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Biochemical Pharmacology (v.76, #4)
HDL-cholesterol: Is it really good? by Carlos G. Santos-Gallego; Borja Ibanez; Juan J. Badimon (pp. 443-452).
Since the very first report showing the regression of established atherosclerotic lesions by means of high-density lipoprotein cholesterol (HDL-C) plasma fraction, much information has been generated about the protective role of HDL-C in atherosclerosis. Nonetheless, this positive point of view about HDL has been nearly surpassed since modern informations concerning torcetrapib have appeared. Disappointment was palpable when its pivotal morbidity-and-mortality clinical trial, ILLUMINATE, was abruptly stopped due to excess mortality amongst the group randomized to receive torcetrapib. In this work we will try to put things in perspective.Lowering low-density lipoprotein cholesterol (LDL-C) levels with statins is a proven strategy for reducing the cardiovascular disease (CVD) risk. Despite the impressive benefits of statins, there remain a significant proportion of treated patients in which cardiovascular events are not prevented. Low HDL-C levels are an important independent risk factor for CVD. There is a need to develop suitable therapies to reduce this residual risk through HDL-C related mechanisms. Therefore, we will first review HDL-C pathways and we will subsequently state the new pharmacological approaches to HDL-C metabolism.
Keywords: Lipids; Cholesterol; Atherosclerosis
Physalin B, a novel inhibitor of the ubiquitin-proteasome pathway, triggers NOXA-associated apoptosis by Isabelle Vandenberghe; Laurent Créancier; Stéphane Vispé; Jean-Philippe Annereau; Jean-Marc Barret; Isabelle Pouny; Arnaud Samson; Yannick Aussagues; Georges Massiot; Frédéric Ausseil; Christian Bailly; Anna Kruczynski (pp. 453-462).
The ubiquitin-proteasome pathway plays a critical role in the degradation of proteins involved in tumor growth and has therefore become a target for cancer therapy. In order to discover novel inhibitors of this pathway, a cellular assay reporter of proteasome activity was established. Human DLD-1 colon cancer cells were engineered to express a 4 ubiquitin-luciferase (DLD-1 4Ub-Luc) reporter protein, rapidly degraded via the ubiquitin-proteasome pathway and designed DLD-1 4Ub-Luc cells. Following treatment with reference proteasome inhibitors, the 4Ub-Luc protein accumulated in DLD-1 4Ub-Luc cells and a 80-fold increase in luciferase-produced bioluminescence signal was measured, as compared to untreated cells. The screening of over 30,000 compounds using this DLD-1 4Ub-Luc assay led to the identification of physalin B as a novel inhibitor of the ubiquitin-proteasome pathway. Indeed, physalin B induced an increase in bioluminescence from DLD-1 4Ub-Luc cells, at concentrations also producing an accumulation of ubiquitinated proteins and inhibiting TNFα-induced NF-κB activation. Physalin B did not inhibit catalytic activities of purified proteasome and interfered with cellular proteasomal catalytic activities at 4- to 8-fold higher concentrations than that required to induce significant increase in bioluminescence and accumulation of ubiquitinated proteins in DLD-1 4Ub-Luc cells. Furthermore, physalin B proved to be cytotoxic, triggered apoptosis in DLD-1 4Ub-Luc cells and induced the proapoptotic protein NOXA, characteristic of the proteasome signaling pathway. Therefore, the use of the DLD-1 4Ub-Luc assay allowed the identification of a novel inhibitor of the ubiquitin-proteasome pathway that might interfere with proteasome functions in a different way from reference proteasome inhibitors.
Keywords: Abbreviations; ATCC; American type cell collection; DEVD-MR; Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-Magic Red; DMSO; dimethyl sulfoxide; DTT; dithiothreitol; FBS; fetal bovine serum; MEM; minimal essential medium; RLU; relative light unit; TNFα; tumor necrosis factor; Ub; ubiquitinUbiquitin-proteasome pathway; Proteasome inhibitor; Natural compound; Physalin; Luciferase-ubiquitin reporter; Apoptosis
Characterisation of the in vitro activity of the depsipeptide histone deacetylase inhibitor spiruchostatin A by Simon J. Crabb; Melanie Howell; Helen Rogers; Muhammad Ishfaq; Alexander Yurek-George; Krystle Carey; Becky M. Pickering; Phil East; Richard Mitter; Satoko Maeda; Peter W.M. Johnson; Paul Townsend; Kazuo Shin-ya; Minoru Yoshida; A. Ganesan; Graham Packham (pp. 463-475).
We recently completed the total synthesis of spiruchostatin A, a depsipeptide natural product with close structural similarities to FK228, a histone deacetylase (HDAC) inhibitor (HDI) currently being evaluated in clinical trials for cancer. Here we report a detailed characterisation of the in vitro activity of spiruchostatin A. Spiruchostatin A was a potent (sub-nM) inhibitor of class I HDAC activity in vitro and acted as a prodrug, requiring reduction for activity. Spiruchostatin A was a potent (low nM) inhibitor of the growth of various cancer cell lines. Spiruchostatin A-induced acetylation of specific lysine residues within histones H3 and H4, and increased the expression of p21cip1/waf1, but did not induce acetylation of α-tubulin. Spiruchostatin A also induced cell cycle arrest, differentiation and cell death in MCF7 breast cancer cells. Like FK228, spiruchostatin A was both an inducer and substrate of the ABCB1 drug efflux pump. Whereas spiruchostatin A and FK228-induced protracted histone acetylation, hydroxamate HDI-induced short-lived histone acetylation. Using a subset of HDI-target genes identified by microarray analysis, we demonstrated that these differences in kinetics of histone acetylation between HDI correlated with differences in the kinetics of induction or repression of specific target genes. Our results demonstrate that spiruchostatin A is a potent inhibitor of class I HDACs and anti-cancer agent. Differences in the kinetics of action of HDI may be important for the clinical application of these compounds.
Keywords: Spiruchostatin A; Depsipeptide; FK228; Histone deacetylase inhibitor; Cancer; Epigenetic
Occurrence and pharmacological characterization of four human tachykinin NK2 receptor variants by Ingela Ahlstedt; Susanna Engberg; John Smith; Chris Perrey; Adrian Moody; John Morten; Maria Lagerström-Fermér; Tomas Drmota; Bengt von Mentzer; Ingrid Påhlman; Erik Lindström (pp. 476-481).
Tachykinin NK2 receptor antagonists are potentially beneficial in treating various disorders including irritable bowel syndrome, urinary incontinence, depression and anxiety. The current study evaluates the frequency of single nucleotide polymorphisms (SNPs) in the human NK2 receptor gene (TACR2). In addition, the potency of the endogenous peptide agonist neurokinin A (NKA), and the small molecule antagonists saredutant (NK2-selective) and ZD6021 (pan-NK antagonist) at the various NK2 receptor protein variants were determined. The TACR2 gene was sequenced from 37 individuals. Two amino acid changing SNPs encoding the NK2 receptor variants Ile23Thr and Arg375His were found. The frequency of the four possible protein variants differed between populations. Site-directed mutagenesis was performed introducing either SNP or both SNPs into the TACR2 gene and the constructs were transfected into CHO cells. NKA-evoked increases in intracellular Ca2+ were monitored by FLIPR. The potency of saredutant and ZD6021 was evaluated by their ability to inhibit NKA-induced increases in intracellular Ca2+. NKA evoked increases in intracellular Ca2+ with a potency ranging between 1 and 5nM in CHO cells expressing the different constructs. Saredutant and ZD6021 blocked NKA-evoked increases in intracellular Ca2+ with p Kb values ranging between 8.8–9.3 and 7.9–8.7, respectively. The current study demonstrates that polymorphisms leading to the Ile23Thr and Arg375His amino acid exchanges are highly prevalent in the human TACR2 gene. These polymorphisms however do not appear to affect the potency of the endogenous agonist NKA or the small molecule antagonists saredutant and ZD6021 with respect to intracellular Ca2+ signalling.
Keywords: Tachykinin receptor antagonist; Neurokinin A; Saredutant; ZD6021; Polymorphism
The A3 adenosine receptor agonist CF502 inhibits the PI3K, PKB/Akt and NF-κB signaling pathway in synoviocytes from rheumatoid arthritis patients and in adjuvant-induced arthritis rats by A. Ochaion; S. Bar-Yehuda; S. Cohen; H. Amital; K.A. Jacobson; B.V. Joshi; Z.G. Gao; F. Barer; R. Patoka; L. Del Valle; G. Perez-Liz; P. Fishman (pp. 482-494).
The A3 adenosine receptor (A3AR) is over-expressed in inflammatory cells and was defined as a target to combat inflammation. Synthetic agonists to this receptor, such as IB-MECA and Cl-IB-MECA, exert an anti-inflammatory effect in experimental animal models of adjuvant- and collagen-induced arthritis.In this study we present a novel A3AR agonist, CF502, with high affinity and selectivity at the human A3AR. CF502 induced a dose dependent inhibitory effect on the proliferation of fibroblast-like synoviocytes (FLS) via de-regulation of the nuclear factor-kappa B (NF-κB) signaling pathway. Furthermore, CF502 markedly suppressed the clinical and pathological manifestations of adjuvant-induced arthritis (AIA) in a rat experimental model when given orally at a low dose (100μg/kg). As is typical of other G-protein coupled receptors, the A3AR expression level was down-regulated shortly after treatment with agonist CF502 in paw and in peripheral blood mononuclear cells (PBMCs) derived from treated AIA animals. Subsequently, a decrease in the expression levels of protein kinase B/Akt (PKB/Akt), IκB kinase (IKK), I kappa B (IκB), NF-κB and tumor necrosis factor-alpha (TNF-α) took place. In addition, the expression levels of glycogen synthase kinase-3 beta (GSK-3β), β-catenin, and poly(ADP-ribose)polymerase (PARP), known to control the level and activity of NF-κB, were down-regulated upon treatment with CF502.Taken together, CF502 inhibits FLS growth and the inflammatory manifestations of arthritis, supporting the development of A3AR agonists for the treatment of rheumatoid arthritis.
Keywords: Abbreviation; AR; adenosine receptorA; 3; adenosine receptor; CF502; Rheumatoid arthritis; PKB/Akt; NF-κB; TNF-α
Effect of flavonoids on rat splenocytes, a structure–activity relationship study by Rocío López-Posadas; Isabel Ballester; Ana Clara Abadía-Molina; María Dolores Suárez; Antonio Zarzuelo; Olga Martínez-Augustin; Fermín Sánchez de Medina (pp. 495-506).
Flavonoids are polyphenols frequently consumed in the diet which have been suggested to exert a number of beneficial actions on human health, including intestinal anti-inflammatory activity. Their properties have been studied in numerous cell types, but little is known about their effect on leukocyte biology. We have selected 9 flavonoids (extended to 14 flavonoids plus the related polyphenol resveratrol in some cases) with different structural features to characterize their effects on leukocyte viability, proliferation, and expression of cyclooxygenase 2 (EC 1.14.99.1), inducible nitric oxide synthase (iNOS, EC 1.14.13.39) and proinflammatory cytokines (TNF-α, IFN-γ, IL-2), as well as to elucidate the structural requirements in each case. Quiescent and concanavalin A-stimulated rat splenocytes were used as a model. Flavonoids (50μM) had a dramatic inhibitory effect on cytokine secretion. Inducible nitric oxide synthase expression was also blocked largely by some flavonoids, especially quercetin, luteolin and apigenin, while cyclooxygenase 2 was downregulated only by apigenin, diosmetin and quercetin. Apigenin, luteolin, genistein and quercetin had substantial cytotoxic/proapoptotic effects, while chrysin, daidzein, hesperetin and kaempferol did not reduce cell viability. In contrast, all flavonoids had powerful antiproliferative effects. However, none of the compounds activated caspase 3 (EC 3.4.22.56), but actually lowered caspase 3 activation and expression in concanavalin A-stimulated cells. The activity of the quercetin metabolite isorhamnetin was generally lower than that of the parent compound. We conclude that flavonoids have powerful effects on lymphocytes with distinct structural requirements that may contribute to their intestinal anti-inflammatory activity. The bioactivity of orally administered flavonoids may be dampened by biotransformation in vivo, particularly in extraintestinal sites.
Keywords: Abbreviations; ConA; concanavalin A; COX2; cyclooxygenase 2; DMSO; dimethyl sulfoxide; FACS; fluorescence-activated cell sorting; IFN-γ; interferon gamma; IL; interleukin; iNOS; inducible nitric oxide synthase; TNF-α; tumor necrosis factor alphaLymphocyte; Flavonoid; Viability; Toxicity; Proliferation
Inhibition of nitric oxide production by the carbazole compound LCY-2-CHO via blockade of activator protein-1 and CCAAT/enhancer-binding protein activation in microglia by Ling-Chu Chang; Lo-Ti Tsao; Chi-Sen Chang; Chun-Jung Chen; Li-Jiau Huang; Sheng-Chu Kuo; Ruey-Hseng Lin; Jih-Pyang Wang (pp. 507-519).
Excessive nitric oxide (NO) production by activated microglia plays a critical role in neurodegenerative disorders. In this study, we found that 9-(2-chlorobenyl)-9 H-carbazole-3-carbaldehyde (LCY-2-CHO) suppressed the NO production in lipopolysaccharide (LPS)/interferon-γ (IFNγ)-stimulated murine microglial N9 and BV-2 cells and in LPS-stimulated N9 cells and rat primary microglia. LCY-2-CHO had no cytotoxic effect on microglia. In activated N9 cells, LCY-2-CHO abolished the expression of inducible nitric oxide synthase (iNOS) protein and mRNA but failed to alter the stability of expressed iNOS mRNA and the enzymatic activity of expressed iNOS protein. LCY-2-CHO did not block DNA-binding activity of nuclear factor-κB (NF-κB) or cyclic AMP response element-binding protein (CREB), but abolished that of activator protein-1 (AP-1), CCAAT/enhancer-binding protein (C/EBP) and nuclear factor IL6 (NF-IL6). LCY-2-CHO attenuated the nuclear levels of c-Jun and C/EBPβ, but not those of p65, p50, C/EBPδ, signal transducer and activator of transcription-1 (STAT-1) or the nuclear expression of IFN regulatory factor-1 (IRF-1). LCY-2-CHO had no effect on the phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), MAPK-activated protein kinase-2 (MAPKAPK-2), STAT-1, CREB or c-Jun in LPS/IFNγ-stimulated N9 cells, whereas it attenuated the phosphorylation of C/EBPβ at Ser105 and Thr235 residues, which occurred concomitantly with LCY-2-CHO inhibition of C/EBPβ expression and phosphorylation. Taken together, these results suggest that LCY-2-CHO inhibits NO production in microglia through the blockade of AP-1 and C/EBP activation.
Keywords: Abbreviations; AP-1; activator protein-1; C/EBP; CCAAT/enhancer-binding protein; CREB; cyclic AMP response element-binding protein; EMSA; electrophoretic mobility shift assay; ERK; extracellular signal-regulated kinase; IFNγ; interferon-γ; iNOS; inducible nitric oxide synthase; IRF-1; IFN regulatory factor-1; JAK; Janus tyrosine kinase; JNK; c-Jun NH; 2; -terminal kinase; LCY-2-CHO; 9-(2-chlorobenyl)-9; H; -carbazole-3-carbaldehyde; LPS; lipopolysaccharide; MAPK; mitogen-activated protein kinase; MAPKAPK-2; MAPK-activated protein kinase-2; NF-IL6; nuclear factor IL-6; NF-κB; nuclear factor-κB; NO; nitric oxide; STAT; signal transducer and activator of transcriptionLCY-2-CHO; Microglial cells; Nitric oxide; Inducible nitric oxide synthase; Activator protein-1; CCAAT/enhancer-binding protein
Functions of acidic transmembrane residues in human melanocortin-3 receptor binding and activation by Shu-Xiu Wang; Zhen-Chuan Fan; Ya-Xiong Tao (pp. 520-530).
The melanocortin-3 receptor (MC3R) is an important regulator of energy homeostasis, inflammation, and cardiovascular function. Inactivating mutations in MC3R gene are associated with childhood obesity. How MC3R binds to its ligands has rarely been studied. In the present study, we systematically mutated all ten acidic residues in transmembrane (TM) domains and measured the cell surface expression levels as well as ligand binding and signaling properties of these mutants. Our results showed that of the 19 mutants stably expressed in HEK293 cells, all were expressed on the cell surface, although some mutants had decreased levels of cell surface expression. We showed that with the superpotent analog [Nle4, D-Phe7]-α-melanocyte stimulating hormone (MSH), E92, E131, D154, D158, D178, and D332 are important for ligand binding. D121 and D332 are important for binding and signaling. Further experiments using other ligands such as D-Trp8-γ-MSH, α-MSH and γ-MSH showed that different ligands induce or select different conformations. In summary, we showed that acidic residues in TMs 1 and 3 are important for ligand binding whereas the acidic residues in TMs 2 and 7 are important for both ligand binding and signaling.
Keywords: Melanocortin-3 receptor; Site-directed mutagenesis; Ligand binding; G protein-coupled receptor; Signaling; Structure–function relationship
Identification of the functional vitamin D response elements in the human MDR1 gene by Mayumi Saeki; Kouichi Kurose; Masahiro Tohkin; Ryuichi Hasegawa (pp. 531-542).
P-glycoprotein, encoded by the multidrug resistance 1 ( MDR1) gene, is an efflux transporter and plays an important role in pharmacokinetics. The expression of MDR1 is induced by a variety of compounds, of which 1α,25-dihydroxyvitamin D3 is known to be an effective inducer. However, it remains unclear how 1α,25-dihydroxyvitamin D3 regulates the expression of MDR1. In this study, we demonstrated that the vitamin D receptor (VDR) induces MDR1 expression in a 1α,25-dihydroxyvitamin D3-dependent manner. Luciferase assays revealed that the region between −7.9 and −7.8kbp upstream from the transcription start site of the MDR1 is responsible for the induction by 1α,25-dihydroxyvitamin D3. Electrophoretic mobility shift assays revealed that several binding sites for the VDR/retinoid X receptor α (RXRα) heterodimer are located between the −7880 and −7810bp region, to which the three molecules of VDR/RXRα are able to simultaneously bind with different affinities. Luciferase assays using mutated constructs revealed that the VDR-binding sites of DR3, DR4(I), MdC3, and DR4(III) contribute to the induction, indicating that these binding sites act as vitamin D response elements (VDREs). The contribution of each VDRE to the inducibility was different for each response element. An additive effect of the individual VDREs on induced luciferase activity by 1α,25-dihydroxyvitamin D3 was also observed. These results indicate that the induction of MDR1 by 1α,25-dihydroxyvitamin D3 is mediated by VDR/RXRα binding to several VDREs located between −7880 and −7810bp, in which every VDRE additively contributes to the 1α,25-dihydroxyvitamin D3 response.
Keywords: Multidrug resistance 1; P-glycoprotein (P-gp); Vitamin D receptor (VDR); 1α,25-Dihydroxyvitamin D; 3; Vitamin D response element (VDRE)
The relative contribution of human cytochrome P450 isoforms to the four caffeine oxidation pathways: An in vitro comparative study with cDNA-expressed P450s including CYP2C isoforms by Marta Kot; Władysława A. Daniel (pp. 543-551).
The aim of the present study was to estimate the relative contribution of cytochrome P450 isoforms (P450s), including P450s of the CYP2C subfamily, to the metabolism of caffeine in human liver. The experiments were carried out in vitro using cDNA-expressed P450s, liver microsomes and specific P450 inhibitors. The obtained results show that (1) apart from the 3-N-demethylation of caffeine – a CYP1A2 marker reaction and the main oxidation pathway of caffeine in man – 1-N-demethylation is also specifically catalyzed by CYP1A2 (not reported previously); (2) 7-N-demethylation is catalyzed non-specifically, mainly by CYP1A2 and, to a smaller extent, by CYP2C8/9 and CYP3A4 (and not by CYP2E1, as suggested previously); (3) C-8-hydroxylation preferentially involves CYP1A2 and CYP3A4 and, to a smaller degree, CYP2C8/9 and CYP2E1 (and not only CYP3A, as suggested previously) at a concentration of 100μM corresponding to the maximum therapeutic concentration in humans. At a higher caffeine concentration, the contribution of CYP1A2 to this reaction decreases in favour of CYP2C8/9. The obtained data show for the first time the contribution of CYP2C isoforms to the metabolism of caffeine in human liver and suggest that apart from 3-N-demethylation, 1-N-demethylation may also be used for testing CYP1A2 activity. Moreover, they indicate that the C-8-hydroxylation is not exclusively catalyzed by CYP3A4.
Keywords: Abbreviations; CYP; cytochrome P450; HPLC; high performance liquid chromatography; DDC; diethyldithiocarbamate; FUR; furafylline; SULF; sulfaphenazole; KET; ketoconazole; P450; cytochrome P450Caffeine metabolism; Human cytochrome P450; cDNA-expressed isoforms; Liver microsomes; Cytochrome P450 inhibitors
Role of mitochondrial dysfunction in cellular responses to S-(1,2-dichlorovinyl)-l-cysteine in primary cultures of human proximal tubular cells by Feng Xu; Irene Papanayotou; David A. Putt; Jian Wang; Lawrence H. Lash (pp. 552-567).
The nephrotoxic metabolite of the environmental contaminant trichloroethylene, S-(1,2-dichlorovinyl)-l-cysteine (DCVC), is known to elicit cytotoxicity in rat and human proximal tubular (rPT and hPT, respectively) cells that involves inhibition of mitochondrial function. DCVC produces a range of cytotoxic and compensatory responses in hPT cells, depending on dose and exposure time, including necrosis, apoptosis, repair, and enhanced cell proliferation. The present study tested the hypothesis that induction of mitochondrial dysfunction is an obligatory step in the cytotoxicity caused by DCVC in primary cultures of hPT cells. DCVC-induced necrosis was primarily a high concentration (≥50μM) and late (≥24h) response whereas apoptosis and increased proliferation occurred at relatively low concentrations (<50μM) and early time points (≤24h). Decreases in cellular DNA content, indicative of cell loss, were observed at DCVC concentrations as low as 1μM. Involvement of mitochondrial dysfunction in DCVC-induced cytotoxicity was supported by showing that DCVC caused modest depletion of cellular ATP, inhibition of respiration, and activation of caspase-3/7. Cyclosporin A protected cells against DCVC-induced apoptosis and both cyclosporin A and ruthenium red protected cells against DCVC-induced loss of mitochondrial membrane potential. DCVC caused little or no activation of caspase-8 and did not significantly induce expression of Fas receptor, consistent with apoptosis occurring only by the mitochondrial pathway. These results support the conclusion that mitochondrial dysfunction is an early and obligatory step in DCVC-induced cytotoxicity in hPT cells.
Keywords: Mitochondrial energetics; Human proximal tubular cells; Apoptosis; Cell proliferation; Cytotoxicity; FasR; Caspase activation