Chinese Medicine, 2009, 1, 30-35
Published Online September 2009 in SciRes (www.SciRP.org/journal/cm)
Original Article Inhibitory Effect of Ginsenoside Rg3 on
Ovarian Cancer Metastasis
ABSTRACT
Ginsenosides are main components extracted from ginseng, and ginsenoside Rg3 is one of the most important parts.
Ginsenoside Rg3 has been found to inhibit several kinds of tumor growth and metastasis. The present study was under-
taken to investigate the effect of ginsenoside Rg3 on human ovarian cancer metastasis and the possible mechanism. The
experimental lung metastasis models of ovarian cancer SKOV-3 and the assay of tumor-induced angiogenesis were
used to observe the inhibitory effects of Rg3 on tumor metastasis and angiogenesis. The effect of Rg3 on invasive ability
of SKOV-3 cells in vitro was detected by Boyden chamber, and immunofluorescence staining was used to recognize the
expression of matrix metalloproteinase 9 (MMP-9) in SKOV-3 cells. Ginsenoside Rg3 can significantly inhibit the me-
tastasis of ovarian cancer. The inhibitory effect is partially due to inhibition of tumor-induced angiogenesis and de-
crease of invasive ability and MMP-9 expression of SKOV-3 cells.
Keywords: ginseng, neoplasm, metastasis, angiogenesis, ovarian cancer
1. Introduction
Ginseng is a medicinal herb widely used in Asian coun-
tries for its wide spectrum of medicinal effects such as
tonic, immunomodulatory, adaptogenic, and anti-aging
activities.1These effects are attributed to the triterpene
glycosides known as ginsenosides and Rg3 is one kind of
the ginsenosides. The molecular formula of ginsenoside
Rg3 is C42H72O13 and its molecular weight is 784.30.
Ginsenoside Rg3 can inhibit catecholamine secretion,
protect cultured cortical cells from glutamate-induced
neurodegeneration, and anti-contraction of vascular
smooth muscle.2-4 Researchers found that ginsenoside
Rg3 can also resist tumor. For example, it inhibited inva-
sion and metastasis of B16 melenoma without impairing
cell growth, and proliferation of tumor cells combined
with cyclophosphamide.5-7 However, the effect of ginse-
noside Rg3 on human ovarian cancer has not been iden-
tified and the mechanism of its anti-tumor is unknown.
The present study was to assess the effects of ginse-
noside Rg3 on angiogenesis and metastasis produced by
human ovarian cancer and on the invasion and the matrix
metalloproteinase 9 (MMP-9) expression of SKOV-3
cells in vitro.
2. Methods
2.1. Drug and Reagents
Ginsenoside Rg3 was provided by Department of Or-
ganic Chemistry of Preclinical Medicine of Jilin Univer-
sity. Its purity is more than 99.5%. Athymic mice were
purchased from the Department of Experimental Ani-
mals of Jilin University. Boyden chamber and matrigel
were purchased from BD Company, USA.
2.2. Cell Culture
Human ovarian cancer SKOV-3 cell line and mouse
NIH3T3 fibroblast cell were obtained from the Tumor
Research Department of Jilin Province and maintained in
RPMI1640 supplemented with 10% fetal bovine serum
(FBS).

T. M. XU ET AL.31
Table 1. Inhibitory effect of ginsenoside Rg3 on experimental lung metastasis and angiogenesis caused by ovarian cancer
*P <0.01, **P <0.001 compared with control group
2.3. Assay for Experimental Lung Metastasis
In 40 mice, each was injected with SKOV-3 cells (2×105)
into the lateral tail vein. The mice were randomly di-
vided into 4 groups (n=10 for each group): Rg3 groups
(0.3, 1.0 and 3.0 mg/kg) and control group. Ginsenoside
Rg3 was injected intraperitoneally at daily doses (0.3,
1.0 and 3.0 mg/kg) to the tumor-bearing mice the next
day after tumor inoculation. The mice were killed 20
days after the inoculation. The lungs were fixed in
Bouin’s solution and the lung tumor colonies were
counted under a dissecting microscope.
2.4. Cell Invasion Assay
The invasive activity of SKOV-3 cells was assayed in
Boyden chambers. The upper surface of the filters in
Boyden chambers was precoated with 60 µl of matrigel.
The SKOV-3 cells were harvested, washed 3 times and
re-suspended to a final concentration of 1×106/ml in
RPMI1640. Then the cells were divided into 3 groups
after pretreatment with ginsenoside Rg3 of different
concentrations (0, 2.5 and 5.0 µg/ml) at 37°C for 30
minutes. 200 µl of each cell suspension was added to the
upper compartment of the chamber and 200 µl of condi-
tioned medium of NIH3T3 cells to the lower compart-
ment. After 5-hour incubation, the cells on the upper
surface of the filters were removed by wiping with cot-
ton swabs and the filters were fixed with FAA solution
and stained with hematoxylin and eosin. The cells that
had invaded through the matrigel and filters into the
lower surface were manually counted under a micro-
scope at a magnification of ×400. Each assay was per-
formed in triplicate. The data were expressed as the
number of invaded cells/field.
2.5. Assay for Tumor-induced Angiogenesis
In 20 athymic mice, each was inoculated intradermally
with SKOV-3 cells (5×105) on the back. They were ran-
domly divided into 4 groups (n=5): Rg3 groups (0.3,
1.0 and 3.0 mg/kg) and control group. Ginsenoside
Rg3 at various doses was injected intraperitoneally to the
mice 1, 2, 3, 4 and 5 days after tumor inoculation. Two
days later, the mice were sacrificed and the skin was
separated from the underlying tissues. Angiogenesis was
quantified by counting the number of vessels oriented
toward the tumor mass under a dissecting microscope.
2.6. MMP-9 Immunofluorescence Studies
The SKOV-3 cells that were seeded onto 24-well plates
and treated with 0, 2.5 and 5.0 µg/ml of ginsenoside Rg3
for 24 hours were used for immunofluoresecent staining.
First, the cells were washed briefly with phosphate buff-
ered saline three times and then fixed in 4% phosphate
buffered paraformaldehyde for 30 minutes. Second, they
underwent permabilization with the addition of phos-
phate-buffered 0.1% Triton X-100 for 10 minutes and
then were incubated in 1% bovine serum albumin for 30
minutes all at room temperature. Third, the cells were
incubated with mouse anti-human MMP-9 monoclonal
antibody (1: 100 dilution) and then with goat anti-mouse
fluorescein isothiocyanate (FITC) antibody respectively
for 1 hour at 4°C. Finally, the coverslips were mounted
and sealed for examination under a confocal microscope.
2.7. Statistical Analysis
Measurement data were evaluated by one-way analysis
of variance (ANOVA) for multiple group comparisons,
and the LSD test for two-group comparisons. Ranked
data were evaluated by the Kruskal-Wallis test for multi-
ple group comparisons and the Nemenyi test for
two-group comparisons. P <0.05 was considered statis-
tically significant.
3. Results
3.1. Effect of Ginsenoside Rg3 on Experimental
lung Metastasis of SKOV-3 cells
As shown in Figure 1, after intraperitoneal injection of
ginsenoside Rg3 at the doses of 0.3, 1.0 and 3.0 mg/kg,
the number of tumor colonies in the lung was lower than
that of the control group (P <0.01). The results indicate
that ginsenoside Rg3 can significantly inhibit the lung
metastasis of SKOV-3 cells.
3.2. Inhibitory Effect of Ginsenoside Rg3 on
Tumor-induced Angiogenesis
The number of vessels oriented toward the tumor mass in
the three groups that the tumor-bearing mice were given

T. M. XU ET AL.
32
intraperitoneal injection of ginsenoside Rg3 at the doses
of 0.3, 1.0 and 3.0 mg/kg was less than that of the control
group (Table). This indicates that ginsenoside Rg3 may
inhibit ovarian tumor-induced angiogenesis.
3.3. Cell invasion Assay
After the SKOV-3 cells were treated with ginsenoside
Rg3 at three concentrations of 0, 2.5 and 5.0 µg/ml, the
number of the cells invading through matrigel and filters
into the lower surface was 157.3±29.4, 110.8±25.6 and
92.5±18.4 respectively. Among the three groups, the
group of 0 µg/ml ginsenoside Rg3 was control group.
Statistical analysis illustrated that the number of cells
invading filters of the ginsenoside groups was less than
that of the control group (P <0.001, Figure 1), and that
ginsenoside Rg3 can depress the ability of invading of
SKOV-3 cells.
3.4. Assay of MMP-9 Immunofluorescence
The SKOV-3 cells were pretreated with ginsenoside Rg3
at doses of 2.5 and 5.0 µg/ml for 24 hours and im-
munofluorescence staining was used to recognize the
expression of MMP-9 in these tumor cells. Figure 2
shows that cytoplasmic fluorescence intensity in the
SKOV-3 cells was depressed as the dose of ginsenoside
Rg3 increased. This result shows that ginsenoside Rg3
can depress the expression of MMP-9 in the SKOV-3
cells.
Figure 1. Inhibitory effect of ginsenoside Rg3 on the invasion of SKOV-3 cells. The SKOV-3 cells were pretreated with gin-
senoside Rg3 at doses of 2.5 and 5.0 µg/ml for 30 minutes and then were added to Boyden chambers. After 5-hour incubation,
the cells that had invaded through the matrigel and filters were manually counted. A: control group; B: 5.0 µg/ml ginsenoside
Rg3 group (HE, original magnification ×400)

T. M. XU ET AL.33
Figure 2. Immunofluorescence staining of MMP-9 antibody in SKOV-3 cells. The SKOV-3 cells were pretreated with ginse-
noside Rg3 at doses of 2.5 and 5.0 µg/ml for 24 hours and immunofluorescence staining was used to recognize the expression
of MMP-9. A: control group; B: 2.5 µg/ml ginsenoside Rg3 group; C: 5.0 µg/ml ginsenoside Rg3 group (original magnifica-
tion ×400)

T. M. XU ET AL.
34
4. Discussion
Ovarian cancer is a severe disease threatening the health
and life of women. The 5-year survival rate of patients
with such disease remains low after conventional treat-
ment.8 Thus it is necessary to develop new agents and
methods for a cure.
With the development of Chinese herbal medicine,
researchers are increasingly interested in detecting
anti-tumor components in Chinese herbal medicine.
Ginsenoside Rg3, a saponin extracted from ginseng, was
found to have a high anti-tumor effect. It inhibited an-
giogenesis of Lewis lung carcinoma,9 invasion and me-
tastasis of intestinal adenocarcinoma and B16 melen-
oma,5,6 and proliferation of prostate cancer cell.10,11
The
present study demonstrated that ginsenoside Rg3 can
also inhibit the metastasis caused by ovarian cancer,
which will benefit patients with ovarian cancer.
The ability to invade tissues and establish colonies at
remote sites is a definite characteristic of malignant neo-
plasms. As a complex cascade it is affected by many
factors. Angiogenesis is a key step in tumor invasion and
metastasis.12 Since massive formation of blood vessels at
the tumor site increases the opportunity for tumor cells to
enter the circulation, the suppression of angiogenesis will
decrease the metastasis of malignant tumor. In this study,
the number of vessels oriented toward the tumor mass
after intraperitoneal injection of ginsenoside Rg3 at the
doses of 0.3, 1.0 and 3.0 mg/kg was less than that of the
untreated group, indicating that ginsenoside Rg3 may
inhibit ovarian tumor-induced angiogenesis, and subse-
quently decrease the metastasis of ovarian cancer.
In addition, tumor invasion and adhesion to an ex-
tracellular matrix and basement membrane components
are important events in the process of tumor metastasis.
In this study, matrigel, an analogue of basement mem-
brane, was used to determine the effect of ginsenoside
Rg3 on invasion of ovarian cancer cells in vitro. Because
the tumor cells adhere to matrigel, then invade and cross
the matrigel, we can evaluate the invasion of tumor cells
by the number of cells passing the matrigel. We observed
that the number of the SKOV-3 cells invading through
the matrigel and filters into the lower surface decreased
after they were treated with ginsenoside Rg3. This indi-
cates that ginsenoside Rg3 can prevent the invasion of
ovarian cancer cells.
However, angiogenesis as well as tumor invasion and
metastasis depend heavily on the controlled interactions
between the cells and the extracellular matrix (ECM).13
These interactions are mediated by integral membrane
proteins and extracellular proteinases. Extracellular pro-
teolysis plays an important role in many aspects, includ-
ing basement degradation and cell migration/ extracellu-
lar matric invasion, and is mediated by metallopro-
teinases (MMPs) and serine proteinases.14,15
MMPs belong to a family of zinc-dependent endopep-
tides.
They secrete as inactive pro-enzymes and are acti-
vated by partial proteolytic cleavage.16 MMP-9 or the
dominant MMPs released by most endothelial cells and
tumor cells appear to play an important role in degrada-
tion of basement membrane type VI collage and other
matrixproteins.17-19 Therefore, MMP-9 is regarded as
marker of tumor invasion and metastasis, and the sup-
pression of its expression may inhibit malignant tumor
invasion and metastasis.20 The results of the present
study demonstrated that ginsenoside Rg3 depressed the
secretion of ovarian cancer cells, which may be the
channel by which ginsenoside Rg3 inhibits the metasta-
sis of ovarian cancer.
In conclusion, ginsenoside Rg3 can significantly in-
hibit the metastasis of ovarian cancer. The inhibitory
effect is partially due to the inhibition of tumor-induced
angiogenesis and decrease of the invasive ability, which
may be related to the depression of MMP-9 expression
of ovarian cancer cells.
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