Crude extract of Rheum palmatum inhibits migration and invasion of
U-2 OS human osteosarcoma cells by suppression of matrix
metalloproteinase-2/-9
Shu-Chun Hsua, Ju-Hwa Lina, Shu-Wen Wengb,c, Fu-Shin Chuehd, Chien-Chih Yue, Kung-Wen Luf,* ,W. Gibson Woodg, and Jing-Gung Chunga,h,*
a Departments of Biological Science and Technology, China Medical University, Taichung 404, Taiwan
b Graduate Institute of Chinese Medicine, China Medical University, Taichung 404, Taiwan
c Department of Chinese Medicine, Taichung Hospital, Department of Health, Executive Yuan, Taichung 403, Taiwan
d Department of Health and Nutrition Biotechnology, Asia University, Taichung 413, Taiwan
e School of Pharmacy, China Medical University, Taichung 404, Taiwan f School of Post-Baccalaureate Chinese Medicine, China Medical University, Taichung 404, Taiwan
g Department of Pharmacology, School of Medicine, Geriatric Research, Education and Clinical Center, VA Medical Center, University of Minnesota, Minneapolis, MN
55455, USA
h Department of Biotechnology, Asia University, Taichung 413, Taiwan
Running title: CERP inhibited migration and invasion of osteosarcoma cells
*Both authors are equal contributions.
*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]
Kung-Wen Lu, School of Post-Baccalaureate Chinese Medicine, China Medical University, No 91, Hsueh-Shih Road, Taichung 40402, Taiwan. Tel: +886-4-22053366 ext 3207,
E-mail: [email protected]
Keywords:
Crude extract of
Rheum palmatum
(CERP) U-2 OS human osteosarcoma cellsmigration invasion
ABSTRACT
Osteosarcoma is the most common primary malignancy of bone which primarily occurs in adolescents and young adults. The crude extract of
Rheum palmatum
L.
(CERP) has been used as Traditional Chinese medicine different diseases in the Chinese population and there is experimental evidence indicating that it may have anti-cancer effects. However, there is no available information showing that CERP can act on cell mobility of human osteosarcoma cells. In the present study, we determined if CERP on would inhibit migration and invasion of U-2 OS human osteosarcoma cells. CERP significantly inhibited migration and invasion of U-2 OS cells. We also found that CERP reduced activity of matrix metalloproteinases-2 (MMP-2) and matrix metalloproteinases-9 (MMP-9). CERP decreased protein levels of FAK, GRP78, PKC, HIF-1α, SOS1, VEGF, PI3K, GRB2, Ras, p-ERK1/2, ERK1/2, p-p38, JNK1/2, p-JNK1/2, MEKK3, MKK7, PERK, p-PERK, iNOS, COX-2, NF-κB p65, IRE-1α, UPA, Rho A, MMP-COX-2, MMP-9 in U-2 OS cells. We also used confocal laser microscope to examine the levels of NF-κB p65, Rho A and Rock 1 and results indicated that CERP decreased the expression of NF-κB p65, Rho A and Rock 1. These in vitro studies suggest that CERP may have novel anti-cancer actions in the treatment of osteosarcoma warranting further studies including animal models1. Introduction
Osteosarcoma is the most common bone tumor, and it occurs predominantly in children, adolescents, and young adults . In children under 15 years of age, osteosarcoma is the 6th leading cancer and it is associated with high pulmonary metastases . Currently, patients with osteosarcoma who have lung metastasis have a five-year survival rateno greater than 30% . Advances in osteosarcoma therapy including neoadjuvant and adjuvant chemotherapy have enhanced patient outcomes , but the prognosis remains poor for most patients with metastatic or recurrent osteosarcoma .
Cancer cells from a primary site give rise to a metastatic tumor involving several regulatory pathways which can be used as biomarkers for predicting metastasis of cancer cells . Matrix metalloproteinases (MMPs) play an initial and important role in cancer cell invasion and metastasis by controlling degradation of the ECM . Currently much effort is focused on identifying MMP inhibitors .
In the Chinese population, the traditional Chinese herbal medicines such as Rheum palmatum L. (RP) have been used for treating different diseases including anti-inflammatory diseases and Rheum undulatum L. components were used for treating chronic liver diseases . Several studies have reported that the major components of Rheum palmatum L such as emodin and aloe-emodin induced
apoptosis in different human cancer cell lines . There are no reports on effects of the crude extract of Rheum palmatum L. on the inhibition of migration and invasion in human osteosarcoma cancer cells. In the present study, we determined if the water extract of Rheum palmatum (CERP) would reduce cell migration and invasion of U-2 OS human bone cancer cells. Our findings showed that CERP inhibited migration and invasion of U-2 OS cells by reducing protein expression of MMP-2 and -9 and
upregulation of NF-kB signaling.
2. Materials and Methods
2.1. Chemicals and reagents
Propidium iodide (PI), Tris-HCl Trypsin, Trypan blue and dimethyl sulfoxide (DMSO) were purchased from Sigma Chemical Co. (St. Louis, MO, USA).. Fetal bovine serum (FBS), McCoy’s 5A medium, L-glutamine, penicillin-streptomycin, and trypsin in-EDTA were purchased from Gibco BRL (Grand Island, NY, USA). Crude extract of
Rheum palmatum L
(CERP) was kindly provided by Dr. Chien-Chih Yu (School of Pharmacy, China Medical University, Taichung 404, Taiwan). All chemicals and reagents used were of analytical grade.2.2. U-2 OS cell culture
The U-2 OS human osteosarcoma cell line was obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were cultured in 90% McCoy’s 5A medium with 10% FBS, 2 mM L-glutamine, 100 Units/ml penicillin and 100 μg/ml streptomycin and maintained onto 75 cm2 tissue culture flasks and were cultured at 37°C under a humidified 5% CO2 atmosphere as described previously .
2.3. Cell viability assay
U-2 OS cells at a density of 2×105 cells/well were placed in 12-well plates for 24 h. The cells were treated with 0, 50, 100, 250, 500 and 750 μg/ml of CERPor a vehicle
1×PBS as control for 24 and 48 h. Cells were harvested from each treatment by centrifugation then were followed by staining with PI (5 μg/ml). Cells were analyzed using a PI exclusion method and flow cytometry (BD Biosciences, FACS Calibur, San Jose, CA, USA) as previously described .
2.4. Wound healing assay
5×105 cells/well were maintained in 10 cm petri dishes for 24 h until they were completely confluent. The cell monolayers in each plate were scraped with a sterile yellow micropipette tip to remove the cells and unscraped cells were washed with PBS three times. Cell in each well were then cultured in medium containing 0-500 μg/ml of CERP for 24 hours then random fields were examined, selected and photographed by using an inverted microscope as described previously .
2.5. Cell invasion and migration assays
The examinations of cell mobility (migration and invasion) were performed by using Matrigel Cell Migration Assay and Invasion System as described previously (31-32). The cell migration assay was performed using transwell (BD Biosciences, Franklin Lakes, New Jersey, USA) cell culture chambers (8 mm pore size; Millipore, Billerica, Massachusetts, USA). U-2 OS cells at a density of 5×104 cells/well were maintained in serum-free medium for 24 h. Cells were then trypsinized and resuspended in serum-free McCoy’s 5A medium and placed in the upper chamber of the transwell insert and incubated with vehicle or CERP (50 and 75 μg/ml). The 90% McCoy’s 5A medium containing 10% FBS was added to the lower chamber. Cells in both chambers were incubated for 24 or 48 hours then non-migrating cells in the upper chamber were removed by wiping
with a cotton swab and the migrating cells in the lower surface of the filter were fixed with 4% formaldehyde in PBS. Cells in the lower chamber were stained with 2% crystal violet in 2% ethanol and then were counted and photographed under a light microscope at 200X. The cell 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 examined and counted under a light microscope at x200 as described previously .
2.6. Gelatin gel zymographic assay for MMP-2 and -9 levels
Gelatin gel zymography was performed for measuring the secretion levels of MMP-2 and MMP-9 from U-2 OS cells after exposure to CERP into the culture medium as described previously (30-31). U-2 OS cells at a density of 5x105 cells/well were maintained in 12-well plates for 24 hours then were treated individually with CERP (50 and 75 μg/ml) for 24 and 48 hours. At the end of the incubation period, cells from each treatment were harvested. The supernatant from the control and CERP treated groups was re-suspended in none reducing loading buffer and incubated at 37ºC for 15 min. The material was then applied to 10% SDS-PAGE cast with 0.1% gelatin and electrophoresed. At the end of electrophoresis, gels were incubated in re-naturing buffer (2.5% Triton X-100) for 30min, followed by incubation for 16 h at 37°C in a
developing buffer (50 mM Tris-HCl (pH7.8) 10 mM CaCl2, 150 mM NaCl). Coomassie Brilliant Blue R 250 was used for subsequent staining and then destained by using 30% methanol, 10% acetic acid to detect gelatinase secretion as described previously .
2.7. Western blotting analysis for examining protein levels associated with cell migration and invasion
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 or without CERP (250 μg/ml) at 37 ºC for 0, 6, 12, 24 and 48 hours. At the end of the incubation period, 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 then centrifuged at 13,000g for 10 min at 4ºC . The supernatant was collected and total protein of each sample was measured using a Bio-Rad protein assay kit (Hercules, California, USA) with bovine serum albumin (BSA) as the standard. Samples (protein 30 g) were loaded onto 12% SDS-poly-acrylamide gels electrophoresed. After electrophoresis, the proteins were electrotransferred to nitrocellulose membranes, blotted with the appropriate primary antibodies (anti- FAK, GRP78, PKC, HIF-1α, SOS1, VEGF, PI3K, GRB2, Ras, p-ERK1/2, ERK1/2, p-p38, JNK1/2, p-JNK1/2, MEKK3, MKK7, PERK, p-PERK, iNOS, COX-2, NF-κB p65,
IRE-1α, UPA, RhoA, MMP-2 and MMP-9).Membranes were then washed and stained with a secondary antibody and detected by an enhanced chemiluminescence reagent (Amersham Biosciences ECL TM). The bands were quantified using a NIH Image analyzer (NIH, Bethesda, MD) .
2.8. Statistical analysis
Results are based on at least 3 independent experiments and expressed as means ± SD. Differences between the CERP -treated and control groups were analyzed by Student’s t test, with values of *p < 0.05 considered significant.
3. Results
3.1. CERP alters the percentage of viable U-2 OS human osteosarcoma cells
In order to determine if CERP had cytotoxic effects U-2 OS cells were treated with or without CERP (100, 250, 500, 750, 1000 and 1500 μg/ml) for 48 h. The total percentage of viable cells were quantified by flow cytometry. It can be seen in Figure 1 That CERP did not significantly decrease the percentage of viable cells at
concentrations between 100-500μg/ml. Higher CERP concentrations caused a significant reduction in cell viability.
3.2. CERP inhibited the migration and invasion of U-2 OS cells
U-2 OS cells were incubated with CERP (0, 100 and 250 μg/ml) for 24 and 48 h and cell migration was examined using a wound healing assay. Data in Figure 2 indicate that CERP inhibited the migration of U-2 OS cells in a dose- and time-dependent manner.
To further establish effects of CERP on cell migration and invasion, U-2 OS cells were treated with or without 100 and 250 μg/ml of CERP for 24 and 48 h and samples were seeded on Millicell chambers with uncoated (for migration) or matrigel-coated (for invasion) filters. Figure 3 shows that CERP significantly inhibited migration of U-2 OS cells and is in agreement with the data in Figure 2. CERP significantly inhibited the invasion of U-2 OS cells and those effects were time-dependent as seen in Figure 4.
3.3. CERP inhibits the matrix metalloproteinases-2 and -9 activities in U-2 OS cells
CERP inhibition of migration and invasion of U-2 OS cells may be associated with effects on MMP-2 and -9 activity. Data in Figure 5 shows that U-2 OS cells constitutively secreted high levels of MMP-2 but low levels of MMP-9. However, after CERP treatment (50, 100, 250, 500 µg/ml) for 24 h, both MMP-2 and -9 levels were reduced (Fig. 5).
3.4. CERP alters levels of proteins associated with migration and invasion
We next examined effects of CERP on levels of proteins that are associated with cell migration and invasion. CERP decreased cytosolic levels of NF-κB p65 (Fig. 6A), Rho A (Fig. 6B) and ROCK-1 (Fig. 6C) . We also showed that CERP reduced protein levels of FAK, GRP78, PKC, HIF-1α, SOS1 (Fig. 7A), VEGF, PI3K, GRB2, Ras (Fig. 7B), p-ERK1/2, ERK1/2, p-p38, JNK1/2, p-JNK1/2 (Fig. 7C), MEKK3, MKK7, PERK, p-PERK (Fig. 7D), iNOS, COX-2, NF-κB p65, IRE-1α (Fig. 7E) and UPA, RhoA, MMP-2, MMP-9 (Fig.ure 7F)
4. Discussion
Several studies reported that
Rheum palmatum
have many biological activity, and its components such as emodin and aloe-emodin have anti-cancer activity . Migration and invasion are key functions in cancer dynamics. In the present study we investigated effects of CERP on the migration and invasion of U-2 OS cells. CERP reduced cell migration and invasion. CERP induced effects on cell motility were associated with MMP-2 and -9. We found that CERP reduced secretion of MMP-2 and -9. It was well documented that both enzymes play important roles in cancer cell migration and invasion .Numerous reports have shown that MMPs (a family of zinc containing proteolytic enzymes) are associated with cancer cell migration and invasion . We
found that CERP reduced protein levels of MMP-2 and -9 . CERP also decreased PI3K and inhibited the expression of FAK and the down-stream kinases ERK1/2, JNK and p38 in U-2 OS cells. It was reported that in tumor metastasis, FAK/Src signaling plays an important role by increasing cell migration and invasiveness . Activated FAK (Tyr 397)/Src (Tyr416) may stimulate signaling through down-stream targets (PI-3K/AKT and Ras/ERK1/2) cascades . We found that CERP decreased the levels of NF-kB p65 in U-2 OS cells. It was reported that NF-kB p65 altered downstream levels of MMP-2 and –9 . CERP in the present study reduced protein levels of UPA and Rho A. UPA is involved in cancer cell migration and invasion . Up-regulation of UPA induces increase cancer cell invasion and metastasis . RhoA also has been shown to stimulate metastasis of tumor cells . Based on those observations, we may propose that CERP diminishes MMP-2 and -9 actions which may involve ERK1/2MAPK, NF-kB and uPA signaling pathway.
CERP was also found to reduce proteins levels of Sos1, PKC, GRB2, and Ras. Those proteins are associated with cell mobility , . It was reported that protein kinase C epsilon (PKCε) (an oncogene) is overexpressed in several human cancers and it is associated with cell proliferation, migration, invasion, and survival . More interesting list indicated that FAK/Src complex allows Src to phosphorylate FAK and then to mediate its interaction with GRB2 then to activate the Ras-ERK signaling pathway . It
was reported that the aberrant regulation of RhoA proteins is associated with metastasis via promoting tumor cell motility CERP may act as an effective inhibitor of ERK/Rho signaling in U-2 OS cells.
In conclusion, CERP effects on U-2 OS cancer cell migration and invasion may involve multiple signaling pathways as depicted in Figure 8. Our results show effectiveness in osteosarcoma cells and future studies need to determine if CERP reduces cell motility in other cancer types and if it can prevent metastasis in animal models of cancer.
ACKNOWLEDGEMENT
This work was supported by grant CMU 101-AWARD-03(1/2) and CMU100-ASIA- 04 from China Medical University, Taichung, Taiwan.
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Figure legends
Fig. 1 - CERP decreases the percentage of viable U-2 OS human osteosacroma cells.
U-2 OS cells were incubated with 0, 100, 250, 500, 750, 1000 and 1500 μg/ml of CERP for 48 h. 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 CERP-treated groups and the control as analyzed by Student’s t test.
Fig. 2 - Wound healing assay for the effects of CERP on the migration of U-2 OS
cell. Cells were maintained on the 6-well dish for 24 h then a wound was introduced by scraping confluent cell layers with a pipette tip. CERP (0, 100 and 250 μg/ml) were individually added to the well then incubation for 24, and 48h. Some of the representative photographs of invading treated and untreated cells are presented.
Fig. 3 - CERP suppressed the migration of U-2 OS cells in vitro. U-2 OS cells at the
density of 5 x104 cells/well, after cells penetrated through to the lower surface of the filter then were stained with crystal violet, examined and were photographed under a light microscope at 200X. Quantification of cells in the lower chambers was performed by counting cells at 200X. Representative columns (mean) from three independent experiments. *p<0.05, significant difference between CERP-treated
groups and the control as analyzed by Student’s t test.
Fig. 4 - CERP suppressed the invasion of U-2 OS cells in vitro. U-2 OS cells at the
density of 5 x104 cells/well, after cells penetrated through the Matrigel of the filter to the lower surface were stained with crystal violet, examined and were photographed under a light microscope at 200X. Quantification of cells in the lower chambers was performed by counting cells at 200X. Representative columns (mean) from three independent experiments. *p<0.05, significant difference between CERP-treated groups and the control as analyzed by Student’s t test.
Fig. 5 - CERP affects the activities of matrix metalloproteinases (MMPs) activities in
U-2 OS cells. Representative zymogram from three independently experiments was used to detect the activity of secreted MMP-2 and MMP-9 by using conditioned medium of U-2 OS cells culture with CERP. 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%).
Fig. 6 - CERP affects the NF-κB p65, Rho A and ROCK-1 expression in U-2 OS
cells. Cells at the density of 2 x105 cells/well were placed on 6-well chamber slides then were treated with 0, 100, 250 µg/ml of CERP for 24 h, fixed and stained using
anti-NF-κB p65 (A), Rho A (B) and ROCK-1 (C) 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.
Fig. 7 - CERP affect the levels of associated proteins in migration and invasion of U-2
OS cells. U-2 OS cells at the density of 1 x106 cells/dish were treated with 250 μg/ml of CERP for 6, 12, 24, 48 hr 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, GRP78, PKC, HIF-1, SOS1 (A); VEGF, PI3K, GRB2, Ras (B); p-ERK, ERK1/2, p-p38, JNK1/2, p-JNK1/2 (C); MEKK3, MKK7, PERK, p-PERK (D); iNOS, COX-2, NF-κB p65, IRE-1α (E) and UPA, Rho A, MMP-2, MMP-9 (F) expressions were estimated by Western blotting as described in materials and methods.
Fig. 8 - The possible signaling pathways for CERP inhibited cell migration and
