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Acta Biochim Biophys |
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doi:10.1111/j.1745-7270.2006.00204.X |
Gene Expression of Ornithine
Decarboxylase in Lung Cancers and Its Clinical Significance
Hui TIAN1*, Qing
HUANG1, Lin LI1, Xian-Xi LIU2, and Yan ZHANG2
1 Department
of Thoracic Surgery, Shandong University Qi Lu Hospital, Jinan 250012, China;
2 Department of Medicine,
Medical Molecular Biology Experimental Center, Shandong University, Jinan
250012, China
Received: March 26,
2006
Accepted: June 3,
2006
This work was
supported by a grant from the National Natural Science Foundation of China (No.
30571844)
*Corresponding
author: Tel, 86-531-82169463; Fax, 86-531-86927544; E-mail,
[email protected]
Abstract Lung cancer is one of the most lethal
cancers in China because of high incidence and high mortality. Ornithine
decarboxylase (ODC), an important enzyme in polyamine biosynthesis, is
increased in cancer cells. Some chemotherapeutic agents aimed at reducing ODC
expression show inhibitory effects on cancer cell growth, so ODC can be useful
in the research of gene diagnosis and gene therapy of cancers. In this study,
we examined the effect of antisense ODC on lung cancer cells. A-549 cells were
infected with rAd-ODC/Ex3as, a recombinant adenovirus containing the
cytomegalovirus promoter, green fluorescent protein gene and 120 bp
antisense ODC. The cell cycle was evaluated by flow cytometry. A nude mouse
xenograft model was used in the tumorigenicity test. Reverse
transcription-polymerase chain reaction, Western blot and immunohistochemistry
were used to study the expressions of ODC on lung cancers. It was found that
the growth of cells infected with rAd-ODC/Ex3as was substantially inhibited and
cells were arrested at G1 phase. Cells infected with rAd-ODC/Ex3as
can suppress tumor formation in a nude mouse xenograft model. The expression of
ODC mRNA and ODC protein levels in lung cancer tissues was significantly higher
than that in normal tissues (P<0.05), which correlated significantly
with the stage of lung cancer (P<0.05). This study suggested that
rAd-ODC/Ex3as has antitumor activity in human lung cancer cells. The ODC
gene might play an important role in lung cancer and the overexpression of ODC
might be related to the occurrence and development of lung cancer.
Key words polyamine biosynthesis; lung neoplasm;
ornithine decarboxylase; A-549 cell line
Recent research has shown that multiple gene changes result in tumor
genesis and progression. In the complicated course of tumor development, many
genes can be involved, including activation of proto-oncogenes and inactivation
of anti-oncogenes. In clinical tumor therapy, the major factors that influence
a patient’s prognosis are the conditions of tumor invasiveness and metastasis,
and tumor biological behavior is also a major element in determining a tumor
therapy plan. Many tumor experts have been working hard on the early diagnosis
of tumor invasiveness and metastasis to improve tumor prognosis and prolong
patient life. Therefore, tumor markers that can provide information on the
conditions of tumor invasiveness and metastasis have always been emphasized by
tumor experts and are a “hot” research subject. The polyamines
spermidine and spermine, and the diamine precursor putrescine, are positively
charged aliphatic amines under physiological conditions. They interact with
various macromolecules, both electrostatically and covalently and, as a
consequence, they have a variety of cellular effects. They are known to be
critically involved in cell growth and have been implicated in the process of
cell transformation [1,2]. However, the levels of polyamines are high in cancer
cells and tissues, and rapid tumor growth has been associated with remarkable
elevation of polyamine biosynthesis and accumulation [3,4].
Ornithine decarboxylase (ODC) is the first and the rate-controlling
enzyme in polyamine biosynthesis. It catalyzes the decarboxylation of L-ornithine
to form diamine putrescine. The complete structure and nucleotide sequence of
the ODC gene from mammals are known [5]. It has 12 exons and 11 introns.
Active mammalian ODC is a homodimer with 2-fold symmetry. Its subunit consists
of 461 amino acids and has a molecular weight of approximately 51 kDa. ODC
becomes active after treatment with chemical carcinogens and tumor promoters,
as well as in cells transformed by various oncogenes, such as v-src, neu
and ras [6–8]. The level of ODC was reported to be elevated in various cancers
[9–11]
and relate to recurrence [12]. Some chemotherapeutic agents, such as
difluoromethylornithine (DFMO), aimed at inhibiting the activity of ODC, have
appeared and taken on inhibitory effects on tumor growth in vitro and in
vivo [13,14], showing dose-dependent toxicity. Stable transfection of human
lung squamous carcinoma cell line LTEP-78 with antisense ODC-expressing plasmid
DNA showed to be related with the reversion of malignant phenotypes of human
lung squamous carcinoma cells [15]. Taken together, these findings suggest that
ODC might be an important target for the development of drugs to inhibit
carcinogenesis and tumor growth.
Lung cancer is one of the most lethal cancers in China because of
high incidence and high mortality. Metastatic lung cancer is essentially
resistant to systemic cytotoxic chemotherapy, and external beam and
radioisotope radiotherapy offer only symptom palliation. The development of
novel therapies, such as gene therapy, is of high priority. Adenoviral vectors
are one of the most promising gene transfer vehicles for direct and in vivo
gene therapy for the treatment of many human diseases [16].
In this study, we used a replication-deficient recombinant
adenovirus to efficiently deliver a 120 bp antisense ODC, which is
complementary to the initiation codon, and investigated its effect on lung
cancer.
Materials and Methods
Cell culture and sample
sources
The A-549 human lung cancer cell line was preserved in our
laboratory. Cells were cultured in DMEM or RPMI 1640 medium supplemented with
10% heat-inactivated fetal bovine serum, 100 U/ml penicillin and 100 mg/ml
streptomycin. Anti-ODC monoclonal antibody was prepared in our laboratory.
Forty-two patients (22 male and 20 female; 60–76 years old,
mean age 66.5 years) with lung cancers (24 squamous cell cancers, 18
adenocarcinomas) were selected from Qi Lu Hospital of Shandong University
(Jinan, China) from October 2003 to September 2004. Before operation, the
patients had not received radiotherapy and/or chemotherapy. In each case, lung
cancer tissues (pathologically confirmed) and normal lung tissues (taken at a
site 5–8 cm from the primary tumors) were sampled. The tissue samples
stored at –80 ºC.
The primers of b-actin were obtained from Shanghai Boya Biotechnology (Shanghai,
China). The goat anti-mouse immunoglobulin (Ig) G antibody conjugated with
horseradish peroxidase (HRP) was purchased from Beijing Zhongshan Biotechnology
Company (Beijing, China). Western blot reagent was from the Institute of
Molecular Biology of Shandong University, School of Medicine, Shandong
University (Jinan, China).
Adenovirus and infection
conditions
The recombinant adenovirus rAd-ODC/Ex3as, containing the
cytomegalovirus promoter and green fluorescent protein gene (GFP), was
constructed by reversely inserting a 120 bp cDNA fragment of ODC into
the multiple clone sites. rAd-ODC/Ex3as was purified by ultracentrifugation
using cesium chloride gradient. The titer of the viral stock, measured in
plaque-forming units (pfu) per milliliter, was determined to be 8.5109 pfu/ml by a method published previously [17], and the frozen stock
was confirmed to have retained its titer. The control virus rAd-GFP was
constructed in the same way but no gene was inserted in the polylinker. Viral
stocks were diluted with serum-free medium to obtain the desired titer, added
to the monolayers of lung cancer cells and incubated at 37 ºC for 2 h. Then a
certain amount of culture medium with 5% fetal bovine serum was added and the
cells were incubated for the desired time.
Flow cytometry analysis
For flow cytometry analysis, A-549 cells were infected with
rAd-ODC/Ex3as at a multiplicity of infection of 50. After 48 h, cells were
washed with phosphate-buffered saline (PBS) twice and harvested by
trypsinization. Cells were washed again with PBS, fixed with 70% cold ethanol
for 1 h, then washed once with PBS and incubated with 4 mg of
ribonuclease A (Roche, Grenzacherstrasse, Switzerland) for 30 min at room
temperature. Propidium iodide was added to the cell suspension at a final
concentration of 20 mg/ml and incubated for 30 min. Cells were then analyzed by flow
cytometry using FACScan (Becton Dickinson, San Jose, USA). The results were
quantified using CellQuest software (Becton Dickinson).
Nude mouse tumorigenicity
study
A-549 cells were infected with rAd-ODC/Ex3as at a multiplicity of
infection value of 50 for 48 h, harvested, washed three times with PBS and
resuspended in RPMI 1640 medium. The cell suspension (approximately 2´106 cells) in a total volume of 100 ml was injected
subcutaneously into 6-week-old BALB/c nude male mice. Tumor was measured every
week and the volume was calculated with Equation 1 according to Rockwell
et al. [18].
Eq. 1
where M1 represents the long axis, and M2 represents the short axis.
ODC mRNA expression detected
by reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA from cancer and normal tissues was extracted according to
the protocols of the Trizol agent kit (Sangon, Shanghai, China). The RNA was
reversely transcribed according to the protocol of cDNA first chain
construction agent kit (Sangon) with random primer oligo(dT). The cDNA was used
as the template for PCR. PCR was carried out using specific primers as
following: 5 min at 94 ºC; 40 s at 94 ºC, 40 s at 56 ºC and 1 min at 72 ºC, 35
cycles; and 7 min at 72 ºC. Ten microliters of total PCR product was
electrophoresed on 1% agarose gel. b-actin gene was used as internal control.
Electrophoresed zones were visualized using ultraviolet light analysis
apparatus, and photographed using a gel imaging analysis system.
Immunohistochemical staining
Immunohistochemical staining was carried out as follows [19].
Tissues were fixed in 96% ethanol for 6 h, embedded in paraffin, and cut into 5
mm
sections. Sections were deparaffinized in xylol, rehydrated through graded
ethanol, and washed in PBS with Tween. Then sections were incubated for 2 h at
room temperature in a humidified chamber with 100 ml of the anti-ODC
monoclonal antibody at 1:500 dilution. The slides were washed and incubated
with HRP-conjugated goat anti-mouse IgG at 1:100 dilution in PBS with 10% (W/V)
BSA for 1 h at room temperature. After washing, the HRP was visualized by
development with chromogenic reagent. The sections of breast carcinoma tissues
known to express ODC protein were used as positive controls and normal mouse
serum and PBS only were used as negative controls. These sections were stained
with 3,3‘-diaminobenzidine-tetrachloride and counterstained with
hematoxylin, then the results were examined under a microscope. ODC protein was
mainly expressed in the cytoplasm of tumor cells and was stained from light
brown to deep brown granule or mass. According to the percentage of the ODC
protein positive cancer cells within the maximum cut-surface specimen of the
tumor tissue, the percentage was categorized into four groups: –, negative; +,
<25%; ++, 25%–75%; +++, 75%–100%.
Western blot analysis of ODC
protein
Tissues were homogenized in RIPA buffer (1´PBS, 1% NP-40, 0.1% SDS and 1 mM EDTA) and centrifuged at 10,000 rpm
for 10 min at 4 ºC [20]. The supernatants were used for Western blot analysis.
One hundred and twenty-five micrograms of each sample was electrophoresed on
12% SDS-PAGE gel. Separated proteins were transferred onto a
nitrocellulose membrane in Tris/glycine buffer (25 mM Tris base, 250 mM glycine, 0.1% SDS, pH 8.3) at 100 V for 2 h. The membrane was blocked for 1 h at room temperature with
PBS containing 5% non-fat milk. It was then incubated at room temperature for 2
h in a solution containing primary anti-ODC antibody, then washed three times
with PBS and incubated with HRP-conjugated goat anti-mouse IgG (1:1000 in
Tris-buffered saline) as secondary antibody for 1 h at room temperature.
Reactive proteins were visualized with a chemiluminescence
detection system.
Statistical analysis
Statistical analysis of flow cytometry and RT-PCR was done by
Student’s t-test and Fisher’s exact test. Statistical analysis of
immunohistochemistry was done by the c2-test.
Results
rAd-ODC/Ex3as infection
induced cell cycle arrest at G1 phase
To examine the mechanism by which rAd-ODC/Ex3as might retard lung
cancer cell growth in vitro, the cell cycle was analyzed using flow
cytometry 48 h post-infection (Fig. 1). The statistical data were shown
in Table 1 which indicated that the proportion of cells in G1 increased significantly in rAd-ODC/Ex3as infected cells compared
with rAd-GFP infected or no virus-treated cells.
Antitumorigenicity effect of
rAd-ODC/Ex3as in nude mouse xenograft model
The potential antitumorigenicity of rAd-ODC/Ex3as was evaluated
using an A-549 xenograft model in nude mice. Expression of antisense ODC
inhibited the growth of A-549 cells in vivo, as shown by the reduction
in tumor incidence and tumor size, when compared with rAd-GFP treated or no
virus-treated tumors [Fig. 2(A)]. Mice that received
rAd-ODC/Ex3as-treated cells did not develop tumors during a 6-week observation
period. The tumor growth rates were 6.80 and 34.82 mm3 per day
for the rAd-GFP-treated and no virus-treated tumors, respectively, as
calculated by an exponential curve [Fig. 2(B)].
ODC mRNA expression detected
by RT-PCR
RT-PCR was used to examine the expression of ODC mRNA in 42 lung
cancer tissues. The results showed that ODC mRNA in lung cancer tissues was
significantly higher than that in normal lung tissues (P<0.05) (Fig.
3 and Table 2). Table 3 showed that it was not significantly
different in differentiation or histology of lung cancers; while the expression
level of ODC mRNA was significantly higher in stage III than in stage I and II
(P<0.05).
Immunohistochemical staining
Immunohistochemical staining showed high level of ODC in lung cancer
tissue (Fig. 4), and the ODC protein level in lung cancer tissues was significantly
higher than in normal ones (Table 4). There was no significant
difference in histology or differentiation of lung cancers; while the
expression of ODC protein was significantly higher in stage III than in stage
I and II (P<0.05) (Table 5).
Western blot analysis
The result of Western blot analysis also showed that the expression
of ODC protein in lung cancer tissues was significantly higher than in normal
lung tissues (Fig. 5).
Discussion
Polyamines are aliphatic cations with multiple functions and are
essential for life. In normal cells, polyamine levels are intricately
controlled by biosynthetic and catabolic enzymes. Multiple abnormalities in
polyamine synthesis, metabolism, uptake and function might be responsible for
increased levels of polyamines in cancer cells compared with those in normal
cells. Polyamine concentration is higher in the prostate than in most other
tissues in the body, making inhibitors of polyamine synthesis desirable
preventive agents for prostate cancer [9]. At the same time, specific molecules
in cells inhibited by antisense were shown to have potential effectiveness on
decreasing the targeted protein expression. ODC is the most important enzyme in
polyamine biosynthesis. More recently, the overexpression of ODC in NIH3T3
cells caused transformation to malignant cells, in essence, ODC
qualified as an oncogene [21]. Inhibition of ODC by DFMO could decrease cell
growth and transformation [22]. Schipper et al.抯 recent in vitro
study using conformationally restricted polyamine analogs showed that these
compounds inhibited cell growth, probably by inducing antizyme-mediated
degradation of ODC [23]. In addition, Alm et al. [24] showed that ODC
was a well-defined target gene for c-myc and other oncogenes. Therefore,
we targeted ODC using an antisense gene delivery strategy with a
replication-deficient rAd vector. In the present study, we showed that
rAd-ODC/Ex3as could inhibit lung cancer growth and lower the invasion of the
A-549 cell line.
To acquire a good result on inhibition of ODC synthesis, first we
scanned different sequences complementary to ODC mRNA by eukaryotic expression
vector pcDNA3.1 in A-549 cells. Sequences of approximately 100 nucleotides in
length which were complementary to regions of the 5‘ untranslated
region, 3‘ untranslated region, the sixth exon region and the initiation
codon region of ODC mRNA were tested for their ability to inhibit translation
of ODC. Compared to any other group, the sequence complementary to the
initiation codon region had a strong reduction of ODC synthesis. This result
was also in accordance with the results of Madhubala et al.’s experiment
using oligodeoxynucleotides [25]. Accordingly, we constructed and packaged the
rAd-ODC/Ex3as which expressed the mRNA complementary to initiation codon of ODC.
We showed that the expression of antisense ODC in lung cancer cells markedly
inhibited cell growth in vitro and in vivo, whereas all
virus-treated control cells possessed antitumor activity.
The mechanism underlying the growth inhibition might be accounted
for in part by the cell cycle arrest of lung cancer cells followed by a
reduction in polyamine pools. Previous studies had demonstrated that cancer
cells underwent cytostasis in the presence of DFMO and this growth arrest could
be prevented by the treatment of cells with putrescine [26]. Recent studies
also showed that polyamine analog N‘,N”-diethylnorspermine also
led to a retardation of S-phase progression [8]. This suggestion was supported
by the data that treatment of A-549 cells with rAd-ODC/Ex3as caused cell cycle
arrest in the G1 phase, compared with the control.
Many studies indicate that polyamines play an important role in
keeping the malignant phenotype of tumors. If the synthesis of polyamines is
restrained, tumor cells will undergo apoptosis and the malignant phenotype
will reverse [27–29]. Although many details must be established in terms of its
antitumor effect, adenoviral vector-mediated antisense ODC could be an option
for lung cancer gene therapy. However, it is known that adenoviral delivery of
genes is transient and lasts for only 4–6 weeks. Our laboratory
will focus on the use of a long-term gene transfer vector, such as a lentiviral
vector or modifying vector with a lung-specific promoter, to specifically
infect lung cancer cells.
In our experiment, ODC mRNA and protein levels in lung cancer and normal lung tissues were compared in order to understand
the relationship between ODC gene expression and the pathogenesis of
lung cancer. The results showed that the expression of ODC mRNA in lung
cancer tissues was significantly higher than in normal lung tissues (P<0.05).
It was not significantly different in differentiation or histology of lung
cancers. The expression of ODC mRNA was significantly higher in stage III than
in stage I and II (P<0.05). The expression of ODC protein in lung
cancer tissues was significantly higher than in normal lung tissues (P<0.05).
There was no significant difference in differentiation or histology of lung
cancers. The expression of ODC protein was significantly higher in stage III
than in stage I and II. Western blot analysis showed that ODC protein is
expressed at low levels in normal lung tissues but is markedly increased in most of the tumor tissues.
The ODC gene might play an important role in lung
carcinogenesis and the upregulated expression of ODC mRNA might be related to
the stages of lung cancer, indicating that ODC expression is associated with
the invasive and aggressive behavior of lung cancer. These
results supported the previous observations and also bettered our
understanding of the molecular mechanisms responsible for lung tumor
development and progression. These findings could help in the design of new therapeutic strategies for the treatment of lung cancer and its
metastases.
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