A549 Cells In Response Research Articles

Salt gradient enhanced sensitivity in nanopores for intracellular calcium ion detection

Intracellular calcium ion detection is of great significance for understanding the cell metabolism and signaling pathways. Most of the current ionic sensors either face the size issue or sensitivity limit for the intracellular solution with high background ion concentrations. In this paper, we proposed a calmodulin (CaM) functionalized nanopore for sensitive and selective Ca2+ detection inside living cells. A salt gradient was created when the nanopore sensor filled with a low concentration electrolyte was in contact with a high background concentration solution, which enhanced the surface charge-based detection sensitivity. The nanopore sensor showed a 10 × sensitivity enhancement by application of a 100-fold salt gradient, and a detection limit of sub nM. The sensor had a wide detection range from 1 nM to 1 mM, and allowed for quick calcium ion quantification in a few seconds. The sensor was demonstrated for intracellular Ca2+ detection in A549 cells in response to ionomycin.

Mechanism of the combined action of green tea polyphenols and concurrent radiochemotherapy in regulating GSK-3β to treat non-small cell lung cancer through the Wnt∕β-catenin pathway.

Green tea, derived from Camellia sinensis, contains polyphenolic active compounds that exhibit diverse pharmacological effects including anticancer, anti-inflammatory, antioxidant, and immunomodulatory properties. Employing various concentrations of green tea polyphenols (GTPs; 0, 100, 200, 300, 400, 500 μg∕mL), human normal lung epithelial cells (BEAS-2B) and non-small cell lung cancer (NSCLC) cells (A549) underwent treatment. The cell viability was assessed using the cell counting kit-8 (CCK-8) assay, proliferation was examined through the colony formation assay, apoptosis was monitored via flow cytometry, cell migration, and epithelial-mesenchymal transition (EMT)-associated proteins (E-cadherin, N-cadherin) were determined by Western blot. A549 cells were subjected to Cisplatin (0, 0.5, 1, 1.5 μM) and X-ray irradiation (0, 2, 4, 6 Gy) for treatment to probe the influence of GTPs on A549 cells in response to chemoradiotherapy. The functioning mechanism of GTPs in the context of NSCLC was validated using lithium chloride (LiCl) [a glycogen synthase kinase-3 beta (GSK-3β) inhibitor], which activates the Wnt∕β-catenin pathway. GTPs suppressed NSCLC cell viability in a concentration-dependent pattern, with a half-maximal inhibitory concentration (IC50) of 362.5 μg∕mL, while showing little impact on BEAS-2B cells' viability (at concentrations not exceeding 500 μg∕mL). Treatment with GTPs dampened colony formation of NSCLC cells, while promoting apoptosis. LiCl treatment vigorously attenuated the inhibitory impact of GTPs on the malignant phenotype of NSCLC cells. Mechanistic studies suggested that GTPs strengthened GSK-3β stability, thereby impeding the Wnt∕β-catenin pathway. Tea polyphenols (TPs) in conjunction with concurrent radiochemotherapy (CRCT) enhance the stability of GSK-3β and dampen the Wnt∕β-catenin pathway, hence exerting anticancer effects in NSCLC.

Identification and characterization of circular RNAs in the A549 cells following Influenza A virus infection

Influenza A virus (IAV) is one of the most dominant zoonotic-pathogen that causes annually recurring epidemic disease. The detailed molecular mechanism underlying IAV infection is still not fully understood. Circular RNAs (circRNAs) are generated from RNA back-splicing and involved in diverse biological processes. Here, we employed high-throughput circRNA microarray technology to profile circRNA expression in A549 cells in response to IAV infection. The analysis data revealed that 178 circRNAs expression were significantly upregulated while 137 downregulated, respectively, compared to the mock (P＜0.05, Fold Change＞2). Subsequently, Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were also conducted. Moreover dysregulated circRNAs were characterized, and of which nine were verified by quantitative real-time PCR (qRT-PCR). We further confirmed that circRNA_0082633 expression was increased following IAV infection. Overexpression of circRNA_0082633 suppressed IAV infection while depletion of circRNA_0082633 promoted viral proliferation. Interestingly, the activation of Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling was involved in IAV-induced circ_0082633 expression. More importantly, we demonstrated that circ_0082633 expression enhanced type I interferon (IFN) signaling by IFN-stimulated response element (ISRE) promoter activity and Ifnb1 mRNA levels. These data firstly provided the expression profile of circRNAs in PR8-infected A549 cells and shed new light on the pathogenesis research of IAV infection. Our findings also suggest that circRNA_0082633 served an important function in IAV infection.

A Repurposed Drug Screen for Compounds Regulating Aquaporin 5 Stability in Lung Epithelial Cells.

Aquaporin 5 (AQP5) is expressed in several cell types in the lung and regulates water transport, which contributes to barrier function during injury and the composition of glandular secretions. Reduced AQP5 expression is associated with barrier dysfunction during acute lung injury, and strategies to enhance its expression are associated with favorable phenotypes. Thus, pharmacologically enhancing AQP5 expression could be beneficial. Here, we optimized a high-throughput assay designed to detect AQP5 abundance using a cell line stably expressing bioluminescent-tagged AQP5. We then screened a library of 1153 compounds composed of FDA-approved drugs for their effects on AQP5 abundance. We show compounds Niclosamide, Panobinostat, and Candesartan Celexitil increased AQP5 abundance, and show that Niclosamide has favorable cellular toxicity profiles. We determine that AQP5 levels are regulated in part by ubiquitination and proteasomal degradation in lung epithelial cells, and mechanistically Niclosamide increases AQP5 levels by reducing AQP5 ubiquitination and proteasomal degradation. Functionally, Niclosamide stabilized AQP5 levels in response to hypotonic stress, a stimulus known to reduce AQP5 levels. In complementary assays, Niclosamide increased endogenous AQP5 in both A549 cells and in primary, polarized human bronchial epithelial cells compared to control-treated cells. Further, we measured rapid cell volume changes in A549 cells in response to osmotic stress, an effect controlled by aquaporin channels. Niclosamide-treated A549 cell volume changes occurred more rapidly compared to control-treated cells, suggesting that increased Niclosamide-mediated increases in AQP5 expression affects functional water transport. Taken together, we describe a strategy to identify repurposed compounds for their effect on AQP5 protein abundance. We validated the effects of Niclosamide on endogenous AQP5 levels and in regulating cell-volume changes in response to tonicity changes. Our findings highlight a unique approach to screen for drug effects on protein abundance, and our workflow can be applied broadly to study compound effects on protein abundance in lung epithelial cells.

Quantitative Proteomic Approach Reveals Altered Metabolic Pathways in Response to the Inhibition of Lysine Deacetylases in A549 Cells under Normoxia and Hypoxia.

Growing evidence is showing that acetylation plays an essential role in cancer, but studies on the impact of KDAC inhibition (KDACi) on the metabolic profile are still in their infancy. Here, we analyzed, by using an iTRAQ-based quantitative proteomics approach, the changes in the proteome of KRAS-mutated non-small cell lung cancer (NSCLC) A549 cells in response to trichostatin-A (TSA) and nicotinamide (NAM) under normoxia and hypoxia. Part of this response was further validated by molecular and biochemical analyses and correlated with the proliferation rates, apoptotic cell death, and activation of ROS scavenging mechanisms in opposition to the ROS production. Despite the differences among the KDAC inhibitors, up-regulation of glycolysis, TCA cycle, oxidative phosphorylation and fatty acid synthesis emerged as a common metabolic response underlying KDACi. We also observed that some of the KDACi effects at metabolic levels are enhanced under hypoxia. Furthermore, we used a drug repositioning machine learning approach to list candidate metabolic therapeutic agents for KRAS mutated NSCLC. Together, these results allow us to better understand the metabolic regulations underlying KDACi in NSCLC, taking into account the microenvironment of tumors related to hypoxia, and bring new insights for the future rational design of new therapies.

Cyclophilin A inhibits A549 cell oxidative stress and apoptosis by modulating the PI3K/Akt/mTOR signaling pathway.

The excessive and inappropriate production of reactive oxygen species (ROS) can cause oxidative stress and is implicated in the pathogenesis of lung cancer. Cyclophilin A (CypA), a member of the immunophilin family, is secreted in response to ROS. To determine the role of CypA in oxidative stress injury, we investigated the role that CypA plays in human lung carcinoma (A549) cells. Here, we showed the protective effect of human recombinant CypA (hCypA) on hydrogen peroxide (H2O2)-induced oxidative damage in A549 cells, which play crucial roles in lung cancer. Our results demonstrated that hCypA substantially promoted cell viability, superoxide dismutase (SOD), glutathione (GSH), and GSH peroxidase (GSH-Px) activities, and attenuated ROS and malondialdehyde (MDA) production in H2O2-induced A549 cells. Compared with H2O2-induced A549 cells, Caspase-3 activity in hCypA-treated cells was significantly reduced. Using Western blotting, we showed that hCypA facilitated Bcl-2 expression and inhibited Bax, Caspase-3, Caspase-7, and PARP-1 expression. Furthermore, hCypA activates the PI3K/Akt/mTOR pathway in A549 cells in response to H2O2 stimulation. Additionally, peptidyl-prolyl isomerase activity was required for PI3K/Akt activation by CypA. The present study showed that CypA protected A549 cells from H2O2-induced oxidative injury and apoptosis by activating the PI3K/Akt/mTOR pathway. Thus, CypA might be a potential target for lung cancer therapy.

Annurca Apple Biophenols’ Effects in Combination with Cisplatin on A549 Cells

Background: The role of a healthy and balanced diet in cancer prevention should never be underestimated; some nutritional factors can reduce the side effects of chemotherapy or radiotherapy and contribute to increasing its effectiveness. Therefore, in recent years, it has been thought to associate the administration of antioxidants to the chemotherapy approach that can protect non-tumor cells from the cytotoxic action of these drugs. However, the protective action of these substances could also limit the chemotherapy effects against the neoplastic cells themselves. Objective: In this context, the goal of this work was to test the viability of cultured human non-small lung cancer A549 cells in response to the combined administration of cisplatin (CDDP) and polyphenols. In particular, Annurca apple flesh polyphenol extract (AFPE) action was examined. Methods: A549 cells were treated with AFPE alone or in combination with CDDP and then cell viability was measured by the MTT assay. The effects of constituent polyphenols (+)-catechin, (-)- epicatechin, and caffeic acid) in AFPE were also evaluated. The cell morphology was observed by an inverted phase-contrast microscope. Results: CDDP reduced A549 cell viability in both concentration- and time-dependent manners. Polyphenols and CDDP co-administration did not interfere with the CDDP efficacy, in fact, the cellular vitality was found to be similar to that detected in the samples treated with CDDP alone. Conclusion: In conclusion, the co-administration of AFPE with CDDP does not interfere with its chemotherapy efficacy. Therefore, Annurca apple could be a good candidate to act as an antioxidant and potentially reduce the side effects of CDDP therapy, although the joint effect of AFPE and CDDP on normal cells is still unclear. More studies will be needed to analyze the molecular mechanism of AFPE and in vivo studies will also be needed to verify the anticancer effects. These findings may represent a starting point for the design of new clinical trials for use in cancer treatment.

RNAseq profiling of circRNA expression in radiation-treated A549 cells and bioinformatics analysis of radiation-related circRNA-miRNA networks.

With the development of new biochemical and computational methods, circular RNAs (circRNAs) have been identified as microRNA sponges. circRNAs are associated with many diseases, particularly cancer. The present study aimed to investigate the expression profile of circRNAs in irradiated A549 lung cancer cells using high-throughput sequencing. Bioinformatics analyses were used to examine the potential functions of circRNAs. RNA sequencing data demonstrated that 1,875 circRNA targets were differentially expressed in A549 cells in response to irradiation. A total of 30 circRNAs were upregulated and 37 circRNAs were downregulated significantly in irradiation-treated A549 cells (fold change ≥2.0; P<0.05). The top 5 upregulated and downregulated circRNAs were successfully validated by reverse transcription-quantitative PCR. In addition, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis suggested that differentially expressed circRNAs might be pivotal in biological irradiation responses to irradiation. circRNA-microRNA co-expression networks highlighted the biological significance of circRNA_0002174 and circRNA_0036627, which require further study. In conclusion, the present study is, to the best of the authors' knowledge, the first to describe the differentially expressed profile of circRNAs in response to irradiation in A549 cells. These results provide a new perspective to elucidate insight into the molecular mechanisms by which A549 cells respond to radiation, and a basis for a more in-depth analysis of the potential application of circRNAs in the treatment of lung cancer therapy.

An across-species comparison of the sensitivity of different organisms to Pb-based perovskites used in solar cells

Organic–inorganic perovskite solar cells (PSCs) are promising candidates as photovoltaic cells. Recently, they have attracted significant attention due to certified power conversion efficiencies exceeding 23%, low–cost engineering, and superior electrical/optical characteristics. These PSCs extensively utilize a perovskite–structured composite with a hybrid of Pb-based nanomaterials. Operation of them may cause the release of Pb-based nanoparticles. However, limited information is available regarding the potential toxicity of Pb-based PSCs on various organisms. This study conducted a battery of in vitro and in vivo toxicity bioassays for three quintessential Pb-based PSCs (CH3NH3PbI3, NHCHNH3PbBr3, and CH3NH3PbBr3) using progressively more complex forms of life. For all species tested, the three different perovskites had comparable toxicities. The viability of Caco–2/TC7 cells was lower than that of A549 cells in response to Pb-based PSC exposure. Concentration–dependent toxicity was observed for the bioluminescent bacterium Vibrio fischeri, for soil bacterial communities, and for the nematode Caenorhabditis elegans. Neither of the tested Pb-based PSCs particles had apparent toxicity to Pseudomonas putida. Among all tested organisms, V. fischeri showed the highest sensitivity with EC50 values (30 min of exposure) ranging from 1.45 to 2.91 mg L-1. Therefore, this study recommends that V. fischeri should be preferably utilized to assess.PSC toxicity due to its increased sensitivity, low costs, and relatively high throughput in a 96–well format, compared with the other tested organisms. These results highlight that the developed assay can easily predict the toxic potency of PSCs. Consequently, this approach has the potential to promote the implementation of the 3Rs (Replacement, Reduction, and Refinement) principle in toxicology and decrease the dependence on animal testing when determining the safety of novel PSCs.

Quantitative proteomic analysis of A549 cells infected with human adenovirus type 2.

Human adenovirus (HAdV-2) is considered a common agent of respiratory tract infection in the human, especially in children. Virus infection is believed to modify host cell expression necessary for its replication and therefore cell proteome can reflect the changes of specific cellular pathways during infection. This study aims to identify differentially expressed proteins of A549 cells in response to HAdV-2 infection using a label-free liquid chromatography-high-resolution tandem mass spectrometry strategy (LC-MS/MS) at 24 and 48 hpi. A total of 248 and 216 proteins were deregulated by 1.35-fold at 24 and 48 hpi, respectively. Among them, 155 were upregulated at 24 hpi and 86 at 48 hpi, whereas 93 and 130 were downregulated at 24 and 48 hpi, respectively. The identified proteins were involved in different pathways as energy, transcription, protein synthesis, cytoskeleton, rescue and defense, cell cycle, DNA processing, transportation, and metabolism. Glycolytic pathway and histone deregulated proteins were further confirmed by chemical testing and immunofluorescence, respectively. The results suggest that the identified proteins influenced HAdV-2 infection in the context of viral replication and propagation. This study complement proteomic data obtained from previous studies and reinforce the understanding of the relationship between HAdV and host cell.

Profiling of apoptosis- and autophagy-associated molecules in human lung cancer A549 cells in response to cisplatin treatment using stable isotope labeling with amino acids in cell culture.

Cis‑diammine‑dichloro‑platinum II‑based adjuvant chemotherapy provides an alternative therapy to improve the survival of patients with lung tumors, especially those with non‑small cell lung cancer (NSCLC). However, drug resistance is a large clinical problem and its underlying mechanism remains unclear. In the present study, NSCLC A549 cells were treated with a low concentration of cisplatin in order to observe and determine the development of chemoresistance, via growth curves, colony forming assays and apoptosis assays. Then the induction of autophagy was examined in the cisplatin‑treated A549 cells with a fluorescence reporter. Profiled proteins in the cisplatin‑treated A549 cells were also assessed using the stable isotope labeling by amino acids in cell culture (SILAC) method, and then the differentially expressed molecules were verified. The results demonstrated that A549 cells became less sensitive to cisplatin [resistant A549 cells (A549R)] following 20passages in the medium containing a low concentration of cisplatin, with less apoptotic cells post‑cisplatin treatment. A549R cells grew more efficiently in the cisplatin medium, with more colony formation and more cells migrating across the baseline. In addition, NSCLC results demonstrated that more autophagy‑related proteins (ATGs) were expressed in the A549R cells. Furthermore, the western blotting results confirmed this upregulation of ATGs in A549R cells. In addition, two repeated SILAC screening experiments recognized 15proteins [glucose‑regulated protein, 78kDa (GRP78), heat shock protein71, pre‑mRNA processing factor19, polypyrimidine tract binding protein1, translationally controlled tumor protein, CathepsinD, Cytochromec, thioredoxin domain containing5, MutS homolog (MSH)6, AnnexinA2 (ANXA2), BRCA2 and Cyclin dependent kinase inhibitor1A interacting protein, MSH2, protein phosphatase2A 55kDa regulatory subunitBα, Rho glyceraldehyde‑3‑phosphate‑dissociation inhibitor1 and ANXA4] that were upregulated by >1.5‑fold in heavy(H)‑ and light(L)‑labeled A549R cells. In addition, 16 and 14proteins were downregulated by >1.5‑fold in the H‑ and L‑labeled A549R cells, respectively. The majority of the downregulated proteins were associated with apoptosis. In conclusion, the present study isolated a cisplatin‑resistant human lung cancer A549 cell clone, with reduced apoptosis and high levels of autophagy, in response to cisplatin treatment. In cisplatin‑resistant A549R cells, SILAC proteomics recognized the high expression of GRP78 and other proteins that are associated with anti‑apoptosis and/or autophagy promotion.

The Class IV human deacetylase, HDAC11, exhibits anti-influenza A virus properties via its involvement in host innate antiviral response.

Histone deacetylase 11 (HDAC11) is most recently discovered deacetylase. Here, we demonstrate that human HDAC11 exhibits anti-influenza A virus (IAV) properties. We found that knockdown of HDAC11 expression augments IAV growth kinetics in human lung epithelial cells A549 by up to 1 log. One of the ways HDAC11 exerts its anti-IAV function is by being a part of IAV-induced host antiviral response. We found that the kinetics of both IAV- and interferon-induced innate antiviral response is significantly delayed in HDAC11-depleted cells. Further, in the absence of HDAC11 expression, there was a significant decrease in the expression of interferon-stimulated genes-IFITM3, ISG15, and viperin-previously implicated in anti-IAV function. One of the ways IAV antagonises HDAC11 is by downregulating its expression in host cells. We found that there was up to 93% reduction in HDAC11 transcript levels in A549 cells in response to IAV infection. HDAC11 is the smallest HDAC with majority of its polypeptide assigned to catalytic domain. Evolutionarily, it seems to be the least evolved and most closely related to common ancestral HDAC gene(s). Furthermore, HDAC11 has also been described as a deacylase. Therefore, our findings present exciting prospects for further investigations into significance of HDAC11 in virus infections.

Catalpol inhibits TGF-β1-induced epithelial-mesenchymal transition in human non-small-cell lung cancer cells through the inactivation of Smad2/3 and NF-κB signaling pathways.

Catalpol, one of the main active ingredients isolated from Rehmannia glutinosa, was reported to possess anticancer activity. However, the role of catalpol in transforming growth factor β1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) in human non-small-cell lung cancer (NSCLC) cells has not been elucidated. The objective of this study was to investigate the effect of catalpol on EMT in human NSCLC cells. Our results showed that catalpol significantly inhibited the TGF-β1-induced cell migration and invasion of A549 cells, as well as repressed matrix metalloproteinase (MMP)2 and MMP9 expression induced by TGF-β1 in A549 cells. In addition, catalpol markedly repressed the EMT process in A549 cells in response to TGF-β1. Furthermore, catalpol prevented the activation of Smad2/3 and nuclear factor κB (NF-κB) signaling pathways induced by TGF-β1 in A549 cells. In conclusion, these findings indicated that catalpol inhibits TGF-β1-induced EMT in human NSCLC cells through the inactivation of Smad2/3 and NF-κB signaling pathways. Thus, catalpol may be a promising agent for the treatment of NSCLC.

Notch signaling pathway suppresses CD8+ T cells activity in patients with lung adenocarcinoma

Evolution and progression of cancer always leads to CD8+ T cells dysfunction/exhaustion. Controversy remains as to the role of Notch signaling pathway in CD8+ T cells regulation in tumorigenesis. Thus, the aim of this study was to investigate the immunomodulatory activity of Notch signaling pathway to peripheral and lung-resident CD8+ T cells in patients with lung adenocarcinoma. Forty-eight lung adenocarcinoma patients and twenty healthy individuals were enrolled in the current study, and CD8+ T cells were purified from both peripheral bloods and bronchoalveolar lavage fluids. Notch receptor mRNA expression was semi-quantified by real-time PCR. Cytolytic and noncytolytic activity of CD8+ T cells evaluated in direct and indirect contact co-culture with A549 cells in response to Notch signaling inhibition by measuring of lactate dehydrogenase release and cytokines production. Expression of Fas ligand (FasL), perforin, and granzyme B were also assessed by flow cytometry. Notch2 mRNA expression was elevated in both peripheral and lung-resident CD8+ T cells in lung adenocarcinoma patients, however, did not correlated with tumor stages or epidermal growth factor receptor mutation. Peripheral CD8+ T cells from healthy individuals exhibited stronger cytotoxicity in direct contact co-culture system, which was not influenced by Notch signaling inhibition. Moreover, suppression of Notch signaling augmented cytotoxicity of peripheral and lung-resident CD8+ T cells from lung adenocarcinoma patients in direct contact co-culture system, and promoted interferon-γ production in both systems. This process was accompanied by increased expression of FasL and perforin within CD8+ T cells. The current data revealed a potential immunosuppressive property of Notch signaling pathway to CD8+ T cells probably via inhibition of FasL and perforin in lung adenocarcinoma patients.

A Radiosensitizing Inhibitor of HIF-1 alters the Optical Redox State of Human Lung Cancer Cells In Vitro

Treatment failure caused by a radiation-resistant cell phenotype remains an impediment to the success of radiation therapy in cancer. We recently showed that a radiation-resistant isogenic line of human A549 lung cancer cells had significantly elevated expression of hypoxia-inducible factor (HIF-1α), and increased glucose catabolism compared with the parental, radiation-sensitive cell line. The objective of this study was to investigate the longitudinal metabolic changes in radiation-resistant and sensitive A549 lung cancer cells after treatment with a combination of radiation therapy and YC-1, a potent HIF-1 inhibitor. Using label-free two-photon excited fluorescence microscopy, we determined changes in the optical redox ratio of FAD/(NADH and FAD) over a period of 24 hours following treatment with YC-1, radiation, and both radiation and YC-1. To complement the optical redox ratio, we also evaluated changes in mitochondrial organization, glucose uptake, reactive oxygen species (ROS), and reduced glutathione. We observed significant differences in the optical redox ratio of radiation-resistant and sensitive A549 cells in response to radiation or YC-1 treatment alone; however, combined treatment eliminated these differences. Our results demonstrate that the optical redox ratio can elucidate radiosensitization of previously radiation-resistant A549 cancer cells, and provide a method for evaluating treatment response in patient-derived tumor biopsies.

XAV939 inhibits the proliferation and migration of lung adenocarcinoma A549 cells through the WNT pathway.

The present study assessed the effects of the tankyrase (TNKS) small molecule inhibitor XAV939 on the proliferation and migration of lung adenocarcinoma A549 cells and the possible underlying mechanism. To do this, the association between TNKS and the WNT/β-catenin signaling pathway in lung acinar adenocarcinoma was investigated. Immunohistochemistry was performed, which demonstrated that TNKS, β-catenin and Myc proto-oncogene protein (c-Myc) proteins are positively expressed in lung adenocarcinoma tissue; this expression was significantly higher than that in normal adjacent non-carcinoma tissues. A549 cell proliferation was inhibited in all XAV939-intervention groups examined. In the wound-healing assay, cells treated with different concentrations of XAV939 exhibited a significantly increased scratch width compared with the control group. Reverse transcription-semi-quantitative polymerase chain reaction analysis revealed that β-catenin mRNA expression was significantly decreased in A549 cells in response to different XAV939 concentrations compared with the control group. Immunofluorescence revealed that β-catenin protein, initially localized in the nucleus/cytoplasm, gradually translocated to the cytoplasm/membrane, an effect that was associated with increased drug concentration. TNKS, β-catenin and c-Myc protein expression in A549 cells treated with XAV939 was reduced compared with that in untreated cells. Therefore, abnormally high TNKS expression may promote the occurrence of lung cancer. The TNKS inhibitor XAV939 inhibited lung adenocarcinoma A549 cell proliferation and migration in vitro. The underlying mechanism by which XAV939 exerted its inhibitory effects may be associated with attenuation of the WNT signaling pathway.

Antiproliferative and antimetastatic action of quercetin on A549 non-small cell lung cancer cells through its effect on the cytoskeleton

To our knowledge, this study is the first to investigate the effect of the dietary flavonoid quercetin on the main cytoskeletal elements, namely microfilaments, microtubules and vimentin intermediate filaments, as well as cytoskeleton-driven processes in A549 non-small cell lung cancer cells. The methyl-thiazol-diphenyl-tetrazolium assay, annexin V/propidium iodide test, electron microscopic examination, cell cycle analysis based on DNA content, real-time PCR assays, in vitro scratch wound-healing assay, fluorescence staining of F-actin, β-tubulin and vimentin were performed to assess the effects of quercetin on A549 cells. Our results showed that quercetin triggered BCL2/BAX-mediated apoptosis, as well as necrosis and mitotic catastrophe, and inhibited the migratory potential of A549 cells. The disassembling effect of quercetin on microfilaments, microtubules and vimentin filaments along with its inhibitory impact on vimentin and N-cadherin expression might account for the decreased migration of A549 cells in response to quercetin treatment. We also suggest that the possible mechanism underlying quercetin-induced mitotic catastrophe involves the perturbation of mitotic microtubules leading to monopolar spindle formation, and, consequently, to the failure of cytokinesis. We further propose that cytokinesis failure could also be a result of the depletion of actin filaments by quercetin. These findings are important to our further understanding of the detailed mechanism of the antitumor activity of quercetin and render this flavonoid a potentially useful candidate for combination therapy with conventional antimicrotubule drugs, nucleic acid-directed agents or novel cytoskeletal-directed agents.

De-novo NAD+ synthesis regulates SIRT1-FOXO1 apoptotic pathway in response to NQO1 substrates in lung cancer cells.

Tryptophan metabolism is essential in diverse kinds of tumors via regulating tumor immunology. However, the direct role of tryptophan metabolism and its signaling pathway in cancer cells remain largely elusive. Here, we establish a mechanistic link from L-type amino acid transporter 1 (LAT1) mediated transport of tryptophan and the subsequent de-novo NAD+ synthesis to SIRT1-FOXO1 regulated apoptotic signaling in A549 cells in response to NQO1 activation. In response to NQO1 activation, SIRT1 is repressed leading to the increased cellular accumulation of acetylated FOXO1 that transcriptionally activates apoptotic signaling. Decreased uptake of tryptophan due to the downregulation of LAT1 coordinates with PARP-1 hyperactivation to induce rapid depletion of NAD+ pool. Particularly, the LAT1-NAD+-SIRT1 signaling is activated in tumor tissues of patients with non-small cell lung cancer. Because NQO1 activation is characterized with oxidative challenge induced DNA damage, these results suggest that LAT1 and de-novo NAD+ synthesis in NSCLC cells may play essential roles in sensing excessive oxidative stress.

Analysis of ISG15-Modified Proteins from A549 Cells in Response to Influenza Virus Infection by Liquid Chromatography-Tandem Mass Spectrometry

Abstract Interferon stimulated gene 15 kDa protein (ISG15) is the first ubiquitin-like protein identified, which plays vital roles in a variety of fields including viral infection and immunological regulation. In this study, liquid chromatography-tandem mass spectrometry was used to analyze ISG15-modified proteins in A549 cells in response to infection by influenza virus, which were enriched by immunoprecipitation. A total of 22 cellular host proteins were identified in A549 cells infected by influenza virus, including ubiquitin-like ISG15 protein, cyclin-T1, heat shock protein 71 kDa, caldesmon, eukaryotic translation initiation factor, and so on. Besides, non-structural protein (NS1) from influenza virus was also identified. Among the 22 host proteins identified, 6 were also identified in the control non-infected A549 cells, including annexin A1, fructose-bisphosphate aldolase A, ATP synthase subunit g, enolase, actin, and tubulin. Bioinformatics analysis revealed that the identified ISG15-modified host proteins induced by influenza virus infection could be classified into 9 protein classes: chaperone, oxidoreductase, enzyme modulator, transferase, nucleic acid binding, transcription factor, kinase, cytoskeletal protein, and structural protein. This study provides a specific and effective tool for analyzing ISG15-modified proteins at proteome level.

NFATc1 regulates the transcription of DNA damage-induced apoptosis suppressor.

DNA damage induced apoptosis suppressor (DDIAS), or human Noxin (hNoxin), is strongly expressed in lung cancers. DDIAS knockdown induced apoptosis in non-small cell lung carcinoma A549 cells in response to DNA damage, indicating DDIAS as a potential therapeutic target in lung cancer. To understand the transcriptional regulation of DDIAS, we determined the transcription start site, promoter region, and transcription factor. We found that DDIAS transcription begins at nucleotide 212 upstream of the DDIAS translation start site. We cloned the DDIAS promoter region and identified NFAT2 as a major transcription factor (Im et al., 2016 [1]). We demonstrated that NFATc1 regulates DDIAS expression in both pancreatic cancer Panc-1 cells and lung cancer cells.