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Biochemical Pharmacology (v.80, #6)

Editorial Advisory Board (pp. iii).

Graphical Abstracts Contents (pp. e1-e6).

Functions and transcriptional regulation of adult human hepatic UDP-glucuronosyl-transferases (UGTs): Mechanisms responsible for interindividual variation of UGT levels by Karl Walter Bock (pp. 771-777).

Ten out of 19 UDP-glucuronosyltransferases (UGTs) are substantially expressed in adult human liver (>1% of total UGTs); 5 UGT1 isoforms (UGT1A1, 1A3, 1A4, 1A6 and 1A9) and 5 UGT2 family members (UGT2B4, 2B7, 2B10, 2B15 and 2B17) (Izukawa et al. ). Surprisingly, UGT2B4 and UGT2B10 mRNA were found to be abundant in human liver suggesting an underestimated role of the liver in detoxification of their major substrates, bile acids and eicosanoids. Among factors responsible for high interindividual variation of hepatic UGT levels (genetic diversity including polymorphisms and splice variants, regulation by liver-enriched transcription factors such as HNF1 and HNF4, and ligand-activated transcription factors) nuclear receptors (PXR, CAR, PPARα, etc.), and the Ah receptor are discussed. Unraveling the mechanisms responsible for interindividual variation of UGT expression will be beneficial for drug therapy but still remains a major challenge.

Keywords: Abbreviations; AhR; Ah receptor; CAR; constitutive androstane receptor; PPAR; peroxisome proliferator-activated receptor; PXR; pregnane X receptor; UGT; UDP-glucuronosyltransferaseHepatic UGTs; Human liver; Interindividual variation; Transcription factors; Xenosensors

Peptide-based inhibitors of the phagocyte NADPH oxidase by Jamel El-Benna; Pham My-Chan Dang; Axel Périanin (pp. 778-785).

*Represents the localization of the inhibitory peptides.Phagocytes such as neutrophils, monocytes and macrophages play an essential role in host defenses against pathogens. To kill these pathogens, phagocytes produce and release large quantities of antimicrobial molecules such as reactive oxygen species (ROS), microbicidal peptides, and proteases. The enzyme responsible for ROS generation is called NADPH oxidase, or respiratory burst oxidase, and is composed of six proteins: gp91phox, p22phox, p47phox, p67phox, p40phox and Rac1/2. The vital importance of this enzyme in host defenses is illustrated by a genetic disorder called chronic granulomatous disease (CGD), in which the phagocyte NADPH oxidase is dysfunctional, leading to life-threatening recurrent bacterial and fungal infections. However, excessive NADPH oxidase activation and ROS over-production can damage surrounding tissues and participate in exaggerated inflammatory processes. As ROS production is believed to be involved in several inflammatory diseases, specific phagocyte NADPH oxidase inhibitors might have therapeutic value. In this commentary, we summarize the structure and activation of the phagocyte NADPH oxidase, and describe pharmacological inhibitors of this enzyme, with particular emphasis on peptide-based inhibitors derived from gp91phox, p22phox and p47phox.

Keywords: Abbreviations; DPI; diphenylene iodonium; CGD; chronic granulomatous disease; fMLF; formyl-methionyl-leucyl-phenylalanine; MPO; myeloperoxidase; phox; phagocyte oxidase; ROS; reactive oxygen speciesPhagocytes; NADPH oxidase; Inhibitors; gp91phox/NOX2; p22phox; p47phox

Accumulation of thymidine-derived sugars in thymidine phosphorylase overexpressing cells by I.V. Bijnsdorp; K. Azijli; E.E. Jansen; M.M. Wamelink; C. Jakobs; E.A. Struys; M. Fukushima; F.A.E. Kruyt; G.J. Peters (pp. 786-792).

Thymidine (TdR) is converted by thymidine phosphorylase (TP) to deoxyribose-1-phosphate (dR-1-P), which can enter various metabolic pathways, which potentially contribute to TP mediated angiogenesis. dR=Deoxyribose, dR-1-P=deoxyribose-1-phosphate, dR-5-P=deoxyribose-5-phosphate, G3P=glyceraldehyde-3-phosphate.Thymidine phosphorylase (TP) is often overexpressed in cancer and potentially plays a role in the stimulation of angiogenesis. The exact mechanism of angiogenesis induction is unclear, but is postulated to be related to thymidine-derived sugars. TP catalyzes the conversion of thymidine (TdR) to thymine and deoxyribose-1-phosphate (dR-1-P), which can be converted to dR-5-P, glyceraldehyde-3-phosphate (G3P) or deoxyribose (dR). However, it is unclear which sugar accumulates in this reaction. Therefore, in the TP overexpressing Colo320 TP1 and RT112/TP cells we determined by LC–MS/MS which sugars accumulated, their subcellular localization (using³H-TdR) and whether dR was secreted from the cells. In both TP-overexpressing cell lines, dR-1-P and dR-5-P accumulated intracellularly at high levels and dR was secreted extensively by the cells. A specific inhibitor of TP completely blocked TdR conversion, and thus no sugars were formed. To examine whether these sugars may be used for the production of angiogenic factors or other products, we determined with³H-TdR in which subcellular location these sugars accumulated. TdR-derived sugars accumulated in the cytoskeleton and to some extent in the cell membrane, while incorporation into the DNA was responsible for trapping in the nucleus. In conclusion, various metabolic routes were entered, of which the TdR-derived sugars accumulated in the cytoskeleton and membrane. Future studies should focus on which exact metabolic pathway is involved in the induction of angiogenesis.

Keywords: Abbreviations; TP; thymidine phosphorlylase; TdR; thymidine; G3P; glyceraldehyde-3-phosphate; dR; deoxyribose; dR-1-P; deoxyribose-1-phosphate; dR-5-P; deoxyribose-5-phosphate; TPI; thymidine phosphorylase inhibitor; AGE; advanced glycation endproducts; VEGF; vascular endothelial growth factor; IL-8; interleukin-8Thymidine phosphorylase; Angiogenesis; Deoxyribose; Thymidine phosphorylase inhibitor

CDC50A plays a key role in the uptake of the anticancer drug perifosine in human carcinoma cells by Francisco Muñoz-Martínez; Cristina Torres; Santiago Castanys; Francisco Gamarro (pp. 793-800).

Functional aminophospholipid translocases are composed of at least two proteins: an alpha subunit from the P4 subfamily of P-type ATPases and a beta subunit from the CDC50-Lem3p family. Over-expression and knockdown of the human beta subunit CDC50A in KB cells enhanced and decreased, respectively, the uptake of both fluorescent aminophospholipid analogues and the anticancer alkyl-phospholipid perifosine. Confocal microscopy showed that CDC50A-V5 was localized at the endoplasmic reticulum and the Golgi complex of both KB (perifosine-sensitive) and KB PER-R (perifosine-resistant, alkyl-phospholipid uptake deficient) cells, but was only widely distributed in the early and late endosomes in KB cells. Biotinylation of cell surface proteins allowed CDC50A-V5 to be detected in the plasma membrane of KB cells but not in KB PER-R cells, thereby suggesting a defect in CDC50A trafficking that could explain the inability of KB PER-R to uptake perifosine. Over-expression of CDC50A in HeLa and HEK293T cells did not increase uptake, since the protein was retained at the endoplasmic reticulum and Golgi. However, when CDC50A was co-expressed with the P4-ATPase Atp8b1, the two proteins co-localized at the plasma membrane and the uptake of aminophospholipids and perifosine increased strikingly in both cell lines. These findings suggest that CDC50A plays a key role in perifosine uptake in human cells, presumably by forming a functional plasma membrane translocator in combination with a P4-ATPase.

Keywords: Perifosine uptake; CDC50A; Atp8b1; P4-ATPase; Aminophospholipid translocase; Resistance to perifosine; Cancer chemotherapy

Inhibition of cellular Shp2 activity by a methyl ester analog of SPI-112 by Liwei Chen; Daniele Pernazza; Latanya M. Scott; Harshani R. Lawrence; Yuan Ren; Yunting Luo; Xin Wu; Shen-Shu Sung; Wayne C. Guida; Said M. Sebti; Nicholas J. Lawrence; Jie Wu (pp. 801-810).

The protein tyrosine phosphatase (PTP) Shp2 ( PTPN11) is an attractive target for anticancer drug discovery because it mediates growth factor signaling and its gain-of-function mutants are causally linked to leukemias. We previously synthesized SPI-112 from a lead compound of Shp2 inhibitor, NSC-117199. In this study, we demonstrated that SPI-112 bound to Shp2 by surface plasmon resonance (SPR) and displayed competitive inhibitor kinetics to Shp2. Like some other compounds in the PTP inhibitor discovery efforts, SPI-112 was not cell permeable, precluding its use in biological studies. To overcome the cell permeation issue, we prepared a methyl ester SPI-112 analog (SPI-112Me) that is predicted to be hydrolyzed to SPI-112 upon entry into cells. Fluorescence uptake assay and confocal imaging suggested that SPI-112Me was taken up by cells. Incubation of cells with SPI-112Me inhibited epidermal growth factor (EGF)-stimulated Shp2 PTP activity and Shp2-mediated paxillin dephosphorylation, Erk1/2 activation, and cell migration. SPI-112Me treatment also inhibited Erk1/2 activation by a Gab1-Shp2 chimera. Treatment of Shp2^E76K mutant-transformed TF-1 myeloid cells with SPI-112Me resulted in inhibition of Shp2^E76K-dependent cell survival, which is associated with inhibition of Shp2^E76K PTP activity, Shp2^E76K-induced Erk1/2 activation, and Bcl-XL expression. Furthermore, SPI-112Me enhanced interferon-γ (IFN-γ)-stimulated STAT1 tyrosine phosphorylation, ISRE-luciferase reporter activity, p21 expression, and the anti-proliferative effect. Thus, the SPI-112 methyl ester analog was able to inhibit cellular Shp2 PTP activity.

Keywords: Abbreviations; AO; acridine orange; DiFMUP; 6,8-difluoro-4-methylumbelliferyl phosphate; DiFMU; 6,8-difluoro-4-methylumbelliferone; DMEM; Dulbecco's modified Eagle's medium; DMSO; dimethyl sulfoxide; dox; doxycycline; DTT; dithiothreitol; EB; Ethidium bromide; EGF; epidermal growth factor; Erk1/2; extracellular signal-regulated kinases 1 and 2; FBS; fetal bovine serum; GM-CSF; granulocyte-macrophage colony-stimulating factor; HPLC; high performance liquid chromatography; LC/MS; liquid chromatography/mass spectrometry; JMML; juvenile myelomonocytic leukemia; IFN; interferon; MAP kinase; mitogen-activated protein kinase; PTP; protein tyrosine phosphatase; SPI-112; (; Z; )-3-(2-(5-(; N; -(4-fluorobenzyl)sulfamoyl)-2-oxoindolin-3-ylidene)hydrazinyl)benzoic acid; SPI-112Me; (; Z; )-3-(2-(5-(; N; -(4-fluorobenzyl)sulfamoyl)-2-oxoindolin-3-ylidene)hydrazinyl)benzoic acid methyl esterPTPN11; Shp2; Protein tyrosine phosphatase; Inhibitor; Erk1/2

Impact of system L amino acid transporter 1 (LAT1) on proliferation of human ovarian cancer cells: A possible target for combination therapy with anti-proliferative aminopeptidase inhibitors by Xuetao Fan; Douglas D. Ross; Hiroshi Arakawa; Vadivel Ganapathy; Ikumi Tamai; Takeo Nakanishi (pp. 811-818).

Amino acids activate nutrient signaling via the mammalian target of rapamycin (mTOR), we therefore evaluated the relationship between amino acid transporter gene expression and proliferation in human ovarian cancer cell lines. Expression of three cancer-associated amino acid transporter genes, LAT1, ASCT2 and SN2, was measured by qRT-PCR and Western blot. The effects of silencing the LAT1 gene and its inhibitor BCH on cell growth were evaluated by means of cell proliferation and colony formation assays. The system L amino acid transporter LAT1 was up-regulated in human ovarian cancer SKOV3, IGROV1, A2780, and OVCAR3 cells, compared to normal ovarian epithelial IOSE397 cells, whereas ASCT2 and SN2 were not. BCH reduced phosphorylation of p70S6K, a down-stream effector of mTOR, in SKOV3 and IGROV1 cells, and decreased their proliferation by 30% and 28%, respectively. Although proliferation of SKOV3 (S1) or IGROV1 (I10) cells was unaffected by LAT1-knockdown, plating efficiency in colony formation assays was significantly reduced in SKOV3(S1) and IGROV1(I10) cells to 21% and 52% of the respective plasmid transfected control cells, SKOV3(SC) and IGROV(IC), suggesting that LAT1 affects anchorage-independent cell proliferation. Finally, BCH caused 10.5- and 4.3-fold decrease in the IC₅₀ value of bestatin, an anti-proliferative aminopeptidase inhibitor, in IGROV1 and A2780 cells, respectively, suggesting that the combined therapy is synergistic. Our findings indicate that LAT1 expression is increased in human ovarian cancer cell lines; LAT1 may be a target for combination therapy with anti-proliferative aminopeptidase inhibitors to combat ovarian cancer.

Keywords: Abbreviations; LAT1; System L amino acid transporter; BCH; 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid; mTOR; mammalian target of rapamycin; p70S6K; p70 S6 kinase; SRB; sulforhodamine B; PI3K; phosphatidylinositol-3-kinase; ASCT2; system ASC amino acid transporter 2; SN2; system N amino acid transporter 2; HPRT; hypoxanthine phosphoribosyltransferase 1; RT-PCR; reverse transcription polymerase chain reaction; XTT; (2,3)-bis-(2-methoxy-4-nitro-5-sulphenyl)-(2H)-tetrazolium-5-carboxanilideAmino acid transporter; LAT1; Ovarian cancer; mTOR (mammalian target of rapamycin); Bestatin

A novel steroidal inhibitor of estrogen-related receptor α (ERRα) by Sarah J. Duellman; Joy M. Calaoagan; Barbara G. Sato; Richard Fine; Boris Klebansky; Wan-Ru Chao; Peter Hobbs; Nathan Collins; Lidia Sambucetti; Keith R. Laderoute (pp. 819-826).

A selective, purely steroidal inhibitor of ERRα has utility both as an experimental anticancer agent and as a chemical probe of ERRα biology. SR16388 inhibits tumor growth of PC3 prostate cancer xenografts in nude mice.The orphan nuclear receptor estrogen-related receptor α (ERRα) has been implicated in the development of various human malignancies, including breast, prostate, ovary, and colon cancer. ERRα, bound to a co-activator protein (e.g., peroxisome proliferator receptor γ co-activator-1α, PGC-1α), regulates cellular energy metabolism by activating transcription of genes involved in various metabolic processes, such as mitochondrial genesis, oxidative phosphorylation, and fatty acid oxidation. Accumulating evidence suggests that ERRα is a novel target for solid tumor therapy, conceivably through effects on the regulation of tumor cell energy metabolism associated with energy stress within solid tumor microenvironments. This report describes a novel steroidal antiestrogen (SR16388) that binds selectively to ERRα, but not to ERRβ or ERRγ, as determined using a time-resolved fluorescence resonance energy transfer assay. SR16388 potently inhibits ERRα’s transcriptional activity in reporter gene assays, and prevents endogenous PGC-1α and ERRα from being recruited to the promoters or enhancers of target genes. Representative in vivo results show that SR16388 inhibited the growth of human prostate tumor xenografts in nude mice as a single agent at 30mg/kg given once daily and 100mg/kg given once weekly. In a combination study, SR16388 (10mg/kg, once daily) and paclitaxel (7.5mg/kg, twice weekly) inhibited the growth of prostate tumor xenografts in nude mice by 61% compared to untreated xenograft tumors. SR16388 also inhibited the proliferation of diverse human tumor cell lines after a 24-h exposure to the compound. SR16388 thus has utility both as an experimental antitumor agent and as a chemical probe of ERRα biology.

Keywords: Abbreviations; 4-OHT; 4-hydroxytamoxifen; ChIP; chromatin immunoprecipitation; ERR; estrogen-related receptor; ER; estrogen receptor; ERRE; estrogen-related receptor response element; ERE; estrogen response element; EC; ₅₀; one-half maximal effective concentration; E2; 17β-estradiol; FBS; fetal bovine serum; LBD; ligand binding domain; NR; nuclear receptor; PGC; peroxisome-proliferator activated receptor coactivator; PDB; protein data bank; RLU; relative light units; TR-FRET; time-resolved fluorescence resonance energy transferEstrogen-related receptor α; Steroid; Antitumor agent; Metabolism

Knockdown of NAPA using short-hairpin RNA sensitizes cancer cells to cisplatin: Implications to overcome chemoresistance by Zchong-Zcho Wu; Chuck C.-K. Chao (pp. 827-837).

Cisplatin is a widely used anti-cancer drug which targets DNA in replicating cells. In the present study, we found that NAPA—a protein found in the endoplasmic reticulum (ER) and implicated in protein trafficking—protects cells against cisplatin. Accordingly, knockdown of NAPA using lentivirus-encoded shRNA (shNAPA) induced ER stress similar to cisplatin treatment in HEK293 cells. A low dose of cisplatin also elicited a mild ER stress response associated with the accumulation of the protective proteins BiP and NAPA. Remarkably, knockdown of NAPA induced apoptosis and enhanced cisplatin-induced cytotoxicity/apoptosis, thereby sensitizing cancer cells to cisplatin. On the other hand, overexpression of NAPA increased resistance to cisplatin by reducing cisplatin-induced ER stress and apoptosis. The modulatory effects of shNAPA required the tumor suppressor p53 since the effects of NAPA knockdown were reduced by the p53 inhibitor PFT-α and in H1299 cells which are p53-null. A partial reversal of cisplatin resistance was also observed in cisplatin-resistant HeLa cells following knockdown of NAPA. Our results also indicated that calpain is required for ER-mediated apoptosis. Importantly, combined cisplatin/shNAPA treatment suppressed tumor growth in vivo in xenograph experiments performed in nude mice. Taken together, these observations suggest that NAPA represents a target of cisplatin, and that knockdown of NAPA may improve cisplatin-based cancer therapy.

Keywords: Abbreviations; ALLN; N-acetyl-leucyl-leucyl-norleucinal; BH3; BCL-2 homology domain 3; BiP; binding immunoglobulin protein; BNIP1; BCL2/adenovirus E1B 19-kDa-interacting protein 1; CDDP; cisplatin; DAPI; 4′,6-diamidino-2-phenylindole; DMEM; Dulbecco's modified Eagle's medium; ER; endoplasmic reticulum; ERAD; ER-associated degradation; GFP; green fluorescent protein; HPV; human papillomavirus; Luc; luciferase; MDM2; murine double minute 2; MF; modification factor; MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NAPA; NSF attachment protein α; NSF; N-ethylmaleimide-sensitive factor; ORF; open-reading frame; PARP; poly-ADP ribose polymerase; PCR; polymerase chain reaction; PFT-α; pifithrin-α; PIRH2; p53-induced RING-H2 domain protein; RT; reverse transcription; shRNA; short-hairpin RNA; SNAP; soluble NSF attachment protein; SNARE; SNAP receptor; VSVG; vesicular stomatitis virus protein GApoptosis; Cisplatin; ER stress; NAPA; p53

PGE₂ inhibits natural killer and γδ T cell cytotoxicity triggered by NKR and TCR through a cAMP-mediated PKA type I-dependent signaling by Ludovic Martinet; Christine Jean; Gilles Dietrich; Jean-Jacques Fournié; Rémy Poupot (pp. 838-845).

Natural killer (NK) and unconventional γδ T cells, by their ability to sense ligands induced by oncogenic stress on cell surface and to kill tumor cells without a need for clonal expansion, show a great therapeutic interest. They use numerous activating and inhibitory receptors which can function with some independence to trigger or inhibit destruction of target cells. Previous reports demonstrated that PGE₂ is able to suppress the destruction of some tumor cell lines by NK and γδ T cells but it remained uncertain if PGE₂ interferes with the different activating receptors governing the cytolytic responses of NK and γδ T cells. In this report, using the model of specific redirected lysis of the mouse FcγR⁺ cell line P815, we clearly demonstrate that the major NK receptors (NKR): NKG2D, CD16 and natural cytotoxicity receptors (NCR: NKp30, NKp44, NKp46) and γδ T cell receptors TCR Vγ9Vδ2, NKG2D and CD16 are all inhibited by PGE₂. As is the case with γδ T cells, we show that PGE₂ binds on E-prostanoid 2 (EP2) and EP4 receptors on NK cells. Finally, we delineate that the signaling of the blockade by PGE₂ is mediated through a cAMP-dependent activation of PKA type I which inhibits early signaling protein of cytotoxic cells. In the discussion, we focused on how these data should impact particular approaches in the treatment of cancer.

Keywords: NK cells; γδ T cells; PGE; ₂; PKA type I; Cytotoxicity receptors

Cyclooxygenase-2 and tissue inhibitor of matrix metalloproteinases-1 confer the antimigratory effect of cannabinoids on human trabecular meshwork cells by Robert Ramer; Burkhard Hinz (pp. 846-857).

Cannabinoids have received considerable attention as potential antiglaucomatous drugs. Recently, prostaglandins (PG) have been suggested to contribute to this effect. Within the factors conferring the development of glaucoma, depletion of the aqueous humor outflow-regulating trabecular meshwork (TM) cells elicited by migration from the outflow system is considered to play a pivotal role. This study therefore investigates the impact of two cannabinoids, Δ⁹-tetrahydrocannabinol (THC) and R(+)-methanandamide (MA), on the migration of human TM cells and the involvement of the PG-synthesizing enzyme cyclooxygenase-2 (COX-2) and one of its potential downstream targets, the tissue inhibitor of matrix metalloproteinases-1 (TIMP-1), to this response. Using Boyden chamber assays cannabinoids were shown to elicit an antimigratory effect that was reversed by antagonists for CB₁ as well as CB₂ receptors and accompanied by upregulation of COX-2 and TIMP-1 expression and PGE₂ synthesis. Knockdown of cannabinoid-induced COX-2 or TIMP-1 expression by siRNA or inhibition of COX-2 activity by NS-398 led to a significant suppression of this antimigratory action. Migration was also diminished by the major COX-2 product PGE₂ and by recombinant TIMP-1. Experiments using selective E prostanoid (EP) receptor agonists and antagonists revealed that decreased migration by PGE₂, THC and MA was mediated via EP₂ and EP₄ receptors. Finally, the cannabinoid-mediated increases of TIMP-1 levels were abolished by NS-398, and PGE₂ was shown to elicit a concentration-dependent increase of TIMP-1. Collectively, this data demonstrate a COX-2-dependent upregulation of TIMP-1 conferring the antimigratory action of cannabinoids. A decreased migration reducing TM cell loss in glaucoma might be involved in the antiglaucomatous action of cannabinoids.

Keywords: Abbreviations; AH-6809; 6-isopropoxy-9-oxoxanthene-2-carboxylic acid; AM-251; [N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide]; AM-630; [(6-Iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl) (4-methoxyphenyl)methanone]; CB; ₁; cannabinoid receptor 1; CB; ₂; cannabinoid receptor 2; COX; cyclooxygenase; FCS; fetal calf serum; GW627368X; 4-(4,9-diethoxy-1,3-dihydro-1-oxo-2H-benz[f]isoindol-2-yl)-N-(phenylsulfonyl)-benzeneacetamide; IOP; intraocular pressure; MA; R(+)-methanandamide (R-(+)-arachidonyl-1′-hydroxy-2′-propylamide); MMP; matrix metalloproteinase; mPGES; microsomal prostaglandin E synthase; NSAID; non-steroidal anti-inflammatory drug; NS-398; N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide; PG; prostaglandin; POAG; primary open-angle glaucoma; RT-PCR; reverse transcriptase-polymerase chain reaction; SC19220; 8-chloro-dibenz[b,f][1,4]oxazepine-10(11H)-carboxy-(2-acetyl)hydrazide; THC; Δ; ⁹; -tetrahydrocannabinol; TIMP; tissue inhibitor of matrix metalloproteinase; U-46619; 9,11-dideoxy-9α,11α-methanoepoxy-prosta-5Z,13E-dien-1-oic acid; WST-1; (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1.6-benzene disulfonate)Cannabinoids; Trabecular meshwork cells; Migration; Cyclooxygenase-2; Prostaglandins; Tissue inhibitor of matrix metalloproteinases-1; Antiglaucomatous drugs

Immunomodulatory effects of diclofenac in leukocytes through the targeting of Kv1.3 voltage-dependent potassium channels by Núria Villalonga; Miren David; Joanna Bielańska; Teresa González; David Parra; Concepció Soler; Núria Comes; Carmen Valenzuela; Antonio Felipe (pp. 858-866).

Kv1.3 plays a crucial role in the activation and proliferation of T-lymphocytes and macrophages. While Kv1.3 is responsible for the voltage-dependent potassium current in T-cells, in macrophages this K⁺ current is generated by the association of Kv1.3 and Kv1.5. Patients with autoimmune diseases show a high number of effector memory T cells that are characterized by a high expression of Kv1.3 and Kv1.3 antagonists ameliorate autoimmune disorders in vivo. Diclofenac is a non-steroidal anti-inflammatory drug (NSAID) used in patients who suffer from painful autoimmune diseases such as rheumatoid arthritis. In this study, we show that diclofenac impairs immune response via a mechanism that involves Kv1.3. While diclofenac inhibited Kv1.3 expression in activated macrophages and T-lymphocytes, Kv1.5 remained unaffected. Diclofenac also decreased iNOS levels in Raw 264.7 cells, impairing their activation in response to lipopolysaccharide (LPS). LPS-induced macrophage migration and IL-2 production in stimulated Jurkat T-cells were also blocked by pharmacological doses of diclofenac. These effects were mimicked by Margatoxin, a specific Kv1.3 inhibitor, and Charybdotoxin, which blocks both Kv1.3 and Ca²⁺-activated K⁺ channels (K_Ca3.1). Because Kv1.3 is a very good target for autoimmune therapies, the effects of diclofenac on Kv1.3 are of high pharmacological relevance.

Keywords: Abbreviations; BMDM; bone marrow-derived macrophages; COX; cyclooxygenase; CTX; Charybdotoxin; Kv; voltage-dependent K; ⁺; channels; LPS; lipopolysaccharide; MgTx; Margatoxin; NSAID; non-steroidal anti-inflammatory drug; PHA; phytohematogglutinin; PMA; phorbol ester; T; _EM; effector memory T-cellsVoltage-dependent K; ⁺; channels; Inflammation; Leukocytes; Immunomodulation; Non-steroidal anti-inflammatory drugs

Regulation of Nur77 protein turnover through acetylation and deacetylation induced by p300 and HDAC1 by Shin-Ae Kang; Hyelin Na; Hyun-Jin Kang; Sung-Hye Kim; Min-Ho Lee; Mi-Ock Lee (pp. 867-873).

Schematic model for regulation of Nur77 protein turnover with modulation of acetylation status by p300 and HDAC1.Although the roles of Nur77, an orphan member of the nuclear hormone receptor superfamily, in the control of cellular proliferation, apoptosis, inflammation, and glucose metabolism, are well recognized, the molecular mechanism regulating the activity and expression of Nur77 is not fully understood. Acetylation of transcription factors has emerged recently as a major post-translational modification that regulates protein stability and transcriptional activity. Here, we examined whether Nur77 is acetylated, and we characterized potential associated factors. First, Nur77 was found to be an acetylated protein when examined by immunoprecipitation and western blotting using acetyl protein-specific antibodies. Second, expression of p300, which possesses histone acetyltransferase activity, enhanced the acetylation and protein stability of Nur77. Treatment with a histone deacetylase (HDAC) inhibitor, trichostatin A, also increased Nur77 acetylation. Among the several types of HDACs, HDAC1 was found as the major enzyme affecting protein level of Nur77. HDAC1 decreased the acetylation level, protein level, and transcriptional activity of Nur77. Interestingly, overexpression of Nur77 induced expression of both p300 and HDAC1. Finally, the expression of Nur77 increased along with that of p300, but decreased with induction of HDAC1 after treatment with epithelial growth factor, nerve growth factor, or 6-mercaptopurine, suggesting that the self-control of the acetylation status contributes to the transient induction of Nur77 protein. Taken together, these results demonstrate that acetylation of Nur77 is modulated by p300 and HDAC1, and suggest that acetylation is an important post-translational modification for the rapid turnover of Nur77 protein.

Keywords: Abbreviations; HAT; histone acetyltransferase; HDAC; histone deacetylase; IP; immunoprecipitation; TSA; trichostatin A; DBD; DNA-binding domain; LBD; ligand-binding domain; NGF; nerve growth factor; EGF; epithelial growth factor; PMA; phorbol myristate acetate; CHX; cycloheximide; 6-MP; 6-mercaptopurinNur77; Acetylation; p300; HDAC1; Protein stability

Tiam1/Rac1 signaling pathway mediates palmitate-induced, ceramide-sensitive generation of superoxides and lipid peroxides and the loss of mitochondrial membrane potential in pancreatic β-cells by Ismail Syed; Bhavaani Jayaram; Wasanthi Subasinghe; Anjaneyulu Kowluru (pp. 874-883).

Proposed mechanism for palmitate induced NADPH oxidase leading to mitochondrial dysfunction in the pancreatic beta-cell.The phagocytic NADPH oxidase [NOX] has been implicated in the generation of superoxides in the pancreatic β-cell. Herein, using normal rat islets and clonal INS 832/13 cells, we tested the hypothesis that activation of the small G-protein Rac1, which is a member of the NOX holoenzyme, is necessary for palmitate [PA]-induced generation of superoxides in pancreatic β-cells. Incubation of isolated β-cells with PA potently increased the NOX activity culminating in a significant increase in the generation of superoxides and lipid peroxides in these cells; such effects of PA were attenuated by diphenyleneiodonium [DPI], a known inhibitor of NOX. In addition, PA caused a transient, but significant activation [i.e., GTP-bound form] of Rac1 in these cells. NSC23766, a selective inhibitor of Rac1, but not Cdc42 or Rho activation, inhibited Rac1 activation and the generation of superoxides and lipid peroxides induced by PA. Fumonisin B-1 [FB-1], which inhibits de novo synthesis of ceramide [CER] from PA, also attenuated PA-induced superoxide and lipid peroxide generation and NOX activity implicating intracellularly generated CER in the metabolic effects of PA; such effects were also demonstrable in the presence of the cell-permeable C2-CER. Further, NSC23766 prevented C2-CER-induced Rac1 activation and production of superoxides and lipid peroxides. Lastly, C2-CER, but not its inactive analogue, significantly reduced the mitochondrial membrane potential, which was prevented to a large degree by NSC23766. Together, our findings suggest that Tiam1/Rac1 signaling pathway regulates PA-induced, CER-dependent superoxide generation and mitochondrial dysfunction in pancreatic β-cells.

Keywords: Abbreviations; C2-CER; C2-Ceramide; DCHFDA; 2′,7′-dichlorodihydrofluorescein diacetate; DHC; Dihydroceramide; DPI; diphenyleneiodonium; FB-1; Fumonisin B-1; GEF; guanine nucleotide exchange factor; MDA; malondialdehyde; MMP; mitochondrial membrane potential; NBT; nitroblue tetrazolium; PA; palmitic acid; Rac1; Ras-related C3 botulinum toxin substrate 1; ROS; reactive oxygen species; Tiam1; T-lymphoma invasion and metastasis 1NADPH oxidase; Rac1; Tiam1; Palmitate; Ceramide; Oxidative stress; Pancreatic β-cells

Olesoxime prevents microtubule-targeting drug neurotoxicity: Selective preservation of EB comets in differentiated neuronal cells by Amandine Rovini; Manon Carré; Thierry Bordet; Rebecca M. Pruss; Diane Braguer (pp. 884-894).

Mechanism of olesoxime neuroprotection against microtubule-targeting agents (MTAs)-induced damages in human and rat neuron-like cells.Microtubule-targeting agents (MTAs), anticancer drugs widely used in the clinic, often induce peripheral neuropathy, a main dose-limiting side effect. The mechanism for this neurotoxicity remains poorly understood and there are still no approved therapies for neuropathies triggered by MTAs. Olesoxime (cholest-4-en-3-one, oxime; TRO19622) has shown marked neuroprotective properties in animals treated with paclitaxel and vincristine. The purpose of this study was to investigate its mechanism of neuroprotection against MTA neurotoxicity by using rat and human differentiated neuronal cells. We first showed that olesoxime prevented neurite shrinkage induced by MTAs in differentiated PC-12 and SK-N-SH neuroblastoma cell lines by up to 90%. This neuroprotective effect was correlated with enhanced EB1 accumulation at microtubule plus-ends, increased growth cone microtubule growing rate (20%) and decreased microtubule attenuation duration (54%). The effects of olesoxime on EB comets were specific for differentiated neuronal cells and were not seen either in proliferating neuroblastoma cells, glioblastoma cells or primary endothelial cells. Importantly, olesoxime did not alter MTA cytotoxic properties in a wide range of MTA-sensitive tumor cells, a prerequisite for future clinical application. Finally, olesoxime also counteracted MTA inhibition of microtubule-dependent mitochondria trafficking. These results provide additional insight into the neuroprotective properties of olesoxime, highlighting a role for microtubule dynamics in preservation of neurite architecture and axoplasmic transport, which are both disturbed by MTAs. The neuron-specific protective properties of olesoxime support its further development to treat MTA-induced neuropathy.

Keywords: Neuroprotection; Microtubule; EB proteins; Mitochondria; Taxanes; Vinca alkaloids

Isolation and characterisation of P-EPTX-Ap1a and P-EPTX-Ar1a: Pre-synaptic neurotoxins from the venom of the northern ( Acanthophis praelongus) and Irian Jayan ( Acanthophis rugosus) death adders by Janeyuth Chaisakul; Nicki Konstantakopoulos; A. Ian Smith; Wayne C. Hodgson (pp. 895-902).

The neurotoxicity observed following death adder envenoming has been thought to be solely due to the presence of potent post-synaptic neurotoxins. Clinically, these effects are often poorly reversed by death adder antivenom or anticholinesterase, particularly when patients present with established paralysis. This suggests that either the post-synaptic neurotoxins are irreversible/‘pseudo’ irreversible, or the venom contains pre-synaptic neurotoxins that do not respond to antivenom. To support the later hypothesis, a pre-synaptic neurotoxin (P-EPTX-Aa1a) has recently been isolated from the venom of Acanthophis antarcticus. We examined Acanthophis praelongus and Acanthophis rugosus venoms for the presence of pre-synaptic neurotoxins. P-EPTX-Ap1a (40,719Da) and P-EPTX-Ar1a (40,879Da) were isolated from A. praelongus and A. rugosus venoms, respectively. P-EPTX-Ap1a and P-EPTX-Ar1a are comprised of three different subunits, α, β1 and β2. The two toxins displayed similar levels of PLA₂ activity which was almost solely attributed to the α subunit in both toxins. P-EPTX-Ap1a (20–100nM) and P-EPTX-Ar1a (20–100nM) caused inhibition of indirect twitches of the skeletal muscle preparation without affecting contractile responses to nicotinic receptor agonists. Interestingly, only the α subunit of both toxins (300nM) displayed neurotoxic activity. Inhibition of PLA₂ activity markedly reduced the effect of the toxins on muscle twitch height. These results confirm that P-EPTX-Ap1a and P-EPTX-Ar1a are pre-synaptic neurotoxins and represent the second and third such toxins to be isolated from death adder venom. The presence of pre-synaptic neurotoxins in Acanthophis sp. venoms indicates that treatment strategies for envenoming by these snakes needs to be reassessed given the likelihood of irreversible neurotoxicity.

Keywords: Abbreviations; ACh; acetylcholine; BSA; bovine serum albumin; CCh; carbachol; MALDI-TOF; matrix-assisted laser desorption ionization time-of-fight; PLA; ₂; phospholipase A; ₂; P-EPTX-Aa1a; P-elapitoxin-Aa1a; RP-HPLC; reverse-phase high-pressure liquid chromatographyDeath adder; Pre-synaptic neurotoxin; Chick biventer cervicis nerve–muscle; Phospholipase A; ₂; Venom

The relative importance of CYP26A1 in hepatic clearance of all-trans retinoic acid by Jayne E. Thatcher; Alex Zelter; Nina Isoherranen (pp. 903-912).

All-trans retinoic acid (RA) is a critical signaling molecule and its concentration is tightly regulated. Several P450 enzymes including CYP26A1, CYP2C8, and CYP3A4 have been proposed to be responsible for RA clearance in the liver but their quantitative importance has not been demonstrated. To determine the contribution of CYP26A1 to hepatic clearance of RA, CYP26A1 protein was quantified in 37 human liver microsomes (HLMs). CYP26A1 expression ranged from not detectable to 2.80pmol/mg microsomal protein. RA clearance by P450 enzymes abundant in human liver was measured in Supersomes^®. CYP2C8, CYP3A4, CYP3A5 and CYP3A7 metabolized RA with unbound K_m values of 3.4–7.2μM and V_max values of 2.3–4.9pmol/min/pmol P450, but were less efficient than CYP26A1 in clearing RA. Simulations performed for livers with varying P450 expression levels over a range of RA concentrations demonstrated that at both endogenous and therapeutic concentrations of RA, CYP26A1 is the primary enzyme responsible for 4-OH RA formation clearance. HLM incubation data showed that 4-OH RA formation velocity varied from 0.2 to 15.3pmol/min/mg microsomal protein and velocity in HLMs was significantly correlated ( p<0.01) to CYP26A1, CYP3A4, and CYP3A5 protein content, but not to CYP2C8. When experimental data were scaled to in vivo clearances, the predicted hepatic clearance of RA (0.07L/min using combined Supersome^® data) was similar to the published in vivo clearance of RA. These findings suggest that CYP26A1 is the P450 isoform that should be targeted when designing RA metabolism blocking agents.

Keywords: CYP26A1; Retinoic acid metabolism; Vitamin A; Hepatic clearance

Increased responsiveness to JNK1/2 mediates the enhanced H₂O₂-induced stimulation of Cl⁻/HCO₃⁻ exchanger activity in immortalized renal proximal tubular epithelial cells from the SHR by S. Simão; P. Gomes; P.A. Jose; P. Soares-da-Silva (pp. 913-919).

H₂O₂-induced stimulation of Cl⁻/HCO₃-exchanger activity is regulated by JNK1/2 in SHR cells. The imbalance between oxidant–antioxidant mechanisms in SHR cells enhances the response of JNK1/2 to H₂O₂, which contributes to their increased sensitivity.We have previously demonstrated that exogenous H₂O₂ stimulates Cl⁻/HCO₃⁻ exchanger activity in immortalized renal proximal tubular epithelial (PTE) cells from both the Wistar-Kyoto (WKY) rat and the spontaneously hypertensive rat (SHR), this effect being more pronounced in SHR cells. The aim of the present study was to examine the mechanism of H₂O₂-induced stimulation of Cl⁻/HCO₃⁻ exchanger activity in WKY and SHR cells. It is now reported that the SHR PTE cells were endowed with an enhanced capacity to produce H₂O₂, comparatively with WKY cells and this was accompanied by a decreased expression of SOD2, SOD3, and catalase in SHR PTE cells. The stimulatory effect of H₂O₂ on the exchanger activity was blocked by SP600125 (JNK inhibitor), but not by U0126 (MEK1/2 inhibitor) or SB203580 (p38 inhibitor) in both cell lines. Basal JNK1 and JNK2 protein expression was higher in SHR PTE cells than in WKY PTE cells. H₂O₂ had no effect on p-JNK1/2 in WKY PTE cells over time. By contrast, H₂O₂ treatment resulted in a rapid and sustained increase in JNK1/2 phosphorylation in SHR PTE cells, which was completely abolished by apocynin. Treatment of SHR PTE cells with apocynin significantly decreased the H₂O₂-induced stimulation of Cl⁻/HCO₃⁻ exchanger activity. It is concluded that H₂O₂-induced stimulation of Cl⁻/HCO₃⁻ exchanger activity is regulated by JNK1/2, particularly by JNK2, in SHR PTE cells. The imbalance between oxidant and antioxidant mechanisms in SHR PTE cells enhances the response of JNK1/2 to H₂O₂, which contributes to their increased sensitivity to H₂O₂.

Keywords: Abbreviations; BCECF-AM; acetoxymethyl ester of 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein; BSA; bovine serum albumin; DTT; dithiothreitol; EDTA; ethylenediamine tetraacetic acid; ERK; extracellular signal-regulated kinase; HEPES; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; JNK; c-Jun-NH; ₂; -terminal kinase; MAPK; mitogen-activated protein kinase; NADPH; nicotinamide adenine dinucleotide phosphate, reduced form; NHE; Na; ⁺; /H; ⁺; exchanger; Nox; NADPH oxidase; PBS; phosphate buffer saline; PKA; protein kinase A; PKC; protein kinase C; PT; proximal tubule; PTE; proximal tubular epithelial; ROS; reactive oxygen species; SDS-PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis; SOD; superoxide dismutase; SHR; spontaneously hypertensive rat; WKY; Wistar-Kyoto ratCl; ⁻; /HCO; ₃; ⁻; exchanger; Hypertension; Oxidative stress; H; ₂; O; ₂; JNK

Heat shock protein 70 protects against bleomycin-induced pulmonary fibrosis in mice by Ken-Ichiro Tanaka; Yuta Tanaka; Takushi Namba; Arata Azuma; Tohru Mizushima (pp. 920-931).

Idiopathic pulmonary fibrosis (IPF) involves infiltration of leucocytes, pulmonary injury, fibrosis and resulting pulmonary dysfunction. Myofibroblasts and transforming growth factor (TGF)-β1 have been suggested to play a major role in the pathology and the myofibroblasts are derived from both lung epithelial cells through epithelial–mesenchymal transition (EMT) and activation of lung fibroblasts. Heat shock protein 70 (HSP70) confers protection against various stressors and has the anti-inflammatory activity. In this study, we examined the effect of expression of HSP70 on bleomycin-induced pulmonary fibrosis in mice, a tentative animal model of IPF. Bleomycin-induced pulmonary injury and inflammatory response were ameliorated in transgenic mice overexpressing HSP70 compared to wild-type mice, even though bleomycin-induced pulmonary fibrosis and dysfunction were also suppressed in the transgenic mice. The production of TGF-β1 and expression of pro-inflammatory cytokines was lower in cells from the transgenic mice than wild-type mice after the administration of bleomycin. In vitro, the suppression of HSP70 expression stimulated TGF-β1-induced EMT-like phenotypes of epithelial cells but did not affect the TGF-β1-dependent activation of fibroblasts. Orally administered geranylgeranylacetone (GGA), a clinically used drug with HSP-inducing activity, conferred protection against bleomycin-induced pulmonary injury, as well as against the inflammatory response, fibrosis and dysfunction. These results suggest that HSP70 plays a protective role against bleomycin-induced pulmonary injury, inflammation, fibrosis and dysfunction through cytoprotective effects and by inhibiting the production of TGF-β1, TGF-β1-dependent EMT of epithelial cells and expression of pro-inflammatory cytokines. Results also suggest that HSP70-inducing drugs, such as GGA, could be beneficial in the prophylaxis of IPF.

Keywords: Heat shock protein 70; Bleomycin; Idiopathic pulmonary fibrosis; Geranylgeranylacetone; Epithelial–mesenchymal transition; Transforming growth factor-β1

Ube2l3 gene expression is modulated by activation of the aryl hydrocarbon receptor: Implications for p53 ubiquitination by O.D. Reyes-Hernández; A. Mejía-García; E.M. Sánchez-Ocampo; M.A. Cabañas-Cortés; P. Ramírez; L. Chávez-González; F.J. Gonzalez; G. Elizondo (pp. 932-940).

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a halogenated aromatic hydrocarbon and environmental contaminant, results in several deleterious effects, including fetal malformation and cancer. These effects are mediated by the aryl hydrocarbon receptor (AhR), a ligand-activated receptor that regulates the expression of genes encoding xenobiotic-metabolizing enzymes. Several reports suggest that AhR function is beyond the adaptive chemical response. In the present study, we analyzed and compared gene expression profiles of C57BL/6N wild-type (WT) and Ahr-null mice. DNA microarray and quantitative RT-PCR analyses revealed changes in the expression of genes involved in the ubiquitin–proteasome system (UPS). UPS has an important role in cellular homeostasis control and dysfunction of this pathway has been implicated in the development of several human pathologies. Protein ubiquitination is a multi-step enzymatic process that regulates the stability, function, and/or localization of the modified proteins. This system is highly regulated post-translationally by covalent modifications. However, little information regarding the transcriptional regulation of the genes encoding ubiquitin (Ub) proteins is available. Therefore, we investigated the role of the AhR in modulation of the UPS and regulation of Ube2l3 transcription, an E2 ubiquitin-conjugating enzyme, as well as the effects on p53 degradation. Our results indicate that AhR inactivation decreases on liver proteasome activity, probably due to a down-regulation on the expression of several proteasome subunits. On the other hand, AhR activation increases Ube2l3 mRNA and protein levels by controlling Ube2l3 gene expression, resulting in increased p53 ubiquitination and degradation. In agreement with this, induction of apoptosis was attenuated by the AhR activation.

Keywords: AhR; Ubiquitin–proteasome system; Ube2l3; p53; TCDD

Kinetic analysis of interactions between alkylene-linked bis-pyridiniumaldoximes and human acetylcholinesterases inhibited by various organophosphorus compounds by Timo Wille; Fredrik Ekström; Jong-Cheol Lee; Yuan-Ping Pang; Horst Thiermann; Franz Worek (pp. 941-946).

Basic structure and structure–activity-relationship of the second-order rate constant k_r2 for the reactivation of OP-inhibited AChE and Ortho oxime linker length.The therapeutic approach of organophosphorus compound (OP) intoxications is to reactivate the inhibited enzyme acetylcholinesterase (AChE). Numerous studies demonstrated a limited efficacy of standard oxime-based reactivators against different nerve agents such as tabun and cyclosarin. This emphasizes research for more effective oximes. In the present study, reactivation kinetics of tabun-, sarin-, cyclosarin-, VX- or paraoxon-ethyl-inhibited human AChE (hAChE) with a homologous series of bis-ortho-pyridiniumaldoximes, Ortho-4 - Ortho-9, was investigated with a robot-assisted setting, allowing determination of second-order reactivation rate constants as well as model calculations. The reactivation constants of Ortho-4 - Ortho-9 resulted in marked differences of affinity and reactivity depending on the OP structure and the linker length of the oximes. In general, the K_D values decreased with increasing linker length. Reactivity increased from Ortho-4 to Ortho-6 for PXE- and VX-inhibited hAChE and from Ortho-4 to Ortho-7 for GA-inhibited hAChE and decreased again with Ortho-8 and Ortho-9. In contrast, k_r decreased with increasing linker length for sarin- and cyclosarin-inhibited hAChE. In view of the pronounced decrease of K_D from Ortho-4 to Ortho-9, the k_r2 values increased with all tested OP. Hence, the ratios of K_I/ K_D and of K_I/ k_r2 showed that in almost all cases the affinity of Ortho- N to the native hAChE was higher than to OP-inhibited enzyme. Model calculations indicated that Ortho-6 - Ortho-9 could be superior to obidoxime in reactivating tabun-inhibited hAChE. Finally, these data emphasize the need to develop oximes with a higher selective affinity towards OP-inhibited hAChE in order to minimize possible side effects.

Keywords: Organophosphorus compounds; Acetylcholinesterase; Oxime; Reactivation; Kinetics; Structure–activity-relationship

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Biochemical Pharmacology (v.80, #6)