Stable transfection of extrinsic Smac gene enhances apoptosis-inducing effects of chemotherapeutic drugs on gastric cancer cells

Abstract

To explore the feasibility of enhancing apoptosis-inducing effects of chemotherapeutic drugs on human gastric cancer cells by stable transfection of extrinsic Smac gene.
After Smac gene was transferred into gastric cancer cell line MKN-45, subclone cells were obtained by persistent G418 selection. Cellular Smac gene expression was determined by RT-PCR and Western blotting. After treatment with mitomycin (MMC) as an apoptotic inducer, in vitro cell growth activities were investigated by trypan blue-staining method and MTT colorimetry. Cell apoptosis and its rates were determined by electronic microscopy, annexin V-FITC and propidium iodide staining flow cytometry. Cellular caspase-3 protein expression and its activities were assayed by Western blotting and colorimetry.
When compared with MKN-45 cells, the selected subclone cell line MKN-45/Smac had significantly higher Smac mRNA (3.12+/-0.21 vs 0.82+/-0.14, t = 7.52, P<0.01) and protein levels (4.02+/-0.24 vs 0.98+/-0.11, t = 8.32, P<0.01). After treatment with 10 microg/mL MMC for 6-24 h, growth inhibition rate of MKN-45/Smac (15.8+/-1.2-54.8+/-2.9%) was significantly higher than that of MKN-45 (5.8+/-0.4- 24.0+/-1.5%, t = 6.42, P<0.01). Partial MKN-45/Smac cancer cells presented characteristic morphological changes of apoptosis under the electronic microscope with an apoptosis rate of 36.4+/-2.1%, which was significantly higher than that of MKN-45 (15.2+/-0.8%, t = 9.25, P<0.01). Compared with MKN-45, caspase-3 expression levels in MKN-45/Smac were improved significantly (3.39+/-0.42 vs 0.96+/-0.14, t = 8.63, P<0.01), while its activities were 3.25 times as many as those of MKN-45 (0.364+/-0.010 vs 0.112+/-0.007, t = 6.34, P<0.01).
Stable transfection of extrinsic Smac gene and its over-expression in gastric cancer cell line can significantly enhance cellular caspase-3 expression and activities, ameliorate apoptosis-inducing effects of mitomycin C on cancer cells, which is a novel strategy to improve chemotherapeutic effects on gastric cancer.

Full text

• GASTRIC CANCER •
Stable transfection of extrinsic Smac gene enhances apoptosis-inducing
effects of chemotherapeutic drugs on gastric cancer cells
Li-Duan Zheng, Qiang-Song Tong, Liang Wang, Jun Liu, Wei Qian
PO Box 2345, Beijing 100023, China World J Gastroenterol 2005;11(1):79-83
Fax: +86-10-85381893 World Journal of Gastroenterology ISSN 1007-9327
E-mail: wjg@wjgnet.com www.wjgnet.com © 2005 The WJG Press and Elsevier Inc. All rights reserved.
ELSEVIER
Li-Duan Zheng, Department of Pathology, Union Hospital of Tongji
Medical College, Huazhong University of Science and Technology,
Wuhan 430022, Hubei Province, China
Qiang-Song Tong, Liang Wang, Department of Surgery, Union
Hospital of Tongji Medical College, Huazhong University of Science
and Technology, Wuhan 430022, Hubei Province, China
Jun Liu, Wei Qian, Department of Gastroenterology, Union Hospital
of Tongji Medical College, Huazhong University of Science and
Technology, Wuhan 430022, Hubei Province, China
Correspondence to: Dr. Qiang-Song Tong, Department of Surgery,
Union Hospital of Tongji Medical College, Huazhong University of
Science and Technology, Wuhan 430022, Hubei Province,
China.
qs_tong@hotmail.com
Telephone: +86-27-85726129
Received: 2004-03-03 Accepted: 2004-04-05
Abstract
AIM: To explore the feasibility of enhancing apoptosis-
inducing effects of chemotherapeutic drugs on human gastric
cancer cells by stable transfection of extrinsic Smac gene.
METHODS:
After Smac gene was transferred into gastric
cancer cell line MKN-45, subclone cells were obtained by
persistent G
418
selection. Cellular Smac gene expression
was determined by RT-PCR and Western blotting. After
treatment with mitomycin (MMC) as an apoptotic inducer,
in vitro cell growth activities were investigated by trypan
blue-staining method and MTT colorimetry. Cell apoptosis
and its rates were determined by electronic microscopy,
annexin V-FITC and propidium iodide staining flow cytometry.
Cellular caspase-3 protein expression and its activities were
assayed by Western blotting and colorimetry.
RESULTS:
When compared with MKN-45 cells, the selected
subclone cell line MKN-45/Smac had significantly higher
Smac mRNA (3.12±0.21 vs 0.82±0.14, t = 7.52, P<0.01) and
protein levels (4.02±0.24 vs 0.98±0.11, t = 8.32, P<0.01).
After treatment with 10 µg/mL MMC for 6-24 h, growth
inhibition rate of MKN-45/Smac (15.8±1.2-54.8±2.9%)
was significantly higher than that of MKN-45 (5.8±0.4-
24.0±1.5%, t = 6.42, P<0.01). Partial MKN-45/Smac cancer
cells presented characteristic morphological changes of
apoptosis under the electronic microscope with an apoptosis
rate of 36.4±2.1%, which was significantly higher than that
of MKN-45 (15.2±0.8%, t = 9.25, P<0.01). Compared with
MKN-45, caspase-3 expression levels in MKN-45/Smac were
improved significantly (3.39±0.42 vs 0.96±0.14, t = 8.63,
P<0.01), while its activities were 3.25 times as many as those
of MKN-45 (0.364±0.010 vs 0.112±0.007, t = 6.34, P<0.01).
CONCLUSION:
Stable transfection of extrinsic Smac gene
and its over-expression in gastric cancer cell line can
significantly enhance cellular caspase-3 expression and
activities, ameliorate apoptosis-inducing effects of mitomycin
C on cancer cells, which is a novel strategy to improve
chemotherapeutic effects on gastric cancer.
© 2005 The WJG Press and Elsevier Inc. All rights reserved.
Key words: Gastric cancer; Mitomycin C; Extrinsic Smac
gene; Apoptosis; Transfection
Zheng LD, Tong QS, Wang L, Liu J, Qian W. Stable transfection
of extrinsic Smac gene enhances apoptosis-inducing effects
of chemotherapeutic drugs on gastric cancer cells. World J
Gastroenterol 2005; 11(1):79-83
http://www.wjgnet.com/1007-9327/11/79.asp
INTRODUCTION
Up to now, chemotherapy is still the important adjuvant treatment
for postoperative and advanced gastric cancer. Its research
focuses on how to reduce the side effects of chemotherapeutic
drugs and improve the sensitivities of tumor cells to them
[1-4]
. A
series of researches indicate chemotherapeutic drugs, radiotherapy
and thermotherapy could all induce apoptosis to various extents
by exerting their anti-tumor effects
[5-7]
. The second mitochondria-
derived activator of caspases (Smac) or DIABLO gene is a recently
identified and novel proapoptotic molecule, which is released from
mitochondria into the cytosol when cell apoptosis undergoes, to
enhance activities of caspase-3 through eliminating the functions
of inhibitors of apoptosis proteins (IAPs)
[8]
. It was reported that
the apoptosis-inducing effects of chemotherapeutic drugs on ovary
cancer and leukemia could be significantly enhanced by gene
transfer of Smac into cancer cells
[9,10]
. In this study, we
investigated the effects of stable transfection of extrinsic Smac
gene on apoptosis of gastric cancer cells induced by
chemotherapeutic drugs, to explore a novel strategy to improve
chemotherapeutic effects on gastric cancers.
MATERIALS AND METHODS
Genes and main reagents
Eukaryotic vector pcDNA3.1-Smac containing a full-length
human Smac cDNA (719 bp) was kindly provided by Professor
Xiao-Dong Wang (USA). Blank vector pcDNA3.1 was
preserved
by our central laboratory. Polyclonal rabbit anti-human Smac
was a kind gift from Professor Emma (Ireland). Monoclonal mouse
anti-human caspase-3 and its activity detection kit were purchased
from Santa Cruz Biotechnology and Clontech Company
respectively. Annexin V-FITC reagent kit was purchased from
Jingmei Biotech Company. Liposome GeneSHUTTLE-40 was
purchased from Q-bio Gene Limited Company. Newborn calf
serum, RPMI 1640, Trizol
TM
reagent kit and G
418
were all
purchased from Gibco Company. Dimethyl sulfoxide (DMSO)
and 3, [4,5- dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
(MTT) were both purchased from Clontech Company. Mitomycin
C (MMC) was a product from Japan Union Ferment Industry
Company, which was prepared into 1 g/L stock solution with
PBS, preserved at 4 and kept from light.
Cell culture
Human gastric cancer cell line MKN-45 was kindly provided by
Professor Ji-Hua Dong, Department of Virology, Union Hospital

of Tongji Medical College, Huazhong University of Science
and Technology, China. Cells were cultured in RPMI 1640 medium
supplemented with penicillin/streptomycin (100 units/mL and
100 µg/mL respectively) and 10% neonatal bovine serum at 37
in a humidified atmosphere of 50 mL/L CO
2
and passaged every
three days.
Gene transfection and selection of subclone cell line
The MKN-45 cells at exponential phases of growth were
inoculated into a 24-well plate. At the same time, non-
transfection, pcDNA3.1 and pcDNA3.1-Smac transfection
groups were designed for this experiment. Gene transfection was
conducted according to the protocol of liposome GeneSHUTTLE-
40 (G40). In brief, 4-6 µL G40 was mixed with 1 µg pcDNA3.1-Smac
or pcDNA3.1 and the mixture was incubated for 30 min, then
DNA/liposome complex solutions were added into the wells.
After incubated at 37
in a mosphere containing 5 mL/L CO
2
for 48 h, the cells were digested with 0.01%EDTA, then seeded
into a 6-well plate (35 mm in diameter) at 1×10
8
/L density, and
then selected with 600 mg/L G
418
for 2 wk. When most of the
non-transfected cells were dead, the concentration of G
418
was
decreased to 300 mg/L and maintained for another 2 wk. After
cellular clones were formed, subclones were chosen at random
and amplified. The subclone cells expressing Smac and neo genes
were named as MKN-45/Smac and MKN-45/neo respectively.
Detection of cellular Smac mRNA expression
Cellular Smac mRNA expression levels were assayed by reverse
transcription polymerase chain reaction (RT-PCR). Total cellular
RNA extraction was conducted with Trizol
TM
reagent kit,
according to the protocol of the manufacturer. The reverse
transcription was conducted at 42 for 30 min in 25 µL total
volume containing 2 µg template RNA, 1 µL 10 mmol/L dNTP,
20 U RNasin, 1 µL Oligo dT
18
, 200 U AMV, 5 µL 5×AMV
buffer. The PCR primers for Smac gene were designed by Primer
5.0 software: upstream 5’-CTGTGACGATTGGCTTTG-3’,
downstream 5’-GTGATT CCTGGCGGTTAT-3’, which were
synthesized by Shanghai GeneBase Company. The anticipated
product length was 425 bp. α-tubulin (310 bp) served as an
internal control. Touchdown PCR (TD PCR) was used for
amplification reaction. Amplified products were separated with
2% agarose electrophoresis. The brightness ratio between Smac
and α-tubulin was evaluated with a gel computer image system
(MGIAS-1000, Bio-Rad Company).
Detection of cellular Smac protein expression
Cellular Smac protein expression levels were assayed by
Western blotting. The extraction, quantification and separation
of proteins were conducted as previously described in Molecular
Cloning. Blots were incubated sequentially with 1% nonfat
dry milk, rabbit polyclonal anti-Smac antibody and goat radish
peroxidase-conjugated immunoglobulin G, and evaluated using
an ECL Western blotting kit. Smac protein band intensities
were determined densitometrically using the video imaging
CMIASWIN system.
Cell growth curve
MKN-45, MKN-45/neo and MKN-45/Smac cells were seeded at
2×10
8
/L density into the 24-well chamber slides (each group had
five wells). After cells were attached to the wells, 10 mg/L MMC
was added into each well. Then cells were incubated with RPMI
1640 at 37 in at mospbere containg 5 mL/L CO
2
for 0 h, 6 h,
12 h, 18 h and 24 h respectively. Then cells were digested by
0.125% trypsinase + 0.01% EDTA, stained with trypan blue and
counted under an inversion microscope. For each well, cell count
was repeated 3 times to draw the cell growth curve.
Detection of cellular chemotherapeutic sensitivity
MTT colorimetry was used. MKN-45, MKN-45/neo and MKN-
45/Smac cells were seeded at 3×10
4
/L density into 96-well
chamber slides. For each cell line, untreated control group,
0.1 mg/L MMC group, 1 mg/L MMC and 10 mg/L MMC group
were designed, with each group having five wells. After treating
with MMC for 24 h, 20 µL MTT (5 g/L) was added into each well
and cultured for another 4 h, the supernatant was discarded,
then 100 µL DMSO was added. When the crystals were dissolved,
the optical density A values of the slides were read on the enzyme-
labeled minireader II at the wavelength of 490 nm. Cell proliferation
inhibitory rate (%) = (1 - average A
490 nm
value of experimental
group/average A
490 nm
value of control group) ×100%. For each
detection, the total procedure was repeated 3 times.
Cell ultrastructure observation
After treated with 10 mg/L MMC for 24 h, three kinds of cancer
cells were collected, sequentially rinsed in PBS, fixed with 2.5%
glutaraldehyde for 30 min, and then washed with PBS. After
routine embedment and section, cells were observed under an
electron microscope.
Detection of cell apoptosis
After treatment with 10 mg/L MMC for 24 h, apoptotic ratios of
three kinds of cells were determinated by annexin V-FITC and
propidium iodide staining flow cytometry. Cells from the above
groups were collected, washed twice with cold PBS,
resuspended with 100 µL binding buffer (10 mmol/L HEPES,
140 mmol/L NaCl, 2.5 mmol/L CaCl
2
, pH 7.4) into 2 - 5×10
5
cells /mL
density, and incubated with annexin V-FITC at room temperature
for 10 min. After washing with binding buffer, the cells were
resuspended with 400 µL binding buffer containing 10 µL
propidium iodide (20 µg/mL), and incubated on ice for 15 min.
Apoptosis was analyzed by flow cytometry (BD Company,
USA) at a wavelength of 488 nm.
Detection of caspase-3 protein expression and activities
After treatment with 10 µg/mL MMC for 24 h, cellular caspase-3
protein expression levels were assayed by Western blotting
(as the same method above). The caspase-3 expression levels
were analyzed with the computer imaging system. 2×10
5
cells
from above groups were respectively collected, added into
50 µL cellular lysis buffer, then incubated on ice for 10 min.
After centrifugation (12 000 r/min) at 4 for 3 min, the
supernatant was collected and added sequentially into 50 µL
2×reaction buffer, 5 µL 1.0 mmol/L caspase-3 substrate DEVD-
pNA, and incubated at 37 for 1 h. After
transferred into 96
wells, the optical density A values of the slides were read on
the enzyme-labeled minireader II at the wavelength of 405 nm
(A
405 nm
), which stood for the relative activities of caspase-3.
Statistical analysis
Data was expressed as mean±SD and analyzed using SPSS10.0
statistical software.
RESULTS
Establishment of Smac stably-transfected subclone cells
All the untransfected MKN-45 cells were dead after G
418
(600 µg/mL) selection for 1 wk. The pcDNA3.1 and pcDNA3.1-
Smac transfected cells were continuously selected with G
418
for
4 wk, until magnificent clones could be observed. The clones
were respectively amplified. The subclone MKN-45/neo and
MKN-45/Smac cells were obtained, stably expressing neo and
Smac genes respectively.
80 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol January 7, 2005 Volume 11 Number 1

Zheng LD
et al. Smac enhances chemotherapeutic effects 81
Cellular Smac mRNA expression
As shown in Figure 1A, after electrophoresis of RT-PCR
products, Smac (425 bp) amplification bands could be observed
in MKN-45, MKN-45/neo and MKN-45/Smac cells. There were
only weak bands in MKN-45 and MKN-45/neo cells, and
brighter bands in MKN-45/Smac cells, amplified with the same
amount of RNA template. MGIAS-1000 gel computer image
system proved that the Smac/α-tubulin ratio in MKN-45/Smac
was 3.8 times, 3.7 times as many as that of MKN-45 (3.12±0.21 vs
0.82±0.14, t = 7.52, P<0.01), MKN-45/neo (3.12±0.21 vs 0.84±0.11,
t = 8.26, P<0.01) respectively. The brightness of Smac bands
between MKN-45 and MKN-45/neo had no significant
difference (P>0.05).
Figure 1
Cellular Smac expression detected by RT-PCR and
Western blotting. A: Cellular Smac mRNA expression detected
by RT-PCR. Lane 1: MKN-45 cells; lane 2: MKN-45/neo cells;
lane 3: MKN-45/Smac cells; lane 4: PCR marker (100 bp, 200 bp,
300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp).
B: Cellular Smac protein expression detected by Western
blotting. Lane 1: MKN-45/Smac cells; lane 2: MKN-45/neo
cells; lane 3: MKN-45 cells.
Figure 2 In vitro growth curves of gastric cancer cells after
treatment with 10 mg/L MMC.
Cellular Smac protein expression
As shown in Figure 1B, M
r
27 000 protein bands could be detected by
Western blotting in MKN-45, MKN-45/neo and MKN-45/Smac cells.
Computer image system indicated that Smac protein band brightness
of MKN-45/Smac cells was 4.1 times and 4.2 times as many as
that of MKN-45 (4.02±0.24 vs 0.98±0.11, t = 8.32, P<0.01) and
MKN-45/neo (4.02±0.24 vs 0.96±0.13, t = 8.84, P<0.01) respectively.
There was no significant difference in Smac protein band brightness
between MKN-45 and MKN-45/neo cells (P>0.05).
Cell growth curve
After treated with 10 mg/L MMC for 6-24 h, the in vitro growth
activities of MKN-45, MKN-45/neo and MKN-45/Smac cells
were all decreased. The growth inhibitory rates were 5.8±0.4-
24.0±1.5%, 7.1±0.6-26.8±1.2% and 15.8±1.2-54.8±2.9%
respectively. The differences in growth activities between
MKN-45 and MKN-45/neo cells were not significant (P>0.05),
while the growth activities of MKN-45/Smac cells were reduced
by 10.0±0.9-30.8±1.5% (t = 6.42, P<0.01), when compared with
those of MKN-45 cells (Figure 2).
Cellular sensitivity to MMC
After treated with 0.1 mg/L, 1 mg/L, 10 mg/L MMC, the growth
activities of MKN-45, MKN-45/neo and MKN-45/Smac cells
were reduced in a time- and dose-dependent manner. After
treatment with 10 mg/L MMC for 24 h, the inhibitory rate of
MKN-45 cells was 21.85±1.64%, while that of MKN-45/Smac
cells reached 43.71±3.12%, and the difference between these two
groups was significant (t = 7.56, P<0.01). The difference in growth
inhibitory rate of MMC between MKN-45 and MKN-45/neo
cells was not significant (P>0.05) (Figure 3).
Cellular morphological features
After treating with 10 mg/L MMC for 24 h, some cells had
characteristic morphological changes of apoptosis under an
electron microscope, such as cellular volume reduction, nuclear
shrinkage, chromatin congregation around the nuclear
membrane and integrity of cellular membranes (Figure 4).
Cell apoptosis detection
After treated with 10 mg/L MMC for 24 h, the apoptotic rates of
MKN-45 and MKN-45/neo cells were 15.2±0.8% and 16.5±1.1%
respectively, with no significant difference (P>0.05). The
apoptotic MKN-45/Smac cells increased and the apoptotic rate
reached 36.4±2.1%, with a significant difference compared to
that of MKN-45 (t = 9.25, P<0.01) and MKN-45/neo (t = 7.72,
P<0.01) (Figure 5).
Figure 3 Growth inhibitory effects of various concentrations
of MMC on gastric cancer cells.
Caspase-3 protein expression and activities
As shown in Figure 6, after treated with 10 mg/L MMC for
24 h, 17 ku (p17) and 20 ku (p20) protein bands, subunits of
caspase-3, could all be detected in MKN-45, MKN-45/neo and
MKN-45/Smac cells. Computer image system indicated that there
was a significantly higher p20 expression in MKN-45/Smac
compared to MKN-45 (3.39±0.42 vs 0.96±0.14, t = 8.63, P<0.01)
and MKN-45/neo (3.39±0.42 vs 0.94±0.11, t = 9.43, P<0.01). There
was no significant difference in p20 expression levels between
MKN-45 and MKN-45/neo cells (P>0.05). The A
405 nm
values of
MKN-45, MKN-45/neo and MKN-45/Smac cells were 0.055±0.008,
0.052±0.012 and 0.060±0.011 respectively, while differences
among them were not significant (P>0.05). After treatment
with 10 µg/mL MMC for 24 h, the A
405 nm
value reached 0.364±0.010
in MKN-45/Smac cells, which was 3.25 times as many as that in
MKN-45 cells (0.112±0.007, t = 6.34, P<0.01) (Figure 7).
1 2 3 4
Smac (425 bp)
α-tubulin (310 bp)
600 bp
500 bp
400 bp
300 bp
200 bp
100 bp
1 2 3
Smac 27 ku
A
B
6 12 18 24
12
10
8
6
4
2
0
MKN-45
MKN-45/neo
MKN-45/Smac
Treatment time (h)
Survival cells (10
4
/mL)
50
45
40
35
30
25
20
15
10
5
0
MKN-45
MKN-45/neo
MKN-45/Smac
0.1 µg/L 1 µg/L 10 µg/L
MMC MMC MMC
Cellular growth
inhibition rates (%)

Figure 6 Cellular caspase-3 expression detected by Western
blotting. Lane 1: MKN-45 cells untreated with MMC; lane 2:
MKN-45/neo cells untreated with MMC; lane 3: MKN-45/Smac
cells untreated with MMC; lane 4: MKN-45 cells treated with
10 mg/L MMC; lane 5: MKN-45/neo cells treated with 10 mg/L
MMC; lane 6: MKN-45/Smac cells treated with 10 mg/L MMC.
DISCUSSION
Gastric cancer is one of the most common malignant neoplasms
in alimentary tract. Because of the side effects of chemotherapy
on normal cells and drug resistance of tumor cells, it has been
a research focus on how to ameliorate the chemotherapeutic
effects on gastric cancer
[11]
. Recent researches indicate that
abnormal blockage of apoptosis is an important factor for the
occurrence and development of cancer
[12-14]
. To understand
apoptosis mechanisms is hopeful for improving the sensitivities
of tumor cells to chemotherapeutic drugs, and overcoming drug
resistance
[15,16]
.
The mechanisms of apoptosis are highly conserved in all
sorts of species, including a series of processes. Apoptosis
usually has three phases: initiation, effectors and execution
[17-19]
.
Figure 7
Caspase-3 activities detected in gastric cancer cells
before and after treatment with 10 mg/L MMC.
When external stimuli induce cell apoptosis by different pathways,
such as death receptor-mediated and stress-dependent pathways,
imbalance between activators and inhibitors of apoptosis occurs,
thus activating caspase family and changing mitochondrial outer
membrane permeability, finally resulting in cell apoptosis
[20]
.
Mitochondria are regarded as the key regulation element of cell
death and the target of many proapoptotic signal pathways
[21,22]
.
Some researches demonstrate that there are inhibitors of
apoptosis proteins (IAPs) in mammalian cells, which suppress
apoptosis by inhibiting procaspase activation and catalytic
activity of mature caspases
[23,24]
. IAPs, including MIHA
(mammalian IAP homolog A, or called XIAP), c-IAP1, c-IAP2
and survivin, could bind directly to caspase-3, caspase-7 and
caspase-9, and inhibit their activities, which are the downstream
effectors during cascades of caspase family. MIHA could also
suppress apoptosis induced by chemotherapeutic agents,
UV-irradiation and Bax
[25-27]
. It is suggested that cellular IAPs
accumulation is one of the reasons for cancer cells to escape
Figure 4 Cellular morphological changes of MKN-45/Smac before and after treatment with MMC under electron microscope,
×5 000. A: Cellular ultrastructure before treatment with MMC; B: Cellular chromatin congregating around nuclear membrane
after treatment with 10 mg/L MMC for 24 h; C: Cellular nuclear shrinking after treatment with 10 mg/L MMC for 24 h.
Figure 5 Apoptosis determination in gastric cancer cells by annexin V-FITC and propidium iodide staining flow cytometry. A:
MMC untreated control; B: MKN-45 cells treated with 10 mg/L MMC for 24 h; C: MKN-45/neo cells treated with 10 mg/L MMC
for 24 h; D: MKN-45/Smac cells treated with 1 0 mg/L MMC for 24 h.
ABC
10
0
10
1
10
2
10
3
10
4
10
4
10
3
10
2
10
1
10
0
A
Annexin Y FITC
PIPE
10
0
10
1
10
2
10
3
10
4
10
4
10
3
10
2
10
1
10
0
Annexin Y FITC
PIPE
10
0
10
1
10
2
10
3
10
4
10
4
10
3
10
2
10
1
10
0
Annexin Y FITC
PIPE
10
0
10
1
10
2
10
3
10
4
10
4
10
3
10
2
10
1
10
0
Annexin Y FITC
PIPE
BCD
1 2 3 4 5 6
p32
p20
p17
MKN-45 MKN-45/neo MKN-45/Smac
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
A
405nm
Value
Untreated control
Treated with MMC
82 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol January 7, 2005 Volume 11 Number 1

the killing effects of anti-cancer agents
[28]
.
Smac or DIABLO gene is a proapoptotic molecule, which is
released from mitochondria into the cytosol, along with
cytochrome C (cyt-C) during apoptosis. As an important apoptotic
modulator, Smac functions as eliminating the caspase-inhibitory
properties of IAPs
[29]
. Some researches have found that Smac
could promote apoptosis via two pathways, which are dependent
on the interaction between Smac and IAPs
[30]
. One hydrolyzes
caspase-3 protein, and the other enhances the catalytic activity
of mature caspase-3. McNeish et al.
[9]
constructed the adenoviral
vector for Smac gene, and transfected it into ovarian cancer
cells. They found that apoptotic cancer cells increased remarkably,
and cell apoptosis mediated by Smac was not dependent on
cyt-C and Bcl-2, but realized through Caspase-9. Jia et al.
[10]
stably transfected full-length (FL) and mature (MT) Smac genes
into K562 and CEM leukemic cell lines, and they concluded that
both FL and MT Smac transfection could promote the sensitivity
of leukemic cells to UV light, and activate cellular caspase-9 and
caspase-3.
In this study, an extrinsic Smac gene was transfected into
gastric cancer cells which induced its over-expression. We found
that Smac overexpression could enhance the apoptosis-
inducing effects of MMC, by electronic microscopy and annexin
V-FITC and propidium iodide staining flow cytometry. These
results are consistent with those recently reported by Guo et al.
[31]
(2002) and Fulda et al.
[32]
(2002), in which Smac could enhance
apoptosis in leukemia and malignant neuroglioma cells induced
by chemistry or immunology in vivo. Western blotting and
colorimetry were sequentially used to assay the cellular caspase-
3 protein expression and its activities, and it was found that
stable transfection of an extrinsic Smac gene could increase the
cellular activity levels of caspase-3 after treating with MMC,
which accords with the functional mechanisms of Smac. These
results provide a novel strategy to improve chemotherapeutic
sensitivity in gastric caner patients and reduce their side effects,
thus establishing a basis for further exploring the roles of Smac
gene in apoptosis regulation of gastric cancer.
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Assistant Editor Guo SY
Edited by Zhang JZ and Wang XL
Zheng LD
et al. Smac enhances chemotherapeutic effects 83