Download Survivin Status Affects Prognosis and Chemosensitivity in Epithelial

ORIGINAL STUDY
Survivin Status Affects Prognosis and Chemosensitivity
in Epithelial Ovarian Cancer
Lifeng Chen, MD,* Lizhi Liang, MD,Þ Xiaojian Yan, MD,* Naihua Liu, PhD,þ Lihua Gong, MD,*
Shishi Pan, MD,* Feng Lin, MD,* Qian Zhang, MD,* Hongqin Zhao, MD,* and Feiyun Zheng, MD*
Objective: The objective of this study was to explore the clinical significance of survivin
expression in epithelial ovarian cancer (EOC) and the effect of survivin small hairpin RNA
(shRNA) on survivin expression, apoptosis, and chemosensitivity in the human ovarian
cancer cell line OVCAR3.
Methods: A retrospective review of 90 consecutive EOC patients with a median follow-up
time of 51 months was conducted. Survivin expression was examined by immunohistochemistry. OVCAR3 cells were transfected in vitro with survivin shRNA. Survivin mRNA
expression levels were detected using reverse transcriptionYpolymerase chain reaction. Flow
cytometry was applied to determine survivin protein expression levels and cell apoptotic
rates. The MTT method was used to examine the effects of survivin shRNA on chemosensitivity in OVCAR3 cells.
Results: Positive cytoplasmic expression of survivin was associated with advanced International Federation of Gynecology and Obstetrics (FIGO) stage, nonmucinous type, high
grade, and recurrence. Positive survivin expression was also associated with platinum resistance (r = 0.306, P = 0.003). Statistical results indicated that FIGO stage (hazard rate =
1.649, P = 0.047) and cytoplasmic expression of survivin (hazard rate = 1.734, P = 0.010)
were independent prognostic factors. Survivin mRNA and protein levels were lower in
OVCAR3S (ovarian cancer cells transfected with a survivin recombinant vector) cells at 24
hours after transfection as compared with controls. The flow cytometric analysis revealed
that survivin shRNA induced accumulation of cells in the G0/Gl phase, with a decrease in
G2/M phase cells following 24 hours of culture as compared with a nontransfected group
(P G 0.01). Furthermore, survivin shRNA increased the sensitivity of OVCAR3 cells to
paclitaxel 15-fold (P G 0.05), whereas it had no significant effect on cisplatin (P 9 0.05).
Conclusions: In addition to FIGO stage, cytoplasmic survivin protein expression is an
independent molecular marker for predicting EOC prognosis. Sequence-specific shRNA
targeting survivin can effectively suppress survivin expression, enhance apoptosis, and
increase the sensitivity of ovarian cancer cells to paclitaxel but not to cisplatin.
Key Words: ovarian neoplasms, survivin, RNA interference, short hairpin RNA
Abbreviations: EOC, epithelial ovarian cancer, IC50, 50% cell growth inhibition, shRNA,
small hairpin RNA, OVCAR3WT, wild-type human ovarian cancer cells, OVCAR3S, ovarian
cancer cells transfected with a survivin recombinant vector, OVCAR3L, ovarian cancer cells
transfected with a liposome, OVCAR3B, ovarian cancer cells transfected with a blank vector
*Department of Gynecology, the First Affiliated Hospital of
Wenzhou Medical College, Wenzhou, Zhejiang; †Department of
Gynecology, Cancer Center, Sun Yat-Sen University, Guangzhou,
Guangdong; and ‡Department of Pharmacy, Wenzhou Medical
College, Wenzhou, Zhejiang, China.
Copyright * 2013 by IGCS and ESGO
ISSN: 1048-891X
DOI: 10.1097/IGC.0b013e31827ad2b8
256
Address correspondence and reprint requests to Xiaojian Yan, MD,
Department of Gynecology, the First Affiliated Hospital of
Wenzhou Medical College, Wenzhou, Zhejiang, 325000, China.
E-mail: [email protected].
The main financial support was from Zhejiang Provincial Education
Department (grant Y201016398) and from Guangdong Natural
Science Foundation (grant 2003-31746).
The authors declare no conflicts of interest.
International Journal of Gynecological Cancer
& Volume 23, Number 2, February 2013
Copyright © 2013 by IGCS and ESGO. Unauthorized reproduction of this article is prohibited.
International Journal of Gynecological Cancer
& Volume 23, Number 2, February 2013
Survivin Status in Epithelial
Ovarian Cancer
Received July 15, 2012, and in revised form October 23, 2012.
Accepted for publication October 24, 2013.
(Int J Gynecol Cancer 2013;23: 256Y263)
ovarian cancer (EOC) is the leading cause of death
E pithelial
from gynecologic malignancy. Over the past 3 decades,
important advances in EOC treatment have been made. However, the majority of patients succumb to the disease. The 5-year
survival rate is still about 30%.1 Despite the activity of initial
chemotherapy with a combination platinum-based regimen in
advanced ovarian cancer, most patients with advanced disease
will ultimately have a relapse because of the development of
drug resistance. Second- or third-line chemotherapeutic agents
have been shown to have a relatively high response rate in shortterm therapy, but the long-term effects are poor because of
acquired drug resistance. Chemotherapeutic agents also have
severe toxicities. It is necessary to identify new methods or
novel agents for the treatment of ovarian cancer. Survivin is a
member of the IAP (inhibitors of apoptosis protein) family. It is
undetectable in normal adult tissues, but highly expressed in
several types of cancer.2,3 Survivin expression in the primary
tumor has been associated with a worse prognosis, resistance
to anticancer agents,4,5 and metastasis.6 Furthermore, attenuation of survivin expression can induce apoptosis and sensitize cancer cells to conventional chemotherapeutics. The
inhibition of apoptosis by survivin is associated with the
mitotic spindle assembly checkpoint.7,8 Therefore, survivin
may be a potential target for EOC therapy. However, the
prognostic significance of survivin in ovarian cancer is still
unknown; more data are needed to confirm its value.9Y12
A variety of survivin antagonists, such as antisense oligonucleotides, ribozymes, small interfering RNAs (siRNAs),13
dominant negative mutants, and cyclin-dependent kinase
inhibitors, have been tested.14 Gene-specific inhibitors of survivin have become a new trend as novel anticancer targets
because of their wide spectrum and minimum toxicity. In this
study, we explored whether survivin plays an important role in
ovarian cancer and whether silencing survivin could enhance
therapeutic efficacy. We investigated the expression of survivin
in 90 Chinese female patients with untreated EOC and then
determined clinical pathological parameters and the impact on
chemosensitivity, recurrence, and prognosis. We used an
in vitro RNA interference technique to explore the effects of
survivin small hairpin RNA (shRNA) on survivin expression,
apoptosis, and chemosensitivity in human ovarian cancer cell
line OVCAR3 to broaden the list of available therapeutic
modalities in the treatment of ovarian cancer. This would
allow us to screen out high-risk cases and modify our treatment strategy to improve chemotherapeutic efficacy.
MATERIALS AND METHODS
Clinical Data and Immunohistochemistry
A retrospective review of 90 consecutive EOC patients
from January 1993 to December 2000 at the Cancer Center of
Sun Yat-Sen University was conducted. The study was approved by the Ethical Review Committee of Sun Yat-Sen
University Cancer Center. Inclusion criteria were as follows:
(1) no patients were treated with preoperative chemotherapy;
(2) the first surgeries were performed in this cancer center; (3)
the pathological results were diagnosed as EOC; (4) the adjuvant therapy administered after surgery included platinumbased chemotherapy; (5) follow-up data were available.
All cases were followed up to November 2004. The median
follow-up time was 51 months. The median age was 50 years
(range, 22Y75 years). Platinum sensitivity refers to the diseasefree or treatment-free interval more than 6 months after primary
platinum treatment. Platinum resistance refers to platinumresistant recurrence (tumor progression within 6 months of
completion of platinum-based therapy), persistent disease (clinically measurable disease with best response as stable disease
at the completion of planned first-line therapy), and refractory
disease (progressive disease during platinum therapy). A
rabbit polyclonal antisurvivin antibody was used in the immunohistochemical analysis. Two investigators independently
scored all of the samples. In discordant cases, a third investigator scored the samples, and the final intensity score was
determined by majority vote. Cytoplasmic staining for survivin was scored as positive when a staining reaction was
noted in more than 10% of the tumor cells and as negative
when it was noted in less than 10% of the tumor cells, as
described previously.15
Statistical Analysis
Continuous variables were reported as the mean (SD) or
the median and first and third quartiles. Categorical variables
were described using proportions. Group differences were
determined using a t test, analysis of variance, Kruskal-Wallis
test, or W2 test as appropriate. The survival of ovarian cancer
patients with positive and negative survivin expression was
compared by Kaplan-Meier method and log-rank test. A
univariate analysis by W2 test was applied to assess prognostic
significance of the different variables. To compare the effect
of survivin expression, we determined the associations of
multiple variables with prognosis using Cox multivariate regression models. The proportional hazards assumption for all
confounding variables was tested visually using log-negativelog survival plots. All statistical analyses were performed
using the Statistical Package for the Social Sciences software
program version 13.0 (SPSS, Inc, Chicago, IL), and P G 0.05
was accepted as statistically significant.
Reagents and Instruments
The human ovarian cancer cell line OVCAR3 was
obtained from China Medical University, Institute of Cell Biology. The survivin shRNA expression vector Mu6/survivin
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Copyright © 2013 by IGCS and ESGO. Unauthorized reproduction of this article is prohibited.
257
International Journal of Gynecological Cancer
Chen et al
& Volume 23, Number 2, February 2013
plasmids were acquired from Prof Luo CQ of Guangdong
Medical College. The pEGFPC2 plasmids were purchased from
Clonetech (Palo Alto, CA). The Plasmid Extraction Kit was
purchased from Qiagen (Valencia, CA). Lipofectamine TM2000
was purchased from Invitrogen (Carlsbad, CA). MTT was
purchased from Sigma (St Louis, MO). A reagent kit was
purchased from Promega (Madison, WI). The rabbit polyclonal antibody against human survivin was purchased from
Santa Cruz (Santa Cruz, CA).
twice, fixed in 1 mL of 0.1% formaldehyde for 30 minutes, and
then incubated with 0.5 Kg survivin rabbit antiYhuman polyclonal antibody for 30 minutes after the cell membranes were
broken. Irrelevant isotype-matched mAb was used as a negative
control. Goat antiYrabbit immunoglobulin GYfluorescein isothiocyanate was then added at a final dose of 5 Kg, and the
mixture was incubated for 30 minutes at room temperature away
from light. The cells were then analyzed using FCM (Beckman
coulter Epics ELITE).
Cells and Culture Conditions
Cell Cycle and Apoptosis
Human ovarian cancer cell line OVCAR3 was grown in
Dulbecco modified Eagle medium (GIBCO) supplemented
with 10% fetal bovine serum, 100 U/mL penicillin, and 100
Kg/mL streptomycin and incubated at 37-C in a humidified
chamber supplemented with 5% CO2.
Cell Transfection and Optimization
In a 24-well plate, human ovarian cancer cell line
OVCAR3 cells were seeded at a density of 3 105 cells/mL in
growth medium with free fetal bovine serum before transfection. To determine the optimal conditions necessary for
higher-efficiency cationic lipid-mediated cell transfection, 4
experimental groups were established: wild-type human ovarian
cancer cells (OVCAR3WT), ovarian cancer cells transfected with
a survivin recombinant vector (OVCAR3S), ovarian cancer cells
transfected with a liposome (OVCAR3L), and ovarian cancer
cells transfected with a blank vector (OVCAR3B). The ratios of
pEGFPC2 plasmid to liposome were 1:1, 1:2, 1:3, and 1:4, respectively. The transfection efficiencies of the 4 groups were
identified by flow cytometry (FCM) and fluorescence microscopy 24 hours after transfection to identify the optimization ratios. OVCAR3 cells were then trypsinized and plated in 24 wells
at 3 105 cells per well 1 day before transfection. Twenty-four
hours later, Mu6/survivin plasmids were mixed with liposome
at a 1:2 ratio (which was asserted by the last step). The mixture
was incubated for 20 minutes at room temperature and then
added to the cells. Cells were collected 24 hours later to assess
the down-regulation of survivin. In addition, a blank plasmid
and liposome were used as negative control groups.
Reverse TranscriptionYPolymerase Chain
Reaction Analysis
Total RNA from transfected cells was isolated using the
RNeasy Kit according to the manufacturer’s instructions.
Reverse transcription was performed at 42-C for 60 minutes
using oligo(dT). Sequences for polymerase chain reaction
(PCR) amplification used in this study were as follows: survivin, forward primer 5¶-CAG ATT TGA ATC GCG GGA
CCC-3¶, reverse primer 5¶-CCA AGT CTG GCT CGT TCT
CAG-3¶; GAPDH(internal control), forward primer 5¶-GTA
GAG GCA GGG ATG ATG TTC-3¶, reverse primer 5¶-GAG
TTA GCT CAC TCA TTA GGC-3¶. The specificity of PCR
products was checked on agarose gel.
Fluorescence-Activated Cell Sorting
Survivin Protein Expression
Briefly, after expressing either survivin shRNA or an
empty vector, cells were washed in phosphate-buffered saline
258
Cells were selected after transfection at 12, 24, 36, or 48
hours and then fixed with 70% ice-cold ethanol overnight.
RNA enzyme was added at a final concentration of 50 Kg/mL.
One hour later, cells were resuspended in phosphate-buffered
saline and incubated with propidium iodide according to the
manufacturer’s instructions. Cells were analyzed by fluorescenceactivated cell sorting, and the data were analyzed using the
Multicycle software (San Diego, CA).
MTT Assay
Cells (4 103) were seeded into two 96-well plates
after transfection for 24 hours and subsequently were treated
with 20, 15, 10, or 5 Kmol/L paclitaxel and 165, 82, 41, or
20 Kmol/L cisplatin. Control cells were treated with 0.1%
dimethyl sulfoxide in culture medium. After treatment for
68 hours, the cells were incubated with MTT reagent (0.5 mg/mL;
[3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide])
at 37-C for 4 hours. The resulting formazan crystals were
solubilized by the addition of 150 KL dimethyl sulfoxide to each
well. The optical densities at 540 and 655 nm were measured,
and the cell inhibition rate was determined based on the following formula: cell inhibition rate (%) = (1 j absorbance of
the treated wells / absorbance of the blank control wells)
100%.
RESULTS
Survivin Expression and Its Correlation
With Clinical Parameters in EOC
To investigate the status of survivin in EOC, we examined the expression of survivin in 90 EOC tissues by immunohistochemistry (Figs. 1AYD). The positive rate of
survivin expression was 60%. Among the 90 patients, 40
(44.44%) relapsed, and the median survival time was 46
months. The 1-, 3-, and 5-year survival rates of all patients
were 84.54%, 79.75%, and 73.62%, respectively. Table 1
shows the distribution of cytoplasmic survivin positivity
according to clinical pathological characteristics. Positive
survivin staining was associated with a lower 5-year survival
rate when compared with negative staining (56.64% vs
94.2%, P = 0.003). Cytoplasmic survivin expression was
associated with advanced International Federation of Gynecology and Obstetrics (FIGO) stage (72.92% vs 45.24%, P =
0.007), nonmucinous type (66.67% vs 40.74%, P = 0.022),
high grade (74.47% vs 47.62% vs 40.91%, P = 0.012), and
recurrence (90% vs 36%, P G 0.001). Survivin status had no
relationship with age, family tumor history, blood type, and
preoperative CA-125 (P 9 0.05). A univariate analysis showed
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International Journal of Gynecological Cancer
& Volume 23, Number 2, February 2013
Survivin Status in Epithelial
Ovarian Cancer
FIGURE 1. Survivin immunostaining in EOC. A, Serous cystadenocarcinoma (negative). B, Adenocarcinoma
(positive). C, Endometrioid carcinoma (positive). D, Mucinous carcinoma (positive) (original magnification 400).
that the factors associated with 5-year survival rate were FIGO
stage, pathological grade, survivin protein expression (Fig. 2),
and recurrence (P G 0.05) (Table 2). Both nonrecurrence (n =
50) and platinum-sensitive recurrent EOCs (n = 18) were
classified as the chemotherapy-sensitive group, with a total
of 68 cases. Platinum-resistant (n = 11), persistent (n = 2),
and refractory (n = 9) recurrent EOCs were regarded as a
chemotherapy-resistant group, totaling 22 cases. Positive
survivin expression was associated with platinum resistance
based on the Spearman test (r = 0.306, P = 0.003). Cox
proportional hazards regression model analysis indicated that
FIGO stage (hazard rate = 1.649, P = 0.047) and cytoplasmic
expression of the survivin protein (hazard rate = 1.734,
P = 0.010) were independent poor prognostic factors in all
patients (Table 3).
Survivin shRNA Silenced Survivin
Expression in OVCAR3 Cells
To regulate the expression of survivin, we transfected
the survivin shRNA expression vector Mu6/survivin and
control plasmids into OVCAR3 cells. Cells were collected 24
hours after transfection for analysis by reverse transcription
(RT)YPCR and FCM. Reverse transcriptionYPCR results
TABLE 1. Correlations between survivin expression and
clinicopathological parameters in 90 EOCs
Survivin
Parameter
FIGO stage
I + II
III + IV
Histology
Mucinous
Nonmucinous
Grade
G1
G2
G3
Recurrence
Yes
No
Total
Negative
Positive
P
42
48
23
13
19
35
0.007
27
63
16
21
11
42
0.022
22
21
47
13
11
12
9
10
35
0.012
40
50
4
32
36
18
0.000
FIGURE 2. Five-year survival after a median follow-up
time of 51 months in 90 EOCs according to survivin
expression.
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259
Chen et al
International Journal of Gynecological Cancer
TABLE 2. Univariate analysis of the prognostic factors
of EOC (n = 90)
Characteristics
No.
5-y Survival
Patients
Rate, %
W2
P
Age, y
G50
46
77.68
Q50
44
69.74
1.12 0.2893
Family tumor history
No
80
69.96
Yes
10
100.00
2.86 0.0907
Blood type
A
27
68.82
B
24
66.23
AB
7
71.43
O
32
83.40
2.89 0.4089
Preoperative CA-125
Unknown
12
67.46
G35
16
100.00
35Y200
17
85.23
9200
45
63.44
7.39 0.605
FIGO stage
IYII
42
84.97
IIIYIV
48
63.63
4.09 0.0431
Histology
Serous
34
73.09
Mucinous
27
81.27
Endometrioid
4
100.00
Adenocarcinoma
25
61.44
3.38 0.2736
Grade
G1
22
100.00
G2
21
75.96
G3
47
60.15
8.92 0.0116
Postoperative residual tumors diameters, cm
e2
78
76.29
92
12
47.34
2.93 0.0869
Recurrence
Yes
40
45.25
No
50
94.52
22.78 0.0000
Survivin
Negative
36
94.20
Positive
54
56.64
9.07 0.0026
Bold numbers indicate statistical significance.
showed that the intensity of OVCAR3S was weaker than that
in control groups (Fig. 3A), indicating that survivin shRNA
down-regulated survivin expression in OVCAR3 at the RNA
level. Flow cytometry results showed that survivin protein in
OVCAR3S decreased as compared with the control groups
(Fig. 3B).
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& Volume 23, Number 2, February 2013
Silencing Survivin Induced Cell Apoptosis
and Cell Cycle Arrest in OVCAR3 Cells
To evaluate the effect of survivin shRNA on OVACR3
cells, we examined cell apoptosis and cell cycle distribution
after silencing survivin. The apoptotic rates of OVCAR3 cells
at 12, 24, 36, and 48 hours after transfection were 20.7%,
31.9%, 39.0%, and 46.7%, respectively, and were significantly higher than those of control groups (Fig. 3C). Flow
cytometric results showed that 24 hours after culture, survivin
shRNA induced the accumulation of cells in the G0/Gl phase
with a decrease in cells in the G2/M phase as compared with
nontransfected groups (P G 0.01) (Table 4).
Silencing Survivin Enhanced the Sensitivity
of Paclitaxel But Not Cisplatin Treatment
in OVCAR3 Cells
We performed a sensitivity analysis of the effect of
paclitaxel and cisplatin after silencing survivin in OVACR3
cells by MTT assay. For OVCAR3WT, OVCAR3L, OVCAR3B,
and OVCAR3S, the IC50 of paclitaxel was 0.305 (SD, 0.032)
Kmol/L, 0.157 (SD, 0.031) Kmol/L, 0.175 (SD, 0.010) Kmol/L,
and 0.019 (SD, 0.001) Kmol/L, respectively, and the IC50 of
cisplatin was 9.4100 (SD, 0.796) Kmol/L, 6.675 (SD, 1.739)
Kmol/L, 6.930 (SD, 1.273) Kmol/L, and 7.862 (SD, 0.081)
Kmol/L, respectively. Survivin shRNA increased the sensitivity of OVCAR3 cells to paclitaxel 15-fold (P G 0.05) but did
not have a significant effect on cisplatin (P 9 0.05) (Fig. 3D).
DISCUSSION
The effect of drug resistance on the long-term effectiveness of paclitaxel and cisplatin in cancer chemotherapy is a
major problem. In fact, most deaths from cancer are attributable
to acquired drug resistance in the clinic. It has been reported that
patients with positive survivin expression are more likely to be
chemoresistant and have higher mortality rates than those of
negative expression.16 In our data set, the positive rate of
survivin expression in female Chinese cancer tissue was 60%,
which was in accordance with published results.10
The relationship between immunohistochemical assessment of cytoplasmic survivin status and clinicopathological
parameters in ovarian cancer is inconsistent. Ferrandina et al9
did not show any relationship between survivin positivity rate
and clinicopathological parameters in ovarian cancer. However, several studies have shown that positive survivin expression
in ovarian cancer was associated with disease aggressiveness, unfavorable clinical outcomes, and chemotherapy
resistance,11,12,17Y19 which is consistent with our results.
Furthermore, recent research showed that serum survivin was
positively correlated with advanced stage and poor diseasefree survival in ovarian cancer.20 Survivin has been proposed
as a chemoresistance and radioresistance factor, and survivin
inhibitors are intensively investigated for cancer therapy.21
Therefore, it is feasible that survivin inhibitors can be used for
at least a subgroup of ovarian cancer patients. On the other
hand, for the therapeutic application of siRNA technology, it
may be critical to use an efficient gene delivery system for
the transduction of siRNA into target cells. Therefore, we used
the vector-based shRNA technique and survivin shRNA
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Copyright © 2013 by IGCS and ESGO. Unauthorized reproduction of this article is prohibited.
International Journal of Gynecological Cancer
Survivin Status in Epithelial
Ovarian Cancer
& Volume 23, Number 2, February 2013
TABLE 3. Multivariate analysis of the prognostic factors of EOC (n = 90)
95% Confidence
Interval for Exp(B)
Survivin protein expression
FIGO stage
A
SE
Wald
P
Exp(B)
Lower
Upper
0.552
0.500
0.214
0.252
6.646
3.929
0.010
0.047
1.737
1.649
1.142
1.006
2.643
2.705
FIGURE 3. A, mRNA expression of survivin in OVCAR3 cells detected by RT-PCR. M: DNA marker; 1: OVCAR3WT,
2: OVCAR3L; 3: OVCAR3B (mU6 transfected) 24 h; 4: OVCAR3S (survivin shRNA transfected) 24 hours. B, Protein
expression of survivin in OVCAR3 cells detected by FCM. C, The cell apoptotic rate in OVCAR3 cells examined by FCM.
A, OVCAR3WT. B, OVCAR3L. C, OVCAR3S transfected 12 hours; D: OVCAR3S transfected 24 hours. E, OVCAR3S
transfected 36 hours. F, OVCAR3S transfected 48 hours. D, The chemosensitivity in OVCAR3 cells among different groups.
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International Journal of Gynecological Cancer
Chen et al
& Volume 23, Number 2, February 2013
TABLE 4. Results of cell apoptosis rate and cell cycle after silence survivin
Groups
OVCAR3WT
OVCAR3L
OVCAR3B
OVCAR3S
Cell No., 104
Apoptotic Rate, %
G0/G1, %
30
30
30
30
4.9 (0.7)
5.6 (0.5)
5.9 (0.2)
31.9 (1.2)
60.6 (0.4)
61.5 (3.1)
61.9 (1.4)
72.0 (3.4)*
S, %
26.7
27.1
26.3
24.9
(0.5)
(2.5)
(0.7)
(3.5)
G2/M, %
12.6 (0.2)
11.4 (0.6)
11.8 (0.7)
3.0 (2.5)*
Values are mean (SD).
*P G 0.05, compared with OVCAR3WT.
expression vector Mu6/survivin plasmids to transfect OVCAR3
cells to further define the role of survivin in ovarian cancers.
The results of RT-PCR and FCM in our study demonstrated
that specific survivin shRNA obviously inhibited survivin
mRNA and its corresponding protein expression in OVCAR3S
cells as compared with OVCAR3B and OVCAR3L cells. Recent research on SKOV3 ovarian cancer cells showed similar
results.22
Cell apoptosis plays a key role in the maintenance of
homeostasis of multicultural organisms, which still represents
one of the main prognostic indicators in neoplasia.23 Deregulated cell apoptosis leads to clinically important malignancies.
Survivin participates in the complex network by regulating both
programmed cell death and cell division.24 Previous reports
found that survivin was associated with cell cycle period and
could control microtubule movement.25 Recently, it has been
demonstrated that survivin overexpression in tumors is associated with poor prognosis because it may overcome the G1-S
checkpoint, imposing progression of cells through the cell cycle
and thus conferring a proliferative advantage.26 It can block
cells in the G0/G1 phase and sharply reduce those in the G2/M
phase.27 In our study, we found that the cell cycle of OVCAR3S
cells changed significantly, as the G0/G1 phase cells increased
and the G2/M phase cells decreased. Our data showed that silencing of survivin in OVCAR3 cells induced cell growth arrest
in vitro, which confirmed previous reports.28
Combination chemotherapy of paclitaxel with platinum (TP) is one of the criterion standard chemotherapeutic
agents to treat ovarian cancer.29,30 Paclitaxel strongly binds to the
N-terminal region of A-tubulin and promotes stabilization of
microtubules resulting in the inhibition of mitosis, which consequently induces cell death.31 Although TP increases overall
survival rate, approximately 60% to 70% of advanced ovarian
cancer patients had a relapse, mainly because of drug resistance.32 In this study, we aimed to evaluate whether survivin
down-regulation improves the efficacy of paclitaxel and cisplatin
against human ovarian cancer. To achieve this goal, 4 groups
were treated with different concentrations of paclitaxel/cisplatin
24 hours after transfection. After 68 hours of incubation, an MTT
cytotoxicity assay was performed. The results showed that survivin shRNA increased the sensitivity of OVCAR3 cells to
paclitaxel 15-fold, supporting the conclusion that inhibition
of survivin activation promotes paclitaxel-induced apoptosis
in vitro, which is consistent with the study results of Zaffaroni
et al.14 There are 3 reasons for enhancing OVCAR3 cell
sensitivity to paclitaxel following transfection of survivin
262
shRNA: (1) survivin and paclitaxel both affect tubulin, exhibiting a competitive inhibition relationship. Once survivin
expression is inhibited, it will significantly increase the sensitivity of OVCAR3 cells to paclitaxel. (2) Paclitaxel-induced
apoptosis in cells proceeds via caspases 3 and 8. The downregulation of survivin also activates caspase, resulting in
killing of cancer cells by a reduced dose of paclitaxel.33 (3)
Survivin may bind to the activating factor of caspase during
paclitaxel chemotherapy, thus obstructing the binding of the
latter and inhibitors of apoptosis proteins and inhibiting the
apoptosis of cells.34
The therapeutic effectiveness of cisplatin is thought to
be mainly regulated by inhibition of DNA replication and/or
delay of transcription through the formation of cisplatin-DNA
adducts.35 Although our immunohistochemical findings indicate that the expression of survivin in ovarian cancer is
strongly correlated with chemoresistance to cisplatin, the
cellular experiment showed that survivin shRNA had no significant effect on sensitivity to cisplatin in OVCAR3 cells.
Therefore, the role of survivin in chemotherapy with cisplatin
requires further investigations.
In summary, our results strongly support the hypothesis
that survivin expression is a sensitive and valuable independent molecular prognostic indicator; positive survivin expression in EOC is associated with high chemoresistance and
recurrence and reduced survival. Survivin may be used
clinically for targeted therapy. We introduced an shRNAsurvivin vector into OVCAR3 cells, leading to a significant
decrease in survivin expression. Furthermore, paclitaxelinduced apoptosis was significantly increased. These results
suggest that shRNA targeting survivin may have therapeutic
potential for the treatment of ovarian cancers, especially those
with drug-resistant characteristics. To improve the overall
survival of ovarian cancer patients, our results warrant further
preclinical and clinical studies to assess the value of both
survivin and shRNA in overcoming drug resistance. The
detailed mechanism of action and extensive validation need to
be further addressed.
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