Publications by authors named "Bingfang Yan"

77 Publications

The COVID-19 Medicine Remdesivir Is Therapeutically Activated by Carboxylesterase-1, and Excessive Hydrolysis Increases Cytotoxicity.

Hepatol Commun 2021 Sep 16;5(9):1622-1623. Epub 2021 Jul 16.

Division of Pharmaceutical Sciences, James L. Winkle College of Pharmacy, University of Cincinnati, Cincinnati, OH, USA.

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http://dx.doi.org/10.1002/hep4.1736DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8250894PMC
September 2021

The COVID-19 medicine remdesivir is therapeutically activated by carboxylesterase-1 AND excessive hydrolysis increases cytotoxicity.

Hepatol Commun 2021 Apr 21. Epub 2021 Apr 21.

Division of Pharmaceutical Sciences James L. Winkle College of Pharmacy University of Cincinnati Cincinnati OH 45229 USA.

The pandemic of coronavirus disease 2019 (COVID-19) has become a global health crisis with the death toll of over 14 million people. So far, there are limited options to treat COVID-19. Remdesivir was granted emergency use authorization earlier, and full use recently. Remdesivir was originally developed against Ebola viral infection and has since been shown to exert a broad antiviral activity against as many as seven viral families [1]. On the other hand, serious adverse events and mortality remained high even with remdesivir among COVID-19 patients [2-4].
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http://dx.doi.org/10.1002/hep4.1736DOI Listing
April 2021

Pig Liver Esterases Hydrolyze Endocannabinoids and Promote Inflammatory Response.

Front Immunol 2021 17;12:670427. Epub 2021 May 17.

State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.

Endocannabinoids are endogenous ligands of cannabinoid receptors and activation of these receptors has strong physiological and pathological significance. Structurally, endocannabinoids are esters (e.g., 2-arachidonoylglycerol, 2-AG) or amides (e.g., N-arachidonoylethanolamine, AEA). Hydrolysis of these compounds yields arachidonic acid (AA), a major precursor of proinflammatory mediators such as prostaglandin E. Carboxylesterases are known to hydrolyze esters and amides with high efficiency. CES1, a human arboxylterase, has been shown to hydrolyze 2-AG, and shares a high sequence identity with pig carboxylesterases: PLE1 and PLE6 (ig iver sterase). The present study was designed to test the hypothesis that PLE1 and PLE6 hydrolyze endocannabinoids and promote inflammatory response. Consistent with the hypothesis, purified PLE1 and PLE6 efficaciously hydrolyzed 2-AG and AEA. PLE6 was 40-fold and 3-fold as active as PLE1 towards 2-AG and AEA, respectively. In addition, both PLE1 and PLE6 were highly sensitive to bis(4-nitrophenyl) phosphate (BNPP), an aryl phosphodiester known to predominately inhibit carboxylesterases. Based on the study with BNPP, PLEs contributed to the hydrolysis of 2-AG by 53.4 to 88.4% among various organs and cells. Critically, exogenous addition or transfection of PLE6 increased the expression and secretion of proinflammatory cytokines in response to the immunostimulant lipopolysaccharide (LPS). This increase was recapitulated in cocultured alveolar macrophages and PLE6 transfected cells in transwells. Finally, BNPP reduced inflammation trigged by LPS accompanied by reduced formation of AA and proinflammatory mediators. These findings define an innovative connection: PLE-endocannabinoid-inflammation. This mechanistic connection signifies critical roles of carboxylesterases in pathophysiological processes related to the metabolism of endocannabinoids.
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http://dx.doi.org/10.3389/fimmu.2021.670427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8165269PMC
May 2021

DEC1 deficiency results in accelerated osteopenia through enhanced DKK1 activity and attenuated PI3KCA/Akt/GSK3β signaling.

Metabolism 2021 05 17;118:154730. Epub 2021 Feb 17.

Department of Pharmacology, Nanjing Medical University, China.

Background: Human differentiated embryonic chondrocyte expressed gene 1 (DEC1) has been implicated in enhancing osteogenesis, a desirable outcome to counteract against deregulated bone formation such as retarded bone development, osteopenia and osteoporosis.

Methods And Results: DEC1 knockout (KO) and the age-matched wild-type (WT) mice were tested for the impact of DEC1 deficiency on bone development and osteopenia as a function of age. DEC1 deficiency exhibited retarded bone development at the age of 4 weeks and osteopenic phenotype in both 4- and 24-week old mice. However, the osteopenia was more severe in the 24-week age groups. Mechanistically, DEC1 deficiency downregulated the expression of bone-enhancing genes such as Runx2 and β-catenin accompanied by upregulating DKK1, an inhibitor of the Wnt/β-catenin signaling pathway. Consistently, DEC1 deficiency favored the attenuation of the integrated PI3KCA/Akt/GSK3β signaling, a pathway targeting β-catenin for degradation. Likewise, the attenuation was greater in the 24-week age group. These changes, however, were reversed by in vivo treatment with lithium chloride, a stabilizer of β-catenin, and confirmed by gain-of-function study with DEC1 transfection into DEC1 KO bone marrow mesenchymal stem cells and loss-of-function study with siDEC1 lentiviral infection into the corresponding WT cells.

Conclusion: DEC1 is a positive regulator with a broad activity spectrum in both bone development and maintenance, and the osteopenic phenotype accelerated by DEC1 deficiency is achieved by enhanced DKK1 activity and attenuated PI3KCA/Akt/GSK3β signaling.
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http://dx.doi.org/10.1016/j.metabol.2021.154730DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8311383PMC
May 2021

Remdesivir potently inhibits carboxylesterase-2 through covalent modifications: signifying strong drug-drug interactions.

Fundam Clin Pharmacol 2021 Apr 6;35(2):432-434. Epub 2021 Jan 6.

Division of Pharmaceutical Sciences, James L. Winkle College of Pharmacy, University of Cincinnati, Cincinnati, OH, USA.

Remdesivir was recently approved to treat COVID-19. While this antiviral agent delivers clinical benefits, several safety concerns in many cases have been raised. This study reports that remdesivir at nanomolar concentrations inhibits carboxylesterase-2 (CES2) through covalent modifications. CES2 is a major drug-metabolizing enzyme. The combination of high potency with irreversible inhibition concludes that cautions must be exercised when remdesivir is used along with drugs hydrolyzed by CES2.
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http://dx.doi.org/10.1111/fcp.12643DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8284663PMC
April 2021

Dechlorination and demethylation of ochratoxin A enhance blocking activity of PXR activation, suppress PXR expression and reduce cytotoxicity.

Toxicol Lett 2020 Oct 10;332:171-180. Epub 2020 Jul 10.

Division of Pharmaceutical Sciences, James L. Winkle College of Pharmacy, University of Cincinnati, Cincinnati, OH, 45229, USA. Electronic address:

The pregnane X receptor (PXR) has been established to induce chemoresistance and metabolic diseases. Ochratoxin A (OTA), a mycotoxin, decreases the expression of PXR protein in human primary hepatocytes. OTA is chlorinated and has a methylated lactone ring. Both structures are associated with OTA toxicity. The study was to test the hypothesis that structural modifications differentially impact PXR blocking activity over cytotoxicity. To test this hypothesis, OTA-M and OTA-Cl/M were synthesized. OTA-M lacked the methyl group of the lactone-ring, whereas OTA-Cl/M had neither the methyl group nor the chlorine atom. The blocking activity of PXR activation was determined in a stable cell line, harboring both PXR (coding sequence) and its luciferase element reporter. OTA-Cl/M showed the highest blocking activity, followed by OTA-M and OTA. OTA-Cl/M was 60 times as potent as the common PXR blocker ketoconazole based on calculated IC values. OTA-Cl/M decreased by 90 % the expression of PXR protein and was the least cytotoxic among the tested compounds. Molecular docking identified that OTA and its derivatives interacted with different sets of residues in PXR, providing a molecular basis for selectivity. Excessive activation of PXR has been implicated in chemoresistance and metabolic diseases. Downregulation of PXR protein expression likely delivers an effective mechanism against structurally diverse PXR agonists.
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http://dx.doi.org/10.1016/j.toxlet.2020.07.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8279013PMC
October 2020

Copper Sulfide Facilitates Hepatobiliary Clearance of Gold Nanoparticles through the Copper-Transporting ATPase ATP7B.

ACS Nano 2019 05 18;13(5):5720-5730. Epub 2019 Apr 18.

Key Laboratory of Smart Drug Delivery, Ministry of Education, & State Key Laboratory of Molecular Engineering of Polymers, School of Pharmacy & Minhang Hospital , Fudan University , Shanghai 201203 , China.

Metallic gold (Au) nanoparticles have great potential for a wide variety of biomedical applications. Yet, slow clearance of Au nanoparticles significantly hinders their clinical translation. Herein, we describe a strategy of utilizing the endogenous copper (Cu) clearance to improve the elimination of Au nanoparticles. Our mechanistic study reveals that a Cu-transporting P-type ATPase, ATP7B, mediates the exocytosis of CuS nanoparticles into bile canaliculi for their rapid hepatobiliary excretion. The efflux of CuS nanoparticles is adopted to facilitate the hepatobiliary clearance of Au nanoparticles through CuS-Au conjugation. Using two different CuS-Au nanoconjugates, we demonstrate that CuS increases the biliary Au excretion of CuS-Au nanospheres or CuS-Au nanorods in mice or rats in comparison to that of their respective unconjugated Au nanoparticles postintravenous injection. The current CuS-Au conjugation approach provides a feasible strategy to enhance the hepatobiliary clearance of Au nanoparticles that may be applicable to various structures.
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http://dx.doi.org/10.1021/acsnano.9b01154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8325778PMC
May 2019

Gemcitabine nanoparticles promote antitumor immunity against melanoma.

Biomaterials 2019 01 20;189:48-59. Epub 2018 Oct 20.

Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Kingston, RI 02881, USA.

Myeloid-derived suppressor cells (MDSCs) promote tumor-mediated immunosuppression and cancer progression. Gemcitabine (Gem) is a MDSC-depleting chemotherapeutic agent; however, its clinical use is hampered by its drug resistance and inefficient in vivo delivery. Here we describe a strategy to formulate a Gem analogue gemcitabine monophosphate (GMP) into a lipid-coated calcium phosphate (LCP) nanoparticle, and investigate its antitumor immunity and therapeutic effects after systemic administrations. In the syngeneic mouse model of B16F10 melanoma, compared with free Gem, the LCP-formulated GMP (LCP-GMP) significantly induced apoptosis and reduced immunosuppression in the tumor microenvironment (TME). LCP-GMP effectively depleted MDSCs and regulatory T cells, and skewed macrophage polarization towards the antitumor M1 phenotype in the TME, leading to enhanced CD8 T-cell immune response and profound tumor growth inhibition. Thus, engineering the in vivo delivery of MDSC-depleting agents using nanotechnology could substantially modulate immunosuppressive TME and boost T-cell immune response for enhanced antitumor efficacy.
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http://dx.doi.org/10.1016/j.biomaterials.2018.10.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6281175PMC
January 2019

Interleukin-6 Induces DEC1, Promotes DEC1 Interaction with RXRα and Suppresses the Expression of PXR, CAR and Their Target Genes.

Front Pharmacol 2017 28;8:866. Epub 2017 Nov 28.

Department of Pharmacology, Nanjing Medical University, Nanjing, China.

Inflammatory burden is a primary cellular event in many liver diseases, and the overall capacity of drug elimination is decreased. PXR (pregnane X receptor) and CAR (constitutive androstane receptor) are two master regulators of genes encoding drug-metabolizing enzymes and transporters. DEC1 (differentiated embryonic chondrocyte-expressed gene 1) is a ligand-independent transcription factor and reportedly is induced by many inflammatory cytokines including IL-6. In this study, we used primary hepatocytes (human and mouse) as well as HepG2 cell line and demonstrated that IL-6 increased DEC1 expression and decreased the expressions of PXR, CAR, and their target genes. Overexpression of DEC1 had similar effect as IL-6 on the expression of these genes, and knockdown of DEC1 reversed their downregulation by IL-6. Interestingly, neither IL-6 nor DEC1 altered the expression of RXRα, a common dimerization partner for many nuclear receptors including PXR and CAR. Instead, DEC1 was found to interact with RXRα and IL-6 enhanced the interaction. These results conclude that DEC1 uses diverse mechanisms of action and supports IL-6 downregulation of drug-elimination genes and their regulators.
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http://dx.doi.org/10.3389/fphar.2017.00866DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5712319PMC
November 2017

17β-estradiol suppresses carboxylesterases by activating c-Jun/AP-1 pathway in primary human and mouse hepatocytes.

Eur J Pharmacol 2018 Jan 23;819:98-107. Epub 2017 Nov 23.

Department of Pharmacology, Nanjing Medical University, Nanjing 211166, China. Electronic address:

In order to study the influence of estrogen on carboxylesterases, we investigated the effects of 17β-estradiol on CES1 (Ces1d) and CES2 (Ces1e) in human and mouse hepatocytes. After being treated with 17β-estradiol, the mRNA levels of CES1 and CES2 decreased by 29-39% and 28-55%, respectively, in the human hepatocytes from four donors. Consistently, the hydrolysis of para-nitrophenylacetate decreased markedly by 32% induced by 17β-estradiol. Moreover, 17β-estradiol decreased CES1 and CES2 by 45% and 47% respectively at protein levels. The response of altered expression of Ces1d (CES1) and Ces1e (CES2) to 17β-estradiolin in mouse hepatocytes was very similar to that in the human hepatocytes. Further, the decreased Ces1d and Ces1e expression induced by 17β-estradiol could be abolished by SP600125, an inhibitor of AP-1, both at mRNA and protein levels. Likewise, the increased c-Jun expression induced by 17β-estradiol could almost be abolished by SP600125. In vivo, the expression of Ces1d, Ces1e and the hydrolytic activity of liver were higher in the ovariectomized female mice(OVX) than those in control mice(SHAM). However, when 17β-estradiol was administrated, the expression of Ces1d, Ces1e and the hydrolytic activity of liver in the ovariectomized female mice (OVX+E2) became restored to their normal levels. Taken together, 17β-estradiol suppresses carboxylesterases by activating c-Jun/AP-1 pathway in primary human and mouse hepatocytes. The findings can offer the potential gains in the safety and efficacy of pharmacotherapy for women, especially for pregnant and menopausal women.
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http://dx.doi.org/10.1016/j.ejphar.2017.11.036DOI Listing
January 2018

Enhanced reactive oxygen species through direct copper sulfide nanoparticle-doxorubicin complexation.

Nanotechnology 2017 Dec;28(50):505101

Department of Pharmaceutics, School of Pharmacy, Key Laboratory of Smart Drug Delivery, Ministry of Education, & State Key Laboratory of Molecular Engineering of Polymers, Fudan University, Shanghai 201203, People's Republic of China. Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, The University of Rhode Island, Kingston, Rhode Island 02881, United States of America.

CuS-based nanostructures loading the chemotherapeutic agent doxorubicin (DOX) exerted excellent cancer photothermal chemotherapy under multi-external stimuli. The DOX loading was generally designed through electrostatic interaction or chemical linkers. However, the interaction between DOX molecules and CuS nanoparticles has not been investigated. In this work, we use PEGylated hollow copper sulfide nanoparticles (HCuSNPs) to directly load DOX through the DOX/Cu chelation process. Distinctively, the synthesized PEG-HCuSNPs-DOX release the DOX/Cu complexes into surrounding environment, which generate significant reactive oxygen species (ROS) in a controlled manner by near-infrared laser. The CuS nanoparticle-mediated photothermal ablation facilitates the ROS-induced cancer cell killing effect. Our current work reveals a DOX/Cu-mediated ROS-enhanced cell-killing effect in addition to conventional photothermal chemotherapy through the direct CuS nanoparticle-DOX complexation.
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http://dx.doi.org/10.1088/1361-6528/aa967bDOI Listing
December 2017

Circadian rhythmicity: A functional connection between differentiated embryonic chondrocyte-1 (DEC1) and small heterodimer partner (SHP).

Arch Biochem Biophys 2017 10 8;631:11-18. Epub 2017 Aug 8.

Department of Biomedical and Pharmaceutical Sciences, Center for Integrated Drug Development, University of Rhode Island, Kingston, RI 02881, United States. Electronic address:

Circadian rhythm misalignment has been increasingly recognized to pose health risk for a wide range of diseases, particularly metabolic disorders. The liver maintains metabolic homeostasis and expresses many circadian genes, such as differentiated embryo chondrocyte-1 (DEC1) and small heterodimer partner (SHP). DEC1 is established to repress transcription through E-box elements, and SHP belongs to the superfamily of nuclear receptors and has multiple E-box elements in its promoter. Importantly, DEC1 and SHP are inversely oscillated. This study was performed to test the hypothesis that the SHP gene is a target gene of DEC1. Cotransfection demonstrated that DEC1 repressed the SHP promoter and attenuated the transactivation of the classic circadian activator complex of Clock/Bmal1. Site-directed mutagenesis, electrophoretic mobility shift assay and chromatin immunoprecipitation established that the repression was achieved through the E-box in the proximal promoter. Transfection of DEC1 suppressed the expression of SHP. In circadian-inducing cells, the epileptic agent valproate inversely altered the expression of DEC1 and SHP. Both DEC1 and SHP are involved in energy balance and valproate is known to induce hepatic steatosis. Our findings collectively establish that DEC1 participates in the negative loop of SHP oscillating expression with potential implications in metabolic homeostasis.
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http://dx.doi.org/10.1016/j.abb.2017.08.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5599142PMC
October 2017

Icariin protects against glucocorticoid induced osteoporosis, increases the expression of the bone enhancer DEC1 and modulates the PI3K/Akt/GSK3β/β-catenin integrated signaling pathway.

Biochem Pharmacol 2017 07 11;136:109-121. Epub 2017 Apr 11.

Department of Pharmacology, Nanjing Medical University, Nanjing 210029, China. Electronic address:

Osteoporosis is a serious public health concern worldwide. Herba epimedii has been used for centuries and even thousands of years to treat osteoporotic conditions. Icariin, a flavonol glycoside, is one of the major active ingredients. In this study, we have shown that icariin protected against glucocorticoid-induced osteoporotic changes in SaoS-2 cells and mice. We have also shown that dexamethasone (a glucocorticoid) suppressed and icariin induced DEC1, a structurally distinct helix-loop-helix protein. DEC1 overexpression promoted whereas DEC1 knockdown decreased osteogenic activity. Likewise, DEC1 overexpression and knockdown inversely regulated the expression of β-catenin and PIK3CA, an essential player in the Wnt/β-catenin and PI3K/Akt signaling pathways, respectively. Interestingly, DKK1, an inhibitor of Wnt/β-catenin signaling inhibitor, and LY294002, an inhibitor of PI3K/Akt signaling, abolished the induction of DEC1 by icariin. It is established that these two pathways are interconnected by the phosphorylation status of GSK3β. Dexamethasone decreased but icariin increased GSK3β phosphorylation. Finally, DEC1 deficient mice developed osteoporotic phenotypes. Taken together, it is concluded that DEC1 likely supports the action of icariin against glucocorticoid induced osteoporosis with an involvement of the PI3K/Akt/GSK3β/β-catenin integrated signaling pathway.
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http://dx.doi.org/10.1016/j.bcp.2017.04.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8316728PMC
July 2017

Covalent inhibition of carboxylesterase-2 by sofosbuvir and its effect on the hydrolytic activation of tenofovir disoproxil.

J Hepatol 2017 03 10;66(3):660-661. Epub 2016 Dec 10.

Department of Biomedical Sciences, Center for Integrated Drug Development, University of Rhode Island, Kingston, RI 02881, USA. Electronic address:

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http://dx.doi.org/10.1016/j.jhep.2016.11.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8297416PMC
March 2017

MicroRNA-30c-1-3p is a silencer of the pregnane X receptor by targeting the 3'-untranslated region and alters the expression of its target gene cytochrome P450 3A4.

Biochim Biophys Acta 2016 09 13;1859(9):1238-1244. Epub 2016 Apr 13.

Department of Biomedical and Pharmaceutical Sciences, Center for Integrated Drug Development, University of Rhode Island, Kingston, RI 02881, United States. Electronic address:

The pregnane X receptor (PXR) is a master regulator of genes involved in drug elimination. Recently, activation of PXR has also been linked to the development of many disease conditions such as metabolic disorders and malignancies. MicroRNAs (miRs) emerge as important molecular species involved in these conditions. This study was undertaken to test a large number of miRs for their ability to regulate PXR expression. As many as 58 miRs were tested and miR-30c-1-3p was identified to suppress PXR expression. The suppression was achieved by targeting the 3'-untranslated region, 438 nucleotides from the stop codon. The suppression was detected in multiple cell lines from different organ origins. In addition, miR-30c-1-3p altered basal and induced expression of cytochrome P450 3A4 (CYP3A4), a prototypical target gene of PXR. The alteration varied depending on the time and amounts of miR-30c-1-3p. CYP3A4 is responsible for the metabolism of more than 50% medicines. The interconnection between miR-30c-1-3p and PXR signifies a role of miRs in drug-drug interactions and chemosensitivity. This article is part of a Special Issue entitled: Xenobiotic nuclear receptors: New Tricks for An Old Dog, edited by Dr. Wen Xie.
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http://dx.doi.org/10.1016/j.bbagrm.2016.03.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4975670PMC
September 2016

Ontogenic expression of human carboxylesterase-2 and cytochrome P450 3A4 in liver and duodenum: postnatal surge and organ-dependent regulation.

Toxicology 2015 Apr 24;330:55-61. Epub 2015 Feb 24.

Department of Biomedical and Pharmaceutical Sciences, Center for Pharmacogenomics and Molecular Therapy, University of Rhode Island, Kingston, RI 2881, United States. Electronic address:

Human carboxylesterase-2 (CES2) and cytochrome P450 3A4 (CYP3A4) are two major drug metabolizing enzymes that play critical roles in hydrolytic and oxidative biotransformation, respectively. They share substrates but may have opposite effect on therapeutic potential such as the metabolism of the anticancer prodrug irinotecan. Both CES2 and CYP3A4 are expressed in the liver and the gastrointestinal tract. This study was conducted to determine whether CES2 and CYP3A4 are expressed under developmental regulation and whether the regulation occurs differentially between the liver and duodenum. A large number of tissues (112) were collected with majority of them from donors at 1-198 days of age. In addition, multi-sampling (liver, duodenum and jejunum) was performed in some donors. The expression was determined at mRNA and protein levels. In the liver, CES2 and CYP3A4 mRNA exhibited a postnatal surge (1 versus 2 months of age) by 2.7 and 29 fold, respectively. CYP3A4 but not CES2 mRNA in certain pediatric groups reached or even exceeded the adult level. The duodenal samples, on the other hand, showed a gene-specific expression pattern at mRNA level. CES2 mRNA increased with age but the opposite was true with CYP3A4 mRNA. The levels of CES2 and CYP3A4 protein, on the other hand, increased with age in both liver and duodenum. The multi-sampling study demonstrated significant correlation of CES2 expression between the duodenum and jejunum. However, neither duodenal nor jejunal expression correlated with hepatic expression of CES2. These findings establish that developmental regulation occurs in a gene and organ-dependent manner.
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http://dx.doi.org/10.1016/j.tox.2015.02.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4496585PMC
April 2015

Estrogen and Estrogen Receptor-α-Mediated Transrepression of Bile Salt Export Pump.

Mol Endocrinol 2015 Apr 12;29(4):613-26. Epub 2015 Feb 12.

Department of Biomedical and Pharmaceutical Sciences, Center for Pharmacogenomics and Molecular Therapy, College of Pharmacy, University of Rhode Island, Kingston, Rhode Island 02881.

Among diseases unique to pregnancy, intrahepatic cholestasis of pregnancy is the most prevalent disorder with elevated serum bile acid levels. We have previously shown that estrogen 17β-estradiol (E2) transrepresses bile salt export pump (BSEP) through an interaction between estrogen receptor (ER)-α and farnesoid X receptor (FXR) and transrepression of BSEP by E2/ERα is an etiological contributing factor to intrahepatic cholestasis of pregnancy. Currently the mechanistic insights into such transrepression are not fully understood. In this study, the dynamics of coregulator recruitment to BSEP promoter after FXR activation and E2 treatment were established with quantitative chromatin immunoprecipitation assays. Coactivator peroxisome proliferator-activated receptor-γ coactivator-1 was predominantly recruited to the BSEP promoter upon FXR activation, and its recruitment was decreased by E2 treatment. Meanwhile, recruitment of nuclear receptor corepressor was markedly increased upon E2 treatment. Functional evaluation of ERα and ERβ chimeras revealed that domains AC of ERα are the determinants for ERα-specific transrepression on BSEP. Further studies with various truncated ERα proteins identified the domains in ERα responsible for ligand-dependent and ligand-independent transrepression. Truncated ERα-AD exhibited potent ligand-independent transrepressive activity, whereas ERα-CF was fully capable of transrepressing BSEP ligand dependently in vitro in Huh 7 cells and in vivo in mice. Both ERα-AD and ERα-CF proteins were associated with FXR in the coimmunoprecipitation assays. In conclusion, E2 repressed BSEP expression through diminishing peroxisome proliferator-activated receptor-γ coactivator-1 recruitment with a concurrent increase in nuclear receptor corepressor recruitment to the BSEP promoter. Domains AD and CF in ERα mediated ligand-independent and ligand-dependent transrepression on BSEP, respectively, through interacting with FXR.
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http://dx.doi.org/10.1210/me.2015-1014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4399276PMC
April 2015

Suppression of the pregnane X receptor during endoplasmic reticulum stress is achieved by down-regulating hepatocyte nuclear factor-4α and up-regulating liver-enriched inhibitory protein.

Toxicol Sci 2015 Apr 22;144(2):382-92. Epub 2015 Jan 22.

Department of Biomedical and Pharmaceutical Sciences, Center for Pharmacogenomics and Molecular Therapy, University of Rhode Island, Kingston, Rhode Island 02881

Endoplasmic reticulum (ER) stress is recognized as a common theme in the development of metabolic syndrome and other diseases. Chronic liver diseases develop ER stress and also show decreased capacity of drug metabolism. The pregnane X receptor (PXR) is a master regulator of genes involved in drug elimination. This study was performed to determine whether ER stress condition decreases the expression of PXR and whether the decrease alters the induction of cytochrome P450 3A4 (CYP3A4). Human primary hepatocytes and HepG2 cell line (human hepatocellular carcinoma) were treated with brefeldin A and thapsigargin, 2 well-established ER stressors. Without exceptions, both stressors significantly decreased the expression of PXR. The decrease led to reduced induction of CYP3A4. Reporter dissection study, electrophoretic mobility shift assay, and chromatin immunoprecipitation located in the PXR promoter region 2 adjacent elements recognized by hepatocyte nuclear factor-4α (HNF-4α) and cytidine-cytidine-adenosine-adenosine-thymidine enhanced binding proteins (C/EBPs), respectively. Additional studies demonstrated that HNF-4α was down-regulated during ER stress but the expression of C/EBPβ varied depending on a particular form of C/EBPβ. Liver-enriched activator protein (LAP) was down-regulated but liver-enriched inhibitory protein (LIP) was highly induced. Nevertheless, over-expression of HNF-4α or LAP restored the expression of PXR. Interestingly, the very same sequence also responded to interleukin-6 (IL-6), and primary hepatocytes treated with thapsigargin significantly increased the level of IL-6 mRNA. These findings establish a functional interconnection between ER stress and signaling of proinflammatory cytokines in the regulation of PXR expression.
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http://dx.doi.org/10.1093/toxsci/kfv008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4372669PMC
April 2015

Deficiency in Nrf2 transcription factor decreases adipose tissue mass and hepatic lipid accumulation in leptin-deficient mice.

Obesity (Silver Spring) 2015 Feb 29;23(2):335-44. Epub 2014 Nov 29.

Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, Kingston, Rhode Island, USA; Institute of Biochemistry and Molecular Biology, College of Life and Health Sciences, Northeastern University, Shenyang, People's Republic of China.

Objective: To evaluate whether Nrf2 deficiency impacts insulin resistance and lipid accumulation in liver and white adipose tissue.

Methods: Lep(ob/ob) mice (OB) with targeted Nrf2 deletion (OB-Nrf2KO) were generated. Pathogenesis of obesity and type 2 diabetes was measured in C57BL/6J, Nrf2KO, OB, and OB-Nrf2KO mice. Hepatic lipid content, lipid clearance, and very low-density lipoprotein (VLDL) secretion were determined between OB and OB-Nrf2KO mice.

Results: OB-Nrf2KO mice exhibited decreased white adipose tissue mass and decreased adipogenic and lipogenic gene expression compared with OB mice. Nrf2 deficiency prolonged hyperglycemia in response to glucose challenge, which was paralleled by reduced insulin-stimulated Akt phosphorylation. In OB mice, Nrf2 deficiency decreased hepatic lipid accumulation, decreased peroxisome proliferator-activated receptor γ expression and nicotinamide adenine dinucleotide phosphate (NADPH) content, and enhanced VLDL secretion. However, this observation was opposite in lean mice. Additionally, OB-Nrf2KO mice exhibited increased plasma triglyceride content, decreased HDL-cholesterol content, and enhanced apolipoprotein B expression, suggesting Nrf2 deficiency caused dyslipidemia in these mice.

Conclusions: Nrf2 deficiency in Lep(ob/ob) mice reduced white adipose tissue mass and prevented hepatic lipid accumulation but induced insulin resistance and dyslipidemia. This study indicates a dual role of Nrf2 during metabolic dysregulation-increasing lipid accumulation in liver and white adipose tissue but preventing lipid accumulation in obese mice.
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http://dx.doi.org/10.1002/oby.20929DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4310781PMC
February 2015

Combinatorial photothermal and immuno cancer therapy using chitosan-coated hollow copper sulfide nanoparticles.

ACS Nano 2014 Jun 13;8(6):5670-81. Epub 2014 May 13.

Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, The University of Rhode Island , Kingston, Rhode Island 02881, United States.

Near-infrared light-responsive inorganic nanoparticles have been shown to enhance the efficacy of cancer photothermal ablation therapy. However, current nanoparticle-mediated photothermal ablation is more effective in treating local cancer at the primary site than metastatic cancer. Here, we report the design of a near-infrared light-induced transformative nanoparticle platform that combines photothermal ablation with immunotherapy. The design is based on chitosan-coated hollow CuS nanoparticles that assemble the immunoadjuvants oligodeoxynucleotides containing the cytosine-guanine (CpG) motifs. Interestingly, these structures break down after laser excitation, reassemble, and transform into polymer complexes that improve tumor retention of the immunotherapy. In this "photothermal immunotherapy" approach, photothermal ablation-induced tumor cell death reduces tumor growth and releases tumor antigens into the surrounding milieu, while the immunoadjuvants potentiate host antitumor immunity. Our results indicated that combined photothermal immunotherapy is more effective than either immunotherapy or photothermal therapy alone against primary treated and distant untreated tumors in a mouse breast cancer model. These hollow CuS nanoparticles are biodegradable and can be eliminated from the body after laser excitation.
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http://dx.doi.org/10.1021/nn5002112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4072412PMC
June 2014

Transcriptional dynamics of bile salt export pump during pregnancy: mechanisms and implications in intrahepatic cholestasis of pregnancy.

Hepatology 2014 Dec 19;60(6):1993-2007. Epub 2014 May 19.

Department of Biomedical and Pharmaceutical Sciences, Center for Pharmacogenomics and Molecular Therapy, College of Pharmacy, University of Rhode Island, Kingston, RI.

Unlabelled: Bile salt export pump (BSEP) is responsible for biliary secretion of bile acids, a rate-limiting step in the enterohepatic circulation of bile acids and transactivated by nuclear receptor farnesoid X receptor (FXR). Intrahepatic cholestasis of pregnancy (ICP) is the most prevalent disorder among diseases unique to pregnancy and primarily occurs in the third trimester of pregnancy, with a hallmark of elevated serum bile acids. Currently, the transcriptional regulation of BSEP during pregnancy and its underlying mechanisms and involvement in ICP are not fully understood. In this study the dynamics of BSEP transcription in vivo in the same group of pregnant mice before, during, and after gestation were established with an in vivo imaging system (IVIS). BSEP transcription was markedly repressed in the later stages of pregnancy and immediately recovered after parturition, resembling the clinical course of ICP in human. The transcriptional dynamics of BSEP was inversely correlated with serum 17β-estradiol (E2) levels before, during, and after gestation. Further studies showed that E2 repressed BSEP expression in human primary hepatocytes, Huh 7 cells, and in vivo in mice. Such transrepression of BSEP by E2 in vitro and in vivo required estrogen receptor α (ERα). Mechanistic studies with chromatin immunoprecipitation (ChIP), protein coimmunoprecipitation (Co-IP), and bimolecular fluorescence complementation (BiFC) assays demonstrated that ERα directly interacted with FXR in living cells and in vivo in mice.

Conclusion: BSEP expression was repressed by E2 in the late stages of pregnancy through a nonclassical E2/ERα transrepressive pathway, directly interacting with FXR. E2-mediated repression of BSEP expression represents an etiological contributing factor to ICP and therapies targeting the ERα/FXR interaction may be developed for prevention and treatment of ICP.
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http://dx.doi.org/10.1002/hep.27171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4194188PMC
December 2014

A comparative study of hollow copper sulfide nanoparticles and hollow gold nanospheres on degradability and toxicity.

ACS Nano 2013 Oct 1;7(10):8780-93. Epub 2013 Oct 1.

Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, The University of Rhode Island , Kingston, Rhode Island 02881, United States.

Gold and copper nanoparticles have been widely investigated for photothermal therapy of cancer. However, degradability and toxicity of these nanoparticles remain concerns. Here, we compare hollow CuS nanoparticles (HCuSNPs) with hollow gold nanospheres (HAuNS) in similar particle sizes and morphology following intravenous administration to mice. The injected pegylated HCuSNPs (PEG-HCuSNPs) are eliminated through both hepatobiliary (67 percentage of injected dose, %ID) and renal (23 %ID) excretion within one month postinjection. By contrast, 3.98 %ID of Au is excreted from liver and kidney within one month after iv injection of pegylated HAuNS (PEG-HAuNS). Comparatively, PEG-HAuNS are almost nonmetabolizable, while PEG-HCuSNPs are considered biodegradable nanoparticles. PEG-HCuSNPs do not show significant toxicity by histological or blood chemistry analysis. Principal component analysis and 2-D peak distribution plots of data from matrix-assisted laser desorption ionization-time-of-flight imaging mass spectrometry (MALDI-TOF IMS) of liver tissues demonstrated a reversible change in the proteomic profile in mice receiving PEG-HCuSNPs. This is attributed to slow dissociation of Cu ion from CuS nanoparticles along with effective Cu elimination for maintaining homeostasis. Nonetheless, an irreversible change in the proteomic profile is observed in the liver from mice receiving PEG-HAuNS by analysis of MALDI-TOF IMS data, probably due to the nonmetabolizability of Au. This finding correlates with the elevated serum lactate dehydrogenase at 3 months after PEG-HAuNS injection, indicating potential long-term toxicity. The comparative results between the two types of nanoparticles will advance the development of HCuSNPs as a new class of biodegradable inorganic nanomaterials for photothermal therapy.
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http://dx.doi.org/10.1021/nn403202wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3870179PMC
October 2013

Mechanistic insights into isoform-dependent and species-specific regulation of bile salt export pump by farnesoid X receptor.

J Lipid Res 2013 Nov 3;54(11):3030-44. Epub 2013 Sep 3.

Department of Biomedical and Pharmaceutical Sciences, Center for Pharmacogenomics and Molecular Therapy, University of Rhode Island, Kingston, RI 02881.

Expression of bile salt export pump (BSEP) is regulated by the bile acid/farnesoid X receptor (FXR) signaling pathway. Two FXR isoforms, FXRα1 and FXRα2, are predominantly expressed in human liver. We previously showed that human BSEP was isoform-dependently regulated by FXR and diminished with altered expression of FXRα1 and FXRα2 in patients with hepatocellular carcinoma. In this study, we demonstrate that FXRα1 and FXRα2 regulate human BSEP through two distinct FXR responsive elements (FXRE): IR1a and IR1b. As the predominant regulator, FXRα2 potently transactivated human BSEP through IR1a, while FXRα1 weakly transactivated human BSEP through a newly identified IR1b. Relative expression of FXRα1 and FXRα2 affected human BSEP expression in vitro and in vivo. Electrophoretic mobility shift and chromatin immunoprecipitation assays confirmed the binding and recruitment of FXRα1 and FXRα2 to IR1b and IR1a. Sequence analysis concluded that IR1b was completely conserved among species, whereas IR1a exhibited apparent differences across species. Sequence variations in IR1a were responsible for the observed species difference in BSEP transactivation by FXRα1 and FXRα2. In conclusion, FXR regulates BSEP in an isoform-dependent and species-specific manner through two distinct FXREs, and alteration of relative FXR isoform expression may be a potential mechanism for FXR to precisely regulate human BSEP in response to various physiological and pathological conditions.
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http://dx.doi.org/10.1194/jlr.M038323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3793607PMC
November 2013

Regulation of carboxylesterase-2 expression by p53 family proteins and enhanced anti-cancer activities among 5-fluorouracil, irinotecan and doxazolidine prodrug.

Br J Pharmacol 2013 Apr;168(8):1989-99

Department of Biomedical and Pharmaceutical Sciences, Center for Pharmacogenomics and Molecular Therapy, University of Rhode Island, Kingston, RI 02881, USA.

Background And Purpose: For four decades, 5-fluorouracil (5-FU) has been a major anti-cancer medicine. This drug is increasingly used with other anti-cancer agents such as irinotecan. Irinotecan and many others such as PPD (pentyl carbamate of p-aminobenzyl carbamate of doxazolidine) require activation by carboxylesterase-2 (CES2). 5-FU, on the other hand, reportedly induces CES2 in colorectal tumour lines. The aims of this study were to determine the molecular basis for the induction and to ascertain interactive cell-killing activity between 5-FU and ester prodrugs.

Experimental Approach: Colorectal and non-colorectal lines and xenografts were treated with 5-FU and the expression of CES2 was determined. Cell-killing activity of irinotecan and PPD were determined in the presence or absence of CES2 inhibitor. Several molecular experiments were used to determine the molecular basis for the induction.

Key Results: Without exceptions, robust induction was detected in cell lines expressing functional p53. High-level induction was also detected in xenografts. 5-FU pretreatment significantly increased cell-killing activity of irinotecan and PPD. Molecular experiments established that the induction was achieved by both transactivation and increased mRNA stability through p53. Either p63 or p73, functionally related to p53, did not support the transactivation.

Conclusions And Implications: The results in this study suggest that FOLFIRI, a common regimen combining irinotecan and 5-FU, should switch the dosing sequence, namely from 5-FU to irinotecan, to enhance hydrolytic activation of irinotecan. This modified order likely reduces the dose of anti-cancer agents, thus minimizing overall toxicity. The results also conclude that p53 family members act differently in regulating gene expression.
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http://dx.doi.org/10.1111/bph.12125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3623067PMC
April 2013

Carboxylesterase-2 is a highly sensitive target of the antiobesity agent orlistat with profound implications in the activation of anticancer prodrugs.

Biochem Pharmacol 2013 Feb 7;85(3):439-47. Epub 2012 Dec 7.

Department of Biomedical Sciences, Center for Pharmacogenomics and Molecular Therapy, University of Rhode Island, Kingston, RI 02881, USA.

Orlistat has been the most used anti-obesity drug and the mechanism of its action is to reduce lipid absorption by inhibiting gastrointestinal lipases. These enzymes, like carboxylesterases (CESs), structurally belong to the α/β hydrolase fold superfamily. Lipases and CESs are functionally related as well. Some CESs (e.g., human CES1) have been shown to hydrolyze lipids. This study was designed to test the hypothesis that orlistat inhibits CESs with higher potency toward CES1 than CES2, a carboxylesterase with little lipase activity. Liver microsomes and recombinant CESs were tested for the inhibition of the hydrolysis of standard substrates and the anticancer prodrugs pentyl carbamate of p-aminobenzyl carbamate of doxazolidine (PPD) and irinotecan. Contrary to the hypothesis, orlistat at 1 nM inhibited CES2 activity by 75% but no inhibition on CES1, placing CES2 one of the most sensitive targets of orlistat. The inhibition varied among some CES2 polymorphic variants. Pretreatment with orlistat reduced the cell killing activity of PPD. Certain mouse but not rat CESs were also highly sensitive. CES2 is responsible for the hydrolysis of many common drugs and abundantly expressed in the gastrointestinal track and liver. Inhibition of this carboxylesterase probably presents a major source for altered therapeutic activity of these medicines if co-administered with orlistat. In addition, orlistat has been linked to various types of organ toxicities, and this study provides an alternative target potentially involved in these toxicological responses.
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http://dx.doi.org/10.1016/j.bcp.2012.11.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4102434PMC
February 2013

Bile salt export pump is dysregulated with altered farnesoid X receptor isoform expression in patients with hepatocellular carcinoma.

Hepatology 2013 Apr 15;57(4):1530-41. Epub 2013 Feb 15.

Department of Biomedical and Pharmaceutical Sciences, Center for Pharmacogenomics and Molecular Therapy, College of Pharmacy, University of Rhode Island, 41 Lower College Road, Kingston, RI 02881, USA.

Unlabelled: As a canalicular bile acid effluxer, the bile salt export pump (BSEP) plays a vital role in maintaining bile acid homeostasis. BSEP deficiency leads to severe cholestasis and hepatocellular carcinoma (HCC) in young children. Regardless of the etiology, chronic inflammation is the common pathological process for HCC development. Clinical studies have shown that bile acid homeostasis is disrupted in HCC patients with elevated serum bile acid level as a proposed marker for HCC. However, the underlying mechanisms remain largely unknown. In this study, we found that BSEP expression was severely diminished in HCC tissues and markedly reduced in adjacent nontumor tissues. In contrast to mice, human BSEP was regulated by farnesoid X receptor (FXR) in an isoform-dependent manner. FXR-α2 exhibited a much more potent activity than FXR-α1 in transactivating human BSEP in vitro and in vivo. The decreased BSEP expression in HCC was associated with altered relative expression of FXR-α1 and FXR-α2. FXR-α1/FXR-α2 ratios were significantly increased, with undetectable FXR-α2 expression in one third of the HCC tumor samples. A similar correlation between BSEP and FXR isoform expression was confirmed in hepatoma Huh7 and HepG2 cells. Further studies showed that intrahepatic proinflammatory cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α), were significantly elevated in HCC tissues. Treatment of Huh7 cells with IL-6 and TNF-α resulted in a marked increase in FXR-α1/FXR-α2 ratio, concurrent with a significant decrease in BSEP expression.

Conclusion: BSEP expression is severely diminished in HCC patients associated with alteration of FXR isoform expression induced by inflammation. Restoration of BSEP expression through suppressing inflammation in the liver may reestablish bile acid homeostasis.
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http://dx.doi.org/10.1002/hep.26187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3608797PMC
April 2013

Hollow copper sulfide nanoparticle-mediated transdermal drug delivery.

Small 2012 Oct 25;8(20):3143-50. Epub 2012 Jul 25.

Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, 41 Lower College Road, Kingston, RI 02881, USA.

A photothermal ablation-enhanced transdermal drug delivery methodology is developed based on hollow copper sulfide nanoparticles (HCuSNPs) with intense photothermal coupling effects. Application of nanosecond-pulsed near-infrared laser allows rapid heating of the nanoparticles and instantaneous heat conduction. This provides very short periods of time but extremely high temperatures in local regions, with focused thermal ablation of the stratum corneum. The depth of skin perforations can be controlled by adjusting the laser power. Skin disruption by HCuSNP-mediated photothermal ablation significantly increases the permeability of human growth hormone. This technique offers compelling opportunities for macromolecular drug and vaccine delivery.
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http://dx.doi.org/10.1002/smll.201200783DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3485307PMC
October 2012

Antioxidant sulforaphane and sensitizer trinitrobenzene sulfonate induce carboxylesterase-1 through a novel element transactivated by nuclear factor-E2 related factor-2.

Biochem Pharmacol 2012 Sep 6;84(6):864-71. Epub 2012 Jul 6.

Department of Biomedical Sciences, Center for Pharmacogenomics and Molecular Therapy, University of Rhode Island, Kingston, RI 02881, USA.

Carboxylesterase-1 (CES1), the most versatile human carboxylesterase, plays critical roles in drug metabolism and lipid mobilization. This enzyme is highly induced by antioxidants and sensitizers in various cell lines. These compounds are known to activate nuclear factor-E2 related factor-2 (Nrf2) by reacting to kelch-like ECH-associated protein-1 (Keap1). The aims of this study were to determine whether antioxidant sulforaphane (SFN) and sensitizer trinitrobenzene sulfonate (TNBS) target Keap1 similarly and whether they use the same element for CES1 induction. Cells over-expressing Keap1 were treated with TNBS or SFN and the formation of disulfide bonds among Keap1 molecules were determined. SFN promoted intramolecular disulfide formation whereas TNBS promoted intermolecular disulfide formation of Keap1. Two elements, sensitizing/antioxidant response element (S/ARE) and ARE4, were identified to support Nrf2 in the regulated expression of CES1A1. Both elements were bound by Nrf2, however, the S/ARE element supported, whereas the ARE4 element repressed Nrf2 transactivation. The repression required higher amounts of Nrf2, suggesting that the transactivation through the S/ARE element dominates the trans-repression through the ARE4 element under normal antioxidative condition. These findings conclude that compounds, although triggering the Keap1-Nrf2 pathway, may differ in the mode of reacting with Keap1. These findings also conclude that both positive and negative Nrf2 elements exist even within the same gene, and such opposing mechanisms provide fine-tuning in transcriptional regulation by the Keap1-Nrf2 pathway. High levels of CES1 are linked to lipid retention. Excessive induction of CES1 by antioxidants and sensitizers likely provides a mechanism for potential detrimental effect on human health.
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http://dx.doi.org/10.1016/j.bcp.2012.06.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4096214PMC
September 2012

DEC1 binding to the proximal promoter of CYP3A4 ascribes to the downregulation of CYP3A4 expression by IL-6 in primary human hepatocytes.

Biochem Pharmacol 2012 Sep 21;84(5):701-711. Epub 2012 Jun 21.

Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, Kingston, Rhode Island, 02881, USA.

In this study, we provided molecular evidences that interleukin-6 (IL-6) contributed to the decreased capacity of oxidative biotransformation in human liver by suppressing the expression of cytochrome P450 3A4 (CYP3A4). After human hepatocytes were treated with IL-6, differentially expressed in chondrocytes 1 (DEC1) expression rapidly increased, and subsequently, the CYP3A4 expression decreased continuously. Furthermore, the repression of CYP3A4 by IL-6 occurred after the increase of DEC1 in primary human hepatocytes. In HepG2 cells, knockdown of DEC1 increased the CYP3A4 expression and its enzymatic activity. In addition, it partially abolished the decreased CYP3A4 expression as well as its enzymatic activity induced by IL-6. Consistent with this, overexpression of DEC1 markedly reduced the CYP3A4 promoter activity and the CYP3A4 expression as well as its enzymatic activity. Using sequential truncation and site directed mutagenesis of CYP3A4 proximal promoter with DEC1 construct, we showed that DEC1 specifically bound to CCCTGC sequence in the proximal promoter of CYP3A4, which was validated by EMSA and ChIP assay. These findings suggest that the repression of CYP3A4 by IL-6 is achieved through increasing the DEC1 expression in human hepatocytes, the increased DEC1 binds to the CCCTGC sequence in the promoter of CYP3A4 to form CCCTGC-DEC1 complex, and the complex downregulates the CYP3A4 expression and its enzymatic activity.
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http://dx.doi.org/10.1016/j.bcp.2012.06.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4088276PMC
September 2012

Age-related inducibility of carboxylesterases by the antiepileptic agent phenobarbital and implications in drug metabolism and lipid accumulation.

Biochem Pharmacol 2012 Jul 10;84(2):232-9. Epub 2012 Apr 10.

Department of Biomedical Sciences, Center for Pharmacogenomics and Molecular Therapy, University of Rhode Island, Kingston, RI 02881, USA.

Carboxylesterases (CES) constitute a class of hydrolytic enzymes that play critical roles in drug metabolism and lipid mobilization. Previous studies with a large number of human liver samples have suggested that the inducibility of carboxylesterases is inversely related with age. To directly test this possibility, neonatal (10 days of age) and adult mice were treated with the antiepileptic agent phenobarbital. The expression and hydrolytic activity were determined on six major carboxylesterases including ces1d, the ortholog of human CES1. Without exception, all carboxylesterases tested were induced to a greater extent in neonatal than adult mice. The induction was detected at mRNA, protein and catalytic levels. Ces1d was greatly induced and found to rapidly hydrolyze the antiplatelet agent clopidogrel and support the accumulation of neutral lipids. Phenobarbital represents a large number of therapeutic agents that induce drug metabolizing enzymes and transporters in a species-conserved manner. The higher inducibility of carboxylesterases in the developmental age likely represents a general phenomenon cross species including human. Consequently, individuals in the developmental age may experience greater drug-drug interactions. The greater induction of ces1d also provides a molecular explanation to the clinical observation that children on antiepileptic drugs increase plasma lipids.
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http://dx.doi.org/10.1016/j.bcp.2012.04.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4107352PMC
July 2012
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