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

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.

Figure 1

Loss of the Growth-Inhibitory Effect of TGF-β1 on CNE2 cells. CNE2 and/or NP69 cells were seeded in 96-well plate at 5 × 10³ cells/well. (A) 2.5-12.5 ng/ml or (B) only 10 ng/mlTGFβ1 was added after 24, 48, 72, and 96 hours. Cell counting assay was used to indicate the degree of cell growth. Results were presented as the spectrophotometrical absorbance of cells treated with CCK-8 solution at the wavelength of 450 nm. * Statistically significant (P < 0.05, t-test) as compared with NP69 group. The values are expressed as means ± SD of six repeated experiments.

Figure 2

The mRNA level of the TGF-β receptor II and the Smads in CNE2 and NP69 cells. (A) Expression level of the TβRII, Smad 2, Smad 3, Smad 4, Smad 7 in CNE2 cells and NP69 cells by RT-PCR using specific primers. β-actin was used as a control and was further to normalize. (B) Bar diagram of the TβRII, Smad 2, Smad 3, Smad 4, Smad 7 mRNA level from densitometric measurement of three real-time quantitative PCR from three separate treatments. * Statistically significant (P < 0.05, t-test) as compared with NP69 group.** Statistically significant (P < 0.01, t-test) as compared with NP69 group.

Figure 3

The expression of the TGF-β receptor II and the Smads in CNE2 and NP69 cells. Expression level of the TβRII, Smad 2, Smad 3, Smad 4, Smad 7 in CNE2 cells and NP69 cells by western blot. Actin was used as a protein loading control and was further to normalize. Relative density was account between NP69 and CNE2 group.

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.