All methods were performed in accordance with the relevant guidelines and regulations.
Cells and cell culture
OSCC cell lines. Cal27 cells and SCC9 cells both were obtained directly from BRJ Biological Technology Company, Ltd (Nanjing, China). Cal27 cells were cultured in dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin (50 units/mL)/streptomycin (50 μg/mL) in a humidiﬁed atmosphere (5%CO2) at 37 °C. SCC9 cells were cultured in dulbecco's modified eagle medium and ham's F-12 nutrient mixture (DMEM/F12) supplemented with 10% FBS and penicillin (50 units/mL)/streptomycin (50 μg/mL) in a humidiﬁed atmosphere (5%CO2) at 37 °C. The two cell lines were both identified by short tandem repeat (STR) profiling (Supplementary Figure1,2).
The primary OSCC cells for the study were from tongue squamous cell carcinoma tissue of one patient. The patient, a 29-year-old man (T4N2M0); had left tongue low-differentiated squamous cell carcinoma with bilateral cervical lymph node metastases. Fresh specimens from tongue carcinoma were dissected promptly after operations carried out at the Department of Oral and Maxillofacial Surgery, Guangxi Medical University School and Hospital of Stomatology. Cells were cultured in DMEM supplemented with 14% FBS and penicillin (50 units/mL)/streptomycin (50 μg/mL) in a humidiﬁed atmosphere (5% CO2) at 37 °C.
Reagents and their administration
To block β2-AR activation, we used the selective β2-AR blocker ICI118,551 (MedChemExpress, USA). The drug was dissolved in dimethyl sulfoxide (DMSO) and added to a mixture of culture medium to 5μmol/L concentration for vitro assay. In vivo assay, it was dissolved in water and diluted to 0.5 mg/ml concentration.
The semi-selective COX-2 inhibitor Etodolac (MACKLIN, China) was used to inhibit COX-2 activation. It was dissolved in DMSO and added to a mixture of culture medium to 5μmol/L concentration for vitro assay. In vivo assay, it was dissolved in corn oil and diluted to 12.5 mg/ml concentration.
Animals and counterbalancing
Balb/c nude mice, purchased from the Animal Research Laboratory of Guangxi Medical University, were distributed randomly into a control group, ICI118,551 treatment group, etodolac treatment group and combined treatment group, 6 mice in each group. Cal27 cells (1 × 107/mL) in 50 μL phosphate buffer saline (PBS) were subcutaneously injected into in the flank of the tongue at day 0. Tumor growth was monitored in an assessor-blind trial, and tumor size was measured every 2 days. Tumor volume was estimated using the following equation: tumor volume = ((A × B2)/2) mm3, where A is the largest dimension and B is the largest dimension perpendicular to A. ICI118,551 (5 mg/kg in a 0.5 mg/ml concentration) and Etodolac (50 mg/kg in a 12.5 mg/ml concentration) were given orally to the mice daily. PBS was given orally to the control group daily. Mice were euthanized 24 h after completion of treatment. The tumor and submandibular lymphatic node histology were analyzed. The Kaplan–Meier method was used to evaluate survival. All experimental procedures received approval by the Laboratory Animal Care and Use Committees of Guangxi Medical University.
Wound healing assay
OSCC cells were grown in 6-well plates to until reaching 100% confluence. A cell scratch-wound was established by a 200µl pipette tip. After this, the culture medium was changed to different drugs, which were diluted in DMEM or DMEM/F12. After 24h, the wound width was photographed, and the percentage of wound closure was determined based on the wound width at 0 h and the areas were measured using ImageJ software.
Transwell invasion assay
Cell invasion assays were performed using a modified 24-well chamber with a membrane which was pre-coated with Matrigel (Corning, USA). 100 µl of cells (1 × 105/ml) in FBS-free medium were seeded into the upper chamber, while different drugs were added 100μL into the upper chamber. In each lower chamber, 500μL medium containing 10% or 14% FBS was placed. After 24h, invasive cells on the undersurface of the membrane were stained with crystal violet (Thermo Fisher Scientific, USA) staining solution for 20 minutes. Quantification was measured by counting the invasive cells in five randomly selected fields.
Real time quantitative polymerase chain reaction
Total ribonucleic acid (RNA) was isolated using RNAiso Plus (Takara Bio, Japan) and according to the and reverse transcribed using PrimeScript™RT reagent Kit (Takara Bio, Japan) according to the instructions. We used SYBR®Premix Ex Taq™II Kit (Takara Bio, Japan) to detect expression of messenger ribonucleic acid (mRNA). The polymerase chain reaction (PCR) primer sequences used for each gene were showed in Table1. PCR reaction was carried out with Step One Plus TM Real-Time PCR System (Takara Bio, Japan): the first step is initial denaturation followed by 95°C for 30s, the second step is denaturation followed by 40 cycles of 95°C for 5s and 60°C for 30s. All the values were standardized with 2-ΔΔCT method.
Western blot analysis
Total protein was extracted from cells or tissues using RIPA buffer in the presence of protease inhibitor (Fdbio science, China) and phosphatase inhibitor cocktail (Fdbio science, China). Proteins at 60μg per lane. Target proteins were separated based on their molecular weight on 10% SDS-PAGE gels (Fdbio science, China) and then transferred to poly vinylidene fluoride membranes (Millipore, USA). The membranes were incubated with the appropriate primary antibody overnight at 4°C. The primary antibody used for each gene were as follows: Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (FD0063, Fdbio science, China) at 1/5000 dilution, Anti-epidermal growth factor receptor (EGFR) (ab52894, abcam, USA) at 1/1000 dilution, Anti-transforming growth factor-β1 (TGF-β1) (ab179695, abcam, USA) at 1/1000 dilution, Anti-matrix metalloproteinase 2 (MMP2) (ab92536, abcam, USA) at 1/1000 dilution, Anti-VEGF-A (ab52917, abcam, USA) at 1/5000 dilution. Then membranes were incubated in corresponding species secondary antibodies for 1h. GAPDH was used as an internal control. Protein expression levels were detected by ChemiDocM universal imager system (Bio-Rad, USA) and gray value was analyzed by Image lab software. The relative expression of target protein (%) = gray value of target protein / gray value of GAPDH.
Enzyme-linked immunosorbent assay
Cytokine interleukin-1β (IL-1β) from culture supernatants of treated cells and serum of xenograft mice were determined by an enzyme-linked immunosorbent assay (ELISA) method using an ELISA kit (Solarbio, China) according to manufactures’ introduction.
Immunohistochemical staining was performed according to a previously described protocol, with minor modifications. Tissues were processed as formalin-fixed paraffin-embedded tissue blocks, the sections were then cut into 4 μm-thick sections and dewaxed in xylene, rehydrated using an ethanol gradient in water, and rinsed with PBS. Antigen retrieval was performed by boiling in sodium citrate buffer for 15 min. The cut sections were treated with 3% hydrogen peroxide and blocked with normal goat serum at 37℃ for 30 min. The sections were incubated overnight at 4°C with anti-EGFR (bsm-33050M, Bioss, China) at 1:400, anti-TGF-β1 (bs-0086R, Bioss, China) at 1:300, anti-IL-1β (bs-6319R, Bioss, China) at 1:300, anti-MMP2 (bs-0412R, Bioss, China) at 1:400, anti-VEGFA (bs-4572R, Bioss, China) at 1:400 and anti-cytokeratin (BH0149, Bioss, China) at 1:200, followed by a conjugated secondary antibodies (ZSGB-BIO, OriGene Technologies) for 20 min at 37 °C and DAB (ZSGB-BIO, OriGene Technologies) staining.
Statistical analysis was carried out using SPSS software (version 22.0, IBM, USA) and GraphPad Prism (version 7.00, GraphPad Software, Inc., La Jolla, USA). The data are presented as mean ± standard deviation. Analysis of Variance (ANOVA) was used to compare the means of multiple groups. P-values less than 0.05 were considered statistically significant.