Resveratrol inhibits MUC5AC expression by regulating SPDEF in lung cancer cells.

BACKGROUND: MUC5AC was recently identified to play important roles in the proliferation and metastasis of malignant mucinous lung tumor cells. Resveratrol (Res), a natural compound with anticancer effects in lung cancer cells, has been reported to inhibit mucin production in airway epithelial cells. This study aimed to investigate the inhibitory effect of Res on MUC5AC expression in lung mucinous adenocarcinoma cells and the potential mechanisms. METHODS: Mucus-producing A549 human lung carcinoma cells were used to test the effects of Res on SPDEF and MUC5AC expression. Gene and protein expression was assessed by real-time quantitative PCR (qPCR), immunofluorescence and western blotting assays. SPDEF lentivirus was used to upregulate SPDEF expression levels in mucus-producing A549 human lung carcinoma cells. Cell proliferation was assessed by Cell Counting Kit-8 (CCK-8) assay. RESULTS: Res decreased MUC5AC expression in an SPDEF-dependent manner in mucus-producing A549 human lung carcinoma cells, and this change was accompanied by decreased ERK expression and AKT pathway activation. Moreover, SPDEF was found to be overexpressed in lung adenocarcinoma (LUAD), especially in mucinous adenocarcinoma. In-vitro functional assays showed that overexpression of SPDEF reduced the chemosensitivity of A549 cells to cisplatin (DDP). In addition, Res treatment increased A549 cell chemosensitivity to DDP by inhibiting the SPDEF-MUC5AC axis. CONCLUSION: Our results indicate that the SPDEF-MUC5AC axis is associated with DDP sensitivity, and that Res decreases SPDEF and MUC5AC expression by inhibiting ERK and AKT signaling in A549 cells, which provides a potential pharmacotherapy for the prevention and therapeutic management of mucinous adenocarcinoma.

Investigation of the Active Ingredients and Mechanism of Polygonum cuspidatum in Asthma Based on Network Pharmacology and Experimental Verification.

BACKGROUND: Polygonum cuspidatum is a Chinese medicine commonly used to treat phlegm-heat asthma. However, its anti-asthmatic active ingredients and mechanism are still unknown. The aim of this study was to predict the active ingredients and pathways of Polygonum cuspidatum and to further explore the potential molecular mechanism in asthma by using network pharmacology. METHODS: The active ingredients and their targets related to Polygonum cuspidatum were seeked out with the TCM systematic pharmacology analysis platform (TCMSP), and the ingredient-target network was constructed. The GeneCards, DrugBank and OMIM databases were used to collect and screen asthma targets, and then the drug-target-disease interaction network was constructed with Cytoscape software. A target protein-protein interaction (PPI) network was constructed using the STRING database to screen key targets. Finally, GO and KEGG analyses were used to identify biological processes and signaling pathways. The anti-asthmatic effects of Polygonum cuspidatum and its active ingredients were tested in vitro for regulating airway smooth muscle (ASM) cells proliferation and MUC5AC expression, two main symptoms of asthma, by using Real-time PCR, Western blotting, CCK-8 assays and annexin V-FITC staining. RESULTS: Twelve active ingredients in Polygonum cuspidatum and 479 related target proteins were screened in the relevant databases. Among these target proteins, 191 genes had been found to be differentially expressed in asthma. PPI network analysis and KEGG pathway enrichment analysis predicted that the Polygonum cuspidatum could regulate the AKT, MAPK and apoptosis signaling pathways. Consistently, further in vitro experiments demonstrated that Polygonum cuspidatum and resveratrol (one active ingredient of Polygonum cuspidatum) were shown to inhibit ASM cells proliferation and promoted apoptosis of ASM cells. Furthermore, Polygonum cuspidatum and resveratrol inhibited PDGF-induced AKT/mTOR activation in ASM cells. In addition, Polygonum cuspidatum decreased H2O2 induced MUC5AC overexpression in airway epithelial NCI-H292 cells. CONCLUSION: Polygonum cuspidatum could alleviate the symptoms of asthma including ASM cells proliferation and MUC5AC expression through the mechanisms predicted by network pharmacology, which provides a basis for further understanding of Polygonum cuspidatum in the treatment of asthma.

Phloretin, an Apple Polyphenol, Inhibits Pathogen-Induced Mucin Overproduction.

SCOPE: Bacterial infection induces mucus overproduction, contributing to acute exacerbations and lung function decline in chronic respiratory diseases. A diet enriched in apples may provide protection from pulmonary disease development and progression. This study examined whether phloretin, an apple polyphenol, inhibits mucus synthesis and secretion induced by the predominant bacteria associated with chronic respiratory diseases. METHODS AND RESULTS: The expression of mucus constituent mucin 5AC (MUC5AC) in FVB/NJ mice and NCI-H292 epithelial cells is analyzed. Nontypeable Haemophilus influenzae (NTHi)-infected mice developed increased MUC5AC mRNA, which a diet containing phloretin inhibited. In NCI-H292 cells, NTHi, Moraxella catarrhalis, Streptococcus pneumoniae, and Pseudomonas aeruginosa increased MUC5AC mRNA, which phloretin inhibited. Phloretin also diminished NTHi-induced MUC5AC protein secretion. NTHi-induced increased MUC5AC required toll-like receptor 4 (TLR4) and NADH oxidase 4 (NOX4) signaling and subsequent activation of the epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK) pathway. Phloretin inhibited NTHi-induced TLR4/NOX4 and EGFR/MAPK signaling, thereby preventing increased MUC5AC mRNA. EGFR activation can also result from increased EGFR ligand synthesis and subsequent ligand activation by matrix metalloproteinases (MMPs). In NCI-H292 cells, NTHi increased EGFR ligand and MMP1 and MMP13 mRNA, which phloretin inhibited. CONCLUSIONS: In summary, phloretin is a promising therapeutic candidate for preventing bacterial-induced mucus overproduction.

Arsenic represses airway epithelial mucin expression by affecting retinoic acid signaling pathway.

Arsenic is a ubiquitous environmental toxicant, found in high concentrations worldwide. Although abundant research has dealt with arsenic-induced cancers, studies on mechanisms of non-malignant lung diseases have not been complete. In addition, decades of research have mostly concentrated on high-dose arsenic exposure, which has very limited use in modeling the biological effects of today's low-dose exposures. Indeed, accumulated evidence has shown that low-dose arsenic exposure (i.e. ≤100 ppb) may also alter lung homeostasis by causing host susceptibility to viral infection. However, the underlying mechanism of this alteration is unknown. In this study, we found that low-dose sodium arsenite (As (III)) repressed major airway mucins-MUC5AC and MUC5B at both mRNA and protein levels. We further demonstrated that this repression was not caused by cellular toxicity or mediated by the reduction of a common mucin-inducing pathway-EGFR. Other established mucin activators- dsRNA, IL1ß or IL17 were not able to override As (III)-induced mucin repression. Interestingly, the suppressing effect of As (III) appeared to be partially reversible, and supplementation of all trans retinoic acid (t-RA) doses dependently restored mucin gene expression. Further analyses indicated that As (III) treatment significantly reduced the protein level of retinoic acid receptors (RARα, γ and RXRα) as well as RARE promoter reporter activity. Therefore, our study fills in an important knowledge gap in the field of low-dose arsenic exposure. The interference of RA signaling, and mucin gene expression may be important pathogenic factors in low-dose arsenic induced lung toxicity.

DFSG, a novel herbal cocktail with anti-asthma activity, suppressed MUC5AC in A549 cells and alleviated allergic airway hypersensitivity and inflammatory cell infiltration in a chronic asthma mouse model.

AIM OF THE STUDY: To develop a novel anti-asthma drug. DFSG is a novel herbal cocktail composed of 4 types of herbal medicines. This study explored whether DFSG has the potential to attenuate asthma symptom severity and aimed to determine the immunomodulatory mechanism of DFSG using a chronic asthmatic mouse model induced by repeated challenges with Dermatogoides pteronyssinus (Der p). MATERIALS AND METHODS: BALB/c mice were intratracheally inoculated with Der p (50 µl, 1 mg/ml) once a week for 5 weeks. In addition, 30 min before Der p challenge, the mice were orally administered 1x DFSG (1 g/kg) or 1/2x DFSG (0.5 g/kg). Three days after the final challenge, the mice were sacrificed to evaluate inflammatory cell infiltration, lung histological features, blood total IgE, and cytokine levels in pulmonary alveolar lavage fluid. Furthermore, 30 min after the addition of DFSG, caffeic acid, p-coumaric acid or chlorogenic acid to A549 cells, 10 ng/ml IL-1ß was added to evaluate the effect of the drug on mucin 5AC (MUC5AC) gene expression after stimulation of A549 cells by IL-1ß. RESULTS: DFSG significantly reduced Der p-induced airway hyperresponsiveness, bronchial inflammatory cell infiltration, and total IgE and IgG1 serum levels. Furthermore, DFSG significantly inhibited TH2 cytokines and increased the expression of TH1 cytokines. In addition, immunohistochemical staining demonstrated that DFSG inhibited MUC5AC expression in the bronchial epithelial cells. DFSG and a mixture of caffeic acid, p-coumaric acid, and chlorogenic acid inhibited MUC5AC gene expression in A549 cells after stimulation with IL-1ß. CONCLUSION: These results suggest that by regulating TH1 and TH2 cytokines and MUC5AC expression, DFSG exhibits anti-airway inflammatory cell infiltration and anti-hyperresponsiveness activity and inhibits specific immunity in a chronic asthmatic mouse model. Therefore, DFSG has potential for development into a drug for chronic asthma treatment.

Depletion of mucin in mucin-producing human gastrointestinal carcinoma: Results from in vitro and in vivo studies with bromelain and N-acetylcysteine.

Aberrant expression of membrane-associated and secreted mucins, as evident in epithelial tumors, is known to facilitate tumor growth, progression and metastasis, and to provide protection against adverse growth conditions, chemotherapy and immune surveillance. Emerging evidence provides support for the oncogenic role of MUC1 in gastrointestinal carcinomas and relates its expression to an invasive phenotype. Similarly, mucinous differentiation of gastrointestinal tumors, in particular increased or de novo expression of MUC2 and/or MUC5AC, is widely believed to imply an adverse clinicopathological feature. Through formation of viscous gels, too, MUC2 and MUC5AC significantly contribute to the biology and pathogenesis of mucin-secreting gastrointestinal tumors. Here, we investigated the mucin-depleting effects of bromelain (BR) and N-acetylcysteine (NAC), in nine different regimens as single or combination therapy, in in vitro (MKN45, KATOIII and LS174T cell lines) and in vivo (female nude mice bearing intraperitoneal MKN45 and LS174T) settings. The inhibitory effects of the treatment on cancer cell growth and proliferation were also evaluated in vivo. Our results suggest that a combination of BR and NAC with dual effects on growth and mucin products of mucin-expressing tumor cells is a promising candidate towards the development of novel approaches to gastrointestinal malignancies with the involvement of mucin pathology. This capability supports the use of this combination formulation in locoregional approaches for reducing the adverse effects of the aberrantly secreted gel-forming mucins, as in pseudomyxoma peritonei and similar pathologies with ectopic production of mucin.

Inhibition of secretion, production and gene expression of mucin from cultured airway epithelial cells by prunetin.

This study investigated whether prunetin significantly affects the secretion, production and gene expression of mucin from cultured airway epithelial cells. Confluent primary rat tracheal surface epithelial (RTSE) cells were pretreated with adenosine triphosphate (ATP) for 5 min and then chased for 30 min in the presence of prunetin to assess the effect on mucin secretion using enzyme-linked immunosorbent assay (ELISA). At the same time, confluent NCI-H292 cells were pretreated with prunetin for 30 min and then stimulated with epidermal growth factor (EGF) or phorbol 12-myristate 13-acetate (PMA) for 24 h, respectively. The MUC5AC mucin gene expression and mucin protein production were measured by reverse transcription-polymerase chain reaction (RT-PCR) and ELISA. The results were as follows: (1) prunetin significantly suppressed ATP-induced mucin secretion from cultured RTSE cells; (2) prunetin inhibited the production of MUC5AC mucin protein induced by EGF or PMA from NCI-H292 cells; (3) prunetin also inhibited the expression of MUC5AC mucin gene induced by EGF or PMA from NCI-H292 cells. This result suggests that prunetin can regulate the secretion, production and gene expression of mucin, by directly acting on airway epithelial cells.

Inhibition of airway MUC5AC mucin production and gene expression induced by epidermal growth factor or phorbol ester by glycyrrhizin and carbenoxolone.

BACKGROUND AND AIM: In this study, we investigated whether glycyrrhizin and carbenoxolone affect MUC5AC mucin production and gene expression induced by epidermal growth factor (EGF) or phorbol ester (PMA) from human airway epithelial cells. METHODS: Confluent NCI-H292 cells were pretreated with each agent for 30 min and then stimulated with EGF and PMA for 24h, respectively. MUC5AC mucin gene expression and mucin protein production were measured by RT-PCR and ELISA. RESULTS: Glycyrrhizin and carbenoxolone were found to inhibit the production of MUC5AC mucin protein induced by EGF or PMA, and both compounds also inhibited the expression of MUC5AC mucin gene induced by EGF or PMA. CONCLUSION: These results suggest that glycyrrhizin and carbenoxolone can inhibit mucin gene expression and production of mucin protein, by directly acting on airway epithelial cells.

Genistein and curcumin suppress epidermal growth factor-induced MUC5AC mucin production and gene expression from human airway epithelial cells.

This study investigated whether genistein and curcumin affect epidermal growth factor (EGF)-induced MUC5AC mucin production and gene expression from human airway epithelial cells. Confluent NCI-H292 cells were pretreated with each agent for 30 min and then stimulated with EGF for 24 h. The MUC5AC mucin gene expression and mucin protein production were measured by reverse transcription - polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. The results were as follows: (1) genistein and curcumin inhibited the production of MUC5AC mucin protein induced by EGF, dose-dependently; (2) genistein and curcumin also inhibited the expression of MUC5AC mucin gene induced by EGF. This result suggests that genistein and curcumin can regulate mucin gene expression and production of mucin protein induced by EGF, by directly acting on airway epithelial cells.