Carboxylesterases expressed in human colon tumor tissue and their role in CPT-11 hydrolysis.

PURPOSE: The purpose is to develop new analytical methods to study the expression profile of CPT-11 carboxylesterases and topoisomerase I in colon tumor samples and understand the impact of their expression on CPT-11 metabolism in chemotherapy. EXPERIMENTAL DESIGN: We investigated 24 colon tumors for expression of carboxylesterases CES1A1, CES2, CES3, hBr-3, and topoisomerase I genes by real-time PCR and correlated the gene expression with activity assays. The relative abundance of the carboxylesterase isoenzymes and topoisomerase I genes was determined by real-time PCR. Activity assays performed on colon tumor extracts included CPT-11 hydrolase, 4-methylumbelliferyl acetate hydrolase, and topoisomerase I activity assays. Additionally, nondenaturing activity gel electrophoresis with activity staining showed the distribution of carboxylesterases. RESULTS: We detect the expression of CES1A1, CES2, and CES3 carboxylesterase genes in human colon tumors. We were unable to detect the hBr-3 (also called hCE-3) in human liver, colon, or brain. We find large interindividual variation, >/=150-fold, for both CES1A1 and CES3 genes, 23-fold for CES2, and 66-fold for topoisomerase I. Only CES2 gene expression correlated with the carboxylesterase activity assays (P < 0.01) with CPT-11 and 4-methylumbelliferyl acetate as substrates. Nondenaturing activity gel electrophoresis showed that CES2 was the most predominant activity. Topoisomerase I gene expression significantly correlated with topoisomerase I activity (P < 0.01) in the colon tumors, but interindividual variation was very high. CONCLUSIONS: We conclude that CES2 is the most abundant carboxylesterase in colon tumors that is responsible for CPT-11 hydrolysis. This pilot study reinforces the hypothesis that there is a large interindividual variation in expression of carboxylesterases that may contribute to variation in therapeutic outcome and/or toxicity of CPT-11 therapy for colon cancer.

Kinetic and cellular characterization of novel inhibitors of S-nitrosoglutathione reductase.

S-Nitrosoglutathione reductase (GSNOR) is an alcohol dehydrogenase involved in the regulation of S-nitrosothiols (SNOs) in vivo. Knock-out studies in mice have shown that GSNOR regulates the smooth muscle tone in airways and the function of beta-adrenergic receptors in lungs and heart. GSNOR has emerged as a target for the development of therapeutic approaches for treating lung and cardiovascular diseases. We report three compounds that exclude GSNOR substrate, S-nitrosoglutathione (GSNO) from its binding site in GSNOR and cause an accumulation of SNOs inside the cells. The new inhibitors selectively inhibit GSNOR among the alcohol dehydrogenases. Using the inhibitors, we demonstrate that GSNOR limits nitric oxide-mediated suppression of NF-kappaB and activation of soluble guanylyl cyclase. Our findings reveal GSNOR inhibitors to be novel tools for regulating nitric oxide bioactivity and assessing the role of SNOs in vivo.

Expression and characterization of a human carboxylesterase 2 splice variant.

CPT-11 [7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin or Irinotecan] is a carbamate prodrug that is activated in vivo by carboxylesterase (CES)-2 to SN-38 (7-ethyl-10-hydroxycamptothecin), a potent topoisomerase I inhibitor. There is high interindividual variation when CPT-11 is used in the treatment of colorectal cancer. Several splice variants of CES2 are reported in the expressed sequence tag database. Real-time polymerase chain reaction was used to determine the abundance of the CES2 and splice variant of human carboxylesterase 2 (CES2Delta(458-473)) transcripts in 10 paired samples of human tumor and normal colon tissue. The results showed that the CES2Delta(458-473) transcript accounts for an average of 6% of total CES2 transcripts in colon tissue, and there is large interindividual variation in CES2 expression in both tumor and normal colon samples. The carboxylesterase activity of the colon samples was determined by 4-methylumbelliferyl acetate hydrolysis assays and nondenaturing polyacrylamide gel electrophoresis followed by activity staining. Significant, positive correlations were found between CES2 expression levels and both measures of carboxylesterase activity. We cloned and expressed the CES2Delta(458-473) protein in Sf9 insect cells. The purification profiles and preliminary characterization of the CES2Delta(458-473) protein indicated that the expressed protein is folded and glycosylated like CES2. However, in vitro assays show that the CES2Delta(458-473) protein lacks 4-methylumbelliferyl acetate and irinotecan hydrolase activities. In conclusion, we found that the CES2Delta(458-473) protein is an inactive splice variant of CES2 and that its transcript is spliced at a relatively constant rate in tumor and normal colon tissue.

Structure-function relationships in human glutathione-dependent formaldehyde dehydrogenase. Role of Glu-67 and Arg-368 in the catalytic mechanism.

The active-site zinc in human glutathione-dependent formaldehyde dehydrogenase (FDH) undergoes coenzyme-induced displacement and transient coordination to a highly conserved glutamate residue (Glu-67) during the catalytic cycle. The role of this transient coordination of the active-site zinc to Glu-67 in the FDH catalytic cycle and the associated coenzyme interactions were investigated by studying enzymes in which Glu-67 and Arg-368 were substituted with Leu. Structures of FDH.adenosine 5'-diphosphate ribose (ADP-ribose) and E67L.NAD(H) binary complexes were determined. Steady-state kinetics, isotope effects, and presteady-state analysis of the E67L enzyme show that Glu-67 is critical for capturing the substrates for catalysis. The catalytic efficiency (V/K(m)) of the E67L enzyme in reactions involving S-nitrosoglutathione (GSNO), S-hydroxymethylglutathione (HMGSH) and 12-hydroxydodecanoic acid (12-HDDA) were 25 000-, 3000-, and 180-fold lower, respectively, than for the wild-type enzyme. The large decrease in the efficiency of capturing GSNO and HMGSH by the E67L enzyme results mainly because of the impaired binding of these substrates to the mutant enzyme. In the case of 12-HDDA, a decrease in the rate of hydride transfer is the major factor responsible for the reduction in the efficiency of its capture for catalysis by the E67L enzyme. Binding of the coenzyme is not affected by the Glu-67 substitution. A partial displacement of the active-site zinc in the FDH.ADP-ribose binary complex indicates that the disruption of the interaction between Glu-67 and Arg-368 is involved in the displacement of active-site zinc. Kinetic studies with the R368L enzyme show that the predominant role of Arg-368 is in the binding of the coenzyme. An isomerization of the ternary complex before hydride transfer is detected in the kinetic pathway of HMGSH. Steps involved in the binding of the coenzyme to the FDH active site are also discerned from the unique conformation of the coenzyme in one of the subunits of the E67L.NAD(H) binary complex.

Maternal separation and handling affects cocaine self-administration in both the treated pups as adults and the dams.

Repeated maternal separation of pups from dams is often used as an early life stressor that causes profound neurochemical and behavioral changes in the pups that persist into adulthood. The effects of maternal separation on both the dams and the treated pups as adults on cocaine self-administration were examined using four separation conditions: 15- or 180-min separation (MS15 and MS180), brief handling without separation (MS0), and a nonhandled group (NH). The separations and handling occurred daily on postnatal days 2 to 15. The acquisition of cocaine self-administration (0.0625-1.0 mg/kg/infusion) was evaluated in the treated pups as adults. The MS180 group acquired cocaine self-administration at the lowest dose tested (0.0625 mg/kg/infusion), whereas the MS15s did not respond for cocaine at rates greater than that seen with saline administration. The NH group received the greatest number of infusions and intake at the highest doses. After self-administration, no differences were observed between groups in activity of two liver carboxylesterases involved in the inactivation of cocaine, ES10 and ES4. Maternal separation affected cocaine self-administration in the dams as well. Although there was an overall significant affect of treatment on cocaine self-administration, the length of separation (15 or 180 min) did not affect cocaine self-administration on the dams. The MS0 dams averaged a greater number of infusions per session than NH group during the 1st week of acquisition. These data suggest that in addition to the profound changes that occur in pups as result of maternal separation, the dams are also susceptible to alterations in behaviors.

Identification of microsomal rat liver carboxylesterases and their activity with retinyl palmitate.

Retinyl esters are a major endogenous storage source of vitamin A in vertebrates and their hydrolysis to retinol is a key step in the regulation of the supply of retinoids to all tissues. Some members of nonspecific carboxylesterase family (EC 3.1.1.1) have been shown to hydrolyze retinyl esters. However, the number of different isoenzymes that are expressed in the liver and their retinyl palmitate hydrolase activity is not known. Six different carboxylesterases were identified and purified from rat liver microsomal extracts. Each isoenzyme was identified by mass spectrometry of its tryptic peptides. In addition to previously characterized rat liver carboxylesterases ES10, ES4, ES3, the protein products for two cloned genes, AB010635 and D50580 (GenBank accession numbers), were also identified. The sixth isoenzyme was a novel carboxylesterase and its complete cDNA was cloned and sequenced (AY034877). Three isoenzymes, ES10, ES4 and ES3, account for more than 95% of rat liver microsomal carboxylesterase activity. They obey Michaelis-Menten kinetics for hydrolysis of retinyl palmitate with Km values of about 1 micro m and specific activities between 3 and 8 nmol.min-1.mg-1 protein. D50580 and AY034877 also hydrolyzed retinyl palmitate. Gene-specific oligonucleotide probing of multiple-tissue Northern blot indicates differential expression in various tissues. Multiple genes are highly expressed in liver and small intestine, important tissues for retinoid metabolism. The level of expression of any one of the six different carboxylesterase isoenzymes will regulate the metabolism of retinyl palmitate in specific rat cells and tissues.

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.

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.

Methylphenidate is stereoselectively hydrolyzed by human carboxylesterase CES1A1.

Methylphenidate is an important stimulant prescribed to treat attention-deficit hyperactivity disorder. It has two chiral centers, but most current commercial formulations consist of the racemic mixture of the threo pair of methylphenidate isomers (d-, l-threo-methylphenidate). The d-isomer is the pharmacologically active component. Numerous studies reported that oral administration of the methylphenidate racemate undergoes first-pass, stereoselective clearance in humans with l-methylphenidate being eliminated faster than d-methylphenidate. Accordingly, the kinetics of hydrolysis of individual enantiomers by purified native and recombinant human liver carboxylesterases CES1A1 and CES2 and a colon isoenzyme CES3 were examined with a liquid chromatography/mass spectrometry assay. The expression of CES1A1, CES2, and CES3 in Sf9 cells and the methods for purification of the three isoenzymes are reported. CES1A1 has a high catalytic efficiency for both d- and l-enantiomers of methylphenidate. No catalytic activity was detected with CES2 and CES3 for either enantiomer. The catalytic efficiency of CES1A1 for l-methylphenidate (k(cat)/K(m) = 7.7 mM(-1) min(-1)) is greater than that of d-methylphenidate (k(cat)/K(m) = 1.3-2.1 mM(-1) min(-1)). Hence, the catalytic efficiency of CES1A1 for methylphenidate enantiomers agrees with stereoselective clearance of methylphenidate reported in human subjects. Both enantiomers of methylphenidate can be fit into the three-dimensional model of CES1A1 to form productive complexes in the active site. We conclude that CES1A1 is the major enzyme responsible for the first-pass, stereoselective metabolism of methylphenidate.