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Biochemical Pharmacology (v.73, #4)

Editorial Advisory Board (pp. iii).

Partial agonists as therapeutic agents at neuronal nicotinic acetylcholine receptors by Ron C. Hogg; Daniel Bertrand (pp. 459-468).

Improved understanding of how brain function is altered in neurodegenerative disease states, pain and conditions, such as schizophrenia and attention deficit disorder, has highlighted the role of nicotinic acetylcholine receptors (nAChRs) in these conditions and identified them as promising therapeutic targets. nAChRs are widely expressed throughout the peripheral and central nervous system, and this widespread nature underlines the need for new ligands with different selectivities and pharmacological profiles if we are to avoid the adverse side effects associated with many of the nAChR modulators currently identified. Partial agonists have the unique property of being able to act both as agonists or antagonists depending on the concentration of endogenous neurotransmitter. Moreover, the agonist action of partial agonists has a ‘ceiling’ effect, giving them a large safety margin and making them an attractive proposition for therapeutic molecules. Partial agonists of nAChRs are currently being developed as a nicotine replacement therapy for smoking cessation and for the treatment of a number of neurological diseases associated with a loss of cholinergic function. This commentary will discuss the pharmacological properties of partial agonists and review recent research developments in the field of partial agonists acting at nicotinic receptors.

Keywords: Partial agonist; Acetylcholine; Nicotinic acetylcholine receptor; Agonist; Neurodegeneration; Ion channels

Irofulven induces replication-dependent CHK2 activation related to p53 status by Yutian Wang; Timothy Wiltshire; Jamie Senft; Eddie Reed; Weixin Wang (pp. 469-480).

CHK2 and p53 are frequently mutated in human cancers. CHK2 is known to phosphorylate and stabilize p53. CHK2 has also been implicated in DNA repair and apoptosis induction. However, whether p53 affects CHK2 activation and whether CHK2 activation modulates chemosensitivity are unclear. In this study, we found that in response to the DNA damage agent, irofulven, CHK2 activation, rather than its expression, is inversely correlated to p53 status. Irofulven inhibits DNA replication and induces chromosome aberrations (breaks and radials) and p53-dependent cell cycle arrest. Pretreatment of cells with the DNA polymerase inhibitor, aphidicolin, resulted in reduction of irofulven-induced CHK2 activation and foci formation, indicating that CHK2 activation by irofulven is replication-dependent. Furthermore, by using ovarian cancer cell lines expressing dominant-negative CHK2 and CHK2-knockout HCT116 cells, we found that CHK2 activation contributes to the control of S and G2/M cell cycle arrests, but not chemosensitivity to irofulven. Overall, this study demonstrates that in response to irofulven-induced DNA damage, the activation of CHK2 is dependent on DNA replication and related to p53 status. By controlling cell cycle arrest and DNA replication, p53 affects CHK2 activation. CHK2 activation contributes to cell cycle arrest, but not chemosensitivity.

Keywords: CHK2; p53; Replication; Chemosensitivity; Cell cycle; Irofulven

Inducible overexpression of c-Jun in MCF7 cells causes resistance to vinblastine via inhibition of drug-induced apoptosis and senescence at a step subsequent to mitotic arrest by Lingling Duan; Kristen Sterba; Sergey Kolomeichuk; Heetae Kim; Powel H. Brown; Timothy C. Chambers (pp. 481-490).

c-Jun is a major component of the AP-1 transcription factor and plays a key role in regulation of diverse biological processes including proliferation and apoptosis. Treatment of a wide variety of cells with the microtubule inhibitor vinblastine leads to a robust increase in c-Jun expression, JNK-mediated c-Jun phosphorylation, and activation of AP-1-dependent transcription. However, the role of c-Jun induction in the response of cells to vinblastine remains obscure. In this study we used MCF7 breast cancer cell lines that express the dominant-negative form of c-Jun, TAM-67, as well as cells that overexpress c-Jun, under the control of an inducible promoter. Vinblastine induced c-Jun protein expression, c-Jun phosphorylation, and AP-1 activation in MCF7 cells, and these parameters were strongly inhibited by inducible TAM-67 expression and strongly enhanced by inducible c-Jun expression. Vinblastine-induced cell death was not affected by TAM-67 expression whereas cells were protected by c-Jun overexpression. Further investigation revealed that apoptotic and senescent cells were observed after vinblastine treatment and that both outcomes were strongly inhibited by c-Jun overexpression. Although c-Jun expression inhibited cell death, it did not affect the ability of vinblastine to induce mitotic arrest. These results indicate that c-Jun expression plays a protective role in the cellular response to vinblastine and operates post-mitotic block to inhibit drug-induced apoptosis and senescence.

Keywords: Abbreviations; AP-1; activator or activating protein 1; JNK; c-Jun NH; ₂; -terminal protein kinase; FBS; fetal bovine serum; MEM; minimal essential medium; DMSO; dimethyl sulphoxide; DTT; dithiothreitolc-Jun; TAM-67; AP-1; Vinblastine; Apoptosis; Senescence

Peripheral-type benzodiazepine receptor overexpression and knockdown in human breast cancer cells indicate its prominent role in tumor cell proliferation by Wenping Li; Matthew J. Hardwick; Dean Rosenthal; Martine Culty; Vassilios Papadopoulos (pp. 491-503).

The peripheral-type benzodiazepine receptor (PBR), an 18-kDa high affinity drug and cholesterol binding protein, is expressed at high levels in various cancers. Its expression is positively correlated with aggressive metastatic behavior in human breast cancer cells. To determine the role of PBR in tumor progression, two human mammary carcinoma cell lines were utilized: the non-aggressive MCF-7 cell line, which expresses extremely low PBR levels, and the highly aggressive MDA-MB-231 cell line, which has much higher PBR levels. We have generated stably transfected lines of the tetracycline-repressible MCF-7 cell line (MCF-7 Tet-Off) with inducible human PBR cDNA. Induction of PBR expression in MCF-7 Tet-Off cells increased PBR ligand binding and cell proliferation. Transfection of MDA-MB-231 cells with multiple siRNAs complementary to PBR (PBR–siRNAs) led to different levels of PBR mRNA knockdown. Lentiviral-mediated PBR RNA interference in MDA-MB-231 cells decreased PBR levels by 50%. Decreased PBR expression was associated with cell cycle arrest at G2 phase, decreased cell proliferation, and significant increases in the protein levels of the cyclin-dependent kinase inhibitor p21^WAF/CIP1. These changes were accompanied by p53 activation seen as increased p53 phosphorylation (Ser15). In parallel, increased proteolytic activation of caspase-3 was also observed. Taken together these results suggest that PBR protein expression is directly involved in regulating cell survival and proliferation in human breast cancer cells by influencing signaling mechanisms involved in cell cycle control and apoptosis.

Keywords: MCF-7; MDA-MB-231; RNA interference; Cell cycle; p21; ^WAF/CIP1; p53; Caspase-3

Myricetin induces human osteoblast differentiation through bone morphogenetic protein-2/p38 mitogen-activated protein kinase pathway by Ya-Ling Hsu; Jiunn-Kae Chang; Chu-Hung Tsai; Tzu-Tsung Chang Chien; Po-Lin Kuo (pp. 504-514).

Myricetin (3,3′,4′,5,5′,7-hexahydroxyflavone), a flavonoid compound, is present in vegetables and fruits. By means of alkaline phosphatase (ALP) activity, osteocalcin, and type I collagen enzyme-linked immunosorbent assay (ELISA), we have shown that myricetin exhibits a significant induction of differentiation in MG-63 and hFOB human osteoblasts. Alkaline phosphatase and osteocalcin are phenotypic markers for early-stage differentiated osteoblasts and terminally differentiated osteoblasts, respectively. Our results indicate that myricetin stimulates osteoblast differentiation at various stages, from maturation to terminally differentiated osteoblasts. Induction of differentiation by myricetin is associated with increased bone morphogenetic protein-2 (BMP-2) production. The BMP-2 antagonist noggin blocked myricetin-mediated ALP activity and osteocalcin secretion enhancement, indicating that BMP-2 production is required in myricetin-mediated osteoblast maturation and differentiation. Induction of differentiation by myricetin is associated with increased activation of SMAD1/5/8 and p38 mitogen-activated protein kinases. Cotreatment of p38 inhibitor SB203580 inhibited myricetin-mediated ALP upregulation and osteocalcin production. In conclusion, myricetin increased BMP-2 synthesis, and subsequently activated SMAD1/5/8 and p38 MAPK, and this effect may contribute to its action on the induction of osteoblast maturation and differentiation, followed by an increase of bone mass.

Keywords: Abbreviations; MAPK; mitogen-activated protein kinase; phospho-p38; phosphorylated p38; BMP; bone morphogenetic protein; ELISA; enzyme-linked immunosorbent assay; PNPP; 4-nitrophenyl phosphate; SB203580; 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazoleMyricetin; Osteoblasts; Differentiation; Bone morphogenetic protein-2; p38

Rottlerin inhibits human T cell responses by Cécile Springael; Séverine Thomas; Souad Rahmouni; Arnaud Vandamme; Michel Goldman; Fabienne Willems; Olivier Vosters (pp. 515-525).

Rottlerin is a pharmacological inhibitor of protein kinase C (PKC) θ, a novel PKC selectively expressed in T lymphocytes. PKC θ is known to regulate T cell receptor (TCR)/CD28 signalling pathways in T lymphocytes, but the impact of PKC θ inhibition on human T cell responses remains undefined. In this work, we describe the effects of rottlerin on the responses of CD4+ and CD8+ human T lymphocytes upon polyclonal activation. We observed a dose-dependent inhibition of CD4+ and CD8+ T cell proliferation in response to anti-CD3/anti-CD28 antibodies stimulation in the presence of rottlerin. This inhibition was associated with impaired CD25 expression and decreased interleukin (IL)-2 production in activated T cells. In contrast, rottlerin did not alter IL-2-induced T cell proliferation. Furthermore, we demonstrated that rottlerin blocked interferon (IFN) γ, IL-10 and IL-13 mRNA expression in TCR/CD28 activated CD4+ T cells. These findings place rottlerin as a potent immunosuppressive agent for the development of novel therapies in T cell mediated immune disorders.

Keywords: Abbreviations; AP-1; activator protein-1; APC; antigen-presenting cell; BSA; bovine serum albumin; Ca; ²⁺; calcium; DAG; diacylglycerol; DMSO; dimethyl sulphoxide; EAE; experimental autoimmune encephalomyelitis; HePTP; hematopoietic protein tyrosine phosphatase; IFN; interferon; K.O.; knockout; IL; interleukin; MFI; median fluorescence intensity; NFAT; nuclear factor of activated T cells; NF-κB; nuclear factor κB; PBMC; peripheral blood mononuclear cells; PKC; protein kinase C; PI; propidium iodide; PMA; phorbol myristate acetate; rhIL-2; recombinant human IL-2; SEB; staphylococcal enterotoxin B; TCR; T cell receptor; Th; T helperRottlerin; Human T cells; TCR/CD28 stimulation; Protein kinase C θ

The autocrine TNFα signalling loop in keratinocytes requires atypical PKC species and NF-κB activation but is independent of cholesterol-enriched membrane microdomains by Steen Lisby; Annesofie Faurschou; Robert Gniadecki (pp. 526-533).

Tumor necrosis factor α (TNFα) is involved in the pathogenesis of many inflammatory skin diseases. Epidermal keratinocytes produce and respond to TNFα via the cognate type 1 receptor (TNFR1). Little is known about regulation of TNFα signalling in this cell type. In this study, we report that in keratinocytes TNFα upregulates its own mRNA synthesis in an autocrine manner. This response peaks at approximately 1h of stimulation with TNFα but sustained elevated levels of TNFα mRNA are observed for up to 24h after stimulation and are dependent on the presence of the soluble cytokine. This autocrine response is mediated by the signalling cascade comprising TNFR1, atypical protein kinase C (aPKC) species and the transcription factor NF-κB, but is not dependent on the integrity of cholesterol-enriched membrane microdomains (lipid rafts). TNFα-stimulated keratinocytes produced the membrane-bound form of TNFα. It is conceivable that the described autocrine signalling loop contributes to the proinflammatory TNFα effect in the skin. The discovery of the crucial roles of aPKC and NF-κB might have consequences for the development of more selective anti-TNFα therapies for inflammatory skin diseases.

Keywords: Tumor necrosis factor α; Keratinocyte; Protein kinase C; NF-κB; Lipid rafts

Cholesterol reduction by methyl-β-cyclodextrin attenuates the delta opioid receptor-mediated signaling in neuronal cells but enhances it in non-neuronal cells by Peng Huang; Wei Xu; Su-In Yoon; Chongguang Chen; Parkson Lee-Gau Chong; Lee-Yuan Liu-Chen (pp. 534-549).

Opioid receptors have been shown to be located in and regulated by lipid rafts/caveolae in caveolin-rich non-neuronal cells. Here, we found that caveolin-1 level was very low in rat brain and undetectable in NG108-15 cells, which endogenously express delta opioid receptors (DOR). Rat caudate putamen (CPu) membranes, NG108-15 cells and CHO cells stably transfected with FLAG-mouse-DOR (CHO-FLAG-mDOR) were homogenized, sonicated in a detergent-free 0.5M Na₂CO₃ buffer and fractionated through discontinuous or continuous sucrose density gradients. About 70% of opioid receptors in CPu and DOR in both cell lines were present in low-density (5–20% sucrose) membrane domains enriched in cholesterol and ganglioside M1 (GM1), characteristics of lipid rafts in plasma membranes. In both cells, stimulation with permeable or non-permeable full agonists, but not with partial or inverse agonists, for 30min shifted ∼25% of DORs out of rafts, by a naloxone-reversible and pertussis toxin-insensitive mechanism, which may undergo internalization. Methyl-β-cyclodextrin (MCD) treatment greatly reduced cholesterol and shifted DOR to higher density fractions and decreased DPDPE affinities. MCD treatment attenuated DPDPE-induced [³⁵S]GTPγS binding in CPu and NG108-15 cells, but enhanced it in CHO-FLAG-mDOR cells. In CHO-FLAG-mDOR cells, G_αi co-immunoprecipitated with caveolin-1, which was shown to inhibit G_αi/o, and MCD treatment dramatically reduced the association leading to disinhibition. Thus, although localization in rafts and agonist-induced shift of DOR are independent of caveolin-1, lipid rafts sustain DOR-mediated signaling in caveolin-deficient neuronal cells, but appear to inhibit it in caveolin-enriched non-neuronal cells. Cholesterol-dependent association of caveolin-1 with and the resulting inhibition of G proteins may be a contributing factor.

Keywords: Abbreviations; CPu; caudate putamen; CHO cells; Chinese hamster ovary cells; CHO-FLAG-mDOR; CHO cells stably transfected with FLAG-mDOR cDNA; DPDPE; [D-Pen; ²; ,D-Pen; ⁵; ]-Enkephalin; DTT; dithiothreitol; FLAG epitope; (DYKDDDDK); FLAG-mDOR; FLAG-tagged mouse δ opioid receptor; GM1; ganglioside M1; GPCRs; G protein-coupled receptors; HRP; horseradish peroxidase; MCD; methyl-β-cyclodextrin; MES; 2-morpholinoethanesulfonic acid; PMSF; phenylmethylsulfonyl fluoride; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TBS-T; 10; mM Tris–HCl, 159; mM NaCl, 0.1% Tween-20, pH 7.4Lipid rafts; Opioid receptors; Cholesterol; Methyl-β-cyclodextrin; Caveolin; Brain; Neuron

Resveratrol protects dopaminergic neurons in midbrain slice culture from multiple insults by Mitsugi Okawara; Hiroshi Katsuki; Emi Kurimoto; Haruki Shibata; Toshiaki Kume; Akinori Akaike (pp. 550-560).

Increasing lines of evidence show that resveratrol, a polyphenol compound contained in several dietary products, exhibits cytoprotective actions. Notably, resveratrol activates sirtuin family of NAD-dependent histone deacetylases implicated in regulation of various cellular processes including gene transcription, DNA repair and apoptosis. Here we examined neuroprotective effect of resveratrol on dopaminergic neurons in organotypic midbrain slice culture. Resveratrol and quercetin, another sirtuin-activating polyphenol, prevented the decrease of dopaminergic neurons and the increase of propidium iodide uptake into slices induced by a dopaminergic neurotoxin 1-methyl-4-phenyl pyridinium (MPP⁺). Resveratrol also provided concentration-dependent neuroprotective effects against sodium azide, a mitochondrial complex IV inhibitor, and thrombin (EC number 3.4.21.5), a microglia-activating agent. Sirtuin inhibitors such as nicotinamide and sirtinol did not attenuate the protective effect of resveratrol against MPP⁺ cytotoxicity. Instead, we found that resveratrol prevented accumulation of reactive oxygen species, depletion of cellular glutathione, and cellular oxidative damage induced by MPP⁺, suggesting involvement of antioxidative properties in the neuroprotective action of resveratrol. On the other hand, resveratrol as well as a sirtuin activator NAD inhibited dopaminergic neurotoxicity of a DNA alkylating agent , N-methyl- N′-nitro- N-nitrosoguanidine (MNNG). Moreover, MNNG-induced increase in acetylation of p53, a representative target of sirtuin deacetylase activity, was suppressed by resveratrol. These results indicate that resveratrol can exert neuroprotective actions in dopaminergic neurons. Either antioxidative activity or sirtuin-activating potential may play an important role in the neuroprotectice actions of resveratrol against different kinds of insults.

Keywords: Parkinson's disease; Dopamine neuron; Mitochondrial dysfunction; Neuroinflammation; DNA damage; Oxidative stress

The metabolomics of (±)-arecoline 1-oxide in the mouse and its formation by human flavin-containing monooxygenases by Sarbani Giri; Kristopher W. Krausz; Jeffrey R. Idle; Frank J. Gonzalez (pp. 561-573).

The alkaloid arecoline is a main constituent of areca nuts that are chewed by approximately 600 million persons worldwide. A principal metabolite of arecoline is arecoline 1-oxide whose metabolism has been poorly studied. To redress this, synthetic (±)-arecoline 1-oxide was administered to mice (20mg/kg p.o.) and a metabolomic study performed on 0–12h urine using ultra-performance liquid chromatography-coupled time-of-flight mass spectrometry (UPLC-TOFMS) with multivariate data analysis. A total of 16 mass/retention time pairs yielded 13 metabolites of (±)-arecoline 1-oxide, most of them novel. Identity of metabolites was confirmed by tandem mass spectrometry. The principal pathways of metabolism of (±)-arecoline 1-oxide were mercapturic acid formation, with catabolism to mercaptan and methylmercaptan metabolites, apparent CC double-bond reduction, carboxylic acid reduction to the aldehyde (a novel pathway in mammals), N-oxide reduction, and de-esterification. Relative percentages of metabolites were determined directly from the metabolomic data. Approximately, 50% of the urinary metabolites corresponded to unchanged (±)-arecoline 1-oxide, 25% to other N-oxide metabolites, while approximately, 30% corresponded to mercapturic acids or their metabolites. Many metabolites, principally mercapturic acids and their derivatives, were excreted as diastereomers that could be resolved by UPLC-TOFMS. Arecoline was converted to arecoline 1-oxide in vitro by human flavin-containing monooxygenases FMO1 ( K_M: 13.6±4.9μM; V_MAX: 0.114±0.01nmolmin⁻¹μg⁻¹ protein) and FMO3 ( K_M: 44.5±8.0μM; V_MAX: 0.014±0.001nmolmin⁻¹μg⁻¹ protein), but not by FMO5 or any of 11 human cytochromes P450. This report underscores the power of metabolomics in drug metabolite mining.

Keywords: Arecoline; Metabolomics; Flavin-containing monooxygenase; Ultra-performance liquid chromatography; Tandem mass spectrometry; Coupled time-of-flight mass spectrometry

Activation of PPARα lowers synthesis and concentration of cholesterol by reduction of nuclear SREBP-2 by Bettina König; Alexander Koch; Julia Spielmann; Christian Hilgenfeld; Gabriele I. Stangl; Klaus Eder (pp. 574-585).

To elucidate the mechanisms underlying the cholesterol lowering effects of PPARα agonists we investigated key regulators of cholesterol synthesis and uptake in rats and in the rat hepatoma cell line Fao after treatment with the PPARα agonists clofibrate and WY 14,643, respectively. In rat liver as well as in Fao cells, PPARα activation led to a decrease of transcriptionally active nuclear SREBP-2. mRNA concentrations of the key regulators of SREBP processing, Insig-1 in rat liver and Insig-1 and Insig-2a in Fao cells, were increased upon PPARα activation. Thus we suggest, that the observed reduction of the amount of nuclear SREBP-2 was due to an inhibition of the processing of the precursor protein. Both, in rat liver and in Fao cells, mRNA concentrations of the SREBP-2 target genes HMG-CoA reductase (EC1.1.1.34) and LDL receptor were reduced after treatment with the PPARα agonists. Furthermore, treatment of Fao cells with WY 14,643 reduced cholesterol synthesis. As a result, the amount of total cholesterol in liver, plasma and lipoproteins of clofibrate treated rats and in WY 14,643 treated Fao cells was decreased compared to control animals and cells, respectively. In conclusion, we could show a novel link between PPARα and cholesterol metabolism by demonstrating that PPARα activation lowers cholesterol concentration by reducing the abundance of nuclear SREBP-2.

Keywords: Abbreviations; ACO; acyl-CoA oxidase; Cyp; cytochrome P450; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; HDL; high density lipoprotein; HMG-CoA; 3-hydroxy-3-methylglutaryl-CoA; Insig; insulin-induced gene; LDL; low density lipoprotein; PPAR; peroxisome proliferator-activated receptor; SCAP; SREBP cleavage activating protein; SREBP; sterol regulatory element binding protein; VLDL; very low density lipoproteinPeroxisome proliferator-activated receptor (PPAR)-α; Cholesterol; Sterol regulatory element binding protein (SREBP)-2; Insulin-induced gene (Insig); HMG-CoA reductase; LDL receptor

Involvement of alcohol and aldehyde dehydrogenase activities on hepatic retinoid metabolism and its possible participation in the progression of rat liver regeneration by Verónica López-Valencia; Pablo Rangel; Sandra Rodríguez; Rolando Hernández-Muñoz (pp. 586-596).

Liver alcohol dehydrogenase (ADH) activity is decreased towards exogenous substrates after partial hepatectomy (PH), probably due to putative endogenous substrates acting as ADH inhibitors. Hence, retinoids could be suitable candidates as such endogenous substrates. Therefore, cytosolic ADH kinetic analysis using several substrates, liver cytosolic and mitochondrial aldehyde dehydrogenase (ALDH) activities, retinal and retinol content, as well as expression of proteins for ADH and CRBPI (a retinol carrier protein) were determined in liver samples, at two stages of liver regeneration (one- or two-thirds PH). The effect of inhibiting in vivo liver ADH by 4-methylpyrazole (4-MP) was also evaluated after 70%-PH. With 70%-PH, in vitro ADH activity towards exogenous alcohols and aldehydes was diminished, but retinol oxidation was increased and retinal reduction was decreased. These activities that be due to the participation of an ADH type which did not correlate with the amount of immunoreactive ADH protein. Cytosolic and mitochondrial ALDH activities oxidized actively retinal, whereas retinol and CBRP-I expression were reduced in these animals. With 30%-PH, these changes were less evident and sometimes opposite to those found with 70%-PH. In addition, retinol readily inhibited ADH-mediated ethanol oxidation. Interestingly, in vivo 4-MP administration inhibited ADH activity in a dose-dependent manner correlating with a progressive inhibition of liver regeneration. In conclusion, PH-induced inhibition of ADH (mainly type I) seems to be related to ADH-mediated retinoid metabolism during liver proliferation. Thus, results suggest a role of ADH in retinoid metabolism, which is apparently required during rat liver regeneration.

Keywords: Liver proliferation; Retinol; Cellular retinal-binding protein (CRBP); 4-Methylpyrazole; Retinol/retinal ratio

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Biochemical Pharmacology (v.73, #4)

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Biochemical Pharmacology (v.73, #4)