I. Introduction
The polyamines, spermidine, spermine and the diamine
precursor, putrescine, are positively charged aliphatic amines at physiological
conditions, have a low-molecular weight and a simple chemical structure. They
interact with various macromolecules, both electrostatically and covalently
and, as a consequence, 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 (Auvinen et al, 1992; Moshier et al, 1993). On the other
hand, the level of polyamine is high in cancer cell and tissues, and rapid
tumor growth has been associated with remarkable elevation of polyamine
biosynthesis and accumulation (Marton and Pegg, 1995; Pegg et al, 1998).
Ornithine decarboxylase (ODC) is the first and the
rate-controlling enzyme in polyamine biosynthesis. It decarboxylates
L-ornithine to form diamine putrescine. Complete structure and nucleotide
sequence of ODC gene from mammalians is known for human (Moshier et al, 1990),
which have 12 exons and 11 introns. Active mammalian ODC is homodimer with
2-fold symmetry. Subunits have molecular weight of about 51kDa 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 oncogens, such as v-src, neu and ras (Pegg et al, 1988;
Sistonen et al, 1989; Auvinen et al, 1992). The level of ODC was reportedly
elevated in various cancers (Glikman et al, 1987; Upp JR, Jr. et al, 1988; Love
et al, 2003) and related to recurrence (Love et al, 2003). Some
chemotherapeutic agents, such as Difluoromethylornithine
(DFMO), which aimed to inhibit the activity of ODC have appeared and taken on
inhibitory effects on tumor growth in
vitro and in vivo (Umemoto, 1989;
Zagaja et al, 1998), though showing dose-limiting toxicity. Stable transfection
of human lung squamous carcinoma cell line LTEP-78 with antisense
ODC-expressing plasmid DNA has been shown too related with the reversion of
malignant phenotypes of human lung squamous carcinoma cells (Guan et al, 1996).
Taken together, these findings suggest that ODC may provide an important target
for the development agents that inhibit carcinogenesis and tumor growth.
Esophageal cancer is one of
the most frequently diagnosed cancers in the world. Metastatic esophageal
cancer is essentially resistant to systemic cytotoxic chemotherapy, while
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 had proved that lung cancer had greater elevated
polyamine levels (Carlisle et al, 2002). Because Adenoviral vectors are among
the most promising gene transfer vehicles for direct, in vivo gene therapy for
the treatment of a diverse array of human disease (Meager, 1999). In this
study, we used a replication-deficient recombinant adenovirus to efficiently
deliver a 120bp antisense ODC which is complementary to initiation codon and
tested the effect of antisense ODC on esophageal cancer. The data presented
here show that adenovirus-mediated gene transfer of antisense ODC could significantly
inhibit growth of esophageal cancer cells.
II.
Materials and methods
A. Cell culture and reagents
Esophageal
cancer Eca109 cell line was obtained from Chinese Academy of Science. Cells
were cultured in DMEM or RPMI 1640 medium supplemented with 10% heat-inactived
fetal bovine serum (FBS), 100U/ml penicillin, and 100μg/ml streptomycin.
MTT was purchased from Sigma, MO. β-actin antibody and ECL Western
blotting detection system were obtained from Santa Cruz, CA. Monoclonal
antibody of ODC was made in our lab. Other reagents were all of reagent grade
and obtained from Chinese companies.
B. Adenovirus
and infection condition
The recombinant
adenovirus rAd-ODC/Ex3as, containing the cytomegalovirus (CMV) promoter and GFP
gene, was constructed by reversely inserting a 120bp cDNA fragment of ODC into
the multiple clone sites (Zhang et al, 2003), rAd-ODC/Ex3as was purified by
ultracentrifugation in cesium chloride step gradients (Prevec et al, 1991). The
titer of the viral stock, measured in plaque-forming unit (pfu)/ml, was
determined to be 8.5×109pfu/ml by a method published previously (Wei et
al, 2000),and the frozen stock was confirmed to have retained their titer. The
control virus rAd-GFP was same to rAd-ODC/Ex3as but no 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℃ for 2 hours. The necessary amount of culture medium with 5% fetal
bovine serum was then added and the cells were incubated for the desired times.
C. MTT
assay
Firstly
MTT assay was employed to assess transduction efficiency of rAd-ODC/Ex3as in Eca109 cell line.
Briefly, cells were seeded at density of 5000 cells/well in 96-well plates and
grown overnight. On the next day the cells were infected by a wide range of
viral titres, from 1 to 100 pfu/cell (MOI, multiplicity of infection). After 48
hours of incubation, 3-(4,5-methylthiazol-2-yl)-2,5-diphenyl-tetrazolium
bromide (MTT) was added (50mg/well)
for 4 hours. Formazan products were solubilized with DSMO, and the optical
density was measured at 570nm. To observe the effect of 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 different infective efficiency, A-549 cells were infected by 50
and 25 MOI respectively. All experiments were performed in sextuple. After 24,
48, 72, 96 and 120 hours, cell viability was measured by absorbance at 570nm as
described previously.
D. Western blotting analysis of
ODC proteins
Eca109 cell line was
infected with rAd-ODC/E3as by 50 MOI in 1640 medium containing 5%FCS for 48
hours. The cells were washed three times with ice-cold PBS and collected with a
cell scraper. Total cell lysates were prepared in extraction buffer containing
0.05M Tris (pH8.0), 0.15M NaCl, 0.02% Sodium Azide, 0.1% SDS, 100mg/ml PMFS (phenylmethylsulfonyl fluoride), 1mg/ml aprotinin and 1%NP-40. The extracts were
subjected to 12% SDS-PAGE and transferred to a nitrocellulose membrane. The
membrane was blocked for 1h at room temperature in PBS containing 1% powdered
milk. Mouse anti-ODC monoclonal antibody was added at a dilution of 1/500 and
incubation was continued overnight at 4℃. The secondary antibody was
horseradish peroxidase-conjugated antimouse IgG antibody (Zhongshan Beijing
China). Antibody reactive bands were revealed using the ECL Western blotting
detection system (Santa Cruz CA). The content of each protein sample was
controlled by means of β-actin. For quantitation of bands, we used Nikon
digital camera and SmartView analysis software.
E. HPLC analysis of polyamine pools
Eca109 cell line was infected with rAd-ODC/Ex3as at the MOI of 50. After 48 hours,
cells were trypsined and washed with PBS twice. Intracellular polyamine were
extracted from cell pellets with 10% trichloroacetic acid, dansylated, and
measured by reverse phase HPLC as described previously (Fu et al, 1998).
F. TUNEL was used to analyze cell
apoptosis
Terminal
deoxynu-cleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick
end-labeling (TUNEL) was used to detect apoptotic cells. TUNEL was performed
with the kit according to the manufactureÕs instruction.
G. Statistical analysis
Statistical
analysis was performed using Statview J 5.0 software (SAS Institute Inc., San
Francisco, CA). A significant difference was defined as p<0.05.
III. Results
A. Inhibitory effects of
rAd-ODC/Ex3as on Eca109 cell line
There was dose-dependent growth inhibition in Eca109 cell line, which
reflected the transduction efficiency of the adenovirus to esophageal cancer
cell line. We chose 50 MOI of adenovirus to infect Eca109 cell line. Under
these conditions, rAd-ODC/Ex3as was more suppressive of growth than the control
rAd-GFP virus, while rAd-GFP had no obviously toxic effect on cells. We
examined the in vitro growth
inhibition of rAd-ODC/Ex3as in Eca109 cell line using cell growth curves as
described in ÒMaterial and MethodsÓ. Antisense ODC had an impact on the growth
of esophageal cancer cells. RAd-ODC/Ex3as in both of the cells inhibited their
proliferation by ~50% when compared with the control virus and no virus-treated
groups (Figure 1).
B. Effect of rAd-ODC/Ex3as on
expression of the ODC and polyamine pools in the cell lysate
The ODC
proteins produced from Eca109 cell line after infection with rAd-ODC/Ex3as were examined by Western immunoblot
analysis. The ODC expression in the cells infected with rAd-ODC/E3as
substantially more reduced than in the cells infected with rAd-GFP or no
virus-treated cells. The results analyzed by SmartView software showed that ODC
expression in Eca109 cell line infected with rAd-ODC/Ex3as accounted for 40% of that in cells treated
with rAd-GFP (Figure 2). HPLC also exhibited a decrease of
concentrations of the three polyamines: putrescine (put), spermidine (spd),spermine
(spm), especially of putrescine (Table 1).
C. TUNEL assay for apoptosis
To examine the mechanism by which rAd-ODC/Ex3as may
retard esophageal cancer cell growth in
vitro, we used TUNEL to detect the effect of the rAd-ODC/Ex3as on apoptotic
cells at 48 (Figure 3) and 72 hours
after infection. As shown in Table 2,
the rate of apoptosis in cells infected by rAd-ODC/Ex3as was significantly high
in comparison to infected by rAd-GFP or no virus-treated cells (p<0.05).
IV. 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 as compared to that of normal cells, especially in lung cancer cells (Carlisle
et al, 2002). At the same time, 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. More recently, the overexpression of ODC in NIH3T3 cells caused
transformation of these cells to a malignant phenotype, in essence qualifying
ODC as an oncogene (Auvinen et al, 1997). Inhibition of ODC by DFMO could
compromise cell growth and transformation (Metcalf et al, 1978). SchipperÕs
recent in vitro studies using
conformationally restricted polyamine analogues showed that these compounds
inhibited cell growth, probably by inducing antizyme-mediated degradation of
ODC (Schipper et al, 2000). In addition, Alm and colleageus showed in 2000 that
ODC was a well-defined target gene for c-myc and other oncogenes. Therefore, we
targeted the ODC by using an antisense gene delivery strategy with a
replication-deficient recombinant Ad vector. In the present study, we
demonstrated that rAd-ODC/Ex3as could inhibit esophageal cancer growth and lead
to the apoptosis of Eca109 cell lines.
MTT assay showed antisense ODC had an impact on the
growth of esophageal cancer cells. RAd-ODC/Ex3as in both of the cells inhibited
their proliferation by ~60% when compared with the control virus and no
virus-treated groups. At the same time, Western blotting showed the ODC
expression in the cells infected with rAd-ODC/E3as substantially more reduced
than in the cells infected with rAd-GFP or no virus-treated cells. On the other
hand a substantial decrease in ODC expression resulted in the reduction of
polyamine biosynthesis. In addition, the reduction of polyamines may contribute
to the marked suppression of cancer cell growth and tumor formation. Resent
studies also showed inhibiting mRNA expression of ODC can effectively inhibits
the growth of some cancer cells, such as breast, prostate, colorectal,
pancreatic cancer and bladder carcinoma cell (Weeks et al, 2000; Love et al,
2003; Subhi et al, 2004; Wolter et al, 2004). These findings suggest that
polyamine metabolism and ODC could be potential therapeutic targets in the
treatment of some cancer.
To examine the mechanism of antisense ODC inhibiting
the growth of esophageal cancer cells, we demonstrated rAd-ODC/Ex3as infection
can contribute significantly to cell apoptosis in comparison to rAd-GFP
infected or no virus-treated cells by TUNEL. In the last years, some studies
had demonstrated the inhibition of ODC could lead to induction of apoptosis of
some cancer cells (Feith et al, 2005; Seiler and Raul, 2005; Stanic et al, 2006).
So, our previous study indicated the induction of apoptosis was the mechanism
of antisense ODC inhibiting the growth of esophageal cancer cells.
In general, Our data suggest that adenoviral vector
mediated antisense ODC can lead to induction of apoptosis and inhibition of
growth of esophageal cancer cells in
vitro. The rAd-ODC/Ex3as could be a potential agent against esophageal
cancer, however, further in-depth in vivo
studies must be warranted.
References
Alm K, Berntsson PS, Kramer
DL, Porter CW, Oredsson SM (2000)
Treatment of cells with the polyamine analog N,N11-diethylnorspermine retards S
phase progression within one cell cycle. Eur
J Biochem 267, 4157-4164.
Auvinen M, Laine A,
Paasinen-Sohns A, Kangas A, Kangas L, Saksela O, Andersson LC, Holtta E (1997) Human ornithine decarboxylase-overproducing
NIH3T3 cells induce rapidly growing, highly vascularized tumors in nude mice. Cancer Res 57, 3016-3025.
Auvinen M, Paasinen A,
Andersson LG, Holtta E (1992)
Ornithine decarboxylase activity is critical for cell transformation. Nature 360, 355-358.
Carlisle DL, Devereux WL,
Hacker A, Woster PM, Casero RA Jr (2002)
Growth status significantly affects the response of human lung cancer cells to
antitumor polyamine-analogue exposure[J]. Clin
Cancer Res ,8, 2684-9.
Devens BH, Weeks RS, Burns
MR, Carlson CL, Brawer MK (2003)
Polyamine depletion therapy in prostate cancer. Prostate Cancer Prostatic Dis 3, 275-279.
Feith DJ, Bol DK, Carboni JM,
Lynch MJ, Sass-Kuhn S, Shoop PL, Shantz LM (2005) Induction of ornithine decarboxylase activity is a necessary
step for mitogen-activated protein kinase kinase-induced skin tumorigenesis. Cancer Res 65, 572-8.
Fu S, Zou X, Wang X, Liu X (1998) Determination of polyamine in
human prostate by high-performance liquid chromatography with fluorescence
detection. J Chromatogr B Biomed Sci
Appi 709, 297-300.
Glikman P, Vegh I, Pollina
MA, Mosto AH, Levy CM (1987)
Ornithine decarboxylase activity, prolactin blood levels, and estradiol and
progesterone receptors in human breast cancer. Cancer 60, 2237-2243.
Guan J, Fan M, Cao S (1996) Reversion of malignant phenotypes
of human lung squamous carcinoma cells by ornithine decarboxylase antisense
RNA. Zhonghua Zhong Liu Za Zhi 18,
81-83.
Love RR, Astrow SH, Cheeks
AM, Havighurst TC (2003) Orinithine
decarboxylase(ODC) as a prognostic factor in operable breast cancer. Breast Cancer Res Treat 79, 329-334.
Marton LJ, Pegg AE (1995) Polyamines as targets for
therapeutic intervention. Annu Rev
Pharmacol Toxicol 35, 55-91.
Meager A (1999) Gene Therapy Technologies,
Applications and Regulations: John Wiley & Sons, Ltd; 81
Metcalf B, Bey P, Danzin C,
Jung M, Casara P, Vevert J (1978)
Catalytic irreversible inhibition of mammalian ornithine decarboxylase by
substrate and product analogues. J Am
Chem Soc 100, 2551-2553.
Moshier JA, Dosescu J, Skunca
M, Luk GD (1993) Transformation of
NIH/3T3 cells by ornithine decarboxylase overexpression. Cancer Res 53, 2618-2622.
Moshier JA, Gilbert JD,
Skunca M, Dosescu J, Almodovar KM, Luk GD (1990)
Isolation and expression of a human ornithine decarboxylase gene. J Biol Chem 265, 4884-4892.
Pegg AE, Madhubala R, Kameji
T, Bergeron RJ (1988) Control of
ornithine decarboxylase activity in alpha-difluoromethylornithine-resistant
L1210 cells by polyamines and synthetic analogues. J Biol Chem 263, 11008-11014.
Pegg AE, Xiong H, Feith DJ,
Shantz LM (1998)
S-adenosylmethionine decarboxylase:structure, function and regulation by
polyamines. Biochem Soc Trans 26,
580-586.
Prevec L, Christie BS, Laurie
KE, Bailey MM, Graham FL, Rosenthal KL (1991)
Immune response to HIV-1 gag antigens induced by recombinant adenovirus vectors
in mice and rhesus macaque monkeys. J
Acquir Immune Defic Syndr 4, 568-576.
Schipper RG, Deli G, Deloyer
P, Lange WP, Schalken JA, Verhofstad AA (2000)
Antitumor activity of the polyamine analog N(1),N(11)-diethylnorspermine
against human prostate carcinoma cells. Prostate
44, 313-321.
Seiler N, Raul F (2005) Polyamines and apoptosis. J Cell Mol Med 9, 623-42.
Sistonen L, Holtta E,
Lehvaslaiho H, Lehtola L, Alitalo K (1989)
Activation of the neu tyrosine kinase induced the fos/jun transcription factor
complex, the glucose transporter and ornithine decarboxylase. J Cell Biol 109, 1911-1919.
Stanic I, Facchini A, Borz“
RM, Vitellozzi R, Stefanelli C, Goldring MB, Guarnieri C, Facchini A, Flamigni
F (2006) Polyamine depletion
inhibits apoptosis following blocking of survival pathways in human
chondrocytes stimulated by tumor necrosis factor-alpha. J Cell Physiol 206, 138-46.
Subhi AL, Tang B, Balsara BR,
Altomare DA, Testa JR, Cooper HS, Hoffman JP, Meropol NJ, Kruger WD (2004) Loss of methylthioadenosine
phosphorylase and elevated ornithine decarboxylase is common in pancreatic cancer[J]. Clin Cancer Res 10, 7290-6.
Umemoto S (1989) Antitumor effect of
alpha-difluoromethylornithine(DFMO) changes in ornithine decarboxylase(ODC)
activity and polyamine(PA) levels in human tumor transplanted into nude mice. Nippon Geka Gakkai Zasshi 90, 650-660.
Upp JR, Jr., Saydjari R,
Townsend CM, Jr., Singh P, Barranco SC, Thompson JC (1988) Polyamine levels and gastrin receptors in colon cancers. Ann Surg 207, 662-669.
Weeks RS, Vanderwerf SM,
Carlson CL, Burns MR, O'Day CL, Cai F, Devens BH, Webb HK (2000) Novel lysine-spermine conjugate inhibits polyamine transport
and inhibits cell growth when given with DFMO[J]. Exp Cell Res 261, 293-302.
Wei D, Tang Z, Chen S (2000) Construction of recombinant
adenovirus vector containing mIL-12 using the method of homogenous
recombination in Bacteria and its expression in vitro with high efficient. Chin J Biochem Mol Biol 16, 716-721.
Wolter F, Ulrich S, Stein J (2004) Molecular mechanisms of the
chemopreventive effects of resveratrol and its analogs in colorectal cancer:
key role of polyamines?[J]. J Nutr 134,
3219-22.
Zagaja GP, Shrivastav M,
Fleig MJ, Marton LJ, Rinker-Schaeffer CW, Dolan ME (1998) Effects of polyamine analogues on prostatic adenocarcinoma
cells in vitro and in vivo. Cancer
Chemother Pharmacol 41, 505-512.
Zhang Y, Liu X, Hu H, Geng Z,
Wang X, Zhang B (2003) Construction
of an antisense RNA recombinant adenovirus vector of the third extron in ODC
gene. Journal of Shandong University
(Health Sciences) 41, 371-374.
