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The effect of transforming growth factor-beta1 on nasopharyngeal carcinoma cells: insensitive to cell growth but functional to TGF-beta/Smad pathway.

Xiao J, Xiang Q, Xiao YC, Su ZJ, Huang ZF, Zhang QH, Tan Y, Li XK, Huang YD - J. Exp. Clin. Cancer Res. (2010)

Bottom Line: We found that the growth of CNE2 cells was not suppressed by TGF-beta1.The signaling proteins TbetaRII, Smad 7 were expressed normally, while Smad2, Smad3, and Smad4 increased significantly at the mRNA level.The results suggested that CNE2 cells are not sensitive to growth suppression by TGF-beta1, but the TGF-beta/Smad signaling transduction is functional.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Pharmacy, Wenzhou Medical College, Wenzhou 325035, China.

ABSTRACT

Objectives: This study explored the response of nasopharyngeal carcinoma cells to TGF-beta1-induced growth suppression and investigated the roles of the TGF-beta/Smad signaling pathway in nasopharyngeal carcinoma cells.

Methods: The cells of nasopharyngeal carcinoma cell line CNE2 were treated with TGF-beta1. The growth responses of CNE2 cells were analyzed by MTT assay. The mRNA expression and protein subcellular localization of the TGF-beta/Smad signaling components in the CNE2 were determined by real time RT-PCR and immunocytochemical analysis.

Results: We found that the growth of CNE2 cells was not suppressed by TGF-beta1. The signaling proteins TbetaRII, Smad 7 were expressed normally, while Smad2, Smad3, and Smad4 increased significantly at the mRNA level. TGF-beta type II receptor and Smad7 had no change compared to the normal nasopharyngeal epithelial cells. In addition, Smad2 was phosphorylated to pSmad2, and the activated pSmad2 translocated into the nucleus from the cytoplasm, while the inhibitory Smad-Smad7 translocated from the nucleus to the cytoplasm after TGF-beta1 stimulation.

Conclusion: The results suggested that CNE2 cells are not sensitive to growth suppression by TGF-beta1, but the TGF-beta/Smad signaling transduction is functional. Further work is needed to address a more detailed spectrum of the TGF-beta/Smad signaling pathway in CNE2 cells.

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Localization of expression of the TβR-II, Smad2, Smad3, Smad4, Smad7 and phosphorylated Smad2 in CNE2 cells. (A) The TβR-II was located mainly in the cell membrane, and positive staining Smad2, Smad3, Smad4, was found in regions of both cytoplasm and nucleus, while the staining of Smad7 was mainly in the area of nucleus. (B) Phosphorylated Smad2 was undetectable in CNE2 cells without TGF-β1, after stimulation with TGF-β1, phosphorylated Smad2 could be detected in the cytoplasm of CNE2 cells, while Smad7 located originally in nuclear without TGF-β1, and it could be detected in the cytoplasm after stimulation of TGF-β1.
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Figure 4: Localization of expression of the TβR-II, Smad2, Smad3, Smad4, Smad7 and phosphorylated Smad2 in CNE2 cells. (A) The TβR-II was located mainly in the cell membrane, and positive staining Smad2, Smad3, Smad4, was found in regions of both cytoplasm and nucleus, while the staining of Smad7 was mainly in the area of nucleus. (B) Phosphorylated Smad2 was undetectable in CNE2 cells without TGF-β1, after stimulation with TGF-β1, phosphorylated Smad2 could be detected in the cytoplasm of CNE2 cells, while Smad7 located originally in nuclear without TGF-β1, and it could be detected in the cytoplasm after stimulation of TGF-β1.

Mentions: To investigate alterations of the TGF-β/Smad signaling pathway in CNE2 cells, the TGF-β type II receptor (TβR-II) and the TGF-β/Smad signaling components-Smads signal transduction were explored at both mRNA level and protein level by real time RT-PCR, using specific primers according to GenBank database sequences, western blotting and immunocytochemical analysis, respectively. First, we investigated TβR-II mRNA expression which is an upstream signaling partner of the TGF-β/Smad signaling pathway, while the normal nasopharyngeal epithelial cells were used as control. Under the same culture conditions, we found that TβR-II was significantly up-regulated in CNE2 cells compared to the levels observed in NP69 cells. We further evaluated the Smads which are the principal intracellular components of the TGF-β signaling pathway, and the results showed that Smad2, Smad3 and Smad4 mRNA all increased significantly in CNE2 cells compared to the levels observed in NP69 cells. However, the mRNA level of smad7, known as an inhibitory Smad, remained at same level as that observed for the normal nasopharyngeal cells (Figure 2A, 2B). To investigate the protein expression of the TβR-II receptor and Smads, western blotting was performed in NP69 and CNE2 cells. We found that Smad2, Smad3, Smad4 and TβR-II were also up-regulated in protein levels, but Smad7 protein level were no different compared to that observed in NP69 cells (Figure 3). To further localize the expression of the above signaling components in CNE2 cells, immunocytochemical staining was conducted. A positive staining of TβR-II was found in most CNE2 cells, and the cell membrane was the main localization of the protein. The positive staining of Smad2, Smad3 and Smad4 was found in regions of both the cytoplasm and nucleus, while the staining of Smad7 was mainly in the nucleus (Figure 4A).


The effect of transforming growth factor-beta1 on nasopharyngeal carcinoma cells: insensitive to cell growth but functional to TGF-beta/Smad pathway.

Xiao J, Xiang Q, Xiao YC, Su ZJ, Huang ZF, Zhang QH, Tan Y, Li XK, Huang YD - J. Exp. Clin. Cancer Res. (2010)

Localization of expression of the TβR-II, Smad2, Smad3, Smad4, Smad7 and phosphorylated Smad2 in CNE2 cells. (A) The TβR-II was located mainly in the cell membrane, and positive staining Smad2, Smad3, Smad4, was found in regions of both cytoplasm and nucleus, while the staining of Smad7 was mainly in the area of nucleus. (B) Phosphorylated Smad2 was undetectable in CNE2 cells without TGF-β1, after stimulation with TGF-β1, phosphorylated Smad2 could be detected in the cytoplasm of CNE2 cells, while Smad7 located originally in nuclear without TGF-β1, and it could be detected in the cytoplasm after stimulation of TGF-β1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2865451&req=5

Figure 4: Localization of expression of the TβR-II, Smad2, Smad3, Smad4, Smad7 and phosphorylated Smad2 in CNE2 cells. (A) The TβR-II was located mainly in the cell membrane, and positive staining Smad2, Smad3, Smad4, was found in regions of both cytoplasm and nucleus, while the staining of Smad7 was mainly in the area of nucleus. (B) Phosphorylated Smad2 was undetectable in CNE2 cells without TGF-β1, after stimulation with TGF-β1, phosphorylated Smad2 could be detected in the cytoplasm of CNE2 cells, while Smad7 located originally in nuclear without TGF-β1, and it could be detected in the cytoplasm after stimulation of TGF-β1.
Mentions: To investigate alterations of the TGF-β/Smad signaling pathway in CNE2 cells, the TGF-β type II receptor (TβR-II) and the TGF-β/Smad signaling components-Smads signal transduction were explored at both mRNA level and protein level by real time RT-PCR, using specific primers according to GenBank database sequences, western blotting and immunocytochemical analysis, respectively. First, we investigated TβR-II mRNA expression which is an upstream signaling partner of the TGF-β/Smad signaling pathway, while the normal nasopharyngeal epithelial cells were used as control. Under the same culture conditions, we found that TβR-II was significantly up-regulated in CNE2 cells compared to the levels observed in NP69 cells. We further evaluated the Smads which are the principal intracellular components of the TGF-β signaling pathway, and the results showed that Smad2, Smad3 and Smad4 mRNA all increased significantly in CNE2 cells compared to the levels observed in NP69 cells. However, the mRNA level of smad7, known as an inhibitory Smad, remained at same level as that observed for the normal nasopharyngeal cells (Figure 2A, 2B). To investigate the protein expression of the TβR-II receptor and Smads, western blotting was performed in NP69 and CNE2 cells. We found that Smad2, Smad3, Smad4 and TβR-II were also up-regulated in protein levels, but Smad7 protein level were no different compared to that observed in NP69 cells (Figure 3). To further localize the expression of the above signaling components in CNE2 cells, immunocytochemical staining was conducted. A positive staining of TβR-II was found in most CNE2 cells, and the cell membrane was the main localization of the protein. The positive staining of Smad2, Smad3 and Smad4 was found in regions of both the cytoplasm and nucleus, while the staining of Smad7 was mainly in the nucleus (Figure 4A).

Bottom Line: We found that the growth of CNE2 cells was not suppressed by TGF-beta1.The signaling proteins TbetaRII, Smad 7 were expressed normally, while Smad2, Smad3, and Smad4 increased significantly at the mRNA level.The results suggested that CNE2 cells are not sensitive to growth suppression by TGF-beta1, but the TGF-beta/Smad signaling transduction is functional.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Pharmacy, Wenzhou Medical College, Wenzhou 325035, China.

ABSTRACT

Objectives: This study explored the response of nasopharyngeal carcinoma cells to TGF-beta1-induced growth suppression and investigated the roles of the TGF-beta/Smad signaling pathway in nasopharyngeal carcinoma cells.

Methods: The cells of nasopharyngeal carcinoma cell line CNE2 were treated with TGF-beta1. The growth responses of CNE2 cells were analyzed by MTT assay. The mRNA expression and protein subcellular localization of the TGF-beta/Smad signaling components in the CNE2 were determined by real time RT-PCR and immunocytochemical analysis.

Results: We found that the growth of CNE2 cells was not suppressed by TGF-beta1. The signaling proteins TbetaRII, Smad 7 were expressed normally, while Smad2, Smad3, and Smad4 increased significantly at the mRNA level. TGF-beta type II receptor and Smad7 had no change compared to the normal nasopharyngeal epithelial cells. In addition, Smad2 was phosphorylated to pSmad2, and the activated pSmad2 translocated into the nucleus from the cytoplasm, while the inhibitory Smad-Smad7 translocated from the nucleus to the cytoplasm after TGF-beta1 stimulation.

Conclusion: The results suggested that CNE2 cells are not sensitive to growth suppression by TGF-beta1, but the TGF-beta/Smad signaling transduction is functional. Further work is needed to address a more detailed spectrum of the TGF-beta/Smad signaling pathway in CNE2 cells.

Show MeSH
Related in: MedlinePlus