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ABSTRACT: Chemotherapy is the main strategy for the treatment of lung cancer. However, sensitivity to chemotherapy, one of the main factors affecting the survival rate of patients with lung cancer, is extremely poor. Forkhead box P3 (Foxp3) is the key regulatory gene for the development and function of CD4+CD25+ regulatory T cells (Tregs). Increased levels of Tregs and Foxp3 expression in the peripheral blood and tumour specimens of cancer patients are associated with tumour progression and poor prognosis. In addition, certain studies have suggested that Tregs may be resistant to conventional chemotherapy and thus, enhance tumour immune evasion. Previous studies have demonstrated that Foxp3 is also expressed within tumour cells and that it may mimic the function of Tregs. Currently, the correlation between the tumour cell expression of Foxp3 and sensitivity to chemotherapy is unclear. Therefore, it was hypothesised that Foxp3 causes resistance to chemotherapeutic agents in lung cancer cells and that it may consequently promote the progression of lung cancer. In the current study, the expression of Foxp3 in mouse Lewis lung cancer (LLC) cells was detected using RT-PCR and immunocytochemistry. The overexpression of Foxp3, which was accomplished by the transient transfection of recombinant pcDNA3.1-Foxp3 or empty plasmids into LLC cells, was confirmed by RT-PCR and western blot analysis. The inhibition of cell proliferation was measured using MTT assay. The expression of multidrug resistance protein 1 (mdr1) mRNA and its protein product, P-glycoprotein (P-gp), were detected by RT-PCR and flow cytometry, respectively. The results revealed that Foxp3 was expressed by LLC cells. The inhibitory rate of cell proliferation in Foxp3-overexpressing LLC cells compared with those transfected with an empty plasmid was significantly decreased following adriamycin (ADM) and mitomycin C (MMC) treatment. The IC50 values of ADM and MMC in Foxp3-overexpressing LLC cells were increased. The expression levels of mdr1 mRNA and P-gp were significantly upregulated in Foxp3 overexpressing LLC cells. These results suggest that Foxp3 reduces the sensitivity of LLC cells to ADM and MMC, thus promoting tumour progression, by upregulating the expression of mdr1 mRNA and P-gp.
Molecular Medicine Reports 11/2012; 6(5):977-82. · 0.42 Impact Factor
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ABSTRACT: Foxp3 is a master regulator of the development and function of CD4+CD25+ regulatory T cells (Tregs). Previous studies have reported that Foxp3 is also expressed in tumour cells and promotes tumour immune evasion. However, the regulation of the expression of Foxp3 in tumour cells remains unclear. Toll-like receptor 4 (TLR4), a member of the pattern recognition receptor family, is also expressed in tumour cells. Previous studies have found that the TLR4 signaling pathway is involved in tumour immune evasion in lung cancer cells, and that the transcription factor, nuclear factor-κB (NF-κB), plays a key role in the TLR4 signaling pathway. Moreover, recent studies found that NF-κB promotes the transcription of Foxp3. We hypothesised that TLR4 may also be involved in the regulation of Foxp3 in A549 cells through NF-κB. Therefore, we examined the effect of TLR4 and NF-κB on the expression of Foxp3 in the A549 lung cancer cell line. The results showed that Foxp3 and TLR4 are expressed in A549 cells; the expression of Foxp3 increased after lipopolysaccharide (LPS) stimulation and decreased after blocking the TLR4 signaling pathway. In addition, the expression of NF-κB (p65) increased after LPS stimulation and the expression of Foxp3 decreased after blocking NF-κB. These results suggest that TLR4 is involved in the regulation of Foxp3 in A549 cells through NF-κB.
Molecular Medicine Reports 07/2012; 6(1):167-72. · 0.42 Impact Factor
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ABSTRACT: The autoimmune regulator (Aire) is a key mediator of the central tolerance for peripheral tissue self-antigen (PTAs) and is involved in the transcriptional control of many antigens in thymic medullary epithelial cells (mTECs). However, the function of Aire in peripheral lymphoid tissues and haematopoietic cells, particularly in monocytes and macrophages, remains poorly understood. We previously found that the expression of Toll-like receptor (TLR) 1, TLR3 and TLR8 was notably upregulated in pEGFPC1/Aire stably transfected RAW264.7 (GFP-Aire/RAW) cells, while the expressions of other TLRs were not significantly changed. The mechanism by which Aire affects TLR1, TLR3 and TLR8 expression is not clear. Interactions with other proteins, such as DNA-dependent protein kinase (DNA-PK), are crucial for regulating the transcriptional activity of Aire. In this study, we found that Aire and DNA-PK catalytic subunit (DNA-PKcs) were co-located in the nucleus of GFP-Aire/RAW cells, and they interact with each other. Small interfering RNA knock-down of DNA-PKcs in these cells decreased the expression of TLR1, TLR3 and TLR8, but no change was observed in pEGFPC1 stably transfected RAW264.7 (GFP/RAW) cells. We did not observe any change in the expressions of other TLRs after DNA-PKcs knock-down in GFP-Aire/RAW or GFP/RAW cells. A similar observation has been made in pEGFPC1/Aire or pEGFPC1 transiently transfected primary peritoneal macrophages. Using a luciferase activity assay, we found the that the transcriptional activity of TLR1, TLR3 and TLR8 promoters was also decreased after knock-down of DNA-PKcs in GFP-Aire/RAW cells. In conclusion, our results suggest that DNA-PKcs may interact with Aire to promote the expression of TLRs in RAW264.7 cells.
Scandinavian Journal of Immunology 01/2012; 75(5):479-88. · 2.23 Impact Factor
