Previous studies have indicated that proteins that bind hyaluronan can also inhibit the growth of tumor cells. To determine if synthetic peptides also possessed these properties, we tested a series of polypeptides containing structural motifs from different proteins for their ability to bind [(3)H]hyaluronan, and identified one compound termed P4 that had a particularly strong interaction. Further studies revealed that P4 also inhibited the growth of tumor cells in tissue culture as well as on the chorioallantoic membranes of chicken embryos. In addition, expression vectors for P4 caused tumor cells to grow slower in nude mice and reduced their vascularization. The P4 peptide also inhibited VEGF-induced angiogenesis in the chorioallantoic membranes of chicken embryos. Studies on cultured cells indicated that P4 induced apoptosis, which was blocked by a pan-caspase inhibitor. Confocal microscopy revealed that shortly after its uptake, P4 became associated with mitochondria. Immunoprecipitation indicated that P4 could bind to Bcl-2 and Bcl-x(L), which are associated with mitochondria and regulate apoptosis. This was also supported by the fact that P4 induced the release of cytochrome c from preparations of mitochondria. Taken together, these results suggest that P4 binds to Bcl-2 and related proteins and this activates the apoptotic cascade.
Copyright 2003 Wiley-Liss, Inc.