Development of a cytotoxic peptide based on the C-terminal ...
Transcript of Development of a cytotoxic peptide based on the C-terminal ...
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
DD
-CT
20 L
L
DD
-CT
20 R
R B.
*
DD
-FL
-Bax
DD
-CT
20
DD
-CT
20 E
E
DD
-CT
20 L
L
DD
-CT
20 R
R
DD
-Vec
tor
CO
NT
RO
L
DD
-FL
-Bax
DD
-CT
20
DD
-CT
20 E
E
DD
-Vec
tor
CO
NT
RO
L
p38
DD-FL-Bax
Prohibitin
Endogenous Bax
DD-Bax-CT20
36 kD 36 kD
22 kD
22 kD
16 kD
A.
Control A. CT20p CT20p-LL CT20p-EE
B. Control CT20p CT20p-LL CT20p-EE
Triton-X100
LUVs
LUVs+CT20
LUVs LUVs+CT20-LL
LUVs
LUVs+CT20-EE
LUVs
Cal
cein
rel
ease
L
ight
scat
teri
ng
A.
B.
C.
Bax
+/+
HC
T 1
16
B
ax -/
- H
CT
116
0hr 24hr (DiI 5hr (DiI + CT20p ) 4hr (DiI + CT20p)
0hr 24hr (DiI) 5hr (DiI + CT20p) 24hr (DiI + CT20p)
Bax +/+ HCT 116 Bax -/- HCT 116
A.
B. Untreated
Treatment +Z-VAD +Bcl-2
SY
TO
X
Membrane Asymmetry
SY
TO
X
Membrane Asymmetry
CT20p-
NPs
CDDP
CDDP
+
CT20p
NPs
4.6% 1.7%
4.6% 89.2%
3.0% 39.5%
55.7% 1.9%
6.8% 17.3%
8.9% 67.0%
21.3% 38.5%
14.9% 25.2%
9.0% 48.9%
5.7% 36.4%
7.8% 22.3%
2.0% 67.9%
3.0% 3.0%
2.6% 91.3%
17.5% 41.1%
2.4% 39.0%
25.0% 22.2%
10.3% 42.5%
2.4% 1.7%
1.2% 94.7%
SY
TO
X
Membrane Asymmetry
Live
Necrotic
(Dead Cells)
Apop
C.
H
I L
56%
36%
37%
52%
71%
22%
Untreated Empty NPs CT20p-NPs m
A.
B.
2.1mm
8.9mm 9.2 5.4mm
Day 0 (IV Inject) Day 2 Day 9
Day 0 (IT Inject) Day 2 (IT Inject) Day 4
CT
20
p-N
Ps
CT
20
p-N
Ps
Em
pty
-NP
s
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.
MC
F-7
0hr 12hr 24hr
0hr 24hr
Mit
otr
acker
D
IC
Mit
otr
acker
A.
DIC
12hr
Empt y NPs CT20p/Am-NPs CT20p/
COOH-NPs
Empty NPs CT20p/AM- NPs CT20p/COOH-NPs B.
MD
A-M
B-2
31
0hr 12hr 19hr
0hr 12hr 21hr
C.
Empty NPs CT20p/AM- NPs CT20p/COOH-NPs D.
0hr 12hr 24hr
0hr 24hr 12hr
Empt y NPs CT20p/Am-NPs CT20p/
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.