Cell culture and treatment. Human squamous cell carcinoma of head and neck cell line Tu686 and its corresponding high metastasis potential cell line M2 (Shanghai NuoChen Biotechnology Co., Ltd) were cultured in 10% fetal bovine serum (FBS) and Dulbecco’s Modified Eagle Medium (DMEM) F12 medium (Gibco, Grand Island, NY, USA), 100 IU/ml penicillin and 100 IU/ml streptomycin at 37 ˚C in a humidified atmosphere with 5% CO2. The appropriate treatment concentration of TGF-β1 (Recombinant human TGF-β1, Peprotech, USA) was 10 ng/ml according to previous study (7). OL (CAS no. 10596-60-1, molecular weight 540.514, Shanghai Yeyuan Biology) powder, was resuspended by precooled sterile PBS, with a final concentration of 1 mg/ml (stored at -20 ˚C), and diluted with PBS and the appropriate concentration was selected by cell viability assay. The morphological changes of cells were observed under an inverted fluorescence microscope (Leica DMI3000B, German).
MTT assay. Cell viability was detected with the 3-(4,5-dimethyl-thiazol-2-yl)-2,5 diphenyltetrazolium (MTT) assay. Tu686 cells (1×104 cells per well) were cultured in 96-well plates. The cells were adhered to the wall on the next day and were starved in serum-free medium overnight. Then the cells were exposed to preset concentrations of OL. After another 24 h, 150 µl of MTT (2 µg/ml; Sigma-Aldrich) was added for incubation at 37 ˚C. Formazan crystals were dissolved in DMSO and the absorbance was measured at 570 nm using a Beckman Coulter microplate reader.
Apoptosis assay. Cells were inoculated into six-well plates in a density of 1×105 cells/well. The next day, the medium was replaced after 5 h of TGF-β1 (10 ng/ml) and/or OL (25 µg/ml) treatment. After another 24 h, the cells were collected, centrifuged at 200 g and resuspended. Cells were then stained by Annexin V-FITC according to the protocol of Annexin V-PI apoptosis detection kit (Kaiji Biology, Jiangsu). Twenty min later, propid iumiodide (PI) was added for another 5 min. Then the cells were analyzed by flow cytometry.
Scratch and invasion assay. Tu686 cells were seeded onto each well of the six-well plates (2×105 cells per well) and allowed to grow to 90% confluence, then they were placed into serum-free medium for 24 h. The cells were scratched with a 200-µl pipette tip and the non-adherent cells were washed off with medium. Cells were then treated with PBS, TGF-β1 (10 ng/ml) and TGF-β1 (10 ng/ml) + OL (25 µg/ml). Migration of wounded cells was observed and photographed at 0 and 24 h with a microscope previously described. Three different areas in each assay were chosen to measure the distance of migrating cells. For invasion assay, 3×104 Tu686 cells in 100 µl of serum-free medium, treated with 10 ng/ml TGF-β1, or with 10 ng/ml TGF-βl + 25 µg/ml OL, were seeded in the upper chamber of Matrigel coated inserts (8 µm pores; BD Bioscience). Following incubation for 24 h, the cells which penetrated the filters were stained with gentian violet. The number of invasive cells was determined by counting all cells attached to the bottom of the inserts under an inverted microscope at ×100 magnification. Both assays were carried out in triplicate.
Western blotting. The assay was performed as previously described (17). Briefly, a total of 50 µg proteins were extracted from the cells or tissues of each group. After electrophoresis, transmembrane and blocking, the blotted membranes were incubated with anti-E-cadherin (ab40772, abcam, 1:500), anti-Vimentin (ab92547, abcam, 1:500), anti-Snail (ab216347, abcam, 1:1,000), anti-Smad2 (ab280888, abcam, 1:1000), anti-p-Smad2 (3140, Cell Signaling, 1:500), anti-Smad4 (ab40759, abcam, 1:1000), anti-MMP9 (3852, Cell Signaling, 1:1000), anti-N-Cadherin (ab76011, abcam, 1:1000), anti-HIF-1α (ab1, abcam, 1:1000), anti-AKT (9271, Cell Signaling, 1:500), anti-p-AKT (9271, Cell Signaling, 1:500), anti-PHD2 (3293, Cell Signaling, 1:1000) antibodies at 4 ˚C overnight. Subsequent to being washed, the membranes were incubated with HRP-labeled secondery antibodies (Boster Biological Engineering, Wuhan) for 1 h at room temperature. Bands were visualized by employing the BeyoECL Plus Detection system (Beyotime Institute of Biotechnology, Jiangsu, China). The intensity of protein fragments was quantified with Quantity One software (4.5.0 Basic; Bio-Rad, Hercules, CA, USA) and represented as the densitometric ratio of the targeted proteins to β-actin. All cell protein lysates were assayed in triplicate.
Xenograft tumor experiments. Male, 5 to 7-week-old BALB/c nude mice were purchased from Changzhou Cavens Animal Experiment Company and raised in SPF animal experiment center. Ten mice were divided into control and OL treatment groups. Preparation of cells for transplantation injection: the M2 cells for control group and OL group were treated with PBS and 1200 µg/ml of OL, respectively, after incubation for 24 h, the cells were collected. On the day of inoculation, tumor cells at 70%-80% confluence were trypsinized and resuspended in FBS-free culture medium. A volume of 100 µl single cell suspension was injected into the subcutaneous area of mice, and whether there were colliculus and redness in the injection area was observed. After inoculation, they were kept in SPF animal room. The lengths and widths of the tumors were measured with vernier calipers, and tumor volumes were calculated using the following formula: tumor volume = length × width2 × 0.5. Two weeks later, the mice were sacrificed and the tissues were obtained for Western blotting assay. The use of animals in the present study was complied with the Guide for the Care and Use of Laboratory Animals. This study was approved by the Ethics Committee of Wuxi Second People’s Hospital. The approval number is (2021) ethical review NO. (Y-13). All methods were reported in accordance with the ARRIVE guidelines. The tissue sections were viewed at ×100 magnification, and images were captured with a digital camera.
Statistical analysis. The statistical analysis software package (SPSS 11.5, Inc., Chicago, IL, USA) was employed for data analysis. The Student’s t-test and Mann-Whitney U test were used for the statistical analysis of data. Difference at P < 0.05 was considered to indicate a statistical significance.