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1.
Cell Mol Life Sci ; 80(9): 249, 2023 Aug 14.
Article in English | MEDLINE | ID: mdl-37578563

ABSTRACT

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.


Subject(s)
Glucocorticoids , Multiple Myeloma , Humans , Glucocorticoids/pharmacology , Receptors, Mineralocorticoid/genetics , Dexamethasone/pharmacology , Dexamethasone/metabolism , Dexamethasone/therapeutic use , Multiple Myeloma/drug therapy , Multiple Myeloma/genetics , Receptors, Glucocorticoid/genetics , Receptors, Glucocorticoid/metabolism , Spironolactone/therapeutic use
3.
Mar Drugs ; 18(1)2020 Jan 10.
Article in English | MEDLINE | ID: mdl-31936833

ABSTRACT

Respiratory exposure to marine phycotoxins is of increasing concern. Inhalation of sea spray aerosols (SSAs), during harmful Karenia brevis and Ostreopsis ovata blooms induces respiratory distress among others. The biogenics hypothesis, however, suggests that regular airborne exposure to natural products is health promoting via a downregulation of the mechanistic target of rapamycin (mTOR) pathway. Until now, little scientific evidence supported this hypothesis. The current explorative in vitro study investigated both health-affecting and potential health-promoting mechanisms of airborne phycotoxin exposure, by analyzing cell viability effects via cytotoxicity assays and effects on the mTOR pathway via western blotting. To that end, A549 and BEAS-2B lung cells were exposed to increasing concentrations (ng·L-1 - mg·L-1) of (1) pure phycotoxins and (2) an extract of experimental aerosolized homoyessotoxin (hYTX). The lowest cell viability effect concentrations were found for the examined yessotoxins (YTXs). Contradictory to the other phycotoxins, these YTXs only induced a partial cell viability decrease at the highest test concentrations. Growth inhibition and apoptosis, both linked to mTOR pathway activity, may explain these effects, as both YTXs were shown to downregulate this pathway. This proof-of-principle study supports the biogenics hypothesis, as specific aerosolizable marine products (e.g., YTXs) can downregulate the mTOR pathway.


Subject(s)
Aerosols/pharmacology , Environmental Exposure , Lung/drug effects , Marine Toxins/pharmacology , TOR Serine-Threonine Kinases/metabolism , A549 Cells , Aerosols/toxicity , Cell Line , Cell Survival/drug effects , Enzyme Activation/drug effects , Harmful Algal Bloom , Humans , In Vitro Techniques , Marine Toxins/toxicity , Mollusk Venoms , Oxocins/pharmacology , Signal Transduction/drug effects
4.
Front Immunol ; 10: 1769, 2019.
Article in English | MEDLINE | ID: mdl-31447832

ABSTRACT

Glucocorticoids (GCs) act via the glucocorticoid receptor (NR3C1, GRα) to combat overshooting responses to infectious stimuli, including lipopolysaccharide (LPS). As such, GCs inhibit the activity of downstream effector cytokines, such as tumor necrosis factor (TNF). PPARα (NR1C1) is a nuclear receptor described to function on the crossroad between lipid metabolism and control of inflammation. In the current work, we have investigated the molecular mechanism by which GCs and PPARα agonists cooperate to jointly inhibit NF-κB-driven expression in A549 cells. We discovered a nuclear mechanism that predominantly targets Mitogen- and Stress-activated protein Kinase-1 activation upon co-triggering GRα and PPARα. In vitro GST-pull down data further support that the anti-inflammatory mechanism may additionally involve a non-competitive physical interaction between the p65 subunit of NF-κB, GRα, and PPARα. Finally, to study metabolic effector target cells common to both receptors, we overlaid the effect of GRα and PPARα crosstalk in mouse primary hepatocytes under LPS-induced inflammatory conditions on a genome-wide level. RNA-seq results revealed lipid metabolism genes that were upregulated and inflammatory genes that were additively downregulated. Validation at the cytokine protein level finally supported a consistent additive anti-inflammatory response in hepatocytes.


Subject(s)
Inflammation/immunology , PPAR alpha/immunology , Receptors, Glucocorticoid/immunology , A549 Cells , Animals , Dexamethasone/pharmacology , Glucocorticoids/pharmacology , Hepatocytes/drug effects , Hepatocytes/metabolism , Humans , Lipid Metabolism/genetics , Lipopolysaccharides , Male , Mice, Inbred C57BL , NF-kappa B/immunology , PPAR alpha/agonists
5.
PLoS One ; 13(5): e0197000, 2018.
Article in English | MEDLINE | ID: mdl-29738549

ABSTRACT

Glucocorticoids (GCs) are a cornerstone in the treatment of lymphoid malignancies such as multiple myeloma (MM) and acute lymphoblastic leukemia (ALL). Yet, prolonged GC use is hampered by deleterious GC-related side effects and the emergence of GC resistance. To tackle and overcome these GC-related problems, the applicability of selective glucocorticoid receptor agonists and modulators was studied, in search of fewer side-effects and at least equal therapeutic efficacy as classic GCs. Compound A (CpdA) is a prototypical example of such a selective glucocorticoid receptor modulator and does not support GR-mediated transactivation. Here, we examined whether the combination of CpdA with the classic GC dexamethasone (Dex) may improve GC responsiveness of MM and ALL cell lines. We find that the combination of Dex and CpdA does not substantially enhance GC-mediated cell killing. In line, several apoptosis hallmarks, such as caspase 3/7 activity, PARP cleavage and the levels of cleaved-caspase 3 remain unchanged upon combining Dex with CpdA. Moreover, we monitor no additional inhibition of cell proliferation and the homologous downregulation of GR is not counteracted by the combination of Dex and CpdA. In addition, CpdA is unable to modulate Dex-liganded GR transactivation and transrepression, yet, Dex-mediated transrepression is also aberrant in these lymphoid cell lines. Together, transrepression-favoring compounds, alone or combined with GCs, do not seem a valid strategy in the treatment of lymphoid malignancies.


Subject(s)
Aziridines/administration & dosage , Dexamethasone/administration & dosage , Multiple Myeloma/drug therapy , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Apoptosis/drug effects , Aziridines/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Glucocorticoids/administration & dosage , Glucocorticoids/chemistry , Humans , Multiple Myeloma/pathology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Receptors, Glucocorticoid/genetics , Transcriptional Activation/drug effects
6.
Exp Cell Res ; 362(2): 332-342, 2018 01 15.
Article in English | MEDLINE | ID: mdl-29196164

ABSTRACT

Cancer-associated fibroblasts (CAFs) support cancer growth, invasion, and metastasis. Glucocorticoids (GCs), drugs often administered together with chemotherapy, are steroidal ligands of the glucocorticoid receptor (GR), a transcription factor which upon activation regulates expression of multiple genes involved in suppression of inflammation. We have previously shown that in dexamethasone (Dex)-treated CAFs derived from colon cancer, production and secretion of several factors related to cancer progression, such as tenascin C (TNC) and hepatocyte growth factor (HGF), were strongly suppressed. In this study we show that GCs can neutralize the cancer cell-promoting properties of CAFs. Conditioned medium from solvent-treated CAFs (CMCTRL) stimulates proliferation, motility and stretched morphotype of GR-deficient HCT8/E11 colon cancer cells. Yet, HCT8/E11 proliferation and stretched morphotype are impaired upon treatment with conditioned medium from Dex-treated CAFs (CMDEX), but HCT8/E11 cell migration is slightly increased under these conditions. Moreover, expression and potential activity of MMP-2 is also reduced in CMDEX compared with CMCTRL. These combined in vitro results concur with the results from in vivo chick chorioallantoic membrane assays, where the co-cultures of CAFs with colon cancer cells displayed impaired tumor formation and cancer cell invasion due to Dex administration. Combined, GC treatment influences cancer cell behavior indirectly through effects on CAFs.


Subject(s)
Cancer-Associated Fibroblasts/drug effects , Cell Proliferation/drug effects , Colonic Neoplasms/drug therapy , Glucocorticoids/administration & dosage , Animals , Cancer-Associated Fibroblasts/pathology , Cell Line, Tumor , Cell Movement/drug effects , Coculture Techniques , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , Dexamethasone/administration & dosage , Gene Expression Regulation, Neoplastic/drug effects , Hepatocyte Growth Factor/genetics , Humans , Matrix Metalloproteinase 2/genetics , Neoplasm Invasiveness/genetics , Neoplasm Invasiveness/pathology , Tenascin/genetics
7.
Vascul Pharmacol ; 89: 19-30, 2017 02.
Article in English | MEDLINE | ID: mdl-27717848

ABSTRACT

Angiogenesis is important in cancer progression and can be influenced by tumor-associated myofibroblasts. We addressed the hypothesis that glucocorticoids indirectly affect angiogenesis by altering the release of pro-angiogenic factors from colon cancer-derived myofibroblasts. Our study shows that glucocorticoids reduced prostanoids, urokinase-type plasminogen activator (uPA) and angiopoietin-like protein-2 (ANGPTL2) levels, but increased angiogenin (ANG) in supernatant from human CT5.3hTERT colon cancer-derived myofibroblasts. Conditioned medium from solvent- (CMS) and dexamethasone (Dex)-treated (CMD) myofibroblasts increased human umbilical vein endothelial cell (HUVEC) proliferation, but did not affect expression of pro-angiogenic factors or tube-like structure formation (by HUVECs or human aortic ECs). In a HUVEC scratch assay CMS-induced acceleration of wound healing was blunted by CMD treatment. Moreover, CMS-induced neovessel growth in mouse aortic rings ex vivo was also blunted using CMD. The latter effect could be ascribed to both Dex-driven reduction of secreted factors and potential residual Dex present in CMD (indicated using a dexamethasone-spiked CMS control). A similar control in the scratch assay, however, revealed that altered levels of factors in the CMD, and not potential residual Dex, were responsible for decreased wound closure. In conclusion, our results suggest that glucocorticoids indirectly alter endothelial cell function during tumor development in vivo.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Cancer-Associated Fibroblasts/drug effects , Cell Movement/drug effects , Dexamethasone/pharmacology , Endothelial Cells/drug effects , Glucocorticoids/pharmacology , Myofibroblasts/drug effects , Stromal Cells/drug effects , Angiopoietin-Like Protein 2 , Angiopoietin-like Proteins , Angiopoietins/genetics , Angiopoietins/metabolism , Animals , Cancer-Associated Fibroblasts/metabolism , Cancer-Associated Fibroblasts/pathology , Cell Line, Transformed , Cell Proliferation/drug effects , Colonic Neoplasms/drug therapy , Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Culture Media, Conditioned/metabolism , Endothelial Cells/metabolism , Endothelial Cells/pathology , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Male , Mice, Inbred C57BL , Myofibroblasts/metabolism , Myofibroblasts/pathology , Neovascularization, Pathologic , Neovascularization, Physiologic/drug effects , Paracrine Communication/drug effects , Ribonuclease, Pancreatic/genetics , Ribonuclease, Pancreatic/metabolism , Signal Transduction/drug effects , Stromal Cells/metabolism , Stromal Cells/pathology , Time Factors , Tissue Culture Techniques , Tumor Microenvironment , Urokinase-Type Plasminogen Activator/genetics , Urokinase-Type Plasminogen Activator/metabolism
8.
Curr Drug Targets ; 18(8): 964-982, 2017.
Article in English | MEDLINE | ID: mdl-27804849

ABSTRACT

Historically, the word cancer is derived from the Latin cancer, as the red swollen arteries near a tumor reminded the physician Galenus and his fellow Romans of a red crab. Currently, cancer remains the disease to beat as it remains a leading cause of death worldwide (WHO). Tumors do not simply consist of cancer cells, as they can recruit normal cells, which will form the tumor-associated stroma. These stromal cells together with the extracellular matrix, constitute the tumor microenvironment. Reciprocal communication between tumor-associated stromal cells and cancer cells is important for the induction of epithelial-to-mesenchymal transition and invasion. A detailed knowledge of this communication can spark the development of novel therapeutic strategies aimed at tackling yet unaddressed regulators of invasion and thus metastasis. Therefore, this review will focus not only on epithelial-to-mesenchymal transition and invasion, but also on communication between tumor cells, in particular colon cancer cells, and their stroma, with a primordial focus on cancer-associated fibroblasts, and lastly this review will discuss how this communication can affect the cancer cell's ability to invade its surroundings and form metastases.


Subject(s)
Cancer-Associated Fibroblasts/metabolism , Colonic Neoplasms/metabolism , Myofibroblasts/metabolism , Cell Communication , Disease Progression , Epithelial-Mesenchymal Transition , Gene Expression Regulation, Neoplastic , Gene Regulatory Networks , Humans , Neoplasm Invasiveness , Tumor Microenvironment
9.
Oncoscience ; 3(7-8): 188-202, 2016.
Article in English | MEDLINE | ID: mdl-27713909

ABSTRACT

Although adverse effects and glucocorticoid resistance cripple their chronic use, glucocorticoids form the mainstay therapy for acute and chronic inflammatory disorders, and play an important role in treatment protocols of both lymphoid malignancies and as adjuvant to stimulate therapy tolerability in various solid tumors. Glucocorticoid binding to their designate glucocorticoid receptor (GR), sets off a plethora of cell-specific events including therapeutically desirable effects, such as cell death, as well as undesirable effects, including chemotherapy resistance, systemic side effects and glucocorticoid resistance. In this context, selective GR agonists and modulators (SEGRAMs) with a more restricted GR activity profile have been developed, holding promise for further clinical development in anti-inflammatory and potentially in cancer therapies. Thus far, the research into the prospective benefits of selective GR modulators in cancer therapy limped behind. Our review discusses how selective GR agonists and modulators could improve the therapy regimens for lymphoid malignancies, prostate or breast cancer. We summarize our current knowledge and look forward to where the field should move to in the future. Altogether, our review clarifies novel therapeutic perspectives in cancer modulation via selective GR targeting.

10.
Trends Pharmacol Sci ; 37(1): 4-16, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26603477

ABSTRACT

Since the 1950s, glucocorticoids (GCs) have been a mainstay therapy for acute and chronic inflammatory disorders, although adverse effects limit their chronic use. Following the notion that the anti-inflammatory therapeutic and metabolic endocrine adverse effects of GCs may be based on different glucocorticoid receptor (GR)-dependent mechanisms, subsequent attempts to separate these mechanisms by trying to develop selective GR agonists and modulators (SEGRAMs) with an improved therapeutic benefit have yielded only a few molecules effective in clinical use. Recent new insights into the pro- and anti-inflammatory activities of GR support a more sophisticated drug discovery model. Here, we suggest that the way forward may include a need to redefine the pharmacological SEGRAM concept into selective monomerizing GR agonists and modulators (SEMOGRAMs) and selective dimerizing GR agonists or modulators (SEDIGRAMs) for selective therapeutic applications against chronic or acute inflammatory disorders, respectively.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Inflammation/drug therapy , Inflammation/metabolism , Receptors, Glucocorticoid/metabolism , Acute Disease , Animals , Humans
11.
Pharmacol Ther ; 152: 28-41, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25958032

ABSTRACT

Glucocorticoids remain the frontline treatment for inflammatory disorders, yet represent a double-edged sword with beneficial therapeutic actions alongside adverse effects, mainly in metabolic regulation. Considerable efforts were made to improve this balance by attempting to amplify therapeutic beneficial anti-inflammatory actions and to minimize adverse metabolic actions. Most attention has focused on the development of novel compounds favoring the transrepressing actions of the glucocorticoid receptor, assumed to be important for anti-inflammatory actions, over the transactivating actions, assumed to underpin the undesirable actions. These compounds are classified as selective glucocorticoid receptor agonists (SEGRAs) or selective glucocorticoid receptor modulators (SEGRMs). The latter class is able to modulate the activity of a GR agonist and/or may not classically bind the glucocorticoid receptor ligand-binding pocket. SEGRAs and SEGRMs are collectively denominated SEGRAMs (selective glucocorticoid receptor agonists and modulators). Although this transrepression vs transactivation concept proved to be too simplistic, the developed SEGRAMs were helpful in elucidating various molecular actions of the glucocorticoid receptor, but have also raised many novel questions. We discuss lessons learned from recent mechanistic studies of selective glucocorticoid receptor modulators. This is approached by analyzing recent experimental insights in comparison with knowledge obtained using mutant GR research, thus clarifying the current view on the SEGRAM field. These insights also contribute to our understanding of the processes controlling glucocorticoid-mediated side effects as well as glucocorticoid resistance. Our perspective on non-steroidal SEGRAs and SEGRMs considers remaining opportunities to address research gaps in order to harness the potential for more safe and effective glucocorticoid receptor therapies.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Glucocorticoids/pharmacology , Receptors, Glucocorticoid/agonists , Receptors, Glucocorticoid/physiology , Animals , Binding Sites/physiology , Humans , Immunosuppressive Agents/pharmacology , Signal Transduction/drug effects , Signal Transduction/physiology
12.
PLoS One ; 10(4): e0123068, 2015.
Article in English | MEDLINE | ID: mdl-25875480

ABSTRACT

BACKGROUND: Glucocorticoid Receptor agonists, particularly classic glucocorticoids, are the mainstay among treatment protocols for various chronic inflammatory disorders, including nasal disease. To steer away from steroid-induced side effects, novel GR modulators exhibiting a more favorable therapeutic profile remain actively sought after. Currently, the impact of 2-(4-acetoxyphenyl)-2-chloro-N-methylethylammonium chloride a plant-derived selective glucocorticoid receptor modulator named compound A, on cytokine production in ex vivo human immune cells and tissue has scarcely been evaluated. METHODS AND RESULTS: The current study aimed to investigate the effect of a classic glucocorticoid versus compound A on cytokine and inflammatory mediator production after stimulation with Staphylococcus aureus-derived enterotoxin B protein in peripheral blood mononuclear cells (PBMCs) as well as in inferior nasal turbinate tissue. To this end, tissue fragments were stimulated with RPMI (negative control) or Staphylococcus aureus-derived enterotoxin B protein for 24 hours, in presence of solvent, or the glucocorticoid methylprednisolone or compound A at various concentrations. Supernatants were measured via multiplex for pro-inflammatory cytokines (IL-1ß, TNFα) and T-cell- and subset-related cytokines (IFN-γ, IL-2, IL-5, IL-6, IL-10, and IL-17). In concordance with the previously described stimulatory role of superantigens in the development of nasal polyposis, a 24h Staphylococcus aureus-derived enterotoxin B protein stimulation induced a significant increase of IL-2, IL-1ß, TNF-α, and IL-17 in PBMCs and in inferior turbinates and of IL-5 and IFN-γ in PBMCs. CONCLUSION: Notwithstanding some differences in amplitude, the overall cytokine responses to methylprednisolone and compound A were relatively similar, pointing to a conserved and common mechanism in cytokine transrepression and anti-inflammatory actions of these GR modulators. Furthermore, these results provide evidence that selective glucocorticoid receptor modulator-mediated manipulation of the glucocorticoid receptor in human tissues, supports its anti-inflammatory potential.


Subject(s)
Cytokines/metabolism , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Receptors, Glucocorticoid/metabolism , Acetates/pharmacology , Adolescent , Adult , Anti-Inflammatory Agents/pharmacology , Cell Survival/drug effects , Female , Glucocorticoids/pharmacology , Humans , Inflammation Mediators/metabolism , Male , Methylprednisolone/pharmacology , Middle Aged , Mifepristone/pharmacology , Receptors, Glucocorticoid/agonists , Receptors, Glucocorticoid/antagonists & inhibitors , Th1 Cells/drug effects , Th1 Cells/metabolism , Th2 Cells/drug effects , Th2 Cells/metabolism , Tyramine/analogs & derivatives , Tyramine/pharmacology , Young Adult
13.
J Steroid Biochem Mol Biol ; 149: 92-105, 2015 May.
Article in English | MEDLINE | ID: mdl-25666906

ABSTRACT

The glucocorticoid receptor functions as a ligand-dependent transcription factor that positively or negatively regulates the transcription of various specific target genes. Not only steroidal glucocorticoids can bind and activate the glucocorticoid receptor, but also the intensively examined non-steroidal selective glucocorticoid receptor modulators can do so, albeit with a select effector profile skewed to glucocorticoid receptor transrepression. Glucocorticoids are widely used to treat inflammatory afflictions, but also as anti-cancer therapies or adjuvants thereof. As the impact of glucocorticoids and selective glucocorticoid receptor modulators has scarcely been researched in this setting, we focused on colon cancer and its stromal environment, in particular the stromal myofibroblasts, which are known to influence cancer cells via paracrine signaling. In these myofibroblasts, the glucocorticoid dexamethasone is able to drive the glucocorticoid receptor into the nucleus and thus negatively regulates the expression of particular pro-inflammatory genes in TNFα-stimulated cells. The selective glucocorticoid receptor modulator compound A has an impaired ability to translocate GR, presumably underpinning its modest anti-inflammatory properties in these cells. Only dexamethasone, and not compound A, can upregulate the glucocorticoid receptor transactivation-dependent GILZ expression. Neither dexamethasone, nor compound A affects myofibroblast cell viability. However, compound A retards the growth of this myofibroblast cell line. Additionally, dexamethasone can inhibit the expression of Tenascin C, hepatocyte growth factor, and TGFß, which are all factors known for their impact on colon cancer cell invasion, in a glucocorticoid receptor-dependent manner. In contrast, compound A can only slightly diminish the expression of just hepatocyte growth factor, and not tenascin C or TGFß. Combined, our results expose new tumor microenvironment-modulating effects of glucocorticoids and the selective GR modulator compound A.


Subject(s)
Acetates/pharmacology , Anti-Inflammatory Agents/pharmacology , Colonic Neoplasms/drug therapy , Glucocorticoids/immunology , Myofibroblasts/drug effects , Receptors, Glucocorticoid/immunology , Tyramine/analogs & derivatives , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Colonic Neoplasms/immunology , Colonic Neoplasms/pathology , Dexamethasone/pharmacology , Gene Expression Regulation/drug effects , Humans , Myofibroblasts/immunology , Myofibroblasts/pathology , NF-kappa B/immunology , Receptors, Glucocorticoid/genetics , Transcriptional Activation/drug effects , Tyramine/pharmacology
14.
Methods Mol Biol ; 1204: 83-94, 2014.
Article in English | MEDLINE | ID: mdl-25182763

ABSTRACT

The Microarray Assay for Realtime Coregulator-Nuclear receptor Interaction (MARCoNI) technology allows the identification of nuclear receptor-coregulator interactions via flow-through microarrays. As such, differences in the coregulator profile between distinct nuclear receptors or of a single receptor in agonist or antagonist mode can be investigated, even in a single run. In this chapter, the method how to perform these peptide microarrays with cell lysates containing the overexpressed glucocorticoid receptor is described, as well as the influence of assay parameters, variations to the protocol, and data analysis.


Subject(s)
Protein Array Analysis/methods , Receptors, Glucocorticoid/metabolism , Animals , HEK293 Cells , Humans , Software
15.
PLoS One ; 9(2): e87850, 2014.
Article in English | MEDLINE | ID: mdl-24498382

ABSTRACT

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.


Subject(s)
Apoptosis/drug effects , Biomarkers, Tumor/genetics , Phytotherapy , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/pathology , Withanolides/pharmacology , Biomarkers, Tumor/metabolism , Blotting, Western , Cell Cycle/drug effects , Cell Proliferation/drug effects , Female , Gene Expression Profiling , Humans , Oligonucleotide Array Sequence Analysis , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Triple Negative Breast Neoplasms/genetics , Tumor Cells, Cultured
16.
Cell Mol Life Sci ; 71(1): 143-63, 2014 Jan.
Article in English | MEDLINE | ID: mdl-23784308

ABSTRACT

Glucocorticoids (GCs) block inflammation via interference of the liganded glucocorticoid receptor (GR) with the activity of pro-inflammatory transcription factors NF-κB and AP-1, a mechanism known as transrepression. This mechanism is believed to involve the activity of GR monomers. Here, we explored how the GR monomer-favoring Compound A (CpdA) affects AP-1 activation and activity. Our results demonstrate that non-steroidal CpdA, unlike classic steroidal GCs, blocks NF-κB- but not AP-1-driven gene expression. CpdA rather sustains AP-1-driven gene expression, a result which could mechanistically be explained by the failure of CpdA to block upstream JNK kinase activation and concomitantly also phosphorylation of c-Jun. In concordance and in contrast to DEX, CpdA maintained the expression of the activated AP-1 target gene c-jun, as well as the production of the c-Jun protein. As for the underlying mechanism, GR is a necessary intermediate in the CpdA-mediated gene expression of AP-1-regulated genes, but seems to be superfluous to CpdA-mediated JNK phosphorylation prolongation. The latter phenomenon concurs with the inability of CpdA to stimulate DUSP1 gene expression. ChIP analysis demonstrates that DEX-activated GR, but not CpdA-activated GR, is recruited to AP-1-driven promoters. Furthermore, in mice we observed that CpdA instigates a strong enhancement of TNF-induced AP-1-driven gene expression. Finally, we demonstrate that this phenomenon coincides with an increased sensitivity towards TNF lethality, and implicate again a role for JNK2. In conclusion, our data support the hypothesis that a ligand-induced differential conformation of GR yields a different transcription factor cross-talk profile.


Subject(s)
NF-kappa B/metabolism , Receptors, Glucocorticoid/metabolism , Transcription Factor AP-1/metabolism , Animals , Cell Line, Tumor , Dexamethasone/pharmacology , Dual Specificity Phosphatase 1/genetics , Dual Specificity Phosphatase 1/metabolism , Gene Expression/drug effects , Humans , Interleukin-6/genetics , Interleukin-6/metabolism , JNK Mitogen-Activated Protein Kinases/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitogen-Activated Protein Kinase 9/deficiency , Mitogen-Activated Protein Kinase 9/genetics , Mitogen-Activated Protein Kinase 9/metabolism , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation/drug effects , Promoter Regions, Genetic , Proto-Oncogene Proteins c-jun/genetics , Proto-Oncogene Proteins c-jun/metabolism , RNA Interference , RNA, Small Interfering/metabolism , Receptors, Glucocorticoid/antagonists & inhibitors , Receptors, Glucocorticoid/genetics , Staurosporine/pharmacology , Transcription Factor AP-1/genetics , Transcriptional Activation/drug effects , Triazoles/pharmacology , Tumor Necrosis Factor-alpha/pharmacology
17.
PLoS One ; 8(7): e69115, 2013.
Article in English | MEDLINE | ID: mdl-23935933

ABSTRACT

Compound A possesses glucocorticoid receptor (GR)-dependent anti-inflammatory properties. Just like classical GR ligands, Compound A can repress NF-κB-mediated gene expression. However, the monomeric Compound A-activated GR is unable to trigger glucocorticoid response element-regulated gene expression. The heat shock response potently activates heat shock factor 1 (HSF1), upregulates Hsp70, a known GR chaperone, and also modulates various aspects of inflammation. We found that the selective GR modulator Compound A and heat shock trigger similar cellular effects in A549 lung epithelial cells. With regard to their anti-inflammatory mechanism, heat shock and Compound A are both able to reduce TNF-stimulated IκBα degradation and NF-κB p65 nuclear translocation. We established an interaction between Compound A-activated GR and Hsp70, but remarkably, although the presence of the Hsp70 chaperone as such appears pivotal for the Compound A-mediated inflammatory gene repression, subsequent novel Hsp70 protein synthesis is uncoupled from an observed CpdA-induced Hsp70 mRNA upregulation and hence obsolete in mediating CpdA's anti-inflammatory effect. The lack of a Compound A-induced increase in Hsp70 protein levels in A549 cells is not mediated by a rapid proteasomal degradation of Hsp70 or by a Compound A-induced general block on translation. Similar to heat shock, Compound A can upregulate transcription of Hsp70 genes in various cell lines and BALB/c mice. Interestingly, whereas Compound A-dependent Hsp70 promoter activation is GR-dependent but HSF1-independent, heat shock-induced Hsp70 expression alternatively occurs in a GR-independent and HSF1-dependent manner in A549 lung epithelial cells.


Subject(s)
Gene Expression Regulation , HSP70 Heat-Shock Proteins/genetics , Promoter Regions, Genetic , Receptors, Glucocorticoid/metabolism , Transcriptional Activation , Animals , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/metabolism , Anti-Inflammatory Agents/pharmacology , Cell Line , DNA-Binding Proteins/metabolism , Dose-Response Relationship, Drug , Female , Gene Expression Regulation/drug effects , HSP70 Heat-Shock Proteins/metabolism , HSP90 Heat-Shock Proteins/metabolism , Heat Shock Transcription Factors , Humans , Mice , Models, Biological , NF-kappa B/antagonists & inhibitors , NF-kappa B/metabolism , Protein Binding , Receptors, Glucocorticoid/agonists , Response Elements , Transcription Factors/metabolism
18.
Eur J Pharmacol ; 715(1-3): 1-9, 2013 Sep 05.
Article in English | MEDLINE | ID: mdl-23831393

ABSTRACT

The glucocorticoid receptor is a nuclear receptor, and can be activated by glucocorticoid ligands. Mitogen- and stress-activated protein kinase (MSK1), when activated by p38 and ERK mitogen-activated protein kinases (MAPKs), plays a major role in chromatin relaxation via phosphorylation of histone H3 S10. The glucocorticoid receptor can target MSK1 as part of its anti-inflammatory mechanism. Here, we studied the converse mechanism, i.e. the impact of MSK1 on glucocorticoid receptor-mediated transactivation. Upstream MSK1-activating kinases concentration-dependently enhanced glucocorticoid response element (GRE)-regulated promoter activity. Correspondingly, MSK1 inhibition, via H89, or combined p38 and ERK MAPK inhibition, via SB203580 and U0126, diminished maximally stimulated GRE-regulated promoter activity using high concentrations of glucocorticoids. Concomitantly, the combination of these agents does not seem to alter site-specific phosphorylations of murine glucocorticoid receptor S212 or S220. Paradoxically, we reveal that a sub-maximally activated GRE-mediated promoter activity, by using lower concentrations of glucocorticoids, is consistently enhanced by H89 or a combination of SB203580 and U0126, irrespective of the GRE promoter context. Furthermore, we show that the glucocorticoid-induced nucleocytoplasmic translocation of MSK1 occurs in a glucocorticoid concentration-dependent manner. The observed glucocorticoid concentration-dependent effect of MSK1 or MAPK inhibition on glucocorticoid receptor transactivation warrants further research into the applicability of combined glucocorticoid and kinase inhibitor strategies for anti-inflammatory purposes.


Subject(s)
Glucocorticoids/metabolism , Glucocorticoids/pharmacology , Response Elements/genetics , Ribosomal Protein S6 Kinases, 90-kDa/metabolism , Animals , Cell Line, Tumor , Cytoplasm/drug effects , Cytoplasm/metabolism , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Isoquinolines/pharmacology , MAP Kinase Kinase 6/genetics , MAP Kinase Kinase 6/metabolism , Mice , Protein Transport/drug effects , Receptors, Glucocorticoid/genetics , Receptors, Glucocorticoid/metabolism , Response Elements/drug effects , Ribosomal Protein S6 Kinases, 90-kDa/antagonists & inhibitors , Sulfonamides/pharmacology , Transcription, Genetic/drug effects , Transcriptional Activation/drug effects , Transfection , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/genetics , p38 Mitogen-Activated Protein Kinases/metabolism
19.
Mol Cell Endocrinol ; 380(1-2): 41-54, 2013 Nov 05.
Article in English | MEDLINE | ID: mdl-23267834

ABSTRACT

The activity of the glucocorticoid receptor (GR), a nuclear receptor transcription factor belonging to subclass 3C of the steroid/thyroid hormone receptor superfamily, is typically triggered by glucocorticoid hormones. Apart from driving gene transcription via binding onto glucocorticoid response elements in regulatory regions of particular target genes, GR can also inhibit gene expression via transrepression, a mechanism largely based on protein:protein interactions. Hereby GR can influence the activity of other transcription factors, without contacting DNA itself. GR is known to inhibit the activity of a growing list of immune-regulating transcription factors. Hence, GCs still rule the clinic for treatments of inflammatory disorders, notwithstanding concomitant deleterious side effects. Although patience is a virtue when it comes to deciphering the many mechanisms GR uses to influence various signaling pathways, the current review is testimony of the fact that groundbreaking mechanistic work has been accumulating over the past years and steadily continues to grow.


Subject(s)
Receptors, Glucocorticoid/physiology , Transcriptional Activation , Animals , Chromatin/genetics , Chromatin/metabolism , Epigenetic Repression , Humans , Protein Kinases/physiology , Protein Processing, Post-Translational , Response Elements , Transcription Factors/physiology
20.
Cell Mol Life Sci ; 68(23): 3823-41, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21744067

ABSTRACT

It has been known for several decades that cyclic AMP (cAMP), a prototypical second messenger, transducing the action of a variety of G-protein-coupled receptor ligands, has potent immunosuppressive and anti-inflammatory actions. These actions have been attributed in part to the ability of cAMP-induced signals to interfere with the function of the proinflammatory transcription factor Nuclear Factor-kappaB (NF-κB). NF-κB plays a crucial role in switching on the gene expression of a plethora of inflammatory and immune mediators, and as such is one of the master regulators of the immune response and a key target for anti-inflammatory drug design. A number of fundamental molecular mechanisms, contributing to the overall inhibitory actions of cAMP on NF-κB function, are well established. Paradoxically, recent reports indicate that cAMP, via its main effector, the protein kinase A (PKA), also promotes NF-κB activity. Indeed, cAMP actions appear to be highly cell type- and context-dependent. Importantly, several novel players in the cAMP/NF-κB connection, which selectively direct cAMP action, have been recently identified. These findings not only open up exciting new research avenues but also reveal novel opportunities for the design of more selective, NF-κB-targeting, anti-inflammatory drugs.


Subject(s)
Cyclic AMP/metabolism , NF-kappa B/metabolism , Animals , Cyclic AMP/immunology , Cyclic AMP/pharmacology , Humans , NF-kappa B/chemistry , NF-kappa B/immunology , Signal Transduction/drug effects
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