2.1 Cell line and treatment
Human laryngeal carcinoma Hep-2 cells, tongue squamous cell carcinoma Cal-27 cells, cervical cancer HeLa cells were purchased from American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM (Gibco/Thermo Fisher Scientific, Beijing, China) supplemented with 10% fetal bovine serum (Gibco/Thermo Fisher Scientific), 100 U/ml penicillin and 100 µg/ml streptomycin at 37 ℃ and 5% CO2 in an atmosphere of 100% humidity.
DHA (TCI, Japan), etoposide (Sigma-Aldrich, St Louis MO, USA), 3-MA (Sigma-Aldrich), and rapamycin (Sigma-Aldrich) were dissolved in DMSO (Sigma-Aldrich) and stored at -20℃.
2.2 Bioinformatics Prediction
The Oncomine database (www.oncomine.com) was used to predict the DNA levels of inflammasome sensors in HNSCC and normal tissues. Then, Gene Expression Profiling Interactive Analysis (GEPIA) (http://gepia.cancer-pku.cn/) was employed to forecast the potential correlation between mRNA expression levels in HNSCC.
2.3 Cell Viability Assay
Hep-2 cells were seeded in 96-well plates (1 × 104 cells/well) and treated with DHA at different concentrations (5, 10, 20 and 40 µM) for 12, 24, 36, and 48 h. Cell viability was determined with Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technology, Japan) according to the manufacturer's protocol. Finally, optical density (OD) was monitored by a Multiskan Spectrum Microplate Reader (Thermo Fisher Scientific, Inc.) at 450 nm, with 650 nm as the reference wavelength. The cell viability values were calculated as previously described . IC50 values were obtained from the cytotoxicity curves using the SOFTmax PRO software.
2.4 Colony Formation Assay
Hep-2 cells were treated with or without 20.2 µM DHA for 24 h. After treatment, cells were trypsinized and replated into 60 mm dishes at 600 cells per dish. After they were cultured for 14 days, the cell colonies were fixed with chilled methanol, colored by Giemsa staining, and counted under the anatomical microscope. Cloning with a diameter not less than 60 µm is considered a clone.
2.5 Transmission Electron Microscopy
Hep-2 cells were treated with 20.2 µM DHA for 24 h, then the treated cells were collected, and fixed with 3% glutaraldehyde, postfixed with 1% OsO4 (Sangon Biotech), dehydrated in acetone, and embedded in Epon 812 (Nissin EM, Tokyo). Ultrathin sections were stained with 2.0% uranyl acetate/lead citrate, and observed under transmission electron microscope (Hitachi, Ltd., Tokyo).
2.6 Immunofluorescence Assay
Hep-2 cells were cultured for 24 h on glass coverslips in 24-well plates (2 × 105 cells/well) with or without treatment with DHA. The samples were fixed, perforated, blocked, and incubated with primary antibody at 37 ℃ for 1 h and then with corresponding secondary antibody at 37 ℃ for 1 h. The primary antibody used in this study included rabbit anti-LC3B antibody (#2775, CST, diluted at 1:400). The used secondary antibodies were Alexa Fluor1 488-conjugated donkey anti-rabbit IgG antibody (Invitrogen Life Technologies, 1:400). Cytoskeleton was stained with phalloidine (Sigma, St Louis, MO, USA) and incubated at 37℃ for 1 h. Cells were counterstained with 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI) (10 µg/ml) (Sigma, USA). Images were captured via a fluorescence microscope (Olympus BX51, Japan), and assessed by confocal microscopy.
2.7 Western Blot Analysis
These cells were seeded in 6-well plates (3 × 105 cells/well), treated as described above. Whole-cell extracts were directly lysed in SDS sample buffer (50 mM Tris-HCl pH 6.8, 1% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.01% bromophenol blue). Total protein was isolated using RIPA lysis buffer (Solarbio, China) from xenografts in mice, 3 fresh biopsy specimens of laryngeal carcinoma tissues and adjacent normal laryngeal tissues. Protein concentrations were determined by the BCA method. The primary antibodies were mouse anti-IFI16 monoclonal antibody (ab50004, Abcam, diluted at 1:1000), rabbit anti-Caspase-1 antibody(#2225, CST, diluted at 1:500), rabbit anti-IL-1β monoclonal antibody (ab2105, Abcam, diluted at 1:1000), rabbit anti-LC3B antibody (#2775, CST, diluted at 1:400), rabbit anti-Beclin-1 antibody (#3495, CST, diluted at 1:400), rabbit anti-USP33 antibody (ab71716, Abcam, diluted at 1:2000), rabbit anti-RalB antibody (ab129077, Abcam, diluted at 1:1000), rabbit anti-GAPDH polyclonal antibody (#2118, CST, diluted at 1:1000), and rabbit anti-β-actin antibody (BE0021, Bioeasy, diluted at 1:5000). The secondary antibody was goat anti-rabbit IgG-HRP (ZB-2301, ZSGB-BIO, diluted at 1:5000) and goat anti-mouce IgG-HRP (ZB-2305, ZSGB-BIO, diluted at 1:5000). The bands were detected by ECL (enhanced chemiluminescence) detection systems (Vilber, Fusion FX5 Spectra, France). The band intensity was measured by the Image-Pro Plus v6.0 software (Media Cybemetic, USA).
2.8 Patients And Tissue Specimens
Paraffin‑embedded tissue samples from 36 patients with LSCC were obtained from the dissected tissues in the archives of the Department of Otolaryngology‑Head and Neck Surgery, Bethune International Peace Hospital (Shijiazhuang, China) between 2014 and 2015.
The inclusion criteria of the patients were as follows: i) A definite pathological diagnosis of LSCC; ii) no anticancer treatment (including chemoradiotherapy or biotreatment) before laryngectomy; iii) the absence of common diseases such as diabetes, hypertension, coronary heart disease (CHD), and no history of long‑term drug use; iv) the availability of formalin‑fixed, paraffin‑embedded tissues; and v) the availability of complete clinicopathological and follow‑up data.
The 36 patients with HSCC were aged from 31 to 79 years with the mean age of 58 years. The clinicopathological characteristics of the HSCC patients are summarized in Table 2. The clinical stage of tumors was evaluated on the basis of the laryngeal cancer staging system of the American Joint Committee on Cancer (AJCC) in 2017. The surgical procedures for local LSCC involved the resection of tumor with or without the preservation of laryngeal function.
The study was approved by the Medical Ethics Institute of Bethune International Peace Hospital (Permit number: 2017-KY-02). All the samples were anonymous. Moreover, the fresh tissue specimens from 3 LSCC and the corresponding adjacent normal laryngeal tissues were collected for Western blotting at our institute in 2016. The corresponding adjacent normal laryngeal tissue with a 0.5 cm of cancer resection margin was selected during surgery. Postoperative pathology confirmed that this tissue was non‑cancerous.
2.9 Establishment Of Xenograft Tumors And Treatment Of Animals
Female BALB/c nude mice (Vital River Laboratory Animal Technology Co. Ltd., Beijing) at the age of 5–6 weeks were used. Each mouse was subcutaneously inoculated with 1 × 107 Hep-2 cells in the left inguinal area to establish the xenograft tumor. When the average tumor size reached 5 mm in diameter, the tumor-bearing mice were randomly distributed into four different groups with six animals in each group. The mice in the DHA group received intraperitoneal injection of DHA in DMSO (25 mg/kg), once daily for five consecutive days per week for 21 d. The mice in the DDP group were injected DDP (2 mg/kg) intraperitoneally once every 2 days. DHA + DDP After 2 hours of DHA injection (25 mg/kg), DDP (2 mg/kg) was injected intraperitoneally . The mice in the normal control (NC) group were intraperitoneally injected with 0.1% DMSO in physiological saline. Tumor size and body weight of each animal were measured every 5 days throughout the study. Tumor volume was calculated by the formula: V (mm3) = width2 (mm2) × length (mm) × 0.5. The inhibition rate of tumor growth was calculated by the formula (1-the average tumor weight of the experimental group / the average tumor weight of NC group) × 100%. During the treatment, no mice died from tumor loading. After 21 days of treatment, all animals were sacrificed by cervical dislocation at the termination of experiments, and the tumors were removed, weighed, fixed in 4% paraformaldehyde, and embedded in paraffin.
All animals were maintained in SPF facility with the constant temperature (22–24℃) and a dark-light cycle of 12 h/12 h, and housed in plastic cages. The protocol was approved by the Ethics Committee for Animal Experiment of Bethune International Peace Hospital (Permit number: 2017-KY-18).
2.10 Immunohistochemistry (IHC)
For histological examination, all paraffin‑embedded tissue samples were cut into 4 µm serial sections on glass slides, and baked at 70℃ for 15 min, followed by dehydration with gradient ethanol. Then, antigen retrieval was performed by heating to 121 ℃ for 3 min in 10 mmol/l citrate buffer (pH 6.0) with an autoclave. After the endogenous enzyme was inactivated by hydrogen peroxide (0.3%), the sections were incubated with normal goat serum at room temperature for 15 min, and incubated with mouse anti-IFI16 monoclonal antibody (ab50004, Abcam, diluted at 1:200), rabbit anti-Caspase-1 antibody(#2225, CST, diluted at 1:200), or rabbit anti-IL-1β monoclonal antibody (ab2105, Abcam, diluted at 1:200) overnight at 4 °C. PBS was used as the negative control for the primary antibody. The sections were rinsed with PBS for 3 times, and then incubated with the secondary antibody at 37 °C for 45 min. After they were rinsed with PBS, the sections were developed with 3, 3-diaminobenzidine (DAB) Kit (ZLI-9018, ZSGB-BIO, China) for 5–10 min, and washed with tap water. Next, the sections were counterstained with hematoxylin, desalinated by dilute hydrochloric acid, and rinsed for 5 min. Subsequently, the sections were dehydrated, cleared, mounted, and examined with a microscope. The results of immunohistochemistry were examined by 2 senior histopathologists using the double blind method. Cell cytomembrane/ cytoplasm stained with light yellow or tan were regarded as positive cells.
IHC staining was scored according to the following method: According to the staining intensity of immunohistochemistry, negative was scored as 0 point, light yellow as 1 point, moderate yellow as 2 points, and tan as 3 points. The percentage of positive cells in total cells of ≤ 5% was scored as 0, that of 6%-25% was scored as 1, that of 26%-50% was scored as 2, and that of > 50% was scored as 3 points. The judgment of protein expression is based on both the staining intensity and positive cell rate, and the product of these two values was calculated. After the multiplication of the two scores, they were divided into two groups: the high expression group was defined as the product was ≥ 3 points, and the low expression group was defined as the product was < 3 points.
2.11 Statistical Analysis
All statistical tests were performed by SPSS19.0 statistics software (SPSS, Chicago, IL). All in vitro experiments were repeated for at least three times. Data were presented as means ± SD. When more than two groups were enrolled, the mean values were compared between each two groups with one-way ANOVA or student’s t test. The IFI16 and caspase-1 expressions in laryngeal carcinoma tissues and that of IL-1β protein in tumor-xenograft from mice were analyzed by Pearson’s χ2 test. Differences with P < 0.05 were considered statistically significant.