- Email: [email protected]
Transforming growth factor-β transiently induces vimentin expression and invasive capacity in a canine mammary gland tumor cell line
Research in Veterinary Science 94 (2013) 539–541
Contents lists available at SciVerse ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Transforming growth factor-b transiently induces vimentin expression and invasive capacity in a canine mammary gland tumor cell line K. Yoshida, T. Saito, A. Kamida, K. Matsumoto, K. Saeki, M. Mochizuki, N. Sasaki, T. Nakagawa ⇑ Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan
a r t i c l e
i n f o
Article history: Received 4 June 2012 Accepted 19 October 2012
Keywords: Dog Mammary gland tumor EMT TGF-b MET xCELLigence RTCA
a b s t r a c t The epithelial–mesenchymal transition (EMT) is a crucial event that occurs during cancer metastasis and can be induced by transforming growth factor-b (TGF-b) in various tumor cells in vitro. However, little is known about the effects of TGF-b in canine mammary gland tumors (CMGTs). Here, we investigated the role of TGF-b in CMGT. We observed that treatment of the CMGT cell line CHMp13a with TGF-b1 leads to transient induction of the mesenchymal marker vimentin. Real-time measurements of cellular electrical impedance also showed that CMGT invasiveness is transiently increased by TGF-b1 treatment, but is reversed after prolonged stimulation. This phenomenon is similar to the mesenchymal–epithelial transition (MET, the reverse phenomenon of EMT), and a process that is implicated in the establishment of secondary metastatic lesions. Ó 2012 Elsevier Ltd. All rights reserved.
Canine mammary gland tumors (CMGTs) are one of the most frequent tumors in female dogs. Histopathologically, approximately half of CMGTs are considered malignant (Brodey et al., 1983; Gilbertson et al., 1983), and prognosis of such tumors is poor because of distant metastasis, especially to the lungs (Misdorp and Hart, 1979). Many researchers have suggested various proteins as prognostic factors in CMGTs. For example, loss of E-cadherin which is an epithelial marker was reported to be related to a poor prognosis (Matos et al., 2006; Sarli et al., 2004; Rodo and Malicka, 2008). The epithelial–mesenchymal transition (EMT) is the differentiation switch from polarized epithelial cells to the fibroblastic mesenchymal cells, and it is a crucial event during physiological and disease processes, including development, fibrosis, and cancer (Thiery, 2002; Kalluri and Weinberg, 2009; Tsuji et al., 2009). When tumor cells invade the surrounding tissues, their loss of cell–cell interaction and acquisition of invasive properties are associated with EMT. EMT is characterized by the loss of epithelial markers such as E-cadherin and up-regulation of mesenchymal markers such as vimentin (Kokkinos et al., 2007). EMT is regulated by various growth factors and cytokines (Moustakas and Heldin, 2007; Yao et al., 2011). Among these, transforming growth factor-b (TGF-b) is a key factor controlling the induction of EMT (Miettinen et al., 1994; Xu et al., 2009). ⇑ Corresponding author. Address: Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Tel.: +81 3 5841 5414; fax: +81 3 5841 8996. E-mail address: [email protected] (T. Nakagawa). 0034-5288/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rvsc.2012.10.016
Secreted TGF-b binds to the complex composed of TGF-b type I receptor (TbRI) and TGF-b type II receptor (TbRII). This complex activates both Smad-dependent and Smad-independent pathways (Aomatsu et al., 2011; Heldin and Moustakas, 2012). In the Smad-dependent pathway, receptor-regulated Smads (R-Smads, Smad2/3) are phosphorylated and interact with a common partner Smad, Smad4, which translocates into the nucleus and acts as transcriptional factor (Shi and Massagué, 2003). TGF-b was reported to induce EMT and promote cell invasion and metastasis in some human and mouse tumor cells (Lenferink et al., 2004; Zavadil and Böttinger, 2005). However, the relationship between TGF-b and the EMT phenotype in CMGT remains unclear. The purpose of this study was to evaluate EMT in a CMGT cell line in response to TGF-b by measuring change in epithelial and mesenchymal markers as well as cellular invasive capacity, and discuss the role of TGF-b induced EMT in metastasis. A clonal CMGT cell line CHMp13a was maintained in RPMI-1640 containing L-glutamine medium (Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) and 50 mg/L gentamicin sulfate (Sigma–Aldrich Corporation, St. Louis, MO, USA) at 37 °C in a humidified atmosphere of 5% CO2. Recombinant human TGF-b1 was purchased from PeproTech (Rocky Hill, NJ, USA). The primary antibodies used for epithelial markers in the western blot analysis were mouse anti-E-cadherin (1:1000, BD Transduction Laboratories, Lexington, KY, USA) and mouse anti-b-catenin (1:2000, BD Transduction Laboratories). Mouse anti-vimentin (1:1500, Millipore, Bedford, MA, USA) and mouse anti-N-cadherin (1:1000, United States Biological,
540
K. Yoshida et al. / Research in Veterinary Science 94 (2013) 539–541
Swampscott, MA, USA) were used for mesenchymal markers. To evaluate downstream TGF-b signaling, mouse anti-Smad2/3 (1:1000, BD Transduction Laboratories) and rabbit anti-phosphorylated Smad2 (1:1000, Millipore) were used. Mouse anti-pan-actin (1:10,000, Cell Signaling, Danvers, MA, USA) was used as a loading control. Secondary antibodies used were anti-mouse IgG conjugated to horseradish peroxidase (HRP) (1:20,000, GE Healthcare UK Ltd., Buckinghamshire, UK) and anti-rabbit IgG conjugated to HRP (1:10,000, GE Healthcare). Recombinant human TGF-b1 was added to a final concentration of 1 ng/mL, and cells were incubated at 37 °C for 1, 2, 4, 8, and 12 d. Cells were washed with TBS buffer (0.1 M Tris–HCl, pH 7.4, 150 mM NaCl) and lysed in RIPA buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton-X, 0.1% SDS, 5 lg/ml aprotinin (Roche, Branchburg, NJ, USA), 5 lg/ml Leupeptin (Roche), 1 mg/ml pefabloc SC (Roche), protease inhibitor cocktail tablets (Roche), 10 mM NaF, 2 mM Na3VO4. Whole cell lysates were used in mixture with sample buffer (100 mM Tris–HCl, pH 6.8, 2% SDS, 12% 2-mercaptoethanol, 20% glycerol, bromophenol blue). Samples were run on SDS–PAGE gels containing 10% acrylamide and blotted to PVDF membranes (Bio-Rad, Hercules, CA, USA). The blots were shaken and blocked in 0.1% Tween-20 in TBS (TBS-T) containing 5% nonfat dry milk and probed with the primary antibodies, followed by incubation with HRP-conjugated secondary antibodies. Visualization was performed with the ECL-Plus detection system (GE Healthcare). We used the xCELLigence RTCA with CIM-Plate 16 to evaluate the invasive capacity of CHMp13a after TGF-b1 stimulation. The principle of the RTCA invasion assay is very similar to that of invasion assays performed with a Boyden chamber (Albini et al., 1987). Briefly, the CIM-plate is composed of an upper chamber (UC) and a lower chamber (LC). The underside of UC measures electrical impedance caused by cell attachment, whereas the LC contains chemoattractants such as FBS. Therefore, an increase of electrical impedance over time is indicative of migration into the LC. The data obtained was represented as an arbitrary unit, which represented migrated cell number. First, RPMI containing 10% FBS was added to the LC, and then a matrigel-coated UC was attached to it. Next, 4 104 cells treated with TGF-b1 for 24 h were seeded in the UC. TGF-b1 stimulation was present for the duration of the experiment and cell invasion was measured continuously for 21 h. All measurements were run in triplicate. The Mann–Whitney U test was used to evaluate differences in the real-time invasion assays. Data analysis was carried out for each time point, with a p-value of <0.05 considered significant. Western blot analysis was performed to evaluate the changes in the expression of epithelial and mesenchymal markers after TGF-b1 stimulation. TGF-b1 treatment up-regulated the mesenchymal marker vimentin on days 1 and 2; however, down-regulations of the epithelial markers E-cadherin and b-catenin were not observed (Fig. 1). Phosphorylation of Smad2 was detected on days 1 and 2, consistent with the role of Smad2 activation in the induction of vimentin expression. The changes were transient, and these molecular signatures were lost in CHMp13a following prolonged stimulation. We next evaluated cellular invasive capacity which is one of the characteristics of EMT using the xCELLigence RTCA with CIM-Plate 16. As shown in Fig. 2, TGF-b1 caused a significant enhancement of invasive capacity was observed after 6, 7, 8 and 9 h. However, there was no statistical difference except those time points. EMT has several key features, including changes in epithelial and mesenchymal markers, morphology, and invasive capacity (Kalluri and Weinberg, 2009). Many reports show that TGF-b1 plays a key role in mediating EMT in several cell lines. However, one report has shown that the frequency of EMT across multiple cell lines is low. Specifically, EMT was only found in 2 of 20 cell
Fig. 1. CHMp13a was treated with 1 ng/mL TGF-b1 for 1, 2, 4, 8, and 12 d, and the expression of epithelial and mesenchymal markers were determined by western blot analysis. A transient increase of vimentin expression was observed, and this change corresponded to the phosphorylation of Smad2.
Fig. 2. The xCELLigence RTCA was used to observe the invasive capacity in realtime. CHMp13a (4 104 cells/well) treated with or without 1 ng/ml TGF-b1 for 24 h were seeded in a matrigel-coated CIM-Plate 16, and incubated for 21 h. CHMp-13a acquired transient invasive capacity upon TGF-b stimulation. The data are represented as the mean cell index.
lines that were tested following TGF-b1 treatment (Brown et al., 2004). In the present study, loss of epithelial markers was not observed but up-regulation of vimentin and acquisition of invasive capacity were observed in CHMp13a, and this change was thought to be ‘‘partial EMT’’ (p-EMT). TGF-b-mediated EMT is mediated by both Smad-dependent and Smad-independent pathways. In this study, we observed the expression of vimentin and phosphorylation of Smad2 on days 1 and 2. These data suggest that the Smad pathway is involved in TGF-b-mediated p-EMT in CHMp13a. However, the changes we observed were not stable, suggesting the existence of additional mechanism that limit EMT. In this regard, there are reports that TGF-b signaling is subject to negative feedback regulation. For example, Smad7 is induced following TGF-b stimulation and can act as inhibitor of TGF-b signaling by competing with receptor-associated Smad molecules (Heldin and Moustakas, 2012). Thus, the disappearance of vimentin and phospho-Smad2 after day 4 may be related to this negative feedback loop, although further studies are required to confirm this hypothesis. In this study, we also observed a change of the invasive capacity of CMGTs after TGF-b stimulation. The acquisition of invasive capacity is a key feature of EMT and is a crucial step in metastasis. Quantitating the invasive capacity in vitro has been performed by many researchers since Albini et al. reported matrigel-coated Boyden chamber assay in 1987 (Albini et al., 1987). In this method, the
K. Yoshida et al. / Research in Veterinary Science 94 (2013) 539–541
cells that have migrated through the membrane are stained and counted. The potential disadvantage of this method is that the process of migration cannot be followed. In contrast, the xCELLigence RTCA system is an excellent approach for monitoring cell migration in real-time, and as such it has advantages over traditional migration assays (Hamada et al., 1998). Because the induction of vimentin by TGF-b1 in CHMp13a was transient, we inferred that enhancement of invasive capacity was also likely to be transient. Indeed, real-time monitoring using xCELLigence RTCA revealed the transient nature of enhanced invasion capacity following TGF-b stimulation in these cells. This change would otherwise have been undetected in the absence of real-time monitoring. Although CHMp13a acquired partial mesenchymal characteristics temporarily, sustained stimulation with TGF-b led to a reversion of the phenotype. This phenomenon is similar to the mesenchymal–epithelial transition (MET). MET, the reverse of EMT, was considered to take place at distal sites during the formation of secondary tumors, and is equally important as EMT for the establishment of metastasis (Hugo et al., 2007). Although the mechanisms underlying MET are still unknown, we suggest that the transient p-EMT observed in CHMp13a contributes to the process. In conclusion, we show that TGF-b1 transiently induced transient expression of vimentin and enhanced invasive capacity in CHMp13a. This phenomenon was similar to MET and may be advantageous for the formation of new tumor clusters at the side of metastatic lesions. To clarify the mechanism of metastasis is very important for the development of treatments. This study could be a first step towards a future vision in the EMT targeting treatments of CMGTs.
References Albini, A., Iwamoto, Y., Kleinman, H.K., Martin, G.R., Aaronson, S.A., Kozlowski, J.M., McEwan, R.N., 1987. A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Research 47, 3239–3245. Aomatsu, K., Arao, T., Sugioka, K., Matsumoto, K., Tamura, D., Kudo, K., Kaneda, H., Tanaka, K., Fujita, Y., Shimomura, Y., Nishio, K., 2011. TGF-b induces sustained upregulation of SNAI1 and SNAI2 through Smad and non-Smad pathways in a human corneal epithelial cell line. Investigative Ophthalmology & Visual Science 14, 2437–2443. Brodey, R.S., Goldschmidt, M.H., Roszel, J.R., 1983. Canine mammary gland neoplasms. Journal of the American Animal Hospital Association 19, 61–90. Brown, K.A., Aakre, M.E., Gorska, A.E., Price, J.O., Eltom, S.E., Pietenpol, J.A., Moses, H.L., 2004. Induction by transforming growth factor-beta 1 of epithelial to
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mesenchymal transition is a rare event in vitro. Breast Cancer Research 6, R215–231. Gilbertson, S.R., Kurzman, I.D., Zachrau, R.E., Hurvitz, A.I., Black, M.M., 1983. Canine mammary epithelial neoplasms: biologic implications of morphologic characteristics assessed in 232 dogs. Veterinary Pathology 20, 127–142. Hamada, J., Nagayasu, H., Kawano, T., Mizutani, T., Nakata, D., Hosokawa, M., Takeichi, N., 1998. Characterization of the progressive sublines derived from a weakly malignant cloned cell line, ER-1, co-inoculated subcutaneously with a foreign body. Clinical & Experimental Metastasis 16, 291–298. Heldin, C.H., Moustakas, A., 2012. Role of Smads in TGFb signaling. Cell and Tissue Research 347, 21–36. Hugo, H., Ackland, M.L., Blick, T., Lawrence, M.G., Clements, J.A., Williams, E.D., Thompson, E.W., 2007. Epithelial–mesenchymal and mesenchymal–epithelial transitions in carcinoma progression. Journal of Cellular Physiology 213, 374– 383. Kalluri, R., Weinberg, R.A., 2009. The basics of epithelial–mesenchymal transition. Journal of Clinical Investigation 119, 1420–1428. Kokkinos, M.I., Wafai, R., Wong, M.K., Newgreen, D.F., Thompson, E.W., Waltham, M., 2007. Vimentin and epithelial–mesenchymal transition in human breast cancer – observations in vitro and in vivo. Cells Tissues Organs 185, 191–203. Lenferink, A.E., Magoon, J., Cantin, C., O’Connor-McCourt, M.D., 2004. Investigation of three new mouse mammary tumor cell lines as models for transforming growth factor (TGF)-beta and Neu pathway signaling studies: identification of a novel model for TGF-beta-induced epithelial-to-mesenchymal transition. Breast Cancer Research 6, R514–530. Matos, A.J.F., Lopes, C., Carvalheira, J., Santos, M., Rutteman, G.R., Gärtner, F., 2006. E-cadherin expression in canine malignant mammary tumours: relationship to other clinico-pathological variables. Journal of Comparative Pathology 134, 182–189. Miettinen, P.J., Ebner, R., Lopez, A.R., Derynck, R., 1994. TGF-b induced transdifferentiation of mammary epithelial cells to mesenchymal cells: involvement of type I receptors. Journal of Cell Biology 127, 2021–2036. Misdorp, W., Hart, A.A.M., 1979. Canine mammary cancer. II. Therapy and causes of death. Journal of Small Animal Practice 20, 395–404. Moustakas, A., Heldin, C.H., 2007. Signaling networks guiding epithelial– mesenchymal transitions during embryogenesis and cancer progression. Cancer Science 98, 1512–1520. Rodo, A., Malicka, E., 2008. E-cadherin immunohistochemical expression in mammary gland neoplasms in bitches. Polish Journal of Veterinary Sciences 11, 47–54. Sarli, G., Preziosi, R., Tolla, L.D., Brunetti, B., Benazzi, C., 2004. E-cadherin immunoreactivity in canine mammary tumors. Journal of Veterinary Diagnostic Investigation 16, 542–547. Shi, Y., Massagué, J., 2003. Mechanisms of TGF-b signaling from cell membrane to the nucleus. Cell 113, 685–700. Thiery, J.P., 2002. Epithelial–mesenchymal transition in tumour progression. Nature Reviews Cancer 2, 442–454. Tsuji, T., Ibaragi, S., Hu, G., 2009. Epithelial–mesenchymal transition and cell cooperativity in metastasis. Cancer Research 15, 7135–7139. Xu, J., Lamouille, S., Derynck, R., 2009. TGF-b-induced epithelial to mesenchymal transition. Cell Research 19, 156–172. Yao, D., Dai, C., Peng, S., 2011. Mechanism of the mesenchymal–epithelial transition and its relationship with metastatic tumor formation. Molecular Cancer Research 9, 1608–1620. Zavadil, J., Böttinger, E.P., 2005. TGF-b and epithelial-to-mesenchymal transitions. Oncogene 24, 5764–5774.
Contents lists available at SciVerse ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Transforming growth factor-b transiently induces vimentin expression and invasive capacity in a canine mammary gland tumor cell line K. Yoshida, T. Saito, A. Kamida, K. Matsumoto, K. Saeki, M. Mochizuki, N. Sasaki, T. Nakagawa ⇑ Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan
a r t i c l e
i n f o
Article history: Received 4 June 2012 Accepted 19 October 2012
Keywords: Dog Mammary gland tumor EMT TGF-b MET xCELLigence RTCA
a b s t r a c t The epithelial–mesenchymal transition (EMT) is a crucial event that occurs during cancer metastasis and can be induced by transforming growth factor-b (TGF-b) in various tumor cells in vitro. However, little is known about the effects of TGF-b in canine mammary gland tumors (CMGTs). Here, we investigated the role of TGF-b in CMGT. We observed that treatment of the CMGT cell line CHMp13a with TGF-b1 leads to transient induction of the mesenchymal marker vimentin. Real-time measurements of cellular electrical impedance also showed that CMGT invasiveness is transiently increased by TGF-b1 treatment, but is reversed after prolonged stimulation. This phenomenon is similar to the mesenchymal–epithelial transition (MET, the reverse phenomenon of EMT), and a process that is implicated in the establishment of secondary metastatic lesions. Ó 2012 Elsevier Ltd. All rights reserved.
Canine mammary gland tumors (CMGTs) are one of the most frequent tumors in female dogs. Histopathologically, approximately half of CMGTs are considered malignant (Brodey et al., 1983; Gilbertson et al., 1983), and prognosis of such tumors is poor because of distant metastasis, especially to the lungs (Misdorp and Hart, 1979). Many researchers have suggested various proteins as prognostic factors in CMGTs. For example, loss of E-cadherin which is an epithelial marker was reported to be related to a poor prognosis (Matos et al., 2006; Sarli et al., 2004; Rodo and Malicka, 2008). The epithelial–mesenchymal transition (EMT) is the differentiation switch from polarized epithelial cells to the fibroblastic mesenchymal cells, and it is a crucial event during physiological and disease processes, including development, fibrosis, and cancer (Thiery, 2002; Kalluri and Weinberg, 2009; Tsuji et al., 2009). When tumor cells invade the surrounding tissues, their loss of cell–cell interaction and acquisition of invasive properties are associated with EMT. EMT is characterized by the loss of epithelial markers such as E-cadherin and up-regulation of mesenchymal markers such as vimentin (Kokkinos et al., 2007). EMT is regulated by various growth factors and cytokines (Moustakas and Heldin, 2007; Yao et al., 2011). Among these, transforming growth factor-b (TGF-b) is a key factor controlling the induction of EMT (Miettinen et al., 1994; Xu et al., 2009). ⇑ Corresponding author. Address: Laboratory of Veterinary Surgery, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Tel.: +81 3 5841 5414; fax: +81 3 5841 8996. E-mail address: [email protected] (T. Nakagawa). 0034-5288/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rvsc.2012.10.016
Secreted TGF-b binds to the complex composed of TGF-b type I receptor (TbRI) and TGF-b type II receptor (TbRII). This complex activates both Smad-dependent and Smad-independent pathways (Aomatsu et al., 2011; Heldin and Moustakas, 2012). In the Smad-dependent pathway, receptor-regulated Smads (R-Smads, Smad2/3) are phosphorylated and interact with a common partner Smad, Smad4, which translocates into the nucleus and acts as transcriptional factor (Shi and Massagué, 2003). TGF-b was reported to induce EMT and promote cell invasion and metastasis in some human and mouse tumor cells (Lenferink et al., 2004; Zavadil and Böttinger, 2005). However, the relationship between TGF-b and the EMT phenotype in CMGT remains unclear. The purpose of this study was to evaluate EMT in a CMGT cell line in response to TGF-b by measuring change in epithelial and mesenchymal markers as well as cellular invasive capacity, and discuss the role of TGF-b induced EMT in metastasis. A clonal CMGT cell line CHMp13a was maintained in RPMI-1640 containing L-glutamine medium (Wako, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) and 50 mg/L gentamicin sulfate (Sigma–Aldrich Corporation, St. Louis, MO, USA) at 37 °C in a humidified atmosphere of 5% CO2. Recombinant human TGF-b1 was purchased from PeproTech (Rocky Hill, NJ, USA). The primary antibodies used for epithelial markers in the western blot analysis were mouse anti-E-cadherin (1:1000, BD Transduction Laboratories, Lexington, KY, USA) and mouse anti-b-catenin (1:2000, BD Transduction Laboratories). Mouse anti-vimentin (1:1500, Millipore, Bedford, MA, USA) and mouse anti-N-cadherin (1:1000, United States Biological,
540
K. Yoshida et al. / Research in Veterinary Science 94 (2013) 539–541
Swampscott, MA, USA) were used for mesenchymal markers. To evaluate downstream TGF-b signaling, mouse anti-Smad2/3 (1:1000, BD Transduction Laboratories) and rabbit anti-phosphorylated Smad2 (1:1000, Millipore) were used. Mouse anti-pan-actin (1:10,000, Cell Signaling, Danvers, MA, USA) was used as a loading control. Secondary antibodies used were anti-mouse IgG conjugated to horseradish peroxidase (HRP) (1:20,000, GE Healthcare UK Ltd., Buckinghamshire, UK) and anti-rabbit IgG conjugated to HRP (1:10,000, GE Healthcare). Recombinant human TGF-b1 was added to a final concentration of 1 ng/mL, and cells were incubated at 37 °C for 1, 2, 4, 8, and 12 d. Cells were washed with TBS buffer (0.1 M Tris–HCl, pH 7.4, 150 mM NaCl) and lysed in RIPA buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton-X, 0.1% SDS, 5 lg/ml aprotinin (Roche, Branchburg, NJ, USA), 5 lg/ml Leupeptin (Roche), 1 mg/ml pefabloc SC (Roche), protease inhibitor cocktail tablets (Roche), 10 mM NaF, 2 mM Na3VO4. Whole cell lysates were used in mixture with sample buffer (100 mM Tris–HCl, pH 6.8, 2% SDS, 12% 2-mercaptoethanol, 20% glycerol, bromophenol blue). Samples were run on SDS–PAGE gels containing 10% acrylamide and blotted to PVDF membranes (Bio-Rad, Hercules, CA, USA). The blots were shaken and blocked in 0.1% Tween-20 in TBS (TBS-T) containing 5% nonfat dry milk and probed with the primary antibodies, followed by incubation with HRP-conjugated secondary antibodies. Visualization was performed with the ECL-Plus detection system (GE Healthcare). We used the xCELLigence RTCA with CIM-Plate 16 to evaluate the invasive capacity of CHMp13a after TGF-b1 stimulation. The principle of the RTCA invasion assay is very similar to that of invasion assays performed with a Boyden chamber (Albini et al., 1987). Briefly, the CIM-plate is composed of an upper chamber (UC) and a lower chamber (LC). The underside of UC measures electrical impedance caused by cell attachment, whereas the LC contains chemoattractants such as FBS. Therefore, an increase of electrical impedance over time is indicative of migration into the LC. The data obtained was represented as an arbitrary unit, which represented migrated cell number. First, RPMI containing 10% FBS was added to the LC, and then a matrigel-coated UC was attached to it. Next, 4 104 cells treated with TGF-b1 for 24 h were seeded in the UC. TGF-b1 stimulation was present for the duration of the experiment and cell invasion was measured continuously for 21 h. All measurements were run in triplicate. The Mann–Whitney U test was used to evaluate differences in the real-time invasion assays. Data analysis was carried out for each time point, with a p-value of <0.05 considered significant. Western blot analysis was performed to evaluate the changes in the expression of epithelial and mesenchymal markers after TGF-b1 stimulation. TGF-b1 treatment up-regulated the mesenchymal marker vimentin on days 1 and 2; however, down-regulations of the epithelial markers E-cadherin and b-catenin were not observed (Fig. 1). Phosphorylation of Smad2 was detected on days 1 and 2, consistent with the role of Smad2 activation in the induction of vimentin expression. The changes were transient, and these molecular signatures were lost in CHMp13a following prolonged stimulation. We next evaluated cellular invasive capacity which is one of the characteristics of EMT using the xCELLigence RTCA with CIM-Plate 16. As shown in Fig. 2, TGF-b1 caused a significant enhancement of invasive capacity was observed after 6, 7, 8 and 9 h. However, there was no statistical difference except those time points. EMT has several key features, including changes in epithelial and mesenchymal markers, morphology, and invasive capacity (Kalluri and Weinberg, 2009). Many reports show that TGF-b1 plays a key role in mediating EMT in several cell lines. However, one report has shown that the frequency of EMT across multiple cell lines is low. Specifically, EMT was only found in 2 of 20 cell
Fig. 1. CHMp13a was treated with 1 ng/mL TGF-b1 for 1, 2, 4, 8, and 12 d, and the expression of epithelial and mesenchymal markers were determined by western blot analysis. A transient increase of vimentin expression was observed, and this change corresponded to the phosphorylation of Smad2.
Fig. 2. The xCELLigence RTCA was used to observe the invasive capacity in realtime. CHMp13a (4 104 cells/well) treated with or without 1 ng/ml TGF-b1 for 24 h were seeded in a matrigel-coated CIM-Plate 16, and incubated for 21 h. CHMp-13a acquired transient invasive capacity upon TGF-b stimulation. The data are represented as the mean cell index.
lines that were tested following TGF-b1 treatment (Brown et al., 2004). In the present study, loss of epithelial markers was not observed but up-regulation of vimentin and acquisition of invasive capacity were observed in CHMp13a, and this change was thought to be ‘‘partial EMT’’ (p-EMT). TGF-b-mediated EMT is mediated by both Smad-dependent and Smad-independent pathways. In this study, we observed the expression of vimentin and phosphorylation of Smad2 on days 1 and 2. These data suggest that the Smad pathway is involved in TGF-b-mediated p-EMT in CHMp13a. However, the changes we observed were not stable, suggesting the existence of additional mechanism that limit EMT. In this regard, there are reports that TGF-b signaling is subject to negative feedback regulation. For example, Smad7 is induced following TGF-b stimulation and can act as inhibitor of TGF-b signaling by competing with receptor-associated Smad molecules (Heldin and Moustakas, 2012). Thus, the disappearance of vimentin and phospho-Smad2 after day 4 may be related to this negative feedback loop, although further studies are required to confirm this hypothesis. In this study, we also observed a change of the invasive capacity of CMGTs after TGF-b stimulation. The acquisition of invasive capacity is a key feature of EMT and is a crucial step in metastasis. Quantitating the invasive capacity in vitro has been performed by many researchers since Albini et al. reported matrigel-coated Boyden chamber assay in 1987 (Albini et al., 1987). In this method, the
K. Yoshida et al. / Research in Veterinary Science 94 (2013) 539–541
cells that have migrated through the membrane are stained and counted. The potential disadvantage of this method is that the process of migration cannot be followed. In contrast, the xCELLigence RTCA system is an excellent approach for monitoring cell migration in real-time, and as such it has advantages over traditional migration assays (Hamada et al., 1998). Because the induction of vimentin by TGF-b1 in CHMp13a was transient, we inferred that enhancement of invasive capacity was also likely to be transient. Indeed, real-time monitoring using xCELLigence RTCA revealed the transient nature of enhanced invasion capacity following TGF-b stimulation in these cells. This change would otherwise have been undetected in the absence of real-time monitoring. Although CHMp13a acquired partial mesenchymal characteristics temporarily, sustained stimulation with TGF-b led to a reversion of the phenotype. This phenomenon is similar to the mesenchymal–epithelial transition (MET). MET, the reverse of EMT, was considered to take place at distal sites during the formation of secondary tumors, and is equally important as EMT for the establishment of metastasis (Hugo et al., 2007). Although the mechanisms underlying MET are still unknown, we suggest that the transient p-EMT observed in CHMp13a contributes to the process. In conclusion, we show that TGF-b1 transiently induced transient expression of vimentin and enhanced invasive capacity in CHMp13a. This phenomenon was similar to MET and may be advantageous for the formation of new tumor clusters at the side of metastatic lesions. To clarify the mechanism of metastasis is very important for the development of treatments. This study could be a first step towards a future vision in the EMT targeting treatments of CMGTs.
References Albini, A., Iwamoto, Y., Kleinman, H.K., Martin, G.R., Aaronson, S.A., Kozlowski, J.M., McEwan, R.N., 1987. A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Research 47, 3239–3245. Aomatsu, K., Arao, T., Sugioka, K., Matsumoto, K., Tamura, D., Kudo, K., Kaneda, H., Tanaka, K., Fujita, Y., Shimomura, Y., Nishio, K., 2011. TGF-b induces sustained upregulation of SNAI1 and SNAI2 through Smad and non-Smad pathways in a human corneal epithelial cell line. Investigative Ophthalmology & Visual Science 14, 2437–2443. Brodey, R.S., Goldschmidt, M.H., Roszel, J.R., 1983. Canine mammary gland neoplasms. Journal of the American Animal Hospital Association 19, 61–90. Brown, K.A., Aakre, M.E., Gorska, A.E., Price, J.O., Eltom, S.E., Pietenpol, J.A., Moses, H.L., 2004. Induction by transforming growth factor-beta 1 of epithelial to
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