Antiproliferative Effects of Gastrin Receptor Antagonists and Antibodies to Gastrin on Human Colon Carcinoma Cell Lines1

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[CANCER RESEARCH 48, 7179-7183, December 15, 1988| Antiproliferative Effects of Gastrin Receptor Antagonists and Antibodies to Gastrin on Human Colon Carcinoma Cell Lines1

Naseema M. Hoosein, Peter A. Kiener, Robert C. Curry, Lucio C. Rovati, Donnie K. McGilbra, and Michael G. Brattain Bristol-Baylor Laboratory, Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030 [N. M. H., D. K. M., M. G. B.J; Pharmaceutical Research and Development Division, Bristol-Myers Company, Wallingford, Connecticut 06492 [P. A. K., R. C. C., M. G. B.J; and Rotta Research Laboratorium, 20050 S. Fruttuoso Manza, Milano, Italy [L. C. R,]

ABSTRACT vivo of MC-26 mouse colon cancer could be due to a direct effect on the tumor cells or due to an indirect effect via the The gastrointestinal hormone gastrin has been shown to stimulate the tumor host. In order to ascertain this, it is necessary to examine growth of normal colonie mucosa. To examine for a possible role of the effect of proglumide on colon carcinoma cells in culture. gastrin in the proliferation of cultured colon tumor cells, we have studied the effects of two gastrin receptor antagonists, proglumide and benzotript, Also, to assess the potential use of gastrin antagonists in colon and of antibodies to gastrin. We find that proglumide (50% effective cancer treatment, their effects on more colon tumor cells, concentration, 2 to 5 DIM)and benzotript (50% effective concentration, preferably of human origin, need to be examined. 0.4 to 0.8 HIM)inhibit the monolayer growth of six human colon cancer We have previously established a large bank of human colon cell lines. Addition of exogenous gastrin abrogated the growth-inhibitory carcinoma cell lines which reflects the high degree of hetero effect of proglumide. The anchorage-independent growth of colon carci geneity observed in patients with this disease, thereby providing noma cells was also inhibited by the two gastrin antagonists. Also, a a good model system for the evaluation of therapeutic agents dose-dependent increase in carcinoembryonic antigen secretion was ob (19, 20). The colon carcinoma cell lines in this bank have been served upon treatment with proglumide and benzotript in three cell lines divided into three groups based on characterizations such as examined. Half-maximal inhibition of labeled gastrin binding was ob tumorigenicity in athymic mice, ability to grow with anchorage served at concentrations of 0.4 mM benzotript and 8.6 HIMproglumide. In addition, antipasti-in antiserum added to HCT 116 cells adapted to independence, growth rate in monolayer culture, formation of growth in serum-free medium resulted in a concentration-dependent transport domes, and secretion of carcinoembryonic antigen inhibition of cellular proliferation. These data suggest that gastrin may (19-21). In this study we have used several colon cancer cell function as an autocrine growth factor in colon carcinoma. lines to examine effects of the gastrin antagonists, proglumide and benzotript, on the growth of colon tumor cells. We have also tested the effect of antigastrin antiserum on cellular prolif INTRODUCTION eration. Our results described below show that the two gastrin Gastrin is a gastrointestinal peptide (1, 2) that has been antagonists as well as antigastrin antiserum have strong unti reported to have trophic effects on the mucosa of the digestive proliferative effects, suggesting an autocrine growth-stimula tract (3, 4). A pleiotypic response including stimulation of tory role for gastrin in these cells. RNA, protein, and DNA synthesis has been shown to occur in response to gastrin in mammalian gastric, duodenal, as well as MATERIALS AND METHODS colonie mucosa (3-5). In addition, gastrin promotes growth of Proglumide and benzotript were from Rotta Research Laboratorium, the exocrine pancreas (6). Proglumide, a derivative of glutar- Monza (Milano), Italy. Pentagastrin and nonsulfated CCK-8 were amic acid (7, 8), inhibits gastrin-stimulated acid secretion (7) purchased from Peninsula Laboratories (Belmont, CA). and the trophic effect of gastrin on normal gut mucosa (9). Cell Culture. The HCT 116, RKO, MOSER, JVC, FET, and CBS Benzotript, a tryptophan derivative, has been shown to have colon cancer cell lines were established in vitro from separate human actions similar to those of proglumide (10, 11). Gastrin, CCK,2 tumor specimens as previously described (19). Working cultures of the lines were maintained at 37 Â°Cina humidified atmosphere of 5% COz and the amphibian skin peptide caerulein are members of a in McCoy's Medium 5A (Flow Laboratories) supplemented with 10% family of peptides that share a common COOH-terminal pen- FBS and antibiotics (streptomycin-penicillin). Cells grown in the ab tapeptide amide (1) and exert their effects on a particular target sence of serum were cultured in McCoy's Medium 5A containing twice tissue by interacting with the same class of receptors (10). The the normal concentrations of sodium pyruvate, vitamins, and amino antagonistic effects of proglumide and benzotript are thought acids. Also included in the serum-free medium were transferrin (4 ÃŸg/ to be due to their ability to inhibit binding of these peptides to ml), insulin (20 /in/ml), epidermal growth factor (10 ng/ml), sodium their cell surface receptors (9, 10, 12). selenite (1 x 10~8M), hydrocortisone (2 Ãcholecystokinin; CEA, carcinoembryonic medium with or without 5 mM proglumide. Starting 2 days after plating, antigen; FBS, fetal bovine serum; IC5o, half-maximal concentration; EC50, 50% cells were counted using a hemocytometer at 24-h intervals for the next effective concentration. 5 days. 7179

Soft agarose assays were performed as described previously (22). O HCT 116 A HOSEH O FET HCT 116-10% FBS cells were plated at 3500 cells/well with proglumide â€¢¿RKO A JVC "CBS and benzotript at the indicated concentrations. Cells were stained with (5% FBS) a vital dye after 9 days, and colonies containing greater than 20 to 25 cells were scored with the aid of an inverted microscope. CEA Assay. A solid-phase direct binding radioimmunoassay was used to determine relative CEA content in serum-free conditioned medium. The procedure used was essentially similar to one described previously for fibronectin radioimmunoassay (22) with the following adaptations: 50 /il of conditioned medium were assayed; and the pri mary antiserum was antihuman CEA antiserum (Dako Corporation, Santa Barbara, CA) at a 1:1000 dilution. Relative CEA increases observed using the above radioimmunoassay procedure were similar to those obtained using a monoclonal enzyme-linked immunoassay for 123456789 10 0.4 0.8 1.2 1.0 2.0 " 3.2 CEA (Abbott Laboratories, N. Chicago, IL). [PROGLUMIDE], mM [BENZOTRIPT], mM Gastrin Labeling. [Leu'5]gastrini7 (Bachern) was labeled using Enzy- Fig. 1. Antiproliferative effects of proglumide and benzotript on six human mobeads (Bio-Rad) according to the manufacturer's instructions. La colon cancer cell lines growing in monolayer cultures in serum-supplemented beled peptide was purified by gel filtration on a Sephadex G-10 column growth medium. Mean values from three separate assays are shown. Variation in followed by adsorption to a C-18 Sep-Pak (Waters). The bound peptide cell counts was between 7 and 12%. was eluted with wateracetonitrile (33:67) and evaporated to dryness on a speed vac (Savant). The labeled gastrin was dissolved in the sterile binding buffer, divided into aliquots, and stored frozen until used. The OHCT 116 AMOSER DCBS specific activity of the peptide prepared this way was 3.8 x IO7 to 1.1 (SERUM FREE) x 10*cpm/Mg. Binding Assay. Cells were trypsinized, plated out at 4 x IO7cells per 175-cm flask, 24 h prior to initiation of the assay. One h before the assay the cells were harvested by scraping, dispersed by passage through a pipet, and then assessed for viability (greater than 85% by trypan blue exclusion). The cells were sedimented, 200 x g for 10 min. and then resuspended in sterile binding buffer (RPMI containing 5 mg/ml of bovine serum albumin and 20 mM 4-(2-hydroxyethyl)-l-piperazineeth- anesulfonic acid, pH 7.4) at about 5 x IO6cells/ml. For the competition binding assay tubes were set up in triplicate, containing 0.1 ng of '"!- gastrin, 5 x IO5cells, and various concentration of ligands, at a final volume of 220 /il. The samples were incubated for l h at 25Â°C,200 M' were then removed and layered over 750 p\ of phthalates (dibu- 34567 0.6 0.8 1.2 tyhdioctyl, 1:1) in 1.5-ml Eppendorf tubes at 4Â°C,and then samples [PROGLUMIDE],mM [BENZOTRIPT], mM were centrifuged for 1 min at 12,000 x g. Following this the tubes were Fig. 2. Antiproliferative effects of proglumide and benzotript on three human immediately frozen in dry ice and then transferred to liquid nitrogen. colon cancer cell lines adapted to grow in defined, serum-free medium. Mean values from three assays are given. Variation in cell counts was 10%. The cell pellets were obtained by cutting of tips off the tubes, still frozen, and then counted in an 1KB gamma counter. Nonspecific binding determined in the presence of 225 /IM [Leu"]gastrinn was Group I cell line HCT 116 in the presence and absence of 5 about 20% of total binding. mM proglumide indicated that the doubling time of untreated In preliminary experiments, gastrin binding affinity obtained with cells was 22.6 h, and this was increased to 33.6 h in cells treated monolayers of HCT 116 cells attached to the substratum was of the with 5 HIM proglumide. Removal of cells from proglumide- same order as that obtained with the method using cells in suspension containing growth medium after 4 and 5 days of treatment described above. resulted in acceleration of the rate of cellular proliferation to Antigastrin Antiserum Preparation. Antibody to gastrinn was raised in rabbits by immunization with a gastrin-rabbit albumin conjugate. control untreated levels (data not shown), indicating a reversal The polyclonal serum was purified by chromatography on a Protein A- of the antiproliferative effect. agarose column, dialyzed against phosphate-buffered saline, and filter Growth in soft agarose of HCT 116 cells was also inhibited sterilized prior to use. The protein concentration of the affinity-purified in a dose-dependent manner by both proglumide and benzotript antigastrin antibody was 1 mg/ml. Normal rabbit IgG (Sigma) (1 mg/ (Fig. 3). There were 81%, 50%, and 33% reductions in colony ml) was used as control. formation at 10 HIM, 5 mM, and 2.5 HIM proglumide, respec tively, compared to untreated HCT 116 cells. With benzotript RESULTS (Fig. 3, bottom row) the reductions in number of colonies were 92.2%, 50%, and 18% at concentrations of 1 mM, 0.5 mM, and Inhibition of Proliferation. The effects of proglumide and 0.25 mM, respectively, compared to untreated cells. Thus, near- benzotript on the growth of six human colon carcinoma cell maximal inhibition in anchorage-independent growth is ob lines in monolayer culture are shown in Fig. 1. Both compounds served at a concentration of 10 mM for proglumide and at a 10- inhibited growth in a dose-dependent manner. Half-maximal fold lower concentration of 1 mM benzotript. inhibition of growth was observed between 2 and 5 mM pro The antiproliferative effects of proglumide on HCT 116 cells glumide and between 0.4 and 0.8 mM benzotript. Thus, benzo (serum free) growing in monolayer cultures were blocked by tript was the more potent growth-inhibitory agent. exogenously added gastrinn (Fig. 4). In the presence of 50 /Â¿M Similar results (Fig. 2) were obtained when colon carcinoma exogenous gastrin, no effect of 2 mM proglumide could be cells adapted to growth in serum-free, defined medium were observed. treated with proglumide and benzotript. Thus, the presence of A polyclonal antigastrin antibody also inhibited the growth serum in the growth medium did not affect the response ob of the poorly differentiated HCT 116 cells (serum free) in a served. Growth curves obtained with the poorly differentiated dose-dependent fashion (Fig. 5), whereas nonimmune rabbit 7180

Fig. 3. Anchorage-independent growth of HCT 116 cells in the presence of proglumide (lop) and benzotript (bottom). Top, control cells (a) and cells treated with 2.5 m\i (/>), 5 mM (c), and 10 mM (d) proglumide. Bottom, control cells (a) and cells treated with 0.5 mM (b), 1 HIM (c), and 2 mM (

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Fig. 5. Antiproliferative effects of antigastrin antiserum. HCT 116 (serum free) cells were plated at 10,000 cells/well in quadruplicate wells (24-well plates). Ã¯^5Ã¶gÃ¬Ã®I Ã¯Ã® After 24 h cells were treated with antisera as indicated. Cells were enumerated IO; fe O IO IO Q IO o5 1=) nc Â« Ã¶ Â» after 4 days. Columns, mean; bars, SD (n = 4). Assays were repeated 3 more ~H55'0 times with similar results. g +2mM PROGLUMIDE displayed a 52-fold and 110-fold increase after exposure to 10

Fig. 4. Abrogation of the antiproliferative effect of proglumide by exogenous mM proglumide and 1.6 mM benzotript, respectively. In the gastrini7. HCT 116 cells were plated at 10,000 cells/well in quadriplicate wells poorly differentiated HCT 116 cells, CEA secretion was en (24-well plates). After 24 h cells were treated as shown. Five days later cells were hanced 27-fold and 48-fold by 10 mM proglumide and 1.6 mM counted using a hemocytometer. Columns, mean: bars, SD (n = 4). Similar results were obtained with two additional experiments. benzotript, respectively. Somewhat lower increases of 5-fold (10 mM proglumide) and 43-fold (1.6 mM benzotript) were observed with the moderately well-differentiated MOSER cell IgG had no significant effect on proliferation. Preincubation of line. the antibody with gastrinn abolished its inhibitory activity. Inhibition of Radiolabeled Gastrin Binding. In competition Dose-dependent inhibition by antigastrin antibodies was also studies proglumide and benzotript inhibited the binding of I25I- observed with the well-differentiated CBS (serum free) cells [Leu15]gastrinn to HCT 116 colon carcinoma cells in a dose- (data not shown). dependent manner (Fig. 7). The half-maximal concentration of Elevation of Carcinoembryonic Antigen Secretion. Higher proglumide (IC5o= 8.6 mM) required for inhibition of gastrin expression of the glycoprotein CEA has been shown to correlate binding to HCT 116 cells was higher than that of benzotript with better differentiated colorectal tumor cells (20, 23-26). (ICso = 0.4 HIM). For both antagonists the half-maximal con We therefore determined CEA levels in the conditioned me centrations required for inhibiting growth (proglumide, EC5o = dium of a representative cell line from each of the 3 groups of 2 to 5 mM; benzotript, EC50 = 0.4 to 0.8 mM; Fig. 1) are close colon carcinoma cells. Results are shown in Fig. 6. In all three to those required for competing with radiolabeled gastrin bind cell lines examined a concentration-dependent elevation in CEA ing. Benzotript and proglumide also inhibited gastrin binding secretion was observed. Basal levels of CEA secretion were the to MOSER cells with IC50sof 0.4 mM and 5.9 mM, respectively highest in the well-differentiated CBS cells (Fig. 6) which (not shown). Half-maximal inhibition of I25l-labeled [Leu15]- 7181

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Fig. 6. Increase in conditioned medium CEA levels upon treatment with Fig. 7. Inhibition of '"I-|Leu'!)gastrin,7 binding by [Leu"]gastrinn, nonsul proglumide and benzotript. HCT 116 cells (1 x IO4cells/well), MOSER (1 x 10" fated cholecystokinin8. and pentagastrin (bottom) by proglumide and benzotript cells/well), and CBS (2 x IO4 cells/well) that had been adapted to growth and (lupi Binding assays were repeated at least twice with similar results. A repre continuously maintained in serum-free medium were plated in triplicate 35-mm sentative assay is shown. wells. Two days later cells were treated with proglumide and benzotript at the indicated concentrations. Five days later conditioned medium was collected, and cells were counted by a hemocytometer. Relative CEA levels in the conditioned medium were determined by a solid-phase radioimmunoassay. Points, mean; tmr\. (14). Abnormal expression and/or response to gastrin by colon SD (n = 3). An additional assay gave similar fold increases in CEA levels in the carcinoma therefore needs further examination. proglumide- and benzotript-treated cells. In receptor studies with radiolabeled gastrin, benzotript dis played an approximately 10-fold higher binding affinity to the gastrinn binding to HCT 116 cells was observed at 1.6 /IM for gastrin binding site compared to proglumide (Fig. 7). This [Leu'5]gastririi7, at 13.2 Â¡IMfor nonsulfated cholecystokining, correlates well with the greater potency of benzotript in inhib and 100 J/M for pentagastrin (Fig. 7). The antiproliferative iting growth of HCT 116 cells (Figs. 1 to 3) as well as the other effects of 2 mM proglumide were blocked by gastrinp partially cell lines examined (Figs. 1 and 2). The order of potency at 0.5 fiM and 5 UMand completely at a concentration of 50 n\t obtained with the gastrin agonists [Leu'5]gastrini7 > cholecys- (Fig. 4). This correlates well with micromolar [Leul5]gastrini7 tokinin > pentagastrin (Fig. 7) concurs with that described for binding affinity. normal rat gastric mucosa! membranes (33). However, in con trast to micromolar binding affinity (ICso =1.6 Ã•/M)ofgastrinn DISCUSSION to colon tumor cells, gastrin receptors in normal gastrointes tinal tissue have high nanomolar affinity for binding gastrin Antiproliferative effects of gastrin antagonists on colon car (33-35). In this regard, there is some evidence that the gastrin cinoma cells growing in defined, serum-free growth medium receptor may exist in two conformational states with different (Fig. 2) suggest that gastrin or a gastrin-like peptide which may binding affinities (36, 37). In agreement with our data Wein be synthesized and secreted by these cells could function as an stock and Baldwin (36) have recently reported the presence of autocrine growth factor. The role of gastrin as an autocrine low affinity (KA0.2 to 3.1 n\t) gastrin receptors on gastric and growth factor in these cells is also suggested by the results that colonie carcinoma cell lines. antibodies to gastrin can inhibit cell proliferation (Fig. 4). As it is true for solid tumors, a high degree of heterogeneity Malignant transformation of cells has been linked to aberrant is observed in colon tumors (20, 38, 39). Subpopulations of production and/or response to growth-modulating polypeptides many tumor cells (38) including those from colon carcinoma (27-29). Immunohistochemical studies have demonstrated the (39) differ in their susceptibilities to various antineoplastic presence of gastrin in human antral mucosa (30). Gastrin-like agents, providing an obstacle to effective therapy. However, we immunoreactivity is also found in the proximal small intestine, find that colon carcinoma cell lines of heterogeneous pheno- but not in the oxyntic mucosa, ileum, or colon (31). Thus, it is types examined in this study displayed nearly equal sensitivities possible that ectopie production of gastrin in the colon as to the growth-inhibitory effects of the two gastrin antagonists described for human chorionic gonadotropin (32) is involved in (Figs. 1 and 2). Also, CEA levels, often used as a marker for the pathogenesis of colon cancer. Also, hypergastrinemia in induction of differentiation in cultured colorectal tumor cells rats, induced by antral exclusion or by administration of pen (23-25), were increased in the poorly, moderately, and well- tagastrin, is known to promote chemically induced colon cancer differentiated colon carcinoma cells examined. 7182

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Naseema M. Hoosein, Peter A. Kiener, Robert C. Curry, et al.

Cancer Res 1988;48:7179-7183.

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