Human SW620 colorectal cancer cells were cultured in DMEM (Hyclone; GE Healthcare Life Sciences) containing 10% FBS (Gibco; Thermo Fisher Scientific Inc.) and 1% penicillin/streptomycin (Invitrogen; Thermo Fisher Scientific Inc.), in a cell culture chamber at 37°C with 5% CO₂. Following counting and inoculation in plates, the cells grew to the required cell density of 80–90% and were treated accordingly. For determining the optimal concentration of flurbiprofen for treating SW620 cells; 2, 4, 10 and 20 nM of flurbiprofen were used for 12, 24 or 48 h at 37°C. Cells treated with 0.1% DMSO were the control group.

A total of 3×10⁵ SW620 cells (cell density, 5×10⁴ cells/well) were plated in a 6-well plate, and transfection with an overexpression plasmid was performed when the cells grew to 70–80% density. During transfection, the original cell culture medium was removed and the cells were cultured in medium without serum and antibiotics. The overexpression plasmid pcDNA-COX2 (20 nM) and negative control pcDNA (1 µg) from Shanghai Genechem Co., Ltd., were diluted with 125 µl Opti-MEM medium (Gibco; Thermo Fisher Scientific Inc.), and Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was diluted with 125 µl Opti-MEM medium at room temperature for 5 min. These were then mixed for 15 min before addition to the cell culture medium. The liquid was changed after 4–6 h, and the cells were collected for further experiments after 24–48 h.

RNA was extracted from the treated cells using TRIzol (Invitrogen; Thermo Fisher Scientific Inc.). RT was performed with PrimeScript™ RT Reagent kit with gDNA Eraser (Takara Bio, Inc.) at 37°C for 15 min and at 98°C for 5 min. qPCR was performed using SYBR reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Thermocycling conditions were as follows: 95°C for 3 min, 40 cycles of 95°C for 5 sec followed by 60°C for 30 sec. GAPDH was used as the internal reference and quantification was performed using the 2^−ΔΔCq method (29). The primer sequences used were: COX2 forward, 5′-ATCATAAGCGAGGGCCAGCT-3′ and reverse, 5′-AAGGCGCAGTTTACGCTGTC-3′; GAPDH forward, 5′-GACAGTCAGCCGCATCTTCT-3′ and reverse, 5′-GCGCCCAATACGACCAAATC-3′.

SW620 cells were seeded into a 96-well plate 24 h post-transfection at a density of 3,000 cells/well. CCK-8 was used to assess the cell viability at 12, 24 and 48 h. A total of 10 µl CCK-8 solution (Dojindo Molecular Technologies) was added to each well and the cells were cultured for a further 4 h according to the manufacturer's protocol. An enzyme-linked immune detector was used to measure the absorbance value of each well at a wavelength of 450 nm.

The treated cells were lysed with RIPA lysis buffer (cat. no. 9806; Cell Signaling Technology, Inc.), placed in a 1.5-ml EP tube, and centrifuged at 4°C and 12,000 × g for 10 min. The supernatant was isolated and the protein concentration was measured using a BCA Protein assay kit (cat. no. 23227; Thermo Fisher Scientific, Inc.). Next, the protein was denatured at 100°C for 10 min with 10% SDS-polyacrylamide gel electrophoresis, and 25 µg protein sample per lane was separated. Following membrane transfer, the PVDF membrane was incubated with non-fat milk for 30 min at room temperature, incubated with primary antibody for 4°C overnight, and washed with TBS containing 0.05% Tween-20 (TBST) 3 times for 10 min each time. Primary antibodies were all purchased from Abcam and were against COX2 (1:1,000; cat. no. ab179800), cyclin E (1:1,000; cat. no. ab33911), CDK2 (1:1,000; cat. no. ab32147), p21 (1:1,000; cat. no. ab109520), Bcl-2 (1:5,000; cat. no. ab196495), Bax (1:1,000; cat. no. ab199677), cleaved caspase-3 (1:500; cat. no. ab49822), caspase-3 (1:5,000; cat. no. ab32351), MMP2 (1:1,000; cat. no. ab181286), MMP9 (1:1,000; cat. no. ab137867) and GAPDH (1:3,000; cat. no. ab9485). The membrane was then incubated with horseradish peroxidase-conjugated secondary antibody goat anti-rabbit IgG (1:10,000; cat. no. ab205718; Abcam) at room temperature for 2 h and washed with 1X TBST three times for 10 min each time. Enhanced chemiluminescence reagents (Beyotime Institute of Biotechnology) was added for visualization and next quantified with Quantity One version 4.1 software (Bio-Rad Laboratories Inc.).

In the immunofluorescence assay, changes in COX2 expression were examined. SW620 cells were subjected to fixation with 4% paraformaldehyde for 15 min at room temperature and permeabilization with 0.5% Triton X-100. Subsequently, 5% normal goat serum (PH0424; Scientific Phygene) was used to block cells for 20 min at 37°C, followed by incubation overnight at 4°C with anti-COX2 (1:1,000; cat. no. 4842; Cell Signaling Technology, Inc.). Subsequently, the cells were probed with an Alexa Fluor 488 labeled-goat anti-rabbit IgG (1:500; cat. no. A0423; Beyotime Institute of Biotechnology) for nearly 2 h at room temperature. Nuclei were counterstained using DAPI (Invitrogen; Thermo Fisher Scientific Inc.) at room temperature for 5 min, and images were captured using a fluorescence microscope (magnification, ×20).

Human IL-6 Quantikine (cat. no. D6050), human IL-1β Quantikine (cat. no. DLB50) and human TNF-α Quantikine ELISA kits (cat. no. DTA00D) (all R&D Systems, Inc.) were used to measure the levels of IL-6, IL-1β and TNF-α in cell supernatants of SW620 according to the manufacturer's protocols.

A total of 200 SW620 cells/well were plated in 12-well plates and cultured in DMEM with 10% FBS for 14 days. PBS was used to wash the cells twice, and then the cells were fixed with methanol for ~10 min at room temperature. Following fixation, the cells were washed twice with PBS, stained with crystal violet for 30 min at room temperature, and then washed with double-distilled water. The number of the colonies ≥50 was counted using the naked eye.

For flow cytometric analysis, FITC Annexin V Apoptosis Detection kit (Guangzhou RiboBio Co., Ltd.) was used. After 30 min of culture in the dark, SW620 cells (1×10⁵ cells/well) in 200 µl of binding buffer were mixed with 5 µl of propidium iodide (PI) for 15 min at 4°C and apoptosis was detected using a BD Accuri C6 Plus personal flow cytometer (Becton, Dickinson and Company) and analyzed using FlowJo version 10 software (Tree Star, Inc.).

Cell migratory ability was analyzed using a wound healing assay. Cells in control, Fp 10 nM, Fp 10 nM + pcDNA and Fp 10 nM + pcDNA-COX2 groups were cultured and then a 5-mm horizontal scratch was made using a pipette tip. Subsequently, cells were incubated for 1 day at 37°C in a serum-free medium, and the gap width was assessed and recorded at 0 and 24 h using a light microscope (Leica Microsystems) with magnification, ×100. Relative migration rate was calculated as the percentage of wound healing.

The cell invasion assay was performed using 24-well Transwell inserts (Corning, Inc.) pre-coated with Matrigel (BD Biosciences at room temperature for 15–30 min. Following cell suspension in 200 ml serum-free DMEM, cells (1×10⁵) were added to the upper chamber and medium supplemented with 10% FBS was placed in the bottom chamber. Subsequently, cells on the upper surface were removed, and cells on the bottom surface were fixed and stained with crystal violet at room temperature for 20 min. Cell numbers in five random fields were calculated using a light microscope (Nikon Corporation) with magnification, ×100.

Data obtained from triplicated independent experiments were presented as mean ± standard deviation (SD). Two-sided unpaired Student's t-test was used to assess the differences between two groups. For multiple groups, one-way ANOVA followed by Tukey's post hoc test was used. GraphPad Prism 5.0 software (GraphPad Software, Inc.) and SPSS 13.0 software (SPSS, Inc.) were employed to analyze data. P<0.05 was considered to indicate a statistically significant difference.

To evaluate the impact of flurbiprofen on colorectal cancer, SW620 cells were treated with different concentrations of flurbiprofen (2, 4, 10 and 20 nM) and the cell proliferation was assessed by a CCK-8 assay at 12, 24 and 48 h, respectively. As expected, flurbiprofen significantly decreased SW620 cell proliferation in a concentration- and time-dependent manner (Fig. 1A). In the colony formation assay, it was observed that with an increasing concentration of flurbiprofen, the number of colonies gradually decreased (Fig. 1B-C). When the cells were treated with 10 nM flurbiprofen, the cell viability (at 24 h) and colony formation ability were reduced to ~50% of the levels observed in the control group, therefore 10 nM flurbiprofen was selected for the following experiments. These data suggest that flurbiprofen inhibited colorectal cancer cell proliferation.

It has been reported that flurbiprofen is a non-selective COX inhibitor (30). Therefore, the present study aimed to evaluate whether flurbiprofen has an effect on COX2 in SW620 colorectal cancer cells. Western blotting was used to measure the protein level of COX2 after cells were treated with 10 nM flurbiprofen (Fig. 2A). In addition, RT-qPCR was used to assess the mRNA level of COX2 following treatment with 10 nM flurbiprofen (Fig. 2B). As expected, the protein and mRNA levels of COX2 were significantly decreased. To confirm this result, an immunofluorescence assay with SW620 cells treated with 10 nM flurbiprofen or DMSO was performed (Fig. 2C). The fluorescence intensity of COX2 was markedly reduced when treated with 10 nM flurbiprofen. The data confirmed that flurbiprofen decreased COX2 expression.

COX2 was overexpressed in SW620 cells, and western blotting and RT-qPCR were used to assess transfection efficiency (Fig. 3A and B). It has been reported that COX2 is a highly expressed inflammatory factor (31). Thus, the present study investigated whether flurbiprofen inhibits the expression of inflammatory factors by inhibiting COX2. ELISA was used to measure the levels of TNF-α, IL-1β and IL-6. As presented in the Fig. 3C, the levels of TNF-α, IL-1β and IL-6 were significantly downregulated in cells treated with flurbiprofen compared with control cells. However, when cells were co-treated with flurbiprofen and COX2 overexpression plasmid, the levels of TNF-α, IL-1β and IL-6 were significantly higher compared with in cells treated with flurbiprofen alone. These results suggested that flurbiprofen inhibited the expression of inflammatory factors via repression of COX2.

To determine whether flurbiprofen has an effect on colorectal cancer cell proliferation, a colony formation assay was performed, and it was identified that transfection of COX2 overexpression plasmid reversed the suppressed cell colony formation ability by flurbiprofen alone (Fig. 4A-B). Subsequently, western blotting was used to evaluate the changes of proliferation-associated proteins, including cyclin E, cyclin-dependent kinase 2 (CDK2) and p21. The results demonstrated that in cells treated with flurbiprofen, the expression levels of cyclin E and CDK2 were significantly decreased, and the expression of p21 was significantly increased. However, in cells co-treated with flurbiprofen and COX2 overexpression plasmid, the flurbiprofen-induced expression level changes of cyclin E, CDK2 and p21 were significantly reversed (Fig. 4C). These results suggested that flurbiprofen could inhibit the proliferation of colorectal cancer cells by inhibiting COX2.

It has been reported that COX2 can inhibit apoptosis (32), therefore the present study investigated whether flurbiprofen has an effect on colorectal cancer cell apoptosis. The results indicated that the apoptosis of cells treated with flurbiprofen was significantly increased. However, in cells co-treated with flurbiprofen and COX2 overexpression vector, the level of apoptosis was significantly reduced compared with in cells treated with flurbiprofen alone (Fig. 5A). Furthermore, the expression levels of apoptosis-associated proteins, including Bcl2, Bax, cleaved-caspase-3 and total caspase-3, were evaluated. The results demonstrated that flurbiprofen significantly decreased the expression of Bcl2 and significantly increased the expression of Bax and cleaved-caspase3, with no effect on total caspase-3 (Fig. 5B). However, following co-treatment with flurbiprofen and COX2 overexpression vector, the flurbiprofen-induced changes of Bcl2, Bax and cleaved-caspase-3 expression levels were significantly reversed.

The aforementioned data demonstrated the effects of flurbiprofen on cell proliferation and apoptosis in colorectal cancer, therefore the effects of flurbiprofen on colorectal cancer cell invasion and migration were then examined. Transwell and wound healing assays with SW620 cells were performed (Fig. 6A and B). When the cells were treated with flurbiprofen, the invasion and migration abilities were significantly reduced compared with the control cells. However, when cells were co-treated with flurbiprofen and COX2 overexpression plasmid, the invasion and migration abilities of cells were significantly reversed compared with the effect of flurbiprofen alone. Since matrix metalloproteinase (MMP)2 and MMP9 are key proteins associated with invasion and migration (33), whether flurbiprofen effected MMP2 and MMP9 expression was then investigated. The western blotting results demonstrated that the expression levels of MMP2 and MMP9 were significantly downregulated by flurbiprofen. However, when cells were co-treated with flurbiprofen and COX2 overexpression plasmid, the expression levels of MMP2 and MMP9 were significantly increased compared with in cells treated with flurbiprofen alone (Fig. 6C). In summary, flurbiprofen inhibited the invasion and migration of colorectal cancer cells by inhibiting COX2.