Hydrolysis of irinotecan and its oxidative metabolites, 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxycamptothecin and 7-ethyl-10-[4-(1-piperidino)-1-amino]-carbonyloxycamptothecin, by human carboxylesterases CES1A1, CES2, and a newly expressed carboxylesterase isoenzyme, CES3.
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Carboxylesterases metabolize ester, thioester, carbamate, and amide compounds to more soluble acid, alcohol, and amine products. They belong to a multigene family with about 50% sequence identity between classes. CES1A1 and CES2 are the most studied human isoenzymes from class 1 and 2, respectively. In this study, we report the cloning and expression of a new human isoenzyme, CES3, that belongs to class 3. The purified recombinant CES3 protein has carboxylesterase activity. Carboxylesterases metabolize the carbamate prodrug 7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin (CPT-11; irinotecan) to its active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38), a potent topoisomerase I inhibitor. CYP3A4 oxidizes CPT-11 to two major oxidative metabolites, 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino] carbonyloxycamptothecin (APC) and 7-ethyl-10-[4-(1-piperidino)-1-amino]-carbonyloxycamptothecin (NPC). In this study, we investigate whether these oxidative metabolites, NPC and APC, can be metabolized to SN-38 by purified human carboxylesterases, CES1A1, CES2, and CES3. We find that CPT-11, APC, and NPC can all be metabolized by carboxylesterases to SN-38. CES2 has the highest catalytic activity of 0.012 min(-1) microM(-1) among the three carboxylesterases studied for hydrolysis of CPT-11. NPC was an equally good substrate of CES2 in comparison to CPT-11, with a catalytic efficiency of 0.005 min(-1) microM(-1). APC was a very poor substrate for all three isoenzymes, exhibiting a catalytic activity of 0.015 x 10(-3) min(-1) microM(-1) for CES2. Catalytic efficiency of CES3 for CPT-11 hydrolysis was 20- to 2000-fold less than that of CES1A1 and CES2. The relative activity of the three isoenzymes was CES2 > CES1A1 >> CES3, for all three substrates. (+info)
Carboxyl ester lipase cofractionates with scavenger receptor BI in hepatocyte lipid rafts and enhances selective uptake and hydrolysis of cholesteryl esters from HDL3.
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Cholesteryl esters are selectively removed from high density lipoproteins by hepatocytes and steroidogenic cells through a process mediated by scavenger receptor BI. In the liver this cholesterol is secreted into bile, primarily as free cholesterol. Previous work showed that carboxyl ester lipase enhanced selective uptake of cholesteryl ether from high density lipoprotein by an unknown mechanism. Experiments were performed to determine whether carboxyl ester lipase plays a role in scavenger receptor BI-mediated selective uptake. When added to cultures of HepG2 cells, carboxyl ester lipase cofractionated with scavenger receptor BI and [(3)H]cholesteryl ether-labeled high density lipoprotein in lipid raft fractions of cell homogenates. Confocal microscopy of immunostained carboxyl ester lipase and scavenger receptor BI showed a close association of these proteins in HepG2 cells. The enzyme and receptor also cofractionated from homogenates of mouse liver using two different fractionation methods. Antibodies that block scavenger receptor BI function prevented carboxyl ester lipase stimulation of selective uptake in primary hepatocytes from carboxyl ester lipase knockout mice. Heparin blockage of cell-surface proteoglycans also prevented carboxyl ester lipase stimulation of cholesteryl ester uptake by HepG2 cells. Inhibition of carboxyl ester lipase activity in HepG2 cells reduced hydrolysis of high density lipoprotein-cholesteryl esters approximately 40%. In vivo, hydrolysis was similarly reduced in lipid rafts from the livers of carboxyl ester lipase-null mice compared with control animals. Primary hepatocytes from these mice yielded similar results. The data suggest that carboxyl ester lipase plays a physiological role in hepatic selective uptake and metabolism of high density lipoprotein cholesteryl esters by direct and indirect interactions with the scavenger receptor BI pathway. (+info)
Cellular parameters predictive of the clinical response of colorectal cancers to irinotecan. A preliminary study.
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BACKGROUND: We have examined, in this study, the feasibility of determining cellular factors contributing to irinotecan activity in colorectal cancers. Irinotecan is a camptothecin derivative requiting carboxylesterase activation to SN-38, which interacts with its target enzyme, topoisomerase I. MATERIALS AND METHODS: In 9 surgical or biopsy samples of colorectal tumours and corresponding normal tissue, kept in a tumour bank, we evaluated topoisomerase I expression and activity, respectively by Western blotting and DNA relaxation assay, carboxylesterase activity using two different substrates and p53 status by immunohistochenistry. RESULTS: Topoisomerase I expression and activity were significantly correlated, as were the two types of determinations for carboxylesterase activity. Topoisomerase I was significantly more active in tumours than in corresponding normal tissue. The three samples presenting the highest topoisomerase I expression and activity originated from the patients who responded to irinotecan treatment. No such features were apparent for carboxylesterase activity and p53 staining. CONCLUSION: Topoisomerase I expression appeared as the parameter most likely to predict response to irinotecan therapy in the clinical setting. (+info)
Dietary antibiotic growth promoters enhance the bioavailability of alpha-tocopheryl acetate in broilers by altering lipid absorption.
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The influence of intestinal microbial bile salt deconjugation on absorption of fatty acids and alpha- and gamma-tocopherol was investigated in a trial with Ross 208 broilers. Birds (n = 1600) were assigned to 4 dietary treatments: no supplementation or supplementation of antibiotics (salinomycin, 40 mg/kg feed and avilamycin, 10 mg/kg feed), and inclusion of either animal fat (10 g/100 g feed) or soybean oil (10 g/100 g feed) in the diet. At d 7, 14, 21, and 35 of age, the intestinal number of the bile salt hydrolase-active bacteria Clostridium perfringens, the concentration of conjugated and unconjugated bile salts, the ileal absorption of fatty acids and tocopherols, and the blood plasma concentrations of tocopherols were measured. All variables were significantly influenced by bird age. C. perfringens counts were lower and bile salt concentrations were greater in birds fed soybean oil. The supplementation of antibiotics reduced the numbers of C. perfringens in the small intestine and reduced the concentration of unconjugated bile salts. The ileal absorption of fatty acids and alpha-tocopherol, as well as the plasma concentration of alpha-tocopherol, was greater in birds fed antibiotics. The absorption and plasma concentration of gamma-tocopherol were not influenced by antibiotics. Unlike gamma-tocopherol, which is present solely as the free alcohol, the major proportion of dietary alpha-tocopherol is present as alpha-tocopheryl acetate, which requires a bile salt-dependent enzymatic hydrolysis before absorption. In conclusion, proper digestion of lipid-soluble compounds is highly dependent on an adequate concentration of bile salts in the small intestine to provide proper lipid emulsification and activation of lipolytic enzymes. (+info)
Carboxylesterase 3 (EC 3.1.1.1) is a major adipocyte lipase.
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Hydrolysis of triglycerides is central to energy homeostasis in white adipose tissue (WAT). Hormone-sensitive lipase (HSL) was previously felt to mediate all lipolysis in WAT. Surprisingly, HSL-deficient mice show active HSL-independent lipolysis, suggesting that other lipase(s) also mediate triglyceride hydrolysis. To clarify this, we used functional proteomics to detect non-HSL lipase(s) in mouse WAT. After cell fractionation of intraabdominal WAT, most non-HSL neutral lipase activity is localized in the 100,000 x g infranatant and fat cake fractions. By oleic acid-linked agarose chromatography of infranatant followed by elution in a 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid gradient, we identified two peaks of esterase activity using p-nitrophenyl butyrate as a substrate. One of the peaks contained most of the lipase activity. In the corresponding fractions, gel permeation chromatography and SDS-PAGE, followed by tandem mass spectrometric analysis of excised Coomassie Blue-stained peptides, revealed carboxylesterase 3 (triacylglycerol hydrolase (TGH); EC 3.1.1.1). TGH is also the principle lipase of WAT fat cake extracts. Partially purified WAT TGH had lipase activity as well as lesser but detectable neutral cholesteryl ester hydrolase activity. Western blotting of subcellular fractions of WAT and confocal microscopy of fibroblasts following in vitro adipocytic differentiation are consistent with a distribution of TGH to endoplasmic reticulum, cytosol, and the lipid droplet. TGH is responsible for a major part of non-HSL lipase activity in WAT in vitro and may mediate some or all HSL-independent lipolysis in adipocytes. (+info)
Identification of the cytosolic carboxylesterase catalyzing the 5'-deoxy-5-fluorocytidine formation from capecitabine in human liver.
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Capecitabine, a prodrug of 5-fluorouracil, is first metabolized to 5'-deoxy-5-fluorocytidine (5'-DFCR) by carboxylesterase (CES), which is mainly expressed in microsomes. Recently, we clarified that 5'-DFCR formation was catalyzed by the enzyme in cytosol as well as microsomes in human liver. In the present study, the cytosolic enzyme involved in 5'-DFCR formation from capecitabine was identified. This enzyme was purified in the cytosolic preparation by ammonium sulfate precipitation, Sephacryl S-300 gel filtration, Mono P chromatofocusing, and Superdex 200 gel filtration. The purified enzyme was identified by the amino acid sequence analysis to be CES1A1 or a CES1A1 precursor. Based on the result of the N-terminal amino acid sequence analysis, the purified enzyme has no putative signal peptide, indicating that it was CES1A1. The apparent Km and Vmax values of 5'-DFCR formation were 19.2 mM and 88.3 nmol/min/mg protein, respectively. The 5'-DFCR formation catalyzed by the purified enzyme was inhibited by both diisopropylfluorophosphate and bis(p-nitrophenyl)phosphate in a concentration-dependent manner. 7-Ethyl-10-hydroxycamptothecin (SN-38) formation from irinotecan also occurred in the purified enzyme, cytosol, and microsomes. In conclusion, the cytosolic enzyme involved in 5'-DFCR formation from capecitabine would be CES1A1. It is suggested that the cytosolic CES has significant hydrolysis activity and plays an important role as the microsomal CES in drug metabolism. It is worthy to investigate the metabolic enzyme in cytosol involved in the activation of ester-type prodrugs such as capecitabine. (+info)
Characterization of inhibitors of specific carboxylesterases: development of carboxylesterase inhibitors for translational application.
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Carboxylesterases, expressed at high levels in human liver and intestine, are thought to detoxify xenobiotics. The anticancer prodrug 7-ethyl-10-[4-1-piperidino)-1-piperidino]carbonyloxycamptothecin (CPT-11) is also metabolized by carboxylesterases to produce the active drug 7-ethyl-10-hydroxycamptothecin. Activation of CPT-11 by human intestinal carboxylesterase (hiCE) in the human intestine may contribute to delayed onset diarrhea, a dose-limiting side effect of this drug. The goal of this study was to develop small molecule inhibitors selective for hiCE to circumvent or treat the toxic side effects of CPT-11. A secondary goal was to develop molecules that specifically inhibit activation of CPT-11 by a rabbit liver carboxylesterase (rCE). rCE is the most efficient CPT-11-activating enzyme thus far identified, and this enzyme is being developed for viral-directed enzyme prodrug therapy applications. Based on in vitro assays with partially purified hiCE and rCE proteins and on growth inhibition assays using U373MG human glioma cells transfected to express hiCE or rCE (U373pIREShiCE or U373pIRESrCE), we identified specific inhibitors of each enzyme. Lead compounds are derivatives of nitrophenol having 4-(furan-2-carbonyl)-piperazine-1-carboxylic acid or 4-[(4-chlorophenyl)-phenylmethyl]-piperazine-1-carboxylic acid substitutions in the p position. Kinetic analysis of each compound for hiCE compared with rCE showed that the Ki values of the most selective of these inhibitors differed by 6- to 10-fold. In growth inhibition assays, nontoxic, low micromolar concentrations of these inhibitors increased the EC50 of CPT-11 for U373pIREShiCE or U373pIRESrCE cells by 13- to >1,500-fold. The four compounds characterized in this study will serve as lead compounds for a series of inhibitors to be constructed using a combinatorial approach. (+info)
The use of transient chromatin immunoprecipitation assays to test models for E2F1-specific transcriptional activation.
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The E2F family of transcription factors regulates the expression of genes involved in cell cycle progression, DNA synthesis, repair, and recombination, and a variety of other cellular processes. Although E2F proteins are often redundant in function, specificity of binding and activity can occur. For example, E2F1, but not other E2F family members, was shown previously to bind the murine carboxylesterase promoter in chromatin immunoprecipitation studies. This promoter region lacks a consensus E2F binding site, suggesting that E2F1 may be recruited to the DNA in a unique fashion. To further investigate this E2F1-specific binding, we have employed a "transient chromatin immunoprecipitation" approach. Using various deletions and mutations of the promoter region, we localized the E2F1-specific binding site and demonstrated that it was required for E2F1-mediated transcription of the carboxylesterase promoter. The identified site was similar to the 8-bp consensus E2F site but differed from the consensus at a crucial position. To address whether E2F1 directly bound to this non-consensus site, we demonstrated that the DNA binding domain of E2F1 is necessary for E2F1-mediated activation of the carboxylesterase promoter. Interestingly, a "UP" mutation of the site, making it more similar to the consensus element, did not improve the ability of E2F1 to bind the promoter. Rather, E2F1 could no longer bind to the carboxylesterase promoter that contained the consensus E2F site. We propose a model in which E2F1-specific regulation of the carboxylesterase promoter requires both E2F1/DNA interactions and protein-protein interaction between E2F1 and a factor that binds adjacent to the non-consensus site. (+info)