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Coronavirus 2019 (COVID-19) disease management is highly dependent on the immune status of the infected individual. An increase in the incidence of depression has been observed during the ongoing COVID-19 pandemic. Autoantibodies against in vitro reactive oxygen species (ROS) modified BSA and Lys as well as antibodies against receptor binding domain subunit S1 (S1-RBD) (S1-RBD-Abs) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were estimated using direct binding and competition ELISA. Serum samples were also tested for fasting blood glucose (FBG), malondialdehyde (MDA), carbonyl content (CC), interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). Significant structural changes were observed in ROS modified BSA and Lys. Female depressed subjects who were also smokers (F-D-S) showed the highest levels of oxidative stress (MDA and CC levels). Similarly, increased levels of autoantibodies against ROS modified proteins were detected in F-D-S subjects, in males who were depressed and in smokers (M-D-S) compared to the other subjects from the rest of the groups. However, contrary to this observation, levels of S1-RBD-Abs were found to be lowest in the F-D-S and M-D-S groups. During the pandemic, large numbers of individuals have experienced depression, which may induce excessive oxidative stress, causing modifications in circulatory proteins. Thus, the formation of neo-antigens is induced, which lead to the generation of autoantibodies. The concomitant effect of increased autoantibodies with elevated levels of IFN-γ and TNF-α possibly tilt the immune balance toward autoantibody generation rather than the formation of S1-RBD-Abs. Thus, it is important to identify individuals who are at risk of depression to determine immune status and facilitate the better management of COVID-19.
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Ziziphus spina-christi L. (ZSC-L) is a tree with thorny branches, belongs to the family Rhamnaceae and grows in the sub-tropics. The purpose of this research is to isolate and partially purify bioactive components from the crude ethanol extract of the stem bark of ZSC-L. Besides, bioassay-guided fractionation of ZSC-L stem bark was conducted using different solvents. The solvents were reutilized to minimize the production cost and environmental harm. In addition, the antimicrobial activities of the fractions were analyzed, followed by metabolic profiling using LC-HRMS. The n-butanol fraction showed the highest antimicrobial efficacy, so it was subjected to further purification. For the first time, two major compounds were isolated from the stem bark of ZSC-L and identified as lupane-type pentacyclic triterpenoids betulinic acid and betulin. Both compounds were used as antibacterial and anticancer agents and considered as a green product as the extraction procedure reduced the use of hazardous chemicals. Metabolic characterization of ZSC-L and its bioactive fractions were performed using LC-HR-ESI-MS and the results revealed the dereplication of 36 compounds belonging to different chemical classes. Flavonoids and triterpenes were the most prominent metabolite classes in the different fractions. The molecular docking results were obtained by studying the interaction of betulin and betulinic acid with antimicrobial receptors (4UYM, 1IYL, 1AJ2, 6J7L, 1AD4, 2VEG) to support the in vitro results. Our study highlights that Ziziphus spina-christi and its phytoconstituents, especially triterpenoids, act as a promising antimicrobial candidate in pharmaceutical and clinical applications.
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Anti-Infecciosos , Triterpenos , Ziziphus , Antibacterianos/análise , Antibacterianos/farmacologia , Anti-Infecciosos/análise , Anti-Infecciosos/farmacologia , Simulação de Acoplamento Molecular , Compostos Fitoquímicos/análise , Compostos Fitoquímicos/farmacologia , Casca de Planta/química , Triterpenos/análise , Triterpenos/farmacologia , Ziziphus/químicaRESUMO
Bacteria evolved to survive in the available environmental chemosphere via several cellular mechanisms. A rich pool of antioxidants and stress regulators plays a significant role in the survival of bacteria in unfavorable environmental conditions. Most of the microbes exhibit resistant phenomena in toxic environment niches. Naturally, bacteria possess efficient thioredoxin reductase, glutaredoxin, and peroxiredoxin redox systems to handle environmental oxidative stress. Further, an array of transcriptional regulators senses the oxidative stress conditions. Transcription regulators, such as OxyR, SoxRS, PerR, UspA, SsrB, MarA, OhrR, SarZ, etc., sense and transduce bacterial oxidative stress responses. The redox-sensitive transcription regulators continuously recycle the utilized antioxidant enzymes during oxidative stress. These regulators promote the expression of antioxidant enzymes such as superoxide dismutase, catalase, and peroxides that overcome oxidative insults. Therefore, the transcriptional regulations maintain steady-state activities of antioxidant enzymes representing the resistance against host cell/environmental oxidative insults. Further, the redox system provides reducing equivalents to synthesize biomolecules, thereby contributing to cellular repair mechanisms. The inactive transcriptional regulators in the undisturbed cells are activated by oxidative stress. The oxidized transcriptional regulators modulate the expression of antioxidant and cellular repair enzymes to survive in extreme environmental conditions. Therefore, targeting these antioxidant systems and response regulators could alter cellular redox homeostasis. This review presents the mechanisms of different redox systems that favor bacterial survival in extreme environmental oxidative stress conditions.
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Antioxidantes , Proteínas de Bactérias , Antioxidantes/metabolismo , Proteínas de Bactérias/metabolismo , Estresse Oxidativo/fisiologia , Oxirredução , Bactérias/genética , Bactérias/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Garlic and ginger are well known as safe alternatives to traditional therapies. Limited information exists regarding antioxidant, antibacterial and antiviral capabilities of the combination of ginger and garlic. Standard methodologies were employed to determine the phytochemical compositions. Antioxidant activities were evaluated through DPPH and FRAP assays. Notably, in DPPH assay, combination of ginger and garlic extracts displayed significantly higher (85.44%, p < 0.005) antioxidant activity even at lower concentrations (6 mg/ml) compared to ginger and garlic extracts alone. Similar findings were observed for FRAP assay. At low concentration of extracts (25 µg/ml), combination of ginger and garlic exhibited significant (p < 0.005) increase in reducing activity (51%) compared to ginger or garlic extracts alone. Significant antibacterial and antiviral activities were exhibited by the combination of both ginger and garlic extracts as compared to ginger and garlic extracts alone. The combined effect of garlic and ginger exhibited a synergistic effect in bacterial and viral growth inhibition. These findings suggest that the diverse phytochemical compositions of the ginger and garlic varieties contribute to their strong antioxidant properties, potentially positioning them as valuable therapeutics for bacterial and viral infections. Further analysis will be required for their widespread utilization and pharmaceutical applications.
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The ribosomal protein S6 kinase beta-1 (RPS6KB1), also known as p70S6 kinase, plays a crucial role in various disease-related conditions such as diabetes, obesity, and cancer. Its activity is regulated by phosphorylation events, including phosphorylation of Threonine 389 in the hydrophobic motif by the mammalian target of rapamycin complex 1 (mTORC1) and phosphorylation of Threonine 229 in the activation loop by PDK1 (phosphoinositide-dependent kinase 1). However, other phenomena connected to RPS6KB1 remain unknown. In this study, we employed virtual screening and molecular docking techniques on the molecules in the ZINC library to identify potential inhibitors. Molecular dynamics (MD) simulations and MMGBSA calculations were carried out on promising compounds to determine their binding affinity and stability. We also evaluated the drug-likeness properties of the selected compounds. A comparative study between the native RPS6KB1 structure, co-crystal ligands, and the shortlisted molecules from the ZINC dataset was carried out. The identified molecules possess significant potential for future in vitro and in vivo studies, paving the way for developing effective cancer treatments.Communicated by Ramaswamy H. Sarma.
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Four new hybrid compounds (H1-H4) bearing pyrazole (S1 and S2) and chalcone (P1 and P2) fragments were synthesized and characterized. Compounds were assayed for their ability to inhibit the proliferation of human lung (A549) and colon (Caco-2) cancer cell lines. Besides, toxicity against normal cells was determined using the human umbilical vein endothelial cells (HUVEC). In silico molecular docking, molecular dynamics (MD) simulation and absorption, distribution, metabolism, excretion, and toxicity (ADMET) studies were carried out to predict the binding modes, protein stability, drug-likeness and toxicity of the reported compounds. The in vitro anticancer activity of the tested compounds revealed dose-dependent cell-specific cytotoxicity. In silico studies revealed that the compounds have a good binding affinity, possess appropriate drug-likeness properties and have low toxicity profiles.Communicated by Ramaswamy H. Sarma.
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Antineoplásicos , Chalcona , Chalconas , Humanos , Simulação de Dinâmica Molecular , Simulação de Acoplamento Molecular , Estrutura Molecular , Relação Estrutura-Atividade , Chalconas/farmacologia , Linhagem Celular Tumoral , Chalcona/farmacologia , Células CACO-2 , Células Endoteliais , Antineoplásicos/química , Desenho de Fármacos , Proliferação de Células , Pirazóis/farmacologia , Pirazóis/químicaRESUMO
Background: Lung cancer is the foremost cause of cancer-related death globally, with non-small cell lung cancer (NSCLC) accounting for 85-90% of cases. Targeted therapy is the most essential therapeutic option for NSCLC, other common treatments include radiation therapy, surgery, chemotherapy, and immunotherapy. Objective: Our study objective was to estimate whether progression-free survival (PFS) is an outcome of NSCLC extracted from 18 randomized control trials (RCTs) with docetaxel as experimental group and antineoplastic agent, kinase inhibitor, and monoclonal antibodies as a control group. Methods: We selected relevant studies published between 2011 and 2022 using Google Scholar, PubMed, Scopus, Science Direct, and Cochrane Library. Advanced NSCLC, chemotherapy, RCT, docetaxel, and second-line treatment were the terms included in the search. A total of 9738 patients were evaluated from the 18 identified studies. We used the meta package of R Studio to perform the meta-analysis. Graphical funnel plots were used to evaluate publication bias visually. Results: Patients who underwent docetaxel-based therapy had a considerably longer PFS than those who got antineoplastic agents, kinase inhibitors, or monoclonal antibodies-based treatment. Patients in the standard treatment arm had a slightly longer PFS than those in the experimental therapy arm in the overall meta-analysis. Conclusion: Docetaxel outperformed monoclonal antibodies, antineoplastic agents, and kinase inhibitors in the second-line therapy of advanced NSCLC since PFS was extensively utilized.
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Forkhead box class O 3a (FOXO3) is a member of the FoxO transcription factor subfamily, which regulates the expression of target genes not only through DNA binding as a transcription factor, but also through protein-protein interaction. Although FoxO3 is a well-known transcription factor involved in diverse biological processes, the role of FoxO3 in cigarette smoke (CS)-induced lung inflammation and injury has not been studied. It is, therefore, hypothesized that deficiency of FoxO3 leads to increased susceptibility to CS-induced lung inflammatory response and airspace enlargement. In this article, we show that the levels of FOXO3 are significantly decreased in lungs of smokers and patients with chronic obstructive pulmonary disease, as well as in lungs of mice exposed to CS. Genetic ablation of FoxO3 led to pulmonary emphysema and exaggerated inflammatory response in lungs of mice exposed to CS. We further showed that CS induced the translocation of FoxO3 into the nucleus where FoxO3 interacted with NF-κB and disrupted NF-κB DNA-binding ability, leading to inhibition of its activity. Targeted disruption of FoxO3 also resulted in downregulation of antioxidant genes in mouse lungs in response to CS exposure. These results suggest that FoxO3 plays a pivotal role in regulation of lung inflammatory response and antioxidant genes, and deficiency of FoxO3 results in development of chronic obstructive pulmonary disease/emphysema.
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Fatores de Transcrição Forkhead/deficiência , Predisposição Genética para Doença , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/patologia , Enfisema Pulmonar/imunologia , Enfisema Pulmonar/patologia , Fumar/imunologia , Fumar/patologia , Animais , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/antagonistas & inibidores , Fatores de Transcrição Forkhead/genética , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Camundongos Transgênicos , Doença Pulmonar Obstrutiva Crônica/genética , Enfisema Pulmonar/genética , Fumar/genéticaRESUMO
Cancer management is highly dependent on the immune status of the patient. During the COVID-19 pandemic, a large number of people suffered from anxiety and depression, especially cancer patients. The effect of depression on breast cancer (BC) and prostate cancer (PC) patients, during the pandemic has been analyzed in this study. Levels of proinflammatory cytokines (IFN-γ, TNF-α, and IL-6) and oxidative stress markers malondialdehyde (MDA) and carbonyl content (CC) were estimated in patients' serum samples. Serum antibodies against in vitro hydroxyl radical (â¢OH) modified pDNA (â¢OH-pDNA-Abs) were estimated using direct binding and inhibition ELISA. Cancer patients showed increased levels of proinflammatory cytokines (IFN-γ, TNF-α, and IL-6) and oxidative stress markers (MDA and CC levels), which were further significantly enhanced in cancer patients with depression compared to normal healthy (NH) individuals. Increased levels of â¢OH-pDNA-Abs were detected in breast cancer (0.506 ± 0.063) and prostate cancer (0.441 ± 0.066) patients compared to NH subjects. Serum antibodies were found to be significantly elevated in BC patients with depression (BCD) (0.698 ± 0.078) and prostate cancer patients with depression (PCD) (0.636 ± 0.058). Inhibition ELISA also exhibited significantly high percent inhibition in BCD (68.8% ± 7.8%) and PCD (62.9% ± 8.3%) subjects compared to BC (48.9% ± 8.1%), and PC (43.4% ± 7.5%) subjects. Cancer is characterized by enhanced oxidative stress and increased inflammation, which may be exaggerated with COVID-19 related depression. High oxidative stress and compromised antioxidant homeostasis exerts alterations in DNA, leading to formation of neo-antigens, subsequently leading to the generation of antibodies. COVID-19 pandemic related depression needs to be addressed globally for improved cancer patient care and cancer disease management.
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This study explains the importance of cellular redox system in preserving the proteome of the radioresistant Deinococcus radiodurans. The thioredoxin reductase (TrxR) redox system was inhibited by ebselen (10 µM), and then the bacterium was exposed to 4 kGy of ionizing radiation. The differentially expressed proteins were analyzed using label-free quantitative (LFQ) proteomics. The 4 kGy radiation treatment increases the expression of stress response proteins like osmotically inducible protein OsmC, catalase, and metallophosphoesterase compared to control. Ebselen plus radiation treatment augments oxidoreductases proteins in D. radiodurans. Further, the proteins involved in glycolysis, tricarboxylic acetic acid (TCA) and proteins like proteases, peptidase, and peptide transporters were significantly decreased in the ebselen plus radiation group compared to radiation treated group. Further, ebselen plus radiation treatment increases the ATP-binding cassette (ABC) transporters involved in the efflux of toxic chemicals and nutrient uptake and the stress response related membrane protein like S-layer homology domain-containing protein in D. radiodurans. Thus, the results show that the altered redox status via inhibition of TrxR redox system significantly affects the expression of essential cellular proteins for the survival. The cellular content of D. radiodurans may be used to handle redox imbalances in the normal cells during cancer radiotherapy. SIGNIFICANCE: Deinococcus radiodurans is a popular radioresistance organism with efficient antioxidant systems and DNA repair mechanisms. There are many antioxidant systems and small molecules that responsible for its resistance. The importance of thiol based antioxidant systems in its resistance property has not fully studied yet. Thioredoxin reductase is an important disulfide containing protein that involved in maintaining redox homeostasis. The TrxR inhibition affects the cell survival and synthesis of molecules against ionizing radiation. In this study we are reporting the effects of TrxR inhibitor on proteome of D. radiodurans upon ionizing radiation. This study reveals the significance of TrxR antioxidant system on the proteome of D. radiodurans. The inhibition of TrxR antioxidant system and the subsequent disturbances in the proteome content makes the organism vulnerable to oxidative stress.
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Deinococcus , Tiorredoxina Dissulfeto Redutase , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Proteínas de Bactérias/metabolismo , Deinococcus/genética , Deinococcus/metabolismo , Deinococcus/efeitos da radiação , Proteínas de Choque Térmico/metabolismo , Proteoma/metabolismo , Proteômica , Radiação Ionizante , Tiorredoxina Dissulfeto Redutase/química , Tiorredoxina Dissulfeto Redutase/genética , Tiorredoxina Dissulfeto Redutase/metabolismoRESUMO
Sirtuin1 (SIRT1) deacetylase levels are decreased in chronic inflammatory conditions and aging where oxidative stress occurs. We determined the mechanism of SIRT1 redox post-translational modifications leading to its degradation. Human lung epithelial cells exposed to hydrogen peroxide (150-250 microM), aldehyde-acrolein (10-30 microM), and cigarette smoke extract (CSE; 0.1-1.5%) in the presence of intracellular glutathione-modulating agents at 1-24 h, and oxidative post-translational modifications were assayed in cells, as well as in lungs of mice lacking and overexpressing glutaredoxin-1 (Glrx1), and wild-type (WT) mice in response to cigarette smoke (CS). CSE and aldehydes dose and time dependently decreased SIRT1 protein levels, with EC(50) of 1% for CSE and 30 microM for acrolein at 6 h, and >80% inhibition at 24 h with CSE, which was regulated by modulation of intracellular thiol status of the cells. CS decreased the lung levels of SIRT1 in WT mice, which was enhanced by deficiency of Glrx1 and prevented by overexpression of Glrx1. Oxidants, aldehydes, and CS induced carbonyl modifications on SIRT1 on cysteine residues concomitant with decreased SIRT1 activity. Proteomics studies revealed alkylation of cysteine residue on SIRT1. Our data suggest that oxidants/aldehydes covalently modify SIRT1, decreasing enzymatic activity and marking the protein for proteasomal degradation, which has implications in inflammatory conditions.
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Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Nicotiana/efeitos adversos , Oxidantes/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Sirtuína 1/metabolismo , Fumaça/efeitos adversos , Acroleína/farmacologia , Animais , Linhagem Celular , Glutarredoxinas/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Immunoblotting , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Carbonilação Proteica/efeitos dos fármacos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
NF-kappaB-mediated proinflammatory response to cigarette smoke (CS) plays a pivotal role in the pathogenesis of chronic obstructive pulmonary disease (COPD). The heterodimer of RelA/p65-p50 (subunits of NF-kappaB) is involved in transactivation of NF-kappaB-dependent genes, but interestingly p50 has no transactivation domain. The endogenous role of p50 subunit, particularly in regulation of CS-mediated inflammation in vivo, is not known. We therefore hypothesized that p50 subunit plays a regulatory role on RelA/p65, and genetic ablation of p50 (p50(-/-)) leads to increased lung inflammation and lung destruction in response to CS exposure in mouse. To test this hypothesis, p50-knockout and wild-type (WT) mice were exposed to CS for 3 days to 6 mo, and inflammatory responses as well as air space enlargement were assessed. Lungs of p50-deficient mice showed augmented proinflammatory response to acute and chronic CS exposures as evidenced by increased inflammatory cell influx and proinflammatory mediators release such as monocyte chemoattractant protein-1 (MCP-1) and interferon-inducible protein-10 (IP-10) compared with WT mice. IKK2 inhibitor (IMD-0354), which reduces the nuclear translocation of RelA/p65, attenuated CS-mediated neutrophil influx in bronchoalveolar lavage fluid and cytokine (MCP-1 and IP-10) levels in lungs of WT but not in p50-deficient mice. Importantly, p50 deficiency resulted in increased phosphorylation (Ser276 and Ser536), acetylation (Lys310), and DNA binding activity of RelA/p65 in mouse lung, associated with increased chromatin remodeling evidenced by specific phosphoacetylation of histone H3 (Ser10/Lys9) and acetylation of H4 (Lys12) in response to CS exposure. Surprisingly, p50-null mice showed spontaneous air space enlargement, which was further increased after CS exposure compared with WT mice. Thus our data showed that p50 endogenously regulates the activity of RelA/p65 by decreasing its phosphoacetylation and DNA binding activity and specific histone modifications and that genetic ablation of p50 leads to air space enlargement in mouse.
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Montagem e Desmontagem da Cromatina , Subunidade p50 de NF-kappa B/metabolismo , Pneumonia , Enfisema Pulmonar , Fumaça/efeitos adversos , Fumar/efeitos adversos , Animais , Marcação de Genes , Humanos , Quinase I-kappa B/antagonistas & inibidores , Proteínas I-kappa B/metabolismo , Pulmão/citologia , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Inibidor de NF-kappaB alfa , Subunidade p50 de NF-kappa B/genética , Neutrófilos/citologia , Neutrófilos/imunologia , Pneumonia/etiologia , Pneumonia/imunologia , Pneumonia/patologia , Enfisema Pulmonar/etiologia , Enfisema Pulmonar/imunologia , Enfisema Pulmonar/patologia , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismoRESUMO
RATIONALE: IkappaB kinase (IKK) activates NF-kappaB which plays a pivotal role in pro-inflammatory response in the lung. NF-kappaB has been shown to be activated in alveolar macrophages and peripheral lungs of smokers and patients with chronic obstructive pulmonary disease. We investigated the anti-inflammatory effect of a highly selective and novel IKKbeta/IKK2 inhibitor, PHA-408 [8-(5-chloro-2-(4-methylpiperazin-1-yl)isonicotinamido)-1-(4-fluorophenyl)-4,5-dihydro-1H-benzo[gamma]indazole-3-carboxamide], in lungs of rat in vivo. METHODS: Adult Sprague-Dawley rats were administered orally with PHA-408 (15 and 45 mg/kg) daily for 3 days and exposed to LPS aerosol (once on day 3, 2 h post-last PHA-408 administration) or cigarette smoke (CS; 2h after PHA-408 administration for 3 days). Animals were sacrificed at 1, 4 and 24 h after the last exposure, and lung inflammatory response and NF-kappaB activation were measured. RESULTS: Oral administration of IKKbeta/IKK2 inhibitor PHA-408 significantly inhibited LPS- and CS-mediated neutrophil influx in bronchoalveolar lavage (BAL) fluid of rats. The levels of pro-inflammatory mediators in BAL fluid (CINC-1) and lungs (IL-6, TNF-alpha, IL-1beta and GM-CSF) were also reduced by PHA-408 administration in response to LPS or CS exposures. The reduced pro-inflammatory response in PHA-408-administered rats was associated with decreased nuclear translocation and DNA binding activity of NF-kappaB in response to LPS or CS. CONCLUSION: These results suggest that IKKbeta/IKK2 inhibitor PHA-408 is a powerful anti-inflammatory agent against LPS- and CS-mediated lung inflammation.
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Quinase I-kappa B/antagonistas & inibidores , Indazóis/farmacologia , Ácidos Isonicotínicos/farmacologia , Lipopolissacarídeos/efeitos adversos , Pulmão/efeitos dos fármacos , Fumar/efeitos adversos , Animais , Líquido da Lavagem Broncoalveolar/imunologia , Relação Dose-Resposta a Droga , Mediadores da Inflamação/metabolismo , Masculino , NF-kappa B/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Zizyphus spina-christi (Rhamnaceae family) is an edible plant used in folk medicine. Therefore, it is of interest to report the cytotoxic effects of Z. spina-christi bark crude extract on human cell lines. Crude ethanol extract of Z. spina-christi bark was fractionated with increasing polarity (diethyl ether, chloroform, ethyl acetate and butanol fractions). The fractions were examined for their cytotoxicity against human colon cancer (HCT-116 and CACO-2), cervical cancer (HeLa and HEp-2), lung carcinoma (A-549), hepatocellular carcinoma (HepG-2), breast cancer (MCF-7) and prostate cancer (PC-3) cell lines using viability assay. Diethyl ether fraction of Z. spina-christi showed the highest cytotoxic effects among the four extracts of Z. spina-christi. The IC50 of diethyl ether fraction was 7.14, 11.2, 11.6, 15.4, 39.8, 42.2, 84.2 and 153.8 µg/ml on HepG-2, A-549, CACO-2, HCT-116, MCF-7, PC-3, HeLa, and HEp-2 cell lines, respectively. Data shows that the diethyl ether fraction of Z. spina-christi showed effective cytotoxic effects in colon, lung and hepatocellular cancer cell lines.
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The activation of transcription factor NF-kappaB is controlled by two main pathways: the classical canonical (RelA/p65-p50)- and the alternative noncanonical (RelB/p52)-NF-kappaB pathways. RelB has been shown to play a protective role in RelA/p65-mediated proinflammatory cytokine release in immune-inflammatory lymphoid cells. Increased infiltration of macrophages and lymphoid cells occurs in lungs of patients with chronic obstructive pulmonary disease, leading to abnormal inflammation. We hypothesized that RelB, and its signaling pathway, is differentially regulated in macrophages and B cells and in lung cells, leading to differential regulation of proinflammatory cytokines in response to cigarette smoke (CS). CS exposure increased the levels of RelB and NF-kappaB-inducing kinase associated with recruitment of RelB on promoters of the IL-6 and macrophage inflammatory protein-2 genes in mouse lung. Treatment of macrophage cell line, MonoMac6, with CS extract showed activation of RelB. In contrast, RelB was degraded by a proteasome-dependent mechanism in B lymphocytes (human Ramos, mouse WEHI-231, and primary mouse spleen B cells), suggesting that RelB is differentially regulated in lung inflammatory and lymphoid cells in response to CS exposure. Transient transfection of dominant negative IkappaB-kinase-alpha and double mutants of NF-kappaB-inducing kinase partially attenuated the CS extract-mediated loss of RelB in B cells and normalized the increased RelB level in macrophages. Taken together, these data suggest that RelB is differentially regulated in response to CS exposure in macrophages, B cells, and in lung cells by IkappaB-kinase-alpha-dependent mechanism. Rapid degradation of RelB signals for RelA/p65 activation and loss of its protective ability to suppress the proinflammatory cytokine release in lymphoid B cells.
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Linfócitos B/metabolismo , Quinase I-kappa B/metabolismo , Pulmão/metabolismo , Fumar/efeitos adversos , Fator de Transcrição RelB/metabolismo , Animais , Linfócitos T CD4-Positivos/metabolismo , Ligante de CD40/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Regulação da Expressão Gênica , Humanos , Interleucina-1beta/metabolismo , Linfotoxina-beta/metabolismo , Macrófagos Alveolares/metabolismo , Masculino , Camundongos , Infiltração de Neutrófilos , Neutrófilos/metabolismo , Pneumonia/etiologia , Pneumonia/metabolismo , Doença Pulmonar Obstrutiva Crônica/etiologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Transdução de Sinais , Fator de Transcrição RelA/metabolismoRESUMO
Cigarette smoke (CS) induces recruitment of inflammatory cells in the lungs leading to the generation of reactive oxygen species (ROS), which are involved in lung inflammation and injury. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a multimeric system that is responsible for ROS production in mammalian cells. We hypothesized that NADPH oxidase-derived ROS play an important role in lung inflammation and injury and that targeted ablation of components of NADPH oxidase (p47(phox) and gp91(phox)) would protect lungs against the detrimental effects of CS. To test this hypothesis, we exposed p47(phox-/-) and gp91(phox-/-) mice to CS and examined inflammatory response and injury in the lung. Surprisingly, although CS-induced ROS production was decreased in the lungs of p47(phox-/-) and gp91(phox-/-) mice compared with wild-type mice, the inflammatory response was significantly increased and was accompanied by development of distal airspace enlargement and alveolar destruction. This pathological abnormality was associated with enhanced activation of the TLR4-nuclear factor-kappaB pathway in response to CS exposure in p47(phox-/-) and gp91(phox-/-) mice. This phenomenon was confirmed by in vitro studies in which treatment of peritoneal macrophages with a nuclear factor-kappaB inhibitor reversed the CS-induced release of proinflammatory mediators. Thus, these data suggest that genetic ablation of components of NADPH oxidase enhances susceptibility to the proinflammatory effects of CS leading to airspace enlargement and alveolar damage.
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Glicoproteínas de Membrana/fisiologia , NADPH Oxidases/fisiologia , Nicotiana , Pneumonia/metabolismo , Enfisema Pulmonar/metabolismo , Fumaça/efeitos adversos , 8-Hidroxi-2'-Desoxiguanosina , Animais , Células Cultivadas , Desoxiguanosina/análogos & derivados , Desoxiguanosina/biossíntese , Peroxidação de Lipídeos , Pulmão/metabolismo , Pulmão/patologia , Macrófagos Peritoneais/metabolismo , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidase 2 , NADPH Oxidases/genética , NF-kappa B/metabolismo , Pneumonia/etiologia , Pneumonia/patologia , Enfisema Pulmonar/etiologia , Enfisema Pulmonar/patologia , Espécies Reativas de Oxigênio/metabolismo , Receptor 4 Toll-Like/metabolismoRESUMO
Vascular endothelial growth factor (VEGF) induces phosphorylation of VEGF receptor-2 (VEGFR-2) and activates the downstream signaling pathway resulting in endothelial cell migration, proliferation, and survival. Cigarette smoking is associated with abnormal vascular and endothelial function, leading to airspace enlargement. Herein, we investigated the mechanism of cigarette smoke (CS) -induced endothelial dysfunction by studying the VEGF-VEGFR-2 signaling in mouse lung and human endothelial cells. CS exposure caused oxidative stress, as shown by increased levels of 4-hydroxy-2-nonenal-adducts in mouse lung and reactive oxygen species generation in human lung microvascular endothelial cells (HMVEC-Ls). Inhibition of VEGFR-2 by a specific kinase inhibitor (NVP-AAD777) enhanced the CS-induced oxidative stress, causing augmented inflammatory cell influx and proinflammatory mediators release in mouse lung. The levels of endothelial nitric oxide synthase (eNOS) and phosphorylated (p) -eNOS in the lungs of mice exposed to CS and/or treated with VEGFR-2 inhibitor were decreased. CS down-regulated VEGFR-2 expression, eNOS levels, and VEGF-induced VEGFR-2 phosphorylation in HMVEC-Ls, resulting in impaired VEGF-induced endothelial cell migration and angiogenesis. Overall, these data show that inhibition of VEGFR-2 augmented CS-induced oxidative stress and inflammatory responses leading to endothelial dysfunction. This explains the mechanism of endothelial dysfunction in smokers and has implications in understanding the pathogenesis of pulmonary and cardiovascular diseases.
Assuntos
Endotélio Vascular/fisiopatologia , Inflamação/fisiopatologia , Estresse Oxidativo/fisiologia , Fumar/fisiopatologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Líquido da Lavagem Broncoalveolar , Modelos Animais de Doenças , Endotélio Vascular/patologia , Imuno-Histoquímica , Inflamação/patologia , Pulmão/patologia , Pulmão/fisiopatologia , Camundongos , Óxido Nítrico Sintase Tipo III/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
RATIONALE: Abnormal inflammation and accelerated decline in lung function occur in patients with chronic obstructive pulmonary disease (COPD). Human sirtuin (SIRT1), an antiaging and antiinflammatory protein, is a metabolic NAD(+)-dependent protein/histone deacetylase that regulates proinflammatory mediators by deacetylating histone and nonhistone proteins. OBJECTIVES: To determine the expression of SIRT1 in lungs of smokers and patients with COPD, and to elucidate the regulation of SIRT1 in response to cigarette smoke in macrophages, and its impact on nuclear factor (NF)-kappaB regulation. METHODS: SIRT1 and NF-kappaB levels were assessed in lung samples of nonsmokers, smokers, and patients with COPD. Human monocyte-macrophage cells (MonoMac6) were treated with cigarette smoke extract (CSE) to determine the mechanism of CSE-mediated regulation of SIRT1 and its involvement in RelA/p65 regulation and IL-8 release. MEASUREMENTS AND MAIN RESULTS: Peripheral lungs of smokers and patients with COPD showed decreased levels of nuclear SIRT1, as compared with nonsmokers, associated with its post-translational modifications (formation of nitrotyrosine and aldehyde carbonyl adducts). Treatment of MonoMac6 cells with CSE showed decreased levels of SIRT1 associated with increased acetylation of RelA/p65 NF-kappaB. Mutation or knockdown of SIRT1 resulted in increased acetylation of nuclear RelA/p65 and IL-8 release, whereas overexpression of SIRT1 decreased IL-8 release in response to CSE treatment in MonoMac6 cells. CONCLUSIONS: SIRT1 levels were reduced in macrophages and lungs of smokers and patients with COPD due to its post-translational modifications by cigarette smoke-derived reactive components, leading to increased acetylation of RelA/p65. Thus, SIRT1 plays a pivotal role in regulation of NF-kappaB-dependent proinflammatory mediators in lungs of smokers and patients with COPD.
Assuntos
Acetilação/efeitos dos fármacos , Macrófagos/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/metabolismo , Sirtuínas/metabolismo , Fumar/efeitos adversos , Adulto , Idoso , Linhagem Celular Tumoral , Feminino , Humanos , Macrófagos/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/enzimologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Sirtuína 1 , Fumaça/efeitos adversos , Fumaça/análise , Fumar/fisiopatologia , Nicotiana/efeitos adversos , Nicotiana/químicaRESUMO
Chromatin modifications and epigenetic regulation are critical for sustained and abnormal inflammatory response seen in lungs of patients with chronic obstructive pulmonary disease (COPD) because the activities of enzymes that regulate these epigenetic modifications are altered in response to cigarette smoke. Cigarette smoke induces chromatin modifications and epigenetic changes by causing post-translational modifications of histone acetyltransferases, and histone/non-histone deacetylases (HDACs), such as HDAC2 and sirtuin 1 (SIRT1), which leads to chromatin remodeling. In this review, we discussed the current knowledge on cigarette smoke/oxidants-induced post-translational modifications of deacetylases (HDAC2 and SIRT1), disruption of HDAC2/SIRT1-RelA/p65 corepressor complex associated with acetylation of RelA/p65, and chromatin modifications (histone H3 phospho-acetylation) leading to sustained pro-inflammatory gene transcription. Knowledge on molecular mechanisms of epigenetic changes in abnormal lung inflammation will help in understanding the pathophysiology of COPD which may lead to the development of novel epigenetic therapies in the near future.
Assuntos
Cromatina/metabolismo , Histona Desacetilases/metabolismo , Doença Pulmonar Obstrutiva Crônica/enzimologia , Epigênese Genética , Histona Desacetilase 2/metabolismo , Humanos , Inflamação/metabolismo , Oxirredução , Estresse Oxidativo , Fosforilação , Doença Pulmonar Obstrutiva Crônica/etiologia , Doença Pulmonar Obstrutiva Crônica/genética , Sirtuína 1/metabolismo , Fumar/efeitos adversosRESUMO
The cyclin-dependent kinase inhibitor p21(CIP1/WAF1/SDI1) (p21) is an important inhibitory checkpoint regulator of cell cycle progression in response to oxidative and genotoxic stresses. It is known that p21 potentiates inflammatory response and inhibits apoptosis and proliferation, leading to cellular senescence. However, the role of endogenous p21 in regulation of lung inflammatory and injurious responses by cigarette smoke (CS) or other pro-inflammatory stimuli is not known. We hypothesized that p21 is an important modifier of lung inflammation and injury, and genetic ablation of p21 will confer protection against CS and other pro-inflammatory stimuli (lipopolysacchride [LPS] and N-formyl-methionyl-leucyl-phenylalanine [fMLP])-mediated lung inflammation and injury. To test this hypothesis, p21-deficient (p21-/-) and wild-type mice were exposed to CS, LPS, or fMLP, and the lung oxidative stress and inflammatory responses as well as airspace enlargement were assessed. We found that targeted disruption of p21 attenuated CS-, LPS-, or fMLP-mediated lung inflammatory responses in mice. CS-mediated oxidative stress and fMLP-induced airspace enlargement were also decreased in lungs of p21-/- mice compared with wild-type mice. The mechanism underlying this finding was associated with decreased NF-kappaB activation, and reactive oxygen species generation by decreased phosphorylation of p47(phox) and down-modulating the activation of p21-activated kinase. Our data provide insight into the mechanism of pro-inflammatory effect of p21, and the loss of p21 protects against lung oxidative and inflammatory responses, and airspace enlargement in response to multiple pro-inflammatory stimuli. These data may have ramifications in CS-induced senescence in the pathogenesis of chronic obstructive pulmonary disease/emphysema.