(CANCER RESEARCH 48, 1439-1441, March 15, 1988] Effects of Bombesin on Growth of Human Small Cell Lung Carcinoma in Vivo1 Robert W. Alexander, James R. Upp, Jr., Graeme J. Poston,2 Vicram Gupta, Courtney M. Townsend, Jr.,3 and James C. Thompson Department of Surgery [R. W. A., J. R. V., G. J. P., C. M. T., J. C. T.] and Department of Internal Medicine [V. G.J, The University of Texas Medical Branch, Galveston, Texas 77550 ABSTRACT Bombesin-like peptides are found in many different human tumors and are thought to function as an autocrine growth factor for small cell lung cancer in humans. In this study, a human small cell lung carcinoma (NCIH69) was s.c. implanted bilaterally into the flanks of 12 nude mice. The mice were randomized and divided into two groups and given either bombesin (20 Mg/kg) or saline i.p. 3 times a day. Tumor areas were measured twice weekly for 6 wk. At sacrifice, the tumors and normal pancreas were excised, weighed, and assayed for DNA, KN'A,and protein content. Significant stimulation of rumor growth was observed at weeks 4, 5, and 6. Tumor weight at sacrifice was significantly elevated (77%) above the control, as was DNA content (78%). Bombesin significantly increased the weight (42%), DNA (48%), and protein (61%) contents of the normal mouse pancreas. We conclude that bombesin may act as an autocrine growth factor, or indirectly through the release of other growth factors, on human small cell lung carcinoma. INTRODUCTION It is estimated that lung cancer will cause approximately 136,000 deaths in the United States in 1987 (1). SCLC4 or oat léñate (30 HM)], HITES supplemented with bombesin or argi nine vasopressin, as well as in serum-supplemented media, suggesting that SCLC secrete an autocrine growth factor (15). Cuttitta and colleagues (16) developed a monoclonal antibody against a synthetic analogue of amphibian bombesin. This antibody inhibited the growth of a xenografted SCLC (NCIN592) in nude mice and inhibited cloning of SCLC cell lines in soft agarose in vitro. Analysis of membrane preparations of both rat brain and SCLC revealed the antibody blocked a specific bombesin receptor. Specific receptors for bombesin have been demonstrated on the Swiss 3T3 fibroblasts (17) as well as in the rat brain (18), guinea pig pancreatic acini (19), rat pituitary (20), and human small cell tumors and cell lines (5, 16, 21). The purpose of our study was to determine whether exoge nous administration of bombesin would affect the growth of a human SCLC growing in nude mice. MATERIALS AND METHODS cell carcinoma accounts for approximately 20% of the classified lung tumors (2). Many SCLC have been found to contain bombesin-like immunoreactivity (3-10). Bombesin is a 14amino acid peptide initially isolated from two European frogs, Bombina bombino and Bombina veriegata veriegata (10). Bom besin-like immunoreactivity is not specific for SCLC; other tumors of neuroendocrine (bronchial and gastrointestinal carcinoids) and nonneuroendocrine origin (squamous, large cell, and adenocarcinoma of the lung) have been found to contain bombesin-like immunoreactivity (7-10). However, bombesinlike immunoreactivity has been found in elevated concentra tions in SCLC when compared with other tumors (3-6, 9). Bombesin stimulates in vitro proliferation of 3T3 mouse fibroblasts (11) and of epithelial cells from the normal human bronchus (12). GRP, the mammalian counterpart of bombesin, has recently been shown to serve as a mitogen for SCLC cell lines in vitro (13). The carboxyterminal fragment (GRPI4~27), Animals. Male athymic nude BALB/c mice (21-24 g) were purchased from Life Sciences, St. Petersburg, FL. The mice were housed in a specific pathogen-free, temperature-controlled isolation compound and were exposed to a light-controled day (lights on, 0700-1900 h). Diet was composed of standard chow (Autoclavable Rodent Chow No. 5010; Ralston Purina, St. Louis, MO) and water given ad libitum. Tumor. A human SCLC (NCI-H69) (American Type Culture Collec tion, Rockville, MD), which contains bombesin-like immunoreactivity (3, 4), was xenografted as 2-mm2 pieces s.c. through an interscapular which exhibits significant homology to bombesin, appears to be the active site, since GRP1"16 does not act as a mitogen (13). Drugs. Mice with implanted tumors were randomized and divided into two groups, each receiving 0.1 ml i.p. injections 3 times per day. The control mice received saline with 0.1% bovine serum albumin (Calbiochem, La Jolla, CA), and the mice in the treatment group received bombesin tetradecapeptide in 0.1% bovine serum albumin (20 Mg/kg) (Sigma Chemical Co., St. Louis, MO). Biochemical Assays. Tissues were extracted for measurement of DNA, RNA, and protein (22). DNA was measured by the Burton modification of the diphenylamine procedure, with calf thymus DNA as a standard (23). RNA was measured using the orcinol procedure and yeast RNA as a standard (24). Protein was determined by the method of Lowry and colleagues (25). Statistical Analysis. Statistical significance was determined by the Kruskal-Wallis analysis of variance. A value of P< 0.05 was considered to be significant. Bombesin, when added to cultures of SCLC, stimulates colony formation in soft agarose (14). Media taken from established cultures of SCLC stimulated the soft agarose cloning of SCLC in serum-free HITES medium [RPMI Medium 1640 supple mented with hydrocortisone (10 nM), bovine insulin (5 fig/ml), transferrin (10 ^g/rnl), 17/î-estradiol(10 nM), and sodium seReceived 8/31/87; revised 11/30/87; accepted 12/11/87. 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 accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by grants from N1H (ROÕDK 15241, POI DK 35608, and RCDA CA 00854), from the American Cancer Society (PDT-220), and from the National Cancer Institute (CA 17701). 'Visiting Scientist from the Department of Surgery, Royal Postgraduate Medical School, University of London, London, United Kingdom. Supported by the Wellcome Foundation, Ethicon Foundation, and British Digestive Founda tion. 3 To whom requests for reprints should be addressed. 4 The abbreviations used are: SCLC, small cell lung cancers; GRP, gastrinreleasing peptide. incision bilaterally into the flanks (two tumors per mouse) of 12 anesthetized mice. Mice were weighed weekly, tumors were measured twice weekly with calipers, and tumor surface areas were calculated using the product of the two greatest perpendicular tumor diameters. Tumor doubling times were calculated from semilogarithmic graphs of tumor area versus day from implantation. At termination of the exper iment, mice were sacrificed by cervical dislocation followed by excision of tumors and pancreas. Tissues were frozen in liquid nitrogen and stored at —70°C until assay. RESULTS The body weights in both groups increased by approximately 87% over the 6-wk study period and were not significantly 1439 Downloaded from cancerres.aacrjournals.org on July 8, 2017. © 1988 American Association for Cancer Research. BOMBESIN AND HUMAN SMALL CELL LUNG CANCER different from each other. Tumor growth was seen in 8 of 12 tumors implanted in each group. Bombesin reduced the tumor cell doubling time by 24% from 7.5 days in the control to 5.75 days in the treated group. Bombesin produced significant stimulation of tumor growth; the areas increased from weeks 4-6 (Fig. 1). At sacrifice, both tumor weight (77%) (Fig. 2) and tumor DNA content (78%) (Fig. 3) were significantly stimulated above the control group. Tumor RNA and protein content were also increased (67 and 32%), although they were not significantly different from con trol (Table 1). Bombesin significantly increased the weight (42%), protein (61%), and DNA content (48%) of the normal mouse pancreas (Table 1). 150^120¿ :< 90K CO§\1 «0¡ 30*rira lu1234WEEKS.Li*I_ä8iÈ¡LU reu Fig. 1. Tumor area in relation to time from implantation. Values, mean ± SEM (n = 8 in each group; *, P < 0.05). We found that bombesin produced significant stimulation of growth of a human SCLC and the normal mouse pancreas. The tumor cell line that we studied has been reported to contain bombesin-like immunoreactivity (3, 4). The importance of our study is that, to our knowledge, this is the first demonstration that exogenous bombesin stimulates growth of human SCLC in vivo. Our findings support and extend the work of Cuttitta and colleagues (16) who suggested that bombesin-like peptides may act as autocrine growth factors in SCLC. We and others have previously shown that bombesin stimu lates growth of the normal rat pancreas (26-29). In the present study, bombesin (20 ¿tg/kgi-p- 3 times a day) produced hyperplasia of the normal mouse pancreas which was characterized by significant increases in weight, DNA, and protein content. The mechanism by which exogenous bombesin induces growth may involve both a direct effect of bombesin and an indirect effect through bombesin-mediated release of cholecystokinin (27, 29). Although we have demonstrated stimulation of human SCLC growth In vivo, we have not used a bombesin receptor antagonist to show inhibition of bombesin stimulated growth. The syn thetic peptide, spantide [(o-Arg1, o-Pro2, o-Trp7-9, Leu") sub stance P], inhibits the in vitro effects of bombesin (17, 30, 31); however, the nonspecificity of action and lack of potency limit the use of spantide in vivo (32). We have found that bombesin does not stimulate the growth of a human colon cancer growing in nude mice,5 suggesting it may be specific for tumors with bombesin receptors. Bombesin has been implicated as a mitogen or as an autocrine growth factor for normal and malignant cells (11-17). The term "autocrine secretion" has been proposed for self-stimulation 0.6- 0.4 DISCUSSION Õ 0.2- Fig. 2. Tumor weight at sacrifice. Values, mean ±SEM (n = 8 in each group; *,P<0.05). 4-•2 n -JL_j8Iw/mi Fig. 3. Tumor DNA content. Values, mean ±SEM (n = 8 in each group; : P < 0.05). Table 1 Tumor and pancreas weight and content of DNA, RNA, and protein ±SEM TumorWeight ±0.10° ±0.03" (g) ±0.10 ±0.04 DNA (mg) 2.42 ±0.66 4.30 ±0.72° 0.69 ±0.10 1.02 ±0.04° RNA (mg) 1.41 ±0.38 2.35 ±0.42 3.20 ±0.47 3.46 ±0.28 5.7°'F< 70.5 ± Protein (mg)Control0.38 67.7 ±18.4Bombesin0.67 89.1 ±15.4PancreasControl0.25 43.8 ±6.1Bombesin0.35 whereby a cell produces endogenous factors for which they have receptors and to which they are able to respond (33). Bombesin has been found in human SCLC as well as neuroendocrine and other pulmonary tumors (3-10). Moreover, specific receptors for bombesin have been identified in SCLC tissue culture cell lines (5, 16, 21). Bombesin, when added to cultures of SCLC, stimulates colony formation in soft agarose (14). One of the most convincing studies that supports the autocrine hypothesis is that of Cuttitta and colleagues (16). They found that a monoclonal antibody, raised against a synthetic analogue of amphibian bombesin, blocked the binding of bombesin to bom besin receptors and inhibited the clonal growth of SCLC in vitro and the growth of SCLC xenografts in vivo (16). The amphibian peptide, bombesin, and the mammalian coun terpart, GRP, have striking homology, the C-terminal heptapeptides are identical. Sausville and colleagues (34) have re cently described the presence of prepro-GRP gene products in 5 human SCLC. It then appears that the bombesin-like immu noreactivity (and probably the growth-stimulatory agent) is due to GRP. The finding of bombesin-like immunoreactivity in pulmonary tumors of all types (including squamous and adenocarcinoma) may be of major significance. The presence or absence of bombesin-like immunoreactivity in the lung may be related to the developmental stage of cellular growth. Yesner (35) postu lated that all lung cancers reflect a spectrum of differentiation and the degree of ectopie hormone secretion rather than the type of secretion may be associated with stage of cellular mat uration. In Yesner's growth concept, SCLC is more primitive (35); bombesin immunoreactivity appears to diminish as cellu- 0.05. 9 Unpublished data. 1440 Downloaded from cancerres.aacrjournals.org on July 8, 2017. © 1988 American Association for Cancer Research. BOMBESIN AND HUMAN SMALL CELL LUNG CANCER lar differentiation occurs. Further evidence to support this hypothesis includes the finding of elevated levels of bombesinlike immunoreactivity in fetal or neonatal lungs compared to adult lung tissue (36) and differences in endocrine cell distri bution during varying stages of pulmonary development (37). 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Endocrine-like cells of the pulmonary epithelium of the human adult lung. Cell Tissue Res., 178: 39-48, 1977. 1441 Downloaded from cancerres.aacrjournals.org on July 8, 2017. © 1988 American Association for Cancer Research. Effects of Bombesin on Growth of Human Small Cell Lung Carcinoma in Vivo Robert W. Alexander, James R. Upp, Jr., Graeme J. Poston, et al. Cancer Res 1988;48:1439-1441. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/48/6/1439 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on July 8, 2017. © 1988 American Association for Cancer Research.