RESUMO
RESUMEN: Las bacteriocinas son péptidos antimicrobianos de síntesis ribosomal secretadas por bacterias. Dentro de estas destaca nisina que posee potenciales usos en terapias antibióticas, como biopreservante de alimentos y probióticos. También se ha descrito que nisina posee citotoxicidad sobre líneas celulares neoplásicas, pero existe poca información de su efecto sobre células tumorales sanguíneas. Debido al potencial uso que presenta nisina, es relevante determinar la toxicidad que presenta sobre líneas celulares tumorales del tipo sanguíneo. Para esto, se realizaron ensayos de actividad hemolítica sobre eritrocitos humanos y de toxicidad sobre células mononucleares de sangre periférica humanas, determinándose que nisina no posee efecto citotóxico sobre este tipo de células normales humanas sanguíneas. Se realizaron también, ensayos de citotoxicidad con líneas celulares tumorales (K562 y U937), con el fin de determinar dosis, tiempo de exposición y selectividad en el efecto tóxico de nisina sobre las células tumorales humanas. Estos ensayos muestran que nisina presenta actividad citotóxica sobre líneas celulares K562 y U937 a las 72 h de exposición, a una concentración de 40 µg/mL, que corresponde a 100 veces la concentración mínima inhibitoria (MIC) usada para su acción sobre bacterias. Al comparar el efecto de nisina sobre células mononucleares de sangre periférica humanas con las líneas tumorales linfoides y mieloides (K562 y U937 respectivamente), se observa un efecto selectivo de nisina sobre las células tumorales sanguíneas.
SUMMARY: Bacteriocins are antimicrobial peptides of ribosomal synthesis secreted by bacteria. Among these, nisin stands out, which has potential uses in antibiotic therapies, as a food bio preservative and probiotics. Nisin has also been reported to have cytotoxicity on neoplastic cell lines, but there is little information on its effect on blood tumor cells. Due to the potential use that nisin presents, it is relevant to determine the toxicity it presents on tumor cell lines of the blood type. For this, hemolytic activity tests were carried out on human erythrocytes and toxicity on human peripheral blood mononuclear cells, determining that nisin does not have a toxic effect on this type of normal human blood cells. Cytotoxicity tests were also carried out with tumor cell lines (K562 and U937), to determine dose, exposure time and selectivity in the toxic effect of nisin on human tumor cells. These tests show that nisin shows cytotoxic activity on K562 and U937 cell lines at 72 h of exposure, at a concentration of 40 µg / mL, which corresponds to 100 times the minimum inhibitory concentration (MIC) used for its action on bacteria. When comparing the effect of nisin on human peripheral blood mononuclear cells with lymphoid and myeloid tumor lines (K562 and U937 respectively), a selective effect of nisin on blood tumor cells is observed.
Assuntos
Humanos , Linhagem Celular Tumoral/efeitos dos fármacos , Antibacterianos/farmacologia , Nisina/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Bacteriocinas/farmacologia , Técnicas In Vitro , Testes de Sensibilidade Microbiana , Sobrevivência Celular/efeitos dos fármacos , Células K562/efeitos dos fármacos , Células U937/efeitos dos fármacosRESUMO
Acute ischemic stroke is a challenging disease that threatens the life of older people. Dysfunction of brain endothelial cells is reported to be involved in the pathogenesis of acute ischemic stroke. Ramelteon is a novel agonist of melatonin receptor developed for the treatment of insomnia. Recently, the promising protective effect of Ramelteon on brain injury has been widely reported. The present study aims to investigate the protective effect of Ramelteon against free fatty acid (FFA)-induced damages in brain vascular endothelial cells and the underlying mechanism. Firstly, we discovered that Ramelteon administration remarkably reversed the decreased cell viability, increased LDH release, activated oxidative stress, and excessive released inflammatory factors caused by FFAs. Secondly, Ramelteon extensively suppressed the attachment of U937 monocytes to bEnd.3 brain endothelial cells induced by FFAs. In addition, the elevated expression of E-selectin and the reduced expression of KLF2 induced by FFAs were pronouncedly alleviated by Ramelteon. Lastly, silencing of KLF2 abolished the protective effects of Ramelteon against FFA-induced expression of E-selectin and the attachment of U937 monocytes to bEnd.3 brain endothelial cells. In conclusion, Ramelteon mitigated FFA-induced attachment of monocytes to brain vascular endothelial cells by increasing the expression of KLF2 and reducing the expression of E-selectin.
Assuntos
Encéfalo/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Ácidos Graxos não Esterificados/metabolismo , Indenos/farmacologia , Monócitos/efeitos dos fármacos , Western Blotting , Encéfalo/patologia , Morte Celular/efeitos dos fármacos , Células Endoteliais/patologia , Ensaio de Imunoadsorção Enzimática , Ácidos Graxos não Esterificados/antagonistas & inibidores , Humanos , L-Lactato Desidrogenase/metabolismo , Monócitos/patologia , Reação em Cadeia da Polimerase em Tempo Real , Células U937/efeitos dos fármacosRESUMO
Although the medical application of betulin has been presented in previous studies, the potential mechanism of the anti-inflammatory action of betulin should be further investigated. This work aims to confirm the hypothesis that betulin has dexamethasone-like anti-inflammatory action through glucocorticoid receptor (GR)-mediated pathway. Firstly, the binding ability of betulin with GR was measured by a fluorescence polarization-based competitive binding assay, with the IC50 value of 79.18 ± 0.30 mM. Betulin could bind to GR and then induced GR nuclear translocation, but lacked GR transcriptional activity in HeLa cells. Hence, betulin exhibited the potential to be a dissociated modulator for GR, with the loss of glucocorticoid response element (GRE)-associated side effects. In addition, betulin downregulated GRE-driven protein expression of G6P involved in gluconeogenesis, namely side effect. The results of pro-inflammatory cytokines analysis showed that betulin exerted anti-inflammatory action in vitro. Both of the hydrophobic and hydrogen-bonding interactions stabilized the binding between betulin and GR during the simulation process. In conclusion, betulin might be a potential dissociated GR modulator with a reduced side effect profile yet keeping its anti-inflammatory action.
Assuntos
Anti-Inflamatórios/farmacologia , Receptores de Glucocorticoides/efeitos dos fármacos , Triterpenos/farmacologia , Sítios de Ligação , Regulação para Baixo , Gluconeogênese/efeitos dos fármacos , Células HeLa/efeitos dos fármacos , Células Hep G2/efeitos dos fármacos , Humanos , Interleucina-1beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Células U937/efeitos dos fármacosRESUMO
Monocytes and macrophages are involved in a wide range of biological processes and parasitic diseases. The characterization of the molecular mechanisms governing such processes usually requires precise control of the expression of genes of interest. We implemented a tetracycline-controlled gene expression system in the U937 cell line, one of the most used in vitro models for the research of human monocytes and macrophages. Here we characterized U937-derived cell lines in terms of phenotypic (morphology and marker expression) and functional (capacity for phagocytosis and for Leishmania parasite hosting) changes induced by phorbol-12-myristate-13-acetate (PMA). Finally, we provide evidence of tetracycline-inducible and reversible Lamin-A gene silencing of the PMA-differentiated U937-derived cells.
Assuntos
Antibacterianos/administração & dosagem , Expressão Gênica , Marcadores Genéticos , Leishmania braziliensis/fisiologia , Fagocitose , Fenótipo , Tetraciclina/administração & dosagem , Técnicas de Cultura de Células/métodos , Humanos , Células U937/citologia , Células U937/efeitos dos fármacosRESUMO
AIMS: Berberine (BBR) is reported to induce apoptosis and inhibit migration of leukemic cells, but the underlying pharmacological mechanisms have not been fully revealed. This study aims to investigate the possible mechanisms from the perspective of autophagy. MAIN METHODS: P-53-null leukemic cell lines Jurkat and U937 were used for the in vitro study. MDC staining was used for observation of autophagy in leukemic cells, and Western blot analysis was for determination of the expression levels of autophagy-associated proteins. Apoptosis of the leukemic cells was detected by flow cytometry. Cellular location of MDM2 was observed with immunofluorescence staining. Ubiquitination of MDM2 was assessed by immunoprecipitation. Male 6-8-week-old NOD/SCID mice were used for evaluating the effect of BBR on chemotherapy sensitivity in vivo. KEY FINDINGS: BBR induced autophagy in p53-null leukemic cells, which was inhibited by autophagy inhibitors 3-methyladenine. 3-methyladenine also inhibited BBR-induced apoptosis in leukemic cells. In addition, BBR not only decreased MDM2 mRNA expression, but also enhanced MDM2 self-ubiquitination in leukemic cells. Forced overexpression of MDM2 reversed the effect of BBR on autophagy and apoptosis. Furthermore, BBR promoted doxorubicin-induced autophagy and cell death in the leukemic cells and overexpression of MDM2 suppressed these effects. In vivo, BBR combined with doxorubicin achieved better therapeutic effect than doxorubicin alone. SIGNIFICANCE: MDM2 inhibits autophagy and apoptosis in leukemic cells in a p53-independent manner. BBR induces autophagy in p53-null leukemic cells through downregulating MDM2 expression at both transcriptional and post-transcriptional levels, which may contribute to the anti-cancer effect of BBR in leukemia.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Berberina/farmacologia , Células Jurkat/efeitos dos fármacos , Leucemia Experimental/tratamento farmacológico , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Células U937/efeitos dos fármacos , Animais , Western Blotting , Citometria de Fluxo , Imunofluorescência , Humanos , Células Jurkat/metabolismo , Leucemia Experimental/metabolismo , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Reação em Cadeia da Polimerase em Tempo Real , Células U937/metabolismo , UbiquitinaçãoRESUMO
The use of herbs with medicinal value and biomedical effects has increased tremendously in the last years. However, inadequate basic knowledge of their mode of action is the main issue related to phytotherapy, although they have shown promising potential. To provide insights into these important issues, we tested here on appropriate in vitro models the efficacy of Angelica archangelica essential oil (Aa-EO) for anti-inflammatory properties. The results demonstrated that Aa-EO induced significant apoptosis and necrosis at high doses in U937 cells. We used nontoxic concentrations to treat for anti-inflammatory capacity. The results also demonstrated a decreased proinflammatory cytokine interleukin-6 level in human umbilical vein endothelial cells, as senescence in vitro model, when cells are challenged with lipopolysaccharide (LPS), one of the most powerful proinflammatory inducer in the presence of Aa-EO. In addition, down expression of miR-126 and miR-146a (inflammamirs) produced by LPS stimulation was reverted by Aa-EO simultaneous treatment. These results provide noteworthy basis for the development/formulation of new drugs for future clinical uses and new food products or dietary supplements for contrasting inflammation.
Assuntos
Angelica archangelica , Anti-Inflamatórios/uso terapêutico , Inflamação/tratamento farmacológico , Fitoterapia , Óleos de Plantas/uso terapêutico , Células U937/efeitos dos fármacos , Anti-Inflamatórios/farmacologia , Apoptose/efeitos dos fármacos , Humanos , Interleucina-6/metabolismo , Óleos de Plantas/farmacologia , Células U937/metabolismoRESUMO
Lauroyltransferase gene (lpxL), Myristoyltransferase gene (lpxM) and palmitoyltransferase gene (crcA) of Escherichia coli BL21 were independently disrupted by the insertional mutations. The knockout mutant of two transferase genes (lpxL and crcA) produced lipid A with no lauric or palmitic acids and only a little amount of myristic acid. The mutant was susceptible to polymyxin B, but showed comparable growth with the wild-type strain at 30°C. The palmitoyltransferase gene from E. coli (crcA) or Salmonella (pagP) was amplified by PCR, cloned in pUC119, and transferred into the double-knockout mutant by transformation. The transformant contained palmitic acid in the lipid A, and recovered resistance to polymyxin B. Mass spectrometric analysis revealed that palmitic acid was linked to the hydroxyl group of 3-hydroxymyristic acid at C-2 position of proximal (reducing-end) glucosamine. LPS from the double-knockout mutant showed reduced IL-6-inducing activity to macrophage-like line cells compared to that of the wild-type strain, and the activity was only slightly restored by the introduction of palmitic acid to the lipid A. These results suggested that the introduction of one palmitic acid was enough to recover the integrity of the outer membrane, but not enough for the stimulation of macrophages.
Assuntos
Aciltransferases/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Lipídeo A/química , Lipídeo A/genética , Lipídeo A/metabolismo , Animais , Proteínas de Bactérias/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/metabolismo , Técnicas de Inativação de Genes , Humanos , Interleucina-6/metabolismo , Ácidos Láuricos/metabolismo , Macrófagos/metabolismo , Camundongos , Testes de Sensibilidade Microbiana , Mutação , Ácido Mirístico/metabolismo , Ácidos Mirísticos/química , Ácidos Palmíticos/metabolismo , Polimixina B/farmacologia , Células RAW 264.7/efeitos dos fármacos , Salmonella/genética , Células U937/efeitos dos fármacosRESUMO
Magnoflorine, a major bioactive metabolite isolated from Tinospora crispa, has been reported for its diverse biochemical and pharmacological properties. However, there is little report on its underlying mechanisms of action on immune responses, particularly on macrophage activation. In this study, we aimed to investigate the effects of magnoflorine, isolated from T. crispa on the pro-inflammatory mediators generation induced by LPS and the concomitant NF-κB, MAPKs, and PI3K-Akt signaling pathways in U937 macrophages. Differentiated U937 macrophages were treated with magnoflorine and the release of pro-inflammatory mediators was evaluated through ELISA, while the relative mRNA expression of the respective mediators was quantified through qRT-PCR. Correspondingly, western blotting was executed to observe the modulatory effects of magnoflorine on the expression of various markers related to NF-κB, MAPK and PI3K-Akt signaling activation in LPS-primed U937 macrophages. Magnoflorine significantly enhanced the upregulation of TNF-α, IL-1ß, and PGE2 production as well as COX-2 protein expression. Successively, magnoflorine prompted the mRNA transcription level of these pro-inflammatory mediators. Magnoflorine enhanced the NF-κB activation by prompting p65, IκBα, and IKKα/ß phosphorylation as well as IκBα degradation. Besides, magnoflorine treatments concentration-dependently augmented the phosphorylation of JNK, ERK, and p38 MAPKs as well as Akt. The immunoaugmenting effects were further confirmed by investigating the effects of magnoflorine on specific inhibitors, where the treatment with specific inhibitors of NF-κB, MAPKs, and PI3K-Akt proficiently blocked the magnoflorine-triggered TNF-α release and COX-2 expression. Magnoflorine furthermore enhanced the MyD88 and TLR4 upregulation. The results suggest that magnoflorine has high potential on augmenting immune responses.
Assuntos
Aporfinas/farmacologia , Fatores Imunológicos/farmacologia , Inflamação/induzido quimicamente , Lipopolissacarídeos/farmacologia , Fator 88 de Diferenciação Mieloide/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células U937/efeitos dos fármacos , Citocinas/metabolismo , Dinoprostona/metabolismo , Humanos , Immunoblotting , Reação em Cadeia da Polimerase em Tempo Real , Células U937/fisiologiaRESUMO
Hydroquinone (1,4-benzenediol; HQ), a major marrow metabolite of the leukemogen benzene, has been proven to evoke benzene-related hematological disorders and myelotoxicity in vitro and in vivo. The goal of the present study was to explore the role of FOXP3 in HQ-induced malignant progression of U937 human leukemia cells. U937 cells were treated with 5 µM HQ for 24 h, and the cells were re-suspended in serum-containing medium without HQ for 2 days. The same procedure was repeated three times, and the resulting U937/HQ cells were maintained in cultured medium containing 5 µM HQ. Proliferation and colony formation of U937/HQ cells were notably higher than those of U937 cells. Ten-eleven translocation methylcytosine dioxygenase-mediated demethylation of the Treg-specific demethylated region in FOXP3 gene resulted in higher FOXP3 expression in U937/HQ cells than in U937 cells. FOXP3-induced miR-183 expression reduced ß-TrCP mRNA stability and suppressed ß-TrCP-mediated Sp1 degradation, leading to up-regulation of Sp1 expression in U937/HQ cells. Sp1 up-regulation further increased ADAM17 and Lyn expression, and ADAM17 up-regulation stimulated Lyn activation in U937/HQ cells. Moreover, U937/HQ cells showed higher Lyn-mediated Akt activation and cytoplasmic p21 expression than U937 cells did. Abolishment of Akt activation decreased cytoplasmic p21 expression in U937/HQ cells. Suppression of FOXP3, ADAM17, and Lyn expression, as well as Akt inactivation, repressed proliferation and clonogenicity of U937/HQ cells. Together with the finding that cytoplasmic p21 shows anti-apoptotic and oncogenic activities in cancer cells, the present data suggest a role of FOXP3/ADAM17/Lyn/Akt/p21 signaling axis in HQ-induced hematological disorders.
Assuntos
Proteína ADAM17/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Hidroquinonas/toxicidade , Quinases da Família src/metabolismo , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Fatores de Transcrição Forkhead/genética , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia/induzido quimicamente , Leucemia/metabolismo , Leucemia/patologia , Metilação , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição Sp1/metabolismo , Células U937/efeitos dos fármacos , Células U937/metabolismoRESUMO
Kampo formulations used in Japan to treat a wide variety of diseases and to promote health are composed of mixtures of crude extracts from the roots, bark, leaves, and rhizomes of a number of herbs. The present study was aimed at identifying the beneficial biological properties of Daiokanzoto (TJ-84), a Kampo formulation composed of crude extracts of Rhubarb rhizomes and Glycyrrhiza roots, with a view to using it as a potential treatment for periodontal disease. Daiokanzoto dose-dependently inhibited the expression of major Porphyromonas gingivalis virulence factors involved in host colonization and tissue destruction. More specifically, Daiokanzoto reduced the expression of the fimA, hagA, rgpA, and rgpB genes, as determined by quantitative real-time PCR. The U937-3xκB-LUC monocyte cell line transfected with a luciferase reporter gene was used to evaluate the anti-inflammatory properties of Daiokanzoto. Daiokanzoto attenuated the P. gingivalis-mediated activation of the NF-κB signaling pathway. It also reduced the secretion of pro-inflammatory cytokines (IL-6 and CXCL8) by lipopolysaccharide-stimulated oral epithelial cells and gingival fibroblasts. Lastly, Daiokanzoto, dose-dependently inhibited the catalytic activity of matrix metalloproteinases (-1 and -9). In conclusion, the present study provided evidence that Daiokanzoto shows potential for treating and/or preventing periodontal disease. The ability of this Kampo formulation to act on both bacterial pathogens and the host inflammatory response, the two etiological components of periodontal disease, is of high therapeutic interest.
Assuntos
Antibacterianos/farmacologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Glycyrrhiza/química , Medicina Kampo/métodos , Extratos Vegetais/uso terapêutico , Raízes de Plantas/química , Porphyromonas gingivalis/efeitos dos fármacos , Rheum/química , Antibacterianos/isolamento & purificação , Anti-Inflamatórios/isolamento & purificação , Anti-Inflamatórios/farmacologia , Relação Dose-Resposta a Droga , Glycyrrhiza uralensis , Humanos , Metaloproteinase 9 da Matriz/metabolismo , NF-kappa B/metabolismo , Extratos Vegetais/farmacologia , Porphyromonas gingivalis/metabolismo , Porphyromonas gingivalis/patogenicidade , Inibidores de Proteases/isolamento & purificação , Inibidores de Proteases/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Rhus , Transdução de Sinais/efeitos dos fármacos , Células U937/efeitos dos fármacos , Células U937/metabolismo , Fatores de Virulência/biossínteseRESUMO
BACKGROUND AND OBJECTIVE: Increasing evidence suggests that 1,25-dihydroxyvitamin D3 (1,25(OH)2 D3 ), a fat-soluble secosteroid hormone, has a positive impact on periodontal health through diverse mechanisms. The present study was aimed at investigating the effect of 1,25(OH)2 D3 on the growth of and virulence factor gene expression by the periodontopathogenic bacterium Porphyromonas gingivalis. The effect of 1,25(OH)2 D3 on P. gingivalis-mediated activation of nuclear factor kappa B (NF-κB) transcription factor in monocytes was also assessed. MATERIAL AND METHODS: A broth microdilution assay was used to determine the antibacterial activity of 1,25(OH)2 D3 . The modulation of virulence factor gene expression in P. gingivalis was assessed by quantitative reverse transcription-polymerase chain reaction. NF-κB activation was assessed using a human monocytic cell line stably transfected with a luciferase reporter containing NF-κB binding sites. RESULTS: Minimal inhibitory concentrations of 1,25(OH)2 D3 against P. gingivalis ranged from 3.125 to 6.25 µg/mL. Moreover, a partial synergistic effect was observed when 1,25(OH)2 D3 was used in association with metronidazole. 1,25(OH)2 D3 attenuated the virulence of P. gingivalis by reducing the expression of genes coding for important virulence factors, including adhesins (fimA, hagA and hagB) and proteinases (rgpA, rgpB and kgp). 1,25(OH)2 D3 dose-dependently prevented P. gingivalis-induced NF-κB activation in a monocyte model. CONCLUSION: Our study suggested that 1,25(OH)2 D3 selectively inhibits the growth of and virulence factor gene expression by P. gingivalis, in addition to attenuating NF-κB activation by this periodontopathogen. This dual action on P. gingivalis and the inflammatory response of host cells may be of particular interest with a view to developing a novel and inexpensive preventive/therapeutic strategy.
Assuntos
Expressão Gênica/efeitos dos fármacos , Monócitos/metabolismo , NF-kappa B/efeitos dos fármacos , Porphyromonas gingivalis/efeitos dos fármacos , Porphyromonas gingivalis/genética , Fatores de Transcrição/efeitos dos fármacos , Fatores de Virulência/genética , Vitamina D/antagonistas & inibidores , Adesinas Bacterianas/efeitos dos fármacos , Adesinas Bacterianas/genética , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/genética , Linhagem Celular Tumoral/efeitos dos fármacos , Cisteína Endopeptidases/efeitos dos fármacos , Cisteína Endopeptidases/genética , Combinação de Medicamentos , Proteínas de Fímbrias/efeitos dos fármacos , Proteínas de Fímbrias/genética , Cisteína Endopeptidases Gingipaínas , Humanos , Lectinas/efeitos dos fármacos , Lectinas/genética , Metronidazol/farmacologia , Testes de Sensibilidade Microbiana , NF-kappa B/metabolismo , Peptídeo Hidrolases/efeitos dos fármacos , Peptídeo Hidrolases/genética , Porphyromonas gingivalis/metabolismo , Fatores de Transcrição/genética , Células U937/efeitos dos fármacos , Vitamina D/análogos & derivadosRESUMO
RATIONALE: Corticosteroid resistance is a major barrier to the effective treatment of chronic obstructive pulmonary disease (COPD). Several molecular mechanisms have been proposed, such as activations of the phosphoinositide-3-kinase/Akt pathway and p38 mitogen-activated protein kinase. However, the mechanism for corticosteroid resistance is still not fully elucidated. OBJECTIVES: To investigate the role of mammalian target of rapamycin (mTOR) in corticosteroid sensitivity in COPD. METHODS: The corticosteroid sensitivity of peripheral blood mononuclear cells collected from patients with COPD, smokers, and nonsmoking control subjects, or of human monocytic U937 cells exposed to cigarette smoke extract (CSE), was quantified as the dexamethasone concentration required to achieve 30% inhibition of tumor necrosis factor-α-induced CXCL8 production in the presence or absence of the mTOR inhibitor rapamycin. mTOR activity was determined as the phosphorylation of p70 S6 kinase, using Western blotting. MEASUREMENTS AND MAIN RESULTS: mTOR activity was increased in peripheral blood mononuclear cells from patients with COPD, and treatment with rapamycin inhibited this as well as restoring corticosteroid sensitivity. In U937 cells, CSE stimulated mTOR activity and c-Jun expression, but pretreatment with rapamycin inhibited both and also reversed CSE-induced corticosteroid insensitivity. CONCLUSIONS: mTOR inhibition by rapamycin restores corticosteroid sensitivity via inhibition of c-Jun expression, and thus mTOR is a potential novel therapeutic target for COPD.
Assuntos
Corticosteroides/farmacologia , Resistência a Medicamentos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-jun/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/efeitos dos fármacos , Corticosteroides/uso terapêutico , Idoso , Resistência a Medicamentos/imunologia , Feminino , Histona Desacetilase 2/efeitos dos fármacos , Histona Desacetilase 2/fisiologia , Humanos , Imunossupressores/imunologia , Imunossupressores/farmacologia , Imunossupressores/uso terapêutico , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo/fisiologia , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/fisiologia , Proteínas Proto-Oncogênicas c-jun/fisiologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Sirolimo/imunologia , Sirolimo/uso terapêutico , Fumar/efeitos adversos , Fumar/fisiopatologia , Serina-Treonina Quinases TOR/fisiologia , Células U937/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologiaRESUMO
BACKGROUND: Current chemotherapeutic agents based on apoptosis induction are lacking in desired efficacy. Therefore, there is continuous effort to bring about new dimension in control and gradual eradication of cancer by means of ever evolving therapeutic strategies. Various forms of PCD are being increasingly implicated in anti-cancer therapy and the complex interplay among them is vital for the ultimate fate of proliferating cells. We elaborated and illustrated the underlying mechanism of the most potent Andrographolide analogue (AG-4) mediated action that involved the induction of dual modes of cell death-apoptosis and autophagy in human leukemic U937 cells. PRINCIPAL FINDINGS: AG-4 induced cytotoxicity was associated with redox imbalance and apoptosis which involved mitochondrial depolarisation, altered apoptotic protein expressions, activation of the caspase cascade leading to cell cycle arrest. Incubation with caspase inhibitor Z-VAD-fmk or Bax siRNA decreased cytotoxic efficacy of AG-4 emphasising critical roles of caspase and Bax. In addition, AG-4 induced autophagy as evident from LC3-II accumulation, increased Atg protein expressions and autophagosome formation. Pre-treatment with 3-MA or Atg 5 siRNA suppressed the cytotoxic effect of AG-4 implying the pro-death role of autophagy. Furthermore, incubation with Z-VAD-fmk or Bax siRNA subdued AG-4 induced autophagy and pre-treatment with 3-MA or Atg 5 siRNA curbed AG-4 induced apoptosis-implying that apoptosis and autophagy acted as partners in the context of AG-4 mediated action. AG-4 also inhibited PI3K/Akt/mTOR pathway. Inhibition of mTOR or Akt augmented AG-4 induced apoptosis and autophagy signifying its crucial role in its mechanism of action. CONCLUSIONS: Thus, these findings prove the dual ability of AG-4 to induce apoptosis and autophagy which provide a new perspective to it as a potential molecule targeting PCD for future cancer therapeutics.
Assuntos
4-Butirolactona/análogos & derivados , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Diterpenos/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/antagonistas & inibidores , 4-Butirolactona/farmacologia , Caspases/metabolismo , Ativação Enzimática/efeitos dos fármacos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas de Neoplasias/antagonistas & inibidores , Oxirredução , Estresse Oxidativo , RNA Interferente Pequeno/farmacologia , Células U937/efeitos dos fármacosRESUMO
The study aimed to assess in vitro the short-term effects exerted by fluoxetine, sertraline and venlafaxine on certain physiological properties in two different study models: U937 monocytes and erythrocytes isolated from patients treated with the above-mentioned molecules. Results on U937 cell suspensions revealed the depolarization of the cell membrane induced by the three antidepressants. The maximal depolarization effect was registered after 15 minutes of cell exposure and was concentration-dependent, in a non-monotonic manner. The effect was also dependent on the tested compound, fluoxetine presenting the strongest depolarizing effect compared to sertraline and venlafaxine. The erythrocyte susceptibility to lipid peroxidation and glucose-6-phosphate dehydrogenase activity were assessed on red blood cells isolated from patients with depressive disorder. Our results revealed that antidepressant treatment induced the antioxidant defense, by decreasing erythrocyte susceptibility to lipid peroxidation and increasing glucose-6-phosphate dehydrogenase activity. The effect is more intense in the case of severe pathology and less evident in the case of moderate or minor disorder, as expressed by MADRS (Montgomery-Åsberg Depression Rating Scale) score. Our results could indicate that selected antidepressants at therapeutic concentrations, besides their known pharmacological effects, exhibit a protective effect against oxidative stress and also influence cells with immune properties.
Assuntos
Antidepressivos/farmacologia , Membrana Celular/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Antioxidantes/química , Transtorno Depressivo/tratamento farmacológico , Relação Dose-Resposta a Droga , Feminino , Fluoxetina/farmacologia , Glucosefosfato Desidrogenase/química , Humanos , Sistema Imunitário , Peroxidação de Lipídeos , Pessoa de Meia-Idade , Estresse Oxidativo , Sertralina/farmacologia , Células U937/efeitos dos fármacos , Cloridrato de Venlafaxina/farmacologiaRESUMO
The need for non-animal data to assess skin sensitisation properties of substances, especially cosmetics ingredients, has spawned the development of many in vitro methods. As it is widely believed that no single method can provide a solution, the Cosmetics Europe Skin Tolerance Task Force has defined a three-phase framework for the development of a non-animal testing strategy for skin sensitization potency prediction. The results of the first phase systematic evaluation of 16 test methods are presented here. This evaluation involved generation of data on a common set of ten substances in all methods and systematic collation of information including the level of standardisation, existing test data,potential for throughput, transferability and accessibility in cooperation with the test method developers.A workshop was held with the test method developers to review the outcome of this evaluation and to discuss the results. The evaluation informed the prioritisation of test methods for the next phase of the non-animal testing strategy development framework. Ultimately, the testing strategy combined with bioavailability and skin metabolism data and exposure consideration is envisaged to allow establishment of a data integration approach for skin sensitisation safety assessment of cosmetic ingredients.
Assuntos
Alternativas aos Testes com Animais/métodos , Dermatite Alérgica de Contato/etiologia , Linhagem Celular , Cosméticos , Epiderme/efeitos dos fármacos , Humanos , Técnicas In Vitro , Interleucina-18/análise , Queratinócitos/efeitos dos fármacos , Medição de Risco , Pele/efeitos dos fármacos , Células U937/efeitos dos fármacosRESUMO
INTRODUCTION: Leishmaniasis is a major public health problem faced by many countries, including Colombia. Its treatment has limitations such as the toxicity of the drugs used, the emergence of resistant strains, and prolonged and expensive treatments. Thus, there is an urgent need to find alternative solutions. OBJECTIVE: To evaluate the leishmanicidal and cytotoxic activities of three 2-styrylquinolines type compounds: 2-[(E)-2-(2,3-diacetyloxyphenyl)ethenyl]quinolin-8-yl-acetate, E1; 2-[(E)-2-(4-acetyloxy-3-methoxyphenyl)ethenyl] quinoline, E2, and 2-[(E)-2-(2,3-diacetyloxyphenyl)ethenyl] quinoline, E3. MATERIALS AND METHODS: The 2-styrylquinolines were obtained by organic synthesis using Perkin-type condensation reaction from 8-hydroxy quinaldine or quinaldine and aromatic aldehydes. The leishmanicidal activity was evaluated on intracellular amastigotes of Leishmania (Viannia) panamensis by flow cytometry. The results were expressed as lethal concentration 50 (LC 50 ) for cytotoxicity and effective concentration 50 (EC 50 ) for leishmanicidal activity, calculated by the Probit method. RESULTS: E3 showed high activity against L. (V) panamensis with a calculated EC 50 value of 1.4 µg/ml, and a selectivity index of 3.9; E1 and E2 showed higher EC 50 values of 5.6 and 68.1 µg/ml, respectively. For cytotoxicity, LC 50 values ranging from 5.4 to 68.1 µg/ml were calculated. E2 was moderately toxic, showing an LC 50 very similar to that of amphotericin B, a substance used as cytotoxic control. CONCLUSION: The styrylquinoline E3 is a promising compound against L. (V) panamensis , as it was able to significantly inhibit amastigotes inside the cell, reducing infection despite its toxicity.
Assuntos
Antiprotozoários/farmacologia , Leishmania guyanensis/efeitos dos fármacos , Quinolinas/farmacologia , Estirenos/farmacologia , Células U937/efeitos dos fármacos , Antiprotozoários/síntese química , Antiprotozoários/química , Antiprotozoários/toxicidade , Avaliação Pré-Clínica de Medicamentos , Humanos , Dose Letal Mediana , Estrutura Molecular , Quinolinas/síntese química , Quinolinas/química , Quinolinas/toxicidade , Estirenos/síntese química , Estirenos/química , Estirenos/toxicidadeRESUMO
Leukemia is currently one of the most deadly diseases. Ginseng has been used in Asian countries for the treatment and prevention of various diseases, including leukemia, but the molecular mechanism of its antileukemia activity has not been well defined. The aim of this study was to explore the effect of 20-(s)-ginsenoside Rg3 on apoptosis in human leukemic U937 and HL-60 cells and the underlying mechanism. We found that 20-(s)-ginsenoside Rg3 reduced cell viability and induced apoptosis in U937 and HL-60 cells. The induction of apoptosis was accompanied by the downregulation of PI3K/Akt family proteins. Moreover, we observed that 20-(s)-ginsenoside Rg3 treatment resulted in activation of caspase-3 and caspase-9. Taken together, our findings suggest for the first time that 20-(s)-ginsenoside Rg3 can promote apoptosis in U937 and HL-60 cells, at least partly through the downregulation of PI3K/Akt family proteins. Moreover, the triggering of caspase-3 and caspase-9 activation mediated apoptotic induction. All these findings collectively demonstrate that the natural compound 20-(s)-ginsenoside Rg3 effectively induces apoptosis in human leukemic cells, which suggests that this compound may play a role in future therapies for leukemia.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Ginsenosídeos/farmacologia , Proteína Oncogênica v-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Caspase 3/metabolismo , Caspase 9/metabolismo , Proliferação de Células/efeitos dos fármacos , Cromonas/farmacologia , Células HL-60/efeitos dos fármacos , Humanos , Leucemia/tratamento farmacológico , Morfolinas/farmacologia , Fosforilação , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Células U937/efeitos dos fármacosRESUMO
OBJECTIVE: The present study aimed to investigate the mechanisms underlying the anti-inflammatory and anti-angiogenic effects of ethyl-p-methoxycinnamate isolated from Kaempferia galanga. METHODS: The anti-inflammatory effects of ethyl-p-methoxycinnamate were assessed using the cotton pellet granuloma assay in rats, whereby the levels of interleukin-1 and tumor necrosis factor-α were measured in the animals' blood. In addition, the levels of interleukin, tumor necrosis factor, and nitric oxide were measured in vitro using the human macrophage cell line (U937). The analgesic effects of ethyl-p-methoxycinnamate were assessed by the tail flick assay in rats. The anti-angiogenic effects were evaluated first by the rat aortic ring assay and, subsequently, by assessing the inhibitory effects of ethyl-p-methoxycinnamate on vascular endothelial growth factor, proliferation, migration, and tube formation in human umbilical vein endothelial cells. RESULTS: Ethyl-p-methoxycinnamate strongly inhibited granuloma tissue formation in rats. It prolonged the tail flick time in rats by more than two-fold compared with the control animals. The inhibition of interleukin and tumor necrosis factor by ethyl-p-methoxycinnamate was significant in both in vivo and in vitro models; however, only a moderate inhibition of nitric oxide was observed in macrophages. Furthermore, ethyl-p-methoxycinnamate considerably inhibited microvessel sprouting from the rat aorta. These mechanistic studies showed that ethyl-p-methoxycinnamate strongly inhibited the differentiation and migration of endothelial cells, which was further confirmed by the reduced level of vascular endothelial growth factor. CONCLUSION: Ethyl-p-methoxycinnamate exhibits significant anti-inflammatory potential by inhibiting pro-inflammatory cytokines and angiogenesis, thus inhibiting the main functions of endothelial cells. Thus, ethyl-p-methoxycinnamate could be a promising therapeutic agent for the treatment of inflammatory and angiogenesis-related diseases.
Assuntos
Inibidores da Angiogênese/farmacologia , Anti-Inflamatórios/farmacologia , Cinamatos/farmacologia , Extratos Vegetais/farmacologia , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Zingiberaceae/química , Análise de Variância , Inibidores da Angiogênese/isolamento & purificação , Animais , Anti-Inflamatórios/isolamento & purificação , Proliferação de Células/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Interleucina-1/análise , Masculino , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Células U937/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/análiseRESUMO
A non-lectin glycoprotein (HM-3A) isolated from the basidioma of edible mushroom Hypsizygus marmoreus (Peck) Bigelow was shown to be inhibitory against the growth of human myeloid leukemia U937 cells in our previous research. The present study further investigated the mechanisms of inhibition and differentiation in an indirect model. Human mononuclear cells (MNC) were stimulated by HM-3A for 1, 2 and 3days to obtain the conditioned media (MNC-CM1, 2 and 3) that was effective in inhibiting the growth of U937 cells. The percentage of mature monocytes/macrophages among the cells and the total productivity of superoxide increased in the treatment and proved the differentiation-inducing effect of HM-3A-MNC-CM. The secretion of cytokines TNF-α, IL-2 and IFN-γ from the treated MNC were increased. Antibody neutralization tests of HM-3A-MNC-CM1 further showed that the growth inhibition effect of HM-3A can be attributed to the promotion in secretion of cytokines IFN-γ and TNF-α. We propose that HM-3A may stimulate human MNC to secrete cytokines IFN-γ and TNF-α to inhibit the growth of U937 cells, and that HM-3A may be developed into an anti-leukemia ingredient in health food.
Assuntos
Agaricales/química , Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Glicoproteínas/farmacologia , Anticorpos Neutralizantes/farmacologia , Meios de Cultivo Condicionados/farmacologia , Citocinas/imunologia , Citocinas/metabolismo , Humanos , Interferon gama/metabolismo , Leucemia/tratamento farmacológico , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Superóxidos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Células U937/efeitos dos fármacosRESUMO
INTRODUCTION: The plant Solanum nudum (Solanaceae) is extensively used for the treatment of malaria-related symptoms in traditional medicine practices in the Colombian Pacific. Recently, it has become a significant source of promising new molecules for developing a pharmaceutical malaria treatment. OBJECTIVE: This research aimed to evaluate the cytotoxic effect and the genetic damage of standardized extracts of S. nudumon different cells. MATERIALS AND METHODS: Sixty six standardized S. nudum extracts were used, evaluating cytotoxicity in U937 and HepG2 cells and the antiplasmodial activity using both a chloroquine-sensitive (NF54) and a chloroquine-resistant (FCB2) strain. The hemolytic effect on healthy O + erythrocytes, the mutagenic effect on S.Typhimurium TA98 and TA100 strains and the genotoxic effect on U937 cells were evaluated. The extracts that displayed both antiplasmodial activity and low toxicity were selected. RESULTS: Five extracts were selected: 28MA1, 29MA1, 51MA1, 55MA1 and 61MA1. These extracts were active against P. falciparum with IC 50 between 9.8 and 54.8 µg/ml and selectivity indexes were calculated between 0.9 and 4.4, the latter for 29MA1. Also, no hemolytic effects in healthy O + erythrocytes were shown at a concentration of 250 µg/ml, nor did they cause mutations in the TA98 and TA100 strains or generate genotoxic effects in U937cells. CONCLUSION: The use of standardized extracts of S. nudum could contribute to the body of work aimed at developing a new pharmaceutical treatment for malaria using natural products.