RESUMEN
The glucocorticoid receptor (GR) is a crucial drug target in multiple myeloma as its activation with glucocorticoids effectively triggers myeloma cell death. However, as high-dose glucocorticoids are also associated with deleterious side effects, novel approaches are urgently needed to improve GR action in myeloma. Here, we reveal a functional crosstalk between GR and the mineralocorticoid receptor (MR) that plays a role in improved myeloma cell killing. We show that the GR agonist dexamethasone (Dex) downregulates MR levels in a GR-dependent way in myeloma cells. Co-treatment of Dex with the MR antagonist spironolactone (Spi) enhances Dex-induced cell killing in primary, newly diagnosed GC-sensitive myeloma cells. In a relapsed GC-resistant setting, Spi alone induces distinct myeloma cell killing. On a mechanistic level, we find that a GR-MR crosstalk likely arises from an endogenous interaction between GR and MR in myeloma cells. Quantitative dimerization assays show that Spi reduces Dex-induced GR-MR heterodimerization and completely abolishes Dex-induced MR-MR homodimerization, while leaving GR-GR homodimerization intact. Unbiased transcriptomics analyses reveal that c-myc and many of its target genes are downregulated most by combined Dex-Spi treatment. Proteomics analyses further identify that several metabolic hallmarks are modulated most by this combination treatment. Finally, we identified a subset of Dex-Spi downregulated genes and proteins that may predict prognosis in the CoMMpass myeloma patient cohort. Our study demonstrates that GR-MR crosstalk is therapeutically relevant in myeloma as it provides novel strategies for glucocorticoid-based dose-reduction.
Asunto(s)
Glucocorticoides , Mieloma Múltiple , Humanos , Glucocorticoides/farmacología , Receptores de Mineralocorticoides/genética , Dexametasona/farmacología , Dexametasona/metabolismo , Dexametasona/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/genética , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Espironolactona/uso terapéuticoRESUMEN
It has been suggested that glucocorticoid receptor (GR) agonists that promote GR homodimerization more than standard glucocorticoids such as Dexamethasone could be more effective anti-inflammatory molecules against acute and life-threatening inflammatory conditions. To test this hypothesis, we set up a screening pipeline aimed at discovering such Selective Dimerizing GR Agonists and Modulators (SEDIGRAM). The pipeline consists of a reporter gene assay based on a palindromic glucocorticoid responsive element (GRE). This assay represents GR dimerization in human A549 lung epithelial cells. In the pipeline, this is followed by analysis of endogenous GRE-driven gene expression, a FRET assay confirming dimerization, and monitoring of in vitro and in vivo anti-inflammatory activity. In a proof of principle experiment, starting from seven candidate compounds, we identified two potentially interesting compounds (Cortivazol and AZD2906) that confer strong protection in a mouse model of aggressive TNF-induced lethal inflammation. A screening pipeline for SEDIGRAM may assist the search for compounds that promote GR dimerization and limit overwhelming acute inflammatory responses.
Asunto(s)
Antiinflamatorios/farmacología , Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos/métodos , Multimerización de Proteína , Receptores de Glucocorticoides/química , Células A549 , Animales , Antiinflamatorios/química , Dexametasona/farmacología , Modelos Animales de Enfermedad , Descubrimiento de Drogas/métodos , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Humanos , Inflamación/tratamiento farmacológico , Inflamación/etiología , Inflamación/metabolismo , Ratones , Unión Proteica , Piridinas/farmacología , Receptores de Glucocorticoides/agonistas , Elementos de Respuesta , Activación TranscripcionalRESUMEN
BACKGROUND: Laserwort, Laserpitium latifolium L. (Apiaceae), is a European medicinal plant. Its roots and rhizomes were traditionally used as a general tonic and to treat inflammatory and infective diseases. PURPOSE: The anti-inflammatory potential of daucane esters, isolated from underground parts extract of L. latifolium and specific structural features that contribute to their activity were investigated. In addition, we studied their interference with the transactivation capacity of the Glucocorticoid Receptor when added together with a classic glucocorticoid (GC), dexamethasone (DEX). This particular property may be relevant in combination strategies, attempting to circumvent diabetogenic side effects of glucocorticoids upon long-term anti-inflammatory treatments. MATERIALS AND METHODS: Nine L. latifolium daucane esters were isolated and elucidated as derivatives of desoxodehydrolaserpitin, laserpitin and a novel 2ß-esterified laserpitinol analogue. Of all compounds effects on NF-κB- and AP-1-driven pro-inflammatory pathways were assessed using TNF- or PMA-induced reporter gene analysis in A549 cells. Daucanes with a strong and concentration-dependent inhibition of both NF-κB and AP-1, were tested for a potential effect on DEX-stimulated GR-driven Glucocorticoid Response Element (GRE) reporter gene activity. In addition, GRE-driven anti-inflammatory mRNA expression was determined (GILZ and DUSP1). Also anti-inflammatory properties were validated by monitoring effects on CCL-2, IL-6, IL-1ß mRNA expression levels (qPCR) and on CCL-2 chemokine production (ELISA). RESULTS: Daucanes featuring an ester moiety and/or a hydroxy group at positions 2ß, 6α and 10α and especially the novel 2ß-esterified laserpitinol derivative that, in comparison to other isolated compounds, features an additional 9α-hydroxy group, demonstrated suppression of both NF-κB- and AP-1-dependent pro-inflammatory pathways. Remarkably, those entities competitively and concentration-dependently repressed GR-driven GRE-dependent reporter gene activities. The most active compounds inhibited CCL-2 protein excretion and compound 4 downregulated genes coding for IL-1ß and IL-6 induced upon TNF treatment in A549. In absence of TNF, compound 4 upregulated the GRE-mediated anti-inflammatory gene GILZ, but not DUSP1. CONCLUSIONS: Daucane esters are novel anti-inflammatory agents that may, in combination with GCs, potentially improve therapeutic benefit. These results contribute to the ongoing search for novel anti-inflammatory agents as safer alternatives to, or with, GCs.
Asunto(s)
Antiinflamatorios/farmacología , Apiaceae/química , Quimiocinas/metabolismo , Citocinas/metabolismo , Células Epiteliales/efectos de los fármacos , Ésteres/farmacología , Extractos Vegetales/farmacología , Células Epiteliales/metabolismo , Humanos , Pulmón/metabolismo , Raíces de Plantas/química , Rizoma/química , SerbiaRESUMEN
Protein-protein interactions (PPIs) underlie most biological processes. An increasing interest to investigate the unexplored potential of PPIs in drug discovery is driven by the need to find novel therapeutic targets for a whole range of diseases with a high unmet medical need. To date, PPI inhibition with small molecules is the mechanism that has most often been explored, resulting in significant progress towards drug development. However, also PPI stabilization is gradually gaining ground. In this review, we provide a focused overview of a number of PPIs that control critical regulatory pathways and constitute targets for the design of novel therapeutics. We discuss PPI-modulating small molecules that are already pursued in clinical trials. In addition, we review a number of PPIs that are still under preclinical investigation but for which preliminary data support their use as therapeutic targets.
Asunto(s)
Descubrimiento de Drogas/métodos , Redes Reguladoras de Genes/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Animales , Evaluación Preclínica de Medicamentos , Humanos , Mapeo de Interacción de Proteínas , Proteínas/metabolismoRESUMEN
Withaferin A (WA) isolated from Withania somnifera (Ashwagandha) has recently become an attractive phytochemical under investigation in various preclinical studies for treatment of different cancer types. In the present study, a comparative pathway-based transcriptome analysis was applied in epithelial-like MCF-7 and triple negative mesenchymal MDA-MB-231 breast cancer cells exposed to different concentrations of WA which can be detected systemically in in vivo experiments. Whereas WA treatment demonstrated attenuation of multiple cancer hallmarks, the withanolide analogue Withanone (WN) did not exert any of the described effects at comparable concentrations. Pathway enrichment analysis revealed that WA targets specific cancer processes related to cell death, cell cycle and proliferation, which could be functionally validated by flow cytometry and real-time cell proliferation assays. WA also strongly decreased MDA-MB-231 invasion as determined by single-cell collagen invasion assay. This was further supported by decreased gene expression of extracellular matrix-degrading proteases (uPA, PLAT, ADAM8), cell adhesion molecules (integrins, laminins), pro-inflammatory mediators of the metastasis-promoting tumor microenvironment (TNFSF12, IL6, ANGPTL2, CSF1R) and concomitant increased expression of the validated breast cancer metastasis suppressor gene (BRMS1). In line with the transcriptional changes, nanomolar concentrations of WA significantly decreased protein levels and corresponding activity of uPA in MDA-MB-231 cell supernatant, further supporting its anti-metastatic properties. Finally, hierarchical clustering analysis of 84 chromatin writer-reader-eraser enzymes revealed that WA treatment of invasive mesenchymal MDA-MB-231 cells reprogrammed their transcription levels more similarly towards the pattern observed in non-invasive MCF-7 cells. In conclusion, taking into account that sub-cytotoxic concentrations of WA target multiple metastatic effectors in therapy-resistant triple negative breast cancer, WA-based therapeutic strategies targeting the uPA pathway hold promise for further (pre)clinical development to defeat aggressive metastatic breast cancer.
Asunto(s)
Apoptosis/efectos de los fármacos , Biomarcadores de Tumor/genética , Fitoterapia , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patología , Witanólidos/farmacología , Biomarcadores de Tumor/metabolismo , Western Blotting , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Femenino , Perfilación de la Expresión Génica , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias de la Mama Triple Negativas/genética , Células Tumorales CultivadasRESUMEN
Several bamboo species have been used in traditional medicine for the treatment of inflammatory conditions. The present study evaluates the in vitro anti-inflammatory properties of the traditionally used bamboo species Phyllostachys nigra (Lodd.) Munro and Sasa veitchii (Carr.) Rehder to explore their future research opportunities and therapeutic potential as anti-inflammatory agents. The extracts were evaluated for their potential inhibitory activity at the level of NF-κB-induced gene expression and suppression of cyclooxygenase (COX)-1 and COX-2 enzyme activities, representative pharmacological targets for the anti-inflammatory action of glucocorticoids and non-steroidal anti-inflammatory drugs, respectively. The activity of P. nigra (Lodd.) Munro and S. veitchii (Carr.) Rehder was compared with bamboo species without traditional anti-inflammatory indications. High-performance liquid chromatography with diode-array detection and liquid chromatography-tandem mass spectrometry analyses were performed to phytochemically characterize the extracts. P. nigra (Lodd.) Munro leaf extract potently inhibited NF-κB-induced gene expression, while S. veitchii (Carr.) Rehder leaf extract exerted a selective COX-2 inhibition. The crude extracts consistently showed a more potent bioactivity than the solid phase extraction fractions. P. nigra (Lodd.) Munro and S. veitchii (Carr.) Rehder both exert anti-inflammatory properties, but act via a different molecular mechanism.
Asunto(s)
Antiinflamatorios/farmacología , Inhibidores de la Ciclooxigenasa 2/farmacología , FN-kappa B/antagonistas & inhibidores , Extractos Vegetales/farmacología , Poaceae/química , Sasa/química , Animales , Antiinflamatorios/química , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Ciclooxigenasa 1/metabolismo , Ciclooxigenasa 2/metabolismo , Inhibidores de la Ciclooxigenasa 2/química , Expresión Génica/efectos de los fármacos , Ratones , FN-kappa B/genética , FN-kappa B/metabolismo , Extractos Vegetales/química , Hojas de la Planta/química , Prostaglandina-Endoperóxido Sintasas/análisisRESUMEN
The glucocorticoid receptor (GR) is a transcription factor able to support either target gene activation via direct binding to DNA or gene repression via interfering with the activity of various proinflammatory transcription factors. An improved therapeutic profile for combating chronic inflammatory diseases has been reported through selectively modulating the GR by only triggering its transrepression function. We have studied in this paper the activity of Compound A (CpdA), a dissociated GR modulator favoring GR monomer formation, in a predominantly Th2-driven asthma model. CpdA acted similarly to the glucocorticoid dexamethasone (DEX) in counteracting OVA-induced airway hyperresponsiveness, recruitment of eosinophils, dendritic cells, neutrophils, B and T cells, and macrophages in bronchoalveolar lavage fluid, lung Th2, Tc2, Th17, Tc17, and mast cell infiltration, collagen deposition, and goblet cell metaplasia. Both CpdA and DEX inhibited Th2 cytokine production in bronchoalveolar lavage as well as nuclear translocation of NF-κB and its subsequent recruitment onto the IκBα promoter in the lung. By contrast, DEX but not CpdA induces expression of the GR-dependent model gene MAPK phosphatase 1 in the lung, confirming the dissociative action of CpdA. Mechanistically, we demonstrate that CpdA inhibited IL-4-induced STAT6 translocation and that GR is essential for CpdA to mediate chemokine repression. In conclusion, we clearly show in this study the anti-inflammatory effect of CpdA in a Th2-driven asthma model in the absence of transactivation, suggesting a potential therapeutic benefit of this strategy.
Asunto(s)
Antiasmáticos/uso terapéutico , Antiinflamatorios no Esteroideos/uso terapéutico , Asma/tratamiento farmacológico , Hiperreactividad Bronquial/tratamiento farmacológico , Compuestos de Amonio Cuaternario/uso terapéutico , Receptores de Glucocorticoides/efectos de los fármacos , Acetatos , Animales , Hiperreactividad Bronquial/fisiopatología , Líquido del Lavado Bronquioalveolar/citología , Citocinas/biosíntesis , Citocinas/genética , Dexametasona/farmacología , Dexametasona/uso terapéutico , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Fosfatasa 1 de Especificidad Dual/biosíntesis , Fosfatasa 1 de Especificidad Dual/genética , Inducción Enzimática/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Células Caliciformes/patología , Inflamación , Leucocitos/inmunología , Pulmón/inmunología , Pulmón/patología , Mastocitos/inmunología , Metaplasia , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/toxicidad , Compuestos de Amonio Cuaternario/farmacología , Receptores de Glucocorticoides/fisiología , Factor de Transcripción STAT6/metabolismo , Activación Transcripcional/efectos de los fármacos , Tiramina/análogos & derivadosRESUMEN
Glucocorticoids (GCs) are in widespread use to treat inflammatory bone diseases, such as rheumatoid arthritis (RA). Their anti-inflammatory efficacy, however, is accompanied by deleterious effects on bone, leading to GC-induced osteoporosis (GIO). These effects include up-regulation of the receptor activator of NF-κB ligand/osteoprotegerin (RANKL/OPG) ratio to promote bone-resorbing osteoclasts and include inhibition of bone-forming osteoblasts. We previously identified suppression of osteoblast differentiation by the monomer glucocorticoid receptor (GR) via the inhibition of Il11 expression as a crucial mechanism for GIO. Here we show that the GR-modulating substance compound A (CpdA), which does not induce GR dimerization, still suppresses proinflammatory cytokines in fibroblast-like synovial cells from patients with RA and in osteoblasts. In contrast to the full GR agonist dexamethasone, it does not unfavorably alter the RANKL/OPG ratio and does not affect Il11 expression and subsequent STAT3 phosphorylation in these cells. Notably, while dexamethasone inhibits osteoblast differentiation, CpdA does not affect osteoblast differentiation in vitro and in vivo. We describe here for the first time that selective GR modulators can act against inflammation, while not impairing osteoblast differentiation.
Asunto(s)
Glucocorticoides/efectos adversos , Osteoblastos/efectos de los fármacos , Osteoporosis/inducido químicamente , Osteoprotegerina/metabolismo , Receptores de Glucocorticoides/fisiología , Animales , Antiinflamatorios no Esteroideos/farmacología , Aziridinas/farmacología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Dexametasona/farmacología , Femenino , Humanos , Interleucina-11/biosíntesis , Interleucina-11/genética , Masculino , Osteoblastos/metabolismo , Osteoclastos/efectos de los fármacos , Extractos Vegetales/farmacología , Ligando RANK/metabolismoRESUMEN
Hop (Humulus lupulus L.) is an essential ingredient of beer, where it provides the typical bitter taste, but is also applied in traditional folk medicine for sedative and antibacterial purposes. In this study, we demonstrate and compare the anti-inflammatory effect of various classes of hop bitter acids (HBA), including alpha-acids (AA), beta-acids (BA), and iso-alpha-acids (IAA), in fibroblasts, which are important players in the inflammatory response. All three studied classes of HBA blocked the tumor necrosis factor alpha (TNF)-induced production of the cytokine IL6, and inhibited the transactivation of the pro-inflammatory transcription factors nuclear factor kappa B (NF-kappaB), activator protein-1 (AP-1), and cAMP-response element-binding protein (CREB). In this respect, the six-membered ring compounds AA and BA showed equal potency, whereas the five-membered ring compounds, IAA, were effective only when used at higher concentrations. Furthermore, with regard to the mechanism of NF-kappaB suppression, we excluded a possible role for glucocorticoid receptor alpha (GRalpha), peroxisome proliferators-activated receptor alpha/gamma (PPARalpha or PPARgamma), nuclear receptors (NRs) that are also known to inhibit inflammation by directly interfering with the activity of pro-inflammatory transcription factors. Interestingly, combining hop acids and selective agonists for GRalpha, PPARalpha, or PPARgamma resulted in additive inhibition of NF-kappaB activity after TNF treatment, which may open up new avenues for combinatorial anti-inflammatory strategies with fewer side effects. Finally, systemic administration of HBA efficiently inhibited acute local inflammation in vivo.
Asunto(s)
Antiinflamatorios/farmacología , Humulus/química , PPAR alfa/fisiología , PPAR gamma/fisiología , Receptores de Glucocorticoides/fisiología , Animales , Línea Celular Tumoral , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/fisiología , Femenino , Humanos , Interleucina-6/biosíntesis , Ratones , Ratones Endogámicos C57BL , FN-kappa B/antagonistas & inhibidores , Factor de Transcripción AP-1/fisiología , Transcripción Genética/efectos de los fármacosRESUMEN
Hop acids, a family of bitter compounds derived from the hop plant (Humulus lupulus), have been reported to exert a wide range of effects, both in vitro and in vivo. They exhibit potential anticancer activity by inhibiting cell proliferation and angiogenesis, by inducing apoptosis, and by increasing the expression of cytochrome P450 detoxification enzymes. Furthermore, hop bitter acids are effective against inflammatory and metabolic disorders, which makes them challenging candidates for the treatment of diabetes mellitus, cardiovascular diseases, and metabolic syndrome. This review summarizes the current knowledge on hop bitter acids, including both phytochemical aspects, as well as the biological and pharmacological properties of these compounds.
Asunto(s)
Ácidos/farmacología , Antineoplásicos Fitogénicos/farmacología , Sistema Nervioso Central/efectos de los fármacos , Humulus/química , Plantas Medicinales/química , Ácidos/análisis , Animales , Antineoplásicos Fitogénicos/aislamiento & purificación , Apoptosis/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Ratones , RatasRESUMEN
The glucocorticoid receptor (GR) is a transcription factor regulating its target genes either positively, through direct binding to the promoter of target genes, or negatively by the interference with the activity of transcription factors involved in proinflammatory gene expression. The well-known adverse effects of glucocorticoids are believed to be mainly caused by their GR-mediated gene-activating properties. Although dimerization of GR is thought to be essential for gene-activating properties, no compound has yet been described which selectively imposes GR monomer formation and interference with other transcription factors. In the present study, we report on a GR-binding, plant-derived compound with marked dissociative properties in rheumatoid arthritis fibroblast-like synoviocytes, which are important effector cells in inflammation and matrix degradation in rheumatoid arthritis. In addition, these findings could be extended in vivo in murine collagen-induced arthritis, in which joint inflammation was markedly inhibited without inducing hyperinsulinemia. Therefore, we conclude that GR monomers are sufficient for inhibition of inflammation in vivo.
Asunto(s)
Acetatos/metabolismo , Acetatos/farmacología , Artritis Experimental/metabolismo , Artritis Experimental/prevención & control , Etilaminas/metabolismo , Etilaminas/farmacología , Extractos Vegetales/metabolismo , Receptores de Glucocorticoides/antagonistas & inhibidores , Salsola , Transactivadores/antagonistas & inhibidores , Acetatos/administración & dosificación , Transporte Activo de Núcleo Celular/efectos de los fármacos , Animales , Antiinflamatorios no Esteroideos/administración & dosificación , Antiinflamatorios no Esteroideos/metabolismo , Antiinflamatorios no Esteroideos/farmacología , Artritis Experimental/patología , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Línea Celular , Colágeno Tipo II/toxicidad , Dexametasona/metabolismo , Dexametasona/farmacología , Dimerización , Etilaminas/administración & dosificación , Humanos , Ligandos , Masculino , Ratones , Ratones Endogámicos DBA , Extractos Vegetales/administración & dosificación , Extractos Vegetales/farmacología , Conformación Proteica/efectos de los fármacos , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/fisiología , Elementos de Respuesta/efectos de los fármacos , Elementos de Respuesta/genética , Membrana Sinovial/efectos de los fármacos , Membrana Sinovial/metabolismo , Membrana Sinovial/patología , Transactivadores/genética , Transactivadores/fisiología , Tiramina/análogos & derivadosRESUMEN
The identification of selective glucocorticoid receptor (GR) modifiers, which separate transactivation and transrepression properties, represents an important research goal for steroid pharmacology. Although the gene-activating properties of GR are mainly associated with undesirable side effects, its negative interference with the activity of transcription factors, such as NF-kappaB, greatly contributes to its antiinflammatory and immune-suppressive capacities. In the present study, we found that Compound A (CpdA), a plant-derived phenyl aziridine precursor, although not belonging to the steroidal class of GR-binding ligands, does mediate gene-inhibitory effects by activating GR. We demonstrate that CpdA exerts an antiinflammatory potential by down-modulating TNF-induced proinflammatory gene expression, such as IL-6 and E-selectin, but, interestingly, does not at all enhance glucocorticoid response element-driven genes or induce GR binding to glucocorticoid response element-dependent genes in vivo. We further show that the specific gene-repressive effect of CpdA depends on the presence of functional GR, displaying a differential phosphorylation status with CpdA as compared with dexamethasone treatment. The antiinflammatory mechanism involves both a reduction of the in vivo DNA-binding activity of p65 as well as an interference with the transactivation potential of NF-kappaB. Finally, we present evidence that CpdA is as effective as dexamethasone in counteracting acute inflammation in vivo and does not cause a hyperglycemic side effect. Taken together, this compound may be a lead compound of a class of antiinflammatory agents with fully dissociated properties and might thus hold great potential for therapeutic use.