Human Lung Epithelial Cell Line Research Articles

Silica nanoparticles-induced cytotoxicity and genotoxicity in A549 cell lines

Among the myriad of nanoparticles, silica nanoparticles (SiO2NPs) have gained significant attention since they are extensively produced and used across several kinds of industries. Because of its widespread usage, there has been increasing concern about the potential health effects. This study aims to evaluate the effects of SiO2NPs on Interleukin-6 (IL-6) gene expression in human lung epithelial cell lines (A549). In this study, A549 cells were exposed to SiO2NPs at concentrations of 0, 1, 10, 50, 100, and 200 µg/mL for 24 and 48 h. The IL-6 gene expression was assessed using Real-Time RT-PCR. Additionally, the impact of SiO2NPs on the viability of A549 cells was determined by MTT assay. Statistical analysis was performed using GraphPad Prism software 8.0. MTT assay results indicated a concentration-dependent impact on cell survival. After 24 h, survival decreased from 80 to 68% (1–100 µg/mL), rising to 77% at higher concentrations. After 48 h, survival dropped from 97 to 80%, decreasing to 90% at higher concentrations. RT-PCR showed a dose–response relationship in cellular toxicity up to 10 µg/mL. At higher concentrations, there was increased IL-6 gene expression, mitigating SiO2NP-induced cytotoxic effects. The study shows that the viability and proliferation of A549 cells are impacted by different SiO2NPs concentrations. There may be a potential correlation between IL-6 gene expression reduction and a mechanism linked to cellular toxicity. However, at higher concentrations, an unknown mechanism increases IL-6 gene expression, reducing SiO2NPs' cytotoxic effects. These effects are concentration-dependent and not influenced by exposure times. Further investigation is recommended to determine this mechanism's nature and implications, particularly in cancer research.

Pneumococcal hydrogen peroxide regulates host cell kinase activity.

Protein kinases are indispensable reversible molecular switches that adapt and control protein functions during cellular processes requiring rapid responses to internal and external events. Bacterial infections can affect kinase-mediated phosphorylation events, with consequences for both innate and adaptive immunity, through regulation of antigen presentation, pathogen recognition, cell invasiveness and phagocytosis. Streptococcus pneumoniae (Spn), a human respiratory tract pathogen and a major cause of community-acquired pneumoniae, affects phosphorylation-based signalling of several kinases, but the pneumococcal mediator(s) involved in this process remain elusive. In this study, we investigated the influence of pneumococcal H2O2 on the protein kinase activity of the human lung epithelial H441 cell line, a generally accepted model of alveolar epithelial cells. We performed kinome analysis using PamGene microarray chips and protein analysis in Western blotting in H441 lung cells infected with Spn wild type (SpnWT) or with SpnΔlctOΔspxB -a deletion mutant strongly attenuated in H2O2 production- to assess the impact of pneumococcal hydrogen peroxide (H2O2) on global protein kinase activity profiles. Our kinome analysis provides direct evidence that kinase activity profiles in infected H441 cells significantly vary according to the levels of pneumococcal H2O2. A large number of kinases in H441 cells infected with SpnWT are significantly downregulated, whereas this no longer occurs in cells infected with the mutant SpnΔlctOΔspxB strain, which lacks H2O2. In particular, we describe for the first time H2O2-mediated downregulation of Protein kinase B (Akt1) and activation of lymphocyte-specific tyrosine protein kinase (Lck) via H2O2-mediated phosphorylation.

The Efficacy & Molecular Mechanisms of a Terpenoid Compound Ganoderic Acid C1 on Corticosteroid-Resistant Neutrophilic Airway Inflammation: In vivo and in vitro Validation.

Neutrophil predominant airway inflammation is associated with severe and steroid-resistant asthma clusters. Previously, we reported efficacy of ASHMI, a three-herb TCM asthma formula in a steroid-resistant neutrophil-dominant murine asthma model and further identified Ganoderic Acid C1 (GAC1) as a key ASHMI active compound in vitro. The objective of this study is to investigate GAC1 effect on neutrophil-dominant, steroid-resistant asthma in a murine model. In this study, Balb/c mice were systematically sensitized with ragweed (RW) and alum and intranasally challenged with ragweed. Unsensitized/PBS challenged mice served as normal controls. Post sensitization, mice were given 4 weeks of oral treatment with GAC1 or acute dexamethasone (Dex) treatment at 48 hours prior to challenge. Pulmonary cytokines were measured by ELISA, and lung sections were processed for histology by H&E staining. Furthermore, GAC1 effect on MUC5AC expression and on reactive oxygen species (ROS) production in human lung epithelial cell line (NCI-H292) was determined by qRT-PCR and ROS assay kit, respectively. Computational analysis was applied to select potential targets of GAC1 in steroid-resistant neutrophil-dominant asthma. Molecular docking was performed to predict binding modes between GAC1 and Dex with TNF-α. The result of the study showed that chronic GAC1 treatment, significantly reduced pulmonary inflammation (P < 0.01-0.001 vs Sham) and airway neutrophilia (P < 0.01 vs Sham), inhibited TNF-α, IL-4 and IL-5 levels (P < 0.05-0.001 vs Sham). Acute Dex treatment reduced eosinophilic inflammation and IL-4, IL-5 levels, but had no effect on neutrophilia and TNF-α production. GAC1 treated H292 cells showed decreased MUC5AC gene expression and production of ROS (P < 0.001 vs stimulated/untreated cells). Molecular docking results showed binding energy of complex GAC1-TNF was -10.8 kcal/mol. GAC1 may be a promising anti-asthma botanical drug for treatment of steroid-resistant asthma.

CircDDX17 targets miR-223-3p / RIP3 to regulate the proliferation and apoptosis of non-small cell lung cancer cells

Objective: To explore the molecular mechanism of circDDX17 regulating the proliferation and apoptosis of non-small cell lung cancer cells by targeting the miR-223-3p/RIP3 molecular axis. Methods: The expression levels of circDDX17, miR-223-3p, and RIP3 in human normal lung epithelial cell lines BEAS-2B and non-small cell lung cancer cells H1299, A549, and H446 were detected by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR). The plasmids of pcDNA, pcDNA-circDDX17, anti-miR-con, anti-miR-223-3p, pcDNA-circDDX17 and miR-con, pcDNA-circDDX17 and miR-223-3p mimics were transfected into H1299 cells. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) assay was used to detect the cell proliferation. Flow cytometry was used to detect the cell cycle and cell apoptosis. Plate cloning experiment was used to detect cell proliferation ability. The dual luciferase report experiment was applied to verify the targeting relationship between miR-223-3p with circDDX17 and RIP3. Western blot was used to detect the protein expression of cyclinD1, CDK2, cleaved caspase-3 and Bax. Results: The expression levels of circDDX17 and RIP3 mRNA in H1299, A549, and H446 cells were significantly reduced (P<0.05), the expression level of miR-223-3p mRNA was significantly increased (P<0.05) compared with BEAS-2B. The cell viability [(69.46±4.68)%], the number of cell clones (83.49±7.86), the proportion of cells in S phase [(22.52±1.41) %], the protein expression levels of cyclinD1 and CDK2 in PCDNa-CircDDX17 group were lower than those in pcDNA group [(97.54±7.72)%, 205.03±13.37, (28.69±1.49)%, respectively, P<0.05], while the percentage of G0/G1 phase cells [(64.45±3.56)%], apoptosis rate [(18.36±1.63)%], the protein expression levels of cleaved caspase-3 and Bax in pcDNA-circDDX17 group were higher than those of pcDNA group [(51.33±2.76) % and (5.21±0.54) %, respectively, P<0.05]. The viability [(72.64±5.44)%], the number of cell clones (78.16±8.23), the proportion of S-stage cells [(21.34±1.59) %], the protein expression levels of CyclinD1 and CDK2 in anti-miR-223-3p group were lower than those in anti-miR-con group [(103.47±6.25)%, 169.32±14.53, (28.43±1.26)%, respectively, P<0.05]. Percentage of G0/G1 phase cells [(62.86±3.28)%], apoptosis rate [(14.64±1.67)%], the protein expression levels of cleaved caspase-3 and Bax in the anti-miR-223-3p group were higher than those of anti-miR-con group [(51.33±2.71)% and (4.83±0.39)%, respectively, P<0.05]. MiR-223-3p has complementary sites with circDDX17 or RIP3. The viability [(135.45±9.28)%], the number of cell clones (174.64±10.68), the proportion of S-phase cells [(26.39±2.25)%], the protein expression levels of cyclinD1 and CDK2 in pcDNA-circDDX17+miR-223-3p group were higher than those in pcDNA-circDDX17+miR-con group [(101.56±6.68)%, 107.65±7.62, (21.64±1.72)%, P<0.05]. Percentage of G0/G1 phase cells [(56.64±2.76)%], apoptosis rate [(8.34±0.76)%], the protein expression levels of cleaved caspase-3 and Bax in pcDNA-circDDX17+miR-223-3p group were lower than those of pcDNA-circDDX17+miR-con group [(64.03±3.48)% and (15.21±1.18)%, respectively, P<0.05]. Conclusion: circDDX17 could inhibit the proliferation and induce apoptosis of non-small cell lung cancer cells via targeting the miR-223-3p / RIP3 molecular axis.

A novel tumor mutation-related long non-coding RNA signature for predicting overall survival and immunotherapy response in lung adenocarcinoma

BackgroundImmunotherapy has changed the treatment landscape for lung cancer. This study aims to construct a tumor mutation-related model that combines long non-coding RNA (lncRNA) expression levels and tumor mutation levels in tumor genomes to detect the possibilities of the lncRNA signature as an indicator for predicting the prognosis and response to immunotherapy in lung adenocarcinoma (LUAD). MethodsWe downloaded the tumor mutation profiles and RNA-seq expression database of LUAD from The Cancer Genome Atlas (TCGA). Differentially expressed lncRNAs were extracted based on the cumulative number of mutations. Cox regression analyses were used to identify the prognostic lncRNA signature, and the prognostic value of the five selected lncRNAs was validated by using survival analysis and the receiver operating characteristic (ROC) curve. We used qPCR to validate the expression of five selected lncRNAs between human lung epithelial and human lung adenocarcinoma cell lines. The ImmuCellAI, immunophenoscore (IPS) scores and Tumor Immune Dysfunction and Exclusion (TIDE) analyses were used to predict the response to immunotherapy for this mutation related lncRNA signature. ResultsA total of 162 lncRNAs were detected among the differentially expressed lncRNAs between the Tumor mutational burden (TMB)-high group and the TMB-low group. Then, five lncRNAs (PLAC4, LINC01116, LINC02163, MIR223HG, FAM83A-AS1) were identified as tumor mutation-related candidates for constructing the prognostic prediction model. Kaplan‒Meier curves showed that the overall survival of the low-risk group was significantly better than that of the high-risk group, and the results of the GSE50081 set were consistent. The expression levels of PD1, PD-L1 and CTLA4 in the low-risk group were higher than those in the high-risk group. The IPS scores and TIDE scores of patients in the low-risk group were significantly higher than those in the high-risk group. ConclusionOur findings demonstrated that the five lncRNAs (PLAC4, LINC01116, LINC02163, MIR223HG, FAM83A-AS1) were identified as candidates for constructing the tumor mutation-related model which may serve as an indicator of tumor mutation levels and have important implications for predicting the response to immunotherapy in LUAD.

Evaluation of hsa_circ_0000018/let-7f-5p/ FAM96A axis in lung adenocarcinoma progression.

Circular RNAs (circRNAs) are critical regulators of lung adenocarcinoma (LA) progression. Although a molecular marker targeting hsa_circ_0000018 has been developed and used for diagnosing colon cancer, the role of this circRNA in LA progression has not been explored till now. This study aimed to elucidate the role and regulatory mechanisms of hsa_circ_0000018 in LA progression. LA tissues and corresponding adjacent non-tumor tissues were collected from 36 patients to confirm the levels of circRNAs, microRNAs (miRNAs), and messenger RNAs (mRNAs) using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). We also cultured two LA cell lines (A549, PC-9), and the human normal lung epithelial cell line BEAS-2B. Cell function experiments were conducted to assess malignancy in LA cells, including proliferation, migration, and invasion, following forced hsa_circ_0000018 expression. The correlation between hsa_circ_0000018, let-7f-5p, and family with sequence similarity 96 member A (FAM96A) was confirmed by using starBase (miRNA-circRNA interaction database), luciferase assay, and western blotting. Expression of hsa_circ_0000018 and FAM96A was reduced, whereas that of let-7f-5p was upregulated in LA. Cell function assays revealed that upregulation of hsa_circ_0000018 had a suppressive effect on the proliferation, migration, and invasion of LA cells. Additionally, hsa_circ_0000018 sponge binds let-7f-5p, resulting in upregulation of FAM96A expression. Our data reveal hsa_circ_0000018 as a tumor suppressor in LA that targets the let-7f-5p/FAM96A axis. Our findings enrich the known regulatory network of circRNAs in LA.

Signal Transducer and Activator of Transcription 4-Induced Up-Regulated LINC01278 Enhances Proliferation and Invasion of Non-Small Cell Lung Cancer Cells via the MicroRNA-877-5p/Activating Transcription Factor 4 Axis.

The purpose of this study was to investigate the specific effects of signal transducer and activator of transcription 4 (STAT4)-induced long intergenic nonprotein coding RNA 1278 (LINC01278) on the growth of non-small cell lung cancer (NSCLC) cells involved in the microRNA (miR)-877-5p/activated transcription factor 4 (ATF4) axis. NSCLC tumor tissue and adjacent normal tissue were collected. Human normal lung epithelial cell BEAS-2B and human NSCLC cell lines (H1299, H1975, A549, H2228) were collected. The expression levels of STAT4, LINC01278, miR-877-5p, and ATF4 were detected. A549 cells were screened for subsequent experiments. The proliferation ability of cells was detected by colony formation experiment. Cell apoptosis was tested by flow cytometry. Scratch test and transwell assay were used to detect the migration and invasion ability of cells. Biological function of LINC01278 in NSCLC was confirmed by xenograft experiments. Low expression miR-877-5p and high expression of STAT4, LINC01278 and ATF4 were detected in NSCLC. Silenced LINC01278 in A549 cell depressed cell proliferation, migration and invasion, but facilitated cell apoptosis. LINC01278 was positively correlated with STAT4 and could directly bind to miR-877-5p. Upregulating miR-877-5p suppressed NSCLC cell progression, while downregulating miR-877-5p had the opposite effect. Upregulating miR-877-5p abrogated the effects of silenced LINC01278 on NSCLC cell progression. MiR-877-5p targeted ATF4. ATF4 upregulation could partly restore the carcinogenic effect of LINC01278 in vitro and in vivo. Our data supports that STAT4-induced upregulation of LINC01278 promotes NSCLC progression by modulating the miR-877-5p/ATF4 axis, suggesting a novel direction for NSCLC treatment.

Ionic Liquid-Based Polymer Matrices for Single and Dual Drug Delivery: Impact of Structural Topology on Characteristics and In Vitro Delivery Efficiency.

Previously reported amphiphilic linear and graft copolymers, derived from the ionic liquid [2-(methacryloyloxy)ethyl]trimethylammonium chloride (TMAMA_Cl‾), along with their conjugates obtained through modification either before or after polymerization with p-aminosalicylate anions (TMAMA_PAS‾), were employed as matrices in drug delivery systems (DDSs). Based on the counterion type in TMAMA units, they were categorized into single drug systems, manifesting as ionic polymers with chloride counterions and loaded isoniazid (ISO), and dual drug systems, featuring ISO loaded in self-assembled PAS conjugates. The amphiphilic nature of these copolymers was substantiated through the determination of the critical micelle concentration (CMC), revealing an increase in values post-ion exchange (from 0.011-0.063 mg/mL to 0.027-0.181 mg/mL). The self-assembling properties were favorable for ISO encapsulation, with drug loading content (DLC) ranging between 15 and 85% in both single and dual systems. In vitro studies indicated ISO release percentages between 16 and 61% and PAS release percentages between 20 and 98%. Basic cytotoxicity assessments using the 2,5-diphenyl-2H-tetrazolium bromide (MTT) test affirmed the non-toxicity of the studied systems toward human non-tumorigenic lung epithelial cell line (BEAS-2B) cell lines, particularly in the case of dual systems bearing both ISO and PAS simultaneously. These results confirmed the effectiveness of polymeric carriers in drug delivery, demonstrating their potential for co-delivery in combination therapy.

PSMD12 promotes non-small cell lung cancer progression through activating the Nrf2/TrxR1 pathway.

Non-small cell lung cancer (NSCLC) contributes to the vast majority of cancer-related deaths. Proteasome 26S subunit, non-ATPase 12 (PSMD12), a subunit of 26S proteasome complex, is known to play the tumor-promoting role in several types of cancer but its function in NSCLC remains elusive. To explore the role and underlying mechanisms of PSMD12 in NSCLC. The PSMD12 expression in human normal lung epithelial cell line (BEAS-2B) and four NSCLC cell lines (A549, NCI-H1299, NCI-H1975, Calu-1) were determined by qRT-PCR and western blot. Malignant phenotypes of NSCLC cells were detected by CCK-8, EdU staining, immunofluorescence staining for E-cadherin, flow cytometry, and Transwell assays to assess cell viability, proliferation, epithelial-mesenchymal transition (EMT), apoptosis, migration and invasion. Dual luciferase assay was used to verify the regulatory role of transcription factor on the promoter. We identified the upregulation of PSMD12 in NSCLC tissues based on the GEO datasets, which further verified in NSCLC and BEAS-2B cell lines. PSMD12 knockdown significantly suppressed malignant behaviors of NSCLC cells, including cell growth, invasion, and migration, while PSMD12 overexpression presented the opposite effects. Interestingly, we found that PSMD12 upregulated the tumor-promoting factor TrxR1 mRNA expression. For its potential mechanisms, we demonstrated that PSMD12 elevated transcription factor Nrf2 protein level and promoted Nrf2 nuclear translocation. And Nrf2 further increased TrxR1 promoter activity and enhanced TrxR1 transcription. Meanwhile, we proved that TrxR1 overexpression erased the inhibitory effect of PSMD12 knockdown. PSMD12 promotes NSCLC progression by activating the Nrf2/TrxR1 pathway, providing a novel prognostic and therapeutic target for NSCLC treatment.

In Vitro and In Silico Studies on 4-Nitroacetophenone Thiosemicarbazone Potential Cytotoxicity Against A549 Cell Lines.

Lung malignancy is a major worldwide issue that occurs due to the dysregulation of various growth factors. Lung cancer has no apparent signs in the early stages, which makes it harder to catch it in time and leads to a higher fatality rate. So, the goal of this work was to create and analyze a novel chemical molecule called 4-nitro acetophenone thiosemicarbazone (4-NAPTSc) against the lung cancer cell line A549 and human non-tumorigenic lung epithelial cell line BAES-2B. The ligand was synthesized by refluxing the reaction mixture of 4-nitro acetophenone and thiosemicarbazide and was further characterized by UV, FTIR, and 1H and 13C NMR and Differential Scanning Calorimetry (DSC) study. Cytotoxicity assay/MTT (3-(4,5-dimethylthiazol-2-yl))2,5-diphenyltetrazolium bromide) was used to evaluate the cytotoxicity of the compound. Epidermal growth factor receptors (EGFR), polo-like kinase-1 (PLK1), and vascular endothelial growth factor receptors (VEGFR) were chosen as the target proteins for molecular docking to find potential ligand binding sites and inhibit their function. A novel yellow-colored crystalline solid has been synthesized. 4-NAPTSc had an IC50 of 2.93 μg/mL against the A549 lung cancer cells. When the dosage is increased from 5 to 15 μg/mL along with time, the cell viability falls. Docking results showed that the compound binds with the targeted proteins' amino acid residues, and the likeness profile of the compound is also favorable. This study reveals that the compound has the potential for further investigation and can be used in multitargeted cancer therapies.

IFN-γ-mediated control of SARS-CoV-2 infection through nitric oxide.

The COVID-19 pandemic has highlighted the need to identify mechanisms of antiviral host defense against SARS-CoV-2. One such mediator is interferon-g (IFN-γ), which, when administered to infected patients, is reported to result in viral clearance and resolution of pulmonary symptoms. IFN-γ treatment of a human lung epithelial cell line triggered an antiviral activity against SARS-CoV-2, yet the mechanism for this antiviral response was not identified. Given that IFN-γ has been shown to trigger antiviral activity via the generation of nitric oxide (NO), we investigated whether IFN-γ induction of antiviral activity against SARS-CoV-2 infection is dependent upon the generation of NO in human pulmonary epithelial cells. We treated the simian epithelial cell line Vero E6 and human pulmonary epithelial cell lines, including A549-ACE2, and Calu-3, with IFN-γ and observed the resulting induction of NO and its effects on SARS-CoV-2 replication. Pharmacological inhibition of inducible nitric oxide synthase (iNOS) was employed to assess the dependency on NO production. Additionally, the study examined the effect of interleukin-1b (IL-1β) on the IFN-g-induced NO production and its antiviral efficacy. Treatment of Vero E6 cells with IFN-γ resulted in a dose-responsive induction of NO and an inhibitory effect on SARS-CoV-2 replication. This antiviral activity was blocked by pharmacologic inhibition of iNOS. IFN-γ also triggered a NO-mediated antiviral activity in SARS-CoV-2 infected human lung epithelial cell lines A549-ACE2 and Calu-3. IL-1β enhanced IFN-γ induction of NO, but it had little effect on antiviral activity. Given that IFN-g has been shown to be produced by CD8+ T cells in the early response to SARS-CoV-2, our findings in human lung epithelial cell lines, of an IFN-γ-triggered, NO-dependent, links the adaptive immune response to an innate antiviral pathway in host defense against SARS-CoV-2. These results underscore the importance of IFN-γ and NO in the antiviral response and provide insights into potential therapeutic strategies for COVID-19.

A novel G3BP1-GFP reporter human lung cell system enabling real-time monitoring of stress granule dynamics for in vitro lung toxicity assessment

Under various cellular stress conditions, including exposure to toxic chemicals, RNA-binding proteins (RBPs), including Ras GTPase-activating protein-binding protein 1 (G3BP1), aggregate and form stress granule complexes, which serve as hallmarks of cellular stress. The existing methods for analyzing stress granule assembly have limitations in the rapid detection of dynamic cellular stress and ignore the effects of constitutively overexpressed RBP on cellular stress and stress-related processes. Therefore, to overcome these limitations, we established a G3BP1-GFP reporter in a human lung epithelial cell line using CRISPR/Cas9-based knock-in as an alternative system for stress granule analysis. We showed that the G3BP1-GFP reporter system responds to stress conditions and forms a stress granule complex similar to that of native G3BP1. Furthermore, we validated the stress granule response of an established cell line under exposure to various household chemicals. Overall, this novel G3BP1-GFP reporter human lung cell system is capable of monitoring stress granule dynamics in real time and can be used for assessing the lung toxicity of various substances in vitro.

C5a/C5aR1 axis as a key driver promotes epithelial-to-mesenchymal transition in airway epithelial cells in silica nanoparticles-induced pulmonary fibrosis

Previous studies have shown that silica nanoparticles (SiNPs) exposure can affect the respiratory, cardiovascular, reproductive and other systems, with the lung being the primary target organ for the direct effect, causing damage with a central feature of pulmonary inflammation and fibrosis. However, the underlying mechanisms of pulmonary fibrosis due to SiNPs are not fully understood. The aim of the study was to investigate the role of complement anaphylatoxin C5a in SiNPs-induced pulmonary fibrosis. A mouse model of SiNPs-induced pulmonary fibrosis was established, and pulmonary fibrosis-related indicators, epithelial-to-mesenchymal transition (EMT), C5a/C5aR1 and high mobility group protein B1 (HMGB1) proteins were measured. An in vitro study using the human lung epithelial cell line BEAS-2B investigated whether C5a leads to epithelial-to-mesenchymal trans-differentiation. In vivo studies revealed that SiNPs-induced pulmonary fibrosis mainly manifested as EMT trans-differentiation in airway epithelial cells, which subsequently led to excessive deposition of extracellular matrix (ECM). Furthermore, we found that C5a and C5aR1 proteins were also increased in SiNPs-induced pulmonary fibrosis tissue. In vitro studies also showed that C5a directly activated HMGB1/RAGE signaling and induced EMT in BEAS-2B cells. Finally, treatment of SiNPs-exposed mice with the C5aR1 inhibitor PMX205 effectively reduced C5aR1 levels and inhibited the activation of HMGB1/RAGE signaling and the expression of EMT-related proteins, culminating in a significant alleviation of pulmonary fibrosis. Taken together, our results suggest that C5a/C5aR1 is the main signaling pathway for SiNPs-induced pulmonary fibrosis, which induces EMT in airway epithelial cells via the HMGB1/RAGE axis.

Avian influenza A viruses exhibit plasticity in sialylglycoconjugate receptor usage in human lung cells.

It is well known that influenza A viruses (IAV) initiate host cell infection by binding to sialic acid, a sugar molecule present at the ends of various sugar chains called glycoconjugates. These sugar chains can vary in chain length, structure, and composition. However, it remains unknown if IAV strains preferentially bind to sialic acid on specific glycoconjugate type(s) for host cell infection. Here, we utilized CRISPR gene editing to abolish sialic acid on different glycoconjugate types in human lung cells, and evaluated human versus avian IAV infections. Our studies show that both human and avian IAV strains can infect human lung cells by utilizing any of the three major sialic acid-containing glycoconjugate types, specifically N-glycans, O-glycans, and glycolipids. Interestingly, simultaneous elimination of sialic acid on all three major glycoconjugate types in human lung cells dramatically decreased human IAV infection, yet had little effect on avian IAV infection. These studies show that avian IAV strains effectively utilize other less prevalent glycoconjugates for infection, whereas human IAV strains rely on a limited repertoire of glycoconjugate types. The remarkable ability of avian IAV strains to utilize diverse glycoconjugate types may allow for easy transmission into new host species.

Induction of antiviral gene expression by cyclosporine A, but not inhibition of cyclophilin A or B, contributes to its restriction of human coronavirus 229E infection in a lung epithelial cell line

The development of antivirals with an extended spectrum of activity is an attractive possibility to protect against future emerging coronaviruses (CoVs). Cyclosporine A (CsA), a clinically approved immunosuppressive drug, has established antiviral activity against diverse unrelated viruses, including several CoVs. However, its antiviral mechanisms of action against CoV infection have remained elusive, precluding the rational design of non-immunosuppressive derivatives with improved antiviral activities. In this study, we evaluated the mechanisms of CsA against HCoV-229E infection in a human lung epithelial cell line. We demonstrate that the antiviral activity of CsA against HCoV-229E is independent of classical CsA target proteins, cyclophilin A or B, which are not required host factors for HCoV-229E in A549 cells. Instead, CsA treatment induces expression of antiviral genes in a manner dependent on interferon regulatory factor 1, but independent of classical interferon responses, which contributes to its inhibitory effect against HCoV-229E infection. Our results also point to a role for the HCoV-229E nucleoprotein in antagonizing activation of type I interferon, but we show that CsA treatment does not affect evasion of innate immune signalling pathways by HCoV-229E. Overall, our findings further the understanding of the antiviral mechanisms of CsA against CoV infection and highlight a novel immunomodulatory strategy to inhibit CoV infection that may inform future drug development efforts.

FOXA1 prolongs S phase and promotes cancer progression in non-small cell lung cancer through upregulation of CDC5L and activation of the ERK1/2 and JAK2 pathways.

Non-small cell lung cancer (NSCLC) causes high mortality worldwide; however, its molecular pathways have not been fully investigated. The relationship between FOXA1 and CDC5L as well as their roles in NSCLC have not been comprehensively studied. Clinical tissues were collected from 78 NSCLC patients for clinical studies. The BEAS-2B human normal lung epithelial cell line and the A549, Calu-3, H526 and H2170 human NSCLC cell lines were used for in vitro studies. sh-FOXA1 and oe-CDC5L constructs were used to generate knockdown and overexpression models, respectively. The CCK-8 assay was used to analyze cell viability. The cell cycle and apoptosis were evaluated by flow cytometry analysis. The relationship between FOXA1 and CDC5L was demonstrated using dual-luciferase and ChIP assays. Gene levels were examined via immunohistochemistry, qRT-PCR and western blot analysis. FOXA1 levels were increased in NSCLC clinical tissues and cell lines. Depletion of FOXA1 increased the apoptosis rate and increased the proportion of cells in G2/M phase. In addition, we demonstrated that FOXA1 was directly bound to the promoter of CDC5L and that depletion of FOXA1 inhibited CDC5L expression. Overexpression of CDC5L induced ERK1/2 phosphorylation, induced JAK2 phosphorylation, inhibited cell apoptosis, prolonged S phase, and significantly reversed the effects of FOXA1 knockdown on the progression of NSCLC. The present study demonstrated that FOXA1 prolongs S phase and promotes NSCLC progression through upregulation of CDC5L and activation of the ERK1/2 and JAK2 pathways.

Cannabidiol and Aloe vera Extract to Human Cells: Bioactive Molecules as a Promising Anti-Lung Cancer Drug

According to World Health Organisation data, cancer is one of the most influential diseases that goes head to head with heart diseases in the ranking of causes of death, causing approximately 10 million deaths annually and accounting for 20% of all deaths. Lung cancer is a leading cause of cancer-related deaths globally for both men and women. In some pre-clinical studies in cancer cell line in vitro experiments, some results have been obtained that Aloe Vera Extract (AVE) obtained from Aloe vera plant and Cannabindiol (CBD) obtained from Cannabis sativa may have anticancer effect against cancer, but more analysis is required for the reliability of these results. In this study, the cytotoxic and apoptotic effects of AVE and CBD in human lung cancer (A549 and BEAS) cells were studied in a multifaceted manner. Diagnosis of lung cancer disease is approximately 15% in the early stage and 85% in the late stage or metastatic stage. Therefore, despite the use of targeted drugs today, the 5-year survival rate of patients can only be 5-10%. In this study, the cytotoxic and apoptotic effects of AVE and CBD on human lung cancer cells A549 and healthy normal BEAS-2B cells were studied in a versatile and comparative manner. Cytotoxic effect of AVE and CBD was evaluated by xCELLigence RTCA System and AnnexinV-FITC/PI Apoptosis Assay by Flow Cytometry. In the current investigation, we discovered that AVE and CBD might incite a relative higher ratio of cell death in lung cancer cells (A549) than in non-cancer human epithelial lung cell line BEAS-2B, which may be achieved through regulating mitochondrial metabolism. In summary, our research is under Patent submission and shows that CBD and AVE have the potential to develop into a potent new anti-lung cancer drug.

Investigation of the Host Kinome Response to Coronavirus Infection Reveals PI3K/mTOR Inhibitors as Betacoronavirus Antivirals.

Host kinases play essential roles in the host cell cycle, innate immune signaling, the stress response to viral infection, and inflammation. Previous work has demonstrated that coronaviruses specifically target kinase cascades to subvert host cell responses to infection and rely upon host kinase activity to phosphorylate viral proteins to enhance replication. Given the number of kinase inhibitors that are already FDA approved to treat cancers, fibrosis, and other human disease, they represent an attractive class of compounds to repurpose for host-targeted therapies against emerging coronavirus infections. To further understand the host kinome response to betacoronavirus infection, we employed multiplex inhibitory bead mass spectrometry (MIB-MS) following MERS-CoV and SARS-CoV-2 infection of human lung epithelial cell lines. Our MIB-MS analyses revealed activation of mTOR and MAPK signaling following MERS-CoV and SARS-CoV-2 infection, respectively. SARS-CoV-2 host kinome responses were further characterized using paired phosphoproteomics, which identified activation of MAPK, PI3K, and mTOR signaling. Through chemogenomic screening, we found that clinically relevant PI3K/mTOR inhibitors were able to inhibit coronavirus replication at nanomolar concentrations similar to direct-acting antivirals. This study lays the groundwork for identifying broad-acting, host-targeted therapies to reduce betacoronavirus replication that can be rapidly repurposed during future outbreaks and epidemics. The proteomics, phosphoproteomics, and MIB-MS datasets generated in this study are available in the Proteomics Identification Database (PRIDE) repository under project identifiers PXD040897 and PXD040901.

Anticancer activity of manganese dioxide/reduced graphene oxide nanocomposites against A549 human lung adenocarcinoma cell line

A modified version of Hummers’ process was used to synthesize graphene oxide (GO), whereas solar radiation was used to create reduced graphene oxide (rGO) and MnO2/rGO (1%, 3%, 5%) nanostructures were synthesized using a facile hydrothermal method. The X-ray diffraction (XRD) pattern revealed the tetragonal structure of α-MnO2 and determined the crystallite size of MnO2/rGO as 14.36 nm. A scanning electron microscope (SEM) analysis confirms the growth of MnO2 nanorods on the rGO surface. MnO2/rGO (1%, 3%, 5%) contains elements such as carbon, oxygen and manganese, as verified by energy-dispersive X-ray analysis (EDAX). When the rGO percentage was increased, the surface area of MnO2/rGO was decreased to 16.340 m2/g, which enhances the interaction between A549 cells, according to Brunauer–Emmett–Teller (BET) analysis. The allotropes of carbon and the functional groups were examined using Raman spectroscopy and Fourier transform infrared spectroscopy (FTIR). A (dimethyl thiazolyl tetrazolium bromide) MTT assay was used to assess the cytotoxicity of GO, rGO and MnO2/rGO (1%, 3%, 5%) nanostructures against the human lung adenocarcinoma cell line (A549) and human lung epithelial cell line (L132). The half maximum inhibitory concentration (IC50) value for MnO2/5% rGO towards A549 cells was determined to be 120 μg/mL, which shows appreciable results when compared to GO (180 μg/mL), rGO (160 μg/mL), MnO2/1% rGO (180 μg/mL) and MnO2/3% rGO (160 μg/mL). The morphological changes in A549 cells were investigated using an AO/EtBr dual stain, the intracellular reactive oxygen species produced were evaluated using a DCFH-DA stain and the fragmented nuclei were examined using a Hoechst stain. According to the findings, the synthesized MnO2/rGO nanocomposites can be efficiently used for cytotoxicity against A549 cells and possess significant potential for the human lung cancer cell line.

Lactobacillus Probiotic Strains Differ in Their Ability to Adhere to Human Lung Epithelial Cells and to Prevent Adhesion of Clinical Isolates of Pseudomonas aeruginosa from Cystic Fibrosis Lung.

The field of probiotic applications is rapidly expanding, including their use for the control of respiratory tract infections. Nevertheless, probiotics ability to colonize the lung environment and to compete with pulmonary pathogens is still a poorly investigated research area. In this study, we aimed to evaluate the adhesion ability of a number of commercial probiotic strains to the human lung epithelial cell line A549. Furthermore, we assessed probiotic ability to prevent host cell adhesion of one of the major lung pathogens in cystic fibrosis, Pseudomonas aeruginosa, and to reduce the pathogen-induced inflammatory response of human peripheral blood mononuclear cells (PBMCs) in terms of cytokine release. Lactobacillus acidophilus displayed the highest adhesion ability to A549 cells evaluated as percent of adhered bacteria compared to the inoculum. In agreement with such an observation, L. acidophilus was the most efficient in preventing adhesion to A549 cells of a P. aeruginosa isolate from CF sputum. Three-color fluorescence labeling of A549 cells, P. aeruginosa, and L. acidophilus, and confocal microcopy image analyses revealed a likely exclusion effect played by both live and UV-killed L. acidophilus towards P. aeruginosa. Such results were confirmed by CFU count. When co-cultured with PBMCs, both live and UV-killed L. acidophilus reduced the amount of IL-1β and IL-6 in culture supernatants in a statistically significant manner. Overall, the results obtained point to L. acidophilus as an interesting candidate for further studies for a potential aerogenous administration to control P. aeruginosa infections.