Cell-Penetrating Penta-Peptides (CPP5s): Measurement of Cell Entry and Protein-Transduction Activity

Previously, we developed cell-penetrating penta-peptides (CPP5s). In the present study, VPTLK and KLPVM, two representative CPP5s, were used to characterize the cellpenetration and protein-transduction activities of these small molecules. Various inhibitors of endocytosis and pinocytosis (chlorpromazine, cytochalasin D, Filipin III, amiloride, methyl-beta-cyclodextrin, and nocodazole) were tested. Only cytochalasin D showed suppression of CPP5 entry, though the effect was partial. In addition, CPP5s were able to enter a proteoglycandeficient CHO cell line. These results suggest that pinocytosis and endocytosis may play only a minor role in the cell entry of CPP5s. By mass spectrometry, we determined that the intracellular concentration of VPTLK ranged from 20 nM to 6.0 mu M when the cells were cultured in medium containing 1 mu M -1.6 mM VPTLK. To determine the protein-transduction activity of CPP5s, the Tex-LoxP EG cell line, which has a Cre-inducible green fluorescent protein (GFP) gene, was used. VPTLK and KLPVM were added to the N-terminus