title: Bovine lactoferricin causes apoptosis in Jurkat T-leukemia cells by sequential permeabilization of the cell membrane and targeting of mitochondria.
abstract: Bovine lactoferricin (LfcinB) is a cationic antimicrobial peptide that kills Jurkat T-leukemia cells by the mitochondrial pathway of apoptosis. However, the process by which LfcinB triggers mitochondria-dependent apoptosis is not well understood. Here, we show that LfcinB-induced apoptosis in Jurkat T-leukemia cells was preceded by LfcinB binding to, and progressive permeabilization of the cell membrane. Colloidal gold electron microscopy revealed that LfcinB entered the cytoplasm of Jurkat T-leukemia cells prior to the onset of mitochondrial depolarization. LfcinB was not internalized by endocytosis because endocytosis inhibitors did not prevent LfcinB-induced cytotoxicity. Furthermore, intracellular delivery of LfcinB via fusogenic liposomes caused the death of Jurkat T-leukemia cells, as well as normal human fibroblasts. Collectively, these findings suggest that LfcinB caused damage to the cell membrane that allowed LfcinB to enter the cytoplasm of Jurkat T-leukemia cells and mediate cytotoxicity. In addition, confocal microscopy showed that intracellular LfcinB co-localized with mitochondria in Jurkat T-leukemia cells, while flow cytometry and colloidal gold electron microscopy showed that LfcinB rapidly associated with purified mitochondria. Furthermore, purified mitochondria treated with LfcinB rapidly lost transmembrane potential and released cytochrome c. We conclude that LfcinB-induced apoptosis in Jurkat T-leukemia cells resulted from cell membrane damage and the subsequent disruption of mitochondrial membranes by internalized LfcinB.
authors.affiliation: Department of Pathology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada.
authors.forename: Jamie S
authors.initials: JS
authors.lastname: Mader
authors.forename: Angela
authors.initials: A
authors.lastname: Richardson
authors.forename: Jayme
authors.initials: J
authors.lastname: Salsman
authors.forename: Deniz
authors.initials: D
authors.lastname: Top
authors.forename: Roberto
authors.initials: R
authors.lastname: de Antueno
authors.forename: Roy
authors.initials: R
authors.lastname: Duncan
authors.forename: David W
authors.initials: DW
authors.lastname: Hoskin
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D019921:Biotinylation; D002417:Cattle; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects*; D045304:Cytochromes c / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D004705:Endocytosis / Q000187:drug effects; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects; D006801:Humans; D019169:Jurkat Cells; D007781:Lactoferrin / Q000494:pharmacology*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D011956:Receptors, Cell Surface / Q000378:metabolism; D013601:T-Lymphocytes / Q000166:cytology* / Q000187:drug effects* / Q000648:ultrastructure
chemical_list: D011956:Receptors, Cell Surface; C082171:lactoferricin B; D045304:Cytochromes c; D007781:Lactoferrin