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Biochemical Pharmacology (v.74, #5)

Editorial Advisory Board (pp. iii).

HDAC inhibitors: Clinical update and mechanism-based potential by Keith B. Glaser (pp. 659-671).

Recently, the role of transcriptional repression through epigenetic modulation in carcinogenesis has been clinically validated with several inhibitors of histone deacetylases and DNA methyltransferases. It has long been recognized that epigenetic alterations of tumor suppressor genes was one of the contributing factors in carcinogenesis. Inhibitors of histone deacetylase (HDAC) de-repress genes that subsequently result in growth inhibition, differentiation and apoptosis of cancer cells. Vorinostat (SAHA), romidepsin (depsipeptide, FK-228), belinostat (PXD101) and LAQ824/LBH589 have demonstrated therapeutic benefit as monotherapy in cutaneous T-cell lymphoma (CTCL) and have also demonstrated some therapeutic benefit in other malignancies. The approval of the HDAC inhibitor vorinostat (Zolinza™) was based on the inherent sensitivity of this type of lymphoma to alterations in acetylation patterns that resulted in the induction of repressed apoptotic pathways. However, the full potential of these inhibitors (epigenetic modulators) is still on the horizon, as the true breadth of their utility as anti-cancer agents will be determined by the careful analysis of gene expression changes generated by these inhibitors and then combined with conventional chemotherapy to synergistically improve response and toxicity for an overall enhanced therapeutic benefit to the patient. The question that must be considered is whether the current HDACIs are being utilized to their fullest potential in clinical trials based on their mechanism-based alterations in disease processes.

Keywords: Histone deacetylase inhibitors; Chromatin modification; Epigenetic regulation; Vorinostat; Belinostat; Romidepsin; LBH589; MGCD0103; MS-275

New structure model for the ATP-binding cassette multidrug transporter LmrA by Luca Federici; Barbara Woebking; Saroj Velamakanni; Richard A. Shilling; Ben Luisi; Hendrik W. van Veen (pp. 672-678).

Multidrug resistance of pathogenic microorganisms and mammalian tumors can be associated with the overexpression of multidrug transporters. These integral membrane proteins are capable of extruding a wide range of structurally unrelated compounds from the cell. Among the different classes of multidrug transporters are the ATP binding cassette (ABC) transporters, which are dependent on the binding and hydrolysis of ATP. In the past five years, many researchers have built homology models of ABC extrusion systems using the atomic coordinates of crystallized MsbA, a lipopolysaccharide transporter in Gram-negative bacteria. Likewise, we have previously used the Vibrio cholera MsbA structure as a template in the modeling of the multidrug transporter LmrA from Lactococcus lactis. In view of the recently discovered inaccuracies in the MsbA structure, we have remodelled LmrA using the atomic coordinates of the MsbA homologue Sav1866 from Staphylococcus aureus. To compare and test our MsbA-based and Sav1866-based LmrA models we performed cysteine cross-linking at three key positions in LmrA. The pattern of cross-linking at these positions was consistent with the overall topology of transmembrane helices in Sav1866, suggesting that its crystal structure might be physiologically relevant. We recently identified E314 as a residue important in proton conduction by LmrA. The predicted location of this residue at the interface between the two half-transporters in the Sav1866-based homodimer, within the inner leaflet of the phospholipid bilayer, provides a new structural basis for the role of E314 in LmrA-mediated transport.

Keywords: Abbreviations; ABC; ATP-binding cassette; MD; membrane domain; NBD; nucleotide-binding domain; TMH; transmembrane helixABC transporter; Cysteine cross-linking; Homology modelling; LmrA; Multidrug resistance; Sav1866

Mechanism of acridine-based telomerase inhibition and telomere shortening by Mekala Gunaratnam; Olga Greciano; Cristina Martins; Anthony P. Reszka; Christoph M. Schultes; Hamid Morjani; Jean-Francois Riou; Stephen Neidle (pp. 679-689).

The trisubstituted acridine compound BRACO-19 has been developed as a ligand for stabilising G-quadruplex structures. It is shown here that BRACO-19 produces short- and long-term growth arrest in cancer cell lines, and is significantly less potent in a normal cell line. BRACO-19 reduces telomerase activity and long-term telomere length attrition is observed. It is also shown that BRACO-19 binds to telomeric single-stranded overhang DNA, consistent with quadruplex formation, and the single-stranded protein hPOT1 has been shown to be displaced from the overhang in vitro and in cellular experiments. It is concluded that the cellular activity of BRACO-19 can be ascribed both to the uncapping of 3′ telomere ends and to telomere shortening that may preferentially affect cells with short telomeres.

Keywords: G-quadruplex ligands; Telomerase; Telomere targeting

Phorbol 12-myristate 13-acetate (PMA)-induced migration of glioblastoma cells is mediated via p38MAPK/Hsp27 pathway by Naoko Nomura; Motohiro Nomura; Kazuhisa Sugiyama; Jun-Ichiro Hamada (pp. 690-701).

We investigated the mechanism of phorbol 12-myristate 13-acetate (PMA)-induced migration of glioblastoma cells focusing on the p38 mitogen-activated protein kinase (MAPK)/heat shock protein 27 (Hsp27) pathway. PMA-induced cell migration and activation of p38MAPK in A172 glioblastoma cells. PMA-induced formation of lamellipodia and focal complexes was blocked by inhibiting p38MAPK with SB203580 or small interfering RNA (siRNA). Furthermore, activation of p38MAPK resulted in phosphorylation of an F-actin polymerization regulator, Hsp27. Immunohistochemical analysis showed that upon PMA stimulation, both unphosphorylated and phosphorylated Hsp27 were translocated to the lamellipodia. SB203580 or p38MAPK siRNA blocked these phenomena, indicating that Hsp27 phosphorylation and translocation from cytosol to membrane were mediated by p38MAPK. To address the question of whether endogenous Hsp27 participates in PMA-induced migration, we inhibited the expression of Hsp27 using Hsp27 siRNA. Although knockdown of Hsp27 by siRNA had little effect on p38MAPK activation, lamellipodia and focal complex formation was markedly inhibited. Migration was also abolished in Hsp27 siRNA-transfected cells. In conclusion, p38MAPK activation followed by Hsp27 phosphorylation was required for PMA-induced migration. Furthermore, Hsp27 itself played critical roles in PMA-induced migration. Our data provide substantial evidence for a model elucidating the molecular mechanisms of regulation of actin dynamics and migration by PMA-activated protein kinase C in glioblastoma cells.

Keywords: Abbreviations; PMA; phorbol 12-myristate 13-acetate; MAPK; mitogen-activated protein kinase; Hsp; heat shock protein; siRNA; small interfering RNA; PKC; protein kinase C; BIS; bisindolylmaleimide I; DMSO; dimethyl sulfoxidePhorbol 12-myristate 13-acetate; Protein kinase C; Glioblastoma; Migration; p38MAPK; Hsp27

Functional consequences of methionine oxidation of hERG potassium channels by Zhi Su; James Limberis; Ruth L. Martin; Rong Xu; Katrin Kolbe; Stefan H. Heinemann; Toshinori Hoshi; Bryan F. Cox; Gary A. Gintant (pp. 702-711).

Reactive species oxidatively modify numerous proteins including ion channels. Oxidative sensitivity of ion channels is often conferred by amino acids containing sulfur atoms, such as cysteine and methionine. Functional consequences of oxidative modification of methionine in human ether à go-go related gene 1 (hERG1), which encodes cardiac I_Kr channels, are unknown. Here we used chloramine-T (ChT), which preferentially oxidizes methionine, to examine the functional consequences of methionine oxidation of hERG channels stably expressed in a human embryonic kidney cell line (HEK 293) and native hERG channels in a human neuroblastoma cell line (SH-SY5Y). ChT (300μM) significantly decreased whole-cell hERG current in both HEK 293 and SH-SY5Y cells. In HEK 293 cells, the effects of ChT on hERG current were time- and concentration-dependent, and were markedly attenuated in the presence of enzyme methionine sulfoxide reductase A that specifically repairs oxidized methionine. After treatment with ChT, the channel deactivation upon repolarization to −60 or −100mV was significantly accelerated. The effect of ChT on channel activation kinetics was voltage-dependent; activation slowed during depolarization to +30mV but accelerated during depolarization to 0 or −10mV. In contrast, the reversal potential, inactivation kinetics, and voltage-dependence of steady-state inactivation remained unaltered. Our results demonstrate that the redox status of methionine is an important modulator of hERG channel.

Keywords: hERG; K-channel; Chloramine-T; Methionine; Oxidation; Methionine sulfoxide reductase

Neutral and positively charged thiols synergize the effect of the immunomodulator AS101 as a growth inhibitor of Jurkat cells, by increasing its uptake by Gabi M. Frei; Ilana Lebenthal; Michael Albeck; Amnon Albeck; Benjamin Sredni (pp. 712-722).

The immunomodulator amonium trichloro[1,2-ethanediolato-O,O’] tellurate (AS101), a nontoxic tellurium(IV) compound, exhibited antitumoral activity in several preclinical and clinical studies. In this study, we investigated the synergism between thiols and AS101 in its antitumoral activity on Jurkat cells. AS101 induced a G₂/M arrest in the cell cycle after 24h. Addition of the thiols 2-mercaptoethanol or cysteamine led to an induction of apoptosis. Other thiols, including glutathione (GSH) and cysteine, did not potentiate the effect of AS101. We propose that this is due to the alpha-carboxylate group present in the compounds formed between AS101 and these thiols. Programmed cell death was associated with the loss of mitochondrial transmembrane potential and activation of caspase-3 and -9. Elevation of intracellular reactive oxygen species (ROS) production was also demonstrated; the antioxidant catalase significantly reduced the apoptosis, suggesting that ROS play a key role in the apoptosis induced by AS101 and the thiols. Finally, we quantified the intracellular concentration of tellurium, using electron microscopy and energy-dispersive spectroscopy (EDS) analysis. The addition of cysteamine to AS101 significantly increased the concentration of tellurium within the cells. The results indicate that neutral or positively charged thiols but not negatively charged ones, increase the antitumoral effect of AS101 by increasing its uptake into the cells.

Keywords: Abbreviations; AS101; amonium trichloro[1,2-ethanediolato-O,O’] tellurate; PBS; phosphate-buffered saline; BCH; 2-aminobicycloheptane-2-carboxylic acid; GSH; glutathione; 2-ME; 2-mercaptoethanol; EDS; energy dispersive X-ray spectroscopy; SEM; scanning electron microscope; TEM; transmission electron microscope; PE; phycoerythrin; MMP; mitochondrial transmembrane potential; NAC; N; -acetyl-; l; -cysteine; CYSM; l; -cysteine methyl ester; PI; propidium iodide; ROS; reactive oxygen speciesTellurium(IV); Thiols; ROS; Jurkat; Antitumoral; AS101

Heme oxygenase-1 mediates cytoprotective effects of immunostimulation in microglia by Shinrye Lee; Kyoungho Suk (pp. 723-729).

Microglia are brain-resident immune cells playing a pivotal role in the neuroinflammation. Previously, it has been shown that immunostimulation protects microglial cells against nitric oxide toxicity. Herein, we report that heme oxygenase-1 (HO-1) mediates the protective effects of immunostimulation. Pro-inflammatory activation of BV-2 microglial cells with endotoxin lipopolysaccharide (LPS) conferred a protection against various cytotoxic stimuli, whereas anti-inflammatory cytokines such as IL-4 and IL-10 were without effects. The LPS-induced cytoprotection was accompanied by HO-1 induction. The cytoprotective effect of LPS treatment was significantly attenuated by co-treatment with a HO-1 inhibitor, zinc protoporphyrin. Adenoviral expression of HO-1 in microglial cells was similarly cytoprotective, indicating that HO-1 mediates the cytoprotective effects of pro-inflammatory stimulation. Additional experiments revealed the involvement of carbon monoxide (CO) and iron, products of HO-1-mediated heme degradation, in the cytoprotective effect of LPS. Taken together, our results suggest that immunostimulation of microglia with LPS provides cytoprotective effects via HO-1 induction followed by the generation of CO and iron.

Keywords: Lipopolysaccharide; Heme oxygenase-1; Inflammation; Cytoprotection; Microglia; Carbon monoxide; Immunostimulation

The PGE₂-induced inhibition of the PLD activation pathway stimulated by fMLP in human neutrophils is mediated by PKA at the PI3-Kγ level by Chantal Burelout; Nathalie Thibault; Danielle Harbour; Paul H. Naccache; Sylvain G. Bourgoin (pp. 730-741).

Prostaglandin E₂ (PGE₂), an eicosanoid that modulates inflammation, inhibits several chemoattractant-elicited functions in neutrophils such as chemotaxis, production of superoxide anions, adhesion, secretion of cytotoxic enzymes and synthesis of leukotriene B₄. We previously reported that PGE₂ inhibits the fMLP signaling pathway that leads to PLD activation through suppression of PI3-Kγ activity and the decreased recruitment to membranes of PLD activation factors, PKC, Rho and Arf-GTPases. This effect is mediated via the EP₂ receptors known to raise cAMP in cells.The inhibition of most fMLP-induced functional responses by PGE₂ via EP₂ receptors is mediated by PKA, except the chemotactic response. We have investigated the role of PKA in the EP₂-mediated inhibition of the PLD activation pathway. H-89, a selective PKA pharmacological inhibitor suppressed the inhibitory effects of PGE₂ at all stages of the PLD pathway activated by fMLP, i.e. PLD activity, translocation to membranes of PKCα, Rho and Arf-GTPases, calcium influx, tyrosine phosphorylation of proteins and finally translocation of p110γ catalytic subunit of PI3-K to membranes. However, neither PLD nor PI3-Kγ was substrate of PKA. These data provide evidence that PGE₂-stimulated PKA activity regulates the PLD pathway stimulated by fMLP at the level of PI3-Kγ and that the inhibition of PI3-Kγ activation by PKA is a complex mechanism that remains to be completely elucidated.

Keywords: Abbreviations; ADA; adenosine deaminase; AKAP; A-kinase anchoring protein; Arf; ADP-rybosilation factor; CB; cytochalasin B; DFP; di-isopropylfluorophosphate; EP receptor; E prostaglandin receptor; fMLP; N; -formyl-methionyl-leucyl-phenylalanine; GEF; guanine nucleotide exchange factor; PEt; phosphatidylethanol; PH; pleckstrin homology; PI3-K; phosphatidylinositol 3-kinase; PKA; cAMP-dependent protein kinase; PMN; polymorphonuclear neutrophil; PtdIns(3,4,5)P; ₃; phosphatidylinositol 3,4,5-trisphophate; PLD; phospholipase D; PLC; phospholipase CPGE; ₂; EP; ₂; receptor; PKA; PI3-Kγ; fMLP; PLD

Prodelphinidin B-4 3′- O-gallate, a tea polyphenol, is involved in the inhibition of COX-2 and iNOS via the downregulation of TAK1-NF-κB pathway by De-Xing Hou; Dong Luo; Shunsuke Tanigawa; Fumio Hashimoto; Takuhiro Uto; Satoko Masuzaki; Makoto Fujii; Yusuke Sakata (pp. 742-751).

Much is known about the bioactive properties of green tea flavan-3- ol. However, very little work has been done to determine the properties of proanthocyanidins, another kind of polyphenols in green tea. In this study, we have investigated the anti-inflammatory effect of tea prodelphinidin B-4 3′- O-gallate (PDG) by demonstrating the inhibitory effects on cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-activated murine macrophage RAW264 cells. PDG caused a dose-dependent inhibition of COX-2 and iNOS at both mRNA and protein levels with the attendant decrease of prostaglandin E₂ (PGE₂) and nitric oxide (NO) production. Molecular data revealed that PDG downregulated NF-κB signaling pathway. Electrophoretic mobility shift assay (EMSA) showed that PDG reduced the binding complex of NF-κB–DNA in the promoter of COX-2 and iNOS. Immunochemical analysis revealed that PDG suppressed LPS-induced phosphorylation and degradation of IκBα, and subsequent nuclear translocation of p65. Consequently, PDG suppressed phosphorylation of IκB kinase α/β (IKKα/β) and TGF-β-activated kinase (TAK1). Taken together, our data indicated that PDG is involved in the inhibition of COX-2 and iNOS via the downregulation of TAK1-NF-κB pathway, revealing partial molecular basis for the anti-inflammatory properties of tea PDG.

Keywords: Abbreviations; COX-2; cyclooxygenase-2; EMSA; electrophoretic mobility shift assay; IKK; IkappaB kinase; iNOS; inducible nitric oxide synthase; LPS; lipopolysaccharide; NF-κB; nuclear factor kappaB; NO; nitric oxide; PDG; prodelphinidin B-4 3′-; O; -gallate; PGE; ₂; prostaglandin E; ₂; TAK1; TGF-β-activated kinase; TLR4; toll-like receptor 4Proanthocyanidin; COX-2; iNOS; NF-κB; IKKα/β; TAK1

Delay of cutaneous wound closure by morphine via local blockade of peripheral tachykinin release by Jerri M. Rook; Kenneth E. McCarson (pp. 752-757).

Topically applied morphine is routinely used to alleviate pain in cutaneous wounds such as burns and pressure sores. Evidence suggests the topical administration of exogenous opioid drugs may impair wound closure. This study examined the effects of topical morphine on a standardized model of cutaneous wound healing in the rat. Full-thickness 4mm diameter circular skin flaps were excised from the intrascapular region of male Sprague-Dawley rats. IntraSite™^® Gel infused with either morphine-sulfate, neurokinin-1 (NK-1) or neurokinin-2 (NK-2) receptor antagonists, substance P (SP), neurokinin A (NKA), SP+morphine-sulfate, or NKA+morphine-sulfate was applied to the wound twice daily. Results demonstrated a significant overall delay in the time course of wound contraction in morphine-treated animals when compared with gel-only treated controls. The delay in wound contraction seen in morphine-treated animals increased in a concentration-dependent manner. Topical application of NK-1 or NK-2 receptor antagonists mimicked the effects of morphine in delaying wound closure, suggesting topical opioids impair wound closure via the inhibition of SP and NKA release peripherally into the healing wound. Additionally, no significant delays in closure were seen in rats receiving morphine combined with SP or NKA, demonstrating the ability of each neuropeptide to attenuate the effects of morphine in delaying wound closure and restore normal wound closure rates. The combination of SP or NKA and morphine-sulfate for wound therapy may provide local analgesia while maintaining normal closure rates.

Keywords: Abbreviations; NK-1; neurokinin-1; NK-2; neurokinin-2; SP; substance P; NKA; neurokinin A; NEP; neutral endopeptidaseSubstance P; Neurokinin A; Neurokinin-1 receptor; Neurokinin-2 receptor; Opioid; Primary afferent neuron

Intrathecal substance P (1–7) prevents morphine-evoked spontaneous pain behavior via spinal NMDA-NO cascade by Tsukasa Sakurada; Takaaki Komatsu; Hikari Kuwahata; Chizuko Watanabe; Toru Orito; Chikai Sakurada; Minoru Tsuzuki; Shinobu Sakurada (pp. 758-767).

Previous research has shown that injection of high-dose of morphine into the spinal lumbar intrathecal (i.t.) space of rats elicits an excitatory behavioral syndrome indicative of severe vocalization and agitation. Substance P N-terminal fragments are known to inhibit nociceptive responses when injected i.t. into animals. In this study, we investigated the effect of i.t. substance P (1–7) on both the nociceptive response and the extracellular concentrations of glutamate and nitric oxide (NO) metabolites (nitrite/nitrate) evoked by high-dose i.t. morphine (500nmol). The induced behavioral responses were attenuated dose-dependently by i.t. pretreatment with the substance P N-terminal fragment substance P (1–7) (100–400pmol). The inhibitory effect of substance P (1–7) was reversed significantly by pretreatment with [d-Pro²,d-Phe⁷]substance P (1–7) (20 and 40nmol), ad-isomer and antagonist of substance P (1–7). In vivo microdialysis analysis showed a significant elevation of extracellular glutamate and NO metabolites in the spinal cord after i.t. injection of high-dose morphine (500nmol). Pretreatment with substance P (1–7) (400pmol) produced a significant reduction on the elevated concentrations of glutamate and NO metabolites evoked by i.t. morphine. The reduced levels of glutamate and NO metabolites were significantly reversed by the substance P (1–7) antagonist (40nmol). The present results suggest that i.t. substance P (1–7) may attenuate the excitatory behavior (vocalization and agitation) of high-dose i.t. morphine by inhibiting the presynaptic release of glutamate, and reducing NO production in the dorsal spinal cord.

Keywords: High-dose morphine; Substance P (1–7); Intrathecal injection; Vocalization and agitation; Glutamate; Nitric oxide; Spinal microdialysis

Role of adenosine A₃ receptors on CA1 hippocampal neurotransmission during oxygen–glucose deprivation episodes of different duration by Anna Maria Pugliese; Elisabetta Coppi; Rosaria Volpini; Gloria Cristalli; Renato Corradetti; Lak Shin Jeong; Kenneth A. Jacobson; Felicita Pedata (pp. 768-779).

The role of adenosine A₃ receptors in synaptic transmission under severe (7min) and shorter (2–5min) ischemic conditions, obtained by oxygen and glucose deprivation (OGD), was investigated in rat hippocampal slices. The effects of selective A₃ agonists or antagonists were examined on field excitatory postsynaptic potentials (fEPSPs) extracellularly recorded at the dendritic level of the CA1 pyramidal region. The novel, selective A₃ antagonist LJ1251 ((2 R,3 R,4 S)-2-(2-chloro-6-(3-iodobenzylamino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol, 0.1–10nM) protected hippocampal slices from irreversible fEPSP depression induced by severe OGD and prevented or delayed the appearance of anoxic depolarization. Similar results were obtained when severe OGD was carried out with a long, receptor-desensitizing exposure to various selective A₃ agonists: 5′- N-methylcarboxamidoadenosine derivatives Cl-IB-MECA ( N⁶-(3-iodobenzyl)-2-chloro), VT72 ( N⁶-methoxy-2-phenylethynyl), VT158 ( N⁶-methoxy-2-phenylethynyl), VT160 ( N⁶-methoxy-2-(2-pyridinyl)-ethynyl), and VT163 ( N⁶-methoxy-2- p-acetylphenylethynyl) and AR132 ( N⁶-methyl-2-phenylethynyladenosine).The selective A₃ antagonist MRS1523 (3-propyl-6-ethyl-5-[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridine carboxylate, 100nM) reduced fEPSP depression evoked by 2-min OGD and induced a faster recovery of fEPSP amplitude after 5-min OGD. Similar results were obtained for 2- or 5-min OGD applied in the presence of each of the A₃ agonists tested. Shorter exposure to A₃ agonists significantly delayed the recovery of fEPSP amplitude after 5-min OGD.This indicates that A₃ receptors, stimulated by selective A₃ agonists, undergo desensitization during OGD. It is inferred that CA1 hippocampal A₃ receptors stimulated by adenosine released during brief ischemia (2 and 5min) might exert A₁-like protective effects on neurotransmission. Severe ischemia would transform the A₃ receptor-mediated effects from protective to injurious.

Keywords: Abbreviations; AD; anoxic depolarization; aCSF; artificial cerebral spinal fluid; AR132; N; ⁶; -methyl-2-phenylethynyladenosine; AUC; area under curve; CHO; Chinese hamster ovary; Cl-IB-MECA; 1-[2-chloro-6[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-; N; -methyl-ß-; d; -ribofuranuronamide; DMSO; dimethylsulfoxide; fEPSP; field excitatory postsynaptic potential; LJ1251; (2; R; ,3; R; ,4; S; )-2-(2-chloro-6-(3-iodobenzylamino)-9H-purin-9-yl)tetrahydrothiophene-3,4-diol; MRS1523; 3-propyl-6-ethyl-5-[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridine carboxylate; OGD; oxygen–glucose deprivation; PKC; protein kinase C; SD; spreading depression; VT72; N; ⁶; -methoxy-2-phenylethynyladenosine; VT158; N; ⁶; -methoxy-2-phenylethynyl-5′-; N; -methylcarboxamidoadenosine; VT160; N; ⁶; -methoxy-2-(2-pyridinyl)-ethynyl-5′-; N; -methylcarboxamidoadenosine; VT163; N; ⁶; -methoxy-2-; p; -acetylphenylethynyl-5′-; N; -methylcarboxamidoadenosine; ZM241385; 4-(2-[7-amino-2-(2-furyl)(1,2,4)triazolo(2,3-; a; )(1,3,5,)triazin-5-yl amino]ethyl)phenolPurines; G protein-coupled receptors; Cerebral ischemia; Hippocampal slices; Field EPSP; Desensitization

[4- t-Butylphenyl]- N-(4-imidazol-1-yl phenyl)sulfonamide (ISCK03) inhibits SCF/c-kit signaling in 501mel human melanoma cells and abolishes melanin production in mice and brownish guinea pigs by Yong Joo Na; Heung Su Baek; Soo Mi Ahn; Hyun Jung Shin; Ih-Seop Chang; Jae Sung Hwang (pp. 780-786).

It is well known that c-kit is related to pigmentation as well as to the oncology target protein. The objective of this study was to discover a skin-whitening agent that regulates c-kit activity. We have developed a high-throughput screening system using recombinant human c-kit protein. Approximately 10,000 synthetic compounds were screened for their effect on c-kit activity. Phenyl-imidazole sulfonamide derivatives showed inhibitory activity on c-kit phosphorylation in vitro. The effects of one derivative, [4- t-butylphenyl]- N-(4-imidazol-1-yl phenyl)sulfonamide (ISCK03), on stem-cell factor (SCF)/c-kit cellular signaling in 501mel human melanoma cells were examined further. Pretreatment of 501mel cells with ISCK03 inhibited SCF-induced c-kit phosphorylation dose dependently. ISCK03 also inhibited p44/42 ERK mitogen-activated protein kinase (MAPK) phosphorylation, which is known to be involved in SCF/c-kit downstream signaling. However ISCK03 did not inhibit hepatocyte growth factor (HGF)-induced phosphorylation of p44/42 ERK proteins. To determine the in vivo potency of ISCK03, it was orally administered to depilated C57BL/6 mice. Interestingly, oral administration of ISCK03 induced the dose-dependent depigmentation of newly regrown hair, and this was reversed with cessation of ISCK03 treatment. Finally, to investigate whether the inhibitory effect of ISCK03 on SCF/c-kit signaling abolished UV-induced pigmentation, ISCK03 was applied to UV-induced pigmented spots on brownish guinea pig skin. The topical application of ISCK03 promoted the depigmentation of UV-induced hyperpigmented spots. Fontana–Masson staining analysis showed epidermal melanin was diminished in spots treated with ISCK03. These results indicate that phenyl-imidazole sulfonamide derivatives are potent c-kit inhibitors and might be used as skin-whitening agents.

Keywords: c-kit; Skin; Depigmentation

Activation of thromboxane receptor α induces expression of cyclooxygenase-2 through multiple signaling pathways in A549 human lung adenocarcinoma cells by Jingyan Wei; Weili Yan; Xiuling Li; Wen-Chang Chang; Hsin-Hsiung Tai (pp. 787-800).

Human lung adenocarcinoma A549 cells stably transfected with TPα (A549-TPα) were used to study agonist I-BOP-induced expression of cyclooxygenase-2 (COX-2) and the related mechanisms of induced expression. I-BOP, a TP agonist, induced a time- and dose-dependent expression of COX-2 in A549-TPα cells. The signaling pathways of I-BOP-induced COX-2 expression were elucidated by using various inhibitors of the signaling molecules. The effects of these inhibitors were assessed at protein level, enzyme activity and promoter activity of COX-2. Within MAPK family, both ERK and p38 MAPK but not JNK/SAPK pathways were involved in the induction. Other pathways such as JAK/Stat3 pathway and β-catenin/TCF/LEF pathway also participated in the induction. The activation of key signaling molecules, ERK, p38 MAPK, CREB and NF-κB, involved in the COX-2 transcription was further studied at the phosphorylation step. Activation of ERK and p38 MAPK appeared to be mediated primarily by transactivation of EGFR, whereas activation of CREB and NF-κB was mediated by PKA, PKC and ERK. The role of CREB and NF-κB in I-BOP-induced COX-2 expression was further explored at the promoter level. Studies on promoter fragments and mutation of responsive motifs indicated that CRE and NF-κB sites are critical for the COX-2 induction. Distal NF-κB site is essential for the basal induction of the COX-2 transcription, whereas CRE and proximal NF-κB sites are important for the induced transcription. These results indicate that I-BOP-induced COX-2 expression through multiple signaling pathways.

Keywords: Thromboxane receptor; Cyclooxygenase-2; Prostaglandins; Signal transduction; Gene expression

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Biochemical Pharmacology (v.74, #5)