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Biochemical Pharmacology (v.79, #10)

Editorial Advisory Board (pp. iii).

Graphical Abstracts Contents (pp. e1-e5).

Soluble RAGE: Therapy and biomarker in unraveling the RAGE axis in chronic disease and aging by Shi Fang Yan; Ravichandran Ramasamy; Ann Marie Schmidt (pp. 1379-1386).

The multi-ligand Receptor for Advanced Glycation Endproducts (RAGE) is implicated in the pathogenesis and progression of chronic diseases such as diabetes and immune/inflammatory disorders. Recent studies are uncovering the precise mechanisms by which distinct RAGE ligands bind the extracellular (soluble) domain of the receptor at the V-, C1- and/or C2-immunoglobulin like domains. Experiments using soluble RAGE in animals as a ligand decoy have illustrated largely beneficial effects in reducing vascular and inflammatory stress and, thereby, preventing long-term tissue damage in models of diabetes and immune/inflammatory disorders. Measurement of soluble RAGE levels in the human, both “total” soluble RAGE and a splice variant-derived product known as endogenous secretory or esRAGE, holds promise for the identification of potential therapeutic targets and/or biomarkers of RAGE activity in disease. In this article, we review the evidence from the rodent to the human implicating RAGE in the diverse disease states in which its ligands accumulate.

Keywords: RAGE; Diabetes; Aging; Soluble receptors; Inflammation

Increased cytotoxicity of an unusual DNA topoisomerase II inhibitor compound C-1305 toward HeLa cells with downregulated PARP-1 activity results from re-activation of the p53 pathway and modulation of mitotic checkpoints by Michal Sabisz; Józefa Węsierska-Gądek; Andrzej Skladanowski (pp. 1387-1397).

Our previous studies have shown that murine fibroblast cells, in which PARP-1 gene was inactivated by gene disruption, are extremely sensitive to triazoloacridone compound C-1305, an inhibitor of DNA topoisomerase II with unusual properties. Here, we show that pharmacological inhibition of PARP-1 activity by its inhibitor compound NU1025, sensitizes human cervical carcinoma HeLa cells to compound C-1305 compared to treatment with drug alone. Cytotoxic effect of drug/NU1025 of other topoisomerase II inhibitors varied depending on the dose of PARP-1 inhibitor. Increased cytotoxicity of topoisomerase II inhibitor/NU1025 combinations was attributable to the re-activation of the p53 pathway in drug-treated HeLa cells. This lead to a more stringent cell cycle checkpoint control during G2 and M and enhanced cell death by mitotic catastrophe induced by drug/NU1025 combinations. Interestingly, treatment of HeLa cells with NU1025 alone also increased p53 expression. This effect is, at least in part, related to the inhibition of proteasome activity by drug treatments. Together, our results show that concomitant inhibition of topoisomerase II and PARP-1 leads to the synergistic cytotoxic effect toward tumor cells that may be important for combination therapies with NU1025 and topoisomerase II inhibitors. We also confirmed our earlier work and show the important role of PARP-1 activity in the maintenance of the G2 arrest induced by DNA damaging drugs. Finally, based on our studies we propose that NU1025 and possibly other inhibitors of PARP-1 may be used as non-genotoxic agents to activate p53 in tumor cells with non-functional p53 pathways.

Keywords: Abbreviations; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DAPI; 4′,6-diamidino-2-phenyindole diacetate; NU1025; 8-hydroxy-2-methylquinazoline-4-one; m-AMSA; 4’-(9-acridinylamino)methanesulfon-; m; -anisididePARP-1; Topoisomerase II; Combination therapy; Mitotic catastrophe; p53

A novel small-molecule disrupts Stat3 SH2 domain–phosphotyrosine interactions and Stat3-dependent tumor processes by Xiaolei Zhang; Peibin Yue; Steven Fletcher; Wei Zhao; Patrick T. Gunning; James Turkson (pp. 1398-1409).

S3I-201.1066 binds to the Stat3 SH2 domain, disrupts aberrant Stat3 activation and Stat3 function, and induces antitumor effects in vivo in tumors harboring aberrant Stat3 activity.The molecular modeling of the phosphotyrosine (pTyr)–SH2 domain interaction in the Stat3:Stat3 dimerization, combined with in silico structural analysis of the Stat3 dimerization disruptor, S3I-201, has furnished a diverse set of analogs. We present evidence from in vitro biochemical and biophysical studies that the structural analog, S3I-201.1066 directly interacts with Stat3 or the SH2 domain, with an affinity ( K_D) of 2.74μM, and disrupts the binding of Stat3 to the cognate pTyr–peptide, GpYLPQTV–NH₂, with an IC₅₀ of 23μM. Moreover, S3I-201.1066 selectively blocks the association of Stat3 with the epidermal growth factor receptor (EGFR), and inhibits Stat3 tyrosine phosphorylation and nuclear translocation in EGF-stimulated mouse fibroblasts. In cancer cells that harbor aberrant Stat3 activity, S3I-201.1066 inhibits constitutive Stat3 DNA-binding and transcriptional activities. By contrast, S3I-201.1066 has no effect on Src activation or the EGFR-mediated activation of the Erk1/2^MAPK pathway. S3I-201.1066 selectively suppresses the viability, survival, and malignant transformation of the human breast and pancreatic cancer lines and the v-Src-transformed mouse fibroblasts harboring persistently active Stat3. Treatment with S3I-201.1066 of malignant cells harboring aberrantly active Stat3 down-regulated the expression of c-Myc, Bcl-xL, Survivin, the matrix metalloproteinase 9, and VEGF. The in vivo administration of S3I-201.1066-induced significant antitumor response in mouse models of human breast cancer, which correlates with the inhibition of constitutively active Stat3 and the suppression of known Stat3-regulated genes. Our studies identify a novel small-molecule that binds with a high affinity to Stat3, blocks Stat3 activation and function, and thereby induces antitumor response in human breast tumor xenografts harboring persistently active Stat3.

Keywords: Stat3; S3I-201; S3I-201.1066; Small-molecule inhibitor; Antitumor effects; Antitumor cell effects

BFPP, a phloroglucinol derivative, induces cell apoptosis in human chondrosarcoma cells through endoplasmic reticulum stress by Ju-Fang Liu; Wei-Hung Yang; Yi-Chin Fong; Sheng-Chu Kuo; Chih-Shiang Chang; Chih-Hsin Tang (pp. 1410-1417).

Chondrosarcoma is a malignant primary bone tumor that responds poorly to both chemotherapy and radiation therapy. This study is the first to investigate the anticancer effects of the new phloroglucinol derivative (2,4-bis(2-fluorophenylacetyl)phloroglucinol; BFPP) in human chondrosarcoma cells. BFPP induced cell apoptosis in two human chondrosarcoma cell lines, JJ012 and SW1353 but not in primary chondrocytes. BFPP triggered endoplasmic reticulum (ER) stress, as indicated by changes in cytosol calcium levels, and increased glucose-regulated protein 78 (GRP78) expression, but failed to show the same effects on GRP94 expression. BFPP also increased calpain expression and activity. Transfection of cells with GRP78 or calpain siRNA reduced BFPP-mediated cell apoptosis in JJ012 cells. Importantly, animal studies have revealed a dramatic 50% reduction in tumor volume after 21 days of treatment. This study demonstrates novel anticancer activity of BFPP against human chondrosarcoma cells and in murine tumor models.

Keywords: BFPP; Chondrosarcoma; ER; GRP78; Calpain

Differential inhibition of restriction enzyme cleavage by chromophore-modified analogues of the antitumour antibiotics mithramycin and chromomycin reveals structure–activity relationships by Sylvia Mansilla; Irene Garcia-Ferrer; Carmen Méndez; José A. Salas; José Portugal (pp. 1418-1427).

Differential cleavage at three restriction enzyme sites was used to determine the specific binding to DNA of the antitumour antibiotics mithramycin A (MTA), chromomycin A₃ (CRO) and six chromophore-modified analogues bearing shorter side chains attached at C-3, instead of the pentyl chain. All these antibiotics were obtained through combinatorial biosynthesis in the producer organisms. MTA, CRO and their six analogues showed differences in their capacity for inhibiting the rate of cleavage by restriction enzymes that recognize C/G-rich tracts. Changes in DNA melting temperature produced by these molecules were also analyzed, as well as their antiproliferative activities against a panel of colon, ovarian and prostate human carcinoma cell lines. Moreover, the cellular uptake of several analogues was examined to identify whether intracellular retention was related to cytotoxicity. These experimental approaches provided mutually consistent evidence of a seeming correlation between the strength of binding to DNA and the antiproliferative activity of the chromophore-modified molecules. Four of the analogues (mithramycin SK, mithramycin SDK, chromomycin SK and chromomycin SDK) showed promising biological profiles.

Keywords: Mithramycin A; Chromomycin A; ₃; Restriction enzymes; Antitumour antibiotics; Carcinoma cells

N- n-Butyl haloperidol iodide protects against hypoxia/reoxygenation-induced cardiomyocyte injury by modulating protein kinase C activity by Jin-Zhi Wang; Cong-Yi Cai; Yan-Mei Zhang; Jin-Hong Zheng; Yi-Cun Chen; Wei-Qiu Li; Gang-Gang Shi (pp. 1428-1436).

The myocardial protection of F₂ on H/R injury is mediated by inhibition of PKCα and activation of PKCɛ in primary cultured cardiomyocytes. Inhibition of PKCα translocation was associated with downregulation of Egr-1 protein expression and attenuated cell damage, PKCɛ activation is necessary for cardioprotection against cardiomyocyte apoptosis. N- n-Butyl haloperidol iodide (F₂), a novel compound derived from haloperidol, protects against the damaging effects of ischemia/reperfusion (I/R) injury in vitro and in vivo. We tested whether the myocardial protection of F₂ on cardiomyocyte hypoxia/reoxygenation (H/R) injury is mediated by modulating protein kinase C (PKC) activity in primary cultured cardiomyocytes. Primary cultures of ventricular cardiomyocytes underwent 2-h hypoxia and 30-min reoxygenation. Total PKC activity was measured, and the translocation pattern of PKCα, βII, δ and ɛ isoforms was assessed by fractionated western blot analysis. We investigated the association of PKC isoform translocation and H/R-induced injury in the presence and absence of the specific inhibitors and activator. Measurements included cell damage evaluated by creatine kinase (CK) release, and apoptosis measured by annexin V-FITC assay. In primary cultured cardiomyocytes exposed to H/R, PKCα, δ and ɛ were translocated, with no change in PKCβII activity. Total PKC activity, CK release and apoptosis were increased after H/R. Treatment with the conventional PKC inhibitor Gő6976 reduced early growth response-1 (Egr-1) protein expression and attenuated apoptosis. The PKCɛ inhibitor peptide ɛV1–2 increased H/R injury without influencing Egr-1 expression. Pretreatment with F₂ inhibited translocation of PKCα, increased translocation of PKCɛ, and relieved the CK release and apoptosis. The protection of F₂ was blocked in part by the conventional PKC activator thymeleatoxin (TXA) and ɛV1–2 peptide. F₂ significantly alleviated H/R-induced injury, which might be attributed to the combined benefits of inhibiting PKCα and activating PKCɛ.

Keywords: Abbreviations; CK; creatine kinase; DMSO; dimethylsulfoxide; Egr-1; early growth response-1; ERK; extracellular-signal-regulated protein kinase; F; ₂; N; -; n; -butyl haloperidol iodide; Hal; haloperidol; H/R; hypoxia/reoxygenation; I/R; ischemia/reperfusion; IP; ₃; inositol triphosphate; IPC; ischemic preconditioning; JNK; c-Jun N-terminal kinase; PI; propidium iodide; PKC; protein kinase C; PPI; phosphatidylinositol; TXA; thymeleatoxinApoptosis; Cardiomyocytes; Early growth response-1; Hypoxia/reoxygenation; Inflammation; Protein kinase C

A claudin-4 modulator enhances the mucosal absorption of a biologically active peptide by Hiroshi Uchida; Masuo Kondoh; Takeshi Hanada; Azusa Takahashi; Takao Hamakubo; Kiyohito Yagi (pp. 1437-1444).

Biologics, such as peptides, proteins and nucleic acids, are emerging pharmaceuticals. Passage across the epithelium is the first step in the absorption of biologics. Tight junctions (TJ) function as seals between adjacent epithelial cells, preventing free movement of solutes across the epithelium. We previously found that modulation of a key TJ component, claudin-4, is a potent method to enhance jejunal absorption when we used dextran as a model drug and the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) as a claudin-4 modulator. Here, we investigated whether the claudin-4 modulator enhances jejunal, nasal and pulmonary absorption of a biologics human parathyroid hormone derivative, hPTH(1-34). The claudin-4 modulator enhanced nasal but not jejunal and pulmonary absorption of hPTH(1-34). C-CPE is hydrophobic with low solubility of less than 0.3mg/ml, but deletion of 10 amino acids at the N-terminal of C-CPE increased its solubility by 30-fold. Moreover, the N-terminal truncated C-CPE bound to claudin-4, modulated the TJ-barrier and enhanced jejunal absorption of dextran. The N-terminal-truncated C-CPE also enhanced jejunal and pulmonary absorption of hPTH(1-34). This report is the first to indicate that a claudin-4 modulator may be a promising enhancer of the jejunal, pulmonary and nasal absorption of a peptide drug.

Keywords: Abbreviations; TJ; tight junction; C-CPE; the C-terminal fragment of; Clostridium perfringens; enterotoxin; C-CPE184; C-terminal fragment of; Clostridium perfringens; enterotoxin from 184 to 319 amino acids; hPTH; human parathyroid hormone; CPE; Clostridium perfringens; enterotoxin; DDM; n-Dodecyl-β-; d; -maltoside; EDC; N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide; NHS; N-hydroxysuccinimide; C-CPE194; C-terminal fragment of; Clostridium perfringens; enterotoxin from 194 to 319 amino acids; C-CPE205; C-terminal fragment of; Clostridium perfringens; enterotoxin from 205 to 319 amino acids; PBS; phosphate-buffered saline; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; BV; budded baculovirus; TBS; Tris-buffered saline; ELISA; enzyme-linked immunosorbent assay; SPR; surface plasmon resonance; TEER; transepithelial electric resistance; FD-4; fluorescein isothiocyanate-dextran with a molecular weight of 4 kDa; AUC; the area under the plasma concentration; BA; bioavailabilityMucosal absorption; Claudin; Paracellular route; Tight junction; Epithelial barrier

Distinction of microsomal prostaglandin E synthase-1 (mPGES-1) inhibition from cyclooxygenase-2 inhibition in cells using a novel, selective mPGES-1 inhibitor by Gabriel Mbalaviele; Adele M. Pauley; Alexander F. Shaffer; Ben S. Zweifel; Sumathy Mathialagan; Stephen J. Mnich; Olga V. Nemirovskiy; Jeff Carter; James K. Gierse; Jane L. Wang; Michael L. Vazquez; William M. Moore; Jaime L. Masferrer (pp. 1445-1454).

Inflammation-induced microsomal prostaglandin E synthase-1 (mPGES-1) is the terminal enzyme that synthesizes prostaglandin E₂ (PGE₂) downstream of cyclooxygenase-2 (COX-2). The efficacy of nonsteroidal anti-inflammatory drugs and COX-2 inhibitors in the treatment of the signs and symptoms of osteoarthritis, rheumatoid arthritis and inflammatory pain, largely attributed to the inhibition of PGE₂ synthesis, provides a rationale for exploring mPGES-1 inhibition as a potential novel therapy for these diseases. Toward this aim, we identified PF-9184 as a novel mPGES-1 inhibitor. PF-9184 potently inhibited recombinant human (rh) mPGES-1 (IC₅₀=16.5±3.8nM), and had no effect against rhCOX-1 and rhCOX-2 (>6500-fold selectivity). In inflammation and clinically relevant biological systems, mPGES-1 expression, like COX-2 expression was induced in cell context- and time-dependent manner, consistent with the kinetics of PGE₂ synthesis. In rationally designed cell systems ideal for determining direct effects of the inhibitors on mPGES-1 function, but not its expression, PF-9184 inhibited PGE₂ synthesis (IC₅₀ in the range of 0.5–5μM in serum-free cell and human whole blood cultures, respectively) while sparing the synthesis of 6-keto-PGF_1α (PGF_1α) and PGF_2α. In contrast, as expected, the selective COX-2 inhibitor, SC-236, inhibited PGE₂, PGF_1α and PGF_2α synthesis. This profile of mPGES-1 inhibition, distinct from COX-2 inhibition in cells, validates mPGES-1 as an attractive target for therapeutic intervention.

Keywords: Abbreviations; COX-2; cyclooxygenase-2; cPGES; cytosolic PGES; mPGES-1; microsomal prostaglandin E synthase-1; PGE; ₂; prostaglandin E; ₂; NSAIDs; nonsteroidal anti-inflammatory drugs; COXibs; COX-2 inhibitors; RA; rheumatoid arthritis; OA; osteoarthritis; TXB; ₂; thromboxane B; ₂; RASF; synovial fibroblasts derived from patients with rheumatoid arthritis; rh; recombinant human; FLAP; 5-lipoxygenase activating proteinProstaglandins; Cyclooxygenase-2; mPGES-1; Arthritis; Inflammation

Myricetin suppresses UVB-induced wrinkle formation and MMP-9 expression by inhibiting Raf by Sung Keun Jung; Ki Won Lee; Ho Young Kim; Mi Hyun Oh; Sanguine Byun; Sung Hwan Lim; Yong-Seok Heo; Nam Joo Kang; Ann M. Bode; Zigang Dong; Hyong Joo Lee (pp. 1455-1461).

Myricetin inhibited UVB-induced wrinkle formation and MMP-9 expression by directly inhibiting Raf kinase activity in mouse skin.Chronic exposure to solar ultraviolet (UV) light causes skin photoaging. Many studies have shown that naturally occurring phytochemicals have anti-photoaging effects, but their direct target molecule(s) and mechanism(s) remain unclear. We found that myricetin, a major flavonoid in berries and red wine, inhibited wrinkle formation in mouse skin induced by chronic UVB irradiation (0.18J/cm², 3 days/week for 15 weeks). Myricetin treatment reduced UVB-induced epidermal thickening of mouse skin and also suppressed UVB-induced matrix metalloproteinase-9 (MMP-9) protein expression and enzyme activity. Myricetin appeared to exert its anti-aging effects by suppressing UVB-induced Raf kinase activity and subsequent attenuation of UVB-induced phosphorylation of MEK and ERK in mouse skin. In vitro and in vivo pull-down assays revealed that myricetin bound with Raf in an ATP-noncompetitive manner. Overall, these results indicate that myricetin exerts potent anti-photoaging activity by regulating MMP-9 expression through the suppression of Raf kinase activity.

Keywords: Myricetin; Anti-wrinkle; Raf; Photoaging; UVB

Pyrazolo[4,3-c]isoquinolines as potential inhibitors of NF-κB activation by Jérémie Mortier; Raphaël Frederick; Corinne Ganeff; Caroline Remouchamps; Patrice Talaga; Lionel Pochet; Johan Wouters; Jacques Piette; Emmanuel Dejardin; Bernard Masereel (pp. 1462-1472).

Docking of a new inhibitor of TAK1 (IC₅₀=0.56μM) and identification of potential interactions with the hinge region:In this work, we aimed to build a 3D-model of NIK and to study the binding of pyrazolo[4,3-c]isoquinolines with a view to highlight the structural elements responsible for their inhibitory potency. However, in the course of this work, we unexpectedly found that the pyrazolo[4,3-c]isoquinolines initially reported as NIK inhibitors were neither inhibitors of this enzyme nor of the alternative NF-κB pathway, but were in fact inhibitors of another kinase, the TGF-β activated kinase 1 (TAK1) which is involved in the classical NF-κB pathway.

Keywords: NF-κB inducing kinase; TGF-β activated kinase 1; Pyrazoloisoquinolines; Alternative pathway; Rheumatoid arthritis; Molecular modeling

Cyanidin suppresses ultraviolet B-induced COX-2 expression in epidermal cells by targeting MKK4, MEK1, and Raf-1 by Jong-Eun Kim; Jung Yeon Kwon; Sang Kwon Seo; Joe Eun Son; Sung Keun Jung; So Yun Min; Mun Kyung Hwang; Yong-Seok Heo; Ki Won Lee; Hyong Joo Lee (pp. 1473-1482).

Skin cancer is the most frequently diagnosed cancer in the United States. Ultraviolet B (UVB) rays (wavelength: 280–320nm) play a pivotal role in the development of skin cancer by inducing the expression of inflammatory proteins such as cyclooxygenase-2 (COX-2). Cyanidin, the most plentiful of the plant pigments known as anthocyanidins, is a potent chemopreventive agent. In the present study, we examined the molecular mechanisms underlying the chemopreventive activity of cyanidin and identified its molecular targets. Cyanidin inhibited UVB-induced COX-2 expression and prostaglandin E₂ secretion in the epidermal skin cell line JB6 P+ by suppressing the transactivation of nuclear factor-κB and activator protein-1 which are well-known transcription factors regulated by mitogen-activated protein kinase. Cyanidin markedly inhibited the phosphorylation of JNK1/2, ERK1/2, and MEK1/2 than the of MKK4 and Raf-1, two upstream kinases of JNK1/2, ERK1/2, and MEK1/2. Cyanidin significantly suppressed the activities of MKK4, MEK1, and Raf-1 through direct binding. Transient transfection of a small interfering RNA specific for MKK4 inhibited the UVB-induced expression of COX-2 in JB6 P+ cells, as did the expression of a dominant-negative ERK2 mutant. We conclude that MKK4, MEK1, and Raf-1 are targets of cyanidin for the suppression of UVB-induced COX-2 expression.

Keywords: COX-2; Cyanidin; MEK1; MKK4; Raf-1; UVB

Modulation of metalloproteinase-9 in U87MG glioblastoma cells by A₃ adenosine receptors by Stefania Gessi; Valeria Sacchetto; Eleonora Fogli; Stefania Merighi; Katia Varani; Pier Giovanni Baraldi; Mojgan Aghazadeh Tabrizi; Edward Leung; Stephen Maclennan; Pier Andrea Borea (pp. 1483-1495).

In this work, we investigated the biological functions of adenosine (ado) in metalloproteinase-9 (MMP-9) regulation in U87MG human glioblastoma cells. The nucleoside was able to increase both MMP-9 mRNA and protein levels through A₃ receptors activation. We revealed that A₃ receptor stimulation induced an increase of MMP-9 protein levels in cellular extracts of U87MG cells by phosphorylation of extracellular signal-regulated protein kinases (ERK1/2), c-Jun N-terminal kinase/stress-activated protein kinase (pJNK/SAPK), protein kinase B (Akt/PKB) and finally activator protein 1 (AP-1). A₃ receptor activation stimulated also an increase of extracellular MMP-9 in the supernatants from U87MG glioblastoma cells. Finally, the Matrigel invasion assay demonstrated that A₃ receptors, by inducing an increase in MMP-9 levels, was responsible for an increase of glioblastoma cells invasion. Collectively, these results suggest that ado, through A₃ receptors activation, modulates MMP-9 protein levels and plays a role in increasing invasion of U87MG cells.

Keywords: Abbreviations; ADA; adenosine deaminase; Ado; adenosine; AP-1; activator protein 1; Akt/PKB; protein kinase B; CGS 21680; 2-[; p; -(carboxyethyl)-phenethylamino]-NECA; CHA; N; ⁶; -cyclohexyladenosine; Cl-IB-MECA; N; ⁶; (3-iodobenzyl)2-chloroadenosine-5′; N; -methyluronamide; DPA23; 1-deoxy-1-[6-{4-[(phenylcarbamoyl)-methoxy]phenylamino}-9H-purin-9-yl]-; N; -ethyl-β-d-ribofuranuronamide; DPCPX; 1,3-dipropyl-8-cyclopentyl-xanthine; EHNA; erythro-9-(2-hydroxy-3-nonyl)adenine; ERK1/2; extracellular signal-regulated kinases; MMP-9; metalloproteinase-9; MRE 2029F20; N; -benzo[1,3]dioxol-5-yl-2-[5-(1,3-dipropyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yl-oxy]-acetamide]; MRE 3008F20; 5-; N-; (4-methoxyphenylcarbamoyl)-amino-8-propyl-2-(2-furyl)-pyrazolo[4,3e]-1,2,4-triazolo[1,5c]pyrimidine; NDGA; Nordihydroguaiaretic acid; pJNK/SAPK; c-Jun N-terminal kinase/stress-activated protein kinase; SB202190; 4-[4-(4-Fluorophenyl)-5-(4-pyridinyl)-1H-imidazol-2-yl]pieno; SCH 58261; 5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-; e; ]-1,2,4-triazolo[1,5-; c; ]-pyrimidine; SH-5; [d-3-deoxy-2-O-methyl-myo-inositol-1-[(R)-2-methoxy-3-(octadecyloxy)propyl hydrogen phosphate]]; siRNA; small interfering RNA; SP600125; anthra[1-9cd]pyrazol-6(2H)-one; ZM; 241385 (4-(2-[7-amino-2-(2-furyl)-[1,2,4]triazolo-[2,32][1,3,6]triazinyl-amino]ethyl)-phenol); U0126; 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadieneA; ₃; adenosine receptors; MMP-9; Glioblastoma cells; Intracellular signalling; Cell invasion

Irsogladine maleate regulates neutrophil migration and E-cadherin expression in gingival epithelium stimulated by Aggregatibacter actinomycetemcomitans by Tsuyoshi Fujita; Akiyoshi Kishimoto; Hideki Shiba; Kouichi Hayashida; Mikihito Kajiya; Yuushi Uchida; Shinji Matsuda; Katsuhiro Takeda; Kazuhisa Ouhara; Hiroyuki Kawaguchi; Yoshimitsu Abiko; Hidemi Kurihara (pp. 1496-1505).

Irsogladine maleate (IM) counters Aggregatibacter actinomycetemcomitans-induced reduction of the gap junction intercellular communication and the expression of zonula occludens-1, which is a major tight junction structured protein, in cultured human gingival epithelial cells (HGEC). In addition, IM obviates the A. actinomycetemcomitans-induced increase in interleukin (IL)-8 levels in HGEC. Thus, by regulating the intercellular junctional complex and chemokine secretion in HGEC, IM may be useful to prevent periodontal disease. To clarify the effects and regulatory mechanism of IM in vivo and in vitro, we examined the expression of E-cadherin and neutrophil chemotaxis induced by A. actinomycetemcomitans under IM pretreatment. Immunohistochemical studies revealed that A. actinomycetemcomitans application to the gingival sulcus decreased the number of cells positive for E-cadherin and increased those positive for cytokine-induced neutrophil chemoattractant-2α (CINC-2α) in rat gingival epithelium. However, in IM-pretreated rats, A. actinomycetemcomitans application had little effect on CINC-2α and E-cadherin in gingival epithelium. In cultured HGEC, real-time PCR and Western blotting showed that IM and the ERK inhibitor PD98059 abolished the A. actinomycetemcomitans-induced increase in CXCL-1 and IL-8 in HGEC. On the other hand, IM, PD98059, and the p38 MAP kinase inhibitor SB203580 recovered the decrease in E-cadherin expression. In addition, conditioned medium from A. actinomycetemcomitans-stimulated HGEC enhanced human neutrophil chemotaxis, compared to that from un-stimulated HGEC or that from A. actinomycetemcomitans-stimulated HGEC under IM pretreatment. Furthermore, IM down-regulated the p38 MAP kinase and ERK phosphorylations induced by A. actinomycetemcomitans. In conclusion, IM may control A. actinomycetemcomitans-induced gingival inflammation by regulating neutrophil migration and E-cadherin expression in gingival epithelium.

Keywords: Irsogladine maleate; Aggregatibacter actinomycetemcomitans; E-cadherin; Neutrophil migration; CXC-chemokine; Gingival epithelial cells

Blockade of LTB₄-induced chemotaxis by bioactive molecules interfering with the BLT2-Gα_i interaction by Joo-Young Kim; Won-Kyu Lee; Yeon Gyu Yu; Jae-Hong Kim (pp. 1506-1515).

BLT2, a low-affinity leukotriene B₄ (LTB₄) receptor, is a member of the G-protein coupled receptor (GPCR) family and is involved in the pathogenesis of inflammatory diseases such as asthma. Despite its clinical implications, however, no pharmacological inhibitors are available. In the present study, we screened for small molecules that interfere with the interaction between the third intracellular loop region of BLT2 (BLT2_iL3) and the Gα_i3 protein subunit (Gα_i3), using a high-throughput screening (HTS) assay with a library of 1040 FDA-approved drugs and bioactive compounds. We identified two small molecules—purpurin [1,2,4-trihydroxy-9,10-anthraquinone; IC₅₀=1.6μM for BLT2] and chloranil [tetrachloro-1,4-benzoquinone; IC₅₀=0.42μM for BLT2]—as specific BLT2-blocking agents. We found that blockade of the BLT2_iL3-Gα_i3 interaction by these small molecules inhibited the BLT2-downstream signaling cascade. For example, BLT2-signaling to phosphoinositide-3 kinase (PI3K)/Akt phosphorylation was completely abolished by these molecules. Furthermore, we observed that these small molecules blocked LTB₄-induced chemotaxis by inhibiting the BLT2-PI3K/Akt-downstream, Rac1-reactive oxygen species-dependent pathway. Taken together, our results show that purpurin and chloranil interfere with the interaction between BLT2_iL3 and Gα_i3 and thus block the biological functions of BLT2 (e.g., chemotaxis). The present findings suggest a potential application of purpurin and chloranil as pharmacological therapeutic agents against BLT2-associated inflammatory human diseases.

Keywords: Chemotaxis; LTB; ₄; BLT2; Purpurin; Chloranil

Identification of metabolic pattern and bioactive form of resveratrol in human medulloblastoma cells by Xiao-Hong Shu; Hong Li; Zheng Sun; Mo-Li Wu; Jing-Xin Ma; Jian-Min Wang; Qian Wang; Yuan Sun; Yuan-Shan Fu; Xiao-Yan Chen; Qing-You Kong; Jia Liu (pp. 1516-1525).

trans-Resveratrol but not its cis- and sulfate counterparts exhibits anti-medulloblastoma efficacy (A) and medulloblastoma UW228-3 cells show less metabolic capacity due to the lower brain-associated sulfotransferase/SULT1A1, 1C2 and 4A1 levels than that expressed in the rat normal brain (B) (*) p<0.001.Cancer preventive reagent trans-resveratrol is intracellularly biotransformed to different metabolites. However, it is still unclear whether trans-resveratrol exerts its biological effects directly or through its metabolite(s). This issue was addressed here by identifying the metabolic pattern and the bioactive form of resveratrol in a resveratrol-sensitive human medulloblastoma cell line, UW228-3. The cell lysates and condition media of UW228-3 cells with or without 100μM resveratrol treatment were analyzed by HPLC and LC/MS which revealed (1) that resveratrol was chemically unstable and the spontaneous generation of cis-resveratrol reduced resveratrol's anti-medulloblastoma efficacy and (2) that resveratrol monosulfate was the major metabolite of the cells. To identify the bioactive form of resveratrol, a mixture-containing approximately half fraction of resveratrol monosulfate was prepared by incubating trans-resveratrol with freshly prepared rat brain lysates. Medulloblastoma cells treated by 100μM of this mixture showed attenuated cell crisis. The overall levels of the three brain-associated sulfotransferases (SULT1A1, 1C2 and 4A1) were low in medulloblastoma cells in vivo and in vitro in comparison with that in human noncancerous and rat normal cerebella; resveratrol could more or less up-regulate the production of these enzymes in UW228-3 cells but their overall level was still lower than that in normal cerebellum tissue. Our study thus demonstrated for the first time that trans-resveratrol is the bioactive form in medulloblastoma cells in which the expression of brain-associated SULTs was down-regulated, resulting in the increased intracellular bioavailability and anti-medulloblastoma efficacy of trans-resveratrol.

Keywords: Abbreviations; Resveratrol; Res or R; HPLC; high performance liquid chromatography; MS; mass spectrum; LC/MS; liquid chromatography coupled with tandem mass spectrum; SULT; sulfotransferases; MB; medulloblastoma; DMEM; Dulbecco's modified Eagle's essential medium; ICC; immunocytochemical staining; TUNEL; terminal deoxynucleotidyl transferase mediated nick end labeling; PBS; phosphate buffered saline solution; SPE; solid phase extraction; ESI; electrospray ionisation; FWHM; full width at half maximum; TMA; the tissue microarray; IHC; immunohistochemical staining; DAB; 3,3′-diaminobenzidine tetrahydrochloride; SDS-PAGE; sodium dodecylsulfate polyacrylamide gel electrophoresis; N2; nebulizing gas; LC/MS-IT-TOF; ion trap-time-of-flight hybrid mass spectrometer; HRMS; high resolution mass spectrometry; Mops; 3-[N-morpholino] propanesulfonic acid; PAPS; 3′-phosphoadenosine 5′-phosphosulfate; DTT; dithiothreitol; STAT3; signal transducer and activator of transcription 3; RT-PCR; reverse transcription-polymerase chain reaction; TIC; total ion chromatogram; CID; collision induced dissociationResveratrol; Medulloblastoma; Drug metabolism; Sulfotransferase; Chemosensitivity

Stereospecific reduction of a potent kinesin spindle protein (KSP) inhibitor in human tissues by Chunze Li; Bing Lu; Robert M. Garbaccio; Edward S. Tasber; Mark E. Fraley; George D. Hartman; Jingjing Ye; Jane C. Harrelson; Thomayant Prueksaritanont (pp. 1526-1533).

Compound A, 1-{(3 R,3a R)-3-[3-(4-acetylpiperazin-1-yl)propyl]-7-fluoro-3-phenyl-3a,4-dihydro-3 H-pyrazolo[5,1- c][1,4]benzoxazin-2-yl}ethanone, is a novel and potent inhibitor of the mitotic kinesin spindle protein. Metabolism studies with human hepatocytes showed that Compound A underwent significant ketone reduction to its biologically active metabolite M1. Here, we describe the studies that characterized the metabolic interconversion between Compound A and M1 in vitro in human tissues. LC–MS/MS analysis showed that the ketone reduction was stereospecific for M1 with no diastereomer of M1 detected in incubations with human hepatocytes. Interestingly, such stereospecific ketone reduction was not observed with Compound B, the enantiomer of Compound A. No reductive products were observed when Compound B was incubated with human hepatocytes. Studies with human liver subcellular fractions showed that Compound A was reduced to M1 primarily by human liver cytosol with little contribution from human liver microsomes and mitochondria. NADPH was the preferred cofactor for the reduction reaction. Reverse oxidation of M1 back to Compound A was also observed, preferentially in human liver cytosol with NADP⁺ as the cofactor. The interconversion between Compound A and M1 in human liver cytosol was inhibited significantly by flufenamic acid and phenolphthalein (potent inhibitors for aldo–keto reductase 1Cs, p<0.05), but not by menadione, a selective inhibitor for carbonyl reductase. In addition to the liver, S9 from human lung and kidney was also capable of catalyzing this interconversion. Collectively, the results implicated the aldo–keto reductase 1Cs as the most likely enzymes responsible for the metabolic interconversion of Compound A and its active metabolite M1.

Keywords: Abbreviations; AKRs; aldo–keto reductases; HSD; hydroxysteroid dehydrogenase; I.S.; an internal standard; KSP; kinesin spindle protein; NADPH_R; NADPH regeneration system; SDRs; short-chain dehydrogenases/reductasesKetone reduction; Stereospecificity; Cofactor dependency; Aldo–keto reductase; Subcellular and tissue distribution

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Biochemical Pharmacology (v.79, #10)

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Biochemical Pharmacology (v.79, #10)