title: A lectin from Bothrops leucurus snake venom raises cytosolic calcium levels and promotes B16-F10 melanoma necrotic cell death via mitochondrial permeability transition.
abstract: BlL, a galactose-binding C-type lectin purified from Bothrops leucurus snake venom, exhibits anticancer activity. The current study was designed to elucidate the cellular mechanisms by which BlL induces melanoma cell death. The viabilities of B16-F10 melanoma cells and HaCaT keratinocytes treated with BlL were evaluated. Necrotic and apoptotic cell death, cytosolic Ca(2+) levels, mitochondrial Ca(2+) transport and superoxide levels were assessed in B16-F10 melanoma cells exposed to BlL. We found that treatment with BlL caused dose-dependent necrotic cell death in B16-F10 melanoma cells. Conversely, the viability of non-tumorigenic HaCaT cells was not affected by similar doses of BlL. BlL-induced B16-F10 necrosis was preceded by a significant (2-fold) increase in cytosolic calcium concentrations and a significant (3-fold) increase in mitochondrial superoxide generation. It is likely that BlL treatment triggers B16-F10 cell death via mitochondrial permeability transition (MPT) pore opening because the pharmacological MPT inhibitors bongkrekic acid and Debio 025 greatly attenuated BlL-induced cell death. Experiments evaluating mitochondrial Ca(2+) transport in permeabilized B16-F10 cells strongly supported the hypothesis that BlL rapidly stimulates cyclosporine A-sensitive Ca(2+)-induced MPT pore opening. We therefore conclude that BlL causes selective B16-F10 melanoma cell death via dysregulation of cellular Ca(2+) homeostasis and Ca(2+)-induced opening of MPT pore.
authors.affiliation: Department of Clinical Pathology, Faculty of Medical Sciences, State University of Campinas, CEP 13083-887, Campinas, SP, Brazil; Department of Biochemistry, Center of Biological Sciences, Federal University of Pernambuco, Recife, PE, Brazil.
authors.forename: Mary A
authors.initials: MA
authors.lastname: Aranda-Souza
authors.affiliation: Department of Clinical Pathology, Faculty of Medical Sciences, State University of Campinas, CEP 13083-887, Campinas, SP, Brazil.
authors.forename: Franco A
authors.initials: FA
authors.lastname: Rossato
authors.affiliation: Department of Clinical Pathology, Faculty of Medical Sciences, State University of Campinas, CEP 13083-887, Campinas, SP, Brazil.
authors.forename: Rute A P
authors.initials: RA
authors.lastname: Costa
authors.affiliation: Department of Clinical Pathology, Faculty of Medical Sciences, State University of Campinas, CEP 13083-887, Campinas, SP, Brazil.
authors.forename: Tiago R
authors.initials: TR
authors.lastname: Figueira
authors.affiliation: Department of Clinical Pathology, Faculty of Medical Sciences, State University of Campinas, CEP 13083-887, Campinas, SP, Brazil.
authors.forename: Roger F
authors.initials: RF
authors.lastname: Castilho
authors.affiliation: Department of Zoology, Center of Biological Sciences, Federal University of Pernambuco, Recife, PE, Brazil.
authors.forename: Miriam C
authors.initials: MC
authors.lastname: Guarniere
authors.affiliation: Department of Biochemistry, Center of Biological Sciences, Federal University of Pernambuco, Recife, PE, Brazil.
authors.forename: Erika S
authors.initials: ES
authors.lastname: Nunes
authors.affiliation: Department of Biochemistry, Center of Biological Sciences, Federal University of Pernambuco, Recife, PE, Brazil.
authors.forename: Luana C B B
authors.initials: LC
authors.lastname: Coelho
authors.affiliation: Department of Biochemistry, Center of Biological Sciences, Federal University of Pernambuco, Recife, PE, Brazil.
authors.forename: Maria T S
authors.initials: MT
authors.lastname: Correia
authors.affiliation: Department of Clinical Pathology, Faculty of Medical Sciences, State University of Campinas, CEP 13083-887, Campinas, SP, Brazil. Electronic address: anibal@unicamp.br.
authors.forename: Anibal E
authors.initials: AE
authors.lastname: Vercesi
mesh_terms: D000818:Animals; D017837:Bothrops / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects*; D045744:Cell Line, Tumor; D003435:Crotalid Venoms / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D037102:Lectins / Q000737:chemistry / Q000494:pharmacology; D008546:Melanoma, Experimental / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins / Q000187:drug effects*; D000083162:Mitochondrial Permeability Transition Pore; D009336:Necrosis / Q000139:chemically induced / Q000473:pathology; D013481:Superoxides / Q000378:metabolism
chemical_list: D003435:Crotalid Venoms; D037102:Lectins; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D013481:Superoxides; D002118:Calcium
keywords: Bothrops leucurus lectin; Cytosolic Ca(2+); Melanoma; Mitochondrial permeability transition; Reactive oxygen species; Snake venom