC administration reduces the weight loss and improves the survival rate of mice infected with lethal influenza virus. Also, EEC treatment attenuated lung injury and reduced virus titer significantly. In conclusion, we showed that EEC has antiviral activity both in vitro and in vivo, suggesting that the plant C. decapetala has the potential to be further developed as a resource of new anti-influenza drugs.

Ginkgolide B protects human pulmonary alveolar epithelial A549 cells from lipopolysaccharide-induced inflammatory responses by reducing TRIM37-mediated NF-κB activation.

The treatment options for acute stroke combined with pulmonary infection are limited. Clinically, there are several therapies to promote blood circulation and dissipate blood stasis; these treatment options include ginkgolide B (GB), which has PAF (platelet activating factor)-inhibiting effects. PAF-receptor (PAF-R) antagonists are used to treat a variety of inflammatory diseases; however, the potential of PAF-R antagonists as a treatment for lung infections remains unclear. The aim of the present study is to investigate the protective effect of GB on lipopolysaccharide-induced inflammatory responses in A549 human pulmonary alveolar epithelial cells (HPAEpiC) in vitro. Cell viability and apoptosis were measured by CCK-8 and flow cytometry. TRIM37, Caspase-3, and NF-κBp65 expression levels were measured by real-time PCR and Western blotting. The release of tumor necrosis factor-α and interleukin-1β was measured by ELISA. The data indicates that GB may reduce TRIM37 expression by antagonizing the PAF-R pathway, thereby inhibiting the activation of nuclear factor-κB and alleviating the inflammatory response of alveolar epithelial cells. This study is the first to provide insight into the therapeutic potential of GB and suggests that clinical application of GB in acute stroke combined with pulmonary inflammation may be efficacious.

Screening of Cellular Stress Responses Induced by Ambient Aerosol Ultrafine Particle Fraction PM0.5 in A549 Cells.

Effects of airborne particles on the expression status of markers of cellular toxic stress and on the release of eicosanoids, linked with inflammation and oxidative damage, remain poorly characterized. Therefore, we proposed a set of various methodological approaches in order to address complexity of PM0.5-induced toxicity. For this purpose, we used a well-characterized model of A549 pulmonary epithelial cells exposed to a non-cytotoxic concentration of ambient aerosol particle fraction PM0.5 for 24 h. Electron microscopy confirmed accumulation of PM0.5 within A549 cells, yet, autophagy was not induced. Expression profiles of various cellular stress response genes that have been previously shown to be involved in early stress responses, namely unfolded protein response, DNA damage response, and in aryl hydrocarbon receptor (AhR) and p53 signaling, were analyzed. This analysis revealed induction of GREM1, EGR1, CYP1A1, CDK1A, PUMA, NOXA and GDF15 and suppression of SOX9 in response to PM0.5 exposure. Analysis of eicosanoids showed no oxidative damage and only a weak anti-inflammatory response. In conclusion, this study helps to identify novel gene markers, GREM1, EGR1, GDF15 and SOX9, that may represent a valuable tool for routine testing of PM0.5-induced in vitro toxicity in lung epithelial cells.

2599. Studying the Effects of Altering Histone Modification on Aspergillus fumigatus Virulence

BackgroundAs there are few drugs for treating invasive aspergillosis, there is an urgent need for new antifungal agents. Enzymes involved in histone modification are possible antifungal drug targets. We set out to investigate whether genes whose products are involved in histone modifications influence the virulence of Aspergillus fumigatus (Af).MethodsGenes whose products were likely involved in histone modification were deleted in strain Af293 using CRISPR-Cas9. Virulence was assessed in a triamcinolone-treated mouse model of invasive pulmonary aspergillosis. The extent of Af-induced damage to the A549 pulmonary epithelial cell line was determined by Cr51 release assay.ResultsAf genes were selected for investigation based on their homology to genes encoding known histone modifying proteins and their high expression level in vivo. The genes were predicted to encode members of the COMPASS histone methyltransferase complex (cclA/bre2, set2/Afu5g06000), the SAGA histone acetyltransferase complex (spt3, spt8), and the RPDL histone deacetylase complex (hosA). The ΔcclA and Δset2 mutants had significant growth defects on rich media and were not tested further. The Δspt3 and Δspt8 mutants grew normally and had mild conidiation defects. The ΔhosA mutant had wild-type (WT) growth and conidiation in vitro. Mice infected with the WT strain had 100% mortality within 9 days whereas mice infected the Δspt3, Δspt8, and ΔhosA mutants had only 40% mortality by 21 days. The ΔhosA mutant also had impaired capacity to damage pulmonary epithelial cells in vitro.ConclusionCcla and Set2, components of the COMPASS complex, are required for normal growth in vitro. Spt3 and Spt8, members of the SAGA complex, are required for normal conidiation and virulence. HosA, part of the RPD3L complex, is necessary for maximal virulence and induction of host cell damage. Our results suggest that the HosA histone deacetylase may be a promising drug target for treating invasive aspergillosis.Disclosures All authors: No reported disclosures.

Impact and mechanism of PMs-3 to epithelial mesenchymal transformation of non-small cell lung cancer cell line A549

Objective To explore Impact and mechanism of PMs-3 to epithelial mesenchymal transformation (EMT) of non-small cell lung cancer (NSCLC) cell line A549. Methods Nanocomposite structure synthetized by our team was utilized on human NSCLC cell line A549 and human normal pulmonary epithelial cell line BEAS-2B with concentrations of 0, 20, 40, 60, 80, 100, 120 nmol/L for 48 h. Methyl thiazol tetrazolium (MTT) assay was employed to detect the effect of PMs-3 on A549 cells. Wound assay and Transwell chamber assay were used to test the migration and invasion of PMs-3 on A549 cells. EMT related genes were detected with real time PCR (RT-qPCR) and Western blotting. Results Results of MTT assay detected that PMs-3 could inhibit activity of A549 cells significantly, and inhibition rate was (3.203±1.222)% (0 nmol/L), (11.958±4.000)% (20 nmol/L), (25.348±7.710)% (40 nmol/L), (34.775±9.155)% (60 nmol/L), (41.163±10.988)% (80 nmol/L), (50.430±9.743)% (100 nmol/L), (51.895±6.161)% (120 nmol/L), which showed a dose-dependent manner (F=35.451, P 0.05). Conclusion PMs-3 could inhibit NSCLC cells via regulating EMT. Key words: Non-small cell lung cancer; PMs-3; Epithelial mesenchymal transformation; Drug treatment

Effect of thiol antioxidants on lipopolysaccharide-induced cyclooxygenase-2 expression in pulmonary epithelial cells.

Cyclooxygenase-2 (COX-2) plays an important role in pulmonary inflammatory response, and its expression is regulated by several transcription factors including nuclear factor kappa-B (NF-κB), activator protein-1 (AP-1), and signal transducer and activator of transcription-3 (STAT-3), which are activated by oxidative stress. Glutathione (GSH) and N-acetyl cysteine (NAC) are thiol antioxidants that scavenge reactive oxygen species (ROS). The present study investigated whether lipopolysaccharide (LPS) induces COX-2 expression through ROS generation and the activation of oxidant-sensitive transcription factors such as NF-κB, AP-1, and STAT-3 in pulmonary epithelial A549 cells. The cells were pretreated with GSH or NAC for 1 hour prior to LPS stimulation. Intracellular ROS levels, DNA-binding activities of NF-κB, AP-1, and STAT-3, and mRNA and protein levels of COX-2 were determined. Our results showed that LPS increased ROS levels that peaked at 2 hours. LPS activated NF-κB, AP-1, and STAT-3 and induced the expression of COX-2 in A549 cells in a time-dependent manner. Pretreatment of thiol antioxidants GSH and NAC reduced ROS levels and attenuated the increase in ROS, the activation of NF-κB, AP-1, and STAT-3, and the expression of COX-2 in LPS-treated A549 cells. In conclusion, GSH and NAC suppress COX-2 expression by reducing ROS levels and inhibiting the activation of NF-κB, AP-1, and STAT-3 in pulmonary epithelial A549 cells exposed to LPS. Pretreatment with thiol antioxidants GSH and NAC may be beneficial for the treatment of pulmonary inflammation associated with oxidative stress.

Role of kinesins in directed adenovirus transport and cytoplasmic exploration.

Many viruses, including adenovirus, exhibit bidirectional transport along microtubules following cell entry. Cytoplasmic dynein is responsible for microtubule minus end transport of adenovirus capsids after endosomal escape. However, the identity and roles of the opposing plus end-directed motor(s) remain unknown. We performed an RNAi screen of 38 kinesins, which implicated Kif5B (kinesin-1 family) and additional minor kinesins in adenovirus 5 (Ad5) capsid translocation. Kif5B RNAi markedly increased centrosome accumulation of incoming Ad5 capsids in human A549 pulmonary epithelial cells within the first 30 min post infection, an effect dramatically enhanced by blocking Ad5 nuclear pore targeting using leptomycin B. The Kif5B RNAi phenotype was rescued by expression of RNAi-resistant Kif5A, B, or C, and Kif4A. Kif5B RNAi also inhibited a novel form of microtubule-based “assisted-diffusion” behavior which was apparent between 30 and 60 min p.i. We found the major capsid protein penton base (PB) to recruit kinesin-1, distinct from the hexon role we previously identified for cytoplasmic dynein binding. We propose that adenovirus uses independently recruited kinesin and dynein for directed transport and for a more random microtubule-based assisted diffusion behavior to fully explore the cytoplasm before docking at the nucleus, a mechanism of potential importance for physiological cargoes as well.

IL-12+IL-18 Cosignaling in Human Macrophages and Lung Epithelial Cells Activates Cathelicidin and Autophagy, Inhibiting Intracellular Mycobacterial Growth.

The ability of Mycobacterium tuberculosis to block host antimicrobial responses in infected cells provides a key mechanism for disease pathogenesis. The immune system has evolved to overcome this blockade to restrict the infection, but it is not clear whether two key innate cytokines (IL-12/IL-18) involved in host defense can enhance antimycobacterial mechanisms. In this study, we demonstrated that the combination of IL-12 and IL-18 triggered an antimicrobial response against mycobacteria in infected macrophages (THP-1 and human primary monocyte-derived macrophages) and pulmonary epithelial A549 cells. The inhibition of intracellular bacterial growth required p38-MAPK and STAT4 pathways, the vitamin D receptor, the vitamin D receptor-derived antimicrobial peptide cathelicidin, and autophagy, but not caspase-mediated apoptosis. Finally, the ability of IL-12+IL-18 to activate an innate antimicrobial response in human primary macrophages was dependent on the autonomous production of IFN-γ and the CAMP/autophagy pathway. Together, these data suggest that IL-12+IL-18 cosignaling can trigger the antimicrobial protein cathelicidin and autophagy, resulting in inhibition of intracellular mycobacteria in macrophages and lung epithelial cells.

Effects of Ghrelin on iNOS-Derived NO Promoted LPS-Induced Pulmonary Alveolar Epithelial A549 Cells Apoptosis

Background/Aims: In the process of abnormal apoptosis of pulmonary alveolar type II epithelial A549 cells in acute respiratory distress syndrome (ARDS), inducible nitric oxide synthase (iNOS) activity in the lung, nitric oxide (NO) production, and the level of protein S-nitrosylation were increased. However, the role of excessive NO production in sepsis-induced ARDS is controversial. Additionally, ghrelin is a growth hormone that exerts an inhibitory role in cell apoptosis. We examined the effect of NO and S-nitrosylation on apoptosis of A549 cells induced by Lipopolysaccharide (LPS) and molecular mechanism underlying the anti-apoptotic effect of ghrelin in this process. Methods: Flow cytometry and qPCR were used to detect lentiviral infection efficiency and iNOS gene level, respectively. Extracellular and intracellular NO levels were observed by Griess assay kit and DAF-FM DA. Mitochondrial transmembrane potential, apoptosis rate and SNO levels were determined by flow cytometry, Biotin-Switch method and immunofluoresence staining. The expression of iNOS, apoptotic proteins and JNK were assessed by immunoblot analysis. Results: The results showed about two times increase in iNOS expression and intracellular NO levels response to LPS exposure at 24 hours (P< 0.05), while not in extracellular NO levels. NO donors, S-nitroso-N-acetylpenicillamine (SNAP) significantly raised (36.7%, P< 0.05; 38.4%, P< 0.05; 41.8%, P< 0.05) extracellular NO levels without influencing the intracellular NO levels. LPS increased the apoptosis rate (42.4%±2.6% vs 2.8%±1%, P< 0.05) of A549 accompanied by increased Bax levels and decreased Bcl-2 levels through activating JNK signaling, which was reversed when we diminished the iNOS expression in A549 cells using lentiviral vectors encoding iNOS shRNA in the presence of LPS (24.8%±3.8% vs 42.4%±2.6%, P< 0.05). However, the apoptosis rate was increased when SNAP was added (38.8%±1.3% vs 24.8%±3.8%, P< 0.05). Furthermore, we investigated whether ghrelin exert a protective role against LPS-induced apoptosis and the potential mechanism involved in. Ghrelin alone appeared to decrease iNOS expression (32.3%, P< 0.05; 42.3%, P< 0.05), which showed no signifiant difference between LPS+ghrelin group and LPS group. However, this study showed that ghrelin decreased the intracellular NO production (38.9%, P< 0.05), protein S-nitrosylation levels (33.5%, P< 0.05), Bax protein expression (70.2%, P< 0.05), whereas increasing Bcl-2 protein expression (14.1%, P< 0.05) and mitochondrial transmembrane potential (∆ΨM) (20.7%, P< 0.05) in the presence of LPS. Conclusion: The data suggested that NO derived from iNOS induced by LPS stimulation exerts an important role in promoting apoptosis of A549 cells, and ghrelin abolished intracellular NO production and protein S-nitrosylation levels, abrogating the apoptosis of A549 cells partly through inhibiting mitochondrial-dependent pathways.