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Biochemical Pharmacology (v.71, #3)
Expression of γ-glutamyltransferase in cancer cells and its significance in drug resistance by A. Pompella; V. De Tata; A. Paolicchi; F. Zunino (pp. 231-238).
The expression of γ-glutamyltransferase (GGT), a cell surface enzyme involved in cellular glutathione homeostasis, is often significantly increased in human tumors, and its role in tumor progression, invasion and drug resistance has been repeatedly suggested. As GGT participates in the metabolism of cellular glutathione, its activity has been mostly regarded as a factor in reconsitution of cellular antioxidant/antitoxic defences. On this basis, an involvement of GGT expression in resistance of cancer cells to cytotoxic drugs (in particular, cisplatin and other electrophilic agents) has been envisaged. Mechanistic aspects of GGT involvement in antitumor pharmacology deserve however further investigations. Recent evidence points to a more complex role of GGT in modulation of redox equilibria, with effects acting both intracellularly and in the extracellular microenvironment. Indications exist that the protective effects of GGT may be independent of intracellular glutathione, and derive rather from processes taking place at extracellular level and involving reactions of electrophilic drugs with thiol metabolites originating from GGT-mediated cleavage of extracellular glutathione. Although expression of GGT cannot be regarded as a general mechanism of resistance, the involvement of this enzyme in modulation of redox metabolism is expected to have impact in cellular response to several cytotoxic agents. The present commentary is a survey of data concerning the role of GGT in tumor cell biology and the mechanisms of its potential involvement in tumor drug resistance.
Keywords: Gamma-glutamyltransferase expression; Glutathione; Cysteinyl-glycine; Human neoplasia; Antitumor drug resistance
Role of GADD34 in modulation of cisplatin cytotoxicity by Melissa L. Fishel; Cara A. Rabik; Wasim K. Bleibel; Xinmin Li; Robert C. Moschel; M. Eileen Dolan (pp. 239-247).
Cisplatin and carboplatin are widely used clinical chemotherapeutic agents, especially against testicular, ovarian, and head and neck cancers. O6-Benzylguanine (BG) has been shown to result in enhanced cytotoxicity, apoptosis, and DNA platination when used in conjunction with cisplatin and carboplatin in head and neck cancer cell lines. Microarray expression data indicated overexpression of 19 genes and underexpression of 22 genes specific to treatment with the combination of BG±cisplatin compared to cisplatin alone treatment in SQ20b head and neck cancer cells ( p<0.05) using the Affymetrix HG-U133A GeneChip®. Among the overexpressed probe sets were genes involved in DNA damage and apoptosis, including GADD34, DDIT4, ATF4, and PTHLH. A similarly structured analog, 9-CH3-BG, does not enhance cisplatin-induced cytotoxicity or apoptosis nor is there enhanced expression of GADD34 in cisplatin or 9-CH3-BG±cisplatin-treated cells compared to control cells. Analysis of cells exposed to 9-CH3-BG±cisplatin allowed us to focus our array list on 32 probe sets specific to BG+cisplatin versus cisplatin, ruling out differentially expressed probe sets common to 9-CH3-BG+cisplatin versus cisplatin. Similarly, 14 probe sets were specific to BG±cisplatin versus BG, ruling out differentially expressed probe sets common to 9-CH3-BG±cisplatin versus 9-CH3-BG. Quantitative real-time PCR demonstrated a dose dependent increase in GADD34 expression in cells exposed to BG±cisplatin with levels approximately >2-fold for cells exposed to BG+cisplatin compared to cisplatin alone. Levels of GADD34 transcripts were determined with both cisplatin and BG+cisplatin at several different time points concomitant with and following drug treatment. At all timepoints, GADD34 transcript levels are approximately two-fold elevated in cells treated with BG+cisplatin compared to cisplatin alone. Furthermore, significant changes in GADD34 expression levels in SQ20b, SCC35, and SCC61 cells, with approximately three-fold, two-fold, and 3.5-fold increases in expression, respectively, upon treatment with BG±cisplatin compared with control. Elucidation of these molecular pathways will aid in our goal of synthesizing more powerful modulators to increase efficacy of platinum agents.
Keywords: Abbreviations; BG; O; 6; -benzylguanine; 9-CH; 3; -BG; O; 6; -benzyl-9-methylguanine; qRT-PCR; quantitative real time polymerase chain reaction; GADD; growth arrest and DNA damage inducible; ATF4; activating transcription factor 4; XBP1; X-box binding protein 1; ER; endoplasmic reticulum; eIF2α; eukaryotic initiation factor 2αMicroarray; Cisplatin; Modulation; Chemotherapy; GADD34
Enhancing the antiproliferative effect of topoisomerase II inhibitors using a polypeptide inhibitor of c-Myc by Gene L. Bidwell III; Drazen Raucher (pp. 248-256).
Topoisomerase II inhibitors are widely used in cancer chemotherapy. However, their use is limited by severe adverse effects to normal tissues, including cardiotoxicity. One approach to reduce the cytotoxicity in normal tissues may be to sensitize cancer cells to the toxicity of these agents, allowing them to be administered in a lower and safer dose. A hallmark of many types of cancer is overexpression of c-Myc, and a molecule which targets c-Myc will affect the cancer cells more significantly than the normal tissues. This report demonstrates that pretreatment of cells with a polypeptide, which inhibits c-Myc transcriptional function causes cells to be more susceptible to the topoisomerase II inhibitors doxorubicin and etoposide. Inhibition of c-Myc and Max dimerization by this polypeptide leads to as much as a 2-fold reduction in the doxorubicin and etoposide IC50 in three different cell lines tested. Furthermore, the c-Myc inhibitor affects the cell cycle distribution of MCF-7 breast cancer cells by enhancing the G0/G1 accumulation induced by doxorubicin and etoposide. We have shown that this effect is not due to enhanced drug accumulation or inhibited drug efflux. Rather, it is likely due to the transcriptional consequences of c-Myc inhibition, specifically reduction in the levels of the polyamine synthesizing enzyme ornithine decarboxylase. In summary, our results suggest that polypeptides, which inhibit c-Myc transcriptional function, may prove to be a useful tool in combination therapy with topoisomerase II inhibiting drugs.
Keywords: Abbreviations; aa; amino acid; BSA; bovine serum albumin; BrdU; 5-bromo-2′-deoxyuridine; DFMO; α-1,2 difluoromethylornithine; ELP; elastin-like polypeptide; H1; helix 1 of the c-Myc helix-loop-helix domain; ODC; ornithine decarboxylase; Pen; penetratin peptide; PBS; phosphate-buffered physiological saline; RT-PCR; reverse transcriptase polymerase chain reaction; T; t; transition temperatureDoxorubicin; Etoposide; Drug delivery; Elastin-like polypeptide; c-Myc; Topoisomerase II inhibitor
Differential toxicity of antimonial compounds and their effects on glutathione homeostasis in a human leukaemia monocyte cell line by Susan Wyllie; Alan H. Fairlamb (pp. 257-267).
Trivalent antimonial compounds (SbIII), originally used in the treatment of leishmaniasis, are now being proposed as a novel therapy for acute promyelocytic leukaemia (APL). Here, we examine the effects of SbIII and pentavalent antimonial drugs (SbV) on glutathione homeostasis, oxidative stress and apoptosis in the human leukaemia monocyte cell line, THP-1. Although growth of THP-1 macrophages is unaffected by SbV, macrophages are extremely sensitive to SbIII. On exposure to SbIII, intracellular free glutathione (GSH) levels in macrophages decrease linearly by 50% over 4h, associated with efflux of both GSH and accumulation of intracellular glutathione disulphide (GSSG). Together these effects increase the redox potential of the GSSG/GSH couple from −282 to −225mV. SbIII-induced GSH efflux from THP-1 macrophages is accompanied by the concomitant efflux of SbIII at a constant molar ratio of 3 (GSH) to 1 (SbIII), respectively. SbIII directly inhibits glutathione reductase activity in macrophages, significantly retarding the regeneration of GSH from GSSG, following diamide oxidation. SbIII-treated THP-1 macrophages go on to exhibit elevated levels of reactive oxygen species and show the early signs of apoptosis. The absence of these effects in SbV-treated THP-1 cells suggests that macrophages do not efficiently reduce SbV to SbIII. Collectively, these findings suggest that SbIII seriously compromises thiol homeostasis in THP-1 macrophages and that this may be an early defining event in the mode of action of antimonials against leukaemia cells.
Keywords: Abbreviations; 7-AAD; 7-aminoactinomycin D; H; 2; DCFDA; dichlorodihydrofluorescein diacetate; PMA; phorbol 12-myristate 13-acetate; PtdSer; phosphatidylserine; ROS; reactive oxygen species; Sb; III; trivalent antimony; Sb; V; pentavalent antimony; TCEP; tris(2-carboxyethyl) phosphineAntimonials; Macrophage; Glutathione; Oxidative stress; Apoptosis; Leukaemia
Peptide-bond modified glutathione conjugate analogs modulate GSTÏ€ function in GSH-conjugation, drug sensitivity and JNK signaling by Danny Burg; Joey Riepsaame; Chantal Pont; Gerard Mulder; Bob van de Water (pp. 268-277).
Glutathione S-transferase π (GST, E.C.2.5.1.18) overexpression contributes to resistance of cancer cells towards cytostatic drugs. Furthermore, GSTπ is involved in the cellular stress response through inhibition of Jun N-terminal-kinase (JNK), a process that can be modulated by GST inhibitors. GSH conjugates are potent GST inhibitors, but are sensitive towards γ-glutamyltranspeptidase (γGT)-mediated breakdown. In search for new peptidase stable GST inhibitors we employed the following strategy: (1) selection of a suitable (GST inhibiting) peptide-bond isostere from a series of previously synthesized γGT stabilized GSH-analogs. (2) The use of this peptidomimetic strategy to prepare a GSTπ selective inhibitor. Two γGT stable GSH conjugate analogs inhibited human GSTs, although non-selectively. One of these, a urethane-type peptide-bond is well accepted by GSTs and we selected this modification for the development of a γGT stable, GSTπ selective inhibitor, UrPhg-Et2. This compound displayed selectivity for GSTπ compared to α and μ class enzymes. Furthermore, the inhibitor reversed GSTπ-mediated drug resistance (MDR) in breast tumor cells. In addition, short-term exposure of cells to UrPhg-Et2 led to GSTπ oligomerization and JNK activation, suggesting that it activates the JNK-cJun signaling module through GSTπ dissociation. Altogether, we show the successful use of peptidomimetic glutathione conjugate analogs as GST inhibitors and MDR-modifiers. As many MDR related enzymes, such as MRP1, glyoxalase 1 and DNA-pk are also inhibited by GSH conjugates, these peptidomimetic compounds can be used as scaffolds for the development of multi-target MDR drugs.
Keywords: Glutathione; S; -transferase; Peptidomimetic; Multidrug resistance; cJun N-terminal kinase; Glutathione conjugate; Cytostatics
Block of hERG channel by ziprasidone: Biophysical properties and molecular determinants by Zhi Su; Jun Chen; Ruth L. Martin; Jeffrey S. McDermott; Bryan F. Cox; Murali Gopalakrishnan; Gary A. Gintant (pp. 278-286).
Ziprasidone, an antipsychotic agent, delays cardiac repolarization and, thus, prolongs the QT interval of the cardiac ECG. In this study, we examined the biophysical properties and the molecular determinants of the ziprasidone block of wild-type hERG potassium channels stably expressed in HEK-293 cells or wild-type and mutant hERG channels expressed in Xenopus oocytes. In stably transfected HEK-293 cells, ziprasidone blocked wild-type hERG current in a voltage- and concentration-dependent manner (IC50=120nM, 0mV, 37°C). Ziprasidone showed minimal tonic block of hERG current estimated during a depolarizing voltage (−20 or +30mV) or evaluated by the envelope of tails test (+30mV). Rate of the block onset was rapid, but not significantly affected by test potentials ranging from −20 to +30mV (time constant ( τ)=114±14ms at +30mV). The time constant of the slow component of hERG current deactivation (at −50mV) was significantly increased by ziprasidone ( τ=1776±90 versus 1008±71ms, P<0.01). Time course of channel inactivation was slowed by ziprasidone in a voltage-dependent manner. The V1/2 values for steady-state activation and inactivation of hERG channel in HEK-293 cells were not significantly altered by ziprasidone. In Xenopus oocytes, ziprasidone exhibited less potent block of wild-type hERG current (IC50=2.8μM, 0mV, 23°C). Mutation of the aromatic residues (Tyr-652 or Phe-656) located in the S6 domain of hERG dramatically reduced the potency of channel block by ziprasidone (IC50>0.4 and 1mM at 0mV for Y652A and F656A, respectively). In conclusion, ziprasidone preferentially binds to and blocks open hERG channels. Tyr-652 and Phe-656 are two critical residues in the ziprasidone-binding site.
Keywords: Ziprasidone; hERG; Potassium channels; HEK-293 cells; Xenopus; oocytes
Interactions of low-molecular-weight semi-synthetic sulfated heparins with human leukocyte elastase and human Cathepsin G by Claudia Sissi; Lorena Lucatello; Annamaria Naggi; Giangiacomo Torri; Manlio Palumbo (pp. 287-293).
Semi-synthetic low-molecular-weight heparin samples (LMWHs), having homogeneous degree of polymerization and saccharide backbone, but differing in the number and location of sulfate groups, were investigated in their ability to interfere with the pharmacologically relevant targets human leukocyte elastase (EL) and human Cathepsin G (CatG). Spectroscopic studies were performed for a quantitative evaluation of the enzyme-inhibitor dissociation constant, Ki, and of the IC50 values for the inhibition of cleavage of target peptide sequences. Both proteases are inhibited by the tested polysaccharides through a mixed hyperbolic binding process. A non-linear relationship was found between degree of sulfation and binding affinity or enzyme inhibition properties, showing a composite correlation between heparin charge density and interference with EL/CatG activity.
Keywords: Heparin; Sulfation degree; Elastase; Cathepsin G; Inhibition; Interaction
Generation of adenosine A3 receptor functionally humanized mice for the evaluation of the human antagonists by Kazuya Yamano; Miho Inoue; Shigehiro Masaki; Mayumi Saki; Michio Ichimura; Mitsuo Satoh (pp. 294-306).
Although the adenosine A3 receptor (A3AR), which is a Gi/o protein-coupled receptor, has attracted considerable interest as a potential target for drugs against asthma or inflammation, the in vivo evaluation of the antagonists using rodents in the first step of drug development has been hampered by the lack of highly potent antagonists for the rodent A3AR. To evaluate the pharmacological effects of human A3AR antagonists in mice, we previously generated A3AR-humanized mice, in which the mouse A3AR gene was replaced by its human counterpart. However, the human A3AR did not lead to the phosphoinositide 3-kinase (PI3K) γ-signaling pathway such as IgE/antigen-dependent mast cell degranulation, probably due to the uncoupling of the mouse Gi/o protein(s). To overcome the uncoupling, we here generated A3AR functionally humanized mice by replacing the mouse A3AR gene with a human/mouse chimeric A3AR sequence in which whole intracellular regions of the human A3AR were substituted for the corresponding regions of the mouse A3AR. The chimeric A3AR led to intracellular Ca2+ elevation and activation of the PI3Kγ-signaling pathway, which are equivalent to the actions induced by A3AR in wild-type mice. The human A3AR antagonist had the same binding affinities for the chimeric A3AR as the human A3AR and completely antagonized this potentiation. This is the first direct evidence that the uncoupling of mouse G protein(s) to the human A3AR is due to a sequence difference in the intracellular regions of A3AR. The A3AR functionally humanized mice can be widely employed for pharmacological evaluations of the human A3AR antagonists.
Keywords: Abbreviations; A3AR; adenosine A3 receptor; A3AR; c/c; mice; A3AR-chimeric mice; A3AR; h/h; mice; A3AR-humanized mice; BMMCs; bone marrow-derived mast cells; [Ca; 2+; ]; i; intracellular Ca; 2+; concentration; Cl-IB-MECA; 2-chloro-; N; 6; -(3-iodobenzyl)adenosine-5′-; N; -methyluronamide; DT-A; diphtheria toxin A fragment; ERK1/2; extracellular signal-regulated kinase 1/2; ES cells; embryonic stem cells; GPCR; G protein-coupled receptor; HAT; hypoxanthine/aminopterin/thymidine; HPRT; hypoxanthine phosphoribosyltransferase; [; 125; I]AB-MECA; N; 6; -(4-amino-3-[; 125; I]iodobenzyl)adenosine-5′-; N; -methyluronamide; KF26777; 2-(4-bromophenyl)-7,8dihydro-4-propyl-1; H; -imidazo[2,1-; i; ]purin-5(4; H; )-one dihydrochloride; MAPK; mitogen-activated protein kinase; PCR; polymerase chain reaction; PI3Kγ; phosphoinositide 3-kinase γ; PKB; protein kinase B; (; R; )-PIA; (R)-; N; 6; -phenylisopropyladenosine; RT-PCR; reverse transcription-PCR; TNP; 2,4,6-trinitrophenyl; anti-TNP IgE; monoclonal IgE antibody against 2,4,6-trinitrophenylAdenosine A3 receptor; G protein-coupled receptor; Chimeric receptor; Uncoupling; A3AR functionally humanized mice; Species difference
CO from enhanced HO activity or from CORM-2 inhibits both O2− and NO production and downregulates HO-1 expression in LPS-stimulated macrophages by Klaokwan Srisook; Shan-Shu Han; Hyung-Sim Choi; Mei-Hua Li; Hideo Ueda; Chaekyun Kim; Young-Nam Cha (pp. 307-318).
Carbon monoxide (CO) arising from heme degradation, catalyzed particularly by the stress-inducible heme oxygenase-1 (HO-1), has recently been demonstrated to provide cytoprotection against cell death in macrophages stimulated with bacterial lipopolysaccharide (LPS). In the present study, we determined the effects of CO on the production of reactive oxygen species (ROS) and nitric oxide (NO) by the LPS-stimulated RAW 264.7 macrophages. In addition, effect of CO-exposure on the production of superoxide (O2−) in the phorbol myristate acetate (PMA)-stimulated PLB-985 neutrophils was determined. Production of ROS by the LPS-stimulated macrophages pretreated with 50μM [Ru(CO)3Cl2]2, a CO-releasing molecule (CORM-2), was abolished and the production of O2− by the PMA-stimulated neutrophils pretreated with the CORM-2 was decreased markedly. The CORM-2 (50μM) was not cytotoxic to both the unstimulated and LPS-stimulated macrophages when determined by employing mitochondrial reductase function test (MTT assay). In macrophages pretreated with increasing doses of CORM-2, both the LPS-derived upregulations of iNOS (NO production) and HO-1 expression (CO production) were suppressed in a dose-dependent manner. Alternatively, when the macrophages were treated with LPS and CO-donor together, the LPS-derived increase in NO production was decreased. Conversely, when the control and LPS-stimulated macrophages were treated with zinc protoporphyrin IX (ZnPP) to inhibit the HO activity blocking endogenous production of CO (basal and enhanced), macrophages died extensively. Interestingly, production of NO in the LPS-stimulated macrophages increased significantly following the ZnPP treatment. Addition of CORM-2 to the LPS-treated cells that were being treated additionally with ZnPP did not prevent the cell death. However, endogenous overproduction of CO by super-induction of HO-1 (obtained by pretreatment of macrophages with either buthionine sulfoximine or hemin) decreased the LPS-derived iNOS expression without affecting cell survival. Combined, these results indicated that enhanced HO activity is essential for the survival of LPS-stimulated macrophages. Thus, upregulation of HO-1 and overproduction of CO may allow the survival of LPS-stimulated macrophages; first, by eliminating the free heme to prevent Fenton reaction, second, by limiting the availability of free heme required for induction of NO-producing heme enzyme (i.e., iNOS), third, by limiting additional production of O2− and NO via CO-derived inhibition on the activities of heme enzymes like NADPH oxidase and iNOS, respectively. CO may allow the LPS-activated macrophages to return back to the normal quiet state insensitive to additional stimuli causing oxidative stress.
Keywords: Carbon monoxide; CORM-2; Heme oxygenase; Superoxide; Nitric oxide
Identification and characterization of surrogate peptide ligand for orphan G protein-coupled receptor mas using phage-displayed peptide library by Rama Kamesh Bikkavilli; Sup-Yin Tsang; Wai-Man Tang; Jing-Xin Sun; Sai-Ming Ngai; Susanna Sau-Tuen Lee; Wing-Hung Ko; Helen Wise; Wing-Tai Cheung (pp. 319-337).
In the present study, a phage-displayed random peptide library was used to identify surrogate peptide ligands for orphan GPCR mas. Sequence analysis of the isolated phage clones indicated a selective enrichment of some peptide sequences. Moreover, multiple alignments of the isolated phage clones gave two conserved peptide motifs from which we synthesized peptide MBP7 for further evaluation. Characterization of the representative phage clones and the synthetic peptide MBP7 by immunocytochemistry revealed a strong punctate cell surface staining in CHO cells expressing mas-GFP fusion protein. The isolated phage clones and synthetic peptide MBP7 induced mas internalization in a stable CHO cell clone (MC0M80) over-expressing mas. In addition, MBP7-stimulated phospholipase C activity and intracellular calcium mobilization in these same cells. In summary, we have demonstrated a systematic approach to derive surrogate peptide ligands for orphan GPCRs. With this technique, we have identified two conserved peptide motifs which allow us to identify potential protein partners for mas, and have generated a peptide agonist MBP7 which will be invaluable for functional characterization of the mas oncogene.
Keywords: Angiotensin; GPCR; Mas; Oncogene; Peptide library; Phage display
Stereospecific recognition of a spirosuccinimide type aldose reductase inhibitor (AS-3201) by plasma proteins: A significant role of specific binding by serum albumin in the improved potency and stability by Masuo Kurono; Akihito Fujii; Makoto Murata; Buichi Fujitani; Toshiyuki Negoro (pp. 338-353).
AS-3201 [(3 R)-2′-(4-bromo-2-fluorobenzyl)spiro[pyrrolidine-3,4′(1′ H)-pyrrolo[1,2- a]pyrazine]-1′,2,3′,5(2′ H)-tetrone] is a structurally novel and stereospecifically potent aldose reductase (AKR1B; EC 1.1.1.21) inhibitor, which contains a succinimide ring that undergoes ring-opening at physiological pH levels. To delineate intermolecular interactions governing its favorable pharmacokinetic profile, the interaction of AS-3201 ( R-isomer) with plasma proteins, especially human serum albumin (HSA), was examined in comparison with that of the optical antipode ( S-isomer). Fluorescence, kinetic, and high-performance frontal analyses showed that the R-isomer is more strongly bound than the S-isomer to sites I and II on HSA, and the R-isomer is particularly protected from hydrolysis, suggesting that the stable HSA– R-isomer complex contributes to its prolonged activity. The thermodynamic parameters for the specific binding indicated that in addition to hydrophobic interactions, hydrogen bonds contribute significantly to the R-isomer complex formation.13C NMR observations of the succinimide ring (5-13C enriched), which are sensitive to its ionization state, suggested the presence of a hydrogen bond between the R-isomer and HSA, and19F NMR of the pendent benzyl ring (2-19F) evaluated the equilibrium exchange dynamics between the specific sites. Furthermore, fatty acid binding or glycation (both are site II-oriented perturbations) inhibited the binding to one of the specific sites and reduced the stereospecificity of HSA toward the isomers, although the clinical influence of these perturbations on the R-isomer binding ratio seemed to be minor. Thus, the difference in the interaction mode at site II might be a major cause of the stereospecificity; this is discussed on the basis of putative binding modes. The present results, together with preliminary absorption and distribution profiles, provide valuable information on the stereospecific pharmacokinetic and pharmacodynamic properties of the R-isomer relevant for the therapeutic treatment of diabetic complications.
Keywords: Abbreviations; α; 1; -AGP; (human) α1-acid glycoprotein; AKR1B; aldose reductase; Compound 1 (racemate); 2-[(4; R; ,; S; )-2-(4-bromo-2-fluorobenzyl)-1,3-dioxo-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazin-4-yl]acetamide; Compound 2 (racemate); (3; R; ,; S; )-2′-(4-bromo-2-fluorobenzyl)spiro[pyrrolidine-3,4′(1′; H; )-6′-chloropyrrolo[1,2-; a; ]pyrazine]-1′,2,3′,5(2′; H; )-tetrone; DNSA; 5-dimethylaminonaphthalene-1-sulfonamide; DSP; diabetic sensorimotor polyneuropathy; FA-HSA; fatty acid containing HSA; G-HSA; glycated HSA; HSA; fatty acid-free human serum albumin; HPFA; high-performance frontal analysis; PA; palmitic acid; R-; isomer (AS-3201); (3; R; )-2′-(4-bromo-2-fluorobenzyl)spiro[pyrrolidine-3,4′(1′; H; )-pyrrolo[1,2-; a; ]pyrazine]-1′,2,3′,5(2′; H; )-tetrone; RSA; fatty acid-free rat serum albumin; SDS; sodium dodecyl sulfate; S; -isomer; (3; S; )-2′-(4-bromo-2-fluorobenzyl)spiro[pyrrolidine-3,4′(1′; H; )-pyrrolo[1,2-; a; ]pyrazine]-1′,2,3′,5(2′; H; )-tetroneHuman serum albumin; Aldose reductase inhibitor; Drug-binding site; Diabetic plasma; Stereospecificity; Binding mode
Oxygen-insensitive enzymatic reduction of oximes to imines by Sabine Heberling; Ulrich Girreser; Stephanie Wolf; Bernd Clement (pp. 354-365).
The reduction of oximes to imines under anaerobic and aerobic conditions was studied using ( E)- and ( Z)-2,4,6-trimethylacetophenone oxime, benzaldoxime and ( E)-2,4,6-trimethylbenzaldoxime. Pig and human liver microsomes, pig liver mitochondria and cytosol to a minor extent catalyzed the conversion of both isomeric ketoximes to the corresponding stable imine, the ( E)-isomer being the better substrate. All reactions were oxygen-insensitive and required active protein and NADH or NADPH; however, NADH was preferred as cofactor. The reconstituted liver microsomal system of a pig liver CYP2D enzyme (NADH-benzamidoxime reductase), which is known to reduce N-hydroxylated derivatives of strongly basic functional groups, such as amidoximes, is also capable of reducing oximes. As expected, the corresponding imine was detected in relevant amounts when incubating 2,4,6-trimethyl-acetophenone oxime using the reconstituted enzyme system, but reduction rates were significantly lower compared to rates obtained when incubating benzamidoxime. Steric hindrance due to the methyl groups in ortho position to the oxime functionality could be excluded as being responsible for the lower conversion rates according to results obtained in incubations of 2,4,6-trimethylbenzamidoxime. When incubating benzaldoxime, only benzoic acid could be detected as metabolite, since the aldehyde is easily oxidized during incubation procedures, whereas incubations of ( E)-2,4,6-trimethylbenzaldoxime also showed the formation of the corresponding aldehyde. These results allow us to postulate that the metabolism of aldoximes like 2,4,6-trimethylbenzaldoxime most likely proceeds through enzymatic reduction of the oxime to yield the intermediate imine, which is subsequently hydrolyzed to the aldehyde and then oxidized to the corresponding benzoic acid.
Keywords: Aldoxime; Amidoxime; Cytochrome P450; N-reduction; Microsomes; Mitochondria
The 21-aminosteroid U74389G prevents the down-regulation and decrease in activity of CYP1A1, 1A2 and 3A6 induced by an inflammatory reaction by Mehrzad Taherzadeh; Caroline Fradette; Anne-Marie Bleau; Claudia Jomphe; Louis-Éric Trudeau; Patrick du Souich (pp. 366-376).
In vivo, the 21-aminosteroid U74389G prevents the decrease in cytochrome P450 (P450) activity produced by a turpentine-induced inflammatory reaction (TIIR). To investigate the underlying mechanism of action, four groups of rabbits were used, controls receiving or not U74389G, and rabbits with the inflammatory reaction receiving or not U74389G. Hepatocytes were isolated 48h later and incubated for 4 and 24h with the serum of the rabbits. In vivo, the TIIR diminished CYP1A1/2 and 3A6 expression, and enhanced hepatic malondialdehyde (MDA) and nitric oxide (NO) concentrations ( p<0.05). U74389G prevented the increase in MDA, as well as the decrease in CYP1A1/2 amounts and activity, but increased CYP3A6 expression by 40% ( p<0.05). In vitro, compared with serum from control rabbits (SCONT), incubation of serum from rabbits with TIIR (STIIR) for 4 and 24h with hepatocytes from rabbits with TIIR (HTIIR) reduced CYP1A2 and CYP3A6 activity ( p<0.05) and increased the formation of NO and MDA. In rabbits with TIIR pretreated with U74389G, the STIIR+U failed to reduce CYP1A2 activity or to increase MDA, although increased NO and further reduced CYP3A6 activity. On the other hand, in hepatocytes harvested from rabbits with TIIR pretreated with U74389G, STIIR did not decrease CYP1A2 activity and did not enhance MDA, but still increased NO. In vitro, the reduction of CYP1A2 and CYP3A6 activity by STIIR is not associated to NF-κB activation. In conclusion, U74389G prevents CYP1A1/2 down-regulation and decrease in activity by a double mechanism: hindering the release of serum mediators and by averting intracellular events, effect possibly associated with its antioxidant activity. On the other hand, U74389G up-regulates CYP3A6 but inhibits its catalytic activity.
Keywords: Abbreviations; CYP; cytochrome P450 isoform; DFB; 3,4-difluorobenzyloxy-5,5-dimethyl-4-(4-methylsulfonyl phenyl)-(5H)-furan-2-one; DFH,; 3-hydroxy-4-(4-methylsulfonyl phenyl)-(5H)-furan-2-one; 1,3DMU; 1,3-dimethyluric acid; H; CONT; hepatocytes from a control rabbit; H; TIIR; hepatocytes from rabbits with a turpentine-induced inflammatory reaction; H; TIIR+U; hepatocytes from rabbits with a turpentine-induced inflammatory reaction and pretreated with U74389G for 72; h; IFN-γ; interferon-γ; IL; interleukin; l; -NAME; N; ω; -nitro-; l; -arginine methyl ester; LPS; lipopolysaccharide; MDA malondialdehyde; 1MU; 1-methyluric acid; 3MX; 3-methylxanthine; NF-κB; nuclear factor-κB; NO; nitric oxide; NOS; nitric oxide synthase; P450; cytochrome P450; S; CONT; serum from control rabbits; S; TIIR; serum from rabbits with a turpentine-induced inflammatory reaction; S; TIIR+U; serum from rabbits with a turpentine-induced inflammatory reaction and pretreated with U74389G for 72; h; TIIR; turpentine-induced inflammatory reaction; TNF-α; tumor necrosis factor-αCytochrome P450; CYP1A1; CYP1A2; CYP3A6; NF-κB; 21-Aminosteroid U74389G; Inflammation; Turpentine; Hepatocytes; Rabbits
Metabolism of nilutamide in rat lung by Kjetil Ask; Nathalie Décologne; Christian Ginies; Marit Låg; Jean Luc Boucher; Jørn A. Holme; Hélène Pelczar; Philippe Camus (pp. 377-385).
Nilutamide is a non-steroidal anti-androgen drug proposed in the treatment of metastatic prostatic carcinoma. Its therapeutic effects are overshadowed by the occurrence of adverse reactions, mediated by mechanisms that remain elusive. To elucidate possible mechanisms for nilutamide toxicity, we investigated the metabolism of nilutamide in rat lung homogenates, in subcellular fractions and in freshly isolated cells.In whole lung homogenates, the nitro group of nilutamide was reduced to the amine and hydroxylamine moieties. These conversions occurred exclusively in the absence of dioxygen, were increased by the addition of FMN, FAD, or NADPH.Reductive metabolism of nilutamide to the amine and hydroxylamine was further evidenced in subcellular fractions obtained by differential ultracentrifugation. It was found to take place mainly in the cytosol of rat lung and to be stimulated, strongly, upon co-addition of NADPH and FMN. Addition of inhibitors of enzymes involved in the reductive metabolism of nitroaromatic compounds indicated that reduction of nilutamide involved, mainly, soluble flavoproteins. Incubations with freshly isolated lung cells revealed that macrophages were the main players in nitroreduction of nilutamide whereas the epithelial type II cells and the non-ciliated Clara cells were less efficient in catalyzing this reaction.Our results show that nilutamide is extensively reduced by lung tissues in the absence of oxygen, especially by enzymes found in alveolar macrophages. In accordance with recent findings, subcellular localization, oxygen sensitivity, cofactor requirements and inhibitor studies lead us to suggest the involvement of a soluble nitric oxide synthase in lung cytosolic nitroreduction.
Keywords: Abbreviations; 2′AMP; 2′ adenosine monophosphate; DPIC; diphenyliodonium chloride; eNOS; endothelial NOS; iNOS; inducible NOS; NADD; norandrostenedione; NMN; N; -methylnicotinamide; NOS; nitric oxide synthase; nNOS; neuronal NOS; NR; nitroreductase; 2-OHP; 2-hydroxypyrimidine; p; HMB; para; -hydroxymercuribenzoic acid; P450R; NADPH:cytochrome; c; (P450) reductase; R-NO; 2; nitro; R-NO; nitroso; R-NHOH; hydroxylamine; R-NH; 2; amineNilutamide; Toxicity; Drug metabolism; Rat lung; Nitroreduction; Nitric oxide synthase
Serotonin induces pulmonary artery smooth muscle cell migration by Regina M. Day; Abena S. Agyeman; Michael J. Segel; Rubén D. Chévere; Jill M. Angelosanto; Yuichiro J. Suzuki; Barry L. Fanburg (pp. 386-397).
The chronic phase of pulmonary arterial hypertension (PAH) is associated with vascular remodeling, especially thickening of the smooth muscle layer of large pulmonary arteries and muscularization of small pulmonary vessels, which normally have no associated smooth muscle. Serotonin (5-hydroxytryptamine, 5-HT) has been shown to induce proliferation and hypertrophy of pulmonary artery smooth muscle cells (PASMC), and may be important for in vivo pulmonary vascular remodeling. Here, we show that 5-HT stimulates migration of pulmonary artery PASMC. Treatment with 5-HT for 16h increased migration of PASMC up to four-fold as monitored in a modified Boyden chamber assay. Increased migratory responses were associated with cellular morphological changes and reorganization of the actin cytoskeleton. 5-HT-induced alterations in morphology were previously shown in our laboratory to require cAMP [Lee SL, Fanburg BL. Serotonin produces a configurational change of cultured smooth muscle cells that is associated with elevation of intracellular cAMP. J Cell Phys 1992;150(2):396–405], and the 5-HT4 receptor was pharmacologically determined to be the primary activator of cAMP in bovine PASMC [Becker BN, Gettys TW, Middleton JP, Olsen CL, Albers FJ, Lee SL, et al. 8-Hydroxy-2-(di- n-propylamino)tetralin-responsive 5-hydroxytryptamine4-like receptor expressed in bovine pulmonary artery smooth muscle cells. Mol Pharmacol 1992;42(5):817–25]. We examined the role of the 5-HT4 receptor and cAMP in 5-HT-induced bovine PASMC migration. PASMC express 5-HT4 receptor mRNA, and a 5-HT4 receptor antagonist and a cAMP antagonist completely blocked 5-HT-induced cellular migration. Consistent with our previous report that a cAMP-dependent Cl− channel is required for 5-HT-induced morphological changes in PASMC, phenylanthranilic acid, a Cl− channel blocker, inhibited actin cytoskeletal reorganization and migration produced by 5-HT. We conclude that 5-HT stimulates PASMC migration and associated cytoskeletal reorganization through the 5-HT4 receptor and cAMP activation of a chloride channel.
Keywords: 5-HT; Migration; cAMP; Cl; −; channel; Cytoskeleton; 5-HTT; 5-HT4 receptor; 5-HT1B/1D receptor; MAPK