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Li et al. Journal of Translational Medicine 2013, 11:257
http://www.translational-medicine.com/content/11/1/257

RESEARCH Open Access

Antitumor efficacy of a recombinant adenovirus
encoding endostatin combined with an
E1B55KD-deficient adenovirus in gastric
cancer cells
Li-xia Li1,2†, Yan-ling Zhang3,4†, Ling Zhou1, Miao-la Ke1, Jie-min Chen1, Xiang Fu1, Chun-ling Ye4, Jiang-xue Wu1,
Ran-yi Liu1* and Wenlin Huang1,5,6*

Abstract

Background: Gene therapy using a recombinant adenovirus (Ad) encoding secretory human endostatin (Ad-Endo)
has been demonstrated to be a promising antiangiogenesis and antitumor strategy of in animal models and clinical
trials. The E1B55KD-deficient Ad dl1520 was also found to replicate selectively in and destroy cancer cells. In this
study, we aimed to investigate the antitumor effects of antiangiogenic agent Ad-Endo combined with the oncolytic
Ad dl1520 on gastric cancer (GC) in vitro and in vivo and determine the mechanisms of these effects.

Methods: The Ad DNA copy number was determined by real-time PCR, and gene expression was assessed by
ELISA, Western blotting or immunohistochemistry. The anti-proliferation effect (cytotoxicity) of Ad was assessed
using the colorimetry-based MTT cell viability assay. The antitumor effects were evaluated in BALB/c nude mice
carrying SGC-7901 GC xenografts. The microvessel density and Ad replication in tumor tissue were evaluated by
checking the expression of CD34 and hexon proteins, respectively.

Results: dl1520 replicated selectively in GC cells harboring an abnormal p53 pathway, including p53 mutation and
the loss of p14ARF expression, but did not in normal epithelial cells. In cultured GC cells, dl1520 rescued Ad-Endo
replication, and dramatically promoted endostatin expression by Ad-Endo in a dose- and time-dependent manner.
In turn, the addition of Ad-Endo enhanced the inhibitory effect of dl1520 on the proliferation of GC cells. The
transgenic expression of Ad5 E1A and E1B19K simulated the rescue effect of dl1520 supporting Ad-Endo replication
in GC cells. In the nude mouse xenograft model, the combined treatment with dl1520 and Ad-Endo significantly
inhibited tumor angiogenesis and the growth of GC xenografts through the increased endostatin expression and
oncolytic effects.

Conclusions: Ad-Endo combined with dl1520 has more antitumor efficacy against GC than Ad-Endo or dl1520
alone. These findings indicate that the combination of Ad-mediated antiangiogenic gene therapy and oncolytic Ad
therapeutics could be one of promising comprehensive treatment strategies for GC.

Keywords: Endostatin, Adenovirus (Ad) vector, Oncolytic adenovirus (Ad), Viral-gene therapy, Gastric cancer

* Correspondence: [email protected]; [email protected]
†Equal contributors
1State Key Laboratory of Oncology in South China, Collaborative Innovation
Center for Cancer Medicine, Sun Yat-sen University Cancer Center,
Guangzhou 510060, China
5Guangdong Provincial Key Laboratory of Tumor-targeted Drugs,
Guangzhou Doublle Bioproducts Co., Ltd., Guangzhou 510663, China
Full list of author information is available at the end of the article

© 2013 Li 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.

Li et al. Journal of Translational Medicine 2013, 11:257 Page 2 of 13
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Introduction
Gastric cancer (GC) is one of the most common malig-
nancies and a leading cause of cancer-related mortality
worldwide, especially in Asian countries [1-3]. GC pa-
tients at early stage have no associated symptoms, and
most of patients are initially diagnosed in an advanced
stage, except those with GC at very early stage found
predominantly by active screening programs in Asian
countries [4]. Despite the recent development of new
chemotherapy regimens and the introduction of bio-
logical therapies, the 5-year survival for advanced GC is
still very low, and the median overall survival remains
less than 1 year [5]. Therefore, the development of novel
therapeutic approaches is crucial for improving the sur-
vival of GC patients.
The growth and metastasis of solid tumors are always

accompanied by and depend on neovascularization [6-8].
Therefore, antiangiogenic therapy is an attractive strat-
egy for the treatment of cancer [9-12]. Endostatin, a
20 kD C-terminal fragment of collagen XVIII composed
of 184 amino acids, was previously considered the most
potent angiogenesis inhibitor [13-16], and was rapidly
moved to clinical trials [17]. However, the high instabil-
ity and shorter serum half-life of the recombinant
endostatin protein made it inappropriate or inconvenient
for clinical application [18,19]. Daily administration is
needed even for Endostar, a more stable product modi-
fied with a tag at the N-terminus [19,20]. Moreover, the
long-term systemic delivery of a recombinant protein is
an expensive, painful experience for patients and is cum-
bersome for medical staff. Antiangiogenic gene therapy
can overcome these problems and represents a promis-
ing new approach for the treatment of cancer.
An adenoviral (Ad) vector encoding a secretory form

of human endostatin (Ad-Endo, also referred to as
E10A) has been demonstrated to inhibit tumor growth
through antiangiogenic effects [21-24]. The results of
preclinical trials showed that no notable toxicities were
found in the experimental dogs after intramuscular in-
jections of Ad-Endo at the doses equivalent to 30 and
7.5 times of the human curative dose once daily, 6 days/
week, for 3 months [25]. In phase I clinical trials, the re-
sults showed that the treatment of solid tumor with Ad-
Endo is likely a safe and promising approach [26,27].
The phase II clinical trial (ClinicalTrials.gov identifier,
NCT00634595) has demonstrated that the addition of
Ad-Endo improved the outcome of chemotherapy for
the treatment of advanced nasopharyngeal carcinoma and
head and neck cancer (Huang W, et al. unpublished data).
However, Ad-Endo does not present a satisfactory thera-
peutic effect due to the limited expression of the endostatin
protein, especially for tumors with large masses. Determin-
ing how to increase endostatin expression is a very import-
ant goal for further clinical trials.

Oncolytic Ad has been demonstrated to replicate select-
ively in cancer cells but not in normal cells [28-30]. We
presumed that the selective replication of an oncolytic Ad
could rescue the amplification of Ad-Endo genomic DNA
and promote the expression of endostatin. In this study,
we investigated the antitumor effects of the combined
treatment of Ad-Endo and the oncolytic Ad dl1520 [31]
on GC in vitro and in vivo. The results indicate that
dl1520 enhanced the antiangiogenic effect of Ad-Endo by
rescuing the replication of Ad-Endo, thereby dramatically
increasing endostatin expression, when Ad-Endo in turn
enhanced the oncolytic effect of dl1520 by reinforcing
viral replication in GC cells.

Materials and methods
Cells, plasmids and transient transfection
The human GC cell line AGS (ATCC No. CRL-1739)
[32], human embryonic kidney cell line 293 (ATCC No.
CRL-1573) and human normal epithelial cell FHC (ATCC
No. CRL-1831) [33] were obtained from the American
Type Culture Collection (ATCC, Rockville, MD, USA).
The human GC cell lines MGc80-3 [34] and SGC-
7901 [35] were obtained from the Chinese Type Culture
Collection. FHC cells were cultured in DMEM:F12 medium
supplemented with 10% fetal bovine serum, extra 10 mM
HEPES, 10 ng/ml cholera toxin, 0.005 mg/ml insulin, 0.005
mg/ml transferrin and 100 ng/ml hydrocortisone (Gibco,
Paisley, UK). The other cells were all cultured in Dulbecco’s
modified Eagle medium (DMEM) supplemented with 10%
fetal bovine serum (Gibco, Paisley, UK) at 37°C with 5%
CO2 and saturated humidity.
The plasmids expressing Ad2 E1A (pCD-E1A),

E1B19k (pCD-E1B19k) or E1A+E1B19k (pCD-E1AB19k)
were constructed by inserting the relevant gene frag-
ments into the HindIII/EcoRI site of pcDNA3.1(+) vec-
tor (Invitrogen Corporation, Carlsbad, CA, USA). These
gene fragments were amplified with the corresponding
primers (Table 1) and dl1520 genomic DNA as the tem-
plate. pCD-p14ARF was constructed by subcloning full
length of p14ARF cDNA fragment (clone IMAGE: 6173590)
into pcDNA3.1(+) vector. p14ARF siRNA (sc-37622) was
purchased from Santa Cruz Biotechnology, Inc (Santa Cruz,
CA, USA). Plasmid or siRNA transfection was performed
using the Effectene transfection reagent (Qiagen, Hilden,
Germany) according to the manufacturer’s instructions.

Recombinant adenoviruses (Ad) and infection
A replication-defective recombinant Ad vector encoding
the secretory form of human endostatin (Ad-Endo) was
generated in our lab as described previously [23,25]. The
E1B55kD-deficient Ad (dl1520), also named Onyx-015
[31,36,37] was kind gift from Professor Arnold J. Berk
(University of California-Los Angeles). The two viruses
were both propagated in 293 cells, and the viral titers

Table 1 The sequence of primers used in this study

Targets Directions Sequences Notes

Real-time PCR Primers:

Ad-Endo (290 bp) Forward 5′-TGACTGCCTCCAAGTAGGCTAGA-3′ Within the Endostatin fragment

Reverse 5′-CCCAGATCCGCGTTAAGA-3′ At the junction of the poly_A signal and the Ad backbone

dl1520 (264 bp) Forward 5′-TGTTTCCAGAACTGAGACGCAT-3′ E1B region (Ad2/2261-2281 nt)

Reverse 5′ –TCTCATCGTACCTCAGCACCTT-3′ E1B region (Ad2 3330–3351 nt)

Total Ad (237 bp) Forward 5′-TCGAAGCCGTTGATGTTGTG-3′ E2B (Ad2/7519-7538 nt or Ad5/7529-7548 nt)

Reverse 5′- GGCCATAGGTCGCCAGTTTA-3′ E2B (Ad2/7519-7538 nt or Ad5/7529-7548 nt)

β-Actin (266 bp) Forward 5′-CCTTTCCTTCCCAGGGCGTGAT-3′ At the intron 2-exon 3 junction

Reverse 5′-CGGGCCACTCACCTGGGTCAT-3′ Within the exon 3

p53 (298 bp) Forward 5′-GTGGTGGTGCCCTATGAG-3′

Reverse 5′-AGGAGCTGGTGTTGTTGG-3′

p14ARF (282 bp) Forward 5′-CGCGAGTGAGGGTTTTCGT-3′

Reverse 5′-CAGCACCACCAGCGTGTCC-3′

GAPDH (258 bp) Forward 5′-AGAAGGCTGGGGCTCATTTG -3′

Reverse 5′-AGGGGCCATCCACAGTCTTC-3′

Cloning primers (for cloning into HindIII/EcoRI site of pcDNA3.1(+)):

Ad E1A Forward 5′-cccaagcttCGGGACTGAAAATGAGAC-3′ E1A gene (Ad2 548 nt – 1554 nt) (942 bp)

Reverse 5′- ccggaattcCAGGTTTACACCTTATGGC-3′

Ad E1B19k Forward 5′-cccaagcttATCTTGGTTACATCTGACCTC-3′ E1B19 kDa (Ad2 1690 nt – 2255 nt) (566 bp)

Reverse 5′-ccggaattcAGCCACCTGTACAACATTC-3′

Ad E1A + E1B19k Forward 5′-cccaagcttCGGGACTGAAAATGAGAC-3′ E1A+E1B19 kDa (Ad2 548 nt – 2255 nt) (1708 bp)

Reverse 5′-ccggaattcAGCCACCTGTACAACATTC-3′

Li et al. Journal of Translational Medicine 2013, 11:257 Page 3 of 13
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were determined using the hexon immunoassay with the
BD Clontech™ Adeno-X Rapid Titer Kit (San Jose, CA,
USA). For infection, gastric cells seeded 24 hours earlier
were infected with Ad-Endo, dl1520 or Ad-Endo com-
bined with dl1520 in serum-free DMEM for 2 hours,
and then the infection medium was replaced with nor-
mal medium. The indicated time points post-infection
correspond to the time after the medium change.

Quantitative real-time PCR
For the measurement of the Ad DNA copy numbers,
Ad-infected cells, including detached cells, were collected
by scraping and centrifugation and then washed twice
with PBS. The DNA was isolated using the Genomic
DNA Mini Preparation Kit (Axygen, Hangzhou, China).
The viral DNA copy numbers were measured by real-time
PCR using the Platinum SYBR Green qPCR SuperMix-
UDG (Invitrogen, Carlsbad, CA, USA). The primers for
Ad-Endo, dl1520 or total Ad (β-actin was used as an in-
ternal control) are listed in Table 1. Real-time PCR was
performed as follows: 50°C for 2 minutes, 95°C for 2
minutes and 40 cycles of 95°C for 15 seconds and 62°C
for 1 minute. The viral DNA copy numbers are presented
as relative values normalized to that of the internal con-
trol, 2−ΔCt. The change of DNA copy number is shown as

the fold change relative to the DNA copy number at 0
hours post-infection.
For the quantitative detection of the mRNA levels of

p53 and p14ARF, cells in the logarithmic growth phase
were collected, and total RNA was isolated using Trizol
Reagent (Invitrogen, Carlsbad, CA, USA). The RNA was
then reverse transcribed into cDNA using GoScript™
Reverse Transcription System (Promega, Madison, WI,
USA). Real-time PCR was performed as described above
with special primer pairs (Table 1) (GAPDH was used as
the internal control).

Western blot analysis
Western blot analysis was performed as described previ-
ously [38]. Briefly, cell pellets were lysed with TNN-SDS
buffer [38] at 4°C for 30 minutes followed by centrifuga-
tion (10,000 g for 10 minutes at 4°C) to remove the in-
soluble materials. The protein concentrations of the
supernatants were measured using a Protein Assay kit.
The proteins were then separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis, transferred to
PVDF membranes, and probed with specific primary anti-
bodies (p14ARF, Ad2 E1A and human actin antibodies
from Santa Cruz Biotech., CA, USA; Ad2 E1B19K anti-
body from Calbiochem, Merck, Germany). After exposed

Li et al. Journal of Translational Medicine 2013, 11:257 Page 4 of 13
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to the primary antibodies, the membranes were reacted
with relevant HRP-conjugated secondary antibodies, and
the signals were detected with ECL reagents (Amersham
Biosciences, Piscataway, NJ, USA) and x-ray film.

Analysis of endostatin expression by ELISA
The culture supernatants of cells infected with Ad were
collected at different time points and frozen at −80°C. The
endostatin concentration was detected using a human
endostatin ELISA kit (Shanghai ExCell Biology, Inc.,
Shanghai, China) according to the manufacturer’s instruc-
tions. The kit’s minimum detectable level is 30 pg/mL.

In vitro Cytotoxicity assay
The cytotoxicity of Ad to GC cells was assessed by the
MTT cell proliferation assay as previously described
[38,39]. Briefly, cells were seeded in 96-well plates at a
density of 3000 cells/well for 24 hours and then infected
with Ad as described above, followed by incubation for
72 h. Viable cells were stained with MTT (Sigma-Aldrich,
Shanghai, China) for 4 hours. The formazan crystals were
dissolved with DMSO, and the optical density at 570 nm
(OD570nm) was then measured using 630 nm as the refer-
ence wavelength.

Animal models and in vivo antitumor activity
BALB/c-nu/nu mice (5-6 weeks old, 18-20 g) were
obtained from the Experimental Animals Center, Sun
Yat-sen University (Guangzhou, China). The mice were
housed and fed under specific pathogen-free conditions
according to protocols approved by the Sun Yat-sen
University Institutional Animal Care and Use Committee.
All experiments were performed in accordance with the
Guidelines for the Welfare of Animals in Experimental
Neoplasia. Pieces (approximately 1.5 mm in diameter) of
SGC-7901 tumors, which were maintained by subcutane-
ous transplantation in nude mice, were subcutaneously
transplanted into the flanks of mice to construct the xeno-
graft model.
To assess the dynamic expression of endostatin in vivo,

mice were injected intratumorally with Ad-Endo (5×108

pfu/dose) or Ad-Endo plus dl1520 (5×108 pfu/dose for
each virus) when the xenografts reached an approximate
diameter of 7 mm; 100 μL of PBS were used as the nega-
tive control. Blood plasma was sampled before and 1, 2, 3,
4, 6, 8, 13, and 21 days after virus administration (3 mice/
group at each time point), and the endostatin concentra-
tion was determined by ELISA.
To assess the antitumor effects of Ad-Endo in combin-

ation with dl1520 in vivo, mice were randomly assigned
to four groups (6–8 mice/group, half male and half fe-
male) when the xenografts reached 3–5 mm in diameter.
The mice were treated by the intratumoral injection of
100 μL of PBS (control group), 5×108 pfu of Ad-Endo

(Ad-Endo group), 5×108 pfu of dl1520 (dl1520 group),
or 5×108 pfu of Ad-Endo plus 5×108 pfu of dl1520 (Ad-
Endo+dl1520 group) (in 100 μL of PBS) per dose every 4
days for 4 consecutive weeks. Body weight and tumor
size were measured every 4 days, and the tumor volumes
were calculated according to the formula V = 0.52 × L ×
W2 (L, length; W, width) [14,23,39]. The tumor xeno-
grafts were weighed at the end point of the experiments.

Immunohistochemical analysis
Tumor tissue was fixed in buffered formalin and embed-
ded in paraffin. Sections (5 μM thick) were mounted on
poly-L-lysine-treated slides, and immunohistochemical
assays were performed to detect endostatin, CD34 and
Ad hexon protein expression. Sections were probed with
the following primary antibodies: mouse anti-Ad hexon
McAb (MAB805, Chemicon/Millipore, Temecula, CA,
USA), mouse anti-endostatin McAb (sc-32720, Santa
Cruz, CA, USA), and rabbit anti-CD34 PcAb (BA3414,
Boster, Wuhan, China). The protein expression was visual-
ized with DAB using an EnVision™ detection kit (peroxid-
ase/DAB, rabbit/mouse) (Gene Tech, Shanghai, China).
The slides were counterstained with hematoxylin.

Statistical analysis
All in vitro experiments were repeated at least three
times, and the animal experiments were repeated at least
two times. The data were analyzed with one-way ANOVA,
two-way ANOVA or Student’s t test. P < 0.05 was consid-
ered statistically significant. The combined effect of the vi-
ruses was assessed with the Q value using Zheng-Jun Jin’s
method [40]: Q=EAB/[EA+EB(1-EA)] (EA, EB and EAB indi-
cate the effects of A, B and the combination of the two
viruses). According to the Q value, the effect of the
combination of two viruses can be classified as antag-
onistic (Q<0.85), additive (0.85<Q<1.15), or synergistic
(Q>1.15).

Results
p53 pathway and oncolytic effects of dl1520 on GC cells
dl1520 has been reported to replicate in and lyse p53-
mutant (mt-p53) cancer cells. To examine the oncolytic
effects of dl1520 on GC cells and to determine if these
effects depend on the p53 gene status, we first assessed
the p53 gene status by sequencing the RT-PCR products
of p53. The results showed that the MGc80-3 and SGC-
7901 cell lines harbored a heterozygous mutant p53
(mt-p53) gene (codon 72 Pro → Arg), whereas the AGS
and FHC cell line harbored a wild-type p53 (wt-p53) (data
not shown).
To examine the replicative capacity of dl1520 in GC

cells, the copy number of dl1520 DNA in GC cells was
detected after dl1520 infection (FHC used as a normal
cell control). The results showed that the copy numbers

Li et al. Journal of Translational Medicine 2013, 11:257 Page 5 of 13
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of dl1520 DNA at 0 hours after infection, indicating the
infection efficiency of dl1520, are different, but not sig-
nificantly, in the three GC cell lines and FHC cells (p>
0.05) (Figure 1A). The dl1520 DNA copy numbers in-
creased greatly in AGS, MGc80-3 and SGC-7901 cells after
infection compared to those at 0 hours post-infection,
whereas dl1520 DNA copy number decreased slightly in
FHC cells (Figure 1B). And the increased folds of dl1520
DNA in GC cells are not correlative to the infection
efficiencies (data not shown). The titers of dl1520 after
48 hours post-infection were (4.11 ± 0.83) × 107, (2.38 ±
0.41) × 107, (1.27 ± 0.32) × 108 and (9.17 ± 1.26) × 104

pfu/mL in AGS, Mgc80-3, SGC-7901 and FHC cells re-
spectively. These data suggested that dl1520 selectively
replicated in GC cells but not in normal cells, and the rep-
lication of dl1520 was regardless of the p53 status in GC
cells and infection efficiency.
To assess the cytopathic effect (CPE) due to dl1520 rep-

lication, an MTT cell proliferation assay was performed.
The data indicated that dl1520 effectively lysed GC cells
and inhibited their growth in a dose-dependent and p53-
independent manner (Figure 1C, 1D and 1E). The findings
are similar to those presented in Lee B et al’s report [41].
The replication of dl1520 and CPE resulted from dl1520
were stronger in AGS (wt-p53) and SGC-7901 (mt-p53)
cells than those in MGc80-3 (mt-p53) cells, which is not
consistent with the previous assumption.
For this reason, we assessed the expression of p14ARF,

an important molecule in p53 pathway. The results
showed that there was a high-level expression of p14ARF

at both the mRNA and protein levels in MGc80-3 cells
but only a low level of expression in SGC-7901 cells;
and p14ARF expression was not detected in AGS cells
(Figure 1F, 1G). These data indicated that the expression
levels of p14ARF were likely related to the selective repli-
cation of dl1520. To further verify the relation of p14ARF

with the replication of dl1520, we analyzed the replica-
tion of dl1520 after modifying the expression level of
p14ARF by knockdown or overexpression. The results
showed that the knockdown of p14ARF increased the
dl1520 replication in MGc80-3 cells (p<0.05) whereas
the overexpression of p14ARF decreased the dl1520 repli-
cation in AGS cells (p<0.05) (Figure 1H). These findings
suggested that the down-regulation or loss of p14ARF ex-
pression played an important role in the oncolytic effects
of dl1520 in GC cells. So, we concluded dl1520 repli-
cated selectively in GC cells harboring an abnormal p53
pathway, including p53 mutation and the loss of p14ARF

expression.

dl1520 rescues the replication of Ad-Endo in GC cells
Ad-Endo is a replication-deficient recombinant Ad due
to the deletion of the whole E1 region. We presumed
that an oncolytic Ad could rescue the replication of Ad-

Endo by providing some of the early proteins necessary
for Ad replication. To test this hypothesis, the amount
of Ad-Endo DNA in GC cells was a quantitatively deter-
mined by real-time PCR. The results showed that the
Ad-Endo DNA copy number in GC cells infected with
Ad-Endo plus dl1520 was greatly increased by more
than 100-fold after infection, whereas the copy number
decreased gradually in cells infected with Ad-Endo alone
(Figure 2A). The rescue effects were enhanced with the
higher dl1520 doses in a certain range (Figure 2B).
These results indicated that the replication of Ad-Endo
was likely rescued by dl1520 in GC cells.

The rescue of Ad-Endo replication by dl1520 depends on
the E1A and E1B19K proteins
To explore the mechanism involved in the rescue of
dl1520 on Ad-Endo replication, the products of dl1520 E1
region genes (E1A, E1B19K) were ectopically expressed
alone or together into MGc80-3 cells to investigate their
effects on Ad-Endo replication. The results showed that
the expression of the E1A (13S and 12S) and E1B19K
together dramatically promoted Ad-Endo replication
(p<0.01). The expression of E1A alone but not E1B19K
also benefited Ad-Endo replication, but not significantly
(Figure 2C). These data suggested that dl1520 rescued
Ad-Endo replication by providing E1A and E1B19K pro-
teins in GC cells.

dl1520 promoted endostatin expression by Ad-Endo in
GC cells
Since dl1520 can rescue the replication of Ad-Endo, we
next asked whether dl1520 can enhance the antiangiogenic
effects of Ad-Endo by promoting endostatin expression.
Hence, we assessed the effect of dl1520 on endostatin ex-
pression by Ad-Endo in GC cells. The results showed that
the endostatin concentrations in the supernatants from
gastric cells infected with Ad-Endo plus dl1520 were much
higher than those from cells infected with Ad-Endo alone
(p<0.05 or p<0.01) (Figure 3A). In addition, the endostatin
amount increased along with the increases in the dl1520
doses when cells were infected with Ad-Endo at a constant
dose (10 MOIs) (Figure 3B). These results indicated that
dl1520 promoted the expression of endostatin by rescuing
Ad-Endo replication in GC cells.

Ad-Endo enhanced the cytotoxic effects of dl1520 in GC
cells
The above experiments demonstrated that dl1520 likely
enhances the antiangiogenic effect of Ad-Endo by pro-
moting endostatin expression. We then, in turn, assessed
the effects of Ad-Endo on the cytotoxicity of oncolytic
Ad. As shown in Figure 4A, 10 MOIs of Ad-Endo had
almost no cytotoxicity, but this dose of Ad-Endo signifi-
cantly enhanced the inhibitory effects of dl1520 on AGS,

Figure 1 dl1520 inhibited the proliferation of GC cells by selectively replicating in and destroying the cancer cells. A, B) The efficiencies
of infection and replication of dl1520 in GC and normal cells. The infection efficiency (A) of dl1520 was shown as the dl1520 DNA copy number
relative to β-actin at 0 hours after infection. The replication efficiency (B) of dl1520 was presented as the fold of the dl1520 DNA copy number at
indicated time relative to that at 0 hours post-infection (One-way ANOVA, *p<0.05, **p<0.01 compared to that at 0 hours post-infection). C~E)
The cytopathic effect (CPE) of dl1520 on GC cells. MTT cell proliferation assays were used to analyze the CPE of dl1520 on AGS (C), MGc80-3
(D) and SGC-7901 (E) GC cells. The results are presented as the percentages of viable cells related to the negative control (one-way ANOVA,
*p<0.05, **p<0.01 compared to that at 0 MOIs). F) Western blotting analysis of protein levels of p14ARF in GC cells (Actin was used as the internal
control). G) Quantitative RT-PCR analysis of the relative mRNA levels of p14ARF and p53 (normalized to that of GAPDH). H) The replication of
dl1520 after modifying the p14ARF levels by knockdown or overexpression. AGS cells were transfected with pCD-p14ARF plasmid (pcDNA3.1(+)
as a negative control), and MGc80-3 cells were transfected with p14ARF siRNA (si-p14ARF) (scrambled siRNA as a negative control). The cells were
analyzed p14ARF expression by Western blotting after 48 hours post-transfection (H upper). Or the cells were infected with 10 MOIs of dl1520
after 24 hours post-transfection, and dl1520 DNA copy numbers were analyzed in GC cells after 48 hours post-infection (normalized against that
at 0 hours) (H lower). (Student’s t test, *p< 0.05 compared with their respective control).

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Figure 2 dl1520 rescued the replication of Ad-Endo in GC cells
by supplying the E1A and E1B19k gene products. The
replication of Ad-Endo is presented as the increase in the Ad-Endo
DNA copy number, which was determined by real-time PCR. The
results are shown here as the fold change in the Ad-Endo DNA copy
number at the indicated time points relative to that at 0 hours post-
infection. A) Ad-Endo DNA copies in GC cells at different time points
after infection with 10 MOIs of Ad-Endo alone or in combination
with 10 MOIs of dl1520 (two-way ANOVA, *p<0.05, **p<0.01
compared to that at 0 hours post-infection). B) The Ad-Endo DNA
copy number in GC cells at 48 hours after infection with 10 MOIs of
Ad-Endo alone or in combination with increasing MOIs of dl1520
(one-way ANOVA, *p<0.05, **p<0.01 compared to that of dl1520 at
0 MOIs). C) The Ad-Endo DNA copy number in MGc80-3 cells
transiently transfected with the E1A or/and E1B19k genes 48 hours
after infection with 10 MOIs of Ad-Endo (one-way ANOVA, **p<0.01
compared to the control pCD-EGFP). The ectopic expression of the
E1A and E1B19k genes in MGc80-3 cells was analyzed by Western
blotting (actin was used as the internal control) (C, Left).

Figure 3 dl1520 promoted endostatin expression by Ad-Endo
in GC cells. GC cells were infected with Ad-Endo alone or
combination with dl1520, and then the culture supernatants were
collected at different time points. The endostatin concentrations
were measured using a human endostatin ELISA kit (Shanghai ExCell
Biology, Inc., Shanghai, China). The minimum detectable dose using
this kit is 30 pg/mL. A) Endostatin concentrations in the culture
supernatants of GC cells at different time points after infection with
10 MOIs of Ad-Endo alone or in combination with 10 MOIs of
dl1520 (two-way ANOVA, *p<0.05, **p<0.01 compared to Ad-Endo
alone). B) Endostatin concentrations in the culture supernatants at
48 hours after infection with 10 MOIs of Ad-Endo alone or in
combination with increasing MOIs of dl1520 (one-way ANOVA,
*p<0.05, **p<0.01 compared to that of dl1520 at 0 MOIs).

Li et al. Journal of Translational Medicine 2013, 11:257 Page 7 of 13
http://www.translational-medicine.com/content/11/1/257

MGc80-3 and SGC-7901 GC cells (p<0.05). Moreover,
the inhibitory effects of dl1520 increased along with in-
creases in the Ad-Endo dose when the dl1520 dose was
kept constant (10 MOIs), even though the same doses

of Ad-Endo alone had little cytotoxicity (Figure 4B, 4C
and 4D). Further investigations showed that the addition
of Ad-Endo resulted in the increased replication of the
total Ad, both dl1520 and Ad-Endo, especially in AGS
and SGC-7901 cells (p<0.05 or p<0.01) (Figure 4E).

In vivo antitumor effects of Ad-Endo combined with
dl1520 on GC xenografts
To investigate the antitumor effects of combination
treatment with Ad-Endo and dl1520, we first examined
the endostatin concentration in the blood plasma to as-
sess …

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