The role of cell surface attachment and proteolysis in the insulin-like growth factor (IGF)-independent effects of IGF-binding protein-3 on apoptosis in breast epithelial cells.
We have recently reported that insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) can significantly increase ceramide-induced apoptosis in an Hs578T breast carcinoma cell line in an IGF-independent manner. It was observed in that study that IGFBP-3 added to the cultures was proteolytically modified, generating a specific pattern of fragmentation. We have also previously reported that almost all of the IGFBP-3 outside the circulation in extravascular fluids is in a fragmented form, apparently due to the activity of a cation-dependent serine protease. The aim of this study was to investigate the role of proteolysis in the IGFBP-3 enhancement of C2-induced apoptosis. 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 IGF-I completely inhibited the enhancement effect, apparently by inhibiting cell surface association and proteolytic modification. The presence of a serine protease inhibitor [4-(2-aminoethyl)benesulfonyl fluoride] completely inhibited the enhancement effect of IGFBP-3, and Western immunoblotting of conditioned medium and cell surface-associated IGFBP-3 revealed that proteolytic fragmentation of the IGFBP-3 was reduced. In addition, fragments from the incubation of IGFBP-3 with plasmin were able to enhance the susceptibility of Hs578T cells to C2. The effect of these fragments could, however, also be reduced by 4-(2-aminoethyl)benesulfonyl fluoride despite the fact that 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.
Maile
L A
LA
Department of Surgery, University of Bristol, Bristol Royal Infirmary, United Kingdom.
Gill
Z P
ZP
Perks
C M
CM
Holly
J M
JM
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001943:Breast Neoplasms / Q000473:pathology / Q000503:physiopathology*; D002277:Carcinoma / Q000473:pathology / Q000503:physiopathology*; D002462:Cell Membrane / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D004357:Drug Synergism; D004847:Epithelial Cells / Q000187:drug effects / Q000473:pathology / Q000502:physiology; D005260:Female; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000378:metabolism / Q000494:pharmacology*; D010447:Peptide Hydrolases / Q000378:metabolism*; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013002:Somatomedins / Q000502:physiology*; D013110:Sphingosine / Q000031:analogs & derivatives / Q000494:pharmacology; D013450:Sulfones / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects
D018972:Insulin-Like Growth Factor Binding Protein 3; C064769:N-acetylsphingosine; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D013002:Somatomedins; D013450:Sulfones; C002010:4-(2-aminoethyl)benzenesulfonylfluoride; D010447:Peptide Hydrolases; D013110:Sphingosine