The role of cell surface attachment and proteolysis in the insulin-like growth factor (IGF)-independent effects of IGF-binding protein-3 on apoptosisin breast epithelial cells

We have recently reported that insulin-like growth factor (IGF)binding prot ein-3 (IGFBP-3) can significantly increase ceramide-induced apoptosis in an Hs578T breast carcinoma cell line in an IGF-independent manner. It was obs erved in that study that IGFBP-3 added to the cultures was proteolytically modified, generating a specific pattern of fragmentation. We have also prev iously reported that almost all of the IGFBP-3 outside the circulation in e xtravascular fluids is in a fragmented form, apparently due to the activity of a cation-dependent serine protease. The aim of this study was to invest igate the role of proteolysis in the IGFBP-3 enhancement of C2-induced apop tosis. In this study we confirmed that preincubation of Hs578T cells with IGFBP-3 enhances the apoptotic effect of the ceramide analog C2. The presence of IG F-I completely inhibited the enhancement effect, apparently by inhibiting c ell surface association and proteolytic modification. The presence of a ser ine protease inhibitor [4-(2-aminoethyl)benesulfonyl fluoride] completely i nhibited the enhancement effect of IGFBP-3, and Western immunoblotting of c onditioned medium and cell surface-associated IGFBP-3 revealed that proteol ytic fragmentation of the IGFBP-3 was reduced. In addition, fragments from the incubation of IGFBP-3 with plasmin were able to enhance the susceptibil ity of Hs578T cells to C2. The effect of these fragments could, however, al so be reduced by 4-(2-aminoethyl)benesulfonyl fluoride despite the fact tha t IGFBP-3 was already fragmented. This suggests additional roles for serine proteases in the IGFBP-3 effect on C2-induced apoptosis in addition to the cleavage of the binding protein.