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Biochemical Pharmacology (v.83, #1)

Editorial Board (pp. iii).

Relevance of both type-1 and type-2 corticotropin releasing factor receptors in stress-induced relapse to cocaine seeking behaviour by Katia Gysling (pp. 1-5).

The essential role of corticotropin releasing factor (CRF) and its type-1 receptor (CRF1) in stress-induced relapse to drug seeking has been demonstrated. The bed nucleus of the stria terminalis is the major anatomical substrate for this CRF/CRF1 receptor action. More recently, the role of type-2 CRF (CRF2) receptors in stress-induced relapse to cocaine seeking has also has been documented. The ventral tegmental area is the anatomical substrate for this CRF/CRF2 receptor action. The new information involving CRF2 receptors in stress-induced relapse to cocaine seeking has generated a need for a reappraisal of the existing anatomical and pharmacological evidence that have been used to support the critical role of CRF1 receptors. The role of CRF2 receptors in stress-induced relapse to drug seeking also opens the question of the putative role of the other peptides of the CRH family (urocotin-1, urocortin-2 and urocortin-3) that have high affinity for CRF2 receptors. In this commentary, the available evidence supporting the role of both CRF1 and CRF2 receptors in stress-induced relapse to drug seeking is reviewed.

Keywords: CRF receptors; CRF binding protein; Relapse; Noradrenaline; Cocaine

Relevance of both type-1 and type-2 corticotropin releasing factor receptors in stress-induced relapse to cocaine seeking behaviour by Katia Gysling (pp. 1-5).

Keywords: CRF receptors; CRF binding protein; Relapse; Noradrenaline; Cocaine

The flavonoid quercetin in disease prevention and therapy: Facts and fancies by Maria Russo; Carmela Spagnuolo; Idolo Tedesco; Stefania Bilotto; Gian Luigi Russo (pp. 6-15).

Biochemical and genetic studies on cellular and animal models on the mechanism(s) of action of phytochemicals provide a functional explanation of how and why a diet rich in fruits and vegetables is considered healthy. It is not unusual to find molecules that protect against diseases, which greatly differ from a physiopathological point of view, such as cancer and cardiovascular disorders. Quercetin falls into this category and possesses a broad range of biological properties. Uptake, metabolism and circulating concentrations of quercetin and its metabolites suggest that a regular diet provides amounts of quercetin (<1μM) not compatible with its chemopreventive and/or cardioprotective effects. However, it appears relatively easy to increase total quercetin concentrations in plasma (>10μM) by supplementation with quercetin-enriched foods or supplements. Multiple lines of experimental evidence suggest a positive association between quercetin intake and improved outcomes of inflammatory cardiovascular risk. The ameliorating effect of quercetin administration can be extended to other chronic inflammatory disorders but only if supplementation occurs in patients. Quercetin can be considered the prototype of a naturally-occurring chemopreventive agent because of its key roles in triggering the “hallmarks of cancer”. However, several critical points must be taken into account when considering the potential therapeutic use of this molecule: (1) pharmacological versus nutraceutical doses applied, (2) specificity of its mechanism of action compared to other phytochemicals, and (3) identification of “direct” cellular targets. The design of specific clinical trials is extremely warranted to depict possible applications of quercetin in adjuvant cancer therapy.

Keywords: Quercetin; Cancer; Cardiovascular disease; Antioxidant; Bioavailability; Phytochemicals

The flavonoid quercetin in disease prevention and therapy: Facts and fancies by Maria Russo; Carmela Spagnuolo; Idolo Tedesco; Stefania Bilotto; Gian Luigi Russo (pp. 6-15).

Keywords: Quercetin; Cancer; Cardiovascular disease; Antioxidant; Bioavailability; Phytochemicals

MG-2477, a new tubulin inhibitor, induces autophagy through inhibition of the Akt/mTOR pathway and delayed apoptosis in A549 cells by Giampietro Viola; Roberta Bortolozzi; Ernest Hamel; Stefano Moro; Paola Brun; Ignazio Castagliuolo; Maria Grazia Ferlin; Giuseppe Basso (pp. 16-26).

Computer model depicting MG-2477 within the colchicine binding domain.We previously demonstrated that MG-2477 (3-cyclopropylmethyl-7-phenyl-3 H-pyrrolo[3,2- f]quinolin-9(6 H)-one) inhibits the growth of several cancer cell lines in vitro. Here we show that MG-2477 inhibited tubulin polymerization and caused cells to arrest in metaphase. The detailed mechanism of action of MG-2477 was investigated in a non-small cell lung carcinoma cell line (A549). Treatment of A549 cells with MG-2477 caused the cells to arrest in the G2/M phase of the cell cycle, with a concomitant accumulation of cyclin B. Moreover, the compound induced autophagy, which was followed at later times by apoptotic cell death. Autophagy was detected as early as 12h by the conversion of microtubule associated protein 1 light chain 3 (LC3-I) to LC3-II, following cleavage and lipid addition to LC3-I. After 48h of MG-2477 exposure, phosphatidylserine externalization on the cell membrane, caspase-3 activation, and PARP cleavage occurred, revealing that apoptotic cell death had begun. Pharmacological inhibition of autophagy with 3-methyladenine or bafilomycin A1 increased apoptotic cell death, suggesting that the autophagy caused by MG-2477 played a protective role and delayed apoptotic cell death. Additional studies revealed that MG-2477 inhibited survival signaling by blocking activation of Akt and its downstream targets, including mTOR, and FHKR. Treatment with MG-2477 also reduced phosphorylation of mTOR downstream targets p70 ribosomal S6 kinase and 4E-BP1. Overexpression of Akt by transfection with a Myr-Akt vector decreased MG-2477 induced autophagy, indicating that Akt is involved. Taken together, these results indicated that the autophagy induced by MG-2477 delayed apoptosis by exerting an adaptive response following microtubule damage.

Keywords: Autophagy; Apoptosis; Microtubule inhibitors; Cell cycle analysis; Akt/mTOR

Role of tyrosyl-DNA phosphodiesterase 1 and inter-players in regulation of tumor cell sensitivity to topoisomerase I inhibition by Paola Perego; Giacomo Cossa; Stella Tinelli; Elisabetta Corna; Nives Carenini; Laura Gatti; Michelandrea De Cesare; Emilio Ciusani; Franco Zunino; Elena Luison; Silvana Canevari; Nadia Zaffaroni; Giovanni Luca Beretta (pp. 27-36).

Tyrosyl-DNA phosphodiesterase 1 (TDP1) plays a unique function as it catalyzes the repair of topoisomerase I-mediated DNA damage. Thus, ovarian carcinoma cell lines exhibiting increased TDP1 levels and resistance to the topoisomerase I poisons campthotecins were used to clarify the role of this enzyme. The camptothecin gimatecan was employed as a tool to inhibit topoisomerase I because it produces a persistent damage. The resistant sublines displayed an increased capability to repair drug-induced single-strand breaks and a reduced amount of drug-induced double-strand breaks, which was enhanced following TDP1 silencing. In loss of function studies using U2-OS cells, we found that TDP1 knockdown did not produce a change in sensitivity to camptothecin, whereas co-silencing of other pathways cooperating with TDP1 in cell response to topoisomerase I poisons indicated that XRCC1 and BRCA1 were major regulators of sensitivity. No change in cellular sensitivity was observed when TDP1 was silenced concomitantly to RAD17, which participates in the stabilization of collapsed replication forks. The expression of dominant-negative PARP1 in cells with reduced expression of TDP1 due to a constitutively expressed TDP1 targeting microRNA did not modulate cell sensitivity to camptothecin. Mild resistance to gimatecan was observed in cells over-expressing TDP1, a feature associated with decreased levels of drug-induced single-strand breaks. In conclusion, since TDP1 alone can account for mild levels of camptothecin resistance, repair of topoisomerase I-mediated DNA damage likely occurs through redundant pathways mainly implicating BRCA1 and XRCC1, but not RAD17 and PARP1. These findings may be relevant to define novel therapeutic strategies.

Keywords: Tyrosyl-DNA phosphodiesterase 1; Camptothecin; DNA topoisomerase I; Drug resistance; DNA repair

7β-Hydroxycholesterol-induced energy stress leads to sequential opposing signaling responses and to death of c6 glioblastoma cells by Ludovic Clarion; Mathilde Schindler; Jan de Weille; Karine Lolmède; Audrey Laroche-Clary; Emmanuelle Uro-Coste; Jacques Robert; Marcel Mersel; Norbert Bakalara (pp. 37-46).

7β-Hydroxycholesterol cytotoxicity has been shown in vivo and in vitro to be dependent on the accumulation of its esters. We show in our study, using a detergent-free raft preparation and LC/MS lipid content analysis, that membrane microdomains isolated from 7β-hydroxycholesterol-treated C6 cells have a reduced cholesterol: cholesterol ester ratio and accumulate 7keto-hydroxycholesterol, 7β-hydroxycholesterol and 7β-hydroxycholesterol esters. These modifications in lipid content are accompanied by a redistribution of flotillin-1 in the lipid rafts. Transient increases of AMPK phosphorylation and mitochondrial activity during the first 12h of 7β-hydroxycholesterol treatment indicate that C6 cells undergo energy stress and increase oxidative phosphorylation. Even so, ATP levels are maintained during 15h until glucose uptake decreases. The cell's answers to raft modifications and energy stress are sequential activations of different signaling pathways such as ERK, AMPK and PI3K/Akt. These pathways, known to be activated under energy stress conditions, are transiently activated at 6h (ERK, AMPK) and 12h (Akt) of treatment respectively suggesting a shift from cell survival to cell proliferation. The persistence of 7β-hydroxycholesterol-induced stress led after 24h to P38 activation, loss of GSK3β activation and to cell death. Finally we demonstrate that the observed signaling responses depend on 7β-hydroxycholesterol esterification, confirming that esterification of 7β-hydroxycholesterol is essential for cytotoxicity.

Keywords: Abbreviations; 22HC; 22-hydroxycholesterol; 25HC; 25-hydroxycholesterol; 7kCH; 7keto-cholesterol; CH; cholesterol; 7βHC; 7β-hydroxycholesterol; 7βHC ester; 7β-hydroxy-cholesteryl 3 ester; CH ester; cholesterol ester; PL; phospholipid; cav-1; caveolin-1; flot-1; flotillin-1; ACAT; acyl-CoA:cholesterol acyltransferase; THF; tetrahydrofuraneGlioblastoma; Oxysterol; Lipid-raft; Energy stress; AMPK

DIF-1 inhibits the Wnt/β-catenin signaling pathway by inhibiting TCF7L2 expression in colon cancer cell lines by Kentaro Jingushi; Fumi Takahashi-Yanaga; Tatsuya Yoshihara; Fumie Shiraishi; Yutaka Watanabe; Masato Hirata; Sachio Morimoto; Toshiyuki Sasaguri (pp. 47-56).

We previously reported that differentiation-inducing factor-1 (DIF-1), a morphogen in Dictyostelium discoideum, inhibits the proliferation of human cancer cell lines by inducing β-catenin degradation and suppressing the Wnt/β-catenin signaling pathway. To determine whether β-catenin degradation is essential for the effect of DIF-1, we examined the effect of DIF-1 on human colon cancer cell lines (HCT-116, SW-620 and DLD-1), in which the Wnt/β-catenin signaling pathway is constitutively active. DIF-1 strongly inhibited cell proliferation and arrested the cell cycle in the G₀/G₁ phase via the suppression of cyclin D1 expression at mRNA and protein levels without reducing β-catenin protein. TCF-dependent transcriptional activity and cyclin D1 promoter activity were revealed to be inhibited via suppression of transcription factor 7-like 2 (TCF7L2) expression. Luciferase reporter assays and EMSAs using the TCF7L2 promoter fragments indicated that the binding site for the transcription factor early growth response-1 (Egr-1), which is located in the −609 to −601bp region relative to the start codon in the TCF7L2 promoter, was involved in DIF-1 activity. Moreover, RNAi-mediated depletion of endogenous TCF7L2 resulted in reduced cyclin D1 promoter activity and protein expression, and the overexpression of TCF7L2 overrode the inhibition of the TCF-dependent transcriptional activity and cyclin D1 promoter activity induced by DIF-1. Therefore, DIF-1 seemed to inhibit the Wnt/β-catenin signaling pathway by suppressing TCF7L2 expression via reduced Egr-1-dependent transcriptional activity in these colon cancer cell lines. Our results provide a novel insight into the mechanisms by which DIF-1 inhibits the Wnt/β-catenin signaling pathway.

Keywords: Abbreviations; DIF; differentiation-inducing factor; GSK-3β; glycogen synthase kinase-3β; TCF7L2; transcription factor 7-like 2; Egr-1; early growth response-1; APC; adenomatous polyposis coli; RT-PCR; reverse transcription-polymerase chain reaction; RNAi; RNA interference; EMSA; electrophoretic mobility shift assayWnt/β-catenin signaling pathway; Differentiation-inducing factor-1 (DIF-1); TCF7L2; Colon cancer cell; Cyclin D1

Dual inhibitors of P-glycoprotein and tumor cell growth: (Re)discovering thioxanthones by Andreia Palmeira; M. Helena Vasconcelos; Ana Paiva; Miguel X. Fernandes; Madalena Pinto; Emília Sousa (pp. 57-68).

1-Aminated thioxanthones are dual inhibitors of P-glycoprotein and tumor cell growth.For many pathologies, there is a crescent effort to design multiple ligands that interact with a wide variety of targets. 1-Aminated thioxanthone derivatives were synthesized and assayed for their in vitro dual activity as antitumor agents and P-glycoprotein (P-gp) inhibitors. The approach was based on molecular hybridization of a thioxanthone scaffold, present in known antitumor drugs, and an amine, described as an important pharmacophoric feature for P-gp inhibition. A rational approach using homology modeling and docking was used, to select the molecules to be synthesized by conventional or microwave-assisted Ullmann C–N cross-coupling reaction. The obtained aminated thioxanthones were highly effective at inhibiting P-gp and/or causing growth inhibition in a chronic myelogenous leukemia cell line, K562. Six of the aminated thioxanthones had GI₅₀ values in the K562 cell line below 10μM and 1-{[2-(diethylamino)ethyl]amino}-4-propoxy-9 H-thioxanthen-9-one (37) had a GI₅₀ concentration (1.90μM) 6-fold lower than doxorubicin (11.89μM) in the K562Dox cell line. The best P-gp inhibitor found was 1-[2-(1 H-benzimidazol-2-yl)ethanamine]-4-propoxy-9 H-thioxanthen-9-one (45), which caused an accumulation rate of rhodamine-123 similar to that caused by verapamil in the K562Dox resistant cell line, and a decrease in ATP consumption by P-gp. At a concentration of 10μM, compound45 caused a decrease of 12.5-fold in the GI₅₀ value of doxorubicin in the K562Dox cell line, being 2-fold more potent than verapamil. From the overall results, the aminated thioxanthones represent a new class of P-gp inhibitors with improved efficacy in sensitizing a resistant P-gp overexpressing cell line (K562Dox) to doxorubicin.

Keywords: Abbreviations; ABC; ATP-binding cassette; ATP; adenosine-5′-triphosphate; BLAST; basic local alignment search tool; DNA; deoxyribonucleic acid; GI; ₅₀; inhibition of cell growth (the concentration needed to reduce the growth of treated cells to half that of untreated cells); MDR; multidrug resistance; MFI; mean fluorescence intensity; MRP; multridrug resistance proteins; MRP-1; multidrug resistance protein 1; MW; molecular weight; NBD; nucleotide binding domain; P-gp; P-glycoprotein; Rh123; rhodamine-123; RLU; relative light unit; RMSD; root-mean-squared error displacement; SE; standard error; SRB; sulphorhodamine-B; TM; transmembrane (one α-helix); TMD; transmembrane domain (six α-helixes)Thioxanthones; P-glycoprotein; Multidrug resistance; Anticancer; Dual ligands

Therapy-induced carboplatin–DNA adduct levels in human ovarian tumours in relation to assessment of adduct measurement in mouse tissues by Ian W.H. Jarvis; Emma L. Meczes; Huw D. Thomas; Richard J. Edmondson; Gareth J. Veal; Alan V. Boddy; Christopher J. Ottley; D. Graham Pearson; Michael J. Tilby (pp. 69-77).

Despite an increasing understanding of the molecular mechanisms by which platinum drug DNA adducts interact with cellular processes, the relationship between adduct formation in tumours and clinical response remains unclear. We have determined carboplatin–DNA adduct levels in biopsies removed from ovarian cancer patients following treatment. Reliability of DNA adduct measurements in tissues samples were assessed using experimental animals. Platinum–DNA adduct levels were measured using inductively coupled plasma mass spectrometry (ICP-MS) and plasma drug concentrations determined by atomic absorption spectrometry (AAS). Adduct levels in tissues and plasma pharmacokinetics were determined in Balb/c mice exposed to platinum drugs. Comparisons of adduct levels in tumour and normal tissue were made in nu/nu mice carrying human neuroblastoma xenografts. At 30min post-cisplatin administration, adduct levels in DNA from kidney and liver were approximately 10- and 6-fold higher than spleen or tumour. By 60min, levels in liver and kidney, but not spleen or tumour, had fallen considerably. Carboplatin showed high adduct levels only in kidney. Adduct levels in tumour xenografts were comparable to those induced in vitro with similar drug exposures. In clinical samples removed 6h after drug administration, adduct levels ranged from 1.9 to 4.3 and 0.2 to 3.6nmolPt/gDNA for tumour biopsies and peripheral blood mononuclear cells, respectively. No correlation was apparent between these two data sets. The present results demonstrate that reliable measurements of adducts in clinical tumours are feasible. Future results should provide insight into drug resistance.

Keywords: Abbreviations; AAS; atomic absorption spectrometry; AUC; area under the curve; C.V.; coefficient of variation; i.v.; intravenous; i.p.; intraperitoneal; i.u.; international units; ICP-MS; inductively coupled plasma mass spectrometry; LC; liquid chromatography; PBCs; peripheral blood cells; PPB; parts per billion (by mass); PPT; parts per trillion (by mass); s.c.; subcutaneous; S.D.; standard deviation; S.E.; standard error of the meanCisplatin; Carboplatin; DNA adducts; Neuroblastoma; Ovarian cancer; ICP-MS

Sam68 interacts with IRS1 by R. Quintana-Portillo; A. Canfrán-Duque; T. Issad; V. Sánchez-Margalet; C. González-Yanes (pp. 78-87).

Sam68 (Src associated in mitosis) is a RNA binding protein that links cellular signaling to RNA processing. In previous studies we found that insulin promotes Sam68 relocalization in the cytoplasm allowing Sam68 to associate with p85PI3K, Grb2, GAP and probably the insulin receptor (IR), modulating insulin action positively. In the present work, we wanted to define the role of Sam68 in the first stages of IR signaling.Both BRET and co-immunoprecipitation assays have been used for the study of Sam68 binding to IR, IRS1 and p85-PI3K.BRET saturation experiments indicated, for the first time, that Sam68 associates with IRS1 in basal condition. To map the region of Sam68 implicated in the interaction with IRS1, different Sam68 mutants deleted in the proline-rich domains were used. The deletion of P0, P1 and P2 proline rich domains in N-terminus as well as P4 and P5 in C-terminus of Sam68 increased BRET₅₀, thus indicating that the affinity of Sam68 for IRS1 is lower when these domains are missing. Moreover, in IR-transfected HEK-293 cells, BRET saturation experiment indicated that insulin increases the affinity between Sam68-Rluc and IRS1-YFP.In conclusion, our data indicate that Sam68 interacts with IRS-1 in basal conditions, and insulin increases the affinity between these two partners.

Keywords: Sam68; IRS1; Insulin receptor; BRET

Sam68 interacts with IRS1 by R. Quintana-Portillo; A. Canfrán-Duque; T. Issad; V. Sánchez-Margalet; C. González-Yanes (pp. 78-87).

Keywords: Sam68; IRS1; Insulin receptor; BRET

The C-terminal segment of the second extracellular loop of the thromboxane A₂ receptor plays an important role in platelet aggregation by John P. Murad; Enma Veronica Paez Espinosa; Harold J. Ting; Fadi T. Khasawneh (pp. 88-96).

There is considerable interest in discovering novel antiplatelet approaches with an enhanced safety profile. To this end, in our efforts to define new targets for antithrombotic activity, we investigated the utility of antibodies which recognize the ligand binding domains of the platelet thromboxane A₂ receptor (TPR). We hypothesized that an antibody (abbreviated as C-EL2Ab), which interacts with the C-terminus of the second extracellular loop (C-EL2; i.e., ligand binding domain) of TPR exhibits antagonistic activity. Our findings demonstrate that C-EL2Ab did indeed inhibit TPR-mediated platelet aggregation. However, it was devoid of any apparent effects on aggregation triggered by ADP or the thrombin receptor activating peptides 1 or 4. Furthermore, results from radiolabeled ligand binding studies indicate that C-EL2Ab competitively displaced the classical TPR antagonist [³H]SQ29,548 from its binding sites. On the other hand, control experiments indicated that normal rabbit IgG and an antibody which targets a TPR domain separate from those involved in ligand recognition, failed to inhibit aggregation in response to TPR activation. Collectively, these findings demonstrate that C-EL2 of TPR plays a critical role in platelet activation, and establish C-EL2Ab as a function blocking antibody. Furthermore, our data suggest a potential for the therapeutic application of C-EL2Ab, which may serve either as an alternative to, or a complement for current treatments. Finally, the identification of a functionally active TPR sequence should aid molecular modeling study predictions for organic derivatives which possess in vivo activity.

Keywords: Abbreviations; TXA; ₂; thromboxane A; ₂; TPR; thromboxane A; ₂; receptor; TS; thromboxane synthase; TRAP1; thrombin receptor-activating peptide 1; TRAP4; thrombin receptor-activating peptide 4; PAR; protease activated receptor; C-EL2; C-terminus of the second extracellular loop; C-EL2Ab; antibody targeting the C-terminus of the second extracellular loop; PRP; platelet rich plasma; PPP; platelet poor plasmaThromboxane A; ₂; receptor; Platelet; Function-blocking antibody; Antagonist; Ligand binding domain

AQP4 knockout mice manifest abnormal expressions of calcium handling proteins possibly due to exacerbating pro-inflammatory factors in the heart by Yu-Si Cheng; Yi-Qun Tang; De-Zai Dai; Yin Dai (pp. 97-105).

We tested the hypothesis that aquaporin-4 (AQP4) knockout (KO) mice might exhibit abnormal Ca²⁺ modulating proteins resulting from the exacerbation of pro-inflammatory factors in the heart. Downregulation of FKBP12.6, SERCA2a, and CASQ2 and calcium leak in diastole have been recognized as endpoints for assessing cardiac failure and arrhythmias. The AQP4 KO mice and wild-type (WT) mice were randomly divided into 3 groups, such as control, isoproterenol (ISO, β-receptor agonist) injected (1mg/kg, sc, 5d), and treated with aminoguanidine (AMG, 100mg/kg, po, a selective inhibitor of the iNOS) during the last 3d. RT-PCR, western blot and calcium transient measurements were conducted. The results demonstrated that the cardiac weight index was increased in AQP4 KO mice and further increased following treatment with ISO. The expression levels of FKBP12.6, SERCA2a, and CASQ2 were downregulated and diastolic calcium concentrations were elevated in the AQP4 KO mice, indicative of a calcium leak. In the myocardium, expressions of pro-inflammatory biomarkers, including ET_A, pPKCɛ, NADPH oxidase p67^phox were upregulated and associated with downregulation of Cx43. The aforementioned changes were exacerbated in response to ISO medication and were attenuated by AMG; however, its treatment effectiveness was less in the AQP4 KO mice. We concluded AQP4 KO caused abnormalities of calcium modulating proteins leading to an exacerbation of risk for cardiac arrhythmias and failure. These changes are likely due to an increase in pro-inflammatory factors which are exacerbated by stress. Therefore, AQP4 KO mice are prone to cardiac failure and arrhythmias through exacerbating pro-inflammatory factors in the myocardium.

Keywords: Abbreviations; AMG; aminoguanidine; AQP4; aquaporin-4; AQPs; aquaporins; CASQ2; calsequestrin 2; CMC-Na; carboxymethyl cellulose-Na; CPVT; catecholaminergic polymorphism ventricular tachycardia; Cx43; connexin 43; ET; _A; endothelin receptor A; ER; endoplasmic reticulum; FKBP12.6; FK506 binding protein; ISO; isoproterenol injection; KO; knockout; LV; left free wall plus septum; LVW/BW; the weight of the LV divided by body weight; PLB; phospholamban; RNS; reactive nitrosative species; ROS; reactive oxygen species; RT-PCR; Reverse transcriptase polymerase chain reaction; RV; the right ventricle; RyR2; ryanodine receptor type 2; SERCA2a; sarcoplasmic reticulum Ca; ²⁺; -ATPase2a; SPF; Specific Pathogen Free; SR; sarcoplasmic reticulum; pPKCɛ; phosphorylated PKCɛ; WT; wild-typeAQP4; FKBP12.6; CASQ2; SERCA2a; ET-ROS

The Phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 modulates cytokine expression in macrophages via p50 nuclear factor kappa B inhibition, in a PI3K-independent mechanism by Dorit Avni; Yifat Glucksam; Tsaffrir Zor (pp. 106-114).

The Phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002 (LY2), has been previously reported to inhibit nuclear factor κB (NFκB) activity, in a PI3K-independent mechanism. The goals of the current research were to determine the specificity of LY2 regarding NFκB subunits, and to identify relevant modulation of cytokine expression in LPS-stimulated macrophages. We found that LY2 specifically diminished the level of p50, but not p65, NFκB in the nucleus of LPS-stimulated mouse RAW264.7 macrophages and human THP-1 monocytes. This activity of LY2 was mimicked by its PI3K-inert analog LY303511 (LY3), but not by another PI3K inhibitor – wortmannin. We further show that LY2 inhibited LPS-induced IL-10 expression by RAW264.7 macrophages, in a PI3K-independent mechanism. Moreover, using a deletion mutant of an IL-10 promoter reporter gene we demonstrate that the activity of the NFκB enhancer site at the IL-10 promoter is regulated by LY2 in a PI3K-independent manner. Finally, both LY2 and LY3 elevated TNFα production in the LPS tolerant state which is regulated by p50 NFκB homodimers, but not before tolerance development. The effects of LY2 and LY3 on p50 translocation and on cytokine production in LPS-stimulated macrophages are thus consistent with specific PI3K-independent inhibition of p50 NFκB homodimer activity by LY2.

Keywords: Abbreviations; LPS; lipopolysaccharide; TNFα; tumor necrosis factor alpha; IL-10; interleukin-10; PI3K; Phosphatidylinositol 3-kinase; LY2; LY294002; LY3; LY303511; NFκB; nuclear factor κB; Wort; wortmannin; PVDF; polyvinylidene fluoride; wed; wedelolactone; EMSA; electrophoretic mobility shift assay; ChIP; chromatin immunoprecipitation; DMEM; Dulbecco's modified eagle's mediump50 NF-kappa B; LY294002; LY303511; Macrophages; Phosphatidylinositol 3-kinase

Boswellic acids from frankincense inhibit lipopolysaccharide functionality through direct molecular interference by Arne Henkel; Nicole Kather; Bettina Mönch; Hinnak Northoff; Johann Jauch; Oliver Werz (pp. 115-121).

Lipophilic extracts of gum resins of Boswellia species (BSE) are used in folk medicine to treat various inflammatory disorders and infections. The molecular background of the beneficial pharmacological effects of such extracts is still unclear. Various boswellic acids (BAs) have been identified as abundant bioactive ingredients of BSE. Here we report the identification of defined BAs as direct inhibitors of lipopolysaccharide (LPS) functionality and LPS-induced cellular responses. In pull-down experiments, LPS could be precipitated using an immobilized BA, implying direct molecular interactions. Binding of BAs to LPS leads to an inhibition of LPS activity which was observed in vitro using a modified limulus amoebocyte lysate assay. Analysis of different BAs revealed clear structure-activity relationships with the classical β-BA as most potent derivative (IC₅₀=1.8μM). In RAW264.7 cells, LPS-induced expression of inducible nitric oxide synthase (iNOS, EC 1.14.13.39) was selectively inhibited by those BAs that interfered with LPS activity. In contrast, interferon-γ-induced iNOS induction was not affected by BAs. We conclude that structurally defined BAs are LPS inhibiting agents and we suggest that β-BA may contribute to the observed anti-inflammatory effects of BSE during infections by suppressing LPS activity.

Keywords: Abbreviations; Aβ-BA; 3-; O; -acetyl-β-boswellic acid; AKBA; 3-; O; -acetyl-keto-β-boswellic acid; β-BA; β-boswellic acid; BA-seph; β-BA-sepharose; cm-BA; 3-carboxymethylenoxy-β-boswellic acid; cm-KBA; 3-carboxymethylenoxy-11-keto-β-boswellic acid; glut-BA, EU; endotoxin units, 3-glutaroyl-β-boswellic acid; glut-KBA, FCS; fetal calf serum, 3-glutaroyl-11-keto-β-boswellic acid; IFN-γ; interferon-γ; KBA; 11-keto-β-boswellic acid; LAL; limulus; amoebocyte lysate; LPS; lipopolysaccharide; MAPK; mitogen-activated protein kinase; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; ox-BA; 3-oxaloyl-β-boswellic acid; ox-KBA; 3-oxaloyl-11-keto-β-boswellic acid; seph; EAH sepharose 6B; succ-BA; 3-succinoyl-β-boswellic acid; succ-KBA; 3-succinoyl-11-keto-β-boswellic acid; TLR4; toll-like receptor 4Boswellic acid; Frankincense; Lipopolysaccharide; Nitric oxide synthase; Inflammation; Sepsis

Oxidative and nitrosative stress in acute pancreatitis. Modulation by pentoxifylline and oxypurinol by Javier Escobar; Javier Pereda; Alessandro Arduini; Juan Sandoval; Mari Luz Moreno; Salvador Pérez; Luis Sabater; Luis Aparisi; Norberto Cassinello; Juan Hidalgo; Leo A.B. Joosten; Máximo Vento; Gerardo López-Rodas; Juan Sastre (pp. 122-130).

Co-treatment with oxypurinol (O) and pentoxifylline (P) prevents oxidative and nitrosative stress in pancreas in acute pancreatitis (AP). The effect of oxypurinol may be ascribed to inhibition of MEK1/2 activity.Reactive oxygen species are considered mediators of the inflammatory response and tissue damage in acute pancreatitis. We previously found that the combined treatment with oxypurinol – as inhibitor of xanthine oxidase- and pentoxifylline – as inhibitor of TNF-α production-restrained local and systemic inflammatory response and decreased mortality in experimental acute pancreatitis. Our aims were (1) to determine the time-course of glutathione depletion and oxidation in necrotizing pancreatitis in rats and its modulation by oxypurinol and pentoxifylline; (2) to determine whether TNF-α is responsible for glutathione depletion in acute pancreatitis; and (3) to elucidate the role of oxidative stress in the inflammatory cascade in pancreatic AR42J acinar cells.We report here that oxidative stress and nitrosative stress occur in pancreas and lung in acute pancreatitis and the co-treatment with oxypurinol and pentoxifylline prevents oxidative stress in both tissues. Oxypurinol was effective in preventing glutathione oxidation, whereas pentoxifylline abrogated glutathione depletion. This latter effect was independent of TNF-α since glutathione depletion occurred in mice deficient in TNF-α or its receptors after induction of pancreatitis. The beneficial effects of oxypurinol in the inflammatory response may also be ascribed to a partial inhibition of MEK1/2 activity. Pentoxifylline markedly reduced the expression of Icam1 and iNos induced by TNF-α in vitro in AR42J cells. Oxidative stress significantly contributes to the TNF-α-induced up-regulation of Icam and iNos in AR42J cells. These results provide new insights into the mechanism of action of oxypurinol and pentoxifylline as anti-inflammatory agents in acute pancreatitis.

Keywords: Acute pancreatitis; Pentoxifylline; Oxypurinol; Glutathione; TNF-α; MEK1/2

Stereoselective urinary MDMA (ecstasy) and metabolites excretion kinetics following controlled MDMA administration to humans by Andrea E. Schwaninger; Markus R. Meyer; Allan J. Barnes; Erin A. Kolbrich-Spargo; David A. Gorelick; Robert S. Goodwin; Marilyn A. Huestis; Hans H. Maurer (pp. 131-138).

The R- and S-enantiomers of racemic 3,4-methylenedioxymethamphetamine (MDMA) exhibit different dose–concentration curves. In plasma, S-MDMA was eliminated at a higher rate, most likely due to stereoselective metabolism. Similar data were shown in various in vitro experiments. The aim of the present study was the in vivo investigation of stereoselective elimination of MDMA's phase I and phase II metabolites in human urine following controlled oral MDMA administration. Urine samples from 10 participants receiving 1.0 and 1.6mg/kg MDMA separated by at least one week were analyzed blind by liquid chromatography–high resolution-mass spectrometry and gas chromatography–mass spectrometry after chiral derivatization with S-heptafluorobutyrylprolyl chloride. R/S ratios at C_max were comparable after low and high doses with ratios >1 for MDMA, free DHMA, and HMMA sulfate, and with ratios <1 for MDA, free HMMA, DHMA sulfate and HMMA glucuronide. In the five days after the high MDMA dose, a median of 21% of all evaluated compounds were excreted as R-stereoisomers and 17% as S-stereoisomers. Significantly greater MDMA, DHMA, and HMMA sulfate R-enantiomers and HMMA and HMMA glucuronide S-stereoisomers were excreted. No significant differences were observed for MDA and DHMA sulfate stereoisomers. Changes in R/S ratios could be observed over time for all analytes, with steady increases in the first 48h. R/S ratios could help to roughly estimate time of MDMA ingestion and therefore, improve interpretation of MDMA and metabolite urinary concentrations in clinical and forensic toxicology.

Keywords: MDMA; Phase I metabolites; Phase II metabolites; Urine; Chiral; Controlled administration

A conserved antioxidant response element ( ARE) in the promoter of human carbonyl reductase 3 ( CBR3) mediates induction by the master redox switch Nrf2 by Qiuying Cheng; James L. Kalabus; Jianping Zhang; Javier G. Blanco (pp. 139-148).

A conserved antioxidant response element (₋₂₆₉₈ ARE) in the promoter of human carbonyl reductase 3 ( CBR3) mediates gene induction via the master redox switch Nrf2.Carbonyl reductase activity catalyzes the two electron reduction of several endogenous and exogenous carbonyl substrates. Recent data indicate that the expression of human carbonyl reductase 3 ( CBR3) is regulated by the master redox switch Nrf2. Nrf2 binds to conserved antioxidant response elements ( AREs) in the promoters of target genes. The presence of functional AREs in the CBR3 promoter has not yet been reported. In this study, experiments with reporter constructs showed that the prototypical Nrf2 activator tert-butyl hydroquinone (t-BHQ) induces CBR3 promoter activity in cultures of HepG2 (2.7-fold; p<0.05) and MCF-7 cells (22-fold; p<0.01). Computational searches identified a conserved ARE in the distal CBR3 promoter region (₋₂₆₉₈ ARE). Deletion of this ARE from a 4212-bp CBR3 promoter construct impacted basal promoter activity and induction of promoter activity in response to treatment with t-BHQ. Deletion of₋₂₆₉₈ ARE also impacted the induction of CBR3 promoter activity in cells overexpressing Nrf2. Electrophoretic mobility shift assays (EMSA) demonstrated increased binding of specific protein complexes to₋₂₆₉₈ ARE in nuclear extracts from t-BHQ treated cells. The presence of Nrf2 in the specific nuclear protein–₋₂₆₉₈ ARE complexes was evidenced in EMSA experiments with anti-Nrf2 antibodies. These data suggest that the distal₋₂₆₉₈ ARE mediates the induction of human CBR3 in response to prototypical activators of Nrf2.

Keywords: Abbreviations; CBR3; carbonyl reductase 3; ARE; antioxidant response element; Nrf2; nuclear factor [erythroid-derived 2]-like 2 (official symbol:; NFE2L2; ); RT-PCR; reverse transcription-polymerase chain reaction; DMSO; dimethyl sulfoxide; bp; base pairs; EMSA; electrophoretic mobility shift assay; ROS; reactive oxygen species; GPXs; glutathione peroxidases; NQO1; NAD(P)H:quinone oxidoreductase-1; NQO2; NRH:quinone oxidoreductase 2; SOD; superoxide dismutase; GSTs; glutathione S-transferases; GPXs; glutathione peroxidases; GCLC; glutamate-cysteine ligase catalytic subunit; GCLM; glutamate cysteine ligase modifier subunitCarbonyl reductase 3; Antioxidant response element; Nuclear factor [erythroid-derived 2]-like 2

Comparison of the function and expression of CYP26A1 and CYP26B1, the two retinoic acid hydroxylases by Ariel R. Topletz; Jayne E. Thatcher; Alex Zelter; Justin D. Lutz; Suzanne Tay; Wendel L. Nelson; Nina Isoherranen (pp. 149-163).

All-trans-retinoic acid ( atRA) is an important signaling molecule in all chordates. The cytochrome P450 enzymes CYP26 are believed to partially regulate cellular concentrations of atRA via oxidative metabolism and hence affect retinoid homeostasis and signaling. CYP26A1 and CYP26B1 are atRA hydroxylases that catalyze formation of similar metabolites in cell systems. However, they have only 40% sequence similarity suggesting differences between the two enzymes. The aim of this study was to determine whether CYP26A1 and CYP26B1 have similar catalytic activity, form different metabolites from atRA and are expressed in different tissues in adults. The mRNA expression of CYP26A1 and CYP26B1 correlated between human tissues except for human cerebellum in which CYP26B1 was the predominant CYP26 and liver in which CYP26A1 dominated. Quantification of CYP26A1 and CYP26B1 protein in human tissues was in agreement with the mRNA expression and showed correlation between the two isoforms. Qualitatively, recombinant CYP26A1 and CYP26B1 formed the same primary and sequential metabolites from atRA. Quantitatively, CYP26B1 had a lower K_m (19nM) and V_max (0.8pmol/min/pmol) than CYP26A1 ( K_m=50nM and V_max=10pmol/min/pmol) for formation of 4-OH-RA. The major atRA metabolites 4-OH-RA, 18-OH-RA and 4-oxo-RA were all substrates of CYP26A1 and CYP26B1, and CYP26A1 had a 2–10-fold higher catalytic activity towards all substrates tested. This study shows that CYP26A1 and CYP26B1 are qualitatively similar RA hydroxylases with overlapping expression profiles. CYP26A1 has higher catalytic activity than CYP26B1 and seems to be responsible for metabolism of atRA in tissues that function as a barrier for atRA exposure.

Keywords: Vitamin A; Cytochrome P450; Retinoic acid; Metabolism; Tissue expression

Mechanisms of glutathione disulfide efflux from erythrocytes by Irina Ellison; John P. Richie Jr. (pp. 164-169).

Glutathione (GSH) plays numerous critical protective roles in the erythrocyte and GSH turnover is likely an important factor in regulating susceptibility to oxidative stress and toxins. Efflux of glutathione disulfide (GSSG) from erythrocytes is an important component in the regulation of GSH levels; however, little is known of the mechanisms involved. We hypothesize that multidrug resistance associated protein 1 (MRP1) is responsible, in part, for GSSG transport from erythrocytes. To test this, we determined the levels of MRP1 protein in erythrocyte membranes from healthy adults and compared them with intracellular levels of GSH. MRP1 levels varied substantially from person to person and were inversely correlated with levels of GSH ( r=−0.39, P<0.05). In contrast, activity levels of glutamyl cysteine ligase, the rate limiting GSH biosynthetic enzyme, were unrelated to GSH levels. To directly determine the role of MRP1 in GSSG transport, in vitro studies were conducted examining the effects of MRP1 inhibitors MK571 and verapamil on GSSG efflux. Both compounds resulted in significant but not complete inhibition (20–53%) of GSSG efflux from cells. Overall, these findings support a role for MPR1 in the regulation of erythrocyte GSH levels through the transport and elimination of GSSG from cells.

Keywords: Abbreviations; MRP1; multidrug resistance associated protein 1; GSH; glutathione; GSSG; glutathione disulfide; GSX; glutathione S-conjugate; GCL; glutamine cysteine ligase; BSO; buthionine sulfoxamine; MK571; (E)-3-[[[3-[2-(7-Chloro-2-quinolinyl)ethenyl]phenyl]-[[3-dimethylamino)-3-oxopropyl]thio]methyl]-thio]-propanoic acid; GSSP; protein bound glutathione; γGC; γ-glutamyl cysteineGlutathione; Glutathione disulfide; Erythrocyte; MRP1; Transport

Mechanisms of glutathione disulfide efflux from erythrocytes by Irina Ellison; John P. Richie Jr. (pp. 164-169).

Cloning and activity of a novel α-latrotoxin from red-back spider venom by Andis Graudins; Michelle J. Little; Sandy S. Pineda; Peter G. Hains; Glenn F. King; Kevin W. Broady; Graham M. Nicholson (pp. 170-183).

The venom of the European black widow spider Latrodectus tredecimguttatus (Theridiidae) contains several high molecular mass (110–140kDa) neurotoxins that induce neurotransmitter exocytosis. These include a vertebrate-specific α-latrotoxin (α-LTX-Lt1a) responsible for the clinical symptoms of latrodectism and numerous insect-specific latroinsectoxins (LITs). In contrast, little is known about the expression of these toxins in other Latrodectus species despite the fact that envenomation by these spiders induces a similar clinical syndrome. Here we report highly conserved α-LTX, α-LIT and δ-LIT sequence tags in Latrodectus mactans, Latrodectus hesperus and Latrodectus hasselti venoms using tandem mass spectrometry, following bioassay-guided separation of venoms by liquid chromatography. Despite this sequence similarity, we show that the anti-α-LTX monoclonal antibody 4C4.1, raised against α-LTX-Lt1a, fails to neutralize the neurotoxicity of all other Latrodectus venoms tested in an isolated chick biventer cervicis nerve–muscle bioassay. This suggests that there are important structural differences between α-LTXs in theridiid spider venoms. We therefore cloned and sequenced the α-LTX from the Australian red-back spider L. hasselti (α-LTX-Lh1a). The deduced amino acid sequence of the mature α-LTX-Lh1a comprises 1180 residues (∼132kDa) with ∼93% sequence identity with α-LTX-Lt1a. α-LTX-Lh1a is composed of an N-terminal domain and a central region containing 22 ankyrin-like repeats. The presence of two furin cleavage sites, conserved with α-LTX-Lt1a, indicates that α-LTX-Lh1a is derived from the proteolytic cleavage of an N-terminal signal peptide and C-terminal propeptide region. However, we show that α-LTX-Lh1a has key substitutions in the 4C4.1 epitope that explains the lack of binding of the monoclonal antibody.

Keywords: Abbreviations; α-LTX; α-latrotoxin; BSA; bovine serum albumin; 4C4.1; murine monoclonal antibody raised against α-latrotoxin from; L; .; tredecimguttatus; CBCNM; chick biventer cervicis nerve–muscle; CNS; central nervous system; ESI-Q-TOF; electrospray ionization quadrupole time-of-flight; FPLC; fast performance liquid chromatography; gDNA; genomic DNA; HEPES; N; -2-hydroxyethylpiperazine-; N; -2-ethanesulfonic acid; α-LCT; α-latrocrustatoxin; LD; ₅₀; median lethal dose; LIT; latroinsectotoxin; LMWP; low molecular weight protein (also known as latrodectin); MS–MS; tandem mass spectrometry; RP-HPLC; reverse-phase high-performance liquid chromatography; SDS-PAGE; sodium dodecyl sulphate polyacrylamide gel electrophoresis; TFA; trifluoroacetic acid Latrodectus; Latrotoxin; α-LTX-Lh1a; Latroinsectotoxin; Neurotransmitter release

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Biochemical Pharmacology (v.83, #1)