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Original Paper
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Acta Biochim Biophys
Sin 2007, 39: 835�843 |
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doi:10.1111/j.1745-7270.2007.00356.x |
Bcl-2 Small Interfering RNA
Sensitizes Cisplatin-resistant Human Lung Adenocarcinoma A549/DDP Cell to
Cisplatin and Diallyl disulfide
Zexiang HUANG, Xiaoyong LEI*, Miao
ZHONG, Bingyang ZHU, Shengsong TANG, and Duanfang LIAO
Institute
of Pharmacy and Pharmacology, University of South China, Hengyang 421001, China
Received: June 05,
2007�������
Accepted: July 17,
2007
This work was supported
by the grants from the National Natural Science Foundation of China (No.
30300426) and the Youth Foundation of Hunan province
education department (No. 03B034)
*Corresponding
author: Tel, 86-734-8282176; Fax, 86-734-8281306; E-mail, [email protected]
Abstract������� Bcl-2 is overexpressed in a variety of
human tumors and is involved in tumorigenesis and chemoresistance. In this
study, we investigated the inhibitory effect of the hairpin Bcl-2 small
interfering (si)RNA on the expression of the Bcl-2 gene in the cisplatin
(DDP)-resistant human lung adenocarcinoma cell line A549/DDP, and the effect of
Bcl-2 siRNA on drug sensitization in A549/DDP cells. Bcl-2 siRNA and negative
siRNA plasmids were constructed and stably transfected into A549/DDP cells.
Reverse transcription-polymerase chain reaction, immunofluorescence microscopy
and Western blot analysis were used to detect the target gene expression.
Spontaneous cell apoptosis was detected by acridine orange and ethidium bromide
staining. Drug sensitivity of the cells to DDP and diallyl disulfide (DADS) was
analyzed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)
assay and flow cytometry. Expression levels of Bcl-2 mRNA and protein in siRNA
stable transfectants were clearly reduced compared with negative siRNA
transfectants and untreated cells. MTT results indicated that Bcl-2
transfectants had a higher cell inhibition rate after treatment with 0.2-200 mg/ml
DDP or 50-200 mm
DADS. Flow cytometry revealed increased apoptosis in Bcl-2 siRNA cells. After
the addition of 20 mg/ml DDP or 100 mm DADS, siRNA targeting of the Bcl-2
gene specifically down-regulated gene expression in A549/DDP cells, increased
spontaneous apoptosis, and sensitized cells to DDP and DADS.
Keywords������� small interfering RNA; adenocarcinoma; Bcl-2; A549/DDP;
apoptosis
Lung cancer is the leading
cause of cancer-related death in the world [1]. Non-small-cell lung cancer (NSCLC)
constitutes approximately 80% of all lung cancers, 40% of which are at an
advanced stage at the time of diagnosis. Cisplatin (DDP), a commonly used
therapeutic agent in NSCLC, together with a third-generation anticancer drug,
such as vinorelbine, gemcitabine, or the taxanes, is the standard regimen used
in the first-line treatment of advanced NSCLC. Of these regimens, DDP has been
evaluated in multiple phase III trials and showed consistent superior
efficiency. diallyl disulfide
(DADS), an important component of garlic (Allium sativum), has been
recently shown to inhibit the growth of human tumor cells from colon, lung,
skin, and breast origins. The antiproliferative effect of DADS is due to its
ability to suppress the cell division rate and induce apoptosis in human tumor
cells. DADS was also reported to induce apoptosis as determined from
morphological changes, DNA fragmentation, and the increased proportion of cells
in the sub-G1
population, all of which were observed in cells after exposure. The underlying
mechanism involved the up-regulation of apoptotic Bax, and down-regulation of
anti-apoptotic Bcl-2 [2-5]. In this study we have
down-regulated the expression level of the Bcl-2 gene by RNA
interference in order to determine whether cellular drug sensitivity increased
after DADS was combined with Bcl-2 siRNA treatment.
Clinical multidrug resistance
to chemotherapeutic agents is a major obstacle to potentially curative
treatments for advanced NSCLC [6]. Thus, new methods to improve the clinical
response to chemotherapy are required. Gene therapy for malignant disease is a
promising approach.
Cancer cells escape apoptosis
by a number of mechanisms, among which overexpression of anti-apoptotic genes,
such as some members of the Bcl-2 gene family or the IAP and Mcl-1
families, has been shown to play a critical role [7]. The proto-oncogene Bcl-2,
discovered in low-grade Burkitt cell lymphomas, is a critical regulator of
apoptosis [8]. Among the Bcl-2 protein family members, it has been repeatedly
shown that Bcl-2 and Bcl-xl overexpression delays the onset of apoptosis
induced by several cytotoxic drugs. Overexpression of Bcl-2 has been associated
with several malignancies, including NSCLC [9-12].
Substantial research has shown that downregulation of anti-apoptotic gene
expression can sensitize cancer cells to anticancer drugs.
Most chemotherapeutic agents,
including DDP and DADS, induce cell apoptosis. Activation of a family of
cysteine proteases or caspases is essential for apoptotic cell death [13]. It is
believed that DNA damage caused by chemotherapeutic drugs induces the release
of mitochondrial cytochrome c, which facilitates activation of initiator
caspase-9, thereby triggering activation of downstream effector caspases, such
as caspase-3 [14].
Recently, the successful use
of small interfering (si)RNA in down-regulating gene expression in several
model systems has led to many attempts to explore this methodology in a
potentially therapeutic setting [15]. With DDP and DADS as the drugs of choice
for NSCLC treatment and the emergence of drug resistance as a critical problem
in DDP therapy, we examined the influence of Bcl-2 siRNA on drug sensitization
in A549/DDP cells and explored the mechanism of NSCLC cell apoptosis after
treatment with DDP and DADS.
Materials and Methods
siRNA vector construction
pSilencer 3.1-H1 linear vector
was purchased from Ambion (Austin, USA). The Bcl-2 siRNA insert sequence was
equivalent to GenBank accession No. Z23115, with sense (5'-AGTACATCCATTATAAGCT-3')
and antisense (5'-AGCTTATAATGGATGTACT-3') sequences. A negative
control vector that expresses a hairpin siRNA with limited homology to any
known sequences of the human genome was commercially available (Ambion).
Plasmid DNA was purified by cesium chloride bromide gradient centrifugation.
The purified DNA was diluted to 1 mg/ml and stored at -20
�C until used.
Cell culture and transfection
The human lung adenocarcinoma
cell line A549 and the DDP-resistant cell line A549/DDP were purchased from the
Xiangya Cell Center, Central South China University (Changsha, China). The
cells were cultured at 37 �C in a humidified atmosphere containing 5% CO2 in
RPMI-1640 medium (Invitrogen, Carlsbad, USA) supplemented with 10% bovine calf
serum (Hyclone, Logan, USA), and, for A549/DDP, 2 mg/ml
DDP (Sigma-Aldrich, St. Louis, USA) was added. Twelve hours before
transfection, cells were seeded into wells of a 24-well plate that contained
antibiotic-free medium; at the time of transfection, the cell confluence was
routinely 90%-95%. Transfection was carried
out according to the manufacturer's protocol. Bcl-2 siRNA or negative siRNA
plasmid (0.8 ml) was diluted with 50 ml OPTI-MEM (Invitrogen) or 2 ml Lipofectamine 2000 (Invitrogen) with 50
ml OPTI-MEM. After 5 min, the dilutions
were mixed together and incubated at 37 �C for 25 min, then dispensed into each
well. Forty-eight hours after transfection, 700 mg/ml
G418 (Amresco, Solon, USA) was added to the medium to select transfected Bcl-2
siRNA and negative siRNA cells. Three to five cell clones with resistance to
G418 were picked and added to culture medium containing 300 mg/ml G418.
Reverse
transcription-polymerase chain reaction (RT-PCR)
Transfected and untreated cells
were collected and washed with phosphate-buffered saline (PBS). Total RNA were
extracted from the cells using a total RNA isolation kit (Bio Basic, Markham,
Canada) according to the manufacturer's protocol. Three micrograms of total RNA
were used for RT-PCR with a total volume of 20 ml
with the Superscript preamplification system (Promega, Madison, USA). Aliquots
of cDNA (3 ml) were amplified in a total
volume of 50 ml using the GeneAmp PCR kit
(Promega) following the conditions recommended by the manufacturer. The sense
and antisense primers for Bcl-2 were 5'-tggatgttctgtgcctgtaaac-3' and 5'-tg�atgcggaagtcaccgaaa-3'
(amplification product 571 bp), respectively. The cycling conditions were 94 �C
for 4 min, followed by 35 cycles of 94 �C for 30 s, 52 �C for 45 s, and 72 �C
for 30 s, with a final extension of 72 �C for 10 min. The sense and antisense
primers for glyceraldehyde-3-phosphate dehydrogenase were 5'-cgga�gt�ca�ac�gg�atttggtcgtat-3'
and 5'-agccttc�tc�catg�gtg�g��t��gaagac-3'
(amplification product 306 bp), respectively. The cycling conditions were
95 �C for 5 min, followed by 35 cycles of 95 �C for 1 min, 55 �C for 1 min, and
72 �C for 1 min, with a final extension of 72 �C for 10 min. PCR products were
separated on a 1.3% agarose gel and viewed by ethidium bromide (EB; Molecular
Probes, Eugene, USA) staining. The data were analyzed using AlphaImager 2200
software (Alpha Innotech, San Leandro, USA).
Immunofluorescence microscopy
A549/DDP cells were collected,
washed twice with PBS, and fixed with methanol:acetic acid at a 3:1 dilution
for 15 min at room temperature. The cells were permeabilized with PBS
containing 0.25% Triton X-100 and 5% dimethylsulfoxide (Sigma-Aldrich) for 30
min at 37 �C and washed twice with PBS. Then the cells were incubated with the
anti Bcl-2 primary antibody (Santa Cruz Biotechnology, Santa Cruz, USA), at a
dilution of 1:100 in PBS, for 60 min at 37 �C. After three washes, the cells
were incubated with the goat anti-rabbit fluorescein-isothiocyanate-junctured
secondary antibody (Santa Cruz Biotechnology) for 60 min at 37 �C and washed
three times with PBS, then analyzed by fluorescence microscopy (Olympus, Tokyo,
Japan) using the 20 objective. Data were acquired with a Pixera camera (Pixera,
Los Gatos, USA).
Western blot analysis
Cells were lyzed in a lysis
buffer containing 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl
sulfate, 50 mg/ml aprotinin, 100 mg/ml phenyl�methylsulfonyl fluoride, 1 mM
sodium orthovanadate, 50 mM sodium fluoride, and PBS (pH 7.4). Cell lysates
were centrifuged at 10,000 g for 10 min at 4 �C, and the protein content
in the supernatants was determined using a BCA protein assay kit (Pierce,
Rockford, USA). Equal amounts of protein lysate were electrophoretically
separated on 10% or 8% sodium dodecyl sulfate-polyacrylamide gels and
transferred to polyvinylidene difluoride membranes (Millipore, Bedford, USA).
After blocking, each membrane was incubated with rabbit anti-Bcl-2 monoclonal
antibody and goat anti-caspase-3 polyclonal antibody (Santa Cruz
Biotechnology), or rabbit anti-poly(ADP-ribose) polymerase (PARP) polyclonal
antibody (Cell Signaling, Beverly, USA) overnight at 4 �C and further incubated
for 1 h with horseradish peroxidase-conjugated anti-mouse or anti-goat or
anti-rabbit secondary antibody (Santa Cruz Biotechnology). Bound antibodies
were detected by an enhanced chemiluminescence kit (Santa Cruz Biotechnology)
using a Lumino image analyzer (Taitec, Tokyo, Japan).
Apoptosis analysis
Cell apoptosis was identified
by fluorescence staining with acridine orange (AO; Becton Dickinson, Franklin
Lakes, USA) and EB. For the morphological examination of apoptosis, cells were seeded
in a 24-well microplate and washed with PBS, mixed with the same volume of a
dual AO/EB solution consisting of both compounds of 100 mg/ml. The final volume (200 ml) was observed using a fluorescence microscope
at an objective magnification of 20 (Olympus). For quantification, three
different fields were counted and at least 300 cells were enumerated in each
field. All experiments were done in triplicate.
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide (MTT) assay
Cells were incubated for 24 h
in a 96-well microplate with various concentrations of DDP and DADS. After 48
h, 5 mg/ml MTT was added and the cells were further incubated at 37 �C for 4 h.
Dimethylsulfoxide (200 ml) was added into each well
and incubated for 10 min. The reaction was optically monitored at 570 nm (A570)
using a 96-well microtiter plate reader (Pharmacia, Piscataway, USA). All
experiments were carried out in triplicate. The inhibitory rate of A549/DDP
cells was calculated according to Equation:
Eq. 1
where A570(control)
was the absorbance in Bcl-2 siRNA or negative siRNA or control groups, and A570(drug)
was the absorbance in the drug-treated group.
Flow cytometry
All cells were treated with 20
mg/ml DDP or 100 mM
DADS, washed twice in PBS, and fixed with 70% ethanol overnight at 4 �C. The
cells were then washed once with PBS and stained with 800 ml of 50 mg/ml
propidium iodide (Sigma-Aldrich) at room temperature for 30 min. The cell
apoptosis was determined by flow cytometry (Beckman Coulter, Fullerton, USA)
and analyzed with CellQuest software version 3.3 (Becton Dickinson, San Jose,
USA).
Statistical analysis
Statistical analysis was
carried out using SPSS software (version
11.0; SPSS, Chicago, USA). Data were expressed as the mean�SD and analyzed by
one-way ANOVA and the least
significant difference tests. P<0.05 was considered significant.
Results
Overexpression of Bcl-2 in
DDP-resistant cells
To understand the underlying
mechanisms of DDP resistance, RT-PCR and Western blot analysis were used to
evaluate Bcl-2 expression. The results revealed elevated mRNA and protein
levels in A549/DDP cells compared to A549 cells (Fig. 1).
Inhibition of Bcl-2 mRNA
expression by siRNA
A549/DDP cells were
transfected with Lipofectamine 2000 in vitro. After 48 h, G418 was added
to select transfected Bcl-2 siRNA and negative siRNA cells. Cell clones were
formed after 14 d of further incubation, and Bcl-2 mRNA levels were then
determined in A549/DDP cells transfected with Bcl-2 siRNA by RT-PCR. The Bcl-2
transcript of transfected A549/DDP cells was significantly less than that of
the negative siRNA or untreated control groups (Fig. 2).
Bcl-2 siRNA efficiently
inhibited Bcl-2 protein expression and activities of caspase-3 and PARP
To further confirm whether the
Bcl-2 protein level was also decreased by Bcl-2 SiRNA, we measured the Bcl-2
protein expression in A549/DDP cells by immunofluorescence microscopy
and Western blotting. As shown in Fig. 3, the Bcl-2 protein was
expressed at a higher level than in normal cells and cells transfected with
negative vector than cells transfected with Bcl-2 siRNA vector. Caspase-3 and
PARP activities were found increased in cells transfected with Bcl-2 siRNA
compared with cells transfected with the vector control (Fig. 4).
Spontaneous apoptosis induced
by Bcl-2 siRNA
To investigate the effect of
siRNA-induced Bcl-2 down-regulation on cell apoptosis, Bcl-2 siRNA or negative
siRNA stably transfected cells were collected and stained with AO/EB. The
results showed that the cells with Bcl-2 siRNA underwent typical apoptotic
morphological changes of nuclear and cytoplasmic condensation, loss of cell
volume, and nuclear fragmentation. In contrast, untreated control cells and
negative siRNA transfected cells did not show these apoptotic characteristics (Fig.
5).
Influence of Bcl-2
down-regulation on cell susceptibility to DDP and DADS-induced death
MTT assay results showed that Bcl-2 siRNA trans�fectants� had a lower cell viability and higher inhibition rate than that of the negative vector or untreated control cells after treatment with various concentrations of DDP and DADS: 0.2 mg/ml DDP (3.31%�0.78% versus 1.14%� 0.98% and 1.72%�1.02%, respectively); 2 mg/ml DDP (6.52%�0.76% versus 1.34%�0.89% and 2.70%�0.66%, respectively); 20 mg/ml DDP (46.19�3.90% versus 26.96%�6.08% and 27.26�4.12%, respectively); 200 mg/ml DDP (50.51%�9.67% versus 33.88%�5.90% and 33.27%�3.58%, respectively); 50 mM DADS (4.46%�0.97% versus 2.16%�0.86% and 2.95%�1.03%, respectively); 100 mm DADS (17.19%�7.01% versus 11.93%�4.90% and 10.21�5.65%, respectively); 150 mm DADS (25.75%�7.09% versus 20.86%�10.12% and 19.45%�6.78%, respectively); and 200 mm DADS (35.21%�7.90% versus 25.53%�8.57% and 25.81%�11.45%, respectively) (Table 1). The inhibition rate showed a dose-dependent effect of DDP and DADS. Moreover, flow cytometry showed that cells with Bcl-2 siRNA had a markedly increased apoptosis population compared� with negative siRNA or untreated control cells after the addition of 20 mg/ml DDP for 48 h and 100 mm DADS for 24 h (Fig. 6).
Discussion
Bcl-2 is widely used as a target
for cancer chemo�therapeutics. Antisense and small molecule inhibitors of Bcl-2
are being developed to down-regulate Bcl-2 to enhance anticancer drug
sensitivity or to reverse drug resistance [16,17].
In this study, we found that
Bcl-2 mRNA and protein expression was increased in the DDP-resistant A549/DDP
cell line, and the expression level was obviously higher than in the A549 cell
line. Suppression by Bcl-2-specific siRNA led to a greater decrease in the mRNA
and protein expression of Bcl-2 and growth inhibition in DDP-resistant cells.
We also found that the apoptosis was increased in Bcl-2 siRNA transfectants.
DDP and DADS induce cell death
by apoptosis. Activation of a family of cysteine proteases or caspases is
essential for cell death by apoptosis [11]. The activation of executioner
caspases results in the cleavage of critical cellular proteins, such as PARP,
DNA-dependent protein kinase, lamin B, and protein kinase Cy. Apoptosis is
regulated by a complex cellular signaling network and a defect in apoptotic
signaling can contribute to drug resistance.
Our results showed Bcl-2 siRNA
can decrease Bcl-2 protein expression and activate procaspase-3, followed by
the cleavage of their substrate PARP, suggesting that Bcl-2 siRNA induces
cytochrome c-mediated caspase-dependent apoptosis in human NSCLC cells. Broader
caspase inhibitors are being used to confirm this result in our laboratory.
The development of RNA
interference technology has made it possible to suppress the function of
specific molecular targets. This technology will be very useful in developing
new treatments for cancer [18-25] because our knowledge of
molecular targets that demarcate the difference between normal and malignant
cells is increasing. Our study results on A549/DDP suggest Bcl-2 protein as a
good target for cancer therapy, especially in cancers resistant to conventional
chemotherapy. Nevertheless, in vivo delivery and tumor specificity are
challenging issues for the use of Bcl-2 siRNA as an anticancer therapeutic
agent [26]. Development of genetic vectors or formulations for in vivo
delivery of siRNA will be necessary before siRNA can be used as a therapeutic
agent.
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