ABT-510, a modified type 1 repeat peptide of thrombospondin, inhibits malignant glioma growth in vivo by inhibiting angiogenesis.

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Research Paper
ABT-510, a Modified Type 1 Repeat Peptide of Thrombospondin,
Inhibits Malignant Glioma Growth In Vivo by Inhibiting Angiogenesis
[Cancer Biology & Therapy 6:3, 454-462; March 2007]; ©2007 Landes Bioscience
Joshua C. Anderson1
J. Robert Grammer1
Wenquan Wang2
L. Burton Nabors3
Jack Henkin4
Jerry E. Stewart Jr.1
Candece L. Gladson1,*
Departments of 1Pathology, Division of Neuropathology, 2Medicine Hematology-
Oncology Division (Biostatistics Section), and 3Neurology (Neuro-Oncology); the
University of Alabama at Birmingham, Birmingham, Alabama USA
4Abbott Laboratories; Abbott Park, Illinois USA
*Correspondence to: Candece L. Gladson; the University of Alabama at
Birmingham; LHRB 567, 701 S. 19th St; Birmingham, Alabama 35294 USA; Tel.:
205.975.7847; Fax: 205.934.7346; Email: gladson@uab.edu
Original manuscript submitted: 06/20/06
Manuscript accepted: 11/26/06
Previously published online as a Cancer Biology & Therapy E-publication:
http://www.landesbioscience.com/journals/cc/abstract.php?id=3630
KEy WoRdS
ABT-510, glioma, model, angiogenesis,
thrombospondin
ACKNoWLEdGEmENtS
This work was supported by grant #CA97247
(Project 5) from the NIH-NCI P50 to L.B.N.,
W.W. and C.L.G., and grant #CA97110 from
the NIH-NCI to C.L.G. We thank Mrs.
Rhonda Carr for assistance in preparing this
manuscript.
ABStRACt
Anti‑angiogenic therapies would be particularly beneficial in the treatment of malig‑
nant gliomas. Peptides derived from the second type 1 repeat (TSR) of thrombospondin‑1
(TSP‑1) have been shown to inhibit angiogenesis in non–glioma tumor models and a
modified TSR peptide, ABT‑510, has now entered into Phase II clinical trials of its efficacy
in non–glioma tumors. As microvascular endothelial cells (MvEC) exhibit heterogeneity,
we evaluated the ability of the modified TSR peptide (NAcSarGlyValDalloIleThrNvaIleAr
gProNHE, ABT‑510) to inhibit malignant glioma growth in vivo and to induce apoptosis
of brain microvessel endothelial cells (MvEC) propagated in vitro. We found that daily
administration of ABT‑510 until euthanasia (days 7 to 19), significantly inhibited the
growth of human malignant astrocytoma tumors established in the brain of athymic nude
mice. The microvessel density was significantly lower and the number of apoptotic MvEC
was significantly higher (3‑fold) in the tumors of the ABT‑510‑treated animals. Similar
results were found using a model in which the established tumor is an intracerebral malig‑
nant glioma propagated in a syngeneic mouse model. ABT‑510 treatment of primary
human brain MvEC propagated as a monolayer resulted in induction of apoptosis in a
dose‑ and time‑dependent manner through a caspase‑8‑dependent mechanism. It also
inhibited tubular morphogenesis of MvEC propagated in collagen gels in a dose‑ and
caspase‑8 dependent manner through a mechanism that requires the TSP‑1 receptor
(CD36) on the MvEC. These findings indicate that ABT‑510 should be evaluated as a
therapeutic option for patients with malignant glioma.
INtRoduCtIoN
ABT-510, a modified type 1 peptide of thrombospondin 1 (TSP-1), recently was
entered into Phase-II clinical trials for non-glioma malignancies.1,2 We are particularly
interested in the development of more effective approaches for the treatment of malig-
nant glioma tumors. These tumors account for the majority of primary brain tumors in
adults, and are associated with severe morbidity and mortality.3,4 Despite the standard
treatments of surgery, radiation and chemotherapy, the mortality rate for patients with
these tumors has not changed significantly in 20 years.3 It has been established that the
progression of a glioma tumor from a low grade glioma, such as a fibrillary astrocytoma
World Health Organization (WHO) classification grade II, to the most malignant WHO
grade IV glioma, is dependent on a tumor environment that promotes angiogenesis.5-7
Thus, ABT-510 may represent a new therapeutic option for malignant glioma; however,
no studies have addressed the potential anti-angiogenic effects of ABT-510 or type 1 repeat
peptides derived from TSP-1 or TSP-2 on malignant glioma tumors. The lack of informa-
tion in this area is particularly important as MvEC isolated from various types of tumors
exhibit different gene expression profiles and it is therefore possible that their responses to
stimuli also differ.8
ABT-510 was developed in an effort to improve the pharmacodynamic and phar-
macokinetic profile of the type 1 repeat peptide of TSP-1. Both TSP-1 and its highly
homologous family member TSP-2 can promote an anti-angiogenic effect through
sequences found in the TSR and amino-terminal domains.9-11 Peptides from the second
TSR of TSP-1 that contain the sequence GVITRIR inhibit angiogenesis in vitro.12
Substitution of the first Ile of GVITRIR with DIle or DalloIle and the first Arg with
norvaline, as well as capping of the terminal amino- and carboxyl-residues, resulted in the
generation of two peptides with significantly improved serum half-lives, ABT-510 (NAc
SarGlyValDalloIleThrNvaIleArgProNHE) and ABT-526 (NAcSarGlyValDIleThrNvaIle
ArgProNHE).13 Treatment with ABT-510 or ABT-526 has been shown to induce apoptosis of
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ABT-510 Peptide Inhibits Glioma Growth
bovine capillary endothelial cells and of human umbilical artery
endothelial cells, to inhibit tubular morphogenesis induced by
vascular endothelial cell growth factor (VEGF), and to inhibit corneal
neovascularization induced by basic fibroblast growth factor
(bFGF).13,14 Furthermore, it has been demonstrated that the growth
of bladder cancer cells and Lewis lung carcinoma cells propagated
in mice is inhibited by the administration of ABT-510.13,14 Inhibition
of the growth of Lewis lung carcinoma cells, bladder and prostate
cancer cells propagated in mice s.c. and administered ABT-510 is
complemented by the addition of another agent.15,16 Due in part to
its longer half-life, ABT-510 has been selected for further clinical
evaluation.
In the present study, we investigated the potential anti-tumor and
anti-angiogenic effects of administration of ABT-510 on the growth
of established malignant glioma tumors in two intracerebral mouse
models. We found that administration of ABT-510 inhibits malig-
nant glioma growth and angiogenesis in these models by inducing
apoptosis of tumor MvEC. We extended these studies to confirm that
ABT-510 induces apoptosis of the human brain MvEC propagated as
a monolayer on type I collagen and inhibits tubular morphogenesis
of primary human brain MvEC propagated on collagen gels. Taken
together, our data indicate that administration of ABT-510 is a
potential new therapy for patients with malignant glioma.
mAtERIALS ANd mEtHodS
Materials. The ABT-510 peptide and a control peptide (NAc
SarGlyValDAsnThrNvaIleArgProNHE) were supplied by Abbott
Laboratories (Abbott Park, IL). zIETD, a caspase-8 inhibitor,
Ac-LEHD-CHO, a caspase-9 inhibitor, and rabbit anti-Poly-ADP-ri-
bose polymerase (PARP) IgG were purchased from Cell Signaling
(Beverly, MA). FGF-2(bFGF) and VEGF were purchased from R&D
Systems (Minneapolis, MN). Calf-collagen type I was purchased from
ICN, Inc. (Aurora, Ohio). The following antibodies were purchased,
rat anti-mouse CD31 (PECAM-1) (BD Pharmingen, Franklin
Lakes, NJ), blocking anti-CD36 mAb (Beckman-Coulter, Fullerton,
CA), rabbit anti-human and mouse vWf IgG (Stressgen, BC,
Canada), mAbs anti-cortactin and anti-glyceraldehyde 3-phosphate
dehydrogenase (G3PDH) (Upstate, Waltham, MA), and anti-cleaved
caspase-3 and -7 specific IgG’s (Calbiochem, Darmstadt, Germany).
The substrate 3,3’-diaminobenzidine (DAB) and the Vector Red
immunohistochemistry kit were purchased from Biogenex (San
Ramon, CA) and Vector Laboratories (Burlingame, CA), respec-
tively. Low endotoxin fetal bovine serum (FBS) was purchased from
Gibco BRL and used for cell propagation and all experiments with
the human brain MvEC. Human brain MvECs (passage 2) and
the recommended CSC media were purchased from Cell Systems
(Kirkland, WA).
Methods. Cell Culture. The human brain MvECs were utilized
at passages 2–8. For all experiments, 35 mm2 wells were coated
with 10 mg/ml calf collagen type I, cells were plated at a
density of 150,000 cells/well in CSC media with 10% FBS or
in M199 media with 10% FBS; and after 24 h the media
was replaced with M199 media with 2% FBS and the indicated addi-
tions. Mouse malignant glioma cells (GL261), and the U-251 MG
human malignant astrocytoma cells were propagated and harvested
as described previously.17-20
Immunoblotting. Cells were lysed in RIPA lysis buffer with the
following protease inhibitors: 200 mM sodium vanadate, 10 ug/ml
aprotinin, 10 ug/ml leupeptin, 10 ug/ml TLCK, and 200 mM PMSF
(10 min, 4˚C).17,18 Lysates were centrifuged (14,000 rpm 15 min, 4˚C),
and equivalent amount of lysate (typically 130 micrograms) subjected
to electrophoresis on a 15% SDS-PAGE, transferred to Immobilon-P
membrane and blocked in TBS-T + 5% w/v BSA or 5% dehydrated
non-fat dried milk overnight (4˚C), as described previously.18
The primary antibody concentrations used were: 0.12 mg/ml of
anti-cleaved caspase-3 or caspase-7 IgG, 1.0 mg/ml of anti-cleaved
PARP IgG, 1.0 mg/ml of mAb anti-actin, 0.5 mg/ml of mAb anti-cor-
tactin, and 1.0 mg/ml of mAb anti-G3PDH. The HRP-conjugated
secondary antibody was developed with the chemiluminescent kit
from Amersham Biosciences (UK). Band intensities were quantified
by densitometry and the densitometric readings averaged and normal-
ized to the actin, cortactin, or G3PDH loading control band.
Tubular morphogenesis assay. Tube assays were performed
on 2 cm2 grid plates. Collagen gels were prepared as described.19
Human brain MvEC were harvested and resuspended in M199
media with 10% FBS, plated onto collagen gels with the indicated
additions and incubated for 48 h (37˚C, 5% CO2). Subsequently,
wells were photographed at 20X magnification and measurement of
the number of branches as well as mean tube length assessed. Data
were plotted as the mean ± S.E.M.
Animal models. Tumor cells (300,000 in 5 ml of PBS) were
injected with stereotactic assistance at a location 2 mm anterior and,
2 mm lateral to the bregma suture, and at a depth of 2 mm.18 Five
or six mice were used in each group except for the day 7 xenograft
model. All procedures were performed in accordance with the guide-
lines and recommendations of the IACUC with appropriate Animal
Welfare Approval. Intraperitoneal (i.p.) administration of ABT-510,
lactated Ringers solution (102 mM NaCl, 28 mM sodium lactate,
4 mM KCl, and 10 mM CaCl2), or control peptide was begun on
day 7 and continued until euthanasia on day 19. The ABT-510
was administered in 0.5 ml of lactated Ringers solution 2 times
per day over two dosages (AM and PM). At euthanasia the brains
were harvested, fixed in 4% neutral buffered-formalin, embedded in
paraffin, and serially sectioned (8 micron sections) in the coronoal
plane, and in a rostral to caudal direction, as described previously.18
H&E stained sections were photographed at every 70 microns
(1.2X magnification) and the tumor volume quantified digitally
from the pixel-number in Adobe Photoshop, as described recently.20
Immunohistochemical analysis of CD31 and vWf was performed
as described previously.14 Microvessel density was quantified as the
number of microvessels per 0.25 mm2 area (20x view grid) immu-
no-stained with anti-CD31 IgG or anti-vWf IgG.
Double‑labeling for TUNEL‑positive MvEC. Tissue sections
were deparaffinized, subjected to antigen retrieval, and then labeled
using the TdT-FragELtm DNA Fragmentation Detection Kit
(Calbiochem). The sections were then permeabilized with 1% Triton
X-100 (10 min, 22˚C) and blocked with 5% BSA in PBS (pH 7.4),
then incubated sequentially with 2 mg/ml rabbit anti-vWf IgG (20
h, 4˚C), anti-rabbit alkaline phosphatase conjugated secondary
IgG (1 h, 22˚C) and the Vector Red alkaline phosphatase substrate
(Biogenex), and counterstained with Methyl Green.
TUNEL assay on brain MvEC. Brain MvEC were plated on
collagen-coated coverslips and the TUNEL assay performed as
per the instructions in the TdT-FragELtm DNA Fragmentation
Detection Kit.
Statistical analysis. Data were analyzed using a one-sided Wilcoxon
Rank Sum test using normal approximation with a significance level
of 0.05. TUNEL positive MvEC in the syngeneic mouse model were
analyzed using a Chi-square test with a significance level of 0.05. A
simple linear regression analysis was utilized to assess the dose-response
effect of ABT-510 on branching in the tubular morphogenesis assays.

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ABT-510 Peptide Inhibits Glioma Growth
RESuLtS
ABT‑510 administration inhibits growth of an established
intracerebral malignant astrocytoma xenograft through a reduction
in microvessel density associated with induction of apoptosis of
MvEC. Other investigators have shown inhibition of the growth of
Lewis lung carcinoma cells propagated s.c. in the mouse flank and
the growth of bladder cancer cells propagated orthotopically in the
nude mouse on administration of ABT-510 peptide starting on day
3 post-tumor cell injection.13,14 To test the potential therapeutic
efficacy of ABT-510 for malignant glioma tumors, we first used a
xenograft model in which 3 x 105 U-251MG human malignant
astrocytoma cells were injected intracerebrally. The ABT-510 was
Figure 1. ABT‑510 administration inhibits tumor growth and microvessel density of established intracerebral malignant glioma tumors in both xenograft and
syngeneic mouse models. U‑251MG human malignant astrocytoma (A–C) or GL261 mouse malignant glioma (E–G) cells (3 x 105) were injected with ste‑
reotactic assistance into the athymic nude (A–C) or C57BL/6 (E–G) mouse brain, respectively, and allowed to become established for seven days at which
point the animals were euthanized and the brains harvested, or administration of 339 mg ABT‑510/kg mouse weight/day was started using two dosages
i.p. in 0.5 ml lactated Ringers solution (AM and PM) until day 19. On day 7 or 19, the animals were euthanized, the brains harvested, fixed, embedded in
paraffin, serially sectioned and tumor volume digitally recreated as described in the Materials and Methods. (A and E) Tumor volume is plotted as the mean
mm3 ± S.E.M. (B and F) The increase in tumor volume at day 19 over that observed at day 7 is plotted as a percent increase in tumor volume ± S.E.M.
C&G, At day 19 the number of microvessels on anti‑vWf IgG immunostained sections in a 0.25 mm2 area of tumor from four 20x fields is plotted as the
mean ± the S.E.M. for each group. (D and H) U‑251MG (D) and GL261 (H) glioma cells were plated and propagated in M199 media with 2% FBS with
the addition of ABT‑510, or vehicle every second day, and the cells harvested and counted at the indicated time points. Conditions were assayed in replicas
of three and the data plotted as the mean ± S.E.M. These experiments were repeated and highly similar results were obtained.
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ABT-510 Peptide Inhibits Glioma Growth
administered as 339 mg ABT-510/kg mouse weight/
day i.p. over two dosages in 0.5 ml lactated Ringers
solution, with an identical volume of lactated Ringers
solution being used as the control. Administration of
ABT-510 or the control solution was started on day
7 and continued until euthanasia on day 19. No
adverse effects of ABT-510 administration were
observed; the animals gained weight and appeared
healthy. The brains were then harvested from the
euthanized mice and the tumor volume recreated
digitally. As shown in Figure 1A, the tumor volume
in the animals administered the control solution
was significantly greater at day 19 (2.3 mm3 +
0.33 mm3) as compared to that observed in the mice
administered ABT-510 (0.71 mm3 + 0.25 mm3);
p = 0.001. As shown in Figure 1B, the percent
increase in tumor volume at day 19 as compared to
day 7 in the control-treated group was significantly
greater than that found in the ABT-510-treated
group (p = 0.001). In the experiment shown, a larger
group of mice was analyzed at the 7 day time point,
as a greater variability was expected in the tumor
volume than that observed. These data suggested
that ABT-510 inhibits growth of established malig-
nant astrocytoma tumors.
Assessment of microvessel density was accom-
plished by counting four 20x fields of 0.25 mm2
area, as we and others have described previously,11,14
of tumors sections immunostained with antibodies
directed toward either the endothelial cell marker
protein vWf or CD315,6 and using rabbit IgG as
a negative control. As shown in Figure 1C, the
microvessel density as assessed using vWf staining
was significantly greater in the tumors of the animals
administered the control solution as compared to
the tumors of those administered ABT-510 (p =
0.015). The results obtained on immunostaining
with anti-mouse CD31 IgG were highly similar
(data not shown). These data suggest that ABT-510
inhibits the growth of established glioma tumors by
inhibiting angiogenesis. No change in the percentage
of TUNEL-positive tumor cells was observed
with ABT-510 treatment, and ABT-510 treatment
(0.1 and 10 mM) of U-251MG cells propagated
in vitro did not alter cell proliferation over six
days (Fig. 1D), consistent with an effect of ABT-510
on tumor MvEC.
Double labeling of the tumor sections with
anti-vWf IgG and TUNEL indicated a significantly higher number
(3-fold) of TUNEL-positive MvEC in the tumors of the animals
administered ABT-510, as compared to the tumors of the animals
administered the control solution (p = 0.037) (Fig. 2A and B),
suggesting that ABT-510 inhibits angiogenesis of the glioma-associ-
ated MvEC by inducing apoptosis.
ABT‑510 administration inhibits tumor growth of an
established intracerebral malignant glioma tumor in a syngeneic
mouse model. To substantiate our findings, we evaluated the poten-
tial anti-tumor and anti-angiogenic effect of ABT-510 administration
using a second model in which an established intracerebral malig-
nant glioma is propagated in immune competent syngeneic mice.
In this model, 3 x 105 GL261 cells are injected intracerebrally into
the C57BL/6 mouse brain. The experimental protocol was exactly
as described for the xenograft model, except that a control peptide
was administered to the control group. During this experiment, two
groups of mice were injected with tumor for the seven day time point
but as one mouse died during anesthesia, the resultant total number
of mice in this group was 9. As found in the xenograft model, the
animals administered ABT-510 showed no adverse effects; the mice
gained weight and appeared healthy. In the mice administered
ABT-510, we again found a significantly smaller tumor volume at
day 19 (mean tumor volume = 22.4 mm3± 3.53 mm3) as compared
to that observed in the mice administered the control peptide (mean
tumor volume = 36.8 mm3± 5.7 mm3); p = 0.027 (Fig. 1E). In addi-
tion, the percent increase in tumor volume at day 19 as compared
Figure 2. ABT‑510 administration induces apoptosis of tumor MvEC in both intracerebral mouse
models of malignant glioma. Six sections of tumor‑bearing brain from each mouse at day 19
were double‑labeled for apoptotic MvEC with TUNEL and anti‑vWf IgG, as described in the
Materials and Methods. (A and C) In the xenograft and syngeneic models, TUNEL and anti‑vWf
IgG‑ double‑labeled cells in a 0.25 mm2 area is presented as the mean percent positive MvEC
± S.E.M. (B and D) TUNEL and anti‑vWf‑double‑labeled sections; arrows denote double labeled
MvEC ± S.E.M. (B and D) 250x magnification. (E) In the syngeneic model a chi square analysis
of the percent of TUNEL and anti‑vWf IgG double‑labeled cells greater than 10% or less than
10% per tissue section is shown. These experiments were repeated and highly similar results
were obtained.

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ABT-510 Peptide Inhibits Glioma Growth
to day 7 was significantly greater in the control-peptide treated
animals (p = 0.027) (Fig. 1F).
The microvessel density in the tumors of the animals admin-
istered the control peptide was significantly greater than that in
the tumors of the ABT-510-treated animals; p = 0.006 (Fig. 1G).
There was a consistently greater number of TUNEL-positive
MvEC in the ABT-510-treated animals as compared to the
control peptide-treated animals; although the increase was
not statistically significant (Fig. 2C and D). However using
a cut-off value of >10% positive MvEC per tissue section
we found nine of the 29 (31%) of the double-labeled
sections in the ABT-510-treated group had greater than 10%
TUNEL-positive MvEC per tissue section, and only three of
29 (10%) in the control peptide-treated group had greater than
10% TUNEL-positive MvEC per tissue section (Fig. 2E).
This difference was statistically significant (p = 0.03). Similar
to the xenograft glioma model no change in the percentage
of TUNEL-positive tumor cells was observed with ABT-510
treatment in the syngeneic model. ABT-510 treatment (0.1 and
10 mM) of GL261 cells propagated in vitro did not alter cell
proliferation over 6 days (Fig. 1H), consistent with an effect
of ABT-510 on tumor MvEC. These data supported the find-
ings we had generated using the xenograft tumor model (Fig.
1A–D and Fig. 2A and B). Extension of the experiments in the
syngeneic model to analysis of the efficacy of a lower dosage of
ABT-510 (113 mg/kg mouse weight/day) administered daily
over two dosages starting on day 7 indicated that the lower
dosage did not result in significant inhibition of glioma
tumor growth (data not shown).
ABT‑510 treatment of primary human brain MvEC
induces apoptosis in a dose‑, time‑ and caspase‑8‑dependent
manner. Other investigators have shown that treatment of
human umbilical artery endothelial cells and of bovine capil-
lary endothelial cells with ABT-526 induces apoptosis, as
assessed by TUNEL assay and ELISA for histone-associ-
ated DNA fragmentation.13,14 We investigated the effects of
ABT-510 on apoptosis of human brain MvEC cultured in
vitro and extended the previous studies by using the cleavage
of the effector caspases-3 or -7 as indicators of apoptosis. These
cleavage assays are considered to be more rigorous indicators
of apoptosis than TUNEL staining.21 ABT-510 treatment of
primary human brain MvEC propagated as a monolayer on
type I collagen for 18 h in reduced serum (2% FBS) induced
apoptosis in a dose-dependent manner (25–100 nM) that was
maximal at 100 nM (Fig. 3A and B, and Fig. 3E and F). The control
peptide (100 nM) failed to induce significant caspase-3 cleavage
(Fig. 3A and B, lane 2). In a time course experiment (6–18 h),
maximal caspase-7 cleavage was observed at 18 h post-treatment
with 100 nM ABT-510 (Fig. 3C and D). The increase in caspase-3
or caspase-7 cleavage when human brain MvEC were treated with
100 nM ABT-510 for 18 h was 3-fold (p = 0.004); the change in the
cleaved caspase-3 or -7 band intensities was quantified by densitom-
etry after normalizing the band intensity to the densitometric reading
for the loading control. In these experiments TNFa stimulation
(40 nM) was used as a positive control for the induction of apoptosis
(Fig. 3A and B, lane 7). The significantly elevated (3-fold) cleavage
of a caspase-3 substrate, poly (ADP-ribose) polymerase (PARP) in
human brain MvEC treated with 50 or 100 nM ABT-510 for 18 h
(3-fold) confirmed that ABT-510 acts to inhibit MvEC through an
apoptotic mechanism (Fig. 3G and H).
The apoptotic pathway activated on treatment with the modified
TSRs had not been elucidated previously. Activation of a death
receptor pathway (the so-called “extrinsic pathway”) has been
reported for dermal MvEC treated with intact TSP-1.22 We there-
fore examined the ability of a caspase-8 inhibitor (zIETD) to block
apoptosis induced by ABT-510 treatment in the primary human
brain MvEC propagated as a monolayer on type I collagen-coated
coverslips, and utilized a TUNEL assay to assess apoptosis. zIETD
was used at the recommended concentration (10–20 mM) to avoid
nonspecific effects. Treatment with 100 nM ABT-510 for 18 h
resulted in TUNEL positivity in 14% of the brain MvEC (Fig. 4A).
Preincubation of the cells with 10 or 20 mM zIETD significantly
inhibited the pro-apoptotic effect of ABT-510 (Fig. 4A, and data
not shown). Treatment with TNFa (200 ng/ml) was used as a posi-
tive control, and 10 or 20 mM zIETD inhibited the TNFa-induced
TUNEL positivity but did not inhibit the staurosporine-induced
cell death (Fig. 4A, and data not shown). zIETD also partially
but significantly blocked (50% inhibition) the caspase-3 cleavage
induced by 100 nM ABT-510 (Fig. 4B and C, lanes 5 and 6 as
Figure 3. ABT‑510 treatment of primary human brain MvEC induces apoptosis.
Primary human brain MvEC were plated onto wells coated with type I collagen in
CSC media with 10% FBS, at 24 h the media was replaced with serum‑starving
M199 media with 2% FBS along with the indicated additions and the cells incu‑
bated for 6–18 h (37˚C, 5% CO2). Subsequently, the cells in the media were
pelleted and lysed along with the cell monolayer in RIPA lysis buffer with protease
inhibitors, equivalent amount of lysate electrophoresed on a 15% disulfide‑reduced
SDS‑PAGE, the proteins transferred to Immobilon P membrane, immunoblotted with
anti‑cleaved caspase‑3, anti‑cleaved caspase‑7, or anti‑cleaved PARP specific IgG,
and stripped and reprobed with mAb anti‑G3PDH, as described in the Materials
and Methods. TNFa treatment was used as a positive control for the induction of
apoptosis. A control peptide was used as a control for ABT‑510 treatment. The
experiment was repeated 2x and highly similar results were obtained.
458 Cancer Biology & Therapy 2007; Vol. 6 Issue 3