Check out our New Publishers' Select for Free Articles

Biochemical Pharmacology (v.77, #7)

Editorial Advisory Board (pp. iii).

Adenosine receptor agonists for promotion of dermal wound healing by María D. Valls; Bruce N. Cronstein; M. Carmen Montesinos (pp. 1117-1124).

Wound healing is a dynamic and complex process that involves a well-coordinated, highly regulated series of events including inflammation, tissue formation, revascularization and tissue remodeling. However, this orderly sequence is impaired in certain pathophysiological conditions such as diabetes mellitus, venous insufficiency, chronic glucocorticoid use, aging and malnutrition. Together with proper wound care, promotion of the healing process is the primary objective in the management of chronic poorly healing wounds. Recent studies have demonstrated that A_2A adenosine receptor agonists promote wound healing in normal and diabetic animals and one such agonist, Sonedenoson, is currently being evaluated as a prospective new therapy of diabetic foot ulcers. We will review the mechanisms by which adenosine receptor activation affects the function of the cells and tissues that participate in wound healing, emphasizing the potential beneficial impact of adenosine receptor agonists in diabetic impaired healing.

Keywords: Adenosine receptors; Impaired healing; Inflammation; Diabetic foot ulcer; Angiogenesis; Granulation tissue

The experimental chemotherapeutic N⁶-furfuryladenosine (kinetin-riboside) induces rapid ATP depletion, genotoxic stress, and CDKN1A (p21) upregulation in human cancer cell lines by Christopher M. Cabello; Warner B. Bair III; Stephanie Ley; Sarah D. Lamore; Sara Azimian; Georg T. Wondrak (pp. 1125-1138).

Cytokinins and cytokinin nucleosides are purine derivatives with potential anticancer activity. N⁶-furfuryladenosine (FAdo, kinetin-riboside) displays anti-proliferative and apoptogenic activity against various human cancer cell lines, and FAdo has recently been shown to suppress tumor growth in murine xenograft models of human leukemia and melanoma. In this study, FAdo-induced genotoxicity, stress response gene expression, and cellular ATP depletion were examined as early molecular consequences of FAdo exposure in MiaPaCa-2 pancreas carcinoma, A375 melanoma, and other human cancer cell lines. FAdo, but not adenosine or N⁶-furfuryladenine (FA), displayed potent anti-proliferative activity that was also observed in human primary fibroblasts and keratinocytes. Remarkably, massive ATP depletion and induction of genotoxic stress as assessed by the alkaline comet assay occurred within 60–180min of exposure to low micromolar concentrations of FAdo. This was followed by rapid upregulation of CDKN1A and other DNA damage/stress response genes (HMOX1, DDIT3, and GADD45A) as revealed by expression array and Western analysis. Pharmacological and siRNA-based genetic inhibition of adenosine kinase (ADK) suppressed FAdo cytotoxicity and also prevented ATP depletion and p21 upregulation suggesting the importance of bioconversion of FAdo into the nucleotide form required for drug action. Taken together our data suggest that early induction of genotoxicity and energy crisis are important causative factors involved in FAdo cytotoxicity.

Keywords: Abbreviations; 5′-AdA; 5′-amino-5′-deoxyadenosine; ADK; adenosine kinase; Ado; adenosine; AV; annexinV; CDKN1A; cyclin-dependent kinase inhibitor 1A; DDIT3; DNA-damage-inducible transcript 3; FA; N; ⁶; -furfuryladenine; FAdo; N; ⁶; -furfuryladenosine; FITC; fluorescein isothiocyanate; GADD45A; growth arrest and DNA-damage-inducible, alpha; HMOX1; heme oxygenase 1; PI; propidium iodide; SDS-PAGE; sodium dodecylsulfate polyacrylamide gel electrophoresisKinetin; N; ⁶; -furfuryladenosine; Genotoxic stress; ATP depletion; CDKN1A; Cancer

Apoptosis, autophagy, accelerated senescence and reactive oxygen in the response of human breast tumor cells to Adriamycin by Xu Di; Robert P. Shiu; Irene F. Newsham; David A. Gewirtz (pp. 1139-1150).

Although the primary response to Adriamycin (doxorubicin) in p53 mutant MDA-MB231 and p53 null MCF-7/E6 breast tumor cells is apoptotic cell death, the residual surviving population appears to be in a state of senescence, based on cell morphology, beta galactosidase staining, induction of p21^waf1/cip1 and down regulation of cdc2/cdk1. Suppression of apoptosis in MDA-MB231 and MCF-7/E6 cells treated with Adriamycin using the broad spectrum caspase inhibitor, zvad-Fmk, results in substantial induction of autophagy. Overall sensitivity to Adriamycin, measured by clonogenic survival, is not altered in the cells undergoing autophagy, consistent with autophagy contributing to cell death in response to Adriamycin. The free radical scavengers, glutathione and N-acetyl cysteine attenuate the accelerated senescence response to Adriamycin in MCF-7 cells as well as in MDA-MB231 and MCF-7/E6 cells, but protect primarily the MCF-7 cells, indicating that reactive oxygen is unlikely to be directly responsible for Adriamycin toxicity in breast tumor cells. Expression of caspase 3 or induced expression of c-myc in MCF-7 cells fails to abrogate accelerated senescence induced by Adriamycin. Taken together, these studies suggest that accelerated senescence induced by Adriamycin is similar in cells with wild type p53 and in cells lacking functional p53 with regard to the upregulation of p21^waf1/cip1, down regulation of cdc2 and the involvement of reactive oxygen species. Furthermore, accelerated senescence, autophagy and apoptosis all appear to be effective in suppressing self-renewal capacity in breast tumor cells exposed to Adriamycin.

Keywords: Abbreviations; ADR; Adriamycin (doxorubicin); TUNEL; terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling; GSH; glutathione; NAC; N-acetyl cysteineSenescence; Apoptosis; Autophagy; p53; p21; c-myc; Reactive oxygen

The selective aryl hydrocarbon receptor modulator 6-methyl-1,3,8-trichlorodibenzofuran inhibits prostate tumor metastasis in TRAMP mice by Wayne A. Fritz; Tien-Min Lin; Stephen Safe; Robert W. Moore; Richard E. Peterson (pp. 1151-1160).

The aryl hydrocarbon receptor (AhR) is a basic-helix-loop-helix transcription factor that binds halogenated aromatic hydrocarbons, polycyclic aromatic hydrocarbons, and endogenous compounds. We previously reported that AhR null ( Ahr^−/−) transgenic adenocarcinoma of the mouse prostate (TRAMP) mice on a C57BL/6J background develop prostate tumors with much greater frequency than AhR wild-type ( Ahr^+/+) TRAMP mice, suggesting that the AhR has tumor suppressor properties. Because AhR signaling pathway inactivation increased susceptibility to prostate tumorigenesis, we tested the hypothesis that a selective AhR modulator (SAhRM), 6-methyl-1,3,8-trichlorodibenzofuran (6-MCDF), can protect against prostate tumorigenesis. TRAMP mice on the standard C57BL/6J×FVB genetic background were fed 0, 10, or 40mg 6-MCDF/kg diet beginning at 8 weeks of age. Tumor incidence, pelvic lymph node metastasis, and serum vascular endothelial growth factor (VEGF) concentrations were determined at 140 days of age. Prostate tumor incidence and size were not significantly reduced in mice fed 6-MCDF. However, the frequency of pelvic lymph node metastasis was reduced fivefold in mice fed the 40mg 6-MCDF/kg diet. Serum VEGF concentrations were also reduced by 6-MCDF treatment, particularly in mice without prostate tumors, and 6-MCDF was shown to act directly on cultured prostates to inhibit VEGF secretion. Together, these results suggest that 6-MCDF inhibits metastasis, in part, by inhibiting prostatic VEGF production prior to tumor formation. This is the first report that 6-MCDF can confer protection against prostate cancer in vivo.

Keywords: TRAMP mice; Prostate cancer; VEGF; 6-MCDF; Selective aryl hydrocarbon receptor modulator; Metastasis inhibition

Metabolism-blocked antifolates as potential anti-rheumatoid arthritis agents: 4-Amino-4-deoxy-5,8,10-trideazapteroyl-d,l-4′-methyleneglutamic acid (CH-1504) and its analogs by John J. McGuire; William H. Haile (pp. 1161-1172).

4-Amino-4-deoxy-5,8,10-trideazapteroyl-d,l-4′-methyleneglutamic acid (CH-1504) is the prototype of a potentially therapeutically more selective class of antifolates for rheumatoid arthritis treatment. This class is characterized by retention of dihydrofolate reductase (DHFR; EC 1.5.1.3) as their locus of action and transport by the reduced folate carrier (RFC; SLC19A1), but their lack of metabolism by known pathways of antifolate (e.g., methotrexate (MTX)) metabolism. Five new CH-1504 analogs (CHL-001–CHL-005) were synthesized and diastereomers of CH-1504 itself were obtained by preparative chiral HPLC; all were characterized biochemically. The analogs are not metabolized by aldehyde oxidase (EC 1.2.3.1), carboxypeptidase G2 (EC 3.4.17.11), or (excepting CHL-003) folylpolyglutamate synthetase (EC 6.3.2.17) and thus, unlike MTX, are “metabolism-blocked”. All analogs are potent DHFR inhibitors; several are nearly as potent as MTX or CH-1504. Each analog uses the RFC for transport, although with varying apparent affinities. In contrast, each weakly inhibits other enzymes of folate metabolism relevant to rheumatoid arthritis therapy (thymidylate synthase (EC 2.1.1.45), two formyltransferases of purine biosynthesis (EC 2.1.2.2 and EC 2.1.2.3), and 5,10-methylenetetrahydrofolate reductase (EC 1.5.1.20)). Biochemical characterization showed one 4′-diastereomer of racemic CH-1504 was significantly more active than the other. Based on literature data concerning the effect ofd- andl-glutamic acid substitution on antifolate activity, it is likely that the diastereomer containingl-4′-methylene-glutamic acid is the more active. Because of concern about potential pharmacokinetic and biochemical effects ofd-4′-methylene-glutamic acid-containing species, these data suggest that future analogs should contain onlyl-4′-methylene-glutamic acid. Overall, these data provide several interesting new leads for preclinical development.

Keywords: Antifolate; Rheumatoid arthritis; CH-1504; Methotrexate; Drug metabolism; Structure–activity relationship

Cyclophilin B stimulates RNA synthesis by the HCV RNA dependent RNA polymerase by Julie A. Heck; Xiao Meng; David N. Frick (pp. 1173-1180).

Cyclophilins are cellular peptidyl isomerases that have been implicated in regulating hepatitis C virus (HCV) replication. Cyclophilin B (CypB) is a target of cyclosporin A (CsA), an immunosuppressive drug recently shown to suppress HCV replication in cell culture. Watashi et al. recently demonstrated that CypB is important for efficient HCV replication, and proposed that it mediates the anti-HCV effects of CsA through an interaction with NS5B [Watashi K, Ishii N, Hijikata M, Inoue D, Murata T, Miyanari Y, et al. Cyclophilin B is a functional regulator of hepatitis C virus RNA polymerase. Mol Cell 2005;19:111–22]. We examined the effects of purified CypB proteins on the enzymatic activity of NS5B. Recombinant CypB purified from insect cells directly stimulated NS5B-catalyzed RNA synthesis. CypB increased RNA synthesis by NS5B derived from genotype 1a, 1b, and 2a HCV strains. Stimulation appears to arise from an increase in productive RNA binding. NS5B residue Pro540, a previously proposed target of CypB peptidyl-prolyl isomerase activity, is not required for stimulation of RNA synthesis.

Keywords: RNA dependent RNA polymerase; Peptidyl-prolyl isomerase; Hepatitis C virus; Cyclosporin; Cyclophilin

Dissection of the inhibition of cardiac ryanodine receptors by human glutathione transferase GSTM2-2 by Dan Liu; Ruwani Hewawasam; Suzy M. Pace; Esther M. Gallant; Marco G. Casarotto; Angela F. Dulhunty; Philip G. Board (pp. 1181-1193).

The muscle specific glutathione transferase GSTM2-2 inhibits the activity of cardiac ryanodine receptor (RyR2) calcium release channels with high affinity and activates skeletal RyR (RyR1) channels with lower affinity. To determine which overall region of the GSTM2-2 molecule supports binding to RyR2, we examined the effects of truncating GSTM2-2 on its ability to alter Ca²⁺ release from sarcoplasmic reticulum (SR) vesicles and RyR channel activity. The C-terminal half of GSTM2-2 which lacks the critical GSH binding site supported the inhibition of RyR2, but did not support activation of RyR1. Smaller fragments of GSTM2-2 indicated that the C-terminal helix 6 was crucial for the action of GSTM2-2 on RyR2. Only fragments containing the helix 6 sequence inhibited Ca²⁺ release from cardiac SR. Single RyR2 channels were strongly inhibited by constructs containing the helix 6 sequence in combination with adjacent helices (helices 5–8 or 4–6). Fragments containing helices 5–6 or helix 6 sequences alone had less well-defined effects. Chemical cross-linking indicated that C-terminal helices 5–8 bound to RyR2, but not RyR1. Structural analysis with circular dichroism showed that the helical content was greater in the longer helix 6 containing constructs, while the helix 6 sequence alone had minimal helical structure. Therefore the active centre of GSTM2-2 for inhibition of cardiac RyR2 involves the helix 6 sequence and the helical nature of this region is essential for its efficacy. GSTM2-2 helices 5–8 may provide the basis for RyR2-specific compounds for experimental and therapeutic use.

Keywords: Glutathione transferase GSTM2-2; Cardiac RyR2 channels; Skeletal RyR1 channels; Calcium release from cardiac sarcoplasmic reticulum; Calcium release from skeletal sarcoplasmic reticulum; Lipid bilayer single channel experiments

Glucocorticoids and mitogen- and stress-activated protein kinase 1 inhibitors: Possible partners in the combat against inflammation by Ilse M.E. Beck; Wim Vanden Berghe; Sarah Gerlo; Nadia Bougarne; Linda Vermeulen; Karolien De Bosscher; Guy Haegeman (pp. 1194-1205).

In the combat against inflammation, glucocorticoids (GCs) are a widespread therapeutic. These ligands of the glucocorticoid receptor (GR) inhibit the transactivation of various transcription factors, including nuclear factor-κB (NF-κB), and alter the composition of the pro-inflammatory enhanceosome, culminating in the repression of pro-inflammatory gene expression. However, pharmacological usage of GCs in long-term treatment is burdened with a detrimental side-effect profile. Recently, we discovered that GCs can lower NF-κB transactivation and pro-inflammatory gene expression by abolishing the recruitment of mitogen- and stress-activated protein kinase 1 (MSK1) (EC 2.7.11.1) to pro-inflammatory gene promoters and displacing a significant fraction of MSK1 to the cytoplasm. In our current investigation in L929sA fibroblasts, upon combining GCs and MSK1 inhibitors, we discovered a dose-dependent additive repression of pro-inflammatory gene expression, most likely due to diverse and multilayered repression mechanisms employed by GCs and MSK1 inhibitors. Therefore, the combined application of GCs and MSK1 inhibitors enabled a similar level of repression of pro-inflammatory gene expression, using actually a lower concentration of GCs and MSK1 inhibitors combined than would be necessary when using these inhibitors separately. Although H89 can inhibit both MSK1 and PKA, TNF does not activate PKA (EC 2.7.11.11) and as such PKA inhibition does not mediate H89-instigated repression of TNF-stimulated gene expression. Furthermore, the additional repressive effects of liganded GR and inhibition of MSK1, are not mediated via GR transactivation mechanisms. In conclusion, these results could entail a new therapeutic strategy using lower drug concentrations, potentially leading to a more beneficial side-effect profile.

Keywords: Abbreviations; AP-1; activator protein-1; ATF; activating transcription factor; β-gal; β-galactosidase; CBP; CREB-binding protein; COPD; chronic obstructive pulmonary disease; CREB; cAMP response element-binding protein; CpdA; Compound A; DEX; dexamethasone; DUSP1; dual-specificity phosphatase 1; ERK; extracellular signal-regulated kinase; GC; glucocorticoid; GR; glucocorticoid receptor; GRE; GC response element; HDAC; histone deacetylase; HMGN1; high mobility group nucleosomal binding protein 1; IκB; inhibitor of NF-κB; JNK; c-Jun N-terminal kinase; luc; luciferase; MAPK; mitogen-activated protein kinase; MEF; mouse embryonic fibroblast; MSK; mitogen- and stress-activated protein kinase; NF-κB; nuclear factor-κB; PBMC; peripheral blood mononuclear cell; PKA; protein kinase A; PKB; protein kinase B; P-TEFb; positive transcription elongation factor b; ROCK; Rho-dependent protein kinase; RSK; 90; kDa ribosomal S6 kinase; S6K; 70; kDa ribosomal protein S6 kinase; TNF; tumor necrosis factorGlucocorticoids; GR; MSK1; PKA; NF-κB; Inflammation

Everolimus and sirolimus antagonize tacrolimus based calcineurin inhibition via competition for FK-binding protein 12 by Huub H. van Rossum; Fred P.H.T.M. Romijn; Nico P.M. Smit; Johan W. de Fijter; Johannes van Pelt (pp. 1206-1212).

The calcineurin inhibitors cyclosporin A and tacrolimus and the inhibitors of the mTOR, sirolimus and everolimus bind immunophilins that are required for their immunosuppressive action. In contrast to cyclosporin A, tacrolimus and the mTOR inhibitors (MTIs) share common immunophilins, the FK506-binding proteins (FKBPs). We investigated the immunosuppressive interactions of MTIs on tacrolimus based immune suppression, since insights in immunological drug–drug interactions can be very relevant for optimization of immunosuppressive regimens in allograft transplantation medicine.Isolated peripheral blood mononuclear cells from healthy volunteers were incubated with combinations of MTIs and calcineurin inhibitors and when monitored for calcineurin activity and IL-2 excretion after mitogen stimulation, tacrolimus IC₅₀ concentrations shifted to higher concentrations in the presence of MTIs. This antagonism was absent for cyclosporin A, reproducible for 10 healthy volunteers ( p<0.001) and stronger for sirolimus than for everolimus. When cell lysate was treated with and without MTI, tacrolimus and FKBP12, FKBP12 could increase calcineurin inhibition by tacrolimus and reverse the MTI antagonism for both MTIs. These results demonstrate that FKBP12 can be rate limiting for calcineurin inhibition at high tacrolimus concentrations and that the antagonism of sirolimus and everolimus on tacrolimus based immune suppression is mediated via saturation of FKBP12.

Keywords: Abbreviations; CN; calcineurin; TRL; tacrolimus; mTOR; mammalian target of rapamycin; MTIs; mTOR inhibitors; FKBP; FK506-binding protein; PBMCs; peripheral blood mononuclear cells; CsA; cyclosporin A; SRL; sirolimus; EVL; everolimus; PHA; phytohemagglutinin; PMA; phorbol-12-myristate-13-acetate; RPMI; roswell park memorial institute medium; iFCS; heat inactivated fetal calf serum; IC; ₅₀; half maximal inhibition concentration; CI; confidence interval; E; _max; maximal drug effect; IL; interleukin; SEM; standard error of the meanCalcineurin; Tacrolimus; Everolimus; Sirolimus; Transplantation; FK-binding protein

Fyn kinase is a direct molecular target of delphinidin for the inhibition of cyclooxygenase-2 expression induced by tumor necrosis factor-α by Mun Kyung Hwang; Nam Joo Kang; Yong-Seok Heo; Ki Won Lee; Hyong Joo Lee (pp. 1213-1222).

Tumor necrosis factor (TNF)-α-mediated cyclooxygenase (COX)-2 expression plays key roles in inflammation and tumorigenesis, particularly skin carcinogenesis, and hence targeting the TNF-α-mediated signaling pathway might be a promising strategy for developing chemopreventive agents against skin cancer and other skin disorders. Here we report that Fyn kinase – one of the members of the nonreceptor protein tyrosine kinase family – is involved in TNF-α-induced COX-2 expression, and that delphinidin – a major anthocyanidin present in red wine and berries – inhibits these effects by directly inhibiting Fyn kinase activity. Delphinidin strongly inhibited TNF-α-induced COX-2 expression in JB6 P+ mouse epidermal (JB6 P+) cells, whereas two other major phenolic compounds (resveratrol and gallic acid) did not exert significant inhibitory effects. Delphinidin inhibited the TNF-α-induced phosphorylations of JNK, p38 MAP kinase, Akt, p90RSK, MSK1, and ERK, and subsequently blocked the activation of the eukaryotic transcription factors AP-1 and NF-κB. Kinase and pull-down assay data revealed that delphinidin inhibited Fyn kinase activity and directly bound with Fyn kinase noncompetitively with ATP. By using PP2 (a commercial inhibitor of Fyn kinase) and siRNA-Fyn, we confirmed that Fyn kinase is involved in TNF-α-induced COX-2 expression mainly by down-regulating JNK in JB6 P+ cells. Together these findings suggest that the targeted inhibition of Fyn kinase activity and COX-2 expression by delphinidin contributes to the chemopreventive potential of red wine and berries.

Keywords: Abbreviations; AP-1; activator protein-1; COX-2; cyclooxygenase-2; CREB; cyclic AMP response element binding protein; EGFR; epidermal growth factor receptor; ERK; extracellular signal-regulated kinase; JB6 P+; JB6 P+ mouse epidermal; JNK; c-Jun N-terminal kinase; MAP kinase; mitogen-activated protein kinase; MSK1; mitogen- and stress- activated protein kinase 1; NFAT; nuclear factor of activated T cells; NF-κB; nuclear factor-κB; PI3K; phosphatidylinositol 3-kinase; p90RSK; p90 ribosomal S6 kinase; SFK; Src family kinase; TNF-α; tumor necrosis factor-α; TNFR; TNF receptor; PMA; phorbol 12-myristate 13-acetatec-Jun N-terminal kinase; Cyclooxygenase-2; Delphinidin; Fyn kinase; Tumor necrosis factor-α

Mechanisms underlying the nociceptive responses induced by platelet-activating factor (PAF) in the rat paw by Denise M. Marotta; Robson Costa; Emerson M. Motta; Elizabeth S. Fernandes; Rodrigo Medeiros; Nara L.M. Quintão; Maria M. Campos; João B. Calixto (pp. 1223-1235).

Platelet-activating factor (PAF) is an inflammatory mediator widely known to exert relevant pathophysiological functions. However, the relevance of PAF in nociception has received much less attention. Herein, we have investigated the mechanisms underlying PAF-induced spontaneous nociception and mechanical hypersensitivity in the rat paw. PAF injection (1–30nmol/paw) resulted in a dose-related overt nociception, whilst only the dose of 10nmol/paw produced a significant and time-related mechanical hypersensitivity. Local coinjection of PAF antagonist WEB2086 significantly inhibited both spontaneous nociception and mechanical hypersensitivity. Moreover, the coinjection of the natural IL-1β receptor antagonist (IRA) notably prevented both PAF-induced nociceptive responses, whilst these responses were not altered by anti-TNFα coinjection. Interestingly, pretreatment with the ultrapotent vaniloid agonist resiniferotoxin, coinjection of the TRPV1 receptor antagonist SB366791, or mast cell depletion with compound 48/80 markedly prevented PAF-induced spontaneous nociception. Conversely, PAF-elicited mechanical hypersensitivity was strikingly susceptible to distinct antineutrophil-related strategies, namely the antineutrophil antibody, the selectin blocker fucoidin, the chemokine CXCR2 receptor antagonist SB225002, and the C5a receptor antibody anti-CD88. Notably, the same antineutrophil migration strategies significantly prevented the increase of myeloperoxidase activity induced by PAF. The mechanical hypersensitivity caused by PAF was also prevented by the cyclooxygenase inhibitors indomethacin or celecoxib, and by the selective β₁ adrenergic receptor antagonist atenolol. Collectively, the present results provide consistent evidence indicating that distinct mechanisms are involved in the spontaneous nociception and mechanical hypersensitivity caused by PAF. They also support the concept that selective PAF receptor antagonists might constitute interesting targets for the development of new analgesic drugs.

Keywords: Platelet-activating factor (PAF); Spontaneous nociception; Mechanical hypersensitivity; Leukocytes

Suramin inhibits the CD40–CD154 costimulatory interaction: A possible mechanism for immunosuppressive effects by Emilio Margolles-Clark; M. Caroline Jacques-Silva; Lakshmi Ganesan; Oliver Umland; Norma S. Kenyon; Camillo Ricordi; Per-Olof Berggren; Peter Buchwald (pp. 1236-1245).

Suramin is a symmetric polysulfonated naphthylamine–benzamide urea derivative approved for the treatment of trypanosomiasis and onchocerciasis and a known P2 (ATP/UTP purine receptor) antagonist. Here, we report its ability to inhibit the important CD40–CD154 costimulatory interaction required for T cell activation and the development of an effective immune response. In vitro, it inhibited the binding of both human and murine CD154 (CD40L) to their receptor (CD40) even in the presence of protein-containing media and prevented the CD154-induced proliferation of human B cells as well as the corresponding increase in surface expression of CD86, CD80, CD40, and MHC class II in a concentration-dependent manner. Furthermore, in isolated human islets, it also decreased the CD154-induced release of inflammatory cytokines such as IFN-γ, interleukin-6 (IL-6), and IL-8. Suramin was selected for investigation because it has been reported to be an inhibitor of the interaction of TNF-α with its receptor and CD154 is a member of the TNF-family. However, it turned out to be a considerably, about 30-fold, more effective inhibitor of the CD40–CD154 protein–protein interaction than of the corresponding TNF interaction. Its median inhibitory concentration (IC₅₀≈50μM) is somewhat higher than for the P2-receptor, but well within the range of its therapeutic concentration levels. Suramin shows considerable polypharmacology, but its interference with the positive costimulatory interaction might provide a possible, not yet identified mechanism for its ability to suppress T cell activity and induce immunosuppression, which might also have limited its clinical usefulness in the treatment of AIDS and cancer.

Keywords: Abbreviations; ANOVA; analysis of variance; BrdU; bromodeoxyuridine; BSA; bovine serum albumin; DAPI; 4’,6-diamidino-2-phenylindole; FBS; fetal bovine serum; HRP; horseradish peroxidase; IFN; interferon; MLR; mixed lymphocyte reaction; MHC; major histocompatibility complex; PBS; phosphate buffered saline; PPI; protein–protein interaction; RT; room temperature; TNF; tumor necrosis factorSuramin; Immunosuppression; Costimulation; CD40 ligand; Protein–protein interaction

Identification of single nucleotide polymorphisms of the human metabotropic glutamate receptor 1 gene and pharmacological characterization of a P993S variant by Patrick M. Downey; Roberta Petrò; Jason S. Simon; David Devlin; Gianluca Lozza; Alessio Veltri; Massimiliano Beltramo; Rosalia Bertorelli; Angelo Reggiani (pp. 1246-1253).

mGluR1 receptors are believed to play major roles in the pathophysiology of diseases such as anxiety and chronic pain and are being actively investigated as targets for drug development. Sequence polymorphisms can potentially influence the efficacy of drugs in patient populations and are therefore an important consideration in the drug development process. To identify DNA sequence variants of the mGluR1 receptor, comparative DNA sequencing was performed on DNA samples ( n=186) from apparently healthy subjects representing two ethnic groups. In total, eight non-synonymous single nucleotide polymorphisms (SNPs) were identified and one SNP (c2977>T) was found to be particularly common, this SNP results in a proline to serine substitution at residue 993 (P993S). The WT (P993) and S993 variants were expressed in an inducible system which allowed us to titrate gene expression to equivalent levels and were pharmacologically characterized. We determined the potency and affinity of standard antagonist compounds as well as the potency and efficacy of the endogenous ligand glutamate and other agonist compounds at both receptor variants. Agonist evoked increases in intracellular Ca²⁺ were measured by fluorometric imaging plate reader (FLIPR). The potency of mGluR1 antagonists was evaluated by their ability to inhibit quisqualate induced increases in intracellular Ca²⁺, while their affinities were determined by radio-ligand binding studies. This study demonstrates that the Pro993Ser amino acid exchange is highly frequent in the human mGluR1 gene. This polymorphism however, does not appear to affect the potency of agonist compounds or the potencies or affinities of small molecule antagonist compounds.

Keywords: Metabotropic glutamate receptors; Polymorphism; Pharmacogenetics; Chronic pain

Curcumin-induced degradation of PKCδ is associated with enhanced dentate NCAM PSA expression and spatial learning in adult and aged Wistar rats by Lisa Conboy; Andrew G. Foley; Noel M. O’Boyle; Marie Lawlor; Helen C. Gallagher; Keith J. Murphy; Ciaran M. Regan (pp. 1254-1265).

Polysialylation of the neural cell adhesion molecule (NCAM PSA) is necessary for the consolidation processes of hippocampus-based learning. Previously, we have found inhibition of protein kinase C delta (PKCδ) to be associated with increased polysialyltransferase (PST) activity, suggesting inhibitors of this kinase might ameliorate cognitive deficits. Using a rottlerin template, a drug previously considered an inhibitor of PKCδ, we searched the Compounds Available for Purchase (CAP) database with the Accelrys^® Catalyst programme for structurally similar molecules and, using the available crystal structure of the phorbol-binding domain of PKCδ, found that diferuloylmethane (curcumin) docked effectively into the phorbol site. Curcumin increased NCAM PSA expression in cultured neuro-2A neuroblastoma cells and this was inversely related to PKCδ protein expression. Curcumin did not directly inhibit PKCδ activity but formed a tight complex with the enzyme. With increasing doses of curcumin, the Tyr¹³¹ residue of PKCδ, which is known to direct its degradation, became progressively phosphorylated and this was associated with numerous Tyr¹³¹-phospho-PKCδ fragments. Chronic administration of curcumin in vivo also increased the frequency of polysialylated cells in the dentate infragranular zone and significantly improved the acquisition and consolidation of a water maze spatial learning paradigm in both adult and aged cohorts of Wistar rats. These results further confirm the role of PKCδ in regulating PST and NCAM PSA expression and provide evidence that drug modulation of this system enhances the process of memory consolidation.

Keywords: Abbreviations; DMEM; Dulbecco's modified Eagle's medium; NCAM; neural cell adhesion molecule; PKC; protein kinase C; PSA; poly-α2,8-linked sialic acid; PST; polysialyltransferase; curcumin; ([diferuloylmethane] 1,7-bis-[4-hydroxy-3-methoxyphenyl]-1,6-heptadiene-3,5-dione); rottlerin; ([mallotoxin] 5,7-dihydroxy-2,2-dimethyl-6-[2,4,6-trihydroxy-3-methyl-5-acetylbenzyl]-8-cinnamoyl-1,2-chromine)PKCδ; PST; NCAM PSA; Curcumin; Spatial learning

Role of carboxylesterase 1 and impact of natural genetic variants on the hydrolysis of trandolapril by Hao-Jie Zhu; David I. Appel; Julie A. Johnson; Kenneth D. Chavin; John S. Markowitz (pp. 1266-1272).

Carboxylesterase 1 (CES1) and carboxylesterase 2 (CES2) are the major hydrolytic enzymes responsible for the metabolism of numerous therapeutic agents as well as endogenous substrates. CES1 and CES2 differ distinctly in their substrate specificity and tissue distribution. In this study, we investigated the role of CES1 and CES2 in converting the antihypertensive prodrug trandolapril to its more active form trandolaprilat, and determined the influence of two newly identified CES1 mutations p.Gly143Glu and p.Asp260fs on trandolapril metabolism. Western blot analysis demonstrated that CES1 is expressed in human liver microsomes (HLM) but not in human intestinal microsomes (HIM). In vitro incubation studies were conducted to contrast the enzymatic activity of HLM as well as HIM upon trandolapril hydrolysis. Trandolapril was rapidly hydrolyzed to its principal active metabolite trandolaprilat after incubation with HLM. In contrast, in HIM, where CES2 is predominantly expressed, incubations did not produce any detectable trandolapril hydrolysis. Furthermore, hydrolysis of trandolapril catalyzed by wild type (WT) and mutant CES1 were assessed utilizing transfected Flp-In-293™ cells stably expressing WT CES1 and two variants. WT CES1 efficiently hydrolyzed trandolapril to trandolaprilat with V_max and K_m values of 103.6±2.2 nmole/min/mg protein and 639.9±32.9μM, respectively. However, no appreciable trandolapril hydrolysis could be found after incubation with both p.Gly143Glu and p.Asp260fs variants. Thus, trandolapril appears to be a CES1 selective substrate while CES2 exerts little to no catalytic activity towards this compound. CES1 mutations p.Gly143Glu and p.Asp260fs are essentially dysfunctional enzymes with regard to the conversion of trandolapril to its more active metabolite trandolaprilat.

Keywords: Carboxylesterase 1; Carboxylesterase 2; Prodrug activation; Genetic polymorphism; Trandolapril; Trandolaprilat

Oleanolic acid activates Nrf2 and protects from acetaminophen hepatotoxicity via Nrf2-dependent and Nrf2-independent processes by Scott A. Reisman; Lauren M. Aleksunes; Curtis D. Klaassen (pp. 1273-1282).

Oleanolic acid is a plant-derived triterpenoid, which protects against various hepatotoxicants in rodents. In order to determine whether oleanolic acid activates nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor known to induce various antioxidant and cytoprotective genes, wild-type and Nrf2-null mice were treated with oleanolic acid (90mg/kg, i.p.) once daily for 3 days. Oleanolic acid increased nuclear accumulation of Nrf2 in wild-type but not Nrf2-null mice, as determined by Western blot and immunofluorescence. Oleanolic acid-treated wild-type mice had increased hepatic mRNA expression of the Nrf2 target genes NAD(P)H:quinone oxidoreductase 1 (Nqo1); glutamate–cysteine ligase, catalytic subunit (Gclc); heme oxygenase-1 (Ho-1); as well as Nrf2 itself. In addition, oleanolic acid increased protein expression and enzyme activity of the prototypical Nrf2 target gene, Nqo1, in wild-type, but not in Nrf2-null mice. Oleanolic acid protected against acetaminophen hepatotoxicity in wild-type mice but to a lesser extent in Nrf2-null mice. Oleanolic acid-mediated Nrf2-independent protection from acetaminophen is, in part, due to induction of Nrf2-independent cytoprotective genes, such as metallothionein. Collectively, the present study demonstrates that oleanolic acid facilitates Nrf2 nuclear accumulation, causing induction of Nrf2-dependent genes, which contributes to protection from acetaminophen hepatotoxicity.

Keywords: Abbreviations; ALT; alanine transaminase; ARE; antioxidant response element; ATF-4; activating transcription factor-4; Cul3; Cullin3; Gclc; glutamate cysteine ligase catalytic subunit; Ho-1; heme oxygenase-1; MT; metallothionein; Nrf2; nuclear factor-erythroid 2-related factor 2; Keap1; kelch-like ECH-associated protein 1; Maf; musculo-aponeurotic fibrosarcoma; Nqo1; NAD(P)H:quinone oxidoreductase 1; NAPQI; N; -acetyl-; p; -benzoquinoneimine; OA; oleanolic acidNrf2; Oleanolic acid; Hepatoprotection; Oxidative stress; Acetaminophen

Carbon tetrachloride-induced liver damage in asialoglycoprotein receptor-deficient mice by Shana R. Dalton; Serene M.L. Lee; Rachel N. King; Amin A. Nanji; Kusum K. Kharbanda; Carol A. Casey; Benita L. McVicker (pp. 1283-1290).

The asialoglycoprotein (ASGP) receptor is an abundant hepatocyte-specific receptor involved in receptor-mediated endocytosis. This receptor's abundance and function is decreased by chronic ethanol administration prior to the appearance of pathology such as necrosis or inflammation. Hence, this study aimed to determine if ASGP receptor function is required to protect against liver injury by utilizing a knockout mouse model lacking functional ASGP receptor in the setting of carbon tetrachloride (CCl₄) hepatotoxicity. Briefly, ASGP receptor-deficient (RD) mice and wild-type (WT) mice were injected with 1ml/kg body weight of CCl₄. In the subsequent week, mice were monitored for liver damage and pathology (aspartate transaminase (AST), alanine transaminase (ALT) and light microscopy). The consequences of CCl₄ injection were examined by measuring α-smooth muscle actin (α-SMA) deposition, contents of malondialdehyde and the percentage of apoptotic hepatocytes. After CCl₄ injection, RD mice showed increased liver pathology together with significantly increased activities of AST and ALT compared to that in WT mice. There were also significantly more apoptotic bodies, lipid peroxidation and deposition of α-SMA in RD mice versus WT mice following CCl₄ injection. Since these two mouse strains only differ in whether or not they have the ASGP receptor, it can be concluded that proper ASGP receptor function exerted a protective effect against CCl₄ toxicity. Thus, receptor-mediated endocytosis by the ASGP receptor could represent a novel molecular mechanism that is responsible for subsequent liver health or injury.

Keywords: Abbreviations; ASGP; asialoglycoprotein; CCl; ₄; carbon tetrachloride; RD; asialoglycoprotein receptor-deficient; WT; wild-type; AST; aspartate transaminase; ALT; alanine transaminase; α-SMA; α-smooth muscle actinCarbon tetrachloride; Asialoglycoprotein receptor; Asialoglycoprotein receptor knockout; Liver injury; Receptor-mediated endocytosis

The NFκB-mediated control of RS and JNK signaling in vitamin A-treated cells: Duration of JNK–AP-1 pathway activation may determine cell death or proliferation by Alfeu Zanotto-Filho; Daniel P. Gelain; Rafael Schröder; Luís F. Souza; Matheus A.B. Pasquali; Fábio Klamt; José Cláudio F. Moreira (pp. 1291-1301).

Nuclear factor kappa B (NFκB) has emerged as a crucial regulator of cell survival, playing important functions in cellular resistance to oxidants and chemotherapeutic agents. Recent studies showed that NFκB mediates cell survival through suppression of the accumulation of reactive species (RS) and a control of sustained activation of the Jun-N-terminal kinase (JNK) cascade. This work was undertaken in order to evaluate the role of NFκB in modulating the pro-oxidant effects of supplementation with vitamin A (retinol, ROH) in Sertoli cells, a major ROH physiological target. In this work, we reported that ROH treatment increases mitochondrial RS formation leading to a redox-dependent activation of NFκB. NFκB activation played a pivotal role in counteract RS accumulation in ROH-treated cells, since NFκB inhibition with DNA decoy oligonucleotides or pharmacological inhibitors (BAY-117082) potentiated ROH-induced RS accumulation and oxidative damage. In the presence of NFκB inhibition, ROH-induced oxidative stress promoted a prolonged activation of the JNK–activator protein 1 (AP-1) pathway and induced significant decreases in cell viability. Inhibition of JNK–AP-1 with decoy oligonucleotides to AP-1 or JNK inhibitor SP600125 prevented the decreases in cell viability. Antioxidants blocked the persistent JNK–AP-1 activation, cell oxidative damage, and the decreases in cell viability induced by NFκB inhibition. Finally, our data point superoxide dismutase (SOD)2 as a potential antioxidant factor involved in NFκB protective effects against ROH-induced oxidative stress. Taken together, data presented here show that NFκB mediates cellular resistance to the pro-oxidant effects of vitamin A by inhibiting RS accumulation and the persistent and redox-dependent activation of JNK–AP-1 cascade.

Keywords: Retinol; NFκB; Oxidative stress; JNK1/2; AP-1

Sections

Personal tools

Biochemical Pharmacology (v.77, #7)

Sections

Personal tools

Local resources

Biochemical Pharmacology (v.77, #7)