An N-terminal fragment of insulin-like growth factor binding protein-3 (IGFBP-3) induces apoptosis in human prostate cancer cells in an IGF-independent manner

OBJECTIVE IGF-binding protein-3 (IGFBP-3) can induce apoptosis in human prostate cancer cells by direct, IGF-independent mechanisms that are poorly understood. IGFBP-3 undergoes limited proteolysis by plasmin and other proteases to generate small N-terminal fragments (e.g., amino acids 1-97) that have lost their affinity for IGF-I and IGF-II yet still can inhibit mitogenesis. The present study examines whether the N-terminal 1-97-IGFBP-3 fragment can induce apoptosis in human prostate cancer cells in an IGF-independent manner. DESIGN N-terminal 1-97-IGFBP-3 with or without a signal prepeptide was fused to yellow fluorescent protein (YFP) and expressed in PC-3 human prostate cancer cells. In some cases, the N-terminal IGF-binding site was mutated. Subcellular localization was determined by confocal microscopy. Loss of cell viability was determined by Annexin V-APC staining in the presence and absence of a general caspase inhibitor, z-VAD-fmk. RESULTS All of the fusion proteins, including those synthesized with a signal peptide, were predominantly intracellular, suggesting that they had been internalized following secretion. YFP-1-97-IGFBP-3 is present at comparable concentrations in the nucleus and cytoplasm, indicating that it does not contain a nuclear localization signal. Cells transfected with YFP-1-97-IGFBP-3 lost viability. Cell death was blocked by incubation with a caspase inhibitor suggesting that it resulted from apoptosis. Similar results were obtained with YFP-1-97-IGFBP-3 mutants that do not bind IGFs. CONCLUSIONS The N-terminal 1-97-IGFBP-3 fragment induces apoptosis in human prostate cancer cells in an IGF-independent manner. Generation of the fragment might contribute to the proapoptotic activity of IGFBP-3 in vivo.