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Biochemical Pharmacology (v.74, #6)

Editorial Advisory Board (pp. iii).

Hemigramicidin–TEMPO conjugates: Novel mitochondria-targeted anti-oxidants by Mitchell P. Fink; Carlos A. Macias; Jingbo Xiao; Yulia Y. Tyurina; Jianfei Jiang; Natalia Belikova; Russell L. Delude; Joel S. Greenberger; Valerian E. Kagan; Peter Wipf (pp. 801-809).

Oxidative damage to various cellular constituents (such as, proteins and lipids) mediated by reactive oxygen species (ROS) is thought to be an important mechanism underlying the pathogenesis of a variety of acute and chronic diseases. Mitochondria are the main source of ROS within most cells. Accordingly, there is increasing interest in the development of pharmacological ROS scavengers, which are specifically targeted to and concentrated within mitochondria. Numerous compounds with these general characteristics have been synthesized and evaluated in a variety of in vitro and in vivo models of redox stress. Among the more promising of these mitochondria-targeted anti-oxidants are those that employ various peptides (or peptide-like moieties) derived from the antibiotic, gramicidin S, as the targeting construct and employ the stable free radical, 4-amino-2,2,6,6-tetramethylpiperidine- N-oxyl (4-NH₂–TEMPO), as the ROS scavenging “payload.” One of these hemigramicidin–TEMPO conjugates, XJB-5-131, has been shown to ameliorate intestinal mucosal injury and prolong survival in rats subjected to lethal hemorrhage.

Keywords: Superoxide; Oxidant stress; TEMPOL; Hemorrhagic shock; LPS; Caspase

Identification of human reductases that activate the dinitrobenzamide mustard prodrug PR-104A: A role for NADPH:cytochrome P450 oxidoreductase under hypoxia by Chris P. Guise; Anderson T. Wang; Anke Theil; David J. Bridewell; William R. Wilson; Adam V. Patterson (pp. 810-820).

Hypoxia is a common trait found in many solid tumours and thus represents a therapeutic target with considerable potential. PR-104, a hypoxia-activated prodrug currently in clinical trial, is a water-soluble phosphate ester which is converted in vivo to the corresponding alcohol, PR-104A. This 3,5-dinitrobenzamide-2-nitrogen mustard is activated by reduction to the corresponding 5-hydroxylamine (PR-104H) and 5-amine (PR-104M) in hypoxic cells. The clinical effectiveness of PR-104 will depend in part on the expression of reductases within tumours that can effect this reduction. Here, we evaluate the roles of NADPH:cytochrome P450 oxidoreductase (CYPOR; E.C.1.6.2.4) and NAD(P)H:quinone oxidoreductase (NQO1; E.C.1.6.99.2) as candidate PR-104A reductases. A weak correlation was observed between NQO1 activity and aerobic cytotoxicity in a panel of eight tumour cell lines. However, overexpression of human NQO1 did not increase cytotoxicity of PR-104A or the formation of PR-104H/M, showing that PR-104A is not a substrate for NQO1. Overexpression of human CYPOR did, however, increase the hypoxic cytotoxicity of PR-104A, and its metabolism to PR-104H and PR-104M, demonstrating it to be a PR-104A reductase. To assess the contribution of CYPOR to overall activation of PR-104A in hypoxic SiHa cells, a combination of siRNA transfection and antisense expression were used to suppress CYPOR protein by 91% (±3%), a phenotype which conferred 45% (±7%) decrease in cytotoxic potency of PR-104A. Regression analysis of all CYPOR depletion data was found to correlate with cytoprotection and metabolism ( p<0.001). Residual PR-104A reductase activity could be inhibited by the flavoprotein inhibitor diphenyliodonium. We conclude that CYPOR is an important PR-104A reductase, but that other flavoenzymes also contribute to its activation in hypoxic SiHa cells.

Keywords: Oxidoreductase; Reductive; Metabolism; Prodrug; Hypoxia; Cancer

Existence of a distinct concentration window governing daunorubicin-induced mammalian liver mitotoxicity—implication for determining therapeutic window by Manash K. Paul; Minal Patkari; Anup K. Mukhopadhayay (pp. 821-830).

Daunorubicin (DNR) is a well known anticancer drug believed to act mainly by topoisomerase II inhibition and mitochondria-mediated free radical generation. Though several studies were dedicated to elucidate the mechanism of action of DNR, however the mechanism still remains illusive. DNR is reported to affect mitochondrial respiration. However, there are contradictory reports regarding DNR effect on oxygen consumption. Interestingly, DNR at low concentration (<10μM) dose-dependently augments respiration but at higher concentration inhibits respiration. To investigate, if a concentration window exists in which the effect of DNR on mitochondria is optimum, dose-dependent effect of DNR on mitochondria was studied. DNR inhibited electron transfer and generates reactive oxygen species (ROS) at complex I and III but not at complex II. DNR-induced ROS generation was found instrumental in mitochondrial membrane potential collapse and mitochondrial permeability transition (MPT) opening. MPT closure reduced the observed respiratory burst. Thus, at lower DNR concentration, MPT opening leads to a sudden burst of respiration while at higher concentration electron transfer gets inhibited, therefore respiration gets repressed. We for the first time, provide a possible explanation for the reports regarding the differential regulation of respiration by DNR. Thus, further establishing the concept of concentration window and justifying the need for dose optimization for maximal therapeutic effect.

Keywords: Daunorubicin; Concentration window; Mitotoxicity; Respiration; Reactive oxygen species; Mitochondrial pore transition

Inhibition of bacterial cell division protein FtsZ by cinnamaldehyde by Prerna Domadia; Sanjay Swarup; Anirban Bhunia; J. Sivaraman; Debjani Dasgupta (pp. 831-840).

Cinnamaldehyde is a natural product from spices that inhibits cell separation in Bacillus cereus. Cell division is regulated by FtsZ, a prokaryotic homolog of tubulin. FtsZ assembles into the Z-ring at the site of cell division. Here, we report the effect of cinnamaldehyde on FtsZ and hence on the cell division apparatus. Cinnamaldehyde decreases the in vitro assembly reaction and bundling of FtsZ. It is found that cinnamaldehyde perturbs the Z-ring morphology in vivo and reduces the frequency of the Z ring per unit cell length of Escherichia coli. In addition, GTP dependent FtsZ polymerization is inhibited by cinnamaldehyde. Cinnamaldehyde inhibits the rate of GTP hydrolysis and binds FtsZ with an affinity constant of 1.0±0.2μM⁻¹. Isothermal titration calorimetry reveals that binding of cinnamaldehyde to FtsZ is driven by favorable enthalpic interactions. Further, we map the cinnamaldehyde binding region of FtsZ, using the saturation transfer difference–nuclear magnetic resonance and an in silico docking model. Both predict the cinnamaldehyde binding pocket at the C terminal region involving the T7 loop of FtsZ. Our results show that cinnamaldehyde binds FtsZ, perturbs the cytokinetic Z-ring formation and inhibits its assembly dynamics. This suggests that cinnamaldehyde, a small molecule of plant origin, is a potential lead compound that can be developed as an anti-FtsZ agent towards drug design.

Keywords: Abbreviations; FtsZ; filamentation temperature sensitive protein Z; GTP; guanosine 5-triphosphate; MES; 2-morpholinoethanesulfonic acid; GFP; green fluorescent protein; STD NMR; saturation transfer difference–nuclear magnetic resonance; MIC; minimum inhibitory concentration; MBC; minimum bactericidal concentration; MRSA; methicillin resistant; Staphylococcus aureus; FPLC; fast protein liquid chromatography; ITC; isothermal titration calorimetry; T7; tubulin like loop 7; LB; Luria-Bertani; DMSO; dimethyl sulfoxideCinnamaldehyde; Z-ring; FtsZ; Polymerization; GTPase; Cell division

Role of peroxidases, thiols and Bak/Bax in tumor cell susceptibility to Cu[DEDTC]₂ by Maricela Viola-Rhenals; Mary Strasberg Rieber; Manuel Rieber (pp. 841-850).

Copper and two molecules of diethyl dithiocarbamate [DEDTC] form the Cu[DEDTC]₂ complex, which shows cytotoxicity against melanoma and carcinoma cells, making it a potentially useful anti-cancer agent. The differential response to Cu[DEDTC]₂ in susceptible human SKBR3 carcinoma and C8161 melanoma cell variants of moderate and high resistance to this organometallic complex was evaluated in this study. Both cell lines underwent apoptosis-associated PARP cleavage, changes in expression of nuclear NFkB p65, p21WAF1 and cyclin A, with loss of clonogenicity in response to this agent. However, a threefold greater concentration [IC₅₀ 0.6μM DEDTC: 0.3μM Cu] was required to kill moderately resistant C8161 melanoma compared to highly susceptible SKBR3 cells. Decreased susceptibility to Cu[DEDTC]₂ in C8161 melanoma correlated with greater levels of glutathione peroxidase and catalase, and a fourfold lower requirement for N-acetyl cysteine (1mM) to overcome toxicity. Whereas melanoma cells selected for resistance to [0.8μM DEDTC: 0.4μM Cu] showed persistent catalase and GPx activity, melanoma cells with moderate susceptibility showed decreased catalase and Gpx when responding to treatment. Cytotoxic response in moderately susceptible C8161 melanoma cells involved an early accumulation of pro-apoptotic Bax in the G2 cell cycle phase, followed by an increased ratio of pro-apoptotic Bak to anti-apoptotic Mcl-1 in mitochondria. Our data suggests that Cu[DEDTC]₂ toxicity is mediated through an increase in pro-apoptotic Bak/Bax via disruption of the peroxide and thiol metabolism.

Keywords: Abbreviations; DEDTC; diethyl dithiocarbamate; CuCl; ₂; copper chloride (II); Px; peroxidase; GPx; glutathione peroxidaseGlutathione; Peroxidase; Bak; Bax; Mcl-1; Dithiocarbamate; G2

Involvement of CtBP1 in the transcriptional activation of the MDR1 gene in human multidrug resistant cancer cells by Wei Jin; Kathleen W. Scotto; William N. Hait; Jin-Ming Yang (pp. 851-859).

Drug resistance caused by overexpression of P-glycoprotein (P-gp), the MDR1 ( ABCB1) gene product, limits the therapeutic outcome. Expression of MDR1 can be induced by divergent stimuli, and involves a number of transcriptional factors. We found that the expression of CtBP1 (C-terminal-binding protein 1), a transcriptional co-regulator, was increased (∼4-fold) in human multidrug resistant (MDR) cancer cell lines, NCI/ADR-RES and A2780/DX, as compared to their sensitive counterparts. Silencing of CtBP1 expression by RNAi decreased the MDR1 mRNA and P-gp. Knockdown of CtBP1 also enhanced the sensitivity of MDR cells to chemotherapeutic drugs that are transported by P-gp and increased intracellular drug accumulation. In a reporter gene assay, co-transfection of MDR1 promoter constructs with a CtBP1 expression vector resulted in a ∼2–4-fold induction of MDR1 promoter activity. CtBP1 appeared to contribute to the activation of MDR1 transcription through directly interacting with the MDR1 promoter, as evidenced by its physical binding to the promoter region of the MDR1 gene in chromatin immunoprecipitation and electromobility shift assays. Histone modifications at the MDR1 promoter, such as mono-methylation, di-methylation, and acetylation of histone H3, were not found to be affected by silencing of CtBP1 expression. Our results reveal a novel role for CtBP1 as an activator of MDR1 gene transcription, and suggest that CtBP1 might be one of the key transcription factors involved in the induction of MDR1 gene. Therefore, CtBP1 may represent a potentially new target for inhibiting drug resistance mediated by overexpression of the MDR1 gene.

Keywords: Multidrug resistance; MDR1; gene; P-glycoprotein; CtBP1; Transcription; Cancer

Ca²⁺ extrusion in aged smooth muscle cells by Pedro J. Gomez-Pinilla; Maria J. Pozo; Akemishi Baba; Toshio Matsuda; Pedro J. Camello (pp. 860-869).

We investigated the effects of aging in Ca²⁺ extrusion mechanisms in smooth muscle bladder cells from 4 and 20–24-month-old guinea pigs using fluorescence microscopy and fura-2. Cells were challenged with a pulse of KCl immediately before perfusion with a Ca²⁺ free solution containing no inhibitors (control, untreated cells) or inhibitors of plasma membrane Ca²⁺ pump (PMCA, 1mM La³⁺), Na⁺/Ca²⁺ exchanger (NCX, 1μM SEA0400) or the sarcoendoplasmic Ca²⁺ pump (SERCA, 1μM thapsigargin). Treatment of young adult cells with the inhibitors allowed estimating a relative contribution of 55% for NCX, 27% for PMCA and 31% for SERCA. Combination of two inhibitors at the same time showed the presence of interaction between extrusion mechanisms. In aged cells the [Ca²⁺]_i extrusion was impaired due to decrease of PMCA activity, as revealed by the loss of effect of La³⁺, and to inhibitory interactions between NCX and SERCA activities, indicated by acceleration of decay in response to their respective inhibitors. In conclusion, in smooth muscle cells aging decreases the overall Ca²⁺ extrusion activity and modifies the interactions between the activities of the main Ca²⁺ removing mechanisms.

Keywords: Calcium signals; Smooth muscle; PMCA; NCX; SERCA; Guinea pig

2′,4′,6′-Tris(methoxymethoxy) chalcone protects against trinitrobenzene sulfonic acid-induced colitis and blocks tumor necrosis factor-α-induced intestinal epithelial inflammation via heme oxygenase 1-dependent and independent pathways by Sung Hee Lee; Dong Hwan Sohn; Xing Yu Jin; Sang Wook Kim; Suck Chei Choi; Geom Seog Seo (pp. 870-880).

2′,4′,6′-Tris(methoxymethoxy) chalcone (TMMC), a synthesized chalcone derivative, displays potent antiproliferative and anti-inflammatory effects in rat hepatic stellate cells and murine macrophages, respectively. Here we tested the hypothesis that TMMC could ameliorate diseases characterized by mucosal inflammation. Treatment of mice with TMMC significantly protected against trinitrobenzene sulfonic acid (TNBS)-induced colitis, as assessed by reductions in the weight loss, colonic damage and mucosal ulceration that together characterize this symptom. Moreover, TMMC suppressed the expression of intercellular adhesion molecule-1, interleukin 1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the mice treated with TNBS. Pretreatment of human intestinal epithelial HT-29 cells with TMMC also significantly inhibited the IL-8 and extracellular matrix metalloproteinase-7 levels induced by TNF-α. TMMC induced the expression of heme oxygenase 1 (HO-1) in HT-29 cells. TMMC increased extracellular signal-regulated kinase1/2 and p38 kinase phosphorylation levels, which led to the nuclear translocation of nuclear factor-erythroid 2-related factor 2 (Nrf2) and consequently to HO-1 expression. TMMC inhibited TNF-α-induced nuclear factor κB (NF-κB) activation directly and indirectly. Interestingly, the latter is mediated by HO-1, which presumably blocks the TNF-α-induced nuclear translocation of NF-κB p65 without affecting I-κBα degradation. Moreover, we found that the different products of HO-1, carbon monoxide and bilirubin, exerted anti-inflammatory effects that were additive or synergistic in HT-29 cells stimulated with TNF-α. Thus, TMMC might serve to protect against intestinal inflammatory diseases.

Keywords: Abbreviations; CO; carbon monoxide; Erk1/2; extracellular signal-regulated kinase1/2; HO-1; heme oxygenase; IBD; inflammatory bowel disease; IEC; intestinal epithelial cell; MAPK; mitogen-activated protein kinase; MMP; matrix matalloproteinase; NF-κB; nuclear factor κB; Nrf2; nuclear factor-erythroid 2-related factor 2; SnPP; Tin protoporphyrin; TMMC; 2′,4′,6′-tris(methoxymethoxy) chalcone; TNBS; trinitrobenzene sulfonic acid2′,4′,6′-Tris(methoxymethoxy) chalcone; Trinitrobenzene sulfonic acid; Tumor necrosis factor-α; Heme oxygenase 1; Nuclear factor-κB

Analysis of second messenger pathways stimulated by different chemokines acting at the chemokine receptor CCR5 by K. Leach; S.J. Charlton; P.G. Strange (pp. 881-890).

The chemokine receptor, CCR5, responds to several chemokines leading to changes in activity in several signalling pathways. Here, we investigated the ability of different chemokines to provide differential activation of pathways. The effects of five CC chemokines acting at CCR5 were investigated for their ability to inhibit forskolin-stimulated 3′–5′-cyclic adenosine monophosphate (cAMP) accumulation and to stimulate Ca²⁺ mobilisation in Chinese hamster ovary (CHO) cells expressing CCR5. Macrophage inflammatory protein 1α (D26A) (MIP-1α (D26A), CCL3 (D26A)), regulated on activation, normal T-cell expressed and secreted (RANTES, CCL5), MIP-1β (CCL4) and monocyte chemoattractant protein 2 (MCP-2, CCL8) were able to inhibit forskolin-stimulated cAMP accumulation, whilst MCP-4 (CCL13) could not elicit a response. CCL3 (D26A), CCL4, CCL5, CCL8 and CCL13 were able to stimulate Ca²⁺ mobilisation through CCR5, although CCL3 (D26A) and CCL5 exhibited biphasic concentration–response curves. The Ca²⁺ responses induced by CCL4, CCL5, CCL8 and CCL13 were abolished by pertussis toxin, whereas the response to CCL3 (D26A) was only partially inhibited by pertussis toxin, indicating G_i/o-independent signalling induced by this chemokine. Although the rank order of potency of chemokines was similar between the two assays, certain chemokines displayed different pharmacological profiles in cAMP inhibition and Ca²⁺ mobilisation assays. For instance, whilst CCL13 could not inhibit forskolin-stimulated cAMP accumulation, this chemokine was able to induce Ca²⁺ mobilisation via CCR5. It is concluded that different chemokines acting at CCR5 can induce different pharmacological responses, which may account for the broad spectrum of chemokines that can act at CCR5.

Keywords: Abbreviations; ATP; adenosine triphosphate; cAMP; 3′–5′-cyclic adenosine monophosphate; CCR5; CC chemokine receptor 5; CHO; Chinese hamster ovary; ECL; enhanced chemiluminescence; FCS; foetal calf serum; FITC; fluorescein isothiocyanate; FLIPR; fluorimetric imaging plate reader; G; _i/o; guanine nucleotide binding protein type i or o; G; _q; guanine nucleotide binding protein type q; G protein; guanine nucleotide binding protein; [; ³⁵; S]GTPγS; guanosine-5′-; O; -(3-[; ³⁵; S]thio)triphosphate; HBSS; Hank's balanced salt solution; HIV; human immunodeficiency virus; MCP-2/4; monocyte chemoattractant protein 2/4; MIP-1α/β; macrophage inflammatory protein 1α/β; PAGE; polyacrylamide gel electrophoresis; RANTES; regulated on activation, normal T-cell expressed and secretedChemokine receptor CCR5; Signalling; Inhibition of cAMP; Calcium ion release; Chemokines; Rank order

Allosteric effects of antagonists on signalling by the chemokine receptor CCR5 by Ben Haworth; Hong Lin; Mark Fidock; Pat Dorr; Philip G. Strange (pp. 891-897).

Antagonists of the chemokine receptor, CCR5, may provide important new drugs for the treatment of HIV-1. In this study we have examined the mechanism of action of two functional antagonists of the chemokine receptor CCR5 (UK-396,794, UK-438,235) in signalling and internalisation assays using CHO cells expressing CCR5. Both compounds were potent inverse agonists versus agonist-independent [³⁵S]GTPγS binding to membranes of CHO cells expressing CCR5. Both compounds also acted as allosteric inhibitors of CCL5 (RANTES) and CCL8 (MCP-2)-stimulated [³⁵S]GTPγS binding to CHO-CCR5 membranes, reducing the potency and maximal effects of the two chemokines. The data are consistent with effects of the allosteric inhibitors on both the binding and signalling of the chemokines. Both compounds inhibited CCR5 internalisation triggered by chemokines. When CHO-CCR5 cells were treated with either of the two compounds for prolonged periods of time (24h) an increase (∼15%) in cell surface CCR5 was detected.

Keywords: Abbreviations; CHO; cells, Chinese hamster ovary cells; FCS; foetal calf serum; FITC; fluorescein isothiocyanate; GPCR; G protein coupled receptor; GRK; G protein coupled receptor kinase; HIV; human immunodeficiency virus; MCP; monocyte chemoattractant protein; MIP; macrophage inflammatory protein; PBS; phosphate buffered saline; RANTES; regulated on activation, normal T cell expressed and secretedChemokine receptor (CCR5); Chemokines; [; ³⁵; S]GTPγS binding; Antagonists; HIV; Receptor internalisation

Inhibition of HIV replication: A powerful antiviral strategy by IFN-β gene delivery in CD4⁺ cells by Fabienne Brule; Emmanuel Khatissian; Alexandre Benani; Audrey Bodeux; Luc Montagnier; Jacques Piette; Evelyne Lauret; Emmanuel Ravet (pp. 898-910).

In this study, we demonstrated the efficiency and feasibility of a gene therapy protocol against HIV infection using the antiviral effects of IFN-β expression. Lentiviral vectors containing the human or the simian IFN-β sequences under the influence of the murine moderate H2-kb promoter were constructed. To examine the capacity of IFN-β to inhibit the replication of HIV in human CD4⁺ cells, a transduction protocol permitting to efficiently transduce CD4⁺ cells or PBMC (85±12% of CD4⁺-transduced cells) with a moderate expression of IFN-β was developed. Results indicate that enforced expression of IFN-β has no negative effects in terms of apoptosis and proliferation. In human CD4⁺ cells, it drastically inhibits (up to 99.9%) replication after challenging with different strains of HIV-1. The expression of exogenous IFN-β leads to an amplification of the CD4⁺ cells (11-fold) and to a drastic decrease of the p24 protein. Micro-array analyses indicated that antiviral effect of IFN-β could be due to a major regulation of the inflammatory response. These results are encouraging for the development of a clinical study of gene therapy against AIDS using IFN-β.

Keywords: Gene therapy; HIV; Lentiviral vector; IFN-β

Inhibition of osteoclast differentiation and bone resorption by sauchinone by Kyoung-Youn Han; Daum Yang; Eun-Ju Chang; Youngkyun Lee; Hao Huang; Sang Hyun Sung; Zang Hee Lee; Young Choong Kim; Hong-Hee Kim (pp. 911-923).

Osteoclasts are bone-specific multinucleated cells generated by differentiation of monocyte/macrophage lineage precursors. Regulation of osteoclast differentiation is considered an effective therapeutic approach to the treatment of bone-lytic diseases. In this study, we investigated effects of sauchinone, a lignan from Saururus chinensis, on osteoclastogenesis induced by the differentiation factor RANKL (receptor activator of nuclear factor kappa B ligand). Sauchinone strongly inhibited the osteoclastogenesis from primary bone marrow-derived macrophages (BMMs). This effect was accompanied by a significant decrease in the level of carbonic anhydrase II, calcitonin receptor, MMP9, and TRAP, which are normally upregulated during osteoclast differentiation. For the induction of osteoclastogenesis-associated genes, RANKL activates multiple transcription factors through mechanisms involving mitogen-activated protein kinases (MAPK) and reactive oxygen species (ROS). Sauchinone greatly attenuated the activation of ERK and, less prominently, that of p38 MAPKs by RANKL. The RANKL-stimulated induction of c-Fos and NFATc1 transcription factors was also abrogated by sauchinone. In addition, the activation of AP-1, NFAT, and NF-κB transcription factors was alleviated in sauchinone-treated cells. Sauchinone also diminished the RANKL-stimulated increase of ROS production in BMMs. Consistent with the in vitro anti-osteoclastogenic effect, sauchinone inhibited bone destruction and osteoclast formation caused by lipopolysaccharide in an animal model. Taken together, our data demonstrate that sauchinone inhibits RANKL-induced osteoclastogenesis by reducing ROS generation, which attenuates MAPK and NF-κB activation, ultimately leading to the suppression of c-Fos and NFATc1 induction. Also the in vivo effect of sauchinone on bone erosion strengthens the potential usefulness of this compound for diseases involving bone resorption.

Keywords: Abbreviations; BMM; bone marrow-derived macrophage; DCFH-DA; 2′,7′-dichlorofluorescein diacetate; M-CSF; macrophage colony-stimulating factor; NFAT; nuclear factor of activated T cell; RANKL; receptor activator of nuclear factor kappa B ligand; ROS; reactive oxygen species; TRAF6; tumor necrosis factor receptor-associated factor 6; TRAP; tartrate-resistant acid phosphatase; VtD; ₃; Vitamin D; ₃Osteoclast; Sauchinone; Inhibition of differentiation and bone resorption; ERK; ROS production; LPS-induced bone destruction

Involvement of p38^MAPK on the antinociceptive action of myricitrin in mice by Flavia Carla Meotti; Thaís Posser; Fabiana Cristina Missau; Moacir Geraldo Pizzolatti; Rodrigo Bainy Leal; Adair R.S. Santos (pp. 924-931).

Previous studies from our group investigated the analgesic and anti-inflammatory properties of the flavonoid myricitrin. Here, we demonstrated the role of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and mitogen-activated protein kinases (MAPKs) on the antinociceptive action of myricitrin. The nociceptive response was evaluated by monitoring biting behaviour following intratecal (i.t.) administration of IL-1β and TNF-α in mice. Western blot analyses of total and phosphorylated MAPKs: p38^MAPK, extracellular-signal regulated kinase (ERK1/2) and c-Jun amino-terminal kinases (JNK1/2) from the spinal cord of mice injected with cytokines were measured. Myricitrin (0.03–30mg/kg) or vehicle (control) was administered 30min beforehand by intraperitoneal (i.p.) injection. Myricitrin pre-treatment prevented cytokine-induced biting behaviour. The calculated ID₅₀ of myricitrin were 6.8 (4.6–9.0) and 2.6 (0.3–4.9)mg/kg and maximal inhibition of 83±9 and 100±0% for IL-1β and TNF-α, respectively. Intrathecal injection of IL-1β and TNF-α significantly increased p38^MAPK phosphorylation and this was inhibited by myricitrin treatment. Cytokines administration did not alter ERK1/2 and JNK1/2 phosphorylation. Myricitrin prevented cytokine-induced biting behaviour and inhibited p38^MAPK phosphorylation in response to cytokines stimulation. Taken together, it suggests that the mechanism for antinociceptive action of myricitrin in response to cytokines may involve a blockage on p38^MAPK pathway. This finding could explain, at least in part, the antinociceptive action of this flavonoid in process like neuropathic and inflammatory chronic pain.

Keywords: Myricitrin; Nociception; Interleukin-1β; Tumor necrosis factor-α; Extracellular-signal regulated kinase; c-Jun amino-terminal kinases; p38; ^MAPK

Inter-individual variability in esterases in human liver by Christopher Jewell; Phillippa Bennett; Elaine Mutch; Chrisita Ackermann; Faith M. Williams (pp. 932-939).

Human liver has numerous hydrolytic enzymes involved in metabolism of endogenous and exogenous esters. Of these enzymes, carboxylesterases (EC 3.1.1.1) form an important group which hydrolyses many diverse ester substrates, including pro-ester drugs. Carboxylesterase activity was investigated in liver subcellular fractions from 22 individuals using the general carboxylesterase substrate phenylvalerate and the homologous series of esters methyl-, ethyl-, propyl-, butyl- and benzylparaben. The intra- and inter-individual variation in phenylvalerate and paraben metabolism was compared. Rates of hydrolysis were higher in microsomal fractions than cytosolic fractions for all compounds. The rate of paraben hydrolysis varied depending on the size of the paraben alcohol leaving group, showing a decrease with increasing leaving group size. Comparisons showed that individuals with high rates of hydrolysis towards methyl paraben also showed high rates of hydrolysis to the other parabens and phenylvalerate. Phenylvalerate as a non-specific carboxylesterase substrate was hydrolysed mainly by hCE1 in human livers and there was good correlation with small alcohol leaving group parabens, suggesting hCE1 involvement. Lower correlations with larger alcohol leaving group parabens are consistent with more hCE2 involvement.

Keywords: Carboxylesterase; Human; Liver; Metabolism; Parabens

Identification of two herbal compounds with potential cholesterol-lowering activity by Yilin Zhang; Haiqing Zhang; Shineng Hua; Lianghui Ma; Cong Chen; Xiaoyu Liu; Liqun Jiang; Huanming Yang; Peicheng Zhang; Dequan Yu; Yinlong Guo; Xuehai Tan; Jianfeng Liu (pp. 940-947).

Low-density lipoprotein receptor (LDLR) plays a pivotal role in the control of plasma LDL-cholesterol level. This occurs predominantly at the transcriptional level through two gene regulation elements, named SRE: sterol-responsive element and SIRE: sterol-independent responsive element. We have developed a high-throughput screening using LDLR promoter activation-based assay to search for cholesterol-lowering compounds from a Chinese herb-based natural compound library. With this approach, we identified two compounds, named Daphnetoxin and Gniditrin, from Chinese herb Daphne giraldii Nitsche, which could activate LDLR promoter. Characterization of these compounds showed that they increased the level of LDLR mRNA and consequently up-regulate LDLR expression. The structures of these compounds are different from well-known LDLR promoter activating compounds such as GW707. The results suggested that these herbal compounds could represent good candidates for development of new classes of cholesterol-lowering drugs.

Keywords: Abbreviations; LDL; low-density lipoprotein; LDLR; LDL receptor; SRE; sterol-regulatory element; SIRE; sterol-independent regulatory element; HMG-CoA; 3-hydroxy-3-methyglutaryl coenzyme A; Dil-LDL; 3,3′-dioctadecylindocarbocyanine LDL; OM; oncostatin MLDLR; Daphne giraldii Nitsche; Daphnetoxin; Gniditrin; Cholesterol-lowering activity

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Biochemical Pharmacology (v.74, #6)