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Effects of Bombesin Antagonists on the Growth of Small Cell Lung Cancer Cells in Vitro Judith E

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Effects of Bombesin Antagonists on the Growth of Small Cell Lung Cancer Cells in Vitro Judith E [CANCER RESEARCH 48,4783-4789, September 1, 1988! Effects of Bombesin Antagonists on the Growth of Small Cell Lung Cancer Cells in Vitro Judith E. I.avion,' Denis B. Scanion,2 Claire Soveny, and George Morstyn Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch, Victoria, Australia, 3050 [J. E. L., D. B. S., G. M.J; and Prince Henry's Hospital, Melbourne, Australia 3000 ¡C.SJ ABSTRACT stimulated growth may be useful for SCLC treatment (8). However, therapy with monoclonal antibodies would need to Small cell lung cancer (SCLC) produces several neuroendocrine pep- tides, including gastrin-releasing peptide (GRP), the mammalian equiv be prolonged and potential problems include the development of neutralizing antibodies, serum sickness, and other hypersen- alent of bombesin. There is some evidence to support the suggestion that GRP is an autocrine regulator of SCLC growth. Therefore, we have sitivity reactions, and poor penetration of the antibodies within tested the effect of bombesin and two antagonists of bombesin on SCLC the tumor. An alternative to anti-GRP antibodies for the treat cell growth in a serum-free liquid tissue culture system. The antagonists ment of SCLC might be GRP antagonists. Several analogues used were analogues of substance P: spantide and (D-Arg1, D-Pro2, D- of substance P have been shown to inhibit the action of bom Trp '', Leu") substance P. The cell lines used in this study all produced besin in various in vitro biological assays (12-15). The present GRP-related peptides and one line had demonstrable GRP receptors. study examines the effect of GRP antagonists on SCLC cell Exogenous bombesin did not cause any stimulation of growth in the liquid growth. The two antagonists used were spantide, which inhibits culture assay. The bombesin antagonists inhibited SCLC cell growth, but binding of bombesin to brain slices (16), and DAPTL-SP, which apparently not via the bombesin receptor. The bombesin used was bio inhibits bombesin-induced proliferation of Swiss 3T3 cells (13, logically active because it stimulated the proliferation of Swiss 3T3 fibroblasts. The antagonists caused inhibition of this bombesin-induced 15) and amylase secretion from pancreatic acinar cells (12). proliferation, which was reversed by addition of excess bombesin. In The biological activities of the peptides used were confirmed by addition, the antagonists and substance P alone stimulated proliferation testing their activity on Swiss 3T3 fibroblasts. of 3T3 cells, indicating that they may interact with another growth factor receptor on 3T3 cells. We conclude that growth of SCLC cells is not dependent on bombesin under all in vitro culture conditions because MATERIALS AND METHODS bombesin failed to stimulate growth in liquid cultures and the growth Cell Lines. Murine Swiss 3T3 fibroblasts were maintained by twice inhibition caused by bombesin antagonists was probably not mediated by weekly passage in DME containing 10% PCS, and antibiotics. Cultures the bombesin receptor. were incubated in 10% CC«2inair. The establishment and characteristics of the SCLC cell lines used in INTRODUCTION this study have been described previously (17) with the exception of SCLC3 comprises 20% of lung cancers. Although mixed LICM 222 which is a recently established line. LICM 222 was estab lished from a bone marrow biopsy from a female patient at relapse. tumors with adeno and squamous elements occur, SCLC ap Culture supernatant was positive for neuron-specific enolase. All the pears to be a distinct entity. SCLC cells exhibit many neuroen cell lines appear to be of the classic type as defined by Carney et al. docrine characteristics, including the presence of neuron-spe (18). Cell lines were maintained in HITES medium (19) containing cific enolase and dense-core granules, as well as the secretion 2.5% PCS, L-glutamine and antibiotics, with weekly passaging, and of a variety of peptide hormones including ACTH, calcitonin, were incubated in 5% CO: in air. Powdered media and PCS were obtained from Flow Laboratories, North Ryde, Australia. Both cell vasopressin, lipotropin, parathormone, glucagon, somatostatin, types were grown in 75-cm2 tissue culture flasks (Nunc, Denmark). and neurotensin (1-3). Most SCLC cell lines of the classic type Peptides. Peptides were synthesized on an Applied Biosystems (Fos produce GRP, the mammalian equivalent of bombesin (2-7). ter City, ÇA)430A peptide synthesizer and were purified by high- This has led to the suggestion that SCLC is an autocrine tumor, performance liquid chromatography. The peptides were characterized dependent on GRP for growth (8). The evidence supporting and analyzed quantitatively by amino acid analysis. The amino acid this hypothesis is: most SCLC cell lines produce GRP; about sequences of the peptides that were synthesized are given in Table 1. 20% of lines tested bind detectable GRP/bombesin (9); some Small quantities of peptides were purchased from Peninsula Labora SCLC cell lines have been reported to respond to bombesin in tories (Belmont, CA) and their activity was compared with that of the vitro (8, 10, 11); and a monoclonal antibombesin antibody peptides we synthesized. In all cases the activities were similar. The inhibits the growth of some SCLC cell lines in vitro and in nude GRP used in this study was purchased from Peninsula. Peptides were mice (8). dissolved and diluted in normal saline containing 4 HIMHC1 and 0.1% If SCLC is dependent on GRP, then inhibition of GRP- BSA (purified from plasma). Both the 3T3 cells and SCLC cells gave no response to the diluent. Received 7/28/87; revised 12/29/87, 5/12/88; accepted 6/1/88. Assays for Bombesin Production. SCLC cell lines were cultured at The costs of publication of this article were defrayed in part by the payment approximately 106/ml and the culture supernatant was collected after of page charges. This article must therefore be hereby marked advertisement in 4 days for assay. Cell extracts were prepared from 0.7 to 9 x IO7cells accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' To whom requests for reprints should be addressed, at Ludwig Institute for as described by Cuttitta et al. (8). Cell extracts and supernatants were Cancer Research, Melbourne Tumor Biology Branch, Royal Melbourne Hospital, assayed for bombesin-like immunoreactivity by a radioimmunoassay Victoria 3050, Australia. described previously (20). In some cases, culture supernatants were 2 Present address: Auspep, Victorian College of Pharmacy, Parkville. Australia, concentrated by lyophilization and reconstitution in a reduced volume 3052. 'The Abbreviations used are: SCLC, small cell lung cancer; GRP, gastrin- prior to assay. Several dilutions of each sample were tested to confirm releasing peptide; DAPTL-SP, (D-Arg1, D-Pro2, D-Trp7'', Leu") substance P; that the assay was linear for the supernatants and extracts. PCS, fetal calf serum; HITES medium. RPMI 1640 medium containing insulin Proliferation Assays. Swiss 3T3 cells were seeded into 96-well micro- (5 Mg/ml), transferrin (10 Mg/ml), hydrocortisone (10 nM), estradiol (10 nM), and liter plates (Nunc) at IO4 cells/well. Cultures were used after 6 or 7 sodium selenite (30 nM); BSA, bovine serum albumin; ACTH, adrenocorticotropic hormone, DME, Dulbecco's modified Eagle's medium; PBS. phosphate buffered days incubation when they were confluent and relatively quiescent. The saline; ICM, level producing 50% inhibition; BN, bombesin; ITS, insulin, trans cells were washed twice with DME without FCS and responses were ferrin, and sodium selenite in the same ratio as in HITES medium. tested in the same medium. Peptides were added at culture initiation 4783 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1988 American Association for Cancer Research. BOMBESIN ANTAGONISTS ON GROWTH OF SMALL CELL LUNG CANCER Table 1 Amino acid sequences of peptides PeptideBombesinGRP.4-27Substance sequence"pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NHzacid Met-Tyr-Pro-Arg-Gly-Asn-His-Trp-AIa-Val-Gly-His-Uu-Met-NH2 P Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NHi Spantide ARG-Pro-Lys-Pro-Gln-Gln-TRP-Phe-TRP-Lcu-Leu-NH2 (DAPTL)-SPAmino ARGTRO-Lys-Pro-Gln-Gln-TRP-Phe-TRP-Leu-tiu-NHj " D-amino acids are in capital letters. Amino acid substitutions in the substance P analogues are underlined. and DNA synthesis was measured by pulsing the cultures with [3H- not an antagonist (12, 16, 36). Fig. 1 shows that 3T3 cells /nefAy/lthymidine (0.5 ¿iCi/well;specific activity 5 Ci/mmol, Amer- responded to bombesin by increased [3H]thymidine uptake, with sham. Inc., UK) for 4-6 h after 20-24 h incubation. Cells were harvested a half-maximal response occurring at 2 nM bombesin. The by trypsinizing, then using a Skatron cell harvester (Skatron, Norway). response of 3T3 cells to substance P and the antagonists in the In several experiments this procedure was compared with TCA precip absence of bombesin is shown in Fig. 2 (top). It was found that itation and the results were similar. A control group in which the response to 10% PCS was measured was included in every experiment. the antagonists at greater than 25 nM and substance P at greater Because the incorporation of [3H-m?rA>7]thymidine in DM E alone was than l ¿/Mstimulated DNA synthesis. The maximum response relatively high, we subcloned our 3T3 cells and obtained a clone with to substance P was similar in magnitude to the maximum 10-fold lower background [3H-mefA/y/]thymidine incorporation, but response to bombesin, whereas the response to the antagonists this clone gave similar responses to peptides to the uncloned line. All was always lower. Fig. 2 (bottom) shows the effects of the experiments reported here were performed on the uncloned cells. antagonists and substance P on the response to a slightly SCLC cells were used 2 to 4 days after passage.
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