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.


METHODS
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.
Shahjee
H
H
Diabetes Branch, NIDDK, National Institutes of Health, Bldg 10-Room 8D12, 9000 Rockville Pike, MSC 1758, Bethesda, MD 20892, United States.
Bhattacharyya
N
N
Zappala
G
G
Wiench
M
M
Prakash
S
S
Rechler
M M
MM
D017209:Apoptosis; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D008297:Male; D010446:Peptide Fragments / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011471:Prostatic Neoplasms / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013002:Somatomedins / Q000378:metabolism
D018972:Insulin-Like Growth Factor Binding Protein 3; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D013002:Somatomedins