The Natural Cell-Penetrating Peptide Crotamine Targets Tumor Tissue in Vivo and Triggers a Lethal Calcium-Dependent Pathway in Cultured Cells

• Published in: Molecular pharmaceutics 2012-02, Vol.9 (2), p.211-221
• Main Authors: Nascimento, Fabio D, Sancey, Lucie, Pereira, Alexandre, Rome, Claire, Oliveira, Vitor, Oliveira, Eduardo B, Nader, Helena B, Yamane, Tetsuo, Kerkis, Irina, Tersariol, Ivarne L. S, Coll, Jean-Luc, Hayashi, Mirian A. F
• Format: Article
• Language: English
• Subjects: Animals; Antineoplastic Agents; Calcium - metabolism; Calcium Signaling - drug effects; Cell Death - drug effects; Cell Survival - drug effects; Cell-Penetrating Peptides - administration & dosage; Cell-Penetrating Peptides - pharmacokinetics; CHO Cells; Cricetinae; Cricetulus; Crotalid Venoms - administration & dosage; Crotalid Venoms - pharmacokinetics; Crotalus; Endoplasmic Reticulum - drug effects; Endoplasmic Reticulum - metabolism; Flow Cytometry; HEK293 Cells; Humans; Injections, Intraperitoneal; Life Sciences; Lysosomes - drug effects; Lysosomes - metabolism; Membrane Potential, Mitochondrial - drug effects; Mice; Mice, Nude; Microscopy, Confocal; Neoplasms - diagnosis; Neoplasms - drug therapy; Neoplasms - metabolism; Thapsigargin - pharmacology; Tumor Cells, Cultured; Xenograft Model Antitumor Assays
• ISSN: 1543-8384; 1543-8392
• DOI: 10.1021/mp2000605

Our goal was to demonstrate the in vivo tumor specific accumulation of crotamine, a natural peptide from the venom of the South American rattlesnake Crotalus durissus terrificus, which has been characterized by our group as a cell penetrating peptide with a high specificity for actively proliferating cells and with a concentration-dependent cytotoxic effect. Crotamine cytotoxicity has been shown to be dependent on the disruption of lysosomes and subsequent activation of intracellular proteases. In this work, we show that the cytotoxic effect of crotamine also involves rapid intracellular calcium release and loss of mitochondrial membrane potential as observed in real time by confocal microscopy. The intracellular calcium overload induced by crotamine was almost completely blocked by thapsigargin. Microfluorimetry assays confirmed the importance of internal organelles, such as lysosomes and the endoplasmic reticulum, as contributors for the intracellular calcium increase, as well as the extracellular medium. Finally, we demonstrate here that crotamine injected intraperitoneally can efficiently target remote subcutaneous tumors engrafted in nude mice, as demonstrated by a noninvasive optical imaging procedure that permits in vivo real-time monitoring of crotamine uptake into tumor tissue. Taken together, our data indicate that the cytotoxic peptide crotamine can be used potentially for a dual purpose: to target and detect growing tumor tissues and to selectively trigger tumor cell death.