RESUMO
Phosphoantigens (PAgs)-like HMBPP ((E)-4-hydroxy-3-methyl-but-2-enyl diphosphate) and butyrophilin 3 (BTN3A, CD277)-specific monoclonal antibody 20.1 induce TCR-mediated activation of Vγ9Vδ2 T cells. Here, we compared murine reporter cells transduced with Vγ9Vδ2 TCRs G115, D1C55, and MOP for the activation in culture with human RAJI cells and PAgs or mAb 20.1 and its single-chain (sc) derivative. All transductants responded readily to PAg but only TCR MOP γ-chain-expressing cells responded to mAb/sc 20.1. Furthermore, both antagonist and agonist mAb and sc of the agonist mAb inhibited the PAg response of TCR-transduced murine reporter cells. These findings suggest that, in contrast to stimulation by physiological stimulators (PAg), the responsiveness to mAb 20.1 depends strongly on CDR3 sequences of the TCR, and that mAb 20.1 can interfere with the PAg-response. Mouse or human origin of reporter cells might affect the mAb 20.1 response since all three TCR-mediated mAb 20.1-induced activation of TCR-transduced Jurkat cells. The pronounced differences between PAg and mAb 20.1-induced activation observed here help to understand the often contradictory published data. This study provides novel perspectives on the physiological mechanism of Vγ9Vδ2 T-cell activation, and highlights the complex mode of action of BTN3A-specific antibodies as agents in cancer immunotherapy.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos CD/imunologia , Butirofilinas/imunologia , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T/imunologia , Animais , Linhagem Celular , Células HEK293 , Humanos , CamundongosRESUMO
Vγ9Vδ2 T cells play an important role in the cross talk of the innate and adaptive immune system. For their activation by phosphoantigens (PAgs), both cell surface receptors, the eponymous Vγ9Vδ2 T cell antigen receptors (Vγ9Vδ2 TCRs) on Vγ9Vδ2 T cells and butyrophilin 3A1 (BTN3A1) on the phosphoantigen-"presenting" cell, are mandatory. To find yet undetected but further contributing proteins, a biotinylated, photo-crosslinkable benzophenone probe BioBP-HMBPP (2) was synthesized from a known allyl alcohol in nine steps and overall 16 % yield. 2 is based on the picomolar PAg (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP, 1). Laser irradiation of 2 at 308â nm initiated the photo-crosslinking reaction with proteins. When the B30.2 domain of BTN3A1, which contains a positively charged PAg-binding pocket, was exposed to increasing amounts of HMBPP (1), labeling by BioBP-HMBPP (2) was reduced significantly. Because BSA labeling was not impaired, 2 clearly binds to the same site as natural ligand 1. Thus, BioBP-HMBPP (2) is a suitable tool to identify co-ligands or receptors involved in PAg-mediated T cell activation.
Assuntos
Antígenos CD/metabolismo , Biotina/análogos & derivados , Butirofilinas/metabolismo , Compostos Organofosforados/química , Animais , Antígenos/imunologia , Antígenos/metabolismo , Antígenos CD/química , Antígenos de Diferenciação de Linfócitos T/metabolismo , Biotina/síntese química , Biotinilação/efeitos da radiação , Butirofilinas/química , Bovinos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Humanos , Lasers , Lectinas Tipo C/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Compostos Organofosforados/síntese química , Compostos Organofosforados/farmacologia , Ligação Proteica , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Espectrofotometria , Linfócitos T/citologia , Linfócitos T/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
The JAK/STAT pathway is an essential mediator of cytokine signaling, often upregulated in human diseases and therefore recognized as a relevant therapeutic target. We previously identified the synthetic chalcone α-bromo-2',3,4,4'-tetramethoxychalcone (α-Br-TMC) as a novel JAK2/STAT5 inhibitor. We also found that treatment with α-Br-TMC resulted in a downward shift of STAT5 proteins in SDS-PAGE, suggesting a post-translational modification that might affect STAT5 function. In the present study, we show that a single cysteine within STAT5 is responsible for the α-Br-TMC-induced protein shift, and that this modification does not alter STAT5 transcriptional activity. We also compared the inhibitory activity of α-Br-TMC to that of another synthetic chalcone, α-trifluoromethyl-2',3,4,4'-tetramethoxychalcone (α-CF3-TMC). We found that, like α-Br-TMC, α-CF3-TMC inhibits JAK2 and STAT5 phosphorylation in response to interleukin-3, however without altering STAT5 mobility in SDS-PAGE. Moreover, we demonstrate that both α-Br-TMC and α-CF3-TMC inhibit interferon-α-induced activation of STAT1 and STAT2, by inhibiting their phosphorylation and the expression of downstream interferon-stimulated genes. Together with the previous finding that α-Br-TMC and α-CF3-TMC inhibit the response to inflammation by inducing Nrf2 and blocking NF-κB activities, our data suggest that synthetic chalcones might be useful as anti-inflammatory, anti-cancer and immunomodulatory agents in the treatment of human diseases.
Assuntos
Chalconas/farmacologia , Interferon-alfa/antagonistas & inibidores , Interleucina-3/antagonistas & inibidores , Janus Quinase 2/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Linhagem Celular , Humanos , Camundongos , Fosforilação/efeitos dos fármacos , Fator de Transcrição STAT5/químicaRESUMO
Pyrophosphorylated metabolites of isoprenoid-biosynthesis (phosphoantigens, PAgs) activate Vγ9Vδ2 T cells during infections and trigger antitumor activity. This activation depends on expression of butyrophilin 3 A1 (BTN3A1) by antigen-presenting cells. This report defines the minimal genetic requirements for activation of Vγ9Vδ2 T cells by PAgs and mAb 20.1. We compared PAg-presentation by BTN3A1-transduced CHO hamster cells with that of CHO cells containing the complete human chromosome 6 (Chr6). BTN3A1 expression alone was sufficient for activation of Vγ9Vδ2 T-cell receptor transductants by mAb 20.1., while activation by PAgs also required the presence of Chr6. We take this finding as evidence that gene(s) on Chr6 in addition to BTN3A1 are mandatory for PAg-mediated activation of Vγ9Vδ2 T cells. This observation is important for the design of animal models for PAg-mediated immune responses and provokes speculations about the analogy between genes controlling PAg presentation and MHC-localized genes controlling peptide-antigen presentation.
Assuntos
Antígenos CD/imunologia , Cromossomos Humanos Par 6/imunologia , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Terpenos/imunologia , Animais , Antígenos CD/genética , Butirofilinas , Células CHO , Cromossomos Humanos Par 6/genética , Cricetinae , Cricetulus , Humanos , Fosforilação/genética , Fosforilação/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/genética , Transdução GenéticaRESUMO
Inflammatory signaling pathways orchestrate the cellular response to infection and injury. These pathways are known to be modulated by compounds that alkylate cysteinyl thiols. One class of phytochemicals with strong thiol alkylating activity is the chalcones. In this study we tested fourteen chalcone derivatives, α-X-substituted 2',3,4,4'-tetramethoxychalcones (α-X-TMCs, X = H, F, Cl, Br, I, CN, Me, p-NO2-C6H4, Ph, p-OMe-C6H4, NO2, CF3, COOEt, COOH), for their ability to modulate inflammatory responses, as monitored by their influence on heme oxygenase-1 (HO-1) activity, inducible nitric oxide synthase (iNOS) activity, and cytokine expression levels. We confirmed that the transcriptional activity of Nrf2 was activated by α-X-TMCs while for NF-κB it was inhibited. For most α-X-TMCs, anti-inflammatory activity was positively correlated with thiol alkylating activity, i.e. stronger electrophiles (X = CF3, Br and Cl) being more potent. Notably, this correlation did not hold true for the strongest electrophiles (X = CN and NO2) which were found to be ineffective as anti-inflammatory compounds. These results emphasize the idea that chemical fine-tuning of electrophilicity is needed to achieve and optimize desired therapeutic effects.
Assuntos
Anti-Inflamatórios/química , Chalconas/química , Inflamação/metabolismo , Animais , Cisteamina/química , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Glutationa/química , Células HeLa , Heme Oxigenase-1/metabolismo , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Lipopolissacarídeos/química , Macrófagos/metabolismo , Camundongos , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Processos Fotoquímicos , Compostos de Sulfidrila/química , Transcrição GênicaRESUMO
A strategy for the synthesis of natural and non-natural 5-deoxy-6,7-dihydrocurcuminoids (diarylheptanoids) was developed for the preparation of 14 compounds with varying aromatic substituent patterns and a different functionality in the aliphatic seven-carbon chain. The in vitro protective activity against glutamate-induced neuronal cell death was examined in the murine hippocampal cell line HT-22 to find structural motifs responsible for neuroprotective effects in vitro. Among the tested compounds the ferulic acid-like unit, present in the structures of (E)-1,7-bis(4-hydroxy-3-methoxyphenyl)hept-1-en-3-one (5) and (E)-1-(4-hydroxy-3-methoxyphenyl)-7-(4-hydroxyphenyl)hept-1-en-3-one (7), appeared to be an important feature for protection against glutamate-induced neurotoxicity. Both compounds demonstrated significant neuroprotective activity in a concentration range between 1 and 25 µM without showing toxic effects in a cytotoxicity assay with HT-22 cells. Furthermore, (E)-1,7-bis(3,4-dihydroxyphenyl)hept-1-en-3-one (9), exhibiting a caffeic acid-like structural motif, displayed a neuroprotective activity at a nontoxic concentration of 25 µM. In contrast, (1E,6E)-1,7-bis(3,4-dihydroxyphenyl)hepta-1,6-diene-3,5-dione (4, di-O-demethylcurcumin) showed mainly cytotoxic effects. A corresponding single-ring analogue that contains the ferulic acid-like unit as an enone was not active.
Assuntos
Diarileptanoides/síntese química , Diarileptanoides/farmacologia , Ácido Glutâmico/farmacologia , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Animais , Antioxidantes/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Ácidos Cumáricos/química , Ácidos Cumáricos/farmacologia , Glutamatos , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Camundongos , Estrutura MolecularRESUMO
Thiol-mediated processes play a key role to induce or inhibit inflammation proteins. Tailoring the reactivity of electrophiles can enhance the selectivity to address only certain surface cysteines. Fourteen 2',3,4,4'-tetramethoxychalcones with different α-X substituents (X=H, F, Cl, Br, I, CN, Me, p-NO2-C6H4, Ph, p-OMe-C6H4, NO2, CF3, COOEt, COOH) were synthesized, containing the potentially electrophilic α,ß-unsaturated carbonyl unit. The assessment of their reactivity as electrophiles in thia-Michael additions with cysteamine shows a change in the reactivity of more than six orders of magnitude. Moreover, a clear correlation between their reactivity and an influence on the inflammation proteins heme oxygenase-1 (HO-1) and the inducible NO synthase (iNOS) is demonstrated. As the biologically most active compound, the α-CF3 -chalcone is shown to inhibit the NO production in RAW264.7 mouse macrophages in the nanomolar range.
Assuntos
Chalcona/química , Compostos de Sulfidrila/química , Animais , Chalcona/farmacologia , Espectroscopia de Ressonância Magnética , CamundongosRESUMO
The electrophilic nature of chalcones (1,3-diphenylprop-2-en-1-ones) and many other α,ß-unsaturated carbonyl compounds is crucial for their biological activity, which is often based on thiol-mediated regulation processes. To better predict their biological activity a simple screening assay for the assessment of the second-order rate constants (k(2)) in thia-Michael additions was developed. Hence, a clear structure-activity relationship of 16 differentially decorated hydroxy-alkoxychalcones upon addition of cysteamine could be established. Moreover, amongst other naturally occurring α,ß-unsaturated carbonyl compounds k(2) values for curcumin and cinnamaldehyde were gained while cinnamic acids or esters gave no or very slow reactions.
Assuntos
Chalconas/química , Compostos de Sulfidrila/química , CinéticaRESUMO
Complement Factor H-Related 3 (FHR-3) is a major regulator of the complement system, which is associated with different diseases, such as age-related macular degeneration (AMD). However, the non-canonical local, cellular functions of FHR-3 remained poorly understood. Here, we report that FHR-3 bound to oxidative stress epitopes and competed with FH for interaction. Furthermore, FHR-3 was internalized by viable RPE cells and modulated time-dependently complement component (C3, FB) and receptor (C3aR, CR3) expression of human RPE cells. Independently of any external blood-derived proteins, complement activation products were detected. Anaphylatoxin C3a was visualized in treated cells and showed a translocation from the cytoplasm to the cell membrane after FHR-3 exposure. Subsequently, FHR-3 induced a RPE cell dependent pro-inflammatory microenvironment. Inflammasome NLRP3 activation and pro-inflammatory cytokine secretion of IL-1ß, IL-18, IL-6 and TNF-α were induced after FHR-3-RPE interaction. Our previously published monoclonal anti-FHR-3 antibody, which was chimerized to reduce immunogenicity, RETC-2-ximab, ameliorated the effect of FHR-3 on ARPE-19 cells. Our studies suggest FHR-3 as an exogenous trigger molecule for the RPE cell "complosome" and as a putative target for a therapeutic approach for associated degenerative diseases.
Assuntos
Proteínas Sanguíneas/imunologia , Ativação do Complemento/imunologia , Fator H do Complemento/imunologia , Células Epiteliais/imunologia , Epitélio Pigmentado da Retina/citologia , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Linhagem Celular , Ativação do Complemento/genética , Complemento C3/genética , Complemento C3/imunologia , Complemento C3/metabolismo , Fator H do Complemento/genética , Fator H do Complemento/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/genética , Transição Epitelial-Mesenquimal/imunologia , Expressão Gênica/genética , Expressão Gênica/imunologia , Células HEK293 , Humanos , Inflamassomos/genética , Inflamassomos/imunologia , Inflamassomos/metabolismo , Antígeno de Macrófago 1/genética , Antígeno de Macrófago 1/imunologia , Antígeno de Macrófago 1/metabolismo , Degeneração Macular/genética , Degeneração Macular/imunologia , Degeneração Macular/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Polimorfismo de Nucleotídeo Único/imunologiaRESUMO
Tumor-derived lactic acid inhibits T and natural killer (NK) cell function and, thereby, tumor immunosurveillance. Here, we report that melanoma patients with high expression of glycolysis-related genes show a worse progression free survival upon anti-PD1 treatment. The non-steroidal anti-inflammatory drug (NSAID) diclofenac lowers lactate secretion of tumor cells and improves anti-PD1-induced T cell killing in vitro. Surprisingly, diclofenac, but not other NSAIDs, turns out to be a potent inhibitor of the lactate transporters monocarboxylate transporter 1 and 4 and diminishes lactate efflux. Notably, T cell activation, viability, and effector functions are preserved under diclofenac treatment and in a low glucose environment in vitro. Diclofenac, but not aspirin, delays tumor growth and improves the efficacy of checkpoint therapy in vivo. Moreover, genetic suppression of glycolysis in tumor cells strongly improves checkpoint therapy. These findings support the rationale for targeting glycolysis in patients with high glycolytic tumors together with checkpoint inhibitors in clinical trials.
Assuntos
Glicólise/fisiologia , Linfócitos T/fisiologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Glucose/metabolismo , Glicólise/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Xenopus laevisRESUMO
(E)-1-Hydroxy-2-methyl-but-2-enyl 4-diphosphate, a recently discovered intermediate in the deoxyxylulose phosphate pathway of isoprenoid biosynthesis, has been shown to act as a potent immunomodulator. In cultures of human peripheral blood mononuclear cells from eight non-related donors, the compound stimulated the proliferation of Vgamma9/Vdelta2 T lymphocytes with a median EC(50) of 70 pM when 10 U/ml of IL-2 was used as costimulant. Isopentenyl diphosphate (IPP), dimethylallyl diphosphate (DMAPP) and some structural analogs of (E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate also stimulated Vgamma9/Vdelta2 T-cell proliferation, albeit at much higher concentrations. The Vgamma9/Vdelta2 T-cell proliferation is highly dependent on the seeding density used in culture. All phosphoantigens tested elicited the proliferation of two T-lymphocyte populations with different apparent ratios between the expression level of Vdelta2 and Vgamma9 chains.
Assuntos
Antígenos de Bactérias/imunologia , Proliferação de Células , Ativação Linfocitária/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T/imunologia , Terpenos/imunologia , Antígenos de Bactérias/química , Antígenos de Bactérias/metabolismo , Células Cultivadas , Hemiterpenos/imunologia , Humanos , Compostos Organofosforados/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Terpenos/química , Terpenos/metabolismoAssuntos
Butadienos/química , Monóxido de Carbono/metabolismo , Complexos de Coordenação/química , Ferro/química , Óxido Nítrico Sintase Tipo II/metabolismo , Animais , Biocatálise , Monóxido de Carbono/química , Esterases/metabolismo , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Oxirredução , SuínosRESUMO
Inflammation plays a central role in the pathophysiology of many diseases. The inducible enzyme heme oxygenase-1 (HO-1) protects cells against inflammation and can be induced by electrophilic compounds like the chalcones (1,3-diphenylprop-2-enones) from the class of α,ß-unsaturated carbonyl compounds. We hypothesized that the synthetic chalcone E-α-(p-methoxyphenyl)-2',3,4,4'-tetramethoxychalcone (E-α-p-OMe-C6H4-TMC) exerts anti-inflammatory effects in RAW264.7, Jurkat lymphocytes and HK-2 cells via HO-1 induction. RAW264.7 cells were treated with lipopolysaccharide prior to E-α-p-OMe-C6H4-TMC treatment. Subsequently, HO-1 protein induction and activity were analyzed, as well as expression of pro- and anti-inflammatory mediators, transcription factors and mitogen-activated protein kinases to evaluate the possible molecular mechanism. These results were confirmed in human cell lines (Jurkat T-lymphocytes and HK-2 epithelial cells). We found that the E-α-p-OMe-C6H4-TMC exerts significant anti-inflammatory effects in a dose dependent manner, showing no toxic effects in LPS-treated RAW264.7 macrophages. E-α-p-OMe-C6H4-TMC induced HO-1 and SOD-1 protein expression and HO-1 enzyme activity, reduced the upregulation of COX-2 and iNOS, while inducing the translocation of Nrf2. NF-κB activity was attenuated following E-α-p-OMe-C6H4-TMC treatment accompanied by the downregulation of proinflammatory cytokines IL-1ß, IL-6 and MCP-1. Pretreatment with E-α-p-OMe-C6H4-TMC revealed significant changes in phosphorylation of ERK and p38, but not JNK. These anti-inflammatory effects of E-α-p-OMe-C6H4-TMC were approved in Jurkat and HK-2 cells, furthermore revealing a downregulation of IL-8 and IL-10. In conclusion, it is tempting to speculate about E-α-p-OMe-C6H4-TMC as a new and non-toxic agent, inducing HO-1 in cells. This opens up new opportunities regarding the development of therapeutic agents using beneficial effects of HO-1 and its products.
Assuntos
Anti-Inflamatórios/farmacologia , Chalconas/farmacologia , Células Epiteliais/efeitos dos fármacos , Heme Oxigenase-1/metabolismo , Macrófagos/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Animais , Anti-Inflamatórios/síntese química , Chalconas/síntese química , Ciclo-Oxigenase 2/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Células Jurkat , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Camundongos , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos , Superóxido Dismutase-1/metabolismo , Linfócitos T/imunologiaRESUMO
SCOPE: Many phytochemicals with beneficial pharmacological properties contain electrophilic sites, e.g. α,ß-unsaturated carbonyl (enone) groups. There is increasing evidence that many biological effects of electrophilic compounds depend on covalent conjugation to reactive protein thiols. For example, the reaction of electrophiles with cysteinyl residues of the sensor protein Keap1 activates the cell-protective Nrf2 response. Thus it is of interest to identify more generally the proteins to which small molecule electrophiles bind covalently. METHODS AND RESULTS: Here we use a Click chemistry approach to identify target proteins of the chemopreventive phytochemical xanthohumol (XN), an enone-containing chalcone from hops (Humulus lupulus L.). Using an alkynylated analog of XN (XN-alkyne), we purified covalent protein-electrophile conjugates from cell lysates. We confirm the previously described conjugation of XN to Keap1. One of the newly identified candidate target proteins is glucose-6-phosphate dehydrogenase (G6PDH). We confirm that XN attenuates intracellular G6PDH activity at low micromolar concentrations. CONCLUSION: We find support for the notion that XN modulates multiple pathways and processes by covalent modification of proteins with reactive cysteines.
Assuntos
Química Click/métodos , Flavonoides/química , Flavonoides/metabolismo , Propiofenonas/química , Propiofenonas/metabolismo , Proteínas/metabolismo , Alcinos/química , Alcinos/metabolismo , Cisteamina/química , Flavonoides/farmacologia , Glucosefosfato Desidrogenase/antagonistas & inibidores , Glucosefosfato Desidrogenase/química , Glucosefosfato Desidrogenase/metabolismo , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/química , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Espectroscopia de Ressonância Magnética , Peso Molecular , Propiofenonas/farmacologia , Proteínas/química , Solubilidade , Espectrometria de Massas por Ionização por Electrospray , Compostos de Sulfidrila/químicaRESUMO
The upregulation of heme oxygenase-1 (HO-1) has proven to be a useful tool for fighting inflammation. In order to identify new HO-1 inducers, an efficient screening method was developed which can provide new lead structures for drug research. We designed a simple ELISA-based HO-1 enzyme activity assay, which allows for the screening of 12 compounds in parallel in the setting of a 96-well plate. The well-established murine macrophage cell line RAW264.7 is used and only about 26µg of protein from whole cell lysates is needed for the analysis of HO-1 activity. The quantification of HO-1 activity is based on an indirect ELISA using the specific anti-bilirubin antibody 24G7 to quantify directly bilirubin in the whole cell lysate, applying a horseradish peroxidase-tagged antibody together with ortho-phenylenediamine and H2O2 for detection. The bilirubin is produced on the action of HO enzymes by converting their substrate heme to biliverdin and additional recombinant biliverdin reductase together with NADPH at pH 7.4 in buffer. This sensitive assay allows for the detection of 0.57-82pmol bilirubin per sample in whole cell lysates. Twenty-three small molecules, mainly natural products with an α,ß-unsaturated carbonyl unit such as polyphenols, including flavonoids and chalcones, terpenes, an isothiocyanate, and the drug oltipraz were tested at typically 6 or 24h incubation with RAW264.7 cells. The activity of known HO-1 inducers was confirmed, while the chalcones cardamonin, flavokawain A, calythropsin, 2',3,4'-trihydroxy-4-methoxychalcone (THMC), and 2',4'-dihydroxy-3,4-dimethoxychalcone (DHDMC) were identified as new potent HO-1 inducers. The highest inductive power after 6h incubation was found at 10µM for DHDMC (6.1-fold), carnosol (3.9-fold), butein (3.1-fold), THMC (2.9-fold), and zerumbone (2.5-fold). Moreover, the time dependence of HO-1 protein production for DHDMC was compared to its enzyme activity, which was further evaluated in the presence of lipopolysaccharide and the specific HO-1 inhibitor tin protoporphyrin IX. Taken together, we developed a convenient and highly sensitive ELISA-based HO-1 enzyme activity assay, allowing the identification and characterization of molecules potentially useful for the treatment of inflammatory and autoimmune diseases.
Assuntos
Bilirrubina/metabolismo , Bioensaio , Flavonoides/farmacologia , Heme Oxigenase-1/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Animais , Anticarcinógenos/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ativação Enzimática , Ensaio de Imunoadsorção Enzimática , Macrófagos/citologia , Camundongos , Pirazinas/farmacologia , Tionas , TiofenosRESUMO
Cell protection against different noxious stimuli like oxidative stress or chemical toxins plays a central role in the treatment of many diseases. The inducible heme oxygenase isoform, heme oxygenase-1 (HO-1), is known to protect cells against a variety of harmful conditions including apoptosis. Because a number of medium strong electrophiles from a series of α-X-substituted 2',3,4,4'-tetramethoxychalcones (α-X-TMCs, X = H, F, Cl, Br, I, CN, Me, p-NO2-C6H4, Ph, p-OMe-C6H4, NO2, CF3, COOEt, COOH) had proven to activate Nrf2 resulting in HO-1 induction and inhibit NF-κB downstream target genes, their protective effect against staurosporine induced apoptosis and reactive oxygen species (ROS) production was investigated. RAW264.7 macrophages treated with 19 different chalcones (15 α-X-TMCs, chalcone, 2'-hydroxychalcone, calythropsin and 2'-hydroxy-3,4,4'-trimethoxychalcone) prior to staurosporine treatment were analyzed for apoptosis and ROS production, as well as HO-1 protein expression and enzyme activity. Additionally, Nrf2 and NF-κB activity was assessed. We found that amongst all tested chalcones only E-α-(4-methoxyphenyl)-2',3,4,4'-tetramethoxychalcone (E-α-p-OMe-C6H4-TMC) demonstrated a distinct, statistically significant antiapoptotic effect in a dose dependent manner, showing no toxic effects, while its double bond isomer Z-α-p-OMe-C6H4-TMC displayed no significant activity. Also, E-α-p-OMe-C6H4-TMC induced HO-1 protein expression and increased HO-1 activity, whilst inhibition of HO-1 by SnPP-IX abolished its antiapoptotic effect. The only weakly electrophilic chalcone E-α-p-OMe-C6H4-TMC reduced the staurosporine triggered formation of ROS, while inducing the translocation of Nrf2 into the nucleus. Furthermore, staurosporine induced NF-κB activity was attenuated following E-α-p-OMe-C6H4-TMC treatment. Overall, E-α-p-OMe-C6H4-TMC demonstrated its effective cytoprotective potential via a non-toxic induction of HO-1 in RAW264.7 macrophages. The observed cytoprotective effect may partly be related to both, the activation of the Nrf2- and inhibition of the NF-κB pathway.
Assuntos
Chalconas/química , Heme Oxigenase-1/efeitos dos fármacos , Proteínas de Membrana/efeitos dos fármacos , Animais , Apoptose , Linhagem Celular , Chalcona/análogos & derivados , Chalcona/química , Cristalografia por Raios X , Citoproteção/efeitos dos fármacos , Relação Dose-Resposta a Droga , Citometria de Fluxo , Regulação da Expressão Gênica , Macrófagos/efeitos dos fármacos , Camundongos , Fator 2 Relacionado a NF-E2/antagonistas & inibidores , Subunidade p50 de NF-kappa B/antagonistas & inibidores , Estresse Oxidativo , Transporte Proteico , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismoRESUMO
The design, synthesis and application of a new fluorescent-labeling reagent for collagen has been developed as a prerequisite for the design of a photoactivated collagen-crosslinking compound for surgical wound closure. The amine groups in collagen are the targets of a rational design for a new fluorophore because natural collagen crosslinks are formed between primary (1(o)) amine groups of lysine and hydroxylysine. The availability of 1(o) amines for crosslinking in native collagenous tissues was evaluated by reacting tendon and corneal samples with o-phthalaldehyde and dansyl chloride, fluorophores commonly used for the detection of 1(o) and 2(o) amines. The resulting fluorescent collagen fibrils indicated the presence of amines in native tissue. Subsequently, a photoactivated fluorescent label for 1(o) and 2(o) amines, coumaryl gamma-amino-butyric acid diazopyruvate (CGDP), was designed and synthesized. CGDP was first used to photolabel poly-L-lysine, forming a fluorescent, covalent bond to the 1(o) amine. CGDP was then photoreacted with corneal and tendon tissue samples to produce CGDP fluorescent-labeled samples that were statistically significantly more fluorescent than were the controls. These experiments support the postulate that 1(o) or 2(o) (or both) amines in native collagenous tissues are available to serve as targets for photoactivated collagen crosslinkers for wound closure.
Assuntos
Aminas/química , Colágeno Tipo I/química , Córnea/química , Corantes Fluorescentes/química , Tendões/química , Aminobutiratos/química , Animais , Cromatografia Líquida de Alta Pressão , Cumarínicos/química , Compostos de Dansil/química , Corantes Fluorescentes/síntese química , Masculino , Polilisina/química , Piruvatos/química , Coelhos , o-Ftalaldeído/químicaRESUMO
On the basis of the earlier examples of diazopyruvoyl (DAP) groups reported by Lawton for covalent binding and cross-linking of proteins and oligopeptides and our recent demonstration that a coumaryl diazopyruvamide was used to label Type-I collagen, we have extended our investigations to the synthesis and cross-linking capabilities of a bis-DAP polyethylene glycol to cross-link Type-I collagen. The new photoactivated cross-linking agent, N,N'-bis(3-diazopyruvoyl)-2,2'-(ethylenedioxy)bis(ethylamine) (DPD, 2), has been designed and synthesized specifically to "weld" collagenous tissues by cross-linking Type-I collagen. A working model for the photochemical welding studies of collagenous tissues was developed using gelatin strips (gel strips) composed of denatured Type-I collagen. Gel strips are transparent to near-UV and visible light, uniform in thickness, and have reproducible composition. Furthermore, the availability of nucleophilic amine sites in gel strips was demonstrated by reaction with o-phthalaldehyde, producing a fluorescent derivative of the protein. Gel strips were coated with a solution of DPD in chloroform 7 irradiated at 320-390 nm, and the resulting bonded gel strips were tested for the strength of the weld. The welds were generally brittle and had average tensile strengths that exceeded 100 N/cm2. Welds were not formed in the absence of light or DPD. Scanning electron microscopy studies revealed a pockmarked surface from severed welds. Welds of rabbit Achilles tendon were also obtained using the tethered diazopyruvamide. These welds were much weaker, having an average tensile strength of 11.95 N/cm2 for DPD-2,2'-ethylenedioxy(bis)ethylamine comonomers in the cross-linking reaction. In both studies the welds obtained by this method were significantly stronger than the controls.
Assuntos
Colágeno Tipo I/química , Reagentes de Ligações Cruzadas/síntese química , Reagentes de Ligações Cruzadas/farmacologia , Etilaminas/síntese química , Luz , Piruvatos/síntese química , Tendão do Calcâneo/metabolismo , Aminas/química , Animais , Colágeno Tipo I/metabolismo , Reagentes de Ligações Cruzadas/química , Etilaminas/química , Etilaminas/farmacologia , Gelatina/química , Nitrofenóis/química , Peptídeos/metabolismo , Proteínas/metabolismo , Piruvatos/química , Piruvatos/farmacologia , Coelhos , Resistência à TraçãoRESUMO
Signal transducer and activator of transcription STAT5 and its upstream activating kinase JAK2 are essential mediators of cytokine signaling. Their activity is normally tightly regulated and transient. However, constitutive activation of STAT5 is found in numerous cancers and a driving force for malignant transformation. We describe here the identification of the synthetic chalcone α-Br-2',3,4,4'-tetramethoxychalcone (α-Br-TMC) as a novel JAK/STAT inhibitor. Using the non-transformed IL-3-dependent B cell line Ba/F3 and its oncogenic derivative Ba/F3-1*6 expressing constitutively activated STAT5, we show that α-Br-TMC targets the JAK/STAT pathway at multiple levels, inhibiting both JAK2 and STAT5 phosphorylation. Moreover, α-Br-TMC alters the mobility of STAT5A/B proteins in SDS-PAGE, indicating a change in their post-translational modification state. These alterations correlate with a decreased association of STAT5 and RNA polymerase II with STAT5 target genes in chromatin immunoprecipitation assays. Interestingly, expression of STAT5 target genes such as Cis and c-Myc was differentially regulated by α-Br-TMC in normal and cancer cells. While both genes were inhibited in IL-3-stimulated Ba/F3 cells, expression of the oncogene c-Myc was down-regulated and that of the tumor suppressor gene Cis was up-regulated in transformed Ba/F3-1*6 cells. The synthetic chalcone α-Br-TMC might therefore represent a promising novel anticancer agent for therapeutic intervention in STAT5-associated malignancies.
Assuntos
Chalconas/farmacologia , Janus Quinase 2/metabolismo , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Sequência de Bases , Linhagem Celular , Imunoprecipitação da Cromatina , Primers do DNA , Camundongos , Fosforilação , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The first total synthesis of either enantiomer of Arteludovicinolide A and their biological evaluation is reported, featuring a new strategy for the asymmetric construction of γ-butyrolactones with stereogenic side chains in the 4-position. Starting from the renewable resource methyl 2-furoate, the sesquiterpene lactone was synthesized in 9 steps and 4.8% overall yield via an asymmetric cyclopropanation and two diastereoselective nucleophile additions making use of a donor-acceptor-cyclopropane-lactonization cascade. At noncytotoxic concentrations (≤10 µM) (+)-1 was found to have a 15 times higher anti-inflammatory activity (4.87 ± 1.1 µM) than previously reported for concentrations of ≥45 µM.