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Biochemical Pharmacology (v.78, #5)

Contents (pp. e1-e4).

Editorial Advisory Board (pp. iii).

Targeting glial physiology and glutamate cycling in the treatment of depression by Gerald W. Valentine; Gerard Sanacora (pp. 431-439).

Accumulating evidence indicates that dysfunction in amino acid neurotransmission contributes to the pathophysiology of depression. Consequently, the modulation of amino acid neurotransmission represents a new strategy for antidepressant development. While glutamate receptor ligands are known to have antidepressant effects, mechanisms regulating glutamate cycling and metabolism may be viable drug targets as well. In particular, excitatory amino acid transporters (EAATs) that are embedded in glial processes constitute the primary means of clearing extrasynaptic glutamate. Therefore, the decreased glial number observed in preclinical stress models, and in postmortem tissue from depressed patients provides intriguing, yet indirect evidence for a role of disrupted glutamate homeostasis in the pathophysiology of depression. More direct evidence for this hypothesis comes from studies using magnetic resonance spectroscopy (MRS), a technique that non-invasively measures in vivo concentrations of glutamate and other amino acids under different experimental conditions. Furthermore, when combined with the infusion of¹³C-labeled metabolic precursors, MRS can measure flux through discrete metabolic pathways. This approach has recently shown that glial amino acid metabolism is reduced by chronic stress, an effect that provides a link between environmental stress and the decreased EAAT activity observed under conditions of increased oxidative stress in the brain. Furthermore, administration of riluzole, a drug that enhances glutamate uptake through EAATs, reversed this stress-induced change in glial metabolism. Because riluzole has antidepressant effects in both animal models and human subjects, it may represent the prototype for a novel class of antidepressants with the modulation of glial physiology as a primary mechanism of action.By combining magnetic resonance spectroscopy (MRS) with the infusion of isotopically-labeled precursors, in vivo measurements of amino acid cycling and glial metabolism can be measured in psychiatric disease models.

Keywords: Abbreviations; AANt; amino acid neurotransmitters; AMPA; alpha-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionate; BDNF; brain-derived neurotrophic factor; CREB; cyclic AMP response element binding protein; CUS; chronic unpredictable stress; EAAT; excitatory amino acid transporter; GABA; gamma-aminobutyric acid; GFAP; glial fibrillary acidic protein; Gln; glutamine; Glu; glutamate; l; -beta-THA; l; -beta-threohydroxyaspartate; l; -Trans 2,4-PDC; l; -trans-2,4-pyrrolidine dicarboxylate; mGlu; metabotropic glutamate; MRS; magnetic resonance spectroscopy; NMDA; N; -methyl-; d; -aspartate; TCA; tricarboxylic acid; VGLUT; vesicular glutamate transportersDepression; Excitatory amino acid transporter (EAAT); Glia; Magnetic resonance spectroscopy; Riluzole; Stress

A renaissance in marine pharmacology: From preclinical curiosity to clinical reality by Keith B. Glaser; Alejandro M.S. Mayer (pp. 440-448).

Opinions provided by leaders in marine natural products will address potential reasons and perceptions from academic and pharmaceutical communities regarding the development of marine natural products as viable therapeutic entities.Marine pharmacology, the pharmacology of marine natural products, has been for some time more associated with marine natural products chemistry rather than mainstay pharmacology. However, in recent years a renaissance has occurred in this area of research, and has seen the US Food & Drug Administration (FDA) approval in 2004 of Prialt^® (ziconotide, ω-conotoxin MVIIA) the synthetic equivalent of a conopeptide found in marine snails, used for the management of severe chronic pain. Furthermore Yondelis^® (trabectedin, ET-743) an antitumor agent discovered in a marine colonial tunicate, and now produced synthetically, receiving Orphan Drug designation from the European Commission (EC) and FDA for soft tissue sarcomas and ovarian cancer and its registration in 2007 in the EU for the treatment of soft tissue sarcoma. The approval/marketing of so few marine natural products has come after many years of research primarily by the academic community and the sporadic involvement of major pharmaceutical companies. This commentary, through the opinions provided by several leaders in the marine natural products field, will examine the potential reasons and perceptions from both the academic and pharmaceutical communities regarding the development of marine natural products as viable therapeutic entities.

Keywords: Marine pharmacology; Marine natural products; Drug discovery

Selective activation of protein kinase C-δ and -ɛ by 6,11,12,14-tetrahydroxy-abieta-5,8,11,13-tetraene-7-one (coleon U) by I. Coutinho; G. Pereira; M.F. Simões; M. Côrte-Real; J. Gonçalves; L. Saraiva (pp. 449-459).

6,11,12,14-tetrahydroxy-abieta-5,8,11,13-tetraene-7-one (coleon U) is a diterpene compound isolated from Plectranthus grandidentatus with an antiproliferative effect on several human cancer cell lines. Herein, we studied the modulatory activity of coleon U on individual isoforms of the three protein kinase C (PKC) subfamilies, classical (cPKC-α and -βI), novel (nPKC-δ and -ɛ) and atypical (aPKC-ζ), using a yeast PKC assay. The results showed that, whereas the PKC activator phorbol-12-myristate-13-acetate (PMA) activated every PKC tested except aPKC, coleon U had no effect on aPKC and cPKCs. Besides, the effect of coleon U on nPKCs was higher than that of PMA. This revealed that coleon U was a potent and selective activator of nPKCs. The isoform-selectivity of coleon U for nPKC-δ and -ɛ was confirmed using an in vitro PKC assay. Most importantly, while PMA activated nPKCs inducing an isoform translocation from the cytosol to the plasma membrane and a G2/M cell cycle arrest, coleon U induced nPKCs translocation to the nucleus and a metacaspase- and mitochondrial-dependent apoptosis. This work therefore reconstitutes in yeast distinct subcellular translocations of a PKC isoform and the subsequent distinct cellular responses reported for mammalian cells. Together, our study identifies a new isoform-selective PKC activator with promising pharmacological applications. Indeed, since coleon U has no effect on cPKCs and aPKC, recognised as anti-apoptotic proteins, and selectively induces an apoptotic pathway dependent on nPKC-δ and -ɛ activation, it represents a promising compound for evaluation as an anti-cancer drug.

Keywords: Coleon U; Selective PKC activator; PKC-δ; PKC-ɛ; Apoptosis; Yeast

Everolimus restores gefitinib sensitivity in resistant non-small cell lung cancer cell lines by Silvia La Monica; Maricla Galetti; Roberta R. Alfieri; Andrea Cavazzoni; Andrea Ardizzoni; Marcello Tiseo; Marzia Capelletti; Matteo Goldoni; Sara Tagliaferri; Antonio Mutti; Claudia Fumarola; Mara Bonelli; Daniele Generali; Pier Giorgio Petronini (pp. 460-468).

Combination treatment with gefitinib and everolimus might be of value in the treatment of NSCLC cells with high proliferative index.The epidermal growth factor receptor (EGFR) is a validated target for therapy in non-small cell lung cancer (NSCLC). Most patients, however, either do not benefit or develop resistance to specific inhibitors of the EGFR tyrosine kinase activity, such as gefitinib or erlotinib. The mammalian target of rapamycin (mTOR) is a key intracellular kinase integrating proliferation and survival pathways and has been associated with resistance to EGFR tyrosine kinase inhibitors. In this study, we assessed the effects of combining the mTOR inhibitor everolimus (RAD001) with gefitinib on a panel of NSCLC cell lines characterized by gefitinib resistance and able to maintain S6K phosphorylation after gefitinib treatment.Everolimus plus gefitinib induced a significant decrease in the activation of MAPK and mTOR signaling pathways downstream of EGFR and resulted in a growth-inhibitory effect rather than in an enhancement of cell death. A synergistic effect was observed in those cell lines characterized by high proliferative index and low doubling time. These data suggest that treatment with everolimus and gefitinib might be of value in the treatment of selected NSCLC patients that exhibit high tumor proliferative activity.

Keywords: Lung cancer; EGFR; Gefitinib; Everolimus

3,3′-Diindolylmethane induces a G₁ arrest in human prostate cancer cells irrespective of androgen receptor and p53 status by Omar I. Vivar; Chia-Lei Lin; Gary L. Firestone; Leonard F. Bjeldanes (pp. 469-476).

3,3′-Diindolylmethane (DIM) is a potential chemopreventive phytochemical derived from Brassica vegetables. In this study we characterized the effect of DIM on cell cycle regulation in both androgen-dependent LNCaP and androgen receptor negative p53 mutant DU145 human prostate cancer cells. DIM had an anti-proliferative effect on both LNCaP and DU145 cells, as it significantly inhibited [³H]-thymidine incorporation. FACS analysis revealed a DIM-mediated G₁ cell cycle arrest. DIM strongly inhibited the expression of cdk2 and cdk4 protein and increased the expression of the cell cycle inhibitor p27^Kip1 protein in LNCaP and DU145 cells. Promoter deletion studies with p27^Kip1 reporter gene constructs showed that this DIM-mediated increase in p27^Kip1 was dependent on the Sp1 transcription factor. Moreover, using a dominant negative inhibitor of p38 MAPK, we showed that the induction of p27^Kip1 and subsequent G₁ arrest by DIM involve activation of the p38 MAPK pathway in the DU145 cells. Taken together, our results indicate that DIM is able to stop the cell cycle progression of human prostate cancer cells regardless of their androgen-dependence and p53 status, by differentially modulating cell cycle regulatory pathways. The Sp1 and p38 MAPK pathways mediate the DIM cell cycle regulatory effect in DU145 cells.

Keywords: Abbreviations; DIM; 3,3′-diindolylmethane; I3C; indole-3-carbinol; DMSO; dimethyl sulfoxide; RRP; recurrent respiratory papillomatosis; AR; androgen receptor; FACS; fluorescence-activated cell sorter; MAPK; mitogen-activated protein kinase; pRb; retinoblastoma protein; cdk; cyclin-dependent kinase3,3′-Diindolylmethane; Prostate cancer; Cell cycle arrest; p27; ^Kip1; p38 MAPK; Cancer

The natural marine anhydrophytosphingosine, Jaspine B, induces apoptosis in melanoma cells by interfering with ceramide metabolism by Yahya Salma; Elodie Lafont; Nicole Therville; Stéphane Carpentier; Marie-José Bonnafé; Thierry Levade; Yves Génisson; Nathalie Andrieu-Abadie (pp. 477-485).

Marine environment has frequently afforded a variety of biologically active compounds with strong anticancer and cytotoxic properties. In the present study, the mechanism of action of Jaspine B, an anhydrophytosphingosine derivative isolated from the marine sponge Jaspis sp., was investigated. Jaspine B was able to dose- and time-dependently decrease the viability of murine B16 and human SK-Mel28 melanoma cells. On these cells, Jaspine B treatment triggered cell death by typical apoptosis as illustrated by phosphatidylserine externalization, the release of cytochrome c and caspase processing. These effects were associated with increased intracellular ceramide levels owing to perturbed ceramide metabolism. Indeed, Jaspine B exposure strongly inhibited the activity of sphingomyelin synthase (SMS), an enzyme that converts de novo ceramide into the membrane lipid sphingomyelin. Moreover, whereas Jaspine B-induced cell death was enhanced in SMS1-depleted cells, it was strongly inhibited in cells that stably overexpress human SMS1. Finally, the cytotoxic effects of Jaspine B truncated analogs were also shown to be dependent on SMS activity.Altogether, Jaspine B is able to kill melanoma cells by acting on SMS activity and consequently on ceramide formation, and may represent a new class of cytotoxic compounds with potential applications in anticancer melanoma therapy.

Keywords: Abbreviations; C6-NBD ceramide; 6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino) hexanoyl) sphingosine; Ac-DEVD-AMC; Ac-Asp-Glu-Val-Asp-aminomethylcoumarin; FCS; fetal calf serum; GCS; glucosylceramide synthase; GlcCer; glucosylceramide; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SL; sphingolipid; SM; sphingomyelin; SMS; sphingomyelin synthase; TLC; thin layer chromatography; zVAD-fmk; benzyloxycarbonyl valyl-alanyl-aspartyl-(O-methyl)-fluoromethylketoneJaspine B; Ceramide; Sphingomyelin; Melanoma; Apoptosis

A novel δ-lactam-based histone deacetylase inhibitor, KBH-A42, induces cell cycle arrest and apoptosis in colon cancer cells by Moo Rim Kang; Jong Soon Kang; Sang-Bae Han; Jang Hyun Kim; Dong-Myung Kim; Kiho Lee; Chang Woo Lee; Ki Hoon Lee; Chul Ho Lee; Gyoonhee Han; Jong Seong Kang; Hwan Mook Kim; Song-Kyu Park (pp. 486-494).

In this study, we investigated the anti-tumor activity of KBH-A42 [ N-hydroxy-3-(2-oxo-1-(3-phenylpropyl)-1,2,5,6-tetrahydropyridin-3-yl)propanamide], a novel synthetic histone deacetylase (HDAC) inhibitor. KBH-A42 inhibited a variety of HDAC isoforms in enzyme assays and suppressed growth of various cancer cell lines. Among the cell lines examined, colon cancer cells, including SW620, SW480 and HCT-15, were the cell types most sensitive to KBH-A42. KBH-A42 inhibition of cancer cell growth was comparable to or stronger than that of suberoylanilide hydroxamic acid (SAHA), a well-known HDAC inhibitor approved by the FDA to treat cutaneous T cell lymphomas. In SW620 cells, KBH-A42 increased the acetylation of histones, mediated cell cycle arrest (G1 arrest at low doses and G2 arrest at high doses), and induced apoptosis. The cell cycle arrest and apoptosis induced by KBH-A42 might be mediated through up-regulation of p21^Waf1 and activation of caspases, respectively. In addition, KBH-A42 inhibited SW620 tumor growth in a human tumor xenograft model. Taken together, our results indicate that KBH-A42 exerts an anti-tumor activity in vitro and in vivo and is a promising therapeutic candidate to treat human cancers.

Keywords: HDAC; KBH-A42; Colon cancer; Cell cycle arrest; Apoptosis

Novel role of thiadiazolidine derivatives in inducing cell death through Myc-Max, Akt, FKHR, and FasL pathway by Pongali B. Raghavendra; Niteen Pathak; Sunil K. Manna (pp. 495-503).

The 1,2,4-thiadiazolidine derivatives show anti-fungal and anti-inflammatory activities. We previously reported that these derivatives inhibit nuclear factor-kappaB (NF-κB), a transcription factor that induces tumorigenesis through activation of several genes. We have aimed to elucidate the mechanism of apoptosis mediated by these derivatives. In this study we provide evidence that dichlorophenyl form of thiadiazolidine (designated as P₃-25) is a potential inducer of cell death by arresting cell cycle at G1 phase and decreases the amounts of cyclin D1 and cyclin E without interfering p16 and p27. It decreased c-Myc level and thereby inhibited DNA binding ability of Myc-Max complex. P₃-25 dephosphorylated Rb and Akt facilitating nuclear translocation of FKHR that then expressed gene FasL. Activated FasL inhibited cell proliferation and induced cell death. Our results suggest that P₃-25 derivative exerts anti-tumor activities by decreasing Myc-mediated response and increasing FasL expression, which may help in designing drugs for tumor therapy.

Keywords: Abbreviations; Bim; Bcl-2 interacting mediator of cell death; FBS; fetal bovine serum; P; ₃; -25; 5-(4-methoxyarylimino)-2-N-(3,4-dichlorophenyl)-3-Oxo-1,2,4-thiadiazolidine; Rb; retinoblastoma; p-Rb; phospho-Rb; FKHR; forkhead transcription factor1,2,4-Thiadiazolidine; Cell signaling; Apoptosis; FasL; Myc; Cell cycle

STIM1 but not STIM2 is an essential regulator of Ca²⁺ influx-mediated NADPH oxidase activity in neutrophil-like HL-60 cells by S. Bréchard; S. Plançon; C. Melchior; E.J. Tschirhart (pp. 504-513).

STIM1, not STIM2, regulates Ca²⁺ influx-mediated NADPH oxidase activity in neutrophil-like HL-60 cells.Extracellular Ca²⁺ entry, primarily mediated through store-operated Ca²⁺ entry (SOCE), is known to be a critical event for NADPH oxidase (NOX2) regulation in neutrophils. While defective NOX2 activity has been linked to various inflammatory diseases, regulatory mechanisms that control Ca²⁺ influx-induced NOX2 activation are poorly understood in SOCE. The role of STIM1, a Ca²⁺ sensor that transduces the store depletion signal to the plasma membrane, seems well established and supported by numerous studies in non-phagocytic cells. Here, in neutrophil-like HL-60 cells we used a siRNA approach to delineate the effect of STIM1 knock-down on NOX2 activity regulated by Ca²⁺ influx. Because the function of the STIM1 homolog, STIM2, is still unclear, we determined the consequence of STIM2 knock-down on Ca²⁺ and NOX2. STIM1 and STIM2 knock-down was effective and isoform specific when assayed by real-time PCR and Western blotting. Consistent with a unique role of STIM1 in the regulation of SOCE, STIM1, but not STIM2, siRNA significantly decreased Ca²⁺ influx induced by fMLF or the SERCA pump inhibitor thapsigargin. A redistribution of STIM1, originally localized intracellularly, near the plasma membrane was observed by confocal microscopy upon stimulation by fMLF. Inhibition of STIM1-induced SOCE led to a marked decrease in NOX2 activity while STIM2 siRNA had no effect. Thus, our results provide evidence for a role of STIM1 protein in the control of Ca²⁺ influx in neutrophils excluding a STIM2 involvement in this process. It also places STIM1 as a key modulator of NOX2 activity with a potential interest for anti-inflammatory pharmacological development.

Keywords: Abbreviations; 2-APB; 2-aminoethoxydiphenyl borane; [Ca; ²⁺; ]; _c; cytosolic free calcium concentration; fMLF; N-formyl-; l; -methionyl-; l; -leucyl-; l; -phenylalanine; siRNA; small interfering RNA; SOCE; store-operated Ca; ²⁺; entry; SOCs; store-operated Ca; ²⁺; channels; STIM; stromal-interacting moleculeNeutrophils; Chemoattractant; STIM1; STIM2; Store-operated Ca; ²⁺; entry; NADPH oxidase

Types I and II interferons upregulate the costimulatory CD80 molecule in monocytes via interferon regulatory factor-1 by B. Bauvois; J. Nguyen; R. Tang; C. Billard; J.-P. Kolb (pp. 514-522).

CD80/B7.1 expressed on monocytes plays a prominent role in the activation of T cell-mediated immunity and its level is reduced in monocytes from cancer patients. Type I (α/β) and type II (γ) IFNs are widely administered as adjuvant therapy. We show here that both classes of IFNs upregulate CD80 mRNA and protein in primary monocytes ex vivo. The stimulatory action of IFN-α/β on CD80 is accompanied by the activation of both interferon regulatory factors IRF-1 and IRF-7, whereas IFN-γ stimulating effect is associated only with IRF-1 induction. IFNs concomitantly upregulate the transcription of CD40 costimulatory molecule whose activation is known to require IRF-1. In monocytic U937 cells, IRF-1 is activated by IFN-γ but not by IFN-α/β, whereas it is the reverse for IRF-7; in the latter cells, only IFN-γ is capable of stimulating CD80 transcription emphasizing the essential role of IRF-1. Moreover, siRNA against IRF-1 prevents IFN-γ-mediated CD80 activation. In AML cells, IFNs upregulate CD40, CD80 and IRF-1 in the FAB-M4/M5 subtypes but not in the less differentiated M1/M2 subtypes. Monitoring the expression of CD80 on AML cells and its modulation by IFNs could help to predict the patients more susceptible to benefit from therapeutic strategies aimed at eliciting specific T cell responses to leukemia-associated antigens.

Keywords: B7.1; Antigen presenting cells; IRF-1; Acute myeloid leukemia

Identification of a functional peroxisome proliferator-activated receptor (PPAR) response element (PPRE) in the human apolipoprotein A-IV gene by Michiaki Nagasawa; Tomoko Hara; Ai Kashino; Yunike Akasaka; Tomohiro Ide; Koji Murakami (pp. 523-530).

Peroxisome proliferator-activated receptor-α (PPARα) is a key regulator in hepatic lipid metabolism and is a potential therapeutic target for dyslipidaemia. We reported previously that human hepatic apoA-IV is a highly sensitive gene up-regulated by the PPARα agonist KRP-101 (KRP), suggesting that induction of apoA-IV expression is one of the mechanisms underlying the decrease in triglycerides and elevation of HDL observed with PPARα agonist treatment. However, the mechanism of transcriptional regulation of apoA-IV by PPARα activation remains unclear. To clarify whether the apoA-IV promoter is regulated directly by PPARα, we analysed the apoA-IV promoter region by transient transfection assay in the human hepatocellular carcinoma cell line, HepG2. Co-transfection assay of unilateral deletions of apoA-IV promoter construct with human PPARα/RXRα showed that the region from −3279 to −2261 of the apoA-IV promoter includes key sites for transactivation by PPARα/RXRα. Sequence analysis suggested three putative PPAR response elements (PPREs) in this region. Electrophoretic mobility shift assay (EMSA) showed that a PPRE located from −2979 to −2967 can bind to PPARα/RXRα. Moreover, site-directed mutagenesis experiments indicated that the −2979/−2967 PPRE plays an essential role in transcriptional regulation of apoA-IV by PPARα. Chromatin immunoprecipitation (ChIP) assay confirmed that ligand-induced binding of PPARα to endogenous −2979/−2967 PPRE. These results indicate that human apoA-IV is regulated directly by PPARα via the −2979/−2967 PPRE.

Keywords: Abbreviations; apo; apolipoprotein; ChIP; chromatin immunoprecipitation; DMEM; Dulbecco's modified Eagle's medium; HDL; high density lipoprotein; PPAR; peroxisome proliferator-activated receptor; PPRE; PPAR response elementApolipoprotein; HepG2 cells; KRP-101; PPARα; PPRE; RXRα

Extreme variability in the formation of chlorpyrifos oxon (CPO) in patients poisoned by chlorpyrifos (CPF) by Florian Eyer; Darren M. Roberts; Nicholas A. Buckley; Michael Eddleston; Horst Thiermann; Franz Worek; Peter Eyer (pp. 531-537).

Formation of chlorpyrifos oxon (CPO) in self-inflicted poisoning with chlorpyrifos (CPF).Left: time course of plasma concentrations (single patient). Right: ratio of CPO/CPF 2–5 days after poisoning ( n=74).Chlorpyrifos (CPF) is a pesticide that causes tens of thousands of deaths per year worldwide. Chlorpyrifos oxon (CPO) is the active metabolite of CPF that inhibits acetylcholinesterase. However, this presumed metabolite has escaped detection in human samples by conventional methods (HPLC, GC-MS, LC-MS) until now. A recently developed enzyme-based assay allowed the determination of CPO in the nanomolar range and was successfully employed to detect this metabolite. CPO and CPF were analysed in consecutive plasma samples of 74 patients with intentional CPF poisoning. A wide concentration range of CPO and CPF was observed and the ratio of CPO/CPF varied considerably between individuals and over time. The ratio increased during the course of poisoning from a mean of 0.005 in the first few hours after ingestion up to an apparent steady-state mean of 0.03 between 30 and 72h. There was a hundred-fold variation in the ratio between samples and the interquartile range (between individuals) indicated over half the samples had a 5-fold or greater variation from the mean. The ratio was independent of the CPF concentration and the pralidoxime regimen. CPO was present in sufficient quantities to explain any observed acetylcholinesterase inhibitory activity. The effectiveness of pralidoxime in reactivating the inhibited acetylcholinesterase is strongly dependent on the CPO concentration. Differences in clinical outcomes and the response to antidotes in patients with acute poisoning may occur due to inter-individual variability in metabolism.

Keywords: Abbreviations; AChE; acetylcholinesterase (EC 3.1.1.7); BChE; butyrylcholinesterase (EC 3.1.1.8); CPF; chlorpyrifos; CPO; chlorpyrifos oxon; PON 1; paraoxonase 1 (EC 3.1.8.1); RBC; red blood cellsOrganophosphorus; Chlorpyrifos; Poisoning; Toxicokinetics; Pralidoxime

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Biochemical Pharmacology (v.78, #5)