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Biochemical Pharmacology (v.82, #12)

Editorial Board (pp. iii).

Island biogeography effects on microbial evolution may contribute to Crohn's disease by Mac A. Johnson; Raymond J. Winquist (pp. 1801-1806).

Inflammatory bowel diseases (IBDs), such as Crohn's disease (CD), involve a poorly understood and complex immune response to both the biota of the human gut and the gut itself. The role of the gut biota in human health has been ill defined and attitudes toward the intestinal flora have ranged from judging them largely irrelevant to declaring them a human organ system. A better way to view the intestinal flora is as a group of evolutionarily self-interested species that form large, potentially interbreeding populations that utilize human beings as a series of semi-isolated habitats, like islands in an archipelago. Here we propose that the imposition of modern sanitation and hygiene standards has drastically attenuated the connection between the “islands” inhabited by the gut flora, and that existing work drawn from evolutionary biology studies of island ecosystems, rather than medicine, predicts that the evolution of gut flora should now be pushed toward limited-dispersion forms of intestinal microorganisms – a proposition borne out by the discovery of so-called “adherent invasive Escherichia coli.” This pathogenic variant of the gut bacterium E. coli clings to and invades the intestinal epithelium and has been implicated in CD. Gut flora and diseases of the gut should arguably be studied as ecology as much as medicine, and treated within this context.

Keywords: Abbreviations; IBD; inflammatory bowel disease; CD; Crohn's disease; E. coli; Escherichia coli; AIEC; adherent, invasive; E. coli; UC; ulcerative colitis; RA; rheumatoid arthritis; IL-12/23, IL-17; interleukin 12/23, interleukin 17; TNF-α; tumor necrosis factor-alpha; Ig; immunoglobulin; Fim; fimbrial family proteins [FM]; ELISA; enzyme linked immunosorbent assay; NMR; nuclear magnetic ratio; UPEC; uropathogenic; E. coli; CEACAM; carcinoembryonic antigen-related cell adhesion molecule; ZO-2; zona occludens protein 2; PKCζ; protein kinase C zeta; VacA; vacuolating cytotoxin gene A; GWAS; genome wide association studies; NOD2; nucleotide-binding oligomerization domain-containing 2; ATG16L1; autophagy-related 16-like 1; IRGM; immunity-related GTPase family MCrohn's disease; Gut biota; Inflammatory bowel disease; Adhesive invasive; E. coli; Biogeography theory

Green tea catechin, epigallocatechin-3-gallate (EGCG): Mechanisms, perspectives and clinical applications by Brahma N. Singh; Sharmila Shankar; Rakesh K. Srivastava (pp. 1807-1821).

An expanding body of preclinical evidence suggests EGCG, the major catechin found in green tea ( Camellia sinensis), has the potential to impact a variety of human diseases. Apparently, EGCG functions as a powerful antioxidant, preventing oxidative damage in healthy cells, but also as an antiangiogenic and antitumor agent and as a modulator of tumor cell response to chemotherapy. Much of the cancer chemopreventive properties of green tea are mediated by EGCG that induces apoptosis and promotes cell growth arrest by altering the expression of cell cycle regulatory proteins, activating killer caspases, and suppressing oncogenic transcription factors and pluripotency maintain factors. In vitro studies have demonstrated that EGCG blocks carcinogenesis by affecting a wide array of signal transduction pathways including JAK/STAT, MAPK, PI3K/AKT, Wnt and Notch. EGCG stimulates telomere fragmentation through inhibiting telomerase activity. Various clinical studies have revealed that treatment by EGCG inhibits tumor incidence and multiplicity in different organ sites such as liver, stomach, skin, lung, mammary gland and colon. Recent work demonstrated that EGCG reduced DNMTs, proteases, and DHFR activities, which would affect transcription of TSGs and protein synthesis. EGCG has great potential in cancer prevention because of its safety, low cost and bioavailability. In this review, we discuss its cancer preventive properties and its mechanism of action at numerous points regulating cancer cell growth, survival, angiogenesis and metastasis. Therefore, non-toxic natural agent could be useful either alone or in combination with conventional therapeutics for the prevention of tumor progression and/or treatment of human malignancies.

Keywords: Abbreviations; AMPK; adenosine monophosphate-activated protein kinase; AP1; activator protein 1; AREs; antioxidant responsive elements; Bak; Bcl-2 antagonist killer; Bax; Bcl-2 associated x protein; Bcl-2; B-cell lymphoma-2; CAT; catalase; CDKN2A; cyclin dependent kinase 2A; Cdks; cyclin-dependent kinases; c-IAP1; cellular inhibitor of apoptosis protein1; COX-2; cyclooxygenase-2; CpG; cytosine-phosphate-guanine; CSCs; cancer stem cells; CYP; cytochrome P450; DHFR; dihydrofolate reductase; DIABLO; direct inhibitor of apoptosis-binding protein with low pI; DNMTs; DNA methyltransferases; EGCG; (−)-epigallocatechin-3-gallate; EGFR; epidermal growth factor receptor; EpRE; electrophile-responsive element; ERK; extracellular signal-regulated kinase; FAK; focal adhesion kinase; FKHR; forkhead homolog of rhabdosarcoma; GFRs; growth factor receptors; GPx; glutathione peroxidase; GR; glutathione reductase; GST; glutathione; S; -transferase; H; ₂; O; ₂; hydrogen peroxide; HATs; histone acetyl transferases; HDACs; histone deacetylases; HER; human epidermal receptor; HIF; hypoxia inducible factor; hTERT; human telomerase reverse transcriptase; HUVEC; human vascular endothelial cell; IKK; I kappa B kinase; IL-1; interleukin 1; JNK; Jun NH; ₂; -terminal kinase; LPs; Lipopolysaccharides; MAP; mitogen-activated protein; MBD; methyl-CpG binding domain; Mcl-1; myeloid cell leukemia 1; mdm2; mouse double minute 2; MEKK1; mitogen-activated protein/ERK kinase 1; MLH1; MutL homologue 1; MMPs; matrix metalloproteinases; MRLC; myosin regulatory light chain; MT1-MMP; membrane Type 1-matrix metalloproteinase; NFκB; nuclear factor kappaB; NQO; NADPH quinone oxidoreductase; Nrf; NF-E2 p45-related factor; O; ₂; ⁻; superoxide anion radical; OH; hydroxyl radical; p90RSK; 90 kDa ribosomal S6 kinase; PCNA; proliferating cell nuclear antigen; PDGF; platelet-derived growth factor; PGE2; prostaglandins E2; PI3K; phosphatidylinositol-3-kinase; PKA; protein kinase A; PKB; protein kinase B; PKC; protein kinase C; PPAR; peroxisome proliferator-activated receptor; pRb; retinoblastoma protein; PUMA; p53 upregulated modulator of apoptosis; RAR; retinoic acid receptor; RECK; reversion-inducing cysteine-rich protein with Kazal motifs; ROS; reactive oxygen species; RTK; receptor tyrosine kinase; RXRα; retinoid X receptor alpha; SAM; S; -adenosyl-methionine; siRNA; small-interfering RNA; Smac; second mitochondria-derived activator of caspase; SOD; superoxide dismutase; STAT; signal transducers and activators of transcription; TERT; telomerase reverse transcriptase; TIMP; tissue inhibitor of metalloproteinase; TRAIL; tumor necrosis factor-related apoptosis-inducing ligand; TRAMP; transgenic adenocarcinoma of the mouse prostate; TSGs; tumor suppressor genes; uPA; urokinase plasminogen activator; VEGF; vascular endothelial growth factor; VEGFR-2; vascular endothelial growth factor receptor-2; XIAP; X-linked inhibitor of apoptosis protein; α-TNF; alpha-tumor necrosis factorChemoprevention; Cell cycle; Apoptosis; Cellular signaling cascades; EGCG

N, N-Bis(cyclohexanol)amine aryl esters inhibit P-glycoprotein as transport substrates by Annalisa Neri; Maria Frosini; Massimo Valoti; Marcello G. Cacace; Elisabetta Teodori; Giampietro Sgaragli (pp. 1822-1831).

Inhibition of Pgp-mediated R123 efflux from L5178 MDR1 cells by compound 2d as a function of R123 concentration.P-Glycoprotein (Pgp) inhibition by three sets of four isomers of N, N-bis(cyclohexanol)amine aryl esters was assessed on rhodamine 123 (R123) efflux in human MDR1-gene transfected mouse T-lymphoma L5178 cells and on Sf9 ATPase activity. The most active compounds inhibited Pgp with IC₅₀ values much lower than those of either cyclosporin A (CSA) or GF120918. As to R123 efflux inhibition, the role of the bond present in the second aryl moiety appeared important since the triple bond derivatives (3a–d) were the most powerful as compared to the double bond (2a–d) and the single bond (1a–d) counterparts. Concentration–inhibition curves of 2c and 3d exhibited a biphasic behaviour suggesting the existence of two binding sites in the recognition domain of Pgp. Persistence of inhibition by these compounds resulted to be intermediate between that caused by CSA and GF120918. R123 exhibited positive interaction with CSA, 1d, 1c, 2d, 2c and 3c, the concentration–inhibition curves being shifted leftward when R123 concentration was increased, while it exhibited negative interaction with 3d and no effect with GF120918. Sf9 ATPase activity was stimulated in an increasing order of potency by 2c, 3c, 2d, CSA, epirubicin and 3d. In a decreasing order of potency 3d, 2c, GF120918, CSA, 2d and 3c inhibited at sub-nanomolar concentrations epirubicin-stimulated ATPase activity. In conclusion, isomeric geometry and restriction of molecular flexibility of N, N-bis(cyclohexanol)amine aryl esters were crucial for their presentation to and inhibition of Pgp as transport substrates, R123 and epirubicin cooperating with them to this inhibition.

Keywords: Abbreviations; CSA; cyclosporin A; DMSO; dimethylsulfoxide; GF120918; N-(4-(2-(1,2,3,4-Tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl)phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide; MDR; multidrug resistance; MFI; mean fluorescence intensity; L5178 MDR1 cell line; L5178 mouse T-lymphoma MDR1 expressing cell line; Pgp; P-glycoprotein; R123; rhodamine123; Sf9; intestinal cell membranes of; Spodoptera frugiperda; enriched of human Pgp; Vi; sodium orthovanadateMDR reverters; Mouse T-lymphoma MDR1 cells; N; ,; N; -Bis(cyclohexanol)amine aryl esters; Epirubicin; Sf; 9-ATPase activity; Pgp inhibitors; Rhodamine123 cell efflux

Effects of the novel vascular targeting agent MDS-11P on tumor vascularity and its antitumor activity by Zhi-Ting Deng; Teng Feng; Peng Wang; Xin Qi; Xue-Hong Chen; Ying-Xia Li; Chun-Li Song; Mei-Yu Geng; Jing Li (pp. 1832-1842).

Vascular disrupting agents show selective effects on tumor established vasculature, and achieve encouraging results in both pre-clinical and clinical experiments. In the present study, we investigated the effects of a new CA4 derivative MDS-11 and its prodrug MDS-11P on vascular disrupting activity in vitro and in vivo. Surface plasmon resonance (SPR) and tubulin polymerization assay showed that MDS-11 interacted with tubulin directly and inhibited tubulin polymerization in a cell free system, and western blot assay further confirmed the action in the cellular level. MDS-11 was found to significantly disrupt the microtubulin skeleton in proliferating HUVECs than quiescent ones determined by confocal microscopy. Furthermore, MDS-11 was found to damage the HUVEC-formed tube quickly, but did not influence structures of microvessels from aortic ring possessing pericytes and smooth muscle cells until 3h treatment. In A549 xenograft mice, immunohistochemistry staining of tumor sections revealed that a single dose of MDS-11P led to large areas of necrosis within tumor and reduced the number of tumor vessels, which was consolidated by perfused vascular volume assay. Pharmacokinetic studies of MDS-11P indicated that MDS-11P rapidly converted to the active form, MDS-11, and exhibited a much faster elimination in mice. The antitumor analysis using H22 and A549 mice xenograft models revealed that the growth inhibition rates of MDS-11P at 50mg/kg (twice a day for three weeks) reached 59.4%, 60.5% respectively without obvious weight loss. Taken together, these results suggest that MDS-11 is a potential vascular disrupting agent for further development of antitumor drug.

Keywords: Abbreviations; AUC; areas under concentration-time curve; DMSO; dimethyl sulfoxide; MVD; microvessel density; PCNA; proliferating cell nuclear antigen; VDA; vascular-disrupting agentVascular disrupting agents; MDS-11P; Antitumor; CA4 derivative; Vascularity

Cytotoxicity and cell death mechanisms induced by the polyamine-vectorized anti-cancer drug F14512 targeting topoisomerase II by Viviane Brel; Jean-Philippe Annereau; Stéphane Vispé; Anna Kruczynski; Christian Bailly; Nicolas Guilbaud (pp. 1843-1852).

The polyamines transport system (PTS) is usually enhanced in cancer cells and can be exploited to deliver anticancer drugs. The spermine-conjugated epipodophyllotoxin derivative F14512 is a topoisomerase II poison that exploits the PTS to target preferentially tumor cells. F14512 has been characterized as a potent anticancer drug candidate and is currently in phase 1 clinical trials. Here we have analyzed the mechanisms of cell death induced by F14512, compared to the parent drug etoposide lacking the polyamine tail. F14512 proved to be >30-fold more cytotoxic than etoposide against A549 non-small cell lung cancer cells and triggers less but unrecoverable DNA damages. The cytotoxic action of F14512 is extremely rapid (within 3h) and does not lead to a marked accumulation in the S-phase of the cell cycle, unlike etoposide. Interestingly, A549 cells treated with F14512 were less prone to undergo apoptosis (neither caspases-dependent nor caspases-independent pathways) or autophagy but preferentially entered into senescence. Drug-induced senescence was characterized qualitatively and quantitatively by an increased β-galactosidase activity, both by cytochemical staining and by flow cytometry. A morphological analysis by electron microscopy revealed the presence of numerous multi-lamellar and vesicular bodies and large electron-lucent (methuosis-like) vacuoles in F14512-treated cell samples. The mechanism of drug-induced cell death is thus distinct for F14512 compared to etoposide, and this difference may account for their distinct pharmacological profiles and the markedly superior activity of F14512 in vivo. This study suggests that senescence markers should be considered as potential pharmacodynamic biomarkers of F14512 antitumor activity.

Keywords: Abbreviations; AML; acute myeloid leukemia; ATP; adenosine tri-phosphate; BSA; bovine serum albumin; DMSO; dimethyl sulfoxide; DNA; deoxyribonucleic acid; DSB; double-strand breaks; EC; ₅₀; half maximal effective concentration; FBS; fetal bovine serum; MAP-LC3; microtubule-associated protein-light chain 3; MEM; minimum essential medium; PBS; phosphate buffer saline; PS; phosphatidyl serine; PTS; polyamine transport system; SA-β-GAL; senescence associated β galactosidaseF14512; Etoposide; Polyamines; Topoisomerase II; Apoptosis; Senescence

The antibiotics roseoflavin and 8-demethyl-8-amino-riboflavin from Streptomyces davawensis are metabolized by human flavokinase and human FAD synthetase by Danielle B. Pedrolli; Shinobu Nakanishi; Maria Barile; Madina Mansurova; Eleonora C. Carmona; Andreas Lux; Wolfgang Gärtner; Matthias Mack (pp. 1853-1859).

Roseoflavin (RoF) and 8-amino-riboflavin (AF) are antibiotics. Apparently, only RoF (not AF) is converted to a potentially toxic cofactor analog (RoFMN) when riboflavin is present. RoFMN may inactivate human flavoenzymes.The non-pathogenic Gram-positive soil bacterium Streptomyces davawensis synthesizes the riboflavin (vitamin B₂) analogs roseoflavin (RoF) and 8-demethyl-8-amino-riboflavin (AF). Both compounds are antibiotics. Notably, a number of other riboflavin analogs are currently under investigation with regard to the development of novel antiinfectives. As a first step towards understanding the metabolism of riboflavin analogs in humans, the key enzymes flavokinase (EC 2.7.1.26) and FAD synthetase (EC 2.7.7.2) were studied. Human flavokinase efficiently converted RoF and AF to roseoflavin mononucleotide (RoFMN) and 8-demethyl-8-amino-riboflavin mononucleotide (AFMN), respectively. Human FAD synthetase accepted RoFMN but not AFMN as a substrate. Consequently, roseoflavin adenine dinucleotide (RoFAD) was synthesized by the latter enzyme but not 8-demethyl-8-amino-riboflavin adenine dinucleotide (AFAD). The cofactor analogs RoFMN, AFMN and RoFAD have different physicochemical properties as compared to FMN and FAD. Thus, the cofactor analogs have the potential to render flavoenzymes inactive, which may negatively affect human metabolism. RoF, but not AF, was found to inhibit human flavokinase. In summary, we suggest that AF has a lower toxic potential and may be better suited as a lead structure to develop antimicrobial compounds.

Keywords: Antibiotics; Flavin analogs; Human flavokinase; Human FAD synthetase; Streptomyces davawensis

Latent membrane protein 1 of Epstein–Barr virus sensitizes cancer cells to cisplatin by enhancing NF-κB p50 homodimer formation and downregulating NAPA expression by Zchong-Zcho Wu; Kai-Ping N. Chow; Tzu-Ching Kuo; Yu-Sun Chang; Chuck C.-K. Chao (pp. 1860-1872).

Expression of the oncogenic latent membrane protein 1 (LMP1) of Epstein–Barr virus is involved in the pathogenesis of nasopharyngeal carcinoma (NPC) and lymphoma. In previous studies, we found that expression of LMP1 was sufficient to transform BALB/c-3T3 cells. In contrast, other studies have shown that LMP1 induces apoptosis in a NF-κB-dependent manner and also inhibits the growth of tumors in mice, thereby indicating that LMP1 may produce various biological effects depending on the biological and cellular context. Still, the mechanism underlying the pro-apoptotic activity of LMP1 remains unclear. In the present study, we found that LMP1 inhibits the expression of NAPA, an endoplasmic reticulum SNARE protein that possesses anti-apoptotic properties against the DNA-damaging drug cisplatin. Accordingly, LMP1-transformed BALB/c-3T3 cells were sensitized to cisplatin-induced apoptosis, whereas no sensitization effect was noted following treatment with the mitotic spindle-damaging drugs vincristine and taxol. Knockdown of LMP1 with antisense oligonucleotides restored NAPA protein level and rendered the cells resistant to cisplatin. Similarly, overexpression of NAPA reduced the effect of LMP1 and induced resistance to cisplatin. LMP1 was shown to upregulate the NF-κB subunit p50, leading to formation of p50 homodimers on the NAPA promoter. These findings suggest that the viral protein LMP1 may sensitize cancer cells to cisplatin chemotherapy by downregulating NAPA and by enhancing the formation of p50 homodimers which in turn inhibit the expression of NF-κB regulated anti-apoptotic genes. These findings provide an explanatory mechanism for the pro-apoptotic activity of LMP1 as well as new therapeutic targets to control tumor growth.

Keywords: Abbreviations; AP-1; activator protein-1; AS; antisense; ChIP; chromatin immunoprecipitation; CMV; cytomegalovirus; CTAR; C-terminal-activating region; DFF; DNA fragmentation factor; DMEM; Dulbecco's modified Eagle's medium; DMSO; dimethyl sulfoxide; EBV; Epstein–Barr virus; EDTA; ethylenediaminetetraacetic acid; ER; endoplasmic reticulum; ERAD; ER-associated degradation; ERK; extracellular response kinase; FBS; fetal bovine serum; FL; full length; GAPDH; glyceraldehyde 3-phosphate dehydrogenase; GST; glutathione S-transferase; HA; haemagglutinin; HBSS; Hank's balanced salt solution; HBx; hepatitis B virus protein X; HDAC; histone deacetylase; HURP; hepatoma upregulated protein; ICAD; inhibitor of caspase activated DNase; IFN-α; interferon-alpha; IP; immunoprecipitation; JAK; Janus kinase; JNK; c-Jun-N-terminal kinase; LMP1; latent membrane protein 1; LPS; lipopolysaccharide; MAPK; mitogen-activated protein kinase; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NAPA; NSF attachment protein α; NC; negative control; Neo; neomycin; NF-κB; nuclear factor kappa-light-chain-enhancer of activated B cells; NPC; nasopharyngeal carcinoma; NSF; N-ethylmaleimide-sensitive factor; ORF; open-reading frame; PARP; poly-ADP ribose polymerase; PCR; polymerase chain reaction; PI3K; phosphatidylinositol 3 kinase; PVDF; polyvinylidene fluoride; P6; pyridone 6; RF; resistance factor; SD; standard deviation; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SF; sensitization factor; shRNA; short-hairpin RNA; SNAP; soluble NSF attachment protein; SNARE; SNAP receptor; STAT; signal transducers and activators of transcription; TNF-α; tumor necrosis factor-alphaChemotherapy; Cisplatin resistance; LMP1; NAPA

Gambogic acid inhibits tumor cell adhesion by suppressing integrin β1 and membrane lipid rafts-associated integrin signaling pathway by Chenglin Li; Na Lu; Qi Qi; Fanni Li; Yun Ling; Yan Chen; Yansu Qin; Zhiyu Li; Haiwei Zhang; Qidong You; Qinglong Guo (pp. 1873-1883).

Cell adhesion plays an important role in the steps of cancer metastasis. Regulation of cell–cell (intercellular) and cell–matrix adhesion is a promising strategy for cancer progression. Gambogic acid is a xanthone derived from the resin of the Chinese plant Garciania hanburyi, with potent anti-metastasis activity on highly metastatic cells. The aim of this study was to investigate the function and mechanism of gambogic acid on tumor adhesion. We found that gambogic acid strongly inhibited the adhesion of human cancer cells to fibronectin. This inhibition was associated with the deformation of focal adhesion complex, which was mediated by suppressing the expression of integrin β1 and integrin signaling pathway. In vitro, cell lipid rafts clustering was inhibited following treatment of gambogic acid, which induced the suppression of integrin β1 and focal adhesion complex proteins colocalization within rafts. Moreover, gambogic acid significantly decreased cellular cholesterol content, whereas cholesterol replenishment lessened the inhibitory effect of gambogic acid on cell adhesion. Real-time PCR analysis showed that gambogic acid reduced mRNA levels of hydroxymethylglutaryl-CoA reductase and sterol regulatory element binding protein-2, while increased acetyl-CoA acetyltransferase-1/2. Taken together, these results demonstrate that gambogic acid inhibits cell adhesion via suppressing integrin β1 abundance and cholesterol content as well as the membrane lipid raft-associated integrin function, which provide new evidence for the anti-cancer activity of gambogic acid.

Keywords: Abbreviations; ACAT; acetyl-CoA acetyltransferase; BSA; bovine serum albumin; CTxB; cholera toxin B subunit; ECM; extracellular matrix; FAC; focal adhesion complex; FAK; focal adhesion kinase; FITC; fluorescein-5-isothiocyanate; FN; fibronectin; GA; gambogic acid; GC–MS; gas chromatography–mass spectrometry; HMG; hydroxy-methylglutaryl; LDL-r; low density lipoprotein receptor; MMP; matrix metalloproteinase; MBCD; methyl-β-cyclodextrin; PE; phycoerythrin; SREBP; sterol regulatory element binding protein; SR-BI; scavenger receptor class B type IGambogic acid; Adhesion; Integrin β1; Lipid raft; Cholesterol

Vincristine potentiates the anti-proliferative effect of an aurora kinase inhibitor, VE-465, in myeloid leukemia cells by Kozue Yoshida; Tadashi Nagai; Ken Ohmine; Mitsuyo Uesawa; Piyanuch Sripayap; Yoji Ishida; Keiya Ozawa (pp. 1884-1890).

Combination of VE-465 and vincristine caused Chk2-mediated activation of the G2/M checkpoint, resulting in sequential induction of apoptosis.Aurora kinases play an essential role in the regulation of mitosis. The kinases are overexpressed in a variety of cancer cells and are involved in tumorgenesis. Although aurora kinase inhibitors are potential agents for treatment of leukemia, the establishment of efficacious combination therapies is an attractive approach for making good use of these agents. In this study, we examined the effects of a specific aurora kinase inhibitor, VE-465, in combination with various conventional anti-leukemia agents, including doxorubicin, daunorubicin, idarubicin, mitoxantron, cytosine arabinoside, vincristine and etoposide, on acute myeloid leukemia cell lines (HL60, U937, THP-1 and KY821), chronic myeloid leukemia cell lines (KCL22, K562 and KU812) and primary leukemia cells. We found that a combination of VE-465 and vincristine had a synergistic/additive inhibitory effect on the growth of leukemia cells. VE-465 initially increased G2/M-phase cells, followed by induction of sub-G1 cells. Vincristine enhanced this effect of VE-465. The combination of VE-465 and vincristine increased the levels of cleaved caspase 3, cleaved caspase 7, cleaved caspase 9, cleaved PARP and Phospho-Chk2, suggesting that the combination caused Chk2-mediated activation of the G2/M checkpoint, resulting in sequential induction of apoptosis. Interestingly, the combination markedly decreased the level of Phospho-ERK1/2, suggesting that the combination alters a network of cellular signaling pathways. In contrast, combinations of VE-465 and other agents showed no synergistic inhibitory effect but rather had an antagonistic effect. In conclusion, our results indicate the utility of the combination of VE-465 and vincristine as a potential therapy for myeloid leukemia.

Keywords: VE-465; Aurora kinase inhibitor; Vincristine; Leukemia

The association of minocycline and the probiotic Escherichia coli Nissle 1917 results in an additive beneficial effect in a DSS model of reactivated colitis in mice by Natividad Garrido-Mesa; Pilar Utrilla; Mónica Comalada; Pedro Zorrilla; José Garrido-Mesa; Antonio Zarzuelo; María Elena Rodríguez-Cabezas; Julio Gálvez (pp. 1891-1900).

Antibiotics have been empirically used for human inflammatory bowel disease, being limited to short periods. Probiotics are able to attenuate intestinal inflammation due to its immunomodulatory properties, being considered as safe when chronically administered. The aim was to test the association of minocycline, a tetracycline with immunomodulatory properties, and the probiotic Escherichia coli Nissle 1917 (EcN) in a mouse model of reactivated colitis. For this purpose, female C57BL/6J mice were assigned to different groups: non-colitic and dextran sodium sulfate (DSS)-control groups (without treatment), minocycline (50mg/kg/day; p.o.), EcN (5×10⁸CFU/day; p.o.), and minocycline plus EcN treated groups. Colitis was induced by adding DSS in the drinking water (3%) for 5 days; 2 weeks later, colitis was reactivated by subsequent exposure to DSS. The inflammatory status was evaluated daily by a disease activity index (DAI); colonic damage was assessed histologically and biochemically by evaluating mRNA relative expression of different mediators by qPCR. Finally, a microbiological analysis of the colonic contents was performed. Minocycline and EcN exerted intestinal anti-inflammatory effect and attenuated the reactivation of the colitis, as shown by the reduced DAI values, being these effects greater when combining both treatments. This was evidenced histologically and biochemically, by reduced expression of TNFα, IL-1β, IL-2, MIP-2, MCP-1, ICAM-1, iNOS and MMP-9, together with increased MUC-3 and ZO-1 expression. Finally, the altered microbiota composition of colitic mice was partially restored after the different treatments. In conclusion, EcN supplementation to minocycline treatment improves the recovery of the intestinal damage and prevents the reactivation of experimental colitis.

Keywords: Minocycline; Escherichia coli; Nissle 1917; DSS experimental colitis; Pro-inflammatory cytokines; Chemokines; IBD

Suppression of pro-inflammatory and proliferative pathways by diferuloylmethane (curcumin) and its analogues dibenzoylmethane, dibenzoylpropane, and dibenzylideneacetone: Role of Michael acceptors and Michael donors by Preetha Anand; Bokyung Sung; Ajaikumar B. Kunnumakkara; Kallikat N. Rajasekharan; Bharat B. Aggarwal (pp. 1901-1909).

Curcumin, a diferuloylmethane, has been shown to exhibit anti-inflammatory and anti-proliferative activities. Whereas curcumin has both a Michael acceptor and a Michael donor units, its analogues dibenzoylmethane (DBM, a component of licorice) and dibenzoylpropane (DBP) have a Michael donor but not a Michael acceptor unit, and the analogue dibenzylideneacetone (DBA) has a Michael acceptor unit. In the current report, we investigated the potency of DBM, DBP, and DBA in relation to curcumin for their ability to suppress TNF-induced NF-κB activation, NF-κB-regulated gene products, and cell proliferation. We found that all four agents were active in suppressing NF-κB activation; curcumin was most active and DBM was least active. When examined for its ability to inhibit the direct DNA binding activity of p65, a subunit of NF-κB, only DBP inhibited the binding. For inhibition of TNF-induced IKK activation, DBA was most active. For suppression of TNF-induced expression of NF-κB-regulated gene products such as COX-2 (inflammation marker), cyclin D1 (proliferation marker), and VEGF (angiogenesis marker), DBA and curcumin were more active than DBM. Similarly for suppression of proliferation of leukemia (KBM-5), T cell leukemia (Jurkat), prostate (DU145), and breast (MDA-MB-231) cancer cells, curcumin and DBA were most active and DBP was least active. Overall, our results indicate that although curcumin and its analogues exhibit activities to suppress inflammatory pathways and cellular proliferation, a lack of Michael acceptor units in DBM and DBP can reduce their activities.

Keywords: Curcumin analogues; NF-κB; Cell proliferation; Michael acceptor

Influence of the N-terminus and the E2-loop onto the binding kinetics of the antagonist mepyramine and the partial agonist phenoprodifen to H₁R by Hans-Joachim Wittmann; Roland Seifert; Andrea Strasser (pp. 1910-1918).

Numerous competitive radioligand binding studies revealed significant differences between human and guinea pig histamine H₁-receptors (hH₁R and gpH₁R), e.g. for the partial H₁R agonist phenoprodifen. But until now, there are only few studies with regard to binding kinetics at H₁R. Previous studies from our group revealed an influence of the exchange of N-terminus and E2-loop between hH₁R and gpH₁R onto affinity of phenoprodifen to H₁R (Strasser A, Wittmann HJ, Seifert R, J Pharmacol Exp Ther 326:783–791, 2008). The aim of this study was, therefore, to examine the impact of the N-terminus and the E2-loop on binding kinetics of the H₁R. The wild type hH₁R and gpH₁R and the chimeric h_gpE2H₁R (E2-loogp from guinea pig) and h_gpNgpE2H₁R (N-terminus and E2-loop from guinea pig) were co-expressed with regulator of G-protein signaling protein RGS4 in Sf9 insect cells and kinetic binding studies were performed using the antagonist [³H]mepyramine as radioligand. The rate constants for association and dissociation were, in dependence of the ligand, different between hH₁R and gpH₁R. Furthermore, the rate constants for association at h_gpNgpE2H₁R were significantly different compared to hH₁R and gpH₁R. Molecular dynamic simulation studies detected different interactions of amino acid side chains on the extracellular surface of the receptor. Based on these findings, the influence of extracellular surface onto binding kinetics and binding affinity can be explained. Thus, the extracellular surface of G protein-coupled receptors for biogenic amines, exhibits influence onto kinetics of ligand binding, onto ligand recognition and ligand guiding into the binding pocket.

Keywords: Abbreviations; E2 loop; second extracellular loop; gp; guinea-pig; GPCR; G protein-coupled receptor; h; human; h; _gpE2; H; ₁; R; human histamine H; ₁; R with gp E2-loop; h; _gpNgpE2; H; ₁; R; human histamine H; ₁; R with gp N-terminus and gp E2-loop; H; ₁; R; histamine H; ₁; receptor; MEP; mepyramineHistamine H; ₁; receptor; Binding kinetics; Gibbs energy; Extracellular surface; Molecular modelling; Molecular dynamics

High density micromass cultures of a human chondrocyte cell line: A reliable assay system to reveal the modulatory functions of pharmacological agents by K.V. Greco; A.J. Iqbal; L. Rattazzi; G. Nalesso; N. Moradi-Bidhendi; A.R. Moore; M.B. Goldring; F. Dell’Accio; M. Perretti (pp. 1919-1929).

Osteoarthritis is a highly prevalent and disabling disease for which we do not have a cure. The identification of suitable molecular targets is hindered by the lack of standardized, reproducible and convenient screening assays. Following extensive comparisons of a number of chondrocytic cell lines, culture conditions, and readouts, we have optimized an assay utilizing C-28/I2, a chondrocytic cell line cultured in high-density micromasses. Utilizing molecules with known effects on cartilage (e.g. IL-1β, TGFβ1, BMP-2), we have exploited this improved protocol to (i) evoke responses characteristic of primary chondrocytes; (ii) assess the pharmacodynamics of gene over-expression using non-viral expression vectors; (iii) establish the response profiles of known pharmacological treatments; and (iv) investigate their mechanisms of action. These data indicate that we have established a medium- throughput methodology for studying chondrocyte-specific cellular and molecular responses (from gene expression to rapid quantitative measurement of sulfated glycosaminoglycans by Alcian blue staining) that may enable the discovery of novel therapeutics for pharmacological modulation of chondrocyte activation in osteoarthritis.

Keywords: IL-1β; TGFβ1; Micromasses; Anti-inflammatory drugs; Naproxen; Prednisolone

Epigallocatechin-3-gallate induces cytokine production in mast cells by stimulating an extracellular superoxide-mediated calcium influx by Toshio Inoue; Yoshihiro Suzuki; Chisei Ra (pp. 1930-1939).

The green tea polyphenol (−)-epigallocatechin-3- O-gallate (EGCG) has various biological activities, including anti-inflammatory, anti-neoplastic, anti- and pro-apoptotic, and neuroprotective effects. Although these are often associated with increased intracellular reactive oxygen species (ROS) and Ca²⁺ levels, their involvement in biological effects is poorly understood. Here we report that EGCG induces cytokine production in mast cells via Ca²⁺ influx and ROS generation. EGCG at concentrations of ≥50μM induced interleukin-13 and tumor necrosis factor-α production in RBL-2H3 and bone marrow-derived mast cells. The effects were dependent on extracellular Ca²⁺, and EGCG induced Ca²⁺ release from intracellular stores and Ca²⁺ influx. Ca²⁺ influx was suppressed by 2-aminoethoxydiphenyl borate, an inhibitor of store-operated Ca²⁺ (SOC) channels, including Ca²⁺ release-activated Ca²⁺ channels and transient receptor potential canonical channels. EGCG failed to induce Ca²⁺ influx through SOC channels. EGCG-activated Ca²⁺ channels were genetically and pharmacologically distinct from Ca_v1.2 L-type Ca²⁺ channels, another route of Ca²⁺ influx into mast cells. EGCG evoked release of superoxide (O₂⁻) into the extracellular space. Exogenous superoxide dismutase, but not catalase, inhibited EGCG-evoked Ca²⁺ influx and cytokine production, indicating that extracellular O₂⁻ regulates these events. EGCG can serve as a powerful tool for studying O₂⁻-regulated Ca²⁺ channels, which may be selectively involved in the regulation of cytokine production but have yet to be elucidated.

Keywords: Mast cell; EGCG; Calcium; Superoxide; Cytokine

Generation of stable reporter breast cancer cell lines for the identification of ER subtype selective ligands by Erin K. Shanle; John R. Hawse; Wei Xu (pp. 1940-1949).

Estrogen signaling is mediated by two estrogen receptors (ERs), ERα and ERβ, which have unique roles in the regulation of breast cancer cell proliferation. ERα induces proliferation in response to estrogen and ERβ inhibits proliferation in breast cancer cells, suggesting that ERβ selective ligands may be beneficial for promoting the anti-proliferative action of ERβ. Subtype selective ligands can be identified using transcriptional assays, but cell lines in which ERα or ERβ are independently expressed are required. Of the available reporter cell lines, none have been generated in breast cancer cells to identify subtype selective ligands. Here we describe the generation of two isogenic breast cancer cell lines, Hs578T-ERαLuc and Hs578T-ERβLuc, with stable integration of an estrogen responsive luciferase reporter gene. Hs578T-ERαLuc and Hs578T-ERβLuc cell lines are highly sensitive to estrogenic chemicals and ER subtype selective ligands, providing a tool to characterize the transcriptional potency and subtype selectivity of estrogenic ligands in the context of breast cancer cells. In addition to measuring reporter activity, ERβ target gene expression and growth inhibitory effects of ERβ selective ligands can be determined as biological endpoints. The finding that activation of ERβ by estrogen or ERβ selective natural phytoestrogens inhibits the growth of Hs578T-ERβ cells implies therapeutic potential for ERβ selective ligands in breast cancer cells that express ERβ.

Keywords: Abbreviations; BRET; bioluminescence resonance energy transfer; Cos; cosmosiin; Dox; doxycycline; DPN; diarylpropionitrile; E2; 17β-estradiol; ER; estrogen receptor; PPT; propyl pyrazole triol; ERE; estrogen response element; ICI; ICI 182,780; Liq; liquiritigeninEstrogen receptors; Subtype selectivity; Phytoestrogens; Breast cancer

Very low density lipoprotein receptor promotes adipocyte differentiation and mediates the proadipogenic effect of peroxisome proliferator-activated receptor gamma agonists by Huan Tao; Tahar Hajri (pp. 1950-1962).

Very low density lipoprotein receptor (VLDLR) is a member of the low density receptor family, expressed mostly in adipose tissue, heart, and skeletal muscles. VLDLR binds apolipoprotein-E-triglyceride-rich lipoproteins and plays a key role in lipid metabolism. In adipocytes, VLDLR expression increases with differentiation but it is not known whether it plays a role in the adipogenesis. Here we report that VLDLR expression in 3T3-L1 adipocytes is upregulated by PPARγ agonist 15-deoxy-delta^12,14-prostaglandin J₂ (15d-PGJ₂) in dose- and time-dependant manners. Knockdown of peroxisome proliferator-activated receptor-γ (PPARγ) with siRNA abolished pioglitazone- and 15d-PGJ₂-induced VLDLR expression and simultaneously reduced VLDL uptake in adipocytes. In addition, PPARγ-agonist treatment of control mouse adipocytes ( vldlr^+/+) enhanced adipogenesis and VLDL uptake concurrently with the induction of VLDLR expression. However, vldlr deficiency ( vldlr^−/− ) significantly blunted the proadipogenic effects of PPARγ agonists. Sequence analysis revealed the presence of a putative PPARγ responsive sequence (PPRE) within the vldlr promoter, which is responsive to natural (15d-PGJ₂) and synthetic (pioglitazone) PPARγ agonists. Reporter gene assays using serial deletion of the 5′-flanking region showed that this putative PPRE site induced promoter transactivation, while a site-targeted mutation abolished transactivation. Moreover, electrophoresis mobility shift assay (EMSA) and chromatic immunoprecipitation (ChIP) assays showed the specific binding of PPARγ to the PPRE sequence.Together, these results support a crucial function for VLDLR in adipocyte differentiation and mediation of the proadipogenic effect of PPARγ.

Keywords: Abbreviations; PPARγ; peroxisome proliferator-activated receptor-γ; VLDLR; very low density lipoprotein receptor; L-FABP; liver fatty acid binding protein; RXR; retinoid X receptor; FATP; fatty acid transport protein; aP2; adipocyte protein 2; ACRP30; adiponectin; HSL; heparin sensitive lipase; LPL; lipoprotein lipase; CD36; fatty acid transporter/cluster of differentiation 36; TZD; thiazolidinedione; PIO; pioglitazone; 15d-PGJ; ₂; 15-deoxy-delta-12,14-prostaglandin J2; DMSO; dimethyl sulfoxideVery low density lipoprotein receptor; Adipocyte; Adipogenesis; 15-Deoxy-delta; ^12,14; -prostaglandin J; ₂; Peroxisome proliferator-activated receptor gamma

Insulin deficiency induces abnormal increase in intestinal disaccharidase activities and expression under diabetic states, evidences from in vivo and in vitro study by Li Liu; Yun-Li Yu; Can Liu; Xin-Ting Wang; Xiao-Dong Liu; Lin Xie (pp. 1963-1970).

Insulin regulates disaccharidase activities and expression partly via the MAPK-dependent pathway and its deficiency induces abnormal increase in intestinal disaccharidase activities and expression under diabetic states.Structural and functional alterations in the gastrointestinal tract of diabetic patients are often accompanied by increase in absorption of intestinal glucose and activities of brush-border disaccharidases. The purpose of this study was to investigate the role of insulin in regulating intestinal disaccharidases using in vivo and in vitro experiments. Streptozotocin-induced diabetic rats and normal rats received protamine zinc insulin (10IU/kg) subcutaneously twice daily for 5 weeks. Disaccharidase activities and sucrase–isomaltase (SI) complex protein and mRNA expression in intestinal regions were assessed. In addition, Caco-2 cells were cultured in medium containing glucose, insulin or insulin plus some pharmacological inhibitors for 7 days, disaccharidase activities, sucrase–isomaltase (SI) complex and Cdx2 mRNA levels were measured. The animal experiments showed that diabetes increased intestinal disaccharidase activities, accompanied by high mRNA and protein expression of SI complex. Insulin treatment reversed the increases induced by diabetes. The cellular results showed that insulin suppressed disaccharidase activities and down-regulated SI complex and Cdx2 mRNA expression in a concentration-dependent manner. The inhibitor of MAPK signal pathway PD-98059 blocked the suppression of disaccharidase activities and expression of SI complex and Cdx2 mRNA induced by insulin. In conclusion, insulin deficiency induces abnormal increase in intestinal disaccharidase activities and expression under diabetic states. Insulin plays an essential role in regulation disaccharidase activities and expression, at least in part, via the MAPK-dependent pathway.

Keywords: Abbreviations; SI; sucrase–isomaltase; STZ; streptozotocin; FBG; fasted blood glucose; PKC; protein kinase C; PKA; protein kinase A; PI3K; phosphoinositide 3-kinase; MAPK; mitogen-activated protein kinase; Cdx2; caudal type homeobox 2; I-OMe-AG538; α-cyano-(3-methoxy-4-hydroxy-5-iodocinnamoyl)-(3′,4′-dihydroxyphenyl) ketone, H-89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride; LY294002; 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride; PD-98059; 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; ANOVA; analysis of varianceInsulin; Diabetes; Disaccharidases; Sucrase–isomaltase complex; MAPK

3-Hydroxy-2′-methoxy-6-methylflavone: A potent anxiolytic with a unique selectivity profile at GABA_A receptor subtypes by Nasiara Karim; Navnath Gavande; Petrine Wellendorph; Graham A.R. Johnston; Jane R. Hanrahan; Mary Chebib (pp. 1971-1983).

The flavonoid, 3-OH-2′MeO6MF modulates α2β2/3γ2L and directly activates α4β1–3δ GABA_A receptors. In mice, 3-OH-2′MeO6MF is an anxiolytic acting in a flumazenil-insensitive manner.Genetic and pharmacological studies have demonstrated that α2- and α4-containing GABA_A receptors mediate the anxiolytic effects of a number of agents. Flavonoids are a class of ligands that act at GABA_A receptors and possess anxiolytic effects in vivo. Here we demonstrate that the synthetic flavonoid, 3-hydroxy-2′-methoxy-6-methylflavone (3-OH-2′MeO6MF) potentiates GABA-induced currents at recombinant α1/2β2, α1/2/4/6β1–3γ2L but not α3/5β1–3γ2L receptors expressed in Xenopus oocytes. The enhancement was evident at micromolar concentrations (EC₅₀ values between 38 and 106μM) and occurred in a flumazenil-insensitive manner. 3-OH-2′MeO6MF displayed preference for β2/3- over β1-containing receptors with the highest efficacy observed at α2β2/3γ2L, displaying a 4–11-fold increase in efficacy over α2β1γ2L and α1/4/6-containing subtypes. In contrast, 3-OH-2′MeO6MF acted as a potent bicuculline-sensitive activator, devoid of potentiation effects at extrasynaptic α4β2/3δ receptors expressed in oocytes. The affinity of 3-OH-2′MeO6MF for α4β2/3δ receptors (EC₅₀ values between 1.4 and 2.5μM) was 10-fold higher than at α4β1δ GABA_A receptors. 3-OH-2′MeO6MF acted as a full agonist at α4β2/3δ (105% of the maximal GABA response) but as a partial agonist at α4β1δ (61% of the maximum GABA response) receptors. In mice, 3-OH-2′MeO6MF (1–100mg/kg i.p.) induced anxiolytic-like effects in two unconditioned models of anxiety: the elevated plus maze and light/dark paradigms. No sedative or myorelaxant effects were detected using holeboard, actimeter and horizontal wire tests and only weak barbiturate potentiating effects on the loss of righting reflex test. Taken together, these data suggest that 3-OH-2′MeO6MF is an anxiolytic without sedative and myorelaxant effects acting through positive allosteric modulation of the α2β2/3γ2L and direct activation of α4β2/3δ GABA_A receptor subtypes.

Keywords: Abbreviations; GABAγ-; aminobutyric acid; 3-OH-2′MeO6MF; 3-hydroxy-2′-methoxy-6-methylflavone; PTZ; pentylenetetrazole; i.p; .; intraperitoneal; BDZs; benzodiazepinesGABA; _A; receptors; Flavonoids; Anxiolytics; Allosteric modulation; Allosteric activation

Pretreatment with human serum butyrylcholinesterase alone prevents cardiac abnormalities, seizures, and death in Göttingen minipigs exposed to sarin vapor by Ashima Saxena; Wei Sun; Paul A. Dabisch; Stanley W. Hulet; Nicholas B. Hastings; Edward M. Jakubowski; Robert J. Mioduszewski; Bhupendra P. Doctor (pp. 1984-1993).

Pretreatment with 7.5mg/kg of Hu BChE alone protected Göttingen minipigs from GB vapor-induced cardiac abnormalities, seizures, and death by sequestering GB in blood.Human serum butyrylcholinesterase (Hu BChE) is a stoichiometric bioscavenger that is being developed as a prophylactic countermeasure against organophosphorus nerve agents. This study was designed to evaluate the efficacy of Hu BChE against whole-body inhalation exposure to a lethal dose of sarin (GB) vapor. Male Göttingen minipigs were subjected to: air exposure, GB vapor exposure, or pretreatment with Hu BChE followed by GB vapor exposure. Hu BChE was administered by i.m. injection 24h prior to exposure to 4.1mg/m³ of GB vapor for 60min. Electrocardiograms (ECG), electroencephalograms (EEG), and pupil size were recorded throughout exposure. Blood drawn before and throughout exposure was analyzed for blood gases, electrolytes, metabolites, acetylcholinesterase and BChE activities, and amount of GB present. Untreated animals exposed to GB vapor exhibited cardiac abnormalities and generalized seizures, ultimately succumbing to respiratory failure. Pretreatment with 3.0 or 6.5mg/kg of Hu BChE delayed blood gas and acid–base disturbances and the onset of cardiac and neural toxic signs, but failed to increase survivability. Pretreatment with 7.5mg/kg of Hu BChE, however, completely prevented toxic signs, with blood chemistry and ECG and EEG parameters indistinguishable from control during and after GB exposure. GB bound in plasma was 200-fold higher than plasma from pigs that did not receive Hu BChE, suggesting that Hu BChE scavenged GB in blood and prevented it from reaching other tissues. Thus, prophylaxis with Hu BChE alone not only increased survivability, but also prevented cardiac abnormalities and neural toxicity in minipigs exposed to a lethal dose of GB vapor.

Keywords: Abbreviations; ChE; cholinesterase; AChE; acetylcholinesterase; Hu BChE; human serum butyrylcholinesterase; OP; organophosphorus compounds; GB (sarin); O-isopropyl methylphosphonofluoridate; GD (soman); O-pinacolyl methylphosphonofluoridate; GA (tabun); O-ethyl N,N-dimethylphosphoramidocyanate; VX; O-ethyl S-2-N,N-diisopropylaminoethyl methylphosphonothiolate; CNS; central nervous system; ECG; electrocardiogram; EEG; electroencephalogram; LCt; ₅₀; the product of vapor concentration and exposure time that will cause death in 50% of an exposed populationHuman serum butyrylcholinesterase; Minipig; Sarin vapor; Cardiac abnormality; Neuronal toxicity

Use of comprehensive screening methods to detect selective human CAR activators by Jenni Küblbeck; Tuomo Laitinen; Johanna Jyrkkärinne; Timo Rousu; Ari Tolonen; Tobias Abel; Tanja Kortelainen; Jouko Uusitalo; Timo Korjamo; Paavo Honkakoski; Ferdinand Molnár (pp. 1994-2007).

The so-called human xenosensors, constitutive androstane receptor (hCAR), pregnane X receptor (hPXR) and aryl hydrocarbon receptor (hAhR), participate in drug metabolism and transport as well as in several endogenous processes by regulating the expression of their target genes. While the ligand specificities for hPXR and hAhR are relatively well described, this property of hCAR still remains fairly unclear. Identifying hCAR agonists for drug development and for studying hCAR biology are hindered mainly by the unique properties of the receptor, such as the high constitutive activity and complex signaling network but also by the lack of robust and reliable assays and cellular models. Here, validated reporter assays for these three xenosensors are presented and thereafter used to screen a large set of chemicals in order to find novel selective hCAR ligands. We introduce a novel selective hCAR agonist, FL81, which can be used as a stable positive control in hCAR activity assays. Our established receptor-selective ligand identification methods consisting of supporting biological assays and molecular modeling techniques are then used to study FL81 as well as other discovered ligands, such as diethylstilbestrol, o, p′-DDT, methoxychlor and permethrin, for their ability to specifically activate hCAR and to regulate the CYP enzyme expression and function.

Keywords: Human constitutive androstane receptor; Xenosensors; Agonist; Cell-based assays; Molecular modeling

Energy sensing factors PGC-1α and SIRT1 modulate PXR expression and function by Marcin Buler; Sanna-Mari Aatsinki; Réka Skoumal; Jukka Hakkola (pp. 2008-2015).

The pregnane X receptor (PXR), a xenobiotic-sensing nuclear receptor plays a major role in regulation of drug metabolism but also modulates hepatic energy metabolism. PXR interacts with and represses several important transcription factors and coactivators regulating key enzymes in energy metabolism. Much less is known about how energy sensing cellular factors regulate PXR function. In this study we have investigated the effect of two major regulators of hepatic energy homeostasis, the transcriptional coactivator, peroxisome proliferator-activated receptor γ coactivator 1 alpha (PGC-1α) and the NAD-dependent deacetylase protein, sirtuin 1 (SIRT1) on PXR expression and function. Fasting induces PXR expression in liver. Furthermore, glucagon and PGC-1α overexpression upregulate PXR expression level in mouse primary hepatocytes suggesting that PGC-1α, in addition to coactivation of PXR, also transcriptionally regulates PXR gene. Knockdown of peroxisome proliferator-activated receptor α by siRNA attenuates PGC-1α mediated induction of PXR mRNA. PGC-1α overexpression alone has no effect on cytochrome P450 (CYP)3A11 expression but potentiates induction by pregnenolone-16α-carbonitrile (PCN). Pyruvate, a nutrient signal activating SIRT1 abolishes synergistic induction of CYP3A11 by PCN and PGC-1α. Knockdown of SIRT1 prevented this effect of pyruvate. Downregulation of CYP7A1 by PCN was not affected by PGC-1α or pyruvate. Mammalian two hydrid assays indicate that pyruvate and SIRT1 interfere with interaction of PXR and PGC-1α. This may be mediated by well established PGC-1α deacetylation by SIRT1. However, we show by immunoprecipitation that SIRT1 also interacts with PXR. Thus we show that two fasting activated pathways PGC-1α and SIRT1 differentially modify PXR expression and function.

Keywords: Abbreviations; PXR; pregnane X receptor; PGC-1α; peroxisome proliferator-activated receptor γ coactivator 1 alpha; SIRT1; sirtuin 1; CYP; cytochrome P450; PCN; pregnenolone-16α-carbonitrile; PPARα; peroxisome proliferator-activated receptor alpha; DMSO; dimethyl sulfoxide; HNF4α; hepatocyte nuclear factor 4 alphaPXR; SIRT1; PGC-1α; CYP3A11; Regulation; Transcription

Homodimerization of UDP-glucuronosyltransferase 2B7 (UGT2B7) and identification of a putative dimerization domain by protein homology modeling by Benjamin C. Lewis; Peter I. Mackenzie; John O. Miners (pp. 2016-2023).

Homodimerization of UGT2B7 was demonstrated using co-immunoprecipitation. The dimerization domain was identified by protein homology modeling.Although homodimerization of UGT1A proteins is well established, direct evidence for dimerization of UGT2B7, which is arguably the most important enzyme involved in human drug glucuronidation, is currently lacking. This study characterized UGT2B7 homodimerization by co-immunopreciptation and generated a UGT2B7 homology model that identified the dimerization domain. It was demonstrated that co-expressed, solubilized UGT2B7 proteins differentially tagged with hemagglutinin (UGT2B7-HA) and c-MYC (UGT2B7-cMYC) co-immunoprecipitated as active homodimers that catalyzed 4-methylumbelliferone glucuronidation. Substrate binding affinities (assessed as S₅₀ values) of the tagged and co-expressed tagged proteins were essentially identical to that of native UGT2B7. Co-association was not observed in a ‘mixed’ UGT2B7-HA and UGT2B7-cMYC protein preparation. Generation of a UGT2B7 homology model established from plant and human templates was achieved using SYBYLX1.2 with all residues energy minimized using the Tripos Force Field. The UGT2B7 model allowed elucidation of a putative protein dimerization domain within the B′–C loop of each UGT2B7 monomer. The eighteen amino acid dimerization domain is present in all UGT2B enzymes and comprises a proposed dimerization signature motif (FPPSYVPVVMS). Stabilization of the dimer interface is maintained by the formation of two salt bridges, aromatic π–π stacking interactions, two S-aromatic (face) interactions, and the presence of ‘proline brackets’. The homology model further provides important insights into structure–function relationships of this enzyme and the mechanism responsible for the atypical glucuronidation kinetics for substrates of UGT2B7 and other human UGT enzymes.

Keywords: UDP-glucuronosyltransferase; UGT2B7; Homology model; Dimerization; Enzyme kinetics; Glucuronidation

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Biochemical Pharmacology (v.82, #12)