Normal Bronchial Epithelial BEAS-2B Cells Research Articles

Modulatory role of PACAP and VIP on HIFs expression in lung adenocarcinoma

Lung adenocarcinoma is the most frequent form of non-small cell lung cancer. Inside the tumor mass, uncontrolled cell proliferation generates hypoxic areas leading to activation of hypoxia-inducible factors (HIFs) responsible for neovascularization and tumor metastasis. Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are two neuropeptides widely distributed in respiratory organs. Previous studies have demonstrated that these peptides interfere with hypoxic pathways in various diseases, including tumors. However, their modulatory role in HIFs expression in lung adenocarcinomas has not yet been evaluated. In the present paper, we detected the expression profile of PACAP, VIP and related receptors in healthy and adenocarcinoma human lung tissue. To characterize peptides’ modulatory effects on HIFs expression, we also exposed A549 lung adenocarcinoma cells and human normal bronchial epithelial BEAS-2B cells to microenvironmental hypoxia by treating them with deferoxamine (DFX). The results showed that PACAP and VIP significantly reduced HIF-1α and HIF-2α levels in both cell lines following hypoxic stress. The HIF-3α expression profile was related to cellular phenotype as it was lower in BEAS-2B and higher in A549 cells under low oxygen tension. In lung adenocarcinoma cells, peptide treatment restored HIF-3 α expression to control levels. These results suggest that endogenous PACAP and VIP exert controversial roles in cellular hypoxic microenvironments depending on the pathophysiological conditions of the lung tissue.

Soluble fms-like tyrosine kinase-1-enriched exosomes suppress the growth of small cell lung cancer by inhibiting endothelial cell migration.

BackgroundPrevious studies have reported that soluble fms‐like tyrosine kinase‐1 (sFlt‐1) possesses anti‐tumor effects by inhibiting angiogenesis in many cancers. Exosomes can be engineered as delivery vehicles for transferring functional biomolecules, such as proteins, lipids, and nucleic acids (DNA, mRNA, and miRNA) to target cells to affect inflammation, apoptosis, and angiogenesis. The purpose of this study was to investigate whether exosomes can function as efficient carriers of sFlt‐1 in vitro and in vivo, to play a role in SCLC therapy.MethodsWe adopted three different methods: TEM, NTA and western blot analysis to characterize the cell‐derived exosomes from NCI‐H69 SCLC cell line and normal bronchial epithelial BEAS‐2B cell line. we next explored the effects of these exosomes on HUVE cell proliferation and migration in vitro.To verify sFlt‐1‐loaded exosomes suppress the tumor growth in vivo,we established subcutaneous xenografts in nude mice using the NCI‐H69 cell line.ResultsWe observed that NCI‐H69‐exo significantly increased human umbilical vein endothelial cells (HUVEC) migration compared to BEAS‐2B‐exo in vitro and in vivo. sFlt‐1 protein expression was statistically higher in BEAS‐2B‐exo than NCI‐H69‐exo. sFlt‐1 protein or sFlt‐1‐enriched exosomes can inhibit the migration of HUVECs. Furthermore, sFlt‐1‐enriched exosomes exhibited higher inhibition efficacy on pro‐angiogenesis of NCI‐H69‐exo in comparison with the same concentration of sFlt‐1 protein. Intriguingly, sFlt‐1‐loaded exosomes showed marked anti‐tumor activity by inhibiting the growth of NCI‐H69 tumor xenografts. CD31 staining revealed that sFlt‐1‐loaded exosomes significantly reduced the vascular density of experimental mice. sFlt‐1‐loaded exosomes markedly induced tumor apoptosis and inhibited tumor cell proliferation in mice.ConclusionExosomes from a SCLC cell line contain low levels of sFlt‐1 and significantly increased the migration of HUVECs. SFlt‐1‐enriched exosomes can inhibit NCI‐H69‐exo‐induced HUVEC migration. Exosomes enriched in sFlt‐1 have the potential to be effective therapeutic agents for SCLC.

Environmental concentration of spray paint particulate matters causes pulmonary dysfunction in human normal bronchial epithelial BEAS-2B cell

In addition to airborne particulate matter (PM), exposure to spray paint PMs (SPPMs) may also be associated with pulmonary dysfunction. In this study, we employed human normal bronchial epithelial BEAS-2B cells to elucidate the association between pulmonary toxicity and different sizes of SPPMs (SPPM10-3.2, SPPM3.2-1 and SPPM1) under realistic environmental concentrations in a spray paint factory. Results indicated that all SPPMs (20–100μg/cm2) induced significant decreases in cell viability (> 70% compared to the control), except for low-dose SPPM10-3.2 (20 and 50μg/cm2). Almost all SPPMs (20μg/cm2 and 100μg/cm2) induced oxidative stress (2–4 times that of the control), which increased the production of proinflammatory cytokines (1.5–4.5 times that of the control) as well as increased α1-antitrypsin expression (3–4.5 times that of the control). Moreover, we found that almost all SPPMs induced pulmonary epithelial barrier dysfunction (0.77–0.11 times that of the control) through the depletion of zonula occludens proteins (0.8–0.65 times that of the control). In conclusion, smaller SPPMs induced more severe adverse pulmonary adverse effects. Exposure to SPPM1 was a potential major risk factor for pulmonary epithelial barrier dysfunction. Our evidence demonstrates that exposure to SPPMs, especially SPPM1, may increase the risk of pulmonary dysfunction.

Ethyl Acetate Extract of Scindapsus cf. hederaceus Exerts the Inhibitory Bioactivity on Human Non-Small Cell Lung Cancer Cells through Modulating ER Stress.

Unfolded protein response (UPR) is a cytoprotective mechanism that alleviates the protein-folding burden in eukaryotic organisms. Moderate activation of UPR is required for maintaining endoplasmic reticulum (ER) homeostasis and profoundly contributes to tumorigenesis. Defects in UPR signaling are implicated in the attenuation of various malignant phenotypes including cell proliferation, migration, and invasion, as well as angiogenesis. This suggests UPR as a promising target in cancer therapy. The pharmacological effects of the plant Scindapsus cf. hederaceus on human cancer cell lines is not understood. In this study, we identified an ethyl acetate extract from Scindapsus cf. hederaceus (SH-EAE), which markedly altered the protein expression of UPR-related genes in human non-small cell lung cancer (NSCLC) cells. Treatment with the SH-EAE led to the dose-dependent suppression of colony forming ability of both H1299 and H460 cells, but not markedly in normal bronchial epithelial BEAS-2B cells. SH-EAE treatment also attenuated the migration and invasion ability of H1299 and H460 cells. Moreover, SH-EAE strikingly suppressed the protein expression of two ER stress sensors, including inositol requiring enzyme-1α (IRE-1α) and protein kinase R-like ER kinase (PERK), and antagonized the induction of C/EBP homologous protein (CHOP) expression by thapsigargin, an ER stress inducer. SH-EAE induced the formation of massive vacuoles which are probably derived from ER. Importantly, SH-EAE impaired the formation of intersegmental vessels (ISV) in zebrafish larvae, an index of angiogenesis, but had no apparent effect on the rate of larval development. Together, our findings demonstrate, for the first time, that the ability of SH-EAE specifically targets the two sensors of UPR, with significant anti-proliferation and anti-migration activities as a crude extract in human NSCLC cells. Our finding also indicates potential applications of SH-EAE in preventing UPR activation in response to Tg-induced ER stress. We suggest that SH-EAE attenuates UPR adaptive pathways for rendering the NSCLC cells intolerant to ER stress.

Aberrant microRNA-137 promoter methylation is associated with lymph node metastasis and poor clinical outcomes in non-small cell lung cancer.

MicroRNA-137 (miR-137) functions as a tumor suppressor and is silenced by aberrant promoter methylation. Previous studies have demonstrated that miR-137 is downregulated in lung cancer. The purpose of the present study was to investigate miR-137 promoter methylation and to assess its prognostic value in non-small cell lung cancer (NSCLC). The expression of miR-137 was analyzed inhuman lung cancer A549 and H1299 cells and normal bronchial epithelial BEAS-2B cells, 10 paired formalin-fixed paraffin-embedded lung cancer and normal tissue samples, and 56 archived paraffin-embedded lung cancer tissues. Quantitative methylation-specific polymerase chain reaction analysis was used to assess the miR-137 methylation status. The associations between miR-137 promoter methylation and the clinicopathological features and prognosis of patients with NSCLC (n=56) were analyzed using analysis of variance. miR-137 was markedly downregulated in lung cancer cells and lung cancer tissue specimens compared with expression in BEAS-2B cells and matched adjacent normal lung tissues. A significant negative correlation between miR-137 expression and miR-137 promoter methylation was observed in human lung cancer tissues (r=−0.343; P=0.01). Smoking, lymph node metastasis and advanced clinical stage were associated with significantly lower expression of miR-137 in variance analysis. High levels of miR-137 promoter methylation were associated with a significantly poorer disease-free survival rate (P=0.034), but were not associated with overall survival, in Kaplan-Meier analysis and univariate analysis. In conclusion, the results of the present study indicated that miR-137 is downregulated and that its promoter is aberrantly methylated in lung cancer, and that high levels of miR-137 promoter methylation may have prognostic value for poor disease-free survival.

Activating transcription factor 3 promotes malignance of lung cancer cells in vitro.

BackgroundLung cancer remains the most common cause of cancer‐related death, with high rates of recurrence and poor outcomes. An abnormally high expression of activating transcription factor 3 (ATF3) in various cancers suggests an oncogenic role; however, its function in lung cancer is largely unknown.MethodsSixty‐four pairs of lung cancer tissues were collected for ATF3 expression analysis by quantitative real‐time PCR, immunoblotting, and immunohistochemistry staining. Correlations between ATF3 expression with clinicopathological features and overall survival were analyzed. ATF3 expression in a panel of lung cancer cell lines together with normal bronchial epithelial Beas‐2B cells was also determined. Human H1299 and A549 cells were used for ATF3 knockdown and/or overexpression assays. Alterations in cell proliferation, cell cycle attribution, migration, and invasion were all assessed in vitro.ResultsIncreased ATF3 messenger RNA and protein expression were observed in lung cancer tissues/cells compared with normal tissues/cells. High tumorous ATF3 expression was significantly correlated with positive advanced tumor grade, lymph node metastasis, and shorter overall survival. Experimentally, we found that RNA interference mediated knockdown of ATF3 significantly inhibited the cell proliferation, cell cycle progression, migration, and invasion capacities of lung cancer cells in vitro, whereas forced expression of ATF3 did the opposite.ConclusionUpregulation of ATF3 in lung cancer promotes cell proliferation, migration, and invasion, and may represent a novel therapeutic target for lung cancer.

Anti-Cancer Effects of a New Docosahexaenoic Acid Monoacylglyceride in Lung Adenocarcinoma

Lung cancer is the leading cause of cancer-related deaths worldwide. Despite advances in research, diagnosis and treatment, lung cancer remains a highly lethal disease, often diagnosed at advanced stages and with a very poor prognosis. Therefore, new strategies for the prevention and treatment of lung cancer are urgently needed. The aim of the present study was to determine the anti-tumorigenic effects of docosahexaenoic acid monoacylglyceride (MAG-DHA), a newly patented DHA derivative in lung adenocarcinoma. Our results demonstrate that MAG-DHA treatments decreased cell proliferation and induced apoptosis in A549 human lung carcinoma cells whereas MAG-DHA treatment did not induce apoptosis of normal bronchial epithelial BEAS-2B cells. MAG-DHA decreased NFκB activation leading to a reduction in COX-2 expression level in both A549 cells and lung adenocarcinoma tissues. Furthermore, MAG-DHA treatment increased PTEN expression and activation concomitant with a decrease in AKT phosphorylation levels and enhanced apoptosis. Oral administration of MAG-DHA significantly reduced tumor growth in a mouse A549 xenograft model. Lastly, MAG-DHA markedly decreased COX-2 and enhanced PTEN protein expression in tumor tissue sections. Altogether, these data provide new evidence regarding the mode of action of MAG-DHA and strongly suggest that this compound could be of clinical interest in cancer treatment.

A possible gene silencing mechanism: Hypermethylation of the Keap1 promoter abrogates binding of the transcription factor Sp1 in lung cancer cells

Hypermethylation often leads to gene silencing; however, the mechanism responsible for the low expression resulting from hypermethylation of the tumor suppressor gene Kelch-like ECH-associating protein 1 (Keap1) in human lung cancer cell lines remains unclear. In this study, using promoter deletion and site mutagenesis assays, we determined that one transcription factor stimulating protein-1 (Sp1) regulatory element in the Keap1 promoter region was important for the transcription of Keap1 in A549 cells. We demonstrated that the transcription factor Sp1 can directly bind to this element in the normal bronchial epithelial BEAS-2B cell line but not in A549 cells, as assessed with chromatin immunoprecipitation (ChIP). EMSAs and supershift assays also showed that CpG island methylation could abrogate Sp1 binding to the Keap1 promoter. Moreover, Keap1 mRNA decreased by 50% after the knock-down of Sp1 with siRNA in BEAS-2B cells, whereas the over-expression of Sp1 led to a dramatic increase in Keap1 promoter activity. The treatment of A549 cells with 5-aza-2′-deoxycytidine restored the binding of Sp1 to the promoter and Keap1 expression. Our results indicate that Sp1 is essential for Keap1 expression and that promoter methylation blocks Sp1 binding in A549 cells. These results demonstrate that hypermethylation may act as an epigenetic gene silencing mechanism, i.e., the inhibition of Sp1 binding to the hypermethylated Keap1 promoter in lung cancer cells, which suggests new approaches to lung cancer treatment.

Epigallocatechin-3-gallate (EGCG) protects against chromate-induced toxicity in vitro

Hexavalent chromium [Cr(VI)] is a human carcinogen that results in the generation of reactive oxygen species (ROS) and a variety of DNA lesions leading to cell death. Epigallocatechin-3-gallate (EGCG), the major polyphenol present in green tea, possesses potent antioxidative activity capable of protecting normal cells from various stimuli-induced oxidative stress and cell death. Here we demonstrated that co-treatment with EGCG protected human normal bronchial epithelial BEAS-2B cells from Cr(VI)-induced cell death in a dose-dependent manner. Cr(VI) induces apoptosis as the primary mode of cell death. Co-treatment of BEAS-2B cells with EGCG dose-dependently suppressed Cr(VI)-induced apoptosis. Fluorescence microscopic analyses and quantitative measurement revealed that EGCG significantly decreased intracellular levels of ROS induced by Cr(VI) exposure. Using a well-established K +/SDS precipitation assay, we further showed that EGCG was able to dose-dependently reduce DNA–protein cross-links (DPC), lesions that could be partially attributed to Cr(VI)-induced oxidative stress. Finally, analyses of Affymetrix microarray containing 28,869 well-annotated genes revealed that, among the 3412 genes changed more than 1.5-fold by Cr(VI) treatment, changes of 2404 genes (70%) were inhibited by pretreatment of EGCG. Real-time PCR confirmed the induction of 3 genes involved in cell death and apoptosis by Cr(VI), which was eliminated by EGCG. In contrast, Cr(VI) reduced the expression of 3 genes related to cellular defense, and this reduction was inhibited by EGCG. Our results indicate that EGCG protects BEAS-2B cells from Cr(VI)-induced cytotoxicity presumably by scavenging ROS and modulating a subset of genes. EGCG, therefore, might serve as a potential chemopreventive agent against Cr(VI) carcinogenesis.