Insights into the Uptake Mechanism of NrTP, A Cell-Penetrating Peptide Preferentially Targeting the Nucleolus of Tumour Cells

• Published in: Chemical biology & drug design Vol. 79; no. 6; pp. 907 - 915
• Main Authors: Rádis-Baptista, Gandhi, de la Torre, Beatriz G., Andreu, David
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.06.2012; Wiley
• Subjects: Amino Acid Sequence; Biochemistry & Molecular Biology; Cell Cycle Checkpoints - drug effects; Cell Line, Tumor; Cell Nucleolus - metabolism; cell-penetrating peptide; Cell-Penetrating Peptides - chemical synthesis; Cell-Penetrating Peptides - chemistry; Cell-Penetrating Peptides - pharmacology; Chemistry, Medicinal; Clathrin - metabolism; clathrin-mediated endocytosis; Endocytosis - drug effects; HeLa Cells; Humans; Life Sciences & Biomedicine; nucleolar localization sequence; Pharmacology & Pharmacy; Science & Technology; theranostic peptide; tumour cell target; LOCALIZATION; theranostic peptide; PROTEOGLYCANS; nucleolar localization sequence; PROTEIN; CROTAMINE; TRANSDUCTION; IDENTIFICATION; cell-penetrating peptide; PLASMA-MEMBRANE; clathrin-mediated endocytosis; DELIVERY; INTERNALIZATION; tumour cell target; MOLECULAR-MECHANISMS
• ISSN: 1747-0277; 1747-0285
• DOI: 10.1111/j.1747-0285.2012.01377.x

Nucleolar targeting peptides are 14–15 residue‐long sequences designed by structural minimization of a snake toxin (J Med Chem 2008;50:7041). Peptides such as NrTP1 (YKQCHKKGGKK GSG) and analogues are capable of penetrating human cervix epithelial carcinoma cells and homing into their nucleoli. We now show that NrTP1 similarly penetrates and localizes in the nucleolus of tumour cells derived from human pancreatic (BxPC‐3) and human ductal mammary gland (BT‐474) carcinomas. Live cell confocal microscopy imaging, combined with flow cytometry analysis of cells arrested to defined phases of their cycle, confirms that NrTP1 uptake and nucleolar homing are independent of cell cycle phase. Peptide uptake is significantly reduced at low temperature. Also, drugs inhibiting chlatrin‐mediated endocytosis severely decrease uptake, pointing to a clathrin‐dependent route as the primary NrTP1 internalization mechanism. These results highlight nucleolar targeting peptides not only as a novel and efficient class of cell‐penetrating peptides but also for their exceptional ability to target preferentially an essential and dynamic subnuclear structure such as the nucleolus. Nucleolar targeting peptides (NrTPs) are 14–15 residue‐long sequences designed by structural minimization of a rattlesnake toxin, capable of penetrating mammalian cells and homing into their nucleoli. One of such developed sequences (NrTP1) penetrates and localizes in the nucleolus of cell lines derived from aggressive types of tumours. Using a combination of confocal microscopy, flow cytometry and pharmacological inhibitors, we demonstrated that peptide uptake is mediated by a chlatrin‐dependent endocytosis, but the translocation is not associated to the cell cycle phase.