The human esophagus adenocarcinoma cell line OE19 was a gift from the Gastroenterology Department of Southwest Hospital of Third Military Medical University. OE19 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), L-glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin at 37°C, 5% CO₂ air atmosphere.

Surface IL-17 receptor (IL-17R) expression on OE19 cells was examined using flow cytometry as described previously (20). In brief, OE19 cells were washed three times, harvested, and resuspended in cold PBS. Then the cells were incubated with PE-labeled mouse anti-human IL-17RA antibody (eBioscience, CA, USA) in the dark for 1 h at 4°C. Isotype-matched antibody was used as a negative control. After washing, at least 1×10⁵ stained cells were analyzed using flow cytometry (BD Biosciences). Fluorescence-positive cells were quantified.

The effect of IL-17A on OE19 cell proliferation was measured by MTT assay. The cells were seeded in a 96-well plate at a density of 5,000 monolayer cells per well. After 24 h, the cells were incubated with IL-17A (rhIL-17A, R&D System, Minneapolis, MN, USA) in increasing concentrations (0, 1, 5, 10, 50 and 100 ng/ml) for 24, 48 and 72 h, respectively. Subsequently, the cells were washed with PBS and incubated with 20 µl MTT solution (5 g/l) for 4 h. After that, 150 µl DMSO was added to each well to dissolve the crystals and then the plates were shaken for 10 min in the dark. Finally, the optical density (OD) was measured at 490 nm using multifunctional fluorescence microplate reader (Polarstar Otima, BMG, Australia).

Cell migration ability in vitro was assessed by wound-healing assay. OE19 cells were seeded in 12-well flat bottomed plates and grown to ~80–90% monolayer confluence. A sterile pipette tip was used to carefully scratch a straight strip in the center of the well. After washing the cell debris, a wide range of doses of IL-17A (0, 1, 10 and 100 ng/ml) was added to cells. After 24 h incubation, the wound edges of different treatment groups were observed and captured. The migration rate was expressed as the percentage of average migration distance to average starting (0 h) wound distance, respectively.

The in vitro invasive assay was performed with 24-well Transwell (8-µm pore size; Corning, NY, USA) precoated with Matrigel (BD Biosciences, San Jose, CA, USA). Cells (2×10⁵), suspended in 200 µl serum-free medium in the absence or presence of IL-17A (1, 10 and 100 ng/ml), were seeded into the upper chamber, and 500 µl medium supplemented with 10% FBS was placed into the lower chamber. After 24 h incubation, the cells on the upper surface of the membrane were removed by cotton swabs. Subsequently, the cells on the lower surface were fixed with paraformaldehyde, and stained with crystal violet. Finally, invasive cells in five microscopic fields were counted and captured.

Intracellular ROS levels were measured by oxidation-sensitive fluorescent dye DCFH-DA. OE19 cells were preconditioned with NAC (5 mM, Beyotime, Shanghai, China) or vehicle for 2 h, then stimulated with IL-17A (100 ng/ml) for another 6 h. After treatment, cells were washed twice and incubated in 10 µM DCFH-DA (Invitrogen, Carlsbad, CA, USA) for 30 min. Then the cells were washed three times and collected for further analysis. Fluorescence was detected by flow cytometry and fluorescence microscopy, respectively.

OE19 cells grown in 6-well plates were pretreated with NAC or PDTC (100 µM, Sigma-Aldrich, St. Louis, MO, USA) for 2 h, then stimulated with IL-17A (100 ng/ml) for 6 h (for the measurement of NF-κB p65, p50, p-IκB-α), or 24 h (for the measurement of MMP-2/9). After incubation, whole cell proteins were extracted using cell lysis buffer. Nuclear lysates were harvested using NucBuster™ Protein Extraction kit (Novagen, Darmstadt, Germany) according to the manufacturer's instructions. Proteins were boiled for 5 min, separated by 8 or 10% SDS-PAGE, transferred to PVDF membranes, and incubated overnight at 4°C with primary antibodies against NF-κB p65 (1:1,000, Millipore), NF-κB p50 (1:1,000, Millipore), p-IκB-α (1:500, Cell Signaling Technology), MMP-2 and MMP-9 (1:1,000, Abcam), then followed by a secondary antibody for 2 h at room temperature. Histone H1 and GAPDH (1:2,000, Santa Cruz Biotechnology) were used as loading control.

All data are represented as mean ± SD from at least three independent experiments performed in triplicate. One-way ANOVA and Student's t-test were conducted to analyze the difference between groups using SPSS 17.0 software. P<0.05 was considered statistically significant.

In order to assess the biological effects of IL-17A on OE19 cells, firstly we examined the expression of IL-17R on OE19 cells. Flow cytometry confirmed that the cells expressed IL-17RA on the surface at the protein level (Fig. 1A). This result is in accordance with the ubiquitous expression of IL-17R (21). Subsequently, MTT assay was used to test the possibility that IL-17A might modulate OE19 cell proliferation in vitro. Cells were treated with IL-17A at concentrations ranging from 1 to 100 ng/ml. The result showed that the cell viability did not differ significantly among groups with different IL-17A concentrations at the exposure times of 24, 48 or 72 h, respectively (all P>0.05, Fig. 1B).

To determine whether IL-17A could promote OE19 cell migration and invasion, wound healing and Matrigel-coated Transwell invasion assay were performed. The wound healing assay showed that the migration rate of OE19 cells reached ~26% in the absence of IL-17A, however, IL-17A treatment remarkably promoted cell migration rate in a dose-dependent manner (Fig. 2A and B). The following Transwell invasion assay indicated that the number of invasive cells was significantly higher in IL-17A-treated group than the control group, and similarly the number markedly increased along with the increasing concentrations of IL-17A (Fig. 2C and D).

MMPs are known to play crucial roles in cancer metastasis by degrading the extra cellular matrix (22), we next investigated the effect of IL-17A on MMP-2 and MMP-9 expression in OE19 cells. Western blotting showed that the protein levels of MMP-2 and MMP-9 were significantly upregulated in IL-17A-treated cells compared with the untreated control, respectively (Fig. 2E and F). These results suggested that IL-17A promoted the migration and invasion of OE19 cells possibly by upregulating expression of MMP-2 and MMP-9.

There is accumulating evidence that ROS perform an important function in tumor development (23,24), and IL-17A was also reported to cause an increase in intracellular ROS levels (8,9), so we next examined the effect of IL-17A on ROS production in OE19 cells. Fluorescence microscopy showed that IL-17A at the concentration of 100 ng/ml caused ~2-fold increase in intracellular ROS levels (Fig. 3A and C). The promoting effect of IL-17A on ROS production was further confirmed by flow cytometry (Fig. 3B), whereas it was strikingly attenuated by the pretreatment with NAC (Fig. 3).

It has been well-documented that NF-κB is a major transcription factor that mediates migration and invasion of cancer cells, which is also downstream of oxidative stress (25). Therefore, we next detected whether IL-17A could induce NF-κB activation in a ROS-dependent manner, which in turn promotes cell invasion. As shown in Fig. 4A and C, the protein levels of both p65 and p50 in the whole cell did not differ between groups with or without IL-17A, however, they were remarkably elevated in the nucleus in IL-17A-treated cells compared with the untreated cells (Fig. 4B and D). The nuclear/overall ratio of p65 and p50 were also increased by IL-17A (Fig. 4E). Furthermore, the protein level of p-IκB-α was markedly upregulated in IL-17A-treated cells (Fig. 4A and C). Whereas, pretreatment with NAC or PDTC significantly reversed these changes, which indicated that IL-17A could induce NF-κB activation in a ROS-dependent manner.

To address the effect of ROS/NF-κB signaling pathway on IL-17A-induced MMP-2/9 expression and cell invasion, OE19 cells were pretreated with NAC or PDTC for 2 h and then incubated with IL-17A (100 ng/ml) for 24 h. Western blotting showed that the effect of IL-17A on the upregulation of MMP-2/9 expression was both significantly inhibited (Fig. 5A and B). Furthermore, Transwell invasion assay indicated that IL-17A-induced cell invasiveness was also markedly attenuated by NAC or PDTC in vitro (Fig. 5C and D).