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Biochemical Pharmacology (v.77, #3)

Editorial Advisory Board (pp. iii).

Catalase and glutathione peroxidase mimics by Brian J. Day (pp. 285-296).

Overproduction of the reactive oxygen species (ROS) superoxide (O₂⁻) and hydrogen peroxide (H₂O₂) are increasingly implicated in human disease and aging. ROS are also being explored as important modulating agents in a number of cell signaling pathways. Earlier work has focused on development of small catalytic scavengers of O₂⁻, commonly referred to as superoxide dismutase (SOD) mimetics. Many of these compounds also have substantial abilities to catalytically scavenge H₂O₂ and peroxynitrite (ONOO⁻). Peroxides have been increasingly shown to disrupt cell signaling cascades associated with excessive inflammation associated with a wide variety of human diseases. Early studies with enzymatic scavengers like SOD frequently reported little or no beneficial effect in biologic models unless SOD was combined with catalase or a peroxidase. Increasing attention has been devoted to developing catalase or peroxidase mimetics as a way to treat overt inflammation associated with the pathophysiology of many human disorders. This review will focus on recent development of catalytic scavengers of peroxides and their potential use as therapeutic agents for pulmonary, cardiovascular, neurodegenerative and inflammatory disorders.

Keywords: Abbreviations; AP-1; activator protein 1; AD; Alzheimer’s disease; ALS; amyotrophic lateral sclerosis; ARDS; acute respiratory distress syndrome; BPD; bronchopulmonary dysplasia; COPD; chronic obstructive pulmonary disease; CD; cyclodextran; DHLA; dihydrolipoate; DPDS; diphenyl diselenide; GSH; glutathione; GSSG; glutathione disulfide; GPx; glutathione peroxidase; HD; Huntington’s disease; H; ₂; O; ₂; hydrogen peroxide; HO; hydroxyl radical; HOCl; hypochlorous acid; IR; ischemia-reperfusion; LOOH; lipid peroxide; MnTBAP; manganese(III) tetrakis (4-benzoic acid) porphyrin; MPTP; 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; NAC; N-acetylcysteine; NADPH; β-nicotinamide adenine dinucleotide phosphate; NO; nitric oxide; O; ₂; oxygen; PD; Parkinson’s disease; ROOH; peroxides; Prx; peroxiredoxin; ONOO; ⁻; peroxynitrite; RNS; reactive nitrogen species; ROS; reactive oxygen species; O; ₂; ⁻; superoxide; SOD; superoxide dismutase; TAA; tetraaza macrocycle; Trx; thioredoxin; H; ₂; O; waterCatalytic antioxidants; Cell signaling; Drug development; Hydrogen peroxide; Inflammation; Oxidative stress

Targeting RET for thyroid cancer therapy by Cinzia Lanzi; Giuliana Cassinelli; Valentina Nicolini; Franco Zunino (pp. 297-309).

The limited efficacy of conventional treatments in progressive thyroid carcinomas indicates the need for new therapeutic options. Activating mutations of the receptor tyrosine kinase-encoding RET gene have been identified as driving oncogenic events in subsets of papillary (PTC) and medullary (MTC) thyroid carcinomas suggesting the interest of targeted therapy. The role of RET oncogenes and the encoded constitutively active oncoproteins as potential targets has been investigated by different strategies including gene therapy and pharmacological approaches, but targeted treatment for RET-driven cancers is not clinically available in current therapy. Small molecule tyrosine kinase inhibitors, including sorafenib, sunitinib, motesanib and vandetanib, which have already shown efficacy against other neoplastic diseases, are being evaluated in clinical trials for treatment of thyroid carcinomas. Most of them, also described as Ret inhibitors, are multi-kinase inhibitors with antiangiogenic activity related to inhibition of receptor tyrosine kinases, such as the vascular endothelial growth factor receptors. Preclinical evidence supports the relevance of Ret oncoproteins as therapeutic targets for a subset of thyroid neoplastic diseases and, although targeting the original causal genetic change may not be sufficient to control the disease efficiently, the available knowledge outlines therapeutic opportunities for exploiting Ret inhibition.

Keywords: Abbreviations; Cys/C; cysteine; DTC; differentiated thyroid cancer; EGF-R; epidermal growth factor receptor; Erk; extracellular signal-regulated kinase; FAK; focal adhesion kinase; FMTC; familial medullary thyroid carcinoma; GDNF; glial cell line-derived neurotrophic factor; GFRα; GDNF-family receptor α; Hsp90; heat shock protein 90; JNK; c-jun NH2-terminal protein kinase; MEN2; multiple endocrine neoplasia type 2; MTC; medullary thyroid carcinoma; PDGF-R; platelet-derived growth factor receptor; PLC-γ; phospholipase C-γ; PR; partial response; PTC; papillary thyroid carcinoma; RET; rearranged during trasfection; RTK; receptor tyrosine kinase; SD; stable disease; Shc; Src homology 2 domain-containing-trasforming protein C; TK; tyrosine kinase; TKI; tyrosine kinase inhibitor; Tyr/Y; tyrosine; VEGF; vascular endothelial growth factor; VEGF-R; vascular endothelial growth factor receptorRet; Thyroid carcinoma; Tyrosine kinase inhibitor; Protein tyrosine kinase; Targeted therapy

The novel pyrrolo-1,5-benzoxazepine, PBOX-21, potentiates the apoptotic efficacy of STI571 (imatinib mesylate) in human chronic myeloid leukaemia cells by Sandra A. Bright; Lisa M. Greene; Tom F. Greene; Giuseppe Campiani; Stefania Butini; Margherita Brindisi; Mark Lawler; Mary J. Meegan; D. Clive Williams; Daniela M. Zisterer (pp. 310-321).

The Bcr-Abl kinase inhibitor, STI571, is the first line treatment for chronic myeloid leukaemia (CML), but the recent emergence of STI571 resistance has led to the examination of combination therapies. In this report, we describe how a novel non-toxic G1-arresting compound, pyrrolo-1,5-benzoxazepine (PBOX)-21, potentiates the apoptotic ability of STI571 in Bcr-Abl-positive CML cells. Co-treatment of CML cells with PBOX-21 and STI571 induced more apoptosis than either drug alone in parental (K562S and LAMA84) and STI571-resistant cells lines (K562R). This potentiation of apoptosis was specific to Bcr-Abl-positive leukaemia cells with no effect observed on Bcr-Abl-negative HL-60 acute myeloid leukaemia cells. Apoptosis induced by PBOX-21/STI571 resulted in activation of caspase-8, cleavage of PARP and Bcl-2, upregulation of the pro-apoptotic protein Bim and a downregulation of Bcr-Abl. Repression of proteins involved in Bcr-Abl transformation, the anti-apoptotic proteins Mcl-1 and Bcl-_XL was also observed. The combined lack of an early change in mitochondrial membrane potential, release of cytochrome c and cleavage of pro-caspase-9 suggests that this pathway is not involved in the initiation of apoptosis by PBOX-21/STI571. Apoptosis was significantly reduced following pre-treatment with either the general caspase inhibitor Boc-FMK or the chymotrypsin-like serine protease inhibitor TPCK, but was completely abrogated following pre-treatment with a combination of these inhibitors. This demonstrates the important role for each of these protease families in this apoptotic pathway. In conclusion, our data highlights the potential of PBOX-21 in combination with STI571 as an effective therapy against CML.

Keywords: Abbreviations; Abl; abelson; Bcl-2; B-cell leukaemia/lymphoma 2; Bcr; breakpoint cluster region; CI; combination index; CML; chronic myeloid leukaemia; DiOC; ₆; 3,3′-dihexyloxacarbocyanine iodide; DTT; dithiothreitol; FACS; fluorescent activated cell sorting analysis; FBS; foetal bovine serum; Mcl-1; myeloid leukaemia cell differentiation protein-1; MTA; microtubule targeting agents; PARP; poly (ADP-ribose) polymerase; PBOX; pyrrolo-1,5-benzoxazepine; STAT; signal transducer and activator transcription factor; STI571; signal transducer inhibitor-571; TLCK; N; _α; -Tosyl-; l; -lysine chloromethyl ketone hydrochloride; TPCK; N-; p; -Tosyl-; l; -phenylalanine chloromethyl ketonePBOX; STI571; CML; Apoptosis; Serine proteases

Accumulation of artemisinin trioxane derivatives within neutral lipids of Plasmodium falciparum malaria parasites is endoperoxide-dependent by Carmony L. Hartwig; Andrew S. Rosenthal; John D’Angelo; Carol E. Griffin; Gary H. Posner; Roland A. Cooper (pp. 322-336).

The antimalarial trioxanes, exemplified by the naturally occurring sesquiterpene lactone artemisinin and its semi-synthetic derivatives, contain an endoperoxide pharmacophore that lends tremendous potency against Plasmodium parasites. Despite decades of research, their mechanism of action remains unresolved. A leading model of anti-plasmodial activity hypothesizes that iron-mediated cleavage of the endoperoxide bridge generates cytotoxic drug metabolites capable of damaging cellular macromolecules. To probe the malarial targets of the endoperoxide drugs, we studied the distribution of fluorescent dansyl trioxane derivatives in living, intraerythrocytic-stage Plasmodium falciparum parasites using microscopic imaging. The fluorescent trioxanes rapidly accumulated in parasitized erythrocytes, localizing within digestive vacuole-associated neutral lipid bodies of trophozoites and schizonts, and surrounding the developing merozoite membranes. Artemisinin pre-treatment significantly reduced fluorescent labeling of neutral lipid bodies, while iron chelation increased non-specific cytoplasmic localization. To further explore the effects of endoperoxides on cellular lipids, we used an oxidation-sensitive BODIPY lipid probe to show the presence of artemisinin-induced peroxyl radicals in parasite membranes. Lipid extracts from artemisinin-exposed parasites contained increased amounts of free fatty acids and a novel cholesteryl ester . The cellular accumulation patterns and effects on lipids were entirely endoperoxide-dependent, as inactive dioxolane analogs lacking the endoperoxide moiety failed to label neutral lipid bodies or induce oxidative membrane damage. In the parasite digestive vacuole, neutral lipids closely associate with heme and promote hemozoin formation. We propose that the trioxane artemisinin and its derivatives are activated by heme-iron within the neutral lipid environment where they initiate oxidation reactions that damage parasite membranes.

Keywords: Digestive vacuole; Heme; Reactive oxygen species; Lipid peroxidation; Free radicals

trans-Resveratrol inhibits H₂O₂-induced adenocarcinoma gastric cells proliferation via inactivation of MEK1/2-ERK1/2-c-Jun signalling axis by Katia Aquilano; Sara Baldelli; Giuseppe Rotilio; Maria Rosa Ciriolo (pp. 337-347).

In this report we investigate the signalling pathway activated by H₂O₂ in human adenocarcinoma gastric cells (AGS) and we evaluate the anti-proliferative action of the natural stilbene trans-resveratrol. We demonstrate that H₂O₂ accelerates cell growth and induces a prompt MEK1/2-ERK1/2 activation. Such events are also associated with the activation of c-Jun and its translocation into the nuclear compartment. A specific inhibitor of ERK1/2 phosphorylation by MEK1/2 (U0126) abrogates these phenomena. On the contrary, specific inhibition of JNK activity does not influence H₂O₂-mediated growth, suggesting that cell proliferation likely proceeds via MEK1/2-ERK1/2-Jun signalling axis. trans-Resveratrol is also able to completely suppress the increase in proliferation. We demonstrate that this property is not due to its antioxidant capacity but rather due to a specific inhibition of ERK1/2 phosphorylation by MEK1/2 and repression of c-Jun activation.

Keywords: Hydrogen peroxide; ERK1/2; Resveratrol; c-Jun; Cell proliferation

Novel derivatives of spirohydantoin induce growth inhibition followed by apoptosis in leukemia cells by Kavitha C.V.; Mridula Nambiar; Ananda Kumar C.S.; Bibha Choudhary; Muniyappa K.; Kanchugarakoppal S. Rangappa; Sathees C. Raghavan (pp. 348-363).

Hydantoin derivatives possess a variety of biochemical and pharmacological properties and consequently are used to treat many human diseases. However, there are only few studies focusing on their potential as cancer therapeutic agents. In the present study, we have examined anticancer properties of two novel spirohydantoin compounds, 8-(3,4-difluorobenzyl)-1′-(pent-4-enyl)-8-azaspiro[bicyclo[3.2.1] octane-3,4′-imidazolidine]-2′,5′-dione (DFH) and 8-(3,4-dichlorobenzyl)-1′-(pent-4-enyl)-8-azaspiro[bicyclo[3.2.1]octane-3,4′-imidazolidine]-2′,5′-dione (DCH). Both the compounds exhibited dose- and time-dependent cytotoxic effect on human leukemic cell lines, K562, Reh, CEM and 8E5. Incorporation of tritiated thymidine ([³H] thymidine) in conjunction with cell cycle analysis suggested that DFH and DCH inhibited the growth of leukemic cells. Downregulation of PCNA and p-histone H3 further confirm that the growth inhibition could be at the level of DNA replication. Flow cytometric analysis indicated the accumulation of cells at subG1 phase suggesting induction of apoptosis, which was further confirmed and quantified both by fluorescence-activated cell sorting (FACS) and confocal microscopy following annexin V-FITC/propidium iodide (PI) staining. Mechanistically, our data support the induction of apoptosis by activation of the mitochondrial pathway. Results supporting such a model include, elevated levels of p53, and BAD, decreased level of BCL2, activation and cleavage of caspase 9, activation of procaspase 3, poly (ADP-ribosyl) polymerase (PARP) cleavage, downregulation of Ku70, Ku80 and DNA fragmentation. Based on these results we discuss the mechanism of apoptosis induced by DFH and its implications in leukemia therapy.

Keywords: Chemotherapy; Double-strand break; Cytotoxicity; Cell death; Anticancer drugs; Flow cytometry; DNA damage

DNA interactions of dinuclear Ru^II arene antitumor complexes in cell-free media by Olga Nováková; Alexey A. Nazarov; Christian G. Hartinger; Bernhard K. Keppler; Viktor Brabec (pp. 364-374).

We recently synthesized and characterized water-soluble dinuclear Ru^II arene complexes, in which two {( η⁶-p-isopropyltoluene)RuCl[3-(oxo-κO)-2-methyl-4-pyridinonato-κO₄]} units were linked by flexible chains of different length [(CH₂)_n ( n=4, 6, 8, 12)]. These new dinuclear ruthenium drugs were found to exert promising cytotoxic effects in human cancer cells. In the present work DNA modifications by these new dinuclear Ru^II arene compounds, which differed in the length of the linker between the two Ru^II centers, were examined by biochemical and biophysical methods. The complexes bind DNA forming intrastrand and interstrand cross-links in one DNA molecule in the absence of proteins. An intriguing aspect of the DNA-binding mode of these dinuclear Ru^II compounds is that they can cross-link two DNA duplexes and also proteins to DNA—a feature not observed for other antitumor ruthenium complexes. Thus, the concept for the design of interhelical and DNA–protein cross-linking agents based on dinuclear Ru^II arene complexes with sufficiently long linkers between two Ru centers may result in new compounds which exhibit a variety of biological effects and can be also useful in nucleic acids research.

Keywords: Abbreviations; bp; base pair; CD; circular dichroism; cisplatin; cis; -diamminedichloridoplatinum(II); CL; cross-link; CT; calf thymus; DMS; dimethyl sulphate; EtBr; ethidium bromide; FAAS; flameless atomic absorption spectrometry; IC; ₅₀; the concentration of the compound that afforded 50% cell killing; ICD; induced circular dichroism; KF; ⁻; Klenow fragment from DNA polymerase I, exonuclease minus mutated to remove the 3′; →; 5′ proofreading activity; [PtCl(dien)]Cl; chloridodiethylenetriamineplatinum(II) chloride; r; _b; the number of molecules of the metal-based compound bound per nucleotide residue; r; _i; the molar ratio of free metal complex to nucleotides at the onset of incubation with DNA; Ru(4); 1,4-bis{chlorido[3-(oxo-κO)-2-methyl-4-pyridinonato-κO; ₄; ](; η; ⁶; -p-isopropyltoluene)ruthenium}butane; Ru(6); 1,6-bis{chlorido[3-(oxo-κO)-2-methyl-4-pyridinonato-κO; ₄; ](; η; ⁶; -p-isopropyltoluene)ruthenium}hexane; Ru(8); 1,8-bis{chlorido[3-(oxo-κO)-2-methyl-4-pyridinonato-κO; ₄; ](; η; ⁶; -p-isopropyltoluene)ruthenium}octane; Ru(12); 1,12-bis{chlorido[3-(oxo-κO)-2-methyl-4-pyridinonato-κO; ₄; ](; η; ⁶; -p-isopropyltoluene)ruthenium}dodecane; SDS; sodium dodecyl sulphateDinuclear ruthenium complex; Arene; Antitumor; DNA; Cross-links; Cytotoxicity

Hsp90 cleavage by an oxidative stress leads to its client proteins degradation and cancer cell death by Raphael Beck; Julien Verrax; Thomas Gonze; Marianne Zappone; Rozangela Curi Pedrosa; Henryk Taper; Olivier Feron; Pedro Buc Calderon (pp. 375-383).

The heat shock protein 90 (Hsp90) plays a crucial role in the stability of several proteins that are essential for malignant transformation. Hsp90 is therefore an interesting therapeutic target for cancer therapy. In this paper, we investigated whether an oxidative stress generated during ascorbate-driven menadione redox cycling (ascorbate/menadione), affects Hsp90 leading to the degradation of some critical proteins and cell death. Unlike 17-AAG, which inhibits Hsp90 but enhances Hsp70 levels, ascorbate/menadione-treated cells present an additional Hsp90 protein band of about 70kDa as shown by Western blot analysis, suggesting Hsp90 cleavage. This Hsp90 cleavage seems to be a selective phenomenon since it was observed in a large panel of cancer cell lines but not in non-transformed cells. Antibodies raised against either the N-terminus or the C-terminus domains of Hsp90 suggest that the site of cleavage should be located at its N-terminal part. Furthermore, antibodies raised against either the α- or the β-Hsp90 isoform show that Hsp90β is cleaved while the α isoform is down-regulated. We have further shown that different Hsp90 client proteins like Bcr-Abl (a chimerical protein expressed in K562 leukemia cells), RIP and Akt, were degraded when K562 cells were exposed to an oxidative stress. Both Hsp90 cleavage and Bcr-Abl degradation were observed by incubating K562 cells with another H₂O₂-generating system (glucose/glucose oxidase) and by incubating KU812 cells (another leukemia cell line) with ascorbate/menadione. Due to the major role of Hsp90 in stabilizing oncogenic and mutated proteins, these results may have potential clinical applications.

Keywords: Protein chaperone Hsp90; Oxidative stress; Ascorbate-driven menadione redox cycling; Chronic myeloid leukemia (CML); K562 cells; Bcr-Abl oncogene

Regulation of genistein-induced differentiation in human acute myeloid leukaemia cells (HL60, NB4) by Yolanda Sánchez; Donna Amrán; Elena de Blas; Patricio Aller (pp. 384-396).

While it has been reported that genistein induces differentiation in multiple tumour cell models, the signalling and regulation of isoflavone-provoked differentiation are poorly known. We here demonstrate that genistein causes G₂/M cycle arrest and expression of differentiation markers in human acute myeloid leukaemia cells (HL60, NB4), and cooperates with all-trans retinoic acid (ATRA) in inducing differentiation, while ATRA attenuates the isoflavone-provoked toxicity. Genistein rapidly stimulates Raf-1, MEK1/2 and ERK1/2 phosphorylation/activation, but does not stimulate and instead causes a late decrease in Akt phosphorylation/activation which is attenuated by ATRA. Both differentiation and G₂/M arrest are attenuated by MEK/ERK inhibitors (PD98059, U0126) and ERK1-/ERK2-directed small interfering RNAs (siRNAs), and by the PI3K inhibitor LY294002, but not by the p38-MAPK inhibitor SB203580. Genistein stimulates p21^waf1/cip1 and cyclin B1 expression, phosphorylation/activation of ATM and Chk2 kinases, and Tyr15-phosphorylation/inactivation of Cdc2 (Cdk1) kinase, and these effects are attenuated by MEK/ERK inhibitors, while LY294002 also attenuates ERK and ATM phosphorylation. Caffeine abrogates the genistein-provoked G₂/M blockade and alterations in cell cycle regulatory proteins, and also suppresses differentiation. Finally, genistein causes reactive oxygen species (ROS) over-accumulation, but the antioxidant N-acetyl-l-cysteine fails to prevent ERK activation, G₂/M arrest, and differentiation induction. By contrast, N-acetyl-l-cysteine and p38-MAPK inhibitor attenuate the apoptosis-sensitizing (pro-apoptotic) action of genistein when combined with the antileukaemic agent arsenic trioxide. In summary, genistein-induced differentiation in acute myeloid leukaemia cells is a ROS-independent, Raf-1/MEK/ERK-mediated and PI3K-dependent response, which is coupled and co-regulated with G₂/M arrest, but uncoupled to the pro-apoptotic action of the drug.

Keywords: Abbreviations; AMPK; AMP-activated kinase; ATM; ataxia telangiectasia mutated kinase; ATRA; all-trans retinoic acid; DAPI; 4,6-diamino-2-phenylindole; EGCG; epigallocatechin-3-gallate; ERK; extracellular signal-regulated kinase; FITC; fluorescein isothiocyanate; H; ₂; DCFDA; dichlorodihydrofluorescein diacetate; MAPK; mitogen-activated protein kinase; MEK; mitogen-activated protein kinase/extracellular signal-regulated kinase; NAC; N; -acetyl-; l; -cysteine; NBT; nitroblue tetrazolium; PBS; phosphate-buffered saline; PI3K; phosphatidylinositol 3-kinase; ROS; reactive oxygen speciesGenistein; Differentiation; Protein kinases; Oxidative stress; Myeloid leukaemia cells

Tumor necrosis factor α induces γ-glutamyltransferase expression via nuclear factor-κB in cooperation with Sp1 by Simone Reuter; Michael Schnekenburger; Silvia Cristofanon; Isabelle Buck; Marie-Hélène Teiten; Sandrine Daubeuf; Serge Eifes; Mario Dicato; Bharat B. Aggarwal; Athanase Visvikis; Marc Diederich (pp. 397-411).

γ-Glutamyltransferase (GGT) cleaves the γ-glutamyl moiety of glutathione (GSH), an endogenous antioxidant, and is involved in mercapturic acid metabolism and in cancer drug resistance when overexpressed. Moreover, GGT converts leukotriene (LT) C4 into LTD4 implicated in various inflammatory pathologies. So far the effect of inflammatory stimuli on regulation of GGT expression and activity remained to be addressed.We found that the proinflammatory cytokine tumor necrosis factor alpha (TNFα) induced GGT promoter transactivation, mRNA and protein synthesis, as well as enzymatic activity. Remicade, a clinically used anti-TNFα antibody, small interfering RNA (siRNA) against p50 and p65 nuclear factor-kappaB (NF-κB) isoforms, curcumin, a well characterized natural NF-κB inhibitor, as well as a dominant negative inhibitor of kappaB alpha (IκBα), prevented GGT activation at various levels, illustrating the involvement of this signaling pathway in TNFα-induced stimulation. Over-expression of receptor of TNFα-1 (TNFR1), TNFR-associated factor-2 (TRAF2), TNFR-1 associated death domain (TRADD), dominant negative (DN) IκBα or NF-κB p65 further confirmed GGT promoter activation via NF-κB.Linker insertion mutagenesis of 536bp of the proximal GGT promoter revealed NF-κB and Sp1 binding sites at −110 and −78 relative to the transcription start site, responsible for basal GGT transcription. Mutation of the NF-κB site located at −110 additionally inhibited TNFα-induced promoter induction. Chromatin immunoprecipitation (ChIP) assays confirmed mutagenesis results and further demonstrated that TNFα treatment induced in vivo binding of both NF-κB and Sp1, explaining increased GGT expression, and led to RNA polymerase II recruitment under inflammatory conditions.

Keywords: TNFα; NF-κB; GGT; Sp1; Curcumin; Inflammation

MKK4 is a novel target for the inhibition of tumor necrosis factor-α-induced vascular endothelial growth factor expression by myricetin by Jong-Eun Kim; Jung Yeon Kwon; Dong Eun Lee; Nam Joo Kang; Yong-Seok Heo; Ki Won Lee; Hyong Joo Lee (pp. 412-421).

Tumor necrosis factor-α (TNF-α) is a mediator of multiple inflammatory diseases. Vascular endothelial growth factor (VEGF) plays a critical role in TNF-α-mediated diseases. We investigated the inhibitory effects of 3,3′,4′,5,5′,7-hexahydroxyflavone (myricetin), an abundant natural flavonoid, on TNF-α-induced VEGF upregulation and the underlying molecular mechanism. Myricetin is a direct inhibitor of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase 1 (MEK1) and inhibits neoplastic cell transformation. We found that myricetin inhibited TNF-α-induced VEGF expression in JB6 P+ mouse epidermal cells by targeting MAPK kinase 4 (MKK4), as well as MEK1. The activation of activator protein-1 by TNF-α was inhibited by myricetin in a dose-dependent manner. The phosphorylation of c-Jun N-terminal kinase (JNK) and ERK was inhibited by myricetin, but not the phosphorylation of their upstream kinases MKK4 and MEK1. TNF-α-induced VEGF expression was inhibited by SP600125 and U0126, which are inhibitors of JNK and MEK, respectively. Myricetin inhibited TNF-α-induced MKK4 activity and bound glutathione S-transferase-MKK4 directly by competing with ATP. Computer modeling suggested that myricetin docks onto the ATP-binding site in MKK4, which is located between the N- and C-lobes of the kinase domain. Overall, our results indicate that myricetin has potent chemopreventive effects against TNF-α-related disease, mainly by targeting MKK4 and MEK1.

Keywords: Abbreviations; AP; activator protein; ELISA; enzyme-linked immunosorbent assay; ERK; extracellular signal-regulated kinase; FBS; fetal bovine serum; HIF-1; hypoxia inducible factor; JNK; c-Jun N-terminal kinase; MAPK; mitogen-activated protein kinase; MEK1; MAPK/extracellular signal-regulated kinase ERK kinase 1; MEM; Eagle's minimum essential medium; MKK4; MAPK kinase 4; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Myricetin; 3,3′,4′,5,5′,7-hexahydroxyflavone; NF; nuclear factor; Sp-1; specificity protein-1; TNF-α; tumor necrosis factor-α; VEGF; vascular endothelial growth factorMitogen-activated protein kinase kinase 4; Myricetin; Vascular endothelial growth factor

Inhibition of pro-inflammatory cytokine production by the dual p38/JNK2 inhibitor BIRB796 correlates with the inhibition of p38 signaling by Lore M. Gruenbaum; Racheline Schwartz; Joseph R. Woska Jr.; Rodney P. DeLeon; Gregory W. Peet; Thomas C. Warren; Alison Capolino; Lisa Mara; Maurice M. Morelock; Anthony Shrutkowski; Jessi Wildeson Jones; Christopher A. Pargellis (pp. 422-432).

The characterization of the potent p38 inhibitor BIRB796 as a dual inhibitor of p38/Jun N-terminal kinases (JNK) mitogen-activated protein kinases (EC 2.7.11.24) has complicated the interpretation of its reported anti-inflammatory activity. To better understand the contribution of JNK2 inhibition to the anti-inflammatory activities of BIRB796, we explored the relationship between the effects of BIRB796 and analogues on cytokine production and on cellular p38 and JNK signaling. We determined the binding affinity for BIRB796 and structural analogues to p38α and JNK2 and characterized compound 2 as a p38 inhibitor that binds to p38α with an affinity equivalent to BIRB796 but does not bind to any of the JNK isoforms. High-content imaging enabled us to show that the inhibition of p38 signaling by BIRB796 and analogues correlates with the ability of these compounds to inhibit the lipopolysaccharide (LPS)-induced TNF-α production in THP-1 monocytes. This finding was extended to cytokine release by disease-relevant human primary cells: to the production of TNF-α by peripheral blood mononuclear cells, and of IL-8 by neutrophils. Furthermore, BIRB796 and compound 2 inhibited the production of TNF-α in THP-1 monocytes and the IL-12/IL-18-induced production of interferon-γ in human T-cells with similar potencies. In contrast, cellular JNK signaling in response to cytokines or stress stimuli was only weakly inhibited by BIRB796 and analogues and not affected by compound 2. In summary, our data suggest that p38 inhibition alone is sufficient to completely suppress cytokine production and that the added inhibition of JNK2 does not significantly contribute to the effects of BIRB796 on cytokine production.

Keywords: p38; Protein kinase inhibitor; T-cells; Neutrophils; Monocytes; High-content imaging

Inhibition of topoisomerase II by 8-chloro-adenosine triphosphate induces DNA double-stranded breaks in 8-chloro-adenosine-exposed human myelocytic leukemia K562 cells by Sheng-Yong Yang; Xiu-Zhen Jia; Li-Yan Feng; Shu-Yan Li; Guo-Shun An; Ju-Hua Ni; Hong-Ti Jia (pp. 433-443).

8-Chloro-cAMP and 8-chloro-adenosine (8-Cl-Ado) are known to inhibit proliferation of cancer cells by converting 8-Cl-Ado into an ATP analog, 8-chloro-ATP (8-Cl-ATP). Because type II topoisomerases (Topo II) are ATP-dependent, we infer that 8-Cl-Ado exposure might interfere with Topo II activities and DNA metabolism in cells. We found that 8-Cl-Ado exposure inhibited Topo II-catalytic activities in K562 cells, as revealed by decreased relaxation of the supercoiled pUC19 DNA and inhibited decatenation of the kinetoplast DNA (kDNA). In vitro assays showed that 8-Cl-ATP, but not 8-Cl-Ado, could directly inhibit Topo IIα-catalyzed relaxation and decatenation of substrate DNA. Furthermore, 8-Cl-ATP inhibited Topo II-catalyzed ATP hydrolysis and increased salt-stabilized closed clamp. In addition, 8-Cl-Ado exposure decreased bromo-deoxyuridine (BrdU) incorporation into DNA and led to enhanced DNA double-stranded breaks (DSBs) and to increased formation of γ-H2AX nuclear foci in exposed K562 cells. Together, 8-Cl-Ado/8-Cl-ATP can inhibit Topo II activities in cells, thereby inhibiting DNA synthesis and inducing DNA DSBs, which may contribute to 8-Cl-Ado-inhibited proliferation of cancers.

Keywords: Abbreviations; Topo II; topoisomerase II; 8-Cl-Ado; 8-chloro-adenosine; 8-Cl-ATP; 8-chloro-adenosine triphosphate; AMPPNP; adenosine 5′-(β,γ-imido)triphosphate; DSBs; double-stranded breaks; PFGE; pulsed field gel electrophoresisType II topoisomerases; 8-Chloro-adenosine/8-chloro-ATP; Relaxation/decatenation; Double-stranded breaks; ATP hydrolysis; Closed clamp

Investigations on the cytochrome P450 (CYP) isoenzymes involved in the metabolism of the designer drugs N-(1-phenyl cyclohexyl)-2-ethoxyethanamine and N-(1-phenylcyclohexyl)-2-methoxyethanamine by Christoph Sauer; Frank T. Peters; Andrea E. Schwaninger; Markus R. Meyer; Hans H. Maurer (pp. 444-450).

Investigations using insect cell microsomes with cDNA-expressed human cytochrome P450 (CYP)s and human liver microsomes (HLM) are reported on the CYP isoenzymes involved in the metabolism of the designer drugs N-(1-phenylcyclohexyl)-2-ethoxyethanamine (PCEEA) to O-deethyl PCEEA and N-(1-phenylcyclohexyl)-2-methoxyethanamine (PCMEA) to O-demethyl PCMEA. Gas chromatography–mass spectrometry or liquid chromatography–mass spectrometry was used for the analysis of the incubation samples. PCEEA O-deethylation was catalyzed by CYP2B6, CYP2C9, CYP2C19, and CYP3A4, while PCMEA O-demethylation was catalyzed only by CYP2B6 and CYP2C19. Considering the relative activity factor approach, these enzymes accounted for 53%, 25%, 4%, and 18% of net clearance for PCEEA and 91% and 9% of net clearance for PCMEA, respectively. The chemical CYP2B6 inhibitor 4-(4-chlorobenzyl)pyridine (CBP) reduced the metabolite formation in pooled HLM by 63% at 1μM PCEEA. At 10μM PCEEA, CBP reduced metabolite formation by 61%, while inhibition of CYP3A4 by ketoconazole and inhibition of CYP2C9 by sulfaphenazole showed no inhibitory effect. At 1μM PCMEA, CBP reduced metabolite formation in pooled HLM by 70% and at 10μM PCMEA by 78%, respectively. In conclusion, the main metabolic step of both studied drugs was catalyzed by different CYPs.

Keywords: Cytochrome P450; HLM; Designer drugs; N; -(1-Phenylcyclohexyl)-2-ethoxyethanamine; N; -(1-Phenylcyclohexyl)-2-methoxyethanamine

Effects of dexamethasone, administered for growth promoting purposes, upon the hepatic cytochrome P450 3A expression in the veal calf by Michela Cantiello; Mery Giantin; Monica Carletti; Rosa M. Lopparelli; Francesca Capolongo; Frederic Lasserre; Enrico Bollo; Carlo Nebbia; Pascal G.P. Martin; Thierry Pineau; Mauro Dacasto (pp. 451-463).

Dexamethasone (DEX) exerts its known anti-inflammatory and immunosuppressant activities through the interaction with the glucocorticoid receptor (GR). In human liver, DEX is metabolized by cytochrome P450 3A (CYP3A); moreover, it is among those xenobiotics which induce CYP3A itself. The transcriptional regulation of CYP3A involves GR and nuclear receptors (NRs). In cattle, DEX is used at low dosages as a growth promoter; besides, CYP3A is expressed in the liver. In the present study, the effects of two illicit DEX protocols upon liver CYP3A were investigated in the veal calf. Dexamethasone, administered per os (DOS) or injected intramuscularly (DIM) at growth promoting purposes, increased GR mRNA (+25.62% and +73.02% of CTRL for DOS and DIM, respectively), while tyrosine aminotransferase (TAT) and NRs gene expression profiles were unaffected; decreased CYP3A mRNA (−20.64% and −16.07% with Q RT-PCR; −30.55% and −34.31% with Northern blotting); at the post-translational level, decreased TAT activity (−19.84% and 44.34%), CYP3A apoprotein (−27.65% and −42.85%) and CYP3A-dependent enzyme activities (erythromycin N-demethylase, −78.89% and −23.87%; ethylmorphine N-demethylase, −44.26% and −28.37%; testosterone 6β-hydroxylase, −44.60% and −18.07%; testosterone 2β-hydroxylase, −43.95% and −11.69%); by contrast, an increase (about 2-fold) of the urinary 6β-hydroxycortisol:cortisol ratio was observed in vivo. In summary, DEX modulates cattle liver CYP3A at pre- and post-translational level. Species-differences in GR–NRs–CYP3A regulation and in their response to differing DEX dosages might justify present results. Furthermore, the urinary 6β-hydroxycortisol:cortisol ratio is not useful to monitor in vivo CYP3A activity in DEX-treated individuals.

Keywords: Abbreviations; 6β-OHCTS; 6β-hydroxycortisol; 6β-OHCTS/CTS ratio; urinary 6β-hydroxycortisol:cortisol ratio; 2β-OHTST; testosterone 2β-hydroxylase; 6β-OHTST; testosterone 6β-hydroxylase; CAR; constitutive androstan receptor; CTRL; control animals; CTS; cortisol; CYP3A; cytochrome P450 3A; dCTP; deoxycytidine-5-triphosphate; DEX; dexamethasone; DIM; calves intramuscularly injected with DEX; dNTP; deoxynucleotide triphosphate; DOS; animals orally administered with DEX; ERDEM; erythromycin; N; -demethylase; ETDEM; ethylmorphine; N; -demethylase; E; _x; PCR efficiency; F; forward; GC; glucocorticoids; GPs; growth promoters; GR; glucocorticoid receptor; im; intramuscularly; HNF4α; hepatocyte nuclear factor 4-alpha; HPLC; high performance liquid chromatography; NRs; nuclear receptors; PXR; pregnane X receptor; Q RT-PCR; quantitative real time polymerase chain reaction; R; reverse; RXRα; retinoid X receptor alpha; TAT; tyrosine aminotransferase; TST; testosterone; VDR; vitamin D receptorCattle; Dexamethasone; Liver; CYP3A; Illicit protocol; Nuclear receptors

TLR4-dependent and -independent regulation of hepatic cytochrome P450 in mice with chemically induced inflammatory bowel disease by Madhusudana R. Chaluvadi; Beatrice A. Nyagode; Ryan D. Kinloch; Edward T. Morgan (pp. 464-471).

The transcription and protein expression of many cytochrome P450 (P450) genes are down-regulated in animal models of inflammation and infection. We determined previously that hepatic P450 mRNAs are selectively regulated in a mouse model of enteropathogenic bacterial infection, and that this regulation was not dependent on the lipopolysaccharide (LPS) receptor protein toll-like receptor 4 (TLR4). In the dextran sulfate sodium (DSS) model of chemically induced inflammatory bowel disease (IBD), the reduction in activities of several hepatic P450 enzymes were concluded to be partially dependent on LPS from commensal bacteria [Masubuchi Y, Horie T. Endotoxin-mediated disturbance of hepatic cytochrome P450 function and development of endotoxin tolerance in the rat model of dextran sulfate sodium-induced experimental colitis. Drug Metab Dispos 2004;32:437–441]. In the present study, we sought to determine whether colitis induced by LPS regulates hepatic P450 mRNA and protein expression similarly to infectious colitis, and to determine the role of TLR4 in the response to DSS colitis. The role of LPS in the response to DSS was further examined by comparison with the effects of injected LPS. We demonstrate that administration of DSS results in the down-regulation of multiple P450 enzymes in mouse liver. However, there are discernable differences in the pattern of P450 expression in the two models. Some effects of DSS-induced colitis are TLR4-dependent, and others are not. In contrast, the effects of injected LPS on hepatic P450 mRNA expression are entirely TLR4-dependent. Thus, our results indicate that the pattern of hepatic P450 expression, and the mechanism of regulation, during inflammation of the bowel depend on the etiology of the disease.

Keywords: Abbreviations; P450; cytochrome P450; TLR4; toll-like receptor 4; DSS; dextran sulfate sodium; TNBS; trinitrobenzenesulfonic acid; IL; interleukin; TNFα; tumor necrosis factor alpha; LPS; lipopolysaccharide; PMN; polymorphonuclear lymphocytes; PPARα; peroxisome proliferator activated receptor alpha; AGP; α1-acid glycoprotein; FBG; fibrinogen alpha polypeptide; AGT; angiotensinogen; EPEC; enteropathogenic; Escherichia coli; DSS; dextran sulfate sodium; IBD; inflammatory bowel disease; RT-PCR; reverse transcription-polymerase chain reaction; GAPDH; glyceraldehyde phosphate dehydrogenase; PCR; polymerase chain reactionCytochrome P450; Inflammation; Inflammatory bowel disease; Toll-like receptor 4

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Biochemical Pharmacology (v.77, #3)

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Biochemical Pharmacology (v.77, #3)