Microbial indoles have been demonstrated as selective or dual agonists and ligands of the pregnane X receptor (PXR) and aryl hydrocarbon receptor (AhR). However, structural determinants of microbial indoles selectivity towards both receptors remain elusive. Here, we studied the effects of existing and newly synthesized derivatives of indole microbial metabolite tryptamine on the activity of AhR and PXR receptors. We show that the elongation of indolyl-3-alkaneamine chain, indole N-methylation and conversion of indolyl-3-alkaneamines to oleamides resulted in a major increase of PXR activity and in parallel loss of AhR activity. Using reporter gene assays, RT-PCR and TR-FRET techniques, we have characterized in detail the activation of PXR by novel indolyl-3-alkanyl-oleamides, 1-methyltryptamine and 1-methyltryptamine-acetamide. As a proof of concept, we demonstrated anti-inflammatory and epithelial barrier-protective activity of lead derivatives in intestinal Caco-2 cells, employing the measurement of expression of pro-inflammatory chemokines, tight junction genes, trans-epithelial electric resistance TEER, and dextran-FITC permeability assay. In conclusion, we show that a subtle chemical modifications of simple microbial indole metabolite tryptamine, leads to substantial changes in AhR and PXR agonist activities.
The activation of the aryl hydrocarbon receptor (AhR) by xenobiotic compounds was demonstrated to result in the degradation of the androgen receptor (AR). Since prostate cancer is often dependent on AR, it has become a significant therapeutic target. As a result of the emerging concept of bacterial mimicry, we tested whether compounds with indole scaffolds capable of AhR activation have the potential to restrict AR activity in prostate cancer cells. Altogether, 22 indolic compounds were tested, and all of them activated AhR. However, only eight decreased DHT-induced AR luciferase activity. All indoles, which met the AhR-activating and AR-suppressing criteria, decreased the expression of DHT-inducible AR target genes, specifically KLK3 and FKBP5 mRNAs. The reduced AR binding to the KLK3 promoter was confirmed by a chromatin immunoprecipitation (ChIP) assay. In addition, some indoles significantly decreased AR protein and mRNA level. By using CRISPR/Cas9 AhR knockout technology, no relationship between AhR and AR, measured as target gene expression, was observed. In conclusion, some indoles that activate AhR possess AR-inhibiting activity, which seems to be related to the downregulation of AR expression rather than to AR degradation alone. Moreover, there does not seem to be a clear relationship that would connect AhR activation with AR activity suppression in 22Rv1 cells.
- MeSH
- androgenní receptory genetika metabolismus MeSH
- indoly farmakologie MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nádory prostaty * genetika metabolismus MeSH
- receptory aromatických uhlovodíků * genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Exposure to environmental pollutants and endogenous metabolites that induce aryl hydrocarbon receptor (AhR) expression has been suggested to affect cognitive development and, particularly in boys, also motor function. As current knowledge is based on epidemiological and animal studies, in vitro models are needed to better understand the effects of these compounds in the human nervous system at the molecular level. Here, we investigated expression of AhR pathway components and how they are regulated by AhR ligands in human motor neurons. Motor neurons generated from human induced pluripotent stem cells (hiPSCs) were characterized at the molecular level and by electrophysiology. mRNA levels of AhR target genes, CYP1A1 and CYP1B1 (cytochromes P450 1A1/1B1), and AhR signaling components were monitored in hiPSCs and in differentiated neurons following treatment with AhR ligands, 2,3,7,8,-tetrachlodibenzo-p-dioxin (TCDD), L-kynurenine (L-Kyn), and kynurenic acid (KA), by RT-qPCR. Changes in AhR cellular localization and CYP1A1 activity in neurons treated with AhR ligands were also assessed. The neurons we generated express motor neuron-specific markers and are functional. Transcript levels of CYP1B1, AhR nuclear translocators (ARNT1 and ARNT2) and the AhR repressor (AhRR) change with neuronal differentiation, being significantly higher in neurons than hiPSCs. In contrast, CYP1A1 and AhR transcript levels are slightly lower in neurons than in hiPSCs. The response to TCDD treatment differs in hiPSCs and neurons, with only the latter showing significant CYP1A1 up-regulation. In contrast, TCDD slightly up-regulates CYP1B1 mRNA in hiPSCs, but downregulates it in neurons. Comparison of the effects of different AhR ligands on AhR and some of its target genes in neurons shows that L-Kyn and KA, but not TCDD, regulate AhR expression and differently affect CYP1A1 and CYP1B1 expression. Finally, although TCDD does not significantly affect AhR transcript levels, it induces AhR protein translocation to the nucleus and increases CYP1A1 activity. This is in contrast to L-Kyn and KA, which either do not affect or reduce, respectively, CYP1A1 activity. Expression of components of the AhR signaling pathway are regulated with neuronal differentiation and are differently affected by TCDD, suggesting that pluripotent stem cells might be less sensitive to this toxin than neurons. Crucially, AhR signaling is affected differently by TCDD and other AhR ligands in human motor neurons, suggesting that they can provide a valuable tool for assessing the impact of environmental pollutants.
- Publikační typ
- časopisecké články MeSH
Targeting the aryl hydrocarbon receptor (AhR) is an emerging therapeutic strategy for multiple diseases (e.g., inflammatory bowel disease). Thermosporothrix hazakensis microbial metabolite 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) is a putative AhR endogenous ligand. To improve the chemical stability, we synthesized a series of ITE chemical mimics. Using a series of in vitro assays, we identified 2-(1H-indole-3-carbonyl)-N-methyl thiazole-4-carboxamide (ITE-CONHCH3) as a highly potent (EC50 = 1.6 nM) AhR agonist with high affinity (Ki = 88 nM). ITE-CONHCH3 triggered AhR nuclear translocation and dimerization of AhR-ARNT, enhanced AhR binding in the CYP1A1 promoter, and induced AhR-regulated genes in an AhR-dependent manner. The metabolic stability of ITE-CONHCH3 in a cell culture was 10 times higher than that of ITE. Finally, we observed protective effects of ITE-CONHCH3 in mice with DSS-induced colitis. Overall, we demonstrate and validate a concept of microbial metabolite mimicry in the therapeutic targeting of AhR.
- MeSH
- cytochrom P-450 CYP1A1 MeSH
- indoly farmakologie terapeutické užití MeSH
- kolitida * chemicky indukované farmakoterapie MeSH
- myši MeSH
- receptory aromatických uhlovodíků * agonisté genetika metabolismus MeSH
- thiazoly farmakologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
SCOPE: CYP3A4 is the most important drug-metabolizing enzyme regulated via the vitamin D receptor (VDR) in the intestine. However, less is known about VDR in the regulation of CYP3A4 and other drug-metabolizing enzymes in the liver. METHODS AND RESULTS: This study investigates whether 1α,25-dihydroxyvitamin D3 (1α,25(OH)2 D3 ) regulates major cytochrome P450 enzymes, selected phase I and II enzymes, and transporters involved in xenobiotic and steroidal endobiotic metabolism in 2D and 3D cultures of human hepatocytes. The authors found that 1α,25(OH)2 D3 increases hepatic CYP3A4 expression and midazolam 1'-hydroxylation activity in 2D hepatocytes. The results are confirmed in 3D spheroids, where 1α,25(OH)2 D3 has comparable effect on CYP3A4 mRNA expression as 1α-hydroxyvitamin D3 , an active vitamin D metabolite. Other regulated genes such as CYP1A2, AKR1C4, SLC10A1, and SLCO4A1 display only mild changes in mRNA levels after 1α,25(OH)2 D3 treatment in 2D hepatocytes. Expression of other cytochrome P450, phase I and phase II enzyme, or transporter genes are not significantly influenced by 1α,25(OH)2 D3 . Additionally, the effect of VDR activation on CYP3A4 mRNA expression is abolished by natural dietary compound sulforaphane, a common suppressor of pregnane X receptor (PXR) and constitutive androstane receptor (CAR). CONCLUSION: This study proposes that VDR or vitamin D supplementation is unlikely to significantly influence liver detoxification enzymes apart from CYP3A4.
- MeSH
- cytochrom P-450 CYP3A * genetika MeSH
- hepatocyty MeSH
- lidé MeSH
- messenger RNA MeSH
- receptory kalcitriolu genetika MeSH
- stanovení celkové genové exprese MeSH
- systém (enzymů) cytochromů P-450 genetika MeSH
- vitamin D farmakologie MeSH
- xenobiotika * farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cytochrome P450 2A13 is an omitted brother of CYP2A6 that has an important role in the drug metabolism of liver. Due to extrahepatic expression, it has gained less attention than CYP2A6, despite the fact that it plays a significant role in toxicant-induced pulmonary lesions and, therefore, lung cancer. The purpose of this mini-review is to summarize the basic knowledge about this enzyme in relation to the substrates, inhibitors, genetic polymorphisms, and transcriptional regulation that are known so far (September 2021).
- MeSH
- aromatické hydroxylasy antagonisté a inhibitory genetika metabolismus MeSH
- lidé MeSH
- methoxsalen farmakologie MeSH
- nádory plic enzymologie genetika patologie MeSH
- plíce enzymologie metabolismus patologie MeSH
- polymorfismus genetický * MeSH
- regulace genové exprese enzymů * MeSH
- regulace genové exprese u nádorů * MeSH
- substrátová specifita MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Androgen receptor (AR) signalling is triggered by androgens that have lipophilic nature. Since it was indicated that graphene oxide (GO) might facilitate passive diffusion of lipophilic compounds probably via Trojan horse-like mechanism, we tested the hypothesis if this suggestion would apply for androgens as well. Thus, we investigated if GO affects dihydrotestosterone (DHT)-triggered signalling of AR in two prostate cancer-derived cell lines, 22Rv1 and LNCaP. These cell lines differ in number of AR variants, i.e. there are two variants in 22Rv1 cells (full length and truncated) but only one in LNCaP cells (full length). Graphene oxide had no effect on basal luciferase activity but significantly decreased DHT-inducible AR-dependent luciferase activity in stably transfected cells. In 22Rv1 cells, it induced concentration-dependent decrease of DHT-inducible KLK3 mRNA and PSA protein after 24 h. While there was no effect on UBE2C mRNA (regulated by truncated variant), there was synergistic effect of DHT and GO on UBE2C protein level. Translocation of full-length AR (AR-FL) was potentiated by GO in the presence of DHT in 22Rv1 cells but it was suppressed in LNCaP cells. DHT-stimulated enrichment of AR-FL on KLK3 promoter was not significantly affected by GO in any tested cell line neither was KLK3 mRNA at 4 h of incubation. In conclusion, GO affects DHT-triggered signalling in both types of cells in similar manner, but ligand-triggered redistribution of AR-FL is affected differently. One of the reasons may be the presence of truncated variant of androgen receptor.
- MeSH
- androgenní receptory * genetika MeSH
- androgeny MeSH
- dihydrotestosteron farmakologie MeSH
- grafit MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nádory prostaty * MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
The interplays between the metabolic products of intestinal microbiota and the host signaling through xenobiotic receptors, including pregnane X receptor (PXR), are of growing interest, in the context of intestinal health and disease. A distinct class of microbial catabolites is formed from dietary tryptophan, having the indole scaffold in their core structure, which is a biologically active entity. In the current study, we examined a series of ten tryptophan microbial catabolites for their interactions with PXR signaling. Utilizing a reporter gene assay, we identified indole (IND) and indole-3-acetamide (IAD) as PXR agonists. IND and IAD induced PXR-regulated genes CYP3A4 and MDR1 in human intestinal cancer cells. Using time-resolved fluorescence resonance energy transfer, we show that IND (IC50 292 μM) and IAD (IC50 10 μM) are orthosteric ligands of PXR. Binding of PXR in its DNA response elements was enhanced by IND and IAD, as revealed by chromatin immunoprecipitation assay. We demonstrate that tryptophan microbial intestinal metabolites IND and IAD are ligands and agonists of human PXR. These findings are of particular importance in understanding the roles of microbial catabolites in human physiology and pathophysiology. Furthermore, these results are seminal in expanding potential drug repertoire through microbial metabolic mimicry.
- MeSH
- cytochrom P-450 CYP3A genetika metabolismus MeSH
- indoly metabolismus MeSH
- kultivované buňky MeSH
- kyseliny indoloctové metabolismus MeSH
- lidé MeSH
- ligandy MeSH
- nádorové buněčné linie MeSH
- P-glykoproteiny genetika metabolismus MeSH
- pregnanový X receptor agonisté genetika MeSH
- reportérové geny MeSH
- střevní mikroflóra * MeSH
- střevní sliznice * metabolismus mikrobiologie MeSH
- transfekce MeSH
- tryptofan metabolismus MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Prostate cancer (PCa) is the most common cancer and the second leading cause of cancer-related deaths of men in Western countries. Androgen deprivation therapy is initially successful, however eventually fails, and tumors progress to the more aggressive castration-resistant PCa (CRPC). Yet, androgen receptor (AR) usually remains as a major regulator of tumor cell proliferation in CRPC. Interleukin-23 (IL-23) was recently shown to promote the development of CRPC by driving AR transcription. Here we used the androgen-sensitive LNCaP, castration-resistant C4-2, and 22Rv1 cells. Interestingly, cellular senescence is induced in these human cell lines by treatment with the AR antagonists enzalutamide (ENZ) or darolutamide (ODM), which might be one underlying mechanism for inhibition of PCa cell proliferation. Treatment with IL-23 alone did not change cellular senescence levels in these cell lines, whereas IL-23 inhibited significantly cellular senescence levels induced by ENZ or ODM in both CRPC cell lines C4-2 and 22Rv1 but not in LNCaP cells. This indicates a response of IL-23 specific in CRPC cells. Generating LNCaP and C4-2 three-dimensional (3D) spheroids and treatment with AR antagonists resulted in the reduced spheroid volume and thus growth inhibition. However, the combination of AR antagonists with IL-23 did not affect the antagonist-mediated reduction of spheroid volumes. This observation was confirmed with proliferation assays using adherent monolayer cell cultures. Taken together, the data indicate that IL-23 treatment reduces the AR antagonists-induced level of cellular senescence of CRPC cells, which could be one possible mechanism for promoting castration resistance.
- MeSH
- antagonisté androgenních receptorů farmakologie terapeutické užití MeSH
- benzamidy farmakologie terapeutické užití MeSH
- fenylthiohydantoin farmakologie terapeutické užití MeSH
- interleukin-23 metabolismus MeSH
- lidé MeSH
- nitrily farmakologie terapeutické užití MeSH
- protokoly antitumorózní kombinované chemoterapie farmakologie terapeutické užití MeSH
- pyrazoly farmakologie terapeutické užití MeSH
- stárnutí buněk MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The efforts for therapeutic targeting of the aryl hydrocarbon receptor (AhR) have emerged in recent years. We investigated the effects of available antimigraine triptan drugs, having an indole core in their structure, on AhR signaling in human hepatic and intestinal cells. Activation of AhR in reporter gene assays was observed for Avitriptan and to a lesser extent for Donitriptan, while other triptans were very weak or no activators of AhR. Using competitive binding assay and by homology docking, we identified Avitriptan as a low-affinity ligand of AhR. Avitriptan triggered nuclear translocation of AhR and increased binding of AhR in CYP1A1 promotor DNA, as revealed by immune-fluorescence microscopy and chromatin immune-precipitation assay, respectively. Strong induction of CYP1A1 mRNA was achieved by Avitriptan in wild type but not in AhR-knockout, immortalized human hepatocytes, implying that induction of CYP1A1 is AhR-dependent. Increased levels of CYP1A1 mRNA by Avitriptan were observed in human colon carcinoma cells LS180 but not in primary cultures of human hepatocytes. Collectively, we show that Avitriptan is a weak ligand and activator of human AhR, which induces the expression of CYP1A1 in a cell-type specific manner. Our data warrant the potential off-label therapeutic application of Avitriptan as an AhR-agonist drug.
- MeSH
- aktivace enzymů účinky léků MeSH
- cytochrom P-450 CYP1A1 genetika MeSH
- hepatocyty metabolismus MeSH
- kultivované buňky MeSH
- lidé MeSH
- ligandy MeSH
- molekulární modely MeSH
- orgánová specificita MeSH
- přehodnocení terapeutických indikací léčivého přípravku MeSH
- promotorové oblasti (genetika) účinky léků MeSH
- receptory aromatických uhlovodíků agonisté chemie metabolismus MeSH
- simulace molekulového dockingu MeSH
- střevní sliznice metabolismus MeSH
- sulfonamidy farmakologie MeSH
- transkripční faktory bHLH agonisté chemie metabolismus MeSH
- tryptaminy farmakologie MeSH
- upregulace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
