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Biochemical Pharmacology (v.84, #5)

Editorial Board (pp. iii).

Role of NADPH oxidase/ROS in pro-inflammatory mediators-induced airway and pulmonary diseases by I-Ta Lee; Chuen-Mao Yang (pp. 581-590).

Reactive oxygen species (ROS) are products of normal cellular metabolism and are known to act as second messengers. Under physiological conditions, ROS participate in maintenance of cellular ‘redox homeostasis’ in order to protect cells against oxidative stress. In addition, regulation of redox state is important for cell activation, viability, proliferation, and organ function. However, overproduction of ROS, most frequently due to excessive stimulation of either reduced nicotinamide adenine dinucleotide phosphate (NADPH) by pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) or the mitochondrial electron transport chain and xanthine oxidase, results in oxidative stress. Oxidative stress is a deleterious process that leads to airway and lung damage and consequently to several respiratory inflammatory diseases/injuries, including acute respiratory distress syndrome (ARDS), asthma, cystic fibrosis (CF), and chronic obstructive pulmonary disease (COPD). Many of the known inflammatory target proteins, such as matrix metalloproteinase-9 (MMP-9), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), cyclooxygenase-2 (COX-2), and cytosolic phospholipase A₂ (cPLA₂), are associated with NADPH oxidase activation and ROS overproduction in response to pro-inflammatory mediators. Thus, oxidative stress regulates both key inflammatory signal transduction pathways and target proteins involved in airway and lung inflammation. In this review, we discuss mechanisms of NADPH oxidase/ROS in the expression of inflammatory target proteins involved in airway and lung diseases. Knowledge of the mechanisms of ROS regulation could lead to the pharmacological manipulation of antioxidants in airway and lung inflammation and injury.

Keywords: Antioxidants; Inflammation; NADPH oxidase; Reactive oxygen species; Respiratory inflammatory diseases; Signaling pathways

Hyperactivation of protein phosphatase 2A in models of glucolipotoxicity and diabetes: Potential mechanisms and functional consequences by Anjaneyulu Kowluru; Andrea Matti (pp. 591-597).

The protein phosphatase 2A [PP2A] family of enzymes has been implicated in the regulation of a variety of cellular functions including hormone secretion, growth, survival and apoptosis. PP2A accounts for ∼1% of total cellular protein and ∼ 80% of total serine/threonine phosphatases, thus representing a major class of protein phosphatases in mammalian cells. Despite significant advances in our current understanding of regulation of cellular function by PP2A under physiological conditions, little is understood with regard to its regulation under various pathological conditions, such as diabetes. Emerging evidence suggests hyperactivation of PP2A in liver, muscle, retina and the pancreatic islet under the duress of glucolipotoxicity and diabetes. Interestingly, pharmacological inhibition of PP2A or siRNA-mediated depletion of the catalytic subunit of PP2A [PP2Ac] levels largely restored PP2A activity to near normal levels under these conditions. Herein, we provide an overview of PP2A subunit expression and activity in in vitro and in vivo models of glucolipotoxicity and diabetes, and revisit the existing data, which are suggestive of alterations in post-translational methylation, phosphorylation and nitration of PP2Ac under these conditions. Potential significance of hyperactive PP2A in the context of cell function, survival and apoptosis is also highlighted. It is hoped that this commentary will provide a basis for future studies to explore the potential for PP2Ac as a therapeutic target for the treatment of diabetes and other metabolic disorders.

Keywords: Abbreviations; AMPK; AMP-activated protein kinase; BAECs; bovine aortic endothelial cells; CAPP; ceramide-activated protein phosphatase; CER; ceramide; CK; creatine kinase; CML; carboxylmethylation; DM; diabetes mellitus; DPI; diphenylene iodonium; FOXO1; forkhead box O1; IFNγ; interferon γ; IL-1β; interleukin 1β; iNOS; inducible nitric oxide synthase; LPS; lipopolysaccharide; NFκB; nuclear factor kappa-light-chain-enhancer of activated B cells; NO; nitric oxide; NOE; n-oleoylethanolamine; Nox2; phagocyte-like NADPH oxidase; PA; palmitate; PKC; protein kinase C; PP1; protein phosphatase 1; PP2A; protein phosphatase 2A; PP2Ac; catalytic subunit of PP2A; PP2C; protein phosphatase 2C; PN; peroxynitrite; PPM1; protein phosphatase methyltransferase 1; Pten; the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10; OKA; okadaic acid; SIN-1; 3-morpholinosydnonimine; STZ; streptozotocin; TNFα; tumor necrosis factor α; ZDF rat; Zucker diabetic fatty ratProtein phosphatase 2A; Glucolipotoxicity; Diabetes; Superoxides; Nitric oxide; Peroxynitrite; Carboxylmethylation and nitration

Down-regulation of the PTTG1 proto-oncogene contributes to the melanoma suppressive effects of the cyclin-dependent kinase inhibitor PHA-848125 by Simona Caporali; Ester Alvino; Lauretta Levati; Alessia I. Esposito; Marina Ciomei; Maria G. Brasca; Donatella Del Bufalo; Marianna Desideri; Enzo Bonmassar; Ulrich Pfeffer; Stefania D’Atri (pp. 598-611).

We previously demonstrated that PHA-848125, a cyclin-dependent kinase inhibitor presently under Phase II clinical investigation, impairs melanoma cell growth. In this study, gene expression profiling showed that PHA-848125 significantly modulated the expression of 128 genes, predominantly involved in cell cycle control, in the highly drug-sensitive GL-Mel (p53 wild-type) melanoma cells. Up-regulation of 4 selected genes ( PDCD4, SESN2, DDIT4, DEPDC6), and down-regulation of 6 selected genes ( PTTG1, CDC25A, AURKA, AURKB, PLK1, BIRC5) was confirmed at protein levels. The same protein analysis performed in PHA-848125-treated M10 melanoma cells – p53 mutated and less sensitive to the drug than GL-Mel cells – revealed no DEPDC6 expression and no changes of PTTG1, PDCD4 and BIRC5 levels. Upon PHA-848125 treatment, a marked PTTG1 down-modulation was also observed in A375 cells (p53 wild-type) but not in CN-Mel cells (p53 mutated). PTTG1 silencing significantly inhibited melanoma cell proliferation and induced senescence, with effects less pronounced in p53 mutated cells. PTTG1 silencing increased PHA-848125 sensitivity of p53 mutated cells but not that of A375 or GL-Mel cells. Accordingly, in M10 but not in A375 cells a higher level of senescence was detected in PHA-848125-treated/ PTTG1-silenced cells with respect to PHA-848125-treated controls. In A375 and GL-Mel cells, TP53 silencing attenuated PHA-848125-induced down-modulation of PTTG1 and decreased cell sensitivity to the drug. These findings indicate that PHA-848125-induced down-regulation of PTTG1 depends, at least in part, on p53 function and contributes to the antiproliferative activity of the drug. Our study provides further molecular insight into the antitumor mechanism of PHA-848125.

Keywords: Abbreviations; AURKA; Aurora kinase A; AURKB; Aurora kinase B; BIRC5; baculoviral IAP repeat-containing 5; CDC25A; cell division control 25 A; CDK; cyclin-dependent kinase; CM; complete medium; DDIT4; DNA damage-inducible transcript 4; df; degrees of freedom; DEPDC6; DEP domain containing 6; DEPTOR; DEP-domain-containing mTOR-interactive protein; ECL; enhanced chemiluminescence; FDR; false discovery rate; GO; Gene Ontology; IC; ₅₀; concentration producing 50% inhibition; mAb; monoclonal antibody; mTOR; mammalian target of rapamycin; mTORC1; mammalian target of rapamycin complex 1; mTORC2; mammalian target of rapamycin complex 2; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PARP; poly(ADP-ribose) polymerase; PDCD4; programmed cell death 4; PLK1; polo-like kinase 1; PTTG1; pituitary tumor transforming 1; RB; retinoblastoma; SA-β-Gal; senescence associated β-galactosidase; SAM; Significance Analysis of Microarrays; SESN2; sestrin 2; siRNA; small interfering RNA; TMZ; temozolomidePHA-848125; Melanoma; Proliferation; Gene expression profile; PTTG1

The vascular targeting agent Combretastatin-A4 directly induces autophagy in adenocarcinoma-derived colon cancer cells by Lisa M. Greene; Niamh M. O’Boyle; Derek P. Nolan; Mary J. Meegan; Daniela M. Zisterer (pp. 612-624).

Recent clinical data demonstrated that the vascular targeting agent Combretastatin-A4 phosphate (CA-4P) prolonged survival of patients with advanced anaplastic thyroid cancer without any adverse side effects. However, as a single agent CA-4 failed to reduce tumour growth in the murine CT-26 adenocarcinoma colon cancer model. Furthermore, the molecular mechanism of the innate resistance of HT-29 human adenocarcinoma cells to CA-4 is largely unknown. In this report, we demonstrate for the first time that prolonged exposure to CA-4 and an azetidinone cis-restricted analogue, CA-432 (chemical name; 4-(3-Hydroxy-4-methoxyphenyl)-3-phenyl-1-(3,4,5-trimethoxyphenyl)-azetidin-2-one) induced autophagy in adenocarcinoma-derived CT-26, Caco-2 and HT-29 cells but not in fibrosarcoma-derived HT-1080 cells. Autophagy is a fundamental self-catabolic process which can facilitate a prolonged cell survival in spite of adverse stress by generating energy via lysosomal degradation of cytoplasmic constituents. Autophagy was confirmed by acridine orange staining of vesicle formation, electron microscopy and increased expression of LC3-II. Combretastatin-induced autophagy was associated with a loss of mitochondrial membrane potential and elongation of the mitochondria. Furthermore, inhibition of autophagy by the vacuolar H⁺ATPase inhibitor Bafilomycin-A1 (BAF-A1) significantly enhanced CA-432 induced HT-29 cell death. Both CA-4 and its synthetic derivative, CA-432 induced the formation of large hyperdiploid cells in Caco-2 and CT-26 cells. The formation of these polyploid cells was significantly inhibited by autophagy inhibitor, BAF-A1. Results presented within demonstrate that autophagy is a novel response to combretastatin exposure and may be manipulated to enhance the therapeutic efficacy of this class of vascular targeting agents.

Keywords: Combretastatin; Autophagy; Apoptosis; Tubulin and mitochondria

MurD enzymes from different bacteria: Evaluation of inhibitors by Hélène Barreteau; Izidor Sosič; Samo Turk; Jan Humljan; Tihomir Tomašić; Nace Zidar; Mireille Hervé; Audrey Boniface; Lucija Peterlin-Mašič; Danijel Kikelj; Dominique Mengin-Lecreulx; Stanislav Gobec; Didier Blanot (pp. 625-632).

d-Glutamic acid-adding enzyme (MurD ligase) catalyses the addition ofd-glutamic acid to UDP- N-acetylmuramoyl-l-alanine, an essential cytoplasmic step in the pathway for bacterial cell-wall peptidoglycan synthesis. As such, it represents an important antibacterial drug-discovery target enzyme. Recently, several series of compounds have been synthesised and found to inhibit MurD from Escherichia coli, the best one having an IC₅₀ value of 8μM. In the present work, we have tested 20 of these compounds against the MurD enzymes from Staphylococcus aureus, Streptococcus pneumoniae, Borrelia burgdorferi and Mycobacterium tuberculosis. Most of the E. coli MurD inhibitors appeared less efficient against the four other orthologues. This divergent result can be explained by the differences in amino acid sequences and topologies of the active sites of the MurD ligases studied.

Keywords: Abbreviations; HPLC; high-performance liquid chromatography; MurNAc; N; -acetylmuramic acid; Ni-NTA; Ni; ²⁺; -nitrilotriacetate; SDS-PAGE; sodium dodecylsulfate-polyacrylamide gel electrophoresis; TLC; thin-layer chromatography; Subscripts Bb, Ec, Mt, Sa and Sp denote the; B. burgdorferi; ,; E. coli; M. tuberculosis; ,; S. aureus; and; S. pneumoniae; origins, respectively, of genes and enzymesd; -Glutamic acid-adding enzymes; MurD ligase active sites; MurD ligase inhibitors; Pathogenic bacteria; Peptidoglycan biosynthesis

CIL-102 binds to tubulin at colchicine binding site and triggers apoptosis in MCF-7 cells by inducing monopolar and multinucleated cells by K.K. Gireesh; Aijaz Rashid; Soumyananda Chakraborti; Dulal Panda; Tapas Manna (pp. 633-645).

A plant dictamine analog, 1-[4-(furo[2,3- b]quinolin-4-ylamino)phenyl]ethanone (CIL-102) has been shown to exert potent anti-tumor activity. In this study, we examined the mode of interaction of CIL-102 with tubulin and unraveled the cellular mechanism responsible for its anti-tumor activity. CIL-102 bound to tubulin at a single site with a dissociation constant ∼0.4μM. Isothermal titration calorimetry revealed that CIL-102–tubulin interaction is highly enthalpy driven and that the binding affords a large negative heat capacity change (Δ C_p=−790calmol⁻¹K⁻¹) with an enthalpy–entropy compensation. An analysis of the modified Dixon plot suggested that CIL-102 competitively inhibited the binding of podophyllotoxin, a colchicine-binding site agent, to tubulin. Computational modeling indicated that CIL-102 binds exclusively at the β-subunit of tubulin and that CIL-102 and colchicine partially share their binding sites on tubulin. It bound to tubulin reversibly and the binding was estimated to be ∼1000 times faster than that of colchicine. CIL-102 potently inhibited the proliferation of MCF-7 cells, induced monopolar spindle formation and multi-nucleation. At half-maximal inhibitory concentration, the spindle microtubules were visibly depolymerized and disorganized. CIL-102 reduced the inter-polar distances of bipolar mitotic cells indicating that it impaired microtubule–kinetochore attachments. CIL-102-treatment induced apoptosis in MCF-7 cells in association with increased nuclear accumulation of p53 and p21 suggesting that apoptosis is triggered through a p53–p21 dependent pathway. The results indicated that CIL-102 exerted anti-proliferative activity by disrupting microtubule functions through tubulin binding and provided important insights into the differential mode of tubulin binding by CIL-102 and colchicine.

Keywords: Abbreviations; GTP; guanosine triphosphate; PIPES; piperazine-N,N′-bis(2-ethanesulfonic acid); EGTA; ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′ tetraacetic acid; FITC; fluorescein isothiocyanate; DTNB; 5, 5′-dithiobis-2-nitrobenzoic acid; DMSO; dimethyl sulfoxide; TCA; trichloro acetic acid; PI; propidium iodideTubulin; CIL-102; Enthalpy; Entropy; p53; p21

Mechanism of putative neo-antigen formation from N-propionyl-4- S-cysteaminylphenol, a tyrosinase substrate, in melanoma models by Shosuke Ito; Akira Nishigaki; Yasue Ishii-Osai; Makoto Ojika; Kazumasa Wakamatsu; Toshiharu Yamashita; Yasuaki Tamura; Akira Ito; Hiroyuki Honda; Eiichi Nakayama; Kowichi Jimbow (pp. 646-653).

Metastatic melanoma is resistant to conventional therapies. N-propionyl-4- S-cysteaminylphenol (NPrCAP), an N-protected sulfur-amine analog of tyrosine, is a good substrate for tyrosinase and is selectively incorporated into melanoma cells, causing cytotoxicity in vitro and in vivo. We have recently shown that intratumoral injections of NPrCAP suppress not only the growth of primary B16F1 melanoma tumors but also of secondary, re-challenged tumors. The participation of CD8⁺ T cells has been suggested for the NPrCAP-mediated anti-B16 melanoma immunity. In this study, the molecular mechanism of the NPrCAP cytotoxicity and immunogenicity was examined. The phenol NPrCAP was shown to be activated by mushroom tyrosinase to the ortho-quinone N-propionyl-4- S-cysteaminyl-1,2-benzoquinone (NPrCAQ), and the structure was confirmed by reducing it to the corresponding catechol. NPrCAQ reacted rapidly with biologically relevant sulfhydryl compounds such as cysteine, glutathione and bovine serum albumin. The NPrCAQ-thiol adduct formation was proven with a model thiol N-acetylcysteine by spectroscopic methods. The production and release of NPrCAQ-protein adducts was verified in B16F1 melanoma cells in vitro and in B16F1 melanoma-bearing mice in vivo through the detection of 5 -S-cysteaminyl-3- S-cysteinylcatechol after acid hydrolysis of the protein fraction. These results suggest that the phenol NPrCAP, acting as a prohapten, can be activated in melanoma cells by tyrosinase to the quinone-hapten NPrCAQ, which binds to melanosomal proteins through their cysteine residues to form possible neo-antigens, thus triggering the immunological response. NPrCAP thus represents a potential new approach to immunotherapy against metastatic melanoma.

Keywords: Abbreviations; 4-; S; -CAP; 4-; S; -cysteaminylphenol; NAcCAP; N; -acetyl-4-; S; -cysteaminylphenol; NPrCAP; N; -propionyl-4-; S; -cysteaminylphenol; NPrCAQ; N; -propionyl-4-; S; -cysteaminyl-1,2-benzoquinone; CA-CysC; 5-; S; -cysteaminyl-3-; S; -cysteinylcatechol; NAcCys; N; -acetyl-; l; -cysteine; BSA; bovine serum albumin; NPrCAC; N; -propionyl-4-; S; -cysteaminylcatechol; NAcCys-NPrCAC; 3-; S; -(; N; -acetylcysteinyl)-5-; S; -(; N; -propionylcysteaminyl)catechol; ROS; reactive oxygen species N; -propionyl-4-; S; -cysteaminylphenol; Melanoma; Melanogenesis; Tyrosinase; Sulfhydryl group; Hapten

The kinetic mechanism of S. pneumoniae DNA ligase and inhibition by adenosine-based antibacterial compounds by Haris Jahić; Ce Feng Liu; Jason Thresher; Stephania Livchak; Hongming Wang; David E. Ehmann (pp. 654-660).

The NAD-dependent DNA ligase is an excellent target for the discovery of antibacterial agents with a novel mode of action. In this work the DNA ligase from Streptococcus pneumoniae was investigated for its steady-state kinetic parameters and inhibition by compounds with an adenosine substructure. Inhibition by substrate DNA that was observed in the enzyme turnover experiments was verified by direct binding measurements using isothermal titration calorimetry (ITC). The substrate-inhibited enzyme form was identified as deadenylated DNA ligase. The binding potencies of 2-(butylsulfanyl) adenosine and 2-(cyclopentyloxy) adenosine were not significantly affected by the presence of the enzyme-bound DNA substrate. Finally, a mutant protein was prepared that was known to confer resistance to the adenosine compounds’ antibacterial activity. The mutant protein was shown to have little catalytic impairment yet it was less susceptible to adenosine compound inhibition.

Keywords: DNA; Ligase; Enzyme; Inhibition; Antibiotics

Reactive oxygen species are required for β2 adrenergic receptor–β-arrestin interactions and signaling to ERK1/2 by Monalisa Singh; Nader H. Moniri (pp. 661-669).

The β2-adrenergic receptor (β2AR) is the prototypical member of the heptahelical G protein-coupled receptor (GPCR) superfamily and is well-known to elicit biological effects through both G protein-dependent and G protein-independent signaling cascades. Agonism of β2AR has been described to promote phosphorylation and activation of extracellular signal-regulated kinases (ERK1/2) via a G-protein/PKA pathway that transpires rapidly upon receptor agonism, as well as by a distinct β-arrestin-mediated pathway that occurs at later time points. We have previously shown that β2AR agonism promotes generation of intracellular reactive oxygen species (ROS) and that β2AR-associated G protein signaling is dependent on ROS formation. It has also been suggested that β2AR-mediated ROS generation occurs via recruitment of β-arrestins. In this study, we confirm the effects of β-arrestin on β2AR-induced ROS generation, and investigate the ROS-dependency of β-arrestin-linked β2AR signaling. In HEK293 cells, both coimmunoprecipitation and BRET studies reveal that ROS are vital for the physical interaction of β2AR with β-arrestin partner proteins. Using phosphorylation of ERK1/2 as a functional endpoint to assess the role of ROS in β2AR–β-arrestin signaling, our results show that inhibition of intracellular ROS abrogates both the β-arrestin and G protein-mediated phosphorylation of ERK1/2 upon agonism of β2AR. Importantly, both the G protein and β-arrestin components were reversed upon exogenous administration of ROS, suggesting a critical role for oxidants in stabilization of β2AR. Taken together, our data signify that ROS serve purposeful roles in stabilizing both G protein- and β-arrestin-mediated β2AR signaling in HEK293 cells.

Keywords: β2-Adrenergic receptor; β-Arrestin; Extracellular signal-regulated kinase; Reactive oxygen species

Suppression of Akt/Foxp3-mediated miR-183 expression blocks Sp1-mediated ADAM17 expression and TNFα-mediated NFκB activation in piceatannol-treated human leukemia U937 cells by Wen-Hsin Liu; Long-Sen Chang (pp. 670-680).

To address the mechanism of piceatannol in inhibiting TNFα-mediated pathway, studies on piceatannol-treated human leukemia U937 cells were conducted. Piceatannol treatment reduced TNFα shedding and NFκB activation and decreased the release of soluble TNFα into the culture medium of U937 cells. Moreover, ADAM17 expression was down-regulated in piceatannol-treated cells. Over-expression of ADAM17 abrogated the ability of piceatannol to suppress TNFα-mediated NFκB activation. Piceatannol-evoked β-TrCP up-regulation promoted Sp1 degradation, thus reducing transcriptional level of ADAM17 gene in U937 cells. Piceatannol treatment induced p38 MAPK phosphorylation but inactivation of Akt and ERK. In contrast to p38 MAPK inhibitor or restoration of ERK activation, transfection of constitutive active Akt abolished the effect of piceatannol on β-TrCP, Sp1 and ADAM17 expression. Piceatannol-elicited down-regulation of miR-183 expression was found to cause β-TrCP up-regulation. Inactivation of Akt resulted in Foxp3 down-regulation and reduced miR-183 expression in piceatannol-treated cells. Knock-down of Foxp3 and chromatin immunoprecipitating revealed that Foxp3 genetically regulated transcription of miR-183 gene. Taken together, our data indicate that suppression of Akt/Foxp3-mediated miR-183 expression blocks Sp1-mediated ADAM17 expression in piceatannol-treated U937 cells. Consequently, piceatannol suppresses TNFα shedding, leading to inhibition of TNFα/NFκB pathway.

Keywords: Piceatannol; TNFα; Sp1; Foxp3; β-TrCP; miR-183

A novel C(28)-hydroxylated lupeolic acid suppresses the biosynthesis of eicosanoids through inhibition of cytosolic phospholipase A₂ by Moritz Verhoff; Stefanie Seitz; Hinnak Northoff; Johann Jauch; Anja M. Schaible; Oliver Werz (pp. 681-691).

Eicosanoids are potent lipid mediators derived from phospholipase (PL)-released arachidonic acid (AA) coupled to subsequent metabolism by cyclooxygenase (COX)-1/2 or lipoxygenases (LO) which are involved in a variety of homeostatic biological functions and inflammation. We have investigated three lupeolic acids (LA) from the gum resin of Boswellia carterii for their ability to interfere with eicosanoid biosynthesis in human blood cells. A novel, yet unknown C(28)-hydroxylated LA, that is, 3α-acetoxy-28-hydroxylup-20(29)-en-4β-oic acid (Ac-OH-LA) was found to inhibit the biosynthesis of COX-, 5-LO- and 12-LO-derived eicosanoids from endogenous AA in activated platelets, neutrophils, and monocytes from human blood with consistent IC₅₀ values of 2.3–6.9μM. In contrast, two other LAs lacking the C(28)-OH moiety were essentially inactive in this respect. Inhibition of eicosanoids by Ac-OH-LA correlated with reduced release of AA in intact cells. When AA was exogenously provided as substrate for cellular eicosanoid biosynthesis the inhibitory effects of Ac-OH-LA were essentially reversed, even though some inhibition of 5-LO and COX-1 product formation still remained. Finally, by means of a cell-free phospholipid hydrolysis assay using human recombinant cytosolic PLA₂α, we show that Ac-OH-LA may directly interfere with cPLA₂α activity (IC₅₀=3.6μM). Together, we identified a novel, naturally occuring C(28)-hydroxylated LA which acts as efficient inhibitor of cPLA₂α and consequently suppresses eicosanoid biosynthesis in intact cells.

Keywords: Abbreviations; 12-HETE; 12(S)-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid; 12–HHT; 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid; LO; lipoxygenase; AA; arachidonic acid; Ac-LA; 3α-acetoxy-lup-20(29)-en-4β-oic acid; Ac-OH-LA; 3α–acetoxy-28-hydroxylup-20(29)-en-4β-oic acid; BA; boswellic acid; COX; cyclooxygenase; FCS; fetal calf serum; LA; 3α-hydroxylup-20(29)-en-4β-oic acid; LUV; large unilamellar vesicles; PAPC; 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; PL; phospholipase; POG; 1-palmitoyl-2-oleoyl-sn-glycerolLupeolic acid; Triterpenes; Cytosolic phospholipase A; ₂; Eicosanoids; Inflammation; Arachidonic acid

Astaxanthin functions differently as a selective peroxisome proliferator-activated receptor γ modulator in adipocytes and macrophages by Makoto Inoue; Hiroki Tanabe; Akira Matsumoto; Michiyo Takagi; Keizo Umegaki; Sakae Amagaya; Jiro Takahashi (pp. 692-700).

Astaxanthin (ASX), an oxygenated carotenoid (xanthophyll), has previously been shown to exert ameliorative effects on obesity and insulin resistance, but the underlying mechanisms were not clearly elucidated. In the present study, we investigated whether ASX serves as a novel selective peroxisome proliferator-activated receptor (PPAR) γ modulator. Analyses of PPARγ binding by CoA-BAP assays revealed that ASX bound to PPARγ in a dose-dependent manner. However, ASX was unable to activate transcription in PPARγ reporter assays, although it antagonized transcriptional activation by the PPARγ agonist rosiglitazone (RGZ). When the molecular interactions between PPARγ and three coactivators were examined, ASX increased the interactions of PPARγ with transcriptional intermediary factor 2 (TIF2) and steroid receptor coactivator-1 (SRC-1), but not cAMP responsive element-binding protein (CREB)-binding protein (CBP). In addition, ASX effectively blocked the increase in CBP recruitment to PPARγ mediated by RGZ. ASX alone did not stimulate 3T3-L1 cell differentiation, although it antagonized 3T3-L1 cell differentiation and lipid accumulation induced by RGZ, similar to the PPARγ antagonist GW9662. When the effects of cotreatment of 3T3-L1 cells with ASX and RGZ were determined based on the mRNA levels of PPARγ target genes, ASX effectively reduced the mRNA levels of aP2 and lipoprotein lipase, but not CD36. Intriguingly, ASX was capable of inducing PPARγ target genes such as liver X receptor, CD36 and ABCA1 in thioglycollate-elicited peritoneal macrophages. Collectively, the present findings indicate that ASX is a novel selective PPARγ modulator that acts as an antagonist or agonist depending on the cell context.

Keywords: Astaxanthin; Xanthophyll; Selective peroxisome proliferator-activated receptor γ modulator; Adipogenesis; Nuclear receptor

DEC1 binding to the proximal promoter of CYP3A4 ascribes to the downregulation of CYP3A4 expression by IL-6 in primary human hepatocytes by Zhao Mao; Xiaofei Luan; Gang Cao; Wei Liu; Jing Xiong; Gang Hu; Ruini Chen; Rui Ning; Wei Shang; Jian Yang; Bingfang Yan (pp. 701-711).

In this study, we provided molecular evidences that interleukin-6 (IL-6) contributed to the decreased capacity of oxidative biotransformation in human liver by suppressing the expression of cytochrome P450 3A4 (CYP3A4). After human hepatocytes were treated with IL-6, differentially expressed in chondrocytes 1 (DEC1) expression rapidly increased, and subsequently, the CYP3A4 expression decreased continuously. Furthermore, the repression of CYP3A4 by IL-6 occurred after the increase of DEC1 in primary human hepatocytes. In HepG2 cells, knockdown of DEC1 increased the CYP3A4 expression and its enzymatic activity. In addition, it partially abolished the decreased CYP3A4 expression as well as its enzymatic activity induced by IL-6. Consistent with this, overexpression of DEC1 markedly reduced the CYP3A4 promoter activity and the CYP3A4 expression as well as its enzymatic activity. Using sequential truncation and site directed mutagenesis of CYP3A4 proximal promoter with DEC1 construct, we showed that DEC1 specifically bound to CCCTGC sequence in the proximal promoter of CYP3A4, which was validated by EMSA and ChIP assay. These findings suggest that the repression of CYP3A4 by IL-6 is achieved through increasing the DEC1 expression in human hepatocytes, the increased DEC1 binds to the CCCTGC sequence in the promoter of CYP3A4 to form CCCTGC–DEC1 complex, and the complex downregulates the CYP3A4 expression and its enzymatic activity.

Keywords: Abbreviations; DEC1; differentially expressed in chondrocytes 1; IL-6; interleukin-6; CYP450; cytochrome P450; PXR; pregnane X receptor; hPXR; human pregnane X receptor; Rif; rifampicin; TNF-α; tumor necrosis factor α; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; PCR; polymerase chain reaction; DMEM; Dulbecco's modified Eagle's medium; PBS; phosphate-buffered saline; bHLH; basic helix-loop-hellix; HLH; helix-loop-hellix; SHARP; split and hairy related protein; STRA13; stimulated with retinoic acid 13; qRT-PCR; quantitative reverse transcription-polymerase chain reaction; EMSA; electrophoretic mobility shift assay; ChIP; chromatin immunoprecipitationCytochrome P450 3A4 (CYP3A4); Interleukin 6 (IL-6); Differentially expressed in chondrocytes 1 (DEC1); Transcription regulator

Inhibition of sphingosine kinase 1 enhances cytotoxicity, ceramide levels and ROS formation in liver cancer cells treated with selenite by V. Chatzakos; A.K. Rundlöf; D. Ahmed; P.J. de Verdier; J. Flygare (pp. 712-721).

Combined treatment with selenite and the spingosine kinase 1 inhibitor SK1-II induces cell death in liver cancer cells (Huh7) without affecting non-tumorigenic immortalized liver cells (MIHA).High doses of selenite have been shown to induce cell death in acute myeloid leukemia and lung cancer cells. In this study, we combined selenite treatment with modulators of sphingolipid metabolism in the hepatocellular carcinoma cell line Huh7. Treatment with 20μM of selenite reduced the viability of Huh7 cells by half and increased the levels of long chain C14-, C16-, C18- and C18:1- ceramides by two-fold. Inhibition of neutral sphingomyelinase with 3-O-methylsphingosine significantly reduced the cytotoxic effect of selenite. In line with this result, selenite caused a 2.5-fold increase in the activity of neutral sphingomyelinase. The sphingosine kinase 1 (SK1) inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole (SK1-II) sensitized the cells to the cytotoxic effects of selenite. Preincubation with 10μM of SK1-II prior to treatment with 10μM of selenite caused induction of apoptosis and gave rise to a 2.5-fold increase in C14-, C16-, C18- and C18:1- ceramides. Instead, 50μM of SK1-II combined with 10μM of selenite caused accumulation of cells in G1/S phases, but less apoptosis and accumulation of ceramides. The formation of reactive oxygen species (ROS) after treatment with 10μM of selenite was maximally enhanced by 1μM of SK1-II. Moreover, combined treatment with SK1-II and 10μM of selenite synergistically reduced the number of viable Huh7 cells, while the non-tumorigenic hepatocyte cell line MIHA remained unaffected by the same treatment. These results raise the possibility that a combination of selenite and SK1 inhibitors could be used to treat liver cancer cells, that are regarded as drug resistant, at doses that are non-toxic to normal liver cells.

Keywords: Abbreviations; SK1; sphingosine kinase 1; PDMP; 1-Phenyl-2-decanoylamino-3-morpholino-1-propanol; ROS; reactive oxygen species; S1P; sphingosine 1 phosphate; SMase; sphingomyelinases; 3-OMS; 3-O-methylsphingosine; GCS; glycosylceramide synthase; SK1-II; 2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole; HCC; hepatocellular carcinoma; DMEM; Dulbecco's modified Eagle's MediumSelenite; Sphingolipids; Ceramide; ROS; Hepatocellular carcinoma

Genetic and epigenetic regulation of AHR gene expression in MCF-7 breast cancer cells: Role of the proximal promoter GC-rich region by Neal A. Englert; Robert J. Turesky; Weiguo Han; Erin E. Bessette; Simon D. Spivack; Michele Caggana; David C. Spink; Barbara C. Spink (pp. 722-735).

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, contributes to carcinogenesis through its role in the regulation of cytochrome P450 1 (CYP1)-catalyzed metabolism of carcinogens. Here, we investigated genetic and epigenetic mechanisms that affect AhR expression. Analyses of the human AHR proximal promoter in MCF-7 human breast cancer cells using luciferase assays and electrophoretic mobility shift assays revealed multiple specificity protein (Sp) 1 binding sequences that are transcriptional activators in vitro. The regulation of AhR expression was evaluated in long-term estrogen exposed (LTEE) MCF-7 cells, which showed increased AhR expression, enhanced CYP1 inducibility, and increased capacity to form DNA adducts when exposed to the dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine. The increased AhR expression in LTEE cells was found not to result from increased mRNA stability, differential RNA processing, or decreased DNA methylation. Analysis of the AHR proximal promoter region using chromatin immunoprecipitation confirmed that enhanced expression of AhR in LTEE cells involves changes in histone modifications, notably decreased trimethylation of histone 3, lysine 27. Upon further examination of the GC-rich Sp1-binding region, we confirmed that it contains a polymorphic (GGGGC)_n repeat. In a population of newborns from New York State, the allele frequency of (GGGGC)_n was n=4>5≫6, 2. Circular dichroism spectroscopy revealed the ability of sequences of this GC-rich region to form guanine-quadruplex structures in vitro. These studies revealed multiple levels at which AhR expression may be controlled, and offer additional insights into mechanisms regulating AhR expression that can ultimately impact carcinogenesis.

Keywords: Abbreviations; AIP; AhR interacting protein; AhR; aryl hydrocarbon receptor; BaP; benzo[; a; ]pyrene; CD; circular dichroism; ChIP; chromatin immunoprecipitation; CYP; cytochrome P450; dG-C8-PhIP; N; -(deoxyguanosin-8-yl)-PhIP; E; ₂; 17β-estradiol; EMSA; electrophoretic mobility shift assay; ER; estrogen receptor; EROD; ethoxyresorufin-; O; -deethylase; H3K4me2; histone 3 dimethyl lysine 4; H3K27me3; histone 3 trimethyl lysine 27; hnRNA; heterologous nuclear RNA; KLF4; Krüppel-like factor-4; LC-ESI/MS; ³; liquid chromatography-electrospray ionization triple-stage mass spectrometry; LTEE; long-term estrogen exposure; ODN; oligodeoxynucleotide; PAH; polycylic aromatic hydrocarbon; PCR; polymerase chain reaction; PGR; progesterone receptor; PhIP; 2-amino-1-methyl-6-phenylimidazo[4,5-; b; ]pyridine; RNA Pol II; RNA-polymerase II; SE; standard error; Sp; specificity protein; TCDD; 2,3,7,8-tetrachlorodibenzo-; p; -dioxin; TSS; transcriptional start siteAryl hydrocarbon receptor; Long-term estrogen exposure; Epigenetic; (GGGGC); _n; repeat polymorphism; Guanine-quadruplex; 2-Amino-1-methyl-6-phenylimidazo[4,5-; b; ]pyridine

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Biochemical Pharmacology (v.84, #5)