Effect of luteinizing hormone-releasing hormone analogs containing cytotoxic radicals on the function of rat pituitary cells: tests in a long term superfusion system.

Abstract

A novel class of targeted chemotherapeutic agents based on cytotoxic compounds linked to LHRH analogs was tested in a long term superfusion system in order to determine their effects on different types of rat pituitary cells. The compounds investigated included two cytotoxic LHRH agonists, T-98 ([D-Lys6]LHRH coupled to glutaryl-2-(hydroxymethyl)anthraquinone (G-HMAQ)) and T-107 ([D-Lys6]LHRH linked to doxorubicin (DOX) through glutaric acid spacer), and two cytotoxic LHRH antagonists T-121 and T-144, both containing two residues of G-HMAQ. The analogs were infused for 24 h at pharmacological (micromolar) concentrations. The secretions of LH, GH, and PRL cells after a short (2-h) and a long (20-h) recovery period following the treatment were compared to those before the analog infusion. All four cytotoxic LHRH analogs selectively decreased the stimulated LH response, while basal secretions of LH, GH, and PRL as well as the stimulated release of GH and PRL were not influenced in spite of the high doses applied and the long duration of exposure. The inhibitory effects of cytotoxic agonists and antagonists were significantly greater than those of their parent peptides. Equimolar concentrations of cytotoxic compounds alone (DOX or G-HMAQ), however, caused functional damage to all types of cells tested. To evaluate the mechanisms of the observed inhibitory actions, we compared the amount of LH released in response to 1) a specific stimulus to receptors (3 nM LHRH), 2) membrane depolarization with 100 MM KCl, not involving the receptor, and 3) the extraction of cells with 0.01 N HCl at the end of experiment. Cytotoxic LHRH agonists and their carriers caused depletion of LH pools, while cytotoxic LHRH antagonists or their parent peptides decreased the available binding sites for LHRH. The inhibitory effect of DOX on stimulated secretion of LH, GH, and PRL in our system could be due to an action on cell membranes. Our work indicates that in the pituitary cell superfusion system, targeted cytotoxic conjugates, based on LHRH analogs, selectively affect LH cells, in contrast to unconjugated cytotoxic compounds, which also damage GH and PRL cells.