The crude extract of Corni Fructus inhibits the migration and invasion
of U-2 OS human osteosarcoma cells through the inhibition of matrix
metalloproteinase-2/ -9 by MAPK signaling
Ching-Lung Liao1, Ju-Hwa Lin2, Jin-Cherng Lien3, Shu-Chun Hsu2, Fu-Shin Chueh4, Chien-Chih Yu5, Ping-Ping Wu5, Yi-Ping Huang6, Jaung-Geng Lin1,* and Jing-Gung Chung2,7,*
1Graduate Institute of Chinese Medicine, Departments of 2Biological Science and Technology and 6Physiology, 3Graduate Institute of Pharmaceutical Chemistry, 5School of Pharmacy, China Medical University, Taichung 404,4Department of Health and Nutrition
Biotechnology, Asia University, Taichung 413; 7Department of Biotechnology, Asia University, Taichung 413, Taiwan
Running title: CECF inhibited migration and invasions of human osteosarcoma cells
*Correspondence to: Prof. Jing-Gung Chung, Department of Biological Science and Technology, China Medical University, No 91, Hsueh-Shih Road, Taichung 40402, Taiwan. Tel: +886-4-22053366 ext 2161. Fax: +886-4-22053764.
E-mail: [email protected]
Prof. Jaung-Geng Lin, School of Chinese Medicine, China Medical University, No 91, Hsueh-Shih Road, Taichung 40402, Taiwan. Tel: 4-22053366 ext 3113, Fax: +886-4-2205 3764.
E-mail: [email protected]
Abstract. Osteosarcoma is the most common primary malignancy of the bone cancers. In the Chinese population, the crude extract of Corni Fructus (CECF) has been used as Traditional Chinese medicine (TCM) to treat several different diseases for hundreds of years. In the present study, effects of CECF on inhibition of migration and invasion in U-2 OS human osteosarcoma cells were examined. CECF significantly inhibited migration and invasion of U-2 OS human osteosarcoma cells. We also found that CECF inhibited activities of matrix metalloproteinases-2 (MMP-2) and matrix metalloproteinases-9 (MMP-9). CECF decreased protein levels of FAK, PKC, SOS1, MKK7, MEKK3, GRB2, NF-κB p65, COX-2, HIF-1α, PI3K, Rho A, ROCK-1, IRE-1α, JNK1/2, ERK1/2, p-p38, Ras, p-PERK, MMP-2, MMP-9 and VEGF in U-2 OS cells. Results of this study indicate that CECF may have potential as a novel anti-cancer agent for the treatment of osteosarcoma by inhibiting migration and invasion of cancer cells
Key word: Crude extract of Corni Fructus (CECF), U-2 OS human osteosarcoma cells,
Introduction
It is well known that in adolescents and children, osteosarcoma is the most common primary malignant tumor in bone . In human neoplasm, primary bone tumors are relatively uncommon and account for only 0.2% but osteosarcoma is easily allied with other malignancies . Osteosarcoma has a poor prognosis due to distal metastases that are usually developed prior to diagnosis. It was reported that patients with osteosarcoma who have five-year survival rates are not >30% after the detection of lung metastasis .
Cancer cell invasion and metastasis involve changes in the physical coupling of cells to their microenvironment, activation/degradation of extra-cellular matrix (ECM) and break-down of ECM by proteinases . Metastatic cells need to detach from the primary tumor and then attach to a metastatic site through tissue-specific microvessel cell adhesion molecules . Matrix metalloproteinases (MMPs), a family of ECM degrading proteinases, are a group of zinc dependent endo-peptidases involved in mammalian angiogenesis, wound healing, and tissue remodeling, and they play an important role in cell invasion and metastasis through controlling degradation of the ECM . Several studies have been focused on identifying MMP inhibitors as anti-cancer agents .
In Asia, individual from rural communities have used a crude substance from Corni
fructus, the fruit of Cornus officinalis Sieb. Et Zucc. (family Cornaceae) for treatment of
different diseases (tuberculosis, urination, allergy, asthma, hepatitis, lumbago, and chronic nephritis) . The crude substance of Corni fructus has biological activities such as anti-microbial, anti-diabetic in animal models (diabetes rats induced by streptozotocin), and the ability to protect against skin injury induced by X- irradiation in mice . There is no information on crude extract of Corni fructus (CECF) affecting migration and
invasion of cancer cells. In the present study, we investigated the effects of CECF on migration and invasion in U-2 OS human osteosarcoma cell line. We found that CECF inhibited migration and invasion by reduction of gene and protein expression of MMP-2 and -9.
Materials and Methods
Chemicals and reagents. Dimethyl sulfoxide (DMSO), propidium iodide (PI), Tris-HCl
Trypsin, and Trypan blue were purchased from Sigma Chemical Co. (St. Louis, MO, USA). McCoy’s 5A medium, L-glutamine, fetal bovine serum, penicillin-streptomycin, and trypsin-EDTA were purchased from Gibco BRL (Grand Island, NY, USA). Crude extract of Corni Fructus (CECF) was kindly provided by Dr. Chien-Chih Yu (School of Pharmacy, China Medical University, Taichung, Taiwan). All chemicals and reagents
were of analytical grade. DMSO (0.5%) was used as the carrier solvent and vehicle control.
U-2 OS cell culture. The U-2 OS human osteosarcoma cell line was purchased from the
Food Industry Research and Development Institute (Hsinchu, Taiwan). U-2 OS cells were maintained in 90% McCoy’s 5A medium with 10% FBS, 2 mM L-glutamine, 100
Units/ml penicillin and 100 g/ml streptomycin and placed onto 75 cm2 tissue culture flasks. Cells were cultured at 37 C under a humidified 5% CO2 atmosphere as described previously .
density of 2×105 cells/well. Cells were treated with different CECF concentrations (0, 50, 100, 250, 500 and 750 g/ml) or 0.5% DMSO (as a vehicle control) for 24 hours. Cells were then harvested stained with PI (5 g/ml) and analyzed using a PI exclusion method by flow cytometry (BD Biosciences, FACSCalibur, San Jose, CA, USA) as previously described .
Wound healing assay. U-2 OS cells at a density of 5×105 cells/well were maintained in 10 cm petri dishes until they were completely confluent. Cell monolayers were then scraped with a sterile yellow micropipette tip and washed with PBS three times. Cells in each well were then cultured in medium containing 0-500 g/ml of CECF for 24 hours, then random fields were photographed using an inverted microscope as described previously .
Cell invasion and migration assay. Cell mobility (migration and invasion) assay was
conducted using Matrigel Cell Migration Assay and Invasion System as described previously . Cell migration was determined using transwell (BD Biosciences, Franklin Lakes, New Jersey, USA) cell culture chambers (8 mm pore size; Millipore, Billerica, Massachusetts, USA). U-2 OS cells were maintained in serum-free medium for 24 h and then were trypsinized and resuspended in serum-free McCoy’s 5A medium and placed in the upper chamber of the transwell insert (5 x104 cells/well). Cells were then incubated with 0.5% DMSO or CECF (250 and 500 g/ml), and 90% McCoy’s 5A medium containing 10% FBS was added to the lower chamber and incubation for 24 or 48 hours. Non-migrating cells in the upper chamber were removed by wiping with a cotton swab and migrating cells in the lower surface of the filter were fixed with 4% formaldehyde in
PBS and stained with a gap between 2% and crystal violet in 2% ethanol and then were counted under a light microscope at x200. The invasion assay was performed as described for the cell migration assay except that the filter membrane was coated with Matrigel from a BioCoat Matrigel invasion kit. Cells located on the underside of the filter were counted with a light microscope at x200.
Gelatin zymographic assay for MMPs activity. Gelatin zymography were performed to measure the secretions of MMP-2 and MMP-9 into the media from U-2 OS after exposure to CECF as described previously . U-2 OS cells (5x105 cells/well) were maintained in 12-well plates for 24 hours then were treated with CECF (50 - 500 g/ml)
and incubated at 37ºC for 24 hours. Cells were harvested and the supernatant from each concentrated culture was re-suspended in non-reducing loading buffer and incubated at 37ºC for 15 min and then placed on 10% SDS-PAGE cast with 0.1% gelatin electrophoresed. Gels were then incubated in re-naturing buffer (2.5% Triton X-100) for 30 min, followed by incubation for 24 h at 37°C in a developing buffer (50mM Tris-HCl (pH 7.8) 10 mM CaCl2, 150 mM NaCl). Coomassie Brilliant Blue R 250 was used for staining and destained by using 30% methanol, 10% acetic acid in order to detect gelatinase secretion .
Western blotting analysis. Levels of proteins associated with cell migration and invasion
were determined in U-2 OS cells. U-2 OS cells at a density of 1x106 cells/well were placed in 6-well plates for 24 hours. Each well was incubated with 500 g/ml of CECF or DMSO (solvent) alone and incubated at 37 ºC for 0, 6, 12, 24 and 48 hours. Cells were
harvested and lysed with ice-cold 50 mM potassium phosphate buffer (pH 7.4) containing 2 mM EDTA and 0.1% Triton X-100, sonicated and then was centrifuged at 13,000g for 10 min at 4ºC. The supernatant was collected and total protein was determined using a Bio-Rad protein assay kit (Hercules, California, USA) with bovine serum albumin (BSA) as the standard. At the end of electrophoresis, proteins were electro transferred to nitrocellulose membranes, blotted with the relevant primary antibodies such as anti-FAK, PKC, SOS1, MKK7, MEKK3, GRB2, NF-κB p65, COX-2, HIF-1α, PI3K, GRB2, Rho A, ROCK-1, IRE-1α, p-JNK1/2, p-ERK1/2, p-p38, Ras, p-PERK, MMP-2, MMP-9 and VEGF, then were washed and were stained with secondary antibody. Detection of the bands was accomplished using an enhanced chemiluminescence reagent (Amersham Biosciences ECLTM) and quantified using a NIH Image analyzer (NIH, Bethesda, MD) .
Statistical analysis. All data are represented as the means ± standard deviation (S.D.) from at least three independent experiments. Statistical analyses of data were done by Student's t-test, the differences between the CECF-treated and control groups are *P < 0.05, **P < 0.01, ***P < 0.001 that were considered significant.
Results
CECF affects the percentage of viable U-2 OS human osteosarcoma cells. U-2 OS cells
were treated with 0, 50, 100, 250, 500 and 750 g/ml of CECF for 24 h, and the percentage of viable cells was measured. Figure 1 shows that CECF did not significantly decrease the percentage of viable cells at concentrations between 50-750 g/ml.
used for determining if migration of U-2 OS cells would be inhibited after incubation with CECF (0, 250 and 500 g/ml). It can be seen in Figure 2 that CECF inhibited the migration of U-2 OS cells in a dose- and time-dependent manner.
CECF inhibited the migration and invasion of U-2 OS cells in vitro. To confirm and
extend effects of CECF on migration and invasion of U-2 OS cells, cells were treated with 0, 250 and 500 g/ml for 24 and 48 h and seeded on Millicell chambers with uncoated (for migration) or matrigel-coated (for invasion) filters. After incubation, cell migration activity and invasive potential of U-2 OS cells were measured and results are shown in Figure 3 and 4. CECF significantly inhibited migration of U-2 OS cells (Fig. 3) and that finding is consistent with data shown in Figure 2. Figure 4 shows that CECF significantly inhibited the invasion of U-2 OS cells.
CECF inhibited activity of the matrix metalloproteinases-2 and -9 in U-2 OS cells. In
order to examine whether CECF induced inhibited migration and invasion of U-2 OS cells are associated with activity of MMP-2 and -9 gelatin zymography was used and results are shown in Figure 5. CECF treatment of U-2 OS cells significantly reduced activity of both MMP-2 and MMP-9 (Fig. 5).
CECF alters levels of proteins associated with migration and invasion of U-2 OS cells.
Cells were treated with CECF for 24 h and levels of the proteins NF-κB p65, Rho A and ROCK-1were examined by immuno-staining. CECF inhibited NF-κB p65 (Figure 6A), Rho A (Figure 6B) and ROCK-1 (Figure 6C) protein levels in the cytosol. We also
observed that levels of FAK, PKC, SOS1, MKK7, MEKK3, GRB2 (Figure 7A), NF-κB p65, COX-2, HIF-1α, PI3K, GRB2, Rho A, ROCK-1 (Figure 7B), p-JNK1/2, p-ERK1/2, p-p38, Ras, p-PERK (Figure 7C), MMP-2, MMP-9 and VEGF (Figure 7D) were significantly lower in CECF-treated cells than control cells.
Discussion
Several studies showed that CECF has biological activity cytotoxicity and reducing cell numbers. CECF has anti-cancer activity . In the present study we determined if CECF would alter cancer cell migration and invasion. CECF reduced cell migration (Figure 2 and 3) and invasion (Figure 4) of U-2 OS human osteosarcoma cancer cells in vitro. We also investigated the key factors and signaling pathways that are acted upon by CECF and that are associated with U-2 OS cancer cell motility. CECF inhibited the activity of both MMP-2 and -9 as determined by gelatin zymography(Fig. 5). Both enzymes (MMP-2, -9) play important roles in cancer cell migration and invasion . A number of recent studies have demonstrated a correlation between MMPs (a family of zinc containing proteolytic enzymes) and cell migration and invasion implications in cancer cell invasion .
Results from Western blotting showed that CECF reduced the levels of FAK and it also led to affect the down-stream kinases ERK1/2, JNK and p38 in U-2 OS cells.It was reported that FAK/Src signaling plays an important role in tumor metastasis by increasing cell migration and invasion . Our findings suggest that the cooperation of FAK/Src with ERK1/2 may play an important role in CECF-mediated inhibition of cell migration in U-2 OS cells. CECF also inhibited the levels of PI-3K. in U-2 OS cells. In cancer cells it has been reported that activated FAK (Tyr 397)/Src (Tyr 416) may stimulate transduction
signaling through multiple down-stream targets, such as PI-3K/AKT and Ras/ERK1/2. We found that CECF decreased the levels of NF-kB and inhibited MMP-2 and -9 activities that may occur via involved in both ERK1/2MAPK and NF-kB signaling pathways for causing the reduced protein levels of MMP-2 and -9.
CECF protein levels of growth factor receptor-bound protein 2 (GRB2), FAK, Src and Rho A which may also contribute to the down-stream inhibition of MMP-2 and -9. Formation of FAK/Src complex allows Src to phosphorylate FAK and then to mediate its interaction with GRB2 then to activate the Ras-ERK signaling pathway . The aberrant regulation of Rho A proteins is associated with metastasis by promoting tumor cell motility . CECF may act as an effective inhibitor of ERK/Rho signaling in U-2 OS cells.
In conclusion, we show for the first time that CECF in U-2 OS cancer cells inhibits migration and invasion by regulating FAK, PKC, SOS1, MKK7, MEKK3, GRB2, NF-κB p65, COX-2, HIF-1α, PI3K, GRB2, Rho A, ROCK-1, IRE-1α, JNK1/2, ERK1/2, p-p38, Ras, p-PERK and VEGF expression and inhibition of MMP-2 and -9 activities. These results provide the foundation for further and more detailed CECF studies on inhibition of cell migration and invasion bothin vitro and in vivo.
Acknowledgement
This work was supported by grant DOH102-TD-C-111-005 from the Taiwan Department of Health, China Medical University Hospital Cancer Research Center of Excellence.
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Figure legends
Figure 1. CECF affects the percentage of viable U-2 OS human osteosacroma cells. U-2 OS cells were incubated with 0, 50, 100, 250, 500 and 750 g/ml of CECF for 24 h, then cells were examined and harvested for determination the percentage of viable cells by flow cytometry as described in Materials and Methods. *p < 0.05, significant difference between CECF-treated groups and the control as analyzed by Student’s t test.
Figure 2. Wound healing examination for the effects of CECF on the migration of U-2 OS cell. Cells were maintained on the dish for 24 h then a wound was introduced by scraping confluent cell layers with a pipette tip. Various concentrations of CECF were added to the cells at the final concentration were 0, 250 and 500 g/ml then incubation for 24 and 48 h. Some of the representative photographs of invading treated and untreated cells are presented.
Figure 3. CECF suppressed the migration of U-2 OS cells in vitro. U-2 OS cells (5 x104 cells/well) that penetrated through to the lower surface of the filter were stained with crystal violet and were photographed under a light microscope at x200. Quantification of cells in the lower chambers was performed by counting cells at x200. Columns repeat the mean from three independent experiments. *p<0.05, ***p<0.001, significant difference between CECF-treated groups and the control as analyzed by Student’s t test.
Figure 4. CECF suppressed the migration and invasion of U-2 OS cells in vitro. U-2 OS cells (5 x104 cells/well) that penetrated through with the Matrigel to the lower surface of
the filter were stained with crystal violet and were photographed under a light microscope at x200. Quantification of cells in the lower chambers was performed by counting cells at x200. Columns repeat the mean from three independent experiments. *p<0.05, ***p<0.001, significant difference between CECF-treated groups and the control as analyzed by Student’s t test.
Figure 5. CECF affects the activities of matrix metalloproteinases (MMPs) activities in U-2 OS cells. Representative zymogram to detect the activity of secreted MMP-2 and MMP-9 using conditioned medium from U-2 OS cells. The different activity of MMP-2 and MMP-9 were determined by densitometric analysis and results are expressed as a percentage of the control (100%).
Figure 6. CECF affects the NF-κB p65, Rho A and ROCK-1 expression in U-2 OS cells. Cells placed on 6-well chamber slides were treated with 500 g/ml of CECF for 24 h, fixed and stained using anti-NF-κB p65, Rho A and ROCK-1 antibodies (1:100) overnight and then stained with a secondary antibody (FITC-conjugated goat anti-mouse IgG at 1:100 dilution) (green fluorescence) followed by nuclear counterstaining individually performed with PI (red fluorescence). Photomicrographs were obtained using a Leica TCS SP2 confocal spectral microscope as described in Materials and methods.
Figure 7. CECF affect the levels of associated proteins in migration and invasion of U-2 OS cells. U-2 OS cells were treated with CECF at 500 g/ml for different periods of time
and then cells were collected and the total protein extracts were prepared and determined as described in materials and methods section. The levels of FAK, PKC, SOS1, MKK7, MEKK3, GRB2 (A), NF-κB p65, COX-2, HIF-1α, PI3K, Rho A, ROCK-1 (B), p-JNK1/2, p-ERK1/2, p-p38, Ras, p-PERK (C), MMP-2, MMP-9 and VEGF (D) expressions were estimated by Western blotting as described in materials and methods.
Figure 8. The possible signaling pathways for CECF inhibited cell invasion and migration in U-2 OS human osteosacroma cells.
Figure 2 (A)
Figure 3
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Figure 4 (A)
Figure 6 (A)
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Figure 7 (A)
(C) (D)
