Characterization of Raloxifene Glucuronidation: Potential Role of UGT1A8 Genotype on Raloxifene Metabolism <i>In Vivo</i>

Sun, Dongxiao; Jones, Nathan R.; Manni, Andrea; Lazarus, Philip

doi:10.1158/1940-6207.capr-12-0448

Cited by 34 publications

(39 citation statements)

References 42 publications

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“…The glucuronidation assays using homogenates from HuH-7 and Hep3B cells were performed as described previously (Sun et al, 2007(Sun et al, , 2013. HuH-7 and Hep3B cell homogenate (50-300 mg of protein) was incubated at 37°C with 500 mM epirubicin hydrochloride for 60 minutes for UGT2B7, 1 mM codeine for 90 minutes for UGT2B4, and 500 mM nicotine for 14 hours for UGT2B10.…”

Section: Methodsmentioning

confidence: 99%

Regulation of UGT2B Expression and Activity by miR-216b-5p in Liver Cancer Cell Lines

Dluzen

Sutliff

Chen

et al. 2016

Journal of Pharmacology and Experimental Therapeutics

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The UDP-glucuronosyltransferase (UGT) 2B enzymes are important in the detoxification of a variety of endogenous and exogenous compounds, including many hormones, drugs, and carcinogens. Identifying novel mechanisms governing their expression is important in understanding patient-specific response to drugs and cancer risk factors. In silico prediction algorithm programs were used to screen for microRNAs (miRNAs) as potential regulators of UGT2B enzymes, with miR-216b-5p identified as a potential candidate. Luciferase data suggested the presence of a functional miR-216b-5p binding motif within the 39 untranslated regions of UGTs 2B7, 2B4, and 2B10. Overexpression of miR-216b-5p mimics significantly repressed UGT2B7 (P , 0.001) and UGT2B10 (P 5 0.0018) mRNA levels in HuH-7 cells and UGT2B4 (P , 0.001) and UGT2B10 (P 5 0.018) mRNA in Hep3B cells. UGT2B7 protein levels were repressed in both HuH-7 and Hep3B cells in the presence of increasing miR-216b-5p concentrations, corresponding with significant (P , 0.001 and P 5 0.011, respectively) decreases in glucuronidation activity against the UGT2B7-specific substrate epirubicin. Inhibition of endogenous miR-216b-5p levels significantly increased UGT2B7 mRNA levels in HuH-7 (P 5 0.021) and Hep3B (P 5 0.0068) cells, and increased epirubicin glucuronidation by 85% (P 5 0.057) and 50% (P 5 0.012) for HuH-7 and Hep3B cells, respectively. UGT2B4 activity against codeine and UGT2B10 activity against nicotine were significantly decreased in both HuH-7 and Hep3B cells (P , 0.001 and P 5 0.0048, and P 5 0.017 and P 5 0.043, respectively) after overexpression of miR-216b-5p mimic. This is the first evidence that miRNAs regulate UGT 2B7, 2B4, and 2B10 expression, and that miR-216b-5p regulation of UGT2B proteins may be important in regulating the metabolism of UGT2B substrates.

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Section: Methodsmentioning

confidence: 99%

Regulation of UGT2B Expression and Activity by miR-216b-5p in Liver Cancer Cell Lines

Dluzen

Sutliff

Chen

et al. 2016

Journal of Pharmacology and Experimental Therapeutics

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“…The glucuronidation assays using homogenates from HuH-7 and HepG2 cells were performed essentially as described elsewhere (Sun et al, 2007(Sun et al, , 2013. HuH-7 and HepG2 cell homogenate (100-300 mg) was incubated with 50 mM raloxifene for 90 minutes, or 500 mM epirubicin hydrochloride for 60 minutes.…”

Section: Methodsmentioning

confidence: 99%

“…Raloxifene gradient elution conditions were performed as previously described elsewhere (Sun et al, 2013). Epirubicin gradient elution conditions were performed using a flow rate of 0.5 ml/min, starting with 95% buffer A (0.1% formic acid in water) and 5% buffer B (0.1% formic acid in acetonitrile) for 30 seconds, a subsequent linear gradient to 50% buffer B over 2.5 minutes, and then 100% buffer B maintained over the next 2 minutes.…”

Section: Methodsmentioning

confidence: 99%

“…UGTs 1A1, 1A3, 1A4, 1A5, 1A6, and 1A9 are the primary hepatic isoforms; UGTs 1A7, 1A8, and 1A10 are exclusively expressed extrahepatically (Nagar and Remmel, 2006;Nakamura et al, 2008;Izukawa et al, 2009). The UGTs' prime numerous endogenous compounds include bilirubin (Bosma et al, 1994) and steroid hormones (Belanger et al, 2003), as well as exogenous compounds, including drugs, chemotherapeutic agents, and carcinogens (Court et al, 2001;Kemp et al, 2002;Nagar and Blanchard, 2006;Nagar and Remmel, 2006;Chen et al, 2007Chen et al, , 2008Chen et al, , 2010Chen et al, , 2012Balliet et al, 2009;Lazarus et al, 2009;Mizuma, 2009;Rowland et al, 2013;Sun et al, 2013) for excretion from the body by Fig. 1.…”

Section: Introductionmentioning

confidence: 99%

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Regulation of UDP-Glucuronosyltransferase 1A1 Expression and Activity by MicroRNA 491-3p

Dluzen

Sun

Salzberg

et al. 2014

J Pharmacol Exp Ther

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The UDP-glucuronosyltransferase (UGT) 1A enzymes are involved in the phase II metabolism of many important endogenous and exogenous compounds. The nine UGT1A isoforms exhibit high interindividual differences in expression, but their epigenetic regulation is not well understood. The purpose of the present study was to examine microRNA (miRNA) regulation of hepatic UGT1A enzymes and determine whether or not that regulation impacts enzymatic activity. In silico analysis identified miRNA 491-3p (miR-491-3p) as a potential regulator of the UGT1A gene family via binding to the shared UGT1A 39-untranslated region common to all UGT1A enzymes. Transfection of miR-491-3p mimic into HuH-7 cells significantly repressed UGT1A1 (P , 0.001), UGT1A3 (P , 0.05), and UGT1A6 (P , 0.05) mRNA levels. For UGT1A1, this repression correlated with significantly reduced metabolism of raloxifene into raloxifene-6-glucuronide (ral-6-gluc; P , 0.01) and raloxifene-49-glucuronide (ral-49-gluc; P , 0.01). In HuH-7 cells with repressed miR-491-3p expression, there was a significant increase (∼80%; P , 0.01) in UGT1A1 mRNA and a corresponding increase in glucuronidation of raloxifene into ral-6-gluc (50%; P , 0.05) and ral-49-gluc (22%; P , 0.01). Knockdown of endogenous miR-491-3p in HepG2 cells did not significantly alter UGT1A1 mRNA levels but did increase the formation of ral-6-gluc (50%; P , 0.05) and ral-49-gluc (34%; P , 0.001). A significant inverse correlation between miR-491-3p expression and both UGT1A3 (P , 0.05) and UGT1A6 (P , 0.01) mRNA levels was observed in a panel of normal human liver specimens, with a significant (P , 0.05) increase in UGT1A3 and UGT1A6 mRNA levels observed in miR-491-3p nonexpressing versus expressing liver specimens. These results suggest that miR-491-3p is an important factor in regulating the expression of UGT1A enzymes in vivo.

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“…Clinically relevant intestinal UGT substrates include the anticancer agent raloxifene and the cholesterol-lowering agent ezetimibe (Kemp et al, 2002;Ghosal et al, 2004;Sun et al, 2013). Raloxifene intestinal glucuronidation is catalyzed predominately by both UGT1A8 and UGT1A10 .…”

Section: Diet-derived Inhibitors Of Intestinal Glucuronidation 1679mentioning

confidence: 99%

Identification of Diet-Derived Constituents as Potent Inhibitors of Intestinal Glucuronidation

et al. 2014

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Drug-metabolizing enzymes within enterocytes constitute a key barrier to xenobiotic entry into the systemic circulation. Furanocoumarins in grapefruit juice are cornerstone examples of diet-derived xenobiotics that perpetrate interactions with drugs via mechanism-based inhibition of intestinal CYP3A4. Relative to intestinal CYP3A4-mediated inhibition, alternate mechanisms underlying dietary substance-drug interactions remain understudied. A working systematic framework was applied to a panel of structurally diverse diet-derived constituents/extracts (n = 15) as inhibitors of intestinal UDPglucuronosyl transferases (UGTs) to identify and characterize additional perpetrators of dietary substance-drug interactions. Using a screening assay involving the nonspecific UGT probe substrate 4-methylumbelliferone, human intestinal microsomes, and human embryonic kidney cell lysates overexpressing gut-relevant UGT1A isoforms, 14 diet-derived constituents/extracts inhibited UGT activity by >50% in at least one enzyme source, prompting IC 50 determination. The IC 50 values of 13 constituents/extracts (£10 mM with at least one enzyme source) were well below intestinal tissue concentrations or concentrations in relevant juices, suggesting that these dietderived substances can inhibit intestinal UGTs at clinically achievable concentrations. Evaluation of the effect of inhibitor depletion on IC 50 determination demonstrated substantial impact (up to 2.8-fold shift) using silybin A and silybin B, two key flavonolignans from milk thistle (Silybum marianum) as exemplar inhibitors, highlighting an important consideration for interpretation of UGT inhibition in vitro. Results from this work will help refine a working systematic framework to identify dietary substance-drug interactions that warrant advanced modeling and simulation to inform clinical assessment.

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Characterization of Raloxifene Glucuronidation: Potential Role of UGT1A8 Genotype on Raloxifene Metabolism In Vivo

Cited by 34 publications

References 42 publications

Regulation of UGT2B Expression and Activity by miR-216b-5p in Liver Cancer Cell Lines

Regulation of UGT2B Expression and Activity by miR-216b-5p in Liver Cancer Cell Lines

Regulation of UDP-Glucuronosyltransferase 1A1 Expression and Activity by MicroRNA 491-3p

Identification of Diet-Derived Constituents as Potent Inhibitors of Intestinal Glucuronidation

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