Molecular Cancer
BioMed Central
Open Access
Research
Enhancing the anti-angiogenic action of histone deacetylase
inhibitors
Selena Kuljaca1, Tao Liu1, Andrew EL Tee1, Michelle Haber1,
Murray D Norris1, Tanya Dwarte1 and Glenn M Marshall*1,2
Address: 1The Children's Cancer Institute Australia for Medical Research, The University of New South Wales, Sydney, NSW 2031, Australia and
2The Centre for Children's Cancer and Blood Disorders, Sydney Children's Hospital, Randwick, Sydney, NSW 2031, Australia
Email: Selena Kuljaca - skuljaca@ccia.unsw.edu.au; Tao Liu - tliu@ccia.unsw.edu.au; Andrew EL Tee - atee@ccia.unsw.edu.au;
Michelle Haber - mhaber@ccia.unsw.edu.au; Murray D Norris - mnorris@ccia.unsw.edu.au; Tanya Dwarte - dwarte@ccia.unsw.edu.au;
Glenn M Marshall* - g.marshall@unsw.edu.au
* Corresponding author
Published: 25 October 2007
Molecular Cancer 2007, 6:68
doi:10.1186/1476-4598-6-68
Received: 25 July 2007
Accepted: 25 October 2007
This article is available from: http://www.molecular-cancer.com/content/6/1/68
© 2007 Kuljaca et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: Histone deacetylase inhibitors (HDACIs) have many effects on cancer cells, such as
growth inhibition, induction of cell death, differentiation, and anti-angiogenesis, all with a wide
therapeutic index. However, clinical trials demonstrate that HDACIs are more likely to be effective
when used in combination with other anticancer agents. Moreover, the molecular basis for the anticancer action of HDACIs is still unknown. In this study, we compared different combinations of
HDACIs and anti-cancer agents with anti-angiogenic effects, and analysed their mechanism of
action.
Results: Trichostatin A (TSA) and α-interferon (IFNα) were the most effective combination
across a range of different cancer cell lines, while normal non-malignant cells did not respond in the
same manner to the combination therapy. There was a close correlation between absence of basal
p21WAF1 expression and response to TSA and IFNα treatment. Moreover, inhibition of p21WAF1
expression in a p21WAF1-expressing breast cancer cell line by a specific siRNA increased the
cytotoxic effects of TSA and IFNα. In vitro assays of endothelial cell function showed that TSA and
IFNα decreased endothelial cell migration, invasion, and capillary tubule formation, without
affecting endothelial cell viability. TSA and IFNα co-operatively inhibited gene expression of some
pro-angiogenic factors: vascular endothelial growth factor, hypoxia-inducible factor 1α and matrix
metalloproteinase 9, in neuroblastoma cells under hypoxic conditions. Combination TSA and IFNα
therapy markedly reduced tumour angiogenesis in neuroblastoma-bearing transgenic mice.
Conclusion: Our results indicate that combination TSA and IFNα therapy has potent cooperative cytotoxic and anti-angiogenic activity. High basal p21WAF1 expression appears to be acting
as a resistance factor to the combination therapy.
Background
Acetylation and deacetylation of histones by histone
acetyltransferases and histone deacetylases (HDACs) alter
chromatin structure and modulate transcriptional regulation (reviewed in [1-3]. Inhibitors of HDACs (HDACIs)
are emerging as a new class of anticancer agents. HDACIs
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Molecular Cancer 2007, 6:68
induce cancer cell differentiation, growth arrest, programmed cell death, and inhibit tumour-driven angiogenesis [1,3]. Clinical trials with HDACIs in cancer patients
demonstrate that HDACI treatment leads to tumour
regression and symptomatic improvement in some heavily pre-treated and multiply relapsed patients, with a surprisingly low side-effect profile [1,4]. However, a large
proportion of the patients are not sensitive to the treatment, demonstrating the need to examine the effectiveness of HDACIs in combination with other anti-cancer
agents.
Angiogenesis is vital for tumor progression and metastasis
[5,6]. As anti-angiogenic therapy is generally less toxic and
better tolerated than conventional cytotoxic chemotherapy, strategies which combine anti-angiogenic agents with
other anti-cancer drugs have been the focus of current
clinical trials to widen the therapeutic index. The interferons (IFNs) are a family of naturally occurring cytokines
with anti-proliferative and anti-angiogenic effects [7,8].
Through inhibiting pro-angiogenic gene expression and
acting directly on endothelial cells, α-interferon (IFNα)
suppresses angiogenesis and tumour growth in vitro and in
vivo [7,9]. Rapamycin and its derivatives also inhibit
tumour cell proliferation and angiogenesis by acting on
the mammalian target of rapamycin and suppressing the
transcriptional activity of pro-angiogenic hypoxia-inducible factor 1α (HIF1α), (reviewed in [10]). While clinical
trials with IFNα, rapamycin and its derivatives used as single agents have shown some effects, none of the drugs are
effective alone in the majority of patients.
It has been reported that a combination therapy with the
HDACI, valproate (VPA), and IFNα exerts synergistic anticancer effects in neuroblastoma BE(2)-C cells both in vitro
and in vivo [11,12]. Here we evaluated the anticancer
actions of combination therapy with HDACIs (Trichostatin A [TSA] or VPA) and anti-cancer agents with anti-angiogenic function (IFNα, rapamycin), and, sought to
determine their mechanism of action.
Results
TSA and IFNα exerted co-operative cytotoxic effects in
cancer cell lines from a range of different tissue origins
The combination of the HDACI, VPA, and IFNα demonstrated synergistic combinational anti-cancer effects in
neuroblastoma BE(2)-C cells both in vitro and in vivo
[11,12]. We investigated the synergistic anti-cancer effect
of IFNα combined with other HDACIs, and, in cancer cell
lines of other tissue origins. We treated breast, lung, colon
and prostate cancer cells and MRC-5 normal non-malignant fibroblasts with control, 0.02 μM TSA and/or 500
IU/ml IFNα, and, then assessed for cell viability. As shown
in Figure 1A, all of the cancer cell lines tested were sensitive to the cytotoxic effects of the combination, and there
http://www.molecular-cancer.com/content/6/1/68
was a significant cooperative effect of TSA and IFNα in
eight of the nine cell lines tested, with MDA-MB-468 as
the only exception. MCF-7, Calu-6, H460, LNCaP, DU145, HT-29, Caco-2 and BE(2)-C cells were all sensitive to
TSA, generally less sensitive to IFNα, and significantly
more sensitive to TSA and IFNα combined. MDA-MB-468
breast cancer cells were sensitive to IFNα but resistant to
TSA, and no more sensitive to the combination than IFNα
alone. When cell sensitivity to the combination treatment
was calculated as a percentage of TSA alone (or IFNα
alone in case of MDA-MB-468), BE(2)-C, HT-29 and
Calu-6 were found to be the most sensitive (Figure 1A).
Importantly, the normal non-malignant MRC-5 fibroblasts were resistant to the treatment of TSA alone, IFNα
alone and TSA plus IFNα combination therapy (Figure
1B). Immunoblot analysis of acetylated histone H3
revealed that treatment with TSA alone or TSA plus IFNα
for 6 hours induced drastic histone acetylation in the
MRC-5 cells (Figure 1B).
SAHA and IFNα exerted co-operative cytotoxic effects in
cancer cell lines, but not in normal cells
The HDACI, SAHA (vorinostat), is in clinical use for the
treatment of cutaneous T-cell lymphoma. We, therefore,
tested whether a combination of SAHA and INFα exerted
co-operative anti-cancer effects. Neuroblastoma BE(2)-C,
breast cancer MCF-7, and normal lung fibroblast MRC-5
cells were treated with control, 0.5 μM SAHA, 500 IU/ml
INFα or SAHA plus INFα for 3 days. Alamar blue assays
revealed that SAHA and INFα co-operatively reduced the
viability of BE(2)-C and MCF-7 cells, although the magnitude was smaller than TSA and INFα. A combination of
SAHA and INFα did not co-operatively reduce the viability of MRC-5 cells (Figure 2).
The effects of other HDACIs and anti-cancer agents used
in combination
We compared the cytotoxicity of the TSA and IFNα combination (Figure 1A) with combinations of another
HDACI VPA and IFNα (Figure 3A). The effect of VPA and
IFNα combination therapy on cell viability was similar to
TSA and IFNα for the BE(2)-C neuroblastoma cells. However, TSA and IFNα were more effective in MCF-7 and
Calu-6 cells than VPA and IFNα. Similar to TSA and IFNα,
VPA and IFNα did not show any co-operative cytotoxic
effects on normal MRC-5 fibroblasts (Figure 3C). We next
compared the cytoxicity of the TSA and IFNα combination (Figure 1A), the VPA and IFNα combination (Figure
3A) with VPA combined with another emerging anticancer agent with both cytotoxic and anti-angiogenic actions,
rapamycin [10] in BE(2)-C and MCF-7 cells (Figure 3B).
The VPA and rapamycin treatment had significant cytotoxic effects compared with VPA alone, but the magnitude
of these effects was much smaller than VPA and IFNα or
TSA and IFNα.
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Molecular Cancer 2007, 6:68
BE(2)-C
Cont IFNα TSA TSA+IFNα
Percentage viable cells
Caco-2
***
100
75
50
25
**
Percentage viable cells
Cont IFNα TSA TSA+IFNα
DU-145
***
100
75
50
25
Cont IFNα TSA TSA+IFNα
Percentage viable cells
B
**
75
50
25
Cont IFNα TSA TSA+IFNα
Percentage viable cells
25
***
H460
***
100
75
50
25
***
Cont IFNα TSA TSA+IFNα
LNCaP
***
100
***
Cont IFN α TSA TSA+IFN α
MCF-7
***
75
50
25
Cont IFNα TSA TSA+IFNα
MDA-MB-468
Cont IFNα TSA TSA+IFNα
TSA (0.02µM)
-
+
-
+
IFNα (500IU/ml)
-
-
+
+
***
100
75
50
25
***
100
75
50
25
Percentage viable cells
50
***
100
Percentage viable cells
75
Percentage viable cells
***
Percentage viable cells
***
100
HT-29
Calu-6
Percentage viable cells
Percentage viable cells
A
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***
Cont IFNα TSA TSA+IFNα
100
75
50
25
MRC-5
100
75
50
25
0
Cont IFNα TSATSA+IFNα
TSA
mal
Figure
non-malignant
and1IFNα exerted
cells co-operative cytotoxic effects in cancer cell lines from a range of different tissue origins, but not in norTSA and IFNα exerted co-operative cytotoxic effects in cancer cell lines from a range of different tissue origins, but not in normal non-malignant cells. A. Neuroblastoma [BE(2)-C], breast (MCF-7 and MDA-MB-468), lung (H460 and Calu-6), prostate
(DU-145 and LNCaP), and colon (HT-29 and Caco-2) cancer cells were treated with control (Cont), 0.02 μM TSA and/or 500
IU/ml IFNα for 72 hours. Cell viability was examined using the Alamar blue assay, measured as optical density (OD) units of
absorbance, and expressed as the absorbance of treated over control samples (ie., % viable cells). ** p < 0.01, *** p < 0.001. B.
MRC-5 cells were treated with control, 0.02 μM TSA and/or 500 IU/ml IFNα for 72 hours, and cell viability was assessed as
above. Moreover, histone protein was extracted and subject to immunoblot analysis with anti-acetylated histone H3 antibody,
after 6 hour exposure to control, TSA and/or IFNα.
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**
100
75
50
25
0
***
MCF-7
Percentage viable cells
C
on
tro
l
IF
N
α
SA
IF
N
α+ HA
SA
H
A
Percentage viable cells
C
on
tro
l
IF
N
α
IF SAH
N
α+ A
SA
H
A
BE(2)-C
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**
100
75
50
25
0
***
Percentage viable cells
Molecular Cancer 2007, 6:68
MRC-5
125
100
75
50
25
0
C
l
α
tro FN HA HA
I
on
SA +SA
α
N
IF
cancer
SAHA and
Figure
cell
2 IFNα
lines, but
exerted
not in
co-operative
normal non-malignant
cytotoxic effects
cells in
SAHA and IFNα exerted co-operative cytotoxic effects in
cancer cell lines, but not in normal non-malignant cells. Neuroblastoma BE(2)-C, breast cancer MCF-7, and normal nonmalignant lung MRC-5 fibroblasts were treated with control,
0.5 μM SAHA and/or 500 IU/ml IFNα for 72 hours. Cell viability was examined using the Alamar blue assay, measured as
optical density (OD) units of absorbance, and expressed as
the absorbance of treated over control samples (ie., % viable
cells). * p < 0.05, ** p < 0.01, *** p < 0.001.
Absence of p21WAF1 expression correlated with sensitivity
to TSA and IFNα combination therapy
Up-regulation of p21WAF1 expression, and p21WAF1induced cell cycle arrest, have been regarded as one of the
main mechanisms through which HDACIs exert their
anti-cancer effects[13]. We examined the role of p21WAF1
in cancer cell sensitivity to the combination therapy.
Immunoblot analysis of p21WAF1 expression was carried
out with protein extracted from the eight cell lines of
breast, lung, prostate and colon origins (Figure 4A).
p21WAF1 was basally expressed in untreated H460, DU145, LNCaP, MCF-7 and MDA-MB-468 cells, but not
expressed in Calu-6, HT-29 and Caco-2 cells. Compared
with control, TSA induced p21WAF1 expression in H460,
MCF-7 and LNCaP cells. IFNα up-regulated p21WAF1 only
in DU-145 cells, and, combination therapy increased
p21WAF1 in all four cell lines. p21WAF1 protein was
expressed but not altered by treatment with TSA and/or
IFNα in MDA-MB-468 cells. Thus, the cancer cell lines
which did not have basal expression of p21WAF1 were generally more sensitive to the combination therapy than
those cancer cells expressing p21WAF1. This correlation
suggested expression of p21WAF1 might render cancer cells
insensitive to the combination therapy.
To determine the role of p21WAF1 expression in cancer cell
sensitivity to TSA and IFNα combination therapy, MCF-7
cells were transfected with control, scrambled siRNA or
siRNA specifically targeting p21WAF1, and, then treated
with control, TSA and/or IFNα. RT-PCR and immunoblot
analysis revealed that p21WAF1 mRNA and protein were
knocked down by approximately 75% by the p21WAF1
siRNA, compared with scrambled control (Figure 4B). The
p21WAF1 siRNA significantly increased the sensitivity of
MCF-7 cells to TSA and IFNα alone, and, in combination,
as measured by cell viability assays (p < 0.01) (Figure 4C).
HDACI and IFNα co-operatively inhibit endothelial cell
functions and pro-angiogenic gene expression in cancer
cells in vitro
Since HDACIs [2,14] and IFNα [8,9] are known to suppress angiogenesis and tumour growth by acting directly
on endothelial cells, we further investigated whether the
combination of TSA and IFNα could inhibit endothelial
cell function. To exclude the possibility that co-operative
anti-angiogenic effects by TSA and IFNα were due to cytotoxicity, we first determined the optimal dosages of TSA
and IFNα with Alamar blue cell viability assays. After
treatment for 18 hours under normoxic or hypoxic conditions (1% O2), a combination of 0.1 μM TSA and 500 IU/
ml IFNα was found to have no cytotoxicity on endothelial
cells within 18 hours after treatment (Figure 5A). These
doses were, therefore, used in all endothelial cell function
studies. Surprisingly, TSA or IFNα alone stimulated
endothelial cell migration toward the chemoattractant,
vascular endothelial growth factor (VEGF) (Figure 5B). In
contrast, the combination of TSA and IFNα suppressed
endothelial cell migration under both hypoxic (Figure 5B)
or normoxic conditions (data not shown). Compared
with control, IFNα or TSA alone reduced endothelial cell
invasion through Matrigel by 35% and 60%, respectively,
whereas the combination of TSA and IFNα decreased cell
invasion by 80%, under normoxic (data not shown) or
hypoxic conditions (Figure 5C). Under normoxic conditions, compared with control, IFNα or TSA alone
decreased the number of complete branches per branching point by 30% and 50%, respectively, while TSA and
IFNα did not further decrease complete branches per
branching point (data not shown). In contrast, under
hypoxic conditions, the combination of TSA and IFNα
decreased complete branches per branching point by
50%, while TSA or IFNα alone reduced the average numbers of complete branches from a branching point by only
25% (Figure 5D).
We next evaluated whether the combination of TSA
[15,16] and IFNα [7,17] represses pro-angiogenic gene
expression, as measured by RT-PCR, in neuroblastoma
BE(2)-C cells. Compared with treatment with TSA or IFNα
alone, the combination therapy significantly down-regulated gene expression of HIF1α, VEGF and MMP-9 under
normoxic conditions at 72 hours after treatment, while no
co-operative effects were observed on the expression of
MMP-2, activin A, thrombospondin-1, von HippelLindau protein and bFGF (data not shown). Suppression
of HIF1α, VEGF and MMP-9 gene expression by TSA and
IFNα was more significant, when compared with TSA or
IFNα alone, under hypoxic conditions (Figure 5E). In the
case of HIF1α and VEGF, IFNα alone repressed gene
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**
**
50
25
0
25
0
***
**
B
**
**
**
100
75
50
25
0
MCF-7
125
100
**
**
75
**
50
25
0
Control
VPA
0.3nM RAP
0.3nM RAP+VPA
1nM RAP
1nM RAP+VPA
3nM RAP
3nM RAP+VPA
10nM RAP
10nM RAP+VPA
30nM RAP
30nM RAP+VPA
**
Percentage viable cells
125
Control
VPA
0.3nM RAP
0.3nM RAP+VPA
1nM RAP
1nM RAP+VPA
3nM RAP
3nM RAP+VPA
10nM RAP
10nM RAP+VPA
30nM RAP
30nM RAP+VPA
Percentage viable cells
BE(2)-C
***
100
75
50
25
0
C
**
**
Control
500IU/ml IFNα
0.3mM VPA
0.3mM VPA + IFNα
1mM VPA
1mM VPA + IFNα
3mM VPA
3mM VPA +IFNα
5mM VPA
5mM VPA + IFNα
75
100
75
50
Calu-6
Percentage viable cells
**
Percentage viable cells
**
MCF-7
Control
500IU/ml IFNα
0.3mM VPA
0.3mM VPA+IFNα
1mM VPA
1mM VPA + IFNα
3mM VPA
3mM VPA + IFNα
5mM VPA
5mM VPA + IFNα
100
Percentage viable cells
BE(2)-C
Control
500IU/ml IFNα
0.3mM VPA
0.3mM VPA + IFNα
1mM VPA
1mM VPA + IFNα
3mM VPA
3mM VPA +IFNα
5mM VPA
5mM VPA + IFNα
Percentage viable cells
A
MRC-5
125
100
75
50
25
0
Control
500IU/ml IFNα
0.3mM VPA
0.3mM VPA + IFNα
1mM VPA
1mM VPA + IFNα
3mM VPA
3mM VPA+IFNα
5mM VPA
5mM VPA+IFNα
Molecular Cancer 2007, 6:68
Figure
The
cytotoxic
3
effects of other HDACI combination therapies
The cytotoxic effects of other HDACI combination therapies. A. Neuroblastoma [BE(2)-C], breast (MCF-7), and lung (Calu-6)
cancer cell lines were treated with either control, 500 IU/ml IFNα and/or various dosages of VPA for 72 hours B. In separate
experiments, BE(2)-C and MCF-7 cells were treated with control, 1 mM VPA and/or various dosages of rapamycin (RAP) for
72 hours. C. Non-malignant lung fibroblast (MRC-5) cells were treated with a range of VPA doses alone, or in combination
with 500 IU/ml IFNα. Cell viability was examined by the Alamar blue assay, measured as optical density (OD) units of absorbance, and expressed as a percentage of absorbance for treated samples, over that for control samples (ie., % viable cells). ** p
< 0.01, *** p < 0.001.
expression, however, the combination still had a more
significant repressive effect, compared with IFNα alone (p
< 0.05). Although MMP-9 gene expression was stimulated
by IFNα and TSA alone, the combination suppressed its
expression, when compared with control-treated samples
(p < 0.05).
TSA and IFNα co-operatively suppress tumour-driven
angiogenesis in neuroblastoma-bearingN-Myc transgenic
mice
Lastly, we tested whether the combination of TSA and
IFNα could co-operatively inhibit tumor-driven angiogenesis in vivo. Abdominal neuroblastoma first became
palpable in 100% of homozygote N-Myc transgenic mice
at 4 weeks of age [18]. Cohorts of five homozygous MYCN
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A
TSA
IFNα
-
+
-
+
TSA
IFNα
+
+
-
+
-
+
+
+
p21
p21
HT-29
Calu-6
actin
actin
p21
p21
Caco-2
H460
actin
actin
p21
DU-145
p21
LNCaP
actin
actin
**
actin
Scrambled p21
siRNA
siRNA
0
scrambled
control
Immunoblot
25
TSA + IFNα
p21
50
TSA
Scrambled p21
siRNA
siRNA
75
IFNα
β2M
100
Control
p21
RT-PCR
Percentage viable cells
C
TSA + IFNα
B
MB-468
TSA
actin
p21
MDA-
IFNα
p21
MCF-7
actin
p21 siRNA
Figure
Absence4of p21WAF1 expression correlated with sensitivity to TSA and IFNα combination therapy
Absence of p21WAF1 expression correlated with sensitivity to TSA and IFNα combination therapy. A. MCF-7, MDA-MB-468,
H460, Calu-6, DU-145, LNCaP, HT-29 and Caco-2 cells were treated with control, 0.02 μM TSA, 500 IU/ml IFNα, or TSA and
IFNα for 24 hours. Whole cell protein was extracted and subjected to immunoblot with an anti- p21WAF1 antibody, and, an
anti-actin antibody as a loading control. B. MCF-7 cells were transfected with control scrambled or p21WAF1 siRNA for 8 hours,
followed by treatment with control, 0.02 μM TSA and/or 500 IU/ml IFNα for 72 hours. The effect of the siRNAs on p21WAF1
gene and protein expression was analysed by semi-quantitative RT-PCR with the house-keeping gene β-2-microglobulin (β2M)
as a loading control or by immunoblot, with actin as a loading control. C. Cell viability was examined by the Alamar blue assay,
measured as optical density (OD) units of absorbance, and expressed as percentage of absorbance for drug-treated samples
over control-treated samples (% viable cells). ** p < 0.01.
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OD
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
Cell viability assay
B
Cont IFNα TSA TSA+IFNα
Migration
900
C
Invaded cells/20x field
A
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Fluorescence OD
Molecular Cancer 2007, 6:68
*
850
800
750
Invasion
40
30
**
20
10
Cont IFNα TSA TSA+IFNα
0
Cont IFNα TSA TSA+IFNα
D
number of branches
per branching point
Vascular Sprouting
IFNα
α
Control
**
1.0
0.8
0.6
0.4
0.2
0.0
Cont IFNα TSA TSA+IFNα
TSA+IFNα
α
TSA
TSA
IFNα
- - +
+
-
+
+
TSA
IFNα
β2M
2.00
1.75
1.50
1.25
1.00
0.75
0.50
0.25
0.00
- + +
+ - +
VEGF
TSA IFNα MMP-9
*
***
Cont IFNα TSA TSA + IFNα
2.00
1.75
1.50
1.25
1.00
0.75
0.50
0.25
0.00
+
+
-
+
+
β2M
VEGF
HIF1α
α
mRNA fold induction
mRNA fold induction
HIF1α
β2M
-
*
***
mRNA fold induction
E
Cont IFNα TSA TSA + IFNα
28
24
20
16
12
8
4
0
MMP-9
*
***
Cont IFNα TSA TSA+IFNα
hypoxic
HDACI 5and
Figure
conditions
IFNα co-operatively
in vitro
inhibit endothelial cell functions, and pro-angiogenic gene expression in cancer cells under
HDACI and IFNα co-operatively inhibit endothelial cell functions, and pro-angiogenic gene expression in cancer cells under
hypoxic conditions in vitro. A. Human umbilical vein endothelial cells (HUVECs) were treated with control (Cont), 0.1 μM TSA
and/or 500 IU/ml IFNα for 18 hours. Cell viability was evaluated with the Alamar blue assay. B. HUVECs were plated in BD
Biosciences Fluroblok chambers and treated with control, 0.1 μM TSA and/or 500 IU/ml IFNα for 22 hours. Cells were stained
with Cell Tracker Green CMFDA, migrated through chamber filters toward the chemo-attractant VEGF, and then quantified
and expressed as optical density (OD) absorbance units. C. HUVECs were plated into BD BioCoat growth factor-reduced
matrigel invasion chambers and treated with control, 0.1 μM TSA and/or 500 IU/ml IFNα for 18 hours. Cells which invaded
through the Matrigel were fixed, stained with a Diff Quick staining kit, photographed and then quantified. D. HUVECs were
plated onto growth factor-reduced Matrigel in 24 well plates and treated with control, 0.1 μM TSA and/or 500 IU/ml IFNα for
18 hours. Vascular sprouting was quantified by counting the numbers of complete branches per branching point. E. Neuroblastoma BE(2)-C cells were treated with control, 0.02 μM TSA and/or 500 IU/ml IFNα for 72 hours under hypoxic (1% O2) conditions. RNA was extracted and subjected to independent semi-competitive RT-PCR analyses using trans-intron PCR primers,
together with primers for the house-keeping gene β-2 microglobulin (β2M). Representative gels for each gene at the 72 hour
time point were shown, and fold induction of a target gene by treatment was calculated by ascribing the ratio between the level
of expression of a target gene and that of β2M as 1.0 for control treated samples. * p < 0.05, ** p < 0.01, *** p < 0.001.
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transgenic mice at four weeks of age, were treated with
control, IFNα, TSA, or TSA and IFNα for one week after
abdominal tumors were first palpable. After mice were
sacrificed, tumour volume was measured, and microvasculature assessed by immunohistochemical staining for
platelet endothelial cell adhesion molecule 1 (PECAM-1)
expression (Figure 6). When tumour volume was analysed, TSA alone suppressed tumour progression by 87%,
while IFNα alone reduced tumour volume by about 36%,
compared with control treated mice. The combination of
TSA and IFNα reduced tumour volume by more than
92%, although this was not statistically significant compared with TSA treatment alone. When tumour micro-vasculature was assessed by PECAM-1 staining, the use of TSA
or IFNα alone, decreased micro-vasculature formation by
32% and 53%, respectively. However, the combination of
TSA and IFNα exerted co-operative anti-angiogenic
effects, reducing micro-vasculature by almost 90% (Figure
6)
Discussion
HDACIs have shown great promise in clinical trials in cancer patients. However, a majority of patients have been
insensitive to the treatment. In this study, we found that
Control
IFNα
α
TSA
the combination of IFNα with the HDACI TSA induced
co-operative cytotoxic effects in almost all cancer cell lines
of diverse tissue types, and demonstrated little cytoxicity
in normal non-malignant cells. The combination of IFNα
with the HDACI SAHA, already in clinical use, also exerted
co-operative anti-cancer effects, with little effect on normal cells. The combination of IFNα with another HDACI,
VPA, was less effective than IFNα and TSA, but more effective than VPA and rapamycin. These results suggest that
HDACI and IFNα combination therapy may be an effective anti-cancer strategy for future clinical trials.
Our data identified p21WAF1 expression as a key factor
responsible for cancer cell resistance to the cytotoxic
effects of combination HDACI and IFNα therapy. While
IFNα can both induce or suppress p21WAF1 gene transcription in different cells [19], it is the most common transcriptional target of HDACIs (reviewed in [2]). Previous
literature suggested that up-regulation of p21WAF1 by
HDACIs may mediate HDACI-induced cell cycle arrest
and growth inhibition [13]. However, recent publications
have cast doubt on the role of p21WAF1 in the action of
HDACIs, and, conversely demonstrated that inducible
p21WAF1 reduced HDACI-induced cell death [20-24]. Our
Number of micro-vessels/40x field
Molecular Cancer 2007, 6:68
40
35
30
25
20
15
10
5
0
***
Control IFNα
TSA TSA+IFNα
TSA+IFNα
α
Figure
TSA and6IFNα co-operatively suppress tumour-driven angiogenesis in neuroblastoma bearing transgenic MYCN mice
TSA and IFNα co-operatively suppress tumour-driven angiogenesis in neuroblastoma bearing transgenic MYCN mice. A. Photomicrographs of neuroblastoma tumour tissue sections from homozygous MYCN transgenic mice treated with either control,
TSA, IFNα, or TSA and IFNα, which were subject to immunohistochemical studies using an anti-PECAM-1 antibody. Arrows
indicate PECAM-1 positive microvessels (brown colored). B. Quantitation of the number of PECAM positive microvessels per
40× high power field in neuroblastoma tumour cross-sections. *** p < 0.001.
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Molecular Cancer 2007, 6:68
data suggests p21WAF1 expression in some cancer cells acts
as a resistance factor for the cytotoxic effects of TSA and
IFNα combination therapy.
The individual effects of HDACIs and IFNα on angiogenesis predict a co-operative therapeutic role in blocking
tumour angiogenesis. Expression of HDACs is often upregulated under angiogenic stimuli such as hypoxia in
cancer cells, and HDACIs can suppress HIF1α expression
and its down-stream targets, including VEGF[25]. HDACIs have been recently demonstrated to inhibit endothelial cell migration, invasion, vascular sprouting in vitro,
and vasculature formation in animal models of cancer
[14,16,26]. IFNα can repress VEGF and MMP-9 gene
expression, endothelial cell functions, and, inhibit
tumour-driven angiogenesis in vivo [9,27]. In our
endothelial cell migration experiments, we found in contrast, that either TSA or IFNα alone stimulated migration.
We cannot fully explain the discrepancy between our data
and previously published migration assays [14], however,
this may be due to different characteristics of the migration chamber used. Importantly, the combination of
HDACI and IFNα suppressed all endothelial cell functions, indicating a possible role for this drug combination
as a therapy for cancer patients at the point of minimal
residual disease.
Conclusion
In summary, we have found that the combination of
HDACIs, TSA, SAHA and VPA, with IFNα have significant
cytotoxic effects on a wide variety of cancer cells, with little toxicity to normal non-malignant cells. Inhibition of
p21WAF1 expression sensitizes p21WAF1-expressing cancer
cells to the combination therapy. Furthermore, HDACI
and IFNα co-operatively suppress pro-angiogenic gene
expression in cancer cells, multiple endothelial cell functions in vitro, and tumour-driven vasculature formation in
vivo. Our results provide a basis for further in vivo studies
and eventual clinical trials using the combination of
HDACIs and IFNα.
Methods
Cell culture and reagentsThe neuroblastoma cell line,
BE(2)-C, was generously supplied by Dr J Biedler (Memorial Sloan-Kettering Cancer Center, NY, USA). Breast
(MCF-7 and MDA-MB-468), lung (Calu-6 and H460),
prostate (DU-145 and LNCaP), and, colon (HT-29 and
Caco-2) cancer cells were purchased from American Type
Culture Collection (Manassas, VA, USA). All cell lines
were cultured in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum, with the exception of H460 and LNCaP, which were cultured in Roswell
Park Memorial Institute Medium, supplemented with
10% fetal calf serum. All cell lines were maintained in a
humidified incubator at 37°C and 5% CO2 in air.
http://www.molecular-cancer.com/content/6/1/68
TSA (Sigma, St. Luis, MO, USA) was dissolved in ethanol,
and SAHA (BioVision, Mountain View, CA) in dimethylsulfoxide (Sigma). IFNα (Sigma) was diluted in serum
free cell culture medium and aliquoted as a stock solution
of 100 000 units/ml. For studies in animals, TSA was dissolved in dimethyl sulfoxide (Sigma) and further diluted
with saline solution to give the final concentration of 30%
dimethyl sulfoxide and 1 mg/ml TSA.
Endothelial cell culture
Human umbilical vein endothelial cells (HUVECs) were a
gift from Dr K MacKenzie (Children's Cancer Institute
Australia, Sydney, Australia). HUVECs were maintained
in 0.1% gelatin coated tissue culture flasks or wells with
medium 199 (Invitrogen, Carlsbad, CA, USA) supplemented with 20% fetal bovine serum, 5% human serum
(Sigma), 10 U/ml heparin (Pharmacia & Upjohn, Peapack, NJ, USA), 5 ng/ml basic fibroblast growth factor
(bFGF) (Sigma) and 20 ug/ml endothelial growth factor
(Roche, Mannheim, Germany). Only passages 5 and 6
were used in the experiments. Hypoxic conditions were
maintained in a chamber filled with 1% oxygen.
Alamar blue cell viability assay
After plating in 96 well plates, cells were allowed to attach
for 24 hours, followed by treatment with various drugs for
72 hours. Before the end of treatment, cells were incubated with Alamar blue (Invitrogen) for 5 hours, and
plates were then read on a micro-plate reader at 570/595
nm. Relative cell viability was calculated according to the
readings and expressed as optical density (OD) absorbance units.
Immunoblot analysis
Twenty four hours after treatment with control, TSA and/
or IFNα, protein was extracted from whole cells, separated
by electrophoresis, and transferred onto nitrocellulose
membrane. Membranes were incubated with mouse antihuman p21WAF1 antibodies (Santa Cruz Biotechnologies,
Santa Cruz, CA, USA) (1:1000), followed by goat antimouse antibody (1:2000) conjugated with horseradish
peroxidase. Chemiluminescent detection was performed
using SuperSignal reagents (Pierce). Membranes were
then re-probed with an anti-β-actin antibody (Pierce), as
a loading control.
siRNA transfectionMCF7 cells were transfected with a validated scrambled siRNA or siRNA specifically targeting
p21WAF1 (SmartPool siRNA CDKN1A, Dharmacon
Research, Lafayete, CO) with Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer's recommendation. Cells were lysed, and RNA or
protein extracted 24 hours later for Reverse Transcriptionpolymerase Chain Reaction (RT-PCR) or immunoblot
analysis of siRNA transfection efficacy.
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Molecular Cancer 2007, 6:68
Semi-quantitative competitive RT-PCR
Semi-quantitative competitive RT-PCR was carried out as
described previously [28] to analyse siRNA transfection
efficiency in MCF-7 cells and the effect of TSA and/or
IFNα treatment on angiogenic gene expression in BE(2)-C
cells. Specific primers used for PCR were as follows: 5'CAGCAGAGGAAGACCATGTG-3' and 5'-GGCGTTTGGA
GTGGTAGAAA-3' for p21WAF1; 5'-TTACAGCAGCCAGACGATCA-3' and 5'-ATTGCCCCAGCAGTCTACAT-3' for
HIF1α; 5'-CCTTGCTGCTCTACCTCCAC-3' and 5'-ATGA
TTCTGCCCTCCTCCTT-3' for vascular endothelial growth
factor (VEGF); 5'-TTCCCTGGAGACCTGAGAAC-3' and
5'-AGGGACAGTTGCTTCTGGAG-3' for metalloproteinase-9 (MMP-9); 5'-ACCCCCACTGAAAAAGATGA-3' and
5'-ATCTTCAAACCTCCATGATG-3' for β2-microglobulin
(β2M).
Endothelial cell migration assay
HUVEC migration towards the chemo-attractant, VEGF
(Sigma), was tested using a BD Biosciences Fluroblok
(Becton Dickinson) endothelial cell migration system
according to the manufacture's guidelines. Cells were
labeled with 1 μM Cell Tracker Green CMFDA fluorescence solution (Invitrogen) for 30 minutes, and migrated
through filters into 24 well plates. Thereafter the plate was
read with a Fluroscence plate reader at 492/517 nm. The
relative cell number was calculated according to the readings and expressed as optical density (OD) absorbance
units.
Endothelial cell invasion assay
HUVEC invasion through matrigel towards the chemoattractant, VEGF, was investigated using BD BioCoat,
growth factor-reduced Matrigel, endothelial cell invasion
chambers (Becton Dickinson), according to the manufacturer's guidelines. Endothelial cells which invaded
through the matrigel to the other side of the inserts, were
fixed and stained with Diff Quick staining kit (Baxter) and
photographed. The number of cells per 20× objective field
was counted under an inverted microscope.
Vascular sprouting (capillary tubule formation) assay
The vascular sprouting assays were performed on 24 well
plates coated with 250 μl of polymerized, growth factorreduced Matrigel matrix (Becton Dickinson) per well.
HUVECs were plated on Matrigel and treated with control, TSA and/or IFNα for 18 hours. Quantification of vascular sprouting was determined by counting the number
of complete branches per branching point.
Animal model studies
As soon as tumors were confirmed by abdominal palpation, MYCN homozygous transgenic mice [18], were randomized to four groups (n = 5/group) and injected
intraperitoneally daily for 7 days with control, TSA at 20
http://www.molecular-cancer.com/content/6/1/68
mg/kg of body weight, mouse IFNα at 1 × 106 IU/kg body
weight, or TSA and IFNα. Mice were sacrificed at the end
of the week of treatment. Tumors were then removed, formalin-fixed and paraffin-embedded. All studies involving
animals were approved by the animal care and ethics
committee of the University of New South Wales, Sydney,
Australia.
Immunohistochemical studies
Mouse tissue sections were incubated with goat anti-platelet endothelial cell adhesion molecule 1 (PECAM-1) antibody (1:500) (Santa Cruz Biotechnology), followed by
incubation with biotinylated rabbit, anti-goat antibody
(1:500) and streptavidin-horseradish peroxidase.
Endothelial cells were visualised with 3,3'-diaminobenzidine solution, and micro-vessels were quantified as
described previously [29].
Statistical analyses
All data for statistical analyses were presented as mean
±standard error. Differences were analyzed for significance using ANOVA among groups. A probability value of
0.05 or less was considered significant.
Abbreviations
bFGF: basic fibroblast growth factor; Cont: control;
HDAC: histone deacetylase; HDACI: histone deacetylase
inhibitor; HIF1α: hypoxia-inducible factor 1α; HUVEC:
human umbilical vein endothelial cells; IFNα: α-interferon; MMP-9: matrix metalloproteinase 9; OD: optical
density; PECAM-1: platelet endothelial cell adhesion molecule 1; TSA: Trichostatin A; RAP: rapamycin; VEGF: vascular endothelial growth factor; VPA: valproate
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
SK, TL, AT and TD performed experiments and analysed
data. GMM, MH and MN designed experiments. TL and
GMM analysed data and wrote the manuscript. All
authors have read and approved the final version of the
manuscript.
Acknowledgements
This work was supported by National Health and Medical Research Council, Cancer Institute New South Wales and Cancer Council New South
Wales. Children's Cancer Institute Australia for Medical Research is affiliated with the University of New South Wales and Sydney Children's Hospital.
References
1.
Johnstone RW: Histone-deacetylase inhibitors: novel drugs for
the treatment of cancer. Nat Rev Drug Discov 2002, 1(4):287-299.
Page 10 of 11
(page number not for citation purposes)
Molecular Cancer 2007, 6:68
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
Liu T, Kuljaca S, Tee A, Marshall GM: Histone deacetylase inhibitors: multifunctional anticancer agents. Cancer Treat Rev 2006,
32(3):157-165.
Marks P, Rifkind RA, Richon VM, Breslow R, Miller T, Kelly WK: Histone deacetylases and cancer: causes and therapies. Nat Rev
Cancer 2001, 1(3):194-202.
Kelly WK, Marks PA: Drug insight: Histone deacetylase inhibitors--development of the new targeted anticancer agent
suberoylanilide hydroxamic acid. Nat Clin Pract Oncol 2005,
2(3):150-157.
Folkman J, Kalluri R: Cancer without disease. Nature 2004,
427(6977):787.
Bergers G, Benjamin LE: Tumorigenesis and the angiogenic
switch. Nat Rev Cancer 2003, 3(6):401-410.
von Marschall Z, Scholz A, Cramer T, Schafer G, Schirner M, Oberg
K, Wiedenmann B, Hocker M, Rosewicz S: Effects of interferon
alpha on vascular endothelial growth factor gene transcription and tumor angiogenesis.
J Natl Cancer Inst 2003,
95(6):437-448.
Tosato G: Interferon-alpha is implicated in the transcriptional
regulation of vascular endothelial growth factor. J Natl Cancer
Inst 2003, 95(6):420-421.
Brouty-Boye D, Zetter BR: Inhibition of cell motility by interferon. Science 1980, 208(4443):516-518.
Bjornsti MA, Houghton PJ: The TOR pathway: a target for cancer therapy. Nat Rev Cancer 2004, 4(5):335-348.
Cinatl J Jr., Kotchetkov R, Blaheta R, Driever PH, Vogel JU, Cinatl J:
Induction of differentiation and suppression of malignant
phenotype of human neuroblastoma BE(2)-C cells by valproic acid: enhancement by combination with interferonalpha. Int J Oncol 2002, 20(1):97-106.
Michaelis M, Suhan T, Cinatl J, Driever PH, Cinatl J Jr.: Valproic acid
and interferon-alpha synergistically inhibit neuroblastoma
cell growth in vitro and in vivo.
Int J Oncol 2004,
25(6):1795-1799.
Richon VM, Sandhoff TW, Rifkind RA, Marks PA: Histone deacetylase inhibitor selectively induces p21WAF1 expression and
gene-associated histone acetylation. Proc Natl Acad Sci U S A
2000, 97(18):10014-10019.
Kwon HJ, Kim MS, Kim MJ, Nakajima H, Kim KW: Histone deacetylase inhibitor FK228 inhibits tumor angiogenesis. Int J Cancer
2002, 97(3):290-296.
Kim MS, Kwon HJ, Lee YM, Baek JH, Jang JE, Lee SW, Moon EJ, Kim
HS, Lee SK, Chung HY, Kim CW, Kim KW: Histone deacetylases
induce angiogenesis by negative regulation of tumor suppressor genes. Nat Med 2001, 7(4):437-443.
Sasakawa Y, Naoe Y, Noto T, Inoue T, Sasakawa T, Matsuo M, Manda
T, Mutoh S: Antitumor efficacy of FK228, a novel histone
deacetylase inhibitor, depends on the effect on expression of
angiogenesis factors. Biochem Pharmacol 2003, 66(6):897-906.
Slaton JW, Karashima T, Perrotte P, Inoue K, Kim SJ, Izawa J, Kedar
D, McConkey DJ, Millikan R, Sweeney P, Yoshikawa C, Shuin T, Dinney CP: Treatment with low-dose interferon-alpha restores
the balance between matrix metalloproteinase-9 and E-cadherin expression in human transitional cell carcinoma of the
bladder. Clin Cancer Res 2001, 7(9):2840-2853.
Hansford LM, Thomas WD, Keating JM, Burkhart CA, Peaston AE,
Norris MD, Haber M, Armati PJ, Weiss WA, Marshall GM: Mechanisms of embryonal tumor initiation: distinct roles for MycN
expression and MYCN amplification. Proc Natl Acad Sci U S A
2004, 101(34):12664-12669.
Detjen KM, Murphy D, Welzel M, Farwig K, Wiedenmann B, Rosewicz S: Downregulation of p21(waf/cip-1) mediates apoptosis
of human hepatocellular carcinoma cells in response to
interferon-gamma. Exp Cell Res 2003, 282(2):78-89.
Burgess AJ, Pavey S, Warrener R, Hunter LJ, Piva TJ, Musgrove EA,
Saunders N, Parsons PG, Gabrielli BG: Up-regulation of
p21(WAF1/CIP1) by histone deacetylase inhibitors reduces
their cytotoxicity. Mol Pharmacol 2001, 60(4):828-837.
Nguyen DM, Schrump WD, Chen GA, Tsai W, Nguyen P, Trepel JB,
Schrump DS: Abrogation of p21 expression by flavopiridol
enhances depsipeptide-mediated apoptosis in malignant
Clin Cancer Res 2004,
pleural mesothelioma cells.
10(5):1813-1825.
Rosato RR, Almenara JA, Yu C, Grant S: Evidence of a functional
role for p21WAF1/CIP1 down-regulation in synergistic anti-
http://www.molecular-cancer.com/content/6/1/68
23.
24.
25.
26.
27.
28.
29.
leukemic interactions between the histone deacetylase
inhibitor sodium butyrate and flavopiridol. Mol Pharmacol
2004, 65(3):571-581.
Qiu L, Burgess A, Fairlie DP, Leonard H, Parsons PG, Gabrielli BG:
Histone deacetylase inhibitors trigger a G2 checkpoint in
normal cells that is defective in tumor cells. Mol Biol Cell 2000,
11(6):2069-2083.
Ungerstedt JS, Sowa Y, Xu WS, Shao Y, Dokmanovic M, Perez G, Ngo
L, Holmgren A, Jiang X, Marks PA: Role of thioredoxin in the
response of normal and transformed cells to histone
deacetylase inhibitors.
Proc Natl Acad Sci U S A 2005,
102(3):673-678.
Mayo MW, Denlinger CE, Broad RM, Yeung F, Reilly ET, Shi Y, Jones
DR: Ineffectiveness of histone deacetylase inhibitors to
induce apoptosis involves the transcriptional activation of
NF-kappa B through the Akt pathway. J Biol Chem 2003,
278(21):18980-18989.
Qian DZ, Wang X, Kachhap SK, Kato Y, Wei Y, Zhang L, Atadja P,
Pili R: The histone deacetylase inhibitor NVP-LAQ824 inhibits angiogenesis and has a greater antitumor effect in combination with the vascular endothelial growth factor receptor
tyrosine kinase inhibitor PTK787/ZK222584. Cancer Res 2004,
64(18):6626-6634.
Ozawa S, Shinohara H, Kanayama HO, Bruns CJ, Bucana CD, Ellis LM,
Davis DW, Fidler IJ: Suppression of angiogenesis and therapy of
human colon cancer liver metastasis by systemic administration of interferon-alpha. Neoplasia 2001, 3(2):154-164.
Liu T, Bohlken A, Kuljaca S, Lee M, Nguyen T, Smith S, Cheung B,
Norris MD, Haber M, Holloway AJ, Bowtell DD, Marshall GM: The
retinoid anticancer signal: mechanisms of target gene regulation. Br J Cancer 2005, 93(3):310-318.
Qian DZ, Kato Y, Shabbeer S, Wei Y, Verheul HM, Salumbides B,
Sanni T, Atadja P, Pili R: Targeting tumor angiogenesis with histone deacetylase inhibitors: the hydroxamic acid derivative
LBH589. Clin Cancer Res 2006, 12(2):634-642.
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