Cells and cell culture
We purchased SAS, a human tongue squamous cell carcinoma cell line, from the RIKEN Cell Bank (Tsukuba, Japan). FaDu, the cells from a human hypopharyngeal SCC cell line, were kindly gifted by the Department of Cell Biology and Morphology, Akita University Graduate School of Medicine (Akita, Japan). SF-TY, a human fibroblast cell line, was obtained from the Japanese Collection of Research Bioresources Cell Bank (JCRB Cell Bank, Osaka, Japan).
All cells were maintained in the Dulbecco's modified Eagle's medium (DMEM; Merck KGaA, Darmstadt, Germany) supplemented with 10% fetal bovine serum in a humidified atmosphere containing 5% CO2 at 37°C. For neutralization of IL-6, neutralizing mouse anti-IL-6 mAb (MAB406) and isotype control immunoglobulin G1 (IgG1) mAb (MAB005) were purchased from R & D Systems (Minneapolis, MN)
Irradiation
Cells were seeded in 60 mm dishes and cultured to 80% confluence before being irradiated at 2, 5, or 10 Gy at room temperature using a 160 kVp cabinet X-ray system filtered with 0.5 mm Cu by the Faxitron CP-160 (Faxitron X-Ray Corp., Wheeling, IL, USA). After irradiation, cells were cultured for 24 h prior to being harvested and subjected to an IL-6 enzyme-linked immunosorbent assay (ELISA) assay or cell migration assay. To conduct a cell survival assay, cells were cultured for 6 days after irradiation before being fixed and stained with crystal violet and then being observed under a light microscope (Olympus, Tokyo, Japan).
Cell survival assay (crystal violet staining)
The cells were fixed in 4% paraformaldehyde for 20 min at room temperature and then stained with 0.04% crystal violet in 1% ethanol (20 min at room temperature). The plates were subsequently washed extensively under running tap water and air dried. After solubilization of the samples in 1% sodium dodecyl sulfate (SDS), the optical density values were read by plate reader at 550 nm.
Migration assay
We evaluated cell migration in vitro using semipermeable modified Boyden inserts with a pore size of 8 µm (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). In total, 3 × 104 SAS and FaDu cells were plated in the inserts. For the coculture assay, 5 × 104 fibroblast cells were plated in a holding well. Plating was conducted on serum‑free DMEM. We plated the same number of cells from the inserts in 96-well plates to serve as loading controls. Both the inserts and holding wells were filled with serum-free medium composition. Depending on the needs of experiment, 10 ng/ml of IL-6, 20 ng/ml of IL-6 neutralizing antibody, or 20 ng/ml of control IgG were added to the medium. After 24 h of treatment at 37°C in a 5% CO2 incubator, we gently wiped away the cells in the insert using a cotton swab. Cells on the reverse side of the insert were fixed and stained with Diff‑Quik® (Sysmex, Kobe, Japan) according to the manufacturer's instructions. We counted the invading cells in four representative fields using light microscopy at a magnification of 200×. Cells plated in 96-well plates were subjected to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays and we normalized the cell numbers across the groups. We also adjusted the number of migrating cells accordingly.
Western blotting
We detected protein expression using western blot analysis with actin used as an internal control. We lysed cell lines in detergent containing 1% NP-40, 150 mmol/l NaCl, 1 mmol/l EDTA, 0.1 mmol/l phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml aprotinin and then determined the protein levels using the Bio-Rad Protein Assay method (Bio-Rad Laboratories Inc., Hercules, CA, USA). We separated 40 µg of the total protein on 8% SDS-PAGE gels and transferred these to nitrocellulose membranes using a semidry transfer machine (Bio-Rad Laboratories). Next, we blocked membranes with 5% skimmed milk/TBS with Tween-20 solution for 1 h at room temperature, before incubating with primary antibodies in 5% skimmed milk in TBS‑T overnight at 4°C. After washing with TBS-T three times, we incubated the membranes for 1 h with horseradish-peroxidase-conjugated secondary antibody (Bio-Rad Laboratories) at 1:3,000 diluted in 5% skimmed milk in TBS‑T. We then rinsed the filters with TBS‑T three times and developed the blot using Luminol Reagent (Santa Cruz Biotechnology, Santa Cruz, CA, USA) by autoradiography. The band intensities were analyzed using ImageJ (U. S. National Institutes of Health). We used the following primary antibodies: mouse anti-IL-6Rα (1:1,000; Santa Cruz Biotechnology, Santa Cruz, USA), mouse anti-gp130 (1:1,000; Santa Cruz Biotechnology), and mouse anti-actin (1:3,000; Santa Cruz Biotechnology).
ELISA
The serum levels of IL-6 were measured by an ELISA using an ELISA kit (Proteintech, Rosemont, IL, USA.). To evaluate changes in IL-6 expression by irradiation in fibroblasts, 3 × 106 fibroblasts were seeded on 6-well plates in DMEM with 10% fetal bovine serum (FBS) overnight. The medium was changed to serum-free DMEM and irradiated at 10 Gy. Nonirradiated fibroblasts were also prepared as a control. At 24 h after irradiation, supernatants were collected, and subjected to an ELISA assay.
Statistical analysis
Statistical analyses were performed using Statcel 3 (OMS Publishing, Tokorozawa, Japan). A Wilcoxon–Mann–Whitney two-tailed exact test was used to assess the statistically significant differences in cell survival, migration studies, and IL-6 expression. Data are presented as means ± standard deviation (SD) from experiments that were repeated at least three times. We considered differences with P < 0.05 as statistically significant.