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Original Paper
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Acta Biochim Biophys
Sin 2006, 38: 639-645 |
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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 poly�amines 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 �C.
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 �C 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 subcuta�neously 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 �C; 40 s at 94 �C, 40 s at 56 �C and 1 min at 72 �C, 35 cycles; and 7 min at 72 �C. 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 �C [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 anti�sense 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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