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Introduction
Multiple myeloma (MM) is characterized by a clonal
proliferation of neoplastic plasma cells in the bone marrow and
is the second most common hematological
malignancy[1,2]. At present, a cure for multiple myeloma has not been achieved
with chemotherapeutic regimen, and many patients die of
drug-resistant diseases. High-dose chemotherapy with stem
cell support has achieved higher response rates than
conventional therapy, but few patients remain in long-term
remission[3]. Thus, the development of new effective
anticancer targets to treat both early and advanced MM seems
to be an attractive perspective.
Prostaglandins are important mediators implicated in
inflammation and angiogenesis, and support the growth of
several solid tumors[4,5]. Cyclooxygenase 2 (COX-2) is the
key enzyme of prostaglandin synthesis in inflamed
tissues[6]. In most normal tissues, its expression is at a low level, but is
induced by cytokines and growth
factors[7]. Overexpression of COX-2 is related to tumorigenesis by mechanisms of
chronic inflammation, immunosuppression, apoptosis
resistance, and angiogenesis[8]. Overexpression of COX-2
has been reported in various cancers such as breast, stomach,
colon, and lung[5,9].
MM is known to involve a deregulated cytokine network
with the secretion of inflammatory
mediators[10], and it has been recently reported that COX-2 is frequently expressed
in MM[11,12]. In the present study, we investigated the
function of COX-2 by siRNA-mediated gene silencing in human
myeloma RPMI8226 cells. Silencing of COX-2 leads to an
inhibited cell proliferation and induced apoptosis, which
is independent of the Bcl-2 family.
Materials and methods
siRNA for COX-2 RNA interference mediated by
duplexes of 21-nucleotide RNA was performed in RPMI8226
cells. The siRNA sequence was used to target exon 5 of the
COX-2 gene (GenBank accession No NM_000963). The 21
nucleotide RNA for silencing were chemically synthesized
by Ambion RNA Co (Austin, TX, USA). COX-2 siRNA
sequences were as follows: sense, 5'-GCACUUCACGCAUCAGUUUtt-3' and antisense,
5'-AAACUGAUGCGUGAA-GUGCtg-3'.
Cell culture and transfection of siRNA The RPMI8226
human myeloma cell line and one colorectal cell line (HT-29)
were obtained from American Type Culture Collection (ATCC,
Manassas, VA, USA) and maintained in RPMI1640
containing 10% fetal bovine serum (Gibco, Grand Island, NY, USA),
100 units/mL penicillin, and 100 µg/mL streptomycin at 37
oC with 5% CO2. The cells were passaged 2_3d before
nucleofec-tion, and the cells for nucleofection were in their logarithmic
growth phase.
The transfection of siRNA used the Amaxa cell
optimization kit V (Amaxa, Koeln, Germany) and followed the Amaxa
guidelines. Briefly, the cells were resuspended in the
nucleofector V solution. 100 µL of cell suspension at a
density of
2×106_5×106/mL mixed with 2 µg of pmax
green fluorescent protein (GFP) vector plus 1 µL of 100 µmol/L siRNA
were transferred to a cuvette and nucleofected with an Amaxa
nucleofector apparatus. The cells
(RPMI8226siRNA) were transfected using the G-16 pulsing parameter and were
immediately transferred into wells containing 37
oC pre-warmed culture medium in 12-well plates.
Untreated cells (RPMI8226Untreated) and cells
(RPMI8226Blank) in nucleofector
solution, without siRNA and with the application of the electroporation
program, were both used as negative controls. Twenty four
hours after the electroporation, the cells were monitored with
an Olympus BH-2 fluorescence microscope (Tokyo, Japan).
RT-PCR analysis Total RNA was isolated from the
RPMI8226 cells using TRIzol Reagent (Gibco-BRL, Gaithersburg, MD, USA). 4 µg RNA was reverse transcribed
to cDNA by the Thermoscript RT-PCR System reagent
(Gibco-BRL, USA). Primers for PCR reaction were designed
specially to human COX-2 gene. The sequences was
5'-CCGA-GGTGTATGTATGAGTG-3' for the sense and
3'-GGAAGA-GATTGTAGAGAGGA-5' for the antisense. Primers for
control were designed according to the GAPDH gene. The
sequences were as follows:
5'-GTCATCATCTCTGCCCCCTC-TGCT-3' for the sense and
3'-GACGCCTGCTTCACCACCT-TCTTG-5' for the antisense primer. PCR amplification
consisted of 35 cycles: 15 s at 94 oC for denaturing, 30 s at 58
oC for annealing, and 45 s at 72
oC for elongation.
Western blot analysis Treated cells
(1×106) were washed twice with phosphate-buffered saline (PBS) and lysed in
ice-cold cell lysis buffer [50 mmol/L Tris (pH 7.2), 150 mmol/L
NaCl, 1 mmol/L ethylenediaminetetraacetic acid, 1% Triton
X-100, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L sodium
orthovanadate, and 1 µg/mL leupeptin] containing 1 mmol/L
phenylmethylsulfonyl fluoride. Equivalent amounts of
proteins (20 µg) were boiled in Laemmli sample buffers and
fractionated on 10% SDS-PAGE. The separated proteins in the
gel were transferred onto polyvinylidine difluoride (PVDF)
membranes using an electroblot apparatus (Bio-Rad Inc,
Hercules, CA, USA). The filters were blocked for 2 h in PBS
containing 0.1% Tween 20 and 5% low fat milk, and the filters
were then incubated with specific primary Abs (Santa Cruz
Biotechnology, Santa Cruz, CA, USA) at dilution of 1:200 in
TBS/T, 5% low fat milk at 4 oC for 12 h. The membranes were
then washed with PBS-T and incubated with horseradish
peroxidase (HRP) conjugated anti-goat Abs or anti-rabbit
Abs (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at
dilution of 1:1000 in TBS/T, 5% low fat milk for in room
temperature for 1 h. Specific signals were detected on X-ray
films using an enhanced chemiluminescence detection
system (SuperSignal, Pierce, Rockford, IL, USA). The blots were
then stripped and reblotted with the antibody against
β-actin to ensure that equivalent levels of proteins were
present in each lane.
3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazo
lium (MTT) assay RPMI8226siRNA,
RPMI8226Untreated, or
RPMI8226Blank cells were seeded onto 96-well plates at a
density of 5×104/mL after being treated or untreated (200
µL/well) and cultured for 12, 24, 48, and 72 h, respectively.
Sub-sequently, 20 µL MTT (Janssen Chimica Co, New Brunswick,
NJ, USA) at a concentration of 5 g/L was added to each well,
and the cells were incubated for an additional 4 h at 37
oC. After a brief centrifugation, supernatants were carefully
removed and 200 µL DMSO was added to the cells. After the
insoluble crystals were completely dissolved, absorbance at
490 nm was measured using an EL×800 reader (Bio-Tek
Instruments, Winooski, VT, USA).
Annexin-V staining Annexin-V staining was performed
based on the Annexin-V- fluorescein isothiocyanate (FITC)
apoptosis assay kit (Bender MedSystems, Vienna, Austria).
In this assay, Annexin-V-FITC (1:250) and propidium iodide
(PI, 1 µg/mL) were used. FITC can specifically bind to the
phosphatidyl serine (PS) residues on the cell membrane, while
PI can bind to DNA once the cell membrane has become
permeable. The cells were stained and analyzed by FACScan
(Becton Dickinson, Franklin Lakes, NJ, USA). The data were
analyzed using the CellQuest (Becton Dickinson, USA)
software. For each analysis, 10 000 events were recorded.
Terminal deoxynucleotidyl transferase-mediated dUTP
nick end-labeling (TUNEL) staining Silencing of COX-2
induced apoptosis was also confirmed by TUNEL by TACS
(Total Access Cellular System) TdT Kit (R&D Systems, Inc,
Minneapolis, MN, USA). Briefly, the slides were rinsed with
Ca2+, Mg2+, and DNase-free PBS (10 mmol/L PBS, pH7.4) and
permeabilized with proteinase K at room temperature to make
the DNA accessible to the labeling enzyme. For the positive
control, the cells were incubated with TACS nuclease for 30
min, which generated DNA strand breaks in virtually every
cell. Endogenous peroxidase activity was quenched using
5% H2O2 (in methanol,
v/v) for 5 min, and the cells were
incubated with TdT labeling buffer for 5 min before starting the
labeling reaction. Then the cells were incubated with the
TdT enzyme and biotinylated nucleotides (for negative
control, labeling buffer was used instead of TdT enzyme) for
1 h at 37 oC in a humidified chamber. The reaction was stopped
by adding TdT stop buffer for 5 min. The slides were
incubated with streptavidin-conjugated horseradish peroxidase
for 10 min. A brown color was developed with incubation in
the diaminobenzidine (DAB) solution (Sigma, St Louis, MO,
USA) for 7 min at room temperature. The slides were
counterstained in 1% methyl green for 1.5 min and visualized and
scored under a light microscope. The apoptosis was
evaluated by calculating the rate of positive cells (brown-stained
cells) at 10 arbitrarily-selected fields at a magnification of 400
in a double-blinded manner.
Statistical analysis Statistical analysis was performed
using the F and q tests by SPSS10.0 (SPSS Inc, Chicago, IL,
USA), and statistical significance was defined as
P<0.05. Data were expressed as mean± SD for at least 3 experiments.
Results
Silencing COX-2 gene with siRNA duplexes To
estimate transfection efficacy, 2 µg pmaxGFP vector was
co-transfected into RPMI8226 cells with a siRNA duplex
designed against maxGFP. More than 300 cells were detected
for fluorescence (Figure 1). The same system was used as
for the silencing of the COX-2 gene. RT-PCR (Figure 2) and
Western blot (Figure 3) analysis indicated that the
transcription and expression of the COX-2 gene was extremely
attenuated in the cells transfected with the specific COX-2
siRNA. Thus, we demonstrated that COX-2 specific siRNA
can efficiently suppress COX-2 expression.
siRNA mediated silencing of COX-2 inhibits
proliferation of RPMI8226 cells As shown in Figure 4, silencing of
COX-2 led to the inhibition of cell proliferation in RPMI8226
cells. The value of OD (optical density) doubling time of
RPMI8226siRNA was significantly longer than that of
RPMI8226Untreated (P<0.001) and
RPMI8226Blank (P<0.001).
Induced apoptosis of RPMI8226 cells by the silencing of
COX-2 gene Twenty four hours after transfection,
RPMI8226siRNA,
RPMI8226Untreated, and
RPMI8226Blank cells were stained with Annexin-V to investigate whether apoptosis
would occur. The silencing of COX-2 via siRNA led to
significant apoptosis changes in the RPMI8226 cells (Figures 5,
6). Meanwhile, TUNEL assay was used to confirm whether
apoptosis was elevated and draw the same result. This
demonstrated that the silencing of COX-2 via siRNA brought
obvious apoptosis to the RPMI8226siRNA cells.
Silencing the COX-2 gene has no effect on the
expression of Bcl-2 family proteins in RPMI8226 cells
We examined the expression of Bcl-2 family proteins, Bcl-2 and Bax,
which play vital roles in programmed death in various types
of cells. As shown in Figure 7, the result of the Western blot
analysis showed that no significant differences could be
found in the expression of both the Bcl-2 and Bax proteins in
RPMI8226siRNA cells compared with the
RPMI8226Untreated or
RPMI8226Blank cells, which demonstrated that silencing the
COX-2 gene inhibited proliferation and induced apoptosis
in RPMI8226 cells via a Bcl-2 independent way.
Discussion
The nucleofector technique is a new non-viral
transfection method especially designed for hard-to-transfect cell
lines[13]. COX-2 gene silencing has been reported in various
tumor cells[14_16], which suggests that siRNA is an effective
technique for tumor study and treatment. However, there
have been no reports of silencing COX-2 in myeloma cells
until recently. Here, we successfully silenced the COX-2
gene in human RPMI8226 myeloma cells by RNA interfering.
For this study, 3 different pairs of siRNA fragments were
provided and the most effective one was chosen for this
study. Co-transfection of pmaxGFP, which encodes the GFP
from copepod Pontellina p. with an siRNA directed against
maxGFP into RPMI8226 cells helps us to understand the
transfection efficiency. Successful gene silencing is
monitored as a decrease of green fluorescence compared to the
control sample using fluorescence microscopy.
COX and lipoxygenase (LOX) are 2 important enzyme
classes that metabolize polyunsaturated fatty acids and
affect carcinogenesis[17, 18]. We have previously shown that
12-LOX, an important arachidonic acid-metabolizing enzyme,
expressed in RPMI8226 cells and its specific inhibitor,
baicalein, plays an important role in the growth and apoptosis
of RPMI8226 cells[19]. COX-2 is essential for the survival
and proliferation of malignant cells[5]. Recently, COX-2 has
been reported to be frequently expressed in MM and is an
independent predictor of poor
outcome[11]. However, the function of COX-2 in the development of human MM
remains less clear even though its inhibitor celecoxib has been
involved in clinical trials combined with thalidomide for
refractory and relapsed myeloma
therapy[20].
In this study, we demonstrate that successfully
silencing the COX-2 gene using siRNA duplexes can lead to the
inhibition of COX-2 expression in transfected RPMI8226 cells.
Furthermore, significant growth inhibition and apoptosis
induction have been detected in these transfected cells
(Figures 4_6). Interleukin-6, a pleiotropic cytokine known to
play a critical role in the survival and growth of multiple
myeloma cells[6], participates in a cytokine network and can
be induced by prostaglandin E2 (PGE2), a major product of
COX-2[21]. Therefore, COX-2 seems to play an important role
in MM via the cytokine network[22]. However, the direct
effects of COX-2 on MM cells have not been elucidated
[23].
There are 2 main apoptosis signaling pathways: death
receptor-dependent apoptosis and mitochondria-dependent
apoptosis[24]. As anti-apoptosis proteins, Bcl-2 and Bax have
been demonstrated to prevent the release of cytochrome C
into the cytosol and inhibit the subsequent activation of
caspase 9[25]. The mitochondrial-mediated pathway of
apoptosis is regulated by the Bcl-2 family of anti-apoptotic
(Bcl-2, Bcl-xL, and Mcl-1) and pro-apoptotic proteins (Bax,
Bad, and Bak), and Bcl-2 inhibits apoptosis by interacting
and forming inactivating heterodimers with
Bax/Bak[26]. PGE2 had been found to inhibit apoptosis via a Bcl-2-dependent
pathway in a human colonic cancer cell
line[27]. It was found that some antimultiple myeloma agents, such as
dexametha-sone, thalidomide, and proteasome inhibitors (PS-341),
induce apoptosis in myeloma cell lines or patient cells
associated with the downregulation of Bcl-2 or/and upregulation
of Bax[3]; however, in this study, we found that although
COX-2 was knocked down in RPMI8226 cells and obvious
apoptosis was observed, the expression of Bcl-2 and Bax did
not decrease. This demonstrates that apoptosis in RPMI8226
cells, induced by the silencing of COX-2, is independent of
modulation of Bcl-2 and Bax, which is consistent with the
results of Zhang's report[23].
In summary, our study suggests that COX-2 takes
considerable part in the regulation of cellular proliferation and
apoptosis of human myeloma cells, which is independent of
the Bcl-2 pathway. COX-2 could be an important target for
MM treatment, and small interfering RNA of COX-2 could
serve as an effective tool.
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