Insights into the uptake mechanism of NrTP, A cell-penetrating peptide preferentially targetingthe nucleolus of tumour cells

Abstract

Nucleolar targeting peptides are 14–15 residue-longsequences designed by structural minimization of asnake toxin (J Med Chem 2008;50:7041). Peptidessuch as NrTP1 (YKQCHKKGGKK GSG) and ana-logues are capable of penetrating human cervix epi-thelial carcinoma cells and homing into theirnucleoli. We now show that NrTP1 similarly pene-trates and localizes in the nucleolus of tumour cellsderived from human pancreatic (BxPC-3) and humanductal mammary gland (BT-474) carcinomas. Livecell confocal microscopy imaging, combined withflow cytometry analysis of cells arrested to definedphases of their cycle, confirms that NrTP1 uptakeand nucleolar homing are independent of cell cyclephase. Peptide uptake is significantly reduced atlow temperature. Also, drugs inhibiting chlatrin-mediated endocytosis severely decrease uptake,pointing to a clathrin-dependent route as the pri-mary NrTP1 internalization mechanism. Theseresults highlight nucleolar targeting peptides notonly as a novel and efficient class of cell-penetratingpeptides but also for their exceptional ability to tar-get preferentially an essential and dynamic subnu-clear structure such as the nucleolus