Tuberc Respir Dis.  2011 Mar;70(3):206-217. 10.4046/trd.2011.70.3.206.

The Regulation of FOXP3 Expression by the Treatment of TGF-beta and the Modification of DNA Methylation in Lung Cancer Cell Lines

• Um SW
• Lee SH
• Kim H
• Kwon OJ
• Kim H
• Kang JS
• Lee WJ

• KMID: 1442850
• DOI: http://doi.org/10.4046/trd.2011.70.3.206

Abstract

BACKGROUND
Transcription factor FOXP3 characterizes the thymically derived regulatory T cells. FOXP3 is expressed by cancer cell itself and FOXP3 expression was induced by TGF-beta treatment in pancreatic cancer cell line. However, the expression of FOXP3 expression is not well known in patients with lung cancer. This study was conducted to investigate the expression of FOXP3 in patients with lung cancer and to investigate the regulation of FOXP3 expression by the treatment of TGF-beta and DNA methyltransferase inhibitor in lung cancer cell lines.
METHODS
FOXP3 expression in the tissue of patients with resected non-small cell lung cancer (NSCLC) was evaluated by immunohistochemistry. The regulation of FOXP3 expression was investigated by Western blot and RT-PCR after lung cancer cell lines were stimulated with TGF-beta1 and TGF-beta2. The regulation of FOXP3 expression was also investigated by RT-PCR and flow cytometry after lung cancer cell lines were treated with DNA methyltransferase inhibitor (5-AZA-dC).
RESULTS
FOXP3 expression was confirmed in 27% of patients with NSCLC. In NCI-H460 cell line, TGF-beta2 decreased FOXP3 mRNA and protein expressions. In A549 cell line, both TGF-beta1 and TGF-beta2 decreased FOXP3 mRNA and protein expressions. 5-AZA-dC increased FOXP3 mRNA expression in NCI-H460 and A549 cell lines. Moreover, 5-AZA-dC increased intracellular FOXP3 protein expression in A549 cell lines.
CONCLUSION
It was shown that FOXP3 is expressed by cancer cell itself in patients with NSCLC. Treatment of TGF-beta2 and DNA methyltransferase inhibitor seems to be associated with the regulation of FOXP3 expression in lung cancer cell lines.

Keyword

Lung Neoplasms; FOXP3 protein, human; Transforming Growth Factor beta; DNA Methylation

MeSH Terms

Blotting, Western
Carcinoma, Non-Small-Cell Lung
Cell Line
DNA
DNA Methylation
Flow Cytometry
Forkhead Transcription Factors
Humans
Immunohistochemistry
Lung
Lung Neoplasms
Pancreatic Neoplasms
RNA, Messenger
T-Lymphocytes, Regulatory
Transcription Factors
Transforming Growth Factor beta
Transforming Growth Factor beta1
Transforming Growth Factor beta2
DNA
Forkhead Transcription Factors
RNA, Messenger
Transcription Factors
Transforming Growth Factor beta
Transforming Growth Factor beta1
Transforming Growth Factor beta2

Figure

• 

  Figure 1 FOXP3 expression in the tissue of patient with lung cancer and lung cancer cell lines. (A) Immunohistochemical staining of paraffin-embedded non-small cell lung carcinoma tissue revealed FOXP3 expression in lung carcinoma cells (H&E stain, ×400). (B) FOXP3 mRNA expression was confirmed in NCI-H460 and A549 cell lines. (C) FOXP3 protein expression was confirmed in NCI-H460 and A549 cell lines.

• 

  Figure 2 FOXP3 mRNA and protein expression after TGF-β treatment of NCI-H460 cell lines. The expression of FOXP3 mRNA after stimulation with TGF-β1 (A) and TGF-β2 (B) with different concentrations in NCI-H460 cell lines for 48 hours. The expression of FOXP3 mRNA after stimulation with 5 ng/mL TGF-β1 (C) and 5 ng/mL TGF-β2 (D) in NCI-H460 cell lines for given times. (E) The expression of FOXP3 protein after stimulation with 5 ng/mL TGF-β1 and 5 ng/mL TGF-β2 for 48 hours in NCI-H460 cell lines. (F) Mean relative expression of FOXP3 expression after stimulation with 5 ng/mL TGF-β1 and 5 ng/mL TGF-β2 for 48 hours from 3 independent experiments of NCI-H460 cell lines. CON: no treatment; NC: negative control (BEAS-2B cell line); PC: positive control (FOXP3 293T cell transient overexpression lysate).

• 

  Figure 3 FOXP3 mRNA and protein expression after TGF-β treatment of A549 cell lines. (A) The expression of FOXP3 mRNA decreased after stimulation with 5 ng/mL TGF-β1 and 5 ng/mL TGF-β2 in A549 cell lines for 48 hours. (B) The expression of FOXP3 protein after stimulation with 5 ng/mL TGF-β1 and 5 ng/mL TGF-β2 in A549 cell lines for 48 hours. (C) Mean relative expression of FOXP3 expression after stimulation with 5 ng/mL TGF-β1 and 5 ng/mL TGF-β2 for 48 hours from 3 independent experiments of A549 cell lines. CON: no treatment; NC: negative control (BEAS-2B cell line); PC: positive control (FOXP3 293T cell transient overexpression lysate).

• 

  Figure 4 FOXP3 mRNA expression after 5-AZA-dC treatment of NCI-H460 and A549 cell lines. (A) The expression of FOXP3 mRNA after treatment with 5-AZA-dC with given concentrations for 24-, 48-, 72-, and 96 hours in NCI-H460 cell lines. (B) The expression of FOXP3 mRNA after treatment with 5-AZA-dC with given concentrations for 24-, 48-, 72-, and 96 hours in A549 cell lines. 5-AZA-dC treatment did not have significant effects on cell viability of NCI-H460 (C) and A549 (D) cell lines. NCI-H460 and A549 cell lines were incubated with 5-AZA-dC (1 and 10 µM) for 24-, 48-, 72-, and 96 hours. Cell viability was assessed by the MTT assay. CON: control.

• 

  Figure 5 5-AZA-dC treatment and intracellular FOXP3 expression in A549 cell lines. Intracellular FOXP3 expression after treatment of 5-AZA-dC with given concentrations for 48 hours in A549 cell lines. The cells were stained using Alexa FluorR 488 anti-human FOXP3 antibody (solid line) or isotype-matched control antibody (dotted line) and analyzed by flow cytometry.

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