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Biochemical Pharmacology (v.69, #9)

Editorial Advisory Board (pp. iii).

13- cis Retinoic acid and isomerisation in paediatric oncology—is changing shape the key to success? by Jane L. Armstrong; Christopher P.F. Redfern; Gareth J. Veal (pp. 1299-1306).

Retinoic acid isomers have been used with some success as chemotherapeutic agents, most recently with 13- cis retinoic acid showing impressive clinical efficacy in the paediatric malignancy neuroblastoma. The aim of this commentary is to review the evidence that 13- cis retinoic acid is a pro-drug, and consider the implications of retinoid metabolism and isomerisation for the further development of retinoic acid for cancer therapy. The low binding affinity of 13- cis retinoic acid for retinoic acid receptors, low activity in gene expression assays and the accumulation of the all- trans isomer in cells treated with 13- cis retinoic acid, coupled with the more-favourable pharmacokinetic profile of 13- cis retinoic acid compared to other isomers, suggest that intracellular isomerisation to all- trans retinoic acid is the key process underlying the biological activity of 13- cis retinoic acid. Intracellular metabolism of all- trans retinoic acid by a positive auto-regulatory loop may result in clinical resistance to retinoic acid. Agents that block or reduce the metabolism of all- trans retinoic acid are therefore attractive targets for drug development. Devising strategies to deliver 13- cis retinoic acid to tumour cells and facilitate the intracellular isomerisation of 13- cis retinoic acid, while limiting metabolism of all- trans retinoic acid, may have a major impact on the efficacy of 13- cis retinoic acid in paediatric oncology.

Keywords: Abbreviations; AHR; aryl hydrocarbon receptor; APL; acute promyelocytic leukaemia; ATRA; all-; trans; retinoic acid; CEPT; cholesteryl ester transfer protein; CRABP; cellular retinoic acid binding protein; GST; glutathione S-transferase; IGFBP-3; insulin-like growth factor binding protein-3; LRAT; lecithin-retinol acyltransferase; M6P; mannose-6-phosphate; MTD; maximum tolerated dose; PGP; P-glycoprotein; RAG; retinoyl β-glucuronide; 13; cis; RA; 13-; cis; retinoic acid; 9; cis; RA; 9-; cis; retinoic acid; RA; retinoic acid; RAR; retinoic acid receptor; RARE; retinoic acid response element; RXR; retinoid X receptorRetinoic acid; Isomerisation; Pro-drug; Metabolism; Neuroblastoma; Paediatric

Bis(pivaloyloxymethyl) thymidine 5′-phosphate is a cell membrane-permeable precursor of thymidine 5′-phosphate in thymidine kinase deficient CCRF CEM cells by Saeed R. Khan; Billie Nowak; William Plunkett; David Farquhar (pp. 1307-1313).

Bis(pivaloyloxymethyl) thymidine 5-phosphate (POM₂-dTMP) has been investigated as a membrane-permeable prodrugs of dTMP. The growth inhibitory activity of POM₂-TMP has been compared with thymidine (TdR) in wild type CCRF CEM cells (CEM) and a strain that lacks TdR kinase (CEM tk−). After 72h incubation at 37°C, TdR showed significant antiproliferative activity (IC₅₀=27μM) against CEM cells but was weakly effective (IC₅₀=730μM) against the mutant cell line. By comparison, bis(pivaloyloxymethyl) thymidine 5′-monophosphate (POM₂-dTMP) was equally inhibitory (IC₅₀=5μM) to both cell lines. The growth inhibitory effects were reversed by deoxycytidine. Cellular [methyl-³H]dTTP pools increased linearly over 2h during incubation of CEM or CEM tk− with 5μM POM₂-[methyl-³H]dTMP. The incorporation of [methyl-³H]TdR into HClO₄-insoluble cell residue by CEM tk− was <0.1% that of CEM and did not increase over 1h. In contrast, CEM tk− incorporated radioactivity from POM₂-dTMP into acid insoluble residue at a rate 59% that of CEM. These results demonstrate that POM₂-dTMP can penetrate into cells and serve as a source of dTMP.

Keywords: Abbreviations; POM; pivaloyloxymethyl; POM; ₂; bis(pivaloyloxymethyl); TdR; thymidine; ddT; dideoxythymidine; dTMP; thymidine 5′-phosphate; ddTMP; dideoxythymidine 5′-phosphate; dTTP; thymidine 5′-triphosphate; NTP; nucleoside triphosphate; CCRF CEM; human acute leukemia lymphoblastic cells; tk; thymidine kinase; tk-; thymidine kinase deficient; AZTMP; 3′-azido-3′-deoxythymidine 5′-monophosphate; ddUMP; 2′,3′-dideoxyuridine 5′-monophosphate; POM; ₂; -dTMP; bis(pivaloyloxymethyl) thymidine 5′-monophosphate; TLC; thin layer chromatography; HPLC; high performance liquid chromatography; HClO; ₄; perchloric acidThymidine 5′-phosphate; Prodrugs; Antitumor; Membrane-permeable

Involvement of multiple signaling pathways in the post-bariatric induction of IL-6 and IL-8 mRNA and release in human visceral adipose tissue by John N. Fain; Suleiman W. Bahouth; Atul K. Madan (pp. 1315-1324).

The present studies were designed to determine the site of and the mechanism for the rapid increase in IL-6 and IL-8 mRNA observed in human visceral adipose tissue after removal during laparoscopic bariatric surgery. Upregulation of IL-6 and IL-8 mRNA as well as their release were seen within 3h whether one intact piece of tissue or minced pieces of adipose tissue were incubated in vitro. Most of the IL-6 and IL-8 mRNA content of visceral adipose tissue after 3h of incubation was in the non-fat cells. Actinomcyin D markedly reduced the upregulation of IL-6 and IL-8 mRNA. Incubation of adipose tissue explants with a soluble TNFα receptor (etanercept) plus a blocking antibody against IL-lβ reduced by 55% the increase in IL-6 mRNA and by 42% that of IL-8 mRNA seen between 1 and 5h of incubation. The upregulation of IL-8 and IL-6 mRNA accumulation as well as their release over a 2 or 4h incubation was reduced by around 50% in the presence of an inhibitor of the p38 MAPK or an inhibitor of the NFκB pathway and by 85% in the presence of both inhibitors. The data suggest that the relative trauma and/or hypoxia that occurs when adipose tissue is removed results in the release of TNFα and IL-1β. These cytokines, and probably other factors as well, enhance IL-6 and IL-8 mRNA accumulation in human adipose tissue explants through mechanisms involving the p38 MAPK and NFκB pathways

Keywords: Abbreviations; SV; stromovascular; TNFα; tumor necrosis factor alpha; NFκB; nuclear factor κB; p38 MAPK; p38 mitogen-activated protein kinase; JNK; Jun N-terminal kinase; ERK; p44/42 mitogen-activated protein kinase; TGF-β1; transforming growth factor-β1IL-6; IL-8; NFΚB; TNFα; IL-1β; p38 MAPK

Severity of pancreatitis-associated gut barrier dysfunction is reduced following treatment with the PAF inhibitor lexipafant by Per Leveau; Xiangdong Wang; Zhengwu Sun; Anna B�rjesson; Ellen Andersson; Roland Andersson (pp. 1325-1331).

The aim of the present study was to investigate the potential effect of treatment with a platelet-activating factor (PAF) antagonist, lexipafant (BB-882), on gut endothelial and epithelial barrier dysfunction and leukocyte recruitment in rats with acute pancreatitis. Severe acute pancreatitis was induced by the intraductal administration of 5% sodium taurodeoxycholate and pancreatitis-associated gut barrier dysfunction was characterized by increased exudation of radiolabelled albumin into the interstitium and alterations in bidirectional (over both the endothelial and epithelial barrier components) permeability of the intestine at the early stage of bile salt-induced acute pancreatitis. Levels of interleukin 1β and 6, ileal and colonic myeloperoxidase (MPO) content, clearance of radiolabelled albumin from blood to the gut lumen or gut lumen to blood, and leakage of radiolabelled albumin to the ileum or colon were measured 3 and 12h after induction of acute pancreatitis. Treatment with lexipafant 30min and 6h after pancreatitis reduced severity of pancreatitis-associated intestinal dysfunction, associated with a diminish in systemic concentrations of IL-1 and local leukocyte recruitment. The findings imply that PAF plays a critical role in the development of pancreatitis-associated gut barrier dysfunction and that PAF antagonist in some forms may represent potential candidates for future therapeutic intervention.

Keywords: Pancreatitis; Intestine; Permeability; Cytokines; Platelet-activating factor antagonist

G2 arrest and apoptosis by 2-amino- N-quinoline-8-yl-benzenesulfonamide (QBS), a novel cytotoxic compound by Yun-Hee Kim; Kum-Joo Shin; Taehoon G. Lee; Euikyung Kim; Myoung-Shik Lee; Sung Ho Ryu; Pann-Ghill Suh (pp. 1333-1341).

We screened a library of 11,000 small molecular weight chemicals, looking for compounds that affect cell viability. We have identified 2-amino- N-quinoline-8-yl-benzenesulfonamide (QBS) as a potent cytotoxic compound that induces cell cycle arrest and apoptosis. Treatment of Jurkat T cells with QBS increased the levels of cyclin B1 as well as phosphorylated-cdc2, which was accompanied by reduced activity of cdc2 kinase, suggesting that QBS may induce cell cycle arrest at G2 phase. Structural analogues of QBS also exhibited similar effects on cell cycle progression and cell viability. Long-term treatment with QBS resulted in DNA fragmentation, cytochrome C release, and PARP cleavage, and an increase in the number of subdiploidy cells, indicative of cellular apoptosis. Moreover, QBS-induced apoptosis was blocked by z-VAD-fmk, a pan-caspase inhibitor. These results suggest that QBS is a novel and potent compound that induces G2 arrest and subsequent apoptosis, implicating it as a putative candidate for chemotherapy.

Keywords: Abbreviations; QBS; 2-amino-; N; -quinoline-8-yl-benzenesulfonamide; PARP; poly(ADP-ribose)-polymerase; QBS-1; N; -8-quinolinyl-benzenesulfonamide; PQS; N; -phenyl-4-quinolinesulfonamide; QBS-2; N; ,4-dimethyl-; N; -8-quinolinylbenzenesulfonamide; QBS-3; 4-methyl-; N; -4-quinolinylbenzenesulfonamide; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; Cyt C; cytochrom CLibrary screening; Quinolinesulfonamide; DNA damage; G2 arrest; Apoptosis

The effect of quercetin on pro-apoptotic activity of cisplatin in HeLa cells by J. Jakubowicz-Gil; R. Paduch; T. Piersiak; K. Głowniak; A. Gawron; M. Kandefer-Szerszeń (pp. 1343-1350).

It is well known that some tumour cells are very resistant to chemotherapy-induced cell death which indicate poor prognosis for patients. Thus the aim of the present study was to investigate the effect of quercetin on pro-apoptotic activity of cisplatin in human cervix carcinoma cells (HeLa).Three variants of experiments were performed. In the first one cells were incubated with studied drugs separately for 8 and 24h. In the second, drugs were added to the culture medium simultaneously. In third cisplatin or quercetin addition was followed by subsequent quercetin or cisplatin treatment, respectively.We observed different apoptotic effects, dependent on the drug succession. Preincubation of cells with quercetin followed by cisplatin treatment appeared to be the most effective and was correlated with strong activation of caspase-3 and inhibition of both heat shock proteins (Hsp72) and multi-drug resistance proteins (MRP) levels.Our results indicate that quercetin pretreatment sensitizes HeLa cells to cisplatin-induced apoptosis in HeLa cells.

Keywords: Quercetin; Cisplatin; Apoptosis; Hsp72; MRP; HeLa cells

Very stable superoxide radical adducts of 5-ethoxycarbonyl- 3,5-dimethyl-pyrroline N-oxide (3,5-EDPO) and its derivatives by Klaus Stolze; Natascha Rohr-Udilova; Thomas Rosenau; Roswitha Stadtm�ller; Hans Nohl (pp. 1351-1361).

Oxygen radicals are involved in the onset of many diseases. Adequate spin traps are required for identification and localisation of free radical formation in biological systems. Superoxide spin adducts with half-lives up to 20min at physiological pH have recently been reported to be formed from derivatives of the spin trap 5-ethoxycarbonyl-5-methyl-1-pyrroline N-oxide (EMPO). This is a major improvement over DMPO ( t_1/2 ca. 45s), and even DEPMPO ( t_1/2 ca. 14min). In this study, an additional methyl group was introduced into position 3 or 4 of the pyrroline ring which greatly increases the stability of the respective superoxide spin adducts. In addition, the ethoxy group of EMPO was exchanged by either a propoxy- or an iso-propoxy group in order to test the influence of increasing lipophilic properties of the investigated spin traps. The structure of all compounds was confirmed by¹H and¹³C-NMR with full signal assignment. In comparison with EMPO ( t_1/2 ca. 8min) or DEPMPO ( t_1/2 ca. 14min), the superoxide adducts of all novel spin traps were considerably higher ( t_1/2 ca. 12–55min). In addition, various other spin adducts obtained from oxygen-centered as well as carbon-centered radicals (e.g. derived from methanol or linoleic acid hydroperoxide) were also detected.

Keywords: Abbreviations; DEPMPO; 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline; N; -oxide; DMPO; 5,5-dimethylpyrroline; N; -oxide; DTPA; diethylenetriaminepentaacetic acid; 3,5-EDPO; 5-(ethoxycarbonyl)-3,5-dimethyl-1-pyrroline; N; -oxide; 4,5-EDPO; 5-(ethoxycarbonyl)-4,5-dimethyl-1-pyrroline; N; -oxide; 3,5-DIPPO; 3,5-dimethyl-5-(iso-propoxycarbonyl)-1-pyrroline; N; -oxide; 4,5-DIPPO; 4,5-dimethyl-5-(iso-propoxycarbonyl)-1-pyrroline; N; -oxide; 3,5-DPPO; 3,5-dimethyl-5-(propoxycarbonyl)-1-pyrroline; N; -oxide; 4,5-DPPO; 4,5-dimethyl-5-(propoxycarbonyl)-1-pyrroline; N; -oxide; EMPO; 5-(ethoxycarbonyl)-5-methyl-1-pyrroline; N; -oxide; EPR; electron paramagnetic resonance; HFS; hyperfine splitting; LO; ^•; lipoxyl radical; NMR; nuclear magnetic resonance; O; ₂; ^•−; superoxide anion radical; SOD; superoxide dismutaseSpin traps; EPR; EMPO derivatives; Superoxide; Linoleic acid hydroperoxide; Free radicals

Clofibrate and perfluorodecanoate both upregulate the expression of the pregnane X receptor but oppositely affect its ligand-dependent induction on cytochrome P450 3A23 by Yuzhong Ma; Karuna Sachdeva; Jirong Liu; Xiulong Song; Yuxin Li; Dongfang Yang; Ruitang Deng; Clinton O. Chichester; Bingfang Yan (pp. 1363-1371).

The pregnane X receptor (PXR) interacts with a vast array of structurally dissimilar chemicals and confers induction of several major types of drug metabolizing enzymes such as cytochrome P450s (CYP). We previously reported that the expression of PXR was markedly increased in rats treated with clofibrate and perfluorodecanoic acid (PFDA). The present study was undertaken to test the hypothesis that induced expression of PXR increases PXR ligand-dependent induction on CYP3A23. Rat hepatocytes were treated with clofibrate or PFDA individually, or along with PXR ligand pregnenolone 16α-carbonitrile (PCN), and the levels of PXR and CYP3A23 were determined by Western blots. Both clofibrate and PFDA markedly increased the expression of PXR with PFDA being more potent, and the induction was abolished by actinomycin D, an inhibitor for mRNA synthesis. As expected, PCN alone markedly induced the expression of CYP3A23. Interestingly, co-treatment with clofibrate enhanced the induction, whereas co-treatment with PFDA suppressed it. Clofibrate and PFDA represent multi-classes of chemicals called peroxisome proliferators including many therapeutic agents and industrial pollutants. The opposing effects of clofibrate and PFDA on the PCN-induced expression of CYP3A23 suggest that peroxisome proliferators likely increase the expression of PXR but differentially alter its ligand-dependent induction. The interaction between PXR inducer and ligand provides a novel mechanism on how functionally and structurally distinct chemicals cooperatively regulate the expression of xenobiotic-metabolizing enzymes and transporters.

Keywords: Abbreviations; CYP; cytochrome P450; DMSO; dimethyl sulfoxide; MDR-1; multidrug resistance-1; PCR; polymerase chain reaction; PFDA; perfluorodecanoic acid; PXR; pregnane X receptor; RT-PCR; reverse transcription-polymerase chain reaction; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; WME; Williams’ Medium EPXR; CYP3A23; Clofibrate; Perfluorodecanoic acid; Pregnenolone 16α-Carbonitrile; Induction

The role of NMDA receptor upregulation in phencyclidine-induced cortical apoptosis in organotypic culture by Cheng Wang; Jared Fridley; Kenneth M. Johnson (pp. 1373-1383).

Phencyclidine (PCP) is an N-methyl-d-aspartate receptor (NMDAR) antagonist known to cause selective neurotoxicity in the cortex following subchronic administration. The purpose of this study was to test the hypothesis that upregulation of the NMDAR plays a role in PCP-induced apoptotic cell death. Corticostriatal slice cultures were used to determine the effects of NMDAR subunit antisense oligodeoxynucleotides (ODNs) on PCP-induced apoptosis and NMDAR upregulation. NR1, NR2A or NR2B antisense ODNs were incubated alone or with PCP for 48h. One day following washout, it was observed that PCP treatment caused an increase in NR1, NR2A and Bax polypeptides in the cortex, but had no effect on Bcl-xL. These increases were associated with an increase in cortical histone-associated DNA fragments. Co-incubation of PCP with either NR1 or NR2A antisense significantly reduced PCP-induced apoptosis, while neither NR2B antisense ODN nor NR1 sense ODN used as a control had an effect. This effect was exactly correlated with the ability of the antisense ODNs to prevent PCP-induced upregulation of NR subunit proteins and the pro-apoptotic protein, Bax. That is, western analysis showed that antisense ODNs directed against either NR1 or NR2A prevented PCP-induced increases in Bax in addition to preventing the upregulation of the respective receptor proteins. On the other hand, the NR2B antisense ODN had no effect on either NR2B protein or on Bax. These data suggest that NR1 and NR2A antisense ODNs offer neuroprotection from apoptosis, and that upregulation of the NR1 and NR2A subunits following PCP administration is at least partly responsible for the observed apoptotic DNA fragmentation.

Keywords: Abbreviations; NMDA; N; -methyl-; d; -aspartate; PCP; phencyclidine; ODN; oligodeoxy-nucleotide; NR; N; -methyl-; d; -aspartate receptor; TUNEL; terminal d-UTP nick end labeling; DIV; days in vitroPhencyclidine; Apoptosis; Cortex; N; -Methyl-; d; -aspartate receptor; Antisense; Bax

Reduced nicotine distribution from mother to fetal brain in rats vaccinated against nicotine: Time course and influence of nicotine dosing regimen by Daniel E. Keyler; Matthew B. Dufek; Andrew D. Calvin; Thomas J. Bramwell; Mark G. LeSage; Donna E. Raphael; Cathy A. Ross; Chap T. Le; Paul R. Pentel (pp. 1385-1395).

Nicotine is a teratogen in rats and possibly in humans. Vaccination against nicotine is being studied as a possible treatment for nicotine dependence. The safety of maternal vaccination against nicotine during or prior to pregnancy is not known. In this study, female rats were vaccinated and then administered acute or chronic nicotine during pregnancy at doses simulating nicotine exposure in smokers. Maternal vaccination reduced nicotine distribution to both maternal brain (44–47%) and fetal brain (17–39%) for up to 25min after a single maternal nicotine dose administered on gestational day (GD) 20, but had a smaller effect on nicotine distribution to brain after continuous nicotine infusion. Nicotine distribution to maternal or fetal brain after repeated nicotine bolus doses was reduced immediately following an individual dose in vaccinated rats, but the chronic accumulation of nicotine in fetal brain was not altered. Nicotine distribution to whole fetus, in contrast to fetal brain, was generally not altered by vaccination. Nicotine-specific antibody concentration in fetal serum was 10% that of maternal serum, and in fetal brain was <1% of maternal serum. Although nicotine transfer to the whole fetus was not reduced by vaccination, protein binding data suggest that nicotine-specific antibody transferred from mother to fetus served to bind nicotine in fetal serum, reduce the unbound nicotine concentration, and thereby reduce nicotine distribution to fetal brain. These data comment on the safety of vaccination against nicotine during pregnancy, and suggest that vaccination may reduce the distribution of nicotine to fetal brain under some nicotine dosing conditions.

Keywords: Abbreviations; GD; gestational day; Nic-vaccine; nicotine-vaccineNicotine; Vaccine; Antibody; Fetal; Pregnancy; Pharmacokinetics

Serotonin glucuronidation by Ah receptor- and oxidative stress-inducible human UDP-glucuronosyltransferase (UGT) 1A6 in Caco-2 cells by Christoph K�hle; Osama A. Badary; Karl Nill; Barbara S. Bock-Hennig; Karl Walter Bock (pp. 1397-1402).

Caco-2 cells are a widely used model in drug development to study intestinal drug transport and metabolism. Recently, serotonin (5-hydroxytryptamine, 5-HT) has been characterized as a highly selective substrate of human UDP-glucuronosyltransferase UGT1A6 [Krishnaswamy S, Duan SX, von Moltke LL, Greenblatt DJ, Court MH. Validation of serotonin (5-hydroxytryptamine) as an in vitro substrate probe for human UDP-glucuronosyltransferase (UGT) 1A6. Drug Metab Disp 2003; 31:133–9], an isoform which conjugates planar phenols and is inducible by Ah receptor agonists and by oxidative/electrophile stress. To gain more insight into intestinal 5-HT disposition, uptake and metabolism of this neurotransmitter was studied in Caco-2 cell monolayers. It was found that 5-HT was taken up from the basolateral and to a lesser extent from the apical surface. It was mainly excreted basolaterally as 5-HT glucuronide. 5-HT UGT activity and UGT1A6 mRNA were induced by Ah receptor agonists and by oxidative stress generated by tert-butylhydroquinone and by isomeric thymoquinone, a potential antitumor agent and constituent of Nigella sativa seeds, commonly used as a condiment in the Middle East. While UGT1A6 induction was clearly detectable in NAD(P)H:quinone oxidoreductase 1 (NQO1)-deficient Caco-2 cells, it was not induced in NQO1-efficient HT-29 colon adenocarcinoma cells. The results suggest that – in addition to its detoxification function – intestinal UGT1A6 contributes to intestinal homeostasis of 5-HT from dietary sources and from release by enterochromaffin cells.

Keywords: Abbreviations; UGT; UDP-glucuronosyltransferase; 5-HT; 5-hydroxytryptamine; 5-HT-GA; 5-hydroxytryptamine glucuronide; MUF; 4-methylumbelliferone; TCDD; 2,3,7,8-tetrachlorodibenzo-; p; -dioxin; BNF; β-naphthoflavone; t; BHQ; tert; -butylhydroquinone; TQ; thymoquinone; NQO1; NAD(P)H:quinone oxidoreductase 1; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumUDP-glucuronosyltransferase UGT1A6; 5-Hydroxytryptamine; Serotonin; Oxidative stress; Tert; -butylhydroquinone; Thymoquinone

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Biochemical Pharmacology (v.69, #9)