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 anti-microbial 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 of colon and breast cancer cells in vitro 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 Bcl-2 over-expression, suggesting that it was independent 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.

Bak apoptotic pores involve a flexible C-terminal region and juxtaposition of the C-terminal transmembrane domains

“…In addition, we examined whether the α9 helix (or the α9:α9 interface) might be necessary for the step of pore formation, as α9 appears to be the only region that traverses the MOM before and after pore formation, [21][22][23] and α9 peptides have been proposed to be membranolytic with antitumor activity. 43,58 Thus, understanding α9 membrane topology may reveal novel insight for cancer therapy.…”

The CT20 peptide: More than a Piece of Bax

“…The major death response triggered by CT20p does not appear to be a purely apoptotic process as inhibition of caspases only provided a slight increase in cell viability and did not rescue cells treated with CT20p [4] . We surmise that Bax C-terminal derived peptides, while still cytotoxic, may act differently than full-length Bax to trigger cell death.…”

“…Furthermore, localization of CT20p to the mitochondria and cell-killing occurred in cells deficient in Bax or in which Bcl-2 was over expressed, suggesting that CT20p does not require apoptosis-associated proteins for localization or function [4] . The ability of CT20p to localize to the mitochondria may in part be due to the -KKMG amino acid motif which has similarity to consensus mitochondrial targeting sequences [12] .Within hours of cell entry, CT20p promoted mitochondrial hyper polarization and aggregation and impaired mitochondrial movement [11] .…”

“…This approach takes advantage of the information available on the localization and function of the protein from which the peptide is derived and could inform of the potential [4][5][6]11] molecular target and action of the peptide. Recently, we reported that a 20 amino acid -helical peptide developed from the 9 transmembrane domain of the pro-apoptotic protein, Bax, has cytotoxic actions for cancer cells [4] . This peptide, henceforth referred to as CT20p, is amphipathic and shares similarities with anti-microbial peptides (AMPs), such as the presence of a tryptophan and two Cterminal lysines.…”

“…This peptide, henceforth referred to as CT20p, is amphipathic and shares similarities with anti-microbial peptides (AMPs), such as the presence of a tryptophan and two Cterminal lysines. While CT20p is capable of forming pores in mitochondrial-like lipid vesicles, in mammalian cells the peptide targets mitochondria and disrupts the cytoskeleton in a manner that causes cell detachment and death [4][5][6] . Hence, while derived from an apoptotic protein and retaining cytotoxic activity, CT20p, due to its inherent features, may have unique actions unrelated to the parent protein that could have significant clinical impact.…”