Insulin-like Growth Factor Binding Protein (IGFBP)-3 Protease Activity Secreted by MCF-7 Breast Cancer Cells: Inhibition by IGFs Does Not Require IGF-IGFBP Interaction1

The proliferative action of insulin-like growth factors (IGFs) on breast cancer cells is regulated by IGF binding proteins (IGFBPs). This study characterizes the proteolysis of IGFBP-3 by an enzyme secreted by MCF-7 human breast cancer cells. Proteolysis of IGFBP-3 by incubation at 37 C with serum-free medium from MCF-7 cells was maximal at pH 5.0-5.5, with no activity detected below pH 4.5. This enzyme activity resulted in the disappearance of the 40- to 45- and 30-kDa bands of pure plasma-derived IGFBP-3, detectable by immunoblotting after SDS-PAGE, and the appearance of a single 21-kDa immunoreactive species. The 21-kDa protein did not bind IGF-I or IGF-II by ligand blotting. The enzyme activity appeared at 25- to 30-kDa by gel chromatography at pH 6.5 and was inhibited by EDTA and leupeptin, an inhibitor of cysteine and serine proteases, but not by the serine protease inhibitors aprotinin and benzamidine. IGFBP-3 protease activity was inhibited in medium conditioned by cells incubated with 50 ng/ml IGF-I. A similar inhibitory effect was seen under cell-free conditions by adding IGF-I to medium harvested from cells incubated without IGFs. The cell-free inhibition of IGFBP-3 proteolysis by IGFs did not require IGF interaction with the binding protein, because [long Arg3]IGF-I, which binds to IGFBP-3 with less than 0.2% of the potency of IGF-I, inhibited IGFBP-3 proteolysis with 20% of the potency of IGF-I. These results suggest that IGFs may regulate their own activity in breast cancer cells, preventing IGFBP-3 proteolysis by a mechanism that is not receptor mediated and does not require IGF-IGFBP interaction.