Baculovirus Mediated Experimental Research on Targeted Egr1-Kringle 5 Gene Radiotherapy in Lung Adenocarcinoma

doi:10.4236/jct.2012.324052

Paper Menu >>

Journal Menu >>

Journal of Cancer Therapy, 2012, 3, 397-405

http://dx.doi.org/10.4236/jct.2012.324052 Published Online September 2012 (http://www.SciRP.org/journal/jct)

397

Baculovirus Mediated Experimental Research on Targeted

Egr1-Kringle 5 Gene Radiotherapy in Lung

Adenocarcinoma

Haoping Xu, Rui Guo, Biao Li*

Department of Nuclear Medicine, Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.

Email: *[email protected]

Received April 15th, 2012; revised May 27th, 2012; accepted June 15th, 2012

ABSTRACT

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.

Baculovirus Mediated Experimental Research on Targeted Egr1-Kringle 5 Gene Radiotherapy

in Lung Adenocarcinoma

398

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-

ously).

in our laboratory. A 255-bp fragment of human K5 was

obtained by double restriction digestion of plasmid

pET22b-K5 with BgIⅡ/HindⅢ and was cloned into the

plasmid pGL3 downstream the Egr1 promoter with two

cohesive ends.

2.2. Recombinant Plasmid Construction and

Baculovirus Generation

The template is used to format your paper and style the

text. All margins, column widths, line spaces, and text

fonts are prescribed; please do not alter them. You may

note peculiarities. For example, the head margin in this

template measures proportionately more than is custom-

ary. This measurement and others are deliberate, using

specifications that anticipate your paper as one part of the

entire proceedings, and not as an independent document.

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-

ward primer 5’-AGTGCAAGTGCAGGTGCCAGAA-

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).

Baculovirus Mediated Experimental Research on Targeted Egr1-Kringle 5 Gene Radiotherapy

in Lung Adenocarcinoma

399

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

was 5’-GAAGAAGACTGTATGTTTGGGAATGG-3’,

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

system.

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-

tions.

2.4. In Vitro Experiments, the Capacity of Spatial

Regulation

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

Baculovirus Mediated Experimental Research on Targeted Egr1-Kringle 5 Gene Radiotherapy

in Lung Adenocarcinoma

400

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

X-Irradiation

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.

Baculovirus Mediated Experimental Research on Targeted Egr1-Kringle 5 Gene Radiotherapy

in Lung Adenocarcinoma

401

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

(a)

(b)

(c)

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

Baculovirus Mediated Experimental Research on Targeted Egr1-Kringle 5 Gene Radiotherapy

in Lung Adenocarcinoma

402

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

403

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

expression.

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

Baculovirus Mediated Experimental Research on Targeted Egr1-Kringle 5 Gene Radiotherapy

in Lung Adenocarcinoma

404

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.

REFERENCES

[1] J. Folkman, E. Merler, C. Abernathy and G. Williams,

“Isolation of a Tumor Factor Responsible for Angiogene-

sis,” Journal of Experimental Medicine, Vol. 133, No. 2,

1971, pp. 275-288. doi:10.1084/jem.133.2.275

[2] P. Wachsberger, R. Burd and A. P. Dicker, “Tumor Re-

sponse to Ionizing Radiation Combined with Antiangio-

genesis or Vascular Targeting Agents: Exploring Mecha-

nisms of Interaction,” Clinical Cancer Research, Vol. 9,

No. 6, 2003, pp. 1957-1971.

[3] Y. Cao, R. Cao and N. Veitonmaki, “Kringle Structures

and Antiangiogenesis,” Current Medicinal Chemistry—

Anti-Cancer Agents, Vol. 2, No. 6, 2002, pp. 667-681.

doi:10.2174/1568011023353705

[4] D. J. Davidson, C. Haskell, S. Majest, A. Kherzai, D. A.

Egan, K. A. Walter, et al., “Kringle 5 of Human Plasmi-

nogen Induces Apoptosis of Endothelial and Tumor Cells

through Surface-Expressed Glucose-Regulated Protein

78,” Cancer Research, Vol. 65, No. 11, 2005, pp. 4663-

4672. doi:10.1158/0008-5472.CAN-04-3426

[5] L. Bello, C. Giussani, G. Carrabba, M. Pluderi, F. Costa

and A. Bikfalvi, “Angiogenesis and Invasion in Glio-

mas,” Cancer Treatment and Research, Vol. 117, 2004,

pp. 263-284. doi:10.1007/978-1-4419-8871-3_16

[6] G.-H. Jin, D.-Y. Ma, N. Wu, F. M. M. T. Marikar, S.-Z.

Jin, W.-W. Jiang, et al., “Combination of Human Plas-

minogen Kringle 5 with Ionizing Radiation Significantly

Enhances the Efficacy of Antitumor Effect,” Interna-

tional Journal of Cancer, Vol. 121, No. 11, 2007, pp.

2539-2546. doi:10.1002/ijc.22708

[7] M. M. Ahmed, “Regulation of Radiation-Induced Apop-

tosis by Early Growth Response-1 Gene in Solid Tu-

mors,” Current Cancer Drug Targets, Vol. 4, No. 1, 2004,

pp. 43-52. doi:10.2174/1568009043481704

[8] O. Greco, B. Marples, G. U. Dachs, K. J. Williams, A. V.

Patterson and S. D. Scott, “Novel Chimeric Gene Pro-

moters Responsive to Hypoxia and Ionizing Radiation,”

Gene Therapy, Vol. 9, No. 20, 2002, pp. 1403-1411.

doi:10.1038/sj.gt.3301823

[9] M. Y. Wu, X. Y. Wu, Q. S. Li and R. M. Zheng, “Ex-

pression of Egr-1 Gene and Its Correlation with the On-

cogene Proteins in Non-Irradiated and Irradiated Eso-

phageal Squamous Cell Carcinoma,” Diseases of the

Esophagus, Vol. 19, No. 4, 2006, pp. 267-272.

doi:10.1111/j.1442-2050.2006.00575.x

[10] H. B. Ma, X. J. Wang, Z. L. Di, H. Xia, Z. Li, J. Liu, et al.,

“Construction of Targeted Plasmid Vector pcDNA3.1-

Egr.1p-p16 and Its Expression in Pancreatic Cancer

JF305 Cells Induced by Radiation in Vitro,” World Jour-

nal of Gastroenterology, Vol. 13, No. 31, 2007, pp. 4214-

4218.

[11] N. Khalighinejad, H. Hariri, O. Behnamfar, A. Yousefi

and A. Momeni, “Adenoviral Gene Therapy in Gastric

Cancer: A Review,” World Journal of Gastroenterology,

Vol. 14, No. 2, 2008, pp. 180-184.

doi:10.3748/wjg.14.180

[12] F. M. Boyce and N. L. Bucher, “Baculovirus-Mediated

Gene Transfer into Mammalian Cells,” Proceedings of

the National Academy of Sciences USA, Vol. 93, No. 6,

1996, pp. 2348-2352. doi:10.1073/pnas.93.6.2348

[13] H. Matilainen, J. Rinne, L. Gilbert, V. Marjomaki, H.

Baculovirus Mediated Experimental Research on Targeted Egr1-Kringle 5 Gene Radiotherapy

in Lung Adenocarcinoma

405

Reunanen and C. Oker-Blom, “Baculovirus Entry into

Human Hepatoma Cells,” Journal of Virology, Vol. 79,

No. 24, 2005, pp. 15452-15459.

doi:10.1128/JVI.79.24.15452-15459.2005

[14] D. L. Jarvis and A. Garcia Jr., “Long-Term Stability of

Baculoviruses Stored under Various Conditions,” Bio-

techniques, Vol. 16, 1994, pp. 508-513.

[15] W. Huang, X. L. Tian, Y. L. Wu, J. Zhong, L. F. Yu, S. P.

Hu, et al., “Suppression of Gastric Cancer Growth by

Baculovirus Vectormediated Transfer of Normal Epithe-

lial Cell Specific-1 Gene,” World Journal of Gastroen-

terology, Vol. 14, No. 38, 2008, pp. 5810-5815.

doi:10.3748/wjg.14.5810

[16] R. Guo, Y. F. Zhang, S. Liang, H. P. Xu, M. Zhang and B.

Li, “Sodium Butyrate Enhances the Expression of Bacu-

lovirus-Mediated Sodium/Iodide Symporter Gene in

A549 Lung Adenocarcinoma Cells,” Nuclear Medicine

Communications, Vol. 31, No. 10, 2010, pp. 916-921.

[17] J. M. McLoughlin, T. M. McCarty, C. Cunningham, V.

Clark, N. Senzer, J. Nemunaitis, et al., “TNFerade, an

Adenovector Carrying the Transgene forHuman Tumor

Necrosis Factor a, for Patients with Advanced Solid Tu-

mors: Surgical Experience and Long-Term Follow-Up,”

Annals of Surgical Oncology, Vol.12, No.10, 2005, pp.

825-830. doi:10.1245/ASO.2005.03.023

[18] R. P. Dings, B. W. Williams, C. W. Song, A. W. Grif-

fioen, K. H. Mayo and R. J. Griffin, “Anginex Synergizes

with Radiation Therapy to Inhibit Tumor Growth by Ra-

diosensitizing Endothelial Cells,” International Journal

of Cancer, Vol. 115, No. 2, 2005, pp. 312-319.

doi:10.1002/ijc.20850

[19] S. Nasu, K. K. Ang, Z. Fan and L. Milas, “C225 Antiepi-

dermal Growth Factor Receptor Antibody Enhances Tu-

mor Radiocurability,” International Journal of Radiation

Oncology•Biology•Physics, Vol. 51, No. 2, 2001, pp. 474-

477.

[20] J. P. Condreay and T. A. Kost, “Baculovirus Expression

Vectors for Insect and Mammalian Cells,” Current Drug

Targets, Vol. 8, No. 10, 2007, pp. 1126-1131.

doi:10.2174/138945007782151351

[21] C. Hofmann, V. Sandig, G. Jennings, M. Rudolph, P.

Schlag and M. Strauss, “Efficient Gene Transfer into

Human Hepatocytes by Baculovirus Vectors,” Proceed-

ings of the National Academy of Sciences of the USA, Vol.

92, 1995, pp. 10099-10103.

doi:10.1073/pnas.92.22.10099

[22] T. A. Kost and J. P. Condreay, “Recombinant Baculovi-

ruses as Mammalian Cell Gene-Delivery Vectors,” Trends

in Biotechnology, Vol. 20, No. 4, 2002, pp. 173- 180.

doi:10.1016/S0167-7799(01)01911-4

[23] S. Ghosh, M. K. Parvez, K. Banerjee, S. K. Sarin and S. E.

Hasnain, “Baculovirus as Mammalian Cell Expression

Vector for Gene Therapy: An Emerging Strategy,” Mo-

lecular Therapy, Vol. 6, No. 1, 2002, pp. 5-11.

doi:10.1006/mthe.2000.0643