Introduction
CRC is one of the leading causes of death worldwide
(1). Aggressive local invasion and
metastasis make CRC difficult to treat (2). Therefore clarifying the mechanism of
invasion of CRC to find new treatment targets is urgent.
Metastasis of cancer cells is a complex processes
during which the degradation of the extracellular matrix (ECM)
plays an important role (3).
MMP-2/−9 play important roles in degrading ECM and its involvement
is complex in the process of cancer metastasis (3,4).
Overactivity of ERK signaling pathway has been found in almost half
of known human tumor cell lines and in many human primary tumors
derived from different origins (5).
ERK signaling pathway plays a vital role in the synthesis of
MMP-2/−9 (3,6). Overactivity of ERK pathway is observed
in CRC (7,8) and is correlated with the metastasis of
CRC (9,10).
Baicalein is extracted from the roots of
Scutellaria baicalensis or Scutellaria radix. The
chemical structure of baicalein is shown in Fig. 1A. The antitumor biological effect of
baicalein has been found in many tumors (11–13).
Baicalein has been found to possess antitumor effect in
hepatocellular carcinoma, glioma and bladder cancer (3,14–16),
but the anti-metastatic effect and related mechanism(s) in CRC are
still unclear. Thus, the present study investigated the effects of
baicalein in CRC invasion and metastasis and related
mechanisms.
Materials and methods
Cell lines and reagents
CRC HT29 and DLD1 cell lines were purchased from the
American Type Culture Collection (ATCC; Manassas, VA, USA) and
cultured in RPMI-1640 (HyClone Laboratories, Inc., Logan, UT, USA)
medium with 10% fetal bovine serum (FBS; HyClone Laboratories). The
cells were cultured at 37°C with 5% CO2. Anti-MMP-2,
ERK, MMP-9 and p-ERK were purchased from Cell Signaling Technology
(Beverly, MA, USA). Unless otherwise indicated, all the other
chemicals were purchased from Sigma-Aldrich (St. Louis, MO,
USA).
Apoptosis assays
Apoptotic and/or necrotic cells were analysed by
propidium iodide (PI) uptake using Annexin V binding an Annexin
V-FITC/PI kit as previously described (15). Briefly, CRC cells were seeded into
6-well plates at a density of 1×105 cells/well for 12 h
and treated with 0, 40, 60 and 80 µM of baicalein for 48 h. After
washing with cold phosphate-buffered saline (PBS), cells were
resuspended in Annexin V binding buffer. Cells were incubated in
Annexin V-FITC for 15 min, washed and then incubated with PI.
Samples were detected by flow cytometer with CellQuest
software.
Cell growth assay and focus formation
assay
Cell growth rate was detected using the MTT assay.
Briefly, cells were seeded in a 96-well plate at a density of
1.5×103 cells. Cells were treated with baicalein (0, 10,
20, 30, 40, 50, 60, 80, 100 and 120 µM) (R&D Systems, Inc.,
Minneapolis, MN, USA) for 24 h. MTT assay was done in accordance
with the manufacturers instructions. Each experiment was done in
triplicate and data were expressed as mean ± SD. In focus formation
assay, 100 cells were firstly seeded onto a plate and then cultured
with (0, 40, 60 and 80 µM) baicalein for 2 weeks. The plates were
fixed and then stained with 1% crystal violet. All experiments were
performed in triplicate.
Construction of expression plasmids
and transfection
The construction of the expression plasmids and
their transfection was performed in accordance with the
manufacturers instructions (3).
First, the full-length pcDNA3.1 (Invitrogen, Carlsbad, CA, USA)
MEK1 vector was made by cloning the full-length PCR product of MEK1
with KOD® DNA polymerase (Toyobo Co., Ltd., Osaka,
Japan). Then we measured the plasmid sequences to confirm the
sequences of the plasmid. In transient transfection experiments,
cells were plated at a density of 2×105 cells/well in a
6-well plate, 24 h later they were transfected with 4.0 µg
pcDNA3.1(+)-MEK1 vector via Lipofectamine 2000 (Invitrogen) in
accordance with the manufacturers protocol. The 4.0 µg pcDNA3.1(+)
empty vector was used as a negative control.
Cell invasion and migration assay
The migration and invasion ability of cells were
detected by Transwell assays. Migration and invasion assay was
performed with 24-well Transwell chambers coated with Matrigel or
not (Becton-Dicknson, Billerica, MA, USA) as previously described.
For the migration assay, cells were seeded into inner well and
cultured in medium with 0, 10, 20 and 30 µM baicalein. After 16 h,
cells on the bottom side of the inner well were fixed in alcohol,
fixed in crystal violet and counted. Invasion assay was performed
with 24-well Transwell chamber coated with Matrigel as previously
described (3).
Western blotting assay
After treated with U0126 or baicalein,
2×106 cells were added into 200 µl of lysis buffer
(Fermentas, Waltham, MA, USA). By 10% SDS-polyacrylamide gel
electrophoresis, the proteins (60 µg) were separated and bloted
onto PVDF membranes. To block non-specific binding, the PVDF
membranes were subsequently blocked in Tris-buffered saline with
Tween-20 (TBST) containing 5% defatted milk buffer for 1 h at 37°C
and were then incubated with antibodies against ERK, p-ERK, MMP-2,
MMP-9 or β-actin overnight in defatted milk 5% in TBST at 4°C. The
membranes were incubated with a horseradish peroxidase goat
anti-rabbit or anti-mouse IgG antibody at room temperature for 1 h.
The pictures were examined with an enhanced chemiluminescence kit
(ECL Plus; Amersham, Freiburg, Germany) according to the
manufacturers instructions and captured by autoradiography. The
relative photographic density was quantified with ImageJ software
(GE Healthcare, Buckinghamshire, UK) and expressed as arbitrary
units (a.u.).
Tumor xenograft experiments and
animals
To assess the anti-proliferation of baicalein in
vivo, Balb/c athymic nude mice (4- to 6-week-old) were obtained
from Shanghai SLAC Laboratory Animal, Co., Ltd., (Shanghai, China).
Each mouse was subcutenously implanted with 1×106 viable
DLD1 cells in the right flank area. One group was treated with
vehicle dimethyl sulfoxide (DMSO), the other group was treated with
baicalein (20 mg/kg/day) for 3 weeks. At day 21, all the mice were
sacrificed and the tumor tissues were removed and weighed. The
experimental protocols used herein were evaluated and approved by
the Animal Care and Use Committee of the Medical School of Xian
Jiaotong University.
Results
Baicalein inhibits the proliferation
of HT29 and DLD1 cells
The inhibition effects of baicalein on the
proliferation of CRC cells at different concentrations (0 to 120
µM) for 24 h are shown in Fig. 1B.
At concentrations >40 µM, the anti-proliferation effect of
baicalein on HT29 and DLD1 cells was significant so we chose
concentrations <40 µM for all the following experiments.
To determine the effect of baicalein on apoptosis in
detail, HT29 cells were incubated for 48 h with baicalein (40, 60
or 80 µM) and then analysed by flow cytometry. As shown in Fig. 1C, the effect of baicalein on
apoptosis of HT29 cells was concentration-dependent. After
treatment for 48 h, both early and late apoptotic cells increased
significantly in the HT29 cells treated with baicalein. Baicalein
reduced the number of colony HT29 cells in vitro
significantly (Fig. 1D).
Baicalein inhibits the metastasis of
HT29 and DLD1 cells
Fig. 2 shows the
inhibition effect of baicalein (0, 10, 20 and 30 µM) on cell
migration (16 h) and invasion (24 h) in HT29 and DLD1 cells,
respectively. We found that inhibition effect of baicalein on the
migration (Fig. 2A and B) and
invasion (Fig. 2C and D) of HT29
and DLD1 cells was concentration-dependent.
 | Figure 2.Baicalein inhibits the migration and
invasion of CRC cells. (A) HT29 and DLD1 cells were pretreated with
0, 10, 20 and 30 µM baicalein for 24 h. Then seeded onto the upper
wells, FBS (10%) was added to the bottom chambers for 16 h to
induce cell migration. After 16 h, cells on the bottom side of the
filter were fixed, stained and counted. (B) The percentage of
migration rate was expressed as a percentage of control (0 µM). (C)
HT29 and DLD1 cells were pretreated with 0, 10, 20 and 30 µM
baicalein for 24 h. Then seeded onto the upper wells, FBS (10%) was
added to the bottom chambers for 24 h to induce cell invasion.
After 24 h, cells on the bottom side of the filter were fixed,
stained and counted. (D) The percentage of invasive rate was
expressed as a percentage of control (0 µM). Values are presented
as means ± SD of three independent experiments performed in
triplicate. **P<0.01 compared with the control group,
respectively. |
Baicalein inhibits the expression of
MMP-2/−9
We analysed the effect of baicalein on the
expression of MMP-2/−9 in HT29 and DLD1 at various concentrations.
Cells were treated with baicalein (0, 10, 20 and 30 µM) for 24 h
and then analysed by western blotting. Fig. 3A and B show the inhibited effect of
baicalein on the expression of MMP-2/−9 in HT-29 cells. The effect
is concentration-dependent. Fig. 3C and
D show the effect of baicalein on the expression of MMP-2/−9 in
DLD1 cells.
The ERK signaling pathway is involved
in the anti-metastatic mechanism of baicalein in CRC
ERK signaling pathway plays an vital role in the
metastasis of cancer cells by regulating MMP-2/−9 (3); hence, we detected the impact of
baicalein on the activity of ERK signaling pathway in CRC cells.
The results of the western blotting showed that baicalein reduced
the phosphorylation level of ERK1/2 in DLD1 cells treated with
baicalein (Fig. 4A and B). To
further check the role of ERK signaling pathway in the
anti-metastatic effect of baicalein, we upregulated the activity of
ERK singnaling pathway in DLD1 cells via transfecting the plasmid
[pcDNA3.1 (+)-MEK1] expressing human MEK1 and found that the
anti-invasion effect of baicalein was reversed by the high
expression of MEK1 (Fig. 4C). The
positive clones were selected by G418. DLD1 cells were separated
into 3 groups: no treatment as the control group ‘C’, transfected
with an empty vector pcDNA3.1(+) as the negative control group ‘N’,
transfected with a pcDNA3.1(+)-MEK1 as the positive group ‘M’.
The inhibition rate was ~61.3 and 39.3% after 24 h
of treatment with 0 or 30 µM of baicalein (Fig. 4D). Baicalein could also inhibit the
phosphorylation level of ERK in the HT29 cells (data not shown).
Our results also show that the combined treatment with the
baicalein and U0126 (an ERK inhibitor) reduced both the MMP-2/−9
protein expression (Fig. 5A and B)
and the cell invasion significantly (Fig. 5C and D).
Baicalein inhibits the growth of CRC
tumors in vivo
The inhibition effect of baicalein on DLD1 xenograft
growth is shown in Fig. 6A. We
found that baicalein could inhibit CRC growth significantly. At day
21, all the mice were sacrificed and the tumor tissues were removed
and weighed. Compared to the control group, baicalein reduced the
volume of solid tumors significantly (Fig. 6B). We also found that baicalein
significantly inhibited phosphorylation level of ERK and the
expression of MMP-2/−9 in vivo. The inhibition effect of
baicalein on the phosphorylation level of ERK is shown in Fig. 6C and D, and on expression of
MMP-2/−9 is shown Fig. 6E and F
in vivo.
Discussion
Baicalein possesses antitumor effect in many tumors
(17–19), but the reports on baicalein effect
are rare in CRC. We found that baicalein significantly inhibited
the migration and invasion of CRC cells by inhibiting MMP-2/−9
expression via inhibiting the ERK signaling pathway.
Our results showed that baicalein inhibited the
proliferation and colony formation ability of CRC cells and the
mechanism is correlated with apoptosis. Transwell chamber is
generally used as an assay to detect the metastasis ability of
cancer cells. In the study, we chose the concentration below
cytotoxicity to detect the effect of baicalein on the migration of
CRC cells. We found that baicalein could inhibit the migration and
invasion ability of CRC cells. Our finding is similar to previous
research in other cancers (3,11,15,16),
in which baicalein could inhibit the proliferation of hepatocellar
carcinoma cells, glioma and breast cancer cells. As concentrations
are below the cytotoxicity concentration, the inhibition effect is
not correlated with the cytotoxicity of baicalein. To the best of
our knowledge, we first show the effect of baicalein on DLD1 CRC
cells whicn was used for research on proliferation, apoptosis and
invasion of CRC (20–22). The inhibitory properties are
probably related to the specific structural features of baicalein
(as shown in Fig. 1A). It was
reported that the hydroxylation of C5 and C7 in A-ring
significantly improved the anticancer activity of flavonoids over
that of 5-fluorouracil (23).
Moreover, it has been demonstrated that the hydroxyl substitutions
in the A-ring (C7) of baicalein are crucial for its anti-metastatic
effects against human hematoma cells (24).
To date, metastasis of CRC causes thousands of
deaths every year worldwide (25).
Degradation of the ECM of lymph or blood vessels excert important
roles in the metastasis of cancers via allowing the invasion of
cancer cells into the circulation system and invade distant organs
or tissues (26). MMPs, especially
MMP-2/−9, play important roles in degrading ECM (27). Baicalein shows inhibition effect on
the expression of MMP-2/−9 in hepatocellular carcinoma and glioma
(3,16). Our results showed the inhibition
effect of baicalein on MMP-2/−9 expression in CRC cells. The
results suggest that MMP-2/−9 play important roles in the
anti-metastatic effect of baicalein in CRC cells.
Multiple signaling cascades play important roles in
the synthesis of MMPs, especially the ERK signaling pathway
(19,28,29).
ERK signaling pathway plays an important role in tumor invasion via
promoting the degradation of ECM proteins (30,31).
Studies have shown that the inhibition of ERK phosphorylation
reduces the expression of MMP-2/−9 in the CRC cells (32,33).
To investigate the related mechanism(s) of the inhibition effect of
baicalein on CRC metastasis, we detected the activity of ERK
signaling pathway in CRC cells. The results proved that baicalein
reduced the activity of ERK signaling pathway significantly.
Upregulation of ERK signaling pathway activity abolished the
baicaleins antimetastatic effect. The results was similar to a
previous study, in which baicalein inhibited the expression and
activity of MMP-2 and MMP-9 via inhibiting the activity of ERK
signaling pathway (3).
Our results also showed that combined treatment with
baicalein and U0126 (an ERK inhibitor) reduced both the MMP-2/−9
protein expression (Fig. 5A and B)
and the cell invasion significantly (Fig. 5C and D) suggesting that baicalein
directly downregulated the ERK signaling pathway, which were
similar to previous studies conducted in other types of carcinomas
(3). Finally, our results
demonstrated that baicalein can inhibit the growth of CRC
xenografts in vivo.
In conclusion, the present study demonstrated the
anti-metastatic effect of baicalein on CRC. Furthermore, ERK
signaling pathway plays a vitally important role in the
anti-metastatic effect of baicalein on CRC cells by regulating
MMP-2/−9. These findings revealed that baicalein may represent a
new potential anti-metastatic therapy for CRC.
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