Exposed BEAS-2B Cells Research Articles

Antioxidants Amelioration Is Insufficient to Prevent Acrylamide and Alpha-Solanine Synergistic Toxicity in BEAS-2B Cells.

Cells produce free radicals and antioxidants when exposed to toxic compounds during cellular metabolism. However, free radicals are deleterious to lipids, proteins, and nucleic acids. Antioxidants neutralize and eliminate free radicals from cells, preventing cell damage. Therefore, the study aims to determine whether the antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) will ameliorate the maximum dose of acrylamide and alpha (α)-solanine synergistic toxic effects in exposed BEAS-2B cells. These toxic compounds are consumed worldwide by eating potato products. BEAS-2B cells were simultaneously treated with BHA 10 μM and BHT 20 μM and incubated in a 5% CO2 humidified incubator for 24 h, followed by individual or combined treatment with acrylamide (3.5 mM) and α-solanine (44 mM) for 48 h, including the controls. Cell morphology, DNA, RNA, and protein were analyzed. The antioxidants did not prevent acrylamide and α-solanine synergistic effects in exposed BEAS-2B cells. However, cell morphology was altered; polymerase chain reaction (PCR) showed reduced RNA constituents but not DNA. In addition, the toxic compounds synergistically inhibited AKT/PKB expression and its downstream genes. The study showed BHA and BHT are not protective against the synergetic toxic effects of acrylamide and α-solanine in exposed BEAS-2B cells.

Ambient particulate matter (PM2.5) exposure contributes to neurodegeneration through the microbiome-gut-brain axis: Therapeutic role of melatonin

Exposure to ambient particulate matter (PM2.5) has been shown to disturb the gut microbiome homeostasis and cause initiation of neuroinflammation and neurodegeneration via gut-brain bi-directional axis. Polyaromatic hydrocarbons (PAHs), which are carcinogenic and mutagenic, are important organic constituents of PM2.5 that could be involved in the microbiome-gut-brain axis-mediated neurodegeneration. Melatonin (ML) has been shown to modulate the microbiome and curb inflammation in the gut and brain. However, no studies have been reported for its effect on PM2.5-induced neuroinflammation. In the current study, it was observed that treatment with ML at 100 µM significantly inhibits microglial activation (HMC-3 cells) and colonic inflammation (CCD-841 cells) by the conditioned media from PM2.5 exposed BEAS2B cells. Further, melatonin treatment at a dose of 50 mg/kg to C57BL/6 mice exposed to PM2.5 (at a dose of 60 µg/animal) for 90 days significantly alleviated the neuroinflammation and neurodegeneration caused by PAHs in PM2.5 by modulating olfactory-brain and microbiome-gut-brain axis.

Electronic Cigarette Aerosol Is Cytotoxic and Increases ACE2 Expression on Human Airway Epithelial Cells: Implications for SARS-CoV-2 (COVID-19).

Tobacco smoking has emerged as a risk factor for increasing the susceptibility to infection from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via increased expression of angiotensin-converting enzyme-2 (ACE2) in the lung, linked to coronavirus disease 2019 (COVID-19) development. Given the modifiable nature of electronic cigarettes and the delivery of high concentrations of nicotine, we investigate whether electronic cigarette vaping has the potential to increase susceptibility to SARS-CoV-2 infection. We exposed BEAS-2B cells (bronchial epithelium transformed with Ad12-SV40 2B) and primary small airway epithelial cells (SAECs) to electronic cigarette aerosol condensates produced from propylene glycol/vegetable glycerin or commercially bought e-liquid (±added nicotine) and cigarette smoke extract to investigate if electronic cigarette exposure, like cigarette smoke, increases the expression of ACE2 in lung epithelial cells. In BEAS-2B cells, cytotoxicity (CCK-8), membrane integrity (LDH), and ACE2 protein expression (immunofluorescence) were measured for both 4- and 24 h treatments in BEAS-2B cells and 4 h in SAECs; ACE2 gene expression was measured using quantitative polymerase chain reaction (qPCR) for 4 h treatment in BEAS-2B cells. Nicotine-free condensates and higher concentrations of nicotine-containing condensates were cytotoxic to BEAS-2B cells. Higher LDH release and reduced membrane integrity were seen in BEAS-2B cells treated for 24 h with higher concentrations of nicotine-containing condensates. ACE2 protein expression was observably increased in all treatments compared to cell controls, particularly for 24 h exposures. ACE2 gene expression was significantly increased in cells exposed to the locally bought e-liquid condensate with high nicotine concentration and cigarette smoke extract compared with cell controls. Our study suggests that vaping alone and smoking alone can result in an increase in lung ACE2 expression. Vaping and smoking are avoidable risk factors for COVID-19, which, if avoided, could help reduce the number of COVID-19 cases and the severity of the disease. This is the first study to utilize electronic cigarette aerosol condensates, novel and developed in our laboratory, for investigating ACE2 expression in human airway epithelial cells.

A Novel Transcriptome Integrated Network Approach Identifies the Key Driver lncRNA Involved in Cell Cycle With Chromium (VI)-Treated BEAS-2B Cells.

Hexavalent chromium [Cr(VI)] is a well-known occupational carcinogen, but the mechanisms contributing to DNA damage and cell cycle alternation have not been fully characterized. To study the dose-response effects of Cr(VI) on transcription, we exposed BEAS-2B cells to Cr(VI) at concentrations of 0.2, 0.6, and 1.8 μmol/L for 24 h. Here, we identified 1,484 differentially expressed genes (DEGs) in our transcript profiling data, with the majority of differentially expressed transcripts being downregulated. Our results also showed that these DEGs were enriched in pathways associated with the cell cycle, including DNA replication, chromatin assembly, and DNA repair. Using the differential expressed genes related to cell cycle, a weighted gene co-expression network was constructed and a key mRNA-lncRNA regulation module was identified under a scale-free network with topological properties. Additionally, key driver analysis (KDA) was applied to the mRNA-lncRNA regulation module to identify the driver genes. The KDA revealed that ARD3 (FDR = 1.46 × 10–22), SND1 (FDR = 5.24 × 10–8), and lnc-DHX32-2:1 (FDR = 1.43 × 10–17) were particularly highlighted in the category of G2/M, G1/S, and M phases. Moreover, several genes we identified exhibited great connectivity in our causal gene network with every key driver gene, including CDK14, POLA1, lnc-NCS1-2:1, and lnc-FOXK1-4:1 (all FDR < 0.05 in those phases). Together, these results obtained using mathematical approaches and bioinformatics algorithmics might provide potential new mechanisms involved in the cytotoxicity induced by Cr.

P62/SQSTM1 accumulation due to degradation inhibition and transcriptional activation plays a critical role in silica nanoparticle-induced airway inflammation via NF-\u03baB activation

BackgroundMost nanoparticles (NPs) reportedly block autophagic flux, thereby upregulating p62/SQSTM1 through degradation inhibition. p62 also acts as a multifunctional scaffold protein with multiple domains, and is involved in various cellular processes. However, the autophagy substrate-independent role of p62 and its regulation at the transcriptional level upon NPs exposure remain unclear.ResultsIn this work, we exposed BEAS-2b cells and mice to silica nanoparticles (SiNPs), and found that SiNPs increased p62 protein levels in vivo and vitro. Then, we further explored the role and mechanism of SiNPs-stimulated p62 in vitro, and found that p62 degradation was inhibited due to autophagic flux blockade. Mechanistically, SiNPs blocked autophagic flux through impairment of lysosomal capacity rather than defective autophagosome fusion with lysosomes. Moreover, SiNPs stimulated translocation of NF-E2-related factor 2 (Nrf2) to the nucleus from the cytoplasm, which upregulated p62 transcriptional activation through direct binding of Nrf2 to the p62 promoter. Nrf2 siRNA dramatically reduced both the mRNA and protein levels of p62. These two mechanisms led to p62 protein accumulation, thus increasing interleukin (IL)-1 and IL-6 expression. SiNPs activated nuclear factor kappa B (NF-κB), and this effect could be alleviated by p62 knockdown.ConclusionSiNPs caused accumulation of p62 through both pre- and post-translational mechanisms, resulting in airway inflammation. These findings improve our understanding of SiNP-induced pulmonary damage and the molecular targets available to mitigate it.

P62/SQSTM1 Accumulation Resulting from Degradation Inhibition and Transcriptional Activation is Essential in Silica Nanoparticle-Induced Pulmonary Inflammation Through NF-κB Activation

Most nanoparticles (NPs) are reported to block autophagic flux, accompanied by accumulated p62/SQSTM1 resulting from degradation inhibition. p62 also acts as a multifunctional scaffold protein that contains multiple domains, involved in various cellular processes. However, the autophagy substrate-independent role and regulation at a transcriptional level of p62 upon NPs exposure are ignored. Here, we exposed BEAS-2b cells to silica nanoparticles (SiNPs), and found that p62 degradation was inhibited due to autophagic flux blockade. Mechanically, SiNPs blocked autophagy flux through lysosomal capacity impairment rather than defective autophagosome fusion with lysosomes. Moreover, SiNPs stimulated translocation of NF-E2-related factor 2 (Nrf2) to the nucleus from the cytoplasm, and upregulated p62 transcriptional activation through direct binding of Nrf2 to p62 promoter. Nrf2 siRNA dramatically decreased both mRNA and protein levels of p62. Above two mechanisms led to p62 protein accumulation, therefore increasing IL-1 and IL-6 expression. SiNPs activated nuclear Factor kappa B (NF-κB), which can be alleviated by p62 knockdown. In summary, SiNPs accumulated p62 by both pre- and post-translational mechanisms, resulting in pulmonary inflammation. These findings improve our understanding of SiNP-induced pulmonary damage and molecular targets to antagonise it.

Ethyl pyruvate reduces organic dust-induced airway inflammation by targeting HMGB1-RAGE signaling

BackgroundAnimal production workers are persistently exposed to organic dust and can suffer from a variety of respiratory disease symptoms and annual decline in lung function. The role of high mobility group box-1 (HMGB1) in inflammatory airway diseases is emerging. Hence, we tested a hypothesis that organic dust exposure of airway epithelial cells induces nucleocytoplasmic translocation of HMGB1 and blocking this translocation dampens organic dust-induced lung inflammation.MethodsRats were exposed to either ambient air or swine barn (8 h/day for either 1, 5, or 20 days) and lung tissues were processed for immunohistochemistry. Swine barn dust was collected and organic dust extract (ODE) was prepared and sterilized. Human airway epithelial cell line (BEAS-2B) was exposed to either media or organic dust extract followed by treatment with media or ethyl pyruvate (EP) or anti-HMGB1 antibody. Immunoblotting, ELISA and other assays were performed at 0 (control), 6, 24 and 48 h. Data (as mean ± SEM) was analyzed using one or two-way ANOVA followed by Bonferroni’s post hoc comparison test. A p value of less than 0.05 was considered significant.ResultsCompared to controls, barn exposed rats showed an increase in the expression of HMGB1 in the lungs. Compared to controls, ODE exposed BEAS-2B cells showed nucleocytoplasmic translocation of HMGB1, co-localization of HMGB1 and RAGE, reactive species and pro-inflammatory cytokine production. EP treatment reduced the ODE induced nucleocytoplasmic translocation of HMGB1, HMGB1 expression in the cytoplasmic fraction, GM-CSF and IL-1β production and augmented the production of TGF-β1 and IL-10. Anti-HMGB1 treatment reduced ODE-induced NF-κB p65 expression, IL-6, ROS and RNS but augmented TGF-β1 and IL-10 levels.ConclusionsHMGB1-RAGE signaling is an attractive target to abrogate OD-induced lung inflammation.

Regulating temperature and relative humidity in air-liquid interface in vitro systems eliminates cytotoxicity resulting from control air exposures.

VITROCELL® systems permit cell exposures at the air-liquid interface (ALI); however, there are inconsistent methodologies in the literature for their operation. Some studies find that exposure to air (vehicle control) induced cytotoxicity relative to incubator controls; others do not mention if any cytotoxicity was encountered. We sought to test whether temperature and relative humidity (temp/RH) influence cytotoxicity with an unmodified (conditions A & B) and modified (condition C) VITROCELL® 6 CF with temp/RH controls to permit conditioning of the sampled air-flow. We exposed BEAS-2B cells for 1 h to air and measured viability (WST-1 cell proliferation assay) and lactate dehydrogenase (LDH) release 6 h post-exposure. Relative to controls, cells exposed to air at (A) 22 °C and 18% RH had a 47.9% ± 3.2% (p < 0.0001) reduction in cell viability and 10.7% ± 2.0% (p < 0.0001) increase in LDH release (B) 22 °C and 55% RH had a 40.3% ± 5.8% (p < 0.0001) reduction in cell viability and 2.6% ± 2.0% (p = 0.2056) increase in LDH release, or (C) 37 °C and >75% RH showed no changes in cell viability and no increase in LDH release. Furthermore, cells exposed to air at 37 °C and >75% RH 24 h post-exposure showed no changes in viability or LDH release relative to incubator controls. Thus, reductions in cell viability were induced under conditions used typically in the literature (conditions A & B). However, our modifications (condition C) overcome this shortcoming by preventing cell desiccation; regulating temp/RH is essential for conducting adequate ALI exposures.

Oncogenic transformation of human lung bronchial epithelial cells induced by arsenic involves ROS-dependent activation of STAT3-miR-21-PDCD4 mechanism.

Arsenic is a well-documented human carcinogen. The present study explored the role of the onco-miR, miR-21 and its target protein, programmed cell death 4 (PDCD4) in arsenic induced malignant cell transformation and tumorigenesis. Our results showed that treatment of human bronchial epithelial (BEAS-2B) cells with arsenic induces ROS through p47phox, one of the NOX subunits that is the key source of arsenic-induced ROS. Arsenic exposure induced an upregulation of miR-21 expression associated with inhibition of PDCD4, and caused malignant cell transformation and tumorigenesis of BEAS-2B cells. Indispensably, STAT3 transcriptional activation by IL-6 is crucial for the arsenic induced miR-21 increase. Upregulated miR-21 levels and suppressed PDCD4 expression was also observed in xenograft tumors generated with chronic arsenic exposed BEAS-2B cells. Stable shut down of miR-21, p47phox or STAT3 and overexpression of PDCD4 or catalase in BEAS-2B cells markedly inhibited the arsenic induced malignant transformation and tumorigenesis. Similarly, silencing of miR-21 or STAT3 and forced expression of PDCD4 in arsenic transformed cells (AsT) also inhibited cell proliferation and tumorigenesis. Furthermore, arsenic suppressed the downstream protein E-cadherin expression and induced β-catenin/TCF-dependent transcription of uPAR and c-Myc. These results indicate that the ROS-STAT3-miR-21-PDCD4 signaling axis plays an important role in arsenic -induced carcinogenesis.

Hexavalent chromium induces malignant transformation of human lung bronchial epithelial cells via ROS-dependent activation of miR-21-PDCD4 signaling.

Hexavalent chromium [Cr(VI)] is a well-known human carcinogen associated with an increased risk of lung cancer. However, the mechanisms underlying Cr(VI)-induced carcinogenesis remain unclear. MicroRNA-21 (miR-21) is a key regulator of oncogenic processes. Studies have shown that miR-21 exerts its oncogenic activity by targeting the tumor suppressor gene programmed cell death 4 (PDCD4). The present study examined the role of miR-21-PDCD4 signaling in Cr(VI)-induced cell transformation and tumorigenesis. Results showed that Cr(VI) induces ROS generation in human bronchial epithelial (BEAS-2B) cells. Chronic exposure to Cr(VI) is able to cause malignant transformation in BEAS-2B cells. Cr(VI) caused a significant increase of miR-21 expression associated with an inhibition of PDCD4 expression. Notably, STAT3 transcriptional activation by IL-6 is crucial for the Cr(VI)-induced miR-21 elevation. Stable knockdown of miR-21 or overexpression of PDCD4 in BEAS-2B cells significantly reduced the Cr(VI)-induced cell transformation. Furthermore, the Cr(VI) induced inhibition of PDCD4 suppressed downstream E-cadherin protein expression, but promoted β-catenin/TCF-dependent transcription of uPAR and c-Myc. We also found an increased miR-21 level and decreased PDCD4 expression in xenograft tumors generated with chronic Cr(VI)-exposed BEAS-2B cells. In addition, stable knockdown of miR-21 and overexpression of PDCD4 reduced the tumorogenicity of chronic Cr(VI)-exposed BEAS-2B cells in nude mice. Taken together, these results demonstrate that the miR-21-PDCD4 signaling axis plays an important role in Cr(VI)-induced carcinogenesis.

Identification of PM10 characteristics involved in cellular responses in human bronchial epithelial cells (Beas-2B)

Notwithstanding evidence is present that physicochemical characteristics of ambient particles attribute to adverse health effects, there is still some lack of understanding in this complex relationship. At this moment it is not clear which properties (such as particle size, chemical composition) or sources of the particles are most relevant for health effects. This study investigates the in vitro toxicity of PM10 in relation to PM chemical composition, black carbon (BC), endotoxin content and oxidative potential (OP).In 2013–2014 PM10 was sampled (24h sampling, 108 sampling days) in ambient air at three sites in Flanders (Belgium) with different pollution characteristics: an urban traffic site (Borgerhout), an industrial area (Zelzate) and a rural background location (Houtem). To characterize the toxic potential of PM10, airway epithelial cells (Beas-2B cells) have been exposed to particles in vitro. Different endpoints were studied including cell damage and death (cell viability) using the Neutral red Uptake assay, the production of pro-inflammatory molecules by interleukin 8 (IL-8) induction and DNA-damaging activity using the FPG-modified Comet assay. The endotoxin levels in the collected samples were analysed and the capacity of PM10 particles to produce reactive oxygen species (OP) was evaluated by electron paramagnetic resonance (EPR) spectroscopy. Chemical characteristics of PM10 (BC, As, Cd, Cr, Cu, Mn, Ni, Pb, Zn) and meteorological conditions were recorded on the sampling days.PM10 particles exhibited dose-dependent cytotoxicity in Beas-2B cells and were found to significantly induce the release of IL-8 in samples from the three locations. Oxidatively damaged DNA was observed in exposed Beas-2B cells. Endotoxin levels above the detection limit were detected in half of the samples. OP was measurable in all samples.Associations between PM10 characteristics and biological effects of PM10 were assessed by single and multiple regression analyses. The reduction in cell viability was significantly correlated with BC, Cd and Pb. The induction of IL-8 in Beas-2B cells was significantly associated with Cu, Ni and Zn and endotoxin. Endotoxin levels explained 33% of the variance in IL-8 induction. A significant interaction between ambient temperature and endotoxin on the pro-inflammatory activity was seen. No association was found between OP and the cellular responses.This study supports the hypothesis that, on an equal mass basis, PM10 induced biological effects differ due to differences in PM10 characteristics. Metals (Cd, Cu, Ni and Zn), BC, and endotoxin were among the main determinants for the observed biological responses.

House dust mite–induced asthma causes oxidative damage and DNA double-strand breaks in the lungs

Asthma is related to airway inflammation and oxidative stress. High levels of reactive oxygen and nitrogen species can induce cytotoxic DNA damage. Nevertheless, little is known about the possible role of allergen-induced DNA damage and DNA repair as modulators of asthma-associated pathology. We sought to study DNA damage and DNA damage responses induced by house dust mite (HDM) invivo and invitro. We measured DNA double-strand breaks (DSBs), DNA repair proteins, and apoptosis in an HDM-induced allergic asthma model and in lung samples from asthmatic patients. To study DNA repair, we treated mice with the DSB repair inhibitor NU7441. To study the direct DNA-damaging effect of HDM on human bronchial epithelial cells, we exposed BEAS-2B cells to HDM and measured DNA damage and reactive oxygen species levels. HDM challenge increased lung levels of oxidative damage to proteins (3-nitrotyrosine), lipids (8-isoprostane), and nucleic acid (8-oxoguanine). Immunohistochemical evidence for HDM-induced DNA DSBs was revealed by increased levels of the DSB marker γ Histone 2AX (H2AX)foci in bronchial epithelium. BEAS-2Bcells exposed to HDM showed enhanced DNA damage, as measured by using the comet assay and γH2AX staining. In lung tissue from human patients with asthma, we observed increased levels of DNA repair proteins and apoptosis, as shown by caspase-3 cleavage, caspase-activated DNase levels, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining. Notably, NU7441 augmented DNA damage and cytokine production in the bronchial epithelium and apoptosis in the allergic airway, implicating DSBs as an underlying driver of asthma pathophysiology. This work calls attention to reactive oxygen and nitrogen species and HDM-induced cytotoxicity and to a potential role for DNA repair as a modulator of asthma-associated pathophysiology.

Direct aeroallergen exposure disrupts the oxidant and antioxidant balance in human bronchial epithelial cells

Abstract Exposure to aeroallergens is a major risk factor for asthma development. Allergen possessing proteolytic activity is capable of disrupting airway epithelium. Increasing evidence pointing to asthma as an epithelial disease, however, the underlying mechanisms that drive asthma are not fully elucidated. Here, we investigated the DNA damage potential of aeroallergens on human bronchial epithelial cells BEAS-2B, and elucidated the damaging mechanisms. We exposed BEAS-2B cells to house dust mite (HDM), and measured DNA damage by CometChip and DNA double strand breaks (DSBs) by γH2AX staining. Intracellular oxidative stress was evaluated by staining cells with CellROS and MitoTracker. Antioxidant genes expression were determined using RT-PCR. Cell viability was determined by XTT assay. Cell death was studied using flow cytometry. To elucidate the underlying mechanism, cells were treated with Toll-like receptor 4 (TLR4) agonist or antagonist, or antioxidants glutathione or catalase. BEAS-2B cells exposed to HDM showed enhanced DNA damage, elevated ROS production, and increased mitochondrial oxidative stress and nitrite level. Expression of Nrf2-dependent antioxidant genes was reduced by HDM exposure. HDM also reduced cell viability and induced cell death. Importantly, HDM-induced DNA damage could be prevented by antioxidants. Our results suggest that direct HDM exposure to bronchial epithelial cells leads to production of RONS that damage DNA and induce cytotoxicity. Antioxidants can prevent HDM-induced DNA damage, revealing a novel role for antioxidants in preventing genotoxicity. [This study was partially supported by NMRC/CBRG/0027/2012, NUHS Seed Grant R-184-000-238-112, NRF, NIEHS R44-ES0211116 and Trevigen Inc.]

Luteolin inhibits Cr(VI)-induced malignant cell transformation of human lung epithelial cells by targeting ROS mediated multiple cell signaling pathways

Hexavalent chromium [Cr(VI)] is a well-known human carcinogen associated with the incidence of lung cancer. Inhibition of metal induced carcinogenesis by a dietary antioxidant is a novel approach. Luteolin, a natural dietary flavonoid found in fruits and vegetables, possesses potent antioxidant and anti-inflammatory activity. We found that short term exposure of human bronchial epithelial cells (BEAS-2B) to Cr(VI) (5μM) showed a drastic increase in ROS generation, NADPH oxidase (NOX) activation, lipid peroxidation, and glutathione depletion, which were significantly inhibited by the treatment with luteolin in a dose dependent manner. Treatment with luteolin decreased AP-1, HIF-1α, COX-2, and iNOS promoter activity induced by Cr(VI) in BEAS-2B cells. In addition, luteolin protected BEAS-2B cells from malignant transformation induced by chronic Cr(VI) exposure. Moreover, luteolin also inhibited the production of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α) and VEGF in chronic Cr(VI) exposed BEAS-2B cells. Western blot analysis showed that luteolin inhibited multiple gene products linked to survival (Akt, Fak, Bcl-2, Bcl-xL), inflammation (MAPK, NF-κB, COX-2, STAT-3, iNOS, TNF-α) and angiogenesis (HIF-1α, VEGF, MMP-9) in chronic Cr(VI) exposed BEAS-2B cells. Nude mice injected with BEAS-2B cells chronically exposed to Cr(VI) in the presence of luteolin showed reduced tumor incidence compared to Cr(VI) alone treated group. Overexpression of catalase (CAT) or SOD2, eliminated Cr(VI)-induced malignant transformation. Overall, our results indicate that luteolin protects BEAS-2B cells from Cr(VI)-induced carcinogenesis by scavenging ROS and modulating multiple cell signaling mechanisms that are linked to ROS. Luteolin, therefore, serves as a potential chemopreventive agent against Cr(VI)-induced carcinogenesis.

Cytoprotective effect of kaempferol on paraquat-exposed BEAS-2B cells via modulating expression of MUC5AC.

Mucins are highly glycosylated secretary proteins produced by most epithelial cells. Hypersecretion of mucins is one of the prominent symptoms of several airway diseases, including asthma, cystic fibrosis, nasal allergy, rhinitis, and sinusitis. Paraquat (PQ), a common herbicide, has been associated with pulmonary damage and is a potent reactive oxygen species (ROS) producer. However, until now the role of PQ on mucin overproduction has not been studied. The aim of this study is to explore how kaempferol (KM), a widely used dietary flavonoid, affects the protection of human PQ-exposed bronchial epithelium BEAS-2B cells by suppressing Mucin gene expression via nuclear factor-kappa B (NF-κB). We observed that PQ generates intracellular ROS, and also induces lipid peroxidation in BEAS-2B cells. Additionally, we found that PQ effectively induces the expression of the MUC5AC gene; however, co-treatment of PQ with KM drastically reduces its expression. Furthermore, we observed that PQ activates NF-κB, while co-treatment with KM occludes its nuclear translocation, and additionally KM repressed the PQ phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) in BEAS-2B cells. Based on our data, we believe that KM can suppress the over-expression of the MUC5AC gene. This would contribute to the protection of PQ cytotoxicity to exposed BEAS-2B cells, and allow further study toward a better understanding of ROS-associated diseases.

Analytical constraints for the analysis of human cell line secretomes by shotgun proteomics

Human cell line secretome represents a valuable source of therapeutic targets and candidate biomarkers. Secreted proteins found in biological fluids or culture media are by essence highly diluted. Secretome investigation with proteomic approaches is hardly compatible with the high content of proteins found in complete cell culture media. Therefore, many studies are currently done with media containing few or no protein. Such conditions may perturb cell metabolism and proliferation. Here, we compared seventeen different compositions of culture media for the human bronchial epithelial BEAS-2B cell line. Cell viability, proliferation rate and initial protein charge were systematically compared. We have shown that an important difficulty for the proteomic analysis is due to the presence of detergents such as Pluronic F-68 which hinders peptide mass spectrometry. The high glucose containing DMEM medium which is free of proteins was shown to preserve a good viability and proliferation of cells. With this conditioning medium, we identified 81 extracellular proteins in the secretome of BEAS-2B cells. Moreover, to illustrate this approach, we exposed BEAS-2B cells to a low toxic dose of CoCl(2,) and found 24 extracellular proteins modulated by cobalt. This study highlights the possible contribution of such proteomic approach in the field of toxicology.

A tobacco-specific N-nitrosamine or cigarette smoke condensate causes neoplastic transformation of xenotransplanted human bronchial epithelial cells.

Using a xenotransplantation system in which immortalized nontumorigenic human bronchial epithelial cells (BEAS-2B cells) are grown in deepithelialized rat tracheas that are subcutaneously transplanted into athymic nude mice, we exposed BEAS-2B cells either to cigarette smoke condensate or to the tobacco-specific N-nitrosamine 4-(methylnitrosamine)-1-(3-pyridyl)-1- butanone. After 6 mo the carcinogen-exposed BEAS-2B cells were neoplastically transformed to invasive adenocarcinomas. Cell lines obtained from xenografts exposed in vivo to chemicals exhibited several features typical of malignant lung cancer cells, such as increased in vivo invasiveness that correlated well with enhanced type IV collagenolytic activity, resistance to serum-induced growth inhibition, and increased expression of transforming growth factor alpha and its cellular-membrane receptor. Invasiveness, similar to that seen after exposure to phorbol esters, was also detected after in vitro exposure of BEAS-2B cells to cigarette smoke condensate. Collectively, these data indicate that cigarette smoke condensate and N-nitrosamine 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone induce in vivo phenotypic changes in BEAS-2B cells similar to the progressive changes that occur during human lung carcinogenesis.