Antisense to cyclin D1 inhibits vascular endothelial growth factor-stimulated growth of vascular endothelial cells: implication of tumor vascularization.

Abstract

Our aim was to determine the effects of cyclin D1 inhibition on tumor-associated neovascularization and endothelial cell growth.
We have generated adenovirus system for antisense to cyclin D1 (AS CyD1) and evaluated in vitro and in vivo effects. Small interfering RNA against cyclin D1 was also used to analyze cyclin D1 inhibition-associated vascular endothelial growth factor (VEGF) regulation.
The xenografts treated with adenoviral AS CyD1 showed less vessel density and displayed smaller tumor size in colon cancer cell lines HCT116 and DLD1. In vitro studies indicated that AS CyD1 decreased VEGF protein expression in DLD1 but not in HCT116. Cyclin D1 small interfering RNA caused a decrease in VEGF expression at protein and RNA levels in DLD1. A modest decrease was noted in the VEGF promoter activity, with inactivation of the STAT3 transcription factor through dephosphorylation. On the hand, the cyclin D1 inhibition plus STAT3 inhibitor markedly decreased VEGF expression in HCT116, although VEGF did not change by the STAT3 inhibitor alone. In cultures of human umbilical vein endothelial cells (HUVEC), VEGF augmented cyclin D1 expression and cell growth. AS CyD1 significantly inhibited HUVEC growth even in the presence of VEGF. AS CyD1 also significantly suppressed in vitro tube formation in VEGF-treated HUVEC and in vivo macroaneurysm formation in VEGF-treated Matrigel plug.
Our results suggest that cyclin D1 may play a role in the maintenance of VEGF expression and that AS CyD1 could be potentially useful for targeting both cancer cells and their microenvironment of tumor vessels.

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2006;12:4720-4729. Published online August 9, 2006.Clin Cancer Res


Masayoshi Yasui, Hirofumi Yamamoto, Chew Yee Ngan, et al.
Implication of Tumor Vascularization
Stimulated Growth of Vascular Endothelial Cells:
−
Factor
Antisense to Cyclin D1 Inhibits Vascular Endothelial Growth



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Antisense to Cyclin D1InhibitsVascular Endothelial Growth
Factor^Stimulated Growth ofVascular Endothelial Cells:
Implication ofT umorVascularization
Masayoshi Yasui,1Hirofumi Yamamoto,1Chew YeeNgan,1Bazarragchaa Damdinsuren,1Yurika Sugita,1
Hiroki Fukunaga,1Jinyu Gu,1Makiko Maeda,3Ichiro Takemasa,1Masataka Ikeda,1Yasushi Fujio,3
Mitsugu Sekimoto,1Nariaki Matsuura,2I. BernardWeinstein,4and Morito Monden1
Abstract
Purpose: Our aim was to determine the effects of cyclin D1inhibition on tumor-associated
neovascularization and endothelial cell growth.
Experimental Design: We have generated adenovirus system for antisense to cyclin D1 (AS
CyD1) and evaluated in vitro and in vivo effects. Small interfering RNA against cyclin D1was
also used to analyze cyclin D1inhibition-associated vascular endothelial growth factor (VEGF)
regulation.
Results: The xenografts treated with adenoviral AS CyD1showed less vessel density and
displayed smaller tumor sizeincoloncancercelllines HCT116 and DLD1. Invitro studiesindicated
that AS CyD1decreased VEGF protein expression in DLD1but not in HCT116. Cyclin D1small
interfering RNA caused a decrease in VEGF expression at protein and RNA levels in DLD1.
A modest decrease was noted in theVEGF promoter activity, with inactivation of the STAT3
transcription factor through dephosphorylation. On the hand, the cyclin D1inhibition plus STAT3
inhibitor markedly decreased VEGF expression in HCT116, althoughVEGF did not change by
the STAT3 inhibitor alone. In cultures of human umbilical vein endothelial cells (HUVEC),VEGF
augmented cyclin D1expression and cell growth. ASCyD1significantly inhibited HUVEC growth
even in the presence ofVEGF. AS CyD1also significantly suppressed in vitro tube formation in
VEGF-treated HUVEC and invivo macroaneurysm formationinVEGF-treated Matrigelplug.
Conclusions: Our results suggest that cyclin D1may play a role in the maintenance ofVEGF
expression and that ASCyD1could be potentially useful for targeting both cancer cells and their
microenvironment of tumor vessels.
Cyclin D1, a putative G1cyclin, preferentially associates with
CDK4 and positively regulates the cell cycle transition from G1
to S phase (1). Cyclin D1 is considered an oncogene because
forced expression in rodent fibroblasts induces tumorigenicity
in nude mice and cyclin D1 transgenic mice develop tumors of
breast, esophagus, stomach, and tongue (2–4). In human
carcinomas, increased expression of cyclin D1 is one of the most
frequent abnormalities because it is detected in f60% of breast
cancers, 40% of colorectal cancers, 40% of squamous carcino-
mas of the head and neck, and 20% of prostate cancers (5–8).
Furthermore, overexpression of cyclin D1 is associated with
poor prognosis of patients with carcinomas of colorectum, eso-
phagus, stomach, pancreas, and liver (9–13). Therefore, cyclin
D1 is a crucial target for various types of human malignancies.
To suppress the malignant potential of carcinomas, the
strategy of antisense to cyclin D1 (AS CyD1) was first assessed
in human esophageal squamous cell carcinoma and colon
cancer cells (14, 15). These studies clearly showed that AS CyD1
reversed the transformed phenotype of tumor cells, inhibited
cell growth of tumor cells, and resulted in loss of tumorigenic-
ity. Subsequently, AS CyD1 was found to enhance chemo-
sensitivity of 5-fluorouracil, mitoxantrone, and cisplatinum in
pancreatic cancer cells and head and neck cancer cells and to
induce apoptosis and tumor shrinkage in esophageal squamous
carcinoma (16–19). Similar antitumor effects were found in
gastric cancer cells and hepatocellular carcinoma cells (20–22).
Based on these favorable effects, it was considered that AS
CyD1 could be a promising strategy against human pancreatic,
colonic, and esophageal cancers (23).
Cancer Therapy: Preclinical
Authors’Affiliations:1Department of Surgery and Clinical Oncology, Graduate
School of Medicine,2Department of Pathology, School of Allied Health Science,
Faculty of Medicine, and3Department of Clinical Evaluation of Medicine and
Therapeutics, Graduate School of Pharmaceutical Sciences, Osaka University,
Osaka, Japan; and4Herbert Irving Comprehensive Cancer Center, College of
Physicians and Surgeons, Columbia University, NewYork, NewYork
Received 6/6/05; revised1/10/06; accepted 2/13/06.
Grant support: Grant-in Aid for Cancer Research from the Ministry of Education,
Science, Sports, and CultureTechnology, Japan (H.Yamamoto) and grant for the
ThirdTerm Comprehensive Strategy for Cancer Control from the Ministry of Health
LaborandWelfare.
The costs of publicationof this article were defrayedinpartby thepaymentof page
charges.This article must therefore be hereby marked advertisement in accordance
with18 U.S.C. Section1734 solely toindicate this fact.
Requests for reprints: HirofumiYamamoto, Department of Surgery and Clinical
Oncology, Graduate School of Medicine, Osaka University, 2-2 Yamada-oka,
Suita-City, Osaka 565-0871, Japan. Phone: 81-6-6879-3251; Fax: 81-6-6879-
3259; E-mail: kobunyam@surg2.med.osaka-u.ac.jp.
F2006 American Association for Cancer Research.
doi:10.1158/1078-0432.CCR-05-1213
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Angiogenesis is essential for tumor growth and expansion
because the blood vessels supply malignant cells with sufficient
oxygen and nutrients (24, 25). Therefore, interruption of this
process is one strategy to prevent invasion and metastasis.
Although various biological effects by AS CyD1 have been
reported as mentioned above, the antiangiogenic action of AS
CyD1 is unknown. To explore this issue, we generated
adenoviral AS CyD1 (Ad-AS CyD1) system and examined the
effect of AS CyD1 on in vivo tumor-associated neovasculariza-
tion, with special attention to vascular endothelial growth
factor (VEGF), because VEGF is known as a critical growth
factor that promotes endothelial cell proliferation and angio-
genesis (26). We also examined its direct effect on in vitro and
in vivo growth of vascular endothelial cells.
Materials and Methods
Cell lines and animals.
(HCT116, DLD1, and LoVo), and gastric cancer cells (MKN45 and
MKN28) were purchased from the American Type Culture Collection
(Manassas, VA) or the Japanese Cancer Research Resources Bank
(Osaka, Japan). MKN45 was grown in RPMI 1640, whereas the other
cell lines were grown in DMEM, both supplemented with 10% fetal
bovine serum, 100 units/mL penicillin, and 100 Ag/mL streptomycin in
5% CO2at 37jC. Human umbilical vein endothelial cells (HUVEC)
were grown on MCDB131 culture medium (Chlorella, Inc., Tokyo,
Japan) supplemented with 10% fetal bovine serum, antibiotics, and 10
ng/mL basic fibroblast growth factor. Female 4-week-old athymic nude
mice were purchased from Nihon CREA, Inc. (Tokyo, Japan) and were
housed under pathogen-free conditions. The experimental protocol was
approved by the Ethics Review Committee for Animal Experimentation
of Osaka University School of Medicine.
Reagents and antibodies. Human recombinant VEGF was obtained
from IBL Co. (Gunma, Japan). Bromodeoxyuridine (BrdUrd) was
purchased from Sigma-Aldrich (St. Louis, MO). STAT3 inhibitor peptide
was purchased from Calbiochem (Darmstadt, Germany). This reagent is
a cell-permeable analogue of the STAT3-SH2 domain-binding phos-
phopeptide that contains a COOH-terminal membrane-translocating
sequence and acts as a highly selective, potent blocker of STAT3
activation (27). The following antibodies were used at appropriate
concentrations as recommended by the manufacturers: (a) anti-human
polyclonal antibodies for cyclin D1 (Santa Cruz Biotechnology, Santa
Cruz, CA), cyclin A (Upstate Biotechnology, Waltham, MA), VEGF
(Santa Cruz Biotechnology), actin (Sigma-Aldrich), phosphorylated
STAT3 antibody (Tyr705; Cell Signaling Technology, Beverly, MA), and
STAT3 antibody (Cell Signaling Technology); (b) anti-human mono-
clonal antibodies for cyclin E (BD Biosciences, BD PharMingen, San
Diego, CA) and BrdUrd (DAKO, Glostrup, Denmark); and (c) anti-
mouse rat monoclonal antibody for CD31 (Santa Cruz Biotechnology).
Western blot analysis.Western blot analysis was done as described
previously (28). Briefly, the protein samples (25 Ag) were separated by
10% or 12.5% PAGE followed by electroblotting onto a polyvinylidene
difluoride membrane. The membrane was incubated with the primary
antibodies at the appropriate concentrations (1:200 for cyclin D1 and
VEGF and 1:1,000 for total STAT3, phosphorylated STAT3, and actin)
for 1 hour. The protein bands were detected using the Amersham
enhanced chemiluminescence detection system (Amersham Biosciences
Corp., Piscataway, NJ).
Generation of Ad-AS CyD1. Ad-AS CyD1 was constructed using
AdEasy Adenoviral Vector System (ref. 29; a generous gift from Dr. Bert
Vogelstein, Johns Hopkins University School of Medicine, Baltimore,
MD). A 1.1-kb human entire cyclin D1 cDNA that provides 90%
homology of mouse cyclin D1 cDNA (2) was cut out from pcDNA3-
cyclin D1 plasmid and subcloned into the HindIII site of pShuttle
plasmid containing cytomegalovirus promoter in antisense orientation.
HEK293 cells, human colon cancer cells
After confirmation of antisense orientation by appropriate enzymes,
recombination with the E1/E3delete adenoviral backbone vector
(AdEasy-1) was done in Escherichia coli BJ5183 cells by electroporation.
Viral particles were amplified in HEK293 cells and then purified
by CsCl banding. Virus titer was measured by Adeno-X rapid titer kit
(BD Clontech, Palo Alto, CA). Ad-(cytomegalovirus) Mock and Ad-
(cytomegalovirus) green fluorescent protein were prepared as experi-
mental controls.
Infectious efficiency and cytotoxicity of adenovirus in the cell lines.
determine the optimal concentrations that sufficiently realize adenovi-
ral gene transfer, Ad-(cytomegalovirus) green fluorescent protein was
infected at various concentrations for 1 hour with gentle shaking and
then incubated with the complete medium. The virus titer [multiplicity
of infection (MOI)] endowing >90% green fluorescent protein–positive
cells at 24 hours after infection was as follows: HUVEC, 20; MKN45, 40;
MKN28, 20; HCT116, 10; DLD1, 40; and LoVo, 60. At the respective
virus titer, the cell viability of HUVEC, MKN45, MKN28, HCT116, and
DLD1 as indicated by the trypan blue exclusion test was >90%, but the
cell viability of LoVo was <60%.
Growth assays.Cells were uniformly seeded (1 ? 105per well) into
six-well dishes in triplicate. Twenty-four hours later, the culture medium
was removed and replaced with 0.5 mL fresh medium containing
adenovirus at the optimal concentration for 1 hour. The cells were then
grown in the complete medium and counted using a hemocytometer.
Immunohistochemistry.Immunostaining was done as described
previously (28). Briefly, after deparaffinization, heat antigen retrieval
was done in 10 mmol/L citrate buffer (pH 6.0) at 95jC for 40 minutes.
The slides were then processed for immunohistochemistry using the
Vectastain Elite avidin-biotin complex kit (Vector Laboratories, Burlin-
game, CA). Primary antibodies were applied to sections at a dilution of
1:750 for CD31 and incubated overnight at 4jC. For the negative
control, nonimmunized immunoglobulin G (Vector Laboratories) was
used as a substitute for the primary antibody.
Semiquantitative reverse transcription-PCR.
carried out with TRIzol reagent in a single-step method and cDNA
was generated with avian myeloblastosis virus reverse transcriptase
(Promega, Madison, WI). Semiquantitative analyses of the expression
of VEGF RNA were done using the duplex reverse transcription-
PCR technique as described previously (30). h-Actin was used as the
internal standard. PCRs were done in a total volume of 25 AL, which
consisted of 2 AL cDNA template, 1? Perkin-Elmer PCR buffer (Perkin-
Elmer, Foster City, CA), 1.5 mmol/L MgCl2, 0.8 mmol/L deoxynucleo-
tide triphosphates, 1.25 pmol h-actin and 5 pmol VEGF primer, and
1 unit Taq DNA polymerase (AmpliTaq Gold; Roche Molecular
Systems, Branchburg, NJ). PCR amplification was done with a GenAmp
PCR System 9600 (Perkin-Elmer). The primer sequences were as
follows (30, 31): h-actin sense 5¶-GAAAATCTGGCACCACACCTT-3¶
and antisense 5¶-GTTGAAGGTAGTTTCGTGGAT-3¶ and VEGF sense 5¶-
AAGCCATCCTGTGTGCCCCTGATG-3¶ and antisense 5¶-GCGAATTCC-
TCCTGCCCGGCTCAC-3¶.
BrdUrd labeling index. The cells were incubated with 20 Amol/L
BrdUrd (Sigma-Aldrich) at 37jC for 25 minutes and fixed in 70% cold
ethanol for 30 minutes. After quenching the endogenous peroxidase
activity, the chambers were incubated in 4 N HCl at 37jC for
30 minutes and then neutralized by buffered boric acid (pH 9.0) for
5 minutes. After blocking with 10% rabbit serum, anti-BrdUrd antibody
was applied to the chambers at the dilution of 1:20 at room tempera-
ture for 2 hours followed by the avidin-biotin complex method.
In vitro angiogenesis assay.In vitro formation of tubular structure in
HUVEC was examined using In vitro Angiogenesis Assay kit (Chemicon
International, Inc., Temecula, CA). HUVEC infected with Ad-AS CyD1
or Ad-Mock (20 MOI) were seeded on Matrigel-coated well and main-
tained on complete medium. After attachment of the cells on Matrigel,
the medium was changed with fresh medium, either with or without the
recombinant VEGF protein (25 ng/mL), and incubated for 10 hours.
Cells were then observed under the inverted microscope and the number
of capillary connections was counted as reported previously (32).
To
RNA extraction was
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In vivo Matrigel angiogenesis assay.
assayed as growth of blood vessels of mouse s.c. tissue in the exogenous
Matrigel plug. Matrigel was prepared with 100 ng/mL basic fibroblast
growth factor and 64 units/mL heparin with or without 40 ng/mL
VEGF. Ad-AS CyD1 or Ad-Mock was included in the Matrigel plug at a
concentration of 1 ? 109plaque-forming units/mL. The Matrigel was
injected (0.5 mL) into the s.c. tissue of female athymic mice (n = 4 for
each group). On day 7, mice were sacrificed and Matrigel plugs were
removed and fixed in 10% buffered formalin and embedded in
paraffin. Sections were stained with H&E or CD31 antibody and
examined under a light microscope. In vivo angiogenesis was scored
planimetrically through observation of 10 fields at high-power
magnification, and the percentage of vessel area to total Matrigel area
was calculated as reported previously (32, 33).
Treatment of established tumor xenografts by intratumoral injection of
Ad-AS CyD1.S.c. xenografts of colorectal cancer (HCT116 and DLD1)
were established in nude mice (n = 4 for each group) by injection 5 ?
106cells. After 1 week (day 7), when the tumor size reached f100 to
150 mm3, Ad-Mock, Ad-AS CyD1 (0.5 ? 109plaque-forming units/
injection), and saline were injected into tumors and more than two
injections per tumor were done on days 9 and 11. On day 30, the mice
were sacrificed.
Transfection.SiTrio cyclin D1 and negative control small interfering
RNA (siRNA) were purchased from B-Bridge International, Inc.
(Sunnyvale, CA). Each siRNA consisted of three different target
sequences and the sequences are as follows: negative control 5¶-ATCC-
GCGCGATAGTACGTA-3¶, 5¶-TTACGCGTAGCGTAATACG-3¶, and 5¶-
TATTCGCGCGTATAGCGGT-3¶ and siRNA CCND1/human 5¶-CGCU-
GGAGCCCGUGAAAAATT-3¶, 5¶-CCAGAGUGAUCAAGUGUGATT-3¶,
and 5¶-UCCAAUAGGUGUAGGAAAUTT-3¶.
Cells were transfected with 100 nmol/L siRNA using LipofectAMINE
2000 (Invitrogen, Carlsbad, CA) in Opti-MEM I Reduced Serum
Medium (Invitrogen). After 6 hours, medium was replaced by standard
medium. In use of STAT3 inhibitor peptide, it was added at 100 Amol/L
12 hours after transfection.
pcDNA3 (1 Ag; Invitrogen) or STAT3 expression vector [pCAG-
wtSTAT3-IP (wild-type) and pCAG-dnSTAT3-IP (a dominant-negative
mutant of STAT3)] was introduced into cells with LipofectAMINE 2000.
The wild-type STAT3 expression vector and the dominant-negative
STAT3 plasmid were provided from Prof. T. Yokota (Department of
Stem Cell Biology, Kanazawa University, Kanazawa, Japan; ref. 34).
These expression vectors are driven by cytomegalovirus enhancer-
chicken h-actin hybrid promoter (developed by Prof. Jun-ichi Miyazaki,
Department Nutrition and Physiological Chemistry, Osaka University,
Osaka, Japan; ref. 35).
Reporter gene assay. The VEGF gene promoter region (provided by
Dr. Abraham, SIOS, Inc., CA) was inserted upstream of the luciferase
reporter gene in pGVB (Toyo, Inc., Tokyo, Japan) as described
previously (36). Cells were transfected with siRNA and 2 Ag reporter
plasmid pGVB-VEGF and 0.025 Ag pRL-SV40 (36) in Opti-MEM I
Reduced Serum Medium. After 6 hours, medium were replaced by
standard medium. Cells were harvested at 24 hours after transfection
for the dual-luciferase assay (Promega) according to the manufacturer’s
instruction. The firefly luciferase activity of pGVB-VEGF was normalized
by the Renilla luciferase activity of pRL-SV40. The level of luciferase in
control cells was assigned a value of 1.0, and the relative activities were
calculated.
Statistical analysis.Data are expressed as mean F SD. Differences
among three groups were examined by the one-factor ANOVA followed
by post-test of Bonferroni/Dunn. Statistical analysis was done using the
StatView 5.0 (SAS Institute, Inc., Cary, NC).
In vivo angiogenesis was
Results
Effects of AS CyD1 on cyclin D1 expression and growth of tumor
cells. A relatively high expression of the cyclin D1 protein was
noted in HCT116, DLD1, and MKN45 cells and VEGF protein
was expressed in HCT116, DLD1, LoVo, and MKN45 (Fig. 1A).
Compared with Mock control, AS CyD1 decreased cyclin D1
expression in MKN45 cells in a time-dependent manner
(Fig. 1B). In HCT116 and DLD1 cells, AS CyD1 showed
decreased expression of the cyclin D1 protein after 48 hours
(data not shown). In vitro cell proliferation assays showed that
AS CyD1 significantly inhibited tumor cell growth compared
with Mock control or nontreatment cultures of MKN45,
HCT116, and DLD1 (Fig. 1C). AS CyD1 decreased expression
of the VEGF protein in DLD1 but not in HCT116 (Fig. 1D).
Effect of AS CyD1 on tumor vascularization. Tumor size on
day 30 was significantly smaller in AS CyD1 group compared
with Mock control group or saline treatment group in both cell
types (P < 0.01; Fig. 2A). Immunohistochemistry of tumor
tissues revealed that there was a significant reduction in vessel
density in xenografts of both AS CyD1 group and Mock control
group (P < 0.01; Fig. 2B). Especially, significant decrease in
vessel density was noted in Ad-AS CyD1–injected DLD1
xenografts when compared with saline treatment (P < 0.01;
Fig. 2B).
Expression of cyclins in HUVEC. Serial changes in the
expression of cyclin D1 were determined in cultures of HUVEC
refed with 10% fetal bovine serum after 24 hours serum
starvation. Increase in cyclin D1 expression was detected as
early as 1 hour after serum addition and a further increase was
noted at 12 hours and subsequent time points (Fig. 3A). Cyclin
A and cyclin E levels also increased with the highest expression
noted at 24 hours (Fig. 3A).
Treatment of HUVEC with recombinant VEGF and Ad-AS
CyD1. The addition of recombinant VEGF protein to HUVEC
culture further increased the level of cyclin D1 (Fig. 3B). AS
CyD1 markedly decreased cyclin D1 expression in VEGF-treated
HUVEC compared with Mock control cultures as early as 1
hour (Fig. 3C). When cell growth was assessed by BrdUrd
incorporation, VEGF treatment in Mock control cultures caused
a significant increase in BrdUrd labeling index than VEGF
without Mock control cultures (P < 0.01), which was drawn
back to the basal level by AS CyD1 (Fig. 3D). The difference
between VEGF treatment and VEGF plus AS CyD1 was
significant (P < 0.01; Fig. 3D).
Effects of AS CyD1 on in vitro and in vivo angiogenesis. We
then examined effects of AS CyD1 on angiogenesis. In vitro
angiogenesis assay showed that HUVEC formed vessel-
like structures (tubes) when plated on Matrigel-coated wells
(Fig. 4A). The VEGF treatment enhanced HUVEC growth,
resulting in thick tubes and increased steady network formation.
In contrast, AS CyD1 caused thinner or only faint tube-like
structures even in VEGF-treated cultures. There was a significant
difference in the number of capillary connections, defined as
cross-points consisting of three tubes (32) in each combination
(P < 0.01; Fig. 4A).
In vivo angiogenesis assay showed modest vessel formation in
Matrigel plug of the Mock control group (Fig. 4B, I). Mean
vessel area relative to the Matrigel plug area was calculated
(Mock group, 13.2 F 0.73%) with reference to CD31-stained
vascular endothelial cells (Fig. 4B, I, a) and RBC (Fig. 4B, I, b)
as guidance for neovascularization in Matrigel plug. With
treatment of VEGF, Matrigel was expansively enlarged because
of the endothelioma-like structure that grew in the center of
Matrigel and contained a giant aneurysm (Fig. 4B, II, mean
Cancer Therapy: Preclinical
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vessel area, 84.2 F 13.1%). Although the majority of the
aneurysm-like structure dropped off in making paraffin blocks,
CD31 staining showed that this structure was lined by
endothelium (Fig. 4B, II, a, arrows). There were also some
RBC remaining in Matrigel (Fig. 4B, II, b). In contrast, VEGF
plus AS CyD1 resulted in production of numerous micro-
aneurysm-like structures in Matrigel (Fig. 4B, III, mean vessel
area, 61.0 F 9.83%). Each small aneurysm consisted of RBC
(Fig. 4B, III, a) and few microhemorrhages were noted in this
group (Fig. 4B, III, b). Although some small canalized vessels
located at the edge of Matrigel were stained with the CD31
antibody, as indicated by arrows (Fig. 4B, III, c), the endothelial
cells surrounding small aneurysm did not display a CD31+
finding, suggesting that the vessels in this group might be
immature or dying after vascular formation. The mean vessel
area of VEGF plus AS CyD1 group was significantly lower than
that of VEGF plus Mock group (P < 0.01; Fig. 4C).
Role of cyclin D1 in VEGF regulation. Finally, we did
mechanistic studies to elucidate differential effects of AS
CyD1 on VEGF expression in DLD1 and HCT116 cells using
Fig.1. Effects of ASCyD1on cyclin D1expression and growth
of tumor cells. A, cyclin D1andVEGF expressionin colon and
gastric cancer celllines. Actin served as a loading control.
B, Ad-ASCyD1at 40 MOIdecreased cyclin D1at 48 and 72
hours in MKN45. C, in vitro cellproliferation assays. ASCyD1
significantly inhibited tumor cell growth when compared with
Mock control or nontreatment cultures of MKN45, HCT116,
and DLD1at MOI 40,10, and 40, respectively (*, P < 0.01).
Significance was also present betweennontreatment cultures
and Mock controlin each cellline (*, P < 0.01in MKN45 and
HCT116; **, P = 0.011in DLD1). D, effects of ASCyD1onVEGF
expression. Cells were harvested 48 hours after infection at the
indicated concentrations and subjected toWestern blot analysis
for cyclin D1andVEGF levels. A decrease inVEGF proteinlevel
was notedin DLD1but not in HCT116.
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siRNA. siRNA against cyclin D1 decreased expression of the
cyclin D1 protein as early as 24 hours, and decreased level was
maintained until 72 hours in both cell lines (Fig. 5A). There
was no change in VEGF expression in HCT116 at protein, RNA,
and promoter levels (Fig. 5A-C). In DLD1, on the other hand,
expression of the VEGF protein apparently decreased with the
cyclin D1 inhibition, and VEGF RNA decreased to some extent
(Fig. 5A and B). The VEGF promoter activity of cultures treated
with cyclin D1 siRNA but not control siRNA was significantly
decreased compared with nontreatment cultures (P < 0.01). We
then examined expression of the STAT3 transcription factor that
is known as an enhancer of the VEGF gene promoter (37, 38).
Introduction of wild-type STAT3 cDNA but not dominant-
negative construct clearly enhanced VEGF RNA expression in
both cell lines (Fig. 6A). The whole STAT3 levels did not change
by cyclin D1 inhibition in both cell lines, whereas expression of
the phosphorylated STAT3, an activated form of STAT3,
changed when the relative ratio to the actin band was calculated
by densitometry analyses (Fig. 6B). Thus, in HCT116 cell line,
treatment of siRNA against cyclin D1 resulted in a slight
decrease in phosphorylated STAT3 as early as 24 hours later,
which was largely unchanged at 48 hours, and then slightly
enhanced at the late time point of 72 hours, whereas an
apparent decrease was consistently seen in DLD1 cell line from
24 to 72 hours (Fig. 6B). When the STAT3 inhibitor was given
in the HCT116 control cultures without cyclin D1 inhibition,
VEGF expression did not change, whereas the VEGF level
decreased at the RNA level at 24 hours (Fig. 6C) and at the
protein level at 48 hours (Fig. 6D) in cultures treated with
cyclin D1 siRNA and the STAT3 inhibitor.
Discussion
In this study, we selected DLD1 and HCT116 among tumor
cells for investigation of tumor-associated neovascularization
because both these cells displayed a high infection efficiency
without toxicity by adenovirus and expressed both cyclin D1
Fig. 2. Treatment of established tumor
xenografts by intratumoralinjection
of Ad-ASCyD1. A, s.c. xenografts of
colorectal cancer (HCT116 and DLD1) were
establishedinnude mice (n = 4 for each
group) by injection of 5?106cells. After
day 7, when the tumor size reached
f100 to150 mm3, Ad-Mock, Ad-ASCyD1
(0.5?109plaque forming units/injection),
or saline were injectedinto tumors and two
more injections per tumor were applied on
days 9 and11.Tumor size on day 30 was
significantly smaller in ASCyD1group
compared with Mock control group or
saline treatment group in both cell types
(*, P < 0.01). B, CD31staining revealed that
there was a significant reductionin vessel
density in xenografts of both ASCyD1
group and Mock control group
(*, P < 0.01). Especially, significant decrease
in vessel density was notedin Ad-AS
CyD1^ injected DLD1xenografts when
compared with saline treatment
(*, P < 0.01). Magnification,?50.
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and putative angiogenetic factor VEGF. Because AS CyD1
prevents tumorigenicity in nude mice in various tumor cell
systems (14, 15), assessment of tumor-associated neovascula-
rization is impossible with tumorigenicity assay. Therefore,
in the present study, we employed a ‘‘therapeutic model’’ after
establishment of tumor xenograft, which permits detailed
survey of vessel formation in the late stage of tumor xenograft.
The therapeutic model is also useful with regard to assessment
of the value of Ad-AS CyD1 as a clinical tool. It was nice that
only three therapeutic administrations of Ad-AS CyD1 were
sufficient to inhibit the growth of xenografts of both HCT116
and DLD1. These results represent the direct antitumor effects
of AS CyD1 as suggested in tumor growth-inhibitory assay in
monolayer cultures. Immunohistochemical survey of the vessel
counts using CD31 antibody in xenografts showed that AS
CyD1 significantly inhibited tumor vessel formation in both
cell lines. It is notable that there was a marked decrease in vessel
formation in DLD1-xenografts, which displayed a significant
reduction in VEGF expression in in vitro assay.
Our in vitro studies showed that AS CyD1 decreased
expression of VEGF protein in DLD1 but not in HCT116.
Reverse transcription-PCR assay also showed that AS CyD1
decreased VEGF RNA in DLD1 but not in HCT116 (data not
shown). To verify these findings and to explore the underlying
mechanism, we used siRNA against cyclin D1. This system
allowed us to monitor both early and late events in a time-
dependent manner, and it was convenient in combination use
with the STAT3 inhibitor because the inhibitor became toxic
when used with adenovirus particles. Using siRNA against
cyclin D1, we confirmed that VEGF expression was down-
regulated at both protein and RNA levels in DLD1 but not in
HCT116. The reporter assays also showed reduction of VEGF
gene promoter in DLD1. To further investigate the regulation
mechanism of VEGF at promoter level, we focused on STAT3
because this transcription factor is considered an enhancer of
the VEGF gene promoter in pancreatic carcinomas and other
human cancers (37, 38). Transfection assays with STAT3 cDNA
indicated that STAT3 enhanced VEGF promoter activity and
increased the VEGF RNA level in HCT116 and DLD1 cell lines,
suggesting that this pathway is active in both cell lines as an
enhancer of the VEGF gene promoter. Of the two cell lines, we
found that cyclin D1 inhibition led to inactivation of STAT3
through dephosphorylation solely in DLD1. AS CyD1 adeno-
virus system also showed similar inactivation of STAT3 in
DLD1 (data not shown). Therefore, it seems that cyclin D1
inhibition regulates VEGF expression at the promoter level in
DLD1. However, because reduction of the VEGF promoter
activity was only modest in DLD1 (f20%) in contrast to
strong inhibition of VEGF expression at the protein level, we
cannot rule out the possibility that other mechanisms might be
also involved. In this context, the experiments in HCT116 using
STAT3 inhibitor provided an important implication. Thus, we
used siRNA against cyclin D1 with the STAT3 inhibitor and
found that VEGF level decreased in HCT116 as expected.
However, of interest was that the VEGF levels did not change in
HCT116 control cultures without cyclin D1 inhibition even
when the STAT3 inhibitor was applied, suggesting that
although STAT3 is an upstream enhancer in the VEGF promoter
the inhibition of STAT3 alone may be insufficient to block
VEGF expression. Conversely, cyclin D1 inhibition may in part
be involved in maintenance of the VEGF expression in
HCT116. We are still uncertain why cyclin D1 inhibition led
to the inactivation of STAT3 in DLD1 but not in HCT116.
However, as this close correlation between cyclin D1 and
STAT3 was also reported in head and neck squamous cell
Fig. 3. Westernblot analysis of cyclinexpressionin HUVEC. A, expressionof cyclin
D1, cyclin A, and cyclin E in growing cultures of HUVEC. HUVEC were starved for
24 hours, refed with10% fetal bovine serum in complete medium, and harvested
at the indicated time points. Actin expression served as a loading control. B, cyclin
D1expressioninduced by stimulation with recombinantVEGF protein. After 24
hours of serum starvation, HUVEC were grownin the complete medium with or
withoutVEGF (25 ng/mL). C, reduction of cyclin D1expressioninVEGF-treated
HUVEC byAd-ASCyD1. HUVEC wereinfectedwitheitherAd-MockorAd-ASCyD1
(20 MOI) in1hour, starvedfor12 hours,and thenrefedwith10% fetalbovine serum
in complete medium containingVEGF (25 ng/mL). D, BrdUrd proliferation assay.
Treatment of Mock control cultures withVEGF significantly increased BrdUrd
labeling index compared withuntreated Mock control cultures (*, P < 0.01). AS
CyD1significantly decreased BrdUrdlabeling inVEGF-treated cultures (*, P < 0.01).
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Fig. 4. Effects of ASCyD1on in vitro and in vivo angiogenesis.
A, in vitro angiogenesis assay.VEGF treatment enhanced tube
formation and capillary connection of HUVEC. ASCyD1 (20 MOI)
significantly inhibitedVEGF-mediated in vitro angiogenesis
compared with other treatments (*, P < 0.01for both). B, in vivo
angiogenesis assay. Mean vessel area relative to the Matrigel
plug area was calculated with reference to CD31-stained
vascular endothelial cells (I, a) and RBC (I, b) as guidance for
neovascularizationin Matrigelplug.WithtreatmentofVEGF, Matrigel
was expansively enlarged because of the endothelioma-like
structure that grew in the center of Matrigel and contained a giant
aneurysm. CD31staining showed that this structure was linedby
endothelium (arrows; II, a).There were also some RBC remaining
in Matrigel (II, b).VEGF plus ASCyD1resultedin production of
numerous microaneurysm-like structures in Matrigel (III). Each small
aneurysm consistedof RBC (III, a) and few microhemorrhages were
notedin this group (III, b). Although some small canalized vessels
located at the edge of Matrigel were stained with the CD31
antibody (arrows; III, c), the endothelial cells surrounding small
aneurysm did not display a CD31+finding, suggesting that the
vessels in this group might be immature or dying after vascular
formation. Magnifications,?6.25 (I-III),?50 (I, b; II, a; III, a, c),
and?100 (I, a; II, b; III, b). C, difference between Mock plusVEGF
groups and AS-CyD1plusVEGF group was significant (*, P < 0.01).
Cancer Therapy: Preclinical
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carcinoma (39), its occurrence is possibly dependent on the cell
context.
One may suppose why AS CyD1 inhibited tumor angiogen-
esis in HCT116 that maintained VEGF expression. To assess this
issue, we examined the direct effects of AS CyD1 on vessel
formation because several studies suggest that cyclin D1 may
play an essential role in growth of endothelial cells. It is
reported that antiangiogenic endostatin-induced G1 arrest
occurred through inhibition of cyclin D1 in endothelial cells
(40) and that up-regulation of cyclin D1 by cytochrome P450
was associated with proliferation of endothelial cells (41). We
found that VEGF up-regulated cyclin D1 as well as cyclin A and
cyclin E in HUVEC, being consistent with a recent report that
VEGF treatment of HUVEC caused progression of the cell cycle
with increases in cyclin D1, cyclin A, and p42/p44 mitogen-
activated protein kinase (42). As a result, AS CyD1 was enough
to decrease the VEGF-enhanced cyclin D1 expression and
significantly inhibited cell proliferation of HUVEC in mono-
layer culture. It took only a short time to suppress cyclin D1 at
protein level in HUVEC with treatment of AS CyD1 compared
with that in tumor cells (within 1 hour versus 2-3 days),
suggesting that effects of AS CyD1 may be more potent in
endothelial cells. To further explore the role of cyclin D1 in
neovascularization, we did in vitro and in vivo angiogenesis
assays. Even in cells treated with VEGF, AS CyD1 rendered
vessel tubes immature and resulted in a decrease in the number
of capillary connections in in vitro angiogenesis assay. In nude
mice, VEGF-induced Matrigel plug developed macroaneurysms,
which represent migration and extended growth of endothelial
cells, whereas AS CyD1 produced only microaneurysms even in
those treated with VEGF and inhibited macroaneurysm
formation. Of interest was that AS CyD1–treated Matrigel
showed several microhemorrhages, which could represent the
destruction of immature vessels. These findings suggest that
cyclin D1 plays a central role in neovascularization not only
in vitro but also in vivo. Thus, it is likely that AS CyD1 may
contribute to inhibition of tumor angiogenesis via direct
inhibitory effects of vascular endothelial cells.
The present results that AS CyD1 could inhibit VEGF-
mediated angiogenesis have important clinical implication in
therapy of human malignancies. It is known that VEGF is
involved in the development of liver metastasis from colorectal
cancer and is associated with poor prognosis of patients (43).
It is notable that a recent clinical study reported the survival
benefits of anti-VEGF reagent when used in combination with
conventional chemotherapy in metastatic colorectal cancer
(44). As mentioned in Introduction, AS CyD1 have thus far
been reported to have various direct effects against tumor cells
Fig. 5. Effects of siRNA against cyclin D1
onVEGF regulation at protein (A),
RNA (B), and promoter (C) levels. siRNA
(100 nmol/L) was introducedinto cells
to inhibit cyclin D1expression as described
in Materials and Methods. Cells were
harvested at 24 hours for semiquantitative
reverse transcription-PCRand the luciferase
reporter assays at 24, 48, and 72 hours for
Western blot analyses. siRNA against cyclin
D1decreased theVEGF protein expression
in DLD1 (A) andVEGFRNA to some extent
in DLD1 (B). C,VEGF promoter activity
of cultures treated with cyclin D1siRNA
but not control siRNAwas significantly
decreased compared withnontreatment
cultures (P < 0.01).There was no change in
VEGF expression and the promoter activity
in HCT116.
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(e.g., loss of tumorigenicity, reduced growth, induced apopto-
sis, and enhanced chemosensitivity). The present studies
revealed that AS CyD1 inhibits not only tumor cells but also
tumor-associated vessel formation. Thus, our data suggest that
AS CyD1 is a promising tool against tumor organ that consists
of tumor cells and its microenvironment.
Acknowledgments
We thank Dr. Bert Vogelstein for providing the AdEasy system, Prof. T. Yokota
for STAT3 expression vectors, Prof. Jun-ichi Miyazaki for cytomegalovirus
enhancer-chicken h-actin hybrid promoter, and Dr. H. Nakamura (Division of
Hepatobiliary and Pancreatic Medicine, Department of Internal Medicine, Hyogo
College of Medicine, Hyogo, Japan) for valuable advice.
Fig. 6. Role of STAT3 as an enhancer of
theVEGF gene promoter. A, introduction
of wild-type (WT) and dominant-negative
(DN) STAT3 cDNA. pcDNA3, wild-type,
or dominant-negative mutant expression
vector of STAT3 (1 Ag) was introducedinto
cells, and 48 hours later, cells were
harvested forVEGFRNA expression.
Wild-type STAT3 cDNA markedly induced
VEGFRNA expressionin both celllines.
B, effects of cyclin D1siRNA on STAT3
expression andinactivation through
dephosphorylation.The whole STAT3 levels
didnotchangebycyclinD1inhibitioninboth
celllines (top).The phosphorylated STAT3,
an activated form of STAT3, changed
(middle). Densitometry analysis indicated
relative ratio of phosphorylated STAT3 to
actin bandin each sample. Cyclin D1siRNA
consistently decreased phosphorylated
STAT3 expression. C, effects of STAT3
inhibitor onVEGFRNA expression at RNA.
The STAT3 inhibitor decreasedVEGFRNA
at 24 hours in HCT116 cultures treated
with cyclin D1siRNA. D, effects of STAT3
inhibitor onVEGF protein expression.The
STAT3 inhibitor decreasedVEGF protein
at 48 hours in HCT116 cultures treated
with cyclin D1siRNA. Actin served as
loading controls.Theintense nonspecific
cross-reactive band givenbyVEGFantibody
also indicated equivalent loading.
References
1. Sherr CJ. G1phase progression: cycling on cue. Cell
1994;79:551^5.
2. JiangW, Kahn SM, Zhou P, et al. Overexpression of
cyclin D1in rat fibroblasts causes abnormalities in
growth control, cell cycle progression and gene ex-
pression. Oncogene1993;8:3447^57.
3.WangTC, Cardiff RD, Zukerberg L, Lees E, Arnold A,
Schmidt EV. Mammary hyperplasia and carcinoma in
MMTV-cyclin D1transgenic mice. Nature 1994;369:
669^71.
4. Nakagawa H,WangTC, Zukerberg L, et al.Thetarget-
ingof the cyclin D1oncogenebyan Epstein-Barr virus
promoter in transgenic mice causes dysplasia in the
tongue, esophagus and forestomach. Oncogene
1997;14:1185^90.
5.Weinstein IB, Begemann M, Zhou P, et al. Disorders
in cell circuitry associated with multistage carcino-
genesis: exploitable targets for cancer prevention and
therapy. Clin Cancer Res1997;3:2696^702.
6.Weinstein IB, Zhou P, editors. Defects in cell cycle
control genes in human cancer. Encyclopedia of
cancer. Vol. 1. New York: Academic Press; 1997.
p. 256^67.
7.Weinstein IB. Disorders in cell circuitry during multi-
stage carcinogenesis: the role of homeostasis. Carci-
nogenesis 2000;21:857^64.
8. Hall M, Peters G. Genetic alterations of cyclins,
cyclin-dependent kinases, and Cdk inhibitors in
human cancer. Adv Cancer Res1996;68:67^108.
9. BahnassyAA, Zekri AR, El-Houssini S, et al. Cyclin A
and cyclin D1 as significant prognostic markers in
colorectal cancer patients. BMC Gastroenterol 2004;
4:22.
10.Toyoda H, NakamuraT, Shinoda M, et al. Cyclin D1
expression is useful as a prognostic indicator for ad-
vancedesophagealcarcinomas, but not forsuperficial
tumors. Dig Dis Sci 2000;45:864^9.
11. Gao P, Zhou GY, LiuY, LiJS, ZhenJH,YuanYP.Alter-
ation of cyclin D1in gastric carcinoma and its clinico-
pathologic significance.World J Gastroenterol 2004;
10:2936^9.
12. Gansauge S, Gansauge F, Ramadani M, et al. Over-
expressionofcyclinD1inhumanpancreaticcarcinoma
is associated with poor prognosis. Cancer Res 1997;
57:1634^7.
Cancer Therapy: Preclinical
www.aacrjournals.orgClin Cancer Res 2006;12(15) August1, 20064728
American Association for Cancer Research Copyright © 2006
on January 5, 2013clincancerres.aacrjournals.orgDownloaded from
DOI:10.1158/1078-0432.CCR-05-1213

Page 11

13. Nishida N, FukudaY, KomedaT, et al. Amplification
andoverexpressionof thecyclinD1geneinaggressive
human hepatocellular carcinoma. Cancer Res 1994;
54:3107^10.
14. Zhou P, Jiang W, ZhangYJ, et al. AS CyD1inhibits
growth and reverses the transformed phenotype of
human esophageal cancer cells. Oncogene 1995;11:
571^80.
15. Arber N, DokiY, Han EK, et al. AS CyD1inhibits the
growth and tumorigenicity of human colon cancer
cells. Cancer Res1997;57:1569^74.
16. Kornmann M, Arber N, Korc M. Inhibition of basal
and mitogen-stimulatedpancreatic cancercellgrowth
by cyclin D1 antisense is associated with loss of
tumorigenicity and potentiation of cytotoxicity to cis-
platinum. JClin Invest1998;101:344^52.
17. Kornmann M, Danenberg KD, Arber N, Beger HG,
Danenberg PV, Korc M. Inhibitionof cyclin D1 expres-
sion in human pancreatic cancer cells is associated
with increased chemosensitivity and decreased ex-
pression of multiple chemoresistance genes. Cancer
Res1999;59:3505^11.
18. NakashimaT, Clayman GL. Antisense inhibition of
cyclin D1in human head and neck squamous cell car-
cinoma. Arch Otolaryngol Head Neck Surg 2000;126:
957^61.
19. Sauter ER, Nesbit M, Litwin S, Klein-Szanto AJ,
Cheffetz S, Herlyn M. Antisense cyclin D1inducesap-
optosis and tumor shrinkage inhuman squamous car-
cinomas. Cancer Res1999;59:4876^81.
20. Chen B, Zhang XY, ZhangYJ,ZhouP, GuY, FanDM.
AS CyD1reverses the transformed phenotype of hu-
man gastric cancer cells.World J Gastroenterol 1999;
5:18^21.
21. SaikawaY, KubotaT, OtaniY, Kitajima M, Modlin IM.
Cyclin D1 antisense oligonucleotide inhibits cell
growth stimulated by epidermal growth factor and
induces apoptosis of gastric cancer cells. JpnJ Can-
cer Res 2001 ;92:1102^9.
22. Uto H, IdoA, Moriuchi A, et al.Transductionofanti-
sense cyclin D1using two-step gene transfer inhibits
the growth of rat hepatoma cells. Cancer Res 2001 ;
61:4779^83.
23. Gordon D. Cyclin D1regulates proliferation in pan-
creatic, colonic, andesophagealcancers.Gastroenter-
ology1998;114:629.
24. Folkman J. Tumor angiogenesis: therapeutic impli-
cations. NEnglJMed1971 ;285:1182^6.
25. Folkman J.What is the evidence that tumors are
angiogenesis dependent? J Natl Cancer Inst 1990;
82:4^6.
26. Ferrara N, Davis-SmythT. The biology of vascular
endothelial growth factor. Endocr Rev1997;18:4^25.
27. Turkson J, Ryan D, Kim JS, et al. Phosphotyrosyl
peptides block Stat3-mediated DNA binding activity,
gene regulation, and cell transformation. JBiol Chem
2001 ;276:45443^55.
28.YamamotoH,Kondo M,Nakamori S,etal.JTE-522,
a cyclooxygenase-2 inhibitor, is an effective chemo-
preventive agent against rat experimentalliverfibrosis.
Gastroenterology 2003;125:556^71.
29. HeTC, Zhou S, da Costa LT, Yu J, Kinzler KW,
Vogelstein B. A simplified system for generating re-
combinant adenoviruses. Proc Natl Acad Sci U S A
1998;95:2509^14.
30. Ogawa M,Yamamoto H, Nagano H, et al. Hepatic
expression of ANG2 RNA in metastatic colorectal
cancer. Hepatology 2004;39:528^39.
31.TokunagaT,OshikaY,AbeY,etal.Vascularendothelial
growth factor (VEGF) mRNA isoform expression
pattern is correlated with liver metastasis and poor
prognosis in colon cancer. Br J Cancer 1998;77:
998^1002.
32. Bussolati B, Dunk C, Grohman M, Kontos CD,
MasonJ, AhmedA.Vascularendothelialgrowthfactor
receptor-1modulates vascular endothelial growth
factor-mediated angiogenesis via nitric oxide. Am J
Pathol 2001 ;159:993^1008.
33. Kibbey MC, Grant DS, Kleinman HK. Role of the
SIKVAV site of laminin in promotion of angiogenesis
and tumor growth: an in vivo Matrigel model. J Natl
Cancer Inst1992;84:1633^8.
34. AkagiT, Usuda M, MatsudaT, et al. Identification of
Zfp-57 as a downstream molecule of STAT3 and Oct-
3/4 in embryonic stem cells. Biochem Biophys Res
Commun 2005;331:23^30.
35. Niwa H,Yamamura K, MiyazakiJ. Efficient selection
for high-expression transfectants with a novel eukary-
otic vector. Gene1991 ;108:193^9.
36. OkudaY, Nakamura H,Yoshida K, et al. Hepatoma-
derived growth factor induces tumorigenesis in vivo
through both direct angiogenic activity and induction
of vascular endothelial growth factor. Cancer Sci
2003;94:1034^41.
37. Niu G, Wright KL, Huang M, et al. Constitutive
STAT3 activity up-regulates VEGF expression and
tumor angiogenesis. Oncogene 2002;21:2000^8.
38.Wei D, Le X, Zheng L, et al. STAT3 activation regu-
lates the expression of vascular endothelial growth
factor and human pancreatic cancer angiogenesis
and metastasis. Oncogene 2003;22:319^29.
39. Masuda M, Suzui M,Yasumatu R, et al. Constitutive
activationof signal transducers and activators of tran-
scription 3 correlates with cyclin D1overexpression
and may provide a novel prognostic marker in head
and neck squamous cell carcinoma. Cancer Res
2002;62:3351^5.
40. Hanai J, Dhanabal M, Karumanchi SA, et al.
Endostatin causes G1arrest of endothelial cells
through inhibition of cyclin D1. J Biol Chem 2002;
277:16464^9.
41. Potente M, Michaelis UR, Fisslthaler B, Busse R,
Fleming I. Cytochrome P450 2C9-induced endothe-
lial cell proliferation involves induction of mitogen-
activated protein (MAP) kinase phosphatase-1,
inhibition of the c-Jun N-terminal kinase, and up-
regulationofcyclinD1.JBiolChem2002;277:15671^6.
42. Favot L, KeravisT, Lugnier C. Modulation ofVEGF-
induced endothelial cell cycle protein expression
through cyclic AMP hydrolysis by PDE2 and PDE4.
Thromb Haemost 2004;92:634^45.
43. Ishigami SI, Arii S, Furutani M, etal. Predictivevalue
of vascular endothelial growth factor (VEGF) in me-
tastasis and prognosis of human colorectal cancer. Br
JCancer1998;78:1379^84.
44. Hurwitz H, Fehrenbacher L, NovotnyW, et al. Bev-
acizumab plus irinotecan, fluorouracil, and leucovorin
for metastatic colorectal cancer. N Engl JMed 2004;
350:2335^42.
Antisense to Cyclin D1InhibitsTumorAngiogenesis
www.aacrjournals.orgClin Cancer Res 2006;12(15) August1, 2006

4729
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