ABBS 2005,38(06): Antitumor Effect of Antisense Ornithine Decarboxylase Adenovirus on Human Lung Cancer Cells

Abstract        Ornithine decarboxylase (ODC), the first enzyme
of polyamine biosynthesis, was found to increase in cancer cells, especially
lung cancer cells. Some chemotherapeutic agents aimed at decreasing ODC gene
expression showed inhibitory effects on cancer cells. In this study, we
examined the effects of adenoviral transduced antisense ODC on lung cancer
cells. An adenovirus carrying antisense ODC (rAd-ODC/Ex3as) was used to infect
lung cancer cell line A-549. The
3-(4,5-methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay was used to
analyze the effect on cell growth. Expression of ODC and concentration of
polyamines in cells were determined by Western blot analysis and high
performance liquid chromatography. Terminal deoxynucleotidyl
transferase-mediated biotin-dUTP nick-end labeling was used to analyze cell
apoptosis. Expression of ODC in A-549 cells was reduced to 54%, and three
polyamines were also decreased through the rAd-ODC/Ex3as treatment.
Consequently, cell growth was substantially inhibited and terminal
deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling proved that
rAd-ODC/Ex3as could lead to cell apoptosis, with apoptosis index of 46%. This
study suggests that rAd-ODC/Ex3as has an antitumor effect on the human lung
cancer cells.

Key words        polyamine biosynthesis; antisense technology; gene therapy;
A-549 cells

The three polyamines, spermidine, spermine and putrescine, which
have a low molecular weight and a simple chemical structure, are positively charged
aliphatic amines under physiological conditions. The polyamines interact with
various macromolecules, both electrostatically and covalently. Consequently,
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 level of polyamine is 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, which decarboxylates L-ornithine to
form diamine putrescine. The complete structure and nucleotide sequence of the
ODC gene from mammals are known, with 12 exons and 11 introns [5]. Active
mammalian ODC is a homodimer with two-fold symmetry. Subunits have a molecular
weight of approximately 51 kDa and the polypeptide chain consists of 461 amino
acids. ODC becomes activated after treatment with chemical carcinogens and
tumor promoters, as well as in cells transformed by various oncogenes, such as v-src,
neu and ras [1,6,7]. The level of ODC protein was elevated in various cancers [8–10] and related
to recurrence [11]. Some chemotherapeutic agents (e.g.,
difluoromethylornithine), aimed at inhibiting the activity of ODC, have
appeared and taken on inhibitory effects on tumor growth in vitro or in
vivo [12,13], although they show dose-limiting toxicity. Stable
transfection of human lung squamous carcinoma cell line LTEP-78 with antisense
ODC-expressing plasmid has showed to relate with the reversion of malignant
phenotypes of human lung squamous carcinoma cells [15]. Taken together, these
findings suggest that ODC might provide an important target for the development
agents that inhibit carcinogenesis and tumor growth.

Lung cancer is one of the most frequently diagnosed cancers in the
world. Metastatic lung cancer is essentially resistant to systemic cytotoxic
chemotherapy, and external beam and radioisotope radiotherapy offers only
symptom palliation. Clearly the development of novel therapies, such as gene
therapy, is a high priority. Some studies have proved that lung carcinomas have
greater elevated polyamine levels [15]. Adenoviral vectors are among the most
promising gene transfer vehicles for direct, in vivo gene therapy for a
diverse array of human diseases [16]. In this study, we used a
replication-deficient recombinant adenovirus to efficiently deliver a 120 bp
antisense ODC (rAd-ODC/Ex3as), which is complementary to the initiation codon,
and tested the effects of antisense ODC on lung cancer. The data presented here
showed that adenovirus-mediated gene transfer of antisense ODC could
significantly inhibit growth of lung cancer cells.

Materials and Methods

Cell culture and reagents

The A-549 lung cancer cell line was obtained from the  

Adenovirus and infection
condition

The recombinant adenovirus rAd-ODC/Ex3as, containing the
cytomegalovirus promoter and green fluorescent protein (GFP) gene, was
constructed by reversely inserting a 120 bp cDNA fragment of the ODC gene into
the multiple clone sites [17]. rAd-ODC/Ex3as was purified by
ultracentrifugation in cesium chloride step gradients [18]. The titer of the
viral stock, measured in plaque-forming units (pfu)/ml, was determined to be
8.5´109 pfu/ml by a method published
previously [20], and the frozen stock was confirmed to have retained its titer.
The control virus rAd-GFP was constructed in the same way as rAd-ODC/Ex3as but
without the targeted gene inserted in the polylinker. Viral stocks were
suitably diluted in serum-free medium to obtain the desired pfu, added to cell
monolayers of lung cancer cells and incubated at 37 ºC for 2 h. The necessary
amount of culture medium with 5% FBS was then added and the cells were
incubated for the desired time.

MTT assay

MTT assay was used to assess the transduction efficiency of
rAd-ODC/Ex3as in A-549 cells. Briefly, cells were seeded at a density of 5000
cells/well in 96-well plates and grown overnight. The next day, these cells
were infected by a different dose of virus, from 1 to 100 pfu/cell
[multiplicity of infection (MOI)]. After 48 h of incubation, MTT was added (50 mg/well) and
incubated for 4 h. Formazan products were dissolved in dimethylformamide, and
the optical density was measured at 570 nm. To observe the effect of the
adenovirus on cell proliferation, MTT assay was also used to draw cell growth
curves. Cells were inoculated at a density of 4000 cells per well, under which
control cells remained subconfluent and in exponential phase growth for the
duration of the assay. Due to a different infective efficiency, A-549 cells
were infected by 50 MOI. All experiments were carried out six times. After 24,
48, 72, 96 and 120 h, absorbance at 570 nm was measured respectively.

Invasion assay

Invasion was assessed by the ability of A-549 cells infected with
rAd-ODC/Ex3as to traverse a matrigel-coated membrane as follows. Transwell
inserts (8 mm pore size; Corning Costar,  

Western blot analysis of ODC
proteins

A-549 cells were infected with rAd-ODC/Ex3as at an MOI of 50
pfu/cell in RPMI 1640 medium containing 5% FBS for 48 h. The cells were washed
three times using ice-cold phosphate-buffered saline (PBS) and collected with a
cell scraper. Total cell lysates were prepared in extraction buffer containing  

High performance liquid
chromatography (HPLC) analysis of polyamine pools

A-549 cells were infected with rAd-ODC/Ex3as at an MOI of 50. After
48 h, cells were trypsinized and washed twice with PBS. Intracellular
polyamines were extracted from cell pellets with 10% trichloroacetic acid,
dansylated and measured by HPLC using muBondapak C18 column ( 

Cell apoptosis analyzed by
terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling
(TUNEL)

TUNEL assay kit was supplied by Santai Biological Company ( 

Statistical analysis

Statistical analysis was carried out using Statview J 5.0 software
(SAS Institute,  

Results

Dose-dependent growth
inhibition of A-549 cells by rAd-ODC/Ex3as

There was dose-dependent growth inhibition in A-549 cells (Fig. 1).
Due to differential sensitivity, we chose an MOI of 50 for the adenovirus to infect
A-549 cells. Under these conditions, the growth of A-549 cells is fast, but
rAd-ODC/Ex3as was more suppressive of growth (with a suppressive rate of 60%)
than the control rAd-GFP virus, which had no obvious toxic effect on cells.

Time dependence of the
inhibitory effects of rAd-ODC/Ex3as on lung cancer cells

There was dose-dependent growth inhibition in the A-549 cell line,
which reflected the transduction efficiency of the adenovirus to the lung cancer
cell line. We chose an MOI of 50 for adenovirus to infect A-549 cells. Under
these conditions, rAd-ODC/Ex3as was more suppressive of growth than the control
rAd-GFP virus, which had no obvious toxic effect on cells. We examined the in
vitro growth inhibition of rAd-ODC/Ex3as in A-549 cells using cell growth
curves as described in “Material and Methods”. Antisense ODC had an
impact on the growth of lung cancer cells. rAd-ODC/Ex3as inhibited their
proliferation by approximately 50% compared with the control virus or no
virus-treated groups (Fig. 2).

Effect of rAd-ODC/Ex3as on
invasion ability of A-549 cells

We used a matrigel invasion model to study the effect of the
rAd-ODC/Ex3as on invasion. A-549 cells infected with rAd-ODC/Ex3as (18.20±8.62)
invaded the matrigel-coated membrane to a much lesser extent than
rAd-GFP-infected cells (36.40±8.76) or no virus-treated cells (39.80±6.88) (Fig.
3).

TUNEL assay for apoptosis

To examine the mechanism by which rAd-ODC/Ex3as might retard lung
cancer cell growth in vitro, we used TUNEL to detect the effect of
rAd-ODC/Ex3as on apoptotic cells at 48 h (Fig. 4) and 72 h after
infection. As shown in Table 1, the rate of apoptosis in cells infected
by rAd-ODC/Ex3as was significantly higher than in cells infected by rAd-GFP or
no virus-treated cells (P<0.05).

Effect of rAd-ODC/Ex3as on
expression of ODC and polyamine pools in cell lysate

The ODC proteins produced in the A-549 cells infecting with
rAd-ODC/Ex3as were examined by Western blot. The ODC expression in the cells
infected with rAd-ODC/Ex3as was substantially reduced compared with that of the
cells infected with rAd-GFP or no virus-treated cells. The results analyzed by
SmartView software showed that ODC gene expression in A-549 cells infected with
rAd-ODC/Ex3as accounted for 40% of that in cells treated with rAd-GFP (Fig.
5).

Effect of rAd-ODC/Ex3as on
polyamine pools in cell lysate

Three polyamines (putrescine, spermidine and spermine) were analyzed
by HPLC. It was shown that the concentrations of the three polyamines,
especially of putrescine, were significantly decreased (Table 2).

Discussion

Polyamines are aliphatic cations with multiple functions and are essential
for life. In normal cells, ployamine levels are intricately controlled by
biosynthetic and catabolic enzymes. Multiple abnormalities in the control of
polyamine synthesis, metabolism, uptake and function might be responsible for
increased levels of polyamines in cancer cells compared to that of normal
cells, especially in lung cancer cells [15]. Targeting specific molecules in
cells by antisense inhibition was shown to have potential effectiveness in
decreasing the protein expression. ODC is the most important enzyme in
polyamine biosynthesis. The overexpression of ODC in NIH3T3 cells caused
transformation of these cells to a malignant phenotype, in essence qualifying
ODC as an oncogene [21]. Inhibition of ODC by difluoromethylornithine could compromise
cell growth and transformation [23]. In vitro studies by Schipper et
al. using conformationally restricted polyamine analogues showed that these
compounds inhibited cell growth, probably by inducing antizyme-mediated
degradation of ODC [23]. In addition, Alm et al. [24] showed that the
ODC gene was a well-defined target gene for c-myc and other oncogenes.
Therefore, we targeted ODC gene using an antisense gene delivery strategy with
a replication-deficient recombinant adenovirus vector. In the present study, we
demonstrated that rAd-ODC/Ex3as could inhibit lung cancer growth and lead to
the apoptosis of A-549 cells.

MTT assay showed antisense ODC had an impact on the growth of lung
cancer cells. rAd-ODC/Ex3as inhibited their proliferation by approximately 60%
when compared with the control virus and no virus-treated groups. Western blot
analysis showed the expression of the ODC gene was substantially more reduced
in the cells infected with rAd-ODC/Ex3as than in the cells infected with
rAd-GFP or no virus-treated cells. A substantial decrease in ODC gene
expression resulted in the reduction of polyamine biosynthesis. In addition,
the reduction of polyamines might contribute to the marked suppression of
cancer cell growth and tumor formation. Recent studies also showed that
inhibiting mRNA expression of ODC gene can effectively inhibit the growth of
some cancer cells, such as breast, prostate, colorectal, pancreatic cancer and
bladder carcinoma cells [25–28]. These findings suggested that polyamine metabolism and ODC
could be potential therapeutic targets in the treatment of some cancers.

To examine the mechanism of antisense ODC inhibiting the growth of
lung cancer cells, we demonstrated by TUNEL assay that rAd-ODC/Ex3as infection
could contribute significantly to cell apoptosis in comparison to rAd-GFP
infected or no virus-treated cells. Some recent studies have demonstrated the
inhibition of ODC could lead to induction of apoptosis of some cancer cells [29–31]. Our study
indicated the induction of apoptosis was the mechanism of antisense ODC
inhibiting the growth of lung cancer cells.

In general, our data suggested that adenoviral vector mediated
antisense ODC can lead to induction of apoptosis and inhibition of growth of
lung cancer cells in vitro. rAd-ODC/Ex3as could be a potential agent
against lung cancer. However, further in-depth in vivo studies are
required.

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