title: Rational development of a cytotoxic peptide to trigger cell death.
abstract: Defects in the apoptotic machinery can contribute to tumor formation and resistance to treatment, creating a need to identify new agents that kill cancer cells by alternative mechanisms. To this end, we examined the cytotoxic properties of a novel peptide, CT20p, derived from the C-terminal, alpha-9 helix of Bax, an amphipathic domain with putative membrane binding properties. Like many antimicrobial peptides, CT20p contains clusters of hydrophobic and cationic residues that could enable the peptide to associate with lipid membranes. CT20p caused the release of calcein from mitochondrial-like lipid vesicles without disrupting vesicle integrity and, when expressed as a fusion protein in cells, localized to mitochondria. The amphipathic nature of CT20p allowed it to be encapsulated in polymeric nanoparticles (NPs) that have the capacity to harbor targeting molecules, dyes or drugs. The resulting CT20p-NPs proved an effective killer, in vitro, of colon and breast cancer cells, and in vivo, using a murine breast cancer tumor model. By introducing CT20p to Bax deficient cells, we demonstrated that the peptide's lethal activity was independent of endogenous Bax. CT20p also caused an increase in the mitochondrial membrane potential that was followed by plasma membrane rupture and cell death, without the characteristic membrane asymmetry associated with apoptosis. We determined that cell death triggered by the CT20p-NPs was minimally dependent on effector caspases and resistant to Bcl-2 overexpression, suggesting that it acts independently of the intrinsic apoptotic death pathway. Furthermore, use of CT20p with the apoptosis-inducing drug, cisplatin, resulted in additive toxicity. These results reveal the novel features of CT20p that allow nanoparticle-mediated delivery to tumors and the potential application in combination therapies to activate multiple death pathways in cancer cells.
authors.affiliation: Burnett School of Biomedical Sciences, College of Medicine, §Medical Education, College of Medicine, and ∥Nanoscience Technology Center, University of Central Florida , Orlando, Florida 32827, United States.
authors.forename: Rebecca J
authors.initials: RJ
authors.lastname: Boohaker
authors.forename: Ge
authors.initials: G
authors.lastname: Zhang
authors.forename: Michael W
authors.initials: MW
authors.lastname: Lee
authors.forename: Kathleen N
authors.initials: KN
authors.lastname: Nemec
authors.forename: Santimukul
authors.initials: S
authors.lastname: Santra
authors.forename: J Manuel
authors.initials: JM
authors.lastname: Perez
authors.forename: Annette R
authors.initials: AR
authors.lastname: Khaled
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001943:Breast Neoplasms / Q000188:drug therapy / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002945:Cisplatin / Q000494:pharmacology; D045325:HCT116 Cells; D057809:HEK293 Cells; D006801:Humans; D061986:MCF-7 Cells; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D053758:Nanoparticles / Q000008:administration & dosage; D010455:Peptides / Q000494:pharmacology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: D010455:Peptides; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D020169:Caspases; D002945:Cisplatin