Limits...
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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2865451&req=5

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.

Mentions: TGF-β1 is a potent growth inhibitor of epithelial cells. To test the response of human NPC cells to TGF-β1, we examined the growth pattern of CNE2 cells after TGF-β1 treatment. The rate of cell growth and the metabolic activity was indicated the degree of the growth suppression by TGF-β1 and a time course study regarding the growth suppression of CNE2 was performed. The data showed that the effect of growth suppression by TGF-β1 against CNE2 was not observed. Instead of suppression, CNE2 continued to grow after 24 h with TGF-β1 treatment at the various concentrations (2.5, 5, 7.5, 10, and 12.5 ng/ml), and reached a growth peak at 48 h after TGF-β1 treatment. Although TGF-β1 caused a slight increase in proliferation on CNE2 after TGF-β1 treatment by 48 h, no statistical significance was found compared to the untreated controls (Figure 1A). The insensitivity to TGF-β1 implied that the TGF-β1 signaling pathway could be abnormal in the CNE2 cells. To confirm the effect of growth suppression on the normal nasopharyngeal epithelial cells by TGF-β1, we performed the Cell Counting Kit-8 assay on the NP69 cells exposed to TGF-β1. Under the same experimental conditions, we used TGF-β1 at a concentration of 10 ng/ml because this concentration induced a high proliferation rate in the CNE2 cells among all time points tested. We monitored cell growth within 96 h after TGF-β1 treatment, and found that TGF-β1 did have the effect of growth suppression on NP69 cells. Adding TGF-β1 at a concentration of 10 ng/ml to the cell culture medium significantly reduced the viable cell number after 48 h, and the suppression rate of NP69 cells by TGF-β1 was statistically significant compared to the untreated NP69 cells (Figure 1B).


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)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

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.
Mentions: TGF-β1 is a potent growth inhibitor of epithelial cells. To test the response of human NPC cells to TGF-β1, we examined the growth pattern of CNE2 cells after TGF-β1 treatment. The rate of cell growth and the metabolic activity was indicated the degree of the growth suppression by TGF-β1 and a time course study regarding the growth suppression of CNE2 was performed. The data showed that the effect of growth suppression by TGF-β1 against CNE2 was not observed. Instead of suppression, CNE2 continued to grow after 24 h with TGF-β1 treatment at the various concentrations (2.5, 5, 7.5, 10, and 12.5 ng/ml), and reached a growth peak at 48 h after TGF-β1 treatment. Although TGF-β1 caused a slight increase in proliferation on CNE2 after TGF-β1 treatment by 48 h, no statistical significance was found compared to the untreated controls (Figure 1A). The insensitivity to TGF-β1 implied that the TGF-β1 signaling pathway could be abnormal in the CNE2 cells. To confirm the effect of growth suppression on the normal nasopharyngeal epithelial cells by TGF-β1, we performed the Cell Counting Kit-8 assay on the NP69 cells exposed to TGF-β1. Under the same experimental conditions, we used TGF-β1 at a concentration of 10 ng/ml because this concentration induced a high proliferation rate in the CNE2 cells among all time points tested. We monitored cell growth within 96 h after TGF-β1 treatment, and found that TGF-β1 did have the effect of growth suppression on NP69 cells. Adding TGF-β1 at a concentration of 10 ng/ml to the cell culture medium significantly reduced the viable cell number after 48 h, and the suppression rate of NP69 cells by TGF-β1 was statistically significant compared to the untreated NP69 cells (Figure 1B).

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