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• W2011802015 abstract "The selenoenzyme thioredoxin reductase regulates redox-sensitive proteins involved in inflammation and carcinogenesis, including ribonucleotide reductase, p53, NFκB, and others. Little is known about endogenous cellular factors that modulate thioredoxin reductase activity. Here we report that several metabolites of 15-lipoxygenase-1 inhibit purified thioredoxin reductase in vitro. 15(S)-Hydroperoxy-5,8,11-cis-13-trans-eicosatetraenoic acid, a metastable hydroperoxide generated by 15-lipoxygenase-1, and 4-hydroxy-2-nonenal, its non-enzymatic rearrangement product inhibit thioredoxin reductase with IC50 = 13 ± 1.5 μm and 1 ± 0.2 μm, respectively. Endogenously generated metabolites of 15-lipoxygenase-1 also inhibit thioredoxin reductase in HEK-293 cells that harbor a 15-LOX-1 gene under the control of an inducible promoter complex. Conditional, highly selective induction of 15-lipoxygenase-1 caused an inhibition of ribonucleotide reductase activity, cell cycle arrest in G1, impairment of anchorage-independent growth, and accumulation of the pro-apoptotic protein BAX. All of these responses are consistent with inhibition of thioredoxin reductase via 15-lipoxygenase-1 overexpression. In contrast, metabolites of 5-lipoxygenase were poor inhibitors of isolated thioredoxin reductase, and the overexpression of 5-lipoxygenase did not inhibit thioredoxin reductase or cause a G cell cycle arrest. The influences of 15-lipoxygenase-1 on 1inflammation, cell growth, and survival may be attributable, in part, to inhibition of thioredoxin reductase and several redox-sensitive processes subordinate to thioredoxin reductase. The selenoenzyme thioredoxin reductase regulates redox-sensitive proteins involved in inflammation and carcinogenesis, including ribonucleotide reductase, p53, NFκB, and others. Little is known about endogenous cellular factors that modulate thioredoxin reductase activity. Here we report that several metabolites of 15-lipoxygenase-1 inhibit purified thioredoxin reductase in vitro. 15(S)-Hydroperoxy-5,8,11-cis-13-trans-eicosatetraenoic acid, a metastable hydroperoxide generated by 15-lipoxygenase-1, and 4-hydroxy-2-nonenal, its non-enzymatic rearrangement product inhibit thioredoxin reductase with IC50 = 13 ± 1.5 μm and 1 ± 0.2 μm, respectively. Endogenously generated metabolites of 15-lipoxygenase-1 also inhibit thioredoxin reductase in HEK-293 cells that harbor a 15-LOX-1 gene under the control of an inducible promoter complex. Conditional, highly selective induction of 15-lipoxygenase-1 caused an inhibition of ribonucleotide reductase activity, cell cycle arrest in G1, impairment of anchorage-independent growth, and accumulation of the pro-apoptotic protein BAX. All of these responses are consistent with inhibition of thioredoxin reductase via 15-lipoxygenase-1 overexpression. In contrast, metabolites of 5-lipoxygenase were poor inhibitors of isolated thioredoxin reductase, and the overexpression of 5-lipoxygenase did not inhibit thioredoxin reductase or cause a G cell cycle arrest. The influences of 15-lipoxygenase-1 on 1inflammation, cell growth, and survival may be attributable, in part, to inhibition of thioredoxin reductase and several redox-sensitive processes subordinate to thioredoxin reductase. Thioredoxin reductase (TrxR) 1The abbreviations used are: TrxR, thioredoxin reductase; AA, arachidonic acid; COX, cyclooxygenase; DMEM, Dulbecco's modified essential medium; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); ETE, eicosatetraenoic acid; FACL-4, fatty acid CoA ligase-4; HETE, hydroxyeicosatetraenoic acid; HIF, hypoxia-inducible factor; HODE, hydroxyoctadecadienoic acid; 4-HNE, 4-hydroxy-2-nonenal; HpETE, hydroperoxy-eicosatetraenoic acid; HpODE, hydroperoxy-octadecadienoic acid; HRP, horseradish peroxidase; LA, linoleic acid; LOX, lipoxygenase; NSAID, non-steroidal anti-inflammatory drug; PBS, phosphate-buffered saline; PPAR, peroxisome proliferator activator receptor; Trx, thioredoxin; FACS, fluorescent-activated cell sorter. is a homodimeric 56 kDa selenoenzyme that catalyzes NADPH-dependent reduction reactions (1Tamura T. Stadtman T.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1006-1011Crossref PubMed Scopus (477) Google Scholar, 2Mustacich D. Powis G. Biochem. J. 2000; 346: 1-8Crossref PubMed Scopus (764) Google Scholar, 3Arnér E.S.J. Holmgren A. Eur. J. Biochem. 2000; 267: 6102-6109Crossref PubMed Scopus (2000) Google Scholar). Trx, a substrate of TrxR, is a 12 kDa protein disulfide reductase and an electron donor for ribonucleotide reductase (2Mustacich D. Powis G. Biochem. J. 2000; 346: 1-8Crossref PubMed Scopus (764) Google Scholar, 3Arnér E.S.J. Holmgren A. Eur. J. Biochem. 2000; 267: 6102-6109Crossref PubMed Scopus (2000) Google Scholar) and other enzymes. The TrxR-Trx system regulates redox-sensitive processes essential for cell growth, differentiation, and genomic integrity (3Arnér E.S.J. Holmgren A. Eur. J. Biochem. 2000; 267: 6102-6109Crossref PubMed Scopus (2000) Google Scholar, 4Powis G. Gasdaska J.R. Gasdaska P.Y. Berggren M. Kirkpatrick D.L. Engman L. Cotgreave I.A. Angulo M. Baker A. Oncol. Res. 1997; 9: 303-312PubMed Google Scholar). The catalytic mechanism of mammalian TrxR is well understood (5Cenas N. Nivinskas H. Anusevicius Z. Sarlauskas J. Lederer F. Arner E.S. J. Biol. Chem. 2004; 279: 2583-2592Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). However, less is known about endogenous cellular factors that modulate its activity. Recent reports have described novel interactions between TrxR and products of the COX or LOX enzymes (6Björnstedt M. Hamberg M. Kumar S. Xue J. Holmgren A. J. Biol. Chem. 1995; 270: 11761-11764Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 7Shibata T. Yamada T. Ishii T. Kumazawa S. Nakamura H. Masutani H. Yodoi J. Uchida K. J. Biol. Chem. 2003; 278: 26046-26054Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 8Moos P.J. Edes K. Cassidy P. Massuda E. Fitzpatrick F.A. J. Biol. Chem. 2003; 278: 745-750Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). TrxR can directly reduce the lipid hydroperoxide, 15(S)-HpETE, and potentially limit its accumulation in cells that express 15-LOX (6Björnstedt M. Hamberg M. Kumar S. Xue J. Holmgren A. J. Biol. Chem. 1995; 270: 11761-11764Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). In addition, several electrophilic lipids, including 4-HNE, a derivative of lipid peroxidation by 15-LOX-1 or 12-LOX (9Schneider C. Tallman K.A. Porter N.A. Brash A.R. J. Biol. Chem. 2001; 276: 20831-20838Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 10Burcham P.C. Mutagenesis. 1998; 13: 287-305Crossref PubMed Scopus (240) Google Scholar, 11Gardner H.W. J. Agric. Food Chem. 1975; 23: 129-136Crossref PubMed Scopus (211) Google Scholar) can irreversibly inhibit cellular TrxR activity (8Moos P.J. Edes K. Cassidy P. Massuda E. Fitzpatrick F.A. J. Biol. Chem. 2003; 278: 745-750Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 12Brash A.R. J. Biol. Chem. 1999; 274: 23679-23682Abstract Full Text Full Text PDF PubMed Scopus (1147) Google Scholar). Other HpETE regioisomers and reactive lipid carbonyl compounds might also have effects similar to 15(S)-HpETE and 4-HNE. Thus, LOX enzymes (12Brash A.R. J. Biol. Chem. 1999; 274: 23679-23682Abstract Full Text Full Text PDF PubMed Scopus (1147) Google Scholar) might modulate TrxR, and the regulatory control it exerts over other proteins and processes, such as ribonucleotide reductase and the cell cycle. To determine if LOX enzymes modulate TrxR, we developed HEK-293 cells that stably express either 15-LOX-1 or 5-LOX under the control of a ponasterone-responsive, heterologous transcription complex (13Saez E. Nelson M.C. Eshelman B. Banayo E. Koder A. Cho G.J. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14512-14517Crossref PubMed Scopus (97) Google Scholar). Such conditional expression systems have been useful to study other eicosanoid biosynthetic enzymes, such as COX-2 and FACL-4 (14Cao Y. Pearman A.T. Zimmerman G.A. McIntyre T.M. Prescott S.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11280-11285Crossref PubMed Scopus (384) Google Scholar), and to study complex processes like apoptosis (14Cao Y. Pearman A.T. Zimmerman G.A. McIntyre T.M. Prescott S.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11280-11285Crossref PubMed Scopus (384) Google Scholar, 15Pastorino J.G. Chen S.-T. Tafani M. Snyder J.W. Farber J.L. J. Biol. Chem. 1998; 273: 7770-7775Abstract Full Text Full Text PDF PubMed Scopus (534) Google Scholar) and cell cycle regulation (16Stewart Z. Mays D. Pietenpol J. Cancer Res. 1999; 59: 3831-3837PubMed Google Scholar). Here we report that overexpression of 15-LOX-1 inhibits cellular TrxR. Specifically, cellular TrxR activity was inversely related to 15-LOX-1 activity. Several 15-LOX-1 metabolites inhibited purified TrxR in vitro: 4-HNE, 12-oxo-ETE, and 15(S)-HpETE had IC50 = 1 ± 0.2 μm, 2.7 ± 0.7 μm, and 13 ± 1.5 μm, respectively. Induction of 15-LOX-1, and the resulting inhibition of TrxR caused a corresponding inhibition of ribonucleotide reductase activity, cell cycle arrest in G1, impairment of anchorage-independent growth, and accumulation of the proapoptotic protein BAX. These effects are consistent with known regulatory roles for the TrxR-Trx system. In contrast to 15-LOX-1, expression of 5-LOX enzyme did not inhibit TrxR. These findings support the concept that molecular interactions between 15-LOX-1 and TrxR, a vital selenoenzyme, might be relevant to carcinogenesis (17Shureiqi I. Lippman S.M. Cancer Res. 2001; 61: 6307-6312PubMed Google Scholar, 18Kelavkar U. Glasgow W. Eling T. Curr. Urol. Rep. 2002; 3: 207-214Crossref PubMed Scopus (56) Google Scholar) and inflammation (19Levy B.D. Clish C.B. Schmidt B. Gronert K. Serhan C.N. Nat. Immunol. 2001; 2: 612-619Crossref PubMed Scopus (1106) Google Scholar, 20Gromer S. Arscott L.D. Williams C.H. Schirmer R.H. Becker K. J. Biol. Chem. 1998; 273: 20096-20101Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar). Materials—A full-length clone of 15-LOX-1, accession number NM_001140, and 5-LOX, accession number NM_000698 were generous gifts from Dr. Colin Funk, University of Pennsylvania. HEK-293-EcR cell lines with a pVgRXR vector were obtained from Invitrogen, Carlsbad, CA. The pIND vector was used to insert the 15-LOX-1 or 5-LOX gene 3′ to the ecdysone response elements. HEK-293 cells were engineered similarly to permit conditional expression of COX-2 (14Cao Y. Pearman A.T. Zimmerman G.A. McIntyre T.M. Prescott S.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11280-11285Crossref PubMed Scopus (384) Google Scholar). Ponasterone (Invitrogen) was used for the induction of 15-LOX-1,5-LOX or COX-2. G418 (Invitrogen) was used for selection of cells. Fatty acid substrates used included AA and LA (NuChek Prep, Elysian, Minn). LOX metabolites included 5(S)-HETE, 5(R)-HETE, 9(S)-HODE, 13(S)-HODE, 12(S)-HETE, 15(S)-HETE, 15(R)-HETE, 20-HETE, 5-oxo-ETE, 12-oxo-ETE, 15-oxo-ETE, 5(S)-HpETE, 9(S)-HpODE, 13(S)-HpODE, 12(S)-HpETE, 15(S)-HpETE, and 4-HNE (Cayman Chemicals, Ann Arbor, MI). 15-LOX-1 activity was quantified by immunoassay of the reaction product 15(S)-HETE (Cayman Chemical). Supplies to make the complete DMEM media included fetal calf serum, Dulbecco's modified essential medium, penicillin/streptomycin, l-glutamine, sodium pyruvate (Sigma). Escherichia coli Trx (Promega, Madison, WI) was used as substrate in the assay for cellular TrxR activity. Bovine insulin and NADPH (Sigma) were also used in the TrxR assay. DTNB (Sigma) was used as a substrate in the assay for purified TrxR activity. Propidium iodide (Sigma) was used to label DNA for flow cytometry evaluation of the cell cycle. Hydroxyurea, lysolecithin, percholic acid, Na4P207, rGDP, rADP, and rTDP for the ribonucleotide reductase activity assay were obtained from Sigma. 3[H]rCDP was obtained from Amersham Biosciences. 2-(p-Isodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride hydrate (Sigma), 250 mg/l in PBS, was used to stain cell colonies growing on soft agar. Rabbit anti-15-LOX-1 antibody was a kind gift from Dr. Douglas Conrad, UCSD. Monoclonal mouse anti-5-LOX antibody (BD Biosciences Pharmingen, San Diego CA), rabbit anti-BAX antibody, mouse anti-bcl-2 antibody, goat anti-rabbit-IgG-HRP secondary antibody, and goat anti-mouse-IgG-HRP secondary antibody were used according to the supplier (Santa Cruz Biotechnology, Santa Cruz, CA). Rabbit anti-Cox-2 antibody was a kind gift from Dr. Stephen Prescott, University of Utah. ECL reagents were used for antigen-antibody detection (Amersham Biosciences). The cell lysis buffer consisted of 50 mm Tris, pH 7.4, 0.1 m NaCl, 2 mm EDTA, 1% SDS, 1% deoxycholate, 1 mm NaF, 1 mm sodium orthovanadate, and 1× Complete™ protease inhibitors. TBS-T consisted of 20 mm Tris-HCl, pH 7.5, 100 mm sodium chloride, and 0.5% v/v Tween 20. Effect of LOX Metabolites on Purified TrxR—We examined the effects of several LOX metabolites on TrxR, isolated from rat liver as described (21Nordberg J. Zhong L. Holmgren A. Arner E.S. J. Biol. Chem. 1998; 273: 10835-10842Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). TrxR purified > 5000-fold to apparent homogeneity had a specific activity of 30 μmol of NADPH/min/mg. A stock solution of 250 μg of TrxR/ml was diluted 1:40 in the TrxR reaction mixture (described below) with 1 mg/ml of bovine serum albumin. Exact concentrations of ethanolic solutions of the stable test compounds 5(S)-, 5(R)-, 12(S)-, 15(S)-, 15(R)-, and 20-HETE; 9(S)-, and 13(S)-HODE, and the metastable test compounds 5(S)-, 12(S)-, and 15(S)-HpETE, 9(S)- and 13(S)-HpODE, 5-oxo-, 12-oxo-, and 15-oxo-ETE, and 4-HNE were determined with UV spectroscopy. Before addition of the TrxR enzyme reaction mixture, 1–10-μl aliquots of test compound in ethanol were evaporated at 25 °C in a 1.5 ml of conical tube. Each test compound was then incubated for 10 min at 25 °C with 80 μl of a reaction buffer containing 50 μm K2PO4, pH 7, and 200 μm EDTA, 2 μm NADPH and 5 μl of purified rat liver TrxR. 5 mm DTNB substrate was added prior to measurement of absorbance at 412 nm for 5 min. Conditional Expression of 15-LOX-1 and 5-LOX Enzymes—Stable cell lines for the conditional expression of 15-LOX-1 and 5-LOX were created using a system based on VgEcR, a chimeric protein composed of the VP16 activation domain fused to an ecdysone receptor with altered DNA-binding specificity. Upon exposure to ponasterone, this protein dimerizes with the retinoid X receptor (RXR), and the VgEcR/RXR heterodimer induces the expression of any gene inserted 3′ to the ecdysone response element (13Saez E. Nelson M.C. Eshelman B. Banayo E. Koder A. Cho G.J. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14512-14517Crossref PubMed Scopus (97) Google Scholar, 14Cao Y. Pearman A.T. Zimmerman G.A. McIntyre T.M. Prescott S.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11280-11285Crossref PubMed Scopus (384) Google Scholar, 22No D. Yao T.P. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3346-3351Crossref PubMed Scopus (755) Google Scholar). We cloned the full-length cDNA of 15-LOX-1 and 5-LOX into the inducible expression vector pIND. We transfected this vector into EcR 293 cells stably transformed with the regulatory vector pVgRXR. We then isolated a population of G418-resistant cells that stably expressed ponasterone-inducible 15-LOX-1 and 5-LOX. Measurement of Protein Expression and Enzyme Activity—15-LOX-1 protein expression was determined immunochemically. Lox15-1+ cells were incubated with 0–10 μm ponasterone for 0–96 h. Approximately 1 × 106 cells were harvested, treated with lysis buffer, sonicated intermittently for 15 s at 4 °C and centrifuged for 5 min at 10,000 × g.15 μg of supernatant protein was fractionated by 10% SDS-PAGE, and proteins were transferred to polyvinylidine difluoride membranes. Membranes were blocked with 5% w/v nonfat dry milk in TBS-T, then incubated for 1 h at 25 °C with primary antibody (anti-15-LOX-1, 1:1000) and HRP-conjugated, goat anti-rabbit secondary antibody (1: 4000). Antigen-antibody complexes were detected with ECL reagents. 5-LOX expression in Lox5+ cells and COX-2 expression in Cox2+ cells was determined with anti-5-LOX antibody (1: 250) and anti-COX-2 antibody (1:450). 15-LOX-1 activity was quantified by measuring 15(S)-HETE formation. Lox15-1+ cells were incubated with 3 μm ponasterone, added once every 24 h, for 24–96 h. At t = 20, 44, 68, or 92 h, 0–120 μm AA was added and incubated with ∼1 × 106 cells for 1 h at 37 °C. The cell culture medium (supernatant) was collected, diluted 1:10–1:100 with PBS, pH 7.4. 15-HETE in the supernatant was quantified by immunoassay. Results were normalized to the cell number. COX-2 activity was quantified by immunoassay for PGE2 formation. 5-LOX activity was quantified by HPLC for 5(S)-HETE with UV detection at 230 nm. Measurement of TrxR Activity in Lox15-1+, Lox5+, and Cox2+Cells— Lox15-1+ cells were incubated for 0–96 h with vehicle, or with 3 μm ponasterone. This concentration of ponasterone was determined by a dose-response experiment (Fig. 2B). Vehicle or ponasterone was added every 24 h to maintain 15-LOX-1 expression; 20–100 μm AA was added to initiate cellular 15-LOX-1 metabolism. The corresponding cellular TrxR activity was quantified at intervals from 10 min to 8 h after the addition of AA or LA. Cox-2+ and Lox5+ cells were treated analogously to quantify the effect of COX-2 and 5-LOX on cellular TrxR activity. Cells were lysed in the usual lysis buffer, sonicated intermittently for 15 s at 4 °C, centrifuged at 10,000 × g for 5 min, and the supernatant was harvested. 20 μl of supernatant (4 μg protein/μl) was added to 80 μl of a reaction mixture containing 50 mm Trism pH 7.4, 143 μm insulin, 171 μm NADPH, and 1 mm EDTA, then incubated at 25 °C for 10 min. 66.6 μm Trx substrate was then added to start the TrxR enzymatic reaction. The oxidation of NADPH was monitored spectrophotometrically at 340 nm in microcuvettes at 25 °C for 5 min. TrxR activity corresponded to the formation of μmol of NADP+/min/mg of cellular protein. Cell Cycle Analysis—Approximately 6 × 105 cells in 2 ml of complete DMEM media were plated in 6-well plates. Lox15-1+ cells were incubated daily with 3 μm ponasterone or vehicle for 96 h. 20 μm AA was added for 4 h prior to cell fixation, and the distribution of cells in the G1, S, and G2/M phases of the cell cycle was determined by FACS analysis. Adherent cells were released by treatment with 0.25% trypsin. The trypsin was inactivated with 10% serum in complete DMEM media. Each sample was washed twice with 1× PBS and fixed overnight with ice-cold 70% ethanol at 4 °C. Cells were rehydrated with 1× PBS, then stained with 10% propidium iodide in 0.1% Triton X-100, 3.7% EDTA, and 100 units/ml of RNase in 1× PBS for 60 min at 25 °C. Cells were filtered through 35-μm filters before analysis. The flow cytometer was configured to track the number of events with the FL2 parameter (BD Pharmingen FACSCAN®. The DNA content was analyzed using a nonlinear least-squares algorithm (23Bauer K.D. Duque R.E. Shankey T.V. Clinical Flow Cytometry: Principles and Application. Williams & Wilkins, Baltimore1993: 44-46Google Scholar). Lox5+ cells were treated analogously in separate experiments. Growth in Soft Agar—Lox15-1+ and Cox-2+ cells were suspended in 0.5% low melting point agar with complete DMEM medium. Approximately 1 × 104 cells were plated on an 85-mm dish coated with 1% agar, and maintained at 37 °C. On day 10, cells were stained with 1 ml of 2-(p-isodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium chloride hydrate (250 mg/l in PBS) for 24 h (24Kelavkar U.P. Nixon J.B. Cohen C. Dillehay D. Eling T.E. Badr K.F. Carcinogenesis. 2001; 22: 1765-1773Crossref PubMed Scopus (122) Google Scholar). Measurement of Ribonucleotide Reductase Activity in Lox15-1+Cells—Approximately 1 × 105 cells were plated on a T-25 flask and rested overnight. They were then treated with 3 μm ponasterone every 24 h, and harvested after 96 h. Four hours prior to harvesting, cells were treated with 20 μm AA. Cells were treated with 1 mm hydroxyurea, a ribonucleotide reductase inhibitor that served as a procedural control (25Yeh Y.C. Tessman I. J. Biol. Chem. 1978; 253: 1323-1324Abstract Full Text PDF PubMed Google Scholar). Hydroxyurea was added at the same time as the AA. Adherent cells were removed with 0.25% trypsin. To enhance cell permeability, cells were washed twice in solution A (150 mm sucrose, 80 mm KCl, 35 mm HEPES (pH 7.4), 5 mm MgCl2, 0.5 mm CaCl2), then suspended in 500 μl of cold Solution A containing 0.25 mg/ml lysolecithin, and incubated for 1 min at 4 °C. To measure ribonucleotide reductase activity, 1 × 106 cells were incubated at 37 °C for 10 min in 300 μl of reaction mixture containing 50 mm HEPES, pH 7.4, 0.75 mm CaCl2, 10 mm phosphoenolpyruvate, 0.2 mm [3H]rCDP, 0.2 mm rGDP, 0.2 mm rADP, and 0.2 mm dTDP. After 10 min, the incubation mixture was added to 60 μl of 60% percholic acid/0.1% Na4P2O7 to quench ribonucleotide reductase activity. The samples were incubated at 4 °C for 15 min, then diluted with 1 ml of H2O, and centrifuged to precipitate acid-insoluble material. The pellet was extracted with 100 μl of 0.2 n NaOH and incubated at 37 °C for 30 min. 75 μl of each sample was suspended in 5 ml of Ecoscint A, and 2′-deoxy-[3H]rCDP, the radioactive product, was counted using a Wallac Microbeta liquid scintillation counter. Results were normalized to the total number of cells. Bcl-2 and BAX Protein Expression in Lox15-1+Cells—Lox15-1+ cells were treated with vehicle or 3 μm ponasterone for 24–96 h. Approximately 1 × 106 cells were lysed, and the supernatant was harvested. 20 μg of protein from each sample was fractionated by 10% SDS-PAGE, and proteins were transferred onto a polyvinylidene difluoride membrane as described above. Membranes were probed for Bcl-2 (antimouse Bcl-2, 1:400) and BAX (anti-rabbit BAX,1:500). Antigen-antibody complexes were detected with ECL reagents. Immunochemical Analysis 4-HNE:TrxR Protein Adducts—For direct modification of TrxR by 4-HNE, purified TrxR (0.51 μg/ul) in 50 μm K2PO4, pH 6.5, and 200 μm EDTA, 2 μm NADPH was incubated with 10–30 μm of 4-HNE or vehicle for 0.5 min. To facilitate spontaneous formation of 4-HNE, 10–30 μm 15(S)-HpETE was preincubated with NADPH in K2PO4 and EDTA buffer at 37 °C for 10 min. Isolated TrxR (0.51 μg/ul) was then added to the mixture and incubated for 0.5 min before fractionation by 10% SDS-PAGE. In the 4-HNE-treated TrxR samples, 75 ng of protein was added to Laemmli buffer with and without β-mercaptoethanol and boiled at 100 °C for 5 min. Three times as much protein (225 ng) was used in the 15(S)HpETE-treated TrxR samples. Gels were transferred to polyvinylidene difluoride membranes and probed for 4-HNE adducts with rabbit anti-4-HNE (1:1000) and goat anti-rabbit-HRP (1:5000). Antigen-antibody complexes were detected with ECL reagents. Statistics—Statistical significance at a 95% confidence interval was determined by analysis of variance with the Krusky-Wallis non-parametric test and Dunn's post hoc test. Effect of LOX Metabolites on Purified TrxR—Metabolites of 15-LOX-1 inhibited isolated TrxR more potently than metabolites of 5-LOX. Table I lists the concentration for half-maximal inhibition (IC50) by reactive carbonyls (enones); hydroperoxy lipids, and hydroxy lipids. Overall, the most potent inhibitor was 4-HNE, a spontaneous rearrangement product of the HpETEs and HpODEs. The most potent inhibitors among hydroperoxy and hydroxy lipids were 15(S)-HpETE and 15(S)-HETE, respectively. Hydroperoxy substituents conferred ∼2–3-fold more potency than the corresponding hydroxy substituents. The steric configuration of the hydroxy substituent also influenced the IC50: 15(S)-HETE was 4-fold more potent than the 15(R)-stereoisomer. The LOX substrates, AA and LA did not inhibit TrxR at >100 μm.Table IInhibition of purified TrxR by LOX metabolitesCompoundIC50μmReactive carbonyls (Enones)4-HNE1 ± 0.212-oxo-ETE2.7 ± 0.715-oxo-ETE29 ± 45-oxo-ETE37 ± 6Hydroperoxy metabolites15(S)-HpETE13 ± 1.512(S)-HpETE19 ± 2.213(S)-HpODE279(S)-HpODE295(S)-HpETE32 ± 0.3Hydroxy metabolites15(S)-HETE2112(S)-HETE6013(S)-HODE649(S)-HODE6515(R)-HETE765(R)-HETE>1005(S)-HETE>100SubstratesAA>100LA>100 Open table in a new tab 4-HNE formed a covalent adduct with TrxR in vitro, as shown by the appearance of a protein with a 4-HNE epitope co-migrating with TrxR under denaturing conditions (Fig. 1). Maximal formation of the 4-HNE:TrxR adduct occurred within ∼0.5 min. Incubation of TrxR with 15(S)-HpETE generated lesser, but detectable amounts of this adduct, consistent with spontaneous generation of 4-HNE from lipid peroxides (9Schneider C. Tallman K.A. Porter N.A. Brash A.R. J. Biol. Chem. 2001; 276: 20831-20838Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 11Gardner H.W. J. Agric. Food Chem. 1975; 23: 129-136Crossref PubMed Scopus (211) Google Scholar). Boiling samples in Laemmli buffer with β-mercaptoethanol eliminated the 4-HNE adduct, consistent with its formation by a Michael reaction between a redox-sensitive nucleophile on TrxR and the electrophilic β-carbon of 4-HNE. Inhibition of Cellular TrxR Activity by 15-LOX-1 Induction and Catalysis—Ponasterone induced enzymatically competent 15-LOX-1 in a concentration and time-dependent manner (Fig. 2). Half-maximal formation of 15(S)-HETE occurred with 20 μm AA (Fig. 2C). TrxR activity in Lox15-1+ cells declined after induction of 15-LOX-1 (Fig. 3). In Lox15-1+ cells treated with ponasterone and 20 μm AA or LA, TrxR activity was 50% lower than the corresponding control cells without 15-LOX-1 induction (p < 0.05, n = 8). The NADPH oxidation shown in Fig. 3 was due to TrxR activity, because NADP+ formation was indistinguishable from background in the absence of added Trx (Fig. 3, panel A). Kinetic experiments also support the conclusion that 15-LOX-1 metabolism causes the loss of TrxR activity. In Lox15-1+ cells treated with ponasterone, TrxR activity fell within 10 min after the addition of AA. The inhibition was saturable, and it persisted for at least 8 h, consistent with an irreversible, or slowly reversible mechanism (Fig. 3, B and C). Maximal inactivation of TrxR occurred with addition of ∼50 μm AA, from which 15-LOX-1 can generate 15(S)-HpETE, 15(S)-HETE, and 4-HNE, along with lesser amounts of 12(S)-HpETE and 12(S)-HETE (26Bryant R.W. Bailey J.M. Schewe T. Rapoport S.M. J. Biol. Chem. 1982; 257: 6050-6055Abstract Full Text PDF PubMed Google Scholar). Comparable experiments with Cox-2+ cells indicated that the induction of COX-2 had no effect on cellular TrxR activity (Table II). Ponasterone induced COX-2 with a dose-response and time course similar to its induction of 15-LOX-1. Cox-2+ cells converted the majority of AA to PGE2.Table IIConditional expression of COX-2 does not inhibit cellular TrxR activityTreatmentCOX-2+ cellsaValues are mean ± S.E., n = 3.%Vehicle control100 ± 63 μm ponasterone97 ± 33 μm ponasteroneplus 20 μm AA95 ± 2Vehicleplus 20 μm AA108 ± 4a Values are mean ± S.E., n = 3. Open table in a new tab Cell Cycle Arrest, Ribonucleotide Reductase Inhibition, and Anchorage-independent Growth: Relation to 15-LOX-1 Induction and Catalysis—Lox15-1+ cells were arrested in the G1 phase of the cell cycle following the induction of 15-LOX-1 (Fig. 4A). Results were similar with Lox15-1+ cells grown in serum-free medium (CD293), which excludes the possibility that fatty acids or other substances present in the serum caused cell cycle arrest (Fig. 4). Cell cycle arrest in G1 is consistent with inhibition of TrxR, which regulates cellular ribonucleotide reductase activity (3Arnér E.S.J. Holmgren A. Eur. J. Biochem. 2000; 267: 6102-6109Crossref PubMed Scopus (2000) Google Scholar). Measurements affirmed that Lox15-1+ cells treated with ponasterone and AA had a 32 ± 4% decline in ribonucleotide reductase activity compared with control cells (Fig. 4C). Anchorage-independent growth in soft agar is one index of the oncogenic potential of cells. Lox15-1+ cells treated with ponasterone formed fewer colonies in soft agar compared with Lox15-1+ cells treated with vehicle, or compared with Cox-2+ cells treated with vehicle or ponasterone (Fig. 5). Cox-2+ cells treated with ponasterone formed more colonies in soft agar compared with Cox-2+ cells treated with vehicle. Thus, 15-LOX-1 induction opposes anchorage-independent growth; COX-2 induction favors it. Apoptosis: Relationship to 15-LOX-1 Induction—To determine if induction of 15-LOX-1 enhanced apoptosis, we measured bcl-2 and BAX protein expression. Bcl-2 and BAX affect apoptosis in opposite ways (27Knudson C.M. Korsmeyer S.J. Nat. Genet. 1997; 16: 358-363Crossref PubMed Scopus (350) Google Scholar, 28Oltvai Z.N. Milliman C.L. Korsmeyer S.J. Cell. 1993; 74: 609-619Abstract Full Text PDF PubMed Scopus (5864) Google Scholar). Bcl-2 is anti-apoptotic, while BAX is pro-apoptotic. The ratio of BAX to bcl-2 rose in the cells expressing 15-LOX-1, suggesting higher numbers of cells poised for apoptosis (Fig. 6A). The level of BAX expression increased progressively from 0–96 h, while the level of bcl-2 expression remained constant in Lox15–1+ cells treated with ponasterone (Fig. 6A). In contrast, in Cox-2+ cells the ratio of bcl-2 to BAX rose, consistent with fewer cells undergoing apoptosis (Fig. 6B). Bcl-2 protein expression increased from 24 to 96 h, while BAX expression remained constant. Consistent with these observations, Lox15-1+ cells treated with ponasterone displayed a 3-fold higher caspase-3 activity than cells treated with vehicle (data not shown). Effect of 5-LOX E" @default.
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• W2011802015 title "Conditional Expression of 15-Lipoxygenase-1 Inhibits the Selenoenzyme Thioredoxin Reductase" @default.
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• W2011802015 cites W1514733921 @default.
• W2011802015 cites W1521799099 @default.
• W2011802015 cites W1538415171 @default.
• W2011802015 cites W1976161942 @default.
• W2011802015 cites W1980881765 @default.
• W2011802015 cites W1985811862 @default.
• W2011802015 cites W1985841114 @default.
• W2011802015 cites W1987338970 @default.
• W2011802015 cites W1988491976 @default.
• W2011802015 cites W1988607142 @default.
• W2011802015 cites W1996344228 @default.
• W2011802015 cites W2001338101 @default.
• W2011802015 cites W2001858936 @default.
• W2011802015 cites W2008320177 @default.
• W2011802015 cites W2008820074 @default.
• W2011802015 cites W2011133904 @default.
• W2011802015 cites W2013862513 @default.
• W2011802015 cites W2019288974 @default.
• W2011802015 cites W2029423245 @default.
• W2011802015 cites W2031760644 @default.
• W2011802015 cites W2042017985 @default.
• W2011802015 cites W2043284283 @default.
• W2011802015 cites W2049126415 @default.
• W2011802015 cites W2049648341 @default.
• W2011802015 cites W2050057935 @default.
• W2011802015 cites W2062159267 @default.
• W2011802015 cites W2071497993 @default.
• W2011802015 cites W2074203890 @default.
• W2011802015 cites W2081577661 @default.
• W2011802015 cites W2092325522 @default.
• W2011802015 cites W2095715712 @default.
• W2011802015 cites W2103650142 @default.
• W2011802015 cites W2105311935 @default.
• W2011802015 cites W2123015104 @default.
• W2011802015 cites W2134979684 @default.
• W2011802015 cites W2139002144 @default.
• W2011802015 cites W2149423279 @default.
• W2011802015 cites W2154672973 @default.
• W2011802015 cites W2158398965 @default.
• W2011802015 cites W2160641917 @default.
• W2011802015 cites W2164736711 @default.
• W2011802015 cites W2171274653 @default.
• W2011802015 cites W4251161705 @default.
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