12,13,14,15,16,17 Our findings in this study together selleck inhibitor with our earlier report using cell-permeable NM23 metastasis suppressor 18 highlight the potential of protein delivery approaches in cancer therapeutics. The limited vascularization of subcutaneous tumors provides a challenging test of in vivo protein delivery and uptake. The widespread tissue distribution and biological activity of HM103p18 against subcutaneous tumors, particularly after intraperitoneal administration, illustrates the ability of MTD-containing peptides and proteins to penetrate multiple cell and tissue barriers. Previous studies suggest the FGF4 hydrophobic MTS peptide penetrates the plasma membrane directly19 after inserting into the membranes in a ��bent�� configuration with hydrophobic sequences adopting an ��-helical conformation.

20 The present study provides the first evidence that a similar mechanism can mediate MTD-dependent uptake of larger protein cargos. In particular, we show that the uptake of HM103p18 is sensitive to low temperature, does not require microtubule reorganization, is not enhanced by agents that disrupts the plasma membrane, and does not utilize ATP. Furthermore, HM103p18 traverses artificial bilayers consisting of cholesterol and phospholipid and is capable of bidirectional movement across membranes as assessed by cell-to-cell protein transfer. Properties of MTD103 required for efficient cellular uptake and systemic delivery in vivo are clearly shared by a number of peptide sequences ranging from 7�C10 amino acids in size.

However, additional studies will be required to confirm direct transfer to the cytosol and to assess the extent to which protein uptake involves other, potentially competing, mechanisms and is influenced by the cargo and such nonspecific factors as protein concentration, aggregation, and solubility. In addition, we are currently trying to identify the optimal sequence and/or structural determinants for tissue delivery/uptake and assess potential contributions by cargo sequences. Like the FGF4 MTS,20 MTD103 is predicted to adopt a helical conformation (Supplementary Figure S9). In contrast, the PTDs from Tat, Hph-1, and Ant are predicted to adopt random coils. However, any understanding of structure-activity relationships will require experimentally determined protein structures and data on a larger number of MTD sequences.

While the hydrophobic MTD103 sequence was strictly required for efficient cellular uptake and systemic delivery in vivo, potential contributions by protein cargo sequences cannot be excluded and could be important determinants of tissue penetration and/or in vivo bioavailability. The antitumor activity of cell-permeable p18INK4c, although striking when compared to previous cell-permeable CKI proteins, still fell short of Drug_discovery that reported for small-molecule CDK4/6 inhibitors, such as PD 0332991.