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Biochemical Pharmacology (v.69, #3)

Editorial Advisory Board (pp. iii).

Modulating the cytokine response to treat Helicobacter gastritis by Yana Zavros; Juanita L. Merchant (pp. 365-371).

The conventional view of gastric acid secretion is that a negative feedback mechanism arises in response to high acidity, such that somatostatin keeps G-cells and parietal cells from producing more gastrin and acid, respectively. When the stomach becomes infected, for example with Helicobacter pylori (H. pylori), the feedback mechanism is impaired. In animal models, our laboratory has demonstrated that other types of bacteria besides H. pylori can cause gastritis. For example, under conditions of low acidity, gastritis is secondary to bacterial overgrowth, not production of excessive acid, thus suggesting a new paradigm for the regulation of gastric acid secretion under inflammatory conditions. Cytokines, released during the gastric inflammatory response, including IFNγ, TNFα and IL-1β stimulate the G-cell to produce gastrin. Gastrin in turn triggers the release of acid, and hypergastrinemia suppresses somatostatin, the inhibitor of acid. The overall response results in maximal gastric acid output that acts as the stomach's most important anti-microbial agent. The increased acid secretion by the stomach in the presence of H. pylori seems to be part of the innate immune response, in that gastrin and somatostatin are reciprocally regulated by Th1 or Th2 cytokines, respectively. In a mouse model, we showed that octreotide, a somatostatin, analog, is an efficacious treatment for Helicobacter gastritis. In humans, octreotide might accelerate recovery from H. pylori infection, reducing the duration of antibiotic therapy.

Keywords: Abbreviations; IFNγ; interferon gamma; IL-1β; interluekin-1 beta; TNFα; tumor necrosis factor alpha; IL-4; interluekin-4; G-cells; gastrin-secreting cells; D-cells; somatostatin-secreting cells; ECL-cells; enterochromaffin-like cells; H. pylori; Helicobacter pyloriGastrin; Somatostatin; IFNγ; IL-4; Octreotide

High effectiveness of platinum(IV) complex with adamantylamine in overcoming resistance to cisplatin and suppressing proliferation of ovarian cancer cells in vitro by Alois Kozubík; Viktor Horváth; Lenka Švihálková-Šindlerová; Karel Sou�?ek; Jiřina Hofmanová; Petr Sova; Aleš Kroutil; František Žák; Adolf Mistr; Jaroslav Turánek (pp. 373-383).

[( OC-6-43)-bis(acetato)(1-adamantylamine)amminedichloroplatinum(IV)], coded as LA-12, is an octahedral platinum(IV) complex containing a bulky hydrophobic ligand – adamantylamine. The use of bulky hydrophobic amines as non-leaving ligands, may increase uptake of the compound by the cancer cells. Therefore, the effects of LA-12 on sensitive (A2780) and cisplatin resistant (A2780cis) ovarian cancer cell lines were investigated and compared to those of cisplatin. IC₅₀ and IC₉₀ concentrations of LA-12 were 6- (A2780) or 18-fold (A2780cis) lower than those for cisplatin (MTT assay). Equitoxic concentrations (IC₅₀ or IC₉₀) of both compounds caused a significant and similar time- and dose-dependent inhibition of cell proliferation and an increase in the number of floating cells which corresponded to the decrease of total cell viability. A different type and dynamics of cell cycle perturbation after cisplatin and LA-12 treatment were detected. Exposure to LA-12 resulted in transient accumulation of A2780 and A2780cis cells in S phase, while cisplatin caused G₂/M arrest in sensitive and S phase arrest in resistant cells. A relatively low rate of apoptosis after exposure to IC₅₀ or IC₉₀ of both complexes was observed, markedly higher in resistant A2780cis cells. Western blot analysis indicated a concentration-dependent p53 level increase in both lines (higher after cisplatin treatment). PARP cleavage was observed only in A2780cis cells. In conclusion, LA-12 was found to be significantly more efficient than cisplatin, and it was able to overcome the acquired cisplatin resistance (showing resistance factor 2.84-fold lower than those for cisplatin). In spite of the low rate of apoptosis, LA-12 caused increase of p53 level and cell cycle perturbations in the ovarian cancer cell lines studied.

Keywords: Abbreviations; cis; -DDP; cis; -diamminedichloroplatinum(II); LA-12; (; OC; -6-43)-bis(acetato)(1-adamantylamine)amminedichloroplatinum(IV); JM216; (; OC; -6-43)-bis(acetato)amminedichloro(cyclohexylamine)platinum(IV); cisplatin (50 or 90); LA-12 (50 or 90), IC; ₅₀; or IC; ₉₀; drug concentrations that cause 50% or 90% inhibition of cell proliferation; Pt(II); planar and four coordinate platinum complex; Pt(IV); octahedral and six coordinate platinum complex; PI; propidium iodide; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DAPI; 4,6-diamidino-2-phenyl-indole dihydrochloride; DABCO; 1,4-diazabicyclo-[2.2.2]octane; PARP; poly(ADP-ribose)polymerase; HRP; horseradish peroxidase; PAGE; polyacrylamide gel electrophoresisCisplatin; LA-12; Cytotoxicity; Cell cycle perturbations; Apoptosis; Ovarian cancer

Inhibition of interleukin-8 (CXCL8/IL-8) responses by repertaxin, a new inhibitor of the chemokine receptors CXCR1 and CXCR2 by Federica Casilli; Andrea Bianchini; Isabelle Gloaguen; Leda Biordi; Edoardo Alesse; Claudio Festuccia; Barbara Cavalieri; Raffaele Strippoli; Maria Neve Cervellera; Rosa Di Bitondo; Elisabetta Ferretti; Fabrizio Mainiero; Cinzia Bizzarri; Francesco Colotta; Riccardo Bertini (pp. 385-394).

Repertaxin is a new non-competitive allosteric blocker of interleukin-8 (CXCL8/IL-8) receptors (CXCR1/R2), which by locking CXCR1/R2 in an inactive conformation prevents receptor signaling and human polymorphonuclear leukocyte (PMN) chemotaxis. Given the unique mode of action of repertaxin it was important to examine the ability of repertaxin to inhibit a wide range of biological activities induced by CXCL8 in human leukocytes. Our results show that repertaxin potently and selectively blocked PMN adhesion to fibrinogen and CD11b up-regulation induced by CXCL8. Reduction of CXCL8-mediated PMN adhesion by repertaxin was paralleled by inhibition of PMN activation including secondary and tertiary granule release and pro-inflammatory cytokine production, whereas PMN phagocytosis of Escherichia coli bacteria was unaffected. Repertaxin also selectively blocked CXCL8-induced T lymphocyte and natural killer (NK) cell migration. These data suggest that repertaxin is a potent and specific inhibitor of a wide range of CXCL8-mediated activities related to leukocyte recruitment and functional activation in inflammatory sites.

Keywords: Abbreviations; PMN; human polymorphonuclear leukocytes; CXCL8/IL-8; interleukin-8; CXCR1; interleukin-8 receptor 1; CXCR2; interleukin-8 receptor 2; GPCR; G protein-coupled receptor; NK; natural killer; LPS; bacterial endotoxin; C5a; fifth component of complement; fMLP; N; -formyl-methionyl-leucyl-phenylalanine; CXCL12/SDF-1; stromal cell-derived factor-1; CXCL6/GCP-2; granulocyte chemotactic protein-2; PBS; phosphate buffered saline; FBS; fetal bovine serum; MMP-9; matrix metalloproteinase 9; IL-1β; interleukin-1β; EDTA; ethylenediaminetetracetic acid; GAPDH; glyceraldehyde-3-phosphate dehydrogenaseInterleukin-8; Polymorphonuclear leukocytes; Adhesion; Degranulation; Phagocytosis; Repertaxin

Dietary rutin, but not its aglycone quercetin, ameliorates dextran sulfate sodium-induced experimental colitis in mice: attenuation of pro-inflammatory gene expression by Ki Han Kwon; Akira Murakami; Takuji Tanaka; Hajime Ohigashi (pp. 395-406).

Oxidative stress has been shown to play a pivotal role in the onset of inflammatory bowel disease (IBD) and carcinogenesis. We evaluated the effects of two dietary anti-oxidants, rutin and its aglycone quercetin, on dextran sulfate sodium (DSS)-induced experimental colitis in mice. Female ICR mice were fed a diet containing 0.1% rutin or 0.1% quercetin for 2 weeks, and given 5% DSS in drinking water during the second week to induce colitis. We also examined the dose-dependency of rutin and quercetin (0.01% and 0.001% each) as well as their therapeutic efficacy, which was evaluated following DSS administration, on DSS-induced colitis. The protein level of interleukin (IL)-1β in both colonic mucosa and peritoneal macrophages was quantified by enzyme-linked immunosorbent assay. Further, mRNA expression levels of IL-1β, tumor necrosis factor-α, IL-6, granulocyte macrophage-colony stimulating factor, inducible nitric oxide synthase, and cyclooxygenase (COX)-1 and COX-2 in colonic mucosa were determined by reverse transcription-polymerase chain reaction. A diet containing 0.1% rutin, but not quercetin, attenuated DSS-induced body weight loss and shortening of the colorectum ( P<0.01 and <0.05, respectively), and dramatically improved colitis histological scores. Further, DSS-induced increases in colonic mucosal IL-1β levels were blunted significantly in rutin-, but not quercetin-, fed mice ( P<0.01), while dietary rutin attenuated the expressions of IL-1β and IL-6 mRNA in colonic mucosa (each, P<0.01). As for dose dependency, 0.01%, but not 0.001%, dietary rutin significantly reduced mucosal IL-1β levels ( P<0.01). Notably, a 0.1% rutin diet given 3 days after DSS treatment significantly suppressed both colorectal shortening and IL-1β production ( P<0.05 and <0.01, respectively). Dietary rutin ameliorates DSS-induced colitis, presumably by suppressing the induction of pro-inflammatory cytokines. Our results suggest that rutin may be useful for the prevention and treatment of IBD and colorectal carcinogenesis via attenuation of pro-inflammatory cytokine production.

Keywords: Abbreviations; CD; Crohn's disease; COX; cyclooxygenase; DMEM; Dulbecco's Modified Eagle Medium; DMSO; dimethylsulfoxide; DSS; dextran sulfate sodium; ELISA; enzyme-linked immunosorbent assay; FBS; fetal bovine serum; GM-CSF; granulocyte macrophage-colony stimulating factor; HPRT; hypoxanthine guanine phosphoribosyltransferase; IBD; inflammatory bowel disease; ICE; IL-1β-converting enzyme; IL; interleukin; iNOS; inducible nitric oxide synthase; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS; phosphate-buffered saline; pMϕ; peritoneal macrophages; ROS; reactive oxygen species; RT-PCR; reverse transcription-polymerase chain reaction; TNBS; trinitrobenzene sulfonic acid; TNF; tumor necrosis factor; UC; ulcerative colitisInflammatory bowel disease (IBD); Dextran sulfate sodium (DSS); Oxidative stress; Rutin; Pro-inflammatory genes; Experimental colitis

Antimetastatic effect of prodigiosin through inhibition of tumor invasion by Jing Zhang; Yaling Shen; Jianwen Liu; Dongzhi Wei (pp. 407-414).

Prodigiosin, a bacterial metabolite, was reported to have immunosuppressive and anticancer activities. In this study, we investigated novel functions of prodigiosin about anti-metastasis and anti-invasion. Prodigiosin dose-dependently inhibited 95-D cells’ migration and invasion according to wound healing assay and the Transwell assay. The inhibitive effect could reach about 50% when cells were treated with 5μM prodigiosin for 12h. In animal experiment, intraperitoneal administration of 5mgkg⁻¹ prodigiosin decreased the number of metastatic nodules by 53% and elevated the survival rate of mice about one-fold comparing with control group. Results of cell aggregation and adhesion assay showed that prodigiosin could promote cell aggregation and simultaneously inhibit cell from adhering to extracellular matrix (ECM). In addition, prodigiosin suppressed RhoA gene expression, hence, decreased protein level of RhoA in 95-D cells, according to RT-PCR assay and Western blot assay. Gel zymogram assay revealed that prodigiosin could suppress the activity of matrix metalloproteinase-2 (MMP-2). These results demonstrate that prodigiosin effectively inhibit tumor metastasis in vitro and in vivo. The action mechanisms of prodigiosin are associated with the promotion of cell aggregation and the inhibition of various steps in cell invasive process, which include the inhibition of cell adhesion and mobility in a RhoA-dependent way and the suppression of MMP-2 ability.

Keywords: Abbreviations; ECM; extracellular matrix; MMPs; matrix metalloproteinases; BS; bovine serum; i.p.; intraperitoneally; i.v.; intravenously; MTT; 3-[4,5-dimethyl-thiazol-2-yl]2,5-diphenyltetrazolium bromide; EC; ₅₀; half effective concentration; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; RT; reverse transcriptionProdigiosin; Metastasis; Invasion; MMPs; RhoA

Directional trans-epithelial transport of organic anions in porcine LLC-PK1 cells that co-express human OATP1B1 (OATP-C) and MRP2 by Kevin J. Spears; Jillian Ross; Alasdair Stenhouse; Clive J. Ward; Lay-Beng Goh; C. Roland Wolf; Paul Morgan; Andy Ayrton; Thomas H. Friedberg (pp. 415-423).

The transcellular transport of many compounds, which cannot readily cross the lipid bilayer, is mediated by drug uptake and efflux transporters. Human OATP1B1 and MRP2 have been implicated in the hepato-biliary transport of many endogenous and exogenous compounds. Here, we have established epithelial porcine kidney LLC-PK1 derived cell lines, that express both transporters in a polarized fashion, as a model to predict hepato-biliary transport. Immunological identification of OATP1B1 in the recombinant cell lines was greatly facilitated by its C-terminal tagging with a peptide sequence derived from hemagglutinin (HA) avoiding the generation of OATP1B1 specific antibodies. Importantly, the tag did not interfere with the functionality of the transporter. Compared to LLC-PK1 cells and cells which expressed only OATP1B1, the cell line that co-expressed MRP2 and OATP1B1 displayed high directional basolateral-to-apical transport of 17β-estradiol–17β-glucuronide and estrone-3-sulfate. Dehydroepiandrosterone sulfate already displayed a significant basolateral-to-apical transport in the parental cell line, which was further stimulated upon expression of both transporters. Transcellular flux of all steroid conjugates in the opposite direction (apical-to-basolateral) was much lower. By employing this cellular model we were able to demonstrate for the first time that OATP1B1 together with MRP2 mediates the trans-cellular transport of rifampicin. It is anticipated that the models established herein will greatly facilitate the identification of transporters involved in the disposition of novel drug candidates.

Keywords: Abbreviations; BSA; bovine serum albumin; DHEAS; Dehydroepiandrosterone-sulfate; E; ₂; 17βG; 17β-estradiol–17β-glucuronide; E; ₁; S; estrone 3-sulfate; FBS; fetal bovine serum; HA; hemagglutinin; HBSS; Hanks balanced salt solution; MDCK; Mardin Darby canine kidney cells; MDR; multidrug resistance protein; MRP; multidrug resistance-associated protein; OATP; organic anion transporting polypeptideDrug transport; Preclinical drug development; Organic anion transport; Mutidrug resistance

Inhibition of interleukin-12 expression in diltiazem-treated dendritic cells through the reduction of nuclear factor-κB transcriptional activity by Martina Severa; Antonella D’Ambrosio; Luciana Giordani; Francesca Quintieri; Eliana Coccia (pp. 425-432).

Diltiazem is a calcium channel blocker that suppresses the activation of a variety of immune cells, such as T and B cells, NK cells, monocytes and dendritic cells (DCs). It has been used in the treatment of cardiovascular disorders and has been widely included in clinical protocols to prevent rejection after kidney transplantation. In line with these data, we previously showed that diltiazem directly affects maturation of human DCs and the production of IL-12. Here, we extended our analysis studying the effect of diltiazem on the transcription of IL-12 p35 and p40 subunits focusing on the activity of nuclear factor-κB (NF-κB). A marked reduction of NF-κB binding to the κB sequences present within the p35 and p40 subunit promoters was observed in diltiazem-treated DCs following the stimulation with lipopolysaccharide (LPS) or CD40L. In order to examine the mechanisms by which NF-κB binding activity is reduced by diltiazem, we analyzed the NF-κB inhibitor, IκBα. No significant differences were observed in the phosphorylation and/or the degradation of IκBα. On the other hand, the subcellular distribution of NF-κB subunits was clearly affected in diltiazem-treated DCs following LPS stimulation, with a reduced nuclear translocation of p65, and RelB, and a nuclear accumulation of p50 subunit. Thus, all together, our data provided evidence that in addition to the inhibition of p65/p50 nuclear translocation, the selective induction and translocation of p50/p50 homodimers is an important mechanism by which diltiazem inhibits NF-κB activity, and in turn, IL-12 expression.

Keywords: NF-κB; Diltiazem; Dendritic cells; IL-12; CD40L; LipopolysaccharideAbbreviations; DCs; dendritic cells; RPA; RNase protection assay; NF-κB; nuclear factor-κB; LPS; lipopolysaccharide; EMSA; electrophoretic mobility shift assay; IKK; IκB kinase; IκB; inhibitor of NF-κB

Histamine H1 receptor antagonist blocks histamine-induced proinflammatory cytokine production through inhibition of Ca²⁺-dependent protein kinase C, Raf/MEK/ERK and IKK/IκB/NF-κB signal cascades by Masahiro Matsubara; Tadafumi Tamura; Kenji Ohmori; Kazuhide Hasegawa (pp. 433-449).

Histamine H1 receptor (H1R), a therapeutic target for alleviation of acute allergic reaction, may be also involved in mediating inflammatory responses via effects on cytokine production. However, the mechanisms whereby histamine induces cytokine production are poorly defined. In this study, we comprehensively investigated the signaling pathway involved in cytokine expression caused by histamine, using native human epidermal keratinocytes. We confirmed the expression of functional H1R by reverse transcription-polymerase chain reaction (RT-PCR), Western blotting and histamine-induced Ca²⁺ elevation. Histamine induced concentration- and time-dependent production of granulocyte-macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-8 and IL-6, which was completely blocked by olopatadine, an H1 antagonist. Histamine activated the phosphorylation of protein kinase C (PKC), c-Raf, mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK), extracellular signal-regulated kinase (ERK), IκB kinase (IKK), inhibitory κB (IκB)-α and nuclear factor-KB (NF-κB) p65, which was inhibited by Ro-31-8220, a PKC inhibitor. Also, Ro-31-8220 significantly suppressed the expression of these cytokines. BAPTA-AM, an intracellular Ca²⁺ chelator, also reduced PKC phosphorylation and cytokine expression. PD98059, a MEK inhibitor, and BAY 11-8702, an IκB-α inhibitor, reduced ERK and NF-κB cascade activation, respectively, with little effect on PKC phosphorylation. PD98059 preferentially inhibited GM-CSF production whereas BAY 11-8702 prevented IL-8 and IL-6 production. Furthermore, in addition to the above cytokines, histamine stimulated the biosynthesis and/or release of numerous keratinocyte-derived mediators, which are probably regulated by the ERK or NF-κB cascades. Our study suggests that histamine activates Ca²⁺-dependent PKC isoforms that play crucial roles in the activation of Raf/MEK/ERK and IKK/IκB/NF-κB cascades, leading to up-regulation of cytokine expression. Thus, the anti-inflammatory benefit of H1 antagonists may be in part due to prevention of cytokine production.

Keywords: Abbreviations; H1R; histamine H1 receptor; RT-PCR; reverse transcription-polymerase chain reaction; GM-CSF; granulocyte-macrophage-colony stimulating factor; IL; interleukin; olopatadine; olopatadine hydrochloride; PKC; protein kinase C; MEK; mitogen-activated protein/extracellular signal-regulated kinase kinase; ERK; extracellular signal-regulated kinase; IKK; IκB kinase; IκB; inhibitory κB; NF-κB; nuclear factor-κB; PTX; pertussis toxin; DAG; diacylglycerol; MAPK; mitogen-activated protein kinase; cetirizine; cetirizine hydrochloride; fexofenadine; fexofenadine hydrochloride; bepotastine; bepotastine besilate; chlorpheniramine; chlorpheniramine maleate; IBMX; 3-isobutyl-1-methylxanthine; PMA; phorbol 12-myristate 13-acetate; BAPTA-AM; 1,2-bis(; o; -aminophenoxy)ethane-; N; ,; N; ,; N; ′,; N; ′-tetraacetic acid-acetoxymethyl ester; TNF; tumor necrosis factor; ICAM-1; intercellular adhesion molecule-1; MMP; matrix metalloprotease; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; PAGE; polyacrylamide gel electrophoresis; ELISA; enzyme-linked immunosorbent assayHistamine H1 receptor; Ca; ²⁺; PKC; ERK; NF-κB; Inflammation

Role of two adjacent cytoplasmic tyrosine residues in MRP1 (ABCC1) transport activity and sensitivity to sulfonylureas by Gwena�lle Conseil; Roger G. Deeley; Susan P.C. Cole (pp. 451-461).

The human ATP-binding cassette (ABC) protein MRP1 causes resistance to many anticancer drugs and is also a primary active transporter of conjugated metabolites and endogenous organic anions, including leukotriene C₄ (LTC₄) and glutathione (GSH). The sulfonylurea receptors SUR1 and SUR2 are related ABC proteins with the same domain structure as MRP1, but serve as regulators of the K⁺ channel Kir6.2. Despite their functional differences, the activity of both SUR1/2 and MRP1 can be blocked by glibenclamide, a sulfonylurea used to treat diabetes. Residues in the cytoplasmic loop connecting transmembrane helices 15 and 16 of the SUR proteins have been implicated as molecular determinants of their sensitivity to glibenclamide and other sulfonylureas. We have now investigated the effect of mutating Tyr¹¹⁸⁹ and Tyr¹¹⁹⁰ in the comparable region of MRP1 on its transport activity and sulfonylurea sensitivity. Ala and Ser substitutions of Tyr¹¹⁸⁹ and Tyr¹¹⁹⁰ caused a ≥50% decrease in the ability of MRP1 to transport different organic anions, and a decrease in LTC₄ photolabeling. Kinetic analyses showed the decrease in GSH transport was attributable primarily to a 10-fold increase in K_m. In contrast, mutations of these Tyr residues had no major effect on the catalytic activity of MRP1. Furthermore, the mutant proteins showed no substantial differences in their sensitivity to glibenclamide and tolbutamide. We conclude that MRP1 Tyr¹¹⁸⁹ and Tyr¹¹⁹⁰, unlike the corresponding residues in SUR1, are not involved in its differential sensitivity to sulfonylureas, but nevertheless, may be involved in the transport activity of MRP1, especially with respect to GSH.

Keywords: Abbreviations; ABC; ATP-binding cassette; MRP; multidrug resistance protein; BCRP; breast cancer resistance protein; LTC; ₄; leukotriene C4; E; ₂; 17βG; 17β-estradiol-17-β-(; d; -glucuronide); NBD; nucleotide binding domain; MSD; membrane spanning domain; TM; transmembrane; CL; cytoplasmic loop; SUR; sulfonylurea receptor; MTX; methotrexate; HEK; human embryonic kidney; E; ₁; 3SO; ₄; estrone 3-sulfate; WT; wild-typeMRP1; Organic anion transport; Glutathione transport; Sulfonylurea; ATP-binding cassette proteins; Site-directed mutagenesis

Impaired activation of caspases and prevention of mitochondrial dysfunction in the metastatic colon carcinoma CC531s-m2 cell line by Jurjen H.L. Velthuis; Zoran Gavric; Hans J.G.M. de Bont; J. Fred Nagelkerke (pp. 463-471).

In a previous paper we described the properties of a rapidly metastasizing cell line CC531s-m2 derived from the poorly metastasizing CC531s cell. The m2-cell line was relatively resistant to killing by NK cells. Both CD95L and TRAIL mediated apoptosis was decreased in the m2-cell line. Now, by flow cytometrical analysis of intra- and extra-cellular expressed receptors, we show that the localization of the receptors for CD95L and TRAIL was not altered in the CC531s-m2 cells as compared to the parental cell line. Subsequently caspase-activation and mitochondrial function were studied by enzymatic cleavage of fluorescent caspase-substrates and retention of the mitochondrial dye rhodamine-123, respectively. The activation of caspases as well as the loss of the mitochondrial membrane potential (MMP) was less in the CC531s-m2 cell line upon CD95L- and TRAIL-signalling. Furthermore, the sensitivity of the CC531-m2 towards cisplatin-induced apoptosis was strongly decreased. This was consistent with less mitochondrial damage, delayed caspase cleavage and decreased caspase activity. Altogether, we conclude that an Natural Killer-cell insensitive cell is less sensitive to CD95L- and TRAIL-induced apoptosis as well as anti-cancer drug induced apoptosis by prevention of mitochondrial damage and activation of caspases.

Keywords: Abbreviations; CD95L; CD95ligand; CDDP; cis; -diamminedichloroplatinum; DcR; decoy receptor; DR; death receptor; TRAIL; tumor necrosis factor related apoptosis inducing ligandCD95L; TRAIL; Apoptosis; Caspase activity; Cisplatin; Metastasizing cell line

Type 4 cAMP phosphodiesterase (PDE4) inhibitors augment glucocorticoid-mediated apoptosis in B cell chronic lymphocytic leukemia (B-CLL) in the absence of exogenous adenylyl cyclase stimulation by Sanjay Tiwari; Hongli Dong; Eun Jung Kim; Lewis Weintraub; Paul M. Epstein; Adam Lerner (pp. 473-483).

cAMP-mediated signaling potentiates glucocorticoid-mediated apoptosis in lymphoid cells, but an effective means by which to take advantage of this observation in the treatment of lymphoid malignancies has not been identified. The primary objective of the current study was to determine whether PDE4 inhibitors, a class of compounds in late clinical development that raise intracellular cAMP levels by inhibiting type 4 cyclic nucleotide phosphodiesterases (PDE4), increase the efficacy of glucocorticoid-mediated apoptosis in leukemic cells from patients with B cell chronic lymphocytic leukemia (B-CLL). Rolipram, a prototypic PDE4 inhibitor, synergized with glucocorticoids in inducing B-CLL but not T cell apoptosis. Rolipram also augmented glucocorticoid receptor element (GRE) transactivation in B-CLL cells. In contrast, inhibition of protein kinase A (PKA) with the cAMP antagonist Rp-8Br-cAMPS reversed both glucocorticoid-induced apoptosis and GRE transactivation. CCRF-CEM cells, a well-studied model of glucocorticoid and cAMP-induced apoptosis, differed from B-CLL cells in that stimulation of adenylyl cyclase with the diterpene forskolin was required to increase both glucocorticoid-mediated apoptosis and GRE activation, while PDE4 inhibition had no effect. Consistent with these results, inhibition of PDE4 induced cAMP elevation in B-CLL but not CCRF-CEM cells, while forskolin augmented cAMP levels in CCRF-CEM but not B-CLL cells. While rolipram treatment up-regulated PDE4B in B-CLL, forskolin treatment up-regulated PDE4D in CCRF-CEM cells. These studies suggest that PKA is required for and enhances glucocorticoid-induced apoptosis in B-CLL by modulating glucocorticoid receptor signal transduction. Clinical trials that examine whether PDE4 inhibitors enhance the efficacy of glucocorticoid-containing chemotherapy regimens in B-CLL are indicated.

Keywords: Abbreviations; B-CLL; B cell chronic lymphocytic leukemia; PDE4; type 4 cAMP phosphodiesteraseGlucocorticoid therapy; B-CLL; PDE4 inhibitors; cAMP; PKA; GRE

3,5-Di- t-butylcatechol (DTCAT) as an activator of rat skeletal muscle ryanodine receptor Ca²⁺ channel (RyRC) by Fabio Fusi; Donata Iozzi; Giampietro Sgaragli; Maria Frosini (pp. 485-491).

In the present study, the effects of 3,5-di- t-butylcatechol (DTCAT) on ryanodine receptor Ca²⁺ channel (RyRC) of skeletal muscle sarcoplasmic reticulum (SR) vesicles were investigated, both by monitoring extravesicular Ca²⁺ concentration directly with the Ca²⁺ indicator dye arsenazo III and by studying the high-affinity [³H]ryanodine binding. DTCAT stimulated Ca²⁺ release from junctional (terminal cisternae) vesicles in a concentration-dependent manner, with a threshold activating concentration of 30μM and a pEC₅₀ value of 3.43±0.03M. The release of Ca²⁺ induced by DTCAT was antagonized in a concentration-dependent manner by ruthenium red, thus indicating that RyRC is involved in the mechanism of stimulation. A structure–activity relationship analysis carried out on a limited number of compounds suggested that both hydroxy and t-butyl groups in DTCAT were important for the activation of RyRC. DTCAT inhibited [³H]ryanodine binding to SR vesicles with a K_i of 232.5μM, thus indicating that it acted directly at the skeletal muscle ryanodine receptor binding site to stimulate Ca²⁺ release. In conclusion, the ability of DTCAT to release Ca²⁺ from TC vesicles of skeletal muscle is noteworthy in view of its possible use as an alternative compound to either caffeine or halothane for performing the “In vitro contracture test�? to diagnose the susceptibility of some patients to develop malignant hyperthermia under particular pharmacological treatments.

Keywords: Abbreviations; DTCAT; 3,5-di-; t; -butylcatechol; RyRC; ryanodine receptor Ca; ²⁺; channel; SR; sarcoplasmic reticulum; MH; malignant hyperthermia; TC; terminal cisternae; RR; ruthenium red; DIICAT; 3,5-diisopropylcatechol; DTBP; 2,4-di-; t; -butylphenol; TBC; 4-; t; -butyl catechol; DTHB; 3,5-di-; t; -butyl-2-hydroxy-benzaldehyde; CMC; 4-chloro-; m; -cresol; IVCT; in vitro contracture test3,5-Di-; t; -butylcatechol; Ryanodine receptor Ca; ²⁺; channel; Rat skeletal sarcoplasmic reticulum

Attenuation of cyclosporine A toxicity by sublethal heat shock by David Andr�s; Mirandeli Bautista; Mar�a Cascales (pp. 493-501).

Cyclosporine A (CsA) is the immunosuppressor most frequently used in transplant surgery and in the treatment of autoimmune diseases because of its specific inhibiting effect on signal transduction pathways of cell T receptor. It has been shown that CsA is able to generate reactive oxygen species and lipid peroxidation, which are directly involved in the CsA hepatotoxicity. In the present study, we investigated the effect of a sublethal heat pre-treatment (43°C for 30min) on the hepatoma cell line HepG2 exposed to cytotoxic concentrations of CsA (10 and 25μM) for 3 and 24h. Parameters of cytotoxicity were assayed by measuring LDH (lactate dehydrogenase) leakage into the medium. Peroxide concentration was tested by flow cytometry by measuring the fluorescence intensity of DCF (dichlorofluorescein). Gene expression of catalase was detected by measuring the respective mRNA and proteins, as well as protein level of HSP70. The enzymatic activity of catalase was also determined. Heat pre-treatment significantly reduced CsA cytotoxicity as well as the level of peroxide generation. The protective effect of the previous heat treatment (corroborated by the irreversible catalase inhibitor 3-aminotriazole) against the CsA cytotoxicity was due to an increased expression and activity of catalase that was significantly reduced by the effect of CsA. We conclude that heat pre-treatment strongly protects against CsA injury, and the mechanism of this protection is by means of inducing not only the expression of HSP70 but also the expression and activity of catalase, the main enzyme system involved in H₂O₂ elimination.

Keywords: Abbreviations; HSP70; heat shock protein 70; SOD; superoxide dismutase; ROS; reactive oxygen species; CsA; Cyclosporine A; 3-AT; 3-aminotriazoleCsA; Heat shock; Catalase; ROS; HSP70; 3-Aminotriazole; HepG2

Some flavonoids and DHEA-S prevent the cis-effect of expanded CTG repeats in a stable PC12 cell transformant by Hirokazu Furuya; Nobue Shinnoh; Yasumasa Ohyagi; Koji Ikezoe; Hitoshi Kikuchi; Manabu Osoegawa; Yasuyuki Fukumaki; Yusaku Nakabeppu; Toshimitsu Hayashi; Jun-ichi Kira (pp. 503-516).

Expanded CUG triplet repeats carrying mRNA seem to be responsible for myotonic dystrophy type 1 (DM1). To study the pathogenesis of DM1, we constructed a DM1 cell culture model using a PC12 neuronal cell line and screened flavonoids that ameliorate this mRNA gain of function. The expanded 250 CTG repeat was subcloned into the 3′-untranslated region of the luciferase gene yielding a stable transformant of PC12 (CTG-250). The cytotoxicity of CTG-250 was evaluated by intracellular LDH activity, and the cis-effect by luciferase activity. To find agents that alter CTG-250 toxic effects, 235 bioflavonoids were screened. An increased cis-effect and cytotoxicity were found when CTG-250 was treated with nerve growth factor to induce differentiation. Western blotting with anti-caspase-3 antibody suggested that cell death was caused by apoptosis. Screening analysis confirmed that a flavone (toringin), an isoflavones (genistein and formononetin), a flavanone (isosakuranetin), and DHEA-S prevent both the cytotoxicity and cis-effect of CTG-250 and that a flavanone (naringenin), isoflavone (ononin), and xanthylatin strongly inhibit the cis-effect of CTG repeats. In conclusion, we found that this neuronal cell line, which expresses the CUG repeat-bearing mRNA, showed cis-effects through the reporter gene and neuronal death after cell differentiation in vitro. However, some flavonoids and DHEA-S inhibit both the cis-effect and cytotoxicity, indicating that their chemical structures work to ameliorate both these toxic effects. This system makes it easy to evaluate the toxic effects of expanded CTG repeats and therefore should be useful for screening other DM1 treatments for their efficacies.

Keywords: Abbreviations; DM1; myotonic dystrophy type1; DM2; myotonic dystrophy type2; DMPK; DM1 protein kinase; 3′-UTR; 3′-untranslated region; RT-PCR; reverse transcription polymerase chain reaction; MW; molecular weight; kDa; kilo-Dalton; EtBr; ethidium bromide; conc.; concentration; nt; nucleotides length; DHEA-S; dehydroepiandrosterone sulfate; DMSO; dimethyl sulfoxide; DMEM; Dulbecco's modified Eagle's medium; CNS; central nervous systemMyotonic dystrophy type 1; Trinucleotide repeats; PC12; Flavonoid; RNA gain of function; Tauopathy

Inhibition of human CYP2B6 by N, N′, N″-triethylenethiophosphoramide is irreversible and mechanism-based by Tanja Richter; Matthias Schwab; Michel Eichelbaum; Ulrich M. Zanger (pp. 517-524).

The chemotherapeutic agent N, N′, N″-triethylenethiophosphoramide (thioTEPA) is frequently used in high-dose chemotherapy regimens including cyclophosphamide. Previous studies demonstrated partial inhibition by thioTEPA of the cytochrome P4502B6 (CYP2B6)-catalyzed 4-hydroxylation of cyclophosphamide, which is required for its bioactivation. The aim of our study was to investigate the detailed mechanism of CYP2B6 inhibition by thioTEPA. Using human liver microsomes and recombinant P450 enzymes we confirmed potent inhibition of CYP2B6 enzyme activity determined with bupropion as substrate. ThioTEPA was found to inhibit CYP2B6 activity in a time- and concentration-dependent manner. The loss of CYP2B6 activity was NADPH-dependent and could not be restored by extensive dialysis. The maximal rates of inactivation ( K_inact) were 0.16min⁻¹ in human liver microsomes and 0.17min⁻¹ in membrane preparations expressing recombinant CYP2B6. The half-maximal inactivator concentrations ( K_I) were 3.8μM in human liver microsomes and 2.2μM in recombinant CYP2B6. Inhibition was attenuated by the presence of alternative active site ligands but not by nucleophilic trapping agents or reactive oxygen scavengers, further supporting mechanism-based action. Inactivated CYP2B6 did not lose its ability to form a CO-reduced complex suggesting a modification of the apoprotein, which is common for sulfur-containing compounds. Pharmacokinetic consequences of irreversible inactivation are more complicated than those of reversible inactivation, because the drug's own metabolism can be affected and drug interactions will not only depend on dose but also on duration and frequency of application. These findings contribute to better understanding of drug interactions with thioTEPA.

Keywords: Abbreviations; CP; cyclophosphamide; CYP; cytochrome P450; ESI; electrospray ionisation; HPLC; high-performance liquid chromatography; OR; NADPH:cytochrome-P450-oxidoreductase; thioTEPA; N; ,; N; ′,; N; ″-triethylenethiophosphoramideCytochrome P450; CYP2B6; thioTEPA; Mechanism-based inhibition; Suicide-inhibition; Cyclophoshamide

Identification and functional analysis of two rare allelic variants of the thiopurine S-methyltransferase gene, TPMT*16 and TPMT*19 by Rima Hamdan-Khalil; Jean-Luc Gala; Delphine Allorge; Jean-Marc Lo-Guidice; Yves Horsmans; Nicole Houdret; Franck Broly (pp. 525-529).

Human thiopurine S-methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs. TPMT is genetically polymorphic and is associated with large interindividual variations in thiopurine drug toxicity and therapeutic efficacy. During routine genotyping of patients with Crohn's disease, one novel missense mutation, 365A>C ( TPMT*19, Lys¹²²Thr), and a recently described missense mutation, 488G>A ( TPMT*16, Arg¹⁶³His), were identified in a Caucasian and a Moroccan patient, respectively. Using a heterologous yeast expression system, kinetic parameters ( K_m and V_max) of the two variants with respect to 6-thioguanine S-methylation were determined and compared with those obtained with the wild-type enzyme. The Lys¹²²Thr exchange did not significantly decrease the intrinsic clearance value ( V_max/ K_m) of the variant enzyme. In contrast, the Arg¹⁶³His substitution significantly decreased the intrinsic clearance value by three-fold. The Arg¹⁶³ is located in a highly conserved region of the human TPMT protein and, as such, the Arg¹⁶³His substitution is expected to result in a marked reduction of enzyme activity, as confirmed by the in vitro data. Phenotyping by measurement of red blood cell TPMT activity indicated that the patient heterozygous for the Lys¹²²Thr mutation had normal TPMT activity, whereas the patient heterozygous for the Arg¹⁶³His mutation was an intermediate methylator, which demonstrated a positive correlation between TPMT phenotyping and the in vitro data. The identification of a novel non-functional allele of the TPMT gene improves our knowledge of the genetic basis of interindividual variability in TPMT activity. These data further enhance the efficiency of genotyping methods to predict patients at risk of an inadequate response to thiopurine therapy.

Keywords: TPMT; Genetic polymorphism; Azathioprine; 6-Thioguanine; Deficient methylator; Heterologous expressionAbbreviations; RBC; red blood cell; 6-TG; 6-thioguanine; TPMT; thiopurine; S; -methyltransferase

Mechanisms involved in spironolactone-induced choleresis in the rat by Mar�a L. Ruiz; Silvina S. M. Villanueva; Marcelo G. Luquita; Enrique J. S�nchez-Pozzi; Fernando A. Crocenzi; Jos� M. Pellegrino; Justina E. Ochoa; Mary Vore; Aldo D. Mottino; Viviana A. Catania (pp. 531-539).

The mechanisms involved in spironolactone (SL, 200μmol/kg body weight, 3 days i.p.)-induced choleresis were explored in vivo by evaluating bile salt export pump (Bsep)-, multidrug resistance-associated protein 2 (Mrp2)-, and anion exchanger 2 (AE2)-mediated secretory processes in rat liver. Hepatic bile salt metabolism was also analyzed. Total bile flow was significantly increased by SL, primarily due to an increase in bile salt-independent bile flow, whereas bile salt secretion was decreased. SL did not produce any choleresis in TR⁻ rats. SL decreased the de novo bile salt synthesis rate in concordance with impaired microsomal cholesterol 7α-hydroxylase activity, thus leading to a decrease in endogenous bile salt pool size. In contrast, the maximum secretory rate of tauroursodeoxycholate as well as expression of Bsep protein detected by Western blotting were not affected. Thus, decreased bile salt availability for canalicular transport rather than transport capability itself likely explains reduced biliary secretion of bile salts. Biliary secretion of glutathione, an endogenous substrate of Mrp2, and HCO₃⁻, the AE2 substrate, were increased by SL, as a main factor explaining enhanced bile salt-independent bile flow. Western blot studies revealed increased expression of Mrp2 in response to SL whereas AE2 content remained unchanged. Enhanced activity and expression of Mrp2 was confirmed by analyzing the excretion rate of dinitrophenyl S-glutathione, an exogenous substrate of Mrp2, in isolated hepatocytes and by immunofluorescence microscopy, respectively. We conclude that SL increased bile flow mainly by increasing the biliary secretion of glutathione species and HCO₃⁻; increased expression of Mrp2 is also involved.

Keywords: Abbreviations; AE2; anionic exchanger 2; BSDF; bile salt-dependent bile flow; Bsep; bile salt export pump; BSIF; bile salt-independent bile flow; CDNB; 1-chloro-2,4-dinitrobenzene; DNP-SG; dinitrophenyl; S; -glutathione; GSH; reduced glutathione; GSSG; oxidized glutathione; GST; glutathione; S; -transferase; MPM; mixed plasma membrane; Mrp2; multidrug resistance-associated protein 2; PXR; pregnane X-receptor; SL; spironolactone; Tm; maximum secretory rate; TUDC; sodium tauroursodeoxycholate; UGT; UDP-glucuronosyltransferaseSpironolactone; Bile flow; Mrp2; Bsep; AE2; Glutathione

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Biochemical Pharmacology (v.69, #3)