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Alain Belanger - One of the best experts on this subject based on the ideXlab platform.
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Glucuronidation activity of the UGT2B17 enzyme toward xenobiotics. Drug Metab Dispos 2003;31: 670 –6
2020Co-Authors: David Turgeon, Jeansebastien Carrier, And Sarah Chouinard, Alain BelangerAbstract:This article is available online at http://dmd.aspetjournals.org ABSTRACT: UDP-glucuronosyltransferase (UGT) 2B17 is one of the most important conjugating enzymes in androgen metabolism and shares more than 95% homology with UGT2B15. Although UGT2B15 has been fully characterized for its ability to conjugate drugs, environmental pollutants, and dietary components, UGT2B17 received less attention for its capacity to glucuronidate xenobiotics. In the present study, more than 55 exogenous compounds belonging to several categories of compounds were analyzed as potential substrates for UGT2B17. Glucuronidation activity was observed with several coumarins, anthraquinones, and flavonoids. The higher glucuronidation activity was measured with alizarin (125 pmol ⅐ min ؊1 ⅐ mg protein ؊1 ), whereas UGT2B17 conjugated eugenol, scopoletin, and galangin with glucuronidation rates of 102.5, 102, and 58 pmol ⅐ min ؊1 ⅐ mg protein ؊1 , respectively. The characterization of UGT2B17 as a xenobiotics-conjugating enzyme demonstrates that its role is not limited to androgen metabolism and that its specificity for exogenous substrates is different from other UGT2B isoforms. Taken together, these data suggest a role of UGT2B17 for the hepatic detoxification
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multiple roles for udp glucuronosyltransferase ugt 2b15 and UGT2B17 enzymes in androgen metabolism and prostate cancer evolution
The Journal of Steroid Biochemistry and Molecular Biology, 2015Co-Authors: Louis Gauthierlandry, Alain Belanger, Olivier BarbierAbstract:In the prostate, approximately 50% of androgens are from adrenal steroids, mainly dehydroepiandrosterone (DHEA), its sulfate and androstenedione. These compounds are converted first into testosterone, and then into the active hormone dihydrotestosterone (DHT). After having activated the androgen receptor (AR), DHT is reduced into androstane-3α-DIOL (3α-DIOL) and androsterone (ADT), which are subsequently converted into 2 inactive and easily excretable metabolites: 3α-DIOL-17glucuronide (3α-DIOL-17G) and ADT-3glucuronide (ADT-3G). The formation of these last derivatives through the glucuronidation reaction involves 2 UDP-glucuronosyltransferase (UGT) enzymes, namely UGT2B15 and UGT2B17. The present review article aims at providing a comprehensive view of the physiological and pharmacological importance of these 2 enzymes for the control of androgen homeostasis. We will resume: (i) how UGT2B15 and UGT2B17 contribute to androgen elimination; (ii) how their glucuronidation capacity influences the androgen signaling pathway in prostate cells; (iii) how they contribute to the anti-proliferative properties of AR antagonists in prostate cancer cells; and (iv) how AR and its spliced variants regulate the UGT2B15 and/or UGT2B17 genes expression. Finally, whether the unexploited AR-UGT axis could serve as a prognostic maker or a pharmacological target for novel therapeutics in the treatment of prostate cancer is also discussed. This article is part of a special issue entitled 'Essential role of DHEA'.
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differential expression of the androgen conjugating ugt2b15 and UGT2B17 enzymes in prostate tumor cells during cancer progression
The Journal of Clinical Endocrinology and Metabolism, 2012Co-Authors: Sophie Pâquet, Alain Belanger, Paul S Rennie, Ladan Fazli, Laurent Grosse, Melanie Verreault, Bernard Tetu, Olivier BarbierAbstract:Context: Androgens play major roles in prostate cancer initiation and development. In prostate cells, the human uridine diphosphate-glucuronosyltransferase (UGT)2B15 and UGT2B17 enzymes inactivate androgens. Objective: We investigated in vivo how UGT2B15 and UGT2B17 expressions are affected during prostate cancer development. Design: We conducted an observational study of the UGT2B15 and UGT2B17 mRNA and protein levels. Setting: The study was conducted at Laval University (Quebec, Canada) and at the University of British Columbia (Vancouver, Canada). Patients/Participants: Participants were from a cohort of prostate cancer patients from the Hotel-Dieu de Quebec hospital (Quebec; mRNA analyses) and from the Vancouver Prostate Centre tissue bank (Vancouver; tissue microarray experiments). Main Outcome Measures: UGT mRNA and protein levels were determined using real-time PCR and immunohistochemical analyses, respectively. Results: Both UGT2B15 and UGT2B17 mRNA and protein levels were not significantly associ...
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isoform specific regulation of uridine diphosphate glucuronosyltransferase 2b enzymes in the human prostate differential consequences for androgen and bioactive lipid inactivation
Endocrinology, 2006Co-Authors: Sarah Chouinard, Alain Belanger, Georges Pelletier, Olivier BarbierAbstract:Androgens as well as monohydroxy-fatty acids are implicated in the pathogenesis of prostate cancer. Like a huge variety of endo- and xenobiotics, they are eliminated as glucuronide conjugates formed by uridine diphosphate-glucuronosyltransferase (UGT) enzymes. In the present study, we observe that treatment of the prostate cancer cells LNCaP with natural and synthetic androgens, IL-1alpha, or epidermal growth factor (EGF) differently modulates the glucuronidation of androgen and bioactive lipid metabolites. Indeed, glucuronidation of 5alpha-androstane-3alpha,17beta-diol and 13-hydroxyoctadecadienoic acid was drastically reduced, whereas 12-hydroxyeicosatetraenoic acid conjugation by UGT was increased after androgen treatment. These effects reflected the reduction of UGT2B10, -B15, and -B17 enzyme expression, and the activation of the UGT2B11 gene. In human prostate epithelial cells, only UGT2B11 and -B15 mRNAs are detected and are regulated by androgens in a similar manner as in LNCaP cells. In LNCaP cells, IL-1alpha and EGF also regulate UGT2B expression in an isoform-specific manner; IL-1alpha induced UGT2B10 and reduced UGT2B17, while having no effects on UGT2B11 mRNA levels. EGF treatment resulted in a decreased UGT2B17 expression, whereas UGT2B10 and -B11 mRNA remained at their basal levels. Overall, these results demonstrate that in the human prostate, androgens do not only affect their own inactivation but also influence the levels of monohydroxy-fatty acids by regulating the expression of UGT2B enzymes in an isoform-specific manner. These differential effects of androgens, IL-1alpha, and EGF on lipid metabolism likely constitute an additional mechanism by which these endogenous factors promote prostate cancer development.
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cellular specific expression of the androgen conjugating enzymes ugt2b15 and UGT2B17 in the human prostate epithelium
Endocrine Research, 2004Co-Authors: Sarah Chouinard, Alain Belanger, Georges Pelletier, Olivier BarbierAbstract:In humans, 3β‐hydroxysteroid dehydrogenase (3β‐HSD), 17β‐HSD, and 5α‐reductase enzymes convert dehydroepiandrosterone (DHEA), androstenedione, and testosterone into the most potent natural androgen dihydrotestosterone (DHT) in the prostate. This androgen is transformed mainly in situ to two Phase I metabolites, androsterone (ADT) and androstane‐3α,17β‐diol (3α‐DIOL), which can, however, be back‐converted to DHT. Here, we report recent findings on the characterization of specific anti‐UDP‐glucuronosyltransferases (UGT)2B15 and 2B17 antibodies and their use to identify UGT2B expressing‐cells in the human prostate epithelium. We found that UGT2B17 is expressed in basal cells where DHEA is converted into 3α‐DIOL and ADT. By contrast, the expression of UGT2B15 was observed only in luminal cells, where DHT is formed from testosterone. These results demonstrate that, in the human prostate, UGT2B15 and UGT2B17 genes have complementary roles, and are expressed in cells where their specific substrates are synthesiz...
Eric Levesque - One of the best experts on this subject based on the ideXlab platform.
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alternative promoters control UGT2B17 dependent androgen catabolism in prostate cancer and its influence on progression
British Journal of Cancer, 2020Co-Authors: Eric Levesque, Veronique Turcotte, Eric P Allai, Michele Rouleau, Adrie Labrie, Helene Hovingto, Herve Isso, Luciana Melogarcia, Patrick Caro, Lyne VilleneuveAbstract:Perturbation of the major UGT2B17-dependent androgen catabolism pathway has the potential to affect prostate cancer (PCa) progression. The objective was to evaluate UGT2B17 protein expression in primary tumours in relation to hormone levels, disease characteristics and cancer evolution. We conducted an analysis of a high-density prostate tumour tissue microarray consisting of 239 localised PCa cases treated by radical prostatectomy (RP). Cox proportional hazard ratio analysis was used to evaluate biochemical recurrence (BCR), and a linear regression model evaluated variations in circulating hormone levels measured by mass spectrometry. The transcriptome of UGT2B17 in PCa was established by using RNA-sequencing data. UGT2B17 expression in primary tumours was associated with node-positive disease at RP and linked to circulating levels of 3α-diol-17 glucuronide, a major circulating DHT metabolite produced by the UGT2B17 pathway. UGT2B17 was an independent prognostic factor linked to BCR after RP, and its overexpression was associated with development of metastasis. Finally, we demonstrated that distinctive alternative promoters dictate UGT2B17-dependent androgen catabolism in localised and metastatic PCa. The androgen-inactivating gene UGT2B17 is controlled by overlooked regulatory regions in PCa. UGT2B17 expression in primary tumours influences the steroidome, and is associated with relevant clinical outcomes, such as BCR and metastasis.
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inactivation of prostaglandin e2 as a mechanism for UGT2B17 mediated adverse effects in chronic lymphocytic leukemia
Frontiers in Oncology, 2019Co-Authors: Eric P Allai, Veronique Turcotte, Eric Levesque, Lyne Villeneuve, Michele Rouleau, Katrina Vanura, Chantal GuillemetteAbstract:High expression of the metabolic enzyme UDP-glucuronosyltransferase UGT2B17 in chronic lymphocytic leukemia (CLL) cells was associated with poor prognosis in two independent studies. However, the underlying mechanism remains unknown. We hypothesized that UGT2B17 impacts intracellular levels of hormone-like signaling molecules involved in the regulation of gene expression in leukemic cells. We initially confirmed in a third cohort of 291 CLL patients that those with high UGT2B17 displayed poor prognosis (hazard ratio of 2.31, P = 0.015). Consistent with the unfavorable prognostic significance of elevated UGT2B17 expression in CLL patients, high UGT2B17 expression was associated with enhanced proliferation of MEC1 and JVM2 malignant B-cell models. Transcriptomic analyses revealed that high UGT2B17 was linked to a significant alteration of genes related to prostaglandin E2 (PGE2) and to its precursor arachidonic acid, both in cell models and a cohort of 448 CLL patients. In functional assays, PGE2 emerged as a negative regulator of apoptosis in CLL patients and proliferation in cells models, whereas its effect was partially abrogated by high UGT2B17 expression in MEC1 and JVM2 cells. Enzymatic assays and mass-spectrometry analyses established that the UGT2B17 enzyme inactivates PGE2 by its conjugation to glucuronic acid (GlcA) leading to the formation of two glucuronide (G) derivatives. High UGT2B17 expression was further associated with a proficient inactivation of PGE2 to PGE2-G in CLL patient cells and cell models. We conclude that UGT2B17-dependent PGE2 glucuronidation impairs anti-oncogenic PGE2 effects in leukemic cells, thereby partially contributing to disease progression in high UGT2B17 CLL patients.
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copy number variations cnvs of the human sex steroid metabolizing genes UGT2B17 and ugt2b28 and their associations with a ugt2b15 functional polymorphism
Human Mutation, 2009Co-Authors: Vincent Menard, Chantal Guillemette, Olivier Eap, Mario Harvey, Eric LevesqueAbstract:UGT2B17 and UGT2B28 are among the most commonly deleted genes in humans and encode members of the uridine diphosphate (UDP)-glucuronosyltransferase 2B (UGT2B) subfamily. They are involved, along with UGT2B15, in the catabolism of sex-steroid hormones. Despite the recent biomedical interest in UGT2B17 and UGT2B28 copy-number variations (CNVs) within human populations, the impact of their gene dosage has been hampered by the lack of precise molecular identification of the common deletion breakpoints within high homology sequence regions on chromosome 4. We have characterized these common deletions and report their coexistence in Caucasians, along with the p.D85Y (rs1902023:G>T) functional polymorphism of UGT2B15. Segmental duplications of 4.9 kb for UGT2B17 and 6.8 kb for UGT2B28 comprise purine-rich recombination sites located 117 kb and 108 kb apart on both ends of the deletions. CNVs of UGT2B17 and UGT2B28 occur in Caucasians at 27% and 13.5%, respectively. While only 43% have two copies of both genes, 57% harbor at least one deletion. Their co-occurrence on 5% of chromosomes creates a 225-kb genomic gap. CNVs of both UGT2B17 and UGT2B28, with the co-occurrence of UGT2B15:p.D85Y, generate seven distinct haplotypes. Restricting the analyses to CNV of the UGT2B17 gene without evaluating UGT2B28 CNV, along with the genotype of UGT2B15, may over- or underestimate the impact of each gene under physiological conditions or disease states. Hum Mutat 30:1–10, 2009. © 2009 Wiley-Liss, Inc.
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relative enzymatic activity protein stability and tissue distribution of human steroid metabolizing ugt2b subfamily members
Endocrinology, 2001Co-Authors: David Turgeon, Alain Belanger, Eric Levesque, Jeansebastien Carrier, Dean W HumAbstract:Androgens and estrogens play major roles in cell differentiation, cell growth, and peptide secretion in steroid target tissues. In addition to the binding of these hormones to their receptors, formation and metabolism are important in the action of steroids. Metabolism of the potent steroid hormones includes glucuronidation, a major pathway of steroid elimination in liver and several steroid target tissues. Glucuronidation is catalyzed by UDP-glucuronosyltransferases (UGTs), which transfer the polar moiety from UDP-glucuronic acid to a wide variety of endogenous compounds, including steroid hormones. The UGT superfamily of enzymes is subdivided into two families, UGT1 and UGT2, on the basis of sequence homology. To date, six UGT2B proteins have been isolated, namely UGT2B4, UGT2B7, UGT2B10, UGT2B11, UGT2B15, and UGT2B17, all of which have been demonstrated to be active on steroid molecules, except for UGT2B10 and UGT2B11, for which no substrate was found. The relative activity of these enzymes on steroidal compounds remains unknown due to variable levels of UGT2B expression in different in vitro cell line models and various conditions of the enzymatic assays. Comparison of the glucuronidation rates of these enzymes requires a unique system for UGT2B protein expression, protein normalization, and enzymatic assays. In this study we have stably expressed UGT2B4, UGT2B7, UGT2B15, and UGT2B17 in the HK293 cell line, which is devoid of steroid UGT activity; characterized their kinetic properties relative to UGT protein expression; determined their transcript and protein stabilities; and established extensively their tissular distributions. UGT2B7 was demonstrated to glucuronidate estrogens, catechol estrogens, and androstane-3alpha,17beta-diol more efficiently than any other human UGTB isoform. UGT2B15 and UGT2B17 showed similar glucuronidation activity for androstane-3alpha,17beta-diol (30% lower than that of UGT2B7), whereas UGT2B17 demonstrated the highest activity for androsterone, testosterone, and dihydrotestosterone. UGT2B4 demonstrates reactivity toward 5alpha-reduced androgens and catechol estrogens, but at a significantly lower level than UGT2B7, 2B15, and 2B17. Cycloheximide treatment of stably transfected HK293 cells demonstrated that the UGT2B17 protein is more labile than the other enzymes; the protein levels decrease after 1 h of treatment, whereas other UGT2B proteins were stable for at least 12 h. Treatment of stable cells with actinomycin D reveals that UGT2B transcripts are stable for 12 h, except for the UGT2B4 transcript, which was decreased by 50% after the 12-h incubation period. Tissue distribution of the UGT2B enzymes demonstrated that UGT2B isoforms are expressed in the liver as well as in several extrahepatic steroid target tissues, namely, kidney, breast, lung, and prostate. This study clearly demonstrates the relative activities and the major substrates of human steroid-metabolizing UGT2B enzymes, which are expressed in a wide variety of steroid target tissues.
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isolation and characterization of the human ugt2b15 gene localized within a cluster of ugt2b genes and pseudogenes on chromosome 4
Journal of Molecular Biology, 2000Co-Authors: David Turgeon, Eric Levesque, Jeansebastien Carrier, Barbara G Beatty, Alain BelangerAbstract:Abstract Glucuronidation is a major pathway of androgen metabolism and is catalyzed by UDP-glucuronosyltransferase (UGT) enzymes. UGT2B15 and UGT2B17 are 95 % identical in primary structure, and are expressed in steroid target tissues where they conjugate C19 steroids. Despite the similarities, their regulation of expression are different; however, the promoter region and genomic structure of only the UGT2B17 gene have been characterizedX to date. To isolate the UGT2B15 gene and other novel steroid-conjugating UGT2B genes, eight P-1-derived artificial chromosomes (PAC) clones varying in length from 30 kb to 165 kb were isolated. The entire UGT2B15 gene was isolated and characterized from the PAC clone 21598 of 165 kb. The UGT2B15 and UGT2B17 genes are highly conserved, are both composed of six exons spanning approximately 25 kb, have identical exon sizes and have identical exon-intron boundaries. The homology between the two genes extend into the 5′-flanking region, and contain several conserved putative cis -acting elements including Pbx-1, C/EBP, AP-1, Oct-1 and NF/κB. However, transfection studies revealed differences in basal promoter activity between the two genes, which correspond to regions containing non-conserved potential elements. The high degree of homology in the 5′-flanking region between the two genes is lost upstream of −1662 in UGT2B15 , and suggests a site of genetic recombination involved in duplication of UGT2B genes. Fluorescence in situ hybridization mapped the UGT2B15 gene to chromosome 4q13.3-21.1. The other PAC clones isolated contain exons from the UGT2B4 , UGT2B11 and UGT2B17 genes. Five novel exons, which are highly homologous to the exon 1 of known UGT2B genes, were also identified; however, these exons contain premature stop codons and represent the first recognized pseudogenes of the UGT2B family. The localization of highly homologous UGT2B genes and pseudogenes as a cluster on chromosome 4q13 reveals the complex nature of this gene locus, and other novel homologous UGT2B genes encoding steroid conjugating enzymes are likely to be found in this region of the genome.
Moshe Finel - One of the best experts on this subject based on the ideXlab platform.
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clopidogrel carboxylic acid glucuronidation is mediated mainly by ugt2b7 ugt2b4 and UGT2B17 implications for pharmacogenetics and drug drug interactions
Drug Metabolism and Disposition, 2018Co-Authors: Helina Kahma, Katriina E Tarkiainen, Mikko T Holmberg, Anne M. Filppula, Matti K Itkonen, Aleksi Tornio, Mikko Neuvonen, Moshe Finel, Mikko NiemiAbstract:The antiplatelet drug clopidogrel is metabolized to an acyl- β -d-glucuronide, which causes time-dependent inactivation of CYP2C8. Our aim was to characterize the UDP-glucuronosyltransferase (UGT) enzymes that are responsible for the formation of clopidogrel acyl- β -d-glucuronide. Kinetic analyses and targeted inhibition experiments were performed using pooled human liver and intestine microsomes (HLMs and HIMs, respectively) and selected human recombinant UGTs based on preliminary screening. The effects of relevant UGT polymorphisms on the pharmacokinetics of clopidogrel were evaluated in 106 healthy volunteers. UGT2B7 and UGT2B17 exhibited the greatest level of clopidogrel carboxylic acid glucuronidation activities, with a CL int,u of 2.42 and 2.82 µ l⋅min −1 ⋅mg −1 , respectively. Of other enzymes displaying activity (UGT1A3, UGT1A9, UGT1A10-H, and UGT2B4), UGT2B4 (CL int,u 0.51 µ l⋅min −1 ⋅mg −1 ) was estimated to contribute significantly to the hepatic clearance. Nonselective UGT2B inhibitors strongly inhibited clopidogrel acyl- β -d-glucuronide formation in HLMs and HIMs. The UGT2B17 inhibitor imatinib and the UGT2B7 and UGT1A9 inhibitor mefenamic acid inhibited clopidogrel carboxylic acid glucuronidation in HIMs and HLMs, respectively. Incubation of clopidogrel carboxylic acid in HLMs with UDPGA and NADPH resulted in strong inhibition of CYP2C8 activity. In healthy volunteers, the UGT2B17*2 deletion allele was associated with a 10% decrease per copy in the plasma clopidogrel acyl- β -d-glucuronide to clopidogrel carboxylic acid area under the plasma concentration-time curve from 0 to 4 hours (AUC 0–4 ) ratio ( P β -d-glucuronide is impaired in carriers of the UGT2B17 deletion. These findings may have implications regarding the intracellular mechanisms leading to CYP2C8 inactivation by clopidogrel.
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clopidogrel carboxylic acid glucuronidation is mediated mainly by ugt2b7 ugt2b4 and UGT2B17 implications for pharmacogenetics and drug drug interactions
Drug Metabolism and Disposition, 2018Co-Authors: Helina Kahma, Katriina E Tarkiainen, Mikko T Holmberg, Anne M. Filppula, Matti K Itkonen, Aleksi Tornio, Mikko Neuvonen, Moshe Finel, Pertti J Neuvonen, Mikko NiemiAbstract:The antiplatelet drug clopidogrel is metabolized to an acyl-β-d-glucuronide, which causes time-dependent inactivation of CYP2C8. Our aim was to characterize the UDP-glucuronosyltransferase (UGT) enzymes that are responsible for the formation of clopidogrel acyl-β-d-glucuronide. Kinetic analyses and targeted inhibition experiments were performed using pooled human liver and intestine microsomes (HLMs and HIMs, respectively) and selected human recombinant UGTs based on preliminary screening. The effects of relevant UGT polymorphisms on the pharmacokinetics of clopidogrel were evaluated in 106 healthy volunteers. UGT2B7 and UGT2B17 exhibited the greatest level of clopidogrel carboxylic acid glucuronidation activities, with a CLint,u of 2.42 and 2.82 µl⋅min-1⋅mg-1, respectively. Of other enzymes displaying activity (UGT1A3, UGT1A9, UGT1A10-H, and UGT2B4), UGT2B4 (CLint,u 0.51 µl⋅min-1⋅mg-1) was estimated to contribute significantly to the hepatic clearance. Nonselective UGT2B inhibitors strongly inhibited clopidogrel acyl-β-d-glucuronide formation in HLMs and HIMs. The UGT2B17 inhibitor imatinib and the UGT2B7 and UGT1A9 inhibitor mefenamic acid inhibited clopidogrel carboxylic acid glucuronidation in HIMs and HLMs, respectively. Incubation of clopidogrel carboxylic acid in HLMs with UDPGA and NADPH resulted in strong inhibition of CYP2C8 activity. In healthy volunteers, the UGT2B17*2 deletion allele was associated with a 10% decrease per copy in the plasma clopidogrel acyl-β-d-glucuronide to clopidogrel carboxylic acid area under the plasma concentration-time curve from 0 to 4 hours (AUC0-4) ratio (P < 0.05). To conclude, clopidogrel carboxylic acid is metabolized mainly by UGT2B7 and UGT2B4 in the liver and by UGT2B17 in the small intestinal wall. The formation of clopidogrel acyl-β-d-glucuronide is impaired in carriers of the UGT2B17 deletion. These findings may have implications regarding the intracellular mechanisms leading to CYP2C8 inactivation by clopidogrel.
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regiospecificity and stereospecificity of human udp glucuronosyltransferases in the glucuronidation of estriol 16 epiestriol 17 epiestriol and 13 epiestradiol
Drug Metabolism and Disposition, 2013Co-Authors: Nina Sneitz, Johanna Mosorin, Mikko Vahermo, Liisa Laakkonen, Donald Poirier, Moshe FinelAbstract:The glucuronidation of estriol, 16-epiestriol, and 17-epiestriol by the human UDP-glucuronosyltransferases (UGTs) of subfamilies 1A, 2A, and 2B was examined. UGT1A10 is highly active in the conjugation of the 3-OH in all these estriols, whereas UGT2B7 is the most active UGT toward one of the ring D hydroxyls, the 16-OH in estriol and 16-epiestriol, but the 17-OH in 17-epiestriol. Kinetic analyses indicated that the 17-OH configuration plays a major role in the affinity of UGT2B7 for estrogens. The glucuronidation of the different estriols by the human liver and intestine microsomes reflects the activity of UGT1A10 and UGT2B7 in combination with the tissues’ difference in UGT1A10 expression. The UGT1A10 mutant 1A10-F93G exhibited much higher Vmax values than UGT1A10 in estriol and 17-epiestriol glucuronidation, but a significantly lower value in 16-epiestriol glucuronidation. To this study on estriol glucuronidation we have added experiments with 13-epiestradiol, a synthetic estradiol in which the spatial arrangement of the methyl on C18 and the hydroxyl on C17 is significantly different than in other estrogens. In comparison with estradiol glucuronidation, the C13 configuration change decreases the turnover of UGTs that conjugate the 3-OH, but increases it in UGTs that primarily conjugate the 17-OH. Unexpectedly, UGT2B17 exhibited similar conjugation rates of both the 17-OH and 3-OH of 13-espiestradiol. The combined results reveal the strong preference of UGT1A10 for the 3-OH of physiologic estrogens and the equivalently strong preference of UGT2B7 and UGT2B17 for the hydroxyls on ring D of such steroid hormones.
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Glucuronidation of the steroid enantiomers ent-17β-estradiol, ent-androsterone and ent-etiocholanolone by the human UDP-glucuronosyltransferases.
The Journal of steroid biochemistry and molecular biology, 2011Co-Authors: Nina Sneitz, Douglas F Covey, Kathiresan Krishnan, Moshe FinelAbstract:Steroids enantiomers are interesting compounds for detailed exploration of drug metabolizing enzymes, such as the UDP-glucuronosyltransferases (UGTs). We have now studied the glucuronidation of the enantiomers of estradiol, androsterone and etiocholanolone by the 19 human UGTs of subfamilies 1A, 2A and 2B. The results reveal that the pattern of human UGTs of subfamily 2B that glucuronidate ent-17β-estradiol, particularly 2B15 and 2B17, resembles the glucuronidation of epiestradiol (17α-estradiol) rather than 17β-estradiol, the main physiological estrogen. The UGTs of subfamilies 1A and 2A exhibit higher degree of regioselectivity than enantioselectivity in the conjugation of these estradiols, regardless of whether the activity is primarily toward the non-chiral site, 3-OH (UGT1A1, UGT1A3, UGT1A7, UGT1A8 and, above all, UGT1A10), or the 17-OH (UGT1A4). In the cases of etiocholanolone and androsterone, glucuronidation of the ent-androgens, like the conjugation of the natural androgens, is mainly catalyzed by UGTs of subfamilies 2A and 2B. Nevertheless, the glucuronidation of ent-etiocholanolone and ent-androsterone by both UGT2B7 and UGT2B17 differs considerably from their respective activity toward the corresponding endogenous androgens, whereas UGT2A1-catalyzed conjugation is much less affected by the stereochemistry differences. Kinetic analyses reveal that the K(m) value of UGT2A1 for ent-estradiol is much higher than the corresponding value in the other two high activity enzymes, UGT1A10 and UGT2B7. Taken together, the results highlight large enantioselectivity differences between individual UGTs, particularly those of subfamily 2B.
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influence of n terminal domain histidine and proline residues on the substrate selectivities of human udp glucuronosyltransferase 1a1 1a6 1a9 2b7 and 2b10
Drug Metabolism and Disposition, 2009Co-Authors: Oranun Kerdpin, Kushari Bowalgaha, Peter I. Mackenzie, Moshe Finel, John O MinersAbstract:An N-terminal domain histidine [corresponding to position 39 of UDP-glucuronosyltransferase (UGT) 1A1] is conserved in all UGT1A and UGT2B subfamily proteins except UGT1A4 (Pro-40) and UGT2B10 (Leu-34). Unlike most UGT1A and UGT2B xenobiotic-metabolizing enzymes, UGT1A4 and UGT2B10 lack the ability to glucuronidate 4-methylumbelliferone (4MU) and 1-naphthol (1NP), both planar phenols, and naproxen (a carboxylic acid). However, only UGT1A4 glucuronidates the tertiary amines lamotrigine (LTG) and trifluoperazine (TFP). In this study, we sought to elucidate the influence of specific N-terminal histidine and proline residues on UGT enzyme substrate selectivity. The conserved N-terminal domain histidine of UGT1A1, UGT1A6, UGT1A9, and UGT2B7 was mutated to proline and leucine 34 of UGT2B10 was substituted with histidine, and the capacity of the wild-type and mutant proteins to glucuronidate 4MU, 1NP, LTG, TFP, and naproxen was characterized. Whereas UGT1A1(H39P), UGT1A6(H38P), and UGT1A9(H37P) lacked the ability to metabolize 4MU, 1NP, and naproxen, all glucuronidated LTG. K(m) values for UGT1A1(H39P) and UGT1A9(H37P) were 774 and 3812 microM, respectively, compared with 1579 microM for UGT1A4. UGT1A1(H39P) also glucuronidated TFP with a V(max)/K(m) value comparable to that of UGT1A4. In contrast to the wild-type enzyme, UGT2B10(L34H) glucuronidated 4MU and 1NP with respective K(m) values of 260 and 118 microM. UGT2B7(H35P) lacked activity toward all substrates. The data confirm a pivotal role for an N-terminal domain proline in the glucuronidation of the tertiary amines LTG and TFP by UGT1A subfamily proteins, whereas glucuronidation reactions involving proton abstraction generally, although not invariably, require a histidine at the equivalent position in both UGT1A and UGT2B enzymes.
Philip Lazarus - One of the best experts on this subject based on the ideXlab platform.
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role of the UGT2B17 deletion in exemestane pharmacogenetics
Pharmacogenomics Journal, 2018Co-Authors: Gang Chen, Shaman Luo, Cristina I Truica, C C Baird, Kim Leitzel, Philip LazarusAbstract:Exemestane (EXE) is an aromatase inhibitor used for the prevention and treatment of breast cancer. The major metabolic pathway for EXE is reduction to form the active 17β-dihydro-EXE (17β-DHE) and subsequent glucuronidation to 17β-hydroxy-EXE-17-O-β-D-glucuronide (17β-DHE-Gluc) by UGT2B17. The aim of the present study was to determine the effects of UGT2B17 copy number variation on the levels of urinary and plasma 17β-DHE-Gluc and 17β-DHE in patients taking EXE. Ninety-six post-menopausal Caucasian breast cancer patients with ER+ breast tumors taking 25 mg EXE daily were recruited into this study. UGT2B17 copy number was determined by a real-time PCR copy number variant assay and the levels of EXE, 17β-DHE and 17β-DHE-Gluc were quantified by UPLC/MS in patients’ urine and plasma. A 39-fold decrease (P<0.0001) in the levels of creatinine-adjusted urinary 17β-DHE-Gluc was observed among UGT2B17 (*2/*2) subjects vs subjects with the UGT2B17 (*1/*1) genotype. The plasma levels of 17β-DHE-Gluc was decreased 29-fold (P<0.0001) in subjects with the UGT2B17 (*2/*2) genotype vs subjects with UGT2B17 (*1/*1) genotype. The levels of plasma EXE-adjusted 17β-DHE was 28% higher (P=0.04) in subjects with the UGT2B17 (*2/*2) genotype vs subjects with the UGT2B17 (*1/*1) genotype. These data indicate that UGT2B17 is the major enzyme responsible for 17β-DHE-Gluc formation in vivo and that the UGT2B17 copy number variant may play a role in inter-individual variability in 17β-DHE levels in vivo.
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glucuronidation of trans 3 hydroxycotinine by UGT2B17 and ugt2b10
Pharmacogenetics and Genomics, 2012Co-Authors: Gang Chen, Nino E. Giambrone, Philip LazarusAbstract:trans-3′-Hydroxycotinine (3HC) and its glucuronide (3HC-Gluc) are major nicotine metabolites excreted in the urine of smokers and other tobacco users. While several members of the UDP-glucuronosyltransferase (UGT) family of enzymes were previously shown to be active in catalyzing the formation of 3HC-Gluc, a comprehensive screening of all known human UGT1A and 2B enzymes for glucuronidation activity against 3HC was not previously performed. In the present study, 8 UGT1A and 6 UGT2B enzymes were screened for activity against 3HC. UGT2B17 exhibited the highest O-glucuronidation activity, exhibiting a 4-fold lower (p<0.005) KM (8.3 mM) than that observed for UGTs 1A9 (35 mM) or 2B7 (31 mM) and a KM smaller than that observed for human liver microsomes (HLM; 26 mM). The KM for 3HC-O-Gluc formation was 3.1-fold lower (p<0.0005) in HLM from male subjects exhibiting the wild-type genotype UGT2B17 (*1/*1) than that in HLM from subjects homozygous for the UGT2B17 deletion genotype [UGT2B17 (*2/*2)]. Both UGTs 2B10 and 1A4 exhibited 3HC-N-Gluc formation activity, with UGT2B10 exhibiting a 4-fold lower (p<0.05) KM (13 mM) than that observed for UGT1A4 (57 mM) and which was similar to the KM observed in HLM (14 mM). There was a 91% (p<0.0001) and 39% (p<0.001) decrease in 3HC-N-Gluc formation activities in HLM from subjects with the UGT2B10 (*2/*2) and UGT2B10 (*1/*2) genotypes, respectively, compared to that of HLM from subjects with the wild-type UGT2B10 (*1/*1) genotype. These results suggest that UGT2B17 and UGT2B10 play key roles in the glucuronidation of 3HC in the human liver and that functional polymorphisms in UGT2B17 and UGT2B10 are associated with significantly reduced glucuronidation activities against 3HC.
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abstract 2757 characterization of the glucuronidation of the major nicotine metabolite 3 hydroxycotinine potential role for knock out polymorphisms in ugts 2b17 and 2b10
Cancer Research, 2011Co-Authors: Gang Chen, Nino E. Giambrone, Joshua E. Muscat, Philip LazarusAbstract:Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL 3’-Hydroxycotinine (3HC) and its glucuronide (3HC-Gluc) conjugate are major nicotine metabolites excreted in the urine of smokers and other tobacco or nicotine users. Previous studies have demonstrated that human liver microsomes (HLM) were active in catalyzing the formation of both 3HC-O-Gluc and 3HC-N-Gluc. While not all human UDP-glucuronosyltransferases (UGTs) were previously screened, UGTs 2B7, 1A9, 2B4 and 2B15 were shown to be active in the formation of 3HC-O-Gluc and UGT1A4 active in 3HC-N-Gluc formation. In the present study, all known human UGTs except for UGT2B28 were screened for glucuronidation activity against 3HC. Homogenates from cell lines over-expressing UGTs 2B17 and 2B10 exhibited the highest O- and N-glucuronidation activities, respectively for the UGTs tested. Kinetic analysis for the formation of 3HC-O-Gluc demonstrated that the KM for UGT2B17 is 3.7- and 4.2-fold lower than that observed for UGTs 1A9 (p<0.005) and 2B7 (p<0.005); the KM of 3HC-N-Gluc formation was 4.4-fold lower (p<0.05) for UGT2B10 than that observed for UGT1A4. Kinetic analysis demonstrated that HLM from subjects exhibiting the homozygous null UGT2B17 deletion [UGT2B17 (\*2/\*2)] exhibited a KM for 3HC-O-Gluc formation that was 3.1-fold higher (p<0.001) than that observed in HLM from subjects exhibiting the wild-type UGT2B17(\*1/\*1) genotype. The KM of 15.6 μM for 3HC-O-Gluc formation in UGT2B17 (\*2/\*2) HLM was close to that observed for UGT2B17 (8.3 μM). 3HC-N-Gluc formation activity was decreased by 91% (p<0.001) in HLM from subjects with the UGT2B10(\*2/\*2) knock-out genotype and 39% (p<0.001) in HLM from subjects with the UGT2B10(\*1/\*2) genotype as compared to that observed for UGT2B10(\*1/\*1) HLMs. The KM of 13 μM for 3HC-N-Gluc formation in UGT2B10 (\*1/\*1) HLM (n=6) was close to that observed for UGT2B10 (13 μM). These results demonstrate that UGTs 2B17 and 2B10 play key roles in the hepatic glucuronidation of 3HC and that functional polymorphisms in UGT2B17 and UGT2B10 are associated with significantly reduced glucuronidation activities against 3HC in HLM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2757. doi:10.1158/1538-7445.AM2011-2757
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Glucuronidation Genotypes and Nicotine Metabolic Phenotypes: Importance of Functional UGT2B10 and UGT2B17 Polymorphisms
Cancer research, 2010Co-Authors: Gang Chen, Nino E. Giambrone, Joshua E. Muscat, Douglas F. Dluzen, Arthur Berg, Carla J. Gallagher, Philip LazarusAbstract:Glucuronidation is an important pathway in the metabolism of nicotine, with previous studies suggesting that ∼22% of urinary nicotine metabolites are in the form of glucuronidated compounds. Recent in vitro studies have suggested that the UDP-glucuronosyltransferases (UGT) 2B10 and 2B17 play major roles in nicotine glucuronidation with polymorphisms in both enzymes shown to significantly alter the levels of nicotine-glucuronide, cotinine-glucuronide, and trans-3′-hydroxycotinine (3HC)–glucuronide in human liver microsomes in vitro. In the present study, the relationship between the levels of urinary nicotine metabolites and functional polymorphisms in UGTs 2B10 and 2B17 was analyzed in urine specimens from 104 Caucasian smokers. Based on their percentage of total urinary nicotine metabolites, the levels of nicotine-glucuronide and cotinine-glucuronide were 42% (P
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abstract 918 glucuronidation genotypes and nicotine metabolic phenotypes importance of ugt2b10 and UGT2B17 knock out polymorphisms
Cancer Research, 2010Co-Authors: Gang Chen, Nino E. Giambrone, Douglas F. Dluzen, Carla J. Gallagher, Andrea S Blevinsprimeau, Diane E Mccloskey, Philip LazarusAbstract:Glucuronidation is an important pathway in the metabolism of nicotine, with ∼28% of urinary nicotine metabolites in the form of glucuronidated compounds. Recent in vitro studies have suggested that the UGTs 2B10 and 2B17 play major roles in nicotine glucuronidation with polymorphisms in both enzymes shown to significantly alter the levels of nicotine-, cotinine-, and trans-3-hydroxy-cotinine (3HC)-glucuronides in human liver microsomes in vitro. In the present study, the relationship between the levels of urinary nicotine metabolites and functional polymorphisms in UGTs 2B10 and 2B17 were analyzed in urine specimens from 104 Caucasian smokers. The levels of nicotine-glucuronide and cotinine-glucuronide were 47 (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 918.
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alteration of human udp glucuronosyltransferase UGT2B17 regio specificity by a single amino acid substitution
Journal of Molecular Biology, 1999Co-Authors: Stephanie Dubois, Martin Beaulieu, Eric Levesque, Dean W Hum, Alain BelangerAbstract:Abstract The glucuronidation of steroid hormones is catalyzed by a family of UDP-glucuronosyltransferase (UGT) enzymes. Previously, two cDNA clones, UGT2B15 and UGT2B17, which encode UGT enzymes capable of glucuronidating C19 steroids, were isolated and characterized. These proteins are 95 % identical in primary structure; however, UGT2B17 is capable of conjugating C19 steroid molecules at both the 3α and 17β-OH positions, whereas UGT2B15 is only active at the 17β-OH position. To identify the amino acid residue(s) which may account for this difference in substrate specificity, a comprehensive study on the role of 15 residues which differ between UGT2B15 and UGT2B17 was performed by site-directed mutagenesis. The stable expression of UGT2B17 mutant proteins into HK293 cells demonstrated that the mutation of isoleucine 125, valine 181 and valine 455 to the residues found in UGT2B15 did not alter enzyme activity nor substrate specificity. Furthermore, mutation of the variant residues in UGT2B15 (serine 124, asparagine 125, phenylalanine 165) to the amino acid residues found in UGT2B17 did not alter enzyme activity nor substrate specificity. However, mutation of the serine residue at position 121 of UGT2B17 to a tyrosine, as found in UGT2B15, abolished the ability of UGT2B17 to conjugate androsterone at the 3α position, but still retained activity for dihydrotestosterone and 5α-androstane-3α,17β-diol, which have an OH-group at the 17β position. Interestingly, mutation of tyrosine 121 in UGT2B15 to a serine abolished activity for C19 steroids. It is suggested that the serine residue at position 121 in UGT2B17 is required for activity towards the 3α and not for the 17β position of C19 steroids, whereas the tyrosine 121 in UGT2B15 is necessary for UGT activity. Despite the high homology between UGT2B15 and UGT2B17, it is apparent that different amino acid residues in the two proteins are required to confer conjugation of C19 steroid molecules.
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effect of interleukins on ugt2b15 and UGT2B17 steroid uridine diphosphate glucuronosyltransferase expression and activity in the lncap cell line
Endocrinology, 1998Co-Authors: Eric Levesque, Martin Beaulieu, Alain BelangerAbstract:Cytokines are known to modulate the level of both phase 1 and phase 2 drug-metabolizing enzymes in hepatocytes. Although the effects of cytokines on cytochrome P450 (CYP450) enzymes are well understood, there is limited knowledge on how cytokines may affect steroid UDP-glucuronosyltransferase (UGT) phase 2 enzyme activity and expression in different cell types, including hepatocytes and steroid target cells. LNCaP cells, which is a human prostate cancer cell line, is a good model to study the effect of cytokines in steroid target cells because it is known to express steroidogenic enzymes, including UGT2B15 and UGT2B17, which are widely expressed steroid UGT enzymes known to conjugate androgens. In this study, we examined the possible interaction among interleukin-1α (IL-1α), IL-4, IL-6, and steroid UGT enzymes (UGT2B15 and UGT2B17). Treatment of LNCaP cells with IL-1α led to a dose-dependent inhibition of dihydrotestosterone (DHT) glucuronidation. IL-1α decreased both UGT activity and LNCaP cell prolifera...
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effect of interleukins on ugt2b15 and UGT2B17 steroid uridine diphosphate glucuronosyltransferase expression and activity in the lncap cell line
Endocrinology, 1998Co-Authors: Eric Levesque, Martin Beaulieu, Chantal Guillemette, Dean W Hum, Alain BelangerAbstract:Cytokines are known to modulate the level of both phase 1 and phase 2 drug-metabolizing enzymes in hepatocytes. Although the effects of cytokines on cytochrome P450 (CYP450) enzymes are well understood, there is limited knowledge on how cytokines may affect steroid UDP-glucuronosyltransferase (UGT) phase 2 enzyme activity and expression in different cell types, including hepatocytes and steroid target cells. LNCaP cells, which is a human prostate cancer cell line, is a good model to study the effect of cytokines in steroid target cells because it is known to express steroidogenic enzymes, including UGT2B15 and UGT2B17, which are widely expressed steroid UGT enzymes known to conjugate androgens. In this study, we examined the possible interaction among interleukin-1alpha (IL-1alpha), IL-4, IL-6, and steroid UGT enzymes (UGT2B15 and UGT2B17). Treatment of LNCaP cells with IL-1alpha led to a dose-dependent inhibition of dihydrotestosterone (DHT) glucuronidation. IL-1alpha decreased both UGT activity and LNCaP cell proliferation in the absence and presence of DHT (0.5 nM); a maximal inhibition of 70% was observed. IL-6 inhibited LNCaP cell proliferation as well as the DHT-induced proliferation of these cells. However, neither IL-4 nor IL-6 significantly affected the formation of DHT glucuronide. Ribonuclease protection and Western blot analyses demonstrated a specific reduction of UGT2B17 transcript and protein levels in IL-1alpha-treated LNCaP cells. The level of UGT2B15 was not affected by cytokine treatments, indicating a differential regulation between these two UGT enzymes. Transfection experiments performed with the UGT2B17 gene promoter region indicates that the regulation occurs at the transcription level via putative cis-acting elements. This study indicates that cell proliferation and UGT expression in steroid-responsive cancer cells are differentially regulated depending on the cytokines present in the cell microenvironment.
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chromosomal localization structure and regulation of the UGT2B17 gene encoding a c19 steroid metabolizing enzyme
DNA and Cell Biology, 1997Co-Authors: Martin Beaulieu, Alain Belanger, Eric Levesque, Barbara G Beatty, Andre Tchernof, Dean W HumAbstract:UGT2B17 is a UDP-glucuronosyltransferase enzyme expressed in several extrahepatic steroid target tissues, including the human prostate, where it glucuronidates C19 steroids such as dihydrotestosterone (DHT), androsterone (ADT), and androstane-3alpha, 17beta-diol (3alpha-diol). To determine if UGT2B17 is regulated by physiological effectors of the human prostate, DHT and epidermal growth factor (EGF) were demonstrated to specifically down-regulate the steady-state levels of UGT2B17 transcript and protein in LNCaP cells (Guillemette et al., 1997). These results implicate regulation of UGT2B17 at the level of gene transcription, therefore, a P-1-derived artificial chromosome (PAC) clone of 120 kb containing the entire UGT2B17 gene was isolated. The gene is comprised of six exons spanning approximately 30 kb, and fluorescence in situ hybridization of the UGT2B17 PAC clone to normal human lymphocyte chromosomes, mapped the gene to chromosome 4q13. To determine if the 5'-flanking DNA of the UGT2B17 gene is sufficient to confer gene expression, a 2,942-bp fragment was subcloned into a luciferase reporter plasmid and yielded an activity of 25-fold over background when transfected in LNCaP cells. However, transfection of the construct into HK-293, MCF-7, JEG-3, and HepG2 cells yielded only a moderate activity of two- to five-fold over background. Treatment of transfected LNCaP cells with 10 nM R1881, a nonmetabolizable analog of DHT, and 10 ng/ml EGF decreased the luciferase activity by 60%. This suggests that at least part, if not all, of the inhibitory effect of EGF and DHT on UGT2B17 is at the level of transcription. Progressive 5' deletions of the UGT2B17 5'-flanking region in the luciferase constructs alleviated the inhibition by R1881 and EGF, and revealed several potential responsive elements that may confer the observed regulation of the UGT2B17 gene. This study demonstrates regulation of the UGT2B17 gene by physiological effectors of the human prostate and supports the hypothesis that UGT enzymes are involved in steroid metabolism in extrahepatic tissues.
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differential regulation of two uridine diphospho glucuronosyltransferases ugt2b15 and UGT2B17 in human prostate lncap cells
Endocrinology, 1997Co-Authors: Chantal Guillemette, Martin Beaulieu, Eric Levesque, David Turgeon, Dean W Hum, Alain BelangerAbstract:Although androgens are important regulators in the prostate, other effectors such as growth factors may also act to maintain normal function of the gland. Human prostate and human prostate cancer LNCaP cells express steroid conjugating uridine diphospho-glucuronosyltransferase (UGT) enzymes, and it was shown that the level of UGT activities and transcripts is down-regulated by androgens, especially dihydrotestosterone (DHT). In the present study, we examined the interaction between androgen, epidermal growth factor (EGF), and steroid UGT enzymes. The formation of DHT glucuronide (DHT-G) was inhibited by 47% when LNCaP cells were treated for 6 days with 10 ng/ml of EGF. Northern blot analysis also demonstrated a decrease in the steady-state level of UGT2B transcripts. Treatment with both DHT (0.5 nm) and EGF (10 ng/ml) caused a greater decrease of DHT glucuronidation and UGT2B messenger RNA levels than when the cells were treated with either compound alone. RNase protection assays showed that treatment wit...
