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Biochemical Pharmacology (v.69, #7)
Emerging prospects for the disease-modifying treatment of Alzheimer's disease by Lary C. Walker; Chris C. Ibegbu; Charles W. Todd; Harriet L. Robinson; Mathias Jucker; Harry LeVine III; Sam Gandy (pp. 1001-1008).
The currently approved therapies for Alzheimer's disease (AD) in the US are designed to modify the function of specific neurotransmitter systems in the brain. While these palliative treatments can benefit some patients for a period of time, they do not halt the relentless cognitive and behavioral deterioration that characterize this neurodegenerative disorder. Consequently, much current research on AD is directed toward illuminating the disease process itself, particularly the abnormal accumulation of certain proteins in brain: the amyloid-β protein (Aβ) in senile plaques and cerebral blood vessels, and the tau protein in neurofibrillary tangles. Genetic, biochemical and pathologic evidence now favors a primary role of Aβ aggregation in the Alzheimer proteopathic cascade, and studies in mice indicate that lowering the amount of this protein in brain can be beneficial. Recently, Aβ-immunization therapy has emerged as a particularly promising therapeutic option for treating Alzheimer's disease, but unexpected treatment-related side-effects are an overriding issue. These adverse events were not anticipated from preclinical studies with rodents; hence, more biologically relevant models, such as nonhuman primates, are needed to test the safety and efficacy of novel therapies for Alzheimer's disease.
Keywords: Amyloid; Cerebral amyloid angiopathy; Primate; Proteopathy; Senile plaque; Tau; Transgenic mice; Vaccination
Anticancer metal compounds in NCI's tumor-screening database: putative mode of action by Ruili Huang; Anders Wallqvist; David G. Covell (pp. 1009-1039).
Clustering analysis of tumor cell cytotoxicity profiles for the National Cancer Institute (NCI)'s open compound repository has been used to catalog over 1100 metal or metalloid containing compounds with potential anticancer activity. The molecular features and corresponding reactivity of these compounds have been analyzed in terms of properties of their metals, their associated organic components (ligands) and their capacity to inhibit tumor cell growth. Cytotoxic responses are influenced by both the identity of the metal and the properties of its coordination ligand, with clear associations between structural similarities and cytotoxicity. Assignments of mechanisms of action (MOAs) for these compounds could be segregated into four broad response classes according to preference for binding to biological sulfhydryl groups, chelation, generation of reactive oxygen species (ROS), and production of lipophilic ions. Correlations between specific cytotoxic responses and differential gene expression profiles within the NCI's tumor cell panel serve as a validation for candidate biological targets and putative MOA classes. In addition, specific sensitivity toward subsets of metal containing agents has been found for certain tumor cell panels. Taken together, our results expand the knowledge base available for evaluating, designing and developing new metal-based anticancer drugs that may provide the basis for target-specific therapeutics.
Keywords: Data mining; NCI tumor screen; Metal compounds; Mechanism of action; Drug discovery; Cancer
The imidazoline-like drug S23515 affects lipid metabolism in hepatocyte by inhibiting the oxidosqualene:lanosterol cyclase activity by Nicolas Venteclef; Raphaelle Guillard; Marc Issandou (pp. 1041-1048).
Imidazoline-like drugs are centrally-acting antihypertensive agents that inhibit the activity of the sympathetic nervous system by interacting with the α2-adrenoreceptor and also with a non-adrenergic imidazoline binding site called the imidazoline 1 receptor. Recently, these molecules were proposed to play an additional role in cardiovascular diseases by acting on glucose and lipid metabolism. We used S23515, a potent imidazoline-like molecule acting selectively on blood pressure through the imidazoline 1 receptor, to decipher the effects of these drugs on lipid metabolism. We found that S23515 inhibited specifically and dose-dependently cholesterol synthesis in cultured rodent and primate hepatocytes. This hypocholesterolemic effect was likely due to the inhibition of the oxido:lanosterol cyclase (OSC), a rate-limiting enzyme in the cholesterol biosynthetic pathway. Partial OSC inhibition induced by S23515 led to the generation of 24( S),25-epoxycholesterol, a potent ligand for the liver X receptor (LXR). Furthermore, S23515 increased in human macrophages the expression of both ABCA1 and G1, the 2 ATP binding cassette transporters, which play a pivotal role in the reverse cholesterol transport. Thus, these results suggest that S23515, and potentially other imidazoline-like drugs, could exert hypolipidemic effects in addition to their hypotensive activities.
Keywords: Abbreviations; IBS; imidazoline binding site; MOS; 2,3-monoepoxysqualene; DOS; 2,3 ;22,23-diepoxysqualene; OSC; oxidosqualene:lanosterol cyclase; α2-AR; α2-adrenoreceptor; I1R; imidazoline 1 receptor; LXR; liver X receptor; ABC-A1/G1; ATP-binding cassette transporter A1 and G1; HMGCoA reductase; 3-hydroxy-3-methylglutaryl coenzyme A; LDL-r; low density lipoprotein receptor; SREBP; sterol regulatory element binding protein; FCS; fetal calf serumImidazoline; Oxydosquale:lanoterol cyclase; Lipid neo-synthesis
Dual effects of acetylsalicylic acid on mast cell degranulation, expression of cyclooxygenase-2 and release of pro-inflammatory cytokines by Esmaeil Mortaz; Frank A. Redegeld; Frans P. Nijkamp; Ferdi Engels (pp. 1049-1057).
Several studies have demonstrated that nonsteroidal anti-inflammatory drugs, such as acetylsalicylic acid (ASA), can have inhibitory or enhancing effects on inflammatory cell function. These effects seem independent of cyclooxygenase activity and prostaglandin synthesis inhibition. Here, we examined the effect of ASA on bone marrow-derived mast cells in more detail. ASA blocked the expression of cyclooxygenase-2, the production of tumor necrosis factor-α and interleukin-6, and the release of granule mediators from mast cells in a concentration-dependent fashion. Concomitantly, ASA inhibited nuclear factor (NF)-κB activity, as well as the phosphorylation and breakdown of the inhibitory protein IκB-α. We thus propose that the anti-inflammatory effects of ASA in mast cells are due to suppression of IκB kinase activity, thereby inhibiting subsequent phosphorylation and degradation of IκB-α, activation of NF-κB, and transcription of proinflammatory cytokines. The inhibition of BMMC degranulation was independent of NF-κB activation, however. Interestingly, the expression of cyclooxygenase-2 was not inhibited at 1mM ASA, but was even enhanced significantly. The latter might contribute to the adverse effects of ASA in ASA-sensitive asthmatics.
Keywords: Abbreviations; ASA; acetylsalicylic acid; Anti-DNP IgE; anti-dinitrophenol IgE; COX; cyclooxygenase; DNP-HSA; dinitrophenol human serum albumin; HPRT; hypoxanthine-guanine phosphoribosyltransferase; MTT; 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide; NaSal; sodium salicylate; RT-PCR; reverse transcription polymerase chain reactionMast cells; NF-κB; Allergy; Inflammation; IgE and antigen; ASA
Retinoblastoma protein is required for efficient colorectal carcinoma cell apoptosis by histone deacetylase inhibitors in the absence of p21Waf by Sascha Wagner; Klaus Roemer (pp. 1059-1067).
Colorectal cancer accounts for approximately 10% of all new cancer cases reported worldwide. High dietary fiber intake has been associated with a reduced risk for this type of neoplasia, and much of this effect is ascribed to the histone acetylase (HDAC) inhibitor n-butyrate produced in the gastrointestinal tract. Natural chemopreventive and several new synthetic HDAC inhibitors exert multiple effects on tumor cells including the induction of differentiation, cell cycle arrest and apoptosis. Since cancer cells undergo mutational changes, it will be important to understand precisely which pathway gains or losses modulate or compromise HDAC inhibitor efficacy. We have recently documented that n-butyrate can provoke apoptosis in human HCT116 colorectal carcinoma cells independently of the p53 tumor suppressor and p21Waf inhibitor. Here, we have developed cell lines on the basis of HCT116 p21−/− cells and HCT116 cells in which the retinoblastoma tumor suppressor protein Rb has been specifically knocked down by antisense expression. The cells were exposed to the DNA-damaging drugs adriamycin (ADR) and etoposide or the HDAC inhibitors n-butyrate and trichostatin A (TSA). While the maximal apoptotic response, observed in the absence of p21Waf, was unaffected by the additional knockdown of Rb when cells were treated with ADR or etoposide, the toxicity of the HDAC inhibitors was significantly reduced. This indicates that hyperphosphorylated Rb itself, dissociated from E2F1 transcription factor, can contribute – directly or indirectly – to tumor cell apoptosis provoked by HDAC inhibitors.
Keywords: Abbreviations; CRC; colorectal carcinoma; Rb; retinoblastoma protein; HDAC; histone deacetylase; ADR; adriamycin; TSA; trichostatin AHistone deacetylase inhibitors; Human colon cancer cells; Retinoblastoma protein; Apoptosis; p53; p21Waf
Cloning and pharmacological characterization of the cat urotensin-II receptor (UT) by Nambi Aiyar; Douglas G. Johns; Zhaohui Ao; Jyoti Disa; David J. Behm; James J. Foley; Peter T. Buckley; Henry M. Sarau; Harjeet K van-der-Keyl; Nabil A. Elshourbagy; Stephen A. Douglas (pp. 1069-1079).
Urotensin-II (U-II), acting through its G-protein-coupled receptor, UT, is a possible contributor to hypertension. Variable functional responses to U-II, both within and between species studied to date, complicate the characterization of UT antagonists. In the cat, however, U-II causes systemic hypertension and constricts arterial segments isolated from several vascular beds. The purpose of this study was to clone and pharmacologically characterize cat recombinant UT to determine whether this system represents a model for characterizing UT antagonists. Cloned cat UT displayed 74% identity to primate UT, and 77% identity to rodent UT. [125I] hU-II bound in a saturable manner to a single site on recombinant cat UT with high affinity ( KD 288±13pM) and high density ( Bmax 747±66fmol/mg protein). U-II isopeptides displayed equipotent, high affinity binding to cat UT ( Ki 1.8–5.3nM). Cat UT was coupled to intracellular [Ca2+] release (EC50 0.6±0.2nM) and total inositol phosphate (IP) formation (EC50 0.4±0.1nM). Protein kinase C activation desensitized cat, but not human, UT-mediated IP formation. UT mRNA expression was detected in cat blood vessels, trachea, lung, and kidney, where the medulla ( KD 815±34) and cortex and ( KD 316±39pM) displayed high affinity binding for human U-II (hU-II). The cat urotensin-II receptor represents a suitable in vitro model to examine the role of the U-II/UT system in the etiology of hypertension, assisting in the evaluation of the UT antagonists to help treat cardiovascular disease.
Keywords: Urotensin-II; UT antagonist; GPR-14; Feline; Radioligand binding; Signal transduction
Inhibition of CYP2E1 catalytic activity in vitro by S-adenosyl-l-methionine by Andres A. Caro; Arthur I. Cederbaum (pp. 1081-1093).
The objective of this work was to evaluate the possible in vitro interactions of S-adenosyl-l-methionine (SAM) and its metabolites S-(5′-Adenosyl)-l-homocysteine (SAH), 5′-Deoxy-5′-(methylthio)adenosine (MTA) and methionine with cytochrome P450 enzymes, in particular CYP2E1. SAM (but not SAH, MTA or methionine) produced a type II binding spectrum with liver microsomal cytochrome P450 from rats treated with acetone or isoniazid to induce CYP2E1. Binding was less effective for control microsomes. SAM did not alter the carbon monoxide binding spectrum of P450, nor denature P450 to P420, nor inhibit the activity of NADPH-P450 reductase. However, SAM inhibited the catalytic activity of CYP2E1 with typical substrates such as p-nitrophenol, ethanol, and dimethylnitrosamine, with an IC50 around 1.5–5mM. SAM was a non-competitive inhibitor of CYP2E1 catalytic activity and its inhibitory actions could not be mimicked by methionine, SAH or MTA. However, SAM did not inhibit the oxidation of ethanol to α-hydroxyethyl radical, an assay for hydroxyl radical generation. In microsomes engineered to express individual human P450s, SAM produced a type II binding spectrum with CYP2E1-, but not with CYP3A4-expressing microsomes, and SAM was a weaker inhibitor against the metabolism of a specific CYP3A4 substrate than a specific CYP2E1 substrate. SAM also inhibited CYP2E1 catalytic activity in intact HepG2 cells engineered to express CYP2E1. These results suggest that SAM interacts with cytochrome P450s, especially CYP2E1, and inhibits the catalytic activity of CYP2E1 in a reversible and non competitive manner. However, SAM is a weak inhibitor of CYP2E1. Since the Ki for SAM inhibition of CYP2E1 activity is relatively high, inhibition of CYP2E1 activity is not likely to play a major role in the ability of SAM to protect against the hepatotoxicity produced by toxins requiring metabolic activation by CYP2E1 such as acetaminophen, ethanol, carbon tetrachloride, thioacetamide and carcinogens.
Keywords: Abbreviations; SAM; S; -adenosyl-; l; -methionine; POBN; N; -; tert; -Butyl-; α; -(4-pyridyl)nitrone; N; ′-oxide; SAH; S; -(5′-Adenosyl)-; l; -homocysteine; MTA; 5′-Deoxy-5′-(methylthio)adenosine; HER; α; -hydroxyethyl radical; 7-ECOD; 7-ethoxycoumarin; O; -deethylase; PNP; p; -nitrophenol; NDMA; dimethylnitrosamine; 7-MFC; 7-methoxy-4-trifluoromethylcoumarin; 7-HFC; 7-hydroxy-4-trifluoromethylcoumarin; DBF; dibenzylfluorescein; DDTC; diethydithiocarbamate; TCA; trichloroacetic acid; E47 cells; HepG2 cell line derived after transfection with pCI-neo vector containing the human CYP2E1 cDNA S; -adenosyl-; l; -methionine; Cytochrome P450; CYP2E1; Binding spectrum; Substrate oxidation; HepG2 cells
Differential modulation by simvastatin of the metabolic pathways in the n-9, n-6 and n-3 fatty acid series, in human monocytic and hepatocytic cell lines by Patrizia Risé; Silvia Ghezzi; Ilaria Priori; Claudio Galli (pp. 1095-1100).
Statins affect the production of long chain polyunsaturated fatty acids (PUFA), both in vitro and in vivo. Various studies have shown the effects of statins on the pattern of n-6 fatty acids (FA), but limited attention has been paid to the n-3 FA.We investigated, in THP-1 and in HepG2 cells, the effects of simvastatin on the conversion of the 18C FA precursors in the n-3 and n-6 series, [1-14C] α-linolenic acid (α-LNA) and [1-14C] linoleic acid (LA) respectively, and on the metabolism of [1-14C] stearic acid (SA).THP-1 cells, as in the case of LA, actively converted α-LNA to its products, and after simvastatin treatment, the total conversion was significantly increased (from 57.2±7.2 to 74.3±8.5%, p<0.05). HepG2 cells also converted LA and α-LNA, but simvastatin increased significantly only the conversion of LA (9.5±1.9% versus 23.8±5.1%, p<0.02). SA conversion was similar in untreated cells (about 50%), while statin increased the production of oleic acid in HepG2, but in THP-1 cells there was a decrease.In conclusion, LA, α-LNA and SA are differentially metabolized in THP-1 and in HepG2 cells and their increased conversion by simvastatin is lower in HepG2 than in THP-1. These differences may reflect the distinct features of the two cell lines: monocytes, precursors of phagocytic cells, versus hepatocytes with mainly metabolic functions. Substantial differences concern also cellular FA pools: structural in THP-1 cells, and also depot, resulting in sequestering of the substrates, in HepG2. The greater n-3 FA metabolism in THP-1 cells may have favourable functional effects.
Keywords: Abbreviations; α-LNA; α-linolenic acid; AA; arachidonic acid; DHA; docosahexaenoic acid; EPA; eicosapentaenoic acid; FA; fatty acids; LA; linoleic acid; OA; oleic acid; PL; phospholipids; PUFA; polyunsaturated fatty acids; SA; stearic acid; TL; total lipids; TG; triglyceridesSimvastatin; Linoleic acid; α-Linolenic acid; Stearic acid; Fatty acid metabolism; Cultured cells
(+)- And (−)-borneol: efficacious positive modulators of GABA action at human recombinant α1β2γ2L GABAA receptors by Renee E. Granger; Erica L. Campbell; Graham A.R. Johnston (pp. 1101-1111).
(+)-Borneol is a bicyclic monoterpene used for analgesia and anaesthesia in traditional Chinese and Japanese medicine and is found in the essential oils of medicinal herbs, such as valerian. (+)-Borneol was found to have a highly efficacious positive modulating action at GABAA receptors, as did its enantiomer (−)-borneol. The effects of these bicyclic monoterpenes alone and with GABA were evaluated at recombinant human α1β2γ2L GABAA receptors expressed in Xenopus laevis oocytes using two-electrode voltage-clamp electrophysiology. (+)-Borneol (EC50 248μM) and (−)-borneol (EC50 237μM) enhanced the action of low concentrations of GABA by more than 1000%. These enhancing effects were highly dependent on the relative concentrations of the borneol enantiomer and GABA, and were insensitive to flumazenil indicating that (+)- and (−)-borneol were not acting at classical benzodiazepine sites. The maximal responses to GABA were enhanced 19% by (+)-borneol and reduced 21% by (−)-borneol. The borneol analogues isoborneol, (−)-bornyl acetate and camphor, produced less marked effects. At high concentrations (>1.5mM) (+)- and (−)-borneol directly activated GABAA receptors producing 89% and 84%, respectively, of the maximal GABA response indicative of a weak partial agonist action. Although of lower potency, the highly efficacious positive modulatory actions of (+)- and (−)-borneol on GABA responses were at least equivalent to that of the anaesthetic etomidate and much greater than that of diazepam or 5α-pregnan-3α-ol-20-one. The relatively rigid cage structure of these bicyclic monoterpenes and their high efficacy may aid in a greater understanding of molecular aspects of positive modulation of the activation of GABAA receptors.
Keywords: Borneol; GABA; A; receptor; Monoterpene; Valeriana officinalis; Xenopus; oocyte
Silibinin prevents cholestasis-associated retrieval of the bile salt export pump, Bsep, in isolated rat hepatocyte couplets: Possible involvement of cAMP by Fernando A. Crocenzi; Cecilia L. Basiglio; Leonardo M. Pérez; María S. Portesio; Enrique J. Sánchez Pozzi; Marcelo G. Roma (pp. 1113-1120).
Estradiol-17ß-d-glucuronide (E217G) and taurolithocholate (TLC) induce acute cholestasis-associated with retrieval of the bile salt export pump (Bsep), which parallels with alteration in transport activity. cAMP stimulates the apically directed vesicular trafficking of transporters, partially preventing these alterations. The hepatoprotector, silymarin, which inhibits cAMP-phosphodiesterase, prevents the cholestasis induced in vivo by both estrogens and TLC.We aimed to assess the ability of silibinin (Sil), the silymarin active component, to prevent the retrieval of Bsep induced by TLC and E217G, and the associated alteration in its transport function. The possible involvement of cAMP as a second messenger and the intracellular signalling pathways implicated were also evaluated. Functional studies were performed analysing the proportion of isolated rat hepatocyte couplets (IRHC) accumulating the fluorescent bile salt analogue, cholyl-lysylfluorescein (CLF), into their sealed canalicular vacuoles. Cellular localisation of Bsep was assessed by immunofluorescent staining. Intracellular levels of cAMP were measured by ELISA. Sil (2.5μM) elevated by 40±3% intracellular cAMP, and mimicked the ability of dibutyryl-cAMP (10μM) to prevent internalisation of Bsep and the TLC (2.5μM)- and E217G (50μM)-induced impairment in the capacity of IRHC to accumulate CLF apically. Preventive effects of Sil and dibutyryl-cAMP were not abolished by the specific protein kinase A inhibitors, KT5720 and H89. Contrarily, the intracellular Ca2+ chelator, BAPTA/AM, significantly blocked the protective effect of both compounds.We conclude that Sil prevented TLC- and E217G-induced bile salt secretory failure, at least in part, by avoiding redistribution of Bsep, by a mechanism probably involving cAMP-induced cytosolic Ca2+ elevations.
Keywords: Abbreviations; BS; bile salts; Bsep; bile salt export pump; TLC; taurolithocholate; E; 2; 17G; estradiol-17ß-; d; -glucuronide; DBcAMP; N6,2′-; o; -dibutyryladenosine 3′,5′-cyclic monophosphate, dibutyryl-cAMP; IRHC; isolated rat hepatocyte couplets; Sil; silibinin; L-15; Leibovitz-15 culture medium; CLF; cholyl-lysylfluorescein; cVA; canalicular vacuolar accumulation; PKA; protein kinase A; Ntcp; sodium taurocholate cotransporting polypeptideEstradiol-17ß-; d; -glucuronide; Taurolithocholate; Bsep; Ca; 2+; - and PKA-dependent signalling pathways; Dibutyryl-cAMP; Isolated rat hepatocyte couplets
Inhibitors of calpain activation (PD150606 and E-64) and renal ischemia-reperfusion injury by Prabal K. Chatterjee; Zoran Todorovic; Ahila Sivarajah; Helder Mota-Filipe; Paul A.J. Brown; Keith N. Stewart; Emanuela Mazzon; Salvatore Cuzzocrea; Christoph Thiemermann (pp. 1121-1131).
Calpain activation has been implicated in the development of ischemia-reperfusion (I-R) injury. Here we investigate the effects of two inhibitors of calpain activity, PD150606 and E-64, on the renal dysfunction and injury caused by I-R of rat kidneys in vivo. Male Wistar rats were administered PD150606 or E-64 (3mg/kg i.p.) or vehicle (10%, v/v, DMSO) 30min prior to I-R. Rats were subjected to bilateral renal ischemia (45min) followed by reperfusion (6h). Serum and urinary biochemical indicators of renal dysfunction and injury were measured; serum creatinine (for glomerular dysfunction), fractional excretion of Na+ (FENa, for tubular dysfunction) and urinary N-acetyl-β-d-glucosaminidase (NAG, for tubular injury). Additionally, kidney tissues were used for histological analysis of renal injury, immunohistochemical analysis of intercellular adhesion molecule-1 (ICAM-1) expression and nitrotyrosine formation. Renal myeloperoxidase (MPO) activity (for polymorphonuclear leukocyte infiltration) and malondialdehyde (MDA) levels (for tissue lipid peroxidation) were determined. Both PD150606 and E-64 significantly reduced the increases in serum creatinine, FENa and NAG caused by renal I-R, indicating attenuation of renal dysfunction and injury and reduced histological evidence of renal damage caused by I-R. Both PD150606 and E-64 markedly reduced the evidence of oxidative stress (ICAM-1 expression, MPO activity, MDA levels) and nitrosative stress (nitrotyrosine formation) in rat kidneys subjected to I-R. These findings provide the first evidence that calpain inhibitors can reduce the renal dysfunction and injury caused by I-R of the kidney and may be useful in enhancing the tolerance of the kidney against renal injury associated with aortovascular surgery or renal transplantation.
Keywords: Abbreviations; ARF; acute renal failure; FE; Na; fractional excretion of Na; +; ICAM-1; intercellular adhesion molecule-1; I-R; ischemia-reperfusion; MDA; malondialdehyde; MPO; myeloperoxidase; NAG; N; -acetyl-β-; d; -glucosaminidase; NF-κB; nuclear factor-kappaB; PMN; polymorphonuclear leukocyteRenal/kidney; Ischemia; Reperfusion-injury; Calpain inhibitor; PD150606; E-64