Balazs Szoke

Gonadotropin-releasing hormone analogues inhibit cell proliferation and activate signal transduction pathways in MDA-MB-231 human breast cancer cell line

A novel gonadotropin-releasing hormone (GnRH) agonist (folligen) which stimulates follicular maturation has been developed in our laboratory. The direct effect of folligen and a well-known superactive GnRH analogue, buserelin, on the MDA-MB-231 human breast cancer cell line was investigated. Folligen was found to be slightly more active in inhibiting cell proliferation than buserelin, and significant differences were found in the signal transduction pathways activated by these analogues. These results demonstrate for the first time that tyrosine kinases and/or phospholipid turnover together with protein kinase C activation can be directly involved in the antitumor activity of GnRH analogues. The results also suggest that folligen and buserelin might have a different mechanism of action on this human breast cancer cell line.

Gonadotropin releasing hormone (GnRH) is not degraded by intact pituitary tissue in vitro.

The possible degradation of GnRH by intact pituitary tissue was investigated. Trypsin-, collagenase- and mechanically dispersed pituitary cells in culture and fresh pituitaries cut into eight segments were incubated with specifically tritiated GnRH or unlabeled GnRH which were detected by HPLC and RIA in incubation media. Degradation of GnRH could not be detected by either method during incubations with any of the pituitary cell cultures or fresh tissue. The results suggest that pituitary degradation is not involved in the regulation of circulating levels of GnRH.

Proteolytic Degradation of Gonadotropin-Releasing Hormone (GnRH) by Rat Ovarian Fractions In Vitro

GnRH in physiological concentrations is highly degradable by both soluble and particulate fractions of rat ovarian homogenate in vitro. The two proteolytic enzyme activities differ strongly by the soluble activity showing a dithiothreitol optimum, high inhibition by diisopropyl fluorophospate (ki = 0.7 microM), and a relatively high affinity (Km = 1.1 microM) as opposed to the particulate fraction (Ki = 3.5 mM and Km = 150 microM, respectively). The results of this study show that the rat ovary is differently endowed with GnRH-degrading activity at different sites. The involvement of these in terminating the biological activity of the hormone on the ovary may possibly depend on its exact pathway in this GnRH-target organ.

Biodegradation of Luteinizing Hormone-Releasing Hormone

The concept that the proteolytic degradation of peptide hormones could be one of the key mechanisms regulating the homeostasis of the hormone, as proposed by Knights et al. (1973), is widely accepted. However, current knowledge about the physiological mechanisms involved is incomplete. The biodegradation of luteinizing hormone-releasing hormone (LH-RH) has been extensively studied, yet the complexity of the information accumulated does not allow a clear conclusion. In this chapter we shall review current information on the biodegradation of LH-RH and discuss the relevance of the data obtained. We also make an attempt to interpret the biochemical mechanisms involved in the degradation of LH-RH that could be of physiological importance.

New Gaba-containing analogues of human Growth Hormone-Releasing Hormone (1–30)-amide: I. Synthesis and in vitro biological activity

Analogues of human growth hormone-releasing hormone (1–30)-amide have been developed. All analogues have been modified in position 27 with Nle and with Gaba in position 30. Additional D-amino-acids have been inserted in the GHRH(1–30)-NH2 sequenceA-1741: Nle27, Gaba30-GH-RH (1–30)-NH2 A-495: D-Ala2, Nle27, Gaba30-GH-RH (1–30 )-NH2 A-515: D Ala2, Leu15, Nle27, Gaba30-GH-RH (1–30)-NH2 A-527: D-Ala2, D-Arg11, Leu15, Nle27, Gaba30-GH-RH (1–30)-NH2.Our analogues were synthesized by solid phase peptide synthesis and were tested is two different in vitro systems and in rat pituitary cell cultures. A-495 and A-1741 were found to be the most active in releasing GH, however they showed different activities in the two different test systems. A-495 exhibited higher potency in the superfusion system (1.63 fold potency of the GHRH (1–29)-amide), while A-1741 evoked higher GH release from cultured pituitary cells (1.5–2.5 times higher than the GH-RH(1–44)-amide). The other analogues (A-515 and A-527) were found to be equipotent to the standard molecule. We can conclude that Nle27 and Gaba30 substitutions appeared to be a good modification in in vitro test systems, and Gaba30 substitution served as a good spacer during the synthesis, since it made the coupling of the C-terminal amino acids easier and produced quantitative coupling. In addition to the advantageous properties in the synthesis these modifications with or without D-Ala at the N-terminus increased the in vitro biological activity to 1.5–2.5 fold of the GHRH molecule. The additional substitution of Gly15 with Leu and Arg11 with D-Arg did not improve the in vitro GH-releasing activity of our analogues. A detailed in vivo investigation, which is essential for the future clinical use, hasbeen performed and written in Part II of this paper.