Regulation of CYP26A1 expression by selective RAR and RXR agonists in human NB4 promyelocytic leukemia cells

All-trans retinoic acid (ATRA) can induce complete remission in acute promyelocytic leukemia (APL), but resistance to this treatment develops rapidly partly due to increased ATRA metabolism. Among the cytochrome P450s (CYPs) involved in ATRA metabolism, the ATRA-inducible cytochrome P450 26A1 (CYP26A1) is particularly active although the molecular mechanisms involved in its regulation are not well defined in the target leukemia cells. To study CYP26A1 expression and regulation in APL cells, we used the NB4 promyelocytic leukemia cell line. CYP26A1 constitutive expression was barely detectable in NB4 cells, but ATRA could induce high levels of CYP26A1 expression, which reached a maximum at 72h. To further define CYP26A1 induction mechanisms in the NB4 leukemia cells, we used RARs and RXR selective agonists. The RARalpha agonist BMS753 could elicit maturation, as expected, but not CYP26A1 expression. Treatment with the RARbeta agonist BMS641, or the RARbeta/gamma agonist BMS961, could not elicit maturation, as expected, nor induce CYP26A1 expression. Because CYP26A1 expression could not be induced by RAR ligands alone, NB4 cells were then co-treated with the RXR agonist BMS649. The RXR agonist alone could not induce CYP26A1 expression, nor in combination with either the RARbeta agonist or the RARbeta/gamma agonist. However, the combination of the RXR agonist and the RARalpha agonist could elicit a marked induction of CYP26A1 expression. In conclusion, we have shown that CYP26A1 induction is not essential for the granulocytic maturation of NB4 leukemia cells, and that CYP26A1 induction requires the activation of both RARalpha and RXR in these cells.     

Expression and functional characterization of cytochrome P450 26A1, a retinoic acid hydroxylase

Retinoic acid (RA) is a critical signaling molecule that performs multiple functions required to maintain cellular viability. It is also used in the treatment of some cancers. Enzymes in the CYP26 family are thought to be responsible for the elimination of RA, and CYP26A1 appears to serve the most critical functions in this family. In spite of its importance, CYP26A1 has neither been heterologously expressed nor characterized kinetically. We expressed the rCYP26A1 in baculovirus-infected insect cells and purified the hexahistidine tagged protein to homogeneity. Heme incorporation was determined by carbon monoxide difference spectrum and a type 1 spectrum was observed with RA binding to CYP26A1. We found that RA is a tight binding ligand of CYP26A1 with low nM binding affinity. CYP26A1 oxidized RA efficiently (depletion K_m 9.4 ± 3.3 nM and V_max 11.3 ± 4.3 pmoles min⁻¹ pmole P450⁻¹) when supplemented with P450 oxidoreductase and NADPH but was independent of cytochrome b5. 4-Hydroxy-RA (4-OH-RA) was the major metabolite produced by rCYP26A1 but two other primary products were also formed. 4-OH-RA was further metabolized by CYP26A1 to more polar metabolites and this sequential metabolism of RA occurred in part without 4-OH-RA leaving the active site of CYP26A1. The high efficiency of CYP26A1 in eliminating both RA and its potentially active metabolites supports the major role of this enzyme in regulating RA clearance in vivo. These results provide a biochemical framework for CYP26A1 function and offer insight into the role of CYP26A1 as a drug target as well as in fetal development and cell cycle regulation.     

Differential modulation of PI3-kinase/Akt pathway during all-trans retinoic acid- and Am80-induced HL-60 cell differentiation revealed by DNA microarray analysis

All-trans retinoic acid (ATRA) and Am80 are natural and synthetic derivatives of Vitamin A and have been used in the fields of oncology and dermatology for years. Their action was considered to be achieved mainly through binding to nuclear hormone receptors, retinoic acid receptors (RARs), although they have been observed to have different biological effects. For example, the two compounds have similar effects on differentiation but different effects on proliferation in human promyelocytic leukemia cell line HL-60 cells. To elucidate the genes responsible for this and other differences, we attempted for the first time to determine the genes whose expressions were differentially modulated during the time course of HL-60 cell differentiation by ATRA and Am80 treatment up to 72h utilizing DNA microarray and clustering analyses. As a result, the expressions of 204 genes were found to be modulated differentially by ATRA and Am80. Among them, we focused on two components of the PI3-kinase/Akt signal transduction pathway, phosphoinositide-3-kinase, beta-catalytic subunit and ribosomal protein S6 kinase polypeptide 1, which are related to the regulation of cell proliferation and apoptosis. Their expressions were specifically suppressed by ATRA, which coincided with the suppressive effects of ATRA on the HL-60 cell proliferation. Moreover, PI3-kinase inhibitors suppressed the proliferation of Am80-treated cells to the same extent as ATRA did. These results indicated that these gene products play a role in HL-60 cell growth suppression during the late stage of differentiation. The complete data and a list of the genes are available at .     

13-cis retinoic acid and isomerisation in paediatric oncology--is changing shape the key to success?

Retinoic acid isomers have been used with some success as chemotherapeutic agents, most recently with 13-cis retinoic acid showing impressive clinical efficacy in the paediatric malignancy neuroblastoma. The aim of this commentary is to review the evidence that 13-cis retinoic acid is a pro-drug, and consider the implications of retinoid metabolism and isomerisation for the further development of retinoic acid for cancer therapy. The low binding affinity of 13-cis retinoic acid for retinoic acid receptors, low activity in gene expression assays and the accumulation of the all-trans isomer in cells treated with 13-cis retinoic acid, coupled with the more-favourable pharmacokinetic profile of 13-cis retinoic acid compared to other isomers, suggest that intracellular isomerisation to all-trans retinoic acid is the key process underlying the biological activity of 13-cis retinoic acid. Intracellular metabolism of all-trans retinoic acid by a positive auto-regulatory loop may result in clinical resistance to retinoic acid. Agents that block or reduce the metabolism of all-trans retinoic acid are therefore attractive targets for drug development. Devising strategies to deliver 13-cis retinoic acid to tumour cells and facilitate the intracellular isomerisation of 13-cis retinoic acid, while limiting metabolism of all-trans retinoic acid, may have a major impact on the efficacy of 13-cis retinoic acid in paediatric oncology.     

Retinoid X receptor α in human liver is regulated by miR-34a

Retinoid X receptor α (RXRα) forms a heterodimer with numerous nuclear receptors to regulate drug- or lipid-metabolizing enzymes. In this study, we investigated whether human RXRα is regulated by microRNAs. Two potential recognition elements of miR-34a were identified in the RXRα mRNA: one in the coding region and the other in the 3′-untranslated region (3′-UTR). Luciferase assays revealed that miR-34a recognizes the element in the coding region. The overexpression of miR-34a in HepG2 cells significantly decreased the endogenous RXRα protein and mRNA levels. The stability of RXRα mRNA was decreased by the overexpression of miR-34a, indicating that miR-34a negatively regulates RXRα expression by facilitating mRNA degradation. We found that the miR-34a-dependent down-regulation of RXRα decreases the induction of CYP26 and the transactivity of CYP3A4. miR-34a has been reported to be up-regulated by p53, which has an ability to promote liver fibrosis. The p53 activation resulted in an increase of the miR-34a level and a decrease of the RXRα protein level. In addition, the miR-34a levels in eight fibrotic livers were higher than those in six normal livers, and the reverse trend was found for the RXRα protein levels. An inverse correlation was observed between the miR-34a and the RXRα protein levels in the 14 samples. Taken together, the data show that miR-34a negatively regulates RXRα expression in human liver, and affects the expression of its downstream genes. This miR-34a-dependent regulation might be the underlying mechanism responsible for the decreased expression of the RXRα protein in fibrotic livers.     

Proteomic analysis of changes in the protein composition of MCF-7 human breast cancer cells induced by all-trans retinoic acid, 9-cis retinoic acid,and their combination

Retinoic acid (all-trans and 9-cis) isomers represent important therapeutic agents for many types of cancers, including human breast cancer. Changes in protein composition of the MCF-7 human breast cancer cells were induced by all-trans retinoic acid, 9-cis retinoic acid, and their combination and subsequently proteomic strategies based on bottom-up method were applied. Proposed approach was used for the analysis of proteins extracted from MCF-7 human breast cancer cell line utilizing a commercially manufactured kit RIPA and separated on two dimensional (2D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after treatment with both retinoic acid isomers. We found significant differences in occurrence of proteins probably affecting the cell migration process in tumour cells. Heat shock protein 27, ribonucleoprotein SmD3, and cofilin-1 were significantly upregulated after treatment with combination of individual retinoic acid isomers. On the other hand, AP-5 complex subunit beta-1 shows the different response. Thus, the results might help to find the answer to important medical questions on (i) the identification of signaling pathways affected by retinoic acid isomers or (ii) how the observed proteomic pattern might reflect the effectiveness of retinoic acids treatment.     

Relative developmental toxicity potencies of retinoids in the embryonic stem cell test compared with their relative potencies in in vivo and two other in vitro assays for developmental toxicity

The present study determines the relative developmental toxicity potencies of retinoids in the embryonic stem (ES)-D3 cell differentiation assay of the embryonic stem cell test, and compares the outcomes with their relative potencies in in vivo and two other in vitro assays for developmental toxicity. The results reveal that the potency ranking obtained in the ES-D3 cell differentiation assay is similar to the reported potency rankings in the two other in vitro assays for developmental toxicity. TTNPB ((E)-4[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid) was the most potent retinoid, whereas etretinate and retinol had the lowest potency. All-trans-retinoic acid, 13-cis-retinoic acid, 9-cis-retinoic acid and acitretin showed an intermediate potency. In vivo potency rankings of the developmental toxicity of retinoids appear to be dependent on the species and/or exposure regimens used. The obtained in vitro potency ranking does not completely correspond with the in vivo potency rankings, although TTNPB is correctly predicted to be the most potent and retinol the least potent congener. The lack of in vivo kinetic processes in the ES-D3 cell differentiation assay might explain the deviating potency predictions of some retinoids. Therefore, knowledge on the species-dependent in vivo kinetics is essential when using in vitro toxicity data for the estimation of in vivo developmental toxicity potencies within series of related compounds.     

Opposing regulation of cytochrome P450 expression by CAR and PXR in hypothyroid mice

Clinical hypothyroidism affects various metabolic processes including drug metabolism. CYP2B and CYP3A are important cytochrome P450 drug metabolizing enzymes that are regulated by the xenobiotic receptors constitutive androstane receptor (CAR, NR1I3) and pregnane X receptor (PXR, NR1I2). We evaluated the regulation of the hepatic expression of CYPs by CAR and PXR in the hypothyroid state induced by a low-iodine diet containing 0.15% propylthiouracil. Expression of Cyp3a11 was suppressed in hypothyroid C57BL/6 wild type (WT) mice and a further decrement was observed in hypothyroid CAR^−/− mice, but not in hypothyroid PXR^−/− mice. In contrast, expression of Cyp2b10 was induced in both WT and PXR^−/− hypothyroid mice, and this induction was abolished in CAR^−/− mice and in and CAR^−/− PXR^−/− double knockouts. CAR mRNA expression was increased by hypothyroidism, while PXR expression remained unchanged. Carbamazepine (CBZ) is a commonly used antiepileptic that is metabolized by CYP3A isoforms. After CBZ treatment of normal chow fed mice, serum CBZ levels were highest in CAR^−/− mice and lowest in WT and PXR^−/− mice. Hypothyroid WT or PXR^−/− mice survived chronic CBZ treatment, but all hypothyroid CAR^−/− and CAR^−/− PXR^−/− mice died, with CAR^−/−PXR^−/− mice surviving longer than CAR^−/− mice (12.3 ± 3.3 days vs. 6.3 ± 2.1 days, p = 0.04). All these findings suggest that hypothyroid status affects xenobiotic metabolism, with opposing responses of CAR and PXR and their CYP targets that can cancel each other out, decreasing serious metabolic derangement in response to a xenobiotic challenge.     

Identification of monoamine oxidase and cytochrome P450 isoenzymes involved in the deamination of phenethylamine-derived designer drugs (2C-series)

In recent years, several compounds of the phenethylamine-type (2C-series) have entered the illicit drug market as designer drugs. In former studies, the qualitative metabolism of frequently abused 2Cs (2C-B, 2C-I, 2C-D, 2C-E, 2C-T-2, 2C-T-7) was studied using a rat model. Major phase I metabolic steps were deamination and O-demethylation. Deamination to the corresponding aldehyde was the reaction, which was observed for all studied compounds. Such reactions could in principal be catalyzed by two enzyme systems: monoamine oxidase (MAO) and cytochrome P450 (CYP). The aim of this study was to determine the human MAO and CYP isoenzymes involved in this major metabolic step and to measure the Michaelis-Menten kinetics of the deamination reactions. For these studies, cDNA-expressed CYPs and MAOs were used. The formation of the aldehyde metabolite was measured using GC-MS after extraction. For all compounds studied, MAO-A and MAO-B were the major enzymes involved in the deamination. For 2C-D, 2C-E, 2C-T-2 and 2C-T-7, CYP2D6 was also involved, but only to a very small extent. Because of the isoenzymes involved, the 2Cs are likely to be susceptible for drug-drug interactions with MAO inhibitors.     

Influence of phenylalanine 120 on cytochrome P450 2D6 catalytic selectivity and regiospecificity: crucial role in 7-methoxy-4-(aminomethyl)-coumarin metabolism

The polymorphic human debrisoquine hydroxylase, cytochrome P450 2D6 (CYP2D6), is one of the most important phase I drug metabolising enzymes. It is responsible for metabolising a large number of compounds that mostly share similarity in having a basic N-atom and an aromatic moiety. In homology modelling studies, it has been suggested that in fixation of this aromatic moiety, there may be an important role for phenylalanine 120 (Phe(120)). In this study, the role of Phe(120) in ligand binding and catalysis was experimentally examined by mutating it into an alanine. Strikingly, this substitution led to a completely abolished 7-methoxy-4-(aminomethyl)-coumarin (MAMC) O-demethylating activity of CYP2D6. On the other hand, bufuralol metabolism was hardly affected (K(m) of 1-hydroxylation mutant: 1.2 microM, wild-type: 2.9 microM, 4-hydroxylation mutant: 1.5 microM, and wild-type: 3.2 microM) and neither was affected dextromethorphan O-demethylation (K(m) mutant: 1.2 microM, wild-type: 2 microM, k(cat) mutant: 4.5 min(-1), and wild-type: 3.3 min(-1)). However, the Phe(120)Ala mutant also formed 3-hydroxymorphinan, the double demethylated form of dextromethorphan, which was not detected using wild-type CYP2D6. 3,4-Methylenedioxymethamphetamine (MDMA) was demethylenated by both mutant and wild-type CYP2D6 to 3,4-dihydroxymethamphetamine (3,4-OH-MA K(m) of mutant: 55 microM and wild-type: 2 microM). In addition, the mutant formed two additional metabolites; 3,4-methylenedioxyamphetamine (MDA) and N-hydroxy-3,4-methylenedioxymethamphetamine (N-OH-MDMA). Inhibition experiments of dextromethorphan O-demethylation showed a decreased affinity of the Phe(120)Ala mutant for quinidine (IC(50) mutant: 240 nM and wild-type, 40 nM), while IC(50)s for quinine were equal (1 microM). These data indicate the importance of Phe(120) in the selectivity and regiospecificity in substrate binding and catalysis by CYP2D6.     

2,3,7,8-Tetrachlorodibenzo-<Emphasis Type="Italic">p</Emphasis>-dioxin (TCDD) alters the endogenous metabolism of all-<Emphasis Type="Italic">trans</Emphasis>-retinoic acid in the rat

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) is known to influence vitamin A homeostasis. In order to investigate the mechanism behind this retinoid disruption, male Sprague-Dawley rats were exposed to TCDD at doses ranging from 0.1 to 100 micro g/kg body weight, and were killed 3 days after exposure. Additional groups of rats were killed 1 and 28 days after a single oral dose of 10 micro g TCDD/kg body weight. Serum, kidney, and liver were investigated for retinoid levels, as well as gene expression and enzyme activities relevant for retinoid metabolism. Besides the well known effects of TCDD on apolar retinoids, i.e. decreased hepatic and increased renal retinyl ester (RE) levels, we have found dose-dependent elevation of all- trans-retinoic acid (all- trans-RA) levels in all investigated tissues. In the liver, 9- cis-4-oxo-13,14-dihydro-RA was drastically decreased by TCDD in a dose-dependent manner. In serum, cis-isomers of all- trans-RA, including 9,13-di- cis-RA, were significantly reduced already at the lowest dose level. Protein and mRNA levels of cellular retinol binding protein I (CRBP-I) in liver or kidneys were not significantly altered by TCDD exposure at doses at which retinoid levels were affected, making CRBP-I an unlikely candidate to account for the alterations in retinoid metabolism caused by TCDD. The expression and activities of relevant cytochrome P450 (CYP) enzymes with potential roles in all- trans-RA synthesis and/or degradation (CYP1A1, 1A2, and 2B1/2) were also monitored. A possible role of CYP1A1 in TCDD-induced all- trans-RA synthesis is suggested from the time-course relationship between CYP1A1 activity and all- trans-RA levels in liver and kidney. The significant alteration of the all- trans-RA metabolism has the potential to contribute significantly to the toxicity of TCDD.     

Deoxyribonucleic acid binding of 3-hydroxy- and 9-hydroxybenzo[a]pyrene following further metabolism by mouse liver microsomal cytochrome P1-450.

In the presence of NADPH and microsomes from 3-methylcholanthrene-treated C57BL/6N mice, [³H]3-OH-benzo[a]pyrene is metabolized to reactive intermediates which covalently bind to deproteinized salmon sperm DNA in vitro. Enzymatically digested DNA, containing bound [³H]3-OH-benzolajpyrene derivatives, generates an elution profile from Sephadex LH20 chromatography which resembles similar chromatograms with [³H]benzo[a]pyrene. All peaks resulting from [³H]benzo[a]pyrene activation appear to be prominently represented in [³H]3-OH-benzo[a]pyrene activation, except that several peaks which emerge near the end of the eluting gradient of methanol and water are much reduced. Notably, a peak designated E, and attributed to benzo[a]pyrene-7, 8-diol-9, 10-oxide binding in [³H]benzo[a]pyrene incubations, is also prominently represented in incubations with [³H]3-OH-benzo[a]pyrene. Radioactivity in all of these peaks is inhibited effectively if one-seventh the concentration of 1-OH-benzo[a]pyrene is included in the incubation with [³H]3-OH-benzo[a]pyrene. Microsomes from 3-methylcholanthrene-treated DBA/ 2N mice cause insignificant binding. UDP-glucuronic acid markedly reduces all peaks except E, and 1, 2-epoxy-3, 3, 3-trichloropropane reduces all peaks except C and E. 9-Hydroxybenzo[a]pyrene is further metabolized to DNA binding species by microsomes from either 3-methylcholanthrene-treated DBA/2N or C57BL/6N mice. UDP-glucuronic acid prevents about 50 per cent of the binding with microsomes from DBA/2N mice but not with microsomes from C57BL/6N. In contrast, UDP-glucuronic acid does prevent binding in some of these same peaks when [³H]benzotaipyrene is the starting substrate with microsomes from C57BL/6N mice. UDP-glucuronic acid does not prevent binding in peak E in incubations with [³H]benzo[a]pyrene or [³H]3-hydroxybenzo[a]pyrene.     

Role of cytochrome P450-mediated metabolism and identification of novel thiol-conjugated metabolites in mice with phenytoin-induced liver injury

Phenytoin, 5,5-diphenylhydantoin (DPH), is widely used as an anticonvulsant agent. Severe hepatic injury rarely occurs in patients who received DPH. The development of liver injury is thought to be caused by reactive metabolites; however, the metabolites suggested to contribute to hepatotoxicity have not yet been detected in vivo and their effect on developing the liver injury is largely unknown. We recently demonstrated that DPH treatment decreased hepatic glutathione (GSH) contents, and GSH-depleted condition exacerbated DPH-induced liver injury in mice. The aim of the present study was to identify the reactive metabolite and to investigate the role of P450-mediated metabolisms in DPH-induced liver injury. We identified a novel GSH-conjugated (GS)-DPH, a conjugate of putative electrophilic arene oxide intermediate with GSH, in the bile of mice with DPH-induced liver injury. In plasma, cysteine- or N-acetylcysteine-conjugated DPH was detected, and these thiol conjugates levels were correlated with the plasma alanine aminotransferase (ALT) levels. These changes were significantly reduced by pretreatment with P450 inhibitor. Furthermore, the increases of hepatic P450 activities were in parallel with elevation of plasma thiol conjugates levels. These findings suggest that the arene oxide intermediate, which can be converted to thiol conjugates, is involved in DPH-induced liver injury.     

All-trans retinoic acid enhances the transport of phase II metabolites of benzo[a]pyrene by inducing the Breast Cancer Resistance Protein expression in Caco-2 cells

All-trans retinoic acid (atRA) is the most active metabolite of vitamin A. It is a ligand of retinoic acid receptors (RAR) as well as of retinoid X receptors (RXR) and effectively stimulates the RAR/RXR signalling pathway.