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

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[CANCER RESEARCH 52, 1744-1749. April 1. 1992] Eicosanoid Production by the Human Gastric Cancer Cell Line ACS and Its Relation 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 Medical School, Ann Arbor, Michigan 48105

ABSTRACT tumor cell lines. For example, certain members of the PGA, PGE, and PGD: series (including 12-PGJ2, the ultimate metab Eicosanoids have the ability to stimulate or inhibit the proliferation of epithelial cells, and they have been shown to modulate the growth olite of PGD2) are potent inhibitors of growth for certain characteristics of certain tumor cell lines. In addition, many epithelial cultured tumor cells (4-7). PGE?, which is abundantly produced cells have the ability to produce eicosanoids, which may then serve as by gastric tissue, has a significant suppressive effect on the autocrine growth factors. We have measured the eicosanoids produced growth of the human stomach cancer cell line Kato III (8). by the human stomach cell line AGS using reverse-phase high-perform Although not studied previously in gastrointestinal epithelium, ance liquid chromatography. AGS cells were incubated with |MIjarachi- it has been suggested that some lipoxygenase products may donic acid and stimulated to release eicosanoids by the calcium ionophore participate in the stimulation of the human leukemia cell line A23187. Unlike its counterpart from the normal stomach, the AGS tumor HL-60 (9). Work by several laboratories has suggested that cell line produced prominent amounts of the leukotrienes I).4,C4,and B4; 6-keto-prostaglandin !â€¢',.:thromboxane B2; hydroxyeicosatetraenoic tumors of the gastrointestinal tract may synthesize eicosanoid acids; and smaller amounts of other prostaglandins in response to products that differ from those found in the corresponding A23187. Under basal condition (in the absence of calcium ionophore), normal tissue (10, 11). However, these studies have not defini hydroxyeicosatetraenoic acid was produced in greatest relative amount tively ascertained the profile of eicosanoids derived from tumor compared with the other eicosanoids. tissue itself, because of the presence of inflammatory cells in To elucidate the potential autacoid role of these agents, exogenous fresh tumor tissue (12, 13). A cultured cell line permits a eicosanoids were added to AGS cells, and proliferation was measured. Prostaglandins I>;and E2 suppressed the growth of AGS cells in a dose- determination of eicosanoid production by the epithelial cells unambiguously; however, it is possible that cultured tumor cells dependent manner. On the other hand, leukotrienes D4 and (., had a dose-dependent proliferative effect on cell growth. The lipoxygenase do not perfectly reflect the behavior of tumors in vivo. inhibitor nordihydroguaiaretic acid (10 ", II) s M) and hydrocortisone We have hypothesized that some tumor cells may gain a (11) * M) had dose-dependent suppressive effects on growth, whereas growth advantage by losing their ability to synthesize growth- indomethacin (IO"6 M and 10~*M) had no effect. These results suggest suppressive PGs and by producing greater amounts of growth- that AGS cells preferentially metabolize arachidonic acid through the 5- stimulatory eicosanoids. To test this hypothesis, we measured lipoxygenase pathway, which results in the production of growth-stimu the eicosanoid-metabolizing capacities of the human gastric latory autocoids. Agents that selectively block this arm of eicosanoid cancer cell line AGS using [3H]AA as a metabolic precursor metabolism might be useful therapeutic agents in the treatment of certain and identified the eicosanoid metabolites using reverse-phase gastrointestinal cancers. HPLC. We found that AGS cells produced a different profile of eicosanoid metabolites than what has been reported previ INTRODUCTION ously from normal gastric mucosal cells, that members of the PG family suppressed the growth of AGS cells, and that LTs Eicosanoids are important autocoids that are known to reg ulate a wide range of physiological processes in gastrointestinal had a proliferative effect on these cells. epithelia including the secretion of fluid and electrolytes, mucosal blood flow, and cell proliferation (1,2). Cells vary in their ability to metabolize AA,3 and it is becoming apparent that MATERIALS AND METHODS each cell type may have a unique ability to produce PCs, LTs, Materials and other related eicosanoids. It has long been known, for example, that normal gastric epithelium produces certain PGs, The following reagents were purchased from Sigma Chemical Co. but has a relatively limited ability to produce LTs, which may (St. Louis, MO): calcium ionophore A23187; DMSO; PGE2, PGD2, be prominently produced by certain inflammatory cells (3). BSA, HBSS, PCA; trifluoroacetic acid; 6-keto-PGF,â€ž,PGE2, PGD2, Many eicosanoids have been shown to affect the growth of PGF2o, TxB2; trypan blue; indomethacin; NDGA; BrdU; and hydroxyurea. LTB4, LTC4, LTD4, 12-HHT and 12- and 15-HETE were Received 7/3/91; accepted 1/17/92. purchased from Cayman Chemical (Ann Arbor, MI). IMDM and 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 in trypsin (0.25%) EDTA (1 mM) were purchased from GIBCO Labora accordance with 18 U.S.C. Section 1734 solely to indicate this fact. tories (Grand Island, NY). Heated-inactivated FBS was from Hyclone, ' Supported in part by NIH Grants R01DK37489 and P30-DK 34933 and by Logan, UT. ['H]AA (243 mCi/mmol) and ['Hjthymidine were pur the Research Service of the Department of Veterans Affairs. 2To whom requests for reprints should be addressed, at GI Section (HID), chased from New England Nuclear (Wilmington, DE). HPLC-grade VA Medical Center, 2215 Fuller Road, Ann Arbor, MI 48105. acetonitrile and methanol were purchased from Mallinckrodt, Inc. 3The abbreviations used are: AA, arachidonic acid; PG, prostaglandin; LT, (Paris, KY). The tissue culture plates were purchased from Costar leukotriene; HPLC, high-performance liquid chromatography; DMSO, dimethyl sulfoxide; BSA, bovine serum albumin; HBSS, Hanks' balanced salt solution; (Cambridge, MA). Anti-BrdU monoclonal antibody was purchased PCA, perchloric acid; I \B;. thromboxane B:: NGDA, nordihydroguaiaretic acid; from Vector Laboratory (Burlingame, CA). Protein was measured using BrdU, 5-bromo-2'-deoxyuridine; 12-HHT, 12-hydroxyheptadecatrienoic acid; the Coomassie blue reagent of Pierce (Rockford, IL). All reagents were HETE, hydroxyeicosatetraenoic acid; IMDM, Iscove's modified Dulbecco's me dium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; LI, labeling of highest grade available, and all water was deionized and passed index; PLA3, phospholipase A2; HPETE, 15-L-(i)-hydroperoxyeicosatrienoic through a MilliQ purification system (Waters Associates, Milford, MA) acid. prior to use. 1744

Methods 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 Eicosanoids Synthesis by ACS Cells added at a specific activity of 0.5 mCi/ml. To estimate nonspecific Cell Culture and Loading with [3H|AA. Two ml of an ACS cell incorporation or adherence of thymidine, 10 ml of hydroxyurea (10 suspension (2 x IO6cells/ml) were plated in tissue culture wells, loaded mg/ml) were added to some wells. Two h later, 2 ml of ice-cold saline with 0.5 mCi of [3H]AA per well, and incubated at 37Â°Cunder 5% CO2 were added, aliquots were removed, and cells were washed twice with and 95% air. After 18 h of incubation, the labeling medium and floating PBS. After counting cell numbers, 2 ml of 5% PCA were added, and cells were removed, and the cells were washed twice with HBSS con the dpm value in the acid-insoluble fraction was measured. BrdU LI. To measure DNA synthesis, 2 x IO6 ACS ceils were taining 2% BSA and once with HBSS containing 0.1% BSA to remove free unincorporated [3H]AA. prepared as described above, after which Bull was added at a final Extraction of 3H-labeled Eicosanoids. The ACS cell (14) monolayers concentration of 100 mg/ml for 4 h. To terminate BrdU labeling, ice- were incubated in 2 ml of IMDM supplemented with 10% FBS in the cold PBS was added to the wells, and the supernatant was discarded. presence or absence (using vehicle as control) of 2.0 or 5.0 mM A23187 BrdU-labeled cells were washed twice with PBS and removed from the in DMSO (at a final DMSO concentration of 0.02%) for 1 h. The wells with trypsin/EDTA. Cells were attached to glass slides by cen- extraction from culture medium of radiolabeled eicosanoids produced trifugation using a Cytospin-2 centrifuge (Shandon, Pittsburgh, PA) by the [3H]AA-labeled ACS cells was performed by the method of and fixed in 70% ethanol. Cells on the glass slides were treated with 4 Wescott et al. (15). After l h of incubation, culture media from three N HC1 for 30 min to denature the DNA, and immunohistochemical wells were pooled and added to two volumes of ice-cold methanol. The detection of BrdU incorporation was performed using the anti-BrdU mixture was centrifuged at 1500 rpm for 5 min, and the methanolic monoclonal antibody and the avidin-biotin-peroxidase complex method supernatant was diluted in 0.1 M sodium phosphate buffer, pH 7.4, to (16). To calculate the BrdU LI, photographs were taken of at least four yield a final methanol concentration of 20%. This 80% aqueous mixture different highpower fields randomly selected under the light micro was applied to Sep-Pac CIS cartridges (Waters Associates, Milford, scope. Total cells and BrdU-positive nuclei were counted in each field. MA) that had been prewashed sequentially with 20 ml of methanol and The LI was defined as the ratio of BrdU-labeled cells to total cells 20 ml of deionized water. The loaded cartridges were washed with 5 ml counted. of 20% methanol in 0.1 M sodium phosphate buffer followed by 5 ml Data Analysis. The data were expressed as the mean Â±SEM of at of deionized water, both of which were discarded. Eicosanoids were least 3 to 5 different series of experiments. Individual measurements eluted with 3 ml of 80% methanol in water. Extracts were then evapo were done in duplicate or triplicate. Differences between control and rated to dryness under nitrogen, and residues were stored at -70Â°C treatment groups were compared by the Student t test, and P < 0.05 pending eicosanoid separation by HPLC. was regarded significant. Separation of |3H|AA Metabolites by HPLC. Eicosanoid separation was accomplished according the method of Peters-Golden and Thebert RESULTS (13). For identificai ion of radiolabeled eicosanoids, the extracted sam ples were redissolved in 300 Â¡Aofacetonitrile:water:trifluoroacetic acid Profile of Eicosanoids Derived from ACS Cells. Fig. 1 shows (33:67:0.1) and subjected to reverse-phase HPLC using a 30- x 0.4-cm the elution of 'H-labeled eicosanoids after separation on the 5-mm Bondapak C,g column (Waters Associates, Millford, MA). The HPLC column. Relative molar amounts of each metabolite may mobile phase consisted of acetonitrile in water and trifluoroacetic acid be measured from the profiles. The lower curve (thin, dashed at a flow rate of 1 ml/min. Cyclooxygenase metabolites were eluted during an initial isocratic phase (acetonitrile:water:trifluoroacetic acid, line) indicates eicosanoids produced in the unstimulated state, 33:67:0.1); the lipoxygenase metabolites and free AA were eluted using and upper curves (the dashed and solid bold lines) indicate a continuous gradient of increasing acetonitrile to 100:0:0.1. eicosanoid production after stimulation with 2.0 HIM and 5.0 The eluate was continuously monitored for UV absorbance (210 nm HIMcalcium ionophore A23187, respectively. The calcium io- for cyclooxygenase products and free AA, 280 nm for leukotrienes, and nophore produced dose-dependent increases in eicosanoid me 235 nm for mono-HETEs). The retention times (min) for authentic tabolites. Although one cannot measure the total mass of eicos eicosanoids standards were as follows: 6-keto-PGF,,,, 7 to 8; TxB2, 13 anoids, the relative molar proportions of all AA metabolites to 15; PGF2â€ž,16;PGE2, 18 to 19; PGD2, 21 to 22; LTC4, 44; LTD4, may be determined using this method. The major metabolites 46; LTB4, 48 to 49; 12-HHT, 58 to 59; 15-HETE, 72 to 75; 12-HETE, produced by ACS cells after A23187 stimulation were LTD4, 77 to 78; and AA, 97 to 99. Cochromatography with authentic eicosa followed by progressively smaller amounts of 6-keto-PGF,,,, noids standards was used with each sample to control for any variation LTC4, LTB4, TxB2,12-HETE, PGF2â€ž,PGE2,and PGD2. Under in retention and elution. One-mi eluted fractions were collected, and 3H-labeled products were basal conditions, the profile showed 12-HETE > LTB4 > LTC4 > PGF2â€ž>PGE2 > 6-keto-PGF,,, > TxB2 > LTD4 > 15-HETE quantitated by liquid scintillation counting in 5 ml of scintillant (Ecol ite; ICN Biomedicals, Inc., Irvine, CA). > PGD2 (Fig. 1). Influence of Exogenous Eicosanoids on Cell Growth. The time course of ACS cell growth is shown in Fig. 2. Both PGD2 and ACS Cell Growth in Response to Exogenous Eicosanoids PGE2 (5.0 Mg/ml) significantly suppressed cell growth at 24, Count of Cell Number and Protein Assay. Suspensions of 2 x 10s 48, and 96 h (P < 0.01 versus control). However, LTC4 and ACS cells in 3 ml of IMDM supplemented with 10% FBS were plated LTD4 (0.5 mg/ml) significantly stimulated cell growth at 48 in tissue culture plates and incubated at 37Â°Cunder 5% CO2:95% air and 96 h (P < 0.05 versus control). overnight ("the preincubation period"). The next day ("time 0"), the Dose-Response for Modulating AGS Cell Growth by Eicosa medium was replaced with 3 ml of fresh medium containing the noids. The dose-response relationships for growth suppression following eicosanoids: PGD2 or PGE2 (0.5, 1.0, 2.5, 5.0 jig/ml); LTC4 or LTD4 (0.01, 0.05, 0.1, 0.25, 0.5 mg/ml); indomethacin (10~6, IO'5 by PGD2 and PGE2 are depicted in Fig. 3. Increasing the PG M); NDGA (IO'7, IO"6, 10"' M); hydrocortisone (IO'5 M); or vehicle concentration from 0.5 to 5.0 Mg/ml produced a progressive only as control. The medium was adjusted to a final methanol concen inhibition of cell growth by both PGD2 and PGE2. The 50% tration of 0.5%. After 96 h of incubation, cells were harvested using effective dose for growth inhibition was in the range of 2.5 to trypsin/EDTA and counted using a hemocytometer; the protein content 5.0 Mg/ml for each prostanoid. of the cells was measured. The initial cell numbers at time 0 (1.85 Â± The change in protein content of the AGS cells produced by 0.27 x 10') (mean Â±SD, n = 9) and protein contents were not PGD2 and PGE2 is shown in Table 1. Both PCs reduced the 1745

250 increases in cell number for LTD4 which reached significance at a concentration of 0.05 mg/ml, and a 36.7% increase in cell 200 number was observed at 0.5 mg/ml. Significant growth stimu lation was seen for LTC4 only at the 0.5 mg/ml concentration. 150 In contrast, no concentration of LTC4 or LTD4 showed a significant increase in the total protein content of AGS cells (data not shown). 100 Effect of NDGA, Hydrocortisone, and Indomethacin on Cell Growth. NDGA is an inhibitor of 5-lipoxygenase activity, and

1; TxB, 3: PGÃ‹, hydrocortisone inhibits PLA2 and may have a variety of other 2: PGF,, 4; PGD, effects on cell growth in culture. If LTs were important stimu lators of AGS cell growth, NDGA or hydrocortisone might be 0 10 20 30 40 SO 60 70 80 90 100 110 expected to inhibit LT synthesis and inhibit cell proliferation. Elution Fraction # ( min ) Increasing concentrations of NDGA (10~7 M, IO'6 M, IO"5 M) Fig. 1. Profile of eicosanoids derived from ACS cells. The ACS cells were and hydrocortisone (10~5 M) produced no significant effect on incubated for l h in the presence or absence of the calcium Â¡onophoreA23187. The [JH]AA metabolites in the medium released by 6 x IO6 AGS cells were cell growth. Since LTs released into the medium may have separated by reverse-phase HPLC. The lowest line (thin, dashed line) represents influenced cell growth prior to the addition of inhibitors, ex the elution profile in the absence of A23187; the bold dashed line and bold solid periments were designed to limit this effect, in which NDGA line represent the elution profile in the presence of 2.0 /IM and 5.0 MMA23187, (10~s M) or hydrocortisone (10~5 M) was added during the respectively. The arrows point to the elution of authentic standards, as labeled. preincubation period. The results of those experiments are shown in Table 2. Increasing the concentration of NDGA (10~7 M, 10~6 M, or 10~5 M) in cells pretreated with 10~5 M NDGA produced a significant, dose-dependent inhibitory effect on cell growth (P < 0.005). Hydrocortisone (10~5 M), added to cells that 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 Â±5and 97 Â±4% of control wells, respectively. ['I I|I hynwliiu- Incorporation. To further document the effects of exogenous eicosanoids on cell growth, [-'Hjthymidine incor poration per IO6AGS cells was measured. As demonstrated in Table 3, PGD2 and PGE2 (at 1.0 and 5.0 Mg/ml) decreased [3H] 24hrs 48hrs 96hrs thymidine incorporation significantly (P < 0.005), whereas Time Course LTD4 (at 0.05 and 0.5 mg/ml) increased incorporation signifi- Fig. 2. Growth curve of AGS cells incubated with PCs or LTs. Lines represent the numbers of AGS cells after incubation with LTD4, LTC4, control, PGE2, and PGD2 from upper to lower lines, respectively. *,P< 0.05; ", P < 0.01; **Â«,P< Table 1 Growth response of AGS cells to PCs (total protein content as percentage) 0.005, compared with control. (Â¿ig/ml)PGD2 Concentration

Â±7.9Â° Â±9.8* Â±7.2C 120 Â±4.6 PGE20.572.273.3 Â±3.5*1.076.363.1 Â±3.92.561.454.5 Â±9.4*5.045.640.8 Â±5.7C " Mean Â±SEM of control wells (n = 3 to 4). 100100 100 *P < 0.05. ' P < 0.01 compared with protein content in control wells.

Â§ 60

O 40

20 CONI. 0.5 1.0 2.5 5.0 Concentration of Prostaglandms (pg/ml) 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 the percentage of control cell number. â€¢¿andâ€¢¿.PGD2and PGE2, respectively. *, P < 0.05; ", P < 0.01; ***, P < 0.005, compared with control wells. COAT., control.

CONT. 0.01 O.OS 0.1 0.25 0.5 total protein content of AGS cells in a dose-dependent manner. CONCENTRATION OF LEUKOTRIENES (mg/ml) The dose-response relationships for stimulation of cell 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 cell growth by LTC4 and LTD4 are shown in Fig. 4. Increasing the number. â€¢¿andH. LTC4 and LTD4, respectively. Â«,P< 0.05; ", P < 0.01; Â«Â»Â», LT concentration from 0.01 to 0.5 mg/ml produced progressive P < 0.005, compared with control wells (CONT.). 1746

Table 2 Effect of inhibitors ofeicosanoid metabolism on cell number and total protein contents of ACS cells Data are expressed as the percentage of control wells. NDGANonpretreated0 M118Â± M99 M90 10"*M100

No. of cells Â±3* 10 Â±8 Â±4 Â±13 ProteincontentPretreated 100Â±3137 102Â±789 95Â±479 91Â±757 10127 94 Â±

with inhibitors' Â±5" Â±7 Â±3'' Â±3' Â±3 75 Â±4' No. of cells 76 Â±8'HydrocortisoneControl113Â± 10 53Â± \3d Protein contentControl100 99 Â±210~' 91 Â±610-' 82 Â±2'10-' " In the "nonpretreated" group, AGS cells were incubated with NDGA (10 7 to 10 * M) or hydrocortisone (10 ' M) for 96 h. * Mean Â±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 between the "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

{â€žg/ml)dpm/fig PGD2 (Mg/ml)1.08,463

of protein/ml Â±165" 0,086 Â±207 Â±289 Â±715 Â±679 Â±225 Â±423 % of control 1000.51 93 Â±2 88 Â±3 71 Â±7 87 Â±6 78 Â±2 74 Â±4 0.005"P value vs. controlControl10,815 P = 0.051.09,520 P < 0.0055.07,641 JÂ»<0.005509,400 /â€¢=0.10PGE2 P< 0.001508,034 P < Mean Â±SEM.

Table 4 Effect of exogenous LTs on f'HJthymidine incorporation into ACS cells (mg/ml)dpin LTC< (mg/ml)0.0513,798 0.0510,810 ,

Table 5 BrdUrd labeling indices of ACS cells treated with PCs M) showed a nonsignificant effect on proliferation (94 Â±5 and (â€žg/ml)1.0 97 Â±4% of control BrdU, respectively). Contro!Labeling 5.034 index 44*1" Â±182 Â±177 177Â±1 33 Â± DISCUSSION (% of cells labeled) % of control Â±2 Â±2 Â±2 75 Â±3 This work reports the full range of eicosanoids produced by P value0.001Â° vs. con P < 0.005>g/ml)5.036P < 0.005PGE2P< 0.001 P< the human stomach tumor cell line AGS. Under basal condi trolPGD2I1.038 tions, AGS cells produce LTD., as its principal eicosanoid Mean Â±SEM. metabolite, followed by progressively smaller molar quantities of 6-keto-PGF,,,, LTC,, LTB4, 12-HETE, TxB2, PGF2â€ž,PGE2, Table 6 BrdUrd labeling indices of ACS cells treated with LTs 12-HHT, 15-HETE, and PGD2. After stimulation with the (mg/ml)ControlLabeling LTC4 (mg/ml)0.156 calcium ionophore A23187, the principal eicosanoids products were (in order of relative molar abundance): 12-HETE; LTB4; LTC4; PGF2lt; PGE2; 6-keto-PGF,,,; TxB2; LTD4; 15-HETE; index 44 Â±1Â° Â±1123 Â±1127 Â±2129 Â±3147 and PGD2. These results indicate that 5-lipoxygenase is more (% of cells labeled) active than cyclooxygenase under both basal and ionophore- % of control Â±3 Â±3 Â±3 Â±6 stimulated conditions in AGS cells. This is different from what P value0.001Â° vs. con P< 0.0050.556P< 0.001LTD,P< 0.0050.565P< trol0.155 has been reported in normal human gastric tissue in which Mean Â±SEM. PGE2 is the most prominent eicosanoid, followed by LTB4 and the sulfidopeptide-LTs (11). This also differs from reports of the prostanoids produced by gastric cancer tissue, in which 6- cantly at both concentrations (P < 0.005). LTC4 showed no keto-PGF,,, was the principal product, followed by smaller response at 0.05 mg/ml and showed a nonsignificant increase amounts of PGE2, PGF2â€ž,andPGD2 (10). Pelus and Brockman at 0.5 mg/ml (P = 0.065) as shown in Table 4. (17) reported that macrophages from tumor-bearing animals Indomethacin (!()'' M) produced a nonsignificant increase in have a markedly augmented capacity to metabolize arachidonic thymidine incorporation to 129 Â±14% of control wells. acid. Tumor tissue may be infiltrated with inflammatory cells, BrdU LI. Both PGD2 and PGE2 (1.0 and 5.0 Mg/ml) signifi neutrophils, or macrophages, which would affect eicosanoid cantly decreased the BrdU LI (P < 0.005), and both LTC, and production (12). Therefore, the measurement of AA metabo LTD., (0.1 and 0.5 mg/ml) significantly increased LI (P < lism in a tumor cell line would be more likely to give an accurate 0.005) as shown in Tables 5 and 6. Indomethacin (10~6 M, 10~5 measure of the endogenous capacity of the malignant epithelial 1747

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1992 American Association for Cancer Research. EICOSANO1DS SYNTHESIS BY ACS CELLS cells themselves. We did not attempt to measure the total mass ent inhibition in growth. As demonstrated in Table 2, pretreat of eicosanoids made by the tumor cells; given the fact that the ment of AGS cells with inhibitors of eicosanoid metabolism, substances would act as autocrine factors, it is difficult to know followed by a change in the medium, was required to demon how one would interpret these findings. One can indirectly strate inhibition of growth by these agents. This suggests that estimate the dose-response relationships of the cells from the the half-life of LTs may be very long in vitro and that their data obtained using exogenous LTs. persistence in the culture medium must be considered when In this study, the growth and DNA synthesis of ACS cells interpreting experimental results. were suppressed by exogenous PGE2 and PGD2, similar to the Hydrocortisone is an inhibitor of PLA2, which affects both reported findings of other investigators using other tumor cell cyclooxygenase and lipoxygenase metabolites. Because of the lines (4-8). It has been proposed that the antitumor activities intrinsically greater activity of the lipoxygenase pathway in of these prostaglandins are exclusively due to their ultimate AGS cells, hydrocortisone will appear to selectively inhibit this metabolites, PGA2 and 12-PGJ2, which share a cyclopentenone arm of eicosanoid metabolism. Indomethacin had no effect on ring structure (18). The growth-inhibiting effect of these PGs proliferation in AGS cells, although this agent has been shown was independent of cyclic AMP, and its mechanism appeared to stimulate cell growth in HEP-2, L, and HeLa cells, all of to depend upon inhibition of entry into the G, phase of the cell which produce considerable amounts of PGE constitutively cycle (7, 19). Furthermore, Santro et al. (20) reported that a MT (26). Furthermore, at higher concentrations, indomethacin sup 74,000 protein was induced by PGA, or PGJ2 in human K562 presses phospholipase A2 and lipoxygenase activities (27, 28). erythroleukemia cells; the protein has been identified as the Other lipoxygenase metabolites including 12-HETE, 15- heat-shock protein, and its appearance was associated with HETE, and HPETE inhibit cell growth in SK-N-SH, a human changes in cell proliferation. Ishioka et al. (21) reported that neuroblastoma cell line (29), and MCF-7, a human breast PGA: arrested HL-60 cells at the G0-G, phase of the cell cycle, cancer cell line (30). Furthermore, 15-HETE can inhibit the which was associated in a reduction in the mRNA for c-myc. biosynthesis of leukotrienes (31). Therefore, the inhibitory ef We have demonstrated that LTD4 (at concentrations from 0.05 to 0.50 mg/ml, or IO"7 M to 10~6 M) and LTC4 (at 0.50 fect of certain lipoxygenase metabolites on tumor cells may be mg/ml or 10~6 M) significantly increased cell number and mediated by an autoregulatory modification in LT metabolism in those cells. stimulated DNA synthesis in ACS cells. Dawson et al. (22) LTC4 may be cleaved by 7-glutamyltranspeptidase to produce have reported that the 5-lipoxygenase inhibitor benoxaprofen LTD4 (32). LTD4 interacts with highly selective and specific suppressed the growth of various tumor cell lines in vitro and tumor growth in vivo after i.p. injection of mice with the T-cell receptors that can distinguish binding sites for LTB4 or LTC4 (33). LTD4 can increase intracellular calcium and release free lymphoma line EL4. Gali et al. (23) reported a similar inhibi tory effect with the 5-lipoxygenase inhibitor NDGA on human AA by the activation of PLA2 through the LTD4 receptor (34, 35). Therefore, it is possible that these LTs may reinforce their glioma spheroids. Miller et al. (9) showed that NDGA, piriprost growth-stimulating effects by a positive feedback mechanism. (a selective inhibitor of 5-lipoxygenase), and other inhibitors of peptide-LT (LTC4, LTD4, and LTE4) metabolism inhibited the In summary, we have found that the human gastric carcinoma growth of the human leukemia cell line HL-60, which was cell line AGS preferentially produces lipoxygenase rather than completely reversed by the daily addition of IO"9 M LTD4. cyclooxygenase metabolites in response to stimulation by cal cium ionophore. The prostanoids PGD2 and PGE2 inhibit the Ralph and Wojcik (24) have reported that various lipoxygenase growth of AGS cells, whereas LTC4 and LTD4 stimulate their and phospholipase A2 inhibitors (i.e., NDGA, hydrocortisone, and others) inhibited the growth of the P815 murine mastocy- growth. These results suggest that certain leukotrienes are autocrine growth factors for this cell line. Data from other toma cell line, but that 0.16 nM LTC4 or 0.3 nM LTB4 failed to reverse this effect. In our study, we used relatively high dosages laboratories suggest that PGs and LTs may have opposite of LTs (up to 10~6M) and demonstrated that LTD4 was a more effects on cell proliferation in a number of tumor systems. The potent stimulator of AGS cell growth than LTC4, similar to role of eicosanoids in proliferation by tumor cell lines suggests what had been reported by Miller et al. (9). However, it is very that this may be a potential area of therapeutic interest. How difficult to know what represents a physiological concentration ever, the complex effects of inhibitors of eicosanoid metabolism of a paracarine or autocrine substance. and the interactions among the members of the LT and PG Our data showed significant increases in AGS cell numbers autacoid families make it difficult to predict the response of after stimulation with LTs, but we did not demonstrate a any tumor to pharmacological manipulation. Furthermore, tu significant increase in total protein content with any concentra mors are typically infiltrated with inflammatory cells and vas tion of LT. The discrepancy between these two measures of cell cular elements which will have additional impact on the local growth may be a reflection of a reduction in cell size. AGS cells production of eicosanoids, which will further complicate at are somewhat pleomorphic, but after the addition of LTs, the tempts at pharmacological manipulation. Nonetheless, this cell size appeared subjectively smaller than in control wells. work demonstrates that stomach cancer cells produce a different Therefore, LTs may stimulate cell division more rapidly than profile of eicosanoids then do the normal tissue counterparts protein synthesis in these cells. and that these autacoids have potentially important influences Nishizawa et al. (25) reported that the 5-lipoxygenase inhib on the growth of the tumor cells. itors NDGA and AA861 stimulated the growth of transformed murine Leydig cells, and that 5-HETE inhibited proliferation in these cells. In this study a single dose of NDGA (10~7 M, ACKNOWLEDGMENTS IO"6 M, or 10~5M) or hydrocortisone (10~7 M or 10~5M) showed The authors gratefully thank Patricia Shaw and Diana Drescher for no significant effect on cell growth, but pretreatment with their help in preparation of the manuscript. We would like to also NDGA or hydrocortisone inhibited LT production during the thank Eugene Kraus, James Scheiman, and Kenji Yoshimura for ex preincubation period and produced a significant, dose-depend perimental support and advice. 1748

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1992 American Association for Cancer Research. Eicosanoid Production by the Human Gastric Cancer Cell Line AGS and Its Relation to Cell Growth

Shuya Shimakura and C. Richard Boland

Cancer Res 1992;52:1744-1749.

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