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Substance P. a Potent Bombesin

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Substance P. a Potent Bombesin Proc. Nati. Acad. Sci. USA Vol. 85, pp. 1859-1863, March 1988 Cell Biology [D-Arg1,D-Phe5,D-Trp7'9,Leul]substance P. a potent bombesin antagonist in murine Swiss 3T3 cells, inhibits the growth of human small cell lung cancer cells in vitro (gastrin-releasing peptide/vasopressin/growth factors/neuropeptides) PENELLA J. WOLL AND ENRIQUE ROZENGURT* Imperial Cancer Research Fund, P 0 Box 123, Lincoln's Inn Fields, London WC2A 3PX, England Communicated by George R. Stark, November 2, 1987 ABSTRACT In the search for a more potent bombesin an- antagonist also inhibits mitogenesis stimulated by the neu- tagonist, we found [D-Arg',D-Phe5,D-Trp7,9,Leul"Jsubstance P rohypophyseal hormone vasopressin (19). It appears that to be effective in mouse fibroblasts and to inhibit the growth of peptide A interacts with several independent cellular recep- small cell lung cancer, a tumor that secretes bombesin-like tors to block their biological effects. peptides that may act as autocrine growth factors. In murine Bombesin-like peptides are present in high concentrations Swiss 3T3 cells, [D-Arg',D-Phe5,D-Trp7",Leu"Jsubstance P in small cell lung cancer (SCLC) (20-22). They can stimulate proved to be a bombesin antagonist as judged by the following the growth of SCLC cells in vitro (23, 24) and may act as criteria: (i) inhibition of DNA synthesis induced by gastrin- autocrine growth factors (2, 25). The dilutional hyponatrae- releasing peptide and other bombesin-like peptides; (i) inhi- mic syndrome, which commonly complicates SCLC, is bition of '2sI-abeled gastrin-releasing peptide binding to the associated with ectopic production of vasopressin (26, 27). bombesin/gastrin-releasing peptide receptor; (iii) reduction in Thus, the identification of more potent antagonists, which cross-linking of the Mr 75,000-85,000 protein putatively a recognize the receptors for both peptides, could be of con- component of the bombesin/gastrin-releasing peptide recep- siderable interest in future clinical approaches to the treat- tor; (iv) blocking of early cellular events that precede mitogen- ment of esis-calcium mobilization and inhibition of epidermal growth SCLC. factor binding. [D-Arg%,D-Phe5,D-Trp7 9,Leu11]substance P Here we report that [D-Arg1,D-Phe5,D-Trp7'9,Leuillsub- was 5-fold more potent than the antagonist [D-Arg',D-Pro2,D- stance P (peptide D in Fig. 1) is 5-fold more potent than Trp7M9,Leu"]substance P. [D-ArgD-PheD-Trp7",Leu"Jsub- peptide A in preventing the cellular effects of GRP and stance P also inhibits mitogenesis induced by vasopressin but vasopressin in mouse 3T3 cells and in inhibiting the growth not that induced by a variety of other mitogens. Both antag- of SCLC cells in serum-free medium. onists reversibly inhibited the growth of small cell lung cancer in vitro in a concentration-dependent manner. Peptide antag- MATERIALS AND METHODS onists could, therefore, have far-reaching therapeutic implica- tions. Swiss 3T3 cells were maintained in culture, and assays of DNA synthesis were performed by measuring [3H]thymidine Regulatory peptides that act as local hormones or neuro- incorporation as described (2, 28). Receptor binding assays transmitters are increasingly implicated in the control of cell utilized 125I-labeled GRP and EGF (3, 9). Cross-linking of proliferation (1). The amphibian tetradecapeptide bombesin 1251-labeled GRP to a Mr 75,000-85,000 surface protein was and structurally related mammalian peptides, including gas- carried out as described (15). Cytosolic [Ca2+] was mea- trin-releasing peptide (GRP), are potent mitogens for Swiss sured by fluorimetry by using the indicator fura-2 (6). SCLC 3T3 cells (2, 3). The peptides bind to high-affinity cell-sur- lines were obtained from the American Type Culture Col- face receptors in these cells (3) and elicit a complex array of lection. early biological responses (4), including enhanced inositol Radiochemicals were obtained from Amersham. Bombe- phospholipid breakdown and mobilization of Ca2+ from sin, GRP, insulin, and vasopressin were from Sigma. Pep- intracellular stores (5-7), stimulation of Na+/H+ antiport tides A, B, C, D, E, F, and K (Fig. 1) were obtained from activity (6), activation of protein kinase C (8-10), inhibition Peninsula Laboratories (San Carlos, CA), and peptides A, of 125I-labeled epidermal growth factor (EGF) binding (8, 9, G, H, and J were from Bachem Fine Chemicals (Torrance, 11), and induction of the cellular oncogenes c-fos and c-myc CA). (12-14). Zachary and Rozengurt (15) identified a surface pro- tein in Swiss 3T3 cells with Mr 75,000-85,000 as a putative component of the bombesin/GRP receptor. RESULTS The widely distributed tachykinin substance P, which has Peptide D Is a Potent Inhibitor of GRP-Mediated Mitogen- slight amino acid sequence homology with bombesin (Fig. esis. To identify a more potent antagonist of bonibesin-like 1), neither inhibits the binding of GRP nor stimulates DNA peptides, we have tested 10 substance P antagonists at 50 synthesis in Swiss 3T3 cells (2, 3). However, [D-Arg1,D- ,M (Fig. 1) for their ability to inhibit mitogenesis stimulated Pro2,D-Trp7'9,Leu"l]substance P (peptide A in Fig. 1), which by GRP [the mammalian homologue of bombesin (29)] in was synthesized as a substance P antagonist (16), was found Swiss 3T3 cells. Peptide D was clearly the most potent GRP to block the secretory effects of bombesin in pancreatic In and acinar cells (17) and to antagonize the growth-promoting antagonist. contrast, peptides B, C, E, F, G, H, J, K effects of bombesin in Swiss 3T3 cells (3, 6, 9, 17, 18). This Abbreviations: EGF, epidermal growth factor; GRP, gastrin- releasing peptide; PDGF, platelet-derived growth factor; peptide A, The publication costs ofthis article were defrayed in part by page charge [D-Arg',D-Pro2,D-Trp7'9,Leu11]substance P; peptide D, [D-Arg',D- payment. This article must therefore be hereby marked "advertisement" Phe5,D-Trp' 9,Leu"I]substance P; SCLC, small cell lung cancer. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 1859 Downloaded by guest on September 27, 2021 1860 Cell Biology: Woll and Rozengurt Proc. Natl. Acad. Sci. USA 85 (1988) Bombesin pGlu Gln Arg Leu Gly Asn Gln Trp Ala Val Gly His Leu Net NH2 1 2 3 S 6 7 8 9 10 11 Substance P Arg Pro Lys Pro Gln Gln Phe Phe Gly Leu Met NH2 Antagonist A DArg DPro Lys Pro Gln Gin DTrp Phe DTrp Leu Leu NH2 B DArg Pro Lys Pro Gln Gin DTrp Phe DTrp Leu Leu NH2 C Arg DPro Lys Pro Gln Gln DPhe Phe DTrp Leu Met NH2 D Dkrg Pro Lys Pro DPhe Gln DTrp Phe DTrp Leu Leu NH2 E Arg DPro Lys Pro Gln Gln DTrp Phe DTrp Leu Met NH2 F Pro Gln Gln DTrp Phe DTrp DTrp Met NH2 G Arg DTrp MePhe DTrp Leu Met NH2 H DArg DPro Lys Pro Gln Gln DPhe Phe DHis Leu Met NH2 J HArg Gly Gln DTrp Phe Gly Asp (OtBu)2 K DPro Gln Gln DTrp Phe DTrp Leu Met NH2 FIG. 1. Amino acid sequences of the substance P antagonists studied. These peptides were tested as inhibitors of GRP-induced DNA synthesis in Swiss 3T3 cells in the presence of [3H]thymidine at 1 uCi/ml (1 Ci = 37 GBq) (1 ,uM), insulin at 1 JLg/ml, GRP at 2.7 nM, and the substance P antagonist to be tested at 50 ,uM (details as in Fig. 2). The peptides were also screened at 50 juM for inhibition of vasopressin-induced DNA synthesis and binding of 1251-labeled GRP to Swiss 3T3 cells. Peptide D was the most potent in repeated assays. Similar results were obtained with peptide A from Bachem Fine Chemicals and Peninsula Laboratories. Peptide D synthesized in the Imperial Cancer Research Fund Laboratories by J. Rothbard gave similar results to the peptide obtained from Peninsula Laboratories. Substance P itself exhibited no antagonist activity. were less potent than either peptide A or peptide D. Spantide presence of insulin. In contrast, the same concentration of (peptide B) had no antagonist activity even at 100 ,M. None peptide D markedly decreased the stimulation of DNA syn- of the peptides stimulated DNA synthesis when tested at 20 thesis by bombesin and GRP-(14-27)-peptide, which share a ,M with insulin at 1 ,ug/ml-i.e., none exhibited any agonist highly conserved COOH-terminal heptapeptide with GRP. activity. Furthermore, addition of peptide D at 20 uM inhibited the Following the identification of peptide D as the most increase in cell number caused by treatment of serum- promising GRP antagonist, we compared its potency with that depleted cells with GRP and insulin, whereas cell prolifera- of peptide A. Fig. 2 shows that peptide D at 20 ,M markedly tion stimulated by EGF and insulin was only slightly affected increased the concentration of GRP required to produce (Fig. 3 Right). Thus peptide D, like peptide A (3), inhibited half-makimal stimulation of DNA synthesis whereas addition the reinitiation of DNA synthesis and cell division in a se- of peptide A also at 20 ,M had only a slight effect. Inhibition lective manner. of DNA synthesis by peptide D was completely reversed by Peptide A has been shown to block specific binding of vas- high concentrations of GRP, indicating that its inhibitory opressin to its receptor in Swiss 3T3 cells and vasopressin- effect was competitive and reversible. The dose-response induced mitogenesis (18, 19). Peptide D at 50 ,uM inhibited curves for the two antagonists in the presence of 3.6 nM GRP vasopressin-induced DNA synthesis in Swiss 3T3 cells (Fig.
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