Effects of Bombesin on Growth of Human Small Cell Lung Carcinoma in Vivo1

(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 lÃ©Ã±ate(30HM)],HITES supplemented with bombesin or argi nine vasopressin, as well as in serum-supplemented media, Bombesin-like peptides are found in many different human tumors and suggesting that SCLC secrete an autocrine growth factor (15). 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 (NCI- Cuttitta and colleagues (16) developed a monoclonal antibody H69) was s.c. implanted bilaterally into the flanks of 12 nude mice. The against a synthetic analogue of amphibian bombesin. This mice were randomized and divided into two groups and given either antibody inhibited the growth of a xenografted SCLC (NCI- bombesin (20 Mg/kg) or saline i.p. 3 times a day. Tumor areas were N592) in nude mice and inhibited cloning of SCLC cell lines measured twice weekly for 6 wk. At sacrifice, the tumors and normal in soft agarose in vitro. Analysis of membrane preparations of pancreas were excised, weighed, and assayed for DNA, KN'A,and protein both rat brain and SCLC revealed the antibody blocked a content. Significant stimulation of rumor growth was observed at weeks specific bombesin receptor. 4, 5, and 6. Tumor weight at sacrifice was significantly elevated (77%) Specific receptors for bombesin have been demonstrated on above the control, as was DNA content (78%). Bombesin significantly the Swiss 3T3 fibroblasts (17) as well as in the rat brain (18), increased the weight (42%), DNA (48%), and protein (61%) contents of guinea pig pancreatic acini (19), rat pituitary (20), and human the normal mouse pancreas. We conclude that bombesin may act as an small cell tumors and cell lines (5, 16, 21). autocrine growth factor, or indirectly through the release of other growth factors, on human small cell lung carcinoma. 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. INTRODUCTION It is estimated that lung cancer will cause approximately 136,000 deaths in the United States in 1987 (1). SCLC4 or oat MATERIALS AND METHODS cell carcinoma accounts for approximately 20% of the classified Animals. Male athymic nude BALB/c mice (21-24 g) were purchased lung tumors (2). Many SCLC have been found to contain from Life Sciences, St. Petersburg, FL. The mice were housed in a bombesin-like immunoreactivity (3-10). Bombesin is a 14- specific pathogen-free, temperature-controlled isolation compound and amino acid peptide initially isolated from two European frogs, were exposed to a light-controled day (lights on, 0700-1900 h). Diet Bombina bombino and Bombina veriegata veriegata (10). Bom was composed of standard chow (Autoclavable Rodent Chow No. 5010; besin-like immunoreactivity is not specific for SCLC; other Ralston Purina, St. Louis, MO) and water given ad libitum. tumors of neuroendocrine (bronchial and gastrointestinal car- Tumor. A human SCLC (NCI-H69) (American Type Culture Collec tion, Rockville, MD), which contains bombesin-like immunoreactivity cinoids) and nonneuroendocrine origin (squamous, large cell, (3, 4), was xenografted as 2-mm2 pieces s.c. through an interscapular and adenocarcinoma of the lung) have been found to contain bombesin-like immunoreactivity (7-10). However, bombesin- incision bilaterally into the flanks (two tumors per mouse) of 12 anesthetized mice. Mice were weighed weekly, tumors were measured like immunoreactivity has been found in elevated concentra twice weekly with calipers, and tumor surface areas were calculated tions in SCLC when compared with other tumors (3-6, 9). using the product of the two greatest perpendicular tumor diameters. Bombesin stimulates in vitro proliferation of 3T3 mouse Tumor doubling times were calculated from semilogarithmic graphs of fibroblasts (11) and of epithelial cells from the normal human tumor area versus day from implantation. At termination of the exper bronchus (12). GRP, the mammalian counterpart of bombesin, iment, mice were sacrificed by cervical dislocation followed by excision has recently been shown to serve as a mitogen for SCLC cell of tumors and pancreas. Tissues were frozen in liquid nitrogen and lines in vitro (13). The carboxyterminal fragment (GRPI4~27), stored at â€”70Â°Cuntilassay. which exhibits significant homology to bombesin, appears to Drugs. Mice with implanted tumors were randomized and divided be the active site, since GRP1"16 does not act as a mitogen (13). 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 Bombesin, when added to cultures of SCLC, stimulates colony (Calbiochem, La Jolla, CA), and the mice in the treatment group formation in soft agarose (14). Media taken from established received bombesin tetradecapeptide in 0.1% bovine serum albumin (20 cultures of SCLC stimulated the soft agarose cloning of SCLC Mg/kg) (Sigma Chemical Co., St. Louis, MO). in serum-free HITES medium [RPMI Medium 1640 supple Biochemical Assays. Tissues were extracted for measurement of mented with hydrocortisone (10 nM), bovine insulin (5 fig/ml), DNA, RNA, and protein (22). DNA was measured by the Burton transferrin (10 ^g/rnl), 17/Ã®-estradiol(10 nM), and sodium se- modification of the diphenylamine procedure, with calf thymus DNA as a standard (23). RNA was measured using the orcinol procedure and Received 8/31/87; revised 11/30/87; accepted 12/11/87. yeast RNA as a standard (24). Protein was determined by the method 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 of Lowry and colleagues (25). accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Statistical Analysis. Statistical significance was determined by the 1Supported by grants from N1H (ROÃ•DK 15241, POI DK 35608, and RCDA Kruskal-Wallis analysis of variance. A value of P< 0.05 was considered CA 00854), from the American Cancer Society (PDT-220), and from the National to be significant. 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 RESULTS tion. 3To whom requests for reprints should be addressed. 4 The abbreviations used are: SCLC, small cell lung cancers; GRP, gastrin- The body weights in both groups increased by approximately releasing peptide. 87% over the 6-wk study period and were not significantly 1439

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 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 DISCUSSION tumors implanted in each group. Bombesin reduced the tumor cell doubling time by 24% from We found that bombesin produced significant stimulation of 7.5 days in the control to 5.75 days in the treated group. growth of a human SCLC and the normal mouse pancreas. The Bombesin produced significant stimulation of tumor growth; tumor cell line that we studied has been reported to contain the areas increased from weeks 4-6 (Fig. 1). At sacrifice, both bombesin-like immunoreactivity (3, 4). The importance of our tumor weight (77%) (Fig. 2) and tumor DNA content (78%) study is that, to our knowledge, this is the first demonstration (Fig. 3) were significantly stimulated above the control group. that exogenous bombesin stimulates growth of human SCLC in vivo. Our findings support and extend the work of Cuttitta Tumor RNA and protein content were also increased (67 and and colleagues (16) who suggested that bombesin-like peptides 32%), although they were not significantly different from con trol (Table 1). may act as autocrine growth factors in SCLC. Bombesin significantly increased the weight (42%), protein We and others have previously shown that bombesin stimu lates growth of the normal rat pancreas (26-29). In the present (61%), and DNA content (48%) of the normal mouse pancreas study, bombesin (20 Â¿tg/kgi-p-3 times a day) produced hyper- (Table 1). plasia of the normal mouse pancreas which was characterized

150^120Â¿ 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 90K (27, 29). Although we have demonstrated stimulation of human SCLC COÂ§\1 Â«0Â¡ growth In vivo, we have not used a bombesin receptor antagonist to show inhibition of bombesin stimulated growth. The syn 30*rira thetic peptide, spantide [(o-Arg1, o-Pro2, o-Trp7-9, Leu") sub reulu1234WEEKS.Li*I_Ã¤8iÃˆÂ¡LU 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 Fig. 1. Tumor area in relation to time from implantation. Values, mean Â± does not stimulate the growth of a human colon cancer growing SEM (n = 8 in each group; *, P < 0.05). 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 0.6- "autocrine secretion" has been proposed for self-stimulation whereby a cell produces endogenous factors for which they have receptors and to which they are able to respond (33). Bombesin 0.4 Ã• has been found in human SCLC as well as neuroendocrine and other pulmonary tumors (3-10). Moreover, specific receptors 0.2- 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 Fig. 2. Tumor weight at sacrifice. Values, mean Â±SEM (n = 8 in each group; most convincing studies that supports the autocrine hypothesis *,P<0.05). 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). 4-â€¢2 The amphibian peptide, bombesin, and the mammalian coun terpart, GRP, have striking homology, the C-terminal hepta- peptides 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

n -JL_j8Iw/mi to GRP. Fig. 3. Tumor DNA content. Values, mean Â±SEM (n = 8 in each group; : The finding of bombesin-like immunoreactivity in pulmonary P < 0.05). tumors of all types (including squamous and adenocarcinoma) may be of major significance. The presence or absence of Table 1 Tumor and pancreas weight and content of DNA, RNA, and bombesin-like immunoreactivity in the lung may be related to protein Â±SEM the developmental stage of cellular growth. Yesner (35) postu TumorWeight lated that all lung cancers reflect a spectrum of differentiation and the degree of ectopie hormone secretion rather than the (g) Â±0.10 Â±0.10Â° Â±0.04 Â±0.03" type of secretion may be associated with stage of cellular mat DNA (mg) 2.42 Â±0.66 4.30 Â±0.72Â° 0.69 Â±0.10 1.02 Â±0.04Â° uration. In Yesner's growth concept, SCLC is more primitive RNA (mg) 1.41 Â±0.38 2.35 Â±0.42 3.20 Â±0.47 3.46 Â±0.28 (35); bombesin immunoreactivity appears to diminish as cellu- 5.7Â°'F

14. Carney, D. N., Cuttitta, F., Moody, T. W., and Minna, J. D. Selective lar differentiation occurs. Further evidence to support this stimulation of small cell lung cancer clonal growth by bombesin and gastrin- hypothesis includes the finding of elevated levels of bombesin- releasing peptide. Cancer Res., 47: 821-825, 1987. like immunoreactivity in fetal or neonatal lungs compared to 15. Carney, D. N., Cuttitta, F. C., Gazadar, A. F., and Minna, J. D. Autocrine adult lung tissue (36) and differences in endocrine cell distri clonogenic factor(s) (CF) are produced by cell lines (CL) of small cell lung cancer (SCLC) (Abstract). Proc. Am. Soc. Clin. Oncol., 2: 14, 1983. bution during varying stages of pulmonary development (37). 16. Cuttitta, F., Carney, D. N., Mulshine, J., Moody, T. W., Fedorko, J., Fischler, Cellular maturation may explain the differences in bombesin A., and Minna, J. D. Bombesin-like peptides can function as autocrine growth factors in human small-cell lung cancer. Nature (Lond.), 316:823-826,1985. immunoreactivity observed between fetal-neonatal and adult 17. Zachary, !.. and Rozengurt, E. High-affinity receptors for peptides of the lung tissue, as well as the differences seen between the subtypes bombesin family in Swiss 3T3 cells. Proc. Nati. Acad. Sci. USA, 82: 7616- of pulmonary carcinoma. The significance of these findings is 7620, 1985. 18. Moody, T. W., Pert, C. B., Rivier, J., and Brown, M. R. Bombesin: specific the idea that malignant cells may indeed be related to their binding to rat brain membranes. Proc. Nati. Acad. Sci. USA, 75:5372-5376, earlier embryonic states and that malignant transformation 1978. 19. Jensen, R. T., Moody, T., Pert, C., Rivier, J. E., and Gardner, J. D. occurred due to inappropriate expression of the autocrine mech Interaction of bombesin and litorin with spÃ©cifiemembranereceptors on anism. pancreatic acinar cells. Proc. Nati. Acad. Sci. USA, 75:6139-6143, 1978. 20. Westendorf, J. M., and Schonbrunn, A. Characterization of bombesin recep Our study demonstrates the action of bombesin as an exog tors in a rat pituitary cell line. J. Biol. Chem., 258: 7527-7535, 1983. enous growth factor for SCLC in vivo. These data support the 21. Moody, T. W., Carney, D. N., Cuttitta, F., Quattrocchi, K., and Minna, J. possibility that GRP is indeed an autocrine growth factor for D. High affinity receptors for bombesin/GRP-like peptides on human small cell lung cancer. Life Sci., 37: 105-113, 1985. SCLC in vivo. As specific autocrine growth factors are identi 22. Ogur, M.. and Rosen, G. The nucleic acids of plant tissues. I. The extraction fied, specific antagonists may be synthesized to combat or and estimation of desoxypentose nucleic acid and pentose nucleic acid. Arch. control the process of malignant transformation. Biochem., 25: 262-276, 1950. 23. Burton, K. A study of the conditions and mechanism of the diphenylamine reaction for the colorimetrie estimation of deoxyribonucleic acid. 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Robert W. Alexander, James R. Upp, Jr., Graeme J. Poston, et al.

Cancer Res 1988;48:1439-1441.

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