Expression Of Carboxylesterase Research Articles

Near-infrared emission tracks inter-individual variability of carboxylesterase-2 via a novel molecular substrate.

A low-molecular-weight molecule (4-(2-(3-(dicyanomethyl)-5,5-dimethylcyclohex-1-en-1-yl)vinyl)phenyl-benzoate, DDPB) has been developed. Theorganic framework possesses very weak fluorescence. The feasibility of the signal transduction has been performed via fluorometric titrations in solution. DDPB gives rise to responses to carboxylesterase 2 (CES2) based on "off-on" responses. The red emission at 670nm has been derived from the enzyme-induced hydrolysis of ester linkages, thus suppressing the intramolecular charge transfer (ICT) effect and thereby generating the fluorescent segment. The optical excitation window for this probe is extended to thevisible light range (λex = 516nm), and it will induce less harmful influence on biological substances. Thedetection limit for the measurement of CES2 concentration isas low as 2.33mU/mL. The conventional studies concerning the activation process are generally performed within only a single liveing cell system. In this study, it is the first time that expression of carboxylesterase 2 in five kinds of cell lines (HeLa > C1498 > active T cell > Jurkat > unactive T cell) has been clarified by flow cytometry, Western blotting, and confocal microscopy analysis. The elucidation of CES2 and its variability in a variety of cells will open new ways for drug metabolism and disease prevention. Graphical abstract We reported a new "substrate-mediated light-on" strategy based on an ester bond cleavage reaction. Most of prepared nanomaterials and organic fluorophores possessed short wavelength emissions in the blue or green region which will not be difficult for cellular imaging. In this study, a novel functional molecule (DDPB) was considered as the substrate for CES2 and the optical "off-on" response was realized. DDPB was cell permeable and possessed very low cytotoxicity. Moreover, the identification of CES2 and their subtle changes in five different cells afforded the sequence for carboxylesterase-2 as Hela > C1498 > Active T cell > Jurkat > Unactive T cell. Inhibition studies showed that the hydrolysis of DDPB was effectively suppressed by bis-p-nitrophenyl phosphate and the cellular tracking results firmly supported this point. To our knowledge, the inter-individual variability for the CES2 expressions in five different cell lines has never been reported via the substrate induced optical changes.

Hepatocyte-Specific Expression of Human Carboxylesterase 1 Attenuates Diet-Induced Steatohepatitis and Hyperlipidemia in Mice.

Rodents have at least five carboxylesterase 1 (Ces1) genes, whereas there is only one CES1 gene in humans, raising the question as to whether human CES1 and mouse Ces1 genes share the same functions. In this study, we investigate the role of human CES1 in the development of steatohepatitis or dyslipidemia in C57BL/6 mice. Hepatocyte‐specific expression of human CES1 prevented Western diet or alcohol‐induced steatohepatitis and hyperlipidemia. Mechanistically, human CES1 induced lipolysis and fatty acid oxidation, leading to a reduction in hepatic triglyceride and free fatty acid levels. Human CES1 also reduced hepatic‐free cholesterol levels and induced low‐density lipoprotein receptor. In addition, human CES1 induced hepatic lipoprotein lipase and apolipoprotein C‐II expression. Conclusion: Hepatocyte‐specific overexpression of human CES1 attenuates diet‐induced steatohepatitis and hyperlipidemia.

Carboxylesterases: Pharmacological Inhibition Regulated Expression and Transcriptional Involvement of Nuclear Receptors and other Transcription Factors

Carboxylesterases (CESs, E.C.3.1.1.1) constitute a large class of enzymes that determine the therapeutic efficacy and toxicity of ester/amide drugs. Without exceptions, all mammalian species studied express multiple forms of carboxylesterases. Two human carboxylesterases, CES1 and CES2, are major contributors to hydrolytic biotransformation. Recent studies have identified therapeutic agents that potently inhibit carboxylesterases-based catalysis. Some of them are reversible whereas others irreversible. The adrenergic antagonist carvedilol, for example, reversibly inhibits CES2 but this carboxylesterase is irreversibly inhibited by orlistat, a popular anti-obesity medicine. Kinetically, the inhibition occurs competitively, non-competitively or in combination, depending on a carboxylesterase. For example, the calcium channel blocker diltiazem competitively inhibits CES1 but non-competitively inhibits CES2. In addition to inhibited catalysis, several therapeutic agents or disease mediators have been shown to regulate the expression of carboxylesterases. For example, the antiepileptic drug phenobarbital induces both human and rodent carboxylesterases, whereas the antibiotic rifampicin induces human carboxylesterases only. Conversely, the proinflammatory cytokine interleukin-6 (IL-6) suppresses the expression of carboxylesterases across species, but depending on the concentrations of glucose in the culture medium. Transactivation, transrepression and altered mRNA stability contribute to the regulated expression. Several nuclear receptors are established to support the regulation including constitutive androstane receptor, glucocorticoid receptor and pregnane X receptor. In addition, non-ligand transcription factors are also involved in the regulation and exemplified by differentiated embryo chondrocyte-1, nuclear factor (erythroid-derived 2)-like 2 and tumor protein p53. These transcription factors coordinate the regulated expression of carboxylesterases, constituting a regulatory network for the hydrolytic biotransformation.

Effects of enrofloxacin on antioxidant system, microsomal enzymatic activity, and proteomics in porcine liver.

Enrofloxacin (EF) is a widely used fluoroquinolone, usually regarded as a safe and effective treatment for bacterial infections. Adverse effects of EF have previously been demonstrated in some species, but so far there have been no studies looking specifically at the impact of EF on pigs. In this study, three different doses of EF (5, 25 and 125mgkgbw-1 ) were administrated to Bama pigs. The results showed that lipid peroxidation of pig liver tissue occurred with all EF doses. The 125mgkg dose of EF induced catalase (CAT) and glutathione peroxidase (GSH-px) and increased CYP450 content in pig liver microsomes. The activity of microsomal NADPH-cytochrome C reductase (NCCR) was elevated at both the 25 and 125mgkg doses of EF. Microsomal erythromycin N-demethylase (ERND) and aminopyrin N-demethylase (AND) were inhibited by high doses of EF, while aniline-4-hydroxylase (AH) was unaffected. None of the EF treatments affected superoxide dismutase (SOD) or cytochrome b5 content. Antioxidases and microsomal enzymes may work together to resist the adverse effects of EF. Proteomic analysis revealed increased protein expression of carboxylesterase (CES) and alpha-enolase (ENO1) in microsomes as a stress response to EF. These results provide new information about the adverse effect of fluoroquinolones and help guide their usage more effectively in the clinic or animal breeding.

Development of a Single Reaction PCR Genotyping Assay for Two Separate Gene Loci in Mice

There are many variables to consider when optimizing a polymerase chainreaction (PCR) assay for a specific gene—from methods of DNA extraction to choosing an effective combination of primers. A multiplex PCR assay analyzing two genes in the same reaction may require additional optimization. The objective of this experiment was to optimize a single PCR assay to effectively genotype C57BL/6 transgenic mouse strains in which two genes have been modified. By removing a portion of the Es1 gene, the expression of serum carboxylesterase was interrupted, resulting in serum carboxylesterase knock out (Es1 KO) mice. In another location, the gene expressing mouse acetylcholinesterase was replaced by a gene expressing human acetylcholinesterase, resulting in a human acetylcholinesterase knock in (AChEKI) mouse strain. There are several possible genotypes for each mouse strain: for Es1 KO mice the possibilities are wild type (WT +/+), knock out (KO −/−), or heterozygous (Het +/−), and for AChEKI mice the possibilities are human (H/H), mouse (M/M), or heterozygous (M/H). Optimization of the PCR genotyping assays for these strains was achieved by altering the method of DNA extraction, annealing temperatures, and testing multiple primer sets. Initially, DNA was extracted from mouse fecal matter, but this method was later determined to produce unreliable results in the subsequent PCR assays. DNA extracted from tail tissue was found to be much more reliable. Utilizing tail tissue DNA, a single pair of primers was found that could differentiate WT, KO, and Het alleles in the Es1 KO strain. The optimal annealing temperature for these primers was determined using a temperature gradient experiment. Primers for genotyping the AChE KI strain were selected and optimized in a similar manner. Finally, a multiplex PCR assay was developed allowing for the simultaneous determination of the genotype of both genes in mice.Support or Funding Information*This research was supported in part by an appointment to the Postgraduate Research Participation Program at the U.S. Army Medical Research Institute of Chemical Defense administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the U. S. Army Medical Research and Materiel Command.The experimental protocol was approved by the Animal Care and Use Committee at the United States Army Medical Research Institute of Chemical Defense and all procedures were conducted in accordance with the principles stated in the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011), and the Animal Welfare Act of 1966 (P.L. 89‐544), as amended.Threat Reduction Agency – Joint Science and Technology Office, Medical S&T Division.The views expressed in this abstract are those of the author(s) and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibitory Influence of Panax notoginseng Saponins on Aspirin Hydrolysis in Human Intestinal Caco-2 Cells.

Herb-drug interactions are important safety concerns in clinical practice. The interactions occur firstly in the intestinal absorption for orally administered drugs. Aspirin and Panax notoginseng saponins (PNS)-based drugs are often combined in China to prevent larger-artery atherosclerosis. Here, we aimed to characterize the aspirin transport across Caco-2 cell monolayers, a model of the intestinal absorption, and further to evaluate the influence of PNS on aspirin hydrolysis and the relating mechanisms. Transcellular transport of aspirin and the influence of PNS were explored using Caco-2 cell monolayers. The protein expression of human carboxylesterase 1 (hCE1) and hCE2 in Caco-2 cells after PNS treatment was analyzed by ELISA, and the mRNA level were determined by qRT-PCR. In the study, Caco-2 cells showed high level of hydrolase activity, and most aspirin was hydrolyzed inside the cells during the transport process. Interestingly, PNS were demonstrated to inhibit the esterase activities responsible for aspirin hydrolysis in Caco-2 cells. PNS could also decrease the protein expression of hCE1 and hCE2, whereas exhibited minor effect on the mRNA expression. These results indicated that oral administration of PNS-based drugs might inhibit the hydrolysis of aspirin during intestinal absorption thus promoting its bioavailability.

Silencing carboxylesterase 1 in human THP-1 macrophages perturbs genes regulated by PPARγ/RXR and RAR/RXR: down-regulation of CYP27A1-LXRα signaling.

Macrophage foam cells store excess cholesterol as cholesteryl esters, which need to be hydrolyzed for cholesterol efflux. We recently reported that silencing expression of carboxylesterase 1 (CES1) in human THP-1 macrophages [CES1KD (THP-1 cells with CES1 expression knocked down) macrophages] reduced cholesterol uptake and decreased expression of CD36 and scavenger receptor-A in cells loaded with acetylated low-density lipoprotein (acLDL). Here, we report that CES1KD macrophages exhibit reduced transcription of cytochrome P45027A1 (CYP27A1) in nonloaded and acLDL-loaded cells. Moreover, levels of CYP27A1 protein and its enzymatic product, 27-hydroxycholesterol, were markedly reduced in CES1KD macrophages. Transcription of LXRα (liver X receptor α) and ABCA1 (ATP-binding cassette transporter A1) was also decreased in acLDL-loaded CES1KD macrophages, suggesting reduced signaling through PPARγ-CYP27A1-LXRα. Consistent with this, treatment of CES1KD macrophages with agonists for PPARγ, RAR, and/or RAR/RXR partially restored transcription of CYP27A1 and LXRα, and repaired cholesterol influx. Conversely, treatment of control macrophages with antagonists for PPARγ and/or RXR decreased transcription of CYP27A1 and LXRα Pharmacologic inhibition of CES1 in both wild-type THP-1 cells and primary human macrophages also decreased CYP27A1 transcription. CES1 silencing did not affect transcript levels of PPARγ and RXR in acLDL-loaded macrophages, whereas it did reduce the catabolism of the endocannabinoid 2-arachidonoylglycerol. Finally, the gene expression profile of CES1KD macrophages was similar to that of PPARγ knockdown cells following acLDL exposures, further suggesting a mechanistic link between CES1 and PPARγ. These results are consistent with a model in which abrogation of CES1 function attenuates the CYP27A1-LXRα-ABCA1 signaling axis by depleting endogenous ligands for the nuclear receptors PPARγ, RAR, and/or RXR that regulate cholesterol homeostasis.

Shengjiang Xiexin Decoction Alters Pharmacokinetics of Irinotecan by Regulating Metabolic Enzymes and Transporters: A Multi-Target Therapy for Alleviating the Gastrointestinal Toxicity.

Shengjiang Xiexin decoction (SXD), a classic traditional Chinese medical formula chronicled in Shang Han Lun, is used in modern clinical practice to decrease gastrointestinal toxicity induced by the chemotherapeutic drug irinotecan (CPT-11). In this study, the effect of SXD on the pharmacokinetics of CPT-11 and its active metabolites (SN-38 and SN-38G), and the underlying mechanisms were further examined. An ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the simultaneous quantification of CPT-11, SN-38, and SN-38G in the plasma, bile, liver, intestine, and intestinal contents of control and SXD-pre-treated rats after intravenous administration of CPT-11. SXD pretreatment increased the area under the curve (AUC) and the initial plasma concentration (C0) of CPT-11 but decreased the plasma clearance (CL). The AUC and the maximum plasma concentration (Cmax) of SN-38 decreased, whereas the Cmax of SN-38G increased. Compared with that of the control group, the biliary excretion of CPT-11, SN-38, and SN-38G was inhibited. The CPT-11, SN-38, and SN-38G concentrations in the liver, intestine, and intestinal contents were different between the two groups. Furthermore, the hepatic expression of multidrug resistance-associated protein-2 (Mrp-2), P-glycoprotein (P-gp), and carboxylesterase 2 (CES2) was significantly down-regulated by SXD, while the hepatic and jejunal uridine diphosphate (UDP)-glucuronosyltransferase 1A1 (UGT1A1) expression was elevated. The hydrolysis of CPT-11 to SN-38 by CES and the glucuronidation of SN-38 to SN-38G by UGT were affected by liver and jejunum S9 fractions from rats pre-treated with SXD. Therefore, this study demonstrated for the first time that SXD could alter the pharmacokinetics of CPT-11 and its metabolites to alleviate CPT-11-induced diarrhea. And the underlying mechanism of drug interaction between CPT-11 and SXD involves decreasing hepatic Mrp-2 and P-gp expression and altering the activities of CES and UGT.

Effect of alcohol on skin permeation and metabolism of an ester-type prodrug in Yucatan micropig skin

We studied the effect that three alcohols, ethanol (EA), propanol (PA), and isopropanol (IPA), have on the skin permeation of p-hydroxy benzoic acid methyl ester (HBM), a model ester-type prodrug. HBM was applied to Yucatan micropig skin in a saturated phosphate buffered solution with or without 10% alcohol, and HBM and related materials in receptor fluid and skin were determined with HPLC. In the absence of alcohol, p-hydroxy benzoic acid (HBA), a metabolite of HBM, permeated the skin the most. The three alcohols enhanced the penetration of HBM at almost the same extent. The addition of 10% EA or PA to the HBM solution led to trans-esterification into the ethyl ester or propyl ester of HBA, and these esters permeated skin as well as HBA and HBM did. In contrast, the addition of 10% IPA promoted very little trans-esterification. Both hydrolysis and trans-esterification in the skin S9 fraction were inhibited by BNPP, an inhibitor of carboxylesterase (CES). Western blot and native PAGE showed the abundant expression of CES in micropig skin. Both hydrolysis and trans-esterification was simultaneously catalyzed by CES during skin permeation. Our data indicate that the alcohol used in dermal drug preparations should be selected not only for its ability to enhance the solubility and permeation of the drug, but also for the effect on metabolism of the drug in the skin.

Functional analysis of carboxylesterase in human induced pluripotent stem cell-derived enterocytes

Human carboxylesterase (CES) is a key esterase involved in the metabolism and biotransformation of drugs. Hydrolysis activity in the human small intestine is predominantly mediated by CES2A1 rather than CES1A. In drug development studies, Caco-2 cells are commonly used as a model to predict drug absorption in the human small intestine. However, the expression patterns of CES2A1 and CES1A in Caco-2 cells differ from those in the human small intestine. There are also species-specific differences in CES expression patterns between human and experimental animals. Furthermore, it is difficult to obtain primary human intestinal epithelial cells. Therefore, there is currently no system that can precisely predict features of drug absorption, such as CES-mediated metabolism, in the human intestine. To develop a novel system to evaluate intestinal pharmacokinetics, we analyzed CES expression and function in human induced pluripotent stem (iPS) cell-derived enterocytes. CES2A1 mRNA and protein levels in human iPS cell-derived enterocytes were comparable to Caco-2 cells, whereas CES1A levels were lower in human iPS cell-derived enterocytes compared with Caco-2 cells. p-nitrophenyl acetate hydrolysis in human iPS cell-derived enterocytes was significantly inhibited by the CES2A1-specific inhibitor telmisartan. Hydrolysis levels of the CES2A1-specific substrate aspirin were similar in human iPS cell-derived enterocytes and Caco-2 cells, whereas hydrolysis of the CES1A-specific substrate monoethylglycylxylidine was observed in Caco-2 cells but not in human iPS cell-derived enterocytes. These findings demonstrated that the expression and activity of CES isozymes in human iPS cell-derived enterocytes are more similar to the human small intestine compared with Caco-2 cells.

Involvement of pregnane X receptor in the suppression of carboxylesterases by metformin in vivo and in vitro, mediated by the activation of AMPK and JNK signaling pathway

Type 2 diabetes mellitus (T2D) is a complex metabolic disorder requiring polypharmacy treatment in clinic, with metformin being widely used antihyperglycemic drug. However, the mechanisms of metformin as a perpetrator inducing potential drug-drug interactions and adverse drug reactions are scarcely known to date. Carboxylesterases (CESs) are major hydrolytic enzymes highly expressed in the liver, including mouse carboxylesterase 1d (Ces1d) and Ces1e. In the present study, experiments are designed to investigate the effects and mechanisms of metformin on Ces1d and Ces1e in vivo and in vitro. In results, metformin suppresses the expression and activity of Ces1d and Ces1e in a dose- and time-dependent manner. The decreased expression of nuclear receptor PXR and its target gene P-gp indicates the involvements of PXR in the suppressed expression of carboxylesterases by metformin. Furthermore, metformin significantly suppresses the phosphorylation of AMPK and JNK, and the suppression of carboxylesterases induced by metformin is repeatedly abolished by AMPK inhibitor Compound C and JNK inhibitor SP600125. It implies that the activation of AMPK and JNK pathways mediates the suppression of carboxylesterases by metformin. The findings deserve further elucidation including clinical trials and have a potential to make contribution for the rational medication in the treatment of T2D patients.

Abstract 4806: Enhancing the efficacy of tosedostat through carboxylesterase induction

Abstract Malignant cells, including acute myeloid leukaemia (AML), have high protein turnover to support their accelerated cell growth. Aminopeptidases regulate proteolysis, supplying free amino acids for new protein synthesis. Inhibition of aminopeptidases depletes free amino acids, impairing new protein production, leading to impaired cell growth and proliferation. Hence, aminopeptidase inhibition is of particular interest as a therapeutic strategy for AML. Tosedostat (CHR-2797) is an aminopeptidase inhibitor which is converted to its active metabolite, CHR-79888, by intracellular carboxylesterases (CES). This active metabolite is poorly membrane permeable, resulting in intracellular accumulation. Tosedostat has shown promise as a potential therapeutic strategy for AML and is well-tolerated in adult patients. However, its efficacy in paediatric AML has not been established. We set out to investigate the use of tosedostat for paediatric AML and enhance its efficacy through CES1 induction. It is hypothesised that inducing CES will lead to increased CHR-79888 production and subsequently, increased tosedostat efficacy. The IC50 of tosedostat across 11 AML cell lines (6 paediatric and 5 adult), determined by resazurin reduction assay, was approximately 13μM. Biochemical assays demonstrated that tosedostat inhibited cellular aminopeptidase activity in a concentration-dependent manner. However, this inhibitory effect was reversible with rapid, but incomplete, enzyme activity recovery upon drug withdrawal, irrespective of initial concentration or treatment duration. Quantitative PCR and immunoblotting revealed no correlation between CES expression and sensitivity to tosedostat. Cellular CES1 activity was also measured. Benzil and Bis(4-nitrophenyl)phosphate (BNPP) were used to modulate cellular CES1 activity. Assays of CES1 enzyme kinetics demonstrated that following temporary exposure to the reversible CES inhibitor benzil, there is a rebound in CES1 activity in AML cells. Consistent with this observation, pre-treatment by benzil increased CES1 activity, especially in MV4-11 cells. Moreover, reduced cell viability with tosedostat treatment following benzil pre-treatment correlated with the induction of CES1 activity in AML cell lines. Together, these data suggest that enhanced tosedostat efficacy through induction of CES activity is a novel therapeutic approach to treat AML. Citation Format: Priscilla Wei Ling Hong, Amanda Melanie Smith, Lambro Angelo Johnson, Dorothy Loo, Thomas Stoll, Michelle M. Hill, Andrew S. Moore. Enhancing the efficacy of tosedostat through carboxylesterase induction. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4806.

Effects of spirotetramat treatments on fecundity and carboxylesterase expression of Aphis gossypii Glover.

Spirotetramat is a novel tetramic acid-based insecticide, belonging to keto-enol pesticide family, with a novel mode of action; it interferes with lipid biosynthesis. Its insecticide activity against various agricultural pest insects have been demonstrated (e.g. on Myzus persicae, Bemisia tabaci and Tetranychus urticae). However, information available is currently limited on the efficacy of spirotetramat on the cotton aphid, Aphis gossypii, a key cotton pest worldwide. We assessed the spirotetramat toxicity on A. gossypii and evaluated its effects on aphid fecundity when exposed to a sublethal concentration (LC10) and to increasing lethal concentrations (LC25, LC50, and LC75). A key mechanism involved in insecticide resistance in aphids relates to esterase activity. We estimated the CarE activity and a CarE gene expression in aphids in response to spirotetramat exposure, then we tested tolerance of offspring to spirotetramat when the parents were exposed to the highest concentration tested in our study (LC75). Results showed that spirotetramat showed increasing toxicity to A. gossypii with exposure duration to treated leaves; LC50 ranged from 23,675.68 to 12.27mg/L for 1 to 5-days exposure. In addition, spirotetramat reduced aphid daily fecundity, in all concentration treatments, especially with up to 90% reduction in case of exposure to LC75. Total CarE activity increased dramatically and CarE mRNA expression was also up regulated in aphids after exposure to LC75 spirotetramat. Finally, the tolerance to spirotetramat in offspring (when parents were exposed to the LC75) showed a 2.5-fold increase when compared to control aphids. Consequently, spiroteramat showed potential for pest management of cotton aphids owing to both lethal and sublethal activities, notably strong impact on aphid fecundity. However, we also demonstrated that increased tolerance of A. gossypii to spirotetramat may happen through increased CarE- activity and subsequent metabolic degradation of the insecticide in aphids' body.

Expression of Carboxylesterase Isozymes and Their Role in the Behavior of a Fexofenadine Prodrug in Rat Skin

The expression of carboxylesterase (CES) and the transdermal movement of an ester prodrug were studied in rat skin. Ethyl-fexofenadine (ethyl-FXD) was used as a model lipophilic prodrug that is slowly hydrolyzed to its parent drug, FXD (MW 502). Among the CES1 and CES2 isozymes, Hydrolase A is predominant in rat skin and this enzyme was involved in 65% of the cutaneous hydrolysis of ethyl-FXD. The similarity of the permeation behavior of ethyl-FXD in full thickness and stripped skin indicated that the stratum corneum was not a barrier to penetration. However, only FXD was observed in receptor fluid, not ethyl-FXD, presumably because of the high degree of binding of ethyl-FXD in viable skin. The rate of hydrolysis of ethyl-FXD was much faster than steady-state flux, such that the influx rate was the rate-limiting process for transdermal permeation. Although Hydrolase A levels gradually increased in skin taken from rats aged from 8 to 90 weeks, variations in the expression levels of the esterase hardly affected the conversion of prodrug. The present data suggest that the slow hydrolysis of the prodrug of an active ingredient in viable skin followed by slow diffusion of active drug may provide a useful approach to topical application.

Fluoxetine reduces CES1, CES2, and CYP3A4 expression through decreasing PXR and increasing DEC1 in HepG2 cells

1. This study investigated the mechanisms of the decreases of carboxylesterases (CES) and cytochrome P4503A4 (CYP3A4) and the enzymatic activities induced by fluoxetine (FLX) in HepG2 cells. We found that FLX decreased the carboxylesterase 1 (CES1) and carboxylesterase 2 (CES2) expression and the hydrolytic activity.2. FLX decreased the pregnane X receptor (PXR) expression which regulated the target genes such as CYP3A4, whereas increased the differentiated embryonic chondrocyte-expressed gene 1 (DEC1) expression.3. FLX repressed the PXR at transcriptional level.4. Overexpression of PXR alone increased the expression of CES1, CES2, and CYP3A4 and attenuated the decreases of CES1, CES2, and CYP3A4 induced by FLX. On the contrary, knockdown of PXR alone decreased the expression of CES1, CES2, and CYP3A4 and almost abolished the decreases of CES1, CES2, and CYP3A4 induced by FLX.5. Knockdown of DEC1 alone increased the expression of PXR and CYP3A4 and almost abolished the decreases of CES1, CES2, and CYP3A4 induced by FLX.6. Taken together, the decreases of CES and CYP3A4 expression and enzymatic activities induced by FLX are through decreasing PXR and increasing DEC1 in HepG2 cells.

Abstract LB-121: Identification of Carboxylesterase-2 as a determinant of response to irinotecan and neoadjuvant FOLFIRINOX therapy in pancreatic ductal adenocarcinoma

Abstract Background: Serine hydrolases (SHs) are among the largest classes of enzymes in human and play crucial role in many pathophysiological processes of cancer. We have undertaken a comprehensive proteomic analysis to assess the differential expression and cellular localization of SHs, which uncovered distinctive expression of Carboxylesterase 2 (CES2), the most efficient carboxyl esterase in activating the pro-drug irinotecan into SN-38, in pancreatic ductal adenocarcinoma (PDAC). We therefore assessed the extent of heterogeneity in CES2 expression in PDAC and its potential relevance to irinotecan based therapy. Methods: CES2 expression in PDAC and paired non-tumor tissues was evaluated by immunohistochemistry. CES2 activity was assessed by monitoring the hydrolysis of the substrate p-NPA. Kaplan-Meier and Cox regression analyses were applied to assess the association between overall survival and CES2 expression in patients who underwent neoadjuvant FOLFIRINOX treatment. Results: Significant overexpression of CES2, both at the mRNA and protein levels, was observed in PDAC compared to paired non-tumor tissue (P < .0001), with 48/118 (40.7%) tumors exhibiting high CES2 expression. CES2 activity in PDAC cell lines was inversely correlated with irinotecan IC50 values (P = .021), while no molecule involved in irinotecan metabolism yielded a significant correlation between its expression and sensitivity to the drug. Remarkably, we recently found that high CES2 expression in tumor tissue was associated with longer overall survival in resectable and borderline resectable patients who underwent neoadjuvant FOLFIRINOX treatment (P = .024). Conclusion: Our findings suggest that CES2 expression and activity, by mediating the intra-tumoral activation of irinotecan, is a contributor to FOLFIRINOX sensitivity in pancreatic cancer and CES2 assessment may define a subset of patients likely to respond to irinotecan based therapy. Citation Format: Michela Capello, Minhee Lee, Hong Wang, Ingrid Babel, Matthew H. Katz, Jason B. Fleming, Anirban Maitra, Huamin Wang, Weihua Tian, Ayumu Taguchi, Samir M. Hanash. Identification of Carboxylesterase-2 as a determinant of response to irinotecan and neoadjuvant FOLFIRINOX therapy in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-121. doi:10.1158/1538-7445.AM2015-LB-121

Isoform-Specific Regulation of Mouse Carboxylesterase Expression and Activity by Prototypical Transcriptional Activators.

Nuclear receptors and transcription factors regulate the mRNA expression of many drug metabolizing enzymes, including the carboxylesterases (Ces). However, there are few data regarding whether these changes in mRNA expression result in alteration of protein levels or activity. In the present study, we sought to determine the isoform-specific regulation of hepatic Ces mRNA expression and activity following the administration of pharmacological activators of the constitutive androstane receptor (CAR), pregnane X receptor (PXR), and nuclear factor E2-related protein (Nrf2) to mice. The CAR activator 1,4-bis-[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) and PXR ligand pregnenolone-16a-carbonitrile (PCN) increased Ces mRNA expression of various Ces2 isoforms, whereas the Nrf2 activator butylated hydroxyanisole primarily reduced Ces3a mRNA expression and induced Ces1g mRNA. TCPOBOP and PCN increased Ces2 hydrolytic activity in an isoform-specific manner. Taken together, these data demonstrate that activation of CAR, PXR, and Nrf2 regulates not only Ces mRNA expression, but also isoform-specific activity.

Carboxylesterase 2 as a Determinant of Response to Irinotecan and Neoadjuvant FOLFIRINOX Therapy in Pancreatic Ductal Adenocarcinoma.

Serine hydrolases (SHs) are among the largest classes of enzymes in humans and play crucial role in many pathophysiological processes of cancer. We have undertaken a comprehensive proteomic analysis to assess the differential expression and cellular localization of SHs, which uncovered distinctive expression of Carboxylesterase 2 (CES2), the most efficient carboxyl esterase in activating the prodrug irinotecan into SN-38, in pancreatic ductal adenocarcinoma (PDAC). We therefore assessed the extent of heterogeneity in CES2 expression in PDAC and its potential relevance to irinotecan based therapy. CES2 expression in PDAC and paired nontumor tissues was evaluated by immunohistochemistry. CES2 activity was assessed by monitoring the hydrolysis of the substrate p-NPA and correlated with irinotecan IC50 values by means of Pearson's correlation. Kaplan-Meier and Cox regression analyses were applied to assess the association between overall survival and CES2 expression in patients who underwent neoadjuvant FOLFIRINOX treatment. All statistical tests were two-sided. Statistically significant overexpression of CES2, both at the mRNA and protein levels, was observed in PDAC compared with paired nontumor tissue (P < .001), with 48 of 118 (40.7%) tumors exhibiting high CES2 expression. CES2 activity in 11 PDAC cell lines was inversely correlated with irinotecan IC50 values (R = -0.68, P = .02). High CES2 expression in tumor tissue was associated with longer overall survival in resectable and borderline resectable patients who underwent neoadjuvant FOLFIRINOX treatment (hazard ratio = 0.14, 95% confidence interval = 0.04 to 0.51, P = .02). Our findings suggest that CES2 expression and activity, by mediating the intratumoral activation of irinotecan, is a contributor to FOLFIRINOX sensitivity in pancreatic cancer and CES2 assessment may define a subset of patients likely to respond to irinotecan based therapy.

Decreased carboxylesterases expression and hydrolytic activity in type 2 diabetic mice through Akt/mTOR/HIF-1α/Stra13 pathway

1. This study investigated the alteration of carboxylesterases in type 2 diabetes. We found that the carboxylesterase 1d (Ces1d) and carboxylesterase 1e (Ces1e) expression and the capacity of hydrolytic activity of liver and intestine decreased, whereas the Akt/mTOR/HIF-1α/ Stra13 (DEC1) signaling was activated in T2D mice. Consistently, high insulin could give rise to the same results in the high-glucose DMEM condition, which mimicked T2D, in primary mouse hepatocytes.2. Perifosine or rapamycin almost abolished the decrease of the Ces1d and Ces1e expression and the hydrolytic activity induced by the insulin in the primary mouse hepatocytes.3. The responsiveness of human hepatoma (HepG2) cells to high insulin in high-glucose condition was similar to that of primary mouse hepatocytes in terms of the altered expression of carboxylesterases.4. The knockdown of HIF-1α or DEC1 with shRNA construct abrogated the decrease of the CES1 and CES2 expression induced by the insulin in high glucose condition in HepG2 cells.5. Taken together, the decreased carboxylesterases expression and hydrolytic activity in T2D mice are through the Akt/mTOR/HIF-1α/Stra13 (DEC1) pathway.

Phospho-NSAIDs have enhanced efficacy in mice lacking plasma carboxylesterase: implications for their clinical pharmacology.

The purpose of the study was to evaluate the metabolism, pharmacokinetics and efficacy of phospho-NSAIDs in Ces1c-knockout mice. Hydrolysis of phospho-NSAIDs by Ces1c was investigated using Ces1c-overexpressing cells. The rate of phospho-NSAID hydrolysis was compared between wild-type, Ces1c+/- and Ces1c-/- mouse plasma in vitro, and the effect of plasma Ces1c on the cytotoxicity of phospho-NSAIDs was evaluated. Pharmacokinetics of phospho-sulindac was examined in wild-type and Ces1c-/- mice. The impact of Ces1c on the efficacy of phospho-sulindac was investigated using lung and pancreatic cancer models in vivo. Phospho-NSAIDs were extensively hydrolyzed in Ces1c-overexpressing cells. Phospho-NSAID hydrolysis in wild-type mouse plasma was 6-530-fold higher than that in the plasma of Ces1c-/- mice. Ces1c-expressing wild-type mouse serum attenuated the in vitro cytotoxicity of phospho-NSAIDs towards cancer cells. Pharmacokinetic studies of phospho-sulindac using wild-type and Ces1c-/- mice demonstrated 2-fold less inactivation of phospho-sulindac in the latter. Phospho-sulindac was 2-fold more efficacious in inhibiting the growth of lung and pancreatic carcinoma in Ces1c -/- mice, as compared to wild-type mice. Our results indicate that intact phospho-NSAIDs are the pharmacologically active entities and phospho-NSAIDs are expected to be more efficacious in humans than in rodents due to their differential expression of carboxylesterases.