Methanol extract of Elaeagnus glabra, a Korean medicinal plant, inhibits HT1080 tumor cell invasion.

Abstract

Elaeagnus glabra (Thunb.), an evergreen shrub with alternate leaves, has been used as a medicinal plant in Korea. Since many plant-derived molecules have inhibitory effects on tumor cell invasion, primarily via suppression of the activity of matrix metalloproteinases (MMPs), we investigated the effect of the methanol extract of E. glabra on tumor cell invasion. The invasiveness of HT1080 human fibrosarcoma cells were reduced in a dose-dependent manner following 24 h treatment of up to 200 microg/ml of the E. glabra extract, at which concentration no cytotoxicity occurred. Furthermore, gelatinolytic activities, and the protein and mRNA levels of MMP-2 and MMP-9 were also suppressed with increasing concentrations of the extract. Given that MMP-2 and MMP-9 are instrumental in tumor cell invasion, it is very likely that the reduction in tumor cell invasion by E. glabra extract is a consequence, at least in part, of suppressed expression of both MMP-2 and MMP-9. Isolation of a molecule(s) responsible for the extract inhibition of tumor cell invasion would pave the way for the development of a new generation of metastasis inhibitors.

Full text

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Abstract. Elaeagnus glabra (Thunb.), an evergreen shrub
with alternate leaves, has been used as a medicinal plant in
Korea. Since many plant-derived molecules have inhibitory
effects on tumor cell invasion, primarily via suppression of
the activity of matrix metalloproteinases (MMPs), we
investigated the effect of the methanol extract of E. glabra on
tumor cell invasion. The invasiveness of HT1080 human
fibrosarcoma cells were reduced in a dose-dependent
manner following 24 h treatment of up to 200 μg/ml of the
E. glabra extract, at which concentration no cytotoxicity
occurred. Furthermore, gelatinolytic activities, and the
protein and mRNA levels of MMP-2 and MMP-9 were also
suppressed with increasing concentrations of the extract.
Given that MMP-2 and MMP-9 are instrumental in tumor
cell invasion, it is very likely that the reduction in tumor cell
invasion by E. glabra extract is a consequence, at least in
part, of suppressed expression of both MMP-2 and MMP-9.
Isolation of a molecule(s) responsible for the extract
inhibition of tumor cell invasion would pave the way for the
development of a new generation of metastasis inhibitors.
Introduction
Matrix metalloproteinases (MMPs) are a family of zinc-
dependent endopeptidases that remodel and degrade the
extracellular matrix (ECM). More than 25 MMPs have been
identified to date and MMPs are classified based on their
substrate specificity and structural similarity (1-3). The ECM
has a complex structure that influences the function and
migration process of its resident cells by offering specific
contextual information (4). MMP-mediated degradation of
ECM is a hallmark in several pathologic conditions such as
arthritis, inflammation, cancer, angiogenesis, cardiovascular,
pulmonary, ocular, gastrointestinal and oral diseases (5,6).
Invasion and metastasis, both fundamental properties of
malignant cancer cells, are the end result of a complex series
of steps involving multiple tumor-host interactions (7,8).
Cancer cells metastasize through a series of the following
sequential steps: escape from the primary tumor, migration
and invasion of surrounding tissues, entrance into the
vasculature, transport through the circulatory system,
extravasation and growth in a secondary organ (7,9,10).
Among these steps, cancer cell migration and invasion of
surrounding tissues are mediated in part by MMPs, especially
MMP-2 and MMP-9 (11-13).
Many plant-derived compounds possess antitumor
activity (14-16) and many Korean medicinal plants have
been shown to exert an inhibitory effect on MMP-9 (17,18).
Elaeagnus glabra (Thunb.) is an evergreen shrub or small
tree with alternate leaves, belongs to the Elaeagnaceae
family, inhabits East Asia (especially Korea, Japan and
China) and is reported to have anti-bacterial, procoagulant,
anti-asthmatic and anti-diarrheal effects (19). Other species
of the Elaeagnus genus have been documented to possess
therapeutic effects as well. For example, E. angustifolia has
anti-nociceptive, anti-inflammatory and muscle relaxing
activities (20-22) and E. multiflora, anti-oxidant and anti-
inflammatory activities (23).
In this study, we investigated the inhibitory effect of the
methanol extract of the bark derived from E. glabra on tumor
invasion using a human fibrosarcoma cell line HT1080.
Materials and methods
Plant extract. The methanol extract of Elaeagnus glabra
bark was purchased from the Plant Extract Bank (Daejeon,
South Korea).
Cell culture. A human fibrosarcoma cell line HT1080 was
purchased from the American Type Culture Collection
(ATCC, USA). The cells were cultured in minimum essential
medium (MEM; Gibco, USA) containing penicillin (100
U/ml), streptomycin (100 μg/ml) and 10% fetal bovine serum
ONCOLOGY REPORTS 21: 559-563, 2009
559
Methanol extract of Elaeagnus glabra, a Korean
medicinal plant, inhibits HT1080 tumor cell invasion
LI-HUA LI1*, IN KYU BAEK1*, JIN HEE KIM1, KYUNG HO KANG1, YANG SEOK KOH2,
YOUNG DO JUNG1, CHOL KYOON CHO2, SEOK-YONG CHOI1and BOO AHN SHIN1
1The Brain Korea 21 Project, Center for Biomedical Human Resources; 2Department of Surgery,
Chonnam National University Medical School, Gwangju 501-190, Korea
Received October 6, 2008; Accepted November 29, 2008
DOI: 10.3892/or_00000257
_________________________________________
Correspondence to: Dr Boo Ahn Shin or Dr Seok-Yong Choi,
Research Institute of Medical Sciences, Chonnam National
University Medical School, Gwangju 501-190, Korea
E-mail: bashin@chonnam.ac.kr; zebrafish@chonnam.ac.kr
*Contributed equally
Key words: Elaeagnus glabra, matrix metalloproteinase, MMP-2,
MMP-9, cancer, metastasis

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(FBS; Gibco; denoted as complete MEM hereafter) at 37˚C
in 5% CO2air. After the cells adhered, the media was
replaced with serum-free MEM and the indicated con-
centration of the extract was added. Conditioned media
were collected and the cells were harvested after a 24-h
incubation at 37˚C in 5% CO2air.
MTT cell viability assay. HT1080 cells were seeded onto a
96-well culture plate at a density of 4x104cells/well in 200 μl
of complete MEM and incubated overnight. On the second
day of culture, the media was replaced with 200 μl of serum-
free MEM and treated with the indicated concentration of
E. glabra extract (0-200 μg/ml). On the third day, 100 μg of
3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium
bromide (MTT; Sigma, USA) was added to each well and
incubated for 4 h. The media was then discarded and 100 μl
of dimethyl sulfoxide (DMSO; Sigma) was added. Absor-
bance was measured at 490 nm on an ELISA reader.
Invasion assay. HT1080 cells (5x104) in 250 μl of complete
MEM were seeded in the upper chamber of a 10-well
chemotaxis chamber (Neuro Probe, USA) and serum-free
MEM was placed in the lower chamber. A Matrigel-coated
membrane was inserted between the two chambers.
Following overnight incubation at 37˚C, the media in the
upper chamber was replaced with serum-free MEM and
treated with the indicated concentration of E. glabra extract.
Upon an additional 24-h incubation at 37˚C in 5% CO2air,
the membrane was fixed and stained with a Hemacolor rapid
staining kit (Merck, Germany) as per manufacturer's
instructions.
Gelatin zymography. The quantity of protein in the
conditioned media was determined with a BSA protein assay
kit (Pierce, USA). Subsequently, the conditioned media was
mixed with an equal volume of 2X sample loading buffer
(62.5 mM Tris-HCl (pH 6.8), 25% glycerol, 4% sodium
dodecyl sulfate (SDS) and 0.01% bromophenol blue; Bio-
Rad, USA) and loaded onto a 7.5% acrylamide:bis-
acrylamide (29:1; Bio-Rad) gel containing 625 μg/ml gelatin
(Sigma). Upon electrophoresis at 100 V for 2 h, the gel was
soaked in 1X zymogram renaturation buffer (Bio-Rad) on a
rocker for 1.5 h at room temperature to remove residual SDS,
rinsed in distilled water, incubated at 37˚C for 18 h in 1X
zymogram development buffer (Bio-Rad), stained with
0.25% (w/v) coomassie brilliant blue R-250 (Bio-Rad) and
then destained in destaining buffer (10% acetic acid and 20%
methanol).
Western blotting. HT1080 cells were treated with the
indicated concentration of methanol extract of E. glabra for
24 h and the conditioned media were harvested. The cells
were then scraped into 1X cell lysis buffer (Cell Signaling,
USA) and incubated for 10 min on ice. The resulting cell
lysate was cleared by centrifugation at 6,700 x g at 4˚C for
5 min. The supernatant containing cytosolic proteins was
collected and the protein concentration of the supernatant and
the conditioned media was measured with a BSA protein
assay kit. The conditioned media or the cell lysate, with the
same amount of protein, was mixed with an equal volume of
2X sample loading buffer, boiled for 5 min, cooled on ice
for 5 min and then analyzed by 8% SDS polyacrylamide gel
electrophoresis (SDS-PAGE). Subsequently, the separated
proteins were transferred to a nitrocellulose membrane
(Amersham, USA). The membrane was blocked with 5%
skim milk in 1X TBST [0.01 M Tris (pH 7.6), 0.1 M NaCl
and 0.1% Tween-20] for 2 h at room temperature with
shaking and incubated with the indicated primary antibody,
followed by HRP-conjugated secondary antibody. The
immunoreactive protein bands were visualized with enhanced
chemiluminescent reagents (Amersham).
Northern blotting. HT1080 cells treated with the indicated
concentration of methanol extract of E. glabra for 24 h were
washed with ice-cold 1X phosphate buffered saline (PBS)
twice and the total RNA was extracted with TRIzol Reagent
(Invitrogen, USA) as per manufacturer's instructions. The
extracted total RNA (20 μg) was separated on a 1% agarose-
formaldehyde gel in 1X 4-morpholinepropanesulfonic acid
(MOPS) buffer [20 mM MOPS (pH 7.0), 1 mM EDTA (pH 8.0)
and 2 mM sodium acetate]. The separated RNA was
transferred onto a Hybond-N membrane (Amersham) by
capillary transfer in 20X SSC [3 M NaCl and 0.3 M sodium
citrate (pH 7.0)] and immobilized by UV cross-linking. The
membrane was incubated in Rapid-Hyb buffer (Amersham)
harboring human MMP-2 and MMP-9 cDNA probes radio-
labeled with [·-32P]dCTP using a random primer method,
washed and exposed to X-ray film.
Statistical analysis. Intensity of bands in images was
quantitated with the NIH ImageJ software (24). Results are
expressed as mean ± standard deviation. Statistical
significance was determined at p<0.05 using Student's t-test.
Results
E. glabra extract is not toxic to HT1080 cells. HT1080 is a
human fibrosarcoma cell line that has been extensively used
to study the migration and invasion of tumor cells. Moreover,
HT1080 cells express high levels of MMP-2 and MMP-9
(25), which play an important role in tumor cell invasion.
LI et al: INHIBITORY EFFECT OF Elaeagnus glabra ON TUMOR CELL INVASION
560
Figure 1. The methanol extract of E. glabra at concentrations up to 200 μg/ml
does not exert cytotoxicity on HT1080 cells. HT1080 cells in serum-free
MEM were left untreated or treated with the indicated concentration of the
E. glabra extract, incubated for 24 h and subjected to MTT assay for
quantifying cell growth. The bar graph shows the absorbance at 490 nm
measured on an ELISA reader (n=3 independent experiments; mean ±
standard deviation is shown).

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Therefore, we chose HT1080 cells to investigate the effect of
E. glabra on tumor cell invasion.
To test if the E. glabra extract is cytotoxic, HT1080 cells
were cultured with E. glabra extract (0-200 μg/ml) for 24 h
and then subjected to MTT assay. The methanol extract of
E. glabra did not significantly inhibit the growth of HT1080
cells at concentrations ranging between 0 and 200 μg/ml
(Fig. 1), suggesting that the E. glabra extract has no
significant effect on the HT1080 cell survival. Thus, we
performed all subsequent experiments with the extract
concentrations ranging between 0 and 200 μg/ml.
E. glabra extract inhibits HT1080 cell invasion. Since many
natural plant products exhibit an anti-invasiveness effect
(26), we questioned whether the E. glabra extract inhibits
tumor cell invasion. To this end, we carried out a Matrigel
invasion assay with HT1080 cells treated with the E. glabra
extract. The extract inhibited the invasive activities of
HT1080 cells in a dose-dependent manner (Fig. 2), demon-
strating that the E. glabra extract has an anti-invasiveness
effect, at least with respect to HT1080 cells.
E. glabra extract inhibits MMP-2 and MMP-9 activities.
Since MMP-2 and MMP-9 play a pivotal role in tumor cell
invasiveness, we wished to assess the effect of E. glabra
extract on MMP-2 and MMP-9 enzyme activities. For this
goal, we performed gelatin zymography with conditioned
media harvested from the extract treated HT1080 cells. The
gelatinolytic activities of both MMP-2 and MMP-9 were
reduced with increasing concentrations of the extract (Fig. 3),
suggesting that a decrease in HT1080 cell invasion is a
consequence, at least in part, of reduced activities of both
MMP-2 and MMP-9.
E. glabra extract reduces MMP-2 and MMP-9 protein levels.
MMP enzyme activity is regulated at both transcriptional and
post-transcriptional levels (27). To determine whether the
reduced MMP-2 and MMP-9 enzyme activities were caused
by a decrease in MMP-2 and MMP-9 protein levels, HT1080
cells treated with the E. glabra extract were processed for
Western blotting along with their conditioned media. MMP-2
and MMP-9 protein levels were reduced in a dose-dependent
manner in both conditioned media (Fig. 4B and D) and cells
treated with the extract (Fig. 4A and C), indicating that
decreased gelatinolytic activities of MMP-2 and MMP-9 in
the extract treated cells ensue from, at least in part, the down-
regulation of MMP-2 and MMP-9 proteins.
E. glabra extract suppresses MMP-2 and MMP-9 mRNA
levels. To examine whether the decreased MMP-2 and
MMP-9 protein levels in the HT1080 cells treated with the
E. glabra extract result respectively from the reduction in
MMP-2 and MMP-9 mRNA levels, we turned to Northern
blotting to measure their mRNA levels. Expression of both
ONCOLOGY REPORTS 21: 559-563, 2009
561
Figure 3. The methanol extract of E. glabra inhibits gelatinolytic activities
of MMP-2 and MMP-9. (A) The conditioned media harvested from the
HT1080 cells treated for 24 h with the indicated concentration of the E. glabra
extract were analyzed by gelatin zymography. The white bands represent
MMP-mediated gelatin digestion. The image is representative of three
independent experiments. Quantitation of band intensity is shown in B
(n=3). Numbers in the box represent the concentration of the extract in μg/ml
added to the cells. Bars represent the average intensity of each band ± standard
deviation.
Figure 2. The methanol extract of E. glabra impedes invasiveness of HT1080 cells. A 10-well chemotaxis chamber was used to measure HT1080 cell
invasiveness upon treatment with the indicated concentration of the E. glabra extract. A Matrigel-coated membrane inserted between the upper and lower
chambers was stained with a Hemacolor rapid staining kit. The stained area represents cells which have migrated from the upper chamber. The number in
each panel denotes the concentration of the E. glabra extract added. Each image is representative of 3 independent experiments.

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MMP-2 and MMP-9 mRNA decreased in the extract treated
cells with increasing concentrations of the extract (Fig. 5),
signifying that the decrease in MMP-2 and MMP-9 protein
levels in the extract treated cells reflects, at least in part, the
reduction in MMP-2 and MMP-9 mRNA levels, respectively.
Discussion
Herein, we show that the methanol extract of E. glabra
suppresses cell invasion, gelatinolytic activities, and protein
and mRNA expressions of both MMP-2 and MMP-9 in
HT1080 cells. In light of the instrumental role for MMP-2
and MMP-9 in tumor cell invasion, it is very likely that E.
glabra extract inhibition of MMP-2 and MMP-9 expression
leads to a decrease in gelatinolytic activity of MMP-2 and
MMP-9, which in turn dampens tumor cell invasion.
However, we cannot rule out the following possibilities: i)
the E. glabra extract inhibits tumor cell invasion indepen-
dently of reduction in MMP-2 and MMP-9 activity or
expression; ii) the E. glabra extract directly inhibits MMP-2
and MMP-9 proteins independently of a reduction in MMP-2
and MMP-9 expression, leading to suppression of tumor cell
invasion; iii) the E. glabra extract promotes activity of MMP
inhibitors, e.g., tissue inhibitors of metalloproteinase (TIMP),
thereby repressing MMP activity. Further work is required to
clarify this issue.
Even though we demonstrate here that the E. glabra
extract inhibits HT1080 tumor cell invasion, which
molecule(s) in the extract is responsible for the inhibitory
effect remains unknown. There have been several studies
on plant-derived molecules inhibiting MMP or tumor cell
invasion. For instance, polyphenol, epigallocatechin gallate
and epicatechin gallate, all isolated from green tea,
individually inhibit MMP-2 and MMP-9 (28,29); obovatal
isolated from Magnolia obovata, MMP-2 and tumor cell
invasion (30); curcumin, isolated from Curcuma longa,
MMP-2, MMP-9 and tumor metastasis (31); and quercetin, a
flavonol present in many vegetables and fruits including
onions and apples, MMP-2 and MMP-9 (32). Therefore, it
would be of high interest to explore whether it is one of these
molecules, a novel molecule(s), or both that accounts for the
inhibitory effect of the E. glabra extract on tumor cell invasion.
Although MMP is a promising target for anticancer
therapy, efforts to develop MMP inhibitor with few side
effects have been unsuccessful thus far. In this regard,
screening natural products, especially plant products, for
anti-MMP activity followed by determining which molecule
in the product is responsible for the activity would shed light
on the development of a new generation of MMP inhibitors.
Acknowledgements
We thank Cheorl-Ho Kim for critical reading of the manuscript
and Eun Young Choi for encouragement. This work was
supported by Chonnam National University, 2008 and the
Korea Science and Engineering Foundation through the
Medical Research Center for Gene Regulation (R13-2002-
013-06002-0) at Chonnam National University.
LI et al: INHIBITORY EFFECT OF Elaeagnus glabra ON TUMOR CELL INVASION
562
Figure 5. The methanol extract of the E. glabra suppresses expression of
MMP-2 and MMP-9 mRNAs in HT1080 cells. (A) The cells were treated
with the indicated concentration of E. glabra extract for 24 h and then
processed for Northern blotting. Ribosomal RNA 18S was used as a loading
control. Images are representative of 3 independent experiments. Quantitation
of band intensity in A normalized to that of ribosomal RNA 18S is shown in
B (n=3). Numbers in the box represent the concentration of the extract in
μg/ml added to the cells. Bars denote the normalized average intensity of
each band ± standard deviation.
Figure 4. The methanol extract of E. glabra reduces expression of MMP-2
and MMP-9 proteins in HT1080 cells. The cells were treated with the
indicated concentration of the E. glabra extract for 24 h. Subsequently, the
cells (A and C) and their conditioned media (B and D) were processed for
Western blotting. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
was used as a loading control. Each image is representative of 3 independent
experiments. Quantitation of MMP-2 and MMP-9 band intensity in A
normalized to that of GAPDH is shown in C (n=3). Quantitation of band
intensity in B is shown in D (n=3). Numbers in the box in C and D represent
the concentration of the extract in μg/ml added to the cells. Bars indicate the
average intensity of each band ± standard deviation.

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