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Biochemical Pharmacology (v.73, #12)
Coordinate regulation of Phase I and II xenobiotic metabolisms by the Ah receptor and Nrf2
by Christoph Köhle; Karl Walter Bock (pp. 1853-1862).
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor with important roles in metabolic adaptation, normal physiology and dioxin toxicology. Metabolic adaptation is based on coordinate regulation of a set of xenobiotic-metabolizing enzymes (XMEs), termed AhR battery. Coordination is achieved by AhR/Arnt-binding to XREs (xenobiotic response elements), identified in the 5′ upstream region of AhR target genes. The AhR battery encodes Phase I and II enzymes. Interestingly, these Phase II genes are linked to the Nrf2 gene battery that encodes enzymes that are essential in protection against oxidative/electrophile stress. Nrf2 binds to AREs (antioxidant response elements) in the regulatory region of a large and distinct set of target genes. Functionally characterized response elements such as XREs and AREs in the regulatory region of target genes may provide a genetic basis to understand AhR- and Nrf2-induced genes. Linkage between AhR and Nrf2 batteries is probably achieved by multiple mechanisms, including Nrf2 as a target gene of the AhR, indirect activation of Nrf2 via CYP1A1-generated reactive oxygen species, and direct cross-interaction of AhR/XRE and Nrf2/ARE signaling. Linkage appears to be species- and cell-dependent. However, mechanisms linking XRE- and ARE-controlled Phase II genes need further investigation. Tightened coupling between Phases I and II by AhR- and Nrf2-induced XMEs may greatly attenuate health risks posed by CYP1A1-generated toxic intermediates and reactive oxygen species. Better recognition of coordinate Phase I and II metabolisms may improve risk assessment of reactive toxic intermediates in the extrapolation to low level endo- and xenobiotic exposure.
Keywords: Abbreviations; AhR; aryl hydrocarbon receptor; ARE; antioxidant response element; BaP; benzo[; a; ]pyrene; BCRP; breast cancer resistance protein; GCS; glutamylcysteine synthetase; GSH; reduced glutathione; GST; glutathione; S; -transferase; MRP; multidrug resistance-associated protein; NQO; NAD(P)H:quinone oxidoreductase; Nrf2; nuclear factor erythroid 2-related factor 2; PAH; polycyclic aromatic hydrocarbon; ROS; reactive oxygen species; SULT; sulfotransferase; tBHQ; tert; -butylhydroquinone; TCDD; 2,3,7,8-tetrachlorodibenzo-; p; -dioxin; UGT; UDP-glucuronosyltransferase; XMEs; xenobiotic-metabolizing enzymes; XRE; xenobiotic response elementAh receptor; Nrf2, Phase I and II metabolism; Coordinate induction; Response elements; Benzo[; a; ]pyrene quinones
Novel tetra-acridine derivatives as dual inhibitors of topoisomerase II and the human proteasome
by S. Vispé; I. Vandenberghe; M. Robin; J.P. Annereau; L. Créancier; V. Pique; J.P. Galy; A. Kruczynski; J.M. Barret; C. Bailly (pp. 1863-1872).
Acridine derivatives, such as amsacrine, represent a well known class of multi-targeted anti-cancer agents that generally interfere with DNA synthesis and inhibit topoisomerase II. But in addition, these tricyclic molecules often display secondary effects on other biochemical pathways including protein metabolism. In order to identify novel anti-cancer drugs, we evaluated the mechanism of action of a novel series of bis- and tetra-acridines. As expected, these molecules were found to interact with DNA and inhibit the topoisomerase II-mediated DNA decatenation. Interestingly when tested on human tumour cells either sensitive (HL-60) or resistant (HL-60/MX2) to topoisomerase II inhibitors, these molecules proved equicytotoxic against the two cell lines, suggesting that they do not only rely on topoisomerase II inhibition to exert their cytotoxic effects. In order to identify alternative targets, we tested the capacity of acridines1–9 to inhibit the proteasome machinery. Four tetra-acridines inhibited the proteasome in vitro, with IC50 values up to 40 times lower than that of the reference proteasome inhibitor lactacystin. Moreover, unlike peptide aldehydes used as reference inhibitors for the proteasome, these new acridine compounds demonstrated a good selectivity towards the proteasome, when tested against four unrelated proteases. A cellular assay based on the degradation of a proteasome protein substrate indicated that at least two of the tetra-acridines maintained this proteasome inhibition activity in a cellular context. This is the first report of tetra-acridines that demonstrate dual topoisomerase II and proteasome inhibition properties. This new dual activity could represent a novel anti-cancer approach to circumvent certain forms of tumour resistance.
Keywords: Abbreviations; m; -AMSA; amsacrine; TL; trypsin-like; PGPH; peptidyl glutamyl peptide hydrolase; CTL; chymotrypsin-like; kDNA; kinetoplast DNA; CPI; calpain peptide inhibitor; RFU; relative fluorescence unit; RLU; relative luminescence unit; HIV; human immunodeficiency virusCancer; Proteasome; Acridine derivatives; Topoisomerase II; Cytotoxicity; DNA interaction
Multidrug-resistant cancer cells are preferential targets of the new antineoplastic lanthanum compound KP772 (FFC24)
by P. Heffeter; M.A. Jakupec; W. Körner; P. Chiba; C. Pirker; R. Dornetshuber; L. Elbling; H. Sutterlüty; M. Micksche; B.K. Keppler; W. Berger (pp. 1873-1886).
Recently, we have introduced [tris(1,10-phenanthroline)lanthanum(III)] trithiocyanate (KP772, FFC24) as a new lanthanum compound which has promising anticancer properties in vivo and in vitro. Aim of this study was to investigate the impact of ABC transporter-mediated multidrug resistance (MDR) on the anticancer activity of KP772. Here, we demonstrate that all MDR cell models investigated, overexpressing ABCB1 (P-glycoprotein), ABCC1 (multidrug resistance protein 1), or ABCG2 (breast cancer resistance protein) either due to drug selection or gene transfection, were significantly hypersensitive against KP772. Using ABCB1-overexpressing KBC-1 cells as MDR model, KP772 hypersensitivity was demonstrated to be based on stronger apoptosis induction and/or cell cycle arrest at unaltered cellular drug accumulation. KP772 did neither stimulate ABCB1 ATPase activity nor alter rhodamine 123 accumulation arguing against a direct interaction with ABCB1. Accordingly, several drug resistance modulators did not sensitize but rather protect MDR cells against KP772-induced cytotoxicity. Moreover, long-term KP772 treatment of KBC-1 cells at subtoxic concentrations led within 20 passages to a complete loss of drug resistance based on blocked MDR1 gene expression. When exposing parental KB-3-1 cells to subtoxic, stepwise increasing KP772 concentrations, we observed, in contrast to several other metallo-drugs, no acquisition of KP772 resistance. Summarizing, our data demonstrate that KP772 is hyperactive in MDR cells and might have chemosensitizing properties by blocking ABCB1 expression. Together with the disability of tumor cells to acquire KP772 resistance, our data suggest that KP772 should be especially active against notoriously drug-resistant tumor types and as second line treatment after standard chemotherapy failure.
Keywords: Abbreviations; 5-FU; 5-fluorouracil; ABC; ATP-binding cassette; ABCB1; P-glycoprotein; ABCC; multidrug resistance-related protein; ABCG2; breast cancer resistance protein; BSA; bovine serum albumin; CSA; cyclosporin A; DMSO; dimethyl sulfoxide; ICP-MS; inductively coupled plasma mass spectroscopy; FACS; fluorescence-activated cell sorting; KP1255; acetatobis[1-(azepanyl)-4(2-pyridyl)-2,3-diazapenta-1,3-dien-1-thiolato-; N; ′,; N; 3; ,; S; ]bismuth(III); KP772; [tris(1,10-phenanthroline)lanthanum(III)] trithiocyanate; LRP; lung resistance protein; MDR; multidrug resistance; PBS; phosphate-buffered saline; Rh123; rhodamine 123; RT-PCR; reverse transcriptase polymerase chain reaction; PRO; probenecid; TMAH; tetramethylammonium hydroxide; VP; verapamilMultidrug resistance; P-glycoprotein; Collateral sensitivity; Phenanthroline; Lanthanum
DNA interactions of new cytotoxic tetrafunctional dinuclear platinum complex trans, trans-[{PtCl2(NH3)}2(piperazine)]
by Viktor Brabec; Petros Christofis; Martina Slámová; Hana Kostrhunová; Olga Nováková; Yousef Najajreh; Dan Gibson; Jana Kašpárková (pp. 1887-1900).
A new tetrafunctional dinuclear platinum complex trans, trans-[{PtCl2(NH3)}2(piperazine)] with sterically rigid linking group was designed, synthesized and characterized. In this novel molecule, the DNA-binding features of two classes of the platinum compounds with proven antitumor activity are combined, namely trans oriented bifunctional mononuclear platinum complexes with a heterocyclic ligand and polynuclear platinum complexes. DNA-binding mode of this new complex was analyzed by various methods of molecular biology and biophysics. The complex coordinates DNA in a unique way and interstrand and intrastrand cross-links are the predominant lesions formed in DNA in cell-free media and in absence of proteins. An intriguing aspect of trans, trans-[{PtCl2(NH3)}2(piperazine)] is that, using a semi-rigid linker, interstrand cross-linking is diminished relative to other dinuclear platinum complexes with flexible linking groups and lesions that span several base pairs, such as tri- and tetrafunctional adducts, become unlikely. In addition, in contrast to the inability of trans, trans-[{PtCl2(NH3)}2(piperazine)] to cross-link two DNA duplexes, the results of the present work convincingly demonstrate that this dinuclear platinum complex forms specific DNA lesions which can efficiently cross-link proteins to DNA. The results substantiate the view that trans, trans-[{PtCl2(NH3)}2(piperazine)] or its analogues could be used as a tool for studies of DNA properties and their interactions or as a potential antitumor agent. The latter view is also corroborated by the observation that trans, trans-[{PtCl2(NH3)}2(piperazine)] is a more effective cytotoxic agent than cisplatin against human tumor ovarian cell lines.
Keywords: Abbreviations; 1,1/c,c; [{; cis; -[PtCl(NH; 3; ); 2; }H; 2; N(CH; 2; ); 2–6; NH; 2; ]; 2+; 1,1/t,t; [{; trans; -[PtCl(NH; 3; ); 2; }H; 2; N(CH; 2; ); 2–6; NH; 2; ]; 2+; 1,2/c,c; [{; cis; -PtCl(NH; 3; ); 2; }μ-H; 2; N(CH; 2; ); 6; NH; 2; -{; cis; -PtCl; 2; (NH; 3; )}]; BBR3464; [{; trans; -PtCl(NH; 3; ); 2; }; 2; μ-; trans; -Pt(NH; 3; ); 2; {H; 2; N(CH; 2; ); 6; NH; 2; }; 2; ]; 4+; bp; base pair; cisplatin; cis; -diamminedichloridoplatinum(II); CL; cross-link; CT; calf thymus; DMS; dimethyl sulfate; EtBr; ethidium bromide; FAAS; flameless atomic absorption spectrometry; IC; 50; the concentration of the compound that afforded 50% cell killing; KF; Klenow fragment from DNA polymerase I (wild type); KF; −; Klenow fragment from DNA polymerase I, exonuclease minus mutated to remove the 3′; →; 5′ proofreading domain; PAA; polyacrylamide; pz; piperazine; PAGE; polyacrylamide gel electrophoresis; [PtCl(dien)]Cl; chlorodiethylenetriamineplatinum(II) chloride; r; b; the number of molecules of the platinum compound bound per nucleotide residue; transplatin; trans; -diamminedichloridoplatinum(II); r; i; the molar ratio of free platinum complex to nucleotides at the onset of incubation with DNA; t; m; DNA melting temperaturePlatinum antitumor drug; DNA modification; DNA cross-links; DNA–protein cross-links; Cytotoxicity; Dinuclear platinum complex
Enhancement of cisplatin induced apoptosis by suberoylanilide hydroxamic acid in human oral squamous cell carcinoma cell lines
by Jun Shen; Canhua Huang; Lu Jiang; Feng Gao; Zhi Wang; Yuanyuan Zhang; Jingping Bai; Hongmei Zhou; Qianming Chen (pp. 1901-1909).
Both the resistance of tumor cells to cisplatin and dose-related toxicity remain two of the most important problems in the chemotherapy of clinical oral squamous cell carcinoma (OSCC). Researchers have been seeking a combinative treatment regimen to improve the effect of chemotherapy. As potent new anti-cancer drugs, histone deacetylase inhibitors (HDACIS) have been reported to be associated with chromatin modification and display synergistic activities with some traditional chemotherapeutic agents. In this study, we evaluated the potential combinative effect of low dose cisplatin and suberoylanilide hydroxamic acid (SAHA, one of the most potent HDACIS) in OSCC cell lines. Cell viability and apoptotic assay were examined. Compared with either cisplatin (4μg/ml) or SAHA (2μM) treated alone, co-administration of both drugs synergistically induces cytotoxicity and apoptosis in both Tca8113 and KB cell lines. Furthermore, diverse apoptosis-associated proteins, including p53, BID, cytochrome C and caspase-3 were involved in the induction of apoptosis. Our results suggest that concurrent treatment with SAHA enhances tumor cell sensitivity to subtoxic doses of cisplatin. This may be regarded as a novel strategy for treatment of OSCC.
Keywords: Cisplatin; Suberoylanilide hydroxamic acid; Apoptosis; Chemotherapy; Oral squamous cell carcinoma
Activity of piperaquine and other 4-aminoquinoline antiplasmodial drugs against chloroquine-sensitive and resistant blood-stages of Plasmodium falciparum
by David C. Warhurst; John C. Craig; Ipemida S. Adagu; R. Kiplin Guy; Peter B. Madrid; Quinton L. Fivelman (pp. 1910-1926).
Chloroquine (CQ), a 4-aminoquinoline, accumulates in acidic digestive vacuoles of the malaria parasite, preventing conversion of toxic haematin to β-haematin. We examine how bis 4-aminoquinoline piperaquine (PQ) and its hydroxy-modification (OH-PQ) retain potency on chloroquine-resistant (CQ-R) Plasmodium falciparum. For CQ, PQ, OH-PQ and4 and5, representing halves of PQ, β-haematin inhibitory activity (BHIA) was assayed, while potency was determined in CQ-sensitive (CQ-S) and CQ-R P. falciparum. From measured p Kas and the pH-modulated distribution of base between water and lipid (logD), the vacuolar accumulation ratio (VAR) of charged drug from plasma water (pH 7.4) into vacuolar water (pH 4.8) and lipid accumulation ratio (LAR) were calculated. All agents were active in BHIA. In CQ-S, PQ, OH-PQ and CQ were equally potent while4 and5 were 100 times less potent. CQ with two basic centres has a VAR of 143,482, while4 and5, with two basic centres of lower p Kas have VARs of 1287 and 1966. In contrast PQ and OH-PQ have four basic centres and achieve VARs of 104,378 and 19,874. This confirms the importance of VAR for potency against CQ-S parasites. Contrasting results were seen in CQ-R.5, PQ and OH-PQ with LARs of 693; 973,492 and 398,118 (compared with 8.25 for CQ) showed similar potency in CQ-S and CQ-R. Importance of LAR for potency against CQ-R parasites probably reflects ability to block efflux by hydrophobic interaction with PfCRT but may relate to β-haematin inhibition in vacuolar lipid.
Keywords: Plasmodium; 4-Aminoquinolines; Piperaquine; Lysosomotropism; Drug-resistance; Hydrophobicity
An increase of cytochrome C oxidase mediated disruption of gemcitabine incorporation into DNA in a resistant KB clone
by Xiyong Liu; Bingsen Zhou; Shu Mi; Lijun Xue; Jennifer Shih; Janice Lee; Jennifer Chau; Frank Un; Yun Yen (pp. 1927-1938).
Mechanistic aberrations leading to Gemcitabine (2′,2′-dFdCyd,2,2-difluorodeoxycytidine, Gem) resistance may include alteration in its transport, metabolism and incorporation into DNA. To explore the mechanism of Gem resistance, the restriction fragment differential display PCR (RFDD-PCR) was employed to compare the mRNA expression patterns of KBGem (Gem resistant), KBHURs (hydroxyurea resistant) and KBwt (parental KB cell). Nine gene fragments were overexpressed specifically in the KBGem clone. Sequencing and BLAST results showed that three fragments represent cytochrome C oxidase (CCOX, respiration complex IV) subunit III (CCOX3). The cDNA microarray confirmed that the mRNAs of CCOX and ATP synthase subunits were upregulated in KBGem as compared to KBwt and KBHURs. The increase in CCOX1 protein and activity led to the increase of free ATP concentration, which is consistent with the gene expression profile of KBGem. Furthermore, the sensitivity to Gem could be reversed by sodium azide, a CCOX inhibitor. Following the treatment of sodium azide, the cellular accumulation of [3H]-Gem increased in a dose (of azide)-dependent manner, which is associated with increase of [3H]-Gem incorporation into DNA in KBGem. In summary, an increase of CCOX activity and free ATP level may reduce the transport, metabolism and DNA incorporation of Gem, resulting in Gem resistance.
Keywords: Abbreviations; Gem; 2′,2′-dFdCyd,2,2-difluorodeoxycytidine, Gemcitabine; dCK; deoxycytidine kinase; dCMPK; dCMP kinase; NDPK; nucleoside diphosphate kinase; CK; cytidylate kinase; CCOX; cytochrome; C; oxidase; ENT; equilibrative nucleoside transporter; CNT; concentrative nucleoside transporter; OAT; organic anion transporter; OCT; organic cation transporter; PEPT; peptide transporter; MRP; multi-drug resistance protein; KBGem; gemcitabine drug resistant KB clone; KBHURs; hydroxyurea drug resistant KB clone; KBwt; parental KB cell; hRR; human ribonucleotide reductase; hRRM1; human ribonucleotide reductase large subunit M1; hRRM2; human ribonucleotide reductase small subunit M2; p53R2; p53 dependent human ribonucleotide reductase small subunit R2 (M2B); dNTP; deoxyribonucleoside triphosphate; NDP; ribonucleoside diphosphate; RFDD-PCR; restriction fragment differential display PCRGemcitabine; Drug resistance; Cytochrome; C; oxidase; Ribonucleotide reductase; ATP
Activity of “reversed” diamidines against Trypanosoma cruzi “ in vitro”
by C.F. Silva; Marcos Meuser Batista; Renata Alves Mota; Elen Mello de Souza; Chad E. Stephens; Phanneth Som; David Wilson Boykin; Maria de Nazaré C. Soeiro (pp. 1939-1946).
Chagas’ disease is an important parasitic illness caused by the flagellated protozoan Trypanosoma cruzi. The disease affects nearly 17 million individuals in endemic areas of Latin America and the current chemotherapy is quite unsatisfactory based on nitroheterocyclic agents (nifurtimox and benznidazol). The need for new compounds with different modes of action is clear. Due to the broad-spectrum antimicrobial activity of the aromatic dicationic compounds, this study focused on the activity of four such diamidines (DB811, DB889, DB786, DB702) and a closely related diguanidine (DB711) against bloodstream trypomastigotes as well as intracellular amastigotes of T. cruzi in vitro. Additional studies were also conducted to access the toxicity of the compounds against mammalian cells in vitro. Our data show that the four diamidines compounds presented early and high anti-parasitic activity (IC50 in low-micromolecular range) exhibiting trypanocidal dose-dependent effects against both trypomastigote and amastigote forms of T. cruzi 2h after drug treatment. Most of the diamidines compounds (except the DB702) exerted high anti-parasitic activity and low toxicity to the mammalian cells. Our results show the activity of reversed diamidines against T. cruzi and suggested that the compounds merit in vivo studies.
Keywords: Chagas disease; Trypanosoma cruzi; Reversed diamidines; Chemotherapy
Oxidative DNA base damage in MCF-10A breast epithelial cells at clinically achievable concentrations of doxorubicin
by Ewa Gajewski; Shikha Gaur; Steven A. Akman; Linda Matsumoto; Josephus N.A. van Balgooy; James H. Doroshow (pp. 1947-1956).
The cellular metabolism of doxorubicin generates reactive oxygen species with significant potential to damage DNA. Such DNA damage can result in mutations if not adequately repaired by cellular DNA repair pathways. Secondary malignancies have been reported in patients who have received doxorubicin-containing chemotherapeutic regimens; however, the underlying molecular mechanism(s) to explain the development of these tumors remains under active investigation. We have previously demonstrated the presence of DNA bases modified by oxidation in the peripheral blood mononuclear cells of patients with breast cancer following treatment with doxorubicin. In those studies, doxorubicin was administered by continuous infusion over 96h to minimize the risk of cardiac toxicity. To evaluate potential mechanisms underlying doxorubicin-induced DNA base oxidation in non-malignant tissues, MCF-10A breast epithelial cells were cultured for 96h with the same doxorubicin concentration achieved in vivo (0.1μM). During doxorubicin exposure, MCF-10A cells underwent growth arrest and apoptosis, developed elevated levels of reactive oxygen species, and demonstrated a time-dependent and significant increase in the levels of 11 oxidized DNA bases, as determined by gas chromatography/mass spectroscopy. Diminished expression of DNA repair enzymes was also observed over the same time course. Thus, clinically achievable concentrations of doxorubicin induce a level of oxidative stress in MCF-10A cells that is capable of oxidizing DNA bases and significantly altering cellular proliferation.
Keywords: Abbreviations; DOX; doxorubicin; AML; acute myelogenous leukemia; ROS; reactive oxygen species; GC/MS; gas chromatography/mass spectroscopy; PBMCs; peripheral blood mononuclear cells; BER; base excision repair; NLB; nuclear lysis buffer; RMRF; relative molecular response factor; RPA; ribonuclease protection assay; RPA4; subunit of the RPA haloenzyme; L32; ribosomal protein L32; GAPDH; glyceraldehydes-3-phosphate dehydrogenase; 5-OH-Hyd; 5-hydroxyhydantoin; 5-OH-5-MeHyd; 5-hydroxy-5-methylhydantoin; 5-OH-Ura; 5-hydroxyuracil; 5-OH-Cyt; 5-hydroxycytosine; ThyGly; thymine glycol; 5,6diOH-Ura; 5,6-dihydroxyuracil; FapyAde; 4,6-diamino-5-formamidopyrimidine; 8-OH-Ade; 8-hydroxyadenine; Xan; xanthine; FapyGua; 2,6-diamino-4-hydroxy-5-formamidopyrimidine; 2-OH-Ade; 2-hydroxy-6-aminopurine; 8-OH-Gua; 2-amino-6, 8-dihydroxyguanine; APE; apurinic/apyrimidinic endonuclease; hOGG1; 8-oxoguanine DNA-glycosylase; mtOGG1; mitochondrial-8-oxoguanine DNA-glycosylase; MGMT; O; 6; -methylguanine-DNA methyltransferase; TDG; G/T mismatch-specific thymine-DNA glycosylase; UDG; uracil-DNA glycosylase; MPG; methylpurine-DNA glycosylase; ENTG; thymine glycol-DNA glycosylase/AP lyaseDoxorubicin; DNA damage; Reactive oxygen species; Apoptosis; Chemotherapy; Secondary malignancy
A synthetic analog of α-galactosylceramide induces macrophage activation via the TLR4-signaling pathways
by Ling-Chien Hung; Chun-Cheng Lin; Shih-Kai Hung; Bing-Ching Wu; Mi-Dan Jan; Sheng-Hung Liou; Shu-Ling Fu (pp. 1957-1970).
α-Galactosylceramide (α-GalCer), a bioactive glycolipid isolated from the marine sponge Agelas mauritianus, is a potent immunomodulator with therapeutic potential for the treatment of autoimmune diseases and cancer. The Toll-like receptor 4 (TLR4), one of the promising molecular targets for immune-modulating drugs, is commonly expressed in innate immune cells especially macrophages and dendritic cells. Currently, whether α-GalCer can activate TLR4 signaling pathways remains unreported. In this study, we examined the effects of α-GalCer and its various structural analogs, CCL-1∼47, on TLR4 activation. We found that one α-GalCer analog (CCL-34), but not α-GalCer itself, strongly stimulated NF-κB activity in RAW 264.7 cells. CCL-34 activated NF-κB in a TLR4-dependent manner and stimulated TNF-α production in bone marrow cells of TLR4-functional C3H/HeN mice but not in those of TLR4-defective C3H/HeJ mice. Furthermore, CCL-34 treatment stimulated NF-κB activation and IL-8 production in a 293 cell line constitutively expressing human TLR4, MD-2 and CD14. Treatment of RAW 264.7 cells with CCL-34 also activated TLR4-downstream mitogen-activated protein kinases (ERK, JNK and p38), induced expression of TLR4-downstream genes (TNF-α, IL-6, IL-1β and iNOS) and promoted production of cytokines characteristic of activated macrophages. CCL-34-treated RAW 264.7 cells acquired a distinct morphology similar to that of LPS-activated macrophages and exhibited higher phagocytotic activity. Moreover, treatment with a TLR4-neutalizing antibody inhibited the CCL-34-induced morphological alteration. In summary, we identify a novel synthetic compound CCL-34 that can activate macrophages via TLR4-dependent signaling pathways. Our results suggest that CCL-34 is an immune modulator and may serve as a potential drug lead for immunotherapy.
Keywords: Abbreviations; α-GalCer; α-galactosylceramide; TLR4; Toll-like receptor 4; ELAM-1; endothelia leukocyte adhesion molecule-1; LPS; lipopolysaccharide; MAPK; mitogen-activated protein kinase; TNF-α; tumor necrosis factor-α; IL-1; interleukin-1; IL-6; interleukin-6; GM-CSF; granulocyte/macrophage colony-stimulating factor; NF-κB; nuclear transcription factor-κB; iNOS; inducible nitric oxide synthaseTLR4; α-Galactosylceramide; CCL-34; NF-κB; MAPK; Macrophage
L-454,560, a potent and selective PDE4 inhibitor with in vivo efficacy in animal models of asthma and cognition
by Z. Huang; R. Dias; T. Jones; S. Liu; A. Styhler; D. Claveau; F. Otu; K. Ng; F. Laliberte; L. Zhang; P. Goetghebeur; W.M. Abraham; D. Macdonald; D. Dubé; M. Gallant; P. Lacombe; Y. Girard; R.N. Young; M.J. Turner; D.W. Nicholson; J.A. Mancini (pp. 1971-1981).
Type 4 phosphodiesterases (PDE4) inhibitors are emerging therapeutics in the treatment of a number of chronic disorders including asthma, chronic obstructive pulmonary disease (COPD) and cognitive disorders. This study delineates the preclinical profile of L-454,560, which is a potent, competitive and preferential inhibitor of PDE4A, 4B, and 4D with IC50 values of 1.6, 0.5 and 1.2nM, respectively. In contrast to the exclusive binding of cilomilast and the preferential binding of roflumilast to the PDE4 holoenzyme state (Mg2+-bound form), L-454,560 binds to both the apo-(Mg2+-free) and holoenzyme states of PDE4. The intrinsic enzyme potency for PDE4 inhibition by L-454,560 also results in an effective blockade of LPS-induced TNFα formation in whole blood (IC50=161nM) and is comparable to the human whole blood potency of roflumilast. The cytokine profile of inhibition of L-454,560 is mainly a Th1 profile with significant inhibition of IFNγ and no detectable inhibition of IL-13 formation up to 1μM. L-454,560 was also found to be efficacious in two models of airway hyper-reactivity, the ovalbumin (OVA) sensitized and challenged guinea pig and the ascaris sensitized sheep model. Furthermore, L-454560 was also effective in improving performance in the delayed matching to position (DMTP) version of the Morris watermaze, at a dose removed from that associated with potential emesis. Therefore, L-454,560 is a novel PDE4 inhibitor with an overall in vivo efficacy profile at least comparable to roflumilast and clearly superior to cilomilast.
Keywords: Abbreviations; cAMP; cyclic adenosine monophosphate; COPD; chronic obstructive pulmonary disease; CTA; conditioned taste aversion; DMTP; delayed matching to position; EAR; early airway response; FEV1; forced expiratory volume in 1; s; fMLP; f-MET-LEU-PHE; LAR; late airway response; LPS; lipopolysaccharide; LTP; long term potentiation; MED; minimal effective dose; OVA; ovalbumin; PDE; phosphodiesterase; TNF; tumor necrosis factorAsthma; cAMP; Cognition; COPD; Inflammation; Phosphodiesterase; PDE4
Role of IKK and ERK pathways in intrinsic inflammation of cystic fibrosis airways
by Catherine Verhaeghe; Caroline Remouchamps; Benoît Hennuy; Alain Vanderplasschen; Alain Chariot; Sebastien P. Tabruyn; Cecile Oury; Vincent Bours (pp. 1982-1994).
In cystic fibrosis (CF) patients, pulmonary inflammation is a major cause of morbidity and mortality and may precede bacterial colonization. The aim of the present study was to investigate the molecular mechanisms underlying intrinsic inflammation in cystic fibrosis airways. Using different cystic fibrosis cell models, we first demonstrated that, beside a high constitutive nuclear factor of kappaB (NF-κB) activity, CF cells showed a higher activator protein-1 (AP-1) activity as compared to their respective control cells. Gene expression profiles, confirmed by RT-PCR and ELISA, showed over-expression of numerous NF-κB and AP-1-dependent pro-inflammatory genes in CF cells in comparison with control cells. Activation of NF-κB was correlated with higher inhibitor of κB kinase (IKK) activity. In addition, Bio-plex phosphoprotein assays revealed higher extracellular signal-regulated kinase (ERK) phosphorylation in CFT-2 cells. Inhibition of this kinase strongly decreased expression of pro-inflammatory genes coding for growth-regulated proteins (Gro-α, Gro-β and Gro-γ) and interleukins (IL-1β, IL-6 and IL-8). Moreover, inhibition of secreted interleukin-1β (IL-1β) and basic fibroblast growth factor (bFGF) with neutralizing antibodies reduced pro-inflammatory gene expression. Our data thus demonstrated for the first time that the absence of functional cystic fibrosis transmembrane conductance regulator (CFTR) at the plasma membrane leads to an intrinsic AP-1, in addition to NF-κB, activity and consequently to a pro-inflammatory state sustained through autocrine factors such as IL-1β and bFGF.
Keywords: Abbreviations; CF; cystic fibrosis; CFTR; cystic fibrosis transmembrane conductance regulator; ICAM-1; intracellular adhesion molecule-1; COX-2; cycloxygenase-2; NF-κB; nuclear factor-kappaB; AP-1; activator protein-1; IκB; inhibitor of kappaB; IKK; IκB kinase; MAPK; mitogen-activated protein kinase; ERK; extracellular signal-regulated kinase; JNK; c-JUN N-terminal kinase; MEK 1; mitogen-activated protein kinase kinase 1; EMSA; electrophoresis mobility shift assay; PCR; polymerase chain reaction; IL; interleukin; bFGF; basic fibroblast growth factor; ALLN; N; -acetyl-; L; -leucyl-; L; -leucyl-; L; -norleucinal; Gro; growth-regulated protein; MMP; matrix metalloproteinaseNF-κB; AP-1; Transcription factors; Inflammation; CFTR; Gene expression
The effects of antidepressants on cyclic AMP-response element-driven gene transcription in a model cell system
by W. Abdel-Razaq; T.E. Bates; D.A. Kendall (pp. 1995-2003).
The effects of the antidepressant drugs clomipramine (CLOM), desipramine (DMI), tianeptine (TIAN) and of norfluoxetine (NORF, the active metabolite of fluoxetine), were investigated in CHO cells expressing human β2 adrenoceptors and a secreted placental alkaline phosphatase (SPAP) reporter gene to determine their actions on cyclic AMP-driven gene transcription.After 18h of exposure, CLOM, DMI and NORF, but not TIAN, had biphasic effects on 1μM isoprenaline-stimulated SPAP fsproduction with concentrations between 10nM and 1μM enhancing the maximal ( Emax) SPAP response, without changing EC50 values, but higher concentrations produced marked inhibitory effects.At nanomolar concentrations, CLOM and DMI increased expression of phospho-CREB (cyclic AMP response element binding protein). NORF was less effective but did significantly increase phospho-CREB at a concentration of 200nM. TIAN had no effect. None of the antidepressants had any effect on CREB expression, nor on the accumulation of cyclic AMP.After prolonged exposure (7–21 days) to a low concentration (200nM) of the antidepressants, the enhanced Emax values for SPAP production evident after 18h were not maintained but CLOM and DMI induced a significant leftward shift in the isoprenaline EC50 after a 7-day period of treatment and this was sustained at the 21 day time point. TIAN did not produce any significant changes.The results demonstrate that, in vitro, some but not all antidepressants can modify gene transcription via monoamine and cyclic AMP-independent mechanisms. The in vivo adaptive responses to TIAN probably involve alterations in different gene sets to those affected by other antidepressants.
Keywords: Antidepressant; Cyclic AMP; CHO cell; CREB; Gene transcription
Cannabinoids inhibit sodium-dependent, high-affinity excitatory amino acid transport in cultured rat cortical astrocytes
by Amruthesh C. Shivachar (pp. 2004-2011).
Cannabinoids have been shown to increase the extracellular levels of glutamate in vivo and in vitro, but no studies have evaluated the possible involvement of glial glutamate reuptake system. The present study investigates whether cannabinoids and endocannabinoid, anandamide have an effect on astroglial excitatory amino acid (EAA) transport. The kinetics of glutamate transport was studied in rat cortical astrocytes, using the radiolabeled, non-metabolized amino acid,d-[3H] aspartate in the absence or presence of cannabinoid receptor agonists. The results show that in vehicle controls the uptake ofd-aspartate was rapid, sodium-dependent and saturated within the first 5min, resulting in a Km 7.365±1.16μmol/L ( n=5) and the maximum velocity ( Vmax) 1207±51nmol/mgprotein/min. Addition of the synthetic cannabinoid analog R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolol][1,2,3de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone (WIN 55,212-2; 3μmol/L) increased the Km (26.25±4.84μmol/L) without affecting the Vmax (1122±77nmol/mgprotein/min), suggesting the inhibition was competitive and reversible. Various other cannabinoid agonists also inhibitedd-aspartate uptake in a dose-dependent and stereospecific manner. The cannabinoid inhibition of EAA transport was partially blocked by the cannabinoid type-1 (CB1) receptor antagonist N-(piperidin-1-yl-5(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl- 1H-pyrazole-3-carboxamidehydrochloride (SR141716A; 100nmol/L). The inhibitory effects of WIN 55,212-2, or its endogenous counterpart anandamide were reversed by 98,059, an inhibitor of mitogen-activated kinase (MAPK) kinase (MEK). These results suggest that cannabinoids and endocannabinoids may constitute a novel class of inhibitors of astroglial glutamate transport system.
Keywords: Abbreviations; AN; anandamide; CB1; cannabinoid subtype-1 receptor; CP 55,940; (−)-; cis; -3[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-; trans; -4-(3-hydroxypropyl) cyclohexanol; DMEM; Dulbecco's modified Eagles medium; DMSO; dimethyl sulfoxide; EAA; excitatory amino acids; EAAT; excitatory amino acid transporter; GFAP; glial fibrillary acidic protein; GLAST; glutamate aspartate transporter; GLT1; glutamate transporter-1; LDH; lactate dehydrogenase; MAPK; mitogen-activated protein kinase; MEK; MAPK kinase; SR141716A; N; -(piperidin-1-yl-5(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-; 1H; -pyrazole-3-carboxamidehydrochloride; WIN 55,212-2; R; (+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolol][1,2,3de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone; WIN 55,212-3; S; (−)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolol]1,2,3de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanoneAnandamide; Arachidonic acid; Astrocytes; Cannabinoids; Cannabinoid receptor subtype-1; Endocannabinoids; Glutamate transport; MAP kinase; Methanadamide
Kinetics of glyburide metabolism by hepatic and placental microsomes of human and baboon
by Olga L. Zharikova; Selvan Ravindran; Tatiana N. Nanovskaya; Ronald A. Hill; Gary D.V. Hankins; Mahmoud S. Ahmed (pp. 2012-2019).
Glyburide (glibenclamide) is under investigation for treatment of gestational diabetes. Two metabolites of glyburide have been previously identified in patients, namely, 4- trans-(M1) and 3- cis-(M2) hydroxycyclohexyl glyburide. Recently, the metabolism of glyburide by microsomes of liver and placenta from humans and baboons revealed the formation of four additional metabolites: 4- cis-(M2a), 3- trans-(M3), and 2- trans-(M4) hydroxycyclohexyl glyburide, and ethyl-hydroxy glyburide (M5). The aim of this investigation was to determine the kinetics for the metabolism of glyburide by cytochrome P450 (CYP) isozymes of human and baboon placental and hepatic microsomes. The metabolism of glyburide by microsomes from the four organs revealed saturation kinetics and apparent Km values between 4 and 12μM. However, the rates for formation of the metabolites varied between organs and species. M1 was the major metabolite (36% of total), formed by human hepatic microsomes with Vmax of 80±13pmolmgprotein−1min−1, and together with M2, accounted for only 51% of the total. M5 was the major metabolite (87%) formed by human placental microsomes with Vmax of 11pmolmgprotein−1min−1. In baboon liver, M5 had the highest rate of formation ( Vmax 135±32pmolmgprotein−1min−1, 39% of total), and in its placenta, was M4 ( Vmax 0.7±0.1pmolmgprotein−1min−1, 65%). The activity of human and baboon hepatic microsomes in metabolizing glyburide was similar, but the activity of human and baboon placental microsomes was 7% and 0.3% of their respective hepatic microsomes. The data obtained suggest that more than 1 CYP isozyme is responsible for catalyzing the hydroxylation of glyburide.
Keywords: IDT; Enzyme kinetics; Drug metabolismAbbreviations; M; metabolite; IS; internal standard; CYP; cytochrome P450; HPLC; high performance liquid chromatography; MS; mass spectrometryPregnancy; Glyburide (glibenclamide) metabolism; Microsomal enzymes; Enzyme kinetics; Human placenta; Baboon placenta; Human liver; Baboon liver
Roles of CYP3A4 and CYP2C19 in methyl hydroxylated and N-oxidized metabolite formation from voriconazole, a new anti-fungal agent, in human liver microsomes
by Norie Murayama; Naoko Imai; Takahisa Nakane; Makiko Shimizu; Hiroshi Yamazaki (pp. 2020-2026).
Involvement of cytochrome P450 (P450 or CYP) 2C19, 2C9, and 3A4 in N-oxidation of voriconazole, a new triazole antifungal agent, has been demonstrated using human liver microsomes. To confirm the precise roles of P450 isoforms in voriconazole clearance in individuals, we investigated the oxidative metabolism of voriconazole catalyzed by recombinant P450s as well as human liver microsomes genotyped for the CYP2C19 gene. Among recombinant P450 isoforms using Escherichia coli expression systems, CYP2C19 and CYP3A4 had voriconazole N-oxidation activities, but not CYP2C9. Apparent Km and Vmax values of CYP2C19 and CYP3A4 for voriconazole N-oxidation were 14±6μM and 0.22±0.02nmol/min/nmol CYP2C19 and 16±10μM and 0.05±0.01nmol/min/nmol CYP3A4, respectively (mean±S.E.). CYP3A4 produced a new methyl hydroxylated metabolite from voriconazole, detected by LC/UV and LC/MS/MS and confirmed by1H and13C NMR analyses, with Km and Vmax values of 11±3μM and 0.10±0.01nmol/min/nmol CYP3A4. The voriconazole 4-hydroxylation to N-oxidation metabolic ratios in liver microsomes from the wild-type CYP2C19*1/ *1 individuals (0.07) were lower than those observed in other genotypes (0.20–0.27) at a substrate concentration of 25μM based on the reported clinical plasma level. These results suggest that the CYP2C19 genotype, but not CYP2C9 genotype, would be evaluated as a key factor in the pharmacokinetics of voriconazole and that 4-hydroxyvoriconazole formation may become an important pathway for voriconazole metabolism in individuals with poor CYP2C19 catalytic function.
Keywords: Cytochrome P450 3A4; P450 2C19; Poor metabolizer; NMR; LC/MS; Hydroxylation
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