Cytochrome P450 (CYP) expression and activity are not homogeneous in the liver lobules. Indeed, CYPs are mainly expressed and induced in centrilobular hepatocytes. The wingless-type MMTV integration site family (WNT)/β-catenin pathway was identified as a major regulator of this zonal organization. We have recently demonstrated that in primary human hepatocytes (PHHs), the expression of CYP2E1, CYP1A2, and aryl hydrocarbon receptor (AhR), but not of CYP3A4, is regulated by the WNT/β-catenin pathway in response to WNT3a, its canonical activator. Here, we investigated whether glycogen synthase kinase 3β (GSK3β) inhibitors, which mimic the action of WNT molecules, could be used in PHHs to activate the β-catenin pathway to study CYP expression. We assessed the activity of 6BIO (6-bromoindirubin-3'-oxime), CHIR99021 (6-((2-((4-(2,4-dichlorophenyl)-5-(4methyl-1H-imidazol-2-yl)pyrimidin-2-yl)amino)ethyl)amino) nicotinonitrile), and GSK3iXV (Pyridocarbazolo-cyclopentadienyl Ruthenium complex GSK3 inhibitor XV) that belong to structurally different families of GSK3β inhibitors. Using small interfering RNAs, reporter gene assays, and molecular docking predictions, we demonstrated that GSK3β inhibitors can activate the WNT/β-catenin pathway in PHHs to regulate CYP2E1 expression. We also found that 6BIO and GSK3iXV are AhR full agonists that participate, through AhR signaling, to CYP1A2 induction. Conversely, CHIR99021 is an AhR partial agonist, and a pregnane X receptor ligand and partial agonist, thus regulating CYP1A2 and CYP3A4 gene expression in a β-catenin-independent manner. In conclusion, GSK3β inhibitors can activate the WNT/β-catenin pathway in PHHs. Nevertheless, their role in CYP regulation should be analyzed with caution as these molecules can interact with xenosensors.

• MeSH
• beta-katenin agonisté   antagonisté a inhibitory   genetika   metabolismus   MeSH
• enzymová indukce účinky léků   MeSH
• hepatocyty cytologie   účinky léků   metabolismus   MeSH
• indoly farmakologie   MeSH
• induktory cytochromu P450 chemie   metabolismus   farmakologie   MeSH
• inhibitory proteinkinas chemie   metabolismus   farmakologie   MeSH
• kinasa 3 glykogensynthasy antagonisté a inhibitory   metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• organokovové sloučeniny farmakologie   MeSH
• oximy farmakologie   MeSH
• pyridiny farmakologie   MeSH
• pyrimidiny farmakologie   MeSH
• receptory aromatických uhlovodíků agonisté   chemie   genetika   metabolismus   MeSH
• rekombinantní fúzní proteiny chemie   metabolismus   MeSH
• reportérové geny účinky léků   MeSH
• RNA interference MeSH
• signální dráha Wnt účinky léků   MeSH
• simulace molekulového dockingu MeSH
• steroidní receptory agonisté   genetika   metabolismus   MeSH
• systém (enzymů) cytochromů P-450 chemie   genetika   metabolismus   MeSH
• transkripční faktory bHLH agonisté   chemie   genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

We have recently demonstrated that the alkaloid colchicine (COL) inhibits glucocorticoid receptor (GR) transcriptional activity. In addition, we described proteasome-mediated degradation of GR in COL-treated HeLa cells. While these effects were previously attributed to cell cycle arrest in G2/M phase, this explanation is not applicable for nonproliferating cells such as human hepatocytes (HH). In the current study, we compared COL-mediated microtubule disruption and cell cycle arrest with selected GR functions in HeLa cells and HH as models of proliferating and quiescent cells, respectively. Microtubule disruption led to irreversible decrease in GR binding capacity and protein level in HeLa cells. None of the parameters was restored 24 hours after COL withdrawal. In contrast, dexamethasone (DEX) binding was increased in HH at the beginning of the treatment, with following transient activation of extracellular signal-regulated kinase (ERK). The findings of these investigations emphasize the GR-signaling differences between primary and transformed cells.

• MeSH
• aktivace enzymů MeSH
• buněčný cyklus účinky léků   MeSH
• časové faktory MeSH
• dexamethason metabolismus   MeSH
• extracelulárním signálem regulované MAP kinasy metabolismus   MeSH
• fluorescenční mikroskopie MeSH
• HeLa buňky MeSH
• hepatocyty účinky léků   metabolismus   ultrastruktura   MeSH
• kolchicin farmakologie   MeSH
• lidé MeSH
• ligandy MeSH
• mikrotubuly účinky léků   ultrastruktura   MeSH
• modulátory tubulinu farmakologie   MeSH
• proliferace buněk účinky léků   MeSH
• průtoková cytometrie MeSH
• receptory glukokortikoidů metabolismus   fyziologie   MeSH
• signální transdukce účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

Drugs for weight loss have been in use for nearly hundred years. Orlistat (Xenical) is a non-centrally acting anti-obesity drug that inactivates gastric and intestinal lipases, thus, preventing absorption of dietary triglycerides. There are reports indicating that Orlistat reduces bioavailability of Cyclosporin to a clinically relevant degree. Since Cyclosporin is metabolized by cytochrome P450 CYP3A4, we examined whether interaction between Orlistat and Cyclosporin involves induction of CYP3A4. Human Caucasian colon adenocarcinoma cells LS174T and primary cultures of human hepatocytes were used, as in vitro models of intestinal and hepatic cells, respectively. Treatment of LS174T cells for 24h with Orlistat (1-100mg/L) did not cause induction of CYP3A4 mRNA levels as compared to control cells while Orlistat (100mg/L) slightly induced CYP3A4 mRNA in human hepatocytes. Rifampicin, a model CYP3A4 inducer, significantly induced CYP3A4 mRNA in both types of cells. The level of CYP3A4 protein in human hepatocytes was increased by Orlistat after 48h, while rifampicin strongly induced CYP3A4 protein level. In addition, Orlistat moderately dose-independently activated pregnane X receptor (PXR) in LS174T cells transiently transfected with p3A4-luc reporter construct containing the basal promoter (-362/+53) with proximal PXR response element and the distal xenobiotic responsive enhancer module (-7836/-7208) of the CYP3A4 gene 5'-flanking region. In conclusion, we report here that Orlistat is weak PXR activator and CYP3A4 inducer in human hepatocytes, but it has no effect on CYP3A4 in intestinal cells, implying no role of CYP3A4 induction in the interaction between Orlistat and Cyclosporin in absorption process.

• MeSH
• biologická dostupnost MeSH
• cytochrom P-450 CYP3A genetika   metabolismus   MeSH
• hepatocyty cytologie   účinky léků   MeSH
• inhibitory enzymů farmakologie   MeSH
• laktony farmakologie   MeSH
• lidé MeSH
• messenger RNA genetika   MeSH
• nádorové buněčné linie MeSH
• promotorové oblasti (genetika) MeSH
• steroidní receptory agonisté   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

CYP1A1 and CYP1A2 genes encode members of the cytochrome P450 superfamily of enzymes primarily involved in xenobiotic and drug metabolism. In this paper we examined the effects of synthetic glucocorticoid dexamethasone (DEX) on aryl hydrocarbon receptor (AhR)-mediated regulation of CYP1A1 and CYP1A2 genes and their enzymatic activity in primary cultures of human hepatocytes obtained from 17 donors and prepared in 3 countries. Dexamethasone significantly reduced both basal and inducible CYP1A1/2 ethoxyresorufin-O-deethylase (EROD) activities by more than 75 and 50%, respectively. Glucocorticoid receptor (GR) antagonist RU486 abolished this effect suggesting the involvement of GR in the process. In contrast, dexamethasone significantly augmented transcriptional activation of CYP1A2 mRNA but not CYP1A1 gene by prototype AhR ligands 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-methylcholanthrene (3MC). Dexamethasone had no effect on basal and TCDD-inducible levels of CYP1As proteins; however, it reduced the levels of AhR and GRalpha mRNAs and AhR protein levels. In addition, using RT(2) Profiler PCR Array, we found the effect of dexamethasone on the expression of several co-activators of AhR and GR nuclear receptors in the primary human hepatocytes. We conclude that dexamethasone controls CYP1A1 and CYP1A2 expression and activity in human hepatocytes via multiple mechanisms, which remain to be elucidated.

• MeSH
• cytochrom P-450 CYP1A1 genetika   metabolismus   MeSH
• cytochrom P-450 CYP1A2 genetika   metabolismus   MeSH
• dexamethason farmakologie   MeSH
• dospělí MeSH
• financování organizované MeSH
• hepatocyty enzymologie   metabolismus   účinky léků   MeSH
• kultivované buňky MeSH
• lidé středního věku MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• methylcholanthren toxicita   MeSH
• polychlorované dibenzodioxiny toxicita   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• preklinické hodnocení léčiv MeSH
• receptory aromatických uhlovodíků metabolismus   MeSH
• regulace genové exprese enzymů účinky léků   MeSH
• senioři MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Geografické názvy
• Česká republika MeSH
• Francie MeSH
• Maďarsko MeSH

SP600125, a specific inhibitor of c-Jun-N-Terminal kinase (JNK), was reported as a ligand and antagonist of aryl hydrocarbon receptor (AhR) [Joiakim A, Mathieu PA, Palermo C, Gasiewicz TA, Reiners Jr JJ. The Jun N terminal kinase inhibitor SP600125 is a ligand and antagonist of the aryl hydrocarbon receptor. Drug Metab Dispos 2003;31(11):1279-82]. Here we show that SP600125 is not an antagonist but a partial agonist of human AhR. SP600125 significantly induced CYP1A1 and CYP1A2 mRNAs in primary human hepatocytes and CYP1A1 mRNA in human hepatoma cells HepG2. This effect was abolished by resveratrol, an antagonist of AhR. Consistent with the recent report, SP600125 dose-dependently inhibited CYP1A1 and CYP1A2 genes induction by a prototype AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in human hepatocytes. Moreover, SP600125 displayed typical behavior of a partial agonist in HepG2 cells transiently transfected with a reporter plasmid containing two inverted repeats of the dioxin responsive element or with a plasmid containing 5'-flanking region of human CYP1A1 gene. SP600125 transactivated the reporter plasmids with EC(50) of 0.005 and 1.89 microM, respectively. On the other hand, TCDD-dependent transactivation of the reporter plasmids was inhibited by SP600125 with IC(50) values of 1.54 and 2.63 microM, respectively. We also tested, whether the effects of SP600125 are due to metabolism. Using liquid chromatography/mass spectrometry approach, we observed formation of two minor monohydroxylated metabolites of SP600125 in human hepatocytes, human liver microsomes but not in HepG2 cells. These data imply that biotransformation is not responsible for the effects of SP600125 on AhR signaling. In conclusion, we demonstrate that SP600125 is a partial agonist of human AhR, which induces CYP1A genes.

• MeSH
• anthraceny farmakologie   metabolismus   MeSH
• cytochrom P-450 CYP1A1 genetika   MeSH
• cytochrom P-450 CYP1A2 genetika   MeSH
• financování organizované MeSH
• hepatocyty enzymologie   MeSH
• hmotnostní spektrometrie MeSH
• inhibitory proteinkinas farmakologie   MeSH
• jaterní mikrozomy metabolismus   MeSH
• JNK mitogenem aktivované proteinkinasy antagonisté a inhibitory   MeSH
• kultivované buňky MeSH
• lidé MeSH
• polychlorované dibenzodioxiny antagonisté a inhibitory   MeSH
• receptory aromatických uhlovodíků agonisté   MeSH
• regulace genové exprese enzymů účinky léků   MeSH
• Check Tag
• lidé MeSH

Mitogen-activated protein kinases (MAPKs) were extensively studied in cancer-derived cell lines; however, studies in non-transformed human cells are scarce. In the current paper, we studied the effect of SB203580, a pharmacological inhibitor of p38 MAPK, on activation and inhibition of p38 MAPK transduction partway in primary human hepatocytes (in vitro model of differentiated cells) in comparison with several tumor cell lines (proliferating non-differentiated in vitro model). In addition, we analyzed the effect of SB203580 on extracellular-regulated protein kinase (ERK) and c-jun-N-terminal kinase (JNK) pathways both in primary human hepatocytes and tumor cell lines employing primary antibodies detecting phosphorylated kinases. We show that SB203580 activates ERK and JNK in primary cultures of human hepatocytes. The levels of ERK-P(Thr202/Tyr204), JNK-P(Thr183/Tyr185) and c-Jun-P(Ser63/73), a target down-stream protein of JNK, were increased by SB203580. In contrast, SB203580 activated ERK but not JNK in HepG2, HL-60, Saos-2 and HaCaT human cancer cell lines. We tested, whether the effects of SB203580 are due to metabolism. Using liquid chromatography/mass spectrometry, we found one minor metabolite in human liver microsomes but not in HepG2 cells. These data imply that biotransformation could be responsible for the effects of SB203580 in human hepatocytes. This study is the first report on the effects of MAPK activators (sorbitol, anisomycin, EGF) and MAPK inhibitors in primary human hepatocytes. We observed differential effects of these compounds in primary human hepatocytes and in cancer cells, implying the cell-type specificity and the essential differences between the role and function of MAPKs in normal and cancer cells.

• MeSH
• aktivace enzymů účinky léků   MeSH
• buněčné linie MeSH
• extracelulárním signálem regulované MAP kinasy metabolismus   MeSH
• financování organizované MeSH
• hepatocyty enzymologie   účinky léků   MeSH
• HL-60 buňky MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
• imidazoly farmakologie   metabolismus   MeSH
• inhibitory enzymů farmakologie   metabolismus   farmakologie   MeSH
• interakce mezi receptory a ligandy účinky léků   MeSH
• jaterní mikrozomy enzymologie   účinky léků   MeSH
• JNK mitogenem aktivované proteinkinasy metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• mitogenem aktivované proteinkinasy p38 antagonisté a inhibitory   MeSH
• pyridiny farmakologie   metabolismus   MeSH
• signální transdukce účinky léků   MeSH
• viabilita buněk účinky léků   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• western blotting MeSH
• Check Tag
• lidé MeSH

Disruption of microtubules has been shown to cause suppression of inducibility of major cytochromes P450 (CYP) through several nuclear receptors. Here we tested the effects of structurally different clinically used microtubules-interfering agents (MIAs), such as colchicine, vincristine, vinblastine, nocodazole and taxol on aryl hydrocarbon receptor signaling pathway in human hepatocytes. We show that tested MIAs inhibit 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible expression of CYP1A2 mRNA and restrict TCDD-dependent nuclear translocation of aryl hydrocarbon receptor. On the other hand, these MIAs increased the content of aryl hydrocarbon receptor protein and mRNA by transcriptional mechanism. We show that the MIAs activate c-Jun -N-terminal kinase (JNK), partly p38 but not extracellular-regulated protein kinase (ERK). Consistently, sorbitol, a model activator of JNK, inhibited TCDD-mediated induction of CYP1A2 mRNA and down-regulated tyrosine aminotransferase mRNA - a target gene of glucocorticoid receptor. Dexamethasone had the opposite effect on aryl hydrocarbon receptor signaling and decreased aryl hydrocarbon receptor mRNA and augmented the inducibility of CYP1A2 by TCDD. We conclude that the effects of tested MIAs on aryl hydrocarbon receptor-CYP1A2 signaling pathway are dual, i.e. they inhibit transcriptional activity and nuclear translocation of aryl hydrocarbon receptor but in parallel increase aryl hydrocarbon receptor protein and mRNA level. Microtubules destabilizers have the same effects as stabilizer taxol. This implies that aryl hydrocarbon receptor functions depend on microtubules dynamics but not integrity. Perturbation of aryl hydrocarbon receptor-CYP1A2 signaling by MIAs comprises glucocorticoid receptor-JNK and probably aryl hydrocarbon receptor-JNK/glucocorticoid receptor interactions. We also demonstrate that the effects of MIAs in human hepatocytes do not proceed via arresting cell cycle as confirmed by flow cytometry (FACS) analyses.

• MeSH
• buněčný cyklus účinky léků   MeSH
• cytochrom P-450 CYP1A2 metabolismus   účinky léků   MeSH
• financování organizované MeSH
• hepatocyty metabolismus   účinky léků   MeSH
• JNK mitogenem aktivované proteinkinasy metabolismus   účinky léků   MeSH
• kultivované buňky MeSH
• lidé MeSH
• messenger RNA metabolismus   MeSH
• mikrotubuly metabolismus   účinky léků   MeSH
• modulátory tubulinu farmakologie   MeSH
• průtoková cytometrie MeSH
• receptory aromatických uhlovodíků metabolismus   účinky léků   MeSH
• receptory glukokortikoidů metabolismus   účinky léků   MeSH
• regulace genové exprese enzymů účinky léků   MeSH
• signální transdukce MeSH
• Check Tag
• lidé MeSH

Recent findings show that colchicine (COL) in submicromolar concentrations downregulates the expression of major drug-metabolizing P450 enzymes in human hepatocytes. Concomitantly, the expression of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) was diminished by COL, whereas expression of glucocorticoid receptor (GR) remained unaltered. A tentative mechanism is perturbation of the GR-PXR/CAR-CYP2/3 signaling cascade, resulting in restricted transcriptional activity of GR receptor by colchicine. In this work we focused on 10-demethylcolchicine (colchiceine; EIN), a structural analogue and a putative metabolite of COL that lacks tubulin-binding activity. We investigated the effects of EIN on the expression of PXR, CAR, and GR receptors in primary cultures of human hepatocytes. In contrast with the effects of COL, EIN does not alter the expression of PXR, CAR, and/or GR receptors mRNAs. In addition, EIN had no effects on transcriptional activities of PXR, CAR, and GR receptors in reporter gene assays using transfected cell lines. Considering that COL and EIN are structurally very close and differ only in their tubulin-binding activity, the data presented imply that the deleterious effects of COL on the GR-PXR/CAR-CYP2/3 cascade are primarily due to perturbation of the microtubule network. Our data support the idea of replacing COL by EIN, which is less toxic and does not interact with xenoreceptors.

• MeSH
• exprese genu účinky léků   MeSH
• financování organizované MeSH
• genetická transkripce účinky léků   MeSH
• hepatocyty metabolismus   účinky léků   MeSH
• kolchicin analogy a deriváty   toxicita   MeSH
• kultivované buňky MeSH
• léčivé přípravky metabolismus   MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• receptory cytoplazmatické a nukleární genetika   metabolismus   účinky léků   MeSH
• receptory glukokortikoidů genetika   MeSH
• reportérové geny MeSH
• steroidní receptory genetika   MeSH
• transfekce MeSH
• transkripční faktory genetika   MeSH
• Check Tag
• lidé MeSH