Acta Pharmacol Sin 2009 Apr; 30 (4): 435–441
Acta Pharmacologica Sinica ©2009 CPS and SIMM
Sinomenine influences capacity for invasion and migration
in activated human monocytic THP-1 cells by inhibiting the
expression of MMP-2, MMP-9, and CD147
Yang-qiong OU, Li-hua CHEN, Xue-jun LI, Zhi-bin LIN, Wei-dong LI *
Department of Pharmacology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China
Aim: The aim of this study was to investigate the mechanism of the effects of Sinomenine (SIN) on the invasion and migra-
tion ability of activated human monocytic THP-1 cells (A-THP-1). Sinomenine is a pure alkaloid extracted from the Chinese
medical plant Sinomenium acutum.
Methods: Human monocytic THP-1 cells were induced to differentiate into macrophages with phorbol 12-myristate 13-ac-
etate (PMA). Cells were treated with different concentrations of SIN. The invasion and migration ability of cells was tested
by in vitro transwell assays. The levels of CD147 and MMPs were evaluated by flow cytometric analysis and zymographic
analysis, respectively. The mRNA expression of CD147, MMP-2, and MMP-9 was measured by RT-PCR.
Results: The invasion and migration ability of A-THP-1 cells was significantly inhibited by SIN in a concentration-depen-
dent fashion; at the same time, the levels of CD147, MMP-2, and MMP-9 were markedly down-regulated. This inhibitory
effect was most notable at concentrations of 0.25 mmol/L and 1.00 mmol/L (P<0.01).
Conclusion: A possible mechanism of the inhibitory effect of SIN on cell invasion and migration ability is repression of the
expression of MMP-2 and MMP-9, which strongly correlates with the inhibition of CD147 activity.
Keywords: sinomenine; rheumatoid arthritis; invasion; migration; CD147; MMP-2; MMP-9
Acta Pharmacologica Sinica (2009) 30: 435–441; doi: 10.1038/aps.2009.21; published online 23rd March 2009
* Correspondence to Wei-dong LI.
Received 2008-12-19 Accepted 2009-02-10
Sinomenine (SIN, 7,8-didehydro-4-hydroxy-3,7-dime-
thoxy-17-methylmorphinan-6-one, C19H23NO4), an alkaloid
isolated from the Chinese medicinal plant, Sinomenium
acutum, has been utilized to safely treat arthritis for many
years. Previous studies have demonstrated that SIN has a
variety of pharmacological effects, including anti-inflamma-
tory effects, immunosuppression, and prevention of cartilage
destruction[1–4]. As an immunosuppressive agent, SIN has
been used extensively in China for the treatment of rheuma-
toid arthritis[5, 6].
Rheumatoid arthritis (RA) is a systemic, immune and
inflammatory disease characterized by joint swelling, syn-
ovial inflammation and joint destruction, leading to signifi-
cant disability. The regulation of cell migration and inva-
sion is a critical process throughout the development of RA.
Enhanced migration and invasion of peripheral macrophages
contribute to joint destruction in RA by directly degrading
the cartilage matrix and indirectly promoting angiogenesis.
Elevated gene expression of gelatinase A (also called matrix
metalloproteinase-2, MMP-2) and gelatinase B (also called
matrix metalloproteinase-9, MMP-9) is critical for the pro-
gression of RA.
The extracellular matrix metalloproteinase inducer
(EMMPRIN, CD147) is a heavily glycosylated protein
containing two immunoglobulin super family domains. It
is enriched on the surface of macrophages and is associated
with differentiation of human monocytic THP-1 cells when
treated with phorbol 12-myristate 13-acetate (PMA). This
protein stimulates the production of matrix metalloprotei-
nases (MMPs). CD147 has also been reported to play an
important role in the invasion and migration ability of cells
in both animal models and cancer patients.
Although many in vivo studies have demonstrated that
SIN can significantly improve RA[10–12], the specific mecha-
nism of SIN has not been established. The purpose of this
www.nature.com/aps Ou YQ et al
study was to investigate the effects of SIN on the invasion
and migration ability of activated human monocytic THP-1
cells (A-THP-1) and to evaluate the underlying mechanism.
Materials and methods
Materials SIN was donated for use in our laboratory
by Hu-nan Zheng Qing Pharmaceutical Factory. Human
monocytic THP-1 cells were obtained from Peking Union
Medical College. All reagents used for cell culture contain-
ing penicillin/streptomycin, pyruvate, FCS (fetal calf serum)
and RPMI-1640 medium were purchased from Gibco BRL
(Grand Island, NY). Phorbol 12-myristate 13-acetate (PMA)
was purchased from Sigma Chemical Co (St Louis, MO).
Restriction enzymes and Taq polymerase were obtained
from Takara, and FITC-labeled anti-human CD147 antibody
was purchased from the Ancell Corporation (USA).
Cell culture and differentiation Human monocytic
THP-1 cells were cultured in RPMI-1640 medium supple-
mented with 10% heat-inactivated fetal bovine serum, 2
mmol/L, L-glutamine, 5 mmol/L sodium pyruvate, and anti-
biotics (105 U/L penicillin, 105 U/L streptomycin) in culture
flasks. Cells were maintained at 37 °C in a humidified 5%
CO2 atmosphere. Experiments were routinely carried out
using cells in the log phase of growth. PMA was dissolved
in DMSO to obtain a 1 mmol/L stock solution and further
diluted with PBS before use. For all experiments, THP-1
cells were cultured at an initial density of 5×108 cells/L
and treated with PMA at a final concentration of 50 μg/L
for 24 h. After being washed three times with complete
medium, cells were plated at a suitable density and left to
recover and adhere for 24 h in the incubator. A-THP-1 cells
used in these experiments were all treated in the same man-
MTT assay A-THP-1 cells were suspended in RPMI
-1640 medium with 10% FCS, plated onto gelatinized
96-well culture plates (5×108 cells/L, 0.1 mL/well), and
incubated at 37 °C with 5% CO2 for 24 h. Then, the medium
was replaced with 0.1 mL of RPMI-1640, containing dif-
ferent concentrations of SIN (0, 0.01, 0.05, 0.25, and 1.00
mmol/L), and incubated for 48 h at 37 °C with 5% CO2. Cell
viability was determined by MTT assay. In the MTT assay, 20
μL of MTT was added to each well (final MTT concentration
was 0.5 g/L) for 4 h before the addition of 150 μL DMSO
to solubilize the reactive dye. The absorbance value of each
well was recorded at 570 nm using a Bio-Rad microplate
In vitro cell invasion and migration assays In vitro cell
invasion assays were performed in 10-mm diameter and 8 μm
pore polycarbonate filter transwell plates (Millipore). Mem-
branes were precoated with 25 μg of matrigel (Peking Uni-
versity Health Science Center) on the upper surface, which
formed a reconstituted basement membrane at 37 °C. After
treatment with SIN (0, 0.01, 0.05, 0.25, and 1.00 mmol/L)
for 48 h, A-THP-1cells (5×105 cells in 100 μL RPMI-1640)
were seeded onto the upper well of the chamber, and the
lower well was filled to the top (500 μL) with RPMI-1640
containing 10% FCS as a chemoattractant. After incuba-
tion for 24 h at 37 °C in the presence of 5% CO2, the cells
were fixed for 30 min in 4% formaldehyde and stained for 15
min with crystal violet. The non-migrating cells were then
carefully removed from the upper surface (inside) of the
transwell with a wet cotton swab. Cells that had migrated or
invaded to the bottom surface of the filter were counted. Six
evenly spaced fields of cells were counted in each well using
an inverted phase-contrast microscope.
There was no matrigel precoated on membranes of the
upper surface in in vitro invasion assays. Other conditions
were the same as in the migration assays.
Isolation of total RNA and RT-PCR Total RNA was
extracted from A-THP-1cells cells treated with SIN (0, 0.01,
0.05, 0.25, 1.00 mmol/L) for 48 h (5×108 cells/L) using Tri-
zol reagent (Invitrogen, Cergy Pontoise, France) according
to the manufacturer’s instructions.
Reverse-transcription reaction components were as fol-
lows: total RNA (2 μg), random primer (5 pmol/L), M-MLV
reverse transcriptase (0.5 U: 1 μL), dNTP (1 mmol/L), and
1×RT buffer. The reaction volume was brought up to 20 μL
with nuclease-free water. All components were mixed on ice
and then reacted for 60 min at 37 °C and 15 min at 72 °C.
PCR reaction components were as follows: RT product
mix (4 μL), primers (0.5 μmol/L of each, for sequences
see Table 1), Taq polymerase (0.1 U: 1 μL), MgCl2 (1.5
mmol/L), dNTP (0.125 mmol/L of each), and 1×PCR buf-
fer. The usual temperature programs were 5 min at 94 °C,
then 45 s at 94 °C for 24–30 cycles, 1 min at 58–61 °C (Table
2), and 1 min at 72 °C, followed by a final 10-min extension
at 72 °C[17, 18].
The mixture of PCR products was analyzed by electro-
phoresis in 1.5% agarose gels stained with ethidium bromide.
The ratio between the sample RNA and human glyceralde-
hyde 3-phosphate dehydrogenase (GAPDH) was calculated
to normalize for initial variations in the sample concentration
and as a control for reaction efficiency. Images of the RT-
PCR ethidium bromide-stained agarose gels were acquired
with a digital Kodak camera (Eastman Kodak Company,
Rochester, NY, USA). Quantification of the bands was ana-
lyzed by imaging analysis software from Kodak.
www.chinaphar.comOu YQ et al
Flow cytometric analysis After being treated with SIN
(0, 0.01, 0.05, 0.25, and 1.00 mmol/L) for 48 h, A-THP-1
cells (5×108 cells/L) were washed with phosphate-buffered
saline (PBS). Cells were labeled with fluorescein isothiocy-
anate-labeled anti-CD147 antibody for 45 min at 4 °C and
then washed with cold PBS and suspended in PBS. Labeled
cells were analyzed on a Cytoron flow cytometer.
Zymographic analysis Cells were harvested at the
exponential growth phase, washed three times in serum-free
RPMI-1640, plated onto gelatinized 24-well culture plates
(0.5 mL/well) at a density of 5×108 cells/L, and incubated
for 48 h in the absence (control) or presence of SIN (0.01,
0.05, 0.25, and 1.00 mmol/L). The supernatants were col-
lected, centrifuged to remove debris, and analyzed by zymog-
Gelatinolytic activities were assessed under non-reducing
conditions using modified sodium dodecyl sulfate polyacryl-
amide gel electrophoresis (SDS-PAGE). This technique
was performed on a 7.5% polyacrylamide gel copolymerized
with 0.1% gelatin type I. Twenty microliter samples mixed
with 10 μL of loading buffer were run under non-reducing
conditions without prior boiling. After electrophoresis, gels
were washed two times with 2.5% Triton X-100 to remove
SDS and allow proteins to renature and then immersed in
buffer (50 mmol/L Tris, pH 7.5, 5 mmol/L CaCl2, 1 μmol/L
ZnCl2 and 0.01% NaN3) for 18 h at 37 °C. Gels were stained
with 0.2% Coomassie (0.25 % brilliant blue, 40% methanol,
7.5% acetic acid) for 4 h at room temperature, followed by
treatment with destaining buffer (30% methanol, 10% acetic
acid) until the desired contrast was achieved. A clear white
band showing proteolytic activity was detected against a blue
background of undigested gelatin. Gels were documented
using an Epi ChemII darkroom (UVP Inc, Upland, CA)
documentation system, and the intensity of the bands was
quantified using imaging analysis software from Kodak.
Statistical analysis The statistical significance (P<0.05)
of differences was evaluated by ANOVA using the SPSS
(10.0) statistics package. All results were expressed as
means±standard deviation (SD).
Cell differentiation For all experiments, THP-1 cells
were cultured at an initial density of 5×108 cells/L and
treated with PMA at a final concentration of 50 μg/L. After
24 h, over 90% of THP-1 cells adhered to the plastic as mac-
rophage-like cells when observed under a microscope.
The effects of SIN on cell viability Cells (5×108
cells/L) were incubated with various concentrations of
SIN (0, 0.01, 0.05, 0.25, and 1.00 mmol/L) for 48 h. The
absorbance value of each well at 570 nm was read in a Bio-
Rad microplate reader. The results showed no changes in
viability of A-THP-1 cells resulting from the addition of SIN
In vitro cell invasion and migration assays Cell inva-
Table 1. Primer sequences used in PCR.
Forward (5′ → 3′) Reverse (5′ → 3′) Size (bp)
Table 2. Annealing temperature and cycles used in PCR.
PCR annealing temperature ( °C ) Cycles
Figure 1. Growth curve of A-THP-1 cells in RPMI 1640 with 10%
FCS. Cells (5×108 cells/L) were incubated with various concentrations
of SIN (0, 0.01, 0.05, 0.25, and 1.00 mmol/L) for 48 h. The intensity
ratio (%) indicates the cell viability at other drug concentrations as
compared with the control. n =6. Mean±SD. aP>0.05 vs control.
www.nature.com/apsOu YQ et al
sion through the matrigel basement membrane in the cell
invasion assay and cell migration directly through the mem-
brane in the cell migration assay were analyzed in a modified
transwell plate. Cells that invaded to the lower surface of the
membrane in these assays were fixed and stained (Figure 2).
The percentage of migration and invasion of A-THP-1 cells
was significantly lower in the 0.05 mmol/L SIN treatment
group (P<0.05). Migration was also significantly reduced in
the 0.25 and 1.00 mmol/L SIN treatment groups (P<0.01).
The effects of SIN on CD147, MMP-2, and MMP-9
gene expression In order to study the effect of SIN on
CD147, MMP-2, and MMP-9 production at the transcrip-
tional level, A-THP-1 cells were maintained in culture in the
presence of SIN for 48 h. The total RNA of cells was isolated
and RT-PCR was performed as described under Methods.
As shown in Figure 3, the level of CD147 mRNA was down
regulated in the 0.25 mmol/L and 1.00 mmol/L SIN treat-
ment groups (P<0.05). The level of MMP-2 and MMP-9
mRNA was efficiently and dose-dependently downregulated
by SIN; a significant reduction was observed in the 0.05
mmol/L SIN treatment group (P<0.05), and it was highly
significant in the 0.25 mmol/L and 1.00 mmol/L SIN treat-
ment groups (P<0.01).
The effects of SIN on levels of CD147 FITC-labeled
A-THP-1 cells were treated with various concentrations of
SIN for 48 h. SIN-mediated inhibition of CD147 release
was dose-dependent, with the maximal effects obtained in
the 0.25 mmol/L and 1.00 mmol/L SIN treatment groups
(P<0.01). A significant inhibition of CD147 release was also
observed in the 0.05 mmol/L SIN treatment group (P<0.05)
(Figure 4). The data indicate that the decreased CD147
protein levels result from a downregulation of CD147 at the
The effects of SIN on activities of MMP-2 and MMP-9
Gelatinolytic activities of MMP-2 and MMP-9 in condi-
tioned medium were detected by electrophoresis of the
soluble protein on a gelatin containing 7.5% polyacrylamide
gel. These experiments show that the suppression of enzyme
activity of MMP-2 and MMP-9 was more pronounced in
the groups treated with SIN when compared with the con-
trol group. The observed suppression in enzymatic activity
was significant in the 0.05 mmol/L (P<0.05), 0.25 and 1.00
mmol/L SIN treatment groups (P<0.01) (Figure 5).
Matrix metalloproteinases (MMPs), which are also
known as matrixins, comprise a family of zinc-containing
endopeptidases that share common structural domains.
These proteins have the capacity to degrade extracellular
matrix (ECM) components, as well as alter their biological
The MMPs are categorized into three major functional
groups, in part based on substrate specificity. The interstitial
collagenases (MMP-1, -8, and -13), which preferentially have
affinities for collagen types I, II, and III, the stromelysins
(MMP-3, -10, and -11), with specificity for laminin, fibronec-
tin, and proteoglycans, and the gelatinases (MMP-2 and -9),
which are most effective in the cleavage of type IV and V col-
lagen, represent the three main groups.
The regulation of gene expression of most MMPs is con-
trolled by CD147/EMMPRIN[20, 21]. CD147/EMMPRIN
is a highly glycosylated cell surface transmembrane protein
found on the cell surface of many types of cells, including dif-
ferentiated macrophages. It has been implicated in inducing
MMP synthesis in rheumatoid synovium, in the surround-
ing fibroblasts after injury involving stromal remodeling,
and in other situations associated with discontinuity of
the basement membrane. But CD147 has no effect on the
physiological inhibitors of MMP production and activation,
TIMP-1 or TIMP-2, so the effects of TIMP on MMPs are
not changed by CD147[22–24].
MMPs transcripts are generally expressed at low levels,
but these levels rise rapidly when tissues are locally induced
Figure 2. Cell invasion and migration assay in SIN (0, 0.01, 0.05, 0.25,
and 1.00 mmol/L) treated A-THP-1 cells (5×108 cells/L). Mean cell
counts from 6 random fields and data represent the mean±SD of three
independent experiments. aP>0.05, bP<0.05, cP<0.01 vs control.
www.chinaphar.comOu YQ et al
to undergo remodeling in events such as inflammation,
wound healing, cancer, and arthritis. MMPs including
MMP-2 and MMP-9 contribute to joint destruction in RA
by enhancing the migration and invasion ability of mac-
rophages. Therefore, inhibition of MMPs is a primary thera-
peutic target in RA and improvements in therapeutic indices
may be achieved by targeting specific MMPs.
Sinomenine is a promising immunosuppressive drug that
has been widely used for treating autoimmune diseases like
RA because of its excellent therapeutic effect and relatively
insignificant side effect profile. However, the molecular
mechanism of SIN has not yet been completely elucidated.
Due to the crucial role of cell migration and invasion
in the pathology of RA and the important role of CD147
in the synthesis of gelatinases (MMP-2 and -9) in RA, the
effects of SIN on cell migration and invasion and its possible
mechanism were investigated using an in vitro transwell assay.
Human monocytic THP-1 cells were induced to differenti-
ate into macrophages with phorbol 12-myristate 13-acetate
(PMA)[25, 26], and cells were then treated with SIN at vari-
ous concentrations. Cellular invasion and migration ability
was inhibited by SIN, particularly at concentrations of 0.05
mmol/L (P<0.05), 0.25 and 1.00 mmol/L (P<0.01). The
expression of CD147, MMP-2, and MMP-9 mRNA, as mea-
sured by RT-PCR, was down-regulated by SIN at concentra-
tions of 0.25 and 1.00 mmol/L (P<0.05). The protein levels
of CD147, as measured by flow cytometric analysis, were
decreased by SIN at concentrations of 0.05 (P<0.05), 0.25
and 1.00 mmol/L (P<0.01). The enzyme activity of MMP-2
and MMP-9, as measured by Zymographic analysis, was sup-
pressed by SIN at concentrations of 0.05 (P<0.05), 0.25 and
1.00 mmol/L (P<0.01). These data demonstrate that SIN
can significantly repress the invasion and migration ability of
macrophages in a dose-dependent manner, and this repres-
sion strongly correlates with the inhibition of CD147, MMP-
2, and MMP-9 activity. Cell viability assay showed that all of
Figure 3. SIN repressed the mRNA expression of CD147, MMP-2,
and MMP-9 as tested by RT-PCR. (A) A-THP-1 cells (5×108 cells/L)
were treated with various concentrations of SIN for 48 h. The data are
representative of three independent experiments. (B) Quantification of
RT-PCR data. Values correspond to the mean±SD of three independent
experiments. aP>0.05, bP<0.05, cP<0.01 vs control.