aspirin-triggeredlipoxins (15-epi-lx) are generated human ... · aspirin-triggeredlipoxins...

Aspirin-Triggered Lipoxins (15-epi-LX)Are Generated by the Human LungAdenocarcinoma Cell Line (A549)-Neutrophil Interactions and ArePotent Inhibitors of Cell Proliferation

Joan Claria,l Min H. Lee, and Charles N. SerhanCenter for Experimental Therapeutics and Reperfusion Injury,Department of Anesthesia, Brigham and Women's Hospital andHarvard Medical School, Boston, Massachusetts, U.S.A.

ABSTRACT

Background: The mechanism by which aspirin (ASA)acts to protect against human cancer is not yet known.We recently showed that ASA triggers the formation of anew series of potent bioactive eicosanoids, 15-epi-li-poxins (15-epi-LXs or ASA-triggered LX [ATL]), duringinteractions between prostaglandin endoperoxide syn-thase-2 (PGHS-2) in endothelial cells and 5-lipoxygen-ase (LO) in leukocytes. Here, we investigated the trans-cellular biosynthesis of these eicosanoids duringcostimulation of the human tumor A549 cell line (alve-olar type II epithelial cells) and neutrophils, and evalu-ated their impact on cell proliferation.Materials and Methods: A549 cells and isolated neu-trophils were coincubated and mRNA expression levelsof key enzymes in eicosanoid biosynthesis were mea-sured. In addition, product formation was analysed byphysical methods. The effect of LX on cell proliferationwas determined by using a soluble microculture tetrazo-lium (MTT) assay and by measuring [3H] -thymidine in-corporation.Results: Interleukin-1l3 (IL-1,)-primed A549 cellsshowed selective elevation in the levels of PGHS-2mRNA and generated 15-hydroxyeicosatetraenoic acid(1 5-HETE). ASA markedly increased 15-HETE formationby A549 cells, while treatment with an inhibitor of cy-tochrome P450 reduced by -50%, implicating bothPGHS-2- and cytochrome P450-initiated routes in 15-

HETE biosynthesis in these cells. Maximal production of15-HETE from endogenous sources occurred within 24hr of cytokine (IL-1*3) exposure and declined thereafter.Chiral analysis revealed that -85% of ASA-triggeredepithelial-derived 15-HETE carries its carbon 15 alcoholgroup in the R configuration. Costirnulation of ASA-treated A549 cells and polymorphonuclear neutrophilicleukocytes (PMN) led to production of both LXA4 andLXB4, as well as 15-epi-LXA4 and 15-epi-LXB4 (9.5 ±0.5 ng LX/107 A549 cells). 15-epi-LXA4 accounted for-88% of the total amount of LXA4 produced. In addi-tion to LXs, stimulation of A549 cells and PMN alsoliberated substantial amounts (77.2 ± 8.2 ng/107 A549cells) of peptidoleukotrienes (pLTs), which were notgenerated by either cell type alone. Addition of ASA tothese co-incubations led to an increase in the amounts ofLXs generated that was paralleled by a decrease in pLTs.LXA4, LXB4, 15-epi-LXA4 and 15-epi-LXB4, as well asdexamethasone, inhibited cell proliferation at 100 nMrange with a rank order of activity of 15-epi-LXB4 >>>LXB4> dexamethasone ' 15-epi-LXA4 > LXA4.Conclusions: These results indicate that ASA promotesthe formation of antiproliferative 15-epi-LXs by epithe-lial cell-leukocyte interactions. Moreover, they suggestthat these novel eicosanoids, when generated within themicroenvironment of tissues, may contribute to ASA'stherapeutic role in decreasing the risk of human cancer.

INTRODUCTIONAlveolar epithelial cells constitute the vast air-tissue interface of the distal lung and are strate-

Address correspondence and reprint requests to: Charles N.Serhan, Center for Experimental Therapeutics and Reperfu-sion Injury, Brigham and Women's Hospital, 75 FrancisStreet, Boston, MA 02115, U.S.A.'Present address: Hospital Clinic i Provincial, Universitat deBarcelona, Villarroel 170, Barcelona 08036, Spain.

gically located to interact with substances or cellsin both the alveolar space and the pulmonaryinterstitium. These cells play an active role in theimmune response by their ability to generatebiologically active mediators in response to stim-uli (1). In this regard, airway epithelial cells mayalso interact with leukocytes that have been re-cruited from peripheral blood to the lung inter-stitium to produce new mediators by transcellu-

Copyright © 1996, Molecular Medicine, 1076-1551/96/$10.50/0Molecular Medicine, Volume 2, Number 5, September 1996 583-596

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584 Molecular Medicine, Volume 2, Number 5, September 1996

lar routes. We hypothesized that lipid mediatorsgenerated during these cell-cell interactionswithin the local microenvironment may servehomeostatic functions and also modulate patho-physiological pulmonary changes such as hyper-plasia, fibrosis, and neoplasia.

Lipoxins (LXs) are eicosanoids (a class ofcompounds derived from polyunsaturated fattyacids, such as arachidonic acid) that carry a con-jugated tetraene structure and biological activi-ties that clearly distinguish them from other ei-cosanoids such as prostaglandins (PGs) andleukotrienes (LTs) (2). In human tissues, LX for-mation occurs predominantly by cell-cell inter-actions and two established routes of transcellu-lar LX biosynthesis have been identified to date.These include oxygenation of arachidonic acid by1 5-lipoxygenase (1 5-LO) followed by formationof a 5(6)-epoxytetraene intermediate in leuko-cytes to give LXA4 and LXB4 (15-H(p)ETE path-way) and/or transformation of leukocyte-de-rived LTA4 to LXs by platelet 12-LO (LTA4:12-LO pathway). LX generated by these twopathways carry their C1 5 alcohol in the S (>85-90%) configuration (reviewed in Ref. 3). Re-cently, a new biosynthetic route of transcellularLX formation was identified (4). This pathway istriggered by aspirin (ASA) and involves the for-mation of a key intermediate from arachidonicacid, namely 15-R-15-hydroxy-eicosatetraenoicacid (1 5R-HETE), by ASA-acetylated prostaglan-din endoperoxide synthase-2 (PGHS-2, cycloox-ygenase). In cytokine-treated endothelial cells,this intermediate is released and transformed inactivated human leukocytes by 5-LO and subse-quent reactions to produce a new series of 15-epimeric LXs (ASA-PGHS-2 pathway).

Evidence is accumulating that ASA has aprotective role against human cancer (5). Themechanism by which ASA acts to reduce theincidence and risk of human cancers (includinglung, colon, and breast cancers) is not yet knownbut may be related to its ability to inhibit PGHS-dependent PG biosynthesis (for review see Ref.6). Given that ASA does not directly affect LOenzymes (2), eicosanoids generated by transcel-lular biosynthesis in the presence of ASA (i.e.,15-epi-LXs and LX), may be involved in ASA'snewly uncovered chemopreventive therapeuticactions (4,7). Here, we report that ASA stimu-lates the formation of 15-epi-LXs from the tu-mor-derived (A549 cells) epithelial cell-PMN in-teractions and provide evidence for the role ofthese eicosanoids, in particular 15-epi-LXB4, ininhibiting cell proliferation. Moreover, this is the

first report both of formation and of a specificbioaction for 1 5-epi-LXB4.

MATERIALS AND METHODSMaterialsSynthetic (5S,6R, 1 5R) -trihydroxy-7,9, 1 3-trans-11 -cis-eicosatetraenoate:carboxymethyl ester(1 5-epi-LXA4-methyl ester) was prepared by to-tal organic synthesis and was a gift of Prof. N. A.Petasis (Department of Chemistry, University ofSouthern California). 1 5-epi-LXA4 free acid wasobtained by saponification of 1 5-epi-LXA4-methyl ester in tetrahydrofuran with LiOH (0.1M) at 4°C for 24 hr. Synthetic eicosanoid refer-ence samples were from Cascade Biochem Ltd.(Reading, Berkshire, England). Inhibitors of 5-li-poxygenase (Rev 5901 isomer) and cytochromeP450 (1 7-octadecaynoic acid, 17-ODYA) activi-ties were from Biomol (Plymouth Meeting, PA,U.S.A.). Radiolabeled ([32p]) dCTP and ([3H])arachidonic acid and methyl-thymidine werefrom Dupont NEN (Boston, MA, U.S.A.). lono-phore (A23187), ASA, 3,(4,5-dimethylthiazoyl-2-yl) 2,5 (diphenyl-tetrazolium bromide) (MTT),and guanidinium isothyocyanate were pur-chased from Sigma Chemical Co. (St. Louis, MO,U.S.A.). Recombinant human interleukin-l1((IL-1iB) was obtained from R&D Systems (Min-neapolis, MN, U.S.A.). Dulbecco's phosphate-buffered saline containing both CaCl2 (0.6 mM)and MgCl2 (1.0 mM) (pH 7.4) (DPBS2+), fetalbovine serum (FBS), penicillin, and streptomycinwere from Bio Whittaker (Walkersville, MD,U.S.A.). Hanks' balanced salt solution (HBSS)and F-12K nutrient mixture were from GibcoLaboratories (Grand Island, NY, U.S.A.). High-pressure liquid chromatography (HPLC) gradesolvents and cesium chloride were purchasedfrom JT Baker (Phillipsburg, NJ, U.S.A.), methylformate was from Eastman Kodak Co. (Roches-ter, NY, U.S.A.) and Sep-Pak C18 cartridges werefrom Waters Associates (Milford, MA, U.S.A.).Diazomethane was prepared from N-methyl-N'-nitro-N-nitroguanidine purchased from AldrichChemical Co. (Milwaukee, WI, U.S.A.). N,0-bis(trimethylsilyl) trifluoroacetamide (BSTFA) wasfrom Pierce (Rockford, IL, U.S.A.). First strandcDNA synthesis kit and other molecular biologyreagents were from Promega (Madison, WI,U.S.A.). Oligonucleotide primers were purchasedfrom Integrated DNA Technologies (Coralville,IA, U.S.A.).

J. Claria et al.: Aspirin-Triggered Lipoxins Inhibit Cell Proliferation

Cell Isolation and CultureHuman type II epithelial A549 cells from humanlung carcinoma and normal human skin fibro-blast (breast) were obtained from the AmericanType Culture Collection (Rockville, MD, U.S.A.).The A549 cell line originates from a human al-veolar cell carcinoma and has proven to be auseful cell line because they are easily assessableand are maintained in culture without contami-nating tissue macrophages (8). Fibroblasts wereutilized within their limited window of viabilityand their passage numbers were recorded (re-sults are reported for cells from three to fivepassages). Epithelial A549 cells were seeded intoT-75 cm2 tissue culture flasks and maintained inF-12K medium supplemented with 10% heat-inactivated FBS, penicillin (50 U/ml), and strep-tomycin (50 ,ug/ml). Human PMN from healthydonors who had not taken ASA or other medi-cations for at least 2 weeks were obtained byFicoll-Hypaque gradient centrifugation and dex-tran sedimentation (9) and suspended in DPBS+at pH 7.4. Viability of A549 cells and PMN wasdetermined by their ability to exclude trypanblue and were 95 ± 2% and 97 ± 1%, respec-tively. These values were not significantly alteredduring the reported incubations.

Incubation ConditionsIn incubations involving permeabilized A549cells (prepared by a rapid freeze-thaw cycle),IL-1,Btreated (1 ng/ml, 24 hr) A549 cells (1.5 X106 cells/ml) were pretreated for 20 min witheither vehicle (0.1% EtOH), ASA (500 ,uM; usedthroughout), 5 ,uM 17-ODYA, an inhibitor ofcytochrome P450 (10), or 5 mM Rev 5901 iso-mer, a 5-LO inhibitor, subjected to two cycles ofrapid freezing in a dry ice-acetone bath andthawing to room temperature (full cycle <20min), and incubated with arachidonic acid (20,uM) for 20 min at 370C in 4 ml of DPBS2+. Inexperiments involving radiolabeled arachidonicacid, incubations were initiated with the additionof [3H]-arachidonic acid (0.25 ,uCi/ml) plus un-labeled arachidonic acid (20 ,uM) for 20 min at370C.

For time course experiments involving thegeneration of 1 5-HETE from endogenous sources(Fig. 2B), intact A549 cells were exposed to IL-1A(1 ng/ml) for up to 48 hr and then treated withvehicle (containing 0.1% EtOH) or ASA for 20min followed by addition of ionophore A23187 (5,uM) in 4 ml of HBSS for 30 min at 370C.

In co-incubation experiments, confluent

A549 cells were exposed to IL-i 3 (1 ng/ml, 24hr), washed in HBSS and treated with vehiclealone or ASA for 20 min and arachidonic acid(20 ,LM) for 60 sec at 37°C. Co-incubations wereperformed by adding PMN to A549 cell mono-layers followed by costimulation with ionophoreA23187 (5 ttM) in 4 ml of HBSS for 30 min at370C.

Analysis of EicosanoidsIncubations were stopped with two volumes ofcold MeOH containing prostaglandin B2 (200 ng)and products were extracted using Sep-Pak Cl8cartridges. Materials that eluted in the methylformate fractions were concentrated under astream of N2 and scanned for ultraviolet (UV)-absorbing material (in methanol) with a Model8452 spectrophotometer (Hewlett Packard Co.,Palo Alto, CA, U.S.A.) prior to injection into areversed phase (RP)-HPLC system. This systemconsisted of a dual pump gradient (LKB,Bromma, Sweden), a diode array detector(Hewlett-Packard 1040M series II) and a HPLC3DChemStation software. The collected UV datawere recalled at 300 nm to monitor conjugatedtetraenes, at 270 nm for trienes, and 234 formonoHETEs. All UV spectra were acquired usingstep = 4 nm, Bw = 10 nm, and range = 220-360nm with a sampling interval of 0.96 sec.

The monohydroxy eicosanoids (i.e., 5-, 12-,and 15-HETE) from A549 cells were analyzedusing a Ultrasphere-ODS column (5 ,um, 4.6mm X 25 cm) (Beckman Instruments, Fullerton,CA, U.S.A.) was eluted with MeOH/H20/aceticacid (65:35:0.01; v/v/v) as phase one (to - 20min), and a linear gradient with MeOH/aceticacid (99.99:0.01, v/v) as phase two (20-45 min)at a flow rate of 1.0 ml/min. The R- and S-enantiomers of 1 5-HETE were resolved andidentified using a chiral HPLC system similar tothat reported by Hawkins et al. (1 1). Briefly, afterRP-HPLC material eluting beneath 1 5-HETEpeak was extracted with chloroform and con-verted into methyl ester by ethereal diazometh-ane treatment. Chiral analysis was performedwith a Bakerbond DNBPG (covalent) chiral col-umn (5 ,um, 4.6 mm X 25 cm) (JT Baker, Phil-lipsburg, NJ, U.S.A.) eluted with n-hexane/2-propanol (100:0.4; v/v) at a flow rate of 0.8ml/min. When indicated, generation of 1 5-HETEfrom endogenous sources was monitored by ra-dioimmunoassay (RIA). The antibody was raisedagainst 15S-HETE with 0.1% cross-reactivity at50% B/Bo for 5-HETE (PerSeptive Diagnostics,Cambridge, MA, U.S.A.).

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For analysis of lipoxins (including LXs and15-epi-LX) from A549 cell-PMN, co-incubationswere carried out using either a Waters,uBondapak C18 (3.9 X 300 nm) column elutedwith an isocratic mobile phase MeOH/H20/aceticacid (60:40:0.01; v/v/v) with a flow rate of 0.6ml/min or an Altex Ultrasphere ODS column (5,um, 10 mm X 25 cm) eluted with MeOH/H20/acetic acid (65:35:0.01; v/v/v) at a flow of 3ml/min. Peptidoleukotrienes (LTC4 and LTD4)eluted in the MeOH fractions from Sep-Pak car-tridge extractions were resolved with a BeckmanUltrasphere-ODS column eluted with MeOH/H20/acetic acid (65:35:0.01; v/v/v), pH 5.7, at 1ml/min. Incubations of PMN with 15R-HETEwere stopped with MeOH and methyl formatefractions of the Sep-Pak C18 extracted productswere injected into an Altex Ultrasphere ODS col-umn (5 ,um, 10 mm X 25 cm) eluted withMeOH/H20/acetic acid (65:35:0.01; v/v/v) usinga flow of 3 ml/min. The material beneath peaksabsorbing at 300 nm were individually collectedafter RP-HPLC and analyzed by gas chromatog-raphy-mass spectrometry (GC-MS) employing aHewlett-Packard 5890 GC series II equipped witha 5971A mass-selective quadrapole detector as inClaria and Serhan (4).

Reverse Transcription and PCRTotal RNA was obtained from A549 cells by theguanidinium isothiocyanate-cesium chloridemethod and cDNA was produced by reversetranscription (RT). Oligonucleotide primers wereconstructed from published sequences of PGHS-1and PGHS-2 (5'-TGC CCA GCT CCT GGC CCGCCG CTT-3' [sense] and 5'-GTG CAT CAA CACAGG CGC CTC TTC-3' [antisense], and 5'-TTCAAA TGA GAT TGT GGG AAA AIT GCT-3'[sense] and 5'-AGA TCA TCT CTG CCT GAG TATCTT-3' [antisense], respectively) (12); of 1 5-LO(5'-ATG GGT CTC TAC CGC ATC CGC GTG TCCACT-3' [sense] and 5'-CAC CCA GCG GTA ACAAGG GAA CCT GAC CTC-3' [antisense] (13); of12-LO (5'-AGT TCC TCA ATG GTG CCA AC-3'[sense] and 5'-ACA GTG ITG GGG TTG GAGAG-3' [antisense]) (14); and of 5-LO (5'-GAAGAC CTG ATG TTT GGC TACC-3' [sense] and5'-AGG GTT CTC ATC TCC CGG-3' [antisense])(15). Amplification of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers wasperformed with 5'-CCA CCC ATG GCA AAT TCCATG GCA-3' (sense) and 5'-TCT AGA CGG CAGGTC AGG TCC ACC-3' (antisense). PGHS-1,PGHS-2, and GAPDH samples were amplified for

25 cycles of denaturation at 94°C for 1 min,annealing at 58°C for 2 min and extension at720C for 3 min. 5-, 12-, and 15-LO were ampli-fied at 94°C (1 min), 55°C (2 min), and 720C (2.5min) for 35 cycles. PCR products were analyzedby electrophoresis in 2% agarose gel and theiridentity was monitored by restriction enzymeanalysis. For detection of specific PCR-amplifiedtargets, 0.5 ,uCi of [32P]dCTP (3000 Ci/mmol)was added to the PCR mixture and the productswere quantified by phosphorimager using Im-age-Quant programming (Molecular Dynamics,San Lorenzo, CA, U.S.A.).

Cell Proliferation

A microculture 3, (4,5-dimethylthiazoyl-2 -yl)2,5 (diphenyl-tetrazolium bromide) (MTT) assay(16) was used to examine the actions of LXs andother eicosanoids on cell proliferation. A549 cellsand fibroblasts from exponential-phase mainte-nance cultures were counted and dispensedwithin replicate 96-well culture plates in 100-,ulvolumes of medium (-2000 cells/well). Follow-ing 24 hr at 37°C, the culture medium was re-moved, and fresh medium (100 ,ul) containingeither the compounds (5-1000 nM) or vehicle(medium plus 0.15% ethanol) was added to fourreplicates for each condition studied and the cul-ture plates were then incubated for up to 96 hr at370C in a 5% CO2 atmosphere. At the end of thisperiod, 25 ,ul of freshly prepared MIT in HBSS (5mg/ml) was added to the wells and the plateswere incubated for 4 hr at 370C. Dye solutionwas aspirated, wells were washed once withHBSS and dye taken up by the cells was ex-tracted in 100 plI of isopropyl alcohol: 1 N HCG(96:4, v/v) and quantitated at 570 nm using amicroplate reader (Molecular Devices, MenloPark, CA, U.S.A.). In some experiments, cellswere grown in 12-well culture plates, treated asabove and enumerated using a Neubauer hemo-cytometer. Viability was assessed routinely usingtrypan blue exclusion assay. For the A549 cellsand fibroblasts, a linear relation was establishedfor the MTT values and cell number within therange of the experiments shown (r = 0.995; p <0.005). A549 cells grown for 72 hr in the pres-ence of the compounds (5-1000 nM) or vehicle(0.1 5% EtOH) were lysed with 0.2 5 N NaOH andthe cellular protein content was determined byapplying the Bio-Rad (Richmond, CA, U.S.A.)microassay method using bovine serum albuminas standard. The mean cellular protein content inresting A549 cells was 46.6 ± 1.5 pg/cell.


Thymidine Incorporation and DNASynthesisA549 cells (-2 X 104 cells/ml) were seeded in96-well plates, allowed to settle for 24 hr andgrown for an additional 72 hr in the presence ofcompounds (5-1000 nM) or vehicle (mediumplus 0.15% ethanol). Twenty-four hours beforethe assay, 2 ,uCi/ml of methyl- [3H] -thymidine(specific activity 6.7 Ci/mmol) were added toeach well (17). After pulse-labeling, each wellwas washed four times with cold DPBS2+, andthe cells were lysed with NaOH (0.25 N) andradioactivity measured.

The Student's t test was used for statisticalanalysis, and differences were considered signif-icant at a p value of <0.05.

RESULTSEicosanoids are formed by initial oxygenation ofarachidonic acid by PGHS or LO enzymatic path-ways (2). To assess which of the eicosanoid-

generating enzymes are present and/or regulatedby cytokines in A459 cells, mRNA levels ofPGHS-1 and -2, and 5-, 12-, and 15-LO fromA549 cells grown in the presence or absence ofIL-1,3 were monitored by RT-PCR followed byphosphorimager analysis. As illustrated in Fig. 1,mRNA levels for PGHS-2 were significantly in-creased (-2-fold) after stimulation of A549 cellswith IL- I P. In contrast, mRNA levels for PGHS- 1and 5-LO were not significantly altered after ex-posure of the cells to cytokine (Fig. 1). A549 cellsfailed to show either 15- or 12-LO expressionbefore or after cytokine induction (Fig. 1). Theabsence of 15-LO mRNA in A549 cells was fur-ther confirmed by performing the RT-PCR inparallel with human lung tissue and peripheralblood monocyte RNA, which are known positiveand negative sources of 15-LO mRNA (14), re-spectively (Fig. 1, inset).

To characterize the profile of monohydroxyproducts produced by airway epithelial cells, IL-13-stimulated A549 cells (1.5 X 106 cells/ml)

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FIG. 1. PGHS and LO expression and monohydroxy products analysis in A549 cells(A) PGHS and LO expression in A549 cells. Cells were grown for 24 hr at 37°C in T-75 cm2 flasks in the presence

or absence of IL-iI3 (1 ng/ml). Extracted total RNA (1 jig) was taken for RT and PCR using specific oligonucleo-tides for PGHS-1 and -2, 15-, 12-, and 5-LO, and GAPDH. Radioactive bands were quantified directly by phospho-rimager analysis, normalized to the expression of GAPDH, and expressed as fold increase in mRNA levels after ex-

posure to IL-1,i. Inset: 15-LO mRNA expression in human lung tissue and peripheral blood monocytes (PBM). ND,not detected. (B) RP-HPLC profile of [3H]-labeled mono-HETEs from permeabilized IL-lIf-treated A549 cells(1.5 X 106 cells/ml) and exposed to [3H]-arachidonic acid (0.25 ,uCi/ml) plus unlabeled arachidonic acid (20 ,M)for 20 min at 37°C. Products were extracted and chromatographed using a linear gradient of methanol:H20:aceticacid (65:35:0.01; v/v/v) and methanol:acetic acid (99.99:0.01, v/v) at a flow rate of 1.0 ml/min. Arrows denoteco-chromatography of synthetic standards.

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were permeabilized and incubated with arachi-donic acid, and the products formed were ex-tracted and analyzed by RP-HPLC. The chro-matographic profile revealed the presence of amajor product with strong UV absorbance at 234nm which coeluted with synthetic 1 5-HETE(data not shown). Also, when [3H]-arachidonicacid was added to IL-l13-treated A549 cells, ra-diolabeled material was recovered beneath thepeak coeluting with 15-HETE (Fig. iB). In theseexperiments, we did not consistently observe theformation of either 5- or 12-HETE. While ASAtreatment of A549 cells led to a marked increasein the formation of 15-HETE, incubation of per-meabilized A549 cells with 17-ODYA, a reportedinhibitor of P450 eicosanoid metabolism (10),resulted in -50% reduction in 15-HETE(Fig. 2A). 17-ODYA is a potent inhibitor of P450eicosanoid metabolism and does not selectivelyinhibit either cyclooxygenase or lipoxygenase ac-tivity (see Muerhoff et al. [10] and supplier'ssupporting materials). The 5-LO inhibitor (Rev-5901 isomer) did not alter the amount of 15-HETE produced by A549 cells in a statistically

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significant fashion. Heat-denatured A549 cellsreduced the quantities of 15-HETE by -90%,suggesting that an enzymatic component is in-volved in its formation. Intact A549 cells treatedwith IL- 1,B (1 ng/ml) for 24 hr also produced15-HETE from endogenous sources of arachido-nate (25.0 ± 10.0 ng/107 cells). Taken togetherthese results indicate that 15-HETE is the mainmonohydroxy product generated by A549 cellsand suggest that both acetylated PGHS-2 andcytochrome P450 contribute to its biosynthesis.

To investigate the time course for generationof 1 5-HETE from endogenous sources of arachid-onate, intact A549 cells were exposed to IL- 1o (1ng/ml) for up to 48 hr and the amount of im-munoreactive 15-HETE present in the cell super-natant was monitored using a specific RIA. Inresting conditions, A549 cells produced signifi-cant levels of immunoreactive 1 5-HETE (19.1 ±10.5 ng/107 cells; n = 3; d = 2). These valueswere not changed by addition of ionophoreA23187 (5 pM) in the absence of IL-1fB (Fig. 2B).Also, in the absence of ionophore stimulation,addition of IL- 13 to A549 cells for up to 48 hr did

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FIG. 2. 15-HETE generation(A) A549 cells (6 X 106 cells/flask) were treated with IL-i13 (1 ng/ml) for 24 hr, subjected to freeze-thaw (two cy-

cles), exposed for 20 min to either vehicle (0.1% v/v EtOH), ASA, the cytochrome P450 inhibitor (17-ODYA, 5AM) or the 5-LO inhibitor (Rev-5901 isomer, 5 iM) and incubated with arachidonic acid (20 AM) for 20 min at370C. In some experiments, cells were heat-denatured (100°C, 60 min) before incubation. Incubations were

stopped with addition of methanol (2 v), and products were extracted for RP-HPLC. Data are mean ± SEM fromfour to six separate flasks. *p < 0.05 and **p < 0.01 for treatments versus control. (B) Time course of 15-HETEformation from endogenous sources. A549 cells (1.5 X 106 cells/ml) were grown for 48 hr in the absence or pres-ence of IL-1f3 (1 ng/ml) and incubated (30 min at 370C) in 4 ml of HBSS with or without A23187 (5 AM). 15-HETE levels were determined by RIA. Results represent the mean ± SEM of three different experiments deter-mined by duplicate. *p < 0.05 for treatments versus vehicle.

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not result in an augmented generation of 15-HETE (Fig. 2B). In sharp contrast, addition ofIL-1 3 plus A23187 stimulation of A549 cells led toa marked increase in the production of 15-HETE(Fig. 2B). Maximal 15-HETE levels were reachedat 24 hr of exposure to the cytokine and declinedthereafter.

To determine the stereochemistry of the al-cohol in 15-HETE produced by A549 cells, weexamined the relative amounts of individual Rand S enantiomers of 15-HETE generated by IL-1(-treated A549 cells using a chiral phase HPLCanalysis (see Materials and Methods). PGHS-2and cytochrome P450 are two enzymes otherthan 15-LO that can convert arachidonic acid to15-HETE. The carbon (C)-15 alcohol group of15-HETE derived from ASA-acetylated PGHS-2 ismainly in the R configuration (18). Here, 15-HETE produced by IL-I 3--primed A549 cells wasconverted to its methyl ester and subjected toSP-HPLC chiral analysis. The 15-HETE from ac-tivated A549 cells was 65% in the R and 35% inthe S configuration (Fig. 3). Pretreatment ofA549 cells with ASA (for 20 min at 37°C) re-sulted in a 3-fold increase in the amount of 15R-HETE, whereas formation of 15S-HETE was notaltered (Fig. 3). In the presence of ASA, 15R-HETE accounted for 85% of the total amount of15-HETE produced by A549 cells. These resultsindicate that the majority of 15-HETE generatedby IL-I /-primed A549 cells in the presence ofASA was in the R configuration.

Transcellular eicosanoid biosynthesis is animportant means of amplifying lipid mediators aswell as generating new mediators (7). Costimu-lation of human endothelial cells and PMN afterASA treatment results in the formation of a newclass of bioactive eicosanoids (4). These noveleicosanoids were identified as 15-epi-LXs andtheir biosynthesis results from the transforma-tion of ASA-triggered endothelial-derived 15R-HETE by leukocytes. In view of these findings, itis possible that formation of new eicosanoids bytranscellular biosynthesis also occurs during ep-ithelial cell-PMN interactions. To test this hy-pothesis, confluent A549 cells were exposed toIL-1/ (1 ng/ml, 24 hr), treated with ASA andcostimulated with PMN. Figure 4 (left panel)shows a representative HPLC profile of materialobtained from stimulated cells exposed to ASA,which revealed the presence of four major prod-ucts with strong UV absorbance when plotted at300 nm. On-line spectral analysis of these prod-ucts showed that they each displayed a triplet ofabsorbing bands characteristic of conjugated tet-

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015S-HETE 15R-HETE

FIG. 3. Chirality of 15-HETE: ASA triggers 15R-HETE formationA549 cells (107 cells/flask) were exposed to IL-1iB (1ng/ml) for 24 hr, treated with vehicle (0.1% v/vethanol) (E) or ASA (U) for 20 min and then incu-bated (30 min, 37°C) in HBSS containing arachi-donic acid (20 ,uM) and A23187 (5 ,AM). Productswere chromatographed by RP-HPLC (as in Fig. IB)and the region containing 1 5-HETE was collected,extracted with chloroform and treated with dia-zomethane. Chiral analysis was performed with aBakerbond DNBPG (see Materials and Methods fordetails). Results are representative of two separateexperiments showing similar results. Inset: Ratio be-tween A549-derived 15R- and 15S-HETE in the ab-sence (0) or presence (U) of ASA.

raene-containing chromophores indicative of theLX basic structure (maxima at 301 nm andshoulders at 288 and 316 ± 2 nm) (Fig. 4, rightpanels).

LXA4 and 15-epi-LXA4 were identified in thechromatographic profiles on the basis of coelu-tion with synthetic standards and the presence ofthe characteristic chromophore. In these co-in-cubations, 15-epi-LXA4 accounted for -88% ofthe total amount of LXA4 detected, which is inagreement with the observation that the major-ity of 15-HETE generated by IL-I (3-primed A549cells exposed to ASA is predominantly in the Rconfiguration (Figs. 3 and 4). In this RP-HPLCsystem, 15-epi- 11 -trans-LXA4 coeluted withLXB4, and 11-trans-LXA4 with 15-epi-LXB4 (notshown). These LX isomers were not further re-solved by HPLC and are denoted in the profilebeneath peaks labeled A and B, respectively(Fig. 4, left panel). The compounds beneath

589


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FIG. 4. Products from epithelial cell-PMNcostimulationRP-HPLC chromatogram (left panel) and on-lineUV spectra (right panels). Confluent A549 cellswere exposed to IL-113 (1 ng/ml) for 24 hr,treated with ASA (20 min) and arachidonic acid(20 ,uM, 60 s), and each incubated with freshlyisolated PMN (A549 cell:PMN cell ratio of 1:8)followed by stimulation with ionophore A23187 (5,uM) in 4 ml of HBSS for 30 min at 37°C. Prod-ucts were extracted and taken to RP-HPLC as de-scribed in Materials and Methods. The chromato-gram was plotted at 300 nm and is representativeof n = 6 experiments. Displayed spectra are frommaterial eluting beneath Peak B (identified as

predominantly 15-epi-LXB4) and 15-epi-LXA4(see text for details). Similar spectra, each show-ing absorbance consistent with tetraene chro-mophores, were obtained from LXA4 and Peaks Aand C.

Peaks A and B did resolve as OTMS, methyl esterderivatives in GC-MS (vide infra). The productswere present in -8:2 ratio in favor of their 15Repimers (n = 3). Compound C (Fig. 4, left panel)did not coelute with any of the previously iden-tified LXs and was also present in the RP-HPLCprofile from activated PMN incubated with 1 5R-HETE (data not shown, n = 5). Material elutingbeneath Compound C matched the physicalproperties of Compound III that we recently iso-lated from endothelial cell-PMN interactions (4).Although the complete stereochemistry of Com-pound C remains to be determined, the UV spec-tral data and chromatographic mobilities suggest

that it may be the 15-epimer form of 7-cis-11-trans-LXA4 (19). Thus, the lipoxins generatedduring epithelial (A549 cell)-PMN costimulationafter ASA treatment were predominantly 1 5-epi-LX (Fig. 4).

In addition to the ability to produce LXs,co-incubations of activated PMN with A549 cellsalso generate significant amounts (approximate-ly eight times more than tetraene-containingLXs) of peptidoleukotrienes (pLTs; LTC4 andLTD4) in the absence of ASA (Fig. 5). Theamounts of both LXs and pLTs produced in theseco-incubations were dependent upon individualcell ratios (Fig. 5 A and B, insets). Exposure ofairway epithelial A549 cells to ASA 20 min be-fore adding PMN led to an increase in the forma-tion of LXs and a decrease in pLTs (Fig. 5 A andB). Neither PMN nor A549 cells incubated sepa-rately in the absence or presence of ASA gener-ated detectable levels of LXs or pLTs (Fig. 5 anddata not shown). Taken together, these resultsindicate that, during A549 cell-PMN interac-tions, both LXs and pLTs originate from transcel-lular routes.

LXs are vasodilators and potent regulators ofleukocyte responses, such as inhibition of che-motaxis, adhesion to endothelial cells, and trans-migration across epithelium (reviewed in Ref. 3).In contrast, pLTs possess both vasoconstrictorand proinflammatory actions as well as stimulatethe growth of several cell types including fibro-blasts, smooth muscle cells, and glomerular epi-thelial cells (20). LXs reverse the vasoconstrictoraction of LTD4 in rat renal hemodynamics andblock LTC4-stimulated hematopoiesis (reviewedin Ref. 3). Since ASA enhances 15-epi-LX forma-tion and inhibits pLT biosynthesis (Fig. 5), theseeicosanoids may play counterregulatory actionson cell proliferation and contribute to ASA's pro-tective mechanisms in human cancer. To thisend, we tested the effect of these LO products onepithelial cell proliferation (Fig. 6) and comparedtheir actions with that of dexamethasone, a well-established inhibitor, employing a soluble micro-culture tetrazolium (MTT) assay (21). These ex-periments were performed with synthetic LXA4and LXB4, which were available in sufficientquantities for bioassay, rather than the 15-epi-LX, which are the major LX produced by thesecells. As shown in Fig. 6, LXA4 and LXB4 inhib-ited A549 cell proliferation in a time- (Fig. 6A)and dose-dependent (Fig. 6B) fashion. LXA4 andLXB4 as well as dexamethasone (1 ,uM) inhibitedA549 cell proliferation after 72 and 96 hr oftreatment (Fig. 6A). After 72 hr, LXA4 shared the


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Vehicle ASAFIG. 5. ASA modulates both LX (A) and pLT (B) formation during epithelial cell-PMN costimulationA549 cells were exposed to IL- I,B (1 ng/ml, 24 hr) and treated (20 min, 37°C) with either vehicle (0.1% v/v) orASA, before the addition of arachidonic acid (20 ,LM, 1 min) and freshly isolated PMN (A549/PMN cell ratio of1:5). Costimulations were carried out as in Fig. 4. Results represent the mean ± SEM from three to five separatedonors. Insets: Effect of cell ratio on generation of tetraene-containing LXs (LX plus 1 5-epi-LX) (A) and pLTs(LTC4 plus LTD4) (B) during co-incubations of A549 cells with PMN in the absence (0) or presence (M) of ASA.

antiproliferative properties (Fig. 6) observed fordexamethasone with these cells (22). The half-maximum inhibition (IC50) for LXA4 was -80nM and that for dexamethasone was -7 nM.LXB4 at concentrations of 0.5 and 1 ,uM wasapproximately three times more active than ei-ther LXA4 or dexamethasone (Fig. 6B). Interest-ingly, both LXA4 and LXB4 showed essentially

equal potency for blocking A549 cell growthwhen each was added repeatedly to the cells (at24-hr intervals) for three consecutive days (datanot shown; n = 3; d = 4), suggesting that LX maybe inactivated by these epithelial cells. Resultsfrom additional experiments employing directcell enumeration (Fig. 7B) and measurement oftotal cellular protein content (data not shown;

35 q A

24 48 72 96

Time (hours)

0

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4.a00

5 10 50 100 500 1000

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FIG. 6. LX inhibits A549 cell prolif-eration(A) Time-dependent growth arrest. A549cells in 96-well plates were treated witheither vehicle (0.15% EtOH) or equimo-lar concentrations (10-6 M) of LXA4,LXB4, or Dex for up to 96 hr at 370C. Atthe indicated intervals, cells were har-vested for the MIT assay. Data are mean± SEM of three to seven experimentsperformed in quadruplicate. *p < 0.05and **p < 0.005 for compounds versusvehicle. (B) Concentration dependence.A549 cells were exposed to LXA4, LXB4,or Dex at the indicated concentrationsfor 72 hr at 370C. Results are mean ±SEM of five to eight experiments per-formed in quadruplicate. Results are ex-pressed as the percent inhibition of pro-liferation relative to vehicle. *p < 0.05,**p < 0.025, and ***p < 0.005 for com-pounds versus vehicle.

A

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FIG. 7. Lipoxin inhibits cell proliferation(A) DNA synthesis. A549 cells were grown for 72 hrin the presence of test compounds (5-500 nM).Twenty-four hours before the assay, methyl- [3H] -thymidine (2 ,tCi/ml) was added to each well. Cellswere subsequently washed four times with DPBS2+(4°C), lysed with 0.25 N NaOH, and radioactivityincorporation was monitored. Values representmean ± SEM of three different experiments per-formed in quadruplicate. Results are expressed asthe percentage of [3H]-thymidine incorporation rela-tive to vehicle alone. *p < 0.05 and **p < 0.005 forcompounds versus vehicle. (B) Direct cell count.A549 cells were seeded in 12-well culture plates inthe presence of compounds (1 ,uM) and cell countswere obtained at 72 hr by enumerating the trypan-blue excluding cells. Values represent mean ± SEMof three different experiments. Results are expressedas the percentage inhibition of proliferation relativeto buffer. *p < 0.05 for compounds versus vehicle.

n = 3; d = 4) paralleled those obtained with MTTassay, thus confirming the anti-proliferative ac-tions of LXs in A549 cells. Furthermore, DNAsynthesis was blocked, as determined by [3H] -thymidine incorporation, when A549 cells wereexposed for 72 hr to concentrations of 50 nM or

TABLE 1. LX and 15-epi-LX actions on cellproliferation

Compound % p(10-7 M) Inhibition Valuea

Vehicle 1.6 ± 10.3 NSLXA4 16.3 ± 5.2 NS15-epi-LXA4 20.1 ± 1.9 <0.025LXB4 34.0 ± 8.5 <0.00515-epi-LXB4 79.3 ± 0.3 <0.001bDexamethasone 42.1 ± 5.4 <0.00515S-HETE 10.0 ± 8.0 NS15R-HETE 16.8±11.5 NS

Cells (2000 A549 cells/well) were grown in 96-well platesand exposed to vehicle (0.15% v/v in EtOH/F-12K media)or equimolar concentrations (10-7 M) of LXA4, 15-epi-LXA4, LXB4, 1 5-epi-LXB4, or dexamethasone for 72 hr at37°C. Values represent mean ± SEM from three to sevenexperiments performed in quadruplicate and are expressedas percentage inhibition of cell growth.ap values denote statistical differences compared with cellsalone.bp < 0.001 for 15-epi-LXB4 compared with LXB4.

higher of LXB4 or dexamethasone (Fig. 7A). Af-ter incubation of A549 cells with these com-pounds, cells were -98% viable, as determinedby trypan blue exclusion assay, indicating thatthese compounds were not cytotoxic within therange of concentrations used in these experi-ments.

The 15-hydroxy epimeric forms of LXA4 andLXB4 (1 5-epi-LXA4 and 1 5-epi-LXB4, respective-ly), which were the dominant forms of LX iso-lated from these cells, proved to also be potentinhibitors of epithelial cell proliferation (Table 1).At equimolar levels (100 nM), 15-epi-LXA4 in-hibited A549 cell growth to a similar extent asLXA4. On the other hand, 15-epi-LXB4 isolatedfrom conversion of 1 5R-HETE by activated PMNand added back to the A549 cells gave a morepotent antiproliferative activity than LXB4 (-80versus -34% inhibition of proliferation, p <0.001; Table 1). This compound was character-ized by UV, HPLC, and GC-MS (C value: 23.3).Diagnostic ions for the OTMS methyl ester werem/z 173 (base peak), 203, 289, 379, and lessprominent ions at 482 (M+ - 100) (It was notpossible to obtain its molecular ion because ofthe low abundance of this ion). Thus, the pre-dominant material beneath the peak labeled B in


Fig. 4 was consistent with that of 15-epi-LXB4,which gave a shorter C value and separated fromLXB4 as OTMS, methyl ester derivative inGC-MS analysis. The mass spectra of 15-epi-LXB4 and LXB4 were essentially identical (notshown) but their C values were distinct. Materialeluting beneath the peak denoted C (isolatedfrom activated PMN incubated with 15R-HETE)also showed a mild inhibitory action on epithelialcell growth (18 ± 2% inhibition of proliferation;n = 3; d = 4). In sharp contrast, 15-epi-trans-LXA4, 11 -trans-LXB4, 8,9-acetylenic-LXB4, pep-tidoleukotrienes (LTC4 and LTD4), and the LXprecursors (15S- and 1 5R-HETE) each tested at10-6_10-9 M were not able to inhibit signifi-cantly A549 cell proliferation (data not shown;n = 3-5; d = 4). These results indicate that LXand 15-epi-LX gave a stereoselective action inblocking cell proliferation in A549 cells. We alsotested LXA4 and LXB4 with human skin fibro-blasts to determine if they were antiproliferativefor this cell type. At 100 nM, both LXA4 andLXB4 inhibited proliferation of fibroblasts. LXB4gave 38.0 ± 7.5% inhibition and LXA4 10.7 ±1.8% compared with dexamethasone (29.7 ±0.4%) as positive control (n = 3).

DISCUSSIONThe results of the present study demonstrate thatASA triggers the formation of 15-epi-LXs (ATL)during costimulation of the human adenocarci-noma A549 cell line (epithelial cells) and PMN.Their formation involves transcellular biosyn-thetic mechanisms that are similar to those de-scribed recently for human endothelial cell-PMNinteractions (4). These novel eicosanoids weregenerated by PMN that transform ASA-triggeredepithelial-derived 1 5R-HETE (Figs. 1-4). Al-though several routes of 15-HETE biosynthesiswere potentially operative in the A549 cells, theobservation that these cells do not possess 15-LOactivity (if present, 15-LO would have generatedpredominantly 1 5S-HETE) points to the involve-ment of PGHS or other enzymes in its genera-tion. This possibility is supported by the observa-tion that IL-I13-primed A549 cells show anincreased PGHS-2 expression (Fig. 1) and thatformation of 1 5-HETE is enhanced following ei-ther ASA or IL- 13 treatment (Fig. 2). Also, chiralanalysis of the A549-derived 1 5-HETE formed inthe presence of ASA indicated that its C- 15 alco-hol was predominantly in the R configuration(Fig. 3). These results are consistent with find-

Arachidonic Acid

/SOA PGHS-2+ASA

EpithelialCells15R-HETE

TranscellularBiosynthesis

5-LO

* COOH

OH

/

1 5-epi-LXA4

Leukocytes

OH

,OOH

HO OH

1 5-epi-LXB4

FIG. 8. Proposed scheme for generating 15-epi-LXsASA-acetylated PGHS-2 and/or P450 activities con-tribute to 15R-HETE generation. Epithelial-derived1 5R-HETE undergoes transcellular conversion byleukocyte 5-LO to a 15-epi-5(6)-epoxytetraene inter-mediate, which is common to both 1 5-epi-LXA4 and15-epi-LXB4. The complete stereochemistry of 15-epi-LXA4 has been determined (4), and the stereo-chemical assignment of 1 5-epi-LXB4 is tentative.

ings from both human recombinant PGHS-2 andtracheal epithelial cells where ASA acetylatesPGHS-2 and modifies its catalytic properties re-sulting in conversion of arachidonic acid to 1 5R-HETE and inhibition of PG biosynthesis (Ref. 18and for review see Ref. 23).

Cytochrome P450-dependent oxygenationof arachidonic acid also results in the stereose-lective formation (40% R and 60% S configura-tion) of 1 5-HETE (24). The presence of an activecytochrome P450 enzyme system in human air-way A549 cells (25) together with the resultsthat inhibition of P450 as well as heat denaturingblocks 1 5-HETE generation in these cells(Fig. 2A) suggests that this enzyme system inepithelial cells also contributes to 15-HETE bio-synthesis and the generation of 15-epi-LX bytranscellular routes (Fig. 8). Taken together,these observations (Figs. 1-4) establish the exis-tence of two separate enzymatic pathways (i.e.,

593


ASA-acetylated PGHS-2 and cytochrome P450),which can initiate the formation of 15-epi-LXduring airway epithelial cell-PMN interactions(Fig. 8). Also, it should be noted that, in view ofASA's ability to induce P450 enzymes (26), it ispossible that these two independent routes mayact in concert to generate 15-epi-LX.

Evidence is presented for the formation ofboth 15-epi-LXA4 and 15-epi-LXB4 by epithelialcell-PMN interactions (Fig. 4), observations thatextend our recent demonstration of 15-epi-LXA4generation by endothelial-PMN interactions (4).The levels of 15R-HETE produced by epithelialcells were higher than those generated by ASA-treated endothelial cells, which permitted us toconsistently identify the LXB4-like product (i.e.,m/z 173 base peak in GC-MS) from 15R-HETE,vide infra. The transcellular route of 15-epi-LXbiosynthesis is likely to proceed via a 15-epi-5(6)epoxytetraene intermediate (i.e., 15(R)-ep-oxytetraene) produced by leukocyte 5-LO con-

version of epithelial-derived 1 5R-HETE. Thisintermediate would be attacked by putative ep-

oxide hydrolases at either C6 to give 15-epi-LXA4 or C14 to give 15-epi-LXB4 (Fig. 8) (whichis analogous to the mechanisms proposed for thebiosynthesis of LX from 15S oxygenating routes(for review see Ref. 3). By analogy with thephysical properties of LXA4 and LXB4 15-epi-LXA4 and 15-epi-LXB4 are also susceptible toisomerization to give their corresponding all-trans isomers (i.e., 1 5-epi- 11 -trans-LXA4 and 15-epi-8-trans-LXB4) (Fig. 4). The chromatographicbehavior (Fig. 4, Compound B) and diagnosticions were consistent with the 15-epi-LXB4 struc-ture, yet it should be noted that this assignmentof complete stereochemistry of the double bondgeometry for 15-epi-LXB4 is tentative (Fig. 8).The 15-epi-5(6)epoxytetraene is also the likelyintermediate for enzymatic production of Com-pound C, which is consistent with the proposedstructure of 15-epi-7-cis-11-trans-LXA4 (Fig. 4).

In addition to 15-epi-LXs, incubation ofA549 cells together with activated PMN led toformation of pLTs by transcellular routes. Theirbiosynthesis is likely to involve the export of theepoxide LTA4 from PMN and its further transfor-mation to LTC4 and LTD4 in epithelial cells. In-terestingly, ASA inhibited the formation of pLTby co-incubations of epithelial cells and PMN(Fig. 5). This observation is consistent with andmay explain at the cellular level the reduction ofpLT release from gastric mucosa observed duringthe oral administration of ASA to euvolemic rats(27). Although nonspecific effects of ASA on, for

example, the enzymes phospholipase A2 and/or5-LO cannot be excluded, the inhibitory effect ofASA on pLT biosynthesis could be explained by amechanism described earlier for 1 5S-HETE,which serves as a substrate for the 5-LO (28),namely, that ASA-triggered 15R-HETE conver-sion by leukocyte 5-LO blocks LT formation andswitches to 15-epi-LX. This notion is supportedby the result that ASA but not sodium salycilateblocks gastric pLT release in rats, suggesting thatacetylation is a key event in blocking pLT forma-tion (27).

The finding that ATL possess marked biolog-ical activity is of interest (Figs. 6 and 7; Table 1).Recently, we found that 15-epi-LXA4 preventsLTB4-induced adhesion of PMN to endothelialcells (4). The present results indicate that 15-epi-LXs, in particular 15-epi-LXB4, are also potentinhibitors of airway epithelial cell (Figs. 6 and 7)as well as fibroblast proliferation (see Results).Lungs are exposed to noxious insults (includingcarcinogens) that cause leukocyte infiltration,mucosecretory and squamous differentiation,and proliferation of epithelial cells and fibroblasts(29). During the course of this process, severalpro-inflammatory (i.e., PGs and LTs) and anti-inflammatory (i.e., LXs) mediators are likely tobe produced via the PGHS and/or LO pathwayswithin the microenvironment. These eicosanoidscan exert either stimulatory or inhibitory effectson cell proliferation. pLT, for instance, not onlyinfluence epithelial permeability, which can en-hance leukocyte migration as well as antigen andmitogen accumulation, but also promote epithe-lial cell growth (20). ASA, although a well-known blocker of PG biosynthesis, suppressescell proliferation by unknown mechanisms. Ourfindings indicating that ASA is responsible forboth inhibition of pLT biosynthesis and forma-tion of LXs and 15-epi-LXs (Fig. 5) draw atten-tion to a potential role for these LO-derived ei-cosanoids in mediating ASA's antiproliferativeproperties. Along these lines, we have examinedATL's ability to stimulate an increase in intracel-lular cAMP level with the A549 cells. These com-pounds did not, however, stimulate accumula-tion of cAMP in these cells (data not shown).Thus, the cellular site(s) of action for ATL's an-tiproliferative action remain to be identified.

Evidence is also accumulating that pulmo-nary cell phenotype changes are also regulatedby pleotropic mediators such as IL- 1,3, which hasproven to inhibit epithelial cell proliferation (30).Our observation that IL-1,B stimulates PGHS-2enzymes as well as 1 5R-HETE generation in

J. Claria et al.: Aspirin-Triggered Lipoxins Inhibit Cell Proliferation 595

A549 (Fig. 1 and 2), thereby providing substratefor leukocyte formation of 15-epi-LXs, suggeststhat the combination of cytokine gene regulationand PGHS-2 regulation together with ASA couldenhance the antineoplastic properties noted forthis drug. Although additional work is needed todefine further the physiologic and pathophysio-logic roles of ATL in vivo, the results of thepresent investigation suggest the intriguing pos-sibility that formation of 15-epi-LX that displayantiproliferative properties (Figs. 6 and 7) maybe relevant in ASA's preventive role against hu-man lung and breast cancers (31). Also, the for-mation of 1 5-epi-LXs, in particular 1 5-epi-LXB4,could be a component in ASA's protective effectagainst colorectal neoplasia (5), a setting whereintestinal epithelial PGHS-2 expression is knownto be up-regulated (32). Since the therapeuticeffect of ASA in lowering the risk of cancer wasapparent at long durations, in most cases greaterthan 2 years of treatment (31), it is possible thatthe generation of LX and their impact in cellproliferation noted in vitro (Figs. 6 and 7;Table 1) may have a cumulative action withinthe microenvironment in vivo.

In summary, the present results demonstratethat costimulation of epithelial cells and PMN inthe presence of ASA leads to the formation ofboth 1 5-epi-LXA4 and 1 5-epi-LXB4. Their forma-tion involves both epithelial ASA-acetylatedPGHS-2 and cytochrome P450-derived 1 5R-HETE that is substrate for leukocyte 5-LO viatranscellular events. The finding that P450 canalso initiate 15-epi-LX suggests that these com-pounds can be generated in tissues with P450activity without requiring ASA triggering andraises the possibility that 15-epi-LX can be gen-erated de novo, and their pathways of formationare enhanced by the presence of ASA. A potentantiproliferative effect of 15-epi-LXs was estab-lished in vitro with cultured cells (Fig. 7 andTable 1), which now suggests that formation ofthese novel eicosanoids may have implications inASA's protective effect against human cancer.

ACKNOWLEDGMENTSWe thank Prof. N. Petasis (Department of Chem-istry, University of Southern California) for pro-viding reference samples of synthetic 1 5-epi-LXA4-methyl ester and M. Halm Small forassistance in manuscript preparation. These stud-ies were supported by National Institutes ofHealth Grants GM38765 and PO1-DK50305

(CNS). JC was the recipient of a Fulbright/Span-ish Ministry of Education and Science (MEC)fellowship during the present experiments.

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aspirin-triggeredlipoxins (15-epi-lx) are generated human ... · aspirin-triggeredlipoxins...

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