Signaling Pathway In A549 Cells Research Articles

The molecular mechanism of action for the potent antitumor component extracted using supercritical fluid extraction from Croton crassifolius root

Ethnopharmacological relevanceThe root of Croton crassifolius has been used as a traditional Chinese medicine (TCM), called Radix Croton Crassifolius, and commonly known as “Ji Gu Xiang” in Chinese. Its medicinal value has been recorded in several medical books or handbooks, such as “Sheng Cao Yao Xing Bei Yao”, “Ben Cao Qiu Yuan” and “Zhong Hua Ben Cao”. It has been traditional employed for treating sore throat, stomach-ache, rheumatism and cancer. Aim of the studyAt present, there are limited studies on the evaluation of low-polarity extracts of roots in C. crassifolius. Consequently, the aim of this study was to evaluate the antitumor effect of the low-polarity extract of C. crassifolius root. Materials and methodsExtracts were obtained by supercritical fluid extraction. The extracts were tested for antitumor effects in vitro on several cancer cell lines. A CCK-8 kit was used for further analysis of cell viability. A flow cytometer and propidium iodide staining were used to evaluate the cell cycle and apoptosis. Hoechst staining, JC-1 staining and the fluorescence probe DCFH-DA were used to evaluate apoptotic cells. Molecular mechanisms of action were analyzed by quantitative RT‒PCR and Western blotting. Immunohistochemistry was used for the evaluation of xenograft tumors in male BALB/c mice. Finally, molecular docking was employed to predict the bond between the desired bioactive compound and molecular targets. ResultsEleven diterpenoids were isolated from low-polarity C. crassifolius root extracts. Among the compounds, chettaphanin II showed the strongest activity (IC50 = 8.58 μM) against A549 cells. Evaluation of cell viability and the cell cycle showed that Chettaphanin II reduced A549 cell proliferation and induced G2/M-phase arrest. Chttaphanin II significantly induced apoptosis in A549 cells, which was related to the level of apoptosis-related proteins. The growth of tumor tissue was significantly inhibited by chettaphanin II in experiments performed on naked mice. The antitumor mechanism of chettaphanin II is that it can obstruct the mTOR/PI3K/Akt signaling pathway in A549 cells. Molecular docking established that chettaphanin II could bind to the active sites of Bcl-2 and Bax. ConclusionsTaken together, the natural diterpenoid chettaphanin II was identified as the major antitumor active component, and its potential for developing anticancer therapies was demonstrated for the first time by antiproliferation evaluation in vitro and in vivo.

TRIM21 reduces H1N1-induced inflammation and apoptosis by regulating the TBK1-IRF3 signaling pathway in A549 cells.

Triple motif protein 21 (TRIM21) has an antiviral function that inhibits various viral infections. However, its role in the progress of influenza A virus (IAV) infection is unclear. In this study, we investigated the role and molecular mechanism of TRIM21 in IAV infection. RT-qPCR was used to determine the level of TRIM21 mRNA, and ELISA was used to measure the levels of IFN-α, IFN-β, IL-6, and TNF-α. The levels of the TRIM21, NP, TBK1, IRF3, p-TBK1, and p-IRF3 proteins were estimated by Western blot. The results showed that, after IAV infection, TRIM21 was upregulated in clinical patient serum and A549 cells, and this was correlated with the IFN response. Overexpression of TRIM21 reduced IAV replication and transcription in in vitro cell experiments. TRIM21 also increased IFN-α and IFN-β levels and decreased IL-6 and TNF-α levels in A549 cells. In addition, overexpression of TRIM21 inhibited IAV-induced apoptosis. Further experiments demonstrated that TBK1-IRF3 signaling was activated by TRIM21 and was involved in the inhibitory effect of TRIM21 on virus replication. In summary, our study suggests that TRIM21 inhibits viral replication by activating the TBK1-IRF3 signaling pathway, further inhibiting the infection process of IAV.

Exosomal miR-223 promotes ARDS by targeting insulin-like growth factor 1 receptor: A cell communication study

Background Acute respiratory distress syndrome (ARDS) is a respiratory failure syndrome characterized by hypoxemia and changes in the respiratory system. ARDS is the most common cause of death in COVID-19 deaths was ARDS. In this study, we explored the role of miR-223 in exosomes in ARDS. Methods Exosomes were purified from the supernatants of macrophages. qPCR was used to detect relative mRNA levels. A luciferase reporter assay was performed to verify the miRNA target genes. Western blotting was used to detect the activation of inflammatory pathways. Flow cytometry was performed to assess apoptosis. An LPS-induced ARDS mouse model was used to assess the function of miR-223 in ARDS. Results Exosomes secreted by macrophages promoted apoptosis in A549 cells. Macrophages and exosomes contain high levels of miR-223. Exogenous miR-223 can decrease the expression of insulin-like growth factor 1 receptor (IGF-1R) in A549 and promote the apoptosis of A549.Transfection of anti-miR223 antisense nucleotides effectively reduced the level of miR-223 in macrophages and exosomes and eliminated the pro-apoptotic effect of A549. In vivo, LPS stimulation increased inflammatory cell infiltration in the lungs of mice, whereas knockdown of miR-223 in mice resulted in significantly reduced eosinophil infiltration. Conclusions Macrophages can secrete exosomes containing miR-223 and promote apoptosis by targeting the IGF-1R/Akt/mTOR signaling pathway in A549 cells and mouse models, suggesting that miR-223 is a potential target for treating COVID-19 induced ARDS.

Chikusetsusaponin IVa Protects against H9N2 Avian Influenza Virus Infection by Inhibiting Inflammation and TLR4/NF-κB Signaling Pathways in vitro and in vivo

Background: The prevalence of Hemagglutinin 9 Neuraminidase 2 (H9N2) avian influenza virus (AIV) in poultry in Asia has raised serious concerns about its zoonotic transmission and the potential for a pandemic, underscoring the need for effective therapeutics. Chikusetsusaponin IVa (CHS), a bioactive triterpenoid saponin from Radix saposhnikoviae, has exhibited anti-inflammatory activity in preclinical studies. We hypothesized that CHS could mitigate immunopathology during tumor necrosis factor (TNF)-infection by suppressing excessive inflammation. Objectives: CHS is a natural saponin with anti-inflammatory activity. This research aimed to explore the in vitro and in vivo protective effects and mechanisms of CHS against H9N2 AIV infection. Materials and Methods: We employed Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and Gene Ontology (GO) analysis for the prediction of the underlying mechanisms, followed by a network analysis approach. In animal experiments, the toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) signaling pathways and associated proteins were assessed using immunohistochemistry and western blot analysis. Results: In A549 cells, CHS treatment reduced H9N2 AIV-induced apoptosis in a dose-dependent manner. CHS was found to have a suppressive effect on the mRNA expression of various inflammatory cytokines (including TNF-α, interleukin (IL)-6, IL-1β, and IL-8) as well as mediators such as inducible nitric oxide synthase (iNOS) and prostaglandin-endoperoxide synthase 2 (PTGS2). It can be seen that CHS does have anti-inflammatory effects. Bioinformatics analysis indicated that CHS might exhibit anti-inflammatory effects by modulating cytokine–cytokine receptor interaction and the NF-κB signaling pathways. Building upon this, it was found that CHS blocked the activation of TLR4 and NF-κB signaling pathways in A549 cells infected with H9N2 AIV. In vivo, CHS prolonged the survival time and increased the survival rate of H9N2 AIV-infected mice. CHS was found to have a protective effect on the lungs of infected mice, resulting in reduced damage and lower levels of viral load and inflammation. CHS inhibited the production of proinflammatory cytokines (IL-1β, TNF-α, IL-6, and IL-8) and inhibited the activation of TLR4 and NF-κB signaling pathways in the pulmonary tissues of mice that were infected. Conclusion: CHS exerts protective effects against H9N2 AIV infection in vitro and in vivo. The anti-inflammatory mechanisms may involve the inhibition of cytokine production and the blockade of TLR4 and NF-κB signaling pathways.

Melittin kills A549 cells by targeting mitochondria and blocking mitophagy flux

ABSTRACT Melittin, a naturally occurring polypeptide found in bee venom, has been recognized for its potential anti-tumor effects, particularly in the context of lung cancer. Our previous study focused on its impact on human lung adenocarcinoma cells A549, revealing that melittin induces intracellular reactive oxygen species (ROS) burst and oxidative damage, resulting in cell death. Considering the significant role of mitochondria in maintaining intracellular redox levels and ROS, we further examined the involvement of mitochondrial damage in melittin-induced apoptosis in lung cancer cells. Our findings demonstrated that melittin caused changes in mitochondrial membrane potential (MMP), triggered mitochondrial ROS burst (Figure 1), and activated the mitochondria-related apoptosis pathway Bax/Bcl-2 by directly targeting mitochondria in A549 cells (Figure 2). Further, we infected A549 cells using a lentivirus that can express melittin-Myc and confirmed that melittin can directly target binding to mitochondria, causing the biological effects described above (Figure 2). Notably, melittin induced mitochondrial damage while inhibiting autophagy, resulting in abnormal degradation of damaged mitochondria (Figure 5). To summarize, our study unveils that melittin targets mitochondria, causing mitochondrial damage, and inhibits the autophagy-lysosomal degradation pathway. This process triggers mitoROS burst and ultimately activates the mitochondria-associated Bax/Bcl-2 apoptotic signaling pathways in A549 cells.

Meroterpenoids from Daphne genkwa shows promising in vitro antitumor activity via inhibiting PI3K/Akt/mTOR signaling pathway in A549 cells

Phytochemical investigation into the leaves and branches of Daphne genkwa afforded 25 meroterpenoids (1–16) including nine pairs of enantiomers (1a/1b−8a/8b and 12a/12b), among which 20 compounds have been reported in the present work for the first time. The structures with absolute configurations of the new molecules (excluding 10–13) were established via comprehensive spectroscopic analyses especially electronic circular dichroism (ECD) and Mosher’s methods. A preliminary in vitro cell viability assay revealed remarkable cytotoxicities of selective compounds against A549 (lung), Hela (cervical), MDA-MB231 (breast) and MCF-7 (breast) cancer cells, and compound 8a showed the best inhibitory activity with IC50 values in the range of 3.12–4.67 μM toward the four cell lines. Subsequent in vitro antitumor evaluation of 8a disclosed that it could inhibit the proliferation and metastasis, as well as induce significant apoptosis and cycle arrest, of A549 cells. Further mechanistic investigations revealed that 8a could exert its antitumor activity via inhibiting the PI3K/Akt/mTOR signaling pathway.

Synthesis and Antitumor Activity of Panaxadiol Pyrazole and Isooxazole Derivatives.

In this study, we designed and synthesized 19 nitrogen-containing heterocyclic derivatives of panaxadiol (PD). We first reported the antiproliferative activity of these compounds against four different tumor cells. The results of the MTT assay showed that the PD pyrazole derivative (compound 12b) had the best antitumor activity and could significantly inhibit the proliferation of four tested tumor cells. For A549 cells, the IC50 value was as low as 13.44±1.23 μM. Western blot analysis showed that the PD pyrazole derivative was a bifunctional regulator. On the one hand, it can down-regulate the expression of HIF-1α by acting on PI3 K/AKT signaling pathway in A549 cells. On the other hand, it can induce the decrease of CDKs protein family and E2F1 protein expression levels, thus playing a crucial role in cell cycle arrest. According to the results of molecular docking, we found that multiple hydrogen bonds were formed between the PD pyrazole derivative and two related proteins, and the docking score of the derivative was also significantly higher than that of the crude drug. In summary, the study of the PD pyrazole derivative laid a foundation for the development of ginsenoside as an antitumor agent.

Anti-cancer activity of Hypnea valentiae seaweed loaded gold nanoparticles through EMT signaling pathway in A549 cells

The biological synthesis of gold nanoparticles (AuNPs) offers enormous promise for the formulation of a new generation anti-carcinogenic drugs. In this study, AuNPs were eco-friendly synthesized using the marine red algae species, Hypnea valentiae. Multiple spectroscopic methods were used to confirm and characterized the synthesized Hv-AuNPs. UV spectroscopy revealed 540 nm is where Hv-AuNPs' Surface Plasmon Resonance (SPR) peak. Scanning Electron Microscope (SEM) and Transmission Electron Microscope (TEM) showed that Hv-AuNPs were 7–45 nm size ranges with round shapes. With the help of an X-ray Diffraction (XRD) spectrum, the crystalline nature of Hv-AuNPs was found, and FTIR analytical tools were used to find the organic compounds, like phenols and carbonyl molecules, that were involved in the reduction of Hv-AuNPs. The biosynthesized Hv-AuNPs' potential to scavenge free radicals was measured using the DPPH test (84.5 ± 1.45%), the hydrogen peroxide assay (70.6 ± 0.8%) and the FRAP assay (90.54 ± 2.3%). Lung cancer (A549) cells were used to test the anti-cancer effects of Hv-AuNPs through EMT signaling pathway (E-cadherin, Vimentin and SNAIL). The Hv-AuNPs showed strong cytotoxic activity against lung cancer cells (IC50 = 23.68 μg/mL) and the nanoparticles effectively control the cell migration and colony formation at 50 μg/mL. The RT-PCR gene expression analysis confirmed the Hv-AuNPs potentially downregulated the Vimentin expression at 50 μg/mL and demonstrated high anti-cancer activity. Therefore, this investigation found that H. valentiae synthesized AuNPs have high therapeutic potential for lung cancer and suggests their use in drug development.

Betulinaldehyde exhibits effective anti-tumor effects in A549 cells by regulating intracellular autophagy

It is of great significance to find new effective drugs for an adjuvant therapy targeting lung cancer to improve the survival rate and prognosis of patients with the disease. Previous studies have confirmed that certain Chinese herbal extracts have clear anti-tumor effects, and in our preliminary study, betulinaldehyde was screened for its potential anti-tumor effects. The current study thus aimed to confirm the anti-tumor effect of betulinaldehyde, using in vitro experiments to explore its underlying molecular mechanism. It was found that betulinaldehyde treatment significantly inhibited the viability, proliferation, and migration of A549 cells in a dose-dependent manner. In addition, betulinaldehyde inhibited the activation of Akt, MAPK, and STAT3 signaling pathways in A549 cells in a time-dependent manner. More importantly, betulinaldehyde also decreased the expression level of SQSTM1 protein, increased the expression level of LC3 II, and increased the autophagy flux in A549 cells. The pretreatment of A549 cells with the autophagy inhibitor, 3-methyladenine, could partially negate the anti-tumor effects of betulinaldehyde. These findings suggest that betulinaldehyde could significantly inhibit the oncological activity of A549 cells by regulating the intracellular autophagy level, making it a potentially effective option for the adjuvant therapy used to treat lung cancer in the future.

Lutein inhibits tumor progression through the ATR/Chk1/p53 signaling pathway in non-small cell lung cancer.

Lung cancer is the leading cause of cancer-related death. In particular, non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer cases. Due to tumor resistance and the toxicity of chemotherapeutic agents, it is increasingly critical to discover novel, potent antitumorigenic drugs for treating NSCLC. Lutein, a carotenoid, has been reported to exert toxic effects on cells in several tumor types. However, the detailed functions and underlying mechanisms of lutein in NSCLC remain elusive. The present study showed that lutein significantly and dose-dependently inhibited cell proliferation, arrested the cell cycle at the G0/G1 phase, and induced apoptosis in NSCLC cells. RNA-sequencing analysis revealed that the p53 signaling pathway was the most significantly upregulated in lutein-treated A549 cells. Mechanistically, lutein exerted antitumorigenic effects by inducing DNA damage and subsequently activating the ATR/Chk1/p53 signaling pathway in A549 cells. In vivo, lutein impeded tumor growth in mice and prolonged their survival. In conclusion, our findings demonstrate the antitumorigenic potential of lutein and reveal its molecular mechanism of action, suggesting that lutein is a promising candidate for clinical NSCLC treatment.

TRPV3 promotes the angiogenesis through HIF-1α-VEGF signaling pathway in A549 cells

BackgroundAngiogenesis is an essential physiological process in the growth and metastasis of primary tumors. Ca2+ signaling is crucial for tumor angiogenesis. The purpose of this study was to detect the potential role of Ca2+ permeable transient receptor potential vanilloid-3 (TRPV3) in the angiogenesis of non-small cell lung cancer (NSCLC). MethodsSmall interfering RNA was used to down-regulate TRPV3 expression in A549 cells. A laser scanning confocal microscope was used to examine intracellular calcium concentration ([Ca2+]i). Human umbilical vein endothelial cells (HUVECs) tube formation and migration assay, Western blot, MTT and ELISA were performed to detect the potential mechanisms of TRPV3 in tumor angiogenesis. A mouse tumor xenograft model was performed to expound the effects of TRPV3 on tumor cell growth. ResultsInhibition of TRPV3 reduced [Ca2+]i and protein expressions of VEGF and HIF-1α in A549 cells. Moreover, HIF-1α depletion decreased the secretion and expression of VEGF. Depletion of TRPV3 inhibited HUVECs proliferation, tube formation and migration induced by conditioned medium. And TRPV3 inhibition could decrease the volume of xenograft tumors and MVD of CD34+ cells. The expression levels of HIF-1α, VEGF and p-CaMKП in the xenograft tumors in RuR and siTRPV3 groups was reduced. ConclusionsTRPV3 calcium channel protein may play a key role in NSCLC angiogenesis. TRPV3 could promote the angiogenesis through HIF-1α-VEGF signaling pathway. Targeting TRPV3 channel protein by novel approaches would be useful for reversing NSCLC angiogenesis.

75P Biosynthesis and characterization of selenium nanoparticles using marine algae Sargassum wightii: Understanding its anticancer activity through ROS-mediated p53 and Akt signaling pathways in A549 cells

75P Biosynthesis and characterization of selenium nanoparticles using marine algae Sargassum wightii: Understanding its anticancer activity through ROS-mediated p53 and Akt signaling pathways in A549 cells

Perilla Fruit Water Extract Attenuates Inflammatory Responses and Alleviates Neutrophil Recruitment via MAPK/JNK-AP-1/c-Fos Signaling Pathway in ARDS Animal Model.

Airway respiratory distress syndrome (ARDS) is usually caused by a severe pulmonary infection. However, there is currently no effective treatment for ARDS. Traditional Chinese medicine (TCM) has been shown to effectively treat inflammatory lung diseases, but a clear mechanism of action of TCM is not available. Perilla fruit water extract (PFWE) has been used to treat cough, excessive mucus production, and some pulmonary diseases. Thus, we propose that PFWE may be able to reduce lung inflammation and neutrophil infiltration in a lipopolysaccharide (LPS)-stimulated murine model. C57BL/6 mice were stimulated with LPS (10 μg/mouse) by intratracheal (IT) injection and treated with three doses of PFWE (2, 5, and 8 g/kg) by intraperitoneal (IP) injections. To investigate possible mechanisms, A549 cells were treated with PFWE and stimulated with LPS. Our results showed that PFWE decreased airway resistance, neutrophil infiltration, vessel permeability, and interleukin (IL)-6 and chemokine (C-C motif) ligand 2 (CCL2/MCP-1) expressions in vivo. In addition, the PFWE inhibited the expression of IL-6, CCL2/MCP-1, chemokine (CXC motif) ligand 1 (CXCL1/GROα), and IL-8 in vitro. Moreover, PFWE also inhibited the MAPK/JNK-AP-1/c-Fos signaling pathway in A549 cells. In conclusion, we demonstrated that PFWE attenuated pro-inflammatory cytokine and chemokine levels and downregulated neutrophil recruitment through the MAPK/JNK-AP-1/c-Fos pathway. Thus, PFWE can be a potential drug to assist the treatment of ARDS.

Silica nanoparticle exposure inhibits surfactant protein A and B in A549 cells through ROS-mediated JNK/c-Jun signaling pathway.

Exposure to silica nanoparticles (SiNPs) is related to the dysregulation of pulmonary surfactant that maintains lung stability and function. Nevertheless, there are limited studies concerning the interaction and influence between SiNPs and pulmonary surfactant, and the damage and mechanism are still unclear. Herein, we used A549 cells to develop an in vitro model, with which we investigated the effect of SiNPs exposure on the expression of pulmonary surfactant and the potential regulatory mechanism. The results showed that SiNPs were of cytotoxicity in regarding of reduced cell viability and promoted the production of excessive reactive oxygen species (ROS). Additionally, the JNK/c-Jun signaling pathway was activated, and the expression of surfactant protein A (SP-A) and surfactant protein B (SP-B) was decreased. After the cells being treated with N-acetyl-L-cysteine (NAC), we found that the ROS content was effectively downregulated, and the expression of proteins related to JNK and c-Jun signaling pathways was suppressed. In contrast, the expression of SP-A and SP-B was enhanced. Furthermore, we treated the cells with JNK inhibitor and c-Jun-siRNA and found that the expression of protein related to JNK and c-Jun signaling pathways, as well as SP-A and SP-B, changed in line with that of NAC treatment. These findings suggest that SiNPs exposure can upregulate ROS and activate the JNK/c-Jun signaling pathway in A549 cells, thereby inhibiting the expression of SP-A and SP-B proteins.

Interaction between AhR and HIF-1 signaling pathways mediated by ARNT/HIF-1\u03b2

BackgroundThe main causes of lung cancer are smoking, environmental pollution and genetic susceptibility. It is an indisputable fact that PAHs are related to lung cancer, and benzo(a) pyrene is a representative of PAHs. The purpose of the current investigation was to investigate the interaction between AhR and HIF-1 signaling pathways in A549 cells, which provide some experimental basis for scientists to find drugs that block AhR and HIF-1 signaling pathway to prevent and treat cancer.MethodsThis project adopts the CYP1A1 signaling pathways and the expression of CYP1B1 is expressed as a measure of AhR strength index. The expression of VEGF and CAIX volume as a measure of the strength of the signal path HIF-1 indicators. Through the construction of plasmid vector, fluorescence resonance energy transfer, real-time quantitative PCR, western blotting and immunoprecipitation, the interaction between AhR signaling pathway and HIF-1 signaling pathway was observed.ResultsBaP can enhance the binding ability of HIF-1α protein to HIF-1β/ARNT in a dose-dependent manner without CoCl2. However, the binding ability of AhR protein to HIF-1β/ARNT is inhibited by HIF-1α signaling pathway in a dose-dependent manner with CoCl2.ConclusionIt is shown that activation of the AhR signaling pathway does not inhibit the HIF-1α signaling pathway, but activation of the HIF-1α signaling pathway inhibits the AhR signaling pathway.

IC261 inhibits the epithelial-mesenchymal transition induced by TGF-\u03b2 in A549 lung cancer cells

Despite rapid advances in cancer diagnosis and therapy, lung cancer continues to be the primary cause of cancer-related mortality. Epithelial mesenchymal transition has been implicated in drug resistance and cancer metastasis. IC261 mediates various pathophysiological processes, including inflammation and tumorigenesis. Therefore, we analyzed the involvement of IC261 in epithelial mesenchymal transition. Pretreatment with IC261 significantly inhibited the expression of transforming growth factor (TGF)-β1-induced mesenchymal cell markers, including N-cadherin (N-cad), vimentin (Vim), and β-catenin (β-cat), at the mRNA and protein levels in A549 lung cancer cells, which was confirmed using immunofluorescence staining. A migration assay revealed that IC261 treatment strongly inhibited TGF-β1-induced migration activity at 24 and 48 h. Additionally, IC261 treatment suppressed the activation of the TGF-β1 signaling pathway in A549 cells and phosphorylation of Smad2 and Smad3. Our findings demonstrate that IC261, a selective inhibitor of casein kinase 1, inhibits the TGF-β1-induced migration of A549 cells by inhibiting Smad2/3 phosphorylation and downregulating the expression of N-cad, Vim, and β-cat.

Isopanduratin A Inhibits Tumor Necrosis Factor (TNF)-α-Induced Nuclear Factor κB Signaling Pathway by Promoting Extracellular Signal-Regulated Kinase-Dependent Ectodomain Shedding of TNF Receptor 1 in Human Lung Adenocarcinoma A549 Cells

Tumor necrosis factor α (TNF-α) induces the nuclear factor κB (NF-κB) signaling pathway via TNF receptor 1 (TNF-R1). We recently reported that isopanduratin A inhibited the TNF-α-induced NF-κB signaling pathway in human lung adenocarcinoma A549 cells. In the present study, we found that isopanduratin A did not inhibit the interleukin-1α-induced NF-κB signaling pathway in A549 cells. Isopanduratin A down-regulated the expression of TNF-R1 in these cells. We also revealed that isopanduratin A down-regulated the cell surface expression of TNF-R1 by promoting the cleavage of TNF-R1 into its soluble forms. TAPI-2, an inhibitor of TNF-α-converting enzyme, suppressed the inhibitory activity of isopanduratin A against the TNF-α-induced activation of NF-κB. The mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (ERK) kinase inhibitor U0126, but not the p38 MAP kinase inhibitor SB203580, blocked the ectodomain shedding of TNF-R1 induced by isopanduratin A. Consistent with this result, isopanduratin A induced the rapid phosphorylation of ERK, but not p38 MAP kinase. Isopanduratin A also promoted the phosphorylation of eukaryotic initiation factor 2α (eIF2α). The present results indicate that isopanduratin A inhibits TNF-α-induced NF-κB signaling pathway by promoting ERK-dependent ectodomain shedding of cell surface TNF-R1, and also decreases cellular TNF-R1 levels through the phosphorylation of eIF2α in A549 cells.

Traditional Chinese Medicine, Qingfei Paidu Decoction and Xuanfei Baidu Decoction, Inhibited Cytokine Production via NF-κB Signaling Pathway in Macrophages: Implications for Coronavirus Disease 2019 (COVID-19) Therapy.

Background and Aims: Qingfei Paidu decoction (QPD) and Xuanfei Baidu decoction (XBD) are two typical traditional Chinese medicines with proven efficacy for the treatment of SARS-CoV-2, although the underlying mechanism is not well defined. Blunted immune response and enhanced production of pro-inflammatory cytokines (cytokine storm) are two main features observed in patients infected with SARS-CoV-2. Analysis based on network pharmacology has revealed that both QPD and XBD played an important role in the regulation of host immunity. We therefore investigated the role of QPD and XBD in the modulation of innate immunity in vitro, focusing on the type 1 interferon (IFN) signaling pathway in A549 cells and pro-inflammatory cytokine production in macrophages. Methods: A549 cells were treated with QPD or XBD and the production of endogenous IFNα and IFNβ as well as the expression levels of some interferon-stimulated genes (ISGs) were detected by reverse transcriptase-quantitative PCR (RT-qPCR). Macrophages derived from THP-1 cells were treated with QPD or XBD and their pro-inflammatory cytokine expression levels were measured by RT-qPCR, 6 h post LPS stimulation. In addition, the expression levels of some pro-inflammatory cytokines were further analyzed by ELISA. The effect of QPD and XBD on the NF-κB signaling pathway and the pinocytosis activity of THP-1-derived macrophages were evaluated by Western blot and neutral red uptake assay, respectively. Results: Although QPD and XBD showed very little effect on the type 1 IFN signaling pathway in A549 cells, either QPD or XBD markedly inhibited the production of pro-inflammatory markers including interleukin-6, tumor necrosis factor-α, monocyte chemotactic protein-1, and chemokine ligand 10 in THP-1-derived M1 macrophages. In addition, the phosphorylation of IκBα and NF-κB p65 during the process of macrophage polarization was significantly suppressed following QPD or XBD treatment. QPD and XBD also suppressed the pinocytosis activity of macrophages. Conclusion: QPD and XBD have been shown to have robust anti-inflammatory activities in vitro. Our study demonstrated that both QPD and XBD decreased pro-inflammatory cytokine expression, inhibited the activation of the NF-κB signaling pathway, and blunted pinocytosis activity in THP-1-derived macrophages.

Influence of o,p′-DDT on MUC5AC expression via regulation of NF-κB/AP-1 activation in human lung epithelial cells

ABSTRACT o,p’-Dichlorodiphenyltrichloroethane (o,p’-DDT) is a representative endocrine disruptor, and exposure to o,p’-DDT may produce immune disorders and inflammation, leading to various diseases such as cancer. Chronic airway inflammation is characterized by excessive mucus secretion resulting in chronic obstructive pulmonary disease (COPD). Mucin 5AC (MUC5AC), one of the mucus genes, plays an important role in mucus secretion and inflammation in the airways. The aim of this study was to examine the effects of o,p′-DDT on the regulation of MUC5AC expression in human lung epithelial A549 cell line. o,p′-DDT increased mRNA levels and the promoter activity of MUC5AC. Transient transfection with mutation promoter constructs of MUC5AC demonstrated that nuclear factor kappa-b (NF-κB) and activator protein 1(AP-1) response elements were essential for the consequences of o,p’-DDT on MUC5AC expression. In addition, o,p′-DDT induced phosphorylation of ERK, JNK, p38, and Akt, which are involved in the regulation of MUC5AC expression. It is noteworthy that inhibitors of NF-κB, AP-1, Akt, and MAPKs blocked enhanced o,p′-DDT-induced MUC5AC mRNA expression. Data indicate that o,p′-DDT increase in NF-κB, and AP-1 transcriptional activation-dependent MUC5AC expression is associated with stimulation of Akt and MAPK signaling pathways in A549 cells.

Chemopreventive effects and anti-tumorigenic mechanisms of 2,6-dimethoxy-1,4-benzoquinone, a constituent of Vitis coignetiae Pulliat (crimson glory vine, known as yamabudo in Japan), toward 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis in A/J mice

Previously, we isolated and identified anti-mutagenic and anti-inflammatory components from Vitis coignetiae (crimson glory vine, known as yamabudo in Japan) as 2,6-dimethoxy-1,4-benzoquinone (DBQ), fertaric acid and caftaric acid. We also reported that the oral intake of a partially purified fraction from yamabudo juice (yamabudo-fr) or DBQ affords significant protection against two-stage skin carcinogenesis in mice. In this study, we found that oral intake of yamabudo-fr or DBQ affords significant protection against a tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced mouse model of lung tumorigenesis. Furthermore, we investigated the anti-tumorigenic mechanisms of yamabudo juice and DBQ. NNK is known to be a DNA-methylating and alkylating agent; thus, we investigated the anti-tumorigenic mechanisms of yamabudo juice and DBQ in relation to DNA methylation. Pretreatment with yamabudo-fr or DBQ dose-dependently decreased formation of O6-methylguanine and N7-methylguanine in DNA of the A549 human lung epithelial-like cell line treated with a methylating agent, 1-methyl-3-nitro-1-nitrosoguanidine. Yamabudo juice and DBQ inhibited the mutagenicity of NNK in the Ames test using Salmonella typhimurium TA1535 but not S. typhimurium YG7108, an alkylguanine DNA alkyltransferase-deficient strain (same as TA1535 but Δadast::Kmr, Δogtst::Cmr). Yamabudo juice and DBQ might accelerate the repair of DNA damage caused by NNK and reduce DNA damage to cells. We also investigated the effects of yamabudo juice and DBQ on signaling pathways in A549 cells. With or without epidermal growth factor stimulation, phosphorylation of Erk1/2, Akt and Stat3 in A549 cells was significantly decreased in the presence of yamabudo juice or DBQ, indicating that yamabudo juice and DBQ suppressed PI3K/AKT, MAPK/ERK and JAK/STAT3 signaling pathways. These results suggest that both initiation and growth/progression steps in carcinogenesis, especially anti-oxidant effects, stimulation of repair of alkyl DNA adducts and suppressed growth signaling pathways are potential anti-tumorigenic targets of yamabudo juice and DBQ in NNK-induced lung tumorigenesis.