Tumour lineage-homing cell-penetrating peptides as anticancer molecular delivery systems.

Cell-penetrating peptides have gained attention owing to their promise in noninvasive delivery systems. Among the identified cell-penetrating peptides, the TAT peptide has been preferentially used for transduction into cells of diverse origins. However, this activity is nonselective between neoplastic and non-neoplastic cells. Here we describe artificial cell-penetrating peptides that are selectively and efficiently incorporated into human tumour cells, according to their lineage. Ten representative tumour lineage-homing cell-penetrating peptides were obtained by screening of a random peptide library constructed using messenger RNA display technology, and some of the isolates were further modified by amino-acid substitution. Their advantageous tumour cell-targeting ability is corroborated in an in vivo mouse model for imaging and growth suppression of metastatic xenoplant tumours. These cell-penetrating peptides are potentially useful for the efficient targeting of human neoplasms in a tumour origin-dependent manner, and provide a framework for the development of peptide-based anti-tumour technologies.

Isolation of novel cell-penetrating peptides from a random peptide library using in vitro virus and their modifications.

A number of cell-penetrating peptides (CPPs) have been reported, but their transduction efficiencies are too low to be used as intracellular carriers for therapeutic purposes. We conducted a comprehensive search to find novel CPPs using an in vitro virus (IVV) library, which presented random peptides consisting of 15 amino acids (diversity of the library was >10(12)). We found 9 kinds of novel CPPs with an intracellular translocation efficiency higher than that of the TAT peptide (YGRKKKRRQRRR). Interestingly, one of the novel CPPs, No. 14 (KLWMRWYSPTTRRYG), showed a dramatic improvement in translocation activity relative to the TAT peptide in CHO cells (>10-fold efficiency in 50 microM). As the intracellular translocation efficiency of No. 14 was increased by substitution Arg for Lys1 (14-1), we carried out alanine scanning on the basis of 14-1 to determine important amino acids for the intracellular translocation. The Ala substitution analysis showed that both Arg and Trp residues were important for the cell-penetrating activity and that their contribution was in the order Trp3

Amyloid formation in rat transthyretin: effect of oxidative stress.

BACKGROUND: Transgenic mice carrying a human mutant transthyretin (TTR) gene are too small for in vivo experiments. It is necessary to have rat TTR protein and its antibody to overcome this problem. METHODS: Posttranslational modification of purified TTR was analyzed by means of matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI/TOF-MS). Production of amyloid fibrils in vitro was confirmed by thioflavin T test and electron microscopy. Amyloidogenicity of rat TTR from rats with or without challenging paraquat was compared in vitro by thioflavin T test. RESULTS: MALDI/TOF-MS for rat TTR revealed three major modified forms-sulfate-conjugated, Cys-conjugated and glutathione-conjugated-in addition to the unconjugated (free) form of TTR. Although rat TTR in buffer of pH 7.0 could not make amyloid fibrils, rat TTR at pH 2.0-3.5 significantly formed amyloid fibrils, as confirmed by the thioflavin T test and electron microscopy. TTR purified from rats administered 4 mg/kg of paraquat formed much more amyloid fibrils than that from normal rats at pH 2.0-3.5 and significant amyloid fibrils were confirmed even at pH 7.0. CONCLUSIONS: Rat TTR may be a valuable experimental tool for examination of the amyloidogenicity of senile systemic amyloidosis (SSA) as well as familial amyloidotic polyneuropathy (FAP) both in vitro and in vivo.