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Original Paper
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Acta Biochim
Biophys Sin 2009, 41: 223–230 |
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doi: 10.1093/abbs/gmp005. |
Knock-down
of survivin expression by siRNAs
enhances chemosensitivity of prostate cancer cells
and attenuates its tumorigenicity
Jianjun Shen1†, Jiayun Liu1†, Yin Long1, Yinye Miao1, Mingquan
Su1, Qing Zhang1, Hua Han2
and Xiaoke Hao1*
1Department of Clinical Laboratory,
2Department of Medical Genetics and
Developmental Biology,
†These authors contributed equally to this
work.
*Corresponding author: Tel, 86-29-84775959;
Fax, 86-29-82527499; E-mail, [email protected]
Survivin, a member of inhibitor of
apoptosis family protein, has become an attractive therapeutic target in cancer due to its selective expression in tumor cells and
its important roles for tumor cell viability. Here we show that vector-based
small interfering RNAs (siRNA)
silenced survivin expression in prostate cancer
cells, resulting in significantly reduced cell proliferation and enhanced
apoptosis, and increased the sensitivity of prostate cancer cells (PC-3) to
apoptosis-inducing agent platinol. Furthermore, PC-3
cells transfected with siRNA-expressing
vector showed lower tumor formation in nude mice xenografts
in vivo. These results demonstrated that inhibition of survivin
expression by siRNA attenuated the malignant phenotypes
of prostate cancer cells, and may provide a novel approach for gene therapy of
androgen-independent prostate cancer.
Keywords
RNA interference; survivin; prostate cancer; apoptosis; gene therapy
Received:
August 16, 2008
Accepted: December 12, 2008
Introduction
Increases in the susceptibility to
chemotherapy would represent an important advancement in the treatment of
patients with advanced and metastatic prostate cancer [1]. Survivin
is a 16.5 kDa intracellular protein with 142 amino
acid residues, and belongs to the inhibitor of apoptosis (IAP) family [2]. It
is a bifunctional member of the IAP family that
counteracts cell death and controls mitotic progression [3]. Survivin is present during embryonic development, but becomes
undetectable in most adult tissues [4]. In nearly all human cancers examined so
far, survivin expression is re-activated [5-7].
Predictive and prognostic significances of survivin
have been implemented in some types of cancer [8-13]. In general, two considerations
have made survivin an attractive therapeutic target
in cancer, namely its selective expression in tumor cells and its important roles
for tumor cell viability [14].
In recent years considerable efforts have
been made to develop strategies for modulating apoptosis in cells of cancer and
other human diseases [15]. In this context, approaches to counteract survivin in tumor cells have been proposed with the dual
aims to inhibit tumor growth through an increase in spontaneous apoptosis, and
to enhance tumor cell responsiveness to apoptosis-inducing agents [16]. To
date, different kinds of survivin antagonists,
including antisense oligonucleotides, ribozymes, small interfering RNAs
(siRNAs) and dominant-negative mutants, as well as cyclin-dependent kinase
inhibitors have been tested [17].
RNA interference (RNAi)
is a genetic interference phenomenon directed by double-stranded RNA (dsRNA). RNAi specifically and
efficiently degrades homogeneous mRNA, resulting in post-transcriptional gene
silencing [18,19]. Since its discovery in 1998, RNAi has been rapidly developed into one of the most widely
applied technologies with therapeutic potential in molecular and cellular
research [20,21].
In order to explore the feasibility of survivin-targeted siRNAs therapy
for prostate cancer, the expression of survivin in androgen-independent
prostate cancer cell lines PC-3, PC
Materials and Methods
Materials
pSilencer3.1-H1neo and pSilencer3.1-H1neo negative
control vectors were purchased from Ambion Inc. (
Cell lines and culture
The human androgen-independent prostate
cancer cell lines PC-3, PC
Immunocytochemical staining
Immunocytochemical staining was performed using the
SABC method with a SABC kit (Boster,
Construction of plasmids expressing shRNAs
Three siRNA sequences
targeting the human survivin, including SVV1, 5¢-GGACCACCGCATCTCTACA-3¢,
SVV2, 5¢-GAAGCAGTTTGAAGAATTA-3¢, and
SVV3, 5¢-GAATTAACCCTTGGTGAAT-3¢,
were designed using the siRNA target finder tool
(http://www.ambion.com/ techlib/misc/siRNA_finder.html). The sense and anti-sense
oligonucleotide chains expressing siRNA
were synthesized (Sangon,
Transfection of PC-3 cells
Before transfection,
cells (1×105/well) were seeded in 6-well plates and grown to 90–95%
of confluence. Prior to transfection, the medium was
replaced by serum-free RPMI-1640 (
Reverse transcription-PCR and calculation of
relative survivin mRNA levels
Cellular RNA was isolated from transfected PC-3 cells using the Trizol
reagent according to the manufacturer’s instructions. Total RNA (
Western blot analysis
The transfected PC-3
cells were washed with PBS and lysed at 4 ºC for 30
min in PBS containing
Flow cytometry
(FACS)
To examine cell apoptosis, transfected cells were fixed in 70% (V/V) ethanol, stained
with an ANNEXIN V-FITC apoptosis assay kit (Becton Dickinson, San Jose, USA)
according to the instruction manual, and analyzed with a flow cytometer (FACSCaliburTM, Becton
Dickinson, USA). In some experiments, transfected
cells were treated with
For cell cycle analysis, transfected
cells were stained with a CycleTESTTM PLUS DNA cell cycle
assay kit (Becton Dickinson), and then analyzed by FACS.
MTT assay
Transfected cells (2×104) were seeded in
24-well plates and cultured in the presence of MTT [3-(4,5-dimethylthiazol-2-yl)2,5-diphenylteiphenylte-trazolium
bromide]. At each time point, cells were harvested, lysed,
and the absorbance (A) at 490 nm of the supernatants was measured using a Microplate Reader (Becton Dickinson). Cell number was
calculated by comparison with a standard curve of known number of cells. In
some experiments, transfected cells were treated with
platinol (
In vivo inoculation of tumors
Nude mice (8 for each group) were injected
subcutaneously with 0.5 ml (2.0×107/ml) PC-3 cells transfected with pSilencer3.1-SVV1, pSilencer3.1-SVV2,
pSilencer3.1-SVV3, or pSilencer3.1-H1neo negative control vectors. Tumor volumes
were measured every day. Four weeks later, mice were sacrificed and tumor weights
were measured.
Statistical analysis
Data were expressed as mean±SD.
Statistical significance was determined by the Student’s t and the SNK-q test. P<0.05
was considered as statistically significant.
Results
Expression of survivin
in prostate cancer cell lines
We first examined the survivin
expression in prostate cancer cell lines by immunohistochemistry.
The results showed that proportion of survivin-positive
cells were 23% in PC3, 18% in DU145, 15% in LNCaP,
11% in 22RV1, and 12% in PC
Silencing of survivin
by RNAi inhibits cell proliferation and enhances chemosensitivity of prostate cancer cells
The efficiency of transfection
was about 75% as determined by pEGFP-N2 vectors [Fig. 2(A)]. To study the
functions of survivin in prostate cancer cells, we
employed the RNAi strategy. PC-3 cells were transfected with siRNA-expressing
vectors, and survivin mRNA and protein were measured
by RT-PCR and Western blot, respectively. The results indicated that pSilencer3.1-SVV2
and pSilencer3.1-SVV3 vectors were able to knock-down the expression of survivin [Fig. 2(B,C)].
The apoptosis index of PC-3 cells transfected with
pSilencer3.1-SVV2 and pSilencer3.1-SVV3 increased greatly [Fig. 3(A)], while
the proliferation of these cells decreased about 30%, as described previously
[23]. In contrast with the control groups, the number of cells in the G1 phases
of the siRNA group increased 10%,
and cells in the G2 and S phases decreased about 5% [Fig. 3(B)]. Moreover,
after treatment with platinol, the survival of the siRNA vectors-transfected PC-3
cells decreased about 35-45% [Fig. 3(C)], while the
apoptosis index increased about 29-42%, compared with control groups [Fig.
3(A)].
Knock-down of survivin
in PC-3 cells attenuates its tumorigenicity in nude
mice xenografts
To examine the effect of survivin
on tumor formation by prostate cancer cells, we performed tumorogenesis
assay in nude mice. Nude mice were inoculated subcutaneously with PC-3 cells transfected with pSilencer3.1-SVV1, pSilencer3.1-SVV2, or
pSilencer3.1-SVV3 vectors, respectively. Four weeks later, the mean volume and weight
of tumors examined. The results showed that the volume and weight of tumors
derived from PC-3 cells transfected with
pSilencer3.1-SVV2 and pSilencer3.1-SVV3 vectors were 50-55% of those of the control groups
(Fig. 4). These results indicate that the tumor formation and growth in nude
mice xenografts were suppressed by silencing of survivin.
Discussion
Survivin is an anti-apoptotic protein,
which is also involved in mitotic checkpoint control and apoptosis induced by
growth factor in human cancer cells [24]. Survivin is
overexpressed in a variety of human neoplasms. Currently, survivin
expression has been used as a prognostic factor in several human neoplasms. High survivin
expression by neoplasms correlates with more
aggressive behavior, decreased responsiveness to chemotherapeutic agents, and
shortened survival, as compared with cancers that are survivin-negative
[25].
Recently, Zhang et al. [22] have shown that survivin mediates resistance to anti-androgen therapy with flutamide in prostate cancer cells. Specifically, these
authors suggested that up-regulation of survivin via insulin-like
growth factor-1/AKT signaling during androgen blockade may be one of the
mechanisms by which prostate cancer cells develop resistance to antiandrogens. In addition, survivin
expression can be induced by heavy ion beams in some cancer cells, although,
the direct killing effect on cancerous cells by heavy ion radiation might be
more significant than the anti-apoptosis effects of the survivin
overexpression induced [26]. Overall, the results
obtained in different studies indicate survivin to be
a cellular factor potentially involved in the chemo-resistant and
radiation-resistant phenotypes of human tumors cells and suggest that
approaches designed to inhibit survivin expression
may lead to human tumor sensitization to chemical and physical agents. McEleny et al. [27] found that the androgen-independent
PC-3 and DU145 cells were highly resistant to apoptosis and expressed the
highest levels of NAIP, XIAP, and survivin mRNA.
Furthermore, Krajewska et al. [28] found that high
expression of these IAP family proteins was evident in human prostate cancers
and in prostate tissues from transgenic mice expressing SV40 large T antigen
under control of a probasin promoter. Positive survivin immunostaining was found
in many invasive cancers but was not detected in normal prostate epithelium.
Interestingly, positive immunostaining was found in
occasional non-transformed prostatic epithelial cells of both transgenic mice
and normal mice, associated with chromosomes or mitotic structures in cells
apparently undergoing division.
In order to knock down the overexpression of survivin in
cancer cells, various strategies have been investigated. Multiple hammerhead ribozymes targeting the exposed regions of survivin mRNA synergistically produced the most potent anti-cancer
effects and inhibited tumor growth in a hepatocellular
carcinoma xenograft mouse model [29]. Uchida et al. [30]
developed recombinant adenoviral vectors expressing siRNA
against the survivin transcript and explored the
impact of these vectors on three cancer cell lines. In all cell lines tested,
knockdown of the survivin expression resulted in
apoptotic cell death. Cancer cells infected with these vectors almost lost
their tumorigenicity following inoculation into nude
mice. Intratumoral injection with the vectors
significantly suppressed tumor growth in a mouse xenograft
model. This novel strategy may be a promising tool for cancer gene therapy.
Yang et al. [14] also showed that in the absence of survivin,
certain euploid human cells undergo mis-segregation of chromosomes, abortive assembly of
microtubules in late mitosis, failure of cytokinesis,
and arrest of DNA synthesis. These results further indicate the significances
of survivin in cancer.
In a word, survivin
is highly expressed in the PC-3, LNCAP, and DU145 prostate cancer cells. The
two pSilencer3.1-SVV2 and pSilencer3.1-SVV3 constructs could knock down the
expression of survivin with similar manner [31]. PC-3
cells transfected with the siRNA
expression vectors showed increased apoptosis, decreased proliferation, and
increased sensitivity to proapoptotic stimuli platinol. The tumorogenesis
activity of these cells decreased also. These results are important in
revealing the role of survivin in prostate cancer [32].
Due to the poor transfection efficiency, the mRNA and
protein levels of survivin in prostate cancer cells
could not be knocked down obviously enough in the experiments, further methods
such as adenoviral or lentiviral delivery system can
be used to overcome this disadvantage. A full understanding of effects of survivin in prostate cancer may lead to the development of
new treatments for metastatic prostate cancer. Taken together, these findings
indicate that survivin is significantly expressed in
prostate cancer cells. Silencing of survivin
expression by RNAi attenuated the malignant
phenotypes of prostate cancer cells, and may provide a novel approach for gene
therapy of androgen-independent prostate cancer.
Funding:
This work was supported by the grants from the
National High Technology Research and Development Program of China (No. 2006AA
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