Human Bronchial Epithelial 16HBE Cells Research Articles

Effect of mitogen-activated protein kinase signaling pathway on apoptosis induced by chloroacetic acid in human normal bronchial epithelial 16HBE cells

Objective: The aim of this study was to investigate the effect of mitogen-activated protein kinase (MAPK) signaling pathway on apoptosis induced by chloroacetic acid in human normal bronchial epithelial 16HBE cells. Methods: 16HBE cells were exposed to 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5 mmol/L chloroacetic acid for 24 h in vitro. The cytotoxicity induced by chloroacetic acid was assessed by CCK-8 and LDH assays. Cell apoptosis was detected by Annexin V-FITC and PI staining. The protein expression levels of phosphorylation of p38, ERK1/2 and JNK were determined by western blotting. 16HBE cells were pretreated with MAPK signaling pathway specific inhibitors including SB203580, U0126 and SP600125 for 1 h, and these cells were subsequently treated with 2.5 mmol/L chloroacetic acid for 24 h. The expressions of p-p38, p-ERK1/2 and p-JNK as well as the changes of cell viability and apoptosis were measured after pretreated with inhibitors for 1 h. Results: The cell viability by CCK-8 and LDH methods gradually reduced in a dose-dependent manner when chloroacetic acid concentrations elevated (P<0.05) , and their correlation coefficients were -0.902 and -0.825, respectively. The detection efficiency of CCK-8 assay significantly increased compared with LDH assay (P<0.05) . The cell apoptosis rates, which were (17.2±4.0) %, (24.6± 4.2) %, (39.3 ± 5.7) % in 1.5, 2.0, 2.5 mmol/L chloroacetic acid-treated groups, were higher than that of the control group[ (5.6 ± 3.0) %] (P<0.05) . There was a time-or dose-dependent change in the protein expressions of p-p38, p-ERK1/2 and p-JNK. Compared with the control, the levels of p-p38 had 2.1 and 2.6-fold increases in 16 and 24 h treated groups (P<0.01) , while the levels of p-ERK1/2 distinctly decreased by 37% and 52% (P<0.01) . In comparison with the control group, the expressions of p-p38 had 1.9 and 2.6-fold increases in 1.5 and 2.5 mmol/L treatment groups (P<0.01) , whereas the expressions of p-ERK1/2 significantly decreased by 40% and 50% (P<0.01) . No significant change was observed in p-JNK protein expression between the chloroacetic acid-treated and control groups. In comparison with the vehicle control and the exposed group, p-p38, p-ERK1/2, p-JNK protein expressions significantly declined in the inhibitor controls and inhibitor groups. Compared with the controls, the cell survival rates had significant reductions of 28%, 18%, 36% and 26% respectively in chloroacetic acid treated group, SB203580 group, U0126 group and SP600125 group, and the apoptosis rates in the abovementioned groups were 7, 4, 8 and 7 times. Compared with chloroacetic acid-treated group, the cell viability increased by 14% in SB203580 group and decreased by 11% in U0126 group, and the cell apoptosis rates decreased by 36% in SB203580 group and increased by 18% in U0126 group (P<0.05) . But no significant changes were observed in cell viability and apoptosis between SP600125 and chloroacetic acid-treated group. Conclusion: Chloroacetic acid might activate p38 MAPK signaling pathway and inhibit ERK1/2 MAPK signaling pathway. The signaling pathways of p38 and ERK1/2 MAPK are involved in 16HBE cell apoptosis induced by chloroacetic acid, but JNK is not involved in chloroacetic acid-induced 16HBE cell apoptosis.

Retraction of "Antioxidative and Antiinflammatory Activities of Asiatic Acid, Glycyrrhizic Acid, and Oleanolic Acid in Human Bronchial Epithelial Cells".

Protective effects of triterpenic acids, asiatic acid (AA), glycyrrhizic acid (GA), or oleanolic acid (OA), for two human bronchial epithelial cells, 16HBE and BEAS-2B cells, against hydrogen peroxide (H2O2) induced injury were examined. Cells were pretreated by triterpenic acid at 4 or 8 μmol/L and followed by H2O2 treatment. Results showed that H2O2 significantly upregulated both Bax and cleaved caspase-3 expression, and also downregulated Bcl-2 expression in test cells. AA at these doses retained Bcl-2 expression, but GA and OA only at 8 μmol/L reserved Bcl-2 expression. Test triterpenic acids lowered cleaved caspase-3 expression dose-dependently. H2O2 treatment lowered Na+-K+-ATPase activity and mitochondrial membrane potential in cells. Triterpenic acid pretreatments significantly maintained mitochondrial membrane potential and Na+-K+-ATPase activity. H2O2 enhanced reactive oxygen species, interleukin-6, tumor necrosis factor-α, and prostaglandin E2 levels in test cells. Three triterpenic acid treatm...

Comparison analysis of microRNAs in response to EV71 and CA16 infection in human bronchial epithelial cells by high-throughput sequencing to reveal differential infective mechanisms

Hand, foot, and mouth disease (HFMD) mainly caused by Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) infections which presented significantly different clinical manifestations. Nevertheless, the factors underlying these differences remain unclear. Recently, the functions of microRNAs (miRNAs) in pathogen-host interactions have been highlighted. Here, we performed comprehensive miRNA profiling in EV71- and CA16-infected human bronchial epithelial (16HBE) cells at multiple time points using high-throughput sequencing. The results showed that 154 known and 47 novel miRNAs exhibited remarkable differences in expression. Of these, 65 miRNAs, including 58 known and 7 novel miRNAs, presented opposite trends in EV71- and CA16-infected samples. Subsequently, we mainly focused on the 56 known differentially expressed miRNAs by further screening for targets prediction. GO and pathway analysis of these targets demonstrated that 18 biological processes, 7 molecular functions, 1 cellular component and 123 pathways were enriched. Among these pathways, Cadherin signalling pathway, Wnt signalling pathway and angiogenesis showed significant alterations. The regulatory networks of these miRNAs with predicted targets, GOs, pathways and transcription factors were determined, which suggested that miRNAs displayed intricate regulatory mechanisms during the infection phase. Consequently, we specifically analysed the hierarchical GO categories of the predicted targets involved in adhesion. The results indicated that the distinct changes induced by EV71 and CA16 infection may be partly linked to airway epithelial barrier function. Taken together, our data provide useful insights that help elucidate the different host-pathogen interactions following EV71 and CA16 infection and might offer novel therapeutic targets for these infections.

Autophagy Attenuates MnCl2-induced Apoptosis in Human Bronchial Epithelial Cells

ObjectiveTo investigate the role of autophagy in MnCl2-induced apoptosis in human bronchial epithelial 16HBE cells. MethodsCell proliferation was measured by MTT assay. Mitochondrial membrane potential (MMP) and apoptosis were measured by flow cytometry. Autophagic vacuoles were detected by fluorescence microscopy. Cellular levels of apoptosis and autophagy-related proteins were measured by western blotting. Results16HBE cell proliferation was inhibited by MnCl2 in a dose- and time-dependent manner. MnCl2-induced 16HBE cell growth inhibition was related to MMP depolarization prior to the induction of apoptosis. Our data revealed that MnCl2-induced apoptosis in 16HBE cells was mediated by decreased expression of Bcl-2 and increased levels of cleaved caspase-3. It was observed that when we exposed 16HBE cells to MnCl2 in a dose-dependent manner, the formation of autophagic vacuoles and the levels of LC-3B-II were elevated. RNA interference of LC3B in these MnCl2-exposed cells demonstrated that MMP loss and apoptosis were enhanced. Additionally, the pan-caspase inhibitor Z-VAD-FMK increased the cellular levels of Bcl-2 and decreased apoptosis, but did not affect the cellular levels of LC3B in MnCl2-treated 16HBE cells. ConclusionMnCl2 dose- and time-dependently inhibits 16HBE cell proliferation and induces MMP loss and apoptosis. Autophagy acts in a protective role against MnCl2-induced apoptosis in 16HBE cells.

Emulsified isoflurane treatment inhibits the cell cycle and respiration of human bronchial epithelial16HBEcells in a p53-independent manner.

Emulsified isoflurane (EIso), as a result of its rapid anesthetic induction, recovery and convenience, is widely used as a novel intravenous general anesthetic. Treatment with EIso can reduce injuries caused by ischemia/reperfusion (I/R) to organs, including the heart, lung and liver, without knowing understanding the molecular mechanism. The present study hypothesized that treatment with EIso can affect the physiological processes of human lung bronchial epithelial cells (16HBE) prior to I/R. To test this hypothesis, the present study first constructed stable p53knockdown and synthesis of cytochromecoxidase (SCO)2knockdown16HBE cells. The above cells were subsequently treated with EIso at a concentration of0.1and0.2% for 24h. The relevant concentration of fat emulsion was used as a negative control. The expression levels of p53, p21, SCO1, SCO2and Tp53‑induced glycolysis and apoptosis regulator (TIGAR) were detected by reverse transcription‑quantitative polymerase chain reaction and western blotting. Subsequently, the cell proliferation, respiration and glycolysis were investigated. The results revealed that EIso treatment significantly decreased the transcription of TIGAR, SCO1and SCO2, and increased the transcription of p21, which are all p53target genes, in a p53-independent manner. The cell cycle was inhibited by arresting cells at the G0/G1 phase. Respiration was reduced, which caused a decrease in oxygen consumption and the accumulation of lactate and reactive oxygen species. Taken together, EIso treatment inhibited the proliferation and respiration, and promoted glycolysis in16HBE cells. This regulatory pathway may represent a protective mechanism of EIso treatment by inhibiting cell growth and decreasing the oxygen consumption from I/R.

Inhibition of suppressor of cytokine signaling 1 on lipopolysaccharide-induced mucin5AC hypersecretion and the mechanism in human bronchial epithelial cells

To explore the inhibitory role of suppressor of cytokine signaling 1 (SOCS1) in lipopolysaccharide (LPS)-induced mucin5AC (MUC5AC) hypersecretion and the potential mechanism involved in this process. The human bronchial epithelial cells 16HBE were divided into 0, 0.5, 1, 6, 12 and 24 h groups according to the time of LPS challenge. In gain- and loss- of functions experiments, wild-type SOCS1 and SOCS1-targeted siRNA (SOCS1-siRNA) were synthesized to identify the function of SOCS1 in LPS-mediated MUC5AC hypersecretion, and named wild-type SOCS1 group and SOCS1-siRNA group, respectively, and the non-transfected group and non-targeted siRNA group were used as controls. In Filgotinib group, the specific inhibitor of Janus kinase 1 (JAK1), Filgotinib, was used to detect the role of JAK1/signal transducer and activator of transcription 1 (STAT1) signaling pathway in LPS challenge, and the aqueous physiological buffer group was used as the control. The production of MUC5AC protein was measured by enzyme linked immunosorbent assay (ELISA), and the amount of MUC5AC protein was normalized to the total protein in cell lysates and was expressed as μg/mg cell lysates. The proteins expressions of SOCS1, phosphorylation of JAK1 and STAT1 were measured by Western blot, and the total expression of its protein (for JAK1 and STAT1) or β-actin (for SOCS1) was used as the loading control. Compared to 0 h group, LPS induced a robust induction in MUC5AC expression, the expression levels of MUC5AC in 0, 0.5, 1, 6, 12 and 24 h groups were (2.86±0.20), (3.42±0.29), (3.43±0.12), (10.22±0.96), (14.56±1.12), (14.15±1.34) μg/mg, in association with a decrease of SOCS1 expression. And in 6 h group, the expressions of MUC5AC and SOCS1 were both medium up-regulated (all P<0.05). Consequently, the application of LPS for 6 h was selected as the optimal responses period in the ensuing experiments. Compared to the expression of MUC5AC protein in non-transfected group, high level of SOCS1 in wild-type SOCS1 group led to a reduced phosphorylation of JAK1/STAT1, as well as Filgotinib did, thereby suppressing excessive MUC5AC production in wild-type SOCS1 group and Filgotinib group [(4.04±0.65), (7.02±0.83) vs (10.37±1.00) μg/mg] (all P<0.05). Conversely, compared to the expression of MUC5AC in non-targeted siRNA group, down-regulation of SOCS1 in SOCS1-siRNA group promoted the phosphorylation of JAK1/STAT1, and then further increased MUC5AC production [(13.69±1.32) vs (11.01±1.41) μg/mg] (all P<0.05). These results show that SOCS1 suppresses LPS-promoted MUC5AC hypersecretion through the inhibition of JAK1/STAT1 signaling pathway.

Downregulation of selenium-binding protein 1 is associated with poor prognosis in lung squamous cell carcinoma.

BackgroundWe found that selenium-binding protein 1 (SBP1) was progressively decreased in the human bronchial epithelial carcinogenic processes. Knockdown of SBP1 in immortalized human bronchial epithelial cell line 16HBE cells significantly increased the efficiency of B[a]P-induced cell transformation. However, the relationship between SBP1 expression and clinicopathological factors of patients has not been defined completely. The specific role of SBP1 in prognosis of lung squamous cell carcinoma (LSCC) is still unknown.MethodsTissue samples from 82 patients treated by pulmonary lobectomy for LSCC were used. Immunohistochemistry and western blotting were used to detect the expressions of SBP1 protein. The relationships between the expression level of SBP1 and the clinicopathological features of patients were analyzed. Cox proportional hazard regression analysis and Kaplan–Meier method were used to perform survival analysis.ResultsExpressions of SBP1 proteins were significantly lower in LSCC tissues than that in the corresponding normal bronchial epithelium (NBE) tissues (P = 0.000). In LSCC, The expression levels of SBP1 had not correlated with patients’ age, gender, smoking state, primary tumor stages (T), TNM clinical stages, and distant metastasis (M) (P > 0.05). However, downregulation of SBP1 was significantly associated with higher lymph node metastasis and lower overall survival rate (P < 0.05). Cox regression analysis indicated low expressions of SBP1 can be an independent prognostic factor for poor overall survival in LSCC patients (P = 0.002).ConclusionsDownregulation of SBP1 may play a key role in the tumorigenic process of LSCC. SBP1 may be a novel potential prognostic factor of LSCC.

Repression of Biotin-Related Proteins by Benzo[a]Pyrene-Induced Epigenetic Modifications in Human Bronchial Epithelial Cells.

Benzo[a]pyrene (B[a]P) exposure has been associated with the alteration in epigenetic marks that are involved in cancer development. Biotinidase (BTD) and holocarboxylase synthetase (HCS) are 2 major enzymes involved in maintaining the homeostasis of biotinylation, and the deregulation of this pathway has been associated with a number of cancers. However, the link between B[a]P exposure and the dysregulation of BTD/HCS in B[a]P-associated tumorigenesis is unknown. Here we showed that the expression of both BTD and HCS was significantly decreased upon B[a]P treatment in human bronchial epithelial (16HBE) cells. Benzo[a]pyrene exposure led to the global loss of DNA methylation by immunofluorescence, which coincided with the reduction in acetylation levels on histones H3 and H4 in 16HBE cells. Consistent with decreased histone acetylation, histone deacetylases (HDACs) HDAC2 and HDAC3 were significantly upregulated in a dosage-dependent manner. When DNA methylation or HDAC activity was inhibited, we found that the reduction in BTD and HCS was separately regulated through distinct epigenetic mechanisms. Together, our results suggested the potential link between B[a]P toxicity and deregulation of biotin homeostasis pathway in B[a]P-associated cancer development.

Budesonide and fluticasone propionate differentially affect the airway epithelial barrier.

BackgroundCOPD patients have a higher risk of pneumonia when treated with fluticasone propionate (FP) than with placebo, and a lower risk with budesonide (BUD). We hypothesized that BUD and FP differentially affect the mucosal barrier in response to viral infection and/or cigarette smoke.MethodsWe assessed protective effects of equivalent concentrations of BUD and FP on cytokine production and barrier function (electrical resistance) in human bronchial epithelial 16HBE cells and primary bronchial epithelial cells (PBECs) upon exposure to viral mimetic poly-(I:C) and/or cigarette smoke extract (CSE) or epidermal growth factor (EGF).ResultsBUD and FP were equally effective in suppressing poly-(I:C)- and/or CSE-induced IL-8 secretion in 16HBE and PBECs. Poly-(I:C) substantially decreased electrical resistance in 16HBE cells and both BUD and FP fully counteracted this effect. However, FP hardly affected 16HBE barrier dysfunction induced by CSE with/without poly-(I:C), whereas BUD (16 nM) provided full protection, an effect likely mediated by affecting EGFR-downstream target GSK-3β. Similarly, BUD, but not FP, significantly improved CSE-induced barrier dysfunction in PBECs. Finally, BUD, but not FP, exerted a modest but significant protective effect against Streptococcus Pneumoniae-induced barrier dysfunction, and BUD, but not FP, prevented cellular adhesion and/or internalization of these bacteria induced by poly-(I:C) in 16HBE.ConclusionsCollectively, both BUD and FP efficiently control epithelial pro-inflammatory responses and barrier function upon mimicry of viral infection. Of potential clinical relevance, BUD more effectively counteracted CSE-induced barrier dysfunction, reinforcing the epithelial barrier and potentially limiting access of pathogens upon smoking in vivo.

ADAM10 mediates the house dust mite-induced release of chemokine ligand CCL20 by airway epithelium.

House dust mite (HDM) acts on the airway epithelium to induce airway inflammation in asthma. We previously showed that the ability of HDM to induce allergic sensitization in mice is related to airway epithelial CCL20 secretion. As a disintegrin and metalloprotease (ADAM)s have been implicated in chemokine shedding, we sought to determine their involvement in HDM-induced release of chemokines, including CCL20, by airway epithelial cells. We studied the effects of pharmacological ADAM inhibitors as well as ADAM10 and ADAM17 siRNA downregulation on chemokine release using (multiplex) ELISA in supernatants from HDM-exposed human bronchial epithelial 16HBE cells and primary normal human bronchial epithelial cells (NHBE) at 4-24h. House dust) mite markedly increased CCL20 levels in both 16HBE and NHBE cells (16-24h). In 16HBE cells, the HDM-induced increase was observed as early as 4h upon exposure and the use of specific inhibitors indicated the involvement of ADAM10/17-mediated shedding. siRNA knockdown of ADAM10, but not of ADAM17, significantly reduced the HDM-induced release of CCL20 in both 16HBE and NHBE cells. A similar effect was observed for HDM-induced CCL2, CCL5, and CXCL8 release in NHBE cells. The HDM-induced increase in CCL20 levels was not affected by protein synthesis inhibitor cycloheximide nor protein transport inhibitor monensin, indicating that HDM induces surface shedding of chemokines. Our data show for the first time that ADAM10 activity contributes to HDM-induced shedding of chemokines, including CCL20. The ADAM10/CCL20 axis may be a target for novel therapeutic strategies in asthma.

Anti-oncogene of opioid binding protein/cell adhesion molecule-like methylation status in malignant transformation of human bronchial epithelial cells induced by glycidyl methacrylate

To analyze the opioid binding protein/cell adhesion molecule-like (OPCML) methylation status at different stages of malignant transformation of human bronchial epithelial (16HBE) cells induced by Glycidyl Methacrylate (GMA) and to explore the effect of OPCML methylation in the process of malignant transformation. Cells were harvested at different stages (the 10th generation, the 20th generation and the 30th generation). To verify the Methylation chip result of OPCML methylation status in the process of malignant transformation, we detected it by methylation-specific-PCR (MSP); Real-time fluorescence Quantitative PCR (qPCR) were applied to measure the gene expression levels of OPCML at different transformed stage, and compared with the control groups (treated with DMSO). Based on the result of methylation chip, the gene of OPCML methylation occurred in all stages, which was consistent to the result of MSP; qPCR showed that the levels of gene expression decreased in the 20th generation and 30th generation. Methylation status of OPCML gene promoter could be considered as a stable and specific biomarker in the transformation process.

Tert-Butylhydroquinone mobilizes intracellular-bound zinc to stabilize Nrf2 through inhibiting phosphatase activity.

The nuclear factor erythroid 2-related factor 2 (Nrf2) is required to combat increases in oxidative stress. The chemical compound tert-butylhydroquinone (tBHQ) can downregulate Kelch-like ECH-associated protein 1 (Keap1), a repressor of Nrf2, thus maintaining the stability of Nrf2. tBHQ can also increase intracellular "free" zinc in human bronchial epithelial (16HBE) cells. We aim to investigate whether the intracellular free zinc change plays a role in Nrf2 activation. tBHQ exposure dose-dependently increases intracellular free zinc concentrations within 30 min in 16HBE cells by mobilizing intracellular zinc pools. Active Nrf2 and the antioxidant enzyme heme oxygenase-1 (HO-1) increase at 3 h after tBHQ treatment. Chelating intracellular free zinc with tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN) during tBHQ exposure partially abrogates the tBHQ-induced activation of Nrf2 and HO-1 expression, while Keap1 is further decreased. These results indicate that tBHQ-induced stability of Nrf2 is associated with the intracellular free zinc level. Because the activated Nrf2 is phosphorylated, the serine/threonine protein phosphatase activity, which is known to be inhibited by zinc, is assayed. The results showed that tBHQ treatment can suppress cellular protein phosphatase-2A (PP2A) and protein phosphatase-2C (PP2C) activity, which can be abrogated by adding TPEN. This finding is verified in a cell-free protein extract experiment by supplying zinc or by chelating zinc with TPEN. These results provide a novel mechanistic insight into Nrf2 activation in antioxidant enzyme induction involving zinc signaling. The increase of intracellular free zinc may be one mechanism for Nrf2 activation. The inhibition of PP2A and PP2C activity may be involved in Nrf2 phosphorylation modulation.

Antioxidative and antiinflammatory activities of asiatic acid, glycyrrhizic acid, and oleanolic acid in human bronchial epithelial cells.

Protective effects of triterpenic acids, asiatic acid (AA), glycyrrhizic acid (GA), or oleanolic acid (OA), for two human bronchial epithelial cells, 16HBE and BEAS-2B cells, against hydrogen peroxide (H2O2) induced injury were examined. Cells were pretreated by triterpenic acid at 4 or 8 μmol/L and followed by H2O2 treatment. Results showed that H2O2 significantly upregulated both Bax and cleaved caspase-3 expression, and also downregulated Bcl-2 expression in test cells. AA at these doses retained Bcl-2 expression, but GA and OA only at 8 μmol/L reserved Bcl-2 expression. Test triterpenic acids lowered cleaved caspase-3 expression dose-dependently. H2O2 treatment lowered Na(+)-K(+)-ATPase activity and mitochondrial membrane potential in cells. Triterpenic acid pretreatments significantly maintained mitochondrial membrane potential and Na(+)-K(+)-ATPase activity. H2O2 enhanced reactive oxygen species, interleukin-6, tumor necrosis factor-α, and prostaglandin E2 levels in test cells. Three triterpenic acid treatments dose-dependently reversed these changes. H2O2 promoted the protein expression of p47(phox), gp91(phox), cyclooxygenase-2 (COX-2), mitogen-activated protein kinase, and nuclear factor-κB (NF-κB). AA, GA, or OA pretreatments dose-dependently downregulated the expression of p47(phox), COX-2, NF-κB p65, and p-p38 but only at 8 μmol/L decreased gp91(phox) expression. These results support that these triterpenic acids could protect bronchial epithelial cells to attenuate apoptotic, oxidative, and inflammatory stress.

Altered Contractile Machinery in Airway Epithelial Cells in Response to Cigarette Smoke

Chronic obstructive pulmonary disease (COPD) is the 3rd leading cause of death in the US with cigarette smoke being the primary insult leading to disease progression. While the lung epithelium is the site that makes initial and primary contact with the inhaled cigarette smoke, we do not understand the role of cytoskeleton and cell mechanics in the airway response to cigarette smoke. We know that cytoskeletal proteins, such as myosin II, actin, alpha-actinin, the catenins, and E-cadherin, are involved in cell-cell adhesion formation and barrier function of the lung. Furthermore, E-cadherin levels decrease in primary epithelial cells from COPD patients. Therefore, we hypothesized that cigarette smoke drives key cytoskeletal protein changes that lead to alterations in barrier function and thereby influence the development of chronic lung disease. To test this, we studied major cytoskeletal (actin and nonmuscle myosin II isoforms) and cell adhesion proteins (E-cadherin) in normal human bronchial epithelial cells (NHBE, collected directly from human patients) or 16HBE cells (primary human bronchial epithelial cell line) grown on an air-liquid interface. Using a Vitrocell smoke chamber, we found that in response to acute smoke exposure (four cigarettes in 24 hrs), myosin IIB and actin assembled into apical stress fibers, which are not normally found in airway epithelial cells. Cigarette smoke exposure also resulted in decreased total E-cadherin levels as well as decreased E-cadherin at the basolateral surface. Thus, cigarette smoke promotes dramatic acute cytoskeletal changes that likely lead to altered cell mechanical properties (tension, elasticity), which in turn lead to reduced epithelial barrier function and our data suggest that epithelial barrier function is critical in dictating chronic tissue responses that contributes to COPD development and progression.

Microfluidic analysis of PM2.5-induced epithelial–mesenchymal transition in human bronchial epithelial 16HBE cells

Polluting particulate matter suspended in the air is a leading cause of lung carcinogenesis, a process often accompanied by epithelial to mesenchymal transition (EMT) of the bronchial epithelium. By providing a continuous supply of nutrients, microfluidic chips simulate the spatial characteristics of the cellular environment to enable integrated, flexible and high-throughput analysis of cell growth and function. Here, we used a microfluidic chip to evaluate the effects of the air pollutant PM2.5 on EMT in human bronchial epithelial 16HBE cells and on alveolar macrophage chemotaxis. PM2.5 induced NF-κB, PI3K, Snail and N-cadherin levels and repressed E-cadherin levels in 16HBE cells and promoted macrophage chemotaxis. Collectively, our results indicate that PM2.5 causes EMT in bronchial epithelial cells via induction of inflammatory pathways and shed light upon its pathological effects in humans.

P53-Dependent apoptosis induced in human bronchial epithelial (16-HBE) cells by PM2.5 sampled from air in Guangzhou, China

Epidemiological studies have shown that air pollution particulate matter (PM) is associated with increased respiratory morbidity and mortality. However, the mechanisms are not fully understood. Oxidative stress-mediated apoptosis plays an important role in the occurrence of respiratory diseases. In this study, human bronchial epithelial (16-HBE) cells were exposed to different concentrations (16–128 µg/ml) of PM2.5 for 24 h to investigate the apoptosis induced by PM2.5. The results showed that PM2.5 exposure significantly induced apoptosis, DNA strand breaks, and oxidative damage in a dose-dependent manner in 16-HBE cells. The expression of p53 and p73 increased significantly along with the dose of PM2.5 in 16-HBE cells, whereas the expression of p21Cip1/WAF1 decreased; the expression of mdm2 increased and then decreased, but not significantly. Taken together, these observations indicate that PM2.5 may lead to oxidative damage and induce apoptosis through the p53-dependent pathway in 16-HBE cells. p53-dependent apoptosis mediated by DNA strand breaks may be an important mechanism of PM2.5-induced apoptosis in 16-HBE cells.

Effect of hexavalent chromium on histone biotinylation in human bronchial epithelial cells

Chromium is a potent human mutagen and carcinogen. The capability of chromium to cause cancers has been known for more than a century, and numerous epidemiological studies have been performed to determine its carcinogenicity. In the post-genome era, cancer has been found to relate to epigenetic mutations. However, very few researches have focused on hexavalent chromium (Cr(VI))-induced epigenetic alterations. The present study was designed to investigate whether Cr(VI) would affect the level of a newfound epigenetic modification: histone biotinylation. Histone acetylation and histone biotinylation were studied in detail using human bronchial epithelial (16HBE) cells as an in vitro model after Cr(VI) treatment. Our study showed that Cr(VI) treatment decreased histone acetylation level in 16HBE cells. In addition, low doses of Cr(VI) (≤0.6μM) elevated the level of histone biotinylation. Furthermore, immunoblot analysis of biotinidase (BTD), a major protein which maintains homeostasis of histone biotinylation, showed that the distribution of BTD became less even and more concentrated at the nuclear periphery in cells exposed to Cr(VI). Moreover, Cr(VI)-induced histone deacetylation may take part in the regulation of histone biotinylation. Together, our study provides new insight into the mechanisms of Cr(VI)-induced epigenetic regulation that may contribute to the chemoprevention of Cr(VI)-induced cancers and may have important implications for epigenetic therapy.

Role of poly (ADP-ribose) polymerase 1 in DNA methylation changes induced by hydroquinone in human bronchial epithelial cell

To investigate the DNA methylation changes induced by hydroquinone (HQ) in human bronchial epithelial cells and to explore the role of poly (ADP-ribose) polymerase-l (PARP-l) in this process. Human bronchial epithelial 16HBE cells and PARP-l-deficient 16HBE cells (16HBE-shPARP-l cells) were exposed to HQ (10, 20, 40, 60, and 80 µmol/L) for 48h, while control cells were treated with an equal volume of PBS solution. The changes in genomic DNA methylation were investigated by high-performance capillary electrophoresis, and the expression levels of PARP-l and DNA methyltransferase 1 (DNMT1) were measured. The percentages of methylated DNA of overall genome (mCpG%) in 16HBE and 16HBE-shPARP-l cells were 4.89%±0.07% and 9.53%±0.51%, respectively; after treatment with 5-aza-2'-deoxycytidine for 72 h, mCpG% decreased to 3.07±0.12% and 6.34%±0.3%, respectively. The one-way analysis of variance revealed significant differences in mCpG% between the cells exposed to different concentrations of HQ in both 16HBE and 16HBE-shPARP-l groups (F = 61.25, P < 0.01; F = 60.36, P < 0.01). For 16HBE cells treated with HQ (10, 20, 40, 60, and 80 µmol/L), the mRNA expression levels of PARP-1 were 145.0%, 159.0%, 169.0%, 215.0%, and 236.0%, respectively, compared with those in the control group, with significant differences (P < 0.01 for all); for 16HBE-shPARP-l cells treated with HQ (10, 20, 40, 60, and 80 µmol/L), the mRNA expression levels of PARP-l were 170.0%, 223.0%, 264.0%, 327.0%, and 320.0%, respectively, compared with those in the control group, with significant differences (P < 0.01 for all). When the dose of HQ reached 20, 40, 60, and 80 µmol/L, the mRNA expression levels of DNMT1 in 16HBE group were 114.0%, 126.0%, 136.0%, and 162.0%, respectively, compared with those in the control group, with significant differences (P < 0.01 for all); when the dose of HQ reached 10, 20, 40, 60, and 80 µmol/L, the mRNA expression levels of DNMT1 in the 16HBE-shPARP-l group were 141.0%, 165.2%, 186.9%, 202.1%, and 217.3%, respectively, compared with those in the control group, with significant differences (P < 0.01 for all). HQ can induce hypomethylation in 16HBE cells, and PARP-1 can regulate DNA methylation in 16HBE cells by influencing the expression and activity of DNMT1.

Resolvin-D1 inhibits interleukin-8 and hydrogen peroxide production induced by cigarette smoke extract in 16HBE cells via attenuating NF-κB activation

Background Cigarette smoke induced airway inflammation plays a role in pathogenesis of airway inflammation. Resolvin-D1 derived from omega-3 polyunsaturated fatty acids is an endogenous anti-inflammatory and proresolving lipid mediator. Resolvin-D1 ameliorated inflammatory responses in lung injury, asthma, peritonitis and atherosclerosis. We investigated whether resolvin-D1 suppressed the productions of chemokines and oxidative stress induced by cigarette smoke extract (CSE) in vitro and its possible mechanism. Methods We examined the proinflammatory chemokine interleukin-8 and hydrogen peroxide (H2O2) productions induced by CSE in 16 human bronchial epithelial (16HBE) cells after resolvin-D1 treatment and their mechanisms. 16HBE cells were treated with resolvin-D1 at up to 10 nmol/L, for 30 minutes before CSE up to 16% (v/v) exposure. Release of interlukin-8 proteins was assessed by enzyme linked immunosort assay (ELISA) and its mRNA level by RT-PCR. We evaluated extracellular H2O2 expression in the supernatant. Phosphorylation of NF-κB/p65 and degradation of I-κB in 16HBE cells were determined by Western blotting analysis and NF-κB DNA binding activity by electrophoretic mobility shift assay (EMSA). Results 16HBE cells treated with 8% CSE showed significantly higher interlukin-8 production. Resolvin-D1 pretreatment inhibited CSE induced interlukin-8 production (mRNA and protein) in a dose and time dependent manner. Extracellular H2O2 level decreased after resolvin-D1 treatment. Resolvin-D1 attenuated CSE triggered I-κB degradation and NF-κB/p65 activation dose dependently and inhibited NF-κB DNA binding activity. Conclusion Resolvin-D1 inhibits CSE induced interlukin-8 and H2O2 production in 16HBE cells by modulating NF-κB activation and has therapeutic potential for pulmonary inflammation.

Expression of Telomerase Reverse Transcriptase during the Malignant Transformation of Cadmium-Induced Cells

The objective of the present study was to investigate human telomerase reverse transcriptase (hTERT) mRNA and protein expressions during the cadmium chloride-induced malignant transformation of human bronchial epithelial (16HBE) cells. Fluorescence quantitative PCR (FQ-PCR) and Western blot analyses were performed to detect the hTERT mRNA and protein expressions in normal 16HBE cells, cadmium chloride-transformed 16HBE cells, and tumorigenic cells from nude mice inoculated with cadmium chloride-transformed 16HBE cells. Under the inner standard of GAPDH, the hTERT mRNA expression was significantly higher at different stages of malignant transformation (cadmium chloride-transformed 16HBE cells at passages 15 and 35 and tumorigenic cells from nude mice) than in normal 16HBE cells, and increased with the development of malignancy (P < 0.01). In addition, hTERT protein expression increased with the development of malignancy. These findings demonstrate that hTERT expression is related to cadmium chlorideinduced malignant transformation. Cadmium chloride-induced malignant transformation is involved in changes in the hTERT activity, and might be an early event in cadmium chloride-induced malignant transformation.