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Biochemical Pharmacology (v.80, #10)

Editorial Advisory Board (pp. iii).

Graphical Abstracts Contents (pp. e1-e5).

Modulation of cell sensitivity to antitumor agents by targeting survival pathways by Paola Perego; Giacomo Cossa; Valentina Zuco; Franco Zunino (pp. 1459-1465).

The advent of drugs targeting tumor-associated prosurvival alterations of cancer cells has changed the interest of antitumor drug development from cytotoxic drugs to target-specific agents. Although single-agent therapy with molecularly targeted agents has shown limited success in tumor growth control, a promising strategy is represented by the development of rational combinations of target-specific agents and conventional antitumor drugs. Activation of survival/antiapoptotic pathways is a common feature of cancer cells that converge in the development of cellular resistance to cytotoxic agents. The survival pathways implicated in cellular response to drug treatment are primarily PI3K/Akt and Ras/MAPK, which also mediate the signalling activated by growth factors and play a role in the regulation of critical processes including cell proliferation, metabolism, apoptosis and angiogenesis. Inhibitors of PI3K, Akt and mTOR have been shown to sensitize selected tumor cells to cytotoxic drugs through multiple downstream effects. Moreover, the MAPK pathway, also implicated in the regulation of gene expression in response to stress stimuli, can interfere with the chemotherapy-induced proapoptotic signals. Targeting Hsp90, which acts as a molecular chaperone for survival factors including Akt, may have the potential advantage to simultaneously block multiple oncogenic pathways. Overall, the available evidence supports the interest of rationally designed approaches to enhance the efficacy of conventional antitumor treatments through the inhibition of survival pathways and the notion that the concomitant targeting of multiple pathways may be a successful strategy to deal with tumor heterogeneity and to overcome drug resistance of tumor cells.

Keywords: Abbreviations; AMPK; AMP-dependent protein kinase; ER; endoplasmic reticulum; ERK; extracellular signal-regulated kinases; GRP78; glucose-regulated protein 78; HDAC; histone deacetylase; mTOR; mammalian target of rapamycin; MAPK; mitogen-activated protein kinase; PI3K; phosphoinositide 3-kinase; SAPK/JNK; stress-activated protein kinase/c-Jun N-terminal kinases; RTK; receptor tyrosine kinase; UPR; unfolded protein response; VEGF; vascular endothelial growth factorAntitumor agents; Targeted therapy; Survival pathways; Drug combinations

Doxorubicin-induced cell death requires cathepsin B in HeLa cells by S. Bien; C. Rimmbach; H. Neumann; J. Niessen; E. Reimer; C.A. Ritter; D. Rosskopf; J. Cinatl; M. Michaelis; H.W.S. Schroeder; H.K. Kroemer (pp. 1466-1477).

The cysteine protease cathepsin B acts as a key player in apoptosis. Cathepsin B-mediated cell death is induced by various stimuli such as ischemia, bile acids or TNFα. Whether cathepsin B can be influenced by anticancer drugs, however, has not been studied in detail. Here, we describe the modulation of doxorubicin-induced cell death by silencing of cathepsin B expression. Previously, it was shown that doxorubicin, in contrast to other drugs, selectively regulates expression and activity of cathepsin B.Selective silencing of cathepsin B by siRNA or the cathepsin B specific inhibitor CA074Me modified doxorubicin-mediated cell death in Hela tumor cells. Both Caspase 3 activation and PARP cleavage were significantly reduced in cells lacking cathepsin B. Moreover, mitochondrial membrane permeabilization as well as the release of cytochrome C and AIF from mitochondria into cytosol induced by doxorubicin were significantly diminished in cathepsin B suppressed cells. In addition, doxorubicin associated down-regulation of XIAP was not observed in cathepsin B silenced cells. Lack of cathepsin B significantly modified cell cycle regulatory proteins such as cdk1, Wee1 and p21 without significant changes in G₁, S or G₂M cell cycle phases maybe indicating further cell cycle independent actions of these proteins. Consequently, cell viability following doxorubicin was significantly elevated in cells with cathepsin B silencing.In summary, our data strongly suggest a role of cathepsin B in doxorubicin-induced cell death. Therefore, increased expression of cathepsin B in various types of cancer can modify susceptibility towards doxorubicin.

Keywords: Doxorubicin; Cathepsin B; Cell death

Identification of a κ-opioid agonist as a potent and selective lead for drug development against human African trypanosomiasis by Deuan C. Jones; Irene Hallyburton; Laste Stojanovski; Kevin D. Read; Julie A. Frearson; Alan H. Fairlamb (pp. 1478-1486).

Phenotypic screening of the LOPAC library identified several potent and selective inhibitors of African trypanosomes. The κ-opioid agonist (+)-U50,488 represents a novel lead for drug discovery against sleeping sickness.A resazurin-based cell viability assay was developed for phenotypic screening of the LOPAC 1280 ‘library of pharmacologically active compounds’ against bloodstream forms of Trypanosoma brucei in vitro identifying 33 compounds with EC₅₀ values <1μM. Counter-screening vs. normal diploid human fibroblasts (MRC5 cells) was used to rank these hits for selectivity, with the most potent (<70nM) and selective (>700-fold) compounds being suramin and pentamidine. These are well-known antitrypanosomal drugs which demonstrate the robustness of the resazurin cell viability assay. The most selective novel inhibitor was (+)- trans-(1 R,2 R)-U50,488 having an EC₅₀ value of 60nM against T. brucei and 270-fold selectivity over human fibroblasts. Interestingly, (−)-U50,488, a known CNS-active κ-opioid receptor agonist and other structurally related compounds were >70-fold less active or inactive, as were several μ- and κ-opioid antagonists. Although (+)-U50,488 was well tolerated by the oral route and displayed good pharmaceutical properties, including high brain penetration, the compound was not curative in the mouse model of infection. Nonetheless, the divergence of antinociceptive and antitrypanosomal activity represents a promising start point for further exploratory chemistry. Bioinformatic studies did not reveal any obvious candidate opioid receptors and the target of this cytostatic compound is unknown. Among the other potent, but less selective screening hits were compound classes with activity against protein kinases, topoisomerases, tubulin, as well as DNA and energy metabolism.

Keywords: Phenotypic screening; African trypanosomiasis; Target identification; Target validation; U50,488

Effects of a combretastatin A4 analogous chalcone and its Pt-complex on cancer cells: A comparative study of uptake, cell cycle and damage to cellular compartments by Miroslava Zoldakova; Zsuzsanna Kornyei; Andreas Brown; Bernhard Biersack; Emília Madarász; Rainer Schobert (pp. 1487-1496).

A chalcone and its Pt-complex, while both anticancer active, were found to differ distinctly in uptake routes, selectivity for glioma over normal neural cells, efficiency in multi-drug resistant cancer cells, influence on cell cycle progression, and fragmentation of the Golgi apparatus.The combretastatin A4 analogous chalcone (2 E)-3-(3-hydroxy-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one1 and its dichloridoplatinum(II) (6-aminomethylnicotinate) complex2 were previously found to be highly active against a variety of cancer cell lines while differing in their apoptosis induction and long-term regrowth retardation (Schobert et al. ). Further differences were identified now. The cellular uptake of complex2, like that of oxaliplatin, occurred mainly via organic cation transporters (OCT-1/2; ∼32%) and copper transporter related proteins (Ctr1; ∼24%), whereas that of chalcone1 was dependent on endocytosis (∼80%). Complex2 was more tumour-specific than1 concerning neural cells. This was apparent from the ratios of IC₅₀(48h) values against primary astrocytes versus human glioma cells U87 (>7000 for complex2; 55 for compound1). In tubulin-rich neurons and 518A2 melanoma cells complex2 disrupted microtubules and actin filaments. Cancer cells treated with2 could repair the cytoskeletal damage but ceased to proliferate and perished. Complex2 was particularly cytotoxic against P-gp-rich cells. It acted as a substrate for ABC-transporters of types BCRP, MRP3, and MRP1 and so was less active against the corresponding cancer cell lines. Complex2 arrested the cell cycle of the melanoma cells in G₁ and G₂/M phases. A fragmentation of their Golgi apparatus was observed by TEM for incubation with complex2 but not with1. In conclusion, unlike chalcone1, its platinum complex2 is highly cell line specific, is taken up via cell-controlled transporters and induces apoptosis by triggering multiple targets.

Keywords: Chalcones; Platinum; Microtubules disrupting agents; Melanoma 518A2 cells; Organic cation transporters; Golgi apparatus

Marine sponge-derived sipholane triterpenoids reverse P-glycoprotein (ABCB1)-mediated multidrug resistance in cancer cells by Ioana Abraham; Sandeep Jain; Chung-Pu Wu; Mohammad A. Khanfar; Yehong Kuang; Chun-Ling Dai; Zhi Shi; Xiang Chen; Liwu Fu; Suresh V. Ambudkar; Khalid El Sayed; Zhe-Sheng Chen (pp. 1497-1506).

The alignment of docked poses of sipholenone E (red) and sipholenol J (blue) in QZ59-RRR binding site.Previously, we reported sipholenol A, a sipholane triterpenoid from the Red Sea sponge Callyspongia siphonella, as a potent reversal of multidrug resistance (MDR) in cancer cells that overexpressed P-glycoprotein (P-gp). Through extensive screening of several related sipholane triterpenoids that have been isolated from the same sponge, we identified sipholenone E, sipholenol L and siphonellinol D as potent reversals of MDR in cancer cells. These compounds enhanced the cytotoxicity of several P-gp substrate anticancer drugs, including colchicine, vinblastine and paclitaxel, and significantly reversed the MDR-phenotype in P-gp-overexpressing MDR cancer cells KB-C2 in a dose-dependent manner. Moreover, these three sipholanes had no effect on the response to cytotoxic agents in cells lacking P-gp expression or expressing MRP1 (ABCC1) or MRP7 (ABCC10) or breast cancer resistance protein (BCRP/ABCG2). All three sipholanes (IC₅₀ >50μM) were not toxic to all the cell lines that were used. [³H]-Paclitaxel accumulation and efflux studies demonstrated that all three triterpenoids time-dependently increased the intracellular accumulation of [³H]-paclitaxel by directly inhibiting P-gp-mediated drug efflux. Sipholanes also inhibited calcein-AM transport from P-gp-overexpressing cells. The Western blot analysis revealed that these three triterpenoids did not alter the expression of P-gp. However, they stimulated P-gp ATPase activity in a concentration-dependent manner and inhibited the photolabeling of this transporter with its transport substrate [¹²⁵I]-iodoarylazidoprazosin. In silico molecular docking aided the virtual identification of ligand binding sites of these compounds. In conclusion, sipholane triterpenoids efficiently inhibit the function of P-gp through direct interactions and may represent potential reversal agents for the treatment of MDR.

Keywords: ABC transporter; Chemosensitivity; P-glycoprotein; Sipholane triterpenoid; Multidrug resistance

Identification of AKN-032, a novel 2-aminopyrazine tyrosine kinase inhibitor, with significant preclinical activity in acute myeloid leukemia by Anna Eriksson; Martin Höglund; Elin Lindhagen; Anna Åleskog; Sadia Bashir Hassan; Carina Ekholm; Karin Fhölenhag; Annika Jenmalm Jensen; Agneta Löthgren; Martin Scobie; Rolf Larsson; Vendela Parrow (pp. 1507-1516).

Aberrant signal transduction by mutant or overexpressed protein kinases has emerged as a promising target for treatment of acute myeloid leukemia (AML). We here present a novel low molecular weight kinase inhibitor, AKN-032, targeting the FMS-like tyrosine kinase 3 (FLT3) and discovered in a new type of screening funnel combining the target therapy approach with sequential cellular screens. AKN-032 was identified among 150 selected hits from three different high throughput kinase screens. Further characterization showed inhibitory activity on FLT3 enzyme with an IC₅₀ of 70nM. Western blot analysis revealed reduced autophosphorylation of the FLT3-receptor in AML cell line MV4-11 cells after exposure to AKN-032. Flow cytometry disclosed cytotoxic activity against MV4-11, but not against non-malignant 3T3-L1 fibroblast cells. Using a fluorometric microculture cytotoxicity assay, AKN-032 was tested against 15 cell lines and displayed a potent cytotoxic activity in AML cell lines MV4-11 (IC₅₀=0.4μM) and Kasumi-1 (IC₅₀=2.3μM). AKN-032 was also highly cytotoxic in tumor cells from AML patients in vitro. Furthermore, AKN-032 demonstrated significant antileukemic effect in a relatively resistant in vivo hollow fiber mouse model. No major toxicity was observed in the animals. In conclusion, AKN-032 is a promising new kinase inhibitor with significant in vivo and in vitro activity in AML. Results from the hollow fiber mouse assay suggest a favorable toxicity profile. Future studies will focus on pharmacokinetic properties, toxicity as well as further clarifying the mechanisms of action of AKN-032 in AML.

Keywords: Acute myeloid leukemia; New drug development; Tyrosine kinase inhibitor; FLT3

Hypoxia and succinate antagonize 2-deoxyglucose effects on glioblastoma by Francesca Pistollato; Sara Abbadi; Elena Rampazzo; Giampietro Viola; Alessandro Della Puppa; Lucia Cavallini; Chiara Frasson; Luca Persano; David M. Panchision; Giuseppe Basso (pp. 1517-1527).

Glioblastoma multiforme (GBM) are highly proliferative brain tumors characterized by a hypoxic microenvironment which controls GBM stem cell maintenance. Tumor hypoxia promotes also elevated glycolytic rate; thus, limiting glucose metabolism is a potential approach to inhibit tumor growth. Here we investigate the effects mediated by 2-deoxyglucose (2-DG), a glucose analogue, on primary GBM-derived cells maintained under hypoxia. Our results indicate that hypoxia protects GBM cells from the apoptotic effect elicited by 2-DG, which raises succinate dehydrogenase activity thus promoting succinate level decrease. As a consequence hypoxia inducible factor-1α (HIF-1α) degradation occurs and this induces GBM cells to acquire a neuronal committed phenotype. By adding succinate these effects are reverted, as succinate stabilizes HIF-1α and increases GBM stem cell fraction particularly under hypoxia, thus preserving the tumor stem cell niche.2-DG inhibits anaerobic glycolysis altering GBM cell phenotype by forcing tumor cells into mitochondrial metabolism and by inducing differentiation.

Keywords: Abbreviations; GBM; glioblastoma multiforme; BMP2; bone morphogenetic protein2; HIF-1α; hypoxia inducible factor 1α; PHD2; prolyl hydroxylase2; SDH; succinate dehydrogenase2-Deoxyglucose; Hypoxia; Hypoxia inducible factor-1α; Prolyl hydroxylase 2; GBM differentiation

Induction of thymidine kinase 1 after 5-fluorouracil as a mechanism for 3′-deoxy-3′-[¹⁸F]fluorothymidine flare by Seung Jin Lee; Seog Young Kim; Jin Hwa Chung; Seung Jun Oh; Jin Sook Ryu; Yong Sang Hong; Tae Won Kim; Dae Hyuk Moon (pp. 1528-1536).

Imaging the pharmacodynamics of anti-cancer drugs may allow early assessment of anti-cancer effects. Increases in 3′-deoxy-3′-[¹⁸F]fluorothymidine ([¹⁸F]FLT) uptake early after thymidylate synthase inhibition (TS) inhibition, the so-called flare response, is considered to be largely due to an increase in binding sites for type-1 equilibrative nucleoside transporter. We investigated the induction of thymidine kinase 1 (TK1) after 5-fluorouracil (5-FU) treatment as one of mechanisms for [¹⁸F]FLT flare. Exposure of nine cancer cell lines to 5-FU for 24h induced a 2.5- to 3.5-fold increase in [¹⁸F]FLT uptake, significantly higher than the 1.5-fold increase observed 2h after treatment. The increase of [¹⁸F]FLT uptake 24h after 5-FU exposure accompanied TK1 induction in most cell lines. In representative cell lines (A431 and HT29), 5-FU time-dependently increased [¹⁸F]FLT uptake, kinase activity and the levels of protein and mRNA for TK1, sequential cyclin E and A induction, and G₁–S phase transition. Cycloheximide treatment and knockdown of TK1 completely inhibited 5-FU-induced [¹⁸F]FLT flare. On the other hand, HCT8 cells showed a biphasic [¹⁸F]FLT flare with lacked TK1 induction in response to the dosage of 5-FU. Cycloheximide did not inhibit 5-FU-induced [¹⁸F]FLT flare in this cells. In vivo dynamic [¹⁸F]FLT-PET and ex vivo analysis in HT29 tumor-bearing mice showed significantly increased [¹⁸F]FLT flux and TK1 activity of tumor tissue 24h after 5-FU administration ( P<0.05). Conclusively, 5-FU induced TK1 and TK1-mediated high [¹⁸F]FLT flare in most of cell lines. [¹⁸F]FLT-PET may be used to assess pharmacodynamics of TS inhibitor by a mechanism involving TK1 induction.

Keywords: 5-Fluorouracil; Thymidine kinase 1; Thymidylate synthase; Positron emission tomography; [; ¹⁸; F]fluorothymidine

Towards a thermodynamic definition of efficacy in partial agonism: The thermodynamics of efficacy and ligand proton transfer in a G protein-coupled receptor of the rhodopsin class by Kenneth J. Broadley; Shane C. Sykes; Robin H. Davies (pp. 1537-1545).

Enthalpy differences between agonist (Δ H^ijx+Δ H^τ) and antagonist (Δ H^rs) component binding of a partial agonist, prenalterol, on the β₁-adrenoreceptor can be coincident with the energetics of ligand proton delivery to an aspartate ion on α-helix II.The thermodynamic binding profiles of agonist and antagonist complexes of the 4-hydroxypropanolamine partial agonist, prenalterol, on the chronotropic adrenergic response in guinea-pig right atria were determined over a 15°C temperature range. The tissue response was compared with data on the ethanolamine agonist, isoprenaline, given by binding studies in a number of rat tissues. Utilising the residue conservatism surrounding the known active conformers bound to either of two aspartate residues (α-helices II, III) in both receptors (β₁, β₂) and species (guinea-pig, rat and human), no significant deformation in the extended side chain could be found in prenalterol's agonist binding compared to isoprenaline. Antagonist binding gave a highly favourable entropy contribution at 30.0°C of −4.7±1.2kcal/mol. The enthalpy change between bound agonist and antagonist complexes, a function of the efficacy alone, was −6.4±1.1kcal/mol, coincident with the calculated intrinsic preference of a primary/secondary amine–aspartate interaction for a neutral hydrogen-bonded form over its ion pair state, giving values of 6.3–6.6kcal/mol with calculations of good quality, a figure expected to be close to that shown within a hydrophobic environment. Delivery of a proton to a conserved aspartate anion (α-helix II) becomes the critical determinant for agonist action with resultant proton transfer stabilisation dominating the enthalpy change. A proposed monocation-driven ligand proton pumping mechanism within the ternary complex is consistent with the data, delivery between two acid groups being created by the movement of the cation and the counter-movement of the ligand protonated amine moving from Asp 138 (α-helix III) to Asp 104 (α-helix II).

Keywords: Thermodynamics; Efficacy; Agonist; Antagonist binding enthalpies; Ligand proton transfer; Rhodopsin receptors

N-methyl-citalopram: A quaternary selective serotonin reuptake inhibitor by Yona Bismuth-Evenzal; Netta Roz; David Gurwitz; Moshe Rehavi (pp. 1546-1552).

We describe the synthesis and the pharmacological characterization of a new quaternary selective serotonin reuptake inhibitor (SSRI) N-methyl-citalopram (NMC) with periphery restricted action due to its inability to cross the blood brain barrier. NMC recognized and blocked the human platelet serotonin transporter (SERT) with similar affinity to that of citalopram as was evident from competition binding studies with [³H]citalopram and uptake studies with [³H]5-HT. In contrast, the affinity of NMC to rat brain SERT was 10-fold lower than its parent compound citalopram. Similarly to citalopram, NMC did not inhibit dopamine and noradrenaline uptake in rat brain synaptosomes at 10⁻⁷M as well as [³H]ketanserin binding to rat brain membranes at 10⁻⁵M, demonstrating its SSRI profile. A comparison of radioactivity retained in perfused mice brain following in vivo intraperitoneal injections of tritium-labeled NMC or citalopram showed that unlike citalopram, NMC did not penetrate the brain. Taken together, our observations suggest that N-methyl-citalopram is a selective serotonin reuptake inhibitor that does not penetrate the mouse brain. Epidemiological studies have suggested that chronic use of SSRI drugs may confer a protective effect against myocardial infarction (MI) apparently reflecting reduced platelet aggregation secondary to reduced platelet serotonin levels. N-methyl-citalopram may therefore have a potential as a new anti-platelet drug that does not cross the blood brain barrier and is thus devoid of the adverse CNS effects of SSRI drugs.

Keywords: Human serotonin transporter; SLC6A4; SSRI; Cardiovascular diseases; Platelets; Citalopram

Butein downregulates chemokine receptor CXCR4 expression and function through suppression of NF-κB activation in breast and pancreatic tumor cells by Angeline Wei Ling Chua; Hui Sin Hay; Peramaiyan Rajendran; Muthu K. Shanmugam; Feng Li; Pradeep Bist; Evelyn S.C. Koay; Lina H.K. Lim; Alan Prem Kumar; Gautam Sethi (pp. 1553-1562).

Potential novel role of butein as an inhibitor of CXCR4 expression and function in tumor cells.The CXC chemokine receptor-4 (CXCR4), a Gi protein-coupled receptor for the ligand CXCL12/stromal cell-derived factor-1α (SDF-1α), is known to be expressed in various tumors. This receptor mediates homing of tumor cells to specific organs that express the ligand CXCL12 for this receptor and plays an important role in tumor growth, invasion, metastasis, and angiogenesis. Thus , a priori, agents that can downregulate CXCR4/CXCL12 signaling cascade have potential against cancer metastasis. In this study, we report the identification of butein (3, 4, 2′, 4′-tetrahydroxychalcone) as a novel regulator of CXCR4 expression and function. We found that butein downregulated the expression of CXCR4 in HER2-overexpressing breast cancer cells in a dose- and time-dependent manner. The decrease in CXCR4 expression induced by butein was not cell type-specific as the inhibition also occurred in pancreatic, prostate, multiple myeloma, head and neck, and hepatocellular cancer cell lines. When investigated for the molecular mechanism(s), it was found that the downregulation of CXCR4 was not due to proteolytic degradation but rather to transcriptional regulation as indicated by downregulation of mRNA expression, inhibition of NF-κB activation evident by both DNA binding, and reporter assays, and suppression of chromatin immunoprecipitation activity. Suppression of CXCR4 expression by butein correlated with the inhibition of CXCL12-induced migration and invasion of both breast and pancreatic cancer cells. Overall, our results demonstrate for the first time that butein is a novel inhibitor of CXCR4 expression and thus has a potential in suppressing metastasis of cancer.

Keywords: Butein; CXCR4; Metastasis; Invasion; HER2; NF-κB

New insights into tetrahydrobiopterin pharmacodynamics from Pah enu1/², a mouse model for compound heterozygous tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency by Florian B. Lagler; Søren W. Gersting; Clemens Zsifkovits; Alice Steinbacher; Anna Eichinger; Marta K. Danecka; Michael Staudigl; Ralph Fingerhut; Hartmut Glossmann; Ania C. Muntau (pp. 1563-1571).

Pah enu1/² is a mouse model for compound heterozygous phenylalanine hydroxylase deficiency and the pharmacological chaperone 6 R-l- erythro-5,6,7,8-tetrahydrobiopterin rescues in vivo enzyme activity in Pah enu1/¹ and in Pah enu1/² but shows genotype-specific pharmacodynamics.Phenylketonuria (PKU), an autosomal recessive disease with phenylalanine hydroxylase (PAH) deficiency, was recently shown to be a protein misfolding disease with loss-of-function. It can be treated by oral application of the natural PAH cofactor tetrahydrobiopterin (BH₄) that acts as a pharmacological chaperone and rescues enzyme function in vivo. Here we identified Pah enu1/² bearing a mild and a severe mutation (V106A/F363S) as a new mouse model for compound heterozygous mild PKU. Although BH₄ treatment has become established in clinical routine, there is substantial lack of knowledge with regard to BH₄ pharmacodynamics and the effect of the genotype on the response to treatment with the natural cofactor. To address these questions we applied an elaborate methodological setup analyzing: (i) blood phenylalanine elimination, (ii) blood phenylalanine/tyrosine ratios, and (iii) kinetics of in vivo phenylalanine oxidation using¹³C-phenylalanine breath tests. We compared pharmacodynamics in wild-type, Pah enu1/¹, and Pah enu1/² mice and observed crucial differences in terms of effect size as well as effect kinetics and dose response. Results from in vivo experiments were substantiated in vitro after overexpression of wild-type, V106A, and F263S in COS-7 cells. Pharmacokinetics did not differ between Pah enu1/¹ and Pah enu1/² indicating that the differences in pharmacodynamics were not induced by divergent pharmacokinetic behavior of BH₄. In conclusion, our findings show a significant impact of the genotype on the response to BH₄ in PAH deficient mice. This may lead to important consequences concerning the diagnostic and therapeutic management of patients with PAH deficiency underscoring the need for individualized procedures addressing pharmacodynamic aspects.

Keywords: Abbreviations; PKU; phenylketonuria; PAH; phenylalanine hydroxylase; BH; ₄; 6; R; -; l; -; erythro; -5,6,7,8-tetrahydrobiopterin; MIM; Mendelian inheritance in man number; EC; Enzyme Commission number; MHP; mild hyperphenylalaninemia; enu; N-ethyl-N-nitrosourea; BTBR; black and tan brachyuric mouse strain; DTT; dithiothreitol; K; _e; elimination constant; c; ₀; initial concentration; c; _max; peak concentration; t; _max; time to peak concentration; AUC; area under the concentration vs. time curve at 0–180; min; t; _1/2; elimination half-life; ¹³; C-phenylalanine; l; -[1-13]C-phenylalanine; DOB; delta over baseline; DOB; _BH4; delta over baseline after BH; ₄; treatment; DOB; _Placebo; delta over baseline after Placebo; Phe; ₀; initial phenylalanine concentration; Phe; ₁₈₀; phenylalanine concentration at 180; minPhenylketonuria; Tetrahydrobiopterin; Pharmacodynamics; Mouse model; Compound heterozygous

TLN-4601 peripheral benzodiazepine receptor (PBR/TSPO) binding properties do not mediate apoptosis but confer tumor-specific accumulation by T. Bertomeu; V. Zvereff; A. Ibrahim; S.P. Zehntner; A. Aliaga; P. Rosa-Neto; B.J. Bedell; P. Falardeau; H. Gourdeau (pp. 1572-1579).

TLN-4601 is a farnesylated dibenzodiazepinone isolated from Micromonospora sp. with an antiproliferative effect on several human cancer cell lines. Although the mechanism of action of TLN-4601 is unknown, our earlier work indicated that TLN-4601 binds the PBR (peripheral benzodiazepine receptor; more recently known as the translocator protein or TSPO), an 18kDa protein associated with the mitochondrial permeability transition (mPT) pore. While the exact function of the PBR remains a matter of debate, it has been implicated in heme and steroid synthesis, cellular growth and differentiation, oxygen consumption and apoptosis. Using the Jurkat immortalized T-lymphocyte cell line, documented to have negligible PBR expression, and Jurkat cells stably transfected with a human PBR cDNA, the present study demonstrates that TLN-4601 induces apoptosis independently of PBR expression. As PBRs are overexpressed in brain tumors compared to normal brain, we examined if TLN-4601 would preferentially accumulate in tumors using an intra-cerebral tumor model. Our results demonstrate the ability of TLN-4601 to effectively bind the PBR in vivo as determined by competitive binding assay and receptor occupancy. Analysis of TLN-4601 tissue and plasma indicated that TLN-4601 preferentially accumulates in the tumor. Indeed, drug levels were 200-fold higher in the tumor compared to the normal brain. TLN-4601 accumulation in the tumor (176μg/g) was also significant compared to liver (24.8μg/g; 7-fold) and plasma (16.2μg/mL; 11-fold). Taken together our data indicate that while PBR binding does not mediate cell growth inhibition and apoptosis, PBR binding may allow for the specific accumulation of TLN-4601 in PBR positive tumors.

Keywords: Peripheral benzodiazepine receptor; Translocator protein; TLN-4601; Apoptosis; Tumor accumulation

Role of monoamine oxidase, nitric oxide synthase and regional brain monoamines in the antidepressant-like effects of methylene blue and selected structural analogues by Brian H. Harvey; Ingrid Duvenhage; Francois Viljoen; Nellie Scheepers; Sarel F. Malan; Gregers Wegener; Christiaan B. Brink; Jacobus P. Petzer (pp. 1580-1591).

Dual action antidepressants have important therapeutic implications. Methylene blue (MB), a charged compound structurally related to tricyclic antidepressants, acts on both monoamine oxidase (MAO) and the nitric oxide (NO)-cGMP pathway, and has demonstrated antidepressant activity in rodents. We investigated the antidepressant properties of MB and selected structural analogues and whether their actions involve MAO, NO synthase (NOS) and regional brain monoamines.Acute imipramine (IMI, 15mg/kg), saline, MB, acriflavine (ACR), methylene green (MG), methylene violet (MV), thionine (THI) and tacrine (TAC) (1-60mg/kg i.p.) were tested for antidepressant activity in the forced swim test (FST), as well as MAO-A/B inhibitory activity. Active antidepressant compounds were subsequently studied at their most effective dose during sub-chronic treatment, followed by behavioural sampling in the FST and assay of cortico-limbic monoamines and hippocampal nitrate (for NOS activity).Only IMI, MB (15, 30, 60mg/kg) and MG (7.5, 25, 40mg/kg) reduced immobility in the acute FST. MB, MG and ACR were potent inhibitors of especially MAO-A. Following sub-chronic treatment, IMI (15mg/kg) increased noradrenergic behaviour in the FST, while MB (15mg/kg) and MG (15mg/kg) enhanced serotonergic behaviour. MB and MG bolstered cortico-limbic serotonin (5HT) levels and to a lesser extent l-norepinephrine ( l-NE), but did not significantly alter regional dopamine (DA) levels. MB, and to lesser degree MG, reduced hippocampal nitrate levels.MB and MG present with structure-specific antidepressant-like effects following acute and sub-chronic treatment, possibly involving NOS and MAO-A inhibition and cortico-limbic 5HT and l-NE release. A role for MAO-B and DA appears minimal.

Keywords: Dual action; Methylene blue; Structural analogues; Antidepressant; Monoamine oxidase; Nitric oxide; Serotonin; Norepinephrine; Dopamine

IL-4-mediated transcriptional regulation of human CYP2E1 by two independent signaling pathways by Jue Wang; Yin Hu; Jana Nekvindova; Magnus Ingelman-Sundberg; Etienne P.A. Neve (pp. 1592-1600).

Cytochrome P450 2E1 (CYP2E1), the alcohol-inducible member of the cytochrome P450 super family, plays an important role in both physiological and pathophysiological processes. The present study focused on the induction of human CYP2E1 transcription by the anti-inflammatory cytokine interleukin-4 (IL-4) in human hepatoma B16A2 cells and revealed that this regulation is mediated by two independent pathways. RNA interference and overexpression of STAT6, indicated that the JAK–STAT signaling pathway is involved in IL-4-dependent induction and mutagenesis revealed the presence of a STAT6 binding site in CYP2E1 proximal promoter region (−583/−574-bp). However, inhibition of the JAK–STAT6 pathway using JAK1 siRNA constructs could only partially inhibit the induction of CYP2E1 promoter constructs indicating the presence of a second IL-4 responsive element. Indeed by using a series of truncated CYP2E1 promoter constructs a second more distal IL-4 responsive element (−1604/−1428-bp) was identified, which was further shown to involve the activation of IRS1/2. This induction was dependent on the transcription factor NFATc1 as IL-4-induced CYP2E1 expression was altered by silencing or overexpressing NFATc1. A NFATc1 binding site was identified in the second distal IL-4 responsive element (−1551/−1545-bp) by chromatin immunoprecipitation (ChIP) analysis. Finally simultaneous siRNA-mediated down-regulation of both STAT6 and NFATc1 or mutation of both STAT6 and NFATc1 binding sites abolished the IL-4-dependent transcriptional induction of CYP2E1, demonstrating that both pathways are required for maximal activation. In conclusion, the present study indicates that the induction of CYP2E1 transcription by IL-4 is mediated through two independent parallel pathways, involving JAK–STAT6 and IRS1/2 and NFATc1.

Keywords: Abbreviations; ChIP; chromatin immunoprecipitation; CYP; cytochrome P450; Elk1; member of ETS oncogene family; FOXO1; forkhead box protein O1; IL-4; interleukin-4; IL-4R; interleukin-4 receptor; IRS; insulin receptor substrate; JAK; janus protein kinase; MAPK; mitogen-acitvated protein kinase; NFATc1; nuclear factor of activated T-cells cytoplasmic 1; NF-κB; nuclear factor kappa B; PI3K; phosphoinositide-3-kinase; STAT6; signal transducer and activator of transcription 6Cytochrome P450 2E1; B16A2 cell line; Interleukin-4; Transcriptional activation; Transcription factors

Eicosapentaenoic acid improves hepatic steatosis independent of PPARα activation through inhibition of SREBP-1 maturation in mice by Naoki Tanaka; Xiuguo Zhang; Eiko Sugiyama; Hiroyuki Kono; Akira Horiuchi; Takero Nakajima; Hiroki Kanbe; Eiji Tanaka; Frank J. Gonzalez; Toshifumi Aoyama (pp. 1601-1612).

Eicosapentaenoic acid (EPA) in fish oil is known to improve hepatic steatosis. However, it remains unclear whether such action of EPA is actually caused by peroxisome proliferator-activated receptor α (PPARα) activation. To explore the contribution of PPARα to the effects of EPA itself, male wild-type and Ppara-null mice were fed a saturated fat diet for 16 weeks, and highly (>98%)-purified EPA was administered in the last 12 weeks. Furthermore, the changes caused by EPA treatment were compared to those elicited by fenofibrate (FF), a typical PPARα activator. A saturated fat diet caused macrovesicular steatosis in both genotypes. However, EPA ameliorated steatosis only in wild-type mice without PPARα activation, which was evidently different from numerous previous observations. Instead, EPA inhibited maturation of sterol-responsive element-binding protein (SREBP)-1 in the presence of PPARα through down-regulation of SREBP cleavage-activating protein and site-1 protease. Additionally, EPA suppressed fatty acid uptake and promoted hydrolysis of intrahepatic triglycerides in a PPARα-independent manner. These effects were distinct from those of fenofibrate. Although fenofibrate induced NAPDH oxidase and acyl-coenzyme A oxidase and significantly increased hepatic lipid peroxides, EPA caused PPARα-dependent induction of superoxide dismutases, probably contributing to a decrease in the lipid peroxides. These results firstly demonstrate detailed mechanisms of steatosis-ameliorating effects of EPA without PPARα activation and ensuing augmentation of hepatic oxidative stress.

Keywords: Abbreviations; ACC; acetyl-CoA carboxylase; ALT; alanine aminotransferase; apo; apolipoprotein; AOX; acyl-CoA oxidase; AST; aspartate aminotransferase; CoA; coenzyme A; CPT-I; carnitine palmitoyl-CoA transferase-I; EPA; eicosapentaenoic acid; FA; fatty acid; FAS; fatty acid synthase; FAT; fatty acid translocase; FATP; fatty acid transport protein; FF; fenofibrate; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; GPAT; glycerol-3-phosphate acyltransferase; GPx; glutathione peroxidase; 4-HNE; 4-hydroxynonenal; HTGL; hepatic triglyceride lipase; Insig; insulin-induced gene product; LACS; long-chain acyl-CoA synthase; L-FABP; liver fatty acid-binding protein; LXR; liver X receptor; MCAD; medium-chain acyl-CoA dehydrogenase; MDA; malondialdehyde; mRNA; messenger RNA; MTP; microsomal triglyceride transfer protein; NAFLD; nonalcoholic fatty liver disease; NASH; nonalcoholic steatohepatitis; NEFA; non-esterified fatty acid; NL; neutral lipase; Nrf2; nuclear factor-E2-related factor 2; PGC; PPARγ coactivator; PMP; peroxisomal membrane protein; PPAR; peroxisome proliferator-activated receptor; PUFA; polyunsaturated fatty acid; ROS; reactive oxygen species; RT-PCR; reverse transcription-polymerase chain reaction; SCAP; SREBP cleavage-activating protein; S1P; site-1 protease; SD; standard deviation; SOD; superoxide dismutase; SREBP; sterol regulatory element-binding protein; TG; triglyceride; TNF; tumor necrosis factorβ-Oxidation; Fatty acid uptake; SCAP; S1P; Superoxide dismutase

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Biochemical Pharmacology (v.80, #10)