(a,b) Suppression of tumors induced by subcutaneous injection of (a) HCT116 tumor cells or (b) HCT116 tumor explants. After tumors reached a size of ~120 mm3 (start), the mice were injected … Discussion Protein transduction approaches for systemic therapeutic delivery of proteins and peptides have been hampered read me due to inefficient cytoplasmic delivery of internalized proteins and poor tissue penetration.10,11 In the present study, we identified several new hydrophobic MTDs, capable of enhancing protein uptake by cultured cells and in animal tissues. We used one of these sequences, MTD103, to deliver biologically active p18INK4c systemically in mice, and significantly inhibited tumor growth in a colon cancer xenograft model.

This study is the first to describe a functional cell-permeable p18INK4c and establishes MTD103 as a potential delivery vehicle for protein-based therapeutics. Tumor suppression using CP-p18INK4c strictly required the MTD103 sequence and inhibition of tumor growth continued for at least 2 weeks after protein therapy was terminated. The therapeutic response was accompanied by high levels of tumor cell apoptosis including the activation of pro-apoptotic pathways (p53, p21, and Caspase-3), and suppression of prosurvival proteins (Bcl2, XIAP and ICAM-1). In addition to targeting tumor cells, it is possible that the therapeutic activity of CP-p18INK4c may benefit from targeting other cells or processes (e.g., angiogenesis) that influence tumor cell survival.

The antitumor activity of HM103p18 exceeded that of previously described cell-permeable INK4 and Cip/Kip Cyclin-dependent kinase AV-951 inhibitors,24,25,26,27,28,29 all of which employed the HIV Tat PTD. Several considerations suggest the greater in vivo activity of HM103p18 resulted from enhanced protein delivery mediated by MTD103 and not from some special attribute of the p18INK4c cargo as compared to other CKIs. The MTD103 enhanced bidirectional transfer of p18INK4c across the plasma membrane circumventing a major limitation of the cationic PTDs, whose predominant mechanisms of protein uptake��absorptive endocytosis and macropinocytosis��sequester significant amounts of protein into membrane-bound and endosomal compartments. The MTD103 sequence also extended the half-life of recombinant p18INK4c proteins in vivo, presumably due to enhanced stability and/or decreased plasma clearance of internalized p18INK4c as compared to extracellular protein. MTD103 joins a growing number of hydrophobic sequences that have been used to enhance the delivery of proteins into mammalian cells. These include MTD39, MTD41, MTD52, MTD58, MTD68, and MTD101 (Supplementary Table S1); MTD76 and MTD77;18 and signal sequence-derived peptides from integrin ��3 and FGF4.