Cyclooxygenase-2 In A549 Cells Research Articles

Effect of thiol antioxidants on lipopolysaccharide-induced cyclooxygenase-2 expression in pulmonary epithelial cells.

Cyclooxygenase-2 (COX-2) plays an important role in pulmonary inflammatory response, and its expression is regulated by several transcription factors including nuclear factor kappa-B (NF-κB), activator protein-1 (AP-1), and signal transducer and activator of transcription-3 (STAT-3), which are activated by oxidative stress. Glutathione (GSH) and N-acetyl cysteine (NAC) are thiol antioxidants that scavenge reactive oxygen species (ROS). The present study investigated whether lipopolysaccharide (LPS) induces COX-2 expression through ROS generation and the activation of oxidant-sensitive transcription factors such as NF-κB, AP-1, and STAT-3 in pulmonary epithelial A549 cells. The cells were pretreated with GSH or NAC for 1 hour prior to LPS stimulation. Intracellular ROS levels, DNA-binding activities of NF-κB, AP-1, and STAT-3, and mRNA and protein levels of COX-2 were determined. Our results showed that LPS increased ROS levels that peaked at 2 hours. LPS activated NF-κB, AP-1, and STAT-3 and induced the expression of COX-2 in A549 cells in a time-dependent manner. Pretreatment of thiol antioxidants GSH and NAC reduced ROS levels and attenuated the increase in ROS, the activation of NF-κB, AP-1, and STAT-3, and the expression of COX-2 in LPS-treated A549 cells. In conclusion, GSH and NAC suppress COX-2 expression by reducing ROS levels and inhibiting the activation of NF-κB, AP-1, and STAT-3 in pulmonary epithelial A549 cells exposed to LPS. Pretreatment with thiol antioxidants GSH and NAC may be beneficial for the treatment of pulmonary inflammation associated with oxidative stress.

Cyclooxygenase-2 mediates puerarin to inhibit the invasion and metastasis of lung cancer A549 cells

Background: The mechanisms by which puerarin prevents and treats lung cancer remain largely unknown. We aimed to study the roles of puerarin in the invasion and metastasis of lung cancer A549 cells based on cyclooxygenase-2 (COX-2). Methods: The effects of different concentrations of puerarin on the proliferation of A549 cells were evaluated. The in vitro and in vivo effects of puerarin on the invasion and metastasis of A549 cells were assessed by Transwell assay and an animal model of tumor lung metastasis respectively. The influence of puerarin on COX-2 in A549 cells was also analyzed in terms of enzymatic activity and tissue level to explore the role of COX-2 in the inhibitory effects of puerarin on cell invasion and metastasis. Results: Puerarin inhibited the proliferation of A549 cells in time- and dose-dependent manners. Compared with the control group, more cells in the puerarin treatment groups were arrested in the G0/G1 phase, but the proportion of S phase cells decreased. Puerarin blocked the cell cycle dose-dependently. After 24 h of culture with 200 μg/mL puerarin, significantly fewer cells penetrated the basement membrane of Transwell chambers than those in the control group, and the MMP-2/TIMP-2 levels in PUE200, PUE400 and PUE800 groups significantly dropped. For the animal experiments, the puerarin treatment groups had significantly fewer and smaller tumors with pulmonary metastasis than those of the control group. After 24 h of culture with puerarin, like the indomethacin (IN) group, PUE200, PUE400 and PUE800 groups had significantly lower enzymatic activity of COX-2 than that of the control group, but the puerarin treatment groups had similar results. The IN group had lower COX-2 protein expression than that of the control group, but the puerarin treatment groups had similar expressions to that of the control group, indicating that COX-2 protein expression was hardly affected by puerarin in A549 cells. Similar to the IN group, the puerarin treatment groups had significantly lower PGE2 levels than that of the control group (P Conclusions: Puerarin inhibited the enzymatic activity of COX-2 in A549 cells and its expression in tumor tissues with pulmonary metastasis. MMP-2/TIMP-2 may be one of the mechanisms by which puerarin inhibits the invasion of A549 cells.

Methyl syringate, a TRPA1 agonist represses hypoxia-induced cyclooxygenase-2 in lung cancer cells

BackgroundWe have previously found that methyl syringate is a specific and selective agonist of the human transient receptor potential channel ankyrin 1 (TRPA1) and suppresses food intake and gastric emptying in imprinting control region mice. Because TRPA1 has been implicated in inflammatory responses, and inflammation and tumorigenesis are stimulated by the cyclooxygenase-2 (COX-2)/prostaglandin E2 pathway in hypoxic cancer cells. PurposeThis study examined the effects of methyl syringate on hypoxia-induced COX-2 in human distal lung epithelial A549 cells. Study designThe effect of the methyl syringate on suppression of hypoxia-induced COX-2 in A549 cells were determined by Western blot and/or quantitative real-time polymerase chain reaction. The anti-invasive effect of methyl syringate was evaluated on A549 cells using matrigel invasion assay. ResultsMethyl syringate suppressed hypoxia-induced COX-2 protein and mRNA expression and promoter activity and reduced hypoxia-induced cell migration and invasion and secretion of vascular endothelial growth factor. These effects were antagonized by a TRPA1 antagonist, implying their mediation by the TRPA1 pathway. ConclusionTogether, these results indicate that methyl syringate inhibits the hypoxic induction of COX-2 expression and cell invasion through TRPA1 activation. These findings suggest that methyl syringate could be effective to suppress hypoxia-induced inflammation and indicate an additional functional effect of methyl syringate.

Effects of microRNA-146a on Fas-associated factor 2 and inflammatory factors in human lung adenocarcinoma A549 cells under the stimulation of cigarette smoke extract

Under the premise of smoke inhalation injury, to explore the effects of microRNA-146a on Fas-associated factor 2 (FAF-2) and inflammatory factors in human lung adenocarcinoma A549 cells under the stimulation of cigarette smoke extract (CSE). (1) The pMIR-FAF-2 recombinant plasmid and the pMIR-FAF-2 recombinant mutated plasmid were constructed. Human embryonic kidney 293 (HEK-293) cells of the third passage were divided into 3 groups according to the random number table, with 5 wells in each group. Cells in plasmid+ microRNA control group were transfected with pMIR-FAF-2 recombinant plasmid, pRL-TK plasmid, and microRNA control; cells in plasmid+ microRNA-146a group were transfected with pMIR-FAF-2 recombinant plasmid, pRL-TK plasmid, and microRNA-146a mimics; cells in mutated plasmid+ microRNA-146a group were transfected with pMIR-FAF-2 recombinant mutated plasmid, pRL-TK plasmid, and microRNA-146a inhibitor. After culture for 24 h, the relative luciferase activity in cells was assessed by dual-luciferase reporter gene assay. (2) Human lung adenocarcinoma A549 cells of the third passage were divided into 3 groups according to the random number table, with 4 wells in each group. Cells in microRNA control group were transfected with microRNA control; cells in microRNA-146a enhancement group were transfected with microRNA-146a mimics; cells in microRNA-146a inhibition group were transfected with microRNA-146a inhibitor. After culture for 24 h, the mRNA expression levels of microRNA-146a and FAF-2 in cells were determined with real-time fluorescent quantitative reverse transcription-PCR. (3) A549 cells of the third passage were stimulated by 0.8% CSE for 24 h after being divided and treated with the same method used in experiment (2). The mRNA expression levels of FAF-2, IL-8, monocyte chemotactic protein-1 (MCP-1), and growth-regulated oncogene-α (GRO-α) in cells were determined with real-time fluorescent quantitative reverse transcription-PCR. The protein expression levels of IL-8, MCP-1, and GRO-α in A549 cell culture supernatant were determined by enzyme-linked immunosorbent assay. The protein expression level of cyclooxygenase 2 (COX-2) of cells was assessed by Western blotting. Data were processed with one-way analysis of variance and LSD test. (1) The pMIR-FAF-2 recombinant plasmid and pMIR-FAF-2 recombinant mutated plasmid were confirmed with successful construction. The relative luciferase activity in HEK-23 cells of plasmid+ microRNA control group was close to that of mutated plasmid+ microRNA-146a group (P>0.05). The relative luciferase activity in HEK-23 cells of plasmid+ microRNA-146a group was significantly lower than that of plasmid+ microRNA control group and mutated plasmid+ microRNA-146a group (with P values below 0.01). (2) The expression level of microRNA-146a in A549 cells of microRNA control group was close to that of microRNA-146a inhibition group (P>0.05), and they were both significantly lower than the expression level of microRNA-146a in A549 cells of microRNA-146a enhancement group (with P values below 0.01). The mRNA expression level of FAF-2 in A549 cells of microRNA control group was close to that of microRNA-146a inhibition group (P>0.05), and they were both significantly higher than the mRNA expression level of FAF-2 in A549 cells of microRNA-146a enhancement group (with P values below 0.05). (3) After stimulation of CSE, the mRNA expression level of FAF-2 in A549 cells of microRNA control group (1.46±0.21) was close to that of microRNA-146a inhibition group (1.43±0.34, P>0.05), which were both significantly higher than the mRNA expression level of FAF-2 in A549 cells of microRNA-146a enhancement group (0.57±0.11, with P values below 0.05). The mRNA expression levels of IL-8, MCP-1, and GRO-α in A549 cells of microRNA-146a enhancement group were significantly lower than those of microRNA control group and microRNA-146a inhibition group (with P values below 0.01). The mRNA expression levels of IL-8, MCP-1, and GRO-α in A549 cells of microRNA-146a inhibition group were significantly higher than those of microRNA control group (with P values below 0.05). The protein expression levels of IL-8, MCP-1, and GRO-α in A549 cell culture supernatant of microRNA-146a enhancement group were significantly lower than those of microRNA control group and microRNA-146a inhibition group (with P values below 0.05). The protein expression level of IL-8 in A549 cell culture supernatant of microRNA-146a inhibition group was close to that of microRNA control group (P>0.05), while the protein expression levels of MCP-1 and GRO-α in A549 cell culture supernatant of microRNA-146a inhibition group were significantly lower than those of microRNA control group (with P values below 0.05). The protein expression level of COX-2 in A549 cells of microRNA-146a enhancement group was significantly lower than the levels of microRNA control group and microRNA-146a inhibition group (with P values below 0.05). The protein expression level of COX-2 in A549 cells of microRNA control group was close to that of microRNA-146a inhibition group (P>0.05). In A549 cells, after being transfected with microRNA-146a and stimulated by CSE, microRNA-146a can decrease the expression of FAF-2 through integrating with the 3'-untranslated region of target gene FAF-2, thereby decrease the expression of inflammatory factors.

Red ginseng represses hypoxia-induced cyclooxygenase-2 through sirtuin1 activation

BackgroundKorean red ginseng (KRG) is a traditional herbal medicine made by steaming and drying the fresh ginseng, leading to chemical transformation of some components by heat. It ameliorates various inflammatory diseases and strengthens the endocrine, immune, and central nervous systems. The cyclooxygenase-2 (COX-2)/prostaglandin E2 pathway in hypoxic cancer cells has important implications for stimulation of inflammation and tumorigenesis. PurposeIn this study we examined the effects and the mechanism underlying Korean red ginseng water extract (KRG-WE) inhibition of hypoxia-induced COX-2 in human distal lung epithelial A549 cells. Study designThe effect of the KRG on suppression of hypoxia-induced COX-2 in A549 cells were determined by Western blot and/or qRT-PCR. The anti-invasive effect of KRG-WE was evaluated on A549 cells using matrigel invasion assay. The activation of glucocorticoid receptor (GR) and sirtuin1 (Sirt1) was examined by using specific inhibitors. ResultsWe first observed that hypoxia induced COX-2 protein and mRNA levels and promoter activity were suppressed by KRG-WE. Second, we observed that hypoxia-induced cell migration is dramatically reduced by KRG-WE. Third, we found that the effect of KRG-WE was not antagonized by the GR antagonist RU486 implying that the effect is mediated other than GR pathway. Finally, we demonstrated that inhibition of Sirt1 abolished the effect of KRG-WE on hypoxia-induced COX-2 suppression and cell-invasion indicating that the suppression is mediated by Sirt1. ConclusionTaken together, KRG-WE inhibits the hypoxic induction of COX-2 expression and cell invasion through Sirt1 activation. Our results imply that KRG-WE could be effective for suppression of inflammation under hypoxia.

Anesthetic Effects on γ-Aminobutyric Acid A Receptors

Anesthetic Effects on γ-Aminobutyric Acid A Receptors

Redox Factor-1 Inhibits Cyclooxygenase-2 Expression via Inhibiting of p38 MAPK in the A549 Cells

In this study, we evaluated the role of apurinic/apyrimidinic endonuclease1/redox factor-1 (Ref-1) on the tumor necrosis factor-alpha (TNF-alpha) induced cyclooxygenase-2 (COX-2) expression using A549 lung adenocarcinoma cells. TNF-alpha induced the expression of COX-2 in A549 cells, but did not induce BEAS-2B expression. The expression of COX-2 in A549 cells was TNF-alpha dose-dependent (5~100 ng/ml). TNF-alpha-stimulated A549 cells evidenced increased Ref-1 expression in a dose-dependent manner. The adenoviral transfection of cells with AdRef-1 inhibited TNF-alpha-induced COX-2 expression relative to that seen in the control cells (Adbetagal). Pretreatment with 10 microM of SB203580 suppressed TNF-alpha-induced COX-2 expression, thereby suggesting that p38 MAPK might be involved in COX-2 expression in A549 cells. The phosphorylation of p38 MAPK was increased significantly after 5 minutes of treatment with TNF-alpha, reaching a maximum level at 10 min which persisted for up to 60 min. However, p38MAPK phosphorylation was markedly suppressed in the Ref-1-overexpressed A549 cells. Taken together, our results appear to indicate that Ref-1 negatively regulates COX-2 expression in response to cytokine stimulation via the inhibition of p38 MAPK phosphorylation. In the lung cancer cell lines, Ref-1 may be involved as an important negative regulator of inflammatory gene expression.

Proteinase-activated Receptor-2 Induces Cyclooxygenase-2 Expression through β-Catenin and Cyclic AMP-response Element-binding Protein

Chronic inflammation of mucosae is associated with an increased cancer risk. Tumorigenesis in these tissues is associated with the activity of some proteinases, cyclooxygenase-2 (COX-2), and beta-catenin. Serine proteinases participate in both inflammation and tumorigenesis through the activation of proteinase-activated receptor-2 (PAR(2)), which up-regulates COX-2 by an unknown mechanism. We sought to determine whether beta-catenin participated in PAR(2)-induced COX-2 expression and through what cellular mechanism. In A549 epithelial cells, we showed that PAR(2) activation increased COX-2 expression through the beta-catenin/T cell factor transcription pathway. This effect was dependent upon ERK1/2 MAPK, which inhibited the beta-catenin-regulating protein, glycogen synthase kinase-3beta, and induced the activity of the cAMP-response element-binding protein (CREB). Knockdown of CREB by small interfering RNA revealed that PAR(2)-induced beta-catenin transcriptional activity and COX-2 expression were CREB-dependent. A co-immunoprecipitation assay revealed a physical interaction between CREB and beta-catenin. Thus, PAR(2) up-regulated COX-2 expression via an ERK1/2-mediated activation of the beta-catenin/Tcf-4 and CREB pathways. These findings reveal new cellular mechanisms by which serine proteinases may participate in tumor development and are particularly relevant to cancers associated with chronic mucosal inflammation, where serine proteinases are abundant and COX-2 overexpression is a common feature.