2.1. Cell lines
Two LSCC cell lines, AMC-HN-8 and Hep-2, were acquired from the Stem Cell Bank of the Chinese Academy of Science. Both cells were cultured in RPMI-1640 medium (HyClone, Logan, UT) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY), 1% penicillin-streptomycin (Genom Biotechnology, China), and maintained in 5% CO2 at 37°C.
Antibodies against caspase 8, caspase 3, caspase 9, cyclin-D1, cyclin-E2, phospho-Akt (Ser473), Akt, phospho-P70S6K (Thr389), P70S6K, phospho-4E-BP1, 4E-BP1, phospho-S6RP, S6RP, LC3-II, LC3-I, and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) were purchased from Sigma, USA. PI3K/mTOR inhibitors LY294002, rapamycin, NVP-BEZ235, and autophagy inhibitors 3-MA and CQ were purchased from Selleck, USA.
2.3. MTT detection for the calculation of drug IC50
The cell lines were prepared and then treated with different concentrations of LY294002 (1, 5, 10, 20, 40, 80, 160 µM), rapamycin (1, 5, 10, 20, 40, 80, 160 µM), and NVP-BEZ235 (0.1, 0.5, 1, 2, 4, 8, 16 µM) for 72 h. DMSO served as the control group. Twenty microliters of MTT (5 mg/ml) was added to each well, and all plates were further incubated for 4 h. Later, 150 µl of DMSO was added, and the OD490 values were measured. The inhibition rate was selected as the y-coordinate and the drug concentration (lnX) as the x-coordinate. The fitting curve formula Y = A * lnX + B was used to calculate the semi-inhibition rate of cells, IC50 = exp [(0.5-b)/a]. All assays were performed in six replicates, and each experiment was repeated three times.
2.4. Western blotting
Hep-2 cells were treated with or without LY294002 (7.5 µM), rapamycin (15 nM), and NVP-BEZ235 (1.5 µM), while AMC-HN-8 cells were treated with or without LY294002 (6 µM), rapamycin (15 nM), and NVP-BEZ235 (0.75 µM). Whole cell protein lysates were obtained 72 h later using RIPA lysis buffer solution (Beyotime, China). Equivalent quantities of cell lysate protein (25 µg) were exposed to SDS-PAGE and transferred to a PVDF membrane (Millipore, USA), which was blocked (with 5% nonfat milk) and incubated with antibodies described above at 4°C overnight. The protein bands were visualized by using an enhanced chemiluminescent substrate (Millipore, USA). ImageJ software was used to analyze relative protein levels and normalize the internal reference protein GAPDH.
2.5. Apoptosis assay
An Annexin V-FITC/PI Detection Kit (Invitrogen, USA) was used to examine cell apoptosis according to the manufacturer’s instructions. After treatment, the LSCC cells were collected and stained with Annexin V-FITC and PI. After 30 min of incubation in the dark, the cell apoptosis ratio was detected using FCM (Becton Dickinson, USA).
The TUNEL kit (Roche, Switzerland) was used to detect apoptotic cells in tumors according to the manufacturer’s instructions. The proportion of TUNEL-positive cells showing a brown color on each section was counted to determine the occurrence of apoptosis.
2.6. Cell cycle assay
The cell cycle was examined using the propidium iodide (PI) technique. After treatment with LY294002, rapamycin, or NVP-BEZ235 72 h, the LSCC cells were starved for 24 h in RPMI-1640 without serum. Single cell suspensions were then fixed in 75% ethanol overnight at -20°C, washed twice with PBS, and incubated with PI/RNase staining buffer (BD Biosciences). Samples were tested on a flow cytometer (BD Biosciences) and analyzed with FlowJo software (FlowJo LLC).
2.7. Invasion and migration assay with a transwell chamber
The invasive and migratory capabilities of Hep-2 and AMC-HN-8 cells following treatment with the three inhibitors were evaluated by transwell assay. For invasion, 100 µl Matrigel (Becton Dickinson, USA) was added to the bottom of each upper chamber in advance and dried for 4 h at 37°C to a gelatinous form. Then, for both experiments, 5*105/ml cells were placed in the upper chambers with LY294002, rapamycin, and NVP-BEZ235 incubated for 24 h at 37°C. The lower chambers were filled with a culture medium with 600 µl of 20% fetal bovine serum (FBS). Cells in the chambers were later fixed with 4% paraformaldehyde and stained with 0.1% crystal violet (Beyotime, China). Five random fields were selected to count, and the results were averaged and plotted.
2.8. ATG7 siRNA Interference
We transfected cells with siRNA oligos against ATG7 with the purpose of genetically inhibit autophagy. The sequences of ATG7 were as follows:
Atg7-1,5′-GATCCGGTGCTGGTTTCCTTGCTTAACTCGAGTTAAGCAAGGAAACCAGCACCTTTTTG-3′ and antisense, 5′-AATTCAAAAAGGTGCTGGTTTCCTTGCTTAACTCGAGTTAAGCAAGGAAACCAGCACCG-3′; Atg7-2: 5′-GATCCGCAGCCTCTCTATGAGTTTGACTCGAGTCAAACTCATAGAGAGGCTGCTTTTTG-3′ and antisense, 5′-AATTCAAAAAGCAGCCTCTCTATGAGTTTGACTCGAGTCAAACTCATAGAGAGGCTGCG-3′.
A total of 1 ×b105 AMC-HN-8 and Hep-2 cells were seeded in 6-well plates overnight before transfection. Cells were transfected with siRNA oligonucleotides at 4 µg/250 µl medium with 10 µl Lipofectamine 2000 (Invitrogen, USA) each well according to the manufacturer’s instructions.
2.9. Xenograft nude mice of LSCC
All male 6- to 8-week-old BALB/c nude mice (SLAC Laboratory Animal Co., Ltd, Shanghai) were inoculated with 6 ×106 Hep-2 cells before being randomly divided into four groups (n = 6 each) as follows: (1) control group; (2) NVP-BEZ235 group, oral administration of NVP-BEZ235 (30 mg/kg/d); (3) CQ group, intraperitoneal (i.p.) administration of CQ (50 mg/kg/d) ; (4) NVP-BEZ235 + CQ group, rats were treated with NVP-BEZ235 (30 mg/kg/d, p.o.) and CQ (50 mg/kg/d, i.p.) simultaneously. The tumor sizes were measured twice a week with calipers. All surviving rats were sacrificed 28 days later to collect tumors for further detection. Tumor size and tumor cell apoptosis of each group were analyzed.
2.10. Statistical analysis
Data analysis was accomplished using SPSS 22.0 software. Differences between groups were assessed by the Student’s t-test (two-tailed), and the results are shown as means ± SEM. P < 0.05 was considered statistically significant.