Characterization of an amino-terminal fragment of insulin-like growth factor binding protein-3 and its effects in MCF-7 breast cancer cells.
This study describes the purification, characterization and actions of a peptide derived from proteolysis of IGFBP-3 by an enzyme secreted by MCF-7 breast cancer cells. One millilitre of cell-conditioned medium at pH 5.5 fully proteolysed 10 microg plasma-derived IGFBP-3, yielding an immunoreactive fragment of apparent molecular mass 21 kDa by SDS-PAGE. After purification to homogeneity by IGF-I affinity chromatography and reverse-phase HPLC, sequence analysis revealed the amino-terminus of IGFBP-3, and mass spectrometry indicated a molecular mass of 12 295 Da. Analysis of the corresponding fragment generated by proteolysis of a non-glycosylated IGFBP-3 mutant indicated a molecular mass of 9855 Da, consistent with cleavage after Arg97. This suggests that the fragment derived from glycosylated IGFBP-3 contains approximately 2.5 kDa carbohydrate on Asn89. IGFBP-3[1-97] formed binary complexes with IGFs, but with reduced efficiency compared with intact IGFBP-3. IGFBP-3[1-97] at 11 nM inhibited IGF-I-stimulated DNA synthesis by 50-60% in MCF-7 breast cancer cells, similar to the inhibition observed with the intact protein. In the absence of IGF-I, DNA synthesis was inhibited by IGFBP-3[1-97], but not intact IGFBP-3. This suggests that the IGFBP-3 protease in MCF-7 cell medium can generate an inhibitor of IGF-dependent and independent breast cancer cell growth.
Salahifar
H
H
Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St Leonards, New South Wales 2065, Australia.
Firth
S M
SM
Baxter
R C
RC
Martin
J L
JL
D015153:Blotting, Western; D001943:Breast Neoplasms / Q000737:chemistry* / Q000201:enzymology; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000737:chemistry*; D013058:Mass Spectrometry; D011485:Protein Binding; D020539:Sequence Analysis, Protein; D014407:Tumor Cells, Cultured
D018972:Insulin-Like Growth Factor Binding Protein 3