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1Research Center for Cell Fate Control and College of Pharmacy, Sookmyung Women’s University, Seoul 140-742
2Department of Natural Medicine Resources, Semyung University, Jecheon 390-711, Republic of Korea
†The first two authors contributed equally to this work.
Wnt/β-catenin signaling pathway was mutated in about 90% of the sporadic and hereditary colorectal cancers. The abnormally activated β-catenin increases the cancer cell proliferation, differentiation and metastasis through increasing the expression of its oncogenic target genes. In this study, we identified an inhibitor of β-catenin dependent Wnt pathway from rhizomes of
Colorectal cancer is one of the most common cancers in the world and accounts for approximately 10% of all cancer related deaths (Jemal
Galectin-3 is a multifunctional protein and present in nucleus, cytoplasm, surface, and extracellular space of cells. Galectin-3 was reported to be involved in various tumors cells transformation, proliferation, survival, differentiation and metastasis (Inohara
The rhizomes of
Rhizomes of
The dried rhizomes of
Human colorectal adenocarcinoma cell line, SW-480 was purchased from Korea Cell Line Bank (Seoul, Korea). The HEK-293 β-catenin reporter cell line (TOPFlash) and media control L (L-control) cell line were kindly provided by Prof. Sangtaek Oh at Kookmin University (Seoul, Korea). Wnt3a-secreting L (L-Wnt3a) cell line was purchased from American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (10 μg/ml) at 37°C.
SW-480 cells were seeded in 96-well plates at a density of 2.9×103 cells per well and incubated at 37°C in 5% CO2 environment. Cells were treated with indicated concentrations of AC for 24 h, 48 h and 72 h. 50 μl of MTT reagent (5 mg/ml in phosphate buffered saline) was added to each well and incubated for 1.5 h at 37°C. The media were removed and DMSO was added to each well. Absorbance was measured using VERSAMax Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) at 570 nm.
Wnt3a-conditioned medium (Wnt3a CM) was prepared according to the manufacturer's instruction. In brief, Wnt3a-secreting L cells were cultured in DMEM with 10% FBS for 4 days. The medium was harvested and sterilized using a 0.22-μm filter. The cells were added fresh medium and cultured for another 3 days, and the medium was collected and combined with the previous medium.
Cells were seeded in 96-well plates at a density of 1.1×104 cells per well and incubated at 37°C in 5% CO2 environment. Then, cells were treated with 20 μg/ml of AC for 24 h and lysed with passive lysis buffer (Promega, Madison, WI, USA). Cell lysates were incubated with luciferase assay substrate (Promega) and the luciferase activity was measured by luminometer (Molecular Devices).
SW-480 cells were seeded in 6-well plates at density of 5.4× 104 cells per well and incubated at 37°C in 5% CO2 environment for 24 h. Cells were treated with 20 μg/ml of AC for 6, 9, 12, 15 h. The cells were harvested with ice-cold PBS and the cell pellets were incubated with cytosolic lysis buffer (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, pH7.6) and centrifuged to get cytosolic fractions. The insoluble fractions were re-suspended with nuclear lysis buffer (20 mM HEPES, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT) and centrifuged to isolate nuclear fractions. Whole cell lysates were prepared with RIPA buffer (25 mM Tris, 150 mM NaCl, 0.5% Triton X-100). The lysates were quantified with BCA protein assay kit (Pierce, Rockford, IL, USA) and loaded on SDS-PAGE after denaturation. The proteins were blotted to PVDF membrane and probed with the primary antibodies. The membranes were then incubated with horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG (Cell Signaling, Beverly, MA, USA) as the secondary antibody and visualized using the ECL chemiluminescence (GE healthcare, Piscataway, NJ, USA). All the primary antibodies were used in 1:1000 dilution and the secondary antibodies in 1:10000.
To isolate inhibitors of Wnt/β-catenin signaling from the extracts of
As shown in Fig. 2A, treatment of AC inhibits TOPflash activity in Wnt3a CM-treated stable reporter HEK-293 cells in a dose-dependent manner. The activity of FOPFlash, a negative control reporter with mutated β-catenin/TCF binding elements, was not altered by the treatment of AC and Wnt3a CM (data not shown). To test whether the GSK-3β was involved in the inhibition of β-catenin response transcription, we treated HEK cells with LiCl as an inhibitor of GSK-3β. AC treatment also inhibits TOPflash activity in LiCl-treated HEK-293 cells (Fig. 2B).
To further confirm the inhibitory effect of AC on Wnt/β-catenin signaling pathway, β-catenin constitutively activated SW-480 colon cancer cell line were used. Treatment of AC (20 μg/ml) decreased the nuclear level of β-catenin in SW-480 cells in a time-dependent manner. But the levels of β-catenin in cytosol and whole cell lysate were not affected by AC (Fig. 3A and 3B). And a target gene of β-catenin, cyclin D1 was decreased by treatment of AC in SW-480 cells. The level of galectin-3, one of the β-catenin nuclear translocation modulators, was also decreased in nuclear of AC-treated SW-480 cells, but not in cytosolic fraction and whole cell lysates (Fig. 3A, B). These data indicate that AC inhibits Wnt/β-catenin signaling pathway through modulating the nuclear translocation of β-catenin and galectin-3 in colon cancer cells.
As AC inhibits Wnt/β-catenin signaling pathway in SW-480 cells, we have tested the effects of AC on proliferation of colon cancer cells. As shown in Fig. 4, the cell viabilities of SW-480 cells were decreased dramatically by treatment of AC in a dose dependent manner ranging from 5 to 20 μg/ml.
Taken together, AC inhibits Wnt/β-catenin signaling pathway to suppress proliferation of colon cancer cells, and galectin-3 is maybe involved in its action mechanism.
It has been reported that about 90% of Wnt/β-catenin signaling pathway is mutated in sporadic and hereditary colorectal cancers (Miyaki
When Wnt pathway is activated, β-catenin can be released from the APC/Axin/GSK-3β destructive complex and translocated to the nucleus to activate expression of target genes. The nuclear translocation is regulated by several genes, such as FoxM1, importinα/β, nucleoporins and galectin-3. Galectin-3 is associated with the development of several cancers including colorectal cancer (Song
AC has been reported as anti-inflammatory and anti-oxidant agent (Resch
In conclusion, AC purified from
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