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. 2016 Aug 30;7(35):57213-57227.
doi: 10.18632/oncotarget.11127.

Celecoxib and sulindac inhibit TGF-β1-induced epithelial-mesenchymal transition and suppress lung cancer migration and invasion via downregulation of sirtuin 1

Affiliations

Celecoxib and sulindac inhibit TGF-β1-induced epithelial-mesenchymal transition and suppress lung cancer migration and invasion via downregulation of sirtuin 1

Byong-Ki Cha et al. Oncotarget. .

Abstract

The non-steroidal anti-inflammatory drugs (NSAIDs) celecoxib and sulindac have been reported to suppress lung cancer migration and invasion. The class III deacetylase sirtuin 1 (SIRT1) possesses both pro- and anticarcinogenic properties. However, its role in inhibition of lung cancer cell epithelial-mesenchymal transition (EMT) by NSAIDs is not clearly known. We attempted to investigate the potential use of NSAIDs as inhibitors of TGF-β1-induced EMT in A549 cells, and the underlying mechanisms of suppression of lung cancer migration and invasion by celecoxib and sulindac. We demonstrated that celecoxib and sulindac were effective in preventing TGF-β1-induced EMT, as indicated by upregulation of the epithelial marker, E-cadherin, and downregulation of mesenchymal markers and transcription factors. Moreover, celecoxib and sulindac could inhibit TGF-β1-enhanced migration and invasion of A549 cells. SIRT1 downregulation enhanced the reversal of TGF-β1-induced EMT by celecoxib or sulindac. In contrast, SIRT1 upregulation promoted TGF-β1-induced EMT. Taken together, these results indicate that celecoxib and sulindac can inhibit TGF-β1-induced EMT and suppress lung cancer cell migration and invasion via downregulation of SIRT1. Our findings implicate overexpressed SIRT1 as a potential therapeutic target to reverse TGF-β1-induced EMT and to prevent lung cancer cell migration and invasion.

Keywords: EMT; SIRT1; celecoxib; lung cancer; sulindac.

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Conflict of interest statement

No potential conflicts of interest were disclosed.

Figures

Figure 1
Figure 1. Transforming growth factor (TGF)-β1-induced sirtuin 1 (SIRT1) expression in lung cancer
A. Endogenous expression of the epithelial-mesenchymal transition (EMT)-related proteins E-cadherin, N-cadherin, and SIRT1 was assessed in non-small cell lung cancer cell lines. B. A549 cells were treated with TGF-β1 (5 or 10 ng/mL) and epidermal growth factor (50 or 100 ng/mL) for 24 h. EMT hallmarks were examined using western blot analysis. Similar data were obtained from three independent experiments.
Figure 2
Figure 2. Transforming growth factor (TGF)-β1-induced sirtuin 1 (SIRT1) expression
A. and B. A549 cells were treated with TGF-β1, and SIRT1 mRNA was quantified by real-time polymerase chain reaction in a time or dose dependent manner. The data represent the mean ± SD of three independent experiments. *p < 0.05 compared to the control. C and D. A549 cells were treated with TGF-β1, and the expression of E-cadherin, N-cadherin, and SIRT1 was determined by immunoblotting. E. A549 cells were treated with 5 ng/mL TGF-β1, with or without SB431542 for 24h, and SIRT1 protein levels were examined by immunoblotting. Similar data were obtained from three independent experiments.
Figure 3
Figure 3. Celecoxib and sulindac inhibit transforming growth factor (TGF)-β1-induced epithelial-mesenchymal transition in A549 cells
A. A549 cells were stimulated with 5 ng/mL TGF-β1 for 2 h and then incubated with 10 μM celecoxib or 500 μM sulindac for 48 h. Cell morphology was examined, and cells were fixed, permeabilized, and stained with anti-SIRT1, E-cadherin, and N-cadherin monoclonal antibody (green); and DAPI (blue). Cells were analyzed by confocal microscopy. All scale bars represent 60 μm. B. Western blot analysis using specific antibodies was performed to examine protein expression in whole cell lysates. Representative images from more than three independent experiments are shown. C. A549 cells were stimulated with 5 ng/mL TGF-β1 for 2 h and then incubated with 10 μM celecoxib or 500 μM sulindac for 48 h. The supernatants were analyzed by gelatin zymography, and cell lysates was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis to measure the expression of MMP2 and MMP9. D. Cells were treated with 10 μM celecoxib or 500 μM sulindac in the absence or presence of 5 ng/mL TGF-β1. Cell lysates were then prepared and subjected to immunoblot analysis with antibodies to phosphorylated (p) or total forms of smad 2/3. Immunoblots are representative of at least three independent experiments.
Figure 4
Figure 4. Effects of celecoxib and sulindac on transforming growth factor (TGF)-β1-induced A549 cell migration
A. For the electric cell-substrate impedance sensing migration assay, A549 cells were stimulated with 5 ng/mL TGF-β1 for 2h and then incubated with 10 μM celecoxib or 500 μM sulindac for 48 h. Cell migration was then assessed by continuous resistance measurements for 40 h. B. The histogram represents the fold change in migration. The data represent the mean ± SD of three independent experiments. *p < 0.05 compared with the control, **p < 0.05 compared to the TGF-β1 group. C. Cell migration was also evaluated by wound healing assay. The confluent A549 monolayer was scratched with a pipette tip and washed to remove the debris. Fresh medium containing 0.5% serum was then added. Red lines indicate the cell edges at the T0 point. Representative pictures are shown.
Figure 5
Figure 5. Effects of celecoxib and sulindac on transforming growth factor (TGF)-β1-induced A549 cell invasion
A. For the electric cell-substrate impedance sensing invasion assay, resistance changes in the impedance at 4 kHz as confluent layers of HUVEC cells were challenged with A549 cells suspensions. The control curve of HUVEC cells received media without A549 cells. A549 cells were treated as above and changes in resistance were monitored for 40 h. The data represent the mean ± SD of three independent experiments. *p < 0.05 compared with the control (HUVEC + A549), **p < 0.05 compared to the HUVEC + A549 + TGF-β1 group. B. Effect of celecoxib and sulindac on A549 cell invasion in a 200× light microscope after crystal violet staining by matrigel invasion assay as described in Materials and Methods. Matrigel invasion of A549 cells counted in five random views. The data represent the mean ± SD of three independent experiments. *p < 0.01 compared with the control, ** p < 0.05 compared to the TGF-β1 group.
Figure 6
Figure 6. Involvement of sirtuin 1 (SIRT1) in transforming growth factor (TGF)-β1-induced epithelial-mesenchymal transition (EMT) inhibited by celecoxib and sulindac
A. and B. Effect of SIRT1 inhibition on TGF-β1-induced EMT. Cells were transfected with SIRT1 siRNA or treated with the SIRT1 inhibitor EX-527, and then further incubated in the presence of celecoxib or sulindac for 24 h. The cell lysates of each group were prepared and probed for EMT hallmarks by western blot. C and D. Effect of SIRT1 activation on TGF-β1-induced EMT. Cells were transfected with Ad-lacZ or Ad-SIRT1, or treated with the SIRT1 activator SRT-1720, and then further incubated in the presence of celecoxib or sulindac for 24 h. The cell lysates were routinely prepared, and alterations in EMT hallmarks were determined by western blot.
Figure 7
Figure 7. Effect of sirtuin 1 (SIRT1) deletion on transforming growth factor (TGF)-β1-induced A549 cell migration and invasion, inhibited by celecoxib
A. Electric cell-substrate impedance sensing (ECIS) migration assay. A549 cells were transfected with siRNA specific for SIRT1. Control siRNA containing the same number of each nucleotide as the SIRT1 siRNA was used as the transfection control. Transfected cells were incubated in complete medium with TGF-β1 and/or 10 M celecoxib for 24 h. Cell migration was then assessed by continuous resistance measurements for 40 h. The data represent the mean ± SD of three independent experiments. *p < 0.05 and ** p < 0.01 compared with the control siRNA + TGF-β1. B. ECIS invasion assay. Resistance changes in the impedance at 4 kHz were measured as confluent layers of HUVEC cells were challenged with A549 cells suspensions. The control curve of HUVEC cells received media without A549 cells. A549 cells were treated as above and changes in resistance were monitored for 40 h. The histogram represents the fold change in migration or invasion. The data represent the mean ± SD of three independent experiments. *p < 0.05 and ** p < 0.01 compared with HUVEC + control siRNA + TGF-β1.

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