Inhibition of cell proliferation by the cholecystokinin antagonist L-364,718

EXPERIMENTAL

CELL

RESEARCH

192,

189-192

(1991)

Inhibition of Cell Proliferation by the Cholecystokinin Antagonist L-364,71 8 CASSANDRA M. THUMWOOD,' Ludwig

Institute

for

Cancer

Research,

JI HONG,ANDGRAHAMS.BALDWIN

Melbourne

Tumour

Press,

Branch,

Melbourne,

Australia

to document in vitro. In a recent publication the nonspecific gastrin and CCK antagonists, proglumide and benzotript, as well as an anti-serum to gastrin, were found to inhibit the proliferation of a number of colon carcinoma cell lines [8]. Furthermore, the same cell lines were subsequently shown to secrete a gastrin/CCK-like factor as detected by radioimmunoassay [9]. These observations supported the notion that a gastrin/CCKlike autocrine factor was involved in cell growth. To examine this hypothesis further and to establish which, if either, of these two hormones is responsible for the proliferative response, the effects of the specific gastrin receptor antagonist, L-365,260 [lo] and the CCK receptor antagonist, L-364,718 [ll], on several colonic and noncolonic cell lines were observed in vitro.

Specific cholecystokinin (CCK) receptor and gastrin receptor antagonists were used to assess what role, if any, these receptors have in autocrine cell growth. Although the cholecystokinin receptor antagonist, L-364,718, inhibited cell proliferation in a broad spectrum of cell lines, the gastrin antagonist, L-365,260, had no effect on cell proliferation. In addition neither added gastrln,, , nor sulfated cholecystokinin,, could reverse the inhibitory action of L-364,718. It is proposed that L-364,718 inhibits cell proliferation independently of classical gastrin/CCK receptors. :c lssi Academic

Biology

Inc.

INTRODUCTION METHODS

Gastrin and cholecystokinin (CCK)’ are closely related peptide hormones that share a common COOHterminal pentapeptide. Both generate a number ofphysiological responses, such as the stimulation of gastric acid release and pancreatic enzyme secretion, in gastrointestinal tissues. The actions of these peptides are initiated by binding to specific cell surface receptors, which have different affinities for gastrin and CCK (Table l), different antagonist specificities, and different tissue distributions. Three receptor types have so far been described. The pancreatic or CCK-A receptor is also found in the gallbladder and some parts of the central nervous system [l, 21, the CCK-B receptor is found in the cerebral cortex [3, 41, and the gastrin receptor is found on various gastric cells [5]. Although the trophic actions of gastrin and CCK are obvious in whole animal experiments [6, 71, convincing and reproducible stimulatory effects have been difficult

The cell lines used in these experiments were HCT 116 (American Type Culture Collection, Rockville, MD., [12]) lA1, a clone of LIM1215, and LIM1839 cells (Dr. R. H. Whitehead, Ludwig Institute for Cancer Research, Melbourne, [13, 14]), MM200 cells (Dr. P. Parsons, Queensland Institute for Medical Research, Brisbane, [15]), T47D cells (Dr. I. Keydar, The George S. Wise Center for Life Sciences, Department of Microbiology, Tel Aviv University, Ramat Aviv, [16]), H157 cells (Dr. J. D. Minna, NCI-Navy Medical Oncology Branch, Division of Cancer Treatment, National Institute and Naval Hospital, Bethesda, MD, [17]), and Okajima cells (Dr. T. Motoyama, First Department of Pathology, Faculty of Medicine, University of Niigata, [IS]). The tissue of origin and the culture conditions for these experiments are summarized in Table 2. Cells were trypsinized and plated out onto 96-well plates (Nunc, Denmark) at a concentration of 10’ to 5 X lo4 cells per well. Antagonists (gifts from Dr. V. J. Lotti, Merck Sharpe & Dohme, West Point, PA) were dissolved and diluted in DMSO and added to the cells, taking care to keep the concentrat,ion of DMSO (0.250/o) equal in all wells. When testing the effects of gastrin,, or CCK,-SO, (Research Plus, Bayonne, NJ) each was dissolved in phosphate-buffered saline and added either simultaneously or 2 h prior to the L-364,718. The cultures were then pulsed overnight with [3H]thymidine (0.5 rCi/ well, NEN; DuPont, Boston, MA), harvested, and counted using the b-Plate harvester and liquid scintillation counting system (LKBWallac, Finland). Each assay point was done in at least triplicate. Cell number experiments were performed in a similar manner except that cells were grown in 6-well plates. The antagonists were added to the wells giving the same final concentrations as in the [3H]thymidine experiments. After 4 days in culture the cells were

’ To whom correspondence and requests for reprints should be addressed at PO Royal Melbourne Hospital, Melbourne, Victoria, 3050, Australia. * Abbreviations used: G,,, gastrin,,; CCK, cholecystokinin; CCKsSO,, sulfated cholecystokinin s; DMSO, dimethyl sulfoxide; IC,,, half maximal inhibitory concentration; K,,, dissociation constant.

189

0014.4827191

Copyright All

rights

8

1991 of reproduction

$3.00

by Academic

Press, Inc.

in any

reserved.

form

190

JI, AND BALDWIN

THUMWOOD, TABLE

eration, as measured by cell number over a 4-day incubation period, was also assessed.The cell number was affected in an analogous manner to the [3H]thymidine incorporation (Fig. lb, data for MM200 cell line not shown) although the curve was shifted slightly to the right. This may be due to the difference in the end point of the two different assays. The [3H]thymidine experiments were completed over a 24-h timespan and the cell number was assessed after 4 days in culture during which time no additional antagonist was added. This may have resulted in an overall decrease in antagonist concentration due to breakdown in the cell number experiments. The gastrin receptor antagonist L-365,260, however, had no effect on [3H]thymidine uptake or increase in cell number in HCT 116 cells (Figs. la and lb), nor on 1Al or MM200 cells (data not shown). Furthermore, the inhibition of [3H]thymidine incorporation by L-364,718 could not be reversed by CCK,-SO, or gastrin,, added individually at concentrations up to 50 pM either 2 h prior to the antagonists (Table 3) or simultaneously with the antagonists (data not shown).

1

A Summary of the Receptor K, (Hormones) and the IC,, (Antagonists) for the Gastrin and CCK Binding Sites Obtained from the Literature

CCK-A Hormone gastrin,, CCKs-SO, CCK, Antagonist L-364, 718 L-365, 260 Proglumide Benzotript

CCK-B

Gastrin

560 nM”

4nM”

3 nM”

0.17 nM” 140 nM’

0.39 nMd NA

0.88 nMd NA

0.08 280 600 102

245 nMd 2.0 nM” 875 /A4 84 FM’

300 nMd 1.1 nM” 900 pM’ 59 ‘.a’

nMd nM” &f’ PtM’

Gastrini CCK-C

0.24-1.28 /.LM*; 1.6 PM” NA 220 nM*; 16 /.dW NA NA 8.6 mM” 0.4 mM’

Note. Standard deviations were omitted for simplicity. NA, not available. a Lotti and Chang (1989) Eur. J. Pharmacol. 162, 2733280. * Weinstock and Baldwin (1988) Cancer Res. 48, 932-937; value depended on cell line used. ’ Hoosein et al. (1988) Cancer Res. 48,7179-7183, in HCT 116 cells. d Chang et al. (1986) hoc. Natl. Acad. Sci. USA 83, 4923-4926. ’ Chang et al. (1985) Science 320, 177-179. e Chang et al. (1986) Mol. Pharmacol. 30, 212-217.

removed with mocytometer.

trypsin

and counted

using

0.5%

trypan

DISCUSSION

Inhibition of colon carcinoma cell proliferation by the nonselective CCK/gastrin antagonists proglumide and benzotript was observed by Hoosein et al. [8]. The concentrations at which proglumide and benzotript produced half maximal inhibition (IC,,) of proliferation of HCT 116 cells were 2-5 mM and 0.4-0.8 mM, respectively, and were in close agreement with the IC,, values determined for inhibition of ‘251-[Leu15]gastrin,, binding to the same cell line [8]. Furthermore, gastrin in the micromolar range could reverse the inhibition of cell proliferation by either antagonist [8]. A gastrin/CCK binding site with affinities in the micromolar range for both gastrin,, and CCK,-SO, had been described previously on both gastric [ 191and colonic [ 19,201 carci-

blue and a he-

RESULTS

DNA synthesis, as determined by [3H]thymidine uptake, was inhibited in a dose-dependent manner by the CCK antagonist L-364,718 in all the cell lines tested (Table 2, Fig. la). The half maximal inhibitory concentrations ranged from 2.7 to 5.3 pM depending on the cell line used. In two cell lines, HCT 116 and MM200, prolifTABLE A Summary Cell

line

HCT 116 1Al MM200 T47D H157 Okajima LIM1839

Tissue

of the Human

of origin

Media

Colon Colon Melanoma Mammary Large lung cell Gastric Gastric

McCoy’s RPM1 RPM1 RPMI” RPMI” RPMI” RPMI”

2

Cell Lines Used in These Experiments FCS

(%)

10 10 2.5 10 10 10 10

IC,,

L-364,718’ (pM f 1 SD) 3.2 4.7 4.0 5.3 4.7 3.8 2.7

f f f f ? + -t

G,, Binding’

0.2 0.4 1.4 1.1 2.3 1.1 0.9

Note. The CCK receptor antagonist L-364,718 inhibited [3H]thymidine incorporation in all lines tested with the number of gastrin binding sites reported previously [19]. NA, not available. a Supplemented with 10 FM thioglycerol, 25 U/liter insulin, and 1 mg/liter hydrocortisone. * Half maximal inhibition determined from two [3H]thymidine incorporation experiments. ’ Results from Weinstock and Baldwin (1988).

(fmol/lOe

cells)

NA NA 1.92 0 0 0.81 NA and this inhibition

did not correlate

INHIBITION

OF

CELL

PROLIFERATION

a .-: 5

BY

191

L-364,718

b 70

-

L-365.260

L-365.260

L~364.718

Concentration

of Antagonist

(PM)

Concentration

of Antagonist

(PM)

FIG. 1. Inhibition of [“Hlthymidine uptake (a) and cell proliferation after 4 days in culture (b) in HCT 116 cells by the CCK receptor antagonist L-364,718. The same concentrations of the gastrin receptor antagonist, L-365,260, did not inhibit [3H]thymidine incorporation (a) nor proliferation (b) in this cell line. Data from one representative experiment of three are shown here. Points are the mean of triplicates; bars represent 1 SD.

noma cell lines. The results of Hoosein et al. [8, 91 suggested that gastrin or a gastrin-like peptide was acting as an autocrine growth factor via a receptor with low affinity for gastrin,, and CCK,, which we have termed the gastrin/CCK-C receptor (see Table 1). In the experiments presented here it was found that the CCK receptor antagonist L-364,718, but not the gastrin receptor antagonist L-365,260, inhibitedcellproliferation. The inhibition by L-364,718 did not appear to involve either the CCK-A, CCK-B, or the gastrin receptor since the IC,, of 2-5 &4 was far higher than the IC,, values (in the nanomolar range) reported for these receptors in both conventional binding studies andphysiological assays (summarized in Table 1). Furthermore, L-364,718 did not appear to inhibit via the gastrin/

TABLE

3

Effect of 50 pM G,, or CCKs-SO, [3H]Thymidine Incorporation in HCT Receptor Antagonist, L-364,718

on the Inhibition 116 Cells by the

of CCK

Percentage of control proliferation (with no added antagonist) L-364,718

(KM)

2.5

5

no added G,, + 50 fiLM G,,

82 k 6% 8Oi 1%

63&3% 63 k 4%

no added CCK, + 50 &4 CCK,

83 f 2% 88*4%

66 +- 6% 69 f 3%

Note. Data (triplicates -+l SD, from one representative experiment of three) are expressed as a percentage of the maximal [3H]thymidine incorporation with no added antagonist. Peptide hormones were added 2 h prior to the antagonists.

CCK-C receptor, since neither gastrin,, nor CCK,-SO, were able to reverse or prevent inhibition at a concentration of 50 pM which was up to 50-100 times the dissociation constants reported for HCT 116 cells [8, 91. In addition there was no correlation between the number of gastrin binding sites and the extent of inhibition by L-364,718 [ 191, (see Table 2). For example, the proliferation of both the mammary carcinoma line T47D and the small cell lung carcinoma line H157, which had no detectable gastrin binding sites, and the melanoma cell line MM200, which bound the highest levels of gastrin of any line tested, was inhibited to an equal extent by L-364,718. Two possible explanations for these observations exist: First, L-364,718 may act through a receptor with low affinity for L-364,718 and with no detectable affinity for L-365,260. Another feature of this receptor would be its presence on a wide variety of cell types, suggesting again that the classical CCK and gastrin receptors are not involved. Second, the observed inhibition may be receptor independent. Since L-364,718 is lipid soluble it may diffuse into cells and cause a decrease in proliferation by inhibition of intracellular processes in agreement with the broad spectrum of inhibitory activity. However, a purely toxic effect of L-364,718 seems unlikely as neither the chemically closely related gastrin receptor antagonist (Fig. 1) nor the benzodiazepine parent molecule (data not shown) affected cell proliferation at comparable concentrations. The inhibitory effect of L-364,718 was also found to be reversible in wash off experiments if the antagonist was removed up to 12 h before the [3H]thymidine pulse (data not shown). Thus, while the experiments described here make it unlikely that gastrin/CCK receptors play a role in the inhibitory effect of L-364,718, further experiments are required to

192

THUMWOOD,

determine the mechanism cell proliferation.

by which

L-364,718

JI,

inhibits

AND 10.

Brattain, Brattain,

11.

Lotti, V. J., and Chang, 273-280.

12.

Chang, R. S. L., and Lotti, IJSA 83,4923-4926.

13.

Whitehead, R. H., Macrae, (1985) J. Natl. Cancer Inst.

14.

Whitehead, R. H., Novak, U., Thomas, R. d., Lukeis, R. E., Walker, F. E., and Jones, J. (1989) Int. J. Cancer 44, 110-113.

15.

Pope, J. H., Morrison, S. R. (1979) Pathology

16.

Keydar, I., Chen, L., Karby, S., Weiss, F. R., Delarea, J., Radu, M., Chaitcik, S., and Brenner, H. ,J. (1979) Eur. J. Cancer 15,

REFERENCES 1. 2. 3. 4. 5.

Jensen, R. T., Lemp, G. F., and Gardner, J. D. (1980) Proc. Nut/. Acad. Sci. USA 77, 2079-2083. Reisine, T., and Jensen, R. T. (1986) J. I’harm. Ther. 236,621l 626. Hays, S. E., Beinfeld, M. C., Jensen, Paul, S. M. (1980) Neuropeptidrs Saito, A., Sankaran, H., Goldfine, (1980) Science 208, 1155-1156.

R. T., Goodwin,

T. I. C., and

1, 53-62. I. D., and

Williams,

D. A., Thomas, J. C&n. Inuest.

6.

Johnson,

Annu.

7.

Solomon, T. E. (1981) Tract (L. R. Johnson, York.

8.

Hoosein, N. M., Kiener, P. A., Curry, R. C., Rovati, L. C., McGilbra, D. K., and Brattain, M. G. (1988) Cancer Res. 48, 71797183.

9.

L. R. (1977)

Reu. Physiol.

May 14, 1990 version received

July

39,

I,. I’., and

135-158.

17.

18.

23, 1990

R. C., and Brattain,

M. G., Fine, D. E. (1981)

W. D., Khaled, F. M., Thompson, Cancer Res. 41, 1751-1756. R. S. L. (1989)

Eur.

V. J. (1986) F. A., St John, 74, 759-765.

J., and

J. Pharmacol.

Proc.

Natl.

162,

Acad.

Sci.

D. J. B., and Ma,

L., Moss, D. J., Parsons, 11, 1912-1915.

J.

P. J., and Mary,

6S9-670.

in Physiology of the Gastrointestinal Ed.), pp. 873-892, Raven Press, New

Hoosein, N. M., Kiener, P. A., Curry, (1990) Erp. Cell Res. 186, 15-21.

Received Revised

L. P., Reedy, 73, 1434-1447.

J. A.

Soil, A. H., Amirian, Elashoff, J. D. (1984)

BALDWIN

M. G.

Carney, D. M., Gazdar, A. F., Bepler, G., Guccion, J. G., Marangos, P. J., Moody, T. W., Zweig, M. H., and Minna, ,I. D. (1985) Cancer Res. 45, 2913-2923. Motoyama, T., and Suzuki, T. (1980) Chemotherapy 28, 1422 147.

19.

Weinstock, 937.

J., and Baldwin,

20.

Singh, P., Rae-Venter, Thompson, .J. C. (1985)

G. S. (1988)

Cancer

B., Townsend, C. M., Amer. J. Physiol. 249,

Rex

48,

Khalil, T., G761lG769.

932and