Human Bronchial Epithelial Cell Line Research Articles

Role of Carbon Monoxide in Oxidative Stress-Induced Senescence in Human Bronchial Epithelium.

Prolonged or excessive stimulation from inhaled toxins may cause oxidative stress and DNA damage that can lead to stress-induced senescence in epithelial cells, which can contribute to several airway diseases. Mounting evidence has shown carbon monoxide (CO) confers cytoprotective effects. We investigated the effects of CO on oxidative stress-induced senescence in human airway epithelium and elucidated the underlying molecular mechanisms. Here, CO pretreatment reduced H2O2-mediated increases in total reactive oxygen species (ROS) production and mitochondrial superoxide in a human bronchial epithelial cell line (BEAS-2B). H2O2 treatment triggered a premature senescence-like phenotype with enlarged and flattened cell morphology accompanied by increased SA-β-gal activity, cell cycle arrest in G0/G1, reduced cell viability, and increased transcription of senescence-associated secretory phenotype (SASP) genes. Additionally, exposure to H2O2 increased protein levels of cellular senescence markers (p53 and p21), reduced Sirtuin 3 (SIRT3) and manganese superoxide dismutase (MnSOD) levels, and increased p53 K382 acetylation. These H2O2-mediated effects were attenuated by pretreatment with a CO-containing solution. SIRT3 silencing induced mitochondrial superoxide production and triggered a senescence-like phenotype, whereas overexpression decreased mitochondrial superoxide production and alleviated the senescence-like phenotype. Air-liquid interface (ALI) culture of primary human bronchial cells, which becomes a fully differentiated pseudostratified mucociliary epithelium, was used as a model. We found that apical and basolateral exposure to H2O2 induced a vacuolated structure that impaired the integrity of ALI cultures, increased goblet cell numbers, decreased SCGB1A1+ club cell numbers, increased p21 protein levels, and increased SASP gene transcription, consistent with our observations in BEAS-2B cells. These effects were attenuated in the apical presence of a CO-containing solution. In summary, we revealed that CO has a pivotal role in epithelial senescence by regulating ROS production via the SIRT3/MnSOD/p53/p21 pathway. This may have important implications in the prevention and treatment of age-associated respiratory pathologies.

The involvement of copper, circular RNAs, and inflammatory cytokines in chronic respiratory disease.

Exposure to high concentrations of copper is associated with pulmonary inflammation and chronic respiratory disease (CRD). Epigenetic modulation of noncoding RNAs contributes to the development of several CRDs. It is unknown whether epigenetic modulation is involved in copper mediated pulmonary inflammation and CRD. We conducted a case-control study of 101 CRD cases and 161 control subjects in Shijiazhuang, China, and evaluated circRNAs and cytokine levels (IL-6 and IL-8) by qPCR and ELISA. Urinary copper concentration was determined by inductively coupled plasma mass spectrometry. Linear mixed models and generalized linear mixed models were used to assess the associations of circRNAs with CRD, urinary copper, and cytokines. We exposed the human bronchial epithelial cell line, 16HBE, to copper and assessed the functional role of a circRNA, circ_0008882, by RNA overexpression. Cellular location of circ_0008882 was assessed by separation of nuclear and cytoplasmic RNAs. Nine circRNAs were associated with an increased risk for CRDs, while the relative expression of circ_0008882 was decreased after copper exposure in vitro and in vivo. Copper exposure stimulated 16HBE cells to release proinflammatory IL-6 and IL-8. The release of the cytokines was inhibited by overexpression of circ_0008882. These results suggest a role for circ_0008882 in the regulation of CRD associated inflammation following copper exposure.

Biphasic regulation of CFTR expression by ENaC in epithelial cells: The involvement of Ca2+-modulated cAMP production

The regulatory interaction between two typical epithelial ion channels, cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial sodium channel (ENaC), for epithelial homeostasis has been noted, although the underlying mechanisms remain unclear. Here, we report that in a human endometrial epithelial cell line (ISK), shRNA-based stable knockdown of ENaC produced a biphasic effect: a low (∼23%) degree of ENaC knockdown resulted in significant increases in CFTR mRNA and protein levels, CFTR-mediated Cl− transport activity as well as intracellular cAMP concentration, while a higher degree (∼50%) of ENaC knockdown did not further increase but restored CFTR expression and cAMP levels. The basal intracellular Ca2+ level of ISK cells was lowered by ENaC knockdown or inhibition in a degree-dependent manner. BAPTA-AM, an intracellular Ca2+ chelator that lowers free Ca2+ concentration, elevated cAMP level and CFTR mRNA expression at a low (5 µM) but not a high (50 µM) dose, mimicking the biphasic effect of ENaC knockdown. Moreover, KH-7, a selective inhibitor of soluble adenylyl cyclase (sAC), abolished the CFTR upregulation induced by low-degree ENaC knockdown or Ca2+ chelation, suggesting the involvement of sAC-driven cAMP production in the positive regulation. A luciferase reporter to indicate CFTR transcription revealed that all tested degrees of ENaC knockdown/inhibition stimulated CFTR transcription in ISK cells, suggesting that the negative regulation on CFTR expression by the high-degree ENaC deficiency might occur at post-transcription stages. Additionally, similar biphasic effect of ENaC knockdown on CFTR expression was observed in a human bronchial epithelial cell line. Taken together, these results have revealed a previously unidentified biphasic regulatory role of ENaC in tuning CFTR expression involving Ca2+-modulated cAMP production, which may provide an efficient mechanism for dynamics and plasticity of the epithelial tissues in various physiological or pathological contexts.

Carbon nanoparticles adversely affect CFTR expression and toxicologically relevant pathways

Cystic fibrosis is an autosomal recessive disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) that can lead to terminal respiratory failure. Ultrafine carbonaceous particles, which are ubiquitous in ambient urban and indoor air, are increasingly considered as major contributors to the global health burden of air pollution. However, their effects on the expression of CFTR and associated genes in lung epithelial cells have not yet been investigated. We therefore evaluated the effects of carbon nanoparticles (CNP), generated by spark-ablation, on the human bronchial epithelial cell line 16HBE14o− at air–liquid interface (ALI) culture conditions. The ALI-cultured cells exhibited epithelial barrier integrity and increased CFTR expression. Following a 4-h exposure to CNP, the cells exhibited a decreased barrier integrity, as well as decreased expression of CFTR transcript and protein levels. Furthermore, transcriptomic analysis revealed that the CNP-exposed cells showed signs of oxidative stress, apoptosis and DNA damage. In conclusion, this study describes spark-ablated carbon nanoparticles in a realistic exposure of aerosols to decrease CFTR expression accompanied by transcriptomic signs of oxidative stress, apoptosis and DNA damage.

ACEing premature codon termination using anticodon-engineered sup-tRNA-based therapy

ACEing premature codon termination using anticodon-engineered sup-tRNA-based therapy

Exposure to naphthalene and β-pinene-derived secondary organic aerosol induced divergent changes in transcript levels of BEAS-2B cells.

The health effects of exposure to secondary organic aerosols (SOAs) are still limited. Here, we investigated and compared the toxicities of soot particles (SP) coated with β-pinene SOA (SOAβPin-SP) and SP coated with naphthalene SOA (SOANap-SP) in a human bronchial epithelial cell line (BEAS-2B) residing at the air-liquid interface. SOAβPin-SP mostly contained oxygenated aliphatic compounds from β-pinene photooxidation, whereas SOANap-SP contained a significant fraction of oxygenated aromatic products under similar conditions. Following exposure, genome-wide transcriptome responses showed an Nrf2 oxidative stress response, particularly for SOANap-SP. Other signaling pathways, such as redox signaling, inflammatory signaling, and the involvement of matrix metalloproteinase, were identified to have a stronger impact following exposure to SOANap-SP. SOANap-SP also induced a stronger genotoxicity response than that of SOAβPin-SP. This study elucidated the mechanisms that govern SOA toxicity and showed that, compared to SOAs derived from a typical biogenic precursor, SOAs from a typical anthropogenic precursor have higher toxicological potency, which was accompanied with the activation of varied cellular mechanisms, such as aryl hydrocarbon receptor. This can be attributed to the difference in chemical composition; specifically, the aromatic compounds in the naphthalene-derived SOA had higher cytotoxic potential than that of the β-pinene-derived SOA.

Mechanism of miR-338-3p in sepsis-induced acute lung injury via indirectly modulating ATF4

Sepsis is recognized as an inflammation-related syndrome in response to invading pathogens. Many patients suffer from sepsis including transplant recipients. Lipopolysaccharide (LPS) is known to trigger sepsis-related organ dysfunction. This study expounded on the possible effect of microRNA (miR)-338-3p in sepsis-induced acute lung injury (ALI). Firstly, human bronchial epithelial cell line 16HBE received LPS treatment to establish the cell models of sepsis-induced ALI. The expression patterns of miR-338-3p, long non-coding RNA OPA-interacting protein 5 antisense transcript 1 (lncRNA OIP5-AS1), and activating transcription factor 4 (ATF4) in 16HBE cells were examined. Afterwards, 16HBE cell viability, the apoptosis rate, and the levels of inflammation and lactate dehydrogenase (LDH) were determined to assess the degree of cell injury. We disclosed that LPS treatment triggered 16HBE cell injury, downregulated miR-338-3p, and upregulated OIP5-AS1 and ATF4. miR-338-3p overexpression repressed LPS-induced 16HBE cell injury. miR-338-3p diminished OIP5-AS1 stability via binding to OIP5-AS1 and downregulated OIP5-AS1 expression and OIP5-AS1 can enhance ATF4 mRNA stability and upregulate ATF4 mRNA level. The rescue experiments showed that ATF4 overexpression aggravated LPS-induced 16HBE cell injury. Overall, miR-338-3p overexpression decreased OIP5-AS1 expression and stability and further downregulated ATF4 mRNA level, thereby mitigating LPS-induced 16HBE cell injury.

Tumor necrosis factor-α coordinates with transforming growth factor-β1 to induce epithelial-mesenchymal transition and migration via the NF-κB/NOX4 pathway in bronchial epithelial cells.

Epithelial-to-mesenchymal transition (EMT) is the process by which epithelial cells transform into mesenchymal cells, which plays a significant role in lung fibrotic disease. Transforming growth factor-β1(TGF-β1) is considered to be the most effective EMT inducer. The purpose of this study was to investigate the effect of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) on TGF-β1-induced EMT and the underlying mechanisms in the human bronchial epithelial cell line BEAS-2B. Human bronchial epithelial BEAS-2B cells were treated with TGF-β1 and TNF-α separately or in combination for 24h, and qRT-PCR, western blotting, immunofluorescence staining, and migration assays were used to investigate the EMT process. Moreover, to further explore the effect of the NF-κB pathway on the EMT process, inhibitor assays (BAY-117082, NF-κB inhibitor), wound healing assays, and western blotting were performed. The results showed that both cytokines enhanced the transformation of BEAS-2B cells from epithelial to mesenchymal cells. In addition, combined treatment with TNF-α and TGF-β1 further reduced E-cadherin expression, which conversely elevated α-SMA and vimentin mRNA and protein levels. Correspondingly, the migration rate of BEAS-2B cells was also increased. Furthermore, inhibiting the NF-κB signaling pathway blocked the expression of EMT-related markers and NOX4 induced by TGF-β1 and TNF-α, as well as cell migration. Taken together, TNF-α and TGF-β1 cooperatively promoted EMT and cell migration in BEAS-2B cells through the NF-κB/NOX4 signaling pathway.

Exercise-induced myokines downregulates the ACE2 level in bronchial epithelial cells: Implications for SARS-CoV-2 prevention.

BackgroundThe Covid-19 pandemic has emerged as the leading public health challenge of our time (20th century). While vaccinations have finally blunted the death rate, concern has remained about more virulent forms highlighting the need for alternative approaches. Epidemiological studies indicate that physical activity has been shown to decrease the risk of infection of some respiratory viruses. Part of the salutary effects of exercise is believed to be through the elaboration of cytokines by contracting skeletal muscles (termed myokines). The objective of this study was to investigate whether exercise-induced myokines would mitigate the SARS-CoV-2 infectivity of the bronchial epithelium through modulating the SARS-CoV-2 Covid-19 receptor (angiotensin-converting enzyme 2 -ACE2) its priming enzyme, transmembrane serine protease 2 (TMPRSS2).MethodsWe utilized a cell culture model of exercise to generate myokines by differentiating C2C12 cells into myotubules and inducing them to contract via low-frequency electric pulse stimulation. Condition media was concentrated via centrifugation and applied to human immortalized human bronchial epithelium cell line (6HBE14o) along with conditioned media from unstimulated myotubules as controls. Following exposure to myokines, the 16HBE14o cells were harvested and subjected to quantitative RT-PCR and Enzyme-Linked Immunosorbent Assay (ELISA) for assessment of mRNA and protein levels of ACE2 and TMPRSS2, respectively. Pilot proteomic data was performed with isotope barcoding and mass spectroscopy.ResultsQuantitative Real-Time PCR of 16HBE14o with 48 h treated unstimulated vs. stimulated myokine treatment revealed a reduction of ACE2 and TMPRSS2 mRNA by 32% (p<2.69x10-5) and 41% (p<4.57x10-5), respectively. The high sensitivity of ELISAs showed downregulation of ACE2 and TMPRSS2 protein expression in 16HBE14o cells by 53% (p<0.01) and 32% (p<0.03) respectively with 48 h treated. For rigor, this work was replicated in the human lung cancer cell line A549, which mirrored the downregulation. Proteomic analysis showed dramatic alteration in myokine profile between contracted and uncontracted C2C12 tubules.ConclusionsThe current study explores a novel approach of a modified exercise cell culture system and uses ACE2 and TMPRSS2 as a surrogate marker of SARS-CoV-2 infectivity. In conclusion, we demonstrated biological data supporting exercise’s protective effect against Covid-19. These further strengthen myokines’ beneficial role as potential therapeutic targets against SARS-CoV-2 and similar viruses albeit these preliminary cell culture studies will require future validation in animal models.

Loss of Frizzled 9 in Lung Cells Alters Epithelial Phenotype and Promotes Premalignant Lesion Development.

The transmembrane receptor Frizzled 9 (FZD9) is important for fetal neurologic and bone development through both canonical and non-canonical WNT/FZD signaling. In the adult lung, however, Fzd9 helps to maintain a normal epithelium by signaling through peroxisome proliferator activated receptor γ (PPARγ). The effect of FZD9 loss on normal lung epithelial cells and regulators of its expression in the lung are unknown. We knocked down FZD9 in human bronchial epithelial cell (HBEC) lines and found that downstream EMT targets and PPARγ activity are altered. We used a FZD9-/- mouse in the urethane lung adenocarcinoma model and found FZD9-/- adenomas had more proliferation, increased EMT signaling, decreased activation of PPARγ, increased expression of lung cancer associated genes, increased transformed growth, and increased potential for invasive behavior. We identified PPARγ as a transcriptional regulator of FZD9. We also demonstrated that extended cigarette smoke exposure in HBEC leads to decreased FZD9 expression, decreased activation of PPARγ, and increased transformed growth, and found that higher exposure to cigarette smoke in human lungs leads to decreased FZD9 expression. These results provide evidence for the role of FZD9 in lung epithelial maintenance and in smoking related malignant transformation. We identified the first transcriptional regulator of FZD9 in the lung and found FZD9 negative lesions are more dangerous. Loss of FZD9 creates a permissive environment for development of premalignant lung lesions, making it a potential target for intervention.

Multi-omic comparisons between CFBE41o- cells stably expressing wild-type CFTR and F508del-mutant CFTR

BackgroundCystic fibrosis (CF) is characterized by chronic inflammation and excessive cytokines secretion in the lung. Isogenic human CF bronchial epithelial (CFBE41o-) cell lines stably expressing wt-CFTR (WTBE) or F508del mutant (CFBE) are widely used tools in understanding responses to stimuli or drugs and CF pathogenesis in vitro. However, the intrinsic cellular differences in culture are unknown. MethodsWe performed integrative analyses of these isogenic cells at the protein, mRNA, and chromatin levels in the submerged and air-liquid interface (ALI) conditions to determine cell intrinsic effects of mutant versus complemented CFTR expression. ResultsCFBE and WTBE cells displayed different cytokine secretion patterns, including IL-6, IL-8, CXCL1, CXCL10, and CCL5. The ALI culture dramatically increased cytokine secretion in both cells. Assay for transposase-accessible chromatin using sequencing (ATAC-seq) result showed different chromatin landscapes upon polarization and CFBE cells, compared to WTBE cells, exhibited higher genome-wide chromatin accessibility under both culture methods. At the transcriptome level, differentially expressed genes identified by mRNA sequencing between two cell lines were highly concentrated in immunity-related pathways. ConclusionsThis multilayered study shows that expression of wild-type CFTR has an epithelial cell intrinsic effect on the cell's epigenome and transcriptome particularly in immunity relevant activities. These data will serve as a resource for the CF community and may serve as epithelial biomarkers for CFTR mRNA therapy.

Abstract 838: Loss of Arp2/3 activity promotes anchorage independent growth in KRAS;TP53-mutated human bronchial epithelial cells

Abstract Mutated KRAS and TP53 are well recognized drivers of multiple human cancers including lung cancer, the leading cause of cancer-associated deaths. Yet, directly targeting mutated KRAS or restoring wildtype p53 levels in KRAS/p53-mutated tumors still remains a therapeutic challenge. A rational alternative approach involves targeting potentiators of KRAS;p53-driven tumorigenesis, which represents one of the research ambitions pioneered by the National Cancer Institute (NCI) RAS initiative to meet this challenge. Therefore, we aimed to identify genes, that when lost, potentiate cellular transformation of a non-transformed KRAS;TP53-mutated human bronchial epithelial cell line (HBEC-KP). To address this question, a genome-wide selection experiment was performed using CRISPR/Cas9. Anchorage-independent (AI) growth, a hallmark of cancer, was selected as a phenotypic readout for one experiment. Without additional genetic perturbation, wildtype HBEC-KP cells were incapable of AI growth. Only certain mutants were capable of supporting colony formation in soft agar. These clones were isolated and the integrated small guide RNAs (sgRNAs) were identified. Genes that are targeted by the sgRNAs were then individually knocked out for validation. Through this approach, we discovered that loss of ARPC3 is sufficient for promoting AI growth in the HBEC-KP cells. This contribution to AI growth is attributed to the role that ARPC3 plays in regulating Arp2/3 activity, as inactivating the Arp2/3 complex using a small molecule inhibitor, CK666 also promoted colony growth in the HBEC-KP cells. We determined that ARPC3 loss was not sufficient to drive AI – in the absence of KRAS activation and p53 loss colony growth was not observed. This indicates the essential role that mutated KRAS and/or p53 play to support AI. To test if mutated KRAS is essential for the phenotype, the Arp2/3 complex was inactivated in KRAS-mutated HBECs, which supported AI growth. This suggests that mutated KRAS and loss of Arp2/3 activity are sufficient for driving AI growth in HBECs. However, combined loss of p53 and ARPC3 failed to drive the phenotype, implicating that mutated KRAS is required. In addition, the Arp2/3 complex is essential for maintaining lamellipodial shape via actin filament network and cell motility, which was validated in our HBEC-KP-ARPC3 knockout cells. However, this protein complex is also known to promote AI growth and development of various cancers, as opposed to acting as a suppressor for growth. Multiple mechanisms that lead to this growth phenotype are currently being explored. Clinical relevance is also being closely examined to further understand the role of the Arp2/3 complex in cancer. Citation Format: Chennan Li, Andrea L. Kasinski. Loss of Arp2/3 activity promotes anchorage independent growth in KRAS;TP53-mutated human bronchial epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 838.

Antibiotic-Loaded Amphiphilic Chitosan Nanoparticles Target Macrophages and Kill an Intracellular Pathogen.

In this work, levofloxacin (LVX), a third-generation fluoroquinolone antibiotic, is encapsulated within amphiphilic polymeric nanoparticles of a chitosan-g-poly(methyl methacrylate) produced by self-assembly and physically stabilized by ionotropic crosslinking with sodium tripolyphosphate. Non-crosslinked nanoparticles display a size of 29nm and a zeta-potential of +36mV, while the crosslinked counterparts display 45nm and +24mV, respectively. The cell compatibility, uptake, and intracellular trafficking are characterized in the murine alveolar macrophage cell line MH-S and the human bronchial epithelial cell line BEAS-2B in vitro. Internalization events are detected after 10min and the uptake is inhibited by several endocytosis inhibitors, indicating the involvement of complex endocytic pathways. In addition, the nanoparticles are detected in the lysosomal compartment. Then, the antibacterial efficacy of LVX-loaded nanoformulations (50% w/w drug content) is assessed in MH-S and BEAS-2B cells infected with Staphylococcus aureus and the bacterial burden is decreased by 49% and 46%, respectively. In contrast, free LVX leads to a decrease of 8% and 5%, respectively, in the same infected cell lines. Finally, intravenous injection to a zebrafish larval model shows that the nanoparticles accumulate in macrophages and endothelium and demonstrate the promise of these amphiphilic nanoparticles to target intracellular infections.

Vitamin D Improves the Effect of Glucocorticoids on Attenuating Lipopolysaccharide-Induced IL-6 Production via TLR4/NF-κB Pathway in Human Respiratory Epithelial Cells

Background: Glucocorticoid (GC) resistance results in unsatisfactory outcomes in chronic rhinosinusitis with nasal polyps (CRSwNP) patients. Previous studies have shown that calcitriol, the active form of vitamin D, alone or in combination with corticosteroids, exerts crucial immunomodulatory effects on inflammatory responses. However, whether vitamin D can improve the effect of GCs to attenuate inflammation in the epithelium of CRSwNP remains unclear. Methods: A human bronchial epithelial cell line (Beas-2B) and primary human nasal epithelial cells (HNECs) obtained from 10 patients with CRSwNP were exposed to lipopolysaccharide (LPS) for 24 h to establish an inflammation model. LPS-stimulated HNECs and Beas-2B cells were treated with/without dexamethasone in the presence or absence of calcitriol pretreatment for 24 h. The expression levels of interleukin-6 (IL-6) mRNA and protein were determined by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Toll-like receptor 4 (TLR4)/NF-κB pathway related markers were examined by western blotting. One-way ANOVA or the Kruskal-Wallis test with post hoc analysis were used for multiple comparisons among groups. Results: The production of IL-6 in Beas-2B cells and primary HNECs after LPS stimulation was significantly increased, which could be inhibited by dexamethasone or calcitriol alone. However, significant inhibition of IL-6 production was observed in the calcitriol plus dexamethasone group. Further analysis showed that calcitriol could enhance the effect of dexamethasone in inhibiting LPS-induced overexpression of TLR4, Myd88, and phosphorylation of p65. Conclusion: Our findings demonstrated that vitamin D could improve the effect of GCs to alleviate the level of IL-6 induced by LPS via the TLR4/NF-κB pathway in human respiratory epithelial cells.

Long Non-Coding RNA AP000695.2 Acts as a Novel Prognostic Biomarker and Regulates the Cell Growth and Migration of Lung Adenocarcinoma.

Long non-coding RNAs (lncRNAs) are tumor-associated biological molecules and have been found to be implicated in the progression of lung adenocarcinoma (LUAD). LncRNA-AP000695.2 (ENSG00000248538) is a long non-coding RNA (lncRNA) that is widely increased in many tumor types including lung adenocarcinoma (LUAD). However, the aberrant expression profile, clinical significance, and biological function of AP000695.2 in human lung adenocarcinoma (LUAD) need to be further investigated. This study mines key prognostic AP000695.2 and elucidates its potential role and molecular mechanism in regulating the proliferation and metastasis of LUAD. Here, we discovered that AP000695.2 was significantly upregulated in lung adenocarcinoma tissues compared with healthy adjacent lung tissue and higher in LUAD cell lines than in normal human bronchial epithelial cell lines. A higher expression of AP000695.2 was positively correlated with aggressive clinicopathological characteristics, and AP000695.2 served as an independent prognostic indicator for the overall survival, disease-free survival, and progression-free survival in patients with LUAD. Receiver operating curve (ROC) analysis revealed the significant diagnostic ability of AP000695.2 (AUC = 0.838). Our in vivo data confirmed that AP000695.2 promotes the proliferation, migration, and invasion of LUAD cells. GSEA results suggested that AP000695.2 co-expressed genes were mainly enriched in immune-related biological processes such as JAK-STAT signaling pathway and toll-like receptor signaling pathway. Single-sample GSEA analysis showed that AP000695.2 is correlated with tumor-infiltrating immune cells in lung adenocarcinoma. Our findings confirmed that AP000695.2 was involved in the progression of lung adenocarcinoma, providing a novel prognostic indicator and promising diagnostic biomarker in the future.

Evaluating electronic cigarette cytotoxicity and inflammatory responses in vitro.

INTRODUCTIONCigarette smoking poses many health risks and can cause chronic obstructive pulmonary disease (COPD), cardiovascular disease, cancer of the lung and other organs. Smokers can substantially reduce their risks of these diseases by quitting, but nicotine addiction makes this difficult. Alternatives, such as electronic cigarettes (e-cigarettes), may provide a similar dose of nicotine, but expose users to fewer toxic chemicals than traditional cigarettes and may still be harmful especially for dual users, therefore, we sought to develop bioassays that can assess the potential toxicity and inflammatory response induced by e-cigarette liquids (e-liquids) with and without flavors.METHODSE-liquids with varying nicotine content and flavors were aerosolized through growth media and exposed to human bronchial epithelial cell line (BEAS-2B) and human monocyte-macrophage cell line (THP-1) in vitro. Cytotoxicity in response to e-cigarette aerosols was measured by MTT assay in BEAS-2B cells and inflammatory response was measured by TNF-α, IL-6, IL-8, and MCP-1 released from THP-1 cells. In addition, the oxidative stress marker, REDD1, and impact on phagocytosis, was assessed following exposure of BEAS-2B and THP-1 derived macrophages, respectively. Cigarette smoke extract was used as a positive control with known cytotoxicity and impairment of inflammatory response.RESULTSE-cigarette aerosols induced moderate cellular toxicity in bronchial epithelial cells. Our data also show that low nicotine levels are less damaging to the bronchial epithelial cells, and flavors in e-liquids influence the combined inflammatory response markers, phagocytosis, and REDD1 when examined in vitro.CONCLUSIONSOur in vitro bioassays can be utilized to effectively measure flavor and nicotine-induced effects of e-cigarettes on combined inflammatory response and cytotoxicity in human macrophages and human bronchial epithelial cells, respectively.

FHL1 Overexpression as A Inhibitor of Lung Cancer Cell Invasion via Increasing RhoGDIß mRNA Expression.

Objective Four and a half Lin-11, Isl-1, Mac-3 (LIM) protein 1 (FHL1) is one of the FHL protein family, which is regarded as a tumor suppressor in the multiple malignant tumors. In this study, we aimed to explore the regulatory effects and mechanisms of FHL1 on lung cancer cell invasion. Materials and Methods In this experimental study, bioinformatics analysis of FHL1 transcripts in human lung adenocarcinomas of TCGA database was performed. Quantitative real-time polymerasechain reaction (PCR) was performed to detect FHL1 mRNA expression in 15 paired human lung cancer tissues and their adjacent normal lung tissues, or lung cancer cell lines (A549 and H1299) in comparison with human bronchial epithelial cell line (Beas- 2B). Moreover, western blot was used to analyze FHL1 and rho GDP-dissociation inhibitor beta (RhoGDIβ) protein expression in the indicated cell lines. Also, transwell assays were employed to measure the migrated, and invaded of indicated cell lines. Results FHL1 transcripts were downregulated in the human lung adenocarcinoma. The impaired FHL1 transcripts were positively correlated with advanced tumor node metastasis (TNM) stage. Moreover, as compared to the adjacent normal lung tissues, FHL1 mRNA was low expressed in 15 paired human lung cancer tissues than their adjacent normal lung tissues. Besides, FHL1 mRNA and protein expression were also reduced in H1299 and A549 cell lines in comparison with Beas-2B cell line. Overexpressed FHL1 protein inhibited the invasive ability of H1299 and A549 cell lines. Mechanically, FHL1 protein overexpression increased the RhoGDIβ protein and mRNA abundance, while knockdown of RhoGDIβ protein, completely restored the invasion ability of A549 (Flag-FHL1) cell line. ConclusionOur findings indicated that as a key FHL1 downstream regulator, RhoGDIβ is in charge of FHL1 inhibiting lung cancer cell invasion abilities, providing a critical insight into understanding the role of FHL1 for lung cancer development.

Downregulation of epithelial sodium channel (ENaC) activity in cystic fibrosis cells by epigenetic targeting

The pathogenic mechanism of cystic fibrosis (CF) includes the functional interaction of the cystic fibrosis transmembrane conductance regulator (CFTR) protein with the epithelial sodium channel (ENaC). The reduction of ENaC activity may constitute a therapeutic option for CF. This hypothesis was evaluated using drugs that target the protease-dependent activation of the ENaC channel and the transcriptional activity of its coding genes. To this aim we used: camostat, a protease inhibitor; S-adenosyl methionine (SAM), showed to induce DNA hypermethylation; curcumin, known to produce chromatin condensation. SAM and camostat are drugs already clinically used in other pathologies, while curcumin is a common dietary compound. The experimental systems used were CF and non-CF immortalized human bronchial epithelial cell lines as well as human bronchial primary epithelial cells. ENaC activity and SCNN1A, SCNN1B and SCNN1G gene expression were analyzed, in addition to SCNN1B promoter methylation. In both immortalized and primary cells, the inhibition of extracellular peptidases and the epigenetic manipulations reduced ENaC activity. Notably, the reduction in primary cells was much more effective. The SCNN1B appeared to be the best target to reduce ENaC activity, in respect to SCNN1A and SCNN1G. Indeed, SAM treatment resulted to be effective in inducing hypermethylation of SCNN1B gene promoter and in lowering its expression. Importantly, CFTR expression was unaffected, or even upregulated, after treatments. These results open the possibility of CF patients’ treatment by epigenetic targeting.

Arsenic Activates the ER Stress-Associated Unfolded Protein Response via the Activating Transcription Factor 6 in Human Bronchial Epithelial Cells.

Arsenic is a well-known human carcinogen associated with a number of cancers, including lung cancers. We have previously shown that long-term exposure to an environmentally relevant concentration of inorganic arsenic (As3+) leads to the malignant transformation of the BEAS2B cells, and some of the transformed cells show cancer stem-like features (CSCs) with a significant upregulation of glycolysis and downregulation of mitochondrial oxidative phosphorylation. In the present report, we investigate the short-term effect of As3+ on the endoplasmic reticulum (ER) stress response—the “unfolded protein response (UPR)” and metabolism in human bronchial epithelial cell line BEAS-2B cells. Treatment of the cells with inorganic As3+ upregulated both glycolysis and mitochondrial respiration. Analysis of ER UPR signaling pathway using a real-time human UPR array revealed that As3+ induced a significant up-regulation of some UPR genes, including ATF6, CEBPB, MAPK10, Hsp70, and UBE2G2. Additional tests confirmed that the induction of ATF6, ATF6B and UBE2G2 mRNAs and/or proteins by As3+ is dose dependent. Chromosome immunoprecipitation and global sequencing indicated a critical role of Nrf2 in mediating As3+-induced expression of these UPR genes. In summary, our data suggest that As3+ is able to regulate the ER stress response, possibly through activating the ATF6 signaling.

CFTR Modulation Reduces SARS-CoV-2 Infection in Human Bronchial Epithelial Cells.

People with cystic fibrosis should be considered at increased risk of developing severe symptoms of COVID-19. Strikingly, a broad array of evidence shows reduced spread of SARS-CoV-2 in these subjects, suggesting a potential role for CFTR in the regulation of SARS-CoV-2 infection/replication. Here, we analyzed SARS-CoV-2 replication in wild-type and CFTR-modified human bronchial epithelial cell lines and primary cells to investigate SARS-CoV-2 infection in people with cystic fibrosis. Both immortalized and primary human bronchial epithelial cells expressing wt or F508del-CFTR along with CRISPR/Cas9 CFTR-ablated clones were infected with SARS-CoV-2 and samples were harvested before and from 24 to 72 h post-infection. CFTR function was also inhibited in wt-CFTR cells with the CFTR-specific inhibitor IOWH-032 and partially restored in F508del-CFTR cells with a combination of CFTR modulators (VX-661+VX-445). Viral load was evaluated by real-time RT-PCR in both supernatant and cell extracts, and ACE-2 expression was analyzed by both western blotting and flow cytometry. SARS-CoV-2 replication was reduced in CFTR-modified bronchial cells compared with wild-type cell lines. No major difference in ACE-2 expression was detected before infection between wild-type and CFTR-modified cells, while a higher expression in wild-type compared to CFTR-modified cells was detectable at 72 h post-infection. Furthermore, inhibition of CFTR channel function elicited significant inhibition of viral replication in cells with wt-CFTR, and correction of CFTR function in F508del-CFTR cells increased the release of SARS-CoV-2 viral particles. Our study provides evidence that CFTR expression/function is involved in the regulation of SARS-CoV-2 replication, thus providing novel insights into the role of CFTR in SARS-CoV-2 infection and the development of therapeutic strategies for COVID-19.