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Introduction  

Although studies have confirmed that the occurrence 
and development of cancers have the involvement of a 
complex cancer-related gene network, the changes in 
the functions of these genes share the consequence that 
cells develop uncontrolled proliferation and disruption 
of apoptosis. Survivin is a new member of inhibitor of 
apoptosis protein (IAP), has high expression in cancers 
and can exert anti-apoptotic effect (Ambrosini et al., 
1997; Mesri et al., 2010). Survivin is a newly identified 
anti-apoptotic molecule and its over-expression is 
frequently observed in cancers. Thus, the development 
and application of survivin signaling pathway are 
beneficial for the early diagnosis of cancers and the 
determination of prognosis of cancer patients and also 
provide new targets for the biotherapy of cancers. Anti-
sense oligonucleotide targeting survivin (ASODN) can 
induce the apoptosis of hepatic cancer cells and inhibit 
their growth (Grossman et al., 2006; Altieri and Marchi-

sio, 2006; Tanabe et al., 2006). In the present study, 
survivin ASODN was transfected into human gastric 
cancer cells and the anti-tumor effects were then obser-
ved. Our findings may provide theoretical and experi-
mental bases for the treatment of gastric cancer.  

 

Materials and Methods 

Reagent and cell lines 

RPMI 1640 (GIBCO, USA), Annexin V FITC/PI (Jing-
mei, China), protease K (Roche, Swiss), acridine orange, 
ethidium bromide (Sigma, USA), Bcl-2 immunohisto-
chemical detection kit (Boster, China), survivin ASODN 
and unrelated oligonucleotide (N-ODN) (Full phospho-
rothioate; Shanghai Sangong, China), MTT and SDS 
(Shanghai Sangong, China) were used in the present 
study. The sequence of ASODN was 5’-
CCCAGCCTTCCAGCTCC TTG-3’and that of N-ODN 

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Effect of antisense oligodeoxynucleotide targeting survivin on 
growth of human gastric cancer cells 
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Our study investigated the effects of antisense oligodeoxynucleotide targeting 
survivin on human gastric cancer cells. Human gastric cancer cells were 
incubated with antisense oligodeoxynucleotide targeting survivin for pre-
designed durations, and then the cell growth was observed under light and 
electronic microscopes. Electrophoresis of fractured DNA fragments was 
performed to detect the DNA distribution and telomere repeat amplification 
protocol (TRAP) was used for the detection of telomerase activity. Antisense 
oligodeoxynucleotide targeting survivin could induce the apoptosis of human 
gastric cancer cells which were characterized by plasma membrane blistering, 
chromatin condensation, nuclear fragmentation, and formation of apoptotic 
bodies. Electrophoresis showed characteristic DNA ladder. Flow cytometry 
revealed hypo-diploid apoptosis peak before G1 phase and the telomerase 
activity was significantly inhibited. These results demonstrated antisense 
oligodeoxynucleotide targeting survivin can induce the apoptosis of gastric 
cancer cells to inhibit their proliferation.  

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Received:  16 January 2013 
Accepted:  18 January 2013 
Available Online:  28 January 2013  
 

DOI: 10.3329/bjp.v8i1.13337 
 
 
 
 
 
 
 

Cite this article: 
Xu L. Effect of antisense oligodeox-
ynucleotide targeting survivin on 
growth of human gastric cancer cells. 
Bangladesh J Pharmacol. 2013; 8: 73-
77.p 

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ZRUN�LQ�WKH�PDQQHU�VSHFLILHG�E\�WKH�DXWKRU�RU�OLFHQVRU�  



 

 

was 5’-GCCACTCCTCGAC TCTCGTG-3’. Both sequen-
ces have no homology with corresponding genes of 
humans.  

Human gastric cancer cell line (SGC-7901) was purchas-
ed from the Cell Bank of Shanghai Institute of Cell 
Biology in Chinese Academy of Science. Cells in the 
logarithmic growth phase were used in the experi-
ments. In the ASODN groups, cells were treated with 
ASODN at different concentrations (1 × 10-5 mol/L, 2 × 
10-5 mol/L, 3 × 10-5 mol/L, 4 × 10-5 mol/L and 5 × 10-5 

mol/L), and cells were incubated with N-ODN in the N
-ODN group. In the control group, cells were treated 
with normal saline. Incubation was performed at 37°C 
in an environment with 5%CO2.  

Effect of ASODN on growth of SGC-7901 cells 

SGC-7901 cells were seeded into 96-well plates at a den-
sity of 2 × 107/ (100 μL/well), and three wells in each 
group. The final concentrations of ASODN were 1 × 10-5 

mol/L, 2 × 10-5 mol/L, 3 × 10-5 mol/L, 4 × 10-5  mol/L 
and 5 × 10-5 mol/L, and that of N-ODN was 5 × 10-5 

mol/L. Incubation was done for 24, 48, 72 and 96 hours. 
At 4 hours before the detection, 5 g/L MMT (20 μL) was 
added to each well followed by incubation for 4 hours. 
SDS (100 μL) was added to stop the reaction. After 
incubation overnight at 37°C, the absorbance (D) was 
measured at 570 nm with a microplate reader.  

Morphology of SGC-7901 cells before and after ASODN 
treatment 

During the treatment with ASODN, the cell growth and 
morphology were observed under an inverted micros-
cope. In addition, cells which were treatment with 
ASODN for 3 days were collected, and the morphology 
of apoptotic cells was evaluated.   

Detection of mRNA expression of survivin in SGC-7901 
cells 

After treatment with 5 × 10-5 mol/L ASODN for 24 and 
48 hours, cells were harvested and total RNA was 
extracted with Trizol followed by PCR. The products 
were subjected to 1.7% agarose gel electrophoresis, and 
representative photographs were captured.  

DNA extraction and electrophoresis 

Cells at 1 × 109 /L were treated with 5 × 10-5 mol/L 
ASODN for 3 days and then washed with buffer by 
centrifugation. These cells were fixed in ethanol. After 
addition of lysis buffer and protease K, the cells were 
incubation at 37°C for 2 hours. Extraction of DNA was 
done with phenol, chloroform and isoamyl alcohol 
sequentially. The 1/10 volume of 3 mol/L sodium 
acetate and 2 volume of cold ethanol were added to the 
supernatant followed by incubation at -20°C overnight. 
Then, centrifugation was performed at -10°C for 10 min 
at 800 × g and the sediment was collected. After addi-
tion of TE buffer, 5 μL of DNA was subjected to 20 g/L 

agarose gel electrophoresis at 50 V for 3 hours. Bands 
were observed under a UV lamp, and representative 
photographs were obtained.  

Detection of apoptosis of SGC-7901 after ASODN 
treatment by flow cytometry 

Gastric cancer cells at 1 × 109 /L were treated with 5 × 
10-5 mol/L ASODN for 3 days and then washed in 
buffer twice. These cells were fixed in absolute ethanol 
for 24 hours followed by washing. After incubation 
with Tris-HCl buffer (pH= 7.4) containing 10 g/L 

RNAase for 10 min, cells underwent DNA staining with 
propidium iodide and then subjected to flow cyto-
metry.  

Electric microscopy 

Gastric cancer cells at 1 × 109 /L were incubated with 5 
× 10-5 mol/L ASODN for 3 hours and then washed in 
PBS twice. These cells were fixed in 2.5% glutar-
aldehyde for 24 hours and then washed in 0.2 mol/L 
PBS thrice (15 min for each). Cells were subsequently 
fixed in 1% osmic acid for 1 hours followed by washing. 
After dehydration in acetone, cells were embedded in 
Epon 812, and ultrathin sections were obtained with the 
LKP-2088 microtome. Following staining with uranyl 
acetate-citrate aluminum, cells were observed under a 
transmission electron microscope, and representative 
photographs were captured.  

Effect of survivin ASODN on activity of telomerase in 
SGC-7901 cells 

Gastric cancer cells at 1 × 109 /L were treated with 5 × 
10-5 mol/L ASODN for 3 days and washed with buffer 
once. After centrifugation at 4°C for 1 min at 800 × g, 
the sediment washed with 150 μL of washing buffer by 
centrifugation for 1 min. The sediment was re-suspen-
ded in 50 μL of lysis buffer, and this solution was then 
kept on ice for 30 min. After centrifugation at 4°C for 20 
min at 1000 × g, the supernatant (2 μL) was used as the 
template in TRAP. Then, 45 μL of reaction mixture and 
2 μL of processed sample were mixed in a reaction tube. 
After addition of 30 μL of liquid paraffin, the solution 
was incubated at 25°C for 30 min. PCR was performed 
as follows: 94°C for 120 sec, 94°C for 30 sec, 48°C for 30 
sec, 72°C for 90 sec and 72°C for 300 sec. A total of 35 
cycles of PCR were done. Then, hybridization solution 
and 25 μL of products from PCR were added to each 
well followed by incubation at 37°C for 60 min. 
Negative control and blank control were also included. 
After addition of chromogenic agent, incubation was 
done at 37°C in dark for 10 min. Then, stop solution 
was added to stop the reaction, and the absorbance was 
measured at 450 nm. 

Statistical analysis 

Data were expressed as mean ± SD, and comparisons 
were done with analysis of variance. A value of p<0.05 
was considered statistically significant.  

74 Bangladesh J Pharmacol 2013; 8: 73-77 



 

 

 

Results 

Results showed cells in the N-ODN group and control 
group were positive for mRNA expression of survivin. 
However, after ASODN treatment for 24  and 48 hours, 
the mRNA expression of survivin was markedly 
inhibited (Figure 1).  

After treatment with ASODN at different 
concentrations, the cell growth was inhibited to 
different extents, which was in a time and concentration 
dependent manner. In the N-ODN group, the growth of 
gastric cancer cells remained unchanged (Table I).  

Under invert microscope, cells in the N-ODN group 
and control group were epithelium-like and presented 
with adherent growth. These cells had clear borderline, 
closely arranged cellular structure and active growth. In 
the ASODN group, cells became round and suspended, 
the intercellular space increased, the cell growth 
reduced, and the cellular granules also increased. With 
the increase in ASODN concentration, the cellular 
debris increased. After treatment with 5 × 10-5 mol/L 
ASODN for 3 days, cells were observed under an invert 
microscope. The nucleus was dark-stained and crescent, 
and aggregated besides the nuclear membrane. The 
membrane protruded in a blister-like manner. The 
shedding of these blisters formed apoptotic bodies 
containing nucleosome fragments. In the N-ODN group 
and control group, the cell morphology remained 

unchanged (Figure 2) . 

Under electric microscope, the SGC-7901 cells presented 
with chromatin condensation, and chromatins mainly 
aggregated besides the nuclear membrane in a block-
like or crescent manner. Condensation of cytoplasm 
and cellular debris were observed, accompanied by a 
lot of apoptotic bodies with different sizes. Under a 
transmission electric microscope, the electron density of 
cytoplasm and nucleus increased in cells undergoing 
ASODN treatment, and the cell volume reduced. The 
cell membrane shrunk or curled to form bud 
protrusions (budding). In addition, the cytoplasm 
condensed, the density of cytoplasm and organelles 
increased, and the organelles were oppressed. The 
nuclear chromatins became pyknotic and dense, and 
aggregated besides the nuclear membrane (chromatin 
margination). The nucleus was also pyknotic and 
became smaller. In the control group, the cell volume 
was relatively large, the nuclear and cell membranes 
were integrate, the chromatins were light, organelles 
arranged in a loose manner, and the nucleus was large 
and had normal structure (Figure 3). 

In apoptotic cells, the endogenous endonucleases were 
activated, and DNA was cut at the junction between 
nucleosomes forming 180-200 bp oligonucleotide 
fragments. Agarose gel electrophoresis showed the 
typical DNA ladder. After treatment with 5 × 10-5  mol/
L ASODN, agarose gel electrophoresis and ethidium 
bromide staining showed obvious DNA ladder which 
was absent in the control group. 

Flow cytometry showed the apoptotic peak of 
hypodiploid before G1 phase in cells treated with 5 × 10
-5 mol/L ASODN for 3 days. In the control group, 
apoptotic peak was absent. (Figure 4). After ASODN 
treatment, the apoptosis rate was 25.0 ± 0.5% which was 
markedly higher than that in the control group (7.0 ± 
0.2%; p<0.01). Moreover, the apoptosis rate increased 
over the increase in ASODN concentration, and the 
ASODN concentration was positively related to the 
apoptosis rate (r= 0.820; Table II).  

Gastric cancer cells were treated with 5 × 10-5 mol/L 

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Figure 1: Survivin mRNA expression in gastric cancer cells. 1: N
-ODN group; 2: Control group; 3: ASODN group [24 hours]; 4: 
ASODN group [48 hours]; M: Marker 

Figure 2: Morphology of cells in different groups under light microscope (~ 100×). (A) Control group; (B) ASODN group (5 × 10-5 

mol/L) 

 Bangladesh J Pharmacol 2013; 8: 73-77                                                                                    75 



 

 

ASODN for 3 days, and results showed the telomerase 
activity was significantly inhibited. The absorbance in 
the ASODN group, control group and N-ODN group 
was 0.007, 0.15 and 0.14, respectively, showing marked 
difference between ASODN group and control group/
N-ODN group. This suggests that ASODN can 
significantly inhibit the activity of telomerase in SGC-
7901 cells (p<0.01).  

 

Discussion 

To induce the apoptosis of cancer cells has been a 
strategy in the treatment of cancers. The survivin has 
specific anti-apoptotic effect and is highly expressed in 
cancers. Thus, survivin has been a target in the 
treatment of cancers. The activation of caspase cascade 
is a major mechanism underlying the cell apoptosis. 
Survivin can directly act on caspase-3 and -7 to block 
the procession of apoptosis (Nagata, 2005; Thompson, 
2006; Graeber et al., 2005). Antisense technique can be 
used to inhibit the transcription and/or translation of 
target genes. In the present study, ASODN and N-ODN 
were used to treat gastric cancer cells. Our results 
showed ASODN could significantly inhibit the growth 
of gastric cancer cells, induce their apoptosis and inhibit 
their telomerase activity. Thus, survivin can be used as 
a target in the treatment of cancers. Our findings 
provide a new target for the development of anti-tumor 

drugs and present with a novel way for the gene 
therapy of cancers (Shinozawa et al., 2005; Tsuji et al., 
2011; Chang et al., 2009; Miao et al., 2010; Tarnawski et 
al., 2005; Idenoue et al., 2011). 

Cell apoptosis has specific morphological and bioche-
mical characteristics. Morphologically, apoptotic cells 
have small volume, cytoplasm is pyknotic or has 
blisters, nucleus is pyknotic and apoptotic bodies can be 
observed. Molecularly, apoptotic cells present with 
degradation of DNA in the nucleus, which is attributed 
to the Ca2+ and Mg2+ dependent activation of endonu-
cleases. After leavage by endonucleases, DNA is frag-
mented. The shortest DNA fragment is 180-200 bp in 

76 Bangladesh J Pharmacol 2013; 8: 73-77 

Figure 3: Morphology of SGC-7901 cells in different groups under transmission electric microscope. (A) Control; (B) ASODN 
group 

Figure 4: Detection of apoptotic cells by flow cytometry. An apoptotic peak was found before G1 phase. (A) control; (B) ASODN 
group 

Table II 

Apoptosis rate of gastric cancer cells in different 
groups 

Group Apoptosis rate (%) 

Control 7.0 ± 0.2 

Lipofectin 7.0 ± 0.2 

N-ODN 6.9 ± 0.2 

ASODN (1×10-5 mol/L) 10.4 ± 0.4 

ASODN (3×10-5 mol/L)  19.4 ± 0.5a 

ASODN (5×10-5 mol/L)  25.0 ± 0.5a 
ap<0.01 vs control group  

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length, and the remaining DNA fragment is multiples 
of 180-200 bp in length (oligomer). Agarose gel 
electrophoresis can reveal characteristic DNA ladder. In 
the apoptotic cells, the cellular DNA is degraded, and 
flow cytometry can present with apoptotic peak of 
hypodiploid. In the present study, our results showed 
ASODN could induce the apoptosis of gastric cancer 
cells. The development and use of survivin signaling 
pathway are beneficial for the early diagnosis of cancers 
and the determination of prognosis of cancer patients. 
Our findings provide a new target for the biotherapy of 
cancers. 

This study shows that ASODN can induce the apopto-
sis of gastric cancer cells to inhibit their growth and 
suppress their telomerase activity. This suggests that 
antisense oligodeoxynucleotide targeting survivin can 
effective inhibit the growth of cancer cells, which 
provide experimental basis for the gene therapy of 
cancers targeting survivin. In a majority of normal cells, 
the telomerase is inactivated. Thus, therapy targeting 
survivin may avoid the activation of telomerase, which 
reduce the side effects and increase the therapeutic 
specificity. 

  

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 Bangladesh J Pharmacol 2013; 8: 73-77                                                                                    77 

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Lantao Xu (Principal contact) 
e-mail: xulantao@yeah.net 


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