crotamine and cyanine-dye-3

We show here that crotamine, a polypeptide from the South American rattlesnake venom with cell penetrating and selective anti-fungal and anti-tumoral properties, presents a potent anti-plasmodial activity in culture. Crotamine inhibits the development of the Plasmodium falciparum parasites in a dose-dependent manner [IC50 value of 1.87 μM], and confocal microscopy analysis showed a selective internalization of fluorescent-labeled crotamine into P. falciparum infected erythrocytes, with no detectable fluorescence in uninfected healthy erythrocytes. In addition, similarly to the crotamine cytotoxic effects, the mechanism underlying the anti-plasmodial activity may involve the disruption of parasite acidic compartments H(+) homeostasis. In fact, crotamine promoted a reduction of parasites organelle fluorescence loaded with the lysosomotropic fluorochrome acridine orange, in the same way as previously observed mammalian tumoral cells. Taken together, we show for the first time crotamine not only compromised the metabolism of the P. falciparum, but this toxin also inhibited the parasite growth. Therefore, we suggest this snake polypeptide as a promising lead molecule for the development of potential new molecules, namely peptidomimetics, with selectivity for infected erythrocytes and ability to inhibit the malaria infection by its natural affinity for acid vesicles.

Topics: Acridine Orange; Amino Acid Sequence; Animals; Antimalarials; Biological Transport; Carbocyanines; Cell-Penetrating Peptides; Cells, Cultured; Chloroquine; Crotalid Venoms; Crotalus; Dose-Response Relationship, Drug; Erythrocytes; Fluorescent Dyes; Humans; Hydrogen-Ion Concentration; Inhibitory Concentration 50; Plasmodium falciparum; Snake Venoms; Staining and Labeling; Vacuoles

Herein we report that crotamine, a small lysine- and cysteine-rich protein from the venom of the South American rattlesnake, can rapidly penetrate into different cell types and mouse blastocysts in vitro. In vivo crotamine strongly labels cells from mouse bone marrow and spleen and from peritoneal liquid, as shown by fluorescent confocal laser-scanning microscopy. Nuclear localization of crotamine was observed in both fixed and unfixed cells. In the cytoplasm, crotamine specifically associates with centrosomes and thus allows us to follow the process of centriole duplication and separation. In the nucleus, it binds to the chromosomes at S/G2 phase, when centrioles start dividing. Moreover, crotamine appears as a marker of actively proliferating cells, as shown by 5-BrdU cell-proliferation assay. Crotamine in the micromolar range proved nontoxic to any of the cell cultures tested and did not affect the pluripotency of ES cells or the development of mouse embryos.

Topics: Animals; Biomarkers; Carbocyanines; Cell Cycle; Cell Line; Cell Membrane Permeability; Cell Nucleus; Cell Proliferation; Centrioles; Centrosome; Chromosomes, Mammalian; Crotalid Venoms; Crotalus; Embryo, Mammalian; Embryo, Nonmammalian; Humans; Mice; Stem Cells