Transforming growth factor-β1 (TGF-β1), a multi-function polypeptide, is a double-edged sword in cancer. For some tumor cells, TGF-β1 is a potent growth inhibitor and apoptosis inducer. More commonly, TGF-β1 loses its growth-inhibitory and apoptosis-inducing effects, but stimulates the metastatic capacity of tumor cells. It is currently little known about TGF-β 1-promoted cell migration in hepatocellular carcinoma (HCC) cells, let alone its mechanism. In this study, we found that TGF-β1 lost its tumor-suppressive effects, but significantly stimulated cell migration in SMMC-7721 human HCC cells. By FACS and Western blot analysis, we observed that TGF-β1 enhanced the expression of α 5 β1 integrin obviously, and subsequently stimulated cell adhesion onto fibronectin (Fn). Furthermore, we observed that TGF-β1 could also promote SMMC-7721 cells adhesion onto laminin (Ln). Our data also provided evidences that TGF-β1 induced epithelial-to-mesenchymal transformation (EMT) in SMMC-7721 cells. First, SMMC-7721 cells clearly switched to the spindle shape morphology after TGF-β1 treatment. Furthermore, TGF-β1 induced the down-regulation of E-cadherin and the nuclear translocation of β -catenin. These results indicated that TGF-β 1-promoted cell adhesion and TGF-β 1-induced epithelial-to-mesenchymal transformation might be both responsible for TGF-β 1-enhanced cell migration.
TGF-β1, a polypeptide with multi-function, modulates a variety of cellular processes, such as proliferation, differentiation and apoptosis1, 2. TGF-β1 is a potent growth inhibitor and apoptosis inducer for most normal cells. However, many tumor cells are nevertheless sensitive to the tumor-suppressive effects of TGF-β1. It is known that TGF-β1 could strongly stimulate the invasive and metastatic capacity of tumor cells3. The role of TGF-β1 in HCC cells is not well understood. Previous studies demonstrated that TGF-β1 also lost the tumor-suppressive effects in many HCC cells4. Moreover, TGF-β1 concentration increased in the plasma of HCC patients5, 6. The study on the role for TGF-β1 in invasive and metastasis of hepatocellular carcinoma is scarce.
It is well known that TGF-β1 could modify the expression of many different integrins, and subsequently alter cell adhesion and migration7. Furthermore, cell migration is dependent on cell adhesion8. EMT correlates with the metastatic potential of tumor cells3, 9. Previous studies demonstrated that TGF-β1 could induce the EMT of tumor cells with epithelial origin10, 11, 12, 13, 14. However, there are no reports about TGF-β 1-promoted EMT of HCC cells.
Here we investigated the role for TGF-β1 in SMMC-7721 human HCC cells with low differentiation15. We found that TGF-β1 had no effects on the growth and apoptosis of SMMC-7721 cells. At the meantime, TGF-β1 significantly enhanced cell migration on Fn and Ln. We intended to study whether TGF-β 1-enhanced cell migration correlated with TGF-β 1-promoted cell adhesion and EMT in SMMC-7721 cells.
MATERIALS AND METHODS
Cell lines, antibodies and reagents
SMMC-7721 human HCC cell line was obtained from Shanghai No.2 Military Medical University (Shanghai, China)15. Fn and Ln were all obtained from Sigma. Human recombinant TGF-β1 was from PeproTech EC Ltd. Anti-E-cadherin polyclonal antibody, anti-integrin α 5 subunit monoclonal antibody, anti-β -actin monoclonal antibody, anti-β -catenin monoclonal antibody were purchased from Santa Cruze. Anti-integrin β1 subunit monoclonal antibody was from BD Transduction Laboratories. Secondary antibodies conjugated with HRP were purchased from Watson Biotech (Shanghai).
Cell culture, TGF-β1 treatment and cell morphology examination
SMMC-7721 cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum in 37 °C and 5% CO2, and grown to 50-70% confluence prior to TGF-β1 treatment. TGF-β1 was stored in PBS containing2 mg/ml bovine serum albumin (BSA), and added into the serum-free medium to a final concentration. Cell morphology was monitored on a phase contrast microscope equipped with a video camera.
Cell migration assay
In vitro wound healing assay16
Subconfluent cells were detached and 5×105 cells were seeded in Falcon six-well tissue culture plates coated with 10 mg/ml Fn. After 12 h, cells were cultured in serum-free medium for 24 h (grown to confluence to form a monolayer), and were treated with or without 10 ng/ml TGF-β1. In vitro,'Scratch' wounds were created by scraping confluent cell monolayers with a sterile pipette tip to make an approximately 1.0 mm gap. The cells scraped down were washed by serum-free medium. After 24 h and 48 h, migration was quantified by counting cell numbers that had advanced into the cell-free space from a number of randomly chosen 1mm segments of the initial wound border. Each point stands for Mean±SD from at least 4 wounds of every experiment, and 4 separate experiments were carried out.
Assay of random migration using the agarose drop method 17
Cellls to be tested were removed from culture dishes by tryspinization, washed and centrifuged into a pellet. To the cell pellet was added 100 ml RPMI 1640 medium containing 10% serum and 16.7 μ l2% (w/v) agarose to be the agarose-cell suspension containing 0.3% agarose. Prior to use, the agarose-cell suspension was kept in a water bath at 37°C for preventing the solidification. 1.5 μ l droplets of the agarose-cell suspension were delivered with a sterile micropipette into the 24-wells tissue culture plates. The dish was then placed in a refrigerator for 10 min to allow the agarose to solidify. The radius of the droplet ( r ) was measured using inverted microscope fitted with a rule in eyepiece. After cultured in the RPMI 1640 medium containing 1% serum for 6-8 h, the droplets were washed gently with serum-free medium, and were treated with or without TGF-β1 in serum-free medium for 24 and 48 h. At 24 and 48 h, the radius of the droplet was measured under eight sides of each droplet, and the average value ( r') was calculated. Cells' random migration potential was determined by the value of (r'-r). Five drops were used for each point.
Flow cytometric analysis of integrin and E-cadherin expression on cell membrane
1×10 6 cells were incubated with PBS containing 1% BSA alone or with primary antibodies against integrin α5 subunit, integrin β1 subunit and E-cadherin respectively for 45 min in 4 °C. Cells were washed extensively (3 times in PBS) and incubated with appropriate FITC-conjugated second antibody for 45 min in 4°C in the dark. After washing the cells extensively, they were analyzed on a FACScan? (Becton-Dickinson &Co., Mountain View, CA). For each sample, data from 10,000 cells were collected. Cells as control were incubated with secondary antibody alone to show background fluorescence.
Cell adhesion assay
Cell adhesion assaywas carried out as previously described 19. Briefly, 96-well plates (Nunc) were coated at 37°C for 1 with 100 μ l 5 μ g/ml Fn, 20 μ g/ml Ln and 100 μ g/ml Poly-lysine in PBS. The plates were washed twice with PBS and blocked with 100 μ l 1% BSA for 1 h at 37°C. Wells were washed twice with PBS and stored at 4°C before use. Cells were collected and resuspended in complete RPMI without FCS. A total of 10,000 cells in 100 ml were added into each substrate coated well, and plates were incubated for 30 min at 37°C in 5% CO2. Unattached cells were gently washed away with PBS. The attached cells were fixed with 4% formaldehyde, stained with 0.5% crystal violet overnight, destained with distilled water, solubilized in 36% acetic acid and quantified by the micro-titer plate reader.
Nuclear protein extracts
Nuclear protein extracts were prepared by the mini-extraction method as described previously19. Cells were washed with ice cold PBS and harvested by being scraped in 1.5 ml PBS. Cells were then pelleted and resuspended in 400 μ l of 10 mM Hepes-potassium hydroxide, pH 7.9, 1.5 mM magnesium chloride, 10 mM potassium chloride, 0.5 mM dithiothreitol, 0.2 mM PMSF. After 10 min of incubation on ice nuclei were pelleted by being spun for 10s and resuspended in 50 ml of 20 mM Hepes-potassium hydroxide, pH 7.9, 25% glycerol, 420 mM sodium chloride, 1.5mM magnesium chloride, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.2 mM PMSF. Tubes were incubated for 20 min on ice and then centrifuged to clear the cellular debris. Nuclear extracts were stored at −70°C.
Cells were washed with PBS and lysated in modified loading buffer containing 50 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, phosphatase inhibitors (100 mM Na3VO4, 10 mM NaF, 10 mM Na4P2O7), and protease inhibitors (1 mM PMSF). The samples were boiled for 10 min and centrifuged at 12,000 rpm for 10 min, and insoluble material was removed.
Western blot analysis
Equal amount of protein were loaded on a SDS-PAGE and transferred to PVDF membrane. After blocked with 3% BSA in PBS (containing 0.05% Tween 20), the membranes were incubated with the specific primary antibodies, followed by HRP-conjugated secondary antibodies. Proteins were visualized by fluorography using an enhanced chemiluminescence system (Perfect Biotech. Shanghai). To reprobe another primary antibody, membranes were incubated in stripping buffer (62.5 mM Tris pH 6.7, 10 0 mM 2-mercaptoethanel, 2% SDS) at 70 °C for 30 min, washed and then used for further study. Signal intensity of each band was quantified by densiometric analysis using a scanning imager (Pharmacia Co.)
TGF-β1 promoted the migration of SMMC-7721 cells
TGF-β1 could inhibit cell growth and induce apoptosis in normal liver cells. However, for most HCC cells with low differentiation, TGF-β1 lost these functions. For example, we found that TGF-β1 lost its growth-inhibitory and apoptosis-inducing effects in SMMC-7721 cells with low differentiation (Data not shown). Since TGF-β1 lost its tumor-suppressive properties, we wondered whether TGF-β1 could promote cell migration. Agarose drop method and wound healing assay were performed to measure the migration of SMMC-7721 cells in vitro. As shown in Fig 1 by agarose drop method, we found that contrast to control, cell migration was 1.39-fold at 24 h, and 1.42-fold at 48 h with the concentration of 10 ng/ml TGF-β1. Using wound healing assay, we also found that cell migration was 1.61-fold at 24 h, and 1.80-fold at 48 h respectively (Fig 2). These results indicated that TGF-β1 could promote the metastatic potential of SMMC-7721 cells in vitro.
TGF-β1 stimulated the expression of α5 β1 integrin and promoted cell adhesion
Cell migration through ECM is generally mediated by integrins on cell membrane. Efficient evidences emerged that TGF-β1 could modify many integrins and subsequently alter the aspects of cell adhesion, migration. Furthermore, cell migration depends on cell adhesion. By FACS we found that contrast to control, α 5 subunit was about 1.77-fold, and β1 subunit was about 1.65-fold with the concentration of 10ng/ml TGF-β1 for 48 h (Fig 3). By Western blot analysis, we also found that the expression of α 5 subunit and β1 subunit were apparently enhanced with the concentration of 0, 1, 5, 10, 20 ng/ml TGF-β1 for 48 h (Fig 4). The β1 subunit appeared as two bands implying two forms of β1 subunit (seen in discussion).
α 5 β1 integrin is a cell adhesion receptor for Fn, and cell adhesion onto Fn is mediated by α 5 β1 integrin. Since TGF-β1 could enhance the expression of α 5 β1 integrin, we wanted to determine whether TGF-β1 could promote cell adhesion onto Fn. As shown in Fig 5 cell adhesion onto Fn was 1.60-fold at 24 h, and 1.87-fold at 48 h with the concentration of 10 ng/ml TGF-β1. We observed that TGF-β1 could also promote cell adhesion onto another ECM protein Ln.
TGF-β1 induced the EMT of SMMC 7721 cells
EMT could contribute to the metastatic potential of tumor cells. Previous studies demonstrated that TGF-β1 could induce the EMT of tumor cells10, 11, 12, 13, 14. The EMT of cells is characterized by a clear switch to the spindle shape morphology, accompanied by down-regulation of E-cadherin and nuclear translocation of β -catenin. The development of tumor cells metastatic phenotype correlates with down-regulation of E-cadherin expression.
By microscopic examination, we found that SMMC-7721 cells underwent a clear switch to the spindle morphology with the concentration of 10 ng/ml TGF-β1 for 48 h (Fig 6). FACS analysis showed that the expression of E-cadherin on cell membrane was about 62% after treatment with 10 ng/ml TGF-β1 for 48 h (Fig 7). By Western blot analysis, we noted that the expression of nuclear β -catenin increased in a dose-dependen manner with the concentration of 0, 1, 5, 10, 20 ng/ml TGF-β1 for 48 h (Fig 8). In contrast the xpression of total β 1-catenin was not altered. We provided evidences that TGF-β1 induced the EMT of SMMC-7721 cells, which also could contribute to TGF-β 1-promoted cell migration.
TGF-β1 is growth inhibitory and apoptosis inducing to most normal cell types. However, the majority tumor cells are nevertheless sensitive to the tumor-suppressive effects of TGF-β1, due to loss-of-function mutation of components in the TGF-β1 signaling pathways or up-regulation of the MAP kinase pathway during carcinogenesis3. Furthermore, previous evidences demonstrated that TGF-β1 could promote the invasive and metastatic capacity of human tumor, in vivo and in vitro3. It is not currently well understood the role of TGF-β1 in HCC cells. The fact that high serum concentration of TGF-β1 in HCC patients indicates that TGF-β1 loses the tumor-suppressive effects in most HCC cells5, 6. However, whether TGF-β1 could enhance the metastatic capacity of HCC cells is not known yet, let alone its mechanism. Giannelli et al reported that TGF-β1 played an important role in the invasiveness of HCC cells by stimulating the expression of α 3 β1 integrin20.
Previous studies demonstrated that cell adhesion and EMT both played important roles in the matastatic phenotype of tumor cells1, 3. TGF-β1 could regulate several integrins on many types of tumor cells, subsequently affected cell adhesion mediated by integrins7. A role for TGF-β1 to induce EMT has been reported in squamous skin carcinomas12, Ras-transformed mammary carcinoma cells and ovarian adenosarcoma cells14. Although the cellular machinery of TGF-β 1-induced EMT remains unclear, previous studies demonstrated that EMT during embryonic development correlated with down-regulation of E-cadherin and nuclear translocation of β -catenin.
In this repoet, we found that TGF-β1 lost the tumor-suppressive effects in SMMC-7721 human HCC cells. At the meantime, TGF-β1 promoted cell migration in vitro. We focused on investigating the possible mechanism of TGF-β 1-stimulated cell migration and found that SMMC-7721 cells underwent a clear switch to the spindle morphology after treatment with TGF-β1.The fibro- blastoid phenotype correlated with down-regulation of E-cadherin expression and nuclear translocation of β -catenin. These results indicated that TGF-β1 could induce the EMT of SMMC-7721 cells, which possibly contributed to TGF-β 1-promoted cell migration.
By FACS and Western blot analysis, we observed that TGF-β1 obviously increased the expression of integrin α 5 β1. In Western blot analysis, the β1 subunit appeared as two bands suggesting two forms of β1 subunit. It maybe caused by variable post-translational modification (mainly N-glycosylation). The hypog-lycosylated form (lower band in Fig 4C) was tentatively identified as biosynthetic precursor of β1 subunit. The band with lower migration rate (upper band in Fig 4 β1 subunit, which mainly located at the plasma membrane21, 22, 23. The correlation between α 5 β1 integrin and cell migration has been previously reported. For example, Beauvais et al found that over-expression of α 5 β1 integrin in transfected sarcoma S180 cells enhanced their mobility on Fn in vitro, and changes their migratory properties in vivo24. We also found that TGF-β1 could promote SMMC-7721 cells adhesion onto Fn mediated by α 5 β1 integrin. Furthermore, TGF-β1 promoted SMMC-7721 cells adhesion onto another ECM protein Ln. The latter result indicated that some type of integrins (such as α 5 β1 and α 3β1; the receptors of Ln) were possibly also up-regulated by TGF-β1. So we hypothesized that TGF-β1 could regulate the expression of several integrins, and subsequently promote cell adhesion onto ECM, which might also contribute to TGF-β 1-promoted cell migration.
Take together, TGF-b1-promoted EMT and cell adhesion might be both responsible for TGF-β 1-enhanced cell migration in SMMC-7721 cells.
- TGF- 1:
transforming growth factor- 1
flow cytometric analysis
Derynck R, Feng XH . TGF-β receptor signaling. Biochem Biophysic Acta 1997; 1333:F105–50.
Attisano L, Wrana JL . Signal transduction by TGF-β superfamily. Science 2002; 1646–7.
Akhurst RJ, Derynck R . TGF-β signaling in cancer-a-double-edged sword. Trends Cell Biol 2001; 11(11):S44–51.
Jong HS, Lee HS, Kim TY et al. Attenuation of transforming growth factor beta-induced growth inhibition in human hepatocellular carcinoma cell lines by cyclin D1 overexpression. Biochem Biophys Res Commun 2002; 292(2):383–9.
Shirai Y, Kawata S, Tamura S et al. Plasma transforming growth factor-b in patients with hepatocellular carcinoma. Cancer 1994; 3(9):2275–9.
Murawaki Y, Ikuta Y, Nishimura Y . Serum makers for fibrosis and plasma transforming growth factor- β 1 in patients with hepatocellular carcinoma in comparison with patients with liver cirrhosis. J Gastroenterol Hepatol 1996; 11(5):443–50.
Xu Z, Zha XL . Regulation of integrin expression and affinity by transforming growth factor-b. Chemistry of life Sinica 2002; 22:6–8.
Clezardin P . Recent insights into the role of integrins in cancer metastasis. Mol Life Sci 1998; 54:541–8.
Thiery JP, Chopin D . Epithelial cell plasticity in development and tumor progression. Cancer Metastasis Rev 1999; 18:31–42.
Janji B, Melchior C, Gouon V, Vallar L, Kieffer N . Autocrine TGF-β -regulated expression of adhesion receptors and integrin-linked kinase in HT-144 melanoma cells correlates with their metastatic phenotype. Int J Cancer 1999; 83:255–62.
Piek E, Moustakas A, Kurisaki A, Heldin CH, Dijke P . TGF-5 type I receptor/ALK-5 and Smad proteins mediate epithelial to mesenchymal transdifferentiation in NMuMG breast epithelial cells. J cell Sci 1999; 112:4557–68.
Portella G, Cumming SA, Liddell J et al. Transforming growth factor-beta is essential for spindle cell conversion of mouse skin carcinoma in vivo: implications for tumor invasion. Cell Growth Differ 1998; 9:393–404.
Miettinen PJ, Ebner R, Lopez AR, Derynck R . TGF-β. induced transdifferentiation of mammary epithelial cells to mesenchymal cells: involvement of type I receptors. J Cell Biol 1994; 127:2021–36.
Kitagawa K, Murata A, Matsuura N et al. Epithelial-mesenchymal transformation of a newly established cell line from ovarian adenosarcoma by transforming growth factor- β 1. Int J Cancer 1996; 66:91–7.
Huang RC, Zhou, RH, Lu FD et al. Establishment and some biological characteristics of SMMC-7721 human hepatocellular carcinoma cell line. Acta Shanghai No.2 Military Med Univ 1980; 1:5–9.
Pukac L, Huangpu J, Karnovsky MJ . Platelet-drived growth factor-BB, insulin-like growth factor-1, and phorbol ester activate different signaling pathways for stimulating of vascular smooth muscle cell migration. Exp Cell Res 1998; 242:548–60.
Varani J . A comparison of the migration patterns of normal and malignant cells in two assays systems. Am J Pathol 1978; 90:159–65.
Busk M, Pytela R, Sheppard D et al. Characterization of the integrin avb6 as a fibronectin-binding protein. J Biol Chem 1992; 267:5790–6.
Persad S, Troussad AA, Mcphee TR et al. Tumor suppressor PTEN inhibits nuclear accumulation of β -catenin and T cell/ Lymphoid enhancer factor 1-mediated transcriptional activa-tion. J Cell Biol 2001; 153(6):1161–73.
Giannelli G, Fransvea E, Marinosci F et al. Transforming growth factor-beta1 triggers hepatocellular carcinoma invasiveness via alpha3beta1 integrin. Am J Pathol 2002; 161(1):183–93.
Heino J, Ignotz RA, Hemler ME, Crouse C, Massague J . Regulation of cell adhesion receptors by transforming growth factor- β 1 Concomitant regulation of integrins that share a common β 1 subunit. J Biol Chem 1989; 264:380–8.
Bellis SL, Newman E, Friedman EA . Steps in integrin β 1-chain glycosylation mediated by TGF β 1 signaling through Ras. J Cell Physiol 1999; 181:33–44.
Yan Z, Chen M, Perucho M, Friedman E . Oncogenic Ki-ras but not oncogenic Ha-ras blocks integrin β 1-chain maturation in colon epithelial cells. J Biol Chem 1997; 272:30928–36.
Beauvais A, Erickson CA, Goins T et al. Changes in the fibronectin-specific integrin expression pattern modify the migratory behavior of sarcoma S180 cells in vitro and in the embryonic environment. J Cell Biol 1995; 128(4):699–713.
This work is supported by grants from National Nature Science Foundation of China (No. 30000083) and Science and Technology Bureau of Shanghai Municipal Government (No. 00JC 14042). We also want to thank the Chinese Medicine Board (CMB) in New York, USA for the kind support to this research.
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