Autocrine stimulation of growth of AR4-2J rat pancreatic tumour cells by gastrin

Abstract

Summary The control of cell proliferation by gastrin has been investigated in a rat pancreatic tumour cell line, AR4-2J. Exogenous gastrin, 10-12 to 10-8 M, stimulated cell growth of thymidine-synchronised AR4-2J cells cultured over 48 h in serum-free medium. Cell lysates of AR4-2J cells contained an average of 4.5 and 3.5 pg gastrin per 106 cells, when grown in serum-supplemented or serum-free media, respectively, as revealed by radioimmunoassay. In serum-free medium, AR4-2J secrete 34ngl 10-6 cells of gastrin over 48h. Addition of an anti-gastrin immunoglobulin preparation, but not control immunoglobulins, caused a maximum 52% reduction in cell growth. These data are consistent with an autocrine role for gastrin in the control of AR4-2J cell growth. These results were supported by studies with gastrin/CCK receptor antagonists. Six non-peptide gastrin/CCK receptor antagonists inhibited AR4-2J cell growth in a concentration-related manner. The concentration required for 50% inhibition (IC50) of cell growth by the amino acid-derived antagonists proglumide (3.5 x 10-M), benzotript (1.8 x 10-3 M), loxiglumide (1.1 x 10-4 M) and lorglumide (6.7 x 10-5 M) were of the same order and significantly correlated with their IC50 for inhibition of 1251-gastrin binding to AR4-2J cells. Inhibition of cell growth by these antagonists was partially reversed by the addition of exogenous gastrin. In contrast, the ICm for inhibition of cell growth with two benzodiazepine-derived antagonists, the CCK-B receptor antagonist L-365,260 (4.6 x 10-5 M) and the CCK-A receptor antagonist devazepide (1.7 x 10-' M) were two-three orders of magnitude greater than those required to inhibit gastrin binding (10-8-10-7 M). The growth inhibitory effects of L-365,260 and devazepide were not reversed by exogenous gastrin suggesting these benzodiazepine-derived antagonists do not inhibit cell growth by interaction with gastrin receptors. The results are consistent with gastrin being an autocrine growth factor in AR4-2J cells, and that stimulation of cell growth is due to stimulation of the gastrin, rather than CCK-B, receptor sub-type. This study highlights that gastrin receptor antagonists warrant further investigation as agents to control growth of tumours, such as those from the gastrointestinal tract, which express gastrin receptors.

Autocrine and paracrine signaling through neuropeptide receptors in human cancer

“…While the CCK-A receptor mRNA was also widely expressed in most of the tumor samples, neither amidated and sulfated CCK nor CCK precursors were expressed in the carcinomas or normal pancreatic tissues (Goetze et al, 2000;Weinberg et al, 1997). Moreover, coexpression of gastrin and CCK-B receptors is observed in multiple cultured pancreatic carcinoma cell lines and growth of these cell lines is inhibited by CCK-B receptor antagonists and gastrin neutralizing antibodies (Blackmore and Hirst, 1992;Smith et al, 1994Smith et al, , 1996.…”

Antiproliferative gastrin/cholecystokinin receptor antagonists target the 78-kDa gastrin-binding protein.

“…The presence of high-affinity gastrin-binding sites on primary human colon cancer tumors is still controversial, with an early report claiming 57% of 67 specimens were positive (38), and a later report finding no positives in 9 specimens (39 (14,23) (Table 1). The CCK-A receptor-selective antagonist L364,718 and the gastrin/CCK-B receptorselective antagonist L365,260 previously have been reported to have no effect on colon carcinoma cell proliferation at concentrations as high as 1 juM (22).…”

“…tIC50 values for the inhibition of proliferation of HCT 116 (14,22) and HT 29 (23) colon carcinoma cells by antagonists were determined from cell counts (14,22) or by colorimetric assay (23). tICso values (mean ± SD, calculated from at least three sets of data) for inhibition of cross-linking of 125j-[Nle15]gastrin2,17 to the 78-kDa GBP in detergent extracts ofporcine gastric mucosal membranes (24) were determined from the data presented in Fig.…”

The peripheral cholecystokinin receptors

“…These mitogenic effects can be at least partially reversed by certain CCK receptor antagonists [233 -2351. Furthermore, AR4-2J cells secrete immunoreactive gastrin which may be involved in an autocrine stimulation of growth [236]. Very recently, another cancer cell line established from azaserineinduced rat pancreatic carcinoma was shown to possess 34% CCK-B receptors [237].…”