Um, Sang-Won
(Division of Pulmonary and Critical Care Medicine, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine)
,
Lee, Sang-Hee
(Division of Pulmonary and Critical Care Medicine, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine)
,
Kim, Ho-Joong
(Division of Pulmonary and Critical Care Medicine, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine)
,
Kwon, O-Jung
(Division of Pulmonary and Critical Care Medicine, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine)
,
Kim, Hang-Rae
(Department of Anatomy and Tumor Immunity Medical Research Center, Seoul National University College of Medicine)
,
Kang, Jae-Seung
(Department of Anatomy and Tumor Immunity Medical Research Center, Seoul National University College of Medicine)
,
Lee, Wang-Jae
(Department of Anatomy and Tumor Immunity Medical Research Center, Seoul National University College of Medicine)
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 know...
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-${\beta}1$ and TGF-${\beta}2$. 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-${\beta}2$ decreased FOXP3 mRNA and protein expressions. In A549 cell line, both TGF-${\beta}1$ and TGF-${\beta}2$ 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-${\beta}2$ and DNA methyltransferase inhibitor seems to be associated with the regulation of FOXP3 expression in lung cancer cell lines.
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-${\beta}1$ and TGF-${\beta}2$. 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-${\beta}2$ decreased FOXP3 mRNA and protein expressions. In A549 cell line, both TGF-${\beta}1$ and TGF-${\beta}2$ 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-${\beta}2$ and DNA methyltransferase inhibitor seems to be associated with the regulation of FOXP3 expression in lung cancer cell lines.
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제안 방법
Recently, in studies conducted on melanoma, colorectal cancer, lung cancer, prostate cancer, breast cancer, and other various cancer cell lines applying flow cytometry and qRT-PCT, the expression of FOXP3 in tumor cells has been reported8,9. In studies examined the expression of FOXP3 in A549, NCI-H460 and CaLu-6 lung cancer cell lines by flow cytometry, the mean fluorescence intensity was higher than the control group fibroblasts8. In lung cancer cell lines such as CaLu-1, CaLu-6, GILI, ONET, SK-LU-1, NCI-H441, NCI-H460, NCI-H596 and NCI-H661 cells, according to studies examined the expression of FOXP3 mRNA by qRT-PCR, the expression of FOXP3 mRNA was elevated in GILI, NCI-H460 and NCI-H661 in comparison with the control fibroblasts9.
The modulation of the expression of FOXP3 mRNA according to the concentration of 5-AZA-dC treatment (0, 1μM and 10μM) as well as the treatment time (24-, 48-, 72- and 96 hours) was evaluated.
The modulation of the expression of FOXP3 protein during 48 hours of the treatment with different concentrations of 5-AZA-dC (0, 0.1μM, 1μM and 10μM) was examined by flow cytometry analysis.
The purpose of this study was to examine whether FOXP3 is expressed in lung cancer tissues by immunohistochemical staining, and to evaluate the modulation of the expression of FOXP3 in response to TGF-β treatment as well as the treatment with DNA methyl-transferase inhibitor.
To assess the intracellular expression of FOXP3 protein, Alexa FluorR 488 anti-human FOXP3 antibody (BioLegend, San Diego, CA, USA), Alexa FluorR 488 mouse IgG1 (BioLegend), k isotype control (BioLegend), and FOXP3 Fix/Perm buffer set (BioLegend) were used. In each experiment, approximately 2×105 tumor cells were used, and approximately 2×104 cells (10%) were analyzed.
대상 데이터
Selected among patients newly diagnosed as nonsmall cell lung cancer at the Samsung Medical Center from 1994 to 1996, the subjects were patients who underwent radical resection surgery. For immunohistochemical staining, tissue microarray blocks were used.
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.
이론/모형
NCI-H460 and A549 cell lines were incubated with 5-AZA-dC (1 and 10μM) for 24-, 48-, 72-, and 96 hours. Cell viability was assessed by the MTT assay. CON: control.
성능/효과
The expression of FOXP3 protein in tumor cells obtained from lung cancer patient tissues was assessed by immunohistochemical staining (Figure 1A). Of total 96 study subject patients, the expression of FOXP3 was confirmed in 27 patients (28.1%). The expression of FOXP3 was not different according to histological types.
The results of 3 independent experiments showed that in response to TGF-β1 or TGF-β2 stimulation, in comparison with the control group, the reduction of the expression of FOXP3 protein by average 0.4 times or 0.5 times, respectively, was shown (Figure 2F).
후속연구
In regard to the regulation of FOXP3 by TGF-β, additional studies on diverse tumor cell lines should be performed in the future.
참고문헌 (14)
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