Evaluation of binding of cytotoxic analogs of luteinizing hormone-releasing hormone to human breast cancer and mouse MXT mammary tumor

The binding characteristics of several cytotoxic analogs of luteinizing hormone-releasing hormone (LH-RH) developed in our laboratory were examined in membranes from human breast cancer and estrogen independent MXT mammary cancer. Specific binding of [¹²⁵I]D-Trp⁶-LH-RH and the cytotoxic LH-RH analog [¹²⁵I]T-98 ([D-Lys⁶]LH-RH coupled to glutaryl-2-(hydroxymethyl)anthraquinone) (HMAQG) was demonstrated in membrane preparations from human breast and MXT mammary tumor cells. Ligand binding of T-98 was specific, saturable, and dependent on temperature, time, and plasma membrane concentration. Analysis of the binding data showed that in human breast cancer, interaction of [¹²⁵I]T-98 was consistent with the presence of two classes of LH-RH receptors, one class showing high affinity and low capacity, and the other class showing low affinity and high capacity binding. In membranes from MXT mammary cancer, T-98 bound to one class of saturable, specific, noncooperative binding sites with high affinity and low capacity. The rates of association and dissociation for [¹²⁵I]T-98 were calculated to be 4.757×10⁸ M^-1 min^-1 and 0.016 min^-1 (t_1/2=38.7) in membranes from MXT mammary cancer. In human breast cancer, association rate constants (K_1a and K_1b) were 2.3×10⁶ M^-1 min^-1 for binding to high affinity and 1.8×10⁴ M^-1 min^-1 for binding to low affinity binding sites. Dissociation rate constants were K_-1a=0.0801 min^-1 (t_1/2a=63.4 min) and K_-1b=0.0467 min^-1 (t_1/2b=23.5 min), respectively. [¹²⁵I]T-98 was not displaced by either unlabeled somatostatin or epidermal growth factor, but was displaced completely by unlabeled T-98 or [D-Trp⁶]LH-RH. The analysis of displacement curves of [D-Trp⁶]LH-RH by cytotoxic agonists and antagonists of LH-RH synthesized in our laboratory showed that T-121, AJ-11, T-120, T-133, and T-98 were the most potent in displacing [¹²⁵I]D-Trp⁶-LH-RH from breast and MXT cancer membranes. Binding kinetics and analyses of displacement curves of [¹²⁵I]D-Trp⁶-LH-RH and [¹²⁵I]T-98 in membranes of human breast cancer and estrogen independent MXT mouse mammary cancer suggest that binding of the cytotoxic analog T-98 to the LH-RH receptor proceeds reversibly like that of its congeners without cytotoxic radicals. Our findings may provide a stimulus for further studies with LH-RH analogs carrying cytotoxic radicals. Such analogs could be targeted to breast cancer and other cancers that have membrane receptors for LH-RH. Because the antitumor action may be exerted to a greater degree at more selective sites that have the cell membrane receptors, the peripheral toxicity could be reduced.