Nuclear protein NOP2 serves as a poor-prognosis predictor of LUAD and aggravates the malignancy of lung adenocarcinoma cells.

Recent studies have shown that NOP2, a nucleolar protein, is up-regulated in various cancers, suggesting a potential link to tumor aggressiveness and unfavorable outcomes. This study examines NOP2's role in lung adenocarcinoma (LUAD), a context where its implications remain unclear. Utilizing bioinformatics, we assessed 513 LUAD and 59 normal tissue samples from The Cancer Genome Atlas (TCGA) to explore NOP2's diagnostic and prognostic significance in LUAD. Additionally, in vitro experiments compared NOP2 expression between Beas-2b and A549 cells. Advanced databases and analytical tools, including LINKEDOMICS, STRING, and TISIDB, were employed to further elucidate NOP2's association with LUAD. Our findings indicate a significantly higher expression of NOP2 mRNA and protein in A549 cells compared to Beas-2b cells (P < 0.001). In LUAD, elevated NOP2 levels were linked to decreased Overall Survival (OS) and advanced clinical stages. Univariate Cox analysis revealed that high NOP2 expression correlated with poorer OS in LUAD (P < 0.01), a finding independently supported by multivariate Cox analysis (P < 0.05). The relationship between NOP2 expression and LUAD risk was presented via a Nomogram. Additionally, Gene Set Enrichment Analysis (GSEA) identified seven NOP2-related signaling pathways. A focal point of our research was the interplay between NOP2 and tumor-immune interactions. Notably, a negative correlation was observed between NOP2 expression and the immune infiltration levels of macrophages, neutrophils, mast cells, Natural Killer (NK) cells, and CD8 + T cells in LUAD. Moreover, the expression of NOP2 was related to the sensitivity of various chemotherapeutic drugs. In vitro, we found that downregulating NOP2 can decrease the proliferation, migration and invasion of A549 cells. Furthermore, NOP2 can regulate Caspase3-mediated apoptosis. Collectively, particularly regarding prognosis, immune infiltration and vitro experiments, these findings suggest NOP2's potential of serving as a poor-prognostic biomarker for LUAD and aggravating the malignancy of lung adenocarcinoma cells.

iRhom2 regulates ERBB signalling to promote KRAS-driven tumour growth of lung cancer cells.

Dysregulation of the ERBB/EGFR signalling pathway causes multiple types of cancer. Accordingly, ADAM17, the primary shedding enzyme that releases and activates ERBB ligands, is tightly regulated. It has recently become clear that iRhom proteins, inactive members of the rhomboid-like superfamily, are regulatory cofactors for ADAM17. Here, we show that oncogenic KRAS mutants target the cytoplasmic domain of iRhom2 (also known as RHBDF2) to induce ADAM17-dependent shedding and the release of ERBB ligands. Activation of ERK1/2 by oncogenic KRAS induces the phosphorylation of iRhom2, recruitment of the phospho-binding 14-3-3 proteins, and consequent ADAM17-dependent shedding of ERBB ligands. In addition, cancer-associated mutations in iRhom2 act as sensitisers in this pathway by further increasing KRAS-induced shedding of ERBB ligands. This mechanism is conserved in lung cancer cells, where iRhom activity is required for tumour xenograft growth. In this context, the activity of oncogenic KRAS is modulated by the iRhom2-dependent release of ERBB ligands, thus placing the cytoplasmic domain of iRhom2 as a central component of a positive feedback loop in lung cancer cells. This article has an associated First Person interview with the first authors of the paper.

Overexpression of transcription factor TBX5 inhibits the activation of YAP1-TEAD1 pathway to promote ferroptosis in lung cancer cells.

BACKGROUND: Non-small cell lung cancer (NSCLC) accounts for more than 80 % of lung cancer (LC) cases, making it the primary cause of cancer-related mortality worldwide. T-box transcription factor 5 (TBX5) is an important regulator of embryonic and organ development and plays a key role in cancer development. Here, our objective was to investigate the involvement of TBX5 in ferroptosis within LC cells and the underlying mechanisms. METHODS: First, TBX5 expression was examined in human LC cells. Next, overexpression of TBX5 and Yes1-associated transcriptional regulator (YAP1) and knockdown of TEA domain 1 (TEAD1) were performed in A549 and NCI-H1703 cells. The proliferation ability of A549 and NCI-H1703 cells, GSH, MDA, ROS, and Fe2+ levels were measured. Co-immunoprecipitation (Co-IP) was performed to verify whether TBX5 protein could bind YAP1. Then TBX5, YAP1, TEAD1, GPX4, p53, FTH1, SLC7A11 and PTGS2 protein levels were assessed. Finally, we verified the effect of TBX5 on ferroptosis in LC cells in vivo. RESULTS: TBX5 expression was down-regulated in LC cells, especially in A549 and NCI-H1703 cells. Overexpression of TBX5 significantly decreased proliferation ability of A549 and NCI-H1703 cells, downregulated GPX4 and GSH levels, and upregulated MDA, ROS, and Fe2+ levels. Co-IP verified that TBX5 protein could bind YAP1. Moreover, oe-YAP1 promoted proliferation ability of A549 and NCI-H1703 cells transfected with Lv-TBX5, upregulated GPX4 and GSH levels and downregulated MDA, ROS, and Fe2+ levels. Additionally, oe-YAP1 promoted FTH1 and SLC7A11 levels and inhibited p53 and PTGS2 levels in A549 and NCI-H1703 cells transfected with Lv-TBX5. However, transfection with si-TEAD1 further reversed these effects. In vivo experiments further validated that TBX5 promoted ferroptosis in LC cells. CONCLUSIONS: TBX5 inhibited the activation of YAP1-TEAD1 pathway to promote ferroptosis in LC cells.

Respiratory Syncytial Virus Infection Does Not Induce Epithelial-Mesenchymal Transition.

Respiratory syncytial virus (RSV) infection does not cause severe disease in most of us despite suffering from multiple RSV infections during our lives. However, infants, young children, older adults, and immunocompromised patients are unfortunately vulnerable to RSV-associated severe diseases. A recent study suggested that RSV infection causes cell expansion, resulting in bronchial wall thickening in vitro. Whether the virus-induced changes in the lung airway resemble epithelial-mesenchymal transition (EMT) is still unknown. Here, we report that RSV does not induce EMT in three different in vitro lung models: the epithelial A549 cell line, primary normal human bronchial epithelial cells, and pseudostratified airway epithelium. We found that RSV increases the cell surface area and perimeter in the infected airway epithelium, which is distinct from the effects of a potent EMT inducer, transforming growth factor ß1 (TGF-ß1), driving cell elongation-indicative of cell motility. A genome-wide transcriptome analysis revealed that both RSV and TGF-ß1 have distinct modulation patterns of the transcriptome, which suggests that RSV-induced changes are distinct from EMT. IMPORTANCE We have previously shown that RSV infects ciliated cells on the apical side of the lung airway. RSV-induced cytoskeletal inflammation contributes to an uneven increase in the height of the airway epithelium, resembling noncanonical bronchial wall thickening. RSV infection changes epithelial cell morphology by modulating actin-protein 2/3 complex-driven actin polymerization. Therefore, it is prudent to investigate whether RSV-induced cell morphological changes contribute to EMT. Our data indicate that RSV does not induce EMT in at least three different epithelial in vitro models: an epithelial cell line, primary epithelial cells, and pseudostratified bronchial airway epithelium.

Radiosensitization effect of quinoline-indole-schiff base derivative 10E on non-small cell lung cancer cells in vitro and in tumor xenografts.

Radioresistance is an inevitable obstacle in the clinical treatment of inoperable patients with non-small cell lung cancer (NSCLC). Combining treatment with radiosensitizers may improve the efficacy of radiotherapy. Previously, the quinoline derivative 10E as new exporter of Nur77 has shown superior antitumor activity in hepatocellular carcinoma. Here, we aimed to investigate the radiosensitizing activity and acting mechanisms of 10E. In vitro, A549 and H460 cells were treated with control, ionizing radiation (IR), 10E, and 10E + IR. Cell viability, apoptosis, and cycle were examined using CCK-8 and flow cytometry assays. Protein expression and localization were examined using western blotting and immunofluorescence. Tumor xenograft models were established to evaluate the radiosensitizing effect of 10E in vivo. 10E significantly inhibited cell proliferation and increased their radiosensitivity while reducing level of p-BCRA1, p-DNA-PKs, and 53BP1 involved in the DNA damage repair pathway, indicating that its radiosensitizing activity is closely associated with repressing DNA damage repair. A549 cells showed low level of Nur77 and a low response to IR but 10E-treated A549 cells showed high level of Nur77 indicating that Nur77 is a core radiosensitivity factor and 10E restores the expression of Nur77. Nur77 and Ku80 extranuclear co-localization in the 10E-treated A549 cells suggested that 10E-modulated Nur77 nuclear exportation inhibits DNA damage repair pathways and increases IR-triggered apoptosis. The combination of 10E and IR significantly inhibits tumor growth in a tumor xenograft model. Our findings suggest that 10E acts as a radiosensitizer and that combining 10E with radiotherapy may be a potential strategy for NSCLC treatment.

Anti-OmpA antibodies as potential inhibitors of Acinetobacter baumannii biofilm formation, adherence to, and proliferation in A549 human alveolar epithelial cells.

Outer membrane protein A (OmpA) is a critical virulence factor in Acinetobacter baumannii, influencing adhesion, biofilm formation, host immune response, and host cell apoptosis. We investigated the invasion of A549 alveolar epithelial cells by A. baumannii and examined how anti-OmpA antibodies impact these interactions. OmpA was expressed and purified, inducing anti-OmpA antibodies in BALB/c mice. The potential toxicity of OmpA was evaluated in mice by analyzing histology from six organs. A549 cells were exposed to A. baumannii strains 19606 and a clinical isolate. Using cell culture and light microscopy, we scrutinized the effects of anti-OmpA sera on serum resistance, adherence, internalization, and proliferation of A. baumannii in A549 cells. The viability of A549 cells was assessed upon exposure to live A. baumannii and anti-OmpA sera. OmpA-induced antibody demonstrated potent bactericidal effects on both strains of A. baumannii. Both strains formed biofilms, which were reduced by anti-OmpA serum, along with decreased bacterial adherence, internalization, and proliferation in A549 cells. Anti-OmpA serum improved the survival of A549 cells post-infection. Pre-treatment with cytochalasin D hindered bacterial internalization, highlighting the role of actin polymerization in invasion. Microscopic examination revealed varied interactions encompassing adherence, apoptosis, membrane alterations, vacuolization, and damage. A549 cells treated with anti-OmpA serum exhibited improved structures and reduced damage. The findings indicate that A. baumannii can adhere to and proliferate within epithelial cells with OmpA playing a pivotal role in these interactions, and the complex nature of these interactions shapes the intricate course of A. baumannii infection in host cells.

METTL14 enhances the m6A modification level of lncRNA MSTRG.292666.16 to promote the progression of non-small cell lung cancer.

BACKGROUND: m6A modification has close connection with the occurrence, development, and prognosis of tumors. This study aimed to explore the roles of m6A modification and its related mechanisms in non-small cell lung cancer (NSCLC). METHODS: NSCLC tissues and their corresponding para-cancerous tissues were collected to determine the m6A levels of total RNA/lncRNAs and the expression of m6A modification-related genes/lncRNAs. Then, A549 cells were transfected with si-METTL14 or oe-METTL14, and the cell transfection efficiency was assessed. Subsequently, the viability, apoptosis, cell colony formation, migration and invasion of the different cells were determined. Finally, the nude mouse tumorigenicity experiments were performed to observe the effects of METTL14 in vivo. RESULTS: Compared to the para-NSCLC tissues, the m6A level and METTL14 expression were both significantly increased in the NSCLC tissues (P < 0.05). Based on the expression of METTL14 in the different cell lines, A549 cells were chosen for further experiments. Then, the A549 cells with METTL14 knockdown and overexpression were successfully established, as well as it was found that METTL14 knockdown could inhibit the viability, colony formation, migration, and invasion of A549 cells, while facilitate their apoptosis. In vivo experiments also showed that METTL14 knockdown could inhibit tumor formation and growth. Additionally, the m6A level of MSTRG.292666.16 was higher in the NSCLC tissues; and after METTL14 knockdown, the expression and m6A level of MSTRG.292666.16 were both significantly reduced in A549 cells, and vice versa. CONCLUSION: METTL14 may promote the progression of NSCLC through up-regulating MSTRG.292666.16 and enhance its m6A modification level.

Antimicrobial and antitumor properties of anuran peptide temporin-SHf induce apoptosis in A549 lung cancer cells.

Temporin-SHf is a linear, ultra-short, hydrophobic, α-helix, and phe-rich cationic antimicrobial peptide. The antitumor activities and mechanism of temporin-SHf-induced cancer cell death are unknown. The temporin-SHf was synthesized by solid-phase Fmoc chemistry and antimicrobial and antitumor activities were investigated. Temporin-SHf was microbiocidal, non-hemolytic, and cytotoxic to human cancer cells but not to non-tumorigenic cells. It affected the cancer cells' lysosomal integrity and caused cell membrane damage. The temporin-SHf inhibited A549 cancer cell proliferation and migration. It is anti-angiogenic and causes cancer cell death through apoptosis. The molecular mechanism of action of temporin-SHf confirmed that it kills cancer cells by triggering caspase-dependent apoptosis through an intrinsic mitochondrial pathway. Owing to its short length and broad spectrum of antitumor activity, temporin-SHf is a promising candidate for developing a new class of anticancer drugs.

ADAM10 and ADAM17 promote SARS-CoV-2 cell entry and spike protein-mediated lung cell fusion.

The severe-acute-respiratory-syndrome-coronavirus-2 (SARS-CoV-2) is the causative agent of COVID-19, but host cell factors contributing to COVID-19 pathogenesis remain only partly understood. We identify the host metalloprotease ADAM17 as a facilitator of SARS-CoV-2 cell entry and the metalloprotease ADAM10 as a host factor required for lung cell syncytia formation, a hallmark of COVID-19 pathology. ADAM10 and ADAM17, which are broadly expressed in the human lung, cleave the SARS-CoV-2 spike protein (S) in vitro, indicating that ADAM10 and ADAM17 contribute to the priming of S, an essential step for viral entry and cell fusion. ADAM protease-targeted inhibitors severely impair lung cell infection by the SARS-CoV-2 variants of concern alpha, beta, delta, and omicron and also reduce SARS-CoV-2 infection of primary human lung cells in a TMPRSS2 protease-independent manner. Our study establishes ADAM10 and ADAM17 as host cell factors for viral entry and syncytia formation and defines both proteases as potential targets for antiviral drug development.

Photosensitizing effects and physicochemical properties of chlorophyll a derivatives with hydrophilic oligoethylene glycol fragments at the macrocycle periphery.

In this work, screening studies of the cytotoxic effect of chlorins with fragments of di-, tri-, and pentaethylene glycol at the macrocycle periphery in relation to HeLa, A549, and HT29 cells were performed. It is shown that, despite different hydrophobicity, all the compounds studied have a comparable photodynamic effect. The conjugate of chlorin e6 with pentaethylene glycol, which has the lowest tendency to association among the studied compounds with tropism for low density lipoproteins and the best characteristics of the formation of molecular complexes with Tween 80, has a significant difference in dark and photoinduced toxicity (ratio IC50(dark)/IC50(photo) approximately 2 orders of magnitude for all cell lines), which allows to hope for a sufficiently large "therapeutic window". A study of the interaction of this compound with HeLa cells shows that the substance penetrates the cell and, after red light irradiation induces ROS appearance inside the cell, associated, apparently, with the photogeneration of singlet oxygen. These data indicate that photoinduced toxic effects are caused by damage to intracellular structures as a result of oxidative stress. Programmed type of cell death characterized with caspase-3 induction is prevailing. So, the conjugate of chlorin e6 with pentaethylene glycol is a promising antitumor PS that can be successfully solubilized with Tween 80, which makes it suitable for further in vivo studies.

Comparison of the adherence of nontypeable haemophilus influenzae to lung epithelial cells.

OBJECTIVE: Nontypeable Haemophilus influenzae (NTHi) plays an important role in respiratory tract infections, and adherence to lung epithelial cells is the first step in lung infections. To explore the role of NTHi in childhood lung infections, a comparative study was conducted on the adherence of strains isolated from sputum culture and bronchoalveolar lavage fluid to A549 lung epithelial cells. METHODS: Haemophilus influenzae strains were obtained from the sample bank of Shenzhen Children's Hospital, and identified as NTHi via PCR detection of the capsule gene bexA. NTHi obtained from healthy children's nasopharyngeal swabs culture were selected as the control group, and a comparative study was conducted on the adherence of strains isolated from sputum culture or bronchoalveolar lavage fluid of patients to A549 cells. RESULTS: The adherence bacterial counts of NTHi isolated from the nasopharyngeal cultures of healthy children to A549 cells was 58.2 CFU. In patients with lung diseases, NTHi isolated from bronchoalveolar lavage fluid was 104.3 CFU, and from sputum cultures was 115.1 CFU, both of which were significantly higher in their adherence to A549 cells compared to the strains isolated from the healthy control group. There was no significant difference in adherence between the strains isolated from sputum cultures and bronchoalveolar lavage fluid (t = 0.5217, p = 0.6033). CONCLUSION: NTHi played an important role in childhood pulmonary infections by enhancing its adherence to lung epithelial cells.

Synthesis and characterization of organoselenium based BODIPY and its application in living cells.

Phenylselenide based BODIPY probe was successfully synthesized and characterized by NMR spectroscopic techniques (1H, 13C and 77Se NMR), mass spectrometry and single crystal XRD. Surprisingly, crystal packing diagram of the probe showed formation of 1-D strip through intermolecular F---H interaction. The probe was screened with various Reactive Oxygen Species (ROS) and found to be selective for superoxide ion over other ROS via "turn-on" fluorescence response. The probe selectively and sensitively detects superoxide with a lower detection limit (43.34 nM) without interfering with other ROS. The quantum yield of the probe was found to increase from 0.091 % to 30.4 % (334-fold) after oxidation. Theoretical calculations (DFT and TD-DFT) were also performed to understand the sensing mechanism of the probe. The probe was able to effectively detect superoxide inside living cells without any toxic effect.

Pelargonidin inhibits cell growth and promotes oxidative stress-mediated apoptosis in lung cancer A549 cells.

Lung cancer has the worst prognosis with an average 5-year survival rate of only 10%-20%. Lung cancer has the highest prevalence rate and a second most common cause of cancer-associated mortalities worldwide. The present study was planned to explore the anticancer effects of pelargonidin against the lung cancer A549 cells via analyzing oxidative stress-mediated apoptosis. The viability of both control and pelargonidin-treated A549 cells was analyzed using the MTT cytotoxicity assay at different time periods. The levels of endogenous ROS generation, mitochondrial membrane potential (Δψm), and apoptosis were assessed using corresponding fluorescent staining assays. The levels of oxidative stress biomarkers, including TBARS, SOD, CAT, and GSH, in the cell lysates of control and pelargonidin-treated A549 cells were examined using the assay kits. The pelargonidin treatment substantially suppressed the A549 cell growth. Further, pelargonidin promoted the ROS production and depleted the Δψm levels in the A549 cells. The fluorescent staining assays witnessed the occurrence of increased apoptosis in the pelargonidin-treated A549 cells. The pelargonidin also boosted the TBARS and reduced the antioxidant levels thereby promoted the oxidative stress-regulated apoptosis in the A549 cells. In summary, the findings' results of the current study demonstrated an anticancer activity of pelargonidin on A549 cells. The pelargonidin treatment substantially decreased the growth and encouraged the oxidative stress-regulated apoptosis in A549 cells. Therefore, it was evident that the pelargonidin could be employed as an effective anticancer candidate to treat the lung cancer.

Combining analytical techniques to assess the translocation of diesel particles across an alveolar tissue barrier in vitro.

BACKGROUND: During inhalation, airborne particles such as particulate matter ≤ 2.5 µm (PM2.5), can deposit and accumulate on the alveolar epithelial tissue. In vivo studies have shown that fractions of PM2.5 can cross the alveolar epithelium to blood circulation, reaching secondary organs beyond the lungs. However, approaches to quantify the translocation of particles across the alveolar epithelium in vivo and in vitro are still not well established. In this study, methods to assess the translocation of standard diesel exhaust particles (DEPs) across permeable polyethylene terephthalate (PET) inserts at 0.4, 1, and 3 µm pore sizes were first optimized with transmission electron microscopy (TEM), ultraviolet-visible spectroscopy (UV-VIS), and lock-in thermography (LIT), which were then applied to study the translocation of DEPs across human alveolar epithelial type II (A549) cells. A549 cells that grew on the membrane (pore size: 3 µm) in inserts were exposed to DEPs at different concentrations from 0 to 80 µg.mL- 1 ( 0 to 44 µg.cm- 2) for 24 h. After exposure, the basal fraction was collected and then analyzed by combining qualitative (TEM) and quantitative (UV-VIS and LIT) techniques to assess the translocated fraction of the DEPs across the alveolar epithelium in vitro. RESULTS: We could detect the translocated fraction of DEPs across the PET membranes with 3 µm pore sizes and without cells by TEM analysis, and determine the percentage of translocation at approximatively 37% by UV-VIS (LOD: 1.92 µg.mL- 1) and 75% by LIT (LOD: 0.20 µg.cm- 2). In the presence of cells, the percentage of DEPs translocation across the alveolar tissue was determined around 1% at 20 and 40 µg.mL- 1 (11 and 22 µg.cm- 2), and no particles were detected at higher and lower concentrations. Interestingly, simultaneous exposure of A549 cells to DEPs and EDTA can increase the translocation of DEPs in the basal fraction. CONCLUSION: We propose a combination of analytical techniques to assess the translocation of DEPs across lung tissues. Our results reveal a low percentage of translocation of DEPs across alveolar epithelial tissue in vitro and they correspond to in vivo findings. The combination approach can be applied to any traffic-generated particles, thus enabling us to understand their involvement in public health.

Modular air-liquid interface aerosol exposure system (MALIES) to study toxicity of nanoparticle aerosols in 3D-cultured A549 cells in vitro.

We present a novel lung aerosol exposure system named MALIES (modular air-liquid interface exposure system), which allows three-dimensional cultivation of lung epithelial cells in alveolar-like scaffolds (MatriGrids®) and exposure to nanoparticle aerosols. MALIES consists of multiple modular units for aerosol generation, and can be rapidly assembled and commissioned. The MALIES system was proven for its ability to reliably produce a dose-dependent toxicity in A549 cells using CuSO4 aerosol. Cytotoxic effects of BaSO4- and TiO2-nanoparticles were investigated using MALIES with the human lung tumor cell line A549 cultured at the air-liquid interface. Experiments with concentrations of up to 5.93 × 105 (BaSO4) and 1.49 × 106 (TiO2) particles/cm3, resulting in deposited masses of up to 26.6 and 74.0 µg/cm2 were performed using two identical aerosol exposure systems in two different laboratories. LDH, resazurin reduction and total glutathione were measured. A549 cells grown on MatriGrids® form a ZO-1- and E-Cadherin-positive epithelial barrier and produce mucin and surfactant protein. BaSO4-NP in a deposited mass of up to 26.6 µg/cm2 resulted in mild, reversible damage (~ 10% decrease in viability) to lung epithelium 24 h after exposure. TiO2-NP in a deposited mass of up to 74.0 µg/cm2 did not induce any cytotoxicity in A549 cells 24 h and 72 h after exposure, with the exception of a 1.7 fold increase in the low exposure group in laboratory 1. These results are consistent with previous studies showing no significant damage to lung epithelium by short-term treatment with low concentrations of nanoscale BaSO4 and TiO2 in in vitro experiments.

Effects of electromagnetic pulses, exosomes inhibition and their coaction on A549 cells.

Mounting literature indicates that electromagnetic pulses (EMP) is the promising modality to treat cancers with advantages such as noninvasiveness and few side-effects, but its appropriate parameters and underlying mechanisms such as its influence on tumor-derived exosomes (TDEs) are largely unknown. This study aimed to elucidate effects of EMP, exosome inhibition and their coaction on A549 lung adenocarcinoma cells. A549 cells were randomly divided into control group, GW4869 group treated by 20 µM GW4869, vehicle group treated by dimethyl sulfoxide, EMP group treated by EMP exposure, and EMPG group treated by EMP exposure combined with 20 µM GW4869. After EMP exposure, cell proliferation was determined by CCK8 assay, cell cycle and apoptosis was detected by flow cytometry, and cell migration was determined by transwell assay. The results showed that EMP or exosomes inhibition did not affect cell proliferation, cell cycle, apoptosis and cell migration (p > 0.05), but cell migration in EMPG group was significantly decreased compared with vehicle group (p < 0.05). We concluded that under the experimental condition, EMP or GW4869 alone had no effects on behaviors of A549 cells, but their coaction could effectively inhibit the migration of A549 cells.

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.

Performance of 4,5-diphenyl-1H-imidazole derived highly selective 'Turn-Off' fluorescent chemosensor for iron(III) ions detection and biological applications.

Two fluorescent chemosensors, denoted as chemosensor 1 and chemosensor 2, were synthesized and subjected to comprehensive characterization using various techniques. The characterization techniques employed were Fourier-transform infrared (FTIR), proton (1 H)- and carbon-13 (13 C)-nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization (ESI) mass spectrometry, and single crystal X-ray diffraction analysis. Chemosensor 1 is composed of a 1H-imidazole core with specific substituents, including a 4-(2-(4,5-c-2-yl)naphthalene-3-yloxy)butoxy)naphthalene-1-yl moiety. However, chemosensor 2 features a 1H-imidazole core with distinct substituents, such as 4-methyl-2-(4,5-diphenyl-1H-imidazole-2-yl)phenoxy)butoxy)-5-methylphenyl. Chemosensor 1 crystallizes in the monoclinic space group C2/c. Both chemosensors 1 and 2 exhibit a discernible fluorescence quenching response selectively toward iron(III) ion (Fe3+ ) at 435 and 390 nm, respectively, in dimethylformamide (DMF) solutions, distinguishing them from other tested cations. This fluorescence quenching is attributed to the established mechanism of chelation quenched fluorescence (CHQF). The binding constants for the formation of the 1 + Fe3+ and 2 + Fe3+ complexes were determined using the modified Benesi-Hildebrand equation, yielding values of approximately 2.2 × 103 and 1.3 × 104 M-1 , respectively. The calculated average fluorescence lifetimes for 1 and 1 + Fe3+ were 2.51 and 1.17 ns, respectively, while for 2 and 2 + Fe3+ , the lifetimes were 1.13 and 0.63 ns, respectively. Additionally, the applicability of chemosensors 1 and 2 in detecting Fe3+ in live cells was demonstrated, with negligible observed cell toxicity.

Synthesis, Antiproliferative Activity and Molecular Docking Analysis of Both Enantiomerically Pure Decursin Derivatives as Anticancer Agents.

Using (S)-decursinol isolated from root of Angelica gigas Nakai (AGN), we semi-synthesized and evaluated a series of both enantiomerically pure decursin derivatives for their antiproliferative activities against A549 human lung cancer cells. All synthesized compounds showed a broad spectrum of inhibitory activities against the growth of A549 cells. Especially, compound (S)-2d with (E)-(furan-3-yl)acryloyl group showed the most potent activity (IC50: 14.03 µM) against A549 cancer cells as compared with the reference compound, decursin (IC50: 43.55 µM) and its enantiomer, (R)-2d (IC50: 151.59 µM). Western blotting assays indicated that (S)-2d more strongly inhibited Janus kinase 1 (JAK1) and signal transducer and activator of transcription activation 3 (STAT3) phosphorylation than decursin in a dose-dependent manner, while having no effect on CXCR7 overexpression and total STAT3 level. In addition, (S)-2d induced cell cycle arrest at G1 phase and subsequent apoptotic cell death in A549 cancer cells. Our combined analysis of molecular docking studies and biological data suggests that the inhibition of JAK1 with (S)-2d resulted in loss of STAT3 phosphorylation and inhibition of cell growth in A549 cancer cells. These overall results strongly suggest that (S)-2d (MRC-D-004) as a novel JAK1 inhibitor may have therapeutic potential in the treatment of A549 human lung cancers by targeting the JAK1/STAT3 signaling pathway.

Grifolin Induces Cell Death of Human Lung Cancer A549 Cell Line via Inhibiting KRAS-Mediated Multiple Signaling Pathways.

In the current work, grifolin was obtained from the twigs and leaves of Daphne genkwa for the first time and displayed significant growth inhibition against human lung carcinoma A549 cells. Subsequent in vitro antitumor evaluation revealed that grifolin could induce remarkable cell apoptosis and G0/G1 phase arrest, as well as block cell migration and invasion. In addition, grifolin also disrupted cellular energy metabolism by inducing reactive oxygen species, reducing adenosine triphosphate and mitochondrial membrane potential, and damaging DNA synthesis. Further RNA-seq analysis demonstrated that treatment of grifolin on A549 cells led to gene enrichment in MAPK, PI3K/Akt and NF-κB signaling pathways, all of which were inhibited by grifolin according to immunoblotting experiments. Further mechanistical studies disclosed that the expression of a key upstream protein KRAS was also blocked, and the cell death triggered by grifolin could be rescued by a RAS activator ML-099. Moreover, pretreatment of ML-099 on A549 cells could reverse the grifolin-induced downregulation of key proteins in the three aforementioned pathways. These findings indicate that grifolin could induce cell death in A549 cell line by inhibiting KRAS-mediated multiple signaling pathways.