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Antagonistic analogs of GHRH inhibit growth of various human cancers both in vivo and in vitro. To elucidate the mechanism of direct action of the antagonistic analogs of GHRH on tumor cells, cultured human cancer cells were exposed to GHRH, vasoactive intestinal peptide (VIP), secretin, glucagon, neuropeptide-Y (NPY), pituitary adenylate cyclase-activating peptide (PACAP), and VIP analogs in a superfusion system, and changes in cAMP and IGF-II release from the cells were measured. Various human cancer cell lines, such as mammary (MDAMB-468 and ZR-75-1), prostatic (PC-3), pancreatic (SW-1990 and Capan-2), ovarian (OV-1063), and colorectal (LoVo) responded to pulsatile stimuli with GHRH (0.5-20 nM), VIP (0.02-10 nM), and PACAP-38 (0.05-5 nM) with a rapid, transient increase in cAMP release from the cells. The VIP antagonist, PG-97-269, and the adenylate cyclase inhibitor, MDL-12330A, but not SQ-22536 or pertussis toxin, blocked the cAMP responses to these peptides. Stimulation of the cells with 100 nM secretin, glucagon or NPY did not alter the cAMP release. Our results suggest that GHRH receptors different from the type expressed in the pituitary are involved in mediating these effects. As cAMP is a potent second messenger controlling a wide variety of intracellular functions, including those required for cell growth, our results indicate that GHRH might have a direct stimulatory effect on growth of human cancers. Blockade of the autocrine/paracrine action of GHRH with its antagonistic analogs may provide a new approach to tumor control.

GH3 rat pituitary tumor cells produce GH and prolactin (PRL), but lack the GHRH receptor (GHRH-R). We expressed human GHRH-R (hGHRH-R) in GH3 cells using recombinant adenoviral vectors and studied the effects of GHRH antagonists. The mRNA expression of the GHRH-R gene in the cells was demonstrated by RT-PCR. An exposure of the GH3 cells infected with hGHRH-R to 10(-10), 10(-9) and 10(-8) m hGHRH for 1 or 2 h in culture caused a dose-dependent elevation of the intracellular cAMP concentration and the cAMP efflux. Exposure to hGHRH also elicited dose-dependent increases in GH and PRL secretion from these cells. Neither the uninfected nor the antisense hGHRH-R-infected control cells exhibited cAMP, GH and PRL responses to GHRH stimulation. GHRH antagonists JV-1-38 and jv-1-36 applied at 3x10(-8) m for 3 h, together with 10(-9) m GHRH, significantly inhibited the GHRH-stimulated cAMP efflux from the hGHRH-R-infected cells by 36 and 80% respectively. The more potent antagonist JV-1-36 also decreased the intracellular cAMP levels in these cells by 55%. Exposure to JV-1-36 for 1 h nullified the stimulatory effect of GHRH on GH secretion and significantly inhibited it by 64 and 77% after 2 and 3 h respectively. In a superfusion system, GHRH at 10(-10), 10(-9) and 10(-8) m concentrations induced prompt and dose-related high cAMP responses and smaller increases in the spontaneous GH secretion of the hGHRH-R-infected cells. Antagonists JV-1-36 and JV-1-38 applied at 3x10(-8) m for 15 min, together with 10(-9) m GHRH, inhibited the GHRH-stimulated cAMP response by 59 and 35% respectively. This work demonstrates that GHRH antagonists can effectively inhibit the actions of GHRH on the hGHRH-R. Our results support the view that this class of compounds would be active clinically.