1 The Natural Cell-Penetrating Peptide Crotamine Targets Tumor 2 Tissue in Vivo and Triggers a Lethal Calcium-Dependent Pathway 3 in Cultured Cells 4 Fabio D. Nascimento, †,‡ Lucie Sancey, ‡,§,∥ Alexandre Pereira, ⊥ Claire Rome, §,∥ Vitor Oliveira, # 5 Eduardo B. Oliveira, ∇ Helena B. Nader, ○ Tetsuo Yamane, ◆ Irina Kerkis, ⊥ Ivarne L. S. Tersariol, ¶ 6 Jean-Luc Coll,* ,§,∥ and Mirian A. F. Hayashi* ,+ 7 † Grupo de Estudos em Odontologia, Universidade Bandeirante de Sa ̃ o Paulo (UNIBAN), Sã o Paulo, SP, Brazil 8 § INSERM U823, Institut Albert Bonniot, Grenoble, France 9 ∥ University Joseph Fourier, Grenoble, France 10 ⊥ Laboratório de Gene ́ tica, Instituto Butantan, Sã o Paulo, SP, Brasil 11 # Departamento de Biofísica, Universidade Federal de Sa ̃ o Paulo (UNIFESP), Sã o Paulo, Brazil 12 ∇ Departamento de Bioquímica e Imunologia, Universidade de Sa ̃ o Paulo (USP), Ribeirã o Preto, Brazil 13 ○ Departamento de Bioquímica, Universidade Federal de Sa ̃ o Paulo (UNIFESP), Sã o Paulo, Brazil 14 ◆ Laboratório de Bioquímica e Biologia Molecular, Centro de Biotecnologia da Amazônia (CBA), Manaus, AM, Brazil 15 ¶ Centro Interdisciplinar de Investigac ̧ ã o Bioquímica (CIIB), Universidade de Mogi das Cruzes, Mogi das Cruzes, SP, Brazil 16 + Departamento de Farmacologia, Universidade Federal de Sa ̃ o Paulo (UNIFESP), Sã o Paulo, SP, Brazil 17 * S Supporting Information 18 ABSTRACT: Our goal was to demonstrate the in vivo tumor 19 specific accumulation of crotamine, a natural peptide from the 20 venom of the South American rattlesnake Crotalus durissus 21 terrif icus, which has been characterized by our group as a cell 22 penetrating peptide with a high specificity for actively 23 proliferating cells and with a concentration-dependent 24 cytotoxic effect. Crotamine cytotoxicity has been shown to 25 be dependent on the disruption of lysosomes and subsequent 26 activation of intracellular proteases. In this work, we show that 27 the cytotoxic effect of crotamine also involves rapid intra- 28 cellular calcium release and loss of mitochondrial membrane potential as observed in real time by confocal microscopy. The 29 intracellular calcium overload induced by crotamine was almost completely blocked by thapsigargin. Microfluorimetry assays 30 confirmed the importance of internal organelles, such as lysosomes and the endoplasmic reticulum, as contributors for the 31 intracellular calcium increase, as well as the extracellular medium. Finally, we demonstrate here that crotamine injected 32 intraperitoneally can efficiently target remote subcutaneous tumors engrafted in nude mice, as demonstrated by a noninvasive 33 optical imaging procedure that permits in vivo real-time monitoring of crotamine uptake into tumor tissue. Taken together, our 34 data indicate that the cytotoxic peptide crotamine can be used potentially for a dual purpose: to target and detect growing tumor 35 tissues and to selectively trigger tumor cell death. 36 KEYWORDS: mitochondria, cellular uptake, tumor-specific cell-penetrating peptides, actively proliferating cells, intracellular calcium 37 ■ INTRODUCTION 38 Molecular imaging and therapy for cancer would greatly benefit 39 from specific targeting of contrast agents and therapeutic drugs 40 to tumor tissues. 1 Currently, the main strategies are based on 41 antibodies against surface markers or ligands for receptors and 42 proteins preferentially expressed on the surface of cells in the 43 target tissue. 2−4 Although antibodies can successfully target 44 tumors, 5 their use is hindered by their bulky size 6 and by the 45 genetic instability of tumor cells. 7 The payload ratio between 46 the targeting device and its cargo can be amplified by 47 incorporating the probe into polymers or nanoparticles. 8,9 48 Small specific ligands like RGD peptides are also being actively 49 explored and will contribute to the definition of targeted 50 delivery systems. 10,11 Moreover, low cell penetration ratios of 51 anticancer drugs into tumors have been an important limiting Received: February 3, 2011 Revised: October 17, 2011 Accepted: December 5, 2011 Article pubs.acs.org/molecularpharmaceutics © XXXX American Chemical Society A dx.doi.org/10.1021/mp2000605 | Mol. Pharmaceutics XXXX, XXX, XXX−XXX dmadmin | MPSJCA | JCA10.0.1408/W Unicode | mp-2011-000605.3d (R2.0.i4 HF02:2745 | 2.0 alpha 36) 2011/11/15 15:21:00 | PROD-JCA1 | rq_33308 | 12/12/2011 16:59:03 | 11