Journal of Cancer Therapy, 2012, 3, 397-405 Published Online September 2012 (
Baculovirus Mediated Experimental Research on Targeted
Egr1-Kringle 5 Gene Radiotherapy in Lung
Haoping Xu, Rui Guo, Biao Li*
Department of Nuclear Medicine, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Email: *
Received April 15th, 2012; revised May 27th, 2012; accepted June 15th, 2012
Objective: To investigate the feasibility of temporally and spatially restricted Kringle5 expression induced by radiation,
as well as the dual effect of radiotherapy and antiangiogenic therapy in lung adenocarcinoma in vitro. Methods: We
first constructed recombinant baculovirus vectors containing Egr1 promoter and human plasminogen Kringle5 gene
(rhK5), then transfected them into lung adenocarcinoma cells (A549). Transfect efficiency of the baculovirus for gene
transfer in A549 cells and the activity of Egr1 promoter induced by X-radiation were detected by fluorescence micros-
copy. The rhK5 mRNA transcription and rhK5 protein expression were detected by Real-time PCR and Western blot
assay, respectively. The apoptosis asssay of human umbilical veins endothelial cells (HUVEC) was analyzed by flow
cytometry. Results: The recombinant baculovirus were successfully transfected into A549 and HUVEC cells. As for the
temporal regulation, the rhK5 mRNA transcription and rhK5 protein expression were elevated with the irradiation time
significantly. And the HUVEC apoptotic percentage increased in relation to the irradiation time as well. As for the spa-
tial regulation, rhK5 mRNA transcription level of A549 cell lines transfected with recombinant baculovirus Egr1-K5
was significantly higher than that of control groups after the same dose of X-radiation. When we analyzed the dose and
frequency of X-radiation, no difference was observed among each dose after continuously three-times of irradiation.
Conclusion: Baculovirus-mediated Egr1-K5 can be used in gene radiotherapy for its temporary and spatial controllable
rhK5 expression by X-radiation and the consequent HUVEC apoptosis in vitro study. And low dose and more times of
irradiation might be more effective. It would provide a promising way for the tumor treatment.
Keywords: Radiotherapy; Lung Adenocarcinoma; Egr1 Promoter; Kringle 5; Baculovirus
1. Introduction
Radiotherapy is widely used in the treatment of various
types of cancer. However, its application is limited by the
side-effects such as the radiation-induced damages of the
normal tissues nearby and the radiation-resistance of
certain tumors. As for the gene therapy, the results are
less encouraging due to the inability to optimally localize
the activity of therapeutic agents to tumor cells and limit
damage to normal tissue. Also, there is a lack of effective
therapeutic gene.
Angiogenesis plays a key role in tumor progression. It
was hypothesized that inhibition of angiogenesis would
be an effective strategy to treat human cancer, and an
active search for angiogenesis inducers and inhibitors
began in 1971 [1]. Antiangiogenic therapy could destroy
tumor vasculature and inhibit tumor growth. And it’s the-
rapeutic benefit may even be greater when used in com-
bination with established treatment modalities, such as
surgery, chemotherapy, and radiation therapy [2]. Plas-
minogen contains 5 kringles, and kringle 1 - 4 consists of
angiostatin. The kringle 5 (K5) domain of human plas-
minogen with low molecular weight of 14kDa and low
immunogenicity is a specific inhibitor for endothelial cell
proliferation and displays powerful antiangiogenic acti-
vity [3]. Study also showed that kringle 5 could directly
lead to apoptosis of anoxic tumor cells in vitro [4]. These
features make K5 an appealing antitumor biopharmaceu-
tical with combined antitumoral and antiangiogenic pro-
perties. Although angiogenesis inhibition offers several
advantages, it is expected to induce a cytostatic effect
resulting in tumor stabilization not eradication. Further-
more, single-agent antiangiogenic therapy may lead to a
compensatory increase in the production of other angio-
genic factors, which may then sustain angiogenesis [5].
In a recent study, the therapeutic effect of radiotherapy
combined with K5 is investigated in Lewis lung carci-
*Corresponding author.
Copyright © 2012 SciRes. JCT
Baculovirus Mediated Experimental Research on Targeted Egr1-Kringle 5 Gene Radiotherapy
in Lung Adenocarcinoma
noma (LLC) tumor model, which indicated that there is
significant additive effect when radiotherapy was com-
bined with K5. The mechanism of the effect might be
related to the increased sensitivity of both tumor cells
and vascular endothelial cells to ionizing radiation in-
duced by K5 [6]. However, without specificity, this com-
bined treatment made no effort to avoid the damage to
surrounding normal tissue.
In our research, we designed to construct K5 gene
downstream the Egr1 (Early growth response gene-1)
promoter in order to target gene expression with radio-
therapy spatially and temporally to decrease the damage
of normal tissue nearby eventually. Previous studies have
shown that the six 5’CarG[CC(A + T rich)GG] elements
mediate transcriptional induction of the Egr1 gene pro-
moter following ionizing radiation [7,8]. It is an effective
approach that uses the specificity of the Egr1 promoter to
construct the radiation-inducible gene expression system
with destination gene [9,10].
The Recombinant baculoviruses were designed as vec-
tors for specific gene transfer in our research. The vector
for gene transfer is a major challenge in medical research.
And the research has showed that viral vectors are the
most efficient tools for genetic modification of the ma-
jority of somatic cells in vitro and in vivo [11]. Recom-
binant baculoviruses with a mammalian expression pro-
moter have recently been viewed as a new generation of
gene therapy vehicles holding a great promise [12,13].
The baculovirus genome is large and thus large trans-
genes can be accommodated. In addition, they are easy to
scale up and obtain high levels of recombinant gene ex-
pression [14].
In the present study, we developed a recombinant ba-
culovirus vector encoding Egr1 promoter and its down-
stream gene K5 to investigate the feasibility of tempo-
rally and spatially restricted K5 expression induced by
radiation, as well as the dual effect of radiotherapy and
antiangiogenic therapy in lung adenocarcinoma models
(Figure 1). The main idea of our research is as follows.
After Egr1 promoter activated by the radiation, its down-
stream gene, K5 will start its transcription and protein
expression. Thus, suppression of tumor vessels by K5
and the direct killing of tumor cells by radiation will
work simultaneously, providing a promising way for
tumor treatment.
2. Materials and Methods
2.1. Plasmid Construction
The plasmid pGL3-Egr1 promoter was kindly provided
by Professor Gerald Thiel (Department of Medical Bio-
chemistry and Molecular Biology, Germany). The plas-
mid pET22b-K5 (His-tagged) was constructed previously
Figure 1. The main idea of our research (After Egr-1 pro-
moter activated by the radiation, its downstream gene,
Kringle5 (K5), will start its transcription and protein ex-
pression. Suppression of tumor vessels by K5 and the direct
killing of tumor cells by radiation will work simultane-
in our laboratory. A 255-bp fragment of human K5 was
obtained by double restriction digestion of plasmid
pET22b-K5 with BgI/Hind and was cloned into the
plasmid pGL3 downstream the Egr1 promoter with two
cohesive ends.
2.2. Recombinant Plasmid Construction and
Baculovirus Generation
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Please do not revise any of the current designations. The
baculovirus plasmid pFB-NES1 was constructed previ-
ously in our laboratory [15]. The Egr1-K5 fragment was
amplified by polymerase chain reaction using the recon-
structed plasmid pGL3 above as a template with the for-
CATTTC-3’ and the reverse primer 5’-TTCCATGGTG-
GCTTTACCAACAGTACCG-3’. Amplification was per-
formed for 35 cycles at 95˚C for 30 s, 60˚C for 1.5 min,
and 72˚C for 1 min. Then the products were digested by
restriction endonuclease Mlu/SalI and were ligated to the
vector pFB-NES1, replacing the NES1 fragment to create
the recombinant baculovirus plasmid pFB-Egr1-K5. Re-
combinant baculoviruses were generated and propagated
in spodotera frugiperda (Sf-9) insect cells by a Bac-to-
Bac system according to standard manual (Invitrogen).
Copyright © 2012 SciRes. JCT
Baculovirus Mediated Experimental Research on Targeted Egr1-Kringle 5 Gene Radiotherapy
in Lung Adenocarcinoma
Once the viruses were amplified, EGFP expression was
observed under a fluorescent microscope to assess the
activity of Egr1 promoter stimulated by 5Gy X-radiation
and the transfect efficiency of baculovirus gene into
A549 cells or HUVEC cell. The viruses were amplified
to a high titer by propagation in Sf-9 cells and stored in
small aliquots at 80˚C. Viral titers were determined by
plaque assay on Sf-9 insect cells. As we reported previ-
ously [16], a multiplicity of infection (MOI) of 50 was
selected for the following baculovirus experiments.
2.3. In Vitro Experiments, the Capacity of
Temporal Regulation
2.3.1. Cell Line Culture, Recombinant Baculovirus
Infection and Cell Irradiation
Human lung adenocarcinoma cells (A549), HUVEC cells
(human umbilical veins endothelial cells) and Sf-9 cells
were preserved in our laboratory. A549 cells and HU-
VEC cells were maintained in Dulbecco’s modified Ea-
gle’s medium (DMEM, Gibco), supplemented with 10%
FBS (fetal bovine serum, Gibco), under standard cell
culture conditions (5% CO2, 37˚C). Sf-9 cells were cul-
tured at 27˚C in a spinner culture bottle containing Sf-
900II(Gibco) supplemented with 4% FBS.
To infected with the recombinant baculovirus, A549
cells and HUVEC cells were plated at a density of 106
cells per well in 6-well plates in serum-free DMEM. Af-
ter 24 h, the culture was infected with the recombinant
baculovirus at 37˚C for 1 h, with sodium butyrate sup-
plemented to a final concentration of 10 mmol/L to en-
hance the infection efficiency. The medium was replaced
with fresh DMEM containing 10% FBS. After another 24
h of incubation, the cells were irradiated with X-radiation
(6 MV, ELEKTA Precise linear accelerator) of different
doses (0 Gy, 1 Gy, 2 Gy, 4 Gy, 6 Gy, 8 Gy and 10 Gy).
Cells were harvested 24 h after irradiation and ready for
the following experiments.
2.3.2. rhK5 mRNA Transcription under Different
Irradiation Doses
Total RNA of human A549 cells was extracted using
RNeasy Mini Kit (Qiagen) following the protocol pro-
vided by the manufacturer. RNA integrity was checked
by electrophoresis and quantified by absorption at 260
nm and 280 nm using a UV-visible spectrophotometer
(Beckman Coulter Du 800). Reverse transcription reac-
tions were performed on 800 ng total RNA by two steps
of elimination of genomic DNA and reverse transcription
according to the instructions using the Quanti Tect Rev.
Transcription Kit (Qiagen). Quantitative real-time PCR
assay was performed in 96-well plates on a real-time
PCR instrument (ABI 7300). The composition of each
reaction was as follows in 25 μl final volume (SYBR®
PrimeScript® RT-PCR Kit, Takara): 12.5 μl of SYBR
Premix Ex Taq (2×), 0.5 μl of each primers (10 μM each),
5 μl of cDNA and 6.5 μl of ddH2O. The forward primer
and the reverse primer was 5’-GTGGTGGTGGTGGT-
GGTGGGCCGCACACT-3’. And the program was 95˚C
for 5 min, followed by 40 cycles of 95˚C (15 s), 60˚C (15
s), and 72˚C (30 s).
2.3.3. rhK5 Protein Expre ssion under Different
Irradiation Doses
Cell lysates were made with standard methods. The pro-
tein concentration of each sample was measured using a
BCA kit (Pierce). For SDS-PAGE, 30 μg of protein sam-
ples was loaded on 15% polyacrylamide gels. Proteins
were transferred to a polyvinylidene difluoride mem-
brane with a tank transfer system (Bio-Rad Laboratory),
then blocked with a buffer containing 5% low fat skim
milk and 0.1% Tween-20 in Tris-buffered saline(TBST)
at room temperature for 1 h. Primary antibodies were
diluted in TBST containing 5% skim milk. The mem-
brane was incubated with primary antibodies overnight at
4˚C. After washed three times with TBST, the membrane
was incubated with a horseradish peroxidase-conjugated
secondary antibody (0.02 μg/mL in TBST) for 1 h at
room temperature. Chemiluminescence was detected with
an ECL Western blot detection kit (Amersham, Little
Chalfont, UK) according to its manufacturer’s instruc-
tions. And quantitation was performed using the Gel-Pro
2.3.4. HUVEC Cell Apoptosis Test
HUVEC cells were divided into two groups according to
whether they were infected with recombinant baculovirus.
The HUVEC cells were grown to ~80% confluence in
100 mm2 dishes. The way of recombinant baculovirus
infection and irradiation by different doses was described
above. Afterthat, cells were trypsinized, centrifuged, ali-
quoted into tubes and labeled with Annexin V and pro-
pidum iodide using FITC Annexin V Apoptosis Detec-
tion Kit (BD Pharmingen™ company). Annexin V and
PI staining were performed following the manufacturer’s
recommendations. Flow cytometry analysis was per-
formed using a FACSCalibur Flow Cytometer (Becton
Dickinson) following the manufacturer’s recommenda-
2.4. In Vitro Experiments, the Capacity of Spatial
The A549 cells were seeded in 6-well plates before the
experiment to achieve a density of 1 × 106 cells/well. We
chose four out of six wells in each plate to be X-irradi-
ated and numbered 1, 2, 3, 4. The cells in No. 1, 2, 3
Copyright © 2012 SciRes. JCT
Baculovirus Mediated Experimental Research on Targeted Egr1-Kringle 5 Gene Radiotherapy
in Lung Adenocarcinoma
wells were treated with Bac E (the recombinant bacu-
lovirus containing Egr1 promoter only), Bac K (the re-
combinant baculovirus containing K5 only) and Bac EK
(Bac Egr1-K5), respectively. The cells in the No. 4 well
were blank controls. The way of cell culture and recom-
binant baculovirus infection was the same as described
above. Twenty-four hours after infection, cells in these
four wells were exposed to a single dose of 4 Gy of
X-radiation generated by a linear accelerator. Each plate
was irradiated only once. We tested three times with
three 6-wells plates in all. Then K5 mRNA transcription
of A549 cells in totally 12 wells (3 plates) was analyzed
to show the capacity of spatial regulation by Egr1 pro-
moter in our experimental system.
2.5. Analyze the Dose and the Frequency of
X-Radiation Preliminarily
The A549 cells were grown in 6-well plates, and the way
of cell culture and recombinant baculovirus infection was
the same as described above. Twenty-four hours after
infection, the cells were irradiated with X-radiation of 0
Gy, 1 Gy, 2 Gy, 4 Gy, 6 Gy, 8 Gy and 10 Gy respectively
three-times continuously (once a day, three days in all).
Cells were harvested 24 h after irradiation and assayed
for K5 mRNA.
2.6. Statistical Analysis
Data were analyzed using the SPSS 11.0 software. Each
experiment was done in triplicate. The data were pre-
sented as mean ± SD. Comparison among experimental
groups was performed using ANVOA test. P < 0.05 was
considered statistically significant. Spearman’s correla-
tion tests were conducted to compare K5 expression and
apoptosis percentage of HUVEC cells.
3. Results
3.1. Preparation of the Baculovirus
We successfully constructed the plasmid pGL3-Egr1-K5
and the recombinant plasmid. Whereafter, we developed
a baculovirus-derived vector, containing the K5 gene
under control of the Egr1 promoter and named it Bac EK
(Bac Egr1-K5). The control vector, Bac E (the recombi-
nant baculovirus containing Egr1 promoter only), Bac K
(the recombinant baculovirus containing K5 only) and
Bac Egr1-EGFP were also developed. Propagation of
these viruses in Sf-9 cells yielded viral stocks with a titer
of 1 × 109 PFU/mL.
3.2. Transfect Efficiency of Recombinant
Baculovirus in A549 Cells and HUVEC Cells
The transfect efficiency of baculovirus in A549 cells and
HUVEC cells were assessed by fluorescence microscopy
after these cells were infected with Bac Egr1-EGFP and
get irradiated by 5 Gy of X-radiation once thereafter. The
control groups were infected with recombinant bacu-
lovirus only, but not get irradiated. A549 and HUVEC
cells infected with Bac Egr1-EGFP 12, 18, 24 and 36 h
after transfection respectively were examined by fluo-
rescence microscopy (Figures 2 and 3), showing that both
A549 human lung adenocarcinoma cells and HUVEC
cells could be infected with recombinant baculovirus.
From these figures, we found that the strongest EGFP
expression was detected after 24 h. It also identified the
activity of Egr-1 promoter irradiated by X-radiation.
3.3. In Vitro Experiments, the Capacity of
Temporal Regulation
3.3.1. Analysis of rhK5 mRNA Transcription after
Figure 4 showed the results of real-time PCR in Bac EK
group and two control groups 24 h after the infected
A549 cells were treated with single X-irradiation. The
transcription level of rhK5 mRNA was elevated with the
dose of X-radiation (0 Gy - 10 Gy) significantly in Bac
(a) (b) (c)
(d) (e)
Figure 2. EGFP expression in A549 cells detected by fluo-
rescence microscopy. (a) 12 h after the cells get irradiated
by 5 Gy X-radiation; (b) 18 h after the cells get irradiated
by 5 Gy X-radiation; (c) 24 h after the cells get irradiated
by 5 Gy X-radiation; (d) 30 h after the cells get irradiated
by 5 Gy X-radiation; (e) without get irradiated by X-radia-
tion (the control).
(a) (b) (c)
Figure 3. EGFP expression in HUVEC cells detected by
fluorescence microscopy. (a) Without get irradiated by X-
radiation ( the control); (b) 18 h after the cells get irradi-
ated by 5 Gy X-radiation; (c) 24 h after the cells get irradi-
ated by 5 Gy X-radiation.
Copyright © 2012 SciRes. JCT
Baculovirus Mediated Experimental Research on Targeted Egr1-Kringle 5 Gene Radiotherapy
in Lung Adenocarcinoma
Figure 4. Kringle5 mRNA transcription in the infected
A549 cells analyzed by real-time PCR (with single X-irra-
diation). From top to bottom: in Bac EK group, in Bac K
group and in Bac E group.
EK group (P < 0.05), but not in the two control groups (P
> 0.05).
3.3.2. rhK5 Expression in the Infected A549 Cells
after X-Irradiati on
Western Blot analysis was performed using antibodies to
6-histidine (His-tagged K5) for the expression of rhK5 in
A549 cells infected with recombinant baculoviruses (Bac
EK). A polyclonal antibody to 6-histidine detected a sin-
gle protein band at 14kDa in the Bac EK group (Figure
5). Quantitative evaluation showed (Figure 6) that the
rhK5 expression increased with the dose of radiation
significantly (P < 0.05).
3.3.3. Cell Apoptosis Assay Res ul ts of Inf ec ted
HUVEC Cells after X-Radiation
The dual parameter fluorescent dot plots (Figure 7)
shows the viable cell population in the lower left quad-
rant (negative annexin-FITC and negative PI), the cells at
Figure 5. Kringle 5 expression in the infected A549 cells
detected by Western blot 24 h after X-radiation. (a) In EK
group, a single protein band at 14 kDa (Kringle5); (b) In
Bac K group, no kringle5 expression; (c) In Bac E group, no
Kringle5 expression.
Figure 6. The quantitative evaluation of K5 expression: In-
tegrated optical density (IOD) was measured and analyzed
by software Gel-pro 4.0. The value of IODK5/IODGAPDH was
showed in 0 Gy, 1 Gy, 2 Gy, 4 Gy, 6 Gy, 8 Gy and 10 Gy.
Dose dependent K5 expr ession was observed.
the early apoptosis are in the lower right quadrant (posi-
tive annexin-FITC and negative PI) while the ones at the
late apoptosis are in the upper right quadrant (positive
annexin-FITC and positive-PI).
As seen in Table 1, the early apoptotic cells percent-
age increased in relation to the dose of X-radiation (P <
0.05) both in Bac EK plus radiotherapy group and the
control radiotherapy alone group. Moreover, of the same
dose of X-radiation, the apoptotic cells percentage of Bac
EK plus radiotherapy group was significantly higher than
that of radiotherapy alone group (P < 0.05). However, in
Bac E and Bac K groups, no significant differences of the
early apoptotic cells percentage was detected between
Bac E/Bac K plus radiotherapy group and radiotherapy
alone group (P > 0.05). These data suggest that Bac EK
plus radiotherapy do best increase apoptosis of HUVEC
cells compared with radiotherapy alone as the Egr-1
promoter was activated by radiotherapy and K5 played a
Copyright © 2012 SciRes. JCT
Baculovirus Mediated Experimental Research on Targeted Egr1-Kringle 5 Gene Radiotherapy
in Lung Adenocarcinoma
Copyright © 2012 SciRes. JCT
Figure 7. Apoptotic effect of HUVEC cells 24 h after radiotherapy with or without Bac EK infection were determined by flow
cytometry analysis using annexin V-FITC and propidium iodide. Cell population in bottom left, bottom right, top right and
top left quadrants represented the proportion of viable cells, early apoptotic cells, late apoptotic cells and necrotic cells, re-
spectively. Radi otherapy alo ne: (1) 0 Gy; (2) 1 Gy; ( 3) 2 Gy; (4) 4 Gy ; (5) 6 Gy; (6) 8 Gy; (7) 1 0 Gy; Radiotherapy w ith Bac
EK: (8) 0 Gy; (9) 1 Gy; (10) 2 Gy; (11) 4 Gy; (12) 6 Gy; (13) 8 Gy; (14)10 Gy.
Baculovirus Mediated Experimental Research on Targeted Egr1-Kringle 5 Gene Radiotherapy
in Lung Adenocarcinoma
Table 1. The early apoptotic cells percentage detected by
fluorescence microscopy.
Bac Egr-1-K5 with R (%) R alone (%)
0 Gy 10.67 ± 1.36 8.01 ± 1.85
1 Gy 32.16 ± 3.73 13.38 ± 2.51
2 Gy 34.66 ± 3.22 14.4 ± 4.17
4 Gy 41.52 ± 5.26 21.43 ± 1.39
6 Gy 44.42 ± 4.38 23.92 ± 5.76
8 Gy 49.06 ± 5.91 29.93 ± 3.17
10 Gy 52.55 ± 3.24 36.49 ± 4.62
R: radiotherapy.
part in accelerating the cell apoptosis.
3.4. In Vitro Experiments, the Capacity of
Spatial Regulation
The real-time PCR results showed that the transcription
of rhK5 mRNA in No.4 wells was significantly higher
than those in other three wells (P < 0.05). The spatial
regulation could be realized through the control of rhK5
3.5. Analyze the Dose and the Frequency of
X-Radiation Preliminarily
When we analyzed the K5 mRNA transcription in Bac
EK group after the infected A549 cells were treated with
continuously three-times of X-radiation (the same frac-
tionated dose as single irradiation, once a day, 3 days in
all), no significant difference was seen among these
groups of different fractionated doses (Figure 8).
4. Discussion
Most nonsmall cell lung cancer (NSCLC) patients were
diagnosed at advanced stage and radiotherapy remains
the treatment choice in such patients. Chemotherapy in
combination with radiotherapy significantly improves the
survival rate. However, the complete response rates are
low and long-term survival remains poor, and the cyto-
toxicity of chemotherapy is severe. All these indicate that
exploration of more potent and safe therapeutic modali-
ties is needed. The combination of gene therapy and ra-
diotherapy has the potential to overcome many of the
limitations of adverse tumor biology on cancer treatment
and appears to be a promising approach to increase the
therapeutic ratio of cancer therapy. In this study, the
vector containing Egr1 promoter and its downstream K5
gene, an antiangiogenic agent gene, was constructed and
tested by fluorescence microscopy for the ability of Egr1
promoter to regulate heterologous gene expression. The
results of what the fluorescence microscopy showed also
Figure 8. Kringle5 mRNA transcription in the infected
A549 cells analyzed by real-time PCR (with continuously
three-times of X-radiation) of Bac EK group.
identified that radiation can induce the transcription of
Egr1 as reported previously [9,17], and laid the ground-
work for the next research.
There are several potential advantages in using radia-
tion-inducible genetic constructs with therapeutic radia-
tion over other gene therapy delivery systems for cancer
treatment. One advantage is that the already well-deve-
loped technology of radiation targeting might add to the
localization of toxin production provided by genetic
therapy. As our study showed, when infected HUVEC
cells received the same dose of X-radiation, the apoptotic
cells percentage of “Bac EK plus radiotherapy” group
was significantly higher than that of radiotherapy alone
group (P < 0.05). A number of preclinical studies have
indicated that antiangiogenic agents can enhance the tu-
mor response to radiation [18,19]. Under X-radiation-
induced cell stress condition, K5 could produce additive
effect on endothelial cell apoptosis. Another advantage is
that it controls gene expression spatially and temporally.
The system we describe exploits the benefits of a tumor-
specific vector in combination with radiotherapy which
triggers and increases gene expression. Since therapeutic
agents such as K5 might bring sideeffects to normal tis-
sues, spatial control of protein expression can be very
beneficial to limit K5 expressing locally in the tumor.
There are two challenges in this method when it is pro-
jected as a gene therapy strategy against cancer cells.
First, introduction of the Egr1 promoter-driven antian-
giogenic gene to the tumor site would require an appro-
priate vector. The therapeutic gene must be present in the
cancer cells when the tumor is irradiated by the X-radia-
tion. However, specific irradiation of the tumor site with
radiotherapy may give us specific activation of Egr1
promoter in the tumor cells. Autograps californica multi-
ple nucleopolyhedrovirus (AcMNPV)-based vector, tra-
ditionally used as a biopesticide to kill infected inserts, is
recently tested as a new type of delivery vehicle for
transgene expression in mammalian cells [20]. These
viruses can enter but not replicate in mammalian cells.
With mammalian expression promoters, recombinant
Copyright © 2012 SciRes. JCT
Baculovirus Mediated Experimental Research on Targeted Egr1-Kringle 5 Gene Radiotherapy
in Lung Adenocarcinoma
baculoviruses provide a high transduction efficiency in
different cells and tissues, including several tumor cell
lines [21]. One of the attractive advantages of using
AcMNPV as a cancer gene therapy vector is the large
cloning capacity conferred by its 130-kb viral genome,
which may be used to deliver a large functional gene or
multiple genes from a single vector [22]. Other empirical
advantages of baculovirus vectors include easy construc-
tion of a recombinant viral vector and simple procedure
of purifying large quantities of viruses with high titers. It
would be possible to scale up the less labor-intensive
process to pharmaceutical levels [23]. A second problem
is the dose and the frequency of X-radiation to activate
Egr1 promoter and work as radiotherapy at the same time.
We found that the K5 expression activated by Egr1 pro-
moter dose-dependently increased in this vitro study, that
is, the most K5 expression was achieved using 10 Gy, the
highest dose in our study. Since a dose of 10 Gy is not
used in a curative treatment setting, and patients are not
treated with a single dose of irradiation but with fraction-
ated radiotherapy, we tried using continuously three-
times of X-radiation. To our surprise, the study demon-
strated that there’s no significant difference of K5 ex-
pression between high-dose groups and low-dose groups
after three times of irradiation. On the basis of these re-
sults we deduce this daily repeated small irradiation dose
results in the same activation efficiency of Egr1 promoter.
The mechanism has not been available yet. Moreover,
irradiated by 2 Gy daily is suitable for clinical treatment
and may decrease normal tissue toxicity compared with a
single large dose.
In summary, we report one therapeutic system with
radiation-inducible promoter, Egr1, which made it have
the capacity of temporal regulation as well as spatial re-
gulation. In this system, radiotherapy and gene therapy
worked simultaneously, and their effects were greater
than those of either modality alone. Low dose and more
times might be the most efficient therapeutic model of
this combined system. On the basis of the results above,
further study in vivo has been carried on to observe the
effect of this therapeutic system. And the potential bene-
fits of exogenous gene induction by radiotherapy include
increased local tumor control as well as the potential for
treatment of metastatic disease.
5. Acknowledgements
This work was supported by Shanghai Leading Aca-
demic Discipline Project S30203.
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