Human Pulmonary Epithelial Cell Line Research Articles

Staphylococcus aureus Co-Infection in COVID-19 Patients: Virulence Genes and Their Influence on Respiratory Epithelial Cells in Light of Risk of Severe Secondary Infection.

Pandemics from viral respiratory tract infections in the 20th and early 21st centuries were associated with high mortality, which was not always caused by a primary viral infection. It has been observed that severe course of infection, complications and mortality were often the result of co-infection with other pathogens, especially Staphylococcus aureus. During the COVID-19 pandemic, it was also noticed that patients infected with S. aureus had a significantly higher mortality rate (61.7%) compared to patients infected with SARS-CoV-2 alone. Our previous studies have shown that S. aureus strains isolated from patients with COVID-19 had a different protein profile than the strains in non-COVID-19 patients. Therefore, this study aims to analyze S. aureus strains isolated from COVID-19 patients in terms of their pathogenicity by analyzing their virulence genes, adhesion, cytotoxicity and penetration to the human pulmonary epithelial cell line A549. We have observed that half of the tested S. aureus strains isolated from patients with COVID-19 had a necrotizing effect on the A549 cells. The strains also showed greater variability in terms of their adhesion to the human cells than their non-COVID-19 counterparts.

Heat-Killed Lactilactobacillus sakei WB2305 and Lactiplantibacillus plantarum WB2324 Inhibited LPS-Induced Inflammation in Human Airway Epithelial Cells.

Asthma is characterized by inflammation of the airways, including the inflammatory and airway structural cells. Probiotics, which have diverse effects, even within the same species, are being studied to prevent and mitigate the severity of asthma. Lactilactobacillus sakei WB2305 and Lactiplantibacillus plantarum WB2324 were isolated from kimchi. These strains have acceptable probiotic properties and are safe. In addition, the anti-inflammatory potential of the heat-killed isolates against lipopolysaccharide (LPS)-induced inflammation in the human pulmonary epithelial cell line (A549) was investigated. The heat-killed Lact. sakei WB2305 and Lact. plantarum WB2324 reduced the chemokine and cytokines mRNA expression levels, as shown by the results of using real-time polymerase chain reaction. Western blotting results showed that the nuclear factor-kappa B (NF-κB) activation and mitogen-activated protein kinases (MAPK) signaling pathways were suppressed by treatment with the heat-killed strains. The production amounts of eotaxin, tumor necrosis factor-ɑ (TNF-α), and interleukin-6 (IL-6) were lower than those in LPS-only treated cells. Additionally, 2',7'-dichlorofluorescein diacetate (DCFH-DA) staining confirmed decreased reactive oxygen species (ROS) production in A549 cells. Therefore, the results of present study demonstrate the anti-inflammatory and anti-asthmatic activities of heat-killed Lact. sakei WB2305 and Lact. plantarum WB2324 in human airway epithelial cells.

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.

Abstracts from The Aerosol Society Drug Delivery to the Lungs 33.

Abstracts from The Aerosol Society Drug Delivery to the Lungs 33.

Plasma membrane damage triggered by benzalkonium chloride and cetylpyridinium chloride induces G0/G1 cell cycle arrest via Cdc6 reduction in human lung epithelial cells.

Quaternary ammonium compounds, including benzalkonium chloride (BAC) and cetylpyridinium chloride (CPC), are widely used as disinfectants. Increased use of inhalable products containing BAC or CPC has raised concerns for lung toxicity. This study sought to elucidate the microstructure of plasma membrane damage caused by BAC and CPC and the subsequent mechanism by which the damage is mediated, as assessed using two human pulmonary epithelial cell lines (A549 and BEAS-2B). Scanning electron microscopic observation showed that exposure to BAC or CPC for 3 hr reduced the length and density of microvilli on the plasma membrane in A549 cells. Analysis of cell cycle distribution following plasma membrane damage revealed that BAC and CPC promote G0/G1 cell cycle arrest in both cell lines. The protein levels of Cdc6, an essential regulator of DNA replication during G1/S transition, are decreased significantly and dose dependently by BAC or CPC exposure. CPC and BAC decreased the Cdc6 levels that had been increased by a PI3K agonist in A549 cells, and levels of phosphorylated AKT were reduced in response to BAC or CPC. Conversely, exposure to equivalent concentrations of pyridinium chloride (lacking a hydrocarbon tail) induce no changes. These results suggest that plasma membrane damage triggered by BAC or CPC causes Cdc6-dependent G0/G1 cell cycle arrest in pulmonary cells. These effects are attributable to the long alkyl chains of BAC and CPC. The reduction of Cdc6 following plasma membrane damage may be caused, at least in part, by diminished signaling via the PI3K/AKT pathway.

Zerumbone combined with gefitinib alleviates lung cancer cell growth through the AKT/STAT3/SLC7A11 axis.

Zerumbone had been verified as a potential anti-cancer agent. Our research aimed to investigate the effect of zerumbone combined with gefitinib in lung cancer. Human pulmonary alveolar epithelial cells (HPAEpiC), A549, and H460 cell lines were used to detect the efficacy of zerumbone. BALB/c nude mice were randomly divided into five groups, including model, gefitinib (Gef, 10 mg/kg), low dose zerumbone (L-Zer, 20 mg/kg), high dose zerumbone (H-Zer, 40 mg/kg), and H-Zer + Gef groups, and the tumor growth in each group was monitored. TdT-mediated dUTP Nick-End Labeling (TUNEL) was used to detect cell apoptosis. Immunohistochemistry (IHC), immunofluorescence, and western blot were used to analyze the protein expressions in tumor tissues. Glutathione (GSH) and malondialdehyde (MAD) were detected by special kits. Zerumbone inhibited the proliferation of lung cancer cells in vitro. Tumor volume and weight were reduced after gefitinib or zerumbone treatment. Gefitinib and zerumbone treatment significantly promoted the apoptosis of tumor cells. The expression of Bcl-2, Bax, and P53 proteins confirmed cell apoptosis. IHC results indicated that zerumbone and gefitinib treatment decreased tumor angiogenesis. Consistent with this result, the expression of EGFR, VEGFR2, and Ki-67 proteins decreased, while the expression of angiostatin and endostatin proteins increased. Interestingly, zerumbone treatment increased the level of MDA while decreasing GSH. Next, the levels of glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) decreased after zerumbone and gefitinib treatment. Our study suggested that zerumbone combined with gefitinib could effectively inhibit lung cancer for multi-model therapies, including the inhibition of tumor growth, angiogenesis, induce cell apoptosis, and ferroptosis.

Biofilm formation, interaction and survival within A549 pneumocytes of Klebsiella pneumoniae clinical strains: identification of pulsotypes, multidrug-resistance and genes coding for adhesins

Klebsiella pneumoniaehas become one of the major causes of hospital-acquired infections over decades due to the spread of virulent clones harboring resistant genes to multiple antimicrobial agents. The aim of this study was to investigate phenotypic and genotypic features of virulence mechanism expressed by K. pneumoniae clinical isolates of different PFGE types, including biofilm formation, interaction with pneumocytes A549 lineage and experimental infection by using C. elegans nematodes. A total of 17 K. pneumoniae strains were isolated from different clinical specimens including blood, urine and respiratory infections. In this present study, 11 strains presented a varied multidrug-resistance profile harboring resistance genes coding for betalactams, aminoglicosydes, fluorquinolones and carbapenemases. PFGE analysis demonstrated the presence of four distinct pulsotypes among K. pneumoniae strains harboring virulence genes for siderophores and fimbiae type 1 and type 3. High adherence and biofilm formation were positively correlated for both polystyrene and glass surfaces in all K. pneumoniae strains analyzed. K. pneumoniae clinical strains showed the ability of adherence, internalization and persistence within human pulmonary epithelial A549 cell line, at different levels. Respiratory infections demonstrated a higher heterogeneity of PFGE types and levels of adherence, intracellular survival and persistence.K. pneumoniae strains were also submitted to Carnohabidits elegans in vivo infection model and data showed that after 24 hr almost 10% of urine-culture isolates worms were dead evidencing virulence profile. Notably, K. pneumoniae strains, presenting virulence genes, was significantly more virulent than those who did not presented any virulence gene after 5 days (survival >60% and >40%).

Long-Chain Poly-d-Lysines Interact with the Plasma Membrane and Induce Protective Autophagy and Intense Cell Necrosis.

Polylysines have been frequently used in drug delivery and antimicrobial and cell adhesion studies. Because of steric hindrance, chirality plays a major role in the functional difference between poly-l-lysine (PLL) and poly-d-lysine (PDL), especially when they interact with the plasma membranes of mammalian cells. Therefore, it is speculated that the interaction between chiral polylysines and the plasma membrane may cause different cellular behaviors. Here, we carefully investigated the interaction pattern of PLL and PDL with plasma membranes. We found that PDL could be anchored onto the plasma membrane and interact with the membrane lipids, leading to the rapid morphological change and death of A549 cells (a human lung cancer cell line) and HPAEpiCs (a human pulmonary alveolar epithelial cell line). In contrast, PLL exhibited good cytocompatibility and was not anchored onto the plasma membranes of these cells. Unlike PLL, PDL could trigger protective autophagy to prevent cells in a certain degree, and the PDL-caused cell death occurred via intense necrosis (featured by increased intracellular Ca2+ content and plasma membrane disruption). In addition, it was found that the short-chain PDL with a repeat unit number of 9 (termed DL9) could locate in lysosomes and induce autophagy at high concentrations, but it could not elicit drastic cell death, which proved that the repeat unit number of polylysine could affect its cellular action. This research confirms that the interaction between chiral polylysines and the plasma membrane can induce autophagy and intense necrosis, which provides guidance for the future studies of chiral molecules/drugs.

Dietary Supplementation with Biobran/MGN-3 Increases Innate Resistance and Reduces the Incidence of Influenza-like Illnesses in Elderly Subjects: A Randomized, Double-Blind, Placebo-Controlled Pilot Clinical Trial.

Influenza-like illness (ILI) remains a major cause of severe mortality and morbidity in the elderly. Aging is associated with a decreased ability to sense pathogens and mount effective innate and adaptive immune responses, thus mandating the development of protective nutraceuticals. Biobran/MGN-3, an arabinoxylan from rice bran, has potent anti-aging and immunomodulatory effects, suggesting that it may be effective against ILI. The objective of the current study was to investigate the effect of Biobran/MGN-3 on ILI incidence, natural killer (NK) cell activity, and the expressions of RIG-1 (retinoic acid-inducible gene 1), MDA5 (melanoma differentiation-associated protein 5), and their downstream signaling genes ISG-15 (interferon-stimulated genes 15) and MX1 (myxovirus (influenza) resistance 1, interferon-inducible). A double-blind, placebo-controlled clinical trial included eighty healthy older adults over 55 years old, 40 males and 40 females, who received either a placebo or Biobran/MGN-3 (500 mg/day) for 3 months during known ILI seasonality (peak incidence) in Egypt. The incidence of ILI was confirmed clinically according to the WHO case definition criteria. Hematological, hepatic, and renal parameters were assessed in all subjects, while the activity of NK and NKT (natural killer T) cells was assessed in six randomly chosen subjects in each group by the degranulation assay. The effect of Biobran/MGN-3 on RIG-1 and MDA5, as well as downstream ISG15 and MX1, was assessed in BEAS-2B pulmonary epithelial cells using flow cytometry. The incidence rate and incidence density of ILI in the Biobran/MGN-3 group were 5.0% and 0.57 cases per 1000 person-days, respectively, compared to 22.5% and 2.95 cases per 1000 person-days in the placebo group. Furthermore, Biobran/MGN-3 ingestion significantly enhanced NK activity compared to the basal levels and to the placebo group. In addition, Biobran/MGN-3 significantly upregulated the expression levels of RIG-1, MDA5, ISG15, and MX1 in the human pulmonary epithelial BEAS-2B cell lines. No side effects were observed. Taken together, Biobran/MGN-3 supplementation enhanced the innate immune response of elderly subjects by upregulating the NK activity associated with reduction of ILI incidence. It also upregulated the intracellular RIG-1, MDA5, ISG15, and MX1 expression in pulmonary epithelial tissue cultures. Biobran/MGN-3 could be a novel agent with prophylactic effects against a wide spectrum of respiratory viral infections that warrants further investigation.

Absence of Light Exposure Increases Pathogenicity of Pseudomonas aeruginosa Pneumonia-Associated Clinical Isolates.

Simple SummaryAn often-overlooked factor when designing an experiment is light exposure. Bacterial cultures are usually grown in an uncontrolled light environment, subject to natural daylight conditions. However, light as an abiotic factor has been described as a trigger for the switch from environmental to a pathogenic host-associated lifestyle. Pseudomonas aeruginosa possesses features that allow it to sense light and adapt its behavior accordingly. In this study, clinical isolates of P. aeruginosa from the sputum of pneumonia-diagnosed patients were assayed in two extreme light exposure conditions: constant illumination and intensity of full-spectrum light, and complete absence of light. The aim was to understand the influence of this factor on P. aeruginosa pathogenic potential, studied in interface with a human pulmonary epithelial cell line model. The preliminary findings here described evidenced that growth in the dark, which would more closely mimic the conditions in the lung microenvironment, was associated with a higher cytotoxicity against host cells, concordant with an increased pathogenic potential. Given the importance of accurately interpreting bacterial responses in in vitro studies, this study raises awareness of the need to control light exposure conditions to be as similar as possible to in vivo parameters.Pseudomonas aeruginosa can alter its lifestyle in response to changes in environmental conditions. The switch to a pathogenic host-associated lifestyle can be triggered by the luminosity settings, resorting to at least one photoreceptor which senses light and regulates cellular processes. This study aimed to address how light exposure affects the dynamic and adaptability of two P. aeruginosa pneumonia-associated isolates, HB13 and HB15. A phenotypic characterization of two opposing growth conditions, constant illumination and intensity of full-spectrum light and total absence of light, was performed. Given the nature of P. aeruginosa pathogenicity, distinct fractions were characterized, and its inherent pathogenic potential screened by comparing induced morphological alterations and cytotoxicity against human pulmonary epithelial cells (A549 cell line). Growth in the dark promoted some virulence-associated traits (e.g., pigment production, LasA proteolytic activity), which, together with higher cytotoxicity of secreted fractions, supported an increased pathogenic potential in conditions that better mimic the lung microenvironment of P. aeruginosa. These preliminary findings evidenced that light exposure settings may influence the P. aeruginosa pathogenic potential, likely owing to differential production of virulence factors. Thus, this study raised awareness towards the importance in controlling light conditions during bacterial pathogenicity evaluation approaches, to more accurately interpret bacterial responses.

The Spike Glycoprotein of SARS-CoV-2 Binds to β1 Integrins Expressed on the Surface of Lung Epithelial Cells.

The spike glycoprotein attached to the envelope of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds to and exploits angiotensin-converting enzyme 2 (ACE2) as an entry receptor to infect pulmonary epithelial cells. A subset of integrins that recognize the arginyl–glycyl–aspartic acid (RGD) sequence in the cognate ligands has been predicted in silico to bind the spike glycoprotein and, thereby, to be exploited for viral infection. Here, we show experimental evidence that the β1 integrins predominantly expressed on human pulmonary epithelial cell lines and primary mouse alveolar epithelial cells bind to this spike protein. The cellular β1 integrins support adhesive interactions with the spike protein independently of ACE2, suggesting the possibility that the β1 integrins may function as an alternative receptor for SARS-CoV-2, which could be targeted for the prevention of viral infections.

High mobility group box-1 protects against Aflatoxin G1-induced pulmonary epithelial cell damage in the lung inflammatory environment

Aflatoxin G1 (AFG1) is a member of the carcinogenic aflatoxin family. Our previous studies indicated that oral administration of AFG1 caused tumor necrosis factor (TNF)-α-dependent inflammation that enhanced oxidative DNA damage in alveolar epithelial cells, which may be related to AFG1-induced lung carcinogenesis. High mobility group box-1 (HMGB1) is a nuclear DNA-binding protein; the intracellular and extracellular roles of HMGB1 have been shown to contribute to DNA repair and sterile inflammation. The role of HMGB1 in DNA damage in an aflatoxin-induced lung inflammatory environment was investigated in this study. Upregulation of HMGB1, TLR2, and RAGE was observed in AFG1-induced lung inflamed tissues and adenocarcinoma. Blocking AFG1-induced inflammation by neutralization of TNF-α inhibited the upregulation of HMGB1 in mouse lung tissues, suggesting that AFG1-induced TNF-α-dependent inflammation regulated HMGB1 expression. In the in vitro human pulmonary epithelial cell line model, Beas-2b, AFG1 directly enhanced the cytosolic translocation of HMGB1 and its extracellular secretion. The addition of extracellular soluble HMGB1 protected AFG1-induced DNA damage through the TLR2/NF-κB pathway in Beas-2b cells. In addition, blockade of endogenous HMGB1 by siRNA significantly enhanced AFG1-induced damage. Thus, our findings showed that both extracellularly-released and nuclear and cytosolic HMGB1 could protect the cell from AFG1-induced cell damage in a TNF-α-dependent lung inflammatory environment.

Liquid biopsy of lung cancer by deep learning and spectroscopic analysis of circulating exosomes.

e15532 Background: Lung cancer has a high mortality rate because of belated diagnosis at advanced stages beyond the treatable condition. Early detection of lung cancer can improve the survival rate. A liquid biopsy that detects tumor-related biomarkers in body fluids has a great potential for the purpose. Particularly, tumor-derived exosomes in blood have been proposed as a promising biomarker. The tumor-derived exosomes carry molecules of their parental cells; thus, they provide information about the tumor in the body. Unfortunately, exosomal markers conducive to the early detection of lung cancer are still obscure. Therefore, using the molecular fingerprint of exosomes markers can be useful to detect the tumor exosomes. Raman spectroscopy is one of the representative methods for the purpose. However, because the exosomes have a heterogeneous composition in blood, interpreting their spectroscopic signals is hard. Thus, we utilized a deep learning approach to analyze the spectroscopic signal of the exosomes for liquid biopsy of lung cancer. Methods: The basic concept was to evaluate how much the exosomes in human plasma resemble cancer cell exosomes. As a proof of concept, exosomes of 43 non-small cell lung cancer (NSCLC) adenocarcinoma patients and 20 healthy controls were isolated from plasma of peripheral blood. Also, cell exosomes were isolated from culture media of adenocarcinoma cell lines and a human pulmonary alveolar epithelial cell line. Then, the spectroscopic signals were detected using surface-enhanced Raman spectroscopy (SERS). Further, the deep learning algorithm was employed to classify the signals. Then, we calculated the relative similarity to cancerous exosomes against human plasma exosomes. Results: Our method was able to classify cancer and normal cell exosomes with 95% accuracy. Also, Raman signals of cancer patients’ exosomes were more similar to the cancer cell exosomes than those of healthy controls. Notably, the similarity was proportional to cancer stages. Importantly, our method even detected stage I patients. The area under the curve (AUC) of receiver operating characteristic (ROC) curves was 0.912 for stage I and II, and 0.910 for stage I. Conclusions: We reported a novel diagnostic method using deep learning analysis against spectroscopic signals of circulating exosomes. Our method that evaluates the similarity to cancer exosomes accurately identified lung cancer patients, even stage I with high accuracy.

Role of 14-3-3σgene in the regulation of endotoxin/lipopolysaccharide-induced inflammatory responses in human pulmonary epithelial cells

Objective: To explore the mechanism of 14-3-3σgene in regulating inflammatory response of human pulmonary epithelial cells induced by endotoxin/lipopolysaccharide (LPS). Methods: (1) Cells of human normal pulmonary epithelial cell line BEAS-2B cultured in logarithmic growth period were collected and divided into control group and PCMV6-14-3-3σgroup using the random number table, with 3 wells in each group. Cells in control group were transfected with empty plasmid, and cells in PCMV6-14-3-3σgroup were transfected with PCMV6-14-3-3σplasmid. The protein expression of 14-3-3σin cell was detected by Western blotting at 48 hours after transfection. (2) Cells of human normal pulmonary epithelial cell line BEAS-2B cultured in logarithmic growth period were collected and divided into control group, PCMV6-14-3-3σgroup, PCMV6-14-3-3σ+ LPS group, and LPS group using the random number table, with 3 wells in each group. Cells in control group were transfected with empty plasmid for 42 hours. Cells in PCMV6-14-3-3σgroup were transfected with PCMV6-14-3-3σplasmid for 42 hours. Cells in PCMV6-14-3-3σ+ LPS group were stimulated with 1 μg/mL LPS (the same final mass concentration below) for 6 hours after being transfected with PCMV6-14-3-3σplasmid for 42 hours. Cells in LPS group were stimulated by LPS for 6 hours. The protein expressions of Bax and B-cell lymphoma-2 (Bcl-2) were detected by Western blotting, and the ratio of Bax to Bcl-2 was calculated. Apoptotic rate was detected by flow cytometry. The mRNA expressions of tumor necrosis factor alpha (TNF-α) and interleukin 1beta (IL-1β) in cells were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction technique. Content of TNF-α and IL-1β in cell culture supernatant was detected by enzyme-linked immunosorbent assay. Data were statistically analyzed with t test, one-way analysis of variance, and least significant difference test. Results: (1) At 48 hours after transfection, the protein expression of 14-3-3σin cells of PCMV6-14-3-3σgroup (1.05±0.03) was significantly higher than that in control group (0.78±0.04, t=5.41, P<0.01). (2) Compared with those in control group, the ratio of Bax to Bcl-2, apoptotic rate, mRNA expressions of TNF-α and IL-1β, and content of TNF-α and IL-1β in cell supernatant in PCMV6-14-3-3σgroup showed no significant difference (P>0.05); the above-mentioned indexes of cells in LPS group were significantly higher or increased (P<0.01). Compared with those in LPS group, the above-mentioned indexes of cells in PCMV6-14-3-3σ+ LPS group were significantly lower or decreased (P<0.01). Conclusions: 14-3-3σis a key factor in regulating apoptosis. It can alleviate the LPS-induced inflammatory responses by regulating the ratio of apoptotic regulators Bax to Bcl-2 and inhibiting apoptosis of human pulmonary epithelial cells.

Safety assessment of excipients (SAFE) for orally inhaled drug products.

The development of new orally inhaled drug products requires their demonstration of safety, which must be proven in animal experiments. New in vitro methods may replace, or at least reduce, these animal experiments, provided they are able to correctly predict safety or possible toxicity in humans. However, the challenge is to link in vitro data obtained in human cells to human in vivo data. We here present a new approach to the safety assessment of excipients (SAFE) for pulmonary drug delivery. The SAFE model is based on a dose response curve of 23 excipients tested on the human pulmonary epithelial cell lines A549 and Calu-3. The resulting in vitro IC50 values were correlated with the FDA-approved concentrations in pharmaceutical products for either pulmonary (if available) or parenteral administration. Setting a threshold of 0.1% (1 mg/mL) for either value yielded four safety classes and allowed to link IC50 data as measured in human cell cultures in vitro with the concentrations of the same compounds in FDA-approved drug products. The necessary in vitro data for novel excipients can be easily generated, and the SAFE approach allows putting them into context for eventual use in human pulmonary drug products. Excipients that are most likely not safe for use in humans can be excluded early on from further pharmaceutical development. The SAFE approach thus helps to avoid unnecessary animal experiments.

MicroRNA-381 inhibits lung adenocarcinoma cell biological progression by directly targeting LMO3 through regulation of the PI3K/Akt signaling pathway and epithelial-to-mesenchymal transition.

To investigate the role of miR-381 in the progression of lung adenocarcinoma (LA) and its underlying mechanism. A total of 54 pairs of LA tissues and para-carcinoma tissues were obtained from May 2015 to April 2017 in our hospital. Four human LA cell lines (A549, SPC-A1, H1299, and PC-9) and one normal human pulmonary epithelial cell line BEAS-2B were obtained and cultured. The protein and mRNA expression levels were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot, respectively. Additionally, cell proliferation assays and cell migration and invasion assays were used. Furthermore, tumor xenograft model in nude mice was made in this study. miR-381 was notably downregulated in LA tissues. Moreover, low miR-381 expression was confirmed to be strongly correlated with poor prognosis and aggressive clinicopathological characteristics of LA patients. Exogenous miR-381 overexpression was found to notably restrict LA cell proliferation, migration, and invasion; additionally, miR-381 overexpression could significantly reduce tumor growth in vivo. Mechanistically, LMO3 was determined as a novel direct target for miR-381 in LA cells. In clinical LA tissues, the LMO3 expressions were clearly overexpressed. Furthermore, miR-381 overexpression affected the PI3K/Akt pathway and EMT in LA. MiR-381 played key roles in LA progression, partially via directly targeting LMO3 and regulating the PI3K/Akt signaling pathway and EMT. Thus, the miR-381/ LMO3 axis has clinical significance in the therapy of patients with LA.

Glucoside Derivatives Of Podophyllotoxin: Synthesis, Physicochemical Properties, And Cytotoxicity.

BackgroundWidespread concern of the side effects and the broad-spectrum anticancer property of podophyllotoxin as an antitumor agent highlight the need for the development of new podophyllotoxin derivatives. Although some per-butyrylated glucosides of podophyllotoxin and 4β-triazolyl-podophyllotoxin glycosides show good anticancer activity, the per-acetylated/free of podophyllotoxin glucosides and their per-acetylated are not well studied.MethodsA few glucoside derivatives of PPT were synthesized and evaluated for their in vitro cytotoxic activities against five human cancer cell lines, HL-60 (leukemia), SMMC-7721 (hepatoma), A-549 (lung cancer), MCF-7 (breast cancer), and SW480 (colon cancer), as well as the normal human pulmonary epithelial cell line (BEAS-2B). In addition, we investigated the structure–activity relationship and the physicochemical property–anticancer activity relationship of these compounds.ResultsCompound 6b shows the highest cytotoxic potency against all five cancer cell lines tested, with IC50 values ranging from 3.27±0.21 to 11.37±0.52 μM. We have also found that 6b displays higher selectivity than the etoposide except in the case of HL-60 cell line. The active compounds possess similar physicochemical properties: MSA > 900, %PSA < 20, ClogP > 2, MW > 700 Da, and RB > 10.ConclusionWe synthesized several glucoside derivatives of PPT and tested their cytotoxicity. Among them, compound 6b showed the highest cytotoxicity. Further studies including selectivity of active compounds have shown that the selectivity indexes of 6b are much greater than the etoposide except in the case of HL-60 cell line. The active compounds possessed similar physicochemical properties. This study indicates that active glucoside analogs of podophyllotoxin have potential as lead compounds for developing novel anticancer agents.

Effects of chronic PM2.5 exposure on pulmonary epithelia: Transcriptome analysis of mRNA-exosomal miRNA interactions

Epidemiological studies have established the correlations between PM2.5 and a wide variety of pulmonary diseases. However, their underlying pathogeneses have not been clearly elucidated yet. In the present study, the epithelial-mesenchymal transition (EMT) phenotype with enhanced proliferation and migration activity of human pulmonary epithelial cell line BEAS-2B was observed after exposure to low dose PM2.5 exposure (50 μg/ml) for 30 passages. Then, epithelial cells derived-exosomal micro-RNA (miRNA) and intracellular total RNA were extracted, and the differentially expressed exosomal miRNAs (DE-Exo-MiRs) as well as differentially expressed protein coding genes (DEGs) were identified by RNA sequencing (RNA-seq) and transcriptome analysis. We found that chronic PM2.5 exposure stimulated the release of pulmonary epithelium derived exosomes. 45 DE-Exo-MiRs including 32 novelly predicted miRNAs and 843 DEGs between PM2.5 exposed group and the normal control were detected. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that DEGs were significantly enriched in extracellular matrix organization, focal adhesion and cancer related terms. Besides, the enrichment analyses on 7774 mRNA targets of 27 DE-Exo-MiRs predicted by MiRanda software also revealed the potential regulatory role of exosomal miRNAs in pathways in cancer, Wingless/Integrated (Wnt) signaling pathway, focal adhesion related genes and other multiple pathogenic pathways. Moreover, the interactive exosomal miRNA-mRNA pair networks were constructed using Cytoscape software. Our results provided a novel basis for a better understanding of the mechanisms of chronic PM2.5 exposure induced pulmonary disorders including pulmonary fibrosis and cancer, in which exosomal miRNAs (Exo-MiRs) potentially functions by dynamically regulating gene expressions.

Impact and mechanism of PMs-3 to epithelial mesenchymal transformation of non-small cell lung cancer cell line A549

Objective To explore Impact and mechanism of PMs-3 to epithelial mesenchymal transformation (EMT) of non-small cell lung cancer (NSCLC) cell line A549. Methods Nanocomposite structure synthetized by our team was utilized on human NSCLC cell line A549 and human normal pulmonary epithelial cell line BEAS-2B with concentrations of 0, 20, 40, 60, 80, 100, 120 nmol/L for 48 h. Methyl thiazol tetrazolium (MTT) assay was employed to detect the effect of PMs-3 on A549 cells. Wound assay and Transwell chamber assay were used to test the migration and invasion of PMs-3 on A549 cells. EMT related genes were detected with real time PCR (RT-qPCR) and Western blotting. Results Results of MTT assay detected that PMs-3 could inhibit activity of A549 cells significantly, and inhibition rate was (3.203±1.222)% (0 nmol/L), (11.958±4.000)% (20 nmol/L), (25.348±7.710)% (40 nmol/L), (34.775±9.155)% (60 nmol/L), (41.163±10.988)% (80 nmol/L), (50.430±9.743)% (100 nmol/L), (51.895±6.161)% (120 nmol/L), which showed a dose-dependent manner (F=35.451, P 0.05). Conclusion PMs-3 could inhibit NSCLC cells via regulating EMT. Key words: Non-small cell lung cancer; PMs-3; Epithelial mesenchymal transformation; Drug treatment

MicroRNA-218 regulates the epithelial-to-mesenchymal transition and the PI3K/Akt signaling pathway to suppress lung adenocarcinoma progression by directly targeting BMI-1.

To investigate the role of miR-218 in the development of lung adenocarcinoma (LA) and its underlying mechanism. Fifty-two pairs of human LA samples and adjacent para-carcinoma tissue samples were collected from our hospital between June 2015 and March 2017. Meanwhile, one normal human pulmonary epithelial cell line BEAS-2B and four human LA cell lines (H1299, PC-9, A549, and SPC-A1) were cultured. The cells' ability of proliferation and migration was detected by MTT assays and Transwell assays, respectively. The target gene was clarified by dual-luciferase reporter assay. The related protein and mRNA expression levels were detected by immunohistochemistry (IHC), Western blot and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. At last, the tumor xenograft model was made for further exploring the mechanism. MiR218 expressions were notably reduced in LA tissues in comparison with controls. In addition, the declined miR218 expressions were correlated with the poor OS and worse clinicopathological parameters of LA patients. Furthermore, miR218 overexpression could suppress the proliferation, migration and invasion capacities of LA cells via regulation of PI3K/Akt signaling pathway and epithelial-mesenchymal transition (EMT) respectively. Results in the current study also revealed that miR-218 upregulation could suppress the tumor growth rate and tumor size of LA mice. B-lymphoma Moloney murine leukemia virus insertion region-1 (BMI-1) was confirmed to be a direct target for miR-218 and upregulated in LA tissues, which indicated the poor prognosis of LA patients. MiR-218 exerted anti-tumor functions in LA partially via the regulation of BMI-1, suggesting that BMI-1/miR-218 axis may provide a novel insight into tumorigenesis and the basis for the development of miRNA-targeting therapies against LA.