(CANCER RESEARCH 46, 5676-5681, November 1986] Eicosanoid Synthesis by Cultured Human Urothelial Cells: Potential Role in Bladder Cancer1
Abraham Danon, Terry V. Zenser,2 David L. Thomasson, and Bernard B. Davis
Geriatric Research, Education, and Clinical Center, Veterans Administration Medical Center, St. Louis, Missouri 63125 [T. V. Z., B. B. D.]; Departments of Biochemistry IT. V. Z., D. L. T.] and Internal Medicine [T. V. Z., B. B. D.J, St. Louis University School of Medicine, St. Louis, Missouri 63104; and Clinical Pharmacology Unit, Ben-Gurion University of the Negev, Beer-Sheva, Israel [A. D.]
ABSTRACT chemical alterations including increased arachidonic acid me tabolism (6, 7). In vivo and in vitro studies have shown that Prostaglandin (PG) H sym liase and eicosanoid products of arachidonic eicosanoid products of arachidonic acid can regulate the rate of acid metabolism have been implicated in several steps in the carcinogenic proliferation and differentiation of certain mammalian cells (8, process. This study assessed these parameters using primary cultures of 9). In addition, numerous tumors demonstrate increased syn human urothelial cells. To determine the possible presence of permeabil thesis of eicosanoids (10). Eicosanoids may also alter host- ity barriers to agonist stimulation, incubations were performed with adherent cells in the presence or absence of thioglycolate pretreatment tumor cell interactions that can affect host immunocompetence, or with cell suspensions. No evidence for permeability barriers was indirectly facilitating tumor growth and spread (11). observed. With adherent cells in the absence of thioglycolate, radioim- Urothelial cells are capable of metabolizing-activating several munoassayable 1'C.I >was stimulated by epinephrine < 12-0-tetradeca- classes of chemical carcinogens. This has been demonstrated noylphorbol-13-acetate = thrombin < bradykinin = A23187
Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1986 American Association for Cancer Research. EICOSANOIDS IN UROTHELIAL CELLS collagen (Sigma Chemical Co., St. Louis, MO) or with 2% gelatin HPLC analysis of eicosanoids was carried out using a Beckman 112 (Fisher Scientific Co.) as described previously (30, 32). The data indi solvent delivery module, a Beckman 421 controller, and a Waters WISP cated that there were no differences in morphology or metabolism 71OB automatic sample injection system. Separation of eicosanoids between urothelial cells grown on gelatin and those grown on collagen was affected by reversed phase chromatography, using an Altex ultra- substrate (not shown). sphere-ODS 5-^m (250 x 4.6 mm) column with Rheodyne 2-^m Experiments were conducted with 80-90% confluent cultures (3-4 precolumn filter and a Brownlee S-^m Spheri-5 reverse phase precol- x 10* cells) by aspirating growth medium and washing with 2-3 ml of umn (30 x 4.6 mm). Three different solvent systems were used at a phosphate-buffered saline. To assess the possible presence of permea flow rate of 1 ml/min (modified from system in Ref. 37). Solvent system bility barriers to agonist stimulation (33, 34), cells were prepared for A, made of 25% acetonitrile, 74.7% water, 0.2% benzene, and 0.1% analysis by three separate methods. Adherent cells were analyzed with acetic acid, was used initially to separate the prostaglandins and throm- or without a 2-h pretreatment with 0.01 M thioglycolate (33). Cells boxane B2; at 39 min, solvent system B, composed of 65% methanol, were also suspended by treatment for 15 min with 0.1% EDTA. 34.9% water, and 0.1% acetic acid buffered with ammonium hydroxide Regardless of the method of preparation, cells were equilibrated with 1 to pH 5.2, eluted the hydroxyeicosatetraenoic acids and leukotrienes. ml of serum-free medium for 30 min at 37°Cin 5% CO2-95% air. A From 81 to 115 min, solvent system C, consisting of 90% methanol, final 30-min incubation was carried out with serum-free medium in the 9.98% water, and 0.02% acetic acid, eluted arachidonic acid. The presence or absence of test agents. Test agents used were arachidonic column was washed with 30 ml of 3% EDTA in water at the beginning acid purchased from Nu-Chek Prep., Inc., Elysian, MN; calcium iono- of each day to improve peak shape and resolution of the leukotrienes phore A23187 was from CalBiochem, La Jolla, CA; and bradykinin (38). Detection was achieved with a Flo-One Beta radioactive flow triacetate, epinephrine reagent, thrombin (2000 NIH units/mg protein), detector (Radiomatic Instruments and Chemical Co., Inc., Tampa, FL). EDTA (disodium salt), indomethacin, platelet-activating factor, epider The tritium channel had its discriminator set at 1-35 with 125 cpm as mal growth factor, antidiuretic hormone (Grade VI), sodium thiogly the background subtract and 10 cpm as the threshold settings. For the >4C-metabolites, the carbon channel had its discriminator set at 35-100 colate (Grade V), mezerein, and TPA and the other phorbol esters were from Sigma. Final media were examined for the amount of PGE2 with 75 and 10 cpm as the background subtract and threshold settings, synthesized by radioimmunoassay and, when prelabeled with [14C]- respectively. A dual-label quench-corrected curve allowed determina arachidonic acid, for the total eicosanoid synthesis by HPLC. tion of the absolute amount of radioactivity. Identification of metabo Preparation of Culture Medium. Ham's Nutrient Mixture Fl 2 powder lites was made by comparison with tritiated authentic standards that (GIBCO, Grand Island, NY) was reconstituted for use. This medium eluted simultaneously. Data were expressed as percentage of total was supplemented with 1% fetal calf serum and the following (final radioactivity recovered. concentration in parentheses): insulin (10 Mg/ml), hydrocortisone (1 Radioimmunoassay of Prostaglandin l>. The PGE2 content of the ¿ig/ml),and transferrin (5 ^ig/ml) which were obtained from Sigma; media was measured by double-antibody radioimmunoassay (39). Rab nonessential amino acids(0.1 HIM),L-glutamine(2.0 HIM),streptomycin bit antiserum to PGE2 was obtained from Regis Chemical Co., Morton (100 Mg/ml), and penicillin (100 U/ml) which were purchased from Grove, IL. Tritium-labeled PGE2 was supplied by New England Nu clear, and goat antiserum to rabbit 7-globulins was from Antibodies GIBCO; and dextrose (2.7 mg/ml) from Fisher Scientific (30). Supple mented medium was designated F12+. Inc., Davis, CA. Each condition was evaluated in two or three separate Ultrastructural Studies. To prepare urothelial cell cultures for trans experiments. In each experiment, conditions were evaluated in at least mission electron microscopy, plates with attached cells were rinsed with triplicate plates. The medium from each plate was analyzed in duplicate phosphate-buffered saline and fixed for 3 h with 2% glutaraldehyde in and the value of duplicate determinations was averaged and considered 0.1 M Sorensen's buffer at room temperature. After the cells were fixed, as A' = 1. Data were expressed as mean ±SE of ng PGE2 per 10*cells. they were rinsed several times with Sorensen's buffer. Cacodylate buffer Statistical differences between data were evaluated by Student's t test (0. l Msodium cacodylate containing 3.5% sucrose and 1.0 HIMcalcium) for unpaired values. was used for the final rinse. These fixed cells were postfixed for l h with 1% osmium tetroxide buffered with cacodylate buffer. Following RESULTS dehydration in graded alcohols, cells were infiltrated with a graded alcohol-resin mixture and embedded in Polybed 812 resin. After curing Cell Identification. Cultured cells were identified as epithelial overnight at 50-55°C, the embedded cells were separated from the by their morphological and histochemical characteristics. Cul culture dish and fragmented into smaller pieces. These were reembed- tures grew as continuous sheets of tightly adherent cells. Using ded in Polybed 812 and cured overnight at 60V, Thick sections were phase contrast microscopy, cells observed in dense areas were cut at 0.5-1.0 mm with a glass knife and stained with toluidine blue cuboidal, while those growing out from the expiants exhibited for surveying cells. Using a diamond knife, thin sections were cut at a more polygonal appearance and contained 1 to 4 prominent 60-90 nm and doubly stained with uranyl acetate and lead citrate. Cells nucleoli. Transmission electron microscopy showed stratified were studied with a Phillips 200 electron microscope. cells containing tight junctions and desmosomes. Cells exhib Alkaline Phosphatase Staining. This was performed according to standard procedures (35). After the cells were fixed using cold 10% ited strongly positive staining for alkaline phosphatase. formalin-90% methanol, they were incubated for 30 min with Naphthol PGE2 Synthesis. Cultured human urothelial cells prepared by AS-MX phosphate (Sigma) in the dark. Fast Blue RR (Sigma) was three different methods were examined for their ability to used as the diazonium salt. Mayer's hematoxylin solution was used as synthesize PGE2 (Fig. 1). Those manipulations were made in a counterstain. The stained cultures were then examined microscopic an effort to overcome a possible permeability barrier at the ally. Similarly treated fibroblasts were used as negative controls. apical surface of the transitional cells (33, 34). Regardless of Labeling of Urothelial Cells with (MC|Arachidonic Acid and Assess how the cells were prepared for analysis, agonist stimulation ment of Eicosanoid Products. Cultures (17-21 days and approximately followed the same general order of responsiveness. That is, 80% confluent) were rinsed twice with serum-free medium containing TPA < bradykinin = A23187 «:arachidonic acid. Both control 0.5 fiCi [MC]arachidonic acid (Amersham, Arlington Heights, IL; spe and stimulated values for PGE2 synthesis were similar for cific activity, 58 mCi/mmol). Following a 1-h incubation, cells were adherent cells treated with thioglycolate and untreated. In con rinsed twice and allowed to equilibrate for 30 min. Cells were then trast, resuspended cells had higher control l"(•Isynthesis and incubated in fresh media with test agents for 30 min. After EDTA was added (final concentration, 1 HIM)to retard degradation of the leuko- a smaller response to agonists. Epinephrine and thrombin were trienes (36), media were frozen at -20"C. Before chromatography, a also shown to enhance PGE2 synthesis in adherent cells. Epi mixture of 3H-labeled authentic standards (New England Nuclear, nephrine was the weakest agonist tested and thrombin had Boston, MA) as indicated in Fig. 4 was added. These standards were activity similar to that of TPA. On the other hand, adherent made up in 50 HIMbutylated hydroxyanisole in benzene:ethanol (1:1). cell PGE2 synthesis was not increased by antidiuretic hormone 5677
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36 D Control M 32 •AA S A23187 g 28 B BK 'o 24 D TPA n Epi - 20 Thrombin *«
12 S 4
Adherent Cells Thloglycolate Suspended Cells Pre-Treated Fig. l. Profile of agonist Stimulation of PGE2 synthesis. The following ago nists were used: ISO MMarachidonic acid (AA); A23187 (0.5 *ig/ml); bradykinin (BK) (\ fig/ml); 0.1 «\rTPA; 0.1 mM epinephrine (Epi); and llirumili n (1 unit/ ml). Columns, mean of 6 experiments; bars, SE.
Table 1 Stimulation of human urothelial cell /'(./.. synthesis by tumor and non- tumor promoters Cells were equilibrated with 1 ml of serum-free medium for 30 min. Medium was removed and replaced with fresh medium alone (control) or containing the test agents at the concentrations indicated. After a 30-min incubation, medium was removed and assayed for PGE2 (N = 6). synthesis ConditionControl1 G*M)0.1100.10.10.30.4200PGE2(ng/105cells)0.7 ±0.4°5.0 20 30 40 SO 60 2-0-Tetradecanoylphorbol-3-acetate+ 1 ±0.4*0.3 Time, Minutes Fig. 2. Time course for uptake of arachidonic acid by urothelial cells. [1-I4C]- IndomethacinPhorbol- ±0.13.8 Arachidonic acid, 0.5 nCi, was added to cell cultures, and radioactivity was 3-dibutyratePhorbol-12,1 ±0.2*5.3 measured in the media at the times indicated. 3-didecanoate4,12,1 ±1.9*0.4 3-didecanoateMezereinI, 1'luir bol 12, 1 ±0.14.3 ±0.3*3.5 the major lipoxygenase products observed. The synthesis of 12- ±0.1* -Oleoyl-2-acetylglycerolConcentration HETE was at the limit of detection. " Mean ±SE. * P < 0.01 compared to control values. DISCUSSION (1 IU/ml) or by either platelet-activating factor or epidermal This is the first report to demonstrate prostaglandin H syn- growth factor (1 to 0.01 Mg/ml). thase activity in human urothelial cells. Factors that regulate PGE2 synthesis by tumor and non-tumor promoters was eicosanoid synthesis and determine the profile of eicosanoid examined with adherent cells in the absence of thioglycolate products synthesized by human urothelial cells were also as (Table 1). TPA increases in PGE2 synthesis were completely sessed. A variety of test agents were shown to increase synthesis prevented by indomethacin (10 ^M). The TPA type tumor of PGE2. In the presence of those test agents, other prostaglan promoters phorbol-12,13-didecanoate and phorbol-12,13-di din H synthase and lipoxygenase products of arachidonic acid butyrate stimulated PGE2 synthesis. However, the TPA type were also demonstrated. Specific antagonists (indomethacin non-tumor promoter 4a-phorbol-12,13-didecanoate did not in and nordihydroguaiaretic acid) of these pathways inhibited crease PGE2 synthesis. The tumor promoter mezerein increased synthesis of these products (40, 41). Mechanical manipulation PGE2. l-Oleoyl-2-acetylglycerol also increased prostaglandin of rabbit bladder epithelium has been shown to increase pros synthesis. taglandin synthesis (42). This may be the reason for the in Synthesis of Radiolabeled Eicosanoids from [I4C]Arachidonic creased PGE2 synthesis observed in the resuspended control Acid. Resuspended cells were prelabeled with [l4C]arachidonic cells. Eicosanoids the synthesis of which was not verified by acid. After 60 min, approximately 70% of the radiolabel was this study include thromboxane, the leukotrienes, and 12- taken up by the cells (Fig. 2). At this time, media were removed HETE. HHT synthesis was observed and may represent throm and the cells were washed. Following a 30-min equilibration boxane synthase activity. However, in most instances, when period, fresh media were applied containing either A23187 or thromboxane synthesis is observed, similar amounts of both bradykinin for 30 min. A representative HPLC profile illus HHT and TXB2 are reported (43). In a recent study with trating eicosanoid synthesis during bradykinin stimulation is primary cultures of mixed renal cells, a large amount of HHT shown in Fig. 3. The relative synthesis of eicosanoids during synthesis was observed with no detectable TXB2. HHT was bradykinin and A23187 stimulation is shown in Fig. 4. Similar subsequently shown to be derived from the breakdown of pros results were observed with both agonists. Prostaglandins rep taglandin endoperoxides and not by way of thromboxane syn resented the major products of metabolism. PGE2 and 6-keto- thase (44). This distinction was not assessed in the present PGF,„were the major prostaglandins synthesized with only study. Epidermal growth factor (45), platelet-activating factor small amounts of PGF2o and PGD2 observed. The syntheses of (46), and antidiuretic hormone (47) which stimulate eicosanoid leukotrienes and TXB2 were at the limit of detection. HHT, a synthesis in other cells were not stimulatory. Thus, certain breakdown product of both the thromboxane and prostaglandin stimuli including hormonal, pharmacological, and mechanical pathways, was observed. Lipoxygenase products of arachidonic can induce human urothelial cells to synthesize a wide range of acid metabolism were observed. 15-HETE and 5-HETE were eicosanoid products known to affect the carcinogenic process. 5678
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Fig. 3. Reverse phase HPLC separation of [MC]eicosanoids released from urothelial cells stimulated by bradykinin (1 (ig/ml). Chromat ographie conditions as described in 'Materials HHT METESw n-s- and Methods." Tritiated standards were eluted 'H simultaneously and recorded on a separate channel (not shown). ¿7°,leukotriene.
to vs too ios tÃo m Time, min. Solvent 1 Solvent 3
20 zole (59), the proximate carcinogen in FANFT-induced bladder D Bradykinin D A23187 cancer. Thus, a sufficient amount of prostaglandin H synthase 16 may be present in human urothelial cells to initiate aromatic amine-induced bladder cancer. S 12 Previous studies have not reported bladder lipoxygenase ac O tivity. This was probably because this activity was not the focus
8 of the studies. For example, when intact rabbit bladder was o studied, only radioimmunoassayable PGE2 was assessed (60). In addition, dog and rabbit microsomes were shown to make i « a. almost exclusively PGE2 with lipoxygenase products not ob served (17, 60). This was because the assay conditions were optimized for PGE2 synthesis (e.g., high concentration of glu- 6 kelo TXB LTB. LTD, MMT 15 - 12 - 5 • PGF, METE METE METE tathione) and because lipoxygenases are soluble rather than Fig. 4. Profile of eicosanoids released following Stimulation with bradykinin microsomal enzymes. The present study prelabeled the uro (l Mg/ml) or A23187 (0.5 Mg/ml). Ordinate, percentage of total radioactivity thelial cell arachidonic acid pool and thus was able to demon which eluted from the column. Columns, mean; hur\. SE. strate both 15-HETE and 5-HETE lipoxygenase activities. The lipoxygenase responsible for 5-HETE synthesis, an essential Prostaglandin synthesis is one of the pleiotropic biochemical component of the leukotriene pathway (61), was at the limit of responses to tumor promoters (6,7,48). With human urothelial detection in urothelial cells. cells, PGE2 synthesis was observed with tumor-promoting, but The physiological or pathophysiological roles of the various not non-tumor-promoting phorbol esters. Mezerein, a related eicosanoids shown to be synthesized by urothelial cells are not diterpene that is a weak complete promoter but a strong second known. Eicosanoids may play a role in the tone and contraction stage promoter (49), also stimulated urothelial cell prostaglan- of the urinary tract smooth muscle, as suggested by the effects din synthesis. Phorbol esters have been shown to increase of exogenous prostaglandins on the one hand and of prosta synthesis of prostaglandin H synthase but not lipoxygenase glandin synthesis inhibitors on the other (62-65). Inotropic products (40, SO).Certain actions of these tumor promoters are effects of leukotrienes have been reported with isolated urinary thought to be mediated by activation of protein kinase C. In bladders of rat and guinea pig (66). Prostaglandins are thought fact, both TPA and mezerein can substitute for diacylglycerol to be released during stimulation of excitatory nerves to the and can directly activate this protein kinase (51, 52). The cell- detrusor (67, 68) or when the bladder is distended (69-71). permeable analogue of diacylglycerol l-oleoyl-2-acetylglycerol Thus, eicosanoids that are synthesized by urothelial cells may activates protein kinase C (53) and has been shown to stimulate contribute to bladder tone and contraction and they could be prostaglandin synthesis by cultured cells (54, 55) including involved in metabolic or physiological functions of the uro- urothelial cells. Thus, tumor promoters increase urothelial cell thelium itself. PGE2 synthesis. This stimulation may involve protein kinase Eicosanoids may play a role in bladder carcinogenesis. Pros C. taglandin H synthase activation of aromatic amines has been A considerable amount of prostaglandin H synthase appears proposed to be involved in the initiation of bladder cancer. This to be present in urothelial cells. Prostaglandins comprised the is supported by in vitro (described above) and in vivo experi largest portion of eicosanoid products representing as much as ments. In the latter, aspirin was coadministered with the bladder 30% of the recovered radiolabel (Figs. 3 and 4). The hydroper- carcinogen FANFT for 12 weeks to reduce the incidence of oxidase activity of prostaglandin H synthase has been proposed both bladder lesions and tumors (58, 72). A more recent study to activate certain aromatic amine carcinogens to initiate blad has used the two-stage bladder carcinogenesis model to specif der cancer (1-2). The dog is the only species besides humans ically assess effects of aspirin on the initiation and promotion to be susceptible to aromatic amine-induced bladder cancer stages of FANFT-induced bladder cancer. Aspirin was shown (56). Using dog bladder transitional epithelial microsomes, to affect both initiation and promotion stages (73). In other prostaglandin H synthase activity was demonstrated and was two-stage models of carcinogenesis, such as the mouse skin shown to activate several aromatic amine bladder carcinogens model, inhibition of prostaglandin synthesis has been found to to bind DNA (17). Dog bladder prostaglandin H synthase be related to inhibition of promotion by a variety of agents, in activity was severalfold more than that observed in dog renal particular, the phorbol esters (5). This inhibition can be over inner medulla, a tissue in other species reported to be very high come by addition of specific prostaglandins (3, 4). Human in prostaglandin H synthase activity (57). Prostaglandin H urothelial cells contain TPA receptors (26). These receptors synthase was also obesrved in rat (58) and rabbit (59) bladder may participate in TPA-mediated increases in eicosanoid syn epithelium and activated 2-amino-4-(5-nitro-2-furyl)-['4C]thia- thesis. Eicosanoids often exert opposing influences on physio 5679
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logical and pathological parameters. The hypothesis that dif R. Bovine bladder urothelial cell activation of carcinogens to metabolites mutagenic to Chinese hamster V79 cells and Salmonella typhimurium. Can ferent eicosanoids may exert opposing influences on tumor cer Res., 43: 5194-5199, 1983. growth and metastasis (74) may serve to explain some of the 23. Cohen, S. M., Arai, M., Jacobs, J. B., and Friedell, G. H. Promoting effect of saccharin and DL-tryptophan in urinary bladder carcinogenesis. Cancer varied reports (75). Res., 39: 1207-1217, 1979. 24. Hicks, R. M., Wakefield, J. St. J., and Chowaniec, J. Evaluation of a new model to detect carcinogens and co-carcinogens: results obtained with sac ACKNOWLEDGMENTS charin, cyclamate and cyclophosphamide. Chem.-Biol. Interact., 11: 225- 233, 1975. The authors appreciate the expert technical assistance of Mark 25. Nakanishi, K., Hirose, M., Ogiso, T., Hasegawa, R., Arai, M., and Ito, N. 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Abraham Danon, Terry V. Zenser, David L. Thomasson, et al.
Cancer Res 1986;46:5676-5681.
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