Alveolar Epithelial Cell Line Research Articles

Differential Responses by Human Respiratory Epithelial Cell Lines to Respiratory Syncytial Virus Reflect Distinct Patterns of Infection Control.

Respiratory syncytial virus (RSV) infects small foci of respiratory epithelial cells via infected droplets. Infection induces expression of type I and III interferons (IFNs) and proinflammatory cytokines, the balance of which may restrict viral replication and affect disease severity. We explored this balance by infecting two respiratory epithelial cell lines with low doses of recombinant RSV expressing green fluorescent protein (rgRSV). A549 cells were highly permissive, whereas BEAS-2B cells restricted infection to individual cells or small foci. After infection, A549 cells expressed higher levels of IFN-β-, IFN-λ-, and NF-κB-inducible proinflammatory cytokines. In contrast, BEAS-2B cells expressed higher levels of antiviral interferon-stimulated genes, pattern recognition receptors, and other signaling intermediaries constitutively and after infection. Transcriptome analysis revealed that constitutive expression of antiviral and proinflammatory genes predicted responses by each cell line. These two cell lines provide a model for elucidating critical mediators of local control of viral infection in respiratory epithelial cells.IMPORTANCE Airway epithelium is both the primary target of and the first defense against respiratory syncytial virus (RSV). Whether RSV replicates and spreads to adjacent epithelial cells depends on the quality of their innate immune responses. A549 and BEAS-2B are alveolar and bronchial epithelial cell lines, respectively, that are often used to study RSV infection. We show that A549 cells are permissive to RSV infection and express genes characteristic of a proinflammatory response. In contrast, BEAS-2B cells restrict infection and express genes characteristic of an antiviral response associated with expression of type I and III interferons. Transcriptome analysis of constitutive gene expression revealed patterns that may predict the response of each cell line to infection. This study suggests that restrictive and permissive cell lines may provide a model for identifying critical mediators of local control of infection and stresses the importance of the constitutive antiviral state for the response to viral challenge.

Transport of AOPP-Albumin into Human Alveolar Epithelial A549 Cell.

Alveolar clearance of proteins, such as albumin, plays an essential role in recovery from lung injuries. Albumin is known to be oxidized by reactive oxygen species (ROS), leading to generation of advanced oxidation protein products (AOPP)-albumin in the alveolar lining fluid. In this study, we aimed to characterize the uptake of FITC-labeled AOPP-albumin (FITC-AOPP-albumin) into human alveolar epithelial cell line, A549. FITC-AOPP-albumin uptake into A549 cells and its effect of ROS generation was evaluated using fluorescence spectrometer and flow cytometry, respectively. FITC-AOPP-albumin was taken up by A549 cells in a time- and temperature-dependent fashion, and showed saturation kinetics with a Km value of 0.37 mg/mL. The uptake of FITC-AOPP-albumin was suppressed by phenylarsine oxide, a clathrin-mediated endocytosis inhibitor, but not by indomethacin and nystatin, caveolae-mediated endocytosis inhibitors, or 5-(N-ethyl-N-isopropyl) amiloride, a macropinocytosis inhibitor. AOPP-albumin induced ROS generation in A549 cells, suggesting that alveolar clearance of AOPP-albumin should be important to prevent further ROS generation. AOPP-albumin is transported into alveolar epithelial cells through clathrin-mediated endocytosis, which may be important to prevent further ROS generation.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Assessing the bioactivity of crystalline silica in heated high-temperature insulation wools

High-Temperature Insulation Wools (HTIW), such as alumino silicate wools (Refractory Ceramic Fibers) and Alkaline Earth Silicate wools, are used in high-temperature industries for thermal insulation. These materials have an amorphous glass-like structure. In some applications, exposure to high temperatures causes devitrification resulting in the formation of crystalline species including crystalline silica. The formation of this potentially carcinogenic material raises safety concerns regarding after-use handling and disposal. This study aims to determine whether cristobalite formed in HTIW is bioactive in vitro. Mouse macrophage (J774A.1) and human alveolar epithelial (A549) cell lines were exposed to pristine HTIW of different compositions, and corresponding heat-treated samples. Cell death, cytokine release, and reactive oxygen species (ROS) formation were assessed in both cell types. Cell responses to aluminum lactate-coated fibers were assessed to determine if responses were caused by crystalline silica. DQ12 α-quartz was used as positive control, and TiO2 as negative control. HTIW did not induce cell death or intracellular ROS, and their ability to induce pro-inflammatory mediator release was low. In contrast, DQ12 induced cytotoxicity, a strong pro-inflammatory response and ROS generation. The modest pro-inflammatory mediator responses of HTIW did not always coincide with the formation of cristobalite in heated fibers; therefore, we cannot confirm that devitrification of HTIW results in bioactive cristobalite in vitro. In conclusion, the biological responses to HTIW observed were not attributable to a single physicochemical characteristic; instead, a combination of physicochemical characteristics (cristobalite content, fiber chemistry, dimensions and material solubility) appear to contribute to induction of cellular responses.

Upregulation and Nuclear Location of MMP28 in Alveolar Epithelium of Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive aging-associated disease of unknown etiology. A growing body of evidence indicates that aberrant activated alveolar epithelial cells induce the expansion and activation of the fibroblast population, leading to the destruction of the lung architecture. Some matrix metalloproteinases (MMPs) are upregulated in IPF, indicating that they may be important in the pathogenesis and/or progression of IPF. In the present study, we examined the expression of MMP28 in this disease and evaluated its functional effects in two alveolar epithelial cell lines and in human primary bronchial epithelial cells. We found that the enzyme is expressed in bronchial (apical and cytoplasmic localization) and alveolar (cytoplasmic and nuclear localization) epithelial cells in two different groups of patients with IPF. In vitro MMP28 epithelial silencing decreased the proliferation rate and delayed wound closing, whereas overexpression showed opposite effects, protecting from apoptosis and enhanced epithelial-mesenchymal transition. Our findings demonstrate that MMP28 is upregulated in epithelial cells from IPF lungs, where it may play a role in increasing the proliferative and migratory phenotype in a catalysis-dependent manner.

Silica nanoparticles of microrods enter lung epithelial cells.

A novel type of microparticle has recently been engineered. It consists of amorphous silica nanoparticles and has a corncob-like shape. It has already been demonstrated in vivo that alveolar macrophages in the lung are able to engulf this particulate carrier and that it also functions successfully as a gene delivery system. This subsequently raises the question as to whether epithelial cells may also be possible targets for these microrods. For this purpose, the alveolar epithelial cell line A549 was used presently. The epithelial character of these confluent cells was documented by the presence of tight junctions using a freeze-fracture technique and transmission electron microscopy. A toxic effect of the particles incubated with these cells was largely excluded. The interaction of the microparticles with the epithelial cells was observed using confocal microscopy and live cell imaging. Interestingly, the particles entered the epithelial cells within hours. After 1 day, the intracellular particles began to disaggregate and release the silica nanoparticles. Thus, even epithelial cells may serve as targets for this novel carrier and gene delivery system. This is particularly important since safe and effective gene delivery remains an unsolved problem. In addition, delivery of anti-cancer and anti-infective drugs may be an application of this novel particulate carrier.

New Water‐Soluble N‐Heterocyclic Carbene‐Palladium Complexes as Promising Anti‐Tumor Agents: Investigating DNA and Protein Interactions

AbstractThis investigation focuses on the synthesis and characterization of three new, water‐soluble palladium complexes, with potential to overcome the drawbacks of platinum‐based anti‐cancer drugs. The complexes contain small N‐heterocyclic carbene (NHC) ligands, having groups that impart water solubility. The anti‐cancer effects of these complexes were evaluated in two different cancer cell lines, namely,HeLa, a cervical cancer cell line and A549,adenocarcinoma human alveolar basal epithelial cell line. Cytotoxicity of the complexes was compared with cisplatin, an established platinum‐based anti‐cancer moiety, using the MTT assay (MTT is 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide). Aqueous solutions of the synthesized complexes demonstrated significant cytotoxicity in both the cell lines. The synthesized metal complexes resulted in a stronger, cytotoxic effect in A549 cells (lung cancer cells), as compared to cisplatin. Furthermore, interaction of these palladium compounds with DNA was studied using gel electrophoresis and spectrometry methods. The result suggested that the complexes interacted with DNA via intercalation. Interaction with BSA (Bovine Serum Albumin) was investigated using UV–Vis spectroscopy suggesting positive interaction of palladium complex with BSA proteins. Furthermore, fluorescence studies revealed quenching of protein fluorescence thus, indicating dynamic quenching mechanism with the number of association sites being calculated as ∼1.

Echinococcus granulosus cyst fluid enhances epithelial-mesenchymal transition.

Cystic echinococcosis is characterized by fluid-filled hydatid cysts in the liver and lungs. The cysts are surrounded by a host fibrous layer (the pericyst) which acts to isolate the parasite from surrounding tissues. Previous studies in liver cysts have indicated that the parasite may be a stimulating fibrosis. The aim of this study was to investigate whether hydatid cyst fluid (HCF) could influence the potential for fibrosis to occur in lung tissue by stimulating epithelial to mesenchymal transition (EMT) in a human lung epithelial cell line. An adenocarcinoma-derived alveolar basal epithelial cell line (A549) was used as a model for human alveolar epithelial cells (AEC II). These were cultured in vitro with HCF (UK sheep origin). Assays to investigate cell proliferation, cell migration and expression of cytoskeletal markers showed that HCF could stimulate changes indicative of EMT, including enhanced cell proliferation and migration; increased expression of mesenchymal cytoskeletal markers (fibronectin and vimentin) accompanied by a down-regulation of an epithelial marker (E-cadherin). Molecules within hydatid cyst fluid are capable of inducing phenotypic changes in A549 cells indicating that the parasite has the potential to modify lung epithelial cells which could contribute to fibrotic reactions.

SB431542 inhibited cigarette smoke extract induced invasiveness of A549 cells via the TGF-β1/Smad2/MMP3 pathway.

Lung cancer has high morbidity and mortality rates. Smoking is involved in the pathogenesis of lung cancer, and tobacco smoke may increase tumor cell invasion and metastasis. The effects of cigarette smoke extract (CSE) on the carcinoma human alveolar basal epithelial A549 cell line were investigated. A549 cells were exposed to increasing concentrations of CSE for 12, 24 and 48 h, and the transforming growth factor-β1 (TGF-β1) signal pathway was inhibited by addition of SB431542, a TGF-β1 receptor antagonist. The proliferation of A549 cells was assayed by a Cell Counting kit-8, invasiveness was assayed using Transwell chambers, and TGF-β1, phosphorylated mothers against decapentaplegic homolog 2 (p-Smad2), and matrix metalloproteinase 3 (MMP3) levels was assessed by western blot analysis. The invasiveness of A549 cells and the expression of TGF-β1, pSmad2, and MMP-3 were significantly increased by CSE (P<0.05). The effects of CSE were abrogated by SB431542 (P<0.05). In conclusion, CSE increased the invasiveness of A549 cells and its effects were abrogated by SB431542 and the TGF-β1/Smad2/MMP-3 pathway may have been involved.

Knockdown of LncRNA MALAT1 contributes to the suppression of inflammatory responses by up-regulating miR-146a in LPS-induced acute lung injury

ABSTRACTBackground: Acute lung injury (ALI) is a type of severe pulmonary inflammatory disease with high rates of morbidity and mortality. Now, an increasing number of studies suggest that lncRNAs may act as key regulators of the inflammatory response and play a crucial role in the pathogenesis of many inflammatory diseases. Our study firstly explored the function and underlying mechanism of lncRNA metastasis-associated lung adenocarcinoma transcription 1 (MALAT1) in regulating the inflammatory response of lipopolysaccharide (LPS)-induced ALI in rats. Methods: The ALI rats were constructed by intratracheal instillation with LPS. Hematoxylin and eosin (HE) for histological examination were performed to detect histopathological changes in the lung tissues. Enzyme-linked immunosorbent assay (ELISA) was used to determine the concentrations of cytokines TNF-α, IL-6, and IL-1β in the supernatants of the bronchoalveolar lavage fluid (BALF). Quantitative real-time PCR (qRT-PCR) analysis was employed to assess the expression of MALAT1, miR-146a, TNF-α, IL-6, and IL-1β in lung tissues. Luciferase reporter assay and RNA immunoprecipitation (RIP) assay were used to detect the relationship between MALAT1 and miR-146a. Results: The results revealed that MALAT1 knockdown played a protective role in the LPS-induced ALI rat model. In addition, knockdown of MALAT1 in vitro inhibited LPS-induced inflammatory response in murine alveolar macrophages cell line MH-S and murine alveolar epithelial cell line MLE-12. This study found that MALAT1 acts as a molecular sponge for miR-146a and MALAT1 negatively regulated miR-146a expression. Mechanistically, MALAT1 overexpression alleviated the inhibitory effect of miR-146a on LPS-induced inflammatory response in MH-S. Conclusions: Together, our study provided the first evidence that MALAT1 knockdown could suppress inflammatory response by up-regulating miR-146a in LPS-induced ALI, which provided a potential therapeutic target for the treatment of ALI.

The reduction of l-cystine to l-cysteine in the supernatant of A549 cell culture causes imipenem inactivation.

In the course of measuring the intracellular antibacterial activity of antibiotics using a human alveolar epithelial cell line A549, we discovered that the antimicrobial activity of several carbapenems (CPs) decreased in the supernatant of the cells cultured with fetal calf serum (FCS)-free RPMI1640 medium (RPMI). Further investigation revealed A549 culture supernatant inhibited the antibacterial activity of CPs but did not inactivate other types of antibiotics. CE-TOFMS and LC-TOFMS metabolomics analysis of the supernatant revealed the presence of l-cysteine (Cys), which is not an original component in RPMI. Cys is known to hydrolyze and inactivate CPs in a time- and concentration-dependent manner. In this study, the inactivating effects of A549 culture supernatant on the imipenem (IPM) were examined. Antimicrobial activity of 100μg/mL IPM decreased to 25% with two-fold dilution of A549 supernatant incubated for 3h. l-Cystine (CS), the Cys oxide, and an original component in RPMI did not inactivate IPM. However, the inactivating effects of A549 supernatant on IPM corresponds with the Cys concentration and depends on the CS content of the culture medium. Addition of FCS to the culture medium decreased the Cys concentration and reduced inactivation of IPM in a dose-dependent manner. Our data suggest that IPM were inactivated by Cys reduced from CS, and this CS-to-Cys conversion must be considered when evaluating the antimicrobial activity of CPs in cell culture. Further studies are needed to understand if the same inactivation occurs around the cells in the human body.

Increased Primary Cilia in Idiopathic Pulmonary Fibrosis.

Primary cilia are solitary, non-motile, axonemal microtubule-based antenna-like organelles that project from the plasma membrane of most mammalian cells and are implicated in transducing hedgehog signals during development. It was recently proposed that aberrant SHH signaling may be implicated in the progression of idiopathic pulmonary fibrosis (IPF). However, the distribution and role of primary cilia in IPF remains unclear. Here, we clearly observed the primary cilia in alveolar epithelial cells, fibroblasts, and endothelial cells of human normal lung tissue. Then, we investigated the distribution of primary cilia in human IPF tissue samples using immunofluorescence. Tissues from six IPF cases showed an increase in the number of primary cilia in alveolar cells and fibroblasts. In addition, we observed an increase in ciliogenesis related genes such as IFT20 and IFT88 in IPF. Since major components of the SHH signaling pathway are known to be localized in primary cilia, we quantified the mRNA expression of the SHH signaling components using qRT-PCR in both IPF and control lung. mRNA levels of SHH, the coreceptor SMO, and the transcription factors GLI1 and GLI2 were upregulated in IPF compared with control. Furthermore, the nuclear localization of GLI1 was observed mainly in alveolar epithelia and fibroblasts. In addition, we showed that defective KIF3A-mediated ciliary loss in human type II alveolar epithelial cell lines leads to disruption of SHH signaling. These results indicate that a significant increase in the number of primary cilia in IPF contributes to the upregulation of SHH signals.

Folic acid prevents methotrexate-induced epithelial-mesenchymal transition via suppression of secreted factors from the human alveolar epithelial cell line A549

Methotrexate (MTX) often induces serious lung diseases such as pulmonary fibrosis. Although MTX is known to be a folic acid (FA) antagonist, the effect of FA on MTX-induced lung injury remains unclear. Recent studies indicate that epithelial-mesenchymal transition (EMT) is involved in pulmonary fibrosis. Here, we aimed to clarify the effect of FA on MTX-induced EMT in human alveolar epithelial cell line A549 using conditioned medium (CM). CM was prepared from the supernatants of A549 cells treated with MTX in the absence (CMM) or presence (CMMF) of FA. FA suppressed EMT-like morphological changes and elevated mRNA/protein expression levels of α-smooth muscle actin induced by MTX in A549 cells. In addition, CMM induced EMT-like phenotypical changes, whereas CMMF had no effect on the phenotype of A549 cells, indicating that FA may suppress MTX-induced EMT via inhibiting the secretion of certain factors into the supernatant of the cells. Furthermore, FA also prevented CMM-induced EMT-like phenotypical changes in A549 cells. These findings indicate that FA may be a useful pharmaceutical for MTX-induced lung injury.

Genotoxicity of fine and coarse fraction ambient particulate matter in immortalised normal (TT1) and cancer-derived (A549) alveolar epithelial cells.

Human exposure to airborne particulate matter (PM) is associated with adverse cardiopulmonary health effects, including lung cancer. Ambient PM represents a heterogeneous mixture of chemical classes including transition metals, polycyclic aromatic hydrocarbons (PAHs) and their derivatives such as nitro‐PAHs, many of which are classified as putative carcinogens. As the primary site of human exposure to PM is the lungs, we investigated the response of two alveolar epithelial cell lines, the tumour‐derived A549 and newly described TT1 cells, to fine and coarse PM collected from background and roadside locations. We show that coarse PM elicits a genotoxic response in the TT1 cells, with the strongest signal associated with the background sample. This response could be recapitulated using the organic extract derived from this sample. No responses were observed in PM‐challenged A549 cells. Fine PM failed to elicit a genotoxic response in either cell line despite the higher PAH concentrations within this fraction. Consistent with the lack of a simplistic association between PM PAH content and the observed genotoxic response, TT1 cells treated with benzo[a]pyrene (BaP) demonstrated no increase in the selected markers. In contrast, a pattern of response was observed in TT1 cells challenged with 3‐nitrobenzanthrone (3‐NBA) similar to that with coarse PM. Together, these data illustrated the suitability of the TT1 cell line for assessing PM‐induced genotoxicity and challenge the contention that fine roadside PM poses the higher cancer risk. Furthermore, the response to 3‐NBA and not BaP suggests a major contribution of nitro‐PAHs to the overall toxicity of PM. Environ. Mol. Mutagen. 59:290–301, 2018. © 2018 The Authors Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society

Nrf2 antioxidant pathway suppresses Numb-mediated epithelial\u2013mesenchymal transition during pulmonary fibrosis

Epithelial mesenchymal transition (EMT) is a key progression that promotes pulmonary fibrosis (PF). Numb, a phosphotyrosine-binding domain (PTB) protein, is implicated with EMT. Nuclear factor erythroid 2-related factor2 (Nrf2) and its downstream proteins heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1) constitute an important pathway of antioxidant defense signal for protecting against PF. It remains elusive whether Nrf2 antioxidant pathway and Numb have a potential relationship in EMT-mediated PF. Here, we observed the effects of Nrf2 pathway and Numb on bleomycin(BLM)-induced PF in Nrf2-knockout (Nrf2−/−) and wild-type (WT) mice. Meanwhile, rat type II alveolar epithelial cells line (RLE-6TN) and human epithelial cells line (A549) were both treated with an Nrf2 activator sulforaphane (SFN), or transfected siRNAs of Nrf2 and Numb to unravel roles of Nrf2 pathway, Numb and the link between them on transforming growth factor β1 (TGF-β1)-induced EMT. We found BLM-induced lung fibrosis were more severe in Nrf2−/− mice compared to WT mice with reduced expressions of HO-1 and NQO1. Numb was enhanced with down-regulated expressions of Nrf2 in BLM groups and further increased in Nrf2−/− groups. In vitro, given exogenous TGF-β1 on RLE-6TN and A549 up-regulated Numb expressions, accompanied with down-regulations of Nrf2 and its target proteins HO-1 and NQO1. Transfected with Nrf2 and Numb siRNAs further aggravated and relieved the progression of EMT, respectively. Inversely, activating Nrf2 pathway by SFN reduced the expression of Numb and EMT-related protein. Moreover, Numb deficiency by siRNA relieved the protection of activating Nrf2 against EMT. In conclusion, activating Nrf2 antioxidant pathway suppresses EMT during PF via inhibiting the abnormal expression of Numb. These findings provide insight into PF pathogenesis and a basis for novel treatment approaches.

TNF-α increases Staphylococcus aureus-induced death of human alveolar epithelial cell line A549 associated with RIP3-mediated necroptosis

AimTo explore the role of tumor necrosis factor-alpha (TNF-α) on Staphylococcus aureus-induced necroptosis in alveolar epithelial cells. Main methodsThe A549 alveolar epithelial cell line was pretreated with small interfering RNA (siRNA) against receptor interacting protein-3 (RIP3) and then stimulated by S. aureus, where some cells were pretreated with TNF-α or TNF-α with anti-TNF-α antibody simultaneously. A549 cell death was assessed using lactate dehydrogenase (LDH) leakage and flow cytometry analyses. The protein expressions of RIP1, RIP3, cleaved caspase-1, and cleaved caspase-8 were analyzed by western blot. Key findingsS. aureus-induced LDH release was increased significantly by TNF-α. In addition, flow cytometry showed that TNF-α increased A549 cell apoptosis and necrosis in S. aureus-infected cell cultures. Levels of RIP3 and cleaved caspase-1 protein in A549 cells infected with S. aureus increased at 12 h post-infection, as shown by western blot. Significant additional increases in RIP3 expression were observed following the addition of TNF-α. Decreasing RIP3 levels by siRNA significantly suppressed the release of LDH induced by TNF-α and S. aureus. RIP3 siRNA also significantly suppressed A549 cell necrosis induced by S. aureus and TNF-α at 6 and 12 h post-infection as shown by flow cytometry analysis. SignificanceTNF-α enhances the damage of S. aureus on lung epithelial cells, and its mechanism is associated with RIP3 mediated necroptosis.

Antiproliferative effect of saffron and its constituents on different cancerous cell lines

Background: Use of herbs as medicinal plants to treat various clinical ailments has grown for the last two decades. Cancer is one of the most dreadful disease and extracts of various medicinal herbs have been subjected for the analysis to elucidate their possible role in the prevention of proliferation of cancer cells. Crocus sativus is an autumnally flowering plant rich in active ingredients apocarotenoids such as crocin, picrocrocin, and safranal which have been reported to have antiproliferative potential due to their strong antioxidant potential. Objective: To elucidate the antiproliferative potential of C. sativus extract (CSE) and its major constituents crocin and safranal on four different malignant cell lines (Alveolar lung epithelial cancerous cell line [A549], breast epithelial cancerous cell line [T47D], colon colorectal cell line [HCT-116], and prostrate cancerous cell line [PC3]) and nonmalignant cell line (L929). Materials and Methods: High-performance liquid chromatography was used to measure the content of crocin and safranal in saffron extract and antiproliferative effects of CSE, crocin, and safranal were evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. All cells were incubated with different concentrations of CSE, crocin, and safranal for 48 h. In a concentration-dependent manner, both safranal and crocin reduced cell proliferation in all malignant cell lines. Results: The IC50 values ranged between 0.32 and 0.42 mM for safranal, 0.31 and 0.92 mM for crocin, and 0.58 and 0.98 mg/ml for saffron extract. Conclusion: Based on these findings, it can be concluded that saffron and its components can inhibit cell proliferation in cancerous cells. Consequently, these agents could potentially be used as a chemopreventive agent for cancer management in the near future. Abbreviations Used: CSE: Crocus sativus extract; A549: Alveolar lung epithelial cancerous cell line, HCT-116: Colon colorectal cell line, T47d cell line: Breast epithelial cancerous cell line, PC-3 cell line: Prostrate cancerous cell line; HPLC: High-performance liquid chromatography, MTT: 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide.

Deamidation of asparagine residues in Prohibitin1 is involved in lipid droplet formation and cell proliferation in alveolar epithelial cell line

Deamidation of asparagine residues in Prohibitin1 is involved in lipid droplet formation and cell proliferation in alveolar epithelial cell line

The Cytotoxic Constituents ofBetula platyphyllaand their Effects on Human Lung A549 Cancer Cells

During the screening for cytotoxic compounds from plants grown in Korea, Betula platyphylla (BP) showed potent activity against the adenocarcinomic human alveolar basal epithelial A549 cell line. To identify the cytotoxic components from BP, the CH2Cl2 fraction with the most significant cytotoxic effect was applied to the column chromatographies. Seven compounds were isolated: lupeol (1), betulinic acid (2), (-)-rhododendrol (3), platyphyllenone (4), platyphyllone (5), (-)-centrolobol (6), and oleanolic acid (7). Among them, three diarylheptanoids (4 - 6) exhibited cytotoxicity toward A549 cells. Especially, 50 μM of 4 reduced A549 cell viability to 18.93 ± 0.82% compared to control (100.00 ± 21.48%). Lactate dehydrogenase (LDH) leakage and intracellular reactive oxygen species (ROS) production were also induced by 50 μM 4. This is the first report on the cytotoxic effect of BP-derived diarylheptanoids 4-6 against A549 cells. The compound 4 may be useful for the development of early hit compounds for non-small cell lung carcinoma, but the consideration about selectivity of 4 is required since 4 also showed the cytotoxicity in the human normal lung epithelial BEAS-2B cell line.

Nrf2 Antioxidant Pathway Suppresses Numb-mediated Epithelial-mesenchymal Transition during Pulmonary Fibrosis

Epithelial mesenchymal transition (EMT) is a key progression that promotes pulmonary fibrosis (PF). Numb, a phosphotyrosine-binding domain (PTB) protein, is implicated with EMT. Nuclear factor erythroid 2-related factor2 (Nrf2) and its downstream proteins heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1) constitute an important pathway of antioxidant defense signal for protecting against PF. It remains elusive whether Nrf2 antioxidant pathway and Numb have a potential relationship in EMT-mediated PF. Here, we observed the effects of Nrf2 pathway and Numb on bleomycin(BLM)-induced PF in Nrf2-knockout (Nrf2−/−) and wild-type (WT) mice. Meanwhile, rat type II alveolar epithelial cells line (RLE-6TN) and human epithelial cells line (A549) were both treated with an Nrf2 activator sulforaphane (SFN), or transfected siRNAs of Nrf2 and Numb to unravel roles of Nrf2 pathway, Numb and the link between them on transforming growth factor β1 (TGF-β1)-induced EMT. We found BLM-induced lung fibrosis were more severe in Nrf2−/− mice compared to WT mice with reduced expressions of HO-1 and NQO1. Numb was enhanced with down-regulated expressions of Nrf2 in BLM groups and further increased in Nrf2−/− groups. In vitro, given exogenous TGF-β1 on RLE-6TN and A549 up-regulated Numb expressions, accompanied with down-regulations of Nrf2 and its target proteins HO-1 and NQO1. Transfected with Nrf2 and Numb siRNAs further aggravated and relieved the progression of EMT, respectively. Inversely, activating Nrf2 pathway by SFN reduced the expression of Numb and EMT-related protein. Moreover, Numb deficiency by siRNA relieved the protection of activating Nrf2 against EMT. In conclusion, activating Nrf2 antioxidant pathway suppresses EMT during PF via inhibiting the abnormal expression of Numb. These findings provide insight into PF pathogenesis and a basis for novel treatment approaches.

Mechanism of the effect of tanshinone IIA epithelial-mesenchymal transition via the TGF-β1/smad pathway in human alveolar epithelial cell line A549

This study aimed to investigate the inhibitory effects of the different concentrations of Tanshinone IIA (Tan IIA) on the proliferation of human alveolar epithelial cell line A549 and its regulatory mechanism in epithelial-mesenchymal transition (EMT). The proliferation activity of cells was examined using the thiazolyl blue tetrazolium bromide (MTT) method. The changes in the expression of epithelial cell marker protein E-cadherin (E-cad) and interstitial marker protein alpha-smooth muscle actin (α-SMA) were detected using the cellular immunochemical method. The changes in cell morphology and ultrastructure were observed under the inverted microscope and transmission electron microscope, respectively. Western blot analysis was used to detect the expression of E-cad, α-SMA, Smad7, and Smad3. The MTT assay showed that the cell viability in the transforming growth factor beta 1 (TGF-β1) induced group was higher than that in the normal group, but the difference was not obvious. However, the cell viability of the Tan IIA-treated groups obviously decreased compared with the TGF-β1-induced and normal groups. Meanwhile, the expression of E-cad and Smad7 decreased, and the expression of α- SMA and Smad3 increased after A549 cells were induced by 5 ng/mL TGF-β1 for 24 h. However, their expression levels were close to the expression level of the control group after the cells were treated with Tan IIA for 24 h. In conclusion, the results demonstrated that Tan IIA could inhibit EMT of alveolar epithelial cells induced by TGF-β1, probably by regulating the expression of TGF-β/Smad pathway protein. Therefore, Tan IIA might serve as a potential anti-fibrosis drug in treating pulmonary fibrosis.