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Kimmel Cancer Center, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
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ABSTRACT |
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 Top ABSTRACT IntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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-irradiation (4)
.
The members of the Bcl-2 family include both death antagonists such as
Bcl-2 and Bcl-XL and death agonists such as Bax,
Bak, Bid, and Bad. They share at least one of four homologous regions
termed
BH4
domains (BH1 to BH4). The BH3 domain of death agonists is
required for these proteins to heterodimerize with Bcl-2 and to promote
apoptosis (2
, 3)
. The three-dimensional structure of a
death antagonist Bcl-XL, a close homologue of
Bcl-2, as determined by X-ray crystallography and nuclear magnetic
resonance spectroscopy, has revealed a surface pocket for the
interaction with the BH3 domain of death agonists (5
, 6)
.
BH3 domain-derived peptides are shown to have in vitro
activity in inducing apoptosis in cell-free systems (7)
and in HeLa cells (8)
. However, their use for in
vivo control of the Bcl-2-regulated apoptotic process remains a
challenge. In addition, the therapeutic potential of using such small
molecules to intervene in tumor growth in mouse models has yet to be
demonstrated. |
Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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Peptide Synthesis and Modification.
Peptides were prepared by solid phase synthesis using Fmoc-strategy on
a 430A peptide synthesizer (Applied Biosystems, Foster City, CA) and a
9050 Pepsynthesizer Plus (Perseptive Biosystems, Cambridge, MA), as
described previously (9)
. The purity of the peptides was
>99%, as assessed by analytical reverse phase high performance liquid
chromatography, capillary electrophoresis, and matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry. The decanoid
acid (Aldrich) was incorporated into the NH2
terminus of deprotected peptide in DCM at room temperature for 2 h. Biotinylated peptides were chemically synthesized with Biotin
(Sigma)/TBTU/HoBt at room temperature after removal of Dde deprotected
N
-Lys with 2% hydrazine in N,N-dimethylformamide or
1-methyl-2-pyrrolidinone. A fluorescence-labeled peptide, Flu-BakBH3,
used in the Bcl-2 competitive binding assay, was prepared by acylation
of the resin-linked peptide with 5-carboxyfluorescein succinimidyl
ester (4 equivalents; Sigma) and triethylamine (4 equivalents in
dimethyl formamide). The coupling was carried out overnight and
followed by simultaneous side-chain deprotection and resin cleavage
with a mixture of trifluoroacetic acid, thioanisole,
H2O, and phenol.
In Vitro Bcl-2 Binding Assay.
The in vitro binding of peptides to the Bcl-2 protein was
determined by a competitive binding assay based on fluorescence
polarization. For this assay, 5-carboxyfluorescein was coupled to the
NH2 terminus of a peptide GQVGRQLAIIGDDINR,
derived from the BH3 domain of Bak (Flu-BakBH3), which has been shown
to have high-affinity binding (dissociation constant
KD of 
0.34
µM) to a surface pocket of the
Bcl-XL protein that is essential for its death
antagonist function (6)
. According to our molecular
modeling studies and binding measurement using fluorescence
polarization, the Flu-BakBH3 peptide also binds the surface pocket of
Bcl-2 with a similar affinity (dissociation constant
KD of 0.20
µM).5
Bcl-2 used in this assay was a recombinant glutathione
S-transferase-fused soluble protein (Santa Cruz
Biotechnology). Flu-BakBH3 and Bcl-2 protein were mixed in the presence
or absence of peptide inhibitors under standard buffer conditions,
using near-physiological salt concentrations, neutral pH, and a small
amount of reductant. After 30 min incubation, Bcl-2 binding of
Flu-BakBH3 was measured by a LS-50 luminescence spectrometer equipped
with polarizers using a dual path length quartz cell (500 µl;
Perkin-Elmer). The binding affinity of each modified or unmodified
peptide for the Bcl-2 protein was assessed by determining the ability
of different concentrations of the peptides to inhibit Flu-BakBH3
binding.
Immunofluorescence and Confocal Microscopy.
To determine the intake of peptides into cells, HL-60 Bcl-2 cells
were incubated in the presence or absence of biotinylated peptides at
50 µM for different times. Cells were washed with PBS and
fixed with 4% formaldehyde solution. An aliquot of 50 µl of cell
suspension was smeared on slides and dried at room temperature. The
slides were further incubated with 100 µl of 20 µg/ml Streptavidin
Fluorescein (Boehringer Mannheim) for 20 min and counterstained with
propidium iodide. To localize the biotinylated peptides in the cells,
12 images were produced through top to bottom edges of the scanned
cells on each section under Zeiss Axiovert 100 microscope with Bio-Rad
MRC 600 confocal facility. This provided detailed information of the
whole intact cell from the membrane through nucleus. The images were
photographed and stored in the same facility.
DNA Fragmentation.
Cells (1 x 106) were incubated
with peptides for the time indicated. Cells were washed once with PBS
and pelleted by centrifugation. The cell pellets were fixed in 70%
ethanol for 1 h and then resuspended in PC buffer (192
mM Na2HPO4 and
4 mM citric acid, pH 7.8) to extract DNA. After
centrifugation for 5 min at 13,000 RPM, the supernatants were collected
and incubated with 10 µg/ml RNase A for 1 h at 37°C, followed
by digestion with 20 µg/ml proteinase K at 50°C for 3 h. The
DNA fragments were separated by electrophoresis in 2% agarose gel and
visualized by ethidium bromide staining.
Cell Viability Assay.
The effect of the peptides on the viability of tumor cells was
tested using the CellTiter 96AQ kit (Promega Corp., Madison, WI).
Briefly, the cell suspension containing 1x105
cells in 100 µl of medium was plated into 96-well plates and
incubated with peptides at different concentrations. The numbers of
apoptotic cells were determined by measuring the absorbance on a Wallac
Victor (2)
counter (EG&G, Gaithersburg, MD) at 490 nm.
Western Blot Analysis.
Cells were harvested and washed once with cold PBS. The cell pellets
were immediately lysed in 100 µl of ice-cold RIPA buffer with fresh
added protease inhibitors (1 mM phenylmethylsulfonyl
fluoride, 0.2 mM sodium orthovanadate, and 10 µg/ml
aprotinin) and incubated on ice for 30 min. The samples were
transferred to microcentrifuge tubes and centrifuged at
10,000 x g for 10 min at 4°C. The total
cell lysates from supernatant were measured using the Bradford
dye-binding procedure (Bio-Rad). Fifty µg of total protein lysate
were separated on 12% SDS/polyacrylamide gels followed by blotting to
pure nitrocellulose membrane (Bio-Rad). Membranes were blocked in Tris
buffered saline [10 mM Tris-HCl (pH 8.0), 150
mM NaCl) with 5% dry milk and 0.05% Tween 20 on
4°C overnight. Antibodies to detect caspase-3 activation (PharMingen;
65906E, diluted to 1:1000) and PARP activation (Boehringer Mannheim;
1835238, diluted to 1:2000) were used for 1 h of incubation at
room temperature. Immune complexes were detected with horseradish
peroxidase-conjugated secondary antibody and visualized using the ECL
system (Amersham).
Xenogeneic Tumor Mouse Model.
Female CB.17-SCID/NCr (SCID) mice were purchased from the Frederick
Cancer Research and Development Center of the National Cancer Institute
(Frederick, MD). At 6 weeks of age, the mice were challenged with human
HL-60 cells (2 x 107; i.p.), and
either left untreated or treated with cpm-1285 and cpm-1285m peptides
(0.5 mg in 0.2 ml H2O i.p.; daily, days 0–3).
The mice were monitored for signs of tumor growth, such as abdominal
distension and mortality. The data were analyzed using a
multi-ANOVA two-way test, and a probability score of
P < 0.05 was considered statistically
significant.
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Results and Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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. We found that decanoic acid could effectively assist
peptides to pass through the cell membrane. As compared with other
peptide delivery methods based on large internalization peptide
sequence (8)
, a potential advantage of using such a small
molecule as the cpm is that it is more amenable to chemical synthesis
and less likely to affect the structure of the functional peptide
sequence. Other larger fatty acids, such as myristic acid and stearic
acid, have also been used for the intracellular delivery of peptide
inhibitors of protein kinase C (10)
and protein-tyrosine
phosphatase (11)
, respectively.
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Using an in vitro binding assay based on fluorescence
polarization, we confirmed the binding interaction of cpm-1285 with the
surface pocket of Bcl-2 protein. For this assay, a Bak BH3 peptide that
is known to bind the surface pocket of Bcl-2 and
Bcl-XL (6)
was labeled with
fluorescein (designated as Flu-Bak BH3) and used as a competitive
binding probe. cpm-1285 displayed strong binding potency for Bcl-2 with
an IC50 of 130 nM and was
even 2-fold higher than that of wild-type 1285 (Fig. 1B)
.
The cpm itself did not show any interaction with Bcl-2, even in high
concentration of 100 µM (data not shown). The
increase in Bcl-2 binding of cpm-1285 could be contributed to the
formation of additional interaction of the hydrophobic cpm with Bcl-2
surface sites. As a control, the mutant cpm-1285m peptide exhibited a
decrease in Bcl-2 binding affinity with a reduction in
IC50 of 15-fold.
The biological effect of cpm-1285 was studied mostly in human myeloid
leukemia HL-60 cells overexpressing Bcl-2 protein. This cell line was
used as a model system to analyze the inhibition of Bcl-2 function
because Bcl-2 has been shown in this cell line to regulate apoptosis
and resistance to chemotherapeutic drugs (16)
. To verify
the design concept that the cpm facilitates the cellular entry of
peptides, the cells were incubated with cpm-1285 or 1285, both of which
were labeled with biotin. After 15 min of incubation, a significant
intake of cpm-1285 was observed by confocal microscopy in >90% of the
cells (Fig. 1C)
. cpm-1285 peptides were detected inside
cells as early as 5 min after exposure and mostly localized in the
cytoplasm. In contrast, the control 1285 peptide did not show any cell
intake, even after a prolonged 24-h incubation. The mutant cpm-1285m
peptide was also found capable of entering HL-60 cells (data not
shown).
To determine whether cpm-1285 mimicking the natural Bad death domain
could induce apoptosis after entering the cell, HL-60 cells were
incubated with cpm-1285 or various controls at 50 µM for
2 h. Cells treated with cpm-1285 displayed morphological changes
characteristic of apoptotic cell death, i.e., chromatin
condensation, margination, cellular shrinkage, and blebbing, whereas no
such changes were observed in untreated cells or cells treated with cpm
alone, or with the 1285 and mutant cpm-1285m peptides (data not shown).
Because a characteristic feature of apoptotic cells is the presence of
DNA strand breaks, HL-60 cells were further analyzed for DNA
fragmentation in 2% agarose gel under electrophoresis. The
characteristic DNA ladders were found only in cells treated with the
cpm-1285 peptide for 2 h (Fig. 2A)
. Neither 1285 peptide, which could not enter the cell, nor
cpm alone, even in higher concentrations (100
µM), induced DNA ladders during the same period
(Fig. 2A)
or after a prolonged time (24 h, not shown). These
results suggest that the apoptosis-inducing effect of cpm-1285 depends
on the 1285 peptide sequence and its efficient delivery into the tumor
cells. In the case of the mutant cpm-1285m peptide, no apoptosis was
detectable during the 2-h incubation, correlating with its decreased
Bcl-2 binding capability. These data are consistent with the mechanism
by which cpm-1285 induces apoptosis through functional blockade of
intracellular Bcl-2 and related death antagonists.
|
. Taken together,
these data reveal that cpm-1285 internalized during the early stage of
incubation was sufficient for the induction of apoptosis in intact
HL-60 cells.
During apoptosis, the activation of a group of caspases, such as
caspase-3, and subsequent cleavage of cellular substrates, such as
PARP, are crucial components of cell death pathways (17)
.
To assess caspase-3 and PARP activation, Western blot analysis was
performed with total protein lysate from HL-60 cells after 2 h
treatment with the peptides. The appearance of the active
Mr 17,000 subunit of caspase-3,
corresponding with the decrease of its
Mr 32,000 precursor form, was detected
only in cells treated with cpm-1285, but not with 1285, cpm, or
cpm-1285m (Fig. 2B)
. Consistent with this finding, the
Mr 113,000 PARP was found to be
cleaved into Mr 89,000 and
Mr 24,000 fragments in cells treated
with cpm-1285 but not with the other controls (Fig. 2C)
. The
role of caspase activation in apoptosis initiated by cpm-1285 was
further demonstrated by the complete inhibition of DNA ladder formation
by the addition of zVAD-fmk, a broad-spectrum caspase inhibitor
(18)
, to the cells prior to cpm-1285 treatment (Fig. 2D)
.
It has been suggested that oncogenic changes in certain tumor cells
render them more susceptible than normal cells to apoptosis
(19)
, which may allow for the selective induction of
apoptosis in these tumors by Bcl-2 inhibitors. In this regard, we noted
that the cpm-1285 peptide had only a minimal effect on the viability
(marked by trypan blue exclusion and lack of blebbing) of normal human
PBLs after a 1-h incubation at concentrations of between 6.3–50
µM (Fig. 3A)
. In addition, the remaining viable PBL cells exhibited
equivalent proliferative responses as untreated cells after PHA mitogen
stimulation (Fig. 3B)
. In contrast, cpm-1285 concentrations
of between 12.5–50 µM resulted in increasing
loss of viability of HL-60 cells, reaching a 72% loss at the 50
µM level (Fig. 3A)
. Furthermore, to
test whether cpm-1285 would have an effect on activated lymphocytes,
PBL cells were stimulated with PHA mitogen for 48 h in the
presence of peptide at titrated concentrations. Proliferative responses
were measured at the end of the incubation period and were found to be
equivalent between all concentrations of cpm-1285 and the untreated PBL
cells (Fig. 3C)
. Taken together, these data suggest that the
cell permeable peptide at these concentrations could be effective
against tumor cells without endangering normal cells, such as
lymphocytes.
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30% loss of viability in both cell lines (data not
shown). The activity of cpm-1285 in inducing the death of HL-60 cells
overexpressed with Bcl-XL suggests that cpm-1285
can also target other Bcl-2-related antiapoptotic proteins such as
Bcl-XL.
Finally, we investigated the effects of the cpm-1285 peptide on tumor
growth in vivo. SCID mice were challenged with HL-60 cells
(2 x 107; i.p.) and either left
untreated or administered cpm-1285 and cpm-1285m peptides (0.5 mg in
0.2 ml H2O i.p.; daily, days 0–3). Mice in the
untreated control group all developed ascites and succumbed to tumor
growth by day 16 (MST of 11 days; Fig. 4
). Mice treated with the mutant cpm-1285 peptide followed a similar
fatal pattern (P > 0.65) with a MST of 12
days. In contrast, mice treated with the cpm-1285 peptide survived
until day 27, with a significantly prolonged MST of 19 days
(P < 0.01). This result suggested that the
cpm-1285 peptide had in vivo efficacy in reduction of the
tumor burden and thereby slowed the progression of tumor growth. In
addition, the effect of the peptide was dependent on its proper
sequence, consistent with the in vitro findings of
diminished effects with the mutant form of the peptide. In a toxicity
test with normal C57BL/6J mice that were administered i.p. four daily
1.0-mg dosages (twice that used for the experiment in Fig. 4
) of the
cpm-1285 peptide on days 0–3, there was no obvious gross signs of
organ toxicity upon autopsy performed on day 4, compared with untreated
normal mice. In addition, cell yields (mean ± SD) from
spleen and bone marrow samples were equivalent, i.e.,
spleen–91.8 ± 10.4 x 106 (normal) versus 94.9 ± 14.1 x 106 (cpm-1285),
n = 3, P 
0.58;
bone marrow–40.1 ± 1.2 x 106 (normal) versus 34.7 ± 2.4 x 106 (cpm-1285),
n = 3, P 0.10.
Flow cytometric analysis of the splenocytes also indicated that there
were no significant changes in the subset constituency of the
cpm-1285-treated population. It should be pointed out that cpm-1285 may
have a short biological half-life in vivo, as assessed by
the in vitro human serum stability test (data not shown),
which could, at least in part, explain the partial tumor suppression
observed under the current experimental conditions. Further synthetic
modifications of this peptide to increase its proteolytic stability and
the optimization of peptide treatment conditions may hopefully lead to
complete abolishment of tumors. Nevertheless, the data shown here
provide an important indication of the in vivo activity of
the Bcl-2 peptide inhibitor.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants (to Z. H.) from the American
Cancer Society and the Sidney Kimmel Foundation for Cancer Research. 
2 These authors contributed equally to this
work.
3 To whom requests for reprints should be
addressed, at Kimmel Cancer Center, Jefferson Medical College, Thomas
Jefferson University, 802 BLSB, 233 South 10th Street, Philadelphia, PA
19107. Phone: (215) 503-4564; Fax: (215) 923-2117; E-mail: zhuang{at}nana.jci.tju.edu
4 The abbreviations used are: BH, Bcl homology;
cpm, cell permeable moiety; PARP, poly(ADP-ribose) polymerase; SCID,
severe combined immunodeficient; PBL, peripheral blood lymphocyte; PHA,
phytohemagglutinin; MST, median survival time.
5 S. Li, S. Shan, and Z. Huang, unpublished
results and manuscript in preparation.
6 J-L. Wang, D. Liu, Z-J. Zhang, S. Shan, X. Han,
S. M. Srinivasula, C. M. Croce, E. S. Alnemri and Z. Huang.
Structure-based discovery of a novel organic compound that binds Bcl-2
protein and induces apoptosis of tumor cells, submitted for
publication.
Received 11/24/99. Accepted 2/ 3/00.
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REFERENCES |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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, HL-60 cells; 
, PBL cells.
Bars, SE. B, PBL cells
(2 x 104) after the 1-h treatment, as
above, were stimulated in microtiter plates with PHA (1 µg/ml) for
48 h, the last 6 h in the presence of
[3H]thymidine (1 µCi/ml). Proliferation was indicated
by the level of [3H]thymidine incorporation (background
without stimulation was 250 cpm). Bars, SE.
C, PBL cells (5 x 105) were
stimulated in microtiter plates with PHA (1 µg/ml) for 48 h in
the presence of varying concentrations of cpm-1285 peptide.
Proliferation was indicated by the level of [3H]thymidine
(1 µCi/ml) incorporated in the last 6 h of incubation
(background without PHA stimulation was 475 cpm). Bars,
SE.