Mangifera indica L. kernel ethanol extract inhibits cell viability and proliferation with induction of cell cycle arrest and apoptosis in lung cancer cells.

In this study, we investigated the effects of an ethanolic extract of Mangifera indica L. kernel on the viability and proliferation of human lung cancer cells. We utilized MTT and BrdU cell proliferation assays, morphological assessments, cell cycle analyses, and apoptosis assays to investigate the extract's effects on lung cancer (A549 and NCI-H292) and normal lung (MRC-5) cells. The extract demonstrated a toxicity toward cancer cells compared to normal cells with dose-dependent anti-proliferative effect on lung cancer cells. The extract also caused differential effects on the cell cycle, inducing G0/G1 arrest and increasing the Sub-G1 population in both lung cancer and normal lung cells. Notably, the extract induced loss of membrane integrity, shrinkage, membrane blebbing, and apoptosis in lung cancer cells, while normal cells exhibited only early apoptosis. Furthermore, the extract exhibited higher toxicity towards NCI-H292 cells, followed by A549 and normal MRC-5 cells in decreasing order of potency. Our results suggest that the ethanolic extract of M. indica L. kernel has significant potential as a novel therapeutic agent for treating lung cancer cells, given its ability to induce apoptosis in cancer cell lines while causing minimal harm to normal cells.

In vitro dosimetry analyses for acrolein exposure in normal human lung epithelial cells and human lung cancer cells.

Establishing accurate dosimetry is important for assessing the toxicity of xenobiotics as well as for comparing responses between different test systems. In this study, we used acrolein as a model toxicant and defined the concentration-response relationships of the key adverse responses in normal human bronchial epithelial (NHBE) cells and human mucoepidermoid pulmonary carcinoma (NCI-H292) cells. Direct trace analysis of intracellular free acrolein is extremely challenging, if not impossible. Therefore, we developed a new method for indirectly estimating the intracellular uptake of acrolein. A 10-min treatment was employed to capture the rapid occurrence of the key alkylation reactions of acrolein. Responses, including protein carbonylation, GSH depletion, and GSH-acrolein (GSH-ACR) adduct formation, were all linearly correlated with acrolein uptake in both cell types. Compared to the NCI-H292 mucoepidermoid carcinoma cells, NHBE cells were more sensitive to acrolein exposure. Furthermore, results from the time-course studies demonstrated that depletion and conjugation of GSH were the primary adverse events and directly associated with the cytotoxicity induced by acrolein. In summary, these data suggest that cell susceptibility to acrolein exposure is closely associated with acrolein uptake and formation of GSH-ACR adducts. The dosimetric analysis presented in this study may provide useful information for computational modeling and risk assessment of acrolein using different test systems.

Pro-inflammatory effects of extracellular Hsp70 on NCI-H292 human bronchial epithelial cell line.

Extracellular Hsp70 (eHsp70) exerts its biological actions via Toll-like receptors 2 and 4, and is increased in sera of chronic obstructive pulmonary disease (COPD) patients. The aim of this study was to explore the pro-inflammatory effects and cytotoxicity of eHsp70 alone and in combination with bacterial components lipoteichoic acid (LTA) and lipopolysaccharide (LPS) on NCI-H292 airway epithelial cells. NCI-H292 cells were treated with recombinant human Hsp70 protein (rhHsp70), LPS, LTA and their combinations for 4, 12, 24 and 48 hours. IL-6, IL-8 and TNF-α levels were measured by an ELISA method. Cell viability was determined by the MTS method, and caspase-3/7, caspase-8 and caspase-9 assays. rhHsp70 induced secretion of IL-6 and IL-8 in a concentration- and time-dependent manner, with the highest secretion at 24 hours. rhHsp70 combined with LTA had antagonistic and with LPS synergistic effect on IL-6 secretion, while the interactions between rhHsp70 and LPS or LTA on IL-8 were synergistic. TNF-α was not detected in the applied conditions. rhHsp70, LPS or LTA did not affect cell viability, and rhHsp70 even suppressed caspase-3/7 activities. We suggest that pro-inflammatory effects of eHsp70, together with other damaging molecules and/or COPD risk factors, might contribute to the aggravation of chronic inflammation in human bronchial epithelium.

Extracellular Hsp70 modulates the inflammatory response of cigarette smoke extract in NCI-H292 cells.

NEW FINDINGS: What is the central question of this study? Does extracellular heat shock protein 70 (eHsp70) alter cigarette smoke extract (CSE)-induced inflammatory responses in NCI-H292 bronchial epithelial cells? What is the main finding and its importance? eHsp70 modulates inflammatory responses and TLR2, TLR4 and Hsp70 gene expression, and protects NCI-H292 cells against CSE-induced cytotoxicity. eHsp70 might be implicated in development of inflammatory diseases affected by cigarette smoke, such as COPD. ABSTRACT: One of the major risk factors for development of chronic obstructive pulmonary disease (COPD) is cigarette smoke. Extracellular Hsp70 (eHsp70) is increased in sera of COPD patients, and can act as damage-associated molecular pattern (DAMP). In this study, we explored inflammatory parameters (cytokine concentrations, Toll-like receptor (TLR) 2 and 4 and Hsp70 expression, mitogen-activated protein kinases (MAPKs) and nuclear factor κB (NF-κB) activation, and cytotoxicity) after exposure of bronchial-epithelial NCI-H292 cells to cigarette smoke extract (CSE) alone (2.5 and 15%) and in combinations with recombinant human (rh) Hsp70 (0.3, 1 and 3 µg ml-1 ). We applied specific MAPKs, NF-κB and Hsp70 inhibitors to elucidate rhHsp70 inflammation-associated responses. CSE alone and combinations of 15% CSE with rhHsp70 stimulated IL-1α, IL-6 and IL-8 release. However, rhHsp70 applied with 2.5% CSE decreased secretion of cytokines indicating antagonistic effects. Individual and combined treatments with 2.5% CSE suppressed TLR2 expression. CSE at 15% induced TLR2 and TLR4 gene expression, whereas rhHsp70 abolished that effect. rhHsp70 and 15% CSE alone reduced, while their combination increased, intracellular Hsp70 mRNA level. CSE alone and in combination with rhHsp70 activated extracellular signal-regulated kinase and p38 MAPKs, while inhibition of MAPKs, NF-κB and Hsp70 attenuated IL-6 and IL-8 secretion. CSE at 15% reduced cell viability and induced apoptosis, as shown by MTS and caspases-3/7 assays. CSE at 2.5% alone stimulated lactate dehydrogenase release, but cellular membrane integrity remained intact in co-treatments with rhHsp70. rhHsp70 might modulate the inflammatory response of CSE and could also protect NCI-H292 cells against CSE cytotoxicity. Those effects are implemented via MAPK and NF-κB signalling pathways.

Time-course of transcriptome response to respiratory syncytial virus infection in lung epithelium cells.

Respiratory syncytial virus (RSV) is the major cause of acute lower respiratory tract infection in infants. Winter outbreaks in Chile result in 5% of infected children hospitalized, with 0.01% mortality. Increased evidence indicates that viral and host factors modulate the severity of infection. Using DNA microarrays, we characterized the genome-wide transcriptional response of lung mucoepidermoid cells (NCI-H292) at 0, 24, 48, 72 and 96 hours post-infection (hpi) with a single dose of RSV/A. During the whole studied period, a bi-phasic gene expression profile was observed by a total of 330 differentially expressed genes. About 60% of them were up-regulated between 24-72 hpi and then turned-off at 96 hpi. This transient, early gene expression pattern was significantly enriched in biological processes like interferon signaling, antigen processing and presentation, double-stranded RNA binding and chemokine activity. We detected 27 common genes up-regulated between 24-72 hpi, from which IFIT1, IFI44, MX1, CXCL11 and OAS1 had the highest expression. The second pattern comprised over 120 genes, which remained silenced until 72 hpi, but were steeply up-regulated by 96 hpi. Biological processes of this late-response profile included cell cycle division and microtubule cytoskeleton organization. Conversely, the genes belonging to virus response pathway showed a decreased expression at 96 hpi. We conclude that RSV induces an early innate immune activation profile response until 72 hpi. Thereafter, the viral response is inhibited, leading to host cell recovery. The presented cellular model allows to study the specific pathways involved in elimination of infection at prolonged time intervals and their subsequent analysis in severe RSV disease of infants and/or older adults.

Deguelin Impairs Cell Adhesion, Migration and Invasion of Human Lung Cancer Cells through the NF-[Formula: see text]B Signaling Pathways.

Deguelin, a rotenoid, is isolated from a natural plant species, and has biological activities including antitumor function. In the present study, we investigated the effect of deguelin on the cell adhesion, migration and invasion of NCI-H292 human lung cancer cells in vitro. Cell viability was analyzed by using flow cytometer. Cell adhesion was determined by using the cell-matrix adhesion assay. Wound healing assay was used to examine cell migration. Cell migration and invasion were investigated using a Boyden chamber assay. The protein expression was measured by Western blotting and confocal laser microscopy. The electrophoretic mobility shift assay was used to measure NF-[Formula: see text]B p65 binding to DNA.We selected the concentrations of deguelin at 0, 0.5, 1.0, 1.5, 2.0 and 2.5[Formula: see text][Formula: see text]M and we found that those concentrations of deguelin did not induce significant cytotoxic effects on NCI-H292 cells. Thus, we selected those concentrations of deguelin for metastasis assay. We found that deguelin inhibited cell adhesion, migration and invasion in dose-dependent manners that was assayed by wound healing and transwell methods, respectively. Deguelin decreased the expression of MMP-2/-9, SOS 1, Rho A, p-AKT (Thr308), p-ERK1/2, p-p38, p-JNK, NF-[Formula: see text]B (p65) and uPA in NCI-H292 cells. Deguelin suppressed the expression of PI3K, SOS 1, NF-[Formula: see text]B (p65), but did not significantly affect PKC and Ras in the nuclei of NCI-H292 cells that were confirmed by confocal laser microscopy. We suggest that deguelin may be used as a novel anticancer metastasis of lung cancer in the future.

Regulation of Human MUC7 Mucin Gene Expression by Cigarette Smoke Extract or Cigarette Smoke and Pseudomonas aeruginosa Lipopolysaccharide in Human Airway Epithelial Cells and in MUC7 Transgenic Mice.

OBJECTIVE: The human MUC7 gene encodes a low-molecular-weight mucin glycoprotein that functions in lubrication/protection of epithelial surfaces of the oral cavity and respiratory tract. This study was designed to evaluate the effect of cigarette smoke extract (CSE), cigarette smoke (CS), and Pseudomonas aeruginosa lipopolysaccharide (LPS), either alone or in the combination, on MUC7 expression in vitro and in vivo. MATERIALS AND METHODS: qRT-PCR was used to determine the levels of mucin gene transcription in the human lung carcinoma cell line NCI-H292 (in vitro) and MUC7 transgenic mouse tissues (in vivo). ELISA was used to assess mucin glycoprotein levels in the cell line, and immunohistochemistry to assess mucins in lung and trachea sections. RESULTS: In vitro treatment of cells with LPS (10 (microg/ml) or CSE (0.5, 1, 2.5 and 5%) alone, resulted in a statistically significant increase of MUC7 transcripts only with 1%CSE (3.2-fold). The combined CSE/LPS treatment resulted in a synergistic increase of MUC7 with 0.5%CSE/LPS (4.4 fold). MUC7 glycoprotein levels increased only minimally, the highest increase was seen with the 0.5%CSE/LPS combination treatment (1.3-fold). In vivo exposure of MUC7 transgenic mice to CS, LPS or CS/LPS combination resulted in significant increase in MUC7 transcripts only with LPS treatment (in both trachea and lung). Immunohistochemistry indicated variable increase in MUC7 glycoprotein with CS and LPS treatment, both in the trachea and lungs, but CS/LPS exposure appeared to yield the highest increase. CONCLUSION: In vitro, CSE and a combination of CSE/LPS treatment upregulated MUC7 gene transcription. In vivo, LPS upregulated MUC7 transcription, and a combination of CS/LPS appeared to increase MUC7 glycoprotein.

Regulation of IL-1beta-Mediated MUC2 Gene and Mucin in Human Airway Epithelial Cells / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Mucin secretion is regulated by the mucin genes (MUC) in the respiratory, gastrointestinal and reproductive system. Inflammation induces mucin hypersecretion in the human body. This study demonstrates the effects of IL-1beta on the regulation of mucin protein expression as well as the MUC2 gene in cultured airway epithelial cells. MATERIALS AND METHOD: Analysis of MUC2 gene was done by RT-PCR and the protein analysis was done by a flow cytometric analysis and an immunoassay method using cultured human airway epithelial cells, and NCI-H292 cells. RESULTS: The expression of MUC2 mRNA and protein induced by IL-1beta increased in a dose-and time-dependent manner. The maximum mRNA level of the MUC2 gene was approximately 3-fold, compared to that of the control cell. The IL-1beta-mediated MUC2 protein started at 6 hours of exposure to IL-1beta (20 ng/ml) and the maximum level was 12 hours. The MUC2 protein data of flow cytometric analysis corresponded to that of immunoassay analysis. The expression of MUC2 gene was suppressed by actinomycin D, but not attenuated by cycloheximide. CONCLUSION: These results suggest that the IL-1beta-mediated MUC2 gene and protein expression were increased in a dose- and time-dependent pattern and regulated by transcriptional step.

IL-1beta Mediated COX-2 Expression in Human Airway Epithelial Cells / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Prostaglandin is one of the important inflammatory mediator in inflammatory diseases. Cyclooxygenase-2 (COX-2) plays a key role in biosynthesis of prostaglandins. In this study, we aimed to investigate COX-2 expression and prostaglandin E2 (PGE2) production by interleukin-1beta (IL-1beta) in cultured human airway epithelial cells. MATERIALS AND METHOD: COX-2 gene expression, and COX-2 protein, PGE2 production by IL-1beta were analyzed by RT-PCR, Western blot, and enzymeimmunoassay (EIA) in cultured human airway NCI-H292 epithelial cells. RESULTS: The COX-2 protein production was increased when the cells were exposed to IL-1beta in a dose dependent manner. The maximum level of COX-2 protein was detected at 20 ng/ml of IL-1beta. After 4 hours, the production of COX-2 protein was detected by IL-1beta(20 ng/ml) and this was held up to 12 hour. The maximum level of COX-2 protein production reached at 8 hour of exposure to IL-1beta and this was held up to 12 hour. The release of PGE2 occurred in the same pattern as the IL-1beta-mediated COX-2 protein production. The COX-2 gene expression was induced by IL-1beta (20 ng/ml). The IL-1beta-mediated COX-2 expression was suppressed by actinomycin D, but was not affected by cycloheximide. CONCLUSION: These results suggest that the IL-1beta-mediated COX-2 expression and the PGE2 production were increased in dose and time dependent manner and regulated in the transcriptional step.