Survivin expression induced by doxorubicin in cholangiocarcinoma.

Abstract

To study the role of survivin expression induced by chemotherapy agent (doxorubicin) in the development and anti-chemotherapy of cholangiocarcinoma.
Expression of survivin was detected by SP immunohistochemical technique in 33 cases of cholangiocarcinoma, 28 cases of adjacent noncancerous bile duct, and 5 cases of benign bile duct lesions. Low concentration of doxorubicin (0.05 mg/l) was added in cultured cholangiocarcinoma cell line (QBC939). The expression of survivin was detected by RT-PCR and Western blot at 24 h and 48 h after adding doxorubicin.
Survivin was expressed in 24 of 33 cholangiocarcinoma cases (72.7%). In contrast, no expression of survivin in adjacent noncancerous and benign bile duct lesions was observed (P<0.01). No correlation was found between survivin expression and clinical features. Doxorubicin could markedly (P<0.001) up-regulate survivin mRNA and protein expression of QBC939 cells.
Overexpression of survivin in cholangiocarcinomas may play an important role in the development of cholangiocarcinoma, its relationship with prognosis of cholangiocarcinoma deserves further investigation. Higher expression of survivin is induced by doxorubicin in QBC939. Survivin expression may resist apoptosis induced by chemotherapy agents.

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PO Box 2345, Beijing 100023, China World J Gastroenterol 2004;10(3):415-418
Fax: +86-10-85381893 World Journal of Gastroenterology
E-mail: wjg@wjgnet.com www.wjgnet.com Copyright © 2004 by The WJG Press ISSN 1007-9327
• CLINICAL RESEARCH •
Survivin expression induced by doxorubicin in cholangiocarcinoma
Qing Chang, Zheng-Ren Liu, Da-Yu Wang, Manoj Kumar, Yi-Bei Chen, Ren-Yi Qin
Qing Chang, Zheng-Ren Liu, Da-Yu Wang, Manoj Kumar, Yi-
Bei Chen, Ren-Yi Qin, Department of Surgery, Tongji Hospital,
Tongji Medical College, Huazhong University of Science and
Technology, Wuhan 430030, Hubei Province, China
Supported by National Natural Science Foundation of China, No.
30271473
Correspondence to: Professor Ren-Yi Qin, Department of Surgery,
Tongji Hospital, Tongji Medical College, Huazhong University of
Science and Technology, Wuhan 430030, Hubei Province,
China. ryqin@tjh.tjmu.edu.cn
Telephone: +86-27-83662389
Received: 2003-08-06 Accepted: 2003-10-07
Abstract
AIM: To study the role of survivin expression induced by
chemotherapy agent (doxorubicin) in the development and
anti-chemotherapy of cholangiocarcinoma.
METHODS: Expression of survivin was detected by SP
immunohistochemical technique in 33 cases of
cholangiocarcinoma, 28 cases of adjacent noncancerous bile
duct, and 5 cases of benign bile duct lesions. Low
concentration of doxorubicin (0.05 mg/l) was added in
cultured cholangiocarcinoma cell line (QBC939). The
expression of survivin was detected by RT-PCR and Western
blot at 24 h and 48 h after adding doxorubicin.
RESULTS: Survivin was expressed in 24 of 33 cholangiocar-
cinoma cases (72.7%). In contrast, no expression of survivin
in adjacent noncancerous and benign bile duct lesions was
observed (P< 0.01). No correlation was found between
survivin expression and clinical features. Doxorubicin could
markedly (P<0.001) up-regulate survivin mRNA and protein
expression of QBC939 cells.
CONCLUSION: Overexpression of survivin in cholangiocar-
cinomas may play an important role in the development of
cholangiocarcinoma, its relationship with prognosis of
cholangiocarcinoma deserves further investigation. Higher
expression of survivin is induced by doxorubicin in QBC939.
Survivin expression may resist apoptosis induced by
chemotherapy agents.
Chang Q, Liu ZR, Wang DY, Kumar M, Chen YB, Qin RY. Survivin
expression induced by doxorubicin in cholangiocarcinoma. World
J Gastroenterol 2004; 10(3):415-418
http://www.wjgnet.com/1007-9327/10/415.asp
INTRODUCTION
Survivin, a member of the inhibitors of apoptosis protein (IAP)
family, is characterized by a unique structure that discriminates
it from other members of the IAP family. It contains only a
single BIR repeat and lacks a carboxy terminal RING finger
domain. Survivin is expressed in the G2/M phase of cell cycle
in a cycle-regulated manner[1]. It directly binds to and inhibits
both Caspase-3 and Caspase-7 activity, leading to arrest of
apoptosis[2] .
Survivin expression is not detectable in differentiated
normal adult cells of any organ[3], but it is abundantly expressed
in embryonic tissues and in a wide range of cancer tissues[4]
including neuroblastoma[5], colorectal[6], stomach[7] and breast[8]
carcinomas. It has been demonstrated recently that survivin
is also frequently expressed in malignant pancreatic ductal
tumors[9] and pancreatic adenocarcinoma[10]. Furthermore, the
prognostic value of survivin expression has been reported in
several human cancers[11].
Inducing apoptosis is the mechanism of chemotherapy
agents killing tumor cells. But tumor cells resist chemotherapy
agents because not only they overexpress MDR1/P-glycoprotein
(P-gp) but also resist apoptosis induced by chemotherapy
agents. Studies demonstrating resistance of survivin-transfected
cells to anticancer drug-induced apoptosis[2] and sensitization
to chemotherapy by survivin antisense treatment[12] have shown
that survivin is implicated in sensitization to chemotherapy.
But in cholangiocarcinoma, survivin distribution and its
implication for apoptosis inhibition are not clear at present.
This study aimed to study the role of survivin expression induced
by chemotherapy agent (doxorubicin) in the development and
anti-chemotherapy of cholangiocarcinoma.
MATERIALS AND METHODS
Materials
Thirty-three specimens were obtained from patients with
cholangiocarcinoma at the Department of General Surgery,
Tongji Hospital of Tongji Medical College during the period
from 1993 to 2001. There were 21 males and 12 females, and
the mean age of the patients was 55.1 years (range from 34 to
79 years). The patients did not receive chemotherapy, radiation
therapy or immunotherapy before surgery. Five specimens of
benign bile duct lesions were also obtained. Formalin-fixed,
paraffin-embedded blocks of tissue samples were taken from
pathological archives. Serial sections of 4 µm were prepared
from the cut surface of the blocks at the maximum cross-section
of the tissue sample. Representative sections were stained with
H&E in order to confirm the histopathological diagnosis.
Human extrahepatic cholangiocarcinoma cell line QBC939
was established by Professor Wang SG (Third Military Medical
University, China) and offered to us as a gift[13]. The cells were
maintained as monolayers in RPMI 1640 medium supplemented
with 10% fetal bovine serum (FBS, Gibco, USA), 100 units/ml
penicillin and 100 mg/ml streptomycin in a humidified
atmosphere of 50 mL/L CO2 at 37
.
Methods
Immunohistochemical staining Immunohistochemical
staining was carried out with the SP technique using the SP kit
(Zhongshan Biotech Co., Beijing, China) after antigen retrieval
by microwave pretreatment. Briefly, deparaffinized sections
were immersed in a 0.1 M sodium citrate buffer (pH 6.0) and
heated three times for 5 min each at a 15 min interval in a
microwave oven at 600 W. After quenched in 3 % hydrogen
peroxide and blocked, the sections were incubated with rabbit
survivin polyclonal antibody (Neomarkers, USA; dilution 1:200)
overnight at 4 . Biotinylated antirabbit immunoglobulin and
streptavidin conjugated to horseradish peroxidase were

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416 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol February 1, 2004 Volume 10 Number 3
subsequently applied. Finally, 3’, 3’-diaminobenzidine was
used for color development, and hematoxylin was used for
counterstaining. As a negative control, the sections were
processed in the absence of primary antibody. Tissue sections
from a hepatocellular carcinoma with a known strong
expression of survivin were used as a positive control. A scoring
method was used to quantitate the survivin expression in
various samples examined. A mean percentage of positive
tumor cells was determined in at least five areas at ×400
magnification. Patients with scores of less than 5% were
defined as negative, otherwise they were defined as positive.
RT-PCR Low concentration of doxorubicin (0.05 mg/l,
Pharmacia & Upjohn Co. Ltd.) was added in cultured
cholangiocarcinoma cell line (QBC939). Expression of
survivin was detected by RT-PCR before adding doxorubicin
and at 24 h and 48 h after adding doxorubicin. Total RNA was
prepared from subconfluent cultures with TRIzol reagent
(Gibco, USA) according to the manufacture’s instructions. The
primers were designed to amplify a fragment of survivin cDNA
based on the reported sequence for human survivin. To
normalize the amount of input RNA, RT-PCR was performed
with primers for constitutively expressed β-actin gene. The
survivin primers were 5’-CCCCATAGAGAACATAAA-3’
(sense) and 5’-GGAATAAACCCTGGAAGTG-3’ (antisense),
giving rise to a 273 base pair polymerase chain reaction product.
The β-actin primers were 5’ -GTGCGTGACATTAAGGAG-
3’(sense) and 5’-CTAAGTCATAGTCCGCCT- 3’ (antisense),
giving rise to a 520 base pair polymerase chain reaction
product. The first strand cDNA synthesis and the subsequent
PCR were performed with RNA PCR kit (AMV) using a
programmed temperature control system set for 35 cycles, each
consisting of denaturation at 94
for 45 s, and extension at 72
mixture was electrophoresed on a 1.5% agarose gel, and the
PCR products were visualized by ethidium bromide staining
and quantified by an ImageQuant software. Survivin mRNA
expression level was determined by survivin/β-actin protein.
Western blot Low concentration of doxorubicin (0.05 mg/l)
was added in cultured cholangiocarcinoma cell line (QBC939).
Expression of survivin was detected by Western blot before
adding doxorubicin and at 24 h and 48 h after adding
doxorubicin. Total cells were lysed with cell-lysis buffer
[50 mM Tris-Cl, pH 8.0, 150 mM NaCl, 0.02% NaN3, 0.1% SDS,
100 µg/ml PMSF, 1 µg/ml Aprotinin, 1% NP-40]. Twenty µg
of protein was separated on 10% of SDS-PAGE gels and
transferred to NC membranes. After blocked with 5% non-fat
milk, the membranes were incubated with rabbit survivin
polyclonal antibody (1:1 000 dilution) at 4
washed three times the membranes were incubated with goat
anti-rabbit IgG at room temperature for 1 hour. The signals
were developed with the ECL kit (Amersham Pharmacia
Biotechnology Inc.).
for 45 s, annealing at 50
for 45 s. Ten µL reaction
overnight. After
Statistical analysis
Association between survivin expression and various clinical
and pathological variables was examined using χ2 test or
Fisher’s exact test. The data of PCR and Western blot were
expressed as mean±SD. Student’s t-test was used for statistical
analysis. P<0.05 was considered statistically significant.
RESULTS
Expression of survivin and associated clinicopathological
variables
Survivin was prominently found in 24 of 33 cholangiocarcinoma
cases (72.7%) by immunohistochemistry. Positive staining for
survivin was located in the cytoplasm of tumor cells (Figure
1). In contrast, expression of survivin was observed neither
in adjacent noncancerous bile ducts nor in benign bile duct
lesions (P<0.01). No correlation was found between survivin
expression and clinical features (Table 1).
Figure 1 Expression of survivin in cholangiocarcinoma
(×200, SP).
Table 1 Correlation between clinicopathological factors and
survivin expression in cholangiocarcinoma
Survivin expression
Clinical features n χ2
P
n %
All patients
Age (years)

60
>60
Sex
Male
Female
Tumor size (cm)

2
>2
Differentiation level
High & Middle
Low
Metastasis
Positive
Negative
3324 72.7
18
15
13
11
72.2 0.0051 0.9431
73.5
21
12
15
9
71.4 0.0491 0.8246
75.0
13
20
10
14
76.9 0.1904 0.6626
70.6
29
4
21
3
72.4 0.0119 0.9133
75.0
7
26
5
19
71.4 0.0076 0.9307
73.1
Expression level of survivin mRNA
Doxorubicin could markedly (P<0.001) up-regulate survivin
mRNA expression of QBC939 cells (Figure 2, Table 2).
Figure 2 Expression of survivin in a human cholangiocarcinoma
cell line. β-actin served as control. M: DL2 000 marker, 1: Normal
Qbc939, 2: 24 h after adding doxorubicin, 3: 48 h after adding
doxorubicin.
β-actin 520 bp
Survivin 273 bp
M 1 2 3

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Table 2 Expression level of survivin mRNA
Group n Survivin/ β-actin t P
A
B
C
7
7
7
0.4210±0.0551 bt=45.89, dt=12.54
0.8481±0.0713 bt=26.11
1.7034±0.0493
bP, dP<0.001
bP<0.001
Survivin mRNA expression level was determined by survivin/
β-actin protein. Data were expressed as mean±SD, b vs C (48 h
after adding doxorubicin), d vs B (24 h after adding doxorubicin),
A: Normal Qbc939.
Expression level of survivin protein
Doxorubicin could markedly (P<0.001) up-regulate survivin
protein expression of QBC939 cells (Figure 3, Table 3).
Figure 3 Expression of survivin in a human cholangiocarcinoma
cell line. 1 and 2: Normal Qbc939, 3: 24 h after adding
doxorubicin, 4: 48 h after adding doxorubicin.
Table 3 Expression level of survivin protein
Group n OD value t P
A
B
C
6
6
6
204.568±1.387 bt=17.99, dt=11.23
311.105±1.539 bt=11.02
339.989±1.872
bP, dP<0.001
bP<0.001
Data were expressed as mean±SD, b vs C (48 h after adding
doxorubicin), d vs B (24 h after adding doxorubicin), A: Normal
Qbc939.
DISCUSSION
Expression of survivin was detected in 72.7% of
cholangiocarcinomas. In contrast, no expression of survivin
in adjacent noncancerous and benign bile duct lesions was
observed (P<0.01). Using a similar polyclonal antibody, survivin
expression was detected in 93% of malignant melanomas[14],
81% of basal cell carcinomas, 92% of cutaneous squamous
cell carcinomas[15], 70% of hepatocellular carcinomas[16], 88%
of gastric carcinomas[17], 100% of oesophageal cancers[18], 88%
of pancreatic adenocarcinomas[10] and 74% of ovarian
carcinomas[19]. Our study demonstrated a high expression of
survivin in cholangiocarcinoma as in other human malignancies.
There was no correlation between survivin expression and
any clinical or pathological characteristics of cholangiocarcinoma.
A similar absence of correlation was also noted in previous
observations including gastric[7,20], colorectal[6,21] and breast
cancers[8]. Though many reports have shown that survivin was
an independent prognostic factor for various cancers[19,22-27],
such a distribution of clinicopathological features and the
high prevalence of survivin expression in cholangiocarcinoma
might have rendered the power of this study insufficient to
demonstrate any correlation between survivin expression and
any clinical or pathological characteristics. Thus, the relationship
with prognosis deserves further investigation.
Substantial evidences have also shown that during
chemotherapy, changes in expression levels of survivin might
provide information about chemo-sensitivity or chemo-resistance
of tumors[28,29]. In the present study, low concentration of
chemotherapy agent doxorubicin (0.05 mg/l) could markedly
(P<0.001) up-regulate survivin mRNA and protein expression
of QBC939 cells. These results suggested a direct link between
survivin expression and bile duct carcinoma cell susceptibility
to doxorubicin. That is, higher survivin expression may directly
down-regulate chemo-sensitivity. It may be one of the
mechanisms of anti-chemotherapy in cholangiocarcinoma.
In summary, a high expression of survivin in
cholangiocarcinoma may play an important role in the
development of cholangiocarcinoma, the relationship with
prognosis deserves further investigation. Higher expression
of survivin could be induced by doxorubicin in QBC939 and
might resist apoptosis induced by chemotherapy agents.
These results provide several exciting therapeutic possibilities.
Initial evidence in vitro and in vivo has shown that targeting
survivin may provide a viable approach to kill cancer cells
selectively[30,31]. Inhibition of survivin expression by molecular
manipulation has been reported to improve the effectiveness
of chemotherapy[12,31-33] and produce impact on radiation
therapy[34-37].
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Edited by Zhang JZ and Wang XL
418 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol February 1, 2004 Volume 10 Number 3