Inhibitory Effect of Bombesin Receptor Antagonist RC-3095 on the Growth of Human Pancreatic Cancer Cells in Vivo

In this study, we investigated the effect of bombesin/GRP antagonist RC-3095 on the growth of CFPAC-1 human pancreatic cancer cells transplanted to nude mice or cultured in vitro. Nude mice bearing xenografts of the CFPAC-1 cell line received s.c. injections of RC-3095 (10/tg twice a day) or the vehicle (control) for 25 days. Chronic administration of RC3095 inhibited the growth of CFPAC-1 tumors in nude mice as shown by a significant decrease in tumor volume throughout the period of treatment. Tumor volume doubling time was prolonged by RC-3095 treatment from 7.2 days to 10 days, and the tumor growth rate was decreased by 49%. In mice treated with RC-3095, the tumor growth delay time was 5.8 days. ~I~reatment with RC-3095 decreased the final tumor weight by 37% and reduced DNA and protein contents in tumor tissues by 44 and 39.9%, respectively, compared to the controls. In cultures of the CFPAC-I cell line, the addition of bombesin(1-14) (1 p~a-0.1 pM) to the medium induced a dose-dependent increase in cell number. RC-3095 at 1 nM concentration effectively inhibited the bombesin-stimulated growth of CFPAC-I cells in cultures. In the presence of 1 btM RC-3095 in the culture medium, the bombesin-induced growth of CFPAC-1 cells was totally suppressed. Bombesin was also shown to stimulate the DNA synthesis in CFPAC-1 cells in vitro as based on [3H]thymidine incorporation assay. When the cells were cultured in the presence of 1-100 nM bombesin, the uptake of [3H]thymidine by the cells was increased by 89-131%. RC-3095 inhibited both the basal and bombesin-stimulated DNA synthesis of CFPAC-1 cells. Addition of RC-3095 (10-100 nM) alone to the cultures caused a 39-40% decrease in the [3H]thymidine incorporation by the cells. Concomitant addition of RC-3095 (1 p~) and bombesin (1-100 nM) to the cultures induced a significant reduction in the uptake of [aH]thymidine by the cells compared to the values obtained with bombesin alone. Receptor binding assays showed the presence of two classes of specific binding sites for bombesin on CFPAC-1 cells, one with high affinity (Ka = 4.25 _+ 0.77 n~a) and low capacity (B,,ox = 0.268 +_ 0.052 pmol/106 cells) and the other with low affinity (K,t -" 321.70 + 68.46 nM) and high capacity (B .... 3.991 +_ 0.374 pmoi/106 cells). Antagonist RC-3095 inhibited the binding of ~2SI-Tyr 4bombesin to CFPAC-1 cell membranes in a dose-dependent manner. These observations suggest that bombesin acts as a growth factor and stimulates proliferation of CFPAC-1 human pancreatic cancer through specific receptors for bombesin/GRP present on the cells. RC-3095 appears to inhibit the growth of CFPAC-1 cells by blocking the interaction of bombesin with its receptors. Bombesin/GRP receptor antagonist RC-3095 could be considered for the development of new approaches for treatment of human pancreatic cancers. I N T R O D U C T I O N Pancreatic cancer is one of the greatest challenges for oncologists (1-5). Carcinoma of the exocrine pancreas is the fifth leading cause of death from cancer in the United States (1). Most of pancreatic cancers are histologically ductal cell carcinomas, which constitute about 8090% of the cases (2). In the past two decades, great efforts have been made to improve the therapies for pancreatic cancers. However, the overall prognosis of patients with pancreatic cancer is still very poor, and the 5-year survival rate is only 2-5% (2-4). Less than 15-20% of pancreatic tumors are resectable, mostly due to the difficulties in early diagnosis and the frequent occurrence of local or distal metastases, and fewer than 5% of the patients can survive for over 5 years postoperatively (3-5). Radiotherapy and chemotherapy are usually ineffective (2-5). Therefore, an urgent need exists to develop a new and effective therapy for treatment of patients with pancreatic cancers. Recently, various investigations have demonstrated that gastrointestinal hormones and growth factors may play important roles in the regulation of growth of normal and malignant exocrine pancreas (510). In vitro studies have shown that gastrin, CCK, 3 and secretin stimulate the proliferation of pancreatic adenocarcinoma cells in tissue cultures (6, 7). Caerulein combined with secretin promotes the in vivo growth of H-2-T pancreatic ductal adenocarcinoma cells in golden hamsters (8). Gastrointestinal hormones may also influence the phenotypic transformation of pancreatic cells (6, 8, 9). These findings suggest that the growth of pancreatic cancers might be possibly controlled by hormonal manipulations (4-10), and various peptide analogues have been suggested for treatment (5-10). Bombesin and its mammalian counterpart, GRP, are hormonal peptides which can exert diverse physiological or pharmacological actions in various systems (5, 11, 12). In the gastrointestinal tract, bombesin and GRP stimulate gastric and pancreatic secretions, enhance the release of several gastrointestinal hormones, and promote the growth of exocrine pancreas (5, 11, 12). Administration of bombesin to rats produces hyperplasia and hypertrophy of pancreatic acinar cells (13). Bombesin and GRP appear to function as autocrine or paracrine growth factors and stimulate the growth of some normal or malignant cells including Swiss 3T3 fibroblast cells (14), human small cell lung cancer cells (15), human mammary and gastric cancer cells (16), 4 mouse and human colon cancer cells (17, 18), and CAPAN human pancreatic tumor cells (19). The trophic effect of bombesin/ GRP has also been demonstrated on the growth of azaserine-induced pancreatic acinar-cell adenocarcinomas in rats (20, 21) and on the cell proliferation of acinar tumor in primary cultures (22). The action of bombesin/GRP is thought to be mediated through its specific receptors present on the target cells (19, 22, 23). The discovery that bombesin appears to act as an autocrine growth factor in human small cell lung carcinoma (15, 23) and may also be involved in other cancers (16-22) has aroused major interest in the development of competitive bombesin/GRP receptor antagonists. During the past few years, various bombesin/GRP antagonists including RC-3095 have been synthesized in our laboratory and evaluated for antitumor activity (24, 25). Receptor studies showed that these synthetic peptide analogues inhibit the binding of bombesin/ GRP to specific receptors on the membranes of Swiss 3T3 cells, SCLC cells, and human gastric cancer cells (24, 25). 4 Bombesin Received 9/20/93; accepted 12/17/93. 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. 1 This work was supported by NIH Grant CA 40077 and the Medical Research Service of the Veterans Affairs (A. V. S.). 2 To whom requests for reprints should be addressed, at Veterans Affairs Medical Center, 1601 Perdido Street, New Orleans, LA 70146. 3 The abbreviations used are: CCK, cholecystokinin; GRP, gastrin-releasing peptide; FCS, fetal calf serum; IMDM, lscove's modified Dulbecco's medium; PBS, phosphatebuffered saline; GHRH, growth hormone-releasing hormone; Tpi, 2,3,4,9-tetrahydro-lHpyrido[3,4-b]indol-3-carboxylic acid; EGF, epidermal growth factor; [D-Trp6]LH-RH, o-tryptophan-6-1uteinizing hormone-releasing hormone. 4 y. Qin, G. Halmos, R-Z. Cai, B. Szoke, T. Ertl, and A. V. Schally. Inhibition of specific binding of bombesin and bombesin-stimulated growth of human gastric cancer cells by bombesin antagonists, submitted for publication.