[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)

Abstract

In the search for a more potent bombesin antagonist, we found [D-Arg',D-Phe5,D-Trp7,9,Leul"Jsubstance P to be effective in mouse fibroblasts and to inhibit the growth of small cell lung cancer, a tumor that secretes bombesin-like peptides that may act as autocrine growth factors. In murine Swiss 3T3 cells, [D-Arg',D-Phe5,D-Trp7",Leu"Jsubstance P proved to be a bombesin antagonist as judged by the following criteria: (i) inhibition of DNA synthesis induced by gastrinreleasing peptide and other bombesin-like peptides; (i) inhibition of '2sI-abeled gastrin-releasing peptide binding to the bombesin/gastrin-releasing peptide receptor; (iii) reduction in cross-linking of the Mr 75,000-85,000 protein putatively a component of the bombesin/gastrin-releasing peptide receptor; (iv) blocking of early cellular events that precede mitogenesis-calcium mobilization and inhibition of epidermal growth factor binding. [D-Arg%,D-Phe5,D-Trp7 9,Leu11]substance P was 5-fold more potent than the antagonist [D-Arg',D-Pro2,DTrp7M9,Leu"]substance P. [D-ArgD-PheD-Trp7",Leu"Jsubstance P also inhibits mitogenesis induced by vasopressin but not that induced by a variety of other mitogens. Both antagonists reversibly inhibited the growth of small cell lung cancer in vitro in a concentration-dependent manner. Peptide antagonists could, therefore, have far-reaching therapeutic implications. Regulatory peptides that act as local hormones or neurotransmitters are increasingly implicated in the control of cell proliferation (1). The amphibian tetradecapeptide bombesin and structurally related mammalian peptides, including gastrin-releasing peptide (GRP), are potent mitogens for Swiss 3T3 cells (2, 3). The peptides bind to high-affinity cell-surface receptors in these cells (3) and elicit a complex array of early biological responses (4), including enhanced inositol phospholipid breakdown and mobilization of Ca2+ from intracellular stores (5-7), stimulation of Na+/H+ antiport activity (6), activation of protein kinase C (8-10), inhibition of 125I-labeled epidermal growth factor (EGF) binding (8, 9, 11), and induction of the cellular oncogenes c-fos and c-myc (12-14). Zachary and Rozengurt (15) identified a surface protein in Swiss 3T3 cells with Mr 75,000-85,000 as a putative component of the bombesin/GRP receptor. The widely distributed tachykinin substance P, which has slight amino acid sequence homology with bombesin (Fig. 1), neither inhibits the binding of GRP nor stimulates DNA synthesis in Swiss 3T3 cells (2, 3). However, [D-Arg1,DPro2,D-Trp7'9,Leu"l]substance P (peptide A in Fig. 1), which was synthesized as a substance P antagonist (16), was found to block the secretory effects of bombesin in pancreatic acinar cells (17) and to antagonize the growth-promoting effects of bombesin in Swiss 3T3 cells (3, 6, 9, 17, 18). This antagonist also inhibits mitogenesis stimulated by the neurohypophyseal hormone vasopressin (19). It appears that peptide A interacts with several independent cellular receptors to block their biological effects. Bombesin-like peptides are present in high concentrations in small cell lung cancer (SCLC) (20-22). They can stimulate the growth of SCLC cells in vitro (23, 24) and may act as autocrine growth factors (2, 25). The dilutional hyponatraemic syndrome, which commonly complicates SCLC, is associated with ectopic production of vasopressin (26, 27). Thus, the identification of more potent antagonists, which recognize the receptors for both peptides, could be of considerable interest in future clinical approaches to the treatment of SCLC. Here we report that [D-Arg1,D-Phe5,D-Trp7'9,Leuillsubstance P (peptide D in Fig. 1) is 5-fold more potent than peptide A in preventing the cellular effects of GRP and vasopressin in mouse 3T3 cells and in inhibiting the growth of SCLC cells in serum-free medium. MATERIALS AND METHODS Swiss 3T3 cells were maintained in culture, and assays of DNA synthesis were performed by measuring [3H]thymidine incorporation as described (2, 28). Receptor binding assays utilized 125I-labeled GRP and EGF (3, 9). Cross-linking of 1251-labeled GRP to a Mr 75,000-85,000 surface protein was carried out as described (15). Cytosolic [Ca2+] was measured by fluorimetry by using the indicator fura-2 (6). SCLC lines were obtained from the American Type Culture Collection. Radiochemicals were obtained from Amersham. Bombesin, GRP, insulin, and vasopressin were from Sigma. Peptides A, B, C, D, E, F, and K (Fig. 1) were obtained from Peninsula Laboratories (San Carlos, CA), and peptides A, G, H, and J were from Bachem Fine Chemicals (Torrance, CA).

7 Figures and Tables

Cite this paper

@inproceedings{WollDArg1DPhe5DTrp79LeulsubstancePA, title={[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)}, author={Penella Woll and Enrique Rozengurt} }