-
Recombinant human interleukin 24 reverses
Adriamycin resistance in
a human breast
cancer cell line
重组人白细胞介素
24<
/p>
对耐阿霉素人乳腺癌细胞系耐药性的逆转研究
Background:
The
major
cause
of
multidrug
resistance
is
over-expression
of
membrane
P-glycoprotein (P-gp). We investigated
the effect of recombinant human interleukin 24
(rhIL-24)
on the Adriamycin
(ADM)-resistant human breast cancer cell line
MCF-7/ADM.
Methods:The
cytotoxicity of rhIL-24 and ADM was determined by
3-[4,5-dimethylthiazol-2-yl],
5-diphenyl tetrazolium bromide (MTT)
assays. The expression of P-gp was assessed by
confocal
microscopy and Western blot
analysis.
背景：
P
糖蛋白
(P-gp)
的过度表达是产生多药
耐药性的主要原因。我们研究了重组人白细胞
介素
24(rhI
L-24)
对耐阿霉素（
ADM
）人乳
腺癌细胞系
MCF-7/ADM
的作用
方法：通过
3-(4,5-
二甲基噻唑
-2),5-
二苯基四氮唑溴盐
(MT
T)
比色分析法确定
rhIL-24
和
阿霉
素的细胞毒性。利用共聚焦显微镜和蛋白质印迹法来测定
P
-gp
表达。
Results:
The
IC
50
values
for
rhIL-24
in
MCF-7/wild-type
and
MCF-7/ADM
cells
were
0.17
μMand
14.6μM,
respectively. The IC 50 value
of Adriamycin in MCF-7/ADM cells decreased in a
dose-dependent
manner
when
rhIL-24
was
used.
The
resistance
modulating
factor
(RMF)
was
directly proportional to the dose of
rhIL24. ADM accumulation increased while P-gp
expression
decreased at a low dose (4 m
M) of rhIL24 in MCF-7/ADM cells. The expression of
P-gp was
decreased at 4 m M in confocal
microscopy and western blot analysis.
结果：
在野生型
MCF-7/
细胞和
MCF-7/ADM
细胞中
rhIL-24
的半数致死浓度
IC50
值分别为
0.17
和
14
.6
μM
。
在
MCF-7/ADM
细胞中，
阿霉素的
IC50
值呈剂量依赖性下降。
阻力调节因
子（
RMF
）值与
rhIL-24
的剂量成比例。当低剂量（
4μM
）的
rhIL-24
作用于
MCF-7/A
DM
细胞时，
ADM
的蓄积量增加而<
/p>
P-gp
表达下降。共聚焦显微镜和蛋白质印迹法的结果表明
rhIL-24
剂量为
4μM
时，
P-gp
的表达下降。
Conclusions:
rhIL-24
circumvented
the
drug-resistance
of
MCF-7/ADM
cells
via
activation
of
the
transcription factor Stat 3. rhIl24 has potential
to act as a P-gp inhibitor to reverse Adriamycin
resistancein breast cancer.
结论：
rhIL-24
之所以能减弱
M
CF-7/ADM
细胞的耐药性，是因为其激活了转录因子
St
at3
。
rhIL-24
有潜力作为<
/p>
P-gp
抑制剂来逆转人乳腺癌细胞对阿霉素的耐药性。
Introduction
Cancer multidrug resistance is the
cross-resistance or insensitivity of cancer cells
to the cytostatic
or cytotoxic actions
of various anticancer drugs [1].Juliano and Ling
[2] discovered a glycoprotein
(P-gp)
of
170
kDa
that
belongs
to
the
superfamily
of
adenosine
triphosphate-binding
cassette
transporters
that
are
associated
with
the
degree
of
drug
resistance
[3].Overexpression
of
P-gp
actively
pumps
drug
substrates
out
of
cancer
cells
thereby
decreasing
their
intracellular
accumulation
[4].This
is
the
primary
mechanism
in
the
development
of
multidrug
resistance
(MDR) [5]. Hyaluronan (HA; a major
component in the extracellular matrix of most
mammalian
tissues)
and
CD44
(a
cell
surface
receptor
that
belongs
to
a
family
of
multifunctional
transmembrane glycoproteins expressed
in mammary cells) are also involved in
chemotherapeutic
drug
resistance.
HA
binding
is
capable
of
stimulating
MDR1
and
P-gp
expression
and
drug
resistance in breast
tumor cells [6]. Therapeutics targeting HA-
mediated signaling are believed to
be
promising anti-cancer drugs [7]. MDR is recognized
as a major reason for the failure of cancer
therapy, and its reversal has become
one of the primary goals of modern approaches to
therapy [8].
Previously,
it
was
suggested
that
recombinant
human
interleukin
24
(rhIL-24)
has
clinical
potential as an
anti-cancer drug, and it has many advantages over
existing cancer therapeutics[9].
IL-24
has
two
functional
heterodimeric
receptors,
IL-20R1/IL-
20R2
and
IL-22R1/IL-20R2,
in
which R1 has a long cytoplasmic tail
and R2 has a short cytoplasmic tail [10]. IL-24
preferentially
signals
through
IL-22R1/IL-20R2
[11].
The
binding
of
IL-24
to
both
receptors
leads
to
the
activation of Stat-1 and Stat-3 [10].
Stat 3 is a component of the Janus-activated
kinase (JAK)/Stat
signaling pathway,
which plays an important role in regulating a
variety of biological activities in
normal and cancer cells [6]. The aim of
the present study was to determine whether
recombinant
human
IL-24
synergizes
the
anticancer
effect
of
ADM
by
decreasing
the
Pgp
expression
and
reverse Adriamycin
resistance in tumor-resistant cells (MCF-7/ADM).
The results suggest a new
avenue of
research for exploring the mechanisms of tumor
resistance.
引言：
癌细胞的多药耐药性主要是指交叉性耐药或对癌细胞抑制剂不敏感亦或是多种抗癌药物之
间产生了细胞毒作用
[1]
。
Juliano
和
Ling[2]
发现分子量为
170kDa
的
P-
糖蛋白（
P-gp
）与耐
药程度有关，该种糖蛋白属于三磷酸腺苷结合转运蛋白超家族成员
[3
]
。
P-gp
的过度表达使
药物被排出细胞基质，从而降低了药物在细胞内的蓄积量
[4]
。这也是形成多药耐药性的主
要原理
[5]
。透明质酸（
HA
；大多数哺乳动物细胞外基
质的主要成分）
CD44
（一种类属于多
功能跨膜糖蛋白并在乳腺细胞中表达的细胞表面受体）。透明质酸（
HA
）结合物能刺激多
药耐药基因和
P-gp
的表达，同时也能刺激乳腺癌细胞的耐药性
[6]
。透明质酸靶向性治疗药
物被认为是有潜力的抗癌药物
[7]<
/p>
。
多药耐药性
（
MDR
）
被认为是抗癌治疗失败的主要原因，
< br>因此如何逆转其耐药性已成为现代治疗方法的主要目标
[8]
。
此前，
人白细胞介素
24(rh
IL-24)
已作为一种潜在抗癌药物应用于临床，相较于传统抗癌治疗法，它具有许多
优势
[9]
。
IL-24
具有两个功能性的异源二聚体受体，分别是
IL-20R1/IL- 20R
2
和
IL-22R1/IL-20R2
，
R1
是长
胞质尾，
< br>R2
是短胞质尾
[10].
。<
/p>
IL-24
的信号优先通过
IL-20R
1/IL- 20R2[11]
，
IL-24
< br>对两种受
体的结合促使
Stat-1
和
Stat-3
的活化
[10]<
/p>
。
Stat3
是活性酪氨酸激酶
/
信号转导子和转录激活子
（
< br>JAK/STAT
）
信号通路的组件，
< br>在正常细胞和癌细胞的生物活性调节中发挥着重要作用
[6]
。
当前的研究目标是确定重组人白细胞介素（
rhIL-2
4
）与具有抗癌效果的阿霉素协同作用后
能否减弱
P-gp
表达并逆转癌细胞
（
MCF-7/ADM
）
对阿霉素的耐药性。
< br>这为探索肿瘤耐药性
机制提供了一条新的研究途径。
Materials and methods
Bacterial expression, refolding, and
analysis
Escherichia coli strain BL21
(DL3) was transformed with plasmid pET21a (+), in
which the IL-24
gene had been inserted
between the XhoI and BamHI sites. Host cells were
transformed using the
CaCl 2 method.
The rhIL-24 strain was transferred into 250 ml of
modified medium and incubated
with
continuous
shaking
at
180
rpm
at
37
8C
for
8
–
10
h.
The
culture
was
transferred
to
3-L
fermenters
containing fermentation medium. The fermentation
media composition and production
process described in detailed in our
previous work [12]. Cells were harvested by
centrifugation at
8000 rpm for 30 min.
The harvested cell-paste was dissolved in 50 mM
Tris-HCl buffer (pH 8.0)
containing 5
mM EDTA and 1 mM PMSF. Cells were lysed and
inclusion bodies (IBs) recovered
by
centrifugation at 8000 rpm for 30 min at 4 8C. IBs
were washed with 50 mM
Tris
–
HCl buffer
(pH 8.0) containing 5 mM EDTA and
finally washed with distilled water to remove
contaminating
salt and detergent. IBs
were dissolved in 5 ml extraction buffer I and
centrifuged at 12,000 rpm for
30
min
at
4
8C. The
supernatant
was
diluted
in
200
ml
dilution
buffer,
and
the
precipitate
was
collected by centrifugation at 12,000
rpm for 30 min at 4 8C. The precipitate was
further dissolved
in 5 ml extraction
buffer II. The solution was stirred for 20 min and
centrifuged at 12,000 rpm for
30 min at
4 8C. The supernatant was collected and the total
protein concentration measured by the
Bradford assay. The solution was added
drop- wise up to 400 ml of refolding buffer and
stirred
slowly with a magnetic stirrer
at 4 8C overnight. Refolded rhIL-24 was filtered
through a 1.2 m m
filter, concentrated,
and buffer exchanged through a tangential flow
filtration system. The refolded
protein
pH was adjusted to 5
–
6 by
adding 2 M acetic acid and loaded on an SP
sepharose column.
SDS-PAGE (15%) was
performed according to the method of Laemmli.
Western blot analysis was
carried
out
using
anti-
rhIL-24
antibody
(Abcam,
Cambridge,
MA,
USA).
Ultra
pure
water
was
used
throughout the upstream and downstream processed.
Anion Exchange chromatography helps
to
minimize the level of endotoxin by removal of
negatively charge endotoxin during purification.
LAL method (Houshiji, China) was
applied to detect the limit of bacterial endotoxin
from rhIL24
purified product.
材料和方法
细菌表达，复性和分析
大肠杆菌菌株
BL21(DL3)
通过载体
pET2
1a (+)
实现转化，
同时
IL-2
4
的基因也已嵌入到
XhoI
和
BamHI
的酶切位点之间。
宿主细胞通过<
/p>
CaCl
2
方法实现转化。
将
RhIL-24
菌株转移到
250ml
的改性介质中，在
37
℃
、
180rpm
转速条件下连续震荡培养
8
到
10
小时。随后将此培养液
转移到
3-L
的含有发酵培养液的发酵罐中
。
在以前的工作中已详细介绍过发酵介质的组成和
生产工艺
[12]
。经过
30
分钟的离心（
8000rpm
）分离后获得细胞浆。将获得的
细胞浆溶解在
含有
5
μ
M
乙二胺四乙酸
(EDTA)
和
1
μ
M
苯甲
基磺酰氟化物（
PMSF
）的三羟甲基氨基甲烷
-
盐酸缓冲液
（
pH 8.0
）
中。
细胞溶解，
在
4
℃、
8000rmp
转速条件下离心
30
分钟后细胞裂解、
包涵体（
IBs
）复原。将该包涵体（
IBs
）用含有
5
μ<
/p>
M
乙二胺四乙酸
(EDTA)
的三羟甲基氨基
甲烷
-
盐
酸缓冲液（
pH
8.0
）冲洗，再用
蒸馏水去除其表面的洗涤剂和盐。将上清液稀释到
200ml
，
同时在
4
℃、
12000rpm
转速条件下离心
30
分钟得到沉淀，将沉淀溶
解于
5ml
的提
取缓冲液
II
中。
溶液搅拌
20
分钟后再在
4
℃、
12000rpm
条件下离心
30
分钟
，
通过
Bradford
法测定上清液
的总蛋白浓度。
将溶液逐滴加入到
400ml
< br>的复性缓冲溶液中，
并在
4
℃的
条件
下用磁力搅拌棒搅拌一整夜。
随后将复性的
rhIL-24
通过
1.2mm
的滤膜过滤、
浓缩，
最后通
过切向流
过流系统实现缓冲液的交换。
通过
2
μ
M
的乙酸溶液和安装
SP
琼脂糖凝胶柱来调节
复性蛋白的
pH
值，使其
pH
保持在
5-6<
/p>
之间。用十二烷基硫酸钠聚丙烯酰胺凝胶电泳
（
< br>SDS-PAGE
）
(15%)
的测定方法进行检测，该法要参考借鉴
Laemmli
法的；借
助重组人白
细胞介素
24
（
anti-rhIL-24
）抗体来进行蛋白印迹法的分析测定。（
Abcam
公司，剑桥，硕
士，美国）。
上游和下游的操作过程中使用超纯水。
阴离子交换色谱法主要是通过
消除纯化
过程中带负电荷的内霉素来降低内霉素水平。拉尔法（
LAL
）（
Houshiji
，中国）
被用于测
定
rhIL-24
纯化液中细
菌内霉素的限值。
Cells
and cell cultures
The
wild-
type
human
breast
cancer
cell
line
MCF-7/WT
(sensitive
cells)
and
the
Adriamycin
(ADM;
doxorubicin)-resistant
cell
line
MCF-7/ADM
were
cultured
in
RPMI-1640
medium
containing 10% fetal bovine serum, 100
U/ml penicillin, and 100 m g/ml streptomycin.
Long-term
culture was performed in an
incubator at 37 8C with 5% CO 2 and saturated
humidity. MCF-7/
ADM
cells
were
continuously
cultured
in
the
above
medium
containing
1.0
m
g/ml
ADM
to
maintain the drug-resistance. Two weeks
prior to experiments, MCF-7/ADM cells were placed
in
Adriamycin-free medium for culture.
细胞和细胞培养
< br>野生型人乳腺癌细胞株
MCF-7/WT
（敏感细胞）和
耐阿霉素
MCF-7/ADM
细胞株在
RPMI-1640
的介质中进行培养。
该介质包含有
10%
的胎牛血清，
100U/ml
青霉素，
和
100
μ
g/ml
链霉素。
长期培养的环境条件要求
：
5%CO
2
、
饱和湿度和
37
℃。
MCF-7/A
DM
在含有
1.0mg/ml
阿霉素（
ADM
）的上述培养基中连续培养。实验前两周，要将
MCF-7/ADM
置于不含有阿
霉素的环境中
。
Cytotoxicity
assay
Cells were seeded onto 96-well
plates at 1*10
4
MCF-7/ADM
cells/well and 0.5*10
4
MCF-7/WT
cells/well,
and
incubated
in
a
humidified
atmosphere
of
5%
CO
2
overnight,
then
rhIL24
and
Adriamycin
were
added.
For
the
control,
medium
was
added
instead
of
the
drug.
After
24-h
incubation, 20mLMTT (5 g/L in PBS) was
added. The plates were incubated for 4 h and 200 m
L
DMSO/
well
was
added.
The
absorbance
at
570
nm
was
recorded
using
an
enzyme-
linked
immunosorbent assay multiscan
reader. The survival rate was calculated as
follows:
细胞毒性实验
将
1*10
4
个
MCF-7/ADM
细胞和
0.5*
10
4
个
MCF-7/WT
接种于标准的
96
孔板中，
并在
5%CO
2
和湿润环境下孵育，
然后加入
rhIL-24
和阿霉素。为了便于控制，应加入培养
基而不是直接
加入药物。经过
24
小时
的孵育后，加入
20ml
的
3-
（
4
，
5-
二甲基噻唑
-2
）
-2
，
5-
二苯基四氮
唑溴盐
(MTT)
（
在磷酸盐缓冲
液中含量为
5g/L
）
。
接种盘中加入
200ml
的二甲基亚砜
（
DMSO/
well
）
溶液并孵育
4
小时。
< br>利用酶联免疫吸附测定方法来读取
570nm
处的吸光率
。
计算成活
率的公式如下：
where T represents treated
(absorbance determined when tumor cells were
exposed to drugs); U,
untreated
(absorbance
of
untreated
cells);
and
B,
blank
(absorbance
when
neither
the
drug
nor
MTT
was
added).
The
IC
50
for
ADM
in
rhIL-24-treated
and
untreated
tumor
cells
was
determined and the resistance
modulating fraction (RMF) was calculated as
follows [13]:
其中
T
代表处理组（肿瘤细胞暴露于药物中时的吸光度测定值），
U
代表对照组（未经处
理的细胞吸收度测定值），
B
代表空白组（既不暴露于药物中也不加入
MTT
的吸光度测定
值）。经过
rhIL-24<
/p>
处理和未经处理的肿瘤细胞的阿霉素（
ADM
）的半致死浓度（
IC
50
）<
/p>
值与阻力调节因子（
RMF
）的关系计算
式如下
[13]:
In situ
analysis of P-gp expression by confocal laser
scanningmicroscopy
The
MCF-7/ADM
cells
were
cultured
and
prepared
in
confocal
microscopy
dishes.
Growth
medium was added and the cells were
stored at 37 8C with 5% CO 2 for 24 h until they
covered
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