[CANCER RESEARCH 52, 1744-1749. April 1. 1992]

Eicosanoid Production by the Human Gastric Cancer Cell Line ACS and ItsRelation to Cell Growth1

Shuya Shimakura and C. Richard Roland2

Gastroenlerology Section, VA Medical Center, and the Gastrointestinal Peptide Research Center, Department of Internal Medicine, University of Michigan MedicalSchool, Ann Arbor, Michigan 48105

ABSTRACT

Eicosanoids have the ability to stimulate or inhibit the proliferation ofepithelial cells, and they have been shown to modulate the growthcharacteristics of certain tumor cell lines. In addition, many epithelialcells have the ability to produce eicosanoids, which may then serve asautocrine growth factors. We have measured the eicosanoids producedby the human stomach cell line AGS using reverse-phase high-performance liquid chromatography. AGS cells were incubated with |MIjarachi-donic acid and stimulated to release eicosanoids by the calcium ionophoreA23187. Unlike its counterpart from the normal stomach, the AGS tumorcell line produced prominent amounts of the leukotrienes I).4,C4, and B4;6-keto-prostaglandin !•',.:thromboxane B2; hydroxyeicosatetraenoic

acids; and smaller amounts of other prostaglandins in response toA23187. Under basal condition (in the absence of calcium ionophore),hydroxyeicosatetraenoic acid was produced in greatest relative amountcompared with the other eicosanoids.

To elucidate the potential autacoid role of these agents, exogenouseicosanoids were added to AGS cells, and proliferation was measured.Prostaglandins I>;and E2 suppressed the growth of AGS cells in a dose-dependent manner. On the other hand, leukotrienes D4 and (., had adose-dependent proliferative effect on cell growth. The lipoxygenaseinhibitor nordihydroguaiaretic acid (10 ", II) s M) and hydrocortisone(11) * M) had dose-dependent suppressive effects on growth, whereasindomethacin (IO"6 M and 10~*M) had no effect. These results suggest

that AGS cells preferentially metabolize arachidonic acid through the 5-lipoxygenase pathway, which results in the production of growth-stimulatory autocoids. Agents that selectively block this arm of eicosanoidmetabolism might be useful therapeutic agents in the treatment of certaingastrointestinal cancers.

INTRODUCTION

Eicosanoids are important autocoids that are known to regulate a wide range of physiological processes in gastrointestinalepithelia including the secretion of fluid and electrolytes, mu-cosal blood flow, and cell proliferation (1,2). Cells vary in theirability to metabolize AA,3 and it is becoming apparent that

each cell type may have a unique ability to produce PCs, LTs,and other related eicosanoids. It has long been known, forexample, that normal gastric epithelium produces certain PGs,but has a relatively limited ability to produce LTs, which maybe prominently produced by certain inflammatory cells (3).

Many eicosanoids have been shown to affect the growth of

Received 7/3/91; accepted 1/17/92.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' Supported in part by NIH Grants R01DK37489 and P30-DK 34933 and by

the Research Service of the Department of Veterans Affairs.2To whom requests for reprints should be addressed, at GI Section (HID),

VA Medical Center, 2215 Fuller Road, Ann Arbor, MI 48105.3The abbreviations used are: AA, arachidonic acid; PG, prostaglandin; LT,

leukotriene; HPLC, high-performance liquid chromatography; DMSO, dimethylsulfoxide; BSA, bovine serum albumin; HBSS, Hanks' balanced salt solution;

PCA, perchloric acid; I \B;. thromboxane B:: NGDA, nordihydroguaiaretic acid;BrdU, 5-bromo-2'-deoxyuridine; 12-HHT, 12-hydroxyheptadecatrienoic acid;HETE, hydroxyeicosatetraenoic acid; IMDM, Iscove's modified Dulbecco's medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; LI, labelingindex; PLA3, phospholipase A2; HPETE, 15-L-(i)-hydroperoxyeicosatrienoicacid.

tumor cell lines. For example, certain members of the PGA,PGE, and PGD: series (including 12-PGJ2, the ultimate metabolite of PGD2) are potent inhibitors of growth for certaincultured tumor cells (4-7). PGE?, which is abundantly produced

by gastric tissue, has a significant suppressive effect on thegrowth of the human stomach cancer cell line Kato III (8).Although not studied previously in gastrointestinal epithelium,it has been suggested that some lipoxygenase products mayparticipate in the stimulation of the human leukemia cell lineHL-60 (9). Work by several laboratories has suggested that

tumors of the gastrointestinal tract may synthesize eicosanoidproducts that differ from those found in the correspondingnormal tissue (10, 11). However, these studies have not definitively ascertained the profile of eicosanoids derived from tumortissue itself, because of the presence of inflammatory cells infresh tumor tissue (12, 13). A cultured cell line permits adetermination of eicosanoid production by the epithelial cellsunambiguously; however, it is possible that cultured tumor cellsdo not perfectly reflect the behavior of tumors in vivo.

We have hypothesized that some tumor cells may gain agrowth advantage by losing their ability to synthesize growth-suppressive PGs and by producing greater amounts of growth-stimulatory eicosanoids. To test this hypothesis, we measuredthe eicosanoid-metabolizing capacities of the human gastriccancer cell line AGS using [3H]AA as a metabolic precursor

and identified the eicosanoid metabolites using reverse-phaseHPLC. We found that AGS cells produced a different profileof eicosanoid metabolites than what has been reported previously from normal gastric mucosal cells, that members of thePG family suppressed the growth of AGS cells, and that LTshad a proliferative effect on these cells.

MATERIALS AND METHODS

Materials

The following reagents were purchased from Sigma Chemical Co.(St. Louis, MO): calcium ionophore A23187; DMSO; PGE2, PGD2,BSA, HBSS, PCA; trifluoroacetic acid; 6-keto-PGF,„,PGE2, PGD2,PGF2o, TxB2; trypan blue; indomethacin; NDGA; BrdU; and hydrox-yurea. LTB4, LTC4, LTD4, 12-HHT and 12- and 15-HETE were

purchased from Cayman Chemical (Ann Arbor, MI). IMDM andtrypsin (0.25%) EDTA (1 mM) were purchased from GIBCO Laboratories (Grand Island, NY). Heated-inactivated FBS was from Hyclone,Logan, UT. ['H]AA (243 mCi/mmol) and ['Hjthymidine were pur

chased from New England Nuclear (Wilmington, DE). HPLC-gradeacetonitrile and methanol were purchased from Mallinckrodt, Inc.(Paris, KY). The tissue culture plates were purchased from Costar(Cambridge, MA). Anti-BrdU monoclonal antibody was purchasedfrom Vector Laboratory (Burlingame, CA). Protein was measured usingthe Coomassie blue reagent of Pierce (Rockford, IL). All reagents wereof highest grade available, and all water was deionized and passedthrough a MilliQ purification system (Waters Associates, Milford, MA)prior to use.

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EICOSANOIDS SYNTHESIS BY ACS CELLS

Methods

Eicosanoids Synthesis by ACS Cells

Cell Culture and Loading with [3H|AA. Two ml of an ACS cellsuspension (2 x IO6cells/ml) were plated in tissue culture wells, loadedwith 0.5 mCi of [3H]AA per well, and incubated at 37°Cunder 5% CO2

and 95% air. After 18 h of incubation, the labeling medium and floatingcells were removed, and the cells were washed twice with HBSS containing 2% BSA and once with HBSS containing 0.1% BSA to removefree unincorporated [3H]AA.

Extraction of 3H-labeled Eicosanoids. The ACS cell (14) monolayers

were incubated in 2 ml of IMDM supplemented with 10% FBS in thepresence or absence (using vehicle as control) of 2.0 or 5.0 mM A23187in DMSO (at a final DMSO concentration of 0.02%) for 1 h. Theextraction from culture medium of radiolabeled eicosanoids producedby the [3H]AA-labeled ACS cells was performed by the method of

Wescott et al. (15). After l h of incubation, culture media from threewells were pooled and added to two volumes of ice-cold methanol. Themixture was centrifuged at 1500 rpm for 5 min, and the methanolicsupernatant was diluted in 0.1 M sodium phosphate buffer, pH 7.4, toyield a final methanol concentration of 20%. This 80% aqueous mixturewas applied to Sep-Pac CIS cartridges (Waters Associates, Milford,MA) that had been prewashed sequentially with 20 ml of methanol and20 ml of deionized water. The loaded cartridges were washed with 5 mlof 20% methanol in 0.1 M sodium phosphate buffer followed by 5 mlof deionized water, both of which were discarded. Eicosanoids wereeluted with 3 ml of 80% methanol in water. Extracts were then evaporated to dryness under nitrogen, and residues were stored at -70°C

pending eicosanoid separation by HPLC.Separation of |3H|AA Metabolites by HPLC. Eicosanoid separation

was accomplished according the method of Peters-Golden and Thebert(13). For identificai ion of radiolabeled eicosanoids, the extracted samples were redissolved in 300 ¡Aof acetonitrile:water:trifluoroacetic acid(33:67:0.1) and subjected to reverse-phase HPLC using a 30- x 0.4-cm5-mm Bondapak C,g column (Waters Associates, Millford, MA). Themobile phase consisted of acetonitrile in water and trifluoroacetic acidat a flow rate of 1 ml/min. Cyclooxygenase metabolites were elutedduring an initial isocratic phase (acetonitrile:water:trifluoroacetic acid,33:67:0.1); the lipoxygenase metabolites and free AA were eluted usinga continuous gradient of increasing acetonitrile to 100:0:0.1.

The eluate was continuously monitored for UV absorbance (210 nmfor cyclooxygenase products and free AA, 280 nm for leukotrienes, and235 nm for mono-HETEs). The retention times (min) for authenticeicosanoids standards were as follows: 6-keto-PGF,,,, 7 to 8; TxB2, 13to 15; PGF2„,16; PGE2, 18 to 19; PGD2, 21 to 22; LTC4, 44; LTD4,46; LTB4, 48 to 49; 12-HHT, 58 to 59; 15-HETE, 72 to 75; 12-HETE,77 to 78; and AA, 97 to 99. Cochromatography with authentic eicosanoids standards was used with each sample to control for any variationin retention and elution.

One-mi eluted fractions were collected, and 3H-labeled products were

quantitated by liquid scintillation counting in 5 ml of scintillant (Ecolite; ICN Biomedicals, Inc., Irvine, CA).

ACS Cell Growth in Response to Exogenous Eicosanoids

Count of Cell Number and Protein Assay. Suspensions of 2 x 10s

ACS cells in 3 ml of IMDM supplemented with 10% FBS were platedin tissue culture plates and incubated at 37°Cunder 5% CO2:95% airovernight ("the preincubation period"). The next day ("time 0"), the

medium was replaced with 3 ml of fresh medium containing thefollowing eicosanoids: PGD2 or PGE2 (0.5, 1.0, 2.5, 5.0 jig/ml); LTC4or LTD4 (0.01, 0.05, 0.1, 0.25, 0.5 mg/ml); indomethacin (10~6, IO'5M); NDGA (IO'7, IO"6, 10"' M); hydrocortisone (IO'5 M); or vehicle

only as control. The medium was adjusted to a final methanol concentration of 0.5%. After 96 h of incubation, cells were harvested usingtrypsin/EDTA and counted using a hemocytometer; the protein contentof the cells was measured. The initial cell numbers at time 0 (1.85 ±0.27 x 10') (mean ±SD, n = 9) and protein contents were not

significantly different in each series of experiments.|3H]Thymidine Incorporation. ACS cells (2 x 10~5) prepared as de

scribed above were incubated for 24 h, to which [3H]thymidine was

added at a specific activity of 0.5 mCi/ml. To estimate nonspecificincorporation or adherence of thymidine, 10 ml of hydroxyurea (10mg/ml) were added to some wells. Two h later, 2 ml of ice-cold salinewere added, aliquots were removed, and cells were washed twice withPBS. After counting cell numbers, 2 ml of 5% PCA were added, andthe dpm value in the acid-insoluble fraction was measured.

BrdU LI. To measure DNA synthesis, 2 x IO6 ACS ceils were

prepared as described above, after which Bull was added at a finalconcentration of 100 mg/ml for 4 h. To terminate BrdU labeling, ice-cold PBS was added to the wells, and the supernatant was discarded.BrdU-labeled cells were washed twice with PBS and removed from thewells with trypsin/EDTA. Cells were attached to glass slides by cen-trifugation using a Cytospin-2 centrifuge (Shandon, Pittsburgh, PA)and fixed in 70% ethanol. Cells on the glass slides were treated with 4N HC1 for 30 min to denature the DNA, and immunohistochemicaldetection of BrdU incorporation was performed using the anti-BrdUmonoclonal antibody and the avidin-biotin-peroxidase complex method(16). To calculate the BrdU LI, photographs were taken of at least fourdifferent highpower fields randomly selected under the light microscope. Total cells and BrdU-positive nuclei were counted in each field.The LI was defined as the ratio of BrdU-labeled cells to total cellscounted.

Data Analysis. The data were expressed as the mean ±SEM of atleast 3 to 5 different series of experiments. Individual measurementswere done in duplicate or triplicate. Differences between control andtreatment groups were compared by the Student t test, and P < 0.05was regarded significant.

RESULTS

Profile of Eicosanoids Derived from ACS Cells. Fig. 1 showsthe elution of 'H-labeled eicosanoids after separation on the

HPLC column. Relative molar amounts of each metabolite maybe measured from the profiles. The lower curve (thin, dashedline) indicates eicosanoids produced in the unstimulated state,and upper curves (the dashed and solid bold lines) indicateeicosanoid production after stimulation with 2.0 HIM and 5.0HIMcalcium ionophore A23187, respectively. The calcium io-nophore produced dose-dependent increases in eicosanoid metabolites. Although one cannot measure the total mass of eicosanoids, the relative molar proportions of all AA metabolitesmay be determined using this method. The major metabolitesproduced by ACS cells after A23187 stimulation were LTD4,followed by progressively smaller amounts of 6-keto-PGF,,,,LTC4, LTB4, TxB2,12-HETE, PGF2„,PGE2, and PGD2. Underbasal conditions, the profile showed 12-HETE > LTB4 > LTC4> PGF2„> PGE2 > 6-keto-PGF,,, > TxB2 > LTD4 > 15-HETE> PGD2 (Fig. 1).

Influence of Exogenous Eicosanoids on Cell Growth. The timecourse of ACS cell growth is shown in Fig. 2. Both PGD2 andPGE2 (5.0 Mg/ml) significantly suppressed cell growth at 24,48, and 96 h (P < 0.01 versus control). However, LTC4 andLTD4 (0.5 mg/ml) significantly stimulated cell growth at 48and 96 h (P < 0.05 versus control).

Dose-Response for Modulating AGS Cell Growth by Eicosanoids. The dose-response relationships for growth suppressionby PGD2 and PGE2 are depicted in Fig. 3. Increasing the PGconcentration from 0.5 to 5.0 Mg/ml produced a progressiveinhibition of cell growth by both PGD2 and PGE2. The 50%effective dose for growth inhibition was in the range of 2.5 to5.0 Mg/ml for each prostanoid.

The change in protein content of the AGS cells produced byPGD2 and PGE2 is shown in Table 1. Both PCs reduced the

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250

200

150

1; TxB, 3: PGË,2: PGF,, 4; PGD,

100

0 10 20 30 40 SO 60 70 80 90 100 110Elution Fraction # ( min )

Fig. 1. Profile of eicosanoids derived from ACS cells. The ACS cells wereincubated for l h in the presence or absence of the calcium ¡onophoreA23187.The [JH]AA metabolites in the medium released by 6 x IO6 AGS cells wereseparated by reverse-phase HPLC. The lowest line (thin, dashed line) representsthe elution profile in the absence of A23187; the bold dashed line and bold solidline represent the elution profile in the presence of 2.0 /IM and 5.0 MMA23187,respectively. The arrows point to the elution of authentic standards, as labeled.

24hrs 48hrsTime Course

96hrs

Fig. 2. Growth curve of AGS cells incubated with PCs or LTs. Lines representthe numbers of AGS cells after incubation with LTD4, LTC4, control, PGE2, andPGD2 from upper to lower lines, respectively. *,P< 0.05; ", P < 0.01; **«,P <0.005, compared with control.

120

100

80

§ 60

O 40

20

100100

CONI. 0.5 1.0 2.5Concentration of Prostaglandms (pg/ml)

5.0

Fig. 3. Dose-response of AGS cell growth in response to exogenous PCs.Columns express the mean cell counts (ears, SEM) (n = 3 to 5) expressed as thepercentage of control cell number. •¿�and •¿�.PGD2 and PGE2, respectively. *, P< 0.05; ", P < 0.01; ***, P < 0.005, compared with control wells. COAT.,

control.

total protein content of AGS cells in a dose-dependent manner.The dose-response relationships for stimulation of cell

growth by LTC4 and LTD4 are shown in Fig. 4. Increasing theLT concentration from 0.01 to 0.5 mg/ml produced progressive

increases in cell number for LTD4 which reached significanceat a concentration of 0.05 mg/ml, and a 36.7% increase in cellnumber was observed at 0.5 mg/ml. Significant growth stimulation was seen for LTC4 only at the 0.5 mg/ml concentration.In contrast, no concentration of LTC4 or LTD4 showed asignificant increase in the total protein content of AGS cells(data not shown).

Effect of NDGA, Hydrocortisone, and Indomethacin on CellGrowth. NDGA is an inhibitor of 5-lipoxygenase activity, andhydrocortisone inhibits PLA2 and may have a variety of othereffects on cell growth in culture. If LTs were important stimulators of AGS cell growth, NDGA or hydrocortisone might beexpected to inhibit LT synthesis and inhibit cell proliferation.Increasing concentrations of NDGA (10~7 M, IO'6 M, IO"5 M)and hydrocortisone (10~5 M) produced no significant effect on

cell growth. Since LTs released into the medium may haveinfluenced cell growth prior to the addition of inhibitors, experiments were designed to limit this effect, in which NDGA(10~s M) or hydrocortisone (10~5 M) was added during the

preincubation period. The results of those experiments areshown in Table 2. Increasing the concentration of NDGA (10~7M, 10~6 M, or 10~5 M) in cells pretreated with 10~5 M NDGA

produced a significant, dose-dependent inhibitory effect on cellgrowth (P < 0.005). Hydrocortisone (10~5 M), added to cellsthat were pretreated with hydrocortisone (IO"5 M), revealed

similar results, also shown in Table 2.The addition of indomethacin (10~6 M and 10~5 M) to AGS

cells reduced the cell numbers to 94 ±5 and 97 ±4% of controlwells, respectively.

['I I|I hynwliiu- Incorporation. To further document the effectsof exogenous eicosanoids on cell growth, [-'Hjthymidine incorporation per IO6AGS cells was measured. As demonstrated inTable 3, PGD2 and PGE2 (at 1.0 and 5.0 Mg/ml) decreased [3H]

thymidine incorporation significantly (P < 0.005), whereasLTD4 (at 0.05 and 0.5 mg/ml) increased incorporation signifi-

Table 1 Growth response of AGS cells to PCs (total protein content as percentage)

Concentration(¿ig/ml)PGD2

PGE20.572.2

±7.9°73.3 ±3.5*1.076.3

±4.663.1 ±3.92.561.4

±9.8*54.5 ±9.4*5.045.6

±7.2C40.8 ±5.7C

" Mean ±SEM of control wells (n = 3 to 4).*P < 0.05.' P < 0.01 compared with protein content in control wells.

CONT. 0.01 O.OS 0.1 0.25 0.5CONCENTRATION OF LEUKOTRIENES (mg/ml)

Fig. 4. Dose-response of AGS cell growth in response to exogenous LTs.Columns express the mean (bars, SEM) (n = 3) of the percentage of control cellnumber. •¿�and H. LTC4 and LTD4, respectively. «,P < 0.05; ", P < 0.01; «»»,P < 0.005, compared with control wells (CONT.).

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Table 2 Effect of inhibitors ofeicosanoid metabolism on cell number and total protein contents of ACS cellsData are expressed as the percentage of control wells.

NDGANonpretreated0

No. of cellsProteincontentPretreated

with inhibitors'

No. of cellsProtein contentControl100

±3*

100±3137

±5"

99 ±210~'

M118±

10102±789

±7

91 ±610-'

M99

±895±479

±3''

82 ±2'10-'

M90

±491±757

±3'

76 ±8'HydrocortisoneControl

10"*M100

±1394 ±10127

±3 75 ±4'

113± 10 53± \3d

" In the "nonpretreated" group, AGS cells were incubated with NDGA (10 7 to 10 * M) or hydrocortisone (10 ' M) for 96 h.* Mean ±SEM.' In the pretreated group, ACS cells were incubated with NDGA (10"' M) or hydrocortisone (10~! M) during the preincubation period and then the same as the

"nonpretreated" group at 0 time point (see text for details). At the 0 time point, there were no significant differences in cell number or total protein content betweenthe "nonpretreated" and pretreated groups.

<//><0.001 compared with "nonpretreated" control wells.' P <0.005.

Table 3 Effect of exogenous PGDi and PGE2 on f'H/thymidine incorporation into ACS cells

PGD2{„g/ml)dpm/fig

of protein/ml% of controlP value vs. controlControl10,815

±165"

1000.510,086 ±207

93 ±2P = 0.051.09,520

±28988 ±3

P < 0.0055.07,641

±71571 ±7

J»<0.005509,400

±67987 ±6

/•=0.10PGE2

(Mg/ml)1.08,463

±22578 ±2

P< 0.001508,034

±42374 ±4

P <0.005"Mean ±SEM.

Table 4 Effect of exogenous LTs on f'HJthymidine incorporation into ACS cells

LTC<(mg/ml)dpin

,<Kof protein/ml% of controlP value vs. controlControl

0.0510,810±165" 10,761+797

100 ±70.512,974±289

121 ±9.2P = 0.065LTD,

(mg/ml)0.0513,798

±369129 ±3

P < 0.0050.51

4,970 ±178140 ±2

P<0.001°Mean ±SEM.

Table 5 BrdUrd labeling indices of ACS cells treated with PCs

Contro!Labelingindex 44*1"

(% of cellslabeled)

% of controlP value vs. con

trolPGD2I1.038

±182

±2P < 0.005>g/ml)5.036

±177

±2P < 0.005PGE2

(„g/ml)1.0

5.034

±1 33 ±177

±2 75 ±3P< 0.001 P<0.001°

Mean ±SEM.

Table 6 BrdUrd labeling indices of ACS cells treated with LTs

LTC4(mg/ml)ControlLabeling

index 44 ±1°

(% of cellslabeled)

% of controlP value vs. con

trol0.155

±1123

±3P< 0.0050.556

±1127

±3P< 0.001LTD,

(mg/ml)0.156

±2129

±3P< 0.0050.565

±3147

±6P<0.001°

Mean ±SEM.

cantly at both concentrations (P < 0.005). LTC4 showed noresponse at 0.05 mg/ml and showed a nonsignificant increaseat 0.5 mg/ml (P = 0.065) as shown in Table 4.

Indomethacin (!()'' M) produced a nonsignificant increase in

thymidine incorporation to 129 ±14% of control wells.BrdU LI. Both PGD2 and PGE2 (1.0 and 5.0 Mg/ml) signifi

cantly decreased the BrdU LI (P < 0.005), and both LTC, andLTD., (0.1 and 0.5 mg/ml) significantly increased LI (P <0.005) as shown in Tables 5 and 6. Indomethacin (10~6 M, 10~5

M) showed a nonsignificant effect on proliferation (94 ±5 and97 ±4% of control BrdU, respectively).

DISCUSSION

This work reports the full range of eicosanoids produced bythe human stomach tumor cell line AGS. Under basal conditions, AGS cells produce LTD., as its principal eicosanoidmetabolite, followed by progressively smaller molar quantitiesof 6-keto-PGF,,,, LTC,, LTB4, 12-HETE, TxB2, PGF2„,PGE2,12-HHT, 15-HETE, and PGD2. After stimulation with thecalcium ionophore A23187, the principal eicosanoids productswere (in order of relative molar abundance): 12-HETE; LTB4;LTC4; PGF2lt; PGE2; 6-keto-PGF,,,; TxB2; LTD4; 15-HETE;and PGD2. These results indicate that 5-lipoxygenase is moreactive than cyclooxygenase under both basal and ionophore-stimulated conditions in AGS cells. This is different from whathas been reported in normal human gastric tissue in whichPGE2 is the most prominent eicosanoid, followed by LTB4 andthe sulfidopeptide-LTs (11). This also differs from reports ofthe prostanoids produced by gastric cancer tissue, in which 6-keto-PGF,,, was the principal product, followed by smalleramounts of PGE2, PGF2„,and PGD2 (10). Pelus and Brockman(17) reported that macrophages from tumor-bearing animalshave a markedly augmented capacity to metabolize arachidonicacid. Tumor tissue may be infiltrated with inflammatory cells,neutrophils, or macrophages, which would affect eicosanoidproduction (12). Therefore, the measurement of AA metabolism in a tumor cell line would be more likely to give an accuratemeasure of the endogenous capacity of the malignant epithelial

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cells themselves. We did not attempt to measure the total massof eicosanoids made by the tumor cells; given the fact that thesubstances would act as autocrine factors, it is difficult to knowhow one would interpret these findings. One can indirectlyestimate the dose-response relationships of the cells from thedata obtained using exogenous LTs.

In this study, the growth and DNA synthesis of ACS cellswere suppressed by exogenous PGE2 and PGD2, similar to thereported findings of other investigators using other tumor celllines (4-8). It has been proposed that the antitumor activitiesof these prostaglandins are exclusively due to their ultimatemetabolites, PGA2 and 12-PGJ2, which share a cyclopentenonering structure (18). The growth-inhibiting effect of these PGswas independent of cyclic AMP, and its mechanism appearedto depend upon inhibition of entry into the G, phase of the cellcycle (7, 19). Furthermore, Santro et al. (20) reported that a MT74,000 protein was induced by PGA, or PGJ2 in human K562erythroleukemia cells; the protein has been identified as theheat-shock protein, and its appearance was associated with

changes in cell proliferation. Ishioka et al. (21) reported thatPGA: arrested HL-60 cells at the G0-G, phase of the cell cycle,which was associated in a reduction in the mRNA for c-myc.

We have demonstrated that LTD4 (at concentrations from0.05 to 0.50 mg/ml, or IO"7 M to 10~6 M) and LTC4 (at 0.50mg/ml or 10~6 M) significantly increased cell number and

stimulated DNA synthesis in ACS cells. Dawson et al. (22)have reported that the 5-lipoxygenase inhibitor benoxaprofensuppressed the growth of various tumor cell lines in vitro andtumor growth in vivo after i.p. injection of mice with the T-celllymphoma line EL4. Gali et al. (23) reported a similar inhibitory effect with the 5-lipoxygenase inhibitor NDGA on humanglioma spheroids. Miller et al. (9) showed that NDGA, piriprost(a selective inhibitor of 5-lipoxygenase), and other inhibitors ofpeptide-LT (LTC4, LTD4, and LTE4) metabolism inhibited thegrowth of the human leukemia cell line HL-60, which wascompletely reversed by the daily addition of IO"9 M LTD4.

Ralph and Wojcik (24) have reported that various lipoxygenaseand phospholipase A2 inhibitors (i.e., NDGA, hydrocortisone,and others) inhibited the growth of the P815 murine mastocy-toma cell line, but that 0.16 nM LTC4 or 0.3 nM LTB4 failed toreverse this effect. In our study, we used relatively high dosagesof LTs (up to 10~6M) and demonstrated that LTD4 was a more

potent stimulator of AGS cell growth than LTC4, similar towhat had been reported by Miller et al. (9). However, it is verydifficult to know what represents a physiological concentrationof a paracarine or autocrine substance.

Our data showed significant increases in AGS cell numbersafter stimulation with LTs, but we did not demonstrate asignificant increase in total protein content with any concentration of LT. The discrepancy between these two measures of cellgrowth may be a reflection of a reduction in cell size. AGS cellsare somewhat pleomorphic, but after the addition of LTs, thecell size appeared subjectively smaller than in control wells.Therefore, LTs may stimulate cell division more rapidly thanprotein synthesis in these cells.

Nishizawa et al. (25) reported that the 5-lipoxygenase inhibitors NDGA and AA861 stimulated the growth of transformedmurine Leydig cells, and that 5-HETE inhibited proliferationin these cells. In this study a single dose of NDGA (10~7 M,IO"6 M, or 10~5M) or hydrocortisone (10~7 M or 10~5M) showed

no significant effect on cell growth, but pretreatment withNDGA or hydrocortisone inhibited LT production during thepreincubation period and produced a significant, dose-depend

ent inhibition in growth. As demonstrated in Table 2, pretreatment of AGS cells with inhibitors of eicosanoid metabolism,followed by a change in the medium, was required to demonstrate inhibition of growth by these agents. This suggests thatthe half-life of LTs may be very long in vitro and that theirpersistence in the culture medium must be considered wheninterpreting experimental results.

Hydrocortisone is an inhibitor of PLA2, which affects bothcyclooxygenase and lipoxygenase metabolites. Because of theintrinsically greater activity of the lipoxygenase pathway inAGS cells, hydrocortisone will appear to selectively inhibit thisarm of eicosanoid metabolism. Indomethacin had no effect onproliferation in AGS cells, although this agent has been shownto stimulate cell growth in HEP-2, L, and HeLa cells, all ofwhich produce considerable amounts of PGE constitutively(26). Furthermore, at higher concentrations, indomethacin suppresses phospholipase A2 and lipoxygenase activities (27, 28).

Other lipoxygenase metabolites including 12-HETE, 15-HETE, and HPETE inhibit cell growth in SK-N-SH, a humanneuroblastoma cell line (29), and MCF-7, a human breastcancer cell line (30). Furthermore, 15-HETE can inhibit thebiosynthesis of leukotrienes (31). Therefore, the inhibitory effect of certain lipoxygenase metabolites on tumor cells may bemediated by an autoregulatory modification in LT metabolismin those cells.

LTC4 may be cleaved by 7-glutamyltranspeptidase to produceLTD4 (32). LTD4 interacts with highly selective and specificreceptors that can distinguish binding sites for LTB4 or LTC4(33). LTD4 can increase intracellular calcium and release freeAA by the activation of PLA2 through the LTD4 receptor (34,35). Therefore, it is possible that these LTs may reinforce theirgrowth-stimulating effects by a positive feedback mechanism.

In summary, we have found that the human gastric carcinomacell line AGS preferentially produces lipoxygenase rather thancyclooxygenase metabolites in response to stimulation by calcium ionophore. The prostanoids PGD2 and PGE2 inhibit thegrowth of AGS cells, whereas LTC4 and LTD4 stimulate theirgrowth. These results suggest that certain leukotrienes areautocrine growth factors for this cell line. Data from otherlaboratories suggest that PGs and LTs may have oppositeeffects on cell proliferation in a number of tumor systems. Therole of eicosanoids in proliferation by tumor cell lines suggeststhat this may be a potential area of therapeutic interest. However, the complex effects of inhibitors of eicosanoid metabolismand the interactions among the members of the LT and PGautacoid families make it difficult to predict the response ofany tumor to pharmacological manipulation. Furthermore, tumors are typically infiltrated with inflammatory cells and vascular elements which will have additional impact on the localproduction of eicosanoids, which will further complicate attempts at pharmacological manipulation. Nonetheless, thiswork demonstrates that stomach cancer cells produce a differentprofile of eicosanoids then do the normal tissue counterpartsand that these autacoids have potentially important influenceson the growth of the tumor cells.

ACKNOWLEDGMENTS

The authors gratefully thank Patricia Shaw and Diana Drescher fortheir help in preparation of the manuscript. We would like to alsothank Eugene Kraus, James Scheiman, and Kenji Yoshimura for experimental support and advice.

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1992;52:1744-1749. Cancer Res   Shuya Shimakura and C. Richard Boland  AGS and Its Relation to Cell GrowthEicosanoid Production by the Human Gastric Cancer Cell Line

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