Development of a cytotoxic peptide based on the C-terminal domain of Bax

Rebecca J. Boohaker1†, Ge Zhang1†, Michael W. Lee2, Kathleen N. Nemec1, Santimukul Santra3, J. Manuel Perez3 and Annette R. Khaled1*

Burnett School of Biomedical Sciences and 2Medical Education, College of Medicine, and 3Nanoscience Technology Center, University of Central Florida, Orlando, FL 32827.

Abstract: The Bcl-2 family proteins maintain the balance of life and death

in the cell. Bax, a pro-apoptotic member, translocates to the mitochondrial

membrane in response to diverse death stimuli, initiated a cascade of

programmed vents that lead to cell death. Tumor progression involves

deregulation of this process in part through mutations that cause aberrant

expression of anti-apoptotic members of the Bcl-2 family that normally

sequester and inhibit Bax. This study seeks identify functional domains of

Bax that can be developed into therapeutic peptides to promote the death of

cancer cells resistant to existing chemotherapeutics. Our goal was to generate

a peptide, much like antimicrobial peptides, that could target and disrupt

mitochondrial membranes. To this end, we discovered that the C-terminal,

alpha-9 helix of Bax (CT20p), when tagged to EGFP or a destabilization

domain (DD), could bind mitochondria and cause cell death. The peptide’s

lethal mode of action was linked to increased mitochondrial membrane

potential, and eventual membrane rupture without the characteristic

membrane asymmetry associated with apoptosis. Targeted mutagenesis of

two adjacent lysines at the carboxyl end of CT20p demonstrated that these

residues enabled mitochondrial membrane association. Expression of the

CT20 peptide in the presence or absence of endogenous Bax resulted in

cytotoxicity, suggesting that the death mechanism involved could be

independent of the Bcl-2 family. These findings suggest that peptide

composition of the a9-helix of Bax is sufficient to induce mitochondrial

membrane binding and cell death via a mechanism not typically associated

with apoptosis. This indicated that CT20p has tpotential to be developed into

a viable therapeutic agent in the treatment of a broad range of cancer types.

Conclusions:

This research is supported by NIH grant GM083224

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Figure 1. Lysines in CT20p, which is Based on the C-terminus

of Bax, Contribute to Intracellular Localization (A)

Mitochondrial translocation of HA-tagged wild type Bax (Bax-

KK) and K189/K190 mutants was examined by immunoblot.

(B) The mitochondrial translocation of DD-tagged Bax full-

length (FL-Bax) and DD-tagged CT20 peptides, wild-type and

EE, LL and RR mutants, was examined in Bax+/+ HCT-116 cells

by immunoblot.

Figure 2. CT20p causes the Release of Sequestered Contents

from Mitochondrial-like Lipid Vesicles without Loss of

Membrane Integrity. (A) CT20p was commercially synthesized

and calcein-loaded mitochondrial-like LUVs prepared. Calcein

release from CT20p-treated LUVs was measured as described in

Experimental Procedures. Blue lines indicate addition of LUVs.

and Red lines (except for control) indicate treatment with CT20p or

mutants. CT20 is the wild-type peptide and CT20-LL and CT20-EE

are mutations of the double lysines (K189/K190 in the full-length

Bax protein). (B) Light scatter analysis of LUVs from (A).

Figure 3. CT20p can be Encapsulated in NPs for

Delivery to Cells. (A) Schematic representation of the

three dimensional structure of aliphatic hyperbranched

NPs. (B) HCT-116 cells were treated with NPs loaded

with DiI or DiI + CT20p (0.07 nM) for 24 hours. Time-

lapse movies were acquired using a 10x air objective. (C)

Bax+/+ and Bax-/- HCT-116 cells were treated with NPs

loaded with DiI at amounts of 5, 10 and 15 g for 24

hours and cell death was measured using Sytox AAD.

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COOH-NPs

Figure 5. CT20p-NPs Kill Breast Cancer Cells. (A,C) MCF-7 (A) or MDA-MB-

231 (C) cells were treated with AM- or COOH-NPs loaded with CT20p (350 pM) for

24 hours. To visualize mitochondria, cells were treated with MitoTracker Red 580 and

time-lapse movies were acquired as described in Experimental Procedures using a 40x

Oil objective. (B, D) MCF-7 (B) or MDA-BB-231 (D) cells were treated CT20p-NPs

(350 pM) and, after three hours, cell death was measured using Sytox AAD.

Figure 6. Mechanism of Death Mediated by CT20p is Effector

Caspase Independent and Resistant to Bcl-2. (A) MDA-MB-231

cells were treated with COOH-NPs encapsulatd with CT20p (350 pM)

and treated with ZVAD-Fmk and/or CDDP. Cells were also

transiently transfected with Bcl-2. Cell death was assayed with Sytox

AAD. (B) MDA-MB-231 cells were treated and membrane

asymmetry was measured with a violet radiometric probe. Dot blots

show a combination of results from Sytox (cell death) and changes in

membrane symmetry. (C) Mitochondrial membrane potential was

measured by JC1 and cells were assayed for changes in polarization.

Figure 7. CT20p Induces Tumor Cell Death in Situ (A)

MDA-MB-231cells were implanted in nude mice and

changes in tumor volume assessed by ultrasound as

described in Methods. Mice were treated either two

intratumoral (IT) or one intravenous (IV) injections of

unloaded NPs (control) and CT20p-NPs. Graph is

representative of at least 4 mice per group. (B)

Representative ultrasound image of tumor regression

induced by treatment with the CT20 peptide. Results

shown are representative of n=4. #2010

The last 20 residues of the C-terminus of Bax contains a

number of the features of antimicrobial peptides which allow

the CT20p to convey cytotoxicity in the absence of death

signals.

The mechanism of action of the CT20p results in a sequence

of events that includes membrane rupture and changes in

membrane polarization, but does not resemble typical apoptosis

The solubility properties, specifically the amphipathic nature,

of the CT20p allow for encapsulation into HBPE nanoparticles

for delivery into cells.

Cytotoxicity in vitro is not mitigated by the absence of pro-

apoptotic proteins, nor is it inhibited by over expression of Bcl-

2 or caspase inhibition. This cytotoxicity is evident in the

tumor regression see in vivo

Use of CT20p in combination with available apoptosis

inducing agents enhance the cytotoxic effects, indicating it’s

potential as a therapeutic.